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Nerve growth factor enhances the CRE-dependent transcriptional activity activated by nobiletin in PC12 cells.
Takito, Jiro; Kimura, Junko; Kajima, Koji; Uozumi, Nobuyuki; Watanabe, Makoto; Yokosuka, Akihito; Mimaki, Yoshihiro; Nakamura, Masanori; Ohizumi, Yasushi
Prevention and treatment of Alzheimer disease are urgent problems for elderly people in developed countries. We previously reported that nobiletin, a poly-methoxylated flavone from the citrus peel, improved the symptoms in various types of animal models of memory loss and activated the cAMP responsive element (CRE)-dependent transcription in PC12 cells. Nobiletin activated the cAMP/PKA/MEK/Erk/MAPK signaling pathway without using the TrkA signaling activated by nerve growth factor (NGF). Here, we examined the effect of combination of nobiletin and NGF on the CRE-dependent transcription in PC12 cells. Although NGF alone had little effect on the CRE-dependent transcription, NGF markedly enhanced the CRE-dependent transcription induced by nobiletin. The NGF-induced enhancement was neutralized by a TrkA antagonist, K252a. This effect of NGF was effective on the early signaling event elicited by nobiletin. These results suggested that there was crosstalk between NGF and nobiletin signaling in activating the CRE-dependent transcription in PC12 cells.
Cre-dependent DNA recombination activates a STING-dependent innate immune response
Pépin, Geneviève; Ferrand, Jonathan; Höning, Klara; Jayasekara, W. Samantha N.; Cain, Jason E.; Behlke, Mark A.; Gough, Daniel J.; G. Williams, Bryan R.; Hornung, Veit; Gantier, Michael P.
Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell–cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies. PMID:27166376
Imaging Activity in Neurons and Glia with a Polr2a-based and Cre-dependent GCaMP5G-IRES-tdTomato Reporter Mouse
Gee, J. Michael; Smith, Nathan A.; Fernandez, Fernando R.; Economo, Michael N.; Brunert, Daniela; Rothermel, Markus; Morris, S. Craig; Talbot, Amy; Palumbos, Sierra; Ichida, Jennifer M.; Shepherd, Jason D.; West, Peter J.; Wachowiak, Matt; Capecchi, Mario R.; Wilcox, Karen S.; White, John A.; Tvrdik, Petr
SUMMARY New strategies for introducing genetically encoded activity indicators into animal models facilitate the investigation of nervous system function. We have developed the PC::G5-tdT mouse line that expresses the GCaMP5G calcium indicator in a Cre-dependent fashion. Instead of targeting the ROSA26 locus, we inserted the reporter cassette nearby the ubiquitously expressed Polr2a gene without disrupting locus integrity. The indicator was tagged with IRES-tdTomato to aid detection of positive cells. This reporter system is effective in a wide range of developmental and cellular contexts. We recorded spontaneous cortical calcium waves in intact awake newborns and evaluated concentration-dependent responses to odorants in the adult olfactory bulb. Moreover, PC::G5-tdT effectively reports intracellular calcium dynamics in somas and fine processes of astrocytes and microglial cells. Through electrophysiological and behavioral analyses, we determined that GCaMP5G expression had no major impact on nervous system performance. PC::G5-tdT will be instrumental for a variety of brain mapping experiments. PMID:25155958
Solomon, M J; Glotzer, M; Lee, T H; Philippe, M; Kirschner, M W
The gradual accumulation of cyclin in the frog egg induces an abrupt and concerted activation of p34cdc2 that initiates mitosis. Activation is delayed even after the accumulation of cyclin to a critical threshold concentration. We have reproduced these unusual kinetic properties of p34cdc2 activation in vitro using bacterially expressed cyclin proteins and extracts derived from Xenopus eggs. Abrupt activation follows a lag period, the length of which is independent of the concentration of cyclin. The threshold concentration of cyclin and the length of the lag period are regulated by INH, an inhibitor of MPF activation in oocytes recently identified as a type 2A protein phosphatase. Binding to cyclin induces both tyrosine and threonine phosphorylation of the previously unphosphorylated p34cdc2, rendering it inactivated. The concerted transition into mitosis involves both a reduction in the rate of p34cdc2 phosphorylation on tyrosine and an increase in its rate of dephosphorylation.
Phosphate-Activated Cyclin-Dependent Kinase Stabilizes G1 Cyclin To Trigger Cell Cycle Entry
Menoyo, S.; Ricco, N.; Bru, S.; Hernández-Ortega, S.; Escoté, X.; Aldea, M.
G1 cyclins, in association with a cyclin-dependent kinase (CDK), are universal activators of the transcriptional G1-S machinery during entry into the cell cycle. Regulation of cyclin degradation is crucial for coordinating progression through the cell cycle, but the mechanisms that modulate cyclin stability to control cell cycle entry are still unknown. Here, we show that a lack of phosphate downregulates Cln3 cyclin and leads to G1 arrest in Saccharomyces cerevisiae. The stability of Cln3 protein is diminished in strains with low activity of Pho85, a phosphate-sensing CDK. Cln3 is an in vitro substrate of Pho85, and both proteins interact in vivo. More interestingly, cells that carry a CLN3 allele encoding aspartic acid substitutions at the sites of Pho85 phosphorylation maintain high levels of Cln3 independently of Pho85 activity. Moreover, these cells do not properly arrest in G1 in the absence of phosphate and they die prematurely. Finally, the activity of Pho85 is essential for accumulating Cln3 and for reentering the cell cycle after phosphate refeeding. Taken together, our data indicate that Cln3 is a molecular target of the Pho85 kinase that is required to modulate cell cycle entry in response to environmental changes in nutrient availability. PMID:23339867
Activation of cyclin A-dependent protein kinases during apoptosis.
Meikrantz, W; Gisselbrecht, S; Tam, S W; Schlegel, R
Apoptosis was induced in S-phase-arrested HeLa cells by staurosporine, caffeine, 6-dimethylaminopurine, and okadaic acid, agents that activate M-phase-promoting factor and induce premature mitosis in similarly treated hamster cell lines. Addition of these agents to asynchronously growing HeLa cells or to cells arrested in early G1 phase with lovastatin had little or no effect. S-phase arrest also promoted tumor necrosis factor alpha-induced apoptosis, eliminating the normal requirement for simultaneous cycloheximide treatment. For all of the apoptosis-inducing agents tested, the appearance of condensed chromatin was accompanied by 2- to 7-fold increases in cyclin A-associated histone H1 kinase activity, levels approximating the mitotic value. Where examined, both Cdc2 and Cdk2, the catalytic subunits known to associate with cyclin A, were activated. Stable overexpression of bcl-2 suppressed the apoptosis-inducing activity of all agents tested and reduced the amount of Cdc2 and Cdk2 in the nucleus, suggesting a possible mechanism by which bcl-2 inhibits the chromatin condensation characteristic of apoptosis. These findings suggest that at least one of the biochemical steps required for mitosis, activation of cyclin A-dependent protein kinases, is also an important event during apoptosis. Images PMID:8170983
Cyclin B1–Cdk1 Activation Continues after Centrosome Separation to Control Mitotic Progression
Lindqvist, Arne; van Zon, Wouter; Karlsson Rosenthal, Christina; Wolthuis, Rob M. F
Activation of cyclin B1–cyclin-dependent kinase 1 (Cdk1), triggered by a positive feedback loop at the end of G2, is the key event that initiates mitotic entry. In metaphase, anaphase-promoting complex/cyclosome–dependent destruction of cyclin B1 inactivates Cdk1 again, allowing mitotic exit and cell division. Several models describe Cdk1 activation kinetics in mitosis, but experimental data on how the activation proceeds in mitotic cells have largely been lacking. We use a novel approach to determine the temporal development of cyclin B1–Cdk1 activity in single cells. By quantifying both dephosphorylation of Cdk1 and phosphorylation of the Cdk1 target anaphase-promoting complex/cyclosome 3, we disclose how cyclin B1–Cdk1 continues to be activated after centrosome separation. Importantly, we discovered that cytoplasmic cyclin B1–Cdk1 activity can be maintained even when cyclin B1 translocates to the nucleus in prophase. These experimental data are fitted into a model describing cyclin B1–Cdk1 activation in human cells, revealing a striking resemblance to a bistable circuit. In line with the observed kinetics, cyclin B1–Cdk1 levels required to enter mitosis are lower than the amount of cyclin B1–Cdk1 needed for mitotic progression. We propose that gradually increasing cyclin B1–Cdk1 activity after centrosome separation is critical to coordinate mitotic progression. PMID:17472438
Cyclin D1 represses peroxisome proliferator-activated receptor alpha and inhibits fatty acid oxidation
Hanse, Eric A.; Mashek, Douglas G.; Mashek, Mara T.; Hendrickson, Anna M.; Mullany, Lisa K.; Albrecht, Jeffrey H.
Cyclin D1 is a cell cycle protein that promotes proliferation by mediating progression through key checkpoints in G1 phase. It is also a proto-oncogene that is commonly overexpressed in human cancers. In addition to its canonical role in controlling cell cycle progression, cyclin D1 affects other aspects of cell physiology, in part through transcriptional regulation. In this study, we find that cyclin D1 inhibits the activity of a key metabolic transcription factor, peroxisome proliferator-activated receptor α (PPARα), a member of nuclear receptor family that induces fatty acid oxidation and may play an anti-neoplastic role. In primary hepatocytes, cyclin D1 inhibits PPARα transcriptional activity and target gene expression in a cdk4-independent manner. In liver and breast cancer cells, knockdown of cyclin D1 leads to increased PPARα transcriptional activity, expression of PPARα target genes, and fatty acid oxidation. Similarly, cyclin D1 depletion enhances binding of PPARα to target sequences by chromatin immunoprecipitation. In proliferating hepatocytes and regenerating liver in vivo, induction of endogenous cyclin D1 is associated with diminished PPARα activity. Cyclin D1 expression is both necessary and sufficient for growth factor-mediated repression of fatty acid oxidation in proliferating hepatocytes. These studies indicate that in addition to playing a pivotal role in cell cycle progression, cyclin D1 represses PPARα activity and inhibits fatty acid oxidation. Our findings establish a new link between cyclin D1 and metabolism in both tumor cells and physiologic hepatocyte proliferation. PMID:27351284
Cyclin D1 represses peroxisome proliferator-activated receptor alpha and inhibits fatty acid oxidation.
Kamarajugadda, Sushama; Becker, Jennifer R; Hanse, Eric A; Mashek, Douglas G; Mashek, Mara T; Hendrickson, Anna M; Mullany, Lisa K; Albrecht, Jeffrey H
Cyclin D1 is a cell cycle protein that promotes proliferation by mediating progression through key checkpoints in G1 phase. It is also a proto-oncogene that is commonly overexpressed in human cancers. In addition to its canonical role in controlling cell cycle progression, cyclin D1 affects other aspects of cell physiology, in part through transcriptional regulation. In this study, we find that cyclin D1 inhibits the activity of a key metabolic transcription factor, peroxisome proliferator-activated receptor α (PPARα), a member of nuclear receptor family that induces fatty acid oxidation and may play an anti-neoplastic role. In primary hepatocytes, cyclin D1 inhibits PPARα transcriptional activity and target gene expression in a cdk4-independent manner. In liver and breast cancer cells, knockdown of cyclin D1 leads to increased PPARα transcriptional activity, expression of PPARα target genes, and fatty acid oxidation. Similarly, cyclin D1 depletion enhances binding of PPARα to target sequences by chromatin immunoprecipitation. In proliferating hepatocytes and regenerating liver in vivo, induction of endogenous cyclin D1 is associated with diminished PPARα activity. Cyclin D1 expression is both necessary and sufficient for growth factor-mediated repression of fatty acid oxidation in proliferating hepatocytes. These studies indicate that in addition to playing a pivotal role in cell cycle progression, cyclin D1 represses PPARα activity and inhibits fatty acid oxidation. Our findings establish a new link between cyclin D1 and metabolism in both tumor cells and physiologic hepatocyte proliferation.
The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.
Sudakin, V; Ganoth, D; Dahan, A; Heller, H; Hershko, J; Luca, F C; Ruderman, J V; Hershko, A
The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles. Images PMID:7787245
Cyclin F suppresses B-Myb activity to promote cell cycle checkpoint control.
Klein, Ditte Kjærsgaard; Hoffmann, Saskia; Ahlskog, Johanna K; O'Hanlon, Karen; Quaas, Marianne; Larsen, Brian D; Rolland, Baptiste; Rösner, Heike I; Walter, David; Kousholt, Arne Nedergaard; Menzel, Tobias; Lees, Michael; Johansen, Jens Vilstrup; Rappsilber, Juri; Engeland, Kurt; Sørensen, Claus Storgaard
Cells respond to DNA damage by activating cell cycle checkpoints to delay proliferation and facilitate DNA repair. Here, to uncover new checkpoint regulators, we perform RNA interference screening targeting genes involved in ubiquitylation processes. We show that the F-box protein cyclin F plays an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein cyclin F with suppression of the B-Myb/cyclin A pathway to ensure a DNA damage-induced checkpoint response in G2.
The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles.
Honda, Reiko; Lowe, Edward D; Dubinina, Elena; Skamnaki, Vicky; Cook, Atlanta; Brown, Nick R; Johnson, Louise N
Cyclin E, an activator of phospho-CDK2 (pCDK2), is important for cell cycle progression in metazoans and is frequently overexpressed in cancer cells. It is essential for entry to the cell cycle from G0 quiescent phase, for the assembly of prereplication complexes and for endoreduplication in megakaryotes and giant trophoblast cells. We report the crystal structure of pCDK2 in complex with a truncated cyclin E1 (residues 81-363) at 2.25 A resolution. The N-terminal cyclin box fold of cyclin E1 is similar to that of cyclin A and promotes identical changes in pCDK2 that lead to kinase activation. The C-terminal cyclin box fold shows significant differences from cyclin A. It makes additional interactions with pCDK2, especially in the region of the activation segment, and contributes to CDK2-independent binding sites of cyclin E. Kinetic analysis with model peptide substrates show a 1.6-fold increase in kcat for pCDK2/cyclin E1 (81-363) over kcat of pCDK2/cyclin E (full length) and pCDK2/cyclin A. The structural and kinetic results indicate no inherent substrate discrimination between pCDK2/cyclin E and pCDK2/cyclin A with model substrates.
Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos
Boulben, Sandrine; Glippa, Virginie; Morales, Julia; Cormier, Patrick
The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism. PMID:26962866
Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos.
Chassé, Héloïse; Mulner-Lorillon, Odile; Boulben, Sandrine; Glippa, Virginie; Morales, Julia; Cormier, Patrick
The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism.
Reversible phosphorylation controls the activity of cyclosome-associated cyclin-ubiquitin ligase.
Lahav-Baratz, S; Sudakin, V; Ruderman, J V; Hershko, A
Cyclin B/cdc2 is responsible both for driving cells into mitosis and for activating the ubiquitin-dependent degradation of mitotic cyclins near the end of mitosis, an event required for the completion of mitosis and entry into interphase of the next cell cycle. Previous work with cell-free extracts of rapidly dividing clam embryos has identified two specific components required for the ubiquitination of mitotic cyclins: E2-C, a cyclin-selective ubiquitin carrier protein that is constitutively active during the cell cycle, and E3-C, a cyclin-selective ubiquitin ligase that purifies as part of a approximately 1500-kDa complex, termed the cyclosome, and which is active only near the end of mitosis. Here, we have separated the cyclosome from its ultimate upstream activator, cdc2. The mitotic, active form of the cyclosome can be inactivated by incubation with a partially purified, endogenous okadaic acid-sensitive phosphatase; addition of cdc2 restores activity to the cyclosome after a lag that reproduces that seen previously in intact cells and in crude extracts. These results demonstrate that activity of cyclin-ubiquitin ligase is controlled by reversible phosphorylation of the cyclosome complex. Images Fig. 3 PMID:7568122
Binding Activity Prediction of Cyclin-Dependent Inhibitors.
Saha, Indrajit; Rak, Benedykt; Bhowmick, Shib Sankar; Maulik, Ujjwal; Bhattacharjee, Debotosh; Koch, Uwe; Lazniewski, Michal; Plewczynski, Dariusz
The Cyclin-Dependent Kinases (CDKs) are the core components coordinating eukaryotic cell division cycle. Generally the crystal structure of CDKs provides information on possible molecular mechanisms of ligand binding. However, reliable and robust estimation of ligand binding activity has been a challenging task in drug design. In this regard, various machine learning techniques, such as Support Vector Machine, Naive Bayesian classifier, Decision Tree, and K-Nearest Neighbor classifier, have been used. The performance of these heterogeneous classification techniques depends on proper selection of features from the data set. This fact motivated us to propose an integrated classification technique using Genetic Algorithm (GA), Rotational Feature Selection (RFS) scheme, and Ensemble of Machine Learning methods, named as the Genetic Algorithm integrated Rotational Ensemble based classification technique, for the prediction of ligand binding activity of CDKs. This technique can automatically find the important features and the ensemble size. For this purpose, GA encodes the features and ensemble size in a chromosome as a binary string. Such encoded features are then used to create diverse sets of training points using RFS in order to train the machine learning method multiple times. The RFS scheme works on Principal Component Analysis (PCA) to preserve the variability information of the rotational nonoverlapping subsets of original data. Thereafter, the testing points are fed to the different instances of trained machine learning method in order to produce the ensemble result. Here accuracy is computed as a final result after 10-fold cross validation, which also used as an objective function for GA to maximize. The effectiveness of the proposed classification technique has been demonstrated quantitatively and visually in comparison with different machine learning methods for 16 ligand binding CDK docking and rescoring data sets. In addition, the best possible features
Cyclin A2 Mutagenesis Analysis: A New Insight into CDK Activation and Cellular Localization Requirements
Bendris, Nawal; Lemmers, Bénédicte; Blanchard, Jean-Marie; Arsic, Nikola
Cyclin A2 is essential at two critical points in the somatic cell cycle: during S phase, when it activates CDK2, and during the G2 to M transition when it activates CDK1. Based on the crystal structure of Cyclin A2 in association with CDKs, we generated a panel of mutants to characterize the specific amino acids required for partner binding, CDK activation and subcellular localization. We find that CDK1, CDK2, p21, p27 and p107 have overlapping but distinct requirements for association with this protein. Our data highlight the crucial importance of the N-terminal α helix, in conjunction with the α3 helix within the cyclin box, in activating CDK. Several Cyclin A2 mutants selectively bind to either CDK1 or CDK2. We demonstrate that association of Cyclin A2 to proteins such as CDK2 that was previously suggested as crucial is not a prerequisite for its nuclear localization, and we propose that the whole protein structure is involved. PMID:21829545
Cyclin D activates the Rb tumor suppressor by mono-phosphorylation.
Narasimha, Anil M; Kaulich, Manuel; Shapiro, Gary S; Choi, Yoon J; Sicinski, Piotr; Dowdy, Steven F
The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, to release E2F transcription factors. However, this model remains unproven biochemically and the biologically active form(s) of Rb remains unknown. In this study, we find that Rb is exclusively mono-phosphorylated in early G1 phase by cyclin D:Cdk4/6. Mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but show preferential E2F binding patterns. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by quantum hyper-phosphorylation. Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription, whereas cells undergoing differentiation utilize un-phosphorylated Rb. These observations fundamentally change our understanding of G1 cell cycle progression and show that mono-phosphorylated Rb, generated by cyclin D:Cdk4/6, is the only Rb isoform in early G1 phase.
Cyclin I-like (CCNI2) is a cyclin-dependent kinase 5 (CDK5) activator and is involved in cell cycle regulation
Liu, Chengcheng; Zhai, Xiaoyan; Zhao, Bin; Wang, Yanfei; Xu, Zhigang
In contrast to conventional cyclin-dependent kinases that are important for mitotic cell division, cyclin-dependent kinase 5 (CDK5) is predominantly activated in post-mitotic cells and is involved in various cellular events. The kinase activity of CDK5 is tightly regulated by specific activators including p35, p39, and cyclin I (CCNI). Here we show that cyclin I-like (CCNI2), a homolog of CCNI, interacts with CDK5 and activates the kinase activity of CDK5. Different from CCNI, which colocalizes with CDK5 in the nuclei in transfected cells, CCNI2 mainly retains CDK5 in the cytoplasm as well as on the cell membrane. Furthermore, although the expression level of CCNI2 mRNA and CCNI2 protein do not change significantly during cell cycle, depletion of CCNI2 with siRNA affects cell cycle progression as well as cell proliferation. In conclusion, our data strongly suggest that CCNI2 is a novel CDK5 activator and is involved in cell cycle regulation. PMID:28112194
Pesticide Roundup provokes cell division dysfunction at the level of CDK1/cyclin B activation.
Marc, Julie; Mulner-Lorillon, Odile; Boulben, Sandrine; Hureau, Dorothée; Durand, Gaël; Bellé, Robert
To assess human health risk from environmental chemicals, we have studied the effect on cell cycle regulation of the widely used glyphosate-containing pesticide Roundup. As a model system we have used sea urchin embryonic first divisions following fertilization, which are appropriate for the study of universal cell cycle regulation without interference with transcription. We show that 0.8% Roundup (containing 8 mM glyphosate) induces a delay in the kinetic of the first cell cleavage of sea urchin embryos. The delay is dependent on the concentration of Roundup. The delay in the cell cycle could be induced using increasing glyphosate concentrations (1-10 mM) in the presence of a subthreshold concentration of Roundup 0.2%, while glyphosate alone was ineffective, thus indicating synergy between glyphosate and Roundup formulation products. The effect of Roundup was not lethal and involved a delay in entry into M-phase of the cell cycle, as judged cytologically. Since CDK1/cyclin B regulates universally the M-phase of the cell cycle, we analyzed CDK1/cyclin B activation during the first division of early development. Roundup delayed the activation of CDK1/cyclin B in vivo. Roundup inhibited also the global protein synthetic rate without preventing the accumulation of cyclin B. In summary, Roundup affects cell cycle regulation by delaying activation of the CDK1/cyclin B complex, by synergic effect of glyphosate and formulation products. Considering the universality among species of the CDK1/cyclin B regulator, our results question the safety of glyphosate and Roundup on human health.
Characterization of a new family of cyclin-dependent kinase activators
Progression through the cell cycle is regulated by CDKs (cyclin-dependent kinases), which associate with activating partners, named cyclins, to efficiently phosphorylate substrates. We previously reported the identification of RINGO, a Xenopus protein that can activate CDK1 and CDK2 despite lack of sequence similarity to cyclins, which plays a role in the regulation of the meiotic cell cycle in oocytes. In the present study we report the characterization of four mammalian RINGO proteins, which are 53–68% identical with Xenopus RINGO in a central core of about 75 residues. We show that all RINGO family members can bind to and activate CDK1 and CDK2, albeit with different efficiencies, but they do not bind to CDK4 or CDK6. The core RINGO sequences are critical for CDK activation. We also identified key residues in CDK2 that are required for RINGO binding. All RINGO proteins can also bind the CDK inhibitor p27Kip1, but with an inverse efficiency of their ability to bind to CDK1. Our results identify a new family of mammalian proteins that can activate CDKs and therefore potentially function as cell cycle regulators. The ability of RINGO proteins to activate CDK1 and CDK2 suggest also cyclin-independent roles for these kinases. PMID:15574121
Mitochondrial Ribosomal Protein L10 Associates with Cyclin B1/Cdk1 Activity and Mitochondrial Function
Li, Hai-Bo; Wang, Ruo-Xi; Jiang, Hai-Bo; Zhang, En-dong; Tan, Jie-Qiong; Xu, Hui-Zhuo
Mitochondrial ribosomal proteins are important for mitochondrial-encoded protein synthesis and mitochondrial function. In addition to their roles in mitoribosome assembly, several mitochondrial ribosome proteins are also implicated in cellular processes like cell cycle regulation, apoptosis, and mitochondrial homeostasis regulation. Here, we demonstrate that MRPL10 regulates cyclin B1/Cdk1 (cyclin-dependent kinase 1) activity and mitochondrial protein synthesis in mammalian cells. In Drosophila, inactivation of mRpL10 (the Drosophila ortholog of mammalian MRPL10) in eyes results in abnormal eye development. Furthermore, expression of human cyclin B1 suppresses eye phenotypes and mitochondrial abnormality of mRpL10 knockdown Drosophila. This study identified that the physiological regulatory pathway of MRPL10 and providing new insights into the role of MRPL10 in growth control and mitochondrial function. PMID:27726420
c-Jun induces apoptosis of starved BM2 monoblasts by activating cyclin A-CDK2
Vanhara, Petr; Bryja, Vitezslav; Horvath, Viktor; Kozubik, Alois; Hampl, Ales; Smarda, Jan . E-mail: firstname.lastname@example.com
c-Jun is one of the major components of the activating protein-1 (AP-1), the transcription factor that participates in regulation of proliferation, differentiation, and apoptosis. In this study, we explored functional interactions of the c-Jun protein with several regulators of the G1/S transition in serum-deprived v-myb-transformed chicken monoblasts BM2. We show that the c-Jun protein induces expression of cyclin A, thus up-regulating activity of cyclin A-associated cyclin-dependent kinase 2 (CDK2), and causing massive programmed cell death of starved BM2cJUN cells. Specific inhibition of CDK2 suppresses frequency of apoptosis of BM2cJUN cells. We conclude that up-regulation of cyclin A expression and CDK2 activity can represent important link between the c-Jun protein, cell cycle machinery, and programmed cell death pathway in leukemic cells.
Copper Uptake in Mammary Epithelial Cells Activates Cyclins and Triggers Antioxidant Response.
dos Santos, Nathália Villa; Matias, Andreza Cândido; Higa, Guilherme Shigueto Vilar; Kihara, Alexandre Hiroaki; Cerchiaro, Giselle
The toxicologic effects of copper (Cu) on tumor cells have been studied during the past decades, and it is suggested that Cu ion may trigger antiproliferative effects in vitro. However, in normal cells the toxicologic effects of high exposures of free Cu are not well understood. In this work, Cu uptake, the expression of genes associated with cell cycle regulation, and the levels of ROS production and related oxidative processes were evaluated in Cu-treated mammary epithelial MCF10A nontumoral cells. We have shown that the Cu additive is associated with the activation of cyclin D1 and cyclin B1, as well as cyclin-dependent kinase 2 (CDK2). These nontumor cells respond to Cu-induced changes in the oxidative balance by increase of the levels of reduced intracellular glutathione (GSH), decrease of reactive oxygen species (ROS) generation, and accumulation during progression of the cell cycle, thus preventing the cell abnormal proliferation or death. Taken together, our findings revealed an effect that contributes to prevent a possible damage of normal cells exposed to chemotherapeutic effects of drugs containing the Cu ion.
Protocatechualdehyde possesses anti-cancer activity through downregulating cyclin D1 and HDAC2 in human colorectal cancer cells
Jeong, Jin Boo; Lee, Seong-Ho
Highlights: Black-Right-Pointing-Pointer Protocatechualdehyde (PCA) suppressed cell proliferation and induced apoptosis in human colorectal cancer cells. Black-Right-Pointing-Pointer PCA enhanced transcriptional downregulation of cyclin D1 gene. Black-Right-Pointing-Pointer PCA suppressed HDAC2 expression and activity. Black-Right-Pointing-Pointer These findings suggest that anti-cancer activity of PCA may be mediated by reducing HDAC2-derived cyclin D1 expression. -- Abstract: Protocatechualdehyde (PCA) is a naturally occurring polyphenol found in barley, green cavendish bananas, and grapevine leaves. Although a few studies reported growth-inhibitory activity of PCA in breast and leukemia cancer cells, the underlying mechanisms are still poorly understood. Thus, we performed in vitro study to investigate if treatment of PCA affects cell proliferation and apoptosis in human colorectal cancer cells and define potential mechanisms by which PCA mediates growth arrest and apoptosis of cancer cells. Exposure of PCA to human colorectal cancer cells (HCT116 and SW480 cells) suppressed cell growth and induced apoptosis in dose-dependent manner. PCA decreased cyclin D1 expression in protein and mRNA level and suppressed luciferase activity of cyclin D1 promoter, indicating transcriptional downregulation of cyclin D1 gene by PCA. We also observed that PCA treatment attenuated enzyme activity of histone deacetylase (HDAC) and reduced expression of HDAC2, but not HDAC1. These findings suggest that cell growth inhibition and apoptosis by PCA may be a result of HDAC2-mediated cyclin D1 suppression.
Cyclin A-dependent phosphorylation of the ETS-related protein, MEF, restricts its activity to the G1 phase of the cell cycle.
Miyazaki, Y; Boccuni, P; Mao, S; Zhang, J; Erdjument-Bromage, H; Tempst, P; Kiyokawa, H; Nimer, S D
MEF, a recently identified member of the E74 family of ETS-related transcription factors, is a strong transcriptional activator of cytokine gene expression. Using a green fluorescent protein gene reporter plasmid regulated by an MEF-responsive promoter, we determined that the transcriptional activity of MEF is largely restricted to the G1 phase of the cell cycle. MEF-dependent transcription was suppressed by the expression of cyclin A but not by cyclin D or cyclin E. This effect was due to the kinase activity generated by cyclin A expression, as co-expression of the cyclin-dependent kinase inhibitors p21 or p27, or a dominant negative form of CDK2 (DNK2), abrogated the reduction of MEF transcriptional activity by cyclin A. Cyclin A-CDK2 phosphorylated MEF protein in vitro more efficiently than cyclin D-CDK4 or cyclin E-CDK2, and phosphorylation of MEF by cyclin A-CDK2 reduced its ability to bind DNA. We determined one site of phosphorylation by cyclin A-CDK2 at the C terminus of MEF, using mass-spectrometry; mutation of three serine or threonine residues in this region significantly reduced phosphorylation of MEF by cyclin A and reduced cyclin A-mediated suppression of its transactivating activity. These amino acid substitutions also reduced the restriction of MEF activity to G1. Phosphorylation of MEF by the cyclin A-CDK2 complex controls its transcriptional activity during the cell cycle, establishing a novel link between the ETS family of proteins and the cell cycle machinery.
Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation.
Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin
Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kappaB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chk1 and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.
Cell-type specific circuit connectivity of hippocampal CA1 revealed through Cre-dependent rabies tracing
Sun, Yanjun; Nguyen, Amanda; Nguyen, Joseph; Le, Luc; Saur, Dieter; Choi, Jiwon; Callaway, Edward M.; Xu, Xiangmin
Summary We applied a new Cre-dependent, genetically modified rabies-based tracing system to map direct synaptic connections to CA1 excitatory and inhibitory neuron types in mouse hippocampus. We found common inputs to excitatory and inhibitory CA1 neurons from CA3, CA2, entorhinal cortex and the medial septum (MS), and unexpectedly also from the subiculum. Excitatory CA1 neurons receive inputs from both cholinergic and GABAergic MS neurons while inhibitory CA1 neurons receive a great majority of input from GABAergic MS neurons; both cell types also receive weaker input from glutamatergic MS neurons. Comparisons of inputs to CA1 PV+ interneurons versus SOM+ interneurons showed similar strengths of input from the subiculum, but PV+ interneurons receive much stronger input than SOM+ neurons from CA3, entorhinal cortex and MS. Differential input from CA3 to specific CA1 cell types was also demonstrated functionally using laser scanning photostimulation and whole cell recordings. PMID:24656815
Cell-type-specific circuit connectivity of hippocampal CA1 revealed through Cre-dependent rabies tracing.
Sun, Yanjun; Nguyen, Amanda Q; Nguyen, Joseph P; Le, Luc; Saur, Dieter; Choi, Jiwon; Callaway, Edward M; Xu, Xiangmin
We developed and applied a Cre-dependent, genetically modified rabies-based tracing system to map direct synaptic connections to specific CA1 neuron types in the mouse hippocampus. We found common inputs to excitatory and inhibitory CA1 neurons from CA3, CA2, the entorhinal cortex (EC), the medial septum (MS), and, unexpectedly, the subiculum. Excitatory CA1 neurons receive inputs from both cholinergic and GABAergic MS neurons, whereas inhibitory neurons receive a great majority of inputs from GABAergic MS neurons. Both cell types also receive weaker input from glutamatergic MS neurons. Comparisons of inputs to CA1 PV+ interneurons versus SOM+ interneurons showed similar strengths of input from the subiculum, but PV+ interneurons received much stronger input than SOM+ neurons from CA3, the EC, and the MS. Thus, rabies tracing identifies hippocampal circuit connections and maps how the different input sources to CA1 are distributed with different strengths on each of its constituent cell types.
Activation of the EGFR/Akt/NF-κB/cyclinD1 survival signaling pathway in human cholesteatoma epithelium.
Liu, Wei; Yin, Tuanfang; Ren, Jihao; Li, Lihua; Xiao, Zian; Chen, Xing; Xie, Dinghua
Cholesteatoma is a benign keratinizing squamous epithelial lesion characterized by the hyper-proliferation of keratinocytes with abundant production of keratin debris in the middle ear. The epidermal growth factor receptor (EGFR)/Akt/nuclear factor-kappa B (NF-κB)/cyclinD1 signaling pathway is one of the most important pathways in regulating cell survival and proliferation. We hypothesized that the EGFR/Akt/NF-κB/cyclinD1 signaling pathway may be activated and involved in the cellular hyperplasia mechanism in acquired cholesteatoma epithelium. Immunohistochemical staining of phosphorylated EGFR (p-EGFR), phosphorylated Akt (p-Akt), activated NF-κB and cyclinD1 protein was performed in 40 cholesteatoma samples and 20 samples of normal external auditory canal (EAC) epithelium. Protein expression of p-EGFR, p-Akt, activated NF-κB and cyclinD1 in cholesteatoma epithelium was significantly increased when compared with normal EAC epithelium (p < 0.01). In cholesteatoma epithelium, a significant positive association was observed between p-EGFR and p-Akt expression and between the expressions of p-Akt and NF-κB, NF-κB and cyclinD1, respectively (p < 0.01). No significant relationships were observed between the levels of investigated proteins and the degree of bone destruction (p > 0.05). The increased protein expression of p-EGFR, p-Akt, NF-κB and cyclinD1 and their associations in cholesteatoma epithelium suggest that the EGFR/Akt/NF-κB/cyclinD1 survival signaling pathway is active and may be involved in the regulatory mechanisms of cellular hyperplasia in cholesteatoma epithelium.
Cyclin B-cdk activity stimulates meiotic rereplication in budding yeast.
Strich, Randy; Mallory, Michael J; Jarnik, Michal; Cooper, Katrina F
Haploidization of gametes during meiosis requires a single round of premeiotic DNA replication (meiS) followed by two successive nuclear divisions. This study demonstrates that ectopic activation of cyclin B/cyclin-dependent kinase in budding yeast recruits up to 30% of meiotic cells to execute one to three additional rounds of meiS. Rereplication occurs prior to the meiotic nuclear divisions, indicating that this process is different from the postmeiotic mitoses observed in other fungi. The cells with overreplicated DNA produced asci containing up to 20 spores that were viable and haploid and demonstrated Mendelian marker segregation. Genetic tests indicated that these cells executed the meiosis I reductional division and possessed a spindle checkpoint. Finally, interfering with normal synaptonemal complex formation or recombination increased the efficiency of rereplication. These studies indicate that the block to rereplication is very different in meiotic and mitotic cells and suggest a negative role for the recombination machinery in allowing rereplication. Moreover, the production of haploids, regardless of the genome content, suggests that the cell counts replication cycles, not chromosomes, in determining the number of nuclear divisions to execute. PMID:15342503
Signal transduction pathways that contribute to CDK1/cyclin B activation during the first mitotic division in sea urchin embryos.
Salaün, Patrick; Le Breton, Magali; Morales, Julia; Bellé, Robert; Boulben, Sandrine; Mulner-Lorillon, Odile; Cormier, Patrick
In sea urchins, fertilization triggers a rapid rise in protein synthesis necessary for activation of CDK1/cyclin B, the universal cell cycle regulator. It has been shown that FRAP/mTOR is required for eIF4E release from the translational repressor 4E-BP, a process that occurs upstream of de novo cyclin B synthesis. Here, we investigate whether PI 3-kinase acts independently or upstream from FRAP/mTOR in the signal transduction pathway that links fertilization to the activation of the CDK1/cyclin B complex in sea urchin egg. We found that wortmannin, a potent inhibitor of PI 3-kinase, partially inhibited the global increase in protein synthesis triggered by fertilization. Furthermore, wortmannin treatment induced partial inhibition of cyclin B translation triggered by fertilization, in correlation with an intermediate effect of the drug on 4E-BP degradation and on the dissociation of the 4E-BP/eIF4E complex induced by fertilization. Our results presented here suggest that PI 3-kinase activity is required for completion of mitotic divisions of the sea urchin embryo. Incubation of eggs with wortmannin or microinjection of wortmannin or LY 294002 affects drastically mitotic divisions induced by fertilization. In addition, we found that wortmannin treatment inhibits dephosphorylation of the tyrosine inhibitory site of CDK1. Taken together, these data suggest that PI 3-kinase acts upstream of at least two independent targets that function in the CDK1/cyclin B activation triggered by fertilization of sea urchin oocytes. We discuss the significance of these results concerning the cascade of reactions that impinge upon the activation of the CDK1/cyclin B complex that follows sea urchin oocyte fertilization.
Adapalene inhibits the activity of cyclin-dependent kinase 2 in colorectal carcinoma
Cyclin-dependent kinase 2 (CDK2) has been reported to be overexpressed in human colorectal cancer; it is responsible for the G1-to-S-phase transition in the cell cycle and its deregulation is a hallmark of cancer. The present study was the first to use idock, a free and open-source protein-ligand docking software developed by our group, to identify potential CDK2 inhibitors from 4,311 US Food and Drug Administration-approved small molecular drugs with a re-purposing strategy. Among the top compounds identified by idock score, nine were selected for further study. Among them, adapalene (ADA; CD271,6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphtoic acid) exhibited the highest anti-proliferative effects in LoVo and DLD1 human colon cancer cell lines. Consistent with the expected properties of CDK2 inhibitors, the present study demonstrated that ADA significantly increased the G1-phase population and decreased the expression of CDK2, cyclin E and retinoblastoma protein (Rb), as well as the phosphorylation of CDK2 (on Thr-160) and Rb (on Ser-795). Furthermore, the anti-cancer effects of ADA were examined in vivo on xenograft tumors derived from DLD1 human colorectal cancer cells subcutaneously inoculated in BALB/C nude mice. ADA (20 mg/kg orally) exhibited marked anti-tumor activity, comparable to that of oxaliplatin (40 mg/kg), and dose-dependently inhibited tumor growth (P<0.05), while combined administration of ADA and oxaliplatin produced the highest therapeutic effect. To the best of our knowledge, the present study was the first to indicate that ADA inhibits CDK2 and is a potential candidate drug for the treatment of human colorectal cancer. PMID:26398439
Actin interaction and regulation of cyclin-dependent kinase 5/p35 complex activity.
Xu, Jiqing; Tsutsumi, Koji; Tokuraku, Kiyotaka; Estes, Katherine A; Hisanaga, Shin-ichi; Ikezu, Tsuneya
Cyclin-dependent kinase 5 (Cdk5) plays a critical role during neurodevelopment, synaptic plasticity, and neurodegeneration. Cdk5 activity depends on association with neuronal proteins p35 and p25, a proteolytic product of p35. Cdk5 regulates the actin cytoskeletal dynamics that are essential for neuronal migration, neuritic growth, and synaptogenesis. However, little is known about the interaction of actin and Cdk5 and its effect on neuronal Cdk5 activity. In a previous study, we observed that Cdk5/p35 activity is negatively correlated with co-immunoprecipitated F-actin (filamentous actin) amounts in the mouse brain, and suggested that F-actin inhibits the formation of the Cdk5/p35 complex [Journal of Neuroscience (2008) vol. 28, p. 14511]. The experiments reported here were undertaken to elucidate the relationship between actin and the formation of the Cdk5/p35 complex and its activity. Instead of an F-actin-mediated inhibition, we propose that G-actin (globular actin) in the F-actin preparations is responsible for inhibiting Cdk5/p35 and Cdk5/p25 kinase activity. We found that F-actin binds to p35 but not p25 or Cdk5. We have shown that G-actin binds directly to Cdk5 without disrupting the formation of the Cdk5/p35 or Cdk5/p25 complexes. G-actin potently suppressed Cdk5/p35 and Cdk5/p25 activity when either histone H1 or purified human tau protein were used as substrates, indicating a substrate-independent inhibitory effect of G-actin on Cdk5 activity. Finally, G-actin suppressed the activity of Cdk5 immunoprecipitated from wild type and p35-deficient mouse brain, suggesting that G-actin suppresses endogenous Cdk5 activity in a p35-independent manner. Together, these results suggest a novel mechanism of actin cytoskeletal regulation of Cdk5/p35 activity.
[Bacteriostatic and bactericidal activities of cyclines, macrolides and fluoroquinolones against Chlamydia trachomatis].
Dailloux, M; Villemain, P
The in vitro activity of minocycline, doxycycline, erythromycin, roxithromycin, spiramycin, pefloxacin, and ofloxacin against ten C. trachomatis strains recovered from human genital tract specimens was evaluated. Mac Coy cell monolayers in 24-microwell plates were used. The C. trachomatis inoculum was 10(4) IFU/well. Appropriate dilutions of antibiotic were added and inclusions were detected by immunofluorescence using monoclonal antibodies. MICs were determined after 48 hours of exposure to each antimicrobial. The MIC90 for cyclines was 0.2 mg/l. Among tested macrolides, roxithromycin had a lower MIC than erythromycin (0.2 versus 0.4 mg/l) whereas spiramycin inhibited growth only in a concentration of 2 mg/l. Ofloxacin showed better activity than pefloxacin. Bactericidal activity was evaluated by determining two parameters: MBC1 (without transfer to new cells) measured the ability of a C. trachomatis particle to persist in a latent form within cells exposed to an antibiotic and to grow again following removal of the antibiotic, whereas MBC2 (with transfer to new cells) reflected infectivity of the bacteria after 48 hours exposure to the antimicrobial. None of the tested antibiotics was bactericidal according to both parameters. The ability of C. trachomatis to remain within antibiotic-exposed cells in a latent form was clearly demonstrated by the high MBC1 values. This feature may explain why recurrences are common in clinical practice.
Cyclin C regulates adipogenesis by stimulating transcriptional activity of CCAAT/enhancer binding protein alpha.
Song, Ziyi; Xiaoli, Alus M; Zhang, Quanwei; Zhang, Yi; Yang, Ellen S T; Wang, Sven; Chang, Rui; Zhang, Zhengdong D; Yang, Gongshe; Strich, Randy; Pessin, Jeffrey E; Yang, Fajun
Brown adipose tissue (BAT) is important for maintaining energy homeostasis and adaptive thermogenesis in rodents and humans. As disorders arising from dysregulated energy metabolism, such as obesity and metabolic diseases, have increased, so has interest in the molecular mechanisms in adipocyte biology. Using a functional screen, we identified cyclin C (CycC), a conserved subunit of the Mediator complex, as a novel regulator for brown adipocyte formation. siRNA-mediated CycC knockdown (KD) in brown preadipocytes impaired the early transcriptional program of differentiation, and genetic knockout (KO) of CycC completely blocked the differentiation process. RNA-seq analyses of CycC-KD revealed a critical role of CycC in activating genes co-regulated by peroxisome proliferator activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein alpha (C/EBPα). Overexpression of PPARγ2 or addition of the PPARγ ligand rosiglitazone rescued the defects in CycC-KO brown preadipocytes, and efficiently activated the PPARγ-responsive promoters in both wild-type (WT) and CycC-KO cells, suggesting that CycC is not essential for PPARγ transcriptional activity. In contrast, CycC-KO significantly reduced C/EBPα-dependent gene expression. Unlike for PPARγ, overexpression of C/EBPα could not induce C/EBPα target gene expression in CycC-KO cells or rescue the CycC-KO defects in brown adipogenesis, suggesting that CycC is essential for C/EBPα-mediated gene activation. CycC physically interacted with C/EBPα and this interaction was required for C/EBPα transactivation domain activity. Consistent with the role of C/EBPα in white adipogenesis, CycC-KD also inhibited differentiation of 3T3-L1 cells into white adipocytes. Together, these data indicate that CycC activates adipogenesis by stimulating the transcriptional activity of C/EBPα.
The prognostic value of mitotic activity index (MAI), phosphohistone H3 (PPH3), cyclin B1, cyclin A, and Ki67, alone and in combinations, in node-negative premenopausal breast cancer.
Klintman, Marie; Strand, Carina; Ahlin, Cecilia; Beglerbegovic, Sanda; Fjällskog, Marie-Louise; Grabau, Dorthe; Gudlaugsson, Einar; Janssen, Emiel A M; Lövgren, Kristina; Skaland, Ivar; Bendahl, Pär-Ola; Malmström, Per; Baak, Jan P A; Fernö, Mårten
Proliferation, either as the main common denominator in genetic profiles, or in the form of single factors such as Ki67, is recommended for clinical use especially in estrogen receptor-positive (ER) patients. However, due to high costs of genetic profiles and lack of reproducibility for Ki67, studies on other proliferation factors are warranted. The aim of the present study was to evaluate the prognostic value of the proliferation factors mitotic activity index (MAI), phosphohistone H3 (PPH3), cyclin B1, cyclin A and Ki67, alone and in combinations. In 222 consecutive premenopausal node-negative breast cancer patients (87% without adjuvant medical treatment), MAI was assessed on whole tissue sections (predefined cut-off ≥10 mitoses), and PPH3, cyclin B1, cyclin A, and Ki67 on tissue microarray (predefined cut-offs 7th decile). In univariable analysis (high versus low) the strongest prognostic proliferation factor for 10-year distant disease-free survival was MAI (Hazard Ratio (HR)=3.3, 95% Confidence Interval (CI): 1.8-6.1), followed by PPH3, cyclin A, Ki67, and cyclin B1. A combination variable, with patients with MAI and/or cyclin A high defined as high-risk, had even stronger prognostic value (HR=4.2, 95%CI: 2.2-7). When stratifying for ER-status, MAI was a significant prognostic factor in ER-positive patients only (HR=7.0, 95%CI: 3.1-16). Stratified for histological grade, MAI added prognostic value in grade 2 (HR=7.2, 95%CI: 3.1-38) and grade 1 patients. In multivariable analysis including HER2, age, adjuvant medical treatment, ER, and one proliferation factor at a time, only MAI (HR=2.7, 95%CI: 1.1-6.7), and cyclin A (HR=2.7, 95%CI: 1.2-6.0) remained independently prognostic. In conclusion this study confirms the strong prognostic value of all proliferation factors, especially MAI and cyclin A, in all patients, and more specifically in ER-positive patients, and patients with histological grade 2 and 1. Additionally, by combining two proliferation factors
Molecular dynamics simulations on the inhibition of Cyclin-Dependent Kinases 2 and 5 in the presence of activators
NASA Astrophysics Data System (ADS)
Zhang, Bing; Tan, Vincent B. C.; Lim, Kian Meng; Tay, Tong Earn
Interests in CDK2 and CDK5 have stemmed mainly from their association with cancer and neuronal migration or differentiation related diseases and the need to design selective inhibitors for these kinases. Molecular dynamics (MD) simulations have not only become a viable approach to drug design because of advances in computer technology but are increasingly an integral part of drug discovery processes. It is common in MD simulations of inhibitor/CDK complexes to exclude the activator of the CDKs in the structural models to keep computational time tractable. In this paper, we present simulation results of CDK2 and CDK5 with roscovitine using models with and without their activators (cyclinA and p25). While p25 was found to induce slight changes in CDK5, the calculations support that cyclinA leads to significant conformational changes near the active site of CDK2. This suggests that detailed and structure-based inhibitor design targeted at these CDKs should employ activator-included models of the kinases. Comparisons between P/CDK2/cyclinA/roscovitine and CDK5/p25/roscovitine complexes reveal differences in the conformations of the glutamine around the active sites, which may be exploited to find highly selective inhibitors with respect to CDK2 and CDK5.
Molecular dynamics simulations on the inhibition of cyclin-dependent kinases 2 and 5 in the presence of activators.
Zhang, Bing; Tan, Vincent B C; Lim, Kian Meng; Tay, Tong Earn
Interests in CDK2 and CDK5 have stemmed mainly from their association with cancer and neuronal migration or differentiation related diseases and the need to design selective inhibitors for these kinases. Molecular dynamics (MD) simulations have not only become a viable approach to drug design because of advances in computer technology but are increasingly an integral part of drug discovery processes. It is common in MD simulations of inhibitor/CDK complexes to exclude the activator of the CDKs in the structural models to keep computational time tractable. In this paper, we present simulation results of CDK2 and CDK5 with roscovitine using models with and without their activators (cyclinA and p25). While p25 was found to induce slight changes in CDK5, the calculations support that cyclinA leads to significant conformational changes near the active site of CDK2. This suggests that detailed and structure-based inhibitor design targeted at these CDKs should employ activator-included models of the kinases. Comparisons between P/CDK2/cyclinA/roscovitine and CDK5/p25/roscovitine complexes reveal differences in the conformations of the glutamine around the active sites, which may be exploited to find highly selective inhibitors with respect to CDK2 and CDK5.
Lack of cyclin-dependent kinase 4 inhibits c-myc tumorigenic activities in epithelial tissues.
Miliani de Marval, Paula L; Macias, Everardo; Rounbehler, Robert; Sicinski, Piotr; Kiyokawa, Hiroaki; Johnson, David G; Conti, Claudio J; Rodriguez-Puebla, Marcelo L
The proto-oncogene c-myc encodes a transcription factor that is implicated in the regulation of cellular proliferation, differentiation, and apoptosis and that has also been found to be deregulated in several forms of human and experimental tumors. We have shown that forced expression of c-myc in epithelial tissues of transgenic mice (K5-Myc) resulted in keratinocyte hyperproliferation and the development of spontaneous tumors in the skin and oral cavity. Although a number of genes involved in cancer development are regulated by c-myc, the actual mechanisms leading to Myc-induced neoplasia are not known. Among the genes regulated by Myc is the cyclin-dependent kinase 4 (CDK4) gene. Interestingly, previous studies from our laboratory showed that the overexpression of CDK4 led to keratinocyte hyperproliferation, although no spontaneous tumor development was observed. Thus, we tested the hypothesis that CDK4 may be one of the critical downstream genes involved in Myc carcinogenesis. Our results showed that CDK4 inhibition in K5-Myc transgenic mice resulted in the complete inhibition of tumor development, suggesting that CDK4 is a critical mediator of tumor formation induced by deregulated Myc. Furthermore, a lack of CDK4 expression resulted in marked decreases in epidermal thickness and keratinocyte proliferation compared to the results obtained for K5-Myc littermates. Biochemical analysis of the K5-Myc epidermis showed that CDK4 mediates the proliferative activities of Myc by sequestering p21Cip1 and p27Kip1 and thereby indirectly activating CDK2 kinase activity. These results show that CDK4 mediates the proliferative and oncogenic activities of Myc in vivo through a mechanism that involves the sequestration of specific CDK inhibitors.
CARMA3 is overexpressed in colon cancer and regulates NF-{kappa}B activity and cyclin D1 expression
Miao, Zhifeng; Zhao, Tingting; Wang, Zhenning; Xu, Yingying; Song, Yongxi; Wu, Jianhua; Xu, Huimian
Highlights: Black-Right-Pointing-Pointer CARMA3 expression is elevated in colon cancers. Black-Right-Pointing-Pointer CARMA3 promotes proliferation and cell cycle progression in colon cancer cells. Black-Right-Pointing-Pointer CARMA3 upregulates cyclinD1 through NF-{kappa}B activation. -- Abstract: CARMA3 was recently reported to be overexpressed in cancers and associated with the malignant behavior of cancer cells. However, the expression of CARMA3 and its biological roles in colon cancer have not been reported. In the present study, we analyzed the expression pattern of CARMA3 in colon cancer tissues and found that CARMA3 was overexpressed in 30.8% of colon cancer specimens. There was a significant association between CARMA3 overexpression and TNM stage (p = 0.0383), lymph node metastasis (p = 0.0091) and Ki67 proliferation index (p = 0.0035). Furthermore, knockdown of CARMA3 expression in HT29 and HCT116 cells with high endogenous expression decreased cell proliferation and cell cycle progression while overexpression of CARMA3 in LoVo cell line promoted cell proliferation and facilitated cell cycle transition. Further analysis showed that CARMA3 knockdown downregulated and its overexpression upregulated cyclin D1 expression and phospho-Rb levels. In addition, we found that CARMA3 depletion inhibited p-I{kappa}B levels and NF-{kappa}B activity and its overexpression increased p-I{kappa}B expression and NF-{kappa}B activity. NF-{kappa}B inhibitor BAY 11-7082 reversed the role of CARMA3 on cyclin D1 upregulation. In conclusion, our study found that CARMA3 is overexpressed in colon cancers and contributes to malignant cell growth by facilitating cell cycle progression through NF-{kappa}B mediated upregulation of cyclin D1.
Fluorescent peptide biosensor for monitoring CDK4/cyclin D kinase activity in melanoma cell extracts, mouse xenografts and skin biopsies.
Prével, Camille; Pellerano, Morgan; González-Vera, Juan A; Henri, Pauline; Meunier, Laurent; Vollaire, Julien; Josserand, Véronique; Morris, May C
Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16(INK4a)-Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16(INK4a) leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity. Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma.
A tumor suppressor C53 protein antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation
Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin
Cyclin dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint (1). More recently, Wang et al (2007) found that C53/LZAP may function as a tumor suppressor via inhibiting NF-κB signaling (2). We report here identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexrepsssion. Intriguingly, we found that C53 interacts with checkpoint kinase 1 (Chk1) and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell cycle progression and DNA damage response. PMID:19223857
Regulation of Cdc28 Cyclin-Dependent Protein Kinase Activity during the Cell Cycle of the Yeast Saccharomyces cerevisiae
Mendenhall, Michael D.; Hodge, Amy E.
The cyclin-dependent protein kinase (CDK) encoded by CDC28 is the master regulator of cell division in the budding yeast Saccharomyces cerevisiae. By mechanisms that, for the most part, remain to be delineated, Cdc28 activity controls the timing of mitotic commitment, bud initiation, DNA replication, spindle formation, and chromosome separation. Environmental stimuli and progress through the cell cycle are monitored through checkpoint mechanisms that influence Cdc28 activity at key cell cycle stages. A vast body of information concerning how Cdc28 activity is timed and coordinated with various mitotic events has accrued. This article reviews that literature. Following an introduction to the properties of CDKs common to many eukaryotic species, the key influences on Cdc28 activity—cyclin-CKI binding and phosphorylation-dephosphorylation events—are examined. The processes controlling the abundance and activity of key Cdc28 regulators, especially transcriptional and proteolytic mechanisms, are then discussed in detail. Finally, the mechanisms by which environmental stimuli influence Cdc28 activity are summarized. PMID:9841670
Sp1 phosphorylation by cyclin-dependent kinase 1/cyclin B1 represses its DNA-binding activity during mitosis in cancer cells.
Chuang, J-Y; Wang, S-A; Yang, W-B; Yang, H-C; Hung, C-Y; Su, T-P; Chang, W-C; Hung, J-J
Sp1 is important for the transcription of many genes. Our previous studies have shown that Sp1 is degraded in normal cell, but it is preserved in cancer cells during mitosis and exists a priori in the daughter cells, ready to engage in gene transcription and thereby contributes to the proliferation and survival of cancer cells. The mechanism by which Sp1 is preserved in cancer cells during mitosis remains unknown. In this study, we observed that Sp1 strongly colocalized with cyclin-dependent kinase 1 (CDK1)/cyclin B1 during mitosis. Moreover, we showed that Sp1 is a novel mitotic substrate of CDK1/cyclin B1 and is phosphorylated by it at Thr 739 before the onset of mitosis. Phospho-Sp1 reduced its DNA-binding ability and facilitated the chromatin condensation process during mitosis. Mutation of Thr739 to alanine resulted in Sp1 remaining in the chromosomes, delayed cell-cycle progression, and eventually led to apoptosis. Screening of Sp1-associated proteins during mitosis by using liquid chromatography/mass spectrometry indicated the tethering of Sp1 to myosin/F-actin. Furthermore, phospho-Sp1 and myosin/F-actin appeared to exist as a congregated ring at the periphery of the chromosome. However, at the end of mitosis and the beginning of interphase, Sp1 was dephosphorylated by PP2A and returned to the chromatin. These results indicate that cancer cells use CDK1 and PP2A to regulate the movement of Sp1 in and out of the chromosomes during cell-cycle progression, which may benefit cancer-cell proliferation.
Leptin-induced Growth Stimulation of Breast Cancer Cells Involves Recruitment of Histone Acetyltransferases and Mediator Complex to CYCLIN D1 Promoter via Activation of Stat3*
Saxena, Neeraj K.; Vertino, Paula M.; Anania, Frank A.; Sharma, Dipali
Numerous epidemiological studies documented that obesity is a risk factor for breast cancer development in postmenopausal women. Leptin, the key player in the regulation of energy balance and body weight control also acts as a growth factor on certain organs in both normal and disease state. In this study, we analyzed the role of leptin and the molecular mechanism(s) underlying its action in breast cancer cells that express both short and long isoforms of leptin receptor. Leptin increased MCF7 cell population in the S-phase of the cell cycle along with a robust increase in CYCLIN D1 expression. Also, leptin induced Stat3-phosphorylation-dependent proliferation of MCF7 cells as blocking Stat3 phosphorylation with a specific inhibitor, AG490, abolished leptin-induced proliferation. Using deletion constructs of CYCLIN D1 promoter and chromatin immunoprecipitation assay, we show that leptin induced increase in CYCLIN D1 promoter activity is mediated through binding of activated Stat3 at the Stat binding sites and changes in histone acetylation and methylation. We also show specific involvement of coactivator molecules, histone acetyltransferase SRC1, and mediator complex in leptin-mediated regulation of CYCLIN D1 promoter. Importantly, silencing of SRC1 and Med1 abolished the leptin induced increase in CYCLIN D1 expression and MCF7 cell proliferation. Intriguingly, recruitment of both SRC1 and Med1 was dependent on phosphorylated Stat3 as AG490 treatment inhibited leptin-induced recruitment of these coactivators to CYCLIN D1 promoter. Our data suggest that CYCLIN D1 may be a target gene for leptin mediated growth stimulation of breast cancer cells and molecular mechanisms involve activated Stat3-mediated recruitment of distinct coactivator complexes. PMID:17344214
The ω-3 epoxide of eicosapentaenoic acid inhibits endothelial cell proliferation by p38 MAP kinase activation and cyclin D1/CDK4 down-regulation
Cui, Pei H; Petrovic, Nenad; Murray, Michael
BACKGROUND AND PURPOSE Dietary intake of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) like eicosapentaenoic acid (EPA) decreases cancer risk, while arachidonic acid and other ω-6 PUFAs increase risk, but the underlying mechanisms are unclear. Cytochrome P450 (CYP)-derived epoxides contribute to enhanced tumourigenesis due to ω-6 PUFA intake. Thus, ω-6 arachidonic acid epoxides (EETs) inhibit apoptosis and stimulate proliferation by up-regulating cyclin D1 expression in cells. The present study evaluated the corresponding ω-3 PUFA epoxides and assessed their role in the regulation of cell proliferation. EXPERIMENTAL APPROACH Four chemically stable EPA epoxides (formed at the 8,9-, 11,12-, 14,15- and 17,18-olefinic bonds) were synthesized and tested against growth-related signalling pathways in brain microvascular endothelial bEND.3 cells. Cell cycle distribution was determined by flow cytometry and cyclin gene expression by immunoblotting and real-time PCR. The role of the p38 mitogen-activated protein (MAP) kinase in cyclin D1 dysregulation was assessed using specific inhibitors and dominant-negative expression plasmids. KEY RESULTS The ω-3 17,18-epoxide of EPA decreased cell proliferation, interrupted the cell cycle in S-phase and down-regulated the cyclin D1/cyclin-dependent kinase (CDK)-4 complex, whereas the 8,9-, 11,12- and 14,15-epoxides were either inactive or enhanced proliferation. Cyclin D1 down-regulation by 17,18-epoxy-EPA was mediated by activation of the growth-suppressing p38 MAP kinase, but the alternate EPA-epoxides were inactive. CONCLUSIONS AND IMPLICATIONS The present findings suggest that the epoxide formed by CYP enzymes at the ω-3 olefinic bond may contribute to the beneficial effects of ω-3 PUFA by down-regulating cyclin D1 and suppressing cell proliferation. PMID:21077851
Summary Cyclin-dependent kinases (CDKs) are protein kinases characterized by needing a separate subunit - a cyclin - that provides domains essential for enzymatic activity. CDKs play important roles in the control of cell division and modulate transcription in response to several extra- and intracellular cues. The evolutionary expansion of the CDK family in mammals led to the division of CDKs into three cell-cycle-related subfamilies (Cdk1, Cdk4 and Cdk5) and five transcriptional subfamilies (Cdk7, Cdk8, Cdk9, Cdk11 and Cdk20). Unlike the prototypical Cdc28 kinase of budding yeast, most of these CDKs bind one or a few cyclins, consistent with functional specialization during evolution. This review summarizes how, although CDKs are traditionally separated into cell-cycle or transcriptional CDKs, these activities are frequently combined in many family members. Not surprisingly, deregulation of this family of proteins is a hallmark of several diseases, including cancer, and drug-targeted inhibition of specific members has generated very encouraging results in clinical trials. PMID:25180339
Phosphorylation of cyclin-dependent kinase 5 (Cdk5) at Tyr-15 is inhibited by Cdk5 activators and does not contribute to the activation of Cdk5.
Kobayashi, Hiroyuki; Saito, Taro; Sato, Ko; Furusawa, Kotaro; Hosokawa, Tomohisa; Tsutsumi, Koji; Asada, Akiko; Kamada, Shinji; Ohshima, Toshio; Hisanaga, Shin-ichi
Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities.
FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G₂arrest in RCS chondrocytes.
Tran, Tri; Kolupaeva, Victoria; Basilico, Claudio
Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G₁ phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G₂ phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G₁ arrest. The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase, that however appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop, and not the activation of specific phosphatases. the inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling, and not a consequence of the G₂ arrest as it can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and AtM/ATR kinase are known to play essential roles in the G₂ checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G₂ arrest. Additionally our results indicate that the transient G₂ arrest is induced by FGF in RCS cell through mechanisms that are independent of the G₁ arrest, and that the G₂ block is not strictly required for the sustained G₁ arrest but may provide a pausing mechanism that allows the FGF response to be fully established.
Baicalein induces G1 arrest in oral cancer cells by enhancing the degradation of cyclin D1 and activating AhR to decrease Rb phosphorylation
Cheng, Ya-Hsin; Li, Lih-Ann; Lin, Pinpin; Cheng, Li-Chuan; Hung, Chein-Hui; Chang, Nai Wen; Lin, Chingju
Baicalein is a flavonoid, known to have anti-inflammatory and anti-cancer effects. As an aryl hydrocarbon receptor (AhR) ligand, baicalein at high concentrations blocks AhR-mediated dioxin toxicity. Because AhR had been reported to play a role in regulating the cell cycle, we suspected that the anti-cancer effect of baicalein is associated with AhR. This study investigated the molecular mechanism involved in the anti-cancer effect of baicalein in oral cancer cells HSC-3, including whether such effect would be AhR-mediated. Results revealed that baicalein inhibited cell proliferation and increased AhR activity in a dose-dependent manner. Cell cycle was arrested at the G1 phase and the expression of CDK4, cyclin D1, and phosphorylated retinoblastoma (pRb) was decreased. When the AhR was suppressed by siRNA, the reduction of pRb was partially reversed, accompanied by a decrease of cell population at G1 phase and an increase at S phase, while the reduction of cyclin D1 and CDK4 did not change. This finding suggests that the baicalein activation of AhR is indeed associated with the reduction of pRb, but is independent of the reduction of cyclin D1 and CDK4. When cells were pre-treated with LiCl, the inhibitor of GSK-3β, the decrease of cyclin D1 was blocked and the reduction of pRb was recovered. The data indicates that in HSC-3 the reduction of pRb is both mediated by baicalein through activation of AhR and facilitation of cyclin D1 degradation, which causes cell cycle arrest at the G1 phase, and results in the inhibition of cell proliferation. -- Highlights: ► Baicalein causes the G1 phase arrest by decreasing Rb phosphorylation. ► Baicalein modulates AhR-mediated cell proliferation. ► Both AhR activation and cyclin D1 degradation results in hypophosphorylation of Rb. ► Baicalein facilitates cyclin D1 degradation by signalling the GSK-3β pathway.
Expression of retinoblastoma gene product (pRb) in mantle cell lymphomas. Correlation with cyclin D1 (PRAD1/CCND1) mRNA levels and proliferative activity.
Jares, P.; Campo, E.; Pinyol, M.; Bosch, F.; Miquel, R.; Fernandez, P. L.; Sanchez-Beato, M.; Soler, F.; Perez-Losada, A.; Nayach, I.; Mallofré, C.; Piris, M. A.; Montserrat, E.; Cardesa, A.
Mantle cell lymphomas (MCLs) are molecularly characterized by bcl-1 rearrangement and constant cyclin D1 (PRAD-1/CCND1) gene overexpression. Cyclin D1 is a G1 cyclin that participates in the control of the cell cycle progression by interacting with the retinoblastoma gene product (pRb). Inactivation of the Rb tumor suppressor gene has been implicated in the development of different types of human tumors including some high grade non-Hodgkin's lymphomas. To determine the role of the retinoblastoma gene in the pathogenesis of MCLs and its possible interaction with cyclin D1, pRb expression was examined in 23 MCLs including 17 typical and 6 blastic variants by immunohistochemistry and Western blot. Rb gene structure was studied in 13 cases by Southern blot. Cytogenetic analysis was performed in 5 cases. The results were compared with the cyclin D1 mRNA levels examined by Northern analysis, and the proliferative activity of the tumors was measured by Ki-67 growth fraction and flow cytometry. pRb was expressed in all MCLs. The expression varied from case to case (mean, 14.1% of positive cells; range, 1.3 to 42%) with a significant correlation with the proliferative activity of the tumors (mitotic index r = 0.85; Ki-67 r = 0.7; S phase = 0.73). Blastic variants showed higher numbers of pRb-positive cells (mean, 29%) than the typical cases (10%; P < 0.005) by immunohistochemistry and, concordantly, higher levels of expression by Western blot. In addition, the blastic cases also had an increased expression of the phosphorylated protein. No alterations in Rb gene structure were observed by Southern blot analysis. Cyclin D1 mRNA levels were independent of pRb expression and the proliferative activity of the tumors. These findings suggest that pRb in MCLs is normally regulated in relation to the proliferative activity of the tumors. Cyclin D1 overexpression may play a role in the maintenance of cell proliferation by overcoming the suppressive growth control of pRb. Images
CDK1-Cyclin B1 Activates RNMT, Coordinating mRNA Cap Methylation with G1 Phase Transcription
Aregger, Michael; Kaskar, Aneesa; Varshney, Dhaval; Fernandez-Sanchez, Maria Elena; Inesta-Vaquera, Francisco A.; Weidlich, Simone; Cowling, Victoria H.
Summary The creation of translation-competent mRNA is dependent on RNA polymerase II transcripts being modified by addition of the 7-methylguanosine (m7G) cap. The factors that mediate splicing, nuclear export, and translation initiation are recruited to the transcript via the cap. The cap structure is formed by several activities and completed by RNMT (RNA guanine-7 methyltransferase), which catalyzes N7 methylation of the cap guanosine. We report that CDK1-cyclin B1 phosphorylates the RNMT regulatory domain on T77 during G2/M phase of the cell cycle. RNMT T77 phosphorylation activates the enzyme both directly and indirectly by inhibiting interaction with KPNA2, an RNMT inhibitor. RNMT T77 phosphorylation results in elevated m7G cap methyltransferase activity at the beginning of G1 phase, coordinating mRNA capping with the burst of transcription that occurs following nuclear envelope reformation. RNMT T77 phosphorylation is required for the production of cohort of proteins, and inhibiting T77 phosphorylation reduces the cell proliferation rate. PMID:26942677
Regulation of APC(Cdh1) E3 ligase activity by the Fbw7/cyclin E signaling axis contributes to the tumor suppressor function of Fbw7.
Lau, Alan W; Inuzuka, Hiroyuki; Fukushima, Hidefumi; Wan, Lixin; Liu, Pengda; Gao, Daming; Sun, Yi; Wei, Wenyi
Fbw7 and Cdh1 are substrate-recognition subunits of the SCF- and APC-type E3 ubiquitin ligases, respectively. There is emerging evidence suggesting that both Fbw7 and Cdh1 function as tumor suppressors by targeting oncoproteins for destruction. Loss of Fbw7, but not Cdh1, is frequently observed in various human tumors. However, it remains largely unknown how Fbw7 mechanistically functions as a tumor suppressor and whether there is a signaling crosstalk between Fbw7 and Cdh1. Here, we report that Fbw7-deficient cells not only display elevated expression levels of SCF(Fbw7) substrates, including cyclin E, but also have increased expression of various APC(Cdh1) substrates. We further defined cyclin E as the critical signaling link by which Fbw7 governs APC(Cdh1) activity, as depletion of cyclin E in Fbw7-deficient cells results in decreased expression of APC(Cdh1) substrates to levels comparable to those in wild-type (WT) cells. Conversely, ectopic expression of cyclin E recapitulates the aberrant APC(Cdh1) substrate expression observed in Fbw7-deficient cells. More importantly, 4A-Cdh1 that is resistant to Cdk2/cyclin E-mediated phosphorylation, but not WT-Cdh1, reversed the elevated expression of various APC(Cdh1) substrates in Fbw7-deficient cells. Overexpression of 4A-Cdh1 also resulted in retarded cell growth and decreased anchorage-independent colony formation. Altogether, we have identified a novel regulatory mechanism by which Fbw7 governs Cdh1 activity in a cyclin E-dependent manner. As a result, loss of Fbw7 can lead to aberrant increase in the expression of both SCF(Fbw7) and APC(Cdh1) substrates. Our study provides a better understanding of the tumor suppressor function of Fbw7, and suggests that Cdk2/cyclin E inhibitors could serve as effective therapeutic agents for treating Fbw7-deficient tumors.
The Requirement for Cyclin D Function in Tumor Maintenance
Choi, Yoon Jong; Li, Xiaoyu; Hydbring, Per; Sanda, Takaomi; Stefano, Joanna; Christie, Amanda L.; Signoretti, Sabina; Look, A. Thomas; Kung, Andrew L.; von Boehmer, Harald; Sicinski, Piotr
SUMMARY D-cyclins represent components of cell cycle machinery. To test the efficacy of targeting D-cyclins in cancer treatment, we engineered mouse strains which allow acute and global ablation of individual D-cyclins in a living animal. Ubiquitous shutdown of cyclin D1 or inhibition of cyclin D-associated kinase activity in mice bearing ErbB2-driven mammary carcinomas triggered tumor cell senescence, without compromising the animals' health. Ablation of cyclin D3 in mice bearing Notch1-driven T-cell acute lymphoblastic leukemias (T-ALL) triggered tumor cell apoptosis. Such selective killing of leukemic cells can also be achieved by inhibiting cyclin D-associated kinase activity in mouse and human T-ALL models. Inhibition of cyclin D-kinase activity represents a highly-selective anti-cancer strategy that specifically targets cancer cells without significantly affecting normal tissues. PMID:23079655
Cyclin-dependent kinase 5 activity is required for allogeneic T-cell responses after hematopoietic cell transplantation in mice
Pareek, Tej K.; Eid, Saada; Ganguly, Sudipto; Tyler, Megan; Huang, Alex Y.; Letterio, John J.
Molecular intermediates in T-cell activation pathways are crucial targets for the therapy and prevention of graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (allo-HCT). We recently identified an essential role for cyclin-dependent kinase 5 (Cdk5) in T-cell activation and effector function, but the contribution of Cdk5 activity to the development of GVHD has not been explored. Using an established, preclinical, murine, GVHD model, we reveal that Cdk5 activity is increased in key target organs early after allo-HCT. We then generated chimeric mice (Cdk5+/+C or Cdk5−/−C) using hematopoietic progenitors from either embryonic day 16.5 Cdk5+/+ or Cdk5−/− embryos to enable analyses of the role of Cdk5 in GVHD, as germ line Cdk5 gene deletion is embryonically lethal. The immunophenotype of adult Cdk5−/−C mice is identical to control Cdk5+/+C mice. However, transplantation of donor Cdk5−/−C bone marrow and T cells dramatically reduced the severity of systemic and target organ GVHD. This phenotype is attributed to decreased T-cell migration to secondary lymphoid organs (SLOs), reduced in vivo proliferation within these organs, and fewer cytokine-producing donor T cells during GVHD development. Moreover, these defects in Cdk5−/− T-cell function are associated with altered CCR7 signaling following ligation by CCL19, a receptor:ligand interaction critical for T-cell migration into SLOs. Although Cdk5 activity in donor T cells contributed to graft-versus-tumor effects, pharmacologic inhibition of Cdk5 preserved leukemia-free survival. Collectively, our data implicate Cdk5 in allogeneic T-cell responses after HCT and as an important new target for therapeutic intervention. PMID:28064242
Candida albicans Cyclin Clb4 Carries S-Phase Cyclin Activity▿†
Ofir, Ayala; Kornitzer, Daniel
Cyclin-dependent kinases (CDKs) are key regulators of eukaryotic cell cycle progression. The cyclin subunit activates the CDK and also imparts to the complex, at least in some cases, substrate specificity. Saccharomyces cerevisiae, an organism in which the roles of individual cyclins are best studied, contains nine cyclins (three G1 cyclins and six B-type cyclins) capable of activating the main cell cycle CDK, Cdc28. Analysis of the genome of the pathogenic yeast Candida albicans revealed only two sequences corresponding to B-type cyclins, C. albicans Clb2 (CaClb2) and CaClb4. Notably, no homolog of the S. cerevisiae S-phase-specific cyclins, Clb5/Clb6, could be detected. Here, we performed an in vitro analysis of the activity of CaClb2 and CaClb4 and of three G1 cyclins, as well as an analysis of the phenotype of S. cerevisiae cells expressing CaClb2 or CaClb4 instead of Clb5. Remarkably, replacement of CLB5 by CaCLB4 caused rapid diploidization of S. cerevisiae. In addition, both in vivo and in vitro analyses indicate that, in spite of the higher sequence similarity of CaClb2 to Clb5/Clb6, CaClb4 is the functional homolog of Clb5/Clb6. The activity of a CaClb2/CaClb4 cyclin hybrid suggests that the cyclin box domain of CaClb4 carries the functional specificity of the protein. These results have implications for our understanding of the evolution of specificity of the cell cycle cyclins. PMID:20639412
Regulation of the activation of the Fanconi anemia pathway by the p21 cyclin-dependent kinase inhibitor.
Rego, M A; Harney, J A; Mauro, M; Shen, M; Howlett, N G
Fanconi anemia (FA) is a rare disease characterized by congenital defects, progressive bone marrow failure and heightened cancer susceptibility. The FA proteins, BRCA1 and FANCD1/BRCA2 function cooperatively in the FA-BRCA pathway to repair damaged DNA. Activation of the FA-BRCA pathway occurs via the monoubiquitination of the FANCD2 and FANCI proteins, targeting these proteins to discrete nuclear foci where they function in DNA repair. The cellular regulation of FANCD2/I monoubiquitination, however, remains poorly understood. In this study, we have examined the roles of the p53 tumor suppressor protein, as well as its downstream target, the p21(Cip1/Waf1) cyclin-dependent kinase inhibitor, in the regulation of the activation of the FA-BRCA pathway. We demonstrate that, in contrast to p53, p21 has a major role in the regulation of the activation of the FA-BRCA pathway: p21 promotes S-phase and DNA damage-inducible FANCD2/I monoubiquitination and nuclear foci formation. Several lines of evidence establish that this effect is not a consequence of a defective G1-S checkpoint or altered cell-cycle progression in the absence of p21. Instead, we demonstrate that p21 is required for the transcriptional repression of the USP1 deubiquitinating enzyme upon exposure to DNA-damaging agents. In the absence of p21, persistent USP1 expression precludes the DNA damage-inducible accumulation of monoubiquitinated FANCD2 and FANCI. Consequently, p21(-/-) cells exhibit increased levels of mitomycin C-inducible complex chromosomal aberrations and elevated γH2AX nuclear foci formation. Our results demonstrate that p21 has a critical role in the regulation of the activation of the FA-BRCA pathway and suggest a broader role for p21 in the orchestration of DNA repair processes following exposure to DNA crosslinking agents.
Apoptosis in 7-hydroxystaurosporine-treated T lymphoblasts correlates with activation of cyclin-dependent kinases 1 and 2.
Wang, Q; Worland, P J; Clark, J L; Carlson, B A; Sausville, E A
7-Hydroxystaurosporine (UCN-01) is a potent inhibitor of protein kinase C (PKC) isozymes alpha, beta, and gamma [Seynaeve et al., Mol. Pharmacol, 45: 1207-1214, 1994] that also has antitumor effects in vivo. To determine whether inhibition of PKC can be related to inhibition of cell growth with induction of apoptosis, we compared the effects of UCN-01 to those of the highly selective bisindolylmaleimide PKC antagonist GF 109203X in leukemic T-cell lines. Both compounds potently inhibited PKC activity when added to T-cell membrane preparations and reversed phorbol ester-induced c-fos gene expression in intact cells. However, whereas UCN-01 potently inhibited growth of Jurkat, Molt-3, Molt-4, and Hut-78 cells (IC50 = 20-65 nM, irreversible after 24 h of exposure), GF 109203X had IC50s for cell growth of 3.6-5.0 muM. Less than 3 h after addition, UCN-01 but not GF 109203X-treated cells displayed loss of cells with G2-M DNA content, appearance of a hypodiploid DNA fraction, and evidence of internucleosomal DNA fragmentation. Six h after treatment, cells appeared to accumulate with S-phase DNA content. These effects correlated with selective UCN-01 but not GF 109203X-induced decrease in total and tyrosine phosphorylation of cyclin-dependent kinases (cdks) 1 and 2, and with increases in the histone H1 kinase activities of cdk1 and cdk2. UCN-01 was relatively less potent in inhibition of properly activated cdk1 and cdk2 when added in vitro to H1 kinase assays (IC50 = 1000 and 600 nM, respectively). We conclude that inhibition of PKC alone is not sufficient to account for the actions of UCN-01 and are led to the hypothesis that inappropriate cdk activation either correlates with or actually mediates cell growth inhibition with apoptosis in T lymphoblasts exposed to UCN-01.
Nuclear Import of Cdk/Cyclin Complexes: Identification of Distinct Mechanisms for Import of Cdk2/Cyclin E and Cdc2/Cyclin B1
Moore, Jonathan D.; Yang, Jing; Truant, Ray; Kornbluth, Sally
Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence–containing protein, binding to the α adaptor subunit of the importin-α/β heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-β that is distinct from that used to bind importin-α. PMID:9922449
Cell Cycle Regulatory Proteins p27(kip), Cyclins Dl and E and Proliferative Activity in Oncocytic (Hurthle Cell) Lesions of the Thyroid.
Maynes, Lincoln J.; Hutzler, Michael J.; Patwardhan, Nilima A.; Wang, Songtao; Khan, Ashraf
Cyclins are prime cell-cycle regulators central to the control of cell proliferation in eukaryotic cells. The formation of cyclin/cyclin-dependent kinases (CDK) complexes activates the kinases and initiates a cascade of events, which directs cells through the cell cycle. CDK inhibitors (CDKIs) such as p27(kip1) inhibit cyclln-CDK complexes and function as negative regulators of the cell cycle. Previous studies have shown that p27(kip1) is decreased In malignant relative to benign thyroid tumors, but its role and Interaction with other cell cycle regulatory proteins have not been well established In oncocytic lesions of the thyroid. We studied the expression of p27(kip1), cyclins D1 and E, and Ki67 In 20 cases of oncocytic adenoma (AD). 6 cases of oncocytic carcinoma (CA). 8 cases of Hashimoto's thyroiditis (HT). and 9 cases of nodular goiter with oncocytic change (NG) by Immunohistochemlstry. In the latter two lesions only oncocytic cells were evaluated. The positive staining was stratified Into four groups. Statistical analysis was done using the Kruslcal-Wallis one-way analysis of variance test, and, when significant the Dunn multiple-comparisons procedure was used to determine pairwise differences. AllI 20 AD were p27(kip1) posItive, 10 were 4+, 2 were 3+, and the remaining 8 were 1+. In contrast all 6 CA showed 4+ p27(kip1) staining, of the 8 HT 2 were 4+, two 3+, three1+, and I was negative.All 9 NG were p27 positive, 7 showed 4+, one 3+, and one 1+ staining. On pairwise comparison differences in p27(kip1) staining between AD and CA and between HT and CA were statistically significant (p=0.0243 and p=0.0142, respectively). In all but one case Ki67 expression was either very low (<3%) or negative. No significant differences were seen in the expression of cyclin D1 or cyclin E among the groups observed. In conclusion, the increased p27(kip1) expression in malignant oncocytlc tumors relative to benign oncocytic lesions is unlike any other malignant progression
Preclinical Activity of Simvastatin Induces Cell Cycle Arrest in G1 via Blockade of Cyclin D-Cdk4 Expression in Non-Small Cell Lung Cancer (NSCLC)
Liang, Yu-Wei; Chang, Chi-Chang; Hung, Chao-Ming; Chen, Tzu-Yu; Huang, Tzuu-Yuan; Hsu, Yi-Chiang
Lung cancer is the most common cause of cancer-related death. Nonetheless, a decrease in overall incidence and mortality has been observed in the last 30 years due to prevention strategies and improvements in the use of chemotherapeutic agents. In recent studies, Simvastatin (SIM) has demonstrated anti-tumor activity, as well as potent chemopreventive action. As an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA), SIM has been shown to stimulate apoptotic cell death. In this study, an MTT assay revealed the cytotoxic activity of SIM against human large cell lung cancer (Non-small cell lung cancer; NSCLC) cells (NCI-H460); however, induced apoptosis was not observed in NCI-H460 cells. Protein expression levels of cell cycle regulating proteins Cdk4, Cyclin D1, p16 and p27 were markedly altered by SIM. Collectively, our results indicate that SIM inhibits cell proliferation and arrests NCI-H460 cell cycle progression via inhibition of cyclin-dependent kinases and cyclins and the enhancement of CDK inhibitors p16 and p27. Our findings suggest that, in addition to the known effects on hypercholesterolemia therapy, SIM may also provide antitumor activity in established NSCLC. PMID:23481641
A Dual-Specificity Phosphatase Cdc25B Is an Unstable Protein and Triggers p34cdc2/Cyclin B Activation in Hamster BHK21 Cells Arrested with Hydroxyurea
Nishijima, Hitoshi; Nishitani, Hideo; Seki, Takashi; Nishimoto, Takeharu
By incubating at 30°C in the presence of an energy source, p34cdc2/cyclin B was activated in the extract prepared from a temperature-sensitive mutant, tsBN2, which prematurely enters mitosis at 40°C, the nonpermissive temperature (Nishimoto, T., E. Eilen, and C. Basilico. 1978. Cell. 15:475–483), and wild-type cells of the hamster BHK21 cell line arrested in S phase, without protein synthesis. Such an in vitro activation of p34cdc2/cyclin B, however, did not occur in the extract prepared from cells pretreated with protein synthesis inhibitor cycloheximide, although this extract still retained the ability to inhibit p34cdc2/cyclin B activation. When tsBN2 cells arrested in S phase were incubated at 40°C in the presence of cycloheximide, Cdc25B, but not Cdc25A and C, among a family of dual-specificity phosphatases, Cdc25, was lost coincidentally with the lack of the activation of p34cdc2/cyclin B. Consistently, the immunodepletion of Cdc25B from the extract inhibited the activation of p34cdc2/cyclin B. Cdc25B was found to be unstable (half-life < 30 min). Cdc25B, but not Cdc25C, immunoprecipitated from the extract directly activated the p34cdc2/cyclin B of cycloheximide-treated cells as well as that of nontreated cells, although Cdc25C immunoprecipitated from the extract of mitotic cells activated the p34cdc2/cyclin B within the extract of cycloheximide-treated cells. Our data suggest that Cdc25B made an initial activation of p34cdc2/cyclin B, which initiates mitosis through the activation of Cdc25C. PMID:9281587
T3 enhances thyroid cancer cell proliferation through TRβ1/Oct-1-mediated cyclin D1 activation.
Perri, Anna; Catalano, Stefania; Bonofiglio, Daniela; Vizza, Donatella; Rovito, Daniela; Qi, Hongyan; Aquila, Saveria; Panza, Salvatore; Rizza, Pietro; Lanzino, Marilena; Andò, Sebastiano
Several studies have demonstrated that thyroid hormone T3 promotes cancer cell growth, even though the molecular mechanism involved in such processes still needs to be elucidated. In this study we demonstrated that T3 induced proliferation in papillary thyroid carcinoma cell lines concomitantly with an up-regulation of cyclin D1 expression, that is a critical mitogen-regulated cell-cycle control element. Our data revealed that T3 enhanced the recruitment of the TRβ1/Oct-1 complex on Octamer-transcription factor-1 site within cyclin D1 promoter, leading to its transactivation. In addition, silencing of TRβ1 or Oct-1 expression by RNA interference reversed both increased cell proliferation and up-regulation of cyclin D1, underlying the important role of both transcriptional factors in mediating these effects. Finally, T3-induced increase in cell growth was abrogated after knocking down cyclin D1 expression. All these findings highlight a new molecular mechanism by which T3 promotes thyroid cancer cell growth.
Quercetin reduces cyclin D1 activity and induces G1 phase arrest in HepG2 cells
Quercetin is able to inhibit proliferation of malignant tumor cells; however, the exact mechanism involved in this biological process remains unclear. The current study utilized a quantitative proteomic analysis to explore the antitumor mechanisms of quercetin. The leucine of HepG2 cells treated with quercetin was labeled as d3 by stable isotope labeling by amino acids in cell culture (SILAC). The isotope peaks of control HepG2 cells were compared with the d3-labeled HepG2 cells by mass spectrometry (MS) to identify significantly altered proteins. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses were subsequently employed to verify the results of the MS analysis. A flow cytometry assay was designed to observe the influence of various quercetin treatment concentrations on the cell cycle distribution of HepG2 cells. The results indicated that quercetin is able to substantially inhibit proliferation of HepG2 cells and induce an obvious morphological alteration of cells. According to the MS results, the 70 credibly-changed proteins that were identified may play important roles in multiple cellular processes, including protein synthesis, signaling, cytoskeletal processes and metabolism. Among these functional proteins, the expression of cyclin D1 (CCND1) was found to be significantly decreased. RT-PCR and western blot analyses verified the SILAC-MS results of decreased CCND1 expression. In summary, flow cytometry revealed that quercetin is able to induce G1 phase arrest in HepG2 cells. Based on the aforementioned observations, it is suggested that quercetin exerts antitumor activity in HepG2 cells through multiple pathways, including interfering with CCND1 gene expression to disrupt the cell cycle and proliferation of HepG2 cells. In the future, we aim to explore this effect in vivo. PMID:27347174
A novel quantitative model of cell cycle progression based on cyclin-dependent kinases activity and population balances.
Pisu, Massimo; Concas, Alessandro; Cao, Giacomo
Cell cycle regulates proliferative cell capacity under normal or pathologic conditions, and in general it governs all in vivo/in vitro cell growth and proliferation processes. Mathematical simulation by means of reliable and predictive models represents an important tool to interpret experiment results, to facilitate the definition of the optimal operating conditions for in vitro cultivation, or to predict the effect of a specific drug in normal/pathologic mammalian cells. Along these lines, a novel model of cell cycle progression is proposed in this work. Specifically, it is based on a population balance (PB) approach that allows one to quantitatively describe cell cycle progression through the different phases experienced by each cell of the entire population during its own life. The transition between two consecutive cell cycle phases is simulated by taking advantage of the biochemical kinetic model developed by Gérard and Goldbeter (2009) which involves cyclin-dependent kinases (CDKs) whose regulation is achieved through a variety of mechanisms that include association with cyclins and protein inhibitors, phosphorylation-dephosphorylation, and cyclin synthesis or degradation. This biochemical model properly describes the entire cell cycle of mammalian cells by maintaining a sufficient level of detail useful to identify check point for transition and to estimate phase duration required by PB. Specific examples are discussed to illustrate the ability of the proposed model to simulate the effect of drugs for in vitro trials of interest in oncology, regenerative medicine and tissue engineering.
S-nitrosylation of cyclin-dependent kinase 5 (cdk5) regulates its kinase activity and dendrite growth during neuronal development.
Zhang, Peng; Yu, Pei-Chun; Tsang, Anthony H K; Chen, Yu; Fu, Amy K Y; Fu, Wing-Yu; Chung, Kenny K; Ip, Nancy Y
Precise regulation of cyclin-dependent kinase 5 (Cdk5), a member of the cyclin-dependent kinase family, is critical for proper neuronal development and functions. Cdk5 is activated through its association with the neuron-specific activator p35 or p39. Nonetheless, how its kinase activity is regulated in neurons is not well understood. In this study, we found that Cdk5 activity is regulated by S-nitrosylation, a post-translational modification of protein that affects a plethora of neuronal functions. S-nitrosylation of Cdk5 occurs at Cys83, which is one of the critical amino acids within the ATP-binding pocket of the kinase. Upon S-nitrosylation, Cdk5 exhibits reduced kinase activity, whereas mutation of Cys83 to Ala on Cdk5 renders the kinase refractory to such inhibition. Importantly, S-nitrosylated Cdk5 can be detected in the mouse brain, and blocking the S-nitrosylation of Cdk5 in cultured hippocampal neurons enhances dendritic growth and branching. Together, our findings reveal an important role of S-nitrosylation in regulating Cdk5 kinase activity and dendrite growth in neurons during development.
Rac1b enhances cell survival through activation of the JNK2/c-JUN/Cyclin-D1 and AKT2/MCL1 pathways
Wang, Hong; Wei, Si-Si; Chen, Jie; Chen, Yi-He; Xu, Wei-Ping; Jie, Qi-Qiang; Zhou, Qing; Li, Yi-Gang; Wei, Yi-Dong; Wang, Yue-Peng
Rac1b is a constitutively activated, alternatively spliced form of the small GTPase Rac1. Previous studies showed that Rac1b promotes cell proliferation and inhibits apoptosis. In the present study, we used microarray analysis to detect genes differentially expressed in HEK293T cells and SW480 human colon cancer cells stably overexpressing Rac1b. We found that the pro-proliferation genes JNK2, c-JUN and cyclin-D1 as well as anti-apoptotic AKT2 and MCL1 were all upregulated in both lines. Rac1b promoted cell proliferation and inhibited apoptosis by activating the JNK2/c-JUN/cyclin-D1 and AKT2/MCL1 pathways, respectively. Very low Rac1b levels were detected in the colonic epithelium of wild-type Sprague-Dawley rats. Knockout of the rat Rac1 gene exon-3b or knockdown of endogenous Rac1b in HT29 human colon cancer cells downregulated only the AKT2/MCL1 pathway. Our study revealed that very low levels of endogenous Rac1b inhibit apoptosis, while Rac1b upregulation both promotes cell proliferation and inhibits apoptosis. It is likely the AKT2/MCL1 pathway is more sensitive to Rac1b regulation. PMID:26918455
Cyclin D2 Protein Stability Is Regulated in Pancreatic β-Cells
He, Lu Mei; Sartori, Daniel J.; Teta, Monica; Opare-Addo, Lynn M.; Rankin, Matthew M.; Long, Simon Y.; Diehl, J. Alan; Kushner, Jake A.
The molecular determinants of β-cell mass expansion remain poorly understood. Cyclin D2 is the major D-type cyclin expressed in β-cells, essential for adult β-cell growth. We hypothesized that cyclin D2 could be actively regulated in β-cells, which could allow mitogenic stimuli to influence β-cell expansion. Cyclin D2 protein was sharply increased after partial pancreatectomy, but cyclin D2 mRNA was unchanged, suggesting posttranscriptional regulatory mechanisms influence cyclin D2 expression in β-cells. Consistent with this hypothesis, cyclin D2 protein stability is powerfully regulated in fibroblasts. Threonine 280 of cyclin D2 is phosphorylated, and this residue critically limits D2 stability. We derived transgenic (tg) mice with threonine 280 of cyclin D2 mutated to alanine (T280A) or wild-type cyclin D2 under the control of the insulin promoter. Cyclin D2 T280A protein was expressed at much higher levels than wild-type cyclin D2 protein in β-cells, despite equivalent expression of tg mRNAs. Cyclin D2 T280A tg mice exhibited a constitutively nuclear cyclin D2 localization in β-cells, and increased cyclin D2 stability in islets. Interestingly, threonine 280-mutant cyclin D2 tg mice had greatly reduced β-cell apoptosis, with suppressed expression of proapoptotic genes. Suppressed β-cell apoptosis in threonine 280-mutant cyclin D2 tg mice resulted in greatly increased β-cell area in aged mice. Taken together, these data indicate that cyclin D2 is regulated by protein stability in pancreatic β-cells, that signals that act upon threonine 280 limit cyclin D2 stability in β-cells, and that threonine 280-mutant cyclin D2 overexpression prolongs β-cell survival and augments β-cell mass expansion. PMID:19628581
Lupeol induces p53 and cyclin-B-mediated G2/M arrest and targets apoptosis through activation of caspase in mouse skin
Nigam, Nidhi Prasad, Sahdeo; George, Jasmine; Shukla, Yogeshwer
Lupeol, present in fruits and medicinal plants, is a biologically active compound that has been shown to have various pharmacological properties in experimental studies. In the present study, we demonstrated the modulatory effect of lupeol on 7,12-dimethylbenz[a]anthracene (DMBA)-induced alterations on cell proliferation in the skin of Swiss albino mice. Lupeol treatment showed significant (p < 0.05) preventive effects with marked inhibition at 48, 72, and 96 h against DMBA-mediated neoplastic events. Cell-cycle analysis showed that lupeol-induced G2/M-phase arrest (16-37%) until 72 h, and these inhibitory effects were mediated through inhibition of the cyclin-B-regulated signaling pathway involving p53, p21/WAF1, cdc25C, cdc2, and cyclin-B gene expression. Further lupeol-induced apoptosis was observed, as shown by an increased sub-G1 peak (28%) at 96 h, with upregulation of bax and caspase-3 genes and downregulation of anti-apoptotic bcl-2 and survivin genes. Thus, our results indicate that lupeol has novel anti-proliferative and apoptotic potential that may be helpful in designing strategies to fight skin cancer.
BMP6 Regulates Proliferation and Apoptosis of Human Sertoli Cells Via Smad2/3 and Cyclin D1 Pathway and DACH1 and TFAP2A Activation
Wang, Hong; Yuan, Qingqing; Sun, Min; Niu, Minghui; Wen, Liping; Fu, Hongyong; Zhou, Fan; Chen, Zheng; Yao, Chencheng; Hou, Jingmei; Shen, Ruinan; Lin, Qisheng; Liu, Wenjie; Jia, Ruobing; Li, Zheng; He, Zuping
Sertoli cells are essential for regulating normal spermatogenesis. However, the mechanisms underlying human Sertoli cell development remain largely elusive. Here we examined the function and signaling pathways of BMP6 in regulating human Sertoli cells. RT-PCR, immunocytochemistry and Western blots revealed that BMP6 and its multiple receptors were expressed in human Sertoli cells. CCK-8 and EDU assays showed that BMP6 promoted the proliferation of Sertoli cells. Conversely, BMP6 siRNAs inhibited the division of these cells. Annexin V/PI assay indicated that BMP6 reduced the apoptosis in human Sertoli cells, whereas BMP6 knockdown assumed reverse effects. BMP6 enhanced the expression levels of ZO1, SCF, GDNF and AR in human Sertoli cells, and ELISA assay showed an increase of SCF by BMP6 and a reduction by BMP6 siRNAs. Notably, Smad2/3 phosphorylation and cyclin D1 were enhanced by BMP6 and decreased by BMP6 siRNAs in human Sertoli cells. The levels of DACH1 and TFAP2A were increased by BMP6 and reduced by BMP6 siRNAs, and the growth of human Sertoli cells was inhibited by these siRNAs. Collectively, these results suggest that BMP6 regulates the proliferation and apoptosis of human Sertoli cells via activating the Smad2/3/cyclin D1 and DACH1 and TFAP2A pathway. PMID:28387750
Abrogation of p53 by its antisense in MCF-7 breast carcinoma cells increases cyclin D1 via activation of Akt and promotion of cell proliferation
Chhipa, Rishi Raj; Kumari, Ratna; Upadhyay, Ankur Kumar; Bhat, Manoj Kumar
The p53 protein has been a subject of intense research interest since its discovery as about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancers suggesting a critical role of p53 in breast cancer development, growth and chemosensitivity. This report describes the derivation and characterization of MCF-7As53, an isogenic cell line derived from MCF-7 breast carcinoma cells in which p53 was abrogated by antisense p53 cDNA. Similar to MCF-7 and simultaneously selected hygromycin resistant MCF-7H cells, MCF-7As53 cells have consistent basal epithelial phenotype, morphology, and estrogen receptor expression levels at normal growth conditions. Present work documents investigation of molecular variations, growth kinetics, and cell cycle related studies in relation to absence of wild-type p53 protein and its transactivation potential as well. Even though wild-type tumor suppressor p53 is an activator of cell growth arrest and apoptosis-mediator genes such as p21, Bax, and GADD45 in MCF-7As53 cells, no alterations in expression levels of these genes were detected. The doubling time of these cells decreased due to depletion of G0/G1 cell phase because of constitutive activation of Akt and increase in cyclin D1 protein levels. This proliferative property was abrogated by wortmannin, an inhibitor of PI3-K/Akt signaling pathway. Therefore this p53 null cell line indicates that p53 is an indispensable component of cellular signaling system which is regulated by caveolin-1 expression, involving Akt activation and increase in cyclin D1, thereby promoting proliferation of breast cancer cells.
FTY720 Shows Promising In vitro and In vivo Preclinical Activity by Downmodulating Cyclin D1 and Phospho-Akt in Mantle Cell Lymphoma
Liu, Qing; Alinari, Lapo; Chen, Ching-Shih; Yan, Fengting; Dalton, James T.; Lapalombella, Rosa; Zhang, Xiaoli; Mani, Rajeswaran; Lin, Teresa; Byrd, John C.; Baiocchi, Robert A.; Muthusamy, Natarajan
Purpose Despite the progress that has been made in the treatment of mantle cell lymphoma (MCL), all patients invariably relapse with the currently available therapies. Because of the absence of curative therapy for MCL, we explored FTY720 as a novel agent against MCL. Experimental Design The cytotoxic effect of FTY720 in primary MCL tumor cells and cell lines were evaluated in vitro. The effects of FTY720 on caspase activation, generation of reactive oxygen species, and modulation of Cyclin D1 and Akt, which are implied in the pathogenesis of MCL, were investigated. The in vivo efficacy of FTY720 was evaluated in a Jeko-severe combined immunodeficient xenograft model of human MCL. Results FTY720 mediated time- and dose-dependent cytotoxicity in primary MCL tumor cells and MCL cell lines in vitro. FTY720-induced cytotoxicity occured independent of caspase activation but dependent on the generation of ROS in MCL. In addition, FTY720 treatment resulted in the time-dependent downmodulation of Cyclin D1 and accumulation of cells in G0-G1 and G2-M phases of the cell cycle with concomitant decrease in S-phase entry. Furthermore, concentrations of FTY720 that induced cytotoxicity led to decreased phospho-Akt in primary MCL cells and cell lines. Most importantly, the in vivo therapeutic activity of FTY720 was shown in severe combined immunodeficient mice engrafted with the Jeko MCL cell line. Conclusions These results provide the first evidence for a potential use of FTY720 in targeting key pathways that are operable in the pathogenesis of MCL and warrant further investigation of FTY720 in clinical trials to treat patients with MCL. PMID:20460491
Differential regulation of cyclins D1 and D3 in hepatocyte proliferation.
Rickheim, David G; Nelsen, Christopher J; Fassett, John T; Timchenko, Nikolai A; Hansen, Linda K; Albrecht, Jeffrey H
Substantial evidence suggests that cyclin D1 plays a pivotal role in the control of the hepatocyte cell cycle in response to mitogenic stimuli, whereas the closely related protein cyclin D3 has not been extensively evaluated. In the current study, we examined the regulation of cyclins D1 and D3 during hepatocyte proliferation in vivo after 70% partial hepatectomy (PH) and in culture. In contrast to cyclin D1, which was nearly undetectable in quiescent liver and substantially up-regulated after PH, cyclin D3 was constitutively expressed and induced only modestly. In the regenerating liver, the concentration of cyclin D3 was only about 10% of that of cyclin D1. Cyclin D1 formed complexes primarily with cyclin-dependent kinase 4 (cdk4), which were markedly activated in the regenerating liver and readily sequestered the cell cycle inhibitory proteins, p21 and p27. Cyclin D3 bound to both cdk4 and cdk6. Cyclin D3/cdk6 activity was readily detectable in quiescent liver and changed little after PH, and this complex appeared to play a minor role in sequestering p21 and p27. In cultured hepatocytes, epidermal growth factor or insulin had little effect, but the combination of these agents substantially induced cyclin D1 and cell cycle progression. Inhibition of Mek1 or phosphoinositide 3-kinase markedly inhibited cyclin D1 expression and replication. In contrast, cyclin D3 was expressed in the absence of mitogens and was only modestly affected by these manipulations. In addition, growth-inhibitory extracellular matrix conditions inhibited cyclin D1 but not cyclin D3 expression. In conclusion, these results support the concept that cyclin D1 is critically regulated by extracellular stimuli that control proliferation, whereas cyclin D3 is regulated through different pathways and plays a distinct role in the liver.
Cyclin D1 regulates hepatic estrogen and androgen metabolism.
Mullany, Lisa K; Hanse, Eric A; Romano, Andrea; Blomquist, Charles H; Mason, J Ian; Delvoux, Bert; Anttila, Chelsea; Albrecht, Jeffrey H
Cyclin D1 is a cell cycle control protein that plays an important role in regenerating liver and many types of cancer. Previous reports have shown that cyclin D1 can directly enhance estrogen receptor activity and inhibit androgen receptor activity in a ligand-independent manner and thus may play an important role in hormone-responsive malignancies. In this study, we examine a distinct mechanism by which cyclin D1 regulates sex steroid signaling, via altered metabolism of these hormones at the tissue and cellular level. In male mouse liver, ectopic expression of cyclin D1 regulated genes involved in the synthesis and degradation of sex steroid hormones in a pattern that would predict increased estrogen and decreased androgen levels. Indeed, hepatic expression of cyclin D1 led to increased serum estradiol levels, increased estrogen-responsive gene expression, and decreased androgen-responsive gene expression. Cyclin D1 also regulated the activity of several key enzymatic reactions in the liver, including increased oxidation of testosterone to androstenedione and decreased conversion of estradiol to estrone. Similar findings were seen in the setting of physiological cyclin D1 expression in regenerating liver. Knockdown of cyclin D1 in HuH7 cells produced reciprocal changes in steroid metabolism genes compared with cyclin D1 overexpression in mouse liver. In conclusion, these studies establish a novel link between the cell cycle machinery and sex steroid metabolism and provide a distinct mechanism by which cyclin D1 may regulate hormone signaling. Furthermore, these results suggest that increased cyclin D1 expression, which occurs in liver regeneration and liver diseases, may contribute to the feminization seen in these settings.
Regulation of cyclin E stability in Xenopus laevis embryos
NASA Astrophysics Data System (ADS)
Brandt-(Webb), Yekaterina
Cyclin-Cdk complexes positively regulate cell cycle progression. Cyclins are regulatory subunits that bind to and activate cyclin-dependent kinases or Cdks. Cyclin E associates with Cdk2 to mediate G1/S phase transition of the cell cycle. Cyclin E is overexpressed in breast, lung, skin, gastrointestinal, cervical, and ovarian cancers. Its overexpression correlates with poor patient prognosis and is involved in the etiology of breast cancer. We have been studying how this protein is downregulated during development in order to determine if these mechanisms are disrupted during tumorigenesis, leading to its overexpression. Using Xenopus laevis embryos as a model, we have shown previously that during the first 12 embryonic cell cycles Cyclin E levels remain constant yet Cdk2 activity oscillates twice per cell cycle. Cyclin E is abruptly destabilized by an undefined mechanism after the 12th cell cycle, which corresponds to the midblastula transition (MBT). Based on work our work and work by others, we have hypothesized that differential phosphorylation and a change in localization result in Cyclin E degradation by the 26S proteasome at the MBT. To test this, we generated a series of point mutations in conserved threonine/serine residues implicated in degradation of human Cyclin E. Using Western blot analysis, we show that similarly to human Cyclin E, mutation of these residues to unphosphorylatable alanine stabilizes Cyclin E past the MBT when they are expressed in vivo. Cyclin E localization was studied by immunofluorescence analysis of endogenous and exogenous protein in pre-MBT, MBT, and post-MBT embryos. In addition, we developed a novel method of conjugating recombinant His6-tagged Cyclin E to fluorescent (CdSe)ZnS nanoparticles (quantum dots) capped with dihydrolipoic acid. Confocal microscopy was used to visualize His6Cyclin E-quantum dot complexes inside embryo cells in real time. We found that re-localization at the MBT from the cytoplasm to the nucleus
Specific activity of cyclin-dependent kinase I is a new potential predictor of tumour recurrence in stage II colon cancer
Zeestraten, E C M; Maak, M; Shibayama, M; Schuster, T; Nitsche, U; Matsushima, T; Nakayama, S; Gohda, K; Friess, H; van de Velde, C J H; Ishihara, H; Rosenberg, R; Kuppen, P J K; Janssen, K-P
Background: There are no established biomarkers to identify tumour recurrence in stage II colon cancer. As shown previously, the enzymatic activity of the cyclin-dependent kinases 1 and 2 (CDK1 and CDK2) predicts outcome in breast cancer. Therefore, we investigated whether CDK activity identifies tumour recurrence in colon cancer. Methods: In all, 254 patients with completely resected (R0) UICC stage II colon cancer were analysed retrospectively from two independent cohorts from Munich (Germany) and Leiden (Netherlands). None of the patients received adjuvant treatment. Development of distant metastasis was observed in 27 patients (median follow-up: 86 months). Protein expression and activity of CDKs were measured on fresh-frozen tumour samples. Results: Specific activity (SA) of CDK1 (CDK1SA), but not CDK2, significantly predicted distant metastasis (concordance index=0.69, 95% confidence interval (CI): 0.55–0.79, P=0.036). Cutoff derivation by maximum log-rank statistics yielded a threshold of CDK1SA at 11 (SA units, P=0.029). Accordingly, 59% of patients were classified as high-risk (CDK1SA ⩾11). Cox proportional hazard analysis revealed CDK1SA as independent prognostic variable (hazard ratio=6.2, 95% CI: 1.44–26.9, P=0.012). Moreover, CKD1SA was significantly elevated in microsatellite-stable tumours. Conclusion: Specific activity of CDK1 is a promising biomarker for metastasis risk in stage II colon cancer. PMID:22108518
Aberrant expression of cyclin D1 in cancer
Inoue, Kazushi; Fry, Elizabeth A.
Cyclin D1 binds and activates cyclin-dependent kinases 4/6 (Cdk4/6) to phosphorylate the retinoblastoma (RB) family proteins, relieving E2F/DPs from the negative restraint of RB proteins and histone deacetylases. The cyclin D-Cdk4/6 complexes activate cyclin E/Cdk2 through titration of the Cdk inhibitors p21Cip1/p27Kip1. Cyclin E/Cdk2 further phosphorylates RBs, thereby activating E2F/DPs, and cells enter the S phase of the cell cycle. Cyclin D-Cdk4/6 also phosphorylates MEP50 subunit of the protein arginine methyltransferase 5 (PRMT5), which cooperates with cyclin D1 to drive lymphomagenesis in vivo. Activated PRMPT5 causes arginine methylation of p53 to suppress expression of pro-apoptotic and anti-proliferative target genes, explaining the molecular mechanism for tumorigenesis. Cyclin D1 physically interacts with transcription factors such as estrogen receptor, androgen receptor, and Myb family proteins to regulate gene expression in Cdk-independent fashion. Dmp1 is a Myb-like protein that quenches the oncogenic signals from activated Ras or HER2 by inducing Arf/p53-dependent cell cycle arrest. Cyclin D1 binds to Dmp1α to activate both Arf and Ink4a promoters to induce cell cycle arrest or apoptosis in non-transformed cells to prevent them from neoplastic transformation. Dmp1-deficiency significantly accelerates mouse mammary tumorigenesis with reduced apoptosis and increased metastasis. Cyclin D1 interferes with ligand activation of PPARγ involved in cellular differentiation; it also physically interacts with histone deacetylases (HDACs) and p300 to repress gene expression. It has also been shown that cyclin D1 accelerates tumorigenesis through transcriptional activation of miR-17/20 and Dicer1 which, in turn, represses cyclin D1 expression. Identification of cyclin D1-binding proteins/promoters will be essential for further clarification of its biological activities. PMID:28090171
Cyclin-dependent kinase 5 modulates the transcriptional activity of the mineralocorticoid receptor and regulates expression of brain-derived neurotrophic factor.
Kino, Tomoshige; Jaffe, Howard; Amin, Niranjana D; Chakrabarti, Mayukh; Zheng, Ya-Li; Chrousos, George P; Pant, Harish C
Glucocorticoids, major end effectors of the stress response, play an essential role in the homeostasis of the central nervous system (CNS) and contribute to memory consolidation and emotional control through their intracellular receptors, the glucocorticoid and mineralocorticoid receptors. Cyclin-dependent kinase 5 (CDK5), on the other hand, plays important roles in the morphogenesis and functions of the central nervous system, and its aberrant activation has been associated with development of neurodegenerative disorders. We previously reported that CDK5 phosphorylated the glucocorticoid receptor and modulated its transcriptional activity. Here we found that CDK5 also regulated mineralocorticoid receptor-induced transcriptional activity by phosphorylating multiple serine and threonine residues located in its N-terminal domain through physical interaction. Aldosterone and dexamethasone, respectively, increased and suppressed mRNA/protein expression of brain-derived neurotrophic factor (BDNF) in rat cortical neuronal cells, whereas the endogenous glucocorticoid corticosterone showed a biphasic effect. CDK5 enhanced the effect of aldosterone and dexamethasone on BDNF expression. Because this neurotrophic factor plays critical roles in neuronal viability, synaptic plasticity, consolidation of memory, and emotional changes, we suggest that aberrant activation of CDK5 might influence these functions through corticosteroid receptors/BDNF.
The Activators of Cyclin-Dependent Kinase 5 p35 and p39 Are Essential for Oligodendrocyte Maturation, Process Formation, and Myelination
Luo, Fucheng; Zhang, Jessie; Burke, Kathryn
The regulation of oligodendrocyte development and myelin formation in the CNS is poorly defined. Multiple signals influence the rate and extent of CNS myelination, including the noncanonical cyclin-dependent kinase 5 (Cdk5) whose functions are regulated by its activators p35 and p39. Here we show that selective loss of either p35 or p39 perturbed specific aspects of oligodendrocyte development, whereas loss of both p35 and p39 completely inhibited the development of mature oligodendrocytes and myelination. In the absence of p35, oligodendrocyte differentiation was delayed, process outgrowth was truncated in vitro, and the patterning and extent of myelination were perturbed in the CNS of p35−/− mice. In the absence of p39, oligodendrocyte maturation was transiently affected both in vitro and in vivo. However, loss of both p35 and p39 in oligodendrocyte lineage cells completely inhibited oligodendrocyte progenitor cell differentiation and myelination both in vitro and after transplantation into shiverer slice cultures. Loss of p35 and p39 had a more profound effect on oligodendrocyte development than simply the loss of Cdk5 and could not be rescued by Cdk5 overexpression. These data suggest p35 and p39 have specific and overlapping roles in oligodendrocyte development, some of which may be independent of Cdk5 activation. SIGNIFICANCE STATEMENT The development of oligodendrocytes and myelination is essential for normal CNS function and cyclin-dependent kinase 5 (Cdk5) activity is critical for oligodendrocyte maturation, but how Cdk5 activity is controlled is unclear. Here we show that the coactivators of Cdk5, p35 and p39, regulate distinct stages of oligodendrocyte development and myelination. Loss of p35 perturbs oligodendrocyte progenitor cell differentiation, whereas loss of p39 delays oligodendrocyte maturation. Loss of both completely inhibits oligodendrogenesis and myelination. Disruption of oligodendrocyte development was more pronounced in p35−/−;p39
Cyclin E Uses Cdc6 as a Chromatin-Associated Receptor Required for DNA Replication
Furstenthal, Laura; Kaiser, Brett K.; Swanson, Craig; Jackson, Peter K.
Using an in vitro chromatin assembly assay in Xenopus egg extract, we show that cyclin E binds specifically and saturably to chromatin in three phases. In the first phase, the origin recognition complex and Cdc6 prereplication proteins, but not the minichromosome maintenance complex, are necessary and biochemically sufficient for ATP-dependent binding of cyclin E–Cdk2 to DNA. We find that cyclin E binds the NH2-terminal region of Cdc6 containing Cy–Arg-X-Leu (RXL) motifs. Cyclin E proteins with mutated substrate selection (Met-Arg-Ala-Ile-Leu; MRAIL) motifs fail to bind Cdc6, fail to compete with endogenous cyclin E–Cdk2 for chromatin binding, and fail to rescue replication in cyclin E–depleted extracts. Cdc6 proteins with mutations in the three consensus RXL motifs are quantitatively deficient for cyclin E binding and for rescuing replication in Cdc6-depleted extracts. Thus, the cyclin E–Cdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication. During the second phase, cyclin E–Cdk2 accumulates on chromatin, dependent on polymerase activity. In the third phase, cyclin E is phosphorylated, and the cyclin E–Cdk2 complex is displaced from chromatin in mitosis. In vitro, mitogen-activated protein kinase and especially cyclin B–Cdc2, but not the polo-like kinase 1, remove cyclin E–Cdk2 from chromatin. Rebinding of hyperphosphorylated cyclin E–Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase–directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro. These three phases of cyclin E association with chromatin may facilitate the diverse activities of cyclin E–Cdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis. PMID:11257126
Cyclin A is required at two points in the human cell cycle.
Pagano, M; Pepperkok, R; Verde, F; Ansorge, W; Draetta, G
Cyclins play a fundamental role in regulating cell cycle events in all eukaryotic cells. The human cyclin A gene was identified as the site of integration of hepatitis B virus in a hepatocarcinoma cell line; in addition, cyclin A is associated with the E2F transcription factor in a complex which is dissociated by the E1A oncogene product. Such findings suggest that cyclin A is a target for oncogenic signals. We have now found that DNA synthesis and entry into mitosis are inhibited in human cells microinjected with anti-cyclin A antibodies at distinct times. Cyclin A binds both cdk2 and cdc2, giving two distinct cyclin A kinase activities, one appearing in S phase, the other in G2. These results suggest that cyclin A defines novel control points of the human cell cycle. Images PMID:1312467
Regulation of Exit from Quiescence by p27 and Cyclin D1-CDK4
Ladha, Mohamed H.; Lee, Kwang Y.; Upton, Todd M.; Reed, Michael F.; Ewen, Mark E.
The synthesis of cyclin D1 and its assembly with cyclin-dependent kinase 4 (CDK4) to form an active complex is a rate-limiting step in progression through the G1 phase of the cell cycle. Using an activated allele of mitogen-activated protein kinase kinase 1 (MEK1), we show that this kinase plays a significant role in positively regulating the expression of cyclin D1. This was found both in quiescent serum-starved cells and in cells expressing dominant-negative Ras. Despite the observation that cyclin D1 is a target of MEK1, in cycling cells, activated MEK1, but not cyclin D1, is capable of overcoming a G1 arrest induced by Ras inactivation. Either wild-type or catalytically inactive CDK4 cooperates with cyclin D1 in reversing the G1 arrest induced by inhibition of Ras activity. In quiescent NIH 3T3 cells expressing either ectopic cyclin D1 or activated MEK1, cyclin D1 is able to efficiently associate with CDK4; however, the complex is inactive. A significant percentage of the cyclin D1-CDK4 complexes are associated with p27 in serum-starved activated MEK1 or cyclin D1 cell lines. Reduction of p27 levels by expression of antisense p27 allows for S-phase entry from quiescence in NIH 3T3 cells expressing ectopic cyclin D1, but not in parental cells. PMID:9774675
Hierarchy of S-Phase-Promoting Factors: Yeast Dbf4-Cdc7 Kinase Requires Prior S-Phase Cyclin-Dependent Kinase Activation
Nougarède, Romain; Della Seta, Flavio; Zarzov, Patrick; Schwob, Etienne
In all eukaryotes, the initiation of DNA synthesis requires the formation of prereplicative complexes (pre-RCs) on replication origins, followed by their activation by two S-T protein kinases, an S-phase cyclin-dependent kinase (S-CDK) and a homologue of yeast Dbf4-Cdc7 kinase (Dbf4p-dependent kinase [DDK]). Here, we show that yeast DDK activity is cell cycle regulated, though less tightly than that of the S-CDK Clb5-Cdk1, and peaks during S phase in correlation with Dbf4p levels. Dbf4p is short-lived throughout the cell cycle, but its instability is accentuated during G1 by the anaphase-promoting complex. Downregulating DDK activity is physiologically important, as joint Cdc7p and Dbf4p overexpression is lethal. Because pre-RC formation is a highly ordered process, we asked whether S-CDK and DDK need also to function in a specific order for the firing of origins. We found that both kinases are activated independently, but we show that DDK can perform its function for DNA replication only after S-CDKs have been activated. Cdc45p, a protein needed for initiation, binds tightly to chromatin only after S-CDK activation (L. Zou and B. Stillman, Science 280:593–596, 1998). We show that Cdc45p is phosphorylated by DDK in vitro, suggesting that it might be one of DDK's critical substrates after S-CDK activation. Linking the origin-bound DDK to the tightly regulated S-CDK in a dependent sequence of events may ensure that DNA replication initiates only at the right time and place. PMID:10805723
Role of the mTORC1 complex in satellite cell activation by RNA-induced mitochondrial restoration: dual control of cyclin D1 through microRNAs.
Jash, Sukanta; Dhar, Gunjan; Ghosh, Utpalendu; Adhya, Samit
During myogenesis, satellite stem cells (SCs) are induced to proliferate and differentiate to myogenic precursors. The role of energy sensors such as the AMP-activated protein kinase (AMPK) and the mammalian Target of Rapamycin (mTOR) in SC activation is unclear. We previously observed that upregulation of ATP through RNA-mediated mitochondrial restoration (MR) accelerates SC activation following skeletal muscle injury. We show here that during regeneration, the AMPK-CRTC2-CREB and Raptor-mTORC-4EBP1 pathways were rapidly activated. The phosho-CRTC2-CREB complex was essential for myogenesis and activated transcription of the critical cell cycle regulator cyclin D1 (Ccnd1). Knockdown (KD) of either mTORC or its subunit Raptor delayed SC activation without influencing the differentiation program. KD of 4EBP1 had no effect on SC activation but enhanced myofiber size. mTORC1 positively regulated Ccnd1 translation but destabilized Ccnd1 mRNA. These antithetical effects of mTORC1 were mediated by two microRNAs (miRs) targeted to the 3' untranslated region (UTR) of Ccnd1 mRNA: miR-1 was downregulated in mTORC-KD muscle, and depletion of miR-1 resulted in increased levels of mRNA without any effect on Ccnd1 protein. In contrast, miR-26a was upregulated upon mTORC depletion, while anti-miR-26a oligonucleotide specifically stimulated Ccnd1 protein expression. Thus, mTORC may act as a timer of satellite cell proliferation during myogenesis.
The transcriptional network activated by Cln3 cyclin at the G1-to-S transition of the yeast cell cycle
Background The G1-to-S transition of the cell cycle in the yeast Saccharomyces cerevisiae involves an extensive transcriptional program driven by transcription factors SBF (Swi4-Swi6) and MBF (Mbp1-Swi6). Activation of these factors ultimately depends on the G1 cyclin Cln3. Results To determine the transcriptional targets of Cln3 and their dependence on SBF or MBF, we first have used DNA microarrays to interrogate gene expression upon Cln3 overexpression in synchronized cultures of strains lacking components of SBF and/or MBF. Secondly, we have integrated this expression dataset together with other heterogeneous data sources into a single probabilistic model based on Bayesian statistics. Our analysis has produced more than 200 transcription factor-target assignments, validated by ChIP assays and by functional enrichment. Our predictions show higher internal coherence and predictive power than previous classifications. Our results support a model whereby SBF and MBF may be differentially activated by Cln3. Conclusions Integration of heterogeneous genome-wide datasets is key to building accurate transcriptional networks. By such integration, we provide here a reliable transcriptional network at the G1-to-S transition in the budding yeast cell cycle. Our results suggest that to improve the reliability of predictions we need to feed our models with more informative experimental data. PMID:20573214
The Activators of Cyclin-Dependent Kinase 5 p35 and p39 Are Essential for Oligodendrocyte Maturation, Process Formation, and Myelination.
Luo, Fucheng; Zhang, Jessie; Burke, Kathryn; Miller, Robert H; Yang, Yan
The regulation of oligodendrocyte development and myelin formation in the CNS is poorly defined. Multiple signals influence the rate and extent of CNS myelination, including the noncanonical cyclin-dependent kinase 5 (Cdk5) whose functions are regulated by its activators p35 and p39. Here we show that selective loss of either p35 or p39 perturbed specific aspects of oligodendrocyte development, whereas loss of both p35 and p39 completely inhibited the development of mature oligodendrocytes and myelination. In the absence of p35, oligodendrocyte differentiation was delayed, process outgrowth was truncated in vitro, and the patterning and extent of myelination were perturbed in the CNS of p35(-/-) mice. In the absence of p39, oligodendrocyte maturation was transiently affected both in vitro and in vivo. However, loss of both p35 and p39 in oligodendrocyte lineage cells completely inhibited oligodendrocyte progenitor cell differentiation and myelination both in vitro and after transplantation into shiverer slice cultures. Loss of p35 and p39 had a more profound effect on oligodendrocyte development than simply the loss of Cdk5 and could not be rescued by Cdk5 overexpression. These data suggest p35 and p39 have specific and overlapping roles in oligodendrocyte development, some of which may be independent of Cdk5 activation.
Distinct and Overlapping Requirements for Cyclins A, B, and B3 in Drosophila Female Meiosis
Bourouh, Mohammed; Dhaliwal, Rajdeep; Rana, Ketki; Sinha, Sucheta; Guo, Zhihao; Swan, Andrew
Meiosis, like mitosis, depends on the activity of the cyclin dependent kinase Cdk1 and its cyclin partners. Here, we examine the specific requirements for the three mitotic cyclins, A, B, and B3 in meiosis of Drosophila melanogaster. We find that all three cyclins contribute redundantly to nuclear envelope breakdown, though cyclin A appears to make the most important individual contribution. Cyclin A is also required for biorientation of homologs in meiosis I. Cyclin B3, as previously reported, is required for anaphase progression in meiosis I and in meiosis II. We find that it also plays a redundant role, with cyclin A, in preventing DNA replication during meiosis. Cyclin B is required for maintenance of the metaphase I arrest in mature oocytes, for spindle organization, and for timely progression through the second meiotic division. It is also essential for polar body formation at the completion of meiosis. With the exception of its redundant role in meiotic maturation, cyclin B appears to function independently of cyclins A and B3 through most of meiosis. We conclude that the three mitotic cyclin-Cdk complexes have distinct and overlapping functions in Drosophila female meiosis. PMID:27652889
ATM is required for rapid degradation of cyclin D1 in response to {gamma}-irradiation
Choo, Dong Wan; Baek, Hye Jung; Motoyama, Noboru; Cho, Kwan Ho; Kim, Hye Sun; Kim, Sang Soo
The cellular response to DNA damage induced by {gamma}-irradiation activates cell-cycle arrest to permit DNA repair and to prevent replication. Cyclin D1 is the key molecule for transition between the G1 and S phases of the cell-cycle, and amplification or overexpression of cyclin D1 plays pivotal roles in the development of several human cancers. To study the regulation of cyclin D1 in the DNA-damaged condition, we analyzed the proteolytic regulation of cyclin D1 expression upon {gamma}-irradiation. Upon {gamma}-irradiation, a rapid reduction in cyclin D1 levels was observed prior to p53 stabilization, indicating that the stability of cyclin D1 is controlled in a p53-independent manner. Further analysis revealed that irradiation facilitated ubiquitination of cyclin D1 and that a proteasome inhibitor blocked cyclin D1 degradation under the same conditions. Interestingly, after mutation of threonine residue 286 of cyclin D1, which is reported to be the GSK-3{beta} phosphorylation site, the mutant protein showed resistance to irradiation-induced proteolysis although inhibitors of GSK-3{beta} failed to prevent cyclin D1 degradation. Rather, ATM inhibition markedly prevented cyclin D1 degradation induced by {gamma}-irradiation. Our data indicate that communication between ATM and cyclin D1 may be required for maintenance of genomic integrity achieved by rapid arrest of the cell-cycle, and that disruption of this crosstalk may increase susceptibility to cancer.
Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes*
Ramos Gomes, Fernanda; Romaniello, Vincenzo; Sánchez, Araceli; Weber, Claudia; Narayanan, Pratibha; Psol, Maryna; Pardo, Luis A.
KV10.1 is a voltage-gated potassium channel expressed selectively in the mammalian brain but also aberrantly in cancer cells. In this study we identified short splice variants of KV10.1 resulting from exon-skipping events (E65 and E70) in human brain and cancer cell lines. The presence of the variants was confirmed by Northern blot and RNase protection assays. Both variants completely lacked the transmembrane domains of the channel and produced cytoplasmic proteins without channel function. In a reconstituted system, both variants co-precipitated with the full-length channel and induced a robust down-regulation of KV10.1 current when co-expressed with the full-length form, but their effect was mechanistically different. E65 required a tetramerization domain and induced a reduction in the overall expression of full-length KV10.1, whereas E70 mainly affected its glycosylation pattern. E65 triggered the activation of cyclin-dependent kinases in Xenopus laevis oocytes, suggesting a role in cell cycle control. Our observations highlight the relevance of noncanonical functions for the oncogenicity of KV10.1, which need to be considered when ion channels are targeted for cancer therapy. PMID:26518875
The PP2A-B56 phosphatase opposes cyclin E autocatalytic degradation via site-specific dephosphorylation.
Davis, Ryan J; Swanger, Jherek; Hughes, Bridget T; Clurman, Bruce E
Cyclin E, in conjunction with its catalytic partner cyclin-dependent kinase 2 (CDK2), regulates cell cycle progression as cells exit quiescence and enter S-phase. Multiple mechanisms control cyclin E periodicity during the cell cycle, including phosphorylation-dependent cyclin E ubiquitylation by the SCF(Fbw7) ubiquitin ligase. Serine 384 (S384) is the critical cyclin E phosphorylation site that stimulates Fbw7 binding and cyclin E ubiquitylation and degradation. Because S384 is autophosphorylated by bound CDK2, this presents a paradox as to how cyclin E can evade autocatalytically induced degradation in order to phosphorylate its other substrates. We found that S384 phosphorylation is dynamically regulated in cells, and that cyclin E is specifically dephosphorylated at S384 by the PP2A-B56 phosphatase, thereby uncoupling cyclin E degradation from cyclin E-CDK2 activity. Furthermore, the rate of S384 dephosphorylation is high in interphase but low in mitosis. This provides a mechanism whereby interphase cells can oppose autocatalytic cyclin E degradation and maintain cyclin E-CDK2 activity, while also enabling cyclin E destruction in mitosis, when inappropriate cyclin E expression is genotoxic.
Upstream stimulatory factor regulates expression of the cell cycle-dependent cyclin B1 gene promoter.
Cogswell, J P; Godlevski, M M; Bonham, M; Bisi, J; Babiss, L
Progression through the somatic cell cycle requires the temporal regulation of cyclin gene expression and cyclin protein turnover. One of the best-characterized examples of this regulation is seen for the B-type cyclins. These cyclins and their catalytic component, cdc2, have been shown to mediate both the entry into and maintenance of mitosis. The cyclin B1 gene has been shown to be expressed between the late S and G2 phases of the cell cycle, while the protein is degraded specifically at interphase via ubiquitination. To understand the molecular basis for transcriptional regulation of the cyclin B1 gene, we cloned the human cyclin B1 gene promoter region. Using a chloramphenicol acetyltransferase reporter system and both stable and transient assays, we have shown that the cyclin B1 gene promoter (extending to -3800 bp relative to the cap site) can confer G2-enhanced promoter activity. Further analysis revealed that an upstream stimulatory factor (USF)-binding site and its cognate transcription factor(s) are critical for expression from the cyclin B1 promoter in cycling HeLa cells. Interestingly, USF DNA-binding activity appears to be regulated in a G2-specific fashion, supporting the idea that USF may play some role in cyclin B1 gene activation. These studies suggest an important link between USF and the cyclin B1 gene, which in part explains how maturation promoting factor complex formation is regulated. PMID:7739559
Constitutive activation of glycogen synthase kinase-3β correlates with better prognosis and cyclin-dependent kinase inhibitors in human gastric cancer
Background Aberrant regulation of glycogen synthase kinase-3β (GSK-3β) has been implicated in several human cancers; however, it has not been reported in the gastric cancer tissues to date. The present study was performed to determine the expression status of active form of GSK-3β phosphorylated at Tyr216 (pGSK-3β) and its relationship with other tumor-associated proteins in human gastric cancers. Methods Immunohistochemistry was performed on tissue array slides containing 281 human gastric carcinoma specimens. In addition, gastric cancer cells were cultured and treated with a GSK-3β inhibitor lithium chloride (LiCl) for immunoblot analysis. Results We found that pGSK-3β was expressed in 129 (46%) of 281 cases examined, and was higher in the early-stages of pathologic tumor-node-metastasis (P < 0.001). The expression of pGSK-3β inversely correlated with lymphatic invasion (P < 0.001) and lymph node metastasis (P < 0.001) and correlated with a longer patient survival (P < 0.001). In addition, pGSK-3β expression positively correlated with that of p16, p21, p27, p53, APC, PTEN, MGMT, SMAD4, or KAI1 (P < 0.05), but not with that of cyclin D1. This was confirmed by immunoblot analysis using SNU-668 gastric cancer cells treated with LiCl. Conclusions GSK-3β activation was frequently observed in early-stage gastric carcinoma and was significantly correlated with better prognosis. Thus, these findings suggest that GSK-3β activation is a useful prognostic marker for the early-stage gastric cancer. PMID:20704706
Both cyclin I and p35 are required for maximal survival benefit of cyclin-dependent kinase 5 in kidney podocytes.
Taniguchi, Yoshinori; Pippin, Jeffrey W; Hagmann, Henning; Krofft, Ronald D; Chang, Alice M; Zhang, Jiong; Terada, Yoshio; Brinkkoetter, Paul; Shankland, Stuart J
Cyclin-dependent kinase (Cdk)-5 is activated by both cyclin I and the noncyclin activator p35 in terminally differentiated cells such as kidney podocytes and neurons. Cyclin I and p35 are restricted to podocytes in the kidney, and each limit podocyte apoptosis by activating Cdk5. To determine whether both activators are necessary, or whether they serve backup roles, a double cyclin I-p35 null mouse was generated. Experimental glomerular disease characterized by podocyte apoptosis was then induced by administering an anti-podocyte antibody. The results showed that under nonstressed conditions double mutants had normal kidney structure and function and were indistinguishable from wild-type, cyclin I(-/-), or p35(-/-) mice. In contrast, when stressed with disease, podocyte apoptosis increased fourfold compared with diseased cyclin I(-/-) or p35(-/-) mice. This resulted in a more pronounced decrease in podocyte number, proteinuria, and glomerulosclerosis. Under normal states and nephritic states, levels for the prosurvival protein Bcl-2 were lower in double cyclin I(-/-) p35(-/-) mice than the other mice. Similarly, levels of Bcl-xL, another prosurvival member, were lower in normal and nephritic double cyclin I(-/-) p35(-/-) mice but similar to single-cyclin I(-/-) mice. Moreover, levels of ERK1/2 and MEK1/2 activation were lower in nephritic double cyclin I(-/-) p35(-/-) mice but similar to single-cyclin I(-/-) mice. The results demonstrate that the activators of Cdk5, p35, and cyclin I are not required for normal kidney function. However, they play pivotal coordinated roles in maintaining podocyte survival during stress states in disease.
Mechanisms and regulation of the degradation of cyclin B.
Hershko, A
The degradation of the cyclin B subunit of protein kinase Cdk1/cyclin B is required for inactivation of the kinase and exit from mitosis. Cyclin B is degraded by the ubiquitin pathway, a system involved in most selective protein degradation in eukaryotic cells. In this pathway, proteins are targeted for degradation by ligation to ubiquitin, a process carried out by the sequential action of three enzymes: the ubiquitin-activating enzyme E1, a ubiquitin-carrier protein E2 and a ubiquitin-protein ligase E3. In the system responsible for cyclin B degradation, the E3-like function is carried out by a large complex called cyclosome or anaphase-promoting complex (APC). In the early embryonic cell cycles, the cyclosome is inactive in the interphase, but becomes active at the end of mitosis. Activation requires phosphorylation of the cyclosome/APC by protein kinase Cdk1/cyclin B. The lag kinetics of cyclosome activation may be explained by Suc1-assisted multiple phosphorylations of partly phosphorylated complex. The presence of a Fizzy/Cdc20-like protein is necessary for maximal activity of the mitotic form of cyclosome/APC in cyclin-ubiquitin ligation. PMID:10582242
MEK2 controls the activation of MKK3/MKK6-p38 axis involved in the MDA-MB-231 breast cancer cell survival: Correlation with cyclin D1 expression.
Huth, Hugo W; Albarnaz, Jonas D; Torres, Alice A; Bonjardim, Claudio A; Ropert, Catherine
The Ras-Raf-MEK-ERK1/2 signaling pathway regulates fundamental processes in malignant cells. However, the exact contributions of MEK1 and MEK2 to the development of cancer remain to be established. We studied the effects of MEK small-molecule inhibitors (PD98059 and U0126) and MEK1 and MEK2 knock-down on cell proliferation, apoptosis and MAPK activation. We showed a diminution of cell viability that was associated with a downregulation of cyclin D1 expression and an increase of apoptosis marker in MEK2 silenced cells; by contrast, a slight increase of cell survival was observed in the absence of MEK1 that correlated with an augment of cyclin D1 expression. These data indicate that MEK2 but not MEK1 is essential for MDA-MB-231 cell survival. Importantly, the role of MEK2 in cell survival appeared independent on ERK1/2 phosphorylation since its absence did not alter the level of activated ERK1/2. Indeed, we have reported an unrevealed link between MEK2 and MKK3/MKK6-p38 MAPK axis where MEK2 was essential for the phosphorylation of MKK3/MKK6 and p38 MAPK that directly impacted on cyclin D1 expression. Importantly, the MEK1 inhibitor PD98059, like MEK1 silencing, induced an augment of cyclin D1 expression that correlated with an increase of MDA-MB-231 cell proliferation suggesting that MEK1 may play a regulatory role in these cells. In sum, the crucial role of MEK2 in MDA-MB-231 cell viability and the unknown relationship between MEK2 and MKK3/MKK6-p38 axis here revealed may open new therapeutic strategies for aggressive breast cancer.
Two Degradation Pathways of the p35 Cdk5 (Cyclin-dependent Kinase) Activation Subunit, Dependent and Independent of Ubiquitination.
Takasugi, Toshiyuki; Minegishi, Seiji; Asada, Akiko; Saito, Taro; Kawahara, Hiroyuki; Hisanaga, Shin-ichi
Cdk5 is a versatile protein kinase that is involved in various neuronal activities, such as the migration of newborn neurons, neurite outgrowth, synaptic regulation, and neurodegenerative diseases. Cdk5 requires the p35 regulatory subunit for activation. Because Cdk5 is more abundantly expressed in neurons compared with p35, the p35 protein levels determine the kinase activity of Cdk5. p35 is a protein with a short half-life that is degraded by proteasomes. Although ubiquitination of p35 has been previously reported, the degradation mechanism of p35 is not yet known. Here, we intended to identify the ubiquitination site(s) in p35. Because p35 is myristoylated at the N-terminal glycine, the possible ubiquitination sites are the lysine residues in p35. We mutated all 23 Lys residues to Arg (p35 23R), but p35 23R was still rapidly degraded by proteasomes at a rate similar to wild-type p35. The degradation of p35 23R in primary neurons and the Cdk5 activation ability of p35 23R suggested the occurrence of ubiquitin-independent degradation of p35 in physiological conditions. We found that p35 has the amino acid sequence similar to the ubiquitin-independent degron in the NKX3.1 homeodomain transcription factor. An Ala mutation at Pro-247 in the degron-like sequence made p35 stable. These results suggest that p35 can be degraded by two degradation pathways: ubiquitin-dependent and ubiquitin-independent. The rapid degradation of p35 by two different methods would be a mechanism to suppress the production of p25, which overactivates Cdk5 to induce neuronal cell death.
Disruption of transforming growth factor-beta signaling through beta-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation.
Kitisin, K; Ganesan, N; Tang, Y; Jogunoori, W; Volpe, E A; Kim, S S; Katuri, V; Kallakury, B; Pishvaian, M; Albanese, C; Mendelson, J; Zasloff, M; Rashid, A; Fishbein, T; Evans, S R T; Sidawy, A; Reddy, E P; Mishra, B; Johnson, L B; Shetty, K; Mishra, L
Transforming growth factor-beta (TGF-beta) signaling members, TGF-beta receptor type II (TBRII), Smad2, Smad4 and Smad adaptor, embryonic liver fodrin (ELF), are prominent tumor suppressors in gastrointestinal cancers. Here, we show that 40% of elf(+/-) mice spontaneously develop hepatocellular cancer (HCC) with markedly increased cyclin D1, cyclin-dependent kinase 4 (Cdk4), c-Myc and MDM2 expression. Reduced ELF but not TBRII, or Smad4 was observed in 8 of 9 human HCCs (P<0.017). ELF and TBRII are also markedly decreased in human HCC cell lines SNU-398 and SNU-475. Restoration of ELF and TBRII in SNU-398 cells markedly decreases cyclin D1 as well as hyperphosphorylated-retinoblastoma (hyperphosphorylated-pRb). Thus, we show that TGF-beta signaling and Smad adaptor ELF suppress human hepatocarcinogenesis, potentially through cyclin D1 deregulation. Loss of ELF could serve as a primary event in progression toward a fully transformed phenotype and could hold promise for new therapeutic approaches in human HCCs.
PPAR{gamma} ligands suppress the feedback loop between E2F2 and cyclin-E1
Komatsu, Yoko; Ito, Ichiaki; Wayama, Mitsutoshi; Fujimura, Akiko; Akaogi, Kensuke; Machida, Hikaru; Nakajima, Yuka; Kuroda, Takao; Ohmori, Kazuji; Murayama, Akiko; Kimura, Keiji; Yanagisawa, Junn
PPAR{gamma} is a nuclear hormone receptor that plays a key role in the induction of peroxisome proliferation. A number of studies showed that PPAR{gamma} ligands suppress cell cycle progression; however, the mechanism remains to be determined. Here, we showed that PPAR{gamma} ligand troglitazone inhibited G1/S transition in colon cancer cells, LS174T. Troglitazone did not affect on either expression of CDK inhibitor (p18) or Wnt signaling pathway, indicating that these pathways were not involved in the troglitazone-dependent cell cycle arrest. GeneChip and RT-PCR analyses revealed that troglitazone decreased mRNA levels of cell cycle regulatory factors E2F2 and cyclin-E1 whose expression is activated by E2F2. Down-regulation of E2F2 by troglitazone results in decrease of cyclin-E1 transcription, which could inhibit phosphorylation of Rb protein, and consequently evoke the suppression of E2F2 transcriptional activity. Thus, we propose that troglitazone suppresses the feedback loop containing E2F2, cyclin-E1, and Rb protein.
Expression of cyclins E1 and E2 during mouse development and in neoplasia
Geng, Yan; Yu, Qunyan; Whoriskey, Wendy; Dick, Fred; Tsai, Kenneth Y.; Ford, Heide L.; Biswas, Debajit K.; Pardee, Arthur B.; Amati, Bruno; Jacks, Tyler; Richardson, Andrea; Dyson, Nicholas; Sicinski, Piotr
Cyclin E1 (formerly called cyclin E) and the recently described cyclin E2 belong to the family of E-type cyclins that operate during the G1/S phase progression in mammalian cells. The two E-cyclins share a catalytic partner, cyclin-dependent kinase 2 (CDK2), and activate their associated kinase activities at similar times during cell cycle progression. Despite these similarities, it is unknown whether the two proteins perform distinct functions, or, alternatively, they control S-phase entry of different cell types in a tissue-specific fashion. To start addressing in vivo functions of E-cyclins, we determined the expression pattern of cyclins E1 and E2 during normal mouse development. We found that the two E-cyclins showed very similar patterns of expression; both were expressed within the proliferating compartment during embryo development. Analyses of cells and tissues lacking members of the retinoblastoma (pRB) family of proteins revealed that the expression of both cyclins is controlled in a pRB-dependent, but p107- and p130-independent fashion, likely through the pRB-dependent E2F transcription factors. We also found that cyclins E1 and E2 are expressed at high levels in mouse breast tumors driven by the Myc oncogene. Last, we found that cyclin E2 is overexpressed in ≈24% of analyzed human mammary carcinomas. Collectively these findings suggest that the expression of cyclins E1 and E2 is governed by similar molecular circuitry. PMID:11687642
Foci of cyclin A2 interact with actin and RhoA in mitosis
Loukil, Abdelhalim; Izard, Fanny; Georgieva, Mariya; Mashayekhan, Shaereh; Blanchard, Jean-Marie; Parmeggiani, Andrea; Peter, Marion
Cyclin A2 is a key player in the regulation of the cell cycle. Its degradation in mid-mitosis depends primarily on the ubiquitin-proteasome system (UPS), while autophagy also contributes. However, a fraction of cyclin A2 persists beyond metaphase. In this work, we focus on cyclin A2-rich foci detected in mitosis by high resolution imaging and analyse their movements. We demonstrate that cyclin A2 interacts with actin and RhoA during mitosis, and that cyclin A2 depletion induces a dramatic decrease in active RhoA in mitosis. Our data suggest cyclin A2 participation in RhoA activation in late mitosis. PMID:27279564
Cell cycle regulatory effects of retinoic Acid and forskolin are mediated by the cyclin C gene.
Makkonen, Katri M; Malinen, Marjo; Ropponen, Antti; Väisänen, Sami; Carlberg, Carsten
As a partner of cyclin-dependent kinase (CDK) 3, Cyclin C controls cellular proliferation and, together with CDK8, represses gene transcription. In this study, we showed that the highly expressed Cyclin C gene is a direct target of the nuclear hormone all-trans retinoic acid (RA) in HEK293 human embryonal kidney cells. The RA receptor (RAR) gamma associates with a Cyclin C promoter region containing two RAR binding sites. The Cyclin C gene also directly responds to the cAMP activator Forskolin via the transcription factor CREB1 (cAMP response element-binding protein 1), for which we identified four binding sites within the first 2250 bp of its promoter. RARgamma and CREB1 show functional convergence via the corepressor NCoR1, which controls in particular the Forskolin response of Cyclin C. The histone deacetylases 1, 5, 6, 7 and 11 are involved in the basal expression of Cyclin C, but in HEK293 and MCF-7 human breast carcinoma cells the antiproliferative effects of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) are not mediated by Cyclin C. However, cell cycle progressing effects of all-trans RA and Forskolin are dependent on Cyclin C expression levels. This suggests that the primary regulation of Cyclin C by all-trans RA and Forskolin mediates some of the cell cycle control actions of these compounds.
Cyclin B2 and p53 control proper timing of centrosome separation
Nam, Hyun-Ja; van Deursen, Jan M.
Cyclins Bl and B2 are frequently elevated in human cancers and are associated with tumour aggressiveness and poor clinical outcome; however, whether and how B-type cyclins drive tumorigenesis is unknown. Here we show that cyclin Bl and B2 transgenic mice are highly prone to tumours, including tumour types where B-type cyclins serve as prognosticators. Cyclins Bl and B2 both induce aneuploidy when overexpressed but through distinct mechanisms, with cyclin Bl inhibiting separase activation, leading to anaphase bridges, and cyclin B2 triggering aurora-A-mediated Plkl hyperactivation, resulting in accelerated centrosome separation and lagging chromosomes. Complementary experiments revealed that cyclin B2 and p53 act antagonistically to control aurora-A-mediated centrosome splitting and accurate chromosome segregation in normal cells. These data demonstrate a causative link between B-type cyclin overexpression and tumour pathophysiology, and uncover previously unknown functions of cyclin B2 and p53 in centrosome separation that may be perturbed in many human cancers. PMID:24776885
Discovery of [4-Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimidin-5-yl](2,3-difluoro-6-methoxyphenyl)methanone (R547), A Potent and Selective Cyclin-Dependent Kinase Inhibitor with Significiant in Vivo Antitumor Activity
Chu,X.; DePinto, W.; Bartkovitz, D.; So, S.; Vu, B.; Packman, K.; Lukacs, C.; Ding, Q.; Jiang, N.; et al.
The cyclin-dependent kinases (CDKs) and their cyclin partners are key regulators of the cell cycle. Since deregulation of CDKs is found with high frequency in many human cancer cells, pharmacological inhibition of CDKs with small molecules has the potential to provide an effective strategy for the treatment of cancer. The 2,4-diamino-5-ketopyrimidines 6 reported here represent a novel class of potent and ATP-competitive inhibitors that selectively target the cyclin-dependent kinase family. This diaminopyrimidine core with a substituted 4-piperidine moiety on the C2-amino position and 2-methoxybenzoyl at the C5 position has been identified as the critical structure responsible for the CDK inhibitory activity. Further optimization has led to a good number of analogues that show potent inhibitory activities against CDK1, CDK2, and CDK4 but are inactive against a large panel of serine/threonine and tyrosine kinases (K{sub i} > 10 {mu}M). As one of these representative analogues, compound 39 (R547) has the best CDK inhibitory activities (K{sub i} = 0.001, 0.003, and 0.001 M for CDK1, CDK2, and CDK4, respectively) and excellent in vitro cellular potency, inhibiting the growth of various human tumor cell lines including an HCT116 cell line (IC{sub 50} = 0.08 {mu}M). An X-ray crystal structure of 39 bound to CDK2 has been determined in this study, revealing a binding mode that is consistent with our SAR. Compound 39 demonstrates significant in vivo efficacy in the HCT116 human colorectal tumor xenograft model in nude mice with up to 95% tumor growth inhibition. On the basis of its superior overall profile, 39 was chosen for further evaluation and has progressed into Phase I clinical trial for the treatment of cancer.
Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells
Sumrejkanchanakij, Piyamas; Eto, Kazuhiro; Ikeda, Masa-Aki . E-mail: email@example.com
The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16{sup INK4a}, a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.
Distinct proliferative and transcriptional effects of the D-type cyclins in vivo.
Mullany, Lisa K; White, Peter; Hanse, Eric A; Nelsen, Christopher J; Goggin, Melissa M; Mullany, Joseph E; Anttila, Chelsea K; Greenbaum, Linda E; Kaestner, Klaus H; Albrecht, Jeffrey H
The D-type cyclins (D1, D2 and D3) are components of the cell cycle machinery and govern progression through G(1) phase in response to extracellular signals. Although these proteins are highly homologous and conserved in evolution, they contain distinct structural motifs and are differentially regulated in various cell types. Cyclin D1 appears to play a role in many different types of cancer, whereas cyclins D2 and D3 are less frequently associated with malignancy. In this study, we transiently expressed cyclin D1, D2 or D3 in hepatocytes and analyzed transcriptional networks regulated by each. All three D-type cyclins promoted robust hepatocyte proliferation and marked liver growth, although cyclin D3 stimulated less DNA synthesis than D1 or D2. Accordingly, the three D-type cyclins similarly activated genes associated with cell division. Cyclin D1 regulated transcriptional pathways involved in the metabolism of carbohydrates, lipids, amino acids, and other substrates, whereas cyclin D2 did not regulate these pathways despite having an equivalent effect on proliferation. Comparison of transcriptional profiles following 70% partial hepatectomy and cyclin D1 transduction revealed a highly significant overlap, suggesting that cyclin D1 may regulate diverse cellular processes in the regenerating liver. In summary, these studies provide the first comparative analysis of the transcriptional networks regulated by the D-type cyclins and provide insight into novel functions of these key cell cycle proteins. Further study of the unique targets of cyclin D1 should provide further insight into its prominent role in proliferation, growth and cancer.
Suberoylanilide hydroxamic acid (SAHA) inhibits EGF-induced cell transformation via reduction of cyclin D1 mRNA stability
Zhang, Jingjie; Ouyang, Weiming; Li, Jingxia; Zhang, Dongyun; Yu, Yonghui; Wang, York; Li, Xuejun; Huang, Chuanshu
Suberoylanilide hydroxamic acid (SAHA) inhibiting cancer cell growth has been associated with its downregulation of cyclin D1 protein expression at transcription level or translation level. Here, we have demonstrated that SAHA inhibited EGF-induced Cl41 cell transformation via the decrease of cyclin D1 mRNA stability and induction of G0/G1 growth arrest. We found that SAHA treatment resulted in the dramatic inhibition of EGF-induced cell transformation, cyclin D1 protein expression and induction of G0/G1 growth arrest. Further studies showed that SAHA downregulation of cyclin D1 was only observed with endogenous cyclin D1, but not with reconstitutionally expressed cyclin D1 in the same cells, excluding the possibility of SAHA regulating cyclin D1 at level of protein degradation. Moreover, SAHA inhibited EGF-induced cyclin d1 mRNA level, whereas it did not show any inhibitory effect on cyclin D1 promoter-driven luciferase reporter activity under the same experimental conditions, suggesting that SAHA may decrease cyclin D1 mRNA stability. This notion was supported by the results that treatment of cells with SAHA decreased the half-life of cyclin D1 mRNA from 6.95 h to 2.57 h. Consistent with downregulation of cyclin D1 mRNA stability, SAHA treatment also attenuated HuR expression, which has been well-characterized as a positive regulator of cyclin D1 mRNA stability. Thus, our study identifies a novel mechanism responsible for SAHA inhibiting cell transformation via decreasing cyclin D1 mRNA stability and induction of G0/G1 growth arrest in Cl41 cells. -- Highlights: ► SAHA inhibits cell transformation in Cl41 cells. ► SAHA suppresses Cyclin D1 protein expression. ► SAHA decreases cyclin D1 mRNA stability.
Rapamycin disrupts cyclin/cyclin-dependent kinase/p21/proliferating cell nuclear antigen complexes and cyclin D1 reverses rapamycin action by stabilizing these complexes.
Law, Mary; Forrester, Elizabeth; Chytil, Anna; Corsino, Patrick; Green, Gail; Davis, Bradley; Rowe, Thomas; Law, Brian
Rapamycin and its derivatives are promising anticancer agents, but the exact mechanisms by which these drugs induce cell cycle arrest and inhibit tumor growth are unknown. A biochemical analysis of human mammary tumor cell lines indicated that rapamycin-induced antiproliferative effects correlated with down-regulation of cellular p21 levels and the levels of p21 in cyclin-dependent kinase (Cdk) 2 and 4 complexes. Cyclin D1 overexpression reversed rapamycin action and this reversal correlated with increased levels of cellular p21, higher levels of p21 associated with Cdk2, and stabilization of cyclin D1/Cdk2/p21/proliferating cell nuclear antigen (PCNA) complexes. Experiments using a novel cyclin D1-Cdk2 fusion protein or a kinase-dead mutant of the fusion protein indicated that reversal of rapamycin action required not only the formation of complexes with p21 and PCNA but also complex-associated kinase activity. Similar results were observed in vivo. The rapamycin derivative RAD001 (everolimus) inhibited the growth of mouse mammary tumors, which correlated with the disruption of cyclin D1/Cdk2 complexes. The potential implications of these results with respect to the use of rapamycin derivatives in breast cancer therapy are discussed.
Rsf-1 is overexpressed in non-small cell lung cancers and regulates cyclinD1 expression and ERK activity
Li, Qingchang; Dong, Qianze; Wang, Enhua
Highlights: Black-Right-Pointing-Pointer Rsf-1 expression is elevated in non-small cell lung cancers. Black-Right-Pointing-Pointer Rsf-1 depletion inhibits proliferation and increased apoptosis in lung cancer cells. Black-Right-Pointing-Pointer Rsf-1 depletion decreases the level of cyclinD1 and phosphor-ERK expression. -- Abstract: Rsf-1 (HBXAP) was recently reported to be overexpressed in various cancers and associated with the malignant behavior of cancer cells. However, the expression of Rsf-1 in primary lung cancer and its biological roles in non-small cell lung cancer (NSCLC) have not been reported. The molecular mechanism of Rsf-1 in cancer aggressiveness remains ambiguous. In the present study, we analyzed the expression pattern of Rsf-1 in NSCLC tissues and found that Rsf-1 was overexpressed at both the mRNA and protein levels. There was a significant association between Rsf-1 overexpression and TNM stage (p = 0.0220) and poor differentiation (p = 0.0013). Furthermore, knockdown of Rsf-1 expression in H1299 and H460 cells with high endogenous Rsf-1 expression resulted in a decrease of colony formation ability and inhibition of cell cycle progression. Rsf-1 knockdown also induced apoptosis in these cell lines. Further analysis showed that Rsf-1 knockdown decreased cyclin D1 expression and phospho-ERK levels. In conclusion, Rsf-1 is overexpressed in NSCLC and contributes to malignant cell growth by cyclin D1 and ERK modulation, which makes Rsf-1 a candidate therapeutic target in lung cancer.
Understanding and modulating cyclin-dependent kinase inhibitor specificity: molecular modeling and biochemical evaluation of pyrazolopyrimidinones as CDK2/cyclin A and CDK4/cyclin D1 inhibitors
NASA Astrophysics Data System (ADS)
Rossi, Karen A.; Markwalder, Jay A.; Seitz, Steven P.; Chang, Chong-Hwan; Cox, Sarah; Boisclair, Michael D.; Brizuela, Leonardo; Brenner, Stephen L.; Stouten, Pieter F. W.
Cyclin-dependent kinases (CDKs) play a key role in regulating the cell cycle. The cyclins, their activating agents, and endogenous CDK inhibitors are frequently mutated in human cancers, making CDKs interesting targets for cancer chemotherapy. Our aim is the discovery of selective CDK4/cyclin D1 inhibitors. An ATP-competitive pyrazolopyrimidinone CDK inhibitor was identified by HTS and docked into a CDK4 homology model. The resulting binding model was consistent with available SAR and was validated by a subsequent CDK2/inhibitor crystal structure. An iterative cycle of chemistry and modeling led to a 70-fold improvement in potency. Small substituent changes resulted in large CDK4/CDK2 selectivity changes. The modeling revealed that selectivity is largely due to hydrogen-bonded interactions with only two kinase residues. This demonstrates that small differences between enzymes can efficiently be exploited in the design of selective inhibitors.
Colocalization of β-catenin with Notch intracellular domain in colon cancer: a possible role of Notch1 signaling in activation of CyclinD1-mediated cell proliferation.
Gopalakrishnan, Natarajan; Saravanakumar, Marimuthu; Madankumar, Perumal; Thiyagu, Mani; Devaraj, Halagowder
The Wnt and Notch1 signaling pathways play major roles in intestinal development and tumorigenesis. Sub-cellular localization of β-catenin has been implicated in colorectal carcinogenesis. However, the β-catenin and Notch intracellular domain (NICD) interaction has to be addressed. Immunohistochemistries of β-catenin, NICD, and dual immunofluorescence of β-catenin and NICD were analyzed in colorectal tissues and HT29 cell line. Moreover, real-time PCR analysis of CyclinD1, Hes1 and MUC2 was done in HT29 cells upon N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment. Dual staining emphasized the strong interaction of β-catenin and NICD in adenoma and adenocarcinoma than in normal tissues. Hes1 transcript levels were decreased 1.5- and 7.1-fold in 12.5 and 25 µM DAPT-treated HT29 cells. CyclinD1 transcript levels decreased 1.2- and 1.6-fold, and MUC2 transcript level increased 4.3- and 7.5-fold in 12.5 and 25 µM DAPT-treated HT29 cells. The results of this study showed that the sub-cellular localization of β-catenin converges with NICD inducing proliferation through the activation of CyclinD1 and Hes1. Moreover, the inhibition of Notch1 signaling by DAPT leads to the arrest of cell proliferation and induces apoptosis leading to the upregulation of MUC2, a secretory cell lineage marker.
Induction of cyclin A gene expression by homocysteine in vascular smooth muscle cells.
Tsai, J C; Wang, H; Perrella, M A; Yoshizumi, M; Sibinga, N E; Tan, L C; Haber, E; Chang, T H; Schlegel, R; Lee, M E
Homocysteine is an important and independent risk factor for arteriosclerosis. We showed previously that homocysteine stimulates vascular smooth muscle cell proliferation, a hallmark of arteriosclerosis. We show here that homocysteine and serum increased DNA synthesis synergistically in both human and rat aortic smooth muscle cells (RASMCs). Treatment of quiescent RASMCs with 1 mM homocysteine or 2% calf serum for 36 h increased cyclin A mRNA levels by 8- and 14-fold, respectively, whereas homocysteine plus serum increased cyclin A mRNA levels by 40-fold, indicating a synergistic induction of cyclin A mRNA. Homocysteine did not increase the half-life of cyclin A mRNA (2.9 h), but it did increase the transcriptional rate of the cyclin A gene in nuclear run-on experiments. The positive effect of homocysteine on cyclin A gene transcription was confirmed by our finding that homocysteine increased cyclin A promoter activity and ATF-binding protein levels in RASMCs. Finally, 1 mM homocysteine increased cyclin A protein levels and cyclin A-associated kinase activity by threefold. This homocysteine-induced expression lesions by promoting proliferation of vascular smooth muscle cells. PMID:8550827
Identification of extracellular signal-regulated kinase 3 as a new interaction partner of cyclin D3
Sun Maoyun; Wei Yuanyan; Yao Luyang; Xie Jianhui; Chen Xiaoning; Wang Hanzhou; Jiang Jianhai; Gu Jianxin . E-mail: firstname.lastname@example.com
Cyclin D3, like cyclin D1 and D2 isoforms, is a crucial component of the core cell cycle machinery in mammalian cells. It also exhibits its unique properties in many other physiological processes. In the present study, using yeast two-hybrid screening, we identified ERK3, an atypical mitogen-activated protein kinase (MAPK), as a cyclin D3 binding partner. GST pull-down assays showed that cyclin D3 interacts directly and specifically with ERK3 in vitro. The binding of cyclin D3 and ERK3 was further confirmed in vivo by co-immunoprecipitation assay and confocal microscopic analysis. Moreover, carboxy-terminal extension of ERK3 was responsible for its association with intact cyclin D3. These findings further expand distinct roles of cyclin D3 and suggest the potential activity of ERK3 in cell proliferation.
Cytotoxicity of diacetoxyscirpenol is associated with apoptosis by activation of caspase-8 and interruption of cell cycle progression by down-regulation of cdk4 and cyclin B1 in human Jurkat T cells
Jun, Do Youn; Kim, Jun Seok; Park, Hae Sun; Song, Woo Sun; Bae, Young Seuk; Kim, Young Ho . E-mail: email@example.com
To understand the mechanism underlying T-cell toxicity of diacetoxyscirpenol (DAS) from Fusarium sambucinum, its apoptogenic as well as growth retardation activity was investigated in human Jurkat T cells. Exposure to DAS (0.01-0.15 {mu}M) caused apoptotic DNA fragmentation along with caspase-8 activation, Bid cleavage, mitochondrial cytochrome c release, activation of caspase-9 and caspase-3, and PARP degradation, without any alteration in the levels of Fas or FasL. Under these conditions, necrosis was not accompanied. The cytotoxicity of DAS was not blocked by the anti-Fas neutralizing antibody ZB-4. Although the DAS-induced apoptotic events were completely prevented by overexpression of Bcl-xL, the cells overexpressing Bcl-xL were unable to divide in the presence of DAS, resulting from the failure of cell cycle progression possibly due to down-regulation in the protein levels of cdk4 and cyclin B1. The DAS-mediated apoptosis and activation of caspase-8, -9, and -3 were abrogated by either pan-caspase inhibitor (z-VAD-fmk) or caspase-8 inhibitor (z-IETD-fmk). While the DAS-mediated apoptosis and activation of caspase-9 and caspase-3 were slightly suppressed by the mitochondrial permeability transition pore inhibitor (CsA), both caspase-8 activation and Bid cleavage were not affected by CsA. The activated normal peripheral T cells possessed a similar susceptibility to the cytotoxicity of DAS. These results demonstrate that the T-cell toxicity of DAS is attributable to not only apoptosis initiated by caspase-8 activation and subsequent mitochondrion-dependent or -independent activation of caspase cascades, which can be regulated by Bcl-xL, but also interruption of cell cycle progression caused by down-regulation of cdk4 and cyclin B1 proteins.
Transcriptional role of cyclin D1 in development revealed by a "genetic-proteomic" screen
Bienvenu, Frédéric; Jirawatnotai, Siwanon; Elias, Joshua E.; Meyer, Clifford A.; Mizeracka, Karolina; Marson, Alexander; Frampton, Garrett M.; Cole, Megan F.; Odom, Duncan T.; Odajima, Junko; Geng, Yan; Zagozdzon, Agnieszka; Jecrois, Marie; Young, Richard A.; Liu, X. Shirley; Cepko, Constance L.; Gygi, Steven P.; Sicinski, Piotr
Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers1,2. The full repertoire of cyclin D1 functions in normal development and in oncogenesis is currently unclear. Here we developed FLAG- and HA-tagged cyclin D1 knock-in mouse strains that allowed high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, we observed several proteins involved in transcription. Genome-wide location (ChIP-chip) analyses revealed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, we found that in developing mouse retinas – an organ that critically requires cyclin D1 function3,4 – cyclin D1 binds the upstream regulatory region of the Notch1 gene where it serves to recruit CBP histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch transcript and protein in cyclin D1-null retinas. Transduction of an activated allele of Notch1 into cyclin D1−/− retinas increased proliferation of retinal progenitor cells, indicating that upregulating Notch1 signaling alleviates the phenotype of cyclin D1-deficiency. These studies reveal that in addition to its well-established cell cycle roles, cyclin D1 plays an in vivo transcriptional function in mouse development. Our approach, which we term "genetic-proteomic" can be used to study the in vivo function of essentially any protein. PMID:20090754
Med13p prevents mitochondrial fission and programmed cell death in yeast through nuclear retention of cyclin C.
Khakhina, Svetlana; Cooper, Katrina F; Strich, Randy
The yeast cyclin C-Cdk8 kinase forms a complex with Med13p to repress the transcription of genes involved in the stress response and meiosis. In response to oxidative stress, cyclin C displays nuclear to cytoplasmic relocalization that triggers mitochondrial fission and promotes programmed cell death. In this report, we demonstrate that Med13p mediates cyclin C nuclear retention in unstressed cells. Deleting MED13 allows aberrant cytoplasmic cyclin C localization and extensive mitochondrial fragmentation. Loss of Med13p function resulted in mitochondrial dysfunction and hypersensitivity to oxidative stress-induced programmed cell death that were dependent on cyclin C. The regulatory system controlling cyclin C-Med13p interaction is complex. First, a previous study found that cyclin C phosphorylation by the stress-activated MAP kinase Slt2p is required for nuclear to cytoplasmic translocation. This study found that cyclin C-Med13p association is impaired when the Slt2p target residue is substituted with a phosphomimetic amino acid. The second step involves Med13p destruction mediated by the 26S proteasome and cyclin C-Cdk8p kinase activity. In conclusion, Med13p maintains mitochondrial structure, function, and normal oxidative stress sensitivity through cyclin C nuclear retention. Releasing cyclin C from the nucleus involves both its phosphorylation by Slt2p coupled with Med13p destruction.
Cyclin E marks quiescent neural stem cells and caspase-3-positive newborn cells during adult hippocampal neurogenesis in mice.
Ikeda, Yayoi; Ikeda, Masa-Aki
Cyclin E is a key regulator of progression through the G1-phase of the cell cycle. Recently, a cell cycle-independent role for cyclin E in the adult mouse central nervous system has been suggested. In the present study, we examined expression of cyclin E in the mouse hippocampal dentate gyrus (DG), a region of neurogenesis in adulthood, using immunofluorescence. In the adult DG, cyclin E-immunoreactive (cyclin E+) cells was limited to postmitotic cells. In the subgranular zone, cyclin E was detected in the vertical process of radial glia-like cells, which were marked by the neural stem cell markers nestin and GFAP. Cyclin E was also detected in the nucleus of cells, which were labeled with stage-specific neuronal cell markers, including Pax6, Sox2, NeuroD, doublecortin, and NeuN. The densities of cyclin E+ cells in the DG reduced and increased with age and running, respectively. Furthermore, the majority of cyclin E+ cells co-expressed active caspase-3, a marker of apoptosis. Together, the results indicate that cyclin E is expressed in the process of quiescent neural stem cells and in the nucleus of active caspase-3+ cells during neuronal cell differentiation, suggesting that cyclin E has a Cdk-independent function, which might be important for the mechanisms regulating adult hippocampal neurogenesis.
Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death
Woo, Sang Hyeok; Seo, Sung-Keum; An, Sungkwan; Choe, Tae-Boo; Hong, Seok-Il; Lee, Yun-Han; Park, In-Chul
Highlights: • Caspase-1 mediates doxorubicin-induced downregulation of cyclin A1. • Active caspase-1 effectively cleaved cyclin A1 at D165. • Cyclin A1 expression is involved in DNA damage-induced cell death. - Abstract: Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lung cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.
Enhanced expression of cyclins and cyclin-dependent kinases in aniline-induced cell proliferation in rat spleen.
Wang, Jianling; Wang, Gangduo; Ma, Huaxian; Khan, M Firoze
Aniline exposure is associated with toxicity to the spleen leading to splenomegaly, hyperplasia, fibrosis and a variety of sarcomas of the spleen on chronic exposure. In earlier studies, we have shown that aniline exposure leads to iron overload, oxidative stress and activation of redox-sensitive transcription factors, which could regulate various genes leading to a tumorigenic response in the spleen. However, molecular mechanisms leading to aniline-induced cellular proliferation in the spleen remain largely unknown. This study was, therefore, undertaken on the regulation of G1 phase cell cycle proteins (cyclins), expression of cyclin-dependent kinases (CDKs), phosphorylation of retinoblastoma protein (pRB) and cell proliferation in the spleen, in an experimental condition preceding a tumorigenic response. Male SD rats were treated with aniline (0.5 mmol/kg/day via drinking water) for 30 days (controls received drinking water only), and splenocyte proliferation, protein expression of G1 phase cyclins, CDKs and pRB were measured. Aniline treatment resulted in significant increases in splenocyte proliferation, based on cell counts, cell proliferation markers including proliferating cell nuclear antigen (PCNA), nuclear Ki67 protein (Ki67) and minichromosome maintenance (MCM), MTT assay and flow cytometric analysis. Western blot analysis of splenocyte proteins from aniline-treated rats showed significantly increased expression of cyclins D1, D2, D3 and E, as compared to the controls. Similarly, real-time PCR analysis showed significantly increased mRNA expression for cyclins D1, D2, D3 and E in the spleens of aniline-treated rats. The overexpression of these cyclins was associated with increases in the expression of CDK4, CDK6, CDK2 as well as phosphorylation of pRB protein. Our data suggest that increased expression of cyclins, CDKs and phosphorylation of pRB protein could be critical in cell proliferation, and may contribute to aniline-induced tumorigenic response in
Enhanced expression of cyclins and cyclin-dependent kinases in aniline-induced cell proliferation in rat spleen
Wang Jianling; Wang Gangduo; Ma Huaxian; Khan, M. Firoze
Aniline exposure is associated with toxicity to the spleen leading to splenomegaly, hyperplasia, fibrosis and a variety of sarcomas of the spleen on chronic exposure. In earlier studies, we have shown that aniline exposure leads to iron overload, oxidative stress and activation of redox-sensitive transcription factors, which could regulate various genes leading to a tumorigenic response in the spleen. However, molecular mechanisms leading to aniline-induced cellular proliferation in the spleen remain largely unknown. This study was, therefore, undertaken on the regulation of G1 phase cell cycle proteins (cyclins), expression of cyclin-dependent kinases (CDKs), phosphorylation of retinoblastoma protein (pRB) and cell proliferation in the spleen, in an experimental condition preceding a tumorigenic response. Male SD rats were treated with aniline (0.5 mmol/kg/day via drinking water) for 30 days (controls received drinking water only), and splenocyte proliferation, protein expression of G1 phase cyclins, CDKs and pRB were measured. Aniline treatment resulted in significant increases in splenocyte proliferation, based on cell counts, cell proliferation markers including proliferating cell nuclear antigen (PCNA), nuclear Ki67 protein (Ki67) and minichromosome maintenance (MCM), MTT assay and flow cytometric analysis. Western blot analysis of splenocyte proteins from aniline-treated rats showed significantly increased expression of cyclins D1, D2, D3 and E, as compared to the controls. Similarly, real-time PCR analysis showed significantly increased mRNA expression for cyclins D1, D2, D3 and E in the spleens of aniline-treated rats. The overexpression of these cyclins was associated with increases in the expression of CDK4, CDK6, CDK2 as well as phosphorylation of pRB protein. Our data suggest that increased expression of cyclins, CDKs and phosphorylation of pRB protein could be critical in cell proliferation, and may contribute to aniline-induced tumorigenic response in
E2-C, a cyclin-selective ubiquitin carrier protein required for the destruction of mitotic cyclins.
Aristarkhov, A; Eytan, E; Moghe, A; Admon, A; Hershko, A; Ruderman, J V
Ubiquitin-dependent proteolysis of the mitotic cyclins A and B is required for the completion of mitosis and entry into the next cell cycle. This process is catalyzed by the cyclosome, an approximately 22S particle that contains a cyclin-selective ubiquitin ligase activity, E3-C, that requires a cyclin-selective ubiquitin carrier protein (UBC) E2-C. Here we report the purification and cloning of E2-C from clam oocytes. The deduced amino acid sequence of E2-C indicates that it is a new UBC family member. Bacterially expressed recombinant E2-C is active in in vitro cyclin ubiquitination assays, where it exhibits the same substrate specificities seen with native E2-C. These results demonstrate that E2-C is not a homolog of UBC4 or UBC9, proteins previously suggested to be involved in cyclin ubiquitination, but is a new UBC family member with unique properties. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:8633058
Inhibition of cyclin-dependent kinases by p21.
Harper, J W; Elledge, S J; Keyomarsi, K; Dynlacht, B; Tsai, L H; Zhang, P; Dobrowolski, S; Bai, C; Connell-Crowley, L; Swindell, E
p21Cip1 is a cyclin-dependent kinase (Cdk) inhibitor that is transcriptionally activated by p53 in response to DNA damage. We have explored the interaction of p21 with the currently known Cdks. p21 effectively inhibits Cdk2, Cdk3, Cdk4, and Cdk6 kinases (Ki 0.5-15 nM) but is much less effective toward Cdc2/cyclin B (Ki approximately 400 nM) and Cdk5/p35 (Ki > 2 microM), and does not associate with Cdk7/cyclin H. Overexpression of P21 arrests cells in G1. Thus, p21 is not a universal inhibitor of Cdks but displays selectivity for G1/S Cdk/cyclin complexes. Association of p21 with Cdks is greatly enhanced by cyclin binding. This property is shared by the structurally related inhibitor p27, suggesting a common biochemical mechanism for inhibition. With respect to Cdk2 and Cdk4 complexes, p27 shares the inhibitory potency of p21 but has slightly different kinase specificities. In normal diploid fibroblasts, the vast majority of active Cdk2 is associated with p21, but this active kinase can be fully inhibited by addition of exogenous p21. Reconstruction experiments using purified components indicate that multiple molecules of p21 can associate with Cdk/cyclin complexes and inactive complexes contain more than one molecule of p21. Together, these data suggest a model whereby p21 functions as an inhibitory buffer whose levels determine the threshold kinase activity required for cell cycle progression. Images PMID:7626805
Cyclin A2 is required for sister chromatid segregation, but not separase control, in mouse oocyte meiosis.
Touati, Sandra A; Cladière, Damien; Lister, Lisa M; Leontiou, Ioanna; Chambon, Jean-Philippe; Rattani, Ahmed; Böttger, Franziska; Stemmann, Olaf; Nasmyth, Kim; Herbert, Mary; Wassmann, Katja
In meiosis, two specialized cell divisions allow the separation of paired chromosomes first, then of sister chromatids. Separase removes the cohesin complex holding sister chromatids together in a stepwise manner from chromosome arms in meiosis I, then from the centromere region in meiosis II. Using mouse oocytes, our study reveals that cyclin A2 promotes entry into meiosis, as well as an additional unexpected role; namely, its requirement for separase-dependent sister chromatid separation in meiosis II. Untimely cyclin A2-associated kinase activity in meiosis I leads to precocious sister separation, whereas inhibition of cyclin A2 in meiosis II prevents it. Accordingly, endogenous cyclin A is localized to kinetochores throughout meiosis II, but not in anaphase I. Additionally, we found that cyclin B1, but not cyclin A2, inhibits separase in meiosis I. These findings indicate that separase-dependent cohesin removal is differentially regulated by cyclin B1 and A2 in mammalian meiosis.
Identification of a novel cyclin required for the intrinsic apoptosis pathway in lymphoid cells.
Roig, M B; Roset, R; Ortet, L; Balsiger, N A; Anfosso, A; Cabellos, L; Garrido, M; Alameda, F; Brady, H J M; Gil-Gómez, G
We have identified an early step common to pathways activated by different forms of intrinsic apoptosis stimuli. It requires de novo synthesis of a novel cyclin, cyclin O, that forms active complexes primarily with Cdk2 upon apoptosis induction in lymphoid cells. Cyclin O expression precedes glucocorticoid and gamma-radiation-induced apoptosis in vivo in mouse thymus and spleen, and its overexpression induces caspase-dependent apoptosis in cultured cells. Knocking down the endogenous expression of cyclin O by shRNA leads to the inhibition of glucocorticoid and DNA damage-induced apoptosis due to a failure in the activation of apical caspases while leaving CD95 death receptor-mediated apoptosis intact. Our data demonstrate that apoptosis induction in lymphoid cells is one of the physiological roles of cyclin O and it does not act by perturbing a normal cellular process such as the cell cycle, the DNA damage checkpoints or transcriptional response to glucocorticoids.
PKC iota promotes ovarian tumor progression through deregulation of cyclin E
Nanos-Webb, Angela; Bui, Tuyen; Karakas, Cansu; Zhang, Dong; Carey, Jason P.W.; Mills, Gordon B.; Hunt, Kelly K.; Keyomarsi, Khandan
The high frequency of relapse of epithelial ovarian tumors treated with standard chemotherapy has highlighted the necessity to identify targeted therapies that can improve patient outcomes. The dynamic relationship between Cyclin E and PKCiota frequent overexpression in high-grade ovarian tumors poses a novel pathway for therapeutic investigation. We hypothesized that a PI3K dependent signaling pathway activating PKCiota perpetuates cyclin E deregulation during ovarian tumorigenesis. We observed a positive correlation between PKCiota and cyclin E in a panel of 19 ovarian cancer cell lines. Modulation of cyclin E had no effect on PKCiota knockdown/overexpression however PKCiota differentially regulated cyclin E expression. In the serous ovarian cancer cells (IGROV, OVCAR-3), shPKCiota decreased proliferation, caused a G1 arrest, and significantly prolonged overall survival in xenograft mouse models. In vitro shPKCiota decreased the ability of IGROV cells to grow under anchorage independent conditions and form aberrant acini, which was dependent upon Ad-cyclin E or Ad-LMW-E expression. RPPA analysis of PKCiota wild-type, catalytic active, dominant negative protein isoforms strengthened the association between phospho-PKCiota levels and PI3K pathway activation. Inhibitors of PI3K coordinately decreased phospho-PKCiota and Cyclin E protein levels. In conclusion, we have identified a PI3K/PKCiota/Cyclin E signaling pathway as a therapeutic target during ovarian tumorigenesis. PMID:26279297
miR-340 inhibits glioblastoma cell proliferation by suppressing CDK6, cyclin-D1 and cyclin-D2
Li, Xuesong; Gong, Xuhai; Chen, Jing; Zhang, Jinghui; Sun, Jiahang; Guo, Mian
Glioblastoma development is often associated with alteration in the activity and expression of cell cycle regulators, such as cyclin-dependent kinases (CKDs) and cyclins, resulting in aberrant cell proliferation. Recent studies have highlighted the pivotal roles of miRNAs in controlling the development and growth of glioblastoma. Here, we provide evidence for a function of miR-340 in the inhibition of glioblastoma cell proliferation. We found that miR-340 is downregulated in human glioblastoma tissue samples and several established glioblastoma cell lines. Proliferation and neurosphere formation assays revealed that miR-340 plays an oncosuppressive role in glioblastoma, and that its ectopic expression causes significant defect in glioblastoma cell growth. Further, using bioinformatics, luciferase assay and western blot, we found that miR-340 specifically targets the 3′UTRs of CDK6, cyclin-D1 and cyclin-D2, leading to the arrest of glioblastoma cells in the G0/G1 cell cycle phase. Confirming these results, we found that re-introducing CDK6, cyclin-D1 or cyclin-D2 expression partially, but significantly, rescues cells from the suppression of cell proliferation and cell cycle arrest mediated by miR-340. Collectively, our results demonstrate that miR-340 plays a tumor-suppressive role in glioblastoma and may be useful as a diagnostic biomarker and/or a therapeutic avenue for glioblastoma. - Highlights: • miR-340 is downregulated in glioblastoma samples and cell lines. • miR-340 inhibits glioblastoma cell proliferation. • miR-340 directly targets CDK6, cyclin-D1, and cyclin-D2. • miR-340 regulates glioblastoma cell proliferation via CDK6, cyclin-D1 and cyclin-D2.
Helicobacter pylori Induced Phosphatidylinositol-3-OH Kinase/mTOR Activation Increases Hypoxia Inducible Factor-1α to Promote Loss of Cyclin D1 and G0/G1 Cell Cycle Arrest in Human Gastric Cells
Canales, Jimena; Valenzuela, Manuel; Bravo, Jimena; Cerda-Opazo, Paulina; Jorquera, Carla; Toledo, Héctor; Bravo, Denisse; Quest, Andrew F. G.
Helicobacter pylori (H. pylori) is a human gastric pathogen that has been linked to the development of several gastric pathologies, such as gastritis, peptic ulcer, and gastric cancer. In the gastric epithelium, the bacterium modifies many signaling pathways, resulting in contradictory responses that favor both proliferation and apoptosis. Consistent with such observations, H. pylori activates routes associated with cell cycle progression and cell cycle arrest. H. pylori infection also induces the hypoxia-induced factor HIF-1α, a transcription factor known to promote expression of genes that permit metabolic adaptation to the hypoxic environment in tumors and angiogenesis. Recently, however, also roles for HIF-1α in the repair of damaged DNA and inhibition of gene expression were described. Here, we investigated signaling pathways induced by H. pylori in gastric cells that favor HIF-1α expression and the consequences thereof in infected cells. Our results revealed that H. pylori promoted PI3K/mTOR-dependent HIF-1α induction, HIF-1α translocation to the nucleus, and activity as a transcription factor as evidenced using a reporter assay. Surprisingly, however, transcription of known HIF-1α effector genes evaluated by qPCR analysis, revealed either no change (LDHA and GAPDH), statistically insignificant increases SLC2A1 (GLUT-1) or greatly enhance transcription (VEGFA), but in an HIF-1α-independent manner, as quantified by PCR analysis in cells with shRNA-mediated silencing of HIF-1α. Instead, HIF-1α knockdown facilitated G1/S progression and increased Cyclin D1 protein half-life, via a post-translational pathway. Taken together, these findings link H. pylori-induced PI3K-mTOR activation to HIF-1α induced G0/G1 cell cycle arrest by a Cyclin D1-dependent mechanism. Thus, HIF-1α is identified here as a mediator between survival and cell cycle arrest signaling activated by H. pylori infection.
Helicobacter pylori Induced Phosphatidylinositol-3-OH Kinase/mTOR Activation Increases Hypoxia Inducible Factor-1α to Promote Loss of Cyclin D1 and G0/G1 Cell Cycle Arrest in Human Gastric Cells.
Canales, Jimena; Valenzuela, Manuel; Bravo, Jimena; Cerda-Opazo, Paulina; Jorquera, Carla; Toledo, Héctor; Bravo, Denisse; Quest, Andrew F G
Helicobacter pylori (H. pylori) is a human gastric pathogen that has been linked to the development of several gastric pathologies, such as gastritis, peptic ulcer, and gastric cancer. In the gastric epithelium, the bacterium modifies many signaling pathways, resulting in contradictory responses that favor both proliferation and apoptosis. Consistent with such observations, H. pylori activates routes associated with cell cycle progression and cell cycle arrest. H. pylori infection also induces the hypoxia-induced factor HIF-1α, a transcription factor known to promote expression of genes that permit metabolic adaptation to the hypoxic environment in tumors and angiogenesis. Recently, however, also roles for HIF-1α in the repair of damaged DNA and inhibition of gene expression were described. Here, we investigated signaling pathways induced by H. pylori in gastric cells that favor HIF-1α expression and the consequences thereof in infected cells. Our results revealed that H. pylori promoted PI3K/mTOR-dependent HIF-1α induction, HIF-1α translocation to the nucleus, and activity as a transcription factor as evidenced using a reporter assay. Surprisingly, however, transcription of known HIF-1α effector genes evaluated by qPCR analysis, revealed either no change (LDHA and GAPDH), statistically insignificant increases SLC2A1 (GLUT-1) or greatly enhance transcription (VEGFA), but in an HIF-1α-independent manner, as quantified by PCR analysis in cells with shRNA-mediated silencing of HIF-1α. Instead, HIF-1α knockdown facilitated G1/S progression and increased Cyclin D1 protein half-life, via a post-translational pathway. Taken together, these findings link H. pylori-induced PI3K-mTOR activation to HIF-1α induced G0/G1 cell cycle arrest by a Cyclin D1-dependent mechanism. Thus, HIF-1α is identified here as a mediator between survival and cell cycle arrest signaling activated by H. pylori infection.
Cyclin D1 blocks the anti-proliferative function of RUNX3 by interfering with RUNX3-p300 interaction
Iwatani, Kazunori; Fujimoto, Tetsuhiro; Ito, Takaaki
Research highlights: {yields} Cyclin D1 interacts with RUNX3 and inhibits the interaction and collaboration of RUNX3 with coactivator p300. {yields} Cyclin D1 blocks the ability of RUNX3 to induce the expression of cdk inhibitor p21. {yields} Cyclin D1 releases cancer cells from the inhibition of proliferation induced by RUNX3. -- Abstract: Transcriptional function of cyclin D1, whose deregulation is frequently observed in human cancers, has been suggested to contribute to cancer formation. In the present study, we show that cyclin D1 protein inhibits RUNX3 activity by directly binding to it and interfering with its interaction with p300 interaction in lung cancer cells. Cyclin D1 inhibits p300-dependent RUNX3 acetylation and negatively regulates cyclin-dependent kinase (cdk) inhibitor p21 expression. These transcriptional effects of cyclin D1 do not require cdk4/6 kinase activation. We propose that cyclin D1 provides a transcriptional switch that allows the tumor suppressor activity of RUNX3 to be repressed in cancer cells. Since RUNX3 plays tumor suppressive roles in a wide range of cancers, a non-canonical cyclin D1 function may be critical for neoplastic transformation of the epithelial cells in which RUNX3 regulates proliferation.
Cyclin B targets p34cdc2 for tyrosine phosphorylation.
Meijer, L; Azzi, L; Wang, J Y
A universal intracellular factor, the 'M phase-promoting factor' (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase (H1K) originates from the concomitant tyrosine dephosphorylation of the p34cdc2 subunit and the phosphorylation of the cylin Bcdc13 subunit. We have investigated the role of cyclin in the formation of this complex and the tyrosine phosphorylation of p34cdc2, using highly synchronous mitotic sea urchin eggs as a model. As cells leave the S phase and enter the G2 phase, a massive tyrosine phosphorylation of p34cdc2 occurs. This large p34cdc2 tyrosine phosphorylation burst does not arise from a massive increase in p34cdc2 concentration. It even appears to affect only a fraction (non-immunoprecipitable by anti-PSTAIR antibodies) of the total p34cdc2 present in the cell. Several observations point to an extremely close association between accumulation of unphosphorylated cyclin and p34cdc2 tyrosine phosphorylation: (i) both events coincide perfectly during the G2 phase; (ii) both tyrosine-phosphorylated p34cdc2 and cyclin are not immunoprecipitated by anti-PSTAIR antibodies; (iii) accumulation of unphosphorylated cyclin by aphidicolin treatment of the cells, triggers a dramatic accumulation of tyrosine-phosphorylated p34cdc2; and (iv) inhibition of cyclin synthesis by emetine inhibits p34cdc2 tyrosine phosphorylation without affecting the p34cdc2 concentration. These results show that, as it is synthesized, cyclin B binds and recruits p34cdc2 for tyrosine phosphorylation; this inactive complex then requires the completion of DNA replication before it can be turned into fully active MPF. These results fully confirm recent data obtained in vitro with exogenous cyclin added to cycloheximide-treated Xenopus egg extracts.
Cyclin A2 is an RNA binding protein that controls Mre11 mRNA translation
Kanakkanthara, Arun; Jeganathan, Karthik B.; Limzerwala, Jazeel F.; Baker, Darren J.; Hamada, Masakazu; Nam, Hyun-Ja; van Deursen, Willemijn H.; Hamada, Naomi; Naylor, Ryan M.; Becker, Nicole A.; Davies, Brian A.; van Ree, Janine H.; Mer, Georges; Shapiro, Virginia S.; Maher, L. James; Katzmann, David J.; van Deursen, Jan M.
Cyclin A2 activates the cyclin-dependent kinases Cdk1 and Cdk2 and is expressed at elevated levels from S phase until early mitosis. We found that mutant mice that cannot elevate cyclin A2 are chromosomally unstable and tumor-prone. Underlying the chromosomal instability is a failure to up-regulate the meiotic recombination 11 (Mre11) nuclease in S phase, which leads to impaired resolution of stalled replication forks, insufficient repair of double-stranded DNA breaks, and improper segregation of sister chromosomes. Unexpectedly, cyclin A2 controlled Mre11 abundance through a C-terminal RNA binding domain that selectively and directly binds Mre11 transcripts to mediate polysome loading and translation.These data reveal cyclin A2 as a mechanistically diverse regulator of DNA replication combining multifaceted kinase-dependent functions with a kinase-independent, RNA binding–dependent role that ensures adequate repair of common replication errors. PMID:27708105
Study of possible changes in genes expression of mitotic cyclin under clinorotation.
NASA Astrophysics Data System (ADS)
Artemenko, Olga
Cell cycle is regulated by cyclins, destruction and accumulation of which is the main process in cell cycle progress. In previous studies we have shown that slow horizontal clinorotation (2rpm) affects proliferative activity and cell cycle stages in inducted to grow 2-4 day old Pisum sativum seedlings. In the first cell cycle, delay in cell transition to S stage and delay in mitosis occur due to the prolongation of pre-synthetic stage. This observation is supported by accumulation of 2c DNA cells and transcripts of 3 cyclin in meristem cells. 3 cyclins are "plant" version of cyclin D, they regulate pre-synthetic stage of cell cycle. Cyclins A and B, regulated by cyclin-dependent kinases, control the beginning of S-stage and are necessary for prevention of certain delay in cell cycle progression. We suggest that delay in mitosis, observed under clinorotation, may take place not only due to prolongation of pre-synthetic stage but also due to change of cyclin genes expression under above condition. Further investigations will be aimed on establishing the level of cyclin genes expression under clinorotation.
Cyclin-dependent kinase complexes in developing maize endosperm: evidence for differential expression and functional specialization.
Dante, Ricardo A; Sabelli, Paolo A; Nguyen, Hong N; Leiva-Neto, João T; Tao, Yumin; Lowe, Keith S; Hoerster, George J; Gordon-Kamm, William J; Jung, Rudolf; Larkins, Brian A
Endosperm development in maize (Zea mays L.) and related cereals comprises a cell proliferation stage followed by a period of rapid growth coupled to endoreduplication. Regulation of the cell cycle in developing endosperm is poorly understood. We have characterized various subunits of cyclin-dependent kinase (CDK) complexes, master cell cycle regulators in all eukaryotes. A-, B-, and D-type cyclins as well as A- and B-type cyclin-dependent kinases were characterized with respect to their RNA and protein expression profiles. Two main patterns were identified: one showing expression throughout endosperm development, and another characterized by a sharp down-regulation with the onset of endoreduplication. Cyclin CYCB1;3 and CYCD2;1 proteins were distributed in the cytoplasm and nucleus of cells throughout the endosperm, while cyclin CYCD5 protein was localized in the cytoplasm of peripheral cells. CDKB1;1 expression was strongly associated with cell proliferation. Expression and cyclin-binding patterns suggested that CDKA;1 and CDKA;3 are at least partially redundant. The kinase activity associated with the cyclin CYCA1 was highest during the mitotic stage of development, while that associated with CYCB1;3, CYCD2;1 and CYCD5 peaked at the mitosis-to-endoreduplication transition. A-, B- and D-type cyclins were more resistant to proteasome-dependent degradation in endoreduplicating than in mitotic endosperm extracts. These results indicated that endosperm development is characterized by differential expression and activity of specific cyclins and CDKs, and suggested that endoreduplication is associated with reduced cyclin proteolysis via the ubiquitin-proteasome pathway.
Rictor regulates FBXW7-dependent c-Myc and cyclin E degradation in colorectal cancer cells
Guo, Zheng; Zhou, Yuning; Evers, B. Mark; Wang, Qingding
Highlights: Black-Right-Pointing-Pointer Rictor associates with FBXW7 to form an E3 complex. Black-Right-Pointing-Pointer Knockdown of rictor decreases ubiquitination of c-Myc and cylin E. Black-Right-Pointing-Pointer Knockdown of rictor increases protein levels of c-Myc and cylin E. Black-Right-Pointing-Pointer Overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Black-Right-Pointing-Pointer Rictor regulation of c-Myc and cyclin E requires FBXW7. -- Abstract: Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation. Here, we show that rictor associates with FBXW7 to form an E3 complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor-FBXW7 complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of c-Myc and cyclin E while overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7 blunts the effects of rictor, suggesting that rictor regulation of c-Myc and cyclin E requires FBXW7. Our findings identify rictor as an important component of FBXW7 E3 ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation. Importantly, our results suggest that elevated growth factor signaling may contribute to decrease rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.
Cyclin D1 in the Liver: Role of Noncanonical Signaling in Liver Steatosis and Hormone Regulation
Núñez, Kelley G.; Gonzalez-Rosario, Janet; Thevenot, Paul T.; Cohen, Ari J.
Background: Cyclin D1 is an important protein for cell cycle progression; however, functions independent of the cell cycle have been described in the liver. Cyclin D1 is also involved in DNA repair, is overexpressed in many cancers, and functions as a proto-oncogene. The lesser-known roles of Cyclin D1, specifically in hepatocytes, impact liver steatosis and hormone regulation in the liver. Methods: A comprehensive search of PubMed was conducted using the keywords Cyclin D1, steatosis, lipogenesis, and liver transplantation. In this article, we review the results from this literature search, with a focus on the role of Cyclin D1 in hepatic lipogenesis and gluconeogenesis, as well as the impact and function of this protein in hepatic steatosis. Results: Cyclin D1 represses carbohydrate response element binding protein (ChREBP) and results in a decrease in transcription of fatty acid synthase (FAS) and acetyl-coenzyme A carboxylase (ACC). Cyclin D1 also inhibits peroxisome proliferator-activated receptor gamma (PPARγ) which is involved in hepatic lipogenesis. Cyclin D1 inhibits both hepatocyte nuclear factor 4 alpha (HNF4α) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) and represses transcription of lipogenic genes FAS and liver-type pyruvate kinase (Pklr), along with the gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Conclusion: Cyclin D1 represses multiple proteins involved in both lipogenesis and gluconeogenesis in the liver. Targeting Cyclin D1 to decrease hepatic steatosis in patients with nonalcoholic fatty liver disease or alcoholic fatty liver disease may help improve patient health and the quality of the donor liver pool. PMID:28331449
Induced ICER I{gamma} down-regulates cyclin A expression and cell proliferation in insulin-producing {beta} cells
Inada, Akari; Weir, Gordon C.; Bonner-Weir, Susan . E-mail: firstname.lastname@example.com
We have previously found that cyclin A expression is markedly reduced in pancreatic {beta}-cells by cell-specific overexpression of repressor inducible cyclic AMP early repressor (ICER I{gamma}) in transgenic mice. Here we further examined regulatory effects of ICER I{gamma} on cyclin A gene expression using Min6 cells, an insulin-producing cell line. The cyclin A promoter luciferase assay showed that ICER I{gamma} directly repressed cyclin A gene transcription. In addition, upon ICER I{gamma} overexpression, cyclin A mRNA levels markedly decreased, thereby confirming an inhibitory effect of ICER I{gamma} on cyclin A expression. Suppression of cyclin A results in inhibition of BrdU incorporation. Under normal culture conditions endogenous cyclin A is abundant in these cells, whereas ICER is hardly detectable. However, serum starvation of Min6 cells induces ICER I{gamma} expression with a concomitant very low expression level of cyclin A. Cyclin A protein is not expressed unless the cells are in active DNA replication. These results indicate a potentially important anti-proliferative effect of ICER I{gamma} in pancreatic {beta} cells. Since ICER I{gamma} is greatly increased in diabetes as well as in FFA- or high glucose-treated islets, this effect may in part exacerbate diabetes by limiting {beta}-cell proliferation.
Anaphase-Promoting Complex/Cyclosome–Dependent Proteolysis of Human Cyclin a Starts at the Beginning of Mitosis and Is Not Subject to the Spindle Assembly Checkpoint
Geley, Stephan; Kramer, Edgar; Gieffers, Christian; Gannon, Julian; Peters, Jan-Michael; Hunt, Tim
Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The "destruction box" (D-box) of cyclin A is 10–20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase. PMID:11285280
Structure of a cyclin-dependent kinase from Giardia lamblia.
Leibly, David J; Newling, Paul A; Abendroth, Jan; Guo, Wenjin; Kelley, Angela; Stewart, Lance J; Van Voorhis, Wesley
Giardia lamblia is the etiologic agent of giardiasis, a water-borne infection that is prevalent throughout the world. The need for new therapeutics for the treatment of giardiasis is of paramount importance. Owing to the ubiquitous nature of kinases and their vital importance in organisms, they are potential drug targets. In this paper, the first structure of a cyclin-dependent kinase (CDK) from G. lamblia (GlCDK; UniProt A8BZ95) is presented. CDKs are cell-cycle-associated kinases that are actively being pursued as targets for anticancer drugs as well as for antiparasitic chemotherapy. Generally, a CDK forms a complex with its associated cyclin. This CDK-cyclin complex is active and acts as a serine/threonine protein kinase. Typically, CDKs are responsible for the transition to the next phase of the cell cycle. Although the structure of GlCDK with its associated cyclin was not solved, the 1.85 Å resolution structure of apo GlCDK and a 2.0 Å resolution structure of GlCDK in complex with adenosine monophosphate are presented and the structural differences from the orthologous human CDK2 and CDK3 are discussed.
Novel complexes of cyclin-dependent kinases and a cyclin-like protein from Arabidopsis thaliana with a function unrelated to cell division.
Barrôco, R M; De Veylder, L; Magyar, Z; Engler, G; Inzé, D; Mironov, V
Although the majority of cyclin-dependent kinases (CDKs) play a key role in cell cycle progression, recent evidence has shown that CDKs are also implicated in transcription regulation. Here, we describe two Arabidopsis CDKs designated Arath;CDKC;1 and Arath; CDKC;2. These CDKs share a PITAIRE signature in the cyclin-binding domain and the structural characteristics of mammalian CDK9. Yeast two-hybrid screens and immunoprecipitation assays identified CDKC-interacting proteins with homology to the animal cyclin T/cyclin K group. We suggest that these Arabidopsis CDKCs may be part of a kinase complex similar to the animal positive transcription elongation factor b, whose activity is essential for transcription control. Expression studies showed that Arath; CDKC transcripts are mainly confined to epidermal tissues and are most abundant in flower tissues. No expression was detected in actively dividing Arabidopsis tissues, suggesting a role for the CDKC proteins in differentiated cells.
Cyclin D1 expression in prostate carcinoma.
Pereira, R A; Ravinal, R C; Costa, R S; Lima, M S; Tucci, S; Muglia, V F; Reis, R B dos; Silva, G E B
The purpose of this study was to investigate the relationship between cyclin D1 expression and clinicopathological parameters in patients with prostate carcinoma. We assessed cyclin D1 expression by conventional immunohistochemistry in 85 patients who underwent radical prostatectomy for prostate carcinoma and 10 normal prostate tissue samples retrieved from autopsies. We measured nuclear immunostaining in the entire tumor area and based the results on the percentage of positive tumor cells. The preoperative prostate-specific antigen (PSA) level was 8.68±5.16 ng/mL (mean±SD). Cyclin D1 staining was positive (cyclin D1 expression in >5% of tumor cells) in 64 cases (75.4%) and negative (cyclin D1 expression in ≤5% of tumor cells) in 21 cases (including 15 cases with no immunostaining). Normal prostate tissues were negative for cyclin D1. Among patients with a high-grade Gleason score (≥7), 86% of patients demonstrated cyclin D1 immunostaining of >5% (P<0.05). In the crude analysis of cyclin D1 expression, the high-grade Gleason score group showed a mean expression of 39.6%, compared to 26.9% in the low-grade Gleason score group (P<0.05). Perineural invasion tended to be associated with cyclin D1 expression (P=0.07), whereas cyclin D1 expression was not associated with PSA levels or other parameters. Our results suggest that high cyclin D1 expression could be a potential marker for tumor aggressiveness.
The structural protein ODV-EC27 of Autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin.
Belyavskyi, M; Braunagel, S C; Summers, M D
Two major characteristics of baculovirus infection are arrest of the host cell at G2/M phase of the cell cycle with continuing viral DNA replication. We show that Autographa californica nucleopolyhedrovirus (AcMNPV) encodes for a multifunctional cyclin that may partially explain the molecular basis of these important characteristics of AcMNPV (baculovirus) infection. Amino acids 80-110 of the viral structural protein ODV-EC27 (-EC27) demonstrate 25-30% similarity with cellular cyclins within the cyclin box. Immunoprecipitation results using antibodies to -EC27 show that -EC27 can associate with either cdc2 or cdk6 resulting in active kinase complexes that can phosphorylate histone H1 and retinoblastoma protein in vitro. The cdk6-EC27 complex also associates with proliferating cell nuclear antigen (PCNA) and we demonstrate that PCNA is a structural protein of both the budded virus and the occlusion-derived virus. These results suggest that -EC27 can function as a multifunctional cyclin: when associated with cdc2, it exhibits cyclin B-like activity; when associated with cdk6, the complex possesses cyclin D-like activity and binds PCNA. The possible roles of such a multifunctional cyclin during the life cycle of baculovirus are discussed, along with potential implications relative to the expression of functionally authentic recombinant proteins by using baculovirus-infected cells.
SUMO2/3 modification of cyclin E contributes to the control of replication origin firing
Bonne-Andrea, Catherine; Kahli, Malik; Mechali, Francisca; Lemaitre, Jean-Marc; Bossis, Guillaume; Coux, Olivier
The small ubiquitin-like modifier (SUMO) pathway is essential for the maintenance of genome stability. We investigated its possible involvement in the control of DNA replication during S phase by using the Xenopus cell-free system. Here we show that the SUMO pathway is critical to limit the number and, thus, the density of replication origins that are activated in early S phase. We identified cyclin E, which regulates cyclin-dependent kinase 2 (Cdk2) to trigger origin firing, as an S-phase substrate of this pathway. We show that cyclin E is dynamically and highly conjugated to SUMO2/3 on chromatin, independently of Cdk2 activity and origin activation. Moreover, cyclin E is the predominant SUMO2/3 target on chromatin in early S phase, as cyclin E depletion abolishes, while its readdition restores, the SUMO2/3 signal. Together, our data indicate that cyclin E SUMOylation is important for controlling origin firing once the cyclin E–Cdk2 complex is recruited onto replication origins. PMID:23673635
Cyclin D1 is an essential mediator of apoptotic neuronal cell death.
Kranenburg, O; van der Eb, A J; Zantema, A
Many neurons in the developing nervous system undergo programmed cell death, or apoptosis. However, the molecular mechanism underlying this phenomenon is largely unknown. In the present report, we present evidence that the cell cycle regulator cyclin D1 is involved in the regulation of neuronal cell death. During neuronal apoptosis, cyclin D1-dependent kinase activity is stimulated, due to an increase in cyclin D1 levels. Moreover, artificial elevation of cyclin D1 levels is sufficient to induce apoptosis, even in non-neural cell types. Cyclin D1-induced apoptosis, like neuronal apoptosis, can be inhibited by 21 kDa E1B, Bcl2 and pRb, but not by 55 kDa E1B. Most importantly, however, overexpression of the cyclin D-dependent kinase inhibitor p16INK4 protects neurons from apoptotic cell death, demonstrating that activation of endogenous cyclin D1-dependent kinases is essential during neuronal apoptosis. These data support a model in which neuronal apoptosis results from an aborted attempt to activate the cell cycle in terminally differentiated neurons. Images PMID:8598205
Dissection of Cdk1–cyclin complexes in vivo
Ear, Po Hien; Booth, Michael J.; Abd-Rabbo, Diala; Kowarzyk Moreno, Jacqueline; Hall, Conrad; Chen, Daici; Vogel, Jackie; Michnick, Stephen W.
Cyclin-dependent kinases (Cdks) are regulatory enzymes with temporal and spatial selectivity for their protein substrates that are governed by cell cycle-regulated cyclin subunits. Specific cyclin–Cdk complexes bind to and phosphorylate target proteins, coupling their activity to cell cycle states. The identification of specific cyclin–Cdk substrates is challenging and so far, has largely been achieved through indirect correlation or use of in vitro techniques. Here, we use a protein-fragment complementation assay based on the optimized yeast cytosine deaminase to systematically identify candidate substrates of budding yeast Saccharomyces cerevisiae Cdk1 and show dependency on one or more regulatory cyclins. We identified known and candidate cyclin dependencies for many predicted protein kinase Cdk1 targets and showed elusory Clb3–Cdk1-specific phosphorylation of γ-tubulin, thus establishing the timing of this event in controlling assembly of the mitotic spindle. Our strategy can be generally applied to identify substrates and accessory subunits of multisubunit protein complexes. PMID:24019491
Regulation of cyclin D2 gene expression by the Myc/Max/Mad network: Myc-dependent TRRAP recruitment and histone acetylation at the cyclin D2 promoter
Bouchard, Caroline; Dittrich, Oliver; Kiermaier, Astrid; Dohmann, Karen; Menkel, Annette; Eilers, Martin; Lüscher, Bernhard
Myc oncoproteins promote cell cycle progression in part through the transcriptional up-regulation of the cyclin D2 gene. We now show that Myc is bound to the cyclin D2 promoter in vivo. Binding of Myc induces cyclin D2 expression and histone acetylation at a single nucleosome in a MycBoxII/TRRAP-dependent manner. Down-regulation of cyclin D2 mRNA expression in differentiating HL60 cells is preceded by a switch of promoter occupancy from Myc/Max to Mad/Max complexes, loss of TRRAP binding, increased HDAC1 binding, and histone deacetylation. Thus, recruitment of TRRAP and regulation of histone acetylation are critical for transcriptional activation by Myc. PMID:11511535
Crystal Structure of Human Cyclin K, a Positive Regulator of Cyclin-dependent Kinase 9
Baek, Kyuwon; Brown, Raymond S.; Birrane, Gabriel; Ladias, John A.A.
Summary Cyclin K and the closely related cyclins T1, T2a, and T2b interact with cyclin-dependent kinase 9 (CDK9) forming multiple nuclear complexes, collectively referred to as positive transcription elongation factor b (P-TEFb). Through phosphorylation of the C-terminal domain of the RNA polymerase II largest subunit, distinct P-TEFb species regulate the transcriptional elongation of specific genes that play central roles in human physiology and disease development, including cardiac hypertrophy and human immunodeficiency virus-1 pathogenesis. We have determined the crystal structure of human cyclin K (residues 11-267) at 1.5 Å resolution, which represents the first atomic structure of a P-TEFb subunit. The cyclin K fold comprises two typical cyclin boxes with two short helices preceding the N-terminal box. A prominent feature of cyclin K is an additional helix (H4a) in the first cyclin box that obstructs the binding pocket for the cell cycle inhibitor p27Kip1. Modeling of CDK9 bound to cyclin K provides insights into the structural determinants underlying the formation and regulation of this complex. A homology model of human cyclin T1 generated using the cyclin K as a template reveals that the two proteins have similar structures, as expected from their high sequence identity. Nevertheless, their CDK9-interacting surfaces display significant structural differences, which could potentially be exploited for the design of cyclin-targeted inhibitors of the CDK9–cyclin K and CDK9–cyclin T1 complexes. PMID:17169370
Cyclin D1 unbalances the redox status controlling cell adhesion, migration, and drug resistance in myeloma cells
Bustany, Sophie; Bourgeais, Jérôme; Tchakarska, Guergana; Body, Simon; Hérault, Olivier; Gouilleux, Fabrice; Sola, Brigitte
The interactions of multiple myeloma (MM) cells with their microenvironment are crucial for pathogenesis. MM cells could interact differentially with their microenvironment depending on the type of cyclin D they express. We established several clones that constitutively express cyclin D1 from the parental RPMI8226 MM cell line and analyzed the impact of cyclin D1 expression on cell behavior. We performed a gene expression profiling study on cyclin D1-expressing vs. control cells and validated the results by semi-quantitative RT-PCR. The expression of cyclin D1 altered the transcription of genes that control adhesion and migration. We confirmed that cyclin D1 increases cell adhesion to stromal cells and fibronectin, stabilizes F-actin fibers, and enhances chemotaxis and inflammatory chemokine secretion. Both control and cyclin D1-expressing cells were more resistant to acute carfilzomib treatment when cultured on stromal cells than in suspension. However, this resistance was specifically reduced in cyclin D1-expressing cells after pomalidomide pre-treatment that modifies tumor cell/microenvironment interactions. Transcriptomic analysis revealed that cyclin D1 expression was also associated with changes in the expression of genes controlling metabolism. We also found that cyclin D1 expression disrupted the redox balance by producing reactive oxygen species. The resulting oxidative stress activated the p44/42 mitogen-activated protein kinase (or ERK1/2) signaling pathway, increased cell adhesion to fibronectin or stromal cells, and controlled drug sensitivity. Our results have uncovered a new function for cyclin D1 in the control of redox metabolism and interactions of cyclin D1-expressing MM cells with their bone marrow microenvironment. PMID:27286258
The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase.
Brandeis, M; Hunt, T
We have studied how the cell cycle-specific oscillations of mitotic B-type cyclins are generated in mouse fibroblasts. A reporter enzyme comprising the N-terminus of a B-type cyclin fused to bacterial chloramphenicol acetyl transferase (CAT) was degraded at the end of mitosis like endogenous cyclins. Point mutations in the destruction box of this construct completely abolished its mitotic instability. When the destructible reporter was driven by the cyclin B2 promoter, CAT activity mimicked the oscillations in the level of the endogenous cyclin B2. These oscillations were largely conserved when the reporter was transcribed constitutively from the SV40 promoter. Pulse-chase experiments or addition of the proteasome inhibitors lactacystin and ALLN showed that cyclin synthesis continued after the end of mitosis. The destruction box-specific degradation of cyclins normally ceases at the onset of S phase, and is active in fibroblasts arrested in G0 and in differentiated C2 myoblasts. We were able to reproduce this proteolysis in vitro in extracts of synchronized cells. Extracts of G1 cells degraded cyclin B1 whereas p27Kip1 was stable, in contrast, cyclin B1 remained stable and p27Kip1 was degraded in extracts of S phase cells. Images PMID:8895573
CDK10/cyclin M is a protein kinase that controls ETS2 degradation and is deficient in STAR syndrome
Guen, Vincent J.; Gamble, Carly; Flajolet, Marc; Unger, Sheila; Thollet, Aurélie; Ferandin, Yoan; Superti-Furga, Andrea; Cohen, Pascale A.; Meijer, Laurent; Colas, Pierre
Cyclin-dependent kinases (CDKs) regulate a variety of fundamental cellular processes. CDK10 stands out as one of the last orphan CDKs for which no activating cyclin has been identified and no kinase activity revealed. Previous work has shown that CDK10 silencing increases ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2)-driven activation of the MAPK pathway, which confers tamoxifen resistance to breast cancer cells. The precise mechanisms by which CDK10 modulates ETS2 activity, and more generally the functions of CDK10, remain elusive. Here we demonstrate that CDK10 is a cyclin-dependent kinase by identifying cyclin M as an activating cyclin. Cyclin M, an orphan cyclin, is the product of FAM58A, whose mutations cause STAR syndrome, a human developmental anomaly whose features include toe syndactyly, telecanthus, and anogenital and renal malformations. We show that STAR syndrome-associated cyclin M mutants are unable to interact with CDK10. Cyclin M silencing phenocopies CDK10 silencing in increasing c-Raf and in conferring tamoxifen resistance to breast cancer cells. CDK10/cyclin M phosphorylates ETS2 in vitro, and in cells it positively controls ETS2 degradation by the proteasome. ETS2 protein levels are increased in cells derived from a STAR patient, and this increase is attributable to decreased cyclin M levels. Altogether, our results reveal an additional regulatory mechanism for ETS2, which plays key roles in cancer and development. They also shed light on the molecular mechanisms underlying STAR syndrome. PMID:24218572
Modulating the interaction between CDK2 and cyclin A with a quinoline-based inhibitor.
Deng, Yongqi; Shipps, Gerald W; Zhao, Lianyun; Siddiqui, M Arshad; Popovici-Muller, Janeta; Curran, Patrick J; Duca, Jose S; Hruza, Alan W; Fischmann, Thierry O; Madison, Vincent S; Zhang, Rumin; McNemar, Charles W; Mayhood, Todd W; Syto, Rosalinda; Annis, Allen; Kirschmeier, Paul; Lees, Emma M; Parry, David A; Windsor, William T
A new class of quinoline-based kinase inhibitors has been discovered that both disrupt cyclin dependent 2 (CDK2) interaction with its cyclin A subunit and act as ATP competitive inhibitors. The key strategy for discovering this class of protein-protein disrupter compounds was to screen the monomer CDK2 in an affinity-selection/mass spectrometry-based technique and to perform secondary assays that identified compounds that bound only to the inactive CDK2 monomer and not the active CDK2/cyclin A heterodimer. Through a series of chemical modifications the affinity (Kd) of the original hit improved from 1 to 0.005μM.
Cyclin A1 is expressed in mouse ovary.
Wei, Hongquan; Li, Yuanhong; Zhao, Chen; Jiang, Xuejun; Chen, Hongduo; Lang, Ming-Fei; Sun, Jing
Cyclin A1 belongs to the type-A cyclins and participates in cell cycle regulation. Since its discovery, cyclin A1 has been shown mostly in testis. It plays important roles in spermatogenesis. However, there were also reports on ovary expression of cyclin A1. Therefore, we intended to revisit the expression of cyclin A1 in mouse ovary. Our study showed that cyclin A1 was expressed at the mRNA level and the protein level in mouse ovary. Tissue staining revealed that cyclin A1 was expressed in maturating oocytes. With the recent data on the functions of cyclins in somatic and stem cells, we also discussed the possibilities of further studies of cyclin A1 in mouse oocytes and perhaps in the oogonial stem cells. Our findings not only add to the supportive evidence of cyclin A1 expression in oocytes, but also may promote more interest in exploring cyclin A1 functions in ovary.
Low molecular weight cyclin E is associated with p27-resistant, high-grade, high-stage and invasive bladder cancer
Zhang, Xin-Qiao; Bondaruk, Jolanta; Tucker, Susan L; Czerniak, P. Bogdan; Benedict, William F; Keyomarsi, Khandan
Expression of low molecular weight (LMW) isoforms of cyclin E is a strong predictor of poor outcome in patients with breast cancer. The purpose of this study was to examine the expression of full-length and LMW cyclin E in bladder cancer cell lines and patient tumors. We used western blotting, immunoprecipitation and kinase assays to examine the expression and activity of key cell cycle-regulatory proteins in various human bladder cell lines, both tumorigenic and non-tumorigenic. We also analyzed cyclin E expression, kinase activity and immune complex binding partners in 43 tissue samples from grade 2 and 3 transitional cell carcinomas. Cyclin E was overexpressed and LMW isoforms were present only in bladder cancer cells. Overexpression of LMW isoforms of cyclin E and increased cyclin E kinase activity were both significantly associated with tumorigenicity of the bladder cell lines (p = 0.005 and 0.022, respectively). Binding of the cyclin-dependent kinase inhibitors p21 and p27 to LMW cyclin E did not inhibit the kinase activity of cyclin E and cyclin-dependent kinase 2 in primary tumor samples overexpressing LMW cyclin E. Full-length and LMW cyclin E were significantly overexpressed in grade 3 tumors compared with grade 2 tumors (p = 0.004). Finally, LMW cyclin E levels were significantly associated with a non-papillary growth pattern (p = 0.031) and invasiveness (p = 0.021) of the bladder tumors and poor overall survival (p = 0.06). These results suggest that LMW cyclin E can be used as a new prognostic marker for bladder cancer. PMID:22441703
Cytoplasmic polyadenylation elements mediate masking and unmasking of cyclin B1 mRNA.
de Moor, C H; Richter, J D
During oocyte maturation, cyclin B1 mRNA is translationally activated by cytoplasmic polyadenylation. This process is dependent on cytoplasmic polyadenylation elements (CPEs) in the 3' untranslated region (UTR) of the mRNA. To determine whether a titratable factor might be involved in the initial translational repression (masking) of this mRNA, high levels of cyclin B1 3' UTR were injected into oocytes. While this treatment had no effect on the poly(A) tail length of endogenous cyclin B1 mRNA, it induced cyclin B1 synthesis. A mutational analysis revealed that the most efficient unmasking element in the cyclin 3' UTR was the CPE. However, other U-rich sequences that resemble the CPE in structure, but which do not bind the CPE-binding polyadenylation factor CPEB, failed to induce unmasking. When fused to the chloramphenical acetyl transferase (CAT) coding region, the cyclin B1 3' UTR inhibited CAT translation in injected oocytes. In addition, a synthetic 3' UTR containing multiple copies of the CPE also inhibited translation, and did so in a dose-dependent manner. Furthermore, efficient CPE-mediated masking required cap-dependent translation. During the normal course of progesterone-induced maturation, cytoplasmic polyadenylation was necessary for mRNA unmasking. A model to explain how cyclin B1 mRNA masking and unmasking could be regulated by the CPE is presented. PMID:10205182
Cyclin C stimulates β-cell proliferation in rat and human pancreatic β-cells
Jiménez-Palomares, Margarita; López-Acosta, José Francisco; Villa-Pérez, Pablo; Moreno-Amador, José Luis; Muñoz-Barrera, Jennifer; Fernández-Luis, Sara; Heras-Pozas, Blanca; Perdomo, Germán; Bernal-Mizrachi, Ernesto
Activation of pancreatic β-cell proliferation has been proposed as an approach to replace reduced functional β-cell mass in diabetes. Quiescent fibroblasts exit from G0 (quiescence) to G1 through pRb phosphorylation mediated by cyclin C/cdk3 complexes. Overexpression of cyclin D1, D2, D3, or cyclin E induces pancreatic β-cell proliferation. We hypothesized that cyclin C overexpression would induce β-cell proliferation through G0 exit, thus being a potential therapeutic target to recover functional β-cell mass. We used isolated rat and human islets transduced with adenovirus expressing cyclin C. We measured multiple markers of proliferation: [3H]thymidine incorporation, BrdU incorporation and staining, and Ki67 staining. Furthermore, we detected β-cell death by TUNEL, β-cell differentiation by RT-PCR, and β-cell function by glucose-stimulated insulin secretion. Interestingly, we have found that cyclin C increases rat and human β-cell proliferation. This augmented proliferation did not induce β-cell death, dedifferentiation, or dysfunction in rat or human islets. Our results indicate that cyclin C is a potential target for inducing β-cell regeneration. PMID:25564474
Cyclin D1 inhibits hepatic lipogenesis via repression of carbohydrate response element binding protein and hepatocyte nuclear factor 4α.
Hanse, Eric A; Mashek, Douglas G; Becker, Jennifer R; Solmonson, Ashley D; Mullany, Lisa K; Mashek, Mara T; Towle, Howard C; Chau, Anhtung T; Albrecht, Jeffrey H
Following acute hepatic injury, the metabolic capacity of the liver is altered during the process of compensatory hepatocyte proliferation by undefined mechanisms. In this study, we examined the regulation of de novo lipogenesis by cyclin D1, a key mediator of hepatocyte cell cycle progression. In primary hepatocytes, cyclin D1 significantly impaired lipogenesis in response to glucose stimulation. Cyclin D1 inhibited the glucose-mediated induction of key lipogenic genes, and similar effects were seen using a mutant (D1-KE) that does not activate cdk4 or induce cell cycle progression. Cyclin D1 (but not D1-KE) inhibited the activity of the carbohydrate response element-binding protein (ChREBP) by regulating the glucose-sensing motif of this transcription factor. Because changes in ChREBP activity could not fully explain the effect of cyclin D1, we examined hepatocyte nuclear factor 4α (HNF4α), which regulates numerous differentiated functions in the liver including lipid metabolism. We found that both cyclins D1 and D1-KE bound to HNF4α and significantly inhibited its recruitment to the promoter region of lipogenic genes in hepatocytes. Conversely, knockdown of cyclin D1 in the AML12 hepatocyte cell line promoted HNF4α activity and lipogenesis. In mouse liver, HNF4α bound to a central domain of cyclin D1 involved in transcriptional repression. Cyclin D1 inhibited lipogenic gene expression in the liver following carbohydrate feeding. Similar findings were observed in the setting of physiologic cyclin D1 expression in the regenerating liver. In conclusion, these studies demonstrate that cyclin D1 represses ChREBP and HNF4α function in hepatocytes via Cdk4-dependent and -independent mechanisms. These findings provide a direct link between the cell cycle machinery and the transcriptional control of metabolic function of the liver.
Decreased cyclin B1 expression contributes to G2 delay in human brain tumor cells after treatment with camptothecin.
Janss, A. J.; Maity, A.; Tang, C. B.; Muschel, R. J.; McKenna, W. G.; Sutton, L.; Phillips, P. C.
DNA damage produces delayed mitosis (G2/M delay) in proliferating cells, and shortening the delay sensitizes human malignant glioma and medulloblastoma cells to cytotoxic chemotherapy. Although activation of the cyclin-dependent kinase CDC2 mediates G2/M transition in all tumor cells studied to date, regulation of CDC2 varies between tumor types. Persistent hyperphosphorylation of kinase and reduced cyclin expression have been implicated as mediators of treatment-induced G2 delay in different tumor models. To evaluate regulation of G2/M transition in human brain tumors, we studied the expression and/or activity of CDC2 kinase and cyclins A and B1 in U-251 MG and DAOY medulloblastoma cells after their treatment with camptothecin (CPT). Synchronized cells were treated during S phase, then harvested at predetermined intervals for evaluation of cell cycle kinetics, kinase activity mRNA, and protein expression. CPT produced G2 delay associated with decreased CDC2 kinase activity and cyclin B1 expression. Kinase activity was associated with CDC2 bound to cyclin B1, not cyclin A, in both cell lines. Cyclin A mRNA and protein expression were reduced after CPT treatment; however, decreased protein expression was short lived and moderate in the glioma and primitive neuroectodermal tumor/medulloblastoma cells, respectively. We conclude that G2 delay is a common response of brain tumor cells to chemotherapy with topoisomerase I inhibitors and that a mechanism of this delay may be reduced expression of cyclin B1. PMID:11305412
Enantioselective effect of 12(S)-hydroxyeicosatetraenoic acid on 3T6 fibroblast growth through ERK 1/2 and p38 MAPK pathways and cyclin D1 activation.
Nieves, Diana; Moreno, Juan J
Hydroxyeicosatetraenoic acids (HETEs) have numerous physiological effects, including modulation of cell proliferation and differentiation. However, little is known about the selective effects of HETE enantiomers on cell proliferation and cell signalling pathways involved in the regulation of cell growth. Furthermore, information on epithelial and endothelial cells growth is controversial. Recently, we demonstrated that 5-, 12-, and 15-HETE are involved in the control of 3T6 fibroblast growth though serine/treonine Akt/PKB (Akt) pathway. Here we examined the participation of both enantiomers (S and R) of HETEs in the control of 3T6 fibroblast growth. Our results show that HETEs (5-, 12-, and 15-HETE) are enantioselective on protein and DNA synthesis and 3T6 fibroblast growth. Furthermore, we observed that 12(S)-HETE induces the enhancement of cAMP and intracellular calcium concentration, whereas 12(R)-HETE was uneffective. Our findings also demonstrated that 12(S)-HETE exerts these effects through enantiospecific interactions with a cellular element, probably a plasma membrane receptor coupling to a pertussis toxin-sensitive protein G. Moreover, these elements may be involved in the activation of mitogen-activated protein kinase pathways which induce the enhancement of cyclin D(1) levels.
Ubiquitin C-terminal hydrolase L1 (UCH-L1) acts as a novel potentiator of cyclin-dependent kinases to enhance cell proliferation independently of its hydrolase activity.
Kabuta, Tomohiro; Mitsui, Takeshi; Takahashi, Masaki; Fujiwara, Yuuki; Kabuta, Chihana; Konya, Chiho; Tsuchiya, Yukihiro; Hatanaka, Yusuke; Uchida, Kenko; Hohjoh, Hirohiko; Wada, Keiji
Dysregulation of cell proliferation and the cell cycle are associated with various diseases, such as cancer. Cyclin-dependent kinases (CDKs) play central roles in cell proliferation and the cell cycle. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed in a restricted range of tissues, including the brain and numerous types of cancer. However, the molecular functions of UCH-L1 remain elusive. In this study, we found that UCH-L1 physically interacts with CDK1, CDK4, and CDK5, enhancing their kinase activity. Using several mutants of UCH-L1, we showed that this enhancement is dependent upon interaction levels between UCH-L1 and CDKs but is independent of the known ubiquitin-related functions of UCH-L1. Gain- and loss-of-function studies revealed that UCH-L1 enhances proliferation of multiple cell types, including human cancer cells. Inhibition of the interaction between UCH-L1 and cell cycle-associated CDK resulted in the abolishment of UCH-L1-induced enhancement of cell proliferation. RNA interference of UCH-L1 reduced the growth of human xenograft tumors in mice. We concluded that UCH-L1 is a novel regulator of the kinase activities of CDKs. We believe that our findings from this study will significantly contribute to our understanding of cell cycle-associated diseases.
Interaction between cyclin-dependent kinases and human papillomavirus replication-initiation protein E1 is required for efficient viral replication
Ma, Tianlin; Zou, Nianxiang; Lin, Biing Yuan; Chow, Louise T.; Harper, J. Wade
We have identified the human papillomavirus (HPV) DNA replication initiation protein E1 as a tight-binding substrate of cyclin E/cyclin-dependent kinase (Cdk) complexes by using expression cloning. E1, a DNA helicase, collaborates with the HPV E2 protein in ori-dependent replication. E1 formed complexes with cyclin E in insect and mammalian cells, independent of Cdks and E2. Additional cyclins, including A-, B-, and F-type (but not D-type), interacted with the E1/E2 complex, and A- and E-type cyclin kinases were capable of phosphorylating E1 and E2 in vitro. Association with cyclins and efficient phosphorylation of E1 required the presence of a cyclin interaction motif (the RXL motif). E1 lacking the RXL motif displayed defects in E2-dependent HPV ori replication in vivo. Consistent with a role for Cdk-mediated phosphorylation in E1 function, an E1 protein lacking all four candidate Cdk phosphorylation sites still associated with E2 and cyclin E but was impaired in HPV replication in vitro and in vivo. Our data reveal a link between cyclin/Cdk function and activation of HPV DNA replication through targeting of Cdk complexes to the E1 replication-initiation protein and suggest a functional role for E1 phosphorylation by Cdks. The use of cyclin-binding RXL motifs is now emerging as a major mechanism by which cyclins are targeted to key substrates. PMID:9892642
Complexes of D-type cyclins with CDKs during maize germination.
Godínez-Palma, Silvia K; García, Elpidio; Sánchez, María de la Paz; Rosas, Fernando; Vázquez-Ramos, Jorge M
The importance of cell proliferation in plant growth and development has been well documented. The majority of studies on basic cell cycle mechanisms in plants have been at the level of gene expression and much less knowledge has accumulated in terms of protein interactions and activation. Two key proteins, cyclins and cyclin-dependent kinases (CDKs) are fundamental for cell cycle regulation and advancement. Our aim has been to understand the role of D-type cyclins and type A and B CDKs in the cell cycle taking place during a developmental process such as maize seed germination. Results indicate that three maize D-type cyclins-D2;2, D4;2, and D5;3-(G1-S cyclins by definition) bind and activate two different types of CDK-A and B1;1-in a differential way during germination. Whereas CDKA-D-type cyclin complexes are more active at early germination times than at later times, it was surprising to observe that CDKB1;1, a supposedly G2-M kinase, bound in a differential way to all D-type cyclins tested during germination. Binding to cyclin D2;2 was detectable at all germination times, forming a complex with kinase activity, whereas binding to D4;2 and D5;3 was more variable; in particular, D5;3 was only detected at late germination times. Results are discussed in terms of cell cycle advancement and its importance for seed germination.
The cyclin-dependent kinase 5 activators p35 and p39 interact with the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II and alpha-actinin-1 in a calcium-dependent manner.
Dhavan, Rani; Greer, Paul L; Morabito, Maria A; Orlando, Lianna R; Tsai, Li-Huei
Cyclin-dependent kinase 5 (Cdk5) is a critical regulator of neuronal migration in the developing CNS, and recent studies have revealed a role for Cdk5 in synaptogenesis and regulation of synaptic transmission. Deregulation of Cdk5 has been linked to the pathology of neurodegenerative diseases such as Alzheimer's disease. Activation of Cdk5 requires its association with a regulatory subunit, and two Cdk5 activators, p35 and p39, have been identified. To gain further insight into the functions of Cdk5, we identified proteins that interact with p39 in a yeast two-hybrid screen. In this study we report that alpha-actinin-1 and the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKIIalpha), two proteins localized at the postsynaptic density, interact with Cdk5 via their association with p35 and p39. CaMKIIalpha and alpha-actinin-1 bind to distinct regions of p35 and p39 and also can interact with each other. The association of CaMKIIalpha and alpha-actinin-1 to the Cdk5 activators, as well as to each other, is stimulated by calcium. Further, the activation of glutamate receptors increases the association of p35 and p39 with CaMKIIalpha, and the inhibition of CaMKII activation diminishes this effect. The glutamate-mediated increase in association of p35 and CaMKIIalpha is mediated in large part by NMDA receptors, suggesting that cross talk between the Cdk5 and CaMKII signal transduction pathways may be a component of the complex molecular mechanisms contributing to synaptic plasticity, memory, and learning.
Non-canonical functions of cell cycle cyclins and cyclin-dependent kinases
Hydbring, Per; Malumbres, Marcos; Sicinski, Piotr
The role of cyclins and their catalytic partners, the cyclin-dependent kinases (CDKs), as core components of the machinery that drives cell cycle progression is well established. Increasing evidence indicates that mammalian cyclins and CDKs also carry out important roles in other cellular processes such as transcription, DNA damage repair, the control of cell death, differentiation, the immune response and metabolism. Some of these non-canonical functions are performed by cyclins or by CDKs, independent of their respective cell cycle partners, suggesting a substantial divergence in the function of these proteins during evolution. PMID:27033256
A Novel Non-agonist Peroxisome Proliferator-activated Receptor γ (PPARγ) Ligand UHC1 Blocks PPARγ Phosphorylation by Cyclin-dependent Kinase 5 (CDK5) and Improves Insulin Sensitivity*
Choi, Sun-Sil; Kim, Eun Sun; Koh, Minseob; Lee, Soo-Jin; Lim, Donghyun; Yang, Yong Ryoul; Jang, Hyun-Jun; Seo, Kyung-ah; Min, Sang-Hyun; Lee, In Hee; Park, Seung Bum; Suh, Pann-Ghill; Choi, Jang Hyun
Thiazolidinedione class of anti-diabetic drugs which are known as peroxisome proliferator-activated receptor γ (PPARγ) ligands have been used to treat metabolic disorders, but thiazolidinediones can also cause several severe side effects, including congestive heart failure, fluid retention, and weight gain. In this study, we describe a novel synthetic PPARγ ligand UNIST HYUNDAI Compound 1 (UHC1) that binds tightly to PPARγ without the classical agonism and which blocks cyclin-dependent kinase 5 (CDK5)-mediated PPARγ phosphorylation. We modified the non-agonist PPARγ ligand SR1664 chemically to improve its solubility and then developed a novel PPARγ ligand, UHC1. According to our docking simulation, UHC1 occupied the ligand-binding site of PPARγ with a higher docking score than SR1664. In addition, UHC1 more potently blocked CDK5-mediated PPARγ phosphorylation at Ser-273. Surprisingly, UHC1 treatment effectively ameliorated the inflammatory response both in vitro and in high-fat diet-fed mice. Furthermore, UHC1 treatment dramatically improved insulin sensitivity in high-fat diet-fed mice without causing fluid retention and weight gain. Taken together, compared with SR1664, UHC1 exhibited greater beneficial effects on glucose and lipid metabolism by blocking CDK5-mediated PPARγ phosphorylation, and these data indicate that UHC1 could be a novel therapeutic agent for use in type 2 diabetes and related metabolic disorders. PMID:25100724
Cyclin D2 induces proliferation of cardiac myocytes and represses hypertrophy
Busk, Peter K. . E-mail: firstname.lastname@example.com; Hinrichsen, Rebecca; Bartkova, Jirina; Hansen, Ane H.; Christoffersen, Tue E.H.; Bartek, Jiri; Haunso, Stig
The myocytes of the adult mammalian heart are considered unable to divide. Instead, mitogens induce cardiomyocyte hypertrophy. We have investigated the effect of adenoviral overexpression of cyclin D2 on myocyte proliferation and morphology. Cardiomyocytes in culture were identified by established markers. Cyclin D2 induced DNA synthesis and proliferation of cardiomyocytes and impaired hypertrophy induced by angiotensin II and serum. At the molecular level, cyclin D2 activated CDK4/6 and lead to pRB phosphorylation and downregulation of the cell cycle inhibitors p21{sup Waf1/Cip1} and p27{sup Kip1}. Expression of the CDK4/6 inhibitor p16 inhibited proliferation and cyclin D2 overexpressing myocytes became hypertrophic under such conditions. Inhibition of hypertrophy by cyclin D2 correlated with downregulation of p27{sup Kip1}. These data show that hypertrophy and proliferation are highly related processes and suggest that cardiomyocyte hypertrophy is due to low amounts of cell cycle activators unable to overcome the block imposed by cell cycle inhibitors. Cell cycle entry upon hypertrophy may be converted to cell division by increased expression of activators such as cyclin D2.
Cyclin A2 modulates EMT via β-catenin and phospholipase C pathways.
Cheung, Caroline T; Bendris, Nawal; Paul, Conception; Hamieh, Abdallah; Anouar, Youssef; Hahne, Michael; Blanchard, Jean-Marie; Lemmers, Bénédicte
We have previously demonstrated that Cyclin A2 is involved in cytoskeletal dynamics, epithelial-mesenchymal transition (EMT) and metastasis. This phenotype was potentiated by activated oncogenic H-Ras. However, the mechanisms governing EMT in these cells have not yet been elucidated. Here, we dissected the pathways that are responsible for EMT in cells deficient for Cyclin A2. In Cyclin A2-depleted normal murine mammary gland (NMuMG) cells expressing RasV12, we found that β-catenin was liberated from the cell membrane and cell-cell junctions and underwent nuclear translocation and activation. Components of the canonical wingless (WNT) pathway, including WNT8b, WNT10a, WNT10b, frizzled 1 and 2 and TCF4 were upregulated at the messenger RNA and protein levels following Cyclin A2 depletion. However, suppression of the WNT pathway using the acetyltransferase porcupine inhibitor C59 did not reverse EMT whereas a dominant negative form of TCF4 as well as inhibition of phospholipase C using U73122 were able to do so. This suggests that a WNT-independent mechanism of β-catenin activation via phospholipase C is involved in the EMT induced by Cyclin A2 depletion. Our findings will broaden our knowledge on how Cyclin A2 contributes to EMT and metastasis.
Artemisinin triggers a G1 cell cycle arrest of human Ishikawa endometrial cancer cells and inhibits cyclin-dependent kinase-4 promoter activity and expression by disrupting nuclear factor-κB transcriptional signaling.
Tran, Kalvin Q; Tin, Antony S; Firestone, Gary L
Relatively little is known about the antiproliferative effects of artemisinin, a naturally occurring antimalarial compound from Artemisia annua, or sweet wormwood, in human endometrial cancer cells. Artemisinin induced a G1 cell cycle arrest in cultured human Ishikawa endometrial cancer cells and downregulated cyclin-dependent kinase-2 (CDK2) and CDK4 transcript and protein levels. Analysis of CDK4 promoter-luciferase reporter constructs showed that the artemisinin ablation of CDK4 gene expression was accounted for by the loss of CDK4 promoter activity. Chromatin immunoprecipitation demonstrated that artemisinin inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) subunit p65 and p50 interactions with the endogenous Ishikawa cell CDK4 promoter. Coimmunoprecipitation revealed that artemisinin disrupts endogenous p65 and p50 nuclear translocation through increased protein-protein interactions with IκB-α, an NF-κB inhibitor, and disrupts its interaction with the CDK4 promoter, leading to a loss of CDK4 gene expression. Artemisinin treatment stimulated the cellular levels of IκB-α protein without altering the level of IκB-α transcripts. Finally, expression of exogenous p65 resulted in the accumulation of this NF-κB subunit in the nucleus of artemisinin-treated and artemisinin-untreated cells, reversed the artemisinin downregulation of CDK4 protein expression and promoter activity, and prevented the artemisinin-induced G1 cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin antiproliferative effects in endometrial cancer cells is the transcriptional downregulation of CDK4 expression by disruption of NF-κB interactions with the CDK4 promoter.
Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb{sup 2+}-induced neuronal death in cultured hippocampal neurons
Li Chenchen Xing Tairan Tang Mingliang Yong Wu Yan Dan Deng Hongmin Wang Huili Wang Ming Chen Jutao Ruan Diyun
Lead (Pb) is widely recognized as a neurotoxicant. One of the suggested mechanisms of lead neurotoxicity is apoptotic cell death. And the mechanism by which Pb{sup 2+} causes neuronal death is not well understood. The present study sought to examine the obligate nature of cyclin D1/cyclin-dependent kinase 4 (CDK4), phosphorylation of its substrate retinoblastoma protein (pRb) and its select upstream signal phosphoinositide 3-kinase (PI3K)/AKT pathway in the death of primary cultured rat hippocampal neurons evoked by Pb{sup 2+}. Our data showed that lead treatment of primary hippocampal cultures results in dose-dependent cell death. Inhibition of CDK4 prevented Pb{sup 2+}-induced neuronal death significantly but was incomplete. In addition, we demonstrated that the levels of cyclin D1 and pRb/p107 were increased during Pb{sup 2+} treatment. These elevated expression persisted up to 48 h, returning to control levels after 72 h. We also presented pharmacological and morphological evidences that cyclin D1/CDK4 and pRb/p107 were required for such kind of neuronal death. Addition of the PI3K inhibitor LY294002 (30 {mu}M) or wortmannin (100 nM) significantly rescued the cultured hippocampal neurons from death caused by Pb{sup 2+}. And that Pb{sup 2+}-elicited phospho-AKT (Ser473) participated in the induction of cyclin D1 and partial pRb/p107 expression. These results provide evidences that cell cycle elements play a required role in the death of neurons evoked by Pb{sup 2+} and suggest that certain signaling elements upstream of cyclin D1/CDK4 are modified and/or required for this form of neuronal death.
Cyclin E in centrosome duplication and reduplication in sea urchin zygotes.
Schnackenberg, Bradley J; Marzluff, William F; Sluder, Greenfield
When protein synthesis is completely blocked from before fertilization, the sea urchin zygote arrests in first S phase and the paternal centrosome reduplicates multiple times. However, when protein synthesis is blocked starting in prophase of first mitosis, the zygote divides and the blastomeres arrest in a G1-like state. The centrosome inherited from this mitosis duplicates only once in each blastomere for reasons that are not understood. The late G1 rise in cyclin E/cdk2 kinase activity initiates centrosome duplication in mammalian cells and its activity is needed for centrosome duplication in Xenopus egg extracts. Since the half-time for cyclin E turnover is normally approximately 1 h in sea urchin zygotes, the different behaviors of centrosomes during G1 and S phase arrests could be due to differential losses of cyclin E and its associated kinase activities at these two arrest points. To better understand the mechanisms that limit centrosome duplication, we characterize the levels of cyclin E and its associated kinase activity at the S phase and G1 arrest points. We first demonstrate that cyclin E/cdk2 kinase activity is required for centrosome duplication and reduplication in sea urchin zygotes. Next we find that cyclin E levels and cyclin E/cdk2 kinase activities are both constitutively and equivalently elevated during both the S phase and G1 arrests. This indicates that centrosome duplication during the G1 arrest is limited by a block to reduplication under conditions permissive for duplication. The cytoplasmic conditions of S phase, however, abrogate this block to reduplication.
Selective killing of transformed cells by cyclin/cyclin-dependent kinase 2 antagonists
Chen, Ying-Nan P.; Sharma, Sushil K.; Ramsey, Timothy M.; Jiang, Li; Martin, Mary S.; Baker, Kayla; Adams, Peter D.; Bair, Kenneth W.; Kaelin, William G.
Recent studies identified a short peptide motif that serves as a docking site for cyclin/cyclin-dependent kinase (cdk) 2 complexes. Peptides containing this motif block the phosphorylation of substrates by cyclin A/cdk2 or cyclin E/cdk2. Here we report that cell membrane-permeable forms of such peptides preferentially induced transformed cells to undergo apoptosis relative to nontransformed cells. Deregulation of E2F family transcription factors is a common event during transformation and was sufficient to sensitize cells to the cyclin/cdk2 inhibitory peptides. These results suggest that deregulation of E2F and inhibition of cdk2 are synthetically lethal and provide a rationale for the development of cdk2 antagonists as antineoplastic agents. PMID:10200261
Real time detection of cell cycle regulator cyclin A on living tumor cells with europium emission.
Li, Hongguang; Chadbourne, Frances L; Lan, Rongfeng; Chan, Chi-Fai; Chan, Wai-Lun; Law, Ga-Lai; Lee, Chi-Sing; Cobb, Steven L; Wong, Ka-Leung
Six water-soluble europium complexes (Eu-L1-P(n) and Eu-L2-P(n), n = 1, 2 and 3) with one antenna chromophore, two different linkers (L1 and L2) and three proposed cyclin A specific peptides (P1: -GAKRRLIF-NH2; P2: -GGAKRRLIF-NH2; P3: -Hex- GAKRRLIF-NH2) have been synthesized. With structural information available, comparisons of the cyclin grooves of cyclin A and the six europium complexes have been made, and insights have been gained into the determinants for peptide binding and the foundation of differential binding. Experiment-wise, the linear and two-photon induced photophysical properties of these conjugates were monitored in aqueous solution. Numerous in situ/in vitro biological assays have been carried out, such as responsive emission changes in situ/in vitro, Western blot and cellular uptake. As imaging agents, complexes with peptides P3: -Hex-GAKRRLIF-NH2 showed high selectivity to cyclin A in numerous cancer cells. When it comes to responsive optical signal changes, complex Eu-L2-P3 exhibited a threefold emission enhancement upon binding with cyclin A (100 nM cyclin A, ϕ = 8% to 21%, log KB = 5.83, detection limit = 5 nM), and this could be initiated by the shortened distance between the antenna and the lanthanide after they bind/get into cyclin A. It is promising that our compounds (especially compound Eu-L2-P3) could serve as the template for structure-guided efforts to develop potential imaging therapeutics on the basis of selective imaging of CDK2/cyclin A activity.
BRCA1-IRIS regulates cyclin D1 expression in breast cancer cells
Nakuci, Enkeleda; Mahner, Sven; DiRenzo, James; ElShamy, Wael M. . E-mail: firstname.lastname@example.com
The regulator of cell cycle progression, cyclin D1, is up-regulated in breast cancer cells; its expression is, in part, dependent on ER{alpha} signaling. However, many ER{alpha}-negative tumors and tumor cell lines (e.g., SKBR3) also show over-expression of cyclin D1. This suggests that, in addition to ER{alpha} signaling, cyclin D1 expression is under the control of other signaling pathways; these pathways may even be over-expressed in the ER{alpha}-negative cells. We previously noticed that both ER{alpha}-positive and -negative cell lines over-express BRCA1-IRIS mRNA and protein. Furthermore, the level of over-expression of BRCA1-IRIS in ER{alpha}-negative cell lines even exceeded its over-expression level in ER{alpha}-positive cell lines. In this study, we show that: (1) BRCA1-IRIS forms complex with two of the nuclear receptor co-activators, namely, SRC1 and SRC3 (AIB1) in an ER{alpha}-independent manner. (2) BRCA1-IRIS alone, or in connection with co-activators, is recruited to the cyclin D1 promoter through its binding to c-Jun/AP1 complex; this binding activates the cyclin D1 expression. (3) Over-expression of BRCA1-IRIS in breast cells over-activates JNK/c-Jun; this leads to the induction of cyclin D1 expression and cellular proliferation. (4) BRCA1-IRIS activation of JNK/c-Jun/AP1 appears to account for this, because in cells that were depleted from BRCA1-IRIS, JNK remained inactive. However, depletion of SRC1 or SRC3 instead reduced c-Jun expression. Our data suggest that this novel signaling pathway links BRCA1-IRIS to cellular proliferation through c-Jun/AP1 nuclear pathway; finally, this culminates in the increased expression of the cyclin D1 gene.
Gcn5 determines the fate of Drosophila germline stem cells through degradation of Cyclin A.
Liu, Tianqi; Wang, Qi; Li, Wenqing; Mao, Feiyu; Yue, Shanshan; Liu, Sun; Liu, Xiaona; Xiao, Shan; Xia, Laixin
The fluctuating CDK-CYCLIN complex plays a general role in cell-cycle control. Many types of stem cells use unique features of the cell cycle to facilitate asymmetric division. However, the manner in which these features are established remains poorly understood. The cell cycle of Drosophila female germline stem cells (GSCs) is characterized by short G1 and very long G2 phases, making it an excellent model for the study of cell cycle control in stem cell fate determination. Using a Drosophila female GSCs model, we found Gcn5, the first discovered histone acetyltransferase, to maintain germline stem cells in Drosophila ovaries. Results showed that Gcn5 is dispensable for the transcriptional silencing of bam, but interacts with Cyclin A to facilitate proper turnover in GSCs. Results also showed that Gcn5 promotes Cyclin A ubiquitination, which is dependent on its acetylating activity. Finally, results showed that knockdown of Cyclin A rescued the GSC-loss phenotype caused by lack of Gcn5. Collectively, these findings support the conclusion that Gcn5 acts through acetylation to facilitate Cyclin A ubiquitination and proper turnover, thereby determining the fate of GSCs.-Liu, T., Wang, Q., Li, W., Mao, F., Yue, S., Liu, S., Liu, X., Xiao, S., Xia, L. Gcn5 determines the fate of Drosophila germline stem cells through degradation of Cyclin A.
Stress-induced nuclear-to-cytoplasmic translocation of cyclin C promotes mitochondrial fission in yeast.
Cooper, Katrina F; Khakhina, Svetlana; Kim, Stephen K; Strich, Randy
Mitochondrial morphology is maintained by the opposing activities of dynamin-based fission and fusion machines. In response to stress, this balance is dramatically shifted toward fission. This study reveals that the yeast transcriptional repressor cyclin C is both necessary and sufficient for stress-induced hyperfission. In response to oxidative stress, cyclin C translocates from the nucleus to the cytoplasm, where it is destroyed. Prior to its destruction, cyclin C both genetically and physically interacts with Mdv1p, an adaptor that links the GTPase Dnm1p to the mitochondrial receptor Fis1p. Cyclin C is required for stress-induced Mdv1p mitochondrial recruitment and the efficient formation of functional Dnm1p filaments. Finally, coimmunoprecipitation studies and fluorescence microscopy revealed an elevated association between Mdv1p and Dnm1p in stressed cells that is dependent on cyclin C. This study provides a mechanism by which stress-induced gene induction and mitochondrial fission are coordinated through translocation of cyclin C.
Characterization of TcCYC6 from Trypanosoma cruzi, a gene with homology to mitotic cyclins.
Di Renzo, María Agostina; Laverrière, Marc; Schenkman, Sergio; Wehrendt, Diana Patricia; Tellez-Iñón, María Teresa; Potenza, Mariana
Trypanosoma cruzi, the etiologic agent of Chagas disease, is a protozoan parasite with a life cycle that alternates between replicative and non-replicative forms, but the components and mechanisms that regulate its cell cycle are poorly described. In higher eukaryotes, cyclins are proteins that activate cyclin-dependent kinases (CDKs), by associating with them along the different stages of the cell cycle. These cyclin-CDK complexes exert their role as major modulators of the cell cycle by phosphorylating specific substrates. For the correct progression of the cell cycle, the mechanisms that regulate the activity of cyclins and their associated CDKs are diverse and must be controlled precisely. Different types of cyclins are involved in specific phases of the eukaryotic cell cycle, preferentially activating certain CDKs. In this work, we characterized TcCYC6, a putative coding sequence of T. cruzi which encodes a protein with homology to mitotic cyclins. The overexpression of this sequence, fused to a tag of nine amino acids from influenza virus hemagglutinin (TcCYC6-HA), showed to be detrimental for the proliferation of epimastigotes in axenic culture and affected the cell cycle progression. In silico analysis revealed an N-terminal segment similar to the consensus sequence of the destruction box, a hallmark for the degradation of several mitotic cyclins. We experimentally determined that the TcCYC6-HA turnover decreased in the presence of proteasome inhibitors, suggesting that TcCYC6 degradation occurs via ubiquitin-proteasome pathway. The results obtained in this study provide first evidence that TcCYC6 expression and degradation are finely regulated in T. cruzi.
Impaired nuclear export of tumor-derived c-terminal truncated cyclin D1 mutant in ESCC cancer.
Hao, Meili; Chen, Xiangmei; Zhang, Ting; Shen, Tao; Xie, Qing; Xing, Xiujuan; Gu, Hongxi; Lu, Fengmin
Cyclin D1 is a significant regulator of the G1- to S-phase transition and is often aberrant in human tumors of various origins. Although cancer-derived cyclin D1 mutants are potent oncogenes in vitro and in vivo, the mechanisms by which they contribute to neoplasia remaind to be elucidated. We previously identified a cyclin D1 mutation (Δ266-295) in esophageal cancer with deleted codons from 266 to 295 of wild-type cyclin D1, the critical COOH-terminal regulatory sequences necessary for cyclin D1 nuclear export. In the present study, this cancer-derived cyclin D1-Δ266-295 was shown to be a constitutively nuclear cyclin D1 protein with a significantly increased oncogenic potential. Moreover, the cancer-derived cyclin D1-Δ266-295 mutant was found to retain its ability to bind to and activate CDK4, which in turn phosphorylates and inactivates the pRb protein and promotes cell cycle progression. In comparison to wild-type cyclin D1a, D1-Δ266-295 exhibited enforced nuclear accumulation. In addition, the transient transfection and ectopic expression of this nuclear localized D1-Δ266-295 up-regulated endogenous Notch1 expression, indicating that the mutant retained its ability as a transcriptional regulator. Furthermore, data from the flow cytometry assay showed that D1-Δ266-295 fractionally increased >4N cell accumulation, and further analysis suggested the retriggering of DNA replication relevant to its inhibition of Cdt1 proteolysis. Therefore, the inappropriate nuclear localization of this cyclin D1 mutant may interfere with DNA replication in cultured cells, thereby contributing to genomic instability.
Cyclin A degradation by primate cytomegalovirus protein pUL21a counters its innate restriction of virus replication.
Caffarelli, Nicolas; Fehr, Anthony R; Yu, Dong
Cyclin A is critical for cellular DNA synthesis and S phase progression of the cell cycle. Human cytomegalovirus (HCMV) can reduce cyclin A levels and block cellular DNA synthesis, and cyclin A overexpression can repress HCMV replication. This interaction has only been previously observed in HCMV as murine CMV does not downregulate cyclin A, and the responsible viral factor has not been identified. We previously reported that the HCMV protein pUL21a disrupted the anaphase-promoting complex (APC), but a point mutant abrogating this activity did not phenocopy a UL21a-deficient virus, suggesting that pUL21a has an additional function. Here we identified a conserved arginine-x-leucine (RxL) cyclin-binding domain within pUL21a, which allowed pUL21a to interact with cyclin A and target it for proteasome degradation. Homologous pUL21a proteins from both chimpanzee and rhesus CMVs also contained the RxL domain and similarly degraded cyclin A, indicating that this function is conserved in primate CMVs. The RxL point mutation disabled the virus' ability to block cellular DNA synthesis and resulted in a growth defect similar to pUL21a-deficient virus. Importantly, knockdown of cyclin A rescued growth of UL21a-deficient virus. Together, these data show that during evolution, the pUL21a family proteins of primate CMVs have acquired a cyclin-binding domain that targets cyclin A for degradation, thus neutralizing its restriction on virus replication. Finally, the combined proteasome-dependent degradation of pUL21a and its cellular targets suggests that pUL21a may act as a novel suicide protein, targeting its protein cargos for destruction.
Growth arrest by the cyclin-dependent kinase inhibitor p27Kip1 is abrogated by c-Myc.
Vlach, J; Hennecke, S; Alevizopoulos, K; Conti, D; Amati, B
We show here that c-Myc antagonizes the cyclin-dependent kinase (CDK) inhibitor p27Kip1. p27 expressed from recombinant retroviruses in Rat1 cells associated with and inhibited cyclin E/CDK2 complexes, induced accumulation of the pRb and p130 proteins in their hypophosphorylated forms, and arrested cells in G1. Prior expression of c-Myc prevented inactivation of cyclin E/CDK2 as well as dephosphorylation of pRb and p130, and allowed continuous cell proliferation in the presence of p27. This effect did not require ubiquitin-mediated degradation of p27. Myc altered neither the susceptibility of cyclin E/CDK2 to inhibition by p27, nor the intrinsic CDK-inhibitory activity of p27, but induced sequestration of p27 in a form unable to bind cyclin E/CDK2. Neither Myc itself nor other G1-cyclin/CDK complexes were directly responsible for p27 sequestration. Retroviral expression of G1 cyclins (D1-3, E or A) or of the Cdc25A phosphatase did not overcome p27-induced arrest. Growth rescue by Myc required dimerization with Max, DNA binding and an intact transcriptional activation domain, as previously shown for cellular transformation. We propose that this activity is mediated by the product of an as yet unknown Myc-Max target gene(s) and represents an essential aspect of Myc's mitogenic and oncogenic functions. Images PMID:8978686
PKCeta enhances cell cycle progression, the expression of G1 cyclins and p21 in MCF-7 cells.
Fima, E; Shtutman, M; Libros, P; Missel, A; Shahaf, G; Kahana, G; Livneh, E
Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. However, not much is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that the expression of PKCeta in MCF-7 cells, under the control of a tetracycline-responsive inducible promoter, enhanced cell growth and affected the cell cycle at several points. The induced expression of another PKC isoform, PKCdelta, in MCF-7 cells had opposite effects and inhibited their growth. PKCeta expression activated cellular pathways in these cells that resulted in the increased expression of the G1 phase cyclins, cyclin D and cyclin E. Expression of the cyclin-dependent kinase inhibitor p21(WAF1) was also specifically elevated in PKCeta expressing cells, but its overall effects were not inhibitory. Although, the protein levels of the cyclin-dependent kinase inhibitor p27(KIP1) were not altered by the induced expression of PKCeta, the cyclin E associated Cdk2 kinase activity was in correlation with the p27(KIP1) bound to the cyclin E complex and not by p21(WAF1) binding. PKCeta expression enhanced the removal of p27(KIP1) from this complex, and its re-association with the cyclin D/Cdk4 complex. Reduced binding of p27(KIP1) to the cyclin D/Cdk4 complex at early time points of the cell cycle also enhanced the activity of this complex, while at later time points the decrease in bound p21(WAF1) correlated with its increased activity in PKCeta-expressing cells. Thus, PKCeta induces altered expression of several cell cycle functions, which may contribute to its ability to affect cell growth.
The puc1 Cyclin Regulates the G1 Phase of the Fission Yeast Cell Cycle in Response to Cell Size
Martín-Castellanos, Cristina; Blanco, Miguel A.; de Prada, José M.; Moreno, Sergio
Eukaryotic cells coordinate cell size with cell division by regulating the length of the G1 and G2 phases of the cell cycle. In fission yeast, the length of the G1 phase depends on a precise balance between levels of positive (cig1, cig2, puc1, and cdc13 cyclins) and negative (rum1 and ste9-APC) regulators of cdc2. Early in G1, cyclin proteolysis and rum1 inhibition keep the cdc2/cyclin complexes inactive. At the end of G1, the balance is reversed and cdc2/cyclin activity down-regulates both rum1 and the cyclin-degrading activity of the APC. Here we present data showing that the puc1 cyclin, a close relative of the Cln cyclins in budding yeast, plays an important role in regulating the length of G1. Fission yeast cells lacking cig1 and cig2 have a cell cycle distribution similar to that of wild-type cells, with a short G1 and a long G2. However, when the puc1+ gene is deleted in this genetic background, the length of G1 is extended and these cells undergo S phase with a greater cell size than wild-type cells. This G1 delay is completely abolished in cells lacking rum1. Cdc2/puc1 function may be important to down-regulate the rum1 Cdk inhibitor at the end of G1. PMID:10679013
A jumonji (Jarid2) protein complex represses cyclin D1 expression by methylation of histone H3-K9.
Shirato, Haruki; Ogawa, Satoko; Nakajima, Kuniko; Inagawa, Masayo; Kojima, Mizuyo; Tachibana, Makoto; Shinkai, Yoichi; Takeuchi, Takashi
Covalent modifications of histone tails have critical roles in regulating gene expression. Previously, we identified the jumonji (jmj, Jarid2) gene, the jmjC domain, and a Jmj family. Recently, many Jmj family proteins have been shown to be histone demethylases, and jmjC is the catalytic domain. However, Jmj does not have histone demethylase activity because the jmjC domain lacks conserved residues for binding to cofactors. Independently of these studies, we previously showed that Jmj binds to the cyclin D1 promoter and represses the transcription of cyclin D1. Here, we show the mechanisms by which Jmj represses the transcription of cyclin D1. We found that a protein complex of Jmj had histone methyltransferase activity toward histone H3 lysine 9 (H3-K9). We also found that Jmj bound to the H3-K9 methyltransferases G9a and GLP. Expression of Jmj recruited G9a and GLP to the cyclin D1 promoter and increased H3-K9 methylation. Inactivation of both G9a and GLP, but not of only G9a, inhibited the methylation of H3-K9 in the cyclin D1 promoter and repression of cyclin D1 expression by Jmj. These results suggest that Jmj methylates H3-K9 and represses cyclin D1 expression through G9a and GLP, and that Jmj family proteins can regulate gene expression by not only histone demethylation but also other histone modification.
Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of cyclins A, B and B3.
Sigrist, S; Jacobs, H; Stratmann, R; Lehner, C F
While entry into mitosis is triggered by activation of cdc2 kinase, exit from mitosis requires inactivation of this kinase. Inactivation results from proteolytic degradation of the regulatory cyclin subunits during mitosis. At least three different cyclin types, cyclins A, B and B3, associate with cdc2 kinase in higher eukaryotes and are sequentially degraded in mitosis. We show here that mutations in the Drosophila gene fizzy (fzy) block the mitotic degradation of these cyclins. Moreover, expression of mutant cyclins (delta cyclins) lacking the destruction box motif required for mitotic degradation affects mitotic progression at distinct stages. Deltacyclin A results in a delay in metaphase, deltacyclin B in an early anaphase arrest and deltacyclin B3 in a late anaphase arrest, suggesting that mitotic progression beyond metaphase is ordered by the sequential degradation of these different cyclins. Coexpression of deltacyclins A, B and B3 allows a delayed separation of sister chromosomes, but interferes wit chromosome segregation to the poles. Mutations in fzy block both sister chromosome separation and segregation, indicating that fzy plays a crucial role in the metaphase/anaphase transition. Images PMID:7588612
Lapatinib-resistant cancer cells possessing epithelial cancer stem cell properties develop sensitivity during sphere formation by activation of the ErbB/AKT/cyclin D2 pathway.
Ohnishi, Yuichi; Yasui, Hiroki; Kakudo, Kenji; Nozaki, Masami
Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR)/ErbB2, has antiproliferative effects and is used to treat patients with ErbB2-positive metastatic breast cancer. In the present study, we examined the effects of lapatinib on growth of oral and prostate cancer cells. Oral squamous cell carcinoma (OSCC) cell lines HSC3, HSC4 and Ca9-22 were sensitive to the antiproliferative effects of lapatinib in anchorage-dependent culture, but the OSCC cell lines KB and SAS and the prostate cancer cell line DU145 were resistant to lapatinib. Phosphorylation levels of EGFR in all cell lines decreased during lapatinib treatment in anchorage‑dependent culture. Furthermore, the phosphorylation levels of ErbB2, ErbB3 and Akt and the protein levels of cyclin D1 were decreased by lapatinib treatment of HSC3, HSC4 and Ca9-22 cells. ErbB3 was not expressed and cyclin D1 protein levels were not altered by lapatinib treatment in KB, DU145 and SAS cells. The phosphorylation of ErbB2 and AKT was not affected by lapatinib in SAS cells and was not detected in KB and DU145 cells. Lapatinib-resistant cell lines exhibited sphere-forming ability, and SAS cells developed sensitivity to lapatinib during sphere formation. The phosphorylation levels of ErbB2 and AKT and protein levels of cyclin D2 increased during sphere formation of SAS cells and decreased with lapatinib treatment. In addition, sphere formation of SAS cells was inhibited by the AKT inhibitor MK2206. AKT phosphorylation and cyclin D2 levels in SAS spheres were decreased by MK2206 treatment. SAS cells expressed E-cadherin, but not vimentin and KB cells expressed vimentin, but not E-cadherin. DU145 cells expressed vimentin and E-cadherin. These results suggested that phosphorylation of EGFR and ErbB2 by cell detachment from the substratum induces the AKT pathway/cyclin D2-dependent sphere growth in SAS epithelial cancer stem-like cells, thereby rendering SAS spheres sensitive to lapatinib treatment.
Molecular evolution of cyclin proteins in animals and fungi
Background The passage through the cell cycle is controlled by complexes of cyclins, the regulatory units, with cyclin-dependent kinases, the catalytic units. It is also known that cyclins form several families, which differ considerably in primary structure from one eukaryotic organism to another. Despite these lines of evidence, the relationship between the evolution of cyclins and their function is an open issue. Here we present the results of our study on the molecular evolution of A-, B-, D-, E-type cyclin proteins in animals and fungi. Results We constructed phylogenetic trees for these proteins, their ancestral sequences and analyzed patterns of amino acid replacements. The analysis of infrequently fixed atypical amino acid replacements in cyclins evidenced that accelerated evolution proceeded predominantly during paralog duplication or after it in animals and fungi and that it was related to aromorphic changes in animals. It was shown also that evolutionary flexibility of cyclin function may be provided by consequential reorganization of regions on protein surface remote from CDK binding sites in animal and fungal cyclins and by functional differentiation of paralogous cyclins formed in animal evolution. Conclusions The results suggested that changes in the number and/or nature of cyclin-binding proteins may underlie the evolutionary role of the alterations in the molecular structure of cyclins and their involvement in diverse molecular-genetic events. PMID:21798004
Cyclins D, phytoregulators and cell cycle onset in germinating maize.
Vázquez-Ramos, Jorge M; Lara-Nuñez, Aurora
Several different D-type cyclins can be found in plants and in maize, four of these have been characterized: CycD2;1, CycD4;1, CycD5;1 and CycD5;2. These cyclins appear to form complexes with Cdks, with PCNA and also with KRP proteins and in these kinase activity can be measured. The expression of the corresponding genes during maize germination is highly stimulated by phytohormones like auxin and cytokinin, however this is not followed by an equivalent increase in the amount of the corresponding proteins; nonetheless, auxins do stimulate the associated kinase activity, particularly at early germination times. Thus, auxins appear to stimulate the cell cycle during germination at two levels, transcription and kinase activation. Both auxins and cytokinins appear to shorten the G1 phase during germination and stimulate DNA synthesis, but apparently they do it in different ways as the simultaneous addition of both to germinating maize axes eliminates DNA synthesis stimulation. Therefore, similar actions may be achieved by different paths.
Cyclins D, phytoregulators and cell cycle onset in germinating maize
Lara-Nuñez, Aurora
Several different D-type cyclins can be found in plants and in maize, four of these have been characterized: CycD2;1, CycD4;1, CycD5;1 and CycD5;2. These cyclins appear to form complexes with Cdks, with PCNA and also with KRP proteins and in these kinase activity can be measured. The expression of the corresponding genes during maize germination is highly stimulated by phytohormones like auxin and cytokinin, however this is not followed by an equivalent increase in the amount of the corresponding proteins; nonetheless, auxins do stimulate the associated kinase activity, particularly at early germination times. Thus, auxins appear to stimulate the cell cycle during germination at two levels, transcription and kinase activation. Both auxins and cytokinins appear to shorten the G1 phase during germination and stimulate DNA synthesis, but apparently they do it in different ways as the simultaneous addition of both to germinating maize axes eliminates DNA synthesis stimulation. Therefore, similar actions may be achieved by different paths. PMID:19704474
NFAT1 transcription factor regulates cell cycle progression and cyclin E expression in B lymphocytes.
Teixeira, Leonardo K; Carrossini, Nina; Sécca, Cristiane; Kroll, José E; DaCunha, Déborah C; Faget, Douglas V; Carvalho, Lilian D S; de Souza, Sandro J; Viola, João P B
The NFAT family of transcription factors has been primarily related to T cell development, activation, and differentiation. Further studies have shown that these ubiquitous proteins are observed in many cell types inside and outside the immune system, and are involved in several biological processes, including tumor growth, angiogenesis, and invasiveness. However, the specific role of the NFAT1 family member in naive B cell proliferation remains elusive. Here, we demonstrate that NFAT1 transcription factor controls Cyclin E expression, cell proliferation, and tumor growth in vivo. Specifically, we show that inducible expression of NFAT1 inhibits cell cycle progression, reduces colony formation, and controls tumor growth in nude mice. We also demonstrate that NFAT1-deficient naive B lymphocytes show a hyperproliferative phenotype and high levels of Cyclin E1 and E2 upon BCR stimulation when compared to wild-type B lymphocytes. NFAT1 transcription factor directly regulates Cyclin E expression in B cells, inhibiting the G1/S cell cycle phase transition. Bioinformatics analysis indicates that low levels of NFAT1 correlate with high expression of Cyclin E1 in different human cancers, including Diffuse Large B-cell Lymphomas (DLBCL). Together, our results demonstrate a repressor role for NFAT1 in cell cycle progression and Cyclin E expression in B lymphocytes, and suggest a potential function for NFAT1 protein in B cell malignancies.
Synergistic effects of AKAP95, Cyclin D1, Cyclin E1, and Cx43 in the development of rectal cancer
Qi, Fengjie; Yuan, Yangyang; Zhi, Xuehong; Huang, Qian; Chen, Yuexin; Zhuang, Wenxin; Zhang, Dengcheng; Teng, Bogang; Kong, Xiangyu; Zhang, Yongxing
Objective: To explore the expression of A-kinase anchor protein 95 (AKAP95), Cyclin D1, Cyclin E1, and Connexin43 (Cx43) in rectal cancer tissues and assess the associations between each of the proteins and pathological parameters, as well as their inter-relationships. Methods: AKAP95, Cyclin D1, Cyclin E1, and Cx43 protein expression rates were evaluated by immunohistochemistry in 50 rectal cancer specimens and 16 pericarcinoma tissues. Results: The positive rates of AKAP95, Cyclin E1, and Cyclin D1 proteins were 54.00 vs. 18.75%, 62.00 vs. 6.25%, and 72.00 vs. 31.25% in rectal cancer specimens and pericarcinoma tissues, respectively, representing statistically significant differences (P < 0.05). The positive rate of Cx43 protein expression in rectal cancer tissues was 44.00% and 62.50% in pericarcinoma tissues, and the difference between them was not significant (P > 0.05). No significant associations were found between protein expression of AKAP95, Cyclin E1, Cyclin D1, and Cx43, and the degree of differentiation, histological type, and lymph node metastasis of rectal cancer (P > 0.05). However, significant correlations were obtained between the expression rates of AKAP95 and Cyclin E1, Cyclin E1 and Cyclin D1, Cyclin E1 and Cx43 protein, and Cyclin D1 and Cx43, respectively (P < 0.05). Conclusion: AKAP95, Cyclin E1, and Cyclin D1 protein expression rates were significantly higher in rectal cancer tissues compared with pericarcinoma samples, suggesting an association between these proteins and the development and progression of rectal cancer. In addition, the significant correlations between the proteins (AKAP95 and Cyclin E1, Cyclin E1 and Cyclin D1, Cyclin E1 and Cx43 protein, and Cyclin D1 and Cx43) indicate the possible synergistic effects of these factors in the development and progression of rectal cancer. PMID:25973052
Model of the delayed translation of cyclin B maternal mRNA after sea urchin fertilization.
Picard, Vincent; Mulner-Lorillon, Odile; Bourdon, Jérémie; Morales, Julia; Cormier, Patrick; Siegel, Anne; Bellé, Robert
Sea urchin eggs exhibit a cap-dependent increase in protein synthesis within minutes after fertilization. This rise in protein synthesis occurs at a constant rate for a great number of proteins translated from the different available mRNAs. Surprisingly, we found that cyclin B, a major cell-cycle regulator, follows a synthesis pattern that is distinct from the global protein population, so we developed a mathematical model to analyze this dissimilarity in biosynthesis kinetic patterns. The model includes two pathways for cyclin B mRNA entry into the translational machinery: one from immediately available mRNA (mRNAcyclinB) and one from mRNA activated solely after fertilization (XXmRNAcyclinB). Two coefficients, α and β, were added to fit the measured scales of global protein and cyclin B synthesis, respectively. The model was simplified to identify the synthesis parameters and to allow its simulation. The calculated parameters for activation of the specific cyclin B synthesis pathway after fertilization included a kinetic constant (ka ) of 0.024 sec(-1) , for the activation of XXmRNAcyclinB, and a critical time interval (t2 ) of 42 min. The proportion of XXmRNAcyclinB form was also calculated to be largely dominant over the mRNAcyclinB form. Regulation of cyclin B biosynthesis is an example of a select protein whose translation is controlled by pathways that are distinct from housekeeping proteins, even though both involve the same cap-dependent initiation pathway. Therefore, this model should help provide insight to the signaling utilized for the biosynthesis of cyclin B and other select proteins. Mol. Reprod. Dev. 83: 1070-1082, 2016. © 2016 Wiley Periodicals, Inc.
The Rho GTPase effector ROCK regulates cyclin A, cyclin D1, and p27Kip1 levels by distinct mechanisms.
Croft, Daniel R; Olson, Michael F
The members of the Rho GTPase family are well known for their regulation of actin cytoskeletal structures. In addition, they influence progression through the cell cycle. The RhoA and RhoC proteins regulate numerous effector proteins, with a central and vital signaling role mediated by the ROCK I and ROCK II serine/threonine kinases. The requirement for ROCK function in the proliferation of numerous cell types has been revealed by studies utilizing ROCK-selective inhibitors such as Y-27632. However, the mechanisms by which ROCK signaling promotes cell cycle progression have not been thoroughly characterized. Using a conditionally activated ROCK-estrogen receptor fusion protein, we found that ROCK activation is sufficient to stimulate G1/S cell cycle progression in NIH 3T3 mouse fibroblasts. Further analysis revealed that ROCK acts via independent pathways to alter the levels of cell cycle regulatory proteins: cyclin D1 and p21(Cip1) elevation via Ras and the mitogen-activated protein kinase pathway, increased cyclin A via LIM kinase 2, and reduction of p27(Kip1) protein levels. Therefore, the influence of ROCK on cell cycle regulatory proteins occurs by multiple independent mechanisms.
Investigating the Role of Cyclin D1 in the Promotion of Genomic Instability and Breast Cancer
20mM Tris, 40mM MgCl2, 2.5mM EGTA). Beads containing SCFFbx4 complexes 6 were then mixed with Sf9 -produced purified cyclin D1 substrate, ATP...ubiquitylation reactions with Sf9 -purified cyclin D1/CDK4 as substrate, in the presence of E1, E2, 1 ubiquitin, and ATP. 2 3 Figure 6. Fbx4 loss drives cell...kinase/methyltrans- ferase reactions with purified recombinant PRMT5/MEP50 pro- duced in Sf9 cells. PRMT5-dependent methyltransferase activity was
Replication licensing promotes cyclin D1 expression and G1 progression in untransformed human cells
Liu, Peijun; Slater, Damien M.; Lenburg, Marc; Nevis, Kathleen; Cook, Jeanette Gowen; Vaziri, Cyrus
Defects in DNA replication are implicated as early and causal events in malignancy. However, the immediate effects of impaired DNA replication licensing on cell cycle progression of non-malignant human cells are unknown. Therefore, we have investigated the acute effects of Mcm7 ablation using synchronized cultures of untransformed Human Dermal Fibroblasts (HDF). Mcm7 ablation elicited a G1 delay associated with impaired activation of CDK4 and CDK2 and reduced Rb phosphorylation. The cell cycle delay of Mcm7-ablated cells was not associated with a DNA damage response. However, levels of cyclin D1 mRNA were specifically reduced and binding of RNA Polymerase II to the CYCD1 promoter was decreased in Mcm7-depleted cells. Similar to Mcm7-deficiency, Mcm2- or Cdc6-depletion led to impaired cyclin D expression. Ectopic overexpression of Cdc6 in quiescent cells promoted cyclin D1 expression, CDK4 activation and G1 progression. Therefore timely and efficient expression of cyclin D1 during G1 phase requires replication licensing. Reconstitution of cyclin D1 expression was insufficient to correct the G1 delay of Mcm7-depleted cells, indicating that additional cell cycle events during G1 are dependent on replication licensing. However, ectopic expression of the HPV-E7 oncoprotein, and the resulting bypass of the requirement for cyclin D1-Rb signaling enabled Mcm7-depleted cells to enter S-phase. HPV-E7-induced S-phase entry of Mcm7-depleted cells led to a DNA damage response, a hallmark of pre-malignancy. Taken together, our results suggest the existence of a 'replication licensing restriction point' that couples pre-RC assembly with G1 progression in normal cells to minimize replication stress, DNA damage and tumorigenesis. PMID:19106611
Functional regulation of D-type cyclins by insulin-like growth factor-I and serum in multiple myeloma cells.
Glassford, Janet; Rabin, Neil; Lam, Eric W-F; Yong, Kwee L
D-type cyclin genes are universally dysregulated in multiple myeloma (MM), but the functional consequences are unclear as D-type cyclin gene expression does not correlate with proliferation or disease progression. We examined the protein expression and regulation of D-type cyclins and other cell cycle regulators in human myeloma cell lines and primary CD138(+) plasma cells (PCs). Cyclin D1, cyclin D2, cyclin dependent kinase (CDK) 4, CDK6, p27(Kip1) p18(INK4C) and retinoblastoma protein (pRb) were absent in normal PCs, heterogeneously expressed in primary MM cells and positively correlated with disease activity/progression. Cyclins D1 and D2 complexed with both CDK4 and CDK6, suggesting that both phosphorylate pRb in MM. Furthermore, cyclin D2 expressed via either t(14;16) or t(4;14) IgH translocations was functionally upregulated by fetal calf serum or insulin-like growth factor-I, leading to pRb phosphorylation and cell cycle entry/progression, and in some cases inversely correlated with p27(Kip1). However, pRb phosphorylation and cell cycle progression mediated by cyclin D1 expressed via t(11;14) was less dependent on exogenous stimuli. These data suggest that the presence or absence of specific IgH translocations underlying aberrant D-type cyclin expression may influence their response to mitogens in the bone marrow microenvironment. We showed for the first time that D-type cyclins are functionally regulated in MM, differentially responsive to exogenous growth factors and upregulated with disease progression.
Specific overexpression of cyclin E·CDK2 in early preinvasive and primary breast tumors in female ACI rats induced by estrogen.
Weroha, S John; Lingle, Wilma L; Hong, Yan; Li, Sara Antonia; Li, Jonathan J
Overexpressed Aurora A, amplified centrosomes, and aneuploidy are salient features of estrogen-induced mammary preinvasive lesions and tumors in female August--Copenhagen Irish (ACI) rats. Intimately involved in these events are cyclins and their associated cyclin-dependent kinase (CDK) partners. Cyclin E1·CDK2 overexpression plays an important dual role in late G1/S phase of the cell cycle in cancer cells. It increases DNA replication providing growth advantage to cancer cells and facilitates aberrant centrosome duplication, generating chromosomal instability and aneuploidy leading to tumor development. Presented herein, a 24.0- and 45.0-fold elevation in cyclin E1 and CDK2 was found in 17β-estradiol (E(2))-induced ACI rat mammary tumors (MTs), respectively. Cyclin E·CDK2 positive staining was confined to the large round cells found within focal dysplasias, ductal carcinomas in situ, and invasive MTs. Co-immunoprecipitation and in vitro kinase activity of these tumors revealed that these cell cycle entities are functional. When mammary tissue derived from untreated normal, E(2)-induced hyperplasia and primary tumors were normalized to cyclin E1 levels, low molecular weight (LMW) cyclin E1 forms (33- and 45-kDa) were detected in all of these tissue groups. Moreover, increasing concentrations of protease inhibitor in tissue lysates resulted in a marked reduction of LMW forms, indicating that the presence of cyclin E1 LMW forms can be markedly reduced. Significant increases in cyclin E1 mRNA (2.1-fold) were detected in primary ACI rat E(2)-induced breast tumors, and quantitative real-time polymerase chain reaction revealed a 20% amplification of the cyclin E1 gene (CCNE1). Collectively, these results support the involvement of cyclin E1·CDK2 in centrosome overduplication during each stage of E(2)-induced mammary tumorigenesis.
AKAP95 promotes cell cycle progression via interactions with cyclin E and low molecular weight cyclin E
Kong, Xiang-Yu; Zhang, Deng-Cheng; Zhuang, Wen-Xin; Hua, Su-Hang; Dai, Yue; Yuan, Yang-Yang; Feng, Li-Li; Huang, Qian; Teng, Bo-Gang; Yu, Xiu-Yi; Liu, Wen-Zhi; Zhang, Yong-Xing
AKAP95 in lung cancer tissues showed higher expression than in paracancerous tissues. AKAP95 can bind with cyclin D and cyclin E during G1/S cell cycle transition, but its molecular mechanisms remain unclear. To identify the mechanism of AKAP95 in cell cycle progression, we performed AKAP95 transfection and silencing in A549 cells, examined AKAP95, cyclin E1 and cyclin E2 expression, and the interactions of AKAP95 with cyclins E1 and E2. Results showed that over-expression of AKAP95 promoted cell growth and AKAP95 bound cyclin E1 and E2, low molecular weight cyclin E1 (LWM-E1) and LWM-E2. Additionally AKAP95 bound cyclin E1 and LMW-E2 in the nucleus during G1/S transition, bound LMW-E1 during G1, S and G2/M, and bound cyclin E2 mainly on the nuclear membrane during interphase. Cyclin E2 and LMW-E2 were also detected. AKAP95 over-expression increased cyclin E1 and LMW-E2 expression but decreased cyclin E2 levels. Unlike cyclin E1 and LMW-E2 that were nuclear located during the G1, S and G1/S phases, cyclin E2 and LMW-E1 were expressed in all cell cycle phases, with cyclin E2 present in the cytoplasm and nuclear membrane, with traces in the nucleus. LMW-E1 was present in both the cytoplasm and nucleus. The 20 kDa form of LMW-E1 showed only cytoplasmic expression, while the 40 kDa form was nuclear expressed. The expression of AKAP95, cyclin E1, LMW-E1 and -E2, might be regulated by cAMP. We conclude that AKAP95 might promote cell cycle progression by interacting with cyclin E1 and LMW-E2. LMW-E2, but not cyclin E2, might be involved in G1/S transition. The binding of AKAP95 and LMW-E1 was found throughout cell cycle. PMID:27158371
AKAP95 promotes cell cycle progression via interactions with cyclin E and low molecular weight cyclin E.
Kong, Xiang-Yu; Zhang, Deng-Cheng; Zhuang, Wen-Xin; Hua, Su-Hang; Dai, Yue; Yuan, Yang-Yang; Feng, Li-Li; Huang, Qian; Teng, Bo-Gang; Yu, Xiu-Yi; Liu, Wen-Zhi; Zhang, Yong-Xing
AKAP95 in lung cancer tissues showed higher expression than in paracancerous tissues. AKAP95 can bind with cyclin D and cyclin E during G1/S cell cycle transition, but its molecular mechanisms remain unclear. To identify the mechanism of AKAP95 in cell cycle progression, we performed AKAP95 transfection and silencing in A549 cells, examined AKAP95, cyclin E1 and cyclin E2 expression, and the interactions of AKAP95 with cyclins E1 and E2. Results showed that over-expression of AKAP95 promoted cell growth and AKAP95 bound cyclin E1 and E2, low molecular weight cyclin E1 (LWM-E1) and LWM-E2. Additionally AKAP95 bound cyclin E1 and LMW-E2 in the nucleus during G1/S transition, bound LMW-E1 during G1, S and G2/M, and bound cyclin E2 mainly on the nuclear membrane during interphase. Cyclin E2 and LMW-E2 were also detected. AKAP95 over-expression increased cyclin E1 and LMW-E2 expression but decreased cyclin E2 levels. Unlike cyclin E1 and LMW-E2 that were nuclear located during the G1, S and G1/S phases, cyclin E2 and LMW-E1 were expressed in all cell cycle phases, with cyclin E2 present in the cytoplasm and nuclear membrane, with traces in the nucleus. LMW-E1 was present in both the cytoplasm and nucleus. The 20 kDa form of LMW-E1 showed only cytoplasmic expression, while the 40 kDa form was nuclear expressed. The expression of AKAP95, cyclin E1, LMW-E1 and -E2, might be regulated by cAMP. We conclude that AKAP95 might promote cell cycle progression by interacting with cyclin E1 and LMW-E2. LMW-E2, but not cyclin E2, might be involved in G1/S transition. The binding of AKAP95 and LMW-E1 was found throughout cell cycle.
Cyclin D3 critically regulates the balance between self-renewal and differentiation in skeletal muscle stem cells.
De Luca, Giulia; Ferretti, Roberta; Bruschi, Marco; Mezzaroma, Eleonora; Caruso, Maurizia
Satellite cells are mitotically quiescent myogenic stem cells resident beneath the basal lamina surrounding adult muscle myofibers. In response to injury, multiple extrinsic signals drive the entry of satellite cells into the cell cycle and then to proliferation, differentiation, and self-renewal of their downstream progeny. Because satellite cells must endure for a lifetime, their cell cycle activity must be carefully controlled to coordinate proliferative expansion and self-renewal with the onset of the differentiation program. In this study, we find that cyclin D3, a member of the family of mitogen-activated D-type cyclins, is critically required for proper developmental progression of myogenic progenitors. Using a cyclin D3-knockout mouse we determined that cyclin D3 deficiency leads to reduced myofiber size and impaired establishment of the satellite cell population within the adult muscle. Cyclin D3-null myogenic progenitors, studied ex vivo on isolated myofibers and in vitro, displayed impaired cell cycle progression, increased differentiation potential, and reduced self-renewal capability. Similarly, silencing of cyclin D3 in C2 myoblasts caused anticipated exit from the cell cycle and precocious onset of terminal differentiation. After induced muscle damage, cyclin D3-null myogenic progenitors exhibited proliferation deficits, a precocious ability to form newly generated myofibers and a reduced capability to repopulate the satellite cell niche at later stages of the regeneration process. These results indicate that cyclin D3 plays a cell-autonomous and nonredundant function in regulating the dynamic balance between proliferation, differentiation, and self-renewal that normally establishes an appropriate pool size of adult satellite cells.
The HTLV-1 HBZ protein inhibits cyclin D1 expression through interacting with the cellular transcription factor CREB.
Ma, Yunyun; Zheng, Shangen; Wang, Yuanyuan; Zang, Wenqiao; Li, Min; Wang, Na; Li, Ping; Jin, Jing; Dong, Ziming; Zhao, Guoqiang
Human T cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that can cause adult T-cell leukemia (ATL) and other diseases. The HTLV-1 bZIP factor (HBZ), which is encoded by an mRNA of the opposite polarity of the viral genomic RNA, interacts with several transcription factors and is involved in T cell proliferation, viral gene transcription and cellular transformation. Cyclin D1 is a pivotal regulatory protein involved in cell cycle progression, and its depressed expression correlates with cell cycle prolongation or arrested at the G1/S transition. In our present study, we observed that HBZ expression suppressed cyclin D1 level. To investigate the role of HBZ on cyclin D1 depression, we transduced HBZ with lentivirus vector into 293T cells, CEM cells and Jurkat cells. The results of Western blot, RT-PCR and luciferase assays showed that transcriptional activity of the cyclin D1 promoter was suppressed by the bZIP domain of HBZ (HBZ-bZIP) through cyclic AMP response element (CRE) site. Immunoprecipitation and GST pull-down assays showed the binding of HBZ-bZIP to CRE-binding protein (CREB), which confirmed that the cyclin D1 promoter activity inhibition via the CRE-site was mediated by HBZ-bZIP. The results suggested that HBZ suppressed cyclin D1 transcription through interactions with CREB and along with other viral protein, HBZ may play a causal role for leukemogenesis.
Novel alterations in CDK1/cyclin B1 kinase complex formation occur during the acquisition of a polyploid DNA content.
Datta, N S; Williams, J L; Caldwell, J; Curry, A M; Ashcraft, E K; Long, M W
The pathways that regulate the S-phase events associated with the control of DNA replication are poorly understood. The bone marrow megakaryocytes are unique in that they leave the diploid (2C) state to differentiate, synthesizing 4 to 64 times the normal DNA content within a single nucleus, a process known as endomitosis. Human erythroleukemia (HEL) cells model this process, becoming polyploid during phorbol diester-induced megakaryocyte differentiation. The mitotic arrest occurring in these polyploid cells involves novel alterations in the cdk1/cyclin B1 complex: a marked reduction in cdk1 protein levels, and an elevated and sustained expression of cyclin B1. Endomitotic cells thus lack cdk1/cyclin B1-associated H1-histone kinase activity. Constitutive over-expression of cdk1 in endomitotic cells failed to re-initiate normal mitotic events even though cdk1 was present in a 10-fold excess. This was due to an inability of cyclin-B1 to physically associate with cdk1. Nonetheless, endomitotic cyclin B1 possesses immunoprecipitable H1-histone kinase activity, and specifically translocates to the nucleus. We conclude that mitosis is abrogated during endomitosis due to the absence of cdk1 and the failure to form M-phase promoting factor, resulting in a disassociation of mitosis from the completion of S-phase. Further studies on cyclin and its interacting proteins should be informative in understanding endomitosis and cell cycle control. Images PMID:8688553
Identification of New Substrates for Breast Tumor Specific LMW Cyclin E/CDK2 Kinase
Cyclin E/CDK2 phosphorylation of Hbo1 does not affect the HAT activity of Hbo1 CDK1 phosphorylates Hbo1 at T85/T88 to create a docking site for polo ...values first subtracted from a water control and then normalized to the positive control, which used HeLa nuclear extract as a source of HAT
Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D.
Dubey, Raghvendra K; Fingerle, Jürgen; Gillespie, Delbert G; Mi, Zaichuan; Rosselli, Marinella; Imthurn, Bruno; Jackson, Edwin K
The goal of this study was to determine whether and how adenosine affects the proliferation of human coronary artery smooth muscle cells (HCASMCs). In HCASMCs, 2-chloroadenosine (stable adenosine analogue), but not N(6)-cyclopentyladenosine, CGS21680, or N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide, inhibited HCASMC proliferation (A2B receptor profile). 2-Chloroadenosine increased cAMP, reduced phosphorylation (activation) of ERK and Akt (protein kinases known to increase cyclin D expression and activity, respectively), and reduced levels of cyclin D1 (cyclin that promotes cell-cycle progression in G1). Moreover, 2-chloroadenosine inhibited expression of S-phase kinase-associated protein-2 (Skp2; promotes proteolysis of p27(Kip1)) and upregulated levels of p27(Kip1) (cell-cycle regulator that impairs cyclin D function). 2-Chloroadenosine also inhibited signaling downstream of cyclin D, including hyperphosphorylation of retinoblastoma protein and expression of cyclin A (S phase cyclin). Knockdown of A2B receptors prevented the effects of 2-chloroadenosine on ERK1/2, Akt, Skp2, p27(Kip1), cyclin D1, cyclin A, and proliferation. Likewise, inhibition of adenylyl cyclase and protein kinase A abrogated 2-chloroadenosine's inhibitory effects on Skp2 and stimulatory effects on p27(Kip1) and rescued HCASMCs from 2-chloroadenosine-mediated inhibition. Knockdown of p27(Kip1) also reversed the inhibitory effects of 2-chloroadenosine on HCASMC proliferation. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 μmol/L for 7 days) downregulated vascular expression of Skp2, upregulated vascular expression of p27(Kip1), and reduced neointima hyperplasia by 71% (P<0.05; neointimal thickness: control, 37 424±18 371 pixels; treated, 10 352±2824 pixels). In conclusion, the adenosine/A2B receptor/cAMP/protein kinase A axis inhibits HCASMC proliferation by blocking multiple signaling pathways (ERK1/2, Akt, and Skp2) that converge at cyclin D, a key G1 cyclin
Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3
Li, Xuhui; Eilers, Grant; He, Quan; Liu, Lili; Wu, Yeqing; Wu, Yuehong; Yu, Wei; Fletcher, Jonathan A.; Ou, Wen-Bin
The MDM2-p53 pathway has a prominent oncogenic function in the pathogenesis of various cancers. Nutlin-3, a small-molecule antagonist of MDM2-p53 interaction, inhibits proliferation in cancer cells with wild-type p53. Herein, we evaluate the expression of MDM2, both the full length and a splicing variant MDM2-A, and the sensitivity of Nutlin-3 in different cancer cell lines. Included are seven cell lines with wild-type p53 (four mesothelioma, one breast cancer, one chondrosarcoma, and one leiomyosarcoma), two liposarcoma cell lines harboring MDM2 amplification and wild-type p53, and one mesothelioma cell line harboring a p53 point mutation. Nutlin-3 treatment increased expression of cyclin D1, MDM2, and p53 in cell lines with wild-type p53. Additive effects were observed in cells containing wild-type p53 through coordinated attack on MDM2-p53 binding and cyclin D1 by lentivirual shRNA knockdown or small molecule inhibition, as demonstrated by immunoblots and cell viability analyses. Further results demonstrate that MDM2 binds to cyclin D1, and that an increase in cyclin D1 expression after Nutlin-3 treatment is correlated with expression and ubiquitin E3-ligase activity of MDM2. MDM2 and p53 knockdown experiments demonstrated inhibition of cyclin D1 by MDM2 but not p53. These results indicate that combination inhibition of cyclin D1 and MDM2-p53 binding warrants clinical evaluation as a novel therapeutic strategy in cancer cells harboring wild-type p53. PMID:27129163
Phosphorylation of synaptic GTPase-activating protein (synGAP) by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5) alters the ratio of its GAP activity toward Ras and Rap GTPases.
Walkup, Ward G; Washburn, Lorraine; Sweredoski, Michael J; Carlisle, Holly J; Graham, Robert L; Hess, Sonja; Kennedy, Mary B
synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons.
Phosphorylation of Synaptic GTPase-activating Protein (synGAP) by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) and Cyclin-dependent Kinase 5 (CDK5) Alters the Ratio of Its GAP Activity toward Ras and Rap GTPases*
Walkup, Ward G.; Washburn, Lorraine; Sweredoski, Michael J.; Carlisle, Holly J.; Graham, Robert L.; Hess, Sonja; Kennedy, Mary B.
synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons. PMID:25533468
Cyclin-dependent kinase inhibitors in maize endosperm and their potential role in endoreduplication.
Coelho, Cintia M; Dante, Ricardo A; Sabelli, Paolo A; Sun, Yuejin; Dilkes, Brian P; Gordon-Kamm, William J; Larkins, Brian A
Two maize (Zea mays) cyclin-dependent kinase (CDK) inhibitors, Zeama;KRP;1 and Zeama;KRP;2, were characterized and shown to be expressed in developing endosperm. Similar to the CDK inhibitors in Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), the maize proteins contain a carboxy-terminal region related to the inhibitory domain of the mammalian Cip/Kip inhibitors. Zeama;KRP;1 is present in the endosperm between 7 and 21 d after pollination, a period that encompasses the onset of endoreduplication, while the Zeama;KRP;2 protein declines during this time. Nevertheless, Zeama;KRP;1 accounts for only part of the CDK inhibitory activity that peaks coincident with the endoreduplication phase of endosperm development. In vitro assays showed that Zeama;KRP;1 and Zeama;KRP;2 are able to inhibit endosperm Cdc2-related CKD activity that associates with p13(Suc1). They were also shown to specifically inhibit cyclin A1;3- and cyclin D5;1-associated CDK activities, but not cyclin B1;3/CDK. Overexpression of Zeama;KRP;1 in maize embryonic calli that ectopically expressed the wheat dwarf virus RepA protein, which counteracts retinoblastoma-related protein function, led to an additional round of DNA replication without nuclear division.
CDK-Dependent Hsp70 Phosphorylation Controls G1 Cyclin Abundance and Cell-Cycle Progression
Truman, Andrew W.; Kristjansdottir, Kolbrun; Wolfgeher, Donald; Hasin, Naushaba; Polier, Sigrun; Zhang, Hong; Perrett, Sarah; Prodromou, Chrisostomos; Jones, Gary W.; Kron, Stephen J.
Summary In budding yeast, the essential functions of Hsp70 chaperones Ssa1–4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity. PMID:23217712
Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells.
Bhattacharya, Sujoy; Ray, Ramesh M; Johnson, Leonard R
Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco-2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco-2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF-α/CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner.
The ATF site mediates downregulation of the cyclin A gene during contact inhibition in vascular endothelial cells.
Yoshizumi, M; Hsieh, C M; Zhou, F; Tsai, J C; Patterson, C; Perrella, M A; Lee, M E
Contact inhibition mediates monolayer formation and withdrawal from the cell cycle in vascular endothelial cells. In studying the cyclins--key regulators of the cell cycle--in bovine aortic endothelial cells (BAEC), we found that levels of cyclin A mRNA decreased in confluent BAEC despite the presence of 10% fetal calf serum. We then transfected into BAEC a series of plasmids containing various lengths of the human cyclin A 5' flanking sequence and the luciferase gene. Plasmids containing 3,200, 516, 406, 266, or 133 bp of the human cyclin A promoter directed high luciferase activity in growing but not confluent BAEC. In contrast, a plasmid containing 23 bp of the cyclin A promoter was associated with a 65-fold reduction in activity in growing BAEC, and the promoter activities of this plasmid were identical in both growing and confluent BAEC. Mutation of the activating transcription factor (ATF) consensus sequence at bp -80 to -73 of the cyclin A promoter decreased its activity, indicating the critical role of the ATF site. We identified by gel mobility shift analysis protein complexes that bound to the ATF site in nuclear extracts from growing but not confluent BAEC and identified (with antibodies) ATF-1 as a binding protein in nuclear extracts from growing cells. Also, ATF-1 mRNA levels decreased in confluent BAEC. Taken together, these data suggest that the ATF site and its cognate binding proteins play an important role in the downregulation of cyclin A gene expression during contact inhibition. PMID:7760822
Functional Interaction between the Bovine Papillomavirus Virus Type 1 Replicative Helicase E1 and Cyclin E-Cdk2†
Cueille, Nathalie; Nougarede, Romain; Mechali, Francisca; Philippe, Michel; Bonne-Andrea, Catherine
We have found that the replicative helicase E1 of bovine papillomavirus type 1 (BPV-1) interacts with a key cell cycle regulator of S phase, the cyclin E-Cdk2 kinase. The E1 helicase, which interacts with cyclin E and not with Cdk2, presents the highest affinity for catalytically active kinase complexes. In addition, E1, cyclin E, and Cdk2 expressed in Xenopus egg extracts are quantitatively coimmunoprecipitated from crude extracts by either anti-Cdk2 or anti-E1 antibodies. E1 protein is also a substrate of the cyclin E-Cdk2 kinase in vitro. Using the viral components required for in vitro BPV-1 replication and free-membrane cytosol from Xenopus eggs, we show that efficient replication of BPV plasmids is dependent on the addition of E1-cyclin E-Cdk2 complexes. Thus, the BPV initiator of replication and cyclin E-Cdk2 are likely to function together as a protein complex which may be the key to the cell cycle regulation of papillomavirus replication. PMID:9696820
Functional interaction between the bovine papillomavirus virus type 1 replicative helicase E1 and cyclin E-Cdk2.
Cueille, N; Nougarede, R; Mechali, F; Philippe, M; Bonne-Andrea, C
We have found that the replicative helicase E1 of bovine papillomavirus type 1 (BPV-1) interacts with a key cell cycle regulator of S phase, the cyclin E-Cdk2 kinase. The E1 helicase, which interacts with cyclin E and not with Cdk2, presents the highest affinity for catalytically active kinase complexes. In addition, E1, cyclin E, and Cdk2 expressed in Xenopus egg extracts are quantitatively coimmunoprecipitated from crude extracts by either anti-Cdk2 or anti-E1 antibodies. E1 protein is also a substrate of the cyclin E-Cdk2 kinase in vitro. Using the viral components required for in vitro BPV-1 replication and free-membrane cytosol from Xenopus eggs, we show that efficient replication of BPV plasmids is dependent on the addition of E1-cyclin E-Cdk2 complexes. Thus, the BPV initiator of replication and cyclin E-Cdk2 are likely to function together as a protein complex which may be the key to the cell cycle regulation of papillomavirus replication.
Hog1 Targets Whi5 and Msa1 Transcription Factors To Downregulate Cyclin Expression upon Stress
González-Novo, Alberto; Jiménez, Javier; Clotet, Josep; Nadal-Ribelles, Mariona; Cavero, Santiago
Yeast cells have developed complex mechanisms to cope with extracellular insults. An increase in external osmolarity leads to activation of the stress-activated protein kinase Hog1, which is the main regulator of adaptive responses, such as gene expression and cell cycle progression, that are essential for cellular survival. Upon osmostress, the G1-to-S transition is regulated by Hog1 through stabilization of the cyclin-dependent kinase inhibitor Sic1 and the downregulation of G1 cyclin expression by an unclear mechanism. Here, we show that Hog1 interacts with and phosphorylates components of the core cell cycle transcriptional machinery such as Whi5 and the coregulator Msa1. Phosphorylation of these two transcriptional regulators by Hog1 is essential for inhibition of G1 cyclin expression, for control of cell morphogenesis, and for maximal cell survival upon stress. The control of both Whi5 and Msa1 by Hog1 also revealed the necessity for proper coordination of budding and DNA replication. Thus, Hog1 regulates G1 cyclin transcription upon osmostress to ensure coherent passage through Start. PMID:25733686
The tumor suppressor, parafibromin, mediates histone H3 K9 methylation for cyclin D1 repression.
Yang, Yong-Jin; Han, Jeung-Whan; Youn, Hong-Duk; Cho, Eun-Jung
Parafibromin, a component of the RNA polymerase II-associated PAF1 complex, is a tumor suppressor linked to hyperparathyroidism-jaw tumor syndrome and sporadic parathyroid carcinoma. Parafibromin induces cell cycle arrest by repressing cyclin D1 via an unknown mechanism. Here, we show that parafibromin interacts with the histone methyltransferase, SUV39H1, and functions as a transcriptional repressor. The central region (128-227 amino acids) of parafibromin is important for both the interaction with SUV39H1 and transcriptional repression. Parafibromin associated with the promoter and coding regions of cyclin D1 and was required for the recruitment of SUV39H1 and the induction of H3 K9 methylation but not H3 K4 methylation. RNA interference analysis showed that SUV39H1 was critical for cyclin D1 repression. These data suggest that parafibromin plays an unexpected role as a repressor in addition to its widely known activity associated with transcriptional activation. Parafibromin as a part of the PAF1 complex might downregulate cyclin D1 expression by integrating repressive H3 K9 methylation during transcription.
Immunohistochemical expression of the p53, mdm2, p21/Waf-1, Rb, p16, Ki67, cyclin D1, cyclin A and cyclin B1 proteins and apoptotic index in T-cell lymphomas.
Kanavaros, P; Bai, M; Stefanaki, K; Poussias, G; Rontogianni, D; Zioga, E; Gorgoulis, V; Agnantis, N J
Fifty-seven cases of T-cell lymphomas (TCL) including 5 lymphoblastic (T-LBL) and 52 peripheral TCL (PTCL) were analyzed by immunohistochemistry for the expression of p53, mdm2, p21, Rb, cyclin D1, cyclin A, cyclin B1, and Ki67/MIB1 proteins and 39/52 PTCL were also analyzed for the expression of p16 protein and for the presence of apoptotic cells by the TUNEL method. The aim was to search for abnormal immunoprofiles of p53 and Rb growth control pathways and to determine the proliferative activity and the apoptotic index of TCL. Abnormal overexpression of p53, p21 and mdm2, in comparison to normal lymph nodes, was found in 12/57, 10/57 and 2/57 cases of TCL, respectively. Abnormal loss of Rb and p16 expression was found in 1/57 and 2/39 cases, respectively, whereas abnormal overexpression of cyclin D1 was not detected in any of the 57 cases. Our data revealed entity-related p53/p21/mdm2 phenotypes. Indeed, most nodal and cutaneous CD30+ anaplastic large cell lymphomas (ALCL) showed concomitant overexpression of p53 and p21 proteins (7/8 cases), and mdm2 was overexpressed in 2 p53-positive nodal ALCL. In contrast, overexpression of p53 was found in 3/17 cases of nodal peripheral TCL unspecified (PTCL-UC) and 2/7 non-ALCL cutaneous pleomorphic TCL. Overexpression of p21 protein was detected in 2/3 p53-positive PTCL-UC and in 1/2 p53-positive non-ALCL cutaneous pleomorphic TCL. Finally, all the remaining 25 cases of TCL did not show p53 and p21 overexpression. Overall, the p53+/p21+ phenotype in 10/57 TCL suggests wild-type p53 capable of inducing p21 expression. The highest apoptotic index (AI) was found in ALCL and a positive correlation between apoptotic index and Ki67 index (p<0.001) was detected. Ki67, cyclin A and cyclin B1 expression was found in all 57 TCL and on the basis of the combined use of these 3 variables, 3 groups of proliferative activity could be determined: a) high in ALCL and T-LBL, b) low in mycosis fungoides (MF) and gammadelta hepatosplenic TCL
Anticancer effect of icaritin inhibits cell growth of colon cancer through reactive oxygen species, Bcl-2 and cyclin D1/E signaling
Li, Chaofeng; Peng, Weichao; Song, Xin; Wang, Qian; Wang, Wenyue
Icaritin has an advantage in enhancing immunity. Besides, with its anticancer effect, it may be of great help in cancer treatment and recovery of cancer patients. As a result, icaritin is likely to become a novel anticancer drug. However, the anticancer effect of icaritin against colon cancer has not been elucidated thus far. The present study investigated the latent anticancer effect of icaritin on the inhibition of colon cancer cell growth by regulating reactive oxygen species (ROS), B-cell lymphoma (Bcl)-2 and cyclin D1/E signaling. The COLO-205 colon cancer cell line was used as a colon cancer cell model in the present study. First, cell growth and apoptosis were measured to analyze the anticancer effect of icaritin against colon cancer. Next, the possible mechanism of icaritin against colon cancer, including ROS, Bcl-2, cyclin D1, cyclin E and caspase-3/9, was explored. The results revealed that icaritin could inhibit cell growth and induce the apoptosis of COLO-205 cells. In addition, icaritin significantly induced ROS generation, suppressed Bcl-2, cyclin D1 and cyclin E protein expression, and activated caspase-3/9 activity in COLO-205 cells. The present findings demonstrated that icaritin exerted antiproliferative and anticancer effects against colon cancer through the activation of ROS generation and the suppression of Bcl-2, cyclin D1 and cyclin E signaling. PMID:27900033
Anticancer effect of icaritin inhibits cell growth of colon cancer through reactive oxygen species, Bcl-2 and cyclin D1/E signaling.
Li, Chaofeng; Peng, Weichao; Song, Xin; Wang, Qian; Wang, Wenyue
Icaritin has an advantage in enhancing immunity. Besides, with its anticancer effect, it may be of great help in cancer treatment and recovery of cancer patients. As a result, icaritin is likely to become a novel anticancer drug. However, the anticancer effect of icaritin against colon cancer has not been elucidated thus far. The present study investigated the latent anticancer effect of icaritin on the inhibition of colon cancer cell growth by regulating reactive oxygen species (ROS), B-cell lymphoma (Bcl)-2 and cyclin D1/E signaling. The COLO-205 colon cancer cell line was used as a colon cancer cell model in the present study. First, cell growth and apoptosis were measured to analyze the anticancer effect of icaritin against colon cancer. Next, the possible mechanism of icaritin against colon cancer, including ROS, Bcl-2, cyclin D1, cyclin E and caspase-3/9, was explored. The results revealed that icaritin could inhibit cell growth and induce the apoptosis of COLO-205 cells. In addition, icaritin significantly induced ROS generation, suppressed Bcl-2, cyclin D1 and cyclin E protein expression, and activated caspase-3/9 activity in COLO-205 cells. The present findings demonstrated that icaritin exerted antiproliferative and anticancer effects against colon cancer through the activation of ROS generation and the suppression of Bcl-2, cyclin D1 and cyclin E signaling.
A Whi7-anchored loop controls the G1 Cdk-cyclin complex at start.
Yahya, Galal; Parisi, Eva; Flores, Alba; Gallego, Carme; Aldea, Martí
Cells commit to a new cell cycle at Start by activation of the G1 Cdk-cyclin complex which, in turn, triggers a genome-wide transcriptional wave that executes the G1/S transition. In budding yeast, the Cdc28-Cln3 complex is regulated by an ER-retention mechanism that is important for proper cell size control. We have isolated small-cell-size CDC28 mutants showing impaired retention at the ER and premature accumulation of the Cln3 cyclin in the nucleus. The differential interactome of a quintuple Cdc28(wee) mutant pinpointed Whi7, a Whi5 paralog targeted by Cdc28 that associates to the ER in a phosphorylation-dependent manner. Our results demonstrate that the Cln3 cyclin and Whi7 act in a positive feedback loop to release the G1 Cdk-cyclin complex and trigger Start once a critical size has been reached, thus uncovering a key nonlinear mechanism at the earliest known events of cell-cycle entry.
Cannabinoids Regulate Bcl-2 and Cyclin D2 Expression in Pancreatic β Cells
Kim, Jung Seok; Rho, Jun Gi; Shin, Jung Jae; Song, Woo Keun; Lee, Eun Kyung; Egan, Josephine M.; Kim, Wook
Recent reports have shown that cannabinoid 1 receptors (CB1Rs) are expressed in pancreatic β cells, where they induce cell death and cell cycle arrest by directly inhibiting insulin receptor activation. Here, we report that CB1Rs regulate the expression of the anti-apoptotic protein Bcl-2 and cell cycle regulator cyclin D2 in pancreatic β cells. Treatment of MIN6 and βTC6 cells with a synthetic CB1R agonist, WIN55,212–2, led to a decrease in the expression of Bcl-2 and cyclin D2, in turn inducing cell cycle arrest in G0/G1 phase and caspase-3-dependent apoptosis. Additionally, genetic deletion and pharmacological blockade of CB1Rs after injury in mice led to increased levels of Bcl-2 and cyclin D2 in pancreatic β cells. These findings provide evidence for the involvement of Bcl-2 and cyclin D2 mediated by CB1Rs in the regulation of β-cell survival and growth, and will serve as a basis for developing new therapeutic interventions to enhance β-cell function and growth in diabetes. PMID:26967640
HB-EGF directs stromal cell polyploidy and decidualization via cyclin D3 during implantation.
Tan, Yi; Li, Meiling; Cox, Sandra; Davis, Marilyn K; Tawfik, Ossama; Paria, Bibhash C; Das, Sanjoy K
Stromal cell polyploidy is a unique phenomenon that occurs during uterine decidualization following embryo implantation, although the developmental mechanism still remains elusive. The general consensus is that the aberrant expression and altered functional activity of cell cycle regulatory molecules at two particular checkpoints G1 to S and G2 to M in the cell cycle play an important role in the development of cellular polyploidy. Despite the compelling evidence of intrinsic cell cycle alteration, it has been implicated that the development of cellular polyploidy may be controlled by specific actions of extracellular growth regulators. Here we show a novel role for heparin-binding EGF-like growth factor (HB-EGF) in the developmental process of stromal cell polyploidy in mice. HB-EGF, which is one of the earliest known molecular mediators of implantation in mice and humans, promotes stromal cell polyploidy via upregulation of cyclin D3. Adenoviral delivery of antisense cyclin D3 attenuates cyclin D3 expression and abrogates HB-EGF-induced stromal cell polyploidy in vitro and in vivo. Collectively, the results demonstrate that the regulation of stromal cell polyploidy and decidualization induced by HB-EGF depend on cyclin D3 induction.
Cyclin is a component of the sea urchin egg M-phase specific histone H1 kinase.
Meijer, L; Arion, D; Golsteyn, R; Pines, J; Brizuela, L; Hunt, T; Beach, D
A so-called 'growth-associated' or 'M-phase specific' histone H1 kinase (H1K) has been described in a wide variety of eukaryotic cell types; p34cdc2 has previously been shown to be a catalytic subunit of this protein kinase. In fertilized sea urchin eggs the activity of H1K oscillates during the cell division cycle and there is a striking temporal correlation between H1K activation and the accumulation of a phosphorylated form of cyclin. H1K activity declines in parallel with proteolytic cyclin destruction of the end of the first cell cycle. By virtue of the high affinity of the fission yeast p13suc1 for the p34cdc2 protein, H1K strongly binds to p13-Sepharose beads. Cyclin, p34cdc2 and H1K co-purify on this affinity reagent as well as through several conventional chromatographic procedures. Anticyclin antibodies immunoprecipitate the M-phase specific H1K in crude extracts or in purified fractions. Sea urchin eggs appear to contain much less cyclin than p34cdc2, suggesting that p34cdc2 may interact with other proteins. These results demonstrate that cyclin and p34cdc2 are major components of the M-phase specific H1K.
HTLV-1 basic leucine zipper factor downregulates cyclin D1 expression via interactions with NF-κB.
Ma, Yunyun; Zhang, Bo; Wang, Dong; Qian, Lili; Song, Xianmei; Wang, Xueyin; Yang, Chaokuan; Zhao, Guoqiang
Human T cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus. It can cause adult T cell leukemia (ATL) and other diseases. The HTLV-1 basic leucine zipper (bZIP) factor (HBZ), which is encoded by the minus-strand of the provirus, is expressed in all cases of ATL and involved in T cell proliferation. However, the exact mechanism underlying its growth-promoting activity is poorly understood. Herein, we demonstrated that HBZ suppressed cyclin D1 expression by inhibiting the nuclear factor (NF)-κB signaling pathway. Among the potential mechanisms of cyclin D1 inhibition mediated by HBZ, we found that HBZ suppressed cyclin D1 promoter activity. Luciferase assay analysis revealed that HBZ repressed cyclin D1 promoter activity by suppressing NF-κB‑driven transcription mediated by the p65 subunit. Using an immunoprecipitation assay, we found that HBZ could bind to p65, but not p50. Finally, we showed that HBZ selectively interacted with p65 via its AD+bZIP domains. By suppressing cyclin D1 expression, HBZ can alter cell cycle progression of HTLV-1-infected cells, which may be critical for oncogenesis.
Cyclin D2 and the CDK substrate p220(NPAT) are required for self-renewal of human embryonic stem cells.
Becker, Klaus A; Ghule, Prachi N; Lian, Jane B; Stein, Janet L; van Wijnen, Andre J; Stein, Gary S
Self-renewal of pluripotent human embryonic stem (hES) cells utilizes an abbreviated cell cycle that bypasses E2F/pRB-dependent growth control. We investigated whether self-renewal is alternatively regulated by cyclin/CDK phosphorylation of the p220(NPAT)/HiNF-P complex to activate histone gene expression at the G1/S phase transition. We show that cyclin D2 is prominently expressed in pluripotent hES cells, but cyclin D1 eclipses cyclin D2 during differentiation. Depletion of cyclin D2 or p220(NPAT) causes a cell cycle defect in G1 reflected by diminished phosphorylation of p220(NPAT), decreased cell cycle dependent histone H4 expression and reduced S phase progression. Thus, cyclin D2 and p220(NPAT) are principal cell cycle regulators that determine competency for self-renewal in pluripotent hES cells. While pRB/E2F checkpoint control is relinquished in human ES cells, fidelity of physiological regulation is secured by cyclin D2 dependent activation of the p220(NPAT)/HiNF-P mechanism that may explain perpetual proliferation of hES cells without transformation or tumorigenesis.
PKCα TUMOR SUPPRESSION IN THE INTESTINE IS ASSOCIATED WITH TRANSCRIPTIONAL AND TRANSLATIONAL INHIBITION OF CYCLIN D1
Pysz, Marybeth A.; Leontieva, Olga V.; Bateman, Nicholas W.; Uronis, Joshua M.; Curry, Kathryn J.; Threadgill, David W.; Janssen, Klaus-Peter; Robine, Sylvie; Velcich, Anna; Augenlicht, Leonard; Black, Adrian R.; Black, Jennifer D.
Alterations in PKC isozyme expression and aberrant induction of cyclin D1 are early events in intestinal tumorigenesis. Previous studies have identified cyclin D1 as a major target in the antiproliferative effects of PKCα in non-transformed intestinal cells; however, a link between PKC signaling and cyclin D1 in colon cancer remained to be established. The current study further characterized PKC isozyme expression in intestinal neoplasms and explored the consequences of restoring PKCα or PKCδ in a panel of colon carcinoma cell lines. Consistent with patterns of PKC expression in primary tumors, PKCα and δ levels were generally reduced in colon carcinoma cell lines, PKCβII was elevated and PKCε showed variable expression, thus establishing the suitability of these models for analysis of PKC signaling. While colon cancer cells were insensitive to the effects of PKC agonists on cyclin D1 levels, restoration of PKCα downregulated cyclin D1 by two independent mechanisms. PKCα expression consistently (a) reduced steady-state levels of cyclin D1 by a novel transcriptional mechanism not previously seen in non-transformed cells, and (b) re-established the ability of PKC agonists to activate the translational repressor 4E-BP1 and inhibit cyclin D1 translation. In contrast, PKCδ had modest and variable effects on cyclin D1 steady state levels and failed to restore responsiveness to PKC agonists. Notably, PKCα expression blocked anchorage-independent growth in colon cancer cells via a mechanism partially dependent on cyclin D1 deficiency, while PKCδ had only minor effects. Loss of PKCα and effects of its re-expression were independent of the status of the APC/β-catenin signaling pathway or known genetic alterations, indicating that they are a general characteristic of colon tumors. Thus, PKCα is a potent negative regulator of cyclin D1 expression and anchorage-independent cell growth in colon tumor cells, findings that offer important perspectives on the
αB-crystallin is mutant B-RAF regulated and contributes to cyclin D1 turnover in melanocytic cells
Hu, Rong; Aplin, Andrew E.
Summary The serine/threonine kinase, B-RAF, is frequently mutated in melanoma and is required for cell proliferation. Proteasomal turnover of cyclins and cyclin-dependent kinase inhibitors via E3 ubiquitin ligases regulates cell cycle progression. We previously showed that B-RAF regulates Cks1, a co-factor for the F-box protein Skp2. Recently, a second F-box protein cofactor was identified, αB-crystallin, that binds Fbx4 and promotes cyclin D1 degradation. Here, we demonstrate that αB-crystallin is down-regulated in mutant B-RAF melanoma cells compared to melanocytes in a B-RAF and MEK-dependent manner. In a subset of lines, MEK inhibition was sufficient to up-regulate αB-crystallin protein levels; whereas in other lines combined MEK and proteasome inhibition was required. αB-crystallin knockdown partially stabilized cyclin D1 in melanocytes. Expression of αB-crystallin in mutant B-RAF melanoma cells did not promote cyclin D1 turnover under normal conditions, but did enhance turnover following etoposide-induced DNA damage. Together, these data show that αB-crystallin is highly expressed in melanocytes contributing, in part, to cyclin D1 turnover. Furthermore, αB-crystallin is down-regulated in a B-RAF-dependent manner in melanoma cells and its re-expression regulates cyclin D1 turnover after DNA damage. Significance αB-crystallin has been implicated in cellular functions as a heat shock protein and, more recently, as a cofactor for an E3 ligase ubiquitin ligase complex that degrades the cell cycle protein, cyclin D1. In this study we identify αB-crystallin as a target of aberrant B-RAF-MEK signaling that is hyper-activated in the majority of melanomas through mutation of B-RAF. Furthermore, we provide evidence for a functional role of αB-crystallin in contributing to the turnover of cyclin D1 in melanocytes and in melanoma cells following DNA damage inducing signals. These findings further our understanding of the regulation of cyclin D1 in melanocytic
Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition*
Timofeev, Oleg; Cizmecioglu, Onur; Settele, Florian; Kempf, Tore; Hoffmann, Ingrid
Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr161 by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr14 and Tyr15 phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, Cdc25A, -B, and -C. We find that expression of Cdc25A leads to an accelerated G2/M phase transition. In Cdc25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr15. In addition, Tyr15-phosphorylated Cdk1 binds more cyclin B in Cdc25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, Cdc25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of Cdc25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G2 and mitosis. Importantly, we find that Cdc25A overexpression leads to an activation of Cdk7 and increase in Thr161 phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G2/M is tightly coupled and regulated by Cdc25 phosphatases. PMID:20360007
Epigenetically altered miR-193b targets cyclin D1 in prostate cancer
Kaukoniemi, Kirsi M; Rauhala, Hanna E; Scaravilli, Mauro; Latonen, Leena; Annala, Matti; Vessella, Robert L; Nykter, Matti; Tammela, Teuvo L J; Visakorpi, Tapio
Micro-RNAs (miRNA) are important regulators of gene expression and often differentially expressed in cancer and other diseases. We have previously shown that miR-193b is hypermethylated in prostate cancer (PC) and suppresses cell growth. It has been suggested that miR-193b targets cyclin D1 in several malignancies. Here, our aim was to determine if miR-193b targets cyclin D1 in prostate cancer. Our data show that miR-193b is commonly methylated in PC samples compared to benign prostate hyperplasia. We found reduced miR-193b expression (P < 0.05) in stage pT3 tumors compared to pT2 tumors in a cohort of prostatectomy specimens. In 22Rv1 PC cells with low endogenous miR-193b expression, the overexpression of miR-193b reduced CCND1mRNA levels and cyclin D1 protein levels. In addition, the exogenous expression of miR-193b decreased the phosphorylation level of RB, a target of the cyclin D1-CDK4/6 pathway. Moreover, according to a reporter assay, miR-193b targeted the 3'UTR of CCND1 in PC cells and the CCND1 activity was rescued by expressing CCND1 lacking its 3'UTR. Immunohistochemical analysis of cyclin D1 showed that castration-resistant prostate cancers have significantly (P = 0.0237) higher expression of cyclin D1 compared to hormone-naïve cases. Furthermore, the PC cell lines 22Rv1 and VCaP, which express low levels of miR-193b and high levels of CCND1, showed significant growth retardation when treated with a CDK4/6 inhibitor. In contrast, the inhibitor had no effect on the growth of PC-3 and DU145 cells with high miR-193b and low CCND1 expression. Taken together, our data demonstrate that miR-193b targets cyclin D1 in prostate cancer. PMID:26129688
DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1
The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells. PMID:8175885
Tylophorine Analog DCB-3503 Inhibited Cyclin D1 Translation through Allosteric Regulation of Heat Shock Cognate Protein 70
Wang, Ying; Lam, Wing; Chen, Shao-Ru; Guan, Fu-Lan; Dutchman, Ginger E.; Francis, Samson; Baker, David C.; Cheng, Yung-Chi
Tylophorine analog DCB-3503 is a potential anticancer and immunosuppressive agent that suppresses the translation of cellular regulatory proteins, including cyclin D1, at the elongation step. However, the molecular mechanism underlying this phenomenon remains unknown. This study demonstrates that DCB-3503 preferentially binds to heat shock cognate protein 70 (HSC70), which is a determinant for cyclin D1 translation by binding to the 3′-untranslated region (3′ UTR) of its mRNA. DCB-3503 allosterically regulates the ATPase and chaperone activities of HSC70 by promoting ATP hydrolysis in the presence of specific RNA binding motifs (AUUUA) of cyclin D1 mRNA. The suppression of cyclin D1 translation by DCB-3503 is not solely caused by perturbation of the homeostasis of microRNAs, although the microRNA processing complex is dissociated with DCB-3503 treatment. This study highlights a novel regulatory mechanism of protein translation with AUUUA motifs in the 3′ UTR of mRNA by HSC70, and its activity can be allosterically modulated by DCB-3503. DCB-3503 may be used to treat malignancies, such as hepatocellular carcinoma or breast cancer with elevated expression of cyclin D1. PMID:27596272
Decreased cyclin A2 and increased cyclin G1 levels coincide with loss of proliferative capacity in rat Leydig cells during pubertal development.
Ge, R S; Hardy, M P
Postnatal development of Leydig cells can be divided into three distinct stages of differentiation: initially they exist as mesenchymal-like progenitors (PLC) by day 21; subsequently, as immature Leydig cells (ILC) by day 35, they acquire steroidogenic organelle structure and enzyme activities but metabolize most of the testosterone they produce; finally, as adult Leydig cells (ALC) by day 90 they actively produce testosterone. The aims of the present study were to determine whether changes in proliferative capacity are associated with progressive differentiation of Leydig cells, and if the proliferative capacity of Leydig cells is controlled by known hormonal regulators of testosterone biosynthesis: LH, insulin-like growth factor I (IGF-I), androgen, and estradiol (E2). Isolated PLC, ILC, and ALC were cultured in DMEM/F-12 for 24 h followed by an additional 24 h in the presence of LH (1 ng/ml), IGF-I (70 ng/ml), 7alpha-methyl-19-nortestosterone (MENT, 50 nM), a synthetic androgen that is not metabolized by 5alpha-reductase, or E2 (50 nM). Proliferative capacity was measured by assaying [3H]thymidine incorporation and labeling index (LI). Messenger RNA (mRNA) and protein levels for cyclin A2 and G1, which are putative intracellular regulators of Leydig cell proliferation and differentiation, were measured by RT-PCR and immunoblotting, respectively. Thymidine incorporation was highest in PLC (9.24 +/- 0.21 cpm/10(3) cell, mean +/- SE), intermediate in ILC (1.74 +/- 0.07) and lowest in ALC (0.24 +/- 0.03). Similarly, LI was highest in PLC (13.42 +/- 0.30%, mean +/- SE), intermediate in ILC (1.95 +/- 0.08%), and undetectable in ALC. Cyclin A2 mRNA levels, normalized to ribosomal protein S16 (RPS16), were highest in PLC (2.76 +/- 0.21, mean +/- SE), intermediate in ILC (1.79 +/- 0.14), and lowest in ALC (0.40 +/- 0.06). In contrast, cyclin G1 mRNA levels were highest in ALC (1.32 +/- 0.16), intermediate in ILC (0.47 +/- 0.07), and lowest in PLC (0.12 +/- 0.02). The
Transcriptional analysis of an E2F gene signature as a biomarker of activity of the cyclin-dependent kinase inhibitor PHA-793887 in tumor and skin biopsies from a phase I clinical study.
Locatelli, Giuseppe; Bosotti, Roberta; Ciomei, Marina; Brasca, Maria G; Calogero, Raffaele; Mercurio, Ciro; Fiorentini, Francesco; Bertolotti, Matteo; Scacheri, Emanuela; Scaburri, Angela; Galvani, Arturo; Pesenti, Enrico; De Baere, Thierry; Soria, Jean-Charles; Lazar, Vladimir; Isacchi, Antonella
A transcriptional signature of the pan-cyclin-dependent kinase (Cdk) inhibitor PHA-793887 was evaluated as a potential pharmacodynamic and/or response biomarker in tumor and skin biopsies from patients treated in a phase I clinical study. We first analyzed the expression of a number of known E2F-dependent genes that were predicted to be modulated after Cdk2 and Cdk4 inhibition in xenograft tumor and skin samples of mice treated with the compound. This panel of 58 selected genes was then analyzed in biopsies from seven patients treated with PHA-793887 in a phase I dose escalation clinical trial in solid tumors. Quantitative real-time PCR or microarray analyses were done in paired skin and tumor biopsies obtained at baseline and at cycle 1. Analysis by quantitative real-time PCR of the signature in skin biopsies of patients treated at three different doses showed significant transcriptional downregulation with a dose-response correlation. These data show that PHA-793887 modulates genes involved in cell cycle regulation and proliferation in a clinical setting. The observed changes are consistent with its mechanism of action and correlate with target modulation in skin and with clinical benefit in tumors.
Cyclin-A1 represents a new immunogenic targetable antigen expressed in acute myeloid leukemia stem cells with characteristics of a cancer-testis antigen
Ochsenreither, Sebastian; Majeti, Ravindra; Schmitt, Thomas; Stirewalt, Derek; Keilholz, Ulrich; Loeb, Keith R.; Wood, Brent; Choi, Yongiae E.; Bleakley, Marie; Warren, Edus H.; Hudecek, Michael; Akatsuka, Yoshiki; Weissman, Irving L.
Targeted T-cell therapy is a potentially less toxic strategy than allogeneic stem cell transplantation for providing a cytotoxic antileukemic response to eliminate leukemic stem cells (LSCs) in acute myeloid leukemia (AML). However, this strategy requires identification of leukemia-associated antigens that are immunogenic and exhibit selective high expression in AML LSCs. Using microarray expression analysis of LSCs, hematopoietic cell subpopulations, and peripheral tissues to screen for candidate antigens, cyclin-A1 was identified as a candidate gene. Cyclin-A1 promotes cell proliferation and survival, has been shown to be leukemogenic in mice, is detected in LSCs of more than 50% of AML patients, and is minimally expressed in normal tissues with exception of testis. Using dendritic cells pulsed with a cyclin-A1 peptide library, we generated T cells against several cyclin-A1 oligopeptides. Two HLA A*0201-restricted epitopes were further characterized, and specific CD8 T-cell clones recognized both peptide-pulsed target cells and the HLA A*0201-positive AML line THP-1, which expresses cyclin-A1. Furthermore, cyclin-A1–specific CD8 T cells lysed primary AML cells. Thus, cyclin-A1 is the first prototypic leukemia-testis-antigen to be expressed in AML LSCs. The pro-oncogenic activity, high expression levels, and multitude of immunogenic epitopes make it a viable target for pursuing T cell–based therapy approaches. PMID:22529286
A roller coaster ride with the mitotic cyclins.
Fung, Tsz Kan; Poon, Randy Y C
Cyclins are discovered as proteins that accumulate progressively through interphase and disappear abruptly at mitosis during each cell cycle. In mammalian cells, cyclin A accumulates from late G1 phase and is destroyed before metaphase, and cyclin B is destroyed slightly later at anaphase. The abundance of the mitotic cyclins is mainly regulated at the levels of transcription and proteolysis. Transcription is stimulated and repressed by several transcription factors, including B-MYB, E2F, FOXM1, and NF-Y. Elements in the promoter, including CCRE/CDE and CHR, are in part responsible for the cell cycle oscillation of transcription. Destruction of the mitotic cyclins is carried out by the ubiquitin ligases APC/C(CDC20) and APC/C(CDH1). Central to our knowledge is the understanding of how APC/C is turned on from anaphase to early G1 phase, and turned off from late G1 till the spindle-assembly checkpoint is deactivated in metaphase. Reciprocal actions of cyclin-dependent kinases (CDKs) on APC/C, as well as on the SCF complexes ensure that the mitotic cyclins are destroyed only at the proper time.
Inhibition of cyclin dependent kinase 9 by dinaciclib suppresses cyclin B1 expression and tumor growth in triple negative breast cancer
Rajput, Sandeep; Khera, Nimmish; Guo, Zhanfang; Hoog, Jeremy; Li, Shunqiang; Ma, Cynthia X.
Cyclin-dependent kinases (CDKs) are potential cancer therapeutic targets because of their critical role in promoting cell growth. Dinaciclib is a novel CDK inhibitor currently under clinical evaluation for the treatment of advanced malignancies. In this study, we demonstrated the anti-tumor activity of dinaciclib in triple negative breast cancer (TNBC) patient derived xenograft (PDX) and cell lines in vitro and in vivo. Treatment with dinaciclib induced cell cycle arrest at G2/M phase and marked apoptosis. These changes were accompanied by reduced phosphorylation of CDK1 and retinoblastoma (Rb) protein and decreased protein levels of cyclin B1, cMYC and survivin. We further demonstrated that siRNA knockdown of CDK9, the kinase subunit of positive transcription elongation factor b (P-TEFb), instead of CDK1 or CDK2, reduced the levels of cyclin B1 and MYC in TNBC cell lines. These data support the importance of CDK9, in addition to CDK1, in mediating the growth inhibitory effect of dinaciclib in TNBC. Further investigation of CDK9 as a therapeutic target in TNBC is needed. PMID:27486754
The coffee diterpene kahweol suppresses the cell proliferation by inducing cyclin D1 proteasomal degradation via ERK1/2, JNK and GKS3β-dependent threonine-286 phosphorylation in human colorectal cancer cells.
Park, Gwang Hun; Song, Hun Min; Jeong, Jin Boo
Kahweol as a coffee-specific diterpene has been reported to exert anti-cancer properties. However, the mechanism responsible for the anti-cancer effects of kahweol is not fully understood. The main aim of this investigation was to determine the effect of kahweol on cell proliferation and the possible mechanisms in human colorectal cancer cells. Kahweol inhibited markedly the proliferation of human colorectal cancer cell lines such as HCT116, SW480. Kahweol decreased cyclin D1 protein level in HCT116 and SW480 cells. Contrast to protein levels, cyclin D1 mRNA level and promoter activity did not be changed by kahweol treatment. MG132 treatment attenuated kahweol-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in kahweol-treated cells. Kahweol increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated cyclin D1 degradation by kahweol. Inhibition of ERK1/2 by PD98059, JNK by SP600125 or GSK3β by LiCl suppressed cyclin D1 phosphorylation and downregulation by kahweol. Furthermore, the inhibition of nuclear export by LMB attenuated cyclin D1 degradation by kahweol. In conclusion, kahweol-mediated cyclin D1 degradation may contribute to the inhibition of the proliferation in human colorectal cancer cells.
Establishment of an immortalized cell line derived from the prairie vole via lentivirus-mediated transduction of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase
Katayama, Masafumi; Kiyono, Tohru; Horie, Kengo; Hirayama, Takashi; Eitsuka, Takahiro; Kuroda, Kengo; Donai, Kenichiro; Hidema, Shizu; Nishimori, Katsuhiko; Fukuda, Tomokazu
The prairie vole (Microtus ochrogaster) shows social behaviors such as monogamy and parenting of infants with pair bonding. These social behaviors are specific to the prairie vole and have not been observed in other types of voles, such as mountain voles. Although the prairie vole has several unique characteristics, an in vitro cell culture system has not been established for this species. Furthermore, establishment of cultured cells derived from the prairie vole may be beneficial based on the three Rs (i.e., Replacement, Reduction, and Refinement) concept. Therefore, in this study, we attempted to establish an immortalized cell line derived from the prairie vole. Our previous research has shown that transduction with mutant forms of cyclin-dependent kinase 4 (CDK4), cyclin D, and telomerase reverse transcriptase (TERT) could efficiently immortalize cells from multiple species, including humans, cattle, pigs, and monkeys. Here, we introduced these three genes into prairie vole-derived muscle fibroblasts. The expression of mutant CDK4 and cyclin D proteins was confirmed by western blotting, and telomerase activity was detected in immortalized vole muscle-derived fibroblasts (VMF-K4DT cells or VMFs) by stretch PCR. Population doubling analysis showed that the introduction of mutant CDK4, cyclin D, and TERT extended the lifespan of VMFs. To the best of our knowledge, this is the first report describing the establishment of an immortalized cell line derived from the prairie vole through the expression of mutant CDK4, cyclin D, and human TERT. PMID:26496927
p53 is phosphorylated by CDK7-cyclin H in a p36MAT1-dependent manner.
Ko, L J; Shieh, S Y; Chen, X; Jayaraman, L; Tamai, K; Taya, Y; Prives, C; Pan, Z Q
The tumor suppressor protein p53 acts as a transcriptional activator that can mediate cellular responses to DNA damage by inducing apoptosis and cell cycle arrest. p53 is a nuclear phosphoprotein, and phosphorylation has been proposed to be a means by which the activity of p53 is regulated. The cyclin-dependent kinase (CDK)-activating kinase (CAK) was originally identified as a cellular kinase required for the activation of a CDK-cyclin complex, and CAK is comprised of three subunits: CDK7, cyclin H, and p36MAT1. CAK is part of the transcription factor IIH multiprotein complex, which is required for RNA polymerase II transcription and nucleotide excision repair. Because of the similarities between p53 and CAK in their involvement in the cell cycle, transcription, and repair, we investigated whether p53 could act as a substrate for phosphorylation by CAK. While CDK7-cyclin H is sufficient for phosphorylation of CDK2, we show that p36MAT1 is required for efficient phosphorylation of p53 by CDK7-cyclin H, suggesting that p36MAT1 can act as a substrate specificity-determining factor for CDK7-cyclin H. We have mapped a major site of phosphorylation by CAK to Ser-33 of p53 and have demonstrated as well that p53 is phosphorylated at this site in vivo. Both wild-type and tumor-derived mutant p53 proteins are efficiently phosphorylated by CAK. Furthermore, we show that p36 and p53 can interact both in vitro and in vivo. These studies reveal a potential mechanism for coupling the regulation of p53 with DNA repair and the basal transcriptional machinery. PMID:9372954
Formation of mos RNA granules in the zebrafish oocyte that differ from cyclin B1 RNA granules in distribution, density and regulation.
Horie, Mayu; Kotani, Tomoya
Many translationally repressed mRNAs are deposited in the oocyte cytoplasm for progression of the meiotic cell cycle and early development. mos and cyclin B1 mRNAs encode proteins promoting oocyte meiosis, and translational control of these mRNAs is important for normal progression of meiotic cell division. We previously demonstrated that cyclin B1 mRNA forms RNA granules in the zebrafish and mouse oocyte cytoplasm and that the formation of RNA granules is crucial for regulating the timing of translational activation of the mRNA. However, whether the granule formation is specific to cyclin B1 mRNA remains unknown. In this study, we found that zebrafish mos mRNA forms granules distinct from those of cyclin B1 mRNA. Fluorescent in situ hybridization analysis showed that cyclin B1 RNA granules were assembled in dense clusters, while mos RNA granules were distributed diffusely in the animal polar cytoplasm. Sucrose density gradient ultracentrifugation analysis showed that the density of mos RNA granules was partly lower than that of cyclin B1 mRNA. Similar to cyclin B1 RNA granules, mos RNA granules were disassembled after initiation of oocyte maturation at the timing at which the poly(A) tail was elongated. However, while almost all of the granules of cyclin B1 were disassembled simultaneously, a fraction of mos RNA granules firstly disappeared and then a large part of them was disassembled. In addition, while cyclin B1 RNA granules were disassembled in a manner dependent on actin filament depolymerization, certain fractions of mos RNA granules were disassembled independently of actin filaments. These results suggest that cytoplasmic regulation of translationally repressed mRNAs by formation of different RNA granules is a key mechanism for translational control of distinct mRNAs in the oocyte.
Cyclin E-CDK2 protein phosphorylates plant homeodomain finger protein 8 (PHF8) and regulates its function in the cell cycle.
Sun, Liping; Huang, Yan; Wei, Qian; Tong, Xiaomei; Cai, Rong; Nalepa, Grzegorz; Ye, Xin
Cyclin E-CDK2 is a key regulator in G1/S transition. Previously, we identified a number of CDK2-interacting proteins, including PHF8 (plant homeodomain finger protein 8). In this report, we confirmed that PHF8 is a novel cyclin E-CDK2 substrate. By taking the approach of mass spectrometry, we identified that PHF8 Ser-844 is phosphorylated by cyclin E-CDK2. Immunoblotting analysis indicated that WT PHF8 demethylates histone H3K9me2 more efficiently than the cyclin E-CDK2 phosphorylation-deficient PHF8-S844A mutant. Furthermore, flow cytometry analysis showed that WT PHF8 promotes S phase progression more robustly than PHF8-S844A. Real-time PCR results demonstrated that PHF8 increases transcription of cyclin E, E2F3, and E2F7 to significantly higher levels compared with PHF8-S844A. Further analysis by ChIP assay indicated that PHF8 binds to the cyclin E promoter stronger than PHF8-S844A and reduces the H3K9me2 level at the cyclin E promoter more efficiently than PHF8-S844A. In addition, we found that cyclin E-CDK2-mediated phosphorylation of PHF8 Ser-844 promotes PHF8-dependent rRNA transcription in luciferase reporter assays and real-time PCR. Taken together, these results indicate that cyclin E-CDK2 phosphorylates PHF8 to stimulate its demethylase activity to promote rRNA transcription and cell cycle progression.
Targeting the AKT/GSK3{beta}/Cyclin D1/Cdk4 Survival Signaling Pathway for Eradication of Tumor Radioresistance Acquired by Fractionated Radiotherapy
Shimura, Tsutomu; Kakuda, Satoshi; Ochiai, Yasushi; Kuwahara, Yoshikazu; Takai, Yoshihiro; Fukumoto, Manabu
Purpose: Radioresistance is a major cause of treatment failure of radiotherapy (RT) in human cancer. We have recently revealed that acquired radioresistance of tumor cells induced by fractionated radiation is attributable to cyclin D1 overexpression as a consequence of the downregulation of GSK3{beta}-dependent cyclin D1 proteolysis mediated by a constitutively activated serine-threonine kinase, AKT. This prompted us to hypothesize that targeting the AKT/GSK3{beta}/cyclin D1 pathway may improve fractionated RT by suppressing acquired radioresistance of tumor cells. Methods and Materials: Two human tumor cell lines with acquired radioresistance were exposed to X-rays after incubation with either an AKT inhibitor, AKT/PKB signaling inhibitor-2 (API-2), or a Cdk4 inhibitor (Cdk4-I). Cells were then subjected to immunoblotting, clonogenic survival assay, cell growth analysis, and cell death analysis with TUNEL and annexin V staining. In vivo radiosensitivity was assessed by growth of human tumors xenografted into nude mice. Results: Treatment with API-2 resulted in downregulation of cyclin D1 expression in cells with acquired radioresistance. Cellular radioresistance disappeared completely both in vitro and in vivo with accompanying apoptosis when treated with API-2. Furthermore, inhibition of cyclin D1/Cdk4 by Cdk4-I was sufficient for abolishing radioresistance. Treatment with either API-2 or Cdk4-I was also effective in suppressing resistance to cis-platinum (II)-diamine-dichloride in the cells with acquired radioresistance. Interestingly, the radiosensitizing effect of API-2 was canceled by overexpression of cyclin D1 whereas Cdk4-I was still able to sensitize cells with cyclin D1 overexpression. Conclusion: Cyclin D1/Cdk4 is a critical target of the AKT survival signaling pathway responsible for tumor radioresistance. Targeting the AKT/GSK3{beta}/cyclin D1/Cdk4 pathway would provide a novel approach to improve fractionated RT and would have an impact on tumor
D-type Cyclins are important downstream effectors of cytokine signaling that regulate the proliferation of normal and neoplastic mammary epithelial cells.
Zhang, Qian; Sakamoto, Kazuhito; Wagner, Kay-Uwe
In response to the ligand-mediated activation of cytokine receptors, cells decide whether to proliferate or to undergo differentiation. D-type Cyclins (Cyclin D1, D2, or D3) and their associated Cyclin-dependent kinases (CDK4, CDK6) connect signals from cytokines to the cell cycle machinery, and they propel cells through the G1 restriction point and into the S phase, after which growth factor stimulation is no longer essential to complete cell division. D-type Cyclins are upregulated in many human malignancies including breast cancer to promote an uncontrolled proliferation of cancer cells. After summarizing important aspects of the cytokine-mediated transcriptional regulation and the posttranslational modification of D-type Cyclins, this review will highlight the physiological significance of these cell cycle regulators during normal mammary gland development as well as the initiation and promotion of breast cancer. Although the vast majority of published reports focus almost exclusively on the role of Cyclin D1 in breast cancer, we summarize here previous and recent findings that demonstrate an important contribution of the remaining two members of this Cyclin family, in particular Cyclin D3, for the growth of ErbB2-associated breast cancer cells in humans and in mouse models. New data from genetically engineered models as well as the pharmacological inhibition of CDK4/6 suggest that targeting the combined functions of D-type Cyclins could be a suitable strategy for the treatment of ErbB2-positive and potentially other types of breast cancer.
Cyclin-dependent kinase 2 protects podocytes from apoptosis
Saurus, Pauliina; Kuusela, Sara; Dumont, Vincent; Lehtonen, Eero; Fogarty, Christopher L.; Lassenius, Mariann I.; Forsblom, Carol; Lehto, Markku; Saleem, Moin A.; Groop, Per-Henrik; Lehtonen, Sanna
Loss of podocytes is an early feature of diabetic nephropathy (DN) and predicts its progression. We found that treatment of podocytes with sera from normoalbuminuric type 1 diabetes patients with high lipopolysaccharide (LPS) activity, known to predict progression of DN, downregulated CDK2 (cyclin-dependent kinase 2). LPS-treatment of mice also reduced CDK2 expression. LPS-induced downregulation of CDK2 was prevented in vitro and in vivo by inhibiting the Toll-like receptor (TLR) pathway using immunomodulatory agent GIT27. We also observed that CDK2 is downregulated in the glomeruli of obese Zucker rats before the onset of proteinuria. Knockdown of CDK2, or inhibiting its activity with roscovitine in podocytes increased apoptosis. CDK2 knockdown also reduced expression of PDK1, an activator of the cell survival kinase Akt, and reduced Akt phosphorylation. This suggests that CDK2 regulates the activity of the cell survival pathway via PDK1. Furthermore, PDK1 knockdown reduced the expression of CDK2 suggesting a regulatory loop between CDK2 and PDK1. Collectively, our data show that CDK2 protects podocytes from apoptosis and that reduced expression of CDK2 associates with the development of DN. Preventing downregulation of CDK2 by blocking the TLR pathway with GIT27 may provide a means to prevent podocyte apoptosis and progression of DN. PMID:26876672
The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells
Badia, Roger; Pujantell, Maria; Riveira-Muñoz, Eva; Puig, Teresa; Torres-Torronteras, Javier; Martí, Ramón; Clotet, Bonaventura; Ampudia, Rosa M.; Ballana, Ester
Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages. PMID:27541004
Transgenic expression of cyclin-dependent kinase 4 results in epidermal hyperplasia, hypertrophy, and severe dermal fibrosis.
Miliani de Marval, P L; Gimenez-Conti, I B; LaCava, M; Martinez, L A; Conti, C J; Rodriguez-Puebla, M L
In a previous report we have described the effects of expression of D-type cyclins in epithelial tissues of transgenic mice. To study the involvement of the D-type cyclin partner cyclin-dependent kinase 4 (CDK4) in epithelial growth and differentiation, transgenic mice were generated carrying the CDK4 gene under the control of a keratin 5 promoter. As expected, transgenic mice showed expression of CDK4 in the epidermal basal-cell layer. Epidermal proliferation increased dramatically and basal cell hyperplasia and hypertrophy were observed. The hyperproliferative phenotype of these transgenic mice was independent of D-type cyclin expression because no overexpression of these proteins was detected. CDK4 and CDK2 kinase activities increased in transgenic animals and were associated with elevated binding of p27(Kip1) to CDK4. Expression of CDK4 in the epidermis results in an increased spinous layer compared with normal epidermis, and a mild hyperkeratosis in the cornified layer. In addition to epidermal changes, severe dermal fibrosis was observed and part of the subcutaneous adipose tissue was replaced by connective tissue. Also, abnormal expression of keratin 6 associated with the hyperproliferative phenotype was observed in transgenic epidermis. This model provides in vivo evidence for the role of CDK4 as a mediator of proliferation in epithelial cells independent of D-type cyclin expression.
Cancer-associated variant expression and interaction of CIZ1 with cyclin A1 in differentiating male germ cells.
Greaves, Erin A; Copeland, Nikki A; Coverley, Dawn; Ainscough, Justin F X
CIZ1 is a nuclear-matrix-associated DNA replication factor unique to higher eukaryotes, for which alternatively spliced isoforms have been associated with a range of disorders. In vitro, the CIZ1 N-terminus interacts with cyclin E and cyclin A at distinct sites, enabling functional cooperation with cyclin-A-Cdk2 to promote replication initiation. C-terminal sequences anchor CIZ1 to fixed sites on the nuclear matrix, imposing spatial constraint on cyclin-dependent kinase activity. Here we demonstrate that CIZ1 is predominantly expressed as a predicted full-length product throughout mouse development, consistent with a ubiquitous role in cell and tissue renewal. CIZ1 is expressed in proliferating stem cells of the testis, but is notably downregulated following commitment to differentiation. Significantly, CIZ1 is re-expressed at high levels in non-proliferative spermatocytes before meiotic division. Sequence analysis identifies at least seven alternatively spliced variants, including a dominant cancer-associated form and a set of novel isoforms. Furthermore, we show that in these post-replicative cells, CIZ1 interacts with germ-cell-specific cyclin A1, which has been implicated in the repair of DNA double-strand breaks. Consistent with this role, antibody depletion of CIZ1 reduces the capacity for testis extract to repair digested plasmid DNA in vitro. Together, the data imply post-replicative roles for CIZ1 in germ cell differentiation that might include meiotic recombination - a process intrinsic to genome stability and diversification.
Cyclin A/Cdk2 regulates Cdh1 and claspin during late S/G2 phase of the cell cycle.
Oakes, Vanessa; Wang, Weili; Harrington, Brittney; Lee, Won Jae; Beamish, Heather; Chia, Kee Ming; Pinder, Alex; Goto, Hidemasa; Inagaki, Masaki; Pavey, Sandra; Gabrielli, Brian
Whereas many components regulating the progression from S phase through G2 phase into mitosis have been identified, the mechanism by which these components control this critical cell cycle progression is still not fully elucidated. Cyclin A/Cdk2 has been shown to regulate the timing of Cyclin B/Cdk1 activation and progression into mitosis although the mechanism by which this occurs is only poorly understood. Here we show that depletion of Cyclin A or inhibition of Cdk2 during late S/early G2 phase maintains the G2 phase arrest by reducing Cdh1 transcript and protein levels, thereby stabilizing Claspin and maintaining elevated levels of activated Chk1 which contributes to the G2 phase observed. Interestingly, the Cyclin A/Cdk2 regulated APC/C(Cdh1) activity is selective for only a subset of Cdh1 targets including Claspin. Thus, a normal role for Cyclin A/Cdk2 during early G2 phase is to increase the level of Cdh1 which destabilises Claspin which in turn down regulates Chk1 activation to allow progression into mitosis. This mechanism links S phase exit with G2 phase transit into mitosis, provides a novel insight into the roles of Cyclin A/Cdk2 in G2 phase progression, and identifies a novel role for APC/C(Cdh1) in late S/G2 phase cell cycle progression.
Role of Cyclin E as an Early Event in Ovarian Carcinogenesis
characterizes a subset of epithelial ovarian cancers . We hypothesized that this subset of tumors may demonstrate an enhanced response to targeted...therapy with the proteasome inhibitor, bortezomib. Cyclin E also exists in multiple low molecular weight (LMW) isoforms in cancer cells which... cancer cells to bortezomib. This finding has translational potential as bortezomib as a single- agent was found to have minimal activity in a phase II
Characterization of cyclin-dependent kinases and Cdc2/Cdc28 kinase subunits in Trichomonas vaginalis.
Amador, Erick; López-Pacheco, Karla; Morales, Nataly; Coria, Roberto; López-Villaseñor, Imelda
Cyclin-dependent kinases (CDKs) have important roles in regulating key checkpoints between stages of the cell cycle. Their activity is tightly regulated through a variety of mechanisms, including through binding with cyclin proteins and the Cdc2/Cdc28 kinase subunit (CKS), and their phosphorylation at specific amino acids. Studies of the components involved in cell cycle control in parasitic protozoa are limited. Trichomonas vaginalis is the causative agent of trichomoniasis in humans and is therefore important in public health; however, some of the basic biological processes used by this organism have not been defined. Here, we characterized proteins potentially involved in cell cycle regulation in T. vaginalis. Three genes encoding protein kinases were identified in the T. vaginalis genome, and the corresponding recombinant proteins (TvCRK1, TvCRK2, TvCRK5) were studied. These proteins displayed similar sequence features to CDKs. Two genes encoding CKSs were also identified, and the corresponding recombinant proteins were found to interact with TvCRK1 and TvCRK2 by a yeast two-hybrid system. One putative cyclin B protein from T. vaginalis was found to bind to and activate the kinase activities of TvCRK1 and TvCRK5, but not TvCRK2. This work is the first characterization of proteins involved in cell cycle control in T. vaginalis.
Cyclin/CDK Regulates the Nucleocytoplasmic Localization of the Human Papillomavirus E1 DNA Helicase
Deng, Wentao; Lin, Biing Yuan; Jin, Ge; Wheeler, Crystal G.; Ma, Tianlin; Harper, J. Wade; Broker, Thomas R.; Chow, Louise T.
Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program. PMID:15564503
Loss of Cul1 results in early embryonic lethality and dysregulation of cyclin E.
Dealy, M J; Nguyen, K V; Lo, J; Gstaiger, M; Krek, W; Elson, D; Arbeit, J; Kipreos, E T; Johnson, R S
The sequential timing of cell-cycle transitions is primarily governed by the availability and activity of key cell-cycle proteins. Recent studies in yeast have identified a class of ubiquitin ligases (E3 enzymes) called SCF complexes, which regulate the abundance of proteins that promote and inhibit cell-cycle progression at the G1-S phase transition. SCF complexes consist of three invariable components, Skp1, Cul-1 (Cdc53 in yeast) and Rbx1, and a variable F-box protein that recruits a specific cellular protein to the ubquitin pathway for degradation. To study the role of Cul-1 in mammalian development and cell-cycle regulation, we generated mice deficient for Cul1 and analysed null embryos and heterozygous cell lines. We show that Cul1 is required for early mouse development and that Cul1 mutants fail to regulate the abundance of the G1 cyclin, cyclin E (encoded by Ccne), during embryogenesis.
Optimization of non-ATP competitive CDK/cyclin groove Inhibitors through REPLACE mediated Fragment Assembly
Liu, Shu; Premnath, Padmavathy Nandha; Bolger, Joshua K.; Perkins, Tracy; Kirkland, Lindsay O.; Kontopidis, George; McInnes, Campbell
A major challenge in drug discovery is to develop and improve methods for targeting protein-protein interactions. Further exemplification of the REPLACE strategy for generating inhibitors of protein-protein interactions demonstrated that it can be used to optimize fragment alternatives of key determinants, to combine these in an effective way and was achieved for compounds targeting the CDK2 substrate recruitment site on the cyclin regulatory subunit. Phenylheterocyclic isosteres replacing a critical charge-charge interaction provided new structural insights for binding to the cyclin groove. In particular, these results shed light onto the key contributions of a H-bond observed in crystal structures of N-terminally capped peptides. Furthermore the structure-activity relationship of a bisarylether C-terminal capping group mimicking dipeptide interactions, was probed through ring substitutions, allowing increased complementarity with the primary hydrophobic pocket. This study further validates REPLACE as an effective strategy for converting peptidic compounds to more pharmaceutically relevant compounds. PMID:23323521
Cyclin B1/Cdk1 Phosphorylation of Mitochondrial p53 Induces Anti-Apoptotic Response
Nantajit, Danupon; Fan, Ming; Duru, Nadire; Wen, Yunfei; Reed, John C.; Li, Jian Jian
The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53+/+ status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53−/− cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53−/− cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53. PMID:20808790
Proteomic Landscape of Tissue-Specific Cyclin E Functions in Vivo
Odajima, Junko; Jung, Piotr; Ndassa-Colday, Yasmine; Ficaro, Scott; Geng, Yan; Marco, Eugenio; Michowski, Wojciech; Wang, Yaoyu E.; DeCaprio, James A.; Litovchick, Larisa; Marto, Jarrod; Sicinski, Piotr
E-type cyclins (cyclins E1 and E2) are components of the cell cycle machinery that has been conserved from yeast to humans. The major function of E-type cyclins is to drive cell division. It is unknown whether in addition to their 'core' cell cycle functions, E-type cyclins also perform unique tissue-specific roles. Here, we applied high-throughput mass spectrometric analyses of mouse organs to define the repertoire of cyclin E protein partners in vivo. We found that cyclin E interacts with distinct sets of proteins in different compartments. These cyclin E interactors are highly enriched for phosphorylation targets of cyclin E and its catalytic partner, the cyclin-dependent kinase 2 (Cdk2). Among cyclin E interactors we identified several novel tissue-specific substrates of cyclin E-Cdk2 kinase. In proliferating compartments, cyclin E-Cdk2 phosphorylates Lin proteins within the DREAM complex. In the testes, cyclin E-Cdk2 phosphorylates Mybl1 and Dmrtc2, two meiotic transcription factors that represent key regulators of spermatogenesis. In embryonic and adult brains cyclin E interacts with proteins involved in neurogenesis, while in adult brains also with proteins regulating microtubule-based processes and microtubule cytoskeleton. We also used quantitative proteomics to demonstrate re-wiring of the cyclin E interactome upon ablation of Cdk2. This approach can be used to study how protein interactome changes during development or in any pathological state such as aging or cancer. PMID:27828963
Crystal structure of a human cyclin-dependent kinase 6 complexwith a flavonol inhibitor, Fisetin
Lu, Heshu; Chang, Debbie J.; Baratte, Blandine; Meijer, Laurent; Schulze-Gahmen, Ursula
Cyclin-dependent kinases (CDKs) play a central role in cell cycle control, apoptosis, transcription and neuronal functions. They are important targets for the design of drugs with anti-mitotic and/or anti-neurodegenerative effects. CDK4 and CDK6 form a subfamily among the CDKs in mammalian cells, as defined by sequence similarities. Compared to CDK2 and CDK5, structural information on CDK4 and CDK6 is sparse. We describe here the crystal structure of human CDK6 in complex with a viral cyclin and a flavonol inhibitor, fisetin. Fisetin binds to the active form of CDK6, forming hydrogen bonds with the side chains of residues in the binding pocket that undergo large conformational changes during CDK activation by cyclin binding. The 4-keto group and the 3-hydroxyl group of fisetin are hydrogen bonded with the backbone in the hinge region between the N-terminal and C-terminal kinase domain, as has been observed for many CDK inhibitors. However, CDK2 and HCK kinase in complex with other flavone inhibitors such as quercetin and flavopiridol showed a different binding mode with the inhibitor rotated by about 180. The structural information of the CDK6-fisetin complex is correlated with the binding affinities of different flavone inhibitors for CDK6. This complex structure is the first description of an inhibitor complex with a kinase from the CDK4/6 subfamily and can provide a basis for selecting and designing inhibitor compounds with higher affinity and specificity.
Structure-function relationships of the yeast cyclin-dependent kinase Pho85.
Santos, R C; Waters, N C; Creasy, C L; Bergman, L W
The PHO85 gene of Saccharomyces cerevisiae encodes a cyclin-dependent kinase involved in both transcriptional regulation and cell cycle progression. Although a great deal is known concerning the structure, function, and regulation of the highly homologous Cdc28 protein kinase, little is known concerning these relationships in regard to Pho85. In this study, we constructed a series of Pho85-Cdc28 chimeras to map the region(s) of the Pho85 molecule that is critical for function of Pho85 in repression of acid phosphatase (PHO5) expression. Using a combination of site-directed and ethyl methanesulfonate-induced mutagenesis, we have identified numerous residues critical for either activation of the Pho85 kinase, interaction of Pho85 with the cyclin-like molecule Pho80, or substrate recognition. Finally, analysis of mutations analogous to those previously identified in either Cdc28 or cdc2 of Schizosaccharomyces pombe suggested that the inhibition of Pho85-Pho80 activity in mechanistically different from that seen in the other cyclin-dependent kinases. PMID:7565699
Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells.
The, Inge; Ruijtenberg, Suzan; Bouchet, Benjamin P; Cristobal, Alba; Prinsen, Martine B W; van Mourik, Tim; Koreth, John; Xu, Huihong; Heck, Albert J R; Akhmanova, Anna; Cuppen, Edwin; Boxem, Mike; Muñoz, Javier; van den Heuvel, Sander
Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activator Cdh1, completely eliminates the essential requirement of CDK4/6-cyclin D (CDK-4/CYD-1) in C. elegans. CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 amino terminus, resembling inactivating phosphorylations of the human proteins. In human breast cancer cells, simultaneous knockdown of Rb and FZR1 synergistically bypasses cell division arrest induced by the CDK4/6-specific inhibitor PD-0332991. Our data identify FZR1 as a candidate CDK4/6-cyclin D substrate and point to an APC/C(FZR1) activity as an important determinant in response to CDK4/6-inhibitors.
DNA-PKcs Negatively Regulates Cyclin B1 Protein Stability through Facilitating Its Ubiquitination Mediated by Cdh1-APC/C Pathway.
Shang, Zeng-Fu; Tan, Wei; Liu, Xiao-Dan; Yu, Lan; Li, Bing; Li, Ming; Song, Man; Wang, Yu; Xiao, Bei-Bei; Zhong, Cai-Gao; Guan, Hua; Zhou, Ping-Kun
The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a critical component of the non-homologous end-joining pathway of DNA double-stranded break repair. DNA-PKcs has also been shown recently functioning in mitotic regulation. Here, we report that DNA-PKcs negatively regulates the stability of Cyclin B1 protein through facilitating its ubiquitination mediated by Cdh1 / E 3 ubiquitin ligase APC/C pathway. Loss of DNA-PKcs causes abnormal accumulation of Cyclin B1 protein. Cyclin B1 degradation is delayed in DNA-PKcs-deficient cells as result of attenuated ubiquitination. The impact of DNA-PKcs on Cyclin B1 stability relies on its kinase activity. Our study further reveals that DNA-PKcs interacts with APC/C core component APC2 and its co-activator Cdh1. The destruction of Cdh1 is accelerated in the absence of DNA-PKcs. Moreover, overexpression of exogenous Cdh1 can reverse the increase of Cyclin B1 protein in DNA-PKcs-deficient cells. Thus, DNA-PKcs, in addition to its direct role in DNA damage repair, functions in mitotic progression at least partially through regulating the stability of Cyclin B1 protein.
Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells
The, Inge; Ruijtenberg, Suzan; Bouchet, Benjamin P.; Cristobal, Alba; Prinsen, Martine B. W.; van Mourik, Tim; Koreth, John; Xu, Huihong; Heck, Albert J. R.; Akhmanova, Anna; Cuppen, Edwin; Boxem, Mike; Muñoz, Javier; van den Heuvel, Sander
Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activator Cdh1, completely eliminates the essential requirement of CDK4/6-cyclin D (CDK-4/CYD-1) in C. elegans. CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 amino terminus, resembling inactivating phosphorylations of the human proteins. In human breast cancer cells, simultaneous knockdown of Rb and FZR1 synergistically bypasses cell division arrest induced by the CDK4/6-specific inhibitor PD-0332991. Our data identify FZR1 as a candidate CDK4/6-cyclin D substrate and point to an APC/CFZR1 activity as an important determinant in response to CDK4/6-inhibitors. PMID:25562820
DNA-PKcs Negatively Regulates Cyclin B1 Protein Stability through Facilitating Its Ubiquitination Mediated by Cdh1-APC/C Pathway
Shang, Zeng-Fu; Tan, Wei; Liu, Xiao-Dan; Yu, Lan; Li, Bing; Li, Ming; Song, Man; Wang, Yu; Xiao, Bei-Bei; Zhong, Cai-Gao; Guan, Hua; Zhou, Ping-Kun
The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a critical component of the non-homologous end-joining pathway of DNA double-stranded break repair. DNA-PKcs has also been shown recently functioning in mitotic regulation. Here, we report that DNA-PKcs negatively regulates the stability of Cyclin B1 protein through facilitating its ubiquitination mediated by Cdh1 / E 3 ubiquitin ligase APC/C pathway. Loss of DNA-PKcs causes abnormal accumulation of Cyclin B1 protein. Cyclin B1 degradation is delayed in DNA-PKcs-deficient cells as result of attenuated ubiquitination. The impact of DNA-PKcs on Cyclin B1 stability relies on its kinase activity. Our study further reveals that DNA-PKcs interacts with APC/C core component APC2 and its co-activator Cdh1. The destruction of Cdh1 is accelerated in the absence of DNA-PKcs. Moreover, overexpression of exogenous Cdh1 can reverse the increase of Cyclin B1 protein in DNA-PKcs-deficient cells. Thus, DNA-PKcs, in addition to its direct role in DNA damage repair, functions in mitotic progression at least partially through regulating the stability of Cyclin B1 protein. PMID:26221070
G1 phase arrest induced by Wilms tumor protein WT1 is abrogated by cyclin/CDK complexes.
Kudoh, T; Ishidate, T; Moriyama, M; Toyoshima, K; Akiyama, T
WT1, the Wilms tumor-suppressor gene, maps to the human chromosomal region 11p13 and encodes a transcriptional repressor, WT1, implicated in controlling normal urogenital development. Microinjection of the WT1 cDNA into quiescent cells or cells in early to mid G1 phase blocked serum-induced cell cycle progression into S phase. The activity of WT1 varied significantly depending on the presence or absence of an alternatively spliced region located upstream of the zinc finger domain. The inhibitory activity of WT1 was abrogated by the overexpression of cyclin E/CDK2 as well as cyclin D1/CDK4. Furthermore, both CDK4- and CDK2-associated kinase activities were downregulated in cells overexpressing WT1, whereas the levels of CDK4, CDK2, and cyclin D1 expression were unchanged. These findings suggest that inhibition of the activity of cyclin/CDK complexes may be involved in mediating the WT1-induced cell cycle block. Images Fig. 1 Fig. 2 PMID:7753836
Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.
Odemuyiwa, Solomon O; Ilarraza, Ramses; Davoine, Francis; Logan, Michael R; Shayeganpour, Anooshirvan; Wu, Yingqi; Majaesic, Carina; Adamko, Darryl J; Moqbel, Redwan; Lacy, Paige
Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation.
Cyclin E and histone H3 levels are regulated by 5-fluorouracil in a DNA mismatch repair-dependent manner
Chung, Heekyung; Chaudhry, Joy; Lopez, Claudia G
Several studies indicate that the DNA mismatch repair (MMR) system may trigger cytotoxicity upon 5-fluorouracil (5-FU) recognition, but signaling pathways regulated by MMR in response to 5-FU are unknown. We hypothesize that recognition of 5-FU in DNA by MMR proteins trigger specific signaling cascades that results in slowing of the cell cycle and cell death. Whole human genome cDNA microarrays were used to examine relative signaling responses induced in MMR-proficient cells after 5-FU (5 µM) treatment for 24 hours. Analysis revealed 43 pathways differentially affected by 5-FU compared to control (p < 0.05), including cyclin and cell cycle regulation involving G1-S cell cycle transition, activation of Src, MAP K, p53 and base excision repair. In particular, 5-FU upregulated cyclins E1 and E2 (≥1.4-fold) and downregulated cdc25C, cyclins B1 and B2, histone H2A, H2B and H3 (≤-1.4-fold) over control. Cell cycle analysis revealed a G1/S arrest by 5-FU that was congruent with increased cyclin E and decreased cdc25C protein expression. Importantly, with knockdown of hMLH1 and hMSH2, we observed that decreased histone H3 expression by 5-FU was dependent on hMLH1. Additionally, 5-FU treatment dramatically decreased levels of several histone H3 modifications. Our data suggest that 5-FU induces a G1/S arrest by regulating cyclin E and cdc25C expression and MMR recognition of 5-FU in DNA may modulate cyclin E to affect the cell cycle. Furthermore, MMR recognition of 5-FU reduces histone H3 levels that could be related to DNA access by proteins and/or cell death during the G1/S phase of the cell cycle. PMID:20930505
Enrichment of Cdk1-cyclins at DNA double-strand breaks stimulates Fun30 phosphorylation and DNA end resection.
Chen, Xuefeng; Niu, Hengyao; Yu, Yang; Wang, Jingjing; Zhu, Shuangyi; Zhou, Jianjie; Papusha, Alma; Cui, Dandan; Pan, Xuewen; Kwon, Youngho; Sung, Patrick; Ira, Grzegorz
DNA double-strand breaks (DSBs) are one of the most cytotoxic types of DNA lesion challenging genome integrity. The activity of cyclin-dependent kinase Cdk1 is essential for DSB repair by homologous recombination and for DNA damage signaling. Here we identify the Fun30 chromatin remodeler as a new target of Cdk1. Fun30 is phosphorylated by Cdk1 on Serine 28 to stimulate its functions in DNA damage response including resection of DSB ends. Importantly, Cdk1-dependent phosphorylation of Fun30-S28 increases upon DNA damage and requires the recruitment of Fun30 to DSBs, suggesting that phosphorylation increases in situ at the DNA damage. Consistently, we find that Cdk1 and multiple cyclins become highly enriched at DSBs and that the recruitment of Cdk1 and cyclins Clb2 and Clb5 ensures optimal Fun30 phosphorylation and checkpoint activation. We propose that the enrichment of Cdk1-cyclin complexes at DSBs serves as a mechanism for enhanced targeting and modulating of the activity of DNA damage response proteins.
Characterization of cytoplasmic cyclin D1 as a marker of invasiveness in cancer
Santacana, Maria; Fernández-Hernández, Rita; Gatius, Sònia; Pedraza, Neus; Pallarés, Judit; Cemeli, Tània; Valls, Joan; Tarres, Marc; Ferrezuelo, Francisco; Dolcet, Xavier; Matias-Guiu, Xavier; Garí, Eloi
Cyclin D1 (Ccnd1) is a proto-oncogen amplified in many different cancers and nuclear accumulation of Ccnd1 is a characteristic of tumor cells. Ccnd1 activates the transcription of a large set of genes involved in cell cycle progress and proliferation. However, Ccnd1 also targets cytoplasmic proteins involved in the regulation of cell migration and invasion. In this work, we have analyzed by immunohistochemistry the localization of Ccnd1 in endometrial, breast, prostate and colon carcinomas with different types of invasion. The number of cells displaying membranous or cytoplasmic Ccnd1 was significantly higher in peripheral cells than in inner cells in both collective and pushing invasion patterns of endometrial carcinoma, and in collective invasion pattern of colon carcinoma. Also, the cytoplasmic localization of Ccnd1 was higher when tumors infiltrated as single cells, budding or small clusters of cells. To evaluate cytoplasmic function of cyclin D1, we have built a variant (Ccnd1-CAAX) that remains attached to the cell membrane therefore sequestering this cyclin in the cytoplasm. Tumor cells harboring Ccnd1-CAAX showed high levels of invasiveness and metastatic potential compared to those containing the wild type allele of Ccnd1. However, Ccnd1-CAAX expression did not alter proliferative rates of tumor cells. We hypothesize that the role of Ccnd1 in the cytoplasm is mainly associated with the invasive capability of tumor cells. Moreover, we propose that subcellular localization of Ccnd1 is an interesting guideline to measure cancer outcome. PMID:27105504
Targeting Cyclin-Dependent Kinases in Human Cancers: From Small Molecules to Peptide Inhibitors
Peyressatre, Marion; Prével, Camille; Pellerano, Morgan; Morris, May C.
Cyclin-dependent kinases (CDK/Cyclins) form a family of heterodimeric kinases that play central roles in regulation of cell cycle progression, transcription and other major biological processes including neuronal differentiation and metabolism. Constitutive or deregulated hyperactivity of these kinases due to amplification, overexpression or mutation of cyclins or CDK, contributes to proliferation of cancer cells, and aberrant activity of these kinases has been reported in a wide variety of human cancers. These kinases therefore constitute biomarkers of proliferation and attractive pharmacological targets for development of anticancer therapeutics. The structural features of several of these kinases have been elucidated and their molecular mechanisms of regulation characterized in depth, providing clues for development of drugs and inhibitors to disrupt their function. However, like most other kinases, they constitute a challenging class of therapeutic targets due to their highly conserved structural features and ATP-binding pocket. Notwithstanding, several classes of inhibitors have been discovered from natural sources, and small molecule derivatives have been synthesized through rational, structure-guided approaches or identified in high throughput screens. The larger part of these inhibitors target ATP pockets, but a growing number of peptides targeting protein/protein interfaces are being proposed, and a small number of compounds targeting allosteric sites have been reported. PMID:25625291
[Expression of cyclin B in megakaryocytes and cells of other hematopoietic lines].
Gu, X F; Allain, A; Li, L; Cramer, E M; Tenza, D; Caen, J P; Han, Z C
Megakaryocytes are normal bone marrow cells which have the unique ability to become polyploid. This phenomenon is termed endomitosis and its mechanism remains poorly understood at present. It is known that the cell cycle of eukaryotes, particularly at G2/M transition, is regulated by a complex with histone H1 kinase activity, the maturation- or M-phase promoting factor (MPF). We have therefore studied the expression of subunits of MPF, the p34cdc2 and cyclin B in normal bone marrow culture megakaryocytic cells, blood leukocytes and platelets as well as in human megakaryoblastic cell lines Dami, Meg-01, HEL and the promyelocytic cell line HL60. Using immunohistochemistry and electron microscopy we have observed that cyclin B was virtually undetectable in megakaryocytes and platelets, but was abundant in granulocytes, monocytes/macrophages and HL60. Studies by RT-PCR showed the presence in large quantities of mRNA of cyclin B in all cell types studied, even in megakaryocytic-like cells. These observations suggest some important implications in the understanding of the mechanisms of megakaryocyte polyploidization and related endomitosis.
Unexpected reduction of skin tumorigenesis on expression of cyclin-dependent kinase 6 in mouse epidermis.
Wang, Xian; Sistrunk, Christopher; Rodriguez-Puebla, Marcelo L
Cyclin-dependent kinases (CDKs) 4 and 6 are important regulators of the G(1) phase of the cell cycle, share 71% amino acid identity, and are expressed ubiquitously. As a result, it was assumed that each of these kinases plays a redundant role regulating normal and neoplastic proliferation. In previous reports, we have described the effects of CDK4 expression in transgenic mice, including the development of epidermal hyperplasia and increased malignant progression to squamous cell carcinoma. To study the role of CDK6 in epithelial growth and tumorigenesis, we generated transgenic mice carrying the CDK6 gene under the keratin 5 promoter (K5CDK6). Similar to K5CDK4 mice, epidermal proliferation increased substantially in K5CDK6 mice; however, no hyperplasia was observed. CDK6 overexpression also triggered keratinocyte apoptosis in interfollicular and follicular epidermis as a compensatory mechanism to override aberrant proliferation. Unexpectedly, CDK6 overexpression results in decreased skin tumor development compared with wild-type siblings. The inhibition in skin tumorigenesis was similar to that previously reported in K5-cyclin D3 mice. Furthermore, biochemical analysis of the K5CDK6 epidermis showed preferential complex formation between CDK6 and cyclin D3, suggesting that this particular complex plays an important role in tumor restraint. These studies provide in vivo evidence that CDK4 and CDK6 play a similar role as a mediator of keratinocyte proliferation but differ in apoptosis activation and skin tumor development.
Novel plant-specific cyclin-dependent kinase inhibitors induced by biotic and abiotic stresses.
Peres, Adrian; Churchman, Michelle L; Hariharan, Srivaidehirani; Himanen, Kristiina; Verkest, Aurine; Vandepoele, Klaas; Magyar, Zoltan; Hatzfeld, Yves; Van Der Schueren, Els; Beemster, Gerrit T S; Frankard, Valerie; Larkin, John C; Inzé, Dirk; De Veylder, Lieven
The EL2 gene of rice (Oryza sativa), previously classified as early response gene against the potent biotic elicitor N-acetylchitoheptaose and encoding a short polypeptide with unknown function, was identified as a novel cell cycle regulatory gene related to the recently reported SIAMESE (SIM) gene of Arabidopsis thaliana. Iterative two-hybrid screens, in vitro pull-down assays, and fluorescence resonance energy transfer analyses showed that Orysa; EL2 binds the cyclin-dependent kinase (CDK) CDKA1;1 and D-type cyclins. No interaction was observed with the plant-specific B-type CDKs. The amino acid motif ELERFL was identified to be essential for cyclin, but not for CDK binding. Orysa;EL2 impaired the ability of Orysa; CYCD5;3 to complement a budding yeast (Saccharomyces cerevisiae) triple CLN mutant, whereas recombinant protein inhibited CDK activity in vitro. Moreover, Orysa;EL2 was able to rescue the multicellular trichome phenotype of sim mutants of Arabidopsis, unequivocally demonstrating that Orysa;EL2 operates as a cell cycle inhibitor. Orysa;EL2 mRNA levels were induced by cold, drought, and propionic acid. Our data suggest that Orysa;EL2 encodes a new type of plant CDK inhibitor that links cell cycle progression with biotic and abiotic stress responses.
Cyclin A expression is under negative transcriptional control during the cell cycle.
Huet, X; Rech, J; Plet, A; Vié, A; Blanchard, J M
Transcription of the gene coding for cyclin A, a protein required for S-phase transit, is cell cycle regulated and is restricted to proliferating cells. To further explore transcriptional regulation linked to cell division cycle control, a genomic clone containing 5' flanking sequences of the murine cyclin A gene was isolated. When it was fused to a luciferase reporter gene, it was shown to function as a proliferation-regulated promoter in NIH 3T3 cells. Transcription of the mouse cyclin A gene is negatively regulated by arrest of cell proliferation. A mutation of a GC-rich sequence conserved between mice and humans is sufficient to relieve transcriptional repression, resulting in a promoter with constitutively high activity. In agreement with this result, in vivo footprinting reveals a protection of the cell cycle-responsive element in G0/early G1 cells which is not observed at later stages of the cell cycle. Moreover, the footprint is present in dimethyl sulfoxide-induced differentiating and not in proliferating Friend erythroleukemia cells. Conversely, two other sites, which in vitro bind ATF-1 and NF-Y, respectively, are constitutively occupied throughout cell cycle progression. PMID:8668196
Cyclin B2 undergoes cell cycle-dependent nuclear translocation and, when expressed as a non-destructible mutant, causes mitotic arrest in HeLa cells
Cyclin proteins form complexes with members of the p34cdc2 kinase family and they are essential components of the cell cycle regulatory machinery. They are thought to determine the timing of activation, the subcellular distribution, and/or the substrate specificity of cdc2- related kinases, but their precise mode of action remains to be elucidated. Here we report the cloning and sequencing of avian cyclin B2. Based on the use of monospecific antibodies raised against bacterially expressed protein, we also describe the subcellular distribution of cyclin B2 in chick embryo fibroblasts and in DU249 hepatoma cells. By indirect immunofluorescence microscopy we show that cyclin B2 is cytoplasmic during interphase of the cell cycle, but undergoes an abrupt translocation to the cell nucleus at the onset of mitotic prophase. Finally, we have examined the phenotypic consequences of expressing wild-type and mutated versions of avian cyclin B2 in HeLa cells. We found that expression of cyclin B2 carrying a mutation at arginine 32 (to serine) caused HeLa cells to arrest in a pseudomitotic state. Many of the arrested cells displayed multiple mitotic spindles, suggesting that the centrosome cycle had continued in spite of the cell cycle arrest. PMID:1532584
Role of Cyclin E as an Early Event in Ovarian Carcinogenesis
truncated cyclin E isoforms into our mouse model. OVCAR5 cells were transfected with 2 µg pRC- CMV -cyclin E, pcDNA3-cyclin E FL, pcDNA3-cyclin E...Imaging System (LI-COR Biotechnology, Lincoln, NB). The pRC- CMV -cyclin E construct was provided by B. Weinberg and the pcDNA3-cyclin E constructs...over-expressed in OVCAR5 cells by transfection. Whole cell lysates were collected and the protein analyzed by Western blot (Fig. 12). The pRC- CMV
Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription
Hossain, Manzar; Stillman, Bruce
Newly born cells either continue to proliferate or exit the cell division cycle. This decision involves delaying expression of Cyclin E that promotes DNA replication. ORC1, the Origin Recognition Complex (ORC) large subunit, is inherited into newly born cells after it binds to condensing chromosomes during the preceding mitosis. We demonstrate that ORC1 represses Cyclin E gene (CCNE1) transcription, an E2F1 activated gene that is also repressed by the Retinoblastoma (RB) protein. ORC1 binds to RB, the histone methyltransferase SUV39H1 and to its repressive histone H3K9me3 mark. ORC1 cooperates with SUV39H1 and RB protein to repress E2F1-dependent CCNE1 transcription. In contrast, the ORC1-related replication protein CDC6 binds Cyclin E-CDK2 kinase and in a feedback loop removes RB from ORC1, thereby hyper-activating CCNE1 transcription. The opposing effects of ORC1 and CDC6 in controlling the level of Cyclin E ensures genome stability and a mechanism for linking directly DNA replication and cell division commitment. DOI: http://dx.doi.org/10.7554/eLife.12785.001 PMID:27458800
Comprehensive phenotypic analysis of knockout mice deficient in cyclin G1 and cyclin G2
Ohno, Shouichi; Ikeda, Jun-ichiro; Naito, Yoko; Okuzaki, Daisuke; Sasakura, Towa; Fukushima, Kohshiro; Nishikawa, Yukihiro; Ota, Kaori; Kato, Yorika; Wang, Mian; Torigata, Kosuke; Kasama, Takashi; Uchihashi, Toshihiro; Miura, Daisaku; Yabuta, Norikazu; Morii, Eiichi; Nojima, Hiroshi
Cyclin G1 (CycG1) and Cyclin G2 (CycG2) play similar roles during the DNA damage response (DDR), but their detailed roles remain elusive. To investigate their distinct roles, we generated knockout mice deficient in CycG1 (G1KO) or CycG2 (G2KO), as well as double knockout mice (DKO) deficient in both proteins. All knockouts developed normally and were fertile. Generation of mouse embryonic fibroblasts (MEFs) from these mice revealed that G2KO MEFs, but not G1KO or DKO MEFs, were resistant to DNA damage insults caused by camptothecin and ionizing radiation (IR) and underwent cell cycle arrest. CycG2, but not CycG1, co-localized with γH2AX foci in the nucleus after γ-IR, and γH2AX-mediated DNA repair and dephosphorylation of CHK2 were delayed in G2KO MEFs. H2AX associated with CycG1, CycG2, and protein phosphatase 2A (PP2A), suggesting that γH2AX affects the function of PP2A via direct interaction with its B'γ subunit. Furthermore, expression of CycG2, but not CycG1, was abnormal in various cancer cell lines. Kaplan–Meier curves based on TCGA data disclosed that head and neck cancer patients with reduced CycG2 expression have poorer clinical prognoses. Taken together, our data suggest that reduced CycG2 expression could be useful as a novel prognostic marker of cancer. PMID:27982046
Consequence of the tumor-associated conversion to cyclin D1b
Augello, Michael A; Berman-Booty, Lisa D; Carr, Richard; Yoshida, Akihiro; Dean, Jeffry L; Schiewer, Matthew J; Feng, Felix Y; Tomlins, Scott A; Gao, Erhe; Koch, Walter J; Benovic, Jeffrey L; Diehl, John Alan; Knudsen, Karen E
Clinical evidence suggests that cyclin D1b, a variant of cyclin D1, is associated with tumor progression and poor outcome. However, the underlying molecular basis was unknown. Here, novel models were created to generate a genetic switch from cyclin D1 to cyclin D1b. Extensive analyses uncovered overlapping but non-redundant functions of cyclin D1b compared to cyclin D1 on developmental phenotypes, and illustrated the importance of the transcriptional regulatory functions of cyclin D1b in vivo. Data obtained identify cyclin D1b as an oncogene, wherein cyclin D1b expression under the endogenous promoter induced cellular transformation and further cooperated with known oncogenes to promote tumor growth in vivo. Further molecular interrogation uncovered unexpected links between cyclin D1b and the DNA damage/PARP1 regulatory networks, which could be exploited to suppress cyclin D1b-driven tumors. Collectively, these data are the first to define the consequence of cyclin D1b expression on normal cellular function, present evidence for cyclin D1b as an oncogene, and provide pre-clinical evidence of effective methods to thwart growth of cells dependent upon this oncogenic variant. PMID:25787974
Cell cycle–regulated phosphorylation of p220NPAT by cyclin E/Cdk2 in Cajal bodies promotes histone gene transcription
Ma, Tianlin; Van Tine, Brian A.; Wei, Yue; Garrett, Michelle D.; Nelson, David; Adams, Peter D.; Wang, Jin; Qin, Jun; Chow, Louise T.; Harper, J. Wade
Cyclin E/Cdk2 acts at the G1/S-phase transition to promote the E2F transcriptional program and the initiation of DNA synthesis. To explore further how cyclin E/Cdk2 controls S-phase events, we examined the subcellular localization of the cyclin E/Cdk2 interacting protein p220NPAT and its regulation by phosphorylation. p220 is localized to discrete nuclear foci. Diploid fibroblasts in Go and G1 contain two p220 foci, whereas S- and G2-phase cells contain primarily four p220 foci. Cells in metaphase and telophase have no detectable focus. p220 foci contain cyclin E and are coincident with Cajal bodies (CBs), subnuclear organelles that associate with histone gene clusters on chromosomes 1 and 6. Interestingly, p220 foci associate with chromosome 6 throughout the cell cycle and with chromosome 1 during S phase. Five cyclin E/Cdk2 phosphorylation sites in p220 were identified. Phospho-specific antibodies against two of these sites react with p220 within CBs in a cell cycle–specific manner. The timing of p220 phosphorylation correlates with the appearance of cyclin E in CBs at the G1/S boundary, and this phosphorylation is maintained until prophase. Expression of p220 activates transcription of the histone H2B promoter. Importantly, mutation of Cdk2 phosphorylation sites to alanine abrogates the ability of p220 to activate the histone H2B promoter. Collectively, these results strongly suggest that p220NPAT links cyclical cyclin E/Cdk2 kinase activity to replication-dependent histone gene transcription. PMID:10995387
Akt-mediated liver growth promotes induction of cyclin E through a novel translational mechanism and a p21-mediated cell cycle arrest.
Mullany, Lisa K; Nelsen, Christopher J; Hanse, Eric A; Goggin, Melissa M; Anttila, Chelsea K; Peterson, Mark; Bitterman, Peter B; Raghavan, Arvind; Crary, Gretchen S; Albrecht, Jeffrey H
The control of hepatocyte growth is relevant to the processes of liver regeneration, development, metabolic homeostasis, and cancer. A key component of growth control is the protein kinase Akt, which acts downstream of mitogens and nutrients to affect protein translation and cell cycle progression. In this study, we found that transient transfection of activated Akt triggered a 3-4-fold increase in liver size within days but only minimal hepatocyte proliferation. Akt-induced liver growth was associated with marked up-regulation of cyclin E but not cyclin D1. Analysis of liver polyribosomes demonstrated that the post-transcriptional induction of cyclin E was associated with increased translational efficiency of this mRNA, suggesting that cell growth promotes expression of this protein through a translational mechanism that is distinct from the cyclin D-E2F pathway. Treatment of Akt-transfected mice with rapamycin only partially inhibited liver growth and did not prevent the induction of cyclin E protein, indicating that target of rapamycin activity is not necessary for this response. In the enlarged livers, cyclin E-Cdk2 complexes were present in high abundance but were inactive due to increased binding of p21 to these complexes. Akt transfection of p21(-/-) mice promoted liver growth, activation of Cdk2, and enhanced hepatocyte proliferation. In conclusion, growth promotes cyclin E expression through a novel translational mechanism in the liver, suggesting a new link between cell growth and the cell cycle machinery. Furthermore, p21 suppresses proliferation in the overgrown livers and may play a role in preventing cell cycle progression in response to organ size homeostatic mechanisms.
Modulation of p53, c-fos, RARE, cyclin A, and cyclin D1 expression in human leukemia (HL-60) cells exposed to arsenic trioxide
Yedjou, Clement G.; Tchounwou, Paul B.
Arsenic trioxide (As2O3) has recently been successfully used to treat all-trans retinoic acid (ATRA) resistant relapsing acute promyelocytic leukemia. However, its molecular mechanisms of action are poorly understood. In the present study, we used the human leukemia (HL-60) cell line as a test model to study the cellular and molecular mechanisms of anti-cancer properties of As2O3. We hypothesized that As2O3-induced expression of stress genes and related proteins may play a role in the cellular and molecular events leading to cell cycle modulation in leukemic cells. To test this hypothesis, we performed Western blot analysis to assess the expression of specific cellular response proteins including p53, c-fos, RARE, Cyclin A, and Cyclin D1. Densitometric analysis was performed to determine the relative abundance of these proteins. Western Blot and densitometric analyses demonstrated a strong dose-response relationship with regard to p53 and RARE expression within the dose range of 0-8μg/mL. Expression of c-fos was slightly up-regulated at 2μg/mL, and down-regulated within the dose-range of 4-8 μg/mL. A statistically significant down-regulation of this protein was detected at the 6 and 8 μg/mL dose levels. No statistically significant differences (p>0.05) in Cyclin D1 expression was found between As2O3-treated cells and the control. Cyclin A expression in As2O3-treated HL-60 cells was up-regulated at 6μg/mL, suggesting that it is required for S phase and passage through G2 phase in cell cycle progression. Taken together, these results indicate that As2O3 has the potential to induce cell cycle arrest through activation of the 53-kDa tumor suppressor protein and repression of the c-fos transcription factor. Up-regulation of RARE by As2O3 indicates that its cytotoxicity may be mediated through interaction/binding with the retinoic acid receptor, and subsequent inhibition of growth and differentiation. PMID:19444595
RhoA promotes epidermal stem cell proliferation via PKN1-cyclin D1 signaling
Wang, Fan; Zhan, Rixing; Chen, Liang; Dai, Xia; Wang, Wenping; Guo, Rui; Li, Xiaoge; Li, Zhe; Wang, Liang; Huang, Shupeng; Shen, Jie
Objective Epidermal stem cells (ESCs) play a critical role in wound healing, but the mechanism underlying ESC proliferation is not well defined. Here, we explore the effects of RhoA on ESC proliferation and the possible underlying mechanism. Methods Human ESCs were enriched by rapid adhesion to collagen IV. RhoA(+/+)(G14V), RhoA(-/-)(T19N) and pGFP control plasmids were transfected into human ESCs. The effect of RhoA on cell proliferation was detected by cell proliferation and DNA synthesis assays. Induction of PKN1 activity by RhoA was determined by immunoblot analysis, and the effects of PKN1 on RhoA in terms of inducing cell proliferation and cyclin D1 expression were detected using specific siRNA targeting PKN1. The effects of U-46619 (a RhoA agonist) and C3 transferase (a RhoA antagonist) on ESC proliferation were observed in vivo. Results RhoA had a positive effect on ESC proliferation, and PKN1 activity was up-regulated by the active RhoA mutant (G14V) and suppressed by RhoA T19N. Moreover, the ability of RhoA to promote ESC proliferation and DNA synthesis was interrupted by PKN1 siRNA. Additionally, cyclin D1 protein and mRNA expression levels were up-regulated by RhoA G14V, and these effects were inhibited by siRNA-mediated knock-down of PKN1. RhoA also promoted ESC proliferation via PKN in vivo. Conclusion This study shows that the effect of RhoA on ESC proliferation is mediated by activation of the PKN1-cyclin D1 pathway in vitro, suggesting that RhoA may serve as a new therapeutic target for wound healing. PMID:28222172
Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage
Ju, Xiaoming; Vetuschi, Antonella; Sferra, Roberta; Casimiro, Mathew C.; Pompili, Simona; Festuccia, Claudio; Colapietro, Alessandro; Gaudio, Eugenio; Di Cesare, Ernesto; Tombolini, Vincenzo; Pestell, Richard G.
Patients with hormone-resistant prostate cancer (PCa) have higher biochemical failure rates following radiation therapy (RT). Cyclin D1 deregulated expression in PCa is associated with a more aggressive disease: however its role in radioresistance has not been determined. Cyclin D1 levels in the androgen-independent PC3 and 22Rv1 PCa cells were stably inhibited by infecting with cyclin D1-shRNA. Tumorigenicity and radiosensitivity were investigated using in vitro and in vivo experimental assays. Cyclin D1 silencing interfered with PCa oncogenic phenotype by inducing growth arrest in the G1 phase of cell cycle and reducing soft agar colony formation, migration, invasion in vitro and tumor formation and neo-angiogenesis in vivo. Depletion of cyclin D1 significantly radiosensitizes PCa cells by increasing the RT-induced DNA damages by affecting the NHEJ and HR pathways responsible of the DNA double-strand break repair. Following treatment of cells with RT the abundance of a biomarker of DNA damage, γ-H2AX, was dramatically increased in sh-cyclin D1 treated cells compared to shRNA control. Concordant with these observations DNA-PKcs-activation and RAD51-accumulation, part of the DNA double-strand break repair machinery, were reduced in shRNA-cyclin D1 treated cells compared to shRNA control. We further demonstrate the physical interaction between CCND1 with activated-ATM, -DNA-PKcs and RAD51 is enhanced by RT. Finally, siRNA-mediated silencing experiments indicated DNA-PKcs and RAD51 are downstream targets of CCND1-mediated PCa cells radioresistance. In summary, these observations suggest that CCND1 is a key mediator of PCa radioresistance and could represent a potential target for radioresistant hormone-resistant PCa. PMID:26689991
Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage.
Marampon, Francesco; Gravina, Giovanni; Ju, Xiaoming; Vetuschi, Antonella; Sferra, Roberta; Casimiro, Mathew C; Pompili, Simona; Festuccia, Claudio; Colapietro, Alessandro; Gaudio, Eugenio; Di Cesare, Ernesto; Tombolini, Vincenzo; Pestell, Richard G
Patients with hormone-resistant prostate cancer (PCa) have higher biochemical failure rates following radiation therapy (RT). Cyclin D1 deregulated expression in PCa is associated with a more aggressive disease: however its role in radioresistance has not been determined. Cyclin D1 levels in the androgen-independent PC3 and 22Rv1 PCa cells were stably inhibited by infecting with cyclin D1-shRNA. Tumorigenicity and radiosensitivity were investigated using in vitro and in vivo experimental assays. Cyclin D1 silencing interfered with PCa oncogenic phenotype by inducing growth arrest in the G1 phase of cell cycle and reducing soft agar colony formation, migration, invasion in vitro and tumor formation and neo-angiogenesis in vivo. Depletion of cyclin D1 significantly radiosensitizes PCa cells by increasing the RT-induced DNA damages by affecting the NHEJ and HR pathways responsible of the DNA double-strand break repair. Following treatment of cells with RT the abundance of a biomarker of DNA damage, γ-H2AX, was dramatically increased in sh-cyclin D1 treated cells compared to shRNA control. Concordant with these observations DNA-PKcs-activation and RAD51-accumulation, part of the DNA double-strand break repair machinery, were reduced in shRNA-cyclin D1 treated cells compared to shRNA control. We further demonstrate the physical interaction between CCND1 with activated-ATM, -DNA-PKcs and RAD51 is enhanced by RT. Finally, siRNA-mediated silencing experiments indicated DNA-PKcs and RAD51 are downstream targets of CCND1-mediated PCa cells radioresistance. In summary, these observations suggest that CCND1 is a key mediator of PCa radioresistance and could represent a potential target for radioresistant hormone-resistant PCa.
Transcriptional regulation of the cyclin D1 promoter by STAT5: its involvement in cytokine-dependent growth of hematopoietic cells.
Matsumura, I; Kitamura, T; Wakao, H; Tanaka, H; Hashimoto, K; Albanese, C; Downward, J; Pestell, R G; Kanakura, Y
STAT5 is a member of a family of transcription factors that participate in the signal transduction pathways of many hormones and cytokines. Although STAT5 is suggested to play a crucial role in the biological effects of cytokines, its downstream target(s) associated with cell growth control is largely unknown. In a human interleukin-3 (IL-3)-dependent cell line F-36P-mpl, the induced expression of dominant-negative (dn)-STAT5 and of dn-ras led to inhibition of IL-3-dependent cell growth, accompanying the reduced expression of cyclin D1 mRNA. Also, both constitutively active forms of STAT5A (1*6-STAT5A) and ras (H-rasG12V) enabled F-36P-mpl cells to proliferate without added growth factors. In NIH 3T3 cells, 1*6-STAT5A and H-rasG12V individually and cooperatively transactivated the cyclin D1 promoter in luciferase assays. Both dn-STAT5 and dn-ras suppressed IL-3-induced cyclin D1 promoter activities in F-36P-mpl cells. Using a series of mutant cyclin D1 promoters, 1*6-STAT5A was found to transactivate the cyclin D1 promoter through the potential STAT-binding sequence at -481 bp. In electrophoretic mobility shift assays, STAT5 bound to the element in response to IL-3. Furthermore, the inhibitory effect of dn-STAT5 on IL-3-dependent growth was restored by expression of cyclin D1. Thus STAT5, in addition to ras signaling, appears to mediate transcriptional regulation of cyclin D1, thereby contributing to cytokine-dependent growth of hematopoietic cells. PMID:10064602
Expression of δ-cyclins of Brassica rapa L. embryos by clinorotation
NASA Astrophysics Data System (ADS)
Artemenko, O. A.
Cyclins is one of the important regulators of cell cycle. There are several types of cyclins exists. They are responding for different phases of cycle and have high homology in plant's and mammalian's cells. δ -cyclins are specific for plants and controlling the presynthetic phase events. These cyclins likes to mammalian D-cyclins and have similar functions. This class consist three types of cyclins -- δ 1, δ 2 and δ 3. Cyclin δ 1 is responding for events in cell, which take place before exiting from stage of quiet (G0). Cyclin δ 1 is responding for entering and outputting from G0, and cyclin δ 3 -- for events, which happen in cell after stage of quiet, by entering to S-phase (phase of DNA's synthesis). In present research was used δ 1- and δ 3-cyclins. For determination of δ -cyclins gene's expression level was excreted RNA from embryos: 3-days (spherical stage), 6-days (heart-shaped stage) and 9-days (generated stage) seedlings of Brassica rapa L. in control and under clinorotation. For definition the cyclins gene's expression level applied Northern Blot Analysis. Obtained data testify about difference in level of gene's expression of cyclin δ 1 between control and clinorotation variants. After three days by pollination the expression of this gene in embryos was observed in control only. By clinorotation the gene's expression was detected on 6 days later, but it level was lower than in control variant. On 9 days it was gently expressed by clinorotation, where as by control it was not detected absolutely. Cyclin δ 3 gene's expression was observed during all time of the experiment. These data also confirm known one about expression δ 1- cyclin, which expressed on beginning of cell cycle only. And δ 3 --cyclin that express during whole presinthetic phase of cell cycle (Sony et al., 1995, Murray, 1994, Inze et al, 1999, Umeda, 2000).
Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis
Bauer, Andrea; Mylroie, Hayley; Thornton, C. Clare; Calay, Damien; Birdsey, Graeme M.; Kiprianos, Allan P.; Wilson, Garrick K.; Soares, Miguel P.; Yin, Xiaoke; Mayr, Manuel; Randi, Anna M.; Mason, Justin C.
Angiogenesis is an essential physiological process and an important factor in disease pathogenesis. However, its exploitation as a clinical target has achieved limited success and novel molecular targets are required. Although heme oxygenase-1 (HO-1) acts downstream of vascular endothelial growth factor (VEGF) to modulate angiogenesis, knowledge of the mechanisms involved remains limited. We set out identify novel HO-1 targets involved in angiogenesis. HO-1 depletion attenuated VEGF-induced human endothelial cell (EC) proliferation and tube formation. The latter response suggested a role for HO-1 in EC migration, and indeed HO-1 siRNA negatively affected directional migration of EC towards VEGF; a phenotype reversed by HO-1 over-expression. EC from Hmox1−/− mice behaved similarly. Microarray analysis of HO-1-depleted and control EC exposed to VEGF identified cyclins A1 and E1 as HO-1 targets. Migrating HO-1-deficient EC showed increased p27, reduced cyclin A1 and attenuated cyclin-dependent kinase 2 activity. In vivo, cyclin A1 siRNA inhibited VEGF-driven angiogenesis, a response reversed by Ad-HO-1. Proteomics identified structural protein vimentin as an additional VEGF-HO-1 target. HO-1 depletion inhibited VEGF-induced calpain activity and vimentin cleavage, while vimentin silencing attenuated HO-1-driven proliferation. Thus, vimentin and cyclins A1 and E1 represent VEGF-activated HO-1-dependent targets important for VEGF-driven angiogenesis. PMID:27388959
Phylogenetic analysis reveals the evolution and diversification of cyclins in eukaryotes.
Ma, Zhaowu; Wu, Yuliang; Jin, Jialu; Yan, Jun; Kuang, Shuzhen; Zhou, Mi; Zhang, Yuexuan; Guo, An-Yuan
Cyclins are a family of diverse proteins that play fundamental roles in regulating cell cycle progression in Eukaryotes. Cyclins have been identified from protists to higher Eukaryotes, while its evolution remains vague and the findings turn out controversial. Current classification of cyclins is mainly based on their functions, which may not be appropriate for the systematic evolutionary analysis. In this work, we performed comparative and phylogenetic analysis of cyclins to investigate their classification, origin and evolution. Cyclins originated in early Eukaryotes and evolved from protists to plants, fungi and animals. Based on the phylogenetic tree, cyclins can be divided into three major groups designated as the group I, II and III with different functions and features. Group I plays key roles in cell cycle, group II varied in actions are kingdom (plant, fungi and animal) specific, and group III functions in transcription regulation. Our results showed that the dominating cyclins (group I) diverged from protists to plants, fungi and animals, while divergence of the other cyclins (groups II and III) has occurred in protists. We also discussed the evolutionary relationships between cyclins and cyclin-dependent kinases (CDKs) and found that the cyclins have undergone divergence in protists before the divergence of animal CDKs. This reclassification and evolutionary analysis of cyclins might facilitate understanding eukaryotic cell cycle control.
Overexpression of cyclins D1 and D3 during estrogen-induced breast oncogenesis in female ACI rats.
Weroha, S John; Li, Sara Antonia; Tawfik, Ossama; Li, Jonathan J
A common feature of human breast oncogenesis is cell cycle deregulation. The expression of cyclins D1 and D3 was examined during estradiol-17beta (E(2))-induced mammary tumorigenesis in female August Copenhagen Irish (ACI) rats. Low serum E(2) levels ( approximately 60-120 pg/ml) were sufficient to induce mammary gland tumors (MGTs) that remarkably resemble human ductal breast cancer (BC) at the histopathologic and molecular levels. Western blot analysis of the E(2)-induced MGTs revealed a marked rise in cyclins D1 (24-fold), D3 (9-fold) and cdk4 (3-fold) expression compared with age-matched untreated controls. Small focal dysplasias with large, pale staining nuclei were commonly seen at 3-3.6 months, large focal dysplasias, including atypical ductal hyperplasia at 3.6-4.3 months, ductal carcinoma in-situ (DCISs) at 4.3-5.0 months, and 100% incidence of invasive ductal BC/frank tumors at 5-6 months were detected after E(2) treatment. Immunohistochemical analysis of serial sections of focal dysplasias, DCISs and invasive ductal carcinomas showed overexpression of cyclins D1, D3, estrogen receptor-alpha (ERalpha) and progesterone receptor (PR). However, cyclin D3 expression, unlike D1, was confined essentially to early pre-malignant lesions (focal dysplasias and DCISs) and primary MGTs with <1-5% of resting and normal hyperplastic breast cells staining positive. The kinase activity for cyclins D1 and D3, using retinoblastoma (Rb) as a substrate, in E(2)-induced MGTs and their binding to cdk4 was significantly elevated. Semi-quantitative reverse transcriptase PCR analysis of the E(2)-induced MGTs exhibited increased expression of cyclins D1 (2.9-fold) and D3 (1.4-fold) mRNA, indicating that their elevated protein expression was due in part to an increase in mRNA transcription. However, when analyzed by quantitative real-time Q-PCR, these genes were not amplified. These data indicate that in female ACI rat mammary glands, E(2)-induced pre-malignant lesions
Effect of growth factors and steroid hormones on heme oxygenase and cyclin D1 expression in primary astroglial cell cultures.
Bramanti, V; Grasso, S; Tomassoni, D; Traini, E; Raciti, G; Viola, M; Li Volti, G; Campisi, A; Amenta, F; Avola, R
Astrocyte activity may be modulated by steroid hormones and GFs. This study investigates the interaction between glucocorticoids or estrogens and GFs on the expression of heme oxygenase-1 (HO-1) and cyclin D1 in astrocyte cultures at 14 days treated for 48 or 60 hr with dexamethasone (DEX) or 48 hr with 17β-estradiol (E2) alone or with GFs added only in the last 12 or 24 hr. Twelve- or twenty-four-hour epidermal growth factor (EGF) treatment significantly enhanced HO-1 expression in astrocyte cultures pretreated for 48 hr with DEX. A highly significant increase in HO-1 expression was obtained after the last-12-hr EGF treatment in 48-hr E2-pretreated astrocyte cultures; this enhancement was particularly significant in 48-hr E2-pretreated cultures as well as in the last-12-hr insulin-treated ones pretreated for 48 hr with E2. Sixty-hour DEX-alone pretreatment as well as the last-12-hr EGF treatment in 60-hr DEX-pretreated astrocyte cultures showed a significant increase of cyclin D1 expression. A significant decrease of cyclin D1 expression in the last-12-hr insulin-like growth factor-I (IGF-1)-treated cultures pretreated for 60 hr with DEX was observed. A highly significant enhancement in cyclin D1 expression in 14 days in vitro astrocyte cultures pretreated with E2 alone for 48 hr and treated in the last 12 hr with IGF-1 in 48-hr E2-pretreated cultures was found. Finally, the data highlight an interactive dialogue between the growth factors and glucocorticoids or estrogens during the maturation of astroglial cells in culture that may control the HO-1 and cyclin D1 expression as well as proliferating astroglial cells during the cell cycle.
Inhibition of cyclin D1 expression by androgen receptor in breast cancer cells--identification of a novel androgen response element.
Lanzino, Marilena; Sisci, Diego; Morelli, Catia; Garofalo, Cecilia; Catalano, Stefania; Casaburi, Ivan; Capparelli, Claudia; Giordano, Cinzia; Giordano, Francesca; Maggiolini, Marcello; Andò, Sebastiano
Cyclin D1 gene (CCND1) is a critical mitogen-regulated cell-cycle control element whose transcriptional modulation plays a crucial role in breast cancer growth and progression. Here we demonstrate that the non-aromatizable androgen 5-α-dihydrotestosterone (DHT) inhibits endogenous cyclin D1 expression, as evidenced by reduction of cyclin D1 mRNA and protein levels, and decrease of CCND1-promoter activity, in MCF-7 cells. The DHT-dependent inhibition of CCND1 gene activity requires the involvement and the integrity of the androgen receptor (AR) DNA-binding domain. Site directed mutagenesis, DNA affinity precipitation assay, electrophoretic mobility shift assay and chromatin immunoprecipitation analyses indicate that this inhibitory effect is ligand dependent and it is mediated by direct binding of AR to an androgen response element (CCND1-ARE) located at -570 to -556-bp upstream of the transcription start site, in the cyclin D1 proximal promoter. Moreover, AR-mediated repression of the CCND1 involves the recruitment of the atypical orphan nuclear receptor DAX1 as a component of a multiprotein repressor complex also embracing the participation of Histone Deacetylase 1. In conclusion, identification of the CCND1-ARE allows defining cyclin D1 as a specific androgen target gene in breast and might contribute to explain the molecular basis of the inhibitory role of androgens on breast cancer cells proliferation.
Cyclin A2-cyclin-dependent kinase 2 cooperates with the PLK1-SCFbeta-TrCP1-EMI1-anaphase-promoting complex/cyclosome axis to promote genome reduplication in the absence of mitosis.
Ma, Hoi Tang; Tsang, Yiu Huen; Marxer, Miriam; Poon, Randy Y C
Limiting genome replication to once per cell cycle is vital for maintaining genome stability. Inhibition of cyclin-dependent kinase 1 (CDK1) with the specific inhibitor RO3306 is sufficient to trigger multiple rounds of genome reduplication. We demonstrated that although anaphase-promoting complex/cyclosome (APC/C) remained inactive during the initial G(2) arrest, it was activated upon prolonged inhibition of CDK1. Using cellular biosensors and live-cell imaging, we provide direct evidence that genome reduplication was associated with oscillation of APC/C activity and nuclear-cytoplasmic shuttling of CDC6 even in the absence of mitosis at the single-cell level. Genome reduplication was abolished by ectopic expression of EMI1 or depletion of CDC20 or CDH1, suggesting the critical role of the EMI1-APC/C axis. In support of this, degradation of EMI1 itself and genome reduplication were delayed after downregulation of PLK1 and beta-TrCP1. In the absence of CDK1 activity, activation of APC/C and genome reduplication was dependent on cyclin A2 and CDK2. Genome reduplication was then promoted by a combination of APC/C-dependent destruction of geminin (thus releasing CDT1), accumulation of cyclin E2-CDK2, and CDC6. Collectively, these results underscore the crucial role of cyclin A2-CDK2 in regulating the PLK1-SCF(beta-TrCP1)-EMI1-APC/C axis and CDC6 to trigger genome reduplication after the activity of CDK1 is suppressed.
Action of the p53 Effector, p21, on its Targets: Cyclin-cdk and PCNA
Lineweaver - Burke analysis of the inhibition of cyclin E-cdk2 by intact p21 and by p21 without the cyclin binding site. Task 2: Months 24-36: Determination...the peptide sequence was the critical feature of a Cy motif. Task 1: Months 1-24: Lineweaver - Burke analysis of the inhibition of cyclin E-cdk2 by...intact p21 and by p21 without the cyclin binding site. p21 without a Cy motif was ineffective in inhibiting cyclin E-cdk2 [2]. Lineweaver - Burke analysis
p14ARF post-transcriptional regulation of nuclear cyclin D1 in MCF-7 breast cancer cells: discrimination between a good and bad prognosis?
McGowan, Eileen M; Tran, Nham; Alling, Nikki; Yagoub, Daniel; Sedger, Lisa M; Martiniello-Wilks, Rosetta
As part of a cell's inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-β-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation), and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in
Cdh1-APC/C, cyclin B-Cdc2, and Alzheimer's disease pathology
Aulia, Selina; Tang, Bor Luen . E-mail: firstname.lastname@example.com
The anaphase-promoting complex/cyclosome (APC/C) is a key E3 ubiquitin ligase complex that functions in regulating cell cycle transitions in proliferating cells and has, as revealed recently, novel roles in postmitotic neurons. Regulated by its activator Cdh1 (or Hct1), whose level is high in postmitotic neurons, APC/C seems to have multiple functions at different cellular locations, modulating diverse processes such as synaptic development and axonal growth. These processes do not, however, appear to be directly connected to cell cycle regulation. It is now shown that Cdh1-APC/C activity may also have a basic role in suppressing cyclin B levels, thus preventing terminally differentiated neurons from aberrantly re-entering the cell cycle. The result of an aberrant cyclin B-induced S-phase entry, at least for some of these neurons, would be death via apoptosis. Cdh1 thus play an active role in maintaining the terminally differentiated, non-cycling state of postmitotic neurons-a function that could become impaired in Alzheimer's and other neurodegenerative diseases.
Overexpression of serum amyloid A-activating factor 1 inhibits cell proliferation by the induction of cyclin-dependent protein kinase inhibitor p21WAF-1/Cip-1/Sdi-1 expression.
Ray, Alpana; Shakya, Arvind; Kumar, Deepak; Ray, Bimal K
Inflammation-responsive transcription factor, serum amyloid A-activating factor 1 (SAF-1), has been shown to regulate several genes, including serum amyloid A, gamma-fibrinogen, and matrix metalloproteinase 1, whose abnormal expression is associated with the pathogenesis of arthritis, atherosclerosis, and amyloidosis. Prolonged high level expression of SAF-1 in cultured cells failed to produce any stable cell line that overexpresses SAF-1. To test the fate of SAF-1-overexpressing cells, the cells were monitored for growth and morphological changes over time. The cells that were programmed to overproduce SAF-1 were found to undergo growth arrest and reduce DNA synthesis within 3 days after transfection. These cells undergo marked morphological changes from typical fibroblasts to round morphology and gradually cease to exist. Microarray analysis for cell cycle-specific genes in SAF1-transfected cells identified several candidate genes whose expression levels were altered during SAF-1 overexpression. Cdk inhibitor protein p21 was significantly affected by SAF-1; its expression level was highly induced by cellular conditions where SAF-1 is abundant. The increased level of p21 in the cell drives it to a growth arrest mode, a condition previously found to be controlled by p53. In this study we provide evidence that, similar to p53, SAF-1 is able to activate p21 gene expression by promoting transcription directly via its interaction with the p21 promoter. Together these data indicate that SAF-1 controls cell cycle progression via p21 induction, and pathophysiological conditions that favor overexpression of SAF-1, such as an acute inflammatory condition, can trigger cellular growth arrest.
QKI-5 suppresses cyclin D1 expression and proliferation of oral squamous cell carcinoma cells via MAPK signalling pathway.
Fu, X; Feng, Y
Oral squamous cell carcinoma (OSCC) is one of the most frequently occurring malignancies in the world. The RNA-binding protein quaking (QKI) is a newly identified tumour suppressor in multiple cancers, but its role in OSCC is currently unknown. The purpose of the present study was to clarify the relationship between QKI expression and OSCC development. We found QKI-5 expression to be significantly decreased in the oral cancer cell line CAL-27. QKI-5 overexpression also reduced the proliferation of CAL-27 cells, which correlated with cyclin D1. This regulative function of QKI-5 occurs by modulating the phosphorylation level of the mitogen-activated protein kinase (MAPK) pathway. Therefore this study shows that underexpression of tumour suppressor QKI-5 could activate the MAPK pathway and contribute to uncontrolled cyclin D1 expression, thus resulting in increased proliferation of oral cancer cells.
2,3,7,8-Tetrachlorodibenzo-p-dioxin stimulates proliferation of HAPI microglia by affecting the Akt/GSK-3β/cyclin D1 signaling pathway.
Xu, Guangfei; Li, Yuanye; Yoshimoto, Katsuhiko; Wu, Qiyun; Chen, Gang; Iwata, Takeo; Mizusawa, Noriko; Wan, Chunhua; Nie, Xiaoke
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxin that induces apoptosis of neurons and a pro-inflammatory response in microglial cells. First, we found that TCDD induced proliferation of HAPI microglial cells in a dose- and time-dependent manner. Flow cytometry analysis showed that this proliferation by TCDD was due to mainly enhancing the G1 to S phase transition. Next, it was found that TCDD treatment led to up-regulation of cyclin D1, which induces cell cycle progression from G1 to S phase, in a time-dependent manner. As for molecular mechanism, we revealed that TCDD was capable of inducing Akt phosphorylation and activation, resulting in phosphorylation and inactivation of glycogen synthase kinase-3β (GSK-3β). Inactivated GSK-3β attenuated proteasomal degradation of cyclin D1 by reducing Thr(286)-phosphorylated cyclin D1 levels. Moreover, inactivated GSK-3β increased cyclin D1 gene transcription by increasing its transcription factor β-catenin in the nucleus. Further, blockage of phosphoinositide 3-kinase/Akt kinase with their specific inhibitors, LY294002 and Akt 1/2 kinase inhibitor, significantly reduced TCDD-enhanced proliferation of HAPI microglial cells. In conclusion, TCDD stimulates proliferation of HAPI microglial cells by affecting the Akt/GSK-3β/cyclin D1 signaling pathway.
Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase
Townsley, Fiona M.; Aristarkhov, Alexander; Beck, Sharon; Hershko, Avram; Ruderman, Joan V.
Destruction of mitotic cyclins by ubiquitin-dependent proteolysis is required for cells to complete mitosis and enter interphase of the next cell cycle. In clam eggs, this process is catalyzed by a cyclin-selective ubiquitin carrier protein, E2-C, and the cyclosome/anaphase promoting complex (APC), a 20S particle containing cyclin-selective ubiquitin ligase activity. Here we report cloning a human homolog of E2-C, UbcH10, which shares 61% amino acid identity with clam E2-C and can substitute for clam E2-C in vitro. Dominant-negative clam E2-C and human UbcH10 proteins, created by altering the catalytic cysteine to serine, inhibit the in vitro ubiquitination and destruction of cyclin B in clam oocyte extracts. When transfected into mammalian cells, mutant UbcH10 inhibits the destruction of both cyclin A and B, arrests cells in M phase, and inhibits the onset of anaphase, presumably by blocking the ubiquitin-dependent proteolysis of proteins responsible for sister chromatid separation. Thus, E2-C/UbcH10-mediated ubiquitination is involved in both cdc2 inactivation and sister chromatid separation, processes that are normally coordinated during exit from mitosis. PMID:9122200
Lack of cyclin D-Cdk complexes in Rb-negative cells correlates with high levels of p16INK4/MTS1 tumour suppressor gene product.
Parry, D; Bates, S; Mann, D J; Peters, G
D-type cyclins, in association with the cyclin-dependent kinases Cdk4 or Cdk6, regulate events in the G1 phase of the cell cycle and may contribute to the phosphorylation of the retinoblastoma gene product (Rb). However, in cells in which the function of Rb has been compromised, either by naturally arising mutations or through binding to proteins encoded by DNA tumour viruses, Cdk4 and Cdk6 are not associated with D cyclins. Instead, both kinases form binary complexes with a stable 16 kDa protein (p16) encoded by the putative tumour suppressor gene INK4/MTS1 on human chromosome 9p21. Here we show an inverse correlation between Rb status and the expression of p16. Since Rb-negative cells express high levels of p16, we suggest that in these cells p16 competes with D cyclins for binding to Cdk4 and Cdk6 and prevents formation of active complexes. In line with these predictions, DNA tumour virus oncoproteins do not disrupt cyclin D1-Cdk4 complexes in cells lacking p16. Images PMID:7859739
B-Cyclin/CDKs Regulate Mitotic Spindle Assembly by Phosphorylating Kinesins-5 in Budding Yeast
Chee, Mark K.; Haase, Steven B.
Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCFCdc4 ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APCCdh1). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCFCdc4 target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors. PMID:20463882
The Cell Wall Sensors Mtl1, Wsc1, and Mid2 Are Required for Stress-Induced Nuclear to Cytoplasmic Translocation of Cyclin C and Programmed Cell Death in Yeast
Jin, Chunyan; Parshin, Andrey V.; Daly, Ira; Strich, Randy; Cooper, Katrina F.
Mtl1 is a member of a cell wall sensor family that monitors cell wall integrity in budding yeast. In response to cell wall stress, Mtl1 activates the cell wall integrity (CWI) MAP kinase pathway which transmits this signal to the nucleus to effect changes in gene expression. One target of the CWI MAP kinase is cyclin C, a negative regulator of stress response genes. CWI activation results in cyclin C relocalization from the nucleus to the cytoplasm where it stimulates programmed cell death (PCD) before it is destroyed. This report demonstrates that under low oxidative stress conditions, a combination of membrane sensors, Mtl1 and either Wsc1 or Mid2, are required jointly to transmit the oxidative stress signal to initiate cyclin C destruction. However, when exposed to elevated oxidative stress, additional pathways independent of these three sensor proteins are activated to destroy cyclin C. In addition, N-glycosylation is important for Mtl1 function as mutating the receptor residue (Asn42) or an enzyme required for synthesis of N-acetylglucosamine (Gfa1) reduces sensor activity. Finally, combining gfa1-1 with the cyclin C null allele induces a severe synthetic growth defect. This surprising result reveals a previously unknown genetic interaction between cyclin C and plasma membrane integrity. PMID:24260614
Pharmacological cyclin dependent kinase inhibitors: Implications for colorectal cancer.
Balakrishnan, Archana; Vyas, Arpita; Deshpande, Kaivalya; Vyas, Dinesh
Colorectal cancer accounts for a significant proportion of cancer deaths worldwide. The need to develop more chemotherapeutic agents to combat this disease is critical. Cyclin dependent kinases (CDKs), along with its binding partner cyclins, serve to control the growth of cells through the cell cycle. A new class of drugs, termed CDK inhibitors, has been studied in preclinical and now clinical trials. These inhibitors are believed to act as an anti-cancer drug by blocking CDKs to block the uncontrolled cellular proliferation that is hallmark of cancers like colorectal cancer. CDK article provides overview of the emerging drug class of CDK inhibitors and provides a list of ones that are currently in clinical trials.
Phosphorylation of XIAP by CDK1–cyclin-B1 controls mitotic cell death
Hou, Ying; Allan, Lindsey A.
ABSTRACT Regulation of cell death is crucial for the response of cancer cells to drug treatments that cause arrest in mitosis, and is likely to be important for protection against chromosome instability in normal cells. Prolonged mitotic arrest can result in cell death by activation of caspases and the induction of apoptosis. Here, we show that X-linked inhibitor of apoptosis (XIAP) plays a key role in the control of mitotic cell death. Ablation of XIAP expression sensitises cells to prolonged mitotic arrest caused by a microtubule poison. XIAP is stable during mitotic arrest, but its function is controlled through phosphorylation by the mitotic kinase CDK1–cyclin-B1 at S40. Mutation of S40 to a phosphomimetic residue (S40D) inhibits binding to activated effector caspases and abolishes the anti-apoptotic function of XIAP, whereas a non-phosphorylatable mutant (S40A) blocks apoptosis. By performing live-cell imaging, we show that phosphorylation of XIAP reduces the threshold for the onset of cell death in mitosis. This work illustrates that mitotic cell death is a form of apoptosis linked to the progression of mitosis through control by CDK1–cyclin-B1. PMID:27927753
Foxp3 Protein Stability Is Regulated by Cyclin-dependent Kinase 2*
Morawski, Peter A.; Mehra, Parul; Chen, Chunxia; Bhatti, Tricia; Wells, Andrew D.
Foxp3 is a transcription factor required for the development of regulatory T cells (Treg). Mice and humans with a loss of Foxp3 function suffer from uncontrolled autoimmunity and inflammatory disease. Expression of Foxp3 is necessary for the anti-inflammatory capacity of Treg, but whether Foxp3 activity is further subject to regulation by extracellular signals is unclear. The primary structure of Foxp3 contains four cyclin-dependent kinase (CDK) motifs (Ser/Thr-Pro) within the N-terminal repressor domain, and we show that CDK2 can partner with cyclin E to phosphorylate Foxp3 at these sites. Consistent with our previous demonstration that CDK2 negatively regulates Treg function, we find that mutation of the serine or threonine at each CDK motif to alanine (S/T→A) results in enhanced Foxp3 protein stability in CD4+ T cells. T cells expressing the S/T→A mutant of Foxp3 showed enhanced induction (e.g. CD25) and repression (e.g. IL2) of canonical Foxp3-responsive genes, exhibited an increased capacity to suppress conventional T cell proliferation in vitro, and were highly effective at ameliorating colitis in an in vivo model of inflammatory bowel disease. These results indicate that CDK2 negatively regulates the stability and activity of Foxp3 and implicate CDK-coupled receptor signal transduction in the control of regulatory T cell function and stability. PMID:23853094
Functional Analysis of the Cyclin-Dependent Kinase Inhibitor Pho81 Identifies a Novel Inhibitory Domain
Huang, Sidong; Jeffery, Douglas A.; Anthony, Malcolm D.; O'Shea, Erin K.
In response to phosphate limitation, Saccharomyces cerevisiae induces transcription of a set of genes important for survival. A phosphate-responsive signal transduction pathway mediates this response by controlling the activity of the transcription factor Pho4. Three components of this signal transduction pathway resemble those used to regulate the eukaryotic cell cycle: a cyclin-dependent kinase (CDK), Pho85; a cyclin, Pho80; and a CDK inhibitor (CKI), Pho81. Pho81 forms a stable complex with Pho80-Pho85 under both high- and low-phosphate conditions, but it only inhibits the kinase when cells are starved for phosphate. Pho81 contains six tandem repeats of the ankyrin consensus domain homologous to the INK4 family of mammalian CKIs. INK4 proteins inhibit kinase activity through an interaction of the ankyrin repeats and the CDK subunits. Surprisingly, we find that a region of Pho81 containing 80 amino acids C terminal to the ankyrin repeats is necessary and sufficient for Pho81's CKI function. The ankyrin repeats of Pho81 appear to have no significant role in Pho81 inhibition. Our results suggest that Pho81 inhibits Pho80-Pho85 with a novel motif. PMID:11533256
The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention
Alao, John P
Cyclin D1 is an important regulator of cell cycle progression and can function as a transcriptionl co-regulator. The overexpression of cyclin D1 has been linked to the development and progression of cancer. Deregulated cyclin D1 degradation appears to be responsible for the increased levels of cyclin D1 in several cancers. Recent findings have identified novel mechanisms involved in the regulation of cyclin D1 stability. A number of therapeutic agents have been shown to induce cyclin D1 degradation. The therapeutic ablation of cyclin D1 may be useful for the prevention and treatment of cancer. In this review, current knowledge on the regulation of cyclin D1 degradation is discussed. Novel insights into cyclin D1 degradation are also discussed in the context of ablative therapy. A number of unresolved questions regarding the regulation of cellular cyclin D1 levels are also addressed. PMID:17407548
The transcription factor, the Cdk, its cyclin and their regulator: directing the transcriptional response to a nutritional signal.
Hirst, K; Fisher, F; McAndrew, P C; Goding, C R
The Pho80-Pho85 cyclin-cdk complex prevents transcription of PHO5 by inhibiting the ability of the basic-helix-loop-helix transcription factor Pho4 to activate transcription in response to high phosphate conditions. In low phosphate the Pho80-Pho85 complex is inactivated and Pho4 is then able to activate the acid phosphatase gene PHO5. We show here that Pho4 and the homeobox protein Pho2 interact in vivo and act cooperatively to activate the PHO5 UAS, with interaction being regulated by the phosphate switch. In addition, we also demonstrate that an additional factor, Pho81, interacts in high phosphate with both the Pho80 cyclin and with Pho4. In low phosphate, Pho80 and Pho81 dissociate from Pho4, but retain the ability to interact with each other. The evidence presented here supports the idea that Pho81 acts as a phosphate-sensitive trigger that regulates the ability of the Pho80-Pho85 cyclin-cdk complex to bind Pho4, while DNA binding by Pho4 is dependent on the phosphate-sensitive interaction with Pho2. Images PMID:7957107
Notch1-induced mammary tumor development is cyclin D1-dependent and correlates with expansion of pre-malignant multipotent duct-limited progenitors.
Ling, H; Sylvestre, J-R; Jolicoeur, P
Members of the Notch family are involved in the development of breast cancer in animal models and in humans. In young transgenic mice, expressing intracellular activated Notch1 (N1(IC)) in mammary cells, we found that CD24(+) CD29(high) progenitor cells had enhanced survival, and were expanded through a cyclin D1-dependent pathway. This expansion positively correlated with the later cyclin D1-dependent formation of basal-like ductal tumors. This expanded population exhibited abnormal differentiation skewed toward the basal cells, showed signs of pre-malignancy (low PTEN/p53 and high c-myc) and contained stem cells with impaired self-renewal in vivo, and more numerous multipotent, ductal-restricted progenitors. Our data suggest that N1(IC) can favor transformation of progenitor cells early in life through a cyclin D1-dependent pathway.
(R)-roscovitine, a cyclin-dependent kinase inhibitor, enhances tonic GABA inhibition in rat hippocampus.
Ivanov, A; Tyzio, R; Zilberter, Y; Ben-Ari, Yehezkel
Pharmacological agents that mediate a persistent GABAergic conductance are of considerable interest for treatment of epilepsy. (R)-roscovitine is a membrane permeable cyclin-dependent kinase inhibitor, designed to block cell division. It is currently undergoing a phase II clinical trial as an anticancer drug. We show that (R)-roscovitine increases a tonic GABA-mediated current in rat hippocampal neurons. This enhanced tonic current appears independent of synaptic GABA release and requires functional transmembrane GABA transport. The effect of (R)-roscovitine is associated with neither modification of GABAA receptors nor protein kinase activity, but is associated with a significant increase in intracellular GABA concentration in hippocampal GABAergic neurons. (R)-roscovitine-induced tonic inhibition significantly suppresses spontaneous spiking activity of hippocampal pyramidal cells. Therefore, (R)-roscovitine is a potent modulator of neuronal activity in rat hippocampus and may provide a tool for preventing paroxysmal activity.
Drug Design of Cyclin-Dependent Kinase 2 Inhibitor for Melanoma from Traditional Chinese Medicine
Tang, Hsin-Chieh
One has found an important cell cycle controller. This guard can decide the cell cycle toward proliferation or quiescence. Cyclin-dependent kinase 2 (CDK2) is a unique target among the CDK family in melanoma therapy. We attempted to find out TCM compounds from TCM Database@Taiwan that have the ability to inhibit the activity of CDK2 by systems biology. We selected Tetrahydropalmatine, Reserpiline, and (+)-Corydaline as the candidates by docking and screening results for further survey. We utilized support vector machine (SVM), multiple linear regression (MLR) models and Bayesian network for validation of predicted activity. By overall analysis of docking results, predicted activity, and molecular dynamics (MD) simulation, we could conclude that Tetrahydropalmatine, Reserpiline, and (+)-Corydaline had better binding affinity than the control. All of them had the ability to inhibit the activity of CDK2 and might have the opportunity to be applied in melanoma therapy. PMID:25045703
Arabidopsis PCNAs form complexes with selected D-type cyclins
Strzalka, Wojciech K.; Aggarwal, Chhavi; Krzeszowiec, Weronika; Jakubowska, Agata; Sztatelman, Olga; Banas, Agnieszka K.
Proliferating Cell Nuclear Antigen (PCNA) is a key nuclear protein of eukaryotic cells. It has been shown to form complexes with cyclin dependent kinases, cyclin dependent kinase inhibitors and the D-type cyclins which are involved in the cell cycle control. In Arabidopsis two genes coding for PCNA1 and PCNA2 proteins have been identified. In this study by analyzing Arabidopsis PCNA/CycD complexes we tested the possible functional differentiation of PCNA1/2 proteins in cell cycle control. Most out of the 10 cyclins investigated showed only nuclear localization except CycD2;1, CycD4;1, and CycD4;2 which were observed both in the nucleus and cytoplasm. Using the Y2H, BiFC and FLIM-FRET techniques we identified D-type cyclins which formed complexes with either PCNA1 or PCNA2. Among the candidates tested only CycD1;1, CycD3;1, and CycD3;3 were not detected in a complex with the PCNA proteins. Moreover, our results indicate that the formation of CycD3;2/PCNA and CycD4;1/PCNA complexes can be regulated by other as yet unidentified factor(s). Additionally, FLIM-FRET analyses suggested that in planta the distance between PCNA1/CycD4;1, PCNA1/CycD6;1, PCNA1/CycD7;1, and PCNA2/CycD4;2 proteins was shorter than that between PCNA2/CycD4;1, PCNA2/CycD6;1, PCNA2/CycD7;1, and PCNA1/CycD4;2 pairs. These data indicate that the nine amino acid differences between PCNA1 and PCNA2 have an impact on the architecture of Arabidopsis CycD/PCNA complexes. PMID:26379676
Recruitment of Cln3 Cyclin to Promoters Controls Cell Cycle Entry via Histone Deacetylase and Other Targets
Cai, Ying; Wijnen, Herman; Futcher, Bruce
In yeast, the G1 cyclin Cln3 promotes cell cycle entry by activating the transcription factor SBF. In mammals, there is a parallel system for cell cycle entry in which cyclin dependent kinase (CDK) activates transcription factor E2F/Dp. Here we show that Cln3 regulates SBF by at least two different pathways, one involving the repressive protein Whi5, and the second involving Stb1. The Rpd3 histone deacetylase complex is also involved. Cln3 binds to SBF at the CLN2 promoter, and removes previously bound Whi5 and histone deacetylase. Adding extra copies of the SBF binding site to the cell delays Start, possibly by titrating Cln3. Since Rpd3 is the yeast ortholog of mammalian HDAC1, there is now a virtually complete analogy between the proteins regulating cell cycle entry in yeast (SBF, Cln3, Whi5 and Stb1, Rpd3) and mammals (E2F, Cyclin D, Rb, HDAC1). The cell may titrate Cln3 molecules against the number of SBF binding sites, and this could be the underlying basis of the size-control mechanism for Start. PMID:19823669
IRE1α controls cyclin A1 expression and promotes cell proliferation through XBP-1
Thorpe, Jeffery A.
IRE1 is a conserved dual endoribonuclease/protein kinase that is indispensable for directing the endoplasmic reticulum (ER) stress response in yeast, flies, and worms. In mammalian systems, however, the precise biological activities carried out by IRE1α are unclear. Here, molecular and chemical genetic approaches were used to control IRE1 activity in a number of prostate cancer cell lines and the resulting impact on gene transcription, cell survival, and proliferation was examined. Modulating IRE1α activity had no transcriptional effect on the induction of genes classically associated with the ER stress response (Grp78 and CHOP) or cell survival when confronted with ER stress agents. Rather, IRE1α activity was positively correlated to proliferation. Since Xbp-1 mRNA is the sole known substrate for IRE1 endoribonuclease activity, the role of this transcription factor in mediating proliferation was examined. Repressing total Xbp-1 levels by siRNA techniques effectively slowed proliferation. In an effort to identify IRE1/XBP-1 targets responsible for the cell cycle response, genome-wide differential mRNA expression analysis was performed. Consistent with its ability to sense ER stress, IRE1α induction led to an enrichment of ER-Golgi, plasma membrane, and secretory gene products. An increase in cyclin A1 expression was the only differentially expressed cell cycle regulatory gene found. Greater cyclin A protein levels were consistently observed in cells with active IRE1α and were dependent on XBP-1. We conclude that IRE1α activity controls a subset of the ER stress response and mediates proliferation through tight control of Xbp-1 splicing. Electronic supplementary material The online version of this article (doi:10.1007/s12192-009-0163-4) contains supplementary material, which is available to authorized users. PMID:20013084
Cyclin D1 depletion induces DNA damage in mantle cell lymphoma lines.
Mohanty, Suchismita; Mohanty, Atish; Sandoval, Natalie; Tran, Thai; Bedell, Victoria; Wu, Jun; Scuto, Anna; Murata-Collins, Joyce; Weisenburger, Dennis D; Ngo, Vu N
Elevated cyclin D1 (CCND1) expression levels in mantle cell lymphoma (MCL) are associated with aggressive clinical manifestations related to chemoresistance, but little is known about how this important proto-oncogene contributes to the resistance of MCL. Here, we showed that RNA interference-mediated depletion of CCND1 increased caspase-3 activities and induced apoptosis in the human MCL lines UPN-1 and JEKO-1. In vitro and xenotransplant studies revealed that the toxic effect of CCND1 depletion in MCL cells was likely due to increase in histone H2AX phosphorylation, a DNA damage marker. DNA fiber analysis suggested deregulated replication initiation after CCND1 depletion as a potential cause of DNA damage. Finally, in contrast to depletion or inhibition of cyclin-dependent kinase 4, CCND1 depletion increased chemosensitivity of MCL cells to replication inhibitors hydroxyurea and cytarabine. Our findings have an important implication for CCND1 as a potential therapeutic target in MCL patients who are refractory to standard chemotherapy.
Cell type-specific translational repression of Cyclin B during meiosis in males.
Baker, Catherine Craig; Gim, Byung Soo; Fuller, Margaret T
The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, we show that Fest is required for proper execution of meiosis I.
Differentiation-inducing factor-1 induces cyclin D1 degradation through the phosphorylation of Thr{sup 286} in squamous cell carcinoma
Mori, Jun; Takahashi-Yanaga, Fumi . E-mail: firstname.lastname@example.com; Miwa, Yoshikazu; Watanabe, Yutaka; Hirata, Masato; Morimoto, Sachio; Shirasuna, Kanemitsu; Sasaguri, Toshiyuki
Differentiation-inducing factors (DIFs) are morphogens which induce cell differentiation in Dictyostelium. We reported that DIF-1 and DIF-3 inhibit proliferation and induce differentiation in mammalian cells. In this study, we investigated the effect of DIF-1 on oral squamous cell carcinoma cell lines NA and SAS, well differentiated and poorly differentiated cell lines, respectively. Although DIF-1 did not induce the expression of cell differentiation makers in these cell lines, it inhibited the proliferation of NA and SAS in a dose-dependent manner by restricting the cell cycle in the G{sub 0}/G{sub 1} phase. DIF-1 induced cyclin D1 degradation, but this effect was prevented by treatment with lithium chloride and SB216763, the inhibitors of glycogen synthase kinase-3{beta} (GSK-3{beta}). Depletion of endogenous GSK-3{beta} by RNA interference also attenuated the effect of DIF-1 on cyclin D1 degradation. Therefore, we investigated the effect of DIF-1 on GSK-3{beta} and found that DIF-1 dephosphorylated GSK-3{beta} on Ser{sup 9} and induced the nuclear translocation of GSK-3{beta}, suggesting that DIF-1 activated GSK-3{beta}. Then, we examined the effect of DIF-1 on cyclin D1 mutants (Thr286Ala, Thr288Ala, and Thr286/288Ala). We revealed that Thr286Ala and Thr286/288Ala mutants were highly resistant to DIF-1-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr{sup 286} was critical for cyclin D1 degradation induced by DIF-1. These results suggest that DIF-1 induces degradation of cyclin D1 through the GSK-3{beta}-mediated phosphorylation of Thr{sup 286}.
Cell cycle-regulatory cyclins and their deregulation in oral cancer.
Mishra, Rajakishore
Oral cancer is a growth-related disorder, and cyclins are the prime regulators of cell division. Cyclins are associated with the pathogenesis of oral cancer and are considered valuable biomarkers for diagnosis and prognosis. These important molecules are regulated in many ways to achieve a gain in function and are involved in promoting neoplastic growth. While the causes of most cyclin overexpression are varied, these cyclins may be induced by buccal mucosal insult mainly with carcinogens that alter various pathways propelling oral cancer. Substantial experimental evidences support a link between oncogenic signaling pathways and the deregulation of cyclins in oral cancer. This review focuses on the mechanisms by which cyclins are regulated and promote oral oncogenesis.
Early events in DNA replication require cyclin E and are blocked by p21CIP1
Using immunodepletion of cyclin E and the inhibitor protein p21WAF/CIP1, we demonstrate that the cyclin E protein, in association with Cdk2, is required for the elongation phase of replication on single-stranded substrates. Although cyclin E/Cdk2 is likely to be the major target by which p21 inhibits the initiation of sperm DNA replication, p21 can inhibit single-stranded replication through a mechanism dependent on PCNA. While the cyclin E/Cdk2 complex appears to have a role in the initiation of DNA replication, another Cdk kinase, possibly cyclin A/Cdk, may be involved in a later step controlling the switch from initiation to elongation. The provision of a large maternal pool of cyclin E protein shows that regulators of replication are constitutively present, which explains the lack of a protein synthesis requirement for replication in the early embryonic cell cycle. PMID:7642695
The Role of Cyclin D1 in Altering Stromal-Epithelial Interactions in Prostate Carcinogenesis
adenocarcinomas. One study of cyclin D1 expression in esophageal carcinomas indicated that cyclin D1 is strongly expressed in stromal fibroblasts. In this study...proliferate faster than controls in vivo in our tissue recombination model. Although cyclin D1 can increase BPH-1 cell motility and promote cell...Alarid ET, Turner T, Donjacour AA, Boutin EL, Foster BA. Normal and abnormal development of the male urogenital tract: role of androgens, mesenchymal
ARTD1 regulates cyclin E expression and consequently cell-cycle re-entry and G1/S progression in T24 bladder carcinoma cells.
Léger, Karolin; Hopp, Ann-Katrin; Fey, Monika; Hottiger, Michael O
ADP-ribosylation is involved in a variety of biological processes, many of which are chromatin-dependent and linked to important functions during the cell cycle. However, any study on ADP-ribosylation and the cell cycle faces the problem that synchronization with chemical agents or by serum starvation and subsequent growth factor addition already activates ADP-ribosylation by itself. Here, we investigated the functional contribution of ARTD1 in cell cycle re-entry and G1/S cell cycle progression using T24 urinary bladder carcinoma cells, which synchronously re-enter the cell cycle after splitting without any additional stimuli. In synchronized cells, ARTD1 knockdown, but not inhibition of its enzymatic activity, caused specific down-regulation of cyclin E during cell cycle re-entry and G1/S progression through alterations of the chromatin composition and histone acetylation, but not of other E2F-1 target genes. Although Cdk2 formed a functional complex with the residual cyclin E, p27(Kip 1) protein levels increased in G1 upon ARTD1 knockdown most likely due to inappropriate cyclin E-Cdk2-induced phosphorylation-dependent degradation, leading to decelerated G1/S progression. These results provide evidence that ARTD1 regulates cell cycle re-entry and G1/S progression via cyclin E expression and p27(Kip 1) stability independently of its enzymatic activity, uncovering a novel cell cycle regulatory mechanism.
Transcriptional and post-transcriptional down-regulation of cyclin D1 contributes to C6 glioma cell differentiation induced by forskolin.
He, Songmin; Zhu, Wenbo; Zhou, Yuxi; Huang, Yijun; Ou, Yanqiu; Li, Yan; Yan, Guangmei
Malignant gliomas are the most common and lethal intracranial tumors, and differentiation therapy shows great potential to be a promising candidate for their treatment. Here, we have elaborated that a PKA activator, forskolin, represses cell growth via cell cycle arrest in the G0/G1 phase and induces cell differentiation characteristic with elongated processes and restoration of GFAP expression. In mechanisms, we verified that forskolin significantly diminishes the mRNA and protein level of a key cell cycle regulator cyclin D1, and maintenance of low cyclin D1 expression level was required for forskolin-induced proliferation inhibition and differentiation by gain and loss of function approaches. In addition, that forskolin down-regulated the cyclin D1 by proteolytic (post-transcriptional) mechanisms was dependent on GSK-3β activation at Ser9. The pro-differentiation activity of forskolin and related molecular mechanisms imply that forskolin can be developed into a candidate for the future in differentiation therapy of glioma, and cyclin D1 is a promising target for pro-differentiation strategy.
Apoptosis signal-regulating kinase 1 and cyclin D1 compose a positive feedback loop contributing to tumor growth in gastric cancer
Hayakawa, Yoku; Hirata, Yoshihiro; Nakagawa, Hayato; Sakamoto, Kei; Hikiba, Yohko; Kinoshita, Hiroto; Nakata, Wachiko; Takahashi, Ryota; Tateishi, Keisuke; Tada, Motohisa; Akanuma, Masao; Yoshida, Haruhiko; Takeda, Kohsuke; Ichijo, Hidenori; Omata, Masao; Maeda, Shin; Koike, Kazuhiko
Mitogen-activated protein kinase (MAPK) pathways regulate multiple cellular functions and are highly active in many types of human cancers. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream MAPK involved in apoptosis, inflammation, and carcinogenesis. This study investigated the role of ASK1 in the development of gastric cancer. In human gastric cancer specimens, we observed increased ASK1 expression, compared to nontumor epithelium. Using a chemically induced murine gastric tumorigenesis model, we observed increased tumor ASK1 expression, and ASK1 knockout mice had both fewer and smaller tumors than wild-type (WT) mice. ASK1 siRNA inhibited cell proliferation through the accumulation of cells in G1 phase of the cell cycle, and reduced cyclin D1 expression in gastric cancer cells, whereas these effects were uncommon in other cancer cells. ASK1 overexpression induced the transcription of cyclin D1, through AP-1 activation, and ASK1 levels were regulated by cyclin D1, via the Rb–E2F pathway. Exogenous ASK1 induced cyclin D1 expression, followed by elevated expression of endogenous ASK1. These results indicate an autoregulatory mechanism of ASK1 in the development of gastric cancer. Targeting this positive feedback loop, ASK1 may present a potential therapeutic target for the treatment of advanced gastric cancer. PMID:21187402
Mitotic p21Cip1/CDKN1A is regulated by cyclin-dependent kinase 1 phosphorylation
Kreis, Nina-Naomi; Friemel, Alexandra; Zimmer, Brigitte; Roth, Susanne; Rieger, Michael A.; Rolle, Udo; Louwen, Frank; Yuan, Juping
The multifunctional protein p21Cip1/CDKN1A (p21) is an important and universal Cdk-interacting protein. Recently, we have reported that p21 is involved in the regulation of the mitotic kinase Cdk1/cyclin B1 and critical for successful mitosis and cytokinesis. In the present work we show that S130 of p21 is phosphorylated by Cdk1/cyclin B1 during mitosis, which reduces p21′s stability and binding affinity to Cdk1/cyclin B1. Interfering with this phosphorylation results in extended mitotic duration and defective chromosome segregation, indicating that this regulation ensures proper mitotic progression. Given that p53, the major transcriptional activator of p21, is the most frequently mutated gene in human cancer and that deregulated Cdk1 associates with the development of different types of cancer, this work provides new insight into the understanding of how deregulated p21 contributes to chromosomal instability and oncogenesis. PMID:27384476
Translokin (Cep57) interacts with cyclin D1 and prevents its nuclear accumulation in quiescent fibroblasts.
Ruiz-Miró, Maria; Colomina, Neus; Fernández, Rita M H; Garí, Eloi; Gallego, Carme; Aldea, Martí
Nuclear accumulation of cyclin D1 because of altered trafficking or degradation is thought to contribute directly to neoplastic transformation and growth. Mechanisms of cyclin D1 localization in S phase have been studied in detail, but its control during exit from the cell cycle and quiescence is poorly understood. Here we report that translokin (Tlk), a microtubule-associated protein also termed Cep57, interacts with cyclin D1 and controls its nucleocytoplasmic distribution in quiescent cells. Tlk binds to regions of cyclin D1 also involved in binding to cyclin-dependent kinase 4 (Cdk4), and a fraction of cyclin D1 associates to the juxtanuclear Tlk network in the cell. Downregulation of Tlk levels results in undue nuclear accumulation of cyclin D1 and increased Cdk4-dependent phosphorylation of pRB under quiescence conditions. In turn, overexpression of Tlk prevents proper cyclin D1 accumulation in the nucleus of proliferating cells in an interaction-dependent manner, inhibits Cdk4-dependent phosphorylation of pRB and hinders cell cycle progression to S phase. We propose that the Tlk acts as a key negative regulator in the pathway that drives nuclear import of cyclin D1, thus contributing to prevent pRB inactivation and to maintain cellular quiescence.
Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis.
Sicinski, P; Donaher, J L; Geng, Y; Parker, S B; Gardner, H; Park, M Y; Robker, R L; Richards, J S; McGinnis, L K; Biggers, J D; Eppig, J J; Bronson, R T; Elledge, S J; Weinberg, R A
THE D-type cyclins (D1, D2 and D3) are critical governors of the cell-cycle clock apparatus during the G1 phase of the mammalian cell cycle. These three D-type cyclins are expressed in overlapping, apparently redundant fashion in the proliferating tissues. To investigate why mammalian cells need three distinct D-type cyclins, we have generated mice bearing a disrupted cyclin D2 gene by using gene targeting in embryonic stem cells. Cyclin D2-deficient females are sterile owing to the inability of ovarian granulosa cells to proliferate normally in response to follicle-stimulating hormone (FSH), whereas mutant males display hypoplastic testes. In ovarian granulosa cells, cyclin D2 is specifically induced by FSH via a cyclic-AMP-dependent pathway, indicating that expression of the various D-type cyclins is under control of distinct intracellular signalling pathways. The hypoplasia seen in cyclin D2(-/-) ovaries and testes prompted us to examine human cancers deriving from corresponding tissues. We find that some human ovarian and testicular tumours contain high levels of cyclin D2 messenger RNA.
The Yeast Cyclin-Dependent Kinase Routes Carbon Fluxes to Fuel Cell Cycle Progression.
Ewald, Jennifer C; Kuehne, Andreas; Zamboni, Nicola; Skotheim, Jan M
Cell division entails a sequence of processes whose specific demands for biosynthetic precursors and energy place dynamic requirements on metabolism. However, little is known about how metabolic fluxes are coordinated with the cell division cycle. Here, we examine budding yeast to show that more than half of all measured metabolites change significantly through the cell division cycle. Cell cycle-dependent changes in central carbon metabolism are controlled by the cyclin-dependent kinase (Cdk1), a major cell cycle regulator, and the metabolic regulator protein kinase A. At the G1/S transition, Cdk1 phosphorylates and activates the enzyme Nth1, which funnels the storage carbohydrate trehalose into central carbon metabolism. Trehalose utilization fuels anabolic processes required to reliably complete cell division. Thus, the cell cycle entrains carbon metabolism to fuel biosynthesis. Because the oscillation of Cdk activity is a conserved feature of the eukaryotic cell cycle, we anticipate its frequent use in dynamically regulating metabolism for efficient proliferation.
Plant D-type cyclins and the control of G1 progression.
Oakenfull, E Ann; Riou-Khamlichi, Catherine; Murray, James A H
The basic pattern of controls that operate during the G1 phase of the plant cell cycle shows much closer similarity to animals than to the yeasts and other fungi. The activity of D-type cyclin (CycD) kinases is induced in response to stimulatory signals, and these phosphorylate the plant homologue of the retinoblastoma tumour susceptibility (Rb) protein. It is likely that Rb phosphorylation results in the activation of genes under the control of E2F transcription factors, including those required for S phase entry. As the initial triggers of the cascade, attention has focused on the CycDs, and a family of 10 genes is present in Arabidopsis, divided into three major and three minor groups. Analysis to date suggests that these groups are functionally distinct. PMID:12079670
Myt1 inhibition of Cyclin A/Cdk1 is essential for fusome integrity and premeiotic centriole engagement in Drosophila spermatocytes
Varadarajan, Ramya; Ayeni, Joseph; Jin, Zhigang; Homola, Ellen; Campbell, Shelagh D.
Regulation of cell cycle arrest in premeiotic G2 phase coordinates germ cell maturation and meiotic cell division with hormonal and developmental signals by mechanisms that control Cyclin B synthesis and inhibitory phosphorylation of the M-phase kinase, Cdk1. In this study, we investigated how inhibitory phosphorylation of Cdk1 by Myt1 kinase regulates premeiotic G2 phase of Drosophila male meiosis. Immature spermatocytes lacking Myt1 activity exhibit two distinct defects: disrupted intercellular bridges (fusomes) and premature centriole disengagement. As a result, the myt1 mutant spermatocytes enter meiosis with multipolar spindles. These myt1 defects can be suppressed by depletion of Cyclin A activity or ectopic expression of Wee1 (a partially redundant Cdk1 inhibitory kinase) and phenocopied by expression of a Cdk1F mutant defective for inhibitory phosphorylation. We therefore conclude that Myt1 inhibition of Cyclin A/Cdk1 is essential for normal fusome behavior and centriole engagement during premeiotic G2 arrest of Drosophila male meiosis. The novel meiotic functions we discovered for Myt1 kinase are spatially and temporally distinct from previously described functions of Myt1 as an inhibitor of Cyclin B/Cdk1 to regulate G2/MI timing. PMID:27170181
AUREOCHROME1a-mediated induction of the diatom-specific cyclin dsCYC2 controls the onset of cell division in diatoms (Phaeodactylum tricornutum).
Huysman, Marie J J; Fortunato, Antonio E; Matthijs, Michiel; Costa, Benjamin Schellenberger; Vanderhaeghen, Rudy; Van den Daele, Hilde; Sachse, Matthias; Inzé, Dirk; Bowler, Chris; Kroth, Peter G; Wilhelm, Christian; Falciatore, Angela; Vyverman, Wim; De Veylder, Lieven
Cell division in photosynthetic organisms is tightly regulated by light. Although the light dependency of the onset of the cell cycle has been well characterized in various phototrophs, little is known about the cellular signaling cascades connecting light perception to cell cycle activation and progression. Here, we demonstrate that diatom-specific cyclin 2 (dsCYC2) in Phaeodactylum tricornutum displays a transcriptional peak within 15 min after light exposure, long before the onset of cell division. The product of dsCYC2 binds to the cyclin-dependent kinase CDKA1 and can complement G1 cyclin-deficient yeast. Consistent with the role of dsCYC2 in controlling a G1-to-S light-dependent cell cycle checkpoint, dsCYC2 silencing decreases the rate of cell division in diatoms exposed to light-dark cycles but not to constant light. Transcriptional induction of dsCYC2 is triggered by blue light in a fluence rate-dependent manner. Consistent with this, dsCYC2 is a transcriptional target of the blue light sensor AUREOCHROME1a, which functions synergistically with the basic leucine zipper (bZIP) transcription factor bZIP10 to induce dsCYC2 transcription. The functional characterization of a cyclin whose transcription is controlled by light and whose activity connects light signaling to cell cycle progression contributes significantly to our understanding of the molecular mechanisms underlying light-dependent cell cycle onset in diatoms.
p53 Dimers associate with a head-to-tail response element to repress cyclin B transcription.
Lipski, Robert; Lippincott, Daniel J; Durden, Brittany C; Kaplan, Anne R; Keiser, Hilary E; Park, Jung-Ho; Levesque, Aime A
DNA damage induced by the topoisomerase I inhibitor SN38 activates cell cycle checkpoints which promote cell cycle arrest. This arrest can be abrogated in p53-defective cells by the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). Previously, we compared p53 wild-type MCF10A cells with derivatives whose p53 function was inhibited by over-expression of the tetramerization domain (MCF10A/OD) or expression of shRNA against p53 (MCF10A/Δp53). Treatment of SN38-arrested MCF10A/OD cells with UCN-01 abrogated S, but not G2 arrest, while the MCF10A/Δp53 cells abrogated both S and G2 arrest. The MCF10A/OD cells had reduced levels of cyclin B, suggesting that tetramerization of p53 is not required for repression of cyclin B gene expression. In the present study, we analyzed p53 oligomerization status using glutaraldehyde cross-linking. Following SN38 treatment, MCF10A cells contained oligomeric forms of p53 with molecular weights approximating monomers, dimers, trimers, and tetramers. However, MCF10A/OD cells possessed only monomers and dimers suggesting that these complexes may be involved in repression of cyclin B. While genes transcriptionally activated by p53 contain a consensus sequence with elements repeated in a head-to-head orientation, the cyclin B promoter contains similar elements oriented head-to-tail. Chromatin immunoprecipitation (ChIP) assays revealed that p53 associates with this head-to-tail element in both MCF10A and MCF10A/OD. Electrophoretic mobility shift assays (EMSA) using a biotin-labeled probe containing the head-to-tail element showed a shift in mobility consistent with the molecular weight of tetramers and dimers in MCF10A nuclear extract, but only the dimer in MCF10A/OD nuclear extract. Taken together, these results suggest a novel mechanism whereby p53 dimers associate with the head-to-tail element to repress cyclin B transcription.
Selective CREB-dependent cyclin expression mediated by the PI3K and MAPK pathways supports glioma cell proliferation
Daniel, P; Filiz, G; Brown, D V; Hollande, F; Gonzales, M; D'Abaco, G; Papalexis, N; Phillips, W A; Malaterre, J; Ramsay, R G; Mantamadiotis, T
The cyclic-AMP response element binding (CREB) protein has been shown to have a pivotal role in cell survival and cell proliferation. Transgenic rodent models have revealed a role for CREB in higher-order brain functions, such as memory and drug addiction behaviors. CREB overexpression in transgenic animals imparts oncogenic properties on cells in various tissues, and aberrant CREB expression is associated with tumours. It is the central position of CREB, downstream from key developmental and growth signalling pathways, which gives CREB this ability to influence a spectrum of cellular activities, such as cell survival, growth and differentiation, in both normal and cancer cells. We show that CREB is highly expressed and constitutively activated in patient glioma tissue and that this activation closely correlates with tumour grade. The mechanism by which CREB regulates glioblastoma (GBM) tumour cell proliferation involves activities downstream from both the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) pathways that then modulate the expression of three key cell cycle factors, cyclin B, D and proliferating cell nuclear antigen (PCNA). Cyclin D1 is highly CREB-dependent, whereas cyclin B1 and PCNA are co-regulated by both CREB-dependent and -independent mechanisms. The precise regulatory network involved appears to differ depending on the tumour-suppressor phosphatase and tensin homolog status of the GBM cells, which in turn allows CREB to regulate the activity of the PI3K itself. Given that CREB sits at the hub of key cancer cell signalling pathways, understanding the role of glioma-specific CREB function may lead to improved novel combinatorial anti-tumour therapies, which can complement existing PI3K-specific drugs undergoing early phase clinical trials. PMID:24979279
Co-expressed Cyclin D variants cooperate to regulate proliferation of germline nuclei in a syncytium.
Subramaniam, Gunasekaran; Campsteijn, Coen; Thompson, Eric M
The role of the G1-phase Cyclin D-CDK 4/6 regulatory module in linking germline stem cell (GSC) proliferation to nutrition is evolutionarily variable. In invertebrate Drosophila and C. elegans GSC models, G1 is nearly absent and Cyclin E is expressed throughout the cell cycle, whereas vertebrate spermatogonial stem cells have a distinct G1 and Cyclin D1 plays an important role in GSC renewal. In the invertebrate, chordate, Oikopleura, where germline nuclei proliferate asynchronously in a syncytium, we show a distinct G1-phase in which 2 Cyclin D variants are co-expressed. Cyclin Dd, present in both somatic endocycling cells and the germline, localized to germline nuclei during G1 before declining at G1/S. Cyclin Db, restricted to the germline, remained cytoplasmic, co-localizing in foci with the Cyclin-dependent Kinase Inhibitor, CKIa. These foci showed a preferential spatial distribution adjacent to syncytial germline nuclei at G1/S. During nutrient-restricted growth arrest, upregulated CKIa accumulated in arrested somatic endoreduplicative nuclei but did not do so in germline nuclei. In the latter context, Cyclin Dd levels gradually decreased. In contrast, the Cyclin Dbβ splice variant, lacking the Rb-interaction domain and phosphodegron, was specifically upregulated and the number of cytoplasmic foci containing this variant increased. This upregulation was dependent on stress response MAPK p38 signaling. We conclude that under favorable conditions, Cyclin Dbβ-CDK6 sequesters CKIa in the cytoplasm to cooperate with Cyclin Dd-CDK6 in promoting germline nuclear proliferation. Under nutrient-restriction, this sequestration function is enhanced to permit continued, though reduced, cycling of the germline during somatic growth arrest.
Expression of cyclin E in postmitotic neurons during development and in the adult mouse brain.
Ikeda, Yayoi; Matsunaga, Yuko; Takiguchi, Masahito; Ikeda, Masa-Aki
Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain.
Cyclin A regulates a cell-cycle-dependent expression of CKAP2 through phosphorylation of Sp1
Kang, Du-Seock; Hong, Kyeong-Man; Park, Joobae; Bae, Chang-Dae
Highlights: Black-Right-Pointing-Pointer We identified a GC box and a CHR element in human CKAP2 minimal promoter. Black-Right-Pointing-Pointer The CHR element repressed the CKAP2 minimal promoter activity at the G1/S phase. Black-Right-Pointing-Pointer The GC box was essential for the basic promoter activity of human CKAP2. Black-Right-Pointing-Pointer The GC box was also essential for the cyclic expression of human CKAP2. Black-Right-Pointing-Pointer The phosphorylation of Sp1, mediated by Cyclin A, underlies the cyclic expression. -- Abstract: CKAP2 plays crucial roles in proper chromosome segregation and maintaining genomic stability. CKAP2 protein showed cell-cycle-dependent expression, which reached a maximum level at the G2/M phase and disappeared at the onset of G1 phase. To elucidate the mechanisms underlying cell cycle-dependent expression of CKAP2, we cloned and analyzed the human CKAP2 promoter. The upstream 115-bp region from the transcription start site was sufficient for minimal CKAP2 promoter activity. We identified 2 regulatory sequences; a CHR (-110 to -104 bp) and a GC box (-41 to -32 bp). We confirmed Sp1 bound to the GC box using a supershift assay and a ChIP assay. Mutation in the GC box resulted in a near complete loss of CKAP2 promoter activity while mutation in the CHR decreased the promoter activity by 50%. The CHR mutation showed enhanced activity at the G1/S phase, but still retained cyclic activity. The Chromatin IP revealed that the amount of Sp1 bound to the GC box gradually increased and reached a maximum level at the G2/M phase. The amount of Sp1 bound to the GC box was greatly reduced when Cyclin A was depleted, which was restored by adding Cyclin A/Cdk2 complex back into the nuclear extracts. Together, we concluded that the GC box was responsible for the cyclic activity of human CKAP2 promoter through the phosphorylation of Sp1, possibly by Cyclin A/Cdk complex.
Vaccination with cyclin-dependent kinase tick antigen confers protection against Ixodes infestation.
Gomes, Helga; Moraes, Jorge; Githaka, Naftaly; Martins, Renato; Isezaki, Masayoshi; Vaz, Itabajara da Silva; Logullo, Carlos; Konnai, Satoru; Ohashi, Kazuhiko
Among arthropods, ticks lead as vectors of animal diseases and rank second to mosquitoes in transmitting human pathogens. Cyclin-dependent kinases (CDK) participate in cell cycle control in eukaryotes. CDKs are serine/threonine protein kinases and these catalytic subunits are activated or inactivated at specific stages of the cell cycle. To determine the potential of using CDKs as anti-tick vaccine antigens, hamsters were immunized with recombinant Ixodes persulcatus CDK10, followed by a homologous tick challenge. Though it was not exactly unexpected, IpCDK10 vaccination significantly impaired tick blood feeding and fecundity, which manifested as low engorgement weights, poor oviposition, and a reduction in 80% of hatching rates. These findings may underpin the development of more efficacious anti-tick vaccines based on the targeting of cell cycle control proteins.
Selective inhibitors of Cyclin-G associated kinase (GAK) as anti-HCV agents
Kovackova, Sona; Chang, Lei; Bekerman, Elena; Neveu, Gregory; Barouch-Bentov, Rina; Chaikuad, Apirat; Heroven, Christina; Šála, Michal; De Jonghe, Steven; Knapp, Stefan; Einav, Shirit; Herdewijn, Piet
Cyclin-G associated kinase (GAK) emerged as a promising drug target for the treatment of viral infections. However, no potent and selective GAK inhibitors have been reported in the literature to date. This paper describes the discovery of isothiazolo[5,4-b]pyridines as selective GAK inhibitors, with the most potent congeners displaying low nanomolar binding affinity for GAK. Co-crystallization experiments revealed that these compounds behaved as classic type I ATP-competitive kinase inhibitors. In addition, we have demonstrated that these compounds exhibit a potent activity against hepatitis C virus (HCV) by inhibiting two temporally distinct steps in the HCV lifecycle (i.e. viral entry and assembly). Hence, these GAK inhibitors represent chemical probes to study GAK function in different disease areas where GAK has been implicated (including viral infection, cancer and Parkinson's disease). PMID:25822739
Processing of meiotic DNA double strand breaks requires cyclin-dependent kinase and multiple nucleases.
Manfrini, Nicola; Guerini, Ilaria; Citterio, Andrea; Lucchini, Giovanna; Longhese, Maria Pia
Meiotic recombination requires the formation of programmed Spo11-dependent DNA double strand breaks (DSBs). In Saccharomyces cerevisiae, the Sae2 protein and the Mre11-Rad50-Xrs2 complex are necessary to remove the covalently attached Spo11 protein from the DNA ends, which are then resected by so far unknown nucleases. Here, we demonstrate that phosphorylation of Sae2 Ser-267 by cyclin-dependent kinase 1 (Cdk1) is required to initiate meiotic DSB resection by allowing Spo11 removal from DSB ends. This finding suggests that Cdk1 activity is required for the processing of Spo11-induced DSBs, thus providing a mechanism for coordinating DSB resection with progression through meiotic prophase. Furthermore, the helicase Sgs1 and the nucleases Exo1 and Dna2 participate in lengthening the 5'-3' resection tracts during meiosis by controlling a step subsequent to Spo11 removal.
Altered expression of the cyclin D1 and retinoblastoma genes in human esophageal cancer
Jiang, W.; Zhang, Y.J.; Kahn, S.M.; Santella, R.M.; Weinstein, I.B. ); Hollstein, M.C.; Montesano, R. ); Harris, C.C. ); Lu, S.H. )
The authors have examined DNA from four human esophageal carcinoma cell lines and 50 primary esophageal carcinomas obtained from China, Italy, and France for amplification of the cyclin D1 gene. They also examined 36 of these 50 carcinomas for expressions of the cyclin D1 and retinoblastoma (RB) proteins by immunohistochemistry. They found a 3- to 10-fold amplification of the cyclin D1 gene in 16 of the 50 (32%) tumors and in two of the four cell lines. Cyclin D1 protein was overexpressed in 12 of 13 tumors and the two cell lines that showed gene amplification when compared to normal controls. Studies on RB protein expression indicated that 6 of the 36 (17%) tumor samples examined and one cell line did not show detectable expression of this protein. The tumors and cell lines that had cyclin D1 gene amplification and overexpression exhibited normal levels of expression of RB protein. By contrast, the tumors and cell line that did not appear to express the RB protein did not show amplification of the cyclin D1 gene and expressed only low levels of the cyclin D1 protein (P = 0.03). These results suggest that the inhibitory effect of RB on cell cycle progression can be abrogated during tumor development either by loss of expression of the RB gene or by increased expression of the cyclin D1 gene. 46 refs., 5 figs., 2 tabs.
The cyclin A centrosomal localization sequence recruits MCM5 and Orc1 to regulate centrosome reduplication.
Ferguson, Rebecca L; Pascreau, Gaetan; Maller, James L
Centrosomes are the major microtubule-organizing centers in animal cells and regulate formation of a bipolar mitotic spindle. Aberrant centrosome number causes chromosome mis-segregation, and has been implicated in genomic instability and tumor development. Previous studies have demonstrated a role for the DNA replication factors MCM5 and Orc1 in preventing centrosome reduplication. Cyclin A-Cdk2 localizes on centrosomes by means of a modular centrosomal localization sequence (CLS) that is distinct from that of cyclin E. Here, we show that cyclin A interacts with both MCM5 and Orc1 in a CLS-dependent but Cdk-independent manner. Although the MRAIL hydrophobic patch is contained within the cyclin A CLS, binding of both MCM5 and Orc1 to cyclin A does not require a wild-type hydrophobic patch. The same domain in MCM5 that mediates interaction with cyclin E also binds cyclin A, resulting in centrosomal localization of MCM5. Finally, unlike its function in DNA synthesis, MCM5-mediated inhibition of centrosome reduplication in S-phase-arrested CHO cells does not require binding to other MCM family members. These results suggest that cyclins E and A sequentially prevent centrosome reduplication throughout interphase by recruitment of DNA replication factors such as MCM5 and Orc1.
E-type cyclins modulate telomere integrity in mammalian male meiosis.
Manterola, Marcia; Sicinski, Piotr; Wolgemuth, Debra J
We have shown that E-type cyclins are key regulators of mammalian male meiosis. Depletion of cyclin E2 reduced fertility in male mice due to meiotic defects, involving abnormal pairing and synapsis, unrepaired DNA, and loss of telomere structure. These defects were exacerbated by additional loss of cyclin E1, and complete absence of both E-type cyclins produces a meiotic catastrophe. Here, we investigated the involvement of E-type cyclins in maintaining telomere integrity in male meiosis. Spermatocytes lacking cyclin E2 and one E1 allele (E1+/-E2-/-) displayed a high rate of telomere abnormalities but can progress to pachytene and diplotene stages. We show that their telomeres exhibited an aberrant DNA damage repair response during pachynema and that the shelterin complex proteins TRF2 and RAP2 were significantly decreased in the proximal telomeres. Moreover, the insufficient level of these proteins correlated with an increase of γ-H2AX foci in the affected telomeres and resulted in telomere associations involving TRF1 and telomere detachment in later prophase-I stages. These results suggest that E-type cyclins are key modulators of telomere integrity during meiosis by, at least in part, maintaining the balance of shelterin complex proteins, and uncover a novel role of E-type cyclins in regulating chromosome structure during male meiosis.
Defective in Mitotic Arrest 1 (Dma1) Ubiquitin Ligase Controls G1 Cyclin Degradation*
Hernández-Ortega, Sara; Bru, Samuel; Ricco, Natalia; Ramírez, Sara; Casals, Núria; Jiménez, Javier; Isasa, Marta; Crosas, Bernat; Clotet, Josep
Progression through the G1 phase of the cell cycle is controlled by diverse cyclin-dependent kinases (CDKs) that might be associated to numerous cyclin isoforms. Given such complexity, regulation of cyclin degradation should be crucial for coordinating progression through the cell cycle. In Saccharomyces cerevisiae, SCF is the only E3 ligase known to date to be involved in G1 cyclin degradation. Here, we report the design of a genetic screening that uncovered Dma1 as another E3 ligase that targets G1 cyclins in yeast. We show that the cyclin Pcl1 is ubiquitinated in vitro and in vivo by Dma1, and accordingly, is stabilized in dma1 mutants. We demonstrate that Pcl1 must be phosphorylated by its own CDK to efficiently interact with Dma1 and undergo degradation. A nonphosphorylatable version of Pcl1 accumulates throughout the cell cycle, demonstrating the physiological relevance of the proposed mechanism. Finally, we present evidence that the levels of Pcl1 and Cln2 are independently controlled in response to nutrient availability. This new previously unknown mechanism for G1 cyclin degradation that we report here could help elucidate the specific roles of the redundant CDK-cyclin complexes in G1. PMID:23264631
Gene structure, expression, and DNA methylation characteristics of sea cucumber cyclin B gene during aestivation.
Zhu, Aijun; Chen, Muyan; Zhang, Xiumei; Storey, Kenneth B
The sea cucumber, Apostichopus japonicus, is a good model for studying environmentally-induced aestivation by a marine invertebrate. One of the central requirements of aestivation is the repression of energy-expensive cellular processes such as cell cycle progression. The present study identified the gene structure of the cell cycle regulator, cyclin B, and detected the expression levels of this gene over three stages of the annual aestivation-arousal cycle. Furthermore, the DNA methylation characteristics of cyclin B were analyzed in non-aestivation and deep-aestivation stages of sea cucumbers. We found that the cyclin B promoter contains a CpG island, three CCAAT-boxes and three cell cycle gene homology regions (CHRs). Application of qRT-PCR analysis showed significant downregulation of cyclin B transcript levels during deep-aestivation in comparison with non-aestivation in both intestine and longitudinal muscle, and these returned to basal levels after arousal from aestivation. Methylation analysis of the cyclin B core promoter revealed that its methylation level showed significant differences between non-aestivation and deep-aestivation stages (p<0.05) and interestingly, a positive correlation between Cyclin B transcripts expression and methylation levels of the core promoter was also observed. Our findings suggest that cell cycle progression may be reversibly arrested during aestivation as indicated by the changes in cyclin B expression levels and we propose that DNA methylation is one of the regulatory mechanisms involved in cyclin B transcriptional variation.
A cyclin-dependent kinase inhibitor, dinaciclib in preclinical treatment models of thyroid cancer
Lin, Shu-Fu; Lin, Jen-Der; Hsueh, Chuen; Chou, Ting-Chao; Wong, Richard J.
Background We explored the therapeutic effects of dinaciclib, a cyclin-dependent kinase (CDK) inhibitor, in the treatment of thyroid cancer. Materials and methods Seven cell lines originating from three pathologic types of thyroid cancer (papillary, follicular and anaplastic) were studied. The cytotoxicity of dinaciclib was measured using a lactate dehydrogenase assay. The expression of proteins associated with cell cycle and apoptosis was assessed using Western blot analysis and immunofluorescence microscopy. Cell cycle distribution was measured by flow cytometry and immunofluorescence microscopy. Apoptosis and caspase-3 activity were measured by flow cytometry and fluorometric assay. Mice bearing flank anaplastic thyroid cancer (ATC) were treated with intraperitoneal injections of dinaciclib. Results Dinaciclib inhibited thyroid cancer cell proliferation in a dose-dependent manner. Dinaciclib had a low median-effect dose (≤ 16.0 nM) to inhibit cell proliferation in seven thyroid cancer cell lines. Dinaciclib decreased CDK1, cyclin B1, and Aurora A expression, induced cell cycle arrest in the G2/M phase, and induced accumulation of prophase mitotic cells. Dinaciclib decreased Mcl-1, Bcl-xL and survivin expression, activated caspase-3 and induced apoptosis. In vivo, the growth of ATC xenograft tumors was retarded in a dose-dependent fashion with daily dinaciclib treatment. Higher-dose dinaciclib (50 mg/kg) caused slight, but significant weight loss, which was absent with lower-dose dinaciclib (40 mg/kg) treatment. Conclusions Dinaciclib inhibited thyroid cancer proliferation both in vitro and in vivo. These findings support dinaciclib as a potential drug for further studies in clinical trials for the treatment of patients with refractory thyroid cancer. PMID:28207834
Cyclin F/FBXO1 Interacts with HIV-1 Viral Infectivity Factor (Vif) and Restricts Progeny Virion Infectivity by Ubiquitination and Proteasomal Degradation of Vif Protein through SCF(cyclin F) E3 Ligase Machinery.
Augustine, Tracy; Chaudhary, Priyanka; Gupta, Kailash; Islam, Sehbanul; Ghosh, Payel; Santra, Manas Kumar; Mitra, Debashis
Cyclin F protein, also known as FBXO1, is the largest among all cyclins and oscillates in the cell cycle like other cyclins. Apart from being a G2/M cyclin, cyclin F functions as the substrate-binding subunit of SCF(cyclin F) E3 ubiquitin ligase. In a gene expression analysis performed to identify novel gene modulations associated with cell cycle dysregulation during HIV-1 infection in CD4(+) T cells, we observed down-regulation of the cyclin F gene (CCNF). Later, using gene overexpression and knockdown studies, we identified cyclin F as negatively influencing HIV-1 viral infectivity without any significant impact on virus production. Subsequently, we found that cyclin F negatively regulates the expression of viral protein Vif (viral infectivity factor) at the protein level. We also identified a novel host-pathogen interaction between cyclin F and Vif protein in T cells during HIV-1 infection. Mutational analysis of a cyclin F-specific amino acid motif in the C-terminal region of Vif indicated rescue of the protein from cyclin F-mediated down-regulation. Subsequently, we showed that Vif is a novel substrate of the SCF(cyclin F) E3 ligase, where cyclin F mediates the ubiquitination and proteasomal degradation of Vif through physical interaction. Finally, we showed that cyclin F augments APOBEC3G expression through degradation of Vif to regulate infectivity of progeny virions. Taken together, our results demonstrate that cyclin F is a novel F-box protein that functions as an intrinsic cellular regulator of HIV-1 Vif and has a negative regulatory effect on the maintenance of viral infectivity by restoring APOBEC3G expression.
Cyclin Y-mediated transcript profiling reveals several important functional pathways regulated by Cyclin Y in hippocampal neurons
Kim, Hanna; Hong, Jung-Hwa; Kim, Mirang; Park, Mikyoung
Cyclin Y (CCNY), which is a cyclin protein known to play a role in cell division, is unexpectedly and thus interestingly expressed in non-proliferating neuronal cells. There have been only a few studies reporting the neuronal functions of CCNY in synapse remodeling and hippocampal long-term potentiation. Therefore, we here provide global and comprehensive information on the putative functions of CCNY in biological and functional pathways in neuronal systems. We adopted high-throughput RNA-sequencing technology for analyzing transcriptomes regulated by CCNY and utilized bioinformatics for identifying putative molecules, biological processes, and functional pathways that are possibly connected to CCNY functions in hippocampal neuronal cells of rats. We revealed that several enriched annotation terms and pathways associated with CCNY expression within neurons, including apoptosis, learning or memory, synaptic plasticity, actin cytoskeleton, focal adhesion, extracellular matrix-receptor interaction and chemokine signaling pathway are targeted by CCNY. In addition, the mRNA levels of some genes enriched for those annotation terms and pathways or genes reported to be altered in Alzheimer's disease mouse model were further validated by quantitative real-time PCR in hippocampal neuronal cells. The present study provides an excellent resource for future investigations of CCNY functions in neuronal systems. PMID:28241067
Expression of CDK7, Cyclin H, and MAT1 Is Elevated in Breast Cancer and Is Prognostic in Estrogen Receptor–Positive Breast Cancer
Patel, Hetal; Abduljabbar, Rezvan; Lai, Chun-Fui; Periyasamy, Manikandan; Harrod, Alison; Gemma, Carolina; Steel, Jennifer H.; Patel, Naina; Busonero, Claudia; Jerjees, Dena; Remenyi, Judit; Smith, Sally; Gomm, Jennifer J.; Magnani, Luca; Győrffy, Balázs; Jones, Louise J.; Fuller-Pace, Frances; Shousha, Sami; Buluwela, Laki; Rakha, Emad A.; Ellis, Ian O.; Coombes, R. Charles; Ali, Simak
Purpose CDK-activating kinase (CAK) is required for the regulation of the cell cycle and is a trimeric complex consisting of cyclin-dependent kinase 7 (CDK7), Cyclin H, and the accessory protein, MAT1. CDK7 also plays a critical role in regulating transcription, primarily by phosphorylating RNA polymerase II, as well as transcription factors such as estrogen receptor-α (ER). Deregulation of cell cycle and transcriptional control are general features of tumor cells, highlighting the potential for the use of CDK7 inhibitors as novel cancer therapeutics. Experimental Design mRNA and protein expression of CDK7 and its essential cofactors cyclin H and MAT1 were evaluated in breast cancer samples to determine if their levels are altered in cancer. Immunohistochemical staining of >900 breast cancers was used to determine the association with clinicopathologic features and patient outcome. Results We show that expressions of CDK7, cyclin H, and MAT1 are all closely linked at the mRNA and protein level, and their expression is elevated in breast cancer compared with the normal breast tissue. Intriguingly, CDK7 expression was inversely proportional to tumor grade and size, and outcome analysis showed an association between CAK levels and better outcome. Moreover, CDK7 expression was positively associated with ER expression and in particular with phosphorylation of ER at serine 118, a site important for ER transcriptional activity. Conclusions Expressions of components of the CAK complex, CDK7, MAT1, and Cyclin H are elevated in breast cancer and correlate with ER. Like ER, CDK7 expression is inversely proportional to poor prognostic factors and survival. PMID:27301701
Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.
Deverman, Benjamin E; Pravdo, Piers L; Simpson, Bryan P; Kumar, Sripriya Ravindra; Chan, Ken Y; Banerjee, Abhik; Wu, Wei-Li; Yang, Bin; Huber, Nina; Pasca, Sergiu P; Gradinaru, Viviana
Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics. Here we describe a capsid selection method, called Cre recombination-based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9 (refs. 14,15,16,17), and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications.
A robust activity marking system for exploring active neuronal ensembles.
Sørensen, Andreas T; Cooper, Yonatan A; Baratta, Michael V; Weng, Feng-Ju; Zhang, Yuxiang; Ramamoorthi, Kartik; Fropf, Robin; LaVerriere, Emily; Xue, Jian; Young, Andrew; Schneider, Colleen; Gøtzsche, Casper René; Hemberg, Martin; Yin, Jerry Cp; Maier, Steven F; Lin, Yingxi
Understanding how the brain captures transient experience and converts it into long lasting changes in neural circuits requires the identification and investigation of the specific ensembles of neurons that are responsible for the encoding of each experience. We have developed a Robust Activity Marking (RAM) system that allows for the identification and interrogation of ensembles of neurons. The RAM system provides unprecedented high sensitivity and selectivity through the use of an optimized synthetic activity-regulated promoter that is strongly induced by neuronal activity and a modified Tet-Off system that achieves improved temporal control. Due to its compact design, RAM can be packaged into a single adeno-associated virus (AAV), providing great versatility and ease of use, including application to mice, rats, flies, and potentially many other species. Cre-dependent RAM, CRAM, allows for the study of active ensembles of a specific cell type and anatomical connectivity, further expanding the RAM system's versatility.
The dual role of cyclin C connects stress regulated gene expression to mitochondrial dynamics
Strich, Randy; Cooper, Katrina F.
Following exposure to cytotoxic agents, cellular damage is first recognized by a variety of sensor mechanisms. Thenceforth, the damage signal is transduced to the nucleus to install the correct gene expression program including the induction of genes whose products either detoxify destructive compounds or repair the damage they cause. Next, the stress signal is disseminated throughout the cell to effect the appropriate changes at organelles including the mitochondria. The mitochondria represent an important signaling platform for the stress response. An initial stress response of the mitochondria is extensive fragmentation. If the damage is prodigious, the mitochondria fragment (fission) and lose their outer membrane integrity leading to the release of pro-apoptotic factors necessary for programmed cell death (PCD) execution. As this complex biological process contains many moving parts, it must be exquisitely coordinated as the ultimate decision is life or death. The conserved C-type cyclin plays an important role in executing this molecular Rubicon by coupling changes in gene expression to mitochondrial fission and PCD. Cyclin C, along with its cyclin dependent kinase partner Cdk8, associates with the RNA polymerase holoenzyme to regulate transcription. In particular, cyclin C-Cdk8 repress many stress responsive genes. To relieve this repression, cyclin C is destroyed in cells exposed to pro-oxidants and other stressors. However, prior to its destruction, cyclin C, but not Cdk8, is released from its nuclear anchor (Med13), translocates from the nucleus to the cytoplasm where it interacts with the fission machinery and is both necessary and sufficient to induce extensive mitochondria fragmentation. Furthermore, cytoplasmic cyclin C promotes PCD indicating that it mediates both mitochondrial fission and cell death pathways. This review will summarize the role cyclin C plays in regulating stress-responsive transcription. In addition, we will detail this new function
Dephosphorylation of human cyclin-dependent kinases by protein phosphatase type 2C alpha and beta 2 isoforms.
Cheng, A; Kaldis, P; Solomon, M J
We previously reported that the activating phosphorylation on cyclin-dependent kinases in yeast (Cdc28p) and in humans (Cdk2) is removed by type 2C protein phosphatases. In this study, we characterize this PP2C-like activity in HeLa cell extract and determine that it is due to PP2C beta 2, a novel PP2C beta isoform, and to PP2C alpha. PP2C alpha and PP2C beta 2 co-purified with Mg(2+)-dependent Cdk2/Cdk6 phosphatase activity in DEAE-Sepharose, Superdex-200, and Mono Q chromatographies. Moreover, purified recombinant PP2C alpha and PP2C beta 2 proteins efficiently dephosphorylated monomeric Cdk2/Cdk6 in vitro. The dephosphorylation of Cdk2 and Cdk6 by PP2C isoforms was inhibited by the binding of cyclins. We found that the PP2C-like activity in HeLa cell extract, partially purified HeLa PP2C alpha and PP2C beta 2 isoforms, and the recombinant PP2Cs exhibited a comparable substrate preference for a phosphothreonine containing substrate, consistent with the conservation of threonine residues at the site of activating phosphorylation in CDKs.
Cyclin-dependent kinase inhibitor, p21Waf1, regulates vascular smooth muscle cell hypertrophy.
Okamoto, Kenichi; Kato, Seiya; Arima, Nobuyuki; Fujii, Teruhiko; Morimatsu, Minoru; Imaizumi, Tsutomu
In the process of vascular diseases, smooth muscle cells (SMC) undergo not only hyperplasia but also hypertrophy, resulting in vascular remodeling. A cyclin-dependent kinase inhibitor (CDKI), p21Waf1, has been shown to play an important role in SMC hyperplasia. Here we investigated a potential role of p21Waf1 in SMC hypertrophy. An exposure of cultured rat SMC to serum drove the cell cycle progression with up-regulation of various cell cycle markers and increased activities of cyclin-dependent kinases, but did not cause SMC hypertrophy. In contrast, incubation of SMC for 48 h with angiotensin II (AII, 100 nmol/l) resulted in a significant increase in the cell size measured by flowcytometric forward-angle light scatter assay, in association with an increase in the ratio of [3H]leucine/[3H]thymidine uptake, indicating SMC hypertrophy. At 48 h, p21Waf1 expression was up-regulated in SMC exposed to AII but not in those exposed to serum. These results suggest that p21Waf1 may be involved in hypertrophy. To further investigate this issue, two manipulations of the p21Waf1 gene were performed. Adenovirus-mediated over-expression of p21Waf1 not only reduced S-phasic cells but also caused hypertrophy, despite the exposure to serum. Antisense oligodeoxynucleotide for p21Waf1 inhibited the hypertrophy of SMC exposed to AII. Our data suggest that p21Waf1 may play a role in SMC hypertrophy as well.
Ethylene-Mediated Regulation of A2-Type CYCLINs Modulates Hyponastic Growth in Arabidopsis1[OPEN
Polko, Joanna K.; van Rooij, Jop A.; Vanneste, Steffen; Pierik, Ronald; Ammerlaan, Ankie M.H.; Vergeer-van Eijk, Marleen H.; McLoughlin, Fionn; Gühl, Kerstin; Van Isterdael, Gert; Voesenek, Laurentius A.C.J.; Millenaar, Frank F.; Beeckman, Tom; Peeters, Anton J.M.; Marée, Athanasius F.M.; van Zanten, Martijn
Upward leaf movement (hyponastic growth) is frequently observed in response to changing environmental conditions and can be induced by the phytohormone ethylene. Hyponasty results from differential growth (i.e. enhanced cell elongation at the proximal abaxial side of the petiole relative to the adaxial side). Here, we characterize Enhanced Hyponasty-d, an activation-tagged Arabidopsis (Arabidopsis thaliana) line with exaggerated hyponasty. This phenotype is associated with overexpression of the mitotic cyclin CYCLINA2;1 (CYCA2;1), which hints at a role for cell divisions in regulating hyponasty. Indeed, mathematical analysis suggested that the observed changes in abaxial cell elongation rates during ethylene treatment should result in a larger hyponastic amplitude than observed, unless a decrease in cell proliferation rate at the proximal abaxial side of the petiole relative to the adaxial side was implemented. Our model predicts that when this differential proliferation mechanism is disrupted by either ectopic overexpression or mutation of CYCA2;1, the hyponastic growth response becomes exaggerated. This is in accordance with experimental observations on CYCA2;1 overexpression lines and cyca2;1 knockouts. We therefore propose a bipartite mechanism controlling leaf movement: ethylene induces longitudinal cell expansion in the abaxial petiole epidermis to induce hyponasty and simultaneously affects its amplitude by controlling cell proliferation through CYCA2;1. Further corroborating the model, we found that ethylene treatment results in transcriptional down-regulation of A2-type CYCLINs and propose that this, and possibly other regulatory mechanisms affecting CYCA2;1, may contribute to this attenuation of hyponastic growth. PMID:26041787
Cyclin Dependent Kinase 9 Inhibitors for Cancer Therapy.
Sonawane, Yogesh A; Taylor, Margaret A; Napoleon, John Victor; Rana, Sandeep; Contreras, Jacob I; Natarajan, Amarnath
Cyclin dependent kinase (CDK) inhibitors have been the topic of intense research for nearly 2 decades due to their widely varied and critical functions within the cell. Recently CDK9 has emerged as a druggable target for the development of cancer therapeutics. CDK9 plays a crucial role in transcription regulation; specifically, CDK9 mediated transcriptional regulation of short-lived antiapoptotic proteins is critical for the survival of transformed cells. Focused chemical libraries based on a plethora of scaffolds have resulted in mixed success with regard to the development of selective CDK9 inhibitors. Here we review the regulation of CDK9, its cellular functions, and common core structures used to target CDK9, along with their selectivity profile and efficacy in vitro and in vivo.
[Cytotoxicity of chimera peptides incorporating sequences of cyclin kinases inhibitors].
Kharchenko, V P; Kulinich, V G; Lunin, V G; Filiasova, E I; Shishkin, A M; Sergeenko, O V; Riazanova, E M; Voronina, O L; Bozhenko, V K
The study is concerned with proapoptotic properties of chimera peptides which incorporate sequences of inhibitors of cyclin kinases p161NK4a and p21CIP/WAF1 as well as internalized sequences (Antp and tat). Sequences of the p16 type appeared to be more cytotoxic than the p21 one. Cytotoxic effect proved dependent on orientation with respect to the C or N terminal point of a polypeptide chain rather than on chimera sequence extent. Although p16 endogenous synthesis did not influence chimera peptide levels, apoptosis did not take place in certain cellular lines. Due to the rather unsophisticated nature of such synthesis, it might be used in designing individually-tailored chemotherapeutic drugs.
A Novel Intracellular Peptide Derived from G1/S Cyclin D2 Induces Cell Death*
de Araujo, Christiane B.; Russo, Lilian C.; Castro, Leandro M.; Forti, Fábio L.; do Monte, Elisabete R.; Rioli, Vanessa; Gozzo, Fabio C.; Colquhoun, Alison; Ferro, Emer S.
Intracellular peptides are constantly produced by the ubiquitin-proteasome system, and many are probably functional. Here, the peptide WELVVLGKL (pep5) from G1/S-specific cyclin D2 showed a 2-fold increase during the S phase of HeLa cell cycle. pep5 (25–100 μm) induced cell death in several tumor cells only when it was fused to a cell-penetrating peptide (pep5-cpp), suggesting its intracellular function. In vivo, pep5-cpp reduced the volume of the rat C6 glioblastoma by almost 50%. The tryptophan at the N terminus of pep5 is essential for its cell death activity, and N terminus acetylation reduced the potency of pep5-cpp. WELVVL is the minimal active sequence of pep5, whereas Leu-Ala substitutions totally abolished pep5 cell death activity. Findings from the initial characterization of the cell death/signaling mechanism of pep5 include caspase 3/7 and 9 activation, inhibition of Akt2 phosphorylation, activation of p38α and -γ, and inhibition of proteasome activity. Further pharmacological analyses suggest that pep5 can trigger cell death by distinctive pathways, which can be blocked by IM-54 or a combination of necrostatin-1 and q-VD-OPh. These data further support the biological and pharmacological potential of intracellular peptides. PMID:24764300
Drosophila cyclin D/Cdk4 regulates mitochondrial biogenesis and aging and sensitizes animals to hypoxic stress
Icreverzi, Amalia; Flor de la Cruz, Aida; Van Voorhies, Wayne A
Drosophila cyclin D (CycD) is the single fly ortholog of the mammalian cyclin D1 and promotes both cell cycle progression and cellular growth. However, little is known about how CycD promotes cell growth. We show here that CycD/Cdk4 hyperactivity leads to increased mitochondrial biogenesis (mitobiogenesis), mitochondrial mass, NRF-1 activity (Tfam transcript levels) and metabolic activity in Drosophila, whereas loss of CycD/Cdk4 activity has the opposite effects. Surprisingly, both CycD/Cdk4 addition and loss of function increase mitochondrial superoxide production and decrease lifespan, indicating that an imbalance in mitobiogenesis may lead to oxidative stress and aging. In addition, we provide multiple lines of evidence indicating that CycD/Cdk4 activity affects the hypoxic status of cells and sensitizes animals to hypoxia. Both mitochondrial and hypoxia-related effects can be detected at global transcriptional level. We propose that mitobiogenesis and the hypoxic stress response have an antagonistic relationship, and that CycD/Cdk4 levels regulate mitobiogenesis contemporaneous to the cell cycle, such that only when cells are sufficiently oxygenated can they proliferate. PMID:22293404
Berberine Suppresses Cyclin D1 Expression through Proteasomal Degradation in Human Hepatoma Cells
Wang, Ning; Wang, Xuanbin; Tan, Hor-Yue; Li, Sha; Tsang, Chi Man; Tsao, Sai-Wah; Feng, Yibin
The aim of this study is to explore the underlying mechanism on berberine-induced Cyclin D1 degradation in human hepatic carcinoma. We observed that berberine could suppress both in vitro and in vivo expression of Cyclin D1 in hepatoma cells. Berberine exhibits dose- and time-dependent inhibition on Cyclin D1 expression in human hepatoma cell HepG2. Berberine increases the phosphorylation of Cyclin D1 at Thr286 site and potentiates Cyclin D1 nuclear export to cytoplasm for proteasomal degradation. In addition, berberine recruits the Skp, Cullin, F-box containing complex-β-Transducin Repeat Containing Protein (SCFβ-TrCP) complex to facilitate Cyclin D1 ubiquitin-proteasome dependent proteolysis. Knockdown of β-TrCP blocks Cyclin D1 turnover induced by berberine; blocking the protein degradation induced by berberine in HepG2 cells increases tumor cell resistance to berberine. Our results shed light on berberine′s potential as an anti-tumor agent for clinical cancer therapy. PMID:27854312
Amygdalin blocks bladder cancer cell growth in vitro by diminishing cyclin A and cdk2.
Makarević, Jasmina; Rutz, Jochen; Juengel, Eva; Kaulfuss, Silke; Reiter, Michael; Tsaur, Igor; Bartsch, Georg; Haferkamp, Axel; Blaheta, Roman A
Amygdalin, a natural compound, has been used by many cancer patients as an alternative approach to treat their illness. However, whether or not this substance truly exerts an anti-tumor effect has never been settled. An in vitro study was initiated to investigate the influence of amygdalin (1.25-10 mg/ml) on the growth of a panel of bladder cancer cell lines (UMUC-3, RT112 and TCCSUP). Tumor growth, proliferation, clonal growth and cell cycle progression were investigated. The cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1, p19, p27 as well as the mammalian target of rapamycin (mTOR) related signals phosphoAkt, phosphoRaptor and phosphoRictor were examined. Amygdalin dose-dependently reduced growth and proliferation in all three bladder cancer cell lines, reflected in a significant delay in cell cycle progression and G0/G1 arrest. Molecular evaluation revealed diminished phosphoAkt, phosphoRictor and loss of Cdk and cyclin components. Since the most outstanding effects of amygdalin were observed on the cdk2-cyclin A axis, siRNA knock down studies were carried out, revealing a positive correlation between cdk2/cyclin A expression level and tumor growth. Amygdalin, therefore, may block tumor growth by down-modulating cdk2 and cyclin A. In vivo investigation must follow to assess amygdalin's practical value as an anti-tumor drug.
Amygdalin Blocks Bladder Cancer Cell Growth In Vitro by Diminishing Cyclin A and cdk2
Makarević, Jasmina; Rutz, Jochen; Juengel, Eva; Kaulfuss, Silke; Reiter, Michael; Tsaur, Igor; Bartsch, Georg; Haferkamp, Axel; Blaheta, Roman A.
Amygdalin, a natural compound, has been used by many cancer patients as an alternative approach to treat their illness. However, whether or not this substance truly exerts an anti-tumor effect has never been settled. An in vitro study was initiated to investigate the influence of amygdalin (1.25–10 mg/ml) on the growth of a panel of bladder cancer cell lines (UMUC-3, RT112 and TCCSUP). Tumor growth, proliferation, clonal growth and cell cycle progression were investigated. The cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1, p19, p27 as well as the mammalian target of rapamycin (mTOR) related signals phosphoAkt, phosphoRaptor and phosphoRictor were examined. Amygdalin dose-dependently reduced growth and proliferation in all three bladder cancer cell lines, reflected in a significant delay in cell cycle progression and G0/G1 arrest. Molecular evaluation revealed diminished phosphoAkt, phosphoRictor and loss of Cdk and cyclin components. Since the most outstanding effects of amygdalin were observed on the cdk2-cyclin A axis, siRNA knock down studies were carried out, revealing a positive correlation between cdk2/cyclin A expression level and tumor growth. Amygdalin, therefore, may block tumor growth by down-modulating cdk2 and cyclin A. In vivo investigation must follow to assess amygdalin's practical value as an anti-tumor drug. PMID:25136960
A comparative study of the degradation of yeast cyclins Cln1 and Cln2.
Quilis, Inma; Igual, J Carlos
The yeast cyclins Cln1 and Cln2 are very similar in both sequence and function, but some differences in their functionality and localization have been recently described. The control of Cln1 and Cln2 cellular levels is crucial for proper cell cycle initiation. In this work, we analyzed the degradation patterns of Cln1 and Cln2 in order to further investigate the possible differences between them. Both cyclins show the same half-life but, while Cln2 degradation depends on ubiquitin ligases SCF(G)(rr1) and SCF(C)(dc4), Cln1 is affected only by SCF(G)(rr1). Degradation analysis of chimeric cyclins, constructed by combining fragments from Cln1 and Cln2, identifies the N-terminal sequence of the proteins as responsible of the cyclin degradation pattern. In particular, the N-terminal region of Cln2 is required to mediate degradation by SCF(C)(dc4). This region is involved in nuclear import of Cln1 and Cln2, which suggests that differences in degradation may be due to differences in localization. Moreover, a comparison of the cyclins that differ only in the presence of the Cln2 nuclear export signal indicates a greater instability of exported cyclins, thus reinforcing the idea that cyclin stability is influenced by their localization.
Cyclin A2 promotes DNA repair in the brain during both development and aging
Gygli, Patrick E.; Chang, Joshua C.; Gokozan, Hamza N.; Catacutan, Fay P.; Schmidt, Theresa A.; Kaya, Behiye; Goksel, Mustafa; Baig, Faisal S.; Chen, Shannon; Griveau, Amelie; Michowski, Wojciech; Wong, Michael; Palanichamy, Kamalakannan; Sicinski, Piotr; Nelson, Randy J.; Czeisler, Catherine; Otero, José J.
Various stem cell niches of the brain have differential requirements for Cyclin A2. Cyclin A2 loss results in marked cerebellar dysmorphia, whereas forebrain growth is retarded during early embryonic development yet achieves normal size at birth. To understand the differential requirements of distinct brain regions for Cyclin A2, we utilized neuroanatomical, transgenic mouse, and mathematical modeling techniques to generate testable hypotheses that provide insight into how Cyclin A2 loss results in compensatory forebrain growth during late embryonic development. Using unbiased measurements of the forebrain stem cell niche, we parameterized a mathematical model whereby logistic growth instructs progenitor cells as to the cell-types of their progeny. Our data was consistent with prior findings that progenitors proliferate along an auto-inhibitory growth curve. The growth retardation in CCNA2-null brains corresponded to cell cycle lengthening, imposing a developmental delay. We hypothesized that Cyclin A2 regulates DNA repair and that CCNA2-null progenitors thus experienced lengthened cell cycle. We demonstrate that CCNA2-null progenitors suffer abnormal DNA repair, and implicate Cyclin A2 in double-strand break repair. Cyclin A2's DNA repair functions are conserved among cell lines, neural progenitors, and hippocampal neurons. We further demonstrate that neuronal CCNA2 ablation results in learning and memory deficits in aged mice. PMID:27425845
Molecular cloning and functional characterization of cyclin E and CDK2 from Penaeus monodon.
Zhao, C; Fu, M J; Qiu, L H
Reduced reproductive performance of the black tiger shrimp (Penaeus monodon) has caused economic losses and hampered the fishing industry. Detailed investigation of the molecular mechanism by which the cell cycle is regulated in this organism is needed to understand the development and maturation of ovaries and oocytes, with a view to improving reproductive capacity. Cell cycle progression is mainly determined by cyclin-dependent kinase (CDK) and cyclin complexes, the cyclin E/CDK2 complex playing a key role in G1/S transition. However, knowledge of the interplay between cyclin E and CDK2 in invertebrates remains limited. In this study, full-length P. monodon cyclin E (Pmcyclin E) and CDK2 (PmCDK2) sequences were cloned. The open reading frame of Pmcyclin E was 1263 bp in length and encoded a 47.9-kDa protein, while that of PmCDK2 was 921 bp, encoding a protein of 34.9 kDa. Recombinant cyclin E and CDK2 proteins were expressed in Escherichia coli and purified by Ni-chelating affinity chromatography. In addition, a pull-down assay was performed to identify any interaction between Pmcyclin E and PmCDK2. This research provides a basis for the study of the functional mechanisms of the cyclin E/CDK2 complex in shrimp, further enriching our knowledge of invertebrate cell cycle regulation.
Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes.
Roques, Magali; Wall, Richard J; Douglass, Alexander P; Ramaprasad, Abhinay; Ferguson, David J P; Kaindama, Mbinda L; Brusini, Lorenzo; Joshi, Nimitray; Rchiad, Zineb; Brady, Declan; Guttery, David S; Wheatley, Sally P; Yamano, Hiroyuki; Holder, Anthony A; Pain, Arnab; Wickstead, Bill; Tewari, Rita
Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.
B-type cyclins regulate G1 progression in fission yeast in opposition to the p25rum1 cdk inhibitor.
Martin-Castellanos, C; Labib, K; Moreno, S
The onset of S phase in fission yeast is regulated at Start, the point of commitment to the mitotic cell cycle. The p34cdc2 kinase is essential for G1 progression past Start, but until now its regulation has been poorly understood. Here we show that the cig2/cyc17 B-type cyclin has an important role in G1 progression, and demonstrate that p34cdc2 kinase activity is periodically associated with cig2 in G1. Cells lacking cig2 are defective in G1 progression, and this is particularly clear in small cells that must regulate Start with respect to cell size. We also find that the cig1 B-type cyclin can promote G1 progression. Whilst p25rum1 can inhibit cig2/cdc2 activity in vitro, and may transiently inhibit this complex in vivo, cig1 is regulated independently of p25rum1. Since cig1/cdc2 kinase activity peaks in mitotic cells, and decreases after mitosis with similar kinetics to cdc13-associated kinase activity, we suggest that cig2 is likely to be the principal fission yeast G1 cyclin. cig2 protein levels accumulate in G1 cells, and we propose that p25rum1 may transiently inhibit cig2-associated p34cdc2 activity until the critical cell size required for Start is reached. Images PMID:8631305
Dephosphorylation of cyclin-dependent kinases by type 2C protein phosphatases
Cheng, Aiyang; Ross, Karen E.; Kaldis, Philipp; Solomon, Mark J.
Activating phosphorylation of cyclin-dependent protein kinases (CDKs) is necessary for their kinase activity and cell cycle progression. This phosphorylation is carried out by the Cdk-activating kinase (CAK); in contrast, little is known about the corresponding protein phosphatase. We show that type 2C protein phosphatases (PP2Cs) are responsible for this dephosphorylation of Cdc28p, the major budding yeast CDK. Two yeast PP2Cs, Ptc2p and Ptc3p, display Cdc28p phosphatase activity in vitro and in vivo, and account for ∼90% of Cdc28p phosphatase activity in yeast extracts. Overexpression of PTC2 or PTC3 results in synthetic lethality in strains temperature-sensitive for yeast CAK1, and disruptions of PTC2 and PTC3 suppress the growth defect of a cak1 mutant. Furthermore, PP2C-like enzymes are the predominant phosphatases toward human Cdk2 in HeLa cell extracts, indicating that the substrate specificity of PP2Cs toward CDKs is evolutionarily conserved. PMID:10580002
p35 and p39 are essential for cyclin-dependent kinase 5 function during neurodevelopment.
Ko, J; Humbert, S; Bronson, R T; Takahashi, S; Kulkarni, A B; Li, E; Tsai, L H
Cyclin-dependent kinase 5 (Cdk5) plays a pivotal role in brain development and neuronal migration. Cdk5 is abundant in postmitotic, terminally differentiated neurons. The ability of Cdk5 to phosphorylate substrates is dependent on activation by its neuronal-specific activators p35 and p39. There exist striking differences in the phenotypic severity of Cdk5-deficient mice and p35-deficient mice. Cdk5-null mutants show a more severe disruption of lamination in the cerebral cortex, hippocampus, and cerebellum. In addition, Cdk5-null mice display perinatal lethality, whereas p35-null mice are viable. These discrepancies have been attributed to the function of other Cdk5 activators, such as p39. To understand the roles of p39 and p35, we created p39-null mice and p35/p39 compound-mutant mice. Interestingly, p39-null mice show no obvious detectable abnormalities, whereas p35(-/-)p39(-/-) double-null mutants are perinatal lethal. We show here that the p35(-/-)p39(-/-) mutants exhibit phenotypes identical to those of the Cdk5-null mutant mice. Other compound-mutant mice with intermediate phenotypes allow us to determine the distinct and redundant functions between p35 and p39. Our data strongly suggest that p35 and p39 are essential for Cdk5 activity during the development of the nervous system. Thus, p35 and p39 are likely to be the principal, if not the only, activators of Cdk5.
Role of Cyclin E as an Early Event in Ovarian Carcinogenesis
of RCAS-cyclin E virus: The DF-1 chicken fibroblastic cell line was used for RCAS-cyclin E FL and RCAS-cyclin E trunc2 transfection and subsequent...DF-1 cells expressing RCAS viruses with different oncogenes were grown in 10-cm tissue culture dishes in DMEM with 10% FBS, and 1% antibiotics . Once...storing at - 80°C. Mouse Model: We have crossed transgenic mice that express Keratin5-TVA ( chicken retroviral keratin receptor that is expressed
Molecular cloning and chromosomal localization of the human cyclin C (CCNC) and cyclin E (CCNE) genes: Deletion of the CCNC gene in human tumors
Li, Haimin; Lahti, J.M.; Kidd, V.J.
The human G1-phase cyclins are important regulators of cell cycle progression that interact with various cyclin-dependent kinases and facilitate entry into S-phase. We have confirmed the localization of the human cyclin C (CCNC) gene to chromosome 6q21 and of human cyclin E (CCNE to 19q12). The CCNC gene structure was also determined, and we have shown that it is deleted in a subset of acute lymphoblastic leukemias, including a patient sample containing a t(2;6)(p21;q15), with no apparent cytogenetic deletion. Single-strand conformational polymorphism analysis of the remaining CCNC allele from patients with a deletion of one allele established that there were no further mutations within the exons or the flanking intronic sequences. These results suggest either that haploinsufficiency of the cyclin C protein is sufficient to promote tumorigenesis or that the important tumor suppressor gene is linked to the CCNC locus. 48 refs., 4 figs., 1 tab.
Overexpression of Ki-67 and cyclins A and B1 in JC virus-infected cells of progressive multifocal leukoencephalopathy.
Ariza, A; Mate, J L; Isamat, M; Calatrava, A; Fernández-Vasalo, A; Navas-Palacios, J J
Both SV40 and JC virus (JCV) appropriate the host cell replicative machinery to attend to their own reproductive needs. SV40 large T antigen is able to induce the expression of cyclins A, B1, and E (but not of cylin D1) in transfected diploid cells. Whether JCV infection influences cyclin expression in a similar fashion in the setting of progressive multifocal leukoencephalopathy (PML) remains unknown. Brain lesions from 7 PML cases (4 autopsies and 3 biopsies) were immunohistochemically investigated for the expression of Ki-67 and cyclins A, B1, and D1. All 7 cases showed strong positivity for Ki-67 and cyclins A and B1 in JCV-infected oligodendrocytes and astrocytes, the nuclear immunolocalization of cyclin A being in strong contrast to the cytoplasmic distribution of cyclin B1. No immunostaining for cyclin D1 was obtained in any of the 7 cases. These findings suggest that JCV infection is associated with overexpression of Ki-67 and cyclins A and B1 in PML host glial cells. Since cyclin changes in JCV-infected cells recapitulate SV40 T antigen-associated cyclin fluctuations, it appears reasonable to think that JCV T antigen shares some of the previously described capabilities of SV40 T antigen to alter cyclin expression for the sake of viral replication.
A plant-specific cyclin-dependent kinase is involved in the control of G2/M progression in plants.
Porceddu, A; Stals, H; Reichheld, J P; Segers, G; De Veylder, L; Barroco, R P; Casteels, P; Van Montagu, M; Inzé, D; Mironov, V
Cyclin-dependent kinases (CDKs) control the key transitions in the eukaryotic cell cycle. All the CDKs known to control G(2)/M progression in yeast and animals are distinguished by the characteristic PSTAIRE motif in their cyclin-binding domain and are closely related. Higher plants contain in addition a number of more divergent non-PSTAIRE CDKs with still obscure functions. We show that a plant-specific type of non-PSTAIRE CDKs is involved in the control of the G(2)/M progression. In synchronized tobacco BY-2 cells, the corresponding protein, accumulated in a cell cycle-regulated fashion, peaking at the G(2)/M transition. The associated histone H1 kinase activity reached a maximum in mitosis and required a yet unidentified subunit to be fully active. Down-regulation of the associated kinase activity in transgenic tobacco plants using a dominant-negative mutation delayed G(2)/M transition. These results provide the first evidence that non-PSTAIRE CDKs are involved in the control of the G(2)/M progression in plants.
Bacterial and plant signal integration via D3-type cyclins enhances symptom development in the Arabidopsis-Rhodococcus fascians interaction.
Stes, Elisabeth; Biondi, Stefania; Holsters, Marcelle; Vereecke, Danny
The phytopathogenic actinomycete Rhodococcus fascians drives its host to form a nutrient-rich niche by secreting a mixture of cytokinins that triggers plant cell division and shoot formation. The discrepancy between the relatively low amount of secreted cytokinins and the severe impact of R. fascians infection on plant development has puzzled researchers for a long time. Polyamine and transcript profiling of wild-type and cytokinin receptor mutant plants revealed that the bacterial cytokinins directly stimulated the biosynthesis of plant putrescine by activating arginine decarboxylase expression. Pharmacological experiments showed that the increased levels of putrescine contributed to the severity of the symptoms. Thus, putrescine functions as a secondary signal that impinges on the cytokinin-activated pathway, amplifying the hormone-induced changes that lead to the formation of a leafy gall. Exogenous putrescine and treatment with polyamine biosynthesis inhibitors combined with transcript and polyamine analyses of wild-type and mutant plants indicated that the direct target of both the bacterial cytokinins and plant putrescine was the expression of D3-type cyclins. Hence, the activated d-type cyclin/retinoblastoma/E2F transcription factor pathway integrates both external and internal hormonal signals, stimulating mitotic cell divisions and inducing pathological plant organogenesis.
Cell division cycle 6, a mitotic substrate of polo-like kinase 1, regulates chromosomal segregation mediated by cyclin-dependent kinase 1 and separase.
Yim, Hyungshin; Erikson, Raymond L
Defining the links between cell division and DNA replication is essential for understanding normal cell cycle progression and tumorigenesis. In this report we explore the effect of phosphorylation of cell division cycle 6 (Cdc6), a DNA replication initiation factor, by polo-like kinase 1 (Plk1) on the regulation of chromosomal segregation. In mitosis, the phosphorylation of Cdc6 was highly increased, in correlation with the level of Plk1, and conversely, Cdc6 is hypophosphorylated in Plk1-depleted cells, although cyclin A- and cyclin B1-dependent kinases are active. Binding between Cdc6 and Plk1 occurs through the polo-box domain of Plk1, and Cdc6 is phosphorylated by Plk1 on T37. Immunohistochemistry studies reveal that Cdc6 and Plk1 colocalize to the central spindle in anaphase. Expression of T37V mutant of Cdc6 (Cdc6-TV) induces binucleated cells and incompletely separated nuclei. Wild-type Cdc6 but not Cdc6-TV binds cyclin-dependent kinase 1 (Cdk1). Expression of wild-type Plk1 but not kinase-defective mutant promotes the binding of Cdc6 to Cdk1. Cells expressing wild-type Cdc6 display lower Cdk1 activity and higher separase activity than cells expressing Cdc6-TV. These results suggest that Plk1-mediated phosphorylation of Cdc6 promotes the interaction of Cdc6 and Cdk1, leading to the attenuation of Cdk1 activity, release of separase, and subsequent anaphase progression.
The structure and substrate specificity of human Cdk12/Cyclin K
Bösken, Christian A.; Farnung, Lucas; Hintermair, Corinna; Merzel Schachter, Miriam; Vogel-Bachmayr, Karin; Blazek, Dalibor; Anand, Kanchan; Fisher, Robert P.; Eick, Dirk; Geyer, Matthias
Phosphorylation of the RNA polymerase II C-terminal domain (CTD) by cyclin-dependent kinases is important for productive transcription. Here we determine the crystal structure of Cdk12/CycK and analyse its requirements for substrate recognition. Active Cdk12/CycK is arranged in an open conformation similar to that of Cdk9/CycT but different from those of cell cycle kinases. Cdk12 contains a C-terminal extension that folds onto the N- and C-terminal lobes thereby contacting the ATP ribose. The interaction is mediated by an HE motif followed by a polybasic cluster that is conserved in transcriptional CDKs. Cdk12/CycK showed the highest activity on a CTD substrate prephosphorylated at position Ser7, whereas the common Lys7 substitution was not recognized. Flavopiridol is most potent towards Cdk12 but was still 10-fold more potent towards Cdk9. T-loop phosphorylation of Cdk12 required coexpression with a Cdk-activating kinase. These results suggest the regulation of Pol II elongation by a relay of transcriptionally active CTD kinases. PMID:24662513
Endoglin inhibits ERK-induced c-Myc and cyclin D1 expression to impede endothelial cell proliferation
Pan, Christopher C.; Bloodworth, Jeffrey C.; Mythreye, Karthikeyan; Lee, Nam Y.
Highlights: Black-Right-Pointing-Pointer Endoglin inhibits ERK activation in endothelial cells. Black-Right-Pointing-Pointer Endoglin is a regulator of c-Myc and cyclin D1 expression. Black-Right-Pointing-Pointer {beta}-arrestin2 interaction with endoglin is required for ERK/c-Myc repression. Black-Right-Pointing-Pointer Endoglin impedes cellular proliferation by targeting ERK-induced mitogenic signaling. -- Abstract: Endoglin is an endothelial-specific transforming growth factor beta (TGF-{beta}) co-receptor essential for angiogenesis and vascular remodeling. Endoglin regulates a wide range of cellular processes, including cell adhesion, migration, and proliferation, through TGF-{beta} signaling to canonical Smad and Smad-independent pathways. Despite its overall pro-angiogenic role in the vasculature, the underlying mechanism of endoglin action is poorly characterized. We previously identified {beta}-arrestin2 as a binding partner that causes endoglin internalization from the plasma membrane and inhibits ERK signaling towards endothelial migration. In the present study, we examined the mechanistic role of endoglin and {beta}-arrestin2 in endothelial cell proliferation. We show that endoglin impedes cell growth through sustained inhibition of ERK-induced c-Myc and cyclin D1 expression in a TGF-{beta}-independent manner. The down-regulation of c-Myc and cyclin D1, along with growth-inhibition, are reversed when the endoglin/{beta}-arrestin2 interaction is disrupted. Given that TGF-{beta}-induced Smad signaling potently represses c-Myc in most cell types, our findings here show a novel mechanism by which endoglin augments growth-inhibition by targeting ERK and key downstream mitogenic substrates.
The cell cycle, cyclin-dependent kinases, and viral infections: new horizons and unexpected connections.
Schang, Luis M
The genomes of small DNA viruses such as papilloma and polyomaviruses code for few or no DNA replication proteins. Consequently, these viruses depend on cellular DNA replication proteins to replicate their genomes and replicate only when the infected cell progresses into S-phase, when these proteins are active. As a consequence of this strict dependence, the relationship between replication of the small DNA viruses and the cell cycle was obvious from the very early studies. The genomes of larger DNA viruses such as adeno- and herpes-viruses, in contrast, encode many of the proteins required for DNA replication. Some of the larger DNA viruses such as adenoviruses, however, also replicate only in S-phase because expression of viral DNA replication proteins is regulated by cellular factors that are activated in S-phase. Other large DNA viruses such as herpes simplex viruses (HSV) can replicate in arrested cells such as neurons, without inducing progression into S-phase. The relationships between cell cycle and replication of these last viruses are, thus, so subtle that their replication was long thought to be independent from cellular proteins whose activities are regulated in a cell cycle dependent manner. In contrast to this hypothesis, recent studies have shown that replication of HSV and other large DNA viruses requires cellular proteins whose activities are normally regulated in a cell cycle dependent manner, such as the cyclin-dependent kinases (cdks). Many excellent reviews on the interactions between cellular proteins involved in cell cycle regulation and smaller DNA viruses (parvo, papilloma, polyoma and adenoviruses) have been published (for example, see (1, 2)). Many reviews on cell cycle regulation also discuss the interactions between the cell cycle and the smaller DNA viruses (for example, see (3-5)). Herein, we will review these relationships only briefly, while focusing on the interactions between cell cycle proteins such as cdks and herpes-, retro
Cyclin Partners Determine Pho85 Protein Kinase Substrate Specificity In Vitro and In Vivo: Control of Glycogen Biosynthesis by Pcl8 and Pcl10
Huang, Dongqing; Moffat, Jason; Wilson, Wayne A.; Moore, Lynda; Cheng, Christine; Roach, Peter J.; Andrews, Brenda
In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls. In association with the cyclin Pho80, Pho85 controls acid phosphatase gene expression through phosphorylation of the transcription factor Pho4. Pho85 has also been implicated as a kinase that phosphorylates and negatively regulates glycogen synthase (Gsy2), and deletion of PHO85 causes glycogen overaccumulation. We report that the Pcl8/Pcl10 subgroup of cyclins directs Pho85 to phosphorylate glycogen synthase both in vivo and in vitro. Disruption of PCL8 and PCL10 caused hyperaccumulation of glycogen, activation of glycogen synthase, and a reduction in glycogen synthase kinase activity in vivo. However, unlike pho85 mutants, pcl8 pcl10 cells had normal morphologies, grew on glycerol, and showed proper regulation of acid phosphatase gene expression. In vitro, Pho80-Pho85 complexes effectively phosphorylated Pho4 but had much lower activity toward Gsy2. In contrast, Pcl10-Pho85 complexes phosphorylated Gsy2 at Ser-654 and Thr-667, two physiologically relevant sites, but only poorly phosphorylated Pho4. Thus, both the in vitro and in vivo substrate specificity of Pho85 is determined by the cyclin partner. Mutation of PHO85 suppressed the glycogen storage deficiency of snf1 or glc7-1 mutants in which glycogen synthase is locked in an inactive state. Deletion of PCL8 and PCL10 corrected the deficit in glycogen synthase activity in both the snf1 and glc7-1 mutants, but glycogen synthesis was restored only in the glc7-1 mutant strain. This genetic result suggests an additional role for Pho85 in the negative regulation of glycogen accumulation that is independent of Pcl8 and Pcl10. PMID:9584169
The δ-cyclin expression at early stages of embryogenesis of Brassica rapa L. under clinorotation
NASA Astrophysics Data System (ADS)
Artemenko, O. A.; Popova, A. F.
We present some results of comparison studying of Brassica embryo development and the δ-cyclin genes expression under slow horizontal clinorotation and in the laboratory control. Some backlog of the δ1-cyclin genes expression at early stages of embryogenesis under clinorotation was revealed in comparison with the laboratory control. The similar level of the δ3-cyclin expression at all stages of embryo formation (from one to nine days) in both variants is shown. Some delays in the rate of Brassica rapa embryo development under clinorotation in comparison with the laboratory control can be a result of decrease of a level and some backlog of the δ1-cyclin expression at early stages of embryogenesis.
Cyclin D1 G870A polymorphism and risk of colorectal cancer: a case control study.
Sameer, Aga Syed; Parray, Fazl Q; Dar, Manzoor Ahmad; Nissar, Saniya; Banday, Mujeeb Zafar; Rasool, Sabha; Gulzar, G M; Chowdri, Nissar A; Siddiqi, Mushtaq A
The present study aimed to analyse the role of cyclin D1 A870G polymorphism in modulating the susceptibility to colorectal cancer (CRC) in the Kashmiri population. The genotype distribution of the cyclin D1 gene in 130 CRC cases in comparison with 160 healthy controls was investigated. No direct significant association between cyclin D1 genotypes and CRC was observed; however, the AG and AA genotypes were found to be associated with an increased risk of CRC compared to the GG genotype, with an almost 2-fold increase in OR. This study suggests that the cyclin D1 polymorphism is associated with an increased risk of CRC in the Kashmiri population.
Growth dynamics and cyclin expression in cutaneous T-cell lymphoma cell lines
Biskup, Edyta; Manfé, Valentina; Kamstrup, Maria R.; Gniadecki, Robert
We have investigated cell growth dynamics and cyclins B1 and E expression in cell lines derived from mycosis fungoides (MyLa), Sézary syndrome (SeAx), and CD30+ lympho-proliferative diseases (Mac1, Mac2a, JK). Mac1 and Mac2a had the highest growth rate (doubling time 18–28 h, >90% cycling cells) whereas SeAx was proliferating slowly (doubling time 55 h, approximately 35% cycling cells). Expression of cyclin B1 correlated positively with doubling time whereas expression of cyclin E was unscheduled and constant across the investigated cell lines. All cell lines exhibited high expression of PCNA. Thus, we concluded that cyclin B1 could be used for rapid screening of cell proliferation in malignant lymphocytes derived from cutaneous T-cell lymphoma. PMID:25386244
TARANIS Functions with Cyclin A and Cdk1 in a Novel Arousal Center to Control Sleep in Drosophila.
Afonso, Dinis J S; Liu, Die; Machado, Daniel R; Pan, Huihui; Jepson, James E C; Rogulja, Dragana; Koh, Kyunghee
Sleep is an essential and conserved behavior whose regulation at the molecular and anatomical level remains to be elucidated. Here, we identify TARANIS (TARA), a Drosophila homolog of the Trip-Br (SERTAD) family of transcriptional coregulators, as a molecule that is required for normal sleep patterns. Through a forward-genetic screen, we isolated tara as a novel sleep gene associated with a marked reduction in sleep amount. Targeted knockdown of tara suggests that it functions in cholinergic neurons to promote sleep. tara encodes a conserved cell-cycle protein that contains a Cyclin A (CycA)-binding homology domain. TARA regulates CycA protein levels and genetically and physically interacts with CycA to promote sleep. Furthermore, decreased levels of Cyclin-dependent kinase 1 (Cdk1), a kinase partner of CycA, rescue the short-sleeping phenotype of tara and CycA mutants, while increased Cdk1 activity mimics the tara and CycA phenotypes, suggesting that Cdk1 mediates the role of TARA and CycA in sleep regulation. Finally, we describe a novel wake-promoting role for a cluster of ∼14 CycA-expressing neurons in the pars lateralis (PL), previously proposed to be analogous to the mammalian hypothalamus. We propose that TARANIS controls sleep amount by regulating CycA protein levels and inhibiting Cdk1 activity in a novel arousal center.
HBx-dependent cell cycle deregulation involves interaction with cyclin E/A-cdk2 complex and destabilization of p27Kip1.
Mukherji, Atish; Janbandhu, Vaibhao C; Kumar, Vijay
The HBx (X protein of hepatitis B virus) is a promiscuous transactivator implicated to play a key role in hepatocellular carcinoma. However, HBx-regulated molecular events leading to deregulation of cell cycle or establishment of a permissive environment for hepatocarcinogenesis are not fully understood. Our cell culture-based studies suggested that HBx had a profound effect on cell cycle progression even in the absence of serum. HBx presence led to an early and sustained level of cyclin-cdk2 complex during the cell cycle combined with increased protein kinase activity of cdk2 heralding an early proliferative signal. The increased cdk2 activity also led to an early proteasomal degradation of p27(Kip1) that could be reversed by HBx-specific RNA interference and blocked by a chemical inhibitor of cdk2 or the T187A mutant of p27. Further, our co-immunoprecipitation and in vitro binding studies with recombinant proteins suggested a direct interaction between HBx and the cyclin E/A-cdk2 complex. Interference with different signalling cascades known to be activated by HBx suggested a constitutive requirement of Src kinases for the association of HBx with these complexes. Notably, the HBx mutant that did not interact with cyclin E/A failed to destabilize p27(Kip1) or deregulate the cell cycle. Thus HBx appears to deregulate the cell cycle by interacting with the key cell cycle regulators independent of its well-established role in transactivation.
Circadian variation in expression of G1 phase cyclins D1 and E and cyclin-dependent kinase inhibitors p16 and p21 in human bowel mucosa
Griniatsos, John; Michail, Othon P; Theocharis, Stamatios; Arvelakis, Antonios; Papaconstantinou, Ioannis; Felekouras, Evangelos; Pikoulis, Emmanouel; Karavokyros, Ioannis; Bakoyiannis, Chris; Marinos, George; Bramis, John; Michail, Panayiotis O
AIM: To evaluate whether the cellular proliferation rate in the large bowel epithelial cells is characterized by circadian rhythm. METHODS: Between January 2003 and December 2004, twenty patients who were diagnosed as suffering from primary, resectable, non-metastatic adenocarcinoma of the lower rectum, infiltrating the sphincter mechanism, underwent abdominoperineal resection, total mesorectal excision and permanent left iliac colostomy. In formalin-fixed and paraffin-embedded biopsy specimens obtained from the colostomy mucosa every six hours (00:00, 06:00, 12:00, 18:00 and 24:00), we studied the expression of G1 phase cyclins (D1 and E) as well as the expression of the G1 phase cyclin-dependent kinase (CDK) inhibitors p16 and p21 as indicators of cell cycle progression in colonic epithelial cells using immunohistochemical methods. RESULTS: The expression of both cyclins showed a similar circadian fashion obtaining their lowest and highest values at 00:00 and 18:00, respectively (P< 0.001). A circadian rhythm in the expression of CDK inhibitor proteins p16 and p21 was also observed, with the lowest levels obtained at 12:00 and 18:00 (P< 0.001), respectively. When the complexes cyclins D1 - p21 and E - p21 were examined, the expression of the cyclins was adversely correlated to the p21 expression throughout the day. When the complexes the cyclins D1 - p16 and E - p16 were examined, high levels of p16 expression were correlated to low levels of cyclin expression at 00:00, 06:00 and 24:00. Meanwhile, the highest expression levels of both cyclins were correlated to high levels of p16 expression at 18:00. CONCLUSION: Colonic epithelial cells seem to enter the G1 phase of the cell cycle during afternoon (between 12:00 and 18:00) with the highest rates obtained at 18:00. From a clinical point of view, the present results suggest that G1-phase specific anticancer therapies in afternoon might maximize their anti-tumor effect while minimizing toxicity
Identification of New Substrates for Breast Tumor-Specific LMW Cyclin E/CDk2 Kinase
cyclin EL or cyclin E-LMW and CDK2 (F80A) and CDK2 (F80G) from insect cells and carried out a similar 8 Rb kinase assay to test their...Multani, A. S., Wingate , H. F., Pathak, S., Zhang, N., Tucker, S. L., Chang, S., and Keyomarsi, K. (2004). Tumor-specific low molecular weight forms of
Dexamethasone Induces Cardiomyocyte Terminal Differentiation via Epigenetic Repression of Cyclin D2 Gene.
Gay, Maresha S; Dasgupta, Chiranjib; Li, Yong; Kanna, Angela; Zhang, Lubo
Dexamethasone treatment of newborn rats inhibited cardiomyocyte proliferation and stimulated premature terminal differentiation of cardiomyocytes in the developing heart. Yet mechanisms remain undetermined. The present study tested the hypothesis that the direct effect of glucocorticoid receptor-mediated epigenetic repression of cyclin D2 gene in the cardiomyocyte plays a key role in the dexamethasone-mediated effects in the developing heart. Cardiomyocytes were isolated from 2-day-old rats. Cells were stained with a cardiomyocyte marker α-actinin and a proliferation marker Ki67. Cyclin D2 expression was evaluated by Western blot and quantitative real-time polymerase chain reaction. Promoter methylation of CcnD2 was determined by methylated DNA immunoprecipitation (MeDIP). Overexpression of Cyclin D2 was conducted by transfection of FlexiCcnD2 (+CcnD2) construct. Treatment of cardiomyocytes isolated from newborn rats with dexamethasone for 48 hours significantly inhibited cardiomyocyte proliferation with increased binucleation and decreased cyclin D2 protein abundance. These effects were blocked with Ru486 (mifepristone). In addition, the dexamethasone treatment significantly increased cyclin D2 gene promoter methylation in newborn rat cardiomyocytes. 5-Aza-2'-deoxycytidine inhibited dexamethasone-mediated promoter methylation, recovered dexamethasone-induced cyclin D2 gene repression, and blocked the dexamethasone-elicited effects on cardiomyocyte proliferation and binucleation. In addition, the overexpression of cyclin D2 restored the dexamethasone-mediated inhibition of proliferation and increase in binucleation in newborn rat cardiomyocytes. The results demonstrate that dexamethasone acting on glucocorticoid receptors has a direct effect and inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via epigenetic repression of cyclin D2 gene.
Expression of Cyclin D1 and P16 in Esophageal Squamous Cell Carcinoma.
Dey, Biswajit; Raphael, Vandana; Khonglah, Yookarin; GiriLynrah, Kyrshanlang
BACKGROUND Esophageal squamous cell carcinoma (ESCC) is one of the lethal cancers with a high incidence rate in Asia. Many genes including cyclin D1 and p16 play important role in its carcinogenesis. We aimed to analyze the expressions of cyclin D1 and p16 with the various clinicopathological characteristics of ESCC. METHODS We examined 30 biopsy samples of ESCC for cyclin D1 and p16 protein expressions using immunohistochemistry. Immunointensity was classified as no immunostaining (-), weakly immunostaining (+), weak immunostaining (++) and strongly positive immunostaining (+++). RESULTS Out of the 30 cases, positive expression of cyclin D1 was detected in 26 cases (86.7%). The percentage of tumors with invasion to the adventitia (88.2%), lymph node metastasis (87.5%), and tumors which were poorly differentiated (92.9%) were higher in cyclin D1 positive tumors than in the cyclin D1 negative tumors. However no significant association was found between cyclin D1 expression and the different clinicopathological parameters.There were 22 cases of ESCC (73.3 %) which showed negativity for p16. The percentage of tumors with invasion to the adventitia (82.4%) and poorly differentiated tumors (92.9%) were higher in the p16 negative tumors than in the p16 positive tumors. There was significant association between the histological grade and p16 expression (p=0.012). However, there were no significant association with regard to site, size and lymph node status of the tumors and p16 expression. CONCLUSION The study shows that alterations of cyclin D1 and p16 play an important role in ESCC. Loss of p16 expression was associated with poor differentiation.
Fluorescent peptide biosensor for probing the relative abundance of cyclin-dependent kinases in living cells.
Kurzawa, Laetitia; Pellerano, Morgan; Coppolani, J B; Morris, May C
Cyclin-dependant kinases play a central role in coordinating cell growth and division, and in sustaining proliferation of cancer cells, thereby constituting attractive pharmacological targets. However, there are no direct means of assessing their relative abundance in living cells, current approaches being limited to antigenic and proteomic analysis of fixed cells. In order to probe the relative abundance of these kinases directly in living cells, we have developed a fluorescent peptide biosensor with biligand affinity for CDKs and cyclins in vitro, that retains endogenous CDK/cyclin complexes from cell extracts, and that bears an environmentally-sensitive probe, whose fluorescence increases in a sensitive fashion upon recognition of its targets. CDKSENS was introduced into living cells, through complexation with the cell-penetrating carrier CADY2 and applied to assess the relative abundance of CDK/Cyclins through fluorescence imaging and ratiometric quantification. This peptide biosensor technology affords direct and sensitive readout of CDK/cyclin complex levels, and reports on differences in complex formation when tampering with a single CDK or cyclin. CDKSENS further allows for detection of differences between different healthy and cancer cell lines, thereby enabling to distinguish cells that express high levels of these heterodimeric kinases, from cells that present decreased or defective assemblies. This fluorescent biosensor technology provides information on the overall status of CDK/Cyclin complexes which cannot be obtained through antigenic detection of individual subunits, in a non-invasive fashion which does not require cell fixation or extraction procedures. As such it provides promising perspectives for monitoring the response to therapeutics that affect CDK/Cyclin abundance, for cell-based drug discovery strategies and fluorescence-based cancer diagnostics.
Cyclin-Dependent Kinase Inhibitors as Anticancer Therapeutics.
Law, Mary E; Corsino, Patrick E; Narayan, Satya; Law, Brian K
Cyclin-dependent kinases (CDKs) have been considered promising drug targets for a number of years, but most CDK inhibitors have failed rigorous clinical testing. Recent studies demonstrating clear anticancer efficacy and reduced toxicity of CDK4/6 inhibitors such as palbociclib and multi-CDK inhibitors such as dinaciclib have rejuvenated the field. Favorable results with palbociclib and its recent U.S. Food and Drug Administration approval demonstrate that CDK inhibitors with narrow selectivity profiles can have clinical utility for therapy based on individual tumor genetics. A brief overview of results obtained with ATP-competitive inhibitors such as palbociclib and dinaciclib is presented, followed by a compilation of new avenues that have been pursued toward the development of novel, non-ATP-competitive CDK inhibitors. These creative ways to develop CDK inhibitors are presented along with crystal structures of these agents complexed with CDK2 to highlight differences in their binding sites and mechanisms of action. The recent successes of CDK inhibitors in the clinic, combined with the potential for structure-based routes to the development of non-ATP-competitive CDK inhibitors, and evidence that CDK inhibitors may have use in suppressing chromosomal instability and in synthetic lethal drug combinations inspire optimism that CDK inhibitors will become important weapons in the fight against cancer.
Emerging Drug Profile: Cyclin-Dependent Kinase Inhibitors
Blachly, James S.; Byrd, John C.
As the rational application of targeted therapies in cancer supplants traditional cytotoxic chemotherapy, there is an ever-greater need for a thorough understanding of the complex machinery of the cell and an application of this knowledge to the development of novel therapeutics and combinations of agents. Here, we review the current state of knowledge of the class of targeted agents known as cyclin-dependent kinase (CDK) inhibitors, with a focus on chronic lymphocytic leukemia (CLL). Flavopiridol (alvocidib) is the best studied of the CDK inhibitors, producing a dramatic cytotoxic effect in vitro and in vivo, with the principal limiting factor of acute tumor lysis. Unfortunately, flavopiridol has a narrow therapeutic window and is relatively non-selective with several off-target (i.e. non-CDK) effects, which prompted development of the second-generation CDK inhibitor dinaciclib. Dinaciclib appears to be both more potent and selective than flavopiridol, with at least an order of magnitude greater therapeutic index, and is currently in phase III clinical trials. In additional to flavopiridol and dinaciclib, we also review the current state of other members of this class, and provide commentary as to the future direction of combination therapy including CDK inhibitors. PMID:23488658
Prefoldin 1 promotes EMT and lung cancer progression by suppressing cyclin A expression
Wang, D; Shi, W; Tang, Y; Liu, Y; He, K; Hu, Y; Li, J; Yang, Y; Song, J
Prefoldin (PFDN) is a co-chaperone protein that is primarily known for its classic cytoplasmic functions in the folding of actin and tubulin monomers during cytoskeletal assembly. Here, we report a marked increase in prefoldin subunit 1 (PFDN1) levels during the transforming growth factor (TGF)-β1-mediated epithelial-mesenchymal transition (EMT) and in human lung tumor tissues. Interestingly, the nuclear localization of PFDN1 was also detected. These observations suggest that PFDN1 may be essential for important novel functions. Overexpression of PFDN1 induced EMT and cell invasion. In sharp contrast, knockdown of PFDN1 generated the opposite effects. Overexpression of PFDN1 was also found to induce lung tumor growth and metastasis. Further experiments showed that PFDN1 overexpression inhibits the expression of cyclin A. PFDN1 suppressed cyclin A expression by directly interacting with the cyclin A promoter at the transcriptional start site. Strikingly, cyclin A overexpression abolished the above PFDN1-mediated effects on the behavior of lung cancer cells, whereas cyclin A knockdown alone induced EMT and increased cell migration and invasion ability. This study reveals that the TGF-β1/PFDN1/cyclin A axis is essential for EMT induction and metastasis of lung cancer cells. PMID:27694898
Molecular cloning and characterization of the mRNA for cyclin from sea urchin eggs.
Pines, J; Hunt, T
We have isolated a cDNA clone encoding sea urchin cyclin and determined its sequence. It contains a single open reading frame of 409 amino acids which shows homology with clam cyclins. RNA transcribed in vitro from this sequence was efficiently translated in reticulocyte lysates, yielding full-length cyclin. Injection of nanogram amounts of this synthetic mRNA into Xenopus oocytes caused them to mature more rapidly than with progesterone treatment. The sea urchin cyclin underwent two posttranslational modifications in the Xenopus oocytes during maturation. The first occurred at about the time that maturation became cycloheximide-resistant, when a small apparent increase in the molecular weight of cyclin was observed. The second modification involved destruction of the cyclin at about the time of white spot appearance, just as would have occurred at the metaphase/anaphase transition in the natural environment of a cleaving sea urchin embryo. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 7. Fig. 9. PMID:2826125
Glycogen synthase kinase 3β and cyclin D1 expression in cervical carcinogenesis
Park, Hyunsoo; Lee, Myunghwa; Kim, Dae Woon; Hong, Seo Yoo
Objective Glycogen synthase kinase 3β (GSK3β) is a pluripotent protein kinase involved in the development of cancers through regulation of numerous oncogenic molecules. Cyclin D1, an important regulator of G1 to S phase transition in various cells, is one of target proteins that GSK3β regulate. Our objective was to assess the expression of GSK3β and cyclin D1 in cervical neoplasm of different histologic grades and to identify their correlation in cervical carcinogenesis. Methods Immunohistochemical analysis of GSK3β and cyclin D1 was performed in a total of 137 patients with 12 normal, 62 cervical intraepithelial neoplasia (CIN) (31 CIN1 and 31 CIN3) and 63 invasive cancers including 56 squamous cell carcinomas and 7 adenocarcinomas. Results The expression of GSK3β increased in parallel with the lesion grade, while that of cyclin D1 decreased with severity of the lesion (P<0.001). There was a significant inverse correlation between GSK3β and cyclin D1 expression in overall cervical neoplasia (Φ=-0.413, P<0.001). GSK3β expression was higher in squamous cell carcinoma than in adenocarcinoma (P=0.049). Conclusion These results suggest that the expressional increase in GSK3β plays a role in cervical carcinogenesis and has inverse correlation with cyclin D1 expression in this process. In addition, GSK3β expression appears to be associated with the histologic type of cervical cancer, especially squamous cell carcinoma. PMID:27896249
Tight function zonula occludens-3 regulates cyclin D1-dependent cell proliferation.
Capaldo, Christopher T; Koch, Stefan; Kwon, Michael; Laur, Oskar; Parkos, Charles A; Nusrat, Asma
Coordinated regulation of cell proliferation is vital for epithelial tissue homeostasis, and uncontrolled proliferation is a hallmark of carcinogenesis. A growing body of evidence indicates that epithelial tight junctions (TJs) play a role in these processes, although the mechanisms involved are poorly understood. In this study, we identify and characterize a novel plasma membrane pool of cyclin D1 with cell-cycle regulatory functions. We have determined that the zonula occludens (ZO) family of TJ plaque proteins sequesters cyclin D1 at TJs during mitosis, through an evolutionarily conserved class II PSD-95, Dlg, and ZO-1 (PDZ)-binding motif within cyclin D1. Disruption of the cyclin D1/ZO complex through mutagenesis or siRNA-mediated suppression of ZO-3 resulted in increased cyclin D1 proteolysis and G(0)/G(1) cell-cycle retention. This study highlights an important new role for ZO family TJ proteins in regulating epithelial cell proliferation through stabilization of cyclin D1 during mitosis.
A role for cyclin D3 in the endomitotic cell cycle.
Zimmet, J M; Ladd, D; Jackson, C W; Stenberg, P E; Ravid, K
Platelets, essential for thrombosis and hemostasis, develop from polyploid megakaryocytes which undergo endomitosis. During this cell cycle, cells experience abrogated mitosis and reenter a phase of DNA synthesis, thus leading to endomitosis. In the search for regulators of the endomitotic cell cycle, we have identified cyclin D3 as an important regulatory factor. Of the D-type cyclins, cyclin D3 is present at high levels in megakaryocytes undergoing endomitosis and is markedly upregulated following exposure to the proliferation-, maturation-, and ploidy-promoting factor, Mpl ligand. Transgenic mice in which cyclin D3 is overexpressed in the platelet lineage display a striking increase in endomitosis, similar to changes seen following Mpl ligand administration to normal mice. Electron microscopy analysis revealed that unlike such treated mice, however, D3 transgenic mice show a poor development of demarcation membranes, from which platelets are believed to fragment, and no increase in platelets. Thus, while our model supports a key role for cyclin D3 in the endomitotic cell cycle, it also points to the unique role of Mpl ligand in priming megakaryocytes towards platelet fragmentation. The role of cyclin D3 in promoting endomitosis in other lineages programmed to abrogate mitosis will need further exploration. PMID:9372957
The cyclin-dependent kinase (CDK) inhibitor flavopiridol inhibits glycogen phosphorylase.
Kaiser, A; Nishi, K; Gorin, F A; Walsh, D A; Bradbury, E M; Schnier, J B
Flavopiridol has been shown to induce cell cycle arrest and apoptosis in various tumor cells in vitro and in vivo. Using immobilized flavopiridol, we identified glycogen phosphorylases (GP) from liver and brain as flavopiridol binding proteins from HeLa cell extract. Purified rabbit muscle GP also bound to the flavopiridol affinity column. GP is the rate-limiting enzyme in intracellular glycogen breakdown. Flavopiridol significantly inhibited the AMP-activated GP-b form of the purified rabbit muscle isoenzyme (IC50 of 1 microM at 0.8 mM AMP), but was less inhibitory to the active phosphorylated form of GP, GP-a (IC50 of 2.5 microM). The AMP-bound GP-a form was poorly inhibited by flavopiridol (40% at 10 microM). Increasing concentrations of the allosteric effector AMP resulted in a linear decrease in the GP-inhibitory activity of flavopiridol suggesting interference between flavopiridol and AMP. In contrast the GP inhibitor caffeine had no effect on the relative GP inhibition by flavopiridol, suggesting an additive effect of caffeine. Flavopiridol also inhibited the phosphorylase kinase-catalyzed phosphorylation of GP-b by inhibiting the kinase in vitro. Flavopiridol thus is able to interfere with both activating modifications of GP-b, AMP activation and phosphorylation. In A549 NSCLC cells flavopiridol treatment caused glycogen accumulation despite of an increase in GP activity, suggesting direct GP inhibition in vivo rather than inhibition of GP activation by phosphorylase kinase. These results suggest that the cyclin-dependent kinase inhibitor flavopiridol interferes with glycogen degradation, which may be responsible for flavopiridol's cytotoxicity and explain its resistance in some cell lines.
G2 cell cycle arrest, down-regulation of cyclin B, and induction of mitotic catastrophe by the flavoprotein inhibitor diphenyleneiodonium.
Scaife, Robin M
Because proliferation of eukaryotic cells requires cell cycle-regulated chromatid separation by the mitotic spindle, it is subject to regulation by mitotic checkpoints. To determine the mechanism of the antiproliferative activity of the flavoprotein-specific inhibitor diphenyleneiodonium (DPI), I have examined its effect on the cell cycle and mitosis. Similar to paclitaxel, exposure to DPI causes an accumulation of cells with a 4N DNA content. However, unlike the paclitaxel-mediated mitotic block, DPI-treated cells are arrested in the cell cycle prior to mitosis. Although DPI-treated cells can arrest with fully separated centrosomes at opposite sides of the nucleus, these centrosomes fail to assemble mitotic spindle microtubules and they do not accumulate the Thr(288) phosphorylated Aurora-A kinase marker of centrosome maturation. In contrast with paclitaxel-arrested cells, DPI impairs cyclin B1 accumulation. Release from DPI permits an accumulation of cyclin B1 and progression of the cells into mitosis. Conversely, exposure of paclitaxel-arrested mitotic cells to DPI causes a precipitous drop in cyclin B and Thr(288) phosphorylated Aurora-A levels and leads to mitotic catastrophe in a range of cancerous and noncancerous cells. Hence, the antiproliferative activity of DPI reflects a novel inhibitory mechanism of cell cycle progression that can reverse spindle checkpoint-mediated cell cycle arrest.
Transcriptome analysis of tetraploid cells identifies cyclin D2 as a facilitator of adaptation to genome doubling in the presence of p53
Potapova, Tamara A.; Seidel, Christopher W.; Box, Andrew C.; Rancati, Giulia; Li, Rong
Tetraploidization, or genome doubling, is a prominent event in tumorigenesis, primarily because cell division in polyploid cells is error-prone and produces aneuploid cells. This study investigates changes in gene expression evoked in acute and adapted tetraploid cells and their effect on cell-cycle progression. Acute polyploidy was generated by knockdown of the essential regulator of cytokinesis anillin, which resulted in cytokinesis failure and formation of binucleate cells, or by chemical inhibition of Aurora kinases, causing abnormal mitotic exit with formation of single cells with aberrant nuclear morphology. Transcriptome analysis of these acute tetraploid cells revealed common signatures of activation of the tumor-suppressor protein p53. Suppression of proliferation in these cells was dependent on p53 and its transcriptional target, CDK inhibitor p21. Rare proliferating tetraploid cells can emerge from acute polyploid populations. Gene expression analysis of single cell–derived, adapted tetraploid clones showed up-regulation of several p53 target genes and cyclin D2, the activator of CDK4/6/2. Overexpression of cyclin D2 in diploid cells strongly potentiated the ability to proliferate with increased DNA content despite the presence of functional p53. These results indicate that p53-mediated suppression of proliferation of polyploid cells can be averted by increased levels of oncogenes such as cyclin D2, elucidating a possible route for tetraploidy-mediated genomic instability in carcinogenesis. PMID:27559130
c-Jun/AP-1 pathway-mediated cyclin D1 expression participates in low dose arsenite-induced transformation in mouse epidermal JB6 Cl41 cells
Zhang Dongyun; Li Jingxia; Gao Jimin; Huang Chuanshu
Arsenic is a well-documented human carcinogen associated with skin carcinogenesis. Our previous work reveals that arsenite exposure is able to induce cell transformation in mouse epidermal cell JB6 Cl41 through the activation of ERK, rather than JNK pathway. Our current studies further evaluate downstream pathway in low dose arsenite-induced cell transformation in JB6 Cl41 cells. Our results showed that treatment of cells with low dose arsenite induced activation of c-Jun/AP-1 pathway, and ectopic expression of dominant negative mutant of c-Jun (TAM67) blocked arsenite-induced transformation. Furthermore, our data indicated that cyclin D1 was an important downstream molecule involved in c-Jun/AP-1-mediated cell transformation upon low dose arsenite exposure, because inhibition of cyclin D1 expression by its specific siRNA in the JB6 Cl41 cells resulted in impairment of anchorage-independent growth of cells induced by low dose arsenite. Collectively, our results demonstrate that c-Jun/AP-1-mediated cyclin D1 expression is at least one of the key events implicated in cell transformation upon low dose arsenite exposure.
Cyclin-Dependent Kinase Inhibitor 1a (p21) Modulates Response to Cocaine and Motivated Behaviors.
Scholpa, Natalie E; Briggs, Sherri B; Wagner, John J; Cummings, Brian S
This study investigated the functional role of cyclin-dependent kinase inhibitor 1a (Cdkn1a or p21) in cocaine-induced responses using a knockout mouse model. Acute locomotor activity after cocaine administration (15 mg/kg, i.p.) was decreased in p21(-/-) mice, whereas cocaine-induced place preference was enhanced. Interestingly, κ-opioid-induced place aversion was also significantly enhanced. Concentration-dependent analysis of locomotor activity in response to cocaine demonstrated a rightward shift in the p21(-/-) mice. Pretreatment with a 5-hydroxytryptamine receptor antagonist did not alter the enhancement of cocaine-induced conditioned place preference in p21(-/-) mice, indicating a lack of involvement of serotonergic signaling in this response. Cocaine exposure increased p21 expression exclusively in the ventral sector of the hippocampus of rodents after either contingent or noncontingent drug administration. Increased p21 expression was accompanied by increased histone acetylation of the p21 promoter region in rats. Finally, increased neurogenesis in the dorsal hippocampus of p21(-/-) mice was also observed. These results show that functional loss of p21 altered the acute locomotor response to cocaine and the conditioned responses to either rewarding or aversive stimuli. Collectively, these findings demonstrate a previously unreported involvement of p21 in modulating responses to cocaine and in motivated behaviors.
Identification of a novel subgroup of melanomas with KIT/cyclin-dependent kinase-4 overexpression.
Smalley, Keiran S M; Contractor, Rooha; Nguyen, Thiennga K; Xiao, Min; Edwards, Robin; Muthusamy, Viswanathan; King, Alastair J; Flaherty, Keith T; Bosenberg, Marcus; Herlyn, Meenhard; Nathanson, Katherine L
Although many melanomas harbor either activating mutations in BRAF or NRAS, there remains a substantial, yet little known, group of tumors without either mutation. Here, we used a genomic strategy to define a novel group of melanoma cell lines with co-overexpression of cyclin-dependent kinase 4 (CDK4) and KIT. Although this subgroup lacked any known KIT mutations, they had high phospho-KIT receptor expression, indicating receptor activity. Quantitative PCR confirmed the existence of a similar KIT/CDK4 subgroup in human melanoma samples. Pharmacologic studies showed the KIT/CDK4-overexpressing subgroup to be resistant to BRAF inhibitors but sensitive to imatinib in both in vitro and in vivo melanoma models. Mechanistically, imatinib treatment led to increased apoptosis and G(1) phase cell cycle arrest associated with the inhibition of phospho-ERK and increased expression of p27(KIP). Other melanoma cell lines, which retained some KIT expression but lacked phospho-KIT, were not sensitive to imatinib, suggesting that KIT expression alone is not predictive of response. We suggest that co-overexpression of KIT/CDK4 is a potential mechanism of oncogenic transformation in some BRAF/NRAS wild-type melanomas. This group of melanomas may be a subpopulation for which imatinib or other KIT inhibitors may constitute optimal therapy.
Cytoplasmic cyclin D1 regulates cell invasion and metastasis through the phosphorylation of paxillin
Fusté, Noel P.; Fernández-Hernández, Rita; Cemeli, Tània; Mirantes, Cristina; Pedraza, Neus; Rafel, Marta; Torres-Rosell, Jordi; Colomina, Neus; Ferrezuelo, Francisco; Dolcet, Xavier; Garí, Eloi
Cyclin D1 (Ccnd1) together with its binding partner Cdk4 act as a transcriptional regulator to control cell proliferation and migration, and abnormal Ccnd1·Cdk4 expression promotes tumour growth and metastasis. While different nuclear Ccnd1·Cdk4 targets participating in cell proliferation and tissue development have been identified, little is known about how Ccnd1·Cdk4 controls cell adherence and invasion. Here, we show that the focal adhesion component paxillin is a cytoplasmic substrate of Ccnd1·Cdk4. This complex phosphorylates a fraction of paxillin specifically associated to the cell membrane, and promotes Rac1 activation, thereby triggering membrane ruffling and cell invasion in both normal fibroblasts and tumour cells. Our results demonstrate that localization of Ccnd1·Cdk4 to the cytoplasm does not simply act to restrain cell proliferation, but constitutes a functionally relevant mechanism operating under normal and pathological conditions to control cell adhesion, migration and metastasis through activation of a Ccnd1·Cdk4-paxillin-Rac1 axis. PMID:27181366
Cyclin-Dependent Kinase Inhibitor 1a (p21) Modulates Response to Cocaine and Motivated Behaviors
Scholpa, Natalie E.; Briggs, Sherri B.; Wagner, John J.
This study investigated the functional role of cyclin-dependent kinase inhibitor 1a (Cdkn1a or p21) in cocaine-induced responses using a knockout mouse model. Acute locomotor activity after cocaine administration (15 mg/kg, i.p.) was decreased in p21−/− mice, whereas cocaine-induced place preference was enhanced. Interestingly, κ-opioid–induced place aversion was also significantly enhanced. Concentration-dependent analysis of locomotor activity in response to cocaine demonstrated a rightward shift in the p21−/− mice. Pretreatment with a 5-hydroxytryptamine receptor antagonist did not alter the enhancement of cocaine-induced conditioned place preference in p21−/− mice, indicating a lack of involvement of serotonergic signaling in this response. Cocaine exposure increased p21 expression exclusively in the ventral sector of the hippocampus of rodents after either contingent or noncontingent drug administration. Increased p21 expression was accompanied by increased histone acetylation of the p21 promoter region in rats. Finally, increased neurogenesis in the dorsal hippocampus of p21−/− mice was also observed. These results show that functional loss of p21 altered the acute locomotor response to cocaine and the conditioned responses to either rewarding or aversive stimuli. Collectively, these findings demonstrate a previously unreported involvement of p21 in modulating responses to cocaine and in motivated behaviors. PMID:26791604
Polycomb protein SCML2 regulates the cell cycle by binding and modulating CDK/CYCLIN/p21 complexes.
Lecona, Emilio; Rojas, Luis Alejandro; Bonasio, Roberto; Johnston, Andrew; Fernández-Capetillo, Oscar; Reinberg, Danny
Polycomb group (PcG) proteins are transcriptional repressors of genes involved in development and differentiation, and also maintain repression of key genes involved in the cell cycle, indirectly regulating cell proliferation. The human SCML2 gene, a mammalian homologue of the Drosophila PcG protein SCM, encodes two protein isoforms: SCML2A that is bound to chromatin and SCML2B that is predominantly nucleoplasmic. Here, we purified SCML2B and found that it forms a stable complex with CDK/CYCLIN/p21 and p27, enhancing the inhibitory effect of p21/p27. SCML2B participates in the G1/S checkpoint by stabilizing p21 and favoring its interaction with CDK2/CYCE, resulting in decreased kinase activity and inhibited progression through G1. In turn, CDK/CYCLIN complexes phosphorylate SCML2, and the interaction of SCML2B with CDK2 is regulated through the cell cycle. These findings highlight a direct crosstalk between the Polycomb system of cellular memory and the cell-cycle machinery in mammals.
Functional Conservation in the SIAMESE-RELATED Family of Cyclin-Dependent Kinase Inhibitors in Land Plants.
Kumar, Narender; Harashima, Hirofumi; Kalve, Shweta; Bramsiepe, Jonathan; Wang, Kai; Sizani, Bulelani L; Bertrand, Laura L; Johnson, Matthew C; Faulk, Christopher; Dale, Renee; Simmons, L Alice; Churchman, Michelle L; Sugimoto, Keiko; Kato, Naohiro; Dasanayake, Maheshi; Beemster, Gerrit; Schnittger, Arp; Larkin, John C
The best-characterized members of the plant-specific SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors regulate the transition from the mitotic cell cycle to endoreplication, also known as endoreduplication, an altered version of the cell cycle in which DNA is replicated without cell division. Some other family members are implicated in cell cycle responses to biotic and abiotic stresses. However, the functions of most SMRs remain unknown, and the specific cyclin-dependent kinase complexes inhibited by SMRs are unclear. Here, we demonstrate that a diverse group of SMRs, including an SMR from the bryophyte Physcomitrella patens, can complement an Arabidopsis thaliana siamese (sim) mutant and that both Arabidopsis SIM and P. patens SMR can inhibit CDK activity in vitro. Furthermore, we show that Arabidopsis SIM can bind to and inhibit both CDKA;1 and CDKB1;1. Finally, we show that SMR2 acts to restrict cell proliferation during leaf growth in Arabidopsis and that SIM, SMR1/LGO, and SMR2 play overlapping roles in controlling the transition from cell division to endoreplication during leaf development. These results indicate that differences in SMR function in plant growth and development are primarily due to differences in transcriptional and posttranscriptional regulation, rather than to differences in fundamental biochemical function.
Functional Conservation in the SIAMESE-RELATED Family of Cyclin-Dependent Kinase Inhibitors in Land Plants
Wang, Kai; Bertrand, Laura L.; Johnson, Matthew C.; Dale, Renee; Simmons, L. Alice; Sugimoto, Keiko; Kato, Naohiro; Dassanayake, Maheshi; Schnittger, Arp
The best-characterized members of the plant-specific SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors regulate the transition from the mitotic cell cycle to endoreplication, also known as endoreduplication, an altered version of the cell cycle in which DNA is replicated without cell division. Some other family members are implicated in cell cycle responses to biotic and abiotic stresses. However, the functions of most SMRs remain unknown, and the specific cyclin-dependent kinase complexes inhibited by SMRs are unclear. Here, we demonstrate that a diverse group of SMRs, including an SMR from the bryophyte Physcomitrella patens, can complement an Arabidopsis thaliana siamese (sim) mutant and that both Arabidopsis SIM and P. patens SMR can inhibit CDK activity in vitro. Furthermore, we show that Arabidopsis SIM can bind to and inhibit both CDKA;1 and CDKB1;1. Finally, we show that SMR2 acts to restrict cell proliferation during leaf growth in Arabidopsis and that SIM, SMR1/LGO, and SMR2 play overlapping roles in controlling the transition from cell division to endoreplication during leaf development. These results indicate that differences in SMR function in plant growth and development are primarily due to differences in transcriptional and posttranscriptional regulation, rather than to differences in fundamental biochemical function. PMID:26546445
Cyclin-Dependent kinase 5 targeting prevents β-Amyloid aggregation involving glycogen synthase kinase 3β and phosphatases.
Castro-Alvarez, John Fredy; Uribe-Arias, Alejandro; Cardona-Gómez, Gloria Patricia
Inappropriate activation of cyclin-dependent kinase 5 (CDK5) resulting from proteolytic release of the activator fragment p25 from the membrane contributes to the formation of neurofibrillary tangles, β-amyloid (βA) aggregation, and chronic neurodegeneration. At 18 months of age, 3× Tg-AD mice were sacrificed after either 3 weeks (short term) or 1 year (long term) of CDK5 knockdown. In short-term-treated animals, CDK5 knockdown reversed βA aggregation in the hippocampi via inhibitory phosphorylation of glycogen synthase kinase 3β Ser9 and activation of phosphatase PP2A. In long-term-treated animals, CDK5 knockdown induced a persistent reduction in CDK5 and prevented βA aggregation, but the effect on amyloid precursor protein processing was reduced, suggesting that yearly booster therapy would be required. These findings further validate CDK5 as a target for preventing or blocking amyloidosis in older transgenic mice.
Superoxide dismutase induces G1-phase cell cycle arrest by down-regulated expression of Cdk-2 and cyclin-E in murine sarcoma S180 tumor cells.
Liu, Dongyue; Liu, Anjun
As an efficient reactive oxygen species-scavenging enzyme, superoxide dismutase (SOD) has been shown to inhibit tumor growth and interfere with motility and invasiveness of cancer cells. In this study, the molecular mechanisms of cell cycle arrest when S180 tumor cells were exposed to high levels of SOD were investigated. Here, both murine sarcoma S180 tumor cells and NIH-3T3 mouse fibroblasts were respectively treated with varying concentrations of Cu/Zn-SOD for 24, 48 and 72 h to determine optimal dose of SOD, which was a concentration of 800 U/ml SOD for 48 h. It is found that SOD induced S180 cell cycle arrest at G1-phase with decreasing level of superoxide production, whereas SOD had less effect on proliferation of NIH-3T3 cells. Moreover, the expression rate of Proliferating Cell Nuclear Antigen (PCNA) in S180 tumor cells was suppressed after SOD treatment, which indicated the inhibition of DNA synthesis in S180 cells. Besides, there were significant down-regulations of cyclin-E and Cdk-2 in S180 cells after SOD treatment, which contributed to the blockage of G1/S transition in S180 cell cycle. Together, our data confirmed that SOD could notably inhibit proliferation of S180 tumor cell and induce cell cycle arrest at G1-phase by down-regulating expressions of cyclin-E and Cdk-2.
Age-dependent kinetics of dentate gyrus neurogenesis in the absence of cyclin D2
Background Adult neurogenesis continuously adds new neurons to the dentate gyrus and the olfactory bulb. It involves the proliferation and subsequent differentiation of neuronal progenitors, and is thus closely linked to the cell cycle machinery. Cell cycle progression is governed by the successive expression, activation and degradation of regulatory proteins. Among them, D-type cyclins control the exit from the G1 phase of the cell cycle. Cyclin D2 (cD2) has been shown to be required for the generation of new neurons in the neurogenic niches of the adult brain. It is differentially expressed during hippocampal development, and adult cD2 knock out (cD2KO) mice virtually lack neurogenesis in the dentate gyrus and olfactory bulb. In the present study we examined the dynamics of postnatal and adult neurogenesis in the dentate gyrus (DG) of cD2KO mice. Animals were injected with bromodeoxyuridine at seven time points during the first 10 months of life and brains were immunohistochemically analyzed for their potential to generate new neurons. Results Compared to their WT litters, cD2KO mice had considerably reduced numbers of newly born granule cells during the postnatal period, with neurogenesis becoming virtually absent around postnatal day 28. This was paralleled by a reduction in granule cell numbers, in the volume of the granule cell layer as well as in apoptotic cell death. CD2KO mice did not show any of the age-related changes in neurogenesis and granule cell numbers that were seen in WT litters. Conclusions The present study suggests that hippocampal neurogenesis becomes increasingly dependent on cD2 during early postnatal development. In cD2KO mice, hippocampal neurogenesis ceases at a time point at which the tertiary germinative matrix stops proliferating, indicating that cD2 becomes an essential requirement for ongoing neurogenesis with the transition from developmental to adult neurogenesis. Our data further support the notion that adult neurogenesis
Characteristics of Cyclin B and its potential role in regulating oogenesis in the red claw crayfish (Cherax quadricarinatus).
Wang, L M; Lv, W W; Zuo, D; Dong, Z J; Zhao, Y L
Cyclin B is a regulatory subunit of maturation-promoting factor (MPF), which has a key role in the induction of meiotic maturation of oocytes. MPF has been studied in a wide variety of animal species; however, its expression in crustaceans is poorly characterized. In this study, the complete cDNA sequence of Cyclin B was cloned from the red claw crayfish, Cherax quadricarinatus, and its spatiotemporal expression profiles were analyzed. Cyclin B cDNA (1779 bp) encoded a 401 amino acid protein with a calculated molecular weight of 45.1 kDa. Quantitative real-time PCR demonstrated that Cyclin B mRNA was expressed mainly in the ovarian tissue and that the expression decreased as the ovaries developed. Immunofluorescence analysis revealed that the Cyclin B protein relocated from the cytoplasm to the nucleus during oogenesis. These findings suggest that Cyclin B plays an important role in gametogenesis and gonad development in C. quadricarinatus.
Cell Cycle-independent Role of Cyclin D3 in Host Restriction of Influenza Virus Infection
Fan, Ying; Mok, Chris Ka-Pun; Chan, Michael Chi Wai; Zhang, Yang; Nal, Béatrice; Kien, François; Bruzzone, Roberto; Sanyal, Sumana
To identify new host factors that modulate the replication of influenza A virus, we performed a yeast two-hybrid screen using the cytoplasmic tail of matrix protein 2 from the highly pathogenic H5N1 strain. The screen revealed a high-score interaction with cyclin D3, a key regulator of cell cycle early G1 phase. M2-cyclin D3 interaction was validated through GST pull-down and recapitulated in influenza A/WSN/33-infected cells. Knockdown of Ccnd3 by small interfering RNA significantly enhanced virus progeny titers in cell culture supernatants. Interestingly, the increase in virus production was due to cyclin D3 deficiency per se and not merely a consequence of cell cycle deregulation. A combined knockdown of Ccnd3 and Rb1, which rescued cell cycle progression into S phase, failed to normalize virus production. Infection by influenza A virus triggered redistribution of cyclin D3 from the nucleus to the cytoplasm, followed by its proteasomal degradation. When overexpressed in HEK 293T cells, cyclin D3 impaired binding of M2 with M1, which is essential for proper assembly of progeny virions, lending further support to its role as a putative restriction factor. Our study describes the identification and characterization of cyclin D3 as a novel interactor of influenza A virus M2 protein. We hypothesize that competitive inhibition of M1-M2 interaction by cyclin D3 impairs infectious virion formation and results in attenuated virus production. In addition, we provide mechanistic insights into the dynamic interplay of influenza virus with the host cell cycle machinery during infection. PMID:28130444
Cell Cycle Independent Role of Cyclin D3 in Host Restriction of Influenza Virus Infection.
Fan, Ying; Mok, Chris Ka-Pun; Chan, Michael Chi Wai; Zhang, Yang; Nal-Rogier, Béatrice; Kien, François; Bruzzone, Roberto; Sanyal, Sumana
To identify new host factors that modulate the replication of influenza A virus, we performed a yeast two-hybrid screen using the cytoplasmic tail of matrix protein 2 from the highly pathogenic H5N1 strain. The screen revealed a high-score interaction with cyclin D3, a key regulator of cell cycle early G1 phase. M2-cyclin D3 interaction was validated through GST pull-down and recapitulated in influenza A/WSN/33-infected cells. Knockdown of Ccnd3 by small interfering RNA significantly enhanced virus progeny titers in cell culture supernatants. Interestingly, the increase in virus production was due to cyclin D3 deficiency per se, and not merely a consequence of cell cycle deregulation. A combined knockdown of Ccnd3 and Rb1, which rescued cell cycle progression into the S phase, failed to normalize virus production. Infection by IAV triggered redistribution of cyclin D3 from the nucleus to the cytoplasm followed by its proteasomal degradation. When over-expressed in HEK 293T cells cyclin D3 impaired binding of M2 with M1, which is essential for proper assembly of progeny virions, lending further support to its role as a putative restriction factor. Our study describes the identification and characterization of cyclin D3 as a novel interactor of influenza A virus M2 protein. We hypothesize that competitive inhibition of M1-M2 interaction by cyclin D3 impairs infectious virion formation and results in attenuated virus production. In addition, we provide mechanistic insights into the dynamic interplay of influenza virus with the host cell cycle machinery during infection.
Cdk5-mediated inhibition of APC/C-Cdh1 switches on the cyclin D1-Cdk4-pRb pathway causing aberrant S-phase entry of postmitotic neurons.
Veas-Pérez de Tudela, Miguel; Maestre, Carolina; Delgado-Esteban, María; Bolaños, Juan P; Almeida, Angeles
The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression in proliferating cells. To enter the S-phase, APC/C must be inactivated by phosphorylation of its cofactor, Cdh1. In post-mitotic cells such as neurons APC/C-Cdh1 complex is highly active and responsible for the continuous degradation of mitotic cyclins. However, the specific molecular pathway that determines neuronal cell cycle blockade in post-mitotic neurons is unknown. Here, we show that activation of glutamatergic receptors in rat cortical primary neurons endogenously triggers cyclin-dependent kinase-5 (Cdk5)-mediated phosphorylation of Cdh1 leading to its cytoplasmic accumulation and disassembly from the APC3 core protein, causing APC/C inactivation. Conversely, pharmacological or genetic inhibition of Cdk5 promotes Cdh1 ubiquitination and proteasomal degradation. Furthermore, we show that Cdk5-mediated phosphorylation and inactivation of Cdh1 leads to p27 depletion, which switches on the cyclin D1-cyclin-dependent kinase-4 (Cdk4)-retinoblastoma protein (pRb) pathway to allow the S-phase entry of neurons. However, neurons do not proceed through the cell cycle and die by apoptosis. These results indicate that APC/C-Cdh1 actively suppresses an aberrant cell cycle entry and death of neurons, highlighting its critical function in neuroprotection.
A family of cyclin D homologs from plants differentially controlled by growth regulators and containing the conserved retinoblastoma protein interaction motif.
Soni, R; Carmichael, J P; Shah, Z H; Murray, J A
A new family of three related cyclins has been identified in Arabidopsis by complementation of a yeast strain deficient in G1 cyclins. Individual members show tissue-specific expression and are conserved in other plant species. They form a distinctive group of plant cyclins, which we named delta-type cyclins to indicate their similarities with mammalian D-type cyclins. The sequence relationships between delta and D cyclins include the N-terminal sequence LXCXE. This motif was originally identified in certain viral oncoproteins and is strongly implicated in binding to the retinoblastoma protein pRb. By analogy to mammalian cyclin D, these plant homologs may mediate growth and phytohormonal signals into the plant cell cycle. In support of this hypothesis, we show that, on restimulation of suspension-cultured cells, cyclin delta 3 is rapidly induced by the plant growth regulator cytokinin and cyclin delta 2 is induced by carbon source. PMID:7696881
CyPA-CD147-ERK1/2-cyclin D2 signaling pathway is upregulated during rat left ventricular hypertrophy.
Tang, Fu-Cai; Wang, Hong-Yan; Ma, Ming-Ming; Guan, Tian-Wang; Pan, Long; Yao, Dun-Chen; Chen, Ya-Lan; Chen, Wei-Bei; Tu, Yong-Sheng; Fu, Xiao-Dong
The changes of serum cyclophilin A (CyPA), its receptor CD147 and the downstream signaling pathway during the process of cardiac hypertrophy remain unknown. The present study aims to investigate the relationships between CyPA-CD147-ERK1/2-cyclin D2 signaling pathway and the development of cardiac hypertrophy. Left ventricular hypertrophy was prepared by 2-kidney, 2-clip in Sprague-Dawley rats and observed for 1 week, 4 and 8 weeks. Left ventricular hypertrophy was evaluated by ratio of left ventricular heart weight to body weight (LVW/BW) and cardiomyocyte cross sectional area (CSA). CyPA levels in serum were determined with a rat CyPA ELISA kit. Expressions of CyPA, CD147, phospho-ERK1/2 and cyclin D2 in left ventricular myocytes were determined by Western blot and immunostaining. Compared with sham groups, systolic blood pressure reached hypertensive levels at 4 weeks in 2K2C groups. LVW/BW and CSA in 2K2C groups were significantly increased at 4 and 8 weeks after clipping. ELISA results indicated a prominent increase in serum CyPA level associated with the degree of left ventricular hypertrophy. Western blot revealed that the expressions of CyPA, CD147, phospho-ERK1/2 and cyclin D2 in left ventricular tissues were also remarkably increased as the cardiac hypertrophy developed. The results of the present study demonstrates that serum CyPA and CyPA-CD147-ERK1/2-cyclin D2 signaling pathway in ventricular tissues are time-dependently upregulated and activated with the process of left ventricular hypertrophy. These data suggest that CyPA-CD147 signaling cascade might play a role in the pathogenesis of left ventricular hypertrophy, and CyPA might be a prognosticator of the degree of left ventricular hypertrophy.
Inter- and intrachromosomal asynchrony of cell division cycle events in root meristem cells of Allium cepa: possible connection with gradient of cyclin B-like proteins
Polit, Justyna Teresa; Maszewski, Janusz
Alternate treatments of Allium cepa root meristems with hydroxyurea (HU) and caffeine give rise to extremely large and highly elongated cells with atypical images of mitotic divisions, including internuclear asynchrony and an unknown type of interchromosomal asynchrony observed during metaphase-to-anaphase transition. Another type of asynchrony that cannot depend solely on the increased length of cells was observed following long-term incubation of roots with HU. This kind of treatment revealed both cell nuclei entering premature mitosis and, for the first time, an uncommon form of mitotic abnormality manifested in a gradual condensation of chromatin (spanning from interphase to prometaphase). Immunocytochemical study of polykaryotic cells using anti-β tubulin antibodies revealed severe perturbations in the microtubular organization of preprophase bands. Quantitative immunofluorescence measurements of the control cells indicate that the level of cyclin B-like proteins reaches the maximum at the G2 to metaphase transition and then becomes reduced during later stages of mitosis. After long-term incubation with low doses of HU, the amount of cyclin B-like proteins considerably increases, and a significant number of elongated cells show gradients of these proteins spread along successive regions of the perinuclear cytoplasm. It is suggested that there may be a direct link between the effects of HU-mediated deceleration of S- and G2-phases and an enhanced concentration of cyclin B-like proteins. In consequence, the activation of cyclin B-CDK complexes gives rise to an abnormal pattern of premature mitotic chromosome condensation with biphasic nuclear structures having one part of chromatin decondensed, and the other part condensed. PMID:20490501
TORC1 kinase and the S-phase cyclin Clb5 collaborate to promote mitotic spindle assembly and DNA replication in S. cerevisiae
Tran, Lieu T.; Wang'ondu, Ruth W.; Weng, Jessica B.; Wanjiku, Grace W.; Fong, Chi M.; Kile, Andrew C.; Koepp, Deanna M.; Hood-DeGrenier, Jennifer K.
The Target of Rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is inhibited by the drug rapamycin. In the budding yeast Saccharomyces cerevisiae, translational defects associated with TORC1 inactivation inhibit cell cycle progression at an early stage in G1, but little is known about the possible roles for TORC1 later in the cell cycle. We investigated the rapamycin-hypersensitivity phenotype of cells lacking the S phase cyclin Clb5 (clb5Δ) as a basis for uncovering novel connections between TORC1 and the cell cycle regulatory machinery. Dosage suppression experiments suggested that the clb5Δ rapamycin hypersensitivity reflects a unique Clb5-associated cyclin-dependent kinase (CDK) function that cannot be performed by mitotic cyclins and that also involves motor proteins, particularly the kinesin-like protein Kip3. Synchronized cell experiments revealed rapamycin-induced defects in pre-anaphase spindle assembly and S phase progression that were more severe in clb5Δ than in wild type cells but no apparent activation of Rad53-dependent checkpoint pathways. Some rapamycin-treated cells had aberrant spindle morphologies, but rapamycin did not cause gross defects in the microtubule cytoskeleton. We propose a model in which TORC1 and Clb5/CDK act coordinately to promote both spindle assembly via a pathway involving Kip3 and S phase progression. PMID:20697716
Sulforaphane, a Dietary Isothiocyanate, Induces G2/M Arrest in Cervical Cancer Cells through CyclinB1 Downregulation and GADD45β/CDC2 Association
Cheng, Ya-Min; Tsai, Ching-Chou; Hsu, Yi-Chiang
Globally, cervical cancer is the most common malignancy affecting women. The main treatment methods for this type of cancer include conization or hysterectomy procedures. Sulforaphane (SFN) is a natural, compound-based drug derived from dietary isothiocyanates which has previously been shown to possess potent anti-tumor and chemopreventive effects against several types of cancer. The present study investigated the effects of SFN on anti-proliferation and G2/M phase cell cycle arrest in cervical cancer cell lines (Cx, CxWJ, and HeLa). We found that cytotoxicity is associated with an accumulation of cells in the G2/M phases of the cell-cycle. Treatment with SFN led to cell cycle arrest as well as the down-regulation of Cyclin B1 expression, but not of CDC2 expression. In addition, the effects of GADD45β gene activation in cell cycle arrest increase proportionally with the dose of SFN; however, mitotic delay and the inhibition of proliferation both depend on the dosage of SFN used to treat cancer cells. These results indicate that SFN may delay the development of cancer by arresting cell growth in the G2/M phase via down-regulation of Cyclin B1 gene expression, dissociation of the cyclin B1/CDC2 complex, and up-regulation of GADD45β proteins. PMID:27626412
Development of highly potent and selective diaminothiazole inhibitors of cyclin-dependent kinases
Schonbrunn, Ernst; Betzi, Stephane; Alam, Riazul; Martin, Mathew P.; Becker, Andreas; Han, Huijong; Francis, Rawle; Chakrasali, Ramappa; Jakkaraj, Sudhakar; Kazi, Aslamuzzaman; Sebti, Said M.; Cubitt, Christopher L.; Gebhard, Anthony W.; Hazlehurst, Lori A.; Tash, Joseph S.; Georg, Gunda I.
Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that act as key regulatory elements in cell cycle progression. We describe the development of highly potent diaminothiazole inhibitors of CDK2 (IC50 = 0.0009 – 0.0015 µM) from a single hit compound with weak inhibitory activity (IC50 = 15 µM), discovered by high-throughput screening. Structure-based design was performed using 35 co-crystal structures of CDK2 liganded with distinct analogues of the parent compound. The profiling of compound 51 against a panel of 339 kinases revealed high selectivity for CDKs, with preference for CDK2 and CDK5 over CDK9, CDK1, CDK4 and CDK6. Compound 51 inhibited the proliferation of 13 out of 15 cancer cell lines with IC50 values between 0.27 and 6.9 µM, which correlated with the complete suppression of retinoblastoma phosphorylation and the onset of apoptosis. Combined, the results demonstrate the potential of this new inhibitors series for further development into CDK-specific chemical probes or therapeutics. PMID:23600925
The cyclin-dependent kinase family in the social amoebozoan Dictyostelium discoideum.
Huber, Robert J
Cyclin-dependent kinases (Cdk) are a family of serine/threonine protein kinases that regulate eukaryotic cell cycle progression. Their ability to modulate the cell cycle has made them an attractive target for anti-cancer therapies. Cdk protein function has been studied in a variety of Eukaryotes ranging from yeast to humans. In the social amoebozoan Dictyostelium discoideum, several homologues of mammalian Cdks have been identified and characterized. The life cycle of this model organism is comprised of a feeding stage where single cells grow and divide mitotically as they feed on their bacterial food source and a multicellular developmental stage that is induced by starvation. Thus it is a valuable system for studying a variety of cellular and developmental processes. In this review I summarize the current knowledge of the Cdk protein family in Dictyostelium by highlighting the research efforts focused on the characterization of Cdk1, Cdk5, and Cdk8 in this model Eukaryote. Accumulated evidence indicates that each protein performs distinct functions during the Dictyostelium life cycle with Cdk1 being required for growth and Cdk5 and Cdk8 being required for processes that occur during development. Recent studies have shown that Dictyostelium Cdk5 shares attributes with mammalian Cdk5 and that the mammalian Cdk inhibitor roscovitine can be used to inhibit Cdk5 activity in Dictyostelium. Together, these results show that Dictyostelium can be used as a model system for studying Cdk protein function.
Cdc14 phosphatases preferentially dephosphorylate a subset of cyclin-dependent kinase (Cdk) sites containing phosphoserine.
Bremmer, Steven C; Hall, Hana; Martinez, Juan S; Eissler, Christie L; Hinrichsen, Thomas H; Rossie, Sandra; Parker, Laurie L; Hall, Mark C; Charbonneau, Harry
Mitotic cell division is controlled by cyclin-dependent kinases (Cdks), which phosphorylate hundreds of protein substrates responsible for executing the division program. Cdk inactivation and reversal of Cdk-catalyzed phosphorylation are universal requirements for completing and exiting mitosis and resetting the cell cycle machinery. Mechanisms that define the timing and order of Cdk substrate dephosphorylation remain poorly understood. Cdc14 phosphatases have been implicated in Cdk inactivation and are thought to be generally specific for Cdk-type phosphorylation sites. We show that budding yeast Cdc14 possesses a strong and unusual preference for phosphoserine over phosphothreonine at Pro-directed sites in vitro. Using serine to threonine substitutions in the Cdk consensus sites of the Cdc14 substrate Acm1, we demonstrate that phosphoserine specificity exists in vivo. Furthermore, it appears to be a conserved property of all Cdc14 family phosphatases. An invariant active site residue was identified that sterically restricts phosphothreonine binding and is largely responsible for phosphoserine selectivity. Optimal Cdc14 substrates also possessed a basic residue at the +3 position relative to the phosphoserine, whereas substrates lacking this basic residue were not effectively hydrolyzed. The intrinsic selectivity of Cdc14 may help establish the order of Cdk substrate dephosphorylation during mitotic exit and contribute to roles in other cellular processes.
Positive feedback of G1 cyclins ensures coherent cell cycle entry.
Skotheim, Jan M; Di Talia, Stefano; Siggia, Eric D; Cross, Frederick R
In budding yeast, Saccharomyces cerevisiae, the Start checkpoint integrates multiple internal and external signals into an all-or-none decision to enter the cell cycle. Here we show that Start behaves like a switch due to systems-level feedback in the regulatory network. In contrast to current models proposing a linear cascade of Start activation, transcriptional positive feedback of the G1 cyclins Cln1 and Cln2 induces the near-simultaneous expression of the approximately 200-gene G1/S regulon. Nuclear Cln2 drives coherent regulon expression, whereas cytoplasmic Cln2 drives efficient budding. Cells with the CLN1 and CLN2 genes deleted frequently arrest as unbudded cells, incurring a large fluctuation-induced fitness penalty due to both the lack of cytoplasmic Cln2 and insufficient G1/S regulon expression. Thus, positive-feedback-amplified expression of Cln1 and Cln2 simultaneously drives robust budding and rapid, coherent regulon expression. A similar G1/S regulatory network in mammalian cells, comprised of non-orthologous genes, suggests either conservation of regulatory architecture or convergent evolution.
Extracellular alpha-synuclein induces calpain-dependent overactivation of cyclin-dependent kinase 5 in vitro.
Czapski, Grzegorz A; Gąssowska, Magdalena; Wilkaniec, Anna; Cieślik, Magdalena; Adamczyk, Agata
Extracellular alpha-synuclein (ASN) could be involved in the pathomechanism of Parkinson's disease (PD) via disturbances of calcium homeostasis, activation of nitric oxide synthase and oxidative/nitrosative stress. In this study we analyzed the role of cyclin-dependent kinase 5 (Cdk5) in the molecular mechanism(s) of ASN toxicity. We found that exposure of PC12 cells to ASN increases Cdk5 activity via calpain-dependent p25 formation and by enhancement of Cdk5 phosphorylation at Tyr15. Cdk5 and calpain inhibitors prevented ASN-evoked cell death. Our findings, indicating the participation of Cdk5 in ASN toxicity, provide new insight into how extracellular ASN may trigger dopaminergic cell dysfunction in PD.
Regulation of a Myb transcription factor by cyclin-dependent kinase 2 in Giardia lamblia.
Cho, Chao-Cheng; Su, Li-Hsin; Huang, Yu-Chang; Pan, Yu-Jiao; Sun, Chin-Hung
The protozoan Giardia lamblia parasitizes the human small intestine to cause diseases. It undergoes differentiation into infectious cysts by responding to intestinal stimulation. How the activated signal transduction pathways relate to encystation stimulation remain largely unknown. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately up-regulated by a Myb2 transcription factor. Because cell differentiation is linked to cell cycle regulation, we tried to understand the role of cell cycle regulators, cyclin-dependent kinases (Cdks), in encystation. We found that the recombinant Myb2 was phosphorylated by Cdk-associated complexes and the levels of phosphorylation increased significantly during encystation. We have identified a putative cdk gene (cdk2) by searching the Giardia genome database. Cdk2 was found to localize in the cytoplasm with higher expression during encystation. Interestingly, overexpression of Cdk2 resulted in a significant increase of the levels of cwp gene expression and cyst formation. In addition, the Cdk2-associated complexes can phosphorylate Myb2 and the levels of phosphorylation increased significantly during encystation. Mutations of important catalytic residues of Cdk2 resulted in a significant decrease of kinase activity and ability of inducing cyst formation. Addition of a Cdk inhibitor, purvalanol A, significantly decreased the Cdk2 kinase activity and the levels of cwp gene expression and cyst formation. Our results suggest that the Cdk2 pathway may be involved in phosphorylation of Myb2, leading to activation of the Myb2 function and up-regulation of cwp genes during encystation. The results provide insights into the use of Cdk inhibitory drugs in disruption of Giardia differentiation into cysts.
Roscovitine blocks leukocyte extravasation by inhibition of cyclin-dependent kinases 5 and 9
Berberich, Nina; Uhl, Bernd; Joore, Jos; Schmerwitz, Ulrike K; Mayer, Bettina A; Reichel, Christoph A; Krombach, Fritz; Zahler, Stefan; Vollmar, Angelika M; Fürst, Robert
BACKGROUND AND PURPOSE Roscovitine, a cyclin-dependent kinase (CDK) inhibitor that induces tumour cell death, is under evaluation as an anti-cancer drug. By triggering leukocyte apoptosis, roscovitine can also enhance the resolution of inflammation. Beyond death-inducing properties, we tested whether roscovitine affects leukocyte-endothelial cell interaction, a vital step in the onset of inflammation. EXPERIMENTAL APPROACH Leukocyte-endothelial cell interactions were evaluated in venules of mouse cremaster muscle, using intravital microscopy. In primary human endothelial cells, we studied the influence of roscovitine on adhesion molecules and on the nuclear factor-κB (NF-κB) pathway. A cellular kinome array, in vitro CDK profiling and RNAi methods were used to identify targets of roscovitine. KEY RESULTS In vivo, roscovitine attenuated the tumour necrosis factor-α (TNF-α)-induced leukocyte adherence to and transmigration through, the endothelium. In vitro, roscovitine strongly inhibited TNF-α-evoked expression of endothelial adhesion molecules (E-selectin, intercellular cell adhesion molecule, vascular cell adhesion molecule). Roscovitine blocked NF-κB-dependent gene transcription, but not the NF-κB activation cascade [inhibitor of κB (IκB) kinase activity, IκB-α degradation, p65 translocation]. Using a cellular kinome array and an in vitro CDK panel, we found that roscovitine inhibited protein kinase A, ribosomal S6 kinase and CDKs 2, 5, 7 and 9. Experiments using kinase inhibitors and siRNA showed that the decreased endothelial activation was due solely to blockade of CDK5 and CDK9 by roscovitine. CONCLUSIONS AND IMPLICATIONS Our study highlights a novel mode of action for roscovitine, preventing endothelial activation and leukocyte-endothelial cell interaction by inhibition of CDK5 and 9. This might expand its usage as a promising anti-inflammatory compound. PMID:21391976
Synthetic lethal mutations in the cyclin A interface of human cytomegalovirus
Vetter, Barbara
Generally, the antagonism between host restriction factors and viral countermeasures decides on cellular permissiveness or resistance to virus infection. Human cytomegalovirus (HCMV) has evolved an additional level of self-imposed restriction by the viral tegument protein pp150. Depending on a cyclin A-binding motif, pp150 prevents the onset of viral gene expression in the S/G2 cell cycle phase of otherwise fully permissive cells. Here we address the physiological relevance of this restriction during productive HCMV infection by employing a cyclin A-binding deficient pp150 mutant virus. One consequence of unrestricted viral gene expression in S/G2 was the induction of a G2/M arrest. G2-arrested but not mitotic cells supported viral replication. Cyclin A destabilization by the viral gene product pUL21a was required to maintain the virus-permissive G2-arrest. An HCMV double-point mutant where both pp150 and pUL21a are disabled in cyclin A interaction forced mitotic entry of the majority of infected cells, with a severe negative impact on cell viability and virus growth. Thus, pp150 and pUL21a functionally cooperate, together building a cell cycle synchronization strategy of cyclin A targeting and avoidance that is essential for productive HCMV infection. PMID:28129404
Correlation between Cyclin Dependent Kinases and Artemisinin-Induced Dormancy in Plasmodium falciparum In Vitro
Gray, Karen-Ann; Gresty, Karryn J.; Chen, Nanhua; Zhang, Veronica; Gutteridge, Clare E.; Peatey, Christopher L.; Chavchich, Marina; Waters, Norman C.; Cheng, Qin
Background Artemisinin-induced dormancy provides a plausible explanation for recrudescence following artemisinin monotherapy. This phenomenon shares similarities with cell cycle arrest where cyclin dependent kinases (CDKs) and cyclins play an important role. Methods Transcription profiles of Plasmodium falciparum CDKs and cyclins before and after dihydroartemisinin (DHA) treatment in three parasite lines, and the effect of CDK inhibitors on parasite recovery from DHA-induced dormancy were investigated. Results After DHA treatment, parasites enter a dormancy phase followed by a recovery phase. During the dormancy phase parasites up-regulate pfcrk1, pfcrk4, pfcyc2 and pfcyc4, and down-regulate pfmrk, pfpk5, pfpk6, pfcrk3, pfcyc1 and pfcyc3. When entering the recovery phase parasites immediately up-regulate all CDK and cyclin genes. Three CDK inhibitors, olomoucine, WR636638 and roscovitine, produced distinct effects on different phases of DHA-induced dormancy, blocking parasites recovery. Conclusions The up-regulation of PfCRK1 and PfCRK4, and down regulation of other CDKs and cyclins correlate with parasite survival in the dormant state. Changes in CDK expression are likely to negatively regulate parasite progression from G1 to S phase. These findings provide new insights into the mechanism of artemisinin-induced dormancy and cell cycle regulation of P. falciparum, opening new opportunities for preventing recrudescence following artemisinin treatment. PMID:27326764
Cyclin D1 and Ki-67 expression in normal, hyperplastic and neoplastic endometrium
Shevra, CR; Ghosh, A; Kumar, M
Background: Proliferation and differentiation of cancer cells are regulated by various cell cycle promoting and inhibiting factors. Our knowledge about these proteins and mechanisms regulating cell cycle progression has increased dramatically in recent years. Aim: The present study was undertaken to examine the expression profile of cell cycle regulatory proteins in normal proliferative endometrium, hyperplasias (simple, complex and atypical) and endometrial carcinoma in a quantitative approach as also to assess correlations of Cyclin D1 expression with Ki-67 a proliferation marker. Settings and Design: A retrospective case control study in a tertiary referral centre. Materials and Methods: We evaluated and compared the expression profile of Cyclin D1 and Ki-67 expressions in 61 endometrial samples submitted as either endometrial curetting or hysterectomy specimens, which were diagnosed as simple hyperplasia (n =11), complex hyperplasia (n = 13), atypical hyperplasia (n = 7), and endometrial carcinoma (n = 20). Results: There was increased expression of Cyclin D1 and Ki-67 in patients with endometrial carcinoma relative to proliferative endometrium and simple hyperplasia, but there was no such difference between cases of atypical hyperplasia and endometrial carcinoma. Cyclin D1 expression had a positive correlation with Ki-67 expression. Cyclin D1 together with Ki-67 may be a marker for endometrial carcinogenesis and tumor cell proliferation. PMID:25511212
A Chrysin Derivative Suppresses Skin Cancer Growth by Inhibiting Cyclin-dependent Kinases*
Liu, Haidan; Liu, Kangdong; Huang, Zunnan; Park, Chan-Mi; Thimmegowda, N. R.; Jang, Jae-Hyuk; Ryoo, In-Ja; He, Long; Kim, Sun-Ok; Oi, Naomi; Lee, Ki Won; Soung, Nak-Kyun; Bode, Ann M.; Yang, Yifeng; Zhou, Xinmin; Erikson, Raymond L.; Ahn, Jong-Seog; Hwang, Joonsung; Kim, Kyoon Eon; Dong, Zigang; Kim, Bo-Yeon
Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P+ cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P+ cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency. PMID:23888052
A chrysin derivative suppresses skin cancer growth by inhibiting cyclin-dependent kinases.
Liu, Haidan; Liu, Kangdong; Huang, Zunnan; Park, Chan-Mi; Thimmegowda, N R; Jang, Jae-Hyuk; Ryoo, In-Ja; He, Long; Kim, Sun-Ok; Oi, Naomi; Lee, Ki Won; Soung, Nak-Kyun; Bode, Ann M; Yang, Yifeng; Zhou, Xinmin; Erikson, Raymond L; Ahn, Jong-Seog; Hwang, Joonsung; Kim, Kyoon Eon; Dong, Zigang; Kim, Bo-Yeon
Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P(+) cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P(+) cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency.
Role of cyclin D1 as a mediator of c-Met- and beta-catenin-induced hepatocarcinogenesis.
Patil, Mohini A; Lee, Susie A; Macias, Everardo; Lam, Ernest T; Xu, Chuanrui; Jones, Kirk D; Ho, Coral; Rodriguez-Puebla, Marcelo; Chen, Xin
Activation of c-Met signaling and beta-catenin mutations are frequent genetic events observed in liver cancer development. Recently, we demonstrated that activated beta-catenin can cooperate with c-Met to induce liver cancer formation in a mouse model. Cyclin D1 (CCND1) is an important cell cycle regulator that is considered to be a downstream target of beta-catenin. To determine the importance of CCND1 as a mediator of c-Met- and beta-catenin-induced hepatocarcinogenesis, we investigated the genetic interactions between CCND1, beta-catenin, and c-Met in liver cancer development using mouse models. We coexpressed CCND1 with c-Met in mice and found CCND1 to cooperate with c-Met to promote liver cancer formation. Tumors induced by CCND1/c-Met had a longer latency period, formed at a lower frequency, and seemed to be more benign compared with those induced by beta-catenin/c-Met. In addition, when activated beta-catenin and c-Met were coinjected into CCND1-null mice, liver tumors developed despite the absence of CCND1. Intriguingly, we observed a moderate accelerated tumor growth and increased tumor malignancy in these CCND1-null mice. Molecular analysis showed an up-regulation of cyclin D2 (CCND2) expression in CCND1-null tumor samples, indicating that CCND2 may replace CCND1 in hepatic tumorigenesis. Together, our results suggest that CCND1 functions as a mediator of beta-catenin during HCC pathogenesis, although other molecules may be required to fully propagate beta-catenin signaling. Moreover, our data suggest that CCND1 expression is not essential for liver tumor development induced by c-Met and beta-catenin.
Role of p16/MTS1, cyclin D1 and RB in primary oral cancer and oral cancer cell lines
Sartor, M; Steingrimsdottir, H; Elamin, F; Gäken, J; Warnakulasuriya, S; Partridge, M; Thakker, N; Johnson, N W; Tavassoli, M
One of the most important components of G1 checkpoint is the retinoblastoma protein (pRB110). The activity of pRB is regulated by its phosphorylation, which is mediated by genes such as cyclin D1 and p16/MTS1. All three genes have been shown to be commonly altered in human malignancies. We have screened a panel of 26 oral squamous cell carcinomas (OSCC), nine premalignant and three normal oral tissue samples as well as eight established OSCC cell lines for mutations in the p16/MTS1 gene. The expression of p16/MTS1, cyclin D1 and pRB110 was also studied in the same panel. We have found p16/MTS1 gene alterations in 5/26 (19%) primary tumours and 6/8 (75%) cell lines. Two primary tumours and five OSCC cell lines had p16/MTS1 point mutations and another three primary and one OSCC cell line contained partial gene deletions. Six of seven p16/MTS1 point mutations resulted in termination codons and the remaining mutation caused a frameshift. Western blot analysis showed absence of p16/MTS1 expression in 18/26 (69%) OSCC, 7/9 (78%) premalignant lesions and 8/8 cell lines. One cell line, H314, contained a frameshift mutation possibly resulting in a truncated p16/MTS1 protein. pRB was detected in 14/25 (56%) of OSCC but only 11/14 (78%) of these contained all or some hypophosphorylated (active) pRB. In premalignant samples, 6/8 (75%) displayed pRB, and all three normal samples and eight cell lines analysed contained RB protein. p16/MTS1 protein was undetectable in 10/11 (91%) OSCCs with positive pRB. Overexpression of cyclin D1 was observed in 9/22 (41%) OSCC, 3/9 (33%) premalignant and 8/8 (100%) of OSCC cell lines. Our data suggest p16/MTS1 mutations and loss of expression to be very common in oral cancer cell lines and less frequent in primary OSCC tumours. A different pattern of p16/MTS1 mutations was observed in OSCC compared to other cancers with all the detected p16/MTS1 mutations resulting in premature termination codons or a frameshift. The RB protein was expressed
Cyclin D1 expression in acral melanoma: a case control study in Sarawak.
Ibrahim, Zainal Abidin; Narihan, M Zulkarnaen A; Ojep, Dk Norlida A; Soosay, Ashley Edward Roy; Pan, Kok Long
Acral melanoma has been reported to have distinctive clinical presentation and ethnic distribution compared to other histological types of malignant melanoma. Acral melanoma also exhibits distinctive focused gene amplifications, including cyclin D1 overexpression. We reviewed archived histological material of malignant melanoma in the Sarawak General Hospital from year 2004 to 2010. 43 tumours, comprising 28 acral melanoma and 15 non-acral melanoma, had sufficient material to be included in the study. The majority (36%) of acral melanoma tumours occurred in the heel. The tumours were analyzed for cyclin D1 expression by immunohistochemistry. 68% of acral melanoma were cyclin D1 positive compared to a positivity of 33% in non-acral tumours. This difference was statistically significant (p < 0.05). This finding may improve the histological diagnosis of acral melanoma and detection of positive resection margins.
Soluble hyaluronan receptor RHAMM induces mitotic arrest by suppressing Cdc2 and cyclin B1 expression
The hyaluronan (HA) receptor RHAMM is an important regulator of cell growth. Overexpression of RHAMM is transforming and is required for H- ras transformation. The molecular mechanism underlying growth control by RHAMM and other extracellular matrix receptors remains largely unknown. We report that soluble RHAMM induces G2/M arrest by suppressing the expression of Cdc2/Cyclin B1, a protein kinase complex essential for mitosis. Down-regulation of RHAMM by use of dominant negative mutants or antisense of mRNA also decreases Cdc2 protein levels. Suppression of Cdc2 occurs as a result of an increased rate of cdc2 mRNA degradation. Moreover, tumor cells treated with soluble RHAMM are unable to form lung metastases. Thus, we show that mitosis is directly linked to RHAMM through control of Cdc2 and Cyclin B1 expression. Failure to sustain levels of Cdc2 and Cyclin B1 proteins leads to cell cycle arrest. PMID:8666924
A standardized method for quantifying proliferation by Ki-67 and cyclin A immunohistochemistry in breast cancer.
Mu, Kun; Li, Li; Yang, Qingrui; Yun, Haiqin; Kharaziha, Pedram; Ye, Ding-Wei; Auer, Gert; Lagercrantz, Svetlana Bajalica; Zetterberg, Anders
Immunohistochemical analysis of proliferation markers such as Ki-67 and cyclin A is widely used in clinical evaluation as a prognostic factor in breast cancer. The proliferation status of tumors is guiding the decision of whether or not a patient should be treated with chemotherapy because low-proliferative tumors are less sensitive by such treatment. However, the lack of optimal cutoff points and selection of tumor areas hamper its use in clinical practice. This study was performed to compare the Ki-67 and cyclin A expression counted in hot-spot vs average counting based on 5 to 14 random tumor areas in 613 breast carcinomas. We correlated the findings with 10-year follow-up in order to standardize the evaluation of proliferation markers in clinical practice. A significant correlation was found between the percentage of positive cells estimated by Ki-67 and cyclin A both by hot-spot and by average counting. Both methods showed that high expression of Ki-67 and cyclin A is associated with more adverse tumor stage. The cutoff value for Ki-67 for distant metastases was set to 22% and to 15%, using hot-spot and average counting, respectively. For cyclin A, the values were set to 14% and 8% using the respective methods. Survival curves revealed that patients with a high hot-spot proliferation index had a significantly greater risk of shorter tumor-free survival. Our findings suggest that the determination of proliferation markers in breast cancer should be standardized to hot-spot counting and that specific cutoff values for proliferation could be useful as prognostic markers in clinical practice. Moreover, we suggest that expression levels of cyclin A could be used as a complementary marker to estimate the proliferation status in tumors, especially those with "borderline" expression levels of Ki-67, in order to more accurately estimate the proliferations status of the tumors.
Placental estrogen suppresses cyclin D1 expression in the nonhuman primate fetal adrenal cortex.
Dumitrescu, Adina; Aberdeen, Graham W; Pepe, Gerald J; Albrecht, Eugene D
We have previously shown that estrogen selectively suppresses growth of the fetal zone of the baboon fetal adrenal cortex, which produces the C19-steroid precursors, eg, dehydroepiandrosterone sulfate, which are aromatized to estrogen within the placenta. In the present study, we determined whether fetal adrenal expression of cell cycle regulators are altered by estrogen and thus provide a mechanism by which estrogen regulates fetal adrenocortical development. Cyclin D1 mRNA levels in the whole fetal adrenal were increased 50% (P < .05), and the number of cells in the fetal adrenal definitive zone expressing cyclin D1 protein was increased 2.5-fold (P < .05), whereas the total number of cells in the fetal zone and fetal serum dehydroepiandrosterone sulfate levels were elevated 2-fold (P < .05) near term in baboons in which fetal serum estradiol levels were decreased by 95% (P < .05) after maternal administration of the aromatase inhibitor letrozole and restored to normal by concomitant administration of letrozole plus estradiol throughout second half of gestation. However, fetal adrenocortical expression of cyclin D2, the cyclin-dependent kinase (Cdk)-2, Cdk4, and Cdk6, and Cdk regulatory proteins p27(Kip1) and p57(Kip2) were not changed by letrozole or letrozole plus estradiol administration. We suggest that estrogen controls the growth of the fetal zone of the fetal adrenal by down-regulating cyclin D1 expression and thus proliferation of progenitor cells within the definitive zone that migrate to the fetal zone. We propose that estrogen restrains growth and function of the fetal zone via cyclin D1 to maintain estrogen levels in a physiological range during primate pregnancy.
Antisense inhibition of cyclin D1 expression is equivalent to flavopiridol for radiosensitization of zebrafish embryos
McAleer, Mary Frances; Duffy, Kevin T.; Davidson, William R.; Kari, Gabor; Dicker, Adam P.; Rodeck, Ulrich; Wickstrom, Eric . E-mail: firstname.lastname@example.com
Purpose: Flavopiridol, a small molecule pan-cyclin inhibitor, has been shown to enhance Radiation response of tumor cells both in vitro and in vivo. The clinical utility of flavopiridol, however, is limited by toxicity, previously attributed to pleiotropic inhibitory effects on several targets affecting multiple signal transduction pathways. Here we used zebrafish embryos to investigate radiosensitizing effects of flavopiridol in normal tissues. Methods and Materials: Zebrafish embryos at the 1- to 4-cell stage were treated with 500 nM flavopiridol or injected with 0.5 pmol antisense hydroxylprolyl-phosphono nucleic acid oligomers to reduce cyclin D1 expression, then subjected to ionizing radiation (IR) or no radiation. Results: Flavopiridol-treated embryos demonstrated a twofold increase in mortality after exposure to 40 Gy by 96 hpf and developed distinct radiation-induced defects in midline development (designated as the 'curly up' phenotype) at higher rates when compared with embryos receiving IR only. Cyclin D1-deficient embryos had virtually identical IR sensitivity profiles when compared with embryos treated with flavopiridol. This was particularly evident for the IR-induced curly up phenotype, which was greatly exacerbated by both flavopriridol and cyclin D1 downregulation. Conclusions: Treatment of zebrafish embryos with flavopiridol enhanced radiation sensitivity of zebrafish embryos to a degree that was very similar to that associated with downregulation of cyclin D1 expression. These results are consistent with the hypothesis that inhibition of cyclin D1 is sufficient to account for the radiosensitizing action of flavopiridol in the zebrafish embryo vertebrate model.
Placental Estrogen Suppresses Cyclin D1 Expression in the Nonhuman Primate Fetal Adrenal Cortex*
Dumitrescu, Adina; Aberdeen, Graham W.; Pepe, Gerald J.
We have previously shown that estrogen selectively suppresses growth of the fetal zone of the baboon fetal adrenal cortex, which produces the C19-steroid precursors, eg, dehydroepiandrosterone sulfate, which are aromatized to estrogen within the placenta. In the present study, we determined whether fetal adrenal expression of cell cycle regulators are altered by estrogen and thus provide a mechanism by which estrogen regulates fetal adrenocortical development. Cyclin D1 mRNA levels in the whole fetal adrenal were increased 50% (P < .05), and the number of cells in the fetal adrenal definitive zone expressing cyclin D1 protein was increased 2.5-fold (P < .05), whereas the total number of cells in the fetal zone and fetal serum dehydroepiandrosterone sulfate levels were elevated 2-fold (P < .05) near term in baboons in which fetal serum estradiol levels were decreased by 95% (P < .05) after maternal administration of the aromatase inhibitor letrozole and restored to normal by concomitant administration of letrozole plus estradiol throughout second half of gestation. However, fetal adrenocortical expression of cyclin D2, the cyclin-dependent kinase (Cdk)-2, Cdk4, and Cdk6, and Cdk regulatory proteins p27Kip1 and p57Kip2 were not changed by letrozole or letrozole plus estradiol administration. We suggest that estrogen controls the growth of the fetal zone of the fetal adrenal by down-regulating cyclin D1 expression and thus proliferation of progenitor cells within the definitive zone that migrate to the fetal zone. We propose that estrogen restrains growth and function of the fetal zone via cyclin D1 to maintain estrogen levels in a physiological range during primate pregnancy. PMID:25247468
Okadaic acid induced cyclin B1 expression and mitotic catastrophe in rat cortex.
Chen, Bo; Cheng, Min; Hong, Dao-Jun; Sun, Feng-Yan; Zhu, Cui-Qing
Accumulating evidence indicates that the aberrant re-entry of post-mitotic neurons into the G2/M phase of cell cycle and the resulting mitotic catastrophe may contribute to the pathogenesis of Alzheimer's disease. However, the cellular event that drives the differentiated neurons to abnormally enter G2/M phase remains elusive. Similarly, whether mitotic catastrophe is indeed one of the death pathways for differentiated neurons is not clear. Previous studies revealed that okadaic acid (OA), a phosphatase inhibitor that induces AD like pathological changes, evokes mitotic changes in neuroblastoma cells. In this study, we examined the in vivo effects of OA on cyclin B1 expression, the induction of mitosis, and subsequent mitotic catastrophe. We found that cyclin B1 expression in adult neurons was significantly increased after injecting OA into rat frontal cortex, which also increased tau protein phosphorylation. Interestingly, cyclin B1 and phosphorylated tau were well co-localized around the OA injection site, but were only partially co-localized in other brain regions. Staining with toluidine blue, Giemsa dye or propidium iodide revealed typical mitotic and mitotic catastrophe-like morphological changes with irregular arrangement of condensed chromatin and chromosome fibers in a few cells. Furthermore, the strong cyclin B1 staining in these cells suggests that cyclin B1 promoted G2 to M phase transition is required for the mitotic catastrophe. The detection of neuron-specific enolase in a portion of these cells demonstrated that at least part them are neuron. All together, our results suggest that the disturbance of the protein kinase-phosphatase system caused by OA is sufficient to induce neuronal cyclin B1 expression, force neurons into the mitotic phase of cell cycle, and cause mitotic catastrophe.
Evaluation of Cyclin D1 expression in Head and Neck Squamous Cell Carcinoma
Dhingra, Vishal; Verma, Jyoti; Misra, Vatsala; Srivastav, Sapan
Introduction Squamous Cell Carcinoma (SCC) is an aggressive epithelial malignancy of the upper aero digestive tract and comprises 90% of all Head and Neck Squamous Cell Carcinoma (HNSCC). It is the sixth leading cancer worldwide with approximately 600,000 cases reported annually. It is one of the most common cancers in India. Tumour Lymph Node and Metastases (TNM) staging has been the most useful indicator to predict prognosis in HNSCC but recently various biomolecular markers have potentially offered new methods for early diagnosis and treatment alternatives for HNSCC patients; one amongst them being cyclin D1. Aim This study has been undertaken to evaluate expression of cyclin D1 in HNSCC cases and to find out its association with various pathological prognostic factors. Materials and Methods A 48 formalin fixed paraffin embedded tumour sections, stained with Haematoxylin & Eosin were graded and staged. Immunohistochemistry for cyclin D1 was evaluated as Extent Score (ES), Intensity Score (IS) and Total Score (TS) was calculated. Statistical Analysis All the relevant data collected was transferred on to the excel sheet. Chi square test with and without Yate's correction was used to compare various parameters. The p-value ≤ 0.05 was taken as critical level of significance. Results A significant association was seen between TS of Cyclin D1 expression with tumour stage and with lymph node metastasis but not with grade. Conclusion Higher Cyclin D1 expression is associated with higher tumour stage and lymph node metastasis. Therefore, there is value of analysing cyclin D1 amplification and expression, for prognostic evaluation. This may also be further used for targeted therapy in head and neck cancers. PMID:28384866
A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division.
Li, Yubing; Liu, Dianyi; López-Paz, Cristina;