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The traditional view of p53 activation includes three steps—p53 stabilization, DNA binding, and transcriptional activation. However, recent studies indicate that each step of p53 activation is more complex than originally anticipated. Moreover, both genetic studies in mice and in vitro studies with purified components suggest that the classical model may not be sufficient to explain all aspects of p53 activation in vivo. To reconcile these differences, we propose that antirepression, the release of p53 from repression by factors such as Mdm2 and MdmX, is a key step in the physiological activation of p53.
The year 2009 celebrates the 30th anniversary of the discovery of p53 (Lane and Crawford, 1979; Linzer and Levine, 1979; DeLeo et al., 1979) and the 20th anniversary of its characterization as a tumor suppressor (Baker et al., 1989; Finlay et al., 1989). Mutations that perturb p53 function, often in its DNA-binding domain, or disruptions to p53's upstream or downstream regulatory network, have been found in more than half of all cancer cases and are present in cancer-prone families with Li-Fraumeni syndrome (Hainaut and Hollstein, 2000; Vogelstein et al., 2000).
p53 has been called a "cellular gatekeeper" (Levine, 1997) or "the guardian of the genome" (Lane, 1992) because of its central role in coordinating the cellular responses to a broad range of cellular stress factors. p53 functions as a node for organizing whether the cell responds to various types and levels of stress with apoptosis, cell cycle arrest, senescence, DNA repair, cell metabolism, or autophagy. p53-controlled transactivation of target genes is an essential feature of each stress response pathway, although some effects of p53 may be independent of transcription (Vogelstein et al., 2000; Vousden and Lane, 2007; Marchenko and Moll, 2007). As a transcription factor that both activates and represses a broad range of target genes, p53 demands an exquisitely complicated network to control and fine-tune responses to the various stress signals encountered by cells (Brooks and Gu, 2003; Laptenko and Prives, 2006). p53 is regulated by an array of posttranslational modifications both during normal homeostasis and in stress-induced responses. More than 36 different amino acids within p53 have been shown to be modified in various biochemical and cell culture studies (Figure 1) (Kruse and Gu, 2008b).
Classical models for the activation of p53 focus on three simple and rate-limiting steps: p53 stabilization induced by ATM/ATR-mediated phosphorylation, sequence-specific DNA binding, and target gene activation by interacting with the general transcriptional machinery (Figure 2). However, as discussed in this review, the diversity and number of modifications, as well as recent studies from knockin mutant mice, suggest a certain degree of redundancy among these modifications. These findings challenge the importance of a number of the traditional regulatory events of p53 activation (Iwakuma and Lozano, 2007; Marine et al., 2006; Wahl, 2006; Brooks and Gu, 2006) and raise questions that cannot be sufficiently explained by the three steps of the classical model. What is the exact mechanism controlling p53 stabilization in vivo? Does p53 require additional activation to induce binding to DNA or is p53 already bound to DNA yet unable to induce transactivation in vivo? Does p53 require activation, or is it intrinsically active in vivo? How are decisions of cell fate controlled in vivo in response to different types and severities of stress? To incorporate recent findings, we revisit the different stages of p53 activation here. We propose that antirepression is a key mechanism for p53 regulation in vivo. Introducing this step for p53 activation into existing models may help to explain the events leading to p53 activation in vivo.
Phosphorylation of p53 is classically regarded as the first crucial step of p53 stabilization. p53 can be modified by phosphorylation by a broad range of kinases, including ATM/ATR/DNA-PK, and Chk1/Chk2 (see Figure 1). Phosphorylation of serine residues within the N-terminal p53 transactivation domain was among the first posttranslational modifications of p53 identified and has been extensively investigated in in vitro biochemical assays, in tissue culture studies, and recently by using site-specific knockin animals. N-terminal phosphorylation at Ser15 (mouse Ser18) and Ser20 (mouse Ser23) have been generally thought to stabilize p53 by inhibiting the interaction between p53 and Mdm2 (Figure 2). Ser15 and Ser20 are phosphorylated after DNA damage and other types of stress by ATM, ATR, DNA-PK, Chk1, and Chk2 (Appella and Anderson, 2001; Shieh et al., 1997; Shieh et al., 2000). Although it remains unresolved which of the different kinases perform the phosphorylation of these N-terminal regulatory sites in response to the varying stress signals, and to which extent N-terminal phosphorylation affects the p53-MDM2 interaction, the general consensus remains that their phosphorylation occurs rapidly in response to various stress stimuli to activate p53.
However, knockin mouse models with individual mutations of these N-terminal sites question the importance of the individual modifications in p53 stabilization in vivo. Studies with a Ser18Ala point mutant knockin mouse (S18A) indicate that at least some of the in vitro effects may not translate directly to a whole organism (Chao et al., 2003; Sluss et al., 2004). Although some mild effects on p53 levels in the knockin cells are observed, stress-induced p53 stabilization is comparable in thymocytes, splenocytes, and fibroblasts derived from wild-type and mutant knockin mice. The consistency in p53 levels in these studies argues against a dominant role for S18 phosphorylation in the regulation of the p53-MDM2 interaction in vivo, as a failure to phosphorylate S18 should promote this interaction and therefore induce p53 ubiquitination and degradation. Similar results are obtained with p53-serine 23 to alanine (S23A) point mutant knockin animals (MacPherson et al., 2004; Wu et al., 2002). Interestingly, more severe defects on both the levels and function of p53 were found in the S18/23A double-mutant mice, lending support to the physiological importance of these two key phosphorylation sites (Chao et al., 2006a). Nevertheless, these defects are limited to certain tissues and mostly for DNA-damage-induced apoptosis. For example, no obvious effects were observed in mouse embryonic fibroblasts or other tissues from the S18/23A double-mutant mice. These results suggest that under specific conditions, p53 phosphorylation is required for p53 stabilization. However, the fact that no differences in stability were observed in other tissues or in embryonic fibroblasts argues that phosphorylation may not be a universal requirement for p53 stabilization. Indeed, results from a wide range of studies have established that p53 can be activated regardless of its phosphorylation status (Ashcroft et al., 1999; 2000; Blattner et al., 1999; Wu et al., 2002). Together, these genetic and biochemical studies emphasize the notion that p53 stabilization in vivo requires a far more sophisticated regulatory network than can be provided by p53 phosphorylation alone.
The tight control of cellular p53 levels is primarily achieved through its ubiquitin-mediated proteasomal degradation (Brooks and Gu, 2006; Michael and Oren, 2003). Three independent studies identified the mouse double minute protein 2 (Mdm2) as the principal endogenous E3-ligase with high specificity for p53 (Haupt et al., 1997; Honda et al., 1997; Kubbutat et al., 1997). Interestingly, although the level of p53 is elevated in the absence of Mdm2, p53 is still degraded in the cells of Mdm2 null mice (Ringshausen et al., 2006), suggesting the existence of alternative, Mdm2-independent pathways for p53 degradation in vivo. Indeed, the recently discovered E3-ligases COP1 (Dornan et al., 2004), Pirh2 (Leng et al., 2003), Arf-BP1 (Chen et al., 2005), and others have clearly been shown to contribute to the efficient control of p53 levels in tissue culture and in in vitro biochemical experiments. Nevertheless, the precise roles of Mdm2-independent degradation in stress-induced p53 stabilization remain to be elucidated.
There are multiple layers of regulation that connect Mdm2 function with p53 stability during stress responses. A prominent physiological regulator of Mdm2 is the tumor suppressor ARF (Lowe and Sherr 2003). ARF interferes with the Mdm2-p53 interaction, thereby acting to stabilize and activate p53. The low steady-state levels of ARF in normal cells are dramatically induced upon oncogenic stress, which suppresses abnormal cell proliferation by triggering p53-dependent growth arrest or apoptosis (Sherr, 2006). Interestingly, the mechanisms by which ARF activates p53 function are complex. On one hand, ARF is predominately a nucleolar protein. Elegant work has shown that ARF can stabilize nucleoplasmic p53 by binding Mdm2 and sequestering it in the nucleolus (Weber et al., 1999). Yet, equally convincing studies have also shown that nucleoplasmic forms of ARF activate p53 by directly inhibiting the ubiquitin ligase activity of Mdm2 (Llanos et al., 2001; Honda and Yasuda., 1999). Moreover, in addition to suppressing Mdm2-mediated effects on p53, ARF may modulate the activity of other E3 ligases such as ARF-BP1, and our recent studies demonstrate that the ARF/ARF-BP1 interaction is involved in both p53-dependent and p53-independent functions of ARF (Chen et al., 2005). Thus, although the precise molecular mechanisms by which ARF mediates these activities need further elucidation, it is clear that ARF has an essential role in p53 regulation.
The other prominent regulator of Mdm2 activity is MdmX (also known as Mdm4). Like Mdm2, MdmX is a critical negative regulator of p53 (Marine and Jochemsen, 2005). The direct roles played by MdmX on p53 activity are discussed in more detail below; here, we focus on the ability of MdmX to regulate the function of Mdm2. Mdm2 and MdmX interact with each other via their C-terminal RING domains. Interestingly, MdmX stabilizes both Mdm2 and p53, yet MdmX also promotes the E3-ligase activity of Mdm2 (Linares et al., 2003; Poyurovsky et al., 2007; Uldrijan et al., 2007). The ability of MdmX to promote Mdm2 function, as well as its own direct impact on p53, suggests that MdmX, like Mdm2, may be a potential therapeutic target for regulating p53 activity (Shangary and Wang, 2009). In addition, the transcription factor YY1 and the ribosomal proteins L5, L11, and L23 interact with Mdm2 and regulate its function. YY1 promotes the ubiquitination of p53 in vivo and in vitro by strengthening the interaction between p53 and Mdm2 (Sui et al., 2004). Conversely, binding of the ribosomal proteins L5, L11, and L23 to Mdm2 inhibits Mdm2 function and plays a crucial role in p53 activation upon ribosomal stress (Dai et al., 2004; Lohrum et al., 2003; Zhang et al., 2003).
The complexity of the regulation of p53 stability through Mdm2 becomes even more apparent when considering the activity of the deubiquitinating enzyme HAUSP. Mdm2 is an unstable protein, primarily due to autoubiquitination or its ubiquitination by undefined E3 ligases (Fang et al., 2000; Stommel and Wahl, 2004; Itahana et al., 2007). HAUSP not only deubiquitinates p53 (Li et al., 2002a), but also Mdm2 (Cummins et al., 2004; Li et al., 2004). The protein DAXX is an important component of this multiprotein complex that regulates HAUSP-mediated deubiquitination of Mdm2 (Tang et al., 2006a), and recent work now shows that the assembly of this complex is in part regulated by the protein RASSF1A (Song et al., 2008). RASSF1A disrupts the interaction between Mdm2, DAXX, and HAUSP, thereby promoting Mdm2 ubiquitination, and consequently resulting in p53 stabilization. As this example demonstrates, an ever growing number of proteins continue to be defined that affect the levels or activities of Mdm2 and thereby influence p53 levels.
Mdm2 is also subject to regulation by phosphorylation and acetylation. Various phosphorylation sites have been described, and depending on the site and the modifying kinase, these modifications can inhibit or activate the activity of Mdm2 (reviewed in Meulmeester et al., 2005). ATM and c-Abl inhibit Mdm2 activity via the phosphorylation of Ser395 (S395) and Tyr394, respectively, whereas phosphorylation of Ser166 and Ser186 promote its E3-ligase activity (Meek, 2004; Mayo and Donner, 2002). Conversely, dephosphorylation of Mdm2 by WIP1 stabilizes Mdm2 and facilitates p53 ubiquitination and degradation (Lu et al., 2007). Mdm2 activity is also inhibited by CBP/p300-mediated acetylation, resulting in increased p53 levels (Wang et al., 2004). This provides an interesting level of coordinated p53 regulation, as acetylation of the p53 C terminus also blocks at least some of the major p53 ubiquitination sites used by Mdm2 for p53 degradation. The six C-terminal lysines of p53 are acetylated by CBP/p300 (Gu and Roeder, 1997). Ubiquitination and acetylation are mutually exclusive modifications, and competition between these modifications is thought to affect p53 stability. p53 acetylation levels are markedly enhanced in response to stress, promoting p53 stabilization and activation (Brooks and Gu, 2003; Luo et al., 2001; Sakaguchi et al., 1998). Purified acetylated p53 cannot be ubiquitinated by Mdm2 in vitro, and ubiquitination is significantly reduced upon induction of acetylation (Ito et al., 2001; Li et al., 2002b). More recent work has shown that the acetylation of eight different lysine residues on p53 prevents the interaction between p53 and Mdm2 (Tang et al., 2008). Therefore, both phosphorylation and acetylation of p53 can inhibit the Mdm--p53 interaction, suggesting a critical role of posttranslational modifications in affecting the p53/Mdm2 feedback loop.
Due to the importance of the Mdm2-p53 interaction, inhibition of this event with small molecules is regarded as having therapeutic potential. A number of different strategies have been employed to screen for and develop small molecules that bind specifically to the N-terminal region of Mdm2 that interacts with p53 (Shangary and Wang, 2009). The most promising candidates described so far have been Nutlin-3A (Vassilev et al., 2004) and MI-219 (Shangary et al., 2008), and analogs of these lead compounds are being developed or have already entered preclinical development or early clinical trials. As expected, inhibition of the Mdm2-p53 interactions by small molecules results in increased p53 levels. The significance of controlling the ubiquitin ligase activity of Mdm2 is further bolstered by work studying the effects of a site-specific (C462A) knockin mutation of Mdm2 (Itahana et al., 2007). Notably, HLI98, a promising inhibitor of Mdm2 E3 ligase activity, also activates p53 (Yang et al., 2005). Nevertheless, the outcome of raising p53 levels and subsequent p53-mediated apoptosis is very dependent on the cell or tissue type. It will be of great interest to determine whether these compounds when used in combination therapies might selectively target cancer cells in the clinic.
The six C-terminal lysines of p53 are the predominant sites for Mdm2-mediated ubiquitination (Lohrum et al., 2001) (Figure 1). Although the in vitro data certainly demonstrate the importance of the six C-terminal lysines of p53 for Mdm2-mediated ubiquitination, knockin studies in which the equivalent lysines are mutated in analogous fashion to arginines (p53-6KR knockin mice) do not dramatically alter p53 protein levels (Feng et al., 2005; Krummel et al., 2005). Analysis of p53 protein stability in embryonic stem cells, embryonic fibroblasts, and thymocytes derived from p53-6KR mutant knockin animals reveals normal p53 stabilization both before and after DNA damage (Feng et al., 2005). Furthermore, the half-life of an analogous p53-7KR mutant (a mouse p53 construct with the seven C-terminal lysines mutated) showed no difference when compared to wild-type p53 in embryonic fibroblasts and thymocytes derived from p53-7KR knockin mice (Krummel et al., 2005). Together, these two studies support the theory that the C-terminal lysines are not essential for efficient p53 degradation in vivo and that additional E3-ligases, as well as ubiquitination of additional p53 lysines, are required for effective p53 regulation. Recent in vitro data suggest that lysine residues located in the DNA-binding domain and in the N-terminal region may be ubiquitinated (Chan et al., 2006). Thus, the sites of Mdm2-mediated ubiquitination are not as specific as originally anticipated. It is very likely that the C terminus of human p53 is preferentially targeted by Mdm2 for ubiquitination, but Mdm2 is still able to degrade p53 in vivo in the absence of these sites.
The levels of nuclear p53 are also modulated by degradation-independent ubiquitination. Mdm2-mediated p53 monoubiquitination, occurring when Mdm2 activity levels are low, promotes p53 nuclear export and accumulation of cytoplasmic p53 (Lohrum et al., 2001; Li et al., 2003; Marchenko et al., 2007). Although originally thought of as a passive way to block the nuclear function of p53, accumulating evidence suggests that cytoplasmic localization of p53 plays an active role in p53-mediated functions such as apoptosis and autophagy. Once in the cytoplasm, p53 can localize to the mitochondria, where it induces apoptosis (Marchenko and Moll, 2007). p53 interacts with antiapoptotic members of the Bcl family such as Bcl-XL and Bcl-2 at the outer mitochondrial membrane to promote the oligomerization of the proapoptotic factors Bak and Bax. These in turn drive the formation of pores in the mitochondrial membrane, resulting in the release of cytochrome c and other apoptotic activators from the mitochondria (Chipuk et al., 2004; Mihara et al., 2003; Tomita et al., 2006). Alternatively, p53 can directly interact with Bak, releasing Bak from its inhibitory interaction with Mcl1 (Leu et al., 2004). Further evidence shows that changes in subcellular localization of p53 can have profound effects on the cell. Recently, cytoplasmic p53 has been shown to regulate autophagy (Tasdemir et al., 2008). An additional factor that regulates p53 localization is MSL2, another E3-ligase for p53, which can promote its cytoplasmic localization independent of Mdm2 (Kruse and Gu, 2008a). Significantly, this RING-E3 does not affect p53 levels or Mdm2 mediated-p53 monoubiquitination, and targets different lysines (K351, K357) for ubiquitination than Mdm2 (Figure 1). It will be interesting to determine whether MSL2 regulates apoptosis or other roles ascribed to cytoplasmic p53.
Together, the studies using knockin mice with mutations in p53's phosphorylation sites and C-terminal lysine residues indicate that p53 stabilization in vivo involves many different mechanisms. None of the sites tested is able to recapitulate in vivo the dramatic effects on p53 stabilization observed in in vitro biochemical and tissue culture studies. Overall, the model for p53 stabilization in vivo has shifted from a primary emphasis on p53 phosphorylation to a greater appreciation for the multiple layers of Mdm2 regulation. However, it remains incompletely understood how p53 is stabilized in vivo in different tissues and in response to different stress signals.
An early breakthrough for the p53 field was the discovery that p53 is a sequence-specific DNA-binding protein (Bargonetti et al., 1991; Kern et al., 1991). The DNA consensus sequence of p53 response elements was subsequently identified by in vitro assays (El-Deiry et al., 1992) and has been recently refined by chromatin immunoprecipitation (ChIP) sequencing in human cells (Wei et al., 2006). The consensus sequence contains two inverted pentameric sequences with the pattern 5′-RRRC(A/T)|(A/T)GYYY-3′. The importance of sequence-specific DNA binding is underscored by the fact that the vast majority of tumor-associated p53 mutations occur within its DNA-binding domain (Hainaut and Hollstein, 2000), which consists of a beta sandwich with a series of loops and short helices (Cho et al., 1994). Transactivation requires p53 to interact with promoter regions of p53 target genes. In the classical model for p53 activation, the second canonical step is the binding of stress-activated and posttranslationally modified p53 to specific response elements in the promoters of target genes (Figure 2). As discussed below, it is unclear whether DNA binding by p53 is dependent on stress signals or other modes of regulation. Early studies focused on the ability of p53 to bind DNA in a sequence-specific manner. DNA binding was thought to occur via the conserved central core domain, whereas the C-terminal region was thought to act as a negative modulator that had to be modified to allow sequence-specific DNA binding. Phosphorylation, ubiquitination, methylation, sumoylation, neddylation, and acetylation all target and modify the C-terminal region of p53, thereby affecting the ability of p53 to bind DNA (Appella and Anderson, 2001; Brooks and Gu, 2003). In addition to affecting DNA binding via the core domain, these modifications may affect C terminus-mediated DNA binding, direct other regulatory events such as cofactor recruitment, and provide sequence specificity.
The ability of stress-induced, allosteric activation of unmodified p53 to bind sequence specific DNA (Hupp et al., 1992) has recently been questioned by a variety of studies. Experiments using larger DNA molecules or chromatin as targets demonstrate that activation of p53 is not required for p53 to bind to DNA (Cain et al., 2000; Kim et al., 1999). Furthermore, analysis of wild-type and mutant p53 variants using nuclear magnetic resonance show that presumed latent and active p53 have an extremely similar molecular structures and that the C-terminal region does not interact with the central DNA-binding region (Ayed et al., 2001). It is currently believed that both the core DNA-binding domain and the C-terminal domain of p53 possess DNA-binding activities, with the former primarily providing sequence specificity whereas the latter recognizes structural features of target DNA.
In light of its ability to bind DNA both based on sequence specificity and on DNA structure and topology, the question of whether stress is required to induce DNA binding of p53 becomes even more intriguing. A number of recent studies now provide additional evidence that a significant portion of p53 is bound to DNA in unstressed cells. An unbiased global ChIP assay demonstrates that the C-terminal domain facilitates p53 binding to genomic DNA, and only 5% of p53-bound genomic sequence isolated by ChIP contains the p53 consensus sites (Liu et al., 2004). Furthermore, a microarray analysis of p53 binding to genomic DNA also shows that only 2% of p53 binding events are associated with p53 consensus sites (Cawley et al., 2004). In addition to this nonspecific binding to DNA, ChIP assays show that p53 is present at promoters of its transcriptional targets during normal homeostasis (Kaeser and Iggo, 2002; Szak et al., 2001). Quantitative ChIP assays demonstrate that p53 is present at the promoters of the p21 and Mdm2 genes from wild-type, unstressed p53 cells. These findings provide further support for a model in which p53 is capable of binding DNA in unstressed cells but remains inactive probably because of repression by Mdm2 and MdmX.
Overall, there is no doubt that the binding of p53 to DNA is essential for p53 function, yet it may not be a rate-limiting step in the p53 response as originally thought (Hupp et al., 1992). DNA binding received tremendous attention early on, in part due to the poor DNA binding activity of p53 in vitro and the known tumor-derived mutants that affect DNA binding. However, recent work has shown that in many cases p53 is already bound to the p53 target promoters without activating transcription, although it remains possible that acetylation is important for enhancement of DNA binding on some specific p53 proapoptotic targets (Sykes et al., 2006).
Early studies suggest that p53 promotes transcription activation by simply interacting with general transcription factors such as TFIID/TAFs or components of SRB/mediator complexes (Thut et al., 1995; Gu et al., 1999). This simple model for transcriptional activation belies the complexity of achieving promoter specificity. In broad terms, promoter specific transactivation could be achieved by the interaction of p53 with specific repressors and activators. The interaction of repressors and activations might be regulated by the posttranslational modification of p53, or interacting proteins might promote changes in p53 posttranslational modification to achieve promoter-specific transactivation. Although there are many proteins reported to bind and regulate p53, very few have been shown to be physically recruited to the promoters of p53 targets, and genetic evidence identifying them as bona fide regulators of p53 is typically lacking. To provide a structure and an overview for this very complex topic, we discuss the roles of p53 repressors and activators and the impact of various p53 modifications in detail below.
Mdm2 and MdmX are structurally related proteins, and physiological levels of both proteins are required in a nonredundant manner to balance p53 activity during embryonic development (Marine et al., 2006). The functions of p53 are downregulated by the Mdm2 oncoprotein, at least in part by ubiquitin-mediated proteolysis (Brooks and Gu, 2006). The central role of Mdm2 in this process is best illustrated by studies carried out in mice, where inactivation of p53 was shown to completely rescue the embryonic lethality caused by loss of Mdm2 function (Jones et al., 1995; Montes de Oca Luna et al., 1995). Notably, MdmX knockout mice die despite having functional Mdm2, and this lethality is also rescued by inactivation of p53 (Parant et al., 2001; Migliorini et al., 2002). Thus, MdmX is as critical as Mdm2 in repressing p53 function. In addition to promoting p53 degradation, accumulating evidence indicates that Mdm2 and MdmX form a protein complex with p53 on the promoters of specific p53 target genes (Minsky and Oren, 2004; Ohkubo et al., 2006; Tang et al., 2008).
Initial studies indicate that MdmX represses p53-mediated transcriptional activation (Marine and Jochemsen, 2005). Despite its sequence homology with Mdm2 and the presence of a RING domain, MdmX does not have intrinsic E3-ligase activity for p53 but instead inhibits p53-induced transcription. The interplay between p53, Mdm2, and MdmX is an extremely interesting topic, and new studies with mutant mice highlight their intricate relationship (Iwakuma and Lozano, 2007; Marine et al., 2006; Wahl, 2006), arguing that the major role of MdmX lies in its ability to repress but not degrade p53. Mdm2 and MdmX can both be recruited to p53 promoter regions and act via a multitude of mechanisms to repress transcription of p53 target genes (Tang et al., 2008). The various results from animal studies, together with in vitro and tissue culture data, have led to models that describe the distinct yet combinatorial roles played by these two negative regulators of p53 (Marine et al., 2006; Wahl, 2006). Mdm2 and MdmX may determine whether a cell responds to p53 activation with growth arrest or apoptosis, but the molecular mechanism of these differential effects remains unknown (Barboza et al., 2008). It is possible that Mdm2 and MdmX are recruited by p53 to different target promoters to execute distinct functions.
Histone acetyltransferases (HATs) provide an important layer of p53 regulation, particularly in transcription (Brooks and Gu, 2003). The covalent linkage of an acetyl group to a lysine residue was first discovered on histones, and histone acetylation is now known to have a major impact on transcriptional regulation (Jenuwein and Allis, 2001). However, histones are not the only proteins that can be acetylated; p53 was the first nonhistone substrate shown to be functionally regulated by acetylation and deacetylation (Gu and Roeder, 1997; Luo et al., 2000). p53 is acetylated by the histone acetyltransferase CBP/p300, and CBP/p300 mutations are found in several types of human tumors (Goodman and Smolik, 2000; Iyer et al., 2004). p53 acetylation is critically important both for the efficient recruitment of cofactors and for the activation of p53 target genes in vivo. Once localized at promoter regions, CBP/p300 can enhance transcription by acetylating histones in the vicinity of target genes, thereby establishing a more accessible chromatin conformation, and by bridging transcription factors to the pol II holoenzyme (Goodman and Smolik, 2000). In addition, the acetyltransferases Tip60 and hMof are cofactors that can be recruited by p53 and are able to acetylate p53 as well as histones (Tang et al., 2006b; Sykes et al., 2006). The acetyl-transferase activity of Tip60 is implicated in both DNA repair and apoptosis, but, most significantly, evidence suggests that Tip60 is a p53 activator by a large-scale RNA interference screen (Berns et al., 2004).
p53 acetylation is elevated in response to stress, and levels of acetylation correlate well with p53 activation and stabilization (Luo et al., 2000; 2001; Vaziri et al., 2001; Ito et al., 2001; Knights et al., 2006). The transcription phenotypes described for the C-terminal acetylation-deficient p53-6KR knockin mice show that p53-dependent gene expression in embryonic stem cells and thymocytes is impaired in a promoter-specific manner after DNA damage. This finding supports the theory that C-terminal acetylation helps to activate p53 dependent trans-activation after DNA damage (Feng et al., 2005). However, the transcription profiles in cells from the 6KR knockin mice show some cell type specificity, with the most dramatic differences observed in embryonic stem cells, a more selective defect in thymocytes, and no significant differences in embryonic fibro-blasts. Similarly, embryonic fibroblasts generated from the p53-7KR knockin mice did not show substantial differences in p53-mediated cell cycle control or apoptosis (Krummel et al., 2005). Together, these two knockin studies suggest that C-terminal acetylation shows some cell type-specific regulatory effects, but does not have a major general impact on apoptosis or cell cycle control. In addition to CBP/p300, p300/CBP associated factor (PCAF) is another transcriptional coactivator of p53, which acetylates p53 at lysine K320 (K317 in mice) (Sakaguchi et al., 1998; Liu et al., 1999). Analysis of K317 acetylation in a mouse model shows that a K317R mutant p53 protein is stabilized and accumulates at a rate comparable to wild-type p53 in embryonic fibroblasts and thymocytes (Chao et al., 2006b). Although the expression profile of p53 target genes is largely normal in K317R mouse embryonic fibroblasts, the authors surprisingly find an increase in the expression of proapoptotic genes in thymocytes. There is also an increase in the apoptotic response upon irradiation in thymocytes and epithelial cells from the small intestine, indicating that the effects of K320 acetylation by PCAF are far more complicated than originally anticipated.
Although the C-terminal lysine mutant knockin animal models raise some concerns over the importance of C-terminal acetylation, recent studies show that p53 acetylation is not limited to this domain (Sykes et al., 2006; Tang et al., 2006b). Both studies show that lysine K120 is acetylated, providing an example of transcriptional regulation mediated by acetylation that requires the DNA-binding core domain. In contrast to other sites of p53 modification, K120 is conserved in all species that contain functional p53 genes, implying that this modification may have an evolutionarily conserved role. K120 is acetylated by hMOF and TIP60, acetyltransferases of the MYST family that share no homology with the CBP/p300 or PCAF acetyltransferases. K120 acetylation is essential for p53-mediated activation of the proapoptotic target genes puma and bax, whereas expression of p21 and Mdm2 are not affected. Furthermore, K120 modification affects neither p53 stability nor p53-DNA binding. K120 acetylation occurs after DNA damage, as well as after activation of the p19ARF oncogenic stress pathway (Mellert et al., 2007; Tang et al., 2006b). K164 is another newly identified site of acetylation by CBP/p300. Like K120, K164 is located within the DNA-binding domain (Tang et al., 2008), and both residues are recurrent sites for p53 mutation in human cancer (Hainaut and Hollstein, 2000). Simultaneous loss of acetylation at K120, K164, and the six C-terminal lysines completely abolishes the ability of p53 to activate p21 and suppress cell cycle progression (Tang et al., 2008). Interestingly, separate acetylation defects at K120, K164, or the 6KR sites can be compensated by acetylation of the other sites, enabling p53-mediated transcription of p21. Importantly, an acetylation-defective p53-8KR mutant retains the ability to function as a DNA-binding transcription factor and induces the p53-Mdm2 feedback loop, establishing promoter specificity for this regulatory event. The complete inability of the p53-8KR mutants to induce cell cycle and apoptotic regulators demonstrates that acetylation is absolutely required for key aspects of p53-mediated tumor suppression. Both K120 and K164 residues, as well as the majority of the p53 core domain, are highly conserved, and it will be of great interest to see what effect a p53 K120R or K164R mutation would have in a knockin animal model.
The functional consequences of p53 acetylation suggest that the timing of acetylation of the different p53 regions may be important for accurate p53 regulation and cell fate determination. It is very likely that distinct mechanisms activate different types of p53 targets during the course of the stress response. For each p53-dependent stress response, at least three different classes of target genes can be turned on by p53 to execute p53-mediated function at different levels (Figure 3). The first class of p53 target genes protects cells from excessive p53 activation; these include Mdm2, Pirh2, and others. Given that activated p53 is potentially dangerous to cell viability, the feedback mediated by Mdm2 is probably an early step in the p53-mediated stress response. p53 modification (at least, in the case of acetylation) and coactivator recruitment (e.g., Tip60) are not required for this step (Tang et al., 2006b, 2008). The second class of p53 target genes induces growth arrest, and these genes are particularly important for cells to initiate productive DNA repair. The most important target of this class is p21. p53-dependent recruitment of coactivators such as CBP/p300 and Tip60/hMOF is absolutely required for p21 activation (Sykes et al., 2006; Tang et al., 2006b). Nevertheless, p21 induction is probably redundantly regulated via the modification of p53 at multiple sites. Although the complete loss of p21 induction in the absence of p53 acetylation in the 8KR mutant cells demonstrates that this event is indispensable for p53-mediated growth arrest, a partial modification of p53 is sufficient to induce p21 activation (Tang et al., 2006b, 2008). The third class of p53 target genes induces apoptosis, including PUMA, BAX, NOXA, and others. Given that this step initiates an irreversible apoptotic response, the requirement for full activation of proapoptotic targets is stringent. For example, all the key coactivators have to be recruited to the target promoters; specific modification (e.g., K120 acetylation) is required, and p53 is likely modified at many different sites (Figure 3). In addition, the corepressors such as Mdm2 and MdmX must be completely removed from these target promoters.
Conversely, p53 can be deacetylated and acetylation-dependent upregulation of p53 transactivation can be reversed by distinct histone deacetylase (HDAC) complexes containing HDAC1 (Luo et al., 2000) or Sir2α/Sirt1 (Luo et al., 2001; Vaziri et al., 2001). Deacetylation represses p53-dependent transcriptional activation, apoptosis, and growth arrest. p53 deacetylation by the HDAC1 complex strongly represses p53 transcriptional activity and modulates growth arrest and apoptosis (Luo et al., 2000). Sirt1-mediated p53 deacetylation represses p53-dependent apoptosis in response to DNA damage and oxidative stress, whereas expression of a Sirt1 dominant-negative point mutant increases the sensitivity of cells to stress (Luo et al., 2001). This notion is further supported by the observation that p53 is hyperacetylated in Sirt1-deficient mice and cells from these mice display increased radiation-induced apoptosis (Cheng et al., 2003). Sirt1-mediated deacetylation of p53 is suppressed by a putative tumor suppressor, DBC-1 (deleted in breast cancer gene 1), in human cells (Zhao et al., 2008; Kim et al., 2008) and is upregulated by oncogenic viral protein HPV E7 (Allison et al., 2009). A small molecule, tenovin-6, has been recently identified as a potent SIRT1 inhibitor that effectively induces p53 activation and cell growth repression in cancer cells (Lain et al., 2008). Furthermore, Sirt1 colocalizes with p53 in promyelocytic leukemia (PML) nuclear bodies and undergoes a conformational change when bound to acetylated p53 to antagonize the increase in p53 acetylation induced by PML (Pearson et al., 2000). p53 deacetylation may provide a quick-acting mechanism to stop p53 function when transcriptional activation of target genes is no longer required. Targeted deacetylation occurs very quickly amidst a global equilibrium of genomic acetylation and deacetylation. Restoring a steady-state level of p53 targets after completion of DNA repair is crucial to allow cells to return to normal homeostasis. Deacetylation could serve as an important step to allow Mdm2-mediated p53 degradation, given that acetylation of p53 inhibits p53 ubiquitination by Mdm2 in vivo and in vitro (Li et al., 2002b).
It is not only acetylation and ubiquitination that compete for p53's C-terminal lysines. Methylation, sumoylation, and neddylation all occur at specific sites, and the exact interplay of these modifications may contribute to p53 promoter specificity. The many possible variations of these modifications of p53 may act like a barcode, resulting in specific p53 responses.
To this point, three different methyltransferases have been shown to be able to methylate C-terminal lysine residues of p53. Set7/9-mediated monomethylation of p53 at lysine K372 promotes p53 activity, in particular the ability of p53 to transactivate p21 (Chuikov et al., 2004). Monomethylation of p53 at K370 and K382 by Smyd2 and Set8/PR-Set7, respectively, represses p53 activity (Huang et al., 2006; Shi et al., 2007). Regulated dimethylation of K370 and K382 provides a binding site for the DNA repair factor 53BP1, and DNA damage increases p53 dimethylation and therefore promotes the interaction of p53 and 53BP1. Interestingly, the demethylase LSD1 specifically demethylates K370, thereby removing at least one anchoring point for 53BP1 (Huang et al., 2007). Further support for the notion that methylation contributes to the regulation of the p53 response comes from evidence that both p53 (Jansson et al., 2008) and the surrounding histones (An et al., 2004) are arginine methylated. An et al. demonstrate that arginine methylation by PRMT1 and acetylation by p300 and CARM1 entails both direct modification of p53 as well as the controlled modification of the surrounding histones to regulate the expression of p53 responsive genes. Jansson et al. demonstrate that arginine methylation of p53 by PRMT5 regulates target gene specificity of p53 by altering promoter binding.
Modification of p53 by the ubiquitin-like modifications SUMO and Nedd8 further add to the competition for the C-terminal lysines. The exact functional consequences of p53 sumoylation at K386 are still somewhat unclear. Although some studies report that sumoylation promotes p53 transcriptional activity (Melchior and Hengst, 2002), others demonstrate that sumoylation promotes the localization of p53 to the cytoplasm (Carter et al., 2007). The role of C-terminal neddylation is interesting but not well defined. Mdm2-mediated neddylation of K370, K372, and K373 (Xirodimas et al., 2004) and FBXO11-mediated neddylation of K320 and K321 (Abida et al., 2007) appear to inhibit p53-mediated transcriptional activation. It remains to be determined under what circumstances methylation, sumoylation, and neddylation are required for p53 activation in vivo.
There are a number of proteins that affect p53 activity through their selective interaction with the tumor suppressor. The ASPP (ankyrin-repeat-, SH3-domain- and proline-rich-region-containing proteins) family specifically affects the p53-mediated apoptotic response (Trigiante and Lu, 2006). The ASPP family consists of two proapoptotic mediators, ASPP1 and ASPP2, and the antiapoptotic mediator iASSP. ASPP1 and ASPP2 enhance the proapoptotic function of p53 by selectively promoting the binding of p53 to proapoptotic gene targets such as BAX, PUMA, and PIG3 (Samuels-Lev et al., 2001). Conversely, iASSP prevents the transcriptional activity of p53 bound to proapoptotic promoters (Bergamaschi et al., 2003). However, the exact mechanism underlying the promoter specificity of ASPP1, ASPP2, and iASSP is still unknown. Interestingly, the expression levels of ASPP1/ASPP2 and iASSP correlate with cellular sensitivity or resistance to apoptosis. Due to their specific regulation of the p53 response, targeting the ASPP family may have therapeutic potential.
53BP1 binds p53 and mediates promoter specificity by a yet unknown mechanism (Iwabuchi et al., 1994). It was originally identified together with 53BP2, which was later shown to be a truncated N-terminal splice form of ASPP2 and has recently been shown to be a coactivator of p53 mediated proliferation arrest (Brummelkamp et al., 2006). In addition to its role in the regulation of p53, it also has an important function in DNA repair. The fact that 53BP1 is involved in both DNA damage repair and p53-induced cell cycle arrest suggests that 53BP1 could contribute to the coordination of these responses. With an ever growing number of p53-interacting partners being identified, such as HZF, APAK, and hCAS/CSE1L (Das et al., 2007; Tanaka et al., 2007; Tian et al., 2009), it will be increasingly important to characterize cofactors that affect promoter specificity of p53. Are there specific cofactors and combinations of cofactors that regulate specific subsets of target genes? Identifying the specific targets affected by an interaction between p53 and its cofactors remains critical for advancing our understanding of the p53 response in vivo.
In broad terms, the traditional model for stress-induced p53 activation consists of a p53 stabilization step that is followed by an intricate regulatory network to induce DNA binding and transcriptional activation of p53 target genes (Figure 2). The stress signal leads to p53 stabilization, primarily via the release of p53 from its interaction with Mdm2. Phosphorylation of p53 inhibits its interaction with Mdm2 and thereby prevents Mdm2-mediated ubiquitination and the resulting proteasomal degradation of p53. Once stabilized, p53 has been thought to be activated by further posttranslational modifications, leading to increased binding of p53 to DNA at p53-specific promoters. After the binding to specific high- or low-affinity promoter sites, p53 would then act as a transcription factor to regulate the expression of its target genes, at least in part via the recruitment of coactivators and corepressors.
This classic model of stabilization and activation continues to provide an explanation for many modes of p53 regulation, especially via posttranslational modifications as observed in in vitro biochemical and in tissue culture systems. However, recent results from genetic studies introducing point mutations in p53 (Toledo and Wahl, 2006) and observations demonstrating the importance of specific modifications (Tang et al., 2008) reveal aspects of p53 regulation that may not be sufficiently explained by the classic model. A refined and extended model is needed to adequately explain our current understanding of p53 activation in vivo.
In vitro and cell culture studies establish that phosphorylation is a key event in both p53 stabilization and regulation of DNA binding (Appella and Anderson, 2001; Hupp et al., 1992). However, as discussed above, point mutant knockin approaches have shown that the dramatic effects observed in vitro are only partially recapitulated in cells and tissues from knockin animals. These observations point toward a regulatory system for p53 in vivo in which individual phosphorylation events may be somewhat redundant, yet by no means insignificant. It is very likely that the loss of phosphorylation at a particular site can be compensated by phosphorylation at different sites, or by other types of modifications such as acetylation. Various types of stress and different tissues may require different modes for p53 regulation. Regulatory redundancy among posttranslational modifications may ensure that p53 is regulated appropriately in response to different stress stimuli and intensities, as well as with the required tissue specificity.
A number of key observations suggest that p53 may be active in the absence of further activation by stress stimuli. As discussed above, both Mdm2 and MdmX knockout mice are embryonic lethal, and the rescue of this phenotype by loss of p53 indicates that p53 is responsible for the early embryonic lethality (Iwakuma and Lozano, 2007; Marine et al., 2006; Wahl, 2006). The severity of the phenotypes of the Mdm2 and MdmX knockout mice suggests that p53 has significant activity. Given the early embryonic lethality, it may be impossible to address whether additional modifications may be required for the activation of p53. Instead of further p53 activation via modifications, the loss of the repressors Mdm2 and MdmX may be sufficient to elevate p53 function, which results in embryonic lethality. This model is supported by data demonstrating that loss of the p53 interaction with either Mdm2 or MdmX, is essential for the activation of cell cycle-specific target genes (Tang et al., 2008). Interestingly, p53 is spontaneously active in tissues lacking mdm2, which leads to p53-dependent pathologies in radiosensitive tissues (Ringshausen et al., 2006). This study also demonstrates that this activity of p53 occurs in the absence of further posttranslational modifications. The notion that p53 does not absolutely require activating modifications is supported by results obtained from purified in vitro systems (An et al., 2004; Espinosa and Emerson, 2001; Gu et al., 1999). These studies raise a fundamental question: what is the main purpose of p53 modification during the stress response? Why is p53 modification required for some biological settings but not others?
In light of the studies discussed above, it appears that p53 activation in vivo requires not only stabilization and activation by posttranslational modifications, but also release from a repressed state. Including this step of antirepression in a refined model of p53 activation may provide a more accurate way to explain p53 activation in vivo (Figure 4). Both in vitro and genetic data indicate that p53 is intrinsically active, yet is repressed by its key negative regulators, Mdm2 and MdmX. By starting with the view that p53 is intrinsically active, we may be able to explain differences observed between in vitro and in vivo experiments by the absence or aberrant activity of specific cofactors or modifications. Interestingly, knockin animals that carry the p53QS mutation are embryonic lethal even though p53QS retains the ability to bind DNA (Johnson et al., 2005). However, p53QS loses its ability to interact with both Mdm2 and MdmX, which may contribute to the embryonic lethality similar to that observed in Mdm2 null mice. p53QS has deficiencies in transactivation, yet retains the ability to activate some targets. This may be a consequence of very high levels of p53QS because of loss of the interaction with Mdm2. These findings further support the concept that p53 is constitutively active but continuously repressed. In order to observe the role anti-repression plays in p53 regulation, both positive and negative regulators, as well as p53, need to be present at near physiological levels.
Overexpression of positive or negative regulators of p53 could mask the effect of antirepression in vivo. This may explain why in vitro experiments using unacetylated p53 show unexpected transcriptional activity in purified systems (An et al., 2004; Espinosa and Emerson, 2001), as negative regulators Mdm2 and MdmX were not present. This could also explain why overexpression of the various kinases that inhibit the p53-Mdm2 interaction lead to a masking of this effect. It will be very interesting to determine to what extent p53 phosphorylation and p53 acetylation contribute to the stabilization and activation of p53 by synergistically acting to inhibit the binding of p53 to its negative regulators. Another intriguing question that arises from the interaction of p53 with Mdm2 and MdmX on gene promoters is whether p53 can be ubiquitinated by Mdm2 while bound to DNA and what role phosphorylation may play in regulating the p53 interaction with Mdm2 and MdmX when p53 is bound to DNA. Although the N-terminus of p53 was originally thought to be the predominant site of the interaction with Mdm2, both the DNA-binding domain (Wallace et al., 2006; Kulikov et al., 2006; Tang et al., 2008; Yu et al., 2006) and the C-terminal domain (Tang et al., 2008) interact with Mdm2. The presence of multiple Mdm2 binding sites in p53 may suggest that there is an interplay between posttranslational modifications that activate p53 by stabilization and that release it from repression.
It is conceivable that antirepression may have varying degrees of significance in vivo, depending on the relative responsiveness of different p53 promoters. Whereas antirepression may be sufficient for the activation of p53 targets that are highly responsive, such as those that block cell cycle progression, antirepression alone may be insufficient to induce apoptosis, which would require additional p53 posttranslational modifications. In this proposed mechanism, a stress signal would initially result in reversible cell cycle arrest, and the more dramatic and irreversible apoptotic response would require additional stimulation. Such regulatory specificity may explain results showing that the small molecule Nutlin-3 acts primarily to activate cell cycle control genes, whereas the transactivation of proapoptotic p53 targets is comparatively weak (Tovar et al., 2006; Vassilev et al., 2004). Inhibition of the p53-Mdm2 interaction leading to p53 stabilization and removal of Mdm2-mediated repression at the promoter may be sufficient to activate cell cycle arrest upon Nutlin-3 treatment, although certain cofactors may be still needed (Brummelkamp et al., 2006). In contrast, the induction of apoptotic p53 targets may require additional signals and posttranslational modifications of p53. This type of differential regulation of specific targets and the concomitant importance of different cofactors reacting to varying stress signals may explain why phenotypes from knockin mice with specific point mutations are modest compared to in vitro results. In a given knockin animal, cells from different tissues may respond to specific stress signals via a multitude of pathways to activate p53. Although particular aspects of the response may be inhibited by the loss of a specific modification site, overall stress responses may remain intact if p53 can still be released from repression and other modifications augment p53 activation to trigger appropriate responses.
Here, based on the latest advances, we have proposed that p53 activation in vivo consists of three major steps: (1) p53 stabilization, (2) antirepression, and (3) promoter-specific activation (Figure 4). In this refinement of the current model, p53 stabilization involves multiple layers of Mdm2 regulation; anti-repression, which is mediated by p53 acetylation and probably phosphorylation; and promoter-specific activation of p53 target genes involving additional posttranslational modifications and cofactor recruitment.
Although the previous 30 years of p53 research have led to an incredibly detailed understanding of the biology of p53, a number of key questions in the study of p53 activation continually reappear in a new form. Are there unified mechanisms that control p53 stabilization in vivo? Do different tissues and stress factors use different pathways for the stabilization of p53? How does the cell coordinate the status of DNA repair and the levels of p53 activation? What is the in vivo significance of ATM- and ATR-mediated phosphorylation in p53 activation? Are the activities of HATs such as CBP/p300 and Tip60/MOF regulated upon stress? How are the many different types of lysine modification of the C-terminal domain coordinated? How many targets of p53 are repressed by the presence of Mdm2 and MdmX? How does Mdm2 or MdmX repress transcriptional activation on DNA? Do promoters for specific p53 responses have similar modes of regulation? For example, are all proapoptotic target genes under the control of one or more specific regulatory mechanisms that are different from mechanisms regulating senescence, autophagy, or metabolic targets? Is there any specific target gene of p53 directly involved in the control of aging or antiaging processes? As in the past, answers to such questions will mean that models for the activation of p53 will need to continually evolve.
The work in W.G.'s laboratory is supported by the NIH/NCI, the Leukemia and Lymphoma Society, and the Ellison Medical Foundation.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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22 June 2017
European Pensions Awards 2017
Millennium Hotel London Mayfair
Time: 7:00 pm onwards
Celebrating excellence in European pension provision.
Now in their 10th year, the European Pensions Awards have been designed to recognise outstanding achievement in the varied fields of European pension provision, honouring the investment firms, consultancies and pension providers across Europe that have set the professional standards in order to best serve European pension funds in these increasingly challenging times.
Book your table: www.europeanpensions.net/awards
12 October 2017
Money Age Awards 2017
Millennium Hotel, London Mayfair
Time: 7:00 pm
DEADLINE FOR ENTRIES: 16 June 2017
Celebrating excellence within the personal finance space.
Now in its second exciting year, the MoneyAge Awards have been launched to celebrate excellence, professionalism and innovation in the hugely competitive but critically important personal finance space. Honouring the banks, building societies, savings and investment providers, mortgage companies and personal finance specialists that have proved themselves ahead of the game in the field of consumer finance with their product provision, innovation, focus on value and customer service, these awards are free to enter and are open to any financial services provider which serves consumers in the UK.
Register your interest: www.moneyage.co.uk/awards
Pensions Age Spring Conference
27 April 2017
The Waldorf Hilton
Time: 08:30 onwards
The Pensions Age Spring Conference: Aiming High offers pension funds and those working in the pensions space the opportunity to both learn and network alongside their peers at one of the most dynamic eras in the history of UK pensions.
Book your place: www.pensionsage.com/conference
Pensions Age Northern Conference
7 June 2017
Leeds Marriott Hotel
Time: 08:30 onwards
The Pensions Age Northern Conference: Hitting the target offers pension funds and those working in the UK pensions space the tools and information and they need to get it right at a time when managing pension schemes is more challenging than it has perhaps ever been.
Book your place: www.pensionsage.com/northernconference
Pensions Age Seminar
7 April 2017
Hilton London Tower Bridge
Time: 08:30am - 13:00pm onwards
A perfect data storm is approaching for UK pension schemes – and at this half day seminar ITM and our expert speakers will help guide you through what needs to be done to weather that data storm – and have a definitive plan to take away with you.
Book your place: www.pensionsage.com/itmseminar
Irish Pensions Awards
23 November 2017
The Shelbourne Hotel, Dublin
DEADLINE FOR ENTRIES: 28 July 2017
The Irish Pensions Awards give recognition to those pension funds and providers who have proved their excellence, professionalism and dedication to maintaining high standards of Irish pension provision over the past year. The winners will be announced at the gala dinner and awards ceremony on 23 November 2017 at the 5 star Shelbourne Hotel Dublin. The event was attended by over three hundred guests in 2016 and this year is set to be bigger and better than ever.
Register your interest: www.europeanpensions.net/irishawards
Pensions Age Autumn Conference
21 September 2017
The Waldorf Hilton, London
Pensions Age Autumn Conference: Staying on Track offers pension fund managers, trustees and all those working in the pensions space the opportunity to both learn and network alongside their peers at one of the most challenging yet exciting times in UK pensions history.
Find out more: www.pensionsage.com/autumnconference
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Forward David Lucas led the way for the Beavers with 21 points and added six rebounds. "He's our go to guy," sophomore guard Chris Stephens said of Lucas. "When we need points, we can dump it down to him."
Oregon State head coach Jay John agreed, "David was ready to play, both halves," John said after the Beavers second consecutive conference win. "That is, without question, his most solid performance offensively."
"I was just being really patient, being quick with my moves, picking and choosing if I'm gonna shoot or not, or pass it out. I think it was a pretty good game on the offensive side," Lucas said.
Next to the offensive game by Lucas, the main storyline in this game was the combination of the Beaver defense and the poor shooting by the Cougars. Washington State hit only 5-of-24 shots in the first half, and 14-of-49 overall for an abysmal shooting percentage of 28.6%.
"Field goal percentage was really the difference," John said. "If Washington State hit some of those shots, then we have a game that goes down to the wire. We played hard, we played well, and we battled."
Crowd favorite Jim Hanchett thought the Beavers had a solid game defensively as well. "(You) can't take us out of the equation," the forward said. "I think we played solid basketball. Whether or not they hit their shots, you'd like to say it was because of our great defense, but definitely we contributed to that."
With 13:56 to go in the second half, the Cougars Jeff Varem made a layup to give WSU their first field goal in 15 minutes, 18 seconds of action. That bucket made the score 34-20 in favor of OSU. Wazzu had previously missed 12 straight field goals before that shot fell.
Stephens and Hanchett added eight points apiece while no other Beaver had more than four, this truly was a defensive game.
Not eon player on the Washington State team scored in double figures. Forward Ezenwa Ukeagu scored nine points to lead the Cougars. Two of WSU's better players, guard's Marcus Moore and Thomas Kelati, combined to shoot just 2-of-22 from the floor for nine points.
It isn't difficult to understand why the Cougars lost this game. Those two players average 24 points per game together as Moore leads the team in scoring, and Kelati is second.
Kyle Jeffers led Oregon State in rebounds with nine while point guard Lamar Hurd led
with four
Note: J.S. Nash went down late in the second half with an injury. Coach John didn't know what Nash's availability will be for Saturday's 1 p.m. game with the University of Washington will be, but he said it appeared to be a quad
Jake Schubert can be reached at


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Buntarou doesn't know what he wants to do in the future. He does not have any kind of dream that he wants to pursue, so currently he just spends his days hanging around with his friends. One day, his classmate Sayuki asks him if he wants to help with development of a gal game. She says that she had become interested in his help after she read one his works intended for the drama club.
Buntarou doesn't know anything about gal games, but Sayuki claims that she has the ability to make it a success. Can they make the game, and will it be a success like she claims it will be? This is the story of youths taking a daring step into the unknown...
Title: Shoujo-tachi wa Kouya wo Mezasu
Audio: Japanese
Year: 2016
Duration: 24 min approx
Subtitle: English
Resolution: HD - 1280×720
Format: Mp4
Size: 110MB-120MB
Servers: Mega, Mediafire
Genres: Comedy, Drama, Romance, School-Life
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Junio 2016


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text: 6 kitchen shortcuts using a NutriBullet Share this article As well as making quick smoothies, the NutriBullet can speed up other kitchen tasks and save you money. 1. Soup Put all your favourite veggies into the gadget, whizz it up and heat it in the microwave for a fresh soup. Add herbs, seasoning, plus stock or low-fat yogurt to get the consistency you like. Check out these alternative uses for herbs and spices2. Frozen fruit smoothies Buy or pick fruit when it's cheap and in season then pop it in the freezer. When you're craving a cold fruit smoothie, just add a handful of frozen fruit to the Nutribullet cup and top it up with coconut water, juice or milk. 3. Slushy cocktails Fancy a Friday night frozen margarita? Add your liquid ingredients to the cup, along with a few crushed ice cubes, and blast it up into a refreshing, summery drink. Just remember never to blast whole ice cubes in a NutriBullet on their own as the blade may break – always add liquid to the mix.4. Almond butter Way healthier than peanut butter, whizz up almonds, almond oil and a pinch of sea salt in the NutriBullet, removing the blade and stirring a few times, then whizzing the blend until smooth. 5. Spice rubs and marinades Make your own spice mixes by using the milling blade. Blast garlic, coriander seeds and dried chilli to make a rub for meat and fish. Or experiment with your own combinations until you find the perfect blend. 6. Quick pasta sauce Instead of tediously chopping and sautéing your ingredients, chuck them in the NutriBullet. For a quick pasta sauce add fresh or tinned tomatoes, half an onion, a whole garlic clove, oregano, a slug of olive oil, a pinch of sugar and seasoning. Once blitzed, simmer on the hob for 25 minutes with a lid on. Article updated with correction 14 July 2016. More money-saving tips5 amazing frozen food swaps to save you time and moneySmart ways to reduce food wasteSee more tips in Saving Money Share this article


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Progressive Era Presidents and their political agendas
Teddy Roosevelt and the Square Deal 1901-1909
Background Info View on the Presidency Served as Calvary Commander of the Rough Riders and the Governor of New York. Became President of the US in after William McKinley was assassinated Believed that the Pres. Should be a man of action with the citizens best interest. Ex: During a coal miner strike, Roosevelt intervened calling both sides to the White House. When a compromise was not reached, he threatened to use troops the settle dispute "It is the duty of the president to act upon the theory that he is the steward of the people, and…to assume that he has the legal right to do whatever the needs of the people demand, unless the Constitution of the laws explicitly forbid him to do it."
Roosevelt as Trust-Buster
Roosevelt was against big business and monopolies He opposed unfair, anti-competitive practices and business consolidation known as trusts He broke up Rockefeller's Standard Oil Company He also filed 44 law suites against other companies (monopolies) winning numerous cases and breaking other companies such as the Northwestern Railroad Co.
The Square Deal Teddy Roosevelt launched new laws to protect consumer health and prevent false advertising, known as the Square Deal Protecting public health- Congress passed the Meat Inspection Act and the Pure Food and Drug Act in 1906. Regulating Transportation and Communication- Roosevelt increased regulated the railroads Conserving the Nation's Resources- Drew attention to conserving the forests, wildlife and natural resources. He set up national forests and parks and formed the National Conservation Commission to protect the nations natural resources.
Wilson's New Freedom Democrat Woodrow Wilson won the presidential election of 1912. Wilson had a different personality from Roosevelt, he was more cool, reserved and logical. Wilson's plan The New Freedom promised Americans reforms that would also tame big business. He focused more his attention on banking and the economy and high tariffs.
New Freedom Legislation
Underwood Tariff created a law to lower tariffs by 25% Graduated Income Tax Created the 16th Amendment which created the 1st income tax based on your yearly income, wealthier Americans would pay higher taxes than the less wealthy. Federal Reserve Act Reformed the banking industry by creating a separate bank to serve as the bankers bank. The Fed Res controlles the amount of money in cirrculation and established interest rates. Clayton Anti-Trust Act- Provided support to labor unions by creating laws to support labor rights.
National Parks and Labor
Like Roosevelt, Wilson also believed in protecting the environment. He created the National Park Service which brought 40 exisiting parks and monument under federal control The purpose of these parks was to conserve nature and wildlife for the enjoyment of the American people. After the incident of the Triangle Shirtwaste Factory, society pushed for reform to prevent such cases. The Department of Labor-was created as a new cabinet of the president. The purpose of the dept was to promote and develop the welfare of the working people. Child Labor Act- law created to prohibit the sale of goods created by children, this law was overturned 2 years later
Word bank Bull Moose Republican Democrat Anti trust reform
Focused on banking reform Created laws to protect consumers Progressives Helped conserve the environment Favored unions Filed laws suits against major monopolies Reserved and logical Strong personality Tried to reform child labor laws Known as a trust buster Created reforms to improve America Square Deal New Freedom Pure Food and Drug Act
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Join Us
We are constantly on the lookout for volunteers, support and donations. We want to extend the reach of ACF's work, and we can't do that without your help.
Our Volunteers
Volunteers are essential in delivering our work. Without their hard work, we would not be where we are today. There are numerous roles available to volunteers, all of which add important life experience, and are deeply rewarding. At ACF we believe we need to invest in our young people, and by volunteering and investing your time into helping us, we will invest in developing you and your skills.
Available Roles:
- Administration Assistant
- Fundraiser
- Marketing & Communication Officer
- Sessional Staff/Project Staff
Non-Monetary Donations
ACF relies on your generosity to continue operating. Even if you can't afford to donate money on a regular basis, we are always open to any other donations, like old games consoles, sports equipment, boards games and computing equipment etc.. for the Youth Centre or Office. So before you throw out any old stuff, we would appreciate if you could consider whether it might be better to donate it, and help to change someone's life for the better.
Ready to Get Involved?
If you want to make a donation, enquire about volunteering, or would like to take part in community events then email us at: firstname.lastname@example.com
Or pop into the centre and have a chat.


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He may not have so far done it in his real life but Samajwadi Party general secretary Amar Singh is all set to play home minister in a Bollywood film.
"The offer to act as home minister has come to me from none other than Bollywood veteran Dev Anand in his forthcoming film 'Chargesheet'," Amar Singh told reporters in an informal chat at his residence in Lucknow.
Describing himself as a "Dev Anand fan", he said, "It is an honour for me to play the role."
Amar Singh was flanked by film star-turned-Samajwadi Party nominee for the Lucknow Lok Sabha seat Sanjay Dutt, who arrived in Lucknow on Saturday to launch his poll campaign.
When a journalist asked Sanjay Dutt if he will give Amar Singh some lessons in acting, he shot back: "Amar Singhji does not need any training. He is adept in everything."


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Added: Monday, July 02, 2012
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Fly Me to the Moon – Frank Sinatra
- Have you seen our "Fly Me to the Moon (In Other Words)" main feature?
Fly Me to the Moon (Bart Howard) – The song was introduced by Felicia Sanders on the cabaret circuit under the original title In Other Words. Though the first recording, by Kaye Ballard in 1954, retained that title, it was recorded as Fly Me to Moon as early as 1956 (Johnny Mathis). The title was eventually changed officially, though the original is still preferred by some recording artists.
From WICN.org's Song of the Week feature:
In the early 1960s Nat King Cole, Patti Page, Julie London and Doris Day made popular recordings of "Fly Me to the Moon," but its lasting fame and endurance as a jazz standard were assured when Frank Sinatra recorded it on his album for Reprise Records, It Might as Well Be Swing, along with Count Basie. Quincy Jones did the arrangement, and he changed the song from its original 3/4 waltz-time to 4/4 time to give it a swinging feel.
Frank Sinatra recorded [Fly Me to the Moon] on the album It Might as Well Be Swing (1964), accompanied by Count Basie. The arrangement by Quincy Jones has become the rendition most people know. Jones changed the time signature, which was originally 3/4 waltz-time, to 4/4 and gave it a looser, swing feel. Sinatra's recording was a hit and was played by the astronauts of Apollo 10 on their lunar-orbital mission and again on the moon itself by the astronaut Buzz Aldrin during the Apollo 11 landing.[2]
Frank Sinatra – recorded 9 June 1964 with Count Basie and his Orchestra, arrangement: Quincy Jones, released on the 1964 album It Might as Well Be Swing
(below) From Frank Sinatra: A Man and His Music, TV special, 1965
Wikipedia excerpts:
Frank Sinatra: A Man and His Music was a one hour television special in color, first broadcast by NBC on November 24, 1965. It was directed by the multi-Emmy-winning Dwight Hemion. Telecast at a time when television had just switched to full-time color programming (except for feature films shot in black-and-white), the show was an enormous success, so much so that it spawned two follow-ups with virtually the same title, featuring, respectively, Nancy Sinatra, and Ella Fitzgerald along with Antonio Carlos Jobim.
By modern standards, especially, the format of the original show was profoundly simple. It consisted only of Sinatra in a television studio singing many of his hit tunes (such as It Was a Very Good Year) in front of a live audience. There were no guests on this first program. The orchestra was conducted by long-time Sinatra arrangers Nelson Riddle and Gordon Jenkins.


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This site does not currently take bookings here on Pitchup.com.
Find nearby sites with availability…
If you are the owner of this site, start taking bookings through Pitchup.com by adding your business details.
What a great place for camping: Ffos Las Racecourse campsite is based on a 600 acre site just next to The Lodge and within easy reach of all the events and fixtures at the racecourse.
We have a play area, games and TV room. There are shower and toilet facilities with a parent and baby washroom available at The Lodge as well as a café and free wifi. The café as well as Ffos Las Pub onsite are both open Friday and Saturday evenings as well as for Sunday lunch.
We look forward to welcoming you soon!
Open from 01/04/2017 to 31/10/2017
Site type
Languages spoken
✓ English
✓ Welsh
Find nearby sites with availability…
(2 nights)
Many sites set a maximum occupancy of 6/8 people per pitch, so try breaking your booking down into smaller groups to generate more results. For example, if you're a group of 10, enter 5 people in your initial search and then book 2 pitches.
However, where the pitch or accommodation occupancy allows it, you can now make a booking for up to 30 adults and 30 children on one pitch.
To book multiple pitches or accommodation, use the 'Special requests' box on the booking page to ask to be sited together. Complete the booking form for the first booking (you can add a password and save your card so that we remember your details). Then click "Make another booking like this" at the top of the confirmation page to make the next booking.
Large groups: check the listing page for any group restrictions set out in the terms and section titled 'Please note'.
Large tents/caravans: check the restrictions on unit dimensions to ensure your unit is suitable for the space you will be provided with.


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Adata Pd3 Driver Download
Adata Pd3 Driver Details:
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Adata Pd3 ZIP Size: 117.kb
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Diamond Ring for Women in 18K GoldItem Code: DR592 Gross Gold Weight: 4.30 Grams
Product Details
18 Karat Gold Diamond Ring
Gross Gold Weight: 4.300 grams
Total Number of Diamonds: 66 Diamonds
Diamond Weight: 0.55 Carats
Diamond Quality: VVS Quality Diamonds
Diamond Color: E/F Color
Diamond Color & Quality as Certified by IGI (International Gemological Institute)
This item comes with IGI certificate.
Internal Reference Code : RS 6.0
Shipping Time: 10 - 14 Days
- sold out - not in stock Ring Size: Please specify the ring size at the time of check out,
otherwise standard ring size will be shipped
Sold By: Totaram Papalal & Sons Jewellers
This Indian Designer jewellery product is made in India. This product will be shipped to you from New Jersey - USA


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Nez Perce D 6
From $310 / night
balance of due
Description from the manager
Description from the manager
Bedrooms: 3/
Bathrooms: 2/
Sleeps: 8/
Type: Apt. / Condo
Book this 3 bedroom, 2 bathroom apt. / condo in Teton Village today! A total of 8 people can sleep here comfortably.
Guests can enjoy a internet access and DVD among other amenities offered by this rental.
Travelers are sharing their opinions on this property. It has an overall rating of 5/5 thanks to an average of 13 reviews.
The owner responds to 75% of inquiries.
About the manager
About the manager
Apex Real Estate & Property MGMT
Apex Real Estate & Property MGMT
- Response rate: 75%
- Response time: A few hours
Overall rating:
4.7 / 5.0
More properties Call manager +1-307-732-0943
Proximity to TripAdvisor's Top Teton Village Attractions
Proximity to TripAdvisor's Top Teton Village Attractions
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Also consider
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THE SHORE-END cable of the Guyana/Suriname submarine fibre-optic cable was successfully landed here yesterday, thereby paving the way for the further enhancement of the nation's information infrastructure and the transformation of the local telecommunication landscape.
President Bharrat Jadgeo symbolically pulls the submarine cable ashore, as GT&T CEO, Major General (ret'd) Joe Singh, extreme right, looks on.
The historic occasion was witnessed by President Bharrat Jadgeo, Finance Minister Dr. Ashni Singh, Agriculture Minister Mr Robert Persaud, Leader of the Opposition, Mr Robert Corbin, prominent members of the private sector and senior GT&T officials.
A similar activity was recently held in neighbouring Suriname. The main submarine cable will be laid next month, while the installation of equipment for the cable stations, the laying of the terrestrial cable between Georgetown and Suriname, the connecting-up of the electronics and the testing of the system will be completed in the second quarter of this year.
The join project between GT&T and the Surinamese telecommunications company, TELESUR, will see both companies providing technical support to each other via improved fibre-optic cable terrestrial roots that will allow alternative connection points to the new submarine cable for both countries.
Speaking at a ceremony convened at the Kingston seawall, behind the Pegasus Hotel, GT&T CEO, Major General (ret'd) Joe Singh said the occasion marks the realisation of the company's vision in delivering a modern telecommunications network to Guyana.
The gathering at yesterday's ceremony as the cable was brought ashore.
Noting that the laying of the cable is an important milestone in the Guyana/Suriname submarine cable project, Mr Singh said:
"The new cable will have 3000 times our current bandwidth capacity, and will facilitate a multitude of national development opportunities… As the only English-speaking country in South America, local employment opportunities will be enabled, opportunities will also be provided for in region outsource support as large companies sending jobs to call centres in other continents.
"Guyana will also be able to provide disaster recovery facilities to industries such as mining, oil and gas companies active in Guyana and surrounding countries," he said, adding: "Subscriber bandwidth services, there will be increased DSL and broad band to home capabilities through the delivery of cheaper bandwidth, increase multi-media content in the form of digital television to the country and increased ability to deliver bandwidth to the handset."
He said too that the development will facilitate services in telemedicine, distance learning in remote areas where there are no colleges or universities, e-security and e-governance.
"We do believe in the context of the Low Carbon Development Strategy (LCDS) and all that it proposes, from both a company and a national perspective, we are on the right path," Singh said, adding that GT&T fully understands the direction in which industry and technology is heading and is committed to working with Government and all stakeholders to improve telecommunication, internet access and introduce modern service.http://www.guyanachronicle.com/topstory.html#Anchor-----------36797"> Read more........


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2016 Deep Clean & Declutterfeatured
This 2016, I've challenged myself by deep cleaning and decluttering my apartment. I want to begin the new year with a fresh and light atmosphere and how better to achieve this than with your living space? Also, I find it much easier to focus on my resolutions and goals when my surroundings are neat and Read more


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Give us a call to get our secret deals 1-800-468-3578
Property Location
With a stay at ABode Glasgow, you'll be centrally located in Glasgow, steps from Willow Tearooms and Royal Conservatoire of Scotland. This 4-star hotel is within close proximity of Glasgow West End and Theatre Royal.
Make yourself at home in one of the 59 guestrooms featuring DVD players and LCD televisions. Complimentary wireless Internet access is available to keep you connected. Private bathrooms with shower/tub combinations feature designer toiletries and hair dryers. Conveniences include laptop-compatible safes and desks, and housekeeping is provided daily.
Make use of convenient amenities such as complimentary wireless Internet access, concierge services, and wedding services.
Satisfy your appetite at the hotel's restaurant, which serves breakfast, lunch, and dinner, or stay in and take advantage of 24-hour room service. Quench your thirst with your favorite drink at a bar/lounge.
Business, Other Amenities
Featured amenities include dry cleaning/laundry services, a 24-hour front desk, and luggage storage. This hotel has 4 meeting rooms available for events.


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|Authored By: Ellen Sue Spicer of Hands on Nutrition
|Enjoy this veggie version of tuna salad.
|Diet Types: Vegan, Dairy Free
|Serves: 4
|Cooking Time: Any
|Steam Tempeh. While it cools prepare the veggies and place in a bowl with 1/4 cup Nayonnaise. Grate the Tempeh into the bowl, adding salt, pepper and paprika to taste. Add more Nayonnaise if too dry. Chill.
|Printable Version
|E-mail a Friend
|Brown Basmati Rice with Cashews and Raisins
|A heavenly combination of flavors. Serve with kabobs and a cucumber sa...


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201 W Loop 340
Directions Waco, TX 76712
Do you want to be remembered? Do you want to be part of a strong and respected heritage? Do you want to drive a vehicle that awakens the soul?
Test Drive a New Dodge in Waco Today!
Opportunity awaits you here at Allen Samuels Chrysler Dodge Jeep RAM Waco. Bring your inner automotive desires to life with our plethora of new 2015 Dodge models. They're unforgettable and exhilarating. Be a part of the Dodge heritage.
Which Dodge best suits your needs? If an SUV is in your prospects, don't rule out the Dodge Durango. The Durango is far from meek; it sports a muscular body, an eye-catching class-exclusive LED racetrack taillamp, available LED daytime running lamps, and six available wheels. You'll dazzle for 600 highway miles on a single tank, too, thanks to its 24.6 gallon tank and 3.6-liter Pentastar® V6 under the hood.
For those who need to downsize but want a vehicle with attitude, the Dodge Dart is here to win you over with a test drive. The Dart maintains the "Born Dodge" look with its sleek design and aforementioned LED taillamp, but it also stays smart with 60 standard and available safety and security features as well as convenience technologies like Uconnect®.
Is it possible to own a vehicle that lets you be a performance panther on the streets of Temple, Gatesville, Killeen, and Hillsboro, TX and also be of practical use? That's the beauty of both the Dodge Charger and the Dodge Challenger. Both of these iconic muscles bring their A-game when it comes to pumping the power. Whether it's the award-winning 3.6-liter Pentastar® V6 or 5.7-liter V8 HEMI®, there's no doubt fellow drivers will be frequent admirers of your dual exhaust pipes and dust clouds.
Staying with the performance theme, rest assured, nothing turns heads and triggers an adrenaline rush more than the Dodge Viper. Behind the wheel, you'll shoot zero to sixty mph in a low three second range, hit a quarter mile in just 11 seconds, and achieve a top speed of 206 mph. Hang on, it'll be one heck of a ride.
What's more, once you've selected your Dodge vehicle at our Waco, TX dealership, our Finance team will be here to assist you if you're in need of a Dodge lease or loan. Welcome to the Dodge family.


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Clergy Day 2016 July 25, 2016Posted by Fr. Felmar Castrodes Fiel, SVD in Archdiocese of Manila, Diocese of Cubao, Press Release, Priesthood, Vocation Campaign.
Tags: Clergy Day, Fr. Nielo Cantilado SVD
Is your light hiding under the bush? Remember that you are the salt of the earth and light of the world. Let your light shine as the Prophet Jeremiah says, "Before I formed you in the womb I knew you, before you were born I set you apart; I appointed you as a prophet to the nations."
Let us all hear God's calling as we invite you to join and be a part of the
Clergy Day.
Happening on Thursday, 4th day of August, 2016
from 1pm to 10 pm at the
Diocesan Shrine of Jesus, the Divine Word,
Christ the King Mission Seminary,
- Rodriguez Sr. Ave. Quezon City.
A sports activity, thanksgiving mass and a tribute dinner will be held on the said day.
Come share your time, talents and energy. Treat yourself with fun and excitement while the value of faith and love of God is shared.
You won't want to miss this significant and triumphant event.
Hope to see you there.
No comments yet — be the first.


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|Senior man in Sarajevo cemetery, Bosnia
|Man walking through deserted street, Bosnia
|East Jerusalem, 1971
Peaked Cap: for people who don't dare to wear a beret
|Senior man in Sarajevo cemetery, Bosnia
|Man walking through deserted street, Bosnia
|East Jerusalem, 1971
|A Royal Navy submariner recently received US Navy Submarine Service 'dolphins', making him just the second UK officer to qualify on a US Navy submarine.
Similar to the crews of tanks, submariners too found that the beret is the most comfortable piece of headgear in a confined space. No peak that's always in the way, grease and oil don't stain the black beret and it acts very well as a cushion when banging the head.
|Chilean Navy Submarine Crew with Black Beret.
|HMS Talent
|Australian Submarine Service
|French Submarine Service
|Israel Submarine Service
|Netherlands Submarine Service
You'd like your own beret, but don't know where to go for the very best?
At South Pacific Berets we now sell a variety of berets from France, Spain, Italy, the Czech Republic, Argentina, Uruguay, the US and the UK, Japan, Germany... Well, from all the traditional beret manufacturers left on the planet! The absolute top in berets (and pakols, chechias and other related felt hats) you could possibly find.
All berets can be ordered on line through South Pacific Berets, click here.


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Island Traditions
Bradford Drawer End Table
Item #548-950
As breathtakingly detailed as it is truly unique, this piece has the ability to elevate a room's style. Set upon carved lion's paw feet and carved rope base molding, the table is adorned with exquisitely detailed decorative metal framing. Two drawers and porters handles bring functionality to beautiful design.
29¼W x 33¾D x 26½H in.
Contact Store for Availability


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I Don't Wanna Grow Up! August 01 2016, 0 Comments
(Photo Credit: The Charlie Foundation)
Let's face it… the older we get, the more and more we start to notice changes in our bodies. But most especially, in our skin! Fine lines, wrinkles, and dry skin. OH MY! So, it's pretty common for us to spend $30-$40 dollars on a cream each month that what we think is going to make us look younger. But what if I told you that you didn't have to spend that $30-$40 dollars each month?! What if I told you that you could make your own miracle cream and save yourself money? Too good to be true? Wrong! It is entirely possible and the benefits are amazing!
Ozonated Olive Oil is a product that is really taking off! It has a million different uses! Here are some of the uses and what our customers have used it for:
|Athlete's Foot
|Hot Spots
|Skin Detoxify and Healthy Glow
|Bed Sores
|Skin Moisturizer
|Bee Stings
|Improves Skin Tone
|Skin Yeast
|Insect Bites
|Surgery Wounds
|Itch Mites
|Sweat Gland Infections
|Tinea Versicolor
|Leg Ulcers
|Hair follicle infections
|Makeup removal
|Yeast Infection
|Cold Sores
|Muscle Aches
|Diaper Rash
|Poison Oak
|Dry Skin
|Ear Infections
|Dry skin
|Fungal infections
Wow! That is a lot of different uses for one kind of cream!
Curious how to make it? Well, when we were making it in-store, we used our S-32 G and paired that with an Oxygen Generator as well. This would produce about 2 gallons for us. But that isn't the only unit that can be used for making the oil! Our MP-1000 up to our MP-8000 Multi-Purpose Ozone Generator can be used to make Ozonated Olive Oil.
Here is a chart on how long it will take for each unit to make the salve or Ozonated Olive Oil:
|Time Span
|6-10 oz
|3-4 days (salve)
|6-12 oz
|2-3 days (salve)
|6-14 oz
|2-3 days (salve)
|6-18 oz
|2-3 days (salve)
Here is the set-up that one of our customers has when they are making their own Ozonated Olive Oil.
We would be happy to help with any questions that you may have when ordering a unit or making your very own Ozonated Olive Oil! Give us a call at 502-499-4977! Happy Ozonizing!
Resources: The Charlie Foundation, https://www.charliefoundation.org/blog/entry/keto-pantry
We're Going to Hawaii! July 01 2016, 0 Comments
Join us in beautiful Honolulu, Hawaii July 8 and July 9, 2015 for the Lodging, Hospitality & Foodservice Expo. That's next week! We can't wait to see you there.
Why Hawaii?
It only makes sense for the Lodging, Hospitality & Foodservice Expo to be held in Honolulu. It highlights Hawaii's most important economic sector. The hospitality market in Hawaii is huge—there are 70,000 hotel rooms alone, not to mention the many rentals and timeshares.
We want you there!
Come enjoy the fine scenery and learn about the industries covered. You didn't know that ozone generators were of value to the Lodging, Hospitality & Foodservice industries? Our hotel customers use multi purpose units to clean rooms of musty smells. Restaurants make use of the units for sanitizing cooking areas and preparing vegetables. Even our SP Swimming Pool Series benefits our customers who have pools. We also expect to instruct representatives from country clubs, hospitals, and retirement homes on how they may improve their businesses. All can benefit from our products.
Lodging, Hospitality & Foodservice Expo Details:
The Expo will be held at the Neal S. Blaisdell Center located between Waikiki Beach and Honolulu's business district. Hours are Wednesday, July 8 from 10:00 am to 5:00 pm and Thursday, July 9 from 10:00 am to 5:00 pm. There is plenty of parking. Admission to the Expo is free! Visit our booth and see how we can help you with your needs.
Get further details at the Douglas Trade Shows website: http://douglastradeshows.com/expos/