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Although senescence in oncogenesis has been widely studied, little is known regarding the role of this process in chemotherapy resistance. Thus, from the standpoint of enhancing and improving cancer therapy, a better understanding of the molecular machinery involved in chemotherapy-related senescence is paramount. We show for the first time that Mcl-1, a Bcl-2 family member, plays an important role in preventing chemotherapy-induced senescence (CIS). Overexpression of Mcl-1 in p53+ cell lines inhibits CIS. Conversely, downregulation of Mcl-1 makes cells sensitive to CIS. Surprisingly, downregulation of Mcl-1 in p53− cells restored CIS to similar levels as p53+ cells. In all cases where senescence can be induced, we observed increased p21 expression. Moreover, we show that the domain of Mcl-1 responsible for its antisenescent effects is distinct from that known to confer its antiapoptotic qualities. In vivo we observe that downregulation of Mcl-1 can almost retard tumor growth regardless of p53 status, while overexpression of Mcl-1 in p53+ cells conferred resistance to CIS and promoted tumor outgrowth. In summary, our data reveal that Mcl-1 can inhibit CIS in both a p53-dependent and -independent manner in vitro and in vivo and that this Mcl-1-mediated inhibition can enhance tumor growth in vivo.
There is growing evidence that senescence is a major barrier in the development of cancer (1, 4). Recently, a network of secreted factors was shown to enforce senescence as a mechanism to prevent tumorigenesis (12, 23). Many of these senescence pathways rely on either p53 or retinoblastoma protein (Rb) tumor suppressors (7). However, data are accumulating on the existence of alternative pathways, which are poorly understood (19). Senescence-like processes also appear to be important forms of cell death induced by both radiation and chemotherapy in cancer (29). Many chemotherapeutics induce the expression of molecules such as p53, which can induce both apoptosis and senescence (16). However, restoring expression of p53 in actively growing cancer prevents tumor progression, not through apoptosis but rather senescence (14, 16). Finally, there is mounting evidence in patients being treated with current anticancer therapies that senescence is often a consequence of treatment (14). Thus, an understanding of the mechanisms of senescence induction in cancer is crucial for designing more effective treatment regimens.
Of the most robust cancer survival molecules are the antiapoptotic members of the Bcl-2 family. Not surprisingly, these proteins can also prevent nonapoptotic cell death pathways such as autophagy and are strongly linked to modulating prosenescent molecules like p53 (1, 11). Furthermore, the senescent state has been linked to being apoptosis resistant through the upregulation of Bcl-2 family members (14). However, their role in modulating senescence is currently poorly understood.
Mcl-1 has a distinct role in the survival and homeostasis of lymphocytes (28). In addition, Mcl-1 appears to be important in the survival of both hematogenous and solid tumors and is now considered a major oncogene (3). For instance, cancer treatment using a small molecule inhibitor capable of preventing the function of Bcl-2 family members but not Mcl-1 fails through a mechanism involving the upregulation of Mcl-1 (22, 33). Whether Mcl-1's survival function includes inhibition of senescence is not known. We therefore hypothesized that Mcl-1 may play an important role in tumor progression through the inhibition of senescence.
Our data show for the first time that Mcl-1 is indeed a major inhibitor of chemotherapy-induced senescence (CIS). Overexpression of Mcl-1 in multiple p53+ tumor and nontumor cell lines was sufficient to block the induction of senescence. Conversely, downregulation of Mcl-1 in p53+ cells resulted in enhanced susceptibility to CIS. We further show that resistance to drug-induced senescence in cells lacking p53 can be overcome by the knockdown of Mcl-1 expression and that this ability to resist senescence in the absence of p53 is unique to Mcl-1 compared to other Bcl-2 family members. We also find that Mcl-1 appears to work downstream of p53 and prevents senescence-associated upregulation of p21 and loss of phosphorylated Rb (pRb) through a mechanism involving reactive oxygen species (ROS) production. Finally, we show that cancer growth and resistance to chemotherapy treatment in vivo is highly dependent on the expression of Mcl-1 due in part to the inhibition of senescence and that inhibition of reactive oxygen species (ROS) by an antioxidant can lead to outgrowth of p53− tumors with low Mcl-1 expression. Successful cancer therapy requires the killing of chemotherapy-resistant tumor cells. Focusing on Mcl-1 from the standpoint of its antisenescence function promises to broaden current anticancer therapeutic regimens.
The HCT116 human colon cancer cell lines (p53+, p53−, and p21−) were generously provided by Bert Vogelstein (Johns Hopkins University). The HCT116 vector and HCT116 Mcl-1up cell lines were generously provided by Hannah Rabinowich (University of Pittsburgh Cancer Institute). Wild-type murine embryonic fibroblasts (MEFs) were kindly provided by Scott Welford (Case Western Reserve University). p53− murine embryonic fibroblasts were derived from p53− C57BL/6 mice as previously described (32). HCT116, HCT116 p53−, HCT116 p21−, HSC-3, MCF-7, MEF, and MEF p53− were all grown in Dulbecco modified Eagle medium (DMEM) containing penicillin and streptomycin (pen/strep) antibiotics and 10% fetal bovine serum (FBS). MCF-10A and MCF-10A p53− cells were generously provided by David Weber (University of Maryland School of Medicine) and Ben Ho Park (Johns Hopkins University), respectively. The cells were grown in DMEM containing 50 μg/ml of epidermal growth factor (EGF) from stocks of 100 μg/ml in 10 nM acetic acid, 0.5 μg/ml of hydrocortisone from a 1.0 mg/ml stock dissolved in 95% ethanol, 0.1 μg/ml of cholera toxin, 10 μg/ml human insulin (Sigma, CA), the antibiotics pen/strep, and 5% horse serum (18). All cell cultures were incubated at 37°C in a humidified incubator containing 5% CO2.
HCT116 shControl and HCT116 shMcl-1 cells, HCT116 p53− shControl, HCT116 p53− shMcl-1 and HCT116 p53− shBcl-2 cells, and HCT116 p21− shControl, and HCT116 p21− shMcl-1 cells are derivatives of HCT116, HCT116 p53−, and HCT116 p21− cells, respectively, in which Mcl-1, Bcl-2, and control were stably knocked down using transcript-specific short hairpin RNA (shRNA) expression vectors (Open Biosystems). Sequences that target Mcl-1 are as follows: shMcl-1 cl.1, GAAATTCTTTCACTTCATT; shMcl-1 cl.2, GCTTGTAAATGTATTTGTA; shMcl-1 cl.3, AGGATTATGGCTAACAAGA. Sequences that target Bcl-2 are as follows: shBcl-2 cl.1, CCCTGATTGTGTATATTCA; shBcl-2 cl.2, CGAAGAACCTTGTGTGACA; shBcl-2 cl.3, CCTTGAAACATTGATGGAA; shBcl-2 cl.4, CTTGGACAATCATGAAATA. The Mcl-1 BH3 binding domain mutant was generously provided by Hannah Rabinowich (University of Pittsburgh School of Medicine), identical to the one described in Clohessy et al. (13). The Mcl-1ΔC mutant was generously provided by Douglas Green (St. Jude's Hospital) in the pET plasmid (10), from which the open reading frame (ORF) was PCR cloned into the pCR4.1 His-tagged mammalian expression vector purchased from Invitrogen. Doxorubicin (Sigma) was used in vitro at 100 ng/ml and in vivo at 1.2 mg/kg. Paclitaxel (Cell Signaling) was used at 10 μM to induce senescence in MEF and MEF p53−. TW37 (2 nM) was a gift from Shaomeng Wang (University of Michigan, Ann Arbor, MI), ABT737 (10 μM) was provided by Abbott Laboratories (Abbott Park, IL), and AT101 (2.5 μM) and roscovitine (25 μM) (Sigma) were used in the indicated experiments.
Transient plasmid transfection into different cell lines was performed by Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Briefly, cells (2.5 × 105 per well) were seeded into 6-well plates and transfected with 0.5 μg of an Mcl-1 BH3 binding groove mutant, Mcl-1ΔC mutant, Bcl-2, or Bcl-xL expressing construct or the empty vector. Transient control and Bcl-xL small interfering RNA (siRNA) transfections were also performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Medium was changed after 24 h. Cells were incubated at 37°C in a 5% CO2 incubator for 48 h prior to testing for transgene expression or knockdown by Western blotting.
Western blot analyses were performed as described previously (28). In brief, total protein from cell lysates (30 to 50 μg) was loaded for each sample lane. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose or polyvinylidene fluoride membrane (Bio-Rad Laboratories, Hercules, CA). Western blots were developed using the enhanced chemiluminescence (ECL) reagents (GE Healthcare). Primary antibodies used for Western blotting were anti-Mcl-1, anti-p53 (Santa Cruz Biotechnology), anti-p21, anti-pRb (S807/811), anti-Rb (Cell Signaling Technology), anti-Bcl-2 (BD Pharmigen), and anti-β-actin (Sigma). Horseradish peroxidase (HRP)-linked secondary antibodies were purchased from Cell Signaling Technology. Comparable protein loading was ensured by reprobing the blots with a mouse anti-β-actin monoclonal antibody.
Cells were seeded into 6-well plates in a DMEM culture medium, and after 24 h, 100 ng/ml doxorubicin was added. The cells were then incubated for 3 to 6 days. SA-β-gal+ staining was followed by the manufacturer's protocol (Cell Signaling Technology). Several representative fields (n = 20) were randomly selected for the quantification of the percentage of SA-β-gal+ cells. Frozen tumor sections were fixed and stained for SA-β-gal activity using X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactoside) at pH 6.0, following the protocol recommended by the manufacturer (Cell Signaling Technology).
Cell lines were plated at 1.0 × 10^5 to 2.0 × 105 cells per well in each well of a 6-well plate containing a poly-l-lysine-treated coverslip. Plates were then incubated overnight in 3 ml of medium to allow cells to adhere. The next day, the chemotherapeutic agent was added to half the samples. Treated cells were left to incubate for 96 h, at which time the cells were fixed with ice-cold methanol and then washed in phosphate-buffered saline (PBS). The cells were permeabilized by incubation in PBS containing 0.25% Triton X-100 (PBS-T) for 10 min, followed by 3 washes in PBS. The fixed cells were then placed in a blocking buffer containing PBS-T and 1% bovine serum albumin (BSA) for 30 min. Next, coverslips were incubated in the above-described blocking buffer containing a 1:200 dilution of mouse monoclonal antibody to PML nuclear bodies (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. The coverslips were washed three times in PBS-T and then incubated for 1 h in the dark with a goat anti-mouse secondary antibody conjugated with Cy3 (Biolegends, CA). The coverslips were then washed three times with PBS-T and mounted on microscope slides using Vectashield mounting medium containing DAPI (4′,6-diamidino-2-phenylindole) for fluorescence (Vector Laboratories, Burlingame, CA). The slides were then observed using a mercury arc lamp and both Texas Red and DAPI filters. Images were merged using Qcapture software suite. The number of PML nuclear bodies per nucleus was determined using a Leica DM5500 B florescent microscope at both 40× and 100× oil immersion. Multiple experiments were incorporated into the figures, and at least 30 nuclei from random fields were selected to obtain the number of PML bodies.
Cells were grown in 6-well plates for 2 days and then left untreated or treated with 5 μM the antioxidant N-acetyl-l-cysteine (NAC) (Sigma-Aldrich) for 24 h. The cells were then treated with 100 ng/ml doxorubicin or left untreated for 6 days as indicated. ROS production was detected using a cell-permeable fluorescent molecule, RedoxSensor Red CC-1 (R-14060; Molecular Probes), in phenol-red-free medium as described in the manufacturer's protocol. The involvement of ROS production in p21 activation was determined by Western blotting after 6 days of doxorubicin treatment. β-Actin was used as an internal control.
Cells were plated at a known cell density and exposed to doxorubicin at 100 ng/ml the next day. Colonies typically emerged 14 to 21 days following chemotherapy and are scored from triplicate plates. Colonies were fixed with methanol and stained with 0.1% crystal violet in 20% methanol for 15 min. Colony numbers were determined by manual counting.
Cells were treated overnight with BrdU and the extent of BrdU incorporation was assayed using the APC BrdU flow kit (BD Pharmingen), followed by flow cytometric analysis. BrdU flow data were tabulated and analyzed using FlowJo (Tree Star, Inc., Ashland, OR).
Female athymic NCr-nu/nu mice (6 weeks old) were obtained from the National Cancer Institute (NCI). HCT116 shControl, HCT116 shMcl-1, HCT116 vector, HCT116 Mcl-1up, HCT116 p53− shControl, and HCT116 p53− shMcl-1 tumor cells were injected subcutaneously (s.c.) at the right dorsal flank of recipient mice (1 × 107 cells per mouse). Doxorubicin (Sigma) at 1.2 mg/kg suspended in PBS (pH 7) was administered intraperitoneally every third day, starting at day 10, after transplantation of tumor cells. Control animals bearing s.c. flank tumors were treated with PBS. Antioxidant-treated mice received drinking water supplemented with 40 mM N-acetylcysteine (NAC; Sigma) to yield an average dose of 1 g NAC per kg body weight per day (27). NAC-treated water was replaced every third day. The length (L) and width (W) of the tumor were measured with calipers every third day, and the tumor volume (TV) was calculated as TV = (L × W2)/2. All animals were maintained in pathogen-free animal facilities at the University of Maryland School of Medicine and Cleveland Clinic Lerner Research Institute, and all procedures were performed under respective institutional animal care and use committee approved protocols.
Tumor tissues from mice were fixed overnight in cold 4% paraformaldehyde (prepared in PBS) at 4°C, followed by incubation in cold 30% sucrose/PBS solution for 24 h before embedding in optimal cutting temperature (OCT) medium (Tissue-Tek; Sakura) on dry ice and stored at −70°C. Tissue sections (7 μm thick) were mounted on gelatin-coated slides and then stored at −70°C. Immunofluorescence and immunohistochemical staining for Mcl-1 and Ki-67 was performed as described elsewhere (10, 30). Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was performed using the ApopTag kit using the manufacturer's protocol (Millipore) and compared to a commercially prepared positive-control slide (Millipore).
Data are presented as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM) from at least three separate experiments. Differences between various experimental groups were calculated using Student's t test, in which P values of <0.05 were considered significant. The Mann-Whitney nonparametric test established the significance for the change in the number of PML bodies with a P value of <0.05 considered significant.
Mcl-1 is frequently overexpressed in neoplastic tissues and can confer resistance to cancer therapies (31, 40). We first sought to determine if Mcl-1 confers resistance to CIS in cancer by utilizing a well-characterized model of HCT116 colon carcinoma cells expressing an intact copy of p53 but with an inactive INK4A locus that impairs the expression of p16 and p14ARF (26). Previous studies have reported that a low dose of doxorubicin induces senescence in these cells (17). We therefore exposed derivatives of this cell line (37), either overexpressing Mcl-1 (HCT116 Mcl-1up) or an empty vector (HCT116 vector), to a low dose of doxorubicin (100 ng/ml) for 6 days. The level of Mcl-1 expression in these lines was verified by Western blot analysis (Fig. 1A). As expected, HCT116 vector cells treated with doxorubicin adopted a senescent phenotype typified by a large, flattened shape and enhanced expression of SA-β-galactosidase (SA-β-gal+) in about 60% of cells (Fig. 1A and B). Although SA-β-gal is the most common assay used to analyze senescence, others have shown that the formation of PML bodies within the nuclei along with SA-β-gal+ is confirmatory of senescence (28). Our data are consistent, as control cells induced to senesce have significant increases in the number of nuclear PML bodies compared to untreated cells. However, cells overexpressing Mcl-1 had significant abrogation of senescent changes after treatment, including reduced SA-β-gal+ and no increase in PML foci compared to HCT116 empty vector cells (Fig. 1A and B).
To confirm our results, we overexpressed Mcl-1 in other p53+ cell lines: MCF-7 (breast) and MEL526 (melanoma). Both were transiently transfected using either empty vector or one overexpressing Mcl-1. The cells were allowed to rest for 48 h and were then treated with 100 ng/ml of doxorubicin for 6 days. MCF-7 and MEL526 cell lines showed statistically significant reductions in the percentage of SA-β-gal+ cells in the Mcl-1 overexpressing cell lines treated with doxorubicin compared to respective vector controls (Fig. 1C).
We next employed two noncancer cell lines: MCF-10A (immortalized breast epithelium) and mouse embryonic fibroblasts (MEFs) (31, 32). Both were transiently transfected with either the empty or Mcl-1 overexpressing vector. In the case of MCF-10A, after 48 h, cells were exposed to 100 ng/ml of doxorubicin for 6 days. MCF-10A cells containing the empty vector exhibited a senescent morphology when treated with low-dose chemotherapy, while those overexpressing Mcl-1 had a significant reduction in SA-β-gal+ cells compared to control (Fig. 1C). Finally, we examined chemotherapy-induced senescence in primary cells (MEFs). Initially, we attempted to use doxorubicin but found that paclitaxel yielded the most robust induction of senescence. Similar to the previous cell lines described, MEFs overexpressing human Mcl-1 were relatively resistant to CIS (Fig. 1C). Human Mcl-1 similarly has been described to inhibit apoptosis in mice, thus the survival functions of Mcl-1 do not appear to be limited to species from which it was derived (40). Thus, overexpression of Mcl-1 in p53+ cells is sufficient to protect from CIS regardless of cell origin.
In addition to measuring SA-β-gal+ cells and PML bodies to identify senescence, we obtained supporting evidence for the senescent phenotype using a clonogenic escape assay and BrdU incorporation (Fig. 1D and E). In the clonogenic assay, the number of untreated control and Mcl-1 overexpressing colonies was comparable on day 15 of the experiment. In contrast, doxorubicin-treated control cells formed significantly fewer colonies compared to those overexpressing Mcl-1. In addition, using the BrdU incorporation assay, we observe that the proliferation of cells growing in drug-free media was equivalent regardless of the level of Mcl-1 expression and that doxorubicin treatment of control cells resulted in a marked decrease in BrdU incorporation. However, overexpression of Mcl-1 resulted in only a slight decline in BrdU uptake after drug treatment. These results further confirm our observation that doxorubicin treatment reduced the proliferation of control cells but had little effect on those overexpressing Mcl-1.
We next examined whether the addition of low-dose chemotherapy alters Mcl-1 protein levels either in control or in Mcl-1 overexpressing HCT116 cells. Though we have found Mcl-1 expression to decrease slightly and then rebound over the first 24 h of drug treatment (data not shown), we find that Mcl-1 protein levels are not significantly affected by longer-term culture in doxorubicin for up to 6 days (Fig. 1F). The lack of significant loss of Mcl-1 during CIS is in stark contrast to what has been reported to occur during chemotherapy-induced apoptosis, which further distinguishes senescence from this form of cell death (17).
To understand the role of Mcl-1 in the inhibition of CIS, we evaluated the expression of some of the key molecules known to be involved in cellular senescence: p53, p21, Rb, and pRb (S708/711, pRb). We noticed that the accumulation of p53 in cells overexpressing Mcl-1 and treated with doxorubicin is the same as in control cells despite a significant difference in SA-β-gal-activity. However, in contrast to controls, Mcl-1 overexpressing cells had a lower accumulation of p21 and an increased (versus loss in controls) accumulation of Rb and pRb. These data suggest that Mcl-1 inhibits CIS downstream or at the level of p53 and subsequently blocks the accumulation of p21 and loss of pRb (Fig. 1G).
To determine if CIS alters the binding of Mcl-1 to p53, we used coimmunoprecipitation (co-IP) to assess whether there are changes in their binding relationship, as we previously described during apoptosis (18). We observed that low-dose doxorubicin treatment did not alter the binding relationship between p53 and Mcl-1 in cells overexpressing Mcl-1, further distinguishing CIS from changes observed during apoptosis (data not shown).
We next examined if downregulation of Mcl-1 could enhance CIS. We generated a number of HCT116 cell lines stably expressing a Mcl-1-specific short hairpin RNA (shRNA) or an irrelevant control. After verifying the knockdown of Mcl-1 (Fig. 2A), cells were then treated with doxorubicin and analyzed 6 days later for SA-β-gal activity. In all treated cell lines, we observed typical increases in SA-β-gal+ cells (Fig. 2A). However, significantly higher percentages of SA-β-gal+ cells, as well as higher numbers of PML bodies, were observed in doxorubicin-treated shMcl-1-expressing cells compared to controls (Fig. 2A and B). Additionally, Mcl-1 downregulation alone did not significantly affect cell proliferation in clonogenic or BrdU uptake assays, even after low-dose doxorubicin treatment (Fig. 2C and D). We further examined the effect long-term doxorubicin treatment had on control and shMcl-1 cells. Similar to our previous findings, doxorubicin treatment had a minimal effect on Mcl-1 levels in control cells, while a noticeable reduction in Mcl-1 expression was found in shMcl-1 cells over the first 3 days of treatment (Fig. 2E). Though Mcl-1 levels returned in these cells by day 4, the expression continued to be reduced compared to those expressing the control vector. Further, despite the increase in SA-β-gal activity in drug-treated shMcl-1 cells, there was no difference in p53, p21, Rb, or pRb expression in comparison to shControl cells undergoing senescence (Fig. 2F). These data indicate that base levels of Mcl-1 at least partially prevent senescence induction in response to DNA damage in HCT116 cells.
Many tumors have inactive p53, which results in resistance to both apoptosis and senescence (5, 34). Our data show that overexpression of Mcl-1 in p53+ cells inhibits CIS while downregulation can enhance it. Therefore, we wanted to know if downregulation of Mcl-1 in p53− cells also sensitizes them to CIS. We stably transfected a previously developed HCT116 p53− cell line with either control plasmid or shMcl-1 and generated a number of clones (6). After verifying the downregulation of Mcl-1 by Western blotting (Fig. 3A), these cells were treated with doxorubicin and analyzed 6 days later. As expected, HCT116 p53− shControl cells treated with doxorubicin did not show any senescent phenotypic changes, did not lose expression of Mcl-1, had no detectable SA-β-gal activity, and few PML bodies (Fig. 3A and B). In contrast, each HCT116 p53− shMcl-1 clone adopted a senescent phenotype and had increased SA-β-gal activity and PML staining, similar to their p53+ counterparts (Fig. 3A and B). We also observe induction of SA-β-gal activity after knockdown of Mcl-1 in other p53− cell lines, namely, HSC3 and p53− MEFs (Fig. 3D). In addition, we obtained the recently derived MCF-10A immortalized human mammary cell line possessing a somatic deletion of p53. MCF-10A p53− cells have a significantly increased sensitivity to DNA-damaging chemotherapies (35); however, their sensitivity to CIS has not been reported. Unlike HCT116 p53− cells, doxorubicin treatment of MCF-10A p53− cells resulted in a significant increase in the percentage of SA-β-gal+ cells compared to untreated controls (Fig. 3D). This result required a change in our strategy, and we instead overexpressed Mcl-1 to see if enhanced Mcl-1 expression could resist the induction of CIS. Indeed, we did observe a loss of senescence in these cells (Fig. 3D). Thus, Mcl-1 appears to have a significant antisenescent role in cells lacking p53.
Consistent with the increased SA-β-gal+ activity and PML body formation in response to low-dose chemotherapy, HCT116 p53− shMcl-1 cells lost the ability to form colonies (Fig. 3E) and proliferated at a level lower than that observed with treated HCT116 p53− shControl cells (Fig. 3F). To further understand this p53-independent pathway of senescence, we evaluated the expression of p21 and pRb in HCT116 p53− shMcl-1 cells and HCT116 p53− shControl cells in the presence of doxorubicin (Fig. 3G). Cells expressing lower levels of Mcl-1 and treated with doxorubicin had greater p21 expression than the corresponding control cells. Furthermore, pRb and levels of total Rb were suppressed in HCT116 p53− shMcl-1 cells versus controls. These data suggest that Mcl-1 blocks CIS in p53− cell lines and that by depleting Mcl-1, senescence is restored via a concomitant induction of p21 and a reduction of pRb.
To further validate the role of Mcl-1 in the induction of senescence in the p53− cells, we employed a cyclin-dependent kinase (CDK) inhibitor (roscovitine) to block Mcl-1 activity. Data from several laboratories has established that CDK inhibitors act, at least in part, by inhibiting CDK9, a kinase intimately involved in transcription initiation and elongation factor b, resulting in downregulation of several short-lived proteins, including Mcl-1 (9). Similar to HCT116 p53− shMcl-1 cells, roscovitine (25 μM) and low-dose doxorubicin treatment of HCT116 p53− cells results in an increase in SA-β-gal+ activity similar to HCT116 cells (Fig. 3H). Roscovitine suppresses Mcl-1 expression as previously reported (Fig. 3I) (24). Interestingly, the reduction of Mcl-1 was noticed as little as 2 h posttreatment and began to recover by 24 h. This observation suggests that downregulation of Mcl-1 is needed only for a few hours after chemotherapy treatment for initiating senescence. In summary, homeostatic levels of Mcl-1 seem to block a p53- and pRb-independent pathway of senescence, which can be initiated via low-dose doxorubicin treatment in HCT116 p53− shMcl-1 cells.
We next wanted to determine whether other Bcl-2 family members play a similar role in CIS resistance. Using a specific shRNA (shBcl-2), we knocked down the Bcl-2 protein in HCT116 p53− cells. However, unlike Mcl-1, downregulation of Bcl-2 did not induce senescence following doxorubicin treatment (Fig. 4A). We also repeated these experiments using another Bcl-2-specific shRNA and obtained similar results (data not shown). It is interesting to note that overexpression of Bcl-2 alone can prevent chemotherapy-induced apoptosis but does not enhance colony formation (39). In contrast, as shown in Fig. 1D, overexpression of Mcl-1 did enhance colony formation in the presence of low-dose chemotherapy. These observations illustrate a distinct activity for Mcl-1 among Bcl-2 family members.
We next wanted to directly compare overexpression of Mcl-1 in p53− (Fig. 4B) and p53+ (Fig. 4C) cells with other Bcl-2 family members (Bcl-2, Bcl-xL). We observe that all Bcl-2 family members tested have significant anti-CIS properties in p53+ cells (Fig. 4C). In contrast, only overexpression of Mcl-1 significantly abrogates CIS in HCT116 p53− cells (Fig. 4B). These differences highlight a unique role for Mcl-1 in the regulation of a p53-independent senescence pathway.
We also employed another approach to inhibit Bcl-2 family members by using several small molecule inhibitors of Bcl-2 family proteins. ABT-737 (a BH3 mimetic) is designed to promote tumor cell apoptosis by blocking interaction between the prosurvival and proapoptotic (BH3-only) members of the Bcl-2 family. ABT-737 efficiently blocks Bcl-2 and Bcl-xL function but not Mcl-1 and on its own was shown to induce senescence in some but not all tumor cell lines (6, 30). Surprisingly, the fraction of SA-β-gal+ cells was moderately reduced in HCT116 cells after treatment with ABT-737 and doxorubicin (Fig. 4D). This result may be explained by increased levels of Mcl-1 in ABT-737-treated cells (Fig. 4E). ABT-737 is known to cause the release of Bim, which can then stabilize and increase the expression of Mcl-1 protein (8). Additionally, we used a similar small molecule inhibitor, AT-101, which can block Bcl-2, Bcl-xL, and Mcl-1 in a similar fashion as ABT-737 (15). AT-101 did not affect the level of senescence induced by low-dose chemotherapy (Fig. 4D). We did confirm that the dose of AT-101 was sufficient to inhibit these antiapoptotic Bcl-2 family members and that AT-101 sensitized HCT116 cells to apoptosis after increasing doses of doxorubicin (data not shown). Using an additional small molecule inhibitor with similar properties as AT-101, TW-37, we also observed no increases in CIS (data not shown). We also tested all three small molecule inhibitors in HCT116 p53− cells, and none induced or sensitized the induction of senescence (Fig. 3H). These findings are important, as these inhibitors, which only target the binding site of BH3-only proapoptotic molecules, appear not to be involved in senescence promotion.
To further confirm that the antisenescent and antiapoptotic properties of Mcl-1 are unique, we obtained two well-described mutant versions of Mcl-1. First, we procured the recombinant Mcl-1ΔC protein lacking the final C terminus/transmembrane domain. This mutant has marginally reduced antiapoptotic function, because the C terminus is in part responsible for the targeting and retention of Mcl-1 to the mitochondria (10). A second mutant was obtained containing specific point mutations within the BH3 binding pocket that changes the binding specificity to BH3-only proteins and has significantly decreased antiapoptotic activity (13). Therefore, each of these mutants addressed the affect of its respective altered domain on senescence. Similar to normal Mcl-1, overexpression of both mutants protects against CIS in both p53− and p53+ cells (Fig. 4B and C). These results indicate that the mechanism by which Mcl-1 inhibits senescence is neither dependent on its C-terminal transmembrane domain or its BH3 binding groove. These data, along with the results obtained using the small molecule inhibitors, indicate that a unique, still undefined area within Mcl-1 is responsible for its antisenescent function.
We have shown that Mcl-1 can regulate both a p53-dependent and -independent senescence pathway. Previous work has demonstrated that HCT116 p21− cells also do not undergo senescence in the presence of low-dose chemotherapy (18). We next assessed whether Mcl-1 knockdown can restore senescence in these cells. As shown in Fig. 5A, downregulation of Mcl-1 either by shMcl-1 or by roscovitine did not induce senescence after treatment with low-dose doxorubicin. The downregulation of Mcl-1 in HCT116 p21− cells was confirmed by Western blotting (Fig. 5A, inset). Consistent with the lack of detectable senescence, we also did not observe any significant changes in p53 or pRb protein expression under these conditions (Fig. 5B). Thus, Mcl-1 loss, which appears to allow the induction of a p53-independent senescence pathway, did not affect HCT116 p21− cells, indicating that this pathway relies on p21.
Other studies have shown that p21 acts within the DNA damage response and ROS signaling pathway to induce senescence (20, 26). Therefore, we examined the kinetics of ROS production in HCT116 p53− cells left untreated, treated with doxorubicin, or treated with doxorubicin plus the antioxidant N-acetylcysteine (NAC). Inhibition of Mcl-1 in HCT116 p53− cells resulted in an increase in ROS production when treated with doxorubicin, which could be abrogated by the addition of NAC (Fig. 5C). Consistent with our observation that Mcl-1-mediated resistance to senescence is not related to the C terminus or BH3 binding pocket, the expression of our Mcl-1 mutants in HCT116 p53− shMcl-1 cells were just as effective at blocking ROS production as the reexpression of full-length Mcl-1 (Fig. 5C). HCT116 p53− shMcl-1 cells had greatly enhanced p21 expression within 24 h of doxorubicin treatment, which the addition of NAC blocked (Fig. 5D). These data suggest that Mcl-1 acts to suppress ROS production, thereby inhibiting the expression of p21. Further, there was a decrease in SA-β-gal+ cells when HCT116 p53− shMcl-1 cells were treated with doxorubicin plus NAC (Fig. 5E). Thus, we demonstrate that Mcl-1 can suppress ROS production in HCT116 p53− cells, thereby inhibiting p21 expression and ultimately CIS.
To evaluate CIS in vivo, we injected various HCT116 cell lines (HCT116 Mcl-1up, HCT116 vector, HCT116 p53− shMcl-1, HCT116 p53− shControl) into athymic nude mice. Untreated control tumors of both HCT116 and HCT116 p53− cell lines displayed robust growth (Fig. 6A). However, downregulation of Mcl-1 (shMcl-1) in both HCT116 and HCT116 p53− cell lines severely inhibited tumor growth. Enhanced SA-β-gal+ activity was observed in both HCT116 and HCT116 p53− shMcl-1 tumors as well as a reduction in Ki-67 expression (Fig. 6B and C). We additionally confirmed persistent Mcl-1 downregulation in vivo in all shMcl-1 tumors and found little apoptosis in all tumors via TUNEL staining (Fig. 6B). These observations illustrate that forced reduction of Mcl-1 expression retards tumor growth even in HCT116 p53− cells, and this growth reduction is associated with senescence.
Because the production of ROS was found to play a key role in senescence induction in p53− HCT116 cells in vitro, we next determined whether it could also block senescence in vivo. Previous reports found that NAC treatment of p53− xenograft tumors could result in a delay in tumor growth, mostly likely due to the inhibition of ROS-related DNA damage and genomic instability (27). Using a similar treatment regimen in which tumor-bearing mice received oral NAC, we observed that the growth of HCT116 p53− tumors possessing normal Mcl-1 expression could be slightly delayed. In contrast, the growth inhibition caused by knockdown of Mcl-1 expression could be almost completely abrogated by NAC treatment (Fig. 7A). NAC-treated p53− shMcl-1 tumors had reduced SA-β-gal activity and greater Ki-67 expression compared to the untreated shMcl-1 group (Fig. 7B and C). All tumor groups had similar low levels of apoptosis as measured by TUNEL staining. Tumor outgrowth was not due to the loss of Mcl-1 knockdown, as treated and untreated tumors had similarly reduced levels of Mcl-1 expression. Further, the enhanced tumor growth caused by ROS inhibition appears to be specific to the p53-independent senescence pathway, as NAC treatment of p53+ control and shMcl-1 tumors had no observable effect on tumor growth (data not shown). Collectively, these data underscore the key role ROS production plays in p53-independent senescence pathways and the importance of Mcl-1 in their inhibition.
In order to determine if overexpression of Mcl-1 can prevent senescence induced by a low dose of doxorubicin in vivo, HCT116 tumor cells overexpressing Mcl-1 or an empty vector were transplanted into nude mice and then, after 10 days, treated with 1.2 mg/kg doxorubicin once every third day. This dose of doxorubicin was chosen based on previous reports (38). Doxorubicin effectively inhibited growth and induced SA-β-gal+ activity in tumors expressing empty vector (Fig. 8A and B). Similarly, the percentage of cells expressing Ki-67 was significantly lower in doxorubicin-treated tumors expressing empty vector (Fig. 8C). In contrast, tumors overexpressing Mcl-1 grew robustly despite doxorubicin treatment and were, from a growth curve standpoint, almost resistant to treatment (Fig. 8A). We also did not observe any reduction of Ki-67 staining in drug-treated Mcl-1 overexpressing tumors (Fig. 8C). Our data show that overexpression of Mcl-1 in HCT116 cells promotes tumor growth in vivo and creates resistance to chemotherapy treatment and its resultant induction of senescence.
Senescence is a recognized cellular pathway involved in all aspects of cancer biology from carcinogenesis to tumor proliferation and treatment sequela and appears to be a major hurdle for cancer progression (14). Senescence appears to be controlled by distinct pathways but, in general, is initiated by tumor suppressors like p53. Interestingly, various oncogenes induce senescence, a response which appears to be the major barrier to the development of cancer (35). Thus, without evasion or loss of tumor suppressor genes such as p53, cells expressing or even overexpressing oncogenes fail to become cancerous due to senescence (2). In the realm of cancer therapy, apoptosis has been the main form of cell death studied in response to chemotherapy and radiation therapy (25). However, recent studies have described how similar treatments of p53− cells in which p53 expression was restored resulted in tumor regression through the induction of senescence (34, 37). Further, chemotherapies have been reported to induce senescence in a variety of cancer types in human patients, which is associated with treatment success (14). In this regard, tumor senescence escape mechanisms not only have implications in carcinogenesis but treatment efficacy as well.
Our study reveals that a well-known antiapoptotic gene, Mcl-1, which is overexpressed in many cancers and has been studied mainly in hematopoietic malignancies, protects several solid cancer cell lines from CIS (10, 30, 39). Mcl-1's homeostatic and antiapoptotic properties have been well described (28, 39); however, its senescence-modulating functions have not been studied until this report. The antisenescent function of Mcl-1 appears to be distinct from its related Bcl-2 family members. For instance, Bcl-2 was reported to be unable to inhibit senescence (29). The differences between these related molecules is further underscored by the finding that, in complementary mouse models of leukemia, tumor cells dependent on Mcl-1 were more susceptible to chemotherapy than those dependent on Bcl-2 (5). Our data reveal that all major Bcl-2 antiapoptotic molecules (Mcl-1, Bcl-2, and Bcl-xL) are antisenescent in p53+ tumors; however, there appears to be a distinct ability of Mcl-1 to inhibit CIS in p53− cells. These differences underscore the uniqueness of the p53− pathway to senescence and highlight the need for its further characterization.
During the process of senescence induction we observe, in both p53+ and p53− cells, induction and dependence on p21 as well as a loss of pRb. These findings agree with a similar study of doxorubicin- and radiation-induced senescence in p53+ cells, which also required p21 induction and was accompanied by pRb loss (19). This study further found a role for another pocket protein, Rb2, which was strongly upregulated during senescence. Another recent study described how loss of pRb was a senescence escape mechanism in normal human fibroblasts (9). That study further showed that pRb loss triggered an additional proliferation barrier via p21-driven induction of senescence. Based on these studies, we hypothesize that loss of pRb expression in our model may be part of a p21-dependent progression into senescence.
Senescence pathways independent of p53 have been described but are still poorly understood (20). Our results reveal that in p53− cells (which do not normally undergo CIS), chemotherapy treatment can induce senescence after knockdown of Mcl-1 expression. This process causes similar changes to those found in p53+ cells, including increases in p21 expression, loss of pRb, enhanced PML body formation, and reduced BrdU incorporation. This implies that homeostatic levels of Mcl-1 govern an alternate pathway which is not dominant when p53 is active.
Similar experiments in HCT116 p21− cells reveal that p21 is indispensable for senescence induction even when Mcl-1 is knocked down. Others have shown that p21 expression during senescence is dependent on the production of ROS (26, 36). We observe that in the absence of p53, not only does knockdown of Mcl-1 expression result in enhanced ROS production concomitant with SA-β-gal activity after doxorubicin treatment, but the addition of an antioxidant is sufficient to block the induction of both ROS and senescence. This observation is further underscored by the use of NAC to block ROS production in vivo. Whereas we and others have demonstrated the ability of NAC treatment to slow the growth of p53− tumors, we find that NAC can accelerate the growth of p53− tumors also lacking Mcl-1, suggesting that NAC's ability to inhibit senescence and thus promote tumor growth is stronger than the protective effects of preventing ROS-related DNA damage in these cells (27). Collectively, these results imply that in p53− cells, Mcl-1 acts to prevent ROS induction perhaps in a manner similar to that reported for Bcl-2 in p53+ cells and that chemotherapy induces senescence primarily through a p53/p21 axis, but without p53, the loss of Mcl-1 can activate an alternative pathway to senescence that is ROS and p21 dependent (21). The trigger for this pathway is still under investigation but we hypothesize that other tumor suppressors are involved (8).
In order to determine whether the canonical BH3 binding domain of Mcl-1 is involved in resisting the induction of CIS as it is for apoptosis, we blocked interaction with this domain through the use of small molecule inhibitors. BH3 mimetics are in various levels of preclinical and clinical trials and block the interaction between antiapoptotic Bcl-2 molecules and their proapoptotic counterparts, leaving the latter molecule free to induce apoptosis (15). We show for the first time that a gossypol variant, AT-101, which targets most major antiapoptotic Bcl-2 family members, including Mcl-1, did not alter the level of CIS. Interestingly, another BH3 mimetic, ABT-737, which does not target Mcl-1, failed to increase senescence and actually partially decreased it. We subsequently found that ABT-737 increases Mcl-1 expression, likely due to its anti-Bcl-2/Bcl-xL effects, and this would explain the decreased senescence observed in ABT-737-treated cells (33). Similarly, other studies of ABT-737 reveal its inability to induce apoptosis in cells overexpressing or selected to overexpress Mcl-1 (38). Taken together, these data demonstrate that BH3 mimetics do not increase the level of CIS, thus indicating that the BH3 binding domain of Mcl-1 (or other Bcl-2 family members) is not involved in regulating CIS.
In order to conclusively exclude the BH3 binding domain of Mcl-1 from its antisenescent activity, we obtained an Mcl-1 mutant containing an inactive BH3 binding pocket (22). While this mutant possesses severely reduced antiapoptotic activity, we found that it nonetheless confers resistance to CIS similar to wild-type Mcl-1. We also obtained an Mcl-1 mutant containing a deletion of the C-terminal transmembrane/mitochondrial targeting domain, which was reported to have moderately reduced antiapoptotic activity, and likewise found that the deletion had minimal effect on its antisenescent properties (23). These data imply that Mcl-1-mediated inhibition of senescence is distinct from its antiapoptotic function and is not reliant on its ability to bind to BH3 proapoptotic molecules. Further, these data indicate that a distinct domain of the Mcl-1 molecule is responsible for its antisenescent function. Identifying this location within Mcl-1 will be the basis of further drug discovery.
Our results also indicate that human solid cancer cells require Mcl-1 for tumor growth in vivo even in the absence of p53. These data are striking since loss of p53 is sufficient to initiate not only tumorigenesis but also cancer progression (34). Our data suggest that, from a tumor growth retardation standpoint, an Mcl-1-dependent and p53-independent process is dominant. Moreover, reduced levels of Mcl-1 even without any treatment were associated with senescence as indicated by SA-β-gal activity and abrogation of Ki-67 expression. When we examined CIS in vivo, we consistently observed that overexpression of Mcl-1 enhanced not only tumor growth but also resistance to CIS, suggesting that tumors which overexpress Mcl-1 basally or by selection are resistant to cancer treatment by resisting both apoptosis and senescence.
The implications of this study are that targeting Mcl-1 in cancers that either express this molecule or could be selected to express it may be an important aspect of cancer therapy. Further, molecular targeting could also be too specific in that "inhibitors" of a molecule may target only one aspect of its function. In the case of Mcl-1, small molecule inhibitors of apoptosis-associated domains are available; however, our study demonstrates that Mcl-1 also inhibits CIS via an undefined domain for which there is no specific inhibitor currently available. Future studies are required to develop complete inhibitors of Mcl-1 and similar molecules in order to target all of their cancer-promoting and treatment-resistant functions.
This work was supported by NIH grants R01CA132796 (to B.R.G.) and CA105005 (to D.V.K.).
Published ahead of print 26 March 2012
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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My Nonprofit Reviews
Review for Family Education And Support Services, Olympia, WA, USA
I am so grateful for this agency. I am a 64 year old grandmother who did not expect to be raising my grandchild but my daughter and son in law passed away in a boating accident. The folks at the Kinship support group were so kind and welcoming, and I could not have attended if there hadn't been child care. Some nights I am too tired to go.. but my kid's drag me because they enjoy it so much. Always after I feel refreshed and I am so glad I went. Thank you Lynn Urvinat, Kinship Navigator for all you have done for us.
More Feedback
I've personally experienced the results of this organization in...
Yes, they are wonderful. A deep well of emotional and educational support.
If I had to make changes to this organization, I would...
I wish I had known about them sooner.

subset: commoncrawl


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16 October
How to translate a flexicontent site with Joomla native functionBy Yannick Berges
Hello we are happy to annonce a tutorial about using FLEXIcontent and Joomla! native translation system
We create a tutorial AND a the first kickstart with a full FLEXIContent project Complete Job apply site, au menu :
- Last FLEXicontent version + Helix 3 template (flexicontent correction)
- Job list system with filter and search
- Job application form (we can see job apply it, that create an automatic relation between job and application form)
- Free job form (user can chose job type and hours for meeting)
- Front-end administration for HR Manager (search annotation, vote etc)
- Fully native translation
- Slider module
- Google maps module
- Backend optimisation with FLEXIadmin module
- CSV export in front end
You can explore this site for testing and download kickstart file for install and test more !
Tutorial : Create a multilingual site with FLEXIContent
Kickstart : https://com3elles.biz/jdays2017
7 June
FLEXIcontent 3.2.0By Yannick Berges
Hello we are happy to announce FLEXIcontent v3.2
Fixed several small / rare issues of v3.1.1
Following is only a SMALL part of improvements and new features of v3.2.0
Added minimimal-overhead task execution for frontend, only loading Joomla Framework,
- e.g. 2x-5x fast AJAX responce, currently only supporting text-search autocomplete task,
no longer effected by web-sites with overloaded onAfterInitialize event (3rd party plugin)
Revised media manager
- new UI for image-gallery field
- new UI media management
-- list / grid views for both file list and AJAX upload,
-- editing item properties for all uploaded items
-- full page thumb previewing
-- instant thumb size slider
-- assign multiple files at once in any file-using field
- new attractive styling
- ajaxified item / category / types / etc forms
- save as copy for all forms
Revises Items manager UX
- compact display of secondary categories and tags
- toggle all secondary categories and tags at once
- 40%-50% reduced time during browser rendering of HTML (important when listing 500 items)
- Intergrated state selector featured, and preview to btn-group
- State selector with minimal HTML no longer add HTML-render or JS load, during initial page opening
- Filter button button remember state and show active filters as a counter, same for columns button
- Converted multi-item "Change state" button to be drop down
Performance, flexicontent period tasks, are not more "smartly" executed, thus reducing even further their alread minimal impact
More power to the fields
- Added state (published / unpublished / archived !!) for the values of "selection-like" (indexable) fields
Easier more understandable configuration
- better grouing for ACL with headers makes it easier to configure
- revised components parameters configuration into new TABs
Many other, to do compile a list
- e.g. 2x-5x fast AJAX responce, currently only supporting text-search autocomplete task,
20 May
Help FLEXIcontent 3.2By Yannick Berges
Hello we work hard to publish next major FLEXIcontent version 3.2 The feature list : Joomla! 3.7 compatibility with Custom field comlpatibility New uploader image (inline single mode auto assign mult ...
22 November
FLEXIcontent 3.1 is stableBy Yannick Berges
Hello we are very happy of you announced the release of stable new version of FLEXIcontent the series 3.1. We worked long months to provide you with an ever more powerful, robust and simpler to use v ...


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We were so fortunate to have Lynda as our agent, when searching for a home purchase. She truly went over and above to find us a home that exceeded our criteria. Lynda was always prompt, informative and we never felt pressured into anything. It was a great experience and we would recommend Lynda to anyone who wants a friendly and professional real estate experience!
Thanks so much for the quick work! As always, you come through in a bind. BEST REALTOR EVER! I appreciate you.
Have a Blessed Day,
I just wanted to write to you to let you know what an amazing person Lynda is. We have been looking for a house for 7 months with no luck! We had suffered an eviction at the hands of my greedy sister who is in charge of my mother's estate where we lived. My friend was also doubling as my agent acted more like a mom than my real estate agent just couldn't get us in a house. I was frustrated, living away from my other half at a coworker's house and had to do something. I sent out 5 emails to 5 different agents and within 2 hours, Lynda answered my cry for help. We looked at 3 houses with her within 1 week and within 3 weeks of knowing Lynda, I have now bought my first house!!! Lynda was there every step of the way. If I had the silliest question, she would have an answer within minutes via text or email. I felt like whatever I was unsure of, she was right there to help me along. It was the greatest and easiest experience I've had to go through during this time in my life. Even now that we are moving into the house, she is still by my side to help me with anything I need and I couldn't think of anyone else that would do this for us! She truly is a kind heart and we are so grateful for her!!!!
Lynda is one of the very best in the industry! Even though my move changed directions, she continues to be one of my favorite Realtors of all time. She is not like most, she is a connector, and it's evident in your first meeting. Keep up the great work, Lynda! You ROCK!


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Victoria Cruise Night is in the Books for 2015
What a great year for car fans. Victoria Cruise Night kicked off at the beginning of the year on the second Saturday each month as an informal get together of people who love motor vehicles of all kinds. The event grew each month with more and more people bringing out all kinds of cool rides, from Model Ts and custom tractors, to classic 60s and 70s muscle cars, 4x4s, trucks, lowriders and rat rods. Since the weather is getting cooler, we're calling it until March.
Check out some photos from the event below.


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Stock Graphic >> Pattern - Winter sweets pattern 106411 by Eisfrei @ CreativeMarket
File Size: 5.67 MB; DPI: 300 DPI; Vector: Yes.
Christmas ginger bread, tangerine and anise seamless pattern.
100% hand painted watercolor vectorized texture.
High quality 300 DPI Jpeg and EPS8 format.
Keywords: christmas, new year, winter, ginger bread, seamless, pattern, background, holiday, watercolor, vintage, sweets, tangerine, cake, wallpaper, objects, party, home, house, snowflake.


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I'm having a protein shake. That's healthy, right?
Have you ever really read the ingredients list on the back of one of those protein powder mixes or protein bars? You might be surprised by what you find. Ingredients are listed in order of greatest to least – the ingredient that comprises most of the product will be listed first, second, and so on.
The first few ingredients probably won't seem too foreign – whey or soy protein, fructose or sugar, cocoa powder, etc. – but if you keep going you may not be able to pronounce many of the ingredients. The lower the quality of protein powder (i.e. cheap) the worse the ingredients. What surprised me even more was not only the vast array of artificial preservatives and food coloring, but the amount of refined sugar! Especially when sugar is the second ingredient listed. Sure it has 20 grams of protein per serving, but so does a can of tuna fish (25 grams, actually).
Lots of protein won't cancel out lots of sugar, folks.
I'm generalizing a little, and don't want to knock anyone's success with a quality protein supplement (one free of preservatives, artificial colors, and artificial sweeteners). If you want to purchase a high quality, gluten free, soy free protein powder blend, PlantFusion has high quality and healthy ingredients at a reasonable price. You can get it here.
What's wrong with soy?
I prefer to stay away from soy products as much as possible. Many of the powdered protein mixes rely on soy protein as a base as do many vegan foods. There have been many studies on the pros and cons of soy-based products. Personally, I lean more toward the cons and find that my body doesn't like it much.
…soybean contains large quantities of natural toxins or "antinutrients." First among them are potent enzyme inhibitors that block the action of trypsin and other enzymes needed for protein digestion.
These inhibitors are large, tightly folded proteins that are not completely deactivated during ordinary cooking. They can produce serious gastric distress, reduced protein digestion and chronic deficiencies in amino acid uptake. In test animals, diets high in trypsin inhibitors cause enlargement and pathological conditions of the pancreas, including cancer.14 Soybeans also contain haemagglutinin, a clot-promoting substance that causes red blood cells to clump together.
Read the full article here
So I decided, why not make my own!
I decided it may be cheaper to use what I've already got in the pantry and I won't have any concerns about questionable ingredients. Besides, if I'm getting my butt kicked at the gym or on a long run I want to replenish my body with stuff it can really use to recover and get stronger. Not having all that excess sugar will also help if you're trying to lose some extra weight or eat clean.
Instead of some complicated mixture of isolated proteins, I chose things I could find in my home that were already packing the protein. This is a base that when added to your favorite smoothie fixings (or made into protein balls) will up your protein intake in a healthy, real-food kind of way. I broke it down and did a ton of research and found that this combination makes the best tasting, most protein packed combo.
You'll get roughly the same protein amount per serving as those big overly priced fructose and preservative filled ones (approximately 18 grams in a 1/3 cup serving.) You do get more protein from the dairy powdered milk than the coconut milk so if using coconut milk just know that you'll be closer to 14 grams of protein per serving. However, once you add in some PB2, a banana, and spinach in your smoothie you'll have plenty of protein to get your day started and keep you full.
Homemade Protein Powder
Makes about 9 servings of 1/3 cup
- 1/2 cup oats (certified gluten free)
- 1/2 cup almond meal (blanched is best)
- 2/3 cup powdered milk (this brand is low in lactose) or 1 cup Coconut Milk Powder
- 1/3 cup hemp seeds
- 2 tbsp. chia seeds
- 1 tbsp. flax seed
In a food processor combine the oats and pulse until it reaches a flour consistency. Add in remaining ingredients and pulse a few more times until everything is powdery smooth. Now you can leave it like that or you can add in some flavoring. FYI: if you opt to add in some PB2 powder you'll be upping your protein even more, other wise everything else is just calorie free flavoring.
Optional Flavors:
- 1/4 tsp. (or to taste) Stevia Powder
- 3 tbsp. cocao powder
- 4 tbsp. PB2 Powdered Peanut Butter
- 2 tsp. vanilla powder (I get mine through THRIVE Life)
Once everything's blended together just put a lid on it and store in the fridge. The cool temperature will help your protein powder keep longer – no preservatives needed, thank you very much! Just take it out and scoop a 1/3 cup into your smoothie in the morning and return to the fridge. If you religiously have a smoothie every single morning, this one batch will last you just over a week.


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Keyano Mango Moisture Mask
Product Overview
Keyano Aromatics Keyano Mango Moisture Mask
Keyano Mango Moisture Mask
A luxurious anti-aging treatment for both the mind and body. This Moisture Mask features mangoes which provide enhancing skin protection due to a significant amount of polyphenols and tocotrienols found in the fruit.
Water (aqua), Glycerin, Montmorillite (natural clay), Caprylic/Capric Triglyceride, Cetearyl Alcohol, Stearic Acid, Green Bean Coffee Extract, Grape Seed Oil (Vitis Vinifera), Jojoba Oil (Buxus Chinensis), Argan Oil, Allatoin, KL-Panthenol (Vitamin B-5), Olive Oil PEG-7 Esters, DMDM Hydantoin, Iodopropynyl Butylcarbamate, Zanthan Gum, Fragrance (Parfum).
Place Your Order Here
Keyano Aromatics Keyano Mango Moisture Mask
Keyano Mango Moisture Mask Reviews Summary
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Part Number(s): 182 5029 10, 182 5029 05
Mfg Part Number(s): 172, 173
Scrip Part Number(s): 182 5029 05, 182 5029 10
Because our price on this item is lower than the manufacturer's suggested retail price, the manufacturer does not allow us to show you our price until you place the item in your shopping cart. Retailers like Allegro Medical have the legal right to sell products at prices we set. However, adding the item to your cart allows us to show you our lower price consistent with our promise of always offering you the lowest possible prices on the widest selection of medical supplies, medical equipment and life enhancing products.
Adding this item to your shopping cart won't require you to buy it. However, our prices do change. If you decide to purchase this item now you are guaranteed the price now displayed in your shopping cart. Complete your purchase by clicking on the "checkout" button or simply remove the product from your cart if you decide not to make your purchase at this time.


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Express Epping Forest pest control offer an insect control service that is second to none. Epping Forest insects such as house fleas or bed bugs need urgent attention as these types of insects will multiply at an alarming rate. For any Epping Forest insect infestation such as ants, silverfish and cockroaches we can have someone to your door that same day to eliminate the problem and provide preventative measures.
For the health and safety of those near the affected area it is imperative that only a trained local Epping Forest engineer apply industrial pesticides and insecticides. For any Epping Forest insect problem you can rely on us for a safe and efficient service.
Want to speak to an expert?
Call: 0800 051 8640
Bed Bugs
Adults are 5mm long, reddish brown in colour with well developed antennae. The primary hosts are mammals, from cats and dogs to humans. Any household can be invaded by bed bugs, and as they cannot fly, they must either crawl or be passively transported in clothing, luggage, furniture or any other object that can be used as a harbourage. Even pets can unknowingly transport them.
Possible sources may be from areas of high population density such as hostels, hotels and holiday camps. These choose to make their home in warm areas, typically in bed linen, mattresses, and even woodwork near beds, although they can infest sofas and other items of upholstery.
Like fleas, they feed on blood, although most species only feed on humans when there is no other food available. The treatment of choice is a professional-grade water-based insecticide that has been specially selected for its safety profile and mode of action, that only a trained and licensed specialist is authorized to administer.


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Electric Keyboard - We are very excited to present our latest keyboard theme: ELECTRIC KEYBOARD! DOWNLOAD Electric Keyboard NOW and make your smartphone more energetic with these dark blue graphics and incredible background that will make you smile every time you need to use your phone!
- For installation, follow the 3 easy steps: open after download, press "Set as Active Theme" and select the theme from the following page!
- The incredible HD screenshots we prepared will convince you this is a must download theme!
- This theme uses GO Keyboard, so if you do not have it installed, you will be redirected to a download page!
- For your comfort, the menus for this theme come with 48 different language translations!
If you enjoyed this theme, take the time to RATE and COMMENT after you install! Also, contact us at email@example.com with any suggestions or questions you have and keep visiting to check out all our latest surprises!
This theme uses a free font which can be found at http://webneel.com/daily/download/7928/1/Lovelo%20font?title=Download%2026%20Free%20Professional%20Fonts%20for%20Designers&nid=8685 and brushes from http://www.brusheezy.com/brushes/1599-aloud-volumetric-brush-set


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- Please select a date to see the proper prices!
- Meal can be requested on the spot
Best offer
76 € / person / night, no meal
Description of the accommodation:
Rooms, services 30 comfortably furnished rooms are offered by Hotel Burgfrieden Gais to guests looking for accommodation in Gais. If necessary, parking is available nearby. Nearby table tennis and bike rental opportunities may provide an excellent option for those who love to spend their leisure time actively. For meetings and nice evening chats the local bar will provide an ideal location. In the safe you can secure your valuables. In case you'd bring your laptop along, you may go online via wireless Internet connection (WiFi) available at the hotel. If you wish to learn more about the exact scope of services provided by Hotel Burgfrieden Gais (including any available for an extra fee), you will find them on the information sheet below.
Payment Depending on the selected date, room and price, you may need to provide credit card guarantee or advance bank card payment to finalize your reservation.
Arrival, opening hours The earliest check-in time is 14:00, the latest check-out time is 10:00. The personnel is at your service in Italian and English languages.
Hotel Burgfrieden Gais rooms Rooms
- Please select a date to see the proper prices!
Accommodation services
Free WiFi in shared places
Table-tennis, Bike rental at the accommodation
- Finnish sauna
- Pool (Outdoor - seasonally open)
Garden, Bar, Safe deposit box
Hairdryer, Desk in the room


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Dami Duro crooner Davido, and singer Chidinma Ekile have done Nigeria proud at the 2012 Kora awards.
While Davido emerged as the 2012 Best male newcomer, Chidinma, the Kedike crooner won 2012 Best Female West African Act.
The Kora Awards, dubbed "Africa's Grammys", took place Sunday in Abidjan.
American Singer Chris Brown also graced the event.
Source: Entertainment.naija.ng


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Product Description
Telescope Carrying Case for Celestron 14" OTA
This case is for the Optical Tube Assembly (OTA) ONLY. It includes two lockable latches, three steel-reinforced handles, a 2" by 30" steel hinge and die-cut foam lining for the telescope optical tube. It can be carried by two people using the two handles on either end or pulled using the attached Kryptane® wheels. The finder scope, diagonal and 2" extension must be detached and carried separately. The wheels must be attached to the case by the customer which is easily accomplished in a few seconds with two 7/16" wrenches. An optional Large-Wheel Upgrade is available.
Outside dimensions of the case are approximately 36-1/4" x 21-1/4" x 18-3/8" and its empty weight is approximately 20.2 lbs. The shipping box is approximately 26-1/4" x 19" x 38-1/2" (FedEx Ground dimensional weight is at 116 lbs) and the shipping weight is approximately 29.0 lbs. All values are subject to slight changes.
|JMI - Multi-use 14" OTA Case
|Jims Mobile, Inc. (JMI)


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events | photo exhibit of SUE ANNE TAY's "shanghai street stories"
The street photography of Shanghai-based documentary photographer Sue Anne Tay will be on display at an upcoming exhibition titled Demolish, Construct, Repeat: Building the Shanghai Dream. Sue Anne's show is focused on the city's disappearing neighborhoods, contrasting the migrant workers who are building the new Shanghai with the locals who are forced to adapt to the fast-paced changes.
The exhibit will run for a month and will launch this coming Saturday, September 11th. The launch party will be from 3 – 6pm. Venue: Southern Barbarian (Lujiazui) 南蛮子B1/F, DBS Building, 1318 Lujiazui Huan Lu, near Dongyuan Lu (陆家嘴环路1318号B1楼, 近东园路).
We've shared a selection of Sue Anne Tay's photography work below. If you're in or around Shanghai, make sure you don't miss the opening reception – free Yunnan snacks with beer and wine!
For more photography on NeochaEDGE, link here. /// Jellyfish
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[…] I've been interviewed by the local magazines including That's Shanghai, City Weekend, NeochaEDGE (more to […]


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The Bow
Release Date: 12 February 2007 | Certificate: Suitable for 18 years and over
£13.79 includes FREE UK Postage (other delivery options available)
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UK - Shipping Included
United Kingdom
Europe Zone 1 - £1.29
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Falkland Islands
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On a fishing boat out at sea, a 60 year old man has raised a young girl since she was a just a baby. Living a sheltered life, they earn a living by renting their boat out to local fisherman and spend their time performing divination ceremonies. As the girl approaches her seventeenth birthday, the age she has agreed to marry her companion, her life is thrown into chaos with the arrival of a young man who quickly falls for her. Captivating and beautifully shot, The Bow is another high class offering from a prolific yet hugely eclectic director.
Trailer, Film notes, Scene selection
Release Date: 12 February 2007
Certificate: 18 - Suitable for 18 years and over. Not for sale to persons under age 18, by placing an order for this product you are declaring that you are 18 years or over.
Region 2
Main Language: English
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Catalogue No: TVD3712


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The Levis JCC presents the Inaugural Sprint and Sip 5K on Thursday, March 1 @ 7:00 PM. This event will take place at South County Regional Park in Boca Raton, Florida.
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The Coral Springs 911 Remembrance 5K Run will take place on Saturday, November 18 @ 7:00 AM. (Rescheduled Date due to Hurricane Irma). This event will take place at the NorthWest Regional Library • 3151 University Dr Coral Springs, FL 33065.
The Wounded Veteran Relief Fund presents the Running for Heroes 5K on Saturday, November 18 @ 7:45 AM. This event will take place at Riverbend Park in Jupiter, Florida.
Friends of Foster Children presents the SuperHero 5K Run / Walk on Saturday (late afternoon), November 18 @ 5:00 PM. This event will take place John Prince Park in Lake Worth, Florida.
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Accounts Receivable Manager ( 1 - 2 yrs)
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An Accounts Receivable Manager forms part of the financial accounting department and coordinates the daily operations of a company's accounts receivable cycle through implementation, design, dunning and all business functions and procedures. Gener...
Key Skills : banking assistant,finacial manager,english editor,bank operations officer,management trainee,specialist radiologist,accounts receivable manager,...
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Identify 5 methods of acknowledgment that also introduce possibilities.
Discuss 3 methods of inclusion.
List 5 therapeutic biases or habits that interfere with clinical results.
Describe when a therapist should clarify a difficult client's goals.
Session 1:
Acknowledgment and possibility
The two legs of successful therapy
Five methods of acknowledging and validating while opening possibilities: spinning problems into the past, going un-global, shifting from reality claims into perceptions, shifting problems into preferences, creating positive expectancy and hope
Session 2:
Inclusive therapy
Three levels of inclusion: permission, inclusion of seeming opposites, exceptions
Methods of inclusion: tag questions, saying the 'no' for the client, oxymoron, finding the value in the problem or the resistance, revaluing the devalued, disowned and dissociated, self-compassion and self-forgiveness
Session 3:
The resistant therapist
The evil empire: what you as a therapist have devalued and resisted
Delusions of certainty and how to recover from them
The definition of insanity
Embracing and learning from clinical failures
Transcending biases and habits and developing flexibility
When to stop being flexible and take a stand or give up
Session 4:
Unpacking problems
Co-creating achievable goals
Case consultations
Bill O'Hanlon, MS, LMFT,
has authored or co-authored 31 books, the latest being
Quick Steps to Resolving Trauma
(W.W. Norton, 2010). He has published 59 articles or book chapters. His books have been translated into 16 languages. He has appeared on
(with his book
Do One Thing Different
The Today Show,
and a variety of other television and radio programs.
For the past 30+ years, Bill has given over 2,000 talks around the world. He has been a top-rated presenter at many national conferences and was awarded the Outstanding Mental Health Educator of the Year in 2001 by the New England Educational Institute. He is a Licensed Mental Health Professional, Certified Professional Counselor, and a Licensed Marriage and Family Therapist. Bill is clinical member of AAMFT (and winner of the 2003 New Mexico AMFT Distinguished Service Award), certified by the National Board of Certified Clinical Hypnotherapists and a Fellow and a Board Member of the American Psychotherapy Association. Bill is known for his storytelling, irreverent humor, clear and accessible style and his boundless enthusiasm for whatever he is doing. His seminars are as entertaining as they are educational.
Continuing Education Credits
Price includes one CE Certificate. Additional certificates are available for an additional fee; please contact our Customer Service at 1-800-844-8260 for more details.
Listed below are the continuing education credit(s) currently available for this self-study package. Please note, your state licensing board dictates whether self study is an acceptable form of continuing education. Please refer to your state rules and regulations.
**Materials that are included in this course may include interventions and modalities that are beyond the authorized practice of mental health professionals. As a licensed professional, you are responsible for reviewing the scope of practice, including activities that are defined in law as beyond the boundaries of practice in accordance with and in compliance with your professions standards.
This intermediate self-study activity consists of 6.0 clock hours of continuing education instruction. Credit requirements and approvals vary per state board regulations. Please save the course outline, the certificate of completion you receive from the activity and contact your state board or organization to determine specific filing requirements.
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This self-study activity consists of 6.0 clock hours of continuing education instruction. Credit requirements and approvals vary per state board regulations. Please save the course outline, the certificate of completion you receive from this self-study activity and contact your state board or organization to determine specific filing requirements.
PESI, Inc. is approved by the American Psychological Association to sponsor continuing education for psychologists. PESI, Inc. maintains responsibility for this program and its content. PESI, Inc. is offering these self-study materials for 6.0 hours of continuing education credit.
Social Workers
PESI, Inc., #1062, is approved as a provider for social work continuing education by the Association of Social Work Boards (ASWB), www.aswb.org through the Approved Continuing Education (ACE) Program. PESI, Inc. maintains responsibility for the program. ASWB Approval Period: January 27, 2017 - January 27, 2020. Licensed Social Workers should contact their regulatory board to determine course approval. Social Workers will receive 6.0 (Clinical) continuing education clock hours for completing this self-study package. Course Level: Intermediate.
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This self-study activity qualifies for 6.0 clock hours of instructional content as required by many national, state and local licensing boards and professional organizations. Retain your certificate of completion and contact your board or organization for specific filing requirements.
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**It is your ethical responsibility to report accurate hours to your licensing board.
**All self-study participants must complete and pass (80% or better) a post-test/evaluation prior to receiving a certificate of completion. If you require a copy of the test/evaluation for CE purposes, please print at the time you take the test. Or you may call our customer service department and a copy of your test/evaluation will be emailed to you. Please allow 30-45 days.
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Your satisfaction is our goal and our guarantee. Concerns should be addressed to Psychotherapy Networker, 5135 MacArthur Boulevard N.W., Washington, D.C. 20016 or call (888) 851-9498.
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Traveling Luck for Wingfield Shopping Center, Mississippi, United States
Where is Wingfield Shopping Center?What's around Wingfield Shopping Center?
Wikipedia near Wingfield Shopping Center
Where to stay near Wingfield Shopping Center
The timezone in Wingfield Shopping Center is America/Rankin_Inlet
Sunrise at 06:38 and Sunset at 16:57. It's light
Latitude. 32.2833°, Longitude. -90.2350° , Elevation. 112m
Weather near Wingfield Shopping Center; Report from Jackson, Hawkins Field Airport, MS 8.5km away
Weather :
Temperature: 16°C / 61°F
Wind: 4.6km/h South
Cloud: Sky Clear
Weather :
Temperature: 16°C / 61°F
Wind: 4.6km/h South
Cloud: Sky Clear
Satellite map around Wingfield Shopping Center
Loading map of Wingfield Shopping Center and it's surroudings
Geographic features & Photographs around Wingfield Shopping Center, in Mississippi, United States
- church;
- a building for public Christian worship.
- Southside Baptist Church (0.5km)
- Southside Assembly of God Church (1.2km)
- First Independent Methodist Church (1.2km)
- Epworth United Methodist Church (2.1km)
- McDowell Road Baptist Church (2.1km)
- First Cumberland Presbyterian Church (2.1km)
- Fairfield Missionary Baptist Church (2.4km)
- Free Mission Missionary Baptist Church (2.4km)
- Westview Baptist Church (2.4km)
- Alta Woods Baptist Church (2.5km)
- Mount Nebo Baptist Church (2.5km)
- Alta Woods United Methodist Church (2.6km)
- Alta Woods Presbyterian Church (2.6km)
- Christ the King Catholic Church (2.7km)
- Greater Gethsemane Missionary Baptist Church (2.7km)
- Lynch Street Christian Methodist Episcopal Church (2.9km)
- Oak Forest Baptist Church (2.9km)
- United Christian Church (3km)
- Kingdom of God in Christ Church (3km)
- Greater Allen Temple African Methodist Episcopal Church (3.1km)
- Foursquare Church (3.1km)
- Robinson Street Baptist Church (3.1km)
- Sweet Rock Missionary Baptist Church (3.2km)
- Saint Paul Church (3.2km)
- Smith Chapel Freewill Baptist Church (3.2km)
- Local Feature;
- A Nearby feature worthy of being marked on a map..
- Wingfield Shopping Center (0km)
- First Church of the Nazarene (0.1km)
- Ellis Isle Shopping Center (0.6km)
- Interchange 42 (1.1km)
- Southport Mall Shopping Center (1.6km)
- Ellis Avenue Plaza Shopping Center (1.8km)
- Tappleridge Shopping Center (2km)
- Bel Aire Shopping Center (2.1km)
- Interchange 41 (2.1km)
- Carmelite Monastery (2.6km)
- Belvedere Church of Christ (2.7km)
- Interchange 43 (2.8km)
- Interchange 1 (2.8km)
- Greenbriar Shopping Center (3km)
- Southgate Shopping Center (3km)
- Metrocenter Mall Shopping Center (3.1km)
- Jackson Square Shopping Center (3.1km)
- school;
- building(s) where instruction in one or more branches of knowledge takes place.
- Iola Tapley Wilkins Elementary School (0.6km)
- Wingfield High School (0.9km)
- Hill High School (2.2km)
- Isable School (2.3km)
- Laura R Lester Elementary School (2.4km)
- Key Elementary School (2.5km)
- Clausell School (2.5km)
- Christ the King Elementary School (2.6km)
- Southwest Academy (2.6km)
- Saint Theresa School (2.8km)
- Marshall Elementary School (3.2km)
- Hillcrest School (3.2km)
- section of populated place;
- a neighborhood or part of a larger town or city.
- Garden Park (subdivision) (0.5km)
- Wingfield Place (subdivision) (1.4km)
- cemetery;
- a burial place or ground.
- Willow Park Cemetery (2.2km)
- Lincoln Cemetery (3.2km)
Accommodation around Wingfield Shopping Center
- populated place;
- a city, town, village, or other agglomeration of buildings where people live and work.
- Bradie (2.2km)
- administrative division;
- an administrative division of a country, undifferentiated as to administrative level.
- District 5 Supervisor District (2.7km)
- hospital;
- a building in which sick or injured, especially those confined to bed, are medically treated.
- Hinds General Hospital (2.4km)
Wikipedia entries close to
Wingfield Shopping Center
- Jackson (Mississippi) city city e
- Mississippi State Capitol landmark government
- Mississippi Veterans Memorial Stadium landmark sport
- Richland, Mississippi city city o
- LeFleur's Bluff State Park park
- Clinton, Mississippi city city o
- Byram, Mississippi city city o
- Flowood, Mississippi city city o
- Pearl, Mississippi city city o
- Jackson-Evers International Airport airport airport
- WLBT-Sendemast landmark tower
- WLBT Tower
- Raymond, Mississippi city city o
- Florence, Mississippi city city o
- Ridgeland, Mississippi city city o
- Mississippi Authority for Educational Television Tower landmark landmark
Airports close to Wingfield Shopping Center
Jackson international(JAN), Jackson, Usa (19.8km)
Greenwood leflore(GWO), Greenwood, Usa (173.8km)


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Promise Me - Harlan Coben
The 2am phone call is from a young, frightened, lost girl who has called the one adult who had promised to help her if she ever got into any trouble -...
5 reviews from the community
Review of "Promise Me - Harlan Coben"
Just a flying visit...hope all is well with all my Ciao buddies.
"Promises Promises"
One simple promise was all it took. Overhearing the kids talking about the drinking, and the driving home afterwards, Myron Bolitar remembered Debbie Frankl. Debbie was the bright young thing he'd been at school with who didn't get away with it. She was one of the unlucky ones that piled into a car after a party… and never made it home.He wanted to tell Erin and Aimee that story, but he knew they wouldn't listen. Instead he made them promise. If they ever needed a lift, from anywhere, at any time, they should call him. He would come, and he wouldn't tell, if only they would promise.
So when Aimee Biel phoned at 2.17a.m. in the morning – of course he went.He drives down-town and picks her up from a lonely street corner. Then she reminds him the promise was a two-way thing. He cannot tell. Rather than going home, she directs him to her friend's house out in the suburbs. She waves from the back gate.
And disappears.It's only a matter of time before the police connect Bolitar with Aimee's disappearance. He is a friend of the family, and freely admits being there to pick her up. Unfortunately for him, just before he did so Aimee had taken a substantial amount of money out of an ATM: the very same ATM that was the last sighting of Katie Rochester.
Katie is over eighteen, missing a couple of months, there's been no ransom demand. So far as the police are concerned, she's a runaway adult and well within her rights to disappear. When Aimee goes missing from the exact same spot, they start to think again. Unfortunately this information reaches the ears of Katie's father, who is not a very nice person – and knows some even worse ones.Bolitar, ex-basketball star, ex-lawyer, current Sports & Celebrity Agent is up to his neck in the proverbial. He can hold his own in a fight, but he's no superhero. Fortunately, on his side he has the legendary Winsor Horne Lockwood III. Win decided long ago to use his privilege to become the best hero, or anti-hero, that money could train him to be. He fights, mean and dirty: if he has to fight, he means to win, and he has the contacts, the training and the skill to be sure that he does.
Regular readers of Harlan Coben will know his style. For those who don't, he is probably the best modern exponent of the hook-and-twist school of crime-writing. He grabs the reader immediately, and quite simply doesn't let go. He doesn't build tension to allow it to dissipate. He builds it to form the foundation for the next turn of the plot, which heads off down an unexpected avenue, which might be towards the denouement or just another cul-de-sac to be escaped.Tightly plotted as his novels always are, Promise Me sees the return of Bolitar after a gap of six years, with a major role for Win and walk-on parts for the rest of the crew: Esperanza, Big Cyndi, El-Al… It might be part of a series (number 8, out of 9 to date) but works perfectly well on its own for those of us not familiar with the previous episodes. Despite his characters, Coben manages to stay the right side of camp and never descends into farce, but the chosen set-up does allow for more dark humour than you'll find in any of the one-off stories.
For me it isn't Coben's best, some of the clues are too easy too soon, and I actually prefer him when he's not trying to be funny, but he is so far ahead of the current pack in the genre that it's hard to drop him a point.A good fast pace keeps the pages turning: just the thing for those occasions when you get the chance to read the whole book at a sitting.
~Published in paperback by Orion
pp 452
Cover price £6.99
~© Lesley Mason
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Comments on this review
teacherofhooch published 10/07/2010I remember reading his first book -Can't remember what it was called. Then another since then not read any although have picked them up - may try again sounds interesting.
Coloneljohn published 25/04/2010Sounds good, not an author I am familiar with though. Perhaps it is time to broaden out my reading again. John
koshkha published 17/04/2010I really don't know why I've never read any of his books.
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Product Information : Promise Me - Harlan Coben
Manufacturer's product descriptionThe 2am phone call is from a young, frightened, lost girl who has called the one adult who had promised to help her if she ever got into any trouble - she has turned to Myron Bolitar. But Myron's help proves not to be enough.
Product Details
Type: Fiction
Genre: Thriller
Title: Promise Me
Author: Harlan Coben
Publisher: Orion
All Authors: Harlan Coben
Listed on Ciao since: 06/01/2007


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Don't miss out on a lower price on this hotel
Get an overview of this hotelHotel in Lincoln with free breakfast and restaurant
Collecting Nectar Points
Simply enter your Nectar card number when you check out. Your points will be credited 35 days after you return from your trip.
*Terms and conditions apply.
Popular property highlights
Situated in Lincoln, this hotel is 2.6 mi (4.2 km) from Haymarket Park and within 6 mi (10 km) of University of Nebraska-Lincoln and Memorial Stadium. Nebraska State Capitol and Lied Center are also within 6 mi (10 km).
Hotel Features
A restaurant, a fitness center, and a bar/lounge are available at this hotel. Free continental breakfast, free WiFi in public areas, and free self parking are also provided. Other amenities include a spa tub, a business center, and a meeting room.
Room Amenities
All 103 rooms offer free WiFi, coffee makers, and TVs with cable channels. Free local calls, ironing boards, and desks are among the other amenities that guests will find.Information missing or incorrect? Tell us!Opens in a new window
Travelodge Lincoln
Hotel Amenities
Hotel Amenities
Travelodge Lincoln features a spa tub and a fitness center. The hotel offers a restaurant. A bar/lounge is on site where guests can unwind with a drink. A complimentary breakfast is offered. Wireless Internet access is complimentary.
This hotel offers access to a business center and a meeting room. Event facilities measure 400 square feet (37 square meters). This business-friendly hotel also offers express check-in, express check-out, and ATM/banking services. Onsite self parking is complimentary.
- Conference space size (meters) - 37
- Express check-in
- Bar/lounge
- ATM/banking
- Fitness facilities
- Conference space size (feet) - 400
- 24-hour front desk
- Business center
- Free breakfast
- Express check-out
- Restaurant
- Spa tub
- Total number of rooms - 103
- One meeting room
- Free self parking
- Number of floors - 2
- Free WiFi
- RV, bus, truck parking
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi
Free self parking, RV, bus, truck parking
Room Amenities
- Air conditioning
- Free local calls
- Coffee/tea maker
- Daily housekeeping
- Private bathroom
- Free toiletries
- Shower/tub combination
- Iron/ironing board
- Television
- Desk
- Cable TV service
- Free WiFi
- Connecting/adjoining rooms available
Where to Eat
Guests are offered a complimentary continental breakfast.
Travelodge Lincoln has a restaurant on site.
Nearby Things to Do
Recreational amenities at the hotel include a spa tub and a fitness center.
If you have requests for specific accessibility needs, please note them at check-out when you book your room.
Hotel Policies
Check-in time starts at 3 PM
Check-in time ends at 6 AM
Check-out time is 11 AM
Payment types
Children and extra beds
- Children are welcome.
- Rollaway/extra beds are not available.
- Cribs (infant beds) are not available.
- Pets allowed for an extra charge of USD 15.00 per pet, per day
- Specific rooms only, restrictions apply
- Pet-friendly rooms can be requested by contacting the property at the number on the booking confirmation
You need to know
Extra-person charges may apply and vary depending on property policy.
Optional extras
- Pet fee: USD 15.00 per pet, per day
Hotel Name
- Lincoln Travelodge
- Travelodge Hotel Lincoln
- Travelodge Lincoln
- Lincoln Travel Lodge
- Travel Lodge Lincoln
- Travelodge Lincoln Thorpe On The Hill Hotel Thorpe On The Hill
- Travelodge Lincoln Hotel
We should mention
The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation. The property allows pets in specific rooms only and has other pet restrictions (surcharges apply and can be found in the Fees section). Guests can arrange to bring pets by contacting the property directly, using the contact information on the booking confirmation.