P21 Waf1 Research Articles

Carnosine inhibits KRAS-mediated HCT116 proliferation by affecting ATP and ROS production

Carnosine is a natural dipeptide that has generated particular interest for its antioxidant, anti-aging and especially for its antiproliferative properties. In this study, we demonstrate that carnosine inhibits the proliferation of human HCT116 colon cancer cells. In this cell line, the activating KRAS mutation induces mitochondrial ROS, the signaling molecules for cell proliferation. We observed that 50–100 mM carnosine decreases ATP and ROS concentration and induces cell cycle arrest in G1 phase. In HCT116 cells these effects are related to decreased ERK1/2 phosphorylation and increased p21 waf1 protein. Our findings support the concept that carnosine could inhibit HCT116 cell growth via its antioxidant activity and its ability to affect glycolysis.

Human papillomavirus type 16 E6 inhibits p21 WAF1 transcription independently of p53 by inactivating p150 Sal2

HPV16 E6 deregulates G1/S cell cycle progression through p53 degradation preventing transcription of the CDK inhibitor p21 WAF1. However, additional mechanisms independent of p53 inactivation appear to exist. Here, we report that HPV16 E6 targets the cellular factor p150 Sal2, which positively regulates p21 WAF1 transcription. HPV16 E6 associates with p150 Sal2, inducing its functional inhibition by preventing its binding to cis elements on the p21 WAF1 promoter. A HPV16 E6 mutant, L110Q, which was unable to bind p150 Sal2, did not affect the ability of the cellular protein to bind p21 WAF1 promoter, underlining the linkage between these events. These data describe a novel mechanism by which HPV16 E6 induces cell cycle deregulation with a p53-independent pathway. The viral oncoprotein targets p150 Sal2, a positive transcription regulator of p21 WAF1 gene, preventing G1/S arrest and allowing cellular proliferation and efficient viral DNA replication.

The expression research of histone acetylation and p21WAF1 in breast cancer

Objective To research the expression change and the sense of histone acetylation and p21WAF1 protein in breast cancer. Methods Pathological morphologic changes of breast cancer were identified by H. E. staining. Immunohistochemical study for p21WAF1 protein expression was performed on 80 breast cancers and 80 normal breast tissues. The expressions of acetylated histone H3, H4 and p21WAF1 protein in 80 breast cancers and 80 normal breast tissues were analyzed by Western blot. Results H.E. staining discovered that the tissue structure and cell morphous of breast cancer had visible atypia, compared with normal breast tissues. The immunohistochemical results displayed that the positive expression of p21 WAF1 in 80 breast cancers and 80 normal breast tissues was 49 cases(61.25 %) or 3 cases (3.75%), respectively. There was significantly difference in the expression level between the 80 breast cancers and 80 normal breast tissues (P ＜ 0. 05). The expression levels of p21WAF1 protein in breast cancer tissues were higher than that in normal breast tissues, 0. 78 ± 0. 095 or 0. 65 ± 0. 055, respectively. The expression levels of acetylated histone H3, H4 protein in normal breast tissues were higher than those in breast cancer tissues. H3 was 2. 35 ± 0. 340 or 1.07 ± 0. 067, respectively, H4 was 3.44 ± 0. 202 or 1.11 ± 0. 086, respectively. There was significantly difference in the expression levels between the 80 breast cancers and 80 normal breast tissues (P ＜0. 01). Conclusions The occurrence and development of breast cancers may be related to the expression change of histone acetylation and p21WAF1 protein. Key words: Breast neoplasms/ME/PA; Histones/BI; Proto-oncogene proteins p21 (ras)/BI; Acetylation

Suppression of Polo like kinase 1 (PLK1) by p21 Waf1 mediates the p53-dependent prevention of caspase-independent mitotic death

Polo-like kinase 1 (Plk1) plays key roles in many aspects of mitosis. We have previously shown that induction of p21 Waf1 by p53 is responsible for protection of cells against adriamycin-induced polyploidy formation and mitotic catastrophe. Here we show that adriamycin treatment suppressed Plk1 expression in a p53- and p21 Waf1-dependent manner. Ablation of p21 Waf1 inhibited the adriamycin-induced p53 activation, and this inhibition was alleviated by knockdown of Plk1, suggesting that p21 Waf1-dependent suppression of Plk1 expression is responsible for maintaining p53 activation during stress response. Plk1 associated with p53 and disrupted its interaction with target gene promoters in cells treated with adriamycin. Overexpression of Plk1 inhibited the p53-mediated prevention of caspase-independent mitotic death, but not polyploidy formation, in adriamycin-treated cells. Together our results indicate that suppression of Plk1 by p21 Waf1 is responsible for p53-dependent protection against adriamycin-induced caspase-independent mitotic death.

Pro-apoptotic and anticancer properties of Thapring – A Tibetan herbal formulation

To evaluate the pro-apoptotic and anti-tumorigenic properties of Thapring - a Traditional Tibetan Medicine - in hepatoma cells and in a transgenic mouse model of hepatocellular carcinoma. The pro-apoptotic action and growth inhibition property of Thapring were assessed in Huh7, HepG2 and A549 cell lines using flow cytometry and MTT assay, respectively. Confocal microscopy for colocalization of cytochrome c and mitochondria was done using dsRed mitotracker in Huh7 cells. The activation of p38 MAP kinase and p53 pathway was evaluated by Western blotting. Serological studies for liver function, vascular endothelial growth factor and superoxide dismutase were assessed in the serum of X15-myc transgenic mice. Immuno-histochemical studies for Bcl2 and p21(Waf1) expression were also carried out in the liver section of the above mice. Treatment with Thapring inhibited proliferation and accumulation of hepatoma cells in G1 phase. There was increased cytochrome c release from mitochondria and decreased Bcl2 levels - the key markers of apoptotic cell death. Besides activation of p38 MAP kinase and increased p53 expression were also observed. Oral administration of Thapring in transgenic mice lowered serum VEGF levels and conferred hepatoprotection as evident from normal serum ALT levels. Further, immunohistochemical analysis of the liver samples revealed reduced expression of anti-apoptotic protein Bcl2 and over-expression of cell cycle regulator p21(Waf1). The ability of Thapring to impose growth arrest and trigger pro-apoptotic death in cell culture as well as ameliorative effects in vivo provides scientific basis for its usefulness as traditional medicine and its clinical application in adjunct/combination therapy along with other known anticancer drugs.

Zinc induces cell cycle arrest and apoptosis by upregulation of WIG-1 in esophageal squamous cancer cell line EC109

Zinc deficiency was implicated in the etiologies of human esophageal squamous cell carcinoma (ESCC). Wild-type p53-induced gene 1 (WIG-1), a kind of zinc finger protein, was cloned from the human 3q26.3 region and encoded a putative polypeptide of 289 amino acids. Our previous studies have demonstrated that the expression of WIG-1 was downregulated in ESCC tissues. Herein, we investigated the effect of zinc on cell proliferation, apoptosis, as well as expression of WIG-1 in EC109 cells. Meanwhile, an RNAi vector of WIG-1 was transfected into EC109 cells and the effect of zinc on WIG-1 expression was investigated. We found that zinc could suppress cell proliferation and induce G0/G1 cell cycle arrest and apoptosis of EC109, and this efficacy might result from the expression altering of several apoptosis-related genes, such as Bax, p21 ( WAF ), and cyclin D1. In particular, upregulation of WIG-1 was observed after zinc supplementation, indicating that WIG-1 might be involved in the zinc-induced cell cycle arrest and apoptosis of EC109 cells by regulating the expression of Bax, p21 ( WAF ), and cyclin D1.

Histone deacetylase inhibitor augments anti-tumor effect of gemcitabine and pegylated interferon-α on pancreatic cancer cells

Histone deacetylase (HDAC) is strongly associated with epigenetic regulation and carcinogenesis, and its inhibitor can induce the differentiation or apoptosis of cancer cells. We investigated the anticancer effects of the HDAC inhibitor valproic acid (VPA) in combination with gemcitabine (GEM), an antimetabolic, and pegylated interferon-α2b (PEG-IFN-α2b) in a human pancreatic cancer cell line using a cell proliferation assay. The gene expressions of HDAC1, MTA1, p21(Waf1), and HIF-1 were evaluated by reverse transcription-PCR. Valproic acid at 0.5 mM when used alone did not suppress cell proliferation. PEG-IFN-α2b at 10(2 )E/ml weakly suppressed cell proliferation in both the BxPC3 (by 28%) and SUIT-2 (by 17%) human pancreatic cancer cell lines. GEM at 5 nM when used alone suppressed cell proliferation by 36 and 61% in the BxPC3 and SUIT-2 cell lines, respectively. The combination treatment of GEM + PEG-IFN-α2b strongly suppressed cell proliferation in the SUIT-2 (82%) and BxPC3 (51%) cell lines, which was further reinforced by the addition of VPA up to 88 and 67%, respectively. The combination treatment of GEM + PEG-IFN-α2b enhanced the expression of p21(Waf1), which was also reinforced by VPA. VPA augmented the inhibitory effects of PEG-IFN-α2b alone or in combination with PEG-IFN-α2b and GEM on cell proliferation. Such inhibitory effects may be due to the up-regulation of p21(Waf1) expression.

P21 Waf1 is required for complete oncogenic transformation of mouse embryo fibroblasts by E1Aad5 and c-Ha-ras oncogenes

Cyclin-dependent kinase inhibitor p21 Waf1 is known to have alternative functions associated with positive regulation of proliferation, actin cytoskeleton remodeling and suppression of apoptosis. The goal of the present study was to assess the role of p21 Waf1 in the establishment of the transformed phenotype of mouse embryo fibroblasts with stable expression of E1Aad5 and c-Ha-ras complementary oncogenes. Herein, we demonstrate that E1A/c-Ha-Ras-transformed p21 Waf1-null fibroblasts possess some characteristic features of transformed cells, such as loss of contact inhibition, high saturation density, shortened cell cycle, inability to undergo cell-cycle arrest after DNA damage and serum deprivation, but, at the same time, they are not completely transformed in that they are unable to proliferate at clonal density, are anchorage-dependent, retain a fibroblast-like morphology with pronounced actin cytoskeleton and show reduced migration and invasion. Our data support the concept of p21 Waf1 “tumor suppressor” having alternative oncogenic functions in the cytoplasm and for the first time indicate that p21 Waf1 can be indispensable for complete oncogenic transformation.

Abstract 4470: Kevetrin™, a novel small molecule, activates p53, enhances expression of p21, induces cell cycle arrest and apoptosis in a human cancer cell line

Abstract We have previously shown that Kevetrin, a small molecule currently under development, has potent antitumor activity in human multi-drug resistant carcinoma xenograft models while being well tolerated. To investigate the mechanism of action for its potent antitumor activity, we assessed Kevetrin's effect on apoptosis, cell cycle progression, including underlying molecular mechanisms. Here we report that Kevetrin strongly induced cell cycle arrest and apoptosis in a human lung adenocarcinoma cell line (A549) using the TUNEL assay and FACS analysis; similar results were observed using a human breast carcinoma cell line (MDA-MB-231). Treatment of A549 cells with 400 µM of Kevetrin for 48 hours resulted in G2/M phase cell cycle arrest that was associated with a marked decline in levels of G2/M regulatory proteins, including CDK1 and cdc25B, and increased expression of Wee1. Further, Kevetrin-mediated growth inhibition of A549 correlated with apoptosis induction that was characterized by cleavage of procaspase-3 and poly (ADP-ribose) polymerase (PARP). The p53 tumor suppressor is a well characterized transcription factor controlling cell growth and apoptosis during times of cellular stress. The tumor suppressor p53 inhibits tumor growth primarily through its ability to induce apoptosis. In cancer development, the resistance of cells to apoptosis is one of the crucial steps. The reactivation of p53 in tumor cells should trigger massive apoptosis and eliminate the tumor. Interestingly, Kevetrin treatment caused enhanced activated p53 levels in A549 cells. Activation of p53 can lead to cell cycle arrest and apoptosis. Western blotting revealed a concentration dependent increase in phosphorylation of p53 at serine 15. The phosphorylation of p53 at serine 15 leads to reduced interaction between p53 and its negative regulator, the oncoprotein MDM2. We also found that Kevetrin increased expression of p53 target genes such as p21 (Waf1). The tumor suppressor protein p21 (Waf1) acts as inhibitor of cell cycle progression. Finally, we also found that p53 induced apoptosis by inducing the expression of PUMA. Our study shows that Kevetrin activates p53 functions in tumor cells. Thus Kevetrin is a strong candidate as an anticancer drug that targets p53. Based on our studies we plan to initiate a Phase I clinical trial in 2011. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4470. doi:10.1158/1538-7445.AM2011-4470

PI3 Kinase inhibition on TRAIL-induced apoptosis correlates with androgen-sensitivity and p21 expression in prostate cancer cells

TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in many types of cancer cells. TRAIL is considered a therapeutic target, therefore, it was of interest to examine molecular mechanisms that may modulate sensitivity to TRAIL signaling in prostate cancer cells. LNCaP cells were found to be relatively resistant to TRAIL induced cell death while PC3 cells were sensitive. PI3-kinase (PI3K) inhibitors were able to render LNCaP cells sensitive to TRAIL but conferred resistance to PC3 cells. PI3K inhibitors were associated with an increase in p21(waf1, cip1) expression in PC3 cells where as p21 decreases in LNCaP cells suggesting that p21 may impart TRAIL resistance. Since androgen receptor (AR) signaling can be modulated by AKT, and p21 is an AR responsive gene, the impact of PI3K inhibition on TRAIL sensitivity was evaluated in AR transfected PC3 cells (PC3AR). The expression of AR was significantly downregulated by PI3K inhibition in LNCaP cells, which have an intact AR signaling axis. PC3AR cells expressed higher levels of p21 protein and were relatively resistant to TRAIL compared to control cells. Finally, using adenoviral p21 gene transfer we directly demonstrated that p21 can confer resistance to TRAIL-induced cell death. These results suggest that TRAIL resistance is not regulated simply by a PI3K/AKT survival pathway associated with inactivating PTEN mutations but may also be modulated by downstream AR responsive targets such as p21. These findings may have significant clinical implications for the utility of TRAIL in the management of prostate cancer.

RNA interference targeting raptor inhibits proliferation of gastric cancer cells

Mammalian target of rapamycin complex 1 (mTORC1) is dysregulated in gastric cancer. The biologic function of mTORC1 in gastric carcinogenesis is unclear. Here, we demonstrate that disruption of mTORC1 function by RNA interference-mediated downregulation of raptor substantially inhibited gastric cancer cell proliferation through induction of G 0/G 1-phase cell cycle arrest. The anti-proliferative effect was accompanied by concomitant downregulation of activator protein-1 and upregulation of Smad2/3 transcriptional activities. In addition, the expression of cyclin D 3 and p21 Waf1, which stabilizes cyclin D/cdk4 complex for G 1–S transition, was reduced by raptor knockdown. In conclusion, disruption of mTORC1 inhibits gastric cancer cell proliferation through multiple pathways. This discovery may have an implication in the application of mTORC1-directed therapy for the treatment of gastric cancer.

Knockdown of Cyclin-dependent Kinase Inhibitors Induces Cardiomyocyte Re-entry in the Cell Cycle

Proliferation of mammalian cardiomyocytes stops rapidly after birth and injured hearts do not regenerate adequately. High cyclin-dependent kinase inhibitor (CKI) levels have been observed in cardiomyocytes, but their role in maintaining cardiomyocytes in a post-mitotic state is still unknown. In this report, it was investigated whether CKI knockdown by RNA interference induced cardiomyocyte proliferation. We found that triple transfection with p21(Waf1), p27(Kip1), and p57(Kip2) siRNAs induced both neonatal and adult cardiomyocyte to enter S phase and increased the nuclei/cardiomyocyte ratio; furthermore, a subpopulation of cardiomyocytes progressed beyond karyokynesis, as assessed by the detection of mid-body structures and by straight cardiomyocyte counting. Intriguingly, cardiomyocyte proliferation occurred in the absence of overt DNA damage and aberrant mitotic figures. Finally, CKI knockdown and DNA synthesis reactivation correlated with a dramatic change in adult cardiomyocyte morphology that may be a prerequisite for cell division. In conclusion, CKI expression plays an active role in maintaining cardiomyocyte withdrawal from the cell cycle.

Inhibition of signal transducer and activator of transcription 5 by the inhibitor of janus kinases stimulates dormant human leukemia CD34+/CD38− cells and sensitizes them to antileukemia agents

To verify molecular mechanisms by which leukemia stem cells (LSCs) maintain a dormant state, we explored the activity of the major prosurvival signal pathways in CD34(+) /CD38(-) compartment, supposed to contain LSCs, and CD34(+) /CD38(+) counterparts from patients with acute myelogenous leukemia (AML, n = 11) by fluorescence-activated cell sorting (FACS). CD34(+) /CD38(-) cells expressed a greater amount of p-janus kinase 2 (JAK2) and p-signal transducer and activator of transcription 5 (STAT5) than CD34(+) /CD38(+) counterparts in all patients except for one case. In addition, we found that CD34(+) /CD38(-) cells were relatively resistant to cytarabine- and the inhibitor of the fms-like tyrosine kinase 3 (FLT3)-mediated growth inhibition, as measured by the clonogenic assay. Interestingly, blockade of JAK2/STAT5 signaling by the specific JAK2 inhibitor AZ960 stimulated cell cycling in CD34(+) /CD38(-) cells in conjunction with downregulation of cyclin-dependent kinase inhibitor p21(waf1) and sensitized these cells to the growth inhibition mediated by cytarabine and the FLT3 kinase inhibitor. Moreover, exposure of CD34(+) /CD38(-) cells to AZ960 potently induced apoptosis in parallel with downregulation of antiapoptotic protein Bcl-xL, as measured by Western blot analysis. Taken together, JAK2/STAT5 signaling may be a promising molecular target to eradicate CD34(+) /CD38(-) leukemia cells in individuals with AML.

CREB represses p53-dependent transactivation of MDM2 through the complex formation with p53 and contributes to p53-mediated apoptosis in response to glucose deprivation

Recently, we have described that CREB (cAMP-responsive element-binding protein) has the ability to transactivate tumor suppressor p53 gene in response to glucose deprivation. In this study, we have found that CREB forms a complex with p53 and represses p53-mediated transactivation of MDM2 but not of p21 WAF1 . Immunoprecipitation analysis revealed that CREB interacts with p53 in response to glucose deprivation. Forced expression of CREB significantly attenuated the up-regulation of the endogenous MDM2 in response to p53. By contrast, the mutant form of CREB lacking DNA-binding domain (CREBΔ) had an undetectable effect on the expression level of the endogenous MDM2. During the glucose deprivation-mediated apoptosis, there existed an inverse relationship between the expression levels of MDM2 and p53/CREB. Additionally, p53/CREB complex was dissociated from MDM2 promoter in response to glucose deprivation. Collectively, our present results suggest that CREB preferentially down-regulates MDM2 and thereby contributing to p53-mediated apoptosis in response to glucose deprivation.

Angiocidin inhibits breast cancer proliferation through activation of epidermal growth factor receptor and nuclear factor kappa (NF-ĸB)

Angiocidin, a tumor-associated peptide, has been previously shown to inhibit tumor progression by blocking angiogenesis. We now show that angiocidin has a direct inhibitory effect on tumor cell proliferation. MDA-MB-231 breast cancer cells were inhibited from proliferating in the presence of epidermal growth factor (EGF) and angiocidin. Angiocidin transfected breast cancer cells also displayed growth inhibition in vitro and failed to develop significant tumors in mice as compared to vector controls. The anti-proliferative effect of angiocidin was reversed by treating the cells with the epidermal growth factor receptor (EGFR) inhibitor 4557W, a potent tyrosine kinase inhibitor. Consistent with these results, we found that treatment of breast cancer cells with angiocidin induced a 2.3 fold increase in EGFR tyrosine 845 phosphorylation while no change in phosphorylation was observed in the remaining 16 phosphorylation sites of EGFR and those of its family members as measured by a human EGFR phosphorylation array. Treatment of breast cancer cells with angiocidin also resulted in the activation of nuclear factor ĸB (Nf-ĸB) and the de novo up-regulation of many down-stream genes transcribed by Nf-ĸB, including cytokines, inflammatory mediators and the cell cycle inhibitor p21 waf1 . Therefore, angiocidin is a peptide that not only inhibits tumor angiogenesis but also directly induces inhibition of tumor growth progression through the activation of EGFR and down-stream genes transcribed by Nf-ĸB.

Involvement of HDAC1 in E-cadherin expression in prostate cancer cells; its implication for cell motility and invasion

In this study, we investigate the molecular mechanism by which histone deacetylase (HDAC) inhibitors exert anti-invasiveness effect against prostate cancer cells. We first evaluate the growth inhibition effect of HDAC inhibitors in prostate cancer cells, which is accompanied by induction of p21 WAF1 expression and accumulation of acetylated histones. And we found that the migration and invasion of prostate cancer cells is strongly inhibited by treatment with HDAC inhibitors. In parallel, E-cadherin level is highly up-regulated in HDAC inhibitor-treated prostate cancer cells. And siRNA knockdown of E-cadherin significantly diminishes the anti-invasion effect of HDAC inhibitors, indicating that E-cadherin overexpression is one of possible mechanism for anti-invasion effect of HDAC inhibitors. Furthermore, specific downregulation of HDAC1, but not HDAC2, causes E-cadherin expression and subsequent inhibition of cell motility and invasion. Collectively, our data demonstrate that HDAC1 is a major repressive enzyme for E-cadherin expression as well as HDAC inhibitor-mediated anti-invasiveness.

Epigenetic profiling of the antitumor natural product psammaplin A and its analogues

A collection of analogues of the dimeric natural product psammaplin A that differ in the substitution on the (halo)tyrosine aryl ring, the oxime and the diamine connection has been synthesized. The effects on cell cycle, induction of differentiation and apoptosis of the natural-product inspired series were measured on the human leukaemia U937 cell line. Epigenetic profiling included induction of p21 WAF1, effects on global H3 histone and tubulin acetylation levels as well as in vitro enzymatic assays using HDAC1, DNMT1, DNMT3A, SIRT1 and a peptide domain with p300/CBP HAT activity. Whereas the derivatives of psammaplin A with modifications in the length of the connecting chain, the oxime bond and the disulfide unit showed lower potency, the analogues with changes on the bromotyrosine ring exhibited activities comparable to those of the parent compound in the inhibition of HDAC1 and in the induction of apoptosis. The lack of HDAC1 activity of analogues modified on the disulfide bond suggests that its cleavage must occur in cells to produce the monomeric Zn 2+-chelating thiol. This assumption is consistent with the molecular modelling of the complex of psammaplin A thiol with h-HDAC8. Only a weak inhibition of DNMT1, DNMT3A and residual activities with SIRT1 and a p300/CBP HAT peptide were measured for these compounds.

P53 and p21(Waf1) Are Recruited to Distinct PML‐Containing Nuclear Foci in Irradiated and Nutlin‐3a‐Treated U2OS Cells

Promyelocytic leukemia nuclear bodies (PML-NBs) are multiprotein complexes that include PML protein and localize in nuclear foci. PML-NBs are implicated in multiple stress responses, including apoptosis, DNA repair, and p53-dependent growth inhibition. ALT-associated PML bodies (APBs) are specialized PML-NBs that include telomere-repeat binding-factor TRF1 and are exclusively in telomerase-negative tumors where telomere length is maintained through alternative (ALT) recombination mechanisms. We compared cell-cycle and p53 responses in ALT-positive cancer cells (U2OS) exposed to ionizing radiation (IR) or the p53 stabilizer Nutlin-3a. Both IR and Nutlin-3a caused growth arrest and comparable induction of p53. However, p21, whose gene p53 activates, displayed biphasic induction following IR and monophasic induction following Nutlin-3a. p53 was recruited to PML-NBs 3-4 days after IR, approximately coincident with the secondary p21 increase. These p53/PML-NBs marked sites of apparently unrepaired DNA double-strand breaks (DSBs), identified by colocalization with phosphorylated histone H2AX. Both Nutlin-3a and IR caused a large increase in APBs that was dependent on p53 and p21 expression. Moreover, p21, and to a lesser extent p53, was recruited to APBs in a fraction of Nutlin-3a-treated cells. These data indicate (1) p53 is recruited to PML-NBs after IR that likely mark unrepaired DSBs, suggesting p53 may either be further activated at these sites and/or function in their repair; (2) p53-p21 pathway activation increases the percentage of APB-positive cells, (3) p21 and p53 are recruited to ALT-associated PML-NBs after Nutlin-3a treatment, suggesting that they may play a previously unrecognized role in telomere maintenance.

CREB Inhibits AP-2α Expression to Regulate the Malignant Phenotype of Melanoma

BackgroundThe loss of AP-2α and increased activity of cAMP-responsive element binding (CREB) protein are two hallmarks of malignant progression of cutaneous melanoma. However, the molecular mechanism responsible for the loss of AP-2α during melanoma progression remains unknown.Methodology/Principal FindingsHerein, we demonstrate that both inhibition of PKA-dependent CREB phosphorylation, as well as silencing of CREB expression by shRNA, restored AP-2α protein expression in two metastatic melanoma cell lines. Moreover, rescue of CREB expression in CREB-silenced cell lines downregulates expression of AP-2α. Loss of AP-2α expression in metastatic melanoma occurs via a dual mechanism involving binding of CREB to the AP-2α promoter and CREB-induced overexpression of another oncogenic transcription factor, E2F-1. Upregulation of AP-2α expression following CREB silencing increases endogenous p21Waf1 and decreases MCAM/MUC18, both known to be downstream target genes of AP-2α involved in melanoma progression.Conclusions/SignificanceSince AP-2α regulates several genes associated with the metastatic potential of melanoma including c-KIT, VEGF, PAR-1, MCAM/MUC18, and p21Waf1, our data identified CREB as a major regulator of the malignant melanoma phenotype.

Pifithrin-α as a Potential Cytoprotective Agent in Radiotherapy: Protection of Normal Tissue without Decreasing Therapeutic Efficacy in Glioma Cells

Activation of p53 has been causally linked to normal tissue damage after irradiation. Pifithrin-α (PFT-α), a specific inhibitor of p53, has been suggested as a combinatory agent in the treatment of p53-deficient tumors in which inhibition of p53 would not compromise therapeutic efficacy but would decrease p53-mediated side effects in normal tissue. We tested this concept for radiotherapy of p53-deficient and -proficient glioma. We observed significant interaction of PFT-α with radiation-induced G(1) checkpoint activation and plating efficiency only in glioma cells expressing at least one wild-type allele of p53. This interaction was correlated with PFT-α-mediated inhibition of radiation-induced expression of the p53 target gene p21(Waf1). Despite inhibition of p53 function we did not observe significant changes in radiosensitivity after treatment with PFT-α in either p53-deficient or p53-proficient tumor cells. We confirmed these results in p53-proficient lung cancer cells. In contrast, PFT-α significantly increased the fraction of normal astrocytes and fibroblasts surviving irradiation; this was accompanied by improved DNA damage repair, speaking against an accumulation of cells with genetic lesions after PFT-α treatment. In conclusion, PFT-α might prove useful in protecting normal tissue from the side effects of radiotherapy without reducing the efficacy of treatment for both p53-proficient and -deficient tumors.