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DNA-PKCS binding to p53 on the p21WAF1/CIP1 promoter blocks transcription resulting in cell death.

ABSTRACT: A key determinant of p53-mediated cell fate following various DNA damage modalities is p21WAF1/CIP1 expression, with elevated p21 expression triggering cell cycle arrest and repressed p21 expression promoting apoptosis. We show that under pro-death DNA damage conditions, the DNA-dependent protein kinase (DNA-PKCS) is recruited to the p21 promoter where it forms a protein complex with p53. The DNA-PKCS-associated p53 displays post-translational modifications that are distinct from those under pro-arrest conditions, ablating p21 transcription and inducing cell death. Inhibition of DNA-PK activity prevents DNA-PKCS binding to p53 on the p21 promoter, restores p21 transcription and significantly reduces cell death. These data demonstrate that DNA-PKCS negatively regulates p21 expression by directly interacting with the p21 transcription machinery via p53, driving the cell towards apoptosis.

SUBMITTER: Hill R

PROVIDER: S-EPMC3282069 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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DNA-PKCS binding to p53 on the p21WAF1/CIP1 promoter blocks transcription resulting in cell death.

Hill Richard R Madureira Patricia A PA Waisman David M DM Lee Patrick W K PW

Oncotarget 20111201 12

A key determinant of p53-mediated cell fate following various DNA damage modalities is p21WAF1/CIP1 expression, with elevated p21 expression triggering cell cycle arrest and repressed p21 expression promoting apoptosis. We show that under pro-death DNA damage conditions, the DNA-dependent protein kinase (DNA-PKCS) is recruited to the p21 promoter where it forms a protein complex with p53. The DNA-PKCS-associated p53 displays post-translational modifications that are distinct from those under pro ...[more]

PMID: 22190353

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Retraction: DNA-PKCS binding to p53 on the p21WAF1/CIP1 promoter blocks transcription resulting in cell death.

Project description:[This retracts the article DOI: 10.18632/oncotarget.378.].

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p53-Independent regulation of p21Waf1/Cip1 expression and senescence by PRMT6.

Project description:p21 is a potent cyclin-dependent kinase inhibitor that plays a role in promoting G1 cell cycle arrest and cellular senescence. Consistent with this role, p21 is a downstream target of several tumour suppressors and oncogenes, and it is downregulated in the majority of tumours, including breast cancer. Here, we report that protein arginine methyltransferase 6 (PRMT6), a type I PRMT known to act as a transcriptional cofactor, directly represses the p21 promoter. PRMT6 knock-down (KD) results in a p21 derepression in breast cancer cells, which is p53-independent, and leads to cell cycle arrest, cellular senescence and reduced growth in soft agar assays and in severe combined immunodeficiency (SCID) mice for all the cancer lines examined. We finally show that bypassing the p21-mediated arrest rescues PRMT6 KD cells from senescence, and it restores their ability to grow on soft agar. We conclude that PRMT6 acts as an oncogene in breast cancer cells, promoting growth and preventing senescence, making it an attractive target for cancer therapy.

| S-EPMC3479215 | biostudies-literature

The gut-enriched Kruppel-like factor (Kruppel-like factor 4) mediates the transactivating effect of p53 on the p21WAF1/Cip1 promoter.

Project description:An important mechanism by which the tumor suppressor p53 maintains genomic stability is to induce cell cycle arrest through activation of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene. We show that the gene encoding the gut-enriched Krüppel-like factor (GKLF, KLF4) is concurrently induced with p21(WAF1/Cip1) during serum deprivation and DNA damage elicited by methyl methanesulfonate. The increases in expression of both Gklf and p21(WAF1/Cip1) due to DNA damage are dependent on p53. Moreover, during the first 30 min of methyl methanesulfonate treatment, the rise in Gklf mRNA level precedes that in p21(WAF1/Cip1), suggesting that GKLF may be involved in the induction of p21(WAF1/Cip1). Indeed, GKLF activates p21(WAF1/Cip1) through a specific Sp1-like cis-element in the p21(WAF1/Cip1) proximal promoter. The same element is also required by p53 to activate the p21(WAF1/Cip1) promoter, although p53 does not bind to it. Potential mechanisms by which p53 activates the p21(WAF1/Cip1) promoter include a physical interaction between p53 and GKLF and the transcriptional induction of Gklf by p53. Consequently, the two transactivators cause a synergistic induction of the p21(WAF1/Cip1) promoter activity. The physiological relevance of GKLF in mediating p53-dependent induction of p21(WAF1/Cip1) is demonstrated by the ability of antisense Gklf oligonucleotides to block the production of p21(WAF1/Cip1) in response to p53 activation. These findings suggest that GKLF is an essential mediator of p53 in the transcriptional induction of p21(WAF1/Cip1) and may be part of a novel pathway by which cellular responses to stress are modulated.

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Reactive oxygen species and p21Waf1/Cip1 are both essential for p53-mediated senescence of head and neck cancer cells.

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Targeted p21WAF1/CIP1 activation by RNAa inhibits hepatocellular carcinoma cells.

Project description:RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double-stranded RNA (dsRNA), also known as small activating RNA (saRNA). p21(WAF1/CIP1) (p21) is a putative tumor suppressor gene due to its role as a key negative regulator of the cell cycle and cell proliferation. It is frequently downregulated in cancer including hepatocellular carcinoma (HCC), but is rarely mutated or deleted, making it an ideal target for RNAa-based overexpression to restore its tumor suppressor function. In the present study, we investigated the antigrowth effects of p21 RNAa in HCC cells. Transfection of a p21 saRNA (dsP21-322) into HepG2 and Hep3B cells significantly induced the expression of p21 at both the mRNA and protein levels, and inhibited cell proliferation and survival. Further analysis of dsP21-322 transfected cells revealed that dsP21-322 arrested the cell cycle at the G(0)/G(1) phase in HepG2 cells but at G(2)/M phase in Hep3B cells which lack functional p53 and Rb genes, and induced both early and late stage apoptosis by activating caspase 3 in both cell lines. These results demonstrated that RNAa of p21 has in vitro antigrowth effects on HCC cells via impeding cell cycle progression and inducing apoptotic cell death. This study suggests that targeted activation of p21 by RNAa may be explored as a novel therapy for the treatment of HCC.

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Polymorphism rs2395655 affects LEDGF/p75 binding activity and p21WAF1/CIP1 gene expression in esophageal squamous cell carcinoma.

Project description:p21WAF1/CIP1 (p21) plays critical roles in cell-cycle regulation and DNA repair and is transcriptionally regulated through p53-dependent or -independent pathways. Bioinformatic analysis predicated one stress-response element (STRE) implicated in single nucleotide polymorphism (SNP) rs2395655 of the p21 promoter. Here, we investigated the transcriptional regulatory function of rs2395655 variant genotype and analyzed its associations with the p21 expression and clinical outcomes in esophageal squamous cell carcinoma (ESCC) patients. Luciferase assay results showed significantly increased transcriptional activity of the rs2395655 G allele-containing p21 promoter compared with rs2395655 A allele-containing counterpart, especially in ESCC cells with ectopic LEDGF/p75 expression. Furthermore electrophoretic mobility shift assay using the rs2395655 G or A allele-containing probe and chromatin immunoprecipitation assay with specific anti-LEDGF/p75 antibody indicated the potential binding activity of LEDGF/p75 with the STRE element implicated in rs2395655 G allele of the p21 promoter. Subsequent specific RNA interference-mediated depletion or ectopic expression of LEDGF/p75 caused obviously down- or up-regulated expression of p21 mRNA in ESCC cells harboring rs2395655 GG genotype but not cells with rs2395655 AA genotype. Furthermore, rs2395655 GG genotype carriers showed significantly elevated p21 protein expression and conferred survival advantage in both univariate and multivariate analyses in total 218 ESCC patients. Our findings suggest that LEDGF/p75 regulates the p21 expression in ESCC cells through interacting with STRE element implicated in polymorphism rs2395655 and the elevated p21 protein expression and rs2395655 GG genotype may serve as positive prognostic factors for ESCC patients.

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The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest.

Project description:Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology.

| S-EPMC4452790 | biostudies-literature

Protracted p53-independent stimulation of p21WAF1/Cip1 fuels genomic instability by deregulating the replication licensing machinery

Project description:The cyclin-dependent kinase inhibitor p21WAF1/Cip1 is the prototype downstream effector of the tumor suppressor protein p53. Yet, evidence from human cancer and mice models, imply that p21WAF1/Cip1, under certain conditions, can exercise oncogenic activity. The mechanism behind this behavior is still obscure. Within this context we unexpectedly noticed, predominantly in p53 mutant human cancers, that a subset of highly atypical cancerous cells expressing strongly p21WAF1/Cip1 demonstrated also signs of proliferation. This finding suggests either tolerance to high p21WAF1/Cip1 levels or that p21WAF1/Cip1 per se guided a selective process that led to more aggressive off-springs. To address the latter scenario we employed p21WAF1/Cip1-inducible p53-null cellular models and monitored them over a prolonged time period, using high-throughput screening means. After an initial phase characterized by stalled growth, mainly due to senescence, a subpopulation of p21WAF1/Cip1 cells emerged, demonstrating increased genomic instability, aggressiveness and chemo-resistance. At the mechanistic level unremitted p21WAF1/Cip1 production “saturates” the CRL4CDT2 and SCFSkp2 ubiquitin ligase complexes reducing the turn-over of the replication licensing machinery. Deregulation of replication licensing triggered replication stress fuelling genomic instability. Conceptually, the above notion should be considered when anti-tumor strategies are designed, since p21WAF1/Cip1 responds also to p53-independent signals, including various chemotherapeutic compounds.

2016-05-15 | GSE62166 | GEO

Protracted p53-independent stimulation of p21WAF1/Cip1 fuels genomic instability by deregulating the replication licensing machinery

Project description:The cyclin-dependent kinase inhibitor p21WAF1/Cip1 is the prototype downstream effector of the tumor suppressor protein p53. Yet, evidence from human cancer and mice models, imply that p21WAF1/Cip1, under certain conditions, can exercise oncogenic activity. The mechanism behind this behavior is still obscure. Within this context we unexpectedly noticed, predominantly in p53 mutant human cancers, that a subset of highly atypical cancerous cells expressing strongly p21WAF1/Cip1 demonstrated also signs of proliferation. This finding suggests either tolerance to high p21WAF1/Cip1 levels or that p21WAF1/Cip1 per se guided a selective process that led to more aggressive off-springs. To address the latter scenario we employed p21WAF1/Cip1-inducible p53-null cellular models and monitored them over a prolonged time period, using high-throughput screening means. After an initial phase characterized by stalled growth, mainly due to senescence, a subpopulation of p21WAF1/Cip1 cells emerged, demonstrating increased genomic instability, aggressiveness and chemo-resistance. At the mechanistic level unremitted p21WAF1/Cip1 production â??saturatesâ?? the CRL4CDT2 and SCFSkp2 ubiquitin ligase complexes reducing the turn-over of the replication licensing machinery. Deregulation of replication licensing triggered replication stress fuelling genomic instability. Conceptually, the above notion should be considered when anti-tumor strategies are designed, since p21WAF1/Cip1 responds also to p53-independent signals, including various chemotherapeutic compounds. We used microarrays to compare the non-induced and â??escapedâ?? Saos2-p21WAF1/Cip1 Tet-ON cells gene expression profile Multiple arrays for non-induced and â??escapedâ?? Saos2-p21WAF1/Cip1 Tet-ON cells

2016-05-15 | E-GEOD-62166 | biostudies-arrayexpress

Glycogen synthase kinase 3beta phosphorylates p21WAF1/CIP1 for proteasomal degradation after UV irradiation.

Project description:UV irradiation has been reported to induce p21(WAF1/CIP1) protein degradation through a ubiquitin-proteasome pathway, but the underlying biochemical mechanism remains to be elucidated. Here, we show that ser-114 phosphorylation of p21 protein by glycogen synthase kinase 3beta (GSK-3beta) is required for its degradation in response to UV irradiation and that GSK-3beta activation is a downstream event in the ATR signaling pathway triggered by UV. UV transiently increased GSK-3beta activity, and this increase could be blocked by caffeine or by ATR small interfering RNA, indicating ATR-dependent activation of GSK-3beta. ser-114, located within the putative GSK-3beta target sequence, was phosphorylated by GSK-3beta upon UV exposure. The nonphosphorylatable S114A mutant of p21 was protected from UV-induced destabilization. Degradation of p21 protein by UV irradiation was independent of p53 status and prevented by proteasome inhibitors. In contrast to the previous report, the proteasomal degradation of p21 appeared to be ubiquitination independent. These data show that GSK-3beta is activated by UV irradiation through the ATR signaling pathway and phosphorylates p21 at ser-114 for its degradation by the proteasome. To our knowledge, this is the first demonstration of GSK-3beta as the missing link between UV-induced ATR activation and p21 degradation.

| S-EPMC1899930 | biostudies-literature

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