DNA damage, p14ARF, Nucleophosmin (NPM/B23), and cancer

The p53/p14ARF/mdm2 stress response pathway plays a central role in mediating cellular responses to oncogene activation, genome instability, and therapy-induced DNA damage. Abrogation of the pathway occurs in most if not all cancers, and may be essential for tumor development. The high frequency with which the pathway is disabled in cancer and the fact that the pathway appears to be incompatible with tumor cell growth, has made it an important point of focus in cancer research and therapeutics development. Recently, Nucleophosmin (NPM, B23, NO38 and numatrin), a multifunctional nucleolar protein, has emerged as a p14ARF binding protein and regulator of p53. While complex formation between ARF and NPM retains ARF in the nucleolus and prevents ARF from activating p53, DNA damaging treatments promote a transient subnuclear redistribution of ARF to the nucleoplasm, where it interacts with mdm2 and promotes p53 activation. The results add support to a recently proposed model in which the nucleolus serves as a p53-uspstream sensor of stress, and where ARF links nucleolar stress signals to nucleoplasmic effectors of the stress response. A better understanding of ARF’s nucleolar interactions could further elucidate the regulation of the p53 pathway and suggest new therapeutic approaches to restore p53 function.     

Stabilization of p21 (Cip1/WAF1) following Tip60-dependent acetylation is required for p21-mediated DNA damage response

The molecular mechanisms controlling post-translational modifications of p21 have been pursued assiduously in recent years. Here, utilizing mass-spectrometry analysis and site-specific acetyl-p21 antibody, two lysine residues of p21, located at amino-acid sites 161 and 163, were identified as Tip60-mediated acetylation targets for the first time. Detection of adriamycin-induced p21 acetylation, which disappeared after Tip60 depletion with concomitant destabilization of p21 and disruption of G1 arrest, suggested that Tip60-mediated p21 acetylation is necessary for DNA damage-induced cell-cycle regulation. The ability of 2KQ, a mimetic of acetylated p21, to induce cell-cycle arrest and senescence was significantly enhanced in p21 null MEFs compared with those of cells expressing wild-type p21. Together, these observations demonstrate that Tip60-mediated p21 acetylation is a novel and essential regulatory process required for p21-dependent DNA damage-induced cell-cycle arrest.     

SOCS3 regulates p21 expression and cell cycle arrest in response to DNA damage

Genotoxic agents such as ionizing radiation trigger cell cycle arrest at the G1/S and G2/M checkpoints, allowing cells to repair damaged DNA before entry into mitosis. DNA damage-induced G1 arrest involves p53-dependent expression of p21 (Cip1/Waf-1), which inhibits cyclin-dependent kinases and blocks S phase entry. While much of the core DNA damage response has been well-studied, other signaling proteins that intersect with and modulate this response remain uncharacterized. In this study, we identify Suppressor of Cytokine Signaling (SOCS)-3 as an important regulator of radiation-induced G1 arrest. SOCS3-deficient fibroblasts fail to undergo G1 arrest and accumulate in the G2/M phase of the cell cycle. SOCS3 knockout cells phosphorylate p53 and H2AX normally in response to radiation, but fail to upregulate p21 expression. In addition, STAT3 phosphorylation is elevated in SOCS3-deficient cells compared to WT cells. Normal G1 arrest can be restored in SOCS3 KO cells by retroviral transduction of WT SOCS3 or a dominant-negative mutant of STAT3. Our results suggest a novel function for SOCS3 in the control of genome stability by negatively regulating STAT3-dependent radioresistant DNA synthesis, and promoting p53-dependent p21 expression.     

Nuclear Deformation Causes DNA Damage by Increasing Replication Stress

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DNA损伤生物学反应中ATM对p21~(WAF1/CIP1)蛋白的直接磷酸化

毛细血管扩张性共济失调症突变蛋白 (mutatedinataxiatelangiectasia ,ATM)是直接感受DNA双链断裂损伤并起始诸多DNA损伤信号反应通路的主开关分子 .已有研究发现 ,DNA损伤生物学反应中 ,ATM可通过磷酸化活化p5 3,继而转录活化细胞周期检查点蛋白p2 1WAF1 CIP1的表达 ,而对于ATM是否直接参与p2 1WAF1 CIP1的早期活化迄今尚无实验证明 .通过免疫共沉淀反应 ,检测到细胞电离辐射 (ionizingradiation ,IR)反应早期ATM与p2 1WAF1 CIP1蛋白存在相互作用 .将p2 1WAF1 CIP1蛋白编码基因全长克隆入原核表达载体pGEX4T 2 ,经诱导表达及亲和层析纯化获取GST p2 1融合蛋白作为磷酸化底物 .体外磷酸化实验检测证明 ,IR活化的ATM具磷酸化p2 1WAF1 CIP1蛋白的功能 ,并且此磷酸化功能可被PI3K家族特异性抑制剂Wortmannin所抑制 .结果揭示了IR后ATM可通过直接磷酸化p2 1WAF1 CIP1蛋白 ,在IR致DNA损伤生物学反应早期调控p2 1WAF1 CIP1蛋白的快速活化过程     

DNA damage responsive miR-33b-3p promoted lung cancer cells survival and cisplatin resistance by targeting p21WAF1/CIP1

Cisplatin is the most potent and widespread used chemotherapy drug for lung cancer treatment. However, the development of resistance to cisplatin is a major obstacle in clinical therapy. The principal mechanism of cisplatin is the induction of DNA damage, thus the capability of DNA damage response (DDR) is a key factor that influences the cisplatin sensitivity of cancer cells. Recent advances have demonstrated that miRNAs (microRNAs) exerted critical roles in DNA damage response; nonetheless, the association between DNA damage responsive miRNAs and cisplatin resistance and its underlying molecular mechanism still require further investigation. The present study has attempted to identify differentially expressed miRNAs in cisplatin induced DNA damage response in lung cancer cells, and probe into the effects of the misexpressed miRNAs on cisplatin sensitivity. Deep sequencing showed that miR-33b-3p was dramatically down-regulated in cisplatin-induced DNA damage response in A549 cells; and ectopic expression of miR-33b-3p endowed the lung cancer cells with enhanced survival and decreased γH2A.X expression level under cisplatin treatment. Consistently, silencing of miR-33b-3p in the cisplatin-resistant A549/DDP cells evidently sensitized the cells to cisplatin. Furthermore, we identified CDKN1A (p21) as a functional target of miR-33b-3p, a critical regulator of G1/S checkpoint, which potentially mediated the protection effects of miR-33b-3p against cisplatin. In aggregate, our results suggested that miR-33b-3p modulated the cisplatin sensitivity of cancer cells might probably through impairing the DNA damage response. And the knowledge of the drug resistance conferred by miR-33b-3p has great clinical implications for improving the efficacy of chemotherapies for treating lung cancers.     

p21Waf1/Cip1 plays a critical role in modulating senescence through changes of DNA methylation

It has been reported that genomic DNA methylation decreases gradually during cell culture and an organism's aging. However, less is known about the methylation changes of age-related specific genes in aging. p21(Waf1/Cip1) and p16(INK4a) are cyclin-dependent kinase (Cdk) inhibitors that are critical for the replicative senescence of normal cells. In this study, we show that p21(Waf1/Cip1) and p16(INK4a) have different methylation patterns during the aging process of normal human 2BS and WI-38 fibroblasts. p21(Waf1/Cip1) promoter is gradually methylated up into middle-aged fibroblasts but not with senescent fibroblasts, whereas p16(INK4a) is always unmethylated in the aging process. Correspondently, the protein levels of DNA methyltransferase 1 (DNMT1) and DNMT3a increase from young to middle-aged fibroblasts but decrease in the senescent fibroblasts, while DNMT3b decreases stably from young to senescent fibroblasts. p21(Waf1/Cip1) promoter methylation directly represses its expression and blocks the radiation-induced DNA damage-signaling pathway by p53 in middle-aged fibroblasts. More importantly, demethylation by 5-aza-CdR or DNMT1 RNA interference (RNAi) resulted in an increased p21(Waf1/Cip1) level and premature senescence of middle-aged fibroblasts demonstrated by cell growth arrest and high beta-Galactosidase expression. Our results suggest that p21(Waf1/Cip1) but not p16(INK4a) is involved in the DNA methylation mediated aging process. p21(Waf1/Cip1) promoter methylation may be a critical biological barrier to postpone the aging process.     

p21 limits S phase DNA damage caused by the Wee1 inhibitor MK1775

The Wee1 inhibitor MK1775 (AZD1775) is currently being tested in clinical trials for cancer treatment. Here, we show that the p53 target and CDK inhibitor p21 protects against MK1775-induced DNA damage during S-phase. Cancer and normal cells deficient for p21 (HCT116 p21^-/-, RPE p21^-/-, and U2OS transfected with p21 siRNA) showed higher induction of the DNA damage marker γH2AX in S-phase in response to MK1775 compared to the respective parental cells. Furthermore, upon MK1775 treatment the levels of phospho-DNA PKcs S2056 and phospho-RPA S4/S8 were higher in the p21 deficient cells, consistent with increased DNA breakage. Cell cycle analysis revealed that these effects were due to an S-phase function of p21, but MK1775-induced S-phase CDK activity was not altered as measured by CDK-dependent phosphorylations. In the p21 deficient cancer cells MK1775-induced cell death was also increased. Moreover, p21 deficiency sensitized to combined treatment of MK1775 and the CHK1-inhibitor AZD6772, and to the combination of MK1775 with ionizing radiation. These results show that p21 protects cancer cells against Wee1 inhibition and suggest that S-phase functions of p21 contribute to mediate such protection. As p21 can be epigenetically downregulated in human cancer, we propose that p21 levels may be considered during future applications of Wee1 inhibitors.     

Cell Fate Regulation upon DNA Damage: p53 Serine 46 Kinases Pave the Cell Death Road

Mild and massive DNA damage are differentially integrated into the cellular signaling networks and, in consequence, provoke different cell fate decisions. After mild damage, the tumor suppressor p53 directs the cellular response to cell cycle arrest, DNA repair, and cell survival, whereas upon severe damage, p53 drives the cell death response. One posttranslational modification of p53, phosphorylation at Serine 46, selectively occurs after severe DNA damage and is envisioned as a marker of the cell death response. However, the molecular mechanism of action of the p53 Ser46 phospho‐isomer, the molecular timing of this phosphorylation event, and its activating effects on apoptosis and ferroptosis still await exploration. In this essay, the current body of evidence on the molecular function of this deadly p53 mark, its evolutionary conservation, and the regulation of the key players of this response, the p53 Serine 46 kinases, are reviewed and dissected.     

Nuclear translocation of p21 protein prior to its cytosolic degradation by UV enhances DNA repair and survival

We previously reported that UV induced rapid proteasomal degradation of p21 protein in an ubiquitination-independent manner. Here, UV-induced p21 proteolysis was found to occur in the cytosol. Before cytosolic degradation, however, p21 protein translocated to and transiently accumulated in the nucleus. Nuclear translocation of p21 was not required for its degradation, but rather promoted DNA repair and cell survival. Overexpression of the wild type p21, but not the one with defective nuclear localization signal (NLS), reduced UV-induced DNA damage and cell death. Some of p21 protein translocated to the nucleus were associated with chromatin-bound PCNA and saved from UV-induced proteolysis. These data together show that p21 translocates to the nucleus to participate in DNA repair, while the rest is rapidly degraded in the cytosol. We propose that our findings reflect a mechanism to facilitate removal of damaged cells, enhancing DNA repair at the same time.     

Mechanism of p53 downstream effectors p21 and Gadd45 in DNA damage surveillance

Both p21 (WAF1/CIP1) and Gadd45 were activated in a p53-dependent manner in MCF-7 cells after being exposed to ionizing radiation. In order to investigate their roles in DNA damage surveillance, p21~(as)/MCF-7 cells stably transfected by p21 antisense expression plasmid pC-WAF1-AS and Gadd45~(as)/MCF-7 stably transfected by Gadd45 antisense expression plasmid pCMVas45 were established. It was observed that G_1 arrest induced by radiation was significantly reduced in Gadd45~(as)/MCF-7 cells as well as in p21~(as)/MCF-7 cells. Repair of radiation damaged report gene greatly reduced in Gadd45~(as)/MCF-7 and p21~(as)/MCF-7 cells. Apoptosis significantly increased in p21~(as)/MCF-7 after exposure to radiation. These results suggest that both p21 and Gadd45 support cellular survival by taking roles in G_1 arrest and DNA repair, furthermore, p21 protects cells from death by inhibiting apoptosis after exposure to ionizing radiation.     

The fate of p21 in cells surviving UV-irradiation

p21(CDKN1A) is a critical regulator of cell cycle progression in response to DNA damage. There are conflicting conclusions as to whether p21(CDKN1A) levels increase or decrease after ultraviolet (UV)-irradiation and recently it was even reported to disappear entirely following 2.5-30 Jm(-2) of UV-irradiation in the presence of growth medium. The latter would suggest an alternative mechanism for cell cycle arrest after UV-irradiation, since p21(CDKN1A) induction has been considered to be the major mediator of p53-mediated cell cycle arrest after DNA damage. Using physiological UV doses based on cell-killing, we previously observed and here verify that low doses (1.2-6 Jm(-2)) induce p21(CDKN1A) immediately after UV-irradiation, though higher doses cause a latency during which p21(CDKN1A) levels remain fairly constant before increasing. As expected, p53 induction preceded p21(CDKN1A) induction at all doses. Thus, p21(CDKN1A) levels after low doses of UV-irradiation may be controlled in a p53-dependent manner without severe reduction. We propose that physiological relevant UV doses should be determined for each target cell type prior to studying UV-induced responses and that p21(CDKN1A) is indeed critical for cell cycle arrest in cells that survive UV-irradiation.     

UTP18-mediated p21 mRNA instability drives adenoma-carcinoma progression in colorectal cancer

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TNF-alpha promotes Doxorubicin-induced cell apoptosis and anti-cancer effect through downregulation of p21 in p53-deficient tumor cells

p53 is a key regulator in cell apoptosis, and cancer cells deficient in p53 expression fail to respond to chemotherapy. Here we show that effective Doxorubicin (DOX)-induced apoptosis is p53-dependent. However, an alternative treatment of DOX/TNF-alpha/DOX restored sensitivity of p53-deficient cells to DOX-induced apoptosis. Treatment of cells with TNF-alpha resulted in a decrease of p21 (waf1/cip1/sdi1) expression following second dose of DOX. In previous work, we demonstrated that p21 suppressed DOX-induced apoptosis via its (cyclin-dependent kinase) CDK-binding and CDK-inhibitory activity. Thus, we propose that TNF-alpha enhances the anti-cancer effect of DOX through suppressing the anti-apoptotic activity of p21, and that a combined treatment TNF-alpha/Dox is an effective chemotherapeutic strategy for p53-deficient cancers.     

不同月龄p21^HBsAg/HBsAg和p21^HBX/HBX转基因小鼠血清酶含量的测定

目的测定p21^HBsAg/HBsAg和p21^HBX/HBX转基因小鼠纯合型及野生型小鼠的血清酶,探讨各种血清酶在两种纯合型小鼠及野生型小鼠的变化规律。方法采用荷兰半自动生化分析仪Ⅱ对于肝功能相关的7种血清酶进行测定,应用SSPS10统计学软件进行T检验及方差分析比较。结果p2^1HBsAg/HBsAg和p21^HBX/HBX转基因小鼠不同年龄、不同的指标雌雄之间差异显著,并且随着年龄的变化,雌雄之间的变化规律不同。结论p21^HBsAg/HBsAg和p21^HBX/HBX转基因小鼠的血清酶在不同的年龄具有一定的变化规律,说明p21^HBsAg/HBsAg和p21^HBX/HBX转基因小鼠的血清酶具有一定的特征,为其功能性研究及临床检验具有重要参考价值。