14-3-3Sigma is required to prevent mitotic catastrophe after DNA damage.

14-3-3Sigma is a member of a family of proteins that regulate cellular activity by binding and sequestering phosphorylated proteins. It has been suggested that 14-3-3sigma promotes pre-mitotic cell-cycle arrest following DNA damage, and that its expression can be controlled by the p53 tumour suppressor gene. Here we describe an improved approach to the generation of human somatic-cell knockouts, which we have used to generate human colorectal cancer cells in which both 14-3-3sigma alleles are inactivated. After DNA damage, these cells initially arrested in the G2 phase of the cell cycle, but, unlike cells containing 14-3-3sigma, the 14-3-3sigma-/- cells were unable to maintain cell-cycle arrest. The 14-3-3sigma-/- cells died ('mitotic catastrophe') as they entered mitosis. This process was associated with a failure of the 14-3-3sigma-deficient cells to sequester the proteins (cyclin B1 and cdc2) that initiate mitosis and prevent them from entering the nucleus. These results may indicate a mechanism for maintaining the G2 checkpoint and preventing mitotic death.     

力达霉素对人红白血病K562细胞死亡的影响

利用100,10和1nmol·L-1力达霉素(LDM)处理人红白血病K562细胞(p53,p16基因缺失),初步探讨了LDM对K562细胞死亡的影响. 结果表明,LDM可明显抑制K562细胞的活性,并呈现剂量依赖效应. 细胞核染色质呈点状或斑块状凝集.通过流式细胞术对细胞周期时相的分析表明,高剂量LDM处理引起S期细胞的比例明显升高,死亡细胞的比例明显增加,提示LDM通过引起DNA的损伤,可阻断细胞周期的进程,促进细胞的死亡实现抑癌作用.     

The prolyl isomerase Pin1 is a regulator of p53 in genotoxic response

p53 is activated in response to various genotoxic stresses resulting in cell cycle arrest or apoptosis. It is well documented that DNA damage leads to phosphorylation and activation of p53 (refs 1-3), yet how p53 is activated is still not fully understood. Here we report that DNA damage specifically induces p53 phosphorylation on Ser/Thr-Pro motifs, which facilitates its interaction with Pin1, a member of peptidyl-prolyl isomerase. Furthermore, the interaction of Pin1 with p53 is dependent on the phosphorylation that is induced by DNA damage. Consequently, Pin1 stimulates the DNA-binding activity and transactivation function of p53. The Pin1-mediated p53 activation requires the WW domain, a phosphorylated Ser/Thr-Pro motif interaction module, and the isomerase activity of Pin1. Moreover, Pin1-deficient cells are defective in p53 activation and timely accumulation of p53 protein, and exhibit an impaired checkpoint control in response to DNA damage. Together, these data suggest a mechanism for p53 regulation in cellular response to genotoxic stress.     

A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage

The p53 gene is frequently inactivated in human cancers. Here we have isolated a p53-inducible gene, p53R2, by using differential display to examine messenger RNAs in a cancer-derived human cell line carrying a highly regulated wild-type p53 expression system. p53R2 contains a p53-binding sequence in intron 1 and encodes a 351-amino-acid peptide with striking similarity to the ribonucleotide reductase small subunit (R2), which is important in DNA synthesis during cell division. Expression of p53R2, but not R2, was induced by ultraviolet and gamma-irradiation and adriamycin treatment in a wild-type p53-dependent manner. Induction of p53R2 in p53-deficient cells caused G2/M arrest and prevented cells from death in response to adriamycin. Inhibition of endogenous p53R2 expression in cells that have an intact p53-dependent DNA damage checkpoint reduced ribonucleotide reductase activity, DNA repair and cell survival after exposure to various genotoxins. Our results indicate that p53R2 encodes a ribonucleotide reductase that is directly involved in the p53 checkpoint for repair of damaged DNA. The discovery of p53R2 clarifies a relationship between a ribonucleotide reductase activity involved in repair of damaged DNA and tumour suppression by p53.     

Interaction of c-Abl and p73alpha and their collaboration to induce apoptosis.

c-Abl, a non-receptor tyrosine kinase, is activated by agents that damage DNA. This activation results in either arrest of the cell cycle in phase G1 or apoptotic cell death, both of which are dependent on the kinase activity of c-Abl. p73, a member of the p53 family of tumour-suppressor proteins, can also induce apoptosis. Here we show that the apoptotic activity of p73alpha requires the presence of functional, kinase-competent c-Abl. Furthermore, p73 and c-Abl can associate with each other, andthis binding is mediated by a PxxP motif in p73 and the SH3 domain of c-Abl. We find that p73 is a substrate of the c-Abl kinase and that the ability of c-Abl to phosphorylate p73 is markedly increased by gamma-irradiation. Moreover, p73 is phosphorylated in vivo in response to ionizing radiation. These findings define a pro-apoptotic signalling pathway involving p73 and c-Abl.     

The pathological response to DNA damage does not contribute to p53-mediated tumour suppression

The p53 protein has a highly evolutionarily conserved role in metazoans as 'guardian of the genome', mediating cell-cycle arrest and apoptosis in response to genotoxic injury. In large, long-lived animals with substantial somatic regenerative capacity, such as vertebrates, p53 is an important tumour suppressor--an attribute thought to stem directly from its induction of death or arrest in mutant cells with damaged or unstable genomes. Chemotherapy and radiation exposure both induce widespread p53-dependent DNA damage. This triggers potentially lethal pathologies that are generally deemed an unfortunate but unavoidable consequence of the role p53 has in tumour suppression. Here we show, using a mouse model in which p53 status can be reversibly switched in vivo between functional and inactive states, that the p53-mediated pathological response to whole-body irradiation, a prototypical genotoxic carcinogen, is irrelevant for suppression of radiation-induced lymphoma. In contrast, delaying the restoration of p53 function until the acute radiation response has subsided abrogates all of the radiation-induced pathology yet preserves much of the protection from lymphoma. Such protection is absolutely dependent on p19(ARF)--a tumour suppressor induced not by DNA damage, but by oncogenic disruption of the cell cycle.     

KLF4沉默与阿霉素诱导的DNA损伤协同抑制肝癌HepG2细胞的生长

探究KLF4沉默与经不同作用浓度阿霉素处理诱导的DNA损伤对肝癌HepG2细胞增殖凋亡的影响及其作用机制.应用RNA干扰技术,采用siRNA转染HepG2细胞以沉默KLF4基因.采用MTT法检测KLF4沉默前后对HepG2细胞增殖的影响,使用流式细胞术检测KLF4沉默前后对HepG2细胞周期变化影响,应用Western blot法检测转染前后HepG2细胞中KLF4蛋白及细胞周期相关蛋白表达变化.Western blot检测到高浓度的阿霉素促进KLF4的表达,并且低浓度的阿霉素可使得细胞停滞在G2/M期,高浓度的阿霉素则使部分细胞凋亡(19.31%).将KLF4沉默后,发现细胞生长变缓,低浓度的阿霉素处理后,细胞随时间增加而出现更多的细胞凋亡;高浓度的阿霉素处理后,细胞数明显减少,更多的细胞发生凋亡(28.89%),且在KLF4沉默前后均发现低浓度阿霉素促进p53与p21表达,高浓度阿霉素抑制其表达.阿霉素诱导的DNA损伤可提高KLF4的表达,KLF4依赖于DNA损伤激活的p53促进p21的表达,进而引起G1/S期细胞周期阻滞.沉默KLF4与阿霉素诱导的DNA损伤可协同抑制肝癌细胞的增殖、促进凋亡,其在肝癌细胞 HepG2中扮演十分重要的角色.     

DNA-dependent protein kinase is not required for the p53-dependent response to DNA damage.

Damage to DNA in the cell activates the tumour-suppressor protein p53, and failure of this activation leads to genetic instability and a predisposition to cancer. It is therefore crucial to understand the signal transduction mechanisms that connect DNA damage with p53 activation. The enzyme known as DNA-dependent protein kinase (DNA-PK) has been proposed to be an essential activator of p53, but the evidence for its involvement in this pathway is controversial. We now show that the p53 response is fully functional in primary mouse embryonic fibroblasts lacking DNA-PK: irradiation-induced DNA damage in these defective fibroblasts induces a normal response of p53 accumulation, phosphorylation of a p53 serine residue at position 15, nuclear localization and binding to DNA of p53. The upregulation of p53-target genes and cell-cycle arrest also occur normally. The DNA-PK-deficient cell line SCGR11 contains a homozygous mutation in the DNA-binding domain of p53, which may explain the defective response by p53 reported in this line. Our results indicate that DNA-PK activity is not required for cells to mount a p53-dependent response to DNA damage.     

Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas

Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence. Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies. To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma. We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth.     

p63 protects the female germ line during meiotic arrest

Meiosis in the female germ line of mammals is distinguished by a prolonged arrest in prophase of meiosis I between homologous chromosome recombination and ovulation. How DNA damage is detected in these arrested oocytes is poorly understood, but it is variably thought to involve p53, a central tumour suppressor in mammals. While the function of p53 in monitoring the genome of somatic cells is clear, a consensus for the importance of p53 for germ line integrity has yet to emerge. Here we show that the p53 homologue p63 (refs 5, 6), and specifically the TAp63 isoform, is constitutively expressed in female germ cells during meiotic arrest and is essential in a process of DNA damage-induced oocyte death not involving p53. We also show that DNA damage induces both the phosphorylation of p63 and its binding to p53 cognate DNA sites and that these events are linked to oocyte death. Our data support a model whereby p63 is the primordial member of the p53 family and acts in a conserved process of monitoring the integrity of the female germ line, whereas the functions of p53 are restricted to vertebrate somatic cells for tumour suppression. These findings have implications for understanding female germ line fidelity, the regulation of fertility and the evolution of tumour suppressor mechanisms.     

Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint

The checkpoint kinase proteins Mec1 and Rad53 are required in the budding yeast, Saccharomyces cerevisiae, to maintain cell viability in the presence of drugs causing damage to DNA or arrest of DNA replication forks. It is thought that they act by inhibiting cell cycle progression, allowing time for DNA repair to take place. Mec1 and Rad53 also slow S phase progression in response to DNA alkylation, although the mechanism for this and its relative importance in protecting cells from DNA damage have not been determined. Here we show that the DNA-alkylating agent methyl methanesulphonate (MMS) profoundly reduces the rate of DNA replication fork progression; however, this moderation does not require Rad53 or Mec1. The accelerated S phase in checkpoint mutants, therefore, is primarily a consequence of inappropriate initiation events. Wild-type cells ultimately complete DNA replication in the presence of MMS. In contrast, replication forks in checkpoint mutants collapse irreversibly at high rates. Moreover, the cytotoxicity of MMS in checkpoint mutants occurs specifically when cells are allowed to enter S phase with DNA damage. Thus, preventing damage-induced DNA replication fork catastrophe seems to be a primary mechanism by which checkpoints preserve viability in the face of DNA alkylation.     

MDC1 is a mediator of the mammalian DNA damage checkpoint

To counteract the continuous exposure of cells to agents that damage DNA, cells have evolved complex regulatory networks called checkpoints to sense DNA damage and coordinate DNA replication, cell-cycle arrest and DNA repair. It has recently been shown that the histone H2A variant H2AX specifically controls the recruitment of DNA repair proteins to the sites of DNA damage. Here we identify a novel BRCA1 carboxy-terminal (BRCT) and forkhead-associated (FHA) domain-containing protein, MDC1 (mediator of DNA damage checkpoint protein 1), which works with H2AX to promote recruitment of repair proteins to the sites of DNA breaks and which, in addition, controls damage-induced cell-cycle arrest checkpoints. MDC1 forms foci that co-localize extensively with gamma-H2AX foci within minutes after exposure to ionizing radiation. H2AX is required for MDC1 foci formation, and MDC1 forms complexes with phosphorylated H2AX. Furthermore, this interaction is phosphorylation dependent as peptides containing the phosphorylated site on H2AX bind MDC1 in a phosphorylation-dependent manner. We have shown by using small interfering RNA (siRNA) that cells lacking MDC1 are sensitive to ionizing radiation, and that MDC1 controls the formation of damage-induced 53BP1, BRCA1 and MRN foci, in part by promoting efficient H2AX phosphorylation. In addition, cells lacking MDC1 also fail to activate the intra-S phase and G2/M phase cell-cycle checkpoints properly after exposure to ionizing radiation, which was associated with an inability to regulate Chk1 properly. These results highlight a crucial role for MDC1 in mediating transduction of the DNA damage signal.     

DNA damage stress induces the dissociation of Smurf1/2 from MDM2 in a slow manner

The tumor suppressor p53 locates at the key point of cell growth or apoptosis balance, and the expression level of p53 is tightly controlled by ubiquitin ligases including MDM2. Upon DNA damage stresses, p53 was accumulated and activated, leading to cell cycle arrest or apoptosis. We previously showed that Smad ubiquitylation regulatory factor 1/2 (Smurf1/2) promotes p53 degradation by interacting with and stabilizing MDM2, and consequently enhancing MDM2-mediated ubiquitylation of p53. However, it is uncle...     

Mouse p53 inhibits SV40 origin-dependent DNA replication

p53 is a cellular phosphoprotein that is present at elevated concentrations in cells transformed by different agents. p53 complementary DNA expression-constructs immortalize primary cells in vitro and co-operate with an activated ras oncogene in malignant transformation. Several reports have implicated p53 in mammalian cell cycle control and specifically with events occurring at the G0-G1 boundary. p53 forms specific complexes with simian virus 40 (SV40) large-T antigen, and such complexes are found associated with both replicating and mature SV40 DNA in lytically infected cells. In an accompanying paper Gannon and Lane report that in in vitro plate-binding assays, mouse p53 can displace polymerase alpha from complex with T-antigen. We have examined the in vivo consequences of expressing wild-type and mutant p53 proteins from other species in SV40-transformed monkey cells. We report here that expression of mouse p53 results in a substantial and selective inhibition of SV40 origin-dependent DNA replication. In addition to any function in the G0-G1 transition, the data presented suggest that p53 may affect directly the initiation or maintenance of replicative DNA synthesis.     

Chk1 prevents abnormal mitosis of S-phase HeLa cells containing DNA damage

To explore effects of DNA damage on cell-cycle progression in p53-deficient tumor cells, synchronized HeLa cells at G1, S and G2/M phases were treated with methyl methanesulfnate （MMS）. The results showed that the MMS treatment resulted in the cell-cycle arrest or delay in all 3 phases, while the S-phase cells were the most sensitive to MMS. Further studies demonstrated that ATM-Chk2 and p38 MAPK signaling pathways were activated in all 3 phases when the cells were treated with MMS; whereas Chk1 was activated only in S phase under the drug treatment, indicating that Chk1 specifically participated in S-phase checkpoints. To analyze the role of Chk1 in S-phase checkpoints, we administered a specific Chk1 inhibitor, UCN-01, to the S-phase cells. The results showed that the S-phase cells treated with MMS＋UCN-01 could enter aberrant mitosis without finishing DNA replication, indicating that Chk1 mainly functions in the DNA damage checkpoint rather than in the replication checkpoint. In addition, MMS treatment alone inhibited the accumulation of cyclin B1, a key component of M-phase CDK-cyclin complex, in the S-phase cells, whereas the inhibition of Chk1 activation resulted in the accumulation of cyclin B1 in the MMS-treated S-phase cells. This observation further supports the view that DNA-damaged S-phase cells enter abnormal mitosis when Chk1 activation is inhibited. Our results demonstrate that Chk1 is a specific kinase that plays an important role in the MMS-induced S-phase DNA damage checkpoint. As p53 is not involved in this process, Chk1 may be a potential target for p53-deficient tumor therapy.     

Effects of Genistein on Cell Cycle and Apoptosis of Two Murine Melanoma Cell Lines

The effects of genistein on several tumor cell lines were investigated to study the effects of gen- istein on cell growth, cell cycle, and apoptosis of two murine melanoma cell lines, B16 and K1735M2. These two closely related murine melanoma cell lines, however, have different responses to the genistein treat- ment. Genistein inhibits the growth of both the B16 and K1735M2 cell lines and arrests the growth at the G2/M phase. After treatment with 60 μmol/L genistein for 72 h, apoptosis and caspase activities were de- tected in B16 cells, while such effects were not found in K1735M2. Further tests showed that after genistein treatment the protein content and mRNA levels of p53 increased in B16, but remained the same in K1735M2. The protein content and mRNA levels of p21WAF1/CIP1 increased in both cell lines after treatment. The results show that genistein might induce apoptosis in B16 cells by damaging the DNA, inhibiting topoi- somerase II, increasing p53 expression, releasing cytochrome c from the mitochondria, and activating the caspases which will lead to apoptosis.