A DNA damage checkpoint response in telomere-initiated senescence

Most human somatic cells can undergo only a limited number of population doublings in vitro. This exhaustion of proliferative potential, called senescence, can be triggered when telomeres--the ends of linear chromosomes-cannot fulfil their normal protective functions. Here we show that senescent human fibroblasts display molecular markers characteristic of cells bearing DNA double-strand breaks. These markers include nuclear foci of phosphorylated histone H2AX and their co-localization with DNA repair and DNA damage checkpoint factors such as 53BP1, MDC1 and NBS1. We also show that senescent cells contain activated forms of the DNA damage checkpoint kinases CHK1 and CHK2. Furthermore, by chromatin immunoprecipitation and whole-genome scanning approaches, we show that the chromosome ends of senescent cells directly contribute to the DNA damage response, and that uncapped telomeres directly associate with many, but not all, DNA damage response proteins. Finally, we show that inactivation of DNA damage checkpoint kinases in senescent cells can restore cell-cycle progression into S phase. Thus, we propose that telomere-initiated senescence reflects a DNA damage checkpoint response that is activated with a direct contribution from dysfunctional telomeres.     

硫酸铈诱导果蝇氧化应激及细胞凋亡的研究

本实验主要研究了稀土硫酸铈（Ce（SO4）2）对果蝇氧化应激生物标记物和细胞凋亡的影响.果蝇培养在不同质量浓度（1,4,16,64,256,1 024 mg/L）的硫酸铈培养基中,分别测定其SOD,CAT和脂质过氧化产物（即MDA含量）,同时用彗星电泳和体外切割DNA实验来检测果蝇细胞中DNA损伤程度,用DNA Laddering法和TUNEL法测定稀土元素Ce对果蝇细胞凋亡的影响.与对照组相比,当Ce（SO4）2质量浓度低于16 mg/L时,果蝇体内SOD和CAT活性显著增加,MDA含量变化不明显;而当Ce（SO4）2质量浓度高于16 mg/L时,SOD和CAT活性明显下降,MDA含量上升.彗星电泳的结果表现为随着硫酸铈剂量的递增,果蝇中肠细胞的彗星率、彗星尾长和Olive尾矩增加,并表现为明显的剂量效应关系.果蝇体外实验结果表明,硫酸铈能打断DNA,使其片段化;同时,TUNEL结果显示果蝇中肠细胞呈现凋亡细胞的特征性蓝绿色颗粒,但DNA琼脂糖电泳没有表现出细胞凋亡特征性的梯形条带图谱.硫酸铈诱导果蝇的氧化应激可以使果蝇中肠细胞SOD和CAT活性降低,MDA含量上升,使中肠细胞出现凋亡特征.由此推断,硫酸铈可诱导果蝇细胞中遗传物质的损伤,对果蝇有一定的氧化毒性和遗传毒性作用．     

Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1

When telomeres are rendered dysfunctional through replicative attrition of the telomeric DNA or by inhibition of shelterin, cells show the hallmarks of ataxia telangiectasia mutated (ATM) kinase signalling. In addition, dysfunctional telomeres might induce an ATM-independent pathway, such as ataxia telangiectasia and Rad3-related (ATR) kinase signalling, as indicated by the phosphorylation of the ATR target CHK1 in senescent cells and the response of ATM-deficient cells to telomere dysfunction. However, because telomere attrition is accompanied by secondary DNA damage, it has remained unclear whether there is an ATM-independent pathway for the detection of damaged telomeres. Here we show that damaged mammalian telomeres can activate both ATM and ATR and address the mechanism by which the shelterin complex represses these two important DNA damage signalling pathways. We analysed the telomere damage response on depletion of either or both of the shelterin proteins telomeric repeat binding factor 2 (TRF2) and protection of telomeres 1 (POT1) from cells lacking ATM and/or ATR kinase signalling. The data indicate that TRF2 and POT1 act independently to repress these two DNA damage response pathways. TRF2 represses ATM, whereas POT1 prevents activation of ATR. Unexpectedly, we found that either ATM or ATR signalling is required for efficient non-homologous end-joining of dysfunctional telomeres. The results reveal how mammalian telomeres use multiple mechanisms to avoid DNA damage surveillance and provide an explanation for the induction of replicative senescence and genome instability by shortened telomeres.     

The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor

Some stimulatory receptors of the innate immune system, such as the NKG2D receptor (also called KLRK1) expressed by natural killer cells and activated CD8(+)T cells, recognize self-molecules that are upregulated in diseased cells by poorly understood mechanisms. Here we show that mouse and human NKG2D ligands are upregulated in non-tumour cell lines by genotoxic stress and stalled DNA replication, conditions known to activate a major DNA damage checkpoint pathway initiated by ATM (ataxia telangiectasia, mutated) or ATR (ATM- and Rad3-related) protein kinases. Ligand upregulation was prevented by pharmacological or genetic inhibition of ATR, ATM or Chk1 (a downstream transducer kinase in the pathway). Furthermore, constitutive ligand expression by a tumour cell line was inhibited by targeting short interfering RNA to ATM, suggesting that ligand expression in established tumour cells, which often harbour genomic irregularities, may be due to chronic activation of the DNA damage response pathway. Thus, the DNA damage response, previously shown to arrest the cell cycle and enhance DNA repair functions, or to trigger apoptosis, may also participate in alerting the immune system to the presence of potentially dangerous cells.     

The wee1 protein kinase is required for radiation-induced mitotic delay.

Cellular feedback or 'checkpoint' mechanisms maintain the order of completion of essential, cell-cycle related functions. In the budding yeast, for example, the RAD9 gene product is required to delay progression into mitosis in response to DNA damage. Similarly, in fission yeast, the cdc25 and cdc2 gene products influence the ability of cells to delay mitosis in response to the inhibition of DNA synthesis. Because these two checkpoint controls regulate the same event, mitosis, we observed the effect of gamma-irradiation on cell cycle progression in fission yeast, to test whether the two controls require the same cell-cycle regulatory elements. We show that gamma-radiation-induced mitotic delay requires functional wee1 protein kinase but does not seem to involve the cdc25 pathway. Mitotic delay in response to DNA damage is thus distinct from the delay induced by inhibition of DNA synthesis, which involves cdc25 but is not dependent on wee1.     

Pb2 + 和Cd2 + 对日本三角涡虫DNA 损伤及损伤后修复的研究

摘要: 以日本三角涡虫( Dugesia japonica) 为实验材料,采用急性毒性实验研究Pb2 + 、Cd2 + 胁迫下日本三角涡虫体细 胞DNA 损伤情况以及绿豆浸出液对DNA 损伤的保护和修复机制。采用浓度为120 mg /L 的Pb( NO3 ) 2 和1 mg /L 的CdCl2 溶液分别处理涡虫,紫外分光光度法和琼脂糖凝胶电泳检测24 h 后三角涡虫DNA 损伤情况。同时增加 由绿豆浸出液进行修复的2 组对照以研究绿豆对于DNA 重金属损伤后的修复作用和效果。结果表明,Pb2 + 、Cd2 + 胁迫使日本三角涡虫DNA 交联程度增加,并引起DNA 链的断裂; 绿豆浸出液对于由Cd2 + 胁迫引起的DNA 损伤修 复作用较好。而对于Pb2 + 胁迫,在绿豆浸出液与Pb2 + 同时培养的对照组中,推测该浸出液可能使Pb2 + 形成沉淀从 而减小Pb2 + 浓度,因此使DNA 损伤修复具有较好效果,对已经由Pb2 + 胁迫造成损伤的DNA,修复作用不大。     

Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions

DNA damage checkpoint genes, such as p53, are frequently mutated in human cancer, but the selective pressure for their inactivation remains elusive. We analysed a panel of human lung hyperplasias, all of which retained wild-type p53 genes and had no signs of gross chromosomal instability, and found signs of a DNA damage response, including histone H2AX and Chk2 phosphorylation, p53 accumulation, focal staining of p53 binding protein 1 (53BP1) and apoptosis. Progression to carcinoma was associated with p53 or 53BP1 inactivation and decreased apoptosis. A DNA damage response was also observed in dysplastic nevi and in human skin xenografts, in which hyperplasia was induced by overexpression of growth factors. Both lung and experimentally-induced skin hyperplasias showed allelic imbalance at loci that are prone to DNA double-strand break formation when DNA replication is compromised (common fragile sites). We propose that, from its earliest stages, cancer development is associated with DNA replication stress, which leads to DNA double-strand breaks, genomic instability and selective pressure for p53 mutations.     

A stress response pathway regulates DNA damage through β2-adrenoreceptors and β-arrestin-1

The human mind and body respond to stress, a state of perceived threat to homeostasis, by activating the sympathetic nervous system and secreting the catecholamines adrenaline and noradrenaline in the 'fight-or-flight' response. The stress response is generally transient because its accompanying effects (for example, immunosuppression, growth inhibition and enhanced catabolism) can be harmful in the long term. When chronic, the stress response can be associated with disease symptoms such as peptic ulcers or cardiovascular disorders, and epidemiological studies strongly indicate that chronic stress leads to DNA damage. This stress-induced DNA damage may promote ageing, tumorigenesis, neuropsychiatric conditions and miscarriages. However, the mechanisms by which these DNA-damage events occur in response to stress are unknown. The stress hormone adrenaline stimulates β(2)-adrenoreceptors that are expressed throughout the body, including in germline cells and zygotic embryos. Activated β(2)-adrenoreceptors promote Gs-protein-dependent activation of protein kinase A (PKA), followed by the recruitment of β-arrestins, which desensitize G-protein signalling and function as signal transducers in their own right. Here we elucidate a molecular mechanism by which β-adrenergic catecholamines, acting through both Gs-PKA and β-arrestin-mediated signalling pathways, trigger DNA damage and suppress p53 levels respectively, thus synergistically leading to the accumulation of DNA damage. In mice and in human cell lines, β-arrestin-1 (ARRB1), activated via β(2)-adrenoreceptors, facilitates AKT-mediated activation of MDM2 and also promotes MDM2 binding to, and degradation of, p53, by acting as a molecular scaffold. Catecholamine-induced DNA damage is abrogated in Arrb1-knockout (Arrb1(-/-)) mice, which show preserved p53 levels in both the thymus, an organ that responds prominently to acute or chronic stress, and in the testes, in which paternal stress may affect the offspring's genome. Our results highlight the emerging role of ARRB1 as an E3-ligase adaptor in the nucleus, and reveal how DNA damage may accumulate in response to chronic stress.     

Identical V beta T-cell receptor genes used in alloreactive cytotoxic and antigen plus I-A specific helper T cells

T lymphocytes involved in the cellular immune response carry cell-surface receptors responsible for antigen and self recognition. This T-cell receptor molecule is a heterodimeric protein consisting of disulphide-linked alpha- and beta-chains with variable (V) and constant (C) regions. Several complementary DNA and genomic DNA clones have been isolated and characterized. These analyses showed that the genomic arrangement and rearrangement of T-cell receptor genes using VT, diversity (DT), joining (JT) and CT gene segments is very similar to the structure of the known immunoglobulin genes. We have isolated two cDNA clones from an allospecific cytotoxic T cell, one of which shows a productive V beta-J beta-C beta 1 rearrangement without an intervening D beta segment. This V beta gene segment is identical to the V beta gene expressed in a helper T-cell clone specific for chicken red blood cells and H-21. The other clone carries the C beta 2 gene of the T-cell receptor, but the C beta 2 sequence is preceded by a DNA sequence that does not show any similarity to V beta or J beta sequences.     

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 function for cyclin D1 in DNA repair uncovered by protein interactome analyses in human cancers

Cyclin D1 is a component of the core cell cycle machinery. Abnormally high levels of cyclin D1 are detected in many human cancer types. To elucidate the molecular functions of cyclin D1 in human cancers, we performed a proteomic screen for cyclin D1 protein partners in several types of human tumours. Analyses of cyclin D1 interactors revealed a network of DNA repair proteins, including RAD51, a recombinase that drives the homologous recombination process. We found that cyclin D1 directly binds RAD51, and that cyclin D1-RAD51 interaction is induced by radiation. Like RAD51, cyclin D1 is recruited to DNA damage sites in a BRCA2-dependent fashion. Reduction of cyclin D1 levels in human cancer cells impaired recruitment of RAD51 to damaged DNA, impeded the homologous recombination-mediated DNA repair, and increased sensitivity of cells to radiation in vitro and in vivo. This effect was seen in cancer cells lacking the retinoblastoma protein, which do not require D-cyclins for proliferation. These findings reveal an unexpected function of a core cell cycle protein in DNA repair and suggest that targeting cyclin D1 may be beneficial also in retinoblastoma-negative cancers which are currently thought to be unaffected by cyclin D1 inhibition.     

Virus-mediated killing of cells that lack p53 activity

A major goal of molecular oncology is to identify means to kill cells lacking p53 function. Most current cancer therapy is based on damaging cellular DNA by irradiation or chemicals. Recent reports support the notion that, in the event of DNA damage, the p53 tumour-suppressor protein is able to prevent cell death by sustaining an arrest of the cell cycle at the G2 phase. We report here that adeno-associated virus (AAV) selectively induces apoptosis in cells that lack active p53. Cells with intact p53 activity are not killed but undergo arrest in the G2 phase of the cell cycle. This arrest is characterized by an increase in p53 activity and p21 levels and by the targeted destruction of CDC25C. Neither cell killing nor arrest depends upon AAV-encoded proteins. Rather, AAV DNA, which is single-stranded with hairpin structures at both ends, elicits in cells a DNA damage response that, in the absence of active p53, leads to cell death. AAV inhibits tumour growth in mice. Thus viruses can be used to deliver DNA of unusual structure into cells to trigger a DNA damage response without damaging cellular DNA and to selectively eliminate those cells lacking p53 activity.     

自由面对应力波反射诱发层裂过程影响的数值模拟

采用基于细观损伤力学基础上开发的动态版岩石破坏过程分析系统RFPA2D数值模拟软件,对冲击载荷作用下非均匀介质中应力波反射诱发层裂过程进行了数值模拟,重点探讨了不同自由面情况对层裂过程的影响.结果表明：动态版RFPA2D数值模拟软件可以逼真地模拟其过程;在不同自由面情况下,应力波传播至自由面产生反射诱发层裂破坏,除与应力波的峰值、延续时间和波形等有关外,还与应力波传播的方向有关,如果相同应力波垂直入射至自由面比斜入射至自由面更容易诱发层裂破坏的发生;当应力波平行入射至自由面时不会诱发层裂破坏.     

噬菌体展示的白色念珠菌热休克蛋白90特异表位在小鼠体内诱发的免疫应答

将展示白色念珠菌热休克蛋白(HSP)90特异表位的杂合噬菌体作为抗原免疫C57BL/6J小鼠,以检测该抗原在小鼠体内诱发的体液免疫和细胞免疫.实验结果表明:抗原PA刺激机体产生了较强的抗白色念珠菌HSP90特异性抗体;脾CD4 T和CD8 T淋巴细胞出现率明显增高;脾细胞分泌IL-2的能力有增高趋势.此外,将免疫后的小鼠通过尾静脉进行系统性白色念珠菌感染,采用组织病理学方法观察了抗原对小鼠肾脏白色念珠菌寄居数量的影响,实验结果表明,抗原对系统性白色念珠菌感染具有明显的保护作用.     

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 unclear how the Smurf1-MDM2 interaction is regulated in response to DNA damage stress. Here, we show that in response to etoposide treatment Smurf1 dissociates from MDM2, resulting in MDM2 destabilization and p53 accumulation. The negative regulation of Smurf1 on apoptosis is released. Notably, this dissociation is a slow process rather than a rapid response, implicating high expression of Smurf1 might confer the resistance against p53 activation. Consistent with this notion, we observed that Smurf1/2 ligases are highly expressed in colon cancer, esophageal squamous cell carcinoma and pancreatic cancer tissues, suggesting the oncogenic tendency of Smurf1/2.     

hCds1-mediated phosphorylation of BRCA1 regulates the DNA damage response

Mutations in the BRCA1 (ref. 1) tumour suppressor gene are found in almost all of the families with inherited breast and ovarian cancers and about half of the families with only breast cancer. Although the biochemical function of BRCA1 is not well understood, it is important for DNA damage repair and cell-cycle checkpoint. BRCA1 exists in nuclear foci but is hyperphosphorylated and disperses after DNA damage. It is not known whether BRCA1 phosphorylation and dispersion and its function in DNA damage response are related. In yeast the DNA damage response and the replication-block checkpoint are mediated partly through the Cds1 kinase family. Here we report that the human Cds1 kinase (hCds1/Chk2) regulates BRCA1 function after DNA damage by phosphorylating serine 988 of BRCA1. We show that hCds1 and BRCA1 interact and co-localize within discrete nuclear foci but separate after gamma irradiation. Phosphorylation of BRCA1 at serine 988 is required for the release of BRCA1 from hCds1. This phosphorylation is also important for the ability of BRCA1 to restore survival after DNA damage in the BRCA1-mutated cell line HCC1937.