Activation of ATM and Chk2 kinases in relation to the amount of DNA strand breaks

Buscemi, Giacomo; Perego, Paola; Carenini, Nives; Nakanishi, Makoto; Chessa, Luciana; Chen, Junjie; Khanna, Kum Kum; Delia, Domenico

doi:10.1038/sj.onc.1207986

Cited by 93 publications

(82 citation statements)

References 60 publications

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“…In fact, the S-phase DNA damage checkpoint was reported to require a threshold level of damage to activate the checkpoint response (Shimada et al, 2002). Full activation of Chk2, an effector of the S-phase DNA damage checkpoint, was reported to require radiation doses above 2 Gy (Buscemi et al, 2004), indicating the presence of a threshold for the induction of the whole battery of S-phase DNA damage responses. Our data suggested that 1 Gy of gammairradiation is enough to activate p53 to execute the p53-dependent suppression of replication fork progression in MEFs through phosphorylation of PCNA.…”

Section: Discussionmentioning

confidence: 99%

Suppression of replication fork progression in low-dose-specific p53-dependent S-phase DNA damage checkpoint

et al. 2006

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The S-phase DNA damage checkpoint is activated by DNA damage to delay DNA synthesis allowing time to resolve the replication block. We previously discovered the p53-dependent S-phase DNA damage checkpoint in mouse zygotes fertilized with irradiated sperm. Here, we report that the same p53 dependency holds in mouse embryonic fibroblasts (MEFs) at low doses of irradiation. DNA synthesis in p53 wild-type (WT) MEFs was suppressed in a biphasic manner in which a sharp decrease below 2.5 Gy was followed by a more moderate decrease up to 10 Gy. In contrast, p53À/À MEFs exhibited radioresistant DNA synthesis below 2.5 Gy whereas the cells retained the moderate suppression above 5 Gy. DNA fiber analysis revealed that 1 Gy irradiation suppressed replication fork progression in p53 WT MEFs, but not in p53À/À MEFs. Proliferating cell nuclear antigen (PCNA), clamp loader of DNA polymerase, was phosphorylated in WT MEFs after 1 Gy irradiation and redistributed to form foci in the nuclei. In contrast, PCNA was not phosphorylated and dissociated from chromatin in 1 Gy-irradiated p53À/À MEFs. These results demonstrate that the novel low-dosespecific p53-dependent S-phase DNA damage checkpoint is likely to regulate the replication fork movement through phosphorylation of PCNA.

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Section: Discussionmentioning

confidence: 99%

Suppression of replication fork progression in low-dose-specific p53-dependent S-phase DNA damage checkpoint

et al. 2006

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“…Several proteins of the DDR are rapidly recruited to sites of breaks, leading to the formation of nuclear foci 22,23 that are cleared upon DNA repair. We enumerated g-H2AX foci in ihNSCs at D0 treated with 0.25 or 1 Gy IR and collected 5 and 60 min and 24 h later (Figure 4a).…”

Section: Resultsmentioning

confidence: 99%

“…[20][21][22] In ATM-deficient cells, the phosphorylation of the ATM targets is markedly attenuated. 23 To determine the effect of ATM depletion in our cells, we analyzed the genotoxic response on western blots using antibodies specific for ATM phosphoresidues. At D0, a dose of IR as low as 0.5 Gy induced in shCon the phosphorylation of ATM-S1981 and of its substrates Smc1-S966, Chk2-T68 and p53-S15 (Figure 3), though this response was more vigorous with higher doses of IR.…”

Section: Resultsmentioning

confidence: 99%

“…At least 1000 cells/slide were scored, and experiments were independently replicated at least three times. For the nuclear foci, cells were cytocentrifuged onto glass slides, air dried, fixed and labeled with an antiphospho-S139-H2AX antibody (clone JBW301, Upstate Biotechnology) as reported by Buscemi et al 23 Images were collected on a Nikon fluorescence microscope equipped with a CCD camera, and foci enumerated by two operators, on three independent experiments and duplicate slides. For flow cytometry analysis, single cell suspensions from neurospheres and D0 cells were made by gently trypsin treatment and trituration.…”

Section: Methodsmentioning

confidence: 99%

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DNA-damage response, survival and differentiation in vitro of a human neural stem cell line in relation to ATM expression

et al. 2009

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Ataxia-telangiectasia (A-T) is a neurodegenerative disorder caused by defects in the ATM kinase, a component of the DNA-damage response (DDR). Here, we employed an immortalized human neural stem-cell line (ihNSC) capable of differentiating in vitro into neurons, oligodendrocytes and astrocytes to assess the ATM-dependent response and outcome of ATM ablation. The time-dependent differentiation of ihNSC was accompanied by an upregulation of ATM and DNA-PK, sharp downregulation of ATR and Chk1, transient induction of p53 and by the onset of apoptosis in a fraction of cells. The response to ionizing radiation (IR)-induced DNA lesions was normal, as attested by the phosphorylation of ATM and some of its substrates (e.g., Nbs1, Smc1, Chk2 and p53), and by the kinetics of c-H2AX nuclear foci formation. Depletion in these cells of ATM by shRNA interference (shATM) attenuated the differentiation-associated apoptosis and response to IR, but left unaffected the growth, self-renewal and genomic stability. shATM cells generated a normal number of MAP2/b-tubulin III þ neurons, but a reduced number of GalC þ oligodendrocytes, which were nevertheless more susceptible to oxidative stress. Altogether, these findings highlight the potential of ihNSCs as an in vitro model system to thoroughly assess, besides ATM, the role of DDR genes in neurogenesis and/or neurodegeneration.

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“…Recent reports have indicated that ATM and CHK2 are specifically activated by drug (calicheamicin) or radiation-induced DNA double-strand breaks (DSBs), and the level of activation directly correlates to the number of DNA DSBs [58,59]. Our previous studies demonstrated that irofulven activates ATM and its targets, NBS1, SMC1, CHK2 and p53 [42].…”

Section: Discussionmentioning

confidence: 98%

Irofulven induces replication-dependent CHK2 activation related to p53 status

Wang

Wiltshire

Senft

et al. 2007

Biochemical Pharmacology

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CHK2 and p53 are frequently mutated in human cancers. CHK2 is known to phosphorylate and stabilize p53. CHK2 has also been implicated in DNA repair and apoptosis induction. However, whether p53 affects CHK2 activation and whether CHK2 activation modulates chemosensitivity are unclear. In this study, we found that in response to the DNA damage agent, irofulven, CHK2 activation, rather than its expression, is inversely correlated to p53 status. Irofulven inhibits DNA replication and induces chromosome aberrations (breaks and radials) and p53-dependent cell cycle arrest. Pretreatment of cells with the DNA polymerase inhibitor, aphidicolin, resulted in reduction of irofulven-induced CHK2 activation and foci formation, indicating that CHK2 activation by irofulven is replication-dependent. Furthermore, by using ovarian cancer cell lines expressing dominant-negative CHK2 and CHK2-knockout HCT116 cells, we found that CHK2 activation contributes to the control of S and G2/M cell cycle arrests, but not chemosensitivity to irofulven. Overall, this study demonstrates that in response to irofulven-induced DNA damage, the activation of CHK2 is dependent on DNA replication and related to p53 status. By controlling cell cycle arrest and DNA replication, p53 affects CHK2 activation. CHK2 activation contributes to cell cycle arrest, but not chemosensitivity.

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Activation of ATM and Chk2 kinases in relation to the amount of DNA strand breaks

Cited by 93 publications

References 60 publications

Suppression of replication fork progression in low-dose-specific p53-dependent S-phase DNA damage checkpoint

Suppression of replication fork progression in low-dose-specific p53-dependent S-phase DNA damage checkpoint

DNA-damage response, survival and differentiation in vitro of a human neural stem cell line in relation to ATM expression

Irofulven induces replication-dependent CHK2 activation related to p53 status

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