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首页> 外文期刊>Cell cycle >Revised genetic requirements for the decatenation G(2) checkpoint: The role of ATM.
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Revised genetic requirements for the decatenation G(2) checkpoint: The role of ATM.

机译：修改的遗传要求的分界G（2）检查点：ATM的作用。

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摘要

The decatenation G(2) checkpoint is proposed to delay cellular progression from G(2) into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G(2) checkpoint function. Here, we show that the method used to quantify decatenation G(2) checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G(2) arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G(2) checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G(2) checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G(2) checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G(2) checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G(2) checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in Chk2, but no appreciable phosphorylation of Ser139-H2AX or Ser345-Chk1. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G(2) arrest independently of DNA damage.
机译：当缠结的子代染色单体未充分消除时，提出了取消结合G（2）检查点以延迟细胞从G（2）进入有丝分裂的过程。先前的研究表明，G（2）检查点功能需要ATM和Rad3相关（ATR）检查点激酶，但共济失调毛细血管扩张突变（ATM）激酶不是必需的。在这里，我们表明用于量化decatenate G（2）检查点功能的方法可以影响对检查点的遗传要求的标识。正常人二倍体成纤维细胞（NHDF）线对拓扑异构酶II（拓扑II）催化抑制剂ICRF-193进行了严格的G（2）逮捕和有丝分裂指数的降低。虽然siRNA介导的ATR和CHEK1耗竭增加了ICRF-193处理的NHDF系中的有丝分裂指数，但这些蛋白质的耗竭并没有影响有丝分裂进入率，表明脱聚G（2）检查点起作用。这些结果表明，ATR和CHEK1并不需要G（2）检查点，但可能会影响topo II抑制后的有丝分裂退出。使用有丝分裂进入分析对共济失调毛细血管扩张（AT）细胞系进行的重新评估表明，GTM（2）检查点需要ATM。三种NHDF细胞系对ICRF-193的反应平均为98％，抑制了有丝分裂进入率。在用ICRF-193治疗后对AT成纤维细胞中有丝分裂进入率的检查显示，显着减弱了脱界G（2）检查点反应，平均抑制了有丝分裂进入率59％。此外，正常的淋巴母细胞系与ICRF-193孵育后，对有丝分裂进入率的抑制率为95％，而两个AT淋巴母细胞系仅对有丝分裂进入率抑制了36％和20％。正常人成纤维细胞与短发夹RNA的ATM的稳定消耗也使G（2）检查点功能的衰减平均降低了40％。 Western免疫印迹分析表明，ICRF-193处理可诱导Chk2中的Ser15-p53和Thr68发生ATM自磷酸化和ATM依赖性磷酸化，但Ser139-H2AX或Ser345-Chk1没有明显的磷酸化。结果表明，抑制拓扑II诱导ATM磷酸化选定的目标，有助于独立于DNA损伤的G（2）逮捕。

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