Revised genetic requirements for the decatenation G2 checkpoint: the role of ATM.
Revised genetic requirements for the decatenation G2 checkpoint: the role of ATM.
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DOI：
10.4161/cc.9.8.11470
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发表时间：
2010-04-15
期刊：
影响因子：
--
通讯作者：
Kaufmann WK
中科院分区：
文献类型：
--
作者：
Bower JJ;Zhou Y;Zhou T;Simpson DA;Arlander SJ;Paules RS;Cordeiro-Stone M;Kaufmann WK
当缠结的子染色单体未充分脱连环时，脱连环G2检查点被提出来延迟细胞从G2到有丝分裂的进展。先前的研究表明，ATM和Rad 3相关（ATR）检查点激酶，但不是共济失调毛细血管扩张突变（ATM）激酶，是去连环化G2检查点功能所必需的。在这里，我们表明，用于量化去连锁G2检查点功能的方法可以影响检查点的遗传要求的识别。正常的人二倍体成纤维细胞（NHDF）线响应拓扑异构酶II（topo II）催化抑制剂ICRF-193与严格的G2期阻滞和有丝分裂指数的减少。虽然siRNA介导的ATR和CHEK 1的消耗增加了ICRF-193处理的NHDF系中的有丝分裂指数，但这些蛋白质的消耗不影响有丝分裂进入速率，表明去连环化G2检查点是功能性的。这些结果表明，ATR和CHEK 1是不需要的decatenation G2检查点，但可能会影响有丝分裂退出后，抑制topo II。使用有丝分裂进入试验对共济失调毛细血管扩张症（AT）细胞系进行的重新评估表明，去连环化G2检查点需要ATM。三种NHDF细胞系对ICRF-193有反应，有丝分裂进入率的平均抑制率为98%。在用ICRF-193处理后，AT成纤维细胞中的有丝分裂进入率的检查揭示了显著减弱的去连环化G2检查点反应，有丝分裂进入率的平均59%抑制。此外，正常的淋巴母细胞系与ICRF-193孵育后，有丝分裂进入率的抑制率为95%，而两个AT淋巴母细胞系仅显示有丝分裂进入率的抑制率为36%和20%。在正常人成纤维细胞中用短发夹RNA稳定消耗ATM也使去连环化G2检查点功能平均减弱40%。Western免疫印迹分析表明，ICRF-193处理诱导ATM自磷酸化和ATM依赖性的CHEK 2中Ser 15-p53和Thr 68磷酸化，但没有明显的H2 AX上Ser 139磷酸化。结果表明，抑制拓扑异构酶II诱导ATM磷酸化选定的目标，有助于G2期逮捕独立的DNA损伤。
The decatenation G2 checkpoint is proposed to delay cellular progression from G2 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 G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 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 G2 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 G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 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 G2 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 G2 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 G2 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 CHEK2, but no appreciable phosphorylation of Ser139 on H2AX. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage.
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影响因子：
3.7
作者：
Díaz-Martínez LA;Giménez-Abián JF;Azuma Y;Guacci V;Giménez-Martín G;Lanier LM;Clarke DJ
通讯作者：
Clarke DJ
影响因子：
64.8
作者：
Bartkova, Jirina;Rezaei, Nousin;Gorgoulis, Vassilis G.
通讯作者：
Gorgoulis, Vassilis G.
影响因子：
64.8
作者：
Di Micco, Raffaella;Fumagalli, Marzia;di Fagagna, Fabrizio d'Adda
通讯作者：
di Fagagna, Fabrizio d'Adda
影响因子：
64.8
作者：
DOWNES, CS;CLARKE, DJ;JOHNSON, RT
通讯作者：
JOHNSON, RT
影响因子：
8
作者：
Ducray, C;Pommier, JP;Sabatier, L
通讯作者：
Sabatier, L