S phase and G2 arrests induced by topoisomerase I poisons are dependent on ATR kinase function
S phase and G2 arrests induced by topoisomerase I poisons are dependent on ATR kinase function
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DOI：
10.1074/jbc.m106287200
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发表时间：
2002-01-11
影响因子：
4.8
通讯作者：
Kaufmann, SH
中科院分区：
文献类型：
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作者：
Cliby, WA;Lewis, KA;Kaufmann, SH
ATR是一种人磷脂酰肌醇3-激酶相关的激酶，是细胞对DNA损伤反应的重要组成部分。在本研究中，我们评估了ATR在调节细胞对拓扑异构酶毒物引起的S期相关DNA双链断裂的反应中的作用。长期暴露在低剂量的拓扑异构酶I毒物拓扑替康(TPT)中，由于后期复制的DNA合成减少，导致S时相变慢。相反，短暂的TPT暴露，以及长时间暴露于拓扑异构酶11毒物依托泊苷，导致随后的G(2)期停滞。这些反应与检查点激酶Chk1的磷酸化有关。细胞周期反应和Chk1的磷酸化显著地被强制过表达ATR的显性负的、不激活的等位基因所降低。相反，相关激酶ATM的缺失对这些事件没有影响。ATR依赖的检查点功能的丧失使GM847人成纤维细胞对拓扑异构酶I毒药TPT和7-乙基-10-羟基喜树碱的细胞毒性作用敏感，通过抑制克隆形成、增加台盼蓝摄取和发展凋亡形态变化来评估。KdATR的表达也使GM847细胞对长期低剂量依托泊苷和阿霉素的细胞毒作用敏感，尽管程度较小。综上所述，这些结果不仅表明ATR在应对拓扑异构酶毒物引起的复制相关的DNA损伤方面是重要的，而且也支持了ATM和ATR在激活参与细胞周期检查点的下游激酶方面具有独特作用的观点。
ATR, a human phosphatidylinositol 3-kinase-related kinase, is an important component of the cellular response to DNA damage. In the present study, we evaluated the role of ATR in modulating the response of cells to S phase-associated DNA double-stranded breaks induced by topoisomerase poisons. Prolonged exposure to low doses of the topoisomerase I poison topotecan (TPT) resulted in S phase slowing because of diminished DNA synthesis at late-firing replicons. In contrast, brief TPT exposure, as well as prolonged exposure to the topoisomerase 11 poison etoposide, resulted in subsequent G(2) arrest. These responses were associated with phosphorylation of the checkpoint kinase Chk1. The cell cycle responses and phosphorylation of Chk1 were markedly diminished by forced overexpression of a dominant negative, kinase-inactive allele of ATR. In contrast, deficiency of the related kinase ATM had no effect on these events. The loss of ATR-dependent checkpoint function sensitized GM847 human fibroblasts to the cytotoxic effects of the topoisomerase I poisons TPT and 7-ethyl-10-hydroxycamptothecin, as assessed by inhibition of colony formation, increased trypan blue uptake, and development of apoptotic morphological changes. Expression of kdATR also sensitized GM847 cells to the cytotoxic effects of prolonged low dose etoposide and doxorubicin, albeit to a smaller extent. Collectively, these results not only suggest that ATR is important in responding to the replication-associated DNA damage from topoisomerase poisons, but also support the view that ATM and ATR have unique roles in activating the downstream kinases that participate in cell cycle checkpoints.