Defective Mre11-dependent activation of Chk2 by ataxia telangiectasia mutated in colorectal carcinoma cells in response to replication-dependent DNA double strand breaks
Defective Mre11-dependent activation of Chk2 by ataxia telangiectasia mutated in colorectal carcinoma cells in response to replication-dependent DNA double strand breaks
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DOI：
10.1074/jbc.m603747200
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发表时间：
2006-10-13
影响因子：
4.8
通讯作者：
Pommier, Yves
中科院分区：
文献类型：
--
作者：
Takemura, Haruyuki;Rao, V. Ashutosh;Pommier, Yves
Mre11（.）Rad50（.）Nbs 1（MRN）复合物结合DNA双链断裂以修复DNA并激活检查点。我们报告了NCI抗癌药物筛选的7个结肠癌细胞系中的3个MRN缺陷。为了研究MRN参与复制介导的DNA双链断裂，我们检查了对喜树碱的检查点反应，喜树碱在复制叉与拓扑异构酶I切割复合物碰撞后诱导复制介导的DNA双链断裂。MRN缺陷细胞缺乏Chk 2激活，而Chk 1激活是独立的MRN。Chk 2激活是共济失调毛细血管扩张突变（ATM）依赖性的，并与Mre 11和Nbs 1的磷酸化相关。在MRN缺陷的HCT 116细胞中，Mre 11互补恢复了Chk 2活化以及Rad 50和Nbs 1水平。相反，在HT 29细胞中通过小干扰RNA（siRNA）下调Mre 11抑制Chk 2活化并下调Nbs 1和Rad 50。蛋白酶体抑制也恢复了HCT 116细胞中的Rad 50和Nbs 1水平，表明Mre 11稳定了Rad 50和Nbs 1。由于Chk 2水平较低，四种MRN-熟练的结直肠细胞系中有三种的Chk 2活化也有缺陷。因此，来自NCI抗癌药物筛选的7个结肠癌细胞系中的6个在响应复制介导的DNA双链断裂时是功能性Chk 2缺陷的。我们建议，Mre 11稳定Nbs 1和Rad 50和MRN激活Chk 2下游ATM复制介导的DNA双链断裂。HCT 116中Chk 2缺陷与S期检查点缺陷、G（2）阻滞延长和对喜树碱超敏反应有关。MRN和Chk 2缺陷的高频率可能导致结直肠癌的基因组不稳定性和对喜树碱的治疗反应。
The Mre11(.)Rad50(.)Nbs1 (MRN) complex binds DNA double strand breaks to repair DNA and activate checkpoints. We report MRN deficiency in three of seven colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study the involvement of MRN in replication-mediated DNA double strand breaks, we examined checkpoint responses to camptothecin, which induces replication-mediated DNA double strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation was independent of MRN. Chk2 activation was ataxia telangiectasia mutated (ATM)-dependent and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRN-deficient HCT116 cells restored Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 down-regulation by small interference RNA ( siRNA) in HT29 cells inhibited Chk2 activation and down-regulated Nbs1 and Rad50. Proteasome inhibition also restored Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in three of four MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, six of seven colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double strand breaks. We propose that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G(2) arrest, and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers.