A double-strand break repair defect in ATM-deficient cells contributes to radiosensitivity
A double-strand break repair defect in ATM-deficient cells contributes to radiosensitivity
复制标题
DOI：
10.1158/0008-5472.can-03-2384
复制
发表时间：
2004-01-15
期刊：
影响因子：
11.2
通讯作者：
Löbrich, M
中科院分区：
文献类型：
--
作者：
Kühne, M;Riballo, E;Löbrich, M
ATM蛋白在共济失调毛细血管扩张症（AT）患者中发生突变，是DNA双链断裂（DSB）引发的细胞周期检查点反应的核心。ATM在DSB修复中的作用目前尚不清楚，AT细胞放射敏感性的基础也不清楚。我们应用免疫荧光检测γ-H2 AX核灶和脉冲场凝胶电泳，以定量修复DSB后，X射线剂量在0.02和80戈伊之间的融合停滞的原代人成纤维细胞从正常人和患者的ATM和DNA连接酶IV，非同源末端连接（NHEJ）修复途径的核心组成部分突变。在DNA连接酶IV中具有亚型突变的细胞在治疗后表现出高达24 μ l的实质性修复缺陷，但继续修复数天，并最终达到与野生型细胞相似的未修复DSB水平。此外，NHEJ突变体中的修复缺陷具有剂量依赖性。相反，ATM缺陷细胞修复大多数DSB具有正常动力学，但不能修复一个亚组的断裂，无论诱导的病变的初始数量。值得注意的是，在生物学相关的辐射剂量和/或长的修复时间后，AT细胞中的修复缺陷比NHEJ突变体更明显，并且与辐射敏感性相关。在辐射后延迟铺板后分析存活的NHEJ缺陷细胞显示实质性恢复，而AT细胞未能显示任何恢复。这些数据表明，DSB修复缺陷是AT细胞放射敏感性的重要组成部分。
The ATM protein, which is mutated in individuals with ataxia telangiectasia (AT), is central to cell cycle checkpoint responses initiated by DNA double-strand breaks (DSBs). ATM's role in DSB repair is currently unclear as is the basis underlying the radiosensitivity of AT cells. We applied immunofluorescence detection of gamma-H2AX nuclear foci and pulsed-field gel electrophoresis to quantify the repair of DSBs after X-ray doses between 0.02 and 80 Gy in confluence-arrested primary human fibroblasts from normal individuals and patients with mutations in ATM and DNA ligase IV, a core component of the nonhomologous end-joining (NHEJ) repair pathway. Cells with hypomorphic mutations in DNA ligase IV exhibit a substantial repair defect up to 24 It after treatment but continue to repair for several days and finally reach a level of unrepaired DSBs similar to that of wild-type cells. Additionally, the repair defect in NHEJ mutants is dose dependent. ATM-deficient cells,in contrast, repair the majority of DSBs with normal kinetics but fail to repair a subset of breaks, irrespective of the initial number of lesions induced. Significantly, after biologically relevant radiation doses and/or long repair times, the repair defect in AT cells is more pronounced than that of NHEJ mutants and correlates with radiosensitivity. NHEJ-defective cells analyzed for survival following delayed plating after irradiation show substantial recovery while AT cells fail to show any recovery. These data argue that the DSB repair defect underlies a significant component of the radiosensitivity of AT cells.