Chloroethylnitrosourea-induced cell death and genotoxicity Cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ
Chloroethylnitrosourea-induced cell death and genotoxicity Cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ
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DOI：
10.4161/cc.20862
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发表时间：
2012-07-15
期刊：
影响因子：
4.3
通讯作者：
Kaina, Bernd
中科院分区：
文献类型：
--
作者：
Nikolova, Teodora;Hennekes, Frank;Kaina, Bernd
氯乙基亚硝基脲（CNUs）是一种用于癌症治疗的强效dna反应性烷基化剂。在这里，我们分析了尼莫司汀（CNUs的一种代表）在同步细胞和缺乏参与同源重组（HR）或非同源末端连接（NHEJ）的修复蛋白的细胞中的细胞毒性和遗传毒性。我们发现HR突变体对ACNU非常敏感，通过集落形成、诱导细胞凋亡和染色体畸变来测量。NHEJ突变体的敏感性不同，Ku80突变体具有中等敏感性，而DNA-PKcs突变细胞具有抗性。HR突变细胞显示持续高水平的伽马H2AX灶，与Rad51和53BP1共同染色，表明DNA双链断裂（DSB）形成。使用同步细胞，我们分析了ACNU处理后DSB的形成是否依赖于复制。我们发现，γ - H2AX灶在G(1)期未被诱导，但在S期显著增加，并在G(2)期保持高水平，在G(2)期，一小部分细胞被阻滞，并经历了延迟约12小时的细胞凋亡和坏死。Rad51、ATM、MDC-1和RPA-2位点也形成，并与S期诱导的gamma H2AX位点共定位，表明DNA损伤应答被激活。MGMT修复o -6-氯乙基鸟嘌呤，使其转化为DNA交联，消除了观察到的所有效应。我们推断CNUs引起的主要遗传毒性和杀伤损伤是o -6-氯乙基鸟嘌呤引发的交联，它在治疗细胞周期中产生dsb， HR是对这组抗癌药物的主要保护途径，而不是NHEJ。碱基切除修复对acnu诱导的细胞毒性无显著影响。
Chloroethylnitrosureas (CNUs) are powerful DNA-reactive alkylating agents used in cancer therapy. Here, we analyzed cyto- and genotoxicity of nimustine (ACNU), a representative of CNUs, in synchronized cells and in cells deficient in repair proteins involved in homologous recombination (HR) or non-homologous end-joining (NHEJ). We show that HR mutants are extremely sensitive to ACNU, as measured by colony formation, induction of apoptosis and chromosomal aberrations. The NHEJ mutants differed in their sensitivity, with Ku80 mutants being moderately sensitive and DNA-PKcs mutated cells being resistant. HR mutated cells displayed a sustained high level of gamma H2AX foci, which co-stained with Rad51 and 53BP1, indicating DNA double-strand breaks (DSB) to be formed. Using synchronized cells, we analyzed whether DSB formation after ACNU treatment was replication-dependent. We show that gamma H2AX foci were not induced in G(1) but increased significantly in S phase and remained at a high level in G(2), where a fraction of cells became arrested and underwent, with a delay of >12 h, cell death by apoptosis and necrosis. Rad51, ATM, MDC-1 and RPA-2 foci were also formed and shown to co-localize with gamma H2AX foci induced in S phase, indicating that the DNA damage response was activated. All effects observed were abrogated by MGMT, which repairs O-6-chloroethylguanine that is converted into DNA cross-links. We deduce that the major genotoxic and killing lesion induced by CNUs are O-6-chloroethylguanine-triggered cross-links, which give rise to DSBs in the treatment cell cycle, and that HR, but not NHEJ, is the major route of protection against this group of anticancer drugs. Base excision repair had no significant impact on ACNU-induced cytotoxicity.