Adachi Noritaka, Suzuki Hiromi, Iiizumi Susumu, Koyama Hideki
Kihara Institute for Biological Research, Graduate School of Integrated Science, Yokohama City University, Totsuka-ku, Yokohama 244-0813, Japan.
J Biol Chem. 2003 Sep 19;278(38):35897-902. doi: 10.1074/jbc.M306500200. Epub 2003 Jul 3.
A number of clinically useful anticancer drugs, including etoposide (VP-16), target DNA topoisomerase (topo) II. These drugs, referred to as topo II poisons, stabilize cleavable complexes, thereby generating DNA double-strand breaks. Bis-2,6-dioxopiperazines such as ICRF-193 also inhibit topo II by inducing a distinct type of DNA damage, termed topo II clamps, which has been believed to be devoid of double-strand breaks. Despite the biological and clinical importance, the molecular mechanisms for the repair of topo II-mediated DNA damage remain largely unknown. Here, we perform genetic analyses using the chicken DT40 cell line to investigate how DNA lesions caused by topo II inhibitors are repaired. Notably, we show that LIG4-/- and KU70-/- cells, which are defective in nonhomologous DNA end-joining (NHEJ), are extremely sensitive to both VP-16 and ICRF-193. In contrast, RAD54-/- cells (defective in homologous recombination) are much less hypersensitive to VP-16 than the NHEJ mutants and, more importantly, are not hypersensitive to ICRF-193. Our results provide the first evidence that NHEJ is the predominant pathway for the repair of topo II-mediated DNA damage; that is, cleavable complexes and topo II clamps. The outstandingly increased cytotoxicity of topo II inhibitors in the absence of NHEJ suggests that simultaneous inhibition of topo II and NHEJ would provide a powerful protocol in cancer chemotherapy involving topo II inhibitors.
包括依托泊苷（VP - 16）在内的许多临床上有用的抗癌药物作用于DNA拓扑异构酶（topo）II。这些药物被称为topo II毒物，可稳定可切割复合物，从而产生DNA双链断裂。双 - 2,6 - 二氧代哌嗪类化合物如ICRF - 193也通过诱导一种独特的DNA损伤（称为topo II钳）来抑制topo II，人们一直认为这种损伤不存在双链断裂。尽管其具有生物学和临床重要性，但topo II介导的DNA损伤修复的分子机制在很大程度上仍不清楚。在这里，我们使用鸡DT40细胞系进行遗传分析，以研究topo II抑制剂引起的DNA损伤是如何修复的。值得注意的是，我们发现非同源DNA末端连接（NHEJ）存在缺陷的LIG4 - / - 和KU70 - / - 细胞对VP - 16和ICRF - 193都极其敏感。相比之下，同源重组存在缺陷的RAD54 - / - 细胞对VP - 16的超敏感性远低于NHEJ突变体，更重要的是，对ICRF - 193不敏感。我们的结果首次证明NHEJ是topo II介导的DNA损伤（即可切割复合物和topo II钳）修复的主要途径。在缺乏NHEJ的情况下，topo II抑制剂的细胞毒性显著增加，这表明同时抑制topo II和NHEJ将为涉及topo II抑制剂的癌症化疗提供一个有力的方案。
