Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA.
Mol Cancer Res. 2012 Mar;10(3):360-8. doi: 10.1158/1541-7786.MCR-11-0477. Epub 2012 Jan 13.
Treatment of base excision repair-proficient mouse fibroblasts with the DNA alkylating agent methyl methanesulfonate (MMS) and a small molecule inhibitor of PARP-1 results in a striking cell killing phenotype, as previously reported. Earlier studies showed that the mechanism of cell death is apoptosis and requires DNA replication, expression of PARP-1, and an intact S-phase checkpoint cell signaling system. It is proposed that activity-inhibited PARP-1 becomes immobilized at DNA repair intermediates, and that this blocks DNA repair and interferes with DNA replication, eventually promoting an S-phase checkpoint and G(2)-M block. Here we report studies designed to evaluate the prediction that inhibited PARP-1 remains DNA associated in cells undergoing repair of alkylation-induced damage. Using chromatin immunoprecipitation with anti-PARP-1 antibody and qPCR for DNA quantification, a higher level of DNA was found associated with PARP-1 in cells treated with MMS plus PARP inhibitor than in cells without inhibitor treatment. These results have implications for explaining the extreme hypersensitivity phenotype after combination treatment with MMS and a PARP inhibitor.
先前有报道称，用 DNA 烷化剂甲磺酸甲酯（MMS）和 PARP-1 的小分子抑制剂处理碱基切除修复功能正常的小鼠成纤维细胞，会产生明显的细胞杀伤表型。早期研究表明，细胞死亡的机制是细胞凋亡，需要 DNA 复制、PARP-1 的表达和完整的 S 期检查点细胞信号系统。有人提出，失活的 PARP-1 会固定在 DNA 修复中间体上，从而阻断 DNA 修复并干扰 DNA 复制，最终促进 S 期检查点和 G2-M 阻滞。在这里，我们报告了旨在评估以下预测的研究结果：在经历烷化剂诱导损伤修复的细胞中，失活的 PARP-1 仍然与 DNA 相关联。使用抗 PARP-1 抗体的染色质免疫沉淀和 qPCR 进行 DNA 定量，结果发现，用 MMS 和 PARP 抑制剂处理的细胞中与 PARP-1 相关联的 DNA 水平高于未用抑制剂处理的细胞。这些结果对于解释 MMS 和 PARP 抑制剂联合治疗后的极度超敏表型具有重要意义。
