BRCA1 activates a G2-M cell cycle checkpoint following 6-thioguanine-induced DNA mismatch damage.
BRCA1 activates a G2-M cell cycle checkpoint following 6-thioguanine-induced DNA mismatch damage.
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DOI：
10.1158/0008-5472.can-06-2205
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发表时间：
2007-07
期刊：
影响因子：
11.2
通讯作者：
K. Yamane;J. Schupp;T. Kinsella
中科院分区：
文献类型：
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作者：
K. Yamane;J. Schupp;T. Kinsella
人类DNA错配修复（MMR）参与对某些化疗药物的反应，包括6-硫鸟嘌呤（6-TG）。与此一致的是，mmr缺陷的人类肿瘤细胞对6-TG损伤表现出抵抗性，表现为G(2)-M阻滞减少和细胞凋亡减少。在这项研究中，我们研究了BRCA1蛋白在调节6- tg诱导的MMR损伤反应中的作用，使用了一个等基因人乳腺癌细胞系模型，包括BRCA1突变细胞系（HCC1937）及其与野生型BRCA1 cDNA的转染。MMR蛋白MSH2、MSH6、MLH1和PMS2在两种细胞系中检测到相似的表达。在克隆生存试验中，brca1突变细胞比brca1阳性细胞对6-TG的抗性更强，凋亡减少。此外，突变的BRCA1导致6-TG诱导的G(2)-M细胞周期检查点反应几乎完全丧失。在brca1阳性细胞中转染针对MSH2、MLH1、ATR和Chk1的单个特异性小干扰rna （siRNA）可显著降低brca1依赖性G(2)-M检查点反应。有趣的是，在brca1阳性细胞中，ATR和Chk1 siRNA转染显示出与对照转染相似的6-TG细胞毒性水平，而MSH2和MLH1 siRNA转染则显示出预期的6-TG抗性。通过使用碱性彗星试验（+/-z-VAD-fmk共处理）测量6-TG诱导的DNA链断裂数量和碘脱氧尿嘧啶-DNA结合水平，DNA MMR加工独立于BRCA1，表明BRCA1参与G(2)-M检查点对6-TG的反应，但不参与随后MMR对6-TG错配的切除处理。
Human DNA mismatch repair (MMR) is involved in the response to certain chemotherapy drugs, including 6-thioguanine (6-TG). Consistently, MMR-deficient human tumor cells show resistance to 6-TG damage as manifested by a reduced G(2)-M arrest and decreased apoptosis. In this study, we investigate the role of the BRCA1 protein in modulating a 6-TG-induced MMR damage response, using an isogenic human breast cancer cell line model, including a BRCA1 mutated cell line (HCC1937) and its transfectant with a wild-type BRCA1 cDNA. The MMR proteins MSH2, MSH6, MLH1, and PMS2 are similarly detected in both cell lines. BRCA1-mutant cells are more resistant to 6-TG than BRCA1-positive cells in a clonogenic survival assay and show reduced apoptosis. Additionally, the mutated BRCA1 results in an almost complete loss of a G(2)-M cell cycle checkpoint response induced by 6-TG. Transfection of single specific small interfering RNAs (siRNA) against MSH2, MLH1, ATR, and Chk1 in BRCA1-positive cells markedly reduces the BRCA1-dependent G(2)-M checkpoint response. Interestingly, ATR and Chk1 siRNA transfection in BRCA1-positive cells shows similar levels of 6-TG cytotoxicity as the control transfectant, whereas MSH2 and MLH1 siRNA transfectants show 6-TG resistance as expected. DNA MMR processing, as measured by the number of 6-TG-induced DNA strand breaks using an alkaline comet assay (+/-z-VAD-fmk cotreatment) and by levels of iododeoxyuridine-DNA incorporation, is independent of BRCA1, suggesting the involvement of BRCA1 in the G(2)-M checkpoint response to 6-TG but not in the subsequent excision processing of 6-TG mispairs by MMR.