DNA mismatch repair (MMR) mediates 6-thioguanine genotoxicity by introducing single-strand breaks to signal a G2-M arrest in MMR-proficient RKO cells.
DNA mismatch repair (MMR) mediates 6-thioguanine genotoxicity by introducing single-strand breaks to signal a G2-M arrest in MMR-proficient RKO cells.
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发表时间：
2003-06
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通讯作者：
T. Yan;S. E. Berry;Anand B. Desai;T. Kinsella
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作者：
T. Yan;S. E. Berry;Anand B. Desai;T. Kinsella
DNA错配修复（MMR）系统在介导各种化疗药物引起的细胞死亡中起重要作用，但其分子机制尚不清楚。在这项研究中，我们试图确定在6-硫代鸟嘌呤（6-TG）处理后，MMR引入了什么信号，以发出G（2）-M阻滞信号，导致细胞死亡。实验设计使用我们为此研究建立的同基因MMR（+）和MMR（-）人结肠直肠癌RKO细胞系统进行比较研究。将细胞暴露于6-TG（3 μ M × 24 h），然后在接下来的3-6天每天收获用于生长抑制测定。流式细胞术测定细胞周期的影响，并使用脉冲场凝胶电泳和碱性彗星试验测定DNA链断裂。结果我们首次将人MutL同源物1（hMLH 1）cDNA转染到缺失hMLH 1（MMR（-））的RKO细胞系中，建立了MMR（+）RKO细胞系。异位表达的hMLH 1蛋白在hMLH 1（+）转染子中恢复了MMR-熟练的表型，与载体对照相比，6-TG暴露后显示出显著增加和延长的G（2）-M停滞，随后细胞死亡。MMR介导的、6-TG诱导的G（2）-M阻滞在第1天开始，在第3天达到高峰，并持续到6-TG去除后的第6天。我们发现，在我们的MMR（+）和MMR（-）细胞中，DNA双链断裂均不可避免地产生，在6-TG处理的1天内达到峰值。相反，单链断裂（SSB）在MMR（+）细胞中更频繁且持续时间更长，并且SSB形成的持续时间与6-TG诱导的G（2）-M阻滞的时间过程在时间上相关。结论我们的数据表明，MMR介导6-TG诱导的G（2）-M阻滞，通过引入SSB信号持续G（2）-M阻滞，导致细胞死亡增加。
PURPOSE The DNA mismatch repair (MMR) system plays an important role in mediating cell death after treatment with various types of chemotherapeutic agents, although the molecular mechanisms are not well understood. In this study, we sought to determine what signal is introduced by MMR after 6-thioguanine (6-TG) treatment to signal a G(2)-M arrest leading to cell death. EXPERIMENTAL DESIGN A comparison study was carried out using an isogenic MMR(+) and MMR(-) human colorectal cancer RKO cell system, which we established for this study. Cells were exposed to 6-TG (3 micro M x 24 h) and then harvested daily for the next 3-6 days for growth inhibition assays. Cell cycle effects were determined by flow cytometry, and DNA strand breaks were measured using pulsed-field gel electrophoresis and alkaline Comet assays. RESULTS We first established MMR(+) RKO cell lines by transfection of human MutL homologue 1 (hMLH1) cDNA into the hMLH1-deficient (MMR(-)) RKO cell line. The ectopically expressed hMLH1 protein restored a MMR-proficient phenotype in the hMLH1(+) transfectants, showing a significantly increased and prolonged G(2)-M arrest followed by cell death after 6-TG exposure, compared with the vector controls. The MMR-mediated, 6-TG-induced G(2)-M arrest started on day 1, peaked on day 3, and persisted to day 6 after 6-TG removal. We found that DNA double-strand breaks were comparably produced in both our MMR(+) and MMR(-) cells, peaking within 1 day of 6-TG treatment. In contrast, single-strand breaks (SSBs) were more frequent and longer lived in MMR(+) cells, and the duration of SSB formation was temporally correlated with the time course of 6-TG-induced G(2)-M arrest. CONCLUSIONS Our data suggest that MMR mediates 6-TG-induced G(2)-M arrest by introducing SSBs to signal a persistent G(2)-M arrest leading to enhanced cell death.