Clingen P H, Wu J Y-H, Miller J, Mistry N, Chin F, Wynne P, Prise K M, Hartley J A
Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, 72 Huntley Street, London, WC1E 6DD, UK.
Biochem Pharmacol. 2008 Jul 1;76(1):19-27. doi: 10.1016/j.bcp.2008.03.025. Epub 2008 Apr 16.
The aims of this study were to investigate mechanisms of action involved in H2AX phosphorylation by DNA interstrand crosslinking (ICL) agents and determine whether gammaH2AX could be a suitable pharmacological marker for identifying potential ICL cellular chemosensitivity. In normal human fibroblasts, after treatment with nitrogen mustard (HN2) or cisplatin, the peak gammaH2AX response was detected 2-3 h after the peak of DNA ICLs measured using the comet assay, a validated method for detecting ICLs in vitro or in clinical samples. Detection of gammaH2AX foci by immunofluorescence microscopy could be routinely detected with 6-10 times lower concentrations of both drugs compared to detection of ICLs using the comet assay. A major pathway for repairing DNA ICLs is the initial unhooking of the ICL by the ERCC1-XPF endonuclease followed by homologous recombination. HN2 or cisplatin-induced gammaH2AX foci persisted significantly longer in both, ERCC1 or XRCC3 (homologous recombination) defective Chinese hamster cells that are highly sensitive to cell killing by ICL agents compared to wild type or ionising radiation sensitive XRCC5 cells. An advantage of using gammaH2AX immunofluorescence over the comet assay is that it appears to detect ICL chemosensitivity in both ERCC1 and HR defective cells. With HN2 and cisplatin, gammaH2AX foci also persisted in chemosensitive human ovarian cancer cells (A2780) compared to chemoresistant (A2780cisR) cells. These results show that gammaH2AX can act as a highly sensitive and general marker of DNA damage induced by HN2 or cisplatin and shows promise for predicting potential cellular chemosensitivity to ICL agents.
本研究的目的是探究DNA链间交联（ICL）剂诱导H2AX磷酸化的作用机制，并确定γH2AX是否可作为一种合适的药理学标志物，用于识别潜在的ICL细胞化学敏感性。在正常人成纤维细胞中，用氮芥（HN2）或顺铂处理后，使用彗星试验检测到DNA ICL峰值后2 - 3小时，检测到γH2AX反应峰值，彗星试验是一种在体外或临床样本中检测ICL的有效方法。与使用彗星试验检测ICL相比，通过免疫荧光显微镜检测γH2AX灶，两种药物的浓度通常可低6 - 10倍。修复DNA ICL的主要途径是首先由ERCC1 - XPF核酸内切酶解开ICL，然后进行同源重组。与野生型或对电离辐射敏感的XRCC5细胞相比，HN2或顺铂诱导的γH2AX灶在对ICL剂细胞杀伤高度敏感的ERCC1或XRCC3（同源重组）缺陷型中国仓鼠细胞中持续的时间明显更长。使用γH2AX免疫荧光法相对于彗星试验的一个优点是，它似乎能检测ERCC1和HR缺陷细胞中的ICL化学敏感性。对于HN2和顺铂，与化疗耐药（A2780cisR）细胞相比，γH2AX灶在化疗敏感的人卵巢癌细胞（A2780）中也持续存在。这些结果表明，γH2AX可作为HN2或顺铂诱导的DNA损伤的高度敏感且通用的标志物，并有望预测细胞对ICL剂的潜在化学敏感性。
