Hartley J M, Spanswick V J, Gander M, Giacomini G, Whelan J, Souhami R L, Hartley J A
Department of Oncology, UCL Medical School, London, United Kingdom.
Clin Cancer Res. 1999 Mar;5(3):507-12.
The single cell gel electrophoresis comet assay has become established as a sensitive technique for measuring DNA strand breaks. The technique has been modified to allow the sensitive detection and quantitation of DNA interstrand cross-linking at the single cell level. Cells are irradiated immediately before analysis to deliver a fixed level of random strand breakage. After embedding of cells in agarose and lysis, the presence of cross-links retards the electrophoretic mobility of the alkaline denatured cellular DNA. Cross-links are, therefore, quantitated as the decrease in the comet tail moment compared with irradiated controls. Using this method, a linear response of cross-linking versus dose of chlorambucil over a wide dose range was demonstrated in human lymphocytes after drug treatment ex vivo. The method was also sensitive enough to determine cross-linking in clinical samples after chemotherapy. For example, crosslinking was observed in the lymphocytes of patients receiving ifosfamide (3 g/m2/day) as a continuous infusion for 3-5 days or as a 3-h infusion daily for 3 days. Cross-links were detected in all patients within 3 h, with no evidence of DNA single strand break formation. In patients receiving continuous infusion, a plateau of cross-linking was reached by 24 h. In the patients receiving ifosfamide over 3 h, a clear decrease in the peak level of cross-linking was observed before subsequent infusions.
单细胞凝胶电泳彗星试验已成为一种用于测量DNA链断裂的灵敏技术。该技术已被改进，以允许在单细胞水平上灵敏地检测和定量DNA链间交联。在分析前立即对细胞进行照射，以产生固定水平的随机链断裂。将细胞包埋在琼脂糖中并裂解后，交联的存在会阻碍碱性变性细胞DNA的电泳迁移率。因此，交联通过与照射对照相比彗星尾矩的降低来定量。使用这种方法，在体外药物处理后的人淋巴细胞中，在很宽的剂量范围内证明了交联与苯丁酸氮芥剂量之间呈线性反应。该方法也足够灵敏，能够测定化疗后临床样本中的交联情况。例如，在接受异环磷酰胺（3 g/m²/天）持续输注3 - 5天或每天3小时输注3天的患者淋巴细胞中观察到交联。在所有患者中，3小时内检测到交联，没有DNA单链断裂形成的证据。在接受持续输注的患者中，24小时时达到交联平台期。在接受3小时异环磷酰胺输注的患者中，在后续输注前观察到交联峰值水平明显下降。
