McKenna Declan J, Gallus Massimo, McKeown Stephanie R, Downes C Stephen, McKelvey-Martin Valerie J
School of Biomedical Sciences, University of Ulster, Northern Ireland, Coleraine BT52 1SA, UK.
DNA Repair (Amst). 2003 Aug 12;2(8):879-90. doi: 10.1016/s1568-7864(03)00086-7.
The alkaline Comet assay is a simple, sensitive method for measuring the extent of DNA strand breaks in individual cells. Several modifications to the original assay have been developed to increase its applications. One such modification allows the measurement of DNA cross-links by assessing the relative reduction in DNA migration induced by a strand-breaking agent. Another modification includes the application of fluorescent in situ hybridisation (FISH) to investigate the localisation of specific gene domains within a cell. Although several studies have used these approaches separately, no report to date has combined these two versions of the Comet assay. The current study describes the modification of the Comet assay, to allow both measurement of mitomycin C (MMC)-induced cross-links and the subsequent application of FISH to study repair in the TP53 gene region. RT4 human bladder cancer cells were treated with 0, 5, 50 and 200 microg/ml MMC to study dose response, whilst for cross-link repair studies, they were treated with 50 microg/ml MMC and allowed to repair for up to 24 h. A clear dose response to MMC was displayed, demonstrable by a marked reduction in DNA migration, whilst repair studies showed that MMC-induced cross-links take at least 24 h to repair fully in RT4 cells. For Comet-FISH experiments, the number and location of TP53 hybridisation spots was also recorded for each cell. In dose response experiments, the number of spots per cell, and per Comet tail, decreased as MMC dose increased. In repair experiments, the number of spots, particularly in the Comet tail, increased as repair time increased. Furthermore, our results suggest that repair of the TP53 gene region is most rapid within the first 4 h following MMC treatment. We conclude that the novel experimental protocol presented here has considerable potential in evaluating DNA damage and sequence-related repair responses to cross-linking agents.
碱性彗星试验是一种用于测量单个细胞中DNA链断裂程度的简单、灵敏的方法。人们已对原始试验进行了多种改进以扩大其应用范围。其中一种改进是通过评估链断裂剂诱导的DNA迁移相对减少来测量DNA交联。另一种改进包括应用荧光原位杂交（FISH）来研究特定基因结构域在细胞内的定位。尽管已有多项研究分别使用了这些方法，但迄今为止尚无将这两种彗星试验版本结合起来的报告。本研究描述了对彗星试验的改进，使其既能测量丝裂霉素C（MMC）诱导的交联，又能随后应用FISH研究TP53基因区域的修复情况。用0、5、50和200微克/毫升的MMC处理RT4人膀胱癌细胞以研究剂量反应，而在交联修复研究中，用50微克/毫升的MMC处理细胞并使其修复长达24小时。显示出对MMC有明显的剂量反应，可通过DNA迁移的显著减少来证明，而修复研究表明，MMC诱导的交联在RT4细胞中至少需要24小时才能完全修复。对于彗星-FISH实验，还记录了每个细胞中TP53杂交点的数量和位置。在剂量反应实验中，随着MMC剂量的增加，每个细胞和每条彗星尾的杂交点数减少。在修复实验中，杂交点的数量，特别是在彗星尾中的数量，随着修复时间的增加而增加。此外，我们的结果表明，MMC处理后的前4小时内TP53基因区域的修复最为迅速。我们得出结论，本文提出的新实验方案在评估DNA损伤以及对交联剂的序列相关修复反应方面具有相当大的潜力。
