Cisplatin DNA damage formation and repair in non-small cell lung cancer cell lines as determined by the alkaline comet assay
Authors: Gabriela M. Almeida, Peter B. Farmer, William P. Steward, George D. Jones
Journal: Cancer Research (2004)

1544   Cisplatin is one of the most widely used agents in chemotherapy. In DNA, it binds to the N7 position of purines to form intra- and inter-strand crosslinks and monofunctional adducts. These lesions are thought to be responsible for the cytotoxic effects of cisplatin, with higher levels of damage and/or its poorer repair leading to greater cell death. The response rate to chemotherapy varies considerably between individuals. If we could predict a patient’s response to a certain chemotherapy regimen it may be possible to greatly increase treatment success rates. In the present study we are evaluating two DNA damage measuring protocols as means of assessing/predicting cancer cell death due to cisplatin. Using a  32 P-postlabelling protocol we have detected cisplatin-induced GG and AG intra-strand crosslinks in DNA treated  in vitro . However, due to the limited sensitivity of the assay we were unable to detect these lesions in lymphocyte DNA of patients receiving cisplatin-based chemotherapy. Our recent studies have focused on using the alkaline comet assay, which is a rapid and sensitive method for measuring DNA damage formation and repair in individual cells. Response to cisplatin was investigated in a panel of five NSCLC cell lines (three adenocarcinoma, one squamous and one large cell carcinoma). Survival was determined by the clonogenic assay whereby cells were incubated with cisplatin at doses between 2.5μM to 50μM for one hour, then washed free of the drug and incubated until colonies were visible. For alkaline comet analysis, the cells were treated with cisplatin doses ranging from 5μM to 250μM for one hour, washed and then incubated for between 0 and 72 hours. Crosslink formation and repair was determined by a modification of the alkaline comet assay whereby the cells are irradiated (10Gy) immediately prior to analysis and where a decrease in Olive tail moment is indicative of crosslink formation. Similarly, after a period of post-treatment incubation, an increase in Olive tail moment indicates crosslink repair. Measures of survival show a differential response to cisplatin, with one of the adenocarcinoma cell lines being most chemoresistant. Initial comet assay results demonstrate the dose-dependent formation of cisplatin induced lesions, with this formation continuing for several hours after the drug is removed from the media. Maximal DNA crosslink formation occurs 2 to 4 hours post-treatment, and repair of these lesions commences between 10 and 24 hours. These initial studies demonstrate the alkaline comet assay to be a suitable method to detect cisplatin damage in NSCLC cells even at low clinically relevant doses. However, further studies are required to determine if this method can be applied as a predictive test of chemosensitivity for cancer patients receiving cisplatin chemotherapy. The comet assay is ideal as a clinical test as it is rapid, inexpensive and requires only low cell numbers.