Evaluation of mitomycin-C induced DNA damage and repair in RT4 bladder cancer cells using the alkaline comet assay.

The alkaline comet assay is a rapid, sensitive and relatively inexpensive test for the detection of a variety of DNA lesions. Modifications of the standard protocol have been proposed for the simple and sensitive detection of crosslinking agents (Olive et al., 1992, Olive and Banath, 1995, Pfuhler and Wolf, 1996, Miyamae et al, 1997, Merk and Speit, 1999). Since crosslinking agents connect DNA with DNA or DNA with proteins, they inhibit spontaneous and mutagen induced DNA migration. In this study a modified version of the alkaline comet assay was used to evaluate the DNA crosslinks induced by mitomycin C (MMC) by determining the reduction of DNA migration following 5Gy -irradiation. MMC was observed to reduce -radiation-induced DNA migration in the comet assay in a dose dependent manner following -irradiation exposure just prior to cell lysis. Repair of cross-links following MMC treatment was also followed over a two hour period before cells were irradiated and lysed. An increase in DNA migration was observed with increasing MMC repair time, as the radiation induced breaks were released to form comet tails. To ascertain the effect of MMC alone, a slightly modified experimental strategy was employed whereby 5Gy -irradiation was administered after, instead of before, cell lysis. A similar dose response to that described above was observed and further analysis of MMC-induced cross-link repair is ongoing. These results indicate that DNA cross-link damage and repair can be measured in RT4 cells following 5Gy -irradiation performed before cell lysis. In addition, preliminary results indicate that DNA cross-link damage can also be measured by a novel approach whereby -irradiation is administered after cell lysis. This finding is of particular interest as the effect of MMC alone is measured since the radiation can have no effect on cellular function. It may also prove possible to study MMC-induced cross-link repair and this novel approach has considerable potential in utilising FISH techniques to study preferential gene repair. Hence, these findings merit further investigation.