Comet assay and flow cytometry analysis of DNA repair in normal and cancer cells treated with known mutagens with different mechanisms of action.
In order to determine differences in repair after treatment with DNA damaging agents, normal and cancer cells were selected for analysis of single strand breaks and DNA crosslinks using the Comet assay. Normal human lymphocytes, human colorectal adenocarcinoma SW620 cells, lung carcinoma A549, and H460 cell lines were exposed to an ethylating agent (ethylmethane sulfonate [EMS]), and a cross-linking agent (mitomycin C [MMC]). Differences in repair profiles of DNA damage demonstrated using the comet assay were observed in human lymphocytes and tumour cell lines with both mutagens. Results were also indicative that MMC repair is concentration-dependent. It was also apparent that normal cells repair DNA damage more readily than tumour cells. Repair also varied between different cell lines. To investigate the mechanistic differences of these two chemicals, flow cytometry studies were undertaken in tumour cells, namely cell cycle analysis and frequency of micronuclei induction (FMN). A G2M phase block was clearly evident following treatment with EMS at all concentrations tested. With MMC, an initial arrest of cells in G2M was accompanied by a build-up in S-phase over longer exposure periods. Also, at the highest mutagen doses there were different patterns of micronuclei induction. Thus, using the mutagens with different mechanisms of action highlighted the differences in repair patterns between normal and tumour cells.
Suggitt
M
M
Tom Connors Cancer Research Centre, University of Bradford, Bradford, United Kingdom.
Fearnley
J
J
Swaine
D J
DJ
Volpato
M
M
Phillips
R M
RM
Bibby
M C
MC
Loadman
P M
PM
Anderson
D
D
D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D020552:Comet Assay / Q000379:methods*; D003432:Cross-Linking Reagents / Q000633:toxicity; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage; D004260:DNA Repair; D004273:DNA, Neoplasm / Q000187:drug effects*; D005020:Ethyl Methanesulfonate / Q000633:toxicity; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects; D048629:Micronuclei, Chromosome-Defective / Q000187:drug effects; D016685:Mitomycin / Q000633:toxicity; D009153:Mutagens / Q000633:toxicity*; D009369:Neoplasms / Q000235:genetics
D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm; D009153:Mutagens; D016685:Mitomycin; D004247:DNA; D005020:Ethyl Methanesulfonate