POLYMERASE CHAIN-REACTION ANALYSIS OF CISPLATIN-INDUCED MITOCHONDRIAL-DNA DAMAGE IN HUMAN OVARIAN-CARCINOMA CELLS

The purpose of this study was to determine whether the observed synerg istic interaction between cisplatin and valinomycin (VM) in human ovar ian carcinoma is the result of mitochondrial DNA (mtDNA) damage. A pol ymerase chain reaction (PCR)-based method was used to quantitate the l esion frequencies produced by cisplatin, VM and/or drug combination in a 1.1 kbp segment of mtDNA and a 0.536 kbp segment of the nuclear-loc ated beta-globin gene in human ovarian CaOV-3 carcinoma cells. Our dat a indicates that the nuclear DNA (nDNA) received more cisplatin-induce d damage at doses of 25 mu M or less than did mtDNA. At higher cisplat in doses (50 mu M or more), however, the damage was relatively equal i n both segments. VM alone produced little or no damage on mtDNA, yet a significant amount of damage was detected within nDNA. However, when 1 mu M VM was used in combination with low doses of cisplatin (0-40 mu M), extensive mtDNA damage was detected as compared with the absence of detectable damage on nDNA. In mtDNA, the lesion frequency was 5.45 lesions/10 kb/10 mu M cisplatin in the presence of 1 mu M VM, whereas no detectable lesions were induced by cisplatin alone. This drug combi nation produced no detectable damage on DNA, indicating that cisplatin -induced mtDNA damage could be the basis for the observed synergistic interaction with VM. These results also correlate well with our recent in vivo study with the nude mice model of human ovarian cancer treate d with a cisplatin/liposomal VM drug combination. Furthermore, this re port shows evidence for the role of mitochondria and mtDNA as alternat ive targets for drug action in cancer therapy.