Characterization of acquired epipodophyllotoxin resistance in a Chinese hamster ovary cell line: loss of drug-stimulated DNA cleavage activity.

Recent evidence indicates that type II DNA topoisomerases mediate epipodophyllotoxin-induced DNA damage and may be intrinsic to the drug's antitumor effects. Using an epipodophyllotoxin-resistant cell line, we have now further defined the relationship between DNA damage and cell death and delineated the significance of certain drug-enzyme interactions. When compared to wild-type cells, the mutant Chinese hamster ovary cell line, VpmR-5, exhibits marked resistance to both the cytotoxic and DNA cleavage activities of etoposide (VP-16). Steady-state concentrations of radiolabeled VP-16 are identical in both cell lines. Catalytic activity in crude nuclear extracts from wild-type and VpmR-5 cells is equal and is equally sensitive to inhibition by VP-16. However, using an assay that specifically measures generation of 5' protein-linked breaks in 32P-labeled 3' DNA, we have found that DNA cleavage activity in nuclear extract from the VpmR-5 line is profoundly resistant to stimulation by VP-16. Further, a somatic cell hybrid line of VpmR-5 cells and drug-sensitive EOT-3 cells exhibits recovery of VP-16 sensitivity in concert with reconstitution of DNA cleavage activity. These data indicate that stimulation of enzyme-mediated DNA cleavage, rather than loss of normal topoisomerase function, is responsible for epipodophyllotoxin-induced cytotoxicity.