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Characterization of Acquired Epipodophyllotoxin Resistance in a Chinese Hamster Ovary Cell Line: Loss of Drug-stimulated DNA Cleavage Activity¹

Departments of Pharmacology and Medicine, University of Florida College of Medicine, Gainesville, Florida 32610 [B. G., S. S-K., and W. R.], and Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada [R. G.]

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, Vpm^R-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 Vpm^R-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 Vpm^R-5 line is profoundly resistant to stimulation by VP-16. Further, a somatic cell hybrid line of Vpm^R-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.

¹ This work was supported by American Cancer Society Grant CH-261 and NIH Grant CA-24586.

² Pharmaceutical Manufacturer's Association Clinical Pharmacology Fellow. Present address: Division of Medicine, M. D. Anderson Hospital and Tumor Institute, 6723 Bertner Drive, Houston, TX 77030.

³ To whom requests for reprints should be addressed, at Box J-267, JHMHC, University of Florida, Gainesville, FL 32610.

Received 8/ 6/85. Revised 11/26/85. Accepted 12/11/85.