Mediation of multi-drug resistance in a Chinese hamster ovary cell line by a mutant type II topoisomerase

Identification of DNA topoisomerase II as the intracellular target for the DNA cleavage activity of the epipodophyllotoxins and several intercalating agents is well established. In contrast, definite correlation of cleavable complex formation with eventual cell death has been more difficult to document. Our studies with an epipodophyllotoxin resistant cell line not only provide additional evidence that the enzyme is a multi-drug target, but also implicate drug-stimulated cleavage activity as a critical component of cytotoxic effect. When compared to WT (wild type) cells, the mutant Chinese hamster ovary cell line, VpmR-5, exhibits marked resistance to both the cytotoxic and DNA cleavage activity of etoposide as well as various intercalating agents. Steady state concentrations of these drugs in both cell lines is identical. Enzyme content as measured by immunoblot and by total decatenation activity in crude nuclear extracts is equal. Catalytic activity is also equally sensitive to inhibition by etoposide. In contrast, cleavage activity in purified enzyme from VpmR-5 cells is profoundly resistant to stimulation by drug. These data indicate that a multi-drug resistant phenotype may be acquired by a qualitative change in the enzyme that alters its interaction with drug. Further, the data strongly support a direct role for cleavable complex formation in cell death.