Abstract

Drug resistance to inhibitors of DNA topoisomerase II can result from qualitative or quantitative alterations in the target enzyme, topoisomerase II, or from perturbations in drug transport that may or may not involve P-glycoprotein. In the present study, a drug-resistant Chinese hamster ovary cell line, SMR16, was selected in the presence of an epipodophyllotoxin (VP-16) and was found to be cross-resistant to all classes of topoisomerase II inhibitors (3-35-fold). The 3-fold level of resistance of these cells to vincristine is likely due to diminished uptake of this drug, and this is not mediated by overexpression of P-glycoprotein. No alteration in transport of VP-16 was observed. Immunoblotting with several polyclonal anti-topoisomerase II antibodies demonstrated that the resistant cells contain approximately two-thirds of the parental enzyme amount. The topoisomerase II catalytic activity present in 0.35 M NaCl nuclear extracts paralleled this decrease. VP-16- and 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced DNA damage, mediated by topoisomerase II, was found to be decreased 10-12-fold in both intact SMR16 cells and nuclei isolated from these cells, when measured by alkaline filter elution. However, the VP-16-induced DNA cleavage activity present in 0.35 M NaCl nuclear extracts of the resistant cells was attenuated only 2-fold, relative to wild-type cells. Homogeneous preparations of the enzyme obtained from resistant cells demonstrated the same cleavage and catalytic activity as purified wild-type topoisomerase II. Analysis by pulse-field gel electrophoresis of the DNA isolated from VM-26- and 4'-(9-acridinylamino)methanesulfon-m-anisidide-treated sensitive and resistant cells demonstrated significantly less conversion of SMR16 chromosomal DNA into 50-150-kilobase DNA fragments. Chinese hamster ovary SMR16 cells are apparently resistant to topoisomerase II poisons because the topoisomerase II that defines the DNA topological domains is either decreased in amount or insensitive to drug action.