Doxorubicin is a therapeutically useful anticancer drug that exerts multiple biological effects. Its antitumor and cardiotoxic properties have been ascribed to anthracycline-mediated free radical damage to DNA and membranes. Evidence for this idea comes in part from the selection by doxorubicin from stationary phase yeast cells of mutants (petites) deficient in mitochondrial respiration and therefore defective in free radical generation. However, doxorubicin also binds to DNA topoisomerase II, converting the enzyme into a DNA damaging agent through the trapping of a covalent enzyme-DNA complex termed the 'cleavable complex.' We have used yeast to determine whether stabilization of cleavable complexes plays a role in doxorubicin action and cytotoxicity. A plasmid-borne yeast TOP2 gene was mutagenized with hydroxylamine and used to transform drug-permeable yeast strain JN394t2-4, which carries a temperature-sensitive top2-4 mutation in its chromosomal TOP2 gene. Selection in growth medium at the nonpermissive temperature of 35 degrees in the presence of doxorubicin resulted in the isolation of plasmid-borne top2 mutants specifying functional doxorubicin-resistant DNA topoisomerase II. Single-point changes of Gly748 to Glu or Ala642 to Ser in yeast topoisomerase II, which lie in and adjacent to the CAP-like DNA binding domain, respectively, were identified as responsible for resistance to doxorubicin, implicating these regions in drug action. None of the mutants selected in JN394t2-4, which has a rad52 defect in double-strand DNA break repair, was respiration-deficient. We conclude that topoisomerase II is an intracellular target for doxorubicin and that the genetic background and/or cell proliferation status can determine the relative importance of topoisomerase II- versus free radical-killing.

Human topoisomerase II, a nuclear protein involved in chromosome segregation, is the target of amsacrine and other clinically important anticancer drugs. The enzyme is expressed as alpha and beta isoforms whose mutation/down-regulation has been implicated in drug resistance. To understand the role of target mutations in cellular drug resistance, we have used yeast to select and characterize plasmid-borne human topoisomerase IIalpha mutants resistant to amsacrine. Single point changes of Glu571 to Lys (E571K) or Arg486 to Lys (R486K) in the conserved PLRGK motif, both of which reside in the GyrB homology domain of human topoisomerase IIalpha, were frequently selected and could be shown in vivo to confer>25-fold and >100-fold resistance, respectively, to amsacrine and approximately 3-fold cross-resistance to etoposide. Highly purified E571K and R486K human topoisomerase IIalpha proteins required 100-fold higher levels of amsacrine to induce DNA cleavage similar to that of wild-type protein, consistent with a resistance mechanism involving reduced cleavable complex formation. Our functional studies of the R486K mutation, previously identified in two amsacrine-resistant human cell lines and in human biopsy material, establish unequivocally that it confers resistance, and suggest mechanisms for its phenotypic expression in vivo. These results differ significantly from previous work using yeast topoisomerase II as a model system: introduction of the equivalent mutation to R486K (R476K) into the yeast enzyme did not give amsacrine resistance. We conclude that species-specific differences in topoisomerase II enzymes can affect the drug resistance phenotype of particular mutations and highlight the need to study the relevant human homolog.