Mutations at arg486 and glu571 in human topoisomerase II alpha confer resistance to amsacrine: Relevance for antitumor drug resistance in human cells

Human topoisomerase II, a nuclear protein involved in chromosome segregatio n, is the target of amsacrine and other clinically important anticancer dru gs. The enzyme is expressed as alpha and beta isoforms whose mutation/down- regulation has been implicated in drug resistance. To understand the role o f target mutations in cellular drug resistance, we have used yeast to selec t and characterize plasmid-borne human topoisomerase II a mutants resistant to amsacrine. Single point changes of Glu571 to Lys (E571K) or Arg486 to L ys (R486K) in the conserved PLRGK motif, both of which reside in the GyrB h omology domain of human topoisomerase II a, were frequently selected and co uld be shown in vivo to confer >25-fold and >100-fold resistance, respectiv ely, to amsacrine and similar to 3-fold cross-resistance to etoposide. High ly purified E571K and R486K human topoisomerase II a proteins required 100- fold higher levels of amsacrine to induce DNA cleavage similar to that of w ild-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 hu man biopsy material, establish unequivocally that it confers resistance, an d suggest mechanisms for its phenotypic expression in vivo. These results d iffer significantly from previous work using yeast topoisomerase II as a mo del system: introduction of the equivalent mutation to R486K (R476K) into t he yeast enzyme did not give amsacrine resistance. We conclude that species -specific differences in topoisomerase II enzymes can affect the drug resis tance phenotype of particular mutations and highlight the need to study the relevant human homolog.