Identification of Yeast DNA Topoisomerase II Mutants Resistant to the Antitumor Drug Doxorubicin: Implications for the Mechanisms of Doxorubicin Action and Cytotoxicity

Abstract

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-sensitivetop2–4 mutation in its chromosomal TOP2gene. Selection in growth medium at the nonpermissive temperature of 35° 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.