Mutations at arg486 and glu571 in human topoisomerase IIalpha confer resistance to amsacrine: relevance for antitumor drug resistance in human cells.
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.
Patel
S
S
Molecular Genetics Group, Department of Biochemistry, St. George's Hospital Medical School, University of London, London, United Kingdom.
Keller
B A
BA
Fisher
L M
LM
D000677:Amsacrine / Q000494:pharmacology*; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001120:Arginine; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005047:Etoposide / Q000494:pharmacology; D018698:Glutamic Acid; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D012441:Saccharomyces cerevisiae; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D000677:Amsacrine; D018698:Glutamic Acid; D005047:Etoposide; D004247:DNA; D001120:Arginine; D004250:DNA Topoisomerases, Type II