Mutation E522K in human DNA topoisomerase IIbeta confers resistance to methyl N-(4'-(9-acridinylamino)-phenyl)carbamate hydrochloride and methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide but hypersensitivity to etoposide.
Human cells express two isoforms of topoisomerase II, alpha and beta, that are both targeted by anticancer drugs. To investigate acridine resistance mediated by topoisomerase IIbeta, we used a forced molecular evolution approach. A library of mutated topoisomerase IIbeta cDNAs was generated by hydroxylamine mutagenesis and was transformed into the yeast JN394 top2-4. Methyl N-(4'-(9-acridinylamino)-phenyl)carbamate hydrochloride (AMCA) selection identified a resistant transformant able to grow in media containing 76 microg/ml AMCA. Topoisomerase IIbeta with a glutamic acid-to-lysine substitution at position 522 was responsible for the approximately 10-fold resistance to AMCA. The transformant was cross-resistant to methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide (mAMSA) and mAMCA but hypersensitive to etoposide and ellipticine. In vitro, the betaE522K protein was unable to support acridine-stimulated DNA cleavage, suggesting that resistance to these acridines is caused by reduced drug-stimulated DNA cleavage. However, betaE522K showed DNA cleavage with etoposide, and the cleavable complexes formed with etoposide showed greater stability, thus accounting for the hypersensitivity to etoposide. Drug-independent cleavage of an oligonucleotide by betaE522K was reduced compared with the wild-type enzyme. Decatenation and relaxation activities were reduced to 52 and 61% of the wild-type levels, which may explain the slower growth of yeast strain JN394top2-4 expressing betaE522K at the nonpermissive temperature. This study confirms that topoisomerase IIbeta is a target for AMCA and that resistance to AMCA can be mediated by a point mutation at Glu522 in topoisomerase IIbeta. Residue 522 lies within a Rossmann fold in the B' subfragment of topoisomerase II, a region previously implicated in drug interactions.
Leontiou
Chrysoula
C
School of Cell and Molecular BioSciences, The Medical School, University of Newcastle-upon-Tyne, UK.
Lakey
Jeremy H
JH
Austin
Caroline A
CA
D000595:Amino Acid Sequence; D000677:Amsacrine / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D005047:Etoposide / Q000494:pharmacology*; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D059005:Topoisomerase II Inhibitors; D014148:Tranexamic Acid / Q000494:pharmacology*
D000970:Antineoplastic Agents; D059005:Topoisomerase II Inhibitors; D000677:Amsacrine; D018698:Glutamic Acid; D005047:Etoposide; D014148:Tranexamic Acid; D004247:DNA; D004250:DNA Topoisomerases, Type II; D008239:Lysine