Lookup NU author(s): Professor Jeremy Lakey,
Professor Caroline Austin
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Human cells express two isoforms of topoisomerase II, α and β, that are both targeted by anticancer drugs. To investigate acridine resistance mediated by topoisomerase IIβ, we used a forced molecular evolution approach. A library of mutated topoisomerase IIβ 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 μg/ml AMCA. Topoisomerase IIβ with a glutamic acid-to-lysine substitution at position 522 was responsible for the ∼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 βE522K protein was unable to support acridine-stimulated DNA cleavage, suggesting that resistance to these acridines is caused by reduced drug-stimulated DNA cleavage. However, βE522K 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 βE522K 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 βE522K at the nonpermissive temperature. This study confirms that topoisomerase IIβ is a target for AMCA and that resistance to AMCA can be mediated by a point mutation at Glu522 in topoisomerase IIβ. Residue 522 lies within a Rossmann fold in the B′ subfragment of topoisomerase II, a region previously implicated in drug interactions.
Author(s): Leontiou C, Lakey JH, Austin CA
Publication type: Article
Publication status: Published
Journal: Molecular Pharmacology
ISSN (print): 0026-895X
ISSN (electronic): 1521-0111
Publisher: American Society for Pharmacology and Experimental Therapeutics
PubMed id: 15322234