Lookup NU author(s): Chrysoula Leontiou,
Professor Jeremy Lakey,
Professor Caroline Austin
Full text for this publication is not currently held within this repository. Alternative links are provided below where available.
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