QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Leontiou, C. Articles by Austin, C. A. Search for Related Content PubMed PubMed Citation Articles by Leontiou, C. Articles by Austin, C. A.

Mutation E522K in Human DNA Topoisomerase II 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

School of Cell and Molecular BioSciences, The Medical School, University of Newcastle-upon-Tyne, United Kingdom

Human cells express two isoforms of topoisomerase II, and , that are both targeted by anticancer drugs. To investigate acridine resistance mediated by topoisomerase II, we used a forced molecular evolution approach. A library of mutated topoisomerase II 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 II 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 E522K protein was unable to support acridine-stimulated DNA cleavage, suggesting that resistance to these acridines is caused by reduced drug-stimulated DNA cleavage. However, E522K 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 E522K 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 E522K at the nonpermissive temperature. This study confirms that topoisomerase II is a target for AMCA and that resistance to AMCA can be mediated by a point mutation at Glu522 in topoisomerase II. Residue 522 lies within a Rossmann fold in the B' subfragment of topoisomerase II, a region previously implicated in drug interactions.

Address correspondence to: Caroline A. Austin, School of Cell and Molecular BioSciences, The Medical School, University of Newcastle-upon-Tyne, UK. E-mail: caroline.austin{at}ncl.ac.uk

This article has been cited by other articles:

				C. Leontiou, G. P. Watters, K. L. Gilroy, P. Heslop, I. G. Cowell, K. Craig, R. N. Lightowlers, J. H. Lakey, and C. A. Austin Differential Selection of Acridine Resistance Mutations in Human DNA Topoisomerase IIbeta Is Dependent on the Acridine Structure Mol. Pharmacol., April 1, 2007; 71(4): 1006 - 1014. [Abstract] [Full Text] [PDF]

				K. L. Gilroy, C. Leontiou, K. Padget, J. H. Lakey, and C. A. Austin mAMSA resistant human topoisomerase II{beta} mutation G465D has reduced ATP hydrolysis activity Nucleic Acids Res., March 20, 2006; 34(5): 1597 - 1607. [Abstract] [Full Text] [PDF]

				C. Leontiou, J. H. Lakey, R. Lightowlers, R. M. Turnbull, and C. A. Austin Mutation P732L in Human DNA Topoisomerase IIbeta Abolishes DNA Cleavage in the Presence of Calcium and Confers Drug Resistance Mol. Pharmacol., January 1, 2006; 69(1): 130 - 139. [Abstract] [Full Text] [PDF]