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Research Articles: Therapeutics, Targets, and Development

Amino acid substitutions at proline 220 of ß-tubulin confer resistance to paclitaxel and colcemid

Departments of ¹ Integrative Biology and Pharmacology and ² Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas

Requests for reprints: Fernando Cabral, Department of Integrative Biology and Pharmacology, University of Texas Medical School, P. O. Box 20708, Houston, TX 77225. Phone: 713-500-7485; Fax: 713-500-7455. E-mail: Fernando.R.Cabral{at}uth.tmc.edu

Abstract

Chinese hamster ovary cells selected for resistance to paclitaxel have a high incidence of mutations affecting L215, L217, and L228 in the H6/H7 loop region of ß1-tubulin. To determine whether other mutations in this loop are also capable of conferring resistance to drugs that affect microtubule assembly, saturation mutagenesis of the highly conserved P220 codon in ß1-tubulin cDNA was carried out. Transfection of a mixed pool of plasmids encoding all possible amino acid substitutions at P220 followed by selection in paclitaxel produced cell lines containing P220L and P220V substitutions. Similar selections in colcemid, on the other hand, yielded cell lines with P220C, P220S, and P220T substitutions. Site-directed mutagenesis and retransfection confirmed that these mutations were responsible for drug resistance. Expression of tubulin containing the P220L and P220V mutations reduced microtubule assembly, conferred resistance to paclitaxel and epothilone A, but increased sensitivity to colcemid and vinblastine. In contrast, tubulin with the P220C, P220S, and P220T mutations increased microtubule assembly, conferred resistance to colcemid and vinblastine, but increased sensitivity to paclitaxel and epothilone A. The results are consistent with molecular modeling studies and support a drug resistance mechanism based on changes in microtubule assembly that counteract the effects of drug treatment. These studies show for the first time that different substitutions at the same amino acid residue in ß1-tubulin can confer cellular resistance to either microtubule-stabilizing or microtubule-destabilizing drugs. [Mol Cancer Ther 2007;6(10):2798–806]

Grant support: NIH grant CA85935.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

³ Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

Received 12/21/06; revised 8/14/07; accepted 8/30/07.

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