Mutations in beta-tubulin map to domains involved in regulation of microtubule stability in epothilone-resistant cell lines.

Abstract

The epothilones (Epos) are a group of natural products isolated from the myxobacterium, Sorangium cellulosum. They have a mechanism of action similar to that of Taxol, i.e., they stabilize microtubules and induce the formation of microtubule bundles in cells. Because they are simpler in structure than Taxol and preserve their activity in P-glycoprotein-expressing cells, they are being studied as potential antitumor drugs. In this work, a series of Epo-resistant A549 and HeLa cell lines have been selected and analyzed. Class I beta-tubulin, the major isotype of beta-tubulin in these Epo-resistant cell lines, has been sequenced in a search for mutations. In the Epo B-resistant A549 cells, there is a mutation at beta 292 from Gln to Glu, in the Epo A-resistant HeLa cell line there is a mutation at beta 173 from Pro to Ala, and in the Epo B-resistant HeLa cell line there is a heterozygous mutation at beta 422 from Tyr to a mixture of Tyr and Cys. These mutations are close to the M-loop, the nucleotide-binding site, and the microtubule-associated protein binding sites, respectively. It is likely that these mutations in beta-tubulin provide cells with a mechanism of resistance to the Epos and taxanes. Among these resistant cell lines, A549.EpoB40 is hypersensitive to microtubule-destabilizing drugs, such as vinblastine and colchicine, and HeLa.EpoB1.8 is dependent on the Epos or taxanes for growth. Our studies provide evidence that the M-loop, the GTP binding site, and the microtubule-associated protein binding sites at the COOH terminus in beta-tubulin are critical for the regulation of microtubule stability.