A Taxol-resistant cell line, K20T, which does not express P-glycoprotein, was selected with Taxol from human MDA-MB-231 breast adenocarcinoma cells and maintained in the presence of 20 nM Taxol. K20T cells were ∼18-fold resistant to Taxol, displayed cross-resistance to Taxotere and the epothilones, but little cross-resistance to discodermolide. Sequence analysis of the class I β-tubulin indicated that it harbored an A593G mutation resulting in a change from glutamate to glycine at amino acid 198, which is near the intradimer interface within the α/β-tubulin heterodimer. An HA-tagged wild-type class I β-tubulin expression vector was transfected into the K20T cells. Immunofluorescence studies demonstrated that this exogenous tubulin was incorporated into cellular microtubules and Western blot analysis indicated that the K20T transfectants predominantly expressed the exogenous wild-type class I β-tubulin. The transfected cells were only ∼5-fold resistant to Taxol. Our results, plus the knowledge that Glu198 is the target for other anti-tubulin agents, suggest that glutamate198 in β-tubulin is a critical determinant for microtubule stability and Taxol resistance.
ASJC Scopus subject areas
- Cancer Research