Correlation of taxol resistance with beta-tubulin mutations
Wiesen, Kenneth Marc
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Both P-glycoprotein (P-gp) and non-P-gp expressing Taxol-resistant cell lines were selected from MDA-MB-231 (MDA-231) breast adenocarcinoma cells in the presence of Taxol. One subline (K20T), which did not express P-gp, was maintained in the presence of 20 nM Taxol. K20T cells were 17-fold resistant to Taxol and displayed cross resistance to Taxotere and the epothilones, but little cross resistance to discodermolide, vinblastine, or doxorubicin. Western blot analysis of tubulin, the cellular target of Taxol, indicated that class I beta-tubulin was the predominant isoform in the MDA-231 cells. The class I beta-tubulin of the K20T cells displayed an increased electrophoretic mobility when compared to the tubulin from the parental cells. Two-dimensional gel electrophoresis demonstrated that the beta-tubulin from the K20T cells was more basic than the tubulin in the parental cells. Sequence analysis of the class I beta-tubulin indicated that K20T beta-tubulin harbored an A593G mutation resulting in a change from glutamate to glycine at amino acid 198, near the alpha/beta-tubulin heterodimer interface. This alteration would be consistent with a more basic tubulin. To determine if this mutation was responsible for Taxol resistance, an HA-tagged wild-type class I beta-tubulin expression vector (BI-HA) was created and stably transfected into the K20T cells. The K20T transfectants predominantly expressed the exogenous wild type class I beta-tubulin, that was incorporated into cellular microtubules. These transfected cells were sensitized to Taxol, and were now only 4- to 5-fold resistant to the drug. Our results indicate that position 198 in beta-tubulin is a critical determinant of Taxol resistance. Another point mutation, Q292E, has been found in beta-tubulin from a drug-resistant A549 human non-small lung cancer cell line selected in epothilone B. These cells are 95-fold resistant to epothilone B, and 22-fold resistant to Taxol. This mutation, Q292E, is located near the M-loop at the interface between adjacent protofilaments, and near the binding site for Taxol/epothilone. Several Q292E BI-HA stable transfectants have been isolated and shown by cDNA sequencing and Western blot analysis to express the HA-tagged mutant beta-tubulin. Cytotoxicity results indicated that MDA-231 cells expressing Q292E BI-HA were approximately 7-fold resistant to Taxol compared to MDA-231 cells transfected with wild type BI-HA. These results demonstrate that the expression of Q292E class I beta-tubulin, can be a contributing factor in resistance towards microtubule stabilizing drugs.