ALTERED BETA-TUBULIN ISOTYPE EXPRESSION IN PACLITAXEL-RESISTANT HUMANPROSTATE CARCINOMA-CELLS

To investigate the role of beta-tubulin isotype composition in resista nce to paclitaxel, an anti-microtubule agent, human prostate carcinoma (DU-145) cells were intermittently exposed to increasing concentratio ns of paclitaxel, Cells that were selected and maintained at 10 nM pac litaxel (Pac-10) were fivefold resistant to the drug. Pac-10 cells acc umulated radiolabelled paclitaxel to the same extent as DU-145 cells a nd were negative for MDR-1. Analysis of Pac-10 and DU-145 cells by flo w cytometry showed similar cell cycle patterns, Immunofluorescent stai ning revealed an overall increase of alpha- and beta-tubulin levels in Pac-10 cells compared with DU-145 cells, Examination of beta-tubulin isotype composition revealed a significant increase in beta(III) isoty pe in the resistant cells, both by immunofluorescence and by western b lot analysis. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the isotypes confirmed the increase observed for the beta (III) by exhibiting ninefold higher beta(III) mRNA levels and also sho wed fivefold increase of the beta(IVa) transcript. In addition, analys is of paclitaxel-resistant cells that were selected at increasing leve ls of the drug (Pac 2, 4, 6, 8 and 10) exhibited a positive correlatio n between increasing beta(III); levels and increasing resistance to pa clitaxel. Increased expression of specific beta-tubulin isotypes and s ubsequent incorporation into microtubules may alter cellular microtubu le dynamics, providing a defence against the anti-microtubule effects of paclitaxel and other tubulin-binding drugs.