Modulation of endogenous β-tubulin isotype expression as a result of human β(III) cDNA transfection into prostate carcinoma cells

Increases of individual β tubulin isotypes in antimicrotubule drug resistant cell lines have been reported by several laboratories. We have previously described elevations in β(III) and β(IVa) isotypes in estramustine and paclitaxel resistant human prostate carcinoma cells. To investigate further the function of β tubulin isotypes in antimicrotubule drug response, human prostate carcinoma cells that normally have very low to undetectable levels of β(III) were stably transfected with β(III) cDNA in pZeoSV system. An 18 bp haemagglutinin (HA) epitope tag was added at the 3′ end prior to cloning into the vector. Cells were transfected with pZeoSV or pZeoSV-β(III) plasmids and selected in the presence of Zeocin. Immunofluorescent staining of the transfectant cells have shown significant expression and incorporation of HA-tagged β(III) tubulin into cellular microtubules. Quantitation of Western blots revealed the HA-tagged β(III) levels to be approximately 7-fold higher than the vector control cells. RT-PCR analysis confirmed the increase at the transcript level and also revealed a collateral increase of β(II) and β(IVb) transcripts. Cell viability assays indicated that sensitivity of β(III) transfected cells to various antimicrotubule agents was similar to vector transfected cells: IC50 values for estramustine, paclitaxel, colchicine and vinblastine were 4 μM, 4 nM, 22 nM and 2 nM, respectively for both cell lines. Thus, overexpression of β(III) isotype in human prostate carcinoma cells by stable transfection failed to confer antimicrotubule drug resistance to these cells. Counterregulatory increases of endogenous β(II) and β(IVb) tubulin isotypes in these β(III) transfected cells may be a compensatory mechanism used by the cells to overcome the effects of elevated β(III) levels on the cellular microtubules. These results highlight the difficulty in isolating the contribution of single tubulin isotypes in drug response studies. © 2001 Cancer Research Campaign http://www.bjcancer.com