RESISTANCE MECHANISMS IN HUMAN SARCOMA MUTANTS DERIVED BY SINGLE-STEPEXPOSURE TO PACLITAXEL (TAXOL)

A fluctuation analysis experiment was performed by exposing 15 expande d populations of MES-SA sarcoma cells to paclitaxel (Taxol) at a conce ntration of 10 nM for 7 days, The mutation rate was approximately 8 x 10(-7)/cell generation. ANOVA supports a stochastic cell survival mech anism of spontaneous mutation rather than induction of an adaptive res ponse under these selection conditions, Surviving colonies were found in 12 populations, 9 of which had clones that remained resistant to pa clitaxel after a 2-month period of propagation, Analysis of mdr1 gene expression by reverse transcription PCR demonstrated positive clones i n 4 of the 9 populations with stable resistance, Accumulation of [H-3] paclitaxel was decreased in these clones but not in the mdr1-negative clones compared with parental cells. A high degree of resistance to pa clitaxel (36- to 93-fold) was selected by this single drug exposure in all 9 stably resistant mutants, Those with mdr1 activation demonstrat ed a broad cross-resistance to vinblastine, doxorubicin, and etoposide , whereas the other 6 mutants were cross-resistant only to the Vinca a lkaloids, Because tubulins are the target molecules for paclitaxel cyt otoxicity, we evaluated total tubulin content by immunoblotting and pe rformed semiquantitative reverse transcription PCR analysis for expres sion of the alpha-tubulin isotypes B alpha 1, K alpha 1 and H alpha 44 , the beta-tubulin isotypes M40, beta 9, 5 beta, beta 2 and beta 4, an d gamma-tubulin. Total tubulin content was decreased significantly in one of the single-step mutants, All surviving clones, both resistant a nd sensitive to paclitaxel, displayed reduced expression of the 5 beta and the beta 4 beta-tubulin isotype transcripts in comparison with th e parental cell line, These data suggest that stringent exposure to pa clitaxel selected clones with reduced transcript levels of 5 beta and beta 4 beta-tubulin isotypes, but that these reduced levels were not d irectly involved in the resistance of the clones to paclitaxel, The re sults suggest an important role for non-multidrug-resistant mechanisms of resistance to paclitaxel. These mechanisms do not involve reduced drug accumulation and provide cross-resistance among both paclitaxel a nd tubulin depolymerizing agents.