A fluctuation analysis experiment was performed by exposing 15 expanded populations of MES-SA sarcoma cells to paclitaxel (Taxol) at a concentration of 10 nm for 7 days. The mutation rate was approximately 8 × 10-7/cell generation. ANOVA supports a stochastic cell survival mechanism of spontaneous mutation rather than induction of an adaptive response under these selection conditions. Surviving colonies were found in 12 populations, 9 of which had clones that remained resistant to paclitaxel after a 2-month period of propagation. Analysis of mdr1 gene expression by reverse transcription PCR demonstrated positive clones in 4 of the 9 populations with stable resistance. Accumulation of [3H]paclitaxel was decreased in these clones but not in the mdr1-negative clones compared with parental cells. A high degree of resistance to paclitaxel (36-to 93-fold) was selected by this single drug exposure in all 9 stably resistant mutants. Those with mdr1 activation demonstrated a broad cross-resistance to vinblastine, doxorubicin, and etoposide, whereas the other 6 mutants were cross-resistant only to the Vinca alkaloids. Because tubulins are the target molecules for paclitaxel cytotoxicity, we evaluated total tubulin content by immunoblotting and performed semiquantitative reverse transcription PCR analysis for expression of the α-tubulin isotypes Bα1, Kα1 and Hα44, the β-tubulin isotypes M40, β9, 5β, β2 and β4, and γ-tubulin. Total tubulin content was decreased significantly in one of the single-step mutants. All surviving clones, both resistant and sensitive to paclitaxel, displayed reduced expression of the 5β and the β4 β-tubulin isotype transcripts in comparison with the parental cell line. These data suggest that stringent exposure to paclitaxel selected clones with reduced transcript levels of 5β and β4 β-tubulin isotypes, but that these reduced levels were not directly involved in the resistance of the clones to paclitaxel. The results 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 and tubulin depolymerizing agents.
This work was supported by American Cancer Society Grant DHP-76, Department of the Army Grant DAMD 17-94-J-4352, NIH Grant CA-52168 (B. I. S.), the Association pour la Recherche contre le Cancer, the Ligue Nationale Contre le Cancer, the Ligue contre le Cancer de la Drôme, and the Philippe Foundation, Inc. (C. D.).