Dumontet C, Duran G E, Steger K A, Beketic-Oreskovic L, Sikic B I
Division of Oncology, Department of Medicine, Stanford University School of Medicine, California 94305-5306, USA.
Cancer Res. 1996 Mar 1;56(5):1091-7.
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 multiplied by 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 [(3)H]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 alpha-tubulin isotypes B alpha 1, K alpha 1 and H alpha 44, the beta-tubulin isotypes M40, beta9, 5beta, beta2 and beta4, and gamma-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 5beta and beta 4 beta-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 5beta and beta4 beta-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.
进行了一项波动分析实验，将15个扩增的MES-SA肉瘤细胞群体暴露于浓度为10 nM的紫杉醇（泰素）中7天。突变率约为8×10⁻⁷/细胞代。方差分析支持自发突变的随机细胞存活机制，而非在这些选择条件下诱导适应性反应。在12个群体中发现了存活菌落，其中9个群体的克隆在传代2个月后仍对紫杉醇耐药。通过逆转录PCR分析mdr1基因表达，在9个具有稳定耐药性的群体中的4个中发现了阳性克隆。与亲代细胞相比，这些克隆中[(³)H]紫杉醇的积累减少，但mdr1阴性克隆中未减少。通过单次药物暴露在所有9个稳定耐药突变体中均筛选出了对紫杉醇高度耐药（36至93倍）的克隆。那些mdr1激活的克隆对长春花碱、阿霉素和依托泊苷表现出广泛的交叉耐药性，而其他6个突变体仅对长春花生物碱交叉耐药。由于微管蛋白是紫杉醇细胞毒性的靶分子，我们通过免疫印迹评估了总微管蛋白含量，并对α-微管蛋白亚型Bα1、Kα1和Hα44、β-微管蛋白亚型M40、β9、5β、β2和β4以及γ-微管蛋白的表达进行了半定量逆转录PCR分析。在一个单步突变体中总微管蛋白含量显著降低。与亲代细胞系相比，所有存活克隆，无论对紫杉醇耐药还是敏感，均显示5β和β4β-微管蛋白亚型转录本的表达降低。这些数据表明，严格暴露于紫杉醇筛选出了5β和β4β-微管蛋白亚型转录水平降低的克隆，但这些降低的水平并未直接参与克隆对紫杉醇的耐药性。结果表明非多药耐药机制在紫杉醇耐药中起重要作用。这些机制不涉及药物积累减少，并在紫杉醇和微管蛋白解聚剂之间提供交叉耐药性。
