Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of β-tubulin (AsP26Glu) and less stable microtubules
Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of β-tubulin (AsP26Glu) and less stable microtubules
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DOI：
10.1158/1535-7163.mct-05-0190
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发表时间：
2006-02-01
影响因子：
5.7
通讯作者：
Greenberger, LM
中科院分区：
文献类型：
--
作者：
Hari, M;Loganzo, F;Greenberger, LM
对紫杉醇为基础的治疗耐药是经常遇到的临床。内在或获得性紫杉醇耐药的机制还不清楚。我们试图描述在P-糖蛋白逆转剂CL-347099存在下，癌细胞系长期暴露于紫杉醇后产生的耐药机制。表皮样肿瘤细胞系KB-3 - 1暴露于浓度递增的紫杉醇和5 μ mol/L CL-347099长达1年。在15 nmol/L紫杉醇加CL-347099（KB-15-PTX/099）中生长的细胞对紫杉醇产生了18倍的耐药性，并且依赖于紫杉醇以实现最大生长。它们在无胸腺小鼠中生长良好，并保留了对紫杉醇的抗性。在组织培养中观察到对多西他赛、新型紫杉烷MAC-321和埃博霉素B的交叉耐药（3 - 5倍）。观察到对解聚剂长春碱、多拉司他汀-10和HTI-286的附带敏感性（类似于3倍）。与亲本细胞相比，KB-15-PTX/099耐药细胞没有过度表达P-糖蛋白，也没有改变[C-14]紫杉醇蓄积。然而，在Ⅰ类（M40）β-微管蛋白中发现了一个新的点突变（T → A），导致天冬氨酸（26）被谷氨酸取代，基于锌稳定微管蛋白片的电子晶体学结构，紫杉醇的C-3 'NHCO-C_6H_5的苯环与β-微管蛋白的天冬氨酸（26）接触，表明这是一种配体诱导的突变。优化的模型复合物紫杉醇，多西他赛，和MAC-321在β-微管蛋白显示了一种新的氢键模式的谷氨酸突变体和合理化所观察到的耐药概况。然而，紫杉醇结合口袋中的突变并不能完全解释该表型。KB-15-PTX/099细胞具有受损的微管稳定性，如通过微管中微管蛋白的百分比降低所确定的，并通过较少的乙酰化微管蛋白所反映。这些结果表明，微管蛋白的突变可能会影响微管稳定性以及药物结合，并有助于观察到的耐药谱。
Resistance to paclitaxel-based therapy is frequently encountered in the clinic. The mechanisms of intrinsic or acquired paclitaxel resistance are not well understood. We sought to characterize the resistance mechanisms that develop upon chronic exposure of a cancer cell line to paclitaxel in the presence of the P-glycoprotein reversal agent, CL-347099. The epidermoid tumor line KB-3-1 was exposed to increasing concentrations of paclitaxel and 5 mu mol/L CL-347099 for up to 1 year. Cells grown in 15 nmol/L paclitaxel plus CL-347099 (KB-15-PTX/099) developed 18-fold resistance to paclitaxel and were dependent upon paclitaxel for maximal growth. They grew well and retained resistance to paclitaxel when grown in athymic mice. Cross-resistance (3- to 5-fold) was observed in tissue culture to docetaxel, the novel taxane MAC-321, and epothilone B. Collateral sensitivity (similar to 3-fold) was observed to the depolymerizing agents vinblastine, dolastatin-10, and HTI-286. KB-15-PTX/099-resistant cells did not overexpress P-glycoprotein nor did they have an alteration of [C-14] paclitaxel accumulation compared with parental cells. However, a novel point mutation (T to A) resulting in ASp(26) to glutamate substitution in class I (M40) beta-tubulin was found. Based on an electron crystallography structure of Zn-stabilized tubulin sheets, the phenyl ring of C-3' NHCO-C6H5 Of paclitaxel makes contact with Asp(26) of beta-tubulin, suggesting a ligand-induced mutation. Optimized model complexes of paclitaxel, docetaxel, and MAC-321 in beta-tubulin show a novel hydrogen bonding pattern for the glutamate mutant and rationalize the observed resistance profiles. However, a mutation in the paclitaxel binding pocket does not explain the phenotype completely. KB-15-PTX/099 cells have impaired microtubule stability as determined by a reduced percentage of tubulin in microtubules and reflected by less acetylated tubulin. These results suggest that a mutation in tubulin might affect microtubule stability as well as drug binding and contribute to the observed resistance profile.