Wiesen Kenneth M, Xia Shujun, Yang Chia-Ping Huang, Horwitz Susan Band
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Cancer Lett. 2007 Nov 18;257(2):227-35. doi: 10.1016/j.canlet.2007.07.022. Epub 2007 Sep 14.
A Taxol-resistant cell line, K20T, which does not express P-glycoprotein, was selected with Taxol from human MDA-MB-231 breast adenocarcinoma cells and maintained in the presence of 20nM Taxol. K20T cells were approximately 18-fold resistant to Taxol, displayed cross-resistance to Taxotere and the epothilones, but little cross-resistance to discodermolide. Sequence analysis of the class I beta-tubulin indicated that it harbored an A593G mutation resulting in a change from glutamate to glycine at amino acid 198, which is near the intradimer interface within the alpha/beta-tubulin heterodimer. An HA-tagged wild-type class I beta-tubulin expression vector was transfected into the K20T cells. Immunofluorescence studies demonstrated that this exogenous tubulin was incorporated into cellular microtubules and Western blot analysis indicated that the K20T transfectants predominantly expressed the exogenous wild-type class I beta-tubulin. The transfected cells were only approximately 5-fold resistant to Taxol. Our results, plus the knowledge that Glu198 is the target for other anti-tubulin agents, suggest that glutamate198 in beta-tubulin is a critical determinant for microtubule stability and Taxol resistance.
从人MDA-MB-231乳腺腺癌细胞中用紫杉醇筛选出一种不表达P-糖蛋白的耐紫杉醇细胞系K20T，并在20nM紫杉醇存在的情况下进行培养。K20T细胞对紫杉醇的耐药性约为18倍，对多西他赛和埃坡霉素表现出交叉耐药性，但对盘尼西林几乎没有交叉耐药性。I类β-微管蛋白的序列分析表明，它存在A593G突变，导致第198位氨基酸从谷氨酸变为甘氨酸，该位置靠近α/β-微管蛋白异二聚体内的二聚体界面。将带有HA标签的野生型I类β-微管蛋白表达载体转染到K20T细胞中。免疫荧光研究表明，这种外源性微管蛋白被整合到细胞微管中，蛋白质印迹分析表明，K20T转染细胞主要表达外源性野生型I类β-微管蛋白。转染后的细胞对紫杉醇的耐药性仅约为5倍。我们的结果，加上已知Glu198是其他抗微管蛋白药物的作用靶点，表明β-微管蛋白中的谷氨酸198是微管稳定性和紫杉醇耐药性 的关键决定因素。
