Relation ship between the emergence of drug-resistant tumor cells and transformation of β-tubulin isotype mRNA expression

抗微管药物暴露于肿瘤细胞后，往往会产生耐药肿瘤细胞。长春花生物碱通过调控p53肿瘤抑制基因在第一内含子区域的结合，增强小鼠B16F10黑色素瘤细胞II类β-微管蛋白同型的表达。长春新碱耐药黑色素瘤细胞β-微管蛋白II类同型mRNA水平升高，III类和IVa同型mRNA水平受到抑制。由于长春新碱的短暂暴露也增加了表明药物直接作用的II类同型mRNA水平，我们试图在小鼠II类β-微管蛋白基因中寻找对长春新碱的反应元件。缺失分析和定点突变发现Sp1、CREB和p53结合位点是II类同型表达的重要区域。电泳迁移迁移和超迁移实验表明，p53从结合位点的释放是vinka生物碱介导的II类微管蛋白同型上调的关键作用。此外，野生型p53表达载体的共转染消除了vinka生物碱的作用。这些结果表明，在耐药肿瘤细胞出现过程中，p53和vinka生物碱负调控的II类同型基因的表达可导致p53功能丧失。

The exposure of anti-microtubule drugs to tumor cells often results in the emergence of drug-resistance tumor cells. Vinca alkaloid enhanced expression of class II β-tubulin isotype in mouse B16F10 melanoma cells through the regulation of binding of p53 tumor suppressor gene in the region of the first intron. Vincristine-resistant melanoma cells elevated the mRNA level of class II isotype of β-tubulin, while suppressed class III and IVa isotype. As transient exposure of vincristine also increased mRNA level of class II isotype indicating direct effect of the drug, we tried to find the response element to vinca alkaloid in mouse class II β-tubulin gene. Deletion analyses and site-directed mutagenesis identified Sp1, CREB and p53 binding site as the important regions for class II isotype expression. Electrophoretic mobility shift assay and supershift assay showed release of p53 from the binding site was the critical role in vinka alkaloid-mediated upregulation of class II □-tubulin isotype. Furthermore, co-transfection of expression vector for wild-type p53 canceled the effect of vinka alkaloid. These results indicated that expression of class II isotype negatively regulated by p53 and vinka alkaoid induced loss of function of p53 during the emergence of drug-resitant tumor cells.