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往往导致耐药肿瘤细胞的出现。苦参碱通过调节p53抑癌基因第一内含子区域的结合增强小鼠B16 F10黑色素瘤细胞中II类β-微管蛋白同种型的表达对阿贝多斯汀耐药的黑色素瘤细胞升高β-微管蛋白II类同种型的mRNA水平，同时抑制III类和IVa类同种型。由于长春新碱的短暂暴露也增加了II类同种型的mRNA水平，表明药物的直接作用，因此我们试图在小鼠II类β-微管蛋白基因中找到对长春碱的反应元件。缺失分析和定点突变鉴定Sp1、CREB和p53结合位点为II类同种型表达的重要区域。电泳迁移率变动分析和超迁移分析表明，p53从结合位点的释放是vinka p53介导的II类微管蛋白同种型上调的关键作用。此外，野生型p53表达载体的共转染取消了长春花生物碱的影响。这些结果表明，在耐药肿瘤细胞的出现过程中，由p53和长春花生物碱负调控的II类同种型的表达导致了p53功能的丧失。

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.