Yu L, Cohen D, Piekarz R L, Horwitz S B
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461.
J Biol Chem. 1993 Apr 5;268(10):7520-6.
Multidrug resistance in mammalian cells is often associated with the overproduction of a membrane glycoprotein, P-glycoprotein, that is encoded by mdr genes. Multidrug resistance cell lines selected with either vinblastine, colchicine, or taxol from the drug-sensitive murine macrophage-like cell line J774.2 overexpress the mdr1a and/or mdr1b genes, and overproduce P-glycoprotein. To elucidate the mechanisms of mdr1b gene expression, the mdr1b 5'-flanking sequences have been isolated from a normal mouse liver genomic library and analyzed by gel shift and DNase I footprinting assays. These analyses have demonstrated three nuclear protein binding sites, from -82 to -59 (site 1), from -123 to -101 (site 2), and from -272 to -249 (site 3), which interact with proteins present in nuclear extracts from both sensitive and resistant cells. Although site 1 contains a partially conserved AP-2 consensus sequence, our results indicate that the nuclear protein binding to site 1 is not AP-2 protein. The sequence of site 2 is conserved in the murine mdr1a, human mdr1, and hamster pgp1 promoters. Such conservation suggests that this sequence may have an important role in mdr gene expression. The use of a transient chloramphenicol acetyltransferase expression vector containing the basal promoter for herpes simplex virus thymidine kinase (tkCAT) and either site 1 or site 2 or both revealed that the sequences of sites 1 and 2 enhanced tkCAT activity. DNase I footprinting analyses demonstrated that site 3 is recognized by human AP-1 protein, indicating that the nuclear protein binding to this site is an AP-1-like protein. These observations suggest that mdr1b gene expression is mediated by preexisting transcription factors present in sensitive and resistant cells.
哺乳动物细胞中的多药耐药性通常与一种膜糖蛋白——P-糖蛋白的过量产生有关，P-糖蛋白由mdr基因编码。从对药物敏感的鼠巨噬细胞样细胞系J774.2中，用长春碱、秋水仙碱或紫杉醇筛选出的多药耐药细胞系，会过度表达mdr1a和/或mdr1b基因，并过量产生P-糖蛋白。为了阐明mdr1b基因表达的机制，已从正常小鼠肝脏基因组文库中分离出mdr1b 5'-侧翼序列，并通过凝胶迁移和DNase I足迹分析进行了研究。这些分析表明，存在三个核蛋白结合位点，分别位于-82至-59（位点1）、-123至-101（位点2）和-272至-249（位点3），它们与敏感细胞和耐药细胞的核提取物中的蛋白质相互作用。虽然位点1包含一个部分保守的AP-2共有序列，但我们的结果表明，与位点1结合的核蛋白不是AP-2蛋白。位点2的序列在鼠mdr1a、人mdr1和仓鼠pgp1启动子中是保守的。这种保守性表明该序列可能在mdr基因表达中起重要作用。使用含有单纯疱疹病毒胸苷激酶基础启动子（tkCAT）以及位点1或位点2或两者的瞬时氯霉素乙酰转移酶表达载体，结果显示位点1和位点2的序列增强了tkCAT活性。DNase I足迹分析表明，位点3可被人AP-1蛋白识别，这表明与该位点结合的核蛋白是一种类AP-1蛋白。这些观察结果表明，mdr1b基因表达是由敏感细胞和耐药细胞中预先存在的转录因子介导的。
