Transcriptional suppression of multidrug resistance-associated protein (MRP) gene expression by wild-type p53.
Transcriptional suppression of multidrug resistance-associated protein (MRP) gene expression by wild-type p53.
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发表时间：
1998-12
期刊：
影响因子：
11.2
通讯作者：
Qingjian Wang;William T. Beck
中科院分区：
文献类型：
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作者：
Qingjian Wang;William T. Beck
多重耐药性是癌症化疗成功的主要障碍。多药耐药相关蛋白（MRP）已被证明可赋予多药耐药性。为了在转录水平研究MRP基因的表达，我们将MRP基因启动子与荧光素酶报告基因融合并研究其调控。在 p53 缺失的人 H1299 和小鼠 (10)1 细胞中将 MRP 启动子构建体与 p53 表达质粒共转染表明，野生型 (wt) p53 显着抑制 MRP 启动子活性，而突变型 p53 几乎没有抑制作用。使用 MRP 启动子 5' 缺失突变体构建体的转染表明，wt p53 对启动子活性的抑制主要位于 -91 至 +103 bp 区域，其中有几个 Sp1 转录因子结合位点。将 MRP 启动子与 Sp1 表达载体共转染到果蝇 SL2 细胞中，启动子活性以剂量相关的方式增加了约 200 倍。 Sp1 对 MRP 启动子活性的刺激通过共转染 wt p53 表达质粒而减弱。此外，我们已经确定，在人肺癌细胞系中，通过恢复wt p53表达，内源MRP mRNA水平被下调。在耐药细胞系 CEM/VM-1-5 中研究了 MRP 调节与耐药性的相关性，该细胞系对表鬼臼毒素替尼泊苷 (VM-26) 的耐药性比亲代 CEM 细胞高约 140 倍。 CEM/VM-1-5 细胞表达的 MRP mRNA 和蛋白质含量比 CEM 细胞高得多，表明耐药表型至少部分是由于 MRP 产生增加所致。启动子构建体的瞬时转染表明，CEM/VM-1-5 细胞具有比 CEM 细胞更高（7 倍）的 MRP 启动子活性。 wt p53 表达质粒的共转染导致 CEM 和 CEM/VM-1-5 细胞中 MRP 启动子活性降低，但与 CEM 细胞相比，CEM/VM-1-5 细胞中的抑制作用增加了一倍以上。我们的结果表明，wt p53 作为 MRP 基因转录的负调节因子，至少部分是通过减弱强大的转录激活因子 Sp1 的作用而发挥作用。因此，肿瘤细胞中wt p53功能的丧失和/或Sp1活性的增加可能导致MRP基因的上调。
Multidrug resistance is a major obstacle to the success of cancer chemotherapy. The multidrug resistance-associated protein (MRP) has been shown to confer multidrug resistance. To study MRP gene expression at the transcriptional level, we have fused the MRP gene promoter with the luciferase reporter gene and studied its regulation. Cotransfection of MRP promoter constructs with p53 expression plasmids in p53-null human H1299 and mouse (10)1 cells demonstrated that the wild-type (wt) p53 markedly suppressed MRP promoter activity, whereas mutant p53 had little inhibitory effect. Transfections using 5' deletion mutant constructs of the MRP promoter showed that inhibition of the promoter activity by wt p53 mainly resided in the region from -91 to +103 bp, where several Sp1 transcription factor binding sites are localized. Cotransfection of the MRP promoter into Drosophila SL2 cells with an Sp1 expression vector increased the promoter activity in a dose-related manner up to approximately 200-fold. The stimulation of MRP promoter activity by Sp1 was attenuated by the cotransfection of a wt p53-expression plasmid. Furthermore, we have determined that endogenous MRP mRNA levels were down-regulated by restoration of wt p53-expression in a human lung cancer cell line. The relevance of MRP regulation in drug resistance was studied in a drug-resistant cell line, CEM/VM-1-5, that is approximately 140-fold more resistant to the epipodophyllotoxin, teniposide (VM-26), than the parental CEM cells. CEM/VM-1-5 cells express a much higher amount of MRP mRNA and protein than CEM cells, indicating that the resistant phenotype is at least partly due to increased MRP production. Transient transfection of the promoter constructs revealed that CEM/VM-1-5 cells had higher (7-fold) MRP promoter activity than CEM cells. Cotransfection of a wt p53-expression plasmid caused a reduction of MRP promoter activity in both CEM and CEM/VM-1-5 cells, but the inhibition was more than double in CEM/VM-1-5 cells compared with CEM cells. Our results demonstrated that wt p53 acts as a negative regulator of MRP gene transcription, at least in part by diminishing the effect of a powerful transcription activator Sp1. Therefore, a loss of wt p53 function and/or an increase in Sp1 activity in tumor cells could contribute to an up-regulation of the MRP gene.