Transcriptional suppression of multidrug resistance-associated protein (MRP) gene expression by wild-type p53

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.