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 chemother apy. The multidrug resistance-associated protein (MRP) has been shown to co nfer multidrug resistance. To study MRP gene expression at the transcriptio nal 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 us ing 5' deletion mutant constructs of the MRP promoter showed that inhibitio n of the promoter activity by wt p53 mainly resided in the region from -91 to +103 bp, where several Spl transcription factor binding sites are locali zed. Cotransfection of the MRP promoter into Drosophila SL2 cells with an S pl expression vector increased the promoter activity in a dose-related mann er up to similar to 200-fold. The stimulation of MRP promoter activity by S pl was attenuated by the cotransfection of a wt p53-expression plasmid. Fur thermore, we have determined that endogenous MRP mRNA levels were down-regu lated 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-r esistant cell line, CEM/VM-1-5, that is similar to 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 MRF production. Transient transfection of the promoter constru cts revealed that CEM/VM-1-5 cells had higher (7-fold) MRP promoter activit y than CEM cells. Cotransfection of a wt p53-expression plasmid caused a re duction 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 powerfu l transcription activator Spl. Therefore, a loss of wt p53 function and/or an increase in Spl activity in tumor cells could contribute to an up-regula tion of the MRP gene.