Promoter characterization and genomic organization of the human breast cancer resistance protein (ATP-binding cassette transporter G2) gene

Abstract

The Breast Cancer Resistance Protein (BCRP) gene encodes an ABC half transporter that causes resistance to certain cancer chemotherapeutic drugs when transfected and expressed in drug sensitive cancer cells. We identified the genomic sequence of BCRP and its promoter by screening a human genomic lambda phage library, a BAC library, and by searching the human genome database. The BCRP gene spans over 66 kb and consists of 16 exons and 15 introns. BCRP promoter activity was characterized by a luciferase reporter assay using transient transfection of the human breast cancer cell line MCF7, and the human choriocarcinoma cell lines JAR, BeWo and JEG-3, which have high endogenous expression of BCRP. The sequence 312 by directly upstream from the BCRP transcriptional start site conferred basal promoter activity: The 5' region upstream of the basal promoter is characterized by both positive and negative regulatory domains. The BCRP gene is transcribed by a TATA-less promoter 06 several putative Sp1 sites, an AP1 site and a CCAAT box, all of which are downstream from a putative CpG island. Electrophoretic mobility shift assays were done using probes for the major transcription how binding sites identified in the core promoter region. This analysis revealed specific protein-DNA complexes at four of the sites tested. We next measured the activity of the BCRP promoter-luciferase constructs in three drug resistant lines that overexpress BCRP; human breast carcinoma MCF-7/AdrVp cells, and human ovarian carcinoma Igrov1/T8 and Igrov1/MX3 cells. Our results showed extremely low BCRP promoter activity in the drug resistant cell lines. This indicated the increase in BCRP expression was not due to transcriptional upregulation through the BCRP promoter constructs tested. To check for nontranscriptional control mechanisms that may regulate the expression of BCRP in the drug selected cell lines we tested the mRNA and protein half-lives of BCRP in the MCF7/AdrVp cells to compare the results with those of the parental cell line MCF-7. Although the MCF7/AdrVp cells had longer BCRP mRNA and protein half-lives, the differences were not significant.