Negative regulation of MDR1 promoter activity in MCF-7, but not in multidrug resistant MCF-7/Adr, cells by cross-coupled NF-kappa B/p65 and c-Fos transcription factors and their interaction with the CAAT region.

Abstract

In this study, the possible involvement of repressor protein(s) in suppressing MDR1 promoter activity in the sensitive MCF-7 human breast cancer cell line and its drug resistant variant MCF-7/Adr was investigated. RT-PCR revealed that MDR1 mRNA is under detectable levels in MCF-7, while it is highly expressed in MCF-7/Adr cells. After treatment of MCF-7 cells with cycloheximide (CHX), MDR1 mRNA reached detectable levels, suggesting that MDR1 mRNA expression might be controlled by a labile negative regulatory protein(s) in MCF-7 cells. In electrophoretic mobility shift assays (EMSA) using a 5'-end-labeled 241 bp MDR1 promoter DNA fragment (residues -198 to +43) as a probe, one protein complex that specifically binds to the CAAT region of the MDR1 promoter was detected in MCF-7, but not MCF-7/Adr. In addition, following transient transfections of MCF-7 and MCF-7/Adr cells with a pGL3-Basic plasmid construct containing a CAAT-deleted MDR1 promoter DNA fragment, a significant increase in luciferase activity was observed compared to the 241 bp MDR1 promoter in MCF-7 but not MCF-7/Adr cells. Moreover, a ds CAAT oligomer, cloned upstream of the SV-40 promoter in the pGL3-Promoter vector, resulted in a 70-80% decrease in luciferase activity in MCF-7 cells. To identify the CAAT binding protein complex, EMSA and SDS-PAGE were performed. Two proteins with molecular masses of about 65 and 60 kDa were detected by silver staining. Western blot analysis revealed that this complex consists of NF-kappa B/p65 and c-Fos transcription factors. Moreover, incubating MCF-7 nuclear extracts with antibodies specific for NF-kappa B/p65 or c-Fos in EMSAs almost completely inhibited formation of the complex, supporting the association of NF-kappa B/p65 and c-Fos. Therefore, this study provides evidence that molecular interplay between the NF-kappa B/p65 and c-Fos transcription factors exhibits a negative regulatory function on MDR1 promoter by interacting with the CAAT region in MCF-7.