Negative Regulation of MDR1 Promoter Activity in MCF-7, but Not in Multidrug Resistant MCF-7/Adr, Cells by Cross-Coupled NF-κB/p65 and c-Fos Transcription Factors and Their Interaction with the CAAT Region

Publisher Website
Google Scholar
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-κB/p65 and c-Fos transcription factors. Moreover, incubating MCF-7 nuclear extracts with antibodies specific for NF-κB/p65 or c-Fos in EMSAs almost completely inhibited formation of the complex, supporting the association of NF-κB/p65 and c-Fos. Therefore, this study provides evidence that molecular interplay between the NF-κB/p65 and c-Fos transcription factors exhibits a negative regulatory function on MDR1 promoter by interacting with the CAAT region in MCF-7.