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
B. Ogretmen et Ar. Safa, 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, BIOCHEM, 38(7), 1999, pp. 2189-2199
In this study, the possible involvement of repressor protein(s) in suppress
ing 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 reve
aled 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 cyclohex
imide (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 p
robe, 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, follo
wing 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 24
1 bp MDR1 promoter in MCF-7 but not MCF-7/Adr cells. Moreover, a ds CAAT ol
igomer, 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 ide
ntify the CAAT binding protein complex, EMSA and SDS-PAGE were performed. T
wo proteins with molecular masses of about 65 and 60 kDa were detected by s
ilver staining. Western blot analysis revealed that this complex consists o
f 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 evi
dence that molecular interplay between the NF-kappa B/p65 and c-Fos transcr
iption factors exhibits a negative regulatory function on MDR1 promoter by
interacting with the CAAT region in MCF-7.