Identification and characterization of the MDR1 promoter-enhancing factor 1 (MEF1) in the multidrug resistant HL60/VCR human acute myeloid leukemia cell line

In this report, the molecular mechanisms involved in the overexpression of MDR1 mRNA in the multidrug resistant variant of the HL60 human acute myeloi d leukemia cell line, HL60/VCR, were investigated. RT-PCR and nuclear run-o n assays revealed that the expression of MDR1 mRNA is regulated by increase d transcriptional initiation in HL60/VCR cells. Transient transfections wit h a 241 bp MDR1 promoter (spanning the -198 to +43 region) DNA fragment/pG1 3-basic plasmid construct resulted in about B-fold increased luciferase act ivity in HL60/VCR but not in HL60 cells. Moreover, ds CAAT-oligomer from th e MDR1 promoter cloned upstream of the SV-40 promoter in the pGL3-promoter plasmid caused about a 7-fold increase in luciferase activity compared with plasmid constructs containing CAAT-deleted, GC-box, and nonspecific oligom ers in HL60/VCR transfectants. These results were confirmed by transfecting HL60/VCR cells with the pGL3-basic plasmid containing a 237 bp mutated MDR 1 proximal promoter lacking the CAAT sequence in which no change in lucifer ase activity was observed. However, a 5-6-fold increase in luciferase activ ity was measured in these cells when transfected with the wt MDR1 promoter DNA/pGL3-basic plasmid constructs. These results show that the CAAT-region is involved in upregulating the MDR1 promoter in HL60/VCR cells. A nuclear factor binding to the CAAT-region of the MDR1 promoter specifically was det ected in electrophoretic mobility shift assays (EMSAs) in HL60/VCR but not in HL60 extracts. Two MDR1 promoter-associated polypeptides with molecular masses of about 130 and 162 kDa were identified in HL60/VCR cells by electr oelution, specific DNA-affinity chromatography, and silver staining. Intere stingly, cross-linking and Southwestern analysis indicate that only the 130 kDa protein, which we refer to as MDR1-promoter enhancing factor 1 (MEF1), has a strong DNA-binding ability, interacting with the 5'-GTCAATCC-3' elem ent of the MDR1 promoter, as determined by DNase I protection assay. These data reveal that MEF1 upregulates the MDR1 promoter activity.