| Abstract
| - In this report, the molecular mechanisms involved in the overexpression of MDR1 mRNA inthe multidrug resistant variant of the HL60 human acute myeloid leukemia cell line, HL60/VCR, wereinvestigated. RT-PCR and nuclear run-on assays revealed that the expression of MDR1 mRNA is regulatedby increased transcriptional initiation in HL60/VCR cells. Transient transfections with a 241 bp MDR1promoter (spanning the −198 to +43 region) DNA fragment/pGL3-basic plasmid construct resulted inabout 6-fold increased luciferase activity in HL60/VCR but not in HL60 cells. Moreover, ds CAAT-oligomer from the MDR1 promoter cloned upstream of the SV-40 promoter in the pGL3-promoter plasmidcaused about a 7-fold increase in luciferase activity compared with plasmid constructs containing CAAT-deleted, GC-box, and nonspecific oligomers in HL60/VCR transfectants. These results were confirmedby transfecting HL60/VCR cells with the pGL3-basic plasmid containing a 237 bp mutated MDR1 proximalpromoter lacking the CAAT sequence in which no change in luciferase activity was observed. However,a 5−6-fold increase in luciferase activity was measured in these cells when transfected with the wt MDR1promoter DNA/pGL3-basic plasmid constructs. These results show that the CAAT-region is involved inupregulating the MDR1 promoter in HL60/VCR cells. A nuclear factor binding to the CAAT-region ofthe MDR1 promoter specifically was detected in electrophoretic mobility shift assays (EMSAs) in HL60/VCR but not in HL60 extracts. Two MDR1 promoter-associated polypeptides with molecular masses ofabout 130 and 162 kDa were identified in HL60/VCR cells by electroelution, specific DNA-affinitychromatography, and silver staining. Interestingly, cross-linking and Southwestern analysis indicate thatonly the 130 kDa protein, which we refer to as MDR1-promoter enhancing factor 1 (MEF1), has a strongDNA-binding ability, interacting with the 5‘-GTCAATCC-3‘ element of the MDR1 promoter, as determinedby DNase I protection assay. These data reveal that MEF1 upregulates the MDR1 promoter activity.
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