title: Identification and characterization of the MDR1 promoter-enhancing factor 1 (MEF1) in the multidrug resistant HL60/VCR human acute myeloid leukemia cell line.
abstract: In this report, the molecular mechanisms involved in the overexpression of MDR1 mRNA in the multidrug resistant variant of the HL60 human acute myeloid leukemia cell line, HL60/VCR, were investigated. RT-PCR and nuclear run-on assays revealed that the expression of MDR1 mRNA is regulated by increased transcriptional initiation in HL60/VCR cells. Transient transfections with a 241 bp MDR1 promoter (spanning the -198 to +43 region) DNA fragment/pGL3-basic plasmid construct resulted in about 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 plasmid caused 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 confirmed by transfecting HL60/VCR cells with the pGL3-basic plasmid containing a 237 bp mutated MDR1 proximal promoter 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 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 detected 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 electroelution, specific DNA-affinity chromatography, and silver staining. Interestingly, 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' element of the MDR1 promoter, as determined by DNase I protection assay. These data reveal that MEF1 upregulates the MDR1 promoter activity.
authors.affiliation: Medical University of South Carolina, Hollings Cancer Center, Charleston 29425, USA.
authors.forename: B
authors.initials: B
authors.lastname: Ogretmen
authors.forename: A R
authors.initials: AR
authors.lastname: Safa
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003850:Deoxyribonuclease I; D018432:Drug Resistance, Multiple; D019450:Genes, MDR; D018922:HL-60 Cells; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism* / Q000473:pathology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014750:Vincristine / Q000494:pharmacology*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004268:DNA-Binding Proteins; C403209:MEF1 protein, human; D014157:Transcription Factors; D014750:Vincristine; D003850:Deoxyribonuclease I