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

Besim Ogretmen, Ahmad Safa

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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.

Original languageEnglish
Pages (from-to)194-204
Number of pages11
JournalBiochemistry
Volume39
Issue number1
DOIs
StatePublished - Jan 11 2000

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Videocassette recorders
HL-60 Cells
Myeloid Cells
Acute Myeloid Leukemia
Luciferases
Plasmids
Cells
Cell Line
DNA
Assays
Messenger RNA
Oligomers
Silver Staining
Deoxyribonuclease I
Electrophoretic Mobility Shift Assay
Affinity Chromatography
Genetic Promoter Regions
Transfection
Affinity chromatography
Up-Regulation

ASJC Scopus subject areas

  • Biochemistry

Cite this

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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.",
author = "Besim Ogretmen and Ahmad Safa",
year = "2000",
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N2 - 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.

AB - 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.

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