Identification and characterization of the MDR1 promoter-enhancing factor 1 (MEF1) in the multidrug resistant HL60/VCR human acute myeloid leukemia cell line
B. Ogretmen et Ar. Safa, Identification and characterization of the MDR1 promoter-enhancing factor 1 (MEF1) in the multidrug resistant HL60/VCR human acute myeloid leukemia cell line, BIOCHEM, 39(1), 2000, pp. 194-204
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.