(Application Abstract) P-glycoprotein (P-gp), the product of the MDR1 gene, causes multidrug resistance (MDR) in cancer cells. In acute myelogenous leukemia (AML) where P-gp occurs in 20 percent of de novo and about 75 percent of secondary cases, expression of P-gp is a negative prognostic feature. We recently discovered a novel 130 kDa transcription factor, referred to as the MDR1 promoter-enhancing factor 1 (MEF1), which binds to the GTCAATCC element of the MDR 1 promoter and increases its activity in HL-60/VCR cells, a MDR variant of the AML cell line HL-60. Moreover, a 162 kDa MEF1-associated protein (MEF1-AP) interacts with MEF1 but its function remains to be found. The overall goal of the application is to unravel the structures of and mechanism(s) by which these proteins enhance the activity of the MDR1 promoter, and use this knowledge to prevent MDR1 gene expression.
The Specific Aims are to (1) purify, clone, and further characterize MEF1 and MEF1-AP; (2) continue to investigate the roles of MEF1 and MEF1-AP in regulating MDR1 promoter activity; and (3) determine whether MEF1 and MEF1-AP play roles in the transcriptional activation of the MDR1 gene by MDR-related drugs and modulators, and analyze their subcellular localization and normal tissue expression.
Specific Aim 1 is designed to (a) purify these proteins for amino acid sequencing; (b) clone the full-length cDNAs for MEF1 and MEF1-AP by screening the cDNA library from HL-60/VCR cells; and (c) produce antibodies for these proteins.
In Specific Aim 2 we will (a) determine whether MEF1 interacts with MEF1-AP in vivo using the yeast-two hybrid screening system; and (b) transfect cancer cells with MEF1 and MEF1-AP cDNAs cloned into bicistronic expression vectors and determine whether MEF1 alone or with MEF1-AP enhances MDR1 promoter/luciferase activity, MDR1 mRNA and P-gp expression.
Specific Aim 3 is designed to (a) transfect HL-60 and HL-60/VCR cells with MEF1 and MEF-AP cDNAs cloned in the sense or antisense orientation into expression vectors and determine whether these factors are involved in the activation of MDR1 gene transcription by MDR-related drugs and modulators; (b) examine their subcellular localization in cells transfected with inducible MEF1 and MEF1-AP expression vectors by confocal microscopy; and (c) determine whether these proteins are expressed in various MDR cells and certain normal tissues. Unraveling the molecular structures of and mechanism(s) by which these proteins enhance the MDR1 promoter activity should aid in the design of strategies to prevent MDR1 expression.
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