(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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA090878-03
Application #
6634028
Study Section
Special Emphasis Panel (ZRG1-ET-2 (02))
Program Officer
Forry, Suzanne L
Project Start
2001-03-05
Project End
2005-02-28
Budget Start
2003-03-01
Budget End
2004-02-29
Support Year
3
Fiscal Year
2003
Total Cost
$278,258
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Pharmacology
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Safa, Ahmad R (2016) Resistance to Cell Death and Its Modulation in Cancer Stem Cells. Crit Rev Oncog 21:203-219
Safa, Ahmad R (2013) Roles of c-FLIP in Apoptosis, Necroptosis, and Autophagy. J Carcinog Mutagen Suppl 6:
Safa, A R (2012) c-FLIP, a master anti-apoptotic regulator. Exp Oncol 34:176-84
Safa, Ahmad R; Pollok, Karen E (2011) Targeting the Anti-Apoptotic Protein c-FLIP for Cancer Therapy. Cancers (Basel) 3:1639-71
Park, Soo-Jung; Bijangi-Vishehsaraei, Khadijeh; Safa, Ahmad R (2010) Selective TRAIL-triggered apoptosis due to overexpression of TRAIL death receptor 5 (DR5) in P-glycoprotein-bearing multidrug resistant CEM/VBL1000 human leukemia cells. Int J Biochem Mol Biol 1:90-100
Choi, Mi-Ran; Najafi, Farhad; Safa, Ahmad R et al. (2008) Analysis of changes in the proteome of HL-60 promyeloid leukemia cells induced by the proteasome inhibitor PSI. Biochem Pharmacol 75:2276-88
Safa, Ahmad R; Day, Travis W; Wu, Ching-Huang (2008) Cellular FLICE-like inhibitory protein (C-FLIP): a novel target for cancer therapy. Curr Cancer Drug Targets 8:37-46
Wu, Ching-Huang; Kao, Ching-Hai; Safa, Ahmad R (2008) TRAIL recombinant adenovirus triggers robust apoptosis in multidrug-resistant HL-60/Vinc cells preferentially through death receptor DR5. Hum Gene Ther 19:731-43
Zhong, Xiaoling; Safa, Ahmad R (2007) Phosphorylation of RNA helicase A by DNA-dependent protein kinase is indispensable for expression of the MDR1 gene product P-glycoprotein in multidrug-resistant human leukemia cells. Biochemistry 46:5766-75
Park, Soo-Jung; Wu, Ching-Huang; Choi, Mi-Ran et al. (2006) P-glycoprotein enhances TRAIL-triggered apoptosis in multidrug resistant cancer cells by interacting with the death receptor DR5. Biochem Pharmacol 72:293-307

Showing the most recent 10 out of 15 publications