Acute exposure of cells to methotrexate (MTX) in both in vitro and in vivo results in a rapid increase in DHFR levels. Because such increases may reduce the therapeutic effectiveness of MTX and other antifolates, there is considerable interest in determining the mechanism that leads to this rapid increase. This increase is not due to gene amplification or an increased rate of transcription of the enzyme or due to stabilization of DHFR protein bound to MTX. In collaboration with Dr. Joseph R. Bertino, we have shown that DHFR mRNA translation, is inhibited by its own protein and that this inhibition is relieved by MTX. Translational inhibition of DHFR is accomplished by DHFR protein by binding to its cognate mRNA. This binding occurs within the coding region of mRNA. Recently, we have also identified several variants of DHFR that are no longer upregulated by MTX. We are investigating further the binding of DHFR to its cognate mRNA, and defining further the region and bases in mRNA involved in binding, using biophysical measurements (fluorometry, circular dichroism and isothermal tiration calorimetry) in collaboration with Dr. Daniel Pilch from the Department of Pharmacology, University of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School. Furthermore, Professor Aleem Gangjee from Mylan School of Pharmacy (Duquesne University, PA) is supplying us with new antifolates that we are testing for their inability to relieve DHFR translational inhibition. Finally, we purified the mutants with attenuated response to MTX and sent them to Dr. Vivian Cody (State University of New York at Buffalo, NY) for crystallography.>>mTOR inhibition as a single agent and / or in combination therapy with antifolates for cancer treatment:>>The mammalian target of rapamycin (mTOR) is an important player in the regulation of cell proliferation, growth, differentiation and survival. mTOR regulates both the activity of the 40S ribosomal protein S6 kinase and the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). This pathway also regulates the translation of mRNAs (e.g. cyclin D1) that are important for the G1/S transition. Inhibition of mTOR pathway increases the rate of turnover of cyclin D1. The cyclin D/CDKs phosphorylate pRb and release the E2Fs which mediate the transcription of proteins required for DNA synthesis, such as dihydrofolate reductase (DHFR). In a variety of cancers, including leukemia and lymphomas the Cyclin D-Cdk4,6/INK4/Rb/E2F pathway is deregulated and contributes to tumor development and also drug resistance towards antifolates. Moreover, the PI3K/Akt/PTEN, the upstream regulators of mTOR pathway are also deregulated in leukemia and lymphomas. Expression of DHFR is regulated both at the transcriptional level through the Rb pathway and at the translational levels. Our preliminary data indicates that by inhibiting mTOR pathway DHFR levels are downregulated leading to increased sensitivity to methotrexate. We are currently investigating whether the sensitization to antifolates in the presence of rapamycin in cell lines is due to changes in the transcription or translational regulation of dihydrofolate reductase.>>Regulation of dihydrofolate reductase to reduce oxidative stress in endothelial cells:>>The endothelial cell has a central role in remodeling of the arterial vessel wall in atherosclerosis and is responsible for the synthesis of nitric oxide synthase (eNOS). Production of nitric oxide (NO) catalyzed by eNOS promotes vasodilatation of vascular smooth muscle, counteracts smooth muscle cell proliferation, decreases platelet adhesiveness, and has antioxidant and anti-inflammatory effects. A decrease in the synthesis of nitric oxide (NO) is one of the major characteristics of many cardiac and coronary diseases; therefore restoration of NO synthesis is critical for prevention and treatment of cardiovascular diseases. Tetrahydrobiopterin (BH4) is the obligatory cofactor for eNOS. During vascular oxidative stress, BH4 is oxidized to dihydrobiopterin (BH2) making BH4 limiting, hence instead of producing protective NO, eNOS starts to generate peroxides that are responsible for endothelial dysfunction. It is therefore essential to reduce BH2 to BH4 using the enzyme dihydrofolate reductase (DHFR). Under oxidative stress DHFR levels are downregulated resulting in a reduction in the bioavailability of BH4 and uncoupling of eNOS (Chalupsky and Cai 2005). We demonstrated that translation of DHFR mRNA is negatively regulated by its protein and exposure to certain folates or antifolates relieves this inhibition by preventing DHFR protein from binding to its cognate mRNA, thereby resulting in elevated levels of DHFR protein. Therefore, we are investigating whether by upregulating DHFR levels through the relief of translational regulation, we can increase BH4 bioavailability and restoring eNOS function. Our long-term goal is to test the promising compounds that are effective in our in vitro system in in vivo experimental models.
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Skacel N, Menon LG, Prasunkumar J. Misra, Peters R, Banerjee D, Bertino JR and Ercikan-Abali,E. (2005) Identification of amino acids required for the functional upregulation of human dihydrofolate reductase protein in response to antifolate treatment. J Biol Chem. 280 (24):22721-22731.
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Ercikan Abali, E.A., Banerjee, D., Waltham, M.C., Scotto, K., Bertino, J.R. (1997) Dihydrofolate reductase protein inhibits its own translation by binding to dihydrofolate reductase mRNA sequences within the coding region. Biochemistry 36:12317-12322.
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Hochhauser, D., Schneiders, B., Ercikan Abali, E.A., Gorlick, R., Muise-Helmericks, R., Li, W.W., Fan, J., Banerjee, D., Bertino, J.R. (1996) Effect of cyclin D1 overexpression on drug sensitivity in a human fibrosarcoma cell line. J Natl Cancer Inst. 88:1269-1275.
Ercikan Abali, E.A., Waltham, M., Dicker, A.P., Gritsman, H., Banerjee, D., Bertino, J.R. (1996) Variants of human dihydrofolate reductase with substitutions at leucine-22: effect on catalytic and inhibitor binding properties. Mol Pharmacol. 49:430-437.