Mechanism of Action of Folate Antagonist

The goal of this revised renewal application is to understand in detail the transcriptional and translational regulation of two key enzymes, dihydrofolate reductase (DHFR) and thymidylate synthase (TS), that are targets for well established anticancer agents, methotrexate and 5- fluorouracil respectively. The hypothesis that drives these studies is that this understanding will generate new targets for drug development and more rational use for these agents, alone or in combination. There are three specific aims: Speeifie aim#1 describes our studies of the translational regulation of DHFR. Biophysical measurements (fluorometry,circular dichroism and isothermal calorimetry) and x-ray crystallography will be used to study the binding of DHFR to its cognate mRNA, and regions of mRNA involved in binding to the protein. Mutational analysis of single amino acids and regions of DHFR protein will be evaluated to reveal areas of DHFR protein involved in its binding to DHFR mRNA. This information will be used to generate peptide aptamers that block the binding to DHFR following MTX treatment. Specific aim#2 will further characterize a peptide that we have shown inhibits E2F binding to its recognition sequence in DNA and has cytoxic activity. Optimization of the peptide sequence to improve stability and increase binding are planned in collaboration with Dr. Breslauer and colleagues of Rutgers University. The effect of down regulation of activating E2Fs (E2F 1,2,3) will be studied using siRNAs specific to each of these transcription factors. A goal of this specific aim is to measure the effect of the down regulation of each of these transcription factors, alone and in combination on levels of DHFR, TS and ribonucleotide reductase, and corresponding sensitivity to specific inhibitors of these enzymes. Specific aim#3 explores further our previous observations, namely that forced expression of p 14ARF in cells with non-functional p53 down regulates DHFR. The use of MEF cells null for p53, p19 ARF and both will be utilized to test in a more well defined system the function of these proteins on DHFR levels.