This application would investigate folate-based control of CpG site methylation in the promoter region of the thymidylate synthase gene leading to attenuation of expression. During previous cycles a series of methods were developed to evaluate tissue and tumor reduced folates which have been used to investigate the metabolic basis for the activity of antitumor drugs. Most recently a stable inducible intisense expression system for folylpolyglutamate synthetase down-regulation was developed and used to investigate this enzyme as a potential therapeutic target. The current application would extend these earlier studies to establish the basis for folate-controlled methylation of DNA and the impact of this methylation on expression of the important drug target, thymidylate synthase. Preliminary studies have shown that low dietary folic acid leads to hypomethylation of DNA in implanted mouse tumors, and the elevated levels of thymidylate synthase. This model would now be used to establish the means by which folate controls methylation and how this altered methylation is associated with thymidylate synthase expression. This goal would be addressed through the following four Specific Aims: 1. Fully characterize the dose and kinetic dependence of DNA methylation on folate, 2. Determine the metabolic basis for folate control of DNA methylation, 3. Establish the role of specific transcription factor site methylation on regulation of thymidylate synthetase expression, and 4. Determine the ability of folate antagonists to alter methylation and thymidylate synthase transcription. It has been shown that as little as two-fold evaluation of thymidylate synthase message in patient tumor samples is related to fluorouracil response failure. Hence, a better understanding of how expression is regulated, particularly by folates and antifolates, is vital to improved use of a very important class of anticancer drugs that target this enzyme.
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