During Fiscal Year 1986 our work continued on the mechanism of action of antifolates. Our major projects were the following: 1. Further elucidation of the effects of antifolates on intracellular folate pools. We developed methods for assay of 5,10-methylene FH4 and showed that methotrexate or trimetrexate treatment led to approximately 50% depletion of this cofactor, at a time when the thymidylate synthesis pathway was completely inhibited. This provides further evidence that antifolate effects are mediated by direct inhibition of TS and purine synthesis by dihydrofolate and methotrexate polyglutamates. 2. Studies of isolated bone marrow myeloblasts revealed that methotrexate produced the same perturbations of intracellular folate pools in this cell population as seen in malignant cells (MCF-7), that is, partial depletion of reduced folate cofactors, a marked rise in dihydrofolate, and the appearance of a new folate, 10-formyl dihydrofolate, a compound that we found to strongly inhibit thymidylate synthase but to serve as a cofactor for AICAR transformylase, the key purine step inhibited by the antifolates. 3. The rise in FH2 can be modulated by blockade of TS by 5-FUdR, thus preventing an increase in FH2. Using this system, we have shown that the activity of AICAR transformylase, as indicated by de novo purine biosynthesis, is inversely correlated with the FH2 concentration and not with 10-formyl FH4 levels.
