Inhibitors of the folate pathways have proven to be useful in the chemotherapy of human cancer and are often used in the treatment of childhood leukemias and of some adult cancers, including metastatic breast carcinomas. In recent years, new classes of folate antagonists have been discovered which inhibit enzymes other than dihydrofolate reductase and which have a different pattern of therapeutic activity and toxicity in animal models of human cancers. We propose to study the biochemical, cellular, and molecular aspects of the processes underlying the action of one such class of new agents, typified by 5,10- Dideazatetrahydrofolate (DDATHF). DDATHF has served as a critical new probe of phenomena not even suspected before its introduction but which underlie tetrahydrofolate antimetabolite chemotherapy. The clinical formulation of DDATHF, Lometrexol, has shown antitumor activity in phase I trials, but has also shown a pattern of cell lineage-specific and cumulative toxicity never experienced before with folate antimetabolites. We now propose to clarify the bases for the specific toxicities, cumulative toxicity, and therapeutic activity of DDATHF through a series of fundamental studies on the proteins involved in its action and the genes that encode them. The thermodynamics of binding of DDATHF and its polyglutamates to recombinant mouse trifunctional GART will be studied as an issue central to the understanding of these compounds and these studies will then be extended to an analysis of the interaction of DDATHF and its metabolites with GART in vivo. Our study of the mechanism of resistance of the L1210/D3 cell to DDATHF showed that the expanded pools of folates in these cells resulted in a blockade of polyglutamation, presumably by a direct effect on FPGS. We will determine the exact mechanism of this effect and its generality as a feedback control of folylpolylutamate homeostasis, and the role of this phenomenon in the cumulative toxicity of DDATHF-FPGS levels are controlled by at least two mechanisms, one of which it tissue specific, the other is tied to proliferation and/or differentiation, we will determine the basis of these effects. We will determine when FPGS is expressed during the maturation of platelets, erythrocytes, and leucocytes in an effort to understand the tissue specific toxicities of DDATHF. We will seek an explanation of the apparent limitation on the cell kill achievable with DDATHF, and will attempt to unravel why cells die after cessation of purine synthesis. Finally, we will seek an explanation of the function of the monofunctional GARS protein and the advantage of trifunctionality of GARS-AIRS-GART.
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