This proposal focuses on the biological consequence of purine starvation in order to elucidate the mechanism of the antitumor effects of purine antimetabolites. These agents cause either general purine starvation or selective depletion of adenine or guanine alone. We have found that each class of purine depleting agents produces distinct biological effects, most notably the dramatic inhibition of DNA synthesis that occurs with guanine starvation. One portion of the project proposal deals with the interaction among the cellular toxicities caused by combined or individual adenine and guanine starvation in order to provide a biochemical rationale for the selection of purine antimetabolites as anticancer agents. The other portion addresses specifically the effects of guanine depletion alone. Inhibitors of de novo guanine nucleotide formation (via IMP dehydrogenase) rank among the most potent antiviral and antitumor agents. Yet it remains unknown why ribavirin for example is a potent antiviral agent with rather poor antitumor efficacy, while tiazofurine is a potent antitumor drug, at least in animal models. The toxicity of both agents can be reversed by the addition of a guanine source via the salvage bypass, which suggests that guanine starvation is indeed the major mode of action. Howeve, we have observed differences in the biochemical mechanism of action of these agents that may result from differential effect on guanine nucleotide metabolism. Indeed, GMP, GDP and GTP each serve different cellular functions, with GDP possibly representing the functional DNA precursor pool that feeds into the """"""""replitase"""""""" system. The replitase is thought to be a multienzyme complex that utilizes distant DNA precursors and channels substrates directly to the site of replication forks. The proposal thus presents a novel approach to study the mechanism of the cellular toxicity of guanine depleting agents, that takes into account the possible compartmentation of guanine nucleotides (mono-, di and tri-phosphates) into general cellular pools and functional pools for specific purposes.
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