This project is divided into two broad areas that include the development of strategies for the treatment of solid tumors and the development of therapies for the treatment of opportunistic infections in patients with AIDS. The antineoplastic investigations revolve around the development of a complete understanding of the mechanisms of action and resistance to the antimetabolite class of agents. These studies have identified that the treatment of patients and malignant cells in in vitro model systems with fluoropyrimidines and antifolate agents results in an acute induction of thymidylate synthase and dihydrofolate reductase respectively. Since the level of intracellular thymidylate synthase is an important determinant of sensitivity to fluoropyrimidine agents, the acute induction of this enzyme following fluoropyrimidine exposure is a central cause of resistance to these agents both in vivo and in vitro. Interferon gamma has been shown to repress the acute thymidylate synthase induction resulting from fluoropyrimidines. This repression results in enhanced sensitivity of malignant cells. While the precise mechanism of interaction between interferon represents an ongoing investigative effort, we have identified an unprecedented mechanism of autoregulatory translational control as a means by which cells regulate the intracellular levels of thymidylate synthase. Given the importance of thymidylate synthase as a chemotherapeutic target, we have developed sensitive assays for the quantitation of this enzyme in cells and human tissues using a monoclonal antibody directed against thymidylate synthase. The use of monoclonal antibodies has resulted in ultrasensitive detection of thymidylate synthase, quantitation of enzyme free and bound by fluoropyrimidines and quantitation on a per cell basis in human tissues and cells. The availability of these sensitive assays will help delineate the role of thymidylate synthase as a prognosticator of survival and response. The investigations of therapies for opportunistic infections is focussed on the interactions of antifolate agents on the metabolic pathways in T. gondii, P. carinii and Mycobacteria (MTb, MAI). We are currently using the tools of molecular biology to clone, sequence and express clinically relevant target enzymes for characterization and as an aide in the search for new therapeutic agents.
