The goal of this research is to define the mechanism responsible for the resistance to cancer chemotherapy agents due to expression of the bcl-2 oncogene. Bcl-2, the prototypic member of a family of proteins that regulate apoptosis, is abnormally expressed in many different types of human cancer including lymphomas, and tumors of the breast, prostate, lung and colon. Bcl-2 blocks apoptosis and, thus, causes tumor cells to become resistant to anticancer treatments such as radiotherapy and chemotherapy. The mechanism by which bcl-2 blocks apoptosis is not understood, but it appears to be related to bcl-2's localization in intracellular membranes including mitochondrial membranes and the nuclear envelope. At these sites, bcl-2 appears to regulate the transport of substances or factors involved in the apoptotic cascade. However, additional activities of bcl-2 also require explanation. Other observations indicate that bcl-2 may have a role in the cell's antioxidant pathways, specifically those involving thiols such as glutathione (GSH). Progress related to this application has demonstrated that bcl-2, at the level of the nuclear envelope, redistributes GSH into the nucleus by active transport where it apparently inhibits the proteases that carry out the apoptotic process. If this model is further validated to indicate that GSH is the substance blocking apoptosis, strategies may be devised to overcome such a block and restore anticancer drug-sensitivity. In this application, the following questions that serve to test predictions of this model, will be addressed. 1. Are other members of the bcl-2 family of proteins also involved in GSH metabolism? 2. Does bcl-2-expression also alter GSH levels in mitochondria? 3. Will newly developed strategies that target bcl-2 function or GSH itself act to sensitize tumor cells to anticancer drugs? 4. Is bcl-2 expression in tumor cells under the control of the NF-kappaB transcription factor? The methods to be used include quantitative measurements of GSH in intracellular pools and determinations of relative expression of bcl-2 and other proteins by immunoblot analysis. Apoptosis and clonogenic survival will be used as relevant endpoints for cell response to chemotherapy agents.
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