The overall objective of this proposal is to examine the role of the OPPC (specific aim 1) in regulating thioltransferase and thioredoxin/thioredoxin reductase. These enzymes are involved in maintaining protein thiol redox homeostasis (specific aim 2), essential for signal transduction and repair pathways (specific aim 3). We expect to produce cell lines (i.e., K1, A549, MCF7, HeLa) deficient in glucose- 6-phosphate dehydrogenase (G6PD), by expressing a blocking anti- G6PD antibody fragment in situ. The direct effects of radiation on these cell lines will be determined. The altered cells are then used to study the role of protein thiol redox status in the X-ray induced necrosis and apoptosis. Protein thiols are oxidized prior to irradiation by exposure to hydroxyethyldisulfide (HEDS). HEDS and lipoate are also used as substrates in a novel assay designed to measure cellular capacity to reduce disulfides. This assay allows us to rapidly determine which of the genetic and/or biochemical manipulations, outlined in aim 2, are the most effective in blocking disulfide reduction. The inhibition of disulfide reduction is used as a criterion for rapidly determining the effects of GSH depletion, BCNU, dehydroepiandrosterone and phenyklarsine oxide on protein disulfide status. In addition, we will use HeLa cells expressing dominant negative thioredoxin reductase on MCF7 cells deficient in thioltransferase activity to determine how each of these enzymes effect radiation-induced apoptosis and clonogenic cell kill. Finally, we will determine if the biochemical or genetic manipulations that result in a lower capacity to reduce disulfides effect specific X-ray and redox-sensitive protein thiols, including; pp59fyn, a cytoplasmic signaling protein; caspase-3, a pro-apoptotic caspase; and Ku70, a component of the DNA damage recognition and repair machinery. Our research program will advance knowledge regarding the biochemistry of sulfhydryl homeostasis in human tumor cells as it relates to radiation damage and repair. Ultimately, we hope to develop specific strategies for modifying thiol redox homeostasis in vivo, to make human tumors more sensitive to ionizing radiation.
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