ROLE OF HYPOXIA IN REGULATING GENE EXPRESSION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This research is focused on understanding mechanisms regulating transcription of genes following oxidative stress. We have been using the self-referencing oxygen sensor to investigate the mechanism of action of the redox active chemicals menadione and the chemotherapeutic drug adriamycin. These chemicals are known to induce cellular oxidative stress including oxidative DNA damage and lipid peroxidation. In initial studies, efforts were focused on characterizing oxygen utilization potential target cells including murine macrophages and lung epithelial cells. In both cell types are gradient of oxygen flux could be measured in single cells as well as in cell monolayers. Oxygen flux was continuous over time and could be detected at 0.5-1 mm distances from cell monolayers. Oxygen flux was also found to be inhibited with mitrochondrial inhibitors including potassium cyanide, antimycin A, a complex III inhibitor, and an uncoupler of oxidative phosphorylation, carbonyl cyanide p-trifluoromethoxy- phenylhydrazone ( FCCP). Both menadioine and adriamycin were found to increase oxygen flux in the cells. However, the mitrochondrial inhibitors were found to only partially inhibit this activity suggesting that these compounds function at alternative stes in cells. Treatment of the cell with diphenylene iodonium, a membrane permeable NADPH oxidase inhibitor was also found to partially inhibit drug-induced increases in oxygen flux. Taken together these data suggest that NADPH oxidase may be a critical mediator of menadione- and adriamycin-induced cellular oxidative stress. Studies are in progress to determine if NADPH oxidase can be knocked-down in target cells using siRNA and if this alters oxygen flux in macrophages and epithelial cells.