ROLE OF HYPOXIA IN REGULATING GENE EXPRESSION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 这项研究的重点是了解氧化应激后基因转录的调节机制。 我们一直在使用自参考氧传感器研究氧化还原活性化学品甲萘醌和化疗药物阿霉素的作用机制。 已知这些化学物质诱导细胞氧化应激，包括氧化性DNA损伤和脂质过氧化。 在最初的研究中，努力集中在表征氧利用潜力靶细胞，包括鼠巨噬细胞和肺上皮细胞。 在两种细胞类型中，可以在单细胞以及细胞单层中测量氧通量的梯度。 氧通量是连续的，随着时间的推移，可以检测到在0.5-1毫米的距离细胞单层。 氧通量也被发现被抑制与线粒体抑制剂，包括氰化钾，抗霉素A，复合物III抑制剂，和解偶联剂的氧化磷酸化，羰基氰对三氟甲氧基苯腙（FCCP）。 甲萘醌和阿霉素都被发现增加细胞中的氧通量。 然而，发现线粒体抑制剂仅部分抑制这种活性，表明这些化合物在细胞中的替代性状态下起作用。 还发现用二亚苯基碘鎓（一种膜渗透性NADPH氧化酶抑制剂）处理细胞可部分抑制药物诱导的氧通量增加。 总之，这些数据表明，NADPH氧化酶可能是甲萘醌和阿霉素诱导的细胞氧化应激的关键介质。 研究正在进行中，以确定是否可以使用siRNA在靶细胞中敲低NADPH氧化酶，以及这是否改变巨噬细胞和上皮细胞中的氧通量。

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.