TNF-[alpha]/cycloheximide-induced apoptosis in intestinal epithelial cells requires Racl-regulated reactive oxygen species

Previously we have shown that both Rac 1 and c-Jun [NH.sub.2]-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor TNF-[alpha]/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Racl-mediated ROS production is involved in TNF-[alpha]-induced apoptosis. In this study, we showed that TNF-[alpha]/CHX-induced ROS production and hydrogen peroxide ([H.sub.2][O.sub.2])-induced oxidative stress increased apoptosis. Inhibition of Racl by a specific inhibitor NSC23766 prevented TNF-[alpha]-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Racl by expression of dominant negative Racl decreased TNF-[alpha]-induced mitochondrial ROS production. Moreover, TNF-[alpha]-induced cytosolic ROS production was inhibited by Racl inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-[alpha]-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Racl or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-[alpha]-induced apoptosis requires Racl-dependent ROS production in intestinal epithelial cells. intestinal epithelial cells-6; N17Rac1; diphenyleneiodonium; JNK 1/2; oxidative stress