Nerve growth factor (NGF) stimulation of pheochromocytoma PC12 cells transiently increased the intracellular concentration of reactive oxygen species (ROS).This increase was blocked by the chemical antioxidant N-acetylcysteine and a flavoprotein inhibitor, diphenylene iodonium. NGF responses of PC12 cells, including neurite outgrowth, tyrosine phosphorylation, and AP-1 activation, was inhibited when ROS production was prevented by N-acetylcysteine and diphenylene iodonium. The expression of dominant negative Rac1N17 blocked induction of both ROS generation and morphological differentiation by NGF. The ROS produced appears to be H 2 O 2 , because the introduction of catalase into the cells abolished NGF-induced neurite outgrowth, ROS production, and tyrosine phosphorylation. These results suggest that the ROS, perhaps H 2 O 2 , acts as an intracellular signal mediator for NGF-induced neuronal differentiation and that NGF-stimulated ROS production is regulated by Rac1 and a flavoprotein-binding protein similar to the phagocytic NADPH oxidase.Reactive oxygen species (ROS) 1 that cause oxidative stress have generally been viewed as cytotoxic depending on the dose (1, 2). ROS are responsible for the host defense mechanism in neutrophils (3) and possess carcinogenic potential associated with tumor promotion (4, 5). Recent studies, however, indicate that small nontoxic amounts of ROS may play a normal role as a second messenger in the various signaling pathways (1). The production of ROS such as superoxide (O 2 . ) and hydrogen peroxide (H 2 O 2 ) was observed in a number of cells stimulated with cytokines such as transforming growth factors-␤1 (6, 7), interleukin-1 (8), and tumor necrosis factor ␣ (9) or peptide growth factors such as platelet-derived growth factor (PDGF) (10) and epidermal growth factor (EGF) (11). H 2 O 2 has been shown to mediate PDGF-induced cellular DNA synthesis of rat vascular smooth muscle cells (10). Ras-dependent cell growth requires generation of the O 2 . free radical through a pathway involving Rac1 (12). Although the role of ROS has been extensively studied in mitogenesis, inflammation, and apoptosis (1), little is known about its functional role in the differentiation process. The differentiation process in the nervous system is regulated by the action of differentiation and growth factors including NGF. NGF induces the growth arrest of PC12 cells and promotes their differentiation into sympathetic neuron-like cells (13). NGF binding to its receptor tyrosine kinase, TrkA, initiates various molecular interactions including tyrosine phosphorylation of proteins and the action of the Ras/Raf/MEK/MAPK pathway (14,15). NGF induces the production of reactive nitric oxide (NO), and NO is required for NGF-induced cytostasis and differentiation (16), suggesting that free radical molecules such as NO and ROS may exert a regulatory role in certain types of cellular differentiation. In the current study, we focused on the role of ROS and a small GTP-binding protein, Rac1, in the NGF-induced neuron...

Interleukin-1 (IL-1) and tumor necrosis factor (TNF) selectively induce mitochondrial manganese superoxide dismutase (MnSOD) production in various cell types. We have evaluated the capacity of tumor cells that overexpress MnSOD to recover from the cytostatic and cytotoxic effects of cytokines (IL-1 and TNF), chemotherapeutic agents, and ionizing irradiation. Clones of human melanoma cell line, A375, which overexpressed MnSOD after sense MnSOD cDNA transfection, showed increased recovery from treatment with cytostatic and cytotoxic doses of IL-1 alpha and TNF alpha, whereas clones of A375 cells that were transfected with anti-sense MnSOD cDNA recovered less well than normal cells from IL-1 alpha and TNF alpha. In addition, Chinese hamster ovary (CHO) cells transfected with sense MnSOD cDNA showed increased survival after treatment with doxorubicin, mitomycin C, and gamma (gamma) radiation in vitro. It is hypothesized that mitochondrial MnSOD, by scavenging oxygen radicals induced by cytokines, some cytotoxic drugs, and ionizing radiation, is protective and promotes the survival of cells from the lethal effects of these treatments.

Pancreatic adenocarcinoma is an aggressive human malignancy and is characterized by resistance to apoptosis. Recently, NADPH oxidase (Nox) 4-mediated generation of intracellular reactive oxygen species (ROS) was proposed to confer antiapoptotic activity and thus a growth advantage to pancreatic cancer cells. The signaling mechanism by which Nox4 transmits cell survival signals remains unclear. Here, we show that both a flavoprotein inhibitor, diphenylene iodonium (DPI), and small interfering RNAs designed to target Nox4 mRNA (siNox4R-NAs) inhibited superoxide production in PANC-1 pancreatic cancer cells, and depletion of ROS by DPI or siNox4RNAs induced apoptosis. Parallely, DPI treatment and siNox4RNA transfection blocked activation of the cell survival kinase AKT by attenuating phosphorylation of AKT. Furthermore, AKT phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) on Ser-83 was reduced by DPI and siNox4RNAs. When ASK1Ser83Ala (an AKT phosphorylation-defective ASK1 mutant) was introduced into PANC-1 cells, this mutant alone induced apoptosis. But, addition of DPI or co-transfection of siNox4RNA had no additive effect, indicating that the mutant can substitute for these reagents in apoptosis induction. Taken together, these findings suggest that ROS generated by Nox4, at least in part, transmit cell survival signals through the AKT-ASK1 pathway in pancreatic cancer cells and their depletion leads to apoptosis.

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