The activated Ras oncogene can transform various mammalian cells and has been implicated in development of a high population of malignant human tumors. Recent studies suggest that generation of reactive oxygen species such as superoxide and H 2 O 2 is involved in cell transformation by the activated Ras. However, the nature of an oxidase participating in Ras-transformation is presently unknown. Here, we report that Ras oncogene up-regulates the expression of Nox1, a homologue of the catalytic subunit of the superoxide-generating NADPH oxidase, via the mitogenactivated protein kinase kinase-mitogen-activated protein kinase pathway, and that small interfering RNAs designed to target Nox1 mRNA effectively blocks the Ras transformed phenotypes including anchorageindependent growth, morphological changes, and production of tumors in athymic mice. Therefore, we propose that increased reactive oxygen species generation by Ras-induced Nox1 is required for oncogenic Ras transformation.

Ha-ras is a member of a multigene family in man which encode highly related proteins of 189 amino acids (p21). In vitro, ras proteins bind GTP, and p21 mutants with treonine at position 59 autophosphorylate at that residue. Mutation (at amino acids 12 or 61) and elevated expression of ras genes result in cell transformation in culture, and are also observed in many types of human tumours. Normal and mutant transforming ras proteins show no differences in localization, lipidation or GTP binding. However, mutations at position 12 in recombinant (Thr 59) p21 molecules were observed to affect autophosphorylation. We have expressed the full-length normal and T24 transforming (Gly----Val at position 12) Ha-ras proteins in Escherichia coli and have purified them to homogeneity (ref. 19 and M.G. et al., in preparation); these proteins bound GTP with approximately molar stoichiometry and with an affinity comparable to partially purified mammalian proteins. Microinjection of the T24 protein into quiescent rodent fibroblasts resulted in a rapid alteration in cell morphology, stimulation of DNA synthesis and cell division; in contrast, little response was observed with the normal protein. We now report that the normal ras protein has an intrinsic GTPase activity, yielding GDP and Pi. In contrast, the T24 transforming protein is reduced 10-fold in this activity. We suggest that this deficiency in GTPase is the probable cause for the transforming phenotype of the T24 protein.

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...

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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.