The finding of frequent nitric oxide synthase expression in human cancers indicates that nitric oxide has a pathophysiological role in carcinogenesis. To determine the role of nitric oxide in tumor progression, we generated human carcinoma cell lines that produced nitric oxide constitutively. Cancer cells expressing inducible nitric oxide synthase that had wild-type p53 had reduced tumor growth in athymic nude mice, whereas those with mutated p53 had accelerated tumor growth associated with increased vascular endothelial growth factor expression and neovascularization. Our data indicate that tumor-associated nitric oxide production may promote cancer progression by providing a selective growth advantage to tumor cells with mutant p53, and that inhibitors of inducible nitric oxide synthase may have therapeutic activity in these tumors.

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High concentrations of nitric oxide (NO) cause DNA damage and apoptosis in many cell types. Thus, regulation of NO synthase (NOS) activity is essential for minimizing effects of cytotoxic and genotoxic nitrogen oxide species. We have shown previously that NO-induced p53 protein accumulation down-regulates basal and cytokinemodulated inducible NOS (NOS2) expression in human cells in vitro. To further characterize the feedback loop between NOS2 and p53, we have investigated NO production, i.e., urinary nitrate plus nitrite excretion, and NOS2 expression in homozygous p53 knockout (KO) mice. We report here that untreated p53 KO mice excreted 70% more nitrite plus nitrate than mice with wild-type (wt) p53. NOS2 protein expression was constitutively detected in the spleen of untreated p53 KO mice, whereas it was undetectable in the spleen of wt p53 controls. Upon treatment with heat-inactivated Corynebacterium parvum, urinary nitrite plus nitrate excretion of p53 KO mice exceeded that of wt controls by approximately 200%. C. parvum treatment also induced p53 accumulation in the liver. Splenectomy reduced the NO output of C. parvum-treated p53 KO mice but not of wt p53 controls. Although NO production and NOS2 protein expression were increased similarly in KO and wt p53 mice 10 days after injection of C. parvum, NOS2 expression returned to baseline levels only in wt p53 controls while remaining up-regulated in p53 KO mice. These genetic and functional data indicate that p53 is an important transrepressor of NOS2 expression in vivo and attenuates excessive NO production in a regulatory negative feedback loop.NO, a chemical radical, and its toxic derivatives (1-3) can cause DNA damage (4-7) and cell death (8-11) in a variety of cell types. NO also has the ability to directly modify intracellular targets such as proteins and lipid-peroxidation products (12)(13)(14)(15)(16). NO produces cytotoxicity at high concentrations, whereas NO at lower concentrations may have the opposite effect and protect against apoptotic cell death from various stimuli (17-19). Therefore, regulation of NO production is vital for both cell survival and genome integrity. Among the three NO synthase (NOS) isoforms, NOS1 and NOS3 are controlled by calcium fluxes and produce only nanomolar NO concentrations (20)(21)(22). NOS2 is active at resting concentrations of calcium and is capable of producing micromolar NO concentrations (23). NOS2 expression can be stimulated by cytokines (23, 24) that act by transcriptional activation of the promoter region (25-27). Functional regulatory elements that are responsive to interferon ␥ and lipopolysaccharide have been characterized in the first 1 kb of the murine promoter region (25,26). In contrast, no functional elements have been identified in the proximal 1 kb of the human NOS2 promoter. In fact, a 16-kb segment of the human promoter was required to produce a response similar to the murine 1.7-kb promoter fragment (27,28). Several inhibitors of human NOS2 expression have been identified. Glucocortic...