INHIBITORS OF ENDOGENOUS NITROGEN-OXIDE FORMATION BLOCK THE PROMOTION OF NEOPLASTIC TRANSFORMATION IN C3H 10T1/2 FIBROBLASTS

The endogenous production of nitric oxide (NO) and its role in the neoplastic transformation of C3H 10T1/2 mouse fibroblasts were investigated. NO production, as indicated by NO2- in the culture medium, was increased in cells initiated with 3-methylcholanthrene or stimulated with the combination of interferon-gamma (IFN-gamma ng/ml) plus bacterial lipopolysaccharide (LPS, 1 mug/ml). NO2 was detectable within 24-48 h of IFNgamma/LPS treatment and accumulated to micromolar concentrations within 4 days. NO production was inhibited in a dose-dependent manner by analogs of L-arginine in which the terminal guanidino nitrogen is blocked, consistent with NO production by the oxidative deamination of L-arginine by nitric oxide synthase (NOS). IFNgamma/LPS-stimulated cells expressed a 4.4 kb mRNA which hybridized to a probe for the mouse macrophage-inducible NOS. Expression of the rat cerebellar constitutive NOS was not detected in these cells. Arginine analogs added to the culture medium during the post-confluence promotional stages of the C3H 10T1/2 transformation assay blocked the formation of transformed foci in a dose-dependent manner comparable to their inhibition of NO production. These data demonstrate that C3H 10T1/2 mouse fibroblasts are a useful model for the study of the effects of endogenous NO production in carcinogenesis and suggest that NO plays a significant role in the promotional phase of neoplastic transformation of these cells.