Cocultured with Interferon- but not Tumor Promoter-resistant, JB6 Mouse Epidermal Cells Nitric Oxide Synthase Is Induced in Tumor Promoter-sensitive,

Expression of inducible nitric oxide synthase (iNOS) has been reported to be involved in certain organs of potential tumorigenesis, including the stomach and colon. The mechanisms for iNOS expression in epithelial cells, however, are not fully understood. In the present study, we investigated the role of macrophages in epithelial iNOS expression by coculturing a stimulated murine macrophage-like cell line, RAW 264.7, with either tumor promoter-sensitive (P1) or promoter-resistant (P2) JB6 murine epidermal cells. After monoculture, treatment of RAW 264.7 cells with IFN-g for 24 h generated a large amount of nitrite (NO2 ), as reported previously, whereas no increase in NO2 2 concentration was observed in the IFN-g-treated P1 or P2 subclones. Interestingly, when IFN-g-treated RAW 264.7 cells were cocultured with P1 but not P2 cells, we observed a marked increase in NO2 2 concentration (30.8 6 3.6 mM), which significantly exceeded (P < 0.01) the sum of the concentrations (20.0 6 2.3 mM) added from each cell line monoculture. Western blotting analysis revealed that, after coculture, iNOS protein was up-regulated 55-fold more than the control in JB6 P1 but not in P2 cells. IFN-g-treated RAW 264.7 cells secreted proinflammatory cytokines, including tumor necrosis factor (TNF)-a and interleukin (IL)-1b. The addition of IFN-g-treated RAW 264.7 cell-conditioned media to P1 subclones led to a significant enhancement of NO2 2 formation that was diminished by the TNF-aspecific but not IL-1b-specific antibody. When combined with IFN-g, the recombinant TNF-a (1–100 ng/ml) enhanced NO2 2 formation in JB6 P1 cells, whereas IL-1b (1–100 ng/ml) did not. These results led us to conclude that IFN-g-treated RAW 264.7 cells release TNF-a to induce iNOS expression in promoter-sensitive JB6 cells. Thus, we propose the hypothesis that macrophages stimulate neoplastic cells with TNF-a via a paracrine loop to induce epithelial iNOS protein expression.