Nitric oxide is a short lived, lipophilic molecule made by a variety of cell types in the body. It's actions are as varied as it sources and range from the endogenous regulation of blood vessels to the control of cell growth. The fact that nitric oxide can inhibit cell growth or induce cell death has interesting implications for the study of cancer because induction and inhibition of nitric oxide can be pharmacologically manipulated. Solid tumors are frequently infiltrated by at least two cell types which are capable of making nitric oxide in response to immunologic stimuli. These are, the endothelial cells that are present in solid tumors undergoing neovascularization, and tumor infiltrating macrophages. The temporal relationship of nitric oxide to expression of effector molecules by the immune system has significant bearing on the cancer problem. A short falling of standard chemotherapy is the inability of the drug(s) to kill all the tumor cells. This gives rise to the phenomenon of the residual tumor burden We have shown that nitric oxide renders a cell transiently resistant to cytotoxic agents whose action is directed towards actively dividing cells. In turn, these cells may give rise to new tumors. The purpose of this application is to expand these findings to the influence of nitric oxide on natural cytolytic agents produced during the immune response to tumors. To study this cells that are known to be sensitive to interleukin-1 and tumor necrosis factor alpha will be exposed to these cytokines in the presence and absence of the nitric oxide generating compound spermine-NO- NOate. Cell death will be determined by a dye exclusion assay and a cloning assay. The ability of nitric oxide to alter the efficacy of natural killer cells will be addressed by co-culturing human natural killer cells with human K562 cells in the presence and absence of spermine NO-NOate. Killing will be quantitated by measuring 51Cr release from the prelabelled K562 cells. The ability of nitric oxide to influence the collagen synthesis stimulator transforming growth factor beta (TGF-beta) will be addressed by incubating rat lung fibroblasts with TGF-beta in the presence and absence of spermine-NO-NOate and then measuring induction of collagen mRNA by Northern blot analysis. Finally, the role of stress proteins in the induction of growth inhibition by nitric oxide will be studied by metabolic labelling, western blot and northern blot analysis.
