Cytokine activated macrophages have potent effects against tumor cells in vitro, including inhibition of mitochondrial respiration and DNA synthesis, via high-output nitric oxide (NO.) synthesis. It is not yet known whether NO can act as a host defense against cancers in vivo. Our studies have demonstrated that NO. synthesis is activated within many murine tumors in vivo, and may serve to decrease tumor growth. Furthermore, treatment of mice or humans with IL-2 strongly induces NO. synthesis. Recent murine studies have suggested that the induction of NO. synthesis during lL-2 treatment can mediate tumor regression and improved survival. Exposure of tumor cells to NO. may also induce tumor cell apoptosis (programmed cell death ), a novel cytolytic mechanism. The goal of the current application is to further evaluate the mechanisms by which cytokine induced NO. synthesis acts as an anticancer effector mechanism in vivo, using lL-2 treatment of tumor hearing mice as a paradigm. A better understanding of the role of NO. synthesis during immune response to tumors is necessary to determine whether future attempts should he made to augment or block the function of iNOS enzyme with pharmacologic agents during cytokine treatment of human cancers.
In Specific Aim 1 we propose to characterize the time and dose relationships of IL-2 induced NO. production by serial measurements of serum and urine nitrite and nitrate excretion. The induction of NO. synthesis within tumors in IL-2 treated mice will be-evaluated by analyzing target cell nitrosylation by electron paramagnetic resonance spectroscopy. Further experiments will evaluate iNOS mRNA and protein induction in tumor specimens using RT-PCR and immunoblotting techniques. We have obtained a strain of C57/B16 mice that have had the iNOS gene inactivated by homologous recombination. These mice will be used to further evaluate whether NO. synthesis during IL-2 treatment is derived from the inducible or constitutive enzyme isoforms.
In Specific Aim 2 we plan to identify cellular sources of cytokine induced NO. synthesis within tumors and normal tissues of normal mice treated with IL-2. The proposed experiments will employ immunohistology, flow cytometry, and immunomagnetic separation techniques to identify activated cell populations within tumors and in normal tissues of wild type and iNOS knockout mice. A specific focus will be to determine if iNOS is expressed by tumor infiltrating macrophages or neoplastic cells (or both). Experiments using B-16 melanoma (a syngeneic tumor containing an intact iNOS gene) implanted into C57/B16 iNOS knockout mice will be used to evaluate the role of cytokine-induced iNOS production by tumor cells.
In Specific Aim 3 we will evaluate potential cellular mechanisms by which activated macrophages may mediate cytotoxicity against cancers. Proposed studies will evaluate-target cell damage by reactive oxygen and nitrogen intermediates using assays for hydrogen peroxide, superoxide, peroxynitrites, as well as nitric oxide. The role of NO. induced apoptosis during IL-2 treatment will be evaluated using TdT mediated nick-end labeling assays, as well as DNA electrophoresis on agarose gels.
