Nitric oxide (NO) is a major cytotoxic molecule, and inducible NO synthase (iNOS) can generate high-output NO. Tumor-associated iNOS activity can be induced in tumor and host cells during the interaction of tumor with host cells and may suppress tumor growth and metastasis. However, due to lack of proper model systems, the as yet unknown mechanisms of iNOS expression and its causal antitumor activity must be clearly understood to logically design an effective therapeutic strategy through targeting tumor-associated iNOS, which is the long-term goal for our currently proposed studies. To test this hypothesis, we propose: #1. To determine the antitumor activity of physiologic iNOS activity in tumor cells, iNOS expression will be determined in iNOS+/+ and iNOS-/- tumor cells growing at the different stages of growth and metastasis and their tumorigenic and metastatic potential will be systematically compared in syngeneic iNOS-/- mice. The use of iNOS-/- mice can clearly demonstrate iNOS expression and its kinetics in tumor cells other than host cells and its exclusive antitumor activity. Unique cell lines differing in p53 status will be used to test the influence of p53 on NO sensitivity. We expect that disruption of iNOS gene and a lack of iNOS expression in tumor cells will lead to increased tumor growth and metastasis, and will have more pronounced effect on tumor cells with wild-type p53 gene than on those with mutant p53 gene. #2. To determine the antitumor activity of physiologic iNOS activity in host cells, iNOS+/+ and iNOS-/- mice will be used in parallel to determine iNOS expression and its kinteics in tumors at various stages formed by iNOS-/- cell lines differing in p53 status. The use of iNOS-/- cell lines can determine the exclusive antitumor activity of iNOS in host cells. Unique cell lines differing in p53 status will be used to test the influence of p53 on the NO sensitivity. We expect that disruption of iNOS gene and a lack of iNOS expression in host cells will lead to increased tumor growth and metastasis, and will have more pronounced effect on tumor cells with wild-type p53 gene than on those with mutant p53 gene. #3. To determine the antitumor activity of increasing the level of iNOS activity in the tumor environment, adenoviral delivery system (Ad.CMV-iNOS) will be used to transduce the iNOS gene directly into the cells within a tumor. We expect that direct iNOS gene transfer will lead to an increased tumor-associated iNOS activity within and around tumors and inhibit their growth and metastasis. These effects will be more pronounced on tumor cells with wild-type p53 gene than on those with mutant p53 gene. The causal effect will be confirmed by blocking NO production with aminoguanidine. #4. To investigate the mechanisms of the antitumor activity of tumor-associated iNOS activity, cell death and proliferation in tumor at their different stages formed by iNOS+/+ and iNOS-/- tumor cells will be systematically compared in iNOS+/+ and iNOS-/- mice. The Ad.CMV-iNOS will be used to increase NO production in the tumors. We expect that disruption of tumor cells or host iNOS gene will increase tumor cell survival, whereas elevated iNOS expression upon Ad.CMV-iNOS treatment will do the opposite. These events will correlate with altered expression of genes important to cell survival and proliferation, e.g., p53, Bcl-2, Bcl-X, and Bax. Collectively, all of these insightful studies will be crucial to effectively targeting the iNOS/NO pathway in designing novel therapeutic strategies or improving current therapeutic modalities against cancer.
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