Antitumor Activity of Inducible Nitric Oxide Synthase

描述（申请人提供）：一氧化氮（NO）是一种主要的细胞毒性分子，诱导型NO合成酶（iNOS）可以产生高产量的NO。在肿瘤与宿主细胞相互作用过程中，肿瘤相关的iNOS活性可在肿瘤和宿主细胞中被诱导，并可能抑制肿瘤的生长和转移。然而，由于缺乏合适的模型系统，必须清楚地了解iNOS表达的未知机制及其抗肿瘤的因果活性，以便通过靶向肿瘤相关的iNOS逻辑地设计有效的治疗策略，这是我们目前提出的研究的长期目标。为了验证这个假设，我们提出：#1。为了确定肿瘤细胞中生理性iNOS活性的抗肿瘤活性，我们将在生长和转移不同阶段的iNOS+/+和iNOS-/-肿瘤细胞中检测iNOS的表达，并在同基因iNOS-/-小鼠中系统比较它们的致瘤和转移潜能。使用iNOS-/-小鼠可以清楚地证明iNOS在宿主细胞以外的肿瘤细胞中的表达及其动力学以及其独有的抗肿瘤活性。我们将使用不同p53状态的独特细胞系来测试p53对NO敏感性的影响。我们预计，肿瘤细胞中iNOS基因的破坏和iNOS表达的缺乏会导致肿瘤生长和转移的增加，并且对野生型p53基因的肿瘤细胞的影响比突变型p53基因的肿瘤细胞更明显。# 2。为了确定宿主细胞中生理性iNOS活性的抗肿瘤活性，我们将并行使用iNOS+/+和iNOS-/-小鼠来测定不同p53状态的iNOS-/-细胞系形成的肿瘤中不同阶段iNOS的表达及其动力学。利用iNOS-/-细胞系可以确定iNOS在宿主细胞中的特异性抗肿瘤活性。我们将利用不同p53状态的独特细胞系来检测p53对NO敏感性的影响。我们预计，宿主细胞中iNOS基因的破坏和iNOS表达的缺乏会导致肿瘤生长和转移增加，并且对携带野生型p53基因的肿瘤细胞的影响比携带突变型p53基因的肿瘤细胞更明显。# 3。为了确定增加肿瘤环境中iNOS活性水平的抗肿瘤活性，将使用腺病毒传递系统（Ad.CMV-iNOS）将iNOS基因直接转导到肿瘤细胞内。我们预计，直接的iNOS基因转移将导致肿瘤内部和肿瘤周围的肿瘤相关iNOS活性增加，并抑制其生长和转移。这些作用在携带野生型p53基因的肿瘤细胞中比携带突变型p53基因的肿瘤细胞更为明显。因果效应将通过用氨基胍阻断NO的产生来证实。# 4。为了探讨肿瘤相关iNOS活性的抗肿瘤作用机制，系统比较iNOS+/+和iNOS-/-肿瘤细胞在不同阶段形成的肿瘤细胞的死亡和增殖情况。这则广告。CMV-iNOS将用于增加肿瘤中NO的产生。我们预计破坏肿瘤细胞或宿主iNOS基因会增加肿瘤细胞的存活率，而Ad时iNOS表达升高。CMV-iNOS治疗则会起到相反的作用。这些事件将与对细胞存活和增殖重要的基因表达的改变相关，例如p53、Bcl-2、Bcl-X和Bax。总的来说，所有这些富有洞察力的研究将对有效靶向iNOS/NO途径设计新的治疗策略或改善目前针对癌症的治疗方式至关重要。

DESCRIPTION (provided by applicant): 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.