Antitumor Activity of Inducible Nitric Oxide Synthase

描述（由申请人提供）：一氧化氮（NO）是一种主要的细胞毒性分子，诱导型NO合酶（iNOS）可产生高输出NO。肿瘤相关的iNOS活性可在肿瘤与宿主细胞相互作用期间在肿瘤和宿主细胞中被诱导，并可抑制肿瘤生长和转移。然而，由于缺乏适当的模型系统，iNOS的表达和其因果抗肿瘤活性的机制尚未清楚地了解，逻辑上设计一个有效的治疗策略，通过靶向肿瘤相关的iNOS，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，将在生长和转移的不同阶段生长的iNOS+/+和iNOS-/-肿瘤细胞中确定iNOS表达，并将在同基因iNOS-/-小鼠中系统地比较它们的致瘤和转移潜力。使用iNOS-/-小鼠可以清楚地证明iNOS在肿瘤细胞而不是宿主细胞中的表达及其动力学，以及其独有的抗肿瘤活性。将使用p53状态不同的独特细胞系来测试p53对NO敏感性的影响。我们预期，肿瘤细胞中iNOS基因的破坏和iNOS表达的缺乏将导致肿瘤生长和转移的增加，并且将对具有野生型p53基因的肿瘤细胞比具有突变型p53基因的肿瘤细胞具有更显著的影响。#2.为了确定宿主细胞中生理性iNOS活性的抗肿瘤活性，将平行使用iNOS+/+和iNOS-/-小鼠来确定由p53状态不同的iNOS-/-细胞系形成的不同阶段的肿瘤中的iNOS表达及其动力学。iNOS-/-细胞系的使用可以确定iNOS在宿主细胞中的排他性抗肿瘤活性。p53状态不同的独特细胞系将用于测试p53对NO敏感性的影响。我们预期，iNOS基因的破坏和宿主细胞中iNOS表达的缺乏将导致肿瘤生长和转移的增加，并且将对具有野生型p53基因的肿瘤细胞比具有突变型p53基因的肿瘤细胞具有更显著的影响。#3.为了确定增加肿瘤环境中iNOS活性水平的抗肿瘤活性，将使用腺病毒递送系统（Ad.CMV-iNOS）将iNOS基因直接递送到肿瘤内的细胞中。我们预期，直接的iNOS基因转移将导致肿瘤内和周围的肿瘤相关的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基因的肿瘤细胞上比在携带突变型p53基因的肿瘤细胞上更明显。将通过用氨基胍阻断NO产生来证实因果效应。#4.为了研究肿瘤相关iNOS活性的抗肿瘤作用机制，将在iNOS+/+和iNOS-/-小鼠中系统地比较由iNOS +/+和iNOS-/-肿瘤细胞形成的不同阶段的肿瘤中的细胞死亡和增殖。Ad.CMV-iNOS将用于增加肿瘤中NO的产生。我们预期肿瘤细胞或宿主iNOS基因的破坏将增加肿瘤细胞存活，而Ad.CMV-iNOS处理后iNOS表达的升高将起相反的作用。这些事件将与对细胞存活和增殖重要的基因的表达改变相关，例如，p53、Bcl-2、Bcl-X和Bax。总的来说，所有这些有见地的研究将是至关重要的，以有效地针对iNOS/NO通路在设计新的治疗策略或改善目前的治疗方式对癌症。

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.