Antitumor Activity of Inducible Nitric Oxide Synthase

说明书(申请人提供)：一氧化氮(NO)是一种主要的细胞毒性分子，诱导型一氧化氮合酶(INOS)能产生高产量的NO。肿瘤与宿主细胞相互作用过程中可在肿瘤和宿主细胞中诱导肿瘤相关的iNOS活性，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表达及其因果抗肿瘤活性的未知机制，以便通过靶向肿瘤相关iNOS来合理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。为了验证这一假设，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，我们将检测生长在不同生长和转移阶段的iNOS/和iNOS-/-肿瘤细胞中iNOS的表达，并系统地比较它们在同基因iNOS-/-小鼠中的致瘤和转移潜能。使用iNOS-/-小鼠可以清楚地显示iNOS在宿主细胞以外的肿瘤细胞中的表达及其动力学，并显示其独特的抗肿瘤活性。不同P53状态的独特细胞株将被用来测试P53对NO敏感性的影响。我们预计iNOS基因的破坏和iNOS在肿瘤细胞中的表达缺失将导致肿瘤生长和转移的增加，并且对携带野生型p53基因的肿瘤细胞的影响将比携带突变型p53基因的肿瘤细胞更明显。2.为了确定iNOS生理活性在宿主细胞中的抗肿瘤活性，将iNOS/和iNOS-/-小鼠平行用于检测不同p53状态的iNOS-/-细胞系在不同阶段形成的肿瘤中iNOS的表达及其运动情况。使用iNOS-/-细胞系可以确定iNOS在宿主细胞中的专一性抗肿瘤活性。不同P53状态的细胞株将被用来测试P53对NO敏感性的影响。我们预计iNOS基因的破坏和宿主细胞中iNOS表达的缺失将导致肿瘤生长和转移的增加，并且对携带野生型p53基因的肿瘤细胞的影响将比携带突变型p53基因的肿瘤细胞更明显。#3.为了确定在肿瘤环境中增加iNOS活性的抗肿瘤活性，将使用腺病毒递送系统(Ad.CMV-iNOS)将iNOS基因直接转导到肿瘤内的细胞中。我们预计，直接的iNOS基因转移将导致肿瘤内和肿瘤周围肿瘤相关的iNOS活性增加，并抑制肿瘤的生长和转移。这些作用在携带野生型p53基因的肿瘤细胞上将比在携带突变型p53基因的肿瘤细胞上更加明显。其因果效应将通过用氨基胍阻断NO的产生来证实。4.为探讨肿瘤相关诱导型一氧化氮合酶(INOS)活性的抗肿瘤作用机制，对iNOS/和iNOS-/-小鼠肿瘤细胞形成不同阶段的细胞死亡和增殖进行了系统的比较。Ad.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基因将增加肿瘤细胞的存活率，而在Ad.CMV-iNOS治疗中iNOS表达增加将起相反的作用。这些事件将与对细胞生存和增殖至关重要的基因的表达变化有关，例如，P53、Bcl2、BclX和Bax。总之，所有这些富有洞察力的研究对于有效地针对iNOS/NO途径设计新的治疗策略或改进目前的癌症治疗方式将是至关重要的。

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.