NITRIC OXIDE AS A TUMOR DEFENSE

细胞因子激活的巨噬细胞对肿瘤细胞具有强有力的作用 体外，包括抑制线粒体呼吸和DNA 合成，通过高输出一氧化氮（NO.）合成.目前尚不 目前还不清楚NO是否能在体内作为宿主防御癌症的机制。我们 研究表明，NO的合成在许多细胞中被激活， 小鼠肿瘤，并可用于减少肿瘤生长。 此外，用IL-2处理小鼠或人强烈诱导NO。 合成.最近的小鼠研究表明，诱导NO。 IL-2治疗期间的合成可以介导肿瘤消退和改善 生存肿瘤细胞暴露于NO也可诱导肿瘤细胞增殖。 细胞凋亡（程序性细胞死亡），一种新的细胞溶解机制。目标 本申请的目的是进一步评估 细胞因子诱导的NO合成作为抗癌效应机制 在体内，使用IL-2治疗肿瘤听力小鼠作为范例。一 更好地了解NO合成在免疫反应中的作用 肿瘤是必要的，以确定未来的尝试是否应该 用于增强或阻断iNOS酶的功能， 在细胞因子治疗人类癌症期间的药物。具体目标1， 建议表征IL-2诱导的时间和剂量关系， 号通过连续测量血清和尿亚硝酸盐的产生， 硝酸盐排泄IL-2诱导肿瘤内NO合成 通过分析靶细胞的亚硝基化来评价处理的小鼠， 电子顺磁共振谱进一步的实验将 用RT-PCR评价肿瘤标本中诱导型一氧化氮合酶mRNA和蛋白的诱导 和免疫印迹技术。我们获得了一个C57/B16小鼠品系， 通过同源重组使iNOS基因失活。 这些小鼠将用于进一步评估在哺乳动物中NO合成是否与哺乳动物中的NO合成有关。 IL-2治疗来源于诱导型或组成型酶 同种型。在具体目标2中，我们计划确定细胞来源， 细胞因子诱导的肿瘤和正常组织中的NO合成 用IL-2处理的小鼠。拟议的实验将采用 免疫组织学、流式细胞术和免疫磁性分离技术 识别肿瘤和正常组织中的活化细胞群， 野生型和iNOS敲除小鼠的组织。一个具体的重点将是 确定iNOS是否由肿瘤浸润巨噬细胞表达， 肿瘤细胞（或两者）。使用B-16黑色素瘤（一种同源黑色素瘤）的实验 含有完整iNOS基因的肿瘤）植入C57/B16 iNOS中 将使用基因敲除小鼠来评估精氨酸诱导的iNOS的作用。 由肿瘤细胞产生。在具体目标3中，我们将评估潜力 活化的巨噬细胞可能介导的细胞机制 对癌症的细胞毒性。拟定的研究将评价靶细胞 活性氧和氮中间体的损伤 过氧化氢、超氧化物、过氧亚硝酸盐以及一氧化氮。 将评估IL-2治疗期间NO诱导细胞凋亡的作用 使用TdT介导的切口末端标记分析，以及DNA 琼脂糖凝胶电泳。

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.