NITRIC OXIDE AS A TUMOR DEFENSE

细胞因子激活的巨噬细胞对肿瘤细胞有很强的杀伤作用 在体外，包括抑制线粒体呼吸和DNA 合成，通过高产量的一氧化氮(No.)综合。现在还不是 已知NO在体内是否可以作为抵抗癌症的宿主防御。我们的 研究表明，没有。合成是在许多情况下被激活的 小鼠体内的肿瘤，并可能起到减少肿瘤生长的作用。 此外，用IL-2处理小鼠或人会强烈地诱导NO。 综合。最近的小鼠研究表明，NO的诱导。 IL-2治疗期间的合成可介导肿瘤的消退和改善 生死存亡。肿瘤细胞暴露于NO。也可能诱导肿瘤细胞 细胞凋亡(程序性细胞死亡)，一种新的细胞溶解机制。目标是 当前应用的关键是进一步评估 细胞因子诱导NO。合成作为一种抗癌效应机制 在体内，以IL-2治疗肿瘤听力的小鼠为范式。一个 更好地理解NO的作用。免疫应答过程中的合成 对肿瘤的诊断是必要的，以确定未来的尝试是否应该 用药理作用增强或阻断iNOS酶的功能 细胞因子治疗人类癌症的药物。在具体目标1中，我们 建议刻画IL-2诱导的时间和剂量关系 不是的。通过连续测定血清和尿中亚硝酸盐和 硝酸盐排泄物。NO的诱导。IL-2在肿瘤内的合成 治疗后的小鼠将通过分析靶细胞亚硝化来进行评估 电子顺磁共振波谱。进一步的实验将 RT-PCR检测肿瘤标本诱导型一氧化氮合酶基因和蛋白的表达 和免疫印迹技术。我们获得了一株C57/B16小鼠 通过同源重组使iNOS基因失活的基因。 这些小鼠将被用来进一步评估是否没有。合成过程中 IL-2治疗是从诱导酶或构成酶衍生出来的 异构体。在具体目标2中，我们计划确定细胞来源 细胞因子诱导NO。正常人体肿瘤及正常组织内合成 用IL-2处理的小鼠。拟议的实验将使用 免疫组织学、流式细胞术和免疫磁分离技术 确定肿瘤内和正常组织中激活的细胞群 野生型和iNOS基因敲除小鼠的组织。具体的重点将是 确定iNOS是由肿瘤浸润性巨噬细胞还是 肿瘤细胞(或两者都有)。使用B-16黑色素瘤(一种同基因黑色素瘤)的实验 含完整iNOS基因的肿瘤)植入C57/B16 iNOS 基因敲除小鼠将被用来评估细胞因子诱导的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 激活的巨噬细胞可能介导的细胞机制 对癌症的细胞毒性。建议的研究将评估-目标细胞 使用活性氧和氮中间体的分析方法进行损害 过氧化氢、超氧化物、过氧亚硝酸盐以及一氧化氮。 NO的作用。将评估在IL-2治疗过程中诱导的细胞凋亡 使用TdT介导的缺口末端标记分析以及DNA 琼脂糖凝胶上的电泳法。

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.