Radiation-induced Nitric Oxide & Cellular Radiosensitivity

描述（由申请人提供）：在过去的资助期内，对细胞感知氧化事件但通过NO 7依赖性蛋白质翻译后机制发出信号的假设进行了测试。两个信号转导机制重要的细胞保护反应电离辐射（IR）进行了检查：酪氨酸激酶信号和激活的转录因子NF-？B。临床相关剂量（<5戈伊）的IR激活Ca 2+依赖性NOS，导致PTP活性位点Cys的瞬时S-亚硝化和细胞PTP的抑制。自分泌调节的肿瘤细胞中的一个结果是IR增强的生长因子受体Tyr激酶信号传导。这些发现证明了解释IR和其他轻度氧化应激激活细胞保护性Tyr激酶信号通路的常见观察结果的第一种机制。这些相同的IR剂量也刺激NK-？B活性的机制，涉及瞬时硝化的抑制剂蛋白，我？B？后一个发现促使本发明提出检验以下假设：关键调节蛋白的IR诱导的Tyr硝化是响应于温和氧化/亚硝化应激（例如IR）的特异性翻译后修饰。eNOS和iNOS在MCF-7中的表达和活性水平7乳腺癌细胞和肿瘤内皮细胞的体外和体内监测作为剂量（1-10戈伊）和时间（IR后最多48小时）的函数。免疫细胞化学和亚细胞分级方法确定硝化和NOS定位的网站。表达高水平iNOS的浸润性炎性细胞的作用通过用抗GR-1耗尽肿瘤的浸润性细胞来检查。具体目标2：通过质谱鉴定IR后硝化的关键调控蛋白的全球方法，以及鉴定预测硝化的结构基序的物理化学建模和遗传验证研究。特定目标3测试p53的特定硝化是否在细胞对辐射的反应中起作用。初步研究表明，p53是瞬时硝化后IR。质谱鉴定出两个可硝化酪氨酸，包括Tyr 327在四聚体结构域和Try 107在DNA结合域。他们的硝化线粒体和核定位，与Bcl分子的相互作用，和转录反应方面的功能后果进行检查。证明一个功能的后果是至关重要的，在建立翻译后修饰的生理意义。这些研究将验证酪氨酸硝化作为响应氧化/亚硝化应激的信号转导机制的作用，并可能提供新的策略来提高癌症治疗的治疗率。公共卫生相关性：该提案评估了一种新的信号机制，涉及一氧化氮和辐射和其他轻度氧化应激激活。了解这种信号传导机制的功能后果对于开发新的策略以提高放射治疗效果非常重要。还可以确定有助于氧化/亚硝化诱导的致癌作用的新机制。

DESCRIPTION (provided by applicant): In the past funding period, the hypothesis that cells sense an oxidative event but signal through NO7 dependent protein post-translational mechanisms was tested. Two signal transduction mechanisms important in the cytoprotective response to ionizing radiation (IR) were examined: Tyr kinase signaling and activation of the transcription factor NF-?B. IR at clinically relevant doses (<5 Gy), activates Ca2+ dependent NOS, resulting in the transient S-nitrosation of PTP active site Cys and inhibition of cellular PTPs. One consequence in autocrine-regulated tumor cells is IR-enhanced growth factor receptor Tyr kinase signaling. These findings demonstrated the first mechanism that accounts for the common observation that IR and other mild oxidative stresses activate cytoprotective Tyr kinase signaling pathways. These same IR doses also stimulated NK-?B activity by a mechanism involving the transient nitration of the inhibitor protein, I?B?. The latter finding has prompted the present proposal to test the hypothesis that IR-induced Tyr nitration of key regulatory proteins is a specific post-translational modification responsive to mild oxidative/nitrosative stresses such as IR. Specific Aim 1 is focused on Herein, the expression and activity levels of eNOS and iNOS in MCF-7 breast carcinoma cells and in tumor endothelial cells in vitro and in vivo are monitored as a function of dose (1-10 Gy) and time (up to 48 hrs post-IR). Immunocytochemical and subcellular fractionation methods identifies sites of nitration and NOS localization. The role of infiltrating inflammatory cells that express high levels of iNOS is examined by depleting tumors of the infiltrating cells with anti-GR-1. Specific Aim 2 a global approach to identify by mass spectroscopy key regulatory proteins nitrated after IR and a physical chemical modeling and genetic validation study to identify structural motifs predictive of nitration. Specific Aim 3 tests whether specific nitration of p53 has a role in the cellular response to radiation. Preliminary studies demonstrated that p53 is transiently nitrated after IR. Mass spectroscopy identified two nitratable tyrosine including Tyr327 in the tetramerization domain and Try107 in the DNA binding domain. Functional consequences of their nitration in terms of mitochondrial and nuclear localization, interaction with Bcl molecules, and transcriptional responses are examined. Demonstrating a functional consequence is critical in establishing the physiological significance of the post-translational modification. These studies will validate the role of Tyr nitration as a signal transduction mechanism responsive to oxidative/nitrosative stresses and may provide new strategies to enhance the therapeutic ratio in the treatment of cancer. PUBLIC HEALTH RELEVANCE: This proposal evaluates a novel signaling mechanism involving nitric oxide and activated by radiation and other mild oxidative stresses. Understanding the functional consequences of this signaling mechanism is important in the development of new strategies to enhance the therapeutic efficacy of radiation. New mechanisms contributing to oxidative/nitrosative induced carcinogenesis may also be identified.