Radiation-induced Nitric Oxide & Cellular Radiosensitivity

描述（由申请人提供）：在过去的资助期内，我们验证了细胞感知氧化事件但通过NO7依赖蛋白翻译后机制发出信号的假设。研究了两个在电离辐射（IR）细胞保护反应中重要的信号转导机制：Tyr激酶信号转导和转录因子NF- B的激活。临床相关剂量（<5 Gy）的IR激活Ca2+依赖性NOS，导致PTP活性位点Cys的短暂s -亚硝化和细胞PTP的抑制。自分泌调节肿瘤细胞的一个后果是ir增强生长因子受体Tyr激酶信号。这些发现证明了第一个解释IR和其他轻度氧化应激激活细胞保护性Tyr激酶信号通路的机制。同样的红外剂量也刺激了NK-？B活性的机制涉及抑制剂蛋白I?B的瞬时硝化作用。后一项发现促使本研究提出了一种假设，即IR诱导的关键调节蛋白的Tyr硝化是一种特定的翻译后修饰，可响应温和的氧化/亚硝化应激，如IR。本文在体外和体内监测MCF-7乳腺癌细胞和肿瘤内皮细胞中eNOS和iNOS的表达和活性水平，作为剂量（1-10 Gy）和时间（ir后48小时）的函数。免疫细胞化学和亚细胞分离方法确定硝化和NOS定位的位置。表达高水平iNOS的浸润性炎症细胞的作用是通过用抗gr -1消耗浸润细胞的肿瘤来检测的。具体目标2：通过质谱鉴定IR后硝化的关键调节蛋白的全球方法，以及物理化学建模和遗传验证研究，以确定预测硝化的结构基序。特异性Aim 3测试p53的特异性硝化是否在细胞对辐射的反应中起作用。初步研究表明，p53在IR后会短暂硝化。质谱鉴定出两种可硝化酪氨酸，包括四聚域Tyr327和DNA结合域Try107。它们的硝化作用在线粒体和核定位、与Bcl分子的相互作用和转录反应方面的功能后果进行了检查。证明功能后果是建立翻译后修饰的生理意义的关键。这些研究将验证Tyr硝化作为一种响应氧化/亚硝化应激的信号转导机制的作用，并可能为提高癌症治疗中的治疗率提供新的策略。公共卫生相关性：本提案评估了一种涉及一氧化氮并由辐射和其他轻度氧化应激激活的新型信号机制。了解这一信号机制的功能后果对于开发新的策略以提高放射治疗效果是重要的。氧化/亚硝化诱导致癌的新机制也可能被发现。

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.