Radiation-induced Nitric Oxide & Cellular Radiosensitivity

描述(由申请人提供)：在过去的资助期间，细胞感觉到氧化事件，但通过N7依赖的蛋白质翻译后机制发出信号的假设得到了验证。在细胞对电离辐射(IR)的保护反应中，有两种重要的信号转导机制：酪氨酸激酶信号转导和转录因子NF-B的激活。临床剂量的IR可激活钙依赖的一氧化氮合酶，导致PTP活性部位半胱氨酸的S亚硝化和细胞PTPs的抑制。在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。这些发现证明了第一个机制，解释了普遍观察到的IR和其他温和的氧化应激激活细胞保护性Tyr激酶信号通路的机制。同样剂量的IR还通过抑制蛋白I？B？的瞬时硝化作用来刺激NK-B活性。后一项发现促使目前的建议检验这一假说，即IR诱导的关键调节蛋白的Tyr硝化是对温和的氧化/亚硝化压力(如IR)做出反应的特定的翻译后修饰。具体目的1在此，监测eNOS和iNOS在MCF-7乳腺癌细胞和体内外肿瘤内皮细胞中的表达和活性水平，作为剂量(1-10GY)和时间(直到IR后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一氧化氮合酶定位的位置。通过用抗GR-1抗体清除肿瘤的浸润性细胞来检测高水平表达iNOS的浸润性炎症细胞的作用。具体目标2通过质谱学鉴定IR后硝化的关键调节蛋白的全球方法，以及确定预测硝化作用的结构基序的物理化学模拟和遗传验证研究。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发挥作用。初步研究表明，在IR后，P53被瞬时硝化。质谱学鉴定了两个硝化酪氨酸，包括四聚化区域的Tyr327和DNA结合区的Try107。他们的硝化作用在线粒体和核定位，与Bcl分子的相互作用，以及转录反应方面的功能后果进行了研究。证明功能结果对于确定翻译后修饰的生理意义至关重要。这些研究将验证酪氨酸硝化作为一种响应氧化/亚硝化压力的信号转导机制的作用，并可能为提高癌症治疗的治疗比率提供新的策略。与公共健康相关：该提案评估了一种新的信号机制，该机制涉及一氧化氮，并由辐射和其他温和的氧化应激激活。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的疗效是重要的。氧化/亚硝化诱导致癌的新机制也可能被识别出来。

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.