Radiation-induced Nitric Oxide & Cellular Radiosensitivity

辐射诱导的一氧化氮

基本信息

批准号：
8268474
负责人：
ROSS B MIKKELSEN
金额：
$ 26.62万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-07-10 至 2013-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8268474
关键词：
Abbreviations Accounting Active Sites Breast Carcinoma Cell Fractionation Cell Nucleus Cells Chemical Models Complex DNA Binding Domain Detergents Development Dissociation Dose Endoplasmic Reticulum Endothelial Cells Epidermal Growth Factor Receptor Event Funding Growth Factor Receptors Health Hydrogen Peroxide In Vitro Inflammatory Infiltrate Ionizing radiation Location MAP Kinase Gene MCF7 cell Malignant Epithelial Cell Mass Spectrum Analysis Methods Mitochondria Mitogen-Activated Protein Kinases Molecular Monitor Nitrates Nitric Oxide Nitric Oxide Synthase Nitroarginine Nitrosation Nuclear Oxidative Stress Phosphoric Monoester Hydrolases Phosphotransferases Physiological Post-Translational Protein Processing Process Proteins Publishing Radiation Radiation Tolerance Reactive Nitrogen Species Reactive Oxygen Species Roentgen Rays Role Signal Pathway Signal Transduction Site Specificity Structure Study models Subcellular Fractions Superoxide Dismutase Superoxides TNFRSF5 gene Testing Therapeutic Time Tissues Treatment Efficacy Tyrosine Work autocrine base cancer therapy carcinogenesis chemical genetics clinically relevant genetic regulatory protein in vivo inhibitor/antagonist interest irradiation neoplastic cell nitration nitrosative stress novel response transcription factor tumor validation studies

项目摘要

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.

描述（由申请人提供）：在过去的资助期间，测试了细胞感知氧化事件但通过 NO7 依赖性蛋白翻译后机制发出信号的假设。检查了电离辐射 (IR) 细胞保护反应中重要的两种信号转导机制：Tyr 激酶信号传导和转录因子 NF-κB 的激活。临床相关剂量 (<5 Gy) 的 IR 会激活 Ca2+ 依赖性 NOS，导致 PTP 活性位点 Cys 瞬时 S-亚硝化并抑制细胞 PTP。自分泌调节肿瘤细胞的结果之一是 IR 增强的生长因子受体 Tyr 激酶信号传导。这些发现证明了第一个机制，可以解释普遍观察到的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。这些相同的 IR 剂量还通过涉及抑制剂蛋白 IκBβ 瞬时硝化的机制刺激 NK-κB 活性。后一个发现促使本提案检验以下假设：IR 诱导的关键调节蛋白的酪氨酸硝化是对温和氧化/亚硝化应激（如 IR）做出反应的特定翻译后修饰。具体目标 1 重点关注在此，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）和时间的函数（ IR 后最多 48 小时）。免疫细胞化学和亚细胞分级分离方法可识别硝化和 NOS 定位的位点。通过用抗 GR-1 消除浸润细胞的肿瘤来检查表达高水平 iNOS 的浸润炎症细胞的作用。具体目标 2 一种通过质谱鉴定红外后硝化的关键调节蛋白的全局方法，以及物理化学模型和遗传验证研究来鉴定预测硝化的结构基序。 Specific Aim 3 测试 p53 的特异性硝化是否在细胞对辐射的反应中发挥作用。初步研究表明，IR 后 p53 被短暂硝化。质谱鉴定出两个可硝化酪氨酸，包括四聚结构域中的 Tyr327 和 DNA 结合结构域中的 Try107。研究了它们硝化在线粒体和核定位、与 Bcl 分子相互作用以及转录反应方面的功能后果。展示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比率。公共健康相关性：该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传导机制。了解这种信号机制的功能后果对于制定增强放射治疗功效的新策略非常重要。还可能确定导致氧化/亚硝化诱发致癌的新机制。