ROLE OF RAD9 IN BYSTANDER EFFECTS

RAD9 在旁观者效应中的作用

• 批准号: 8281638
• 负责人: HOWARD B. LIEBERMAN
• 金额: $ 33.03万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8281638
• 关键词: Address; Apoptosis; Binding; Biological; Biological Assay; Bystander Effect; Cell Cycle; Cell Cycle Checkpoint; Cell Nucleus; Cell physiology; Cells; Collaborations; Communication; Connexins; Cytoplasm; DNA Repair; DNA Repair Gene; Female; Gene Expression; Gene Targeting; Genes; Genomic Instability; Genotype; Goals; Growth; Health; Hela Cells; Human; In Vitro; Incidence; Ionizing radiation; Lead; Lung; Mammary gland; Mediating; Molecular; Mus; Mutagenesis; Mutation; Nuclear; Pathway interactions; Play; Population; Process; Production; Promoter Regions; Protein Binding Domain; Proteins; RNA; Radiation; Radiation therapy; Regulation; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Risk; Role; Scanning; Signal Transduction; Small Interfering RNA; Testing; Thinking; Time; Tissues; Transactivation; Transgenic Organisms; Up-Regulation; Work; base; connexin 32; embryonic stem cell; gene induction; improved; in vivo; irradiation; knock-down; micronucleus; mutant; null mutation; overexpression; protein function; rad9 protein; radiation effect; radiation resistance; response

The overall cellular response to ionizing radiation exposure is not limited to cells directly irradiated, but includes neighboring "bystanders". Essentially every endpoint used routinely to test for changes in cells direcfiy exposed to radiation, such as mutation, apoptosis, micronuclei formation or cell cycle checkpoints, has been studied in bystanders. This phenomenon has caused a paradigm shift in thinking about biological effects and associated health risks of radiafion exposure; the impacted cell population is larger than once believed. The mechanism regulating bystander effects is not well understood. However, certain gap junction proteins, i.e., Connexin 32, and proteins like Cox-2 control the overall process, whereas p21 just regulates the radiation-induced Gl to S checkpoint. We have shown that Rad9 regulates the bystander response to radiation, since Rad9 null enhances bystander apoptosis and micronuclei formation. Rad9 protein, which controls cell cycle checkpoints, DNA repair, apoptosis and transactivation of downstream target genes in directly irradiated cells, likely impacts on the bystander response via more than one pathway. In collaboration with investigators in Projects 2 and 3, we show that Rad9 is essential for bystander induction of Cox-2 and p21 (Project 2), and production of Connexin 32 in HeLa cells, which are normally devoid of the protein, increases bystander radiation resistance and levels of Rad9 as well as TCTP (Project 3), which both function in the bystander process. We also show that TCTP and Rad9 physically interact (Project 3). Our overarching hypothesis is that Rad9 plays an important role in radiation-induced bystander effects via mechanisms involving at least Cox-2, p2I, Connexin 32 and TCTP. Specific aims to address this hypothesis are: I. Determine the activity of Rad9 in nuclear versus cytoplasmic irradiation-induced bystander effects; 2. Analyze Rad9 functional domains and protein-protein interactions for roles in the bystander process; 3. Establish the significance of Rad9-dependent bystander induction of Cox-2, p21 and other genes for bystander regulation; 4. Determine if Rad9 is critical for bystander mutagenesis; and 5. Define the significance of junctional communication on the regulation and funcfion of Rad9. These studies will reveal mechanisms by which Rad9 regulates radiation-induced bystander effects, and will impact on understanding the biological response to radiation exposure, with implications for improving radiotherapy and better defining health risk.

细胞对电离辐射暴露的总体反应不限于直接照射的细胞，而是 包括邻近的“旁观者”。基本上每个端点都被例行地用来测试细胞的变化 直接暴露于辐射，如突变、凋亡、微核形成或细胞周期检查点， 已经在旁观者中进行了研究。这一现象导致了生物学思维的范式转变。 辐射照射的影响和相关的健康风险；受影响的细胞数量不止一次 相信。对旁观者效应的调控机制还不是很清楚。然而，某些缝隙结 连接蛋白32和COX-2等蛋白质控制着整个过程，而p21只是调节 辐射诱发G1至S检查站。我们已经证明，Rad9调节旁观者的反应 辐射，因为RAD9缺失增加了旁观者的凋亡和微核的形成。RAD9蛋白，它 控制细胞周期检查点、DNA修复、细胞凋亡和下游靶基因的反式激活 直接照射的细胞，可能通过不止一条途径影响旁观者的反应。在协作中 在项目2和项目3中的研究人员中，我们表明Rad9在旁观者诱导COX-2和 P21(项目2)，以及在HeLa细胞中产生连接蛋白32，而HeLa细胞通常不含连接蛋白， 增加旁观者的辐射抵抗力和RAD9和TCTP的水平(项目3)，这两者都起作用 在旁观者的过程中。我们还证明了TCTP和Rad9在物理上相互作用(项目3)。我们最重要的是 假设Rad9通过机制在辐射诱导的旁观者效应中发挥重要作用 至少涉及COX-2、P2i、连接蛋白32和TCTP。解决这一假设的具体目标是：I。 目的：1.测定核内与胞质内辐射诱导的旁观者效应中Rad9的活性； 分析RAD9功能结构域和蛋白质-蛋白质相互作用在旁观者过程中的作用； 建立依赖Rad9的旁观者诱导COX-2、p21等基因对旁观者的意义 调控；4.确定Rad9是否是旁观者突变的关键；以及5.定义 RAD9的调节和功能的连接通讯。这些研究将通过以下方式揭示机制 其中RAD9调节辐射诱导的旁观者效应，并将影响对生物学 对辐射暴露的反应，对改进放射治疗和更好地确定健康风险的影响。