MECHANISM OF BYSTANDER MUTAGENESIS

项目总结(见说明)： 辐射诱导的旁观者效应已经通过使用哺乳动物细胞培养和3D人体组织的各种终点进行了演示。然而，旁观者对人类健康的反应的机制和相关性都不清楚。虽然缝隙连接通讯和可溶性介体(S)的存在在旁观者反应中起着重要作用，但确切的信号分子尚不清楚。该项目的总体目标是明确体内辐射诱导的旁观者突变反应(非靶向反应)的发生率和机制；阐明环氧合酶-2(COX-2)信号通路在这一过程中的作用；以及检测野生型和遗传易感动物旁观者组织中基因组不稳定性的发生率。中心的可检验假设是COX-2在体内介导辐射诱导的旁观者突变，并且在ATM纯合子突变的动物中旁观者细胞在遗传上是不稳定的。为实现这些目标，提出了一系列五个相互关联的具体目标。新的gpt Delta转基因小鼠和来自这些动物的胚胎成纤维细胞将在体外和体内条件下用于总结这一发现。下腹部1 cm×1 cm的小区域将接受分级剂量的X射线照射，并将检测非靶向肺和乳腺组织中COX-2的表达水平以及SPI(缺失)和GPT(点突变)的发生率和类型。野生型和COX-2基因敲除小鼠的MEF细胞由于细胞核和细胞质靶向而产生的旁观者反应将用微束进行，以描绘特定的基因信号通路。为了进一步确定旁观者组织中的基因组不稳定性，将在野生型和y47M型基因敲除小鼠的照射后几周内检查gpt和spi突变的发生率。计划与项目1和项目3进行广泛的项目互动，因为将检查Rad9(项目1)和连接蛋白(TCTP，项目3)在调节COX-2功能中的作用。辐射诱导的旁观者效应代表着我们对电离辐射的基本放射生物学原理和靶理论的理解的范式转变。更好地理解旁观者效应的机制对于准确评估 与低剂量辐射暴露相关的癌症风险。

PROJECT SUMMARY (See instructions): Radiation induced bystander effects have been demonstrated with a variety of endpoints using mammalian cell cultures as well as 3D human tissues. However, neither the mechanism nor the relevance of the bystander response to human health is clear. While gap junction communication and presence of soluble mediator(s) are known to play an important role in the bystander response, the precise signaling molecules have yet to be identified. The overall goals of this project are to define the incidence and mechanism of radiation-induced bystander mutagenic response in vivo (non-targeted response); to clarify the role of cyclooxygenase-2(COX-2) signaling pathways in the process; and to examine the incidence of genomic instability in bystander tissues in wild type and in genetic susceptible animals. The central testable hypothesis is that COX-2 mediates radiation induced bystander mutagenesis in vivo and that the bystander cells are genomically unstable in ATM homozygously mutated animals. A series of five inter-related specific aims are proposed to address these goals. The novel gpt delta ttansgenic mice and embryo fibroblasts from these animals will be used to conelate the findings under both in vitro and in vivo conditions. A small 1 cm by 1 cm zone in the lower abdominal area will be inadiated with graded doses of X-rays and the expression of COX-2 levels as well as incidence and types of Spi (deletions) and gpt (point mutations) mutations will be examined in the non-targeted lung and breast tissues. Bystander response as a result of nuclei and cytoplasmic targeting in MEF cells from wild type and COX-2knock out mice will be conducted with a microbeam to delineate specific gene signaling pathways. To further define genomic instability in bystander tissues, incidence of gpt and Spi mutations will be examined over a period of several weeks post-irradiation in wild type as well as y47Mknock out mice. Extensive program interaction with Projects 1 and 3 are planned in that the role of Rad9 (Project 1) and the connexin protein (TCTP, Project 3) in modulating COX-2 function will be examined. Radiation induced bystander effects represent a paradigm shift in our understanding of the basic radiobiological principle and target theory of ionizing radiation. A better understanding of the mechanism of the bystander effect is important for an accurate assessment of cancer risk associated with low dose radiation exposure.