MECHANISMS OF BYSTANDER MUTAGENESIS

描述(由申请人提供)：使用精确的带电粒子微束，申请人和他的合作研究人员已经证明，在某些已知核片段被击中的人群中，诱发突变和染色体变化的频率与对反应有显著贡献的非击中细胞是一致的。事实上，用每个细胞单一的阿尔法粒子照射10%的哺乳动物细胞群体，会产生与照射群体中所有细胞时观察到的突变产量相似的突变产量。虽然缝隙连接通讯已被证明在旁观者反应中起着重要作用，但确切的机制尚不清楚。利用基因芯片，申请人已经证明COX-2酶在旁观者细胞中持续升高。此外，初步证据表明，活性氮物种可能参与了信号转导过程。这引发了以下问题：过氧亚硝酸根阴离子是否参与了旁观者效应？这种激进的生成过程是否涉及线粒体损伤？COX-2酶的增加是如何 与旁观者进程有关吗？细胞质照射能否像申请人最近证明的那样，在哺乳动物细胞中诱导旁观者突变效应，就像申请人最近展示的正常人类支气管上皮(NHBE)细胞的核穿越一样？最后，旁观者信号能否导致哺乳动物细胞的基因组不稳定？为了解决这些问题，提出了一系列8个相互关联的具体目标，以解决5个可检验的假设。AL细胞的CD59位点突变评分，NHBE细胞的G2PCC评分。建议的研究将有助于解决哺乳动物细胞中旁观者突变的机制。与细胞质遗传毒性研究一起，该项目将在该计划项目的背景下，解决与目标和辐射剂量效应有关的一些基本问题，并可能对我们目前对辐射风险评估的理解产生重大影响。

DESCRIPTION (provided by applicant): Using a precision charged particle microbeam, the applicant and his co-investigators have shown that the frequencies of induced mutations and chromosomal changes in populations where some known fractions of nuclei were hit are consistent with non-hit cells contributing significantly to the response. In fact, irradiation of 10% of a mammalian cell population with a single alpha particle per cell results in a mutant yield similar to that observed when all of the cells in the population are irradiated. While gap junctional communication has been shown to play an important role in the bystander response, the precise mechanism is not known. Using cDNA microarrays, the applicant has shown that the COX-2 enzyme is consistently elevated in bystander cells. Furthermore, preliminary evidence suggests that reactive nitrogen species may be involved in the signaling process. This raised the following questions: Are peroxynitrite anions involved in the bystander effect? Does this radical generating process involve mitochondrial damage? How does an increase in COX-2 enzymes relate to the bystander process? Can cytoplasmic irradiation induce bystander mutagenic effect in mammalian cells in a manner similar to what the applicant has recently demonstrated with nuclear traversal of normal human bronchial epithelial (NHBE) cells? And finally, can bystander signal induce genomic instability in mammalian cells? To address these issues, a series of 8 inter-related specific aims are proposed to address the 5 testable hypotheses. Mutations will be scored at the CD59 locus of the AL cells and G2PCC will be scored in NHBE cells. The proposed studies will help to address the mechanism of bystander mutagenesis in mammalian cells. Together with the cytoplasmic genotoxicity study, this project will address, in the context of this program project, some of the fundamental issues regarding both the target and radiation dose effect and are likely to have a significant impact on our current understanding of radiation risk assessment.