FANCONI ANEMIA GENE PATHWAY IN RADIATION RESPONSES

该项目的目标是了解细胞使用的分子调控过程，以最大限度地减少与内源过程或电离辐射(IR)引起的活性氧(ROS)相关的遗传损害和遗传不稳定。这个目标是通过对FANCG/XRCC9的研究来实现的，FANCG/XRCC9是一种存在缺陷的基因。癌症易感疾病范可尼贫血(FA)G组。由于FancG蛋白在仓鼠细胞中具有IR抗性，因此人类同系物有望参与人类细胞的IR反应。从历史上看，FA基因和辐射反应之间的联系一直不清楚，一些研究表明FA的主要缺陷在于去除DNA链间的交联键。需要检验的一般假设是，FANCG蛋白作为多蛋白复合体的一员，通过协调包括调节ROS水平、细胞凋亡和细胞周期进展在内的动态平衡过程，保护哺乳动物细胞免受内源性和IR产生的氧化损伤，并维持基因组的完整性。拟议的研究将提供FANCG蛋白对生化和与正常细胞增殖和对IR暴露的反应相关的细胞终点的高度定量表征。在仓鼠CHO细胞和人类淋巴母细胞中都将获得突变和FANCG互补的同基因对细胞。我们将分析这些配对的染色体畸变率、细胞存活率、HPRT基因突变、细胞凋亡、ROS和细胞周期参数。FANCG互补的FA-G淋巴母细胞将用于检测细胞周期中的基因和蛋白质调节，以及有和没有IR损伤的蛋白质的亚细胞定位。将对初步研究中与FANCG相互作用的三种候选蛋白质进行评估，以确定它们可能参与FA途径。最后，将对美国人群中已确定的人类FANCG高频等位基因变异进行功能障碍程度的评估。这些研究的结果将导致更具体的模型，以了解FA蛋白“途径”的性质及其对IR介导的氧化损伤相关的多种生物学效应的定量贡献。

The project's objective is to understand the molecular regulatory processes cells use to minimize genetic damage and genetic instability associated with reactive oxygen species (ROS) arising from endogenous processes or ionizing radiation (IR). This goal is addressed through studies of FANCG/XRCC9, the gene that is defective in. group G of the cancer-prone disorder Fanconi anemia (FA). Because FancG protein confers IR resistance in hamster cells, the human homolog is expected to participate in IR responses in human cells. Historically, a link between the FA genes and radiation responses has been unclear, with some studies suggesting that the primary defect in FA lies in removing DNA interstrand crosslinks. The general hypothesis to be tested is that the FANCG protein, as a member of a multiprotein complex, protects mammalian cells against endogenous and IR-generated oxidative damage and maintains genomic integrity by coordinating homeostasis processes that include regulation of ROS levels, apoptosis, and cell cycle progression. The proposed studies will provide a highly quantitative characterization of FANCG protein's contribution to biochemical and cellular endpoints associated with both normal cell proliferation and responses to IR exposure. Isogenic pairs of mutant and FANCG-complemented cells will be derived in both hamster CHO cells and human lymphoblasts. These pairs will be analyzed with respect to chromosomal aberrations, cell survival, hprt gene mutations, apoptosis, ROS, and cell cycle parameters with and without IR exposure. The FANCG-complemented FA-G lymphoblasts will be used to examine gene and protein regulation during the cell cycle as well as the subcellular localization of the protein with and without IR damage. Three proteins that are candidate interactors with FANCG from preliminary studies will be evaluated for possible involvement in the FA pathway. Finally, already identified high-frequency human allelic variants of FANCG in the US population will be evaluated for degree of dysfunction. The results of these studies will lead to more specific models of the nature of the FA protein "pathway" and its quantitative contributions to multiple biological effects associated with IR-mediated oxidative damage.