Genetic analysis of genomic instability and cancer in zebrafish

描述（由申请人提供）：癌症是由癌症基因（包括肿瘤抑制基因）的遗传和表观遗传变化的积累引起的；这些变化导致正常体细胞进化成癌细胞。在Knudson的双重打击范式中，肿瘤抑制基因功能的丧失分两个步骤发生。第二个步骤可以通过多种机制发生，包括染色体丢失、重组、插入、删除、点突变或表观遗传修饰。为了找到参与这些步骤的脊椎动物基因，这些基因有助于保持基因组的稳定性，我们使用斑马鱼（Danio rerio）来筛选基因组不稳定性突变体，以模拟这第二次袭击。该筛检使用马赛克眼试验，在黑色背景的视网膜色素上皮细胞（RPE）中出现苍白细胞，这些细胞是隐性色素突变goldenb1的杂合细胞。12个ENU诱导的基因组不稳定性（gin）或体细胞突变在这个筛选中被分离出来，表明可能存在200个这样的基因。大多数突变在杂合子中表现为弱显性，在纯合子中表现为强显性。最强的突变体gin-10在与其他gin突变（“gin-10相互作用组”）反式遗传时，表现出明显的（~10倍）纯合表型增强；Gin-10携带者对自发性癌症的易感性增加了9.6倍。这些发现代表了我们长期目标的第一步，即将斑马鱼发展为脊椎动物模型，以寻找参与控制脊椎动物基因组稳定性的基因。既然我们已经证明斑马鱼可以用来发现易患癌症的体细胞突变体，我们建议阐明组成gin-10相互作用组的基因之间惊人相互作用的本质，并更好地表征相关的肿瘤易感性。这将通过三个具体目标来实现。对于Specific Aim 1，我们将使用gin-10和gin-12的位置克隆克隆gin-10相互作用组的成员，并使用带有新型“基因断裂”To12转座子载体的敏化插入筛选。具体目标2是利用微卫星、间期FISH分析和镶嵌眼金色RPE细胞中金色基因的外显子测序，以及对gin-10相关肿瘤中p53的类似分析，来表征基因组不稳定性的机制。特异性目的3是确定强镶嵌型gin-10/gin-12反杂合子的肿瘤易感性。提出的研究将有助于我们了解基因功能体细胞丧失中涉及的基因相互作用，并阐明它们如何促进癌症易感性。我们希望这种理解将导致预防或治疗人类癌症的新方法。公共卫生相关性：癌症是一种正常细胞在进化为癌细胞时DNA发生变化的疾病。我们现在知道控制DNA稳定性的基因在癌症中起着关键作用。我们正在利用斑马鱼作为脊椎动物模型系统的独特力量来寻找和研究在遗传稳定性和癌症中重要的基因，希望我们对遗传稳定性和癌症的了解的增加将导致治疗和预防人类癌症的措施。

DESCRIPTION (provided by applicant): Cancer results from an accumulation of genetic and epigenetic changes in cancer genes, including tumor suppressor genes; these changes cause normal somatic cells to evolve into cancer cells. In Knudson's two-hit paradigm, loss of tumor suppressor gene function occurs in two steps. The second of those steps can occur by multiple mechanisms including chromosome loss, recombination, insertion, deletion, point mutation, or epigenetic modification. In order to find vertebrate genes involved in those steps, which serve to maintain genome stability, we have used the zebrafish (Danio rerio) to perform a screen for genomic instability mutants that models this second hit. This screen used the mosaic eye assay, in which pale cells appear in a black background of retinal pigmented epithelial (RPE) cells that are heterozygous for a recessive pigment mutation, goldenb1. Twelve ENU- induced genomic instability (gin), or somatic mutator mutations were isolated in this screen at a rate suggesting the potential existence of 200 such genes. Most of the gin mutations showed weak dominance in heterozygotes, and all showed a stronger phenotype in homozygotes. The strongest mutant, gin-10, showed a striking (~10-fold) phenotypic enhancement over homozygous phenotype when inherited maternally in trans with other gin mutations (the "gin-10 interacting group"); gin-10 carriers show a 9.6-fold increase in susceptibility to spontaneous cancer. These findings represent first steps in our long-term goal of developing the zebrafish as a vertebrate model for finding genes involved in the control of genomic stability in vertebrates. Now that we have shown that the zebrafish can be used to find somatic mutators that are susceptible to cancer, we propose to elucidate the nature of the striking interactions between the genes comprising the gin-10 interacting group, and to better-characterize the associated tumor susceptibility. This will be accomplished in three Specific Aims. For Specific Aim 1, we will clone members of the gin-10 interacting group using positional cloning for gin-10 and gin-12, and using a sensitized insertional screen with a novel "gene breaking" To12 transposon-based vector. Specific Aim 2 is to characterize the mechanism of genomic instability using microsatellite, interphase FISH analysis, and exon sequencing of the golden gene in golden RPE cells from mosaic eyes and by similar analysis of p53 in gin-10-associated tumors. Specific Aim 3 is to determine the tumor susceptibility of the strongly mosaic gin-10/gin-12 trans-heterozygotes. The proposed studies will contribute to our understanding of gene interactions involved in somatic loss of gene function, and to clarify how they contribute to cancer susceptibility. It is our hope that such understanding will lead to novel ways to prevent or treat human cancer. PUBLIC HEALTH RELEVANCE: Cancer is a disease in which normal cells accumulate changes in their DNA as they evolve into cancer cells. We now know that the genes controlling the stability of our DNA play a key role in cancer. We are using the unique power of the zebrafish as a vertebrate model system to find and study genes that are important in genetic stability and cancer, in the hope that our increased understanding will lead to measures to treat and prevent human cancer.