Genetic analysis of genomic instability and cancer in zebrafish

斑马鱼基因组不稳定性和癌症的遗传分析

• 批准号: 7845016
• 负责人: Keith Chi Cheng
• 金额: $ 31.07万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7845016
• 关键词: Biological Assay; Biological Models; Cells; Chromatin; Cloning; Collection; DNA; DNA Modification Process; Disease; Embryo; Employee Strikes; Epigenetic Process; Epithelial; Ethylnitrosourea; Event; Exhibits; Exons; Eye; Family; Fishes; Fluorescence; Fluorescent in Situ Hybridization; Gene Conversion; Gene Deletion; Gene Expression; Gene Mutation; Genes; Genetic; Genetic Recombination; Genetic Screening; Genome Stability; Genomic Instability; Genotype; Goals; Heterozygote; Homozygote; Human; Inherited; Insertional Mutagenesis; Interphase; Investigation; Lead; Malignant Neoplasms; Maps; Measures; Mediating; Methods; Microsatellite Repeats; Modeling; Modification; Molecular; Mutate; Mutation; Nature; Normal Cell; Oncogenes; Penetrance; Phenotype; Pigments; Play; Point Mutation; Predisposition; RNA Splicing; Retinal Pigments; Retinoblastoma; Role; Siblings; Site; Somatic Cell; Structure of retinal pigment epithelium; TP53 gene; Testing; Tissues; Transgenic Organisms; Translations; Tumor Suppressor Genes; Tumor Tissue; Vertebrates; Zebrafish; base; cancer cell; chromosome loss; gene function; gene interaction; genetic analysis; genome-wide; insertion/deletion mutation; knock-down; loss of function; member; mutant; novel; novel strategies; positional cloning; premature; prevent; public health relevance; tumor; vector

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.

描述（由申请人提供）：癌症是由癌症基因（包括肿瘤抑制基因）中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演变为癌细胞。在Knudson的两次打击范例中，肿瘤抑制基因功能的丧失分两步发生。这些步骤中的第二个可以通过多种机制发生，包括染色体丢失、重组、插入、缺失、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，这些基因用于维持基因组稳定性，我们使用斑马鱼（Danio rerio）进行基因组不稳定突变体的筛选，以模拟第二次打击。该筛选使用了马赛克眼试验，其中苍白细胞出现在视网膜色素上皮（RPE）细胞的黑色背景中，这些细胞是隐性色素突变goldenb 1的杂合细胞。在该筛选中分离出12个ENU诱导的基因组不稳定性（gin）或体细胞增变因子突变，其速率表明潜在存在200个这样的基因。大多数gin突变在杂合子中表现为弱显性，在纯合子中均表现为较强的表型。最强的突变体，gin-10，表现出显着的（~10倍）的表型增强超过纯合子表型时，遗传的母系在反式与其他gin突变（“gin-10相互作用组”）; gin-10携带者表现出9.6倍的自发性癌症的易感性增加。这些发现代表了我们长期目标的第一步，即开发斑马鱼作为脊椎动物模型，以寻找脊椎动物中控制基因组稳定性的基因。现在，我们已经表明，斑马鱼可用于寻找对癌症易感的体细胞突变体，我们建议阐明组成gin-10相互作用组的基因之间惊人的相互作用的性质，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。对于特定目标1，我们将使用定位克隆gin-10和gin-12克隆gin-10相互作用组的成员，并使用具有新型“基因断裂”To 12转座子载体的敏化插入筛选。具体目标2是使用微卫星，间期FISH分析，和外显子测序的黄金基因在黄金RPE细胞从马赛克的眼睛和类似的分析，在杜松子酒-10相关的肿瘤的p53基因组不稳定性的机制。具体目标3是确定强嵌合gin-10/gin-12反式杂合子的肿瘤易感性。拟议的研究将有助于我们了解基因功能体细胞丢失所涉及的基因相互作用，并阐明它们如何有助于癌症易感性。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：癌症是一种疾病，其中正常细胞在进化成癌细胞时积累其DNA的变化。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能力来寻找和研究在遗传稳定性和癌症中重要的基因，希望我们对这些基因的了解将导致治疗和预防人类癌症的措施。