Cancer Biology and Chemical Genetics in the Zebrafish

描述(申请人提供)：约50万美国人死于癌症 每年都是。我们对癌症的认识已经取得了很大的进展 通过使用动物模型进行生物学研究。发展斑马鱼系统作为 癌症模型将创造一种新的、强大的研究工具。斑马鱼是 在正向遗传学和癌症方面有良好历史的脊椎动物 毒理学，并已知会患上类似人类癌症的肿瘤。 Zon实验室的成员已经开发出一种策略来筛选诱变物 斑马鱼在胚胎细胞周期中的缺陷，假设一些 这些突变体会更容易罹患癌症。七个斑马鱼细胞周期 突变体已经被鉴定出来，并且是胚胎致死的。成年杂合子 因为七个突变中的五个表现出癌症患病率的增加。至 为了进一步表征细胞周期缺陷，我们将利用化学物质(例如， 诺康唑)使野生型和突变型胚胎的细胞周期同步。 将观察同步队列在细胞周期中的进展 使用细胞周期标记物。此外，我们还将测试化学物质 改变突变体表型能力的检查点(如咖啡因) 表现出细胞周期停滞。基于这一细胞周期特征和 关于癌症易感性的数据，一个细胞周期/癌症易感性突变将是 被选作候选基因或位置克隆的。基因识别将会 为传统的遗传和化学遗传策略奠定基础 识别与该途径相互作用的基因或化合物。在那 在这方面，第二个目标是利用细胞周期/癌症易感性突变来开发 斑马鱼作为一种化学遗传模型系统，用于识别 扰乱致癌途径。这一目标将在合作中实现 与两位化学遗传学专家蒂莫西·米奇森和兰德尔·金 哈佛医学院化学和细胞生物学研究所。 斑马鱼是基于全胚胎的小分子筛查的理想选择，因为它们 是对化学物质敏感的水生生物吗？ 小胚胎的数量。基于完整胚胎的筛查具有以下优势 选择对多细胞生物体无毒的化合物和 避免使用转化的细胞系。使用基于整个生物体的 方法，一个单一的屏幕将能够检测到抑制化学物质 细胞周期突变表型，即具有合成致命性的突变，或 有选择性的有毒物质。然后，这些分子将被检查是否有 能够改变成鱼的肿瘤率或大小。令人不安的化学物质 特定致癌途径将成为研究肿瘤的有价值的工具 斑马鱼肿瘤模型系统的研究进展及可能的先导化合物 用于人类癌症化疗和/或化学预防。

DESCRIPTION (provided by applicant): About 500,000 Americans die of cancer every year. Much progress has been made in our understanding of cancer biology through the use of animal models. Developing the zebrafish system as a cancer model would create a new, powerful research tool. Zebrafish are vertebrates with a well established history in forward genetics and cancer toxicology and are known to develop neoplasms that resemble human cancers. Members of the Zon lab have developed a strategy to screen mutagenized zebrafish for defects in the embryonic cell cycle, hypothesizing that some of these mutants would be more susceptible to cancer. Seven zebrafish cell cycle mutants have been identified and are embryonic lethal. Adult heterozygotes for five of the seven mutations exhibit an increased cancer prevalence. To further characterize the cell cycle defects, we will utilize chemicals (e.g., nocodazole) to synchronize the cell cycle in wild-type and mutant embryos. Progression of the synchronized cohort through the cell cycle will be observed using cell cycle markers. In addition, we will test chemicals that override checkpoints (e.g., caffeine) for the ability to alter the phenotype of mutants exhibiting cell cycle arrest. Based on this cell cycle characterization and data on cancer susceptibility, one cell cycle/cancer-prone mutant will be chosen for candidate gene or positional cloning. Gene identification would lay the foundation for traditional genetic and chemical genetic strategies for identifying genes or compounds that interact with the pathway. In that regard, a second aim is to use a cell cycle/cancer prone mutant to develop zebrafish as a chemical genetic model system for identifying drugs that perturb oncogenic pathways. This goal will be accomplished in collaboration with two experts in chemical genetics, Timothy Mitchison and Randall King of the Institute of Chemistry and Cell Biology at Harvard Medical School. Zebrafish are ideal for whole-embryo-based small molecule screens because they are water-dwellers that are sensitive to chemicals and give rise to large numbers of small embryos. Intact-embryo-based screens have the advantages of selecting for compounds that are not toxic to multicellular organisms and of avoiding the use of transformed cell lines. Using a whole-organism-based approach, a single screen will be able to detect chemicals that suppress the cell cycle mutant phenotype, that are synthetic lethal with the mutation, or that are selectively toxic. Such molecules will then be examined for the ability to alter tumor rate or size in adult fish. Chemicals that perturb specific oncogenic pathways will be valuable tools for studying tumor progression in the zebrafish cancer model system and could be lead compounds for human cancer chemotherapy and/or chemoprevention.