MOLECULAR BASIS OF RETINOBLASTOMA FORMATION

视网膜母细胞瘤形成的分子基础

• 批准号: 2159571
• 负责人: Wen-Hwa Lee
• 金额: $ 29.87万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2159571
• 关键词: Escherichia coli; Retroviridae; binding proteins; cell differentiation; chimeric proteins; developmental genetics; embryo /fetus cell /tissue; gene expression; gene interaction; genetic library; genetic manipulation; human fetus tissue; in situ hybridization; laboratory mouse; laboratory rabbit; microinjections; molecular cloning; molecular oncology; monoclonal antibody; mutant; neoplasm /cancer genetics; neoplastic cell; nucleic acid sequence; oncoproteins; phosphorylation; protein structure; protein structure function; proteolysis; retinoblastoma; site directed mutagenesis; transcription factor; transfection; tumor suppressor genes; yeasts

Retinoblastoma, the most common intraocular tumor in children, is a prototypical model for studying tumor suppression. The identification of the first human cancer suppressor gene (retinoblastoma susceptibility gene) marked a new era of cancer research. Introducing the wild type RB gene into several RB deficient human tumor cells suppresses their neoplastic phenotypes including tumorigenic ability in nude mice. This finding substantiated the claim that RB is a tumor suppressor gene. Based on these important discoveries, we propose three interrelated projects to further explore the biochemical and cellular function of the retinoblastoma protein in order to fully understand how RB suppresses tumor formation. The major aims of these projects are as follows: I. To analyze the RB protein biochemically. RB proteins will be expressed in bacteria and purified to homogeneity for structural/functional domains mapping by partial proteolysis and for characterizing its polymerization property. II. To determine biochemical and biological significance of RB phosphorylation. By using site-directed mutagenesis the 12 potential cdc2 phosphorylation sites will be systematically altered either individually or in combination. Mutants will be tested in vitro for their ability to bind T antigen and other associated proteins or in vivo for their ability to suppress neoplastic phenotypes. III. To elucidate the RB protein interactive network by cloning and identifying its associated proteins (Aps). Two complementary cloning approaches either in vitro by direct screening expression libraries with RB protein as probe or in vivo using yeast GAL4 DNA binding and transcriptional activation domain system will be taken. Among dozens of clones isolated, five were chosen to be characterized first by sequence analysis of entire cDNAs, defining RB-binding domains of the Aps, generating antibodies against each Aps and exploring their roles in the progression of cell growth and differentiation. Results of these studies will contribute significantly to a basic understanding of retinoblastoma genesis as well as human oncogenesis in general.

视网膜母细胞瘤是儿童最常见的眼内肿瘤，是 用于研究肿瘤抑制的原型模型。 标识 第一个人类癌症抑制基因（视网膜细胞瘤 敏感性基因标志着癌症研究的新时代。 介绍 野生型Rb基因进入几个RB缺乏的人类肿瘤细胞 抑制其肿瘤表型，包括肿瘤能力 在裸鼠。 这一发现证实了RB是肿瘤的说法 抑制基因。 根据这些重要发现，我们提出 三个相互关联的项目，以进一步探索生化和 视网膜母细胞瘤蛋白的细胞功能，以便完全 了解RB如何抑制肿瘤的形成。 这些的主要目的 项目如下：I。在生化上分析RB蛋白。 RB蛋白将在细菌中表达并纯化至均匀性 用于通过部分蛋白水解映射的结构/功能域和 用于表征其聚合特性。 ii。确定 RB磷酸化的生化和生物学意义。 经过 使用定向诱变12势CDC2磷酸化 站点将对单独或在系统中进行系统更改 组合。 突变体将在体外测试其结合能力 T抗原和其他相关蛋白或体内的能力 抑制肿瘤表型。 iii。阐明RB蛋白 通过克隆和识别其相关蛋白的交互式网络 （APS）。 直接在体外进行两种互补的克隆方法 用Rb蛋白作为探针或体内筛选表达文库 使用酵母Gal4 DNA结合和转录激活结构域 系统将被采用。 在数十个孤立的克隆中，有五个是 选择首先通过整个cDNA的序列分析来表征 定义AP的RB结合域，生成针对的抗体 每个AP并探索它们在细胞生长发展中的作用 和分化。 这些研究的结果将有助于 显着对视网膜细胞瘤起源的基本理解为 以及人类的肿瘤发生。