The GOF Mutant p53 Beacon System

GOF 突变体 p53 信标系统

• 批准号: 9233416
• 负责人: Sumitra Deb
• 金额: $ 22.32万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-24 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9233416
• 关键词: Affect; Alleles; Alpha Cell; Animals; Biochemistry; Cancer Etiology; Cancerous; Cell Count; Cell Line; Cell physiology; Cell surface; Cells; Custom; Darkness; Defect; Development; Elements; Event; Evolution; Fluorescence; Gene Mutation; Genetic; Image; Invaded; Knowledge; Light; Location; Malignant Neoplasms; Monitor; Mus; Mutation; Neoplasm Metastasis; Pathway interactions; Phase; Population; Premalignant; Property; Proteins; Puromycin; Reporter Genes; Resistance; Screening for cancer; Stem cells; Streptavidin; System; Systems Development; TP53 gene; Testing; Therapeutic; Time; Transgenic Mice; cancer cell; cancer type; gain of function; genetic evolution; high throughput screening; improved; inhibitor/antagonist; luminescence; mutant; neoplastic cell; novel; novel therapeutics; promoter; small molecule; small molecule inhibitor; stem-like cell; therapeutic target; tool; tumor; tumor growth; tumor progression; vector

Project Summary We have developed a system in which Gain-Of-Function (GOF) mutant p53 makes living cells fluoresce and luminesce (GOF Mutant p53 Beacon System). This powerful tool allows us to instantly assess the presence or absence of mutant p53 in living cells and is not affected by wild-type p53. It also provides a means to assess activity and properties for a large variety of mutant p53 alleles in cell lines. We propose to test the utility of the Beacon System for assessing small molecule mutant p53 inhibitors and wild-type p53 reactivators. We will adapt the Beacon System for use in high throughput screening of inhibitors such that when mutant p53 is inhibited, the cells will fluoresce and luminesce. The utility of the system for identifying cellular modulators of mutant p53 will also be investigated by adapting the system so that when mutant p53 is inhibited, the cells become resistant to puromycin. The ultimate phase of developing this tool is to make transgenic mice in which cells that acquire p53 mutations fluoresce and luminesce. This advance should allow us to visualize any tumor growth in the living mouse as cells acquire p53 mutations and as the tumor cells grow, evolve, invade, and metastasize and establish glowing cell populations at new locations. The mouse system will also be adapted such that cells that acquire p53 mutations will express a cell surface streptavidin tag making it easy to purify the cells at various stages of cancer development to study cellular properties, stem-cell properties, genetic events, and the evolution of these events at early to late stages of cancer. Development of this system should create opportunities for a simple and rapid screening for cancer therapeutics that target mutant p53, one of the most prevalent cancer-causing dominant defects in all cancers. A mouse in which the tumor glows in the living mouse will provide a powerful real-time analysis of cancer progression. This tool will advance our knowledge of how tumors grow and tumor cells migrate, and how they respond to therapeutics.

项目概要 我们开发了一种系统，其中功能获得 (GOF) 突变体 p53 使活细胞发出荧光并 luminesce（GOF 突变体 p53 信标系统）。这个强大的工具使我们能够立即评估存在或 活细胞中不存在突变型 p53，并且不受野生型 p53 的影响。它还提供了一种评估方法 细胞系中多种突变 p53 等位基因的活性和特性。我们建议测试该功能的实用性 用于评估小分子突变型 p53 抑制剂和野生型 p53 重新激活剂的 Beacon 系统。我们将 使 Beacon 系统适用于抑制剂的高通量筛选，以便当突变 p53 被筛选时 受到抑制，细胞会发出荧光和发光。该系统用于识别细胞调节剂的效用 还将通过调整系统来研究突变型 p53，以便当突变型 p53 受到抑制时，细胞 对嘌呤霉素产生耐药性。开发该工具的最终阶段是制造转基因小鼠，其中 获得 p53 突变的细胞会发出荧光和发光。这一进展将使我们能够可视化任何肿瘤 当细胞获得 p53 突变以及肿瘤细胞生长、进化、侵袭和扩散时，活体小鼠的生长 在新位置转移并建立发光细胞群。鼠标系统也将进行调整 这样获得 p53 突变的细胞将表达细胞表面链霉亲和素标签，使其易于纯化 癌症发展各个阶段的细胞，以研究细胞特性、干细胞特性、遗传 事件，以及这些事件在癌症早期到晚期的演变。 该系统的开发将为简单快速的癌症筛查创造机会 针对突变 p53 的疗法，突变 p53 是所有癌症中最常见的致癌缺陷之一。 活体小鼠肿瘤发光的小鼠将为癌症提供强大的实时分析 进展。该工具将增进我们对肿瘤如何生长和肿瘤细胞迁移以及它们如何迁移的了解。 对治疗有反应。