The GOF Mutant p53 Beacon System

项目摘要 我们已经开发了一种系统，其中功能获得（GOF）突变体p53使活细胞发出荧光， 发光（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， 细胞系中大量突变p53等位基因的活性和性质。我们建议测试的效用 用于评估小分子突变型p53抑制剂和野生型p53再激活剂的信标系统。我们将 使Beacon系统适用于抑制剂的高通量筛选，使得当突变型p53 细胞会发出荧光和发光。本发明提供了用于鉴定细胞调节剂的系统的实用性， 还将通过调整系统来研究突变型p53，使得当突变型p53被抑制时，细胞 对嘌呤霉素产生抗药性开发这种工具的最终阶段是制造转基因小鼠， 获得p53突变的细胞发出荧光和发光。这一进展可以让我们看到任何肿瘤 当细胞获得p53突变时，以及当肿瘤细胞生长、进化、侵袭和 转移并在新的位置建立发光细胞群。鼠标系统也将适应 使得获得p53突变的细胞将表达细胞表面链霉抗生物素蛋白标签， 在癌症发展的各个阶段的细胞，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。 这一系统的发展将为简单快速的癌症筛查创造机会 靶向突变型p53的治疗剂，突变型p53是所有癌症中最普遍的致癌显性缺陷之一。 一只肿瘤在活体小鼠体内发光的小鼠将提供强大的癌症实时分析 进展这一工具将推进我们对肿瘤生长和肿瘤细胞迁移的认识，以及肿瘤细胞如何生长和迁移。 对治疗有反应。

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.