The GOF Mutant p53 Beacon System

项目摘要 我们已经开发出一种系统，在该系统中，功能获得(GOF)突变体P53使活细胞发光并 LIGHTESCE(GOF突变型P53信标系统)这一强大的工具使我们能够立即评估是否存在或 活细胞中没有突变型P53，不受野生型P53的影响。它还提供了一种方法来评估 多种突变型p53等位基因在细胞系中的活性和性质。我们建议测试 用于评估小分子突变型P53抑制剂和野生型P53激活因子的信标系统。我们会 使信标系统用于高通量筛选抑制剂，以便当突变的p53被 被抑制后，细胞会发出荧光并发光。用于识别细胞调节器的系统的效用 突变的p53也将通过调整系统进行研究，以便当突变的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，在其中 获得P53突变的细胞发出荧光和发光。这一进展应该能让我们看到任何肿瘤 在活着的小鼠中，随着细胞获得p53突变以及随着肿瘤细胞的生长、进化、侵袭和 转移并在新的位置建立发光的细胞群。鼠标系统也将进行调整 这样获得p53突变细胞将表达细胞表面的链霉亲和素标签，从而易于纯化 在癌症发展的不同阶段的细胞研究细胞特性，干细胞特性，基因 事件，以及这些事件在癌症早期到晚期的演变。 这一系统的开发将为癌症的简单快速筛查创造机会 针对突变型p53的治疗，突变型p53是所有癌症中最常见的致癌缺陷之一。 活着的小鼠体内的肿瘤发光的小鼠将提供强大的癌症实时分析 进步。这一工具将促进我们对肿瘤如何生长和肿瘤细胞迁移以及它们如何 对治疗学有反应。

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.