DOMINANT-NEGATIVE AND GAIN OF FUNCTION P53 MUTATIONS

P53 显性失活和功能获得突变

• 批准号: 2376977
• 负责人: ERIC J. STANBRIDGE
• 金额: $ 20.03万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-10 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2376977
• 关键词: DNA binding protein; DNA damage; SCID mouse; angiogenesis; apoptosis; athymic mouse; enzyme linked immunosorbent assay; flow cytometry; gene expression; gene mutation; genetic transcription; loss of heterozygosity; metastasis; molecular pathology; neoplasm /cancer invasiveness; neoplastic growth; neoplastic process; phenotype; protein structure function; site directed mutagenesis; transfection; tumor suppressor genes

The p53 tumor suppressor gene is the most commonly altered gene in human cancer and its loss of function is considered to be a critical event in neoplastic progression for many cancers. As such, restoration of function of wild type (Wt) p53 in cancer cells that lack such function is considered an attractive approach to cancer therapy. One possible obstacle is that certain mutant p53s have been considered to be dominant-negative, i.e. they interfere with Wt p53 function. The data that have led to this conclusion are based primarily on rodent model systems. The few studies that have been performed with human cancer cells have led to conflicting conclusions. The intent of this proposal is to definitively evaluate the notion of loss-of-function in human cancer cells. Experimental approaches will involve the use of human cancer cells that lack expression of any p53 protein. To investigate dominant-negative function, p53 null cells will be co- transfected with wild type (Wt) and mutant p53 cDNA expression vectors. Using both constitutive and inducible (sheep metallothionein or tetracycline-responsive) promoter constructs, we will manipulate the relative levels of mutant:Wt p53 ranging from 1:1 and 10:1 ratios. Dominant-negative effects will be assayed using transactivation and transrepression transcriptional assays, in vitro growth inhibition assays, and in vivo tumor formation. In order to more rigorously obtain 1:1 mutant:Wt ratios, we will use bicistronic vectors that will facilitate equal levels of transcription of Wt and mutant p53. All of the above experiments (and those published by others) will result in overexpression of p53 - whether Wt and/or mutant - because of the use of strong heterologous promoters. In order to simulate physiological expression levels of p53, we will use bicistronic vectors that contain the homologous p53 promoter. At physiological levels of expression, Wt p53 should not suppress growth in vitro unless DNA damage occurs. Thus, we will be able to determine potential dominant-negative effects on the important parameters of G1/S block and growth suppression following exposure to gamma-irradiation and PALA - an inducer of genomic instability and amplification. To investigate possible gain-of-function mutants, p53 null cells will be stably transfected with various mutant p53 expression vectors. The stable transfectants will be examined for alteration of function in transcriptional assays, more aggressive growth in vitro - both as adherent populations and in soft agar - and for more aggressive growth in vivo during tumor formation. Particular attention will be placed on orthotopic implantation since this represents the optimal conditions for neoplastic growth, specifically in xenogenic systems that will be used in this study. We will measure the kinetics of primary tumor growth, local invasiveness, metastasis and relative angiogenesis. Resolution of the controversy regarding dominant-negative and gain-of- function mutations in p53 will have distinct significance for therapeutic strategies involving restoration of Wt p53 function as well as possible prognostic outcome of tumors that express certain mutant p53s.

p53抑癌基因是人类最常见的变异基因， 癌症及其功能丧失被认为是一个关键事件， 许多癌症的肿瘤进展。因此，恢复功能 野生型（Wt）p53在缺乏这种功能的癌细胞中的作用， 被认为是一种有吸引力的癌症治疗方法。一个可能的障碍 某些突变型p53被认为是显性阴性的， 即它们干扰野生型p53的功能。导致这一结果的数据 结论主要基于啮齿动物模型系统。毒药作用的研究进行的 已经用人类癌细胞进行了实验， 结论.本提案的目的是明确评估 人类癌细胞功能丧失的概念。实验方法 将涉及使用缺乏任何p53表达的人类癌细胞 蛋白 为了研究显性负性功能，p53无效细胞将被共培养。 用野生型（Wt）和突变型p53 cDNA表达载体转染。 使用组成型和诱导型（绵羊金属硫蛋白或 四环素响应）启动子构建体，我们将操纵 突变体：野生型p53的相对水平范围为1：1至10：1。 将使用反式激活和 反式阻遏转录测定，体外生长抑制测定， 和体内肿瘤形成。为了更严格地获得1：1 突变体：重量比，我们将使用双顺反子载体，这将促进 Wt和突变型p53的转录水平相等。所有上述 实验（以及其他人发表的实验）将导致过度表达 p53 -无论是野生型和/或突变型-由于使用强 异源启动子为了模拟生理表达 水平的p53，我们将使用双顺反子载体，含有同源 p53启动子。在生理表达水平，野生型p53不应 抑制体外生长，除非发生DNA损伤。因此，我们将能够 以确定潜在的显性-负面影响的重要 暴露后G1/S阻滞和生长抑制的参数 γ-辐射和PALA -基因组不稳定性的诱导剂， 放大 为了研究可能的功能获得性突变体，将使用p53无效细胞。 用各种突变型p53表达载体稳定转染。稳定 将检查转染子的功能改变， 转录测定，更积极的生长在体外-无论是作为粘附 群体和软琼脂中-以及体内更具侵略性的生长 在肿瘤形成过程中。将特别注意原位 植入，因为这代表了肿瘤的最佳条件 生长，特别是在本研究中使用的异种系统中。 我们将测量原发肿瘤生长的动力学，局部侵袭性， 转移和相关的血管生成。 关于显性否定和增益的争议的解决 p53基因功能突变对肿瘤的治疗具有重要意义。 尽可能恢复Wt p53功能的策略 表达某些突变p53的肿瘤的预后结果。