DOMINANT-NEGATIVE AND GAIN OF FUNCTION P53 MUTATIONS

P53抑癌基因是人类最常见的突变基因 癌症及其功能丧失被认为是 许多癌症的肿瘤进展。因此，功能的恢复 在缺乏野生型(Wt)P53功能的癌细胞中 被认为是一种有吸引力的癌症治疗方法。一个可能的障碍 某些突变的p53基因被认为是显性阴性的， 即它们干扰Wt 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。为数不多的研究 对人类癌细胞进行的研究导致了相互矛盾的 结论。这项提案的目的是最终评估 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-负功能，P53零细胞将与 野生型(Wt)和突变型P53基因表达载体。 同时使用结构性和诱导性(绵羊金属硫蛋白或 四环素反应)启动子的构建，我们将操纵 突变型：WTP53的相对水平介于1：1和10：1之间。 显性-负性效应将使用反式激活和 转录抑制实验，体外生长抑制实验， 并在体内形成肿瘤。为了更严格地获得1：1 突变：WT比率，我们将使用双顺反子载体，这将有助于 Wt和突变型P53的转录水平相等。所有这些都是 实验(以及其他人发表的实验)将导致过度表达 P53--无论是Wt和/或突变体--因为使用了Strong 异源启动子。为了模拟生理表情 水平的P53，我们将使用包含同源基因的双顺反子载体 P53启动子。在生理表达水平上，Wt P53不应该 抑制体外生长，除非发生DNA损伤。因此，我们将能够 确定对重要事务的潜在显性负面影响 G1期/S期阻滞参数与暴露后的生长抑制 伽玛射线和PALA-基因组不稳定的诱导剂和 放大。 为了研究可能的功能获得突变，P53零细胞将 稳定转染各种突变型P53表达载体。马厩 将对转染者进行功能改变检查 转录分析，在体外更具侵略性的生长-两者都是贴壁的 种群和软琼脂中--以及体内更具侵略性的生长 在肿瘤形成过程中。将特别注意矫形外科 植入，因为这代表了肿瘤的最佳条件 生长，特别是在本研究将使用的异种系统中。 我们将测量原发肿瘤生长的动力学，局部侵袭性， 转移和相对血管生成。 关于显性-否定性和收益性的争议的解决 P53的功能突变将对治疗具有明显的意义 尽可能恢复Wt P53功能的策略 表达某些突变型p53基因的肿瘤的预后。

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.