CONTROL OF P53 DEPENDENT APOPTOSIS

人类DNA肿瘤病毒腺病毒编码两个转化基因E1a 和E1B，它们合作转化原代啮齿动物细胞。E1a基因 产品有效地刺激细胞增殖，但无法转化为 由于诱导细胞程序性死亡(凋亡)而导致的细胞死亡。 E1a诱导的细胞凋亡是由p53肿瘤的产物介导的 抑癌基因提示P53可作为肿瘤抑制基因 通过启动细胞死亡。E1B基因或人bcl2基因的表达 原癌基因，阻断E1a诱导的p53依赖的细胞凋亡 转型。因此，必须同时放松对细胞生长控制的管制。 为了有效地抑制固有的细胞自杀反应 原代细胞的转化。E1B基因编码两个独特的19 kDa 和55 kDa蛋白，这两种蛋白都通过干扰 P53的功能。E1B 55K蛋白与p53结合并使其失活 直接，而E1B 19K蛋白利用的机制 抑制P53介导的细胞凋亡尚不清楚。的过度表达 人的Bcl2蛋白同样会阻断P53依赖的细胞凋亡，并将 在所有功能检测中替代E1B 19K蛋白。它是高度的 E1B 19K蛋白可能代表了病毒中的等价物-- 2.我打算确定E1B 19K和Bcl-2蛋白在 生化水平阻断P53依赖的细胞凋亡。一个重要的方面 这项工作的一部分是鉴定E1B 19K与细胞蛋白 和Bcl2蛋白相互作用。我们有证据表明E1B 19K蛋白 不直接与P53结合，表明19K蛋白是一种 P53功能的间接修饰物。首先，细胞内的蛋白质 将确定与E1B 19K蛋白相互作用。第二，我会 确定相互作用如何阻止P53的凋亡活性。这个 酵母双杂交系统将被用来鉴定细胞蛋白质 它们与E1B 19K蛋白相互作用。这项工作将是对 美国国立卫生研究院正在资助的项目，通过严格的 生化手段。这些目标的目标是开发一个完整的 细胞控制P53活性的机制。它正在成为 越来越明显的是，对死亡的监管可能与 对增殖的调节，作为对病毒的细胞内防御 感染和癌症。确定P53如何诱导以及如何诱导细胞凋亡 DNA肿瘤病毒的转化蛋白干预和颠覆了这一点 过程对于理解原因和 预防癌症。

The human DNA tumor virus adenovirus encodes two transforming genes, E1A and E1B, which cooperate to transform primary rodent cells. The E1A gene products efficiently stimulate cell proliferation but fail to transform cells due to the induction of programmed cell death (apoptosis). Induction of apoptosis by E1A is mediated by the product of the p53 tumor suppressor gene indicating that p53 can function as a tumor suppressor by initiating cell death. Expression of the E1B gene or the human bcl-2 proto-oncogene, blocks E1A-induced p53-dependent apoptosis to produce transformation. Thus, deregulation of cell growth control must be coupled to suppression of an intrinsic cell suicide response for the efficient transformation of primary cells. The E1B gene encodes two unique l9kDa and 55kDa proteins, both of which block apoptosis by interfering with the function of p53. The E1B 55K protein binds to and inactivates p53 directly, whereas the.mechanism utilized by the E1B l9K protein to inhibit p53-mediated apoptosis is not yet known. Overexpression of the human Bcl-2 protein will similarly block p53-dependent apoptosis and will substitute for the E1B l9K protein in all functional assays. It is highly probable that the E1B l9K protein represents the viral equivalent of Bcl- 2. I intend to determine how the E1B l9K and Bcl-2 proteins act at the biochemical level to block p53-dependent apoptosis. An essential aspect of this work is to identify the cellular proteins with which the E1B l9K and Bcl-2 proteins interact. We have evidence that the E1B l9K protein does not bind p53 directly, indicating that the l9K protein is an indirect modifier of p53 function. First, the cellular proteins which interact with the E1B l9K protein will be identified. Second, I will determine how the interaction prevents the apoptotic activity of p53. The two-hybrid system in yeast will be utilized to identify cellular proteins which interact with the E1B l9K protein. This work will complement the ongoing NIH funded project to identify l9K binding proteins by strictly biochemical means. The goal of these aims is to develop a complete mechanism by which the cell controls the activity of p53. It is becoming increasingly apparent the regulation of death may be as important as regulation of proliferation, as an intracellular defense against viral infection and cancer. Determining how apoptosis is induced by p53 and how the transforming proteins of DNA tumor viruses intervene and subvert this process is of fundamental importance to understanding the cause and prevention of cancer.