CONTROL OF P53 DEPENDENT APOPTOSIS

人DNA肿瘤病毒腺病毒编码两个转化基因，E1 A 和E1 B，它们合作转化原代啮齿动物细胞。e1 a基因 产品有效地刺激细胞增殖，但不能转化 由于诱导程序性细胞死亡（凋亡）， E1 A诱导细胞凋亡是由p53肿瘤产物介导的 抑制基因，表明p53可以作为肿瘤抑制因子发挥作用 通过启动细胞死亡。E1 B基因或人bcl-2的表达 原癌基因，阻断E1 A诱导的p53依赖性凋亡， 转型因此，细胞生长控制的放松管制必须与 本发明涉及抑制内在细胞自杀反应， 原代细胞的转化。E1 B基因编码两个独特的19 kDa的 和55 kDa蛋白质，这两种蛋白质都通过干扰细胞凋亡来阻断细胞凋亡。 p53的功能E1 B 55 K蛋白结合并灭活p53 直接，而E1 B19 K蛋白利用的机制， 抑制p53介导细胞凋亡是未知的。过表达 人Bcl-2蛋白将类似地阻断p53依赖性细胞凋亡， 在所有功能测定中替代E1 B19 K蛋白。极有 E1 B19 K蛋白可能代表Bcl-2的病毒等价物。 2.我打算确定E1 B19 K和Bcl-2蛋白如何在细胞内起作用。 生化水平阻断p53依赖性凋亡。一个重要方面 这项工作的目的是鉴定与E1 B19 K 和Bcl-2蛋白相互作用。我们有证据表明E1 B19 K蛋白 不直接结合p53，表明19 K蛋白是一种 p53功能的间接修饰物。首先，细胞蛋白质， 将鉴定与E1 B19 K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。的 酵母双杂交系统将用于鉴定细胞蛋白质 与E1 B19 K蛋白相互作用。这项工作将补充 正在进行的NIH资助的项目，以确定19 K结合蛋白， 生化手段。这些目标的目标是制定一个完整的 细胞控制p53活性的机制。它正在成为 越来越明显的是，对死亡的调控可能与 调节增殖，作为细胞内防御病毒 感染和癌症。确定p53如何诱导细胞凋亡以及 DNA肿瘤病毒的转化蛋白质介入并破坏了这一点， 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.