E1A Oncoprotein Induced Deregulation of Replication Origin Firing

描述(申请人提供)：本申请的重点是确定腺病毒(Ad)转化E1a蛋白以p300结合依赖的方式诱导静止细胞进入S期的机制。E1a与细胞激活的ras或编码E1B19K或55K蛋白的病毒协同作用，结合p300/CBP和Rb家族蛋白，可转化啮齿动物细胞。在静止期细胞中，E1a与p300/CBP和Rb蛋白的相互作用导致S期的快速诱导。虽然Rb蛋白的失活会诱导E2F和S期，但E1A结合对p300的作用还没有得到深入的研究。先前我们已经证明，p300通过负调控c-Myc来阻止静止细胞过早进入S时相。P300与转录因子YY1和组蛋白脱乙酰酶3在YY1的上游结合部位协同作用，使c-Myc处于抑制状态，这有助于维持细胞在G0期。E1a通过干扰这一机制诱导c-Myc。最近，我们发现在静止的细胞中，一些与DNA合成和细胞周期进程相关的额外基因也被p300抑制，这些基因是由E1a以p300结合依赖的方式诱导的。无论是shRNA下调p300，还是野生型E1a的表达而不是p300结合缺陷E1a突变体的表达，都会导致c-Myc的诱导，这与快速诱导S期、激活DNA损伤反应和S期停滞有关。这些细胞含有增加的细胞DNA复制起始活性。我们现在建议进行研究，以确定E1A如何以p300结合依赖的方式解除对起始活性的调控。这些研究有望对E1a介导的异常S期进展及其在病毒和细胞转化中的作用提供新的线索。 公共卫生相关性：通过各种机制放松对细胞生长的控制是人类癌症发展的关键一步。腺病毒编码的E1a是一种历史悠久的模式癌蛋白。了解其在异常S期进展中的作用将促进我们对癌基因如何通过破坏细胞蛋白的生长抑制途径而促进肿瘤转化的理解。

DESCRIPTION (provided by applicant): The focus of this application is to determine the mechanisms by which adenovirus (Ad) transforming E1A protein induces progression to S phase in quiescent cells, in a p300 binding dependent manner. In cooperation with cellular activated ras or virus encoded E1B19K or 55K proteins and by binding to p300/CBP and Rb family proteins, E1A can transform rodent cells. Interactions of E1A with p300/CBP and Rb proteins in quiescent cells result in rapid induction of S phase. While the inactivation of Rb proteins is well known to induce E2F and S phase, the contribution of E1A binding to p300 has not been thoroughly studied. Previously we showed that p300 prevents premature entry of quiescent cells into S phase by negatively regulating c-Myc. p300 cooperates with transcription factor YY1 and histone deacetylase 3 at an upstream YY1 binding site to keep c-Myc in a repressed state and that this contributes to maintaining cells in G0 phase. E1A induces c-Myc by interfering with this mechanism. Recently, we have discovered that a number of additional genes related to DNA synthesis and cell cycle progression are also repressed by p300 in quiescent cells and these genes are induced by E1A in a p300 binding dependent manner. Either downregulation of p300 by shRNAs or expression of wild type E1A but not p300 binding defective E1A mutants leads to the induction of c-Myc that is linked to rapid induction of S phase, activation of DNA damage response and S phase arrest. These cells contain increased cellular DNA replication origin activity. We now propose studies to determine how the origin activity is deregulated by E1A in a p300 binding dependent manner. These studies are expected to shed new light on E1A mediated aberrant S phase progression and its role in viral and cellular transformation. PUBLIC HEALTH RELEVANCE: Deregulation of cell growth control by a variety of mechanisms is a key step in the development of human cancer. Adenovirus encoded E1A is a time honored model oncoprotein. Understanding its effects on aberrant S phase progression will advance our understanding of how an oncogene by disrupting the growth inhibitory pathways of a cellular protein contributes to neoplastic transformation.