p53-Regulation in Liver Regeneration

描述（由申请人提供）：在手术切除（部分肝切除术，PHx）或化学物质或疾病破坏肝组织后，刺激完全分化肝脏的更新器官发生。一个诱导的信号转导通路网络触发肝细胞（随后是非实质细胞）重新进入细胞周期、增殖和代偿生长，并在一个精确调节的终点终止。我们假设p53肿瘤抑制蛋白促进细胞周期阻滞和细胞凋亡的关键功能在部分肝切除术后一定被阻断或减弱。我们的目标是定义这一监管过程的机制。关于p53的调控和功能的丰富信息主要来自肿瘤来源细胞、长期连续培养细胞和/或表达功能失调、突变或外源性p53的细胞的研究。尽管越来越多的证据表明p53在正常细胞的发育、衰老和衰老过程中起作用，但我们对内源性p53调控机制的了解仍存在重大空白。肿瘤抑制因子p53低水平表达，受多种途径的严格控制。这对内源性p53的生化纯化和机制分析提出了相当大的挑战，特别是在正常细胞中。我们已经开发了一种新的小鼠模型来解决我们知识上的这些空白。通过敲入方法创建的小鼠模型表达内源性p53与c端表位标签（TAP-p53）融合在框架内。TAP或串联亲和纯化是一种在相对生理条件下纯化低丰度、大蛋白复合物的有效方法。TAP-p53纯化和随后的质谱多肽分析将用于确定p53-蛋白相互作用（p53“蛋白质组”），并确定p53的翻译后修饰。p53的表位标记进一步通过染色质免疫沉淀（ChIP）和p53靶点全基因组分析（ChIP- ChIP）促进了p53-染色质相互作用的研究。我们将使用这些方法和其他方法来建立正常肝细胞中p53功能和调控的基态，并确定这些功能在肝脏再生过程中如何改变。拟议的研究将解决p53如何在正常分化细胞中发挥作用，这一领域在肿瘤抑制活性的研究中通常被忽视。这项工作将进一步从分子角度理解组织再生，长期目标是了解如何暂时控制p53网络的监视状态，以促进细胞更新和组织再生。

DESCRIPTION (provided by applicant): Renewed organogenesis of the fully differentiated liver is stimulated in response to surgical removal (partial hepatectomy, PHx) or destruction of liver tissue by chemicals or disease. An induced network of signal transduction pathways triggers re-entry of hepatocytes (followed by non-parenchymal cells) into cell cycle, proliferation and compensatory growth, which terminates at a precisely regulated endpoint. We hypothesize that critical functions of the p53 tumor suppressor protein, in promoting cell cycle arrest and apoptosis, must be blocked or attenuated in response to partial hepatectomy. Our goal is to define the mechanisms of this regulatory process. The wealth of information, regarding regulation and functions of p53, stems primarily from studies of tumor-derived cells, cells under long term, continuous culture, and/or cells expressing dysfunctional, mutant or exogenous p53. Although evidence continues to mount that p53 acts in normal cells during development, aging and senescence, major gaps in our knowledge exist regarding mechanisms of endogenous p53-regulation. Tumor suppressor p53 is expressed at low levels, which are tightly controlled by multiple pathways. This offers a considerable challenge to biochemical purification and mechanistic analyses of endogenous p53, especially in normal cells. We have developed a new mouse model to address these gaps in our knowledge. The mouse model, created by knock-in methodology, expresses endogenous p53 fused in-frame with a C-terminal epitope-tag (TAP-p53). TAP or Tandem Affinity Purification is a proven means of purifying low-abundance, large protein complexes under relatively physiological conditions. TAP-p53 purification and subsequent peptide analyses by mass spectrometry will be used to define p53-protein interactions (the p53 "proteome") and to determine post-translational modifications of p53. Epitope-tagging of p53 further facilitates studies of p53-chromatin interactions by chromatin immunoprecipitation (ChIP) and genome-wide analysis of p53-targets (ChIP-chip). We will use these and other approaches to establish the ground state of p53 function and regulation in normal hepatic cells and to determine how these functions may be altered during liver regeneration. The proposed research will address how p53 functions in normal, differentiated cells, an area generally overlooked in investigations of tumor suppressor activities. This work will further molecular understanding of tissue regeneration with a long-term goal of understanding how the surveillance status of the p53-network may be temporally controlled to facilitate cellular renewal and tissue regeneration. PROJECT NARRATIVE: These studies are focused on understanding how a fully differentiated tissue circumvents regulation and surveillance by tumor suppressor p53 to regenerate itself in response to partial hepatectomy. We believe that p53, which normally stops growth and proliferation, is temporarily blocked from functioning during regeneration and then restored to normal capacities when regeneration is finished. How p53 functions in normal cells and during regeneration is essentially unknown.