Role of Uhrf1 in Liver Development, Regeneration and Carciogenesis

描述(由申请人提供)：胚胎中的肝脏发育和成人中的再生以受控的肝细胞增殖为特征。这与许多慢性肝病和肝细胞癌(肝细胞癌)伴随的不受控制的肝细胞增殖形成了鲜明对比。虽然有迹象表明，共同的遗传途径同时调节生理性和病理性肝细胞的增殖，但只有少数哺乳动物研究阐明了这一假说。通过斑马鱼胚胎的正向遗传筛选，结合成体肝再生的功能研究和人肝细胞癌标本的表达分析，我们发现含有PHD和RING FING结构域-1(Uhrf1)基因的泛素样蛋白对生理性肝细胞的增殖是必不可少的。我们还认为，uhrf1表达和/或调控的改变也有助于癌症的非调控增殖。这项建议中的工作将结合斑马鱼的发育和遗传学、人类癌症基因组分析和哺乳动物组织培养细胞周期研究和生物化学来解决uhrf1与肝细胞增殖相关的三个方面的功能。在特定的目标1中，我们将研究uhrf1在斑马鱼胚胎中调节肝细胞增殖的机制。在特定的目标2中，我们将阐明uhrf1是如何通过细胞周期蛋白依赖性激酶2的磷酸化来调节的。这种磷酸化的功能相关性将通过生物化学和细胞生物学技术在培养细胞上进行评估。在特定的目标3中，我们将确定uhrf1在肝癌发生中的作用。通过对人类肝细胞癌样本的分析，我们将评估uhrf1基因扩增在癌症中促进其上调的可能性。其次，我们将在斑马鱼中进行遗传和致癌研究，并确定uhrf1是否对肝脏肿瘤的形成是必要的和充分的。综上所述，这项建议将把控制胚胎和肝再生期间肝细胞增殖的机制与控制肝癌发生的机制联系起来。这项建议与肝病领域有直接关系。由于肝脏疾病的负担仍然巨大，识别在肝细胞增殖和肝细胞癌进展中起关键作用的基因具有重要的科学和临床意义。其目标是确定新的肝细胞增殖机制，以帮助开发用于治疗包括肝癌在内的慢性肝病患者的化疗药物。与公共健康相关：我们感兴趣的是肝脏如何发育，受伤后如何自我恢复，以及肝癌是如何发生的。我们认为，这三个过程都是相互联系的，了解正常的肝脏生长将有助于护理肝癌患者。我们相信，我们已经发现了一种名为uhrf1的基因，它参与了所有这三个过程，并将研究它如何在每种情况下发挥作用。PHS 398/2590(09/04版，2006年4月4日重新发布)页面续格式页面

DESCRIPTION (provided by applicant): Liver development in embryos and regeneration in adults are characterized by regulated hepatocyte proliferation. This contrasts with the unregulated hepatocyte proliferation that accompanies many chronic hepatic diseases and hepatocellular carcinoma (HCC). While there is suggestion that common genetic pathways regulate both physiologic and pathologic hepatocyte proliferation, only a few studies in mammals illustrate this hypothesis. By forward genetic screening in zebrafish embryos combined with functional studies on liver regeneration in adults and expression analysis of human HCC samples, we have found that the ubiquitin-like, containing PHD and RING finger domains-1 (uhrf1) gene is essential for physiologic hepatocyte proliferation. We also believe that alterations in UHRF1 expression and/or regulation also contribute to deregulated proliferation in cancer. The work in this proposal will use a combination of zebrafish development and genetics, human cancer genomic analysis and mammalian tissue culture cell cycle studies and biochemistry to address 3 aspects of UHRF1 function in relation to hepatocyte proliferation. In Specific Aim 1, we will examine the mechanism by which UHRF1 functions in regulating hepatocyte proliferation in zebrafish embryos. In Specific Aim 2, we will elucidate how UHRF1 is regulated through phosphorylation by cyclin dependent kinase 2. The functional relevance of this phosphorylation will be assessed on cultured cells using biochemical and cell biological techniques. In Specific Aim 3, we will determine the role of UHRF1 in hepatocarcinogenesis. By analysis of human HCC samples, we will evaluate the possibility that amplification of the UHRF1 locus contributes to its upregulation in cancer. Secondly, we will perform genetic and oncogenic studies in zebrafish and determine if UHRF1 is necessary and sufficient for hepatic tumor formation. In summary, this proposal will link together mechanisms that control hepatocyte proliferation in the embryo and during liver regeneration with those that control hepatocarcinogenesis. This proposal has direct relevance to the field of liver disease. Because the burden of liver disease remains enormous, the identification of genes that play critical roles in hepatocyte proliferation and in HCC progression are of significant scientific and clinical importance. The goal is to identify novel mechanisms of hepatocyte proliferation that can aid in the development of chemotherapeutic agents for use in management of patients with chronic liver diseases including HCC. PUBLIC HEALTH RELEVANCE: We are interested in how the liver develops, how it restores itself after injury and how liver cancer occurs. We believe that all three processes are linked and that understanding of normal liver growth will help in caring for patients with liver cancer. We believe that we have discovered a gene called UHRF1 that is involved in all the three processes and will study how it plays a role in each situation. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page