Role of CREB/ATF Proteins in Hepatocyte Growth Control

描述（由申请人提供）：CREB/ATF蛋白是有丝分裂ras-raf-MAPK、JNK和p38MAPK通路的效应蛋白，介导增殖、应激反应、分化和凋亡所需的基因表达。CREB/ATF蛋白通过bZip结构域与乙型肝炎病毒（HBV） X蛋白（pX）相互作用，参与肝细胞癌（HCC）的发展。由于病毒通过解除关键的细胞生长控制机制来影响共蛋白的转化，我们假设pX与CREB/ATF的相互作用在肝细胞转化中很重要。在之前的资助期内，我们已经测试了这一假设的具体方面，并有：1)描绘了与CREB/ATF蛋白相互作用所需的pX的最小区域；2)建立并表征了一种新的、可比较的px介导的肝癌发生的体外细胞模型。该细胞模型由两个四环素调控的、表达px的细胞系组成，这些细胞系来源于aml12永生化肝细胞系。3pX-1细胞系是被pX转化的分化肝细胞；另一种是4pX-1，它是一种去分化的肝细胞细胞系，不显示px介导的转化，但对px介导的凋亡敏感。本研究的目的是为了更好地了解px介导的肝细胞转化机制，以及CREB/ATF蛋白在px介导的肝细胞转化和凋亡中的作用。由于我们最早的研究定义了增加CREB/ATF转录效能所需的最小pX区域，在Aim 1中，我们将描述分化肝细胞转化与去分化肝细胞凋亡所需的最小pX区域。在Aim 2中，我们将研究pX使去分化的4pX-1细胞致敏凋亡的机制，以及pX介导的使4pX-1细胞免于凋亡的机制，从而产生转化的肝细胞。因此，我们将研究去分化的4pX-1细胞模拟HCC癌前前体的假设。在Aim 3中，我们将描述在px介导的肝细胞转化过程中表达的两个克隆的新型ESTs。本研究将进一步阐明痘介导转化的机制；pX介导细胞凋亡的机制及肝癌细胞癌前前体；并将表征新分子，这些分子有可能成为人类HCC发展的早期诊断标志物。

DESCRIPTION (provided by applicant): CREB/ATF proteins, the effectors of the mitogenic ras-raf-MAPK, JNK and p38MAPK pathways, mediate gene expression required for proliferation, response to stress, differentiation and apoptosis. CREB/ATF proteins, via the bZip domain, interact with Hepatitis B virus (HBV) X protein (pX), implicated in hepatocellular carcinoma (HCC) development. Since viral on co-proteins effect transformation by deregulating key, cellular growth control mechanisms, we hypothesize that pX interaction with CREB/ATF is important in hepatocyte transformation. In the previous funding period we have tested specific aspects of this hypothesis and have: 1) delineated the minimal region of pX required for interaction with CREB/ATF proteins; and 2) developed and characterized a novel, comparative in vitro cellular model of pX-mediated hepatocarcinogenesis. This cellular model is comprised of two tetracycline-regulated, pX-expressing cell lines derived from the AML12immortalized hepatocyte cell line. The 3pX-1 cell line is a differentiated hepatocyte that becomes transformed by pX; the other, 4pX-1, is a de-differentiated hepatocyte cell line that does not display pX-mediated transformation, but is sensitive to pX-mediated apoptosis. The goal of this proposal is to gain better understanding of the mechanism of pX-mediated hepatocyte transformation, and the role of CREB/ATF proteins in pX-mediated hepatocyte transformation and apoptosis. Since our earliest studies defined the minimal pX region required for increased CREB/ATF transcriptional efficacy, in Aim 1 we will delineate the minimal pX region required for transformation in differentiated hepatocytes vs. apoptosis in de-differentiated hepatocytes. In Aim 2 we will investigate the mechanism by which pX sensitizes de-differentiated 4pX-1 cells to apoptosis, and the pX-mediated mechanism(s) that rescue 4pX-1 cells from apoptosis, resulting in transformed hepatocytes. Thus, we will investigate the hypothesis that the de-differentiated 4pX-1 cells model a precancerous precursor for HCC. In Aim 3 we will characterize two cloned, novel ESTs expressed during pX-mediated hepatocyte transformation. The proposed studies will elucidate further the mechanism of pox-mediated transformation; the mechanism of pX mediated apoptosis, and the cellular precancerous precursor of HCC; and will characterize new molecules, which have the potential of being early diagnostic markers in human HCC development.