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通路的效应者，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结构域与乙肝病毒X蛋白(Px)相互作用，参与肝细胞癌的发生发展。由于病毒共蛋白通过解除对关键的细胞生长控制机制的调控而影响转化，我们假设Px与CREB/ATF的相互作用在肝细胞转化中是重要的。在之前的资助期间，我们已经测试了这一假说的特定方面，并已经：1)描绘了与CREB/ATF蛋白相互作用所需的PX的最小区域；以及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节的、PX表达的细胞系组成，这些细胞系来自AML12永生化肝细胞系。3px-1细胞系为Px诱导转化的分化肝细胞系；4px-1细胞系为去分化肝细胞系，不表现Px介导的转化，但对Px介导的细胞凋亡敏感。本研究的目的是为了更好地了解PX介导的肝细胞转化的机制，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提高CREB/ATF转录效率所需的最小Px区域，在目标1中，我们将描述分化肝细胞转化所需的最小Px区域与去分化肝细胞中的凋亡。在目标2中，我们将研究PX使去分化的4PX-1细胞对凋亡敏感的机制，以及PX介导的拯救4PX-1细胞免于凋亡、导致肝细胞转化的机制(S)。因此，我们将研究去分化的4PX-1细胞是肝癌癌前体细胞模型的假设。在目标3中，我们将鉴定两个克隆的、在PX介导的肝细胞转化过程中表达的新EST。这些研究将进一步阐明POX介导的转化机制；PX介导的细胞凋亡机制，以及肝癌的细胞癌前体物；并将表征新的分子，这些分子有可能成为人类肝癌发生的早期诊断标志物。

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.