Role of CREB/ATF Proteins in Hepatocyte Growth Control

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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.