Hepatitis B virus (HBV) X protein (pX) is implicated in Epidemiological evidence (1) links chronic hepatitis B virus (HBV)1 infection in humans to development of hepatocellular carcinoma (HCC). Evidence derived from comparative studies of mammalian and avian hepadnaviruses, transgenic animal studies, and cell culture transformation studies collectively (2, 3) support a role for the 16.5-kDa HBV X protein (pX) as a weak oncogene, implicated in HCC development. However, the mechanism(s) by which pX effects hepatocyte transformation is not yet understood. Also not entirely understood is the cell type in the liver, which is the precancerous precursor giving rise to HBV-mediated HCC. The accumulating evidence derived from rat models of hepatocarcinogenesis (4 -7), woodchuck hepatitis virus-mediated HCC (8), and human liver pathologies (9, 10) point to the undifferentiated (oval cell) or less-differentiated (transitional) hepatocyte as the precancerous precursor in HCC development.Activities ascribed to pX probably linked to HBV-mediated pathology include activation of the Ras-Raf-MAPK (11-14), JNK (15), and STAT3 pathways (16, 17); direct interactions with specific components of the basal transcriptional apparatus (18 -21) and with the CREB/ATF family of transcription factors (22-25); interaction with DNA repair proteins (26); and activation of the proteasome complex (27). Importantly, many studies have demonstrated that pX expression in different cell types (Chang cells, NIH3T3 cells, immortalized differentiated AML12 hepatocytes) results in distinct and opposing cellular responses, including cell cycle progression (28), G 1 /S phase arrest (29, 30), transformation (31, 32), and apoptosis (32-35). However, despite evidence supporting the growth-promoting (36) versus the antiproliferative or apoptotic function of pX (37), the molecular mechanisms by which pX effects these processes, for the most part, remain to be deciphered. Likewise, the significance of these pX-mediated processes in HCC development is poorly understood.In our studies, we employ a cellular model system linked to pX-mediated hepatocyte transformation (32) and suitable to molecular analyses. It is composed of two tetracycline-regulated, pX-expressing cell lines, a differentiated hepatocyte 3pX-1 cell line, and a dedifferentiated hepatocyte 4pX-1 cell line. Conditional pX expression selectively transforms the 3pX-1 cell line. We recently demonstrated (36) that an early pX-mediated event in 3pX-1 cells is sustained activation of the Ras-Raf-MAPK pathway, an activation that is causally linked to pX-mediated transformation. In the pX-nontransforming 4pX-1 cell line, pX expression results in sustained activation of the JNK pathway and only transient activation of the Ras-Raf-MAPK pathway. Since pX expression mediates distinct growth characteristics between the 3pX-1 and 4pX-1 cell lines (i.e. transformation in the differentiated 3pX-1 cells versus absence of transformation in the less differentiated 4pX-1 cells and differential activation of the Ras-Raf-M...

The engagement of antigen receptors on lymphocytes leads to the activation of phospholipase C-γ, the mobilization of intracellular calcium and the activation of the NFAT transcription factor. The coupling of antigen receptors to the activation of NFAT is modulated by numerous cellular effectors including phosphoinositide 3-kinase (PI3K), which is activated following receptor cross-linking. The activation of PI3K has both positive and negative effects on the receptor-mediated activation of NFAT. An increase in the level and activity of Akt2, a target of activated PI3K, potently inhibits the subsequent activation of NFAT. In contrast, an elevation in Akt1 has no effect on signaling. Signaling pathways operating both upstream and downstream of inositol 1,4,5-trisphosphate (IP3)-stimulated calcium release from intracellular stores are unaffected by Akt2. An increase in the level of Akt2 has no significant effect on the initial amplitude, but substantially reduces the duration of calcium mobilization. The ability of Akt2 to inhibit prolonged calcium mobilization is abrogated by the administration of a cell permeable peptide that blocks the interaction between Bcl-2 and the IP3 receptor. Thus, Akt2 is a negative regulator of NFAT activation through its ability to inhibit calcium mobilization from the ER.