Studies on the mechanism of the hepatitis B virus X protein (pX)-mediated hepatocarcinogenesis

Abstract

The Hepatitis B Virus (HBV) X protein (pX), required for the viral life cycle, is implicated in HBV-mediated hepatocarcinogenesis by an unknown mechanism. pX is a multifunctional protein, with activities affecting transcription, cell growth, and apoptosis. pX activates transcription by two mechanisms: via direct interaction with the basal transcriptional apparatus and cellular bZip transcription factors, and via activation of cytoplasmic signaling pathways. To understand the mechanism by which pX transforms hepatocytes, conditional, tetracycline-controlled mouse hepatocyte AML 12 cell lines were constructed which express HBV pX, via the tetracycline-controlled expression system. One such cell line (3pX-1) displays characteristics of adult hepatocytes and pX-dependent oncogenic transformation. Another clonal cell line (4pX-1) is a de-differentiated hepatocyte cell line, which lacks pX-dependent transformation. Comparative analyses of this cellular model system were applied to investigate the early signaling events activated by pX, while the hepatocytes are becoming transformed. The following pX-dependent differences are observed between the pX-transforming (3pX-1) and non-transforming (4pX-1) cell lines: (1) Differential immediate early gene (IEG) expression: Selective Activating Transcription Factor 3 (ATF3) messenger Ribonucleic Acid (mRNA) induction and prolonged c-fos expression in 3pX-1 cells. (2) Differential mitogenic pathway activation: Sustained activation of the Ras-Raf-MAPK pathway and transient activation of the c-Jun N-terminal Kinase (JNK) pathway in 3pX-1 cells. The inverse is observed with the non-transforming 4pX-1 cell line. (3) Differential phosphorylation of endogenous CREB and c-Jun: prolonged phosphorylation of cAMP Response Element Binding Protein (CREB) in 3pX-1 cells and prolonged phosphorylation of c-Jun in 4pX-1 cells. To demonstrate the functional significance of the mitogenic pathway activation by pX, a variant of pX tagged with Nuclear Localization Sequence (pX-NLS) was used to construct conditional cell lines, via the Tet-off system. These cell lines display markedly reduced pX-dependent hepatocyte transformation and minimal mitogenic pathway activation. Taken together, these results support the significance of the mitogenic pathway activation by pX in hepatocyte transformation