Hepatitis B Virus X Protein Differentially Regulates Cell Cycle Progression in X-transforming Versus Nontransforming Hepatocyte (AML12) Cell Lines

Abstract

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...

Hepatitis B virus X protein affects S phase progression leading to chromosome segregation defects by binding to damaged DNA binding protein 1

“…2). Furthermore, it is unclear how centrosome overduplication or a defect in the mitotic checkpoint would lead to the prolonged S-phase that we and others 18,24 observe on HBx expression, nor does it easily explain the high occurrence of lagging chromosomes in mitotic cells showing normal bipolar spindles (Fig. 1).…”

“…[18][19][20][21][22] We further show that, unexpectedly, despite causing multiple mitotic defects, HBx neither prevents bipolar spindle cells from proceeding normally through mitosis nor impairs the mitotic spindle checkpoint, but instead delays cells in S-phase because of activation of the DNA replication checkpoint. The use of an HBx point mutant, HBx(R96E), that is specifically impaired for DDB1 binding 9 showed that all these events are dependent on HBx interacting with DDB1, thus suggesting a causal link and providing an explanation for the DDB1-binding dependent cytotoxic activity of HBx reported previously.…”

HBV X protein targets hBubR1, which induces dysregulation of the mitotic checkpoint

“…Similarly, HBx-induced multinucleated cells under microtubule-damaging conditions (Lee et al, 2002) could be potentially mediated through defects in the mitotic checkpoint. To examine this possibility, we first used a targeted yeast two-hybrid assay to detect binding between HBx and any of the MCC components.…”

“…HBx is a multifunctional protein (Murakami, 2001), which deregulates cell cycle checkpoint controls (Madden and Slagle, 2001) potentially including the G 2 /M checkpoint (Lee et al, 2002). Notably, HBx-expressing cells become multinucleated under microtubule-damaging conditions (Lee et al, 2002), raising the possibility of mitotic checkpoint defects.…”

“…Notably, HBx-expressing cells become multinucleated under microtubule-damaging conditions (Lee et al, 2002), raising the possibility of mitotic checkpoint defects.…”