Chronic Hepatitis B Virus Infection: The Relation between Hepatitis B Antigen Expression, Telomere Length, Senescence, Inflammation and Fibrosis.

BACKGROUND: Chronic Hepatitis B virus (HBV) infection can lead to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. We hypothesized that HBV might accelerate hepatocyte ageing and investigated the effect of HBV on hepatocyte cell cycle state and biological age. We also investigated the relation between inflammation, fibrosis and cell cycle phase. METHODS: Liver samples from patients with chronic HBV (n = 91), normal liver (n = 55) and regenerating liver (n = 15) were studied. Immunohistochemistry for cell cycle phase markers and HBV antigens was used to determine host cell cycle phase. Hepatocyte-specific telomere length was evaluated by quantitative fluorescent in-situ hybridization (Q-FISH) in conjunction with hepatocyte nuclear area and HBV antigen expression. The effects of induced cell cycle arrest and induced cellular senescence on HBV production were assessed in vitro. RESULTS: 13.7% hepatocytes in chronic HBV had entered cell cycle, but expression of markers for S, G2 and M phase was low compared with regenerating liver. Hepatocyte p21 expression was increased (10.9%) in chronic HBV and correlated with liver fibrosis. Mean telomere length was reduced in chronic HBV compared to normal. However, within HBV-affected livers, hepatocytes expressing HBV antigens had longer telomeres. Telomere length declined and hepatocyte nuclear size increased as HBV core antigen (HBcAg) expression shifted from the nucleus to cytoplasm. Nuclear co-expression of HBcAg and p21 was not observed. Cell cycle arrest induced in vitro was associated with increased HBV production, in contrast to in vitro induction of cellular senescence, which had no effect. CONCLUSION: Chronic HBV infection was associated with hepatocyte G1 cell cycle arrest and accelerated hepatocyte ageing, implying that HBV induced cellular senescence. However, HBV replication was confined to biologically younger hepatocytes. Changes in the cellular location of HBcAg may be related to the onset of cellular senescence.

Hepatocyte senescence predicts progression in non-alcohol-related fatty liver disease.

BACKGROUND & AIMS: Models of non-alcohol-related fatty liver disease (NAFLD) reveal features of accelerated ageing, such as impaired regeneration, and an increased risk of hepatocellular carcinoma. The relation between accelerated ageing, disease progression and clinical outcome has not been previously investigated and is the subject of the current study. METHODS: Liver sections from 70 patients with NAFLD (105 biopsies) and 60 controls were studied for telomere length, nuclear area, DNA damage and cell cycle phase markers, using quantitative fluorescent in situ hybridization and immunohistochemistry. RESULTS: Hepatocyte telomeres were shorter in NAFLD than controls (p <0.0001). Hepatocytes in NAFLD demonstrated lack of cell cycle progression beyond G1/S phase and high-level expression of p21, the universal cell cycle inhibitor (p=0.001). γ-H(2)AX expression increased with steatosis (p=0.01), indicating DNA damage, and was associated with shorter hepatocyte telomeres (p <0.0001). Hepatocyte p21 expression correlated with fibrosis stage and diabetes mellitus, independently (p <0.001 and p=0.002, respectively). Further analysis revealed that an adverse liver-related outcome was strongly associated with higher hepatocyte p21 expression and greater hepatocyte nuclear area (p=0.02 and p=0.006), but not with telomere length. In paired biopsies, changes in hepatocyte p21 expression and nuclear area mirrored changes in fibrosis stage (p=0.01 and p=0.006, respectively). CONCLUSIONS: These findings are consistent with hepatocyte senescence and permanent cell cycle arrest in NAFLD. Hepatocyte senescence correlated closely with fibrosis stage, diabetes mellitus, and clinical outcome. Hepatocyte p21 expression could be used as a prognostic marker and for stratification in clinical studies.

Sustained telomere length in hepatocytes and cholangiocytes with increasing age in normal liver.

UNLABELLED: Telomeres, a validated biomarker of aging, comprise multiple nucleotide repeats capping chromosomes that shorten with each cell cycle until a critical length is achieved, precipitating cell senescence. Only two previous studies focused on the effect of aging in "normal" liver tissue, but these studies were compromised by small sample size, limited age range, tissue derived from individuals with an increased risk of senescence, and the use of liver homogenates. We developed a robust large-volume, four-color quantitative fluorescent in situ hybridization technique to measure telomere length in large numbers of hepatocytes, Kupffer cells, hepatic stellate cells, CD4-positive and CD8-positive lymphocytes, and cholangiocytes. Following validation against the gold standard (Southern blotting), the technique was applied to normal archived paraffin-embedded liver tissue obtained following reperfusion of implanted donor liver. We studied 73 highly selected donors aged 5-79 years with a short medical illness preceding death and no history of liver disease, reperfusion injury, or steatosis and normal graft function 1-year posttransplantation. Cholangiocytes had significantly longer telomeres compared with all other intrahepatic lineages over a wide age range (P < 0.05). Age-related telomere attrition was restricted to sinusoidal cells (i.e., Kupffer cells [P = 0.0054] and stellate cells [P = 0.0001]). Cholangiocytes and hepatocytes showed no age-related telomere shortening. CONCLUSION: In normal liver and over a broad age range, cholangiocytes have longer telomeres than all other intrahepatic lineages. Age-related telomere length decline is restricted to Kupffer cells and stellate cells.

mRNA export from mammalian cell nuclei is dependent on GANP.

Bulk nuclear export of messenger ribonucleoproteins (mRNPs) through nuclear pore complexes (NPCs) is mediated by NXF1. It binds mRNPs through adaptor proteins such as ALY and SR splicing factors and mediates translocation through the central NPC transport channel via transient interactions with FG nucleoporins. Here, we show that mammalian cells require GANP (germinal center-associated nuclear protein) for efficient mRNP nuclear export and for efficient recruitment of NXF1 to NPCs. Separate regions of GANP show local homology to FG nucleoporins, the yeast mRNA export factor Sac3p, and the mammalian MCM3 acetyltransferase. GANP interacts with both NXF1 and NPCs and partitions between NPCs and the nuclear interior. GANP depletion inhibits mRNA export, with retention of mRNPs and NXF1 in punctate foci within the nucleus. The GANP N-terminal region that contains FG motifs interacts with the NXF1 FG-binding domain. Overexpression of this GANP fragment leads to nuclear accumulation of both poly(A)(+)RNA and NXF1. Treatment with transcription inhibitors redistributes GANP from NPCs into foci throughout the nucleus. These results establish GANP as an integral component of the mammalian mRNA export machinery and suggest a model whereby GANP facilitates the transfer of NXF1-containing mRNPs to NPCs.

AURORA-A amplification overrides the mitotic spindle assembly checkpoint, inducing resistance to Taxol.

The serine-threonine kinase gene AURORA-A is commonly amplified in epithelial malignancies. Here we show that elevated Aurora-A expression at levels that reflect cancer-associated gene amplification overrides the checkpoint mechanism that monitors mitotic spindle assembly, inducing resistance to the chemotherapeutic agent paclitaxel (Taxol). Cells overexpressing Aurora-A inappropriately enter anaphase despite defective spindle formation, and the persistence of Mad2 at the kinetochores, marking continued activation of the spindle assembly checkpoint. Mitosis is subsequently arrested by failure to complete cytokinesis, resulting in multinucleation. This abnormality is relieved by an inhibitory mutant of BUB1, linking the mitotic abnormalities provoked by Aurora-A overexpression to spindle checkpoint activity. Consistent with this conclusion, elevated Aurora-A expression causes resistance to apoptosis induced by Taxol in a human cancer cell line.