Transforming growth factor-beta1 (TGF-␤1) is a pleiotropic cytokine with pivotal roles in the regulation of cellular functions and immune responses. In this study, we found that TGF-␤1 was able to effectively suppress hepatitis B virus (HBV) replication. In the presence of TGF-␤1, the level of viral replicative intermediates was dramatically decreased, both in actively dividing cells and in confluent cells. At the same time, the levels of viral transcripts, core protein, and nucleocapsid were significantly diminished by TGF-␤1 treatment. Interestingly, the inhibitory activity of TGF-␤1 was associated with preferential reduction of the level of pregenomic RNA compared with pre-C mRNA. [3][4][5] It has been demonstrated that during HBV infection viral clearance occurs before destruction of infected hepatocytes in both the chimpanzee and the transgenic mouse model. 6,7 Some experimental results have implied that cytokines are involved in the suppression of HBV replication noncytopathically. Clinical observations indeed have revealed several cytokines to be elevated during the progression of hepatitis. 8 These cytokines are likely to be involved in the regulation of immune responses against viral infection and may directly inhibit viral replication. Recently, several cytokines including IL-18, IFNs, and TNF-␣ have been reported to be effective in the suppression of HBV replication both in the livers of HBV transgenic mice and in a cell culture system. 9-12 However, the effect of TGF-␤1, which is the major cytokine secreted by hepatocytes and nonparenchymal cells, on HBV replication has not been studied. Abbreviations: cccDNA, covalently closed circular DNA; HBcAg, HBV core protein; HBeAg, HBV e antigen; pgRNA, pregenomic RNA; From the

BackgroundThe covalently closed-circular DNA (cccDNA) of hepatitis B virus (HBV) is associated with viral persistence in HBV-infected hepatocytes. However, the regulation of cccDNA and its transcription in the host cells at different growth stages is not well understood.MethodsWe took advantages of a stably HBV-producing cell line, 1.3ES2, and examine the dynamic changes of HBV cccDNA, viral transcripts, and viral replication intermediates in different cellular growth stages.ResultsIn this study, we showed that cccDNA increased suddenly in the initial proliferation phase of cell growth, probably attributable to its nuclear replenishment by intracellular nucleocapsids. The amount of cccDNA then decreased dramatically in the cells during their exponential proliferation similar to the loss of extrachromosomal plasmid DNA during cell division, after which it accumulated gradually while the host cells grew to confluency. We found that cccDNA was reduced in dividing cells and could be removed when proliferating cells were subjected to long term of lamivudine (3TC) treatment. The amounts of viral replicative intermediates were rapidly reduced in these proliferating cells and were significantly increased after cells reaching confluency. The expression levels of viral transcripts were increased in parallel with the elevated expression of hepatic transcription factors (HNF4α, CEBPα, PPARα, etc.) during cell growth confluency. The HBV transcripts were transcribed from both integrated viral genome and cccDNA, however the transcriptional abilities of cccDNA was less efficient then that from integrated viral genome in all cell growth stages. We also noted increases in the accumulation of intracellular viral particles and the secretion of mature virions as the cells reached confluency and ceased to grow.ConclusionsBased on the dynamics of HBV replication, we propose that HBV replication is modulated differently in the different stages of cell growth, and can be divided into three phases (initial proliferation phase, exponential proliferation phase and growth confluency phase) according to the cell growth curve. The regulation of cccDNA in different cell growth phase and its importance regarding HBV replication are discussed.

The risk of liver cancer in patients infected with the hepatitis B virus (HBV) and their clinical response to interferon alpha therapy vary based on the HBV genotype. The mechanisms underlying these differences in HBV pathogenesis remain unclear. In HepG2 cells transfected with a mutant HBV G2335A expression plasmid that does not transcribe the 2.2-kb doubly spliced RNA (2.2DS-RNA) expressed by wild-type HBV genotype A, the level of HBV pregenomic RNA (pgRNA) was higher than that in cells transfected with an HBV genotype A expression plasmid. By using cotransfection with HBV genotype D and 2.2DS-RNA expression plasmids, we found that a reduction of pgRNA was observed in the cells even in the presence of small amounts of the 2. T he hepatitis B virus (HBV) causes acute and chronic liver diseases in humans. Millions of people worldwide suffer from HBV-induced liver disorders, and HBV infection increases the risk of hepatic cirrhosis and hepatocellular carcinoma (1, 2). Type I interferons (IFNs), including IFN-␣ and IFN-␤, exert antiviral activity and important immunomodulatory effects in the innate immune response against HBV infection (3-5). However, the mechanism through which HBV evades the host immune response in chronically infected patients has not been fully elucidated.In addition to the factors such as viral load and naturally occurring mutants, the HBV genotype, classified as A through J based on genomic sequence, has been shown to be associated with disease progression and responses to IFN-based therapy (6). Previous studies have shown that chronic hepatitis B patients infected with genotype A or B exhibit higher rates of seroclearance of HBV e antigen (HBeAg) and viral DNA in response to IFN-␣ therapy than patients with genotype C or D infection (7-9) and that chronically infected children with HBV genotype A infection have lower viral DNA loads and exhibit less severe symptoms than patients with genotype D infection (10, 11). Clinical studies have also shown that differences between HBV genotypes correlate with deoxycytidine analog resistance (12) and hepatic pathogenesis (13).

Hepatitis B virus reactivation is an important medical issue in cancer patients who undergo systemic chemotherapy. Up to half of CHB carriers receiving chemotherapy develop hepatitis and among these cases a notable proportion are associated with HBV reactivation. However, the molecular mechanism(s) through which various chemotherapeutic agents induce HBV reactivation is not yet fully understood. In this study, we investigated the role of the cell cycle regulator p21 (Waf1/Cip1) in the modulation of HBV replication when a common chemotherapeutic agent, doxorubicin, is present. We showed that p21 expression was increased by doxorubicin treatment. This elevation in p21 expression enhanced the expression of CCAAT/enhancer-binding protein α (C/EBPα); such an increase is likely to promote the binding of C/EBPα to the HBV promoter, which will contribute to the activation of HBV replication. Our current study thus reveals the mechanism underlying doxorubicin modulation of HBV replication and provides an increased understanding of HBV reactivation in CHB patients who are receiving systemic chemotherapy.

Previous studies have identified that the expression of UK114 is tissue specific and the protein has been found to be most abundant in liver and kidney. However, the expression of UK114 in human hepatocellular carcinoma and its relationship to differentiation and transformation of hepatocellular carcinoma have not been studied. In this study, the expression of UK114 in human hepatocellular carcinoma was examined by Northern and Western blot analyses. We found that UK114 was significantly down-regulated in most of hepatocellular carcinoma tissues compared with adjacent nontumor tissues (72.7%) at both mRNA and protein levels. We looked into the possibility that this decreased expression of UK114 in the hepatocellular carcinoma tissues may play a role in the differentiation or tumorigenicity of hepatocellular carcinoma. Immunohistochemical staining showed that the reduced expression of UK114 in hepatocellular carcinoma tissues was correlated with the tumor differentiation status as graded by the Edmondson-Steiner classification. On the other hand, overexpression of UK114 was not able to suppress the proliferation of human hepatoma cells and tumorigenicity in nude mice. These results suggest that UK114 does not seem to act as a tumor suppressor gene; however, it may useful as a biomarker that will assist in the grading of the differentiation status of hepatocellular carcinoma samples. (Cancer Epidemiol Biomarkers Prev 2008;17(3):535 -42)