Kyung Lib Jang

Hepatitis B virus X protein represses E-cadherin expression via activation of DNA methyltransferase 1

E-cadherin is a key cell adhesion molecule implicated as a tumor suppressor, which is frequently altered in hepatocellular carcinoma, especially in hepatitis B virus (HBV)-related tumors. Here, we report that HBV X protein (HBx) represses E-cadherin expression at the transcription level. Based on the differential effects of HBx natural variants, we determined that Lys-130 in the transactivation domain of HBx is critical for the E-cadherin repression. The repression effect of HBx was abolished after treatment with DNA methyltransferase inhibitor, 5′-Aza-2′dC. In addition, methylation-specific PCR analysis revealed that the CpG island 1 of E-cadherin promoter is hypermethylated by HBx. Furthermore, HBx induces DNA methyltransferase 1 expression by stimulating its transcription. Therefore, we conclude that HBx represses E-cadherin expression by inducing methylation-mediated promoter inactivation. The reduced E-cadherin expression results in dramatic morphological changes of the HBx-expressing cells. In addition, HBx-expressing cells aggregate poorly in suspension culture, reflecting their altered intercellular interactions. The biological significance was further demonstrated by the increased collagen invasion ability of HBx-expressing cells. Therefore, the present study suggests that HBx plays a role during hepatocellular carcinogenesis by favoring cell detachment from the surrounding cells and migration outside of the primary tumor site.

MUC1 Induced by Epstein-Barr Virus Latent Membrane Protein 1 Causes Dissociation of the Cell-Matrix Interaction and Cellular Invasiveness via STAT Signaling

ABSTRACT Disruption of cellular adhesion is an essential pathobiologic step leading to tumor dissemination. Mucin 1 (MUC1) is a mucinous glycoprotein expressed at the surfaces of epithelial cells in many tissues and their carcinomas. MUC1 plays crucial roles in tumor invasion and metastasis, especially in opposing cell adhesion. We have shown that virus infection, specifically by the human tumor virus Epstein-Barr virus (EBV) induces a spectrum of cellular invasiveness and metastasis factors. Here we show that expression of MUC1 is increased in diverse latently EBV-infected cell lines that express latent membrane protein 1 (LMP1), the main viral oncoprotein, and that the level of MUC1 was suppressed by expression of a dominant-negative mutant of LMP1. Expression of LMP1 in EBV-negative nasopharyngeal cell lines induces expression of MUC1 through activation of the MUC1 promoter via binding of STAT1 and STAT3. Finally, LMP1 reduces cell adhesion ability, which is restored by inhibition of MUC1 expression with MUC1 small interfering RNA (siRNA). In addition, LMP1 increases cell invasiveness, which is suppressed by MUC1 siRNA. Thus, LMP1 induces MUC1, a factor important in an early step of detachment and release of tumor cells, which along with induction of other invasiveness and angiogenic factors may combine to act in a complex sequential process that culminates in metastasis of EBV-infected tumor cells.

Nationwide Surveillance for Pathogenic Microorganisms in Groundwater near Carcass Burials Constructed in South Korea in 2010

Widespread outbreaks of foot-and-mouth disease and avian influenza occurred in South Korea during 2010. In response to the culling of many animals to attenuate the spread of disease, South Korea used mass burial sites to dispose of the large number of carcasses; consequently, concerns about groundwater contamination by leachate from these burial sites are increasing. Groundwater is one of the main sources of drinking water, and its cleanliness is directly related to public health. Thus, this study aimed to evaluate the safety of groundwater around the burial sites (total of 600 sites). A total of 1,200 groundwater samples were collected though the country, and microbial analysis was conducted during two time periods: during the spring (n = 600; April to June 2012) and after rainfall (n = 600; August to October, 2012; fall). Fecal coliform and Escherichia coli were detected in 173 (14.4%) and 85 (7.1%) of the 1,200 samples, respectively. Salmonella spp. and Shigella spp. each were detected only once (0.083%). Clostridium perfringens was detected from 7 groundwater samples (0.583%), and E. coli O157:H7 was not detected. With respect to norovirus, only the GII type was detected from six groundwater samples (0.5%), and enterovirus was detected in 15 groundwater samples (1.25%). The frequency of E. coli that we detected was lower than that found in previous studies conducted in South Korea, but we detected higher frequency of fecal coliform than that observed in a previous report. The contamination frequencies of Salmonella spp. and Shigella spp. were very low, but C. perfringens, which could be an indicator of fecal pollution, was detected in seven regions. Overall, the results of the present study indicate a low possibility of contamination from burial sites. However, consistent monitoring is required to prevent microbial contamination of groundwater near the burial sites.

Hepatitis B virus X protein activates E3 ubiquitin ligase Siah-1 to control virus propagation via a negative feedback loop

The seven in absentia homologue 1 (Siah-1) protein is an E3 ubiquitin ligase that induces ubiquitin-dependent proteasomal degradation of HBx, the principal regulatory protein of hepatitis B virus (HBV); however, its role in HBV propagation remains unknown. Here, we found that HBx upregulates Siah-1 levels in HepG2 but not in Hep3B cells, in which p53 is absent. For this effect, HBx sequentially activated ataxia telangiectasia mutated kinase and checkpoint kinase 2 via phosphorylation at the Ser-1981 and Thr-68 residues, respectively, which led to the activation of p53 via phosphorylation at the Ser-15 and Ser-20 residues. As a result, HBx was heavily ubiquitinated by Siah-1 and degraded by the ubiquitin-proteasome system in HepG2 cells, whereas this effect was marginal or undetectable in Hep3B cells. Knock-down of p53 in HepG2 cells downregulated Siah-1 levels and subsequently upregulated HBx levels, whereas ectopic p53 expression in Hep3B cells upregulated Siah-1 levels and subsequently downregulated HBx levels. In addition, Siah-1 knock-down impaired the ubiquitination and proteasomal degradation of HBx in HepG2 cells, whereas ectopic Siah-1 expression induced ubiquitin-dependent proteasomal degradation of HBx in Hep3B cells. The effects of HBx on p53 and Siah-1 were exactly reproduced in a 1.2-mer HBV replicon system, mimicking the natural course of HBV infection. In particular, Siah-1 knock-down upregulated the levels of HBx derived from the HBV replicon, resulting in an increase in HBV production. In conclusion, HBx modulates its own protein level via a negative feedback loop involving p53 and Siah-1 to control HBV propagation.

Hepatitis C virus Core activates proteasomal activator 28 gamma expression via upregulation of p53 levels to control virus propagation.

The proteasomal activator 28γ (PA28γ), frequently overexpressed in hepatocellular carcinoma, is believed to play several important roles in hepatitis C virus (HCV) replication and viral pathogenesis. However, the underlying mechanism for PA28γ overexpression in hepatocellular carcinoma and its role during HCV replication are still unclear. In the present study, we found that HCV core derived from either ectopic expression or HCV infection upregulates PA28γ levels in p53-positive human hepatocytes. For this effect, HCV core sequentially activated ataxia telangiectasia mutated and checkpoint kinase 2 via phosphorylation at Ser-1981 and Thr-68 residues, respectively, resulting in stabilization of p53 via phosphorylation at Ser-15 and Ser-20 residues and subsequent transcriptional activation of PA28γ expression. The elevated PA28γ in turn downregulated HCV core levels by either inducing its ubiquitination-dependent proteasomal degradation via upregulation of E6AP levels in the presence of p53 or activating an ubiquitin-independent proteasomal degradation pathway in the absence of p53, which ultimately led to a decrease in HCV propagation. HCV core modulates its own protein level via a negative feedback loop involving p53 and PA28γ to control HCV replication in p53-positive hepatocytes, which may help HCV evade immune responses and establish chronic infection.

Hepatitis B virus X protein activates proteasomal activator 28 gamma expression via upregulation of p53 levels to stimulate virus replication

Proteasomal activator gamma (PA28γ), frequently overexpressed in hepatocellular carcinoma, is believed to play important roles in tumourigenesis. However, the underlying mechanism of PA28γ overexpression and its possible roles in hepatitis B virus (HBV) replication are largely unknown. In the present study, we found that hepatitis B virus X protein (HBx) activates PA28γ expression by upregulating p53 levels in human hepatoma cells. The elevated PA28γ levels in turn repressed seven in absentia homologue 1 expression via downregulation of p53 levels, thereby inhibiting ubiquitin-dependent proteasomal degradation of HBx, which ultimately led to upregulation of HBx levels. The correlation among HBx, p53 and PA28γ was exactly reproduced in a 1.2-mer HBV replicon system, mimicking the natural course of HBV infection. In particular, knockdown of either p53 or PA28γ in HepG2 cells downregulated HBx levels and thereby inhibited HBV replication, whereas overexpression of p53 or PA28γ in Hep3B cells upregulated HBx levels, which stimulated HBV replication, indicating that p53 and PA28γ act as activators of HBV replication. In conclusion, HBx levels are upregulated via a positive feedback loop involving p53 and PA28γ to stimulate HBV propagation.

Sodium Butyrate Alters Cell-Cell Interactions through Up-Regulation of E-Cadherin in Human Hepatocellular Carcinoma Cells

Sodium butyrate (NaBt), a naturally occurring short chain fatty acid derived from carbohydrate metabolism in the gut, is known to exhibit strong anti-cancer potentials in various human cancer cells; however, its action mechanism is poorly understood. In the present study, we demonstrated that NaBt up-regulates levels of E-cadherin, a key cell adhesion molecule implicated as a tumor suppressor, in a cell type-specific manner. Although levels of p21, a potential activator for E-cadherin expression, were also up-regulated by treatment with NaBt in several types of cells, it does not seem to be associated with the activation of E-cadherin in the NaBt-treated cells. Instead, the data from promoter analysis suggest that NaBt up-regulates expression of E-cadherin at the transcription level by enhancing its promoter strength via a CCAAT-box. The elevated E-cadherin in the presence of NaBt was primarily localized at the cell-cell contacts, converting Hep3B cells into a more differentiated form.

Cooperative stimulation of cisplatin-mediated apoptosis by hepatitis B virus X Protein and hepatitis C virus core Protein

The co-infection with hepatitis B virus (HBV) and hepatitis C Virus (HCV) is associated with a more severe liver disease and increased frequency in the development of hepatocellular carcinoma compared to those with single infection. Here, we demonstrated that HBV X protein (HBx) and HCV Core cooperatively up-regulated the level of p53 in human hepatoma HepG2 cells. The elevated p53 subsequently stimulated the expression of proapoptotic Bax whereas it repressed the expression of anti-apoptotic Bcl2. These effects, however, were not observed in p53-negative Hep3B cells. Consistently to their cooperative regulation of apoptotic effectors, HBx and HCV Core additively stimulated cisplatin-mediated apoptotic cell death of HepG2 but not of Hep3B cells. These results may help to explain the development of a more severe liver disease in patients co-infected with HBV and HCV as well as some contradictory results on the roles of HBx and Core in apoptosis.