Hepatitis B virus X (HBx) protein has been known to play an important role in development of hepatocellular carcinoma (HCC). The aim of this study is to find out whether HBx protein expression affects antiproliferative effect of an epidermal growth factor receptor-tyrosine kinase (EGFR-TK) inhibitor and a MEK inhibitor in HepG2 and Huh-7 cell lines. We established HepG2 and Huh-7 cells transfected stably with HBx gene. HBx protein expression increased pERK and pAkt expression as well as beta-catenin activity in both cells. Gefitinib (EGFR-TK inhibitor) inhibited pERK and pAkt expression and beta-catenin activity in both cells. Selumetinib (MEK inhibitor) reduced pERK level and beta-catenin activity but pAkt expression was rather elevated by selumetinib in these cells. Reduction of pERK levels was much stronger with selumetinib than gefitinib in both cells. The antiproliferative efficacy of selumetinib was more potent than that of gefitinib. However, the antiproliferative effect of gefitinib, as well as selumetinib, was not different between cell lines with or without HBx expression. Signal pathway activation by HBx might not be strong enough to attenuate the antiproliferative effect of EGFR-TK inhibitor. Future experiments are needed to understand the role of HBx protein expression in HCC treatment using molecular targeting agent.
Animals ; Antineoplastic Agents/*pharmacology ; Benzimidazoles/*pharmacology ; Carcinoma, Hepatocellular/*metabolism ; Cell Line, Tumor/drug effects ; Cell Proliferation ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Liver Neoplasms/*metabolism ; Mitogen-Activated Protein Kinase Kinases/*antagonists & inhibitors ; Protein Kinase Inhibitors/*pharmacology ; Proto-Oncogene Proteins c-akt ; Quinazolines/*pharmacology ; Receptor, Epidermal Growth Factor/antagonists & inhibitors ; Signal Transduction/*drug effects ; Trans-Activators/*metabolism ; beta Catenin/metabolism

Hepatitis B virus (HBV) is regarded as a stealth virus, invading and replicating efficiently in human liver undetected by host innate antiviral immunity. Here, we show that type I interferon (IFN) induction but not its downstream signaling is blocked by HBV replication in HepG2.2.15 cells. This effect may be partially due to HBV X protein (HBx), which impairs IFNβ promoter activation by both Sendai virus (SeV) and components implicated in signaling by viral sensors. As a deubiquitinating enzyme (DUB), HBx cleaves Lys63-linked polyubiquitin chains from many proteins except TANK-binding kinase 1 (TBK1). It binds and deconjugates retinoic acid-inducible gene I (RIG I) and TNF receptor-associated factor 3 (TRAF3), causing their dissociation from the downstream adaptor CARDIF or TBK1 kinase. In addition to RIG I and TRAF3, HBx also interacts with CARDIF, TRIF, NEMO, TBK1, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IKKi) and interferon regulatory factor 3 (IRF3). Our data indicate that multiple points of signaling pathways can be targeted by HBx to negatively regulate production of type I IFN.
Animals ; B-Lymphocytes ; immunology ; metabolism ; Cell Line ; DEAD Box Protein 58 ; DEAD-box RNA Helicases ; antagonists & inhibitors ; immunology ; metabolism ; Hep G2 Cells ; Hepatitis B virus ; immunology ; metabolism ; Humans ; I-kappa B Kinase ; antagonists & inhibitors ; immunology ; metabolism ; Immune Evasion ; Immunity, Innate ; Interferon Regulatory Factor-3 ; antagonists & inhibitors ; immunology ; metabolism ; Interferon Type I ; antagonists & inhibitors ; immunology ; metabolism ; Mice ; Molecular Targeted Therapy ; Polyubiquitin ; antagonists & inhibitors ; metabolism ; Protein Binding ; immunology ; Sendai virus ; immunology ; metabolism ; Signal Transduction ; immunology ; TNF Receptor-Associated Factor 3 ; antagonists & inhibitors ; immunology ; metabolism ; Trans-Activators ; immunology ; metabolism

BACKGROUNDS/AIMS: Hepatitis B virus (HBV) replicates via RNA intermediates, which could serve as targets for RNA interference (RNAi). Vector-mediated short-hairpin RNA (shRNA) can induce sustained RNAi in comparison to small interfering RNA. Lentiviral vector is known to induce prolonged RNAi with high transduction efficiency. In this study, we sought to test the in vitro efficacy of shRNA delivered by a lentiviral vector in suppressing the replication of HBV. METHODS: Two shRNA sequences against the hepatitis B viral protein HBx (sh1580 and sh1685) were cloned downstream of the U6 promoter in an HIV-based plasmid to generate third-generation lentiviral vectors. HepAD38 cells were transduced with anti-HBx lentiviral vectors, and HBV replication was induced for 5 days. HBV DNA was isolated and quantified using real-time PCR. RESULTS: Lentiviral vectors encoding the shRNA against HBV transduced HepAD38 cells with high efficacy. The total intracellular HBV DNA content was significantly reduced by both sh1580 and sh1685 (2.9% and 12.0%, respectively; P<0.05). HBV covalently closed circular DNA (cccDNA) was also suppressed significantly (19.7% and 25.5%, respectively; P<0.05). CONCLUSIONS: Lentivirus-mediated delivery of shRNA against HBx can effectively suppress the replication of HBV and reduce HBV cccDNA in cell culture systems.
Cell Line, Tumor ; Genetic Vectors ; Hepatitis B virus/*genetics/growth & development/physiology ; Humans ; Lentivirus/*genetics ; *RNA Interference ; RNA, Viral/metabolism ; Trans-Activators/*antagonists & inhibitors/genetics/metabolism ; *Virus Replication

No abstract available.
Genetic Vectors ; Hepatitis B virus/*genetics/growth & development/physiology ; Humans ; Lentivirus/genetics ; *RNA Interference ; RNA, Viral/metabolism ; Trans-Activators/antagonists & inhibitors/genetics ; *Virus Replication

OBJECTIVETo explore the signal pathway mediating the regulatory effect of Hepatitis B virus X protein (HBX) on c-met gene promoter in HepG2 cells.

METHODSThe expression of c-met in HBX-transfected HepG2 cells treated with different signal pathway inhibitors was detected by western blot, the invasion capability of cells was determined by Matrigel invasion assay.

RESULTSERK inhibitor U0126 inhibited the expression of the c-Met in HBx-transfected HepG2 cells. However, both p38MAPK inhibitor SB203580 and PI-3K inhibitor wortmanin had no effect on expression of the c-Met in HBx-transfected HepG2 cells. Furthermore, the ERK inhibitor U0126 also inhibited the invasiveness of HBX-transfected HepG2 cells.

CONCLUSIONHBx induces invasion of HCC via activation of ERK pathway.

Blotting, Western ; Butadienes ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Hep G2 Cells ; Hepatitis B virus ; genetics ; Humans ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Nitriles ; pharmacology ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; Proto-Oncogene Proteins c-met ; metabolism ; Signal Transduction ; Trans-Activators ; genetics ; metabolism ; Transfection

Two LAL family regulatory genes, gdmRI and gdmRII, were identified in the geldanamycin biosynthetic gene cluster of Streptomyces hygroscopicus 17997. Disruption of the two regulatory genes resulted in absolute elimination of geldanamycin biosynthesis. The complementation experiments using a single wild-type gene could restore geldanamycin production. These results indicated that both gdmRI and gdmRII were positive regulatory genes of the geldanamycin biosynthesis.
Anti-Bacterial Agents ; biosynthesis ; Benzoquinones ; metabolism ; Gene Expression Regulation, Bacterial ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Lactams, Macrocyclic ; metabolism ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Repressor Proteins ; genetics ; Streptomyces ; genetics ; metabolism ; Trans-Activators ; genetics

The present study was purposed to investigate the relation and difference of expression phase between class II transactivator (CIITA) and HLA-DR antigens after IFN-gamma induction, and the inhibition of CIITA and HLA-DR by STAT1-alpha antisense oligonucleotides (STAT1-alpha AS); and to explore the potential effect and significance of CIITA and STAT1-alpha AS in transplantation immunity. T lymphocytes from peripheral blood of healthy subjects were incubated with IFN-gamma at different doses. RT-PCR was used to detect CIITA mRNA and Western blot was used to analyze HLA-DR antigen. Then the optimum dose of IFN-gamma was chosen for the experiment. CIITA mRNA and HLA-DR antigen were detected at various time points. Different doses of STAT1-alpha AS and sense oligonucleotides (STAT1-alpha S) were added to T lymphocytes followed by IFN-gamma. After incubation with IFN-gamma, the expression of CIITA mRNA and HLA-DR was detected once again. The results showed that CIITA mRNA was detectable at 5 hours after IFN-gamma incubation and reached the peak at 14 hours, then declined, but the CIITA mRNA was still found at 23 hours. HLA-DR antigen was detectable at 28 hours after IFN-gamma incubation and reached a peak at 52 hours, then declined. CIITA mRNA expression was positively correlated to HLA-DR expression, and was earlier than the latter. The expression of CIITA mRNA in the AS groups was significantly lower than that in the control group after 5 micromol/L, 10 micromol/L and 20 micromol/L STAT1-alpha AS treatment (P < 0.01). The expression of CIITA mRNA in the S groups was higher than that in the AS groups (P < 0.01), but there was no significant difference between the S group and the control group. The expression of HLA-DR antigen was significantly inhibited by STAT1-alpha AS, and the expression level of HLA-DR protein in the AS group was about 64.3% of that in the control group (P < 0.01), while there was no significant difference in HLA-DR expression between the S group and the control group. The changes in HLA-DR expression were similar to those in CIITA expression after STAT1-alpha AS treatment. It is concluded that CIITA expression is positively correlated with HLA-DR expression, and was detectable earlier than that of latter after IFN-gamma incubation. Stat1-alpha antisense oligonucleotides may have a sequence-specific inhibiting effect on the expression of CIITA and HLA-DR antigen after IFN-gamma incubation in vitro culture, and can prevent T lymphocyte activation. CIITA may play an important role in pathogenesis of transplantation immunity.
Cells, Cultured ; HLA-DR Antigens ; biosynthesis ; genetics ; Humans ; Interferon-gamma ; pharmacology ; Nuclear Proteins ; biosynthesis ; genetics ; Oligonucleotides, Antisense ; antagonists & inhibitors ; RNA, Messenger ; biosynthesis ; genetics ; STAT1 Transcription Factor ; antagonists & inhibitors ; T-Lymphocytes ; cytology ; Trans-Activators ; biosynthesis ; genetics ; Transplantation Immunology ; immunology

Replication of the hepatitis B virus is suppressed by deficiency of the X protein. Although several molecules that block cellular targets of X protein reduce the production of hepatitis B virus progeny, the effect of a specific inhibitor of X protein on viral replication has not been investigated. To block X protein specifically, we adopted an intracellular expression approach using H7 single chain variable fragment (H7scFv), an antibody fragment against X protein. We previously demonstrated that cytoplasmic expression of H7scFv inhibits X protein-induced tumorigenicity and transactivation. In this study, intracellular H7scFv expression inhibits reporter gene transactivation but not viral replication determined by endogenous hepatitis B virus polymerase activity assay and real-time PCR. Our findings imply that intracellular expression of antibody fragment against X protein may not be an alternative therapeutic modality for inhibition of hepatitis B virus replication.
Virus Replication/*drug effects ; Trans-Activators/*antagonists & inhibitors/immunology ; Immunoglobulin Variable Region/genetics/metabolism/*pharmacology ; Hepatitis B virus/*drug effects/physiology ; Hepatitis B e Antigens/metabolism ; Cell Line

BACKGROUNDTransforming growth factor-beta1 (TGF-beta1) exerts strong fibrogenic potential in culture-activated HSCs. Smad4 is a key intracellular mediator for the transforming growth factor-beta (TGF-beta) superfamily of growth factors. The aim of this study was to assess the effects of the antisense Smad4 gene on Ito cell line, LI90.

METHODSThe recombinant retroviral vector pLXSN-Smad4 was constructed by cloning the rat antisense Smad4 cDNA into the retroviral vector pLXSN. Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad4 and pLXSN vectors into PA317 cells. Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418. The expression of Smad4 was detected by Northern and Western blots. Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed.

RESULTSmRNA and protein expressions of Smad4 in LI90 cells transfected with retrovirus containing the antisense Smad4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells. Cells hypoexpressing the Smad4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline (P < 0.01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus.

CONCLUSIONSThe antisense Smad4 gene can suppress the expression of the Smad4 gene, reduce endogenous production of Smad4 mRNA and protein, block TGF-beta1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM). Our results may provide a basis for the development of antifibrotic gene therapy.

Cell Line ; DNA, Antisense ; pharmacology ; DNA-Binding Proteins ; antagonists & inhibitors ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Liver Cirrhosis ; therapy ; Retroviridae ; genetics ; Smad4 Protein ; Trans-Activators ; antagonists & inhibitors ; genetics ; Transfection ; Transforming Growth Factor beta ; physiology ; Transforming Growth Factor beta1

OBJECTIVETo study the role of epidermal growth factor receptor (EGFR) in the proliferation and survival of human myeloma cells.

METHODSThe inhibitor of EGFR, PD153035, was used to block the signal transduction of EGFR. The proliferation and apoptosis of myeloma cell line, XG-1, were detected by 3H-TCR incorporation assay and Annexin V staining analysis, respectively. The phosphatation of STAT3, the key activate signal to the myeloma cell proliferation, was detected with Western blot.

RESULTSPD153035 decreased the proliferation of XG-1 and induced an obvious apoptosis in XG-1. The phosphatation of STAT3 induced by HB-EGF but not by IL-6 was blocked by PD153035.

CONCLUSIONThe proliferation and survival of myeloma cells may be suppressed by PD153035 due to the blockage of phosphatation of STAT3 induced by the activation of EGFR.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Division ; Cell Line, Tumor ; DNA-Binding Proteins ; metabolism ; Humans ; Multiple Myeloma ; pathology ; Quinazolines ; pharmacology ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; STAT3 Transcription Factor ; Trans-Activators ; metabolism