The production of interleukin-6 (IL-6) has been discovered in a variety of human tumors. Here we report the expression of IL-6, IL-6 receptor a (IL-6Ra), and gp130 in human esophageal carcinoma tissues. We further demonstrate that IL-6 protects an esophageal carcinoma cell line CE48T/VGH from apoptosis induced by staurosporine. IL-6 stimulation induced a rapid phosphorylation of gp130 and STAT3, and a dominant-negative STAT3 completely abolished the antiapoptotic effect. IL-6 also activated ERK 1/2 in CE48T/VGH cells. Inhibition of the ERK activation by PD98059 and transfection of a dominant-negative ERK2 completely blocked the protection of IL-6 against apoptosis. Thus, both STAT and MAP kinase pathways are responsible for the IL-6-delivered survival signal in human esophageal carcinoma cells. In contrast, PI3-K inhibitors only partially attenuated the effect of IL-6, suggesting that PI3-K does not play a major role in the antiapoptotic signal of IL-6 in our system. To investigate whether IL-6 could induce the production of antiapoptotic molecules, proteins of the Bcl-2 family were measured. While Bcl-2, Bcl-x L, , and Bax were not affected, Mcl-1 was induced by IL-6 in human esophageal carcinoma cells. Our results suggest that IL-6 may contribute to the progression of esophageal cancers in an autocrine or paracrine manner.

Transfection of human hepatoma cell lines with cloned HBV DNA resulted in the secretion of large amounts of hepatitis B surface antigen (HBsAg) and core‐related antigens (HBc/HBeAg) if well‐differentiated cell lines were employed. Synthesis of both viral antigens was the highest in cell line HuH‐7 and continued for approximately 25 days. Particles resembling hepatitis B virions (Dane particles) by morphology, density and by the presence of the preS1 surface antigen were released from the transfected HuH‐7 cells into the culture medium. These particles produced in vitro were also indistinguishable from the naturally occurring hepatitis B virions in containing the virus‐associated DNA polymerase and mature HBV genomes. Restriction analysis of these DNA molecules was compatible with the nucleotide sequence of the transfecting HBV DNA sequence. Viral surface antigens and core proteins present in the culture medium were fractionated and characterized by immunoprecipitation and SDS‐‐PAGE after labeling with [35S]methionine. Antisera specific for X‐gene products identified in cell extracts two hitherto unknown HBV gene products. This system thus provides a new approach to open questions regarding HBV‐related gene function and HBV replication.

Overexpression of epidermal growth factor receptor (EGFR) and establishment of transforming growth factor a (TGF a)/EGF autocrine system are frequently detected in tumor cells. In addition to mitogenic ability, we demonstrate in this report that EGF protects a human esophageal carcinoma (CE) cell line, CE81T/VGH, from staurosporine-induced apoptosis. The anti-apoptotic signal of EGF is alleviated by a MEK inhibitor PK98059 or an ERK2 dominant negative mutant but not by a phosphatidylinositol-3'-kinase (PI-3K) inhibitor wortmannin. Furthermore, v-raf blocks apoptosis induced by staurosporine. This evidence implies that the survival signal of EGF is mediated via the Raf-MEK-ERK pathway but not the PI3-K pathway. The survival e ect of EGF is coincident with the induction of mcl-1, an antiapoptotic gene in the bcl-2 family. PD98059 also suppresses the induction of Mcl-1 by EGF, implying that EGF may up-regulate Mcl-1 via the MAP kinase pathway. Overexpression of mcl-1 is su cient to protect against apoptosis, while transfection of a mcl-1 antisense plasmid causes cell death. The expression of mcl-1 antisense plasmid also suppresses the anti-apoptotic e ect of EGF. Taken together, these results indicate that EGF may up-regulate Mcl-1 through the MAP kinase pathway to suppress apoptosis.

Interleukin-6 (IL-6) is a pleiotropic cytokine with pivotal functions in the regulation of the biological responses of several target cells including hepatocytes. The level of serum IL-6 has been reported to be elevated in patients with chronic hepatitis B, cirrhosis and hepatocellular carcinoma and represents the best marker of HBV-related clinical progression as compared with several other cytokines. In this study, we found that IL-6 was able to effectively suppress hepatitis B virus (HBV) replication and prevent the accumulation of HBV covalently closed circular DNA (cccDNA) in a human hepatoma cell line. We also demonstrated that the suppression of HBV replication by IL-6 requires concurrently a moderate reduction of viral transcripts/core proteins and a marked decrease in viral genome-containing nucleocapsids. Studies on the stability of existing viral capsids suggest that the IL-6 effect on the reduction of genome-containing nucleocapsids is mediated through the prevention of the formation of genome-containing nucleocapsids, which is similar to the effect of interferons. However, IFN-α/β and IFN-γ did not participate in the IL-6-induced suppression of HBV replication. Taken together, our results will provide important information to better understand the role of IL-6 in the course of HBV infection.

Hepatitis delta virus (HDV) superinfection causes a poor prognosis in hepatitis B virus-infected patients and effective therapy is lacking. Cytotoxic T-lymphocyte (CTL) responses play an important role in the pathogenesis of chronic viral hepatitis; however, the CD8 + T-cell epitopes of HDV have never been defined. Potential HLA-A*0201-restricted HDV peptides were selected from the SYFPEITHI database and screened by T2 cell-stabilization assay. HLA-A*0201 transgenic mice on a C57BL/6 background were injected intramuscularly with an HDV DNA vaccine. Splenocytes were stained directly ex vivo with HLA-A*0201-peptide tetramers after immunization. Epitope-specific CTL responses were confirmed by cytotoxic assays. HLA-A2, chronically infected HDV patients were also enrolled, to assess the existence of HDV-specific CD8 + T cells, based on findings in animals. Following HDV DNA vaccination, nearly 0?9 % of the total splenic CD8 + T cells were specific for peptides HDV 26-34 and HDV 43-51 in HLA-A*0201 transgenic mice, which was significantly higher than the number found in non-transgenic mice or in transgenic mice that had been immunized with control plasmid. HDV 26-34-and 43-51-specific CTL lines were able to produce CTL responses to each peptide. Interestingly, HDV 26-34-and HDV 43-51-specific CD8 + T cells were also detectable in two chronically infected HDV patients in the absence of active HDV replication. In conclusion, HDV 26-34 and 43-51 are novel HLA-A*0201-restricted CTL epitopes on genotype I HDV. HDV 26-34-and 43-51-specific CTLs have been detected in chronic hepatitis delta patients without active disease. Evoking CTL responses to HDV may be an alternative approach to controlling HDV viraemia in patients with chronic hepatitis delta.

Virus persistence in chronic hepatitis B patients is due to the sustaining level of covalently closed circular DNA (cccDNA) within the nuclei of infected hepatocytes. In this study, we used a modified 1.3-fold hepatitis B virus (HBV) genome, with a BclI genetic marker embedded in the redundancy region, to examine the transcriptional activity of cccDNA and the effect of the HBx protein on transcriptional regulation. After harvesting total RNA from transfected cells or stable lines, we specifically identified and monitored the transcripts from cccDNA by using reverse transcription-PCR (RT-PCR) combined with the restriction enzyme digestion method. In this approach, we have found that (i) RT-PCR combined with detection of the BclI marker is a highly specific method for distinguishing cccDNA-derived transcripts from the original integrated viral genome, (ii) the transcriptional ability of cccDNA was less efficient than that from the integrated viral genome, and (iii) the transcriptional activity of cccDNA was significantly regulated by the HBx protein, a potential transcription activator. In conclusion, we provided a tool with which to elucidate the transcriptional regulation of cccDNA and clarified the transcriptional regulation mechanism of HBx on cccDNA. The results obtained may be helpful in the development of a clinical intervention for patients with chronic HBV infections.

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