Unknown,Transcriptomics,Genomics,Proteomics

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Interferon alpha induced gene expression in SOCS1 and SOCS3 overexpressing melanoma and hepatoma cell lines.

ABSTRACT: Interferon-alpha (pegylated interferon and ribavirin) is used as standard-of-care therapeutic for chronic hepatitis C virus infection. Besides good cure in some patients other patients do not benefit from the treatment dependent on the virus type and host factors. One class of putative effector proteins is the family of Suppressors of cytokine signalling (SOCS). They act in a classical negative feedback-loop against the action of interferons and many other cytokines. It has been proven that some of them, in particular SOCS1 and SOCS3, inhibit the expression of interferon induced antiviral proteins. Their mode of action depends on the signal they are interfering with. In relation to the interferon-gamma pathway, they are thought to act on the interferon-alpha receptors by masking its recognition site for the Janus kinases (JAK), by blocking the kinase activity of the JAKs and coincidentally hindering STAT molecules from binding to the kinases. They are also thought to ubiquitinate the JAKs resulting in their proteosomal degradation. The function of SOCS proteins in suppressing the interferon-alpha pathway has not yet been characterized exhaustively. This study should unveil links to understand the resistance in interferon-alpha therapy. As results we got almost complete silencing of JAK-STAT signaling in SOCS1 over-expressing cells and tissue-dependent partially suppressed gene induction in SOCS3 over-expressing cell lines. Two human cancer cell lines (ME-15, HuH-7) were stably transfected with pcDNA3.1-SOCS plasmids in presence of geneticin and daughter cell lines were generated after singularization of cells. Next, original cell lines as well as SOCS1 and SOCS3 over-expressing cell lines were treated with 1000 U/ml interferon-alpha for 4 or 24 hours or in normal culture medium. Cells lines obtained from SOCS4 plasmid transfections were screened as additional control. Gene expression levels of cell cultured in control (0 for 4 hours, 2 for 24 hours) or interferon-alpha supplemented medium for 4 hours (1) or 24 hours (4) were analyzed. mRNA abundance was measured in triplicates using 12x8-sample commercial Illumina microarrays (HumanRef 8, version 3) and scanner system (iScan) as well as reagents recommend by Illumina (IlluminaM-BM-. TotalPrep Kit).

ORGANISM(S): Homo sapiens

SUBMITTER: Fredy Siegrist

PROVIDER: E-GEOD-22801 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Oral administration of nucleotide analogues and injection of interferon-alpha (IFNalpha) are used to achieve immediate suppression in replication of hepatitis B virus (HBV). Nucleotide analogs and IFNalpha inhibit viral polymerase activity and cause long-term eradication of the virus at least in part through removing covalently closed circular DNA (cccDNA) via induction of the APOBEC3 deaminases family of molecules, respectively. This study aimed to explore whether the orally administrable low molecular weight agent CDM-3008 (RO8191), which mimics IFNalpha through the binding to IFNalpha receptor 2 (IFNAR2) and the activation of the JAK/STAT pathway, can suppress HBV replication and reduce cccDNA levels. In primary cultured human hepatocytes, HBV DNA levels as well as cccDNA levels were decreased after CDM-3008-treatment in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) value of 0.1 micro M, and this was accompanied by significant reductions in both HBeAg and HBsAg levels in the cell culture medium. Using a microarray we comprehensively analyzed and compared changes in gene (mRNA) expression in CDM-3008- and IFNalpha-treated primary cultured human hepatocytes. As reported previously, CDM-3008 mimicked the induction of　molecules that participate in the interferon signaling pathway. OAS1 and ISG20 mRNA expression was similarly enhanced by both CDM-3008 and IFNalpha. Thus, CDM-3008 could suppress pgRNA expression to show anti-HBV activity. APOBEC3F and 3G mRNA expression was induced by CDM-3008 and IFNalpha treatment suggesting that cccDNA could be degraded through induced APOBEC3 family proteins. Finally, we identified the genes whose expression was specifically enhanced in CDM-3008-treated cells compared to IFNalpha-treated cells. The expression of SOCS1, SOCS2, SOCS3, and CISH, which inhibit STAT activation, was enhanced in CDM-3008-treated cells suggesting that a feedback inhibition of the JAK/STAT pathway was enhanced in CDM-3008-treated cells compared to IFNalpha-treated cells. In addition, CDM-3008 showed an additive effect with entecavir on inhibition of HBV replication. In summary, CDM-3008 showed anti-HBV activity through activation of the JAK/STAT pathway, inducing the expression of interferon-stimulated genes (ISGs), with greater feedback inhibition than IFNalpha.

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Interferon alpha induced gene expression in SOCS1 and SOCS3 overexpressing melanoma and hepatoma cell lines.

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