OBJECTIVETo investigate the roles of STAT1 and STAT2 in growth inhibition induced by phosphatidylethanolamine (PE) in human hepatoma HepG2 cells.

METHODSThe growth of HepG2 cells exposed to 0.125, 0.25, 0.5 and 1.0 mmol/L PE was assessed by MTT assay, and the expressions of STAT1 and STAT2 were analyzed using immunocytochemical assay.

RESULTSPE inhibited the growth of HepG2 cells in a dose-dependent manner and increased the expression of STAT1 and STAT2 in comparison with those in the control group. AG490, an inhibitor of JAKs, partially reversed PE-induced growth inhibition of HepG2 cells.

CONCLUSIONSTAT1 and STAT2 are involved in the growth inhibition of human hepatoma HepG2 cells induced by PE.

Apoptosis ; drug effects ; Cell Proliferation ; Hep G2 Cells ; Humans ; Phosphatidylethanolamines ; pharmacology ; STAT1 Transcription Factor ; metabolism ; STAT2 Transcription Factor ; metabolism

To study the gene expression profile in K562 cells treated by IFN-alpha, so as to provide some information about the potential mechanism of IFN-alpha curing CML, the changes of gene expression were examined with the DNA array in K562 cells before and after treatment with IFN-alpha. The results showed that no gene expression difference more than 2.5 times in K562 cells was found on the first day after treatment with IFN-alpha (200 U/ml), then the genes significant expression difference increased step by step, and reached the peak on the forth day. In all examined genes, 97 genes significant expression difference were detected, 86.60% (84/97) gene of interest out of those gene were up-regulated, 13.40% (13/97) were down-regulated. In these 97 genes with significant expression difference, cell regulator protein genes accounted to 23.71% (23/97), surface receptor genes 14.43% (14/97), oncogenes and tumor suppressors 11.34% (11/97), extracellular communication proteins 9.28% (9/97), cell adhesion molecular genes 8.25% (8/97) and the other genes accounted to 32.99% (32/97). JAK1 was up-regulated to 3.78 times, JAK2 to 15.43, STAT1 and STAT2 were up-regulated to 11.98 and 8.11 times respectively, and these genes are components of JAK-STAT pathway. The number of different genes began to decrease on the fifth day. There were still 9 genes that had expression difference more than 3 times on the twenty-first day. It is concluded that when concentration of IFN-alpha was 200 U/ml, the forth day should be considered as the best time to examine change of gene expression in K562 cells treated by IFN-alpha. IFN-alpha realizes its biological functions through the JAK-STAT pathways and it may be one of the mechanisms for curing CML with IFN-alpha.
Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Interferon-alpha ; pharmacology ; Janus Kinase 1 ; genetics ; K562 Cells ; Oligonucleotide Array Sequence Analysis ; STAT1 Transcription Factor ; genetics ; STAT2 Transcription Factor ; genetics

OBJECTIVETo investigate the effects of different subtypes IFN alpha (IFN alpha2b, IFN alpha2a, and IFN alpha1b) transduction molecular STAT1, STAT2, IFNAR, PKR, and RNase L, and to study the differences of their antiviral effects and to evaluate the key signaling transduction molecules.

METHODS(1) After HepG2 cells were treated with IFN alpha2b, IFN alpha2a, or IFN alpha1b, the mRNA levels of STAT1, STAT2, IFNAR, PKR, and RNase L were detected by RT-PCR. (2) After HepG2 cells were treated with 1000 U/ml IFN alpha2b, IFN alpha2a, or IFN alpha1b, the protein expression levels of STAT1 and IFNAR were examined by Western blot.

RESULTSRT-PCR results: (1) IFNAR, STAT1, and STAT2 mRNA expression levels were slightly higher in the IFN alpha1b group than those in the IFN alpha2b group (P > 0.05). The mRNA expression levels in IFN alpha1b or IFN alpha2b groups were significantly higher than in the IFN alpha2a group (P < 0.05). (2) The PKR mRNA expression showed no significant differences among IFN alpha1b, IFN alpha2b, and IFN alpha2a groups. (3) The RNase L mRNA expression was very weak. We could not compare the differences of the RNase L mRNA levels in different groups by RT-PCR. Western blot results: (1) The IFNAR, and STAT1 protein expressions were greatly up-regulated after IFN alpha induction compared with the untreated group (P < 0.05). (2) The IFNAR, and STAT1 protein expression levels in IFN alpha1b group were slightly higher than the IFN alpha2b group. IFNAR, and STAT1 protein levels of IFN alpha1b or IFN alpha2b group were significantly higher than IFN alpha2a group (P < 0.05).

CONCLUSIONSTAT1, STAT2, IFNAR mRNA and protein expressions could all be markedly up-regulated after IFN alpha treatment. Effects of IFN alpha1b or IFN alpha2b were greatly stronger than IFN alpha2a. The PKR mRNA expression also was greatly up-regulated after IFN alpha treatment. Expression levels of PKR in IFN alpha1b, IFN alpha2b, and IFN alpha2a groups were all similar. The mRNA level results were consistent with the protein level results. Our results showed that the antiviral activity of IFN alpha1b or IFN alpha2b were stronger than that of IFN alpha2a. The signal transduction molecules STAT1, STAT2, and IFNAR could be regarded as a key index to evaluate antiviral activity of IFN alpha. Further confirmation is still needed to see whether PKR could be regarded as a key index.

Antiviral Agents ; pharmacology ; Carcinoma, Hepatocellular ; virology ; Humans ; Interferon-alpha ; pharmacology ; Liver Neoplasms ; virology ; Recombinant Proteins ; STAT1 Transcription Factor ; biosynthesis ; genetics ; STAT2 Transcription Factor ; biosynthesis ; genetics ; Signal Transduction ; Tumor Cells, Cultured

OBJECTIVETo study the regulatory role of interferon-stimulated response elements (ISREs) located on the retinoic acid-induced gene G (RIG-G) promoter in RIG-G expression.

METHODSBy using point mutation technique, the authors constructed the wide type and site mutant reporter gene plasmids according to the ISRE sequence on RIG-G promoter, and detected the functional activities by luciferase reporter assay.

RESULTSMutation in ISRE II alone had no obvious effect on the expression of the reporter gene, whereas mutation in ISRE I dramatically inhibited the transactivity of RIG-G promoter. Mutation in both ISRE I and ISRE II resulted in complete loss of its response to the transcription factors for the reporter gene.

CONCLUSIONBoth ISRE I and ISRE II on the RIG-G promoter are the binding sites for the complex of transcription factors. They are required for RIG-G expression, and ISRE I has a preferential role over ISRE II.

Cell Line, Tumor ; Humans ; Interferon Regulatory Factor-1 ; genetics ; metabolism ; Interferons ; genetics ; metabolism ; Intracellular Signaling Peptides and Proteins ; genetics ; Mutation ; Promoter Regions, Genetic ; genetics ; Response Elements ; genetics ; STAT2 Transcription Factor ; genetics ; metabolism

To study the effect of HCV core protein on the interferon-induced antiviral genes expression and its mechanisms. Methods HepG2 cells were transiently transfected with HCV core protein expression plasmid and the blank plasmid respectively. RT-PCR was used to analyze the effect of HCV core protein on PKR and 2'-5'OAS expression. The effect of HCV core protein on ISRE-medicated gene expression was detected by luciferase activity assay. Western-blot assay was performed to observe the change of mRNA and protein levels of SOCS3, STAT1 and p-STAT1 following HCV core expression. In the presence of HCV core protein, the transcription of PKR and 2'-5' OAS are down-regulated. ISRE-medicated reporter gene expression and STAT1 phosphorylation were inhibited. The transcription and expression of SOCS3 were induced compared with blank plasmid-transfected group. In HepG2 cells, HCV core protein can down-regulate the expression of some interferon-induced antiviral genes, which involves the induction of SOCS3 and the inhibition of STAT1 phosphorylation.
2',5'-Oligoadenylate Synthetase ; genetics ; metabolism ; Carcinoma, Hepatocellular ; pathology ; Down-Regulation ; Hepacivirus ; genetics ; metabolism ; Humans ; Interferon-Stimulated Gene Factor 3 ; genetics ; metabolism ; Interferon-alpha ; genetics ; immunology ; Liver Neoplasms ; pathology ; Protein Kinases ; genetics ; metabolism ; STAT1 Transcription Factor ; genetics ; metabolism ; STAT2 Transcription Factor ; genetics ; metabolism ; Transcription, Genetic ; Transfection ; Tumor Cells, Cultured ; Viral Core Proteins ; genetics ; metabolism ; physiology

OBJECTIVETo investigate the molecular mechanisms by which IFN-alpha regulated retinoic acid-induced gene G (RIG-G) expression.

METHODSThe expression of STAT1, p-STAT1 and RIG-G in IFN-alpha-treated NB4 cells was detected by Western blot. The roles of STAT1, STAT2 and IRF-9 in IFN-alpha-induced RIG-G expression were analyzed in STAT1-null U3A cells by cell transfection, reporter gene assay, co-immunoprecipitation and chromatin immunoprecipitaion.

RESULTSIn U3A cells, only when STAT2 and IRF-9 were co-transfected, the luciferase activities of RIG-G promoter-containing reporter gene could be highly increased about 8-fold compared with that in the control group. Moreover, in the absence of IFN-alpha, similar effects were observed in either IRF-9 co-transfected with wild type or mutant form of STAT2, whereas IFN-alpha could increase the transactivation activity of wild type STAT2 and IRF-9 by 6-fold compared with that without IFN-alpha, but had no effect on mutant STAT2. In addition, STAT2 could interact with IRF-9 and bind to the RIG-G promoter.

CONCLUSIONSTAT2 may interact with IRF-9 in a STAT1-independent manner. The complex STAT2/IRF-9 is the key factor mediating the expression of RIG-G gene regulated by IFN-alpha. This is a novel signal transduction cascade for IFN which is different from the classical JAK-STAT pathway.

Cell Line, Tumor ; Fibrosarcoma ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Immunoprecipitation ; Interferon-Stimulated Gene Factor 3, gamma Subunit ; genetics ; metabolism ; Interferon-alpha ; pharmacology ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Phosphorylation ; Plasmids ; STAT1 Transcription Factor ; genetics ; metabolism ; STAT2 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; Transfection

To investigate the molecular mechanisms of all-trans retinoic acid (ATRA)-induced rig-g gene expression and to better understand the signal transduction of ATRA during acute promyelocytic leukemia (APL) cell differentiation, the luciferase reporter assay, co-immunoprecipitation and chromatin immunoprecipitation were used to clarify the basic transcriptional factors, which directly initiated the expression of rig-g gene. The results showed that the expression of STAT2, IRF-9 and IRF-1 could be upregulated by ATRA with different kinetics in NB4 cells. IRF-9 was able to interact with STAT2 to form a complex, which could bind the rig-g gene promoter and trigger the rig-g expression. IRF-1 alone could also activate the reporter gene containing rig-g gene promoter, but C/EBPalpha could strongly inhibit this transcription activity of IRF-1. It is concluded that during ATRA-induced APL cell differentiation, IRF-1 is first upregulated by ATRA, and then IRF-1 increases the protein levels of IRF-9 and STAT2 with the downregulation of C/EBPalpha. The complex of IRF-9 and STAT2 is the primary transcriptional factor for rig-g gene induction. This study will be helpful for better understanding the signal transduction networks of ATRA during the course of APL cell differentiation.
CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Gene Expression Regulation, Leukemic ; drug effects ; Genes, Regulator ; drug effects ; Humans ; Interferon Regulatory Factor-1 ; metabolism ; Interferon-Stimulated Gene Factor 3, gamma Subunit ; metabolism ; Intracellular Signaling Peptides and Proteins ; genetics ; Leukemia, Promyelocytic, Acute ; genetics ; STAT2 Transcription Factor ; metabolism ; Signal Transduction ; Tretinoin ; pharmacology ; Tumor Cells, Cultured

OBJECTIVETo study the mechanism of signal transduction in anti-HBV action of IFN-alpha.

METHODSThe HBV DNA in HepG 2.2.15 cell line supernatant with/without IFNalpha-2b were monitored by fluorescence real-time quantitive PCR. STAT1, STAT2, ISGF3-gamma, PKR, 2'5'-OAS mRNA levels from HepG 2 and HepG 2.2.15 cell lines that were treated with/without IFNalpha-2b at different times were detected by semi-quantitive RT-PCR. And the ISGF3-gamma protein was detected by Western blot. Then, these items were detected again after inhibiting the JAK-STAT pathway with genistein.

RESULTSThe HBV DNA in 2215 supernatant that were treated with IFNalpha-2b for 8 hours decreased 0.72 log 10 copies/ml. But the basal levels of DNA in cells pretreated with genistein? followed by IFNalpha-2b did not decrease. The STAT1, STAT2, ISGF3-gamma, 2'5'-OAS, PKR mRNA levels were upregulated by IFNalpha-2b. The same phenomena were observed with STAT1, STAT2, ISGF3-gamma mRNA when pretreated with genistein then treated with IFNalpha-2b, but the levels of 2'5'-OAS, PKR mRNA were decreased in this situation. The expression of the protein of ISGF3-gamma was also augmented by IFNalpha-2b, and was blocked by genistein.

CONCLUSIONThe JAK-STAT pathway seems to be a critical pathway in IFNalpha-2b action against HBV? and ISGF3 is most probably a key factor of the route.

Antiviral Agents ; pharmacology ; Blotting, Western ; Cell Line, Tumor ; DNA, Viral ; genetics ; isolation & purification ; Gene Expression Regulation, Neoplastic ; drug effects ; Hepatitis B virus ; drug effects ; genetics ; growth & development ; Humans ; Interferon-Stimulated Gene Factor 3 ; genetics ; metabolism ; Interferon-Stimulated Gene Factor 3, gamma Subunit ; genetics ; metabolism ; Interferon-alpha ; pharmacology ; Polymerase Chain Reaction ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; STAT1 Transcription Factor ; genetics ; metabolism ; STAT2 Transcription Factor ; genetics ; metabolism ; Signal Transduction