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CD95 and caspase 8 in human liver cell lines
S 116
POSTERS
PAO, through its control of intracellular polyamine levels, seems to be a potential target for therapy.
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THE APOPTOSIS OF PERIPHERAL BLOOD M O N O N U C L E A R CELLS DURING VIRAL HEPATITIS IN CHILDREN, AND ITS CORRECTION BY U R S O D E O X Y C H O L I C ACID
A.R. Reizisl~ N.V. Matanina 1, D.A. Shmarov 2. 1 The Central Institute for Epidemiology of the Russian Federation, 2The Research Center for Hematology, Moscow, Russia Background and Aims: Apoptosis plays an important role in the pathogenesis of viral hepatitis (VH), affecting not only hepatocytes but also immune cells. However, the occurrence of apoptosis in peripheral blood lymphocytes (PBL) during the course of VH in children has not been described. The aim of this study was to measure the level of PBL apoptosis during VH in children, and to test its possible correction by ursodeoxycholic acid (UDCA). Methods: An open, randomized, placebo-controlled study encompassed 145 children with VH A, B or C, receiving either UDCA (10 15mg/kg daily) or placebo. Apoptosis was determined by flow cytometry using propidium iodide (PI) staining for hypodiploid cells. Results: The normal level of PBL apoptosis in children was determined (0.77• and found similar to that in adults. The apoptosis was 20 22fold higher at the peak of acute VH A and B (17.1• and 15.3• respectively), and subsequently decreased during early recovery (6.7• 1.4 and 7.3• During the prolonged course of VH A and B apoptosis remained high for 3 4 months (44.7• p <0.05), whereas chronic VH B and C manifested constitutively elevated apoptosis levels (14.08 and 13.8 for VH B and C, respectively). The levels of PBL apoptosis showed significant correlation with the severity of the disease, including elevated bilimbin and ALT levels and viral replication. A significant inverse correlation between apoptosis and leukopenia was observed (r 0.62), suggesting a possible causal relationship between the two phenomena. Both the course of the disease and the apoptosis of PBL were improved by UDCA administration: the apoptosis levels in acute VH A and B were 10.35• in the UDCA group versus 27.05• in the placebo group (p <0.05); in prolonged disease 7.1• versus 44.7• in chronic VH B and C 5.4• versus 13.8• (p <0.001). Conclusions: The apoptosis of PBL is closely correlated with the severity and progression of hepatitis A, B and C in children. Furthermore, the improved course and outcomes of the disease following UDCA treatment are associated with the reduction of PBL apoptosis.
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GLYCOCHENODEOXYCHOLIC ACID AT MODERATE LEVELS INDUCES A P O P T O S I S INDEPENDENT OF FAS/CD95 AND CASPASE 8 IN HUMAN LIVER CELL LINES
C. Rust, C. Bernt, T. Vennegeerts, U. Beuers. Department of Medicine II
Grosshadern, University of Munieh, Germany Background and Aims: Apoptosis induced by hydrophobic bile acids is thought to contribute to liver injury during cholestasis. Ligand-independent activation of the death receptor Fas/CD95 and subsequent activation of caspase 8 by hydrophobic bile acids was shown to initiate bile acidinduced apoptosis in vitro. In cell free systems, hydrophobic bile acids may directly cause mitochondrial damage leading to cytochrome C leakage, a downstream inducer of apoptosis. The aims of our study were to investigate the role of (i) Fas/CD95 and (ii) caspase 8 in human hepatoma cells for the induction of apoptosis by the major human hydrophobic bile acid, glycochenodeoxycholic acid (GCDCA).
Methods: Human Fas-containing HepG2, and Fas-deficient Hep3B and HuH7 hepatoma cell lines were stably transfected with the bile acid transporter Ntcp. Caspase 8 was downregulated by specific small interfering RNA (siRNA) for caspase 8. Caspase 8 activity was also inhibited using the specific inhibitor Z-IETD-FMK. Full length and cleaved caspase 3, 7, 8, and 9 protein levels were determined by immunoblot using specific antibodies. Apoptosis was quantified by measuring caspase-3/7 activity using the fluorogenic substrate Z-DEVD-R110. Results: GCDCA (75mM, 4h) induced apoptosis in Fas-containing HepG2-Ntcp as well as in Fas-deficient Hep3B-Ntcp and HuH7-Ntcp hepatoma cells, whereas no induction of apoptosis was observed in native cell lines lacking Ntcp. Caspase 8 was distinctly downregulated by synthetic caspase 8 siRNA (10 nM) 24 72 h after transfection in HepG2-Ntcp cells, but GCDCA-induced apoptosis was downregulated by only 26% in these caspase 8-deficient cells. In contrast, apoptosis induced by TNFa (50 ng/ml) and actinomycin D (0.2 gg/ml) was downregulated by 79% in the identical experimental setting. Likewise, cleaved forms of caspases 3, 7 and 9 were detected at comparable levels in caspase 8 siRNA transfected and sham-transfected cells treated with GCDCA, but not in those treated with TNFa/actinomycin D. In parallel, the Caspase 8-inhibitor Z-IETDFMK (20 gM) prevented induction of apoptosis in HepG2-Ntcp cells exposed to TNFa/actinomycin D, but not GCDCA. Conclusion: GCDCA at moderate doses induces apoptosis in human hepatoma cell lines by mainly Fas/CD95- and caspase 8-independent mechanisms.
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HUMAN TRANSCRIPTION FACTOR AP-1 IS A MEDIATOR OF BILE ACID-INDUCED LIVER CELL A P O P T O S I S
C. Bernt, T. Vennegeerts, U. Beuers, C. Rust. Department of Medicine II
Grosshadern, University of Munieh, Germany Background and Aims: Bile acid-induced apoptosis is discussed as a key mechanism of liver injury in cholestasis. Transcriptional regulation by bile acids of genes involved in apoptosis is incompletely elucidated. The transcription factor activator protein-1 (AP-1) is a regulator of apoptosis. Thus, the aims of our study were to investigate (i) the efibct of the major human hydrophobic bile acid, glycochenodeoxycholic acid (GCDCA), on expression of AP-1 components and AP-1 activation, and (ii) the role of AP-1 for GCDCA-induced apoptosis in human hepatoma cells. Methods: Transcription of AP-1 proteins cFos and JunB was analyzed by microarray analysis and confrmed by quantitative real-time PCR (RT-PCR) in human HepG2 hepatoma cells stably transfected with the bile acid carrier Ntcp. AP-1 activity was assessed by electromobility gel shift assay (EMSA) and luciferase reporter gene assays. Apoptosis was quantified by measuring caspase-3/7 activity using the fluorogenic substrate Z-DEVD-R110. Results: GCDCA (75 mM, 4h) upregulated cFos 4-fold and JunB 2.5-fold as shown by microarray analysis in HepG2-Ntcp hepatoma cells. RT-PCR confirmed upregulation of cFos-mRNA (26.3-fold) and JunB-mRNA (3.1-fold) by GCDCA. GCDCA (75mM) induced AP-1 activation as determined by EMSA that was most distinct after 30 min. In parallel, AP-1 transcriptional activity increased by 40% after exposure to GCDCA (10 mM, 1 h) as demonstrated by luciferase reporter gene assay. The AP-1 inhibitor, curcumin, dose-dependently reduced (1 25 mM) or completely abolished (50 mM) the apoptotic effect of GCDCA (75 mM) in HepG2Ntcp hepatoma cells. Conclusion: GCDCA significantly enhanced transcription of the human transcription factor AP-1 proteins cFos and JunB and activated AP-1. Inhibition of AP-1 by curcumin antagonized GCDCA-induced apoptosis. Thus, AP-1 (cFos/JunB) may represent a key nuclear mediator of GCDCAinduced liver cell apoptosis.
POSTERS
PAO, through its control of intracellular polyamine levels, seems to be a potential target for therapy.
~-]
THE APOPTOSIS OF PERIPHERAL BLOOD M O N O N U C L E A R CELLS DURING VIRAL HEPATITIS IN CHILDREN, AND ITS CORRECTION BY U R S O D E O X Y C H O L I C ACID
A.R. Reizisl~ N.V. Matanina 1, D.A. Shmarov 2. 1 The Central Institute for Epidemiology of the Russian Federation, 2The Research Center for Hematology, Moscow, Russia Background and Aims: Apoptosis plays an important role in the pathogenesis of viral hepatitis (VH), affecting not only hepatocytes but also immune cells. However, the occurrence of apoptosis in peripheral blood lymphocytes (PBL) during the course of VH in children has not been described. The aim of this study was to measure the level of PBL apoptosis during VH in children, and to test its possible correction by ursodeoxycholic acid (UDCA). Methods: An open, randomized, placebo-controlled study encompassed 145 children with VH A, B or C, receiving either UDCA (10 15mg/kg daily) or placebo. Apoptosis was determined by flow cytometry using propidium iodide (PI) staining for hypodiploid cells. Results: The normal level of PBL apoptosis in children was determined (0.77• and found similar to that in adults. The apoptosis was 20 22fold higher at the peak of acute VH A and B (17.1• and 15.3• respectively), and subsequently decreased during early recovery (6.7• 1.4 and 7.3• During the prolonged course of VH A and B apoptosis remained high for 3 4 months (44.7• p <0.05), whereas chronic VH B and C manifested constitutively elevated apoptosis levels (14.08 and 13.8 for VH B and C, respectively). The levels of PBL apoptosis showed significant correlation with the severity of the disease, including elevated bilimbin and ALT levels and viral replication. A significant inverse correlation between apoptosis and leukopenia was observed (r 0.62), suggesting a possible causal relationship between the two phenomena. Both the course of the disease and the apoptosis of PBL were improved by UDCA administration: the apoptosis levels in acute VH A and B were 10.35• in the UDCA group versus 27.05• in the placebo group (p <0.05); in prolonged disease 7.1• versus 44.7• in chronic VH B and C 5.4• versus 13.8• (p <0.001). Conclusions: The apoptosis of PBL is closely correlated with the severity and progression of hepatitis A, B and C in children. Furthermore, the improved course and outcomes of the disease following UDCA treatment are associated with the reduction of PBL apoptosis.
•
GLYCOCHENODEOXYCHOLIC ACID AT MODERATE LEVELS INDUCES A P O P T O S I S INDEPENDENT OF FAS/CD95 AND CASPASE 8 IN HUMAN LIVER CELL LINES
C. Rust, C. Bernt, T. Vennegeerts, U. Beuers. Department of Medicine II
Grosshadern, University of Munieh, Germany Background and Aims: Apoptosis induced by hydrophobic bile acids is thought to contribute to liver injury during cholestasis. Ligand-independent activation of the death receptor Fas/CD95 and subsequent activation of caspase 8 by hydrophobic bile acids was shown to initiate bile acidinduced apoptosis in vitro. In cell free systems, hydrophobic bile acids may directly cause mitochondrial damage leading to cytochrome C leakage, a downstream inducer of apoptosis. The aims of our study were to investigate the role of (i) Fas/CD95 and (ii) caspase 8 in human hepatoma cells for the induction of apoptosis by the major human hydrophobic bile acid, glycochenodeoxycholic acid (GCDCA).
Methods: Human Fas-containing HepG2, and Fas-deficient Hep3B and HuH7 hepatoma cell lines were stably transfected with the bile acid transporter Ntcp. Caspase 8 was downregulated by specific small interfering RNA (siRNA) for caspase 8. Caspase 8 activity was also inhibited using the specific inhibitor Z-IETD-FMK. Full length and cleaved caspase 3, 7, 8, and 9 protein levels were determined by immunoblot using specific antibodies. Apoptosis was quantified by measuring caspase-3/7 activity using the fluorogenic substrate Z-DEVD-R110. Results: GCDCA (75mM, 4h) induced apoptosis in Fas-containing HepG2-Ntcp as well as in Fas-deficient Hep3B-Ntcp and HuH7-Ntcp hepatoma cells, whereas no induction of apoptosis was observed in native cell lines lacking Ntcp. Caspase 8 was distinctly downregulated by synthetic caspase 8 siRNA (10 nM) 24 72 h after transfection in HepG2-Ntcp cells, but GCDCA-induced apoptosis was downregulated by only 26% in these caspase 8-deficient cells. In contrast, apoptosis induced by TNFa (50 ng/ml) and actinomycin D (0.2 gg/ml) was downregulated by 79% in the identical experimental setting. Likewise, cleaved forms of caspases 3, 7 and 9 were detected at comparable levels in caspase 8 siRNA transfected and sham-transfected cells treated with GCDCA, but not in those treated with TNFa/actinomycin D. In parallel, the Caspase 8-inhibitor Z-IETDFMK (20 gM) prevented induction of apoptosis in HepG2-Ntcp cells exposed to TNFa/actinomycin D, but not GCDCA. Conclusion: GCDCA at moderate doses induces apoptosis in human hepatoma cell lines by mainly Fas/CD95- and caspase 8-independent mechanisms.
•
HUMAN TRANSCRIPTION FACTOR AP-1 IS A MEDIATOR OF BILE ACID-INDUCED LIVER CELL A P O P T O S I S
C. Bernt, T. Vennegeerts, U. Beuers, C. Rust. Department of Medicine II
Grosshadern, University of Munieh, Germany Background and Aims: Bile acid-induced apoptosis is discussed as a key mechanism of liver injury in cholestasis. Transcriptional regulation by bile acids of genes involved in apoptosis is incompletely elucidated. The transcription factor activator protein-1 (AP-1) is a regulator of apoptosis. Thus, the aims of our study were to investigate (i) the efibct of the major human hydrophobic bile acid, glycochenodeoxycholic acid (GCDCA), on expression of AP-1 components and AP-1 activation, and (ii) the role of AP-1 for GCDCA-induced apoptosis in human hepatoma cells. Methods: Transcription of AP-1 proteins cFos and JunB was analyzed by microarray analysis and confrmed by quantitative real-time PCR (RT-PCR) in human HepG2 hepatoma cells stably transfected with the bile acid carrier Ntcp. AP-1 activity was assessed by electromobility gel shift assay (EMSA) and luciferase reporter gene assays. Apoptosis was quantified by measuring caspase-3/7 activity using the fluorogenic substrate Z-DEVD-R110. Results: GCDCA (75 mM, 4h) upregulated cFos 4-fold and JunB 2.5-fold as shown by microarray analysis in HepG2-Ntcp hepatoma cells. RT-PCR confirmed upregulation of cFos-mRNA (26.3-fold) and JunB-mRNA (3.1-fold) by GCDCA. GCDCA (75mM) induced AP-1 activation as determined by EMSA that was most distinct after 30 min. In parallel, AP-1 transcriptional activity increased by 40% after exposure to GCDCA (10 mM, 1 h) as demonstrated by luciferase reporter gene assay. The AP-1 inhibitor, curcumin, dose-dependently reduced (1 25 mM) or completely abolished (50 mM) the apoptotic effect of GCDCA (75 mM) in HepG2Ntcp hepatoma cells. Conclusion: GCDCA significantly enhanced transcription of the human transcription factor AP-1 proteins cFos and JunB and activated AP-1. Inhibition of AP-1 by curcumin antagonized GCDCA-induced apoptosis. Thus, AP-1 (cFos/JunB) may represent a key nuclear mediator of GCDCAinduced liver cell apoptosis.