- Email: [email protected]
[435] INTRACELLULAR LOCATION AND SHIFT OF HEPATITIS B VIRUS CORE PROTEIN IN HepG2.2.15 CELL-LINE
POSTERS
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the antioxidant nature of FK506, which blocks oxidative stress production in hepatocytes expressing the core protein both in vitro and in vivo. Conclusions: FK506 reversed the effect of the core protein in the pathogenesis of HCV infection. This result may provide a new therapeutic aid for chronic hepatitis C, in which oxidative stress and metabolic abnormalities in lipid and glucose contribute to liver pathogenesis.
14341 MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN (MTP) GENE POLYMORPHISM AND LIPID METABOLISM IN HCV-INFECTED PATIENTS S. Mirandola', M. Marcolongo', S. Realdon',2, F. Stickel', C. Datz4, E. Aigner4, F, Noventa3, F. Dal Pero', L. Franceschini', G. Bortoletto', M. Gerotto' , A. Alberti' VIMM, Padovu; 2Department ofSzirgical und Gastroenterologic Sciences, University of Padova, Padova; 3Departnwnt of' Clinical and Experinzental Medicine, liniversiw of' Padova, Padova, Italy; 4Krankenhaus Oberndoyf; Salzburg, Austria; "Institute of Clinical Pharmacology, Univer~sih~ of Bern, Bern, Switzerlund E-mail: [email protected] s3.
Aims and Methods: Microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of apoB- lipoproteins in liver and intestine. Genetic variability can modulate MTP gene expression and its lipid transport activity leading to an impairment of cholesterol, apoB serum levels and liver steatosis. Recently we have demonstrated that virus mediated MTP inhibition is involved in liver steatosis in HCV infection particularly with HCV-3. To investigate the influence of genetic functional polymorphism in the promoter region of the MTP gene (-493 G/T) on the development and degree of liver steatosis during HCV infection, 301 HCV infected patients were genotyped for the MTP -493 GIT polymorphism. Results: Results were correlated with virological, biochemical and clinical parameters and with levels of serum lipids. The frequency of homozygosis for the MTP -493 T variant (that is associated with increased MTP gene transcription) was 0.06 and the allele frequency of MTP -493 T was 0.26. By logistic regression multivariate analysis HCV-3 was the most influent factor for severe steatosis (OR= 5.4; 95'X C1: 2.7-10.8; P=0.0001) while the MTP genotype had no effect (OR=0.8; 95'XCI: 0.27-2.36; P=0.68). HCV infected patients with MTP genotype T/T had higher HDL cholesterol (65.6&14.6 vs 56.1f16.2 mg/dL; P=0.003) and ApoAl (1.80f0.3 vs 1.60&0.3g/L; P=0.005) compared to those with G/G and GIT genotypes. There was also a trend for higher total cholesterol, LDL and ApoB in TT patients. When patients were divided according to the HCV genotype, G/G and GIT (MTP) patients infected with HCV-3 had significant lower serum levels of total cholesterol (P <0.001), LDL (P<0.001), HDL (P=0.007), ApoB (P<0.001) and ApoAl (P<0.001) compared to GIG-GIT (MTP) patients infected with other HCV genotypes (HCV I , HCV 2, HCV 4). Conclusions: These results suggest that MTP gene polymorphism does not play a significant role in the development of HCV related steatosis while HCV-3 has a pivotal role. However modulation of ApoAl and HDL levels by MTP polymorphism deserves further investigation during HCV infection
14351 INTRACELLULAR LOCATION AND SHIFT OF HEPATITIS B VIRUS CORE PROTEIN IN HepG2.2.15 CELL-LINE X.B. Pan, L. Wei, G.Y. Wu, Y. Gao. Hepatology Institute, Peking Universih~People 's Hospital, Beijing, China E-mail: [email protected] Background and Aims: The hepatitis B virus core protein (HBcAg) has been found in nucleus, cytoplasm, or both of hepatocytes transfected with HBVDNA. Whether intact core particle could through the nuclear pore and the mechanism that regulates the nuclear localization of the core protein remained unclear. Here, hepatocyte inducer dimethyl sulphoxide
(DMSO) and the heteroaryldihydropyrimidine (HAP-I ), which interferes the assembly of core particle, were added into HepG2.2.15 cell culture system to observe the potential changes of HBcAg location and expression. Methods: To synchronize cell cycle, the established clonal cell line HepG2.2.15 was cultured with serum-free medium or aphidicolin for 2 days, and cell cycle was initiated with medium containing 20% FBS. During GO phase, 2% DMSO and/or 1 pM HAP-1 were added into medium for 12 hours, 24 hours or 2 days. HBcAg was detected by fluorescent immunocytochemical method and analyzed by using image and the nucleus was labeled by DAPI. lntranuclear (covaluently closed circular DNA) cccDNA was detected by southern blot. Results: The HBcAg was mostly expressed in cytoplasm in HepG2.2.15, except for the cells at phase ofmitosis, in which HBcAg could be observed both in cytoplasm and nucleus. Using of DMSO slightly enhanced the expression of HBcAg in nucleus, whereas HBcAg was still mainly expressed in cytoplasm and no obvious signal of intranuclear cccDNA could be detected. In HAP-I group, the signal intensity of HBcAg increasingly enhanced in nucleus and weakened in cytoplasm, whereas the intensity in nucleus was still no more than that in cytoplasm, and the signal of cccDNA could be detected. When the HepG2.2.15 cells were treated with DMSO and HAP-I for 24h, we found that the HBcAg mainly expressed in nucleus and strong cccDNA signal could be detected. Conclusion: In HepG2.2.15 cells, the HBcAg was assembled as formation of core particle in cytoplasm and could not penetrate the nuclear membrane. HAP-I interfered the assembly of core particle and render to more dissociative core proteins in cytoplasm. DMSO promotes the nuclear entry of dissociate core protein but can not lead to the core particle nuclear entry.
14361 THE HEPATITIS C VIRUS CORE PROTEIN OF GENOTYPES 3a AND 1b DOWN-REGULATES INSULIN RECEPTOR SUBSTRATE 1 VIA GENOTYPE-SPECIFIC MECHANISMS
V. Pazienza' , S. Clement', P. Pugnale' , S. Conzelman' , M. Foti2, A. Mangia3, F. Negro',4. 'Dioisions of Clinical Pathology, Unioersiiy Hospital, Geneou; 'Departnzent of Medical Genetics and Developnzent, University of Geneva; 'Division of Gastroenterology, Ospedale; Gastroenterology and Hepatology, University Hospital, Geneva, Switzerland E-mail: [email protected] Background and Aims: Both molecular and clinical evidence support a link between the hepatitis C virus (HCV) infection and insulin resistance. We examined the in vitro interaction between the HCV core protein of genotypes 3a and l b with the insulin signaling pathway. Methods: We measured the expression levels of insulin receptor substrate 1 (TRS-I), TRS-2 and other factors involved in the insulin signal transduction in a human hepatoma cell line (Huh-7) transiently expressing the HCV core protein of genotypes 3a or I b by both molecular biology and biochemical techniques. Results: IRS-1 (but not 1RS-2) protein level was significantly reduced in Huh-7 expressing the core protein of both genotype 3a (P= 0.0067) and 1 b (P= 0.04) as compared to cells transfected with the empty pTRES2EGFP vector. However, while the core protein of genotype 3a promoted IRS-1 degradation through the downregulation of peroxisome proliferatoractivated receptor y (PPAR:,) and by upregulating the suppressor of cytolcine signal 7 (SOCS-7), the core protein of genotype 1 b activated the mammalian target of rapamycin (mTOR). These findings were confirmed by using agonists for PPARy (rosiglitazone) or short interfering RNAs for SOCS-7. Conclusions: Despite the little sequence divergence of the HCV core proteins of genotypes 3a and I b, the two proteins appear to interfere with the insulin signaling pathway using genotype-specific mechanisms. This coupled with clinical mounting evidence, suggest an avolutionary advantage for HCV to maintain an insulin resistance state.
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the antioxidant nature of FK506, which blocks oxidative stress production in hepatocytes expressing the core protein both in vitro and in vivo. Conclusions: FK506 reversed the effect of the core protein in the pathogenesis of HCV infection. This result may provide a new therapeutic aid for chronic hepatitis C, in which oxidative stress and metabolic abnormalities in lipid and glucose contribute to liver pathogenesis.
14341 MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN (MTP) GENE POLYMORPHISM AND LIPID METABOLISM IN HCV-INFECTED PATIENTS S. Mirandola', M. Marcolongo', S. Realdon',2, F. Stickel', C. Datz4, E. Aigner4, F, Noventa3, F. Dal Pero', L. Franceschini', G. Bortoletto', M. Gerotto' , A. Alberti' VIMM, Padovu; 2Department ofSzirgical und Gastroenterologic Sciences, University of Padova, Padova; 3Departnwnt of' Clinical and Experinzental Medicine, liniversiw of' Padova, Padova, Italy; 4Krankenhaus Oberndoyf; Salzburg, Austria; "Institute of Clinical Pharmacology, Univer~sih~ of Bern, Bern, Switzerlund E-mail: [email protected] s3.
Aims and Methods: Microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of apoB- lipoproteins in liver and intestine. Genetic variability can modulate MTP gene expression and its lipid transport activity leading to an impairment of cholesterol, apoB serum levels and liver steatosis. Recently we have demonstrated that virus mediated MTP inhibition is involved in liver steatosis in HCV infection particularly with HCV-3. To investigate the influence of genetic functional polymorphism in the promoter region of the MTP gene (-493 G/T) on the development and degree of liver steatosis during HCV infection, 301 HCV infected patients were genotyped for the MTP -493 GIT polymorphism. Results: Results were correlated with virological, biochemical and clinical parameters and with levels of serum lipids. The frequency of homozygosis for the MTP -493 T variant (that is associated with increased MTP gene transcription) was 0.06 and the allele frequency of MTP -493 T was 0.26. By logistic regression multivariate analysis HCV-3 was the most influent factor for severe steatosis (OR= 5.4; 95'X C1: 2.7-10.8; P=0.0001) while the MTP genotype had no effect (OR=0.8; 95'XCI: 0.27-2.36; P=0.68). HCV infected patients with MTP genotype T/T had higher HDL cholesterol (65.6&14.6 vs 56.1f16.2 mg/dL; P=0.003) and ApoAl (1.80f0.3 vs 1.60&0.3g/L; P=0.005) compared to those with G/G and GIT genotypes. There was also a trend for higher total cholesterol, LDL and ApoB in TT patients. When patients were divided according to the HCV genotype, G/G and GIT (MTP) patients infected with HCV-3 had significant lower serum levels of total cholesterol (P <0.001), LDL (P<0.001), HDL (P=0.007), ApoB (P<0.001) and ApoAl (P<0.001) compared to GIG-GIT (MTP) patients infected with other HCV genotypes (HCV I , HCV 2, HCV 4). Conclusions: These results suggest that MTP gene polymorphism does not play a significant role in the development of HCV related steatosis while HCV-3 has a pivotal role. However modulation of ApoAl and HDL levels by MTP polymorphism deserves further investigation during HCV infection
14351 INTRACELLULAR LOCATION AND SHIFT OF HEPATITIS B VIRUS CORE PROTEIN IN HepG2.2.15 CELL-LINE X.B. Pan, L. Wei, G.Y. Wu, Y. Gao. Hepatology Institute, Peking Universih~People 's Hospital, Beijing, China E-mail: [email protected] Background and Aims: The hepatitis B virus core protein (HBcAg) has been found in nucleus, cytoplasm, or both of hepatocytes transfected with HBVDNA. Whether intact core particle could through the nuclear pore and the mechanism that regulates the nuclear localization of the core protein remained unclear. Here, hepatocyte inducer dimethyl sulphoxide
(DMSO) and the heteroaryldihydropyrimidine (HAP-I ), which interferes the assembly of core particle, were added into HepG2.2.15 cell culture system to observe the potential changes of HBcAg location and expression. Methods: To synchronize cell cycle, the established clonal cell line HepG2.2.15 was cultured with serum-free medium or aphidicolin for 2 days, and cell cycle was initiated with medium containing 20% FBS. During GO phase, 2% DMSO and/or 1 pM HAP-1 were added into medium for 12 hours, 24 hours or 2 days. HBcAg was detected by fluorescent immunocytochemical method and analyzed by using image and the nucleus was labeled by DAPI. lntranuclear (covaluently closed circular DNA) cccDNA was detected by southern blot. Results: The HBcAg was mostly expressed in cytoplasm in HepG2.2.15, except for the cells at phase ofmitosis, in which HBcAg could be observed both in cytoplasm and nucleus. Using of DMSO slightly enhanced the expression of HBcAg in nucleus, whereas HBcAg was still mainly expressed in cytoplasm and no obvious signal of intranuclear cccDNA could be detected. In HAP-I group, the signal intensity of HBcAg increasingly enhanced in nucleus and weakened in cytoplasm, whereas the intensity in nucleus was still no more than that in cytoplasm, and the signal of cccDNA could be detected. When the HepG2.2.15 cells were treated with DMSO and HAP-I for 24h, we found that the HBcAg mainly expressed in nucleus and strong cccDNA signal could be detected. Conclusion: In HepG2.2.15 cells, the HBcAg was assembled as formation of core particle in cytoplasm and could not penetrate the nuclear membrane. HAP-I interfered the assembly of core particle and render to more dissociative core proteins in cytoplasm. DMSO promotes the nuclear entry of dissociate core protein but can not lead to the core particle nuclear entry.
14361 THE HEPATITIS C VIRUS CORE PROTEIN OF GENOTYPES 3a AND 1b DOWN-REGULATES INSULIN RECEPTOR SUBSTRATE 1 VIA GENOTYPE-SPECIFIC MECHANISMS
V. Pazienza' , S. Clement', P. Pugnale' , S. Conzelman' , M. Foti2, A. Mangia3, F. Negro',4. 'Dioisions of Clinical Pathology, Unioersiiy Hospital, Geneou; 'Departnzent of Medical Genetics and Developnzent, University of Geneva; 'Division of Gastroenterology, Ospedale; Gastroenterology and Hepatology, University Hospital, Geneva, Switzerland E-mail: [email protected] Background and Aims: Both molecular and clinical evidence support a link between the hepatitis C virus (HCV) infection and insulin resistance. We examined the in vitro interaction between the HCV core protein of genotypes 3a and l b with the insulin signaling pathway. Methods: We measured the expression levels of insulin receptor substrate 1 (TRS-I), TRS-2 and other factors involved in the insulin signal transduction in a human hepatoma cell line (Huh-7) transiently expressing the HCV core protein of genotypes 3a or I b by both molecular biology and biochemical techniques. Results: IRS-1 (but not 1RS-2) protein level was significantly reduced in Huh-7 expressing the core protein of both genotype 3a (P= 0.0067) and 1 b (P= 0.04) as compared to cells transfected with the empty pTRES2EGFP vector. However, while the core protein of genotype 3a promoted IRS-1 degradation through the downregulation of peroxisome proliferatoractivated receptor y (PPAR:,) and by upregulating the suppressor of cytolcine signal 7 (SOCS-7), the core protein of genotype 1 b activated the mammalian target of rapamycin (mTOR). These findings were confirmed by using agonists for PPARy (rosiglitazone) or short interfering RNAs for SOCS-7. Conclusions: Despite the little sequence divergence of the HCV core proteins of genotypes 3a and I b, the two proteins appear to interfere with the insulin signaling pathway using genotype-specific mechanisms. This coupled with clinical mounting evidence, suggest an avolutionary advantage for HCV to maintain an insulin resistance state.