Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 119
Filter
Add more filters










Publication year range
1.
Toxicol Appl Pharmacol ; 254(3): 256-66, 2011 Aug 01.
Article in English | MEDLINE | ID: mdl-21569786

ABSTRACT

In this study, our goal was to develop an efficient in situ test adapted to screen hepatotoxicity of various chemicals, a process which remains challenging during the early phase of drug development. The test was based on functional human hepatocytes using the HepaRG cell line, and automation of quantitative fluorescence microscopy coupled with automated imaging analysis. Differentiated HepaRG cells express most of the specific liver functions at levels close to those found in primary human hepatocytes, including detoxifying enzymes and drug transporters. A triparametric analysis was first used to evaluate hepatocyte purity and differentiation status, mainly detoxication capacity of cells before toxicity testing. We demonstrated that culturing HepaRG cells at high density maintained high hepatocyte purity and differentiation level. Moreover, evidence was found that isolating hepatocytes from 2-week-old confluent cultures limited variations associated with an ageing process occurring over time in confluent cells. Then, we designed a toxicity test based on detection of early mitochondrial depolarisation associated with permeability transition (MPT) pore opening, using JC-1 as a metachromatic fluorescent dye. Maximal dye dimerization that would have been strongly hampered by efficient efflux due to the active, multidrug-resistant (MDR) pump was overcome by coupling JC-1 with the MDR inhibitor verapamil. Specificity of this test was demonstrated and its usefulness appeared directly dependent on conditions supporting hepatic cell competence. This new hepatotoxicity test adapted to automated, image-based detection should be useful to evaluate the early MPT event common to cell apoptosis and necrosis and simultaneously to detect involvement of the multidrug resistant pump with target drugs in a human hepatocyte environment.


Subject(s)
Benzimidazoles/analysis , Carbocyanines/analysis , Fluorescent Dyes/analysis , Hepatocytes/chemistry , Image Processing, Computer-Assisted/methods , Mitochondria/chemistry , Toxicity Tests/methods , ATP Binding Cassette Transporter, Subfamily B/analysis , ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Line, Tumor , Cells, Cultured , Coculture Techniques , Drug-Related Side Effects and Adverse Reactions/diagnosis , Drug-Related Side Effects and Adverse Reactions/metabolism , Hepatocytes/drug effects , Humans , Image Processing, Computer-Assisted/standards , Microscopy, Fluorescence/methods , Mitochondria/drug effects , Toxicity Tests/standards , Verapamil/analysis , Verapamil/pharmacology
2.
J Gen Virol ; 90(Pt 1): 127-35, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19088281

ABSTRACT

The recently described hepatic cell line HepaRG is the sole hepatoma cell line susceptible to hepatitis B virus (HBV) infection. It provides a unique tool for investigating some unresolved issues of the virus' biology, particularly the formation of the viral mini-chromosome believed to be responsible for the persistence of infection. In this study, we characterized the main features of HBV infection: it is restricted to a subpopulation of differentiated hepatocyte-like cells that express albumin as a functional marker and represents around 10 % of all differentiated HepaRG cells. Infection may persist for more than 100 days in cells maintained at the differentiated state. Even though infected cells continued to produce infectious viral particles, very limited or no spreading of infection was observed. Low genetic variation was also observed in the viral DNA from viruses found in the supernatant of infected cells, although this cannot explain the lack of reinfection. HBV infection of HepaRG cells appears to be a very slow process: viral replication starts at around day 8 post-infection and reaches a maximum at day 13. Analysis of viral DNA showed slow and inefficient conversion of the input relaxed circular DNA into covalently closed circular (CCC) DNA, but no further amplification. Continuous lamivudine treatment inhibited viral replication, but neither prevented viral infection nor initial formation of CCC DNA. In conclusion, HBV infection in differentiated HepaRG cells is characterized by long-term persistence without a key feature of hepadnaviruses, the so-called 'CCC DNA amplification' described in the duck hepatitis B model.


Subject(s)
DNA, Circular/metabolism , DNA, Viral/metabolism , Hepatitis B virus/physiology , Virus Replication , Cell Line, Tumor , Hepatocytes/virology , Humans
3.
J Virol ; 75(23): 11565-72, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11689638

ABSTRACT

Infection by hepatitis B virus (HBV) is mainly restricted to humans. This species specificity is likely determined at the early phase of the viral life cycle. Since the envelope proteins are the first viral factors to interact with the cell, they represent attractive candidates for controlling the HBV host range. To investigate this assumption, we took advantage of the recent discovery of a second virus belonging to the primate Orthohepadnavirus genus, the woolly monkey HBV (WMHBV). A recombinant plasmid was constructed for the expression of all WMHBV envelope proteins. In additional constructs, N-terminal sequences of the WMHBV large envelope protein were substituted for their homologous HBV counterparts. All wild-type and chimeric WMHBV surface proteins were properly synthesized by transfected human hepatoma cells, and they were competent to replace the original HBV proteins for the production of complete viral particles. The resulting pseudotyped virions were evaluated for their infectious capacity on human hepatocytes in primary culture. Virions pseudotyped with wild-type WMHBV envelope proteins showed a significant loss of infectivity. By contrast, infectivity was completely restored when the first 30 residues of the large protein originated from HBV. Analysis of smaller substitutions within this domain limited the most important region to a stretch of only nine amino acids. Reciprocally, replacement of this motif by WMHBV residues in the context of the HBV L protein significantly reduced infectivity of HBV. Hence this short region of the L protein contributes to the host range of HBV.


Subject(s)
Hepatitis B virus/physiology , Viral Envelope Proteins/physiology , Amino Acid Sequence , Animals , Cell Line , Humans , Molecular Sequence Data , Sequence Homology, Amino Acid , Species Specificity , Viral Envelope Proteins/chemistry , Virion
4.
Dev Dyn ; 222(2): 206-17, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11668598

ABSTRACT

Annexin (Anx) 1, 2, 5, and 6 expressions were determined at the transcriptional and translational levels in the rat hepatocytes from gestational day 15 to postnatal day 17. Dramatic shifts were observed in Anx 1 and 2 levels, which peaked at day 1 and gestational day 20, respectively, and reached low levels thereafter. However, Anx 5 and 6 rates were more constant. Prenatal administration of dexamethasone (dex) resulted in a decrease of Anx 1 mRNA levels, and a strong increase in Anx 2 mRNA contents. In adult hepatocytes cultured in the presence of EGF or HGF, Anx 1 and 2 expressions resumed. By immunohistochemistry, Anx 1 was detected only in the cytoplasm of hepatocytes of 1- to 3-day-old rats, Anx 2 and 6 both exhibited a redistribution from the cytoplasm toward the plasma membrane, and Anx 5 was present in the nucleus, cytoplasm, and plasma membrane. Thus, Anx 1, 2, 5, and 6 have individual modes of expression and localization in the differentiating hepatocytes, where they might play unique roles at well defined phases of liver ontogeny.


Subject(s)
Annexins/genetics , Gene Expression Regulation, Developmental/physiology , Hepatocytes/cytology , Liver/embryology , Animals , Annexin A1/genetics , Annexin A2/genetics , Annexin A5/genetics , Annexin A6/genetics , Cell Differentiation , Cells, Cultured , Dexamethasone/pharmacology , Epidermal Growth Factor/pharmacology , Female , Gene Expression Regulation, Developmental/drug effects , Glucocorticoids/pharmacology , Hepatocyte Growth Factor/pharmacology , Liver/cytology , Male , Pregnancy , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley
5.
Biochem Pharmacol ; 61(10): 1293-303, 2001 May 15.
Article in English | MEDLINE | ID: mdl-11322933

ABSTRACT

In this study, we investigated the combined effects of EGF and collagen I gel on the phenotype of cultured rat hepatocytes and we focussed our investigations on the regulation of xenobiotic-mediated induction of CYP, cell cycle progression and activation of capases 8 and 3. We found that EGF, added to basal culture medium or phenobarbital (3.2 mM) containing medium, provoked a moderate decrease of CYP1A1 and CYP2B1/2 activities. However, EGF did not exert any inhibitory effect on 3-methylcholantrene (5 microM) and beta-naphtoflavone (25 microM) induction of CYP1A1 activities. In collagen gel sandwich cultures, hepatocytes remained arrested in mid-G1 phase of the cell cycle, even in the presence of EGF. In conventional primary cultures, caspases 8 and 3 were activated at 3 and 5 days after plating respectively. In collagen gel sandwich cultures, we found that neither collagen I nor EGF prevented activation of caspase 8 while collagen I gel inhibited activation of caspase 3, preventing spontaneous apoptosis of cultured rat hepatocytes. In contrast, EGF transiently increased caspase 3 activity at day 1 after plating. Altogether, our data demonstrate that collagen I gel triggers intracellular signals which strongly affect cultured hepatocyte phenotype, leading to a cell cycle arrest in G1 phase and long-term survival through the inhibition of caspase 3 activation and that EGF-free medium improves survival and liver-specific gene expression in hepatocytes maintained in collagen I gel sandwich cultures.


Subject(s)
Caspases/metabolism , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP2B1/metabolism , Epidermal Growth Factor/pharmacology , Hepatocytes/drug effects , Xenobiotics/pharmacology , Animals , Caspase 3 , Cell Cycle/drug effects , Cell Survival/drug effects , Collagen/pharmacology , Cytochrome P-450 CYP1A1/drug effects , Cytochrome P-450 CYP2B1/drug effects , DNA Replication/drug effects , Drug Interactions , Enzyme Activation , Enzyme Induction/drug effects , Hepatocytes/enzymology , In Vitro Techniques , Male , Rats , Rats, Sprague-Dawley , Serum Albumin/metabolism
6.
Mol Biol Cell ; 12(3): 725-38, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11251083

ABSTRACT

Cell shape plays a role in cell growth, differentiation, and death. Herein, we used the hepatocyte, a normal, highly differentiated cell characterized by a long G1 phase, to understand the mechanisms that link cell shape to growth. First, evidence was provided that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade is a key transduction pathway controlling the hepatocyte morphology. MEK2/ERK2 activation in early G1 phase did not lead to cell proliferation but induced cell shape spreading and demonstration was provided that this MAPK-dependent spreading was required for reaching G1/S transition and DNA replication. Moreover, epidermal growth factor (EGF) was found to control this morphogenic signal in addition to its mitogenic effect. Thus, blockade of cell spreading by cytochalasin D or PD98059 treatment resulted in inhibition of EGF-dependent DNA replication. Our data led us to assess the first third of G1, is exclusively devoted to the growth factor-dependent morphogenic events, whereas the mitogenic signal occurred at only approximately mid-G1 phase. Moreover, these two growth factor-related sequential signaling events involved successively activation of MEK2-ERK2 and then MEK1/2-ERK1/2 isoforms. In addition, we demonstrated that inhibition of extracellular matrix receptor, such as integrin beta1 subunit, leads to cell arrest in G1, whereas EGF was found to up-regulated integrin beta1 and fibronectin in a MEK-ERK-dependent manner. This process in relation to cytoskeletal reorganization could induce hepatocyte spreading, making them permissive for DNA replication. Our results provide new insight into the mechanisms by which a growth factor can temporally control dual morphogenic and mitogenic signals during the G1 phase.


Subject(s)
Cell Size/drug effects , Cell Size/physiology , Epidermal Growth Factor/pharmacology , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , S Phase/drug effects , S Phase/physiology , Animals , Base Sequence , Cells, Cultured , DNA Primers/genetics , Enzyme Activation/drug effects , Female , Fibronectins/genetics , Flavonoids/pharmacology , G1 Phase/drug effects , G1 Phase/physiology , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/metabolism , In Vitro Techniques , Integrin beta1/genetics , Liver Regeneration/drug effects , Liver Regeneration/physiology , Phosphorylation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Signal Transduction/physiology , Up-Regulation/drug effects
7.
Eur J Biochem ; 268(5): 1448-59, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11231298

ABSTRACT

The BC2 cell line derived from the human hepatocarcinoma, HGB, undergoes a spontaneous sharp differentiation process in culture as it becomes confluent, remains stably differentiated for several weeks, and may return to proliferation thereafter under appropriate density conditions. The relevance of the line as an hepatic model has been evaluated. Cells synthesize a large number of plasma proteins, and rates of glycogen and urea synthesis increase with time of confluency and become sensitive to insulin, reflecting the process of differentiation. Differentiated BC2 cells express the most relevant cytochrome P-450 (CYP) isozyme activities (CYP1A1/2, 2A6, 2B6, 2C9, 2E1, and 3A4) and conjugating enzymes (glutathione S-transferase and UDP-glucuronyltransferase) and also respond to model inducers. Methylcholanthrene induced an increase in CYP1A1/2 enzyme activity (eightfold), phenobarbital induced CYP2B6 activity (1.7-fold), and dexamethasone induced CYP3A4 activity (fivefold). In parallel, expression of the most relevant liver-enriched transcription factors, HNF-4, HNF-1, C/EBP-alpha and C/EBP-beta mRNAs, was significantly increased in differentiated cultures. This increase was largest in HNF-1 and HNF-4, which supports the idea that a redifferentiation process towards the hepatic phenotype takes place. BC2 is an hepatic cell line that is able to express most hepatic functions, especially the drug-biotransformation function, far more efficiently than any previously described human hepatoma cell line.


Subject(s)
Biotransformation , Cell Differentiation , Gene Expression Regulation, Neoplastic , Liver Neoplasms/metabolism , Albumins/biosynthesis , Ammonia/metabolism , Cell Count , Cell Division , Cell Size , Cytochrome P-450 Enzyme System/biosynthesis , Cytochrome P-450 Enzyme System/metabolism , DNA, Neoplasm/analysis , DNA, Neoplasm/biosynthesis , Enzyme Induction , Glutathione/metabolism , Glycogen/metabolism , Humans , Liver Neoplasms/enzymology , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Organ Specificity , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/genetics , Tumor Cells, Cultured , Urea/metabolism
8.
J Hepatol ; 34(1): 108-13, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11211885

ABSTRACT

BACKGROUNDS/AIMS: The effects of iron-depletion on hepatitis B virus (HBV) replication were examined in HepG2.2.15 cells. METHODS: Proliferating cells were iron-depleted with desferrioxamine (DFO), at 20 or 100 microM for 48 h. Levels of viral mRNAs, cytoplasmic DNA replicative intermediates and virion production were examined. A comparative study was performed with hydroxyurea, a specific inhibitor of ribonucleotide reductase. RESULTS: In desferrioxamine treated cells, virion production is dramatically decreased, while viral replicative intermediates accumulate in the cytoplasm. DFO, like hydroxyurea, blocks cell cycle progression in the G1/S transition or S phase with a corresponding 2-fold increase of viral mRNAs. As expected, hydroxyurea leads to a strong reduction of virion production associated with low levels of intracellular replicative intermediates. CONCLUSIONS: These results strongly suggest that iron depletion affects the HBV life cycle indirectly through the cell cycle arrest and directly through the inhibition of the viral DNA secretion. They also indicate the need to re-evaluate with caution the iron depletion protocols on HBV infected patients since a decrease of viral markers in the serum following iron-depletion may not reflect a decrease of viral replicative forms, but on the contrary, could be associated with active viral DNA synthesis.


Subject(s)
DNA, Viral/analysis , Deferoxamine/pharmacology , Hepatitis B virus/physiology , Iron/physiology , Virus Replication , Cell Cycle/drug effects , Cell Line , DNA, Viral/biosynthesis , Humans , Hydroxyurea/pharmacology , RNA, Messenger/analysis , RNA, Viral/analysis
9.
Genomics ; 67(1): 40-7, 2000 Jul 01.
Article in English | MEDLINE | ID: mdl-10945468

ABSTRACT

F-box proteins are critical components of the SCF ubiquitin-protein ligase complex and are involved in substrate recognition and recruitment for ubiquitination and consequent degradation by the proteasome. We have isolated cDNAs encoding a further 10 mammalian F-box proteins. Five of them (FBL3 to FBL7) share structural similarities with Skp2 and contain C-terminal leucine-rich repeats. The other 5 proteins have different putative protein-protein interaction motifs. Specifically, FBS and FBWD4 proteins contain Sec7 and WD40-repeat domains, respectively. The C-terminal region of FBA shares similarity with bacterial protein ApaG while FBG2 shows homology with the F-box protein NFB42. The marked differences in F-box gene expression in human tissues suggest their distinct role in ubiquitin-dependent protein degradation.


Subject(s)
Multigene Family/genetics , Peptide Synthases/genetics , Amino Acid Motifs , Amino Acid Sequence , Animals , Caenorhabditis elegans , Cloning, Molecular , Databases, Factual , Humans , Leucine Zippers , Molecular Sequence Data , Multigene Family/physiology , Peptide Synthases/classification , Peptide Synthases/metabolism , Proteins/chemistry , Proteins/classification , Proteins/genetics , Rats , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Tissue Distribution , Ubiquitins/metabolism , Yeasts
10.
Am J Pathol ; 157(2): 537-48, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10934156

ABSTRACT

gamma-Glutamyl transpeptidase (GGT), a major enzyme of glutathione (GSH) homeostasis, is often used as a biliary marker to follow the differentiation of hepatic precursor cells. The expression of the GGT gene is driven by different promoters and yields multiple mRNAs, depending on the cell type or the stage of differentiation. In the present study, we analyzed the GGT mRNA expression pattern by quantitative reverse transcriptase-polymerase chain reaction or by in situ hybridization i) in the liver, in vivo, at early stages of development; ii) in oval cells, which proliferate and differentiate into hepatocytes in response to galactosamine injury in vivo; and finally, iii) during hepatoblast differentiation, in vitro. We show that GGT gene transcription originates from promoters P3, P4, and P5 in rat hepatic precursor cells. Differentiation of these cells induces profound alterations in GGT gene expression, leading to extinction of promoters P4 and P5, when they differentiate into the hepatocytic pathway, and to extinction of promoters P3 and P5 when they differentiate into the biliary pathway. This diversity in GGT mRNA expression provides unique molecular probes to follow hepatic precursor cell differentiation. Furthermore, the identification of factors governing GGT P5 and P4 promoter expression should provide further insight into the molecular events that occur as the liver precursor cell differentiates into the hepatic lineages.


Subject(s)
Bile Ducts/cytology , Liver/cytology , Promoter Regions, Genetic , gamma-Glutamyltransferase/genetics , Animals , Animals, Newborn , Bile Ducts/embryology , Bile Ducts/enzymology , Cell Differentiation , Cell Lineage , Cells, Cultured , Female , Galactosamine/pharmacology , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Enzymologic/drug effects , In Situ Hybridization , Liver/embryology , Liver/enzymology , Male , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction
11.
Ann Pharm Fr ; 58(4): 260-5, 2000 Jul.
Article in French | MEDLINE | ID: mdl-10915974

ABSTRACT

In the adult liver, the plasma membrane Liver Regulating Protein (LRP) has been found to participate in the recognition signals between hepatocytes and rat liver epithelial cells (RLECs) and to play a critical role in the functional stability of hepatocytes. Here, we report the involvement of LRP in hematopoiesis. First, LRP was evidenced in the hematopoietic fetal rat liver and in adult rat bone marrow. Then, the involvement of LRP in adult rat and human hematopoiesis was investigated in coculture of hematopoietic cells with RLECs. In the rat, RLECs sustain long-term production of hematopoietic cells and committed progenitors as well as bone marrow stroma cells. These effects can be specifically blocked by addition of anti-LRP antibodies to both culture systems. The supportive activity of RLECs on human hematopoiesis was assessed by comparison to the reference MS-5 cell line from both unifractioned and purified CD34(+)CD38(-) hematopoietic cells. Taken together, our results argue for the involvement of LRP in heterotypic interaction signals mediated by RLEC and bone marrow stromal cells which lead to hematopoietic primitive progenitors development. They might lead to interesting applications in fundamental research, pharmacology and therapeutics.


Subject(s)
Cell Differentiation/physiology , Hematopoietic Stem Cells/cytology , Liver/cytology , Receptors, Immunologic/physiology , Stem Cells/cytology , Animals , Bone Marrow Cells/cytology , Fetus , Humans , Low Density Lipoprotein Receptor-Related Protein-1 , Rats
12.
Cytokine ; 12(7): 951-9, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10880240

ABSTRACT

Fetal liver is the main site of haematopoiesis during mid-gestation. The adult liver still provides a favourable environment for extramedullary haematopoiesis. Nevertheless, regulation of liver haematopoiesis by cell-cell contacts or by cytokines remains poorly understood. Recently, we have shown that rat liver epithelial cells (RLECs) support long-term survival and multilineage differentiation of adult human CD34(+)and CD34(+)/CD38(-)haematopoietic cells obtained from granulocyte-colony stimulating factor mobilized peripheral blood and from bone marrow respectively. In addition, the importance of physical proximity between haematopoietic cells and RLECs was clearly demonstrated. Here, our findings give evidence that RLECs belonging to the epithelial but non-parenchymal liver compartment also sustain the long-term production of progenitors from human CD34(+)umbilical cord blood cells. Moreover, to better analyse the regulation of haematopoiesis in this RLEC coculture model, we have investigated the cytokine expression by RLECs alone and by RLECs coming from coculture with CD34(+)cells from umbilical cord blood. We demonstrated that a broad spectrum of cytokines acting at different stages of haematopoiesis is produced by RLECs. Interestingly, an upregulation of leukemia inhibitory factor expression by RLECs in presence of CD34(+)haematopoietic cells was observed. These data suggest an important role of cell-cell interactions in the regulation of haematopoiesis.


Subject(s)
Antigens, CD34 , Cytokines/genetics , Fetal Blood/cytology , Interleukin-6 , Liver/metabolism , Animals , Cells, Cultured , Coculture Techniques/methods , Cytokines/biosynthesis , Epithelial Cells/cytology , Epithelial Cells/metabolism , Gene Expression , Growth Inhibitors/genetics , Hematopoiesis/physiology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Leukemia Inhibitory Factor , Liver/cytology , Liver/embryology , Lymphokines/genetics , Macrophage Colony-Stimulating Factor/metabolism , Rats , Rats, Sprague-Dawley , Time Factors , Up-Regulation
13.
Virology ; 270(1): 9-16, 2000 Apr 25.
Article in English | MEDLINE | ID: mdl-10772975

ABSTRACT

We identified the epitopes on the preS1 which induce antibodies that neutralize both ad and ay subtypes of hepatitis B virus (HBV). Previously we generated murine monoclonal antibodies KR359 and KR127 that bind specifically to the preS1 of HBV. In this study we have performed fine mappings of the epitopes of the antibodies by examining their reactivity with GST fusion proteins, which contain a series of deletion mutants of the preS1. KR359 and KR127 specifically recognize aa 19-26 and 37-45 of the preS1, respectively. The antibodies neutralized both adr and ayw subtypes of the virus in an in vitro neutralization assay using in vitro infection of adult human hepatocyte primary culture by HBV. The epitopes showed little sequence divergence and the antibodies bound to the preS1 of all the HBV subtypes and variants tested.


Subject(s)
Antibodies, Monoclonal/immunology , Epitope Mapping , Epitopes/immunology , Hepatitis B Surface Antigens/immunology , Hepatitis B virus/immunology , Protein Precursors/immunology , Viral Envelope Proteins/immunology , Amino Acid Sequence , Animals , Antibody Specificity/immunology , Cells, Cultured , DNA, Viral/biosynthesis , DNA, Viral/genetics , Genetic Variation/genetics , Genetic Variation/immunology , Hepatitis Antigens/chemistry , Hepatitis Antigens/genetics , Hepatitis Antigens/immunology , Hepatitis B Surface Antigens/chemistry , Hepatitis B Surface Antigens/genetics , Hepatitis B virus/classification , Hepatitis B virus/genetics , Hepatitis B virus/physiology , Humans , Liver/cytology , Liver/virology , Mice , Molecular Sequence Data , Neutralization Tests , Protein Precursors/chemistry , Protein Precursors/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Sequence Alignment , Sequence Deletion/genetics , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Virus Replication
14.
J Virol ; 74(1): 110-6, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10590097

ABSTRACT

It has been suggested that hepatitis B virus (HBV) binds to a receptor on the plasma membrane of human hepatocytes via the pre-S1 domain of the large envelope protein as an initial step in HBV infection. However, the nature of the receptor remains controversial. In an attempt to identify a cell surface receptor for HBV, purified recombinant fusion protein of the pre-S1 domain of HBV with glutathione S-transferase (GST), expressed in Escherichia coli, was used as a ligand. The surface of human hepatocytes or HepG2 cells was biotinylated, and the cell lysate (precleared lysate) which did not bind to GST and glutathione-Sepharose beads was used as a source of receptor molecules. The precleared lysate of the biotinylated cells was incubated with the GST-pre-S1 fusion protein, and the bound proteins were visualized by Western blotting and enhanced chemiluminescence. An approximately 80-kDa protein (p80) was shown to bind specifically to the pre-S1 domain of the fusion protein. The receptor binding assay using serially or internally deleted segments of pre-S1 showed that amino acid residues 12 to 20 and 82 to 90 are essential for the binding of pre-S1 to p80. p80 also bound specifically to the pre-S1 of native HBV particles. Analysis of the tissue and species specificity of p80 expression in several available human primary cultures and cell lines of different tissue origin showed that p80 expression is not restricted to human hepatocytes. Taken together the results suggest that p80 may be a component of the viral entry machinery.


Subject(s)
Hepatitis B Surface Antigens/metabolism , Hepatitis B virus/immunology , Protein Precursors/metabolism , Adult , Animals , Binding Sites , Cells, Cultured , Humans , Rats
15.
FEBS Lett ; 459(1): 75-9, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10508920

ABSTRACT

The F-box protein Skp2 is important for S phase entry and binds to Skp1 and the cyclin A-Cdk2 complex. Here we report the cloning, analysis of genomic organization and characterization of a novel gene product related to Skp2 named FBL2. The human FBL2 gene was found to be a highly interrupted gene of at least 126.6 kb located on chromosome 17 in close proximity to the TRAP220 gene in a head-to-tail orientation. The predicted protein contains an F-box and six perfect C-terminal leucine-rich repeats. Similar to Skp2, this protein interacts with Skp1 and deletion of the F-box inhibits this association. However, in contrast to Skp2, FBL2 was detected in non-proliferating hepatocytes and its expression increased in growth-arrested liver epithelial cells. In addition, FBL2 was localized primarily in the cytoplasm concentrated around the nucleus. Overall, our data indicate that although FBL2 shares strong structural homology with Skp2 as well as having a similar ability to associate with Skp1, these proteins likely play distinct roles and target different substrates to the SCF complex.


Subject(s)
Cell Cycle Proteins/genetics , F-Box Proteins , Leucine/chemistry , Amino Acid Sequence , Animals , Binding Sites , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Cloning, Molecular , Conserved Sequence , DNA, Complementary/analysis , Exons , Humans , Introns , Leucine-Rich Repeat Proteins , Male , Molecular Sequence Data , Protein Binding , Protein Conformation , Proteins/chemistry , Proteins/genetics , Rats , Rats, Sprague-Dawley , S-Phase Kinase-Associated Proteins , Sequence Homology, Amino Acid
16.
In Vitro Cell Dev Biol Anim ; 35(5): 289-95, 1999 May.
Article in English | MEDLINE | ID: mdl-10475275

ABSTRACT

Primary cultures of Pecten maximus heart cells, isolated by an enzymatic procedure, were routinely obtained with a high level of reproducibility in a simple medium based on sterile seawater. Cells attached to the plastic substratum without the need to add a special factor. The number of adhering cells gradually increased with the time of culture. Two types of adhering cells were observed: epitheliallike cells and fibroblastlike cells, which were more numerous. The latter cells were identified as myocytes by electron microscopy and immunofluorescent staining. Results obtained by autoradiography, after incorporation of [14C]leucine, [3H]thymidine, and [14C]acetate, confirmed functional activity of the cells. These cultures were maintained viable in vitro during at least 1 mo.


Subject(s)
Mollusca/cytology , Animals , Autoradiography , Cell Adhesion , Cell Differentiation , Cell Division , Cell Separation , Cells, Cultured , Culture Media , DNA/biosynthesis , Epithelial Cells/cytology , Fibroblasts/cytology , Fluorescent Antibody Technique , Microscopy, Electron , Myocardium/cytology , Seawater , Time Factors
17.
Mol Cell Biol ; 19(9): 6003-11, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10454547

ABSTRACT

In this study, activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling pathway was analyzed in proliferating rat hepatocytes both in vivo after partial hepatectomy and in vitro following epidermal growth factor (EGF)-pyruvate stimulation. First, a biphasic MEK/ERK activation was evidenced in G(1) phase of hepatocytes from regenerating liver but not from sham-operated control animals. One occurred in early G(1) (30 min to 4 h), and the other occurred in mid-late G(1), peaking at around 10.5 h. Interestingly, the mid-late G(1) activation peak was located just before cyclin D1 induction in both in vivo and in vitro models. Second, the biological role of the MEK/ERK cascade activation in hepatocyte progression through the G(1)/S transition was assessed by adding a MEK inhibitor (PD 98059) to EGF-pyruvate-stimulated hepatocytes in primary culture. In the presence of MEK inhibitor, cyclin D1 mRNA accumulation was inhibited, DNA replication was totally abolished, and the MEK1 isoform was preferentially targeted by this inhibition. This effect was dose dependent and completely reversed by removing the MEK inhibitor. Furthermore, transient transfection of hepatocytes with activated MEK1 construct resulted in increased cyclin D1 mRNA accumulation. Third, a correlation between the mid-late G(1) MEK/ERK activation in hepatocytes in vivo after partial hepatectomy and the mitogen-independent proliferation capacity of these cells in vitro was established. Among hepatocytes isolated either 5, 7, 9, 12 or 15 h after partial hepatectomy, only those isolated from 12- and 15-h regenerating livers were able to replicate DNA without additional growth stimulation in vitro. In addition, PD 98059 intravenous administration in vivo, before MEK activation, was able to inhibit DNA replication in hepatocytes from regenerating livers. Taken together, these results show that (i) early induction of the MEK/ERK cascade is restricted to hepatocytes from hepatectomized animals, allowing an early distinction of primed hepatocytes from those returning to quiescence, and (ii) mid-late G(1) MEK/ERK activation is mainly associated with cyclin D1 accumulation which leads to mitogen-independent progression of hepatocytes to S phase. These results allow us to point to a growth factor dependency in mid-late G(1) phase of proliferating hepatocytes in vivo as observed in vitro in proliferating hepatocytes and argue for a crucial role of the MEK/ERK cascade signalling pathway.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , G1 Phase/physiology , Liver/cytology , Liver/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Cell Cycle/physiology , Cell Division/drug effects , Cells, Cultured , Cyclin D1/biosynthesis , Cyclin D1/genetics , DNA Replication , Enzyme Activation , Enzyme Inhibitors/pharmacology , Female , Flavonoids/pharmacology , Hepatectomy , Liver/drug effects , Liver Regeneration , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction , Transfection
18.
FEBS Lett ; 452(3): 247-53, 1999 Jun 11.
Article in English | MEDLINE | ID: mdl-10386600

ABSTRACT

The changes in phosphoproteins purified with the affinity peptide p9CKShs1 were analyzed from extracts of regenerating rat livers in order to define some G1 and G1/S regulations characteristic of mature hepatocytes stimulated to proliferate. We observed a 47 kDa phosphoprotein that occurred first at the end of G1 before peaking in the S phase. P47 was also found to be phosphorylated in late G1 in primary hepatocyte cultures stimulated with mitogens. P47 was still phosphorylated in extracts depleted of Cdc2, but to a lesser extent after Cdk2 depletion. This phosphoprotein was identified as Skp2. (i) P47 shared the same electrophoretic mobility than Skp2, a cell cycle protein essential for S phase entry in human fibroblasts; (ii) Skp2, like P47, started to be expressed and was highly phosphorylated during the G1/S transition of hepatocytes stimulated to proliferate in vivo and in vitro; (iii) P47 was specifically immunoprecipitated by an antibody directed against Skp2. In addition, cyclin A/Cdk2 complexes from regenerating liver clearly interacted with Skp2. This is the first demonstration that Skp2 is induced and phosphorylated in the late G1 and S phase of hepatocytes in vivo in regenerating liver as well as in vitro in mitogen-stimulated hepatocytes.


Subject(s)
Cell Cycle , Liver/cytology , Liver/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Animals , Cells, Cultured , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , DEAD-box RNA Helicases , Epidermal Growth Factor/pharmacology , G1 Phase , Gene Expression Regulation , Humans , Phosphates/metabolism , Phosphorylation , Rats , S Phase
19.
J Virol ; 73(3): 2052-7, 1999 Mar.
Article in English | MEDLINE | ID: mdl-9971786

ABSTRACT

During the life cycle of hepatitis B virus (HBV), the large envelope protein (L) plays a pivotal role. Indeed, this polypeptide is essential for viral assembly and probably for the infection process. By performing mutagenesis experiments, we have previously excluded a putative involvement of the pre-S2 domain of the L protein in viral infectivity. In the present study, we have evaluated the role of the pre-S1 region in HBV infection. For this purpose, 21 mutants of the L protein were created. The entire pre-S1 domain was covered by contiguous deletions of 5 amino acids. First, after transfection into HepG2 cells, the efficient expression of both glycosylated and unglycosylated L mutant proteins was verified. The secretion rate of envelope proteins was modified positively or negatively by deletions, indicating that the pre-S1 domain contains several regulating sequences able to influence the surface protein secretion. The ability of mutant proteins to support the production of virions was then studied. Only the four C-terminal deletions, covering the 17 amino acids suspected to interact with the cytoplasmic nucleocapsids, inhibited virion release. Finally, the presence of the modified pre-S1 domain at the external side of all secreted virions was confirmed, and their infectivity was assayed on normal human hepatocytes in primary culture. Only a short sequence including amino acids 78 to 87 tolerates internal deletions without affecting viral infectivity. These results confirm the involvement of the L protein in the infection step and demonstrate that the sequence between amino acids 3 and 77 is involved in this process.


Subject(s)
Hepatitis B Surface Antigens/physiology , Hepatitis B virus/physiology , Protein Precursors/physiology , Humans , Liver/virology , Mutation , Tumor Cells, Cultured , Virion/physiology
20.
J Virol ; 72(7): 5573-8, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9621015

ABSTRACT

Among the three viral proteins present in the hepatitis B virus (HBV) envelope, both the small and large polypeptides, but not the middle polypeptide, are necessary for the production of complete viral particles. Whereas it has been established that the C-terminal extremity of the pre-S1 region is required for HBV morphogenesis, whether the pre-S2 region of the large surface protein plays a critical role remains questionable. In the present study, we have analyzed the role of the large-polypeptide pre-S2 region in viral maturation and infectivity. For this purpose, mutants bearing contiguous deletions covering the entire pre-S2 domain were generated. First, the efficient expression of all the mutant large envelope proteins was verified and their ability to substitute for the wild-type form in virion secretion was tested. We found that distinct deletions covering the domain between amino acids 114 and 163 still allowed virion production. In contrast, the polypeptide lacking the first 5 amino acids of pre-S2 (amino acids 109 to 113) was unable to support viral secretion. This result shows that the domain of the large surface protein, required for this process, must be extended to the N-terminal extremity of pre-S2. We then demonstrated that all the mutants competent for virion release were able to infect normal human hepatocytes in primary culture. Taken together, these results indicate that only 10% of the large-protein pre-S2 region at its N-terminal extremity is essential for virion export and that the remaining part, dispensable for viral secretion, is also dispensable for infectivity.


Subject(s)
Hepatitis B Surface Antigens/physiology , Hepatitis B virus/physiology , Protein Precursors/physiology , Virus Assembly , Adult , Humans , Mutation , Nucleocapsid/physiology , Tumor Cells, Cultured , Virion/physiology
SELECTION OF CITATIONS
SEARCH DETAIL
...