72 results on '"Rogler, Ce"'
Search Results
2. The role of Ito cells in the biosynthesis of HGF-SF in the liver
- Author
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Schirmacher, P., Geerts, A., Jung, W., Antonello Pietrangelo, Rogler, Ce, and Dienes, Hp
- Published
- 1993
3. Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from hepatocellular carcinomas of woodchucks
- Author
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Rogler Ce, Susan M. Astrin, Jonak Gj, Jesse Summers, and Ogston Cw
- Subjects
Genes, Viral ,Hepatitis B virus DNA polymerase ,viruses ,DNA, Recombinant ,medicine.disease_cause ,General Biochemistry, Genetics and Molecular Biology ,law.invention ,Nucleic acid thermodynamics ,Restriction map ,Liver Neoplasms, Experimental ,law ,Hepatitis Viruses ,medicine ,Animals ,Cloning, Molecular ,Southern blot ,Hepatitis B virus ,biology ,Base Sequence ,Woodchuck hepatitis virus ,Nucleic Acid Hybridization ,DNA ,DNA Restriction Enzymes ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Virology ,Molecular biology ,digestive system diseases ,Restriction site ,Marmota ,DNA, Viral ,Recombinant DNA - Abstract
Woodchuck hepatitis virus (WHV), like the related hepatitis B virus, induces in its natural host hepatocellular carcinomas that contain integrated viral sequences. As a first step in determining whether and how the integrated sequences contribute to formation of the tumors in which they are found, we have cloned two such integrations of WHV and have determined their structure by restriction mapping and heteroduplex electron microscopy. The identity of the cloned sequences was confirmed by comparison of restriction sites in the clones with those located by Southern blot analysis of tumor DNA. Viral sequences in both integrations are extensively rearranged, and in neither were all parts of the viral genome represented. In this respect, the behavior of WHV in vivo is similar to that of other DNA tumor viruses that have been studied in vitro. We discuss the implications of these results in relation to possible mechanisms for tumor induction by WHV.
- Published
- 1982
4. A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing
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Astrid Novosel, Markus Landthaler, Julia Schliwka, Sabina Chiaretti, Miklós Palkovits, Roberto Di Lauro, Nicola Iovino, Mirabela Rusu, Roman-Ulrich Müller, Giuseppe Macino, James W. Nagle, Andreas Bosio, Mihaela Zavolan, Peter Wernet, Alice O. Kamphorst, Charles E. Rogler, Chris Sander, Veit Hornung, James J. Russo, Wayne Tam, Thomas Benzing, Alexei A. Aravin, Uta Fuchs, Thomas Tuschl, Pablo Landgraf, Sébastien Pfeffer, Alain Sewer, Hans Ingo Trompeter, Jason M. Inman, Ruchi Choksi, Amanda J. Rice, Arndt Borkhardt, Robert L. Sheridan, Valerio Fulci, Carolina Lin, Leandro C. Hermida, Michael J. Brownstein, Ute Bissels, Bernhard Schermer, Daniela Frezzetti, Minchen Chien, Grace Teng, Jingyue Ju, Quang Phan, Gunther Hartmann, F. Nina Papavasiliou, Robin Foà, Nicholas D. Socci, David B. Weir, Gabriella De Vita, Peter Lichter, Landgraf, P, Rusu, M, Sheridan, R, Sewer, A, Iovino, N, Aravin, A, Pfeffer, S, Rice, A, Kamphorst, Ao, Landthaler, M, Lin, C, Socci, Nd, Hermida, L, Fulci, V, Chiaretti, S, Foa, R, Schliwka, J, Fuchs, U, Novosel, A, Muller, Ru, Schermer, B, Bissels, U, Inman, J, Phan, Q, Chien, M, Weir, Db, Choksi, R, DE VITA, Gabriella, Frezzetti, D, Trompeter, Hi, Hornung, V, Teng, G, Hartmann, G, Palkovits, M, DI LAURO, Roberto, Wernet, P, Macino, G, Rogler, Ce, Nagle, Jw, Ju, J, Papavasiliou, Fn, Benzing, T, Lichter, P, Tam, W, Brownstein, Mj, Bosio, A, Borkhardt, A, Russo, Jj, Sander, C, Zavolan, M, Tuschl, T., Institut de biologie moléculaire des plantes (IBMP), and Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)
- Subjects
Small RNA ,moleneuro ,molimmuno ,rna ,Molecular Sequence Data ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Biology ,Genome ,General Biochemistry, Genetics and Molecular Biology ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,IsomiR ,Mirtron ,Sequence Homology, Nucleic Acid ,microRNA ,Animals ,Humans ,Cell Lineage ,RNA, Messenger ,MOLIMMUNO ,Conserved Sequence ,Phylogeny ,MOLENEURO ,Gene Library ,030304 developmental biology ,Genetics ,Regulation of gene expression ,0303 health sciences ,Base Sequence ,Biochemistry, Genetics and Molecular Biology(all) ,Gene Expression Profiling ,RNA ,Hematopoietic Stem Cells ,Rats ,Gene expression profiling ,MicroRNAs ,Gene Expression Regulation ,Hematologic Neoplasms ,030220 oncology & carcinogenesis - Abstract
SummaryMicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.
- Published
- 2007
5. Knockdown of miR-23, miR-27, and miR-24 Alters Fetal Liver Development and Blocks Fibrosis in Mice.
- Author
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Rogler CE, Matarlo JS, Kosmyna B, Fulop D, and Rogler LE
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- Actins genetics, Animals, Bile Ducts pathology, Cell Differentiation genetics, Cell Line, Cell Proliferation genetics, Hepatic Stellate Cells pathology, Hepatocytes pathology, Mice, Mice, Inbred C57BL, Mice, Transgenic, Transforming Growth Factor beta1, Fetal Development genetics, Liver pathology, Liver Cirrhosis genetics, Liver Cirrhosis pathology, MicroRNAs genetics, Organogenesis genetics
- Abstract
MicroRNAs (miRNAs) regulate cell fate selection and cellular differentiation. miRNAs of the miR23b polycistron (miR-23b, miR-27b, and miR-24) target components of the TGF-β signaling pathway and affect murine bile ductular and hepatocyte cell fate selection in vitro. Here we show that miR-23b polycistron miRNAs directly target murine Smad4, which is required for TGF-β signaling. Injection of antagomirs against these miRNAs directly into E16.5 murine fetuses caused increased cytokeratin expression in sinusoids and primitive ductular elements throughout the parenchyma of newborn mice. Similar antagomir injection in newborn mice increased bile ductular differentiation in the liver periphery and reduced hepatocyte proliferation. Antagomir injection in newborn Alb/TGF-β1 transgenic mice that develop fibrosis inhibited the development of fibrosis, and injection of older mice caused the resolution of existing fibrosis. Furthermore, murine stellate cell activation, including ColA1 and ACTA2 expression, is regulated by miR-23b cluster miRNAs. In summary, knockdown of miR-23b cluster miRNAs in fetal and newborn liver promotes bile duct differentiation and can block or revert TGF-β-induced liver fibrosis that is dependent on stellate cell activation. These data may find practical application in the highly needed development of therapies for the treatment of fibrosis.
- Published
- 2017
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6. Triple Staining Including FOXA2 Identifies Stem Cell Lineages Undergoing Hepatic and Biliary Differentiation in Cirrhotic Human Liver.
- Author
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Rogler CE, Bebawee R, Matarlo J, Locker J, Pattamanuch N, Gupta S, and Rogler LE
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- Adult, Aged, Bile Ducts cytology, Cell Differentiation, Cell Lineage, Female, Hepatocytes cytology, Humans, Infant, Keratin-19 analysis, Liver cytology, Male, Staining and Labeling methods, Stem Cells cytology, Bile Ducts pathology, Hepatocyte Nuclear Factor 3-beta analysis, Hepatocytes pathology, Immunohistochemistry methods, Liver pathology, Stem Cells pathology
- Abstract
Recent investigations have reported many markers associated with human liver stem/progenitor cells, "oval cells," and identified "niches" in diseased livers where stem cells occur. However, there has remained a need to identify entire lineages of stem cells as they differentiate into bile ducts or hepatocytes. We have used combined immunohistochemical staining for a marker of hepatic commitment and specification (FOXA2 [Forkhead box A2]), hepatocyte maturation (Albumin and HepPar1), and features of bile ducts (CK19 [cytokeratin 19]) to identify lineages of stem cells differentiating toward the hepatocytic or bile ductular compartments of end-stage cirrhotic human liver. We identified large clusters of disorganized, FOXA2 expressing, oval cells in localized liver regions surrounded by fibrotic matrix, designated as "micro-niches." Specific FOXA2-positive cells within the micro-niches organize into primitive duct structures that support both hepatocytic and bile ductular differentiation enabling identification of entire lineages of cells forming the two types of structures. We also detected expression of hsa-miR-122 in primitive ductular reactions expected for hepatocytic differentiation and hsa-miR-23b cluster expression that drives liver cell fate decisions in cells undergoing lineage commitment. Our data establish the foundation for a mechanistic hypothesis on how stem cell lineages progress in specialized micro-niches in cirrhotic end-stage liver disease., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
- Published
- 2017
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7. Small RNAs derived from lncRNA RNase MRP have gene-silencing activity relevant to human cartilage-hair hypoplasia.
- Author
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Rogler LE, Kosmyna B, Moskowitz D, Bebawee R, Rahimzadeh J, Kutchko K, Laederach A, Notarangelo LD, Giliani S, Bouhassira E, Frenette P, Roy-Chowdhury J, and Rogler CE
- Subjects
- Alternative Splicing, Cell Line, HEK293 Cells, Hematopoiesis genetics, High-Throughput Nucleotide Sequencing, Humans, Liver pathology, Nucleic Acid Conformation, Osteochondrodysplasias genetics, Patched Receptors, Patched-2 Receptor, Phenotype, Primary Immunodeficiency Diseases, Receptors, Cell Surface metabolism, SOXC Transcription Factors metabolism, Endoribonucleases genetics, Hair abnormalities, Hirschsprung Disease genetics, Immunologic Deficiency Syndromes genetics, Liver metabolism, MicroRNAs genetics, Osteochondrodysplasias congenital, RNA Interference, RNA, Long Noncoding genetics
- Abstract
Post-transcriptional processing of some long non-coding RNAs (lncRNAs) reveals that they are a source of miRNAs. We show that the 268-nt non-coding RNA component of mitochondrial RNA processing endoribonuclease, (RNase MRP), is the source of at least two short (∼20 nt) RNAs designated RMRP-S1 and RMRP-S2, which function as miRNAs. Point mutations in RNase MRP cause human cartilage-hair hypoplasia (CHH), and several disease-causing mutations map to RMRP-S1 and -S2. SHAPE chemical probing identified two alternative secondary structures altered by disease mutations. RMRP-S1 and -S2 are significantly reduced in two fibroblast cell lines and a B-cell line derived from CHH patients. Tests of gene regulatory activity of RMRP-S1 and -S2 identified over 900 genes that were significantly regulated, of which over 75% were down-regulated, and 90% contained target sites with seed complements of RMRP-S1 and -S2 predominantly in their 3' UTRs. Pathway analysis identified regulated genes that function in skeletal development, hair development and hematopoietic cell differentiation including PTCH2 and SOX4 among others, linked to major CHH phenotypes. Also, genes associated with alternative RNA splicing, cell proliferation and differentiation were highly targeted. Therefore, alterations RMRP-S1 and -S2, caused by point mutations in RMRP, are strongly implicated in the molecular mechanism of CHH.
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- 2014
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8. Overexpression of miR-21 promotes an in vitro metastatic phenotype by targeting the tumor suppressor RHOB.
- Author
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Connolly EC, Van Doorslaer K, Rogler LE, and Rogler CE
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- Carcinoma, Hepatocellular genetics, Cell Line, Tumor, Female, Genes, Tumor Suppressor physiology, Hep G2 Cells, Humans, Liver Neoplasms pathology, MicroRNAs physiology, Neoplasm Invasiveness genetics, Neoplasm Invasiveness physiopathology, Phenotype, Up-Regulation genetics, rhoB GTP-Binding Protein genetics, rhoB GTP-Binding Protein physiology, Carcinoma, Hepatocellular metabolism, Carcinoma, Hepatocellular secondary, Gene Expression Regulation, Neoplastic genetics, Liver Neoplasms genetics, Liver Neoplasms metabolism, MicroRNAs biosynthesis, MicroRNAs genetics, rhoB GTP-Binding Protein metabolism
- Abstract
Metastasis is a multistep process that involves the deregulation of oncogenes and tumor suppressors beyond changes required for primary tumor formation. RHOB is known to have tumor suppressor activity, and its knockdown is associated with more aggressive tumors as well as changes in cell shape, migration, and adhesion. This study shows that oncogenic microRNA, miR-21, represses RHOB expression by directly targeting the 3' untranslated region. Loss of miR-21 is associated with an elevation of RHOB in hepatocellular carcinoma cell lines Huh-7 and HepG2 and in the metastatic breast cancer cell line MDA-MB-231. Using in vitro models of distinct stages of metastasis, we showed that loss of miR-21 also causes a reduction in migration, invasion, and cell elongation. The reduction in migration and cell elongation can be mimicked by overexpression of RHOB. Furthermore, changes in miR-21 expression lead to alterations in matrix metalloproteinase-9 activity. Therefore, we conclude that miR-21 promotes multiple components of the metastatic phenotype in vitro by regulating several important tumor suppressors, including RHOB., ((c)2010 AACR.)
- Published
- 2010
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9. MicroRNA-23b cluster microRNAs regulate transforming growth factor-beta/bone morphogenetic protein signaling and liver stem cell differentiation by targeting Smads.
- Author
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Rogler CE, Levoci L, Ader T, Massimi A, Tchaikovskaya T, Norel R, and Rogler LE
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- Animals, Bile Ducts cytology, Bone Morphogenetic Proteins metabolism, Cell Line, Gene Expression Profiling, Hepatocytes cytology, Liver cytology, Liver embryology, Mice, Oligonucleotide Array Sequence Analysis, RNA, Small Interfering metabolism, Smad Proteins metabolism, Cell Differentiation, Hepatocytes metabolism, Liver metabolism, MicroRNAs metabolism, Transforming Growth Factor beta metabolism
- Abstract
Unlabelled: Transforming growth factor-beta / bone morphogenetic protein (TGFbeta/BMP) signaling has a gradient of effects on cell fate choice in the fetal mouse liver. The molecular mechanism to understand why adjacent cells develop into bile ducts or grow actively as hepatocytes in the ubiquitous presence of both TGFbeta ligands and receptors has been unknown. We hypothesized that microRNAs (miRNAs) might play a role in cell fate decisions in the liver. miRNA profiling during late fetal development in the mouse identified miR-23b cluster miRNAs comprising miR-23b, miR-27b, and miR-24-1 and miR-10a, miR-26a, and miR-30a as up-regulated. In situ hybridization of fetal liver at embryonic day 17.5 of gestation revealed miR-23b cluster expression only in fetal hepatocytes. A complementary (c)DNA microarray approach was used to identify genes with a reciprocal expression pattern to that of miR-23b cluster miRNAs. This approach identified Smads (mothers against decapentaplegic homolog), the key TGFbeta signaling molecules, as putative miR-23b cluster targets. Bioinformatic analysis identified multiple candidate target sites in the 3' UTRs (untranslated regions) of Smads 3, 4, and 5. Dual luciferase reporter assays confirmed down-regulation of constructs containing Smad 3, 4, or 5, 3' UTRs by a mixture of miR-23b cluster mimics. Knockdown of miR-23b miRNAs during hepatocytic differentiation of a fetal liver stem cell line, HBC-3, promoted expression of bile duct genes, in addition to Smads, in these cells. In contrast, ectopic expression of miR-23b mimics during bile duct differentiation of HBC-3 cells blocked the process., Conclusion: Our data provide a model in which miR-23b miRNAs repress bile duct gene expression in fetal hepatocytes while promoting their growth by down-regulating Smads and consequently TGFbeta signaling. Concomitantly, low levels of the miR-23b miRNAs are needed in cholangiocytes to allow TGFbeta signaling and bile duct formation.
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- 2009
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10. MicroRNAs make inroads into liver development.
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Rogler CE
- Subjects
- Animals, Humans, Liver cytology, Liver embryology, Liver physiology, MicroRNAs physiology, Stem Cells physiology
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- 2009
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11. Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype.
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Connolly E, Melegari M, Landgraf P, Tchaikovskaya T, Tennant BC, Slagle BL, Rogler LE, Zavolan M, Tuschl T, and Rogler CE
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- Animals, Apoptosis physiology, Blotting, Northern, Blotting, Western, Carcinoma, Hepatocellular virology, Cell Proliferation, Cell Transformation, Neoplastic genetics, Flow Cytometry, Humans, Liver Neoplasms virology, Marmota, Reverse Transcriptase Polymerase Chain Reaction, Transfection, Carcinoma, Hepatocellular genetics, Hepatitis B genetics, Liver Neoplasms genetics, MicroRNAs biosynthesis
- Abstract
Alterations in microRNA (miRNA) expression in both human and animal models have been linked to many forms of cancer. Such miRNAs, which act directly as repressors of gene expression, have been found to frequently reside in fragile sites and genomic regions associated with cancer. This study describes a miRNA signature for human primary hepatitis B virus-positive human hepatocellular carcinoma. Moreover, two known oncomiRs--miRNAs with known roles in cancer--the miR-17-92 polycistron and miR-21, exhibited increased expression in 100% of primary human and woodchuck hepatocellular carcinomas surveyed. To determine the importance of these miRNAs in tumorigenesis, an in vitro antisense oligonucleotide knockdown model was evaluated for its ability to reverse the malignant phenotype. Both in human and woodchuck HCC cell lines, separate treatments with antisense oligonucleotides specific for either the miR-17-92 polycistron (all six members) or miR-21 caused a 50% reduction in both hepatocyte proliferation and anchorage-independent growth. The combination of assays presented here supports a role for these miRNAs in the maintenance of the malignant transformation of hepatocytes.
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- 2008
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12. Clonal, cultured, murine fetal liver hepatoblasts maintain liver specification in chimeric mice.
- Author
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Rogler CE, Zhou HC, LeVoci L, and Rogler LE
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- Animals, Bile Ducts physiology, Cell Line, Fetus, Liver cytology, Liver physiology, Mice, Mice, Knockout, Microdissection, Models, Animal, Stem Cell Transplantation, Cell Differentiation physiology, Chimera, Clone Cells physiology, Hepatocytes physiology, Stem Cells physiology
- Abstract
Unlabelled: Recent studies have shown a pluripotential nature of stem cells that were previously thought to be committed to specific lineages. HBC-3 cells are a clonal fetal murine hepatoblast cell line derived from an e9.5 murine embryo, and these cells can be induced to form hepatocytes and bile ducts in vitro and when transplanted into adult mouse livers. To determine whether HBC-3 cells can exhibit a pluripotential phenotype, we created chimeric mice by injection of enhanced green fluorescent protein (EGFP)-marked HBC-3 cells into wild-type or dipeptidyl dipeptidase IV (DPPIV) knockout blastocysts. Genetically labeled HBC-3 cells were identified by EGFP polymerase chain reaction (PCR) in all the major organs of many chimeric mice and visualized in chimeras as bright red DPPIV-positive cells in the DPPIV knockout chimeric mice. Strikingly, the HBC-3 cells maintained phenotypic and biochemical features of liver specification in every case in which they were identified in nonliver organs, such as brain, mesenchyme, and bone. In adult liver they were present as small foci of hepatocytes and bile ducts in the chimeras. Additional major histocompatibility complex (MHC) marker analysis and X and Y chromosome content analysis further demonstrated that HBC-3 cells did not acquire the phenotype of the organs in which they resided and that they were not present because of fusion with host cells., Conclusion: In contrast to other stem cell types, these data demonstrate that cultured murine fetal liver stem cells appear to maintain their liver specification in the context of nonliver organs in chimeric mice.
- Published
- 2007
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13. A mammalian microRNA expression atlas based on small RNA library sequencing.
- Author
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Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, Pfeffer S, Rice A, Kamphorst AO, Landthaler M, Lin C, Socci ND, Hermida L, Fulci V, Chiaretti S, Foà R, Schliwka J, Fuchs U, Novosel A, Müller RU, Schermer B, Bissels U, Inman J, Phan Q, Chien M, Weir DB, Choksi R, De Vita G, Frezzetti D, Trompeter HI, Hornung V, Teng G, Hartmann G, Palkovits M, Di Lauro R, Wernet P, Macino G, Rogler CE, Nagle JW, Ju J, Papavasiliou FN, Benzing T, Lichter P, Tam W, Brownstein MJ, Bosio A, Borkhardt A, Russo JJ, Sander C, Zavolan M, and Tuschl T
- Subjects
- Animals, Cell Lineage genetics, Conserved Sequence genetics, Hematologic Neoplasms genetics, Hematopoietic Stem Cells metabolism, Humans, Mice, Molecular Sequence Data, Phylogeny, RNA, Messenger genetics, Rats, Sequence Homology, Nucleic Acid, Base Sequence genetics, Gene Expression Profiling methods, Gene Expression Regulation genetics, Gene Library, MicroRNAs genetics
- Abstract
MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.
- Published
- 2007
- Full Text
- View/download PDF
14. Identification of hepatocytic and bile ductular cell lineages and candidate stem cells in bipolar ductular reactions in cirrhotic human liver.
- Author
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Zhou H, Rogler LE, Teperman L, Morgan G, and Rogler CE
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- Antibodies metabolism, Bile Ducts metabolism, Hepatocytes metabolism, Humans, Immunohistochemistry, Liver Cirrhosis metabolism, Liver Regeneration physiology, Neural Cell Adhesion Molecules immunology, Neural Cell Adhesion Molecules metabolism, Proteins immunology, Proteins metabolism, Stem Cells metabolism, Bile Ducts pathology, Cell Lineage, Hepatocytes pathology, Liver Cirrhosis pathology, Stem Cells pathology
- Abstract
Hepatocyte function and regeneration are severely compromised in severe liver disease, and a common sequela is cirrhosis. Structural changes caused by cirrhosis create a cellular environment conducive to the formation of ductular reactions (DRs). Ductular reactions are primarily composed of oval cells also known as "intermediate hepatobiliary cells". We have conducted single, double, and triple staining to study lineages of oval cells present in DRs. Staining with NCAM, CK19, and HepPar1 has revealed a distinctly bipolar structure to DRs that are embedded in cirrhotic tissue. Spatial analysis of cells that are singly HepPar1-positive, or CK19-positive, has revealed hepatocytic and biliary poles, respectively, in the DRs. Also, the location of singly NCAM-positive cells in DRs suggests that they may be bipotent liver stem/progenitor cells. The locations of other intermediate hepatobiliary cells, which have combinations of markers, suggest that CK19+/NCAM+ cells are transitional cells in the biliary lineage and that rare cells that are negative for all three markers are transitional cells in the hepatocytic lineage. A working cell lineage model for DRs is presented.
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- 2007
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15. Multi-miRNA hairpin method that improves gene knockdown efficiency and provides linked multi-gene knockdown.
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Sun D, Melegari M, Sridhar S, Rogler CE, and Zhu L
- Subjects
- Base Sequence, Cell Line, Cell Line, Tumor, Genetic Vectors, Humans, MicroRNAs genetics, Models, Genetic, Molecular Sequence Data, Oligonucleotides genetics, Plasmids metabolism, RNA metabolism, Genetic Techniques
- Abstract
A number of natural microRNA (miRNA) hairpins have been found in clusters of multiple identical or different copies, suggesting that effects of miRNAs can be enhanced and multiple genes can be regulated together by encoding multiple miRNA hairpins in a single transcript. Here, we report a simple and effective artificial multi-hairpin method that stimulates production of mature 22-nucleotide small RNAs from modified miRNA hairpins, improves gene knockdown over single-hairpin constructs, and provides linked multi-gene knockdowns.
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- 2006
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16. cDNA microarray analysis of HBV transgenic mouse liver identifies genes in lipid biosynthetic and growth control pathways affected by HBV.
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Hajjou M, Norel R, Carver R, Marion P, Cullen J, Rogler LE, and Rogler CE
- Subjects
- Animals, DNA, Complementary analysis, Disease Models, Animal, Hepatitis B genetics, Hepatitis B immunology, Hepatitis B virology, Hepatitis B virus immunology, Hepatitis B virus pathogenicity, Hepatitis B virus physiology, Liver virology, Mice, Mice, Transgenic, Hepatitis B virus genetics, Lipids biosynthesis, Liver metabolism, Oligonucleotide Array Sequence Analysis
- Abstract
Hepatitis B virus (HBV) transgenic mice that replicate HBV in the liver generally do not exhibit gross liver pathology, while maintaining a high level (10(7) or greater) of viral titer in the blood. We have used this model to determine the minimum effects of HBV replication in the liver on cellular gene transcription, using cDNA microarrays. cDNA microarray data from sets of HBV versus control cDNA microarrays revealed a very small impact of HBV on the cellular transcriptome. After deletion of genes that were variable in control cDNA microarrays and applying significance analysis of microarrays (SAM), an application to detect statistically significantly regulated genes, we identified 18 upregulated genes and 14 downregulated genes. Most of the regulated genes show a change in expression with respect to control of less than 40% in either direction, demonstrating small effects of HBV. The largest functional category for upregulated genes was lipid biosynthesis, in which ATP citrate lyase, fatty acid synthase, sterol regulatory element binding factor 2, and retinol binding protein 1 were all upregulated. The most strongly downregulated genes were in the cytochrome p450 group, particularly p450, 4a14. Several growth regulatory genes including cyclin D1, IGF binding protein 3, and PCNA were moderately upregulated. These data are the first to specifically identify enzymes involved in fatty acid and NADPH-electron transport pathways that are altered by the presence of HBV. The data also demonstrates that HBV is well adapted to non-cytopathic replication in hepatocytes. Cellular genes expected to be affected by viral secretion from membranes are clearly upregulated, and upregulation of growth regulatory genes may facilitate replacement of dying hepatocytes during persistent infection.
- Published
- 2005
- Full Text
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17. Hit-and-run mechanism of HBV-mediated progression to hepatocellular carcinoma.
- Author
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Hessein M, Saad el G, Mohamed AA, Kamel el AM, Abdel Hady AM, Amina M, and Rogler CE
- Subjects
- Carcinoma, Hepatocellular etiology, DNA, Viral analysis, DNA, Viral genetics, Humans, Liver Neoplasms etiology, Loss of Heterozygosity, Microsatellite Repeats, Mutagenesis, Polymorphism, Genetic, Tumor Cells, Cultured, Carcinoma, Hepatocellular physiopathology, Carcinoma, Hepatocellular virology, Cell Transformation, Neoplastic, Hepatitis B complications, Hepatitis B virus pathogenicity, Liver Neoplasms physiopathology, Liver Neoplasms virology, Virus Integration
- Abstract
Aim and Background: Hepatitis B virus is implicated in the development of hepatocellular caracinoma. No oncogenes have been identified within the viral genome. Furthermore, it frequently fragments after integration into the hepatocyte genome. Simultaneous investigations of hepatitis B virus integration patterns and genetic changes in precancerous tissues are important to understand the role played by hepatitis B virus integration in hepatocellular caracinoma., Method: We used a combination approach of dual characterization of highly polymorphic loci and the change in hepatitis B virus-DNA integration pattern. Large regenerative nodules were dissected from 6 explanted hepatitis B virus infected cirrhotic livers. Nodules within each liver segment were schematically mapped and histopathologically analyzed. Genomic DNA from each nodule was analyzed for hepatitis B virus integration and the genetic stability of 12 microsatellite loci including D3S2321, D8S1022, D17S1159, D4S2281, D5S1/2, D16S675, D16S685, D16S490, D16S526, D16S673, D16S677 and D16S690., Results: Data from different liver segments revealed few viral integrations and average allele loss. The most exciting results came from a segment containing a set of clonally and spatially related nodules having similar histologic features, a progressive lineage of allele loss, HBV integration and loss of integration., Conclusions: This model portrait, a scenario of genetic events that precede tumor formation where the acquisition and loss of hepatitis B virus integrations in clonally related regenerative nodules, might explain how the virus acts as a hit-and-run mutagen.
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- 2005
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18. RNA expression microarrays (REMs), a high-throughput method to measure differences in gene expression in diverse biological samples.
- Author
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Rogler CE, Tchaikovskaya T, Norel R, Massimi A, Plescia C, Rubashevsky E, Siebert P, and Rogler LE
- Subjects
- Animals, DNA, Complementary biosynthesis, Female, Gene Expression Profiling standards, Mice, Mice, Inbred C57BL, Neoplasms genetics, Neoplasms metabolism, Oligonucleotide Array Sequence Analysis standards, Organ Specificity, Polymerase Chain Reaction, RNA, Messenger analysis, Reference Standards, Gene Expression Profiling methods, Oligonucleotide Array Sequence Analysis methods, RNA, Messenger metabolism
- Abstract
We have developed RNA expression microarrays (REMs), in which each spot on a glass support is composed of a population of cDNAs synthesized from a cell or tissue sample. We used simultaneous hybridization with test and reference (housekeeping) genes to calculate an expression ratio based on normalization with the endogenous reference gene. A test REM containing artificial mixtures of liver cDNA and dilutions of the bacterial LysA gene cDNA demonstrated the feasibility of detecting transcripts at a sensitivity of four copies of LysA mRNA per liver cell equivalent. Furthermore, LysA cDNA detection varied linearly across a standard curve that matched the sensitivity of quantitative real-time PCR. In REMs with real samples, we detected organ-specific expression of albumin, Hnf-4 and Igfbp-1, in a set of mouse organ cDNA populations and c-Myc expression in tumor samples in paired tumor/normal tissue cDNA samples. REMs extend the use of classic microarrays in that a single REM can contain cDNAs from hundreds to thousands of cell or tissue samples each representing a specific physiological or pathophysiological state. REMs will extend the analysis of valuable samples by providing a common broad based platform for their analysis and will promote research aimed at defining gene functions, by broadening our understanding of their expression patterns in health and disease.
- Published
- 2004
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19. Gene expression pattern in hepatic stem/progenitor cells during rat fetal development using complementary DNA microarrays.
- Author
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Petkov PM, Zavadil J, Goetz D, Chu T, Carver R, Rogler CE, Bottinger EP, Shafritz DA, and Dabeva MD
- Subjects
- Animals, Animals, Newborn growth & development, Animals, Newborn physiology, Embryonic and Fetal Development, Fetus physiology, Oligonucleotide Array Sequence Analysis, Aging genetics, Gene Expression, Liver embryology, Liver physiology, Rats, Stem Cells physiology
- Abstract
To identify new and differentially expressed genes in rat fetal liver epithelial stem/progenitor cells during their proliferation, lineage commitment, and differentiation, we used a high throughput method-mouse complementary DNA (cDNA) microarrays-for analysis of gene expression. The gene expression pattern of rat hepatic cells was studied during their differentiation in vivo: from embryonic day (ED) 13 until adulthood. The differentially regulated genes were grouped into two clusters: a cluster of up-regulated genes comprised of 281 clones and a cluster of down-regulated genes comprised of 230 members. The expression of the latter increased abruptly between ED 16 and ED 17. Many of the overexpressed genes from the first cluster fall into distinct, differentially expressed functional groups: genes related to development, morphogenesis, and differentiation; calcium- and phospholipid-binding proteins and signal transducers; and cell adhesion, migration, and matrix proteins. Several other functional groups of genes that are initially down-regulated, then increase during development, also emerged: genes related to inflammation, blood coagulation, detoxification, serum proteins, amino acids, lipids, and carbohydrate metabolism. Twenty-eight genes overexpressed in fetal liver that were not detected in adult liver are suggested as potential markers for identification of liver progenitor cells. In conclusion, our data show that the gene expression program of fetal hepatoblasts differs profoundly from that of adult hepatocytes and that it is regulated in a specific manner with a major switch at ED 16 to 17, marking a dramatic change in the gene expression program during the transition of fetal liver progenitor cells from an undifferentiated to a differentiated state. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).
- Published
- 2004
- Full Text
- View/download PDF
20. Hepatitis B viruses : a triple threat for malignant transformation of hepatocytes.
- Author
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Rogler CE
- Subjects
- Hepatocytes cytology, Mutagenesis, Cell Transformation, Neoplastic, Hepatitis B virus physiology, Hepatocytes virology
- Published
- 2004
- Full Text
- View/download PDF
21. Transplantation of human hepatocytes in immunodeficient UPA mice: a model for the study of hepatitis B virus.
- Author
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Petersen J, Burda MR, Dandri M, and Rogler CE
- Subjects
- Animals, Base Sequence, DNA analysis, DNA Primers, Humans, Liver chemistry, Mice, Polymerase Chain Reaction, Transplantation, Heterologous, Cell Transplantation, Hepatocytes transplantation, Immunologic Deficiency Syndromes therapy
- Published
- 2004
- Full Text
- View/download PDF
22. Reduced hepatocyte proliferation is the basis of retarded liver tumor progression and liver regeneration in mice lacking N-acetylglucosaminyltransferase III.
- Author
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Yang X, Tang J, Rogler CE, and Stanley P
- Subjects
- Acetylglucosamine blood, Acetylglucosamine metabolism, Animals, Apoptosis physiology, Carbohydrate Sequence, Carcinogens, Cell Division physiology, Diethylnitrosamine, Disease Progression, Female, Gene Expression Regulation, Neoplastic, Glycoproteins blood, Glycoproteins metabolism, Hepatectomy, Hepatocytes cytology, Hepatocytes enzymology, Hepatocytes pathology, Liver Neoplasms, Experimental blood, Male, Mice, Mice, Inbred C57BL, Mice, Inbred CBA, Molecular Sequence Data, N-Acetylglucosaminyltransferases biosynthesis, N-Acetylglucosaminyltransferases genetics, N-Acetylglucosaminyltransferases metabolism, Phenobarbital, Vascular Endothelial Growth Factor A blood, Liver Neoplasms, Experimental enzymology, Liver Neoplasms, Experimental pathology, Liver Regeneration physiology, N-Acetylglucosaminyltransferases deficiency
- Abstract
Mice lacking N-acetylglucosaminyltransferase III (GlcNAc-TIII) exhibit slightly but significantly retarded liver tumor progression after a single injection of 10 micro g/g diethylnitrosamine (DEN) and continued administration of phenobarbital (PB) in drinking water. A key question is whether the absence of GlcNAc-TIII inhibits cell proliferation or induces apoptosis. Because PB aids tumor progression, we tested whether it diminished the difference in tumor progression between Mgat3(+/+) and Mgat3(Delta/Delta) mice. Here, we show that in the absence of PB, control males developed about twice as many liver tumor nodules as males lacking GlcNAc-TIII. Both the size of liver tumors and liver weights were significantly greater in DEN-treated wild-type or heterozygous mice. Apoptosis assays performed monthly after DEN treatment showed no differences between mutant and wild-type. However, there was a marked retardation in liver regeneration after partial (70%) hepatectomy (PH). Wild-type mice incorporated bromodeoxyuridine in approximately 15% of hepatocyte nuclei at 48 h after PH, whereas mice lacking GlcNAc-TIII had only approximately 5% positive nuclei. This was not because of enhanced apoptosis in mutant mice after PH. Expression of the Mgat3 gene remained undetectable in wild-type liver by Northern analysis after tumor induction or after PH. In addition, transgenic overexpression of GlcNAc-TIII in hepatocytes did not enhance tumor progression in Mgat3(Delta/Delta) mice, and there were no differences in tumor progression or liver regeneration after PH between control and transgenic mice overexpressing GlcNAc-TIII in liver. Therefore, the nonhepatic action of GlcNAc-TIII promotes hepatocyte proliferation after PH, as well as the progression of DEN-induced tumors, providing evidence for a functional role of the bisecting GlcNAc on circulating glycoprotein growth factor(s) that stimulate hepatocyte proliferation.
- Published
- 2003
23. Increase in de novo HBV DNA integrations in response to oxidative DNA damage or inhibition of poly(ADP-ribosyl)ation.
- Author
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Dandri M, Burda MR, Bürkle A, Zuckerman DM, Will H, Rogler CE, Greten H, and Petersen J
- Subjects
- Apoptosis, Blotting, Southern, Cloning, Molecular, DNA Repair drug effects, DNA Restriction Enzymes metabolism, Deoxyribonucleases, Type II Site-Specific metabolism, Enzyme Inhibitors pharmacology, Hepatoblastoma, Humans, Hydrogen Peroxide pharmacology, In Situ Nick-End Labeling, Liver Neoplasms, Poly Adenosine Diphosphate Ribose antagonists & inhibitors, Tumor Cells, Cultured, Virus Replication, DNA Damage, DNA, Viral genetics, Hepatitis B virus genetics, Oxidative Stress, Virus Integration
- Abstract
Chronic infection with hepatitis B virus (HBV) is associated with an increased risk for the development of cirrhosis and hepatocellular carcinoma (HCC). Although clonal HBV DNA integrations are detected in nearly all HCCs the role of these integrations in hepatocarcinogenesis is poorly understood. We have used a cloning protocol that allows studying the frequency and the natural history of HBV DNA integrations in cell culture. Southern blot analysis of the genomic DNA of HepG2 2.2.15 subclones, which replicate HBV, enabled us to detect new HBV DNA integrations in approximately 10% of the HepG 2.2.15 subclones over 4 rounds of sequential subcloning, whereas no loss of any preexisting HBV DNA integrations was observed. Treatments of HepG2 cells with H(2)O(2), designed to increase DNA damage, increased the frequency of HBV integrations to approximately 50% of the subclones and treatments designed to inhibit DNA repair, by inhibiting Poly(ADP-ribosyl)ation, also increased the frequency of HBV integration to 50%. These findings suggest that DNA strand breaks induced by oxidative stress during persistent HBV infection in humans may increase HBV DNA integration events, whereas PARP-1 activity may function to limit the occurrence of de novo HBV DNA integrations.
- Published
- 2002
- Full Text
- View/download PDF
24. Woodchuck hepatocytes remain permissive for hepadnavirus infection and mouse liver repopulation after cryopreservation.
- Author
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Dandri M, Burda MR, Gocht A, Török E, Pollok JM, Rogler CE, Will H, and Petersen J
- Subjects
- Animals, Cell Division, Cells, Cultured, DNA, Viral analysis, Hepatocytes transplantation, Marmota, Mice, Cryopreservation, Hepatitis B Virus, Woodchuck physiology, Hepatocytes physiology, Hepatocytes virology
- Abstract
Isolated hepatocytes represent a relevant model of the liver and are highly required both for research and therapeutic applications. However, sources of primary liver cells from human beings and from some animal species are limited. Therefore, cryopreservation of hepatocytes could greatly facilitate advances in various research areas. The aim of this study was to evaluate whether cryopreserved primary woodchuck hepatocytes could be used for woodchuck hepatitis B virus (WHV) infection studies, and whether they could maintain their regenerative potential in vivo after thawing. Critical steps for good quality of cryopreserved hepatocytes included the use of University of Wisconsin (UW) solution as a main component of the freezing medium, stepwise reduction of dimethylsulfoxide (DMSO) to avoid osmotic shock, and maintenance of low concentrations of DMSO in the culture medium. After cryopreservation, cell viability was still high (70% to 80%), and 50% to 60% of thawed cells attached to the plates. The appearance of covalently closed circular (ccc)DNA and of WHV-replicative forms a few days after in vitro infection demonstrated that thawed woodchuck hepatocytes were still susceptible to viral infection, thus proving maintenance of a very high hepatocyte-specific differentiation status. Furthermore, transplantation of woodchuck hepatocytes into the liver of urokinase-type plasminogen activator (uPA)/recombination activation gene-2 (RAG-2) mice, a model of liver regeneration, demonstrated that cryopreserved cells retained the ability to divide and to extensively repopulate a xenogenic liver. Notably, in vivo susceptibility to infection with WHV and proliferative capacity of frozen/thawed woodchuck hepatocytes in recipient mice were identical to those observed by transplanting fresh hepatocytes.
- Published
- 2001
- Full Text
- View/download PDF
25. Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus.
- Author
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Dandri M, Burda MR, Török E, Pollok JM, Iwanska A, Sommer G, Rogiers X, Rogler CE, Gupta S, Will H, Greten H, and Petersen J
- Subjects
- Adult, Animals, Cell Survival, Chimera, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Hepatocytes physiology, Humans, Liver metabolism, Mice, Mice, Knockout genetics, Mice, Transgenic genetics, Nuclear Proteins, Time Factors, Urokinase-Type Plasminogen Activator genetics, Urokinase-Type Plasminogen Activator metabolism, Hepatitis B pathology, Hepatocytes transplantation, Liver pathology, Transplantation, Heterologous
- Abstract
Mice containing livers repopulated with human hepatocytes would provide excellent in vivo models for studies on human liver diseases and hepatotropic viruses, for which no permissive cell lines exist. Here, we report partial repopulation of the liver of immunodeficient urokinase-type plasminogen activator (uPA)/recombinant activation gene-2 (RAG-2) mice with normal human hepatocytes isolated from the adult liver. In the transplanted mice, the production of human albumin was demonstrated, indicating that human hepatocytes remained functional in the mouse liver for at least 2 months after transplantation. Inoculation of transplanted mice with human hepatitis B virus (HBV) led to the establishment of productive HBV infection. According to human-specific genomic DNA analysis and immunostaining of cryostat liver sections, human hepatocytes were estimated to constitute up to 15% of the uPA/RAG-2 mouse liver. This is proof that normal human hepatocytes can integrate into the mouse hepatic parenchyma, undergo multiple cell divisions, and remain permissive for a human hepatotropic virus in a xenogenic liver. This system will provide new opportunities for studies on etiology and therapy of viral and nonviral human liver diseases, as well as on hepatocyte biology and hepatocellular transplantation.
- Published
- 2001
- Full Text
- View/download PDF
26. Duck hepatitis B virus DNA integration induced by oxidative radicals and its transmission from parent cells to offspring ones.
- Author
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Zhang L and Rogler CE
- Subjects
- Animals, Ducks, Free Radicals, DNA, Viral analysis, Hepatitis B Virus, Duck genetics, Hydrogen Peroxide pharmacology, Virus Integration drug effects
- Abstract
Objective: To investigate the role of oxidative radicals in duck hepatitis B virus DNA (DHBV DNA) integration and the transmission of integrated DHBV DNA from parent cells to their offspring cells., Methods: TUNEL assay and Southern blot hybridization were used to detect the cellular DNA strand breaks and the incidence of new type of DHBV DNA integration into cellular DNA of LMHD(21-6) cell line after the cells treated by low dose of hydrogen peroxide (10.0 micromol/L) during its growth from one cell to 3 +/- 10(7) to 5 +/- 10(7) cells., Results: The rate of cell death induced by low dose of H(2)O(2) (10.0 micromol/L) was 32.0%, but that induced by high dose (>10.0 micromol/L) was more than 50.0%. At the same time, the incidence of new DHBV DNA integration into cellular DNA induced by 10.0 micromol/L of H(2)O(2) was 50.0% (6/12) and that in control group was 8.33% (1/12). There was a significant difference between the two groups (P<0.05). Besides, the new integrated DHBV-DNA in the parent cells could be found in the DNA of their offspring cells with the same size of base pair., Conclusion: Oxidative radicals is one of the important inducing factors in DHBV-DNA integration into cellular DNA and the integrated DHBV-DNA is able to transmit from the parent cells to their offspring cells stably.
- Published
- 2000
27. New evidence for an extra-hepatic role of N-acetylglucosaminyltransferase III in the progression of diethylnitrosamine-induced liver tumors in mice.
- Author
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Yang X, Bhaumik M, Bhattacharyya R, Gong S, Rogler CE, and Stanley P
- Subjects
- Alleles, Animals, Blotting, Northern, Blotting, Southern, Blotting, Western, Diethylnitrosamine, Disease Progression, Excitatory Amino Acid Antagonists pharmacology, Exons, Gene Deletion, Genotype, Liver Neoplasms, Experimental chemically induced, Mice, Mice, Transgenic, N-Acetylglucosaminyltransferases genetics, Organ Size, Phenobarbital pharmacology, Phenotype, Promoter Regions, Genetic, Proteins genetics, Transcription, Genetic, Liver enzymology, Liver Neoplasms, Experimental enzymology, N-Acetylglucosaminyltransferases physiology
- Abstract
N-acetylglucosaminyltransferase III (GlcNAc-TIII) is encoded by the Mgat3 gene and catalyzes the addition of the bisecting GlcNAc to the core of N-glycans. Mice lacking GlcNAc-TIII due to the insertion mutation Mgat3tmlPst (termed Mgat3neo), exhibit retarded progression of liver tumors induced by diethylnitrosamine (DEN; M. Bhaumik et al, Cancer Res., 58: 2881-2887, 1998). This phenotype seemed to be due to a reduction, in activity or amount, of a circulating glycoprotein(s) that enhances DEN-induced liver tumor progression. Here, we provide new evidence to support this hypothesis. First, we show that mice with a deletion mutation of the Mgat3 gene coding exon (Mgat3tmlJxm, termed Mgat3delta) also exhibit retarded progression of DEN-induced liver tumors. At 7 months there was a significant decrease in liver weight (approximately 27%; P < 0.01), reflecting reduced tumor burden in Mgat3delta/delta mice. In addition, tumors were generally fewer and smaller, and histological changes were less severe in Mgat3delta/delta livers. Therefore, tumor progression is retarded in mice with two different null mutations in the Mgat3 gene. Second, we show that the development of DEN-induced tumors is unaltered by high levels of GlcNAc-TIII in the liver of transgenic mice. The Mgat3 gene coding exon under the control of the major urinary protein (MUP) promoter was used to generate transgenic mice that express GlcNAc-TIII in liver. Following DEN injection and phenobarbitol treatment, however, no significant differences were observed between MUP/Mgat3 transgenic and control mice in either tumor numbers or liver weight. The combined data provide strong evidence that retarded progression of tumors in mice lacking GlcNAc-TIII is due to the absence of the bisecting GlcNAc residue on N-glycans of a circulating glycoprotein(s) from a tissue other than liver.
- Published
- 2000
28. Lessons From Genetically Engineered Animal Models VI. Liver repopulation systems and study of pathophysiological mechanisms in animals.
- Author
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Gupta S and Rogler CE
- Subjects
- Animals, Hepatitis B surgery, Hepatitis B virology, Hepatitis, Animal surgery, Hepatitis, Animal virology, Liver cytology, Disease Models, Animal, Hepatitis B physiopathology, Hepatitis, Animal physiopathology, Liver Transplantation, Organisms, Genetically Modified
- Abstract
The ability to localize transplanted hepatocytes in the liver offers exciting new opportunities. Transplanted hepatocytes enter liver plates, form hybrid plasma membrane structures with adjacent hepatocytes, express liver genes correctly, and survive indefinitely. The transplanted cell mass is regulated, such that cell proliferation is limited in the normal adult liver, whereas the liver is repopulated extensively when proliferation rates in transplanted and host hepatocytes become dissociated or host hepatocytes are ablated selectively. Transplanted hepatocytes are susceptible to hepatitis viruses. These aspects of transplanted hepatocyte biology indicate that liver repopulation systems can help address questions concerning pathophysiological mechanisms.
- Published
- 1999
- Full Text
- View/download PDF
29. Human DNA topoisomerase I-mediated cleavage and recombination of duck hepatitis B virus DNA in vitro.
- Author
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Pourquier P, Jensen AD, Gong SS, Pommier Y, and Rogler CE
- Subjects
- Animals, Base Sequence, Binding Sites, Chickens, Chromosome Mapping, DNA, Single-Stranded, Ducks virology, Humans, Molecular Sequence Data, Sequence Analysis, DNA, Tumor Cells, Cultured, Virion, DNA Topoisomerases, Type I metabolism, DNA, Viral metabolism, Hepatitis B Virus, Duck genetics, Recombination, Genetic
- Abstract
In this study, we report that eukaryotic topoisomerase I (top1) can linearize the open circular DNA of duck hepatitis B virus (DHBV). Using synthetic oligonucleotides mimicking the three-strand flap DR1 region of the DHBV genome, we found that top1 cleaves the DNA plus strand in a suicidal manner, which mimics the linearization of the virion DNA. We also report that top1 can cleave the DNA minus strand at specific sites and can linearize the minus strand via a non-homologous recombination reaction. These results are consistent with the possibility that top1 can act as a DNA endo-nuclease and strand transferase and play a role in the circularization, linearization and possibly integration of viral replication intermediates.
- Published
- 1999
- Full Text
- View/download PDF
30. Double-stranded linear duck hepatitis B virus (DHBV) stably integrates at a higher frequency than wild-type DHBV in LMH chicken hepatoma cells.
- Author
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Gong SS, Jensen AD, Chang CJ, and Rogler CE
- Subjects
- Animals, Base Sequence, Chickens, Molecular Sequence Data, Repetitive Sequences, Nucleic Acid, Telomere, Tumor Cells, Cultured, Virion, DNA, Circular, DNA, Viral, Hepatitis B Virus, Duck genetics, Virus Integration
- Abstract
Integration of hepadnavirus DNAs into host chromosomes can have oncogenic consequences. Analysis of host-viral DNA junctions of DHBV identified the terminally duplicated r region of the viral genome as a hotspot for integration. Since the r region is present on the 5' and 3' ends of double-stranded linear (DSL) hepadnavirus DNAs, these molecules have been implicated as integration precursors. We have produced a LMH chicken hepatoma cell line (LMH 66-1 DSL) which replicates exclusively DSL duck hepatitis B virus (DHBV) DNA. To test whether linear DHBV DNAs integrate more frequently than the wild type open circular DHBV DNAs, we have characterized the integration frequency in LMH 66-1 DSL cells by using a subcloning approach. This approach revealed that 83% of the LMH 66-1 DSL subclones contained new integrations, compared to only 16% of subclones from LMH-D2 cells replicating wild-type open circular DHBV DNA. Also, a higher percentage of the LMH 66-1 DSL subclones contained two or more new integrations. Mathematical analysis suggests that the DSL DHBV DNAs integrated stably once every three generations during subcloning whereas wild-type DHBV integrated only once every four to five generations. Cloning and sequencing of new integrations confirmed the r region as a preferred integration site for linear DHBV DNA molecules. One DHBV integrant was associated with a small deletion of chromosomal DNA, and another DHBV integrant occurred in a telomeric repeat sequence.
- Published
- 1999
- Full Text
- View/download PDF
31. Metabolic labeling of woodchuck hepatitis B virus X protein in naturally infected hepatocytes reveals a bimodal half-life and association with the nuclear framework.
- Author
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Dandri M, Petersen J, Stockert RJ, Harris TM, and Rogler CE
- Subjects
- Animals, Cell Compartmentation, Cell Nucleus metabolism, Cell Nucleus virology, Cytosol metabolism, Cytosol virology, Half-Life, Hepatitis B metabolism, Hepatitis B virology, Hepatitis B Virus, Woodchuck pathogenicity, Kinetics, Solubility, Subcellular Fractions metabolism, Subcellular Fractions virology, Hepatitis B veterinary, Hepatitis B Virus, Woodchuck metabolism, Liver metabolism, Liver virology, Marmota, Trans-Activators metabolism, Viral Proteins metabolism
- Abstract
In order to identify potential sites of hepadnavirus X protein action, we have investigated the subcellular distribution and the stability of woodchuck hepatitis virus (WHV) X protein (WHx) in primary hepatocytes isolated from woodchucks with persistent WHV infection. In vivo cell labeling and cell fractionation studies showed that the majority of WHx is a soluble cytoplasmic protein while a minor part of newly synthesized WHx is associated with a nuclear framework fraction (20%) and with cytoskeletal components (5 to 10%). Pulse-chase experiments revealed that cytoplasmic WHx has a short half-life and decays with bimodal kinetics (approximately 20 min and 3 h). The rates of association and turnover of nucleus-associated WHx suggest that compartmentalization may be responsible for the bimodal turnover observed in the cytoplasm.
- Published
- 1998
- Full Text
- View/download PDF
32. Antisense downregulation of N-myc1 in woodchuck hepatoma cells reverses the malignant phenotype.
- Author
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Wang HP, Zhang L, Dandri M, and Rogler CE
- Subjects
- Animals, Base Sequence, Carcinoma, Hepatocellular, Cell Line, DNA, Viral, Gene Expression, Genetic Vectors, Marmota, Mice, Mice, Nude, Molecular Sequence Data, Nucleic Acid Conformation, Phenotype, Tumor Cells, Cultured, Virus Integration, Gene Expression Regulation, Viral, Genes, myc, Hepatitis B Virus, Woodchuck genetics, RNA, Antisense, RNA, Viral
- Abstract
Cell line WH44KA is a highly malignant woodchuck hepatoma cell line. WH44KA cells contain a single woodchuck hepatitis virus (WHV) DNA integration in the 3' untranslated region of exon 3 of the woodchuck N-myc1 gene. The highly rearranged WHV DNA contains WHV enhancers which activate the N-myc promoter, and a hybrid N-myc1-WHV mRNA is produced, which leads to a high steady-state level of N-myc1 protein. To investigate whether continuous N-myc1 expression is required to maintain the tumor phenotype, we knocked out N-myc expression using a WHV-N-myc1 antisense vector. We identified two WH44KA antisense cell lines, designated 4-5 and 4-11, in which steady-state N-mycl protein levels were reduced by 95 and 80%, respectively. The growth rates of both antisense cell lines were reduced in comparison to those of wild-type and vector controls. The phenotype of 4-5 and 4-11 cells changed to a flattened appearance, and the cells exhibited contact inhibition. Colony-forming ability in soft agar was reduced by 92% for 4-5 cells and by 88% for 4-11 cells. Cell line 4-11 formed only small, slow-growing tumors in nude mice, consistent with a low level of N-myc1 remaining in the cells. In contrast, 4-5 cells, in which N-myc protein was reduced by greater than 95%, failed to form tumors in nude mice. The integrated WHV DNA contained the complete WHV X gene (WHx) and its promoter; however, we did not detect any WHx protein in the cells by using a sensitive assay. These data demonstrate that N-myc overexpression is required to maintain the malignant phenotype of WH44KA woodchuck hepatoma cells and provide a direct function for integrated WHV DNA in hepatocarcinogenesis.
- Published
- 1998
- Full Text
- View/download PDF
33. Reactivation of the maternally imprinted IGF2 allele in TGFalpha induced hepatocellular carcinomas in mice.
- Author
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Harris TM, Rogler LE, and Rogler CE
- Subjects
- Animals, Mice, Mice, Transgenic, Polymerase Chain Reaction, Promoter Regions, Genetic, Transforming Growth Factor alpha physiology, Alleles, Carcinoma, Hepatocellular genetics, Genomic Imprinting, Insulin-Like Growth Factor II genetics, Liver Neoplasms genetics, Transforming Growth Factor alpha genetics
- Abstract
The Insulin like growth factor 2 (IGF2) gene is expressed in several types of tumors in humans and mice and has been implicated as an important growth factor in tumor progression. IGF2 expression in the TGF alpha transgenic mice was analysed in liver and tumors from animals which also contained one or two functional IGF2 alleles. In a two by two mating experiment using transgenic mice containing either a TGF alpha transgene or a IGF2 gene knockout, we have investigated whether IGF2 imprinting is reversed during hepatocarcinogenesis and the consequences of IGF2 expression for tumor growth. We observed that: (1) 100% of the hepatocellular carcinomas expressed IGF2 (2) the normally imprinted maternal allele is active in the tumors in which the paternal allele is knocked out and (3) all three of the murine IGF2 promoters upstream of the reactivated maternal alleles are transcriptionally active in tumors. We also observed that the total tumor burden of animals with two wild type IGF-2 alleles (paternal and maternal) was the same as the tumor burden in animals which contained only a single reactivated maternal allele. The 100% incidence of reactivation of the imprinted maternal allele suggests that IGF2 expression is selected during murine hepatocarcinogenesis and can substitute for the paternal allele when it is inactivated.
- Published
- 1998
- Full Text
- View/download PDF
34. Liver repopulation with xenogenic hepatocytes in B and T cell-deficient mice leads to chronic hepadnavirus infection and clonal growth of hepatocellular carcinoma.
- Author
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Petersen J, Dandri M, Gupta S, and Rogler CE
- Subjects
- Animals, Antineoplastic Agents pharmacology, Chronic Disease, Clone Cells, DNA-Binding Proteins genetics, DNA-Binding Proteins physiology, Dexamethasone pharmacology, Interferon-alpha pharmacology, Liver Neoplasms, Experimental genetics, Liver Transplantation immunology, Marmota, Mice, Transplantation, Heterologous, Urokinase-Type Plasminogen Activator genetics, Urokinase-Type Plasminogen Activator physiology, Virus Replication, B-Lymphocytes physiology, Hepadnaviridae Infections pathology, Liver pathology, Liver Neoplasms, Experimental pathology, Liver Transplantation pathology, T-Lymphocytes physiology
- Abstract
To investigate host and viral mechanisms determining hepadnaviral persistence and hepatocarcinogenesis, we developed a mouse model by transplanting woodchuck hepatocytes into the liver of mice that contain the urokinase-type plasminogen activator transgene (uPA) and lack mature B and T lymphocytes due to a recombination activation gene 2 (RAG-2) gene knockout. The woodchuck hepatocytes were transplanted via intrasplenic injection and were found to integrate into the recipient mouse liver cord structure. Normal adult woodchuck hepatocytes proliferated and reconstituted up to 90% of the uPA/RAG-2 mouse liver. uPA/RAG-2 mice containing woodchuck hepatocytes were infectable with woodchuck hepatitis virus (WHV) and showed WHV replication for at least 10 months with titers up to 1 x 10(11) virions per ml in the peripheral blood. WHV-infected hepatocytes from chronic carrier woodchucks also established a persistent infection in uPA/RAG-2 mice after an 8- to 12-week lag period of viremia. Although WHV envelope, core, and X proteins were produced in the uPA/RAG-2 mice, no inflammatory host immune response was observed in the liver of WHV-replicating mice. A first antiviral test demonstrated a greater than four orders of magnitude drop in WHV titer in response to interferon alpha treatment. WHV replication was up-regulated by dexamethasone treatment. Comparison of precancerous lesions in donor woodchucks versus recipient uPA/RAG-2 mice revealed an enrichment of dysplastic precancerous hepatocytes in transplanted mice. Clonal amplification of hepatocytes from a woodchuck with hepatocellular carcinomas was demonstrated by the detection of unique WHV DNA integration patterns in hepatocellular carcinomas that arose in uPA/RAG-2 mice. In the absence of B or T cell-mediated immune responses, WHV establishes a persistent noncytotoxic infection of woodchuck hepatocytes in uPA/RAG-2 chimeric mouse livers. Further studies of the kinetics of hepadnavirus infection and replication in quiescent and proliferating hepatocytes should increase our understanding of hepadnavirus spread and aid in the design of therapies to block or cure persistent infection.
- Published
- 1998
- Full Text
- View/download PDF
35. Increase in the frequency of hepadnavirus DNA integrations by oxidative DNA damage and inhibition of DNA repair.
- Author
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Petersen J, Dandri M, Bürkle A, Zhang L, and Rogler CE
- Subjects
- Animals, Benzamides pharmacology, Cell Division drug effects, Chickens, Enzyme Inhibitors pharmacology, Oxidative Stress, Poly(ADP-ribose) Polymerase Inhibitors, Tumor Cells, Cultured, DNA Damage, DNA Repair, DNA, Viral, Hepatitis B Virus, Duck genetics, Hydrogen Peroxide pharmacology, Oxidants pharmacology, Virus Integration drug effects
- Abstract
Persistent hepadnavirus infection leads to oxidative stress and DNA damage through increased production of toxic oxygen radicals. In addition, hepadnaviral DNA integrations into chromosomal DNA can promote the process of hepatocarcinogenesis (M. Feitelson, Clin. Microbiol. Rev. 5:275-301, 1992). While previous studies have identified preferred integration sites in hepadnaviral genomes and suggested integration mechanisms (M. A. Buendia, Adv. Cancer Res. 59:167-226, 1992; C. E. Rogler, Curr. Top. Microbiol. Immunol. 168:103-141, 1991; C. Shih et al., J. Virol. 61:3491-3498, 1987), very little is known about the effects of agents which damage chromosomal DNA on the frequency of hepadnaviral DNA integrations. Using a recently developed subcloning approach to detect stable new integrations of duck hepatitis B virus (DHBV) (S. S. Gong, A. D. Jensen, and C. E. Rogler, J. Virol. 70:2000-2007, 1996), we tested the effects of increased chromosomal DNA damage induced by H2O2, or of the disturbance in DNA repair due to the inhibition of poly(ADP-ribose) polymerase (PARP), on the frequency of DHBV DNA integrations. Subclones of LMH-D21-6 cells, which replicate DHBV, were grown in the presence of various H2O2 concentrations and exhibited up to a threefold increase in viral DNA integration frequency in a dose-dependent manner. Moreover, inhibition of PARP, which plays a role in cellular responses to DNA breakage, by 3-aminobenzamide (3-AB) resulted in a sevenfold increase in the total number of new DHBV DNA integrations into host chromosomal DNA. Removal of either H2O2 or 3-AB from the culture medium in a subsequent cycle of subcloning was accompanied by a reversion back towards the original lower frequency of stable DHBV DNA integrations for LMH-D21-6 cells. These data support the hypothesis that DNA damage sites can serve as sites for hepadnaviral DNA integration, and that increasing the number of DNA damage sites dramatically increases viral integration frequency.
- Published
- 1997
- Full Text
- View/download PDF
36. The hepatitis B virus (HBV) precore protein inhibits HBV replication in transgenic mice.
- Author
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Guidotti LG, Matzke B, Pasquinelli C, Shoenberger JM, Rogler CE, and Chisari FV
- Subjects
- Animals, Mice, Mice, Transgenic, Trans-Activators genetics, Gene Expression Regulation, Viral, Genes, Viral, Hepatitis B virus physiology, Viral Core Proteins genetics, Viral Envelope Proteins genetics, Virus Replication genetics
- Abstract
In this study, we examined the ability of the hepatitis B virus (HBV) precore, envelope, and X gene products to modulate HBV replication in the livers of transgenic mice that replicate the virus. Hepatic HBV replication was not affected by overexpression of the envelope or X gene products when these animals were crossed with transgenic mice that express the corresponding viral genes in the hepatocyte. Overexpression of the precore protein, however, eliminated nucleocapsid particles from the cytoplasm of the hepatocytes and abolished HBV replication without affecting the hepatic steady-state content of pregenomic HBV RNA. These observations suggest that the precore protein can exert a dominant negative effect on HBV replication, presumably at the level of nucleocapsid particle maturation or stability, suggesting an important role for this enigmatic viral protein in the HBV life cycle.
- Published
- 1996
- Full Text
- View/download PDF
37. Insulin-like growth factor II blocks apoptosis of N-myc2-expressing woodchuck liver epithelial cells.
- Author
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Yang D, Faris R, Hixson D, Affigne S, and Rogler CE
- Subjects
- Animals, Carcinoma, Hepatocellular, Cell Division, Cell Line, Culture Media, Serum-Free, DNA analysis, DNA drug effects, Epithelial Cells, Epithelium drug effects, Epithelium physiology, Humans, Kinetics, Liver physiology, Liver Neoplasms, Marmota, Recombinant Proteins biosynthesis, Recombinant Proteins pharmacology, Transfection, Apoptosis, Insulin-Like Growth Factor II pharmacology, Liver cytology, Proto-Oncogene Proteins c-myc biosynthesis
- Abstract
N-myc2 and insulin-like growth factor II (IGF-II) are coordinately overexpressed in the great majority of altered hepatic foci, which are the earliest precancerous lesions observed in the liver of woodchuck hepatitis virus carrier woodchucks, and these genes continue to be overexpressed in hepatocellular carcinomas (HCCs). We have investigated the function of these genes in woodchuck hepatocarcinogenesis by using a woodchuck liver epithelial cell line (WC-3). WC-3 cells react positively with a monoclonal antibody (12.8.5) against woodchuck oval cells, suggesting a lineage relationship with oval cells. Overexpression of N-myc2 in three WC-3 cell lines caused their morphological transformation and increased their growth rate and saturation density in medium containing 10% serum. Removal of serum from the medium increased cell death of the N-myc2-expressing lines, whereas cell death in control lines was minimal. The death of N-myc2-expressing WC-3 cells was accompanied by nucleosomal fragmentation of cellular DNA, and DAPI (4',6-diamidino-2-phenylindole) staining revealed condensation and fragmentation of the nuclei, suggesting that N-myc2-expressing WC-3 cells undergo apoptosis in the absence of serum. In colony regression assays, conducted in the absence of serum, control colonies were stable, while N-myc2-expressing colonies regressed to various degrees. Addition of recombinant human IGF-II to the serum-free medium blocked both cell death and colony regression in all the N-myc2-expressing lines. Therefore, coordinate overexpression of N-myc2 and IGF-II in woodchuck altered hepatic foci may allow cells which otherwise might die to survive and progress to hepatocellular carcinoma.
- Published
- 1996
- Full Text
- View/download PDF
38. Woodchuck hepatitis virus X protein is present in chronically infected woodchuck liver and woodchuck hepatocellular carcinomas which are permissive for viral replication.
- Author
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Dandri M, Schirmacher P, and Rogler CE
- Subjects
- Animals, Carcinoma, Hepatocellular pathology, Carcinoma, Hepatocellular virology, Hepatitis B pathology, Hepatitis B virology, Liver Neoplasms, Experimental pathology, Liver Neoplasms, Experimental virology, Marmota, Mice, Carcinoma, Hepatocellular metabolism, Gene Products, tax biosynthesis, Hepatitis B metabolism, Hepatitis B Virus, Woodchuck physiology, Liver Neoplasms, Experimental metabolism, Virus Replication
- Abstract
The woodchuck hepatitis virus (WHV) X gene (WHx) is required for infectivity of WHV in woodchucks, and the gene encodes a broadly acting transcription factor. Several lines of evidence from cell culture and transgenic mice suggest that X proteins can promote hepatocarcinogenesis. To determine whether WHx-encoded proteins are present during persistent infection and hepatocellular carcinoma (HCC) in woodchucks, we surveyed livers and HCCs from a panel of WHV carrier woodchucks for the presence of WHx by utilizing an immunoprecipitation-Western blot (immunoblot) procedure. We detected a single 15.5-kDa WHx gene product in 100% of the persistently infected livers but not in livers from animals which had recovered from acute infection or in those of uninfected woodchucks. Analysis of HCCs revealed that all of the tumors which contained WHV replication intermediates were also positive for WHx. In contrast, WHx was undetectable in HCCs which did not contain replicative intermediates. Subcellular localization studies detected WHx in the cytoplasm but not in the nuclei of primary woodchuck hepatocytes. Comparative immunoprecipitation experiments revealed that there were 4 X 10(4) to 8 X 10(4) molecules of WHx per primary woodchuck hepatocyte. Four lines of WHx transgenic mice did not develop HCC spontaneously. However, when one line was treated with diethylnitrosamine, the occurrence of precancerous lesions was enhanced compared with that in diethylnitrosamine-treated nontransgenic controls. The apparent absence of WHx in some woodchuck HCCs indicates that WHx may not be required to maintain the tumor phenotype, whereas its presence in all persistently infected livers leaves open the possibility that it plays a role in hepatocarcinogenesis.
- Published
- 1996
- Full Text
- View/download PDF
39. Loss and acquisition of duck hepatitis B virus integrations in lineages of LMH-D2 chicken hepatoma cells.
- Author
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Gong SS, Jensen AD, and Rogler CE
- Subjects
- Animals, Carcinoma, Hepatocellular pathology, Chickens, Chromosomes, DNA, Viral, Gene Rearrangement, Genetic Vectors genetics, Hepatitis B Virus, Duck genetics, Plasmids, Tumor Cells, Cultured, Carcinoma, Hepatocellular virology, Hepatitis B Virus, Duck physiology, Virus Integration
- Abstract
Hepatocellular carcinoma is the culmination of a series of genetic events which progressively alter the phenotype of a hepatocyte toward malignancy. Hepadnaviral DNA integrations are agents of genetic change which can promote the process of hepatocarcinogenesis. We previously characterized episomally derived duck hepatitis B virus (DHBV) integrations in LMH-D2 cells that replicate wild-type DHBV. In an effort to understand how integrations function as agents of progressive genetic change, we have studied integrations of DHBV DNA in three lineages of LMH-D2 cells through three generations of subclones. Our data have established several features of the integration process. First, single and multiple integrations occur continuously through successive cell generations. Second, the integration frequency can vary dramatically in subclones of the same cell line. Third, integrations can be lost from successive generations of cells and loss of an integration can be accompanied by loss of cellular DNA associated with the integration. Finally, certain subclones which acquire greater plating efficiency have been distinguished by unique new integration patterns. These results provide a basis for DHBV integrations to function as activators of protooncogenes, as well as agents of the loss of tumor suppressor genes during hepatocellular carcinogenesis.
- Published
- 1996
- Full Text
- View/download PDF
40. Hepatic overexpression of insulin-like growth factor-II in adulthood increases basal and insulin-stimulated glucose disposal in conscious mice.
- Author
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Rossetti L, Barzilai N, Chen W, Harris T, Yang D, and Rogler CE
- Subjects
- Adult, Animals, Glucokinase metabolism, Glucose Clamp Technique, Glucose-6-Phosphatase metabolism, Glycogen metabolism, Glycogen Synthase metabolism, Humans, Insulin-Like Growth Factor II genetics, Liver enzymology, Liver metabolism, Mice, Mice, Inbred C57BL, Mice, Inbred CBA, Mice, Transgenic, Muscle, Skeletal metabolism, Phosphoenolpyruvate Carboxykinase (GTP) metabolism, Tritium, Glucose metabolism, Insulin metabolism, Insulin-Like Growth Factor II metabolism
- Abstract
The physiological role of circulating insulin-like growth factor-II (IGF-II) in adult humans is poorly understood. We recently generated an IGF-II transgenic murine model of persistent IGF-II production (plasma IGF-II approximately 30-fold increased above normal) through over-expression of the transgene driven by the major urinary protein promoter (Rinderknecht, E., and Humbel, R. E. (1978) J. Biol. Chem. 269, 13779-13784). To determine whether in vivo insulin action is improved in these transgenic mice, we performed euglycemic insulin (18 milliunits/kg.min) clamp studies in conscious IGF-II transgenic and in age- and weight-matched control mice. Plasma glucose and insulin concentrations were significantly lower in the IGF-II transgenic compared with both control grouoff Despite decreased plasma glucose concentration, basal hepatic glucose production (HGP) and glucose clearance were increased. During the insulin clamp studies in IGF-II transgenic mice compared with control mice (a) the rates of glucose infusion and glucose uptake were increased by approximately by 65 and approximately 55%, respectively; (b) glycolysis was increased by approximately 12% while glycogen synthesis was approximately 2-fold higher; (c) while the suppression of plasma free fatty acid was similar, the increment in plasma lactate concentration was significantly higher; (d) although HGP was similarly inhibited by insulin, phosphoenolpyruvate gluconeogenesis was enhanced and accounted for a larger portion of HGP (64% versus approximately 40% in control mice). Our data suggest that the persistence of circulating IGF-II in adult mice to levels commonly observed in adult humans (50-70 nM) causes a marked improvement in peripheral (skeletal muscle) insulin action, which is not due to changes in body composition. These results suggest that circulating IGF-II may exert a regulatory role on insulin sensitivity and body composition in humans.
- Published
- 1996
- Full Text
- View/download PDF
41. Duck hepatitis B virus integrations in LMH chicken hepatoma cells: identification and characterization of new episomally derived integrations.
- Author
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Gong SS, Jensen AD, Wang H, and Rogler CE
- Subjects
- Animals, Base Sequence, Blotting, Southern, Carcinoma, Hepatocellular genetics, Carcinoma, Hepatocellular virology, Chickens, Chromosome Mapping, Clone Cells, Cloning, Molecular, DNA Primers, DNA, Viral analysis, Genome, Viral, Liver Neoplasms genetics, Liver Neoplasms virology, Molecular Sequence Data, Polymerase Chain Reaction, Proviruses genetics, Restriction Mapping, Tumor Cells, Cultured, Bird Diseases, Carcinoma, Hepatocellular veterinary, Hepatitis Virus, Duck genetics, Liver Neoplasms veterinary, Virus Integration
- Abstract
While the cytoplasmic phase of the hepadnavirus replication cycle is well understood, very little is known about the nuclear phase. In contrast to retroviruses, proviral integration is not required for hepadnavirus replication; however, some of the viral DNAs in the nucleus are diverted into an integration pathway. Under certain conditions these integrations function as carcinogenic agents. In order to study the integration process, we have utilized LMH-D2 cells, which replicate wild-type duck hepatitis B virus (DHBV), to develop the first protocol to detect and characterize integrations of DHBV originating from episomal viral DNAs. Contrary to expectations, our results showed that stable new integrations are readily detectable in subclones of LMH-D2 cells. Complete characterization of one integration revealed a single-genome-length integrant with the structure of double-stranded linear (DSL) DHBV DNAs which are produced by in situ priming during viral replication. The integration contained a terminal redundancy of 6 bp from the r region of the virus DNA minus strand as well as a direct repeat of 70 bp of cellular DNA. On the basis of the structure of the integrant and the cellular DNA target site, we propose a molecular model for the integration mechanism that has some similarities to that of retroviruses. Identification of DSL hepadnavirus DNA integration suggests the possibility that modified DSL viral DNAs may be the precursors to a class of simple, unrearranged hepadnavirus integrations.
- Published
- 1995
- Full Text
- View/download PDF
42. Sequences and structures at hepadnaviral integration: recombination sites implicate topoisomerase I in hepadnaviral DNA rearrangements and integration.
- Author
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Schirmacher P, Wang H, Stahnke G, Will H, and Rogler CE
- Subjects
- Base Sequence, Humans, Molecular Sequence Data, Nucleic Acid Conformation, Sequence Homology, Nucleic Acid, DNA Topoisomerases, Type I physiology, DNA, Viral genetics, Gene Rearrangement, Hepadnaviridae genetics, Recombination, Genetic, Virus Integration genetics
- Abstract
We have previously shown that eukaryotic topoisomerase I (topo I) can mediate hepadnaviral integration in vitro. To investigate further the possible in vivo significance of topo I in hepadnaviral integration and to detect additional important factors, we have generated an extensive compilation of hepadnaviral recombination sites from chronically infected liver tissues and hepatocellular carcinomas. These sequences were subjected to various established sequence and structural analyses. Our investigation provides evidence that topo I can mediate hepadnaviral integration and rearrangement in vivo. During integration, free ends can be exposed to other nuclear enzymes, resulting in the addition of 'filler DNA'. In other cases, junctional homologies between viral and cellular DNA may facilitate integration. Structural analysis suggests that torsional stress may act on the cellular target sites, possibly promoting the integration process. A mechanism is proposed by which hepadnavirus integration into the host chromosomes is primarily mediated by topo I.
- Published
- 1995
43. Hepatitis B virus nucleocapsid particles do not cross the hepatocyte nuclear membrane in transgenic mice.
- Author
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Guidotti LG, Martinez V, Loh YT, Rogler CE, and Chisari FV
- Subjects
- Animals, Base Sequence, Cell Compartmentation, Cell Cycle, Cell Nucleus microbiology, DNA Primers chemistry, Hepatitis B Core Antigens metabolism, Hepatitis B virus ultrastructure, Mice, Mice, Transgenic, Mitosis, Molecular Sequence Data, RNA, Viral metabolism, Time Factors, Virus Replication, Capsid metabolism, Hepatitis B virus growth & development, Liver microbiology, Viral Core Proteins metabolism
- Abstract
Transgenic mice that express the hepatitis B virus core protein were used to examine factors that influence the intracellular localization of nucleocapsid particles in the primary hepatocyte in vivo. In this model, viral nucleocapsid particles are strictly localized to the nucleus of the hepatocyte except when the nuclear membrane dissolves during cell division, at which time they enter the cytoplasm. The cytoplasmic nucleocapsid particles do not reenter the nucleus, however, when the nuclear membrane re-forms after cell division. The data support the notion that nucleocapsid particles can form de novo within the nucleus, and they suggest that performed nucleocapsid particles cannot be transported across the intact nuclear membrane in either direction. The results imply that nucleocapsid disassembly is probably required for entry of the hepadnaviral genome into the nucleus, and they question the role of the intranuclear viral nucleocapsid particle during the viral life cycle.
- Published
- 1994
- Full Text
- View/download PDF
44. Altered body composition and increased frequency of diverse malignancies in insulin-like growth factor-II transgenic mice.
- Author
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Rogler CE, Yang D, Rossetti L, Donohoe J, Alt E, Chang CJ, Rosenfeld R, Neely K, and Hintz R
- Subjects
- Animals, Carrier Proteins genetics, Carrier Proteins metabolism, Female, Heterozygote, Homozygote, Incidence, Insulin-Like Growth Factor Binding Proteins, Insulin-Like Growth Factor II genetics, Male, Mice, Mice, Transgenic, Neoplasms, Experimental epidemiology, Neoplasms, Experimental genetics, Somatomedins genetics, Somatomedins metabolism, Body Composition genetics, Insulin-Like Growth Factor II physiology, Neoplasms, Experimental etiology
- Abstract
The physiological role of insulin-like growth factor (IGF) II (IGF-II) in adult humans is poorly understood. Rather high levels of IGF-II persist in adult human serum, whereas, in rodents, IGF-II levels are very low. To investigate the physiological and carcinogenic effects of persistently elevated IGF-II in adults, we have produced two lines of transgenic mice in which high levels of IGF-II (20- or 30-fold increase above normal) are persistently maintained in the blood. The transgene is driven by the major urinary protein promoter, and it is highly expressed in the liver and perputial glands in both lines. The adult transgenic mice are smaller than controls, and their body composition is altered. Their lean body mass is reduced by 5-8%, whereas fat mass is reduced between 44 and 77%. The mice expressing the highest level of IGF-II (30x) develop hypoglycemia and hypoinsulinemia and IGF-I levels are normal. Mice in the lower expression line (20-fold elevated IGF-II) develop hypoglycemia progressively over their lifetime. Mice from both lines also develop a diverse spectrum of tumors at a higher frequency than controls after 18 months of age, and the most frequent types of tumors are hepatocellular carcinomas and lymphomas. Squamous cell carcinoma, sarcoma, and thyroid carcinomas also occurred in our test group. The long latent period before tumors arise and the wide spectrum of tumor types suggest that IGF-II may function primarily as a tumor progression factor in mice via autocrine and endocrine mechanisms of action.
- Published
- 1994
45. Coordinate expression of N-myc 2 and insulin-like growth factor II in precancerous altered hepatic foci in woodchuck hepatitis virus carriers.
- Author
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Yang D, Alt E, and Rogler CE
- Subjects
- Animals, Gene Expression, Genes, Viral, Hepadnaviridae genetics, Histones genetics, In Situ Hybridization, Liver microbiology, Liver Diseases microbiology, Liver Neoplasms genetics, Liver Neoplasms, Experimental genetics, RNA, Messenger genetics, S Phase, Viral Structural Proteins genetics, Genes, myc, Hepadnaviridae pathogenicity, Insulin-Like Growth Factor II genetics, Liver Diseases genetics, Marmota genetics, Precancerous Conditions genetics
- Abstract
Over 50% of the hepatocellular carcinomas (HCCs) arising in the livers of woodchucks with persistent woodchuck hepatitis virus (WHV) infection contain integrations of WHV DNA within, or immediately adjacent to, a unique and functional N-myc 2 retroposon [G. Fourel et al., Nature (Lond.), 347: 294-298, 1990; Y. Wei et al., J. Virol., 66: 5265-5276, 1992]. The integrations are believed to activate the expression of N-myc 2 by an enhancer insertion mechanism [Y. Wei et al., J. Virol., 66: 5265-5276, 1992]. Since the fetal growth factor insulin-like growth factor II (IGF-II) is also expressed in woodchuck HCCs [X. X. Fu et al., J. Virol., 62: 3422-3430, 1988; D. Yang and C. E. Rogler, Carcinogenesis (Lond.), 12: 1893-1901, 1991] we sought to determine the earliest stage in hepatocarcinogenesis at which overexpression of N-myc and IGF-II could be detected. The earliest precancerous lesions so far identified in woodchucks are altered hepatic foci (AHFs) [K. Abe et al., Jpn. J. Cancer Res., 79: 466-472, 1988; H. Popper et al., Hepatology (Baltimore), 1: 91-98, 1981]. Using in situ hybridization, we have demonstrated that both the N-myc and IGF-II genes are coordinately overexpressed in nearly all AHFs in precancerous woodchuck livers. In contrast, WHV replication was either repressed or undetectable in the same AHFs. The use of probes selective for N-myc 2 versus N-myc 1 (the normal mammalian homologue) revealed nearly exclusive expression of N-myc 2 in AHFs. Cells within AHFs were generally slow growing, as determined by frequency of histone III-expressing hepatocytes; however, a few fast-growing AHFs, with growth rates nearly equivalent to those of HCCs, were identified. Furthermore, very highly elevated N-myc 2 or IGF-II expression was detected in a few subregions within AHFs which otherwise exhibited a uniformly moderate expression, suggesting that selection for higher levels of N-myc or IGF-II expression may occur within AHFs. These data suggest that coordinate expression of N-myc 2 and IGF-II and repression of WHV replication may be functionally involved in the development of AHFs and that cells expressing very high levels of N-myc and IGF-II may be selectively enriched as AHFs progress to HCC, since high levels of N-myc and IGF-II are common in HCCs.
- Published
- 1993
46. The role of Ito cells in the biosynthesis of HGF-SF in the liver.
- Author
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Schirmacher P, Geerts A, Jung W, Pietrangelo A, Rogler CE, and Dienes HP
- Subjects
- Acute Disease, Animals, Chronic Disease, Hepatocyte Growth Factor metabolism, Humans, Liver cytology, Liver Diseases metabolism, Models, Biological, Proto-Oncogene Proteins c-met, Receptors, Cell Surface metabolism, Hepatocyte Growth Factor biosynthesis, Liver metabolism
- Abstract
Hepatocyte Growth Factor-Scatter Factor (HGF-SF) is produced by sinusoidal cells in normal rat liver. Analysis of isolated cells has proven that Ito cells (fat-storing cells, hepatic lipocytes) are the source of hepatic HGF. In diseased liver the HGF-expression pattern is more complex. In acute liver injury HGF is expressed by an increased number of resident liver cells, which may be due to recruitment of other nonparenchymal liver cells as well as an increased number of Ito cells due to cell division. In cirrhotic rat liver tissue, the number of HGF-expressing cells is decreased. This may be explained by the complete loss of HGF-expression in myofibroblast-like cells derived from Ito cells. Quiescent Ito cells seem to be in a strategic position to control parenchymal cell proliferation. Activation of Ito cells, which occurs in chronic fibrotic liver disease, may lead to the loss of this control function.
- Published
- 1993
47. Current pathogenetic and molecular concepts in viral liver carcinogenesis.
- Author
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Schirmacher P, Rogler CE, and Dienes HP
- Subjects
- Animals, Carcinoma, Hepatocellular pathology, Disease Models, Animal, Genes, Tumor Suppressor, Growth Substances physiology, Hepatitis B complications, Hepatitis C complications, Humans, Liver Neoplasms pathology, Liver Neoplasms, Experimental etiology, Liver Neoplasms, Experimental pathology, Precancerous Conditions etiology, Precancerous Conditions pathology, Proto-Oncogenes, Risk Factors, Carcinoma, Hepatocellular etiology, Liver Neoplasms etiology
- Abstract
Hepatocellular carcinoma (HCC) is one of the most frequent malignancies in humans and in most cases a consequence of chronic infection of the liver by hepatotropic viruses (Hepatitis B Virus (HBV) and possibly Hepatitis C Virus (HCV)). Formation of HCC results from a stepwise process involving different preneoplastic lesions that reflect multiple genetic events, like protooncogene activation, tumor suppressor gene inactivation, and growth factor over- or reexpression. Recent investigations have gained new insights into how these factors are activated and may interact. In addition, improved knowledge of the molecular biology of HBV has led to better understanding of its pleiotropic effects on induction and progression in hepatocarcinogenesis.
- Published
- 1993
- Full Text
- View/download PDF
48. Cellular and molecular mechanisms of hepatocarcinogenesis.
- Author
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Rogler CE and Chisari FV
- Subjects
- Animals, DNA, Viral genetics, Hepadnaviridae genetics, Hepadnaviridae pathogenicity, Hepatitis B complications, Hepatocyte Growth Factor physiology, Humans, Insulin-Like Growth Factor II physiology, Liver injuries, Virus Diseases complications, Virus Integration, Liver Neoplasms etiology
- Abstract
For colorectal carcinomas, the rate of tumor development is proportional to the fourth to sixth power of elapsed time, suggesting that four to six independent events are necessary. Although similar calculations have not been made for HBV-associated HCCs, it is likely that this is also the case for HCCs, since individuals with persistent HBV infection do not usually develop HCC until they are 45 or greater years old. As evidence for specific genetic and epigenetic changes in HCCs accumulate, the important players in multistep hepatocarcinogenesis are becoming clearer. However, even though Myc family oncogenes are clearly implicated in woodchuck HCC, similar integrations have not been found in human HCCs. Therefore, although rodent and human systems have many similarities, we must realize that important differences may also exist. Regarding tumor suppressor genes, the evidence for p53 alterations in HCC is strong. A growing body of evidence suggests further that alterations in the retinoblastoma gene and one or more tumor suppressor genes on chromosome 11 are also involved in HCC. HBV integrations may certainly play a role in the generation of chromosome aberrations leading to loss of tumor suppressor alleles, since chromosomes 11 and 17 are the most common integration sites. Finally, the role of X proteins as participants in malignant transformation has been demonstrated for certain immortalized, nontransformed hepatocytes. Altered autocrine mechanisms of cell growth control, possibly involving IGF-II, are clearly implicated in HCC. Paracrine mechanisms for the control of hepatocyte growth and differentiated functions may also be altered as a result of the synthesis and secretion of a complex array of interleukins, HGF, and basic and acidic FGFs by cells in the inflammatory and cirrhotic lesions of precancerous livers. Whether the order of molecular changes in the hepatocyte is important for malignant progression is presently not clear. What is clear, however, is that hepatocarcinogenesis involves alterations in the concerted action of protooncogenes, growth factor, and tumor suppressor genes. How these factors interact will provide a more complete understanding of the mechanism or mechanisms of hepatic oncogenesis.
- Published
- 1992
- Full Text
- View/download PDF
49. Reactivation of insulin-like growth factor II during hepatocarcinogenesis in transgenic mice suggests a role in malignant growth.
- Author
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Schirmacher P, Held WA, Yang D, Chisari FV, Rustum Y, and Rogler CE
- Subjects
- Animals, Blotting, Northern, Cell Division, Histones metabolism, Liver metabolism, Liver Neoplasms, Experimental etiology, Liver Neoplasms, Experimental pathology, Mice, S Phase, Antigens, Polyomavirus Transforming metabolism, Insulin-Like Growth Factor II metabolism, Liver Neoplasms, Experimental metabolism, RNA, Messenger metabolism
- Abstract
We have studied the expression of insulin-like growth factor II (IGF-II) during hepatocarcinogenesis in four independent transgenic mouse lines. In all four lines liver-directed transgene expression induces a stepwise and relatively synchronized tumorigenesis. IGF-II reexpression occurs in all four lines irrespective of the mechanism of tumor induction. Reexpression is chronologically associated with late progression steps toward hepatocellular carcinoma and correlated with the respective tumor progression rate in each line. IGF-II activation is focal and topographically associated with high replicative activity. IGF-II mRNAs in hepatocellular carcinomas show similarities to the expression pattern in fetal liver, and a M(r) 15,000 IGF-II polypeptide accumulates intracellularly in distinct cytoplasmic preferentially perinuclear compartments. These data indicate that IGF-II reexpression is a marker for progression to hepatocellular carcinoma and may contribute to hepatocarcinogenesis via an autocrine mechanism.
- Published
- 1992
50. Hepatocyte growth factor/hepatopoietin A is expressed in fat-storing cells from rat liver but not myofibroblast-like cells derived from fat-storing cells.
- Author
-
Schirmacher P, Geerts A, Pietrangelo A, Dienes HP, and Rogler CE
- Subjects
- Animals, Blotting, Northern, Cell Line, Fibroblasts physiology, Gene Expression, Growth Substances genetics, Hepatocyte Growth Factor, Lipid Metabolism, Liver cytology, Male, Nucleic Acid Hybridization, RNA, Messenger analysis, Rats, Rats, Inbred Strains, Transcription, Genetic, Fibroblasts metabolism, Growth Substances metabolism, Liver metabolism, Muscle, Smooth cytology
- Abstract
Hepatocyte growth factor/hepatopoietin A is a complete mitogen for parenchymal liver cells, and its expression is increased as an early response to acute liver injury. To identify the liver cell population responsible for hepatocyte growth factor gene expression, we investigated tissue sections and isolated and purified cell fractions from normal rat liver by in situ and Northern blot hybridization. Hepatocyte growth factor transcripts were present in sinusoidal liver cells, which were preferentially located in the periportal parenchyma. Northern hybridization analysis of RNA isolated from purified liver cell fractions demonstrated that HGF messenger RNA is present only in fat-storing cells. No specific hepatocyte growth factor gene expression was detected in parenchymal cells, endothelial cells and Kupffer cells. Myofibroblast-like transition of fat-storing cells, which is linked to fibrogenesis in chronic liver disease, results in the loss of hepatocyte growth factor expression. Hepatocyte growth factor gene expression in the normal liver, a new function of fat-storing cells, suggests that this growth factor may play a role in the physiological balance between cell death and replacement in the liver and that hepatocyte growth factor may also act in a paracrine manner. Furthermore, loss of hepatocyte growth factor expression in myofibroblast-like cells derived from fat-storing cells may be responsible for reduced parenchymal cell regeneration in chronic liver disease.
- Published
- 1992
- Full Text
- View/download PDF
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