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1
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Lambrecht R, Delgado ME, Gloe V, Schuetz K, Plazzo AP, Franke B, San Phan T, Fleming J, Mayans O, Brunner T. Liver receptor homolog-1 (NR5A2) orchestrates hepatic inflammation and TNF-induced cell death. Cell Rep 2023; 42:113513. [PMID: 38039134 DOI: 10.1016/j.celrep.2023.113513] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2023] [Revised: 10/09/2023] [Accepted: 11/14/2023] [Indexed: 12/03/2023] Open
Abstract
The nuclear receptor liver receptor homolog-1 (LRH-1) has been shown to promote apoptosis resistance in various tissues and disease contexts; however, its role in liver cell death remains unexplored. Hepatocyte-specific deletion of LRH-1 causes mild steatosis and inflammation but unexpectedly shields female mice from tumor necrosis factor (TNF)-induced hepatocyte apoptosis and associated hepatitis. LRH-1-deficient hepatocytes show markedly attenuated estrogen receptor alpha and elevated nuclear factor κB (NF-κB) activity, while LRH-1 overexpression inhibits NF-κB activity. This inhibition relies on direct physical interaction of LRH-1's ligand-binding domain and the Rel homology domain of NF-κB subunit RelA. Mechanistically, increased transcription of anti-apoptotic NF-κB target genes and the proteasomal degradation of pro-apoptotic BCL-2 interacting mediator of cell death prevent mitochondrial apoptosis and ultimately protect mice from TNF-induced liver damage. Collectively, our study emphasizes LRH-1 as a critical, sex-dependent regulator of cell death and inflammation in the healthy and diseased liver.
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Affiliation(s)
- Rebekka Lambrecht
- Biochemical Pharmacology, Department of Biology, University of Konstanz, Universitätsstrasse 10, 78464 Konstanz, Germany
| | - M Eugenia Delgado
- Biochemical Pharmacology, Department of Biology, University of Konstanz, Universitätsstrasse 10, 78464 Konstanz, Germany
| | - Vincent Gloe
- Biochemical Pharmacology, Department of Biology, University of Konstanz, Universitätsstrasse 10, 78464 Konstanz, Germany
| | - Karina Schuetz
- Biochemical Pharmacology, Department of Biology, University of Konstanz, Universitätsstrasse 10, 78464 Konstanz, Germany
| | - Anna Pia Plazzo
- Biochemical Pharmacology, Department of Biology, University of Konstanz, Universitätsstrasse 10, 78464 Konstanz, Germany
| | - Barbara Franke
- Biophysics and Structural Biology, Department of Biology, University of Konstanz, Universitätsstrasse 10, 78464 Konstanz, Germany
| | - Truong San Phan
- Biochemical Pharmacology, Department of Biology, University of Konstanz, Universitätsstrasse 10, 78464 Konstanz, Germany
| | - Jennifer Fleming
- Biophysics and Structural Biology, Department of Biology, University of Konstanz, Universitätsstrasse 10, 78464 Konstanz, Germany
| | - Olga Mayans
- Biophysics and Structural Biology, Department of Biology, University of Konstanz, Universitätsstrasse 10, 78464 Konstanz, Germany
| | - Thomas Brunner
- Biochemical Pharmacology, Department of Biology, University of Konstanz, Universitätsstrasse 10, 78464 Konstanz, Germany.
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2
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Voloshin N, Tyurin-Kuzmin P, Karagyaur M, Akopyan Z, Kulebyakin K. Practical Use of Immortalized Cells in Medicine: Current Advances and Future Perspectives. Int J Mol Sci 2023; 24:12716. [PMID: 37628897 PMCID: PMC10454025 DOI: 10.3390/ijms241612716] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2023] [Revised: 07/23/2023] [Accepted: 08/09/2023] [Indexed: 08/27/2023] Open
Abstract
In modern science, immortalized cells are not only a convenient tool in fundamental research, but they are also increasingly used in practical medicine. This happens due to their advantages compared to the primary cells, such as the possibility to produce larger amounts of cells and to use them for longer periods of time, the convenience of genetic modification, the absence of donor-to-donor variability when comparing the results of different experiments, etc. On the other hand, immortalization comes with drawbacks: possibilities of malignant transformation and/or major phenotype change due to genetic modification itself or upon long-term cultivation appear. At first glance, such issues are huge hurdles in the way of immortalized cells translation into medicine. However, there are certain ways to overcome such barriers that we describe in this review. We determined four major areas of usage of immortalized cells for practical medicinal purposes, and each has its own means to negate the drawbacks associated with immortalization. Moreover, here we describe specific fields of application of immortalized cells in which these problems are of much lesser concern, for example, in some cases where the possibility of malignant growth is not there at all. In general, we can conclude that immortalized cells have their niches in certain areas of practical medicine where they can successfully compete with other therapeutic approaches, and more preclinical and clinical trials with them should be expected.
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Affiliation(s)
- Nikita Voloshin
- Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia; (N.V.); (P.T.-K.); (M.K.)
| | - Pyotr Tyurin-Kuzmin
- Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia; (N.V.); (P.T.-K.); (M.K.)
| | - Maxim Karagyaur
- Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia; (N.V.); (P.T.-K.); (M.K.)
| | - Zhanna Akopyan
- Medical Research and Education Center, Lomonosov Moscow State University, 119234 Moscow, Russia;
| | - Konstantin Kulebyakin
- Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia; (N.V.); (P.T.-K.); (M.K.)
- Medical Research and Education Center, Lomonosov Moscow State University, 119234 Moscow, Russia;
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3
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Esposito R, Lanzós A, Uroda T, Ramnarayanan S, Büchi I, Polidori T, Guillen-Ramirez H, Mihaljevic A, Merlin BM, Mela L, Zoni E, Hovhannisyan L, McCluggage F, Medo M, Basile G, Meise DF, Zwyssig S, Wenger C, Schwarz K, Vancura A, Bosch-Guiteras N, Andrades Á, Tham AM, Roemmele M, Medina PP, Ochsenbein AF, Riether C, Kruithof-de Julio M, Zimmer Y, Medová M, Stroka D, Fox A, Johnson R. Tumour mutations in long noncoding RNAs enhance cell fitness. Nat Commun 2023; 14:3342. [PMID: 37291246 PMCID: PMC10250536 DOI: 10.1038/s41467-023-39160-7] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2022] [Accepted: 06/01/2023] [Indexed: 06/10/2023] Open
Abstract
Long noncoding RNAs (lncRNAs) are linked to cancer via pathogenic changes in their expression levels. Yet, it remains unclear whether lncRNAs can also impact tumour cell fitness via function-altering somatic "driver" mutations. To search for such driver-lncRNAs, we here perform a genome-wide analysis of fitness-altering single nucleotide variants (SNVs) across a cohort of 2583 primary and 3527 metastatic tumours. The resulting 54 mutated and positively-selected lncRNAs are significantly enriched for previously-reported cancer genes and a range of clinical and genomic features. A number of these lncRNAs promote tumour cell proliferation when overexpressed in in vitro models. Our results also highlight a dense SNV hotspot in the widely-studied NEAT1 oncogene. To directly evaluate the functional significance of NEAT1 SNVs, we use in cellulo mutagenesis to introduce tumour-like mutations in the gene and observe a significant and reproducible increase in cell fitness, both in vitro and in a mouse model. Mechanistic studies reveal that SNVs remodel the NEAT1 ribonucleoprotein and boost subnuclear paraspeckles. In summary, this work demonstrates the utility of driver analysis for mapping cancer-promoting lncRNAs, and provides experimental evidence that somatic mutations can act through lncRNAs to enhance pathological cancer cell fitness.
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Affiliation(s)
- Roberta Esposito
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland.
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland.
- Institute of Genetics and Biophysics "Adriano Buzzati-Traverso", CNR, 80131, Naples, Italy.
| | - Andrés Lanzós
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
- Graduate School of Cellular and Biomedical Sciences, University of Bern, 3012, Bern, Switzerland
| | - Tina Uroda
- School of Biology and Environmental Science, University College Dublin, Dublin, D04 V1W8, Ireland
- Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Dublin, D04 V1W8, Ireland
| | - Sunandini Ramnarayanan
- School of Biology and Environmental Science, University College Dublin, Dublin, D04 V1W8, Ireland
- Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Dublin, D04 V1W8, Ireland
- The SFI Centre for Research Training in Genomics Data Science, Dublin, Ireland
| | - Isabel Büchi
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
- Department of Visceral Surgery and Medicine, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland
| | - Taisia Polidori
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
| | - Hugo Guillen-Ramirez
- School of Biology and Environmental Science, University College Dublin, Dublin, D04 V1W8, Ireland
- Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Dublin, D04 V1W8, Ireland
| | - Ante Mihaljevic
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
| | - Bernard Mefi Merlin
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
| | - Lia Mela
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
| | - Eugenio Zoni
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
- Department of Urology, Inselspital, Bern University Hospital, Bern, Switzerland
| | - Lusine Hovhannisyan
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
- Department of Radiation Oncology, Inselspital, Bern University Hospital and University of Bern, Bern, Switzerland
| | - Finn McCluggage
- School of Molecular Sciences, University of Western Australia, Crawley, WA, Australia
- School of Human Sciences, University of Western Australia, Crawley, WA, Australia
| | - Matúš Medo
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
- Department of Radiation Oncology, Inselspital, Bern University Hospital and University of Bern, Bern, Switzerland
| | - Giulia Basile
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
| | - Dominik F Meise
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
| | - Sandra Zwyssig
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
| | - Corina Wenger
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
| | - Kyriakos Schwarz
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
| | - Adrienne Vancura
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
| | - Núria Bosch-Guiteras
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
- Graduate School of Cellular and Biomedical Sciences, University of Bern, 3012, Bern, Switzerland
| | - Álvaro Andrades
- GENYO, Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government, Granada, 18016, Spain
- Instituto de Investigación Biosanitaria, Granada, 18014, Spain
- Department of Biochemistry and Molecular Biology I, University of Granada, Granada, 18071, Spain
| | - Ai Ming Tham
- School of Biology and Environmental Science, University College Dublin, Dublin, D04 V1W8, Ireland
- Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Dublin, D04 V1W8, Ireland
| | - Michaela Roemmele
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
| | - Pedro P Medina
- GENYO, Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government, Granada, 18016, Spain
- Instituto de Investigación Biosanitaria, Granada, 18014, Spain
- Department of Biochemistry and Molecular Biology I, University of Granada, Granada, 18071, Spain
| | - Adrian F Ochsenbein
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
| | - Carsten Riether
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
| | - Marianna Kruithof-de Julio
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
- Department of Urology, Inselspital, Bern University Hospital, Bern, Switzerland
| | - Yitzhak Zimmer
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
- Department of Radiation Oncology, Inselspital, Bern University Hospital and University of Bern, Bern, Switzerland
| | - Michaela Medová
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
- Department of Radiation Oncology, Inselspital, Bern University Hospital and University of Bern, Bern, Switzerland
| | - Deborah Stroka
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland
- Department of Visceral Surgery and Medicine, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland
| | - Archa Fox
- School of Molecular Sciences, University of Western Australia, Crawley, WA, Australia
- School of Human Sciences, University of Western Australia, Crawley, WA, Australia
| | - Rory Johnson
- Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, 3010, Bern, Switzerland.
- Department for BioMedical Research, University of Bern, 3008, Bern, Switzerland.
- School of Biology and Environmental Science, University College Dublin, Dublin, D04 V1W8, Ireland.
- Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Dublin, D04 V1W8, Ireland.
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4
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Sepehrinezhad A, Shahbazi A, Sahab Negah S, Joghataei MT, Larsen FS. Drug-induced-acute liver failure: A critical appraisal of the thioacetamide model for the study of hepatic encephalopathy. Toxicol Rep 2021; 8:962-970. [PMID: 34026559 PMCID: PMC8122178 DOI: 10.1016/j.toxrep.2021.04.011] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2021] [Revised: 04/17/2021] [Accepted: 04/27/2021] [Indexed: 12/12/2022] Open
Abstract
Hepatic encephalopathy (HE) following acute and chronic liver failure is defined as a complex of neuropsychiatric abnormalities, such as discrete personal changes, sleep disorder, forgetfulness, confusion, and decreasing the level of consciousness to coma. The use and design of suitable animal models that represent clinical features and pathological changes of HE are valuable to map the molecular mechanisms that result in HE. Among different types of animal models, thioacetamide (TAA) has been used extensively for the induction of acute liver injury and HE. This agent is not directly hepatotoxic but its metabolites induce liver injury through the induction of oxidative stress and produce systemic inflammation similar to that seen in acute HE patients. In this short review article, we shortly review the most important pathological findings in animal models of acute HE following the administration of TAA.
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Key Words
- ALT, alanine aminotransferase
- AQP4, aquaporin 4 water channel
- AST, aspartate aminotransferase
- Acute liver failure
- Animal model
- B7, B7 molecules (CD80+CD86)
- BBB, blood-brain barrier
- CBF, cerebral blood flow
- CCL2, chemokine ligand 2
- CNS, central nervous system
- CTLA4, Cytotoxic T-lymphocyte-associated Protein 4
- CYP2E1, Cytochrome P450 family 2 subfamily E member 1
- GFAP, glial fibrillary acidic protein
- HE, hepatic encephalopathy
- Hepatic encephalopathy
- IL-6, interleukin 6
- IL-β, interleukin 1 β
- Iba1, ionized calcium-binding adaptor molecule 1
- JNK, c-Jun N-terminal kinase
- NAC, N-acetylcysteine
- NF-κB, nuclear factor κB
- OA, L-ornithine-l-aspartate
- ROS, reactive oxygen species
- TAA, thioacetamide
- TASO, thioacetamide sulfoxide
- TASO2, thioacetamide sulfdioxide
- TLR-2, toll-like receptor 2
- TLR-4, toll-like receptor 4
- TNFα, tumor necrosis factor α
- Thioacetamide
- Toxicity pathway
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Affiliation(s)
- Ali Sepehrinezhad
- Department of Neuroscience, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
- Neuroscience Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Ali Shahbazi
- Department of Neuroscience, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
- Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Sajad Sahab Negah
- Neuroscience Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Shefa Neuroscience Research Center, Khatam Alanbia Hospital, Tehran, Iran
- Department of Neuroscience, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Mohammad Taghi Joghataei
- Department of Neuroscience, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
- Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Fin Stolze Larsen
- Department of Hepatology CA-3163, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, 2100, Copenhagen, Denmark
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Perrone F, Craparo EF, Cemazar M, Kamensek U, Drago SE, Dapas B, Scaggiante B, Zanconati F, Bonazza D, Grassi M, Truong N, Pozzato G, Farra R, Cavallaro G, Grassi G. Targeted delivery of siRNAs against hepatocellular carcinoma-related genes by a galactosylated polyaspartamide copolymer. J Control Release 2021; 330:1132-1151. [PMID: 33212117 DOI: 10.1016/j.jconrel.2020.11.020] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2020] [Revised: 11/07/2020] [Accepted: 11/11/2020] [Indexed: 02/08/2023]
Abstract
Given the lack of effective treatments for Hepatocellular carcinoma (HCC), the development of novel therapeutic approaches is very urgent. Here, siRNAs were delivered to HCC cells by a synthetic polymer containing α,β-poly-(N-2-hydroxyethyl)-D,L-aspartamide-(PHEA) derivatized with diethylene triamine (DETA) and bearing in the side chain galactose (GAL) linked via a polyethylene glycol (PEG) to obtain (PHEA-DETA-PEG-GAL, PDPG). The GAL residue allows the targeting to the asialo-glycoprotein receptor (ASGPR), overexpressed in HCC cells compared to normal hepatocytes. Uptake studies performed using a model siRNA or a siRNA targeted against the enhanced green fluorescence protein, demonstrated the PDPG specific delivery of siRNA to HuH7 cells, a human cellular model of HCC. GAL-free copolymer (PHEA-DETA-PEG-NH2, PDP) or the chemical block of ASGPR, impaired PDPG targeting effectiveness in vitro. The specificity of PDPG delivery was confirmed in vivo in a mouse dorsal skinfold window chamber assay. Functional studies using siRNAs targeting the mRNAs of HCC-related genes (eEF1A1, eEF1A2 and E2F1) delivered by PDPG, significantly decreased HuH7 vitality/number and down regulated the expression of the target genes. Only minor effectiveness was in contrast observed for PDP. In IHH, a human model of normal hepatocytes with reduced ASGPR expression, PDPG barely reduced cell vitality. In a subcutaneous xenograft mouse model of HCC, PDPG-siRNAs reduced HCC tumor growth compared to controls without significant toxic effects. In conclusion, our study demonstrates the valuable potentials of PDPG for the specific delivery of siRNAs targeting HCC-related genes.
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Affiliation(s)
- Francesca Perrone
- Department of Life Sciences, Cattinara University Hospital, Trieste University, Strada di Fiume 447, Trieste I-34149, Italy
| | - Emanuela Fabiola Craparo
- Department of Scienze e Tecnologie Biologiche, Chimiche, Farmaceutiche (STEBICEF), Lab of Biocompatible Polymers, University of Palermo, via Archirafi 32, Palermo 90123, Italy
| | - Maja Cemazar
- Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, Ljubljana SI-1000, Slovenia; Faculty of Health Sciences, University of Primorska, Polje 42, SI-, Izola 6310, Slovenia
| | - Urska Kamensek
- Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, Ljubljana SI-1000, Slovenia
| | - Salvatore Emanuele Drago
- Department of Scienze e Tecnologie Biologiche, Chimiche, Farmaceutiche (STEBICEF), Lab of Biocompatible Polymers, University of Palermo, via Archirafi 32, Palermo 90123, Italy
| | - Barbara Dapas
- Department of Life Sciences, Cattinara University Hospital, Trieste University, Strada di Fiume 447, Trieste I-34149, Italy
| | - Bruna Scaggiante
- Department of Life Sciences, Cattinara University Hospital, Trieste University, Strada di Fiume 447, Trieste I-34149, Italy
| | - Fabrizio Zanconati
- Department of Medical, Surgical and Health Sciences, University of Trieste, Cattinara Hospital, Strada di Fiume, Trieste 447, Italy
| | - Debora Bonazza
- Department of Medical, Surgical and Health Sciences, University of Trieste, Cattinara Hospital, Strada di Fiume, Trieste 447, Italy
| | - Mario Grassi
- Department of Engineering and Architecture, University of Trieste, Via Valerio 6/A, Trieste I 34127, Italy
| | - Nhung Truong
- Stem Cell Research and Application Laboratory - VNUHCM - University of Science, Ho Chi Minh city, Viet Nam
| | - Gabriele Pozzato
- Department of Medical, Surgical and Health Sciences, University of Trieste, Cattinara Hospital, Strada di Fiume, Trieste 447, Italy
| | - Rossella Farra
- Department of Life Sciences, Cattinara University Hospital, Trieste University, Strada di Fiume 447, Trieste I-34149, Italy.
| | - Gennara Cavallaro
- Department of Scienze e Tecnologie Biologiche, Chimiche, Farmaceutiche (STEBICEF), Lab of Biocompatible Polymers, University of Palermo, via Archirafi 32, Palermo 90123, Italy.
| | - Gabriele Grassi
- Department of Life Sciences, Cattinara University Hospital, Trieste University, Strada di Fiume 447, Trieste I-34149, Italy; Department of Medical, Surgical and Health Sciences, University of Trieste, Cattinara Hospital, Strada di Fiume, Trieste 447, Italy
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6
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Tu T, Zhang H, Urban S. Hepatitis B Virus DNA Integration: In Vitro Models for Investigating Viral Pathogenesis and Persistence. Viruses 2021; 13:v13020180. [PMID: 33530322 PMCID: PMC7911709 DOI: 10.3390/v13020180] [Citation(s) in RCA: 51] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2020] [Revised: 01/12/2021] [Accepted: 01/21/2021] [Indexed: 02/07/2023] Open
Abstract
Hepatitis B virus (HBV) is a globally-distributed pathogen and is a major cause of liver disease. HBV (or closely-related animal hepadnaviruses) can integrate into the host genome, but (unlike retroviruses) this integrated form is replication-defective. The specific role(s) of the integrated HBV DNA has been a long-standing topic of debate. Novel in vitro models of HBV infection combined with sensitive molecular assays now enable researchers to investigate this under-characterised phenomenon with greater ease and precision. This review covers the contributions these systems have made to understanding how HBV DNA integration induces liver cancer and facilitates viral persistence. We summarise the current findings into a working model of chronic HBV infection and discuss the clinical implications of this hypothetical framework on the upcoming therapeutic strategies used to curb HBV-associated pathogenesis.
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Affiliation(s)
- Thomas Tu
- Storr Liver Centre, Faculty of Medicine and Health, Westmead Clinical School and Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW 2145, Australia;
- Centre for Infectious Diseases and Microbiology, Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney at Westmead Hospital, Westmead, NSW 2145, Australia
- Correspondence:
| | - Henrik Zhang
- Storr Liver Centre, Faculty of Medicine and Health, Westmead Clinical School and Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW 2145, Australia;
| | - Stephan Urban
- Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany;
- German Center for Infection Research (DZIF), Heidelberg Partner Site, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany
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8
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Sato M, Saitoh I, Inada E, Nakamura S, Watanabe S. Potential for Isolation of Immortalized Hepatocyte Cell Lines by Liver-Directed In Vivo Gene Delivery of Transposons in Mice. Stem Cells Int 2019; 2019:5129526. [PMID: 31281376 PMCID: PMC6589260 DOI: 10.1155/2019/5129526] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2019] [Accepted: 05/06/2019] [Indexed: 12/14/2022] Open
Abstract
Isolation of hepatocytes and their culture in vitro represent important avenues to explore the function of such cells. However, these studies are often difficult to perform because of the inability of hepatocytes to proliferate in vitro. Immortalization of isolated hepatocytes is thus an important step toward continuous in vitro culture. For cellular immortalization, integration of relevant genes into the host chromosomes is a prerequisite. Transposons, which are mobile genetic elements, are known to facilitate integration of genes of interest (GOI) into chromosomes in vitro and in vivo. Here, we proposed that a combination of transposon- and liver-directed introduction of nucleic acids may confer acquisition of unlimited cellular proliferative potential on hepatocytes, enabling the possible isolation of immortalized hepatocyte cell lines, which has often failed using more traditional immortalization methods.
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Affiliation(s)
- Masahiro Sato
- Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan
| | - Issei Saitoh
- Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata 951-8514, Japan
| | - Emi Inada
- Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan
| | - Shingo Nakamura
- Division of Biomedical Engineering, National Defense Medical College Research Institute, Saitama 359-8513, Japan
| | - Satoshi Watanabe
- Animal Genome Unit, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki 305-0901, Japan
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Fang SY, Hu CQ, Liu MN, Tao L, Wang Y, Gong MJ, He Y, He TC, Bi Y. Reversibly immortalized hepatic progenitor cell line containing double suicide genes. Int J Mol Med 2018; 42:1977-1986. [PMID: 30085335 PMCID: PMC6108856 DOI: 10.3892/ijmm.2018.3803] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2018] [Accepted: 08/01/2018] [Indexed: 01/14/2023] Open
Abstract
A large number of functional hepatocytes is required for bioartificial liver therapy. Simian virus 40 T-antigen (SV40T) has been previously reported to improve the immortalized proliferation of primary hepatocytes to generate a sufficient number of cells; however, these long-term immortalized hepatocytes may induce further malignant transformation in vivo. In the present study, the SV40T immortalization gene and two suicide genes, herpes simplex virus thymidine kinase (HSV-tk) and cytosine deaminase (CD), were transducted into primary hepatocytes to construct a novel type of Cre/LoxP-mediated reversible immortalized hepatocyte line. Polymerase chain reaction analysis and western blotting confirmed that the SV40T, HSV-tk and CD genes were successfully inserted into hepatic progenitor cells and their expression was controlled by Cre/LoxP recombination. Total removal of SV40T could be achieved via the ganciclovir (GCV)/HSV-tk suicide system. Cells maintained their biosafety in vivo with CD gene expression and 5-fluoro-cytosine (5-FC) induced cell death. Following transplantation into the carbon tetrachloride (CCl4) model group, the majority of cells had survived after 14 days post-implantation and a number of the cells had transported into the liver parenchyma. When compared with the CCl4 model group, the transplanted cells repaired the liver biochemical index and pathological structure markedly. Thus, the present study reports a novel reversible immortalized hepatocyte with double suicide genes, which exhibited the cellular phenotype and recovery function of normal liver cells. This method maximally guaranteed the biological safety of immortalized hepatocytes for in vivo application, providing a reliable, safe and ideal cell material for the artificial liver technique.
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Affiliation(s)
- Shu-Yu Fang
- Department of Pediatric Surgery, Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Stem Cell Therapy Engineering Technical Center, The Children's Hospital of Chongqing Medical University, Chongqing 400014, P.R. China
| | - Chao-Qun Hu
- Department of Pediatric Surgery, Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Stem Cell Therapy Engineering Technical Center, The Children's Hospital of Chongqing Medical University, Chongqing 400014, P.R. China
| | - Meng-Nan Liu
- Department of Pediatric Surgery, Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Stem Cell Therapy Engineering Technical Center, The Children's Hospital of Chongqing Medical University, Chongqing 400014, P.R. China
| | - Li Tao
- Department of Pediatric Surgery, Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Stem Cell Therapy Engineering Technical Center, The Children's Hospital of Chongqing Medical University, Chongqing 400014, P.R. China
| | - Yi Wang
- Department of Pediatric Surgery, Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Stem Cell Therapy Engineering Technical Center, The Children's Hospital of Chongqing Medical University, Chongqing 400014, P.R. China
| | - Meng-Jia Gong
- Department of Pediatric Surgery, Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Stem Cell Therapy Engineering Technical Center, The Children's Hospital of Chongqing Medical University, Chongqing 400014, P.R. China
| | - Yun He
- Department of Pediatric Surgery, Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Stem Cell Therapy Engineering Technical Center, The Children's Hospital of Chongqing Medical University, Chongqing 400014, P.R. China
| | - Tong-Chuan He
- Department of Pediatric Surgery, Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Stem Cell Therapy Engineering Technical Center, The Children's Hospital of Chongqing Medical University, Chongqing 400014, P.R. China
| | - Yang Bi
- Department of Pediatric Surgery, Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Stem Cell Therapy Engineering Technical Center, The Children's Hospital of Chongqing Medical University, Chongqing 400014, P.R. China
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10
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Hang HL, Liu XY, Wang HT, Xu N, Bian JM, Zhang JJ, Xia L, Xia Q. Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival. Exp Cell Res 2017; 360:81-93. [PMID: 28870599 DOI: 10.1016/j.yexcr.2017.08.020] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2017] [Revised: 08/10/2017] [Accepted: 08/12/2017] [Indexed: 12/22/2022]
Abstract
Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application. This study aimed to determine the role of hepatocyte nuclear factor 4A (HNF4A) in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with acute liver failure (ALF). Primary human hepatocytes were transduced with lentivirus-mediated catalytic subunit of human telomerase reverse transcriptase (hTERT) to establish IHH. Cells were analyzed for telomerase activity, proliferative capacity, hepatocyte markers, and tumorigenicity (c-myc) expression. Hepatocyte markers, hepatocellular functions, and morphology were studied in the HNF4A-overexpressing IHH. Hepatocyte markers and karyotype analysis were completed in the primary hepatocytes using shRNA knockdown of HNF4A. Nuclear translocation of β-catenin was assessed. Rat models of ALF were treated with encapsulated IHH or HNF4A-overexpressing IHH. A HNF4A-positive IHH line was established, which was non-tumorigenic and conserved properties of primary hepatocytes. HNF4A overexpression significantly enhanced mRNA levels of genes related to hepatic differentiation in IHH. Urea levels were increased by the overexpression of HNF4A, as measured 24h after ammonium chloride addition, similar to that of primary hepatocytes. Chromosomal abnormalities were observed in primary hepatocytes transfected with HNF4A shRNA. HNF4α overexpression could significantly promote β-catenin activation. Transplantation of HNF4A overexpressing IHH resulted in better liver function and survival of rats with ALF compared with IHH. HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF.
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Affiliation(s)
- Hua-Lian Hang
- Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China
| | - Xin-Yu Liu
- Department of General Surgery, Nanjing Hospital Affiliated to NanJing Medical University, Nanjing 210006, China
| | - Hai-Tian Wang
- Department of General Surgery, Nanjing Hospital Affiliated to NanJing Medical University, Nanjing 210006, China
| | - Ning Xu
- Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China
| | - Jian-Min Bian
- Department of General Surgery, Nanjing Hospital Affiliated to NanJing Medical University, Nanjing 210006, China
| | - Jian-Jun Zhang
- Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China
| | - Lei Xia
- Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.
| | - Qiang Xia
- Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.
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11
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Di Maso V, Mediavilla MG, Vascotto C, Lupo F, Baccarani U, Avellini C, Tell G, Tiribelli C, Crocè LS. Transcriptional Up-Regulation of APE1/Ref-1 in Hepatic Tumor: Role in Hepatocytes Resistance to Oxidative Stress and Apoptosis. PLoS One 2015; 10:e0143289. [PMID: 26624999 PMCID: PMC4666459 DOI: 10.1371/journal.pone.0143289] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2015] [Accepted: 11/03/2015] [Indexed: 01/11/2023] Open
Abstract
OBJECTIVE Human Hepatocellular Carcinoma (HCC) is the fifth most frequent neoplasm worldwide and the most serious complication of long-standing chronic liver diseases (CLD). Its development is associated with chronic inflammation and sustained oxidative stress. Deregulation of apurinic apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1), a master regulator of cellular response to oxidative stress, has been associated with poor prognosis in several cancers including HCC. DESIGN In the present study we investigated the APE1/Ref-1 mRNA levels in cirrhotic and HCC tissues obtained during HCC resection. The possible protective role of APE1/Ref-1 against oxidative stress and apoptosis was evaluated in vitro in immortalized human hepatocytes (IHH) over-expressing APE1/Ref-1. RESULTS APE1/Ref-1 was up-regulated in HCC, regulation occurring at the transcriptional level. APE1/Ref-1 mRNA content increased with the progression of liver disease with the transcriptional up-regulation present in cirrhosis significantly increased in HCC. The up-regulation was higher in the less differentiated cancers. In vitro, over-expression of APE1/Ref-1 in normal hepatocytes conferred cell protection against oxidative stress and it was associated with BAX inhibition and escape from apoptosis. CONCLUSION APE1/Ref-1 is up-regulated in HCC and this over-expression correlates with cancer aggressiveness. The up-regulation occurs at the transcriptional level and it is present in the earliest phases of hepatocarcinogenesis. The APE-1/Ref-1 over-expression is associated with hepatocyte survival and inhibits BAX activation and apoptosis. These data suggest a possible role of APE1/Ref-1 over-expression both in hepatocyte survival and HCC development calling attention to this molecule as a promising marker for HCC diagnosis and treatment.
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Affiliation(s)
- Vittorio Di Maso
- FIF- Fondazione Italiana Fegato Area Science Park Basovizza. Trieste, Italia
| | | | - Carlo Vascotto
- Dipartimento Scienze Mediche e Biologiche, Università di Udine. Udine, Itala
| | - Francesco Lupo
- Chirurgia Generale 2 Centro Trapianto Fegato Universitá Torino. Torino, Italia
| | - Umberto Baccarani
- Dipartimento Scienze Mediche e Biologiche, Università di Udine. Udine, Itala
| | - Claudio Avellini
- Dipartimento Scienze Mediche e Biologiche, Università di Udine. Udine, Itala
| | - Gianluca Tell
- FIF- Fondazione Italiana Fegato Area Science Park Basovizza. Trieste, Italia
- Dipartimento Scienze Mediche e Biologiche, Università di Udine. Udine, Itala
| | - Claudio Tiribelli
- FIF- Fondazione Italiana Fegato Area Science Park Basovizza. Trieste, Italia
- Dipartimento Scienze Mediche, Università di Trieste. Trieste, Italia
| | - Lory Saveria Crocè
- FIF- Fondazione Italiana Fegato Area Science Park Basovizza. Trieste, Italia
- Dipartimento Scienze Mediche, Università di Trieste. Trieste, Italia
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12
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Iansante V, Choy PM, Fung SW, Liu Y, Chai JG, Dyson J, Del Rio A, D'Santos C, Williams R, Chokshi S, Anders RA, Bubici C, Papa S. PARP14 promotes the Warburg effect in hepatocellular carcinoma by inhibiting JNK1-dependent PKM2 phosphorylation and activation. Nat Commun 2015; 6:7882. [PMID: 26258887 PMCID: PMC4918319 DOI: 10.1038/ncomms8882] [Citation(s) in RCA: 172] [Impact Index Per Article: 17.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2015] [Accepted: 06/23/2015] [Indexed: 02/07/2023] Open
Abstract
Most tumour cells use aerobic glycolysis (the Warburg effect) to support anabolic growth and evade apoptosis. Intriguingly, the molecular mechanisms that link the Warburg effect with the suppression of apoptosis are not well understood. In this study, using loss-of-function studies in vitro and in vivo, we show that the anti-apoptotic protein poly(ADP-ribose) polymerase (PARP)14 promotes aerobic glycolysis in human hepatocellular carcinoma (HCC) by maintaining low activity of the pyruvate kinase M2 isoform (PKM2), a key regulator of the Warburg effect. Notably, PARP14 is highly expressed in HCC primary tumours and associated with poor patient prognosis. Mechanistically, PARP14 inhibits the pro-apoptotic kinase JNK1, which results in the activation of PKM2 through phosphorylation of Thr365. Moreover, targeting PARP14 enhances the sensitization of HCC cells to anti-HCC agents. Our findings indicate that the PARP14-JNK1-PKM2 regulatory axis is an important determinant for the Warburg effect in tumour cells and provide a mechanistic link between apoptosis and metabolism.
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Affiliation(s)
- Valeria Iansante
- Cell Signaling and Cancer Laboratory, Institute of Hepatology, Foundation for Liver Research, London WC1E 6HX, UK
| | - Pui Man Choy
- Cell Signaling and Cancer Laboratory, Institute of Hepatology, Foundation for Liver Research, London WC1E 6HX, UK
| | - Sze Wai Fung
- Department of Medicine, Section of Inflammation and Signal Transduction, Imperial College, London W12 0NN, UK
| | - Ying Liu
- The Sol Goldman Pancreatic Cancer Research Center, Division of Gastrointestinal and Liver Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA
- Department of Medical Oncology, Henan Cancer Hospital, Zhengzhou, Henan 450000, China
| | - Jian-Guo Chai
- Department of Medicine, Section of Molecular Immunology, Imperial College, London W12 0NN, UK
| | - Julian Dyson
- Department of Medicine, Section of Molecular Immunology, Imperial College, London W12 0NN, UK
| | - Alberto Del Rio
- Institute of Organic Synthesis and Photoreactivity, National Research Council, Bologna 40129, Italy
| | - Clive D'Santos
- Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Cambridge CB2 0RE, UK
| | - Roger Williams
- Cell Signaling and Cancer Laboratory, Institute of Hepatology, Foundation for Liver Research, London WC1E 6HX, UK
- Viral Hepatitis Laboratory, Institute of Hepatology, Foundation for Liver Research, London WC1E 6HX, UK
| | - Shilpa Chokshi
- Viral Hepatitis Laboratory, Institute of Hepatology, Foundation for Liver Research, London WC1E 6HX, UK
| | - Robert A Anders
- The Sol Goldman Pancreatic Cancer Research Center, Division of Gastrointestinal and Liver Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA
| | - Concetta Bubici
- Department of Medicine, Section of Inflammation and Signal Transduction, Imperial College, London W12 0NN, UK
| | - Salvatore Papa
- Cell Signaling and Cancer Laboratory, Institute of Hepatology, Foundation for Liver Research, London WC1E 6HX, UK
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Ramboer E, De Craene B, De Kock J, Berx G, Rogiers V, Vanhaecke T, Vinken M. Development and characterization of a new human hepatic cell line. EXCLI JOURNAL 2015; 14:875-89. [PMID: 26869867 PMCID: PMC4747020 DOI: 10.17179/excli2015-424] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/22/2015] [Accepted: 06/29/2015] [Indexed: 12/20/2022]
Abstract
The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics.
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Affiliation(s)
- Eva Ramboer
- In Vitro Toxicology and Dermato-Cosmetology research group, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium
| | - Bram De Craene
- Unit of Molecular and Cellular Oncology, Inflammation Research Center, VIB, Technologiepark 927, 9052 Zwijnaarde, Belgium; Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium
| | - Joey De Kock
- In Vitro Toxicology and Dermato-Cosmetology research group, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium
| | - Geert Berx
- Unit of Molecular and Cellular Oncology, Inflammation Research Center, VIB, Technologiepark 927, 9052 Zwijnaarde, Belgium; Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium
| | - Vera Rogiers
- In Vitro Toxicology and Dermato-Cosmetology research group, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium
| | - Tamara Vanhaecke
- In Vitro Toxicology and Dermato-Cosmetology research group, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium
| | - Mathieu Vinken
- In Vitro Toxicology and Dermato-Cosmetology research group, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium
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14
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Goodson WH, Lowe L, Carpenter DO, Gilbertson M, Manaf Ali A, Lopez de Cerain Salsamendi A, Lasfar A, Carnero A, Azqueta A, Amedei A, Charles AK, Collins AR, Ward A, Salzberg AC, Colacci AM, Olsen AK, Berg A, Barclay BJ, Zhou BP, Blanco-Aparicio C, Baglole CJ, Dong C, Mondello C, Hsu CW, Naus CC, Yedjou C, Curran CS, Laird DW, Koch DC, Carlin DJ, Felsher DW, Roy D, Brown DG, Ratovitski E, Ryan EP, Corsini E, Rojas E, Moon EY, Laconi E, Marongiu F, Al-Mulla F, Chiaradonna F, Darroudi F, Martin FL, Van Schooten FJ, Goldberg GS, Wagemaker G, Nangami GN, Calaf GM, Williams GP, Wolf GT, Koppen G, Brunborg G, Lyerly HK, Krishnan H, Ab Hamid H, Yasaei H, Sone H, Kondoh H, Salem HK, Hsu HY, Park HH, Koturbash I, Miousse IR, Scovassi A, Klaunig JE, Vondráček J, Raju J, Roman J, Wise JP, Whitfield JR, Woodrick J, Christopher JA, Ochieng J, Martinez-Leal JF, Weisz J, Kravchenko J, Sun J, Prudhomme KR, Narayanan KB, Cohen-Solal KA, Moorwood K, Gonzalez L, Soucek L, Jian L, D’Abronzo LS, Lin LT, Li L, Gulliver L, McCawley LJ, Memeo L, Vermeulen L, Leyns L, Zhang L, Valverde M, Khatami M, Romano MF, Chapellier M, Williams MA, Wade M, et alGoodson WH, Lowe L, Carpenter DO, Gilbertson M, Manaf Ali A, Lopez de Cerain Salsamendi A, Lasfar A, Carnero A, Azqueta A, Amedei A, Charles AK, Collins AR, Ward A, Salzberg AC, Colacci AM, Olsen AK, Berg A, Barclay BJ, Zhou BP, Blanco-Aparicio C, Baglole CJ, Dong C, Mondello C, Hsu CW, Naus CC, Yedjou C, Curran CS, Laird DW, Koch DC, Carlin DJ, Felsher DW, Roy D, Brown DG, Ratovitski E, Ryan EP, Corsini E, Rojas E, Moon EY, Laconi E, Marongiu F, Al-Mulla F, Chiaradonna F, Darroudi F, Martin FL, Van Schooten FJ, Goldberg GS, Wagemaker G, Nangami GN, Calaf GM, Williams GP, Wolf GT, Koppen G, Brunborg G, Lyerly HK, Krishnan H, Ab Hamid H, Yasaei H, Sone H, Kondoh H, Salem HK, Hsu HY, Park HH, Koturbash I, Miousse IR, Scovassi A, Klaunig JE, Vondráček J, Raju J, Roman J, Wise JP, Whitfield JR, Woodrick J, Christopher JA, Ochieng J, Martinez-Leal JF, Weisz J, Kravchenko J, Sun J, Prudhomme KR, Narayanan KB, Cohen-Solal KA, Moorwood K, Gonzalez L, Soucek L, Jian L, D’Abronzo LS, Lin LT, Li L, Gulliver L, McCawley LJ, Memeo L, Vermeulen L, Leyns L, Zhang L, Valverde M, Khatami M, Romano MF, Chapellier M, Williams MA, Wade M, Manjili MH, Lleonart ME, Xia M, Gonzalez Guzman MJ, Karamouzis MV, Kirsch-Volders M, Vaccari M, Kuemmerle NB, Singh N, Cruickshanks N, Kleinstreuer N, van Larebeke N, Ahmed N, Ogunkua O, Krishnakumar P, Vadgama P, Marignani PA, Ghosh PM, Ostrosky-Wegman P, Thompson PA, Dent P, Heneberg P, Darbre P, Leung PS, Nangia-Makker P, Cheng Q(S, Robey R, Al-Temaimi R, Roy R, Andrade-Vieira R, Sinha RK, Mehta R, Vento R, Di Fiore R, Ponce-Cusi R, Dornetshuber-Fleiss R, Nahta R, Castellino RC, Palorini R, Hamid RA, Langie SA, Eltom SE, Brooks SA, Ryeom S, Wise SS, Bay SN, Harris SA, Papagerakis S, Romano S, Pavanello S, Eriksson S, Forte S, Casey SC, Luanpitpong S, Lee TJ, Otsuki T, Chen T, Massfelder T, Sanderson T, Guarnieri T, Hultman T, Dormoy V, Odero-Marah V, Sabbisetti V, Maguer-Satta V, Rathmell W, Engström W, Decker WK, Bisson WH, Rojanasakul Y, Luqmani Y, Chen Z, Hu Z. Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead. Carcinogenesis 2015; 36 Suppl 1:S254-S296. [PMID: 26106142 PMCID: PMC4480130 DOI: 10.1093/carcin/bgv039] [Show More Authors] [Citation(s) in RCA: 186] [Impact Index Per Article: 18.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2014] [Revised: 01/23/2015] [Accepted: 01/31/2015] [Indexed: 02/07/2023] Open
Abstract
Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.
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Affiliation(s)
- William H. Goodson
- *To whom correspondence should be addressed. William H.Goodson III, California Pacific Medical Center Research Institute, 2100 Webster Street #401, San Francisco, CA 94115, USA. Tel: +41 59 233925; Fax: +41 57 761977;
| | - Leroy Lowe
- Getting to Know Cancer, Room 229A, 36 Arthur Street, Truro, Nova Scotia B2N 1X5, Canada
- Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster LA1 4AP, UK
| | - David O. Carpenter
- Institute for Health and the Environment, University at Albany, 5 University Pl., Rensselaer, NY 12144, USA
| | | | - Abdul Manaf Ali
- School of Biotechnology, Faculty of Agriculture Biotechnology and Food Sciences, Sultan Zainal Abidin University, Tembila Campus, 22200 Besut, Terengganu, Malaysia
| | | | - Ahmed Lasfar
- Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers, State University of New Jersey, Piscataway, NJ 08854, USA
| | - Amancio Carnero
- Instituto de Biomedicina de Sevilla, Consejo Superior de Investigaciones Cientificas. Hospital Universitario Virgen del Rocio, Univ. de Sevilla., Avda Manuel Siurot sn. 41013 Sevilla, Spain
| | - Amaya Azqueta
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Navarra, Pamplona 31008, Spain
| | - Amedeo Amedei
- Department of Experimental and Clinical Medicine, University of Firenze, Florence 50134, Italy
| | - Amelia K. Charles
- School of Biological Sciences, University of Reading, Hopkins Building, Reading, Berkshire RG6 6UB, UK
| | | | - Andrew Ward
- Department of Biochemistry and Biology, University of Bath, Claverton Down, Bath BA2 7AY, UK
| | - Anna C. Salzberg
- Department of Public Health Sciences, College of Medicine, Pennsylvania State University, Hershey, PA 17033, USA
| | - Anna Maria Colacci
- Center for Environmental Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency, 40126 Bologna, Italy
| | - Ann-Karin Olsen
- Department of Chemicals and Radiation, Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo N-0403, Norway
| | - Arthur Berg
- Department of Public Health Sciences, College of Medicine, Pennsylvania State University, Hershey, PA 17033, USA
| | | | - Binhua P. Zhou
- Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40508, USA
| | - Carmen Blanco-Aparicio
- Spanish National Cancer Research Centre, CNIO, Melchor Fernandez Almagro, 3, 28029 Madrid, Spain
| | - Carolyn J. Baglole
- Department of Medicine, McGill University, Montreal, Quebec H4A 3J1, Canada
| | - Chenfang Dong
- Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40508, USA
| | - Chiara Mondello
- Istituto di Genetica Molecolare, CNR, Via Abbiategrasso 207, 27100 Pavia, Italy
| | - Chia-Wen Hsu
- Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Bethesda, MD 20892–3375, USA
| | - Christian C. Naus
- Department of Cellular and Physiological Sciences, Life Sciences Institute, Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia V5Z 1M9, Canada
| | - Clement Yedjou
- Department of Biology, Jackson State University, Jackson, MS 39217, USA
| | - Colleen S. Curran
- Department of Molecular and Environmental Toxicology, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Dale W. Laird
- Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario N6A 3K7, Canada
| | - Daniel C. Koch
- Stanford University Department of Medicine, Division of Oncology, Stanford, CA 94305, USA
| | - Danielle J. Carlin
- Superfund Research Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27560, USA
| | - Dean W. Felsher
- Department of Medicine, Oncology and Pathology, Stanford University,Stanford, CA 94305, USA
| | - Debasish Roy
- Department of Natural Science, The City University of New York at Hostos Campus, Bronx, NY 10451, USA
| | - Dustin G. Brown
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523–1680, USA
| | - Edward Ratovitski
- Department of Head and Neck Surgery/Head and Neck Cancer Research, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Elizabeth P. Ryan
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523–1680, USA
| | - Emanuela Corsini
- Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, 20133 Milan, Italy
| | - Emilio Rojas
- Department of Genomic Medicine and Environmental Toxicology, Institute for Biomedical Research, National Autonomous University of Mexico, Mexico City 04510, México
| | - Eun-Yi Moon
- Department of Bioscience and Biotechnology, Sejong University, Seoul 143–747, Korea
| | - Ezio Laconi
- Department of Biomedical Sciences, University of Cagliari, 09124 Cagliari, Italy
| | - Fabio Marongiu
- Department of Biomedical Sciences, University of Cagliari, 09124 Cagliari, Italy
| | - Fahd Al-Mulla
- Department of Pathology, Kuwait University, Safat 13110, Kuwait
| | - Ferdinando Chiaradonna
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 Milan, Italy
- SYSBIO Centre of Systems Biology, Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 Milan, Italy
| | - Firouz Darroudi
- Human Safety and Environmental Research, Department of Health Sciences, College of North Atlantic, Doha 24449, State of Qatar
| | - Francis L. Martin
- Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster LA1 4AP, UK
| | - Frederik J. Van Schooten
- Department of Toxicology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University, Maastricht 6200, The Netherlands
| | - Gary S. Goldberg
- Department of Molecular Biology, School of Osteopathic Medicine, Rowan University, Stratford, NJ 08084, USA
| | - Gerard Wagemaker
- Hacettepe University, Center for Stem Cell Research and Development, Ankara 06640, Turkey
| | - Gladys N. Nangami
- Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208, USA
| | - Gloria M. Calaf
- Center for Radiological Research, Columbia University Medical Center, New York, NY 10032, USA
- Instituto de Alta Investigacion, Universidad de Tarapaca, Arica, Chile
| | - Graeme P. Williams
- School of Biological Sciences, University of Reading, Reading, RG6 6UB, UK
| | - Gregory T. Wolf
- Department of Otolaryngology - Head and Neck Surgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Gudrun Koppen
- Environmental Risk and Health Unit, Flemish Institute for Technological Research, 2400 Mol, Belgium
| | - Gunnar Brunborg
- Department of Chemicals and Radiation, Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo N-0403, Norway
| | - H. Kim Lyerly
- Department of Surgery, Pathology, Immunology, Duke University Medical Center, Durham, NC 27710, USA
| | - Harini Krishnan
- Department of Molecular Biology, School of Osteopathic Medicine, Rowan University, Stratford, NJ 08084, USA
| | - Hasiah Ab Hamid
- Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, 43400 Universiti Putra Malaysia, Serdang, Selangor, Malaysia
| | - Hemad Yasaei
- Department of Life Sciences, College of Health and Life Sciences and the Health and Environment Theme, Institute of Environment, Health and Societies, Brunel University Kingston Lane, Uxbridge, Middlesex UB8 3PH, UK
| | - Hideko Sone
- National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibraki 3058506, Japan
| | - Hiroshi Kondoh
- Department of Geriatric Medicine, Kyoto University Hospital 54 Kawaharacho, Shogoin, Sakyo-ku Kyoto, 606–8507, Japan
| | - Hosni K. Salem
- Department of Urology, Kasr Al-Ainy School of Medicine, Cairo University, El Manial, Cairo 11559, Egypt
| | - Hsue-Yin Hsu
- Department of Life Sciences, Tzu-Chi University, Hualien 970, Taiwan
| | - Hyun Ho Park
- School of Biotechnology, Yeungnam University, Gyeongbuk 712-749, South Korea
| | - Igor Koturbash
- Department of Environmental and Occupational Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
| | - Isabelle R. Miousse
- Department of Environmental and Occupational Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
| | - A.Ivana Scovassi
- Istituto di Genetica Molecolare, CNR, Via Abbiategrasso 207, 27100 Pavia, Italy
| | - James E. Klaunig
- Department of Environmental Health, Indiana University, School of Public Health, Bloomington, IN 47405, USA
| | - Jan Vondráček
- Department of Cytokinetics, Institute of Biophysics Academy of Sciences of the Czech Republic, Brno, CZ-61265, Czech Republic
| | - Jayadev Raju
- Regulatory Toxicology Research Division, Bureau of Chemical Safety, Food Directorate, Health Canada, Ottawa, Ontario K1A 0K9, Canada
| | - Jesse Roman
- Department of Medicine, University of Louisville, Louisville, KY 40202, USA
- Robley Rex VA Medical Center, Louisville, KY 40202, USA
| | - John Pierce Wise
- Department of Applied Medical Sciences, University of Southern Maine, 96 Falmouth St., Portland, ME 04104, USA
| | - Jonathan R. Whitfield
- Mouse Models of Cancer Therapies Group, Vall d’Hebron Institute of Oncology (VHIO), 08035 Barcelona, Spain
| | - Jordan Woodrick
- Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC 20057, USA
| | - Joseph A. Christopher
- Cancer Research UK. Cambridge Institute, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK
| | - Josiah Ochieng
- Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208, USA
| | | | - Judith Weisz
- Departments of Obstetrics and Gynecology and Pathology, Pennsylvania State University College of Medicine, Hershey PA 17033, USA
| | - Julia Kravchenko
- Department of Surgery, Pathology, Immunology, Duke University Medical Center, Durham, NC 27710, USA
| | - Jun Sun
- Department of Biochemistry, Rush University, Chicago, IL 60612, USA
| | - Kalan R. Prudhomme
- Environmental and Molecular Toxicology, Environmental Health Science Center, Oregon State University, Corvallis, OR 97331, USA
| | | | - Karine A. Cohen-Solal
- Department of Medicine/Medical Oncology, Rutgers Cancer Institute of New Jersey, New Brunswick, NJ 08903, USA
| | - Kim Moorwood
- Department of Biochemistry and Biology, University of Bath, Claverton Down, Bath BA2 7AY, UK
| | - Laetitia Gonzalez
- Laboratory for Cell Genetics, Vrije Universiteit Brussel, 1050 Brussels, Belgium
| | - Laura Soucek
- Mouse Models of Cancer Therapies Group, Vall d’Hebron Institute of Oncology (VHIO), 08035 Barcelona, Spain
- Catalan Institution for Research and Advanced Studies (ICREA), Barcelona 08010, Spain
| | - Le Jian
- School of Public Health, Curtin University, Bentley, WA 6102, Australia
- Department of Urology, University of California Davis, Sacramento, CA 95817, USA
| | - Leandro S. D’Abronzo
- Department of Urology, University of California Davis, Sacramento, CA 95817, USA
| | - Liang-Tzung Lin
- Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan
| | - Lin Li
- School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, The People’s Republic of China
| | - Linda Gulliver
- Faculty of Medicine, University of Otago, Dunedin 9054, New Zealand
| | - Lisa J. McCawley
- Department of Biomedical Engineering and Cancer Biology, Vanderbilt University, Nashville, TN 37235, USA
| | - Lorenzo Memeo
- Department of Experimental Oncology, Mediterranean Institute of Oncology, Via Penninazzo 7, Viagrande (CT) 95029, Italy
| | - Louis Vermeulen
- Center for Experimental Molecular Medicine, Academic Medical Center, Meibergdreef 9, Amsterdam 1105 AZ, The Netherlands
| | - Luc Leyns
- Laboratory for Cell Genetics, Vrije Universiteit Brussel, 1050 Brussels, Belgium
| | - Luoping Zhang
- Division of Environmental Health Sciences, School of Public Health, University of California, Berkeley, CA 94720-7360, USA
| | - Mahara Valverde
- Department of Genomic Medicine and Environmental Toxicology, Institute for Biomedical Research, National Autonomous University of Mexico, Mexico City 04510, México
| | - Mahin Khatami
- Inflammation and Cancer Research, National Cancer Institute (NCI) (Retired), National Institutes of Health, Bethesda, MD 20892, USA
| | - Maria Fiammetta Romano
- Department of Molecular Medicine and Medical Biotechnology, Federico II University of Naples, 80131 Naples, Italy
| | - Marion Chapellier
- Centre De Recherche En Cancerologie,De Lyon, Lyon, U1052-UMR5286, France
| | - Marc A. Williams
- United States Army Institute of Public Health, Toxicology Portfolio-Health Effects Research Program, Aberdeen Proving Ground, Edgewood, MD 21010-5403, USA
| | - Mark Wade
- Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia, Via Adamello 16, 20139 Milano, Italy
| | - Masoud H. Manjili
- Department of Microbiology and Immunology, Virginia Commonwealth University, Massey Cancer Center, Richmond, VA 23298, USA
| | - Matilde E. Lleonart
- Institut De Recerca Hospital Vall D’Hebron, Passeig Vall d’Hebron, 119–129, 08035 Barcelona, Spain
| | - Menghang Xia
- Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Bethesda, MD 20892–3375, USA
| | - Michael J. Gonzalez Guzman
- University of Puerto Rico, Medical Sciences Campus, School of Public Health, Nutrition Program, San Juan 00921, Puerto Rico
| | - Michalis V. Karamouzis
- Department of Biological Chemistry, Medical School, University of Athens, Institute of Molecular Medicine and Biomedical Research, 10676 Athens, Greece
| | | | - Monica Vaccari
- Center for Environmental Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency, 40126 Bologna, Italy
| | | | - Neetu Singh
- Advanced Molecular Science Research Centre (Centre for Advanced Research), King George’s Medical University, Lucknow, Uttar Pradesh 226 003, India
| | - Nichola Cruickshanks
- Departments of Neurosurgery and Biochemistry and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Nicole Kleinstreuer
- Integrated Laboratory Systems Inc., in support of the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods, RTP, NC 27709, USA
| | - Nik van Larebeke
- Analytische, Milieu en Geochemie, Vrije Universiteit Brussel, Brussel B1050, Belgium
| | - Nuzhat Ahmed
- Department of Obstetrics and Gynecology, University of Melbourne, Victoria 3052, Australia
| | - Olugbemiga Ogunkua
- Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208, USA
| | - P.K. Krishnakumar
- Center for Environment and Water, Research Institute, King Fahd University of Petroleum and Minerals, Dhahran 3126, Saudi Arabia
| | - Pankaj Vadgama
- School of Engineering and Materials Science, Queen Mary University of London, Mile End Road, London, E1 4NS, UK
| | - Paola A. Marignani
- Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada
| | - Paramita M. Ghosh
- Department of Urology, University of California Davis, Sacramento, CA 95817, USA
| | - Patricia Ostrosky-Wegman
- Department of Genomic Medicine and Environmental Toxicology, Institute for Biomedical Research, National Autonomous University of Mexico, Mexico City 04510, México
| | - Patricia A. Thompson
- Department of Pathology, Stony Brook School of Medicine, Stony Brook University, The State University of New York, Stony Brook, NY 11794-8691, USA
| | - Paul Dent
- Departments of Neurosurgery and Biochemistry and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA
| | - Petr Heneberg
- Charles University in Prague, Third Faculty of Medicine, CZ-100 00 Prague 10, Czech Republic
| | - Philippa Darbre
- School of Biological Sciences, The University of Reading, Whiteknights, Reading RG6 6UB, England
| | - Po Sing Leung
- School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, The People’s Republic of China
| | | | - Qiang (Shawn) Cheng
- Computer Science Department, Southern Illinois University, Carbondale, IL 62901, USA
| | - R.Brooks Robey
- White River Junction Veterans Affairs Medical Center, White River Junction, VT 05009, USA
- Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA
| | - Rabeah Al-Temaimi
- Human Genetics Unit, Department of Pathology, Faculty of Medicine, Kuwait University, Jabriya 13110, Kuwait
| | - Rabindra Roy
- Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC 20057, USA
| | - Rafaela Andrade-Vieira
- Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada
| | - Ranjeet K. Sinha
- Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Rekha Mehta
- Regulatory Toxicology Research Division, Bureau of Chemical Safety, Food Directorate, Health Canada, Ottawa, Ontario K1A 0K9, Canada
| | - Renza Vento
- Department of Biological, Chemical, and Pharmaceutical Sciences and Technologies, Polyclinic Plexus, University of Palermo, Palermo 90127, Italy
- Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA 19122, USA
| | - Riccardo Di Fiore
- Department of Biological, Chemical, and Pharmaceutical Sciences and Technologies, Polyclinic Plexus, University of Palermo, Palermo 90127, Italy
| | | | - Rita Dornetshuber-Fleiss
- Department of Pharmacology and Toxicology, University of Vienna, Vienna A-1090, Austria
- Institute of Cancer Research, Department of Medicine, Medical University of Vienna, Wien 1090, Austria
| | - Rita Nahta
- Departments of Pharmacology and Hematology and Medical Oncology, Emory University School of Medicine and Winship Cancer Institute, Atlanta, GA 30322, USA
| | - Robert C. Castellino
- Division of Hematology and Oncology, Department of Pediatrics, Children’s Healthcare of Atlanta, GA 30322, USA
- Department of Pediatrics, Emory University School of Medicine, Emory University, Atlanta, GA 30322, USA
| | - Roberta Palorini
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 Milan, Italy
- SYSBIO Centre of Systems Biology, Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 Milan, Italy
| | - Roslida A. Hamid
- Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, 43400 Universiti Putra Malaysia, Serdang, Selangor, Malaysia
| | - Sabine A.S. Langie
- Environmental Risk and Health Unit, Flemish Institute for Technological Research, 2400 Mol, Belgium
| | - Sakina E. Eltom
- Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208, USA
| | - Samira A. Brooks
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, NC 27599, USA
| | - Sandra Ryeom
- Department of Cancer Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Sandra S. Wise
- Department of Applied Medical Sciences, University of Southern Maine, 96 Falmouth St., Portland, ME 04104, USA
| | - Sarah N. Bay
- Program in Genetics and Molecular Biology, Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, GA 30322, USA
| | - Shelley A. Harris
- Population Health and Prevention, Research, Prevention and Cancer Control, Cancer Care Ontario, Toronto, Ontario, M5G 2L7, Canada
- Departments of Epidemiology and Occupational and Environmental Health, Dalla Lana School of Public Health, University of Toronto, Toronto, Ontario, M5T 3M7, Canada
| | - Silvana Papagerakis
- Department of Otolaryngology - Head and Neck Surgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Simona Romano
- Department of Molecular Medicine and Medical Biotechnology, Federico II University of Naples, 80131 Naples, Italy
| | - Sofia Pavanello
- Department of Cardiac, Thoracic and Vascular Sciences, Unit of Occupational Medicine, University of Padova, Padova 35128, Italy
| | - Staffan Eriksson
- Department of Anatomy, Physiology and Biochemistry, The Swedish University of Agricultural Sciences, PO Box 7011, VHC, Almas Allé 4, SE-756 51, Uppsala, Sweden
| | - Stefano Forte
- Department of Experimental Oncology, Mediterranean Institute of Oncology, Via Penninazzo 7, Viagrande (CT) 95029, Italy
| | - Stephanie C. Casey
- Stanford University Department of Medicine, Division of Oncology, Stanford, CA 94305, USA
| | - Sudjit Luanpitpong
- Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
| | - Tae-Jin Lee
- Department of Anatomy, College of Medicine, Yeungnam University, Daegu 705–717, South Korea,
| | - Takemi Otsuki
- Department of Hygiene, Kawasaki Medical School, Matsushima Kurashiki, Okayama 701-0192, Japan,
| | - Tao Chen
- Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, United States Food and Drug Administration, Jefferson, AR 72079, USA
| | - Thierry Massfelder
- INSERM U1113, team 3 ‘Cell Signalling and Communication in Kidney and Prostate Cancer’, University of Strasbourg, Faculté de Médecine, 67085 Strasbourg, France
| | - Thomas Sanderson
- INRS-Institut Armand-Frappier, 531 Boulevard des Prairies, Laval, QC H7V 1B7, Canada,
| | - Tiziana Guarnieri
- Department of Biology, Geology and Environmental Sciences, Alma Mater Studiorum Università di Bologna, Via Francesco Selmi, 3, 40126 Bologna, Italy
- Center for Applied Biomedical Research, S. Orsola-Malpighi University Hospital, Via Massarenti, 9, 40126 Bologna, Italy
- National Institute of Biostructures and Biosystems, Viale Medaglie d’ Oro, 305, 00136 Roma, Italy
| | - Tove Hultman
- Department of Biosciences and Veterinary Public Health, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, PO Box 7028, 75007 Uppsala, Sweden
| | - Valérian Dormoy
- INSERM U1113, team 3 ‘Cell Signalling and Communication in Kidney and Prostate Cancer’, University of Strasbourg, Faculté de Médecine, 67085 Strasbourg, France
- Department of Cell and Developmental Biology, University of California, Irvine, CA 92697, USA
| | - Valerie Odero-Marah
- Department of Biology/Center for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, GA 30314, USA
| | - Venkata Sabbisetti
- Harvard Medical School/Brigham and Women’s Hospital, Boston, MA 02115, USA
| | - Veronique Maguer-Satta
- United States Army Institute of Public Health, Toxicology Portfolio-Health Effects Research Program, Aberdeen Proving Ground, Edgewood, MD 21010-5403, USA
| | - W.Kimryn Rathmell
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, NC 27599, USA
| | - Wilhelm Engström
- Department of Biosciences and Veterinary Public Health, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, PO Box 7028, 75007 Uppsala, Sweden
| | | | - William H. Bisson
- Environmental and Molecular Toxicology, Environmental Health Science Center, Oregon State University, Corvallis, OR 97331, USA
| | - Yon Rojanasakul
- Department of Pharmaceutical Sciences, West Virginia University, Morgantown,WV, 26506,USA
| | - Yunus Luqmani
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kuwait University, PO Box 24923, Safat 13110, Kuwait and
| | - Zhenbang Chen
- Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208, USA
| | - Zhiwei Hu
- Department of Surgery, The Ohio State University College of Medicine, The James Comprehensive Cancer Center, Columbus, OH 43210, USA
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15
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Carnero A, Blanco-Aparicio C, Kondoh H, Lleonart ME, Martinez-Leal JF, Mondello C, Ivana Scovassi A, Bisson WH, Amedei A, Roy R, Woodrick J, Colacci A, Vaccari M, Raju J, Al-Mulla F, Al-Temaimi R, Salem HK, Memeo L, Forte S, Singh N, Hamid RA, Ryan EP, Brown DG, Wise JP, Wise SS, Yasaei H. Disruptive chemicals, senescence and immortality. Carcinogenesis 2015; 36 Suppl 1:S19-S37. [PMID: 26106138 PMCID: PMC4565607 DOI: 10.1093/carcin/bgv029] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2014] [Revised: 08/04/2014] [Accepted: 08/05/2014] [Indexed: 12/16/2022] Open
Abstract
Carcinogenesis is thought to be a multistep process, with clonal evolution playing a central role in the process. Clonal evolution involves the repeated 'selection and succession' of rare variant cells that acquire a growth advantage over the remaining cell population through the acquisition of 'driver mutations' enabling a selective advantage in a particular micro-environment. Clonal selection is the driving force behind tumorigenesis and possesses three basic requirements: (i) effective competitive proliferation of the variant clone when compared with its neighboring cells, (ii) acquisition of an indefinite capacity for self-renewal, and (iii) establishment of sufficiently high levels of genetic and epigenetic variability to permit the emergence of rare variants. However, several questions regarding the process of clonal evolution remain. Which cellular processes initiate carcinogenesis in the first place? To what extent are environmental carcinogens responsible for the initiation of clonal evolution? What are the roles of genotoxic and non-genotoxic carcinogens in carcinogenesis? What are the underlying mechanisms responsible for chemical carcinogen-induced cellular immortality? Here, we explore the possible mechanisms of cellular immortalization, the contribution of immortalization to tumorigenesis and the mechanisms by which chemical carcinogens may contribute to these processes.
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Affiliation(s)
- Amancio Carnero
- *To whom correspondence should be addressed. Tel: +34955923111; Fax: +34955923101;
| | - Carmen Blanco-Aparicio
- Spanish National Cancer Research Center, Experimental Therapuetics Department, Melchor Fernandez Almagro, 3, 28029 Madrid, Spain
| | - Hiroshi Kondoh
- Department of Geriatric Medicine, Kyoto University Hospital, 54 Kawaharacho, Shogoin, Sakyo-ku Kyoto 606-8507, Japan
| | - Matilde E. Lleonart
- Institut De Recerca Hospital Vall D’Hebron, Passeig Vall d’Hebron, 119–129, 08035 Barcelona, Spain
| | | | - Chiara Mondello
- Istituto di Genetica Molecolare, CNR, Via Abbiategrasso 207, 27100 Pavia, Italy
| | - A. Ivana Scovassi
- Istituto di Genetica Molecolare, CNR, Via Abbiategrasso 207, 27100 Pavia, Italy
| | - William H. Bisson
- Environmental and Molecular Toxicology, Environmental Health Science Center, Oregon State University, Corvallis, OR 97331, USA
| | - Amedeo Amedei
- Department of Experimental and Clinical Medicine, University of Firenze, Italy, Florence 50134, Italy
| | - Rabindra Roy
- Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
| | - Jordan Woodrick
- Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA
| | - Annamaria Colacci
- Center for Environmental Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency, Bologna 40126, Italy
| | - Monica Vaccari
- Center for Environmental Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency, Bologna 40126, Italy
| | - Jayadev Raju
- Toxicology Research Division, Bureau of Chemical Safety Food Directorate, Health Products and Food Branch Health Canada, Ottawa, Ontario K1A0K9, Canada
| | - Fahd Al-Mulla
- Department of Pathology, Kuwait University, Safat 13110, Kuwait
| | | | - Hosni K. Salem
- Urology Department, kasr Al-Ainy School of Medicine, Cairo University, El Manial, Cairo 12515, Egypt
| | - Lorenzo Memeo
- Mediterranean Institute of Oncology, Viagrande 95029, Italy
| | - Stefano Forte
- Mediterranean Institute of Oncology, Viagrande 95029, Italy
| | - Neetu Singh
- Centre for Advanced Research, King George’s Medical University, Chowk, Lucknow, Uttar Pradesh 226003, India
| | - Roslida A. Hamid
- Department of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor 43400, Malaysia
| | - Elizabeth P. Ryan
- Department of Environmental and Radiological Health Sciences, Colorado State University/Colorado School of Public Health, Fort Collins, CO 80523-1680, USA
| | - Dustin G. Brown
- Department of Environmental and Radiological Health Sciences, Colorado State University/Colorado School of Public Health, Fort Collins, CO 80523-1680, USA
| | - John Pierce Wise
- The Wise Laboratory of Environmental and Genetic Toxicology, Maine Center for Toxicology and Environmental Health, Department of Applied Medical Sciences, University of Southern Maine, 96 Falmouth Street, Portland, ME 04104, USA and
| | - Sandra S. Wise
- The Wise Laboratory of Environmental and Genetic Toxicology, Maine Center for Toxicology and Environmental Health, Department of Applied Medical Sciences, University of Southern Maine, 96 Falmouth Street, Portland, ME 04104, USA and
| | - Hemad Yasaei
- Brunel Institute of Cancer Genetics and Pharmacogenomics, Health and Environment Theme, Institute of Environment, Health and Societies, Brunel University London, Kingston Lane, Uxbridge, UB8 3PH, UK
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16
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Ramboer E, Vanhaecke T, Rogiers V, Vinken M. Immortalized Human Hepatic Cell Lines for In Vitro Testing and Research Purposes. Methods Mol Biol 2015; 1250:53-76. [PMID: 26272134 PMCID: PMC4579543 DOI: 10.1007/978-1-4939-2074-7_4] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The ubiquitous shortage of primary human hepatocytes has urged the scientific community to search for alternative cell sources, such as immortalized hepatic cell lines. Over the years, several human hepatic cell lines have been produced, whether or not using a combination of viral oncogenes and human telomerase reverse transcriptase protein. Conditional approaches for hepatocyte immortalization have also been established and allow generation of growth-controlled cell lines. A variety of immortalized human hepatocytes have already proven useful as tools for liver-based in vitro testing and fundamental research purposes. The present chapter describes currently applied immortalization strategies and provides an overview of the actually available immortalized human hepatic cell lines and their in vitro applications.
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Affiliation(s)
- Eva Ramboer
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, Brussels, 1090, Belgium,
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17
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Podrini C, Koffas A, Chokshi S, Vinciguerra M, Lelliott CJ, White JK, Adissu HA, Williams R, Greco A. MacroH2A1 isoforms are associated with epigenetic markers for activation of lipogenic genes in fat-induced steatosis. FASEB J 2014; 29:1676-87. [PMID: 25526730 DOI: 10.1096/fj.14-262717] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2014] [Accepted: 11/24/2014] [Indexed: 01/14/2023]
Abstract
The importance of epigenetic changes in the development of hepatic steatosis is largely unknown. The histone variant macroH2A1 under alternative splicing gives rise to macroH2A1.1 and macroH2A1.2. In this study, we show that the macroH2A1 isoforms play an important role in the regulation of lipid accumulation in hepatocytes. Hepatoma cell line and immortalized human hepatocytes transiently transfected or knocked down with macroH2A1 isoforms were used as in vitro model of fat-induced steatosis. Gene expressions were analyzed by quantitative PCR array and Western blot. Chromatin immunoprecipitation analysis was performed to check the association of histone H3 lysine 27 trimethylation (H3K27me3) and histone H3 lysine 4 trimethylation (H3K4me3) with the promoter of lipogenic genes. Livers from knockout mice that are resistant to lipid deposition despite a high-fat diet were used for histopathology. We found that macroH2A1.2 is regulated by fat uptake and that its overexpression caused an increase in lipid uptake, triglycerides, and lipogenic genes compared with macroH2A1.1. This suggests that macroH2A1.2 is important for lipid uptake, whereas macroH2A1.1 was found to be protective. The result was supported by a high positivity for macroH2A1.1 in knockout mice for genes targeted by macroH2A1 (Atp5a1 and Fam73b), that under a high-fat diet presented minimal lipidosis. Moreover, macroH2A1 isoforms differentially regulate the expression of lipogenic genes by modulating the association of the active (H3K4me3) and repressive (H3K27me3) histone marks on their promoters. This study underlines the importance of the replacement of noncanonical histones in the regulation of genes involved in lipid metabolism in the progression of steatosis.
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Affiliation(s)
- Christine Podrini
- *Foundation for Liver Research, Institute of Hepatology, London, United Kingdom; University College London (UCL)--Institute for Liver & Digestive Health, UCL Medical School, London, United Kingdom, Mouse Genetics Project, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom; and Physiology and Experimental Medicine Research Program, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
| | - Apostolos Koffas
- *Foundation for Liver Research, Institute of Hepatology, London, United Kingdom; University College London (UCL)--Institute for Liver & Digestive Health, UCL Medical School, London, United Kingdom, Mouse Genetics Project, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom; and Physiology and Experimental Medicine Research Program, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
| | - Shilpa Chokshi
- *Foundation for Liver Research, Institute of Hepatology, London, United Kingdom; University College London (UCL)--Institute for Liver & Digestive Health, UCL Medical School, London, United Kingdom, Mouse Genetics Project, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom; and Physiology and Experimental Medicine Research Program, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
| | - Manlio Vinciguerra
- *Foundation for Liver Research, Institute of Hepatology, London, United Kingdom; University College London (UCL)--Institute for Liver & Digestive Health, UCL Medical School, London, United Kingdom, Mouse Genetics Project, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom; and Physiology and Experimental Medicine Research Program, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
| | - Christopher J Lelliott
- *Foundation for Liver Research, Institute of Hepatology, London, United Kingdom; University College London (UCL)--Institute for Liver & Digestive Health, UCL Medical School, London, United Kingdom, Mouse Genetics Project, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom; and Physiology and Experimental Medicine Research Program, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
| | - Jacqueline K White
- *Foundation for Liver Research, Institute of Hepatology, London, United Kingdom; University College London (UCL)--Institute for Liver & Digestive Health, UCL Medical School, London, United Kingdom, Mouse Genetics Project, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom; and Physiology and Experimental Medicine Research Program, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
| | - Hibret A Adissu
- *Foundation for Liver Research, Institute of Hepatology, London, United Kingdom; University College London (UCL)--Institute for Liver & Digestive Health, UCL Medical School, London, United Kingdom, Mouse Genetics Project, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom; and Physiology and Experimental Medicine Research Program, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
| | - Roger Williams
- *Foundation for Liver Research, Institute of Hepatology, London, United Kingdom; University College London (UCL)--Institute for Liver & Digestive Health, UCL Medical School, London, United Kingdom, Mouse Genetics Project, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom; and Physiology and Experimental Medicine Research Program, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
| | - Azzura Greco
- *Foundation for Liver Research, Institute of Hepatology, London, United Kingdom; University College London (UCL)--Institute for Liver & Digestive Health, UCL Medical School, London, United Kingdom, Mouse Genetics Project, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom; and Physiology and Experimental Medicine Research Program, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
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Eva R, Bram DC, Joery DK, Tamara V, Geert B, Vera R, Mathieu V. Strategies for immortalization of primary hepatocytes. J Hepatol 2014; 61:925-43. [PMID: 24911463 PMCID: PMC4169710 DOI: 10.1016/j.jhep.2014.05.046] [Citation(s) in RCA: 76] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2014] [Revised: 04/17/2014] [Accepted: 05/30/2014] [Indexed: 02/06/2023]
Abstract
The liver has the unique capacity to regenerate in response to a damaging event. Liver regeneration is hereby largely driven by hepatocyte proliferation, which in turn relies on cell cycling. The hepatocyte cell cycle is a complex process that is tightly regulated by several well-established mechanisms. In vitro, isolated hepatocytes do not longer retain this proliferative capacity. However, in vitro cell growth can be boosted by immortalization of hepatocytes. Well-defined immortalization genes can be artificially overexpressed in hepatocytes or the cells can be conditionally immortalized leading to controlled cell proliferation. This paper discusses the current immortalization techniques and provides a state-of-the-art overview of the actually available immortalized hepatocyte-derived cell lines and their applications.
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Affiliation(s)
- Ramboer Eva
- Department of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel Laarbeeklaan 103, 1090 Brussel, Belgium
| | - De Craene Bram
- Unit of Molecular and Cellular Oncology, Inflammation Research Center, VIB, Technologiepark 927, 9052 Zwijnaarde, Belgium
,Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium
| | - De Kock Joery
- Department of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel Laarbeeklaan 103, 1090 Brussel, Belgium
| | - Vanhaecke Tamara
- Department of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel Laarbeeklaan 103, 1090 Brussel, Belgium
| | - Berx Geert
- Unit of Molecular and Cellular Oncology, Inflammation Research Center, VIB, Technologiepark 927, 9052 Zwijnaarde, Belgium
,Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium
| | - Rogiers Vera
- Department of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel Laarbeeklaan 103, 1090 Brussel, Belgium
| | - Vinken Mathieu
- Department of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel Laarbeeklaan 103, 1090 Brussel, Belgium
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Kouprina N, Tomilin AN, Masumoto H, Earnshaw WC, Larionov V. Human artificial chromosome-based gene delivery vectors for biomedicine and biotechnology. Expert Opin Drug Deliv 2014; 11:517-35. [DOI: 10.1517/17425247.2014.882314] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
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Chávez-Tapia NC, Rosso N, Uribe M, Bojalil R, Tiribelli C. Kinetics of the inflammatory response induced by free fatty acid accumulation in hepatocytes. Ann Hepatol 2014; 13:113-120. [DOI: 10.1016/s1665-2681(19)30912-3] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 08/30/2023]
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Abstract
BACKGROUND Orthotopic liver transplantation (OLT) is the most effective therapy for liver failure. However, OLT is severely limited by the shortage of liver donors. Bioartificial liver (BAL) shows great potential as an alternative therapy for liver failure. In recent years, progress has been made in BAL regarding genetically engineered cell lines, immortalized human hepatocytes, methods for preserving the phenotype of primary human hepatocytes, and other functional hepatocytes derived from stem cells. DATA SOURCES A systematic search of PubMed and ISI Web of Science was performed to identify relevant studies in English language literature using the key words such as liver failure, bioartificial liver, hepatocyte, stem cells, differentiation, and immortalization. More than 200 articles related to the cell sources of hepatocyte in BAL were systematically reviewed. RESULTS Methods for preserving the phenotype of primary human hepatocytes have been successfully developed. Many genetically engineered cell lines and immortalized human hepatocytes have also been established. Among these cell lines, the incorporation of BAL with GS-HepG2 cells or alginate-encapsulated HepG2 cells could prolong the survival time and improve pathophysiological parameters in an animal model of liver failure. The cBAL111 cells were evaluated using the AMC-BAL bioreactor, which could eliminate ammonia and lidocaine, and produce albumin. Importantly, BAL loading with HepLi-4 cells could significantly improve the blood biochemical parameters, and prolong the survival time in pigs with liver failure. Other functional hepatocytes differentiated from stem cells, such as human liver progenitor cells, have been successfully achieved. CONCLUSIONS Aside from genetically modified liver cell lines and immortalized human hepatocytes, other functional hepatocytes derived from stem cells show great potential as cell sources for BAL. BAL with safe and effective liver cells may be achieved for clinical liver failure in the near future.
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Affiliation(s)
- Xiao-Ping Pan
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China
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22
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Chavez-Tapia NC, Rosso N, Tiribelli C. Effect of intracellular lipid accumulation in a new model of non-alcoholic fatty liver disease. BMC Gastroenterol 2012; 12:20. [PMID: 22380754 PMCID: PMC3313845 DOI: 10.1186/1471-230x-12-20] [Citation(s) in RCA: 98] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/14/2011] [Accepted: 03/01/2012] [Indexed: 02/08/2023] Open
Abstract
Background In vitro exposure of liver cells to high concentrations of free fatty acids (FFA) results in fat overload which promotes inflammatory and fibrogenic response similar to those observed in patients with Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH). Since the mechanisms of this event have not been fully characterized, we aimed to analyze the fibrogenic stimuli in a new in vitro model of NASH. Methods HuH7 cells were cultured for 24 h in an enriched medium containing bovine serum albumin and increasing concentrations of palmitic and oleic acid at a molar ratio of 1:2 (palmitic and oleic acid, respectively). Cytotoxic effect, apoptosis, oxidative stress, and production of inflammatory and fibrogenic cytokines were measured. Results FFA induces a significant increment in the intracellular content of lipid droplets. The gene expression of interleukin-6, interleukin-8 and tumor necrosis factor alpha was significantly increased. The protein level of interleukin-8 was also increased. Intracellular lipid accumulation was associated to a significant up-regulation in the gene expression of transforming growth factor beta 1, alpha 2 macroglobulin, vascular endothelial growth factor A, connective tissue growth factor, insulin-like growth factor 2, thrombospondin 1. Flow cytometry analysis demonstrated a significant increment of early apoptosis and production of reactive oxygen species. Conclusions The exposure of hepatocytes to fatty acids elicits inflammation, increase of oxidative stress, apoptosis and production of fibrogenic cytokines. These data support a primary role of FFA in the pathogenesis of NAFLD and NASH.
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Affiliation(s)
- Norberto C Chavez-Tapia
- Fondazione Italiana Fegato- Centro Studi Fegato, AREA SCIENCE Park Basovizza, Bldg Q, Trieste, Italy.
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23
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Farra R, Dapas B, Pozzato G, Scaggiante B, Agostini F, Zennaro C, Grassi M, Rosso N, Giansante C, Fiotti N, Grassi G. Effects of E2F1-cyclin E1-E2 circuit down regulation in hepatocellular carcinoma cells. Dig Liver Dis 2011; 43:1006-1014. [PMID: 21831731 DOI: 10.1016/j.dld.2011.07.007] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/24/2011] [Revised: 06/30/2011] [Accepted: 07/07/2011] [Indexed: 02/07/2023]
Abstract
BACKGROUND No effective therapy is available for hepatocellular carcinoma. To identify novel therapeutic strategies, we explored the effects of the depletion of E2F1, cyclin E1-E2 whose inter-relationships in hepatocellular carcinoma cell proliferation have never been defined. METHODS siRNA-mediated depletion of the targets was studied in the hepatocellular carcinoma cells HepG2, HuH7 and JHH6 characterized by high, medium and low hepatocyte differentiation grade, respectively; a model of normal human hepatocytes was also considered. RESULTS The depletion of each target mRNA reduced the levels of the other two mRNAs, thus demonstrating a close regulatory control, also confirmed by over-expression experiments. At the protein level, an exception to this trend was observed for cyclinE1 whose amount increased upon cyclin E2 (HepG2, HuH7, JHH6) and E2F1 (HepG2) depletion. In HepG2, reduced cyclinE1 proteolysis accounted for this observation. Additionally, cyclin E1-E2-E2F1 targeting decreased the levels of cyclin A2 mRNA and of the hyper-phosphorylated form of pRb thus leading to an S-phase cell decrease; migration was impaired as well. Finally, the model of human hepatocytes used was clearly less affected by target mRNAs depletion than hepatocellular carcinoma cells. CONCLUSION Our data provide novel mutual relationships amongst cyclin E1-E2-E2F1 and indicate their role in sustaining hepatocellular carcinoma cell proliferation/migration, validating the concept of an anti-cyclin E1-E2-E2F1 therapeutic approach for hepatocellular carcinoma.
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Affiliation(s)
- Rossella Farra
- Department of Medical, Technological and Translational Sciences, University Hospital of Cattinara, Trieste, Italy
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Gong M, Bi Y, Jiang W, Zhang Y, Chen L, Hou N, Liu Y, Wei X, Chen J, Li T. Immortalized mesenchymal stem cells: an alternative to primary mesenchymal stem cells in neuronal differentiation and neuroregeneration associated studies. J Biomed Sci 2011; 18:87. [PMID: 22118013 PMCID: PMC3239243 DOI: 10.1186/1423-0127-18-87] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2011] [Accepted: 11/25/2011] [Indexed: 12/28/2022] Open
Abstract
Background Mesenchymal stem cells (MSCs) can be induced to differentiate into neuronal cells under appropriate cellular conditions and transplanted in brain injury and neurodegenerative diseases animal models for neuroregeneration studies. In contrast to the embryonic stem cells (ESCs), MSCs are easily subject to aging and senescence because of their finite ability of self-renewal. MSCs senescence seriously affected theirs application prospects as a promising tool for cell-based regenerative medicine and tissue engineering. In the present study, we established a reversible immortalized mesenchymal stem cells (IMSCs) line by using SSR#69 retrovirus expressing simian virus 40 large T (SV40T) antigen as an alternative to primary MSCs. Methods The retroviral vector SSR#69 expressing simian virus 40 large T (SV40T) antigen was used to construct IMSCs. IMSCs were identified by flow cytometry to detect cell surface makers. To investigate proliferation and differentiation potential of IMSCs, cell growth curve determination and mesodermal trilineage differentiation tests were performed. Neuronal differentiation characteristics of IMSCs were detected in vitro. Before IMSCs transplantation, we excluded its tumorigenicity in nude mice firstly. The Morris water maze tests and shuttle box tests were performed five weeks after HIBD models received cells transplantation therapy. Results In this study, reversible IMSCs were constructed successfully and had the similar morphology and cell surface makers as primary MSCs. IMSCs possessed better ability of proliferation and anti-senescence compared with primary MSCs, while maintained multilineage differentiation capacity. Neural-like cells derived from IMSCs had similar expressions of neural-specific genes, protein expression patterns and resting membrane potential (RMP) compared with their counterparts derived from primary MSCs. There was no bump formation in nude mice subcutaneously injected with IMSCs. IMSCs played same role as primary MSCs to improve learning ability and spatial memory of HIBD rats. Conclusions IMSCs not only retain their features of primary MSCs but also possess the ability of high proliferation and anti-senescence. IMSCs can definitely be induced to differentiate into neuronal cells in vitro and take the place of primary MSCs for cell transplantation therapy without tumorigenesis in vivo. The stable cell line is particularly useful and valuable as an alternative to MSCs in neuronal differentiation and neuroregeneration associated studies.
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Affiliation(s)
- Min Gong
- Children's Hospital of Chongqing Medical University, Chongqing, China
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Risal P, Cho BH, Sylvester KG, Kim JC, Kim HT, Jeong YJ. The establishment and characterization of immortal hepatocyte cell lines from a mouse liver injury model. In Vitro Cell Dev Biol Anim 2011; 47:526-34. [PMID: 21805231 DOI: 10.1007/s11626-011-9445-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2011] [Accepted: 07/11/2011] [Indexed: 12/18/2022]
Abstract
Hepatocytes are an important research tool used for numerous applications. However, a short life span and a limited capacity to replicate in vitro limit the usefulness of primary hepatocyte cultures. We have hypothesized that in vivo priming of hepatocyte could make them more susceptible to growth factors in the medium for continuous proliferation in vitro. Here, a novel approach used to establish hepatocyte cell lines that included hepatocyte priming in vivo prior to culture with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet was attempted. The cell line grew in a monolayer while maintaining a granular cytoplasm and a round nucleus. Electron microscopy displayed hepatocyte-like features including mitochondria, glycogen granules, and the presence of bile canaliculi. This cell line expressed many mature hepatocyte-specific genes including albumin, alpha1-antitrypsin, glucose 6-phosphatase, and tyrosine aminotransferase. Functional characteristic of hepatocytes like the ability to store glycogen, lipid, and synthesis of urea is well demonstrated by this cell line. These cells demonstrated anchorage dependent growth properties in soft agar and did not form tumors after transplantation into nude mice. This cell line can be sustained in culture for more than 100 passages (>1.5 years) without undergoing noticeable morphological changes or transformation. This novel method resulted in the establishment of an immortal, non-transformed hepatocyte cell line with functional characteristics that may aid research of cell metabolism, toxicology, and hepatocyte transplantation.
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Affiliation(s)
- Prabodh Risal
- Laboratory of Liver Regeneration, Research Institute of Clinical medicine, Chonbuk National University Hospital, 634-18 Geuman-dong, Duckjin-gu, Jeonju, 516-712, South Korea.
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Sgroi A, Mai G, Morel P, Baertschiger RM, Gonelle-Gispert C, Serre-Beinier V, Buhler LH. Transplantation of encapsulated hepatocytes during acute liver failure improves survival without stimulating native liver regeneration. Cell Transplant 2011; 20:1791-803. [PMID: 21396154 DOI: 10.3727/096368911x564976] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
The aim of this study was to evaluate the effects of intraperitoneal transplantation of encapsulated human hepatocytes on liver metabolism and regeneration of mice with acute liver failure. Primary human hepatocytes were immortalized using lentiviral vectors coding for antiapoptotic genes and microencapsulated using alginate-polylysine polymers. In vitro, immortalized human hepatocytes showed low, but stable, synthetic and catabolitic functions over time, when compared to primary hepatocytes. In vivo, mice with acute liver failure and transplanted with encapsulated immortalized human hepatocytes had a significantly improved survival and biochemical profile, compared to mice transplanted with empty capsules. Serum levels of cytokines implicated in liver regeneration were lower in mice transplanted with hepatocytes compared to mice receiving empty capsules. This decrease was significant for IL-6 and HGF at 3 h. Measurement of liver regeneration showed no significant difference between mice transplanted with hepatocytes compared to control groups. Intraperitoneal transplantation of encapsulated immortalized hepatocytes significantly improved survival of mice with acute liver failure by providing metabolic support and without modifying liver regeneration. The lower levels of cytokines implicated in liver regeneration suggest that the metabolic support provided by the encapsulated hepatocytes reduced the inflammatory stress on the liver and herein decreased the regenerative trigger on residual hepatocytes. These data emphasize that metabolic function and regeneration of hepatocytes are two distinct aspects that need to be studied and approached separately during acute liver failure.
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Affiliation(s)
- Antonino Sgroi
- Surgical Research Unit, Department of Surgery, University Hospital Geneva, Geneva, Switzerland
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27
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De Gottardi A, Spahr L, Ravier-Dall'Antonia F, Hadengue A. Cannabinoid receptor 1 and 2 agonists increase lipid accumulation in hepatocytes. Liver Int 2010; 30:1482-9. [PMID: 20602678 DOI: 10.1111/j.1478-3231.2010.02298.x] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
BACKGROUND Cannabinoid receptors CB1 and CB2 are expressed in the liver, but their regulation in fatty hepatocytes is poorly documented. The aim of this study was to investigate the effects of selective CB1 or CB2 agonists on the expression of key regulators of lipid metabolism. METHODS We used an in vitro model of fatty liver by treating immortalized human hepatocytes and HepG2 cells with oleic acid and the selective agonists arachidonyl-2-chloroethylamide (ACEA) (CB1, 12 nM) and (2-iodo-5-nitrophenyl)-[1-(1-methylpiperidin-2-ylmethyl)-1H-indol-3-yl]-methanone (AM1241) (CB2, 16 nM). The quantity of intracellular lipids was assessed using Oil-Red-O and a biochemical triglyceride assay. The expression of several proteins regulating endocannabinoid signalling and lipid metabolism was quantified by real-time polymerase chain reaction and by Western blot. RESULTS Both CB1 and CB2 agonists dose-dependently increased the degree of steatosis of oleic acid-treated fatty hepatocytes. Cannabinoid receptors were downregulated in the presence of steatosis, and treatment with a CB2 agonist increased the expression of CB1. Carnitine palmitoyltransferase 1 was significantly overexpressed and sterol response element-binding protein (SREBP)-1c, fatty acid synthase and lecithin-cholesterol acetyltransferase (LCAT) were downregulated in fatty immortalized human hepatocytes. Treatment with the CB agonists ACEA and AM1241 partially reversed these changes, except for SREBP-1c. CB2, but not CB1, agonism decreased the expression of apolipoprotein B. In HepG2 cells, only LCAT resulted increased after treatment with CB agonists. CONCLUSIONS Not only CB1 but also CB2 participated in the regulation of lipid metabolism in human-derived immortalized hepatocytes by regulating the expression of key enzymes of lipid synthesis and transport.
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Affiliation(s)
- Andrea De Gottardi
- Division of Gastroenterology and Hepatology, University Hospital of Geneva, Geneva, Switzerland
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Synergistic induction of cell death in liver tumor cells by TRAIL and chemotherapeutic drugs via the BH3-only proteins Bim and Bid. Cell Death Dis 2010; 1:e86. [PMID: 21368859 PMCID: PMC3035907 DOI: 10.1038/cddis.2010.66] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Although death receptors and chemotherapeutic drugs activate distinct apoptosis signaling cascades, crosstalk between the extrinsic and intrinsic apoptosis pathway has been recognized as an important amplification mechanism. Best known in this regard is the amplification of the Fas (CD95) signal in hepatocytes via caspase 8-mediated cleavage of Bid and activation of the mitochondrial apoptosis pathway. Recent evidence, however, indicates that activation of other BH3-only proteins may also be critical for the crosstalk between death receptors and mitochondrial triggers. In this study, we show that TNF-related apoptosis-inducing ligand (TRAIL) and chemotherapeutic drugs synergistically induce apoptosis in various transformed and untransformed liver-derived cell lines, as well as in primary human hepatocytes. Both, preincubation with TRAIL as well as chemotherapeutic drugs could sensitize cells for apoptosis induction by the other respective trigger. TRAIL induced a strong and long lasting activation of Jun kinase, and activation of the BH3-only protein Bim. Consequently, synergistic induction of apoptosis by TRAIL and chemotherapeutic drugs was dependent on Jun kinase activity, and expression of Bim and Bid. These findings confirm a previously defined role of TRAIL and Bim in the regulation of hepatocyte apoptosis, and demonstrate that the TRAIL–Jun kinase–Bim axis is a major and important apoptosis amplification pathway in primary hepatocytes and liver tumor cells.
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Pan X, Du W, Yu X, Sheng G, Cao H, Yu C, Lv G, Huang H, Chen Y, Li J, Li LJ. Establishment and characterization of immortalized porcine hepatocytes for the study of hepatocyte xenotransplantation. Transplant Proc 2010; 42:1899-1906. [PMID: 20620546 DOI: 10.1016/j.transproceed.2009.11.043] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2009] [Accepted: 11/23/2009] [Indexed: 10/19/2022]
Abstract
BACKGROUND In light of the critical shortage of donor livers, xenogeneic sources offer the best alternative to human hepatocytes for the treatment of acute liver failure. This study investigated whether a combination of simian virus 40 large T antigen (SV40 LT) and human telomerase catalytic subunit (hTERT) genes could immortalize primary porcine hepatocytes that could reverse acute liver failure (ALF) in rats. METHODS We cotransfected SV40 LT and hTERT genes into primary porcine hepatocytes to examine the features of the transfected cell lines. We characterized the potentially therapeutic effect of immortalized porcine hepatocytes in a rat model of ALF induced by 90% hepatectomy. RESULTS An immortalized porcine hepatocyte cell line, HepLi, was expanded by >250 passages. HepLi cells maintained the defining characteristics of primary porcine hepatocytes, including porcine albumin secretion, urea production, and diazepam metabolism. Intrasplenic transplantation of HepLi cells significantly improved liver function, and significantly prolonging the survival of rats with ALF. CONCLUSIONS Cotransfection of SV40 LT and hTERT immortalized primary porcine hepatocytes without tumorigenicity in vitro. The Immortalized porcine hepatocytes served as a potential cell resource for xenotransplantation.
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Affiliation(s)
- X Pan
- First Affiliated Hospital, College of Medicine, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Zhejiang, China
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Harding MJ, Lepus CM, Gibson TF, Shepherd BR, Gerber SA, Graham M, Paturzo FX, Rahner C, Madri JA, Bothwell ALM, Lindenbach BD, Pober JS. An implantable vascularized protein gel construct that supports human fetal hepatoblast survival and infection by hepatitis C virus in mice. PLoS One 2010; 5:e9987. [PMID: 20376322 PMCID: PMC2848675 DOI: 10.1371/journal.pone.0009987] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2009] [Accepted: 02/22/2010] [Indexed: 01/16/2023] Open
Abstract
Background Widely accessible small animal models suitable for the study of hepatitis C virus (HCV) in vivo are lacking, primarily because rodent hepatocytes cannot be productively infected and because human hepatocytes are not easily engrafted in immunodeficient mice. Methodology/Principal Findings We report here on a novel approach for human hepatocyte engraftment that involves subcutaneous implantation of primary human fetal hepatoblasts (HFH) within a vascularized rat collagen type I/human fibronectin (rCI/hFN) gel containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVEC) in severe combined immunodeficient X beige (SCID/bg) mice. Maturing hepatic epithelial cells in HFH/Bcl-2-HUVEC co-implants displayed endocytotic activity at the basolateral surface, canalicular microvilli and apical tight junctions between adjacent cells assessed by transmission electron microscopy. Some primary HFH, but not Huh-7.5 hepatoma cells, appeared to differentiate towards a cholangiocyte lineage within the gels, based on histological appearance and cytokeratin 7 (CK7) mRNA and protein expression. Levels of human albumin and hepatic nuclear factor 4α (HNF4α) mRNA expression in gel implants and plasma human albumin levels in mice engrafted with HFH and Bcl-2-HUVEC were somewhat enhanced by including murine liver-like basement membrane (mLBM) components and/or hepatocyte growth factor (HGF)-HUVEC within the gel matrix. Following ex vivo viral adsorption, both HFH/Bcl-2-HUVEC and Huh-7.5/Bcl-2-HUVEC co-implants sustained HCV Jc1 infection for at least 2 weeks in vivo, based on qRT-PCR and immunoelectron microscopic (IEM) analyses of gel tissue. Conclusion/Significance The system described here thus provides the basis for a simple and robust small animal model of HFH engraftment that is applicable to the study of HCV infections in vivo.
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Affiliation(s)
- Martha J Harding
- Section of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut, United States of America.
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Getachew Y, James L, Lee WM, Thiele DL, Miller BC. Susceptibility to acetaminophen (APAP) toxicity unexpectedly is decreased during acute viral hepatitis in mice. Biochem Pharmacol 2009; 79:1363-71. [PMID: 20036646 DOI: 10.1016/j.bcp.2009.12.019] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2009] [Revised: 12/17/2009] [Accepted: 12/17/2009] [Indexed: 01/27/2023]
Abstract
Acetaminophen (APAP) hepatotoxicity results from cytochrome P450 metabolism of APAP to the toxic metabolite, n-acetyl-benzoquinone imine (NAPQI), which reacts with cysteinyl residues to form APAP adducts and initiates cell injury. As APAP is commonly used during viral illnesses there has been concern that APAP injury may be additive to that of viral hepatitis, leading physicians to advise against its use in such patients; this has not been investigated experimentally. We infected C57BL/6 male mice with replication-deficient adenovirus to produce moderately severe acute viral hepatitis and observed that APAP doses that were hepatotoxic or lethal in control mice produced neither death nor additional increase in serum ALT when administered to infected mice at the peak of virus-induced liver injury. Moreover, the concentration of hepatic APAP-protein adducts formed in these mice was only 10% that in control mice. Protection from APAP hepatotoxicity also was observed earlier in the course of infection, prior to the peak virus-induced ALT rise. Hepatic glutathione limits APAP-protein adduct formation but glutathione levels were similar in control and infected mice. Cyp1a2 (E.C. 1.14.14.1) and Cyp2e1 (E.C. 1.14.13.n7) mRNA expression decreased by 3 days post-infection and hepatic Cyp2e1 protein levels were reduced almost 90% at 7 days, when adduct formation was maximally inhibited. In vitro, hepatocytes from virally infected mice also were resistant to APAP-induced injury but sensitive to NAPQI. Rather than potentiating APAP-induced liver injury, acute viral hepatitis in this model resulted in selective down-regulation of APAP metabolizing P450s in liver and decreased the risk of APAP hepatotoxicity.
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Affiliation(s)
- Yonas Getachew
- Division of Liver and Digestive Diseases, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9151, USA
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Deurholt T, van Til NP, Chhatta AA, ten Bloemendaal L, Schwartlander R, Payne C, Plevris JN, Sauer IM, Chamuleau RA, Elferink RPO, Seppen J, Hoekstra R. Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes. BMC Biotechnol 2009; 9:89. [PMID: 19845959 PMCID: PMC2770505 DOI: 10.1186/1472-6750-9-89] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2008] [Accepted: 10/21/2009] [Indexed: 01/15/2023] Open
Abstract
Background A clonal cell line that combines both stable hepatic function and proliferation capacity is desirable for in vitro applications that depend on hepatic function, such as pharmacological or toxicological assays and bioartificial liver systems. Here we describe the generation and characterization of a clonal human cell line for in vitro hepatocyte applications. Results Cell clones derived from human fetal liver cells were immortalized by over-expression of telomerase reverse transcriptase. The resulting cell line, cBAL111, displayed hepatic functionality similar to the parental cells prior to immortalization, and did not grow in soft agar. Cell line cBAL111 expressed markers of immature hepatocytes, like glutathione S transferase and cytokeratin 19, as well as progenitor cell marker CD146 and was negative for lidocaine elimination. On the other hand, the cBAL111 cells produced urea, albumin and cytokeratin 18 and eliminated galactose. In contrast to hepatic cell lines NKNT-3 and HepG2, all hepatic functions were expressed in cBAL111, although there was considerable variation in their levels compared with primary mature hepatocytes. When transplanted in the spleen of immunodeficient mice, cBAL111 engrafted into the liver and partly differentiated into hepatocytes showing expression of human albumin and carbamoylphosphate synthetase without signs of cell fusion. Conclusion This novel liver cell line has the potential to differentiate into mature hepatocytes to be used for in vitro hepatocyte applications.
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Affiliation(s)
- Tanja Deurholt
- AMC Liver Center, Meibergdreef 69-71, 1105 BK Amsterdam, the Netherlands.
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Mei J, Sgroi A, Mai G, Baertschiger R, Gonelle-Gispert C, Serre-Beinier V, Morel P, Bühler LH. Improved survival of fulminant liver failure by transplantation of microencapsulated cryopreserved porcine hepatocytes in mice. Cell Transplant 2009; 18:101-10. [PMID: 19476213 DOI: 10.3727/096368909788237168] [Citation(s) in RCA: 70] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The aim of this study was to establish hepatocyte isolation in pigs, and to evaluate function of isolated hepatocytes after encapsulation, cryopreservation, and transplantation (Tx) in a mouse model of fulminant liver failure (FLF). After isolation, porcine hepatocytes were microencapsulated with alginate-poly-L-Lysine-alginate membranes and cryopreserved. In vitro, albumin production of free and encapsulated hepatocytes were measured by enzyme linked-immunoadsorbent assay. In vivo, encapsulated hepatocytes were transplanted into different groups of mice with FLF and the following experimental groups were performed: group 1, Tx of empty capsules; group 2, Tx of free primary porcine hepatocytes; group 3, Tx of fresh encapsulated porcine hepatocytes; group 4, Tx of cryopreserved encapsulated porcine hepatocytes. In vitro, fresh or cryopreserved encapsulated porcine hepatocytes showed a continuous decreasing metabolic function over 1 week (albumin and urea synthesis, drug catabolism). In vivo, groups 1 and 2 showed similar survival (18% and 25%, respectively, p > 0.05). In groups 3 and 4, Tx of fresh or cryopreserved encapsulated porcine hepatocytes significantly increased survival rate to 75% and 68%, respectively (p < 0.05). Primary porcine hepatocytes maintained metabolic functions after encapsulation and cryopreservation. In mice with FLF, Tx of encapsulated xenogeneic hepatocytes significantly improved survival. These results indicate that porcine hepatocytes can successfully be isolated, encapsulated, stored using cryopreservation, and transplanted into xenogeneic recipients with liver failure and sustain liver metabolic functions.
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Affiliation(s)
- Jie Mei
- Surgical Research Unit, Department of Surgery, University Hospital Geneva, Geneva, Switzerland
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Abstract
Estimates of hepatitis C virus infection include 170 million people worldwide, who face increased risk of development of cirrhosis, liver failure, and hepatocellular carcinoma. Standard of care therapy with pegylated interferon and ribavirin is effective in just half of patients, is challenged by substantial treatment-related morbidity, and is prohibitively expensive in most parts of the world. New therapeutics for treatment and prevention are clearly needed. Development of effective therapies has been significantly hampered by difficulties in establishing in vitro and in vivo models of viral replication. This chapter reviews development, validation, and early application of a mouse model with a chimeric human liver.
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Affiliation(s)
- Norman M Kneteman
- Department of Surgery Faculty of Medicine and Oral Health Sciences, University of Alberta, Edmonton, Canada
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Baertschiger RM, Serre-Beinier V, Morel P, Bosco D, Peyrou M, Clément S, Sgroi A, Kaelin A, Buhler LH, Gonelle-Gispert C. Fibrogenic potential of human multipotent mesenchymal stromal cells in injured liver. PLoS One 2009; 4:e6657. [PMID: 19684854 PMCID: PMC2722022 DOI: 10.1371/journal.pone.0006657] [Citation(s) in RCA: 98] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2009] [Accepted: 07/07/2009] [Indexed: 12/26/2022] Open
Abstract
Multipotent mesenchymal stromal cells (MSC) are currently investigated clinically as cellular therapy for a variety of diseases. Differentiation of MSC toward endodermal lineages, including hepatocytes and their therapeutic effect on fibrosis has been described but remains controversial. Recent evidence attributed a fibrotic potential to MSC. As differentiation potential might be dependent of donor age, we studied MSC derived from adult and pediatric human bone marrow and their potential to differentiate into hepatocytes or myofibroblasts in vitro and in vivo. Following characterization, expanded adult and pediatric MSC were co-cultured with a human hepatoma cell line, Huh-7, in a hepatogenic differentiation medium containing Hepatocyte growth factor, Fibroblast growth factor 4 and oncostatin M. In vivo, MSC were transplanted into spleen or liver of NOD/SCID mice undergoing partial hepatectomy and retrorsine treatment. Expression of mesenchymal and hepatic markers was analyzed by RT-PCR, Western blot and immunohistochemistry. In vitro, adult and pediatric MSC expressed characteristic surface antigens of MSC. Expansion capacity of pediatric MSC was significantly higher when compared to adult MSC. In co-culture with Huh-7 cells in hepatogenic differentiation medium, albumin expression was more frequently detected in pediatric MSC (5/8 experiments) when compared to adult MSC (2/10 experiments). However, in such condition pediatric MSC expressed alpha smooth muscle more strongly than adult MSC. Stable engraftment in the liver was not achieved after intrasplenic injection of pediatric or adult MSC. After intrahepatic injection, MSC permanently remained in liver tissue, kept a mesenchymal morphology and expressed vimentin and alpha smooth muscle actin, but no hepatic markers. Further, MSC localization merges with collagen deposition in transplanted liver and no difference was observed using adult or pediatric MSC. In conclusion, when transplanted into an injured or regenerating liver, MSC differentiated into myofibroblasts with development of fibrous tissue, regardless of donor age. These results indicate that MSC in certain circumstances might be harmful due to their fibrogenic potential and this should be considered before potential use of MSC for cell therapy.
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Affiliation(s)
- Reto M. Baertschiger
- Surgical Research Unit, Department of Surgery, University Hospital Geneva, Geneva, Switzerland
| | - Véronique Serre-Beinier
- Surgical Research Unit, Department of Surgery, University Hospital Geneva, Geneva, Switzerland
| | - Philippe Morel
- Surgical Research Unit, Department of Surgery, University Hospital Geneva, Geneva, Switzerland
| | - Domenico Bosco
- Cell Isolation and Transplantation Center, Department of Surgery, University Hospital Geneva, Geneva, Switzerland
| | - Marion Peyrou
- Surgical Research Unit, Department of Surgery, University Hospital Geneva, Geneva, Switzerland
| | - Sophie Clément
- Department of Pathology and Immunology, Medical School of Geneva, Geneva, Switzerland
| | - Antonino Sgroi
- Surgical Research Unit, Department of Surgery, University Hospital Geneva, Geneva, Switzerland
| | - André Kaelin
- Department of Pediatric Orthopedics, Children's Hospital, University Hospital of Geneva, Geneva, Switzerland
| | - Leo H. Buhler
- Surgical Research Unit, Department of Surgery, University Hospital Geneva, Geneva, Switzerland
| | - Carmen Gonelle-Gispert
- Surgical Research Unit, Department of Surgery, University Hospital Geneva, Geneva, Switzerland
- * E-mail:
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Tuñón MJ, Alvarez M, Culebras JM, González-Gallego J. An overview of animal models for investigating the pathogenesis and therapeutic strategies in acute hepatic failure. World J Gastroenterol 2009; 15:3086-98. [PMID: 19575487 PMCID: PMC2705730 DOI: 10.3748/wjg.15.3086] [Citation(s) in RCA: 161] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/24/2009] [Revised: 05/23/2009] [Accepted: 05/30/2009] [Indexed: 02/06/2023] Open
Abstract
Acute hepatic failure (AHF) is a severe liver injury accompanied by hepatic encephalopathy which causes multiorgan failure with an extremely high mortality rate, even if intensive care is provided. Management of severe AHF continues to be one of the most challenging problems in clinical medicine. Liver transplantation has been shown to be the most effective therapy, but the procedure is limited by shortage of donor organs. Although a number of clinical trials testing different liver assist devices are under way, these systems alone have no significant effect on patient survival and are only regarded as a useful approach to bridge patients with AHF to liver transplantation. As a result, reproducible experimental animal models resembling the clinical conditions are still needed. The three main approaches used to create an animal model for AHF are: surgical procedures, toxic liver injury and infective procedures. Most common models are based on surgical techniques (total/partial hepatectomy, complete/transient devascularization) or the use of hepatotoxic drugs (acetaminophen, galactosamine, thioacetamide, and others), and very few satisfactory viral models are available. We have recently developed a viral model of AHF by means of the inoculation of rabbits with the virus of rabbit hemorrhagic disease. This model displays biochemical and histological characteristics, and clinical features that resemble those in human AHF. In the present article an overview is given of the most widely used animal models of AHF, and their main advantages and disadvantages are reviewed.
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Establishment of a human neonatal hepatocyte cell line. In Vitro Cell Dev Biol Anim 2009; 45:535-42. [PMID: 19565302 DOI: 10.1007/s11626-009-9219-0] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2009] [Accepted: 05/28/2009] [Indexed: 01/05/2023]
Abstract
Hepatocytes are routinely used to generate and identify drug metabolites and hepatic toxicity. Primary cultures of human hepatocytes are the model cell of choice for most of these pharmacological and toxicological studies. However, major problems are encountered with primary liver cell cultures: the dwindling availability of viable livers, hepatocytes having a limited life span, the loss of liver-specific functions in culture, and the donor to donor variability. These limitations have created a significant need for an in vitro hepatocyte system, which has both the potential for use in toxicological and pharmaceutical studies as well as clinical applications. Ectopic expression of human telomerase reverse transcriptase (hTERT) is one of the major strategies used to develop immortalized cells. Immortalization of primary cells using hTERT allows retention of the original cellular characteristics and functions and avoids some of the genetic and phenotypic instabilities associated with using known oncogenes. In the present study, we developed a cell line from human neonatal hepatocytes by transduction with a recombinant retrovirus expressing the hTERT gene. Induction of stable expression of hTERT in the neonatal cells led to immortalization of these cells. The cell line was cultured continuously for more than 25 passages, equivalent to >25 population doublings, whereas the parental cells senesced within five passages. Analysis of telomerase activity as measured by telomeric repeat amplification protocol assay indicated elevated levels of telomerase activity in immortalized cells compared to the parental cells. These immortalized human hepatocytes cells maintained a normal diploid karyotype as well as the gene expression profile similar to that of human normal neonatal hepatocytes. The data suggest that these immortalized cells preserved some of the biological characteristics of hepatic progenitor cells and might be useful as an in vitro model for pharmacological and toxicity studies.
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Correia MP, Cardoso EM, Pereira CF, Neves R, Uhrberg M, Arosa FA. Hepatocytes and IL-15: a favorable microenvironment for T cell survival and CD8+ T cell differentiation. THE JOURNAL OF IMMUNOLOGY 2009; 182:6149-59. [PMID: 19414768 DOI: 10.4049/jimmunol.0802470] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Human intrahepatic lymphocytes are enriched in CD1d-unrestricted T cells coexpressing NKR. Although the origin of this population remains controversial, it is possible to speculate that the hepatic microenvironment, namely epithelial cells or the cytokine milieu, may play a role in its shaping. IL-15 is constitutively expressed in the liver and has a key role in activation and survival of innate and tissue-associated immune cells. In this in vitro study, we examined whether hepatocyte cell lines and/or IL-15 could play a role in the generation of NK-like T cells. The results show that both HepG2 cells and a human immortalized hepatocyte cell line increase survival and drive basal proliferation of T cells. In addition, IL-15 was capable of inducing Ag-independent up-regulation of NKR, including NKG2A, Ig-like receptors, and de novo expression of CD56 and NKp46 in CD8(+)CD56(-) T cells. In conclusion, our study suggests that hepatocytes and IL-15 create a favorable microenvironment for T cells to growth and survive. It can be proposed that the increased percentage of intrahepatic nonclassical NKT cells could be in part due to a local CD8(+) T cell differentiation.
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Ignatova ID, Kostadinova RM, Goldring CE, Nawrocki AR, Frey FJ, Frey BM. Tumor necrosis factor-alpha upregulates 11beta-hydroxysteroid dehydrogenase type 1 expression by CCAAT/enhancer binding protein-beta in HepG2 cells. Am J Physiol Endocrinol Metab 2009; 296:E367-77. [PMID: 19088256 DOI: 10.1152/ajpendo.90531.2008] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the conversion of inactive to active glucocorticoids. 11beta-HSD1 plays a crucial role in the pathogenesis of obesity and controls glucocorticoid actions in inflammation. Several studies have demonstrated that TNF-alpha increases 11beta-HSD1 mRNA and activity in various cell models. Here, we demonstrate that mRNA and activity of 11beta-HSD1 is increased in liver tissue from transgenic mice overexpressing TNF-alpha, indicating that this effect also occurs in vivo. To dissect the molecular mechanism of this increase, we investigated basal and TNF-alpha-induced transcription of the 11beta-HSD1 gene (HSD11B1) in HepG2 cells. We found that TNF-alpha acts via p38 MAPK pathway. Transient transfections with variable lengths of human HSD11B1 promoter revealed highest activity with or without TNF-alpha in the proximal promoter region (-180 to +74). Cotransfection with human CCAAT/enhancer binding protein-alpha (C/EBPalpha) and C/EBPbeta-LAP expression vectors activated the HSD11B1 promoter with the strongest effect within the same region. Gel shift and RNA interference assays revealed the involvement of mainly C/EBPalpha, but also C/EBPbeta, in basal and only of C/EBPbeta in the TNF-alpha-induced HSD11B1 expression. Chromatin immunoprecipitation assay confirmed in vivo the increased abundance of C/EBPbeta on the proximal HSD11B1 promoter upon TNF-alpha treatment. In conclusion, C/EBPalpha and C/EBPbeta control basal transcription, and TNF-alpha upregulates 11beta-HSD1, most likely by p38 MAPK-mediated increased binding of C/EBPbeta to the human HSD11B1 promoter. To our knowledge, this is the first study showing involvement of p38 MAPK in the TNF-alpha-mediated 11beta-HSD1 regulation, and that TNF-alpha stimulates enzyme activity in vivo.
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Affiliation(s)
- Irena D Ignatova
- Depts. of Nephrology and Hypertension and Clinical Research, Freiburgstrasse 15, Univ. Hospital, Berne, CH-3010 Berne, Switzerland
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40
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Abstract
More than 30 years after the first hepatocyte transplant to treat the Gunn rat, the animal model for Crigler-Najjar syndrome, there are still a number of impediments to hepatocyte transplantation. Numerous animal models are still used in work aimed at improving hepatocyte engraftment and/or long-term function. Although other cell sources, particularly hepatic and extrahepatic stem cells, are being explored, adult hepatocytes remain the cells of choice for the treatment of liver diseases by cell therapy. In recent years, diverse approaches have been developed in various animal models to enhance hepatocyte transduction and amplification in vitro and cell engraftment and functionality in vivo. They have led to significant progress in hepatocyte transplantation for the treatment of patients with metabolic diseases and for bridging patients with acute injury until their own livers regenerate. This review presents and considers the results of this work with a special emphasis on procedures that might be clinically applicable.
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Affiliation(s)
- Anne Weber
- Institut National de la Santé et de la Recherche Médicale Unité 804, Université Paris-Sud, Bicêtre Hospital, Kremlin-Bicêtre, France.
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Tsuruga Y, Kiyono T, Matsushita M, Takahashi T, Kasai H, Todo S. Establishment of Immortalized Human Hepatocytes by Introduction of HPV16 E6/E7 and hTERT as Cell Sources for Liver Cell-Based Therapy. Cell Transplant 2008; 17:1083-1094. [DOI: 10.3727/096368908786991542] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
For future cell-based therapies for liver diseases, the shortage of cell sources must be resolved. Immortalized human hepatocytes are expected to be among the new sources. In addition to telomerase activation by the introduction of human telomerase reverse transcriptase (hTERT), inactivation of the p16/RB pathway and/or p53 by E6/E7 of human papillomavirus type 16 (HPV16) has been shown to be useful for efficient immortalization of several human cell types. Here we report the immortalization of human hepatocytes by the introduction of HPV16 E6/E7 and hTERT. Human adult hepatocytes were lentivirally transduced with HPV16 E6/E7 and hTERT. Two human immortalized hepatocyte cell lines were established and were named HHE6E7T-1 and HHE6E7T-2. Those cells proliferated in culture beyond 200 population doublings (PDs). Albumin synthesis and expression of liver-enriched genes were confirmed, but gradually decreased as passages progressed. Karyotype analysis showed that HHE6E7T-1 cells remained near diploid but that HHE6E7T-2 cells showed severe aneuploidy at 150 PDs. Subcutaneous injection of these cells into severe combined immunodeficiency (SCID) mice did not induce tumor development. Intrasplenic transplantation of dedifferentiated HHE6E7T-1 cells over 200 PDs significantly improved the survival of acetaminophen-induced acute liver failure SCID mice. In conclusion, we successfully established immortalized human hepatocytes that retain the characteristics of differentiated hepatocytes. We also showed the reduction of hepatocyte-specific functions in long-term culture. However, the results of intrasplenic transplantation to SCID mice with acetaminophen-induced acute liver failure showed the possibility of HHE6E7T-1 serving as a cell source for hepatocyte transplantation.
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Affiliation(s)
- Yosuke Tsuruga
- Department of General Surgery, Hokkaido University Graduate School of Medicine, Sapporo 060-8648, Japan
| | - Tohru Kiyono
- Virology Division, National Cancer Center Research Institute, Tokyo 104-0045, Japan
| | - Michiaki Matsushita
- Department of General Surgery, Hokkaido University Graduate School of Medicine, Sapporo 060-8648, Japan
| | - Tohru Takahashi
- Department of General Surgery, Hokkaido University Graduate School of Medicine, Sapporo 060-8648, Japan
| | - Hironori Kasai
- Department of General Surgery, Hokkaido University Graduate School of Medicine, Sapporo 060-8648, Japan
| | - Satoru Todo
- Department of General Surgery, Hokkaido University Graduate School of Medicine, Sapporo 060-8648, Japan
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Quasdorff M, Hösel M, Odenthal M, Zedler U, Bohne F, Gripon P, Dienes HP, Drebber U, Stippel D, Goeser T, Protzer U. A concerted action of HNF4α and HNF1α links hepatitis B virus replication to hepatocyte differentiation. Cell Microbiol 2008; 10:1478-90. [DOI: 10.1111/j.1462-5822.2008.01141.x] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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Human Health Effects Workgroup Report. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2008; 619:579-606. [DOI: 10.1007/978-0-387-75865-7_26] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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Haker B, Fuchs S, Dierlamm J, Brümmendorf TH, Wege H. Absence of oncogenic transformation despite acquisition of cytogenetic aberrations in long-term cultured telomerase-immortalized human fetal hepatocytes. Cancer Lett 2007; 256:120-7. [PMID: 17630152 DOI: 10.1016/j.canlet.2007.06.007] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2007] [Revised: 05/10/2007] [Accepted: 06/05/2007] [Indexed: 10/23/2022]
Abstract
As a culture model to study hepatocarcinogenesis, telomerase-immortalized human fetal hepatocytes were monitored for karyotype changes evolving in long-term culture and development of functional defects in DNA damage response. G-banding revealed acquisition of characteristic karyotype abnormalities, e.g., trisomy 7 and monosomy X, in two independently immortalized and cultured populations after 80-100 population doublings. Interestingly, the detected aneuploidies resemble some of the genetic events observed in hepatocellular cancer. However, these genetic changes were not sufficient to induce oncogenic transformation reflected by absence of anchorage-independent growth. Furthermore, long-term cultured telomerase-immortalized cells preserved p53 expression levels and effective p53-mediated damage response.
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Affiliation(s)
- Björn Haker
- Department of Gastroenterology and Hepatology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany
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De Gottardi A, Vinciguerra M, Sgroi A, Moukil M, Ravier-Dall'Antonia F, Pazienza V, Pugnale P, Foti M, Hadengue A. Microarray analyses and molecular profiling of steatosis induction in immortalized human hepatocytes. J Transl Med 2007; 87:792-806. [PMID: 17558421 DOI: 10.1038/labinvest.3700590] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Hepatic steatosis is an important risk factor for the development of inflammation, fibrosis and impaired liver regeneration. The factors regulating lipid accumulation and driving hepatic steatosis toward inflammation, fibrosis and impaired regeneration are largely unknown. The aim of this study was to identify major alterations in gene expression occurring in steatotic hepatocytes, and to analyze how these changes impact cellular processes associated with steatosis. Microarray gene chips and RT-PCR were performed to analyze changes in gene expression induced in fatty human immortalized hepatocytes after treatment with 50 muM oleic acid for 7 days. Lipid metabolism and triglyceride accumulation in these cells was examined by Oil-Red-O staining, thin-layer chromatography (TLC) and immunofluorescence. Caspase 3 activity, BrdU incorporation and trypan blue exclusion were used to study apoptosis, proliferation and cell viability. Finally, quantitative analysis of signalling induced by insulin was performed by Western blot. Characterization of steatosis in three hepatocyte-derived cell lines indicated that the immortalized human hepatocytes (IHH) line was the most appropriate cell line for this study. Gene expression analysis showed significant alterations in the transcription of two major classes of genes involved either in cholesterol and fatty acid biosynthesis, as well as lipid export, or in apoptosis and cell proliferation. Such changes were functionally relevant, since TLC indicated that synthesis and accumulation of triglycerides were increased in steatotic cells, while synthesis of cholesterol and fatty acids were decreased. Lipid accumulation in IHH was associated with an increased apoptosis and an inhibition of cell proliferation and viability. No detectable changes in genes associated with insulin resistance were observed in steatotic cells, but signalling induced by insulin was more efficient in steatotic IHH as compared to control cells. We conclude that IHH represent a new valuable model of steatosis, not associated with insulin resistance, to study at both the genetic and functional level factors involved in the process of lipid accumulation and steatosis-associated liver injury.
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Affiliation(s)
- Andrea De Gottardi
- Division of Gastroenterology and Hepatology, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
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Dansako H, Ikeda M, Kato N. Limited suppression of the interferon-beta production by hepatitis C virus serine protease in cultured human hepatocytes. FEBS J 2007; 274:4161-76. [PMID: 17651439 DOI: 10.1111/j.1742-4658.2007.05942.x] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Toll-like receptors and RNA helicase family members [retinoic acid-inducible gene I (RIG-I) and melanoma differentiation associated gene-5 (MDA5)] play important roles in the induction of interferon-beta as a major event in innate immune responses after virus infection. TRIF (adaptor protein of Toll-like receptor 3)-mediated and Cardif (adaptor protein of RIG-I or MDA5)-mediated signaling pathways contribute rapid induction of interferon-beta through the activation of interferon regulatory factor-3 (IRF-3). Previously, it has been reported that the hepatitis C virus NS3-4A serine protease blocks virus-induced activation of IRF-3 in the human hepatoma cell line HuH-7, and that NS3-4A cleaves TRIF and Cardif molecules, resulting in the interruption of antiviral signaling pathways. On the other hand, it has recently been reported that non-neoplastic human hepatocyte PH5CH8 cells retain robust TRIF- and Cardif-mediated pathways, unlike HuH-7 cells, which lack a TRIF-mediated pathway. In the present study, we further investigated the effect of NS3-4A on antiviral signaling pathways. Although we confirmed that PH5CH8 cells were much more effective than HuH-7 cells for the induction of interferon-beta, we obtained the unexpected result that NS3-4A could not suppress the interferon-beta production induced by the TRIF-mediated pathway, although it suppressed the Cardif-mediated pathway by cleaving Cardif at the Cys508 residue. Using PH5CH8, HeLa, and HuH-7-derived cells, we further showed that NS3-4A could not cleave TRIF, in disagreement with a previous report describing the cleavage of TRIF by NS3-4A. Taken together, our findings suggest that the blocking of the interferon production by NS3-4A is not sufficient in HCV-infected hepatocyte cells.
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Affiliation(s)
- Hiromichi Dansako
- Department of Molecular Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Japan
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47
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Jost S, Turelli P, Mangeat B, Protzer U, Trono D. Induction of antiviral cytidine deaminases does not explain the inhibition of hepatitis B virus replication by interferons. J Virol 2007; 81:10588-96. [PMID: 17652382 PMCID: PMC2045472 DOI: 10.1128/jvi.02489-06] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Interferons (IFNs) play a major role in the control of hepatitis B virus (HBV), whether as endogenous cytokines limiting the spread of the virus during the acute phase of the infection or as drugs for the treatment of its chronic phase. However, the mechanism by which IFNs inhibit HBV replication has so far remained elusive. Here, we show that type I and II IFN treatment of human hepatocytes induces the production of APOBEC3G (A3G) and, to a lesser extent, that of APOBEC3F (A3F) and APOBEC3B (A3B) but not that of two other cytidine deaminases also endowed with anti-HBV activity, activation-induced cytidine deaminase (AID), and APOBEC1. Most importantly, we reveal that blocking A3B, A3F, and A3G by combining RNA interference and the virion infectivity factor (Vif) protein of human immunodeficiency virus does not abrogate the inhibitory effect of IFNs on HBV. We conclude that these cytidine deaminases are not essential effectors of IFN in its action against this pathogen.
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Affiliation(s)
- Stéphanie Jost
- School of Life Sciences and Frontiers in Genetics National Center for Competence in Research, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
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Yuan RH, Chen HL, Chen HL, Hsu MK, Lee PH, Chang MH. Attenuation of Kupffer Cell Function in Acute on Chronic Liver Injury Enhanced Engraftment of Transplanted Hepatocytes. World J Surg 2007; 31:1270-7; discussion 1278-9. [PMID: 17437157 DOI: 10.1007/s00268-007-9044-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
BACKGROUND The present study was designed to elucidate the relationship of engraftment efficiency of transplanted cells and Kupffer cell function in mice with acute on chronic liver injury and acute liver injury. METHODS The recipient dipeptidyl peptidase IV knockout (DPPIV(-/-)) mice were divided into two groups: (1) the acute on chronic liver injury group (CCl(4)/APAP group) that received CCl(4) (1 ml/kg) twice a week for 4 weeks following one dose of acetaminophen (APAP), 600 mg/kg; (2) the acute liver injury group (APAP-only group) that received a single dose of APAP at 600 mg/kg. DPPIV(+/+) hepatocytes were transplanted 24 h after APAP intoxication. Engraftment efficiency was evaluated at day 7 and day 14 after transplantation. The tumor necrosis factor-alpha (TNF-alpha) mRNA expression level of Kupffer cells immediately before cell transplantation was compared between the two groups before and after lipopolysaccharide (LPS, 100 ng/ml) stimulation. RESULTS The number of transplanted cells and clusters in each 100x microscopic field were higher in the CCl(4)/APAP group at both day 7 (21.5 +/- 6.3 versus 8.3 +/- 4.0, p < 0.001; 14.9 +/- 4.6 versus 6.6 +/- 3.4, p < 0.001, respectively) and day 14 (17.3 +/- 4.4 versus 10.2 +/- 3.3, p = 0.001; 12.6 +/- 3.2 versus 7.9 +/- 1.6, p = 0.004, respectively). After LPS stimulation, the expression level of TNF-alpha was lower (175.7 +/- 54.6 versus 465.6 +/- 64.2, p = 0.002), and the increment of TNF-alpha expression was also less significant in the CCl(4)/APAP group (1.5-fold versus 6.5-fold, p = 0.014). CONCLUSIONS Chronic liver injury desensitized Kupffer cells and reduced TNF-alpha expression, two results that correlated with the increased engraftment of transplanted cells.
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Affiliation(s)
- Ray-Hwang Yuan
- Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
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Durantel D, Zoulim F. Going towards more relevant cell culture models to study the in vitro replication of serum-derived hepatitis C virus and virus/host cell interactions? J Hepatol 2007; 46:1-5. [PMID: 17112625 DOI: 10.1016/j.jhep.2006.10.005] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
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Detry O, De Roover A, Honore P, Meurisse M. Brain edema and intracranial hypertension in fulminant hepatic failure: Pathophysiology and management. World J Gastroenterol 2006; 12:7405-12. [PMID: 17167826 PMCID: PMC4087583 DOI: 10.3748/wjg.v12.i46.7405] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Intracranial hypertension is a major cause of morbidity and mortality of patients suffering from fulminant hepatic failure. The etiology of this intracranial hypertension is not fully determined, and is probably multifactorial, combining a cytotoxic brain edema due to the astrocytic accumulation of glutamine, and an increase in cerebral blood volume and cerebral blood flow, in part due to inflammation, to glutamine and to toxic products of the diseased liver. Validated methods to control intracranial hypertension in fulminant hepatic failure patients mainly include mannitol, hypertonic saline, indomethacin, thiopental, and hyperventilation. However all these measures are often not sufficient in absence of liver transplantation, the only curative treatment of intracranial hypertension in fulminant hepatic failure to date. Induced moderate hypothermia seems very promising in this setting, but has to be validated by a controlled, randomized study. Artificial liver support systems have been under investigation for many decades. The bioartificial liver, based on both detoxification and swine liver cells, has shown some efficacy on reduction of intracranial pressure but did not show survival benefit in a controlled, randomized study. The Molecular Adsorbents Recirculating System has shown some efficacy in decreasing intracranial pressure in an animal model of liver failure, but has still to be evaluated in a phase III trial.
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