RESUMEN
BACKGROUND & AIMS: C/EBPbeta is an important mediator of several cellular processes, such as differentiation, proliferation, and survival of hepatic cells. However, a complete catalog of the targets of C/EBPbeta or the mechanism by which this transcription factor regulates certain liver-dependent pathways has not been clearly determined. Two major natural isoforms of this transcription factor exist: the liver-enriched activating protein (LAP) and the liver-enriched inhibitory protein (LIP), a functional LAP antagonist. In this study, we used the opposing transcriptional effects driven by LAP and LIP to determine the genuine C/EBPbeta molecular signature in the Hep3B human hepatoma cell line. We subsequently investigated the role of each of the LAP and LIP isoforms in drug-induced Hep3B cell death. METHODS: We engineered Hep3B cells with regulated LAP or LIP expression using the Tet-off expression system. The genes that showed inverse regulation by LAP and LIP were identified by cDNA array analysis. The cohort of direct-C/EBPbeta-targets was distinguished from indirect-targets by ChIP-on-chip analysis. RESULTS: We characterized 676 genes by this approach. Among these genes, 39 are novel direct targets of C/EBPbeta. Eleven of these new direct targets are involved in cell survival, suggesting critical roles for LAP/LIP isoforms in this cellular process. Therefore, we examined the effects of LAP and LIP over-expression on cell survival. We show that LIP promotes survival in staurosporine- or taxol-induced Hep3B cell death. CONCLUSIONS: Our study provides new molecular and cellular insights into the role of C/EBPbeta in cells of hepatic origin.
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Proteína beta Potenciadora de Unión a CCAAT/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteína beta Potenciadora de Unión a CCAAT/fisiología , Carcinoma Hepatocelular/patología , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Muerte Celular/fisiología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Perfilación de la Expresión Génica , Ingeniería Genética , Humanos , Neoplasias Hepáticas/patología , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Paclitaxel/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Estaurosporina/farmacología , Transcripción GenéticaRESUMEN
OBJECTIVES: The overall non-response rate to biologics remains 30-40% for patients with RA resistant to MTX. The objective of this study was to predict responsiveness to the anakinra-MTX combination by peripheral blood mononuclear cell gene profiling in order to optimize treatment choice. METHODS: Thirty-two patients treated with anakinra (100 mg/day s.c.) and MTX were categorized as responders when their 28-joint DAS (DAS-28) had decreased by ≥1.2 at 3 months. Pre-treatment blood samples had been drawn. RESULTS: For seven responders and seven non-responders, 52 microarray-identified mRNAs were expressed as a function of the response to treatment, and unsupervised hierarchical clustering correctly separated responders from non-responders. The levels of seven of these 52 transcripts, as assessed by real-time, quantitative RT-PCR, were able to accurately classify 15 of 18 other patients (8 responders and 10 non-responders), with 87.5% specificity and 77.8% negative-predictive value for responders. Among the 52 genes, 56% were associated with IL-1ß. CONCLUSION: This predictive gene expression profile was obtained with a non-invasive procedure. After further validation in other cohorts of patients, it could be proposed and used on a large scale to select likely RA responders to combined anakinra-MTX. Trial registration. Clinical Trials; NCT00213538 (http://www.clinicaltrials.gov).
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Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Perfilación de la Expresión Génica , Proteína Antagonista del Receptor de Interleucina 1/administración & dosificación , Metotrexato/administración & dosificación , Adulto , Anciano , Artritis Reumatoide/genética , Quimioterapia Combinada , Femenino , Humanos , Masculino , Persona de Mediana Edad , Resultado del Tratamiento , Estudios de Validación como AsuntoRESUMEN
Liver biopsy is considered the gold-standard method for the assessment of liver fibrosis during follow-up of hepatitis C virus-infected patients, but this invasive procedure is not devoid of complications. The aim of the present study was to identify novel non-invasive markers of fibrosis progression. By microarray analysis, we compared transcript levels in two extreme stages of fibrosis from 16 patients. Informative transcripts were validated by real-time PCR and used for the assessment of fibrosis in 23 additional patients. Sixteen transcripts were found to be dysregulated during the fibrogenesis process. Among them, some were of great interest because their corresponding proteins could be serologically measured. Thus, the protein levels of inter-alpha inhibitor H1, serpin peptidase inhibitor clade F member 2, and transthyretin were all significantly different according to the four Metavir stages of fibrosis. In conclusion, we report here that dysregulation, at both the transcriptional and protein levels, exists during the fibrogenesis process. Our description of three novel serum markers and their potential use as serological tests for the non-invasive diagnosis of liver fibrosis open new opportunities for better follow-up of hepatitis C virus-infected patients.
Asunto(s)
Biomarcadores/sangre , Hepatitis C/sangre , Hepatitis C/patología , Cirrosis Hepática/sangre , Cirrosis Hepática/patología , alfa-Globulinas/biosíntesis , alfa-Globulinas/genética , Western Blotting , Progresión de la Enfermedad , Femenino , Hepacivirus , Hepatitis C/genética , Humanos , Cirrosis Hepática/virología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Prealbúmina/biosíntesis , Prealbúmina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , alfa 2-Antiplasmina/biosíntesis , alfa 2-Antiplasmina/genéticaRESUMEN
AIMS: Hepatocellular carcinoma (HCC) results from cirrhosis and, in Western Europe, hepatitis C virus and alcoholism are the predominant causes of this disease. We recently documented a global transcript repression in hepatocarcinoma nodules. The tumour suppressor activated pathway-6 (TSAP6) transcript codes for a transmembrane molecule that is an inducer of a caspase-3-dependent apoptotic pathway. The down-regulation of TSAP6 transcripts in HCC and perinodular cirrhosis, which contrasts with a sustained transcript level in HCC-free cirrhosis, has suggested that this hepatic protein level may provide a prognostic marker for HCC occurrence. METHODS AND RESULTS: This protein was quantified by semiquantitative assessment of immunohistochemistry on samples from 42 cases HCC-free cirrhosis, 49 cases cirrhosis with HCC, 43 HCC associated with healthy liver and 31 controls. TSAP6 expression was linked to the liver state, healthy or cirrhotic without or with an HCC and to tumour grade. CONCLUSIONS: With biopsies periodically performed for surveillance purposes, the decreased expression of TSAP6 in cirrhotic tissue could reflect a decrease in the apoptotic process and could be interpreted as a warning sign. This evaluation of the TSAP6 level in cirrhotic liver conveys predictive information for the development of HCC.
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Carcinoma Hepatocelular/patología , Regulación hacia Abajo/genética , Cirrosis Hepática/patología , Neoplasias Hepáticas/patología , Hígado/patología , Proteínas Oncogénicas/genética , Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular , Expresión Génica , Inmunohistoquímica , Hígado/metabolismo , Cirrosis Hepática/genética , Neoplasias Hepáticas/genética , Proteínas Oncogénicas/metabolismo , OxidorreductasasRESUMEN
AIM: To look at a comprehensive picture of etiology-dependent gene abnormalities in hepatocellular carcinoma in Western Europe. METHODS: With a liver-oriented microarray, transcript levels were compared in nodules and cirrhosis from a training set of patients with hepatocellular carcinoma (alcoholism, 12; hepatitis C, 10) and 5 controls. Loose or tight selection of informative transcripts with an abnormal abundance was statistically valid and the tightly selected transcripts were next quantified by qRTPCR in the nodules from our training set (12 + 10) and a test set (6 + 7). RESULTS: A selection of 475 transcripts pointed to significant gene over-representation on chromosome 8 (alcoholism) or -2 (hepatitis C) and ontology indicated a predominant inflammatory response (alcoholism) or changes in cell cycle regulation, transcription factors and interferon responsiveness (hepatitis C). A stringent selection of 23 transcripts whose differences between etiologies were significant in nodules but not in cirrhotic tissue indicated that the above dysregulations take place in tumor but not in the surrounding cirrhosis. These 23 transcripts separated our test set according to etiologies. The inflammation-associated transcripts pointed to limited alterations of free iron metabolism in alcoholic vs hepatitis C tumors. CONCLUSION: Etiology-specific abnormalities (chromo-some preference; differences in transcriptomes and related functions) have been identified in hepatocellular carcinoma driven by alcoholism or hepatitis C. This may open novel avenues for differential therapies in this disease.
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Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Hepatitis C/complicaciones , Cirrosis Hepática Alcohólica/complicaciones , Cirrosis Hepática/complicaciones , Neoplasias Hepáticas/genética , Adulto , Anciano , Carcinoma Hepatocelular/virología , Cromosomas Humanos Par 2 , Cromosomas Humanos Par 8 , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica/métodos , Hepatitis C/genética , Humanos , Cirrosis Hepática/genética , Cirrosis Hepática/virología , Cirrosis Hepática Alcohólica/genética , Neoplasias Hepáticas/virología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Reproducibilidad de los ResultadosRESUMEN
AIM: To evaluate the effect of glutamine on intestinal mucosa integrity, glutathione stores and acute phase response in protein-depleted rats during an inflammatory shock. METHODS: Plasma acute phase proteins (APP), jejunal APP mRNA levels, liver and jejunal glutathione concentrations were measured before and one, three and seven days after turpentine injection in 4 groups of control, protein-restricted, protein-restricted rats supplemented with glutamine or protein powder. Bacterial translocation in mesenteric lymph nodes and intestinal morphology were also assessed. RESULTS: Protein deprivation and turpentine injection significantly reduced jejunal villus height, and crypt depths. Mucosal glutathione concentration significantly decreased in protein-restricted rats. Before turpentine oil, glutamine supplementation restored villus heights and glutathione concentration (3.24 +/- 1.05 vs 1.72 +/- 0.46 mumol/g tissue, P<0.05) in the jejunum, whereas in the liver glutathione remained low. Glutamine markedly increased jejunal alpha1-acid glycoprotein mRNA level after turpentine oil but did not affect its plasma concentration. Bacterial translocation in protein-restricted rats was not prevented by glutamine or protein powder supplementation. CONCLUSION: Glutamine restored gut glutathione stores and villus heights in malnourished rats but had no preventive effect on bacterial translocation in our model.
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Reacción de Fase Aguda/metabolismo , Glutamina/metabolismo , Glutatión/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/efectos de los fármacos , Desnutrición/metabolismo , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/metabolismo , Animales , Traslocación Bacteriana/efectos de los fármacos , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Suplementos Dietéticos , Modelos Animales de Enfermedad , Glutamina/administración & dosificación , Glutatión/administración & dosificación , Glicoproteínas/genética , Glicoproteínas/metabolismo , Enfermedades Inflamatorias del Intestino/microbiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Irritantes/efectos adversos , Hígado/metabolismo , Masculino , Orosomucoide , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Trementina/efectos adversosRESUMEN
Fetuin-A is an hepatic protein whose mRNA transiently falls during the inflammatory acute phase via unknown transcriptional mechanisms. Various FETUA promoter/cat constructs transiently transfected in the Hep3B hepatoma cell line allowed us to identify four NF-1 and C/EBP binding sites (N, C) arranged in a 5'-N2-C2-N1-C1-3' order and required for basal promoter activity. Mutant constructs demonstrated that C1 and C2 but not N1 nor N2 are required for the cytokine-driven down-regulation of the promoter. A variable spacing between C2 and N1 showed that the alignment of the (C1+N1) and (C2+N2) areas is critical for the promoter activity in quiescent but not cytokine-stimulated cells. Co-transfection of a plasmid only producing either a long or short C/EBPbeta isoform prevented FETUA regulation by cytokines. Electromobility shift assays with liver nuclear extracts showed that during the acute phase the complexes formed over N1 and N2 are not modified whereas short C/EBPalpha and -beta isoforms replace the long isoforms bound to C1 and C2 in the quiescent liver. Therefore the basal promoter activity requires an interaction between the long C/EBP isoforms bound to C1 and C2 whereas the inflammation-induced down-regulation results from the loss of interaction between the cytokine-induced, short C/EBP isoforms.
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Proteínas Sanguíneas/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Inflamación/fisiopatología , Hígado/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Proteínas Sanguíneas/genética , Proteínas Potenciadoras de Unión a CCAAT/genética , Línea Celular Tumoral , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Citocinas/farmacología , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Hígado/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Factores de Transcripción NFI , Regiones Promotoras Genéticas/genética , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta/efectos de los fármacos , Elementos de Respuesta/genética , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/metabolismo , Transfección , alfa-2-Glicoproteína-HSRESUMEN
BACKGROUND: B and T cells play a key role in rheumatoid arthritis (RA) pathophysiology. RasGRP1 and RasGRP3 are involved in T and B cell receptors signaling, and belong to gene combination able to predict infliximab responsiveness, leading to the question of RasGRP1 and RasGRP3 involvement in RA. METHODS: RasGRP1 and RasGRP3 expression levels were measured by qRT-PCR and/or western-blot in peripheral blood mononuclear cells (PBMCs), in T and B cells from untreated RA patients and in RA patients treated by TNFα inhibitors. T and B cells from healthy controls (HC) were cultured with TNFα, and TNFα receptors neutralizing antibodies to highlight the TNFα effects on RasGRP1 and RasGRP3 pathways. MAPK pathways and apoptosis were respectively analyzed using the Proteome Profiler arrays and flow cytometry. RESULTS: In PBMCs from RA patients, gene expression levels of RasGRP1 were invariant while RasGRP3 was downregulated under TNFα inhibitors and upregulated under TNFα. In T cells from RA patients, RasGRP1 was decreased and its gene expression level was correlated with disease activity. In T cells from HC, TNFα stimulation increased RasGRP1 gene expression level while it reduced RasGRP1 protein expression level. Bryostatin-1 experiments have confirmed that the TNFα effect observed on T cells proliferation was due to the decrease of RasGRP1 expression. Besides, RasGRP3 expression level increased in PBMCs from RA patients under TNFα and in B cells from HC leading us to conclude that RasGRP3 in B cells was modulated by TNFα. CONCLUSION: This study demonstrates RasGRP1 dysregulation in RA patients while RasGRP3 is characterized as a biomarker linked to TNFα inhibitors. After binding to TNFR1, TNFα reduced RasGRP1 protein expression resulting in inhibition of T cell activation. TRIAL REGISTRATION: Clinicaltrials.gov NCT00234234 , registered 04 November 2008; NCT00767325 , registered 05 October 2005.
Asunto(s)
Artritis Reumatoide/sangre , Proteínas de Unión al ADN/sangre , Factores de Intercambio de Guanina Nucleótido/sangre , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/sangre , Adalimumab/farmacología , Adalimumab/uso terapéutico , Adulto , Anciano , Artritis Reumatoide/tratamiento farmacológico , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Biomarcadores/sangre , Células Cultivadas , Etanercept/farmacología , Etanercept/uso terapéutico , Femenino , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Adulto Joven , Factores de Intercambio de Guanina Nucleótido rasRESUMEN
AIM: To search for transcription dysregulation that could (1) differentiate hepatocellular carcinoma (HCC)-free from HCC-related cirrhosis (2) differentiate HCC-free cirrhosis related to HCV from that related to alcohol intake. METHODS: Using microarray analysis, we compared transcript levels in HCC-free cirrhosis (alcoholism: 7; hepatitis C: 7), HCC-associated cirrhosis (alcoholism: 10; hepatitis C: 10) and eight control livers. The identified transcripts were validated by qRT-PCR in an independent cohort of 45 samples (20 HCC-free cirrhosis; 15 HCC-associated cirrhosis and 10 control livers). We also confirmed our results by immunohistochemistry. RESULTS: In HCC-free livers, we identified 70 transcripts which differentiated between alcoholic-related cirrhosis, HCV-related cirrhosis and control livers. They mainly corresponded to down-regulation. Dysregulation of Signal Transduction and Activator of Transcription-3 (STAT-3) was found along with related changes in STAT-3 targets which occurred in an etiology-dependent fashion in HCC-free cirrhosis. In contrast, in HCC, such transcription dysregulations were not observed. CONCLUSION: We report that transcriptional dysregulations exist in HCC-free cirrhosis, are transiently observed prior to detectable HCC onset and may be appear like markers from cirrhosis to HCC transition.
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Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Cirrosis Hepática/etiología , Cirrosis Hepática/genética , Neoplasias Hepáticas/genética , Transcripción Genética , Adulto , Anciano , Carcinoma Hepatocelular/patología , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Cirrosis Hepática/patología , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Plasminógeno/genética , Plasminógeno/metabolismoRESUMEN
INTRODUCTION: Rheumatoid arthritis (RA) is a heterogeneous disease and its underlying molecular mechanisms are still poorly understood. Because previous microarray studies have only focused on long-standing (LS) RA compared to osteoarthritis, we aimed to compare the molecular profiles of early and LS RA versus control synovia. METHODS: Synovial biopsies were obtained by arthroscopy from 15 patients (4 early untreated RA, 4 treated LS RA and 7 controls, who had traumatic or mechanical lesions). Extracted mRNAs were used for large-scale gene-expression profiling. The different gene-expression combinations identified by comparison of profiles of early, LS RA and healthy synovia were linked to the biological processes involved in each situation. RESULTS: Three combinations of 719, 116 and 52 transcripts discriminated, respectively, early from LS RA, and early or LS RA from healthy synovia. We identified several gene clusters and distinct molecular signatures specifically expressed during early or LS RA, thereby suggesting the involvement of different pathophysiological mechanisms during the course of RA. CONCLUSIONS: Early and LS RA have distinct molecular signatures with different biological processes participating at different times during the course of the disease. These results suggest that better knowledge of the main biological processes involved at a given RA stage might help to choose the most appropriate treatment.
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Artritis Reumatoide/genética , Artritis Reumatoide/patología , Perfilación de la Expresión Génica/métodos , Líquido Sinovial/química , Líquido Sinovial/fisiología , Adulto , Artritis Reumatoide/metabolismo , Femenino , Marcadores Genéticos/genética , Humanos , Masculino , Análisis por Micromatrices/métodos , Persona de Mediana Edad , Líquido Sinovial/metabolismo , Factores de Tiempo , Adulto JovenRESUMEN
As indicators of responsiveness to a tumour necrosis factor (TNF)alpha blocking agent (infliximab) are lacking in rheumatoid arthritis, we have used gene profiling in peripheral blood mononuclear cells to predict a good versus poor response to infliximab. Thirty three patients with very active disease (Disease Activity Score 28 >5.1) that resisted weekly methotrexate therapy were given infliximab at baseline, weeks 2 and 6, and every 8th week thereafter. The patients were categorized as responders if a change of Disease Activity Score 28 = 1.2 was obtained at 3 months. Mononuclear cell RNAs were collected at baseline and at three months from responders and non-responders. The baseline RNAs were hybridised to a microarray of 10,000 non-redundant human cDNAs. In 6 responders and 7 non-responders, 41 mRNAs identified by microarray analysis were expressed as a function of the response to treatment and an unsupervised hierarchical clustering perfectly separated these responders from non-responders. The informativeness of 20 of these 41 transcripts, as measured by qRT-PCR, was re-assessed in 20 other patients. The combined levels of these 20 transcripts properly classified 16 out of 20 patients in a leave-one-out procedure, with a sensitivity of 90% and a specificity of 70%, whereas a set of only 8 transcripts properly classified 18/20 patients. Trends for changes in various transcript levels at three months tightly correlated with treatment responsiveness and a down-regulation of specific transcript levels was observed in non-responders only. Our gene profiling obtained by a non-invasive procedure should now be used to predict the likely responders to an infliximab/methotrexate combination.
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Anticuerpos Monoclonales/uso terapéutico , Antirreumáticos/uso terapéutico , Artritis Reumatoide/sangre , Artritis Reumatoide/tratamiento farmacológico , Perfilación de la Expresión Génica , Monocitos/metabolismo , Adulto , Anciano , Femenino , Estudios de Seguimiento , Humanos , Infliximab , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Valor Predictivo de las Pruebas , ARN Mensajero/sangre , Factores de Tiempo , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
BACKGROUND/AIMS: Whether the transcriptional reprogramming process induced by hepatocellular carcinoma recapitulates that of the developing liver is at present unclear. METHODS: With a complete coverage of the liver transcriptome by microarray using adult livers as controls, we searched for similarities and differences in mRNA abundances between hepatocellular carcinoma nodules and fetal livers taken before (early) or after (late) the 22-24th week of gestation. Changes in some mRNA levels were studied in further liver samples by quantitative RT-PCR. RESULTS: Altered gene expression in hepatocellular carcinoma mostly results in down-regulated mRNAs which largely overlap with those repressed in the late fetal liver. Different frequencies of transcription factor binding sites in the down-regulated genes vs control genes as well as changes in abundance of mRNAs for relevant transcription factors point to a transcriptional repression. The down-regulated mRNAs code for proteins involved in (i) transcription and translation, (ii) specific functions of the differentiated hepatocyte or (iii) activation of proliferation and apoptosis. CONCLUSIONS: Apoptosis limitation is likely to predominate over active proliferation during liver development and hepatocellular carcinoma. Repression of the apoptosis-associated dudulin-2 mRNA points to a potential marker for the transition from a carcinoma-free to carcinoma-associated cirrhosis.
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Carcinoma Hepatocelular/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Hígado/fisiología , Proteínas Oncogénicas/genética , Anciano , Anciano de 80 o más Años , Sitios de Unión/genética , Biomarcadores de Tumor/genética , Proteínas de Ciclo Celular , Regulación hacia Abajo/genética , Femenino , Humanos , Hígado/embriología , Cirrosis Hepática/genética , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidorreductasas , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Given the unknown timing of the onset of an acute systemic inflammation in humans, the fine tuning of cascades and pathways involved in the associated hepatocyte response cannot be appraised in vivo. Therefore, the authors used a genome-wide and kinetic analysis in the human Hep3B hepatoma cell line challenged with a conditioned medium from bacterial lipopolysaccharide-stimulated macrophages. A complete coverage of the liver transcriptome disclosed 648 mRNAs whose change in abundance allowed for their clustering in mRNA subsets with an early, intermediate, or late regulation. The contribution of transcription, stability, or translation was appraised with genome-wide studies of the changes in nuclear primary transcripts, mRNA decay, or polysome-associated mRNAs. A predominance of mRNAs with decreased stability and the fact that translation alone controls a significant number of acute phase-associated proteins are prominent findings. Transcription and stability act independently or, more rarely, cooperate or even counteract in a gene-by-gene manner, which results in a unidirectional change in mRNA abundance. Waves of mRNAs for groups of functionally related proteins are up- or downregulated in an ordered fashion. This includes an early regulation of transcription-associated proteins, an intermediate repression of detoxication and metabolism proteins, and finally an enhanced translation and transport of a number of membranous or secreted proteins along with an enhanced protein degradation. In conclusion, this study provides a comprehensive and simultaneous overview of events in the human hepatocyte during the inflammatory acute phase.
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Citocinas/inmunología , Genómica , Hepatocitos/inmunología , Hepatocitos/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción de Fase Aguda/genética , Carcinoma Hepatocelular , Línea Celular Tumoral , Genoma Humano , Humanos , Cinética , Neoplasias Hepáticas , Polirribosomas/fisiología , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/inmunología , Estabilidad del ARN , ARN Mensajero/metabolismo , Transcripción Genética/genética , Transcripción Genética/inmunologíaRESUMEN
Activation of the complement system generates the anaphylatoxin C5a whose activities are mediated through its binding to the widely expressed C5aR. C5aR mRNA and protein expressions are known to be induced in rat hepatocytes under inflammatory conditions. However, little is known about the role of the C5a/C5aR complex in liver and its involvement during a proliferative process. We have evaluated the expression of C5aR in regenerating rat hepatocytes following a partial hepatectomy and in hepatocyte cultures. C5aR induction was observed in hepatocytes from regenerating liver, as well as in normal hepatocytes under a culture-induced stress. The effect of a stimulation by a C5a agonist upon the synthesis of a growth factor/receptor pair (hepatocyte growth factor/c-Met) was also evaluated. Our data demonstrated an up-regulated expression of hepatocyte growth factor and c-Met mRNAs, but we failed to observe a direct mitogenic effect of C5a in culture. However, a significantly increased expression of cyclin E and D1mRNA levels, as well as an increased BrdU incorporation, were observed in rats given an i.v. C5a agonist injection following an 80% partial hepatectomy. These studies demonstrate for the first time that: 1) C5aR is up-regulated during liver regeneration, 2) the binding of C5a to C5aR promotes a growth response, and 3) C5aR is involved in a cell cycle signaling pathway. Taken together, these findings point to a novel role for the hepatic C5aR implicating this complement system in the context of normal or abnormal proliferative pathways.
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Hepatocitos/metabolismo , Regeneración Hepática/fisiología , Receptor de Anafilatoxina C5a/metabolismo , Transducción de Señal/fisiología , Animales , División Celular/fisiología , Complemento C5a/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Hepatocitos/citología , Hepatocitos/inmunología , Inflamación/inmunología , Hígado/inmunología , Hígado/cirugía , Proteínas Proto-Oncogénicas c-met/metabolismo , ARN Mensajero/metabolismo , Ratas , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/inmunologíaRESUMEN
A change in the balance between proliferation and apoptosis in the course of hepatocellular carcinoma (HCC) development and progression has been suspected. We wanted to identify related genes whose mRNA levels could provide markers of severity and prognosis after resection. The extent of cell apoptosis, proliferation, and differentiation was measured with a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick-end labeling assay, and the Ki-67 index was determined in paired tumor and cirrhotic tissue samples from patients who had undergone HCC resection after diagnosis of hepatitis C-related or alcoholism-related cirrhosis. These patients included two groups with highly versus poorly differentiated tumor cells, and the latter was split into two subgroups of those with versus without early recurrence. The mRNA levels for various apoptosis-related or proliferation-related genes and those for the growth factor/receptor systems were measured by quantitative reverse transcriptase-polymerase chain reaction in paired tumor and cirrhotic liver samples from every patient, and some of the corresponding proteins were detected by immunohistochemistry. In all instances, protein expression was highly heterogeneous within groups and similar between groups. In contrast, some differences in mRNA level between tumor and cirrhotic tissues were quite informative. Low levels of hepatocyte growth factor and transforming growth factor alpha mRNAs were found concomitantly in highly differentiated tumors, whereas overexpression of mRNAs for the cognate receptors c-met and epidermal growth factor receptor were found in poorly differentiated tumors and primarily in patients with early tumor recurrence. These results argue for growth factor-dependent HCC development and provide novel and combined prognosis markers after HCC surgery.