RESUMO
We assessed, in real-life practice, viral, demographic, genetic and metabolic factors influencing the sustained virologic response (SVR), with a gender-oriented analysis, in patients with chronic hepatitis C virus (HCV) treated with pegylated interferon and ribavirin. Six hundred and seventy naïve patients were treated with dual therapy and evaluated by gender and HCV genotype. Associations between baseline variables and SVR were assessed by multivariate logistic regression analysis. Among 362 genotype 1 patients, SVR was achieved in 158 patients (44%), and SVR was independently associated with age less than 50 years (OR 2.12; 95% CI 1.09-4.30; P=0.039) and C/C genotype rs12979860 SNP (OR 2.83; 1.19-6.74; P=0.002) in 163 females, while absence of visceral obesity (OR 2.491; 1.131-5.487; P=0.023), HCV-RNA lower than 400,000 IU/mL (OR 2.66; 1.273-5.558; P=0.009) and C/C genotype rs12979860 SNP (OR 4.969; 2.401-10.283; P<0.001) were independently associated with SVR in 199 males. Combining favourable baseline variables, the probability of obtaining SVR ranged from 27.6% to 84.2% in females, and from 14.3% to 85.7% in males. The rate of SVR was 81.1% in 175 genotype 2 patients, and 69% in 100 genotype 3 patients. Rapid virologic response was the only valid predictor of SVR regardless of other features. In conclusions, in the setting of HCV genotype 1, chronic hepatitis, combining rapid virologic response and predictive factors, which are different for females and males, allows clinicians to single out a group of patients whose likelihood of SVR exceeds 80%. For these patients, triple therapy with first-generation protease inhibitors may be unwarranted.
Assuntos
Hepatite C Crônica/tratamento farmacológico , Interferons/uso terapêutico , Ribavirina/uso terapêutico , Adulto , Idoso , Estudos de Coortes , Quimioterapia Combinada/métodos , Feminino , Hepacivirus/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores Sexuais , Resultado do Tratamento , Carga ViralRESUMO
The human homologue of Drosophila Toll (hToll) is a recently cloned receptor of the interleukin 1 receptor (IL-1R) superfamily, and has been implicated in the activation of adaptive immunity. Signaling by hToll is shown to occur through sequential recruitment of the adapter molecule MyD88 and the IL-1R-associated kinase. Tumor necrosis factor receptor-activated factor 6 (TRAF6) and the nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK) are both involved in subsequent steps of NF-kappaB activation. Conversely, a dominant negative version of TRAF6 failed to block hToll-induced activation of stress-activated protein kinase/c-Jun NH2-terminal kinases, thus suggesting an early divergence of the two pathways.
Assuntos
Antígenos de Diferenciação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno , NF-kappa B/metabolismo , Proteínas/metabolismo , Receptores de Superfície Celular , Receptores Imunológicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Modelos Imunológicos , Dados de Sequência Molecular , Monócitos/imunologia , Fator 88 de Diferenciação Mieloide , Receptores do Fator de Necrose Tumoral/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fator 6 Associado a Receptor de TNF , Receptores Toll-LikeRESUMO
Mutations that abrogate recognition of a viral epitope by class I-restricted cytotoxic T lymphocyte (CTL) can lead to viral escape if the CTL response against that epitope is crucial for viral clearance. The likelihood of this type of event is low when the CTL response is simultaneously directed against multiple viral epitopes, as has been recently reported for patients with acute self-limited hepatitis B virus (HBV) infection. The CTL response to HBV is usually quite weak, however, during chronic HBV infection, and it is generally acknowledged that this is a major determinant of viral persistence in this disease. If such individuals were to produce a mono- or oligospecific CTL response, however, negative selection of the corresponding mutant viruses might occur. We have recently studied two HLA-A2-positive patients with chronic hepatitis B who, atypically, developed a strong HLA-A2-restricted CTL response against an epitope (FLPSDFFPSV) that contains an HLA-A2-binding motif located between residues 18-27 of the viral nucleocapsid protein, hepatitis B core antigen (HBcAg). These patients failed, however, to respond to any of other HLA-A2-restricted HBV-derived peptides that are generally immunogenic in acutely infected patients who successfully clear the virus. Interestingly, DNA sequence analysis of HBV isolates from these two patients demonstrated alternative residues at position 27 (V --> A and V --> I) and position 21 (S --> N, S --> A, and S --> V) that reduced the HLA and T cell receptor-binding capacities of the variant sequences, respectively. Synthetic peptides containing these alternative sequences were poorly immunogenic compared to the prototype HBc18-27 sequence, and they could not be recognized by CTL clones specific for the prototype peptide. While we do not know if the two patients were originally infected by these variant viruses or if the variants emerged subsequent to infection because of immune selection, the results are most consistent with the latter hypothesis. If this is correct, the data suggest that negative selection of mutant viral genomes might contribute to viral persistence in a subset of patients with chronic HBV infection who express a narrow repertoire of anti-HBV CTL responses.
Assuntos
Epitopos , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Linfócitos T Citotóxicos/imunologia , Doença Aguda , Sequência de Aminoácidos , Sequência de Bases , Doença Crônica , Antígeno HLA-A2/imunologia , Humanos , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
p73 has been recently identified as a new structural and functional homologue of the transcription factor p53. It is expressed in either a full-length form, alpha, or a shorter beta mRNA variant, with exon 13 spliced out. Here we report the identification and functional characterization of two new p73 splicing variants, gamma (splicing out exon 11) and delta (splicing out exons 11, 12, and 13). Both gamma and delta p73 variants are expressed in human peripheral blood lymphocytes, primary keratinocytes, and different tumor cell lines, including neuroblastoma, glioblastoma, melanoma, hepatoma, and leukemia. The expression pattern of the four p73 splicing variants differs in both primary cells of different lineage and established cell lines even within the same type of tumor. A two-hybrid assay was used to characterize the homodimeric and heterodimeric interactions between the p73 variants, and showed that neither p73gamma nor p73delta interact with p53, whereas p73gamma showed strong interactions with all p73 isoforms, and p73delta binds efficiently p73alpha and p73gamma but only weakly p73beta. At the functional level, p73gamma is significantly less efficient in activating transcription of the p21(Waf1/Cip1) promoter than p53 or p73beta, whereas the effect of p73delta is intermediate and comparable to that of p73alpha. The ability of the different p73 variants to affect cell growth in p53 null osteosarcoma SAOS-2 cells correlates with their transcriptional activity on the p21(Waf1/Cip1) promoter: p73beta is the most efficient in inhibiting colony formation, whereas p73gamma is almost ineffective. Our results suggest that p73 isoforms may be differentially regulated, with four different isoforms capable of interacting among themselves and with p53. The relative expression level of each splice variant may modulate p73 transcriptional and growth suppression activities by affecting heterodimer formation.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Apoptose , Sequência de Bases , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Primers do DNA/genética , Proteínas de Ligação a DNA/química , Dimerização , Genes Supressores de Tumor , Variação Genética , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/química , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Transfecção , Células Tumorais Cultivadas , Proteína Tumoral p73 , Proteínas Supressoras de TumorRESUMO
The current study sought to evaluate the characteristics of chronic hepatitis B virus (HBV) infection and current management practices in the European Union by surveying physician and patient records. A detailed survey of physician practices and management of patients with CHB was conducted between July and October 2006 in France, Germany, Italy and Spain. A total of 200 physicians participated in the survey, and data were collected from 2023 patients with chronic HBV infection. Most patients were men (69%), who had hepatitis B e antigen (HBeAg)-negative disease (64%), and demonstrated evidence of significant disease [53%; moderate fibrosis (35%), compensated cirrhosis (14%), or decompensated cirrhosis (4%)]. Among the 1665 HBV-monoinfected patients surveyed, 1184 (71%) were currently receiving treatment for chronic HBV infection. At treatment initiation, 70% of HBeAg-positive patients had both pretreatment serum HBV DNA levels Assuntos
Hepatite B Crônica/diagnóstico
, Hepatite B Crônica/terapia
, Adulto
, Alanina Transaminase/sangue
, Estudos Transversais
, DNA Viral/sangue
, União Europeia
, Feminino
, Antígenos E da Hepatite B/sangue
, Humanos
, Cirrose Hepática/patologia
, Masculino
, Pessoa de Meia-Idade
, Carga Viral
RESUMO
The balance between antiapoptotic and proapoptotic proteins of the Bcl-2 family is critical in determining the fate of T cells in response to death stimuli. Proapoptotic genes, such as bax, are generally regulated by the p53 family of transcription factors, whereas NF-kappaB subunits can activate the transcription of antiapoptotic Bcl-2 members. Here, we show that CD28 activation protects memory T cells from irradiation-induced apoptosis by both upregulating bcl-xL and inhibiting bax gene expression. We found that p73, but not p53, binds to and trans-activates the bax gene promoter in irradiated T cells. The activation of RelA/NF-kappaB subunit in CD28 costimulated T cells and its binding onto the bax gene promoter results in suppression of bax transcription and decrease in both p73 and RNA polymerase II recruitment in vivo. RelA recruitment on the bax gene promoter is also accompanied by the lost of p300 binding and the parallel appearance of histone deacetylase-1-containing complexes. These findings identify RelA/NF-kappaB as a critical regulator of T-cell survival by affecting the balance of Bcl-2 family members.
Assuntos
Apoptose , Linfócitos T CD4-Positivos/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Linfócitos T/fisiologia , Fator de Transcrição RelA/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína X Associada a bcl-2/genética , Apoptose/efeitos da radiação , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/efeitos da radiação , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Memória Imunológica , Células Jurkat , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Polimerase II/metabolismo , Linfócitos T/efeitos da radiação , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Interaction of the p55 tumor necrosis factor receptor 1 (TNF-R1)-associated signal transducer TRADD with FADD signals apoptosis, whereas the TNF receptor-associated factor 2 protein (TRAF2) is required for activation of the nuclear transcription factor nuclear factor kappa B. TNF-induced activation of the stress-activated protein kinase (SAPK) was shown to occur through a noncytotoxic TRAF2-dependent pathway. TRAF2 was both sufficient and necessary for activation of SAPK by TNF-R1; conversely, expression of a dominant-negative FADD mutant, which blocks apoptosis, did not interfere with SAPK activation. Therefore, SAPK activation occurs through a pathway that is not required for TNF-R1-induced apoptosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , MAP Quinase Quinase Quinase 1 , Proteínas Quinases Ativadas por Mitógeno , Proteínas/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Acetilcisteína/farmacologia , Apoptose , Proteínas de Transporte/metabolismo , Linhagem Celular , Dactinomicina/farmacologia , Ativação Enzimática , Proteína de Domínio de Morte Associada a Fas , Sequestradores de Radicais Livres/farmacologia , Células HeLa , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Fator 1 Associado a Receptor de TNF , Fator 2 Associado a Receptor de TNF , TransfecçãoRESUMO
OBJECTIVE: We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability. METHODS: This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry. RESULTS: F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity). CONCLUSIONS: F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.
Assuntos
Apoptose , Carcinoma Hepatocelular/virologia , Vírus da Hepatite B/genética , Neoplasias Hepáticas/virologia , Transativadores/genética , Replicação Viral , Adulto , Idoso , DNA Viral/genética , Feminino , Genótipo , Células Hep G2 , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/virologia , Humanos , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Homologia Estrutural de Proteína , Transativadores/química , Proteínas Virais Reguladoras e AcessóriasRESUMO
Satellite cells are the main source of myogenic progenitors in postnatal skeletal muscle, but their use in cell therapy for muscle disorders is limited because these cells cannot be delivered through circulation and they are rapidly exhausted in severe myopathies. The search for alternative donor cells is ongoing, but none of the candidates so far show all the features required for successful colonization and repair of diseased muscle. In this study, we show that bisperoxovanadium, a phospho-tyrosine phosphatase inhibitor, induces myogenic cells to acquire a gene expression profile and a differentiation potential consistent with the phenotype of a circulating precursors, while maintaining their myogenic potential. These effects are mediated, at least in part, by NF-kappaB activation through the Tyr42-IkappaB-alpha phosphorylation, as shown by the expression of the dominant negative mutant form of the p50 NF-kappaB subunit. Moreover, when bisperoxovanadium-treated cells are injected into the femoral artery of alpha-sarcoglican null dystrophic mice, they are able to circulate and to reach muscle tissue; importantly, they contribute to muscle regeneration, as shown by the expression of alpha-sarcoglican in some fibers. Our observations indicate that bisperoxovanadium, or similar compounds, may prove very valuable to obtain and to expand, from committed cells, multipotent cell populations suitable for gene-cell therapy applications and may help to understand the molecular basis of genome reprogramming and "stem-ness."
Assuntos
Inibidores Enzimáticos/farmacologia , Coração/efeitos dos fármacos , Miocárdio/citologia , Células-Tronco Pluripotentes/citologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Compostos de Vanádio/farmacologia , Animais , Sequência de Bases , Ciclo Celular , Linhagem Celular , Primers do DNA , Citometria de Fluxo , Imunofluorescência , Expressão Gênica , Camundongos , Miocárdio/metabolismo , Fenótipo , Células-Tronco Pluripotentes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Chronic infection with the hepatitis C virus (HCV) is a major risk factor for the development of hepatocellular carcinoma (HCC) worldwide. The pathogenesis of HCC in HCV infection has extensively been analysed. Hepatitis C virus-induced chronic inflammation and the effects of cytokines in the development of fibrosis and liver cell proliferation are considered as one of the major pathogenic mechanisms. Increasing experimental evidence suggests that HCV contributes to HCC by directly modulating pathways that promote the malignant transformation of hepatocytes. Hepatitis C virus is an RNA virus that does not integrate into the host genome but HCV proteins interact with many host-cell factors well beyond their roles in the viral life cycle and are involved in a wide range of activities, including cell signaling, transcription, cell proliferation, apoptosis, membrane rearrangements, vesicular trafficking and translational regulation. At least four of the HCV gene products, namely HCV core, NS3, NS4B and NS5A, have been shown to exhibit transformation potential in tissue culture and several potentially oncogenic pathways have been shown to be altered by the expression of HCV proteins. Both HCV core and NS5A induce the accumulation of wild-type beta-catenin and the Wnt-beta-catenin pathway emerges as a common target for HCV (and HBV) in human HCCs, also independently from axin/beta-catenin gene mutations. Induction of both endoplasmic reticulum stress and oxidative stress by HCV proteins might also contribute to HCV transformation. Most of the putative transforming functions of the HCV proteins have been defined in artificial cellular systems, which may not be applicable to HCV infection in vivo, and still need to be established in relevant infection and disease models.
Assuntos
Carcinoma Hepatocelular/virologia , Hepacivirus/patogenicidade , Hepatite C Crônica/virologia , Neoplasias Hepáticas/virologia , Animais , Carcinoma Hepatocelular/patologia , Modelos Animais de Doenças , Hepacivirus/genética , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/patologia , Humanos , Neoplasias Hepáticas/patologiaRESUMO
The P73 gene is transcribed from two promoters, P1 and P2, that direct the expression of multiple transactivation competent (TA) and dominant negative (DN) isoforms. TAp73 transcription factors mediate cell cycle arrest and/or apoptosis in response to DNA damage and are involved in developmental processes. P73 mRNA levels increase and the P1p73 promoter is upregulated during myogenic differentiation of C2C12 skeletal muscle satellite cells. The DNp73 proteins act as trans-repressors of p53- and p73-dependent transcription, and possess both antiapoptotic and pro-proliferative potential. Here, we show that DNp73alpha is expressed in proliferating C2C12 myoblasts, rapidly accumulates in differentiating myocytes and remains elevated in C2C12 myotubes. By combining transactivation assays and chromatin immunoprecipitation analysis, we could show that the upregulation of the P2p73 promoter during myogenic differentiation is mediated by the coordinated recruitment and activity of MyoD and p53/p73. Abrogation of DNp73 expression by specific siRNA led to a strong potentiation of the spontaneous apoptosis of C2C12 myoblasts induced to differentiate. Finally, unlike TAp73 that contributes to DNA damage-induced apoptosis of myotubes, endogenous DNp73 mediates the relative resistance of differentiated myotubes to DNA damage. Altogether, our findings identify DNp73alpha as an important target in designing strategies aimed at the potentiation of the regenerative potential of skeletal satellite cells.
Assuntos
Apoptose/fisiologia , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica , Células Musculares/citologia , Proteínas Nucleares/metabolismo , Animais , Immunoblotting , Camundongos , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno , Ativação Transcricional , Transfecção , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
The molecular and cellular basis of long-term T cell memory against viral antigens is still largely undefined. To characterize anti-viral protection by memory T cells against non-cytopathic viruses able to cause acute self-limited and chronic infections, such as the hepatitis B virus (HBV), we studied HLA class II restricted responses against HBV structural antigens in 17 patients with acute hepatitis B, during the acute stage of infection and 2.2 to 13 yr after clinical resolution of disease. Results indicate that: (a) significant T cell proliferative responses to HBV nucleocapsid antigens were detectable in all patients during the acute phase of infection and in 14/17 also 2-13 yr after clinical resolution of disease; b) long-lasting T cell responses were sustained by CD45RO+T cells, predominantly expressing the phenotype of recently activated cells; c) limiting dilution analysis showed that in some patients the frequency of HBV-specific T cells was comparable to that observed in the acute stage of infection and, usually, higher than in patients with chronic HBV infection; d) the same amino acid sequences were recognized by T cells in the acute and recovery phases of infection; and e) HBV-DNA was detectable by nested-PCR in approximately half of the subjects. to conclusion, our results show that vigorous anti-viral T cell responses are detectable in vitro several years after clinical recovery from acute hepatitis B. Detection of minute amounts of virus in some recovered subjects suggests that long-term maintenance of an active anti-viral T cell response could be important not only for protection against reinfection but also for keeping the persisting virus under tight control.
Assuntos
Antígenos da Hepatite B/imunologia , Hepatite B/imunologia , Memória Imunológica , Subpopulações de Linfócitos T/imunologia , Viremia/imunologia , Doença Aguda , Adulto , Citocinas/metabolismo , Epitopos , Feminino , Antígenos HLA , Antígenos de Superfície da Hepatite B/imunologia , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Nucleocapsídeo/imunologia , Fatores de TempoRESUMO
The hepatitis B virus (HBV) X protein (pX) is capable of activating transcription regulated by viral and cellular promoters containing binding sites for different transcription factors, including AP1. In this study we have analyzed the mechanisms of AP1 induction by pX. The hepatitis B virus transactivator was able to activate TRE (12-O-tetradecanoylphorbol-13-acetate response element)-directed transcription in different cell lines, including HepG2, HeLa, CV1, and PLC/PRF/5 cells. pX-induced AP1 activation in HepG2 cells was associated with an increase in the DNA-binding activity of c-Jun/c-Fos heterodimers, which was not dependent either on an increase in the overall amount of c-Fos and c-Jun proteins in the cells or on formation of dimers between pX and the two proteins, thus suggesting the involvement of posttranslational modifications of the transcription factor. The observation that the overexpression of c-Jun and c-Fos in the cells results in a strong augmentation of the effect of pX on TRE-directed transcription is additional evidence indicating the involvement of posttranscriptional modifications of c-Jun/c-Fos heterodimers. The increased AP1 binding observed in the presence of pX was unaffected by the protein kinase C inhibitors calphostin C and sphingosine and by the protein kinase A inhibitor HA1004, while it was almost completely blocked by staurosporine, a potent and nonspecific protein kinase inhibitor, suggesting that protein kinase C- and A-independent phosphorylation events might play a role in the phenomenon. The ability of pX also to increase TRE-directed transcription in cell lines in which AP1-binding activity is not increased (i.e., HeLa, CV1, and PLC/PRF/5 cells) suggests that pX can activate canonical TRE sites by different mechanisms as well.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Vírus da Hepatite B/metabolismo , Naftalenos , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Sulfonamidas , Transativadores/metabolismo , Ativação Transcricional , Alcaloides/farmacologia , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Células HeLa , Humanos , Isoquinolinas/farmacologia , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/metabolismo , Fosforilação , Compostos Policíclicos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-jun/biossíntese , Esfingosina/farmacologia , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Transfecção , Células Tumorais Cultivadas , Vaccinia virus/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
Hepatitis C virus (HCV) infection is a leading cause of hepatocellular carcinoma (HCC), mainly through cirrhosis induction, spurring research for a deeper understanding of HCV versus host interactions in cirrhosis. The present study investigated crosstalks between HCV infection and UNC5A, a netrin-1 dependence receptor that is inactivated in cancer. UNC5A and HCV parameters were monitored in patients samples (n=550) as well as in in vitro. In patients, UNC5A mRNA expression is significantly decreased in clinical HCV(+) specimens irrespective of the viral genotype, but not in (HBV)(+) liver biopsies, as compared to uninfected samples. UNC5A mRNA is downregulated in F2 (3-fold; P=0.009), in F3 (10-fold, P=0.0004) and more dramatically so in F4/cirrhosis (44-fold; P<0.0001) histological stages of HCV(+) hepatic lesions compared to histologically matched HCV(-) tissues. UNC5A transcript was found strongly downregulated in HCC samples (33-fold; P<0.0001) as compared with non-HCC samples. In vivo, association of UNC5A transcripts with polyribosomes is decreased by 50% in HCV(+) livers. Consistent results were obtained in vitro showing HCV-dependent depletion of UNC5A in HCV-infected hepatocyte-like cells and in primary human hepatocytes. Using luciferase reporter constructs, HCV cumulatively decreased UNC5A transcription from the UNC5 promoter and translation in a UNC5A 5'UTR-dependent manner. Proximity ligation assays, kinase assays, as well as knockdown and forced expression experiments identified UNC5A as capable of impeding autophagy and promoting HCV restriction through specific impact on virion infectivity, in a cell death-independent and DAPK-related manner. In conclusion, while the UNC5A dependence receptor counteracts HCV persistence through regulation of autophagy in a DAPK-dependent manner, it is dramatically decreased in all instances in HCC samples, and specifically by HCV in cirrhosis. Such data argue for the evaluation of the implication of UNC5A in liver carcinogenesis.
Assuntos
Hepatite C/metabolismo , Receptores de Superfície Celular/metabolismo , Autofagia , Linhagem Celular Tumoral , Expressão Gênica , Hepacivirus/fisiologia , Hepatócitos/fisiologia , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Receptores de Netrina , Lesões Pré-Cancerosas/metabolismo , Receptores de Superfície Celular/genética , Replicação ViralRESUMO
The ability of the hepatitis B virus (HBV)-encoded X protein (HBx) to coactivate transcription of viral and cellular genes has been implicated in the development of HBV-related liver cancer. To dissect the transformation and the transcription activation properties of HBx, we generated REV2 cell lines expressing the wild-type and different truncated versions of the protein. Full-length HBx-expressing REV-2 cells display an altered morphology and form large colonies in soft agar. A similar transformation efficiency has been obtained with a truncated version of HBx, which contains only the first 50 NH2-terminal amino acids (HBx 1-50). In contrast, HBx mutants that lack the NH2-terminal segment but retain most of the transactivating function, as compared to the full length HBx, were unable to alter the growth characteristic of REV-2 cells. Furthermore, abrogation of full-length HBx transcriptional activation by the insertion of two amino acids (Arg-Pro) at position 68 did not affect REV-2 cells transformation. These results demonstrate that the transactivation activity of HBx is neither essential nor sufficient for tumor promotion.
Assuntos
Transformação Celular Viral , Vírus da Hepatite B , Transativadores/fisiologia , Ativação Transcricional/genética , Proteínas Virais/fisiologia , Antígenos Virais de Tumores/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
E2F/DP heterodimers play a pivotal role in the regulation of cell growth and differentiation. A decrease in E2F/DP activity occurs during cell cycle arrest and differentiation. However, very little is known about the specific role of the various E2F/DP members along the transition from proliferation to terminal differentiation. We have previously shown that E2F4 accounts for the vast majority of the endogenous E2F in differentiating muscle cells. Here, we show that E2F4, which lacks a nuclear localization signal (nls), is distributed in both the nucleus and the cytoplasm, in either asynchronously growing myoblasts or differentiated myotubes. E2F4 nuclear accumulation is induced by the binding in the cytoplasm with specific partners p107, pRb2/p130, and DP3delta, an nls-containing spliced form of DP3, which provide the nls. Although overexpression of E2F4/DP3delta reactivates the cell cycle in quiescent cells, the E2F4 nuclear accumulation induced by pRb2/p130 and p107 correlates with cell growth arrest Moreover, E2F4/DP3delta-induced cell cycle reactivation is efficiently counteracted by either p107 or pRb2/p130 overexpression. Reinduction in quiescent cells of DNA synthesis by E2F1/DP1 overexpression is abrogated by coexpression of pRb and is hampered by MyoD overexpression. Both pRb2/p130 and pRb, as well as MyoD, are up-regulated in myotubes. Accordingly, multinucleated myotubes, which are induced to reenter the S-phase by oncoviral proteins, are refractory to cell cycle reactivation by forced expression of E2F4/DP3delta or E2F1/DP1. Thus, E2F/DP repression represents only one of multiple redundant circuits that control the postmitotic state in terminally differentiated cells and that are targeted by adenovirus E1A and SV40 large T antigen.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila , Substâncias de Crescimento/fisiologia , Músculos/citologia , Músculos/metabolismo , Fatores de Transcrição/fisiologia , Translocação Genética/fisiologia , Animais , Ciclo Celular/fisiologia , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , DNA/biossíntese , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Dimerização , Fator de Transcrição E2F4 , Vetores Genéticos , Substâncias de Crescimento/biossíntese , Substâncias de Crescimento/metabolismo , Camundongos , Microinjeções , Músculos/fisiologia , Frações Subcelulares/metabolismo , Transativadores/biossíntese , Transativadores/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Chronic hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and hepatocellular carcinoma (HCC) worldwide. The HCV capside core is a multifunctional protein with regulatory functions that affects transcription and cell growth in vitro and in vivo. Here, we show that both HCV genotype 1a and 3 core proteins activate MEK1 and Erk1/2 MAP kinases and that the costitutive expression of the HCV core results in a high basal activity of Raf1 and MAP/kinase/kinase, as determined by endogenous Raf1 in vitro kinase assay and immunodetection of hyperphosphorylated Erk1 and Erk2 even after a serum starvation. Moreover, the activation of both Erk1/2 and the downstream transcription factor Elk-1 in response to the mitogenic stimulus EGF is significantly prolonged. The sustained response to EGF in cells expressing the HCV core occurs despite a normal induction of the MAP phosphatases MKP regulatory feedback and is likely due to the costitutive activation of Raf-1 activity. The ability of HCV core proteins to directly activate the MAP kinase cascade and to prolong its activity in response to mitogenic stimuli may contribute to the neoplastic transformation of HCV infected liver cells.
Assuntos
MAP Quinase Quinase Quinase 1 , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas do Core Viral/fisiologia , Ativação Enzimática , Genótipo , Hepacivirus/genética , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-raf/fisiologia , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/genéticaRESUMO
The mechanisms by which pX, the transactivator of the Hepatitis B Virus (HBV), exerts its effects on transcription of viral and cellular genes have not yet been fully clarified. While previous reports suggested the possibility of a direct interaction of pX, which lacks intrinsic DNA-binding activity, with components of the cellular transcription machinery, more recent investigations support the hypothesis that pX might activate cellular kinases involved in transcriptional regulation and growth control. We analysed the mechanisms of c-Jun transcription factor activation by pX and in particular the role of cellular proteins involved in the transduction of mitogenic signals (namely Ha-Ras and Raf-1). In both HeLa and undifferentiated F9 cells pX was able to increase the activity of exogenous transfected c-Jun but not of c-Jun proteins bearing mutations in the serine residues located in the amino-terminal transcriptional activation domain. We show by use of Ha-Ras and Raf-1 dominant negative mutants that both Ha-Ras and Raf-1 are required for pX-induced activation of c-Jun transcriptional activity. In addition we show that pX is able to cooperate with Raf-1 in c-Jun activation. Our results are consistent with the hypothesis that at least one site of action of pX is peripheral and is located upstream of the Ras genes products.
Assuntos
Vírus da Hepatite B/genética , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Oncogênicas de Retroviridae/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Genes ras , Células HeLa , Humanos , Proteínas Oncogênicas v-raf , Transdução de Sinais , Proteínas Virais Reguladoras e AcessóriasRESUMO
CTLs- and lymphokine-induced apoptosis of infected hepatocytes during the course of chronic viral hepatitis is thought to be important for both disease termination and prevention of hepatocellular transformation. We therefore studied apoptosis induced by Fas (APO-1 or CD95)-a widely expressed cell surface receptor whose ligand is involved in lymphocyte cytotoxicity-in a set of human hepatoma cell lines. As normal hepatocytes, all of the human hepatoma cell lines tested do express detectable amounts of Fas on their surface. Nevertheless, only PLC/PRF/5 cells undergo apoptosis following treatment with anti-Fas. Systematic cloning and sequence analysis of the Fas cDNA did not show mutations in the Fas gene in any of the cells lines tested. However, due to alternative splicing, 5 to 10% of the Fas cDNAs are deleted of 63 internal nucleotides corresponding to the transmembrane domain, thus encoding for a soluble and secreted form of Fas (Fas delta TM), potentially able to neutralize anti-Fas or Fas-Ligand. Although we could not demonstrate a direct correlation between resistance of different hepatoma cell lines to Fas mediated death and endogenous expression of this transcript, we show that PLC/PRF 5 stable transfectants overexpressing Fas delta TM are less sensitive to anti-Fas than control cells. In three different cell lines, resistance to anti-Fas was overcome by treatment with the protein synthesis inhibitor cycloheximide. Although this could suggest the existence of short-lived repressors of the Fas-activated apoptotic signalling pathway(s), we show that translational inhibition is not required for the synergistic effect of cycloheximide to take place, and that resistant hepatoma cells can be sensitized to anti-Fas by subinhibitory concentrations of this protein synthesis inhibitor. Since cycloheximide is able to activate intracellular signalling independently on its effects on protein synthesis, we suggest that it might provide a costimulatory signal that cooperates with Fas in the induction of cell death and that, at least in the cells we tested, resistance to Fas is not an active process involving gene transcription and translation but only the consequence of an inadequate apoptotic stimulation.
Assuntos
Apoptose , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Receptor fas/fisiologia , Adulto , Sequência de Aminoácidos , Sequência de Bases , Cicloeximida/farmacologia , DNA Complementar/química , Humanos , Masculino , Dados de Sequência Molecular , Proteínas Repressoras/fisiologia , Células Tumorais CultivadasRESUMO
Withdrawal from the cell cycle of differentiating myocytes is regulated by the myogenic basic helix-loop-helix (bHLH) protein MyoD and the pocket proteins pRb, p107 and pRb2/p130. Downstream effectors of 'pocket' proteins are the components of the E2F family of transcription factors, which regulate the G1/S-phase transition. We analysed by EMSA the composition of E2F complexes in cycling, quiescent undifferentiated and differentiated C2C12 skeletal muscle cells. An E2F complex containing mainly E2F4 and pRb2/p130 (E2F-G0/G1 complex) appears when DNA synthesis arrests, replacing the cyclinA/cdk2 containing E2F complex of proliferating myoblasts (E2F-G1/S complex). Serum stimulation reinduces DNA synthesis and the re-appearance of E2F-G1/S complexes in quiescent myoblasts but not in differentiated C2C12 myotubes. In differentiating C2C12 cells, E2F complexes switch and DNA synthesis in response to serum are prevented when MyoD DNA binding activity and the cdks inhibitor MyoD downstream effector p21 are induced. Thus, during myogenic differentiation, formation of E2F4 and pRb2/p130 containing complexes is an early event, but not enough on its own to prevent the reactivation of DNA synthesis. Using a subclone of C3H10T1/2 mouse fibroblasts stably expressing Estrogen Receptor-MyoD (ER-MyoD) chimerae, we found that estrogen directed MyoD activation prevents the reassociation of cyclinA/cdk2 to the E2F4 containing complex following serum stimulation and this correlates with suppression of E2F activity and the inability of cells to re-enter the cell cycle. Our data indicate that, in differentiating myocytes, one mechanism through which MyoD induces permanent cell cycle arrest involves p21 upregulation and suppression of the proliferation-associated cdks-containing E2F complexes formation.