RESUMO
BACKGROUND AND AIM: Blood pressure is an independent predictor of target organ damage (TOD). Recent data from literature suggest that TOD can be present also in pre-hypertensive subjects, diagnosed with pressure monitoring (PM). Aim of this study is to clarify whether an augmentation of the carotid Intima-Media Thickness (cIMT) in office prehypertensives is a TOD associated to monitoring prehypertension (MP). PATIENTS AND METHODS: We have analyzed our database of individuals office normotensives showing an increase of cIMT. The ambulatory blood pressure monitoring (ABPM) of these was compared with those of office monitoring normotensives, matched by age and gender, antropometric characteristics, negative for familial hypertension and other risk factors (true normotensives, TN). RESULTS: We have selected 15 presumable prehypetensives (PP) and 8 TN subjects. The ABPM (ambulatory blood pressure monitoring) analysis confirmed that neither the PP nor TN showed systolic (S) and diastolic (D) BP within-day values above their day-night upper reference limits. However the statistical comparison between PP and TN revealed that the first group had a significant elevation of SBP and DBP Daily Mean Level (DML(SBP/DBP): 121 ± 2/81 ± 2 vs 112 ± 2/70 ± 2 mmHg, respectively, p = 0.007 and p = 0.002), confirming the MP diagnosis. CONCLUSIONS: These results demonstrate that cIMT increase in PP fulfill the criteria for MP diagnosis, suggesting that MP should be undertaken in all PP with altered cIMT, but larger prospective studies are needed.
Assuntos
Pressão Arterial , Determinação da Pressão Arterial , Doenças das Artérias Carótidas/diagnóstico por imagem , Artéria Carótida Primitiva/diagnóstico por imagem , Espessura Intima-Media Carotídea , Visita a Consultório Médico , Pré-Hipertensão/diagnóstico , Adulto , Determinação da Pressão Arterial/métodos , Monitorização Ambulatorial da Pressão Arterial , Distribuição de Qui-Quadrado , Feminino , Humanos , Masculino , Valor Preditivo dos Testes , Pré-Hipertensão/fisiopatologia , Estudos RetrospectivosRESUMO
Orocecal transit time (OCTT) is one of the main determinant of the hunger ratings and gastrointestinal sensitivity. While marked-isotopes scintigraphy is the gold standard in its determination in the clinical frame, breath tests are cheap, well-tolerated and non-invasive alternatives. In fact C-13 and C-14 stable isotopes breath tests can be used to assess gastric emptying and OCTT in the clinical and research frames. Moreover, hydrogen (H2) lactulose breath test can be used to assess OCTT in the research frame only due to its laxative action; inulin breath test, devoid of this bias, could be replacing it. However, the main limitation in the use of breath tests in the OCTT determination is their low reproducibility.
Assuntos
Testes Respiratórios , Gastroenteropatias/diagnóstico , Trânsito Gastrointestinal , Animais , Biomarcadores/metabolismo , Gases , Esvaziamento Gástrico , Gastroenteropatias/metabolismo , Gastroenteropatias/fisiopatologia , Humanos , Inulina , Lactulose , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
The management of HBV or HCV has improved dramatically over the last decade with the development of new drugs. This paper provides a review of new available and developing treatment options for HBV and HCV associated liver diseases. In the closer future the most realistic therapeutical option for most of the patients with HBV and HCV infection will be combination and/or long-term usage of the new, stronger antiviral drugs, if they maintain good safety profiles, achieve low resistance rates and will be available at lower prices.
Assuntos
Antivirais/uso terapêutico , Hepatite B/tratamento farmacológico , Hepatite C/tratamento farmacológico , Vacinas contra Hepatite Viral/uso terapêutico , Antivirais/química , Antivirais/economia , Farmacorresistência Viral , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Conformação Molecular , Estereoisomerismo , Vacinas contra Hepatite Viral/química , Vacinas contra Hepatite Viral/economiaRESUMO
Myogenic differentiation is a highly orchestrated multistep process controlled by extracellular growth factors that modulate largely unknown signals into the cell affecting the muscle-transcription program. P38MAPK-dependent signalling, as well as PI3K/Akt pathway, has a key role in the control of muscle gene expression at different stages during the myogenic process. P38MAPK affects the activities of transcription factors, such as MyoD and myogenin, and contributes, together with PI3K/Akt pathway, to control the early and late steps of myogenic differentiation. The aim of our work was to better define the role of PKR, a dsRNA-activated protein kinase, as potential component in the differentiation program of C2C12 murine myogenic cells and to correlate its activity with p38MAPK and PI3K/Akt myogenic regulatory pathways. Here, we demonstrate that PKR is an essential component of the muscle development machinery and forms a functional complex with p38MAPK and/or Akt, contributing to muscle differentiation of committed myogenic cells in vitro. Inhibition of endogenous PKR activity by a specific (si)RNA and a PKR dominant-negative interferes with the myogenic program of C2C12 cells, causing a delay in activation of myogenic specific genes and inducing the formation of thinner myofibers. In addition, the construction of three PKR mutants allowed us to demonstrate that both N and C-terminal regions of PKR are critical for the interaction with p38MAPK and Akt. The novel discovered complex permits PKR to timely regulate the inhibition/activation of p38MAPK and Akt, controlling in this way the different steps characterizing skeletal muscle differentiation.
Assuntos
Diferenciação Celular , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , eIF-2 Quinase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Ativação Enzimática , Camundongos , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/enzimologia , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Transfecção , eIF-2 Quinase/genéticaRESUMO
HCV chronic infection leads to liver diseases and also to a wide range of extrahepatic disorders including benign, but pre-lymphomatous forms (mixed cryoglobulinemia) to frank hematological neoplasia (non-Hodgkin's lymphoma). Recent data showed the involvement of p53 superfamily members in the pathogenesis of different lymphatic malignancies. In fact, tymomas and a subset of non-Hodgkin's lymphomas (NHLs) express high levels of p63. Thus, we analyzed whether alterations in p53 superfamily gene expression are observable in B lymphocytes isolated from HCV-infected patients with and without lymphoproliferative disorders. We showed, by real-time PCR, a significant induction of DNp63 mRNAs in B lymphocytes obtained from HCV-positive low grade non-Hodgkin's lymphoma patients. Since our current understanding of HCV proteins emphasizes the ability of the HCV core protein to deregulate the expression and activity of p53-related proteins, we established different B lymphocyte cell lines (Wil2-ns, Daudi and Ramos) stably expressing HCV core protein, in order to investigate the possible involvement of the viral protein in the upregulation of DNp63 in B lymphocytes. The analysis of p63 family transcripts showed no transcriptional changes for the p63 TA isoforms, whereas an increase (>5 times) of DNp63 mRNA occurred. In all cell lines, this abnormal expression was associated with a significant increase of cell proliferation that was specifically inhibited by silencing DNp63 mRNA. These findings suggest a pathogenetic role of the HCV core in HCV-related lymphomagenesis, through the induction of DNp63's pro-proliferative effects.
Assuntos
Linfócitos B/fisiologia , Hepacivirus/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Core Viral/fisiologia , Apoptose/fisiologia , Divisão Celular/fisiologia , Células Cultivadas , Humanos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
Hepatocellular carcinoma (HCC) is a common cancer worldwide. A majority of HCC patients present with an unresectable or metastatic HCC and systemic therapy with cytotoxic agents provides marginal benefits. Some evidence suggests that certain natural phytochemical compounds used in conjunction with chemotherapeutic agents could enhance therapeutic efficacy by sensitizing cells to treatment.
Assuntos
Anticarcinógenos/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Resistência a Medicamentos , Sinergismo Farmacológico , Humanos , Resultado do TratamentoRESUMO
Hepatitis C virus (HCV) is the major causative viral agent of cirrhosis and hepatocarcinoma (HCC). HCV core protein affects cell homeostasis, playing an important role in viral pathogenesis of HCC. We investigate the effects of HCV core protein expression on cell growth in HCC cell lines. Cell cycle distribution analysis of HepG2 polyclonal core positive cells reveals a peculiar accumulation of cells in G2/M phase. Different pathways mediate G2/M arrest: such as p53 and double strand RNA protein kinase (PKR). Flow cytometry in p53-null cells demonstrates that p53 plays only a marginal role in inducing HCV core-dependent G2/M phase accumulation that seems to be significantly affected by the functional inactivation of PKR. HCC core positive cells are characterized by a significant PKR phosphorylation in Thr 446 residue, which leads deregulation of mitosis. Moreover, we observe that the overexpression of the viral protein induces an upregulation of PKR activity, which does not correlate with an increased eIF-2 phosphorylation. This uncommon behavior of PKR suggests that its activation by HCV core protein could involve alternative PKR-dependent pathways, implicated in core-dependent G2/M accumulation. The described biological effects of HCV core protein on cell cycle could be an additional viral mechanism for both HCV resistance to interferon (IFN) and HCC HCV-related pathogenesis.
Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Divisão Celular , Fase G2 , Fosfotreonina/metabolismo , Proteínas do Core Viral/metabolismo , eIF-2 Quinase/metabolismo , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular , Hepacivirus , Humanos , Camundongos , Camundongos Knockout , Fosforilação , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas do Core Viral/genética , eIF-2 Quinase/química , eIF-2 Quinase/genéticaRESUMO
BACKGROUND: Infection with transfusion transmitted virus, a new member of the Parvoviridae family, has been found in patients both with chronic and fulminant post-transfusion cryptogenic hepatitis. AIM: To evaluate the prevalence and clinical impact of transfusion transmitted virus infection in Italy. PATIENTS AND METHODS: Studies were carried out on 256 patients and control subjects from three centres from Northern, Central and Southern Italy (92 nonA-nonC chronic hepatitis, 10 acute non fulminant cryptogenic hepatitis, 41 hepatitis C virus-related chronic hepatitis and 113 blood donors). Serum transfusion transmitted virus was detected by nested polymerase chain reaction using two overlapping sets of primers. RESULTS: A total of 52 of the 92 patients (54.3%) with chronic cryptogenic liver disease and 17 of the 41 hepatitis C virus chronic hepatitis patients (41.4%) were transfusion transmitted virus-DNA positive. Transfusion transmitted virus co-infection in hepatitis C virus patients was not associated with either a higher severity of liver histology or higher alanine transaminase levels or signs of cholestasis, transfusion transmitted virus was found in 48 out of 113 (42.4%) blood donors. In the majority of samples, transfusion transmitted virus DNA was detected with only one of the two sets of primers used. Genotyping and phylogenetic analysis performed on 21 randomly selected viral isolates showed the presence of both type 1 and type 2 transfusion transmitted virus and allowed identification of two isolates with high homology to genotype 6, described, so far, mostly in Japan. CONCLUSIONS: Transfusion transmitted virus type 1 and 2 infection is common among blood donors and patients with liver disease in Italy. The pathogenic potential of transfusion transmitted virus type 1 and 2 in nonA-nonC hepatitis patients is unlikely but further studies are needed to evaluate the epidemiological and clinical impact of other transfusion transmitted virus subtypes.
Assuntos
Doadores de Sangue , Infecções por Vírus de DNA/epidemiologia , Hepatite Crônica/virologia , Torque teno virus/genética , Adolescente , Adulto , Idoso , DNA Viral/análise , Feminino , Hepatite C Crônica/virologia , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência , Torque teno virus/isolamento & purificaçãoRESUMO
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
Many studies have shown that hepatitis B virus infection may also occur in hepatitis B surface antigen-negative patients. This occult infection has been identified both in patients with cryptogenic liver disease and in patients with hepatitis C virus-related chronic hepatitis, and much evidence suggests that it may be a risk factor of hepatocellular carcinoma development. However several aspects of this occult infection remain unclear such as its prevalence and the factor(s) involved in the lack of circulating hepatitis B surface antigen. Moreover, it is uncertain whether the occult hepatitis B virus infection may contribute to chronic liver damage, considering that it is usually associated with a suppressed viral replication. Evidence and hypotheses concerning this fascinating field of bio-medical research are reviewed.
Assuntos
Carcinoma Hepatocelular/epidemiologia , Antígenos de Superfície da Hepatite B/análise , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/epidemiologia , Neoplasias Hepáticas/epidemiologia , Carcinoma Hepatocelular/diagnóstico , Comorbidade , DNA Viral/análise , Diagnóstico Diferencial , Progressão da Doença , Feminino , Humanos , Incidência , Neoplasias Hepáticas/diagnóstico , Masculino , Prognóstico , Medição de Risco , Índice de Gravidade de DoençaRESUMO
A sustained response to standard interferon therapy for chronic hepatitis C has been demonstrated in no more than 25% of patients. To improve interferon alfa (IFN-alpha) antiviral effect, a number of combination therapies with IFNs plus other drugs have been proposed for both relapser and nonresponder hepatitis C virus (HCV)-infected patients. Although the causes of IFN resistance in subsets of HCV-infected patients are unknown, both viral and host factors have been involved, including defects in IFN signal transduction and IFN-alpha/beta receptor down-regulation. Here, we report that nonsteroidal anti-inflammatory drugs (NSAIDs), which have been proposed for IFN-alpha combination therapy in nonresponders, potentiate IFN-alpha signaling. We found that, in the hepatoma cell lines, CCL13/Chang and HepG2, indomethacin, a selective cyclo-oxygenase 1 and 2 (COX-1 and COX-2) inhibitor, increases IFN-alpha stimulation of interferon-stimulated response element (ISRE)-dependent transcription in a dose-dependent manner. Interestingly, maximal potentiation was observed with suboptimal IFN-alpha concentrations. Indomethacin exerts its effects by synergizing with IFN-alpha in inducing STAT1 activation by phosphorylation, without affecting concurrent Jak1 phosphorylation. Our data indicate that blockade of arachidonic acid (AA) metabolism by indomethacin activates a signaling pathway that converges on STAT1 activation to potentiate IFN-alpha-dependent gene activation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Proteínas de Ligação a DNA/metabolismo , Indometacina/farmacologia , Interferon-alfa/farmacologia , Transativadores/metabolismo , Ácido Araquidônico/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Humanos , Fosfolipases A/fisiologia , Fosforilação , Elementos de Resposta , Fator de Transcrição STAT1 , Ativação Transcricional/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Inherited mutations of the Fas/Apo1/CD95 gene, a cell-surface receptor involved in cell death signaling and in the control of self-reactivity, characterize the recently identified autoimmune lymphoproliferative syndromes. A patient with type 2 autoimmune hepatitis with the immunologic and genetic features of autoimmune lymphoproliferative syndrome is described. The clinical picture was dominated by liver disease with hepatosplenomegaly and positivity for anti-liver-kidney microsome 1 and anti-liver-cytosol 1 antibodies. A marked increase in CD3+CD4-CD8-T lymphocytes and inherited mutations in Fas alleles that led to the expression of a soluble form of the protein were also found. Fas-mediated apoptosis was deficient in the patient as it was in her mother and her sister, who carried the same allele 2 mutation. This observation links type 2 autoimmune hepatitis, an organ-specific disease, with a genetically determined defect in peripheral tolerance control.
Assuntos
Doenças Autoimunes/imunologia , Hepatite/imunologia , Transtornos Linfoproliferativos/imunologia , Mutação Puntual , Receptor fas/genética , Alelos , Apoptose , Autoanticorpos/sangue , Doenças Autoimunes/sangue , Doenças Autoimunes/genética , Pré-Escolar , Feminino , Hepatite/sangue , Hepatite/genética , Hepatomegalia , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Testes de Função Hepática , Transtornos Linfoproliferativos/sangue , Transtornos Linfoproliferativos/genética , Masculino , Linhagem , Esplenomegalia , Síndrome , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologiaRESUMO
Like other members of the tumor necrosis factor (TNF) receptor family, p55 TNF receptor 1 (TNF-R1) lacks intrinsic signaling capacity and transduces signals by recruiting associating molecules. The TNF-R1 associated death domain protein interacts with the p55 TNF-R1 cytoplasmic domain and recruits the Fas-associated death domain protein (which directly activates the apoptotic proteases), the protein kinase receptor interacting protein, and TNF receptor-associated factor 2 (TRAF2). TRAF2 has previously been demonstrated to activate both transcription factor nuclear factor kappaB (NFkappaB) and the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway, which in turn stimulates transcription factor activating protein 1 (AP1) mainly via phosphorylation of the c-Jun component. We have investigated the signaling properties of NFkappaB-inducing kinase (NIK), a TRAF2-associated protein kinase that mediates NFkappaB induction. NIK was found to be unable to activate JNK/SAPK, mitogen-activated protein kinase, or p38 kinase. Moreover, NIK was not required for JNK/SAPK activation by TNF-R1, thus representing the first TNF-R1 complex component to dissect the NFkappaB and the JNK/SAPK pathways. Despite being unable to activate JNK/SAPK and mitogen-activated protein kinase, NIK strongly activated AP1 and was required for TNF-R1-induced AP1 activation. Therefore, NIK links TNF-R1 to a novel, JNK/SAPK-independent, AP1 activation pathway.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Ativadas por Mitógeno , NF-kappa B/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Linhagem Celular , Ativação Enzimática , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Fator 2 Associado a Receptor de TNF , Quinase Induzida por NF-kappaBRESUMO
The human hepatitis B virus (HBV) protein pX is a multifunctional regulatory protein that is known to affect both transcription and cell growth. Here we describe induction of apoptosis in NIH 3T3 polyclonal cell lines upon stimulation of pX expression from a dexamethasone inducible mouse mammary tumor virus (MMTV)-X expression vector. The effect of long-term pX expression on the cell survival of mouse fibroblasts was confirmed in colony generation assays. This effect is not shared either by the other HBV products and it is c-myc mediated, as shown by the use of a dominant negative deletion mutant of c-myc. pX also sensitize cells to programmed cell death after exposure to DNA damaging agents. Taking advantage of stable transfectants carrying the p53val135 temperature-sensitive allele, we directly demonstrate that induction of apoptosis by pX requires p53. In p53 null mouse embryo fibroblasts pX activates transcription and confers an evident growth advantage without loss of cell viability. Although pX protein was not detectable in the experimental conditions we used, our results indicate that its expression affects both cell growth and cell death control.
Assuntos
Apoptose/genética , Regulação da Expressão Gênica/genética , Antígenos da Hepatite B/genética , Transativadores/genética , Proteína Supressora de Tumor p53/genética , Células 3T3 , Animais , Sobrevivência Celular/genética , Meios de Cultura Livres de Soro , Replicação do DNA/genética , Camundongos , Camundongos Endogâmicos BALB C , Ativação Transcricional , Proteínas Virais Reguladoras e AcessóriasRESUMO
Doxorubicin (Dox, Adriamicin), a potent broad spectrum anthracycline anticancer drug, selectively inhibits muscle specific gene expression in cardiac cells in vivo and prevents terminal differentiation of skeletal muscle cells in vitro. By inducing the expression of the helix-loop-helix (HLH) transcriptional inhibitor ld2, Dox represses the myogenic function of the MyoD family of muscle regulatory factors (MRFs). In many cell types, terminal differentiation is coupled to an irreversible exit from the cell cycle and MyoD plays a critical role in the permanent cell cycle arrest of differentiating myocytes by upregulating the cyclin dependent kinase inhibitor (cdki) p21. Here, we correlate Dox effects on cell cycle with changes of E2F/DP complexes and activity in differentiating C2C12 myocytes. In Dox-treated quiescent myoblasts, which fail to differentiate into myotubes under permissive culture conditions, serum re-stimulation induces cyclin/cdk re-association on the E2F/DP complexes and this correlates with an evident increase in E2F/DP driven transcription and re-entry of myoblasts into the cell cycle. Despite Dox ability to activate the DNA-damage dependent p53/p21 pathway, when induced in the absence of MyoD or other MRFs, p21 fails to maintain the postmitotic state in Dox-treated myocytes induced to differentiate. Thus, uncoupling p21 induction and MyoD activity results in a serum-reversible cell cycle arrest, indicating that MRF specific activation of cdki(s) is required for permanent cell cycle arrest in differentiating muscle cells.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Proteínas de Transporte , Proteínas de Ciclo Celular , Ciclo Celular/efeitos dos fármacos , Ciclinas/metabolismo , Doxorrubicina/farmacologia , Músculos/citologia , Proteína MyoD/metabolismo , Proteínas Repressoras , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Proteína 2 Inibidora de Diferenciação , Camundongos , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
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.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteínas de Ligação a DNA , Músculo Esquelético/metabolismo , Proteína MyoD/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Quinase 2 Dependente de Ciclina , DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F4 , Fase G1/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Músculo Esquelético/citologia , Proteína MyoD/genética , Fase de Repouso do Ciclo Celular/fisiologia , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Regulação para CimaRESUMO
The nuclear phosphoprotein p300 is a new member of a family of 'co-activators' (which also includes the CREB binding protein CBP), that directly modulate transcription by interacting with components of the basal transcriptional machinery. Both p300 and CBP are targeted by the adenovirus E1A protein, and binding to p300 is required for E1A to inhibit terminal differentiation in both keratinocytes and myoblasts. Here we demonstrate that, in differentiating skeletal muscle cells, p300 physically interacts with the myogenic basic helix-loop-helix (bHLH) regulatory protein MyoD at its DNA binding sites. During muscle differentiation, MyoD plays a dual role: besides activating muscle-specific transcription, it induces permanent cell cycle arrest by up-regulating the cyclin-dependent kinase inhibitor p21. We show that p300 is involved in both these activities. Indeed, E1A mutants lacking the ability to bind p300 are greatly impaired in the repression of E-box-driven transcription, and p300 overexpression rescues the wild-type E1A-mediated repression. Moreover, p300 potentiates MyoD- and myogenin-dependent activation of transcription from E-box-containing reporter genes. We also provide evidence, obtained by microinjection of anti-p300 antibodies, that p300 is required for MyoD-dependent cell cycle arrest in either myogenic cells induced to differentiate or in MyoD-converted C3H10T1/2 fibroblasts, but is dispensable for maintenance of the postmitotic state of myotubes.
Assuntos
Ciclo Celular , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , Proteína MyoD/fisiologia , Proteínas Nucleares/fisiologia , Transativadores , Fatores de Transcrição/fisiologia , Animais , Western Blotting , Diferenciação Celular , DNA/metabolismo , Proteína p300 Associada a E1A , Sequências Hélice-Alça-Hélice , Camundongos , Microinjeções , Miogenina/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.