RESUMEN
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.
Asunto(s)
Apoptosis/genética , Regulación de la Expresión Génica/genética , Antígenos de la Hepatitis B/genética , Transactivadores/genética , Proteína p53 Supresora de Tumor/genética , Células 3T3 , Animales , Supervivencia Celular/genética , Medio de Cultivo Libre de Suero , Replicación del ADN/genética , Ratones , Ratones Endogámicos BALB C , Activación Transcripcional , Proteínas Reguladoras y Accesorias ViralesRESUMEN
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.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas Portadoras , Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteínas de Unión al ADN , Músculo Esquelético/metabolismo , Proteína MioD/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular/fisiología , Línea Celular , Quinasa 2 Dependiente de la Ciclina , ADN/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F4 , Fase G1/fisiología , Ratones , Ratones Endogámicos C3H , Músculo Esquelético/citología , Proteína MioD/genética , Fase de Descanso del Ciclo Celular/fisiología , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Regulación hacia ArribaRESUMEN
In this study, we characterized the molecular events involved in the activation of the ubiquitous transcription factor NF-kappa B by the viral transactivator pX. pX expression in HeLa cells determines a manyfold increase in NF-kappa B-dependent transcription, which is associated with an increase in p50/p65 heterodimer DNA-binding activity. Since the I kappa B-alpha inhibitory subunit proteolytic degradation, which follows its phosphorylation/modification, is a key event in NF-kappa B activation by different stimuli (such as growth factors, phorbol esters, tumor necrosis factor, UV irradiation, and oxygen radicals), we investigated pX effects on I kappa B-alpha, as well as the possible involvement of known signalling pathways in pX-induced NF-kappa B-dependent transcription. We observed that although pX had no direct effect on p50 or p65, it was able to restore the I kappa B-alpha-suppressed p50/p65 activity. More directly, the stable expression of pX in HeLa cells resulted in reduced levels of I kappa B-alpha in the cytoplasm. Pretreatment of the cells with H7, calphostin C, tyrphostin 25, or N-acetylcysteine did not impair the effects of pX on NF-kappa B, thus ruling out the involvement of protein kinase C, tyrosine kinases, and oxygen radicals. Finally, while most of the known NF-kappa B-activating agents converge on Raf-1 protein kinase, when Raf-1 activity is blocked by overexpression of a dominant negative mutant, the effects of pX on NF-kappa B are not impaired. Thus, we suggest that although pX is able to activate the Ras/Raf-1-signalling pathway, it triggers NF-kappa B activation by an as yet unidentified Raf-1-independent pathway.
Asunto(s)
Proteínas I-kappa B , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Activación Transcripcional , Secuencia de Bases , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , FN-kappa B/genética , Subunidad p50 de NF-kappa B , Proteínas Proto-Oncogénicas c-raf , Transducción de Señal , Factor de Transcripción ReIA , Células Tumorales Cultivadas , Proteínas Reguladoras y Accesorias ViralesRESUMEN
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.
Asunto(s)
Apoptosis , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Receptor fas/fisiología , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Cicloheximida/farmacología , ADN Complementario/química , Humanos , Masculino , Datos de Secuencia Molecular , Proteínas Represoras/fisiología , Células Tumorales CultivadasRESUMEN
Angiotensin II (Ang-II) receptor engagement activates many immediate early response genes in both vascular smooth muscle cells and cardiomyocytes whether a hyperplastic or hypertrophic response is taking place. Although the signaling pathways stimulated by Ang-II in different cell lines have been widely characterized, the correlation between the generation of different second messengers and specific physiological responses remains relatively unexplored. In this study, we report how in both C2C12 quiescent myoblasts and differentiated myotubes Ang-II significantly stimulates AP1-driven transcription and c-Jun.c-Fos heterodimer DNA binding activity. Using a set of different protein kinase inhibitors, we could demonstrate that Ang-II-induced increase in AP1 binding is not mediated by the cAMP-dependent pathway and that both protein kinase C and tyrosine kinases are involved. The observation that in quiescent myoblasts Ang-II increase of AP1 binding and induction of DNA synthesis and, in differentiated myotubes, Ang-II stimulation of protein synthesis are abolished by the cysteine-derivative and glutathione precursor N-acetyl-L-cysteine strongly suggests a role for reactive oxygen intermediates in the intracellular transduction of Ang-II signals for immediate early gene induction, cell proliferation, and hypertrophic responses.
Asunto(s)
Angiotensina II/fisiología , Proteínas de Unión al ADN/metabolismo , Músculos/fisiología , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción AP-1/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Acetilcisteína/farmacología , Animales , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , ADN/biosíntesis , Expresión Génica/efectos de los fármacos , Isoquinolinas/farmacología , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Piperazinas/farmacología , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/genética , Transducción de SeñalRESUMEN
Increasing evidence suggests that angiotensin II may act as a growth factor for several muscle cell types. Angiotensin II stimulation activates many immediate early response genes like c-Fos, c-Jun, c-Myc and Egr-1 in both vascular smooth muscle cells and cardiomyocytes, independently of whether a hyperplastic or hypertrophic response is taking place. In this study we report that angiotensin II significantly stimulates AP1-driven transcription in mouse skeletal muscle cells C2C12 stably transfected with a TRE-tk-CAT plasmid in a dose-dependent manner (peak stimulation at 10(-5) M of angiotensin II). Moreover, angiotensin II increases the binding of the AP1 complex to its DNA target in both quiescent C2C12 myoblasts and in differentiated C2C12 myotubes. Most of the TRE-bound complexes in both unstimulated and angiotensin II-treated cells consist of c-jun/c-fos heterodimers. Using a set of different protein kinase inhibitors, including HA1004, H7, tyrphostin, genistein and staurosporine, we could demonstrate that the angiotensin II-induced AP1 binding increase is not mediated by the cAMP-dependent pathway and that protein kinase C and tyrosine kinases are involved. Treatment of C2C12 cells with H2O2 induces a dose-dependent increase in c-jun/c-fos heterodimer binding, specifically reverted by the cysteine derivative and glutathione precursor N-acetyl-L-cysteine (NAC). The observation that the induction by angiotensin II of both the AP1 DNA binding activity and DNA synthesis in quiescent C2C12 myoblasts is abolished by NAC strongly suggests a role for reactive oxygen intermediates (ROIs) in the intracellular transduction of angiotensin II signals for immediate early gene induction and for cell proliferation.
Asunto(s)
Angiotensina II/farmacología , Expresión Génica/efectos de los fármacos , Músculo Esquelético/fisiología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Transducción de Señal/fisiología , Animales , Antioxidantes/farmacología , Secuencia de Bases , Diferenciación Celular , Línea Celular , Genes Inmediatos-Precoces/efectos de los fármacos , Corazón/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocardio/metabolismo , Oligodesoxirribonucleótidos , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Transducción de Señal/efectos de los fármacos , Factor de Transcripción AP-1/metabolismo , TransfecciónRESUMEN
The mechanisms by which pX, the transactivator of the hepatitis B virus (HBV), exerts its effects on transcription of viral and cellular genes and affects cell-growth regulation have not yet been fully defined. 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 characterized the mechanisms of AP-1 transcription factor activation by pX and, in particular, the role of cellular proteins involved in the intracellular signal transduction of growth-factor receptors. The observation that the overexpression of c-fos and c-jun in the cells results in a clear augmentation of the effects of pX on TRE-directed transcription and the induction of the DNA-binding activity of c-jun/c-fos heterodimers by AP1-depleted nuclear extracts from pX-expressing cells strongly supports the involvement of post-translational modifications. In both HeLa and undifferentiated F9 cells, pX was able to increase the activity of exogenous transfected c-jun but not of c-jun mutants bearing mutations in the serine residues located in the amino-terminal transcriptional activation domain. Moreover, by use of Ha-ras and Raf-1 dominant negative mutants, we show that both Ha-ras and Raf-1 are required for pX-induced activation of c-jun transcriptional activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Genes jun/fisiología , Genes pX/fisiología , Virus de la Hepatitis B/genética , Transducción de Señal/fisiología , Activación Transcripcional , Animales , Humanos , Transducción de Señal/genética , Factores de Transcripción/fisiología , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Virus de la Hepatitis B/genética , Proteína Oncogénica p21(ras)/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Oncogénicas de Retroviridae/metabolismo , Transactivadores/metabolismo , Activación Transcripcional , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Genes ras , Células HeLa , Humanos , Proteínas Oncogénicas v-raf , Transducción de Señal , Proteínas Reguladoras y Accesorias ViralesRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Virus de la Hepatitis B/metabolismo , Naftalenos , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Sulfonamidas , Transactivadores/metabolismo , Activación Transcripcional , Alcaloides/farmacología , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferasa/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Células HeLa , Humanos , Isoquinolinas/farmacología , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/metabolismo , Fosforilación , Compuestos Policíclicos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Esfingosina/farmacología , Estaurosporina , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Transfección , Células Tumorales Cultivadas , Virus Vaccinia/genética , Proteínas Reguladoras y Accesorias ViralesRESUMEN
The hepatitis B virus (HBV) X protein (pX) stimulates transcription regulated by cis-acting elements that control many viral and cellular genes, including the c-myc and the c-fos proto-oncogenes. Using several c-fos promoter deletion mutants, we found the serum-responsive element (SRE) located at -315, the modified TPA-responsive element located at -296 (fos-AP-1 binding site, FAP) and the region spanning from nucleotide -220 to -120, which contains an NF1-like site and several stretches of sequence homologous to the AP-2 consensus binding sites, to be responsive to pX. pX does not modify the pattern of the retarded complexes bound to the SRE/FAP region which, in our system, appears to be occupied by SRE-binding factors. The activation of the SRE does not involve complex formation between SRE-binding factors and pX, it is not associated with an increase in serum response factor binding to the SRE and it does not determine changes in SRE mobility-shift pattern.
Asunto(s)
Genes fos , Virus de la Hepatitis B/genética , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Transactivadores/metabolismo , Activación Transcripcional , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Regulación Viral de la Expresión Génica , Células HeLa , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Plásmidos , Proteínas Quinasas/metabolismo , Mapeo Restrictivo , Transcripción Genética , Transfección , Células Vero , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Clinical and experimental evidence suggests the possible existence of one or more extrahepatic sites of HCV infection. In order to demonstrate the "in vivo" infection of lymphoid cells by HCV, we applied a nested PCR to total cytoplasmic RNA extracted from fresh or cultured peripheral blood mononuclear cells (PBMCs) of HCV chronically infected patients, using primers derived from the highly conserved 5' untranslated region of the HCV genome. The presence of virions in PBMCs occurs frequently, if not always, and is often accompanied by active viral replication. Moreover, the appearance of replicative intermediates after stimulation of cellular growth with mitogens suggests that latent genomes could undergo replication upon cellular activation and/or proliferation.