Asunto(s)
Neoplasias de la Mama/metabolismo , Diferenciación Celular , Resistencia a Antineoplásicos , Proteínas Musculares/metabolismo , Tolerancia a Radiación , Proteínas Reguladoras de la Apoptosis , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/radioterapia , Línea Celular , Doxorrubicina/farmacología , HumanosRESUMEN
Childhood ataxia with diffuse central nervous system hypomyelination syndrome (CACH) is a recently described leukodystrophy of unknown etiology. To characterize the neuropathological features and gain insight as to the pathogenesis of this disorder, we studied cerebral tissue from six patients with the CACH syndrome. Evaluation of toluidine blue-stained, semithin sections of white matter from CACH patients disclosed unusual cells with "foamy" cytoplasm, small round nuclei and fine chromatin. Electron microscopy (EM) revealed cells in the white matter with abundant cytoplasm containing many mitochondria and loosely clustered, membranous structures, but lacking the lysosomal structures seen in macrophages. Further analysis of tissue sections with antibodies and special stains demonstrated that the abnormal cells with abundant cytoplasm labeled with oligodendroglial markers, but did not react with macrophage or astrocytic markers. Double immunolabeling with macrophage and oligodendroglial markers clearly distinguished macrophages from the "foamy" oligodendroglial cells (FODCs). Proteolipid protein (PLP) mRNA in situ hybridization demonstrated PLP mRNA transcripts in a high proportion of oligodendrocytes in CACH patients compared to control patients, and PLP mRNA transcript signal in cells, morphologically consistent with FODCs. Normal and pathological brain control tissues did not contain FODCs. These neuropathological findings will be useful pathological identifiers of CACH, and may provide clues to the pathogenesis of this disorder.
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Ataxia/complicaciones , Ataxia/patología , Encéfalo/patología , Células Espumosas/patología , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/complicaciones , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Oligodendroglía/patología , Ataxia/metabolismo , Biomarcadores , Biopsia , Encéfalo/metabolismo , Encéfalo/fisiopatología , Niño , Preescolar , Femenino , Células Espumosas/metabolismo , Células Espumosas/ultraestructura , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Humanos , Lactante , Masculino , Oligodendroglía/metabolismo , Oligodendroglía/ultraestructura , Fenotipo , ARN Mensajero/metabolismoRESUMEN
Fibroblast growth factor 2 (FGF2) is an excellent candidate to regulate remyelination based on its proposed actions in oligodendrocyte lineage cell development in conjunction with its involvement in CNS regeneration. To assess the potential for FGF2 to play a role in remyelination, we examined the expression pattern of FGF2 and FGF receptors (FGFRs) in an experimental demyelinating disease with extensive remyelination. Adult mice were intracranially injected with murine hepatitis virus strain A-59 (MHV-A59) to induce focally demyelinated spinal cord lesions that spontaneously remyelinate, with corresponding recovery of motor function. Using kinetic RT-PCR analysis of spinal cord RNA, we found significantly increased levels of FGF2 mRNA transcripts, which peaked during the initial stage of remyelination. Analysis of tissue sections demonstrated that increased levels of FGF2 mRNA and protein were localized within demyelinated regions of white matter, including high FGF2 expression associated with astrocytes. The expression of corresponding FGF receptors was significantly increased in lesion areas during the initial stage of remyelination. In normal and lesioned white matter, oligodendrocyte lineage cells, including progenitors and mature cells, were found to express multiple FGFR types (FGFR1, FGFR2, and/or FGFR3). In addition, in lesion areas, astrocytes expressed FGFR1, FGFR2, and FGFR3. These findings indicate that, during remyelination, FGF2 may play a role in directly regulating oligodendrocyte lineage cell responses and may also act through paracrine or autocrine effects on astrocytes, which are known to synthesize other growth factors and immunoregulatory molecules that influence oligodendrocyte lineage cells.
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Factor 2 de Crecimiento de Fibroblastos/metabolismo , Vaina de Mielina/metabolismo , Neuroglía/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Coronavirus , Femenino , Hepatitis Viral Animal , Ratones , Ratones Endogámicos C57BL , Trastornos de la Destreza Motora/inducido químicamente , Esclerosis Múltiple/metabolismo , ARN Mensajero/metabolismoRESUMEN
Expression of the wild-type alpha subunit of Gq stimulates phospholipase C and induces hypertrophy in cardiomyocytes. Addition of Gq-coupled receptor agonists additionally activates phospholipase C, as does expression of a constitutively active mutant form of Galphaq. Under these conditions, hypertrophy is rapidly succeeded by apoptotic cellular and molecular changes, including myofilament disorganization, loss of mitochondrial membrane potential, alterations in Bcl-2 family protein levels, DNA fragmentation, increased caspase activity ( approximately 4-fold), cytochrome c redistribution, and nuclear chromatin condensation in approximately 12% of the cells. We used various interventions to define the molecular relationships between these events and identify potential sites at which these features of apoptosis could be rescued. Treatment with caspase inhibitors prevented DNA fragmentation and promoted myocyte survival; however, cytochrome c release and loss of mitochondrial membrane potential still occurred. In contrast, treatment with bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore, not only prevented DNA fragmentation and reduced nuclear chromatin condensation but also preserved mitochondrial membrane potential and limited cytochrome c redistribution to only approximately 2% of cells. These data demonstrate the central role of mitochondrial membrane potential in initiation of caspase activation and downstream apoptotic events and suggest that preservation of mitochondrial integrity is crucial for prolonging the life and function of cardiomyocytes exposed to pathological levels of stress.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/fisiología , Mitocondrias/fisiología , Miocardio/citología , Adenoviridae/genética , Animales , Antibacterianos/farmacología , Apoptosis , Ácido Bongcréquico/farmacología , Caspasas/metabolismo , Células Cultivadas , Grupo Citocromo c/metabolismo , Activación Enzimática , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Permeabilidad/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Fosfolipasas de Tipo C/metabolismoRESUMEN
CONTEXT: The dysembryoplastic neuroepithelial tumor (DNT) is an uncommon lesion characterized by a heterogeneous population of neurons, astrocytes, and oligodendroglia-like cells (OLCs). The basic nature of the DNT and its constituent cells, particularly the OLCs, remains unresolved; some authors favor a neuronal origin, and others propose a glial or mixed origin for these cells. DESIGN: We examined 11 DNTs with antibodies to myelin oligodendrocyte glycoprotein, a marker of mature oligodendrocytes. RESULTS: All DNTs studied (7 from males, 4 from females; age range of patients, 2-37 years) were composed of varying proportions of neurons, astrocytes, and OLCs. Membrane or cytoplasmic immunoreactivity for myelin oligodendrocyte glycoprotein was found in many OLCs in 9 of 11 cases. The number of myelin oligodendrocyte glycoprotein-positive OLCs was variable: >75% of the OLCs were positive in 5 cases, 25% to 75% of the OLCs were positive in 2 cases, and <25% of the OLCs were positive in 2 cases. CONCLUSION: These findings suggest that many of the OLCs represent mature oligodendrocytes and support the notion that DNTs are heterogenous lesions composed of multiple, mature cell types.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Glicoproteína Asociada a Mielina/metabolismo , Tumores Neuroectodérmicos Periféricos Primitivos/metabolismo , Oligodendroglía/metabolismo , Adolescente , Adulto , Neoplasias Encefálicas/patología , Niño , Preescolar , Femenino , Humanos , Técnicas para Inmunoenzimas , Masculino , Proteínas de la Mielina , Glicoproteína Mielina-Oligodendrócito , Tumores Neuroectodérmicos Periféricos Primitivos/patologíaRESUMEN
BACKGROUND: Septic shock is a leading cause of mortality in intensive care units. No new interventions in the last 20 years have made a substantial impact on the outcome of patients with septic shock. Identification of inhibitable pathways that mediate death in shock is an important goal. MATERIALS AND METHODS: Two novel caspase inhibitors, (2-indolyl)-carbonyl-Ala-Asp-fluoromethylketone (IDN 1529) and (1-methyl-3-methyl-2-indolyl)-carbonyl-Val-Asp-fluoromethylketone (IDN 1965), were studied in a murine model of endotoxic shock. RESULTS: IDN 1529 prolonged survival when given before or up to 3 hr after high-dose LPS (p < 0.01) and increased by 2.2-fold the number of animals surviving longterm after a lower dose of LPS (p < 0.01). Despite its similar chemical structure, IDN 1965 lacked these protective effects. Both compounds inhibited caspases 1, 2, 3, 6, 8, and 9, and both afforded comparable reduction in Fas- and LPS-induced caspase 3-like activity and apoptosis. Paradoxically, administration of IDN 1529 but not IDN 1965 led to an increase in the LPS-induced elevation of serum cytokines related directly (IL-1beta, IL-18) or indirectly (IL-1alpha, IL-1Ra) to the action of caspase 1. CONCLUSIONS: A process that appears to be distinct from both apoptosis and the release of inflammatory cytokines is a late-acting requirement for lethality in endotoxic shock. Inhibition of this process can rescue mice even when therapy is initiated after LPS has made the mice severely ill.
Asunto(s)
Inhibidores de Cisteína Proteinasa/farmacología , Indoles/farmacología , Oligopéptidos/farmacología , Choque Séptico/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Caspasa 1/efectos de los fármacos , Caspasa 1/genética , Caspasa 1/metabolismo , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Citocinas/sangre , Citocinas/efectos de los fármacos , Femenino , Interleucina-1/metabolismo , Lipopolisacáridos , Hígado/efectos de los fármacos , Hígado/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Choque Séptico/mortalidad , Tasa de Supervivencia , Receptor fas/inmunologíaRESUMEN
The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is unknown. Here we characterize an intracellular, membrane-associated form of procaspase-3 whose activation is controlled by Bcl-2. Heavy membranes isolated from control cells contained a spontaneously activatable caspase-3 zymogen. In contrast, in Bcl-2 overexpressing cells, although the caspase-3 zymogen was still associated with heavy membranes, its spontaneous activation was blocked. However, Bcl-2 expression had little effect on the levels of cytoplasmic caspase activity in unstimulated cells. Furthermore, the membrane-associated caspase-3 differed from cytosolic caspase-3 in its responsiveness to activation by exogenous cytochrome c. Our results demonstrate that intracellular membranes can generate active caspase-3 by a Bcl-2-inhibitable mechanism, and that control of caspase activation in membranes is distinct from that observed in the cytoplasm. These data suggest that Bcl-2 may control cytoplasmic events in part by blocking the activation of membrane-associated procaspases.
Asunto(s)
Caspasas/metabolismo , Precursores Enzimáticos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Apoptosis , Caspasa 3 , Inhibidores de Caspasas , Línea Celular , Cumarinas/metabolismo , Grupo Citocromo c/farmacología , Activación Enzimática , Precursores Enzimáticos/antagonistas & inhibidores , Humanos , Hidrólisis , Oligopéptidos/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
We have examined the potential roles of intracellular Ca2+ regulation and of multiple cytoskeletal elements in control of the directed migration of cultured oligodendrocyte progenitor cells (OPs). OPs were found to migrate in response to platelet-derived growth factor (PDGF) or to a lesser extent to basic fibroblast growth factor (FGF) in a non-additive manner. This response was inhibited by chelation of intracellular Ca2+ by using BAPTA-AM. OP migration was not evoked by the neurotransmitter agonists phenylephrine or methacholine, which elevate OP Ca2+ levels. Inhibition of the MAP kinase pathway with PD 098059 did not affect OP migration to PDGF. Within growth cone-like leading edges of migratory OP processes, monomeric and filamentous actin were found to be colocalized with myosin and filamentous actin was prominent in filopodia extending beyond the leading edge. Tubulin was distributed throughout OP processes and cell bodies. Inhibition of actin or tubulin polymerization, by using cytochalasin B or nocodazole, respectively, altered OP morphology and markedly impaired migration. Inhibition of the myosin ATPase by BDM, which prevents force-generating actin/myosin interactions, greatly inhibited the chemotaxic response at concentrations that did not disrupt cell morphology. These results indicate that growth factors stimulate OP migration by activating pathways which include intracellular Ca2+ regulation, and characterize the distribution of multiple cytoskeletal elements involved in the generation of directed OP movement.
Asunto(s)
Encéfalo/fisiología , Citoesqueleto/fisiología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Oligodendroglía/fisiología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Transducción de Señal/fisiología , Células Madre/fisiología , Animales , Animales Recién Nacidos , Becaplermina , Encéfalo/citología , Calcio/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Movimiento Celular/fisiología , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Humanos , Oligodendroglía/citología , Oligodendroglía/efectos de los fármacos , Proteínas Proto-Oncogénicas c-sis , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
Endogenous oligodendrocyte lineage cells spontaneously remyelinate focal areas of demyelination induced by murine hepatitis virus A59 infection of C57Bl/6 mice. We used this model to examine the potential for platelet-derived growth factor (PDGF) to have a role in repopulating demyelinated lesions, and in doing so we also further characterized the in vivo responses of oligodendrocyte lineage cells following demyelination. Very early in the progress of remyelination, we administered a 4-h in vivo pulse of bromodeoxyuridine (BrdU) and subsequently performed in situ hybridization for PDGF-alpha receptor (PDGFalphaR), an established marker for oligodendrocyte progenitors in vivo, or for proteolipid protein (PLP), to identify oligodendrocytes. Sections of lesioned spinal cords had a 14.5-fold increase in the number of BrdU-labeled oligodendrocyte progenitor cells (PDGFalphaR+), while BrdU-labeled oligodendrocytes (PLP+) were extremely rare. Immunocytochemistry of similar sections demonstrated that immunoreactivity for both PDGFalphaR and NG2, another marker of oligodendrocyte progenitors, was locally increased in areas of white-matter lesions. High-resolution immunofluorescence imaging was used to detect oligodendrocyte progenitor cells expressing receptors for both PDGF and fibroblast growth factor. In addition, expression of PDGF-A mRNA transcripts was increased in sections of lesioned spinal cords and reactive astrocytes in lesions exhibited immunoreactivity for PDGF ligand. Our findings indicate that during the initial stages of remyelination, oligodendrocyte progenitors proliferate locally, and that this response may potentially involve PDGF.
Asunto(s)
Vaina de Mielina/fisiología , Regeneración Nerviosa/fisiología , Oligodendroglía/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Animales , Bromodesoxiuridina/metabolismo , División Celular/fisiología , Línea Celular , Femenino , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Proteína Proteolipídica de la Mielina/genética , Oligodendroglía/citología , ARN Mensajero/análisis , Receptores de Factores de Crecimiento de Fibroblastos/biosíntesis , Factores de TiempoRESUMEN
Internal initiation of translation, a mechanism infrequently used by cellular messages, avoids the requirement of a methyl cap structure for translation of messenger RNAs. The mRNA transcript encoding the DNA-binding protein MYT2 represents one of the exceptional cellular messages that contains an internal ribosome entry site (IRES). The RNA pseudoknot structure located in the 5' untranslated region of MYT2 functions to promote translation in vivo. MYT2 was cloned by its specific binding to a TTCCA motif in the promoter region of a glial-specific gene, myelin proteolipid protein. MYT2 also recognizes single-stranded nucleic acids. In the central nervous system, MYT2 protein is found in oligodendrocyte progenitor cells, subsets of neurons, and cells of the choroid plexus together with ciliated ependymal cells. MYT2 protein can also be secreted from cells, an atypical event for a DNA-binding protein. The presence of an internal ribosome entry site in MYT2, together with the unusual localization of MYT2, suggests that this nucleic acid-binding protein may be in the class of proteins involved in cellular growth control and survival in the nervous system.
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Encéfalo/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Biosíntesis de Proteínas/genética , Ribosomas/fisiología , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Secuencia de Bases , Línea Celular , Humanos , Ratones , Datos de Secuencia Molecular , Ácidos Nucleicos/metabolismo , Oligodendroglía/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Distribución TisularRESUMEN
The Rad51 gene of Saccharomyces cerevisiae is required for genetic recombination and recombinational repair of DNA strand breaks. In higher eukaryotes Rad51 is essential for embryonic development, and is involved in cell proliferation and DNA repair. Here we show that human Rad51 (HsRad51) is proteolytically cleaved during apoptosis in two T-lymphocyte cell lines, Jurkat and PFI-285. Apoptosis was induced by camptothecin or anti-Fas monoclonal antibody (anti-Fas mAb). HsRad51 was cleaved with similar kinetics as human poly(ADP-ribose) polymerase (HsPARP) after treatment with either agent. The time course of cleavage coincided with internucleosomal DNA fragmentation. The HsRad51 fragments observed in apoptotic cells were identical to those generated from in vitro translated (IVT) HsRad51 exposed to activated Jurkat S-100 extract in a cell-free system. In each case, cleavage of HsRad51 was abolished by acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). However, cleavage of IVT HsRad51 could not be demonstrated using purified caspase-2, -3 or -6 to -10, and the identity of the responsible protease thus remains to be determined. In summary, we have shown that HsRad51 belongs to a group of repair proteins, including PARP and DNA-dependent protein kinase, which are specifically cleaved during the execution phase of apoptosis.
Asunto(s)
Apoptosis/fisiología , Proteínas de Unión al ADN/metabolismo , Linfocitos T/enzimología , Anticuerpos Monoclonales , Camptotecina/farmacología , Línea Celular Transformada , Sistema Libre de Células , Inhibidores de Cisteína Proteinasa/farmacología , ADN-Topoisomerasas de Tipo I/farmacología , Proteína Quinasa Activada por ADN , Humanos , Células Jurkat/enzimología , Proteínas Nucleares , Oligopéptidos/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinasa Rad51 , Inhibidores de Serina Proteinasa/farmacología , Clorometilcetona de Tosilfenilalanila/farmacología , Receptor fas/fisiologíaRESUMEN
The gene encoding human IAP-like protein (hILP) is one of several mammalian genes with sequence homology to the baculovirus inhibitor-of-apoptosis protein (iap) genes. Here we show that hILP can block apoptosis induced by a variety of extracellular stimuli, including UV light, chemotoxic drugs, and activation of the tumor necrosis factor and Fas receptors. hILP also protected against cell death induced by members of the caspase family, cysteine proteases which are thought to be the principal effectors of apoptosis. hILP and Bcl-xL were compared for their ability to affect several steps in the apoptotic pathway. Redistribution of cytochrome c from mitochondria, an early event in apoptosis, was not blocked by overexpression of hILP but was inhibited by Bcl-xL. In contrast, hILP, but not Bcl-xL, inhibited apoptosis induced by microinjection of cytochrome c. These data suggest that while Bcl-xL may control mitochondrial integrity, hILP can function downstream of mitochondrial events to inhibit apoptosis.
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Apoptosis/genética , Proteínas Bacterianas/genética , Grupo Citocromo c/genética , Proteínas de Escherichia coli , Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Grupo Citocromo c/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína bcl-XRESUMEN
Using in vitro systems, the proliferation, migration, differentiation, and survival of immature oligodendrocyte lineage cells can be examined to elucidate the cellular and molecular interactions that regulate this lineage. The ability to monitor progressive stages of differentiation within the lineage by immunophenotyping and to manipulate the cellular responses with growth factors makes these cultures advantageous as both a method for studying the cell biology of myelination and as a model system for lineage analysis in the mammalian central nervous system. In addition, cultured oligodendrocytes carry out the normal in vivo sequence of expression of a set of cell type-specific genes, some of which are extremely highly expressed, and so provide advantages for analysis of gene regulation. This paper describes commonly used methods for the preparation of mixed glial cell cultures from perinatal rodent brain. Although these cultures are most commonly derived from perinatal rat brain, a protocol for preparation from mouse brain is also provided because of the increasing number of studies that use mice to facilitate molecular biological techniques. Methods to prepare secondary cultures of different stages of oligodendrocyte lineage cells are detailed. As examples of methods to use for the characterization of these cells, immunophenotypes of each stage of the oligodendrocyte lineage are illustrated, incorporation of [3H]thymidine for analysis of cell proliferation is illustrated, and detailed methods are provided for analysis of migration in a microchemotaxis chamber.
Asunto(s)
Separación Celular/métodos , Oligodendroglía/fisiología , Adulto , Animales , Técnicas de Cultivo de Célula/métodos , Sistema Nervioso Central/fisiología , Quimiotaxis , Enfermedades Desmielinizantes , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Microglía/química , Neuroglía/química , Oligodendroglía/química , Ratas , Células Madre/químicaRESUMEN
Bcl-xL, an antiapoptotic member of the Bcl-2 family, inhibits programmed cell death in a broad variety of cell types. Recent reports have demonstrated that cytochrome c is released from mitochondria during apoptosis and have suggested that this release may be a critical step in the activation of proapoptotic caspases and subsequent cell death. Furthermore, it has been demonstrated that Bcl-2 can prevent the release of cytochrome c from mitochondria in cells triggered to undergo apoptosis. This has led to the hypothesis that the antiapoptotic effects of Bcl-2 family members are due specifically to their ability to prevent cytochrome c release thus preventing subsequent cytochrome c-dependent caspase activation. In the present report, we use microinjection techniques to investigate the relationship between cytochrome c release, induction of apoptosis, and Bcl-xL activity in intact cells. We demonstrate that microinjection of cytochrome c into the cytosol of human kidney 293 cells results in a dose-dependent induction of apoptosis. In contrast, MCF7 breast carcinoma cells (stably transfected to express the Fas antigen CD95, and denoted MCF7F) that lack detectable levels of caspase 3 (CPP32), are totally resistant to microinjection of cytochrome c. However, transfection of MCF7F cells with an expression plasmid coding for pro-caspase 3, but not other pro-caspases, restores cytochrome c sensitivity. Although MCF7F cells are insensitive to cytochrome c microinjection, they rapidly undergo apoptosis in a caspase-dependent manner in response to either tumor necrosis factor or anti-Fas plus cycloheximide, and these deaths are strongly inhibited by Bcl-xL expression. Furthermore, microinjection of cytochrome c does not overcome these antiapoptotic effects of Bcl-xL. Our results support the concept that the release of cytochrome c into the cytoplasm can promote the apoptotic process in cells expressing pro-caspase 3 but that cytochrome c release is not sufficient to induce death in all cells. Importantly, the ability of Bcl-xL to inhibit cell death in the cytochrome c-insensitive MCF7F cells cannot be due solely to inhibition of cytochrome c release from mitochondria.
Asunto(s)
Apoptosis , Caspasas , Grupo Citocromo c/administración & dosificación , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Caspasa 3 , Línea Celular , Cisteína Endopeptidasas/fisiología , Citocinas/farmacología , Humanos , Microinyecciones , Factores de Tiempo , Células Tumorales Cultivadas , Proteína bcl-XRESUMEN
Fas- and tumor necrosis factor receptor 1 (TNFR1)-induced apoptosis is mediated by the interaction of FADD with caspase-8. Here, we report that the bovine herpesvirus 4 (BHV4) BORFE2 gene encodes a protein that inhibits Fas- and TNFR1-induced apoptosis and contains death effector domains (DEDs). Using the yeast two-hybrid system, we found that the BORFE2 protein interacts with the prodomain of caspase-8. Furthermore, we show that BHV4 BORFE2 is a member of a family of DED-containing proteins that includes other gamma-2 herpesviruses, such as Kaposi's sarcoma-associated herpesvirus and herpesvirus saimiri.
Asunto(s)
Apoptosis , Gammaherpesvirinae/patogenicidad , Proteínas Virales/química , Secuencia de Aminoácidos , Animales , Caspasa 1 , Bovinos/virología , Cisteína Endopeptidasas/metabolismo , Gammaherpesvirinae/química , Gammaherpesvirinae/genética , Unión Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas Virales/genéticaRESUMEN
Detection and characterization of distinct central nervous system (CNS) tumor cell types is clinically important since distinct tumor types are associated with different prognoses and treatments. However, there is currently a lack of markers to identify certain glioma types and insufficient understanding as to which cells give rise to different glioma cell types. In the present study, biopsy specimens from human brain tumors were analyzed for expression of Myelin Transcription Factor 1 (MYT1) to explore the extent to which glioma cells reflect characteristic expression of MYT1 in developing glial progenitor cells. Immunostaining with an antibody against MYT1 revealed widespread immunoreactivity that was most prominent in high-grade oligodendrogliomas, astrocytomas, and mixed oligoastrocytomas as well as in a dysembryoplastic neuroepithelial tumor. MYT1 immunoreactivity in tumor regions generally correlated with the prevalence of cells exhibiting nuclear immunolabeling with an antibody against Ki-67, suggesting an association of MYT1 with cell proliferation that was also observed in normal adult human and rat brain in the germinal subependymal zone. The MYT1 immunoreactivity was frequently nuclear, appearing as dotted or punctate, but in some cases it was localized to the cytoplasm. In combination with histopathological studies and analysis of Ki-67 immunoreactivity, examination of MYT1 immunolabeling may provide additional information to aid in the detection and diagnosis of CNS tumors.
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Astrocitoma/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/metabolismo , Proteínas de Unión al ADN/metabolismo , Oligodendroglioma/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factores de Transcripción , Dedos de Zinc , Adulto , Animales , Humanos , Técnicas para Inmunoenzimas , Antígeno Ki-67/metabolismo , Masculino , Proteínas de la Membrana , RatasRESUMEN
We identified and cloned a novel human protein that contains FADD/Mort1 death effector domain homology regions, designated FLAME-1. FLAME-1, although most similar in structure to Mch4 and Mch5, does not possess caspase activity but can interact specifically with FADD, Mch4, and Mch5. Interestingly, FLAME-1 is recruited to the Fas receptor complex and can abrogate Fas/TNFR-induced apoptosis upon expression in FasL/tumor necrosis factor-sensitive MCF-7 cells, possibly by acting as a dominant-negative inhibitor. These findings identify a novel endogenous control point that regulates Fas/TNFR1-mediated apoptosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis , Proteínas Portadoras/metabolismo , Caspasas , Péptidos y Proteínas de Señalización Intracelular , Proteínas/metabolismo , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/química , Proteínas Portadoras/genética , Caspasa 10 , Caspasa 8 , Caspasa 9 , Clonación Molecular , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Proteína Ligando Fas , Proteína de Dominio de Muerte Asociada a Fas , Humanos , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Factor 1 Asociado a Receptor de TNF , Distribución Tisular , Rayos UltravioletaRESUMEN
To identify novel antiapoptotic proteins encoded by DNA viruses, we searched viral genomes for proteins that might interfere with Fas and TNFR1 apoptotic signaling pathways. We report here that equine herpesvirus type 2 E8 protein and molluscum contagiosum virus MC159 protein both show sequence similarity to the death effector domains (DEDs) of the Fas/TNFR1 signaling components FADD and caspase-8. Yeast two-hybrid analysis revealed that E8 protein interacted with the caspase-8 prodomain whereas MC159 protein interacted with FADD. Furthermore, expression of either E8 protein or MC159 protein protected cells from Fas- and TNFR1-induced apoptosis indicating that certain herpesviruses and poxviruses use DED-mediated interactions to interfere with apoptotic signaling pathways. These findings identify a novel control point exploited by viruses to regulate Fas- and TNFR1-mediated apoptosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Antígenos CD/metabolismo , Apoptosis/fisiología , Virus ADN , Receptores del Factor de Necrosis Tumoral/metabolismo , Proteínas Virales/metabolismo , Receptor fas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteína de Dominio de Muerte Asociada a Fas , Herpesviridae , Modelos Biológicos , Datos de Secuencia Molecular , Virus del Molusco Contagioso , Unión Proteica , Receptores Tipo I de Factores de Necrosis Tumoral , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
The effects of the expression of the human Bcl-2 family proteins Bax, Bak, Bcl-2, and Bcl-XL were examined in the fission yeast Schizosaccharomyces pombe and compared with Bax-induced cell death in mammalian cells. Expression of the proapoptotic proteins Bax and Bak conferred a lethal phenotype in this yeast, which was strongly suppressed by coexpression of the anti-apoptotic protein Bcl-XL. Bcl-2 also partially abrogated Bax-mediated cytotoxicity in S. pombe, whereas a mutant of Bcl-2 (Gly145Ala) that fails to heterodimerize with Bax or block apoptosis in mammalian cells was inactive. However, other features distinguished Bax- and Bak-induced death in S. pombe from animal cell apoptosis. Electron microscopic analysis of S. pombe cells dying in response to Bax or Bak expression demonstrated massive cytosolic vacuolization and multifocal nuclear chromatin condensation, thus distinguishing this form of cell death from the classical morphological features of apoptosis seen in animal cells. Unlike Bax-induced apoptosis in 293 cells that led to the induction of interleukin-1 beta-converting enzyme (ICE)/CED-3-like protease activity, Bax- and Bak-induced cell death in S. pombe was accompanied neither by internucleosomal DNA fragmentation nor by activation of proteases with specificities similar to the ICE/CED-3 family. In addition, the baculovirus protease inhibitor p35, which is a potent inhibitor of ICE/CED-3 family proteases and a blocker of apoptosis in animal cells, failed to prevent cell death induction by Bax or Bak in fission yeast, whereas p35 inhibited Bax-induced cell death in mammalian cells. Taken together, these findings suggest that Bcl-2 family proteins may retain an evolutionarily conserved ability to regulate cell survival and death but also indicate differences in the downstream events that are activated by overexpression of Bax or Bak in divergent cell types.
Asunto(s)
Apoptosis , Caspasas , Proteínas de la Membrana/fisiología , Proteínas Proto-Oncogénicas/fisiología , Schizosaccharomyces/fisiología , Proteínas de Caenorhabditis elegans , Línea Celular Transformada , Cisteína Endopeptidasas/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis , Proteínas de la Membrana/genética , Fenotipo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Schizosaccharomyces/genética , Factores de Tiempo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
Neuronal apoptosis occurs during nervous system development and after pathological insults to the adult nervous system. Inhibition of CED3/ICE-related proteases has been shown to inhibit neuronal apoptosis in vitro and in vivo, indicating a role for these cysteine proteases in neuronal apoptosis. We have studied the activation of the CED3/ICE-related protease CPP32 in two in vitro models of mouse cerebellar granule neuronal cell death: K+/serum deprivation-induced apoptosis and glutamate-induced necrosis. Pretreatment of granule neurons with a selective, irreversible inhibitor of CED3/ICE family proteases, ZVAD-fluoromethylketone, specifically inhibited granule neuron apoptosis but not necrosis, indicating a selective role for CED3/ICE proteases in granule neuron apoptosis. Extracts prepared from apoptotic, but not necrotic, granule neurons contained a protease activity that cleaved the CPP32 substrate Ac-DEVD-aminomethylcoumarin. Induction of the protease activity was prevented by inhibitors of RNA or protein synthesis or by the CED3/ICE protease inhibitor. Affinity labeling of the protease activity with an irreversible CED3/ICE protease inhibitor, ZVK(biotin)D-fluoromethylketone, identified two putative protease subunits, p20 and p18, that were present in apoptotic but not necrotic granule neuron extracts. Western blotting with antibodies to the C terminus of the large subunit of mouse CPP32 (anti-CPP32) identified p20 and p18 as processed subunits of the CPP32 proenzyme. Anti-CPP32 specifically inhibited the DEVD-amc cleaving activity, verifying the presence of active CPP32 protease in the apoptotic granule neuron extracts. Western blotting demonstrated that the CPP32 proenzyme was expressed in granule neurons before induction of apoptosis. These results demonstrate that the CED3/ICE homolog CPP32 is processed and activated during cerebellar granule neuron apoptosis. CPP32 activation requires macromolecular synthesis and CED3/ICE protease activity. The lack of CPP32 activation during granule neuron necrosis suggests that proteolytic processing and activation of CED3/ICE proteases are specific biochemical markers of apoptosis.