RESUMO
BACKGROUND: Degranulation of mast cells is stimulated by store-operated Ca(2+) -entry (SOCE). In other cell types, Ca(2+) -entry is modified by ceramide. Exogenously added ceramide has been shown to trigger mast cell apoptosis. Effects of endogenously produced ceramide in mast cells remained, however, elusive. Ceramide may be produced from sphingomyelin by acid sphingomyelinase (Asm). OBJECTIVE: This study explored the impact of Asm on mast cell functions. METHODS: Mast cells were isolated from bone marrow (BMMCs) or peritoneal lavage of gene-targeted mice lacking Asm (asm(-/-)) and their wild-type littermates (asm(+/+)). BMMC maturation and apoptosis-associated annexin V binding were determined by flow cytometry. Asm activity was assessed enzymatically, cytosolic Ca(2+) activity ([Ca(2+)]i) utilizing Fura-2 fluorescence, current across the cell membrane by whole-cell patch clamp, degranulation from hexosaminidase-release and migration utilizing a transwell chamber. In vivo anaphylaxis was derived from decrease in body temperature. RESULTS: Peritoneal mast cell number, BMMC phenotype, spontaneous BMMC apoptosis as well as BMMC CD117, CD34 and FcεRI expression were similar in both genotypes. In asm(+/+) BMMCs, stimulation with antigen resulted in a fast ~2.5-fold increase in Asm activity. Release of Ca(2+) from internal stores and hence several Ca(2+) -dependent functions were strongly impaired in asm(-/-) BMMCs. Thus, antigen-induced increase in [Ca(2+)]i in IgE-sensitized cells, antigen- but not ionomycin-induced currents through Ca(2+) -activated K(+) -channels (KCa 3.1), IgE/antigen-triggered ß-hexosaminidase release, and antigen-induced migration were all lower in asm(-/-) BMMCs than in asm(+/+) BMMCs. Pharmacological inhibition of Asm by amitriptyline (500 nm, 3 h) in asm(+/+) BMMCs similarly decreased antigen-induced increase in [Ca(2+)]i , KCa 3.1 currents, ß-hexosaminidase release and migration. The decrease in body temperature upon the induction of systemic anaphylaxis was significantly less pronounced in asm(-/-) mice than in asm(+/+) mice, an observation pointing to in vivo significance of Asm. CONCLUSIONS AND CLINICAL RELEVANCE: Asm is a novel, powerful regulator of mast cell function and thus a potential target in the treatment of allergic reactions.
Assuntos
Mastócitos/imunologia , Mastócitos/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Anafilaxia/genética , Anafilaxia/imunologia , Anafilaxia/metabolismo , Animais , Antígenos/imunologia , Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/genética , Degranulação Celular/imunologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Movimento Celular/imunologia , Ativação Enzimática/efeitos dos fármacos , Imunofenotipagem , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Mastócitos/ultraestrutura , Camundongos , Camundongos Knockout , Fenótipo , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/genéticaRESUMO
Over the past decade a number of murine models of Graves' disease (GD) have been described. The full symptom complex, including typical orbital changes, however, could not yet be induced. In this report, we examined the influence of modified immunization protocols on orbital pathology. C57BL/6 and BALB/c mice were immunized against the human TSH receptor (TSHR), using either a TSHR encoding plasmid or a TSHR A-subunit adenovirus. Prior to immunization with the TSHR plasmid, regulatory T cells were depleted in one group of each strain. TSHR-stimulating antibodies (TSAbs) were evaluated and orbits were stained immunohistochemically for F4/80, uncoupling protein-1 (UCP-1) and the TSHR. We found that after depletion of regulatory T cells, incidence of TSAb was increased in TSHR plasmid immunized C57BL/6 mice. Examination of early immunized mice showed no antibody production. However, a TSHR epitope-specific cellular immune response could be detected by tetramer-analyses. Adenoviral immunization lead to TSAb production in all but one animal. Analysis of F4/80 positive cells in retrobulbar fat revealed no significant macrophage infiltration in the orbits of immunized mice. Immunohistochemical staining shows co-localization of F4/80 positive cells, UCP-1 and the TSHR in retrobulbar fat. Though targets for TSHR autoimmunity could clearly be shown, immunization methods were not efficient enough to cause clear signs of orbital inflammation.
Assuntos
Tecido Adiposo/metabolismo , Doença de Graves/genética , Canais Iônicos/genética , Proteínas Mitocondriais/genética , Órbita/metabolismo , Receptores da Tireotropina/genética , Animais , Modelos Animais de Doenças , Feminino , Doença de Graves/metabolismo , Humanos , Canais Iônicos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Receptores da Tireotropina/metabolismo , Proteína Desacopladora 1RESUMO
Despite the high prevalence and devastating impact of psychiatric disorders, little is known about their etiopathology. In this review, we provide an overview on the participation of sphingolipids and enzymes responsible for their metabolism in mechanisms underlying psychiatric disorders. We focus on the pathway from sphingomyelin to proapoptotic ceramide and the subsequent metabolism of ceramide to sphingosine, which is in turn phosphorylated to yield anti-apoptotic sphingosine-1-phosphate (S1P).The sphingomyelinase/ceramide system has been linked to effects of reactive oxygen species and proinflammatory cytokines in the central nervous system as well as to synaptic transmission. Compared to ubiquitously expressed acid sphingomyelinase, acid and neutral ceramidase and neutral sphingomyelinase are highly active in brain regions. Depressed patients show elevated plasma ceramide levels and increased activities of acid sphingomyelinase which is functionally inhibited by many anti-depressive drugs. Exposure to alcohol is associated with an activation of acid and neutral sphingomyelinase observed in cell culture, mouse models and in alcohol-dependent patients and with increased concentrations of ceramide in various organs.Levels of sphingomyelin and ceramide are altered in erythrocytes and post-mortem brain tissues of schizophrenic patients in addition to changes in expression patterns for serine palmitoyltransferase and acid ceramidase leading to impaired myelination. After induction of anxiety-like behavior in animal models, higher serum levels of S1P were reported to lead to neurodegeneration. Correspondingly, S1P infusion appeared to increase anxiety-like behavior. Significantly upregulated levels of the endogenous ceramide catabolite N,N-dimethylsphingosine were observed in rat models of allodynia. Conversely, rats injected intrathecally with N,N-dimethylsphingosine developed mechanical allodynia. Moreover, S1P has been implicated in spinal nociceptive processing.The increasing interest in lipidomics and improved analytical methods led to growing insight into the connection between psychiatric and neurological disorders and sphingolipid metabolism and may once provide new targets and strategies for therapeutic intervention.
Assuntos
Encéfalo/metabolismo , Transtornos Mentais/metabolismo , Dor/metabolismo , Transdução de Sinais , Esfingolipídeos/metabolismo , Animais , Ceramidas/metabolismo , Humanos , Transtornos Mentais/terapia , Dor/prevenção & controle , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , SíndromeRESUMO
AIMS: To examine the association between benzodiazepine receptor agonist (BZRA) use and mortality in patients hospitalised for coronavirus disease 2019 (COVID-19). METHODS: A multicentre observational study was performed at Greater Paris University hospitals. The sample involved 14 381 patients hospitalised for COVID-19. A total of 686 (4.8%) inpatients received a BZRA at hospital admission at a mean daily diazepam-equivalent dose of 19.7 mg (standard deviation (s.d.) = 25.4). The study baseline was the date of admission, and the primary endpoint was death. We compared this endpoint between patients who received BZRAs and those who did not in time-to-event analyses adjusted for sociodemographic characteristics, medical comorbidities and other medications. The primary analysis was a Cox regression model with inverse probability weighting (IPW). RESULTS: Over a mean follow-up of 14.5 days (s.d. = 18.1), the primary endpoint occurred in 186 patients (27.1%) who received BZRAs and in 1134 patients (8.3%) who did not. There was a significant association between BZRA use and increased mortality both in the crude analysis (hazard ratio (HR) = 3.20; 95% confidence interval (CI) = 2.74-3.74; p < 0.01) and in the IPW analysis (HR = 1.61; 95% CI = 1.31-1.98, p < 0.01), with a significant dose-dependent relationship (HR = 1.55; 95% CI = 1.08-2.22; p = 0.02). This association remained significant in sensitivity analyses. Exploratory analyses indicate that most BZRAs may be associated with an increased mortality among patients hospitalised for COVID-19, except for diazepam, which may be associated with a reduced mortality compared with any other BZRA treatment. CONCLUSIONS: BZRA use may be associated with an increased mortality among patients hospitalised for COVID-19, suggesting the potential benefit of decreasing dose or tapering off gradually these medications when possible.
Assuntos
COVID-19 , Antagonistas de Receptores de GABA-A/efeitos adversos , COVID-19/mortalidade , Hospitalização , Humanos , Modelos de Riscos ProporcionaisRESUMO
Pseudomonas aeruginosa infection is a serious complication in patients with cystic fibrosis and in immunocompromised individuals. Here we show that P. aeruginosa infection triggers activation of the acid sphingomyelinase and the release of ceramide in sphingolipid-rich rafts. Ceramide reorganizes these rafts into larger signaling platforms that are required to internalize P. aeruginosa, induce apoptosis and regulate the cytokine response in infected cells. Failure to generate ceramide-enriched membrane platforms in infected cells results in an unabated inflammatory response, massive release of interleukin (IL)-1 and septic death of mice. Our findings show that ceramide-enriched membrane platforms are central to the host defense against this potentially lethal pathogen.
Assuntos
Ceramidas/metabolismo , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/fisiologia , Esfingomielina Fosfodiesterase/metabolismo , beta-Ciclodextrinas , Animais , Apoptose/fisiologia , Transplante de Medula Óssea , Células Cultivadas , Ciclodextrinas/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Ativação Enzimática , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Filipina/farmacologia , Corantes Fluorescentes/metabolismo , Humanos , Ionóforos/farmacologia , Microdomínios da Membrana/química , Camundongos , Nistatina/farmacologia , Infecções por Pseudomonas/imunologia , Transdução de Sinais/fisiologia , Esfingomielina Fosfodiesterase/genética , Receptor fas/metabolismoRESUMO
Osmotic cell swelling activates Cl- channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (ICl-swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of ICl-swell is defective in p56lck-deficient cells. Retransfection of p56lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of ICl-swell. Addition of purified p56lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56lck washed into the cytoplasm activates ICl-swell in native and p56lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56lck, slow single-channel gating events can be observed revealing a unitary conductance of 25-28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56lck. We conclude that osmotic swelling activates ICl-swell in lymphocytes via the tyrosine kinase p56lck.
Assuntos
Canais de Cloreto/fisiologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Linfócitos T/fisiologia , Benzoquinonas , Linhagem Celular , Canais de Cloreto/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Soluções Hipotônicas , Células Jurkat , Lactamas Macrocíclicas , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/biossíntese , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/deficiência , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Fenóis/farmacologia , Quinonas/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Rifabutina/análogos & derivados , Linfócitos T/citologia , TransfecçãoRESUMO
The hematopoietically expressed product of the vav proto-oncogene, Vav, shared homology with guanine nucleotide releasing factors (GRFs) [also called guanosine diphosphate-dissociation stimulators (GDSs)] that activate Ras-related small guanosine triphosphate (GTP)-binding proteins. Human T cell lysates or Vav immunoprecipitates possessed GRF activity that increased after T cell antigen receptor (TCR)-CD3 triggering; an in vitro-translated Vav fragment that contained the putative GRF domain was also active. Vav-associated GRF stimulation after TCR-CD3 ligation paralleled its tyrosine phosphorylation; both were blocked by a protein tyrosine kinase (PTK) inhibitor. Vav also was a substrate for the p56lck PTK. Thus, Vav is a PTK-regulated GRF that may be important in TCR-CD3-initiated signal transduction through the activation of Ras.
Assuntos
Proteínas de Ciclo Celular , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/imunologia , Benzoquinonas , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Lactamas Macrocíclicas , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Muromonab-CD3/farmacologia , Fosforilação , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-vav , Quinonas/farmacologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Rifabutina/análogos & derivados , Transdução de Sinais , Linfócitos T/metabolismo , Células Tumorais Cultivadas , Proteínas rap de Ligação ao GTPRESUMO
Pseudomonas aeruginosa causes severe infections, particularly of the lung, that are life threatening. Here, we show that P. aeruginosa infection induces apoptosis of lung epithelial cells by activation of the endogenous CD95/CD95 ligand system. Deficiency of CD95 or CD95 ligand on epithelial cells prevented apoptosis of lung epithelial cells in vivo as well as in vitro. The importance of CD95/CD95 ligand-mediated lung epithelial cell apoptosis was demonstrated by the rapid development of sepsis in CD95- or CD95 ligand-deficient mice, but not in normal mice, after P. aeruginosa infection.
Assuntos
Apoptose , Células Epiteliais/imunologia , Pneumopatias/imunologia , Pulmão/imunologia , Glicoproteínas de Membrana/metabolismo , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/patogenicidade , Receptor fas/metabolismo , Animais , Transplante de Medula Óssea , Linhagem Celular , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Proteína Ligante Fas , Humanos , Marcação In Situ das Extremidades Cortadas , Pulmão/microbiologia , Pulmão/patologia , Pneumopatias/microbiologia , Pneumopatias/patologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/imunologia , Sepse/microbiologia , Baço/microbiologia , Receptor fas/genéticaRESUMO
We have previously shown that activation of the acid sphingomyelinase (ASM), the release of ceramide and the formation of ceramide-enriched membrane domains are central for the induction of apoptosis by CD95. Here, we demonstrate that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95 activate the ASM via a redox mechanism resulting in release of ceramide and formation of ceramide-enriched membrane platforms. Ceramide-enriched membrane platforms serve to cluster DR5 upon stimulation. Antioxidants prevent TRAIL-mediated stimulation of ASM, the release of ceramide, the formation of ceramide-enriched membrane platforms and the induction of apoptosis by TRAIL. Further, ASM-deficient splenocytes fail to cluster DR5 in ceramide-enriched membrane domains upon TRAIL stimulation and resist TRAIL-induced apoptosis, events that were restored by addition of natural C(16)-ceramide. A dose-response analysis indicates that ceramide-enriched membrane platforms greatly sensitized tumor cells to TRAIL-induced apoptosis. Our data indicate that ceramide-enriched membrane platforms are required for the signaling of TRAIL-DR5 complexes under physiological conditions.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose/fisiologia , Ceramidas/metabolismo , Glicoproteínas de Membrana/fisiologia , Esfingomielina Fosfodiesterase/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Animais , Linhagem Celular , Ativação Enzimática , Humanos , Imuno-Histoquímica , Camundongos , Oxirredução , Ligante Indutor de Apoptose Relacionado a TNFRESUMO
We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.
Assuntos
Proteínas de Ciclo Celular , Diglicerídeos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Naftalenos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Alcaloides/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Benzoquinonas , Linhagem Celular , Ceramidas/farmacologia , Chlorocebus aethiops , Primers do DNA , Guanosina Difosfato/metabolismo , Humanos , Cinética , Lactamas Macrocíclicas , Dados de Sequência Molecular , Muromonab-CD3/farmacologia , Mutagênese Sítio-Dirigida , Dibutirato de 12,13-Forbol/metabolismo , Mutação Puntual , Compostos Policíclicos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-vav , Quinonas/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Rifabutina/análogos & derivados , Estaurosporina , Linfócitos T/efeitos dos fármacos , Transfecção , Células Tumorais CultivadasRESUMO
We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) for Ras. Vav is enzymatically activated by lymphocyte antigen receptor-coupled protein tyrosine kinases or independently by diglycerides. To further evaluate the physiological role of Vav, we assessed its GDP-GTP exchange activity against several Ras-related proteins in vitro and determined whether Vav activation in transfected NIH 3T3 fibroblasts correlates with the activity status of Ras and mitogen-activated protein (MAP) kinases. In vitro translated purified Vav activated by phorbol myristate acetate (PMA) or phosphorylation with recombinant p56lck displayed GEF activity against Ras but not against recombinant RacI, RacII, Ral, or RhoA proteins. Expression of vav or proto-vav in stably transfected NIH 3T3 cells led to a approximately 10-fold increase in basal or PMA-stimulated Ras exchange activity, respectively, in total-cell lysates and Vav immunoprecipitates. Elevated GEF activity was paralleled in each case by a significant increase in the proportion of active, GTP-bound Ras. PMA had a minimal effect on the low Ras. GTP level in untransfected control fibroblasts but increased it from 20 to 37% in proto-vav-transfected cells. vav-transfected cells displayed a constitutively elevated Ras. GTP level (35%), which was not increased further by PMA treatment. MAP kinases, known downstream intermediates in Ras-dependent signaling pathways, similarly exhibited increased basal or PMA-stimulated activity in Vav-expressing cells by comparison with normal NIH 3T3 cells. These results demonstrate a physiologic interaction between Vav and its target, Ras, leading to MAP kinase activation.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação ao GTP/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Células 3T3 , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Células Clonais , Fibroblastos/metabolismo , Proteínas de Ligação ao GTP/biossíntese , Guanosina Difosfato/metabolismo , Humanos , Cinética , Camundongos , Oncogenes , Biossíntese de Proteínas , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas c-vav , Proto-Oncogenes , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
To convert the concept already successful in mice into clinical practice and commercialize it, a human anti-CD95-antibody must be produced. In a second step experiments must be performed on various normal healthy cells and tissues to determine whether these human anti-CD95-antibodies administered in very low doses have any effect on human cells (particularly hepatocytes) or at least cause only minimal side effects. If these studies yield positive results, then clinical trials can be conducted in which increasing doses are given to exclude an acute hepatotoxic effect and then the effect exerted by the antibody in combination with irradiation on tumor growth can be investigated.The advantage of this concept lies in the fact that systemic stimulus (low doses of anti-CD95-antibodies) is highly intensified by local radiotherapy and only then initiates cell death. Since the anti-CD95-antibodies trigger apoptosis primarily in tumor endothelia, this approach could be employed not only for prostate cancer and melanomas, which have already been tested, but also for many other tumors.
Assuntos
Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Ceramidas/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos da radiação , Receptor fas/imunologia , Animais , Anticorpos/toxicidade , Linhagem Celular Tumoral , Terapia Combinada , Relação Dose-Resposta a Droga , Endotélio/efeitos dos fármacos , Endotélio/patologia , Endotélio/efeitos da radiação , Humanos , Masculino , Melanoma Experimental , Camundongos , Neoplasias da Próstata/patologia , Esfingomielina Fosfodiesterase/metabolismoRESUMO
Coexpression of the proto-oncogenes c-myc and bcl-2 under the control of the immunoglobulin enhancer E mu provokes the rapid development of primitive lymphoid tumors in transgenic mice. In the present study we show that the myc family members N-myc and L-myc also cooperate with bcl-2 in oncogenesis and can provoke the development of more mature pre-B, B and T cell type lymphomas. The analysis of prelymphomatous B-cells from single E mu N-myc and bcl-2-Ig transgenic animals and from young, tumor free, double transgenic E mu N-myc/bcl-2-Ig mice revealed that E mu directed expression of N-myc leads to very rapid apoptosis after explantation and culturing compared to B-cells from normal mice. As expected, B-cells from bcl-2-Ig transgenics were protected to a certain degree from apoptosis. Strikingly however, B-cells from E mu N-myc/bcl-2-Ig double transgenic animals were found to be almost completely resistant towards a number of different apoptotic stimuli. Furthermore, after treatment with H2O2, which can trigger apoptosis, B-cells from E mu N-myc animals reach levels of intracellular free Ca2+ concentrations that are comparable to B-cells from normal mice, whereas B-cells from bcl-2-Ig or E mu N-myc/bcl-2-Ig double transgenic mice show no increase in intracellular Ca2+ concentrations after stimulation with H2O2. These findings suggest that the prevention of apoptosis conferred by bcl-2 correlates with the inhibition of intracellular Ca2+ fluxes whereas induction of apoptosis mediated by N-myc requires normal Ca2+ levels. We hypothesize therefore that the regulation of intracellular Ca2+ concentrations represent one important parameter in the oncogenic cooperation between bcl-2 and N-myc.
Assuntos
Apoptose/fisiologia , Linfócitos B/metabolismo , Cálcio/metabolismo , Genes myc/fisiologia , Linfoma de Células B/genética , Lesões Pré-Cancerosas/genética , Proteínas Proto-Oncogênicas/genética , Animais , Linfócitos B/citologia , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Feminino , Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Líquido Intracelular/metabolismo , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas c-bcl-2RESUMO
Death ligands such as CD95 ligand (CD95L) or tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) induce apoptosis in radiochemotherapy-resistant human malignant glioma cell lines. The death-signaling TRAIL receptors 2 (TRAIL-R2/death receptor (DR) 5) and TRAIL-R1/DR4 were expressed more abundantly than the non-death-inducing (decoy) receptors TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in 12 human glioma cell lines. Four of the 12 cell lines were TRAIL/Apo2L-sensitive in the absence of a protein synthesis inhibitor, cycloheximide (CHX). Three of the 12 cell lines were still TRAIL/Apo2L-resistant in the presence of CHX. TRAIL-R2 expression predicted sensitivity to apoptosis. Coexposure to TRAIL/Apo2L and cytotoxic drugs such as topotecan, lomustine (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, CCNU) or temozolomide resulted in synergistic killing. Synergistic killing was more often observed in cell lines retaining wild-type p53 activity (U87MG, LN-229) than in p53 mutant cell lines (LN-18, T98G, U373MG). Drug exposure resulted in enhanced TRAIL-R2 expression, but decreased TRAIL-R4 expression in U87MG cells. Ectopic expression of dominant-negative p53(V135A) abrogated the drug-induced changes in TRAIL-R2 and TRAIL-R4 expression, but had no effect on synergy. Thus, neither wild-type p53 function nor changes in TRAIL receptor expression were required for synergy. In contrast, synergy resulted possibly from drug-induced cytochrome c release from mitochondria, serving as an amplifier of the TRAIL/Apo2L-mediated cascade of caspase activation. These data provide novel insights into the role of the TRAIL/Apo2L system in malignant gliomas and illustrate that TRAIL/Apo2L-based immunochemotherapy may be an effective therapeutic strategy for these lethal neoplasms.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Apoptose/fisiologia , Neoplasias Encefálicas/patologia , Grupo dos Citocromos c/metabolismo , Glioma/patologia , Lomustina/farmacologia , Glicoproteínas de Membrana/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Proteínas Reguladoras de Apoptose , Western Blotting , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/metabolismo , Caspase 8 , Caspase 9 , Caspases/metabolismo , Citometria de Fluxo , Glioma/enzimologia , Glioma/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais CultivadasRESUMO
Induction of apoptosis is a hallmark of cytostatic drug and radiation-induced cell death in human lymphocytes and lymphoma cells. However, the mechanisms leading to apoptosis are not well understood. We provide evidence that ionizing radiation induces a rapid activation of caspase-8 (FLICE) followed by apoptosis independently of CD95 ligand/receptor interaction. The radiation induced cleavage pattern of procaspase-8 into mature caspase-8 resembled that following CD95 crosslinking and resulted in cleavage of the proapoptotic substrate BID. Overexpression of dominant-negative caspase-8 interfered with radiation-induced apoptosis. Caspase-8 activation by ionizing radiation was not observed in cells genetically defective for the Src-like tyrosine kinase Lck. Cells lacking Lck also displayed a marked resistance towards apoptosis induction upon ionizing radiation. After retransfection of Lck, caspase-8 activation and the capability to undergo apoptosis in response to ionizing radiation was restored. We conclude that radiation activates caspase-8 via an Lck-controlled pathway independently of CD95 ligand expression. This is a novel signaling event required for radiation induced apoptosis in T lymphoma cells.
Assuntos
Apoptose/efeitos da radiação , Caspases/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Transdução de Sinais/efeitos da radiação , Receptor fas/metabolismo , Anticorpos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteínas de Transporte/metabolismo , Caspase 8 , Caspase 9 , Caspases/genética , Ativação Enzimática/efeitos da radiação , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Proteína Ligante Fas , Genes Dominantes/genética , Humanos , Células Jurkat , Cinética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutação , Processamento de Proteína Pós-Traducional/efeitos da radiação , RNA Mensageiro/análise , RNA Mensageiro/genética , Radiação Ionizante , Solubilidade , TransfecçãoRESUMO
A variety of pathological conditions lead to swelling of astrocytes, which in turn stimulates ion release by activation of ion channels at the plasma membrane. In the present study, acridine orange and fluorescein isothiocyanate coupled to dextran (FITC-dextran) have been used to examine the effect of cell swelling on pH in acidic compartments of cultured astroglial cells. Both NH4Cl (2 mM) and chloroquine (10 microM), known to alkalinize acidic cellular compartments, led to the expected increase in acridine orange fluorescence intensity. Similar, albeit smaller, effects were elicited by a reduction of extracellular osmolarity (-80 mOsm) and treatment of the cells with glutamate (l mM), manoeuvres which enhanced cell volume. Determination of changes in the FITC-dextran fluorescence ratio (485/440 nm) allowed quantification of the pH changes in lysosomal compartments. Treatment with NH4Cl, reduced extracellular osmolarity and glutamate increased lysosomal pH by 0.65 +/- 0.07, 0.85 +/- 0.14 and 0.25 +/- 0.07, respectively. Measurement of cytosolic pH using 2',7',-bis-(2-carboxyethyl)-5- (and -6) carboxyfluorescein (BCECF) demonstrated a pronounced acidification following cell swelling, observed with both reduced extracellular osmolarity (by 0.23 +/- 0.05 pH units) and 1 mM glutamate (by 0.26 +/- 0.02 pH units). In conclusion, pH within lysosomes and possibly other acidic cellular compartments of astrocytes is increased by cell swelling, which may have important consequences for astrocyte function.
Assuntos
Astrócitos/metabolismo , Tamanho Celular , Líquido Intracelular/metabolismo , Laranja de Acridina/metabolismo , Cloreto de Amônio/farmacologia , Animais , Cloroquina/farmacologia , Dextranos/metabolismo , Espaço Extracelular/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Fluorometria , Ácido Glutâmico/farmacologia , Concentração de Íons de Hidrogênio , Soluções Hipotônicas/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Concentração Osmolar , RatosRESUMO
Most human malignant glioma cell lines are susceptible to CD95 ligand (CD95L)-induced apoptosis. Here, we report that glioma cells are also susceptible to the cytotoxic effects of exogenous C2-ceramide. This form of cell death exhibits some morphological features of apoptosis as assessed by electron microscopy, but is unaffected by the broad spectrum caspase inhibitor, zVAD-fmk. Further, CD95L-induced apoptosis is synergistically enhanced by coexposure of the glioma cells to CD95L and C2-ceramide. CD95L-induced caspase 3-like activity, cytochrome c release and cleavage of caspases 3, 8, 9 and poly(ADP-ribose)polymerase (PARP) increase substantially after cotreatment with CD95L and C2-ceramide compared with CD95L treatment alone. None of these events occur in response to cytotoxic concentrations of C2-ceramide alone. C2-ceramide does not alter CD95 expression. Gene transfer-mediated enhancement of CD95 expression results not only in increased susceptibility to CD95L, but also in increased sensitivity to C2-ceramide. We conclude that (i) synergistic induction of apoptosis by C2-ceramide and CD95L depend on a cross-talk between the two signal transduction pathways and that (ii) C2-ceramide, independently of its sensitizing effects on CD95-dependent caspase activation, is also capable of triggering an apoptotic signaling cascade that is unaffected by zVAD-fmk-mediated caspase inhibition, but promoted by high levels of CD95 expression.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Neuroglia/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Esfingosina/farmacologia , Receptor fas/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Inibidores de Caspase , Grupo dos Citocromos c/metabolismo , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Proteína Ligante Fas , Humanos , Immunoblotting , Glicoproteínas de Membrana/metabolismo , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neuroglia/patologia , Neuroglia/ultraestrutura , Poli(ADP-Ribose) Polimerases/metabolismo , Esfingosina/análogos & derivados , Células Tumorais CultivadasRESUMO
Susceptibility to CD95 (Fas/APO-1)-mediated apoptosis in human glioma cells depends on CD95 expression and unknown factors that regulate signal transduction. Thus, LN-18 cells are highly sensitive to CD95 ligand (CD95L) whereas LN-229 cells require coexposure to inhibitors of RNA or protein synthesis for induction of apoptosis. Here, we report that caspase 8 and 3 activation, poly(ADP-ribose)polymerase cleavage and apoptosis are inhibited by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA), or ectopic expression of crm-A or bcl-2. CD95L-induced glioma cell apoptosis does not involve ceramide generation. Apoptosis induced by exogenous ceramide resembles CD95-mediated apoptosis in that bcl-2 is protective but differs in that NDGA and crm-A have no effect and in that cycloheximide (CHX) inhibits rather than potentiates ceramide-induced cell death. We conclude that caspase 8 and caspase 3 activation, but not ceramide generation, are required for CD95 ligand-induced apoptosis of glioma cells and that bcl-2, crm-A and NDGA all act upstream of caspases to inhibit apoptosis.
Assuntos
Apoptose , Caspases/metabolismo , Glioma/enzimologia , Glioma/patologia , Masoprocol/farmacologia , Glicoproteínas de Membrana/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Serpinas/metabolismo , Proteínas Virais , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Caspase 8 , Caspase 9 , Sobrevivência Celular , Ceramidas/metabolismo , Cicloeximida/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática , Proteína Ligante Fas , Humanos , Camundongos , Serpinas/genética , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas , Receptor fas/fisiologiaRESUMO
Fas/CD95/Apo-I has been shown to stimulate a variety of molecules including several members of the caspase family and the acidic sphingomyelinase (Martin and Green 1995; Gulbins et al, 1995). Here, we demonstrate that Fas receptor-triggered activation of the acidic sphingomyelinase, consumption of sphingomyelin, release of ceramide, and subsequent activation of JNK and p38-K are regulated by caspases. Inhibition of caspases by Ac-YVAD-chloromethylketone or transient CrmA transfection prevented stimulation of acidic sphingomyelinase, release of ceramide and activation of JNK and p38-K upon Fas-receptor crosslinking. Likewise, Fas triggered apoptosis was almost completely blocked by Ac-YVAD-chloromethylketone or CrmA mediated inhibition of caspases. The results suggest a new signalling cascade from the Fas receptor via caspases to acidic sphingomyelinase, ceramide and JNK/p38-K.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Células Jurkat/citologia , Proteínas Quinases Ativadas por Mitógeno , Esfingomielina Fosfodiesterase/metabolismo , Proteínas Virais , Receptor fas/metabolismo , Ácidos/metabolismo , Inibidores da Captação Adrenérgica/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ceramidas/metabolismo , Inibidores de Cisteína Proteinase/genética , Inibidores de Cisteína Proteinase/farmacologia , Diglicerídeos/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Imipramina/farmacologia , Células Jurkat/enzimologia , Serpinas/genética , Transdução de Sinais/fisiologia , Linfócitos T/citologia , Linfócitos T/enzimologia , Transfecção , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
To rapidly respond to invading microorganisms, humans call on their innate immune system. This occurs by microbe-detecting receptors, such as CD14, that activate immune cells to eliminate the pathogens. Here, we link the lipopolysaccharide receptor CD14 with Alzheimer's disease, a severe neurodegenerative disease resulting in dementia. We demonstrate that this key innate immunity receptor interacts with fibrils of Alzheimer amyloid peptide. Neutralization with antibodies against CD14 and genetic deficiency for this receptor significantly reduced amyloid peptide induced microglial activation and microglial toxicity. The observation of strongly enhanced microglial expression of the LPS receptor in brains of animal models of Alzheimer's disease indicates a clinical relevance of these findings. These data suggest that CD14 may significantly contribute to the overall neuroinflammatory response to amyloid peptide, highlighting the possibility that the enormous progress currently being made in the field of innate immunity could be extended to research on Alzheimer's disease.