RESUMEN
Malignant mesothelioma (MM) remains a highly deadly malignancy with poor treatment option. The MM cells further promote a highly inflammatory microenvironment, which contributes to tumor initiation, development, severity and propagation. We reasoned that the anti-inflammatory actions of mesenchymal stromal cells (MSCs) and further antitumor effects of MSCs engineered to overexpress tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein (MSC-TRAIL) would effectively inhibit mesothelioma growth. Using a mouse xenograft model of intraperitoneal human mesothelioma, native mouse (mMSCs) or human (hMSC) MSCs were administered either systemically (intravenously or intraperitoneally) at various times following tumor inoculation. Both mMSCs and hMSCs localized at the sites of MM tumor growth in vivo and decreased local inflammation. Further, a trend towards decrease in tumor burden was observed. Parallel studies of in vitro exposure of nine primary human mesothelioma cell lines to mMSCs or hMSCs demonstrated reduced tumor cell migration. MSC-TRAIL exposure induced apoptosis of TRAIL-sensitive MM cells in vitro, and both mouse and human MSC-TRAIL significantly reduced the inflammatory tumor environment in vivo. Moreover, human MSC-TRAIL administration significantly reduced peritoneal tumor burden in vivo and increased tumor cell apoptosis. These proof-of-concept studies suggest that TRAIL-expressing MSCs may be useful against malignant mesothelioma.
Asunto(s)
Expresión Génica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Mesotelioma/genética , Mesotelioma/terapia , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Mesotelioma/metabolismo , Mesotelioma/patología , Mesotelioma Maligno , Ratones , Ratones SCID , Carga Tumoral , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
An exposure chamber is described for the quantifiable addition of fine and ultrafine aerosol particulate matter directly to cells and used to demonstrate the in vitro cytotoxicity of fine 1,4-naphthoquinone particles to murine lung epithelial cells. The electrostatic particulate dosage and exposure system (EPDExS) operates on the principle of electrostatic precipitation and is shown to deposit fine and ultrafine aerosol particles directly to cells with 100% efficiency for particle diameters in the range of 40-530nm. This range is not limited by the EPDExS, but rather by the aerosolization method used for this study. Numbers of particles deposited onto the cells are counted with a condensation particle counter, negating any need to calculate or estimate particle exposure. The process of particle introduction, assessed using Trypan blue dye exclusion, had no effect on cell viability. In combination with a differential mobility classifier, the EPDExS can deliver select particle diameters to cells. The ability to control the diameter and number of particles deposited permits in vitro toxicity studies of particulate matter using different particle dosage metrics, i.e., particle number and size, surface area and mass. Finally, because EPDExS introduces particles directly from the aerosol, it can be used to expose cells grown at air/liquid interfaces.
Asunto(s)
Aerosoles/administración & dosificación , Material Particulado/administración & dosificación , Electricidad Estática , Aerosoles/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Precipitación Química , Células Epiteliales/efectos de los fármacos , Ratones , Naftoquinonas/administración & dosificación , Naftoquinonas/toxicidad , Tamaño de la Partícula , Material Particulado/toxicidad , Alveolos Pulmonares/citología , Alveolos Pulmonares/efectos de los fármacos , Propiedades de SuperficieRESUMEN
During extensive inflammation, neutrophils undergo secondary necrosis causing myeloperoxidase (MPO) release that may damage resident lung cells. Recent observations suggest that MPO has pro-inflammatory properties, independent of its enzymatic activity. The aims of the present study were to characterise MPO internalisation by lung epithelial cells and to investigate the effect of MPO on oxidative stress, DNA damage and cytokine production by lung epithelial cells. Human alveolar and bronchial epithelial cells were stimulated with MPO, with or without priming the cells with pro-inflammatory stimuli. MPO protein was detected in the cell cytoplasm. Expression of haemoxygenase (HO)-1 and DNA strand breakage were determined. The production of interleukin (IL)-8 and -6 were measured. Analyses of MPO-stimulated cells demonstrated MPO presence in the cells. HO-1 expression was increased after MPO stimulation and increased further when cells were primed before MPO stimulation. MPO exposure also induced DNA strand breakage. Interestingly, MPO inhibited IL-8 production in bronchial, but not alveolar epithelium. In conclusion, alveolar and bronchial epithelial cells can internalise myeloperoxidase. Stimulation with myeloperoxidase increases haemoxygenase-1 expression and DNA strand breakage, suggesting cell damaging capacity of myeloperoxidase. In addition, myeloperoxidase inhibited interleukin-8 production by bronchial epithelial cells, indicating a negative feedback loop for neutrophil recruitment.
Asunto(s)
Daño del ADN/efectos de los fármacos , Hemo-Oxigenasa 1/metabolismo , Estrés Oxidativo/efectos de los fármacos , Peroxidasa/efectos adversos , Especies Reactivas de Oxígeno/metabolismo , Análisis de Varianza , Western Blotting , Células Cultivadas , Ensayo Cometa , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Hemo-Oxigenasa 1/efectos de los fármacos , Humanos , Inmunohistoquímica , Técnicas In Vitro , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolisacáridos/farmacología , Pulmón/citología , Peroxidasa/farmacología , Reacción en Cadena de la Polimerasa , Probabilidad , Alveolos Pulmonares/citología , Sensibilidad y EspecificidadRESUMEN
Old age is associated with a higher prevalence of cardiovascular disease and diabetes mellitus. Vascular smooth muscle cells (VSMC) play a role in the pathogenesis of vascular diseases, often a complication of diabetes mellitus. We examined in explanted aortic VSMC from young vs. older rats glucose-related activation of nuclear factor kappaB (NF-kappaB), a transcription factor induced by many oxidants. Data demonstrate that old age is associated with enhanced NF-kappaB activity in unstimulated VSMC that is further increased after exposure to high glucose medium. Furthermore, VSMC from old animals exhibit increased levels of protein carbonyls, an indicator of oxidative stress, and less apoptosis in response to glucose than VSMC isolated from young animals. These changes are accompanied by increased expression of NF-kappaB-related genes, gamma-glutamylcysteine synthetase, inhibitor of apoptosis protein-1 (IAP-1), and inducible nitric oxide synthase (iNOS). Results suggest that high glucose, a putative oxidative stress, causes apoptosis in VSMC from young animals and is associated with greater induction of NF-kappaB in VSMC from older animals. Increases in IAP-1 and decreased apoptosis implicate NF-kappaB as a survival factor in VSMC.
Asunto(s)
Apoptosis , Senescencia Celular/fisiología , Glucosa/farmacología , Músculo Liso Vascular/efectos de los fármacos , Animales , Aorta/citología , Glutamato-Cisteína Ligasa/biosíntesis , Glutamato-Cisteína Ligasa/genética , Proteínas Inhibidoras de la Apoptosis , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Biosíntesis de Proteínas , Proteínas/genética , RatasRESUMEN
Cell-imaging approaches using new laser-based technologies have a wide applicability to thefields of pathology and cell biology. Here, we present the application of several of these techniques, including confocal scanning laser microscopy (CSLM), laser scanning cytometry (LSC), and laser capture microdissection (LCM), to studies of cell signaling by environmental agents in lung disease. Using both cells in culture and lung tissue, we show that these technologies are powerful tools for understanding signal transduction cascades elicited by toxic agents, such as oxidants and asbestosfibers, and their relationship to the development of cell injury and proliferation, responses leading to lung disease and/or repair.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/fisiopatología , Microscopía Confocal/métodos , Animales , Biotecnología , Comunicación Celular , Citometría de Flujo/métodos , Rayos Láser , Enfermedades Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Reacción en Cadena de la Polimerasa , Transducción de Señal , TransfecciónRESUMEN
Crystalline silica has been classified as a group 1 human carcinogen in the lung. However, its mechanisms of action on pulmonary epithelial cells which give rise to lung cancers are unclear. Using a nontransformed alveolar type II epithelial cell line (C10), we show that alpha-quartz silica causes persistent dose-related increases in phosphorylation of c-Jun-NH2-terminal amino kinases (JNKs) that are inhibited by antioxidants (P < or = 0.05). Increases in activator protein-1 (AP-1) binding to DNA and transactivation of AP-1-dependent gene expression by silica were accompanied by increases in steady-state mRNA levels of the AP-1 family members, c-jun, junB, fra-1, and c-fos at 8 h and elevated mRNA levels of fra-1 at 24 h (P < or = 0.05). Addition of tetramethylthiourea inhibited silica-associated increases infra-1 and proportions of cells in S-phase (P < or = .05). Our findings indicate that silica induces JNK activity, AP-1-dependent gene expression, ie., fra-1, and DNA synthesis via oxidative stress. Moreover, they suggest that silica may act mechanistically as a mitogen or tumor promoter, rather than a genotoxic carcinogen, in the development of lung cancers.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estrés Oxidativo/fisiología , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Alveolos Pulmonares/efectos de los fármacos , Fase S/efectos de los fármacos , Dióxido de Silicio/toxicidad , Tiourea/análogos & derivados , Animales , Línea Celular , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Depuradores de Radicales Libres/farmacología , Radical Hidroxilo/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Fosforilación/efectos de los fármacos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-fos/genética , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Dióxido de Silicio/antagonistas & inhibidores , Tiourea/farmacología , Factor de Transcripción AP-1/metabolismoRESUMEN
Macrophage inflammatory protein 2 (MIP-2) is a chemotactic cytokine which mediates neutrophil recruitment in the lung and other tissues. Pneumotoxic particles such as quartz increase MIP-2 expression in rat lung and rat alveolar type II epithelial cells. Deletion mutant analysis of the rat MIP-2 promoter demonstrated quartz-induction depended on a single NFkappaB consensus binding site. Quartz activation of NFkappaB and MIP-2 gene expression in RLE-6TN cells was inhibited by anti-oxidants suggesting the responses were dependent on oxidative stress. Consistent with anti-oxidant effects, quartz was demonstrated to increase RLE-6TN cell production of hydrogen peroxide. Rotenone treatment of RLE-6TN cells attenuated hydrogen peroxide production, NFkappaB activation and MIP-2 gene expression induced by quartz indicating that mitochondria-derived oxidants were contributing to these responses. Collectively, these findings indicate that quartz and crocidolite induction of MIP-2 gene expression in rat alveolar type II cells results from stimulation of an intracellular signaling pathway involving increased generation of hydrogen peroxide by mitochondria and subsequent activation of NFkappaB.
Asunto(s)
Expresión Génica , Peróxido de Hidrógeno/metabolismo , Mitocondrias/metabolismo , Monocinas/genética , Oxidantes/metabolismo , Animales , Antioxidantes/farmacología , Línea Celular , Quimiocina CXCL2 , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Depuradores de Radicales Libres/farmacología , Expresión Génica/efectos de los fármacos , FN-kappa B/genética , FN-kappa B/metabolismo , Alveolos Pulmonares/citología , Cuarzo/farmacología , RatasRESUMEN
High levels of ambient air pollution are associated with exacerbation of asthma and respiratory morbidity, yet little is known concerning the mechanisms of inflammation and toxicity by components of inhaled particulate matter (PM). Brief inhalation of PM(2.5) (particles of an aerodynamic diameter of < 2.5 microns) (300 microg/m(3) air for 6 h followed by a period of 24 h in clean air) by either C3H/HeJ or C57/BL6 mice caused significant (P
Asunto(s)
Contaminantes Atmosféricos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , FN-kappa B/efectos de los fármacos , Oxidantes/fisiología , Animales , Carbono/farmacología , Línea Celular , ADN/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fluoresceínas/metabolismo , Exposición por Inhalación , Interferón gamma/genética , Interleucina-6/genética , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Linfotoxina-alfa/genética , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Oxidantes/metabolismo , Tamaño de la Partícula , Unión Proteica/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Activación Transcripcional/efectos de los fármacos , Factor de Crecimiento Transformador beta/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The relationship between crystalline silica and lung cancer has been the subject of many recent publications, conferences, and regulatory considerations. An influential, international body has determined that there was sufficient evidence to conclude that quartz and cristobalite are carcinogenic in humans. The present authors believe that the results of these studies are inconsistent and, when positive, only weakly positive. Other, methodologically strong, negative studies have not been considered, and several studies viewed as providing evidence supporting the carcinogenicity of silica have significant methodological weaknesses. Silica is not directly genotoxic and is a pulmonary carcinogen only in the rat, a species that seems to be inappropriate for assessing particulate carcinogenesis in humans. Data on humans demonstrate a lack of association between lung cancer and exposure to crystalline silica. Exposure-response relationships have generally not been found. Studies in which silicotic patients were not identified from compensation registries and in which enumeration was complete did not support a causal association between silicosis and lung cancer, which further argues against the carcinogenicity of crystalline silica.
Asunto(s)
Carcinógenos/efectos adversos , Neoplasias Pulmonares/epidemiología , Cuarzo/efectos adversos , Dióxido de Silicio/efectos adversos , Silicosis/epidemiología , Animales , Autopsia , Causalidad , Humanos , Neoplasias Pulmonares/inducido químicamente , Radiografía , Ratas , Proyectos de Investigación , Silicosis/patologíaRESUMEN
Coexposures to asbestos and cigarette smoke cause increased risks of lung cancer in asbestos workers. Although these carcinogens cause DNA damage to epithelial cells in vitro via generation of reactive oxygen species (ROS), it is unclear whether they cause injury to bronchiolar epithelial cells (i.e., the target cells of lung cancers in vivo). We exposed rats to amosite asbestos, cigarette smoke, and the two agents in combination for 1, 2, and 14 d. Numbers of cells exhibiting DNA strand breaks in comparison to sham rats were then evaluated in lungs using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end labeling (TUNEL) method and by transmission electron microscopy (TEM). Increases in TUNEL-positive, necrotic epithelial cells occurred after exposure to asbestos alone and in an additive fashion after smoke and asbestos in combination. These results indicate that DNA strand breakage and necrosis are prominent mechanisms of injury by asbestos fibers and cigarette smoke in vivo to epithelial cells of the respiratory tract, thus validating in vitro observations from a number of laboratories.
Asunto(s)
Asbesto Amosita/toxicidad , Bronquios/efectos de los fármacos , Daño del ADN , Nicotiana , Plantas Tóxicas , Humo/efectos adversos , Animales , Apoptosis/efectos de los fármacos , Bronquios/citología , Cocarcinogénesis , Sinergismo Farmacológico , Células Epiteliales/química , Células Epiteliales/efectos de los fármacos , Etiquetado Corte-Fin in Situ , Masculino , Necrosis , Ratas , Ratas Sprague-Dawley , Especies Reactivas de OxígenoRESUMEN
Activation of extracellular signal-regulated kinases (ERK) has been associated with the advent of asbestos-associated apoptosis and proliferation in mesothelial and alveolar epithelial cells and may be linked to the development of pulmonary fibrosis. The objective of studies here was to characterize the development of inflammation, cellular proliferation, and fibrosis in asbestos-exposed C57Bl/6 mice in relationship to patterns of ERK phosphorylation. Inflammation occurred after 10 and 20 days of asbestos exposure as evidenced by increases in total protein and neutrophils in bronchoalveolar lavage fluid. Increases in cell proliferation were observed at 30 days in bronchiolar epithelia and at 4, 14, and 30 days in the alveolar compartment of the lung. Trichrome-positive focal lesions of pulmonary fibrosis developed at 30 days in the absence of elevations in lung hydroxyproline or procollagen mRNA levels. Striking increases in ERK phosphorylation were observed within pulmonary epithelial cells at sites of developing fibrotic lesions after 14 and 30 days of inhalation. In addition to characterizing a murine inhalation model of asbestosis, we provide the first evidence showing activation of ERK signaling within lung epithelium in vivo, following inhalation of asbestos fibers.
Asunto(s)
Asbestos Serpentinas/efectos adversos , Asbestosis/enzimología , Asbestosis/patología , Pulmón/enzimología , Pulmón/patología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Administración por Inhalación , Animales , Asbestos Serpentinas/administración & dosificación , Asbestosis/etiología , Asbestosis/metabolismo , Bromodesoxiuridina/metabolismo , División Celular , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Distribución TisularRESUMEN
For reasons that are unclear, poorly soluble particulates are associated with the development of inflammation, fibrogenesis, and carcinogenesis in the rat. The pathogenesis of these changes may be triggered by distinct or species-specific cellular responses to inhaled particulates in a manner similar to known fibrogenic and carcinogenic fibers, such as asbestos. Data reviewed here suggest that generation of oxidants by poorly soluble particulates is a key factor in the initiation of inflammation and generation of chemokines and cytokines in the rat. These substances then cause hyperplasia of epithelial cells and fibroblasts. The diminished or lack of proliferative responses by poorly soluble particulates in mice and primates, in comparison to rats, may be reflected by intrinsic differences in their oxidant-generating capacities or repair after oxidant injury or DNA damage.
Asunto(s)
Contaminantes Ocupacionales del Aire/toxicidad , Contaminantes Atmosféricos/toxicidad , Neoplasias Pulmonares/etiología , Neumoconiosis/etiología , Fibrosis Pulmonar/etiología , Animales , División Celular/efectos de los fármacos , Daño del ADN , Haplorrinos , Humanos , Hiperplasia/inducido químicamente , Hiperplasia/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Oxidantes/metabolismo , Neumoconiosis/metabolismo , Neumoconiosis/patología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Especificidad de la EspecieRESUMEN
Gene expression changes in rat asbestos-induced malignant mesothelioma (MM) cells were investigated by differential mRNA display. A mRNA transcript identified by this approach was abundant in normal rat mesothelial cells but not expressed in rat MM cell lines. Northern blot analysis confirmed that this transcript is uniformly silenced in rat MM cell lines and primary tumors. Nucleotide sequence analysis revealed that this transcript is encoded by the rat glypican 3 gene (GPC3), whose human homolog is mutated in the Simpson-Golabi-Behmel overgrowth syndrome. Allelic loss at the GPC3 locus was infrequent (6.9%) in MM cell lines, and no mutations were found. GPC3 transcript levels were markedly decreased in 16 of 18 primary tumors and 17 of 22 human MM cell lines. Most of the cell lines were shown to have aberrant methylation of the GPC3 promoter region. In two of four human MM cell lines tested, GPC3 expression was restored after 2-deoxy 5-azacytidine (DAC)-mediated demethylation of its promoter region. Ectopic expression of GPC3 inhibited in vitro colony formation of human MM cells. Collectively, these data suggest that down-regulation of GPC3 is a common occurrence in MM and that GPC3, an X-linked recessive overgrowth gene, may encode a negative regulator of mesothelial cell growth.
Asunto(s)
Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/genética , Mesotelioma/genética , Proteoglicanos/genética , Alelos , Animales , Línea Celular , Metilación de ADN , ADN Complementario/análisis , Regulación hacia Abajo , Femenino , Glipicanos , Humanos , Mesotelioma/patología , Regiones Promotoras Genéticas , Ratas , Ratas Endogámicas F344RESUMEN
Aging is characterized by an accumulation of oxidative injury to DNA, RNA, proteins, lipids, and carbohydrates. In addition to damage, oxidative stress can initiate cell signaling cascades that modulate cell function, growth, and death. Aging and two common age-related diseases, diabetes mellitus and atherosclerosis, may share common oxidant-related signaling pathways that lead to abnormal transcription factor activation and ultimately to cellular dysfunction, degeneration, or death. This review will focus on approaches to evaluate key redox-sensitive signaling pathways and the transcription factors altered by diabetes, atherosclerosis, and aging.
Asunto(s)
Envejecimiento , Transducción de Señal , Factores de Transcripción/metabolismo , Arteriosclerosis/metabolismo , Diabetes Mellitus/metabolismo , Humanos , Modelos Biológicos , Estrés Oxidativo , Oxígeno/metabolismoRESUMEN
We examined the mechanisms of interaction of crocidolite asbestos fibers with the epidermal growth factor (EGF) receptor (EGFR) and the role of the EGFR-extracellular signal-regulated kinase (ERK) signaling pathway in early-response protooncogene (c-fos/c-jun) expression and apoptosis induced by asbestos in rat pleural mesothelial (RPM) cells. Asbestos fibers, but not the nonfibrous analog riebeckite, abolished binding of EGF to the EGFR. This was not due to a direct interaction of fibers with ligand, inasmuch as binding studies using fibers and EGF in the absence of membranes showed that EGF did not adsorb to the surface of asbestos fibers. Exposure of RPM cells to asbestos caused a greater than twofold increase in steady-state message and protein levels of EGFR (P < 0.05). The tyrphostin AG-1478, which inhibits the tyrosine kinase activity of the EGFR, but not the tyrphostin A-10, which does not affect EGFR activity, significantly ameliorated asbestos-induced increases in mRNA levels of c-fos but not of c-jun. Pretreatment of RPM cells with AG-1478 significantly reduced apoptosis in cells exposed to asbestos. Our findings suggest that asbestos-induced binding to EGFR initiates signaling pathways responsible for increased expression of the protooncogene c-fos and the development of apoptosis. The ability to block asbestos-induced elevations in c-fos mRNA levels and apoptosis by small-molecule inhibitors of EGFR phosphorylation may have therapeutic implications in asbestos-related diseases.
Asunto(s)
Apoptosis/fisiología , Asbesto Crocidolita/farmacología , Receptores ErbB/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Animales , Células Cultivadas , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/metabolismo , Receptores ErbB/genética , Receptores ErbB/fisiología , Regulación de la Expresión Génica/fisiología , Homeostasis/efectos de los fármacos , Fosforilación , Pleura/citología , Pleura/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Transducción de Señal/fisiologíaRESUMEN
The transcription factor nuclear factor (NF)-kappaB is activated by oxidative stress or cytokines and is critical to the activation of inflammatory genes. Here, we report that hydrogen peroxide or 3-morpholinosydnonimine, which simultaneously releases nitric oxide and superoxide, synergize with the cytokine tumor necrosis factor (TNF)-alpha to activate NF-kappaB in rat lung epithelial cells, suggesting that signaling pathways elicited by reactive oxygen species (ROS)/reactive nitrogen species (RNS) are different from TNF-induced signaling. These findings were substantiated by observations that levels of IkappaB-alpha did not change after exposure to ROS/RNS, whereas a rapid depletion of IkappaB-alpha was observed in cells exposed to TNF. In addition, the proteosome inhibitor MG132 did not affect activation of NF-kappaB by ROS/RNS, whereas it abolished the TNF response. Transfection of a dominant negative Ras construct prevented the activation of NF-kappaB by ROS/RNS, demonstrating the requirement for Ras in the activation of NF-kappaB by oxidants. In contrast, TNF activated NF-kappaB in a Ras-independent fashion. Evaluation of members of the mitogen-activated protein kinase (MAPK) family as downstream effectors of Ras revealed the requirement of MAPK/ extracellular-regulated kinase (ERK) kinase kinase (MEKK)1 and c-Jun N-terminal kinases in the induction of NF-kappaB by both oxidants and TNF, whereas the MEK-ERK pathway negatively regulates NF-kappaB. Our findings demonstrate that cytokines and oxidants cooperate in the activation of transcription factors through distinct pathways, and suggest that anti-inflammatory and antioxidant therapies may be required in concert to prevent the activation of NF-kappaB-regulated genes important in the development of inflammatory diseases.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Peróxido de Hidrógeno/farmacología , Molsidomina/análogos & derivados , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Animales , Línea Celular Transformada , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Proteínas I-kappa B , Molsidomina/farmacología , Estrés Oxidativo , Ratas , Proteínas ras/metabolismoRESUMEN
The National Institute of Environmental Health Sciences (NIEHS) supports a number of training programs for predoctoral and postdoctoral (D.V.M., M.D., Ph.D.) fellows in toxicology, epidemiology and biostatistics, and environmental pathology. At the Experimental Biology meeting in April 1997, the American Society of Investigative Pathology (ASIP) sponsored a workshop including directors, trainees, and other interested scientists from several environmental pathology programs in medical and veterinary colleges. This workshop and a related session on "Novel Cell Imaging Techniques for Detection of Cell Injury" revealed advances in molecular and cell imaging approaches as reviewed below that have a wide applicability to toxicologic pathology.
Asunto(s)
Enfermedad/etiología , Contaminantes Ambientales/efectos adversos , Animales , Amianto/efectos adversos , Carcinógenos/efectos adversos , Humanos , Patología Clínica/educación , Patología Clínica/instrumentación , Patología Clínica/métodosRESUMEN
In addition to their capacity to damage macromolecules, oxidants play important roles in initiation of a number of signal transduction pathways. These include phosphorylation and dephosphorylation of members of the extracellular-regulated kinase (ERK) family of the mitogen-activated protein kinase (MAPK) cascade and events leading to activation of the transcription factor nuclear factor-kappaB (NF-kappaB). These cascades are key to transcriptional upregulation of genes important for cell survival, apoptosis, proliferation, transformation, and inflammation. To complement biochemical assays, cell-imaging approaches are necessary to detect the phosphorylated proteins of these cascades and their nuclear translocation, i.e., activation in cells. Protocols for these studies are presented, and the advantages of in situ microscopy-based techniques to detect oxidant-induced signaling pathways are discussed.