RESUMEN
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
Epidermal growth factor (EGF) is a potent mitogen for human mesothelial cells, and autophosphorylation of the EGF receptor (EGF-R) occurs in these cell types after exposure to asbestos, a carcinogen associated with the development of mesothelioma. Here, the intensity and distribution of EGF-R protein was documented by immunocytochemistry in a human mesothelial cell line (MET5A) exposed to various concentrations of crocidolite asbestos and man-made vitreous fibers (MMVF-10). Whereas cells in contact with or phagocytizing shorter asbestos fibers (<60 microm length) or MMVF-10 at a range of concentrations showed no increase in EGF-R protein as determined by immunofluorescence, elongated cells phagocytizing and surrounding longer fibers (> or =60 microm) showed intense staining for EGF-R. In contrast, human A549 lung carcinoma cells showed neither elongation nor increased accumulation of EGF-R protein in response to long fibers. Patterns of aggregation and increases in EGF-R protein in mesothelial cells phagocytizing long asbestos fibers were distinct from diffuse staining of phosphotyrosine residues observed in asbestos-exposed cultures. These studies indicate that aggregation of EGF-R by long fibers may initiate cell signaling cascades important in asbestos-induced mitogenesis and carcinogenesis.
Asunto(s)
Amianto/farmacología , Carcinógenos/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Carcinoma/metabolismo , Carcinoma/patología , Línea Celular Transformada , Técnica del Anticuerpo Fluorescente , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Fosfotirosina/metabolismo , Distribución Tisular , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
We developed in situ dual-fluorescence detection techniques for measuring apoptosis and proliferation simultaneously in single dishes of cells. The deoxyribonucleic acid (DNA)-specific labeling method, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), first was used in conjunction with a 4',6-diamidino-2-phenylindole (DAPI) counterstain to detect and measure morphologic characteristics of apoptotic rat pleural mesothelial (RPM) cells isolated from Fischer 344 rats and exposed to 300 microM hydrogen peroxide (H2O2). For this purpose, 100 TUNEL-positive nuclei were measured while being viewed with DAPI counterstaining for area, perimeter, longest diameter, and average diameter, using imaging software and an image-collection apparatus. We then exposed cells to a range of concentrations of crocidolite asbestos and putative apoptotic and mitogenic agents. Exposure to crocidolite asbestos (5 microg/cm2) caused a striking dose-dependent apoptotic response at 24 h, 48 h, and 72 h. The nonfibrous crocidolite analogue riebeckite failed to induce apoptosis. At 24 h, tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml) caused an increase in apoptotic nuclei. A second method, utilizing an antibody to 5'-bromodeoxyridine (BrdU) and oxazole yellow homodimer (YOYO), showed a dose-dependent increase in proliferation occurring in cells exposed to asbestos (5 microg/cm2) at 48 h and 72 h. In addition, increased numbers of rat pleural mesothelial (RPM) cells exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA), TNF-alpha, and epidermal growth factor (EGF) exhibited incorporation of BrdU at these time points, although total numbers of cells per unit area were unchanged. Results indicate a dynamic balance between apoptosis and increased DNA synthesis after exposure of mesothelial cells to asbestos.
Asunto(s)
Apoptosis/efectos de los fármacos , Asbesto Crocidolita/farmacología , Carcinógenos/farmacología , Citometría de Imagen/métodos , Pleura/citología , Animales , Benzoxazoles , Biotina , Bromodesoxiuridina , División Celular/fisiología , Fragmentación del ADN , Nucleótidos de Desoxiuracil , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales , Colorantes Fluorescentes , Procesamiento de Imagen Asistido por Computador/métodos , Indoles , Microscopía Fluorescente/métodos , Mitógenos/farmacología , Compuestos de Quinolinio , Ratas , Ratas Endogámicas F344 , Coloración y Etiquetado , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Asbestos fibers are human carcinogens with undefined mechanisms of action. In studies here, we examined signal transduction events induced by asbestos in target cells of mesothelioma and potential cell surface origins for these cascades. Asbestos fibers, but not their nonfibrous analogues, induced protracted phosphorylation of the mitogen-activated protein (MAP) kinases and extracellular signal-regulated kinases (ERK) 1 and 2, and increased kinase activity of ERK2. ERK1 and ERK2 phosphorylation and activity were initiated by addition of exogenous epidermal growth factor (EGF) and transforming growth factor-alpha, but not by isoforms of platelet-derived growth factor or insulin-like growth factor-1 in mesothelial cells. MAP kinase activation by asbestos was attenuated by suramin, which inhibits growth factor receptor interactions, or tyrphostin AG 1478, a specific inhibitor of EGF receptor tyrosine kinase activity (IC50 = 3 nM). Moreover, asbestos caused autophosphorylation of the EGF receptor, an event triggering the ERK cascade. These studies are the first to establish that a MAP kinase signal transduction pathway is initiated after phosphorylation of a peptide growth factor receptor following exposure to asbestos fibers.
Asunto(s)
Asbesto Crocidolita/farmacología , Amianto/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Receptores ErbB/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos , Pleura/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Activación Enzimática/efectos de los fármacos , Receptores ErbB/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Pleura/metabolismo , Procesamiento Proteico-Postraduccional , Ratas , Ratas Endogámicas F344RESUMEN
Peroxidized low-density lipoprotein (p-LDL) has been previously demonstrated to be preferentially cytotoxic to certain malignant cells compared to normal cells of the same type. We present evidence that p-LDL is at least partially taken up through the LDL receptor and that it becomes localized in lysosomes. The integrity of lysosomes of p-LDL-treated cells is compromised, and leakage of their contents into the cytosol occurs. This leakage occurs early and precedes mitochondrial dysfunction. Brefeldin A inhibits this leakage, perhaps by interfering with the traffic between endosomes and lysosomes. Electron micrographs taken at various times suggest a mechanism of cell death which resembles certain aspects of the broad definition of apoptosis. However, we suggest that the cell death observed following p-LDL-induced release of lysosomal contents is essentially unique, with released lysosomal enzymes degrading the cell from within. We suggest that this process should be described as endopepsis.