RESUMEN
Cytokines trigger the rapid assembly of multimolecular signaling complexes that direct the activation of downstream protein kinase cascades. Two protein kinases that have been linked to growth factor-regulated proliferation and survival are mitogen-activated protein/ERK kinase (MEK) and its downstream target Erk, a member of the mitogen-activated protein kinase family. Using complementary pharmacological and genetic approaches, we demonstrate that MEK and Erk activation requires a phosphatidylinositol 3-kinase (PI3-K)-generated signal in an interleukin (IL)-3-dependent myeloid progenitor cell line. Analysis of the upstream pathway leading to MEK activation revealed that inhibition of PI3-K did not block c-Raf activation, whereas MEK activation was effectively blocked under these conditions. Furthermore, agents that elevated cAMP suppressed IL-3-induced c-Raf activation but did not inhibit MEK activation. Because c-Raf activation and MEK activation were inversely affected by PI3-K- and cAMP-dependent pathways, we examined whether IL-3 activated the alternative Raf isoforms A-Raf and B-Raf. Although IL-3 did not activate B-Raf, A-Raf was activated by the cytokine. Moreover, A-Raf activation, like MEK activation, was blocked by inhibition of PI3-K but was insensitive to cAMP. Experiments with dominant negative mutants of the Raf isoforms showed that overexpression of dominant negative c-Raf did not prevent MEK activation. However, dominant negative A-Raf effectively blocked MEK activation, suggesting that activation of the MEK-Erk signaling cascade is mediated through A-Raf. Taken together, these results suggest that IL-3 receptors engage and activate both c-Raf and A-Raf in hemopoietic cells. However, these intermediates are differentially regulated by upstream signaling cascades and selectively coupled to downstream signaling pathways.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Androstadienos/farmacología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Interleucina-3/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Transducción de Señal , WortmaninaRESUMEN
PURPOSE: Ionizing radiation (IR) triggers several intracellular signaling cascades that have commonly been regarded as mitogenic, including the Raf-MEK-Erk kinase cascade. In addition to promoting proliferation, activated MEK and Erk may also prevent cell death induced by cytotoxic stimuli. Because Raf, MEK, and Erk are activated by IR in some tumor cell lines, this suggests that IR-induced activation of the kinase cascade may enhance the survival of irradiated cells. METHODS AND MATERIALS: IR-induced activation of MEK and Erk was assessed in irradiated UM-SCC-6 cells, a human squamous carcinoma cell line. Activation of MEK and Erk was blocked with the pharmacological inhibitor of MEK activation, PD098059. Clonogenic survival was assessed in irradiated UM-SCC-6 cells that were pretreated with nothing or with the MEK inhibitor. RESULTS: In UM-SCC-6 cells, IR doses as low as 2 Gy rapidly activated MEK and Erk. Pretreatment of the cells with the pharmacological inhibitor of MEK activation, PD098059, effectively blocked IR-induced activation of MEK and Erk. However, inhibition of the kinase cascade did not affect the clonogenic survival of irradiated cells in either early or delayed-plating experiments. CONCLUSION: Taken together, these results suggest that although MEK and Erk are rapidly activated by IR treatment, these protein kinases do not affect the clonogenic survival of irradiated UM-SCC6 cells.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Quinasa 1 de Quinasa de Quinasa MAP , Proteínas de Neoplasias/efectos de la radiación , Proteínas Serina-Treonina Quinasas/efectos de la radiación , Transducción de Señal/efectos de la radiación , Carcinoma de Células Escamosas/radioterapia , Supervivencia Celular/efectos de la radiación , Activación Enzimática/efectos de los fármacos , Humanos , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
Epithelial cells of the gallbladder have potential to represent an important model for studies of ductal epithelial in normal and pathological states. We therefore initiated studies to establish human gallbladder epithelial cells (GBEC) in culture. GBEC were isolated by trypsinization of small tissue fragments from human gallbladders obtained at cholecystectomy; cells were plated on tissue culture dishes and grown in defined MCDB 153 medium containing added growth factors. In this medium, GBEC showed a plating efficiency of approximately 1%; those GBEC that attached formed colonies and proliferated, as demonstrated by autoradiographic analysis of [3H]thymidine incorporation into DNA. Cultured GBEC expressed two markers found on GBEC in situ, i.e., gamma-glutamyl transpeptidase and cytokeratin 19. By using various attachment substrates, with and without added serum, increased plating efficiency and better growth were achieved. When type IV collagen was used as substrate and 10% fetal bovine serum was added to MCDB 153, passage of GBEC was possible, and cells proliferated through five to six population doublings. GBEC in culture under all conditions eventually enlarged, showed vacuolization, and demonstrated irreversible growth arrest. Nonetheless, the culture conditions described here allow for preparation of large quantities of highly enriched human GBEC.
Asunto(s)
Células Cultivadas/fisiología , Vesícula Biliar/citología , División Celular , Separación Celular/métodos , Medios de Cultivo , Medio de Cultivo Libre de Suero , Técnicas Citológicas , Células Epiteliales , HumanosRESUMEN
We have developed a novel technique for the isolation from normal rat liver of morphologically polar, intrahepatic bile duct epithelial cells which exhibit clathrin-coated pits. Using electron microscopic cytochemistry, we demonstrated receptor-mediated endocytosis of EGF by cultured IBDEC. Also, using freshly isolated polar couplets of IBDEC, we demonstrated that these cells participate in fluid-phase endocytosis. Finally, using a novel fluorescence unquenching assay and our isolated bile duct epithelial cell model, we showed that secretin stimulates exocytosis in IBDEC, a finding compatible with the possibility that secretin-induced changes in ductular bile flow may occur by an exocytic process. The availability of a reproducible and reliable technique to prepare liver cell fractions highly enriched in intrahepatic bile duct epithelial cells with morphologic polarity has made it possible to do direct experiments on the functions of intrahepatic bile duct epithelial cells, including the study of plasma membrane movement (i.e., endocytosis and exocytosis). With the availability of this technique, other studies previously impossible to carry out in IBDEC are now feasible. Such studies are too numerous to mention, but would include experiments on ligand binding, transport of macromolecules, assessment of metabolic activities and toxicity studies, to name just a few. Indeed, virtually any question that has been asked about hepatocytes and addressed using isolated hepatocytes can now be directed toward isolated intrahepatic bile duct epithelial cells. Finally, the methodology described here is theoretically applicable to human liver. Indeed, intrahepatic bile duct epithelial cells are considered to be involved in the pathogenesis of several kinds of drug and immunologically induced liver diseases, including allograft rejection, primary biliary cirrhosis, and primary sclerosing cholangitis. The availability of the technology described here should make feasible direct experimental approaches to questions in all of these areas.
Asunto(s)
Conductos Biliares Intrahepáticos/ultraestructura , Animales , Conductos Biliares Intrahepáticos/fisiología , Separación Celular , Invaginaciones Cubiertas de la Membrana Celular/fisiología , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Endocitosis/fisiología , Epitelio/fisiología , Epitelio/ultraestructura , Exocitosis/fisiología , Membranas/fisiología , Membranas/ultraestructura , Microscopía Electrónica , Movimiento/fisiología , RatasRESUMEN
A monoclonal antibody to a fungal protein has been used to demonstrate the presence of the nonhormone binding component of molybdate-stabilized steroid receptors in a variety of vertebrate tissues. We recently identified a steroid receptor in the aquatic fungus Achlya ambisexualis where sexual morphogenesis of the male is directed by the steroid antheridiol. This receptor resembles receptors of higher organisms in exhibiting an 8S, molybdate-stabilized form. In the chick oviduct, a 90 000 molecular weight protein has previously been shown to be associated with the molybdate-stabilized complex of the progesterone receptor. We have isolated a similar protein of molecular weight about 88 000 from A. ambisexualis and have obtained a hybridomal-derived monoclonal antibody directed against it. This mouse anti-Achlya immunoglobulin G1 (IgG1) cross-reacts with the 90 000 molecular weight protein in chick oviduct cytosol and was used to detect analogous 90 000 molecular weight proteins in mammalian tissues. Tissue cytosols were incubated with antibody, and the complexes were isolated onto protein A-Sepharose. The resin-bound proteins were then analyzed by gel electrophoresis. This procedure revealed the presence of 90 000 molecular weight proteins in several mammalian tissues including rat liver, mouse liver and uterus, pig ovarian granulosa cells, human endometrium, and HeLa cells. These results demonstrate that the 90 000 molecular weight protein is not peculiar to the chick oviduct but is present in several different tissues from a variety of animals. This antibody should be a useful probe for further studies on the biological role of these proteins.
Asunto(s)
Oviductos/metabolismo , Receptores de Esteroides/metabolismo , Animales , Anticuerpos Monoclonales , Pollos , Cricetinae , Citosol/metabolismo , Femenino , Proteínas Fúngicas , Hongos , Células de la Granulosa/metabolismo , Células HeLa/metabolismo , Humanos , Inmunoensayo , Hígado/metabolismo , Ratones , Peso Molecular , Ratas , Receptores de Progesterona/metabolismo , Receptores de Esteroides/aislamiento & purificación , Especificidad de la Especie , Porcinos , Distribución Tisular , Útero/metabolismoRESUMEN
Previous studies have shown that the molybdate-stabilized progesterone receptor from the chick oviduct contains a nonhormone binding component with a molecular weight of 90 000. This protein has also been shown to be associated with some other molybdate-stabilized steroid receptors of the oviduct. In order to access this larger pool of the receptor binding protein, we have developed an isolation procedure based on the observation that the protein is selectively shed from proteins adsorbed to heparin-agarose when molybdate is removed. The protein obtained by this procedure is shown to be the same as that isolated from affinity-purified progesterone receptor as compared by protease digestion and one-dimensional peptide mapping. Four immunoglobulin G secreting hybridoma cell lines were generated against the 90 000-dalton antigen. All of the antibodies recognize the 90 000-dalton protein obtained by electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. In addition, two of the antibodies complex the molybdate-stabilized progesterone receptor as demonstrated by sedimentation analysis on sucrose gradients. One of these antibodies was used to show the presence of the 90 000-dalton component in molybdate-stabilized glucocorticoid and androgen receptors and also to show its presence in brain, liver, and skeletal muscle, but not in serum.