ABSTRACT
The epithelial to mesenchymal transition (EMT) consists of a rapid change of cell phenotype, characterized by the loss of epithelial characteristics and the acquisition of a more invasive phenotype. Transcription factors regulating EMT (Snail, Twist and Zeb) are extremely labile proteins, rapidly degraded by the proteasome system. In this review we analyze the current mechanisms controlling degradation of EMT transcription factors, focusing on the role of new E3 ubiquitin-ligases involved in EMT. We also summarize the regulation of the stability of these EMT transcription factors, specially observed in different stress conditions, such as hypoxia, chemotherapeutic drugs, oxidative stress or γ-irradiation.
Subject(s)
Epithelial-Mesenchymal Transition , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Line, Tumor , Homeodomain Proteins/metabolism , Humans , Mice , Phenotype , Protein Stability , Signal Transduction , Snail Family Transcription Factors , Stress, PhysiologicalABSTRACT
Snail1 is a transcriptional factor essential for triggering epithelial-to-mesenchymal transition. Moreover, Snail1 promotes resistance to apoptosis, an effect associated to PTEN gene repression and Akt stimulation. In this article we demonstrate that Snail1 activates Akt at an additional level, as it directly binds to and activates this protein kinase. The interaction is observed in the nucleus and increases the intrinsic Akt activity. We determined that Akt2 is the isoform interacting with Snail1, an association that requires the pleckstrin homology domain in Akt2 and the C-terminal half in Snail1. Snail1 enhances the binding of Akt2 to the E-cadherin (CDH1) promoter and Akt2 interference prevents Snail1 repression of CDH1 gene. We also show that Snail1 binding increases Akt2 intrinsic activity on histone H3 and have identified Thr45 as a residue modified on this protein. Phosphorylation of Thr45 in histone H3 is sensitive to Snail1 and Akt2 cellular levels; moreover, Snail1 upregulates the binding of phosphoThr45 histone H3 to the CDH1 promoter. These results uncover an unexpected role of Akt2 in transcriptional control and point out to phosphorylation of Thr45 in histone H3 as a new epigenetic mark related to Snail1 and Akt2 action.
Subject(s)
Cadherins/genetics , Promoter Regions, Genetic , Proto-Oncogene Proteins c-akt/metabolism , Transcription Factors/metabolism , Antigens, CD , Cadherins/metabolism , Cell Line, Tumor , Enzyme Activation , Epithelial-Mesenchymal Transition , Fibronectins/genetics , Fibronectins/metabolism , Gene Expression Regulation, Neoplastic , Histones/metabolism , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-akt/genetics , Snail Family Transcription FactorsABSTRACT
BACKGROUND: Overexpression of tissue plasminogen activator (t-PA) in pancreatic cancer cells promotes invasion and proliferation in vitro and tumour growth and angiogenesis in vivo. AIMS: To understand the mechanisms by which t-PA favours cancer progression, we analysed the surface membrane proteins responsible for binding specifically t-PA and studied the contribution of this interaction to the t-PA promoted invasion of pancreatic cancer cells. METHODS: The ability of t-PA to activate plasmin and a fluorogenic plasmin substrate was used to analyse the nature of the binding of active t-PA to cell surfaces. Specific binding was determined in two pancreatic cancer cell lines (SK-PC-1 and PANC-1), and complex formation analysed by co-immunoprecipitation experiments and co-immunolocalisation in tumours. The functional role of the interaction was studied in Matrigel invasion assays. RESULTS: t-PA bound to PANC-1 and SK-PC-1 cells in a specific and saturable manner while maintaining its activity. This binding was competitively inhibited by specific peptides interfering with the interaction of t-PA with annexin II. The t-PA/annexin II interaction on pancreatic cancer cells was also supported by co-immunoprecipitation assays using anti-t-PA antibodies and, reciprocally, with antiannexin II antibodies. In addition, confocal microscopy showed t-PA and annexin II colocalisation in tumour tissues. Finally, disruption of the t-PA/annexin II interaction by a specific hexapeptide significantly decreased the invasive capacity of SK-PC-1 cells in vitro. CONCLUSION: t-PA specifically binds to annexin II on the extracellular membrane of pancreatic cancer cells where it activates local plasmin production and tumour cell invasion. These findings may be clinically relevant for future therapeutic strategies based on specific drugs that counteract the activity of t-PA or its receptor annexin II, or their interaction at the surface level.
Subject(s)
Annexin A2/metabolism , Pancreatic Neoplasms/metabolism , Plasminogen/metabolism , Tissue Plasminogen Activator/metabolism , Binding, Competitive , Cell Membrane/metabolism , Humans , Neoplasm Invasiveness , Neoplasm Proteins/metabolism , Neoplasm Proteins/physiology , Pancreatic Neoplasms/pathology , Tissue Plasminogen Activator/physiology , Tumor Cells, CulturedABSTRACT
UEV proteins are enzymatically inactive variants of the E2 ubiquitin-conjugating enzymes that regulate noncanonical elongation of ubiquitin chains. In Saccharomyces cerevisiae, UEV is part of the RAD6-mediated error-free DNA repair pathway. In mammalian cells, UEV proteins can modulate c-FOS transcription and the G2-M transition of the cell cycle. Here we show that the UEV genes from phylogenetically distant organisms present a remarkable conservation in their exon-intron structure. We also show that the human UEV1 gene is fused with the previously unknown gene Kua. In Caenorhabditis elegans and Drosophila melanogaster, Kua and UEV are in separated loci, and are expressed as independent transcripts and proteins. In humans, Kua and UEV1 are adjacent genes, expressed either as separate transcripts encoding independent Kua and UEV1 proteins, or as a hybrid Kua-UEV transcript, encoding a two-domain protein. Kua proteins represent a novel class of conserved proteins with juxtamembrane histidine-rich motifs. Experiments with epitope-tagged proteins show that UEV1A is a nuclear protein, whereas both Kua and Kua-UEV localize to cytoplasmic structures, indicating that the Kua domain determines the cytoplasmic localization of Kua-UEV. Therefore, the addition of a Kua domain to UEV in the fused Kua-UEV protein confers new biological properties to this regulator of variant polyubiquitination.
Subject(s)
Biopolymers/metabolism , Ligases/genetics , Recombination, Genetic , Saccharomyces cerevisiae Proteins , Transcription Factors , Ubiquitins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Caenorhabditis elegans/genetics , Conserved Sequence/genetics , Gene Expression Profiling , HeLa Cells , Humans , Introns/genetics , Jurkat Cells , Ligases/isolation & purification , Mice , Molecular Sequence Data , Multigene Family/genetics , Polyubiquitin , Tumor Cells, Cultured , Ubiquitin-Conjugating EnzymesABSTRACT
Because hepatocyte growth factor (HGF) is a potent mitogen for normal human exocrine pancreas cells (NPCs) in vitro, we have analyzed the expression of HGF and its receptor, Met, in NPC and pancreas cancer cells and studied its effects in vitro. Using immunohistochemistry, Northern blotting, and reverse transcription-polymerase chain reaction, we examined the expression of HGF and Met in normal pancreas and pancreas cancer. Scatter assays, wound-healing assays, and migration through transwell filters were used to study HGF-stimulated motility of IMIM-PC-2 cancer cells. In tumors, HGF is mainly detected in stromal cells, whereas Met is overexpressed in cancer cells with an unpolarized distribution. In vitro, HGF stimulates motogenesis but not proliferation in cancer cells. Cell motility is accompanied by a rapid decrease in the cytoskeleton-bound E-cadherin, an acceleration of cellular adhesion to the substrate, an up-regulation of urokinase plasminogen activator (u-PA) RNA and protein, and a change in the solubility and proteolysis of the u-PA receptor. Cell motility is significantly reduced by inhibitors of u-PA proteolytic activity such as antibodies neutralizing u-PA activity, plasminogen activator inhibitor 1, and amiloride. These results show that a paracrine loop of HGF activation may participate in the development or progression of pancreas cancer. In vitro, the HGF-stimulated motogenesis of pancreas cancer cells involves the activation of the u-PA/u-PA receptor proteolytic system, suggesting its role in the invasive stages of tumor progression.
Subject(s)
Cadherins/metabolism , Hepatocyte Growth Factor/metabolism , Pancreatic Neoplasms/metabolism , Urokinase-Type Plasminogen Activator/physiology , Antibodies/pharmacology , Blotting, Northern , Blotting, Western , Cell Division/drug effects , Cell Movement/drug effects , Cells, Cultured , Hepatocyte Growth Factor/pharmacology , Humans , Immunoenzyme Techniques , Microscopy, Confocal , Pancreas/metabolism , Plasminogen/pharmacology , Plasminogen Activator Inhibitor 1/pharmacology , Polymerase Chain Reaction , Proto-Oncogene Proteins c-met/metabolism , Receptors, Cell Surface/metabolism , Receptors, Urokinase Plasminogen Activator , Tissue Plasminogen Activator/antagonists & inhibitors , Tissue Plasminogen Activator/metabolism , Tumor Cells, Cultured , Urokinase-Type Plasminogen Activator/antagonists & inhibitors , Urokinase-Type Plasminogen Activator/metabolismABSTRACT
Plasminogen activators (PAs) play an important role in tumor cell invasion. We have analysed the expression of tissue-type PA (t-PA), urokinase-type PA (u-PA), and their respective receptors, annexin II and u-PAR, in normal and neoplastic cultures of pancreatic cells, as well as in pancreatic tissues, and have examined their role in tumor invasiveness in vitro. Using Northern blotting, Western blotting, and ELISA, t-PA is detected in cultured pancreas cancer cells displaying a well differentiated phenotype but it is undetectable in less differentiated cells and in normal pancreatic cultures. In contrast, u-PA transcripts, protein, and enzymatic activity are detected both in cancer cells and in normal cultures. Higher levels of u-PAR and annexin II are present in cancer cells than in normal cultures and, in SK-PC-1 cells, both receptors are localized in the basolateral membrane. In vitro invasion assays indicate that both t-PA and u-PA contribute to the invasiveness of SK-PC-1 cells through reconstituted extracellular matrix. To determine the relevance of these studies to pancreas cancer, immunohistochemical assays have been used to examine the expression of t-PA, u-PA, and their receptors in normal and neoplastic tissues. t-PA is absent from normal pancreas and from tumor associated pancreatitis, whereas it is detected in the majority of pancreas cancer tissues (16/17). Annexin II is also overexpressed in some tumors (5/13). u-PAR is overexpressed in most tumor samples examined (14/15), while u-PA is weakly detected in a low number of cases (3/14); both u-PAR and u-PA are overexpressed in areas of tumor associated pancreatitis. Indirect evidences indicate that K-ras and p53 mutated proteins can regulate the expression of PAs. In pancreatic cancer we have found an association between codon 12 K-ras mutations and t-PA expression (P=0.04). These results support the contention that, in the exocrine pancreas, activation of t-PA is more specifically associated to neoplastic transformation and to the invasive phenotype, whereas the induction of u-PA/u-PAR system might be more relevant to inflammatory or non-neoplastic events.
Subject(s)
Pancreatic Neoplasms/enzymology , Pancreatic Neoplasms/pathology , Tissue Plasminogen Activator/physiology , Annexin A2/biosynthesis , Blotting, Northern , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Humans , Immunohistochemistry , Neoplasm Invasiveness , Pancreas/enzymology , Pancreatic Neoplasms/genetics , Phenotype , Receptors, Cell Surface/biosynthesis , Receptors, Urokinase Plasminogen Activator , Tissue Plasminogen Activator/biosynthesis , Tumor Cells, Cultured , Urokinase-Type Plasminogen Activator/biosynthesis , Urokinase-Type Plasminogen Activator/physiologyABSTRACT
La contribución del Centro Panamericano de Fiebre Aftosa para el conocimiento de las enfermedades vesiculares de los animales, especialmente la fiebre aftosa, ha sido de gran transcedencia para los programas de control iniciados durante las últimas cuatro décadas. En esta revisión se examina la importancia de la ingestigación en el pasado y los enfoques futuros para la investigacion y el desarrollo sobre el control y erradicación de la enfermedad de las Américas
Subject(s)
Foot-and-Mouth Disease , Health Programs and Plans , AmericasABSTRACT
Se efectuaron pruebas de pirogenicidad in vivo e in vitro en una serie de lotes de vacuna oleosa antiaftosa. Los hallazgos demostraron que normalmente estos inmunógenos no son reactivos en estas técnicas. Se sugiere emplear pruebas de pirógenos en aquellos casos en que se sospecha de problemas de contaminación bacteriana en vacunas antiaftosa, y para la caracterización de las reacciones posvacunales.
Subject(s)
Foot-and-Mouth Disease , Aphthovirus , Pyrogens , Freund's Adjuvant , Vaccines , Serial Passage , Injections, IntravenousABSTRACT
La contribución del Centro Panamericano de Fiebre Aftosa para el conocimiento de las enfermedades vesiculares de los animales, especialmente la fiebre aftosa, ha sido de gran trascendencia para los programas de control iniciados durante las últimas cuatro décadas. En esta revisión se examina la importancia de la investigación en el pasado y los enfoques futuros para la investigación y el desarrollo sobre el control y erradicación de la enfermedad de las Américas.
The contribution made by the Pan American Foot-and Mouth Disease Center to our knowledge of vesicular diseases of animals and, in particular, foot-and-mouth disease, has assisted greatly the control programs initiated during the last four decades. This review examines the relevance of past research and explores future directions for research and development in relation to the control and eradication of the disease from the Americas.
Subject(s)
Foot-and-Mouth Disease , Health Programs and Plans , Pan American Foot-and-Mouth Disease Center , Health Programs and Plans , Foot-and-Mouth DiseaseABSTRACT
Este artículo informa los resultados de un estudio para seleccionar las condiciones óptimas de trabajo para la utilización de la cromatografía en capa fina (TLC) en la determinación de los niveles de penicilina, neomicina y polimixina en vacuna oleosa antiaftosa. Se describen los procedimientos de elección para el quiebre de la emulsión y para extraer, purificar, concentrar, identificar y cuantificar los antibióticos. Se presentan los resultados obtenidos al exminar por TLC una serie de vacunas oleosas antiaftosa disponibles comercialmente. Los hallazgos se analizan en términos de la aplicación de esta metodología para el control de la calidad de estos inmunógenos y para el estudio de las reacciones posvacunación.
This paper reports the results of a study to select the optimal working conditions for rendering thin layer chromatography (TLC) useful for determining the levels of penicillin, neomycin and polymyxin in FMD oil vaccines. Procedures are described for breaking vaccine emulsions and for extracting, purifying, concentrating, identifying and quantifying the antibiotics contained in them. Subsequently, commercially avaiable FMD oil vaccines were examined by these procedures. Findings are discussed in terms of the application of TLC for quality control of immunogens and for studying post-vaccinal reactions.
Subject(s)
Foot-and-Mouth Disease , Aphthovirus , Anti-Bacterial Agents , Serial Passage , Serologic Tests , Freund's Adjuvant , Foot-and-Mouth Disease , Serial PassageABSTRACT
El análisis de la bibliografía disponible sobre el aislamiento del virus de la fiebre aftosa reveló que la inoculación de animales de laboratorio con fines diagnósticos no ha sido uniformada. Los estudios realizados sobre la efectividad de las distintas especies difieren metodológicamente entre sí, en lo que respecta a varios parámetros que incluyen la especie, edad, sexo y cepa del huésped, así como la dosis, vía de inoculación y volumen del inóculo. El empleo de virus adaptados a cultivos o a diferentes especies animales representa otra variable, si se considera que su infectividad no sería comparable a la de las cepas de campo. Por ende, se desconoce la susceptibilidad comparativa de las distintas especies de animales de laboratorio para aislar el virus de la fiebre aftosa y consecuentemente, su efectividad diagnóstica en muestras de bovinos infectados naturalmente.
Analysis of the available literature on the isolation of foot-and-mouth disease virus revealed that the inoculation of laboratory animals for diagnostic purposes has not been uniform. Studies carried out on the effectiveness of a variety of species differ from each other methodologically in several parameters which include the species, age, sex and strain of host as well as the dose, route of administration, and volume of the inoculum. The use of viruses adapted to culture or to different laboratory animal species represents another variable if it is considered that their infectivity may not be comparable to that of field strains. On this basis, the comparative susceptibility of the different species of laboratory animals to isolate Aphthovirus, and consequently, their disgnostic effectiveness in samples from bovines with natural infections, remains to be determined.
Subject(s)
Foot-and-Mouth Disease , Aphthovirus , Animals, Laboratory , Injections, Subcutaneous , Injections, Intramuscular , Injections, Intraperitoneal , Foot-and-Mouth Disease , Animals, Laboratory , Injections, Subcutaneous , Injections, Intramuscular , Injections, IntraperitonealABSTRACT
Los hallazgos del presente estudio demuestran que el hamster es más susceptible a la infección por el virus de la fiebre aftosa que el ratón lactante, habitualmente empleado para efectuar aislamientos de este agente con fines diagnósticos. El hamster mostró una susceptibilidad superior a la inoculación de Aphthovirus obtenidos de bovinos infectados naturalmente. La comparación se basó en las manifestaciones clínicas, el tiempo medio de supervivencia, el porcentaje de mortalidad, la relación entre título y mortalidad, y el comportamiento de la infección en animales destetados. Le siguieron en orden descendente de susceptibilidad los meriones, conejos y cobayos lactantes, mientras que las ratas resistieron a la infección. Los resultados se analizan en términos de su implicancia diagnóstica para estudios epidemiológicos y el control de la enfermedad.
The present findings demonstrate that the hamster is more susceptible to infection with foot-and-mouth disease virus than the sucking mouse, traditionally used for isolating this agent. Hamsters were more sensitive to the inoculation of Aphthovirus obtained from bovines with natural infections. The comparison was based on clinical manifestations, mean survival time, percent mortality, relationship between titer and mortality, and evolution of infection in weanlings. Following in decreasing order of sisceptibility were suckling gerbils, rabbits and guinea pigs, while rats were refractory. The results are discussed in terms of their diagnostic implications for epidemiologic studies and disease control.
Subject(s)
Foot-and-Mouth Disease , Aphthovirus , Animals, Laboratory , Serial Passage , Epidemiologic Studies , Foot-and-Mouth Disease , Animals, Laboratory , Serial Passage , Epidemiologic StudiesABSTRACT
Con el propósito de evaluar la efectividad del programa CADEL en mejorar el crecimiento y desarrollo de sus beneficiarios se estudian 366 preescolares desnutridos o en riesgo de desnutrición que asistían a 22 Centros ubicados en tres regiones del país. Al ingreso, 4 y 8 meses después se midió el peso y la talla en condiciones estandarizadas y se calculó la adecuación a los estándares NCHS/OMS (peso-edad, talla-edad y peso-talla). En la Región Metropolitana se evaluó además el desarrollo psicomotor por medio del test TEPSI, al ingreso y 8 meses después. El incremento promedio mensual de peso y talla fue 144 +- 83 g y 0,54 +- 0,16 cm lo que representa el 72 y 90% respectivamente del crecimiento normal, lo que fue insuficiente para corregir el estado nutricional (p N.S.). El desarrollo psicomotor en cambio se modificó positivamente (coordinación, motricidad y global p < 0,05) y no así el lenguaje. Se concluye que el programa representa una alternativa no tradicional, de bajo costo, que permite ampliar la cobertura de los servicios de atención preescolar en las familias de extrema pobreza. La incorporación más afectiva de la familia y el refuerzo de las actividades educativas y de estimulación podrían mejorar la eficiencia del programa
Subject(s)
Child, Preschool , Humans , Male , Female , Child Development , Applied Nutrition Programs/trends , Program Evaluation , PovertySubject(s)
Echinococcosis/diagnosis , Cysticercosis/diagnosis , Diagnosis, Differential , Echinococcosis/diagnostic imaging , Echinococcosis/immunology , Echinococcus/immunology , Humans , Immunologic Techniques , Liver/diagnostic imaging , Radionuclide Imaging , Tomography, X-Ray Computed , UltrasonographyABSTRACT
The antibody response to rabies virus was studied in twenty volunteers immunized with different schemes of suckling mouse brain and human diploid cell culture rabies vaccines. Throughout the study period, titers in serum neutralization and indirect fluorescent antibody tests, as well as the class of immunoglobulins with antirabies activity, varied in different individuals with the treatment scheme and the antigenic potency of the vaccine. The results suggest that measurement of the IgG class of antirabies antibodies, and possibly IgA as well, may be a more adequate criterion to assess the immunogenicity of rabies vaccines than the determination of SN titers alone.
Subject(s)
Rabies Vaccines/immunology , Rabies/immunology , Animals , Antibodies, Viral/analysis , Brain , Culture Techniques , Epitopes/immunology , Humans , Immunization Schedule , Immunoglobulins/analysis , Mice , Rabies Vaccines/therapeutic useABSTRACT
Fluid was collected from cysts of Taenia hydatigena in 60 adult sheep and fluid from each animal pooled separately. By double diffusion antigen 5 was demonstrated in all pools but one. The criteria are described for selection and standardization of these preparations for use as antigens for the immunodiagnosis of human hydatid disease. Sera from 50 persons harbouring hydatid cysts and from 50 patients with other disease conditions were examined by the arc-5 double-diffusion test, using two antigens prepared from Echinococcus granulosus and T. hydatigena cyst fluids, respectively. The results showed that a higher diagnostic sensitivity was obtained with the hydatid antigen. The significance of the findings is discussed in terms of their application to human immunodiagnosis in areas where hydatidosis, but not cysticercosis, is rare in livestock.
Subject(s)
Antigens, Helminth , Echinococcosis/diagnosis , Taenia/immunology , Animals , Antibodies/analysis , Antigen-Antibody Reactions , Antigens, Helminth/immunology , Cross Reactions , Echinococcosis/immunology , Echinococcus/immunology , Humans , Immunodiffusion , Sheep , Sheep Diseases/immunology , Taeniasis/immunology , Taeniasis/veterinaryABSTRACT
Hydatid disease was diagnosed by the arc 5 double diffusion (DD5) test in a patient with concomitant pulmonary disease. The localization of the cysts could not be determined by radiologic and scintillographic studies of the lung and abdomen. The hydatid nature of fluid collected by pleural puncture required for the concomitant infection, was established by using the aspirate as antigen in the DD5 test and five small pulmonary hydatid cysts were found at surgery.
Subject(s)
Echinococcosis, Pulmonary/diagnosis , Adult , Humans , Immunodiffusion , MaleABSTRACT
A diagnosis of hydatid disease was established by the arc 5 double diffusion (DD5) test in two persons who had no symptoms, of whom one had had prior surgery for this parasitic infection. The location of some cysts which were removed surgically from these patients could not be determined in preoperative radiological and scintillographic studies. Postoperative serological monitoring by DD5 showed that one of these patients, though without symptoms, was harbouring an additional cyst. Abdominal x-rays showed no abnormalities, a partially calcified 2.7 cm cyst was detected in the right lobe of the liver by computerized axial tomography.
