Regulation of the protein stability of EMT transcription factors.

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.

Akt2 interacts with Snail1 in the E-cadherin promoter.

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.

Specific interaction of tissue-type plasminogen activator (t-PA) with annexin II on the membrane of pancreatic cancer cells activates plasminogen and promotes invasion in vitro.

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.

Fusion of the human gene for the polyubiquitination coeffector UEV1 with Kua, a newly identified gene.

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.

Activation of the urokinase plasminogen activator/urokinase plasminogen activator receptor system and redistribution of E-cadherin are associated with hepatocyte growth factor-induced motility of pancreas tumor cells overexpressing Met.

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.

The plasminogen activator system in pancreas cancer: role of t-PA in the invasive potential in vitro.

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.

La importancia de PANAFTOSA en los programasa de investigación y de desarrollo para el control y erradicación de la fiebre aftosa en las Américas / The relevance of the research and development programs of PANAFTOSA to the control and eradication of foot and mouth disease in the Americas

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

Evaluación de un programa de alimentación y estimulación para preescolares de sectores de extrema pobreza / Evaluation of \"feeding and language stimulation centers\" for urban poor preschool children

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

Serologic and immune responses to rabies virus during different human treatments with tissue culture and suckling mouse brain vaccines.

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.

Immunodiagnosis of pulmonary hydatid disease in a patient with negative radiologic and scintillographic findings.

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.

Application of antigens from Taenia hydatigena cyst fluid for the immunodiagnosis of human hydatidosis.

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.

Immunodiagnosis of abdominal hydatid disease. Difficulties in the radiological and scintillographic localization of cysts.

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.

Limitaciones de la tomografia axial computada en la localizacion de quistes hidatidicos abdominales en pacientes con confirmacion inmunodiagnostica. / Limitations of computerized axial tomography in the localization of abdominal hydatid cysts in patients with immunodiagnostic confirmation

Se describe el aporte de la prueba de doble disfusion arco 5 (DD5) al diagnostico de la hidatidosis en dos pacientes con operaciones previas. En una de ellas, asintomatica, la reaccion positiva a los cinco anos de su ultima intervencion quirurgica por hidatidosis, permitio diagnosticar la presencia de quistes cuya localizacion abdominal fue establecida por la tomografia axial computada (TAC). En el otro caso, sintomatico, aunque operado de hidatidosis nueve anos antes, la positividad a la DD5 confirmo la etiologia de la enfermedad que se habia sospechado por centellografia, TAC y su historia clinica. En ambos casos, sin embargo, se hallaron en el acto quirurgico otros quistes abdominales cuyas imagenes no habian sido reveladas en los estudios a los que se las habia sometido

Immunodiagnosis of human hydatid disease: applications and contributions to a control program in Argentina.

The sequential development of the hydatid immunodiagnostic activities of the control program of the Province of Neuquén, Argentina is described. Test results were used to obtain immunological confirmation of clinical cases and to detect asymptomatic cyst carriers amongst residents of rural endemic areas. The information was also valuable for improving the accuracy of prevalence estimates of human hydatidosis and the quality of surveillance data in different areas of the Province, characterized by varying degrees of environmental contamination by Echinococcus granulosus. In population groups examined by radiologic and immunologic methods, the latter detected more cases. When only immunodiagnostic surveys were carried out, mostly liver but also pulmonary hydatidosis cases were detected. This experience illustrates the advantages which may be obtained in endemic areas through the local application of hydatid immunodiagnosis based on arc 5 positivity.

Significance of hydatid immunodiagnostic surveys to health care and estimation of prevalence in the Argentine Province of Chubut.

More hydatidosis cases were detected in an immunodiagnostic survey of rural schoolchildren from an endemic area in Argentina by the arc 5 double diffusion (DD5) test than by indirect hemagglutination (IHA) and latex agglutination (LA) tests. Evidence of infection was obtained by clinical, radiologic, echographic, and/or computerized axial tomography examinations in only one of three DD5-negative individuals, and in 2 of 4 students showing a questionable reaction of identity with arc 5 in DD5, who were positive in LA and/or IHA. In contrast, cysts could be demonstrated in all DD5-positive cases, whether positive or negative in the agglutination procedures. These cysts in DD5-positive cases included the smallest cysts (1-1.2 cm wide) detected in surveys to date. Comparison of data obtained in the immunodiagnostic survey and in a review of hospital records was used to assess the relative contribution of each method in estimating the prevalence of infection. The findings are discussed in terms of the advantages and limitations of carrying out immunodiagnostic surveys of residents of endemic areas for purposes of primary medical care and surveillance.