RESUMEN
We evaluated the process of inferential revision during text comprehension in adults. Participants with high or low working memory read short texts, in which the introduction supported two plausible concepts (e.g., 'guitar/violin'), although one was more probable ('guitar'). There were three possible continuations: a neutral sentence, which did not refer back to either concept; a no-revise sentence, which referred to a general property consistent with either concept (e.g., ' beautiful curved body'); and a revise sentence, which referred to a property that was consistent with only the less likely concept (e.g., ' matching bow'). Readers took longer to read the sentence in the revise condition, indicating that they were able to evaluate their comprehension and detect a mismatch. In a final sentence, a target noun referred to the alternative concept supported in the revise condition (e.g., 'violin'). ERPs indicated that both working memory groups were able to evaluate their comprehension of the text (P3a), but only high working memory readers were able to revise their initial incorrect interpretation (P3b) and integrate the new information (N400) when reading the revise sentence. Low working memory readers had difficulties inhibiting the no-longer-relevant interpretation and thus failed to revise their situation model, and they experienced problems integrating semantically related information into an accurate memory representation.
Asunto(s)
Comprensión/fisiología , Potenciales Evocados/fisiología , Memoria a Corto Plazo/fisiología , Lectura , Pensamiento/fisiología , Adolescente , Adulto , Electroencefalografía , Humanos , Adulto JovenRESUMEN
The von Hippel-Lindau (VHL) tumor suppressor gene regulates extracellular matrix deposition. In VHL negative renal cancer cells, VHL(-), the lack of fibronectin matrix assembly is thought to promote and maintain tumor angiogenesis allowing vessels to infiltrate tumors. Therefore, and considering the importance of this process in tumor growth, we aimed to study why VHL(-) renal cancer cells fail to form a proper extracellular matrix. Our results showed that VHL(-) cells were not defective in fibronectin production and that the fibronectin produced by these cells was equally functional in promoting cell adhesion and matrix assembly as that produced by VHL+ cells. We have previously reported that VHL(-) cells fail to form beta1 integrin fibrillar adhesions and have a diminished organization of actin stress fibers; therefore, we aimed to study if the small GTPase family is involved in this process. We found that activation of the RhoA GTPase was defective in VHL(-) cells, and this was possibly mediated by an increased activation of its inhibitor, p190RhoGAP. Additionally, the expression of constitutively active RhoA in VHL(-) cells resulted in formation of a fibronectin matrix. These results strongly suggest an important role for RhoA in some of the defects observed in renal cancer cells.
Asunto(s)
Matriz Extracelular/metabolismo , Fibronectinas/biosíntesis , Neoplasias Renales/enzimología , Neovascularización Patológica/enzimología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau , Proteína de Unión al GTP rhoA/metabolismo , Adhesión Celular/genética , Línea Celular Tumoral , Activación Enzimática/genética , Matriz Extracelular/genética , Matriz Extracelular/patología , Fibronectinas/genética , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/patología , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Fibras de Estrés/genética , Fibras de Estrés/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/genéticaRESUMEN
The defense mechanisms of endothelial cells (EC) against reactive oxygen species (ROS) are insufficiently characterized. We have addressed the hypothesis that vascular endothelial growth factor (VEGF) and its receptors are relevant elements in this response. Cell viability, VEGF and VEGF receptor (VEGFR1 and VEGFR2) expression, and transcription factor activation were studied on transient exposure of monolayer EC to H2O2. Wild-type and mutant inhibitors of kappaBalpha (IkappaBalpha) constructions were used to further assess the role of NF-kappaB in the induction of VEGFR2 expression. A concentration of H2O2>or=60 microM elicited clear-cut damaging effects on EC, whereas lower concentrations (2-4 microM) were cytoprotective. The cytoprotective effect was shifted to an EC-damaging pattern by means of specific VEGF blockade, therefore revealing a major role of autologous VEGF. Exposure to H2O2 increased VEGF and VEGFR2 mRNA and protein in EC, without affecting VEGFR1 expression. Also, H2O2 challenge was accompanied by increased NF-kappaB, activator protein-1, and specific protein-1 nuclear binding. A role of NF-kappaB as the mediator of the H2O2 induction of VEGFR2 mRNA expression was supported by inhibition by the ROS scavenger pyrrolidine dithiocarbamate and by the blocking effect of transfected IkappaBalpha. Exposure to exogenous VEGF also increased VEGFR2 and induced NF-kappaB in EC. In summary, autologous VEGF is instrumental for EC protection induced by low concentrations of ROS. ROS induce expression not only of VEGF but also of VEGFR2. VEGFR2 increase by ROS is mainly driven through a NF-kappaB-dependent pathway.
Asunto(s)
Endotelio Vascular/fisiología , Peróxido de Hidrógeno/farmacología , Especies Reactivas de Oxígeno/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Antioxidantes/farmacología , Bovinos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteínas I-kappa B/farmacología , Inhibidor NF-kappaB alfa , FN-kappa B/fisiología , Estrés Oxidativo/fisiología , Pirrolidinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tiocarbamatos/farmacología , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Mutations in von Hippel-Lindau tumor suppressor gene (VHL) underlie the VHL hereditary cancer syndrome and also occur in most sporadic clear cell renal cell cancers (CCRCC). Currently, the mechanism(s) by which VHL loss of function promotes tumor development in the kidney are not fully elucidated. Here, we show that VHL inactivation in precancerous lesions in kidneys from patients with VHL disease correlates with marked down-regulation of the intercellular adhesion molecule E-cadherin. Moreover, in VHL-defective cell lines (RCC4 and RCC10) derived from sporadic CCRCC, reexpression of VHL was found to restore E-cadherin expression. The product of the VHL gene has multiple reported functions, the best characterized of which is its role as the recognition component of an ubiquitin E3 ligase complex responsible for mediating oxygen-dependent destruction of hypoxia-inducible factor-alpha (HIF-alpha) subunits. We show that HIF activation is necessary and sufficient to suppress E-cadherin in renal cancer cells. Given the fundamental role of E-cadherin in controlling epithelial behavior, our findings give insight into how VHL inactivation/HIF activation may lead to kidney cancer and also indicate a mechanism by which reduced oxygenation could alter E-cadherin expression in other cancers and influence normal homeostasis in other epithelia.
Asunto(s)
Cadherinas/biosíntesis , Carcinoma de Células Renales/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Renales/metabolismo , Lesiones Precancerosas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Enfermedad de von Hippel-Lindau/metabolismo , Adulto , Cadherinas/genética , Carcinoma de Células Renales/genética , Transformación Celular Neoplásica/genética , Regulación hacia Abajo , Femenino , Humanos , Neoplasias Renales/genética , Túbulos Renales/metabolismo , Túbulos Renales/patología , Masculino , Persona de Mediana Edad , Lesiones Precancerosas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/biosíntesis , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Enfermedad de von Hippel-Lindau/genéticaRESUMEN
Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is responsible for the development of renal cell cancers (RCC), pheochromocytomas, and tumors in other organs. The best known function of VHL protein (VHL) is to target the hypoxia-inducible factor (HIF) for proteasome degradation. VHL is also required for the establishment of an epithelial-like cell shape in otherwise fibroblastic-like RCC cell lines. However, the underlying mechanisms and whether this is linked to HIF remain undetermined. Because the breakage of intercellular junctions induces a fibroblastic-like phenotype in multiple cancer cell models, we hypothesized that VHL may be required for the assembly of intercellular junctions in RCC cells. Our experiments showed that VHL in RCC cell lines is necessary for the normal organization of adherens and tight intercellular junctions, the maintenance of cell polarity, and control of paracellular permeability. Additionally, 786-O cells reconstituted with wild-type VHL and with a constitutively active form of HIF-2alpha did not reproduce any of the phenotypic alterations of VHL-negative cells. In summary, we show that VHL inactivation in RCC cells disrupts intercellular junctions and cell shape through HIF-independent events, supporting the concept that VHL has additional functions beside its role in the regulation of HIF.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Carcinoma de Células Renales/patología , Uniones Intercelulares/patología , Neoplasias Renales/patología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/fisiología , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Línea Celular Tumoral , Fibronectinas/antagonistas & inhibidores , Fibronectinas/metabolismo , Humanos , Uniones Intercelulares/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Transfección , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/biosíntesis , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
Hypoxia-inducible factors (HIF) are heterodimeric (alpha/beta) transcription factors that play a fundamental role in cellular adaptation to low oxygen tension. In the presence of oxygen, the HIF-alpha subunit becomes hydroxylated at specific prolyl residues by prolyl hydroxylases. This post-translational modification is recognized by the von Hippel-Lindau (VHL) protein, which targets HIF-alpha for degradation. In the absence of oxygen, HIF-alpha hydroxylation is compromised and this subunit is stabilized. We have previously shown that the hypoxia-induced accumulation of HIF-alpha protein is strongly impaired by the inhibitor of diacylglycerol kinase, R59949. Here, we have investigated the mechanisms through which this inhibitor exerts its effect. We found that R59949 inhibits the accumulation of HIF-1/2alpha protein without affecting the expression of their mRNAs. We also determined that R59949 could only block the accumulation of HIF-alpha in the presence of VHL protein. In agreement with this, the binding of VHL to endogenous HIF-alpha was significantly enhanced after R59949 treatment, even under hypoxic conditions. In addition, we found that R59949 could stimulate prolyl hydroxylase both at 21% O2 as well as at 1% O2. Taken together, these results reveal that R59949 is an activator of HIF prolyl hydroxylases. This is of particular interest when we consider that, to date, mainly inhibitors of these enzymes have been described.
Asunto(s)
Diacilglicerol Quinasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Piperidinas/farmacología , Procolágeno-Prolina Dioxigenasa/metabolismo , Quinazolinas/farmacología , Línea Celular , Activación Enzimática , Humanos , Inmunoprecipitación , Procolágeno-Prolina Dioxigenasa/genética , Quinazolinonas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene are responsible for a hereditary cancer syndrome characterized by high susceptibility to hemangioblastomas of the retina and central nervous system, pheochromocytomas, and renal cell carcinomas. In agreement with its role as a tumor suppressor, the vast majority of spontaneous clear cell carcinomas of the kidney present loss of heterozygosity at the VHL locus. Recently, it has been shown that VHL works as the substrate recognition component of an E3 ubiquitination complex that targets the hypoxia-inducible factor (HIF) for proteosomal degradation. Under normal oxygen tension, the half-life of HIF transcription factors is extremely short because of its high degradation rate by the proteasome, resulting in undetectable HIF activity in normal cells. However, in VHL-deficient tumor cells, the HIF transcriptional pathway is constitutively activated because of impaired ubiquitination of this transcription factor. To target VHL-deficient tumors, we have exploited this feature to develop a conditionally replicative adenovirus (Ad9xHRE1A), the replication of which is HIF dependent. In this new oncolytic adenovirus, the expression of the E1A gene is controlled by an optimized minimal promoter containing HIF recognition elements. Here, we show that the induction of the E1A gene, as well as the viral replication and cytolytic effect of Ad9xHRE1A, are dependent on HIF activity. As a consequence, this virus efficiently kills VHL-deficient cells both in vitro and in vivo, as well as cells growing under hypoxic conditions. These data suggest that Ad9xHRE1A could be used as a highly specific therapy for VHL-deficient cancers and probably many other tumors that show extensive hypoxic areas or increased HIF activity by genetic alterations other than VHL loss.
Asunto(s)
Adenovirus Humanos/fisiología , Carcinoma de Células Renales/terapia , Carcinoma de Células Renales/virología , Neoplasias Renales/terapia , Neoplasias Renales/virología , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Ubiquitina-Proteína Ligasas/deficiencia , Proteínas E1A de Adenovirus/biosíntesis , Proteínas E1A de Adenovirus/genética , Adenovirus Humanos/genética , Secuencia de Bases , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Replicación Viral , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
Most of the genes induced by hypoxia are regulated by a family of transcription factors termed hypoxia-inducible factors (HIF). Under normoxic conditions, HIFalpha proteins are very unstable due to hydroxylation by a recently described family of proline hydroxylases termed EGL-Nine homologs (EGLN). Upon hydroxylation, HIFalpha is recognized by the product of the tumor suppressor vhl and targeted for proteosomal degradation. Since EGLNs require oxygen to catalyze HIF hydroxylation, this reaction does not efficiently occur under low oxygen tension. Thus, under hypoxia, HIFalpha escapes from degradation and transcribes target genes. The mRNA levels of two of the three EGLNs described to date are induced by hypoxia, suggesting that they might be novel HIF target genes; however, no proof for this hypothesis has been reported. Here we show that the induction of EGLN1 and -3 by hypoxia is found in a wide range of cell types. The basal levels of EGLN3 are always well below those of EGLN1 and EGLN2, and its induction by hypoxia is larger than that found for EGLN1. The inhibitor of transcription, actinomycin D, prevents the increase of EGLN3 mRNA induced by hypoxia, indicating that it is due to enhanced gene expression. Interestingly, EGLN1 and EGLN3 mRNAs were also triggered by EGLN inhibitors, suggesting the involvement of HIFalpha in the control of its transcription. In agreement with this possibility, pVHL-deficient cell lines, which present high HIF activity under normoxia, also showed dramatically increased normoxic levels of EGLN3. Moreover, the overexpression of an oxygen-insensitive mutant form of HIFalpha resulted in increased normoxic levels of EGLN3 mRNA. Finally, hypoxic induction of EGLNs was not observed in cells lacking functional HIFalpha.