RESUMEN
Multiple levels of representation are involved in reading single words: visual representations of letter shape, orthographic representations of letter identity and order, phonological representations of the word's pronunciation, and semantic representations of its meaning. Previous lesion and neuroimaging studies have identified a network of regions recruited during word reading, including ventral occipital-temporal regions and the angular gyrus (AG). However, there is still debate about what information is being represented and processed in these regions. This study has two aims. The first is to help adjudicate between competing hypotheses concerning the role of ventral occipital cortex in reading. The second is to adjudicate between competing hypotheses concerning the role of the AG in reading. Participants read words in the scanner while performing a proper name detection task and we use a multivariate pattern analysis technique for analyzing fMRI data - representational similarity analysis (RSA) - to decode the type of information being represented in these regions based on computationally explicit theories. Distributed patterns of activation in the left ventral occipitotemporal cortex (vOT) and the AG show evidence of some type of orthographic processing, while the right hemisphere homologues of the vOT supports visual, but not orthographic, information processing of letter strings. In addition, there is evidence of left-lateralized semantic processing in the lvOT and evidence of top-down feedback in the lvOT. Taken together, these results suggest an interactive activation theory of visual word processing in which both the lvOT and lAG are neural loci of an orthographic level of representations.
Asunto(s)
Mapeo Encefálico , Reconocimiento Visual de Modelos/fisiología , Lectura , Lóbulo Temporal/fisiología , Adolescente , Adulto , Femenino , Lateralidad Funcional/fisiología , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Masculino , Lóbulo Occipital/fisiología , Estimulación Luminosa/métodos , Adulto JovenRESUMEN
Prostate specific antigen, the clinical marker for prostate cancer, is a neutral serine protease whose function is to lyse seminal proteins. Recent work by our laboratory has suggested that prostate specific antigen stimulates the generation of reactive oxygen species in prostate cancer cells. Using 2',7'-dichlorofluorescin diacetate, a dye that fluoresces in the presence of hydrogen peroxide or hydroxyl radicals, we found that prostate specific antigen markedly stimulated reactive oxygen species generation in LNCaP cells. The effect was concentration dependent and its specificity was supported by the fact that anti-prostate specific antigen antibodies abolished the response. Since testosterone stimulates the production of prostate specific antigen, we considered that the reactive oxygen species response to testosterone may be linked to prostate specific antigen. We found that the testosterone effect on reactive oxygen species was blocked by flutamide and by anti-prostate specific antigen antibody. Additionally, though PC3 and DU145 could not respond to testosterone, they readily increased reactive oxygen species in response to prostate specific antigen. Focusing on the mechanism of the prostate specific antigen effect, we tested two other serine proteases, trypsin and chymotrypsin, but found no effect on reactive oxygen species in LNCaP cells. Nevertheless, serine protease inhibitors, alpha(1)-antichymotrypsin, alpha(2)-macroglobulin and Bowman-Birk inhibitor, blocked reactive oxygen species generation stimulated by prostate specific antigen. This apparent paradox was investigated with the use of a specific anti-'prostate specific antigen' antibody which recognizes an epitope away from the catalytic site and which does not inhibit protease activity. Despite the lack of inhibition of proteolytic activity, this antibody blocked the effect of prostate specific antigen on reactive oxygen species generation. These findings suggest that although the integrity of the prostate specific antigen molecule is necessary for stimulating reactive oxygen species generation, its proteolytic activity is not. The underlying mechanism is currently under investigation.
Asunto(s)
Antígeno Prostático Específico/farmacología , Neoplasias de la Próstata/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Testosterona/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Western Blotting , Flutamida/farmacología , Humanos , Immunoblotting , Masculino , Oxidación-Reducción , Péptido Hidrolasas/metabolismo , Receptores Androgénicos/metabolismo , Testosterona/antagonistas & inhibidores , Células Tumorales Cultivadas/metabolismoRESUMEN
The p53-dependent initiation of apoptosis is accompanied by the induction of proline oxidase (POX), a mitochondrial enzyme catalyzing the conversion of proline to pyrroline-5-carboxylate with the concomitant transfer of electrons to cytochrome c. However, the contribution of increased POX activity to apoptosis, if any, remains unknown. Using Adriamycin to initiate p53-dependent apoptosis, we showed that the expression of POX is up-regulated in a time- and dose-dependent manner in a human colon cancer cell line (LoVo). In cells expressing POX, the addition of proline increases reactive oxygen species (ROS) generation in a concentration-dependent manner; glutamate, a downstream product of proline oxidation, had no effect. Induction of POX was dependent on the p53 status of the cell. In the conditionally immortalized murine colonic epithelial cell line YAMC, where the p53 phenotype can be modulated by temperature, proline oxidase expression and ROS production could only be induced when the cells were phenotypically p53-positive. To confirm that the observed ROS production was not secondary to some other effect of p53, we also conditionally expressed POX in a p53-negative colon cancer line. Again, we found a proline-dependent ROS increase with POX expression. We hypothesize that proline oxidation supports the generation of ROS by donating reducing potential to an electron transport chain altered either by p53-dependent mechanisms or by overexpression of POX.
Asunto(s)
Neoplasias del Colon/enzimología , Regulación Neoplásica de la Expresión Génica/fisiología , Prolina Oxidasa/metabolismo , Prolina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Catálisis , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Doxorrubicina/farmacología , Inducción Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Prolina/farmacología , Prolina Oxidasa/biosíntesis , Prolina Oxidasa/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Increased cytoplasmic beta-catenin levels and the associated nuclear beta-catenin/T-cell factor (Tcf)-lymphoid enhancer factor (LEF) complex formation have been frequently found in colon cancer. In this context, overproduction of nitric oxide (NO) attributable to inflammatory stimuli in diseases such as ulcerative colitis and Crohn's disease may-contribute to colonic carcinogenesis. Therefore, we examined the modulation by NO of cytoplasmic beta-catenin levels and the formation of the nuclear beta-catenin/LEF-1 DNA binding complex in conditionally immortalized mouse colonic epithelial cells that differed in adenomatous polyposis coli (Apc) genotype, namely young adult mouse colon (YAMC; Apc+/+) and immortal mouse colon epithelium (IMCE; ApcMin/+). Unlike most colon cancer cell lines, this pair of cell lines has either nondetectable or low basal level of beta-catenin when they are cultured under nonpermissive and nonproliferative conditions. Using electrophoretic mobility shift assays, we found that NO-releasing agents (E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide and S-nitroso-N-acetylpenicillamine greatly enhanced the formation of beta-catenin/LEF-1 DNA binding complex in a concentration- and time-dependent fashion in YAMC and IMCE cells. Significantly, IMCE cells showed a markedly greater amount of nuclear beta-catenin/LEF-1 DNA binding complex in response to NO. Super shift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Western blot analysis of the soluble cytoplasmic fractions demonstrated that these NO donors caused differential accumulation of cytoplasmic beta-catenin in YAMC and IMCE. In conclusion, this study indicates that the defective beta-catenin degradation machinery attributable to ApcMin/+ mutation in IMCE cells not only affects basal levels but also contributes to NO-induced dysregulation of cytoplasmic beta-catenin and nuclear beta-catenin/LEF-1 DNA binding complex formation.
Asunto(s)
Colon/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Genes APC , Mucosa Intestinal/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico/fisiología , Nitrocompuestos/farmacología , Penicilamina/análogos & derivados , Transactivadores , Factores de Transcripción/metabolismo , Animales , Línea Celular Transformada , Núcleo Celular/metabolismo , Colon/efectos de los fármacos , Proteínas del Citoesqueleto/aislamiento & purificación , Proteínas de Unión al ADN/aislamiento & purificación , Genotipo , Mucosa Intestinal/efectos de los fármacos , Factor de Unión 1 al Potenciador Linfoide , Ratones , Penicilamina/farmacología , S-Nitroso-N-Acetilpenicilamina , Factores de Transcripción/aislamiento & purificación , beta CateninaRESUMEN
Increased expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) has been implicated in pathological conditions such as inflammatory bowel diseases and colon cancer. Recently, it has been demonstrated that inducible nitric oxide synthase (NOS II) expression and nitric oxide (NO) production are up-regulated in these diseases as well. However, the apparent link between PGHS-2 and NOS II has not been thoroughly investigated in nontransformed and nontumorigenic colonic epithelial cells. In the present study, we examined the concomitant expression of PGHS-2 and NOS II as well as the production of prostaglandin E2 (PGE2) and NO in conditionally immortalized mouse colonic epithelial cells, namely YAMC (Apc(+/+)). We found that the induction of PGHS-2 and generation of PGE2 in these cells by IFN-gamma and lipopolysaccharide (LPS) were greatly reduced by two selective NOS II inhibitors, L-NIL and SMT. To ascertain the effect of NO on PGHS-2 overexpression, we tested NO-releasing compounds, NOR-1 and SNAP, and found that they caused PGHS-2 expression and PGE2 production. This effect was abolished by hemoglobin, a NO scavenger. Using electrophoretic mobility shift assays, we found that both NOR-1 and SNAP caused beta-catenin/LEF-1 DNA complex formation. Super-shift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Cell fractionation studies indicated that NO donors caused an increase in free soluble cytoplasmic beta-catenin. This is further corroborated by the immunocytochemistry data showing the redistribution of beta-catenin from the predominantly membrane localization into the cytoplasm and nucleus after treatment with NO donors. To further explore the possible connection between PGHS-2 expression and beta-catenin/LEF-1 DNA complex formation, we studied IMCE (Apc(Min/+)) cells, a sister cell line of YAMC with similar genetic background but differing in Apc genotype and, consequently, their beta-catenin levels. We found that IMCE cells, in comparison with YAMC cells, had markedly higher beta-catenin/LEF-1 DNA complex formation under both resting conditions as well as after induction with NO. In parallel fashion, IMCE cells expressed significantly higher levels of PGHS-2 mRNA and protein, and generated more PGE2. Overall, this study suggests that NO may be involved in PGHS-2 overexpression in conditionally immortalized mouse colonic epithelial cells. Although the molecular mechanism of the link is still under investigation, this effect of NO appears directly or indirectly to be a result of the increase in free soluble beta-catenin and the formation of nuclear beta-catenin/LEF-1 DNA complex.
Asunto(s)
Enterocitos/enzimología , Isoenzimas/biosíntesis , Óxido Nítrico/farmacología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Transactivadores , Proteína de la Poliposis Adenomatosa del Colon , Animales , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Ciclooxigenasa 2 , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Dinoprostona/biosíntesis , Dinoprostona/metabolismo , Enterocitos/citología , Enterocitos/efectos de los fármacos , Enterocitos/metabolismo , Inducción Enzimática/efectos de los fármacos , Depuradores de Radicales Libres/metabolismo , Depuradores de Radicales Libres/farmacología , Interferón gamma/farmacología , Isoenzimas/genética , Lipopolisacáridos/farmacología , Factor de Unión 1 al Potenciador Linfoide , Ratones , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/antagonistas & inhibidores , Donantes de Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , Penicilamina/análogos & derivados , Penicilamina/antagonistas & inhibidores , Penicilamina/metabolismo , Penicilamina/farmacología , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Solubilidad/efectos de los fármacos , Factores de Transcripción/metabolismo , beta CateninaRESUMEN
Mutations in Apc underlie the intestinal lesions in familial adenomatous polyposis and are found in >85% of sporadic colon cancers. They are frequently associated with overexpression of prostaglandin endoperoxide H synthase-2 (PGHS-2) in colonic adenomas. It has been suggested that Apc mutations are linked mechanistically to increased PGHS-2 expression by elevated nuclear accumulation of beta-catenin-Tcf-LEF transcription complex. In the present study, we show that PGHS-2 is differentially expressed in mouse colonic epithelial cells with distinct Apc status. Cells with a mutated Apc expressed markedly higher levels of PGHS-2 mRNA and protein and produced significantly more prostaglandin E2 than cells with normal Apc. Using electrophoretic mobility shift assays, we demonstrate that DNA-beta-catenin-LEF-1 complex formation is differentially induced in these two cell lines in an Apc-dependent manner. Our data indicate that the differential induction of beta-catenin-LEF-1 complex correlates closely with differential expression of PGHS-2. These findings support the hypothesis that the differential expression of PGHS-2 is mediated through the proposed beta-catenin/Tcf-LEF signaling pathway.