RESUMEN
Netrin-1 was recently proposed to control tumorigenesis by inhibiting apoptosis induced by the dependence receptors DCC (Deleted in colorectal cancer) and UNC5H. Although the loss of these dependence receptors' expression has been described as a selective advantage for tumor growth and progression in numerous cancers, recent observations have shown that some tumors may use an alternative strategy to block dependence receptor-induced programmed cell death: the autocrine expression of netrin-1. This alternative strategy has been observed in a large fraction of aggressive breast cancers, neuroblastoma, pancreatic adenocarcinoma, and lung cancer. This observation is of potential interest regarding future targeted therapy, as in such cases interfering with the ability of netrin-1 to inhibit DCC or UNC5H-induced cell death is associated with apoptosis of netrin-1-expressing tumor cells in vitro, and with inhibition of tumor growth or metastasis in different animal tumor models. The understanding of the mechanism by which netrin-1 inhibits cell death is therefore of interest. Here, we show that netrin-1 triggers the multimerization of both DCC and UNC5H2 receptors, and that multimerization of the intracellular domain of DCC and UNC5H2 is the critical step to inhibit the proapoptotic effects of both of these receptors. Taking advantage of this property, we utilized a recombinant specific domain of DCC that (i) interacts with netrin-1 and (ii) inhibits netrin-1-induced multimerization, to trigger apoptosis in netrin-dependent tumor cells.
Asunto(s)
Apoptosis , Neoplasias/metabolismo , Factores de Crecimiento Nervioso/farmacología , Receptores de Superficie Celular/metabolismo , Proteínas Supresoras de Tumor/farmacología , Animales , Línea Celular , Pollos , Receptor DCC , Modelos Animales de Enfermedad , Humanos , Receptores de Netrina , Netrina-1 , Multimerización de Proteína/efectos de los fármacos , Proteínas Recombinantes/farmacología , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The involvement of reactive oxygen species (ROS) in neuronal death has been determined in culture, and in association with several neurodegenerative disorders. We examined whether ROS participate in the cell death observed during spinal cord development. We found that the general pattern of high ROS levels, gene expression for some antioxidant enzymes, and motoneuron death correlated positively along spinal cord development. ROS were reduced in spinal cords cultured in the presence of a synthetic superoxide dismutase and catalase mimetic, with a concomitant reduction in cell death and an increase in the number of motoneurons. The number of motoneurons was higher in spinal cords treated with the antioxidant than in those treated with caspase inhibitors. In general, the increase in motoneuron survival did not correlate with the reduction in cells undergoing DNA degradation in the motoneuronal region. These results suggest that ROS are signaling molecules controlling caspase-dependent and caspase-independent programmed motoneuron death, and support the hypothesis that this mechanism is abnormally turned on in some neurodegenerative disorders and aging.
Asunto(s)
Neuronas Motoras/citología , Estrés Oxidativo/fisiología , Médula Espinal/citología , Animales , Antioxidantes/farmacología , Autofagia , Caspasa 8 , Caspasa 9 , Inhibidores de Caspasas , Catalasa/metabolismo , Recuento de Células , Muerte Celular/fisiología , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Ratones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Compuestos Organometálicos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Salicilatos/farmacología , Médula Espinal/embriología , Médula Espinal/metabolismo , Superóxido Dismutasa/metabolismo , Tórax , Técnicas de Cultivo de TejidosRESUMEN
Several receptors that mediate apoptosis have been identified, such as Fas and tumor necrosis factor receptor I. Studies of the signal transduction pathways utilized by these receptors have played an important role in the understanding of apoptosis. Here we report the first ligand-receptor pair-the neuropeptide substance P and its receptor, neurokinin-1 receptor (NK(1)R)-that mediates an alternative, non-apoptotic form of programmed cell death. This pair is widely distributed in the central and peripheral nervous systems, and has been implicated in pain mediation and depression, among other effects. Here we demonstrate that substance P induces a non-apoptotic form of programmed cell death in hippocampal, striatal, and cortical neurons. This cell death requires gene expression, displays a non-apoptotic morphology, and is independent of caspase activation. The same form of cell death is induced by substance P in NK(1)R-transfected human embryonic kidney cells. These results argue that NK(1)R activates a death pathway different than apoptosis, and provide a signal transduction system by which to study an alternative, non-apoptotic cell death program.
Asunto(s)
Apoptosis/fisiología , Células Epiteliales/metabolismo , Riñón/metabolismo , Neuronas/metabolismo , Prosencéfalo/metabolismo , Receptores de Neuroquinina-1/metabolismo , Sustancia P/metabolismo , Triptófano/análogos & derivados , Animales , Anexina A5/metabolismo , Inhibidores de Caspasas , Caspasas/genética , Caspasas/metabolismo , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Feto , Humanos , Inmunohistoquímica , Riñón/ultraestructura , Microscopía Electrónica , Antagonistas del Receptor de Neuroquinina-1 , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Piperidinas/farmacología , Prosencéfalo/ultraestructura , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Ratas Sprague-Dawley , Sustancia P/farmacología , Triptófano/farmacologíaRESUMEN
The endoplasmic reticulum (ER) is the site of assembly of polypeptide chains destined for secretion or routing into various subcellular compartments. It also regulates cellular responses to stress and intracellular Ca(2+) levels. A variety of toxic insults can result in ER stress that ultimately leads to apoptosis. Apoptosis is initiated by the activation of members of the caspase family and serves as a central mechanism in the cell death process. The present study was carried out to determine the role of caspases in triggering ER stress-induced cell death. Treatment of cells with ER stress inducers such as brefeldin-A or thapsigargin induces the expression of caspase-12 protein and also leads to translocation of cytosolic caspase-7 to the ER surface. Caspase-12, like most other members of the caspase family, requires cleavage of the prodomain to activate its proapoptotic form. Caspase-7 associates with caspase-12 and cleaves the prodomain to generate active caspase-12, resulting in increased cell death. We propose that any cellular insult that causes prolonged ER stress may induce apoptosis through caspase-7-mediated caspase-12 activation. The data underscore the involvement of ER and caspases associated with it in the ER stress-induced apoptotic process.
Asunto(s)
Caspasas/metabolismo , Muerte Celular , Retículo Endoplásmico/metabolismo , Animales , Apoptosis , Western Blotting , Brefeldino A/farmacología , Calcio/metabolismo , Caspasa 12 , Caspasa 7 , Caspasa 9 , Caspasas/biosíntesis , Catálisis , Línea Celular , Sistema Libre de Células , ADN Complementario/metabolismo , Activación Enzimática , Humanos , Ratones , Mutación , Plásmidos/metabolismo , Pruebas de Precipitina , Transporte de Proteínas , Proteínas Recombinantes/metabolismo , Estrés Fisiológico , Fracciones Subcelulares , Tapsigargina/farmacología , TransfecciónRESUMEN
We have previously described a novel cancer chemotherapeutic approach based on the induction of apoptosis in targeted cells by homing pro-apoptotic peptides. In order to improve this approach we developed a computational method (approach for detecting potential apoptotic peptides, APAP) to detect short PAPs, based on the prediction of the helical content of peptides, the hydrophobic moment, and the isoelectric point. PAPs are toxic against bacteria and mitochondria, but not against mammalian cells when applied extracellularly. Among other peptides, substance P was identified as a PAP and subsequently demonstrated to be a pro-apoptotic peptide experimentally. APAP thus provides a method to detect and ultimately improve pro-apoptotic peptides for chemotherapy.
Asunto(s)
Apoptosis/efectos de los fármacos , Biología Computacional/métodos , Reconocimiento de Normas Patrones Automatizadas , Sustancia P/química , Sustancia P/farmacología , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Antibacterianos/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Caspasa 3 , Caspasas/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Bases de Datos como Asunto , Activación Enzimática/efectos de los fármacos , Escherichia coli/citología , Escherichia coli/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Punto Isoeléctrico , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Dilatación Mitocondrial/efectos de los fármacos , Estructura Secundaria de Proteína , Ratas , Programas Informáticos , Sustancia P/toxicidadRESUMEN
Programmed cell death or apoptosis is an essential process during the morphogenesis of a large number of structures. Evidence obtained over the past few years indicates that, in some cases, the generation of reactive oxygen species (ROS) is an important event during the course of apoptosis. Using an in vitro culture system in which digit individualization of developing limbs normally occurs, we assayed the effect of different antioxidants on the cell death that takes place at interdigits. The addition of phenol, dimethyl sulfoxide, or 2',7'-dichlorodihydrofluorescein diacetate (DCDHF-DA) to murine developing limbs in culture prevented digit individualization as well as the typical interdigital cell death. Two ROS-sensitive dyes, 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide and DCDHF-DA, stained interdigits and the so-called "necrotic zones," implying that they contain cells under oxidative stress. Very few interdigital cells were doubly stained with the ROS probes and two cell death indicators (i.e., acridine orange and propidium iodide), suggesting that they detect a different stage during the course of apoptosis. Furthermore, we found cells stained for ROS that did not express a specific macrophage marker and in a few cases were seen surrounded by a macrophage. Surprisingly, many regions of the midgestation mouse embryo that are undergoing cell death correlated with those that have a markedly higher level of ROS. Our data suggest that the generation of oxidative stress is a common requirement for cell death that occurs during mouse embryonic development.
Asunto(s)
Antioxidantes/farmacología , Apoptosis , Fluoresceínas/farmacología , Esbozos de los Miembros/citología , Esbozos de los Miembros/fisiología , Especies Reactivas de Oxígeno/fisiología , Animales , Apoptosis/efectos de los fármacos , Dimetilsulfóxido/farmacología , Embrión de Mamíferos , Desarrollo Embrionario y Fetal/efectos de los fármacos , Desarrollo Embrionario y Fetal/fisiología , Colorantes Fluorescentes , Esbozos de los Miembros/ultraestructura , Macrófagos/citología , Macrófagos/fisiología , Macrófagos/ultraestructura , Ratones , Microscopía Confocal , Morfogénesis/efectos de los fármacos , Técnicas de Cultivo de Órganos , Fenol/farmacologíaRESUMEN
To understand the mechanisms regulating the tissue non-specific alkaline phosphatase (TNAP) activity during development, we characterized cis-transcriptional regulatory elements. In embryonic cells and tissues, TNAP expression was driven preferentially by the exon 1A (E1A) promoter, one of the two promoters previously defined. Transcriptional activity of E1A promoter was up-regulated by retinoic acid (RA) through a putative RA-responsive element. Transgenic mice analysis with lacZ reporter constructs revealed negative regulatory elements within 8.5 kb of E1A promoter. Promoter sequences of endogenous TNAP in non-expressing tissues and those carried by the 8.5 kb-lacZ transgene were found to be highly methylated. A 1 kb fragment of E1A promoter increased the methylation level of lacZ and promoter sequences. The role of RA and DNA methylation in defining the embryonic expression pattern of TNAP is discussed.
Asunto(s)
Fosfatasa Alcalina/genética , Metilación de ADN , Regulación Enzimológica de la Expresión Génica , Genes Reguladores/fisiología , Tretinoina/farmacología , Animales , Secuencia de Bases , Células Cultivadas , Embrión de Mamíferos/fisiología , Genes Reguladores/efectos de los fármacos , Células Germinativas/fisiología , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/efectos de los fármacosRESUMEN
Embryonic stem (ES) cells are a suitable system to study events occurring during development. In the present work we show that the apoptotic program was activated in ES cells, either by simple removal of the reducing agent 2-mercaptoethanol (2-ME), or by addition of all trans-retinoic (ATRA) to embryoid bodies. In these two conditions, there was an increase in reactive oxygen species and antioxidants such as catalase, superoxide dismutase or phenol prevented ATRA-induced cell death. Neuronal differentiation was observed when undifferentiated ES cells were treated with ATRA in the absence of serum and the presence of 2-ME.