RESUMEN
Recent studies have established miR-34a as a key effector of the p53 signaling pathway and have implicated its role in multiple cancer types. Here, we establish that miR-34a induces apoptosis, G2 arrest, and senescence in medulloblastoma and renders these cells more sensitive to chemotherapeutic agents. These effects are mediated in part by the direct post-transcriptional repression of the oncogenic MAGE-A gene family. We demonstrate that miR-34a directly targets the 3' untranslated regions of MAGE-A genes and decreases MAGE-A protein levels. This decrease in MAGE-A results in a concomitant increase in p53 and its associated transcriptional targets, p21/WAF1/CIP1 and, importantly, miR-34a. This establishes a positive feedback mechanism where miR-34a is not only induced by p53 but increases p53 mRNA and protein levels through the modulation of MAGE-A genes. Additionally, the forced expression of miR-34a or the knockdown of MAGE-A genes by small interfering RNA similarly sensitizes medulloblastoma cells to several classes of chemotherapeutic agents, including mitomycin C and cisplatin. Finally, the analysis of mRNA and micro-RNA transcriptional profiles of a series of primary medulloblastomas identifies a subset of tumors with low miR-34a expression and correspondingly high MAGE-A expression, suggesting the coordinate regulation of these genes. Our work establishes a role for miR-34a in modulating responsiveness to chemotherapy in medulloblastoma and presents a novel positive feedback mechanism involving miR-34a and p53, via direct targeting of MAGE-A.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/metabolismo , MicroARNs/fisiología , Proteínas de Neoplasias/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Antígenos de Neoplasias/genética , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología , Cisplatino/farmacología , Perfilación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Luciferasas/metabolismo , Meduloblastoma/patología , Antígenos Específicos del Melanoma , Mitomicina/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Neuralized (Neurl) is a highly conserved E3 ubiquitin ligase, which in Drosophila acts upon Notch ligands to regulate Notch pathway signaling. Human Neuralized1 (NEURL1) was investigated as a potential tumor suppressor in medulloblastoma (MB). The gene is located at 10q25.1, a region demonstrating frequent loss of heterozygosity in tumors. In addition, prior publications have shown that the Notch pathway is functional in a proportion of MB tumors and that Neurl1 is only expressed in differentiated cells in the developing cerebellum. In this study, NEURL1 expression was downregulated in MB compared with normal cerebellar tissue, with the lowest levels of expression in hedgehog-activated tumors. Control of gene expression by histone modification was implicated mechanistically; loss of 10q, sequence mutation, and promoter hypermethylation did not play major roles. NEURL1-transfected MB cell lines demonstrated decreased population growth, colony-forming ability, tumor sphere formation, and xenograft growth compared with controls, and a significant increase in apoptosis was seen on cell cycle and cell death analysis. Notch pathway inhibition occurred on the exogenous expression of NEURL1, as shown by decreased expression of the Notch ligand, Jagged1, and the target genes, HES1 and HEY1. From these studies, we conclude that NEURL1 is a candidate tumor suppressor in MB, at least in part through its effects on the Notch pathway.
Asunto(s)
Apoptosis , Neoplasias Cerebelosas/patología , Regulación hacia Abajo , Meduloblastoma/patología , Receptores Notch/genética , Ubiquitina-Proteína Ligasas/fisiología , Adolescente , Adulto , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Western Blotting , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Cerebelo/metabolismo , Cerebelo/patología , Niño , Preescolar , Metilación de ADN , Proteínas de Drosophila , Epigénesis Genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Técnicas para Inmunoenzimas , Lactante , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1 , Meduloblastoma/genética , Meduloblastoma/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fenotipo , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Receptores Notch/antagonistas & inhibidores , Receptores Notch/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Serrate-Jagged , Transducción de Señal , Factor de Transcripción HES-1RESUMEN
The neonatal immune environment and the events that occur during this time have profound effects for the adult period. While protective immune responses can develop, the neonatal immune system, particularly the skin immune system (SIS), tends to promote tolerance. With this information we undertook a number of studies to identify unique aspects of skin during the neonatal period. Proteomics revealed proteins uniquely expressed in neonatal, but not adult, skin (e.g. Stefin A, peroxiredoxins) and these may have implications in the development of SIS. Vitamin D was found to have a modulating role on SIS and this was apparent from the early neonatal period. Exposure of the neonatal skin to UV radiation altered the microenvironment resulting in the generation of regulatory T cells, which persisted in adult life. As the development of UV radiation-induced melanoma can occur following a single high dose (equivalent to burning in adults) to transgenic mice (hepatocyte growth factor/scatter factor or TPras) during the neonatal period, the early modulating events which lead to suppression may be relevant for the development of UV radiation-induced human melanoma. Any attempt to produce effective melanoma immunotherapy has to accommodate and overcome these barriers. Margaret Kripke's pioneering work on UV-induced immunosuppression still remains central to the understanding of the development of melanoma and how it frequently escapes the immune system.
Asunto(s)
Sistema Inmunológico/inmunología , Sistema Inmunológico/efectos de la radiación , Melanoma/inmunología , Neoplasias Cutáneas/inmunología , Rayos Ultravioleta , Animales , Animales Recién Nacidos , Antígenos/inmunología , Humanos , Recién Nacido , Melanoma/patología , Neoplasias Cutáneas/patologíaRESUMEN
Abnormalities of chromosome 10 are frequently observed in the development of medulloblastoma, the most common malignant brain tumor of childhood. To identify critical genetic loci involved, we performed detailed physical mapping of regions of allelic loss on this chromosome. 18% of cases (5/32 primary tumors, 2/8 cell lines) harbored allelic losses on 10q. Refined mapping identified a 21.7Mb common interval, affecting the region 10q23.3-10q25.3. This region contains three genes, MXI1, SUFU and BTRC, which represent putative medulloblastoma tumor suppressor (TS) genes on the basis of either (i) negative regulation of critical medulloblastoma pathways, or (ii) mutation in other cancer types. We therefore sought evidence of their genetic inactivation in 46 cases, by mutational analysis of their entire coding regions. A MXI1 mutation was identified which abolishes its translation initiation site (A1G; MET1VAL), however no further tumor-specific sequence variations were detected. We next identified and characterised CpG islands associated with 5' regions of the MXI1, SUFU and BTRC genes; analysis of these regions for evidence of DNA hypermethylation, alongside expression analysis of their respective transcripts, revealed no evidence to support epigenetic inactivation of any gene. These findings implicate the inactivation of critical TS loci at 10q23.3-25.3 in medulloblastoma, however comprehensive analysis of SUFU, BTRC and MXI1 indicates they are unlikely to represent major targets of these allelic losses. MXI1 mutation appears to play a role in the pathogenesis of a small subset of cases, and suggests an alternative mechanism to MYC amplification for disruption of the MYC/MAD/MAX network in medulloblastoma.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Cromosomas Humanos Par 10 , Genes Supresores de Tumor , Meduloblastoma/genética , Proteínas Supresoras de Tumor/genética , Adolescente , Adulto , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Silenciador del Gen , Humanos , Lactante , Masculino , Meduloblastoma/etiología , Mapeo Físico de Cromosoma , Proteínas Represoras/genética , Proteínas con Repetición de beta-Transducina/genéticaRESUMEN
Australia has the highest incidence of skin cancer in the world and ultraviolet (UV)-B radiation has been implicated as its major aetiological agent. Despite the link between melanoma and exposure to UV-B radiation in childhood, little work has been carried out to determine the effects of UV-B on neonatal skin. In this study, we investigated the response of adult and neonatal Langerhans cells (LC) to UV-B radiation to determine whether exposure in the neonatal period impairs the development of the skin immune system, thus having implications for the immune response later in life. Neonatal and adult mice were irradiated with a single dose of UV-B radiation and epidermal sheets prepared to determine the number of LC present. In addition, antigen carriage and T-cell proliferation assays were carried out to assess the immune response when the mice reached maturity. Results showed that neonatal LC were more susceptible than adult LC to depletion at 2 kJ/m(2) UV-B exposure; however, there was similar susceptibility at lower doses. When mice that were irradiated as neonates were analysed at maturity, there was an increased ability to respond to cutaneously applied antigen as more antigen was transported to the lymph node and the lymph node dendritic cells had an enhanced ability to stimulate T-cell proliferation. In addition, this response was skewed towards a Th2 type response. Thus, single high-dose UV-B exposure alters the development of neonatal LC, resulting in a short-term reduction in the number of LC but an enhanced immune response when assessed at maturity.
Asunto(s)
Células de Langerhans/efectos de la radiación , Activación de Linfocitos/fisiología , Piel/inmunología , Linfocitos T/inmunología , Rayos Ultravioleta , Animales , Animales Recién Nacidos , Recuento de Células , Epidermis/efectos de la radiación , Femenino , Células de Langerhans/patología , Ganglios Linfáticos/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos BALB C , Piel/efectos de la radiación , Bazo/efectos de la radiación , Irradiación Corporal TotalRESUMEN
Monoclonal antibodies are reproducible, specific, and cost-effective molecular probes; use outside the laboratory is, however, restricted by technical limitations. Addressing these constraints, the first self-signalling antibodies are now described, where specific antigen binding causes release of bound reporter from bispecific antibodies (BsAb) to generate a detectable signal. The report examines the concept that two different antibody binding sites in close proximity can promote interaction between molecules recognised by these sites, generating a signal by molecular crowding. Signal strength is found to increase with increasing homogeneity for a BsAb reactive with multimeric surfactant antigen; signal response is linear for a BsAb reactive with univalent small analyte deoxypyridinoline. Self-signalling is consistent with intramolecular steric hindrance. This is the first report detailing integration of two different functions, molecular detection and signal response, into BsAbs and with detection of large and small analytes, has generic application to antibody-based systems.