RESUMEN
Resistance to anticancer drugs is widely observed in vitro and in cancer patients, but its prevalence is too high to be solely explained by the acquisition of mutations. Sharma et al. (2010) now report that dynamic chromatin modifications may be an independent route to drug resistance in cancer cells that can be reversed by epigenetic drugs.
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DOT1L methylates histone H3K79 and is aberrantly regulated in MLL-rearranged leukemia. Inhibitors have been developed to target DOT1L activity in leukemia, but cellular mechanisms that regulate DOT1L are still poorly understood. We have identified the histone deacetylase Rpd3 as a negative regulator of budding yeast Dot1. At its target genes, the transcriptional repressor Rpd3 restricts H3K79 methylation, explaining the absence of H3K79me3 at a subset of genes in the yeast genome. Similar to the crosstalk in yeast, inactivation of the murine Rpd3 homolog HDAC1 in thymocytes led to an increase in H3K79 methylation. Thymic lymphomas that arise upon genetic deletion of Hdac1 retained the increased H3K79 methylation and were sensitive to reduced DOT1L dosage. Furthermore, cell lines derived from Hdac1Δ/Δ thymic lymphomas were sensitive to a DOT1L inhibitor, which induced apoptosis. In summary, we identified an evolutionarily conserved crosstalk between HDAC1 and DOT1L with impact in murine thymic lymphoma development.
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Histona Desacetilasa 1/genética , Histona Desacetilasa 2/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Linfoma/metabolismo , Neoplasias del Timo/metabolismo , Acetilación , Animales , Línea Celular Tumoral , Eliminación de Gen , Histona Desacetilasas/genética , Humanos , Linfoma/genética , Metilación , Ratones , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Neoplasias del Timo/genéticaRESUMEN
At present, little information is available in the scientific literature related to the durability (weathering resistance) of fire-retarded wood and natural fiber-reinforced thermoplastics. In this work, thermoplastic profiles for façade applications based on high-density polyethylene, wheat straw particles, and fire-retardants were extruded and their reaction-to-fire performance before and after artificial weathering evaluated. Profile geometries were either solid or hollow-core profiles, and fire-retardants (FR) were added either in the co-extruded layer or in the bulk. Various FR for inclusion in the co-extruded layer were screened based on UL-94 tests. For profile extrusion, two types of FR were chosen: a coated intumescent combination based on ammonium polyphosphate (APP) and an APP coated with melamine and without formaldehyde. Before weathering, the peak heat release rate (pHRR) and the total heat release (THR), which were determined using cone calorimeter measurements, were reduced by up to 64% and 67% due to the FR. However, even before weathering, pHRR of the profiles was relatively high, with best (lowest) values between 230 and 250 kW/m2 under the test conditions. After 28 days of artificial weathering, changes in reaction-to-fire performance and color were evaluated. Use of the APP in the co-extruded layer worsened color change compared to the formulation without APP but the pHRR was not significantly changed. The influence of weathering on the fire behavior was small compared to the difference between fire-retarded and non-fire-retarded materials. Results from the cone calorimeter were analyzed with regard to ETAG 028, which provides requirements related to the durability of fire performance of building products. In many formulations, increase in THR was less than 20% compared to before weathering, which would place some of the profiles in class C or better (EN 13501-1). However, due to the high pHRR, at best, class D was obtained under the conditions of this study. In addition to cone calorimeter measurements, results from the single flame source test, limiting oxygen index determination and thermogravimetric analysis, are shown and discussed. Strength properties, water uptake and swelling of the profiles, thermal conductivity, and energy dispersive X-ray data are also presented.
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Retardadores de Llama , Fosfatos/química , Polietileno/química , Triticum/metabolismo , Compuestos de Amonio/química , Calorimetría/métodos , Celulosa/química , Técnicas de Química Analítica , Color , Calor , Lignina/química , Ensayo de Materiales , Tamaño de la Partícula , Polifosfatos/química , Polisacáridos/química , Reproducibilidad de los Resultados , Dióxido de Silicio/química , Temperatura , Conductividad Térmica , AguaRESUMEN
MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression in physiological processes as well as in diseases. Currently miRs are already used to find novel mechanisms involved in diseases and in the future, they might serve as diagnostic markers. To identify miRs that play a role in glomerular diseases urinary miR-screenings are a frequently used tool. However, miRs that are detected in the urine might simply be filtered from the blood stream and could have been produced anywhere in the body, so they might be completely unrelated to the diseases. We performed a combined miR-screening in pooled urine samples from patients with different glomerular diseases as well as in cultured human podocytes, human mesangial cells, human glomerular endothelial cells and human tubular cells. The miR-screening in renal cells was done in untreated conditions and after stimulation with TGF-ß. A merge of the detected regulated miRs led us to identify disease-specific, cell type-specific and cell stress-induced miRs. Most miRs were down-regulated following the stimulation with TGF-ß in all cell types. Up-regulation of miRs after TGF-ß was cell type-specific for most miRs. Furthermore, urinary miRs from patients with different glomerular diseases could be assigned to the different renal cell types. Most miRs were specifically regulated in one disease. Only miR-155 was up-regulated in all disease urines compared to control and therefore seems to be rather unspecific. In conclusion, a combined urinary and cell miR-screening can improve the interpretation of screening results. These data are useful to identify novel miRs potentially involved in glomerular diseases.
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Enfermedades Renales/metabolismo , Glomérulos Renales/metabolismo , Túbulos Renales/metabolismo , MicroARNs/orina , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/orina , Regulación hacia Abajo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Humanos , Enfermedades Renales/genética , Enfermedades Renales/orina , Glomérulos Renales/efectos de los fármacos , Túbulos Renales/citología , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Persona de Mediana Edad , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia ArribaRESUMEN
The pathophysiology of many proteinuric kidney diseases is poorly understood, and microRNAs (miRs) regulation of these diseases has been largely unexplored. Here, we tested whether miR-378a-3p is a novel regulator of glomerular diseases. MiR-378a-3p has two predicted targets relevant to glomerular function, the glomerular basement membrane matrix component, nephronectin (NPNT), and vascular endothelial growth factor VEGF-A. In zebrafish (Danio rerio), miR-378a-3p mimic injection or npnt knockdown by a morpholino oligomer caused an identical phenotype consisting of edema, proteinuria, podocyte effacement, and widening of the glomerular basement membrane in the lamina rara interna. Zebrafish vegf-A protein could not rescue this phenotype. However, mouse Npnt constructs containing a mutated 3'UTR region prevented the phenotype caused by miR-378a-3p mimic injection. Overexpression of miR-378a-3p in mice confirmed glomerular dysfunction in a mammalian model. Biopsies from patients with focal segmental glomerulosclerosis and membranous nephropathy had increased miR-378a-3p expression and reduced glomerular levels of NPNT. Thus, miR-378a-3p-mediated suppression of the glomerular matrix protein NPNT is a novel mechanism for proteinuria development in active glomerular diseases.
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Proteínas de la Matriz Extracelular/genética , Membrana Basal Glomerular/metabolismo , Glomerulonefritis Membranosa/genética , Glomeruloesclerosis Focal y Segmentaria/genética , MicroARNs/metabolismo , Regiones no Traducidas 3'/genética , Animales , Biopsia , Modelos Animales de Enfermedad , Regulación hacia Abajo , Proteínas de la Matriz Extracelular/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Membrana Basal Glomerular/patología , Glomerulonefritis Membranosa/patología , Glomerulonefritis Membranosa/orina , Glomeruloesclerosis Focal y Segmentaria/patología , Glomeruloesclerosis Focal y Segmentaria/orina , Humanos , Masculino , Ratones , MicroARNs/genética , Morfolinos/metabolismo , Podocitos/metabolismo , Podocitos/patología , Proteinuria/genética , Proteinuria/patología , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Histone deacetylases (HDACs) are posttranslational modifiers that deacetylate proteins. Despite their crucial role in numerous biological processes, the use of broad-range HDAC inhibitors (HDACi), has shown clinical efficacy. However, undesired side effects highlight the necessity to better understand the biology of different HDACs and target the relevant HDACs. Using a novel mouse model, in which HDAC1 and HDAC2 can be simultaneously deleted in the intestine of adult mice, we show that the simultaneous deletion of HDAC1 and HDAC2 leads to a rapid loss of intestinal homeostasis. Importantly, this deletion cannot be sustained, and 8 days after initial ablation, stem cells that have escaped HDAC1 or HDAC2 deletion swiftly repopulate the intestinal lining. In vitro ablation of HDAC1 and HDAC2 using intestinal organoid cultures resulted in a down-regulation of multiple intestinal stem cell markers and functional loss of clonogenic capacity. Importantly, treatment of wild-type organoids with class I-specific HDACi MS-275 also induced a similar loss of stemness, providing a possible rationale for the gastrointestinal side effects often observed in HDACi-treated patients. In conclusion, these data show that HDAC1 and HDAC2 have a redundant function and are essential to maintain intestinal homeostasis.
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Histona Desacetilasa 1/fisiología , Histona Desacetilasa 2/fisiología , Homeostasis/fisiología , Intestinos/citología , Células Madre/citología , Animales , Benzamidas/farmacología , Biomarcadores/metabolismo , Western Blotting , Diferenciación Celular , Células Cultivadas , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 2/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/farmacología , Homeostasis/efectos de los fármacos , Humanos , Técnicas para Inmunoenzimas , Intestinos/efectos de los fármacos , Intestinos/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Piridinas/farmacología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/efectos de los fármacos , Células Madre/enzimologíaRESUMEN
Histone deacetylases (HDACs) are epigenetic erasers of lysine-acetyl marks. Inhibition of HDACs using small molecule inhibitors (HDACi) is a potential strategy in the treatment of various diseases and is approved for treating hematological malignancies. Harnessing the therapeutic potential of HDACi requires knowledge of HDAC-function in vivo. Here, we generated a thymocyte-specific gradient of HDAC-activity using compound conditional knockout mice for Hdac1 and Hdac2. Unexpectedly, gradual loss of HDAC-activity engendered a dosage-dependent accumulation of immature thymocytes and correlated with the incidence and latency of monoclonal lymphoblastic thymic lymphomas. Strikingly, complete ablation of Hdac1 and Hdac2 abrogated lymphomagenesis due to a block in early thymic development. Genomic, biochemical and functional analyses of pre-leukemic thymocytes and tumors revealed a critical role for Hdac1/Hdac2-governed HDAC-activity in regulating a p53-dependent barrier to constrain Myc-overexpressing thymocytes from progressing into lymphomas by regulating Myc-collaborating genes. One Myc-collaborating and p53-suppressing gene, Jdp2, was derepressed in an Hdac1/2-dependent manner and critical for the survival of Jdp2-overexpressing lymphoma cells. Although reduced HDAC-activity facilitates oncogenic transformation in normal cells, resulting tumor cells remain highly dependent on HDAC-activity, indicating that a critical level of Hdac1 and Hdac2 governed HDAC-activity is required for tumor maintenance.
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Dosificación de Gen/fisiología , Genes Supresores de Tumor/fisiología , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , Proteínas Proto-Oncogénicas c-myc/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Células Cultivadas , Epistasis Genética/fisiología , Dosificación de Gen/genética , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Genes/fisiología , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 1/fisiología , Histona Desacetilasa 2/metabolismo , Histona Desacetilasa 2/fisiología , Linfoma/genética , Linfoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Chromatin modifications, especially histone-tail acetylation, have been implicated in memory formation. Increased histone-tail acetylation induced by inhibitors of histone deacetylases (HDACis) facilitates learning and memory in wild-type mice as well as in mouse models of neurodegeneration. Harnessing the therapeutic potential of HDACis requires knowledge of the specific HDAC family member(s) linked to cognitive enhancement. Here we show that neuron-specific overexpression of HDAC2, but not that of HDAC1, decreased dendritic spine density, synapse number, synaptic plasticity and memory formation. Conversely, Hdac2 deficiency resulted in increased synapse number and memory facilitation, similar to chronic treatment with HDACis in mice. Notably, reduced synapse number and learning impairment of HDAC2-overexpressing mice were ameliorated by chronic treatment with HDACis. Correspondingly, treatment with HDACis failed to further facilitate memory formation in Hdac2-deficient mice. Furthermore, analysis of promoter occupancy revealed an association of HDAC2 with the promoters of genes implicated in synaptic plasticity and memory formation. Taken together, our results suggest that HDAC2 functions in modulating synaptic plasticity and long-lasting changes of neural circuits, which in turn negatively regulates learning and memory. These observations encourage the development and testing of HDAC2-selective inhibitors for human diseases associated with memory impairment.
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Sinapsis Eléctricas/fisiología , Histona Desacetilasas/metabolismo , Memoria/fisiología , Proteínas Represoras/metabolismo , Animales , Butiratos/farmacología , Espinas Dendríticas/fisiología , Femenino , Regulación de la Expresión Génica , Hipocampo/metabolismo , Histona Desacetilasa 1 , Histona Desacetilasa 2 , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/deficiencia , Histona Desacetilasas/genética , Ácidos Hidroxámicos/farmacología , Aprendizaje/efectos de los fármacos , Masculino , Memoria/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Sodio/farmacología , VorinostatRESUMEN
Chromatin modifier Swi-independent 3a (SIN3A), together with associated histone deacetylases, influences gene expression during development and differentiation through a variety of transcription factors in a cell-specific manner. Sin3a is essential for the maintenance of inner cell mass cells of mouse blastocysts, embryonic fibroblasts, and myoblasts, but is not required for the survival of trophectoderm or Sertoli cells. To better understand how this transcriptional regulator modulates cells at different developmental stages within a single lineage, we used conditional gene targeting in mice to ablate Sin3a from perinatal quiescent male gonocytes and from postnatal differentiating spermatogonia. Mitotic germ cells expressing stimulated by retinoic acid gene 8 (Stra8) that lacked Sin3a exhibited increased DNA damage and apoptosis, yet collectively progressed through meiosis and spermiogenesis and generated epididymal sperm at approximately 50% of control levels, sufficient for normal fertility. In contrast, perinatal gonocytes lacking Sin3a underwent rapid depletion that coincided with cell cycle reentry, exhibiting 2.5-fold increased histone H3 phosphorylation upon cycling that suggested a prophase/metaphase block; germ cells were almost entirely absent two weeks after birth, resulting in sterility. Gene expression profiling of neonatal testes containing Sin3a-deleted gonocytes identified upregulated transcripts highly associated with developmental processes and pattern formation, and downregulated transcripts involved in nuclear receptor activity, including Nr4a1 (Nur77). Interestingly, Nr4a1 levels were elevated in testes containing Stra8-expressing, Sin3a-deleted spermatogonia. SIN3A directly binds to the Nr4a1 promoter, and Nr4a1 expression is diminished upon spermatogonial differentiation in vitro. We conclude that within the male germline, Sin3a is required for the mitotic reentry of gonocytes, but is dispensable for the maintenance of differentiating spermatogonia and subsequent spermatogenic processes.
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Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Células Germinativas/crecimiento & desarrollo , Proteínas Represoras/metabolismo , Espermatogonias/crecimiento & desarrollo , Proteínas Adaptadoras Transductoras de Señales , Animales , Apoptosis/genética , Linaje de la Célula/fisiología , Inmunoprecipitación de Cromatina , Daño del ADN/genética , Cartilla de ADN/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Marcación de Gen/métodos , Células Germinativas/citología , Histonas/metabolismo , Inmunohistoquímica , Masculino , Ratones , Análisis por Micromatrices , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Fosforilación , Proteínas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Complejo Correpresor Histona Desacetilasa y Sin3 , Testículo/citología , Testículo/metabolismoRESUMEN
Histone deacetylases (HDACs) counterbalance acetylation of lysine residues, a protein modification involved in numerous biological processes. Here, Hdac1 and Hdac2 conditional knock-out alleles were used to study the function of class I Hdac1 and Hdac2 in cell cycle progression and haematopoietic differentiation. Combined deletion of Hdac1 and Hdac2, or inactivation of their deacetylase activity in primary or oncogenic-transformed fibroblasts, results in a senescence-like G(1) cell cycle arrest, accompanied by up-regulation of the cyclin-dependent kinase inhibitor p21(Cip). Notably, concomitant genetic inactivation of p53 or p21(Cip) indicates that Hdac1 and Hdac2 regulate p53-p21(Cip)-independent pathways critical for maintaining cell cycle progression. In vivo, we show that Hdac1 and Hdac2 are not essential for liver homeostasis. In contrast, total levels of Hdac1 and Hdac2 in the haematopoietic system are critical for erythrocyte-megakaryocyte differentiation. Dual inactivation of Hdac1 and Hdac2 results in apoptosis of megakaryocytes and thrombocytopenia. Together, these data indicate that Hdac1 and Hdac2 have overlapping functions in cell cycle regulation and haematopoiesis. In addition, this work provides insights into mechanism-based toxicities observed in patients treated with HDAC inhibitors.
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Ciclo Celular , Hematopoyesis , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Anemia/enzimología , Animales , Apoptosis , Biocatálisis , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Histona Desacetilasa 1/deficiencia , Histona Desacetilasa 2/deficiencia , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trombocitopenia/enzimología , Trombocitopenia/patología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Class I histone deacetylases are critical regulators of gene transcription by erasing lysine acetylation. Targeting histone deacetylases using relative non-specific small molecule inhibitors is of major interest in the treatment of cancer, neurological disorders and acquired immune deficiency syndrome. Harnessing the therapeutic potential of histone deacetylase inhibitors requires full knowledge of individual histone deacetylases in vivo. As hematologic malignancies show increased sensitivity towards histone deacetylase inhibitors we targeted deletion of class I Hdac1 and Hdac2 to hematopoietic cell lineages. Here, we show that Hdac1 and Hdac2 together control hematopoietic stem cell homeostasis, in a cell-autonomous fashion. Simultaneous loss of Hdac1 and Hdac2 resulted in loss of hematopoietic stem cells and consequently bone marrow failure. Bone-marrow-specific deletion of Sin3a, a major Hdac1/2 co-repressor, phenocopied loss of Hdac1 and Hdac2 indicating that Sin3a-associated HDAC1/2-activity is essential for hematopoietic stem cell homeostasis. Although Hdac1 and Hdac2 show compensatory and overlapping functions in hematopoiesis, mice expressing mono-allelic Hdac1 or Hdac2 revealed that Hdac1 and Hdac2 contribute differently to the development of specific hematopoietic lineages.
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Hematopoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Histona Desacetilasa 1/deficiencia , Histona Desacetilasa 2/deficiencia , Homeostasis/fisiología , Proteínas Represoras/deficiencia , Animales , Células de la Médula Ósea/fisiología , Linaje de la Célula/fisiología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Complejo Correpresor Histona Desacetilasa y Sin3RESUMEN
Resistance of cancer cells to chemotherapeutics and emerging targeted drugs is a devastating problem in the treatment of cancer patients. Multiple mechanisms contribute to drug resistance such as increased drug efflux, altered drug metabolism, secondary mutations in drug targets, and activation of downstream or parallel signal transduction pathways. The rapid kinetics, the reversibility of acquired drug resistance and the absence of genetic mutations suggest an epigenetic basis for drug insensitivity. Similar to the cellular variance seen in the human body, epigenetic mechanisms, through reversible histone modifications and DNA methylation patterns, generate a variety of transcriptional states resulting in a dynamic heterogeneous tumor cell population. Consequently, epigenomes favoring survival in the presence of a drug by aberrant transcription of drug transporters, DNA-repair enzymes and pro-apoptotic factors render cytotoxic and targeted drugs ineffective and allow selection of rare drug-resistant tumor cells. Recent advances in charting cancer genomes indeed strongly indicate a role for epigenetic regulators in driving cancer, which may result in the acquisition of additional (epi)genetic modifications leading to drug resistance. These observations have important clinical consequences as they provide an opportunity for "epigenetic drugs" to change reversible drug-resistance-associated epigenomes to prevent or reverse non-responsiveness to anti-cancer drugs.
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Transformación Celular Neoplásica/genética , Resistencia a Antineoplásicos/genética , Epigénesis Genética , Neoplasias/genética , Acetilación , Animales , Antineoplásicos/uso terapéutico , Metilación de ADN , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Heterogeneidad Genética , Histonas/genética , Histonas/metabolismo , Humanos , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Microenvironments support the maintenance of stem cells and the growth of tumors through largely unknown mechanisms. While cell-autonomous chromatin modifications have emerged as important determinants for self-renewal and differentiation of stem cells, a role for non-cell autonomous epigenetic contributions is not well established. Here, we genetically ablated the chromatin modifier Swi-independent 3a (Sin3a) in fetal Sertoli cells, which partly comprise the niche for male germline stem cells, and investigated its impact on spermatogenic cell fate and teratoma formation in vivo. Sertoli cell-specific Sin3a deletion resulted in the formation of few undifferentiated spermatogonia after birth while initially maintaining spermatogenic differentiation. Stem cell-associated markers Plzf, Gfra1, and Oct4 were downregulated in the mutant fetal gonad, while Sertoli cell markers Steel and Gdnf, which support germ cells, were not diminished. Following birth, markers of differentiating spermatogonia, Kit and Sohlh2, exhibited normal levels, but chemokine-signaling molecules chemokine (C-X-C motif) ligand 12 (CXCL12)/stromal cell-derived factor 1 (SDF1) and chemokine (C-X-C motif) receptor 4 (CXCR4), expressed in Sertoli cells and germ cells, respectively, were not detected. In the juvenile, mutant testes exhibited a progressive loss of differentiating spermatogonia and a block in spermatid elongation, followed by extensive germ cell degeneration. Sertoli cell-specific Sin3a deletion also suppressed teratoma formation by fetal germ cells in an in vivo transplantation assay. We conclude that the epigenome of Sertoli cells influences the establishment of a niche for germline stem cells as well as for tumor initiating cells.
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Neoplasias de Células Germinales y Embrionarias/metabolismo , Proteínas Represoras/metabolismo , Células de Sertoli/metabolismo , Espermátides/metabolismo , Espermatogonias/metabolismo , Animales , Diferenciación Celular/fisiología , Femenino , Inmunohistoquímica , Masculino , Ratones , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Sertoli/citología , Complejo Correpresor Histona Desacetilasa y Sin3 , Espermátides/citología , Espermatogonias/citologíaRESUMEN
AIM: This paper is a report of a study of the attitudes of Dutch nursing students towards people with physical or intellectual disabilities. BACKGROUND: Attitudes of healthcare professionals are a major factor in the rehabilitation and self-acceptance of persons with disabilities. Consequently, it is important that nurses develop or maintain positive attitudes towards people with disabilities during their education. However, more knowledge is needed about current attitudes of nursing students and factors influencing these attitudes. METHODS: A sample of Dutch nursing students (n = 81) and an age-matched group of non-nursing peers (n = 48) completed standardized scales measuring attitudes about physically or intellectually disabled people. Data were collected in 2006. FINDINGS: Nursing students were more positive towards physically disabled people than their peers, and more strongly endorsed empowerment and similarity of intellectually disabled people. These attitudinal differences generally remained statistically significant after multivariate adjustment for demographic variables and experience and contact with individuals with disabilities. An important independent determinant of a positive attitude towards physically disabled people in the total sample was having a relative or friend with a physical disability. This association, however, was not apparent in attitudes towards intellectually disabled persons. CONCLUSION: Educational interventions aimed at improving attitudes towards people with disabilities should include focus on forms of contact beyond the context of formal care relationships.
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Actitud del Personal de Salud , Personas con Discapacidad , Discapacidad Intelectual/psicología , Grupo Paritario , Estudiantes de Enfermería/psicología , Adolescente , Actitud Frente a la Salud , Femenino , Humanos , Masculino , Percepción Social , Adulto JovenRESUMEN
The transcription factor nuclear factor-κB (NF-κB) is crucial for the maintenance of homeostasis. It is incompletely understood how nuclear NF-κB and the crosstalk of NF-κB with other transcription factors are controlled. Here, we demonstrate that the epigenetic regulator histone deacetylase 2 (HDAC2) activates NF-κB in transformed and primary cells. This function depends on both, the catalytic activity and an intact HDAC2 sumoylation motif. Several mechanisms account for the induction of NF-κB through HDAC2. The expression of wild-type HDAC2 can increase the nuclear presence of NF-κB. In addition, the ribosomal S6 kinase 1 (RSK1) and the tumor suppressor p53 contribute to the regulation of NF-κB by HDAC2. Moreover, TP53 mRNA expression is positively regulated by wild-type HDAC2 but not by sumoylation-deficient HDAC2. Thus, sumoylation of HDAC2 integrates NF-κB signaling involving p53 and RSK1. Since HDAC2-dependent NF-κB activity protects colon cancer cells from genotoxic stress, our data also suggest that high HDAC2 levels, which are frequently found in tumors, are linked to chemoresistance. Accordingly, inhibitors of NF-κB and of the NF-κB/p53-regulated anti-apoptotic protein survivin significantly sensitize colon carcinoma cells expressing wild-type HDAC2 to apoptosis induced by the genotoxin doxorubicin. Hence, the HDAC2-dependent signaling node we describe here may offer an interesting therapeutic option.
Asunto(s)
Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasa 2/metabolismo , FN-kappa B/metabolismo , Sumoilación , Animales , Apoptosis , Catálisis , Línea Celular Tumoral , Núcleo Celular/metabolismo , Neoplasias del Colon/metabolismo , Fibroblastos/metabolismo , Genes Reporteros , Células HEK293 , Homeostasis , Humanos , Ratones , Mutágenos/química , Transducción de Señal , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The retinoblastoma protein pRB and its two homologs p130 and p107 form the family of pocket proteins and play a major role in cell-cycle regulation and suppression of human and mouse tumorigenesis. Pocket proteins regulate the activity of E2F transcription factors during G1-S transition. Two mechanisms have been described: (i) pocket protein binding blocks the transactivation domain of activator E2Fs, inhibiting E2F-dependent transcription and (ii) E2F-bound pocket proteins can recruit chromatin remodeling proteins containing an LxCxE motif (x encoding any amino acid), resulting in active repression of E2F target genes. To investigate the importance of pRB's LxCxE-interacting motif in cell-cycle control and tumor suppression, we generated mouse embryonic fibroblasts and mice expressing a mutant pRB protein carrying an asparagine for phenylalanine substitution at position 750, abrogating LxCxE binding. Because p130 may compensate for loss of pRB, we studied pRB(N750F) activity in the presence and absence of p130. The pRB-LxCxE interaction was not required for cell-cycle arrest upon mitogen deprivation and cell-cell contact, but did contribute to RAS(V12)- and radiation-induced cell-cycle arrest. Remarkably, the pRB-LxCxE interaction was not required for suppression of in vitro and in vivo transformation, even in the absence of p130. These results indicate that pRB's tumor suppressor activity is not effectuated by active silencing of E2F target genes, but rather by regulation of activator E2Fs or another unidentified mechanism. Furthermore, the in vitro response of pocket protein-perturbed cells to mitogen deprivation and cell-cell contact seems a better predictor of tumor development than the response to ectopic RAS(V12) expression. Cancer Res; 74(18); 5266-76. ©2014 AACR.
Asunto(s)
Factores de Transcripción E2F/genética , Proteína de Retinoblastoma/genética , Animales , Procesos de Crecimiento Celular/genética , Factores de Transcripción E2F/metabolismo , Silenciador del Gen , Humanos , Ratones , Proteína de Retinoblastoma/metabolismo , TransfecciónRESUMEN
We previously found that the adamantyl-substituted retinoid-related molecules bind to the small heterodimer partner (SHP) as well as the Sin3A complex. In this report, we delineated the role of SHP and the Sin3A complex in 4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC)-mediated inhibition of cell growth and apoptosis. We examined the effect of loss of SHP and Sin3A expression in a number of cell types on 3-Cl-AHPC-mediated growth inhibition and apoptosis induction, 3-Cl-AHPC-mediated nuclear factor-kappaB (NF-kappaB) activation, and 3-Cl-AHPC-mediated increase in c-Fos and c-Jun expression. We found that loss of SHP or Sin3A expression, while blocking 3-Cl-AHPC-mediated apoptosis, had little effect on 3-Cl-AHPC inhibition of cellular proliferation. We have previously shown that 3-Cl-AHPC-mediated NF-kappaB activation is necessary for apoptosis induction. We have now shown that 3-Cl-AHPC-enhanced c-Fos and c-Jun expression is also essential for maximal 3-Cl-AHPC-mediated apoptosis. 3-Cl-AHPC induction of c-Fos and c-Jun expression as well as NF-kappaB activation was dependent on SHP protein levels. In turn, SHP levels are regulated by Sin3A because ablation of Sin3A resulted in a decrease in SHP expression. Thus, SHP and Sin3A play an important role in adamantyl-substituted retinoid-related induction of cellular apoptosis.
Asunto(s)
Adamantano/análogos & derivados , Apoptosis/efectos de los fármacos , Cinamatos/farmacología , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/metabolismo , Adamantano/farmacología , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Histona Desacetilasas/metabolismo , Humanos , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Interferente Pequeño/genética , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Complejo Correpresor Histona Desacetilasa y Sin3 , TransfecciónRESUMEN
Since its discovery in 1986, as the first tumor suppressor gene, the retinoblastoma gene (Rb) has been extensively studied. Numerous biochemical and genetic studies have elucidated in great detail the function of the Rb gene and placed it at the heart of the molecular machinery controlling the cell cycle. As more insight was gained into the genetic events required for oncogenic transformation, it became clear that the retinoblastoma gene is connected to biochemical pathways that are dysfunctional in virtually all tumor types. Besides regulating the E2F transcription factors, pRb is involved in numerous biological processes such as apoptosis, DNA repair, chromatin modification, and differentiation. Further complexity was added to the system with the discovery of p107 and p130, two close homologs of Rb. Although the three family members share similar functions, it is becoming clear that these proteins also have unique functions in differentiation and regulation of transcription. In contrast to Rb, p107 and p130 are rarely found inactivated in human tumors. Yet, evidence is accumulating that these proteins are part of a "tumor-surveillance" mechanism and can suppress tumorigenesis. Here we provide an overview of the knowledge obtained from studies involving the retinoblastoma gene family with particular focus on its role in suppressing tumorigenesis.
Asunto(s)
Ciclo Celular , Regulación Neoplásica de la Expresión Génica , Genes de Retinoblastoma/genética , Genes Supresores de Tumor , Proteína de Retinoblastoma/genética , Retinoblastoma/genética , Animales , HumanosRESUMEN
mSin3A is a core component of a large multiprotein corepressor complex with associated histone deacetylase (HDAC) enzymatic activity. Physical interactions of mSin3A with many sequence-specific transcription factors has linked the mSin3A corepressor complex to the regulation of diverse signaling pathways and associated biological processes. To dissect the complex nature of mSin3A's actions, we monitored the impact of conditional mSin3A deletion on the developmental, cell biological, and transcriptional levels. mSin3A was shown to play an essential role in early embryonic development and in the proliferation and survival of primary, immortalized, and transformed cells. Genetic and biochemical analyses established a role for mSin3A/HDAC in p53 deacetylation and activation, although genetic deletion of p53 was not sufficient to attenuate the mSin3A null cell lethal phenotype. Consistent with mSin3A's broad biological activities beyond regulation of the p53 pathway, time-course gene expression profiling following mSin3A deletion revealed deregulation of genes involved in cell cycle regulation, DNA replication, DNA repair, apoptosis, chromatin modifications, and mitochondrial metabolism. Computational analysis of the mSin3A transcriptome using a knowledge-based database revealed several nodal points through which mSin3A influences gene expression, including the Myc-Mad, E2F, and p53 transcriptional networks. Further validation of these nodes derived from in silico promoter analysis showing enrichment for Myc-Mad, E2F, and p53 cis-regulatory elements in regulatory regions of up-regulated genes following mSin3A depletion. Significantly, in silico promoter analyses also revealed specific cis-regulatory elements binding the transcriptional activator Stat and the ISWI ATP-dependent nucleosome remodeling factor Falz, thereby expanding further the mSin3A network of regulatory factors. Together, these integrated genetic, biochemical, and computational studies demonstrate the involvement of mSin3A in the regulation of diverse pathways governing many aspects of normal and neoplastic growth and survival and provide an experimental framework for the analysis of essential genes with diverse biological functions.
Asunto(s)
Supervivencia Celular , Neoplasias Experimentales/patología , Proteínas Represoras/fisiología , Transcripción Genética/fisiología , Animales , Ciclo Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Neoplasias Experimentales/fisiopatología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Complejo Correpresor Histona Desacetilasa y Sin3RESUMEN
The retinoblastoma gene family consists of three genes: RB, p107, and p130. While loss of pRB causes retinoblastoma in humans and pituitary gland tumors in mice, tumorigenesis in other tissues may be suppressed by p107 and p130. To test this hypothesis, we have generated chimeric mice from embryonic stem cells carrying compound loss-of-function mutations in the Rb gene family. We found that Rb/p107- and Rb/p130-deficient mice were highly cancer prone. We conclude that in a variety of tissues tumor development by loss of pRB is suppressed by its homologs p107 and p130. The redundancy of the retinoblastoma proteins in vivo is reflected by the behavior of Rb-family-defective mouse embryonic fibroblasts in vitro.