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Beclin 1, an autophagy and haploinsufficient tumor-suppressor protein, is frequently monoallelically deleted in breast and ovarian cancers. However, the precise mechanisms by which Beclin 1 inhibits tumor growth remain largely unknown. To address this question, we performed a genome-wide CRISPR/Cas9 screen in MCF7 breast cancer cells to identify genes whose loss of function reverse Beclin 1-dependent inhibition of cellular proliferation. Small guide RNAs targeting CDH1 and CTNNA1, tumor-suppressor genes that encode cadherin/catenin complex members E-cadherin and alpha-catenin, respectively, were highly enriched in the screen. CRISPR/Cas9-mediated knockout of CDH1 or CTNNA1 reversed Beclin 1-dependent suppression of breast cancer cell proliferation and anchorage-independent growth. Moreover, deletion of CDH1 or CTNNA1 inhibited the tumor-suppressor effects of Beclin 1 in breast cancer xenografts. Enforced Beclin 1 expression in MCF7 cells and tumor xenografts increased cell surface localization of E-cadherin and decreased expression of mesenchymal markers and beta-catenin/Wnt target genes. Furthermore, CRISPR/Cas9-mediated knockout of BECN1 and the autophagy class III phosphatidylinositol kinase complex 2 (PI3KC3-C2) gene, UVRAG, but not PI3KC3-C1-specific ATG14 or other autophagy genes ATG13, ATG5, or ATG7, resulted in decreased E-cadherin plasma membrane and increased cytoplasmic E-cadherin localization. Taken together, these data reveal previously unrecognized cooperation between Beclin 1 and E-cadherin-mediated tumor suppression in breast cancer cells.
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Beclina-1/metabolismo , Neoplasias de la Mama/metabolismo , Cadherinas/metabolismo , Genes Supresores de Tumor , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Proteínas Relacionadas con la Autofagia/metabolismo , Neoplasias de la Mama/patología , Sistemas CRISPR-Cas/genética , Membrana Celular/metabolismo , Proliferación Celular/genética , Femenino , Genoma Humano , Humanos , Interferones/metabolismo , Células MCF-7 , Ratones Endogámicos NOD , Ratones SCID , Transporte de Proteínas , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , alfa Catenina/metabolismoRESUMEN
CDKN2A is an evolutionarily conserved gene encoding proteins implicated in tumor suppression, ocular development, aging, and metabolic diseases. Like the human form, mouse Cdkn2a encodes two distinct proteins-p16Ink4a, which blocks cyclin-dependent kinase activity, and p19Arf, which is best known as a positive regulator of the p53 tumor suppressor-and their functions have been well-studied in genetically engineered mouse models. Relatively little is known about how expression of the two transcripts is controlled in normal development and in certain disease states. To better understand their coordinate and transcript-specific expression in situ, we used a transposase-aided approach to generate a new BAC transgenic mouse model in which the first exons encoding Arf and Ink4a are replaced by fluorescent reporters. We show that mouse embryo fibroblasts generated from the transgenic lines faithfully display induction of each transgenic reporter in cell culture models, and we demonstrate the expected expression of the Arf reporter in the normal testis, one of the few places where that promoter is normally expressed. Interestingly, the TGFß-2-dependent induction of the Arf reporter in the eye-a process essential for normal eye development-does not occur. Our findings illustrate the value of BAC transgenesis in mapping key regulatory elements in the mouse by revealing the genomic DNA required for Cdkn2a induction in cultured cells and the developing testis, and the apparent lack of elements driving expression in the developing eye.
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Factores de Ribosilacion-ADP/genética , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Testículo/metabolismo , Transposasas/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Animales , Línea Celular , Cromosomas Artificiales Bacterianos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones TransgénicosRESUMEN
The analysis of patient blood transcriptional profiles offers a means to investigate the immunological mechanisms relevant to human diseases on a genome-wide scale. In addition, such studies provide a basis for the discovery of clinically relevant biomarker signatures. We designed a strategy for microarray analysis that is based on the identification of transcriptional modules formed by genes coordinately expressed in multiple disease data sets. Mapping changes in gene expression at the module level generated disease-specific transcriptional fingerprints that provide a stable framework for the visualization and functional interpretation of microarray data. These transcriptional modules were used as a basis for the selection of biomarkers and the development of a multivariate transcriptional indicator of disease progression in patients with systemic lupus erythematosus. Thus, this work describes the implementation and application of a methodology designed to support systems-scale analysis of the human immune system in translational research settings.
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Perfilación de la Expresión Génica/métodos , Genómica/métodos , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Adolescente , Niño , Biología Computacional/métodos , Progresión de la Enfermedad , Femenino , Humanos , MasculinoRESUMEN
Blood monocytes from children with systemic lupus erythematosus (SLE) behave similar to dendritic cells (DCs), and SLE serum induces healthy monocytes to differentiate into DCs in a type I IFN-dependent manner. In this study, we found that these monocytes display significant transcriptional changes, including a prominent IFN signature, compared with healthy controls. Few of those changes, however, explain DC function. Exposure to allogeneic T cells in vitro reprograms SLE monocytes to acquire DC phenotype and function, and this correlates with both IFN-inducible (IP10) and proinflammatory cytokine (IL-1ß and IL6) expression. Furthermore, we found that both IFN and SLE serum induce the upregulation of CCR7 transcription in these cells. CCR7 protein expression, however, requires a second signal provided by TLR agonists such as LPS. Thus, SLE serum "primes" a subset of monocytes to readily (<24 h) respond to TLR agonists and acquire migratory DC properties. Our findings might explain how microbial infections exacerbate lupus.
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Movimiento Celular/inmunología , Células Dendríticas/inmunología , Interferón Tipo I/inmunología , Lupus Eritematoso Sistémico/inmunología , Adolescente , Adulto , Niño , Citocinas/inmunología , Células Dendríticas/patología , Femenino , Humanos , Lipopolisacáridos/farmacología , Lupus Eritematoso Sistémico/patología , Receptores CCR7/inmunología , Receptores Toll-Like/agonistas , Receptores Toll-Like/inmunologíaRESUMEN
In castration-resistant prostate cancer (CRPC), increased glucocorticoid receptor (GR) expression and ensuing transcriptional activity have been proposed as an oncogenic "bypass" mechanism in response to androgen receptor (AR) signaling inhibition (ARSi). Here, we report that GR transcriptional activity acquired following ARSi is associated with the upregulation of cyclic adenosine monophosphate (cAMP)-associated gene expression pathways in both model systems and metastatic prostate cancer patient samples. In the context of ARSi, the expression of GR-mediated genes encoding cAMP signaling pathway-associated proteins can be inhibited by treatment with selective GR modulators (SGRMs). For example, in the context of ARSi, we found that GR activation resulted in upregulation of protein kinase inhibitor beta (PKIB) mRNA and protein levels, leading to nuclear accumulation of the cAMP-dependent protein kinase A catalytic subunit (PKA-c). Increased PKA-c, in turn, is associated with increased cAMP response element-binding protein phosphorylation and activity. Furthermore, enzalutamide and SGRM combination therapy in mice bearing CRPC xenografts delayed CRPC progression compared with enzalutamide therapy alone, and reduced tumor PKIB mRNA expression. Supporting the clinical importance of GR/PKA signaling activation in CRPC, we found a significant enrichment of both cAMP pathway signaling-associated gene expression and high NR3C1 (GR) activity in patient-derived xenograft models and metastatic human CRPC samples. These findings suggest a novel mechanism linking CRPC-induced GR transcriptional activity with increased cAMP signaling in AR-antagonized CRPC. Furthermore, our findings suggest that GR-specific modulation in addition to AR antagonism may delay GR+ CRPC time to recurrence, at least in part, by inhibiting tumor cAMP/PKA pathways.
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Benzamidas , Feniltiohidantoína , Neoplasias de la Próstata Resistentes a la Castración , Masculino , Humanos , Animales , Ratones , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores de Glucocorticoides/metabolismo , Glucocorticoides/uso terapéutico , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Línea Celular Tumoral , Nitrilos/uso terapéutico , Transducción de Señal , ARN MensajeroRESUMEN
Systemic onset juvenile idiopathic arthritis (SoJIA) represents up to 20% of juvenile idiopathic arthritis. We recently reported that interleukin (IL) 1 is an important mediator of this disease and that IL-1 blockade induces clinical remission. However, lack of specificity of the initial systemic manifestations leads to delays in diagnosis and initiation of therapy. To develop a specific diagnostic test, we analyzed leukocyte gene expression profiles of 44 pediatric SoJIA patients, 94 pediatric patients with acute viral and bacterial infections, 38 pediatric patients with systemic lupus erythematosus (SLE), 6 patients with PAPA syndrome, and 39 healthy children. Statistical group comparison and class prediction identified genes differentially expressed in SoJIA patients compared with healthy children. These genes, however, were also changed in patients with acute infections and SLE. An analysis of significance across all diagnostic groups identified 88 SoJIA-specific genes, 12 of which accurately classified an independent set of SoJIA patients with systemic disease. Transcripts that changed significantly in patients undergoing IL-1 blockade were also identified. Thus, leukocyte transcriptional signatures can be used to distinguish SoJIA from other febrile illnesses and to assess response to therapy. Availability of early diagnostic markers may allow prompt initiation of therapy and prevention of disabilities.
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Artritis Juvenil/diagnóstico , Artritis Juvenil/epidemiología , Interleucina-1/antagonistas & inhibidores , Leucocitos Mononucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Adolescente , Adulto , Edad de Inicio , Anciano , Artritis Juvenil/genética , Artritis Juvenil/terapia , Estudios de Casos y Controles , Niño , Canales de Cloruro/genética , Enfermedades Transmisibles/genética , Diagnóstico Diferencial , Perfilación de la Expresión Génica , Salud , Humanos , Lactante , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/patología , Persona de Mediana Edad , Transcripción Genética/efectos de los fármacosRESUMEN
Estrogen receptor-positive (ER+) invasive lobular breast cancer (ILC) comprises about ~15% of breast cancer. ILC's unique genotypic (loss of wild type E-cadherin expression) and phenotypic (small individual round cancer cells that grow in discontinuous nests) are thought to contribute to a distinctive pattern of metastases to serosal membranes. Unlike invasive ductal carcinoma (IDC), ILC metastases often intercalate into the mesothelial layer of the peritoneum and other serosal surfaces. While ER activity is a known driver of ILC proliferation, very little is known about how additional nuclear receptors contribute to ILC's distinctive biology. In ER+ IDC, we showed previously that glucocorticoid receptor (GR) activity inhibits pro-proliferative gene expression and cell proliferation. Here we examined ER+ ILC models and found that GR activation similarly reduces S-phase entry gene expression and ILC proliferation. While slowing tumor growth rate, our data also suggest that GR activation results in an enhanced metastatic phenotype through increasing integrin-encoding gene expression, extracellular matrix protein adhesion, and mesothelial cell clearance. Moreover, in an intraductal mouse mammary gland model of ILC, we found that GR expression is associated with increased bone metastases despite slowed primary mammary tumor growth. Taken together, our findings suggest GR-mediated gene expression may contribute to the unusual characteristics of ILC biology.
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A key mediator of macroautophagy/autophagy induction is the class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) consisting of PIK3C3/VPS34, PIK3R4/VPS15, BECN1, and ATG14. Although several proteins are known to enhance or decrease PtdIns3K-C1 activity, our understanding of the molecular regulation of PtdIns3K-C1 is still incomplete. Previously, we identified a Golgi-associated protein, GLIPR2, in a screen for proteins that interact with amino acids 267-284 of BECN1, a region of BECN1 sufficient to induce autophagy when fused to a cell penetrating leader sequence. In this study, we used CRISPR-Cas9-mediated depletion of GLIPR2 in cells and mice to investigate the role of GLIPR2 in the regulation of autophagy and PtdIns3K-C1 activity. Depletion of GLIPR2 in HeLa cells increased autelophagic flux and generation of phosphatidylinositol 3-phosphate (PtdIns3P). GLIPR2 knockout resulted in less compact Golgi structures, which was also observed in autophagy-inducing conditions such as amino acid starvation or Tat-BECN1 peptide treatment. Importantly, the binding of GLIPR2 to purified PtdIns3K-C1 inhibited the in vitro lipid kinase activity of PtdIns3K-C1. Moreover, the tissues of glipr2 knockout mice had increased basal autophagic flux as well as increased recruitment of the PtdIns3P-binding protein, WIPI2. Taken together, our findings demonstrate that GLIPR2 is a negative regulator of PtdIns3K-C1 activity and basal autophagy.Abbreviations: ATG14: autophagy related 14; Baf A1: bafilomycin A1; BARA: ß-α repeated, autophagy-specific; CQ: chloroquine; GFP: green fluorescent protein; GLIPR2: GLI pathogenesis related 2; HBSS: Hanks' balanced salt solution; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PBS: phosphate-buffered saline; PtdIns3K-C1: phosphatidylinositol 3-kinase complex I; PtdIns3P: phosphatidylinositol-3-phosphate; SEM: standard error of the mean; WIPI2: WD repeat domain, phosphoinositide interacting 2.
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Autofagia , Fosfatidilinositol 3-Quinasas Clase III , Proteínas de la Membrana , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Autofagia/fisiología , Proteínas Relacionadas con la Autofagia/metabolismo , Beclina-1/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/metabolismo , Ratones , FosforilaciónRESUMEN
Systemic lupus erythematosus (SLE) is a prototype systemic autoimmune disease characterized by flares of high morbidity. Using oligonucleotide microarrays, we now show that active SLE can be distinguished by a remarkably homogeneous gene expression pattern with overexpression of granulopoiesis-related and interferon (IFN)-induced genes. Using the most stringent statistical analysis (Bonferroni correction), 15 genes were found highly up-regulated in SLE patients, 14 of which are targets of IFN and one, defensin DEFA-3, a major product of immature granulocytes. A more liberal correction (Benjamini and Hochberg correction) yielded 18 additional genes, 12 of which are IFN-regulated and 4 granulocyte-specific. Indeed immature neutrophils were identified in a large fraction of SLE patients white blood cells. High dose glucocorticoids, a standard treatment of disease flares, shuts down the interferon signature, further supporting the role of this cytokine in SLE. The expression of 10 genes correlated with disease activity according to the SLEDAI. The most striking correlation (P < 0.001, r = 0.55) was found with the formyl peptide receptor-like 1 protein that mediates chemotactic activities of defensins. Therefore, while the IFN signature confirms the central role of this cytokine in SLE, microarray analysis of blood cells reveals that immature granulocytes may be involved in SLE pathogenesis.
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Perfilación de la Expresión Génica , Granulocitos/fisiología , Interferón-alfa/metabolismo , Leucopoyesis , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/genética , Niño , Femenino , Regulación de la Expresión Génica , Granulocitos/citología , Granulocitos/inmunología , Humanos , Interferón-alfa/inmunología , Lupus Eritematoso Sistémico/inmunología , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Estadística como AsuntoRESUMEN
High-throughput microarray technologies were used to study DNA methylation accompanied by transcriptional changes in follicular lymphoma (FL). Using Methylated CpG Island Amplification with Microarrays to study CpG Island DNA methylation in FL, we discovered widespread hypermethylation of homeobox genes and previously identified targets of polycomb repressive complex 2 (PRC2) in cell lines and primary tumors, but not in benign follicular hyperplasia (BFH). DNA methylation for HOXA11, HOXD10, HOXB7, HOXC12, PAX6, LHX9, SFMBT2, EN2, and PAX7 was independently validated in the RL cell line and HOXA11, HOXD10, PAX6, and EN2 in primary tumors. Combined Bisulfite Restriction Analysis (COBRA) also established DNA methylation for the previously identified PRC2 targets DCC, DES, GAD2, AQP5, GPR61, GRIA4, GJD2, and AMPH in FL but not in BFH. Gene expression analyses revealed 411 genes that were hypermethylated and transcriptionally repressed in RL, 74% of which were reactivated by the demethylating agent 5-aza-2'-deoxycytidine (5-azaD) plus or minus the histone deacetylase inhibitor trichostatin A (TSA). Forty genes were also downregulated in primary FL. Our results suggest that extensive hypermethylation in promoters of polycomb target genes is a characteristic of FL and that loss of expression of certain SUZ12 target genes could be functionally relevant for lymphomagenesis.
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Metilación de ADN , Linfoma Folicular/genética , Proteínas Portadoras/genética , Línea Celular Tumoral , Análisis por Conglomerados , Islas de CpG , Ciclina D1/genética , Epigénesis Genética , Femenino , Genes Homeobox , Proteínas de Homeodominio/genética , Humanos , Hiperplasia/genética , Ganglios Linfáticos/patología , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Reproducibilidad de los Resultados , Factores de Transcripción , Transcripción GenéticaRESUMEN
Disruption of the CDKN2A (INK4A/ARF) and B (INK4B) genes, which encode three function-independent tumor suppressors, is one of the most common events in human cancer. Because their relative importance in tumor prevention appears to be species- and context-specific, studying their regulation can shed light on mechanisms by which they are bypassed in malignant transformation. We previously unveiled a new pathway in which TGFß selectively induces Arf at mouse Cdkn2a in eye development and cultured fibroblasts. As TGFß signaling is often derailed in cancer development or progression, we investigated its control of CDKN2A/B in human cancer. Computational analyses of sequencing and array data from nearly 11,000 patients with cancer in TCGA showed discordant expression of ARF and INK4A in most cancer subtypes, with gene copy-number loss and promoter methylation involved in only a subset. Using HeLa cells as a model, we found that exogenous TGFß induced ARF mRNA and protein, and ARF knockdown limited TGFß-mediated growth suppression. TGFß-mediated ARF mRNA induction required SMAD2/3, p38MAPK, and SP1, and ARF mRNA was induced without added RNAPII recruitment. Chromatin immunoprecipitation unveiled a remote enhancer element engaged by TGFß by a mechanism that partially depended on p38MAPK. CRISPR-based editing of this enhancer limited induction of ARF and INK4B by TGFß, but not by oncogenic RAS. IMPLICATIONS: Our findings reveal new molecular mechanisms by which CDKN2A/B regulation is coupled to external cues, and those findings represent entry points to further explore pharmacologic strategies to restore their expression in cancer.
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Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Elementos de Facilitación Genéticos , Neoplasias/genética , Factor de Crecimiento Transformador beta/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Modelos Biológicos , Neoplasias/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia ArribaRESUMEN
The utilization of gene-expression microarrays in patient-based research creates new prospects for the discovery of diagnostic biomarkers and the identification of genes or pathways linked to pathogenesis. Gene-expression signatures in peripheral blood mononuclear cells isolated from over one hundred patients with conditions presenting a strong immunological component (patient with autoimmune, graft versus host and infectious diseases, as well as immunosuppressed transplant recipients) were generated. This dataset provides the opportunity to carry out comparative analyses and define disease signatures in a broader context. Transcriptional changes of 22,283 probe sets were evaluated through statistical group comparison performed systematically for seven diseases versus their respective healthy control group. Patterns of significance were generated by hierarchical clustering of P-values. This approach led to the identification of a SLE-specific "diagnostic signature," formed by genes that did not change compared to healthy subjects in the other six diseases. Conversely, a "sentinel signature" that was common to all seven diseases was characterized. These findings bring new perspectives for the application of blood leukocyte expression signatures for diagnosis and early disease detection.
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Perfilación de la Expresión Génica , Leucocitos Mononucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Adulto , Niño , Infecciones por Escherichia coli/diagnóstico , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/inmunología , Humanos , Gripe Humana/diagnóstico , Gripe Humana/genética , Gripe Humana/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/patología , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Metaanálisis como Asunto , Infecciones Neumocócicas/diagnóstico , Infecciones Neumocócicas/genética , Infecciones Neumocócicas/inmunología , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/inmunologíaRESUMEN
The immune system is controlled by dendritic cells (DCs). Just as lymphocytes comprise different subsets, DCs comprise several subsets that differentially control lymphocyte function. In humans, the myeloid pathway includes Langerhans cells (LCs) and interstitial DCs (intDCs). While both subsets produce IL-12, only intDCs make IL-10 and induce B cell differentiation. Another pathway includes plasmacytoid DCs, which promptly secrete large amounts of IFN-alpha/beta viral encounter. Thus, insights into in vivo DC functions are important to understand the launching and modulation of immunity.
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Células Dendríticas/inmunología , Inmunoterapia , Humanos , Tolerancia Inmunológica , Vacunas/inmunologíaRESUMEN
Studies on the role of the RNA receptor TLR8 in inflammation have been limited by its different function in human versus rodents. We have generated multiple lines of transgenic mice expressing different levels of human TLR8. The high copy number chimeras were unable to pass germline; developed severe inflammation targeting the pancreas, salivary glands, and joints; and the severity of the specific phenotypes closely correlated with the huTLR8 expression levels. Mice with relatively low expression levels survived and bred successfully but had increased susceptibility to collagen-induced arthritis, and the levels of huTLR8 correlated with proinflammatory cytokines in the joints of the animals. At the cellular level, huTLR8 signaling exerted a DC-intrinsic effect leading to up-regulation of co-stimulatory molecules and subsequent T cell activation. A pathogenic role for TLR8 in human diseases was suggested by its increased expression in patients with systemic arthritis and the correlation of TLR8 expression with the elevation of IL-1ß levels and disease status. We found that the consequence of self-recognition via TLR8 results in a constellation of diseases, strikingly distinct from those related to TLR7 signaling, and points to specific inflammatory diseases that may benefit from inhibition of TLR8 in humans.
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Artritis Juvenil/metabolismo , Autoinmunidad , Inflamación/patología , ARN/química , Receptor Toll-Like 8/metabolismo , Animales , Artritis Experimental/metabolismo , Artritis Experimental/fisiopatología , Artritis Juvenil/fisiopatología , Niño , Colágeno/química , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ensayos Clínicos Controlados Aleatorios como Asunto , Transducción de Señal , Linfocitos T/inmunología , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/genética , TransgenesRESUMEN
PURPOSE OF REVIEW: The purpose of review is to describe the recent advances in the field of human epigenetics. RECENT FINDINGS: With the completion of the genome project in 2003, high expectations existed for the DNA sequence information to provide answers about the causative mutations for common diseases. However, this was not completely the case. Another interesting finding that resulted from the genome project was that the perceived level of complexity of humans was not accompanied with a relative increase in the number of genes when compared to 'lower species'. Epigenetics is able to provide answers to previously unanswered health-related questions and can explain differences in level of complexity between organisms. Epigenetic studies accomplished in the last few years have exposed a very complex multilayered regulatory mechanism that is able to answer previously puzzling questions in biology. SUMMARY: Understanding and interpretation of the role for epigenetic modifications in the human genome has progressed rapidly over the past decade with the advancement of microarray-based and sequence-based technologies. The complex interaction between DNA methylation, histone modifications, protein complexes and microRNAs has become better appreciated in the context of both local and long range epigenetic control of transcription in both normal cellular differentiation and tumorigenesis.
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Epigénesis Genética , Epigenómica/tendencias , Animales , Metilación de ADN , Epigenómica/métodos , Genoma Humano , Histonas/metabolismo , Humanos , MicroARNs/fisiologíaRESUMEN
Certain WNT and WNT network target genes are expressed at higher or lower levels in chronic lymphocytic leukemia compared with normal B-cells. This includes upregulation of nuclear complex genes, as well as genes for cytoplasmic proteins and WNT ligands and their cognate receptors. In addition, epigenetic silencing of several negative regulators of the WNT pathway have been identified. The balance between epigenetic downregulation of negative effector genes and increased expression of positive effector genes demonstrate that the epigenetic downregulation of WNT antagonists is one mechanism, perhaps the main mechanism, that is permissive to active WNT signaling in chronic lymphocytic leukemia. Moreover, constitutive activation of the WNT network and target genes is likely to impact on additional interacting signaling pathways. Based on published studies, we propose a model of WNT signaling that involves mainly permissive expression, and sometimes overexpression, of positive effectors and downregulation of negative regulators in the network. In this model, DNA methylation, histone modifications and altered expression of microRNA molecules interact to allow continuous WNT signaling.
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PAPA syndrome (Pyogenic Arthritis, Pyoderma gangrenosum, and Acne) is an autosomal dominant, hereditary auto-inflammatory disease arising from mutations in the PSTPIP1/CD2BP1 gene on chromosome 15q. These mutations produce a hyper-phosphorylated PSTPIP1 protein and alter its participation in activation of the "inflammasome" involved in interleukin-1 (IL-1ß) production. Overproduction of IL-1ß is a clear molecular feature of PAPA syndrome. Ongoing research is implicating other biochemical pathways that may be relevant to the distinct pyogenic inflammation of the skin and joints characteristic of this disease. This review summarizes the recent and rapidly accumulating knowledge on these molecular aspects of PAPA syndrome and related disorders.
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BACKGROUND: Follicular lymphoma (FL) is a form of non-Hodgkin's lymphoma (NHL) that arises from germinal center (GC) B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL. METHODOLOGY/PRINCIPAL FINDINGS: We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19(+) B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19(+) B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs) were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19(+) B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells. CONCLUSIONS/SIGNIFICANCE: This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples.
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Metilación de ADN , Genoma Humano , Estudio de Asociación del Genoma Completo , Linfoma Folicular/genética , Análisis de Secuencia de ADN/métodos , Linfocitos B/metabolismo , Humanos , Linfoma Folicular/metabolismo , Regiones Promotoras Genéticas , Sulfitos/químicaRESUMEN
AIMS: B-cell chronic lymphocytic leukemia (CLL) is a heterogeneous malignancy that clinically ranges from indolent to rapidly progressive. CLL, like other cancers, can be affected by epigenetic alterations. MATERIALS & METHODS: A microarray discovery-based study was initiated to determine DNA methylation in CLL cases with a range of CD38 expression (192%). RESULTS: Many loci were either methylated or unmethylated across all CD38 levels, but differential methylation was also observed for some genes. Genomic sequencing of DLEU7 confirmed extensive cytosine methylation preferentially in patient samples with low CD38 expression, whereas NRP2, SFRP2 and ADAM12 were more commonly methylated in those with high CD38 expression. CONCLUSION: This study demonstrates that CLL is affected by CpG island methylation in some genes that segregate with CD38 expression levels, while most others show similar methylation patterns across all levels. The CpG island methylation in certain functional gene groups and pathway-associated genes that are known to be deregulated in CLL provides additional insights into the CLL methylome and epigenetic contribution to cellular dysfunction. It will now be useful to investigate the effectiveness of epigenetic therapeutic reversal of these alterations to develop effective treatments for the disease.
Asunto(s)
Metilación de ADN , ADN/análisis , Leucemia Linfocítica Crónica de Células B/genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM12 , ADP-Ribosil Ciclasa 1/metabolismo , Línea Celular Tumoral , Análisis por Conglomerados , Islas de CpG , Epigénesis Genética , Sitios Genéticos , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Proteínas de Neoplasias , Neuropilina-2/genética , Neuropilina-2/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Secuencia de ADN , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Dendritic cells (DCs) loaded with killed allogeneic melanoma cells can cross-prime naive CD8(+) T cells to differentiate into melanoma-specific CTLs in 3-wk cultures. In this study we show that DCs loaded with killed melanoma cells that were heated to 42 degrees C before killing are more efficient in cross-priming of naive CD8(+) T cells than DCs loaded with unheated killed melanoma cells. The enhanced cross-priming was demonstrated by several parameters: 1) induction of naive CD8(+) T cell differentiation in 2-wk cultures, 2) enhanced killing of melanoma peptide-pulsed T2 cells, 3) enhanced killing of HLA-A*0201(+) melanoma cells in a standard 4-h chromium release assay, and 4) enhanced capacity to prevent tumor growth in vitro in a tumor regression assay. Two mechanisms might explain the hyperthermia-induced enhanced cross-priming. First, heat-treated melanoma cells expressed increased levels of 70-kDa heat shock protein (HSP70), and enhanced cross-priming could be reproduced by overexpression of HSP70 in melanoma cells transduced with HSP70 encoding lentiviral vector. Second, hyperthermia resulted in the increased transcription of several tumor Ag-associated Ags, including MAGE-B3, -B4, -A8, and -A10. Thus, heat treatment of tumor cells permits enhanced cross-priming, possibly via up-regulation of both HSPs and tumor Ag expression.