RESUMEN
Colorectal cancers (CRCs) form a heterogenous group classified into epigenetic and transcriptional subtypes. The basis for the epigenetic subtypes, exemplified by varying degrees of promoter DNA hypermethylation, and its relation to the transcriptional subtypes is not well understood. We link cancer-specific transcription factor (TF) expression alterations to methylation alterations near TF-binding sites at promoter and enhancer regions in CRCs and their premalignant precursor lesions to provide mechanistic insights into the origins and evolution of the CRC molecular subtypes. A gradient of TF expression changes forms a basis for the subtypes of abnormal DNA methylation, termed CpG-island promoter DNA methylation phenotypes (CIMPs), in CRCs and other cancers. CIMP is tightly correlated with cancer-specific hypermethylation at enhancers, which we term CpG-enhancer methylation phenotype (CEMP). Coordinated promoter and enhancer methylation appears to be driven by downregulation of TFs with common binding sites at the hypermethylated enhancers and promoters. The altered expression of TFs related to hypermethylator subtypes occurs early during CRC development, detectable in premalignant adenomas. TF-based profiling further identifies patients with worse overall survival. Importantly, altered expression of these TFs discriminates the transcriptome-based consensus molecular subtypes (CMS), thus providing a common basis for CIMP and CMS subtypes.
Asunto(s)
Neoplasias Colorrectales , Lesiones Precancerosas , Humanos , Factores de Transcripción , Regulación de la Expresión Génica , Metilación de ADN , Epigénesis GenéticaRESUMEN
Ovarian cancer is the most lethal of all gynecological cancers, and there is an urgent unmet need to develop new therapies. Epithelial ovarian cancer (EOC) is characterized by an immune suppressive microenvironment, and response of ovarian cancers to immune therapies has thus far been disappointing. We now find, in a mouse model of EOC, that clinically relevant doses of DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi, respectively) reduce the immune suppressive microenvironment through type I IFN signaling and improve response to immune checkpoint therapy. These data indicate that the type I IFN response is required for effective in vivo antitumorigenic actions of the DNMTi 5-azacytidine (AZA). Through type I IFN signaling, AZA increases the numbers of CD45+ immune cells and the percentage of active CD8+ T and natural killer (NK) cells in the tumor microenvironment, while reducing tumor burden and extending survival. AZA also increases viral defense gene expression in both tumor and immune cells, and reduces the percentage of macrophages and myeloid-derived suppressor cells in the tumor microenvironment. The addition of an HDACi to AZA enhances the modulation of the immune microenvironment, specifically increasing T and NK cell activation and reducing macrophages over AZA treatment alone, while further increasing the survival of the mice. Finally, a triple combination of DNMTi/HDACi plus the immune checkpoint inhibitor α-PD-1 provides the best antitumor effect and longest overall survival, and may be an attractive candidate for future clinical trials in ovarian cancer.
Asunto(s)
Epigénesis Genética/efectos de los fármacos , Inmunomodulación/efectos de los fármacos , Interferón Tipo I/metabolismo , Neoplasias Ováricas/etiología , Neoplasias Ováricas/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos Inmunológicos , Azacitidina/farmacología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Inhibidores de Histona Desacetilasas/farmacología , Ratones , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Carga Tumoral/efectos de los fármacos , Carga Tumoral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Breast cancers are comprised of molecularly distinct subtypes that may respond differently to pathway-targeted therapies now under development. Collections of breast cancer cell lines mirror many of the molecular subtypes and pathways found in tumors, suggesting that treatment of cell lines with candidate therapeutic compounds can guide identification of associations between molecular subtypes, pathways, and drug response. In a test of 77 therapeutic compounds, nearly all drugs showed differential responses across these cell lines, and approximately one third showed subtype-, pathway-, and/or genomic aberration-specific responses. These observations suggest mechanisms of response and resistance and may inform efforts to develop molecular assays that predict clinical response.
Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Neoplasias de la Mama/genética , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Dosificación de Gen/genética , Humanos , Modelos Biológicos , Transducción de Señal/genética , Transcripción Genética/efectos de los fármacosRESUMEN
Biallelic inactivation of cancer susceptibility gene BRCA1 leads to breast and ovarian carcinogenesis. Paradoxically, BRCA1 deficiency in mice results in early embryonic lethality, and similarly, lack of BRCA1 in human cells is thought to result in cellular lethality in view of BRCA1's essential function. To survive homozygous BRCA1 inactivation during tumorigenesis, precancerous cells must accumulate additional genetic alterations, such as p53 mutations, but this requirement for an extra genetic "hit" contradicts the two-hit theory for the accelerated carcinogenesis associated with familial cancer syndromes. Here, we show that heterozygous BRCA1 inactivation results in genomic instability in nontumorigenic human breast epithelial cells in vitro and in vivo. Using somatic cell gene targeting, we demonstrated that a heterozygous BRCA1 185delAG mutation confers impaired homology-mediated DNA repair and hypersensitivity to genotoxic stress. Heterozygous mutant BRCA1 cell clones also showed a higher degree of gene copy number loss and loss of heterozygosity in SNP array analyses. In BRCA1 heterozygous clones and nontumorigenic breast epithelial tissues from BRCA mutation carriers, FISH revealed elevated genomic instability when compared with their respective controls. Thus, BRCA1 haploinsufficiency may accelerate hereditary breast carcinogenesis by facilitating additional genetic alterations.
Asunto(s)
Mama/citología , Células Epiteliales/fisiología , Genes BRCA1 , Inestabilidad Genómica/genética , Haploinsuficiencia/genética , Femenino , Silenciador del Gen , Inestabilidad Genómica/fisiología , Heterocigoto , Humanos , Hibridación Fluorescente in Situ , Polimorfismo de Nucleótido Simple , Eliminación de Secuencia/genéticaRESUMEN
We attempted to answer two central questions about epigenetic silencing of the tumor suppressor gene p16(INK4a) in this study: (1) whether the maintenance of associated histone modifications is dependent on DNA methylation and (2) whether such histone modifications can occur prior to DNA methylation. By coupling chromatin immunoprecipitation with gene targeting and the analysis of specific alleles, we found that elimination of DNA methylation from a p16(INK4a) allele resulted in profound changes in surrounding histones. After continued passage of such cells, methylation of histone H3 lysine-9 occurred in conjunction with re-silencing in the absence of DNA methylation. These results have important implications for understanding the biochemical events underlying the silencing of tumor suppressor genes and the resultant growth suppression.
Asunto(s)
Metilación de ADN , Silenciador del Gen , Genes p16 , Histonas/metabolismo , División Celular/genética , Cromatografía Líquida de Alta Presión , Neoplasias Colorrectales/genética , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , Marcación de Gen , Histonas/genética , Humanos , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Células Tumorales Cultivadas , ADN Metiltransferasa 3BRESUMEN
The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. Here we used gene targeting to "knock in" PIK3CA mutations into human breast epithelial cells to identify new therapeutic targets associated with oncogenic PIK3CA. Mutant PIK3CA knockin cells were capable of epidermal growth factor and mTOR-independent cell proliferation that was associated with AKT, ERK, and GSK3beta phosphorylation. Paradoxically, the GSK3beta inhibitors lithium chloride and SB216763 selectively decreased the proliferation of human breast and colorectal cancer cell lines with oncogenic PIK3CA mutations and led to a decrease in the GSK3beta target gene CYCLIN D1. Oral treatment with lithium preferentially inhibited the growth of nude mouse xenografts of HCT-116 colon cancer cells with mutant PIK3CA compared with isogenic HCT-116 knockout cells containing only wild-type PIK3CA. Our findings suggest GSK3beta is an important effector of mutant PIK3CA, and that lithium, an FDA-approved therapy for bipolar disorders, has selective antineoplastic properties against cancers that harbor these mutations.
Asunto(s)
Mutación , Oncogenes , Fosfatidilinositol 3-Quinasas/genética , Animales , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Técnicas de Sustitución del Gen , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Glándulas Mamarias Humanas/metabolismo , Ratones , Ratones Desnudos , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR , Trasplante HeterólogoRESUMEN
We aimed to investigate the expression pattern of phosphatase and tensin homolog (PTEN), to evaluate the relationship between PTEN expression and clinicopathological characteristics, including fatty acid synthase (FAS) expression, and to determine the correlations of PTEN and FAS expression with survival in Chinese patients with hepatocellular carcinoma (HCC). The expression patterns of PTEN and FAS were determined using tissue microarrays and immunohistochemistry. The expression of PTEN was compared with the clinicopathological characteristics of HCC, including FAS expression. Receiver operator characteristic curves were used to calculate the clinical sensitivity and specificity of PTEN expression. Kaplan-Meier survival curves were constructed to evaluate the correlations of PTEN loss and FAS overexpression with overall survival. We found that the loss of PTEN expression occurred predominantly in the cytoplasm, while FAS was mainly localized to the cytoplasm. Cytoplasmic and total PTEN expression levels were significantly decreased in HCC compared with adjacent non-neoplastic tissue (both, p < 0.0001). Decreased cytoplasmic and total PTEN expression showed significant clinical sensitivity and specificity for HCC (both, p < 0.0001). Downregulation of PTEN in HCC relative to non-neoplastic tissue was significantly correlated with histological grade (p = 0.043 for histological grades I-II versus grade III). Loss of total PTEN was significantly correlated with FAS overexpression (p = 0.014). Loss of PTEN was also associated with poor prognosis of patients with poorly differentiated HCC (p = 0.049). Moreover, loss of PTEN combined with FAS overexpression was associated with significantly worse prognosis compared with other HCC cases (p = 0.011). Our data indicate that PTEN may serve as a potential diagnostic and prognostic marker of HCC. Upregulating PTEN expression and inhibiting FAS expression may offer a novel therapeutic approach for HCC.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Acido Graso Sintasa Tipo I/metabolismo , Neoplasias Hepáticas/metabolismo , Fosfohidrolasa PTEN/metabolismo , Adulto , Anciano , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/secundario , China , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Metástasis Linfática , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica , Estadificación de Neoplasias , Pronóstico , Tasa de SupervivenciaRESUMEN
Tamoxifen is widely used for the treatment of hormonally responsive breast cancers. However, some resistant breast cancers develop a growth proliferative response to this drug, as evidenced by tumor regression upon its withdrawal. To elucidate the molecular mediators of this paradox, tissue samples from a patient with tamoxifen-stimulated breast cancer were analyzed. These studies revealed that loss of the cyclin-dependent kinase inhibitor p21 was associated with a tamoxifen growth-inducing phenotype. Immortalized human breast epithelial cells with somatic deletion of the p21 gene were then generated and displayed a growth proliferative response to tamoxifen, whereas p21 wild-type cells demonstrated growth inhibition upon tamoxifen exposure. Mutational and biochemical analyses revealed that loss of p21's cyclin-dependent kinase inhibitory property results in hyperphosphorylation of estrogen receptor-alpha, with subsequent increased gene expression of estrogen receptor-regulated genes. These data reveal a previously uncharacterized molecular mechanism of tamoxifen resistance and have potential clinical implications for the management of tamoxifen-resistant breast cancers.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Receptor alfa de Estrógeno/metabolismo , Tamoxifeno/farmacología , Línea Celular Tumoral , Proliferación Celular , Metilación de ADN , Análisis Mutacional de ADN , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Persona de Mediana Edad , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Resultado del TratamientoRESUMEN
Selective cyclooxygenase (COX)-2 inhibitors have been extensively studied for colorectal cancer (CRC) chemoprevention. Celecoxib has been reported to reduce the incidence of colorectal adenomas and CRC but is also associated with an increased risk of cardiovascular events. Here, we report a series of gut-restricted, selective COX-2 inhibitors characterized by high colonic exposure and minimized systemic exposure. By establishing acute ex vivo 18F-FDG uptake attenuation as an efficacy proxy, we identified a subset of analogues that demonstrated statistically significant in vivo dose-dependent inhibition of adenoma progression and survival extension in an APCmin/+ mouse model. However, in vitro-in vivo correlation analysis showed their chemoprotective effects were driven by residual systemic COX-2 inhibition, rationalizing their less than expected efficacies and highlighting the challenges associated with COX-2-mediated CRC disease chemoprevention.
Asunto(s)
Antineoplásicos/farmacología , Celecoxib/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa 2/farmacología , Ciclooxigenasa 2/metabolismo , Etoricoxib/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Celecoxib/química , Celecoxib/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Inhibidores de la Ciclooxigenasa 2/química , Inhibidores de la Ciclooxigenasa 2/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Etoricoxib/química , Etoricoxib/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Inactivation of the Fanconi anemia (FA) pathway occurs in diverse human tumors among the general population and renders those tumors hypersensitive to DNA interstrand-cross-linking (ICL) agents. The identification of novel agents to which FA pathway-deficient cells were hypersensitive could provide new therapeutic opportunities and improve our molecular understanding of the FA genes. Using high-throughput screening, we assessed the growth of isogenic human cancer cells that differed only in the presence or absence of single FA genes upon treatment with 880 active drugs and 40,000 diverse compounds. We identified several compounds to which FA pathway-deficient cells were more sensitive than FA pathway-proficient cells, including two groups of structurally related compounds. We further investigated the compound eliciting the strongest effect, termed 80136342. Its mechanism of action was distinct from that of ICL agents; 80136342 did not cause increased chromosomal aberrations, enhanced FANCD2 monoubiquitination, H2AX phosphorylation, p53 activation, or ICL induction. Similar to ICL agents, however, 80136342 caused a pronounced G(2) arrest in FA pathway-deficient cells. When applied in combination with ICL agents, 80136342 had at least additive toxic effects, excluding interferences on ICL-induced toxicity and facilitating a combinational application. Finally, we identified one particular methyl group necessary for the effects of 80136342 on FA-deficient cells. In conclusion, using high-throughput screening in an isogenic human FA cancer model, we explored a novel approach to identify agents eliciting hypersensitivity in FA pathway-deficient cells. We discovered several attractive candidates to serve as lead compounds for evaluating structure-activity relationships and developing therapeutics selectively targeting FA pathway-deficient tumors.
Asunto(s)
Antineoplásicos/análisis , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Neoplasias/genética , Transducción de Señal/efectos de los fármacos , Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Rotura Cromosómica/efectos de los fármacos , Reactivos de Enlaces Cruzados/farmacología , Evaluación Preclínica de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Histonas/metabolismo , Humanos , Modelos Biológicos , Neoplasias/tratamiento farmacológico , Piridinas/uso terapéutico , Quinolinas/uso terapéutico , Células Tumorales CultivadasRESUMEN
The oncogenic function of mutant ras in mammalian cells has been extensively investigated using multiple human and animal models. These systems include overexpression of exogenous mutant ras transgenes, conditionally expressed knock-in mouse models, and somatic cell knockout of mutant and wild-type ras genes in human cancer cell lines. However, phenotypic discrepancies between knock-in mice and transgenic mutant ras overexpression prompted us to evaluate the consequences of targeted knock-in of an oncogenic K-ras mutation in the nontumorigenic human breast epithelial cell line MCF-10A and hTERT-immortalized human mammary epithelial cells. Our results show several significant differences between mutant K-ras knock-in cells versus their transgene counterparts, including limited phosphorylation of the downstream molecules extracellular signal-regulated kinase and AKT, minor proliferative capacity in the absence of an exogenous growth factor, and the inability to form colonies in semisolid medium. Analysis of 16 cancer cell lines carrying mutant K-ras genes indicated that 50% of cancer cells harbor nonoverexpressed heterozygous K-ras mutations similar to the expression seen in our knock-in cell lines. Thus, this system serves as a new model for elucidating the oncogenic contribution of mutant K-ras as expressed in a large fraction of human cancer cells.
Asunto(s)
Neoplasias de la Mama/genética , Transformación Celular Neoplásica/genética , Genes ras/genética , Mutación , Alelos , Mama/metabolismo , Mama/patología , Mama/fisiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Células Epiteliales/fisiología , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Telomerasa/genética , Transgenes , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas ras/biosíntesis , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Although ovarian cancer has a low incidence rate, it remains the most deadly gynecologic malignancy. Previous work has demonstrated that the DNMTi 5-Azacytidine (5AZA-C) activates type I interferon signaling to increase IFNγ+ T cells and natural killer (NK) cells and reduce the percentage of macrophages in the tumor microenvironment. To improve the efficacy of epigenetic therapy, we hypothesized that the addition of α-difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor, may further decrease immunosuppressive cell populations improving outcome. We tested this hypothesis in an immunocompetent mouse model for ovarian cancer and found that in vivo, 5AZA-C and DFMO, either alone or in combination, significantly increased survival, decreased tumor burden, and caused recruitment of activated (IFNγ+) CD4+ T cells, CD8+ T cells, and NK cells. The combination therapy had a striking increase in survival when compared with single-agent treatment, despite a smaller difference in recruited lymphocytes. Instead, combination therapy led to a significant decrease in immunosuppressive cells such as M2 polarized macrophages and an increase in tumor-killing M1 macrophages. In this model, depletion of macrophages with a CSF1R-blocking antibody reduced the efficacy of 5AZA-C + DFMO treatment and resulted in fewer M1 macrophages in the tumor microenvironment. These observations suggest our novel combination therapy modifies macrophage polarization in the tumor microenvironment, recruiting M1 macrophages and prolonging survival. SIGNIFICANCE: Combined epigenetic and polyamine-reducing therapy stimulates M1 macrophage polarization in the tumor microenvironment of an ovarian cancer mouse model, resulting in decreased tumor burden and prolonged survival.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Cistadenocarcinoma Seroso/inmunología , Modelos Animales de Enfermedad , Inmunidad Innata/inmunología , Macrófagos/inmunología , Neoplasias Ováricas/inmunología , Microambiente Tumoral/inmunología , Animales , Azacitidina/administración & dosificación , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patología , Eflornitina/administración & dosificación , Femenino , Inmunidad Innata/efectos de los fármacos , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Poliaminas/metabolismo , Células Tumorales Cultivadas , Microambiente Tumoral/efectos de los fármacosRESUMEN
MCT-1 is an oncogene that was initially identified in a human T cell lymphoma and has been shown to induce cell proliferation as well as activate survival-related pathways. MCT-1 contains the PUA domain, a recently described RNA-binding domain that is found in several tRNA and rRNA modification enzymes. Here, we established that MCT-1 protein interacts with the cap complex through its PUA domain and recruits the density-regulated protein (DENR/DRP), containing the SUI1 translation initiation domain. Through the use of microarray analysis on polysome-associated mRNAs, we showed that up-regulation of MCT-1 was able to modulate the translation profiles of BCL2L2, TFDP1, MRE11A, cyclin D1, and E2F1 mRNAs, despite equivalent levels of mRNAs in the cytoplasm. Our data establish a role for MCT-1 in translational regulation, and support a linkage between translational control and oncogenesis.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Biosíntesis de Proteínas/fisiología , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , Animales , Proteínas de Ciclo Celular/biosíntesis , Transformación Celular Neoplásica/genética , Factores Eucarióticos de Iniciación/genética , Factores Eucarióticos de Iniciación/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Oncogénicas/biosíntesis , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
A major obstacle toward understanding how patterns of abnormal mammalian cytosine DNA methylation are established is the difficulty in quantitating the de novo methylation activities of DNA methyltransferases (DNMT) thought to catalyze these reactions. Here, we describe a novel method, using native human CpG island substrates from genes that frequently become hypermethylated in cancer, which generates robust activity for measuring de novo CpG methylation. We then survey colon cancer cells with genetically engineered deficiencies in different DNMTs and find that the major activity against these substrates in extracts of these cells is DNMT1, with minor contribution from DNMT 3b and none from DNMT3a, the only known bona fide de novo methyltransferases. The activity of DNMT1 against unmethylated CpG rich DNA was further tested by introducing CpG island substrates and DNMT1 into Drosophila melanogaster cells. The exogenous DNMT1 methylates the integrated mammalian CpG islands but not the Drosophila DNA. Additionally, in human cancer cells lacking DNMT1 and DNMT3b and having nearly absent genomic methylation, gene-specific de novo methylation can be initiated by reintroduction of DNMT1. Our studies provide a new assay for de novo activity of DNMTs and data suggesting a potential role for DNMT1 in the initiation of promoter CpG island hypermethylation in human cancer cells.
Asunto(s)
Neoplasias del Colon/genética , Islas de CpG , ADN (Citosina-5-)-Metiltransferasas/fisiología , Metilación de ADN , Animales , Neoplasias del Colon/patología , ADN (Citosina-5-)-Metiltransferasa 1 , Drosophila/genética , Ingeniería Genética , Humanos , Células Tumorales CultivadasRESUMEN
A model of B cell affinity selection is proposed, and an explanation of peripheral tolerance mechanisms through antibody repertoire editing is presented. We show that affinity discrimination between B cells is driven by a competition between obtaining T cell help and removal of B cells from the light zone, either through apoptosis or by a return to the dark zone of germinal centers. We demonstrate that this mechanism also allows for the negative selection of self reactive B cells and maintenance of B cell tolerance during the Germinal Center reaction. Finally, we demonstrate that clonal expansion upon return to the Germinal Center dark zone amplifies differences in the antigen affinity of B cells that survive the light zone.
Asunto(s)
Afinidad de Anticuerpos , Linfocitos B/inmunología , Centro Germinal/inmunología , Modelos Inmunológicos , Linfocitos T/inmunología , Animales , Antígenos/metabolismo , Apoptosis , Simulación por Computador , Epítopos de Linfocito B/metabolismo , Epítopos de Linfocito T/metabolismo , Tolerancia InmunológicaRESUMEN
Epigenetics contributes to the pathogenesis of immune-mediated diseases like rheumatoid arthritis (RA). Here we show the first comprehensive epigenomic characterization of RA fibroblast-like synoviocytes (FLS), including histone modifications (H3K27ac, H3K4me1, H3K4me3, H3K36me3, H3K27me3, and H3K9me3), open chromatin, RNA expression and whole-genome DNA methylation. To address complex multidimensional relationship and reveal epigenetic regulation of RA, we perform integrative analyses using a novel unbiased method to identify genomic regions with similar profiles. Epigenomically similar regions exist in RA cells and are associated with active enhancers and promoters and specific transcription factor binding motifs. Differentially marked genes are enriched for immunological and unexpected pathways, with "Huntington's Disease Signaling" identified as particularly prominent. We validate the relevance of this pathway to RA by showing that Huntingtin-interacting protein-1 regulates FLS invasion into matrix. This work establishes a high-resolution epigenomic landscape of RA and demonstrates the potential for integrative analyses to identify unanticipated therapeutic targets.
Asunto(s)
Artritis Reumatoide/genética , Epigénesis Genética , Fibroblastos/metabolismo , Sinoviocitos/metabolismo , Adulto , Anciano , Artritis Reumatoide/metabolismo , Cromatina/genética , Cromatina/metabolismo , Metilación de ADN , Femenino , Código de Histonas , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Metilación , Persona de Mediana Edad , Regiones Promotoras GenéticasRESUMEN
RNA interference (RNAi) has become a popular tool for analyzing gene function in cancer research. The feasibility of using RNAi in cellular and animal models as an alternative to conventional gene knock out approaches has been demonstrated. Although these studies show that RNAi can recapitulate phenotypes seen in knock out animals and their derived cell lines, a systematic study rigorously comparing downstream effector genes between RNAi and gene knock out has not been performed. Here we present data contrasting the phenotypic and genotypic changes that occur with either stable knock down via RNAi of the cyclin dependent kinase inhibitor p21 versus its somatic cell knock out counterpart in the human mammary epithelial cell line MCF-10A. Our results demonstrate that p21 knock down clones display a growth proliferative response upon exposure to Transforming Growth Factor-Beta Type 1 (TGFbeta) similar to p21 knock out clones. However, gene expression profiles were significantly different in p21 knock down cells versus p21 knock out clones. Importantly p21 knock down clones did not display increased gene expression of interleukin-1alpha (IL-1alpha), a critical effector of this growth response previously validated in p21 knock out cells. We conclude that gene knock out can yield additional vital information that may be missed with gene knock down strategies.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Línea Celular Tumoral , Humanos , Interferencia de ARN , ARN Interferente Pequeño/genética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
DNA promoter hypermethylation has been shown to be a functional mechanism of transcriptional repression. This epigenetic gene silencing is thought to involve the recruitment of chromatin-remodeling factors, such as histone deacetylases, to methylated DNA via a family of proteins called methyl-CpG binding proteins (MBD1 to -4). MBD1, a member of this family, exhibits transcription-repressive activity, but to this point no interacting protein partners have been identified. In this study, we demonstrate that MBD1 partners with the p150 subunit of chromatin assembly factor 1 (CAF-1), forming a multiprotein complex that also contains HP1alpha. The MBD1-CAF-1 p150 interaction requires the methyl-CpG binding domain of MBD1, and the association occurs in the C terminus of CAF-1 p150. The two proteins colocalize to regions of dense heterochromatin in mouse cells, and overexpression of the C terminus of CAF-1 p150 prevents the targeting of MBD1 in these cells without disrupting global heterochromatin structure. This interaction suggests a role for MBD1 and CAF-1 p150 in methylation-mediated transcriptional repression and the inheritance of epigenetically determined chromatin states.
Asunto(s)
Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN/metabolismo , Proteínas Represoras/metabolismo , Células 3T3 , Animales , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Factor 1 de Ensamblaje de la Cromatina , Homólogo de la Proteína Chromobox 5 , Islas de CpG , Metilación de ADN , Proteínas de Unión al ADN/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Sustancias Macromoleculares , Mamíferos , Ratones , Complejos Multiproteicos , Unión Proteica , Proteínas Represoras/genética , Factores de TranscripciónRESUMEN
The dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) is a heterocyclic amine and is a common byproduct of cooked meat and fish. Although most cells undergo apoptosis when exposed to this mutagen, subsets develop resistance. Rather than die, these resistant cells persist and accumulate mutations, thereby driving tumorigenesis of exposed organs within the gastrointestinal tract. By applying a high-throughput cell-based screen of 32,000 small molecules, we have identified a family of compounds that specifically inhibit the growth of PhIP-resistant cancer cells. These compounds may prove useful for the treatment or prevention of gastrointestinal tumors arising after exposure to PhIP and related carcinogens.