RESUMEN
DNA methylation analysis is increasingly used in stress research. Available methods are expensive, laborious and often limited by either the analysis of short CpG stretches or low assay sensitivity. Here, we present a cost-efficient next generation sequencing-based strategy for the simultaneous investigation of multiple candidate genes in large cohorts. To illustrate the method, we present analysis of four candidate genes commonly assessed in psychoneuroendocrine research: Glucocorticoid receptor (NR3C1), Serotonin transporter (SLC6A4), FKBP Prolyl isomerase 5 (FKBP5), and the Oxytocin receptor (OXTR). DNA methylation standards (100 %; 75 %; 50 %; 25 % and 0 %) and DNA of a female and male donor were bisulfite treated in three independent trials and were used to generate sequencing libraries for 42 CpGs from the NR3C1 1 F promoter region, 84 CpGs of the SLC6A4 5' regulatory region, 5 CpGs located in FKBP5 intron 7, and additional 12 CpGs located in a potential enhancer element in intron 3 of the OXTR. In addition, DNA of 45 patients with borderline personality disorder (BPD) and 45 healthy controls was assayed. Multiplex libraries of all samples were sequenced on a MiSeq system and analyzed for mean methylation values of all CpG sites using amplikyzer2 software. Results indicated excellent accuracy of the assays when investigating replicates generated from the same bisulfite converted DNA, and very high linearity (R2 > 0.9) of the assays shown by the analysis of differentially methylated DNA standards. Comparing DNA methylation between BPD and healthy controls revealed no biologically relevant differences. The technical approach as described here facilitates targeted DNA methylation analysis and represents a highly sensitive, cost-efficient and high throughput tool to close the gap between coverage and precision in epigenetic research of stress-associated phenotypes.
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Metilación de ADN/genética , Epigenómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuencia de Bases/genética , Islas de CpG/genética , ADN/química , Humanos , Regiones Promotoras Genéticas/genética , Receptores de Glucocorticoides/análisis , Receptores de Oxitocina/análisis , Receptores de Oxitocina/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/análisis , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Sulfitos/química , Proteínas de Unión a Tacrolimus/análisis , Proteínas de Unión a Tacrolimus/genéticaRESUMEN
Classical Hodgkin lymphoma (cHL) and anaplastic large cell lymphoma (ALCL) feature high expression of activator protein-1 (AP-1) transcription factors, which regulate various physiological processes but also promote lymphomagenesis. The AP-1 factor basic leucine zipper transcription factor, ATF-like 3 (BATF3), is highly transcribed in cHL and ALCL; however, its functional importance in lymphomagenesis is unknown. Here we show that proto-typical CD30+ lymphomas, namely cHL (21/30) and primary mediastinal B-cell lymphoma (8/9), but also CD30+ diffuse large B-cell lymphoma (15/20) frequently express BATF3 protein. Mass spectrometry and co-immunoprecipitation established interactions of BATF3 with JUN and JUNB in cHL and ALCL lines. BATF3 knockdown using short hairpin RNAs was toxic for cHL and ALCL lines, reducing their proliferation and survival. We identified MYC as a critical BATF3 target and confirmed binding of BATF3 to the MYC promoter. JAK/STAT signaling regulated BATF3 expression, as chemical JAK2 inhibition reduced and interleukin 13 stimulation induced BATF3 expression in cHL lines. Chromatin immunoprecipitation substantiated a direct regulation of BATF3 by STAT proteins in cHL and ALCL lines. In conclusion, we identified STAT-mediated BATF3 expression that is essential for lymphoma cell survival and promoted MYC activity in cHL and ALCL, hence we recognized a new oncogenic axis in these lymphomas.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Enfermedad de Hodgkin/genética , Linfoma Anaplásico de Células Grandes/genética , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción STAT/genética , Regulación hacia Arriba/genética , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular , Regulación Neoplásica de la Expresión Génica/genética , Enfermedad de Hodgkin/patología , Humanos , Janus Quinasa 2/genética , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Linfoma Anaplásico de Células Grandes/patología , Oncogenes/genética , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/genética , Transducción de Señal/genética , Factor de Transcripción AP-1/genética , Activación Transcripcional/genéticaRESUMEN
BACKROUND: Pneumocystis jirovecii pneumonia (PCP) is an opportunistic fungal infection that is associated with a high morbidity and mortality in immunocompromised individuals. In this study, we analysed the microbiome of the lower respiratory tract from critically ill intensive care unit patients with and without pneumocystosis. METHODS: Broncho-alveolar fluids from 65 intubated and mechanically ventilated intensive care unit patients (34 PCP+ and 31 PCP- patients) were collected. Sequence analysis of bacterial 16S rRNA gene V3/V4 regions was performed to study the composition of the respiratory microbiome using the Illumina MiSeq platform. RESULTS: Differences in the microbial composition detected between PCP+ and PCP- patients were not statistically significant on class, order, family and genus level. In addition, alpha and beta diversity metrics did not reveal significant differences between PCP+ and PCP- patients. The composition of the lung microbiota was highly variable between PCP+ patients and comparable in its variety with the microbiota composition of the heterogeneous collective of PCP- patients. CONCLUSIONS: The lower respiratory tract microbiome in patients with pneumocystosis does not appear to be determined by a specific microbial composition or to be dominated by a single bacterial species.
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Pulmón/microbiología , Microbiota , Neumonía por Pneumocystis/microbiología , ARN Ribosómico 16S/análisis , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Líquido del Lavado Bronquioalveolar/microbiología , Estudios de Casos y Controles , Femenino , Humanos , Unidades de Cuidados Intensivos , Intubación Intratraqueal , Masculino , Persona de Mediana Edad , Respiración Artificial , Estudios Retrospectivos , Adulto JovenRESUMEN
Genetic and epigenetic aberrations contribute to the initiation and progression of acute myeloid leukemia (AML). GFI1, a zinc-finger transcriptional repressor, exerts its function by recruiting histone deacetylases to target genes. We present data that low expression of GFI1 is associated with an inferior prognosis of AML patients. To elucidate the mechanism behind this, we generated a humanized mouse strain with reduced GFI1 expression (GFI1-KD). Here we show that AML development induced by onco-fusion proteins such as MLL-AF9 or NUP98-HOXD13 is accelerated in mice with low human GFI1 expression. Leukemic cells from animals that express low levels of GFI1 show increased H3K9 acetylation compared to leukemic cells from mice with normal human GFI1 expression, resulting in the upregulation of genes involved in leukemogenesis. We investigated a new epigenetic therapy approach for this subgroup of AML patients. We could show that AML blasts from GFI1-KD mice and from AML patients with low GFI1 levels were more sensitive to treatment with histone acetyltransferase inhibitors than cells with normal GFI1 expression levels. We suggest therefore that GFI1 has a dose-dependent role in AML progression and development. GFI1 levels are involved in epigenetic regulation, which could open new therapeutic approaches for AML patients.
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Proteínas de Unión al ADN/biosíntesis , Epigénesis Genética , Leucemia Mieloide Aguda/metabolismo , Síndromes Mielodisplásicos/metabolismo , Factores de Transcripción/biosíntesis , Acetilación , Animales , Carcinogénesis/genética , Proteínas de Unión al ADN/deficiencia , Progresión de la Enfermedad , Inhibidores Enzimáticos/uso terapéutico , Histona Acetiltransferasas/antagonistas & inhibidores , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Síndromes Mielodisplásicos/genética , Proteínas de Fusión Oncogénica , Pronóstico , Factores de Transcripción/deficienciaRESUMEN
CONTEXT: Multiple endocrine neoplasia type 2 (MEN2) is usually caused by missense mutations in the proto-oncogene, RET. OBJECTIVE: This study aimed to determine the mutation underlying MEN2A in a female patient diagnosed with bilateral pheochromocytoma at age 31 years and with medullary thyroid carcinoma (MTC) 6 years later. METHODS: Leukocyte DNA was used for exome and Sanger sequencing. Wild-type (WT) RET and mutants were expressed in HEK293 cells. Activation of MAPK/ERK and PI3K/AKT was analyzed by Western blotting and luciferase assay. The effect of RET mutants on cell proliferation was tested in a colony forming assay. RESULTS: Exome sequencing revealed a 6-nucleotide/2-amino acid in-frame deletion in exon 7 of RET (c.1512_1517delGGAGGG, p.505_506del). In vitro expression showed that phosphorylation of the crucial tyrosine 905 was much stronger in the p.505_506del RET mutant compared with WT RET, indicating ligand-independent autophosphorylation. Furthermore, the p.505_506del RET mutant induced a strong activation of the MAPK/ERK pathway and the PI3K/AKT pathway. Consequently, the p.505_506del RET mutant cells increased HEK293 colony formation 4-fold compared with WT RET. CONCLUSION: The finding of bilateral pheochromocytoma and MTC in our patient was highly suspicious of a RET mutation. Exome sequencing revealed a 6-base-pair deletion in exon 7 of RET, an exon not yet associated with MEN2. Increased ligand-independent phosphorylation of the p.505_506del RET mutant, increased activation of downstream pathways, and stimulation of cell proliferation demonstrated the pathogenic nature of the mutation. We therefore recommend screening the whole sequence of RET in MTC and pheochromocytoma patients with red flags for a genetic cause.
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Emparejamiento Base/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Neoplasia Endocrina Múltiple Tipo 2a/genética , Proteínas Proto-Oncogénicas c-ret/genética , Eliminación de Secuencia/genética , Neoplasias de las Glándulas Suprarrenales/genética , Neoplasias de las Glándulas Suprarrenales/patología , Adulto , Carcinoma Neuroendocrino/genética , Activación Enzimática , Exones/genética , Femenino , Mutación de Línea Germinal , Células HEK293 , Humanos , Mutación Missense , Feocromocitoma/genética , Feocromocitoma/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-ret/metabolismo , Análisis de Secuencia de ADN , Neoplasias de la Tiroides/genéticaRESUMEN
Neuroblastoma, a childhood cancer that originates from neural crest-derived cells, is the most common deadly solid tumor of infancy. Amplification of the MYCN oncogene, which occurs in approximately 20-25% of human neuroblastomas, is the most prominent genetic marker of high-stage disease. The availability of valid preclinical in vivo models is a prerequisite to develop novel targeted therapies. We here report on the generation of transgenic mice with Cre-conditional induction of MYCN in dopamine ß-hydroxylase-expressing cells, termed LSL-MYCN;Dbh-iCre. These mice develop neuroblastic tumors with an incidence of >75%, regardless of strain background. Molecular profiling of tumors revealed upregulation of the MYCN-dependent miR-17-92 cluster as well as expression of neuroblastoma marker genes, including tyrosine hydroxylase and the neural cell adhesion molecule 1. Gene set enrichment analyses demonstrated significant correlation with MYC-associated expression patterns. Array comparative genome hybridization showed that chromosomal aberrations in LSL-MYCN;Dbh-iCre tumors were syntenic to those observed in human neuroblastomas. Treatment of a cell line established from a tumor derived from a LSL-MYCN;Dbh-iCre mouse with JQ1 or MLN8237 reduced cell viability and demonstrated oncogene addiction to MYCN. Here we report establishment of the first Cre-conditional human MYCN-driven mouse model for neuroblastoma that closely recapitulates the human disease with respect to tumor localization, histology, marker expression and genomic make up. This mouse model is a valuable tool for further functional studies and to assess the effect of targeted therapies.
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Evaluación Preclínica de Medicamentos/métodos , Integrasas/genética , Neuroblastoma/patología , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Regiones Promotoras Genéticas , Transgenes , Animales , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Análisis por Micromatrices , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/genética , Células Tumorales CultivadasRESUMEN
Nager syndrome (MIM #154400) is the best-known preaxial acrofacial dysostosis, mainly characterized by craniofacial and preaxial limb anomalies. The craniofacial abnormalities mainly consist of downslanting palpebral fissures, malar hypoplasia, micrognathia, external ear anomalies, and cleft palate. The preaxial limb defects are characterized by radial and thumb hypoplasia or aplasia, duplication of thumbs and proximal radioulnar synostosis. Haploinsufficiency of SF3B4 (MIM *605593), which encodes SAP49, a component of the pre-mRNA spliceosomal complex, has recently been identified as the underlying cause of Nager syndrome. In our study, we performed exome sequencing in two and Sanger sequencing of SF3B4 in further ten previously unreported patients with the clinical diagnosis of Nager syndrome, including one familial case. We identified heterozygous SF3B4 mutations in seven out of twelve patients. Four of the seven mutations were shown to be de novo; in three individuals, DNA of both parents was not available. No familial mutations were discovered. Three mutations were nonsense, three were frameshift mutations and one T > C transition destroyed the translation start signal. In three of four SF3B4 negative families, EFTUD2 was analyzed, but no pathogenic variants were identified. Our results indicate that the SF3B4 gene is mutated in about half of the patients with the clinical diagnosis of Nager syndrome and further support genetic heterogeneity for this condition.
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Exoma/genética , Disostosis Mandibulofacial/genética , Mutación/genética , Precursores del ARN/genética , Proteínas de Unión al ARN/genética , Empalmosomas/genética , Adolescente , Adulto , Preescolar , Femenino , Estudios de Asociación Genética , Humanos , Lactante , Masculino , Disostosis Mandibulofacial/diagnóstico , Factores de Empalme de ARNRESUMEN
Soft tissue sarcomas (STS) are a heterogeneous group of malignant tumours representing 1% of all malignancies in adults. Therapy for STS should be individualised and multimodal, but complete surgical resection with clear margins remains the mainstay of therapy. Disseminated soft tissue sarcoma still represents a therapeutic dilemma. Commonly used chemotherapeutic agents such as doxorubicin and ifosfamide have proven to be effective in fewer than 30% in these cases. Therefore, we tested the apoptotic and anti-proliferative in vitro effects of TNF-related apoptosis-inducing ligand (TRAIL) and taurolidine (TRD) on rhabdomyosarcoma (A-204), leiomyosarcoma (SK-LMS-1) and epithelioid cell sarcoma (VA-ES-BJ) cell lines. Viability, apoptosis and necrosis were quantified by FACS analysis (propidium iodide/Annexin V staining). Gene expression was analysed by DNA microarrays and the results validated for selected genes by rtPCR. Protein level changes were documented by western blot analysis. Cell proliferation was analysed by BrdU ELISA assay. The single substances TRAIL and TRD significantly induced apoptotic cell death and decreased proliferation in rhabdomyosarcoma and epithelioid cell sarcoma cells. The combined use of TRAIL and TRD resulted in a synergistic apoptotic effect in all three cell lines, especially in rhabdomyosarcoma cells leaving 18% viable cells after 48 h of incubation (p<0.05). Analysis of the differentially regulated genes revealed that TRD and TRAIL influence apoptotic pathways, including the TNF-receptor associated and the mitochondrial pathway. Microarray analysis revealed remarkable expression changes in a variety of genes, which are involved in different apoptotic pathways and cross talk to other pathways at multiple levels. This in vitro study demonstrates that TRAIL and TRD synergise in inducing apoptosis and inhibiting proliferation in different human STS cell lines. Effects on gene expression differ relevantly in the sarcoma entities. These results provide experimental support for in vivo trials assessing the effect of TRAIL and TRD in STS and sustain the approach of individualized therapy.
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Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/farmacología , Sarcoma/tratamiento farmacológico , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Taurina/análogos & derivados , Tiadiazinas/farmacología , Apoptosis/efectos de los fármacos , Proteínas de Ciclo Celular/biosíntesis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Expresión Génica/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/biosíntesis , Humanos , Leiomiosarcoma/tratamiento farmacológico , Proteínas Nucleares/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína Fosfatasa 1/biosíntesis , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Recombinantes/farmacología , Rabdomiosarcoma/tratamiento farmacológico , Taurina/farmacologíaRESUMEN
BACKGROUND: Disseminated fibrosarcoma still represents a therapeutic dilemma because of lack of effective cytostatics. Therefore we tested tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and taurolidine, in combination with established and new chemotherapeutic agents on human fibrosarcoma (HT1080). MATERIALS AND METHODS: Human fibrosarcoma cells (HT1080) were incubated with doxorubicin, mafosfamide and trabectedin both alone and in combination with taurolidine and TRAIL. Vital, apoptotic and necrotic cells were quantified using flow cytometric analysis. Cell proliferation was analysed using a bromodeoxyuridine (BrdU) ELISA assay. RESULTS: Single application of doxorubicin and trabectedin induced apoptotic cell death and significantly reduced the proliferation of HT1080 cells. In combination treatment, the addition of taurolidine and TRAIL resulted in a stronger reduction in the degree of cell viability when compared to single treatment. Trabectedin and taurolidine displayed a greater potential for inhibiting proliferation than did doxorubicin alone. CONCLUSION: When combined with TRAIL and taurolidine, treatment with doxorubicin and trabectedin demonstrated stronger apoptosis-inducing and antiproliferative effects.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Fibrosarcoma/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Ciclofosfamida/administración & dosificación , Ciclofosfamida/análogos & derivados , Ciclofosfamida/farmacología , Dioxoles/administración & dosificación , Dioxoles/farmacología , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Sinergismo Farmacológico , Fibrosarcoma/patología , Humanos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Taurina/administración & dosificación , Taurina/análogos & derivados , Taurina/farmacología , Tetrahidroisoquinolinas/administración & dosificación , Tetrahidroisoquinolinas/farmacología , Tiadiazinas/administración & dosificación , Tiadiazinas/farmacología , TrabectedinaRESUMEN
SPC2996 is a novel locked nucleic acid phosphorothioate antisense molecule targeting the mRNA of the Bcl-2 oncoprotein. We investigated the mechanism of action of SPC2996 and the basis for its clinically observed immunostimulatory effects in chronic lymphocytic leukemia (CLL). Patients with relapsed CLL were treated with a maximum of six doses of SPC2996 (0.2-6 mg/kg) in a multicenter phase I trial. Microarray-based transcriptional profiling of circulating CLL cells was carried out before and after the first infusion of SPC2996 in 18 patients. Statistically significant transcriptomic changes were observed at doses î¶4 mg/kg and occurred as early as 24 h after the first infusion of the oligonucleotide. SPC2996 induced the upregulation of 466 genes including a large number of immune response and apoptotic regulator molecules, which were enriched for Toll-like receptor response genes. Serum measurements confirmed the release of pro-inflammatory cytokines including chemokine (C-C motif) ligand 3 (macrophage inflammatory protein 1α) and tumor necrosis factor-α, thereby validating the in vivo transcriptomic data at the protein level. SPC2996 caused a î¶50% reduction of circulating lymphocytes in five of 18 (28%) patients, which was found to be independent of its immunostimulatory and anti-Bcl-2 effects.
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Biomarcadores de Tumor/genética , Citocinas/metabolismo , Regulación Leucémica de la Expresión Génica/genética , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/terapia , Oligorribonucleótidos Antisentido/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/inmunología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Recently developed molecular prognostic tests in patients with early Binet stage chronic lymphocytic leukemia (B-CLL) are costly and often require a high level of technologic expertise. Recent data give evidence for the prognostic relevance of the percentage of smudge cells in B-CLL. In our study we analysed the prognostic potential of this novel marker in a cohort of 100 CLL patients. The percentage of smudge cells ranged from 0% to 70% (median 21%). Patients with
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Linfocitos B/ultraestructura , Leucemia Linfocítica Crónica de Células B/sangre , ADP-Ribosil Ciclasa 1/sangre , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Muerte Celular , Aberraciones Cromosómicas , Pruebas Diagnósticas de Rutina , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Péptidos y Proteínas de Señalización Intracelular/sangre , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/mortalidad , Leucemia Linfocítica Crónica de Células B/patología , Recuento de Linfocitos , Masculino , Glicoproteínas de Membrana/sangre , Persona de Mediana Edad , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/sangre , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Vimentina/sangre , Vimentina/fisiología , Proteína Tirosina Quinasa ZAP-70/sangreRESUMEN
Myxoid/round cell liposarcoma are characterized by typical chromosomal translocations. This genetic alteration might result in specific gene-expression profiles in this tumor entity. To identify over-expressed genes in myxoid/round cell liposarcoma DNA microarray analysis was performed on four tumors and four samples of adult fat tissue. Genes ret, cdk4, cyclin D2 and c-myc showed over-expression by means of microarray analysis and Northern blotting. Immunohistochemistry demonstrated cytoplasmic localization of associated proteins in 36 different tumors. The localization of ret was seen in endothelial cells of plexiform vasculature in addition to its accumulation in tumor cells (25% of cases). The results show an over-expression of cdk4, cyclin D2, c-myc and ret on both the transcriptional and protein level in myxoid/round cell liposarcoma. For cyclin D2 and ret this finding has not been reported in this tumor type. The increase of ret on transcriptional level might be explained by its expression in endothelium in intratumoral plexiform blood vessels. For the molecular pathogenesis of myxoid/round cell liposarcoma our findings imply the involvement of these four genes in the deregulation of the cell cycle, especially as cdk4 and cyclin D2 are target genes of c-myc.
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Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica/genética , Marcadores Genéticos/genética , Liposarcoma Mixoide/genética , Liposarcoma Mixoide/patología , Liposarcoma/genética , Liposarcoma/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias de los Tejidos Blandos/genética , Neoplasias de los Tejidos Blandos/patología , Ciclina D2/genética , Quinasa 4 Dependiente de la Ciclina/genética , Perfilación de la Expresión Génica , Humanos , Liposarcoma/clasificación , Liposarcoma Mixoide/clasificación , Técnicas de Diagnóstico Molecular , Pronóstico , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-ret/genética , Neoplasias de los Tejidos Blandos/clasificación , Translocación Genética/genéticaAsunto(s)
Amidohidrolasas/deficiencia , Emparejamiento Base/genética , Enfermedad de Canavan/genética , Cromosomas Humanos Par 17/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Polimorfismo de Nucleótido Simple/genética , Eliminación de Secuencia/genética , Alelos , Amidohidrolasas/genética , Enfermedad de Canavan/enzimología , Niño , Exones/genética , Homocigoto , Humanos , Masculino , Reacción en Cadena de la PolimerasaRESUMEN
Microarray-based gene expression profiling (GEP) was used to study how stroma modulates the survival of CLL cells in an in vitro coculture model employing the murine fibroblast cell line M2-10B4. CLL cells cultured in direct contact with the stromal layer (STR) showed a significantly better survival than cells cultured in transwell (TW) inserts above the M2-10B4 cells. STR as compared to TW conditions induced a significant up-regulation of PI3K/NF-kappaB pro-survival pathway genes and mediated a pro-angiogenetic switch in the CLL cells by up-regulation of vascular endothelial growth factor (VEGF) and osteopontin (OPN) and down-regulation of the anti-angiogenetic molecule thrombospondin-1 (TSP-1).
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Proteínas Angiogénicas/metabolismo , Fibroblastos/fisiología , Leucemia Linfocítica Crónica de Células B/metabolismo , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Apoptosis , Células de la Médula Ósea/citología , Comunicación Celular , Ciclo Celular/genética , Línea Celular , Técnicas de Cocultivo , Reparación del ADN/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/genética , Ratones , Fenotipo , Transducción de Señal/genética , Células del Estroma/fisiología , Células Tumorales CultivadasRESUMEN
To identify specific markers of rectovaginal endometriotic nodule vasculature, highly enriched preparations of vascular endothelial cells and pericytes were obtained from endometriotic nodules and control endometrial and myometrial tissue by laser capture microdissection (LCM), and gene expression profiles were screened by microarray analysis. Of the 18 400 transcripts on the arrays, 734 were significantly overexpressed in vessels from fibromuscular tissue and 923 in vessels from stromal tissue of endometriotic nodules, compared with vessels dissected from control tissues. The most frequently expressed transcripts included known endothelial cell-associated genes, as well as transcripts with little or no previous association with vascular cells. The higher expression in blood vessels was further corroborated by immunohistochemical staining of six potential markers, five of which showed strong expression in pericytes. The most promising marker was matrix Gla protein, which was found to be present in both glandular epithelial cells and vascular endothelial cells of endometriotic lesions, although it was barely expressed at all in normal endometrium. LCM, combined with microarray analysis, constitutes a powerful tool for mapping the transcriptome of vascular cells. After immunohistochemical validation, markers of vascular endothelial and perivascular cells from endometriotic nodules could be identified, which may provide targets to improve early diagnosis or to selectively deliver therapeutic agents.
Asunto(s)
Antígenos/inmunología , Vasos Sanguíneos/inmunología , Endometriosis/inmunología , Adulto , Antígenos/genética , Antígenos/metabolismo , Vasos Sanguíneos/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Endometriosis/genética , Endometriosis/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Humanos , Inmunohistoquímica , Microdisección , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Gla de la MatrizRESUMEN
T-cell prolymphocytic leukemia (T-PLL) is a rare aggressive lymphoma derived from mature T cells, which is, in most cases, characterized by the presence of an inv(14)(q11q32)/t(14;14)(q11;q32) and a characteristic pattern of secondary chromosomal aberrations. DNA microarray technology was employed to compare the transcriptomes of eight immunomagnetically purified CD3+ normal donor-derived peripheral blood cell samples, with five highly purified inv(14)/t(14;14)-positive T-PLL blood samples. Between the two experimental groups, 734 genes were identified as differentially expressed, including functionally important genes involved in lymphomagenesis, cell cycle regulation, apoptosis and DNA repair. Notably, the differentially expressed genes were found to be significantly enriched in genomic regions affected by recurrent chromosomal imbalances. Upregulated genes clustered on chromosome arms 6p and 8q, and downregulated genes on 6q, 8p, 10p, 11q and 18p. High-resolution copy-number determination using single nucleotide polymorphism chip technology in 12 inv(14)/t(14;14)-positive T-PLL including those analyzed for gene expression, refined chromosomal breakpoints as well as regions of imbalances. In conclusion, combined transcriptional and molecular cytogenetic profiling identified novel specific chromosomal loci and genes that are likely to be involved in disease progression and suggests a gene dosage effect as a pathogenic mechanism in T-PLL.
Asunto(s)
Inversión Cromosómica , Mapeo Cromosómico/métodos , Cromosomas Humanos Par 14 , Perfilación de la Expresión Génica , Leucemia Prolinfocítica/genética , Leucemia de Células T/genética , Polimorfismo de Nucleótido Simple , Apoptosis , Complejo CD3/biosíntesis , Aberraciones Cromosómicas , Reparación del ADN , Progresión de la Enfermedad , Dosificación de Gen , Humanos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Drug resistance constitutes a considerable problem in the therapy of acute myeloid leukemia (AML). In order to identify genes which might be related to drug resistance, we retrospectively studied gene expression patterns in blast populations of 14 patients with de novo AML, focusing on known or potential resistance mechanisms against cytosine arabinoside and anthracyclines. Following induction and postremission chemotherapy, 7 patients achieved a complete remission (CR) for more than 1 year, while 7 patients showed blast persistence (BP) after induction and salvage chemotherapy. Gene expression analysis was performed using RNA extracted from archived guanidine extracts and Affymetrix HGU133A gene chips. We utilized the Gene Ontology category Biological Process to select genes implicated in DNA metabolism, nucleoside and nucleotide metabolism and transport, reactive oxygen species metabolism, apoptosis and response to drugs and identified 32 differentially expressed genes. From this functional perspective, we found differences between the CR and BP groups with regard to nucleotide metabolism (PBEF1, G6PD; p = 0.048), apoptosis (TNFAIP3, TNFAIP8, MPO, BCL2A1, BAX, SON, BNIP3L; p = 0.039) and reactive oxygen species metabolism (SOD2, KIAA0179; p = 0.048). However, the attempt to construct a predictive model of chemoresistance failed. BP samples had a 2-fold higher expression of CD34 than CR samples. Thus, our findings are in line with reports describing differences in apoptosis resistance between CD34+ and CD34- blast populations. Taken together, our results suggest that drug resistance in AML is a heterogenous phenomenon that might be better defined by means of disturbed biological processes than by focusing on the alteration of the expression of distinct genes.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Leucemia Mieloide/genética , Proteínas de Neoplasias/biosíntesis , Células Madre Neoplásicas/metabolismo , Enfermedad Aguda , Adolescente , Adulto , Antígenos CD34/análisis , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/patología , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/química , Nucleósidos/metabolismo , Nucleótidos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Inducción de Remisión , Terapia Recuperativa , Superóxidos/metabolismo , Resultado del TratamientoRESUMEN
Tobacco smoking is the leading cause of lung cancer worldwide. Gene expression in surgically resected and microdissected samples of non-small-cell lung cancers (18 squamous cell carcinomas and nine adenocarcinomas), matched normal bronchial epithelium, and peripheral lung tissue from both smokers (n = 22) and non-smokers (n = 5) was studied using the Affymetrix U133A array. A subset of 15 differentially regulated genes was validated by real-time PCR or immunohistochemistry. Hierarchical cluster analysis clearly distinguished between benign and malignant tissue and between squamous cell carcinomas and adenocarcinomas. The bronchial epithelium and adenocarcinomas could be divided into the two subgroups of smokers and non-smokers. By comparison of the gene expression profiles in the bronchial epithelium of non-smokers, smokers, and matched cancer tissues, it was possible to identify a signature of 23 differentially expressed genes, which might reflect early cigarette smoke-induced and cancer-relevant molecular lesions in the central bronchial epithelium of smokers. Ten of these genes are involved in xenobiotic metabolism and redox stress (eg AKR1B10, AKR1C1, and MT1K). One gene is a tumour suppressor gene (HLF); two genes act as oncogenes (FGFR3 and LMO3); two genes are involved in matrix degradation (MMP12 and PTHLH); three genes are related to cell differentiation (SPRR1B, RTN1, and MUC7); and five genes have not been well characterized to date. By comparison of the tobacco-exposed peripheral alveolar lung tissue of smokers with non-smokers and with adenocarcinomas from smokers, it was possible to identify a signature of 27 other differentially expressed genes. These genes are involved in the metabolism of xenobiotics (eg GPX2 and FMO3) and may represent cigarette smoke-induced, cancer-related molecular targets that may be utilized to identify smokers with increased risk for lung cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Fumar/efectos adversos , Adenocarcinoma/etiología , Adenocarcinoma/genética , Adenocarcinoma/patología , Adulto , Anciano , Bronquios/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/etiología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Alveolos Pulmonares/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Fumar/metabolismoRESUMEN
B-cell chronic lymphocytic leukaemia (B-CLL) is a heterogenous disease with a highly variable clinical course and analysis of zeta-associated protein 70 (ZAP-70) and CD38 expression on B-CLL cells allowed for identification of patients with good (ZAP-70-CD38-) and poor (ZAP-70+CD38+) prognosis. DNA microarray technology was employed to compare eight ZAP-70+CD38+ with eight ZAP-70-CD38- B-CLL cases. The expression of 358 genes differed significantly between the two subgroups, including genes involved in B-cell receptor signaling, angiogenesis and lymphomagenesis. Three of these genes, that is, immune receptor translocation-associated protein 4 (IRTA4)/Fc receptor homologue 2 (FcRH2), angiopoietin 2 (ANGPT2) and Pim2 were selected for further validating studies in a cohort of 94 B-CLL patients. IRTA4/FcRH2 expression as detected by flow cytometry was significantly lower in the poor prognosis subgroup as compared to ZAP-70-CD38- B-CLL cells. In healthy individuals, IRTA4/FcRH2 protein expression was associated with a CD19+CD27+ memory cell phenotype. ANGPT2 plasma concentrations were twofold higher in the poor prognosis subgroup (P<0.05). Pim2 was significantly overexpressed in poor prognosis cases and Binet stage C. Disease progression may be related to proangiogenic processes and strong Pim2 expression.