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1.
Lab Invest ; 93(5): 611-21, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23459372

RESUMEN

Metabolic adaptations and changes in the expression of nutrient transporters are known to accompany tumorigenic processes. Nevertheless, in the context of solid tumors, studies of metabolism are hindered by a paucity of tools allowing the identification of cell surface transporters on individual cells. Here, we developed a method for the dissociation of human breast cancer tumor xenografts combined with quantification of cell surface markers, including metabolite transporters. The expression profiles of four relevant nutrient transporters for cancer cells' metabolism, Glut1, ASCT2, PiT1 and PiT2 (participating to glucose, glutamine and inorganic phosphate, respectively), as detected by new retroviral envelope glycoprotein-derived ligands, were distinctive of each tumor, unveiling underlying differences in metabolic pathways. Our tumor dissociation procedure and nutrient transporter profiling technology provides opportunities for future basic research, clinical diagnosis, prognosis and evaluation of therapeutic responses, as well as for drug discovery and development.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Citometría de Flujo/métodos , Proteínas de Transporte de Membrana/metabolismo , Análisis de Varianza , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Supervivencia Celular/fisiología , Femenino , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Inmunohistoquímica/métodos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Reproducibilidad de los Resultados , Trasplante Heterólogo
2.
Eur J Immunol ; 39(7): 1966-76, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19548248

RESUMEN

Complete lack of function of the tyrosine kinase ZAP70 in humans results in a severe immunodeficiency, characterized by a lack of mature CD8(+) T cells and non-functional CD4(+) T cells. We report herein an immunodeficiency with an inherited hypomorphic mutation of ZAP70 due to a single G-to-A substitution in a non-coding intron. This mutation introduces a new acceptor splice site and allows low levels of normal alternative splicing and of WT ZAP70 expression. This partial deficiency results in a compromised TCR signaling that was totally restored by increased expression of ZAP70, demonstrating that defective activation of the patient T cells was indeed caused by the low level of ZAP70 expression. This partial ZAP70 deficiency was associated with an attenuated clinical and immunological phenotype as compared with complete ZAP70 deficiency. CD4(+) helper T-cell populations including, follicular helper T cells, Th1, Th17 and Treg were detected in the blood. Finally, the patient had no manifestation of autoimmunity suggesting that the T-cell tolerogenic functions were not compromised, in contrast to what has been observed in mice carrying hypomorphic mutations of Zap70. This report extends the phenotype spectrum of ZAP70 deficiency with a residual function of ZAP70.


Asunto(s)
Síndromes de Inmunodeficiencia/genética , Mutación Puntual , Proteína Tirosina Quinasa ZAP-70/genética , Secuencia de Aminoácidos , Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Calcio/metabolismo , Niño , Análisis Mutacional de ADN , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Síndromes de Inmunodeficiencia/inmunología , Síndromes de Inmunodeficiencia/metabolismo , Recuento de Linfocitos , Masculino , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Transfección , Proteína Tirosina Quinasa ZAP-70/metabolismo
3.
Glia ; 57(13): 1450-7, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19243017

RESUMEN

Neural crest (NC) cells are a multipotent, highly migratory cell population that generates most of the components of the peripheral nervous system (PNS), including the glial Schwann cells (SC) and boundary cap (BC) cells. These latter cells are located at the interface between the central nervous system and PNS, at the exit/entry points of ventral motor/dorsal sensory axons and give rise to all SC in the nerve roots and to a subset of nociceptive neurons and satellite cells in the dorsal root ganglia. In the present study we have compared BC cells with two closely related cell types, NC and Schwann cell precursors (SCpr), by RNA profiling. This led to the definition of a set of 10 genes that show specific expression in BC cells and/or in their derivatives along the nerve roots. Analysis of the expression of these genes during mouse development revealed novel features, of those most important are: (i) dorsal and ventral nerve root BC cell derivatives express different sets of genes, suggesting that they have distinct properties; (ii) these cells undergo major modifications in their gene expression pattern between embryonic days 14.5 and 17.5, possibly linked to the SCpr-immature Schwann cell transition; (iii) nerve roots SC differ from more distal SC not only in their origins and locations, but also in their gene expression patterns. In conclusion, the identification of these novel makers opens the way for a detailed characterization of BC cells in both mouse and man.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Cresta Neural/embriología , Cresta Neural/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores/metabolismo , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN/metabolismo , Receptores del Factor Natriurético Atrial/genética , Receptores del Factor Natriurético Atrial/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Células de Schwann/fisiología , Raíces Nerviosas Espinales/embriología , Raíces Nerviosas Espinales/fisiología , Células Madre/fisiología
4.
Curr Protoc Immunol ; 117: 5.4.1-5.4.38, 2017 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-28369683

RESUMEN

Multicolor flow cytometry is a rapidly evolving technology that uses multiple fluorescent markers to identify and characterize cellular subpopulations of interest, allowing rapid analysis on tens of thousands of cells per second, with the possibility of isolating pure, viable populations by cell sorting for further experimentation. This unit covers the tools needed by the beginning immunologist to plan and run multicolor experiments, with information on fluorochromes and their characteristics, spectral spillover, compensation and spread, instrument and reagent variables, and the basic elements of multicolor panel design. Protocols to quantify and maximize sensitivity by titration of reagents and optimization of instrument settings, as well as basic surface and intracellular cell staining, are included. © 2017 by John Wiley & Sons, Inc.


Asunto(s)
Citometría de Flujo/métodos , Animales , Anticuerpos Monoclonales , Biomarcadores , Citometría de Flujo/normas , Colorantes Fluorescentes , Humanos , Coloración y Etiquetado
5.
PLoS One ; 11(7): e0157670, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27388901

RESUMEN

Drug discovery efforts have focused on the tumor microenvironment in recent years. However, few studies have characterized the stroma component in patient-derived xenografts (PDXs) and genetically engineered mouse models (GEMs). In this study, we characterized the stroma in various models of breast cancer tumors in mice. We performed transcriptomic and flow cytometry analyses on murine populations for a series of 25 PDXs and the two most commonly used GEMs (MMTV-PyMT and MMTV-erBb2). We sorted macrophages from five models. We then profiled gene expression in these cells, which were also subjected to flow cytometry for phenotypic characterization. Hematopoietic cell composition, mostly macrophages and granulocytes, differed between tumors. Macrophages had a specific polarization phenotype related to their M1/M2 classification and associated with the expression of genes involved in the recruitment, invasion and metastasis processes. The heterogeneity of the stroma component of the models studied suggests that tumor cells modify their microenvironment to satisfy their needs. Our observations suggest that such models are of relevance for preclinical studies.


Asunto(s)
Neoplasias de la Mama/fisiopatología , Macrófagos/citología , Neoplasias Mamarias Animales/fisiopatología , Animales , Separación Celular , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Metástasis de la Neoplasia , Fenotipo , Receptor ErbB-2/metabolismo , Transcriptoma , Microambiente Tumoral/genética
6.
Cancer Cytopathol ; 119(6): 411-23, 2011 Dec 25.
Artículo en Inglés | MEDLINE | ID: mdl-21774092

RESUMEN

BACKGROUND: The use of cytological material for diagnosis and prognosis in patients with neuroblastic tumors is poorly described in the literature. METHODS: A total of 129 patients with primary neuroblastic tumors underwent sampling with fine-needle aspiration for diagnosis and the evaluation of prognostic parameters. Of these, 125 (97%) were neuroblastomas or ganglioneuroblastomas and 4 (3%) were ganglioneuromas. Cellularity of the smears was assessed, as well as the percentage of neuroblasts versus stroma, maturation (differentiating/mature vs immature cells), the mitosis-karyorrhexis index (MKI), and the number of Homer Wright rosettes. The cytology samples were also analyzed for MYCN amplification, flow cytometric DNA index, and array comparative genomic hybridization. RESULTS: MYCN was found to be amplified in 35 (27%) cases. Strong correlations with overall survival were found for MYCN amplification in localized stages including IVs (P < .001), the percentage of neuroblasts versus stroma (P < .001), maturation (P = .002), MKI (P < .001), and DNA index (P < .03). CONCLUSIONS: Poorly differentiated tumors with few stromal components and low numbers of differentiating/mature cells were found to be MYCN amplified. The authors propose a "cytology prognostic score" in which neuroblastomas comprised of predominantly neuroblastic elements with no differentiating cells and an MKI >; 2% are classified as high-risk tumors, regardless of their MYCN amplification status. The use of this proposed score would ensure accurate and optimal diagnostic and prognostic classifications of neuroblastic tumors in cases in which the histological biopsy is absent or inadequate for analysis.


Asunto(s)
Biopsia con Aguja Fina/métodos , Hibridación Genómica Comparativa/métodos , Ganglioneuroblastoma , Ganglioneuroma , Neuroblastoma , Adulto , Anciano , Anciano de 80 o más Años , Recuento de Células , Diferenciación Celular , Transformación Celular Neoplásica , ADN de Neoplasias/análisis , Femenino , Citometría de Flujo , Ganglioneuroblastoma/diagnóstico , Ganglioneuroblastoma/patología , Ganglioneuroma/diagnóstico , Ganglioneuroma/patología , Humanos , Masculino , Neuroblastoma/diagnóstico , Neuroblastoma/patología , Pronóstico , Adulto Joven
7.
Cell Cycle ; 4(12): 1842-6, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16294040

RESUMEN

Neuroblastoma (NB) is a frequent paediatric extra cranial solid tumor characterized by the occurrence of unbalanced chromosome translocations, frequently, but not exclusively, involving chromosomes 1 and 17. We have used a 1 Mb resolution BAC array to further refine the mapping of breakpoints in NB cell lines. Replication timing profiles were evaluated in 7 NB cell lines, using DNAs from G1 and S phases flow sorted nuclei hybridised on the same array. Strikingly, these replication timing profiles were highly similar between the different NB cell lines. Furthermore, a significant level of similarity was also observed between NB cell lines and lymphoblastoid cells. A segmentation analysis using the Adaptative Weights Smoothing procedure was performed to determine regions of coordinate replication. More than 50% of the breakpoints mapped to early replicating regions, which account for 23.7% of the total genome. The breakpoints frequency per 10(8) bases was therefore 10.84 for early replicating regions, whereas it was only 2.94 for late replicating regions, these difference being highly significant (p < 10(-4)). This strong association was also observed when chromosomes 1 and 17, the two most frequent translocation partners in NB were excluded from the statistical analysis. These results unambiguously establish a link between unbalanced translocations, whose most likely mechanism of occurrence relies on break-induced replication, and early replication of the genome.


Asunto(s)
Rotura Cromosómica/genética , Momento de Replicación del ADN/genética , Neuroblastoma/genética , Línea Celular Tumoral , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 17/genética , Genoma Humano/genética , Humanos , Neuroblastoma/patología , Fase S/genética , Translocación Genética/genética , Células Tumorales Cultivadas
8.
Blood ; 105(12): 4776-83, 2005 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-15718417

RESUMEN

Nonhomologous end-joining (NHEJ) DNA factors maintain genomic stability through their DNA double-strand break (DSB) repair and telomere-associated activities. Unrepaired or misrepaired DSBs can lead to apoptotic death or chromosomal damage. The B cells of some B-chronic lymphocytic leukemia (B-CLL) patients are resistant to radiation-induced apoptosis in vitro. We show here that the novel DNA-dependent protein kinase (DNA-PK) inhibitor, NU7026 (2-(morpholin-4-yl)-benzo[h]chomen-4-one), and the phosphatidylinositol 3 (PI-3) kinase inhibitor, wortmannin, restored sensitivity to DNA damage-induced apoptosis of otherwise resistant cells. These resistant malignant B cells also escaped DSB-induced apoptosis following exposure to etoposide or neocarzinostatin. We found that at 15 minutes after irradiation, the levels of NHEJ (as measured by an in vitro DSB end-ligation assay) and DNA-PK catalytic subunit (DNA-PKcs) activity were, respectively, 2-fold and 4-fold higher in radio-resistant than in radio-sensitive B-CLL cells or Epstein-Barr virus (EBV)-transformed B cells. Ku70/Ku80 heterodimer DNA end-binding activity was also 2- to 3-fold higher in the resistant B-CLL cell subset compared with the sensitive B-CLL cell subset. Our results provide the first evidence that overactivating the NHEJ DNA repair pathway impairs DNA damage-induced apoptosis in malignant B cells and that this may contribute to their resistance to current chemotherapy.


Asunto(s)
Apoptosis , Reparación del ADN , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/patología , Androstadienos/farmacología , Antibióticos Antineoplásicos/farmacología , Antígenos Nucleares/metabolismo , Antineoplásicos Fitogénicos/farmacología , Linfocitos B/patología , Western Blotting , Línea Celular Transformada , Línea Celular Tumoral , Sistema Libre de Células , Cromonas/farmacología , ADN/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Dimerización , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Etopósido/farmacología , Rayos gamma , Humanos , Autoantígeno Ku , Morfolinas/farmacología , Ácido Ocadaico/farmacología , Fosfatidilinositol 3-Quinasas/farmacología , Unión Proteica , Telómero/ultraestructura , Factores de Tiempo , Wortmanina , Cinostatina/farmacología
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