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1.
Mol Cancer Ther ; 18(6): 1137-1148, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30926633

RESUMEN

Besides the detection of somatic receptor tyrosine kinases (RTK) mutations in tumor samples, the current challenge is to interpret their biological relevance to give patients effective targeted treatment. By high-throughput sequencing of the 58 RTK exons of healthy tissues, colorectal tumors, and hepatic metastases from 30 patients, 38 different somatic mutations in RTKs were identified. The mutations in the kinase domains and present in both tumors and metastases were reconstituted to perform an unbiased functional study. Among eight variants found in seven RTKs (EPHA4-Met726Ile, EPHB2-Val621Ile, ERBB4-Thr731Met, FGFR4-Ala585Thr, VEGFR3-Leu1014Phe, KIT-Pro875Leu, TRKB-Leu584Val, and NTRK2-Lys618Thr), none displayed significantly increased tyrosine kinase activity. Consistently, none of them induced transformation of NIH3T3 fibroblasts. On the contrary, two RTK variants (FGFR4-Ala585Thr and FLT4-Leu1014Phe) caused drastic inhibition of their kinase activity. These findings indicate that these RTK variants are not suitable targets and highlight the importance of functional studies to validate RTK mutations as potential therapeutic targets.

2.
Leukemia ; 33(2): 348-357, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30089916

RESUMEN

Despite constant progress in prognostic risk stratification, children with acute myeloid leukemia (AML) still relapse. Treatment failure and subsequent relapse have been attributed to acute myeloid leukemia-initiating cells (LSC), which harbor stem cell properties and are inherently chemoresistant. Although pediatric and adult AML represent two genetically very distinct diseases, we reasoned that common LSC gene expression programs are shared and consequently, the highly prognostic LSC17 signature score recently developed in adults may also be of clinical interest in childhood AML. Here, we demonstrated prognostic relevance of the LSC17 score in pediatric non-core-binding factor AML using Nanostring technology (ELAM02) and RNA-seq data from the NCI (TARGET-AML). AML were stratified by LSC17 quartile groups (lowest 25%, intermediate 50% and highest 25%) and children with low LSC17 score had significantly better event-free survival (EFS: HR = 3.35 (95%CI = 1.64-6.82), P < 0.001) and overall survival (OS: HR = 3.51 (95%CI = 1.38-8.92), P = 0.008) compared with patients with high LSC17 scores. More importantly, the high LSC17 score was an independent negative EFS and OS prognosticator determined by multivariate Cox model analysis (EFS: HR = 3.42 (95% CI = 1.63-7.16), P = 0.001; OS HR = 3.02 (95%CI = 1.16-7.85), P = 0.026). In conclusion, we have demonstrated the broad applicability of the LSC17 score in the clinical management of AML by extending its prognostic relevance to pediatric AML.


Asunto(s)
Biomarcadores de Tumor/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , Transcriptoma , Adolescente , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Lactante , Recién Nacido , Leucemia Mieloide Aguda/clasificación , Masculino , Células Madre Neoplásicas/metabolismo , Pronóstico , Tasa de Supervivencia
3.
BMC Cancer ; 18(1): 1219, 2018 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-30514258

RESUMEN

BACKGROUND: Metastatic melanoma is one of the most aggressive forms of cancer in humans. Among its types, mucosal melanomas represent one of the most highly metastatic and aggressive forms, with a very poor prognosis. Because they are rare in Caucasian individuals, unlike cutaneous melanomas, there has been fewer epidemiological, clinical and genetic evaluation of mucosal melanomas. Moreover, the lack of predictive models fully reproducing the pathogenesis and molecular alterations of mucosal melanoma makes its treatment challenging. Interestingly, dogs are frequently affected by melanomas of the oral cavity that are characterized, as their human counterparts, by focal infiltration, recurrence, and metastasis to regional lymph nodes, lungs and other organs. In dogs, some particular breeds are at high risk, suggesting a specific genetic background and strong genetic drivers. Altogether, the striking homologies in clinical presentation, histopathological features, and overall biology between human and canine mucosal melanomas make dogs invaluable natural models with which to investigate tumor development, including tumor ætiology, and develop tailored treatments. METHODS: We developed and characterized two canine oral melanoma cell lines from tumors isolated from dog patients with distinct clinical profiles; with and without lung metastases. The cells were characterized using immunohistochemistry, pharmacology and genetic studies. RESULTS: We have developed and immunohistochemically, genetically, and pharmacologically characterized. Two cell lines (Ocr_OCMM1X & Ocr_OCMM2X) were produced through mouse xenografts originating from two clinically contrasting melanomas of the oral cavity. Their exhaustive characterization showed two distinct biological and genetic profiles that are potentially linked to the stage of malignancy at the time of diagnosis and sample collection of each melanoma case. These cell lines thus constitute relevant tools with which to perform genetic and drug screening analyses for a better understanding of mucosal melanomas in dogs and humans. CONCLUSIONS: The aim of this study was to establish and characterize xenograft-derived canine melanoma cell lines with different morphologies, genetic features and pharmacological sensitivities that constitute good predictive models for comparative oncology. These cell lines are relevant tools to advance the use of canine mucosal melanomas as natural models for the benefit of both veterinary and human medicine.


Asunto(s)
Melanoma/diagnóstico por imagen , Melanoma/genética , Neoplasias de la Boca/diagnóstico por imagen , Neoplasias de la Boca/genética , Neoplasias Cutáneas/diagnóstico por imagen , Neoplasias Cutáneas/genética , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Perros , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Melanoma/tratamiento farmacológico , Ratones , Ratones Desnudos , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto/métodos
4.
J Thorac Oncol ; 13(12): 1873-1883, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30195702

RESUMEN

INTRODUCTION: Genomic alterations affecting splice sites of MNNG HOS transforming gene (MET) exon 14 were recently identified in NSCLC patients. Objective responses to MET tyrosine kinase inhibitors have been reported in these patients. Thus, detection of MET exon 14 splice site mutations represents a major challenge. So far, most of these alterations were found by full-exome sequencing or large capture-based next-generation sequencing (NGS) panels, which are not suitable for routine diagnosis. METHODS: Aiming to provide a molecular testing method applicable in routine practice, we first developed a fragment-length analysis for detecting deletions in introns flanking MET exon 14. Second, we designed an optimized targeted NGS panel called CLAPv1, covering the MET exon 14 and flanking regions in addition to the main molecular targets usually covered in genomic testing. In patients with MET exon 14 mutations, MET gene amplification, gene copy number and MET receptor expression were also determined. RESULTS: Among 1514 formalin-fixed paraffin-embedded NSCLC samples, nonoptimized NGS allowed detection of MET exon 14 mutations in only 0.3% of the patients, and fragment length analysis detected deletions in 1.1% of the patients. Combined, the optimized CLAPv1 panel and fragment-length analysis implemented for routine molecular testing revealed MET exon 14 alterations in 2.2% of 365 additional NSCLC patients. MET gene amplification or high gene copy number was observed in 6 of 30 patients (20%) harboring MET exon 14 mutations. CONCLUSIONS: These results show that optimized targeted NGS and fragment-length analysis improve detection of MET alterations in routine practice.


Asunto(s)
Adenocarcinoma del Pulmón/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Pruebas Diagnósticas de Rutina/normas , Neoplasias Pulmonares/diagnóstico , Mutación , Proteínas Proto-Oncogénicas c-met/genética , Empalme del ARN , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/metabolismo , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Variaciones en el Número de Copia de ADN , Femenino , Estudios de Seguimiento , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Persona de Mediana Edad , Pronóstico , Proteínas Proto-Oncogénicas c-met/metabolismo
5.
Immunol Lett ; 192: 27-34, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-29030252

RESUMEN

BACKGROUND: In acute myeloid leukaemia (AML)-affected patients, the presence of heterogeneous sub-clones at diagnosis has been shown to be responsible for minimal residual disease and relapses. The role played by the immune system in this leukaemic sub-clonal hierarchy and maintenance remains unknown. As leukaemic sub-clone immunogenicity could not be evaluated in human AML xenograft models, we assessed the sub-clonal diversity of the murine C1498 AML cell line and the immunogenicity of its sub-clones in immune-competent syngeneic mice. METHODOLOGY: The murine C1498 cell line was cultured in vitro and sub-clonal cells were generated after limiting dilution. The genomic profiles of 6 different sub-clones were analysed by comparative genomic hybridization arrays (CGH). The sub-clones were then injected into immune-deficient and - competent syngeneic mice. The immunogenicities of the sub-clones was evaluated through 1) assessment of mouse survival, 2) determination of leukaemic cell infiltration into organs by flow cytometry and the expression of a fluorescent reporter gene, 3) assessment of the CTL response ex vivo and 4) detection of residual leukaemic cells in the organs via amplification of the genomic reporter gene by real-time PCR (qPCR). RESULTS: Genomic analyses revealed heterogeneity among the parental cell line and its derived sub-clones. When injected individually into immune-deficient mice, all sub-clones induced cases of AML with different kinetics. However, when administered into immune-competent animals, some sub-clones triggered AML in which no mice survived, whereas others elicited reduced lethality rates. The AML-surviving mice presented efficient anti-leukaemia CTL activity ex vivo and eliminated the leukaemic cells in vivo. CONCLUSION: We showed that C1498 cell sub-clones presented genomic heterogeneity and differential immunogenicity resulting either in immune escape or elimination. Such findings could have potent implications for new immunotherapeutic strategies in patients with AML.


Asunto(s)
Antígenos de Neoplasias/inmunología , Inmunoterapia/métodos , Leucemia Mieloide Aguda/genética , Animales , Antígenos de Neoplasias/genética , Biodiversidad , Línea Celular Tumoral , Células Clonales , Citotoxicidad Inmunológica , Femenino , Humanos , Vigilancia Inmunológica , Leucemia Mieloide Aguda/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Polimorfismo Genético
6.
J Thorac Oncol ; 12(4): 724-733, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28034829

RESUMEN

INTRODUCTION: Malignant mesothelioma is a deadly disease that is strongly associated with asbestos exposure. Peritoneal mesotheliomas account for 10% of all the cases. BRCA1 associated protein 1 (BAP1) is a deubiquitinating hydrolase that plays a key role in various cellular processes. Germline and somatic inactivation of BRCA1 associated protein 1 gene (BAP1) is frequent in pleural mesothelioma; however, little is known about its status in peritoneal mesothelioma. METHODS: Taking advantage of the extensive French National Network for the Diagnosis of Malignant Pleural Mesothelioma and Rare Peritoneal Tumors and the French National Network for the Treatment of Rare Peritoneal Surface Malignancies, we collected biological material and clinical and epidemiological data for 46 patients with peritoneal mesothelioma. The status of BAP1 was evaluated at the mutational and protein expression levels and combined with our previous data on copy number alterations assessed in the same samples. RESULTS: We detected mutations in 32% of the malignant peritoneal mesotheliomas analyzed. In addition, we have previously reported that copy number losses occurred in 42% of the samples included in this series. Overall, 73% of the malignant peritoneal mesotheliomas analyzed carried at least one inactivated BAP1 allele, but only 57% had a complete loss of its protein nuclear expression. Better overall survival was observed for patients with BAP1 mutations (p = 0.04), protein expression loss (p = 0.016), or at least one of these alterations (p = 0.007) independently of tumor histological subtype, age, and sex. CONCLUSIONS: As in pleural mesothelioma, inactivation of BAP1 is frequent in peritoneal mesotheliomas. We found that BAP1 protein nuclear expression is a good prognostic factor and a more reliable marker for the complete loss of BAP1 activity than mutation or copy number loss.


Asunto(s)
Variaciones en el Número de Copia de ADN , Neoplasias Pulmonares/patología , Mesotelioma/patología , Mutación , Neoplasias Peritoneales/patología , Neoplasias Pleurales/patología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Adolescente , Adulto , Anciano , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Mesotelioma/genética , Mesotelioma/metabolismo , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Peritoneales/genética , Neoplasias Peritoneales/metabolismo , Neoplasias Pleurales/genética , Neoplasias Pleurales/metabolismo , Pronóstico , Tasa de Supervivencia , Adulto Joven
7.
Neurobiol Dis ; 96: 312-322, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27663142

RESUMEN

CAG triplet expansions in Ataxin-2 gene (ATXN2) cause spinocerebellar ataxia type 2 and have a role that remains to be clarified in Parkinson's disease (PD). To study the molecular events associated with these expansions, we sequenced them and analyzed the transcriptome from blood cells of controls and three patient groups diagnosed with spinocerebellar ataxia type 2 (herein referred to as SCA2c) or PD with or without ATXN2 triplet expansions (named SCA2p). The transcriptome profiles of these 40 patients revealed three main observations: i) a specific pattern of pathways related to cellular contacts, proliferation and differentiation associated with SCA2p group, ii) similarities between the SCA2p and sporadic PD groups in genes and pathways known to be altered in PD such as Wnt, Ephrin and Leukocyte extravasation signaling iii) RNA metabolism disturbances with "RNA-binding" and "poly(A) RNA-binding" as a common feature in all groups. Remarkably, disturbances of ALS signaling were shared between SCA2p and sporadic PD suggesting common molecular dysfunctions in PD and ALS including CACNA1, hnRNP, DDX and PABPC gene family perturbations. Interestingly, the transcriptome profiles of patients with parkinsonian phenotypes were prevalently associated with alterations of translation while SCA2c and PD patients presented perturbations of splicing. While ATXN2 RNA expression was not perturbed, its protein expression in immortalized lymphoblastoid cells was significantly decreased in SCA2c and SCA2p versus control groups assuming post-transcriptional biological perturbations. In conclusion, the transcriptome data do not exclude the role of ATXN2 mutated alleles in PD but its decrease protein expression in both SCA2c and SCA2p patients suggest a potential involvement of this gene in PD. The perturbations of "RNA-binding" and "poly(A) RNA-binding" molecular functions in the three patient groups as well as gene deregulations of factors not yet described in PD but known to be deleterious in other neurological conditions, suggest the existence of RNA-binding disturbances as a continuum between spinocerebellar ataxia type 2 and Parkinson's disease.


Asunto(s)
Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/metabolismo , ARN/metabolismo , Ataxias Espinocerebelosas/complicaciones , Ataxias Espinocerebelosas/metabolismo , Adulto , Anciano , Ataxina-2/metabolismo , Estudios de Casos y Controles , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , Transcriptoma , Expansión de Repetición de Trinucleótido/genética
9.
Sci Rep ; 6: 29636, 2016 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-27404661

RESUMEN

Mannose-binding lectin, together with mannose-associated serine proteases, activates the lectin pathway of the complement system and subsequent inflammatory mechanisms. An association between mannose-binding lectin deficiency and anti-Saccharomyces cerevisiae antibody levels is observed in Crohn's disease and this deficiency is frequently associated with a severe Crohn's disease phenotype. In the present study, we assessed the relationship between serum concentrations of mannose-binding lectin, mannose-binding lectin functional activity, MBL2 and NOD2 polymorphisms, anti-S. cerevisiae antibody levels and clinical Crohn's disease phenotype in 69 Crohn's disease patients and 30 age- and sex-matched healthy controls. The results show that the MBL2 variant rs5030737 at codon 52 was associated with a low level of mannose-binding lectin and impaired mannose-binding lectin-mannose-associated serine protease (MBL-MASP) functional activity in Crohn's disease patients. This MBL2 variant was also associated with a higher level of anti-S. cerevisiae antibodies. In addition, the NOD2 variant rs2066844, which is associated with susceptibility to Crohn's disease, was significantly correlated with an impairment in MBL-MASP functional activity. These results provide evidence that Crohn's disease patients have an impairment in MBL-MASP functional activity and that this defect is associated with MBL2 and NOD2 variants.


Asunto(s)
Enfermedad de Crohn/genética , Lectina de Unión a Manosa/genética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Proteína Adaptadora de Señalización NOD2/genética , Enfermedad de Crohn/sangre , Femenino , Genotipo , Humanos , Masculino , Lectina de Unión a Manosa/sangre , Cordón Nucal , Fenotipo , Polimorfismo de Nucleótido Simple , Saccharomyces cerevisiae/inmunología
10.
Hum Pathol ; 55: 72-82, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27184482

RESUMEN

Malignant peritoneal mesotheliomas (MPM) are rare, accounting for approximately 8% of cases of mesothelioma in France. We performed comparative genomic hybridization (CGH) on frozen MPM samples using the Agilent Human Genome CGH 180 K array. Samples were taken from a total of 33 French patients, comprising 20 men and 13 women with a mean (range) age of 58.4 (17-76) years. Asbestos exposure was reported in 8 patients (24.2%). Median (range) overall survival (OS) was 39 (0-119) months. CGH analysis demonstrated the presence of chromosomal instability in patients with MPM, with a genomic pattern that was similar to that described for pleural mesothelioma, including the loss of chromosomal regions 3p21, 9p21, and 22q12. In addition, novel genomic copy number alterations were identified, including the 15q26.2 region and the 8p11.22 region. Median OS was associated with a low peritoneal cancer index (P=.011), epithelioid subtype (P=.038), and a low number of genomic aberrations (P=.015), all of which constitute good prognostic factors for MPM. Our results provide new insights into the genetic and genomic background of MPM. Although pleural and peritoneal mesotheliomas have different risk factors, different therapeutics, and different prognosis; these data provide support to combine pleural and peritoneal mesothelioma in same clinical assays.


Asunto(s)
Biomarcadores de Tumor/genética , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , Amplificación de Genes , Dosificación de Gen , Neoplasias Pulmonares/genética , Mesotelioma/genética , Neoplasias Peritoneales/genética , Adolescente , Adulto , Anciano , Asbestos/efectos adversos , Inestabilidad Cromosómica , Femenino , Francia , Predisposición Genética a la Enfermedad , Humanos , Inmunohistoquímica , Exposición por Inhalación/efectos adversos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Masculino , Mesotelioma/mortalidad , Mesotelioma/patología , Mesotelioma/terapia , Persona de Mediana Edad , Neoplasias Peritoneales/mortalidad , Neoplasias Peritoneales/patología , Neoplasias Peritoneales/terapia , Fenotipo , Valor Predictivo de las Pruebas , Pronóstico , Modelos de Riesgos Proporcionales , Factores de Riesgo , Adulto Joven
11.
Clin Cancer Res ; 22(6): 1480-8, 2016 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-26490317

RESUMEN

PURPOSE: Whole-genome sequencing has revealed MYD88 L265P and CXCR4 mutations (CXCR4(mut)) as the most prevalent somatic mutations in Waldenström macroglobulinemia. CXCR4 mutation has proved to be of critical importance in Waldenström macroglobulinemia, in part due to its role as a mechanism of resistance to several agents. We have therefore sought to unravel the different aspects of CXCR4 mutations in Waldenström macroglobulinemia. EXPERIMENTAL DESIGN: We have scanned the two coding exons of CXCR4 in Waldenström macroglobulinemia using deep next-generation sequencing and Sanger sequencing in 98 patients with Waldenström macroglobulinemia and correlated with SNP array landscape and mutational spectrum of eight candidate genes involved in TLR, RAS, and BCR pathway in an integrative study. RESULTS: We found all mutations to be heterozygous, somatic, and located in the C-terminal domain of CXCR4 in 25% of the Waldenström macroglobulinemia. CXCR4 mutations led to a truncated receptor protein associated with a higher expression of CXCR4. CXCR4 mutations pertain to the same clone as to MYD88 L265P mutations but were mutually exclusive to CD79A/CD79B mutations (BCR pathway). We identified a genomic signature in CXCR4(mut) Waldenström macroglobulinemia traducing a more complex genome. CXCR4 mutations were also associated with gain of chromosome 4, gain of Xq, and deletion 6q. CONCLUSIONS: Our study panned out new CXCR4 mutations in Waldenström macroglobulinemia and identified a specific signature associated to CXCR4(mut), characterized with complex genomic aberrations among MYD88L265P Waldenström macroglobulinemia. Our results suggest the existence of various genomic subgroups in Waldenström macroglobulinemia.


Asunto(s)
Estudio de Asociación del Genoma Completo , Genómica , Mutación , Receptores CXCR4/genética , Macroglobulinemia de Waldenström/genética , Alelos , Sustitución de Aminoácidos , Biomarcadores , Análisis por Conglomerados , Análisis Citogenético , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo/métodos , Genómica/métodos , Genotipo , Humanos , Inmunofenotipificación , Masculino , Fenotipo , Pronóstico , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Transcriptoma , Macroglobulinemia de Waldenström/metabolismo , Macroglobulinemia de Waldenström/mortalidad
12.
Anticancer Res ; 35(11): 5983-91, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26504021

RESUMEN

BACKGROUND: The combination of irinotecan, a topoisomerase I inhibitor with cetuximab, an antibody against epidermal growth factor receptor, produces synergistic and beneficial effects in patients with irinotecan-refractory colorectal cancer. Our hypothesis was that synergistic effects could be due to anti-angiogenesis and anti-invasion, but not to cytotoxicity. MATERIALS AND METHODS: Cytotoxicity was assessed by viability test and flow cytometry. Anti-angiogenesis, anti-invasion were studied by the endothelial cell capillary-like network formation and transmigration through an extracellular matrix. Protein kinase B (PKB, frequently cited as AKT), and extracellular signal-regulated kinases (ERK) activation was assayed by cell-based enzyme-linked immunosorbent assay (ELISA). RESULTS: Combinations of SN-38 (the active of irinotecan) and cetuximab did not induce any synergistic cytotoxicity confirmed by viability test and cell-cycle analyses. Interestingly, their combination produced synergistic anti-angiogenesis and anti-invasion activities revealed by endothelial cell capillary-like network formation and cell invasion tests. Subsequently, their combination attenuated either expression or phosphorylation of AKT and ERK1/2 using cell-based ELISA. CONCLUSION: SN-38/cetuximab combination has synergistic anti-angiogenesis and anti-invasion activities mediated by down-regulation of phosphatidylinositol-3-kinases/AKT and mitogen-activated protein kinase/ERK pathways.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Movimiento Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Sinergismo Farmacológico , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Western Blotting , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Adhesión Celular/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cetuximab/administración & dosificación , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/patología , Citometría de Flujo , Humanos , Irinotecán , Invasividad Neoplásica , Neovascularización Patológica/patología , Fosforilación/efectos de los fármacos
13.
Neurobiol Dis ; 63: 165-70, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24269915

RESUMEN

The leucine-rich repeat kinase 2 (LRRK2) G2019S mutation is a common genetic cause of Parkinson's disease (PD). Although patients with sporadic PD and individuals with LRRK2-linked PD display the classical PD phenotype, it is not known whether or not the same biological pathways are deregulated in each context. By using transcriptome profiling, we investigated the deregulation of various biological pathways in a total of 47 peripheral blood mononuclear cell (PBMC) samples from patients with sporadic PD, patients heterozygous for the LRRK2 G2019S mutation compared to healthy controls. We found that the deregulation patterns were indeed similar in PBMCs obtained from patients with sporadic PD and from LRRK2 G2019S carriers, with dysfunctions in mitochondrial pathways, cell survival signaling, cancerization, endocytosis signaling and iron metabolism. Analysis of our PBMC data and other publicly available transcriptome datasets (for whole blood samples) showed that deregulation of the immune system, endocytosis and eukaryotic initiation factor 2 (EIF2) signaling are the main features of transcriptome profiles in PD (since they are also present in the transcriptome of dopaminergic neurons from patients). Transcriptome analysis of PBMCs is thus valuable for (i) characterizing the pathophysiological pathways shared by genetic and sporadic forms of PD and (ii) identifying potential biomarkers and therapeutic targets. This minimally invasive approach opens up tremendous perspectives for better diagnosis and therapy of neurodegenerative diseases because it can be applied from the earliest stages of the disease onwards.


Asunto(s)
Endocitosis/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Sistema Inmunológico/fisiopatología , Enfermedad de Parkinson , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Perfilación de la Expresión Génica , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana Edad , Mutación/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/inmunología , Enfermedad de Parkinson/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética
14.
Am J Hematol ; 88(4): 306-11, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23460398

RESUMEN

Germline heterozygous alterations of the tumor-suppressor gene neurofibromatosis-1 (NF1) lead to neurofibromatosis type 1, a genetic disorder characterized by a higher risk to develop juvenile myelomonocytic leukemia and/or acute myeloid leukemia (AML). More recently, somatic 17q11 deletions encompassing NF1 have been described in many adult myeloid malignancies. In this context, we aimed to define NF1 involvement in AML. We screened a total of 488 previously untreated de novo AML patients for the NF1 deletion using either array comparative genomic hybridization (aCGH) or real-time quantitative PCR/fluorescence in situ hybridization approaches. We also applied massively parallel sequencing for in depth mutation analysis of NF1 in 20 patients including five NF1-deleted patients. We defined a small ∼0.3 Mb minimal deleted region involving NF1 by aCGH and an overall frequency of NF1 deletion of 3.5% (17/485). NF1 deletion is significantly associated with unfavorable cytogenetics and with monosomal karyotype notably. We discovered six NF1 variants of unknown significance in 7/20 patients of which only one out of four disappeared in corresponding complete remission sample. In addition, only one out of five NF1-deleted patients has an acquired coding mutation in the remaining allele. In conclusion, direct NF1 inactivation is infrequent in de novo AML and may be a secondary event probably involved in leukemic progression.


Asunto(s)
Eliminación de Gen , Leucemia Mieloide Aguda/genética , Neurofibromatosis 1/genética , Neurofibromina 1/genética , Adolescente , Adulto , Anciano , Alelos , Hibridación Genómica Comparativa , Femenino , Frecuencia de los Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Persona de Mediana Edad , Tasa de Mutación , Neurofibromina 1/deficiencia , Reacción en Cadena en Tiempo Real de la Polimerasa
15.
Am J Hum Genet ; 89(3): 398-406, 2011 Sep 09.
Artículo en Inglés | MEDLINE | ID: mdl-21907011

RESUMEN

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.


Asunto(s)
Cromosomas Humanos Par 3/genética , Factor 4G Eucariótico de Iniciación/genética , Enfermedad de Parkinson/genética , Biosíntesis de Proteínas/genética , Secuencia de Bases , Clonación Molecular , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , Citometría de Flujo , Ligamiento Genético , Genotipo , Humanos , Inmunoprecipitación , Mitocondrias/fisiología , Datos de Secuencia Molecular , Mutación Missense/genética , Linaje
16.
Hum Mutat ; 32(4): E2079-90, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21412942

RESUMEN

Genomic multiplication of the alpha-synuclein gene (SNCA) locus is one cause of familial Parkinson disease (PD). We performed detailed genomic, SNCA expression level, clinical, neuropsychological and functional imaging analyses of a parkinsonian kindred with a known duplication of the SNCA locus. We demonstrated that the duplication spanned 4.928 Mb (encompassing 31 known and putative genes) and was the largest to have been described at this locus. The presence of several repetitive long interspersed nuclear elements (LINEs) flanking the potential break area suggested that the duplication resulted from a genomic recombination between LINEs. We sequenced the break junction and confirmed the involvement of L1PA2 and L1PA4 in a non-allelic, homologous recombination. An analysis of mRNA levels in immortalized lymphoblastoid cells and peripheral blood mononuclear cells showed SNCA overexpression in subjects with the duplication, as well as overexpression of 13 other genes highlighting the usefulness of such cell models to study this duplication. Interestingly, abnormal tracer uptake in DaTSCAN(®) imaging correlated with the severity of the clinical symptoms. Our detailed genomic analysis and clinical exploration enabled us to specify the genotype-phenotype relationship, identify a case of presymptomatic PD and gain insight into the role of LINEs in SNCA locus duplication.


Asunto(s)
Duplicación de Gen , Elementos de Nucleótido Esparcido Largo/genética , Enfermedad de Parkinson/genética , Fenotipo , alfa-Sinucleína/genética , Secuencia de Bases , Predisposición Genética a la Enfermedad , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Linaje
17.
J Cell Biochem ; 112(5): 1277-85, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21308741

RESUMEN

Hematopoietic cancer stem cells preserve cellular hierarchy in a manner similar to normal stem cells, yet the underlying regulatory mechanisms are poorly understood. It is known that both normal and malignant stem/progenitor cells express CD34. Here, we demonstrate that several cell lines (HL-60, U266) derived from hematopoietic malignancies contain not only CD34(-) but also CD34(+) subpopulations. The CD34(+) cells displayed a stem/progenitor-like phenotype since, in contrast to CD34(-) cells, they frequently underwent cellular division and rapidly formed colonies in methylcellulose-based medium. Strikingly, a constant fraction of the CD34(+) and CD34(-) cell subpopulations, when separated, rapidly switched their phenotype. Consequently, both separated fractions could generate tumors in immunocompromised NOD/LtSz-scid/scid mice. Cultures in vitro showed that the proportion of CD34(+) stem/progenitor-like cells in the population was decreased by cell-cell contact and increased by soluble factors secreted by the cells. Using cytokine arrays, we identified some of these factors, notably thymopoietin that was able to increase the proportion of CD34(+) cells and overall colony-forming capacity in tested cell lines. This action of thymopoietin was conserved in mononuclear cells from bone marrow. Therefore, we propose that hematopoietic cancer cell lines containing subpopulations of CD34(+) cells can provide an in vitro model for studies of cancer stem/progenitor cells.


Asunto(s)
Antígenos CD34/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Nucleares/metabolismo , Timopoyetinas/metabolismo , Animales , Antígenos CD34/genética , Biomarcadores/metabolismo , Línea Celular Tumoral , Células Clonales , Células HL-60 , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/efectos de los fármacos , Proteínas Nucleares/farmacología , Timopentina/farmacología , Timopoyetinas/farmacología
18.
Neurobiol Aging ; 32(10): 1839-48, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20096956

RESUMEN

To gain insight into systemic molecular events associated with an age-related neurodegenerative disorder, we compared gene expression patterns in peripheral blood mononuclear cells (PBMCs) sampled from elderly, healthy controls and from Parkinson's disease (PD) patients carrying the most frequently found mutation of the LRRK2 gene (G2019S). A transcriptomic approach enabled us to detect differentially expressed genes and revealed perturbations of pathways known to be involved in PD-related neurodegeneration: the ubiquitin-proteasome system, the mitochondrial oxidation system, inflammation, axonal guidance, calcium signalling and apoptosis. Moreover, alterations of the MAP kinase pathway, the actin cytoskeleton, the ephrin receptor system and vesicular transport - all recently associated with the LRRK2 G2019S mutation pathogenesis - were noted. Furthermore, we acquired new evidences of dysregulation in leukocyte extravasation signalling and immune system pathways in PD. These data show that the G2019S mutation affects the entire body and highlight some of the molecular events observed in the brain. This PBMC transcriptomic approach could be used to better understand neurodegeneration in PD and decipher new pathogenetic mechanisms, even at early stages of the disease.


Asunto(s)
Moléculas de Adhesión Celular Neuronal/genética , Leucocitos Mononucleares/metabolismo , Mutación/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Factores de Transcripción/metabolismo , Anciano , Anciano de 80 o más Años , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Componente Principal , Transducción de Señal/genética , Estadística como Asunto , Factores de Transcripción/genética , Adulto Joven
19.
Leuk Res ; 35(4): 448-58, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20684991

RESUMEN

To ascertain genomic alterations associated with Imatinib resistance in chronic myeloid leukaemia, we performed high resolution genomic analysis of CD34(+) cells from 25 Imatinib (IM) resistant and 11 responders CML patients. Using patients' T-cells as reference, we found significant association between number of acquired cryptic copy number alterations (CNA) and disease phase (p=0.036) or loss of IM response for patients diagnosed in chronic phase (CP) (p=0.04). Recurrent cryptic losses were identified on chromosomes 7, 12 and 13. On chromosome 7, recurrent deletions of the IKZF1 locus were detected, for the first time, in 4 patients in CP.


Asunto(s)
Resistencia a Antineoplásicos/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Antígenos CD34/metabolismo , Antineoplásicos/uso terapéutico , Benzamidas , Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 13/genética , Cromosomas Humanos Par 7/genética , Hibridación Genómica Comparativa , Dosificación de Gen , Genes abl/genética , Estudio de Asociación del Genoma Completo , Humanos , Factor de Transcripción Ikaros/genética , Mesilato de Imatinib , Hibridación Fluorescente in Situ , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Mutación , Linfocitos T/metabolismo
20.
Eur J Med Genet ; 51(4): 373-81, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18463015

RESUMEN

We report on a 6-years-old boy with psychomotor retardation, mild dysmorphic features and behavioral disturbances associated with epilepsy. Conventional cytogenetic analysis concluded to an interstitial de novo 6p21.2p22.3 duplication. Molecular cytogenetic analysis, including array-CGH technology, allows characterization of this 7.3Mb interstitial tandem duplication. The phenotype of this small 6p duplication reported to date is compared to other cases in the literature. Presence of epilepsy, although rare in patients with 6p duplication may be linked to genes involved in brain function and synaptic transmission in the 6p21.2p22.1 duplicated region (GABBR1, BRD2 and GRM4).


Asunto(s)
Cromosomas Humanos Par 6/genética , Discapacidades del Desarrollo/genética , Epilepsia/genética , Duplicación de Gen , Niño , Preescolar , Análisis Citogenético , Discapacidades del Desarrollo/diagnóstico , Genoma Humano , Humanos , Hibridación Fluorescente in Situ , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos
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