RESUMEN

Human breast cancers are a highly heterogeneous group of tumours consisting of several molecular subtypes with a variable profile of hormone, growth factor receptors and cytokeratins [1]. Here, the data shows immunofluorescence profiling of four different cell lines belonging to distinct clinical subtypes of breast cancer. Post revival, the cell lines were passaged in culture and immunophenotyping was done for ER, HER-2, AR and EGFR. Data for the markers from early passage (5th) through passages as late as 25 for the different cell lines is presented.

RESUMEN

Resistance to anthracycline based chemotherapy is a major limitation in the treatment of breast cancer, particularly of the triple negative sub-type that lacks targeted therapies. Resistance that arises from tumor-stromal interaction facilitated by integrins provides the possibility of targeted disruption. In the present study, we demonstrate that integrin ß3 signaling inhibits apoptosis induced by a DNA-damaging chemotherapeutic agent, epirubicin, in MDA-MB-231 breast cancer cells. Drug efflux based mechanisms do not contribute to this effect. We show that integrin ß3 employs the PI3K-Akt and the MAPK pathway for enabling cell survival and proliferation. Further, our results indicate that integrin ß3 helps inhibit epirubicin induced cytotoxicity by repression of the pro-apoptotic protein BAD, thus promoting an anti-apoptotic response. Myristoylated RGT peptide and a monoclonal antibody against integrin ß3 brought about a reversal of this effect and chemosensitized the cells. These results identify ß3 integrin signaling via repression of BAD as an important survival pathway used by breast cancer cells to evade chemotherapy induced stress.

Asunto(s)

Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Epirrubicina/farmacología , Integrina beta3/metabolismo , Proteína Letal Asociada a bcl/metabolismo , Anticuerpos Bloqueadores/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colágeno/metabolismo , Citometría de Flujo , Humanos , Concentración 50 Inhibidora , Ligandos , Ácido Mirístico/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

PURPOSE: Apart from germ-line BRCA1-mutated breast cancers, a significant proportion of women with sporadic triple negative breast cancer (TNBC) sub-type are known to harbour varying levels of BRCA1-dysfuction. There is currently no established diagnostic method to identify these patients. METHODS: The analysis was performed on 183 primary breast cancer tumor specimens from our longitudinal case-series archived as formalin-fixed-paraffin-embedded (FFPE) blocks comprising 71 TNBCs and 112 Hormone receptor positive HER2 negative (HR+HER2-) tumors. Transcript levels of BRCA1 and two of its repressors ID4 and microRNA182 were determined by TaqMan quantitative PCR. BRCA1 protein was detected immunohistochemically with the MS110 antibody. RESULTS: The representation of BRCA1 and its repressor ID4 as a ratio led to improved separation of TNBCs from HR+HER2- compared to either measure by itself. We then dichotomised the continuous distribution of each of the three measurements (Protein, MIRNA and transcript:repressor ratio) into categories of deficient (0) and adequate (1). A composite BRCA1 Deficiency Score (BDS) was computed by the addition of the score for all three measures. Samples deficient on 2 or more measures were deemed to be BRCA1 deficient; and 40% of all TNBCs met this criterion. CONCLUSION: We propose here a simple multi-level assay of BRCA1 deficiency using the BRCA1:ID4 ratio as a critical parameter that can be performed on FFPE samples in clinical laboratories by the estimation of only 3 bio-markers. The ease of testing will hopefully encourage adoption and clinical validation.

Asunto(s)

Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Formaldehído , Adhesión en Parafina , Fijación del Tejido , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Carboplatino/farmacología , Carboplatino/uso terapéutico , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Inhibidoras de la Diferenciación/genética , MicroARNs/genética , Persona de Mediana Edad , Estadificación de Neoplasias , ARN Mensajero/genética , ARN Mensajero/metabolismo , Resultado del Tratamiento , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo

RESUMEN

Estimations of RNA abundance and DNA methylation by quantitative PCR (qPCR) from formalin-fixed, paraffin-embedded (FFPE) tissue specimens are not yet routine in clinical laboratory practice. Excluding specimens with poorly preserved nucleic acids is an important quality-control step for avoiding unreliable results. Because the assays for RNA abundance and DNA methylation have different critical limiting factors, we examined the extent of overlap of excluded specimens for RNA abundance versus methylated DNA. The transcript abundance of three reference genes and of the test gene, estrogen receptor 1 (ESR1), was estimated by SYBR Green qPCR in 250 breast cancer specimens. The estrogen receptor (ER) protein was identified by IHC, and concordance between ESR1 and ER was estimated by Cohen's κ. TaqMan PCR for the ALU-C4 sequence was performed with bisulfite-treated DNA to determine usability in the MethyLight assay. Excluding specimens with mean reference gene CT values exceeding the group mean by >1 SD led to significant improvement of the concordance of ESR1 and ER. Specimens with usable DNA after bisulfite treatment likewise had ALU-C4 CT values of less than the group mean + 1 SD. Samples with low-quality RNA and DNA were partly nonoverlapping. RNA and DNA extracted from the same FFPE block need separate exclusion criteria for qPCR assays of transcript abundance and methylated DNA.

Asunto(s)

Metilación de ADN , ADN/genética , ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , ADN/normas , Femenino , Formaldehído , Humanos , Adhesión en Parafina , Control de Calidad , ARN/normas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Fijación del Tejido

RESUMEN

To re-examine the correlation between mtDNA variability and longevity, we examined mtDNAs from samples obtained from over 2200 ultranonagenarians (and an equal number of controls) collected within the framework of the GEHA EU project. The samples were categorized by high-resolution classification, while about 1300 mtDNA molecules (650 ultranonagenarians and an equal number of controls) were completely sequenced. Sequences, unlike standard haplogroup analysis, made possible to evaluate for the first time the cumulative effects of specific, concomitant mtDNA mutations, including those that per se have a low, or very low, impact. In particular, the analysis of the mutations occurring in different OXPHOS complex showed a complex scenario with a different mutation burden in 90+ subjects with respect to controls. These findings suggested that mutations in subunits of the OXPHOS complex I had a beneficial effect on longevity, while the simultaneous presence of mutations in complex I and III (which also occurs in J subhaplogroups involved in LHON) and in complex I and V seemed to be detrimental, likely explaining previous contradictory results. On the whole, our study, which goes beyond haplogroup analysis, suggests that mitochondrial DNA variation does affect human longevity, but its effect is heavily influenced by the interaction between mutations concomitantly occurring on different mtDNA genes.

Asunto(s)

ADN Mitocondrial/genética , Longevidad/genética , Fosforilación Oxidativa , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Mutación

RESUMEN

The breast cancer type 1 susceptibility gene (BRCA1) is a tumor suppressor gene, mutations or loss of which lead to genomic instability and breast cancer. BRCA1 protein is part of a large multi-protein complex involved in a variety of DNA repair and transcription regulatory functions. At least four splice variants have been described and these differ in their function and tissue and spatio-temporal expression patterns. Structural analysis has revealed the presence of two nuclear localization signals (NLS) located in exon 11 of BRCA1. Interestingly, a splice variant of the protein that lacks both of the known NLS still manages to gain entry to the nucleus. While there is experimental proof for the translocation of these proteins by binding to other established nuclear proteins, we examined the possibility of a hitherto unidentified NLS in this particular variant. In this paper, we present evidence for the existence of a previously unreported non-canonical NLS contained within the first 39 amino acids of exon 11. A fusion protein with this 39mer and a reporter green fluorescent protein translocated into the nucleus when it was expressed in breast epithelial cells. We demonstrate the presence of a hitherto unreported noncanonical NLS in exon 11a of BRCA1. This NLS might aid proteins that were encoded by splice variants and lack the canonical NLS to localize to the nucleus.

Asunto(s)

Empalme Alternativo/genética , Proteína BRCA1/química , Proteína BRCA1/metabolismo , Núcleo Celular/metabolismo , Señales de Localización Nuclear/metabolismo , Secuencia de Aminoácidos , Proteína BRCA1/genética , Línea Celular Tumoral , Exones/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Datos de Secuencia Molecular , Señales de Localización Nuclear/química , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad

RESUMEN

This paper illustrates the advantages that a fuzzy-based aggregation method could bring into the validation of a multiplex method for GMO detection (DualChip GMO kit, Eppendorf). Guidelines for validation of chemical, bio-chemical, pharmaceutical and genetic methods have been developed and ad hoc validation statistics are available and routinely used, for in-house and inter-laboratory testing, and decision-making. Fuzzy logic allows summarising the information obtained by independent validation statistics into one synthetic indicator of overall method performance. The microarray technology, introduced for simultaneous identification of multiple GMOs, poses specific validation issues (patterns of performance for a variety of GMOs at different concentrations). A fuzzy-based indicator for overall evaluation is illustrated in this paper, and applied to validation data for different genetically modified elements. Remarks were drawn on the analytical results. The fuzzy-logic based rules were shown to be applicable to improve interpretation of results and facilitate overall evaluation of the multiplex method.

Asunto(s)

Lógica Difusa , Técnicas Genéticas/estadística & datos numéricos , Organismos Modificados Genéticamente/genética , Estudios de Validación como Asunto , Algoritmos , Animales , Recolección de Datos/métodos , Recolección de Datos/estadística & datos numéricos , Interpretación Estadística de Datos , Análisis por Micromatrices/métodos , Análisis por Micromatrices/estadística & datos numéricos

RESUMEN

Lymphomas are classified according to the World Health Organisation (WHO) classification which defines subtypes on the basis of clinical, morphological, immunophenotypic, molecular and cytogenetic criteria. Differential diagnosis of the subtypes is sometimes difficult, especially for small B-cell lymphoma (SBCL). Standardisation of molecular genetic assays using multiple gene expression analysis by microarrays could be a useful complement to the current diagnosis. The aim of the present study was to develop a low density DNA microarray for the analysis of 107 genes associated with B-cell non-Hodgkin lymphoma and to evaluate its performance in the diagnosis of SBCL. A predictive tool based on Fisher discriminant analysis using a training set of 40 patients including four different subtypes (follicular lymphoma n = 15, mantle cell lymphoma n = 7, B-cell chronic lymphocytic leukemia n = 6 and splenic marginal zone lymphoma n = 12) was designed. A short additional preliminary analysis to gauge the accuracy of this signature was then performed on an external set of nine patients. Using this model, eight of nine of those samples were classified successfully. This pilot study demonstrates that such a microarray tool may be a promising diagnostic approach for small B-cell non-Hodgkin lymphoma.

Asunto(s)

Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Linfoma de Células B/diagnóstico , Análisis de Secuencia por Matrices de Oligonucleótidos , Inteligencia Artificial , Diagnóstico Diferencial , Humanos , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/genética , Linfoma de Células B/clasificación , Linfoma de Células B/genética , Linfoma de Células B de la Zona Marginal/diagnóstico , Linfoma de Células B de la Zona Marginal/genética , Linfoma Folicular/diagnóstico , Linfoma Folicular/genética , Linfoma de Células del Manto/diagnóstico , Linfoma de Células del Manto/genética , Proyectos Piloto , ARN Neoplásico/análisis , ARN Neoplásico/aislamiento & purificación

RESUMEN

RNA-mediated gene silencing (RNA interference) is a powerful way to knock down gene expression and has revolutionized the fields of cellular and molecular biology. Indeed, the transfection of cultured cells with small interfering RNAs (siRNAs) is currently considered to be the best and easiest approach to loss-of-function experiments. However, several recent studies underscore the off-target and potential cytotoxic effects of siRNAs, which can lead to the silencing of unintended mRNAs. In this study, we used a low-density microarray to assess gene expression modifications in response to five different siRNAs in various cell types and transfection conditions. We found major differences in off-target signature according to: (a) siRNA sequence; (b) cell type; (c) duration of transfection; and (d) post-transfection time before analysis. These results contribute to a better understanding of important parameters that could impact on siRNA side effects in knockdown experiments.

Asunto(s)

Regulación de la Expresión Génica , Silenciador del Gen , ARN Interferente Pequeño/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica , Técnicas Genéticas , Humanos , Cinética , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , ARN Mensajero/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Factores de Tiempo , Transfección

RESUMEN

BACKGROUND: it is now well established that hypoxia renders tumor cells resistant to radio- but also chemotherapy. However, few elements are currently available as for the mechanisms underlying this protection. RESULTS: in this study, physiological hypoxia was shown to inhibit apoptosis induced in HepG2 cells by etoposide. Indeed, hypoxia reduced DNA fragmentation, caspase activation and PARP cleavage. The DNA binding activity of 10 transcription factors was followed while the actual transcriptional activity was measured using specific reporter plasmids. Of note is the inhibition of the etoposide-induced activation of p53 under hypoxia. In parallel, data from low density DNA microarrays indicate that the expression of several pro- and anti-apoptotic genes was modified, among which are Bax and Bak whose expression profile paralleled p53 activity. Cluster analysis of data unravels several possible pathways involved in the hypoxia-induced protection against etoposide-induced apoptosis: one of them could be the inhibition of p53 activity under hypoxia since caspase 3 activity parallels Bax and Bak expression profile. Moreover, specific downregulation of HIF-1alpha by RNA interference significantly enhanced apoptosis under hypoxia possibly by preventing the hypoxia mediated decrease in Bak expression without altering Bax expression. CONCLUSION: these results are a clear demonstration that hypoxia has a direct protective effect on apoptotic cell death. Moreover, molecular profiling points to putative pathways responsible for tumor growth in challenging environmental conditions and cancer cell resistance to chemotherapeutic agents.

Asunto(s)

Apoptosis/efectos de los fármacos , Citoprotección/efectos de los fármacos , Etopósido/farmacología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factores de Transcripción/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Análisis por Conglomerados , ADN de Neoplasias/metabolismo , Silenciador del Gen , Genes Reporteros , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección

RESUMEN

A key issue in the treatment of many cancers is the development of resistance to chemotherapeutic drugs. Resistance mechanisms are numerous and complex. One of them is mediated by the overexpression of ATP-binding cassette (ABC) transporters able to efflux drugs out of the tumor cell. The last two decades have seen notable growth of knowledge concerning the involvement of ABC transporters in resistance to chemotherapy. This review emphasizes these transporters, their clinical relevance and the diagnostic methods and strategies to circumvent multidrug resistance (MDR) mediated by ABC transporters.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/genética , Antineoplásicos/efectos adversos , Resistencia a Medicamentos , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/clasificación , Cristalografía por Rayos X , Terapia Genética , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Conformación Proteica , Células Madre/fisiología

RESUMEN

The aim of the 5-year European Union (EU)-Integrated Project GEnetics of Healthy Aging (GEHA), constituted by 25 partners (24 from Europe plus the Beijing Genomics Institute from China), is to identify genes involved in healthy aging and longevity, which allow individuals to survive to advanced old age in good cognitive and physical function and in the absence of major age-related diseases. To achieve this aim a coherent, tightly integrated program of research that unites demographers, geriatricians, geneticists, genetic epidemiologists, molecular biologists, bioinfomaticians, and statisticians has been set up. The working plan is to: (a) collect DNA and information on the health status from an unprecedented number of long-lived 90+ sibpairs (n = 2650) and of younger ethnically matched controls (n = 2650) from 11 European countries; (b) perform a genome-wide linkage scannning in all the sibpairs (a total of 5300 individuals); this investigation will be followed by linkage disequilibrium mapping (LD mapping) of the candidate chromosomal regions; (c) study in cases (i.e., the 2650 probands of the sibpairs) and controls (2650 younger people), genomic regions (chromosome 4, D4S1564, chromosome 11, 11.p15.5) which were identified in previous studies as possible candidates to harbor longevity genes; (d) genotype all recruited subjects for apoE polymorphisms; and (e) genotype all recruited subjects for inherited as well as epigenetic variability of the mitochondrial DNA (mtDNA). The genetic analysis will be performed by 9 high-throughput platforms, within the framework of centralized databases for phenotypic, genetic, and mtDNA data. Additional advanced approaches (bioinformatics, advanced statistics, mathematical modeling, functional genomics and proteomics, molecular biology, molecular genetics) are envisaged to identify the gene variant(s) of interest. The experimental design will also allow (a) to identify gender-specific genes involved in healthy aging and longevity in women and men stratified for ethnic and geographic origin and apoE genotype; (b) to perform a longitudinal survival study to assess the impact of the identified genetic loci on 90+ people mortality; and (c) to develop mathematical and statistical models capable of combining genetic data with demographic characteristics, health status, socioeconomic factors, lifestyle habits.

Asunto(s)

Envejecimiento/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , ADN Mitocondrial/genética , Europa (Continente) , Unión Europea , Ligamiento Genético , Genoma , Humanos , Desequilibrio de Ligamiento , Persona de Mediana Edad , Modelos Biológicos , Modelos Genéticos

RESUMEN

Premature senescence of IMR-90 human diploid fibroblasts expressing telomerase (hTERT) establishes after exposure to an acute sublethal concentration of H2O2. We showed herein that p38(MAPK) was phosphorylated after exposure of IMR-90 hTERT cells to H2O2. Selective inhibition of p38(MAPK) activity attenuated the increase in the proportion of cells positive for senescence associated beta-galactosidase activity. We generated a low density DNA array to study gene expression profiles of 240 senescence-related genes. Using this array, p38(MAPK) inhibitor and p38(MAPK) small interferent RNA, we identified several p38(MAPK)-target genes differentially expressed in H2O2-stressed IMR-90 hTERT fibroblasts.

Asunto(s)

Senescencia Celular/efectos de los fármacos , Fibroblastos/enzimología , Regulación de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Telomerasa/biosíntesis , Línea Celular , Senescencia Celular/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/genética , Telomerasa/genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

A major issue in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) is resistance to chemotherapeutic drugs. Multidrug resistance can be caused by ATP-binding cassette (ABC) transporters. The majority of these proteins have not yet been examined in T-ALL. Using a newly developed microarray for the simultaneous quantification of 38 ABC transporter genes, we observed a consistent overexpression of ABCA2/ABCA3 in clinical samples of ALL. Therefore, we analyzed the association of these two genes with drug resistance. Treatment of CCRF-CEM and Jurkat cells with methotrexate, vinblastine, or doxorubicin led to an induction of ABCA3 expression, whereas a significant increase of ABCA2 expression was only observed in Jurkat cells. To study the causal relationship of ABCA2/A3 overexpression with drug resistance, we applied RNA interference (RNAi) technology. RNAi specific for ABCA2 or ABCA3 led to a partial decrease of expression in these two ABC transporters. Upon cotreatment of RNAi for ABCA2 with methotrexate and vinblastine, a partial decrease of ABCA2 expression as well as a simultaneous increase of ABCA3 expression was observed. Vice versa, ABCA3 RNAi plus drugs decreased ABCA3 and increased ABCA2 expression. This indicates that down-regulation of one ABC transporter was compensated by the up-regulation of the other. Application of RNAi for both ABCA2 and ABCA3 resulted in a more efficient reduction of the expression of both transporters. As a consequence, a significant sensitization of cells to cytostatic drugs was achieved. In conclusion, ABCA2 and ABCA3 are expressed in many T-ALL and contribute to drug resistance.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/genética , Resistencia a Antineoplásicos/genética , Leucemia-Linfoma de Células T del Adulto/genética , Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Adolescente , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Niño , Preescolar , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica , Humanos , Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Masculino , Modelos Teóricos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Interferencia de ARN , Células Tumorales Cultivadas

RESUMEN

BACKGROUND: A major issue in the treatment of acute myeloid leukemia (AML) is resistance to chemotherapeutic drugs. Multidrug resistance can be caused by ATP-binding cassette (ABC) transporters that function as drug efflux pumps. The majority of these proteins have not yet been examined in malignant diseases. EXPERIMENTAL DESIGN: A newly developed microarray for the simultaneous quantification of 38 ABC transporter genes and Taqman real-time PCR was used to analyze the expression of ABC transporters in pediatric AML and healthy bone marrow. Small interfering RNA was used to verify the role of ABCA3 in drug resistance. RESULTS: Using the microarray, we identified four new ABC transporters, which were overexpressed in many AML samples compared with healthy bone marrow: ABCA2, ABCA3, ABCB2, and ABCC10. The overexpression of these four genes was verified by real-time PCR in 42 samples from children with AML and 18 samples of healthy bone marrow. The median expression of ABCA3 was three times higher in 21 patients who had failed to achieve remission after the first course of chemotherapy than in a well-matched group of 21 patients who had achieved remission at this stage (P = 0.023). Incubation of cell lines with a number of different cytostatic drugs induced an up-regulation of ABCA3. Down-regulation of ABCA3 by small interfering RNA sensitized cells to doxorubicin. CONCLUSION: Our results show that ABCA2, ABCA3, ABCB2, and ABCC10 are overexpressed in childhood AML compared with healthy bone marrow. ABCA3 is the most likely transporter to cause drug resistance.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/fisiología , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adolescente , Adulto , Células de la Médula Ósea/metabolismo , Niño , Preescolar , Análisis por Conglomerados , Resistencia a Múltiples Medicamentos , Femenino , Humanos , Células Jurkat , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Interferente Pequeño/metabolismo

RESUMEN

Premature senescence of human fibroblasts is established after exposure to an acute sublethal concentration of H(2)O(2). Overexpression of transforming growth factor-beta1 (TGF-beta1) was shown to be responsible for the appearance of the biomarkers of senescence in these conditions. Other studies have shown that incubation of human fibroblasts with TGF-beta1 leads to overexpression of H(2)O(2). In this work, we show an increased production of H(2)O(2) by human fibroblasts as premature senescence is established after an initial exposure to H(2)O(2).

Asunto(s)

Senescencia Celular/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/metabolismo , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Células Cultivadas , Humanos , Peróxido de Hidrógeno/análisis , Peróxido de Hidrógeno/metabolismo , Pulmón/citología , Pulmón/embriología , Modelos Biológicos , Oxidantes/análisis , Oxidantes/metabolismo , Estrés Oxidativo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1

RESUMEN

A multiplex DNA microarray chip was developed for simultaneous identification of nine genetically modified organisms (GMOs), five plant species and three GMO screening elements, i.e. the 35S promoter, the nos terminator and the nptII gene. The chips also include several controls, such as that for the possible presence of CaMV. The on-chip detection was performed directly with PCR amplified products. Particular emphasis was placed on the reduction of the number of PCR reactions required and on the number of primers present per amplification tube. The targets were biotin labelled and the arrays were detected using a colorimetric methodology. Specificity was provided by specific capture probes designed for each GMO and for the common screening elements. The sensitivity of the assay was tested by experiments carried out in five different laboratories. The limit of detection was lower than 0.3% GMO for all tests and in general around 0.1% for most GMOs. The chip detection system complies with the requirements of current EU regulations and other countries where thresholds are established for the labelling of GMO.

Asunto(s)

Alimentos Modificados Genéticamente , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Plantas Modificadas Genéticamente/genética , Agrobacterium tumefaciens/genética , Beta vulgaris/genética , Brassica rapa/genética , Caulimovirus/genética , Unión Europea , Solanum lycopersicum/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Glycine max/genética , Zea mays/genética

RESUMEN

Over the last 5 years, the emergence of gene expression profiling using high-density DNA microarrays led to a better understanding of tumor development and identified new prognostic markers. However, high-density microarrays failed to leap from the researcher's bench to the clinical practice due to their cost, data management and lack of standardization. DualChip low-density DNA microarrays were developed as a new flexible tool that is able to reliably quantify the expression of a limited number of genes of clinical relevance. This review will illustrate how DualChip technology can be applied to tumor diagnosis and tumor-acquired drug resistance.

Asunto(s)

Biomarcadores de Tumor , Técnicas de Diagnóstico Molecular , Neoplasias/diagnóstico , Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Antineoplásicos/farmacología , Línea Celular Tumoral , Análisis por Conglomerados , ADN de Neoplasias , Resistencia a Antineoplásicos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/tratamiento farmacológico , Pronóstico

RESUMEN

Several mitochondrial pathologies are characterized by lipid redistribution and microvesicular cell phenotypes resulting from triglyceride accumulation in lipid-metabolizing tissues. However, the molecular mechanisms underlying abnormal fat distribution induced by mitochondrial dysfunction remain poorly understood. In this study, we show that inhibition of respiratory complex III by antimycin A as well as inhibition of mitochondrial protein synthesis trigger the accumulation of triglyceride vesicles in 3T3-L1 fibroblasts. We also show that treatment with antimycin A triggers CREB activation in these cells. To better delineate how mitochondrial dysfunction induces triglyceride accumulation in preadipocytes, we developed a low-density DNA microarray containing 89 probes, which allows gene expression analysis for major effectors and/or markers of adipogenesis. We thus determined gene expression profiles in 3T3-L1 cells incubated with antimycin A and compared the patterns obtained with differentially expressed genes during the course of in vitro adipogenesis induced by a standard pro-adipogenic cocktail. After an 8-day treatment, a set of 39 genes was found to be differentially expressed in cells treated with antimycin A, among them CCAAT/enhancer-binding protein alpha (C/EBPalpha), C/EBP homologous protein-10 (CHOP-10), mitochondrial glycerol-3-phosphate dehydrogenase (GPDmit), and stearoyl-CoA desaturase 1 (SCD1). We also demonstrate that overexpression of two dominant negative mutants of the cAMP-response element-binding protein CREB (K-CREB and M1-CREB) and siRNA transfection, which disrupt the factor activity and expression, respectively, inhibit antimycin-A-induced triglyceride accumulation. Furthermore, CREB knockdown with siRNA also downregulates the expression of several genes that contain cAMP-response element (CRE) sites in their promoter, among them one that is potentially involved in synthesis of triglycerides such as SCD1. These results highlight a new role for CREB in the control of triglyceride metabolism during the adaptative response of preadipocytes to mitochondrial dysfunction.

Asunto(s)

Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Mitocondrias/patología , Triglicéridos/biosíntesis , Células 3T3-L1 , Adipocitos/citología , Animales , Antimicina A/farmacología , Western Blotting , Diferenciación Celular , Cloranfenicol/farmacología , ADN/análisis , ADN/genética , ADN Complementario/genética , Ensayo de Inmunoadsorción Enzimática , Fluoresceínas , Técnica del Anticuerpo Fluorescente Indirecta , Colorantes Fluorescentes , Perfilación de la Expresión Génica , Silenciador del Gen , Genes Reporteros , Hibridación in Situ , Metabolismo de los Lípidos , Luciferasas/análisis , Luciferasas/metabolismo , Ratones , Mitocondrias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

We present a novel readout scheme for gold nanoparticle-based DNA microarrays relying on "Laser-Induced Scattering around a NanoAbsorber". It provides direct counting of individual nanoparticles present on each array spot and stable signals, without any silver enhancement. Given the detection of nanometer-sized particles, which minimize the steric hindrance, the linear dynamic range of the method is particularly large and well suited for microarray detection.

Asunto(s)

Oro/química , Nanoestructuras/química , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Plata/química , Acústica , ADN/química , Rayos Láser , Microscopía Fluorescente , Hibridación de Ácido Nucleico , Óptica y Fotónica , Reproducibilidad de los Resultados , Sensibilidad y Especificidad