RESUMEN
Double-strand breaks (DSB) in genomic DNA are induced by ionizing radiation or radiomimetic drugs but also occur spontaneously during the cell cycle at quite significant frequencies. In vertebrate cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of DSB repair which is able to rejoin two broken DNA termini directly end-to-end irrespective of sequence and structure. Genetic studies in various radiosensitive and DSB repair-deficient cell lines yielded insight into the factors involved in NHEJ. Studies in cell-free systems derived from Xenopus eggs and mammalian cells allowed the dissection of the underlying mechanisms. In the present chapter, we describe a protocol for the preparation of whole cell extracts from mammalian cells and a plasmid-based in vitro assay which permits the easy analysis of the efficiency and fidelity of DSB repair via NHEJ in different cell types.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Animales , Secuencia de Bases , Extractos Celulares/genética , Línea Celular , Sistema Libre de Células , Electroforesis en Gel de Agar , Escherichia coli , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Plásmidos/genéticaRESUMEN
In the childhood tumor neuroblastoma, high expression of the TrkA neurotrophin receptor is associated with a favorable prognosis and a lack of structural chromosomal changes, whereas TrkB is expressed in aggressive neuroblastomas demonstrating high genomic instability. The ability to repair DNA double-strand breaks (DSBs) is considered a central determinant of chromosomal stability with nonhomologous end joining (NHEJ) being the major pathway of DSB repair in vertebrates. Here, we used the SH-SY5Y human neuroblastoma cell line ectopically expressing either TrkA or TrkB as a model system to analyze the impact of Trk receptor expression on NHEJ-mediated DSB repair. In a cell-free NHEJ assay, SY5Y-TrkA cells displayed a significantly higher efficiency for NHEJ compared to SY5Y-TrkB cells. To detect possible underlying mechanisms, gene expression data (Affymetrix U95A microarray chips) obtained from the same SY5Y-TrkA/TrkB model system were reanalyzed focussing on genes involved in DNA repair. Expression of XRCC4, a central component of NHEJ, was significantly upregulated in SY5Y-TrkA compared to SY5Y-TrkB cells. Expression data were confirmed using real-time PCR and western blotting. Additionally, XRCC4 expression was enhanced in most primary neuroblastomas with high TrkA expression. The TrkA-induced increase in NHEJ activity could be reverted by XRCC4 knock-down, confirming the induction of XRCC4 by TrkA to be essential for the observed phenotype. Our data provide the first evidence for a functional relationship between tyrosine kinase receptor signaling and NHEJ-mediated DSB repair in cancer cells, potentially contributing to their genomic stability.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Neuroblastoma/enzimología , Neuroblastoma/patología , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Línea Celular Tumoral , Sistema Libre de Células , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Cinética , Neuroblastoma/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recombinación Genética , Regulación hacia ArribaRESUMEN
To study the relationships between different DNA repair pathways, we established a set of clones in which one specific DNA repair gene was silenced using long-term RNA interference in HeLa cell line. We focus here on genes involved in either nucleotide excision repair (XPA and XPC) or nonhomologous end joining (NHEJ; DNA-PKcs and XRCC4). As expected, XPA(KD) (knock down) and XPC(KD) cells were highly sensitive to UVC. DNA-PKcs(KD) and XRCC4(KD) cells presented an increased sensitivity to various inducers of double-strand breaks (DSBs) and a 70% to 80% reduction of in vitro NHEJ activity. Long-term silencing of XPC gene expression led to an increased sensitivity to etoposide, a topoisomerase II inhibitor that creates DSBs through the progression of DNA replication forks. XPC(KD) cells also showed intolerance toward acute gamma-ray irradiation. We showed that XPC(KD) cells exhibited an altered spectrum of NHEJ products with decreased levels of intramolecular joined products. Moreover, in both XPC(KD) and DNA-PKcs(KD) cells, XRCC4 and ligase IV proteins were mobilized on damaged nuclear structures at lower doses of DSB inducer. In XPC-proficient cells, XPC protein was released from nuclear structures after induction of DSBs. By contrast, silencing of XPA gene expression did not have any effect on sensitivity to DSB or NHEJ. Our results suggest that XPC deficiency, certainly in combination with other genetic defects, may contribute to impair DSB repair.
Asunto(s)
Daño del ADN , Reparación del ADN/fisiología , Proteínas de Unión al ADN/genética , Silenciador del Gen , ADN Ligasa (ATP) , ADN Ligasas/metabolismo , Reparación del ADN/genética , Reparación del ADN/efectos de la radiación , Proteína Quinasa Activada por ADN/biosíntesis , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Etopósido/farmacología , Rayos gamma , Células HeLa , Humanos , Interferencia de ARNRESUMEN
Non-homologous end joining (NHEJ), the major pathway of double-strand break (DSB) repair in mammalian cells, comprises two subpathways: one that requires the three core factors Ku70/80, DNA-PKcs and XRCC4/LigIV (DNA-PK-dependent NHEJ) and the other that is independent of these factors. Using a cell-free NHEJ assay, we have investigated the ability of three Chinese hamster ovary (CHO) mutants deficient in Ku80 (xrs6), DNA-PKcs (XR-C1) and XRCC4 (XR-1) in comparison with CHO-K1 wild-type cells to rejoin non-compatible DSB ends. Both NHEJ efficiency and fidelity are strongly reduced in the mutants with xrs6 and XR-1 exhibiting the strongest reduction and XR-C1 displaying a phenotype intermediate between the wild-type and the other two mutants indicating a non-essential but facilitating role of DNA-PKcs in NHEJ. The decrease in fidelity in the mutants is expressed by an increase of deletion junctions formed at microhomologies (microhom) near the DSB (microhomology-mediated non-homologous end joining: microhomNHEJ). Using a novel microhomNHEJ assay, we show that microhom regions of 6-10 bp that are located directly at the DSB termini strongly enhance the mutagenic microhomNHEJ reaction even in the wild type. Due to its error proneness, DNA-PK-independent microhomNHEJ may actively promote genome instability. It will, therefore, be of increasing importance to examine NHEJ fidelity in the context with tumorigenesis and cellular senescence for which we here provide two efficient and reliable tools.
Asunto(s)
Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN/deficiencia , Reparación del ADN/fisiología , Proteína Quinasa Activada por ADN/deficiencia , Proteínas de Unión al ADN/deficiencia , Animales , Antígenos Nucleares/genética , Western Blotting , Células CHO , Extractos Celulares , Clonación Molecular , Cricetinae , Cricetulus , Reparación del ADN/genética , Enzimas Reparadoras del ADN/genética , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/genética , Electroforesis en Gel de Agar , Inmunohistoquímica , Cinética , Autoantígeno Ku , Pruebas de Mutagenicidad/métodos , Oligonucleótidos/genéticaRESUMEN
The Trk family consists of three receptor tyrosine kinases, each of which can be activated by one or more of four neurotrophins-NGF, BDNF, NT3 and NT4. Neurotrophins mediate their multiple effects through a number of distinct intracellular signaling cascades regulating such diverse biological responses as cell survival, proliferation and differentiation in normal and neoplastic neuronal cells. Expression of Trk receptors also plays an important role in the biology and clinical behavior of neuroblastomas. High expression of TrkA is present in neuroblastomas with favorable biological features and highly correlated with patient survival, whereas TrkB is mainly expressed on unfavorable, aggressive neuroblastomas. This short review discusses recent data on the biological roles of TrkA and TrkB signaling in neuroblastoma.
Asunto(s)
Neuroblastoma/metabolismo , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Transducción de Señal , Humanos , Receptor trkA/fisiología , Receptor trkB/fisiologíaRESUMEN
Double-strand breaks (DSBs) in genomic DNA are induced by ionizing radiation or radiomimetic drugs, but they also occur spontaneously during the cell cycle at quite significant frequencies. In vertebrate cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of DSB repair. NHEJ is able to rejoin two broken DNA termini directly end-to-end irrespective of sequence and structure. Genetic studies in various radiosensitive and DSB repair-deficient hamster cell lines have yielded insights into the factors involved in NHEJ. Studies in cell-free systems derived from Xenopus eggs and mammalian cells have allowed the dissection of the underlying mechanisms. In the present chapter, we describe a protocol for the preparation of whole cell extracts from mammalian cells and a plasmid-based in vitro assay that permits the easy analysis of the efficiency and fidelity of DSB repair via NHEJ in different cell types.
Asunto(s)
Sistema Libre de Células , Reparación del ADN , Recombinación Genética , Animales , Secuencia de Bases , Células CHO , Extractos Celulares/química , Cricetinae , Cricetulus , ADN/análisis , ADN/química , Daño del ADN , Humanos , Datos de Secuencia Molecular , Plásmidos/genéticaRESUMEN
Structural abnormalities of chromosomes, including translocations and deletions, are extremely frequent in human cancer cells and particularly in breast cancer cells. One hypothesis to account for these alterations is a deficiency in the repair of DNA double-strand breaks (DSB). This repair process relies on two distinct pathways, homologous recombination (HR) and non-homologous DNA end joining (NHEJ). To investigate this latter pathway, we have studied the ability of cell-free extracts from a variety of human cells to rejoin different types of DSBs. The end joining activity of eleven sporadic breast cancer cell lines (BCCLs) was compared with that of control cells including primary human fibroblasts and cells harbouring a limited number of chromosome abnormalities. In vitro rejoining activity was not detected in extracts from MO59J DNA-PKcs-deficient cells and was strongly inhibited by wortmannin in control extracts. In contrast, most sporadic BCCLs and BRCA1 or BRCA2 deficient cells demonstrated similar efficiencies and accuracies of in vitro NHEJ than control cells. Only two BCCLs, SKBR3 and MDA-MB-453 exhibited decreased in vitro NHEJ. This study therefore indicates that a major defect in the NHEJ pathway is unlikely to account for the high number of chromosomes abnormalities observed in sporadic and hereditary BCCLs.
Asunto(s)
Proteína BRCA1/deficiencia , Proteína BRCA1/metabolismo , Proteína BRCA2/deficiencia , Proteína BRCA2/metabolismo , Neoplasias de la Mama/genética , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/metabolismo , Reparación del ADN/genética , Recombinación Genética/genética , Androstadienos/farmacología , Proteína BRCA1/genética , Proteína BRCA2/genética , Proteínas de Unión al Calcio/genética , Aberraciones Cromosómicas , ADN/química , ADN/genética , ADN/metabolismo , Daño del ADN/genética , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Células Tumorales Cultivadas , WortmaninaRESUMEN
In mammalian cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair. Rejoining of DSB produced by decay of (125)I positioned against a specific target site in plasmid DNA via a triplex-forming oligonucleotide (TFO) was investigated in cell-free extracts from Chinese hamster ovary cells. The efficiency and quality of NHEJ of the "complex" DSB induced by the (125)I-TFO was compared with that of "simple" DSB induced by restriction enzymes. We demonstrate that the extracts are indeed able to rejoin (125)I-TFO-induced DSB, although at approximately 10-fold decreased efficiency compared with restriction enzyme-induced DSB. The resulting spectrum of junctions is highly heterogeneous exhibiting deletions (1-30 bp), base pair substitutions, and insertions and reflects the heterogeneity of DSB induced by the (125)I-TFO within its target site. We show that NHEJ of (125)I-TFO-induced DSB is not a random process that solely depends on the position of the DSB but is driven by the availability of microhomology patches in the target sequence. The similarity of the junctions obtained with the ones found in vivo after (125)I-TFO-mediated radiodamage indicates that our in vitro system may be a useful tool to elucidate the mechanisms of ionizing radiation-induced mutagenesis and repair.
Asunto(s)
Daño del ADN , Reparación del ADN , ADN/efectos de la radiación , Recombinación Genética , Animales , Secuencia de Bases , Sitios de Unión , Células CHO , Línea Celular , Sistema Libre de Células , Cricetinae , Escherichia coli/metabolismo , Eliminación de Gen , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Homología de Secuencia de Ácido Nucleico , XenopusRESUMEN
Se trata de un estudio analítico-observacional de casos y controles, realizado sobre una muestra de 290 gestantes atendidas en la maternidad del Hospital Central de Valencia, de las cuales 145 presentaron parto prematuro (grupo experimental) y las restantes (145) fueron utilizadas como grupo control. Se evaluaron 24 factores de riesgo, de los que sólo 11 (45,83 por ciento) mostraron asociación estadísticamente significativa (Chi con p<0,05): Antecendentes de P.P. (OR: 2,94), antecedentes de amenaza de P.P (OR: 2,10), antecedentes de aborto (OR:2,04), antecedentes de amenaza de aborto (OR: 2,42) placenta previa (OR: 3,89), anamia (OR: 1,73), embarazo no controlado (OR: 3,69), infecciones génito-urinarias (OR: 2,83), amenaza de parto prematuro en el embarazo actual (OR: 3,76), y ruptura prematura de membranas en el embarazo actual (OR: 3,50). El control, modificación o erradicación de los factores (RAP por ciento), embarazo no controlado, amenaza de parto prematuro en embarazo actual, la solteria, la RPM, las infecciones génitourinarias y el patrón de la función sexual durante el embarazo, resultaría en una disminución del problema (parto prematuro) en un 50,5 por ciento, 41,9 por ciento, 39,2 por ciento, 38,4 por ciento, 29,2 por ciento y 27,7 por ciento respectivamente. Se concluye afirmando que a pesar de los esfuerzos realizados hasta el presente, el parto prematuro continúa siendo un problema irresoluto, en el que la aplicación del enfoque de riesgo podría contribuir de manera eficaz, al identificar los factores involucrados en su etiología y estimar el impacto favorable de su control mediante políticas de salud bien orientadas