RESUMEN
Artificial metallo-nucleases (AMNs) are promising DNA damaging drug candidates. Here, we demonstrate how the 1,2,3-triazole linker produced by the Cu-catalysed azide-alkyne cycloaddition (CuAAC) reaction can be directed to build Cu-binding AMN scaffolds. We selected biologically inert reaction partners tris(azidomethyl)mesitylene and ethynyl-thiophene to develop TC-Thio, a bioactive C3 -symmetric ligand in which three thiophene-triazole moieties are positioned around a central mesitylene core. The ligand was characterised by X-ray crystallography and forms multinuclear CuII and CuI complexes identified by mass spectrometry and rationalised by density functional theory (DFT). Upon Cu coordination, CuII -TC-Thio becomes a potent DNA binding and cleaving agent. Mechanistic studies reveal DNA recognition occurs exclusively at the minor groove with subsequent oxidative damage promoted through a superoxide- and peroxide-dependent pathway. Single molecule imaging of DNA isolated from peripheral blood mononuclear cells shows that the complex has comparable activity to the clinical drug temozolomide, causing DNA damage that is recognised by a combination of base excision repair (BER) enzymes.
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Química Clic , Cobre , Cobre/química , Leucocitos Mononucleares/metabolismo , Ligandos , ADN/química , Azidas/químicaRESUMEN
DNA-damaging agents, such as radiation and chemotherapy, are common in cancer treatment, but the dosing has proven to be challenging, leading to severe side effects in some patients. Hence, to be able to personalize DNA-damaging chemotherapy, it is important to develop fast and reliable methods to measure the resulting DNA damage in patient cells. Here, we demonstrate how single DNA molecule imaging using fluorescence microscopy can quantify DNA-damage caused by the topoisomerase II (TopoII) poison etoposide. The assay uses an enzyme cocktail consisting of base excision repair (BER) enzymes to repair the DNA damage caused by etoposide and label the sites using a DNA polymerase and fluorescently labeled nucleotides. Using this DNA-damage detection assay we find a large variation in etoposide induced DNA-damage after in vitro treatment of blood cells from healthy individuals. We furthermore used the TopoII inhibitor ICRF-193 to show that the etoposide-induced damage in DNA was TopoII dependent. We discuss how our results support a potential future use of the assay for personalized dosing of chemotherapy.
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Daño del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/efectos de los fármacos , ADN de Cadena Simple/efectos de los fármacos , ADN de Cadena Simple/genética , Dicetopiperazinas/farmacología , Etopósido/farmacología , Imagen Individual de Molécula , Antineoplásicos Fitogénicos/farmacología , ADN/efectos de los fármacos , Reparación del ADN , Relación Dosis-Respuesta a Droga , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Microscopía Fluorescente , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
Optical DNA mapping (ODM) allows visualization of long-range sequence information along single DNA molecules. The data can for example be used for detecting long range structural variations, for aiding DNA sequence assembly of complex genomes and for mapping epigenetic marks and DNA damage across the genome. ODM traditionally utilizes sequence specific marks based on nicking enzymes, combined with a DNA stain, YOYO-1, for detection of the DNA contour. Here we use a competitive binding approach, based on YOYO-1 and netropsin, which highlights the contour of the DNA molecules, while simultaneously creating a continuous sequence specific pattern, based on the AT/GC variation along the detected molecule. We demonstrate and validate competitive-binding-based ODM using bacterial artificial chromosomes (BACs) derived from the human genome and then turn to DNA extracted from white blood cells. We generalize our findings with in-silico simulations that show that we can map a vast majority of the human genome. Finally, we demonstrate the possibility of combining competitive binding with enzymatic labeling by mapping DNA damage sites induced by the cytotoxic drug etoposide to the human genome. Overall, we demonstrate that competitive-binding-based ODM has the potential to be used both as a standalone assay for studies of the human genome, as well as in combination with enzymatic approaches, some of which are already commercialized.
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Benzoxazoles/química , Mapeo Cromosómico/métodos , ADN/química , Genoma Humano , Netropsina/química , Compuestos de Quinolinio/química , Análisis de Secuencia de ADN/métodos , Antineoplásicos Fitogénicos/farmacología , Sitios de Unión , Unión Competitiva , Cromosomas Artificiales Bacterianos/química , ADN/genética , Etopósido/farmacología , Colorantes Fluorescentes/química , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Imagen Individual de Molécula/métodosRESUMEN
FBXO31 was originally identified as a putative tumor suppressor gene in breast, ovarian, hepatocellular, and prostate cancers. By screening a set of cell cycle-regulated proteins as potential FBXO31 interaction partners, we have now identified Cdt1 as a novel substrate. Cdt1 DNA replication licensing factor is part of the pre-replication complex and essential for the maintenance of genomic integrity. We show that FBXO31 specifically interacts with Cdt1 and regulates its abundance by ubiquitylation leading to subsequent degradation. We also show that Cdt1 regulation by FBXO31 is limited to the G2 phase of the cell cycle and is independent of the pathways previously described for Cdt1 proteolysis in S and G2 phase. FBXO31 targeting of Cdt1 is mediated through the N terminus of Cdt1, a region previously shown to be responsible for its cell cycle regulation. Finally, we show that Cdt1 stabilization due to FBXO31 depletion results in re-replication. Our data present an additional pathway that contributes to the FBXO31 function as a tumor suppressor.
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Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Proteínas F-Box/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas F-Box/genética , Fase G2 , Humanos , Unión Proteica , Proteolisis , Proteínas Supresoras de Tumor/genética , UbiquitinaciónRESUMEN
BACKGROUND: DNA repair deficiency disorders are rare inherited diseases arising from pathogenic (disease-causing) variants in genes involved in DNA repair. There are no standardized diagnostic assays for the investigation of pathological significance of unknown variants in DNA repair genes. We hypothesized that our assays for measuring in vitro patient blood cell hypersensitivity to DNA-damaging agents can be used to establish the pathological significance of unknown variants in DNA repair genes. Six patients with variants in the DNA repair genes PRKDC (two siblings), DCLRE1C (two siblings), NBN, and MSH6 were included. Here, we used the cell division assay (CDA) and the γ-H2AX assay, which were both developed and clinically validated by us, to measure patient cell hypersensitivity in response to ionizing radiation, mitomycin C, cytarabine and doxorubicin. RESULTS: Radiation hypersensitivity was detected in the two patients with variants in the PRKDC gene (p < 0.0001 for both at 3.5 Gy), and the two patients with DCLRE1C variants (p < 0.0001 at 3.5 Gy for sibling 1 and p < 0.0001 at 1 Gy for sibling 2). The cells from the patients with the PRKDC variant were also deficient in removing γ-H2AX (p < 0.001). The cells from the patient with variants in the NBN gene were hypersensitive to mitomycin C (p = 0.0008) and deficient in both induction and removal of γ-H2AX in response to radiation. CONCLUSIONS: The combination of the CDA and the γ-H2AX assay is useful in investigating the significance of unknown variants in some DNA repair genes.
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Reparación del ADN , Histonas , Línea Celular , Daño del ADN/genética , Reparación del ADN/genética , Fibroblastos/metabolismo , Histonas/genética , Histonas/metabolismo , HumanosRESUMEN
INTRODUCTION: Analysis of measurable residual disease (MRD) is increasingly being implemented in the clinical care of children and adults with acute myeloid leukaemia (AML). However, MRD methodologies differ and discordances in results lead to difficulties in interpretation and clinical decision-making. The aim of this study was to compare results from reverse transcription quantitative polymerase chain reaction (RT-qPCR) and multiparameter flow cytometry (MFC) in childhood AML and describe the kinetics of residual leukaemic burden during induction treatment. METHODS: In 15 children who were treated in the NOPHO-AML 2004 trial and had fusion transcripts quantified by RT-qPCR, we compared MFC with RT-qPCR for analysis of MRD during (day 15) and after induction therapy. Eight children had RUNX1::RUNX1T1, one CBFB::MYH11 and six KMT2A::MLLT3. RESULTS: When ≥0.1% was used as cut-off for positivity, 10 of 22 samples were discordant. The majority (9/10) were MRD positive with RT-qPCR but MRD negative with MFC, and several such cases showed the presence of mature myeloid cells. Fusion transcript expression was verified in mature cells as well as in CD34 expressing cells sorted from diagnostic samples. CONCLUSIONS: Measurement with RT-qPCR suggests slower response kinetics than indicated from MFC, presumably due to the presence of mature cells expressing fusion transcript. The prognostic impact of early measurements with RT-qPCR remains to be determined.
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Leucemia Mieloide Aguda , Trastornos Mieloproliferativos , Niño , Humanos , Citometría de Flujo/métodos , Cinética , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Recurrencia Local de Neoplasia , Neoplasia Residual/diagnóstico , Pronóstico , Ensayos Clínicos como AsuntoRESUMEN
The Epstein-Barr virus (EBV)-encoded LMP1 oncogene has a role in transformation, proliferation, and metastasis of several EBV-associated tumors. Furthermore, LMP1 is critically involved in transformation and growth of EBV-immortalized B cells in vitro. The oncogenic properties of LMP1 are attributed to its ability to upregulate anti-apoptotic proteins and growth signals. The transcriptional regulation of LMP1 is dependent on the context of cellular and viral proteins present in the cell. Here, we investigated the effect of several signaling pathways on the regulation of LMP1 expression. Inhibition of p38 signaling, using p38-specific inhibitors SB203580 and SB202190, downregulated LMP1 in estrogen-induced EREB2.5 cells. Similarly, p38 inhibition decreased trichostatin A-induced LMP1 expression in P3HR1 cells. Exogenous expression of p38 in lymphoblastoid cell lines (LCLs) led to an increase in LMP1 promoter activity in reporter assays, and this activation was mediated by the previously identified CRE site in the promoter. Inhibition of p38 by SB203580 and p38-specific small interfering RNA (siRNA) also led to a modest decrease in endogenous LMP1 expression in LCLs. Chromatin immunoprecipitation indicated decreased binding of CREB-ATF1 to the CRE site in the LMP1 promoter after inhibition of the p38 pathway in EREB2.5 cells. Taken together, our results suggest that an increase in p38 activation upregulates LMP1 expression. Since p38 is activated in response to stimuli such as stress or possibly primary infection, a transient upregulation of LMP1 in response to p38 may allow the cells to escape apoptosis. Since the p38 pathway itself is activated by LMP1, our results also suggest the presence of an autoregulatory loop in LMP1 upregulation.
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Regulación Viral de la Expresión Génica , Herpesvirus Humano 4 , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Oncogénicas Virales , Proteínas de la Matriz Viral , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Regiones Promotoras Genéticas , Proteínas/genética , Proteínas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Regulación hacia Arriba , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Quantification of the DNA damage induced by chemotherapy in patient cells may aid in personalization of the dose used. However, assays to evaluate individual patient response to chemotherapy are not available today. Here, we present an assay that quantifies single-stranded lesions caused by the chemotherapeutic drug Bleomycin (BLM) in peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals. We use base excision repair (BER) enzymes to process the DNA damage induced by BLM and then extend the processed sites with fluorescent nucleotides using a DNA polymerase. The fluorescent patches are quantified on single DNA molecules using fluorescence microscopy. Using the assay, we observe a significant variation in the in vitro induced BLM damage and its repair for different individuals. Treatment of the cells with the BER inhibitor CRT0044876 leads to a lower level of repair of BLM-induced damage, indicating the ability of the assay to detect a compromised DNA repair in patients. Overall, the data suggest that our assay could be used to sensitively detect the variation in BLM-induced DNA damage and repair in patients and can potentially be able to aid in personalizing patient doses.
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Bleomicina/farmacología , Roturas del ADN de Cadena Simple , Reparación del ADN , Leucocitos Mononucleares/efectos de los fármacos , ADN/metabolismo , Humanos , Indoles , Leucocitos Mononucleares/metabolismo , Microscopía Fluorescente , Imagen Individual de MoléculaRESUMEN
The recently reported cell division assay (CDA) was optimized to measure the relative sensitivity of cells to cytotoxic drugs in vitro. Here, we investigated the in vitro hypersensitivity of lymphocytes from Fanconi anemia (FA) patients, to cytotoxic drugs using CDA. Peripheral blood mononuclear cells (PBMC) as well as cell lines derived from FA patients were treated with two DNA interstrand crosslinking (ICL) agents, mitomycin C and cyclophosphamide. Our data indicate that the CDA detects hypersensitivity of cells from FA patients to mitomycin C. Further, cell lines derived from FA-patients were also hypersensitive to mitomycin C as well as cyclophosphamide, when assayed by the CDA. This study suggests that the CDA is a useful alternative for the diagnosis of FA patients' hypersensitivity to ICL agents.
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División Celular/efectos de los fármacos , Anemia de Fanconi/tratamiento farmacológico , Mitomicina/farmacología , Antineoplásicos/farmacología , Línea Celular , Ciclofosfamida/farmacología , Citometría de Flujo/métodos , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Linfocitos/efectos de los fármacosRESUMEN
The latent membrane protein 1 (LMP1) oncogene carried by Epstein-Barr virus (EBV) is essential for transformation and maintenance of EBV-immortalized B cells in vitro, and it is expressed in most EBV-associated tumor types. The activation of the NF-kappaB pathway by LMP1 plays a critical role in the upregulation of antiapoptotic proteins. The EBV-encoded EBNA2 transactivator is required for LMP1 activation in latency III, while LMP1 itself appears to be critical for its activation in the latency II gene expression program. In both cases, additional viral and cellular transcription factors are required in mediating transcription activation of the LMP1 promoter. Using DNA affinity purification and chromatin immunoprecipitation assay, we showed here that members of the NF-kappaB transcription factor family bound to the LMP1 promoter in vitro and in vivo. Electrophoretic mobility shift assay analyses indicated the binding of the p50-p50 homodimer and the p65-p50 heterodimer to an NF-kappaB site in the LMP1 promoter. Transient transfections and reporter assays showed that the LMP1 promoter is activated by exogenous expression of NF-kappaB factors in both B cells and epithelial cells. Exogenous expression of NF-kappaB factors in the EBNA2-deficient P3HR1 cell line induced LMP1 protein expression. Overall, our data are consistent with the presence of a positive regulatory circuit between NF-kappaB activation and LMP1 expression.
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FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Regulación hacia Arriba , Proteínas de la Matriz Viral/genética , Secuencia de Bases , Sitios de Unión , Línea Celular , Inmunoprecipitación de Cromatina , Cromatografía de Afinidad , Cartilla de ADN , Dimerización , Ensayo de Cambio de Movilidad Electroforética , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Ionizing radiation (IR) is a common mode of cancer therapy, where DNA damage is the major reason of cell death. Here, we use an assay based on fluorescence imaging of single damaged DNA molecules isolated from radiated lymphocytes, to quantify IR induced DNA damage. The assay uses a cocktail of DNA-repair enzymes that recognizes and excises DNA lesions and then a polymerase and a ligase incorporate fluorescent nucleotides at the damage sites, resulting in a fluorescent "spot" at each site. The individual fluorescent spots can then be counted along single stretched DNA molecules and the global level of DNA damage can be quantified. Our results demonstrate that inclusion of the human apurinic/apyrimidinic endonuclease 1 (APE1) in the enzyme cocktail increases the sensitivity of the assay for detection of IR induced damage significantly. This optimized assay also allowed detection of a cooperative increase in DNA damage when IR was combined with mild hyperthermia, which is sometimes used as an adjuvant in IR therapy. Finally, we discuss how the method may be used to identify patients that are sensitive to IR and other types of DNA damaging agents.
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BACKGROUND: The phosphorylation of histone H2AX (γ-H2AX) at the DNA double-strand break (DSB) site is frequently used for quantifying DSBs and may be useful as a biomarker for clinical applications. We have previously reported a flow cytometry-based quantification of γ-H2AX for clinical routine. One major challenge, however, is the lack of a control sample for normalization of the day-to-day variation of the flow cytometry γ-H2AX assay. METHODS: Here, we report development of a mix-control sample containing peripheral blood mononuclear cells (PBMC) from 10 control individuals, for normalization of day-to-day variation of the flow cytometry-γ-H2AX assay. RESULTS: We showed that control individuals sampled on different days show an average day-to-day variation (CV) of 34%, which was reduced to 12% after normalization to the control sample. The normalization allowed detection of radiosensitivity of lymphoblastoid cell lines from ataxia telangiectasia patients, sampled over three days. CONCLUSION: The mix-control sample, consisting of 10 control individuals' PBMC, can be used as a control sample to normalize for day-to-day variation of the γ-H2AX assay. The use of this sample will facilitate integration of the γ-H2AX assay into clinical routine. © 2018 International Clinical Cytometry Society.
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Citometría de Flujo , Histonas/sangre , Células Cultivadas , Humanos , Leucocitos Mononucleares/patologíaRESUMEN
Endothelin receptor type A (EDNRA) is known as a mediator of cell proliferation and survival. Aberrant regulation of EDNRA has been shown to play a role in tumor growth and metastasis. Using a global gene expression screen, we found that expression of Ednra was upregulated in murine leukemia inducing cells co-expressing Hoxa9 and Meis1 compared to cells only expressing Hoxa9. The aim of this study was to explore the role of Ednra in leukemogenesis further. In a murine bone marrow transplantation model, mice transplanted with cells overexpressing Ednra and Hoxa9 succumbed to leukemia significantly earlier than mice transplanted with cells overexpressing Hoxa9 only. Furthermore, overexpression of Ednra led to increased proliferation and resistance to apoptosis of bone marrow cells in vitro. We could also show that Meis1 binds to the Ednra promoter region, suggesting a regulatory role for Meis1 in Ednra expression. Taken together, our results suggest a role for Ednra in Hoxa9/Meis1-driven leukemogenesis.
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Regulación Leucémica de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/metabolismo , Receptor de Endotelina A/metabolismo , Animales , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BLRESUMEN
Chondrocytes are effectively involved in the pathophysiological processes of inflammation in joints. They form cellular processes in the superficial layer of the articular cartilage and form gap junction coupled syncytium to facilitate cell-to-cell communication. However, very little is known about their physiological cellular identity and communication. The aim with the present work is to evaluate the physiological behavior after stimulation with the inflammatory inducers interleukin-1ß and lipopolysaccharide. The cytoskeleton integrity and intracellular Ca2+ release were assessed as indicators of inflammatory state. Cytoskeleton integrity was analyzed through cartilage oligomeric matrix protein and actin labeling with an Alexa 488-conjugated phalloidin probe. Ca2+ responses were assessed through the Ca2+ sensitive fluorophore Fura-2/AM. Western blot analyses of several inflammatory markers were performed. The results show reorganization of the actin filaments. Glutamate, 5-hydoxytryptamine, and ATP evoked intracellular Ca2+ release changed from single peaks to oscillations after inflammatory induction in the chondrocytes. The expression of toll-like receptor 4, the glutamate transporters GLAST and GLT-1, and the matrix metalloproteinase-13 increased. This work demonstrates that chondrocytes are a key part in conditions that lead to inflammation in the cartilage. The inflammatory inducers modulate the cytoskeleton, the Ca2+ signaling, and several inflammatory parameters. In conclusion, our data show that the cellular responses to inflammatory insults from healthy and inflammatory chondrocytes resemble those previously observed in astrocyte and cardiac fibroblasts networks.
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BACKGROUND: The nucleosomal histone protein H2AX is specifically phosphorylated (γ-H2AX) adjacent to DNA double-strand breaks (DSBs) and is used for quantifying DSBs. Many chemotherapies and ionizing radiation (IR) used in cancer treatment result in DSBs. Therefore, γ-H2AX has a significant potential as a biomarker in evaluating patient sensitivity and responsiveness to IR and chemotherapy. METHODS: Here, we report a flow cytometry-based quantification of γ-H2AX (FCM-γ-H2AX assay) customized for clinical practice. RESULTS: We validated that our method is able to detect DNA damage in peripheral blood mononuclear cells (PBMCs) treated with DSB inducing agents. The method also detected the DNA repair deficiency in PBMCs treated with DNA repair inhibitors, as well as the deficiency in DNA repair signaling in PBMCs from two ataxia telangiectasia patients. CONCLUSIONS: The FCM-γ-H2AX assay has sufficient analytical sensitivity and precision to measure levels of DNA damage and DNA repair for clinical purposes. © 2016 International Clinical Cytometry Society.
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Ataxia Telangiectasia/genética , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN/efectos de la radiación , ADN/efectos de la radiación , Citometría de Flujo/normas , Histonas/genética , Aminoglicósidos/farmacología , Ataxia Telangiectasia/metabolismo , Ataxia Telangiectasia/patología , Cromonas/farmacología , ADN/efectos de los fármacos , ADN/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Relación Dosis-Respuesta en la Radiación , Enediinos/farmacología , Citometría de Flujo/métodos , Rayos gamma , Histonas/metabolismo , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Leucocitos Mononucleares/efectos de la radiación , Morfolinas/farmacología , Fosforilación , Cultivo Primario de Células , Pironas/farmacologíaRESUMEN
OBJECTIVES: The clonogenic assay examines cell sensitivity to toxic agents and has been shown to correlate with normal tissue sensitivity to radiotherapy in cancer patients. The clonogenic assay is not clinically applicable due to its intra-individual variability and the time frame of the protocol. We aimed to develop a clinically applicable assay that correlated with the clonogenic assay. DESIGN AND METHODS: We have developed a faster and less labor-intensive cell division assay (CD assay) using flow cytometry and incorporation of a fluorescent thymidine analogue. The CD assay was calibrated to the clonogenic assay and optimized for peripheral blood lymphocytes. RESULTS: Following ionizing radiation of primary human skin fibroblasts, the four-day CD assay gave similar results as the 14-day clonogenic survival assay. In lymphocytes isolated from patient blood samples, the CD assay was able to detect increased radiosensitivity in ataxia telangiectasia patients and increased radiosensitivity after in vitro treatment with DNA-PK and ATM inhibitors. The CD assay found a variation in the intrinsic radiosensitivity of lymphocytes isolated from healthy control samples. The CD assay was able to measure the anti-proliferation effect of different chemotherapeutic drugs in lymphocytes. CONCLUSIONS: Our results indicate that the CD assay is a fast and reliable method to measure the anti-proliferation effect of DNA-damaging agents with a potential to find the most sensitive patients in the work-up before cancer treatment.
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Ataxia Telangiectasia/patología , Técnicas de Laboratorio Clínico/normas , Anemia de Fanconi/patología , Fibroblastos/patología , Citometría de Flujo/métodos , Piel/patología , Ataxia Telangiectasia/tratamiento farmacológico , Ataxia Telangiectasia/radioterapia , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Células Cultivadas , Cromonas/farmacología , Ensayo de Unidades Formadoras de Colonias , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Anemia de Fanconi/tratamiento farmacológico , Anemia de Fanconi/radioterapia , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/efectos de la radiación , Morfolinas/farmacología , Pironas/farmacología , Tolerancia a Radiación , Radiación Ionizante , Piel/efectos de los fármacos , Piel/efectos de la radiaciónRESUMEN
Immunofluorescence quantification of γH2AX foci is a powerful approach to quantify DNA double-strand breaks induced by cancer therapy or accidental exposure to ionizing radiation. Here we report a modification to the γH2AX immunofluorescence labeling method, whereby cells are stained in-solution before being spotted and fixed onto microscope slides. Our modified method allows arraying of 16 patient samples/slide ready for foci counting in 2 h and demonstrated reliably detection of γH2AX foci in mononuclear cells prepared from patients who had undergone radiation therapy.
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Roturas del ADN de Doble Cadena , Técnica del Anticuerpo Fluorescente/métodos , Histonas/análisis , Leucocitos Mononucleares/química , Neoplasias/radioterapia , Análisis de Matrices Tisulares/métodos , Aminoglicósidos/farmacología , Enediinos/farmacología , Fibroblastos/metabolismo , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/efectos de la radiación , Coloración y Etiquetado/métodosRESUMEN
The Epstein-Barr virus (EBV)-encoded tumour-associated latent membrane protein 1 (LMP1) gene expression is transactivated by EBV nuclear antigen 2 (EBNA2) in human B cells. We have previously identified a cyclic AMP-responsive element (CRE) in the B95-8 LMP1 promoter that is essential for transcription activation. Sequencing of LMP1 promoter in the P3HR1-derived EREB2.5 cell line revealed 25 single base pair substitutions in comparison to the B95-8 virus, one of them localized in the CRE element. Sequence variations in this element have been identified in several EBV isolates of both African and Asian origins. The effect of the P3HR1 CRE site variation on binding of factors to the LMP1 promoter sequence (LRS) and promoter activation was investigated with electrophoretic mobility-shift assays and reporter gene transfection assays. ATF1 and CREB1 transcription factors bound with reduced efficiency to the P3HR1 variant and below the detection level to the other tested variants. Accordingly, reporter plasmids carrying the P3HR1 CRE sequence in a B95-8 LRS context displayed 50 % lower activity in all tested cell lines. The impaired ability to activate transcription caused by the C to A substitution in CRE was not apparent when the mutated site was placed in a P3HR1 LRS context and the reporter transfected into Jijoye cells, most likely as a consequence of the other base pair substitutions in P3HR1 LRS. Overall, our results suggest that the mutations in the LRS CRE site have been conserved to adjust LMP1 expression to levels that favour cell survival in certain cellular and environmental contexts.
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Herpesvirus Humano 4/genética , Proteínas de la Matriz Viral/genética , Factor de Transcripción Activador 1 , Secuencia de Bases , Sitios de Unión/genética , Línea Celular Transformada , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Genes Virales , Variación Genética , Herpesvirus Humano 4/patogenicidad , Herpesvirus Humano 4/fisiología , Humanos , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/metabolismoRESUMEN
The Epstein-Barr virus (EBV) tumor-associated latent membrane protein 1 (LMP1) gene expression is transactivated by EBV nuclear antigen 2 (EBNA2) in human B cells. We previously reported that an E-box element at the LMP1 regulatory sequence (LRS) represses transcription of the LMP1 gene through the recruitment of a Max-Mad1-mSin3A complex. In the present study, using deletion/mutation analysis, and electrophoretic mobility shift assays, we show that the promoter region adjacent to the E-box (-59/-67) is required for the full repression conferred by E-box binding proteins. The repressive effect of these factors was overcome by an inhibitor of histone deacetylation, Trichostatin A (TSA), concurring with the reports that histone deacetylation plays an important role in repression mediated by Max-Mad1-mSin3A complex. Furthermore, ChIP analyses showed that histones at the transcriptionally active LMP1 promoter were hyperacetylated, whereas in the absence of transcription they were hypoacetylated. EBNA2 activation of the promoter required a consensus AP-2 sequence in the -103/-95 LRS region. While EMSA results and the low level of AP-2 factors expression in B cells argue against known AP-2 factors binding to this site, several pieces of evidence point to a similar mechanism of promoter activation as seen by AP-2 factors. We conclude that an AP-2 site-binding factor and EBNA2 act in concert to overcome the repression of the LMP1 promoter via the consensus AP-2 site. This activation showed strong correlation with histone hyperacetylation at the promoter, indicating this to be a major mechanism for the EBNA2 mediated LMP1 transactivation.