RESUMEN
BACKGROUND: Tissue clearing technologies have enabled remarkable advancements for in situ characterization of tissues and exploration of the three-dimensional (3D) relationships between cells, however, these studies have predominantly been performed in non-human tissues and correlative assessment with clinical imaging has yet to be explored. We sought to evaluate the feasibility of tissue clearing technologies for 3D imaging of intact human prostate and the mapping of structurally and molecularly preserved pathology data with multi-parametric volumetric MR imaging (mpMRI). METHODS: Whole-mount prostates were processed with either hydrogel-based CLARITY or solvent-based iDISCO. The samples were stained with a nuclear dye or fluorescently labeled with antibodies against androgen receptor, alpha-methylacyl coenzyme-A racemase, or p63, and then imaged with 3D confocal microscopy. The apparent diffusion coefficient and Ktrans maps were computed from preoperative mpMRI. RESULTS: Quantitative analysis of cleared normal and tumor prostate tissue volumes displayed differences in 3D tissue architecture, marker-specific cell staining, and cell densities that were significantly correlated with mpMRI measurements in this initial, pilot cohort. CONCLUSIONS: 3D imaging of human prostate volumes following tissue clearing is a feasible technique for quantitative radiology-pathology correlation analysis with mpMRI and provides an opportunity to explore functional relationships between cellular structures and cross-sectional clinical imaging.
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Imágenes de Resonancia Magnética Multiparamétrica/métodos , Imagen Óptica/métodos , Próstata , Neoplasias de la Próstata , Diagnóstico por Computador/métodos , Humanos , Genómica de Imágenes/métodos , Imagenología Tridimensional/métodos , Masculino , Microscopía Confocal/métodos , Persona de Mediana Edad , Estadificación de Neoplasias , Próstata/diagnóstico por imagen , Próstata/patología , Prostatectomía/métodos , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Coloración y Etiquetado/métodos , Carga TumoralRESUMEN
Purpose To investigate whether non-small cell lung cancer (NSCLC) tumors that express high normalized maximum standardized uptake value (SUVmax) are associated with a more epithelial-mesenchymal transition (EMT)-like phenotype. Materials and Methods In this institutional review board-approved study, a public NSCLC data set that contained fluorine 18 ((18)F) fluoro-2-deoxyglucose positron emission tomography (PET) and messenger RNA expression profile data (n = 26) was obtained, and patients were categorized on the basis of measured normalized SUVmax values. Significance analysis of microarrays was then used to create a radiogenomic signature. The prognostic ability of this signature was assessed in a second independent data set that consisted of clinical and messenger RNA expression data (n = 166). Signature concordance with EMT was evaluated by means of validation in a publicly available cell line data set. Finally, by establishing an in vitro EMT lung cancer cell line model, an attempt was made to substantiate the radiogenomic signature with quantitative polymerase chain reaction, and functional assays were performed, including Western blot, cell migration, glucose transporter, and hexokinase assays (paired t test), as well as pharmacologic assays against chemotherapeutic agents (half-maximal effective concentration). Results Differential expression analysis yielded a 14-gene radiogenomic signature (P < .05, false discovery rate [FDR] < 0.20), which was confirmed to have differences in disease-specific survival (log-rank test, P = .01). This signature also significantly overlapped with published EMT cell line gene expression data (P < .05, FDR < 0.20). Finally, an EMT cell line model was established, and cells that had undergone EMT differentially expressed this signature and had significantly different EMT protein expression (P < .05, FDR < 0.20), cell migration, glucose uptake, and hexokinase activity (paired t test, P < .05). Cells that had undergone EMT also had enhanced chemotherapeutic resistance, with a higher half-maximal effective concentration than that of cells that had not undergone EMT (P < .05). Conclusion Integrative radiogenomic analysis demonstrates an association between increased normalized (18)F fluoro-2-deoxyglucose PET SUVmax, outcome, and EMT in NSCLC. (©) RSNA, 2016 Online supplemental material is available for this article.
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Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Transición Epitelial-Mesenquimal/fisiología , Fluorodesoxiglucosa F18/farmacocinética , Neoplasias Pulmonares/diagnóstico , Tomografía de Emisión de Positrones/métodos , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/fisiopatología , Femenino , Genómica/métodos , Humanos , Pulmón/diagnóstico por imagen , Pulmón/fisiopatología , Neoplasias Pulmonares/fisiopatología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Pronóstico , Radiofármacos/farmacocinéticaRESUMEN
PURPOSE: To perform a radiogenomic analysis of women with breast cancer to study the multiscale relationships among quantitative computer vision-extracted dynamic contrast material-enhanced (DCE) magnetic resonance (MR) imaging phenotypes, early metastasis, and long noncoding RNA (lncRNA) expression determined by means of high-resolution next-generation RNA sequencing. MATERIALS AND METHODS: In this institutional review board-approved study, an automated image analysis platform extracted 47 computational quantitative features from DCE MR imaging data in a training set (n = 19) to screen for MR imaging biomarkers indicative of poor metastasis-free survival (MFS). The lncRNA molecular landscape of the candidate feature was defined by using an RNA sequencing-specific negative binomial distribution differential expression analysis. Then, this radiogenomic biomarker was applied prospectively to a validation set (n = 42) to allow prediction of MFS and lncRNA expression by using quantitative polymerase chain reaction analysis. RESULTS: The quantitative MR imaging feature, enhancing rim fraction score, was predictive of MFS in the training set (P = .007). RNA sequencing analysis yielded an average of 55.7 × 10(6) reads per sample and identified 14 880 lncRNAs from a background of 189 883 transcripts per sample. Radiogenomic analysis allowed identification of three previously uncharacterized and five named lncRNAs significantly associated with high enhancing rim fraction, including Homeobox transcript antisense intergenic RNA (HOTAIR) (P < .05), a known predictor of poor MFS in patients with breast cancer. Independent validation confirmed the association of the enhancing rim fraction phenotype with both MFS (P = .002) and expression of four of the top five differentially expressed lncRNAs (P < .05), including HOTAIR. CONCLUSION: The enhancing rim fraction score, a quantitative DCE MR imaging lncRNA radiogenomic biomarker, is associated with early metastasis and expression of the known predictor of metastatic progression, HOTAIR.
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Biomarcadores de Tumor/biosíntesis , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Imagen por Resonancia Magnética , Adulto , Anciano , Biomarcadores de Tumor/análisis , Medios de Contraste , Femenino , Regulación Neoplásica de la Expresión Génica , Genómica , Humanos , Imagen por Resonancia Magnética/métodos , Persona de Mediana Edad , Metástasis de la Neoplasia , Fenotipo , ARN Largo no Codificante/análisis , ARN Largo no Codificante/biosíntesis , Estudios Retrospectivos , Análisis de Secuencia de ARNRESUMEN
Chemical-induced read through of premature stop codons might be exploited as a potential treatment strategy for genetic disorders caused by nonsense mutations. Despite the promise of this approach, only a few read-through compounds (RTCs) have been discovered to date. These include aminoglycosides (e.g., gentamicin and G418) and nonaminoglycosides (e.g., PTC124 and RTC13). The therapeutic benefits of these RTCs remain to be determined. In an effort to find new RTCs, we screened an additional ~36,000 small molecular weight compounds using a high-throughput screening (HTS) assay that we had previously developed and identified two novel RTCs, GJ071, and GJ072. The activity of these two compounds was confirmed in cells derived from ataxia telangiectasia (A-T) patients with three different types of nonsense mutation in the ATM gene. Both compounds showed activity comparable to stop codons (TGA, TAG, and TAA) PTC124 and RTC13. Early structure-activity relationship studies generated eight active analogs of GJ072. Most of those analogs were effective on all three stop codons. GJ071 and GJ072, and some of the GJ072 analogs, appeared to be well tolerated by A-T cells. We also identified another two active RTCs in the primary screen, RTC204 and RTC219, which share a key structural feature with GJ072 and its analogs.
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Acetanilidas/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/genética , Ataxia Telangiectasia/tratamiento farmacológico , Benzodioxoles/farmacología , Codón sin Sentido , Codón de Terminación/efectos de los fármacos , Tiourea/análogos & derivados , Triazoles/farmacología , Acetanilidas/química , Ataxia Telangiectasia/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Benzodioxoles/química , Células Cultivadas , Proteínas de Unión al ADN/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Terapia Molecular Dirigida , Peso Molecular , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Tiourea/química , Tiourea/farmacología , Triazoles/químicaRESUMEN
Antisense morpholino oligonucleotides (AMOs) can reprogram pre-mRNA splicing by complementary binding to a target site and regulating splice site selection, thereby offering a potential therapeutic tool for genetic disorders. However, the application of this technology into a clinical scenario has been limited by the low correction efficiency in vivo and inability of AMOs to efficiently cross the blood brain barrier and target brain cells when applied to neurogenetic disorders such as ataxia-telangiecatasia (A-T). We previously used AMOs to correct subtypes of ATM splicing mutations in A-T cells; AMOs restored up to 20% of the ATM protein and corrected the A-T cellular phenotype. In this study, we demonstrate that an arginine-rich cell-penetrating peptide, (RXRRBR)(2)XB, dramatically improved ATM splicing correction efficiency when conjugated with AMOs, and almost fully corrected aberrant splicing. The restored ATM protein was close to normal levels in cells with homozygous splicing mutations, and a gene dose effect was observed in cells with heterozygous mutations. A significant amount of the ATM protein was still detected 21 days after a single 5 µm treatment. Systemic administration of an fluorescein isothiocyanate-labeled (RXRRBR)(2)XB-AMO in mice showed efficient uptake in the brain. Fluorescence was evident in Purkinje cells after a single intravenous injection of 60 mg/kg. Furthermore, multiple injections significantly increased uptake in all areas of the brain, notably in cerebellum and Purkinje cells, and showed no apparent signs of toxicity. Taken together, these results highlight the therapeutic potential of (RXRRBR)(2)XB-AMOs in A-T and other neurogenetic disorders.
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Arginina/química , Proteínas de Ciclo Celular/genética , Péptidos de Penetración Celular/farmacología , Cerebelo/metabolismo , Proteínas de Unión al ADN/genética , Técnicas de Transferencia de Gen , Oligonucleótidos Antisentido/farmacología , Proteínas Serina-Treonina Quinasas/genética , Empalme del ARN/genética , Proteínas Supresoras de Tumor/genética , Secuencia de Aminoácidos , Animales , Ataxia Telangiectasia/enzimología , Ataxia Telangiectasia/patología , Proteínas de la Ataxia Telangiectasia Mutada , Péptidos de Penetración Celular/química , Cerebelo/efectos de los fármacos , Fluoresceína-5-Isotiocianato/metabolismo , Ratones , Datos de Secuencia Molecular , Transporte de Proteínas/efectos de los fármacos , Células de Purkinje/efectos de los fármacos , Células de Purkinje/metabolismo , Empalme del ARN/efectos de los fármacos , Tolerancia a Radiación/efectos de los fármacosRESUMEN
Ataxia-telangiectasia mutated (ATM) is a high molecular weight protein serine/threonine kinase that plays a central role in the maintenance of genomic integrity by activating cell cycle checkpoints and promoting repair of DNA double-strand breaks. Little is known about the regulatory mechanisms for ATM expression itself. MicroRNAs are naturally existing regulators that modulate gene expression in a sequence-specific manner. Here, we show that a human microRNA, miR-421, suppresses ATM expression by targeting the 3'-untranslated region (3'UTR) of ATM transcripts. Ectopic expression of miR-421 resulted in S-phase cell cycle checkpoint changes and an increased sensitivity to ionizing radiation, creating a cellular phenotype similar to that of cells derived from ataxia-telangiectasia (A-T) patients. Blocking the interaction between miR-421 and ATM 3'UTR with an antisense morpholino oligonucleotide rescued the defective phenotype caused by miR-421 overexpression, indicating that ATM mediates the effect of miR-421 on cell cycle checkpoint and radiosensitivity. Overexpression of the N-Myc transcription factor, an oncogene frequently amplified in neuroblastoma, induced miR-421 expression, which, in turn, down-regulated ATM expression, establishing a linear signaling pathway that may contribute to N-Myc-induced tumorigenesis in neuroblastoma. Taken together, our findings implicate a previously undescribed regulatory mechanism for ATM expression and ATM-dependent DNA damage response and provide several potential targets for treating neuroblastoma and perhaps A-T.
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Regiones no Traducidas 3' , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neuroblastoma/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Secuencia de Bases , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Humanos , Neuroblastoma/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Fase S/efectos de la radiación , Transducción de Señal , Transcripción Genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
A recent challenge for investigators studying the progressive neurological disease ataxia-telangiectasia (A-T) is to identify mutations whose effects might be alleviated by mutation-targeted therapies. We studied ATM mutations in eight families of Japanese A-T patients (JPAT) and were able to identify all 16 mutations. The probands were compound heterozygotes in seven families, and one (JPAT2) was homozygous for a frameshift mutation. All mutations--four frameshift, two nonsense, four large genomic deletions, and six affecting splicing--were novel except for c.748C>T found in family JPAT6 and c.2639-384A>G found in family JPAT11/12. Using an established lymphoblastoid cell line (LCL) of patient JPAT11, ATM protein was restored to levels approaching wild type by exposure to an antisense morpholino oligonucleotide designed to correct a pseudoexon splicing mutation. In addition, in an LCL from patient JPAT8/9, a heterozygous carrier of a nonsense mutation, ATM levels could also be partially restored by exposure to readthrough compounds (RTCs): an aminoglycoside, G418, and a novel small molecule identified in our laboratory, RTC13. Taken together, our results suggest that screening and functional characterization of the various sorts of mutations affecting the ATM gene can lead to better identification of A-T patients who are most likely to benefit from rapidly developing mutation-targeted therapeutic technologies.
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Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Codón sin Sentido , Proteínas de Unión al ADN/genética , Mutación del Sistema de Lectura , Proteínas Serina-Treonina Quinasas/genética , Eliminación de Secuencia , Proteínas Supresoras de Tumor/genética , Aminoglicósidos/farmacología , Aminoglicósidos/uso terapéutico , Pueblo Asiatico , Ataxia Telangiectasia/tratamiento farmacológico , Proteínas de la Ataxia Telangiectasia Mutada , Secuencia de Bases , Proteínas de Ciclo Celular/agonistas , Línea Celular , Análisis Mutacional de ADN , Proteínas de Unión al ADN/agonistas , Exones , Gentamicinas/farmacología , Gentamicinas/uso terapéutico , Heterocigoto , Humanos , Datos de Secuencia Molecular , Terapia Molecular Dirigida , Morfolinos/farmacología , Morfolinos/uso terapéutico , Oligodesoxirribonucleótidos Antisentido/farmacología , Oligodesoxirribonucleótidos Antisentido/uso terapéutico , Linaje , Empalme del ARN , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Linfocitos T/patología , Proteínas Supresoras de Tumor/agonistasRESUMEN
A structure-activity relationship (SAR) study was carried out to identify novel, small molecular weight compounds which induce readthrough of premature termination codons. In particular, analogs of RTC13, 1, were evaluated. In addition, hypothesizing that these compounds exhibit their activity by binding to the ribosome, we prepared the hybrid analogs 13 containing pyrimidine bases and these also showed good readthrough activity.
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Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/metabolismo , Codón sin Sentido/metabolismo , Proteínas de Unión al ADN/metabolismo , Evaluación Preclínica de Medicamentos , Furanos/síntesis química , Furanos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Tiazoles/síntesis química , Tiazoles/farmacología , Proteínas Supresoras de Tumor/metabolismo , Ataxia Telangiectasia/tratamiento farmacológico , Ataxia Telangiectasia/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Línea Celular , Codón sin Sentido/química , Codón sin Sentido/genética , Codón de Terminación/genética , Codón de Terminación/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Diseño de Fármacos , Furanos/química , Ensayos Analíticos de Alto Rendimiento , Estructura Molecular , Terapia Molecular Dirigida , Mutación , Sistemas de Lectura Abierta , Terminación de la Cadena Péptídica Traduccional/genética , Biosíntesis de Proteínas/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Relación Estructura-Actividad , Tiazoles/química , Activación Transcripcional , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genéticaRESUMEN
BACKGROUND: No rapid reliable method exists for identifying ataxia-telangiectasia (A-T) homozygotes or heterozygotes. Heterozygotes are at an increased risk of cancer and are more sensitive to the effects of ionizing radiation (IR) than the general population. We report a rapid flow cytometry (FC)-based ataxia-telangiectasia mutated (ATM) kinase assay that measures ATM- dependent phosphorylation of structural maintenance of chromosomes 1 (SMC1) following DNA damage (FC-pSMC1 assay). METHODS: After optimizing conditions with lymphoblastoid cell lines (LCLs), we studied peripheral blood mononuclear cells (PBMCs) isolated from 16 healthy donors (unknowns), 10 obligate A-T heterozygotes, and 6 unrelated A-T patients. One hour after DNA damage (by either IR or bleomycin), the cells were fixed and incubated with a primary antibody to SMC1pSer966. We analyzed the stained cells by FC to determine the difference in geometric mean fluorescence intensity (DeltaGMFI) of untreated and treated cells; this difference was expressed as a percentage of daily experimental controls. RESULTS: The FC-pSMC1 assay reliably distinguished ATM heterozygotes and homozygotes from controls. Average DeltaGMFI percentages (SD) of daily controls were, for unknowns, 106.1 (37.6); for A-T heterozygotes, 37.0 (18.7); and for A-T homozygotes; -8.73 (16.2). Values for heterozygotes and homozygotes were significantly different from those of controls (P < 0.0001). CONCLUSIONS: The FC-pSMC1 assay shortens the turnaround time for diagnosing A-T homozygotes from approximately 3 months to approximately 3 h. It also identifies A-T heterozygotes and can be used for prenatal counseling or for screening individuals in large study cohorts for potential ATM heterozygosity, which can then be confirmed by sequencing.
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Ataxia Telangiectasia/diagnóstico , Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Citometría de Flujo/métodos , Heterocigoto , Homocigoto , Ataxia Telangiectasia/genética , Proteínas de la Ataxia Telangiectasia Mutada , Bleomicina/farmacología , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/metabolismo , Ensayo de Inmunoadsorción Enzimática , Reacciones Falso Positivas , Inestabilidad Genómica/genética , Humanos , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Tiempo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Mutations in the ataxia-telangiectasia mutated (ATM) gene are responsible for the autosomal recessive genetic disorder, ataxia-telangiectasia (A-T). Approximately 80% of ATM mutations found in A-T patients results in truncations, which can be detected by Protein Truncation Test (PTT). Conventional PTT uses SDS-PAGE electrophoresis to detect mobility of radiolabeled truncated protein fragments. In this study, we developed a non-radioactive Protein Truncation Test which utilizes an enzyme-linked immunosorbent assay (PTT-ELISA) to detect ATM mutations in eight overlapping fragments. N- and C-terminal epitopes (c-myc and V5, respectively) were introduced into transcription/translation products, which could then be detected by Sandwich ELISA. Using this assay, we screened 9 newly diagnosed A-T patients consecutively. Of the 18 expected mutations, 14 truncating mutations were independently identified by cDNA direct sequencing and/or DNA dHPLC analysis. PTT-ELISA detected all of these 14. Four mutations were novel. The PTT-ELISA provides a rapid method for detecting truncating mutations in large genes and should be considered prior to using more laborious or costly methods, such as direct sequencing.
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Proteínas de Ciclo Celular/genética , Análisis Mutacional de ADN/métodos , Proteínas de Unión al ADN/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/genética , Proteínas de la Ataxia Telangiectasia Mutada , Células Cultivadas , Heterocigoto , Homocigoto , Humanos , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: To investigate the biological effects of overexpression of the human DNA polymerase (pol-beta) on cellular response to DNA damage. METHODS: The cell strain HLFbeta from the stable overexpression of the human pol-beta was contaminated with methyl methanesulfonate (MMS) for investigating the effects of the pol-beta on the cellular responses to DNA damage on the aspects such as the DNA damage, the cell cycle and the induced mutation rate. RESULTS: The cell HLFbeta from the stable overexpression of the human pol-beta was obtained through the screening. The cellular response to DNA damage of HLFbeta induced by the MMS in the intermediate and high dosage group (ranging from 0.5 to 0.8 mmol/L) was significantly lower than that in the control group. The analysis for the cell cycle distribution showed that both the two types of cells contaminated by MMS had retardation at G(2) phase. In the HLFbeta group, the cells had the obvious G(2) phase retardation and 49.0% of the cells were retarded at G(1) phase as well when the MMS was increased to 0.5 mmol/L while in the control, only 20.1% of the cells were retarded at the G(1) phase when the same dosage of MMS was administered. Moreover, the MMS-induced mutagenesis in HLFbeta was increased from 4.5 x 10(-6) to 8.2 x 10(-6), significantly higher than that in the control group (P < 0.05). CONCLUSION: High Pol-beta level decreases cellular DNA damage induced by MMS. Nevertheless, the overexpression of Pol-beta can also increase error-prone DNA synthesis during DNA repair process.
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Ciclo Celular/genética , Daño del ADN/fisiología , ADN Polimerasa beta/biosíntesis , Ciclo Celular/efectos de los fármacos , Línea Celular , Daño del ADN/genética , Análisis Mutacional de ADN , ADN Polimerasa beta/genética , Reparación del ADN , Relación Dosis-Respuesta a Droga , Humanos , Metilmetanosulfonato/toxicidad , Mutágenos/toxicidad , MutaciónRESUMEN
Dysregulated epithelial to mesenchymal transition (EMT) in cancer cells endows invasive and metastatic properties upon cancer cells that favor successful colonization of distal target organs and therefore play a critical role in transforming early-stage carcinomas into invasive malignancies. EMT has also been associated with tumor recurrence and drug resistance and cancer stem cell initiation. Therefore, better understanding of the mechanisms behind EMT could ultimately contribute to the development of novel prognostic approaches and individualized therapies that specifically target EMT processes. As an effort to characterize the central transcriptome changes during EMT, we have developed a Transforming growth factor (TGF)-beta-based in vitro EMT model and used it to profile EMT-related gene transcriptional changes in two different cell lines, a non-small cell lung cancer cell line H358, and a breast cell line MCF10a. After 7 days of TGF-beta/Oncostatin M (OSM) treatment, changes in cell morphology to a mesenchymal phenotype were observed as well as concordant EMT-associated changes in mRNA and protein expression. Further, increased motility was noted and flow cytometry confirmed enrichment in cancer stem cell-like populations. Microarray-based differential expression analysis identified an EMT-associated gene expression signature which was confirmed by RT-qPCR and which significantly overlapped with a previously published EMT core signature. Finally, two novel EMT-regulating transcription factors, IRF5 and LMCD1, were identified and independently validated.
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Transición Epitelial-Mesenquimal/genética , Factores de Transcripción/genética , Transcriptoma , Factor de Crecimiento Transformador beta/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reproducibilidad de los Resultados , Transducción de SeñalRESUMEN
Screening for ATM mutations is usually performed using genomic DNA as a template for PCR amplification across exonic regions, with the consequence that deep intronic sequences are not analyzed. Here we report a novel pseudoexon-retaining deep intronic mutation (IVS28-159A>G; g.75117A>G based on GenBank U82828.1) in a patient with ataxia-telangiectasia (A-T), as well as the identification of a previously unrecognized alternative exon in the ATM gene (exon 28a) expressed in lymphoblastoid cell lines (LCL) derived from normal individuals. cDNA analysis using the A-T patient's LCL showed the retention of two aberrant intronic segments of 112 and 190 nt between exons 28 and 29. Minigenes were constructed to determine the functional significance of two genomic changes in the region of aberrant splicing: IVS28-193C>T (g.75083C>T) and IVS28-159A>G, revealing that: 1) the first is a polymorphism; 2) IVS28-159A>G weakens the 5' splice site of the alternative exon 28a and activates a cryptic 5' splice site (ss) 83 nt downstream; and 3) wild-type constructs also retain a 29-nt segment (exon 28a) as part of both the 112- and 190-nt segments. Maximum entropy estimates of ss strengths corroborate the cDNA and minigene findings. Such mutations may prove relevant in planning therapy that targets specific splicing aberrations.
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Empalme Alternativo , Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Mutación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/genética , Proteínas de la Ataxia Telangiectasia Mutada , Secuencia de Bases , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proteínas de Unión al ADN/metabolismo , Exones , Humanos , Intrones , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/metabolismo , Sitios de Empalme de ARN , Proteínas Supresoras de Tumor/metabolismoRESUMEN
In order to study the genetic polymorphisms of nucleotide repair gene hMTH1 in southern Chinese Han population, the polymorphisms of the gene's promoter and its five exons among peripheral blood lymphocytes of 172 Chinese Han people were analyzed with polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing. The sequences of the promoter and exon 1 of hMTH1 gene were conserved. A T to C polymorphism was detected at the 73th base in exon 2. The genotype frequencies of TT and TC were 93.02% and 6.98%, respectively. The allelic frequencies of T and C were 96.51% and 3.49%, respectively. A T to C polymorphism was detected at codon 45 in exon 3, which was first reported. The genotype frequencies of TT and TC were 95.35% and 4.65%, respectively. The allelic frequencies of T and C were 97.67% and 2.33%, respectively. A G to A polymorphism was detected at codon 83 in exon 4. The genotype frequencies of GG and GA were 89.53% and 10.47%, respectively. The allelic frequencies of G and A were 94.77% and 5.23%, respectively. A C to T polymorphism was detected at codon 119 in exon 5. The genotype frequencies of CC and CT were 95.93% and 4.07%, respectively. The allelic frequencies of C and T were 97.97% and 2.03%, respectively.
Asunto(s)
Pueblo Asiatico/genética , Enzimas Reparadoras del ADN/genética , Monoéster Fosfórico Hidrolasas/genética , Mutación Puntual , Polimorfismo Conformacional Retorcido-Simple , China/etnología , Exones , Frecuencia de los Genes , Genotipo , Humanos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Análisis de Secuencia de ADNRESUMEN
OBJECTIVE: To study DNA and nucleus damage in human embryo lung fibroblast (HLF) exposed to hydroquinone (HQ) and its genotoxicity. METHODS: HLF were treated with HQ (0, 10, 20, 40, 80 micro mol/L, respectively) for 3 h and DNA damage was detected by comet assay. HLF was also treated with the same concentrations of HQ for 1 h and micronucleus test was performed after they were cultured for 24 h. RESULTS: Comet assay showed that percentage of cells with tails in each groups treated with varied doses of HQ was 12%, 19%, 42%, 79% and 95%, respectively, with mean tail length of 7.87, 9.35, 11.03, 19.28 and 23.32 micro m, respectively, in an obvious dose-dependent manner (P < 0.05). Very significant increase in percentage of cells with tails and length of their comet tail were observed in those groups treated with HQ of 20, 40 and 80 micro mol/L (P < 0.01). And, proportion of high and severe DNA damage increased with dose of HQ. HQ could also induce formation of micronucleus and abnormal nucleus in all groups treated by varied doses of HQ, with rates of micronucleus and abnormal nucleus of 2%, 3%, 10%, 9% and 15%, and 6%, 7%, 16%, 27% and 28%, respectively, in a significant dose-dependent manner. There was significant increase in rates of micronuclei and abnormal nuclei in cells treated with HQ at doses of 20, 40 and 80 micro mol/L (P < 0.05). CONCLUSIONS: Exposure to HQ could cause DNA and nucleus damage inducing genotoxic effects on HLF.
Asunto(s)
Daño del ADN/efectos de los fármacos , Fibroblastos/citología , Hidroquinonas/toxicidad , Pulmón/citología , Núcleo Celular/efectos de los fármacos , Ensayo Cometa , Embrión de Mamíferos , Humanos , Pruebas de MicronúcleosRESUMEN
OBJECTIVE: To construct pEGFP-C1-T vector, an eukaryotic expression plasmid of hMTH1 gene antisense RNA. METHODS: The conservative region of hMTH1 gene was amplified by RT-PCR after total RNA being extracted from human embryo lung fibroblast (HLF) and then cloned into pGEM-T vector. After the recombinant plasmid was certified by DNA sequencing, the conservative region of hMTH1 gene was inserted into pEGFP-C1 vector reversedly and pEGFP-C1-T vector was constructed. The efficiency of antisense inhibition was verified by Western blotting after cell transfection. RESULTS: 423 bp fragment including conservative region of hMTH1 gene was obtained by RT-PCR. After cloned by pGEM-T vector and certified by DNA sequencing, pEGFP-C1-T vector was successfully constructed by means of recombinant DNA technology. Additionally pEGFP-C1-T vector could efficiently decrease hMTH1 protein level by 46%. CONCLUSION: The efficient expression vector of hMTH1 gene antisense RNA, pEGFP-C1-T has been constructed successfully.
Asunto(s)
Enzimas Reparadoras del ADN , Vectores Genéticos/genética , Monoéster Fosfórico Hidrolasas/genética , ARN sin Sentido/biosíntesis , Humanos , Plásmidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In an effort to explore the possible causes of human radiosensitivity and identify more rapid assays for cellular radiosensitivity, we interrogated a set of assays that evaluate cellular functions involved in recognition and repair of DNA double-strand breaks: (1) neutral comet assay, (2) radiation-induced γ-H2AX focus formation, (3) the temporal kinetics of structural maintenance of chromosomes 1 phosphorylation, (4) intra-S-phase checkpoint integrity, and (5) mitochondrial respiration. We characterized a unique panel of 19 "radiosensitive" human lymphoblastoid cell lines from individuals with undiagnosed diseases suggestive of a DNA repair disorder. Radiosensitivity was defined by reduced cellular survival using a clonogenic survival assay. Each assay identified cell lines with defects in DNA damage response functions. The highest concordance rate observed, 89% (17/19), was between an abnormal neutral comet assay and reduced survival by the colony survival assay. Our data also suggested that the neutral comet assay would be a more rapid surrogate for analyzing DNA repair/processing disorders.
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Bioensayo/métodos , Línea Celular/fisiología , Línea Celular/efectos de la radiación , Ensayo de Unidades Formadoras de Colonias/métodos , Ensayo Cometa/métodos , Daño del ADN , Tolerancia a Radiación/fisiología , Relación Dosis-Respuesta en la Radiación , Humanos , Dosis de RadiaciónRESUMEN
Highly complementary antisense morpholino oligonucleotides (AMOs) can bind to pre-mRNA and modulate splicing site selection. This offers a powerful tool to regulate the splicing process, such as correcting subtypes of splicing mutations and nonsense mutations and reprogramming alternative splicing processes. Therefore, AMO-mediated splicing modulation represents an attractive therapeutic strategy for genetic disorders. Primary immunodeficiency diseases (PIDs) are a heterogeneous group of genetic disorders that result from mutations in genes involved in development and maintenance of the immune system. Many of these mutations are splicing mutations and nonsense mutations that can be manipulated by AMOs. This review discusses AMO-mediated splicing modulation approaches and their potential applications in treating PIDs.
Asunto(s)
Terapia Genética/métodos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/terapia , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/uso terapéutico , Empalme Alternativo , Codón sin Sentido , Humanos , Mutación , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/química , Precursores del ARN/genética , Sitios de Empalme de ARN/genética , Empalme del ARNRESUMEN
Antisense oligonucleotides (AO) or antisense RNA can complementarily bind to a target site in pre-mRNA and regulate gene splicing, either to restore gene function by reprogramming gene splicing or to inhibit gene expression by disrupting splicing. These two applications represent novel therapeutic strategies for several types of diseases such as genetic disorders, cancers and infectious diseases. In this review, the recent developments and applications of antisense-mediated splicing modulation in molecular therapy are discussed, with emphasis on advances in antisense-mediated splice targeting, applications in diseases and systematic delivery.