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PURPOSE: Craniopharyngiomas are benign tumors of the sellar or parasellar regions. They arise from the remnants of Rathke's pouch and are considered a "developmental disease." microRNAs are short non-coding RNAs that play a key regulatory role in the control of expression of entire gene networks. We performed an extensive analysis of miRNAs in craniopharyngiomas aiming to identify a miRNA expression signature that might aid in the prognosis of disease progression and outcome. METHODS: Thirty-seven craniopharyngioma samples from twenty-three patients, ten age-matched controls from autopsy, and ten infant controls from the developing pituitary from autopsy were evaluated for the expression of 754 miRNAs using TaqMan® Low Density Arrays (TLDAs) v2.0 (Applied Biosystems, Foster City, CA). RESULTS: Among the most differentially expressed miRNAs, downregulation of miR-132 appears to be a marker of aggressiveness and also plays a role in epithelial-mesenchymal transition. CONCLUSIONS: This is the first time that an extensive study of miRNA expression has been performed in craniopharyngiomas. Further research needs to be performed to investigate the potential role of miR-132 in the development and progression of craniopharyngiomas, and its value as a prognostic marker of aggressiveness.
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Biomarcadores de Tumor/genética , Craneofaringioma/diagnóstico , Craneofaringioma/genética , MicroARNs/genética , Neoplasias Hipofisarias/diagnóstico , Neoplasias Hipofisarias/genética , Adolescente , Biomarcadores de Tumor/biosíntesis , Niño , Preescolar , Craneofaringioma/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , MicroARNs/biosíntesis , Neoplasias Hipofisarias/metabolismoRESUMEN
A limited number of reports have investigated the role of microRNAs in osteosarcoma. In this study, we performed miRNA expression profiling of osteosarcoma cell lines, tumor samples, and normal human osteoblasts. Twenty-two differentially expressed microRNAs were identified using high throughput real-time PCR analysis, and 4 (miR-135b, miR-150, miR-542-5p, and miR-652) were confirmed and validated in a different group of tumors. Both miR-135b and miR-150 have been previously shown to be important in cancer. We hypothesize that dysregulation of differentially expressed microRNAs may contribute to tumorigenesis. They might also represent molecular biomarkers or targets for drug development in osteosarcoma.
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Studies are beginning to emerge that demonstrate intriguing differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). Here, we investigated the expression of key members of the Nodal embryonic signaling pathway, critical to the maintenance of pluripotency in hESCs. Western blot and real-time RT-PCR analyses reveal slightly lower levels of Nodal (a TGF-beta family member) and Cripto-1 (Nodal's co-receptor) and a dramatic decrease in Lefty (Nodal's inhibitor and TGF-beta family member) in hiPSCs compared with hESCs. The noteworthy drop in hiPSC's Lefty expression correlated with an increase in the methylation of Lefty B CpG island. Based on these findings, we addressed a more fundamental question related to the consequences of epigenetically reprogramming hiPSCs, especially with respect to maintaining a stable ESC phenotype. A global comparative analysis of 365 microRNAs (miRs) in two hiPSC versus four hESC lines ultimately identified 10 highly expressed miRs in hiPCSs with >10-fold difference, which have been shown to be cancer related. These data demonstrate cancer hallmarks expressed by hiPSCs, which will require further assessment for their impact on future therapies..
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Biomarcadores de Tumor/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes/metabolismo , Biomarcadores de Tumor/genética , Western Blotting , Línea Celular , Epigénesis Genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Células Madre Pluripotentes/citología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Chondrosarcomas are malignant cartilage tumors that do not respond to traditional chemotherapy or radiation. The 5-year survival rate of histologic grade III chondrosarcoma is less than 30%. An animal model of chondrosarcoma has been established--namely, the Swarm Rat Chondrosarcoma (SRC)--and shown to resemble the human disease. Previous studies with this model revealed that tumor microenvironment could significantly influence chondrosarcoma malignancy. METHODS: To examine the effect of the microenvironment, SRC tumors were initiated at different transplantation sites. Pyrosequencing assays were utilized to assess the DNA methylation of the tumors, and SAGE libraries were constructed and sequenced to determine the gene expression profiles of the tumors. Based on the gene expression analysis, subsequent functional assays were designed to determine the relevancy of the specific genes in the development and progression of the SRC. RESULTS: The site of transplantation had a significant impact on the epigenetic and gene expression profiles of SRC tumors. Our analyses revealed that SRC tumors were hypomethylated compared to control tissue, and that tumors at each transplantation site had a unique expression profile. Subsequent functional analysis of differentially expressed genes, albeit preliminary, provided some insight into the role that thymosin-ß4, c-fos, and CTGF may play in chondrosarcoma development and progression. CONCLUSION: This report describes the first global molecular characterization of the SRC model, and it demonstrates that the tumor microenvironment can induce epigenetic alterations and changes in gene expression in the SRC tumors. We documented changes in gene expression that accompany changes in tumor phenotype, and these gene expression changes provide insight into the pathways that may play a role in the development and progression of chondrosarcoma. Furthermore, specific functional analysis indicates that thymosin-ß4 may have a role in chondrosarcoma metastasis.
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Biomarcadores de Tumor/genética , Condrosarcoma/genética , Epigénesis Genética , Perfilación de la Expresión Génica , Neoplasias Pulmonares/etiología , Tibia/patología , Animales , Biomarcadores de Tumor/metabolismo , Western Blotting , Cartílago/metabolismo , Cartílago/patología , Condrosarcoma/metabolismo , Condrosarcoma/patología , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Metilación de ADN , Genes fos/fisiología , Humanos , Inyecciones Subcutáneas , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Desnudos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Ratas , Ratas Sprague-Dawley , Timosina/genética , Timosina/metabolismo , Tibia/metabolismo , Células Tumorales Cultivadas/trasplanteRESUMEN
PURPOSE: Currently, there is no conclusive treatment for brainstem tumor. To facilitate the development of new treatments, it is essential to establish predictive preclinical in vivo models in which therapeutic modalities can be evaluated. Although a few rodent models have been reported, there is no novel approach that can monitor tumor response qualitatively and quantitatively. MATERIALS AND METHODS: Bioluminescence imaging was used to characterize a rat brainstem tumor model. In this model, 9L gliosarcoma cells, transduced with an onco-retroviral vector containing the luciferase coding sequence, were inoculated into Fisher 344 rats. RESULT: Histopathological assessment showed successful cell implantation into the brainstem. There was a strong correlation between pathological tumor volume and luminescence strength. Longitudinal quantitative responses of the tumor after application of a therapeutic agent were also demonstrated. CONCLUSION: This study demonstrates a robust rodent model with the ability to monitor brainstem tumor growth and response to chemotherapeutic agents.
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Neoplasias del Tronco Encefálico/diagnóstico , Diagnóstico por Imagen , Gliosarcoma/diagnóstico , Mediciones Luminiscentes , Animales , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/patología , Línea Celular Tumoral , Diagnóstico por Imagen/métodos , Modelos Animales de Enfermedad , Gliosarcoma/genética , Gliosarcoma/patología , Luciferasas/genética , Mediciones Luminiscentes/métodos , Proteínas Luminiscentes/química , Imagen por Resonancia Magnética , Masculino , Microinyecciones , Trasplante de Neoplasias , Ratas , Ratas Endogámicas F344RESUMEN
Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses, to generate multicistronic retroviral vectors with efficient translation of four cistrons. Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.
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Vectores Genéticos , Péptidos/metabolismo , Retroviridae/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Homología de Secuencia de AminoácidoRESUMEN
miRNAs are noncoding RNAs with abnormal expression in breast cancer; their expression in high-risk benign breast tissue may relate to breast cancer risk. We examined miRNA profiles in contralateral unaffected breasts (CUB) of patients with breast cancer and validated resulting candidates in two additional sample sets. Expression profiles of 754 mature miRNAs were examined using TaqMan Low Density Arrays in 30 breast cancer samples [15 estrogen receptor (ER)-positive and 15 ER-negative] and paired CUBs and 15 reduction mammoplasty controls. Pairwise comparisons identified miRNAs with significantly differential expression. Seven candidate miRNAs were examined using qRT-PCR in a second CUB sample set (40 cases, 20 ER+, 20 ER-) and 20 reduction mammoplasty controls. Further validation was performed in 80 benign breast biopsy (BBB) samples; 40 from cases who subsequently developed breast cancer and 40 from controls who did not. Logistic regression, using tertiles of miRNA expression, was used to discriminate cases from controls. Seven miRNAs were differentially expressed in tumors and CUBs versus reduction mammoplasty samples. Among them, miR-18a and miR-210 were validated in the second CUB set, showing significantly higher expression in tumor and CUBs than in reduction mammoplasty controls. The expression of miR-18a and miR-210 was also significantly higher in BBB cases than in BBB controls. When both miR-18a and miR-210 were expressed in the upper tertiles in BBB, OR for subsequent cancer was 3.20, P = 0.023. miR-18a and miR-210 are expressed at higher levels in CUBs of patients with breast cancer, and in BBB prior to cancer development, and are therefore candidate breast cancer risk biomarkers. Cancer Prev Res; 10(1); 89-97. ©2016 AACR.
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Neoplasias de la Mama/patología , Carcinogénesis/patología , MicroARNs/genética , Adulto , Anciano , Biomarcadores de Tumor/metabolismo , Biopsia , Mama , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/metabolismo , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Estrógenos/metabolismoRESUMEN
It is now well established that tumor growth is angiogenesis dependent. Inhibition of angiogenesis, therefore, is likely to be an effective anticancer approach. A gene therapy-mediated approach to the delivery of antiangiogenic agents using adeno-associated virus (AAV) vectors has a number of advantages, including the potential for sustained expression. We have constructed a rAAV vector in which the expression of a soluble, truncated form of the vascular endothelial growth factor receptor-2 (Flk-1), a known inhibitor of endothelial cell activation, is driven by a composite beta-actin-based promoter. After intraportal injection of this vector, high-level, stable transgene expression was generated in mice. This established a systemic state of angiogenesis inhibition; sera from these mice inhibited endothelial cell activation in vitro and Matrigel plug neovascularization in vivo. Significant antitumor efficacy was observed in two murine models of pediatric kidney tumors. Tumor development was prevented in 10 of 15 (67%) mice, with significant growth restriction of tumors in the remaining mice. For the first time, long-term, in vivo expression of a functional angiogenesis inhibitor has been established using rAAV, with resultant anticancer efficacy in a relevant, orthotopic tumor model. These findings establish the feasibility of using rAAV vectors in antiangiogenic gene therapy.
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Neoplasias Renales/terapia , Hígado/metabolismo , Neovascularización Patológica/terapia , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Factores de Crecimiento/fisiología , Tumor de Wilms/terapia , Animales , División Celular/genética , División Celular/fisiología , Dependovirus/genética , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Factores de Crecimiento Endotelial/biosíntesis , Factores de Crecimiento Endotelial/genética , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Terapia Genética/métodos , Humanos , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/genética , Neoplasias Renales/patología , Hígado/fisiología , Linfocinas/antagonistas & inhibidores , Linfocinas/biosíntesis , Linfocinas/genética , Ratones , Ratones SCID , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Receptores de Factores de Crecimiento/biosíntesis , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento Endotelial Vascular , Transducción Genética , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Tumor de Wilms/irrigación sanguínea , Tumor de Wilms/genética , Tumor de Wilms/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We and others have demonstrated expression of the aldo-keto reductase AKR1C3 in myeloid leukemia cell lines and that inhibitors of the enzyme, including nonsteroidal anti-inflammatory drugs (NSAIDs), promote HL-60 differentiation in response to all-trans retinoic acid (ATRA) and 1alpha,25-dihydroxyvitamin D3 (D3). Here, we demonstrate that overexpression of AKR1C3 reciprocally desensitizes HL-60 cells to ATRA and D3, thus confirming the enzyme as a novel regulator of cell differentiation. AKR1C3 possesses marked 11-ketoreductase activity converting prostaglandin (PG) D2 to PGF2alpha. Supplementing HL-60 cultures with PGD2 mimicked treatment with AKR1C3-inhibitors by enhancing the differentiation of the cells in response to ATRA. However, PGD2 is chemically unstable, being converted first to PGJ2 and then stepwise to 15-deoxy-Delta(12,14)-prostaglandin J2(15Delta-PGJ2), a natural ligand for the peroxisome proliferator-activated receptor-gamma (PPARgamma). Consistent with this, PGD2 was rapidly converted to PGJ2 under normal tissue culture conditions but not in the presence of recombinant AKR1C3 when PGF2alpha was predominantly formed. In addition, PGJ2 but not PGF2alpha recapitulated the potentiation of HL-60 differentiation by PGD2 and AKR1C3 inhibitors. Furthermore, the capacity of all of these treatments to potentiate HL-60 cell differentiation was significantly reduced in the presence of the PPARgamma-antagonist GW 9662. We conclude that AKRIC3 protects HL-60 cells against ATRA and D3-induced cell differentiation by limiting the production of natural PPARgamma ligands via the diversion of PGD2 toward PGF2alpha and away from PGJ2. In addition, these observations identify AKR1C3 as plausible target for the non-cyclooxygenase-dependent antineoplastic actions of NSAIDs.
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3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos/farmacología , Prostaglandina D2/análogos & derivados , 3-Hidroxiesteroide Deshidrogenasas/biosíntesis , 3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , 3-alfa-Hidroxiesteroide Deshidrogenasa (B-Específica) , Androstano-3,17-diol/metabolismo , Androstano-3,17-diol/farmacología , Anilidas/farmacología , Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Dihidrotestosterona/metabolismo , Dihidrotestosterona/farmacología , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/farmacología , Expresión Génica , Células HL-60 , Humanos , Prostaglandina D2/metabolismo , Prostaglandina D2/farmacología , Transgenes , Tretinoina/farmacologíaRESUMEN
Treatment of MSCV-GFP-transduced HL60 promyelocytic cells with all-trans retinoic acid (ATRA) resulted in a significant increase in GFP expression. The increased GFP expression was observed by 16 hr and was dependent on de novo protein production. This effect was specific to ATRA and unrelated to cell differentiation because it was not induced by dimethyl sulfoxide. Furthermore, a similar increase in GFP expression was observed in MSCV-GFP-transfected K562 cells, which do not differentiate when exposed to ATRA. Significantly increased GFP expression was seen at doses as low as 0.5 nM ATRA and was abrogated by AGN193109, an antagonist of retinoid signaling. We therefore conclude that this increase in gene expression is mediated by retinoic acid receptors. The long terminal repeat (LTR) region of MSCV contains candidate retinoic acid response elements and response elements for the ATRA-inducible transcription factor C/EBPalpha. We suggest that the increase in GFP expression is driven by the action of ATRA-activated host cell transcription factors. These findings offer a method to increase the expression of retroviral transgenes either in vitro or in vivo by treatment with low doses of retinoic acid that are clinically achievable and well tolerated. This use of inducible host cell transcription factors offers an alternative to engineering novel LTR regulatory sequences in order to increase transgene expression.
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Antineoplásicos/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Virus de la Leucemia Murina/genética , Transgenes/fisiología , Tretinoina/farmacología , Animales , Secuencia de Bases , Diferenciación Celular , Proteínas Fluorescentes Verdes/genética , Células HL-60 , Humanos , Células K562 , Glicoproteínas de Membrana/genética , Ratones , Datos de Secuencia Molecular , Naftalenos/farmacología , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptores de Ácido Retinoico/metabolismo , Células Madre/virología , Factores de Transcripción , Transducción Genética , Proteínas del Envoltorio Viral/genéticaRESUMEN
MDM2 inhibits transactivation properties of the tumor suppressor protein p53 by binding to and facilitating proteasomal degradation of p53. Because MDM2 targets p53 for degradation, it was anticipated that cells that overexpress MDM2 would not contain functional wild-type p53 (wtp53). However, p53 and MDM2 in cells with damaged DNA can become phosphorylated, and their binding to each other can become inhibited. Thus, p53 remains functional and induces apoptosis of damaged cells. Here we report the results of experiments designed to investigate whether MDM2 amplification and overexpression can inhibit p53-mediated chemosensitivity to DNA-damaging drugs. Two cell lines in which MDM2 is amplified, NB-1691 and Rh18, were transduced with an adenoviral expression vector for p53 (Ad.p53). Although functional wtp53 was detected, no change in chemosensitivity was observed, suggesting that endogenous wtp53 may have been active in the MDM2-amplified cells. The adenoviral vector Ad.MDM2 was used to generate MDM2 expression in a rhabdomyosarcoma cell line, Rh30-CI.27, engineered to express inducible wtp53. When p53 expression was induced, cells became chemosensitive to actinomycin D in the presence or absence of MDM2 expression; this result suggests that MDM2 cannot inhibit p53-mediated chemosensitivity. There was no evidence of a reduced amount of MDM2-p53 binding after drug exposure, but the remaining unbound wtp53 may be functional and capable of potentiating cytotoxicity. In conclusion, MDM2 expression is important in inhibiting p53 function during tumor development but not during the DNA damage-mediated cytotoxic response.
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ADN/efectos de los fármacos , Proteínas Nucleares , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adenoviridae/genética , Antineoplásicos/farmacología , Western Blotting , División Celular , Línea Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , Daño del ADN , Dactinomicina/farmacología , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Genes Reporteros , Vectores Genéticos , Humanos , Concentración 50 Inhibidora , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fenotipo , Pruebas de Precipitina , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Rabdomiosarcoma/metabolismo , Células Tumorales CultivadasRESUMEN
Destruction and remodeling of the extracellular matrix occurs during the formation of new blood vessels that are required for tumor growth. We sought to determine whether gene-therapy mediated in vivo delivery of tissue inhibitor of matrix metalloproteinase-3 (TIMP-3), using retroviral vector-producer cells, could suppress angiogenesis and subsequent tumor growth in a murine neuroblastoma model. Tumor volume 28 days after coinjection of tumor cells with producer cells generating TIMP-3-encoding retroviral vectors was 21% that of controls, as was the mean tumor vascular index, a measure of blood vessel maturity. When tumors were allowed to reach a mean volume of 0.05 cm(3) before treatment, their size 2 weeks later was 47% relative to controls; larger tumors were not significantly affected. When producer cells were injected at surgical sites following excision of subcutaneous tumors, local recurrence 14 days later was only 22% in TIMP-3 producer cell treated mice as compared to 71% in controls. Unsuccessful transduction of melanoma cells in situ, another tumor of neural crest origin, resulted in unimpaired tumor growth, despite the fact that these tumors are susceptible to TIMP-3 overexpression, demonstrating the importance of tumor cell transduction in this approach. Thus, retroviral vector-producer cell-mediated in vivo gene transfer of TIMP-3 to tumor cells can significantly restrict tumor-induced angiogenesis and tumor growth. This approach may be an effective adjuvant in the treatment of neuroblastoma and other solid tumors refractory to traditional therapy, although it appears to be most effective in smaller tumors or in the setting of minimal residual disease, and the tumor cells must be susceptible to retroviral vector-mediated transduction.
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Inhibidores de la Angiogénesis/genética , Vectores Genéticos/administración & dosificación , Neuroblastoma/terapia , Retroviridae/genética , Inhibidor Tisular de Metaloproteinasa-3/genética , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Animales , Línea Celular Tumoral , Matriz Extracelular , Ratones , Ratones SCID , Neoplasia Residual , Neovascularización Patológica , Neuroblastoma/irrigación sanguínea , Neuroblastoma/patologíaRESUMEN
BACKGROUND: Mechanisms underlying the development of virus-induced asthma exacerbations remain unclear. To investigate if epigenetic mechanisms could be involved in virus-induced asthma exacerbations, we undertook DNA methylation profiling in asthmatic and healthy nasal epithelial cells (NECs) during Human Rhinovirus (HRV) infection in vitro. METHODS: Global and loci-specific methylation profiles were determined via Alu element and Infinium Human Methylation 450 K microarray, respectively. Principal components analysis identified the genomic loci influenced the most by disease-status and infection. Real-time PCR and pyrosequencing were used to confirm gene expression and DNA methylation, respectively. RESULTS: HRV infection significantly increased global DNA methylation in cells from asthmatic subjects only (43.6% to 44.1%, p = 0.04). Microarray analysis revealed 389 differentially methylated loci either based on disease status, or caused by virus infection. There were disease-associated DNA methylation patterns that were not affected by HRV infection as well as HRV-induced DNA methylation changes that were unique to each group. A common methylation locus stood out in response to HRV infection in both groups, where the small nucleolar RNA, H/ACA box 12 (SNORA12) is located. Further analysis indicated that a relationship existed between SNORA12 DNA methylation and gene expression in response to HRV infection. CONCLUSIONS: We describe for the first time that Human rhinovirus infection causes DNA methylation changes in airway epithelial cells that differ between asthmatic and healthy subjects. These epigenetic differences may possibly explain the mechanism by which respiratory viruses cause asthma exacerbations.
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Asma/genética , Asma/virología , Metilación de ADN/genética , Células Epiteliales/virología , Nariz/patología , Infecciones por Picornaviridae/genética , Rhinovirus/fisiología , Adulto , Asma/fisiopatología , Demografía , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Regulación de la Expresión Génica , Sitios Genéticos , Genoma Humano/genética , Humanos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Infecciones por Picornaviridae/patología , Infecciones por Picornaviridae/virología , Análisis de Componente Principal , Pruebas de Función Respiratoria , Adulto JovenRESUMEN
In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm, which is manifested by rosette formation, with consecutive differentiation into neural progenitors and early glial-like cells. In this study, we examined the involvement of early neural markers - OTX2, PAX6, Sox1, Nestin, NR2F1, NR2F2, and IRX2 - in the onset of rosette formation, during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation, which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation, when rosettes comprise no more than 3-5 cells, and that its expression precedes that of established markers of early neuronal differentiation. Importantly, the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly, we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro, and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice.
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Diferenciación Celular/genética , Neuronas Dopaminérgicas/fisiología , Células Madre Embrionarias/fisiología , Células Madre Pluripotentes/fisiología , Formación de Roseta/métodos , Neuronas Serotoninérgicas/fisiología , Animales , Biomarcadores/metabolismo , Línea Celular , Neuronas Dopaminérgicas/metabolismo , Regulación hacia Abajo/genética , Células Madre Embrionarias/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Corteza Motora/metabolismo , Corteza Motora/fisiología , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismo , Células Madre Pluripotentes/metabolismo , Neuronas Serotoninérgicas/metabolismoRESUMEN
MicroRNAs (miRNAs) are short non-coding RNA transcripts that have the ability to regulate the expression of target genes, and have been shown to influence the development of various tumors. The purpose of our study is to identify aberrantly expressed miRNAs in retinoblastoma for the discovery of potential therapeutic targets for this disease, and to gain a greater understanding of the mechanisms driving retinoblastoma progression. We report 41 differentially expressed miRNAs (p<0.05) in 12 retinoblastomas as compared to three normal human retinae. Of these miRNAs, many are newly identified as being differentially expressed in retinoblastoma. Further, we report the validations of five of the most downregulated miRNAs in primary human retinoblastomas (p<0.05), human retinoblastoma cell lines, and mouse retinoblastoma cell lines. This serves as the largest and most comprehensive retinoblastoma miRNA analysis to date with corresponding clinical and pathological characteristics. This is an essential step in the discovery of miRNAs associated with retinoblastoma progression, and in the identification of potential therapeutic targets for this disease.
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Regulación Neoplásica de la Expresión Génica , MicroARNs/biosíntesis , ARN Neoplásico/biosíntesis , Neoplasias de la Retina/metabolismo , Retinoblastoma/metabolismo , Adulto , Animales , Línea Celular Tumoral , Femenino , Humanos , Masculino , Ratones , MicroARNs/genética , ARN Neoplásico/genética , Neoplasias de la Retina/genética , Neoplasias de la Retina/patología , Retinoblastoma/genética , Retinoblastoma/patologíaRESUMEN
INTRODUCTION: We have examined expression of microRNAs (miRNAs) in ependymomas to identify molecular markers of value for clinical management. miRNAs are non-coding RNAs that can block mRNA translation and affect mRNA stability. Changes in the expression of miRNAs have been correlated with many human cancers. MATERIALS AND METHODS: We have utilized TaqMan Low Density Arrays to evaluate the expression of 365 miRNAs in ependymomas and normal brain tissue. We first demonstrated the similarity of expression profiles of paired frozen tissue (FT) and paraffin-embedded specimens (FFPE). We compared the miRNA expression profiles of 34 FFPE ependymoma samples with 8 microdissected normal brain tissue specimens enriched for ependymal cells. miRNA expression profiles were then correlated with tumor location, histology and other clinicopathological features. RESULTS: We have identified miRNAs that are over-expressed in ependymomas, such as miR-135a and miR-17-5p, and down-regulated, such as miR-383 and miR-485-5p. We have also uncovered associations between expression of specific miRNAs which portend a worse prognosis. For example, we have identified a cluster of miRNAs on human chromosome 14q32 that is associated with time to relapse. We also found that miR-203 is an independent marker for relapse compared to the parameters that are currently used. Additionally, we have identified three miRNAs (let-7d, miR-596 and miR-367) that strongly correlate to overall survival. CONCLUSION: We have identified miRNAs that are differentially expressed in ependymomas compared with normal ependymal tissue. We have also uncovered significant associations of miRNAs with clinical behavior. This is the first report of clinically relevant miRNAs in ependymomas.
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Biomarcadores de Tumor/genética , Ependimoma/genética , MicroARNs/genética , Adolescente , Biomarcadores de Tumor/metabolismo , Estudios de Casos y Controles , Niño , Preescolar , Biología Computacional , Ependimoma/patología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Lactante , Masculino , MicroARNs/metabolismo , Análisis Multivariante , Pronóstico , Modelos de Riesgos Proporcionales , Análisis de Regresión , Análisis de SupervivenciaRESUMEN
OBJECT: Direct delivery of chemotherapeutic agents for the treatment of brain tumors is an area of focus in the development of therapeutic paradigms because this method of delivery circumvents the blood-brain barrier without causing adverse systemic side effects. Few studies have investigated longitudinal tumor response to this type of therapy. In this study, the authors examined the time course of tumor response to direct delivery of a chemotherapeutic agent in a rodent malignant glioma model. METHODS: To visualize tumor response to chemotherapy, the authors used bioluminescence imaging in a rodent model. Rat 9L gliosarcoma cells expressing a luciferase gene were inoculated into adult male rat striata. Ten days following surgery the animals were randomly divided into 4 groups. Groups 1 and 2 received 20 and 40 microl carboplatin (1 mg/ml), respectively, via convection-enhanced delivery (CED); Group 3 received 60 mg/kg carboplatin intraperitoneally; and Group 4 received no treatment. Tumor growth was correlated with luminescence levels twice weekly. RESULTS: Differential growth curves were observed for the 4 groups. Systemically treated rats showed decreasing photon flux emission at 15.0 + or - 4.7 days; rats treated with 20- or 40-microl CED showed decreased emissions at 4.0 + or - 2.0 and 3.2 + or - 1.3 days after treatment, respectively. Histopathologically, 6 of 12 CED-treated animals exhibited no residual tumor at the end point of the study. CONCLUSIONS: Direct and systemic delivery of carboplatin was examined to determine how the method of drug delivery affects tumor growth. The present report is one of the first in vivo studies to examine the time course of tumor response to direct drug delivery. The results indicate that direct drug delivery may be a promising option for treating gliomas.
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Antineoplásicos/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Carboplatino/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Gliosarcoma/tratamiento farmacológico , Animales , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Gliosarcoma/diagnóstico , Gliosarcoma/patología , Luciferasas , Sustancias Luminiscentes , Masculino , Ratas , Análisis de Supervivencia , Factores de Tiempo , Resultado del TratamientoRESUMEN
Abnormal patterns of DNA methylation are observed in several types of human cancer. While localized DNA methylation of CpG islands has been associated with gene silencing, the effect that genome-wide loss of methylation has on tumorigenesis is not completely known. To examine its effect on tumorigenesis, we induced DNA demethylation in a rat model of human chondrosarcoma using 5-aza-2-deoxycytidine. Rat specific pyrosequencing assays were utilized to assess the methylation levels in both LINEs and satellite DNA sequences following 5-aza-2-deoxycytidine treatment. Loss of DNA methylation was accompanied by an increase in invasiveness of the rat chondrosarcoma cells, in vitro, as well as by an increase in tumor growth in vivo. Subsequent microarray analysis provided insight into the gene expression changes that result from 5-aza-2-deoxycytidine induced DNA demethylation. In particular, two genes that may function in tumorigenesis, sox-2 and midkine, were expressed at low levels in control cells but upon 5-aza-2-deoxycytidine treatment these genes became overexpressed. Promoter region DNA analysis revealed that these genes were methylated in control cells but became demethylated following 5-aza-2-deoxycytidine treatment. Following withdrawal of 5-aza-2-deoxycytidine, the rat chondrosarcoma cells reestablished global DNA methylation levels that were comparable to that of control cells. Concurrently, invasiveness of the rat chondrosarcoma cells, in vitro, decreased to a level indistinguishable to that of control cells. Taken together these experiments demonstrate that global DNA hypomethylation induced by 5-aza-2-deoxycytidine may promote specific aspects of tumorigenesis in rat chondrosarcoma cells.
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Azacitidina/análogos & derivados , Condrosarcoma/genética , Condrosarcoma/patología , Metilación de ADN/efectos de los fármacos , Animales , Azacitidina/farmacología , Línea Celular Tumoral , Citocinas/genética , Citocinas/metabolismo , Decitabina , Epigénesis Genética/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Elementos de Nucleótido Esparcido Largo/genética , Ratones , Repeticiones de Microsatélite/genética , Midkina , Invasividad Neoplásica/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Análisis de Secuencia de ADN , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The efficacy of adoptive T-cell therapy as treatment for malignancies may be enhanced by genetic modification of infused cells. However, oncogenic events due to vector/transgene integration, and toxicities due to the infused cells themselves, have tempered enthusiasm. A safe and efficient means of removing aberrant cells in vivo would ameliorate these concerns. We describe a "safety switch" that can be stably and efficiently expressed in human T cells without impairing phenotype, function, or antigen specificity. This reagent is based on a modified human caspase 9 fused to a human FK506 binding protein (FKBP) to allow conditional dimerization using a small molecule pharmaceutical. A single 10-nM dose of synthetic dimerizer drug induces apoptosis in 99% of transduced cells selected for high transgene expression in vitro and in vivo. This system has several advantages over currently available suicide genes. First, it consists of human gene products with low potential immunogenicity. Second, administration of dimerizer drug has no effects other than the selective elimination of transduced T cells. Third, inducible caspase 9 maintains function in T cells overexpressing antiapoptotic molecules. These characteristics favor incorporation of inducible caspase 9 as a safety feature in human T-cell therapies.
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Caspasas/genética , Inducción Enzimática , Genes Transgénicos Suicidas , Terapia Genética/métodos , Inmunoterapia Adoptiva/métodos , Animales , Apoptosis/efectos de los fármacos , Caspasa 9 , Caspasas/uso terapéutico , Dimerización , Humanos , Ratones , Ratones Endogámicos NOD , Linfocitos T/metabolismo , Linfocitos T/trasplante , Proteínas de Unión a Tacrolimus/uso terapéuticoRESUMEN
To inhibit tumor-induced angiogenesis, the VEGF signaling pathway was targeted using AAV vectors encoding a VEGF decoy receptor, a truncated, soluble form of the murine VEGF receptor-2 (tsFlk-1). This approach initially had significant anti-neuroblastoma efficacy in murine xenograft models of local and metastatic disease, but when higher circulating levels of tsFlk-1 were established, tumor growth was more aggressive than even in control mice. Part of the mechanism for this apparent tumor resistance was increased human VEGF expression by the tumor cells. However, further investigation revealed that although a greater amount of VEGF could be bound by higher levels of tsFlk-1, more VEGF also existed in an unbound state and was, therefore, available to support angiogenesis. This novel, tumor-independent mechanism for resistance to antiangiogenic strategies suggests that careful titering of angiogenesis inhibitors may be required to achieve maximal antitumor efficacy and avoid therapy resistance mediated, in part, by ligand bioavailability. This has important implications for therapeutic strategies that use decoy receptors and other agents, such as antibodies, to bind angiogenic factors, in an attempt to inhibit tumor neovascularization.