RESUMEN
Zinc finger nuclease (ZFN) is an artificially engineered hybrid protein that contains a zinc finger protein (ZFP) domain and a Fok I endonuclease cleavage domain. It has recently emerged as a powerful molecular tool for targeted genome modifications. ZFNs recognize and bind to specific DNA sequences to generate a double-strand break (DSB) by its nuclease activity. Based on this finding, various genetic methods, including gene targeting (gene disruption), gene addition, gene correction etc., are being designed to manipulate the genomes of different species at specific loci. One particular advantage of this new technique is its broad applications, which can be employed to generate desirable inheritable mutations both at the organismal level and at the cellular level. Here, we review the recent progress and prospects of ZFN technology. This article focused on the mechanism of how it works, currently available target assessment, ZFP library construction and screening methods, target modification strategies, as well as a collection of specie and genes that have been successfully modified by ZFN. This review will provide a useful reference for researchers who are interested in applying this new technique in their studies.
Asunto(s)
Proteínas de Unión al ADN/genética , Endonucleasas/genética , Marcación de Gen/instrumentación , Genoma , Ingeniería de Proteínas , Animales , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Endonucleasas/química , Endonucleasas/metabolismo , Humanos , Dedos de ZincRESUMEN
IFN regulatory factor (IRF)-1 and IRF-2 are generally regarded as a tumor suppressor and an oncoprotein, respectively. However, little is known about their expression and function in esophageal squamous cell carcinomas (ESCC). In our present work, IRF-1 expression was decreased and IRF-2 expression was increased in ESCCs compared with matched normal esophageal tissues. Moreover, statistical data indicated that IRF-2 expression was tightly correlated with progression of ESCCs. As expected, overexpression of either IRF-1 or IRF-2 in an ESCC cell line resulted in either suppression or enhancement of cell growth, respectively. Also, proliferation- and apoptosis-related molecules (p21(WAF1/CIP1), cyclin-D1, Bcl-2, and histone H4) were regulated by IRF-1 and IRF-2. Additionally, high levels of IRF-2 blocked the function of IRF-1 by preventing the latter from translocating into the nucleus; in contrast, knock down of IRF-2 by small interfering RNA permitted nuclear localization and activity of IRF-1. In vivo assay using nude mice indicated that the tumorigenicity of ESCC cells was enhanced with IRF-2 overexpression but dramatically attenuated after forced expression of IRF-1. In conclusion, IRF-1 and IRF-2 are able to regulate tumorigenicity of ESCC cells as antioncoprotein and oncoprotein, respectively. Relative amounts of IRF-1 to IRF-2 are functionally very important for the development and progression of ESCCs, and reduction of the ratio of IRF-1/IRF-2 may lead to the enhancement of tumorigenicity of ESCC cells. Therefore, levels of IRF-1 and IRF-2 are useful indicators in diagnosis and prognosis for ESCCs, and these molecules are potential drug targets for ESCC therapy.
Asunto(s)
Neoplasias Esofágicas/metabolismo , Factor 1 Regulador del Interferón/biosíntesis , Factor 2 Regulador del Interferón/biosíntesis , Animales , Apoptosis/fisiología , Procesos de Crecimiento Celular/fisiología , Progresión de la Enfermedad , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Esófago/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Factor 2 Regulador del Interferón/genética , Factor 2 Regulador del Interferón/metabolismo , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transfección , Trasplante HeterólogoRESUMEN
Pancreatic endocrine islets are vital for glucose homeostasis. However, the islet developmental trajectory and its regulatory network are not well understood. To define the features of these specification and differentiation processes, we isolated individual islet cells from TgBAC(neurod1:EGFP) transgenic zebrafish and analyzed islet developmental dynamics across four different embryonic stages using a single-cell RNA-seq strategy. We identified proliferative endocrine progenitors, which could be further categorized by different cell cycle phases with the G1/S subpopulation displaying a distinct differentiation potential. We identified endocrine precursors, a heterogeneous intermediate-state population consisting of lineage-primed alpha, beta and delta cells that were characterized by the expression of lineage-specific transcription factors and relatively low expression of terminally differentiation markers. The terminally differentiated alpha, beta, and delta cells displayed stage-dependent differentiation states, which were related to their functional maturation. Our data unveiled distinct states, events and molecular features during the islet developmental transition, and provided resources to comprehensively understand the lineage hierarchy of islet development at the single-cell level.
Asunto(s)
Antígenos de Diferenciación/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Islotes Pancreáticos/embriología , Análisis de la Célula Individual , Transcripción Genética , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente/embriología , Animales Modificados Genéticamente/genética , Antígenos de Diferenciación/genética , Pez Cebra/genéticaRESUMEN
PURPOSE: RWJ-241947 (MCC-555) is a novel peroxisome proliferator-activated receptor-gamma ligand of the thiazolidinedione class that was recently developed as an antidiabetic drug with unique properties. Some thiazolidinediones have anticancer activity against solid and hematological malignancies; the anticancer potency of RWJ-241947 has not been examined. We, therefore, investigated these effects in vitro and in vivo either alone or in combination with other compounds. EXPERIMENTAL DESIGN: Tumor growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, soft agar colony assay in vitro, and xenografts in nude mice. Its effects on cell cycle, differentiation, and apoptosis were examined. RESULTS: In vitro studies using various solid and hematological tumor cell lines showed that RWJ-241947 had antiproliferative activity against prostate cancer cells, with the strongest effect against the androgen-independent PC-3 prostate cancer cells. It increased expression of cyclin-dependent kinase inhibitor p21(WAF1), deceased cyclin E, and induced apoptosis in PC-3 cells. It increased E-cadherin and lowered protein expression of prostate-specific antigen without down-regulating the androgen receptor in androgen-dependent LNCaP prostate cancer cells. Reporter gene assays showed that this peroxisome proliferator-activated receptor-gamma ligand inhibited androgen activation of the androgen receptor response elements of the prostate-specific antigen gene. Remarkably, in vivo treatment of male beige/nude/X-linked immunodeficient (BNX) mice with RWJ-241947 profoundly suppressed growth of PC-3 prostate cancer xenografts with prominent apoptosis, as well as fibrosis, including inflammatory and giant cell reaction in the remaining tumor tissue. Notably, the experimented mice had a significantly decreased cholesterol. In addition, we studied the combination of arsenic trioxide (As2O3), which is used in the treatment of multiple myeloma, and RWJ-241947; these two reagents together prominently inhibited proliferation and caused apoptosis of multiple myeloma cells. CONCLUSIONS: RWJ-241947 has surprisingly potent antiproliferative effects against prostate cancer cells in vivo, and it enhances the antitumor activity of As2O3 against myeloma cells. Small, well-defined clinical studies using RWJ-241947 are in order for these cancers.
Asunto(s)
Antineoplásicos/farmacología , Arsenicales/farmacología , Mieloma Múltiple/patología , Óxidos/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Tiazoles/farmacología , Factores de Transcripción/metabolismo , Agar/química , Animales , Apoptosis , Trióxido de Arsénico , Western Blotting , Antígenos CD36/biosíntesis , Caspasas/metabolismo , Ciclo Celular , Diferenciación Celular , División Celular , Línea Celular Tumoral , Supervivencia Celular , Ligamiento Genético , Humanos , Síndromes de Inmunodeficiencia/genética , Ligandos , Luciferasas/metabolismo , Masculino , Ratones , Neoplasias de la Próstata/patología , Sales de Tetrazolio/farmacología , Tiazolidinedionas , Factores de Tiempo , Transfección , Células U937 , Cromosoma X/genéticaRESUMEN
Eph receptors, the largest subfamily of transmembrane tyrosine kinase receptors, have been increasingly implicated in various physiologic and pathologic processes, and the roles of the Eph family members during tumorigenesis have recently attracted growing attention. Until now, research on EphB3 function in cancer is limited to focusing on tumor suppression by EphB receptors in colorectal cancer. However, its function in other types of cancer remains poorly investigated. In this study, we explored the function of EphB3 in non-small-cell lung cancer (NSCLC). We found that the expression of EphB3 was significantly upregulated in clinical samples and cell lines, and the expression level correlated with the patient pathologic characteristics, including tumor size, differentiation, and metastasis. Overexpression of EphB3 in NSCLC cell lines accelerated cell growth and migration and promoted tumorigenicity in xenografts in a kinase-independent manner. In contrast, downregulation of EphB3 inhibited cell proliferation and migration and suppressed in vivo tumor growth and metastasis. Furthermore, we showed that silencing of EphB3 inhibited cell growth by reducing DNA synthesis and caspase-8-mediated apoptosis and suppressed cell migration by increasing accumulation of focal adhesion formation. Taken together, our findings suggest that EphB3 provides critical support to the development and progression of NSCLC by stimulating cell growth, migration, and survival, thereby implicating EphB3 as a potential therapeutic target in NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/enzimología , Neoplasias Pulmonares/enzimología , Receptor EphB3/biosíntesis , Animales , Apoptosis/fisiología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Caspasa 8/metabolismo , Adhesión Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Supervivencia Celular/fisiología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , ADN de Neoplasias/biosíntesis , ADN de Neoplasias/genética , Progresión de la Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor EphB3/genéticaRESUMEN
Connective tissue growth factor (CTGF or CCN2), a member of the CCN family, is involved in diverse biological processes such as cell adhesion, proliferation, and angiogenesis. In this study, we show that overexpression of CTGF occurred in a significant proportion of esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and metastatic. Forced expression of CTGF in Eca109 ESCC cells accelerated their growth in culture and significantly increased tumor formation in nude mice, whereas RNA interference-mediated knockdown of CTGF in ESCC cells significantly inhibited cell growth and colony formation, as well as tumorigenicity in vivo. Moreover, overexpression of CTGF in ESCC cells resulted in the accumulation and nuclear translocation of beta-catenin, leading to activation of beta-catenin-T-cell factor (TCF)/Lef signaling. Up-regulation of c-Myc and cyclin D1, two target genes of beta-catenin-TCF/Lef signaling, was also observed in the CTGF-overexpressing cells. These effects of CTGF in ESCC cells were abolished by transfection with either dominant negative beta-catenin or dominant negative TCF4. Furthermore, we identified a beta-catenin-TCF/Lef-binding site (TBE) in the promoter region of CTGF and found that CTGF is a transcriptional target of beta-catenin-TCF/Lef signaling. Taken together, these results revealed that the interaction of CTGF and beta-catenin-TCF/Lef forms a positive feedback loop, which could contribute to the tumorigenicity of ESCC.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Proteínas Inmediatas-Precoces/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Transducción de Señal , Factores de Transcripción TCF/metabolismo , beta Catenina/metabolismo , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Adhesión Celular/genética , Línea Celular Tumoral , Proliferación Celular , Factor de Crecimiento del Tejido Conjuntivo , Ciclina D , Ciclinas/genética , Ciclinas/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , Elementos de Respuesta/genética , Factores de Transcripción TCF/genética , Regulación hacia Arriba/genética , beta Catenina/genéticaRESUMEN
BACKGROUND: CCN family, comprising six members (Cyr61, CTGF, Nov, WISP-1, WISP-2, WISP-3), is involved in the stimulation of cell proliferation, migration, adhesion, angiogenesis, and tumorigenesis. Several studies have shown that expression of Cyr61, CTGF, and WISP-1 affects the tumorigenic potential of lung cancer cells in vitro. However, the correlation of expression of CCN family proteins and clinical features of lung cancer remains unknown. METHODOLOGY AND PRINCIPAL FINDINGS: In the present work, we quantified the mRNA levels of Cyr61, CTGF, and WISP-1 in samples from 60 primary lung cancers and their matched normal lung tissues by quantitative real-time PCR assay. Downregulation of the Cyr61 and CTGF genes and upregulation of the WISP-1 gene were found in primary lung cancers compared to the paired normal lung tissues. Immunohistochemistry analysis also disclosed a similar expression pattern of Cyr61, CTGF, and WISP-1 protein in paired lung cancer tissues. Statistical analysis revealed significant associations between expression of either Cyr61 or CTGF with tumor stage, tumor histology, metastasis, smoking, and family history at diagnosis. A significant correlation also existed between WISP-1 expression with tumor histology, and patient age. Moreover, expression levels of Cyr61 and CTGF correlated with survival of the lung-cancer patients. CONCLUSIONS: Our results suggest that Cyr61, CTGF, and WISP-1 might be implicated in the development and progression of primary lung cancers, and their levels might serve as valuable prognostic markers, as well as potential targets for therapeutic intervention.