RESUMEN
PURPOSE: Dedifferentiated liposarcoma (DDLS), one of the most common and aggressive sarcomas, infrequently responds to chemotherapy. DDLS survival and growth depend on underexpression of C/EBPα, a tumor suppressor and transcriptional regulator controlling adipogenesis. We sought to screen and prioritize candidate drugs that increase C/EBPα expression and may therefore serve as differentiation-based therapies for DDLS. EXPERIMENTAL DESIGN: We screened known bioactive compounds for the ability to restore C/EBPα expression and inhibit proliferation selectively in two DDLS cell lines but not in normal adipose-derived stem cells (ASC). Selected hits' activity was validated, and the mechanism of the most potent, SN-38, was investigated. The in vivo efficacy of irinotecan, the prodrug of SN-38, was evaluated in DDLS xenograft models. RESULTS: Of 3,119 compounds, screen criteria were met by 19. Validation experiments confirmed the DDLS selectivity of deguelin, emetine, and SN-38 and showed that they induce apoptosis in DDLS cells. SN-38 had the lowest IC50 (approximately 10 nmol/L), and its pro-apoptotic effects were countered by knockdown of CEBPA but not of TP53. Irinotecan significantly inhibited tumor growth at well-tolerated doses, induced nuclear expression of C/EBPα, and inhibited HIF1α expression in DDLS patient-derived and cancer cell line xenograft models. In contrast, doxorubicin, the most common treatment for nonresectable DDLS, reduced tumor growth by 30% to 50% at a dose that caused weight loss. CONCLUSIONS: This high-content screen revealed potential treatments for DDLS. These include irinotecan, which induces apoptosis of DDLS cells in a C/EBPα-dependent, p53-independent manner, and should be clinically evaluated in patients with advanced DDLS.
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Proteína alfa Potenciadora de Unión a CCAAT , Proteínas Potenciadoras de Unión a CCAAT , Liposarcoma , Adipocitos/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/análisis , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/análisis , Genes Supresores de Tumor , Humanos , Liposarcoma/tratamiento farmacológico , Liposarcoma/genética , Liposarcoma/patología , Células Madre/metabolismoRESUMEN
A method is described for the determination of the CCAAT/enhancer binding protein alpha (C/EBPα) which is a regulator in adipocyte differentiation. The method is based on quenching of the red fluorescence (with excitation/emission maxima at 548/562 nm) of Cy3-labeled DNA if it becomes adsorbed on positively charged gold nanoparticles (AuNPs). Fluorescently labeled dsDNA that can bind C/EBPα is introduced as a fluorescent probes. The dsDNA is electrostatically adsorbed on the positively charged AuNPs to quench their fluorescence. In the presence of C/EBPα, it will bind dsDNA which then diffuses away. The fluorescence of the AuNPs becomes restored. The fluorescent signal increases linearly in the 0.05 to 600 ng·mL-1 µM C/EBPα concentration range, and the detection limit is 29 pg·mL-1. The method is specific and was applied to analyze cell lysates and in-situ. Graphical abstractSchematic representation of a fluorometric method for determination of the CCAAT/enhancer binding protein alpha (C/EBPα). Fluorescently labeled dsDNA that can bind C/EBPα is introduced as a fluorescent probes. The dsDNA is electrostatically adsorbed on the positively charged AuNPs to quench their fluorescence. In the presence of C/EBPα, it will bind dsDNA which then diffuses away. The fluorescence of the AuNPs becomes restored.
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Proteína alfa Potenciadora de Unión a CCAAT/análisis , Sondas de ADN/química , Colorantes Fluorescentes/química , Fluorometría/métodos , Oro/química , Nanopartículas del Metal/química , Proteína alfa Potenciadora de Unión a CCAAT/química , Línea Celular , Estudios de Factibilidad , HumanosRESUMEN
BACKGROUND: This study aims to investigate the effects of CCAAT/enhancer binding protein alpha (C/EBPα) overexpression on cell proliferation, apoptosis and surfactant protein-C(SP-C) in alveolar epithelial type II (AEC II) cells after exposure to hyperoxia. METHODS: pcDNA3.1(+)-C/EBPα plasmid or air-empty vector were transfected into AEC II cells with or without hyperoxia. AEC II cells were divided into air group, air+pcDNA3.1-C/EBPα group, air-empty vector group, hyperoxia group, hyperoxia+pcDNA3.1-C/EBPα group, and hyperoxia-empty vector group. Cell proliferation was analyzed using Cell Counting Kit-8. The mRNA level and protein expression were measured using PCR and Western blot techniques, respectively. The cell cycle and apoptosis were analyzed using flow cytometry. RESULTS: After 48 h of post-transfection, significantly higher protein expression of C/EBPα was observed in the C/EBPα transfection group with or without hyperoxia compared to the others (P < 0.05). Compared to the air group, hyperoxia decreased cell proliferation, increased apoptosis, decreased SP-C expression, decreased percentage of cells in G1 phase, and increased percentage of cells in the S and G2 phases (P < 0.05); however, reversed by C/EBPα transfection (P < 0.05). No significant changes were observed in cell proliferation, SP-C expression, and apoptosis rates in the C/EBPα transfection group as compared to the controls air-empty vector group. CONCLUSION: C/EBPα overexpression significantly upregulates the expression of SP-C, promotes cell proliferation, and inhibits apoptosis in AEC II cells after exposure to hyperoxia. Hence, this data suggests that C/EBPα overexpression may reverse the damage and exert a protective role in hyperoxia-induced lung injury.
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Células Epiteliales Alveolares/citología , Proteína alfa Potenciadora de Unión a CCAAT/análisis , Hipoxia de la Célula , Proteína C Asociada a Surfactante Pulmonar/análisis , Apoptosis/efectos de los fármacos , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , ARN Mensajero/análisisRESUMEN
Clinical and animal studies have suggested efficacies of common bean (Phaseolus vulgaris) consumption on weight loss. Fermentation of common bean-derived dietary fiber by gut microbiota is proposed to mitigate obesity; however, the mechanism of action is unclear. The objective of this study was to investigate whether and how fecal fermentation of common bean-derived dietary fiber impacts adipogenesis in a cell model. Dietary fiber was generated by in vitro digestion of cooked, lyophilized common bean flour, followed by anaerobic fermentation with the use of fresh feces from healthy mice without antibiotics treatment. The murine 3T3-L1 cells were induced to differentiate in the presence of the fermentation products. Treatment of the fecal fermentation products inhibited adipocyte differentiation and lipid accumulation in a dose- and time-dependent manner. The fermentation products decreased (P<.05) protein levels of two key transcription factors for adipogenesis, CCAAT/enhancer binding protein α and peroxisome proliferator-activated receptor γ by 79-92% and 78-90%, respectively, and one of their downstream targets fatty acid binding protein 4 by 49-86% and 63-98% at protein and mRNA levels, respectively, during the time course. In contrast, the fermentation products increased (P<.05) levels of two proteins promoting energy expenditure, peroxisome proliferator-activated receptor δ (71-91%) on days 2 and 4 and mitochondrial uncoupling protein 2 (1.1-1.2 fold) on days 4-8. Altogether, fecal fermentation of dietary fiber derived from in vitro digestion of common bean temporally and dose-dependently inhibits adipogenesis and key adipogenic transactivators, but activates two energy expenditure proteins in 3T3-L1 cells.
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Adipogénesis/efectos de los fármacos , Fibras de la Dieta/metabolismo , Metabolismo Energético/efectos de los fármacos , Heces/microbiología , Fermentación , Phaseolus/química , Células 3T3-L1 , Adipocitos/fisiología , Animales , Proteína alfa Potenciadora de Unión a CCAAT/análisis , Proteína alfa Potenciadora de Unión a CCAAT/genética , Diferenciación Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Ratones , PPAR delta/análisis , PPAR delta/genética , PPAR gamma/análisis , PPAR gamma/genética , ARN Mensajero/análisis , Proteína Desacopladora 2/análisis , Proteína Desacopladora 2/genéticaRESUMEN
Objective To explore the effect of hyperoxia on the expressions of CCAAT enhancer binding protein α (C/EBPα) and pulmonary surfactant proteins (SP-A, SP-B, SP-C, SP-D) and their correlations in primary type II alveolar epithelial cells (AECIIs) from premature rats. Methods AECIIs were divided into an air control group and a hyperoxia model group. The cells of the two groups were respectively exposed toair and 950 mL/L O2. The cells were harvested at 24, 48, and 72 hours after exposure. Inverted phase-contrast microscope was used to observe morphological changes of the cells. Real-time quantitative PCR and Western blotting were performed to measure the mRNA and protein expressions of C/EBPα, SP-A, SP-B, SP-C and SP-D. Cell Counting Kit-8 (CCK-8) was applied to detect the proliferation of AECIIs. Results With the prolonging incubation time, the air group showed a significantly decreasing mRNA and protein expressions of C/EBPα, and significantly ascending mRNAand protein expressions of SP-A, SP-B, SP-C, SP-D and increasing proliferation of AECIIs. The mRNA and protein expressions of SP-A, SP-B, SP-C, SP-D and the proliferation of AECIIs in the hyperoxia group showed a trend of increasing at first and then decreasing as the culture time went on. Compared with the air group, the hyperoxia group showed significantly increased mRNA and protein expressions of C/EBPα and SP-A, SP-B, SP-C, SP-D and enhanced proliferation of AECIIs at 48 hours. In the hyperoxia group, the protein expression of C/EBPα was positively correlated with the protein expressions of SP-A, SP-B, SP-C, SP-D as well as the proliferation of AECIIs(r=0.96, 0.98, 0.92, 0.97, 0.90). Conclusion In the early stage of hyperoxia exposure, C/EBPα can promote the secretion of pulmonary surfactant protein to participate in the body's protective regulation. However, over the time of hyperoxia exposure, C/EBPα loses compensatory protective effect.
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Proteína alfa Potenciadora de Unión a CCAAT/análisis , Hipoxia de la Célula , Células Epiteliales/química , Surfactantes Pulmonares/análisis , Animales , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proliferación Celular , Células Cultivadas , Células Epiteliales/fisiología , Femenino , Masculino , ARN Mensajero/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Health problems related to the lack of bone formation are a major problem for ageing populations in the modern world. As a part of the ongoing trend to develop natural substances that attenuate bone loss in osteoporosis, the effects of the edible brown alga Sargassum thunbergii and its active contents on adipogenic differentiation in 3T3-L1 fibroblasts and osteoblast differentiation in MC3T3-E1 pre-osteoblasts were evaluated. RESULTS: Treatment with S. thunbergii significantly reduced lipid accumulation and expression of adipogenic differentiation markers such as peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α and sterol regulatory element binding protein 1c. In addition, S. thunbergii successfully enhanced osteoblast differentiation as indicated by increased alkaline phosphatase activity along raised levels of osteoblastogenesis indicators, namely bone morphogenetic protein-2, osteocalcin and collagen type I. Two compounds, sargaquinoic and sargahydroquinoic acid, were isolated from active extract and shown to be active by means of osteogenesis inducement. CONCLUSION: S. thunbergii could be a source for functional food ingredients for improved treatment of osteoporosis and obesity.
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Adipogénesis/efectos de los fármacos , Osteoblastos/fisiología , Osteogénesis/efectos de los fármacos , Quinonas/farmacología , Sargassum/química , Células 3T3-L1 , Alquenos/aislamiento & purificación , Alquenos/farmacología , Animales , Benzoquinonas/aislamiento & purificación , Benzoquinonas/farmacología , Proteína alfa Potenciadora de Unión a CCAAT/análisis , Diferenciación Celular/efectos de los fármacos , Línea Celular , Lípidos/análisis , Ratones , PPAR gamma/análisis , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/análisisRESUMEN
Sunflower-type nanogels carrying the QD 655 nanoprobe can be used for both gene transfection and bioimaging of hMSCs. The entry of sunflower-type nanogels into hMSCs can be possibly controlled by changing the formation of QDs. The physico-chemical properties of sunflower-type nanogels internalized by hMSCs were confirmed by AFM, SEM, TEM, gel retardation, and ζ-potential analyses. The bioimaging capacity was confirmed by confocal laser microscopy, Kodak imaging, and Xenogen imaging. Specifically, we investigated the cytotoxicity of sunflower-type nanogels via SNP analysis. Internalization of sunflower-type nanogels does not cause malfunction of hMSCs.
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Rastreo Celular/métodos , Trasplante de Células Madre Mesenquimatosas/métodos , Polietilenglicoles/administración & dosificación , Polietileneimina/administración & dosificación , Puntos Cuánticos/análisis , Transfección , Animales , Proteína alfa Potenciadora de Unión a CCAAT/análisis , Proteína alfa Potenciadora de Unión a CCAAT/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/análisis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , ADN Complementario/administración & dosificación , ADN Complementario/genética , Composición de Medicamentos , Endosomas , Femenino , Dosificación de Gen , Genes Reporteros , Heparina , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía/métodos , Nanogeles , Poloxámero , Polietilenglicoles/toxicidad , Polietileneimina/toxicidad , Polimorfismo de Nucleótido Simple , Factor de Transcripción SOX9/análisis , Factor de Transcripción SOX9/genética , Electricidad EstáticaRESUMEN
Hypoxia inducible factor-1α (HIF-1α) is overexpressed in various types of solid tumor in humans, including bladder cancer. HIF-1α regulates the expression of a series of genes, which are involved in cell proliferation, differentiation, apoptosis, angiogenesis, migration and invasion and represents a potential therapeutic target for the treatment of human cancer. Despite extensive investigation of the effects of HIF-1α in the progression and metastasis of bladder cancer, the possible regulatory mechanisms underlying the effects of HIF-1α on bladder cancer cell proliferation and differentiation remain to be elucidated. It has been suggested that the transcription factor CCAAT/enhancer binding protein α (C/EBPα) acts as a tumor suppressor in several types of cancer cell, which are involved in regulating cell differentiation, proliferation and apoptosis. The present study confirmed that, in bladder cancer cells, the expression and localization of C/EBPα was regulated by hypoxia through an HIF-1α -dependent mechanism, which may be significant in bladder cancer cell proliferation and differentiation. The 5637 and T24 bladder cancer cell lines were incubated under normoxic and hypoxic conditions. The expression levels of HIF-1α and C/EBPα were detected by reverse transcription-quantitative polymerase chain reaction, western blotting and immunofluorescence analysis. The results revealed that, under hypoxic conditions, the protein expression levels of HIF-1α were markedly upregulated, but the mRNA levels were not altered. However, the mRNA and protein levels of C/EBPα were significantly reduced. The present study further analyzed the subcellular localization of C/EBPα, which was markedly decreased in the nuclei under hypoxic conditions. Following HIF-1α small interference RNA silencing of HIF-1α, downregulation of C/EBPα was prevented in the bladder cancer cells cultured under hypoxic conditions. In addition, groups of cells treated with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole, which inhibits the expression of HIF-1α in hypoxia, contributed to the inhibited expression of HIF-1α and enhanced expression of C/EBPα in hypoxic bladder cancer cells. These results suggested that C/EBPα was a downstream effector regulated by HIF-1α in hypoxic bladder cancer cells and that this regulatory pathway may represent a potential therapeutic target in the treatment of bladder cancer.
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Proteína alfa Potenciadora de Unión a CCAAT/genética , Carcinoma de Células Transicionales/complicaciones , Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Hipoxia/complicaciones , Neoplasias de la Vejiga Urinaria/complicaciones , Proteína alfa Potenciadora de Unión a CCAAT/análisis , Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/patología , Línea Celular Tumoral , Humanos , Hipoxia/genética , Hipoxia/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/análisis , Vejiga Urinaria/metabolismo , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
CCAAT/enhancer binding protein α (C/EBPα) dimerizes via its leucine zipper (LZ) domain to bind DNA via its basic region and activate transcription via N-terminal trans-activation domains. The activity of C/EBPα is modulated by several serine/threonine kinases and via sumoylation, its gene is activated by RUNX1 and additional transcription factors, its mRNA stability is modified by miRNAs, and its mRNA is subject to translation control that affects AUG selection. In addition to inducing differentiation, C/EBPα inhibits cell cycle progression and apoptosis. Within hematopoiesis, C/EBPα levels increase as long-term stem cells progress to granulocyte-monocyte progenitors (GMP). Absence of C/EBPα prevents GMP formation, and higher levels are required for granulopoiesis compared to monopoiesis. C/EBPα interacts with AP-1 proteins to bind hybrid DNA elements during monopoiesis, and induction of Gfi-1, C/EBPε, KLF5, and miR-223 by C/EBPα enables granulopoiesis. The CEBPA ORF is mutated in approximately 10 % of acute myeloid leukemias (AML), leading to expression of N-terminally truncated C/EBPαp30 and C-terminal, in-frame C/EBPαLZ variants, which inhibit C/EBPα activities but also play additional roles during myeloid transformation. RUNX1 mutation, CEBPA promoter methylation, Trib1 or Trib2-mediated C/EBPαp42 degradation, and signaling pathways leading to C/EBPα serine 21 phosphorylation reduce C/EBPα expression or activity in additional AML cases.
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Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Leucemia Mieloide Aguda/genética , Mielopoyesis , Animales , Proteína alfa Potenciadora de Unión a CCAAT/análisis , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , MicroARNs/genética , MicroARNs/metabolismo , Mutación , Procesamiento Proteico-PostraduccionalRESUMEN
INTRODUCTION: Dental pulp has limited capability to regenerate, which happens in the early stage of pulpitis. An ambiguous relationship exists; inflammation may impair or support pulp regeneration. Epigenetics, which is involved in cell proliferation and inflammation, could regulate human dental pulp cell (HDPCs) regeneration. The aim of this study was to determine the role of the epigenetic mark, enhancer of zeste homolog 2 (EZH2), in the inflammation, proliferation, and regeneration of dental pulp. We used trimethylated histone H3 lysine 27(H3K27me3) and its lysine demethylase 6B (KDM6B) to monitor functional effects of altered EZH2 levels. METHODS: We detected epigenetic marks (EZH2, H3K27me3, and KDM6B) in pulp tissue by immunohistochemistry and immunofluorescence. EZH2 levels in HDPCs in inflammatory responses or differentiation were analyzed by quantitative polymerase chain reaction and Western blot. Quantitative polymerase chain reaction was used to assess the effects of EZH2 inhibition on interleukins in HDPCs upon tumor necrosis factor alpha stimulation. Cell proliferation was tested by cell counting kit-8, cell cycle, and apoptosis analysis. HDPC differentiation was investigated by quantitative polymerase chain reaction, alkaline phosphatase activity, and oil red O staining. RESULTS: EZH2 and H3K27me3 were decreased, whereas KDM6B was increased in infected pulp tissue and cells, which were similar to HDPC differentiation. EZH2 inhibition suppressed IL-1b, IL-6, and IL-8 messenger RNA (mRNA) in HDPCs upon inflammatory stimuli and impeded HDPC proliferation by decreasing cell number, arresting cell cycle, and increasing apoptosis. Suppressed EZH2 impaired adipogenesis, peroxisome proliferator-activated receptor r (PPAR-r), and CCAAT-enhancer binding protein a (CEBP/a) mRNA in adipogenic induction while enhancing alkaline phosphatase activity, Osx, and bone sialoprotein (BSP) mRNA in mineralization induction of HDPCs. CONCLUSIONS: EZH2 inhibited HDPC osteogenic differentiation while enhancing inflammatory response and proliferation, suggesting its role in pulp inflammation, proliferation, and regeneration.
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Pulpa Dental/fisiología , Complejo Represivo Polycomb 2/fisiología , Pulpitis/fisiopatología , Regeneración/fisiología , Adipogénesis/fisiología , Fosfatasa Alcalina/análisis , Apoptosis/fisiología , Proteína alfa Potenciadora de Unión a CCAAT/análisis , Calcificación Fisiológica/fisiología , Diferenciación Celular/fisiología , Proliferación Celular , Células Cultivadas , Pulpa Dental/citología , Pulpa Dental/efectos de los fármacos , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética/fisiología , Histonas/análisis , Humanos , Sialoproteína de Unión a Integrina/análisis , Interleucina-1beta/análisis , Interleucina-6/análisis , Interleucina-8/análisis , Histona Demetilasas con Dominio de Jumonji/análisis , Osteogénesis/fisiología , Receptores Activados del Proliferador del Peroxisoma/análisis , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Factor de Transcripción Sp7 , Factores de Transcripción/análisis , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Sinonasal inverted papilloma (SIP) is a rare benign tumor featuring increased cell proliferation, a tendency toward squamous differentiation, recurrence and malignant transformation. The CCAAT enhancer binding proteins, C/EBPs, are transcription factors regulating the proliferation and differentiation of various types of cells, including epithelial cells. We prospectively investigated the production of these transcription factors and the related proliferation and differentiation targets, keratin-10, keratin-15 and cyclin-D1, in 26 SIP patients and 8 sinonasal polyposis cases suspected for SIP. Ten of these patients had one or more recurrences over follow-up periods of one to eight years. C/EBP-alpha and C/EBP-beta proteins were not found in normal-looking sinonasal epithelial cells. The proteins and RNAs were detected in SIP and, occasionally, in polyposis tissues. The production of these factors was not significantly correlated with age, sex, site, tumor size or medical history. By contrast, correlations were found between the levels of C/EBP-alpha and keratin-10 levels and between those of C/EBP-beta and keratin-15. C/EBP-alpha levels were also significantly correlated with cyclin-D1 levels. These data suggested that the C/EBPs are implicated in the regulation of cell proliferation and differentiation in SIP. Finally, recurrent SIP produced significantly larger amounts of C/EBP-alpha than non- recurrent tumors. These results implicate CCAAT enhancer binding proteins in the pathogenesis of SIP and highlight the role of C/EBP-alpha as a candidate marker for tumor recurrence.
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Biomarcadores de Tumor/análisis , Proteína alfa Potenciadora de Unión a CCAAT/análisis , Proteína beta Potenciadora de Unión a CCAAT/análisis , Papiloma Invertido/metabolismo , Neoplasias de los Senos Paranasales/metabolismo , Adolescente , Adulto , Anciano , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/metabolismo , Papiloma Invertido/patología , Neoplasias de los Senos Paranasales/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
Fluorescence lifetime imaging microscopy (FLIM) is now routinely used for dynamic measurements of signaling events inside single living cells, such as monitoring changes in intracellular ions and detecting protein-protein interactions. Here, we describe the digital frequency domain FLIM data acquisition and analysis. We describe the methods necessary to calibrate the FLIM system and demonstrate how they are used to measure the quenched donor fluorescence lifetime that results from Förster Resonance Energy Transfer (FRET). We show how the "FRET-standard" fusion proteins are used to validate the FLIM system for FRET measurements. We then show how FLIM-FRET can be used to detect the dimerization of the basic leucine zipper (B Zip) domain of the transcription factor CCAAT/enhancer binding protein α in the nuclei of living mouse pituitary cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail.
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Proteína alfa Potenciadora de Unión a CCAAT/análisis , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Microscopía Fluorescente/métodos , Mapeo de Interacción de Proteínas , Animales , Calibración , Proteínas Luminiscentes/análisis , Ratones , Unión Proteica , Transducción de SeñalRESUMEN
OBJECTIVE: This study examined the effects of PI3K inhibitor LY294002 on the differentiation of mouse preadipocytes and the expression of CCAAT enhancer binding protein α (C/EBPα) and peroxisome proliferation activated receptor γ (PPARγ), in order to study the possible roles of insulin receptor substrate (IRSs)/PI3K signal pathway in the differentiation of preadipocytes. METHODS: The mouse 3T3-L1 cells were cultured normally and divided into experimental and control groups. 3T3-L1 cells in the experimental group were treated with PI3K inhibitor LY294002 (25 µmol/L) and those in the control group were treated with DMSO culture medium. 3-isobutyl-1-methylxanthine (IBMX) (0.5 mmol/L), dexamethasone (10-6 mol/L) and insulin (5 µg/mL) were used to induce the differentiation of 3T3-L1 preadipocytes in both groups. Before culture, and 2, 4 and 8 days after culture, the cells were collected to detect the expression of C/EBPα and PPARγ by real-time PCR and Western blot assays. The lipid droplets of 3T3-L1 preadipocytes were observed by oil-red O staining. RESULTS: PI3K inhibitor LY294002 did not affect the expression of C/EBPα and PPARγ in un-induced 3T3-L1 preadipocytes (P>0.05), but decreased the expression of C/EBPα and PPARγ during the in vitro induced differentiation of 3T3-L1 preadipocytes compared with the control group (P<0.05 or 0.01). The lipid droplets count was greatly reduced by LY294002. CONCLUSIONS: PI3K inhibitor LY294002 can inhibit the differentiation of mouse 3T3-LI preadipocytes and the expression of C/EBPα and PPARγ in the differentiation of 3T3-LI preadipoeytes, suggesting that IRSs/PI3K signal pathway may play an important role in the differentiation of 3T3-L1 preadipocytes by regulating the expression of C/EBPα and PPARγ.
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Adipocitos/efectos de los fármacos , Proteína alfa Potenciadora de Unión a CCAAT/genética , Cromonas/farmacología , Morfolinas/farmacología , PPAR gamma/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Células 3T3-L1 , Adipocitos/citología , Animales , Proteína alfa Potenciadora de Unión a CCAAT/análisis , Diferenciación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , PPAR gamma/análisis , ARN Mensajero/análisisRESUMEN
Intramuscular fat content is increased by feeding of low lysine diets in pigs. Reduction in dietary lysine intake results in low plasma lysine concentration and low cytosolic lysine concentration in skeletal muscles. From these observations, we hypothesized that low plasma lysine concentration in pigs fed on low lysine diets reduced supply of lysine from blood circulation to preadipocytes, and this limited supply of lysine might promote adipocyte differentiation in porcine muscles. In order to verify the hypothesis, we investigated the effects of low concentrations of lysine in culture medium on differentiation of 3T3-L1 preadipocytes. Low concentration of lysine suppressed lipid accumulation and messenger RNA (mRNA) expression and enzyme activity of fatty acid synthase. mRNA expressions of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) were lower in cells cultured in low lysine medium. On the other hand, mRNA and protein expressions of C/EBPß and C/EBPδ were not inhibited by low concentrations of lysine in culture medium. These results indicate that low lysine concentrations in culture medium inhibit differentiation of 3T3-L1 preadipocytes through inhibiting the mRNA expressions of PPARγ and C/EBPα.
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Adipocitos/citología , Diferenciación Celular/efectos de los fármacos , Lisina/administración & dosificación , Porcinos/fisiología , Células 3T3-L1 , Animales , Western Blotting , Proteína alfa Potenciadora de Unión a CCAAT/análisis , Proteína beta Potenciadora de Unión a CCAAT/análisis , Proteína delta de Unión al Potenciador CCAAT/análisis , Células Cultivadas , Medios de Cultivo , Lípidos/análisis , Lisina/farmacología , Ratones , PPAR gamma/análisis , ARN Mensajero/análisis , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
This study investigated the growth performance and gene expression for muscle development between grass hay-fed (GH) and concentrate-fed (CT) steers. Daily gain and energy intake during the fattening period of the GH group were lower than those of the CT group. Analysis of C/EBPα, PPARγ2, myosin heavy chain (MHC), and myostatin gene expressions was performed by real-time PCR. Expressions of C/EBPα and myostatin in semitendinosus and longissimus lumborum (LL) muscles were higher in the CT group than in the GH group at the end of fattening. In LL muscle, MHC expression at the end of fattening was greater in the GH group than in the CT group. These results suggest that regulation of adipogenesis and myogenesis by the expression of genes involved in muscle development might have occurred in the skeletal muscle of the GH group by the feeding of grass hay and/or because of the low energy intakes.
Asunto(s)
Alimentación Animal , Desarrollo de Músculos/genética , Músculo Esquelético/crecimiento & desarrollo , Adipogénesis/genética , Animales , Proteína alfa Potenciadora de Unión a CCAAT/análisis , Proteína alfa Potenciadora de Unión a CCAAT/genética , Bovinos , Ingestión de Energía , Regulación de la Expresión Génica , Masculino , Músculo Esquelético/efectos de los fármacos , Cadenas Pesadas de Miosina/análisis , Cadenas Pesadas de Miosina/genética , Miostatina/análisis , Miostatina/genética , PPAR gamma/análisis , PPAR gamma/genética , ARN/aislamiento & purificaciónRESUMEN
Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G(1) checkpoint, and diminished or ablated expression of C/EBPα results in G(1) checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/fisiología , Carcinoma de Células Escamosas/etiología , Neoplasias Inducidas por Radiación/etiología , Neoplasias Cutáneas/etiología , Rayos Ultravioleta/efectos adversos , Animales , Proteína alfa Potenciadora de Unión a CCAAT/análisis , Carcinoma de Células Escamosas/química , Ciclo Celular , Células Cultivadas , Genes p53 , Humanos , Queratosis Actínica/metabolismo , Masculino , Ratones , Ratones Pelados , Ratones Endogámicos C57BL , Mutación , Lesiones Precancerosas/química , Piel/metabolismo , Piel/efectos de la radiación , Neoplasias Cutáneas/químicaRESUMEN
After human genome is decoded, the characterization of the proteins is the next challenging task. The study of the complete protein complement of the genome, the 'proteome' referred to as proteomics, is an important tool for the identification of new therapeutic targets. Research efforts are underway to develop the technology necessary to compare the specific protein profiles of diseased versus healthy states. These technologies provide a wealth of information by rapidly generating large quantities of data. These data can be useful for predictive mathematical descriptions of biological systems for rapid identification of novel therapeutic targets and identification of biomarkers in metabolic disorders. In recent years, using proteomics, we and others have identified various interacting as well as target proteins, PTMs and protein markers in myeloid leukemia. This review summarizes the usage of proteomics in recent years as an important technique in defining the proteome of myeloid leukemia, which has helped in elaborate understanding of the disease and has provided new avenues for developing better therapeutics.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/análisis , Marcadores Genéticos , Leucemia Mieloide/diagnóstico , Proteómica/instrumentación , Proteómica/métodos , Proteína alfa Potenciadora de Unión a CCAAT/genética , Técnicas y Procedimientos Diagnósticos , Diseño de Fármacos , Humanos , Leucemia Mieloide/sangre , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/genética , Modelos Moleculares , Mutación/genética , Proteínas , Proteoma/análisis , Proteoma/metabolismoRESUMEN
Lactoferrin accelerates the differentiation of osteogenic and chondrogenic lineage cells, whereas it inhibits the myogenic and adipogenic differentiation of pluripotent mesenchymal cells; however, the effect of lactoferrin on the differentiation of preadipocytes is unknown. In this study, we examined the effect of lactoferrin on adipogenic differentiation using a mouse preadipocyte cell line, MC3T3-G2/PA6. The cells were cultured in differentiation medium with or without lactoferrin to induce cellular differentiation. The cell lineage was then determined by Oil Red O staining, real-time PCR screening for the mRNA expression of phenotype-specific markers, and Western blot analysis. The number of Oil Red O-positive lipid droplets decreased following treatment with lactoferrin, as did the mRNA expression of C/EBPalpha, PPARgamma, aP2, and adiponectin. Furthermore, our Western blot data revealed a decrease in PPARgamma expression attributable to lactoferrin exposure. These results suggest that lactoferrin suppresses the adipogenic differentiation of MC3T3-G2/PA6 cells.
Asunto(s)
Adipocitos/efectos de los fármacos , Lactoferrina/farmacología , Células 3T3 , Adipogénesis/efectos de los fármacos , Adiponectina/análisis , Animales , Compuestos Azo , Biomarcadores/análisis , Western Blotting , Proteína alfa Potenciadora de Unión a CCAAT/análisis , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Proliferación Celular/efectos de los fármacos , Colorantes , Proteínas de Unión a Ácidos Grasos/análisis , Lípidos/análisis , Ratones , PPAR gamma/análisis , Fenotipo , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisisRESUMEN
Rehmannia glutinosa, a Traditional Chinese Medicine (TCM), has been used to increase physical strength. Here, we report that Rehmannia glutinosa extract (RE) inhibits adipocyte differentiation and adipogenesis. RE impairs differentiation of 3T3-L1 preadipocytes in a dose-dependent manner. At the molecular level, treatment with RE inhibits expression of the key adipocyte differentiation regulator C/EBPbeta, as well as C/EBPalpha and the terminal marker protein 422/aP2, during differentiation of preadipocytes into adipocytes. Additionally, RE inhibits the mitotic clonal expansion (MCE) process of adipocyte differentiation, and RE prevents localization of C/EBPbeta to the centromeres. RE also prevents high fat diet (HFD) induced weight gain and adiposity in rats. Taken together, our results indicate that Rehmannia glutinosa extract inhibits preadipocyte differentiation and adipogenesis in cultured cells and in rodent models of obesity.
Asunto(s)
Adipocitos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteína beta Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Obesidad/prevención & control , Extractos Vegetales/farmacología , Células 3T3-L1 , Adipocitos/metabolismo , Adipogénesis/genética , Animales , Proteína alfa Potenciadora de Unión a CCAAT/análisis , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/análisis , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Centrómero/química , Centrómero/metabolismo , Dieta , Modelos Animales de Enfermedad , Grasas/administración & dosificación , Grasas/efectos adversos , Proteínas de Unión a Ácidos Grasos/antagonistas & inhibidores , Proteínas de Unión a Ácidos Grasos/metabolismo , Masculino , Ratones , Mitosis/efectos de los fármacos , Obesidad/inducido químicamente , Obesidad/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Microscopy has become an essential tool for cellular protein investigations. The development of new fluorescent markers such as green fluorescent proteins generated substantial opportunities to monitor protein-protein interactions qualitatively and quantitatively using advanced fluorescence microscope techniques including wide-field, confocal, multiphoton, spectral imaging, lifetime, and correlation spectroscopy. The specific aims of the investigation of protein dynamics in live specimens dictate the selection of the microscope methodology. In this article confocal and spectral imaging methods to monitor the dimerization of alpha enhancer binding protein (C/EBPalpha) in the pituitary GHFT1-5 living cell nucleus have been described. Also outline are issues involved in protein imaging using light microscopy techniques and the advantages of lifetime imaging of protein-protein interactions.