RESUMEN
Polyglutamine diseases are a group of neurodegenerative disorders caused by expansion of a CAG repeat that encodes polyglutamine in each respective disease gene. The transcription factor THAP11, a member of THAP family, is involved in cell growth, ES cell pluripotency and embryogenesis. Previous studies suggest that THAP11 protein contains a 29-residue repeat polyglutamine motif and the number of polyglutamine ranges from 20 to 41 in Indian population. We have investigated the CAG numbers at the THAP11 locus in normal individuals and neurodegenerative disease patients of Chinese Han population and a 38Q expansion (THAP11(38Q)) was found in patients. Using fluorescence confocal-based cell imaging, THAP11(38Q) protein formed intranuclear inclusions easier than THAP11(29Q) in PC12 cells. Enhanced toxicity was investigated in THAP11(38Q)-expressing cells by growth inhibition and G0/G1 arrest. CREB-mediated transcription activity was inhibited by THAP11(38Q). The transcription factor, TBP, coactivator CBP, and chaperon protein, HSP70, could be recruited to THAP11(38Q). These results indicate that expansion of the polyglutamine in THAP11 forms intracellular aggregation and is toxic in PC12 cells, suggesting a putative role of THAP11 in polyglutamine disease.
Asunto(s)
Cuerpos de Inclusión Intranucleares/patología , Péptidos/genética , Proteínas Represoras/genética , Ataxias Espinocerebelosas/genética , Animales , Línea Celular , Proliferación Celular/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Puntos de Control de la Fase G1 del Ciclo Celular , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Células PC12 , Fragmentos de Péptidos/metabolismo , Polimorfismo Genético/genética , Ratas , Sialoglicoproteínas/metabolismo , Proteína de Unión a TATA-Box/metabolismoRESUMEN
AIM: To construct a stable cell strain encoding tumor-associated fused gene which expresses oncoprotein Tpr-Met. METHODS: We transfected Tpr-Met vector into Ba/F3 cells and screened the cell strain stably expressing Tpr-Met. The interleukin 3 (IL-3) independent proliferation of the cells was measured using the MTS assay. The expression of Tpr-Met, the activity of downstream signal transduction pathway and SU11274-induced inhibition of the signal pathway were investigated by Western blotting. RESULTS: We obtained a Ba/F3 cell strain stably expressing Tpr-Met. The cells presented IL-3 independent proliferation, suggesting a malignant transformation of the cell line. In Tpr-Met transformed Ba/F3 cells, the phosphorylation of Met and ERK were enhanced; however, specific c-Met inhibitor SU11274 suppressed the cell proliferation and c-Met phosphorylation. CONCLUSION: Tpr-Met transformed Ba/F3 strain has been successfully constructed.
Asunto(s)
Proteína Oncogénica tpr-met/genética , Transfección , Animales , Proliferación Celular/efectos de los fármacos , Indoles/farmacología , Interleucina-3/farmacología , Ratones , Fosforilación , Piperazinas/farmacología , Transducción de Señal , Sulfonamidas/farmacologíaRESUMEN
Human augmenter of liver regeneration (hALR) is a sulfhydryl oxidase that is highly expressed in spermatogonia and early spermatocytes. To investigate the physiological effects of hALR in spermatogenesis, we generated a hALR transgenic mouse model driven by the human TSPY (testis-specific protein, Y-encoded) promoter that allows the transgene to be specifically activated in the testes. hALR content was found to be increased in both germ cells. The histological and TUNEL analysis of transgenic testes revealed a number of spermatogenetic defects including primary spermatocyte overpopulation followed by depletion through apoptosis, degenerating and detached nucleated germ cells, haploid cell loss and intraepithelial vacuoles of varying sizes. In line with these features, adult transgenic male mice also displayed a reduction in fertility. Our data suggest that regulated spatial and temporal expression of hALR is required for normal testicular development and spermatogenesis, and overexpression of hALR results in influencing the sperm morphology and quantity and the eventual reduction in male fertility. Present findings in the mouse may be of interest to human male fertility.
Asunto(s)
Reductasas del Citocromo/biosíntesis , Infertilidad Masculina/genética , Espermatogénesis/fisiología , Animales , Humanos , Masculino , Ratones , Ratones Transgénicos , Oligospermia , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro , Espermatogénesis/genética , Testículo/metabolismoRESUMEN
PURPOSE: The eukaryotic cytosolic chaperonin containing TCP-1 (CCT) plays an important role in maintaining cellular homeostasis by assisting the folding of many proteins and is also well known for the critical roles in disease. However, the functions of CCT complex have not been established globally, especially when translocating into nuclear. The purpose of this study is to explore the function of CCT in nuclear and present a strategy in clinical proteomics studies. EXPERIMENTAL DESIGN: Blue native polyacrylamide gel electrophoresis (BN-PAGE) combined with mass spectrometry was applied to separate and identify CCT protein complexes. RESULTS: We isolated the CCT complex in K562 nucleus and identified a novel CCT complex containing 40 protein components involved in protein folding, RNA processing, apoptosis, and cell metabolism. The interactions between four candidate proteins and CCT were confirmed by immunoblotting. Computational biological analyses and independent biochemical assays validated the overall quality of interactions. CONCLUSIONS AND CLINICAL RELEVANCE: Our results support clues that CCT might play an unexpected role in various biological processes including RNA processing. And we envision future applications for this system searching for new clues of CCT in disease and readily be applied to the clinic.
Asunto(s)
Chaperonina con TCP-1/metabolismo , Núcleo Celular/metabolismo , Chaperonina con TCP-1/análisis , Biología Computacional , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Células K562 , Mapeo de Interacción de ProteínasRESUMEN
THAP11 is an essential factor involved in ES cell pluripotency and cell growth. Here, we identified THAP11 as a novel physiological binding partner of PCBP1. In HepG2 cells, THAP11 overexpression inhibited CD44 v6 expression and cell invasion. However, when deleting the binding domain with PCBP1 or endogenous PCBP1 was knocked down, THAP11 failed to inhibit CD44 v6 expression, indicating that THAP11 regulates CD44 v6 expression through interacting with PCBP1. In HCC patients, the expression of THAP11 mRNA significantly correlated with PCBP1 mRNA expression. Our results suggest a novel role of THAP11 in CD44 alternative splicing and hepatoma invasion.
Asunto(s)
Empalme Alternativo , Carcinoma Hepatocelular/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Receptores de Hialuranos/metabolismo , Neoplasias Hepáticas/metabolismo , Invasividad Neoplásica , Proteínas Represoras/fisiología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proteínas de Unión al ADN , Técnicas de Silenciamiento del Gen , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Neoplasias Hepáticas/patología , Unión Proteica , ARN Mensajero/genética , Proteínas de Unión al ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Erythroid differentiation-associated gene (EDAG) is a haematopoietic tissue-specific transcription regulator that plays a key role in maintaining the homeostasis of haematopoietic lineage commitment. In acute myeloid leukaemia (AML) patients, the high expression level of EDAG is associated with poor prognosis. NPM1 (nucleophosmin/B23), a ubiquitous nucleolar phosphoprotein, comprises a multifunctional protein that is involved in several cellular processes, including ribosome biogenesis, centrosome duplication, cell cycle progression, cell growth and transformation. Various studies have implicated NPM1 overexpression in promoting tumour cell proliferation, blocking the differentiation of leukaemia cells and resisting apoptosis. In the present study, using co-immunoprecipitation, we characterized EDAG as a physiological binding partner of NPM1; The N-terminal (amino acids 1-124) region of EDAG interacts with the N-terminal (amino acids 118-187) of NPM1. Under cycloheximide treatment, the stability of NPM1 protein was enhanced by EDAG overexpression, whereas knockdown of EDAG by lentivirus-mediated small interfering RNA resulted in an increased degradation rate of NPM1 in K562 cells. During 4ß-phorbol l2-myristate 13-acetate-induced K562 megakaryocytic differentiation, overexpression of EDAG prevented the down-regulation of NPM1 proteins, whereas knockdown of EDAG accelerated the down-regulation of NPM1. EDAG deletion mutant lacking the binding domain with NPM1 lost the ability to stabilize NPM1 protein. Furthermore, knockdown of EDAG in K562 cells led to increased cell apoptosis induced by imatinib, and re-expression of NPM1 attenuated the increased apoptosis. These results suggest that EDAG enhances the protein stability of NPM1 via binding to NPM1, which plays a critical role in the anti-apoptosis of leukaemia cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Nucleares/metabolismo , Benzamidas , Cicloheximida/farmacología , Regulación hacia Abajo/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Mesilato de Imatinib , Inmunoprecipitación , Células K562 , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Nucleofosmina , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The antioxidant response elements (ARE) are a cis-acting enhancer sequence located in regulatory regions of antioxidant and detoxifying genes. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a member of the Cap 'n' Collar family of transcription factors that binds to the ARE and regulates the transcription of specific ARE-containing genes. Under oxidative stress, Nrf2/ARE induction is fundamental to defense against reactive oxygen species (ROS) and serves as a key factor in the protection against toxic xenobiotics. 3-(3-Pyridylmethylidene)-2-Indolinone (PMID) is a derivative of 2-indolinone compounds which act as protein kinase inhibitors and show anti-tumor activity. However, the role of PMID in the oxidative stress remains unknown. In the present study, we showed that PMID induced the activation of ARE-mediated transcription, increased the DNA-binding activity of Nrf2 and then up-regulated the expression of antioxidant genes such as HO-1, SOD, and NQO1. The level of Nrf2 protein was increased in cells treated with PMID by a post-transcriptional mechanism. Under CHX treatment, the stability of Nrf2 protein was enhanced by PMID with decreased turnover rate. We showed that PMID reduced the ubiquitination of Nrf2 and disrupted the Cullin3 (Cul3)-Keap1 interaction. Furthermore, cells treated with PMID showed resistance to cytotoxicity by H(2)O(2) and pro-oxidant 6-OHDA. PMID also up-regulated the antioxidant level in BALB/c mice. Taken together, the compound PMID induces the ARE-mediated gene expression through stabilization of Nrf2 protein and activation of Nrf2/ARE pathway and protects against oxidative stress-mediated cell death.
Asunto(s)
Indoles/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/fisiología , Piridinas/farmacología , Elementos de Respuesta , Animales , Antioxidantes/metabolismo , Supervivencia Celular/efectos de los fármacos , Glutatión/análisis , Glutatión/metabolismo , Células Hep G2 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Factor 2 Relacionado con NF-E2/genética , Superóxido Dismutasa/metabolismo , Activación Transcripcional/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Hepassocin (HPS) is a specific mitogenic active factor for hepatocytes, and inhibits growth by overexpression in hepatocellular carcinoma (HCC) cells. However, the mechanism of HPS regulation on growth of liver-derived cells still remains largely unknown. In this study, we found that HPS was expressed and secreted into the extracellular medium in cultured L02 human hepatic cells; conditional medium of L02 cells promoted proliferation of L02 cells and this activity could be blocked by anti-HPS antibody. Moreover, we identified the presence of receptor for HPS on L02 cells and HepG2 human hepatoma cells. Overproduction of truncated HPS, which signal peptide was deleted, significantly inhibited the proliferation of HCC cells and induced cell cycle arrest. These findings suggest that HPS promotes hepatic cell line L02 cells proliferation via an autocrine mechanism and inhibits HCC cells proliferation by an intracrine pathway.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Anticuerpos Neutralizantes/farmacología , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular Tumoral , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática , Fibrinógeno , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Erythroid differentiation-associated gene (EDAG), a hematopoietic tissue-specific transcription regulator, plays a key role in maintaining the homeostasis of hematopoietic lineage commitment. However, the mechanism and genes regulated by EDAG remain unknown. In this study, we showed that overexpression of EDAG in a myeloid cell line 32D led to an erythroid phenotype with increased number of benzidine-positive cells and up-regulation of erythroid specific surface marker TER119. The megakaryocytic specific marker CD61 was also induced significantly. Using a genome-wide microarray analysis and a twofold change cutoff, we identified 332 genes with reduced expression and 288 genes with increased expression. Among up-regulation genes, transcription factor GATA-1 and its target genes including EKLF, NF-E2, Gfi-1b, hemogen, SCL, hemoglobin alpha, beta and megakaryocytic gene GPIX were increased. Silencing of EDAG by RNA interference in K562 cells resulted in down-regulation of these genes. Taken together, EDAG functions as a positive regulator of erythroid/megakaryocytic differentiation in 32D cells associated with the induction of GATA-1 and its target genes.
Asunto(s)
Eritrocitos/metabolismo , Factor de Transcripción GATA1/genética , Megacariocitos/metabolismo , Proteínas Nucleares/genética , Diferenciación Celular , Eritrocitos/citología , Citometría de Flujo , Silenciador del Gen , Humanos , Interleucina-3/metabolismo , Células K562 , Megacariocitos/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
BACKGROUND: PCBP1 (or alpha CP1 or hnRNP E1), a member of the PCBP family, is widely expressed in many human tissues and involved in regulation of transcription, transportation process, and function of RNA molecules. However, the role of PCBP1 in CD44 variants splicing still remains elusive. RESULTS: We found that enforced PCBP1 expression inhibited CD44 variants expression including v3, v5, v6, v8, and v10 in HepG2 cells, and knockdown of endogenous PCBP1 induced these variants splicing. Invasion assay suggested that PCBP1 played a negative role in tumor invasion and re-expression of v6 partly reversed the inhibition effect by PCBP1. A correlation of PCBP1 down-regulation and v6 up-regulation was detected in primary HCC tissues. CONCLUSIONS: We first characterized PCBP1 as a negative regulator of CD44 variants splicing in HepG2 cells, and loss of PCBP1 in human hepatic tumor contributes to the formation of a metastatic phenotype.
Asunto(s)
Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteínas Nucleares Heterogéneas/genética , Receptores de Hialuranos/genética , Neoplasias Hepáticas/genética , Empalme Alternativo , Western Blotting , Carcinoma Hepatocelular/patología , Proteínas de Unión al ADN , Células Hep G2 , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Neoplasias Hepáticas/patología , Invasividad Neoplásica/genética , Interferencia de ARN , ARN Interferente Pequeño , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
BACKGROUND: Human hepassocin (HPS) was originally detected by subtractive and differential cDNA cloning as a liver-specific gene that was markedly upregulated during liver regeneration. Previous studies suggested that HPS showed mitogenic activity on isolated hepatocytes in vitro. However, its in vivo functions remained largely unknown. Therefore, the function of recombinant human HPS during liver regeneration and chemically induced liver injury was investigated. METHODS: The proliferation of primary hepatocytes was examined by [(3)H]thymidine incorporation and immunohistological staining of proliferating cell nuclear antigen (PCNA). RNA interference was performed to knock down the endogenous expression of HPS. The proliferation of L02 cells was examined by MTS assay. The phosphorylation of ERK1/2 (extracellular signal-regulated kinase 1/2) was investigated by western blotting analysis. Assessment of liver injury (histology, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels) and of apoptosis, by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay, was performed. RESULTS: Purified recombinant human HPS showed specific mitogenic activity on primary hepatocytes and normal liver cell lines in a mitogen-activated protein kinase (MAPK)-dependent manner and stimulated the proliferation of hepatocytes in rats with 70% partial hepatectomy. Administration of HPS to rats after d-galactose and carbon tetrachloride (CCl(4)) treatment protected against liver injury (minimal liver necrosis, depressed ALT and AST levels, and decreased lethality), reduced apoptosis and enhanced proliferation. Knock-down of endogenous HPS in vivo enhanced the liver injury induced by d-galactose by increasing the apoptosis and elevating ALT and AST levels. CONCLUSIONS: HPS is a hepatic growth factor which can accelerate hepatocyte proliferation in vivo and protect against liver injury. These data point to the potential interest of HPS in the treatment of fulminant hepatic failure.
Asunto(s)
Hepatocitos/efectos de los fármacos , Fallo Hepático Agudo/tratamiento farmacológico , Proteínas de Neoplasias/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Modelos Animales de Enfermedad , Fibrinógeno , Hepatocitos/patología , Humanos , Fallo Hepático Agudo/patología , Regeneración Hepática/efectos de los fármacos , Masculino , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Proteínas de Neoplasias/farmacología , Interferencia de ARN , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéuticoRESUMEN
To gain new insight into the biological function of the human augmenter of liver regeneration (hALR) in HCC, we studied its involvement in radiation-induced damage and recovery of HCC cells. We found that hALR expression was up-regulated in both HCC tissues and multiple hepatoma cell lines and correlated significantly with increased radiation clonogenic survival after radiation treatment. Exogenous expression of hALR increased radiation resistance in SMMC-7721 cells, and the increased survival was accompanied by a decrease in apoptosis and a prolonged G(2)-M arrest after irradiation. Overexpression of ALR significantly increased the mitochondrial membrane potential, inhibited cytochrome c release, and opposed the loss of intracellular ATP levels after radiation. Moreover, knockdown of ALR by siRNA resulted in inhibition of viability in the absence of exogenously added oxidative stress and radiation sensitization in HepG2 cells. Importantly, hALR expression was very low in normal hepatocyte L02 cells, and hALR silencing had a minimal effect on L02 viability and radiation sensitivity. These results suggest that human ALR is important for hepatoma cell viability and involved in the protection of hepatoma cells against irradiation-induced damage by its association with mitochondria. Targeting hALR may be a promising novel approach to enhance the radiosensitivity of hepatoma cancers.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Reductasas del Citocromo/metabolismo , Neoplasias Hepáticas/metabolismo , Animales , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de la radiación , Reductasas del Citocromo/biosíntesis , Relación Dosis-Respuesta en la Radiación , Femenino , Rayos gamma , Humanos , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/efectos de la radiación , Datos de Secuencia Molecular , Estrés Oxidativo/efectos de la radiación , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro , Tolerancia a Radiación , Estudios Retrospectivos , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
Hepassocin (HPS), is a liver-specific gene with mitogenic activity on isolated hepatocytes. It is up-regulated following partial hepatectomy and down-regulated frequently in heptocellular carcinoma (HCC). However, very little is known about the HPS transcription regulation mechanism. In this study, we identified HNF1alpha (hepatocyte nuclear factor-1alpha) as an important liver-specific cis-acting element for HPS using in vivo luciferase assays. Deletion of the HNF1 binding site not only led to a complete loss of HPS promoter activity in vivo but also abolished the induction of the HPS promoter by HNF1alpha. An electrophoretic mobility shift assay demonstrated that HNF1alpha interacted with the HPS gene promoter in vitro. Chromatin immunoprecipitation showed that HNF1alpha interacted with HMGB1 and CREB-binding protein, and all of them were recruited to the HPS promoter in vivo. Moreover, HNF1alpha expression was lower in HCC cell lines and tissues and correlated significantly with the down-regulation of HPS expression. Re-expression of HNF1alpha in human hepatoma HepG2 cells reinduced HPS expression. In contrast, knockdown of endogenous HNF1alpha expression by small interfering RNA resulted in a significant reduction of HPS expression. Furthermore, we found that partial hepatectomy and IL-6 significantly induced promoter activity of HPS, depending on STAT3 and HNF1 binding sites in the HPS promoter. These results demonstrate that the HNF1 binding site and HNF1alpha are critical to liver-specific expression of HPS, and down-regulation or loss of HNF1alpha causes, at least in part, the transcriptional down-regulation of HPS in HCC.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Proteínas de Neoplasias/biosíntesis , Elementos de Respuesta , Animales , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Fibrinógeno , Eliminación de Gen , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Factor Nuclear 1-alfa del Hepatocito/genética , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas de Neoplasias/genética , Unión Proteica , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transcripción GenéticaRESUMEN
EDAG, a hematopoietic tissue-specific protein, is involved in the regulation of proliferation, differentiation and apoptosis of hematopoietic cells. In this study, a dose-dependent inhibition of EDAG expression by PMA was observed in K562 cells. The responsive element for the PMA-induced inhibition was contained in the region between -211 and +32bp of the EDAG gene promoter. By oligonucleotide-directed mutagenesis, EMSA, ChIP and transient transfection assays, we found that two tandem repeat GATA-1 sites in the promoter of EDAG gene played an important role in the PMA-mediated down-regulation of the EDAG gene expression in K562 cells. The kinetics of EDAG expression during PMA induction showed that the levels of EDAG expression were down-regulated concomitantly with GATA-1 down-expression. Decreased GATA-1 expression by siRNA reduced expression of EDAG in K562 cells, and restored expression of GATA-1 significantly rescued EDAG expression from PMA-mediated suppression. Overexpression of EDAG in K562 cells inhibited the megakaryocytic differentiation induced by PMA which raised the interesting possibility that PMA induced K562 cells differentiation toward megakaryocytic phenotype through, at least in part, the inhibition of EDAG expression. In vivo analysis confirmed that EDAG was highly expressed in primitive progenitor cells and down-regulated in megakaryocytes which was consistent with the expression pattern of GATA-1. Furthermore, PKC and MAPK specific inhibitors treatment attenuated the down-regulation of EDAG induced by PMA. Taken together, these results suggest that the inhibition of the EDAG gene by PMA is mediated through down-regulation of transcription factor GATA-1 and involved the PKC/MAPK signaling pathway.
Asunto(s)
Factor de Transcripción GATA1/metabolismo , Silenciador del Gen/efectos de los fármacos , Proteínas Nucleares/genética , Acetato de Tetradecanoilforbol/farmacología , Secuencia de Bases , Sitios de Unión , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Dactinomicina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Factor de Transcripción GATA1/fisiología , Humanos , Células K562 , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica/efectos de los fármacos , ARN Mensajero/metabolismo , TransfecciónRESUMEN
Hepatocyte nuclear factor (HNF)-1alpha is one of the liver-enriched transcription factors involved in many tissue-specific expressions of hepatic genes. The molecular mechanisms for determining HNF1alpha-mediated transactivation have not been explained fully. To identify unknown proteins that interact with HNF1alpha, we developed a co-IP-MS strategy to search HNF1alpha interactions, and high mobility group protein-B1 (HMGB1), a chromosomal protein, was identified as a novel HNF1alpha-interacting protein. In vitro glutathione S-transferase pull-down and in vivo co-immunoprecipitation studies confirmed an interaction between HMGB1 and HNF1alpha. The protein-protein interaction was mediated through the HMG box domains of HMGB1 and the homeodomain of HNF1alpha. Furthermore, electrophoretic mobility shift assay and chromatin-immunoprecipitation assay demonstrated that HMGB1 was recruited to endogenous HNF1alpha-responsive promoters and enhanced HNF1alpha binding to its cognate DNA sequences. Moreover, luciferase reporter analyses showed that HMGB1 potentiated the transcriptional activities of HNF1alpha in cultured cells, and downregulation of HMGB1 by RNA interference specifically affected the HNF1alpha-dependent gene expression in HepG2 cell. Taken together, these findings raise the intriguing possibility that HMGB1 is a new cofactor of HNF1alpha and participates in HNF1alpha-mediated transcription regulation through protein-protein interaction.
Asunto(s)
Proteína HMGB1/metabolismo , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Sitios de Unión , Línea Celular , Proteína HMGB1/antagonistas & inhibidores , Proteína HMGB1/química , Factor Nuclear 1-alfa del Hepatocito/química , Humanos , Inmunoprecipitación , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Proteómica , Interferencia de ARN , Activación Transcripcional , alfa-Fetoproteínas/genéticaRESUMEN
BACKGROUND & OBJECTIVE: Embryonic development associated gene 1 (EDAG-1), located at chromosome 9q22, is specially expressed in hematopoietic cells, and related to the regulation of hematopoietic system. This study was designed to explore relationship between pathogenesis of leukemia, lymphoma and EDAG-1 through analyzing the structure of EDAG-1 coding region, and its expression in these cell lines. METHODS: Fifteen leukemia and lymphoma cell lines, HEL, K562, HL-60, Namalwa, Raji, J111, Jurkat, HuT 78, MEG-01, U937, 6T-CEM, HPB-ALL, KG-1a, THP-1, and DAMI, were selected to observe the expression of EDAG-1 by reverse transcriptase-polymerase chain reaction (RT-PCR), EDAG-1 cDNA coding fragments (1.5 kb) were purified to construct the corresponding recombinant plasmid. Then, the plasmid was sequenced to analyze mutation of the coding region. The expression of EDAG-1 protein, and mRNA in these cell lines were detected by Western blot, and Northern blot; the rearrangement and amplification of EDAG-1 genome in these cell lines were detected by Southern blot. RESULTS: EDAG-1 mRNA and protein were highly expressed in erythroleukemia cell lines (K-562, HEL), megakaryoblast leukemia cell lines (DAMI, MEG-01), and T cell leukemia cell line (Jurkat), while no gene mutation was found in coding region, no amplification and rearrangement of genome was detected in these cell lines. EDAG-1 was absent in HL-60 cell line, and rearranged in HuT 78 cells. CONCLUSION: EDAG-1 may relate with pathogenesis of erythroleukemia and megakaryoblast leukemia; its coding region may have no relation with the mechanism of its activation.
Asunto(s)
Linfoma de Burkitt/metabolismo , Cromosomas Humanos Par 9 , Leucemia Eritroblástica Aguda/metabolismo , Oncogenes , Proteínas/metabolismo , Linfoma de Burkitt/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico , Humanos , Leucemia Eritroblástica Aguda/patología , Leucemia Megacarioblástica Aguda/metabolismo , Leucemia Megacarioblástica Aguda/patología , Leucemia de Células T/metabolismo , Leucemia de Células T/patología , Mutación , Proteínas Nucleares , Plásmidos , Proteínas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
The human sprouty 4 (SPRY4) gene was localized to chromosome band 5q32 approximately 33 by screening the Stanford radiation hybrid G3 panel using a SPRY4-specific primer pair for PCR. Northern blot analysis revealed two different mRNAs (5 kb and 2 kb) in liver, skeletal muscle, heart, lung, kidney, spleen, placenta and small intestine. Reverse transcriptase-PCR analysis showed that SPRY4 was expressed in all tested tissues to different levels.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 5 , Proteínas/genética , Northern Blotting , Cartilla de ADN/química , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas del Tejido Nervioso , Proteínas/metabolismo , ARN Mensajero/metabolismo , Mapeo de Híbrido por Radiación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución TisularRESUMEN
OBJECTIVE: To explore the relationship between the proliferation, differentiation of rat hepatic stem like cell line WB-F344 and cytokines in vitro. METHODS: (3)H thymidine labelling of new synthesized DNA was used to examine the mitogenic responsiveness of WB-F344 cells to cytokines, western blot was used to study the expression of cytokines receptors on hepatic stem cells, and apoptotic cells were detected by Flow cytometry. RESULTS: WB-F344 cells showed a proliferative response to the cytokines of hepatocyte growth factor (HGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), Insulin at the dose of 80 ng/ml, and the relative cpm values are 982.95, 906.32, 863.98 and 968.67 respectively, while non response to interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) at the same dose, and an inhibition or apoptosis response to transforming growth factor-beta (TGF-beta) at 80 ng/ml with a 26.89% apoptotic rate. Western blot showed that there were HGF, EGF, FGF, TGF-beta receptors expressed on WB-F344 cells. When WB-F344 cells were cultured in the differential system (DMEM, 10% Fcs, HGF 10 to approximately 50 ng/ml, EGF 20 ng/ml, Insulin 1 microg/ml, Dex 1 micromol/L), the cells could differentiated into hepatocytes. In addition, HGF could scattered WB-F344 cells. CONCLUSION: The proliferation and differentiation of liver stem cells are regulated by various cytokines which may play an important role when liver is damaged seriously.
Asunto(s)
Citocinas/farmacología , Hígado/citología , Células Madre/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Factor de Crecimiento de Hepatocito/farmacología , Insulina/farmacología , Ratas , Ratas Endogámicas F344 , Células Madre/citología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
A novel transcript of human HPO (HPO-205) encoding a 205 amino acid ORF corresponding to a translated product of 23 kD different from the previous HPO that lacks the N-terminal 80 amino acids, was isolated from human fetal liver cDNA library by 5'RACE methods. The dose-dependent stimulation of DNA synthesis of HepG2 hepatoma cells by HPO-205 demonstrated its similar biological activity with HPO in vitro. The level of MAPK (mitogen-activated protein kinase)phosphorylation was further examined by Western blot analysis and the result revealed that HPO-205 protein might have stronger activity on stimulating hepatic cell proliferation than that of HPO. The similar result was observed by FACS technique. Therefore, our results suggest that the HPO-205 has activity to stimulate proliferation of liver derived cells, and this activity may be stronger than HPO.
Asunto(s)
Factor de Crecimiento de Hepatocito/farmacología , ARN/genética , Secuencia de Aminoácidos , Animales , Western Blotting , Células COS , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , ADN Complementario/química , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Expresión Génica , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , ARN/aislamiento & purificación , ARN/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción Genética , Células Tumorales CultivadasRESUMEN
AIM: To isolate a novel isoform of human HPO (HPO-205) from human fetal liver Marathon-ready cDNA and characterize its primary biological function. METHODS: 5'-RACE (rapid amplification of cDNA 5' ends) was used to isolate a novel isoform of hHPO in this paper. The constructed pcDNA(HPO-205), pcDNA(HPO) and pcDNA eukaryotic expression vectors were respectively transfected by lipofectamine method and the stimulation of DNA synthesis was observed by (3)H-TdR incorporation assay. Proteins extracted from different cells were analyzed by Western blot. RESULTS: A novel isoform of hHPO (HPO-205) encoding a 205 amino acid ORF corresponding to a translated production of 23 kDa was isolated and distinguished from the previous HPO that lacked the N-terminal 80 amino acids. The dose-dependent stimulation of DNA synthesis of HepG2 hepatoma cells by HPO-205 demonstrated its similar biological activity with HPO in vitro. The level of MAPK (Mitogen-activated protein kinase) phosphorylation by Western blot analysis revealed that HPO-205 might have the stronger activity of stimulating hepatic cell proliferation than that of HPO. CONCLUSION: A novel isoform of hHPO (HPO-205) was isolated from hepatic-derived cells. The comparison of HPO-205 and HPO will lead to a new insight into the structure and function of hHPO, and provide the new way of thinking to deeply elucidate the biological roles of HPO/ALR.