RESUMEN
Src family kinases (SFKs) are highly expressed and active in clinical glioblastoma multiforme (GBM) specimens. SFKs inhibitors have been demonstrated to inhibit proliferation and migration of glioma cells. However, the role of SFKs in glioma stem cells (GSCs), which are important for treatment resistance and recurrence, has not been reported. Here, we examined the expression pattern of individual members of SFKs and their functional role in CD133⺠GSCs in comparison to primary glioma cells. We found that Fyn, c-Src and Yes were robustly expressed in GSCs while Lck was absent. Knockdown of c-Src, Yes or treatment with the SFK inhibitor dasatinib inhibited the migration of GSCs, but had no impact on their growth or self-renewal. These results suggest that SFKs represent an effective target for GSC migration but not for their growth.
Asunto(s)
Glioma/patología , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/metabolismo , Familia-src Quinasas/metabolismo , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Proteína Tirosina Quinasa CSK , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Dasatinib , Glioma/metabolismo , Glicoproteínas/metabolismo , Humanos , Ratones , Neoplasias Experimentales , Células Madre Neoplásicas/patología , Péptidos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Proteínas Proto-Oncogénicas c-yes/metabolismo , Pirimidinas/farmacología , Tiazoles/farmacología , Regulación hacia ArribaRESUMEN
Protein arginine methyltransferase 5 (PRMT5) catalyzes the formation of ω-NG,N'G-symmetric dimethylarginine residues on histones as well as other proteins. These modifications play an important role in cell differentiation and tumor cell growth. However, the role of PRMT5 in human glioma cells has not been characterized. In this study, we assessed protein expression profiles of PRMT5 in control brain, WHO grade II astrocytomas, anaplastic astrocytomas, and glioblastoma multiforme (GBM) by immunohistochemistry. PRMT5 was low in glial cells in control brain tissues and low grade astrocytomas. Its expression increased in parallel with malignant progression, and was highly expressed in GBM. Knockdown of PRMT5 by small hairpin RNA caused alterations of p-ERK1/2 and significantly repressed the clonogenic potential and viability of glioma cells. These findings indicate that PRMT5 is a marker of malignant progression in glioma tumors and plays a pivotal role in tumor growth.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Proliferación Celular/fisiología , Glioma/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Adulto , Anciano , Arginina/análogos & derivados , Arginina/metabolismo , Neoplasias Encefálicas/patología , Diferenciación Celular/genética , Proliferación Celular/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Ensayo de Unidades Formadoras de Colonias , Epitelio/metabolismo , Epitelio/patología , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioma/patología , Humanos , Sistema de Señalización de MAP Quinasas/genética , Masculino , Persona de Mediana Edad , Neuronas/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
In experimental autoimmune encephalomyelitis (EAE) and other neurodegenerative diseases, astrocytes play an important role in promoting or attenuating the inflammatory response through induction of different cytokines and growth factors. HuR plays a major role in regulating many of these factors by modulating RNA stability and translational efficiency. Here, we engineered transgenic mice to express HuR in astrocytes using the human glial fibrillary acidic protein promoter and found that female transgenic mice had significantly less clinical disability and histopathological changes in the spinal cord. Ovariectomy prior to EAE induction abrogated the protective effect. Our findings support a role for the astrocyte and posttranscriptional regulation in hormonally-mediated attenuation of EAE.
Asunto(s)
Astrocitos/metabolismo , Proteínas ELAV/biosíntesis , Proteínas ELAV/genética , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Congéneres del Estradiol/fisiología , Regulación de la Expresión Génica/inmunología , Animales , Astrocitos/inmunología , Astrocitos/patología , Proteínas ELAV/fisiología , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Humanos , Ratones , Ratones Transgénicos , Médula Espinal/inmunología , Médula Espinal/metabolismo , Médula Espinal/patologíaRESUMEN
Posttranscriptional regulation is a critical control point for the expression of genes that promote or retard tumor growth. We previously found that the mRNA-binding protein, ELAV 1 (HuR), is upregulated in primary brain tumors and stabilizes growth factor mRNAs such as VEGF and IL-8. To better understand the role of HuR in brain tumor growth, we altered levels of HuR in glioma cells by short hairpin RNA or ectopic expression and measured tumor cell phenotype using in vitro and in vivo models. In HuR-silenced cells, we found a significant decrease in anchorage-independent growth and cell proliferation with a concomitant induction of apoptosis. Using an intracranial tumor model with primary glioblastoma cells, HuR silencing produced a significant decrease in tumor volume. In contrast, overexpression of HuR produced in vitro chemoresistance to standard glioma therapies. Because bcl-2 is abundantly expressed in glioma and associated with tumor growth and survival, we determined the impact of HuR on its regulation as a molecular validation to the cellular and animal studies. Using UV cross-linking and RNA immunoprecipitation, we show that HuR bound to the 3'-untranslated region of all bcl-2 family members. Silencing of HuR led to transcript destabilization and reduced protein expression. Polysome profiling indicated loss of HuR from the translational apparatus. In summary, these findings reveal a HuR-dependent mechanism for cancer cell survival and sensitivity to chemotherapeutic drugs suggesting that HuR should be considered as a new therapeutic target.
Asunto(s)
Antígenos de Superficie/metabolismo , Resistencia a Antineoplásicos/genética , Glioma/genética , Glioma/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3'/genética , Animales , Antígenos de Superficie/genética , Secuencia de Bases , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Proteínas ELAV , Proteína 1 Similar a ELAV , Técnicas de Silenciamiento del Gen , Glioma/tratamiento farmacológico , Glioma/patología , Ratones , Datos de Secuencia Molecular , Polirribosomas/genética , Polirribosomas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas de Unión al ARN/genéticaRESUMEN
Down-regulation of vascular endothelial growth factor (VEGF) in the mouse leads to progressive and selective degeneration of motor neurons similar to amyotrophic lateral sclerosis (ALS). In mice expressing ALS-associated mutant superoxide dismutase 1 (SOD1), VEGF mRNA expression in the spinal cord declines significantly prior to the onset of clinical manifestations. In vitro models suggest that dysregulation of VEGF mRNA stability contributes to that decline. Here, we show that the major RNA stabilizer, Hu Antigen R (HuR), and TIA-1-related protein (TIAR) colocalize with mutant SOD1 in mouse spinal cord extracts and cultured glioma cells. The colocalization was markedly reduced or abolished by RNase treatment. Immunoanalysis of transfected cells indicated that colocalization occurred in insoluble aggregates and inclusions. RNA immunoprecipitation showed a significant loss of VEGF mRNA binding to HuR and TIAR in mutant SOD1 cells, and there was marked depletion of HuR from polysomes. Ectopic expression of HuR in mutant SOD1 cells more than doubled the mRNA half-life of VEGF and significantly increased expression to that of wild-type SOD1 control. Cellular effects produced by mutant SOD1, including impaired mitochondrial function and oxidative stress-induced apoptosis, were reversed by HuR in a gene dose-dependent pattern. In summary, our findings indicate that mutant SOD1 impairs post-transcriptional regulation by sequestering key regulatory RNA-binding proteins. The rescue effect of HuR suggests that this impairment, whether related to VEGF or other potential mRNA targets, contributes to cytotoxicity in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Mutación , Proteínas de Unión al ARN/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Antígenos de Superficie/metabolismo , Citoplasma/metabolismo , Proteínas ELAV , Proteína 1 Similar a ELAV , Ratones , Modelos Biológicos , Neuronas/metabolismo , Polirribosomas/metabolismo , ARN Mensajero/metabolismo , Ribonucleasas/metabolismo , Médula Espinal/metabolismo , Superóxido Dismutasa-1RESUMEN
Homozygous ataxia (ax(J)) mice have reduced expression of ubiquitin-specific protease 14 (Usp14), resulting in severe neuromuscular defects and death by 2 months of age. Transgenic expression of Usp14 exclusively in the nervous system of ax(J) mice (ax(J)-Tg) prevents early lethality and restores motor system function to the ax(J) mice, enabling an analysis of the reproductive capabilities of Usp14-deficient mice. Although female ax(J)-Tg mice had a 75% reduction of Usp14 in the ovaries, they were able to produce normal litters. Ovary transfer experiments also demonstrated that the ovaries of ax(J) mice were capable of producing viable pups. In contrast, male ax(J) and ax(J)-Tg mice displayed a 50% reduction in testicular Usp14 levels and were infertile, indicating that Usp14 is required for development and function of the male reproductive system. Immunohistochemistry experiments showed that Usp14 is found in the redundant nuclear envelope and cytoplasmic droplet of epididymal spermatozoa. Analysis of ax(J) testes demonstrated a 50% reduction in testis weight, a 100-fold reduction in sperm number and the presence of abnormal spermatozoa in the epididymis. Histological examination of the Usp14-deficient testes revealed abnormal spermatogenesis and the presence of degenerating germ cells, indicating that Usp14 and the ubiquitin proteasome system are required for spermatid differentiation during spermiogenesis.
Asunto(s)
Ataxia/complicaciones , Ataxia/patología , Infertilidad Masculina/complicaciones , Infertilidad Masculina/patología , Animales , Compensación de Dosificación (Genética) , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Endopeptidasas/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Dosificación de Gen , Perfilación de la Expresión Génica , Heterocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Tamaño de los Órganos , Transporte de Proteínas , Espermatozoides/metabolismo , Testículo/embriología , Testículo/enzimología , Testículo/metabolismo , Testículo/patología , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismoRESUMEN
The ataxia mutation (axJ) is a recessive neurological mutation that results in reduced growth, ataxia, and hindlimb muscle wasting in mice. The axJ gene encodes ubiquitin-specific protease 14 (Usp14), a deubiquitinating enzyme (DUB) that associates with the proteasome via its ubiquitin-like (Ubl) domain and is involved in processing ubiquitin chains. Analysis of Usp14 gene products demonstrated that Usp14 undergoes alternative pre-mRNA splicing to produce a full-length form of Usp14 that is capable of binding proteasomes and a form that contains a deletion in the Ubl domain. The full-length form of Usp14 is the only form that appears to be reduced in the axJ mice. Transgenic rescue of the axJ mice with neuronal-specific expression of Usp14 demonstrated that the full-length form of Usp14 was sufficient to restore viability and motor system function to the axJ mice. Biochemical analysis showed that the ubiquitin hydrolyase activity of this form of Usp14 is dependent on the presence of proteasomes, and neuronal expression of full-length Usp14 was able to restore the levels of monomeric ubiquitin in the brains of axJ mice. However, the axJ-rescued mice still displayed the Purkinje cell axonal swellings that are seen in the axJ mice, indicating that this cerebellar alteration is not the primary cause of the axJ movement disorders. These results show that the motor defects observed in the axJ mice are attributable to a neuropathic disease rather than to a muscular disorder and suggest that changes in proteasomal function may contribute to neurological dysfunction in the axJ mice.
Asunto(s)
Ataxia/enzimología , Ataxia/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Neuronas/enzimología , Ubiquitina Tiolesterasa/biosíntesis , Ubiquitina/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Ratas , Ubiquitina Tiolesterasa/genéticaRESUMEN
Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.
Asunto(s)
Núcleo Celular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Viral de la Expresión Génica , Papillomaviridae/patogenicidad , Proteínas Virales/metabolismo , Transporte Activo de Núcleo Celular , Animales , Células COS , Línea Celular , Chlorocebus aethiops , ADN Helicasas/metabolismo , Replicación del ADN , Humanos , Carioferinas/metabolismo , Fosforilación , Receptores Citoplasmáticos y Nucleares/metabolismo , Replicación Viral , Proteína Exportina 1RESUMEN
BACKGROUND: Moderate alcohol consumption is associated with reduced risk for coronary heart disease. This may due, in part to increased fibrinolysis. Monocytes synthesize fibrinolytic proteins, tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and their receptors. These studies were carried out to determine the effect of low alcohol on monocyte fibrinolytic activity and PA messenger RNA (mRNA) synthesis. METHODS: Peripheral blood monocytes and U937 cells were incubated in absence/presence of low alcohol (0.1%, v/v) for various times (0-1 hr), followed by incubations in the absence of alcohol (0-24 hr) before measurement of fibrinolytic activity and PA mRNA levels (reverse transcriptase polymerase chain reaction). RESULTS: Brief exposure (15 min, 4 degrees C) of U937 cells to low alcohol resulted in an approximately 2- to 3-fold increase (269.0 +/- 5.6 fmol/1 x 10 cells versus 656.0 +/- 94.0 fmol/1 x 10 cells) in fibrinolytic activity. Preincubation of U937 cells and peripheral blood monocytes in low alcohol (1 hr, 37 degrees C) followed by incubation in the absence of alcohol (24 hr) resulted in a sustained approximately 4- to 5-fold increase (414.0 +/- 174.7 vs. 965.33.0 +/- 104.8 fmol/1 x 10 cells) and an approximately 3- to 4-fold (20.5 +/- 2.14 vs. 74 +/- 2.28 fmol/2 x 10 cells, respectively) increase in fibrinolytic activity. Preincubation of monocytes with low alcohol (1 hr, 37 degrees C) followed by incubation in the absence of alcohol (6 hr) resulted in an approximately 5- to 6-fold (0.06 +/- 0.02 vs. 0.42 +/- 0.02) and an approximately 2- to 3-fold (0.89 +/- 0.04 vs. 2.07 +/- 0.29) increase in t-PA and u-PA mRNA (reverse transcriptase polymerase chain reaction; PA/glyceraldehyde-3-phosphate dehydrogenase ratio), respectively. CONCLUSIONS: These data suggest that low alcohol exerts a rapid, direct, and sustained effect on monocyte fibrinolytic activity, which may be, due in part, to increased monocyte t-PA/u-PA expression. These data provide a feasible molecular mechanism by which alcohol effects on monocyte fibrinolysis may contribute to the cardioprotective benefit associated with moderate alcohol consumption.