RESUMO
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.
Assuntos
Neoplasias Encefálicas/metabolismo , Proliferação de Células/fisiologia , Glioma/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Adulto , Idoso , Arginina/análogos & derivados , Arginina/metabolismo , Neoplasias Encefálicas/patologia , Diferenciação Celular/genética , Proliferação de Células/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ensaio de Unidades Formadoras de Colônias , Epitélio/metabolismo , Epitélio/patologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Glioma/patologia , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Pessoa de Meia-Idade , Neurônios/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteína-Arginina N-Metiltransferases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
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.
Assuntos
Esclerose Lateral Amiotrófica/genética , Mutação , Proteínas de Ligação a RNA/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Antígenos de Superfície/metabolismo , Citoplasma/metabolismo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Camundongos , Modelos Biológicos , Neurônios/metabolismo , Polirribossomos/metabolismo , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Medula Espinal/metabolismo , Superóxido Dismutase-1RESUMO
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.
Assuntos
Glioma/patologia , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Quinases da Família src/metabolismo , Antígeno AC133 , Animais , Antígenos CD/metabolismo , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dasatinibe , Glioma/metabolismo , Glicoproteínas/metabolismo , Humanos , Camundongos , Neoplasias Experimentais , Células-Tronco Neoplásicas/patologia , Peptídeos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas Proto-Oncogênicas c-yes/metabolismo , Pirimidinas/farmacologia , Tiazóis/farmacologia , Regulação para CimaRESUMO
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.
Assuntos
Etanol/farmacologia , Fibrinólise/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Ativadores de Plasminogênio/biossíntese , Regulação para Cima/efeitos dos fármacos , Adulto , Fibrinolisina/biossíntese , Fibrinólise/fisiologia , Humanos , Monócitos/metabolismo , Células U937 , Regulação para Cima/fisiologiaRESUMO
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.