RESUMO
The ChlR1 DNA helicase is mutated in Warsaw breakage syndrome characterized by developmental anomalies, chromosomal breakage, and sister chromatid cohesion defects. However, the mechanism by which ChlR1 preserves genomic integrity is largely unknown. Here, we describe the roles of ChlR1 in DNA replication recovery. We show that ChlR1 depletion renders human cells highly sensitive to cisplatin; an interstrand-crosslinking agent that causes stalled replication forks. ChlR1 depletion also causes accumulation of DNA damage in response to cisplatin, leading to a significant delay in resolution of DNA damage. We also report that ChlR1-depleted cells display defects in the repair of double-strand breaks induced by the I-PpoI endonuclease and bleomycin. Furthermore, we demonstrate that ChlR1-depeleted cells show significant delays in replication recovery after cisplatin treatment. Taken together, our results indicate that ChlR1 plays an important role in efficient DNA repair during DNA replication, which may facilitate efficient establishment of sister chromatid cohesion.
Assuntos
RNA Helicases DEAD-box/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Replicação do DNA , Cromátides/efeitos dos fármacos , Cromátides/metabolismo , Cisplatino/toxicidade , Reagentes de Ligações Cruzadas/toxicidade , RNA Helicases DEAD-box/genética , Reparo do DNA por Junção de Extremidades , DNA Helicases/genética , Células HEK293 , Células HeLa , Humanos , RNA Interferente PequenoRESUMO
Tetraspanin CD151 is highly expressed in endothelial cells and regulates pathologic angiogenesis. However, the mechanism by which CD151 promotes vascular morphogenesis and whether CD151 engages other vascular functions are unclear. Here we report that CD151 is required for maintaining endothelial capillary-like structures formed in vitro and the integrity of endothelial cell-cell and cell-matrix contacts in vivo. In addition, vascular permeability is markedly enhanced in the absence of CD151. As a global regulator of endothelial cell-cell and cell-matrix adhesions, CD151 is needed for the optimal functions of various cell adhesion proteins. The loss of CD151 elevates actin cytoskeletal traction by up-regulating RhoA signaling and diminishes actin cortical meshwork by down-regulating Rac1 activity. The inhibition of RhoA or activation of cAMP signaling stabilizes CD151-silenced or -null endothelial structure in vascular morphogenesis. Together, our data demonstrate that CD151 maintains vascular stability by promoting endothelial cell adhesions, especially cell-cell adhesion, and confining cytoskeletal tension.
Assuntos
Comunicação Celular/fisiologia , Citoesqueleto/metabolismo , Células Endoteliais/metabolismo , Tetraspanina 24/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Vasos Sanguíneos/citologia , Vasos Sanguíneos/metabolismo , Adesão Celular/fisiologia , Células Cultivadas , Citoesqueleto/genética , Células Endoteliais/citologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Mutantes , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Tetraspanina 24/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Here, we report the identification of the RNA binding motif protein RBM15B/OTT3 as a new CDK11(p110) binding partner that alters the effects of CDK11 on splicing. RBM15B was initially identified as a binding partner of the Epstein-Barr virus mRNA export factor and, more recently, as a cofactor of the nuclear export receptor NXF1. In this study, we found that RBM15B co-elutes with CDK11(p110), cyclin L2α, and serine-arginine (SR) proteins, including SF2/ASF, in a large nuclear complex of â¼1-MDa molecular mass following size exclusion chromatography. Using co-immunoprecipitation experiments and in vitro pulldown assays, we mapped two distinct domains of RBM15B that are essential for its direct interaction with the N-terminal extension of CDK11(p110), cyclin L2α, and SR proteins such as 9G8 and SF2/ASF. Finally, we established that RBM15B is a functional competitor of the SR proteins SF2/ASF and 9G8, inhibits formation of the functional spliceosomal E complex, and antagonizes the positive effect of the CDK11(p110)-cyclin L2α complex on splicing both in vitro and in vivo.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/antagonistas & inibidores , Ciclinas/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Animais , Ligação Competitiva , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Fatores de Processamento de Serina-Arginina , Spliceossomos/metabolismoRESUMO
The ChlR1 DNA helicase, encoded by DDX11 gene, which is responsible for Warsaw breakage syndrome (WABS), has a role in sister-chromatid cohesion. In this study, we show that human ChlR1 deficient cells exhibit abnormal heterochromatin organization. While constitutive heterochromatin is discretely localized at perinuclear and perinucleolar regions in control HeLa cells, ChlR1-depleted cells showed dispersed localization of constitutive heterochromatin accompanied by disrupted centromere clustering. Cells isolated from Ddx11(-/-) embryos also exhibited diffuse localization of centromeres and heterochromatin foci. Similar abnormalities were found in HeLa cells depleted of combinations of HP1α and HP1ß. Immunofluorescence and chromatin immunoprecipitation showed a decreased level of HP1α at pericentric regions in ChlR1-depleted cells. Trimethyl-histone H3 at lysine 9 (H3K9-me3) was also modestly decreased at pericentric sequences. The abnormality in pericentric heterochromatin was further supported by decreased DNA methylation within major satellite repeats of Ddx11(-/-) embryos. Furthermore, micrococcal nuclease (MNase) assay revealed a decreased chromatin density at the telomeres. These data suggest that in addition to a role in sister-chromatid cohesion, ChlR1 is also involved in the proper formation of heterochromatin, which in turn contributes to global nuclear organization and pleiotropic effects.
Assuntos
RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Heterocromatina/metabolismo , Células Cultivadas , Homólogo 5 da Proteína Cromobox , RNA Helicases DEAD-box/deficiência , DNA Helicases/deficiência , Células HeLa , HumanosRESUMO
Neuroblastoma, the most common paediatric solid tumour, arises from defective neural crest cells. Genetic alterations occur frequently in the most aggressive neuroblastomas. In particular, deletion or suppression of the proapoptotic enzyme caspase-8 is common in malignant, disseminated disease, although the effect of this loss on disease progression is unclear. Here we show that suppression of caspase-8 expression occurs during the establishment of neuroblastoma metastases in vivo, and that reconstitution of caspase-8 expression in deficient neuroblastoma cells suppressed their metastases. Caspase-8 status was not a predictor of primary tumour growth; rather, caspase-8 selectively potentiated apoptosis in neuroblastoma cells invading the collagenous stroma at the tumour margin. Apoptosis was initiated by unligated integrins by means of a process known as integrin-mediated death. Loss of caspase-8 or integrin rendered these cells refractory to integrin-mediated death, allowed cellular survival in the stromal microenvironment, and promoted metastases. These findings define caspase-8 as a metastasis suppressor gene that, together with integrins, regulates the survival and invasive capacity of neuroblastoma cells.
Assuntos
Caspases/deficiência , Caspases/genética , Metástase Neoplásica/patologia , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Animais , Apoptose , Caspase 8 , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Embrião de Galinha , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Integrinas/metabolismo , Rim/patologia , Camundongos , Camundongos Nus , Metástase Neoplásica/genética , Transplante de Neoplasias , Neuroblastoma/genética , Ovário/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Hydroxycarbamide (hydroxyurea) provides laboratory and clinical benefits for adults and children with sickle cell anaemia (SCA). Given its mechanism of action and prior reports of genotoxicity, concern exists regarding long-term toxicities and possible carcinogenicity. We performed cross-sectional analyses of chromosome stability using peripheral blood mononuclear cells (PBMC) from 51 children with SCA and 3-12 years of hydroxycarbamide exposure (mean age 13·2 ± 4·1 years), compared to 28 children before treatment (9·4 ± 4·7 years). Chromosome damage was less for children receiving hydroxycarbamide than untreated patients (0·8 ± 1·2 vs. 1·9 ± 1·5 breaks per 100 cells, P = 0·004). There were no differences in repairing chromosome breaks after in vitro radiation; PBMC from children taking hydroxycarbamide had equivalent 2 Gy-induced chromosome breaks compared to untreated patients (30·8 ± 16·1 vs. 31·7 ± 8·9 per 100 cells, P = not significant). Radiation plus hydroxycarbamide resulted in similar numbers of unrepaired breaks in cells from children on hydroxycarbamide compared to untreated patients (95·8 ± 44·2 vs. 76·1 ± 23·1 per 100 cells, P = 0·08), but no differences were noted with longer exposure (97·9 ± 42·8 breaks per 100 cells for 3-6 years of hydroxycarbamide exposure vs. 91·2 ± 48·4 for 9-12 years of exposure). These observations provide important safety data regarding long-term risks of hydroxycarbamide exposure for children with SCA, and suggest low in vivo mutagenicity and carcinogenicity.
Assuntos
Anemia Falciforme/genética , Antidrepanocíticos/efeitos adversos , Aberrações Cromossômicas/induzido quimicamente , Hidroxiureia/efeitos adversos , Adolescente , Adulto , Anemia Falciforme/tratamento farmacológico , Antidrepanocíticos/administração & dosagem , Células Cultivadas , Criança , Pré-Escolar , Estudos Transversais , Dano ao DNA , Reparo do DNA , Esquema de Medicação , Humanos , Hidroxiureia/administração & dosagem , Pessoa de Meia-IdadeRESUMO
Background: Pancreatic islets are exposed to strong pro-apoptotic stimuli: inflammation and hyperglycemia, during the progression of the autoimmune diabetes (T1D). We found that the Cdk11(Cyclin Dependent Kinase 11) is downregulated by inflammation in the T1D prone NOD (non-obese diabetic) mouse model. The aim of this study is to determine the role of CDK11 in the pathogenesis of T1D and to assess the hierarchical relationship between CDK11 and Cyclin D3 in beta cell viability, since Cyclin D3, a natural ligand for CDK11, promotes beta cell viability and fitness in front of glucose. Methods: We studied T1D pathogenesis in NOD mice hemideficient for CDK11 (N-HTZ), and, in N-HTZ deficient for Cyclin D3 (K11HTZ-D3KO), in comparison to their respective controls (N-WT and K11WT-D3KO). Moreover, we exposed pancreatic islets to either pro-inflammatory cytokines in the presence of increasing glucose concentrations, or Thapsigargin, an Endoplasmic Reticulum (ER)-stress inducing agent, and assessed apoptotic events. The expression of key ER-stress markers (Chop, Atf4 and Bip) was also determined. Results: N-HTZ mice were significantly protected against T1D, and NS-HTZ pancreatic islets exhibited an impaired sensitivity to cytokine-induced apoptosis, regardless of glucose concentration. However, thapsigargin-induced apoptosis was not altered. Furthermore, CDK11 hemideficiency did not attenuate the exacerbation of T1D caused by Cyclin D3 deficiency. Conclusions: This study is the first to report that CDK11 is repressed in T1D as a protection mechanism against inflammation-induced apoptosis and suggests that CDK11 lies upstream Cyclin D3 signaling. We unveil the CDK11/Cyclin D3 tandem as a new potential intervention target in T1D.
Assuntos
Apoptose/efeitos dos fármacos , Glicemia/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Citocinas/farmacologia , Diabetes Mellitus Tipo 1/enzimologia , Inflamação/enzimologia , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/fisiologia , Fator 4 Ativador da Transcrição/metabolismo , Animais , Autoimunidade/efeitos dos fármacos , Ciclina D3/genética , Ciclina D3/metabolismo , Quinases Ciclina-Dependentes/genética , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Inflamação/sangue , Inflamação/genética , Inflamação/patologia , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Tapsigargina/farmacologia , Técnicas de Cultura de Tecidos , Fator de Transcrição CHOP/metabolismoRESUMO
BACKGROUND: Demethylating agents may alter the expression of genes involved in chemotherapy resistance. We conducted a phase I trial to determine the toxicity and molecular effects of the demethylating agent, decitabine, followed by doxorubicin and cyclophosphamide in children with refractory solid tumors. PROCEDURE: Stratum A included children with any solid tumor; Stratum B included neuroblastoma patients only. Patients received a 1-hr decitabine infusion for 7 days, followed by doxorubicin (45 mg/m(2)) and cyclophosphamide (1 g/m(2)) on day 7. Pharmacokinetic studies were performed after the first dose of decitabine. Biological studies included methylation and gene expression analyses of caspase-8, MAGE-1 and fetal hemoglobin (HbF), and expression profiling of pre- and post-treatment peripheral blood and bone marrow cells. RESULTS: The maximum-tolerated dose of decitabine was 5 mg/m(2)/day for 7 days. Dose-limiting toxicities at 10 mg/m(2)/day were neutropenia and thrombocytopenia. Decitabine exhibited rapid clearance from plasma. Three of 9 patients in Stratum A and 4/12 patients in Stratum B had stable disease for > or = 4 months. Sustained MAGE-1 demethylation and increased HbF expression were observed in the majority of patients post-treatment (12/20 and 14/16, respectively). Caspase-8 promoter demethylation and gene expression were seen in 2/7 bone marrow samples. Differentially expressed genes were identified by microarray analysis. CONCLUSION: Low-dose decitabine when combined with doxorubicin/cyclophosphamide has tolerable toxicity in children. However, doses of decitabine capable of producing clinically relevant biologic effects were not well tolerated with this combination. Alternative strategies of combining demethylating agents with non-cytotoxic, biologically targeted agents such as histone deacetylase inhibitors should be explored.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias/tratamento farmacológico , Neuroblastoma/tratamento farmacológico , Adolescente , Adulto , Antígenos de Neoplasias/genética , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Azacitidina/administração & dosagem , Azacitidina/efeitos adversos , Azacitidina/análogos & derivados , Azacitidina/farmacocinética , Caspase 8/genética , Criança , Pré-Escolar , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Ciclofosfamida/farmacocinética , Metilação de DNA , Decitabina , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacocinética , Feminino , Hemoglobina Fetal/genética , Perfilação da Expressão Gênica , Humanos , Lactente , Masculino , Antígenos Específicos de Melanoma , Proteínas de Neoplasias/genéticaRESUMO
Caspase-8 is frequently deleted or silenced in neuroblastoma and other solid tumor such as medulloblastoma and small cell lung carcinoma. Caspase-8 expression can be re-established in neuroblastoma cell lines by treatment with demethylating agents or with IFN-gamma. Here we show that four different retinoic acid (RA) derivatives also increase caspase-8 protein expression in neuroblastoma, medulloblastoma and small cell lung carcinoma cell lines. This increase in protein expression is mirrored by an increase in RNA expression in NB cells. However, the promoter region of the caspase-8 gene was not responsible for the induction of caspase-8 expression. Rather, we identified another intronic region containing a CREB binding site that was required for maximal induction of caspase-8 via RA. DNA-protein interaction assays revealed increased phospho-CREB binding to this response element in RA-treated NB cells. Furthermore, mutations of the CREB binding site completely blocked caspase-8 induction in the luciferase reporter system assay and transfection of dominant-negative form of CREB repressed the up-regulation of caspase-8 by RA. Importantly, RA-released cells maintained caspase-8 expression for at least 2-5 days and were more sensitive to doxorubicin and TNFalpha. Thus, RA treatment in conjunction with TNFalpha and/or subsets of cytotoxic agents may have therapeutic benefits.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 8/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neuroblastoma/patologia , Transcrição Gênica , Tretinoína/farmacologia , Antibióticos Antineoplásicos/farmacologia , Apoptose/fisiologia , Western Blotting , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Caspase 8/metabolismo , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Metilação de DNA , Doxorrubicina/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Íntrons/genética , Luciferases/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/patologia , Mutação/genética , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Transdução de Sinais , Ativação Transcricional , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Although epithelial morphogenesis is tightly controlled by intrinsic genetic programs, the microenvironment in which epithelial cells proliferate and differentiate also contributes to the morphogenetic process. The roles of the physical microenvironment in epithelial morphogenesis, however, have not been well dissected. In this study, we assessed the impact of the microenvironment on epithelial cyst formation, which often marks the beginning or end step of morphogenesis of epithelial tissues and the pathological characteristic of some diseases. Previous studies have demonstrated that Madin-Darby canine kidney (MDCK) epithelial cells form cysts when grown in a three-dimensional (3D) extracellullar matrix (ECM) environment. We have now further demonstrated that the presence of ECM in the 3D scaffold is required for the formation of properly polarized cysts. Also, we have found that the full interface of epithelial cells with the ECM environment (in-3D) is not essential for cyst formation, since partial contact (on-3D) is sufficient to induce cystogenesis. In addition, we have defined the minimal ECM environment or the physical threshold for cystogenesis under the on-3D condition. Only above the threshold can the morphological cues from the ECM environment induce cyst formation. Moreover, cyst formation under the on-3D condition described in this study defines a novel and more feasible model to analyze in vitro morphogenesis. Finally, we have found that, during cystogenesis, MDCK cells generate basal microprotrusions and produce vesicle-like structures to the basal extracellular space, which are specific to and correlated with cyst formation. For the first time, we have systematically and quantitatively elucidated the microenvironmental determinants for epithelial cystogenesis.
Assuntos
Células Epiteliais/citologia , Rim/citologia , Rim/crescimento & desenvolvimento , Morfogênese , Animais , Agregação Celular , Comunicação Celular , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Polaridade Celular , Meios de Cultura , Cães , Células Epiteliais/ultraestrutura , Epitélio/crescimento & desenvolvimento , Epitélio/ultraestrutura , Matriz Extracelular , Concentração de Íons de Hidrogênio , Imageamento Tridimensional , Rim/ultraestrutura , Microscopia Confocal , Microscopia EletrônicaRESUMO
Sister-chromatid cohesion, the machinery used in eukaryote organisms to prevent aneuploidy, tethers sister chromatids together after their replication in S phase until mitosis. Previous studies in fission yeast, Drosophila and mammals have demonstrated the requirement for the heterochromatin formation pathway for proper centromeric cohesion. However, the exact role of heterochromatin protein 1 (HP1) in sister-chromatid cohesion in mammals is still unknown. In this study, we disrupted endogenous HP1 expression in HeLa cells using a dominant-negative mutant of HP1beta and wild-type or mutant forms of HP1alpha. We then examined their effects on chromosome alignment, segregation and cohesion. Enforced expression of these constructs leads to frequent chromosome misalignment and missegregation. Mitotic chromosomes from these cells also exhibit a loosened primary constriction and separated sister chromatids. We further demonstrate that alignment of the cohesin proteins around kinetochores was also aberrant and that cohesin complexes bound less tightly in these cells. Unexpectedly, we observed a "wavy" chromosome morphology resembling that seen upon depletion of condensin proteins in cells with over-expression of HP1alpha, but not in cells expressing the HP1beta mutant. These results indicate that proper HP1 status is required for sister-chromatid cohesion in mammalian cells, and suggest that HP1alpha might be required for chromosome condensation.
Assuntos
Cromátides/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Cromatina/metabolismo , Homólogo 5 da Proteína Cromobox , Segregação de Cromossomos , Humanos , Fase SRESUMO
To develop metastatic capability, tumor cells must evolve the capacity to survive in novel microenvironments. Recently, we showed that metastasis of neuroblastoma cells is enhanced by loss of caspase-8, an event that occurs frequently in this malignancy. In poorly metastatic cells, unligated integrins were found to trigger activation of caspase-8, providing a selective pressure to promote its attenuation and thereby increased survival in foreign adhesive environments. Our findings suggest one mechanism by which the organotropism of metastatic cancer cells can arise.
Assuntos
Caspases/metabolismo , Neoplasias/enzimologia , Neoplasias/patologia , Animais , Caspase 8 , Morte Celular/fisiologia , Humanos , Metástase NeoplásicaRESUMO
Significant caspase-8 activity has been found in normal and certain tumor cells, suggesting that caspase-8 possesses an alternative, nonapoptotic function that may contribute to tumor progression. In this article, we report that caspase-8 promotes cell motility. In particular, caspase-8 is required for the optimal activation of calpains, Rac, and lamellipodial assembly. This represents a novel nonapoptotic function of caspase-8 acting at the intersection of the caspase-8 and calpain proteolytic pathways to coordinate cell death versus cell motility signaling.
Assuntos
Calpaína/metabolismo , Caspases/metabolismo , Movimento Celular/fisiologia , Animais , Apoptose/fisiologia , Calpaína/deficiência , Caspase 8 , Caspases/deficiência , Linhagem Celular , Embrião de Mamíferos , Ativação Enzimática , Fibroblastos/citologia , Fibroblastos/enzimologia , Camundongos , Camundongos KnockoutRESUMO
Loss of caspase-8 expression and resistance to cytotoxic agents occurs frequently in late stage neuroblastoma (NB). Interferon-gamma (IFN-gamma) induces caspase-8 in NB cells, sensitizing them to death receptor mediated apoptosis. This study characterizes the kinetics of this phenomenon and examines the effects of IFN-gamma on global gene expression to determine whether IFN-gamma responses are achievable at physiologically relevant doses and to define the biological effects of this cytokine. Here we examine the IFN-gamma responses of 16 NB cell lines. A single <5-min exposure to IFN-gamma (0.5 ng/ml) induced caspase-8 expression in all non-expressing cell lines and in 3/6 cell lines which already expressed high caspase-8. This increase in caspase-8 proteins was observed within 16 h and persisted for up to 9 days. Furthermore, IFN-gamma pretreatment of NB cells increased doxorubicin-induced apoptosis nearly 3-fold. Microarray analysis was used to identify additional genes involved in proliferation, signaling and apoptosis whose expression was modulated via IFN-gamma. Altered expression of these genes should further enhance the responsiveness of NB cells to chemotherapeutics. Thus, the use of IFN-gamma to sensitize NB cells to cytotoxic agents represents an attractive therapeutic strategy and warrants further investigation.
Assuntos
Regulação Neoplásica da Expressão Gênica , Interferon gama/farmacologia , Neuroblastoma/metabolismo , Apoptose , Caspase 8/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Humanos , Metilação , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
The CDK11(p110) protein kinases are part of large-molecular-weight complexes that also contain RNA polymerase II, transcriptional elongation factors, and general pre-mRNA splicing factors. CDK11(p110) isoforms may therefore couple transcription and pre-mRNA splicing by their effect(s) on certain proteins required for these processes. The CDK11(p58) kinase isoform is generated from the CDK11(p110) mRNA through the use of an internal ribosome entry site in a mitosis-specific manner, suggesting that this kinase may regulate the cell cycle during mitosis. The in vivo role and necessity of CDK11(p110/p58) kinase function during mammalian development were examined by generating CDK11(p110/p58)-null mice through targeted disruption of the corresponding gene using homologous recombination. While heterozygous mice develop normally, disruption of both CDK11(p110/p58) alleles results in early embryonic lethality due to apoptosis of the blastocyst cells between 3.5 and 4 days postcoitus. Cells within these embryos exhibit both proliferative defect(s) and a mitotic arrest. These results are consistent with the proposed cellular functions of the CDK11(p110/p58) kinases and confirm that the CDK11(p110/p58) kinases are essential for cellular viability as well as normal early embryonic development.
Assuntos
Blastocisto/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Mitose/fisiologia , Animais , Apoptose/fisiologia , Blastocisto/citologia , Caspase 3 , Caspases/metabolismo , Divisão Celular/fisiologia , Tamanho Celular , Quinases Ciclina-Dependentes/genética , Viabilidade Fetal , Marcação de Genes , Marcação In Situ das Extremidades Cortadas , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos KnockoutRESUMO
The archetypal TATA-box deficient G+C-rich promoter of the murine adenosine deaminase gene (Ada) requires a 48-bp minimal self-sufficient promoter element (MSPE) for function. This MSPE was used to isolate a novel full-length cDNA clone that encodes a 66-kDa murine G+C-rich promoter binding protein (mGPBP). The mGPBP mRNAs are ubiquitously expressed as either 3.0- or 3.5-kb forms differing in 3' polyadenylation site usage. Purified recombinant mGPBP, in the absence of any other mammalian cofactors, binds specifically to both the murine Ada gene promoter's MSPE and the nonhomologous human Topo IIalpha gene's G+C-rich promoter. In situ binding assays, immunoprecipitation, and Western blot analyses demonstrated that mGPBP is a nuclear factor that can form complexes with TATA-binding protein, TFIIB, TFIIF, RNA polymerase II, and P300/CBP both in vitro and in intact cells. In cotransfection assays, increased mGPBP expression transactivated the murine Ada gene's promoter. Sequestering of GPBP present in HeLa cell nuclear extract by immunoabsorption completely and reversibly suppressed extract-dependent in vitro transcription from the murine Ada gene's G+C-rich promoter. However, transcription from the human Topo IIalpha gene's TATA box-containing G+C-rich promoter was only partially suppressed and the adenovirus major late gene's classical TATA box-dependent promoter is totally unaffected under identical assay conditions. These results implicate GPBP as a requisite G+C-rich promoter-specific transcription factor and provide a mechanistic basis for distinguishing transcription initiated at a TATA box-deficient G+C-rich promoter from that initiated at a TATA box-dependent promoter.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/genética , DNA/metabolismo , Regiões Promotoras Genéticas , Adenosina Desaminase/genética , Animais , Antígenos de Neoplasias , Composição de Bases , Sequência de Bases , Clonagem Molecular , DNA/química , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fases de Leitura Aberta , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TATA Box , Transcrição GênicaRESUMO
Different isoforms of a protein complex termed the apoptosis- and splicing-associated protein (ASAP) were isolated from HeLa cell extract. ASAP complexes are composed of the polypeptides SAP18 and RNPS1 and different isoforms of the Acinus protein. While Acinus had previously been implicated in apoptosis and was recently identified as a component of the spliceosome, RNPS1 has been described as a general activator of RNA processing. Addition of ASAP isoforms to in vitro splicing reactions inhibits RNA processing mediated by ASF/SF2, by SC35, or by RNPS1. Additionally, microinjection of ASAP complexes into mammalian cells resulted in acceleration of cell death. Importantly, after induction of apoptosis the ASAP complex disassembles. Taken together, our results suggest an important role for the ASAP complexes in linking RNA processing and apoptosis.
Assuntos
Apoptose/fisiologia , Proteínas de Transporte , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas Correpressoras , Células HeLa , Humanos , Técnicas In Vitro , Substâncias Macromoleculares , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Cyclin C is a highly conserved protein that is involved in divergent cellular processes. The exact roles of its isoforms are presently not very well defined and it is possible that there is a functional divergence amongst them. We therefore sought to assess the expression pattern of cyclin C1 and C2 isoforms in various human tissues and in cell cycle by using real-time PCR experiments. Our findings strongly suggest that the C2 isoform may play a presently unexplored and important role in mammalian testis and probably this isoform is the one that is mainly implicated in cell cycle regulation.
Assuntos
Ciclinas/genética , Regulação da Expressão Gênica , Animais , Adesão Celular , Ciclo Celular , Sequência Conservada , Ciclina C , Ciclinas/fisiologia , Humanos , Masculino , Mamíferos , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Testículo/citologiaRESUMO
The production of mRNAs in all living organisms is an extremely complex process that includes multiple catalytic activities such as transcription, capping, splicing, polyadenylation, cleavage and export. All of these processes are controlled by a large group of proteins which form very dynamic complexes interacting with DNA and pre-mRNAs to coordinate these activities. Phosphorylations play a central role in regulating formation, activation and inactivation of these complexes. A growing number of protein kinases have been identified that are capable of phosphorylating proteins involved in mRNA production. Among them, Cyclin-dependent Kinases (CDKs) represent a family of serine/threonine protein kinases that become active upon binding to a cyclin regulatory partner. CDK/cyclin complexes were first identified as crucial regulators of cell cycle progression. More recently, CDK/cyclin complexes have also been implicated in transcription and mRNA processing leading to the concept of an intricate network of CDK/cyclin complexes regulating cell cycle, transcription and mRNA processing via cross-talk between multiple CDKs. In this review, we discuss the role of CDK/cyclin-dependent phosphorylation in the regulation of transcription and RNA splicing and highlight recent findings that indicate the involvement of CDK/cyclin complexes in connecting transcription and RNA splicing.
Assuntos
Quinases Ciclina-Dependentes/fisiologia , Ciclinas/fisiologia , Splicing de RNA , Transcrição Gênica , Sequência de Aminoácidos , Animais , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Regulação da Expressão Gênica , Dados de Sequência MolecularRESUMO
Important roles have been suggested for caspase-8, caspase-9 and Apaf-1 in controlling tumor development and their sensitivity to chemotherapeutic agents. Methylation and deletion of Apaf-1 and CASP8 results in the loss of their expression in melanoma and neuroblastoma, respectively, while CASP9 localization to 1p36.1 suggests it is a good candidate tumor suppressor. The status of CASP9 and Apaf-1 expression in numerous neuroblastoma cell lines with/without amplified MYCN and chromosome 1p36 loss-of-heterozygosity (LOH) was therefore examined to test the hypothesis that one or both of these genes are tumor suppressors in neuroblastoma. Although CASP9 is included in the region encompassing 1p36 LOH in all neuroblastoma cell lines examined, the remaining CASP9 allele(s) express a functional caspase-9 enzyme. Apaf-1 is also expressed in all neuroblastoma tumor cell lines examined. Thus, the CASP9 or Apaf-1 genes do not appear to function as tumor suppressors in MYCN amplified neuroblastomas. However, approximately 20% of the neuroblastoma cell lines with methylated CASP8 alleles are also highly resistant to staurosporine (STS)- and radiation-induced cell death, presumably because cytochrome c is not released from mitochondria. This suggests that a second, smaller sub-group of MYCN amplified neuroblastoma tumors exists with defect(s) in apoptotic signaling components upstream of caspase-9 and Apaf-1. Since no consistent differences in Bcl-2, Bcl-x(L) or Bax expression were seen in the STS- and radiation-resistant neuroblastomas, it suggests that a unique mitochondrial signaling factor(s) is responsible for the defect in cytochrome c release in this sub-group of tumors.