RESUMO
mcl-1 is an immediate-early gene activated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) signaling pathways and plays an important role in the viability response of these cytokines. In this study, we demonstrated that cytokine stimulation of mcl-1 mRNA and protein expression were attenuated by pretreatment of cells with phosphatidylinositol 3-kinase (PI3-K) inhibitors. Reporter gene assays further showed that the PI3-K/Akt signaling pathway was involved in IL-3 activation of mcl-1 gene transcription. Analysis of the mcl-1 promoter revealed that both promoter elements, SIE at position -87 and CRE-2 at -70, contribute to IL-3 stimulation of mcl-1 gene expression. Although either the SIE site or the CRE-2 site alone was sufficient to confer IL-3 inducibility on a heterologous promoter, only IL-3 activation of the CRE-2 reporter was mediated via the PI3-K/Akt pathway. The SIE binding activity was constitutively high in cells deprived of or stimulated by IL-3. In contrast, the CRE-2 binding activity was low in cytokine-starved cells and was strongly induced within 1 h following cytokine treatment of cells. In addition, cytokine induction of the CRE-2 but not of the SIE binding activity was dependent on activation of the PI3-K/Akt signaling pathway. Lastly, we showed that CREB was one component of the CRE-2 binding complex and played a role in IL-3 activation of the mcl-1 reporter gene. Taken together, our results suggest that both PI3-K/Akt-dependent and -independent pathways contribute to the IL-3 activation of mcl-1 gene expression. Activation of mcl-1 by the PI3-K/Akt-dependent pathway is through a transcription factor complex containing CREB.
Assuntos
Apoptose/genética , Genes Precoces , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-bcl-2 , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Primers do DNA/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-3/farmacologia , Substâncias Macromoleculares , Dados de Sequência Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
Growth factors and cytokines play an important role in supporting cellular viability of various tissues during development due to their ability to suppress the default cell death program in each cell type. To date, neither the triggering molecule nor the transduction pathway of these default apoptosis programs is understood. In this study, we explored the possibility that cytokine receptors are involved in modulating cytokine withdrawal-induced apoptosis (CWIA) in hematopoietic cells. Expression of the exogenous cytokine receptor common beta chain (betac), but not the alpha chains, accelerated CWIA in multiple cytokine-dependent cell lines. Reduction of the expression level of endogenous betac by antisense transcripts resulted in prolonged survival during cytokine deprivation, suggesting a critical role of betac in modulating CWIA. Fine mapping of the betac subunit revealed that a membrane-proximal cytoplasmic sequence, designated the death enhancement region (DER), was critical to the death acceleration effect of betac. Furthermore, DER accelerated cell death either as a chimeric membrane protein or as a cytosolic protein, suggesting that DER functions independently of the cytokine receptor and membrane anchorage. Cross-linking of the chimeric membrane-bound DER molecules by antibody or of the FK506-binding protein-DER fusion protein by a synthetic dimerizing agent, AP1510, did not abrogate the death acceleration effect. Transient transfection assays further indicated that DER promoted cell death in the absence of serum in the nonhematopoietic 293 cell line. In summary, our data suggest that betac plays an important role in modulating CWIA via an anchorage-independent and aggregation-insensitive mechanism. These findings may facilitate further studies on the signaling pathways of CWIA.
Assuntos
Apoptose , Citocinas/deficiência , Células-Tronco Hematopoéticas/fisiologia , Receptores de Citocinas/metabolismo , Elementos Antissenso (Genética) , Sobrevivência Celular , Células Cultivadas , Reagentes de Ligações Cruzadas/farmacologia , Meios de Cultura , Dimerização , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/deficiência , Humanos , Interleucina-3/deficiência , Interleucina-5/deficiência , Receptores de Citocinas/genética , Tacrolimo/análogos & derivados , Tacrolimo/farmacologiaRESUMO
Ectopic overexpression of v-H-Ras protein in NIH 3T3 cells resulted in cellular transformation and an acceleration of G1 progression of these cells. A shortened G1 phase was found to be associated with an increased level of cyclin D1 but not cyclin E protein. Using an antisense blocking method, reduced synthesis of cyclin D1 in v-H-Ras transformants resulted in a slower G1 progression rate of these cells. Although constitutive overexpression of cyclin D1 in NIH 3T3 cells accelerated G1 progression, cells remained untransformed. Furthermore, inhibition of cyclin D1 synthesis greatly impaired the soft-agar cloning efficiency of v-H-Ras transformants. These results suggest that increased expression of cyclin D1 is necessary but not sufficient for the transforming activity of v-H-Ras. Similar effect on cell cycle progression was also observed in Raf-transformed cells. In addition to cyclin D1, cyclin E protein was found to be elevated in Src transformants. This may account for the further shortening of the G1 phase of these cells. Activation of an additional Ras-independent pathway was suggested to be responsible for the further acceleration of the G1 phase in Src transformants.
Assuntos
Transformação Celular Neoplásica , Ciclinas/biossíntese , Interfase/fisiologia , Proteínas Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Células 3T3 , Animais , Northern Blotting , Células Clonais , Ciclina D1 , Ciclinas/genética , Citometria de Fluxo , Fase G1/fisiologia , Immunoblotting , Camundongos , Proteína Oncogênica pp60(v-src)/biossíntese , Proteína Oncogênica pp60(v-src)/genética , Proteínas Oncogênicas/genética , Proteínas Oncogênicas v-raf , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Mensageiro/análise , Proteínas Oncogênicas de Retroviridae/biossíntese , Proteínas Oncogênicas de Retroviridae/genética , Fase S/fisiologia , Fatores de Tempo , Transformação GenéticaRESUMO
The receptors for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) share a common beta subunit, the distal cytoplasmic domain of which is essential for the promotion of cell survival by these two cytokines. Genes whose expression is specifically induced by signaling through the distal cytoplasmic domain of this receptor beta subunit were screened by a subtraction cloning approach in derivatives of a mouse pro-B-cell line. One gene thus identified was shown to encode a protein highly homologous (with only 7 amino acid substitutions) to murine osteopontin (OPN), a secreted adhesion protein. Conditioned medium from cells expressing wild-type OPN, but not that from cells expressing a deletion mutant lacking residues 79 to 140, increased the viability of a non-OPN-producing cell line in the presence of human GM-CSF. Antibody blocking experiments revealed that OPN produced as a result of IL-3 or GM-CSF signaling was secreted into the medium and, through binding to its cell surface receptor, CD44, contributed to the survival-promoting activities of these two cytokines. Furthermore, coupling of the OPN-CD44 pathway to the survival response to IL-3 was also demonstrated in primary IL-3-dependent mouse bone marrow cells. These results thus show that induction of an extracellular adhesion protein and consequent activation of its cell surface receptor are important for the antiapoptotic activities of IL-3 and GM-CSF.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Receptores de Hialuronatos/fisiologia , Interleucina-3/farmacologia , Sialoglicoproteínas/fisiologia , Transdução de Sinais/fisiologia , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cricetinae , Humanos , Camundongos , Osteopontina , Transdução de Sinais/efeitos dos fármacosRESUMO
The Ces-2/E2A-HLF binding element (CBE) is recognized by Caenorhabditis elegans death specification gene product Ces-2 and human acute lymphocytic leukemia oncoprotein E2A-HLF. In an attempt to identify a cellular CBE-binding protein(s) that may be involved in apoptosis regulation in mammals, multiple nuclear binding complexes of CBE were identified in various mammalian cell lines and tissues by electrophoretic mobility shift assay. Cyclic AMP (cAMP)-responsive element (CRE)-binding protein (CREB) was present in one major CBE complex of Ba/F3 and TF-1 cells, and both in vitro-translated and Escherichia coli-synthesized CREB bound to CBE. Activation of CREB by cAMP-elevating chemicals or the catalytic subunit of protein kinase A (PKAc) resulted in induction of the CBE-driven reporter gene. Stimulation of Ba/F3 cells with interleukin-3 (IL-3) promptly induced phosphorylation of CREB at serine(133) partially via a PKA-dependent pathway. Consistently, Ba/F3 cell survival in the absence of IL-3 was prolonged by activation of PKA. Conversely, treatment of cells with a PKA inhibitor or expression of the dominant negative forms of the regulatory subunit type I of PKA and CREB overrode the survival activity of IL-3. Last, the bcl-2 gene was demonstrated to be one candidate cellular target of the CREB-containing CBE complex, as mutations in the CRE and CBE sites significantly reduced the IL-3 inducibility of the bcl-2 promoter. Together, our results suggest that CREB is one cellular counterpart of Ces-2/E2A-HLF and is part of IL-3 dependent apoptosis regulation in hematopoietic cells.
Assuntos
Apoptose , Proteínas de Caenorhabditis elegans , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Helminto/metabolismo , Interleucina-3/metabolismo , Zíper de Leucina , Proteínas de Fusão Oncogênica/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica , Caenorhabditis elegans , Linhagem Celular , Sobrevivência Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Genes Reporter , Proteínas de Helminto/genética , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Proteínas de Fusão Oncogênica/genética , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fatores de Transcrição/genéticaRESUMO
mcl-1, a bcl-2 family member, was originally identified as an early gene induced during differentiation of ML-1 myeloid leukemia cells. In the present study, we demonstrate that Mcl-1 is tightly regulated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling pathway. Upon deprivation of survival factor from TF-1 myeloid progenitor cells, Mcl-1 levels quickly dropped prior to visible detection of apoptosis of these cells. Upon restimulation of these deprived cells with GM-CSF, the mcl-1 mRNA was immediately induced and its protein product was accordingly resynthesized. Analysis with Ba/F3 cells expressing various truncation mutants of the GM-CSF receptor revealed that the membrane distal region between amino acids 573 and 755 of the receptor beta chain was required for mcl-1 induction. Transient-transfection assays with luciferase reporter genes driven by various regions of the mcl-1 promoter demonstrated that the upstream sequence between -197 and -69 is responsible for cytokine activation of the mcl-1 gene. Overexpression of mcl-1 delayed but did not completely prevent apoptosis of cells triggered by cytokine withdrawal. Its down regulation by antisense constructs overcame, at least partially, the survival activity of GM-CSF and induced the apoptosis of TF-1 cells. Taken together, these results suggest that mcl-1 is an immediate-early gene activated by the cytokine receptor signaling pathway and is one component of the GM-CSF viability response.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Transdução de Sinais , Ativação Transcricional , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA Complementar , Regulação da Expressão Gênica , Humanos , Interleucina-3/farmacologia , Dados de Sequência Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Células Tumorais CultivadasRESUMO
Apoptosis of HepG2 cells triggered by various agents is characterized in an attempt to delineate the common apoptosis signaling pathway in human hepatoma cells. Several hallmarks of apoptosis, including DNA laddering, chromatin condensation and fragmentation, and an apoptosis specific cleavage of 28S and 18S ribosomal RNA were observed after treatment with curcumin. Curcumin treatment however did not alter the expression levels of Bcl-2 and Bax proteins. p53 protein accumulated slowly and decreased abruptly after reaching the maximum. Conversely, c-Myc protein decreased initially and subsequently increased preceding the onset of apoptosis. The accumulation of p53 protein is not due to increased levels of p53 mRNA and does not result in growth arrest. Staurosporine, quinacrine, ultraviolet irradiation, hydrogen peroxide, and cyclohexamide are all capable of triggering apoptosis in HepG2 cells. While most of these agents affect the expression levels of p53 and c-Myc similarly, none of them altered the expression levels of the Bcl-2 and Bax proteins. In conclusion, these data suggest that p53 and c-Myc may play a more important role in the apoptosis signaling pathway in HepG2 cells, than the bcl-2 gene family.
Assuntos
Apoptose/fisiologia , Hepatoblastoma/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/fisiologia , Curcumina/farmacologia , Regulação Neoplásica da Expressão Gênica , Hepatoblastoma/genética , Hepatoblastoma/patologia , Humanos , Neoplasias Hepáticas/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2RESUMO
Human hematopoietic progenitor cells (TF-1) undergo apoptosis upon deprivation of their dependent cytokine. In this report, we have isolated and characterized some spontaneously derived cytokine-independent variants from TF-1 cells. Analysis of several signaling molecules known to be activated by the GM-CSF pathway revealed that two non-autocrine variants were still responsive to GM-CSF stimulation. However, both variants, without ligand stimulation, already had some activated forms of Raf and MAP kinases. Given current knowledge, the activated Raf/MAP kinase pathway was likely to be responsible for the survival of both variants in the cytokine-free medium. However, the growth of hybrids between wild type and either variant was unexpectedly dependent on GM-CSF. Both variants like the wild type cells were still susceptible to apoptosis induced by other stimuli. These results suggest that either the activated Raf/MAP kinase pathway in both variants is not sufficient to repress the 'two-fold' death signals generated from the hybrids or that there is another mechanism that is responsible for the factor-independent growth of both variants.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais/fisiologia , Apoptose/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Ativação Enzimática , Fator Estimulador de Colônias de Granulócitos e Macrófagos/deficiência , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Interleucina-3/deficiência , Interleucina-3/farmacologia , Interleucina-3/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-rafRESUMO
T-cell receptor (TCR)-transduced signaling is critical to thymocyte development at the CD4/CD8 double-positive stage, but the molecules involved in this process are not yet fully characterized. We previously demonstrated that GM-CSF/IL-3/IL-5 receptor common ß-chain-associated protein (CBAP) modulates ZAP70-mediated T-cell migration and adhesion. On the basis of the high expression of CBAP during thymocyte development, we investigated the function of CBAP in thymocyte development using a CBAP knockout mouse. CBAP-deficient mice showed normal early thymocyte development and positive selection. In contrast, several negative selection models (including TCR transgene, superantigen staphylococcal enterotoxin B, and anti-CD3 antibody treatment) revealed an attenuation of TCR-induced thymocyte deletion in CBAP knockout mice. This phenotype correlated with a reduced accumulation of BIM upon TCR crosslinking in CBAP-deficient thymocytes. Loss of CBAP led to reduced TCR-induced phosphorylation of proteins involved in both proximal and distal signaling events, including ZAP70, LAT, PLCγ1, and JNK1/2. Moreover, TCR-induced association of LAT signalosome components was reduced in CBAP-deficient thymocytes. Our data demonstrate that CBAP is a novel component in the TCR signaling pathway and modulates thymocyte apoptosis during negative selection.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética , Timócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Adesão Celular , Diferenciação Celular , Movimento Celular , Feminino , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Timócitos/citologia , Timo/citologia , Timo/crescimento & desenvolvimento , Timo/metabolismo , Proteína-Tirosina Quinase ZAP-70/genética , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
The cytoplasmic domain of the common beta-chain (betac) of the granulocyte-macrophage-colony-stimulating factor (GM-CSF)/interleukin-3 (IL-3)/IL-5 receptor contains a membrane proximal region that is sufficient to mediate ligand-dependent mitogenic activity. Within this region two motifs, designated as box 1 and box 2, are highly conserved among members of the cytokine receptor superfamily. Whereas box 1 is required for the recruitment and phosphorylation of Janus kinase-2, the function of box 2 remains largely unknown. Here, we report the identification of a novel transmembrane protein (common beta-chain associated protein (CBAP)) which directly associated with betac via the box 2 motif. Interestingly, such an association only occurred in the absence of GM-CSF in vivo. Ectopic overexpression of CBAP triggered apoptosis of factor-dependent cells via mitochondrial dysfunction, which could be inhibited by Bcl-2 overexpression. Reduced expression of endogenous CBAP by small interfering RNA did not interfere GM-CSF-activated signaling molecules, but such treatment significantly inhibited apoptosis induced by GM-CSF deprivation, but not other death stimuli. Domain mapping studies indicated that one apoptogenic domain of CBAP correlated with its ability to interact with betac. Taken together, these results suggest that CBAP modulates GM-CSF-deprivation-induced apoptosis possibly via a novel mechanism involving interaction with an un-liganded betac molecule.
Assuntos
Apoptose/fisiologia , Subunidade beta Comum dos Receptores de Citocinas/fisiologia , Interleucina-3/metabolismo , Mitocôndrias/fisiologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Interleucina-3/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Subunidade beta Comum dos Receptores de Citocinas/deficiência , Subunidade beta Comum dos Receptores de Citocinas/genética , Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Ligantes , Proteínas de Membrana/fisiologia , Mitocôndrias/patologia , Estrutura Terciária de Proteína/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Stem cell factor (SCF) has been suggested as essential for optimal production of various hematopoietic lineages mainly because of its apoptosis prevention function when it costimulates with other cytokines. However, the underlying mechanism of this synergism of apoptosis prevention is largely unknown. The present study examined the expression of some Bcl-2 family members, including Bcl-2, Bcl-X(L), Mcl-1, and Bax, in response to cytokine stimulation in TF-1 and JYTF-1 cells in which SCF costimulation is differentially required for optimal proliferation. The results revealed that only the expression of Mcl-1 highly correlated with the antiapoptotic activity of interleukin-5 (IL-5) and the synergistic effect of SCF. In TF-1 cells, the defect of IL-5 in apoptosis suppression and Mcl-1 induction was associated with the incapability to highly phosphorylate Janus kinases (JAK1, JAK2), signal transducer and activator of transcription-5 (STAT5), mitogen-activated protein kinase (MAPK), and Akt/PKB, whereas SCF costimulation restored the potent phosphorylation of MAPK and Akt/PKB, but not STAT5. The importance of MAPK and Akt/PKB signaling pathways in regulating the expression of Mcl-1 and cell survival was further supported by the observation that inhibition of MEK by PD98059 or phosphatidylinositol-3 kinase (PI-3K) by LY294002 independently resulted in the reduction of Mcl-1 expression and loss of cell viability. Therefore, the data suggest that Mcl-1 is a common antiapoptotic target of both early-stage cytokine SCF and late-stage cytokine IL-5. Both MEK/MAPK and PI-3K/Akt signaling pathways are essential in the regulation of Mcl-1 expression and apoptosis prevention. (Blood. 2000;96:1764-1771)
Assuntos
Apoptose/efeitos dos fármacos , Interleucina-5/farmacologia , Proteínas de Neoplasias/efeitos dos fármacos , Fosfotransferases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-bcl-2 , Transdução de Sinais/efeitos dos fármacos , Fator de Células-Tronco/farmacologia , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Cromonas/farmacologia , Citocinas/farmacologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Morfolinas/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Células Tumorais CultivadasRESUMO
Restriction mapping is one of the essential steps in gene analysis and molecular biology studies. Slab gel electrophoresis is the traditional way to separate DNA fragments for restriction mapping. However, slab gel electrophoresis does not provide sufficient resolution as required in many mapping applications, and the use of radioisotopes in traditional mapping methods creates health hazards. In the present study, capillary electrophoresis coupled with laser-induced fluorescence detection and a modified partial digestion mapping procedure was developed to map DNA fragments. By using capillary electrophoresis, a restriction map of genomic lambda phage clone of human interleukin 5 receptor alpha chain (IL5R alpha) gene was constructed. The IL5R alpha gene was analyzed to have five XbaI enzyme cutting sites at locations 1370, 2290, 2950, 5430, and 9330. The system was further characterized by using pBluescript SK(+) phagemid DNA as a model. Using a sequence-derived map as a reference, the pBluescript SK(+) restriction map constructed by capillary electrophoresis had an accuracy greater than 90%.
Assuntos
DNA/química , Eletroforese Capilar/métodos , Mapeamento por Restrição , Humanos , Lasers , Receptores de Interleucina/genética , Receptores de Interleucina-5 , Espectrometria de FluorescênciaRESUMO
Curcumin, which is a widely used dietary pigment and spice, has been demonstrated to be an effective inhibitor of tumor promotion in mouse skin carcinogenesis. We report that curcumin induces cell shrinkage, chromatin condensation, and DNA fragmentation, characteristics of apoptosis, in immortalized mouse embryo fibroblast NIH 3T3 erb B2 oncogene-transformed NIH 3T3, mouse sarcoma S180, human colon cancer cell HT-29, human kidney cancer cell 293, and human hepatocellular carcinoma Hep G2 cells, but not in primary culture of mouse embryonic fibroblast C3H 10T1/2, rat embryonic fibroblast, and human foreskin fibroblast cells in a concentration- and time-dependent manner. Many cellular and biochemical effects of curcumin in mouse fibroblast cells have been reported, such as inhibition of protein kinase C (PKC) activity induced by phorbol 12-myristate 13-acetate treatment, inhibition of tyrosine protein kinase activity, and inhibition of arachidonic acid (AA) metabolism. Treatment of NIH 3T3 cells with the PKC inhibitor staurosporine, the tyrosine kinase inhibitor herbimycin A, and the AA metabolism inhibitor quinacrine induces apoptotic cell death. These results suggest that, in some immortalized and transformed cells, blocking the cellular signal transduction might trigger the induction of apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Curcumina/farmacologia , Células 3T3 , Animais , Carcinoma Hepatocelular/patologia , Cromatina/efeitos dos fármacos , Cromatina/ultraestrutura , Neoplasias do Colo/patologia , Fragmentação do DNA/efeitos dos fármacos , Humanos , Neoplasias Renais/patologia , Neoplasias Hepáticas/patologia , Camundongos , Ratos , Sarcoma Experimental/patologia , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
In vitro proliferation of hematopoietic stem cells requires costimulation by multiple regulatory factors whereas expansion of lineage-committed progenitor cells generated by stem cells usually requires only a single factor. The distinct requirement of factors for proliferation coincides with the differential temporal expression of the subunits of cytokine receptors during early stem cell differentiation. In this study, we explored the underlying mechanism of the requirement of costimulation in a hematopoietic progenitor cell line TF-1. We found that granulocyte-macrophage colony-stimulating factor (GM-CSF) optimally activated proliferation of TF-1 cells regardless of the presence or absence of stem cell factor (SCF). However, interleukin-5 (IL-5) alone sustained survival of TF-1 cells and required costimulation of SCF for optimal proliferation. The synergistic effect of SCF was partly due to its anti-apoptosis activity. Overexpression of the IL-5 receptor alpha subunit (IL5Ralpha) in TF-1 cells by genetic selection or retroviral infection also resumed optimal proliferation due to correction of the defect in apoptosis suppression. Exogenous expression of an oncogenic anti-apoptosis protein, Bcl-2, conferred on TF-1 cells an IL-5-dependent phenotype. In summary, our data suggested SCF costimulation is only necessary when the expression level of IL5Ralpha is low and apoptosis suppression is defective in the signal transduction of IL-5. Expression of Bcl-2 proteins released the growth restriction of the progenitor cells and may be implicated in leukemia formation.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-kit/fisiologia , Fator de Células-Tronco/farmacologia , Diferenciação Celular , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Interleucina-5/farmacologia , Cinética , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fator de Células-Tronco/fisiologia , TransfecçãoRESUMO
We have established a human IL-5 (hIL-5) dependent cell line, JYTF-1, derived from TF-1 parental human erythroleukemic cells by long-term cultivation in the presence of hIL-5. The ED50 values of hIL-5 for both TF-1 and JYTF-1 cell lines remained similar. However, when cells were grown in an optimal concentration of IL-5, some TF-1 cells but not JYTF-1 cells died via apoptosis. Although the rates of DNA synthesis were similar for TF-1 and JYTF-1 cells grown in IL-5, [3-H]thymidine releasing of pulse-labeled DNA analysis indicated that the viable TF-1 cells in IL-5 were more apoptosis-prone than were JYTF-1 cells. Therefore, in the JYTF-1 variant, the ability to suppress apoptosis has apparently been restored. The following findings suggest that overexpression of the hIL-5 receptor alpha-chain may be responsible for restoring the apoptosis suppression ability of IL-5: 1) the growth of JYTF-1 cells remained cytokine-dependent; 2) the proliferation of JYTF-1 cells in IL-5 was not mediated by autocrine secretion; 3) JYTF-1 and TF-1 cells responded similarly to other cytokines such as human erythropoietin; 4) Northern blot analysis revealed that JYTF-1 cells expressed approximately eightfold more IL-5 receptor alpha-chain mRNA than did TF-1. To our knowledge, JYTF-1 represents the first example in which coupling of mitogenesis stimulation and apoptosis suppression from otherwise uncoupled parental cells confers a phenotype of IL-5-dependent growth.