RESUMO
SPIN90 regulates actin dynamics, which is important for cell migration control. CXCL13-mediated B cell migration is essential for B cell immune responses. In this study, we investigated the role of SPIN90 in CXCL13-mediated B cell migration using Spin90 gene-deficient mice. Our chemokinesis analysis and transwell cell migration assay showed that SPIN90 is involved in CXCL13-mediated B cell migration. Moreover, the level of CXCR5, which is CXCL13 receptor, was increased in SPIN90-deficient B cells compared with wild-type B cells. Overall, our data suggest that SPIN90 plays an important role in B cell immune responses through the regulation of CXCL13-mediated B cell migration.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Linfócitos B/imunologia , Movimento Celular/imunologia , Quimiocina CXCL13/imunologia , Proteínas do Tecido Nervoso/imunologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linfócitos B/metabolismo , Movimento Celular/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Quimiocina CXCL13/genética , Quimiocina CXCL13/metabolismo , Citometria de Fluxo , Expressão Gênica/imunologia , Immunoblotting , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Receptores CXCR5/genética , Receptores CXCR5/imunologia , Receptores CXCR5/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Mast cell-mediated allergic inflammation is involved in many diseases such as asthma, sinusitis, and rheumatoid arthritis. Mast cells induce synthesis and production of pro-inflammatory cytokines including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 with immune regulatory properties. The formulated ethanol extract of Artemisia asiatica Nakai (DA-9601) has been reported to have antioxidative and anti-inflammatory activities. In this report, we investigated the effect of DA-9601 on the expression of pro-inflammatory cytokines by the activated human mast cell line HMC-1 and studied its possible mechanisms of action. DA-9601 dose-dependently decreased the gene expression and production of TNF-alpha, IL-1beta, and IL-6 on phorbol 12-myristate 13-acetate (PMA)- and calcium ionophore A23187-stimulated HMC-1 cells. In addition, DA-9601 attenuated PMA- and A23187-induced activation of NF-kappaB as indicated by inhibition of degradation of IkappaBalpha, nuclear translocation of NF-kappaB, NF-kappaB/DNA binding, and NF-kappaB-dependent gene reporter assay. Our in vitro studies provide evidence that DA-9601 might contribute to the treatment of mast cell-derived allergic inflammatory diseases.
Assuntos
Mastócitos/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Extratos Vegetais/farmacologia , Artemisia , Calcimicina/farmacologia , Linhagem Celular , Citocinas/biossíntese , Citocinas/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Mastócitos/fisiologia , NF-kappa B/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
BACKGROUND: Nitric oxide (NO) is known to act cytostatically on several tumor cell when functioning as an effector molecule of activated macrophages, but the differential effects of NO on immortalized and malignant oral keratinocytes have not been examined. METHODS: We investigated the influence of NO on the proliferation, cell cycle, apoptosis, and differentiation of immortalized human oral keratinocytes (IHOK) and primary oral cancer cells (HN4) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, sulforhodamine B (SRB) assay, flow cytometry, nuclear DNA staining, and Western blotting. RESULTS: The MTT and SRB assays indicated inhibited growth of IHOK and HN4 cells that were treated with sodium nitroprusside (SNP) at concentrations higher than 1 mM but not at lower SNP concentrations. The higher concentrations of SNP up-regulated the apoptosis-related protein expression, which is consistent with the analyses of sub-G(1) phase arrest, annexin V-FITC (fluorescein isothiocynate) staining, nuclear staining, and DNA fragmentation. On the other hand, the lower concentrations of SNP enhanced the expression of keratinocyte differentiation markers in IHOK and HN4 cells. CONCLUSIONS: These data suggest that high concentrations of NO can inhibit the growth of IHOK and HN4 cells through the induction of apoptosis, while low concentrations of NO can induce cytodifferentiation. The dual effects of NO, namely, the induction of apoptosis or cytodifferentiation, have important implications for the possible anti-oral cancer treatment.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/metabolismo , Queratinócitos/efeitos dos fármacos , Neoplasias Bucais/metabolismo , Óxido Nítrico/farmacologia , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Transformada , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Humanos , Queratinócitos/citologia , Mucosa Bucal/citologia , Óxido Nítrico/fisiologiaRESUMO
Several methods have been developed for the immortalization of B lymphocytes by Epstein-Barr virus (EBV). We developed an efficient method which reduces the time from culture initiation to immortalization and cryopreservation. Two infections of EBV to lymphocytes, and the use of phorbol ester-induced EBV stock significantly improved immortalization efficiency and reduced the time between initiation and immortalization and cryopreservation. The resulting cell bank was used to produce DNA for genetic studies focusing on the genes involved in immune and autistic disorders.
Assuntos
Linfócitos B/citologia , Transformação Celular Viral , Herpesvirus Humano 4/fisiologia , Adulto , Animais , Transtorno Autístico/imunologia , Linfócitos B/virologia , Linhagem Celular , Criança , Criopreservação , Humanos , Imunofenotipagem , Doenças Inflamatórias Intestinais/imunologia , Fatores de TempoRESUMO
Previous studies have demonstrated dimerization of intercellular adhesion molecule-1 (ICAM-1) on the cell surface and suggested a role for immunoglobulin superfamily domain 5 and/or the transmembrane domain in mediating such dimerization. Crystallization studies suggest that domain 1 may also mediate dimerization. ICAM-1 binds through domain 1 to the I domain of the integrin alpha(L)beta(2) (lymphocyte function-associated antigen 1). Soluble C-terminally dimerized ICAM-1 was made by replacing the transmembrane and cytoplasmic domains with an alpha-helical coiled coil. Electron microscopy revealed C-terminal dimers that were straight, slightly bent, and sometimes U-shaped. A small number of apparently closed ring-like dimers and W-shaped tetramers were found. To capture ICAM-1 dimerized at the crystallographically defined dimer interface in domain 1, cysteines were introduced into this interface. Several of these mutations resulted in the formation of soluble disulfide-bonded ICAM-1 dimers (domain 1 dimers). Combining a domain 1 cysteine mutation with the C-terminal dimers (domain 1/C-terminal dimers) resulted in significant amounts of both closed ring-like dimers and W-shaped tetramers. Surface plasmon resonance studies showed that all of the dimeric forms of ICAM-1 (domain 1, C-terminal, and domain 1/C-terminal dimers) bound similarly to the integrin alpha(L)beta(2) I domain, with affinities approximately 1.5--3-fold greater than that of monomeric ICAM-1. These studies demonstrate that ICAM-1 can form at least three different topologies and that dimerization at domain 1 does not interfere with binding in domain 1 to alpha(L)beta(2).
Assuntos
Molécula 1 de Adesão Intercelular/química , Molécula 1 de Adesão Intercelular/ultraestrutura , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetinae , Cristalografia por Raios X , Cisteína , DNA Complementar , Dimerização , Humanos , Molécula 1 de Adesão Intercelular/genética , Antígeno-1 Associado à Função Linfocitária/química , Antígeno-1 Associado à Função Linfocitária/fisiologia , Microscopia Eletrônica , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestrutura , Ressonância de Plasmônio de Superfície , Propriedades de Superfície , TransfecçãoRESUMO
Dimeric intercellular adhesion molecule-1 (ICAM-1) binds more efficiently to lymphocyte function-associated antigen-1 (LFA-1) than monomeric ICAM-1. However, it is unknown whether dimerization enhances binding simply by providing two ligand-binding sites and thereby increasing avidity, or whether it serves to generate a single "fully competent" LFA-1-binding surface. Domain 1 of ICAM-1 contains both the binding site for LFA-1, centered on residue E34, and a homodimerization interface. Whether the LFA-1-binding site extends across the homodimerization interface has not been tested. To address this question, we constructed four different heterodimeric soluble forms of ICAM-1 joined at the C terminus via an alpha-helical coiled coil (ACID-BASE). These heterodimeric ICAM-1 constructs include, (i) E34/E34 (two intact LFA-1-binding sites), (ii) E34/K34 (one disrupted LFA-1-binding site), (iii) E34/DeltaD1-2 (one deleted LFA-1-binding site), and (iv) K34/K34 (two disrupted LFA-1-binding sites). Cells bearing activated LFA-1 bound similarly to surfaces coated with either E34/K34 or E34/DeltaD1-2 and with an approximately 2-fold reduction in efficiency compared with E34/E34, suggesting that D1 dimerization, which is precluded in E34/DeltaD1-D2, is not necessary for optimal LFA-1 binding. Furthermore, BIAcore (BIAcore, Piscataway, NJ) affinity measurements revealed that soluble open LFA-1 I domain bound to immobilized soluble ICAM-1, E34/E34, E34/K34, and E34/DeltaD1-D2 with nearly identical affinities. These studies demonstrate that a single ICAM-1 monomer, not dimeric ICAM-1, represents the complete, "fully competent" LFA-1-binding surface.
Assuntos
Molécula 1 de Adesão Intercelular/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Adesão Celular , Cricetinae , Dimerização , Humanos , Molécula 1 de Adesão Intercelular/química , Antígeno-1 Associado à Função Linfocitária/química , Dados de Sequência MolecularRESUMO
Water-soluble chitosan oligomer (WSCO) has been reported to have anticancer activity, immuno-enhancing effect and antimicrobial activity. However, other biological activities are unknown. Herein, we have shown that WSCO is able to inhibit proliferation of human leukemia HL-60 cells and induce these cells to differentiate. Treatment with WSCO for 4 days resulted in a concentration-dependent reduction in HL-60 cell growth as measured by cell counting and MTT assay. This effect was accompanied by a marked increase in the proportion of G(0)/G(1) cells as measured by flow cytometry. WSCO also induced differentiation of the cells as measured by phorbol ester-dependent reduction of NBT, morphological changes as examined by Wright-Giemsa staining and expression of CD11b but not of CD14 as analysed by flow cytometry, indicating differentiation of HL-60 cells toward granulocyte-like cells. A combination of low dose of WSCO with all-trans retinoic acid, a differentiating agent toward granulocyte-like cells, exhibited a synergistic effect on the differentiation. In addition, treatment of HL-60 cells with WSCO for 6 or 8 days resulted in the induction of apoptosis as assayed qualitatively by agarose gel electrophoresis and quantitatively by Annexin V technique using flow cytometry. Collectively, there is a potential for WSCO in the treatment of myeloid leukemia.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Quitina/farmacologia , Granulócitos/citologia , Leucemia Promielocítica Aguda/tratamento farmacológico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Quitina/análogos & derivados , Quitosana , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Células HL-60/efeitos dos fármacos , Células HL-60/patologia , Humanos , Leucemia Promielocítica Aguda/patologia , Tretinoína/farmacologiaRESUMO
The effects of water-soluble chitosan oligomers (WSCO) on the synthesis of nitric oxide (NO) by murine peritoneal macrophages and on macrophage-mediated cytotoxicity towards murine fibrosarcoma Meth A cells were investigated. WSCO alone had no effect on NO synthesis and killing of tumor cells. However, treatment of macrophages with a combination of WSCO and interferon-gamma (IFN-gamma) synergically increased NO synthesis and enhanced killing of tumor cells. The synergism between IFN-gamma and WSCO in NO synthesis and tumoricidal activity was mainly dependent on increased secretion of tumor necrosis factor-alpha by WSCO.
Assuntos
Quitina/análogos & derivados , Quitina/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Interferon gama/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Óxido Nítrico/biossíntese , Animais , Anticorpos Monoclonais/farmacologia , Quitosana , Meios de Cultivo Condicionados/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Solubilidade , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
This study was designed to investigate the effect of cAMP on ursolic acid-induced apoptosis of HL-60 cells. Ursolic acid decreased the viability of the cells in a dose-dependent manner, which was revealed as an apototic process characterized by ladder-pattern DNA fragmentation in agarose gel electrophoresis and segmented nuclei in DAPI-sulpharhodamin 101 staining. Ursolic acid-induced apoptosis of the cells was markedly inhibited by the addition of cAMP-elevating agents including DB-cAMP, CPT-cAMP, 8-Br-cAMP and forskolin. These results were further evidenced by the fact that inhibitors of cAMP-dependent protein kinase including H89 and KT5720 completely inhibited the cAMP-mediated rescue of HL-60 cells from ursolic acid-induced apoptosis. In addition, differentiating agents of the cells such as dimethyl sulfoxide and retinoic acid did not affect the ursolic acid-induced apoptosis of HL-60 cells. These results suggest that signaling pathway of cAMP-dependent activation of protein kinase A may affect the responsiveness of tumor cells upon ursolic acid.
Assuntos
Antineoplásicos Fitogênicos/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Carbazóis , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Células HL-60/enzimologia , Sulfonamidas , Triterpenos/antagonistas & inibidores , Antineoplásicos Fitogênicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Dimetil Sulfóxido/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Ágar , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Humanos , Indóis/farmacologia , Isoquinolinas/farmacologia , Pirróis/farmacologia , Tretinoína/farmacologia , Triterpenos/farmacologia , Ácido UrsólicoAssuntos
Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Pele/efeitos dos fármacos , Tionucleotídeos/administração & dosagem , Fator de Crescimento Transformador beta/biossíntese , Administração Cutânea , Animais , Ativação de Macrófagos , Macrófagos/efeitos dos fármacos , Ratos , Fator de Crescimento Transformador beta/genéticaRESUMO
Bioassay-guided fractionation of an H2O extract of the barks of Fraxinus rhynchophylla has furnished two inducible nitric oxide synthase (iNOS) inhibitory compounds, ferulaldehyde (1) and scopoletin (3) together with a coumarin, fraxidin (2). Compounds 1 and 3 showed inhibition of nitric oxide (NO) synthesis in a dose-dependent manner by murine macrophage-like RAW 264.7 cells stimulated with interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS). The inhibition of NO synthesis of 1 was reflected in the decreased amount of iNOS protein, as determined by Western blotting.
Assuntos
Acroleína/análogos & derivados , Cumarínicos/farmacologia , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico/antagonistas & inibidores , Plantas Medicinais/química , Escopoletina/farmacologia , Acroleína/isolamento & purificação , Acroleína/farmacologia , Animais , Linhagem Celular , Cumarínicos/isolamento & purificação , Inibidores Enzimáticos/isolamento & purificação , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II , Escopoletina/isolamento & purificaçãoRESUMO
Bioassay-guided fractionation of an H2O extract of Artemisia feddei has furnished an inducible nitric oxide synthase (iNOS) inhibitory coumarin, scopoletin (1) and one of the inactive sesquiterpenes, achillin (2). Compound 1 showed inhibition of nitric oxide (NO) synthesis in a dose-dependent manner in murine macrophage-like RAW 264.7 cells stimulated with interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS). The inhibition of NO synthesis of 1 was due to suppression of iNOS mRNA and iNOS protein, as determined by Northern and Western blotting, respectively.
Assuntos
Artemisia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Extratos Vegetais/farmacologia , Plantas Medicinais , Escopoletina/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos , Camundongos , Óxido Nítrico Sintase Tipo II , Extratos Vegetais/química , Proteínas Recombinantes , Escopoletina/isolamento & purificaçãoRESUMO
To define the signaling pathways during NO-induced apoptotic events and their possible modulation by two protein kinase systems, we explored the involvement of three structurally related mitogen-activated protein kinase subfamilies. Exposure of HL-60 cells to sodium nitroprusside (SNP) strongly activated p38 kinase, but did not activate c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). In addition, SNP-induced apoptosis was markedly blocked by the selective p38 kinase inhibitor (SB203580) but not by MEK1 kinase inhibitor (PD098059), indicating that p38 kinase serves as a mediator of NO-induced apoptosis. In contrast, treatment of cells with phorbol 12-myristate 13-acetate (PMA) strongly activated not only JNK but also ERK, while not affecting p38 kinase. However, although SNP by itself weakly activated CPP32-like protease, SNP in combination with PMA markedly increased the extent of CPP32-like protease activation. Interestingly, N6,O2-dibutylyl cAMP (DB-cAMP) significantly blocked SNP- or SNP plus PMA-induced activation of CPP32-like protease and the resulting induction of apoptosis. DB-cAMP also blocked PMA-induced JNK activation. Collectively, these findings demonstrate the presence of specific up- or down-modulatory mechanisms of cell death pathway by NO in which (1) p38 kinase serves as a mediator of NO-induced apoptosis, (2) PKC acts at the point and/or upstream of JNK and provides signals to potentiate NO-induced CPP32-like protease activation, and (3) PKA lies upstream of either JNK or CPP32-like protease to protect NO- or NO plus PMA-induced apoptotic cell death in HL-60 cells.
Assuntos
Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Caspases/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Óxido Nítrico/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Bucladesina/farmacologia , Caspase 3 , Ativação Enzimática , Células HL-60 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Mitógenos/farmacologia , Nitroprussiato/farmacologia , Acetato de Tetradecanoilforbol/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The treatment of advanced cancers with paclitaxel (taxol) is hindered by the development of drug resistance. Resistance to taxol is known to be associated with multidrug resistance (MDR) and a mutation affecting either the alpha- or beta-subunit of tubulin. In this study, we demonstrated that an intracellular cAMP level may also play an important role in resistance to taxol in HL-60, acute promyelocytic leukemia cells. Exposure of HL-60 cells to various doses of taxol for 18 hr resulted in cell death. However, pretreatment of the cells with cAMP analogs such as N6:O2-dibutyl cAMP (Db-cAMP), 8-(4-chlorophenylthio) cAMP (CPT-cAMP) and 8-bromo-cAMP (8-Br-cAMP) or an intracellular cAMP elevating agent such as forskolin apparently rendered HL-60 cells more resistant to taxol, but not with dimethyl sulfoxide (DMSO) or retinoic acid (RA), well known differentiating agents. To investigate whether protein kinase A (PKA) activated by an increase in intracellular cAMP level could be involved in increased taxol resistance of the cells, we examined the effects of PKA inhibitors, including H-89 and KT5720, on taxol resistance induced by Db-cAMP. The PKA inhibitors significantly abolished Db-cAMP-induced taxol resistance. These results suggest that cAMP analogs may render tumor cells more resistant to taxol via PKA activation.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , AMP Cíclico/fisiologia , Paclitaxel/farmacologia , Bucladesina/farmacologia , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Dimetil Sulfóxido/farmacologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Células HL-60 , Humanos , Tretinoína/farmacologia , Células U937RESUMO
Nitric oxide (NO) induces apoptotic cell death in murine RAW 264.7 macrophages. To elucidate the inhibitory effects of protein kinase C (PKC) on NO-induced apoptosis, we generated clones of RAW 264.7 cells that overexpress one of the PKC isoforms and explored the possible interactions between PKC and three structurally related mitogen-activated protein (MAP) kinases in NO actions. Treatment of RAW 264.7 cells with sodium nitroprusside (SNP), a NO-generating agent, activated both c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 kinase, but did not activate extracellular signal-regulated kinase (ERK)-1 and ERK-2. In addition, SNP-induced apoptosis was slightly blocked by the selective p38 kinase inhibitor (SB203580) but not by the MAP/ERK1 kinase inhibitor (PD098059). PKC transfectants (PKC-beta II, -delta, and -eta) showed substantial protection from cell death induced by the exposure to NO donors such as SNP and S-nitrosoglutathione (GSNO). In contrast, in RAW 264.7 parent or in empty vector-transformed cells, these NO donors induced internucleosomal DNA cleavage. Moreover, overexpression of PKC isoforms significantly suppressed SNP-induced JNK/SAPK and p38 kinase activation, but did not affect ERK-1 and -2. We also explored the involvement of CPP32-like protease in the NO-induced apoptosis. Inhibition of CPP32-like protease prevented apoptosis in RAW 264.7 parent cells. In addition, SNP dramatically activated CPP32 in the parent or in empty vector-transformed cells, while slightly activated CPP32 in PKC transfectants. Therefore, we conclude that PKC protects NO-induced apoptotic cell death, presumably nullifying the NO-mediated activation of JNK/SAPK, p38 kinase, and CPP32-like protease in RAW 264.7 macrophages.
Assuntos
Apoptose/imunologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Caspases/fisiologia , Macrófagos/enzimologia , Proteínas Quinases Ativadas por Mitógeno , Óxido Nítrico/farmacologia , Proteína Quinase C/biossíntese , Transdução de Sinais/imunologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular , Ativação Enzimática/imunologia , Isoenzimas/biossíntese , Isoenzimas/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno , Macrófagos/efeitos dos fármacos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteína Quinase C/fisiologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Since there is increasing evidence that nitric oxide (NO) plays a crucial role in the pathogenesis of inflammatory diseases, this study was undertaken to address whether the methanol (MeOH) extract and its fractions of the bark of Ulmus davidiana Planch (Ulmaceae) could modulate the expression of inducible NO synthase (iNOS) in thioglycollate-elicited murine peritoneal macrophages and murine macrophage cell line, RAW264.7 cells. Stimulation of the peritoneal macrophages and RAW264.7 cells with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) resulted in increased production of NO in the medium. However, the butanol (BuOH) fraction of the MeOH extract of U. davidiana barks showed marked inhibition of NO synthesis in a dose-dependent manner. The inhibition of NO synthesis was reflected in the decreased amount of iNOS protein, as determined by Western blotting. The BuOH fraction did not affect the viability of RAW264.7 cells, as assessed by methylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide (MTT) assay; rather, it reduced endogenous NO-induced apoptotic cell death via inhibition of NO synthesis in RAW264.7 cells. On the other hand, the BuOH fraction showed no inhibitory effect on the synthesis of NO by RAW264.7 cells, when iNOS was already expressed by the stimulation with IFN-gamma and LPS. Collectively, these results demonstrate that the BuOH fraction inhibits NO synthesis by inhibition of the induction of iNOS in murine macrophages.
Assuntos
Macrófagos Peritoneais/efeitos dos fármacos , Óxido Nítrico/antagonistas & inibidores , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Animais , Butanóis/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/metabolismo , Metanol/química , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Extratos Vegetais/químicaRESUMO
S-nitrosylation by S-nitrosoglutathione (GSNO), a nitric oxide (NO) donor, suppresses the phosphotransferase activity of cJun N-terminal kinase 2 (JNK2)/stress activated protein kinase (SAPK) in dose- and time-dependent manners in vitro. JNK2 activity is significantly decreased at 10 microM of GSNO, which is dramatically reversed by adding 10 mM of DTT. Reduced form of glutathione protects the GSNO-induced suppression of JNK2 activation in a dose-dependent fashion. However, GSNO-treated Sek1 does not affect the JNK2 activity of phosphotransferation toward c-Jun N-terminal1-79 protein. These results indicate that NO may exert a regulatory role of JNK2 activity by S-nitrosylation of the protein in apoptotic signaling pathway. Suppression of JNK2 phosphotransferase activity by NO is also supported by the observation that NO plays an important anti-apoptotic roles in heptocytes, splenocytes, eosinophils and B lymphocytes.
Assuntos
MAP Quinase Quinase 4 , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Óxido Nítrico/farmacologia , Proteínas Quinases/metabolismo , Apoptose/efeitos dos fármacos , Ditiotreitol/farmacologia , Inibidores Enzimáticos/farmacologia , Glutationa/análogos & derivados , Glutationa/metabolismo , Glutationa/farmacologia , Humanos , Proteína Quinase 9 Ativada por Mitógeno , Nitroprussiato/farmacologia , Compostos Nitrosos/metabolismo , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Recombinantes/metabolismo , S-Nitrosoglutationa , Reagentes de Sulfidrila/farmacologiaRESUMO
Several studies have suggested a role for heat shock proteins (hsps) during development and differentiation. However, relatively little is known about the role of hsp70 in controlling human hematopoietic cell differentiation and death. Here, we show that constitutive expression of human inducible 70-kDa heat shock protein (hsp70) promotes differentiation of HL-60 cells and prevents apoptosis that occurred after terminal differentiation or directly by apoptotic agents. After treatment with phorbol 12-myristate 13-acetate (PMA), hsp70-overexpressing cells (HL-60/hsp70) underwent rapid growth arrest and plastic adherence and expressed more CD14 than parental HL-60 or empty vector-transformed cells (HL-60/puro). HL-60/hsp70 cells also rapidly differentiated into granulocytes by addition of all-trans-retinoic acid, as assessed by phenotypic changes after staining with Wright-Giemsa. After differentiation into monocyte/macrophage-like cells or granulocytes, hsp70-overexpressing cells showed little evidence for apoptosis and had a prolonged survival, indicating that the survival-enhancing properties of hsp70 counteract programmed cell death that accompanies terminal differentiation. HL-60/hsp70 cells also showed more resistance than parental cells against apoptotic agents such as sodium nitroprusside, a NO-generating agent, or Taxol, a microtubule stabilizing agent. Further, heat shock of parental HL-60 cells at 42 degrees C for 3 h increased hsp70 levels, promoted plastic adherence (< 6 h) of the cells in respond to PMA, and protected cells from SNP or Taxol. Taken together, these studies demonstrate that hsp70 plays a crucial role in the differentiation of myeloid cells, participating in cell cycle controls and phenotypic changes, with protecting effects on apoptosis induced by different pathways.
Assuntos
Apoptose/fisiologia , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Proteínas de Choque Térmico HSP70/fisiologia , Apoptose/efeitos dos fármacos , Sequência de Bases , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Dano ao DNA , Fragmentação do DNA , DNA Recombinante/genética , Expressão Gênica , Granulócitos/citologia , Granulócitos/metabolismo , Células HL-60 , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/genética , Hematopoese/fisiologia , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Paclitaxel/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Previously we reported that phorbol ester, a protein kinase C (PKC) activator, exhibits a unique pattern of potentiation of nitric oxide (NO)-related apoptosis in HL-60 human promyelocytic leukemia cells. Here we show that elevation of intracellular cAMP could protect HL-60 cells from NO- or NO plus PMA-induced DNA damage. Exposure of cells to sodium nitroprusside (SNP; 0.5 to 4 mM), a NO-generating agent, induced apoptotic cell death as monitored by morphological means, gel electrophoresis, and in situ TdT-apoptosis assay. However, concomitant incubation of the cells with DB-cAMP markedly inhibited SNP-induced apoptotic cell death in a dose-dependent manner. Similar results were obtained with other commonly used cAMP analogs such as CPT-cAMP and 8-C1-cAMP and the intracellular cAMP-elevating agent such as forskolin. In contrast, pretreatment of HL-60 cells with H89 or KT5720, which are known to inhibit cAMP-dependent protein kinase (PKA), abolished the protective effect of cAMP analogs and forskolin on SNP-induced apoptosis. Synergism between SNP and phorbol ester to induce apoptosis was also inhibited by prior treatment of HL-60 cells with DB-cAMP or forskolin. The effect of DB-cAMP in maintaining cell viability was not associated with the onset of G0/G1 cell cycle arrest. In addition, neither dimethyl sulfoxide nor retinoic acid (which produce granulocyte differentiation) could produce cAMP effect. Under the same conditions, DB-cAMP also inhibited NO- or NO plus phorbol ester-induced apoptosis in another transformed cell line, U-937 cells. Taken together, these findings suggest that exposure of HL-60 cells to cAMP analogs renders them more resistant to NO-induced DNA damage and further suggest the existence of specific down-modulatory mechanisms related to NO-induced apoptotic DNA fragmentation.
Assuntos
Apoptose , AMP Cíclico/metabolismo , Óxido Nítrico/fisiologia , Bucladesina/farmacologia , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Dimetil Sulfóxido/farmacologia , Glutationa/análogos & derivados , Glutationa/farmacologia , Células HL-60 , Humanos , Óxido Nítrico/antagonistas & inibidores , Nitroprussiato/farmacologia , Compostos Nitrosos/farmacologia , Penicilamina/análogos & derivados , Penicilamina/farmacologia , S-Nitroso-N-Acetilpenicilamina , S-Nitrosoglutationa , Acetato de Tetradecanoilforbol/farmacologia , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
Taxol, an anticancer drug, has been known not only to block cell division by stabilizing microtubules but also to activate murine macrophages to express TNF-alpha, interleukin-1, and to produce nitric oxide (NO). We therefore reasoned that taxol could activate murine macrophages to generate reactive oxygen intermediates, such as superoxide anion (O2-), which are responsible for intracellular killing of pathogenic microbes. Treatment of RAW264.7 cells, murine macrophage cell line, with taxol increased phorbol ester-induced O2- production in a dose dependent manner (approximately 2 fold). In addition, taxol rapidly (< 1 hr) primed RAW264.7 cells to enhance O2- release stimulated with PMA. Taxol also enhanced stimulation of O2- production by FMLP, but not by Con A. This effect was abolished by prior treatment with both superoxide dismutase (SOD) and N-acetyl-L-cystein, a free radical scavenger. To investigate the mechanism of taxol-induced macrophage stimulation, we evaluated the ability of colchicine, a drug that inhibit tubulin polymerization, and cAMP analogues, which is known to depolymerize microtubule. Taxol-induced O2- production was inhibited by the treatment with both colchicine and DB-cAMP. Taken together, these results demonstrated that taxol provides two signals, "priming" and "enhancing", to generate superoxide anion via the stabilization of microtubules in murine RAW264.7 cells