RESUMO
Copy numbers of mRNAs for GFRalpha-1 and GFRalpha-2, the preferred receptors for glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) were determined by real-time quantitative RT-PCR (QRT-PCR). Receptor expression was assessed in striatum (ST) and substantia nigra (SN) of normal rats and rats acutely or progressively lesioned by 6-OHDA injected into the medial forebrain bundle or ST, respectively. GFRalpha-1 mRNA was clearly detected in normal ST. In normal SN, significantly higher expression of both receptors was observed. At 4 weeks after acute lesion, GFRalpha-2 mRNA was markedly decreased in SN bilaterally, whereas GFRalpha-1 mRNA in SN and ST was not affected. A progressive lesion resulted in a progressive decrease of GFRalpha1 mRNA in ST bilaterally. In SN, levels of GFRalpha-1 mRNA were not significantly affected by a progressive lesion, whereas GFRalpha-2 mRNA was markedly decreased bilaterally. Quantitative western blotting standardized against tyrosine hydroxylase (TH) protein from PC12 cells revealed the expected decrease in TH protein in lesioned SN, but also significant increases in TH protein in contralateral, unlesioned SNs at 4 weeks after both acute and progressive lesions. These data suggest that previously unrecognized compensatory changes in the nigrostriatal system occur in response to unilateral dopamine depletion. Since the changes observed in receptor expression did not always parallel loss of dopamine neurons, cells in addition to the nigral dopamine neurons appear to be affected by a 6-OHDA insult and are potential targets for the neurotrophic factors, GDNF and NTN.
Assuntos
Corpo Estriado/metabolismo , Lateralidade Funcional/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Análise de Variância , Animais , Comportamento Animal , Western Blotting/métodos , Corpo Estriado/lesões , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Masculino , Feixe Prosencefálico Mediano/lesões , Oxidopamina/toxicidade , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos F344 , Receptores Proteína Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Simpatolíticos/toxicidade , Fatores de Tempo , Área Tegmentar Ventral/lesõesRESUMO
Vectors suitable for delivery of therapeutic genes to the CNS for chronic neurodegenerative diseases will require regulatable transgene expression. In this study, three self-regulating rAAV vectors encoding humanized green fluorescent protein (hGFP) were made using the tetracycline (tet)-off system. Elements were cloned in different orientations relative to each other and to the AAV internal terminal repeat (ITRs). The advantage of this vector system is that all infected cells will carry both the 'therapeutic' gene and the tet-regulator. To compare the efficiency of the vectors, 293T cells infected by each vector were grown in the presence or absence of the tet-analog doxycycline (dox). Cells were analyzed by flow cytometry for hGFP protein expression, and quantitative RT-PCR (QRT-PCR) for levels of hGFP mRNA and the tet-activator (tTA) mRNA. In the presence of dox, cells infected with one of the vectors, rAAVS3, showed less than 2% total fluorescent intensity and mRNA copy number than cells grown without dox. The other two vectors were significantly more leaky. Levels of tTA mRNA were not affected by dox. The S3 vector also displayed tight regulation in HeLa and HT1080 cells. To assess regulation in the brain, the S3 vector was injected into rat striatum and rats maintained on regular or dox-supplemented water. At 1 month after vector injection, numerous positive cells were observed in rats maintained on regular water whereas only rare positive cells with very low levels of fluorescence were observed in rats maintained on water containing dox. The QRT-PCR analysis showed that dox inhibited expression of hGFP mRNA in brain by greater than 99%. These results demonstrate that exceedingly tight regulation of transgene expression is possible using the tet-off system in the context of a self-regulating rAAV vector and that the specific orientation of two promoters relative to each other and to the ITRs is important. Regulatable vectors based on this design are ideal for therapeutic gene delivery to the CNS.
Assuntos
Sistema Nervoso Central/metabolismo , Dependovirus/genética , Regulação da Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/genética , Doenças Neurodegenerativas/terapia , Animais , Linhagem Celular , Doxiciclina/farmacologia , Citometria de Fluxo , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/administração & dosagem , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/genética , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetraciclina , TransgenesRESUMO
Pax3 is a transcription factor that is required for the development of embryonic neural tube, neural crest, and somatic derivatives. Our previous study (Mayanil, C. S. K., George, D., Mania-Farnell, B., Bremer, C. L., McLone, D. G., and Bremer, E. G. (2000) J. Biol. Chem. 275, 23259-23266) reveals that overexpression of Pax3 in a human medulloblastoma cell line, DAOY, resulted in an up-regulation in alpha-2,8-polysialyltransferase (STX) gene expression and an increase in polysialic acid on neural cell adhesion molecule. This finding suggests that STX might be a previously undescribed downstream target of Pax3. Because Pax3 is important in diverse cellular functions during development, we are interested in the identification of additional downstream targets of Pax3. We utilized oligonucleotide arrays and RNA isolated from stable Pax3 transfectants to identify potential target genes. A total of 270 genes were altered in the Pax3 transfectants as compared with the vector control and parental cell line. An independent analysis by cDNA expression array and real-time quantitative polymerase chain reaction of several genes confirmed the changes observed by the oligonucleotide microarray data. Of the genes that displayed significant changes in expression, several contain paired and homeodomain binding motifs of Pax3 in their promoter regions. Using promoter-luciferase reporter transfection assays and electromobility shift assays, we showed at least one previously undescribed downstream target, STX, to be a biological downstream target of Pax3. Thus we report several previously undescribed candidate genes to be potential downstream targets of Pax3.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Sialiltransferases/genética , Fatores de Transcrição/metabolismo , Animais , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Genes Reporter , Humanos , Lectinas Tipo C , Meduloblastoma , Camundongos , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , Regiões Promotoras Genéticas , RNA/metabolismo , Reprodutibilidade dos Testes , Sialiltransferases/metabolismo , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Versicanas , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
Whereas previous investigations have shown that pharmacologic addition of gangliosides inhibits keratinocyte proliferation by downregulating epidermal growth factor receptor phosphorylation, the underlying biochemical basis and physiologic relevance are unknown. Using Scatchard and displacement plots, we have shown that supplemental purified gangliosides decrease the binding of (125)I-labeled epidermal growth factor to keratinocyte-derived SCC12 cells. Conversely, SCC12 cells transfected with sialidase and thus depleted of gangliosides show increased ligand binding to the epidermal growth factor receptor, which is consistent with their increased proliferation in response to epidermal growth factor and transforming growth factor-alpha, and increased phosphorylation of the epidermal growth factor receptor, and downstream signal transduction pathway components. The mechanism of the altered binding appears to involve primarily decreased numbers of available receptors within the intact membrane, but not altered receptor protein expression. These studies provide evidence that the effect of gangliosides on keratinocyte proliferation results, at least in part, from the direct binding of ganglioside to the receptor and disruption of the receptor-ligand interaction. Manipulation of membrane ganglioside content may be a powerful new means to alter epidermal growth factor receptor-dependent cell proliferation.
Assuntos
Receptores ErbB/metabolismo , DNA Complementar/genética , Fator de Crescimento Epidérmico/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Gangliosídeos/metabolismo , Gangliosídeos/farmacologia , Humanos , Queratinócitos/química , Ligantes , Neuraminidase/biossíntese , Neuraminidase/genética , Transfecção , Fator de Crescimento Transformador alfa/efeitos dos fármacos , Fator de Crescimento Transformador alfa/metabolismo , Células Tumorais CultivadasRESUMO
Polysialic acid (PSA) is a developmentally regulated carbohydrate found primarily on neural cell adhesion molecules (NCAM) in embryonic tissues. The majority of NCAM in adult tissues lacks this unique carbohydrate, but polysialylated NCAM (PSA-NCAM) is present in adult brain regions where neural regeneration persists and in some pediatric brain tumors such as medulloblastoma, which show greater propensity for leptomeningeal spread. Pax3, a developmentally regulated paired homeodomain transcription factor, is thought to be involved in the regulation of neural cell adhesion molecules. Overexpression of murine Pax3 into a human medulloblastoma cell line (DAOY) resulted in an increase in NCAM polysialylation and a 2-4-fold increase in alpha2, 8-polysialyltransferase type II mRNA levels. No difference was observed in alpha2,8-polysialyltransferase type IV message. The addition of PSA to NCAM changed the adhesive behavior of these Pax3 transfectants. Transfectants expressing high PSA-NCAM show much less NCAM-dependent aggregation than those with less PSA-NCAM. In addition, Pax3 transfectants having high PSA-NCAM show heterophilic adhesion involving polysialic acid to heparan sulfate proteoglycan and agrin. These observations suggest that a developmentally regulated transcription factor, Pax3, could affect NCAM polysialylation and subsequently cell-cell and cell-substratum interaction.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Meduloblastoma/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Ácidos Siálicos/metabolismo , Fatores de Transcrição , Animais , Sequência de Bases , Primers do DNA , Proteínas de Ligação a DNA/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Meduloblastoma/enzimologia , Meduloblastoma/patologia , Camundongos , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , Sialiltransferases/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Gangliosides have been described as modulators of growth factor receptors. For example, GM3 addition in cell culture medium inhibits epidermal growth factor (EGF)-stimulated receptor autophosphorylation. Furthermore, depletion of ganglioside by sialidase gene transfection appeared to increase EGF receptor (EGFR) autophosphorylation. These data suggested that changes in GM3 content may result in different responses to EGF. In this study, the ceramide analog d-threo-1-phenyl-2-decannoylamino-3-morpholino-1-propanol ([D]-PDMP), which inhibits UDP-glucose-ceramide glucosyltransferase, and addition of GM3 to the culture medium were used to study the effects of GM3 on the EGFR. Addition of 10 microM [D]-PDMP to A431 cells resulted in significant GM3 depletion. Additionally, EGFR autophosphorylation was increased after EGF stimulation. When exogenous GM3 was added in combination with [D]-PDMP, the enhanced EGFR autophosphorylation was returned to control levels. [D]-PDMP also increased EGF-induced cell proliferation, consistent with its effect on autophosphorylation. Once again, the addition of GM3 in combination with [D]-PDMP reversed these effects. These results indicate that growth factor receptor functions can be modulated by the level of ganglioside expression in cell lines. Addition of GM3 inhibits EGFR activity and decrease of GM3 levels using [D]-PDMP treatment enhances EGFR activity. Modulation of growth factor receptor function may provide an explanation for how transformation-dependent ganglioside changes contribute to the transformed phenotype.
Assuntos
Inibidores Enzimáticos/farmacologia , Receptores ErbB/fisiologia , Gangliosídeo G(M3)/fisiologia , Gangliosídeos/metabolismo , Morfolinas/farmacologia , Carcinoma de Células Escamosas , Divisão Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/efeitos dos fármacos , Gangliosídeo G(M3)/farmacologia , Glucosiltransferases/antagonistas & inibidores , Humanos , Cinética , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Increased expression of the bisecting GlcNAc has been correlated with tumor progression in several experimental tumor models. Its expression and function in brain tumors are, however, not yet known. In this study, we investigated expression of the bisecting GlcNAc structure in a series of pediatric brain tumors and its relationship to tumor response to vinblastine. A plant lectin (E-PHA) that recognizes the bisecting GlcNAc structure was used for detection of this molecule in a total of 90 pediatric brain tumors and normal brain tissue specimens. Our results showed that, whereas E-PHA staining was undetectable in the normal brain tissue, pediatric brain tumor specimens exhibited different levels of reactivity. Lectin staining was particularly prominent in high-grade astrocytomas (73%) and ependymomas (72%). In astrocytomas, there was a positive correlation with the tumor grade, which suggests that the bisecting GlcNAc may be of particular interest as a tumor marker for diagnosis and/or prognosis. By using a human glioma cell culture model, we have found that treatment of these cells with E-PHA lectin enhances their sensitivity to vinblastine. E-PHA interacted directly with the drug transporter P-glycoprotein and inhibited its drug efflux function. In a drug-resistant glioma cell line transfected with the mdr1 gene, drug resistance was reversed by E-PHA. Our findings indicate that: (a) expression of the bisecting GlcNAc in pediatric brain tumors may have a potential relevance as a tumor marker; and (b) glioma response to chemotherapy may be modulated through the bisecting GlcNAc.
Assuntos
Acetilglucosamina/metabolismo , Neoplasias Encefálicas/patologia , Córtex Cerebral/citologia , Fito-Hemaglutininas , Vimblastina/toxicidade , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Acetilglucosamina/análise , Astrocitoma/patologia , Sequência de Carboidratos , Divisão Celular/efeitos dos fármacos , Neoplasias Cerebelares/patologia , Córtex Cerebral/patologia , Ependimoma/patologia , Glioma/metabolismo , Glioma/patologia , Glicosilação , Humanos , Lectinas , Meduloblastoma/patologia , Dados de Sequência Molecular , Oligossacarídeos/biossíntese , Oligossacarídeos/química , Fito-Hemaglutininas/farmacologia , Células Tumorais CultivadasRESUMO
Glycosphingolipids expressed in cancer cells have been implicated in the modulation of tumor cell growth through their interaction with transmembrane signaling molecules such as growth factor receptors. For glycosphingolipids to interact with growth factor receptors, the presence of sialic acid seems to be essential. Stable transfection of a gene encoding a soluble Mr 42,000 sialidase into a human epidermoid carcinoma cell line (A431) provided an approach by which the level of terminal lipid-bound sialic acid on the cell surface could be altered. In the sialidase-positive clones, the level of ganglioside GM3 was diminished, and little change was observed in protein sialylation. Sialidase-transfected cells grew faster than control cells. Sialidase expression did not modify the binding of epidermal growth factor (EGF) to its receptor but enhanced EGF receptor (EGFR) tyrosine autophosphorylation as compared to that of parental cells or cells transfected with the vector (pcDNA3) alone. Moreover, the phosphorylation of the EGFR, as well as other protein substrates, was observed at low EGF concentrations, suggesting an increase in the receptor kinase sensitivity. These data provided evidence that changes in ganglioside expression in cancer cells by appropriate gene transfection can dramatically affect EGFR kinase activity. Hence, the modulation of ganglioside expression may represent an approach to alter tumor cell growth.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Neuraminidase/genética , Carcinoma de Células Escamosas/genética , Receptores ErbB/genética , Técnicas de Transferência de Genes , Humanos , Transdução de Sinais/genética , Células Tumorais CultivadasRESUMO
Growth factor receptors (GFRs) have been described as overexpressed in several types of brain tumors. Overexpression of these transmembrane proteins is considered to be an important part of tumorigenesis. Genetic as well as epigenetic modulation of the receptors have to be considered when trying to understand the role of GFRs in tumors or as targets for tumor therapy. GFR function can be modulated by membrane components (e.g. gangliosides) or by the change in receptor glycosylation. These types of changes and the occurrence of the expression of mutated receptor expressed in tumor cell can result in altered signaling. In this review, we have focused on GFRs, their expression and mutations in brain tumors. Recently the correlation between GFR expression and patient outcome has suggested that these tyrosine kinases and their signaling might play a decisive role in the course of patients with brain tumors. The importance of GFRs as possible targets for brain tumor therapy is also discussed.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Encéfalo/metabolismo , Glioma/genética , Glioma/terapia , Receptores de Fatores de Crescimento , Animais , Neoplasias Encefálicas/metabolismo , Criança , Gangliosídeos/metabolismo , Expressão Gênica , Glioma/metabolismo , Humanos , Mutação , Ratos , Receptores de Fatores de Crescimento/análise , Receptores de Fatores de Crescimento/genética , Resultado do TratamentoRESUMO
Glycosyltransferase gene transfection into cell lines has been an approach used successfully to elucidate the functional role of cell surface glycoconjugates. We have transfected the rat CMP-NeuAc:Galbeta1,4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) gene into a human, tumorigenic, glioma cell line, U373 MG. This transfection led to a marked inhibition of invasivity, alterations in adhesivity to fibronectin and collagen matrices, and inappropriately sialylated alpha3beta1 integrin. Adhesion-mediated protein tyrosine phosphorylation was reduced in the transfectants despite increased expression of focal adhesion kinase, p125fak. Furthermore, the transfectants showed a distinct cell morphology, an increased number of focal adhesion sites, and different sensitivity to cytochalasin D treatment than control U373 MG cells. These results suggest that inappropriate sialylation of cell surface glycoconjugates, such as integrins, can change focal adhesion as well as adhesion-mediated signal transduction and block glioma cell invasivity in vitro.
Assuntos
Invasividade Neoplásica/fisiopatologia , Sialiltransferases/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , Colágeno/metabolismo , Citoesqueleto/metabolismo , Fibronectinas/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Glioma , Humanos , Integrina alfa3beta1 , Integrinas/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Ratos , Receptor de Insulina/metabolismo , Receptores de Laminina/metabolismo , Ácidos Siálicos/metabolismo , Sialiltransferases/genética , Transdução de Sinais/fisiologia , Transfecção , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/enzimologia , Tirosina/metabolismo , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
CMP-NeuAc: Galbeta1,3(4)GlcNAc alpha2,3-sialyltransferase (alpha2,3-ST) mRNA was expressed in human glioma specimens, human fetal astrocytes, and a panel of brain tumor cell lines. Maackia amurensis agglutinin staining revealed the presence of alpha2,3-linked sialic acids on glioma cell surfaces and extracellular matrices whereas normal human adult astrocytes were negative. Increased expression of alpha2,3-linked glycoprotein sialylation may play a role in glial tumorigenesis.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Glicoproteínas/metabolismo , RNA Mensageiro/metabolismo , Sialiltransferases/genética , Adenocarcinoma/metabolismo , Astrócitos/metabolismo , Astrocitoma/metabolismo , Neoplasias Encefálicas/secundário , Configuração de Carboidratos , Glioblastoma/metabolismo , Humanos , Células Tumorais Cultivadas , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
N-linked oligosaccharides appear to be important for the function of the epidermal growth factor (EGF) receptor. In a previous study (Rebbaa, A., Yamamoto, H., Moskal, J. R., and Bremer, E. G. (1996) J. Neurochem. 67, 2265-2272), we showed that binding of the erythroagglutinating phytohemagglutin lectin from Phaseolus vulgaris to the bisecting structures on the EGF receptor from U373 MG glioma cells blocked EGF binding and receptor autophosphorylation. In this study we examined the consequences of overexpression of the bisecting structure on the EGF receptor by gene transfection of U373 MG cells with the N-acetylglucosaminyltransferase III (GnT-III). This modification leads to a significant decrease in EGF binding and EGF receptor autophosphorylation. In addition, the cellular response to EGF was found to be altered. Proliferation of U373 MG cells in serum-free medium is inhibited by EGF. In contrast, proliferation of the GnT-III-transfected cells was stimulated by EGF. These data demonstrate that changes in EGF receptor glycosylation by GnT-III transfection reduces the number of the active receptors in U373 MG cells and that this change results in change in the cellular response to EGF.
Assuntos
Acetilglucosamina/metabolismo , Receptores ErbB/metabolismo , Glioma/genética , Oligossacarídeos/metabolismo , Transfecção , Fator de Crescimento Epidérmico/metabolismo , Glioma/metabolismo , Glicosilação , Humanos , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Fosforilação , Fito-Hemaglutininas/metabolismo , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
Little is known about the role of the N-linked oligosaccharides in the function of the epidermal growth factor (EGF) receptor (EGF-R). In a human glioma cell line, U373 MG, EGF-Rs contain the bisecting N-linked oligosaccharide sequence recognized by erythroagglutinating phytohemagglutinin lectin from Phaseolus vulgaris (E-PHA). Incubation of E-PHA with cultured U373 MG cells results in inhibition of EGF binding to its receptor and consequently inhibition of EGF-induced autophosphorylation of the receptor. Consistent with the inhibitory effects on the EGF-R, phenotypic events that depend on EGF-R signaling, such as cell spreading and proliferation, were also found to be modified. The effect of this lectin seems to be specific because leukoagglutinating phytohemagglutinin lectin from P. vulgaris (L-PHA), an isolectin of E-PHA, had no effect on EGF-R activity or the biological functions of these cells even though L-PHA was able to bind to the EGF-R. These findings suggest the presence of an important bisecting N-linked oligosaccharide structure in close proximity to the EGF binding site on the receptor. Furthermore, these results suggest the possibility that E-PHA lectin binding may provide an additional approach to blocking EGF-dependent glioma cell growth.
Assuntos
Receptores ErbB/agonistas , Receptores ErbB/fisiologia , Fito-Hemaglutininas/metabolismo , Transdução de Sinais/fisiologia , Divisão Celular/efeitos dos fármacos , Glioma , Humanos , Isomerismo , Oligossacarídeos/análise , Fosforilação , Fito-Hemaglutininas/química , Fito-Hemaglutininas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/fisiologiaRESUMO
Ganglioside GM3 is a membrane component that has been described to modulate cell growth through inhibition of EGF receptor associated tyrosine kinase. In order to determine if the inhibition of cell growth by this ganglioside is specifically mediated through EGF receptor signaling, the effects of GM3 on key enzymes implicated in EGF signaling were determined and compared to another inhibitor of the EGF receptor kinase. Treatment of A1S cells in culture by GM3 or a tyrosine kinase inhibitor, leflunomide, led to the inhibition of MAP kinase and PI3 kinase activities. There was no detectable effect on phosphotyrosine phosphatases. In a cell free system, however, GM3 had no effect on the activity of these signaling intermediates. Leflunomide was able to directly inhibit MAP kinase activity. GM3 and leflunomide were also found to act differently on the expression of the early immediate genes. The expression of c-fos and c-jun was inhibited by both GM3 and leflunomide. The expression of c-myc, however, was only inhibited by leflunomide. These findings suggest that the action of GM3 on cell growth and signaling is specifically mediated by EGF receptor and that this ganglioside does not act directly on the intracellular intermediates of EGF receptor signaling. In addition, soluble small molecule tyrosine kinase inhibitors such as leflunomide can directly affect the activity of MAP kinases and possibly other signaling intermediates. The direct effects of leflunomide on signaling intermediates may explain the differential effects of leflunomide and GM3 on gene expression and cell growth.
Assuntos
Receptores ErbB/antagonistas & inibidores , Gangliosídeo G(M3)/fisiologia , Transdução de Sinais , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular , Receptores ErbB/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Genes Precoces , Humanos , Isoxazóis/farmacologia , LeflunomidaRESUMO
Leflunomide is an immunosuppressive drug capable of inhibiting cellular and humoral mediated responses in vivo. The mechanism responsible for suppression of B cell antibody responses in vivo has not been identified. In this study we demonstrate that leflunomide functions to inhibit murine B cell antibody production by directly acting on the B cell. Experiments performed in vivo showed that both T cell-dependent as well as T cell-independent antigen responses were suppressed by leflunomide. Initial in vitro experiments demonstrated that leflunomide inhibited B cell antibody production by decreasing B cell proliferation. The suppression of B cell proliferation induced by a variety of stimuli that use different signal cascade components suggested that leflunomide was acting on a common component required for B cell proliferation. Kinetic studies with LPS activated B cells revealed that leflunomide retained its inhibitory activity when added as late as 24 hr after stimulation in an 88-hr assay. By analyzing the cell cycle of LPS-stimulated B cells we observed that leflunomide targets two different stages in cell cycle transition: (1) from G1 to S phase and (2) from S phase to G2/M phase. Analysis of one of the cyclin-dependent kinases, Cdk2 protein, by Western blot revealed that Cdk2 levels were decreased, in the presence of leflunomide, 48 hr after stimulation. These data further confirmed that leflunomide inhibited B cell progression through the S phase. We also present evidence that the addition of exogenous uridine reversed the antiproliferative activity of leflunomide. This indicated that leflunomide acted as a pyrimidine synthesis inhibitor, thereby inhibiting B cell proliferation and cell cycle progression.
Assuntos
Linfócitos B/efeitos dos fármacos , Linfócitos B/fisiologia , Imunossupressores/farmacologia , Isoxazóis/farmacologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Leflunomida , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Estimulação Química , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de TempoRESUMO
Leflunomide, a novel immunosuppressive drug, is able to prevent and reverse allograft and xenograft rejection in rodents, dogs, and monkeys. It is also effective in the treatment of several rodent models of arthritis and autoimmune disease. In vitro studies indicate that leflunomide is capable of inhibiting anti-CD3- and interleukin-2 (IL-2)-stimulated T cell proliferation. However, the biochemical mechanism for the inhibitory activity of leflunomide has not been elucidated. In this study, we characterized the inhibitory effects of leflunomide on Src family (p56lck and p59fyn)-mediated protein tyrosine phosphorylation. Leflunomide was able to inhibit p59fyn and p56lck activity in in vitro tyrosine kinase assays. The IC50 values for p59fyn (immunoprecipitated from either Jurkat or CTLL-4 cell lysate) autophosphorylation and phosphorylation of the exogenous substrate, histone 2B, were 125-175 and 22-40 microM respectively, while the IC50 values for p56lck (immunoprecipitated from Jurkat cell lysates) autophosphorylation and phosphorylation of histone 2B were 160 and 65 microM respectively. We also demonstrated the ability of leflunomide to inhibit protein tyrosine phosphorylation induced by anti-CD3 monoclonal antibody in Jurkat cells. The IC50 values for total intracellular tyrosine phosphorylation ranged from 5 to 45 microM, with the IC50 values for the zeta chain and phospholipase C isoform gamma 1 being 35 and 44 microM respectively. Leflunomide also inhibited Ca2+ mobilization in Jurkat cells stimulated by anti-CD3 antibody but not in those stimulated by ionomycin. Distal events of anti-CD3 monoclonal antibody stimulation, namely, IL-2 production and IL-2 receptor expression on human T lymphocytes, were also inhibited by leflunomide. Finally, tyrosine phosphorylation in CTLL-4 cells stimulated by IL-2 was also inhibited by leflunomide. These data collectively demonstrate the ability of leflunomide to inhibit tyrosine kinase activity in vitro, and suggest that inhibition of tyrosine phosphorylation events may be the mechanism by which leflunomide functions as an immunosuppressive agent.
Assuntos
Imunossupressores/farmacologia , Isoxazóis/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Linfócitos T/metabolismo , Animais , Complexo CD3/metabolismo , Cálcio/metabolismo , Células Cultivadas , Humanos , Interleucina-2/metabolismo , Isoenzimas/metabolismo , Leflunomida , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Camundongos , Fosfolipase C gama , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fyn , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Interleucina-2/biossíntese , Transdução de Sinais , Linfócitos T/enzimologia , Fosfolipases Tipo C/metabolismoRESUMO
Excretion of urinary modified nucleosides is frequently elevated in patients with oncogenic disease. Increases of urinary pseudouridine excretion are now demonstrated in patients with a variety of brain tumors. The potential use of urinary modified base excretion as a cancer marker is discussed and possible sources of the elevated nucleosides are detailed. The specific steps in RNA metabolism that result in increased levels of RNA nucleoside excretion are poorly understood. This knowledge will be necessary to understand the molecular mechanism and the clinical significance of urinary nucleoside excretion in treatment and diagnosis of oncogenic disease.
Assuntos
Biomarcadores Tumorais/urina , Neoplasias/metabolismo , RNA Neoplásico/metabolismo , RNA/metabolismo , Ribonucleosídeos/urina , Adulto , Animais , Criança , Feminino , Humanos , Neoplasias/diagnóstico , Neoplasias/terapia , Neoplasias/urina , Neoplasias Experimentais/metabolismoRESUMO
Ganglioside GM3 has been shown to modulate epidermal growth factor receptor function. These observations have lead to the hypothesis that GM3 may bind to the epidermal growth factor receptor. An enzyme-linked immunosorbant assay was designed to test this hypothesis. In these experiments, receptor-rich vesicle preparations were incubated with ganglioside GM1 or GM3 coated 96-well microtiter plates and the amount of bound receptor was compared. Plates coated with GM3 consistently bound more epidermal growth factor receptor than did GM1 coated plates. The binding of epidermal growth factor receptors to GM3 coated wells appeared to be specific and saturable. These results suggest that GM3 may modulate epidermal growth factor receptor function owing to a specific association of the two molecules.
Assuntos
Receptores ErbB/metabolismo , Gangliosídeo G(M3)/metabolismo , Células 3T3 , Animais , Química Encefálica , Bovinos , Linhagem Celular , Membrana Celular/metabolismo , Cães , Receptores ErbB/isolamento & purificação , Gangliosídeo G(M1)/isolamento & purificação , Gangliosídeo G(M1)/metabolismo , Gangliosídeo G(M3)/isolamento & purificação , Humanos , Radioisótopos do Iodo , Camundongos , Ligação Proteica , Células Tumorais CultivadasRESUMO
The active metabolite of leflunomide, A77 1726 inhibits the proliferation of a variety of mammalian cell lines in culture. Epidermal growth factor (EGF)-dependent proliferation is inhibited by A77 1726 at an effective dose of 30-40 microM. A77 1726 appears to directly inhibit the EGF receptor tyrosine-specific kinase activity both in intact cells and purified EGF receptors at the same effective dose. These data suggest that leflunomide inhibits cellular proliferation by the inhibition of tyrosine-specific kinase activities.
Assuntos
Compostos de Anilina/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Hidroxibutiratos/farmacologia , Isoxazóis/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Carcinoma de Células Escamosas , Linhagem Celular , Células Cultivadas , Crotonatos , Fibroblastos , Humanos , Células KB , Leflunomida , Masculino , Nitrilas , Fosforilação , Pele/metabolismo , Toluidinas , Células Tumorais CultivadasRESUMO
We previously showed that gangliosides inhibit DNA synthesis in Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) in a dose-responsive manner. This correlated with the inhibitory effects of several gangliosides (except GM3) on tyrosine phosphorylation of the PDGF receptor (PDGFR). [35S]Methionine-labeled Swiss 3T3 cells were incubated either with or without gangliosides and stimulated with PDGF, and proteins were cross-linked with bis(sulfosuccinimidyl) suberate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that two protein bands (170 and 350 kDa) were specifically immunoprecipitated with an anti-PDGFR antibody. Using both Swiss 3T3 and human glioma U-1242MG cells, western blots with anti-PDGFR and anti-phosphotyrosine antibodies confirmed that these bands were the PDGFR monomer and dimer, respectively, and that phosphotyrosine was present in these bands only after cells were stimulated with PDGF. Of the gangliosides tested, GM1, GM2, GD1a, GD1b, GD3, and GT1b, but not GM3, inhibited the formation of the 350-kDa band. These results demonstrate that all gangliosides tested, except GM3, probably inhibit PDGF-mediated growth by preventing dimerization of PDGFR monomers. Loss of more complex gangliosides in human gliomas would permit unregulated activation of the PDGFR, contributing to uncontrolled growth stimulation. We propose that ganglioside inhibition of receptor dimerization is a novel mechanism for regulating and coordinating several trophic factor-mediated cell functions.