RESUMEN
Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/metabolismo , Neoplasias Ováricas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Western Blotting , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/genética , ARN Interferente Pequeño/farmacología , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transcripción Genética , Trasplante Heterólogo , Células Tumorales Cultivadas , Proteína 1 de Unión a la Caja YRESUMEN
UFT, an anticancer agent that is composed of tegafur (FT) and uracil at a molar ratio of 1:4, is widely used in clinical practice in Japan to treat cancer patients requiring a long-term chemotherapy, and it is associated with few side effects, if any. In this study, we have evaluated the inhibitory effect of UFT against RENCA cell-induced angiogenesis by a dorsal air sac assay. Marked angiogenesis is induced by implantation of a chamber containing RENCA cells into mice. In this model, UFT showed a strong angiogenesis-inhibitory effect, whereas 5-fluorouracil (5-FU) and doxifluridine were less effective. Additional experiments revealed FT to be effective component of UFT; uracil remained ineffective in the inhibition of angiogenesis. Moreover, we have found that gamma-hydroxybutyric acid and gamma-butyrolactone, the metabolites of FT, possess a potent angiogenesis inhibitory effect that is amplified when the compounds are administered by a continuous infusion. This may reflect a transition in blood concentration of each metabolite resulting from the administration of UFT. Similar results were also obtained with respect to 5-FU. It was suggested that UFT has a stronger angiogenesis-inhibitory effect than did other fluorinated pyrimidines, partly due to its pharmacokinetic properties characterized by maintaining of higher and long-lasting blood levels of 5-FU and partly due the inhibitory effects derived from gamma-hydroxybutyric acid and gamma-butyrolactone, UFT-specific metabolites.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Renales/irrigación sanguínea , Neoplasias Renales/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , 4-Butirolactona/uso terapéutico , Animales , División Celular/efectos de los fármacos , Cámaras de Difusión de Cultivos , Relación Dosis-Respuesta a Droga , Floxuridina/uso terapéutico , Fluorouracilo/uso terapéutico , Hidroxibutiratos/uso terapéutico , Masculino , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Tegafur/uso terapéutico , Células Tumorales Cultivadas , Uracilo/uso terapéuticoRESUMEN
Local eosinophilia has been linked to the pathogenesis of the inflammatory aspect of allergic diseases. The present study found that co-injection of D10G4.1 (D10) cells, a murine Th2 clone, with conalbumin (CA) into the peritoneal cavity of AKR/J mice increased the number of peritoneal eosinophils. The accumulation of eosinophils reached a maximum level at 24 to 48 hr and was accompanied by a marked increase in the number of neutrophils and a minor increase in the number of mononuclear cells. D10-induced peritoneal eosinophilia was suppressed by administration of either anti-IL-4 and anti-IL-5 monoclonal antibodies in an additive manner or by cyclosporin A (CsA). Interestingly, suplatast tosilate (IPD-1151T), known to be antiallergic agent capable of suppressing IgE synthesis and chemical mediator release, but not disodium cromoglycate, selectively suppressed eosinophil accumulation. Taken together with the observation that CsA and IPD-1151T suppressed IL-4 and IL-5 production by CA-stimulated D10 cells in vitro, the present results strongly suggest that agents capable of down-regulating Th2 cell cytokine production may attenuate allergic inflammation by impairing the recruitment of eosinophils that is mediated by Th2 cells.
Asunto(s)
Eosinofilia/prevención & control , Antagonistas de los Receptores Histamínicos/farmacología , Células Th2/fisiología , Animales , Arilsulfonatos/farmacología , Células Cultivadas , Cromolin Sódico/farmacología , Ciclosporina/farmacología , Regulación hacia Abajo , Interleucina-4/fisiología , Interleucina-5/fisiología , Masculino , Ratones , Ratones Endogámicos AKR , Cavidad Peritoneal/citología , Compuestos de Sulfonio/farmacologíaRESUMEN
Treatment with UFT for spontaneous lung metastasis of murine renal carcinoma (RENCA) after resection of the primary tumor has resulted in significant prolongation of the life span of tumor-bearing animals. UFT inhibited the growth of metastatic nodules in the lung, apparently via decreased density of microvessels in the metastatic foci. Subsequent experiments used dorsal air sac assay to directly trace newly forming microvessels. UFT abrogated the process of angiogenesis, induced by the RENCA cells, in a dose-dependent manner. The inhibitory effect appeared to originate from tegafur, a component of UFT, and from its known metabolites: fluorouracil (5-FU), gamma-hydroxybutyric acid (GHB), and gamma-butyrolactone (GBL). The inhibition of angiogenesis by UFT appeared to be a common phenomenon, also observed in other human cancer cell lines characterized by an excessive production of vascular endothelial growth factor (VEGF)--such as gastric, lung, and colon cancers. In vitro analysis revealed that 5-FU and gamma-hydroxybutyric acid regulated VEGF-dependent responses of human umbilical vein endothelial cells. Dorsal air sac assay revealed that UFT, 5-FU, and gamma-hydroxybutyric acid strongly inhibited the angiogenesis induced by recombinant human VEGF. These data suggest that the antiangiogenic activity of UFT is at least partially associated with an ability of the metabolites of UFT to interfere with VEGF-dependent responses of vascular endothelial cells.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma de Células Renales/patología , Neoplasias Renales/patología , Neoplasias Pulmonares/secundario , Linfocinas/efectos de los fármacos , Neovascularización Patológica/fisiopatología , Sacos Aéreos/efectos de los fármacos , Sacos Aéreos/patología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/agonistas , Bioensayo , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/fisiopatología , Modelos Animales de Enfermedad , Factores de Crecimiento Endotelial/farmacología , Endotelio/citología , Endotelio/efectos de los fármacos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/fisiopatología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/fisiopatología , Linfocinas/farmacología , Ratones , Tegafur/administración & dosificación , Tegafur/agonistas , Venas Umbilicales/citología , Uracilo/administración & dosificación , Uracilo/agonistas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
We investigated the effect of UFT on a pulmonary metastasis after excision of the primary lesion, which was induced by implantation of murine renal carcinoma cells, RENCA. The cells were implanted into the left kidney of Balb/cA mice, and nephrectomy of the left kidney with a primary tumor was performed on day 10 after implantation. Administration of antitumor drugs, was started on day 13 [UFT (20 mg/kg, p.o., 5'-DFUR (24.6 mg/kg, p.o.), 5-FU (19 mg/kg, i.v.), TNP-470 (30 mg/kg, s.c.)]. In this metastasis model, the estimated mean survival time of the control group was 41.3 +/- 2.9 days. A significant life-prolonging effect was observed for UFT and 5-FU (T/C: 160.8%, 125.7%, respectively). An inhibitory effect on the growth of metastatic tumors in the lung was detected for both UFT and TNP-470 (TWI: 55.5%, 48.7%, respectively), but not 5'-DFUR. In a dorsal air sac (DAS) model, UFT abrogated angiogenesis induced by RENCA in a dose-dependent manner. These data suggest that the life-prolonging effect of UFT results from the continuous exposure of a tumor to its cytotoxic effects and anti-angiogenic activity.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/secundario , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Neovascularización Patológica/prevención & control , Animales , Carcinoma de Células Renales/sangre , Neoplasias Renales/patología , Neoplasias Pulmonares/irrigación sanguínea , Ratones , Ratones Endogámicos BALB C , Tegafur/administración & dosificación , Uracilo/administración & dosificaciónRESUMEN
We investigated the effects of UFT and its metabolites, GHB and GBL, on angiogenesis induced by tumor cells in a dorsal air sac (DAS) assay in mice. Five tumor cell lines (murine renal carcinoma; RENCA, human gastric cancer; 4-1ST, human small-cell lung carcinoma; LX-1, and human colon carcinoma; DLD-1, KM-20C) were used in the DAS assay. In this model, UFT demonstrated a significant anti-angiogenic activity in a dose-dependent manner while 5-FU (19 mg/kg/day) and 5'-DFUR (200 mg/kg/day) were less effective. Moreover, tegafur (FT), a component of UFT, and gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL), in vivo metabolites of UFT, inhibited angiogenesis induced by RENCA cells. The inhibitory effects of 5-FU, GHB, and GBL on angiogenesis were increased with administration by continuous infusion, providing a suitable pharmacokinetic profile. These results suggest that GHB and GBL are involved in the expression of anti-angiogenic activity of UFT.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Renales/irrigación sanguínea , Neoplasias Pulmonares/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , Neoplasias Gástricas/irrigación sanguínea , 4-Butirolactona/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Femenino , Humanos , Neoplasias Renales/patología , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos BALB C , Oxibato de Sodio/metabolismo , Neoplasias Gástricas/patología , Tegafur/metabolismo , Tegafur/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Uracilo/metabolismo , Uracilo/farmacologíaRESUMEN
Nuclear factor-kappa B (NF-kappa B) plays a broad role in gene regulation, but it is not evident whether NF-kappa B acts as a messenger system for germline C epsilon transcription. We report here that the signaling cascade triggered by interleukin-4 (IL-4) or anti-CD40 monoclonal antibody (mAb) participates in NF-kappa B activation responsible for germline C epsilon transcription in a human Burkitt lymphoma B cell line, DND39. Both IL-4 and anti-CD40 mAb induced activation of phosphatidylinositol 3-kinase (PI3-kinase), translocation of a zeta isoform of protein kinase C, and nuclear expression of NF-kappa B. All such events were abrogated by treatment with LY294002, a specific inhibitor of PI3-kinase. In addition, N-acetyl-L-cysteine (NAC), a potent antioxidant, decreased NF-kappa B activation caused by IL-4, anti-CD40 mAb, or their combination. NAC was also effective in diminishing germline C epsilon transcription, and its potency was higher in cultures costimulated with IL-4 and anti-CD40 mAb than in those stimulated with IL-4 alone. These results indicate that IL-4 and ligation of CD40 induce NF-kappa B expression via at least a mechanism dependent on the PI3-kinase pathway and suggest that NF-kappa B sensitive to NAC may play a role in regulating germline C epsilon transcription.
Asunto(s)
Linfocitos B/metabolismo , Regiones Constantes de Inmunoglobulina/genética , FN-kappa B/fisiología , Transcripción Genética , Acetilcisteína/farmacología , Antioxidantes/farmacología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfoma de Burkitt , Antígenos CD40/inmunología , Núcleo Celular/metabolismo , Activación Enzimática , Regulación de la Expresión Génica , Humanos , Interleucina-4/inmunología , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteína Quinasa C/metabolismo , Células Tumorales CultivadasRESUMEN
The binding site for nuclear factor-kappaB (NF-kappaB) is present at the promoter region of the germline Cepsilon gene, but there is little information on whether this factor is involved in regulating IgE synthesis by human B cells. Accordingly, we studied the role of NF-kappaB in germline Cepsilon transcription by using two human Burkitt's lymphoma B cell lines, DND39 and DG75. In both cell lines, n-acetyl-L-cysteine (NAC), a potent thiol antioxidant, inhibited the triggering of the nuclear expression of NF-kappaB by IL-4 and by anti-CD40 monoclonal antibody. Although IL-4 activated signal transducers and activators of transcription (STAT) 6 in addition to NF-kappaB, NAC treatment or the transfection of decoy oligodeoxynucleotides for NF-kappaB or STAT6 only partly blocked IL-4-induced germline Cepsilon transcription. However, these two decoy oligodeoxynucleotides together almost completely abrogated IL-4-induced germline Cepsilon transcription. Of note, CD40-mediated enhancement of IL-4-driven germline Cepsilon transcription was markedly decreased by NAC or by a decoy oligodeoxynucleotide for NF-kappaB. The effect of NAC was also examined on deletional switch recombination underlying the isotype switch to IgE. NAC inhibited the generation of Smu/Sepsilon switch fragments in normal human B cells costimulated with IL-4 and anti-CD40 monoclonal antibody. It also abolished IL-4-induced upregulation of CD40 but promoted upregulation of CD23. These results suggest that coordination of NF-kappaB and STAT6 may be required for induction of germline Cepsilon transcription by IL-4, and that CD40-mediated NF-kappaB activation may be important in regulating both enhancement of germline Cepsilon transcription and class switching to IgE.
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Linfocitos B/metabolismo , Genes de Inmunoglobulinas , Inmunoglobulina E/genética , FN-kappa B/antagonistas & inhibidores , Antígenos CD40/fisiología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes de Cambio , Humanos , Interleucina-4/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Transcripción STAT6 , Transactivadores/metabolismo , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
IL-4 with the IgE-inducing activity is shown to upregulate the expression of IL-4 receptor (IL-4R) on lymphocytes. Antisense strategy was used that aimed at investigating the significance of IL-4-induced upregulation of IL-4R on B cells in human IgE production. When an antisense phosphorothioate oligodeoxynucleotide to IL-4R (S-oligo 1) was added to B cells together with IL-4, the agent selectively abrogated the upregulation of IL-4R without affecting its constitutive level expression. Moreover, S-oligo 1 had a suppressive effect on the T-cell-independent synthesis of IgE by B cells costimulated with IL-4 and anti-CD40 antibody. This suppression was accompanied by inhibition of mature but not germline C epsilon transcription. These findings indicate that constitutively expressed IL-4R provides a signal or signals responsible for the induction of germline C epsilon transcription and suggest that IL-4R upregulation may be required for the subsequent class switch recombination that leads to mature C epsilon transcription and IgE synthesis. The IL-4R signal transduction mechanism underlying germline C epsilon transcription was also analyzed in a human Burkitt lymphoma B-cell line, DND39. Induction of germline C epsilon transcripts in DND39 cells by IL-4 required at least two distinct signaling cascades. One was mediated by enhancement of tyrosine phosphorylation of a 57 kd protein associated with phospholipase C-gamma 1 (PLC-gamma 1) that resulted in PLC-gamma 1 activation, inositol lipid hydrolysis, and protein kinase C delta translocation. The other was dependent on phosphatidylinositol 3-kinase, whose activation induced protein kinase C zeta translocation. In fact, kinase inhibitors such as herbimycin A, K-252a, and wortmannin were effective in inhibiting IL-4-induced germline C epsilon transcription. Therefore, in addition to activation of protein tyrosine kinases, coordinated actions of PLC-gamma 1 and phosphatidylinositol 3-kinase may be involved in IL-4-driven germline C epsilon transcription in DND39 cells.
Asunto(s)
Antígenos CD/fisiología , Linfocitos B/fisiología , Inmunoglobulina E/biosíntesis , Interleucina-4/fisiología , Receptores de Interleucina/fisiología , Linfocitos B/metabolismo , Linfocitos B/ultraestructura , Humanos , Interleucina-4/farmacología , Receptores de Interleucina-4 , Transducción de Señal/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Induction of human IgE synthesis in B cells requires, in addition to IL-4 or IL-13, a second signal provided by CD40 ligand (CD40L) on activated Th2-type CD4+ T cells that do not or weakly express Fas ligand (FasL). Mast cells and basophils also produce IL-4 or IL-13 and express CD40L after immunologic or pharmacologic stimulation, although it is unknown whether these cells express FasL. This study investigated the capacity of KU812 cells, a human basophilic cell line, to produce IL-4 and IL-13, to express CD40L and FasL, and to induce IgE and IgG4 synthesis in human normal B cells. Upon stimulation of KU812 cells with either phorbol myristate acetate (PMA) or ionomycin (Iono), IL-4, but not IL-13, was produced in response to Iono, while IL-13, but not IL-4, was inducible by PMA. Moreover, both the time courses of IL-4 and IL-13 production and their amounts secreted were different; IL-4 production was transient, IL-13 production gradually increased, and IL-13 was heavily secreted as compared with IL-4. The combination of PMA and Iono (PMA/Iono) induced higher production of IL-4 or IL-13 than did Iono or PMA alone. KU812 cell-derived IL-4 and IL-13 had the ability to cause CD23 expression on B cells. PMA/Iono also up-regulated CD40L expression and induced a very low level expression of FasL. KU812 cells that had been activated by PMA/Iono followed by fixation could induce IgE and IgG4 synthesis in B cells in the presence of recombinant IL-4 or IL-13. This contact-dependent induction of IgE was completely abrogated by adding anti-CD40L MoAb or soluble CD40, whereas anti-FasL antibody did not significantly affect IgE production. These results indicate that activated KU812 cells produce biologically active IL-4 and IL-13, express functional CD40L, and exhibit weak induction of FasL, thereby supporting sufficient IgE production by B cells.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Basófilos/metabolismo , Inmunoglobulina E/biosíntesis , Antígenos CD40/biosíntesis , Ligando de CD40 , Proteína Ligando Fas , Humanos , Interleucina-13/biosíntesis , Interleucina-4/biosíntesis , Cinética , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Ligandos , Glicoproteínas de Membrana/biosíntesis , Células Tumorales Cultivadas , Receptor fas/biosíntesisRESUMEN
Nuclear factor-kappa B (NF-kappa B) is a transcription factor that binds to the consensus DNA sequence in the cis-acting elements of various genes. Although NF-kappa B activates the expression of many genes involved in immune and inflammatory responses, little is known about the role of NF-kappa B activation in the induction of IgE synthesis in human B cells. Therefore we first examined the participation of NF-kappa B in germline C epsilon transcription in a human Burkitt lymphoma B cell line, DND39. Stimulation of DND39 cells with IL-4 or anti-CD40 monoclonal antibody (mAb) activated phosphatidylinositol 3-kinase and subsequently induced nuclear expression of NF-kappa B, which was identified by electrophoretic mobility shift assays. n-Acetyl-L-cysteine (NAC), a potent antioxidant, blocked NF-kappa B activation caused by IL-4 and by anti-CD40 mAb. Although inhibition of IL-4-driven germline C epsilon transcription by NAC was not sufficient, the agent remarkably diminished anti-CD40 mAb-mediated up-regulation of germline C epsilon transcription. Second, we studied the effect of NAC on IgE synthesis in human normal B cells costimulated with IL-4 and anti-CD40 mAb. NAC was effective in inhibiting mature C epsilon transcription and IgE synthesis in the T cell-independent culture system. However, NAC did not significantly affect the spontaneous production of IgE by atopic B cells. These results indicate that NF-kappa B activity is commonly inducible in DND39 cells by IL-4 and anti-CD40 mAb and suggest that NF-kappa B sensitive to NAC may play a role in regulating IgE synthesis in B cells.
Asunto(s)
Linfocitos B/metabolismo , Regulación de la Expresión Génica/inmunología , Inmunoglobulina E/biosíntesis , FN-kappa B/genética , FN-kappa B/fisiología , Linfocitos B/efectos de los fármacos , Humanos , FN-kappa B/biosíntesisRESUMEN
Association of interleukin-4 receptor (IL-4R) with phosphatidylinositol 3-kinase (PI3-kinase) has been demonstrated as the proximal event of IL-4 signaling. We investigated the role of this enzyme in the IL-4 signaling pathway in a human Burkitt lymphoma B cell line, DND39, that expresses germline C epsilon transcripts in response to IL-4. Stimulation of DND39 cells with IL-4 resulted in an accumulation of PI-3-monophosphate as well as a decrease of PI-4,5-bisphosphate, which were abrogated by wortmannin, a potent inhibitor of PI3-kinase. Activation of PI3-kinase was further confirmed by the finding that IL-4 caused an increase in PI3-kinase activity coimmunoprecipitated with anti-IL-4R and with anti-JAK3 kinase antibodies. As a possible downstream event of PI3-kinase activation, the translocation of a zeta isoform of protein kinase C (PKC) from the cytosol to the membrane fraction was observed after IL-4 stimulation, and wortmannin also suppressed this translocation. Moreover, IL-4-induced expression of germline C epsilon transcription was inhibited not only by wortmannin, but also by a PKC inhibitor, K252a. These results suggest that the signaling pathway involving PI3-kinase and PKC zeta plays an important role in induction of germline C epsilon transcription in DND39 cells by IL-4.
Asunto(s)
Regiones Constantes de Inmunoglobulina/biosíntesis , Inmunoglobulina E/biosíntesis , Interleucina-4/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Transcripción Genética/genética , Transcripción Genética/fisiología , Secuencia de Aminoácidos , Androstadienos/farmacología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Linfoma de Burkitt/metabolismo , Carbazoles/farmacología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Humanos , Regiones Constantes de Inmunoglobulina/genética , Inmunoglobulina E/genética , Alcaloides Indólicos , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/efectos de los fármacos , Proteína Quinasa C/análisis , Proteína Quinasa C/antagonistas & inhibidores , ARN Mensajero/análisis , Células Tumorales Cultivadas , WortmaninaRESUMEN
BACKGROUND: The role of CD8(+) T cells in IgE synthesis remains unclear. OBJECTIVE: The aim of this study was to investigate IL-4 production and CD40 ligand expression by human CD8(+) T cells. METHODS: We conducted functional and phenotypic analyses of human T cells in peritoneal washings from severe combined immunodeficiency mice reconstituted with PBMCs from normal and atopic human donors. We also examined the expression of IL-4 and CD40 ligand by CD8(+) T cells from a patient with adenosine deaminase deficiency who received autologous T cell-directed gene therapy. RESULTS: Transfer of atopic cells into the mice caused production of IgE and IgG with increased expression of IL-4 and CD40 ligand mRNA. In addition, both intracellular IL-4 and cell surface CD40 ligand were detected in CD8(+) and in CD4(+) T cells. CD8(+) T-cell lines generated from the patient's T cells carrying the adenosine deaminase gene expressed not only IL-4 mRNA and protein but also CD40 ligand mRNA and protein after being stimulated with an anti-CD3 mAb. After anti-CD3 stimulation and paraformaldehyde fixation, CD8(+) T cells induced IgE synthesis by normal human B cells in the presence of recombinant IL-4. CONCLUSION: Taken together, these results demonstrate that IL-4-producing and CD40 ligand-expressing CD8(+) cells are detectable among human T cells and suggest that such cells may promote IgE production by B cells under some conditions.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Interleucina-4/metabolismo , Glicoproteínas de Membrana/biosíntesis , Adenosina Desaminasa/deficiencia , Animales , Ligando de CD40 , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/fisiología , Línea Celular , Terapia Genética , Humanos , Hipersensibilidad Inmediata/sangre , Inmunoglobulina E/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Ratones , Ratones SCID , Cavidad Peritoneal/citología , FenotipoRESUMEN
UFT, a drug composed of uracil and tegafur at the molar ratio of 4:1, is an orally active agent for the treatment of a wide variety of malignant tumours. Using a murine dorsal air sac (DAS) assay, we have previously shown that UFT and its metabolites, gamma-hydroxybutyric acid (GHB) and 5-fluorouracil (5-FU), inhibited the angiogenesis induced by murine renal cell carcinoma. Here we report that UFT was more effective than other fluorinated pyrimidines such as 5-FU and doxifluridine (5'-DFUR) in blocking the angiogenic responses elicited by five human cancer cell lines which produced high levels of vascular endothelial growth factor (VEGF), but no detectable fibroblast growth factor-2 (FGF-2) in vitro. In contrast, UFT was unable to block the angiogenic response to one human gastric cancer cell line which produced both VEGF and FGF-2 in vitro. However, the production or secretion of VEGF by these cells was unaffected by GHB and 5-FU treatment. Interestingly, GHB suppressed the chemotactic migration and tube formation of human umbilical vein endothelial cells (HUVECs) stimulated by VEGF, without inhibiting their DNA synthesis. Since GHB did not affect the FGF-2-driven activities in HUVECs, its action appears to be VEGF-selective. On the other hand, 5-FU inhibited DNA synthesis and migration of HUVECs stimulated by both VEGF and FGF-2, and tube formation driven by VEGF, suggesting that 5-FU is cytotoxic to endothelial cells. The inhibitory effects of 5-FU, and especially those GHB, were reproduced under in vivo condition using the DAS assay. The VEGF-mediated angiogenesis was significantly inhibited by UFT, 5-FU, and especially by GHB. We propose that the selective inhibitory effects of GHB on VEGF-mediated responses of endothelial cells are involved in the anti-angiogenic activity of UFT.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antimetabolitos Antineoplásicos/farmacocinética , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Fluorouracilo/farmacología , Linfocinas/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Neovascularización Patológica/prevención & control , Profármacos/farmacocinética , Oxibato de Sodio/farmacología , Tegafur/farmacocinética , Uracilo/farmacocinética , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma/metabolismo , Carcinoma/patología , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Quimiotaxis/efectos de los fármacos , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Replicación del ADN/efectos de los fármacos , Combinación de Medicamentos , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Femenino , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Floxuridina/farmacología , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Linfocinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Proteínas de Neoplasias/metabolismo , Prótesis e Implantes , Proteínas Recombinantes/antagonistas & inhibidores , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Células Tumorales Cultivadas/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
By generating human mast cells and basophils from umbilical cord blood mononuclear cells cultured in the presence of appropriate cytokines, we investigated whether these two cultured cells could provide the cytokine and cell contact signals that are required to induce IgE synthesis in B cells. To activate cultured mast cells and basophils, cross-linking of cell surface high-affinity IgE receptor (Fc epsilonRI) was performed with specific antigen after sensitization with murine IgE. Upon Fc epsilonRI stimulation, basophils, but not mast cells, secreted significant amounts of immunoreactive IL-4 and IL-13 and expressed detectable CD40 ligand (CD40L) and a very low level of Fas ligand (FasL). These observations at the protein level were consistent with the data obtained at the gene transcriptional level, except for the faint expression of only IL-13 mRNA in mast cells. When added to normal human B cells, activated basophils induced IgE and IgG4 synthesis as well as soluble CD23 release. In contrast, neither IgE nor IgG4 synthesis could be induced by the interaction of B cells with activated mast cells, even in the presence of recombinant IL-4. The induction of IgE synthesis by activated basophils was completely abrogated by two neutralizing MoAbs against IL-4 and IL-13 and by a soluble form of CD40. This abrogation was accompanied by abolished mature C epsilon transcription in both cases. Addition of anti-FasL MoAb, however, did not significantly affect IgE induction mediated by activated basophils. These results demonstrate that unlike cultured mast cells, cultured basophils produce biologically active IL-4 and IL-13 and express functional CD40L after Fc epsilonRI stimulation, thereby contributing to IgE production by B cells, and suggest that relatively weak expression of FasL by cultured basophils is not involved in IgE regulation.
Asunto(s)
Linfocitos B/inmunología , Basófilos/inmunología , Inmunoglobulina E/inmunología , Activación de Linfocitos , Mastocitos/inmunología , Ligando de CD40 , Células Cultivadas , Humanos , Inmunoglobulina E/biosíntesis , Interleucina-13/inmunología , Interleucina-4/inmunología , Glicoproteínas de Membrana/inmunologíaRESUMEN
BACKGROUND: Exposure of atopic patients to a specific allergen evokes an immediate response which is followed, in many cases, by a late phase reaction (LPR) some hours later. Here we have examined the immunological mechanisms required for the expression of cutaneous LPR in mice. METHODS: BALB/c mice were immunized by i.p. injection of ovalbumin (OVA) and alum actively or by i.v. injection of anti-OVA IgE monoclonal antibody (mAb) passively. After challenge by intradermal injection of OVA into ears, the changes in ear thickness, the number of eosinophils, and the levels of IL-4 and IFN-gamma protein at the site of antigen challenge were examined. RESULTS: Actively immunized mice developed a biphasic response at the site of OVA injection, while mice passively immunized with IgE anti-OVA mAb displayed a strong early response but no LPR. Cell transfer experiments using BALB/c nu/nu mice revealed that both OVA-specific IgE mAb and OVA-primed CD4 T cells were required to evoke LPR. Moreover, LPR was associated with increased levels of IL-4 production concomitant with reduced IFN-gamma production and was abolished by pretreatment with anti-IL-4 neutralizing mAb. CONCLUSION: It is suggested that murine cutaneous LPR against OVA is a type 2 inflammatory response in which both IgE antibodies and CD4 T cells play an obligatory role.