RESUMO
Avian leukosis virus subgroup J (ALV-J) is an oncogenic virus causing hemangiomas and myeloid tumors in chickens. Interleukin-6 (IL-6) is a multifunctional pro-inflammatory interleukin involved in many types of cancer. We previously demonstrated that IL-6 expression was induced following ALV-J infection in chickens. The aim of this study is to characterize the mechanism by which ALV-J induces IL-6 expression, and the role of IL-6 in tumor development. Our results demonstrate that ALV-J infection increases IL-6 expression in chicken splenocytes, peripheral blood lymphocytes, and vascular endothelial cells. IL-6 production is induced by the ALV-J envelope protein gp85 and capsid protein p27 via PI3K- and NF-κB-mediated signaling. IL-6 in turn induced expression of vascular endothelial growth factor (VEGF)-A and its receptor, VEGFR-2, in vascular endothelial cells and embryonic vascular tissues. Suppression of IL-6 using siRNA inhibited the ALV-J induced VEGF-A and VEGFR-2 expression in vascular endothelial cells, indicating that the ALV-J-induced VEGF-A/VEGFR-2 expression is mediated by IL-6. As VEGF-A and VEGFR-2 are important factors in oncogenesis, our findings suggest that ALV-J hijacks IL-6 to promote tumorigenesis, and indicate that IL-6 could potentially serve as a therapeutic target in ALV-J infections.
Assuntos
Vírus da Leucose Aviária/metabolismo , Leucose Aviária/enzimologia , Células Endoteliais/enzimologia , Interleucina-6/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Baço/enzimologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Leucose Aviária/genética , Leucose Aviária/virologia , Vírus da Leucose Aviária/genética , Vírus da Leucose Aviária/patogenicidade , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Transformação Celular Viral , Células Cultivadas , Galinhas , Células Endoteliais/virologia , Interações Hospedeiro-Patógeno , Interleucina-6/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Baço/virologia , Fatores de Tempo , Transfecção , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Avian leukosis virus (ALV) is an enveloped and oncogenic retrovirus. Avian leukosis caused by the members of ALV subgroups A, B and J has become one of the major problems challenging the poultry industry in China. However, the cellular factors such as signal transduction pathways involved in ALV infection are not well defined. In this study, our data demonstrated that ALV-J strain NX0101 infection in primary chicken embryo fibroblasts or DF-1 cells was correlated with the activity and phosphorylation of Akt. Akt activation was initiated at a very early stage of infection independently of NX0101 replication. The specific phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin could suppress Akt phosphorylation, indicating that NX0101-induced Akt phosphorylation is PI3K-dependent. ALV-A strain GD08 or ALV-B strain CD08 infection also demonstrated a similar profile of PI3K/Akt activation. Treatment of DF-1 cells with the drug 5-(N, N-hexamethylene) amiloride that inhibits the activity of chicken Na(+)/H(+) exchanger type 1 significantly reduced Akt activation induced by NX0101, but not by GD08 and CD08. Akt activation triggered by GD08 or CD08 was abolished by clathrin-mediated endocytosis inhibitor chlorpromazine. Receptor-mediated endocytosis inhibitor dansylcadaverine had a negligible effect on all ALV-induced Akt phosphorylation. Moreover, viral replication of ALV was suppressed by LY294002 in a dose-dependent manner, which was due to the inhibition of virus infection by LY294002. These data suggest that the activation of the PI3K/Akt signalling pathway by exogenous ALV infection plays an important role in viral entry, yet the precise mechanism remains under further investigation.
Assuntos
Vírus da Leucose Aviária/fisiologia , Leucose Aviária/enzimologia , Fosfatidilinositol 3-Quinase/metabolismo , Doenças das Aves Domésticas/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Leucose Aviária/genética , Leucose Aviária/metabolismo , Leucose Aviária/virologia , Vírus da Leucose Aviária/genética , Linhagem Celular , Embrião de Galinha , Galinhas , China , Fosfatidilinositol 3-Quinase/genética , Fosforilação , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/virologia , Proteínas Proto-Oncogênicas c-akt/genética , Replicação ViralRESUMO
The partially purified RNA polymerase II from chicken leukemia cells (Chuang R. Y., Chuang L. F. & Israel M. (1986) Biochem. Pharmacol. 35, 1293-1297) contained multiple subunits with molecular masses (in Da) ranging from 220,000 to 24,000, as shown by SDS-polyacrylamide gel electrophoresis. The enzyme was further purified through phosphocellulose column and fractions containing the enzyme activity were collected and concentrated 400-fold through a microconcentrator. The microconcentrator contained a membrane with a molecular weight cutoff around 30,000 and, hence, removed the 24,000 Da polypeptide from the enzyme. It was found that the resulting enzyme retained all the catalytic activity as compared to the enzyme preparation before the concentration step, suggesting that the stoichiometric amount of the 24,000 Da polypeptide is not required for RNA synthesis activity with a denatured DNA template.
Assuntos
RNA Polimerase II/metabolismo , Animais , Leucose Aviária/enzimologia , Galinhas , Eletroforese em Gel de Poliacrilamida/métodos , Estabilidade Enzimática , UltrafiltraçãoRESUMO
Avian erythroblastosis virus (AEV) is a replication-defective retrovirus that causes erythroblastosis and sarcomas in chickens and transforms immature erythroid cells and fibroblasts in culture. AEV encodes two oncogenes, v-erbA and v-erbB, whose products are closely related to the thyroxine receptor and the epidermal growth factor receptor, respectively. Since tyrosine protein kinases have been implicated in the process of normal growth signal transduction, we wished to study the possible consequences of the expression of these mutated, growth-regulating receptor genes on the activity of the cellular tyrosine kinase pp60c-src. A continuous cell line from AEV-infected quail embryo fibroblasts was derived that exhibited a typical transformed phenotype and expressed the viral oncogene products, p75gag-erbA and gp66-68erbB. Using an immune-complex kinase assay, we found that the specific activity of pp60c-src in AEV-transformed quail cells was decreased by a factor of 6-30 relative to that found in uninfected quail cells. A concomitant 50-80% reduction of 32Pi incorporation into the pp60c-src protein from radiolabeled, transformed cells was also observed, indicating a relationship between hypophosphorylation and diminished enzyme activity. Partial proteolytic phosphopeptide analysis revealed a decrease in phosphorylation of both serine- and tyrosine-containing peptides, suggesting an activation of specific phosphatases or inhibition of specific kinases in the AEV-transformed quail cells. Similar results were found in pp60c-src precipitated from AEV-transformed chicken and rat cells.
Assuntos
Leucose Aviária/enzimologia , Proteínas Tirosina Quinases/metabolismo , Proteínas dos Retroviridae/metabolismo , Alpharetrovirus , Animais , Galinhas , Coturnix , Regulação da Expressão Gênica , Proteína Oncogênica pp60(v-src) , Oncogenes , Mapeamento de Peptídeos , Fosforilação , Ratos , Serina/análise , Tirosina/análiseRESUMO
The new adriamycin (ADR) derivatives, 3'-deamino-3'-(4"-morpholinyl)adriamycin (MRA) and 3'-deamino-3'-(3"-cyano-4"-morpholinyl)-adriamycin (MRA-CN), were studied, in comparison with ADR, for their inhibitory effects on DNA and RNA syntheses in vitro using isolated DNA and RNA polymerases from both Escherichia coli and chicken (myeloblastosis) leukemia cells. Under standard assay conditions, MRA and ADR demonstrated a similar inhibitory effect on the enzymes, whereas MRA-CN showed a slightly greater inhibitory activity than ADR or MRA at low drug concentrations, but with the inhibitory effect plateauing when drug concentration reached 10 microM. Both the A and B forms of the MRA-CN diastereoisomers were effective as inhibitors. Kinetic studies of the inhibition showed that unlike ADR, MRA-CN inhibition of DNA or RNA synthesis could not be reversed by increasing DNA-template concentration in the reaction mixture. Whereas ADR or MRA relaxed completely 1 microgram of pBR322 supercoiled DNA at a drug concentration of 15 microM, MRA-CN, at 60 microM, produced a mixture of intermediate relaxation forms of the DNA. A complete relaxation was achieved at 300 microM MRA-CN. Both DNA relaxation and fluorescence spectroscopic studies indicated that DNA-drug interactions occurred in the following order: ADR (or MRA) greater than MRA-CN greater than N-trifluoroacetyl-ADR-14-O-hemiadipate (a DNA-nonbinding anthracycline).
Assuntos
Leucose Aviária/enzimologia , DNA Polimerase II/antagonistas & inibidores , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacologia , RNA Polimerase II/antagonistas & inibidores , Animais , Galinhas , DNA/biossíntese , DNA/efeitos dos fármacos , DNA Super-Helicoidal/efeitos dos fármacos , Escherichia coli/enzimologia , RNA/biossíntese , Espectrometria de FluorescênciaRESUMO
Purification of RNA polymerase II from chicken myeloblastosis (leukemia) cells to homogeneity and subsequent structural analysis of the purified enzyme revealed that the enzyme contained seven polypeptides with molecular masses ranging from 27 KDa to 220 KDa. Inclusion of protease inhibitors in the buffer system during purification significantly increased the molar ratio of the largest (220 KDa) polypeptide to the second largest (180 KDa) polypeptide. However, proteolytic conversion of the 220 KDa to 180 KDa polypeptide did not inhibit the DNA binding activity of the enzyme. The enzyme, after dissociation into subunits in a SDS-polyacrylamide gel containing urea was blotted onto a nitrocellulose filter. The filter was incubated with 32P-labeled calf thymus DNA and both the 220 KDa and 180 KDa polypeptides of the enzyme bind DNA, suggesting that the DNA-binding site of the enzyme resides on the 180 KDa polypeptide of the largest subunit.
Assuntos
Leucose Aviária/enzimologia , DNA/metabolismo , RNA Polimerase II/isolamento & purificação , Animais , Sítios de Ligação , Galinhas , Substâncias Macromoleculares , Peso Molecular , Ligação Proteica , RNA Polimerase II/metabolismoRESUMO
The biochemical mechanism of the N-trifluoroacetyladriamycin-14-O-hemiadipate-induced inhibition of RNA synthesis in vitro by chicken (myeloblastosis) leukemia RNA polymerase II was studied. The inhibition was found to be dependent upon preincubation of the drug with the enzyme prior to enzyme assays, suggesting that drug-enzyme interactions occur. A drug-enzyme association complex was subsequently isolated through glycerol gradient sedimentation and further characterized by fluorescent microscopic studies. The drug was dissociated from the complex upon sodium dodecyl sulfate (SDS)-gel electrophoresis, revealing the non-covalent nature of the binding between the drug and the RNA polymerase.
Assuntos
Leucose Aviária/enzimologia , Doxorrubicina/análogos & derivados , RNA Polimerase II/antagonistas & inibidores , Animais , Galinhas , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Eletroforese em Gel de Poliacrilamida , Microscopia de Fluorescência , RNA Polimerase II/metabolismo , RNA Neoplásico/biossíntese , Fatores de TempoRESUMO
An improved indirect ELISA test for detection of antibodies to the reverse transcriptase (revertase) is described. The sensitivity of the revertase ELISA test was compared to that of the revertase inhibition test. Serum samples of various origin and sera of specific pathogen free (SPF) hens were examined for the revertase antibodies. The presence of these antibodies in the sera of SPF chickens is discussed.
Assuntos
Anticorpos Antivirais/análise , Vírus da Leucose Aviária/enzimologia , Vírus da Mieloblastose Aviária/enzimologia , DNA Polimerase Dirigida por RNA/imunologia , Animais , Leucose Aviária/enzimologia , Leucose Aviária/imunologia , Vírus da Mieloblastose Aviária/imunologia , Galinhas , Ensaio de Imunoadsorção Enzimática , Feminino , Organismos Livres de Patógenos EspecíficosRESUMO
Four glycosidases (beta-galactosidase, alpha-mannosidase, alpha-fucosidase and beta-N-acetylglucosaminidase) were studied in chicken normal and regenerating liver, in turkey poult liver and in virus induced avian tumors--chicken hepatoma (strain Mc-29), Rous sarcoma (strain Schmidt-Ruppin) and turkey poult hemocytoblastoma nodules (strain Mc-31). The multiple forms of beta-N-acetylglucosaminidase were assayed as well. A particular enzyme pattern was found in the tumor lines under investigation. A characteristic property of hepatoma cells was the elevation of beta-galactosidase activity and of the former enzyme and that of beta-N-acetylglucosaminidase for the hemocytoblastoma. In Rous sarcoma the glycosidase activities (except that of alpha-fucosidase) were much lower, compared to the other two solid tumors. All enzyme activities were compared with those in the normal liver of the corresponding avian species, and with the liver of tumor bearing fowls and with regenerating chicken liver. Unlike the rat liver in the avian normal and tumor tissues the percentual ratio between the multiple forms A and B of beta-N-acetylglucosaminidase was found to be 30:70%.
Assuntos
Leucose Aviária/enzimologia , Glicosídeo Hidrolases/metabolismo , Neoplasias Hepáticas Experimentais/enzimologia , Regeneração Hepática , Fígado/enzimologia , Sarcoma Aviário/enzimologia , Acetilglucosaminidase/metabolismo , Animais , Galinhas/metabolismo , Neoplasias Hepáticas Experimentais/etiologia , Manosidases/metabolismo , Ratos , Perus/metabolismo , alfa-L-Fucosidase/metabolismo , alfa-Manosidase , beta-Galactosidase/metabolismoRESUMO
In rapidly frozen livers from chickens affected with myeloblastosis and Marek's disease and from unaffected control birds there exists a strong correlation between catalase activity and catalase Electron Paramagnetic Resonance (EPR) signal intensities. The diseased chickens had activities and signals reduced to as little as 10% of control values. There were no changes in the EPR parameters in diseased liver and the data support the hypothesis that the lowering in activity is due to lowered catalase levels rather than to catalase inhibition. The rate of transformation of catalase to catalase-formate in liver was studied by freeze-clamping liver in anaesthetised chickens, then warming to 37 degrees for 1 or 2 minutes anaerobiosis, and then refreezing. The only difference of significance in this transformation between diseased and normal livers was the greater percentage of total catalase present as catalase-formate (approximately + 15%) in aerobic diseased liver, which may indicate a lowered production of hydrogen peroxide, relative to formate, in these livers. The rate of transformation was far faster in chickens (t1/2 less than 1 min) than in the rat (t1/2 = 7.7 min).
Assuntos
Leucose Aviária/enzimologia , Catalase/metabolismo , Fígado/enzimologia , Doença de Marek/enzimologia , Anaerobiose , Animais , Vírus da Mieloblastose Aviária , Galinhas , Espectroscopia de Ressonância de Spin EletrônicaRESUMO
N-Trifluoroacetyladriamycin-14-O- Hemiadipate (AD 143), a new derivative of adriamycin with greater antitumor activity and lower cardiotoxicity, was shown not to interact with DNA and yet inhibit the activities of both RNA polymerases I and II of chicken myeloblastosis cells in vitro with ID50 values equal to 6.5 microM and 7 microM, respectively. On the other hand, an approximately 35-fold higher concentration of AD 143 was required to cause a similar inhibition of the activity of DNA polymerase alpha from chicken myeloblastosis cells. Under the same assay conditions, AD 143 had even less effect on either RNA polymerase or DNA polymerase I of E. coli cells (ID50 greater than 265 microM for both enzymes). These studies suggest that AD 143, in contrast to its parental drug adriamycin, may have a selective inhibitory effect against eukaryotic RNA polymerases.
Assuntos
Antineoplásicos/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Doxorrubicina/análogos & derivados , Animais , Leucose Aviária/enzimologia , Galinhas , Doxorrubicina/farmacologia , Escherichia coli/enzimologia , Hidrólise , Inibidores da Síntese de Ácido Nucleico , Espectrometria de FluorescênciaRESUMO
Only one DNA polymerase is present in the microsomal fraction of the cells producing AMV. Chromatographically purified enzyme shows the properties of revertase, that is it transcribes in DNA the information encoded in natural RNA. The enzyme possesses identical chromatographic characteristics and the same template specificity as the enzyme isolated from pure AMV virus. Thus the virus enzyme and the cellular DNA polymerase from the microsomal fraction cannot be differentiated on the basis of certain properties.
Assuntos
Leucose Aviária/enzimologia , DNA Polimerase Dirigida por DNA/isolamento & purificação , Células-Tronco Hematopoéticas/ultraestrutura , Microssomos/enzimologia , Animais , Vírus da Mieloblastose Aviária , Células Cultivadas , Galinhas , Microscopia Eletrônica , Microssomos/microbiologiaRESUMO
RNA-directed DNA polymerase was isolated from the liver, spleen, and myeloblasts of chickens that had been infected with virus of avian myeloblastosis. The enzyme was chromatographically purified from the myeloblasts and brought to about 1,000-fold concentration. The method consisted in cell fractionation, lysis of the microsomal fraction, chromatography on Sephadex G-200 and phosphocellulose, as well as ultracentrifugation in the glycerol gradient. The cellular DNA polymerases alpha and beta were clearly separated from the DNA polymerase of avian myeloblastosis virus and could be distinguished from one another by template-specific reactions. The viral DNA polymerase activities in the microsomal fractions of liver, spleen, and myeloblasts were compared with one another. The amount of avian myeloblastosis virus and related DNA polymerase recorded from the myeloblasts was about six times that in the liver and four times higher than that in the spleen. The procedure described, together with the use of cell fractionation and gel filtration, is an appropriate method for biochemical detection of avian oncorna viruses in cells.
Assuntos
Vírus da Leucose Aviária/enzimologia , Leucose Aviária/enzimologia , Vírus da Mieloblastose Aviária/enzimologia , DNA Polimerase Dirigida por DNA/isolamento & purificação , DNA Polimerase Dirigida por RNA/isolamento & purificação , Animais , Galinhas , DNA Polimerase I/isolamento & purificação , DNA Polimerase II/isolamento & purificação , Leucócitos/enzimologia , Fígado/enzimologia , Microssomos/enzimologia , Microssomos Hepáticos/enzimologia , Baço/enzimologiaRESUMO
Revertase present in the myeloblasts of chickens infected with avian myeloblastosis virus failed to synthetise DNA in vitro on the endogenous viral template. Enzyme inhibitors were not blocking this synthesis. It was rather impaired by incompletness of viral RNA in immature virus particles.
Assuntos
Vírus da Leucose Aviária/enzimologia , Leucose Aviária/enzimologia , Vírus da Mieloblastose Aviária/enzimologia , Medula Óssea/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Animais , Células da Medula Óssea , Transformação Celular Neoplásica , Galinhas , RNA Viral , Moldes GenéticosAssuntos
Leucose Aviária/imunologia , Vírus Oncogênicos/enzimologia , DNA Polimerase Dirigida por RNA/imunologia , Animais , Leucose Aviária/enzimologia , Vírus da Mieloblastose Aviária/enzimologia , Vírus da Mieloblastose Aviária/imunologia , Galinhas , Vírus Oncogênicos/imunologia , Inibidores da Transcriptase ReversaRESUMO
Chickens were treated with cyclophosphamide in order to induce nonspecific immunosuppression. Treated and untreated animals were injected with avian myeloblastosis virus (AMV) or myeloblasts at the age when a pronounced resistance to the disease is observed. Chickens treated with cyclophosphamide and then challenged with AMV developed acute myeloblastic leukemia in 70 percent. Similarly treated chickens transplanted with fresh AMV producing myeloblasts exhibited 30 percent incidence of myeloblastosis. In contrast, the control animals without treatment showed no myeloblastosis either after myeloblasts application or AMV injection. These results have shown that nonspecific immunosuppression by cyclophosphamide treatment strongly affects the expression of AMV in age-resistant chickens.
Assuntos
Vírus da Leucose Aviária/imunologia , Leucose Aviária/imunologia , Vírus da Mieloblastose Aviária/imunologia , Terapia de Imunossupressão , Adenosina Trifosfatases/metabolismo , Fatores Etários , Animais , Leucose Aviária/enzimologia , Células da Medula Óssea , Transplante de Medula Óssea , Galinhas , Ciclofosfamida , Transplante HomólogoRESUMO
Nuclear RNA polymerases of chicken myeloblastosis cells were solubilized and fractionated by diethylaminoethyl-Sephades A25 column chromatography. Both alpha-amanitin-insenstitive (polymerase I) and- sensitive (polymerase II) species were isolated. Polymerase activity, contained two peaks of enzyme (IIa and IIb), which were further purified by glycerol gradient centrifugation. The partially purified enzymes were characterized by their requirement of four nucleoside triphosphates and metal ions and by their sensitivity to several inhibitors. The enzymes were compared with RNA polymearases derived from normal chickent bone marrow cells,and the total extractable myeloblastosis than in bone marrow cells. Polymearse II from both cell types was shown to be sensitive to cytosine arabinoside triphosphate inhibiton.