RESUMEN
Oncolytic Newcastle disease virus (NDV) replicates selectively in most human tumor cells but not in normal cells. The relationship between tumorigenesis and the selective susceptibility of most tumor cells to oncolytic NDV replication is poorly understood. A multistage skin carcinogenesis model derived from non-tumorigenic HaCaT cells was used to systematically investigate the molecular mechanisms involved in the oncolytic NDV-sensitivity associated with tumorigenic transformation. No significant differences in interferon signaling were observed between the virus-sensitive tumor cells and the virus-resistant non-tumorigenic parental cells. Oncogenic H-Ras, which had been used for tumorigenic transformation, was shown to be necessary for virus replication but was not sufficient to render cells susceptible to NDV replication. By using an siRNA screening approach to search for virus-sensitizing genes in the tumorigenic cells, we could identify the small GTPase Rac1 as an oncogenic protein that is essential for NDV replication and anchorage-independent growth in tumorigenic cells. Furthermore, Rac1 expression was sufficient to render non-tumorigenic cells susceptible to NDV replication and to oncolytic cytotoxicity. This study establishes Rac1 as a link between tumorigenesis and oncolytic virus sensitivity in the HaCaT multistage skin carcinogenesis model.
Asunto(s)
Neoplasias/patología , Neoplasias/virología , Virus de la Enfermedad de Newcastle/fisiología , Virus Oncolíticos/fisiología , Replicación Viral , Proteína de Unión al GTP rac1/metabolismo , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Humanos , ARN Interferente Pequeño/genética , Proteína de Unión al GTP rac1/deficiencia , Proteína de Unión al GTP rac1/genética , Proteínas ras/metabolismoRESUMEN
Targeted disruptions of the mouse genes for cytokines, cytokine receptors, or components of cytokine signaling cascades convincingly revealed the important roles of these molecules in immunologic processes. Cytokines are used at present as drugs to fight chronic microbial infections and cancer in humans, and they are being evaluated as immune response modifiers to improve vaccines. Until recently, only a few avian cytokines have been characterized, and potential applications thus have remained limited to mammals. Classic approaches to identify cytokine genes in birds proved difficult because sequence conservation is generally low. As new technology and high throughput sequencing became available, this situation changed quickly. We review here recent work that led to the identification of genes for the avian homologs of interferon-alpha/beta (IFN-alpha/beta) and IFN-gamma, various interleukins (IL), and several chemokines. From the initial data on the biochemical properties of these molecules, a picture is emerging that shows that avian and mammalian cytokines may perform similar tasks, although their primary structures in most cases are remarkably different.
Asunto(s)
Pollos/inmunología , Citocinas/fisiología , Adyuvantes Inmunológicos/farmacología , Secuencia de Aminoácidos , Animales , Aves/genética , Aves/inmunología , Quimiocinas/genética , Quimiocinas/fisiología , Pollos/genética , Citocinas/genética , Citocinas/farmacología , Modelos Animales de Enfermedad , Humanos , Interferones/genética , Interferones/fisiología , Interleucinas/genética , Interleucinas/fisiología , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Virosis/inmunologíaRESUMEN
By searching a chicken EST database, we identified a cDNA clone that appeared to contain the entire open reading frame (ORF) of chicken interleukin-18 (ChIL-18). The encoded protein consists of 198 amino acids and exhibits approximately 30% sequence identity to IL-18 of humans and various others mammals. Sequence comparisons reveals a putative caspase-1 cleavage site at aspartic acid 29 of the primary translation product, indicating that mature ChIL-18 might consist of 169 amino acids. Bacterially expressed ChIL-18 in which the N-terminal 29 amino acids of the putative precursor molecule were replaced by a histidine tag induced the synthesis of interferon-gamma (IFN-gamma) in cultured primary chicken spleen cells, indicating that the recombinant protein is biologically active.
Asunto(s)
Clonación Molecular , ADN Complementario/aislamiento & purificación , Interleucina-18/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Pollos , Caballos , Humanos , Interleucina-18/fisiología , Ratones , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes/farmacología , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
Growth of tumors induced by Rous sarcoma virus (RSV) is controlled by alleles at the major histocompatibility complex locus in chickens, indicating that immunological host defense mechanisms play a major role. We show here that the resistance phenotype of CB regressor chickens can be partially reverted by treating the animals with a monoclonal antibody that neutralizes the major serotype of chicken type I interferon, ChIFN-alpha. Injection of recombinant ChIFN-alpha into susceptible CC progressor chickens resulted in a dose-dependent inhibition of RSV-induced tumor development. This treatment was not effective, however, in CC chickens challenged with a DNA construct expressing the v-src oncogene, suggesting that the beneficial effect of type I interferon in this system resulted from its intrinsic antiviral activity and probably not from indirect immunmodulatory effects. By contrast, recombinant chicken interferon-gamma strongly inhibited tumor growth when given to CC chickens that were challenged with the v-src oncogene, indicating that the two cytokines target different steps of tumor development.
Asunto(s)
Virus del Sarcoma Aviar/patogenicidad , Genes src , Interferón Tipo I/uso terapéutico , Sarcoma Aviar/prevención & control , Animales , Virus del Sarcoma Aviar/genética , Línea Celular , Pollos , Coturnix , ADN Viral/genética , Interferón gamma/uso terapéutico , Proteínas Recombinantes , Sarcoma Aviar/inmunología , Sarcoma Aviar/patología , Factores de Tiempo , TransfecciónRESUMEN
To counteract the host immune response, poxviruses have evolved secreted factors that bind cytokines and thereby neutralize their biological activities. The vaccinia virus B8R gene encodes a protein that neutralizes interferon-gamma (IFN-gamma) from several mammals including man, cow, rat, and rabbit but not mice. We now report that the activity of the B8R gene product is not restricted to cytokines of mammals: it also efficiently neutralized chicken IFN-gamma. B8R blocked chicken IFN-gamma-mediated induction of guanylate binding protein RNA in the chicken fibroblast cell line CEC-32 and secretion of nitric oxide in HD-11 cells. Radiolabeled baculovirus-expressed B8R efficiently bound to immobilized recombinant chicken IFN-gamma. Scatchard analysis revealed a binding constant of chicken IFN-gamma to B8R of approximately 0.5 nM. A mutant form of chicken IFN-gamma which lacks the 18 C-terminal amino acids and which has lost more than 99% of its biological activity was able to block the IFN-gamma-neutralizing effect of B8R. Binding studies showed that the mutant protein bound radiolabeled B8R only about threefold less well than wild-type chicken IFN-gamma but failed to compete with wild-type chicken IFN-gamma for binding to the cellular receptor. These results suggest that the extreme C terminus of chicken IFN-gamma is crucial for binding to its cellular receptor but less important for recognition by the viral cytokine receptor.