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
The most advanced oncolytic Newcastle disease virus (NDV) strains that are used in clinical trials for the treatment of cancer are wild-type mesogenic strains. These virus strains have an inherent, nongenetically engineered, oncolytic activity and selectively replicate in tumor cells but not in normal human cells. To date no investigations have been performed with genetically engineered mesogenic NDV regarding the oncolytic activity. We describe here the generation of recombinant viruses of the mesogenic naturally oncolytic NDV strain MTH68. We show that not only one, but also two additional transgenes coding for amino-acid chains with a molecular weight of 25 and 50 kDa can be inserted into the viral genome without affecting viral growth, oncolytic potency or tumor-selective replication of the virus. Transgenic expression of the heavy and light chains of a monoclonal antibody, as separate additional transcriptional cassettes, leads to the expression of full immunoglobulin G (IgG) monoclonal antibody by recombinant NDV. Infection of tumor cells with antibody-transgenic viruses results in the efficient production and secretion of a functional full size IgG antibody by the tumor cells, that specifically binds to its target-antigen in tumor tissue. This approach will allow to combine the advantages of oncolytic RNA viruses and monoclonal antibodies in a single powerful anticancer agent with improved or even new therapeutic properties.
Asunto(s)
Terapia Genética/métodos , Inmunoglobulina G/metabolismo , Neoplasias/terapia , Virus de la Enfermedad de Newcastle/genética , Viroterapia Oncolítica/métodos , Animales , Células CHO , Línea Celular Tumoral , Técnicas de Cocultivo , Cricetinae , Cricetulus , Expresión Génica , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Genes de las Cadenas Ligeras de las Inmunoglobulinas , Ingeniería Genética , Inmunoglobulina G/análisis , Inmunohistoquímica , Transfección/métodos , TransgenesRESUMEN
Monoclonal antibodies (McAbs) generated against rhamnogalacturonan I (RG-I) purified from suspension-cultured sycamore maple (Acer pseudoplatanus) cells fall into three recognition groups. Four McAbs (group I) recognize an epitope that appears to be immunodominant and is present on RG-I from maize and sycamore maple, pectin and polygalacturonic acid from citrus, gum tragacanth, and membrane glycoproteins from suspension-cultured cells of maize, tobacco, parsley, bean, and sycamore maple. A second set of McAbs (group II) recognizes an epitope present in sycamore maple RG-I but does not bind to any of the other polysaccharides or glycoproteins recognized by group I. Lastly, one McAb, CCRC-M1 (group III), binds to RG-I and more strongly to xyloglucan (XG) from sycamore maple but not to maize RG-I, citrus polygalacturonic acid, or to the plant membrane glycoproteins recognized by group I. The epitope to which CCRC-M1 binds has been examined in detail. Ligand competition assays using a series of oligosaccharides derived from or related to sycamore maple XG demonstrated that a terminal alpha-(1-->2)-linked fucosyl residue constitutes an essential part of the epitope recognized by CCRC-M1. Oligosaccharides containing this structural motif compete with intact sycamore maple XG for binding to the antibody, whereas structurally related oligosaccharides, which do not contain terminal fucosyl residues or in which the terminal fucosyl residue is linked alpha-(1-->3) to the adjacent glycosyl residue, do not compete for the antibody binding site. The ligand binding assays also indicate that CCRC-M1 binds to a conformationally dependent structure of the polysaccharide. Other results of this study establish that some of the carbohydrate epitopes of the plant extracellular matrix are shared among different macromolecules.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Pared Celular/inmunología , Epítopos/inmunología , Pectinas/inmunología , Plantas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Unión Competitiva , Secuencia de Carbohidratos , Pared Celular/química , Femenino , Fucosa/inmunología , Hibridomas , Fragmentos Fab de Inmunoglobulinas/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pectinas/química , Plantas/ultraestructuraRESUMEN
Two homogeneous, metallic ion-containing pectic polysaccharides with mean M(r)s of 7600 and 15,000 were isolated from dried aerial parts of Achyrocline satureioides by anion exchange column chromatography on DEAE-Sepharose CL-6B and gel filtration column chromatography on Fractogel TSK HW-50 (S). The structures, as determined by methylation analysis, carboxyl reduction, and partial acid hydrolysis, were shown to be rhamnogalacturonans. Both pectins show a pronounced anticomplementary effect in vitro. The larger carbohydrate AS 4 of higher M(r) exerts anti-inflammatory activity and a strong enhancement of phagocytosis in vivo.
Asunto(s)
Pectinas/aislamiento & purificación , Plantas Medicinales/química , Secuencia de Carbohidratos , Proteínas Inactivadoras de Complemento/química , Proteínas Inactivadoras de Complemento/aislamiento & purificación , Metales/química , Datos de Secuencia Molecular , Pectinas/química , Pectinas/inmunología , Plantas Medicinales/inmunologíaRESUMEN
From the nutrition medium of Arnica montana cell cultures two homogeneous polysaccharides, an acidic arabino-3,6-galactan-protein with mean Mr of 100,000 and a neutral fucogalactoxyloglucan with mean Mr of 22,500 have been isolated by DEAE-Sepharose CL-6B and Sephacryl S-400 column chromatography. Their structures were elucidated mainly by methylation analysis, partial acidic and enzymatic hydrolysis and 13C NMR spectroscopy. The fucogalactoxyloglucan shows a pronounced enhancement of phagocytosis in vivo. The arabino-3,6-galactan-protein displays a strong anticomplementary effect and stimulates macrophages to excrete the tumour necrosis factor (TNF alpha).
Asunto(s)
Polisacáridos/inmunología , Secuencia de Carbohidratos , Células Cultivadas , Espectroscopía de Resonancia Magnética , Metilación , Datos de Secuencia Molecular , Estructura Molecular , Plantas/análisis , Plantas/inmunología , Polisacáridos/aislamiento & purificaciónRESUMEN
Ethanolic extracts of Echinacea purpurea, E. pallida and E. angustifolia roots were examined for immunological activity in the carbon clearance test with mice and in the granulocyte test. In the in vivo experiment all extracts, administered orally, were found to enhance phagocytosis significantly. These results correlate with the stimulation of phagocytosis in the in vitro granulocyte test. The lipophilic fractions of the extracts appeared to be more active than the polar fractions. All extracts were analyzed by HPLC in order to correlate the chemical constituents with the immunological activities.