RESUMEN
Guidelines for testing gene therapy products for ecotropic replication-competent retrovirus (Eco-RCR) have not been delineated as they have for amphotropic viruses. To evaluate biologic assays that can detect these viruses, we compared an S(+)/L(-) assay and a marker rescue assay designed specifically for Eco-RCR detection. Moloney murine leukemia virus (Mo-MuLV) obtained from the American Type Culture Collection was used as the positive control. For marker rescue, NIH 3T3 cells were transduced with a retroviral vector expressing the neomycin phosphotransferase gene (3T3/Neo). Inoculation and passage of test material in 3T3/Neo cells for 3 weeks (amplification) and subsequent testing in the S(+)/L(-) assay or the marker rescue assay increased the level of sensitivity for virus detection greater than 10-fold compared with direct inoculation of D56 S(+)/L(-) cells. When serial dilutions of Mo-MuLV stock were evaluated, six of six cultures had detectable virus by the S(+)/L(-) and marker rescue assays at dilutions of 10(-5) and 10(-6). At the 10(-7) dilution, five of six assays had detectable virus in both assays. The ability to detect virus-infected cells was also evaluated in a modification that substituted cells for supernatant. Fifteen 3T3/Neo cultures inoculated with 10(6) 293 cells containing 100 or 10 Mo-MuLV/3T3 cells were all positive by marker rescue. For dilution with 1 virus-infected cell per 10(6) 293 cells, 10 of 15 cultures were positive. At the 0.1-cell dilution only 2 of 15 cultures were positive. If we hope to detect one infected cell in a test article, the probability of detecting virus if the assay is performed in triplicate is 96.3%. In summary, after 3 weeks of amplification the S(+)/L(-) and marker rescue assays can detect virus with similar sensitivities. We prefer the marker rescue assay because of the more reliable growth features of NIH 3T3 cells compared with the D56 cell line. For laboratories analyzing clinical materials, this report may prove useful in establishing detection assays for Eco-RCR.
Asunto(s)
Bioensayo , Terapia Genética/normas , Vectores Genéticos/fisiología , Kanamicina Quinasa/análisis , Retroviridae/aislamiento & purificación , Virus Formadores de Foco en el Bazo/aislamiento & purificación , Replicación Viral , Células 3T3/virología , Animales , Línea Celular/virología , Virus Defectuosos/fisiología , Genes Reporteros , Marcadores Genéticos , Humanos , Kanamicina Quinasa/genética , Riñón , Ratones , Virus de la Leucemia Murina de Moloney/fisiología , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Retroviridae/fisiología , Seguridad , Virus del Sarcoma Murino/fisiología , Sensibilidad y Especificidad , Virus Formadores de Foco en el Bazo/fisiología , Transducción GenéticaRESUMEN
Our previous studies have suggested that colonic epithelium from rodents pretreated in vivo with suboptimal doses of carcinogen could be more easily maintained in explant culture. Transformation of colonic epithelium from these explants may be induced by subsequent exposure to additional genotropic agents. Therefore, we describe the development of a 3-step transformation model which uses (1) in vivo pretreatment with a suboptimal dose of 1,2-dimethylhydrazine (DMH) followed by (2) in vitro organ culture and exposure to xenotropic murine sarcoma virus (X-MSV), and finally (3) xenograft maintenance in nude mice to allow sufficient time for transformation. Male Wistar rats were injected intramuscularly with 20 mg DMH/kg 10 times per week followed by the removal and explant culture of the colon, which was then treated in vitro with X-MSV, and transplanted into nude mice after 1 week of culture. All the nude mice (n = 6) transplanted with rat colon explants contained viable xenograft explant epithelium and 1 of the 6 showed transformation. Our results demonstrate that the epithelium from animals pretreated with suboptimal doses of carcinogens can be easily transformed. We also demonstrate that human colonic epithelium is viable for an extended period of time in this model. Based on these results, we hypothesize that such a 3-step transformation model is applicable for carcinogenesis studies of various organs from different species, including human if one uses dysplastic or 'pre-neoplastic initiated' tissues obtained at surgery (e.g., ulcerative colitis; Barret's esophagus, etc.).
Asunto(s)
Transformación Celular Neoplásica/patología , Colon/citología , Animales , Transformación Celular Neoplásica/efectos de los fármacos , Colon/enzimología , Colon/trasplante , Dimetilhidrazinas/administración & dosificación , Dimetilhidrazinas/farmacología , Relación Dosis-Respuesta a Droga , Células Epiteliales , Epitelio/enzimología , Humanos , Inyecciones Intramusculares , Mucosa Intestinal/citología , Mucosa Intestinal/enzimología , Mucosa Intestinal/patología , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ratones , Ratones Desnudos , Técnicas de Cultivo de Órganos , Ratas , Ratas Endogámicas , Virus del Sarcoma Murino/fisiología , Factores de Tiempo , Trasplante HeterólogoRESUMEN
We recently developed a new progenitor assay using murine fetal liver cells that provides a source of pluripotent progenitors, bipotent progenitors, and committed macrophage, megakaryocyte, erythroid, and mast cell progenitors. This clonal cell culture system was used to examine the direct effects of Harvey sarcoma virus on murine hemopoietic progenitors. Very large erythroid colonies containing 100,000 to 200,000 cells were seen in the infected group. Only small erythroid colonies were seen in the uninfected control cultures. The cells in the large erythroid colonies from infected cultures expressed the ras gene as demonstrated by immunofluorescence with a monoclonal antibody to p21, the ras gene product. The infected cells were not immortal since they did not yield secondary colonies upon replating. Sequential observation of individual colonies showed that maturation was not blocked by infection with the virus. The size of other colony types, including granulocyte/macrophage, mast cell, and mixed, was unaffected even though some of these colonies expressed the ras gene. Thus, infection with Harvey sarcoma virus appears to give a growth advantage primarily to committed erythroid progenitors.
Asunto(s)
Transformación Celular Viral , Células Precursoras Eritroides/fisiología , Virus del Sarcoma Murino de Harvey/fisiología , Virus del Sarcoma Murino/fisiología , Animales , División Celular , Eritropoyetina/farmacología , Feto , Genes ras , Hígado/citología , Ratones , Ratones Endogámicos BALB CRESUMEN
BALB/MK (MK) is a continuous murine keratinocyte line whose cells are strictly dependent on exogenous epidermal growth factor (EGF) for growth in culture. A derivative cell, KC, resulted from Kirsten murine sarcoma virus transformation, and these cells no longer require EGF for their growth. Despite differences in MK and KC growth conditions, both cell lines are growth inhibited by picomolar concentrations of transforming growth factor-beta (TGF-beta). When MK and KC cells were maintained in the presence of TGF-beta, resistant variants eventually proliferated only from the KC population. In an attempt to determine the mechanism of development of TGF-beta resistance, the TGF-beta-resistant cells (KCR cells) were compared with TGF-beta-sensitive KC cells with regard to growth properties, TGF-beta 1 binding characteristics, and gene expression. KCR cells continued to synthesize DNA and proliferated in the presence of TGF-beta 1 concentrations up to 2 nM, which was 500-fold greater than the ED50 for the sensitive cells. Although the KCR cells possess similar receptor numbers and affinity for TGF-beta 1, we observed differences in affinity cross-linking studies. The KCR cells expressed more of the type III, high molecular weight cell surface binding protein and less of the type II than the KC cells. The type I moiety was clearly altered to a smaller size in some, but not all, KCR cells. In gene regulation studies, there was no apparent difference in c-Ki-ras and v-Ki-ras mRNA levels in the KC and KCR cells. Additionally, expression of TGF-alpha and TGF-beta 1 mRNA was similar in MK, KC, and KCR cells. The expression of proliferation-associated genes, such as c-myc and MGSA/c-gro/kc, which were markedly decreased by TGF-beta 1 in the MK and KC cells, was not altered by TGF-beta 1 in the KCR cells. The data suggest that the loss of TGF-beta 1 responsiveness in the KCR cells was due to an alteration in the TGF-beta receptor that did not permit signal transduction, although the existence of postreceptor alterations cannot be excluded.
Asunto(s)
Transformación Celular Viral , Queratinocitos/fisiología , Virus del Sarcoma Murino de Kirsten/fisiología , Virus del Sarcoma Murino/fisiología , Factores de Crecimiento Transformadores/farmacología , Animales , Northern Blotting , División Celular , Células Cultivadas , Resistencia a Medicamentos , Regulación de la Expresión Génica , Genes ras , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , ARN Mensajero/genética , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento Transformadores beta , Factores de Crecimiento Transformadores/genética , Factores de Crecimiento Transformadores/metabolismoRESUMEN
9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP) is a potent inhibitor of the replication of human immunodeficiency virus (HIV) in human T-lymphocyte MT-4 cells (50% effective dose: 2 microM). PMEDAP strongly inhibited Moloney murine sarcoma virus (MSV)-induced transformation of murine C3H/3T3 embryo fibroblasts and caused a dose-dependent suppression of tumor formation and mortality in newborn mice inoculated with MSV. Even at a dose as low as 0.25 mg/kg/day, PMEDAP effected a significant delay in tumor appearance and an enhancement of the survival rate of tumor-bearing mice. PMEDAP proved fivefold more efficacious as an anti-MSV agent than 9-(2-phosphonylmethoxyethyl)-adenine (PMEA), which has been previously shown to exhibit strong antiretroviral efficacy in vivo. However, PMEDAP was also more toxic, so that its therapeutic index was equivalent to that of PMEA.
Asunto(s)
Adenina/análogos & derivados , Antivirales/farmacología , VIH/fisiología , Virus del Sarcoma Murino de Moloney/fisiología , Virus del Sarcoma Murino/fisiología , Linfocitos T/microbiología , Replicación Viral/efectos de los fármacos , Adenina/farmacología , Animales , Línea Celular , Evaluación Preclínica de Medicamentos , Embrión de Mamíferos/citología , Humanos , Ratones , Virus del Sarcoma Murino de Moloney/metabolismoRESUMEN
Previous studies have shown that vimentin, an intermediate filament protein, is reduced in amount in cells acutely infected with Moloney mouse sarcoma virus (Mo-MuSV). In this report, we provide evidence for specific alteration of vimentin in Mo-MuSV-transformed cells and demonstrate specific phosphorylation of vimentin by the p37mos protein kinase in vitro. Specificity of the phosphorylation reaction was demonstrated by using viral mos proteins encoded by various isolates of Mo-MuSV and p37mos produced in yeast. A phosphotransfer domain mutant lacking the ability to autophosphorylate p37mos failed to phosphorylate vimentin. Similarly, vimentin was not phosphorylated by the temperature-sensitive P85gag-mos kinase derived from infected cells maintained at the restrictive temperature. In ts110 MuSV-transformed NRK cells, vimentin was phosphorylated at both the permissive and nonpermissive temperatures for transformation. However, at the permissive temperature, an altered form of vimentin (about 50 kDa) with a more basic isoelectric point and lower apparent molecular weight was detected. This 50-kDa product was highly phosphorylated as compared to the bulk of the normal 55-kDa form of vimentin. On the basis of its mobility in two-dimensional gels, the 50-kDa form of vimentin should lack the carboxy terminus. This type of alteration could conceivably modulate the function of vimentin filaments in the transformed cell.
Asunto(s)
Virus del Sarcoma Murino de Moloney/fisiología , Proteínas Quinasas/metabolismo , Proteínas Oncogénicas de Retroviridae/metabolismo , Virus del Sarcoma Murino/fisiología , Vimentina/metabolismo , Animales , Línea Celular Transformada , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Productos del Gen gag/metabolismo , Punto Isoeléctrico , Peso Molecular , Proteínas Oncogénicas v-mos , Mapeo Peptídico , Fosforilación , Pruebas de Precipitina , TemperaturaRESUMEN
Chondroprogenitor cells present in the apical and lateral parts of the mandibular condyle from neonatal mice differentiate towards the osteoblastic lineage and form bone within 7 days in culture. Infection of condylar explants with the FBR osteosarcoma virus (FBR MSV) results in the transformation of cells in the progenitor zone, previously identified as the target for the virus, and the formation of a transplantable osteosarcoma-like lesion. Morphological and biochemical changes in this system were investigated in the course of tumor development. Virus infection was followed by a significant increase in cell density and 3H-thymidine incorporation within the progenitor zone at the early stage of culture. In later stages, cell density and 3H-thymidine incorporation were lower than in control tissue. The 3H-thymidine labeling index gave similar results in infected and control tissues until day 7. Then, a significantly higher labeling index was found in the progenitor zone of infected condyles. At this stage, the proliferative effect of the virus even affected the cartilagenous core of the tissue. Quantitative alkaline phosphatase activity increased between day 3 and day 7 and was particularly high in the zone of infected cells. In addition, infected tissues consistently revealed a higher uptake of 45Ca, and deposition of the radioisotope along irregularly formed bone trabecules in the transformed tissue. The results suggest that there is an enhancement of tissue maturation following infection with the FBR osteosarcoma virus. Although biochemical investigations of whole condyles showed few differences in the total values of alkaline phosphatase activity, 3H-thymidine incorporation, DNA content, and 45Ca uptake, the histochemical assays revealed clear differences in the distributional pattern of these parameters within infected and control condyles.
Asunto(s)
Neoplasias Óseas/patología , Transformación Celular Neoplásica/patología , Osteogénesis , Osteosarcoma/patología , Virus del Sarcoma Murino/fisiología , Fosfatasa Alcalina/metabolismo , Animales , Animales Recién Nacidos , Neoplasias Óseas/metabolismo , Calcio/metabolismo , Cartílago/patología , Recuento de Células , Cinética , Cóndilo Mandibular , Ratones , Técnicas de Cultivo de Órganos , Osteoblastos/patología , Osteosarcoma/metabolismo , Células Madre/patología , Timidina/metabolismoRESUMEN
Human small cell lung cancers (SCLC) and cell lines derived therefrom are phenotypically heterogeneous concerning neuroendocrine differentiation. Unlike most SCLC tumors and cell lines that express poorly differentiated neuroendocrine phenotypes, the SCLC cell line DMS 53 exhibits mature endocrine differentiation features, including unusually high expression of the gene for the peptide hormone, calcitonin (CT). We now report that introduction of the viral Harvey ras (v-rasH) oncogene into DMS 53 cells via retroviral infection, with resultant constitutive expression, results in increased features of neuroendocrine differentiation. 7-10 d after infection the cells demonstrated altered morphology, increased CT secretion, increased CT gene expression, markedly diminished cellular proliferation, and nearly abolished methylcellulose cloning efficiency. This response of DMS 53 cells to v-rasH is unlike the tumor progression effects we have previously observed in other SCLC lines. Significantly, the differentiation response that follows expression of the virally introduced v-rasH oncogene in DMS 53 cells is similar to that of neoplastic neuroendocrine cell lines derived from adrenal pheochromocytes and thyroid C cells. The effects of constitutive v-rasH expression in DMS 53 SCLC cells and other neuroendocrine cell lines suggest an important role for rasH or related genes in neuroendocrine differentiation.
Asunto(s)
Calcitonina/metabolismo , Carcinoma de Células Pequeñas/patología , Hormonas Ectópicas/metabolismo , Neoplasias Pulmonares/patología , Oncogenes , Northern Blotting , Calcitonina/biosíntesis , Calcitonina/genética , Carcinoma de Células Pequeñas/metabolismo , Carcinoma de Células Pequeñas/microbiología , Diferenciación Celular , Genes Virales , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/microbiología , Hibridación de Ácido Nucleico , Virus del Sarcoma Murino/fisiología , Células Tumorales Cultivadas/microbiología , Células Tumorales Cultivadas/ultraestructura , Replicación ViralRESUMEN
Spontaneous and induced transmission of oncogenic retroviruses from parents to offspring is well documented in mammals, as exemplified by murine leukaemia viruses. In addition, it is now common knowledge that transgenic mice, carrying viral and cellular transforming genes, can show unusual tumour incidences and can transmit this character to successive generations. The present report deals with the occurrence of tumours in rodents exposed directly in utero to murine sarcoma viruses (MSV). Attention was focused on whether MSV are involved in the etiopathogenetic processes leading to early-life and adult-type neoplasms in mice. According to our data, this is a likely possibility, since, for example, Kirsten MSV (Ki-MSV) induced stage-specific tumours in CD-1 mice, including lung tumours and skin papillomas, following intraembryonal injections on gestation days 8 and 10, respectively. When the injections were given in the second half of pregnancy, however, only capillary angiomas and other vascular malformations of the brain, as well as mesenchymal sarcomas, were induced in the newborn. Harvey MSV (Ha-MSV), a close relative of Ki-MSV, and Moloney MSV (Mo-MSV) were much less dependent on the stage of embryogenesis, and mesenchymal sarcomas were frequently detected in prenatally infected animals. Other MSV viruses, including 3611, J-2 and 4070A, were active carcinogens in a way totally independent of the embryonal stage exposed, and tumours in the progeny ranged from mesenchymal sarcomas to lymphoblastomas and rhabdomyosarcomas, respectively. We next looked at the possibility that spontaneous tumours of CD-1 mice might result from the interaction of certain chemical carcinogens with developmentally expressed components of the Ki-MSV virus. The experiments conducted thus far have provided dubious results. An example is given by experiments with N-ethyl-N-nitrosourea (ENU). This compound is a powerful transplacental carcinogen in CD-1 mice, where it induces lung tumours. It was expected that by exposing developing mice to Ki-MSV on day 8 of embryogenesis and to ENU late in pregnancy or in newborns, a great increase in the incidence of lung tumours would have appeared in the offspring. However, this did not happen, and the agents behaved as if different cellular targets were involved. Understanding this difference could be very informative and useful in identifying neoplastic factors in mammalian embryos.
Asunto(s)
Transformación Celular Neoplásica , Transformación Celular Viral/fisiología , Efectos Tardíos de la Exposición Prenatal , Virus del Sarcoma Murino/fisiología , Animales , Animales Recién Nacidos , Transformación Celular Neoplásica/patología , Células Cultivadas , Cocarcinogénesis , Embrión de Mamíferos/patología , Etilnitrosourea/toxicidad , Femenino , Ratones , EmbarazoRESUMEN
Murine sarcoma virus-transformed rat fibroblasts (KNRK cells) undergo marked cytoarchitectural reorganization during in vitro exposure to sodium-n-butyrate (NaB) resulting in restoration of (1) a more typical fibroblastoid morphology, (2) proper cell-to-cell orientation, and (3) substratum adherence. Augmented cell spreading, involving greater than 90% of the population, was a function of culture density and time of exposure to NaB (2 mM final concentration). Induced cell spreading reflected a 2.5- to 3.0-fold increase in both total cellular actin content and deposition of actin into the detergent-resistant cytoskeleton. Cytoskeletal actin deposition in response to NaB was accompanied by the formation of occasionally dense, parallel alignments of F-actin-containing microfilaments and by a dramatic increase in the size and incidence of actin-enriched membrane ruffles. Long-term NaB-treated cells exhibited parallel orientations of microfilaments similar to those found in untransformed fibroblasts. Increased cytoskeletal actin occurred within 24 hr of NaB exposure, correlating with the initial reorganization of actin-containing microfilaments detected microscopically, and reflected concomitant 3-fold increases in cellular alpha-actinin and fibronectin content. In contrast, the amount of vimentin, tropomyosin, and tubulin in NaB-treated cells was significantly decreased. NaB-induced morphologic restructuring of sarcoma virus-transformed fibroblasts, thus, impacts on all three basic cytoskeletal systems. Selective increases, however, were evident in particular cytoskeletal proteins (actin, alpha-actinin, fibronectin) implicated in microfilament networking and cell spreading.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Butiratos/farmacología , Transformación Celular Viral , Citoesqueleto/ultraestructura , Virus del Sarcoma Murino de Kirsten/fisiología , Virus del Sarcoma Murino/fisiología , Citoesqueleto de Actina/efectos de los fármacos , Actinas/metabolismo , Animales , Ácido Butírico , Adhesión Celular , Comunicación Celular , Línea Celular Transformada , Citoesqueleto/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Fibroblastos , Microscopía Fluorescente , RatasRESUMEN
Mature connective tissue mast cells (CTMC) have not been previously available as a cell line from any species. Here we describe 15 novel mast cell lines (KiSV-MC) that were derived by coculturing murine splenocytes with fibroblasts that produce a Ki-ras-containing murine sarcoma virus. Some of the KiSV-MC lines are similar to CTMC in that they synthesize predominantly heparin proteoglycans, and contain up to 35 micrograms of histamine and 2.2 units of carboxypeptidase A/10(6) cells in secretory granules which stain red with Safranin. Other cell lines display phenotypic characteristics intermediate to CTMC and mucosal-like mast cells in being predominantly Safranin-, having lower amounts of histamine and carboxypeptidase A, and in synthesizing chondroitin sulfate E proteoglycans in preference to heparin proteoglycans. When the individual KiSV-MC lines were compared, a linear relationship was found between the number of Safranin+ granules, the cellular contents of histamine and carboxypeptidase A, and the biosynthesis of heparin relative to chondroitin sulfate E proteoglycans. Upon sensitization with monoclonal IgE and exposure to hapten-specific antigen, the cells exocytose the contents of their secretory granules. Thus, these immortalized cells provide the first source of CTMC-like lines for chemical and functional analysis and illustrate that murine mast cells can express a continuum of phenotypes.
Asunto(s)
Células del Tejido Conectivo , Fibroblastos/microbiología , Virus del Sarcoma Murino de Kirsten/fisiología , Mastocitos/metabolismo , Virus del Sarcoma Murino/fisiología , Bazo/fisiología , Animales , Carboxipeptidasas/metabolismo , Carboxipeptidasas A , Diferenciación Celular , División Celular , Línea Celular , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Gránulos Citoplasmáticos/metabolismo , Exocitosis , Fibroblastos/fisiología , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/biosíntesis , Histamina/metabolismo , Histocitoquímica , Inmunoglobulina E/inmunología , Mastocitos/inmunología , Mastocitos/ultraestructura , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos DBA , Microscopía Electrónica , Fenazinas , Fenotipo , Bazo/citología , Coloración y Etiquetado , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
The effect of infecting fibroblasts with Kirsten murine sarcoma virus/murine leukemia virus (Ki-MSV/MLV) on constitutive and IFN-gamma-induced H-2 antigen expression was investigated. The fibroblasts used were two established cell lines (C3H10T1/2 and BALB/c3T3) and fresh embryo fibroblasts from C3H mice. Class I antigens were expressed constitutively by BALB/c3T3; infection with MLV, MSV or the two together had little effect on this constitutive expression. Class I antigens (H-2K, H-2D) were strongly induced on all three types of fibroblast by rIFN-gamma, and infection had little effect on this. None of the fibroblasts expressed constitutively detectable levels of class II antigen; however, C3H10T1/2 fibroblasts could be induced for both H-2A and H-2E by IFN-gamma. Infection of C3H10T1/2 with helper-free Ki-MSV, or MSV together with MLV, completely abolished this induction of class II antigens, while infection with MLV alone had little effect, implying that the abolition of class II induction was due to genomic regions of Ki-MSV not shared with Ki-MLV, probably the v-Ki-ras gene.
Asunto(s)
Fibroblastos/inmunología , Antígenos H-2/análisis , Antígenos de Histocompatibilidad Clase II/análisis , Interferón gamma/farmacología , Virus del Sarcoma Murino de Kirsten/fisiología , Virus del Sarcoma Murino/fisiología , Animales , Línea Celular , Células Clonales/inmunología , Virus de la Leucemia Murina/fisiología , Leucemia Experimental/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Sarcoma Experimental/inmunologíaRESUMEN
Amphotropic murine leukemia virus pseudotypes of murine sarcoma viruses containing the ras or mos oncogenes were constructed to permit efficient introduction of the sarcoma virus genome into early-passage human umbilical vein endothelial cells. The resulting cell lines were morphologically and phenotypically unchanged, retaining properties characteristic of differentiated endothelial cells. For example, the cells in a Kirsten sarcoma virus-modified line were found to biosynthesize and secrete von Willebrand factor in both a constitutive and regulated manner, and they contained ultrastructurally identifiable Weibel-Palade bodies, an endothelial cell-specific organelle. In contrast to the parent cultures, sarcoma virus-modified cells were able to proliferate indefinitely in culture. Examination of both Kirsten sarcoma and Moloney leukemia virus-modified lines indicated that the immortalized cells retained a diploid female karyotype after over 18 months in culture. In addition, the sarcoma virus-modified cells were able to grow independently of added endothelial cell growth factor. This growth factor autonomy does not appear to be due to autocrine production of a biologically cross-reactive growth factor. These immortal, virus-modified endothelial cells express large amounts of sarcoma virus-specific mRNA but no detectable helper virus or transforming virus activity. This technique for immortalization of primary human cells without alteration of the differentiated characteristics of the cell type is readily applied to a variety of human cell types. Moreover, the ability to separate the immortalizing and transforming activities of viral oncogenes should provide further understanding as to mechanisms of oncogene action.
Asunto(s)
Transformación Celular Viral , Endotelio Vascular/citología , Virus del Sarcoma Murino/fisiología , Fenómenos Biomecánicos , Diferenciación Celular , División Celular , Línea Celular , Endotelio Vascular/ultraestructura , Humanos , Recién Nacido , Microscopía Electrónica , Factores de TiempoRESUMEN
We demonstrated that the Kirsten murine sarcoma virus (KiMSV) and the Harvey murine sarcoma virus (HaMSV) converted human skin fibroblasts (HSF) into adipocytes. Adipocytic conversion of HSF by KiMSV and HaMSV was dependent on the presence of glucocorticosteroids. The Kirsten murine leukemia virus, the Harvey murine sarcoma [corrected] virus and the amphotropic helper virus (AP292) were ineffective by themselves. Balb murine sarcoma virus and Moloney murine sarcoma virus were, to a lesser degree, able to effect adipocytic conversion of HSF. In contrast, the feline sarcoma virus and the simian sarcoma virus did not cause this conversion. Together, the results suggest a role for certain oncogenes and glucocorticosteroids in the transformation/neodifferentiation of human cells.
Asunto(s)
Transformación Celular Neoplásica/fisiopatología , Dexametasona/farmacología , Fibroblastos/fisiopatología , Retroviridae/fisiología , Piel/patología , Biopsia , Transformación Celular Viral , Células Cultivadas , Fibroblastos/patología , Genes ras , Humanos , Virus del Sarcoma Murino/fisiología , Piel/fisiopatologíaRESUMEN
Mammalian type C retroviruses can be detected by their induction of foci of cell transformation in S+I - cells. We have noted that certain subtypes of the mouse xenotropic type C retrovirus (MuLV) inhibit this cell alteration. This inhibition, associated with intact virions, is irreversible and gives the infected cells a phenotype of uninfected cells. In comparison to the transformed cells, the inhibited cultures showed primarily a decrease in murine sarcoma virus (MSV) progeny production concomitant with a reduction in the MSV mos RNA expression. No difference in beta-actin RNA production was observed between the inhibited and transformed cultures. This selective effect of mouse xenotropic MuLV on MSV and mos RNA production in these cells focuses attention on the mechanism of transformation in this system.
Asunto(s)
Transformación Celular Viral , Virus Helper/fisiología , Virus de la Leucemia Murina/fisiología , Ratones Endogámicos NZB/microbiología , Retroviridae/fisiología , Actinas/biosíntesis , Animales , Gatos/microbiología , Línea Celular , Virus Defectuosos/fisiología , Perros , Humanos , Virus de la Leucemia Murina/aislamiento & purificación , Ratones , Ratones Endogámicos C57BL/microbiología , Visón , Proteínas Oncogénicas v-mos , Conejos , Ratas , Retroviridae/aislamiento & purificación , Proteínas de los Retroviridae/biosíntesis , Virus del Sarcoma Murino/fisiología , Replicación ViralRESUMEN
A murine helper/inducer T cell clone, D10.G4, has been infected with Kirsten-murine sarcoma virus (KiSV) pseudotyped with an amphotropic murine leukemia virus. The resultant Ki-ras-expressing lines (KiSV-D10) remain dependent on exogenous factors for continued growth but display distinctly different mitotic responses to certain cytokines as compared to the uninfected parent clone. Unlike the parent D10.G4 cells, these KiSV-D10 cells can be maintained in vitro indefinitely in the presence of recombinant interleukin 2 (IL 2), and they all display a maximal proliferative response to purified or recombinant interleukin 1 (IL 1). The IL 1-induced proliferation is shown not to be dependent or secretion of the T cell autocrine growth factors IL 2 or B cell stimulatory factor-1 (BSF-1). The KiSV-D10 lines show certain differences from one another and parent D10.G4 cells in their secretory and proliferative responses to T cell receptor- and BSF-1 mediated signals. These viral oncogene-expressing T cell lines, which remain responsive to and dependent on physiologic growth factors, should prove valuable for analyzing the mechanisms of action of single oncogenes and the intracellular events in T lymphocyte activation.
Asunto(s)
Sustancias de Crecimiento/farmacología , Interleucina-1/farmacología , Interleucina-2/farmacología , Virus del Sarcoma Murino de Kirsten/fisiología , Virus de la Leucemia Murina/fisiología , Linfocinas/farmacología , Virus del Sarcoma Murino/fisiología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Animales , Antígenos de Superficie/análisis , División Celular/efectos de los fármacos , Línea Celular , Interleucina-4 , Virus del Sarcoma Murino de Kirsten/genética , Ratones , Proteína Oncogénica p21(ras) , Proteínas Oncogénicas Virales/genética , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
Expression of proto-oncogene fos is induced in response to a variety of growth factors and differentiation-specific agents. However, the induction of fos gene expression is not influenced by inhibition of protein synthesis. We, therefore, entertained the notion that expression of the fos gene may be governed by posttranslational modification of cellular transcriptional factors. We report here that transcription of the human c-fos gene is modulated by negatively and positively acting cellular factors. The nuclear protein products of the resident oncogene of the FBJ-murine osteosarcoma virus (v-fos) and its corresponding cellular proto-oncogene (c-fos) are stoichiometrically phosphorylated on serine and threonine residues. The c-fos protein is more highly phosphorylated than the v-fos protein due to the phosphorylation of unique sites tentatively localized to the c-terminal 20 amino acid residues. The protein kinase C agonist, TPA, stimulates phosphorylation of the c-fos, but not the v-fos protein.
Asunto(s)
Proteínas Proto-Oncogénicas/biosíntesis , Proto-Oncogenes , Animales , Fenómenos Fisiológicos Sanguíneos , Bovinos , Transformación Celular Neoplásica/genética , Transformación Celular Viral , Células Cultivadas , Fibroblastos , Regulación de la Expresión Génica/efectos de los fármacos , Productos del Gen gag , Fosforilación , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-fos , Ratas , Proteínas de los Retroviridae/biosíntesis , Proteínas de los Retroviridae/genética , Proteínas de los Retroviridae/fisiología , Virus del Sarcoma Murino/genética , Virus del Sarcoma Murino/fisiología , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
Here we have demonstrated that transformation of human skin fibroblasts (SF) by the Kirsten murine sarcoma virus (KiMSV) is associated with their neodifferentiation into preadipose cells. Hydrocortisone (HC) promotes the transformation/neodifferentiation of such preadipocytes into mature fat cells. The effects of HC on the expression of adipocyte-containing foci and on the total number of transformed foci present in KiMSV-treated cultures appeared to be dose-dependent and was optimal at a concentration of about 500 ng/ml, or 1.25 X 10(-6) M. Although increasing serum concentrations (2-15%) increased the total number of transformed foci, it had no effect on the expression of adipocyte-containing foci in the presence of HC. The virus-induced preadipocytes undergoing partial conversion in the presence of HC were capable of clonal expansion and extensive proliferative activity. In contrast, mature adipocytes were terminally differentiated and as such have lost their ability to proliferate. The results suggest a role for a ras oncogene and HC in the transformation/neodifferentiation of human cells that might ultimately lead to cancer in some fraction of such cells.
Asunto(s)
Tejido Adiposo/citología , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Viral/efectos de los fármacos , Fibroblastos/citología , Hidrocortisona/farmacología , Virus del Sarcoma Murino de Kirsten/fisiología , Virus del Sarcoma Murino/fisiología , Adulto , Diferenciación Celular/efectos de los fármacos , División Celular , Línea Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Virus del Sarcoma Murino de Kirsten/genética , Oncogenes , Fenotipo , Piel/citologíaRESUMEN
ts110 Moloney murine sarcoma virus (Mo-MuSV)-nonproductively infected cells (6m2) have a transformed phenotype at 28 to 33 degrees C and a normal phenotype at 39 degrees C. At temperatures permissive for transformation, 6m2 cells contain P58gag produced from the 4.0-kilobase (kb) viral RNA genome and P85gag-mos translated from a 3.5-kb spliced mRNA. At 39 degrees C, only the 4.0-kb RNA and its product P58gag are detected. Two temperature-sensitive defects have been observed in ts110-infected 6m2 cells: (i) the splicing of the 4.0-kb RNA to the 3.5-kb RNA; and (ii) the thermolability of P85gag-mos and its kinase activity relative to the wild-type revertant protein, termed P100gag-mos (R.B. Arlinghaus, J. Gen. Virol. 66:1845-1853, 1985). In the present study, we examined the mos gene products of two cell lines (204-2F6 and 204-2F8) obtained by infection of normal rat kidney cells with ts110 Mo-MuSV as a simian sarcoma-associated virus pseudotype to see whether the temperature-sensitive splicing defect could be transferred by viral infection. Southern blot analysis of these two cell lines showed that viral DNAs containing restriction fragments from cellular DNA are different from those in 6m2 cells, indicating that 204-2F6 and 204-2F8 cells have different ts110 provirus integration sites from those of 6m2 cells. Northern blots, S1 mapping analyses, and immunoprecipitation experiments showed unequivocally that the splicing defect of ts110 Mo-MuSV is virus encoded and is independent of host cell factors.