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At least 325 records · Page 18Linked to original sources
PubMed1992

Involvement of somatically acquired ecotropic viruses in the immunogenicity of nude-transplanted NIH/3T3 transformed cell lines.

Immunogenic tumor variants were previously derived after transplantation in vivo into nude mice of NIH/3T3-transformed cell lines. Nude-passaged cell lines were rejected by immunocompetent H-2q NIH mice, were recognized by specific CTL clones, and expressed new retroviral Ag. The aim of the present work was to investigate whether somatically acquired proviral sequences were present in the genome of nude-passaged cells and to test directly for a causative relationship between murine leukemia virus (MuLV) expression and immunogenicity. Southern blot analysis of PstI-digested DNA indicated that in contrast to the parental NIH/3T3 transformed cell lines (pT, T12N/5a, NS-1) all the nude-passaged immunogenic variants (pT-nude, T12N/5a-nude, NS-1-nude) contained newly acquired ecotropic-related proviruses. Immediately after in vitro establishment, these tumors displayed multiple integration sites as assessed by analysis of 3' proviral-cellular junctions. Long term in vitro culture of one of the cell lines (pT-nude) resulted in a cell line (pT-nude/vitro) that was clonal or oligo-clonal with respect to viral integration. Northern blot analysis established that the new proviruses were actively transcribed in all the immunogenic variants. To assess whether the somatically acquired ecotropic proviral sequences encode for target structures recognized by specific CTL, obtained after immunization of NIH mice with pT-nude, the parental cell line pT was transfected with plasmids containing the entire AKV MuLV genome, the cloned AKV gag or env genes. Screening of transfectants for their ability to stimulate the production of TNF by anti-pT-nude effectors indicated that cells transfected with the entire ecotropic virus or with MuLV-env gene products could be recognized by an NIH anti-pT-nude CTL line and NIH anti-pT-nude Kq-restricted CTL clones as well as the immunizing target pT-nude.

3T3 Cells↗
PubMed2003

Establishment of stably EBV-transformed cell lines from residual clinical blood samples for use in performance evaluation and quality assurance in molecular genetic testing.

Positive control materials for clinical molecular genetic testing applications are currently in critically short supply or non-existent for many genetically based diseases of public health importance. Here we demonstrate that anonymous, residual, clinical blood samples are potential sources of viable lymphocytes for establishing Epstein-Barr virus (EBV)-transformed blood lymphocyte cell lines. We attempted to transform 34 residual blood samples, and analyzed transformation success with respect to sample age, anticoagulant, storage temperature, volume, hemolysis, and patient age and sex. In univariate analysis, sample age was significantly associated with transformation success (P = 0.002). The success rate was 67% (6 of 9) for samples 1 to 7 days old, 38% (3 of 8) for samples 8 to 14 days old and 0% for samples 15 to 21 (0 of 11) days old. When we controlled for sample age in multivariate logistic regression, anticoagulant and storage temperature approached significance (P = 0.070 and 0.087, respectively; samples in acid citrate dextrose (ACD) and refrigerated samples were more likely to transform). Based on these findings, we suggest that samples collected in either ACD or ethylene diamine tetraacetic acid, and up to 14 days old (refrigerated) or 7 days old (stored ambient), are reasonable candidates for EBV transformation. The transformation rate for samples that met these criteria was 63% (10 of 16). Implementation of this process could help alleviate the shortage of positive control materials for clinical molecular genetic testing.

Adult↗
PubMed1997

Establishment and characteristics of a practical and useful astrocyte cell line transformed by a temperature-sensitive mutant of simian virus 40.

A practical mouse astrocyte cell line (A640-IG) was established by transformation with a temperature-sensitive mutant of simian virus 40 (SV40) and the relationship between the function of SV40 large T antigen and the growth and differentiation of A640-IG cells, which are most clearly dependent on temperature that ever established, was reported. A640-IG cells proliferated actively with expression of large T antigen when they were cultured at 33 degrees C. They had a fibroblast-like appearance, and displayed faint immunoreactivity with an antibody against glial fibrillary acidic protein (GFAP). However, when large T antigen expression ceased at 39 degrees C, the cells did not grow actively and differentiated into astrocytes as demonstrated by both their morphological and immunohistochemical characteristics. Differentiation into astrocytes was more obvious when the cells were plated on bacteriological dishes in high density. Western blotting confirmed immunohistochemical observations. A640-IG cells thus showed contrasting behaviour in terms of cell growth and differentiation depending on the temperature. This unique and practical astrocyte cell line is a useful model for investigating the mechanisms of astrocyte growth and differentiation.

Animals↗
PubMed1978

Induction of oncornavirus-like particles from a spontaneously transformed hamster cell line.

A spontaneously transformed hamster cell line, designated Clone B (Cl B), has been activated by halogenated pyrimidines to produce oncornavirus-like particles. The optimal conditions for virus induction were studied by the analysis of culture fluids for particles with polymerase activity. Maximal enzymatic activity was induced when exponentially growing Cl B cells were treated with 20 to 30 microgram/ml 5-iodo-2'-deoxyrudine for a 24-h period. Under these conditions, the kinetics of particle release reached a maximal level between 48 and 72 h following removal of the drug, and then decreased rapidly. The induced particles exhibited features characteristic of oncogenic RNA viruses: actinomycin D was found to inhibit their induction; the particles band in sucrose gradients at a density of 1.15 g/ml; nonionic detergent treatment releases the virion core component exhibiting a buoyant density of 1.21 g/ml in sucrose gradients; the viral particles contain high molecular weight RNA species with sedimentation coefficients of 65 S and 35 S; and the polymerase associated with Cl B particles efficiently transcribes synthetic templates preferred by the reverse transcriptases of known RNA tumor viruses, but demonstrates a very low endogenous activity.

Animals↗
PubMed1989

[Preparation and characteristics of a new transformed cell line G11 by transfection of NIH/3T3 mouse fibroblasts with DNA from the SA7 oncogene of simian adenovirus].

Murine fibroblasts NIH 3T3 were transfected with the plasmid pASP containing simian adenovirus oncogene insertion. Focus forming transformants were cloned with a final dilution technique and a new cell line G11 was created as a result. Transformed status of this cell line is evidenced by changes in morphology, specific cytochemical and adhesion properties, ability to grow in semisolid agar and FCS concentration growth independence. Presence of intact integrated E1a-region of adenovirus SA7 oncogene was shown by blot-hybridization technique. Transformed status of G11 cells can be explained by integration of SA7 oncogene, that is evidenced indirectly by the increased resistance to heat shock.

Adenoviridae↗
PubMed1987

Abelson virus transformation of an interleukin 2-dependent antigen-specific T-cell line.

Abelson murine leukemia virus (A-MuLV) carries the gene v-abl, one of a group of oncogenes with structural and functional (tyrosine kinase) homology to three growth factor receptors. Work in this and other laboratories has shown that A-MuLV infection can render myeloid and lymphoid cells independent of the growth factors interleukin 3 and granulocyte-macrophage colony-stimulating factor. We have now shown that v-abl can also relieve interleukin 2 (IL-2) dependence in T cells. We infected a cloned IL-2-dependent antigen-specific cell line. Transformed cells were generated which were factor independent and tumorigenic. The transformants each bore unique v-abl DNA inserts and expressed v-abl mRNA. No elevation of expression of either IL-2 or its receptor could be detected in these cells. Thus, A-MuLV can short-circuit the dependence of hematopoietic cells on IL-2, IL-3, and possibly granulocyte-macrophage colony-stimulating factor, none of whose receptors are known to be of the tyrosine kinase type.

Abelson murine leukemia virus↗
PubMed1986

Enhanced transcription in a pre-B-like cell line transformed with cloned antibody genes after antigen-specific stimulation.

The transducing vector, pSV2-neo, carrying the rearranged immunoglobulin (Ig) heavy (mu) and light (kappa) chain genes specific for the hapten 2,4,6-trinitrophenyl (TNP) was introduced into a pre-B cell line. The transformants expressed the TNP-specific IgM receptor on the surface. Furthermore, the addition of TNP-bovine serum albumin (hapten-carrier conjugate) to the culture media activated the expression of the transferred Ig genes and several endogenous genes such as v-abl and beta-tubulin. However, expression of the beta2-microglobulin gene was not affected. The results presented in this paper show that transfection of cloned Ig genes into B cells is a useful system for establishing monoclonal B cell lines expressing functional Ig receptor molecules with defined hapten specificity.

Animals↗
PubMed1988

Bovine mononuclear cell lines transformed by Theileria parva or Theileria annulata express different subpopulation markers.

Bovine mononuclear cell lines infected with the protozoan parasites Theileria annulata and T. parva have been studied with a panel of monoclonal antibodies reacting with bovine lymphocyte subpopulation markers. All infected lines are MHC class II positive, though the amount of class II antigen expressed varied between lines, and within individual lines there was variation in the proportion of positive cells from 100% with many, to less than 10%. All lines were negative for a macrophage/monocyte marker and for surface IgM. The T. parva lines tested were all positive for BoT4 or BoT8 or both, whereas T. annulata lines were uniformly negative for both of these markers. These results suggest that the two parasites preferentially infect different lymphocyte subpopulations.

Animals↗
PubMed1997

HTLV-I infection in squirrel monkeys (Saïmiri sciureus) using autologous, homologous, or heterologous HTLV-I-transformed cell lines.

Peripheral blood mononuclear cells (PBMC) from three adult male squirrel monkeys (Saïmiri sciureus) were transformed by human T-cell leukemia/lymphoma virus type I (HTLV-I) by cocultivation with lethally irradiated human MT-2 cells. Three permanent monkey T-cell lines producing HTLV-I were obtained and characterized. Six weeks after inoculation seroconversion was observed in three of three monkeys inoculated with autologous transformed T cells and in two of three monkeys receiving homologous cells. Proviral DNA was detected in their PBMC at various times after inoculation, with the highest proviral load and antibody titers being found in monkeys infected with homologous cells. Monkeys inoculated with heterologous MT-2 cells did not seroconvert, and HTLV-I provirus was detected only transiently in their PBMC. To determine whether in vitro and in vivo HTLV-I infection of squirrel monkey cells led to a selection of monkey-adapted viral mutants, comparative sequencing of the proviral gp21 env between ex vivo monkey HTLV-I-infected PBMC, the inoculum, and MT-2 cells was done and no significant differences were detected. The squirrel monkey, which is naturally free of simian T-cell leukemia/ lymphoma virus, thus appears to be a suitable model for evaluating HTLV-I candidate vaccines and for studying the pathogenesis of HTLV-I.

Animals↗
PubMed1999

Amplification of the Y chromosome in three murine tumor cell lines transformed in vivo by different human prostate cancers.

Conventional and molecular cytogenetic analyses of three murine cancer cell lines that had been induced in male athymic mice by the injection of three different human prostate cancer cell lines revealed selective amplification of the Y chromosome. In particular, analysis of metaphase and interphase nuclei by fluorescence in situ hybridization (FISH) with the mouse Y chromosome-specific DNA painting probe revealed the presence of various numbers of Y chromosomes, ranging from one to eight, with a large majority of nuclei showing two copies (46.5-60.1%). In Interphase nuclei, the Y chromosomes showed distinct morphology, allowing identification irrespective of whether the preparations were treated for 15 min or for 5 h with Colcemid, a chemical known to cause chromosome condensation. However, FISH performed on human lymphocyte cultures with chromosome-specific DNA painting probes other than the Y chromosome did not reveal condensed chromosome morphology in interphase nuclei even after 12 h of Colcemid treatment. Our FISH results indicate that (1) the Y chromosome is selectively amplified in all three cell lines; (2) the mouse Y chromosome number is comparable in both interphase and metaphase cells: (3) the Y chromosome number varies between one and eight, with a large majority of cells showing two or three copies in most interphase nuclei; (4) the condensation of the Y chromosome is not affected by the duration of Colcemid treatment but by its inherent DNA constitution; and (5) the number of copies of the Y chromosome is increased and retained not only in human prostate tumor cell lines but also in murine tumors induced by these prostate tumor cell lines.

Animals↗
PubMed1995

Human fibroblasts (KMST-6/RAS cell line) transformed with 60Co gamma-rays and c-Ha-ras oncogene produce a large amount of granulocyte colony-stimulating factor (G-CSF); production is enhanced by cAMP, theophylline, and butyrate.

Human fibroblasts (KMST-6/RAS cell line), which was malignantly transformed in vitro with 60Co gamma-rays and the c-Ha-ras oncogene, produced a large amount of granulocyte colony-stimulating factor (G-CSF). The production was greater during the logarithmic growth phase than during the stationary phase. cAMP and theophylline, alone or in combination, and butyrate significantly enhanced G-CSF production, but dexamethasone or 5-azacytidine did not. Enhanced production of G-CSF by these agents was regulated at the posttranscriptional level. Neither the expression of the ras oncogene nor the tumorigenicity of the cells correlated with the production of G-CSF.

Butyrates↗
PubMed1974

Mutants of nonproducer cell lines transformed by murine sarcoma virus. 3. Detection and characterization of RNA specific for helper and sarcoma viruses.

BALB/3T3 cells transformed by the Kirsten sarcoma virus (nonvirus producer BALB/3T3 cells) and mutant cell lines derived therefrom by treatment with bromodeoxyuridine (BrdU) were analyzed for expression of virus-specific RNA using single-stranded DNA transcripts of Rauscher leukemia virus (RLV), a virus activated in one of the cell lines (58-2T), and Ki-SV-specific DNA transcript; the latter transcript after removal of all sequences cross-reactive with RLV RNA. The Rauscher virus DNA detected multiple copies of viral RNA in virus-producing cells ( approximately 2.5 x 10(3)/cell) whether infected with RLV or activated to produce virus with BrdU. Nonproducer (NP) cells and normal BALB cells showed small numbers of RNA genomes (70-250/cell) and only partial saturation of the transcript. The intracellular RNA sedimented at 35S (main peak) with a variable minor peak at 20S with the exception of one mutant cell, M-43-2 (main peak at 26-27S). The 58-2T transcript reacted preferentially in NP cells and their derivatives with biphasic kinetics suggesting the possibility of sequences specific for the original transforming virus. The size of Ki-SV specific sequences were 30S in mutant cells whether or not complete virus was being produced and independent of in vivo transplantability.

Animals↗
PubMed2004

Sequential cytogenetic and molecular cytogenetic characterization of an SV40T-immortalized nasopharyngeal cell line transformed by Epstein-Barr virus latent membrane protein-1 gene.

Cytogenetic and molecular cytogenetic analyses were performed on four sublines derived from a newly established, SV40T-immortalized nasopharyngeal (NP) cell line, NP69, with two of the sublines expressing LMP1, an Epstein-Barr virus-encoded gene. A total of seven cytogenetically related subclones were identified, all having highly complex karyotypes with massive numerical and structural rearrangements. Centromeric rearrangements in the form of isochromosomes and whole-arm translocations were prevalent. A cytogenetic sign of gene amplification [i.e., homogeneously staining region (HSR)] was detected at 1q25 in all metaphase cells analyzed. Multicolor combined binary ratio labeling fluorescence in situ hybridization (COBRA-FISH) was used to confirm the karyotypic interpretations. Furthermore, multicolor COBRA-FISH also showed that part of the HSR contained chromosome 20 material. Extensive clonal evolution could be observed by the assessment of karyotypic variation among different subclones and individual metaphase cells. The evaluation of clonal evolution enabled the identification of the temporal order of chromosome aberrations during cell immortalization and malignant transformation. A striking karyotypic similarity was found between sublines expressing LMP1 and an NP carcinoma cell line, with loss of genetic material from chromosome arm 3p being an important recurrent observation. More interestingly, the karyotypic features of NP69 were also similar to those of many epithelial malignancies. Our observations suggest that serial transformation of NP cell lines might provide a useful in vitro model for the study of the multistep neoplastic transformation of NP cells.

Cell Culture Techniques↗