methylcellulose and Cell-Transformation--Viral

Topics: 3T3 Cells; Agar; Animals; Antigens, Polyomavirus Transforming; Cell Adhesion; Cell Communication; Cell Count; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Culture Media; Cytopathogenic Effect, Viral; Methylcellulose; Mice; Mice, Nude; Mutation; Neoplasm Transplantation; Polyhydroxyethyl Methacrylate; Rats; Simian virus 40; Virology

The present studies were undertaken to investigate the ability of Abelson murine leukemia virus (A-MuLV) to transform cells derived in vitro from pluripotent hemopoietic progenitor cells of high proliferative potential. We now report that continuously growing, autonomous cell lines could be obtained from a high proportion of individually infected multilineage colonies generated in assays of spleen cells from normal adult mice if the infected cells were cocultivated for the first two to three months with irradiated NIH-3T3 cells. No lines were obtained if the 3T3 cell feeders were not initially present. Similar results were obtained when the cells exposed to virus were from multilineage colonies originating from isolated single cells obtained by replating small blast colonies. Characterization of the transformants and a number of derivative cloned sublines revealed the consistent presence of a mast cell phenotype, with some suggestion of macrophage differentiation in a few cases. All cell lines tested produced virus, showed a variable pattern of A-MuLV integration, and gave rise directly to tumors when injected subcutaneously, as shown by both Southern analysis and cytogenetic studies. The early absolute but transient dependence of these A-MuLV mast cell transformants on a fibroblast feeder suggests a multistep process in their evolution, in which the acquisition of autonomy from factors of mesenchymal cell origin may play an important role.

Topics: Animals; Bone Marrow; Bone Marrow Cells; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Clone Cells; Culture Media; Mast Cells; Methylcellulose; Mice; Mice, Inbred BALB C; Phenotype; Retroviridae

Topics: Animals; Antigens, Neoplasm; Antigens, Viral; Antigens, Viral, Tumor; Cell Cycle; Cell Division; Cell Line; Cell Nucleus; Cell Transformation, Viral; Clone Cells; Cricetinae; Cytoplasm; Embryo, Mammalian; Fluorescent Antibody Technique; Mesocricetus; Methylcellulose; Neoplasm Transplantation; Neoplasms, Experimental; Simian virus 40

Topics: Adult; Antigens, Neoplasm; Antigens, Viral; Antigens, Viral, Tumor; Blood; Cell Division; Cell Line; Cell Transformation, Viral; Clone Cells; Female; Fibroblasts; Humans; Karyotyping; Kinetics; Methylcellulose; Peptide Hydrolases; Simian virus 40

Whereas methylcellulose cultures have been valuable for biochemical studies on hemopoiesis in vitro, it has been difficult to study cellular morphology in these cultures. We have found that clotting factors can be added at any time during the culture period and non-adherent cells become entrapped in a semisolid clot. The clot can be removed, fixed and stained for detailed microscopic examination.

Topics: Animals; Bone Marrow Cells; Cell Transformation, Viral; Cells, Cultured; Culture Media; Erythropoiesis; Friend murine leukemia virus; Leukemia, Experimental; Methylcellulose; Mice; Staining and Labeling

We have developed a non-enzymatic acetylating procedure, closely resembling the situation in vivo, utilizing acetyl adenylate, an acetylating agent in vivo, that mimics the enzymatic hyperacetylation of specific histone species. Analysis of the acetylated species of calf thymus histones produced from reaction with soluble chromatin yielded the same species generated in vivo and observed during active gene transcription. Four species of histone H4 and three of histone H3 occur with no alteration in histones H2A or H2B. This procedure has been utilized to hyperacetylate simian virus 40 (SV40) minichromatin in vitro in order to study the effect of acetylated compared to non-acetylated minichromatin in cellular transformation of cultured Balb/3T3 cells. Transformed cell foci appeared only in the cultures infected with hyperacetylated SV40 minichromatin. To select for cellular transformation, foci were transferred to agar-lined culture flasks and grown in the suspension of 1% methylcellulose. The selected cells were plated on slides and analyzed for the presence of T-antigen by indirect immunofluorescence. The hyperacetylated-minichromatin-infected cells exhibited T-antigen-specific fluorescence, while non-acetylated-minichromatin-treated cells and normal cells showed no specific fluorescence. These results suggest a major role for histone hyperacetylation in the mechanism of SV40 viral transformation.

Topics: Animals; Antigens, Neoplasm; Antigens, Viral; Antigens, Viral, Tumor; Cell Transformation, Viral; Cells, Cultured; Chromatin; Histones; Lysine; Methylcellulose; Mice; Simian virus 40

Topics: Animals; Antigens, Neoplasm; Antigens, Viral; Antigens, Viral, Tumor; Cell Division; Cell Transformation, Viral; Cells, Cultured; Cricetinae; Culture Media; Cytoskeleton; Methylcellulose; Neoplasms, Experimental; Plasminogen Activators; Simian virus 40; Viral Proteins; Virus Replication

Three adenovirus-2-transformed rat embryo brain cell lines and their methylcellulose-selected sub-clones were examined for fibronectin expression, anchorage-independent growth, saturation density, T antigen expression and morphology. Tumorigenicity studies were carried out on newborn and ATS immunosuppressed syngeneic rats and congenitally athymic nude mice. With one exception the methylcellulose sub-clones contained significantly fewer fibronectin-positive cells than the parent lines; a number of sub-clones contained no fibronectin-positive cells. Methylcellulose selection did not always alter cell morphology, saturation density or anchorage-independent growth as compared with parent lines. However, the methylcellulose sub-clones were considerably more malignant than the parent cell lines as measured by invasion and metastasis in nude mice. No in vitro characteristic correlated with malignant behaviour.

Topics: Adenoviridae; Animals; Antigens, Viral; Antilymphocyte Serum; Brain; Cell Line; Cell Separation; Cell Transformation, Neoplastic; Cell Transformation, Viral; Clone Cells; Culture Media; Female; Fibronectins; Fluorescent Antibody Technique; Male; Methylcellulose; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Rats; T-Lymphocytes; Transplantation, Isogeneic

By the calcium technique, intact DNA of bovine adenovirus type 3 (BAV3) was found to transform A31 cells, a clone of BALB/3T3. Transforming activity was resistant to RNase and Pronase but sensitive to DNase. The efficiency of transformation was approximately 5 to 10 foci per mug of DNA. Attempts were also made to test for transforming activity of BAV3 DNA fragments prepared with restriction endonucleases EcoRI and HindIII. The activity was found to associate exclusively with the EcoRI D fragment mapped in the region of 3.6 and 19.7 units (molecular weight, 3.9 x 10(6)). No transformation could be obtained with three HindIII fragments, J, E, and B, located at the left-hand end of the BAV3 genome. However, the enzymatic joining of J and E fragments (0 to 11.9 map units) with a ligase restored the transforming activity. These results suggest that all the genetic information of BAV3 required for transformation is located in the region between 3.6 and 11.9 units on the viral genome. Some properties of A31 cells transformed by BAV3 DNA EcoRI D fragment (TrD) and the ligated DNA of HindIII J and E fragments (TrJE), as well as those transformed by whole BAV3 DNA (Tr), were examined. As compared to untransformed A31 cells, all the transformed cell lines tested showed rapid growth, high saturation densities, and anchorage-independent growth. Moreover, they contained BAV3-specific T antigen and induced tumors in adult nude and BALB/c mice. These properties of Tr, TrD, and TrJE lines were similar to those of BAV3-transformed cells.

Topics: Adenoviridae; Animals; Cattle; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Culture Media; DNA Restriction Enzymes; DNA, Viral; Methylcellulose; Mice; Neoplasm Transplantation