Transformation in vitro of glial hamster cells by Rous sarcoma virus.

Cell lines from the brains of inbred CF hamster embryos were established in vitro. The morphology of the cells in the light and electron microscopes was that of glial cells, and the cells contained the nervous system-specific protein S-100. Infection with the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup B, resulted in foci of transformation. The transformed cells were virogenic and upon intracerebral and sc inoculations into young hamsters, they developed into histologically typical gliomas.     

Transformation of NIH3T3 cells by A-MuLV proviral DNA: effect of plasmid linearization and carrier DNA on transformation efficiency

We have optimized the conditions for efficient NIH3T3 focus formation by calcium phosphate transfection of proviral Abelson-murine leukemia virus (A-MuLV) plasmid DNA. Linearized pA-MuLV, P120 or P160 strains, when transfected with calf thymus carrier DNA, will produce 40-50 foci/100 ng pA-MuLV without co-transfecting Moloney-murine leukemia virus (Mo-MuLV) plasmid DNA.     

Transformation of chicken chondrocytes by Rous sarcoma virus.

Chicken chondrocytes isolated from 11-day-old chicken vertebrate cartilage were transformed by Rous sarcoma virus ts LA24 of the Prague strain as well as by the wild-type Prague strain of Rous sarcoma virus. The morphology of chondrocytes transformed by Rous sarcoma virus ts LA 24 was dependent on the temperature, and the change was reversible. A similar but irreversible change in morphology was observed with chondrocytes transformed by wild-type virus. Hyaluronic acid production and deoxyglucose transport were markedly increased in the transformed chondrocytes. A marked increase of labeled acetate incorporation was observed with the transformed chondrocytes. In contrast to the normal chondrocytes, the labeled hyaluronic acid synthesized by the transformed chondrocytes was mostly released into the culture medium.     

Electron microscopic observation of simian sarcoma virus transformed NIH 3T3 cells in vitro

For electronic microscopic observation, we found SSV-transformed NIH 3T3 cells were different from non-transformed cells. In SSV-transformed NIH 3T3 cells nuclei/cytoplasma ratio was increased and in cytoplasma the ribosomes (polyribosomes were attached to the swollen rough endoplasmic reticulum. It was likely that ribosomes were lined together functionally and structionally to produce specific protein (PDGF-like protein).     

Persistence of Rous sarcoma virus in transformed non-permissive cells: mechanism of virus induction by association with permissive cells in the absence of Sendai virus

Virus induction by association of non-permissive cells transformed by Rous sarcoma virus (RSV) and permissive chicken cells in the absence of Sendai virus has been studied with two clones of BHK21 hamster fibroblasts transformed respectively by Schmidt-Ruppin strain RSV (clone RS2) and Bryan strain RSV (clone RB12), and with sub-clones derived from these clones after many divisions. The main results have been the following: (1) All the transformed non-permissive (TNP) cells presumably carry at least one complete viral genome since RSV could be recovered from all the subclones tested. (2) The first infective centers which appear in mixed cultures are presumably heterokaryons formed by spontaneous fusion of single TNP cells with chicken cells; and Rous cells (transformed chicken cells) which appear later on, arise by secondary infection of chicken cells. (3) The incidence of appearance of infective centers is low and can vary considerably depending on subclones in the case of RS2 cells. In the most highly inducible subclones, it never exceeds one center per 10³ RS2 cells and it can be much less in other subclones. (4) No induction was observed with two RS2 subclones from which RSV was nevertheless recovered after Sendai virus-mediated cell fusion. (5) In the case of one of these subclones, even heterokaryon formation was inefficient in removing the block of virus production in the TNP cells. (6) No induction was obtained by any other method than cell association and no subviral determinant able to give rise to virus in CE cells was recovered from TNP cells. The reasons for non-permissiveness and the mechanism of induction are discussed.     

Transformation of NIH3T3 cells with synthetic c-Ha-ras genes

Synthetic human c-Ha-ras genes in which amino acid codons were altered to those which are frequently used in highly expressed Escherichia coli genes were ligated to the 3'-end of Rous sarcoma virus long terminal repeat. When NIH3T3 cells were transfected with the plasmids having those genes with valine at codon 12, leucine at codon 61 or arginine at codon 61, transformants were efficiently produced. These results indicated that the synthetic c-Ha-ras genes are expressed in a mammalian system even though their codon usage is altered to correspond with that of E. coli. This expression vector system should be useful for studies on the structure-function relationships of c-Ha-ras, since the synthetic gene can be easily modified to have multiple base alterations, and can also be used simultaneously for the production of large amounts of p21 in E. coli for biochemical and biophysical studies.     

Transformation of human cells by polyoma and Rous sarcoma viruses mediated by inactivated Sendai virus

Transformation of embryonic human fibroblasts was obtained by treatment with inactivated Sendai virus and polyoma virus. Transformed cells differed morphologically from normal cells and formed a stable cell line (P-2). A stable transformed cell line (23) was also formed after absorption of Rous sarcoma virus (Schmidt-Ruppin strain) and inactivated Sendai virus on the embryonic human fibroblasts. Cell line P-2 and cell line 23 contained a human chromosome set, although some karyological abnormalities were apparent. The cell lines P-2 and 23 showed some characteristics of malignant cells growing in vitro (ability to multiply when suspended in semi-solid medium and to form tumours in the cheek pouches and brains of golden hamsters). The potent transforming factor (s) was/were synthesized by these cells. The transforming factors had some properties identical to those of polyoma or Rous viruses and caused rapid transformation of embryonic mouse and human cells growing in vitro. Foci of epithelioid cells appeared in human embryonic fibroblasts after the absorption of human adenovirus type 12 and inactivated Sendai virus.     

Rous sarcoma virus production in mixed cultures of mammalian Rous sarcoma cells and chick embryo cells

The induction of Rous sarcoma virus (RSV) production was analyzed in mixed cultures of mouse or hamster Rous sarcoma cells and chick embryo cells treated with ultraviolet inactivated hemagglutinating virus of Japan (UV-HVJ). The pre-treatment of UV-HVJ with a certain concentration of anti-HVJ serum increased the yield of infectious RSV in mixed cultures enabling a quantitative analysis of this phenomenon to be made. RSV-inducing ability of a mixture of HVJ and anti-HVJ serum was affected by the concentration of anti-HVJ serum and showed changes in parallel with the cell fusion activity of the mixture. The effect of UV-HVJ on RSV induction was not achieved by incubation of the cell mixture at 0° C as it was after shaking at 37° C. These findings suggest that the production of infectious RSV in mixed cultures may be initiated in heterokaryons of mammalian Rous sarcoma cells and chick embryo cells. The number of infective centers in mixed cultures was, however, as small as 1% of that of heterokaryons. It is possible that only a small fraction of the heterokaryon population is productive of infectious RSV.     

Transformation of NIH3T3 cells by transfection with UV-irradiated human c-Ha-ras-1 proto-oncogene DNA

Our previous studies have shown that human skin cancers occurring on sun-exposed body sites frequently contain G----T mutations at the second position of Ha-ras codon 12. In this study, we investigated whether the c-Ha-ras-1 proto-oncogene could be activated by in vitro UV-irradiation of pEC plasmid DNA, which contains the 6.6 kb BamHI fragment of the human c-Ha-ras-1 proto-oncogene. Focus formation and nude mouse tumorigenicity assays showed that UV-irradiated pEC DNA induced morphologic and tumorigenic transformation of NIH3T3 cells in multiple cycles of transfection, whereas unirradiated pEC DNA did not. DNAs from secondary cycle foci and tertiary cycle tumors were analyzed for mutations in Ha-ras codons 12 and 61 using the polymerase chain reaction and synthetic oligonucleotide probes. Eleven of 11 secondary cycle foci analyzed possessed a G----T mutation at the second position of Ha-ras codon 12. However, the nude mouse tumors exhibited a G----A mutation at position 1 of the Ha-ras codon 12. These results suggest that in vitro UV irradiation of the c-Ha-ras-1 proto-oncogene DNA can induce mutations that are similar to those found in human skin cancers that originated on sun-exposed body sites.     

Transformation of NIH/3T3 cells by DNA from a human hepatoma cell line with integrated hepatitis B virus DNA

We have studied by means of DNA-mediated gene transfer the transforming activity of the DNA of the human hepatoma cell line HCC-M, which contains genomes of hepatitis B virus (HBV) in integrated form. DNA from HCC-M induced transformed foci on transfection of NIH/3T3 cells. DNAs from primary transformants were capable of inducing secondary transformants. Most of the DNAs of these transformants were demonstrated to contain both human repetitive sequences and HBV DNA, indicating that the transformants had incorporated exogenous human DNA and HBV DNA as well. These results suggest that transformation occurs as the result of the transfer of oncogene which might be closely associated with HBV genome.     

Specific increase in polyamine levels in chick embryo cells transformed by Rous sarcoma virus.

Chick embryo fibroblasts and chorioallantoic membranes of chick embryos infected with oncogenic or nononcogenic viruses were analyzed for polyamines. Nononcogenic viruses (influenze, Newcastle disease, or vaccinia virus) had no effect on the polyamine content of chorioallantoic membranes. Transformation of chorioallantoic membranes by a wild-type or temperature-sensitive mutant strain of Rous sarcoma virus under permissive conditions (37 degrees) caused a 2- to 4-fold increase in cellular spermidine and putrescine content. Only putrescine accumulated in chick embryo fibroblasts transformed by Rous sarcoma virus at 37 degrees. At the nonpermissive temperature (42 degrees), the temperature-sensitive mutant, unlike the wild-type strain, did not alter cellular morphology or polyamine content.     

Transformation of NIH3T3 Cells with Synthetic c-Ha-ras Genes

Synthetic human c-Ha- ras genes in which amino acid codons were altered to those which are frequently used in highly expressed Escherichia coli genes were ligated to the 3'-end of Rous sarcoma virus long terminal repeat. When NIH3T3 cells were transfected with the plasmids having those genes with valine at codon 12, leucine at codon 61 or arginine at codon 61, transformants were efficiently produced. These results indicated that the synthetic c-Ha- ras genes are expressed in a mammalian system even though their codon usage is altered to correspond with that of E. colt. This expression vector system should he useful for studies on the structure-function relationships of c-Ha- ras , since the synthetic gene can be easily modified to have multiple base alterations, and can also be used simultaneously for the production of large amounts of p21 in E. coli for biochemical and biophysical studies.     

Studies on the loss of growth inhibition in cells infected with Rous sarcoma virus

The loss of density-dependent and anchorage-dependent growth was studied in cells infected with RSV. Transformed cells, but not uninfected cells, will proliferate in soft gel suspension and amongst crowded normal cells. A large proportion of cells freshly infected with RSV acquired the capacity to proliferate in soft agar suspension even when placed in suspension immediately after infection. Cell transformation and viral replication were inhibited by plating freshly infected cells on to crowded mouse embryo cells or 3T3 cells. The inhibition of cell transformation depended on close proximity to crowded mouse cells spread on a solid substratum. Cells became released from density-dependent transformation within 12 h following infection. The change in proliferative behaviour of transformed cells was not influenced by the quantity or envelope properties of virus released from the cell surface. Pre-infection with RAV1 did not affect density-dependent cell transformation by RSV. Freshly infected cells held in the inhibited state for 6 days were able to transform when replated to sparse conditions. It was concluded that cell transformation depended on cell proliferation but that infection with RSV could not initiate the cell division cycle in crowded monolayers.     

Induction of thymidine-3H incorporation in multinucleated myotubes by Rous sarcoma virus

During the differentiation of skeletal muscle, mononucleated myoblasts fuse to form multinucleated myotubes. After fusion, both DNA synthesis and nuclear division cease. When clones of chick embryonic leg muscle consisting of both myoblasts and myotubes are infected with Rous sarcoma virus, DNA synthesis (as measured by thymidine-³H incorporation) is induced in the nuclei of myotubes within 24 hours. On the other hand, nuclear division is not reactivated. These findings indicate that the restriction of DNA synthesis which attends the formation of myotubes is not irreversible since RSV, an RNA virus, reactivates DNA synthesis in mature myotubes.     

Inhibition by methioninyl adenylate of focus formation by Rous sarcoma virus.

Methioninyl adenylate is a specific and potent inhibitor of the enzyme methionyl-tRNA synthetase and, consequently, of protein biosynthesis. In cultures of chick embryo fibroblasts infected with Rous sarcoma virus, incubation for a 2-day period with 1 to 3 mM concentrations of this inhibitor, as late as 4 days after infection, irreversibly prevented subsequent formation of foci of transformed cells. Later addition could also elicit the irreversible disappearance of already existing foci, by phenotypic reversion and/or cell killing. Virus production in transformed cells and replication in newly infected cells were also inhibited but to a lesser degree. Under the same conditions, the same concentrations of methioninyl adenylate caused only a reversible growth arrest of normal cells. The selective toxicity of the inhibitor for transformed cells is not due to a greater affinity for the target enzyme, but it may be due to the fact that inhibition of protein biosynthesis is only partially reversible in these cells, whereas it is fully reversible in normal cells.     

Differential tyrosine-specific phosphorylation of integrin in Rous sarcoma virus transformed cells with differing transformed phenotypes.

We have investigated the localization and phosphorylation of the fibronectin receptor in chick embryo fibroblasts transformed either by wild-type Prague C strain Rous sarcoma virus, which induces a rounded, less adhesive phenotype with a loss of surface fibronectin and gross cytoskeletal changes, or transformed by rASV2234.3, a variant which induces a flat, adhesive morphology with the retention of surface fibronectin and more normal cytoskeleton. Immunofluorescence showed co-distribution of pp60v-src with integrin and fibronectin fibrils in rASV2234.3-transformed cells. Total levels and surface expression of integrin were unchanged in both transformed cell types compared with untransformed cells. However, whereas integrin band 3 (beta 1 subunit) in Prague C-transformed cells was hyperphosphorylated on tyrosine, this was reduced virtually to normal in rASV2234.3-transformed cells. A similar differential phosphorylation of integrin band 3 could be found in membranes phosphorylated in vitro. rASV2234.3 pp60v-src was less efficient in phosphorylating a synthetic peptide containing the putative integrin tyrosine phosphorylation site, indicating that this variant pp60v-src has an altered substrate specificity compared to Prague C pp60v-src. The correlation between tyrosine-specific phosphorylation of integrin and loss of surface fibronectin suggests that this could play an important role in the induction of the transformed phenotype.     

Aggregation of platelets and cell membrane vesiculation by rat cells transformed in vitro by Rous sarcoma virus.

Primary rat embryo cells and established normal rat kidney cells transformed in vitro by Rous sarcoma virus induced the aggregation of rat platelets in vitro. The aggregating activity was shown to be specific for the transformed cells and was absent in the normal parent cells. The aggregation reaction is accompanied by the release of serotonin from the platelets. Further analysis and purification of this activity from the transformed cells demonstrated that the activity is shed from the cells growing in culture and is associated with membrane vesicles of heterogenous size. The normal cells also produced vesicles in culture; however, the level of vesicle productio was less than that from transformed cells, and the platelet aggregation and serotonin release activities were greatly reduced or absent in these vesicles.