Intracellular K and the expression of transformation parameters by chick cells transformed with the Bryan strain of Rous sarcoma virus

As normal chick embryo (CE) cells entered quiescence the intracellular concentrations of both Na+ and K+ declined. Comparable decreases in intracellular concentrations of Na+ and K+ were not observed in CE cells transformed by either the Schmidt-Ruppin (SR) or the Bryan (B) strain of Rous sarcoma virus (RSV). Intracellular concentrations of Na+ were higher in SR-RSV-transformed CE cells than in B-RSV-transformed cells and uninfected CE cells at all times after plating. In contrast, intracellular concentrations of K+ were higher in B-RSV-transformed CE cells than in SR-RSV-transformed cells. Uninfected CE cells incubated in medium containing an elevated concentration of K+ (an increase from 5 to 30 mM) exhibited several, but not all, of the transformation parameters expressed by B-RSV-transformed CE cells.     

Immunological study of a cellular 35K phosphorylated polypeptide detected in Rous sarcoma virus transformed cells

We have immunoprecipitated a phosphoprotein of 35K daltons (35K) common to RSV-transformed chick embryo fibroblasts (CEF) and rodent cells. The phosphorylation of this antigen depends on the expression of the v-src gene and contains phosphotyrosine. The pre-existing 35K protein, of CEF infected de novo, was further shown to become phosphorylated shortly after the appearance of active pp60v-src, and about 1 day before morphological transformation. The experiments with RSV-transformed rodent cells have shown that the 35K phosphoprotein is associated with the cellular framework. Another phosphoprotein of 37K was found in the RSV-transformed rodent cells, but not in the transformed CEF. However, this protein was not phosphorylated at tyrosine residues and its phosphorylation persisted, at the restrictive temperature, in cells transformed by a ts mutant of RSV.     

Preferential inhibition of growth and protein synthesis in Rous sarcoma virus transformed cells by diphtheria toxin.

Protein synthesis in Rous sarcoma virus-transformed cultured chick embryo cells is shown to be more sensitive to diphtheria toxin than protein synthesis in normal chick embryo fibroblasts. Similarly, cells from viral-induced sarcomas are more sensitive to diphtheria toxin than cells from normal tissues of the same chicken. Diphtheria toxin inhibits the growth of virus-transformed chick cells in culture at a concentration that has no effect on control cells.     

Inhibition of glycosylation processing alters the growth parameters of cells transformed by the oncogene of Simian Sarcoma virus

Summary Normal rat kidney (NRK) cells transformed by the v-sis oncogene of Simian Sarcoma virus (SSV) were treated with the glucosidase I inhibitor castanospermine. The inhibitor did not change cell morphology, but specific growth parameters such as serum- and anchorage-independence were lost.     

Expression of viral envelope glycoprotein and transformation genes in cells transformed by a defective Kirsten murine sarcoma virus.

Concurrent expression of transformation properties and virion envelope glycoproteins has been observed as a property of several clones of normal rat kidney cells transformed by, but not producing, Kirsten murine sarcoma virus. The present studies were carried out to determine whether a genetic linkage exists between the viral sarcoma and envelope genes in these cells. Several alternative models for the possible structure and origin of the sarcoma and envelope genes were considered. One possibility, that the viral envelope gene was derived from an endogenous rat virus, was studied by characterization of the interference properties of the transformed cells. The sarcoma virus genome of envelope-positive clones was efficiently rescued by woolly monkey and murine xenotropic but not by murine ecotropic viruses. Thus, the interference properties of cells producing the envelope glycoprotein are analogous to those of a cell producing murine ecotropic virus, indicating that the envelope was of murine viral origin. In these experiments it was also found that sarcoma viruses rescued from envelope-positive cells upon superinfection with primate and xenotropic murine viruses could transform host cells for both xenotropic and ecotropic viruses, indicating that these superinfecting viruses became phenotypically mixed with the ecotropic envelope expressed in transformed, envelope-positive cells. Possible linkage between the envelope and transformation genes was analyzed by the frequency of concurrent rescue of sarcoma and envelope genes. Transfer of the Kirsten sarcoma viral genome to uninfected cells upon rescue by superinfection with woolly monkey virus showed a high frequency of apparent segregation of the transformation and envelope genes [from 29 to 57% for (KSV env⁺)NRK-6]. The model supported by the present data is that the transformed, envelope-positive cells were infected with a virus which contained both the envelope and the sarcoma genes.     

Viral glycoprotein synthesis under conditions of glucosamine block in cells transformed by avian sarcoma viruses.

The synthesis of the viral glycoprotein gp85 in B77-transformed chick embryo cells cultured under conditions of glucosamine block was studied. As determined by long-term labeling with radioactive fucose, no gp85 is formed in the presence of 20 μmoles/ml of glucosamine, although an intracellular component was detected which was recognized by a serum prepared against gp85 but migrated in SDS-polyacrylamide gel with a greater mobility than gp85. The labeling properties of this component suggest that it is a carbohydrate-deficient form of gp85. These data are discussed in terms of a chemical mechanism for glucosamine block.     

Cyclic AMP levels and 2-deoxyglucose uptake in cells transformed by temperature-sensitive class T mutants of Rous sarcoma virus.

Chick embryo fibroblasts, normal and transformed by the Schmidt-Ruppin strain of Rous sarcoma virus or by mutants derived therefrom which were temperature-sensitive for transformation, contain cAMP levels which were broadly similar within each experiment performed. In contrast, 2-deoxyglucose uptake was increased by two- to over fourfold in cells transformed by the wild type strain or by the mutants at the permissive temperature. It appears that while 2-deoxyglucose uptake is a reliable marker of the transformed state, the same does not hold for the cAMP level in this system.     

Novel localization of pp60src in Rous sarcoma virus-transformed rat and goat cells and in chicken cells transformed by viruses rescued from these mammalian cells

We have investigated by indirect immunofluorescence and subcellular fractionation the intracellular location of pp60^src in RSV-transformed mammalian cells and in CEF cells transformed by virus rescued from these cells. Two independently derived cell lines were examined: RR1022 cells isolated from an in vivo sarcoma induced in an Amsterdam rat by infection with SR-RSV-D; and Pcl, cells isolated from a soft agar colony of normal goat skin fibroblasts transformed in vitro by SR-RSV-D. Transforming viruses (RSV-RR and RSV-Pcl) were rescued from RR1022 and Pcl cells by fusion with CEF cells. Immunofluorescence microscopy showed association of pp60^src with the nuclear envelope and the juxtanuclear reticular membranes in the transformed mammalian cells and in CEF cells transformed by the rescued viruses, in contrast to the plasma membrane localization of pp60^src seen in SR-RSV-transformed CEF cells. Results of subcellular fractionation by differential centrifugation and fractionation of particulate fractions by equilibrium centrifugation in discontinuous sucrose gradients were in agreement with the differences in pp60^src distribution observed by immunofluorescence microscopy. Although the mammalian cell lines were independently derived, pp60^srcs isolated from RR1022 and Pcl cells both lacked amino-terminal 21- and 18-kilodalton [³⁵S]methionine S. aureus V8 protease peptides found in SR-RSV-D pp60^src. Proteolytic peptides identical to those of pp60^src from the mammalian cells were obtained from pp60^src proteins isolated from rescued virus-transformed CEF cells, suggesting that the alteration in the amino-terminal half of the src protein represents a stable change, and that an alteration in the primary structure of pp60^src is responsible for the altered intracellular membrane localization of pp60^src in these cells.