Long-term cultures of chick embryo fibroblasts transformed by the Schmidt-Ruppin strain (D) of Rous sarcoma virus.

Attempts have been made to keep in vitro, for extended periods of time, cultures of chick embryo fibroblasts transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D. Roller cultures of transformed chick cells kept in serum-deficient medium can be maintained without subcultivation for up to 6 months. The confluent cultures continuously release viruses and viable tumor cells into the medium. The released cells can be plated and have characteristics of growth and morphology which are relatively stable with time until the culture degenerates. Cells released at later stages of the culture produced substantially more viruses than those released earlier, suggesting that cell selection or differentiation occurs during long-term cultivation in low serum concentration. Long-term cultures of untransformed chick embryo fibroblasts can also be maintained in the same way. The release of viable cells by these confluent cultures, however, is negligible.     

Adenylate cyclase activity is decreased in chick embryo fibroblasts transformed by wild type and temperature sensitive Schmidt-Ruppin Rous sarcoma virus

Chick embryo fibroblasts (CEF) transformed by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV-SR) have decreased adenylate cyclase activity. In cells infected by a temperature-sensitive mutant of this virus (RSV-SR-T5), enzyme activity is near normal when the cells are grown at the non-permissive temperature (41°C) but decreases at the permissive temperature (36°). Adenylate cyclase activity decreases slowly over a 24 hr period to one half normal levels when CEF-RSV-SR-T5 are shifted from 41° to 36°C. The low enzyme activity in CEF-RSV-SR is not due to an alteration in the K_m ATP or a change in the kinetics of Mg⁺⁺ activation, and is not observed when the enzyme is assayed in the presence of NaF. We conclude that transformation by RSV-SR reduces adenylate cyclase activity by a different mechanism than the Bryan high-titer strain of RSV.     

Alterations in components of adenylate cyclase associated with transformation of chicken embryo fibroblasts by Rous sarcoma virus

Regulation of adenylate cyclase coincident with transformation of chicken embryo fibroblasts by Rous sarcoma virus is manifest as a 10-50% decrease in basal, Mg2+-, and forskolin-stimulated activities; activities elicited by fluoride and guanosine 5'-O-(3-thiotriphosphate) are unaltered. The level of the catalytic component of adenylate cyclase, assessed with activated stimulatory guanine nucleotide-binding protein (Gs), increases approximately 1.5-fold. The level of the beta subunit common to Gs and the inhibitory regulatory protein assessed by enzyme-linked immunotransfer blotting, increases 2.7-fold. The isoelectric behavior of the beta subunit is unaltered. The amount of radiolabel incorporated into the alpha subunit of Gs (Mr = 45,000) upon incubation of membranes with 32P-labeled NAD and cholera toxin increases 3-fold upon transformation. Detergent extracts prepared from membranes of untransformed and transformed fibroblasts nevertheless exhibit equivalent abilities to reconstitute fluoride-stimulated activities to membranes of the cyc-variant of mouse S49 lymphoma cells. Islet-activating protein catalyzes incorporation of radiolabel from 32P-labeled NAD into 39,000- and 41,000-dalton proteins; the extent of radiolabel incorporation does not change upon transformation. Modest alterations in the isoelectric behaviors of substrates for cholera toxin and islet-activating protein occur.     

Cooperative transformation studies with temperature-sensitive mutants of Rous sarcoma virus.

Stocks of Rous sarcoma virus Bryan strain were mutagenized using a bromodeoxyuridine treatment immediately after infection. Thirty temperature-sensitive (ts) mutants defective in transformation (td) were isolated by a replica plating technique. Twenty of these mutants were preliminarily characterized and found to be defective in late functions related to transformation. These mutants were used in experiments of cooperative transformation with four Prague strain td ts mutants of different co-transformation group. A small number of Bryan ts mutants were found to cooperate with some of the Prague mutants in transforming chicken embryo cells at the nonpermissive temperature. However, the amount of co-transformation observed was lower than that observed with cooperating Prague ts mutants and no clear-cut pattern of cotransformation was obtained in Prague and Bryan crosses. Indirect evidence indicates that cooperative transformation is the result of recombination events.     

Blockage of morphological transformation of chick embryo fibroblasts infected with Rous sarcoma virus by N-myristoyl glycinal diethylacetal in vitro

N-Myristoyl glycinal diethylacetal strongly inhibited morphological transformation of chick embryo fibroblasts infected with a temperature-sensitive mutant (tsNY68) of Rous sarcoma virus. Myristoylated or nonmyristoylated pp60v-src, which were expressed in tsNY68-infected cells in the absence or presence of the compound, were identified separately by fluorography or immunoblotting analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]myristate-labeled cell lysate. The results taken together suggest that the blockage of morphological transformation was caused by prevention of protein myristoylation of the transforming protein pp60v-src.     

Rous sarcoma virus mutant dlPA105 induces different transformed phenotypes in quail embryonic fibroblasts and neuroretina cells.

dlPA105 is a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion in the N-terminal portion of the v-src gene coding sequence. This virus was isolated on the basis of its ability to induce proliferation of quiescent quail neuroretina cells. The altered v-src gene encodes a phosphoprotein of 45,000 daltons which possesses tyrosine kinase activity. DNA sequencing of the mutant v-src gene has shown that deletion extends from amino acid 33 to 126 of wild-type p60v-src. We investigated the tumorigenic and transforming properties of this mutant virus. dlPA105 induced fibrosarcomas in quails with an incidence identical to that induced by wild-type virus. Quail neuroretina cells infected with the mutant virus were morphologically transformed and formed colonies in soft agar. In contrast, dlPA105 induced only limited morphological alterations in quail fibroblasts and was defective in promoting anchorage-independent growth of these cells. Synthesis and tyrosine kinase activity of the mutant p45v-src were similar in both cell types. These data indicate that the portion of the v-src protein deleted in p45v-src is dispensable for the mitogenic and tumorigenic properties of wild-type p60v-src, whereas it is required for in vitro transformation of fibroblasts. The ability of dlPA105 to induce different transformation phenotypes in quail fibroblasts and quail neuroretina cells is a property unique to this Rous sarcoma virus mutant and provides evidence for the existence of cell-type-specific response to v-src proteins.     

Polyamines and tumor cells: effect of transformation of chick embryo fibroblasts by Rous sarcoma virus on polyamine levels

The effect of transformation of chick embryo fibroblasts, by Rous sarcoma virus, on intracellular polyamine levels has been studied. A good correlation between spermidine and cellular protein content has been demonstrated. Upon changing the medium, a sharp increase in spermidine level was noticed both in normal and transformed cells. This increase was accompanied by enhanced protein synthesis. The intracellular concentrations of spermine and spermidine were very similar in normal and transformed cells. On the other hand, significant differences in putrescine levels were demonstrated: in normal cultures the intracellular concentration of putrescine reached a plateau approximately 6 days after seeding, whereas a continuous rise of the diamine in transformed cells was noticed. These differences, which were observed in cultured cells, may explain the known accumulation of polyamines during neoplastic growth.