Interactions of concanavalin A with chick embryo fibroblasts transformed by Rous sarcoma virus. Study with an RSV mutant thermosensitive for transformation.

The interactions between concanavalin A and chick embryo fibroblasts, normal and infected with Rous sarcoma virus (RSV-BH) or its thermosensitive mutant RSV-BH-Ta, have been studied. Normal chick embryo cells and RSV-BH transformed cells showed at 4 and 25 degrees C a similar number of concanavalin A receptors per cell. Analysis of the binding data by the Scatchard relation showed that apparent changes in binding as a function of temperature are due to the thermodynamic properties of the process and not to endocytosis. The lectin receptors on the cell surface of normal and RSV-BH infected cells showed homogeneity in their binding properties. Chick cells infected with RSV-BH-Ta showed a lectin binding behavior that was dependent on the temperature at which the cells were grown. At the permissive temperature for transformation (37 degrees C), the binding process was similar to that observed for normal and RSV-BH infected cells. At the nonpermissive temperature (41 degrees C), the cells showed at least two sets of concanavalin A receptors. The new set of receptors on the cell surface had a lower lectin affinity than those observed in the same cells at 37 degrees C. Chick cells infected with RSV-BH showed an enhanced agglutinability by concanavalin A, as compared with normal cells. Cells infected with RSV-BH-Ta showed a reversal of the correlation between increased concanavalin A agglutinability and the transformed state. At the permissive temperature for transformation, the cells were not agglutinable, whereas at the nonpermissive temperature they presented agglutinability indexes as high as those observed with RSV-BH infected cells. This enhanced agglutinability observed with cells maintained at the nonpermissive temperature for transformation may be related to the new set of low affinity receptors present at 41 degrees C.     

Interactions of concanavalin A with chick embryo fibroblasts transformed by rous sarcoma virus Study with an RSV mutant thermosensitive for transformation

The interactions between concanvalin A and chick embryo fibroblasts, normal and infected with Rous sarcoma virus (RSV-BH) or its thermosensitive mutant RSV-BH-Ta, have been studied. Normal chick embryo cells and RSV-BH transformed cells showed at 4 and 25 °C a similar number of concanavalin A receptors per cell. Analysis of the binding data by the Scatchard relation showed that apparent changes in binding as a function of temperature are due to the thermodynamic properties of the process and and not to endocytosis. The lectin receptors on the cell surface of normal and RSV-BH infected cells showed homogeneity in their binding properties. Chick cells infected with RSV-BH-Ta showed a lectin binding behavior that was dependent on the temperature at which the cells were grown. At the permissive temperature for transformation (37 °C), the binding process was similar to that observed for normal and RSV-BH infected cells. At the nonpermissive temperature (41 °C), the cells showed at least two sets of concanavalin A receptors. The new set of receptors on the cell surface had a lower lectin affinity than those observed in the same cells at 37 °C.Chick cells infected with RSV-BH showed an enhanced agglutinability by concanavalin A, as compared with normal cells. Cells infected with RSV-BH-Ta showed a reversal of the correlation between increased concanavalin A agglutinability and the transformed state. At the permissive temperature for transformation, the cells were not agglutinable, whereas at the nonpermissive temperature they presented agglutinability indexes as high as those observed with RSV-BH infected cells. This enhanced agglutinability observed with cells maintained at the nonpermissive temperature for transformation may be related to the new set of low affinity receptors present at 41 °C.     

Temperature-Dependent Transformation of Cells Infected with a Mutant of Bryan Rous Sarcoma Virus

Chick embryo cells infected with a mutant (Ta) of the Bryan high-titer strain of Rous sarcoma virus (RSV-BH) are morphologically transformed at 36 C but appear similar to uninfected cells at 41 C. When cells infected with RSV-BH-Ta are switched from 41 to 36 C, morphological changes characteristic of transformation are observable within 10 min. The transformation is reversible; cells shifted from 36 to 41 C have been observed to lose their transformed morphology within 1 hr. The transformation after a shift in temperature is unaffected by inhibition of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein synthesis, demonstrating that the proteins involved in the morphological change are already present. Transformed cells infected with RSV-BH or RSV-BH-Ta take up hexose and synthesize hyaluronic acid at higher rates than uninfected cells or RSV-BH-Ta-infected cells grown at 41 C. However, inhibition of either protein or RNA synthesis, but not DNA synthesis, prevented the induction of increased hexose uptake and hyaluronic acid synthesis after a shift of RSV-BH-Ta-infected cells from 41 to 36 C. Therefore, these biochemical changes are secondary to a more basic change responsible for morphological transformation.     

Polypeptide composition of cell membranes from chick embryo fibroblasts transformed by rous sarcoma virus.

Chick embryo fibroblasts were transformed by the Bryan high-titer strain of Rous sarcoma virus (RSV-BH), or a mutant (RSV-BH-Ta) inducing temperature-dependent transformation. Surface membranes from normal and transformed cells were isolated as membrane vesicles by differential centrifugation, and as cell ghosts after ZnCl2 treatment and separation in an aqueous two-phase system. These preparations were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate or phenol/urea/acetic acid. In general a greater resolution of individual bands was found in gels containing phenol/urea/acetic acid, which separates polypeptides on the bases of size and charge. Electrophoresis of preparations from nontransformed cells showed that two polypeptides (molecular weights 200 000 and 250 000) found in cell ghosts were missing in membrane vesicles. In cell ghosts, transformation by RSV-BH resulted in a significant decrease of the 250 000 molecular weight complex. Also a polypeptide (molecular weight 73 000) prominent in membrane vesicles from nontransformed cells was decreased in transformed cells. Surfaces from cells transformed by RSV-BH-Ta at 37 degrees C presented patterns similar to those for RSV-BH infected cells. Shifting these cells to 41 degrees C resulted in an increase in the 250 000 molecular weight complex, although the amount of this protein(s) never reached that found in noninfected cells. Inhibitors of RNA and protein synthesis failed to block the morphological changes occurring in RSV-BH-Ta cells after temperature shifts from 41 degrees C to 37 degrees C or vice-versa. The same inhibitors caused a reduction in the levels of the 250 000 molecular weight complex at both temperatures. These data indicate that these large membrane-associated polypeptides play little or no role in the morphological changes associated with transformation and its reversal.     

Differentiation of quail myoblasts transformed with a temperature sensitive mutant of Rous sarcoma virus. I. Relationship between differentiation and tyrosine kinase of src gene product.

Quail embryonic pectoral myoblasts fuse with each other at 35.5 degrees C and 41 degrees C to essentially equal extents. When the myoblasts were transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV), their fusion and biochemical processes of differentiation became temperature-sensitive: their fusion occurred at 41 degrees C, the non-permissive temperature, but not at 35.5 degrees C, the permissive temperature, suggesting that the fusion was regulated by the viral transforming gene. Fusion of the transformed cells proceeded more rapidly and synchronously than that of the parent cells at 41 degrees C, and was completely suppressed at the permissive temperature, unlike that of the parent cells. These transformed cells were used to examine the relationship between myogenic differentiation and the tyrosine kinase activity of the src gene product. In spite of the temperature sensitivity of transformation, results showed that expressions of the src gene at 35.5 degrees C and 41 degrees C were similar. However, the level of tyrosine-phosphorylated protein was decreased at 41 degrees C. Moreover, myoblast fusion could occur at 35.5 degrees C in the presence of herbimycin A, an inhibitor of the tyrosine kinase activity of the src gene product. These results indicate that the tyrosine kinase activity of the src gene product is closely associated with regulation of myogenic differentiation of the cells.     

Effect of temperature on arginine incorporation by ribosomeless extracts of cells transformed by a temperature-sensitive mutant of Rous sarcoma virus.

The effect of transformation of normal rat kidney cells by a temperature-sensitive mutant of the Prague strain of Rous sarcoma virus (ts LA 24 PR-A) on the post-translational addition of arginine to the NH2-terminus of preformed acceptor molecules has been studied. Cells maintained at the permissive (35 degrees C) temperature show a high arginine-incorporating activity in ribosome free extracts compared to that found in extracts of cells grown at the non-permissive (40 degrees C) temperature. Temperature shift experiments as well as studies with cells transformed by wild type Rous sarcoma virus suggest that the decreased activity in cells grown at 40 degrees C is not due to a high temperature per se. The lower arginine incorporation in the 40 degrees C cell extracts is partially due to a decrease in the activity of arginyl transferase which catalyses the transfer of arginine from arginyl tRNA to the acceptor protein. Polyacrylamide gel electrophoresis of the radioactive product shows that the acceptor molecules present in extracts of cells grown at 40 degrees C are larger and qualitatively different from those found in extracts of cells grown at 35 degrees C.     

Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of Rous sarcoma virus. Comparison of transport properties of whole cells and membrane vesicles

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37 degrees C) had a 2-fold higher rate of 2-deoxy-D-glucose uptake than the same cells cultured at the non-permissive temperature (41 degrees C). However, both the non-transformed and transformed cells had comparable rates of alpha-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41 degrees C or 37 degrees C, displayed carrier-mediated, intravesicular uptake of D-glucose and alpha-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37 degrees C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41 degrees C. The two types of membrane vesicle had similar uptake rates of alpha-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. Km values for stereospecific D-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37 degrees C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37 degrees C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virally-transformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.     

Agglutination of Cells transformed by Rous Sarcoma Virus by Wheat Germ Agglutinin and Concanavalin A

Chick embryo fibroblasts transformed by Rous sarcoma virus are agglutinated by wheat germ agglutinin and by concanavalin A if the cells are pretreated with purified hyaluronidase. Cells infected by a temperature-sensitive mutant of this virus are agglutinable if grown at the permissive temperature but not if grown at the non-permissive temperature.     

Characterization of a temperature-sensitive membrane alteration in chick embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus

The intramembrane particles of freeze-fractured chick embryo fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (TS68) are distributed differently at the permissive and non-permissive temperatures if, and only if, the cells are treated with glycerol before fixation. Few aggregates of intramembrane particles are present in glycerol-treated cells grown at the permissive temperature for transformation (36 degrees C), while numerous large aggregates of particles are present at the non-permissive temperature (41 degrees C). Changes in the distribution of particles after cells are shifted from 36 to 41 degrees C are observed after 20 min, while a temperature shift from 41 to 26 degrees C causes changes in glycerol-induced redistributions after 1 h. The changes observed in temperature shifts from 36 to 41 degrees C and from 41 to 36 degrees D do not require protein synthesis or RNA synthesis.     

Recombination between a temperature-sensitive mutant and a deletion mutant of Rous sarcoma virus.

Cells doubly infected with two mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), ts68, which is temperature sensitive for cell transformation (srcts), and a deletion mutant, N8, which is deficient in the envelope glycoprotein (env-), produced a recombinant which carried the defects of both parents. The frequency of formation of such a recombinant was exceptionally high and made up 45 to 55% of the progeny carrying the srcts marker. By contrast, the reciprocal recombinant, which is wild type in transformation (srcts) and contains the subgroup A envelope glycoprotein (envA), was almost undetectable. This remarkable difference in the frequency of the formation of the two possible recombinants suggests that a unique mechanism may be involved in the genetic interaction of the two virus genomes, one of which has a large deletion. When an RNA-dependent DNA polymerase-negative variant of the N8 (N8alpha) was crinants also became deficient in the polymerase. Cells infected by the srctsenv- recombinant were morphologically normal at the nonpermissive temperature (41 degrees C) and susceptible to all subgroups of RSV. The rate by which the wild-type RSV transformed the recombinant-preinfected cells was indistinguishable from that of transformation of uninfected chicken cells by the same wild-type virus. This indicates that no detectable interference exists at postpenetration stages between the preinfected and superinfecting virus genomes and confirms that the expression of the transformed state is dominant over the suppressed state.     

Membrane lipid acyl group alterations in cells infected with a temperature-conditional mutant of Rous sarcoma virus.

The increased percentage of oleic acid and decreased percentage of arachidonic acid which occurs in the lipids of chicken embryo fibroblasts transformed by Rous sarcoma virus was shown to be transformation specific rather than a consequence of virus infection. Cells infected with a temperature conditional mutant of Rous sarcoma virus (RSV-T5) had a normal type fatty acid composition when held at the restrictive temperature of 41 degrees C, but had a transformed type fatty acid composition when held at the permissive temperature of 36 degrees C. However, these acyl group changes occurred slowly in the course of transformation, suggesting that they are not a primary event in the genesis of the transformed phenotype.     

Colloidal iron hydroxide-binding to the surfaces of chick embryo fibroblasts transformed by wild-type and a temperature-sensitive mutant of Rous sarcoma virus.

The densities of colloidal iron hydroxide (CIH) particles binding to the surfaces of chick embryo fibroblasts were determined before and after transformation with wild type Rous sarcoma virus and a temperature sensitive (ts) mutant of this virus. On the basis of in vitro behavior, cells transformed by the ts virus manifest a malignant phenotype at 36 degrees C (permissive temperature) and appear normal at 41 degrees C (non-permissive temperature). At the permissive temperatures there is a significant increase in CIH particle-binding to spaces of cell surface between microvilli on the wild type and ts transformed cells. At the non-permissive temperature this significant increase in binding is only observed on the wild type transformant, while the density found on the ts transformant is not significantly different from the untransformed state. Therefore, in vitro characteristics of normalcy and malignancy are reflected in changes in the CIH binding properties of the cell surface spaces between microvilli. The CIH densities observed on the microvilli are significantly different from the density on the spaces between them for each of the classes of cells studied at either temperature. The microvilli are found to bind a lower density of particles in five of the six cases. No correlations between microvilli particle density and transformation to in vitro malignant characteristics were observed.     

Changes in the synthesis and phosphorylation of cellular proteins in chick fibroblasts transformed by two avian sarcoma viruses

35S- and 32P-labeled proteins from control chick embryo fibroblasts and from fibroblasts transformed by UR2 sarcoma virus, or by a temperature-sensitive mutant (tsLA29) of Rous sarcoma virus, were separated by two-dimensional electrophoresis on giant gels to detect transformation-specific changes in protein synthesis and total phosphorylation. A nontransforming avian retrovirus, UR2-associated virus (UR2AV), was also studied. Virus-coded proteins appear in whole cell lysates of all infected cells. The structural proteins can be identified by comparison with proteins immunoprecipitated with antivirus serum. The transforming proteins pp60src and p68ros, present in cells transformed with Rous sarcoma virus and UR2, respectively, are phosphorylated in vivo. Eighteen increases and eight decreases in cellular phosphoproteins are associated with transformation, and revert toward normal levels when cells infected with tsLA29 are incubated at 42 degrees C. These changes are more extensive than previously reported, but none represent new phosphorylations, since all phosphoproteins seen in transformed cells also appear to be phosphorylated to a certain extent in control cells. Fifteen cellular proteins show increased relative rates of synthesis apparently related either to transformation or to growth at 42 degrees C. Four other proteins are increased exclusively in cells incubated at 42 degrees C, but not at 37 degrees C, whether transformed or not. Eleven additional increases in the synthesis of cellular proteins, many quite large, and one seemingly a de novo induction, appear to be specific for transformation. These changes occur in cells transformed by either UR2 or Rous sarcoma virus at 37 degrees C, do not occur with UR2AV infection, and tend to revert in cells infected with tsLA29 incubated at 42 degrees C. These 11 changes may represent increases in cellular gene expression that are related specifically to the maintenance of the transformed state.     

The use of Rous sarcoma virus transformation mutants with differing tyrosine kinase activities to study the relationships between vinculin phosphorylation, pp60v-src location and adhesion plaque integrity

Tyrosine-specific phosphorylation of cellular proteins has been implicated in the neoplastic transformation of cells by Rous sarcoma virus (RSV). One of the putative substrates for the src gene product (pp60v-src) of RSV is the cytoskeletal protein vinculin, giving rise to the hypothesis that tyrosine-specific phosphorylation of vinculin disrupts adhesion plaque integrity, leading to the characteristic rounded morphology of RSV-transformed cells. We have investigated this hypothesis by analysing the properties of fibroblasts transformed by conditional and non-conditional mutants of RSV which confer different morphologies on infected cells, with respect to formation of microfilament bundles, formation of vinculin-containing adhesion plaques, the deposition of a fibronectin-containing extracellular matrix, the localization of pp60v-src and the tyrosine-specific phosphorylation of vinculin. Cells transformed by the temperature-sensitive (ts) RSV mutant LA32 cultured at 41 degrees C were morphologically normal, and contained prominent microfilament bundles and well-developed adhesion plaques. However, these cells had a fully active pp60v-src kinase, had pp60v-src concentrated in their adhesion plaques and contained vinculin which was heavily phosphorylated on tyrosine residues. Cells transformed by a recovered avian sarcoma virus, rASV 2234.3 exhibited a markedly fusiform morphology with pp60v-src concentrated in well-developed adhesion plaques and an elevation of the phosphotyrosine content of vinculin. Cells transformed by LA32 at restrictive temperature comprise morphologically normal cells, indistinguishable from untransformed CEF, yet which contain tyrosine-phosphorylated vinculin and suggest that neither tyrosine-specific phosphorylation of vinculin nor pp60v-src concentration in adhesion plaques is sufficient for the rounded morphology of RSV-transformed cells.     

The influence of higher temperature on dengue-2 virus infected C6/36 mosquito cell line

Dengue-2 virus infection of C6/36 cells was studied at 28 and 37 degrees C. In infected cells maintained at 28 degrees C, syncytial development was seen on day 4 postinfection, whereas at 37 degrees C, extensive syncytial development was seen by 32 h. Extracellular virus titre was found to correlate with the cytopathic changes. Nine Dengue-2 virus specified proteins were observed in polyacrylamide analyses of cytoplasmic extracts of C6/36 infected cells. All the proteins were observed, although in varied intensities by 32 h postinoculation at 37 degrees C and only on day 4 postinoculation at 28 degrees C. The GP60 glycoprotein appeared at 32 h postinfection when the cells were maintained at 37 degrees C and became prominent only on day 5 at 28 degrees C. The results revealed that a higher temperature accelerated the onset of cytopathic effects, hastened the development of virus specified proteins, and also enhanced the titre of extracellular infectious virus. The importance of the accumulation of the envelope protein GP60 for the development of CPE was indicated.     

Dissociation of inositol trisphosphate from diacylglycerol production in Rous sarcoma virus-transformed fibroblasts

The metabolism of phosphatidylinositol (PI) and related intermediates was studied in uninfected and Rous sarcoma virus-(RSV) infected chicken embryo fibroblasts (CEFs). Cells infected with wild-type RSV exhibited twofold increases in steady-state concentrations of inositol trisphosphate (IP3) and inositol bisphosphate (IP2) as compared to uninfected CEFs. In addition, increased concentrations of IP3 and IP2 were observed in CEFs infected with the RSV temperature-sensitive transformation mutant NY72-4 when maintained at the permissive temperature (35 degrees C) for greater than 24 h. Slight increases were observed in the amounts of inositol lipids in RSV-transformed cells. Phosphoinositol metabolic changes were related to transformation and not to viral infection since CEFs infected with NY72-4, maintained at the nonpermissive temperature (41.5 degrees C), revealed amounts of phosphoinositols similar to that of uninfected cells. CEFs infected with a transformation-defective virus exhibited PI metabolic changes intermediate between those of transformed and nontransformed cells. NY72-4 CEF exhibited no increase in phosphoinositol concentrations before 8 h incubation at 35 degrees C, indicating that the transformation-specific changes in inositol metabolism were a delayed event. Furthermore, inositol turnover was not activated during this time. In contrast to the case of inositol metabolism, significant increases in diacylglycerol (DAG) concentrations were observed within 15-30 min after shift of NY72-4 CEFs to 35 degrees C. These findings suggest that (a) the major changes in inositol metabolism are specific for RSV-transformed cells; (b) transformation-specific changes in phosphoinositol content in RSV-infected CEFs are not an early effect of the expression of pp60v-src; and (c) increases in the DAG content of transformed cells occur before changes in inositol metabolism, indicating that DAG may be derived from other lipid sources.     

Properties of mammalian cells transformed by temperature-sensitive mutants of avian sarcoma virus.

Fibroblasts from European field vole (Microtus agrestis) and from normal rat kidney (NRK) have been infected by avian sarcoma virus mutants which are temperature-sensitive for the maintenance of transformation. These cells are transformed at 33 degrees C, but show normal cell characteristics in morphology, colony formation in agar, saturation density, sugar uptake and membrane proteins at 39 degrees C and 40 degrees C, the nonpermissive temperatures. Ts mutant virus was rescued from most of the ts transformed cell lines. NRK cells infected by avian sarcoma virus ts mutants and kept at the nonpermissive temperature can be transformed by wild-type avian sarcoma virus. The susceptibility of the temperature-sensitive NRK lines to this transformation is higher than the susceptibility of uninfected NRK at either permissive or nonpermissive temperature.     

Isolation of cold-sensitive mutants of measles virus from persistently infected murine neuroblastoma cells.

Clone NS20Y of the mouse neuroblastoma C1300 was infected with wild-type Edmonston measles virus, and, after a transition to a carrier culture, became persistently infected. Persistently infected clones were derived and characterized morphologically by the appearance of multinucleate giant cells and nucleocapsid matrices in cytoplasm and nucleus, but very few budding virus particles. Antimeasles antibodies markedly suppressed the expression of viral antigens and giant cells, and the effect was totally reversible. When the cells were cultured at 33 degrees C, the number of giant cells began to diminish and ultimately disappeared; in contrast, when cultured at 39 degrees C, the cultures invariably lysed. Yields at 33 degrees C were ca. 2 logs lower than those at 39 degrees C. Cells cultured at 33 degrees C produced relatively high levels of interferon, whereas those at 39 degrees C produced little or no interferon. When the persistently infected cultures were exposed to anti-interferon alpha/beta serum at a nonpermissive temperature, there was a marked increase in multinucleate cells, suggesting that maintenance of the persistence state and its regulation by temperature may be related to the production of interferon. Viral isolates from cells cultured at 39 degrees C were obtained, and 90% of viral clones were found to be cold sensitive. Complementation studies with different viral clones indicated that the cold-sensitive defect was probably associated with the same genetic function. Western blot analysis of the persistently infected cells indicated a significant diminution and expression of all measles-specific proteins at a nonpermissive temperature. Infection of NS20Y neuroblastoma cells with the cold-sensitive virus isolates resulted in the development of an immediate persistent infection, whereas infection of Vero or HeLa cells resulted in a characteristic lytic infection, suggesting that the cold-sensitive mutants may be selected or adapted for persistent infection in cells of neural origin.     

Temperature-dependent and transformation-associated changes in polyamine metabolism in normal and Rous sarcoma virus-infected chick embryo fibroblasts

Tertiary cultures of chick embryo fibroblasts infected and transformed by the wild-type Rous sarcoma virus, when actively growing at 35 degrees C, had higher putrescine levels than the respective uninfected cells. Transformed cells also had much higher specific activity of ornithine decarboxylase (EC 4.1.1.17) than the normal fibroblasts. At 41 degrees C the difference in putrescine levels between the normal and the transformed cells was less marked, and both cell types showed a relative accumulation of spermine. Cultures infected with the NY68 mutant virus, which is temperature-sensitive for transformation, showed at 41 degrees C normal cell morphology and intermediate polyamine patterns, while at 35 degrees C a transformed phenotype was found in both aspects. In shift-down experiments a change towards the permissive temperature pattern of polyamine metabolism was evident within 2-3 h. Difluoromethylornithine, a specific and irreversible inhibitor of ornithine decarboxylase efficiently reduced the enzyme activity as well as the levels of both putrescine and spermidine in all culture types and temperatures. Incubation of Rous sarcoma virus-transformed cells with 3 mM difluoromethylornithine for 36 h did not affect the maintenance of the transformed state. Likewise, when NY68-infected cultures were exposed to difluoromethylornithine at 41 degrees C for 12 h and then shifted down to 35 degrees C, the appearance of the transformed morphology took place concomitantly with that of the control cultures without respective changes in the polyamine levels. This suggests that the transformation-associated pattern of polyamines in chick embryo fibroblasts is not a prerequisite for morphological transformation of these cells.     

Differentiation of quail myoblasts transformed with a temperature sensitive mutant of Rous sarcoma virus. II. Relationship of myoblast fusion with calcium and temperature.

The effects of calcium and temperature on fusion of quail embryonic myoblasts were examined using cells transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV). The transformed quail myoblasts (QM-RSV) fused to form myotubes at 41 degrees C, the non-permissive temperature, but not at 35.5 degrees C, the permissive temperature. On incubation at 41 degrees C, a period of more than 10 hr was needed for the myoblasts to become fusion-competent, but calcium was not needed for development of fusion-competence. Once the cells had become competent, fusion proceeded even at 35.5 degrees C. These results suggest that the src gene product expressed at 35.5 degrees C may control the fusion of cells in the competent stage by inactivating a component(s) that is associated with fusion-competence. However, fusion of even myoblasts in the competent stage was blocked in calcium-deficient medium, suggesting that calcium is essential for the fusion, probably at a step immediately before membrane union. Unlike fusion, other biochemical processes of differentiation proceeded even in calcium-deficient medium, indicating a distinction of fusion from these other processes during myoblast differentiation.