Neuraminidase activity and cell surface sialic acid turnover in Rous sarcoma virus transformed chick embryo fibroblasts

Neuraminidase activity of Rous sarcoma virus transformed chick embryo fibroblasts (RSV-CEF) was assayed using an exogenous substrate, neuraminlactitol-[3H], and endogenous, cell surface [14C]-N]-acetyl-neuraminic acid. RSV-CEF had higher neuraminidase activity toward both substrates than did chick embryo fibroblasts (CEF) or nontransformed, Rous associated virus infected CEF (RAV-CEF). The total sialic acid content of RSV-CEF was lower than CEF or RAV-CEF, and more of the total sialic acid was accessible to extracellular Clostridium perfringens neuraminidase. Activity of the enzymes synthesizing and degrading the substrate for sialyltransferase, cytidine-5'-monophosphate-N-acetyl-neuraminic acid (CMP-AcNeu) was measured in order to determine whether control of substrate levels for sialyltransferase might contribute to the decreased levels of glycoprotein bound sialic acid. No change in activity of these enzymes was found in RSV-CEF as compared to CEF or RAV-CEF.     

Reduced levels of adenosine deaminase in chick embryo fibroblasts transformed by Rous sarcoma virus.

Adenosine deaminase activities in chick embryo fibroblasts were substantially reduced after infection and transformation by Rous sarcoma virus. Concomitant with the reduction in adenosine deaminase activities, the incorporation of exogenous adenosine into RNA species of the virus transformed cells was moderately increased. The significance between reduction in adenosine deaminase activity and malignant transformation by Rous sarcoma virus remains to be eleucidated.     

Comparison of cAMP-dependent protein kinase in normal and Rous sarcoma virus transformed chick embryo fibroblasts

cAMP-dependent protein kinase was compared in normal and Rous Sarcoma Virus transformed chicken embryo fibroblasts. Total cAMP binding activity and cAMP-dependent histone kinase activity were unaltered by RSV transformation. The apparent Km for activation of histone kinase activity by cAMP was 35 nM in both normal and transformed cells. Using 8-N3-cAMP photoaffinity labeling, normal and transformed cells were also found to contain equal quantities of a single 42,000 Mr regulatory sub-unit isoenzyme of A-kinase. This isoenzyme corresponded to the lower molecular weight isoenzyme of the two enzymes found in normal chicken skeletal muscle. Both avian isoenzymes were about 4,000 Mr smaller than the corresponding bovine type I and type II regulatory subunits. Rous Sarcoma Virus transformation does not directly alter the amount or activity of cAMP-dependent protein kinase.     

Neutral protease activity of Rous sarcoma (RSV) transformed chick embryo fibroblasts.

The proteolytic activities of normal, Schmidt-Ruppin Rous sarcoma virus (SR-RSV) transformed, and infected (RAV) chick embryo fibroblasts (CEF) have been measured by a highly sensitive technique using 3H-acetylated haemoglobin as a substrate.When all 3 types of CEF cells were maintained in serumless media, no differences were detected in the amount of pH 3-4 protease activity released into the media over a 24-h period, and only negligible amounts of pH 7-6 proteolytic activity were found. When normal, transformed, and infected cells were maintained in serumless media and later incubated with 3H-acetylate haemoglobin, a significant proteolysis of the haemoglobin, a 6-fold increase compared to the normal CEF cells, was associated only with plates containing SR-RSV-CEF cells. A fluorescent assay for peptides confirmed that SR-RSV-CEF cells have increased cell-associated proteolytic activity. The net surface charge of the transformed CEF cells was unchanged by maintenance in serumless media but the net surface negativity of the normal and RAV-CEF cells was significantly increased by incubation in media minus serum for 24 h. This suggests that normal CEF cells, maintained in media plus serum, have a substance masking their surface charge which is absent from the surface of transformed cells, possibly because of proteolytic degradation.     

Protein and lipid lateral diffusion in normal and Rous sarcoma virus transformed chick embryo fibroblasts.

We measured the lateral diffusion of the fluorescent lipid analogue dioctadecylindocarbocyanine iodide (DiI) and of membrane glycoproteins labeled with tetramethylrhodamine (TRITC) succinyl concanavalin A (SConA) via fluorescence photobleaching recovery (FPR) at selected times during a temperature downshift experiment on transformation-defective temperature-sensitive (td-ts) Rous sarcoma virus (RSV) NY68-transformed chicken embryo fibroblasts (CEF) and on identically treated CEF and RSV-transformed CEF. There were no significant differences in the lateral diffusion in DiI at any of the times measured. The lateral diffusion of TRITC-SConA on the RSV-transformed CEF, (1.32 +/- 0.12).10(-10) cm2 s-1, was approximately two times faster than that observed in normal CEF, (0.61 +/- 0.06).10(-10) cm2 s-1. In the cells undergoing RSV NY68-mediated transformation, TRITC-SConA diffusion increased over a 24-h period from a value comparable to that observed in normal CEF, (0.72 +/- 0.13).10(-10) cm2 s-1 to a value comparable to the RSV-CEF transformed cells, (1.74 +/- 0.20).10(-10) cm2 s-1. All diffusion measurements reported were made at the permissive temperature for RSV-NY68 (35 degrees C) unless stated otherwise. The changes in the lateral diffusion of TRITC-SConA occurred between the fifth and twelfth hour of the downshift course and could be associated with cytoskeletal disruption and/or fibronectin degradation, both known to occur at this time in RSV-transformed cells. To assess the contribution of extracellular matrix (ECM) degradation, SConA mobility was measured in normal and RSV-transformed cells treated with trypsin. This treatment increased SConA mobility approximately 4-fold in the normal cells relative to untreated controls and only 2-fold in the RSV-CEF transformed cells. No significant difference in SConA mobility between trypsinized spherical normal and transformed cells was apparent.     

Plasma membrane phosphoproteins in normal and Rous sarcoma virus transformed chick embryo fibroblasts: characterization by in vitro phosphorylation.

Plasma membranes isolated from normal and RSV transformed chick embryo fibroblasts were phosphorylated in vitro using endogenous protein kinase and ATP (gamma32P) and the labeled phosphoproteins were analyzed by SDS-PAGE. A number of protein phosphorylation changes were observed following transformation, however in most cases they were relatively small quantitative differences. The four major changes were in proteins of 47,000, 58,000, 75,000 and 135,000 daltons. Decreased phosphorylation of the 47,000 dalton polypeptide was found in transformed cell membranes but this alteration was shown to be due to differences in cell growth rather than transformation. Increase phosphorylation of the 75,000 dalton protein was at least partially related to virus infection. However, increased phosphorylation of the 58,000 and 135,000 dalton polypeptides were entirely transformation specific.     

A similar ribosomal protein S6 kinase activity is found in insulin-treated 3T3-L1 cells and chick embryo fibroblasts transformed by Rous sarcoma virus

Insulin and transformation by Rous sarcoma virus stimulate the phosphorylation of ribosomal protein S6. Soluble fractions containing activated S6 protein kinase from insulin-treated cells and from transformed chick embryo fibroblasts were compared. Based upon several characteristics notably elution from DEAE-cellulose and sedimentation in glycerol gradients, these two S6 protein kinase activities appear to be similar enzymes. Thus insulin and retroviral transformation may activate the same enzyme to regulate the phosphorylation state of S6.     

Attenuation of ribosomal protein S6 phosphatase activity in chicken embryo fibroblasts transformed by Rous sarcoma virus.

In chicken embryo fibroblasts, phosphorylation of the 40S ribosomal protein S6 increases during G1 but returns to basal level by mitosis. In contrast, in Rous sarcoma virus (RSV)-transformed fibroblasts, S6 remains highly phosphorylated throughout mitosis. This study investigated the mechanism by which RSV alters the pattern of S6 phosphorylation. Pulse-chase experiments demonstrate that phosphate turnover in S6 is rapid in normal cells and in cells infected with an RSV transformation-defective virus. In contrast, phosphate turnover in S6 is severely reduced in cells infected with temperature-sensitive RSV at a temperature permissive for transformation, indicating a diminished S6 phosphatase activity. Fractionation of cell lysates by DEAE chromatography showed an almost threefold lower S6 phosphatase activity in RSV-transformed versus normal cells. The S6 phosphatase was sensitive to inhibitor 2 and specifically recognized by an antibody to type 1 phosphatase (PP1). The S6 phosphatase activity recovered by immunoprecipitation of PP1 was threefold lower in transformed cells, but the steady-state level of expression and the rate of synthesis of PP1 were not altered by oncogenic transformation. Together, the results show that transformation by RSV reduced the S6-PP1 activity.     

Specific changes in the synthesis of mitochondrial DNA in chick embryo fibroblasts transformed by Rous sarcoma viruses

In chick-embryo fibroblasts infected with the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A (wild type), or with a thermosensitive mutant of this virus, T5, the rates of mitochondrial DNA synthesis differ in cells that exhibit normal and malignant phenotypes. In wild type virus-infected cells grown at 36 or 41 degrees C, morphological transformation is expressed, the rate of 2-deoxy-D-[3H]glucose uptake is stimulated, and mitochondrial DNA synthesis in vivo is stimulated three- to fivefold over that in uninfected cells. In T5-infected cells these changes occur only at the permissive temperature (36 degrees C); a shift to the nonpermissive temperature (41 degrees C) causes the reversal of these effects, and the specific activity of purified mitochondrial DNA is characteristic of that from uninfected cells. In contrast, the specific activities of nuclear DNA purified from cells maximally transformed under the permissive conditions do not differ between wild type-infected and uninfected with the T5 virus. In parallel experiments with isolated mitochondria, the rate of mtDNA synthesis in vitro is again greater in mitochondria isolated from transformed cells. In addition, mitochondrial DNA synthesis in vitro in mitochondria from nontransformed and virus-transformed cells exhibits differential sensitivity to inhibition by mercaptoethanol. Furthermore, the ntDNAP polymerase activity in mitochondrial extracts prepared from cells with transformed phenotypes is about sevenfold higher than in extracts from cells with nontransformed phenotypes.     

Plasma membrane protein kinase activity in normal and Rous sarcoma virus-transformed chick embryo fibroblasts

A preliminary study has been carried out to investigate the effect of Rous sarcoma virus transformation on plasma membrane protein kinase activity in chick embryo fibroblasts. Enzyme activity was measured using an in vitro phosphorylation method employing [γ-³²P]ATP with isolated plasma membranes serving as the source of both protein kinase and protein substrate. In general, the enzymatic properties observed were similar to those of other known protein kinases. However, for maximal activity a marked dependence on high Mg²⁺ concentrations was noted. Evidence was obtained which showed that cyclic nucleotide-dependent protein kinases were present in membranes from normal cells, but none could be measured in preparations from transformed cells. In addition, transformation appeared to result in a slight increase in basal plasma membrane protein kinase activity.     

Regulation of the proliferative response in Rous sarcoma virus transformed chicken embryo fibroblasts by serum and multiplication-stimulating activity (MSA).

A temperature sensitive mutant of Rous sarcoma virus (tsNY68) was used to obtain cultures of quiescent virus-infected chicken embryo fibroblasts arrested by serum starvation at the non-permissive temperature. Upon shift to the permissive temperature, these cells enter the replicative cell cycle as evidenced by increases in 2-deoxyglucose uptake, 3H-thymidine incorporation and percent labeled nuclei. These changes occur in the absence of serum and the cells become morphologically transformed within eight to ten hours after the temperature shift. Entry into the S phase temporally resembles that of normal quiescent fibroblasts stimulated with serum. This experimental system was used to examine the proliferative response of transformed cells to serum and purified multiplication-stimulating activity (MSA) during the transition from the resting to the growing state. Data are presented which show that the presence of serum in the medium enhances the proliferative response of quiescent infected cells shifted to the permissive temperature over those shifted in the absence of serum. In contrast, the presence of MSA has no additional effect on the response exhibited by infected cells shifted to the permissive temperature in serum-free medium. Labeled MSA binding experiments show that this lack of response is not due to a loss of MSA receptors on the cell surface since transformed cells are still capable of binding MSA at the same level as normal cells. The results are consistent with the hypothesis that the set of biochemical events initiated by MSA in normal cells are turned on in infected cells shifted to the permissive temperature by the activation of the src gene product.     

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.     

Plasma membrane protein kinase activity in normal and Rous sarcoma virus-transformed chick embryo fibroblasts.

A preliminary study has been carried out to investigate the effect of Rous sarcoma virus transformation on plasma membrane protein kinase activity in chick embryo fibroblasts. Enzyme activity was measured using an in vitro phosphorylation method employing [gamma-32P]ATP with isolated plasma membranes serving as the source of both protein kinase and protein substrate. In general, the enzymatic properties observed were similar to those of other known protein kinases. However, for maximal activity a marked dependence on high Mg2+ concentrations was noted. Evidence was obtained which showed that cyclic nucleotide-dependent protein kinases were present in membranes from normal cells, but none could be measured in preparations from transformed cells. In addition, transformation appeared to result in a slight increase in basal plasma membrane protein kinase activity.     

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.