Rous Sarcoma Virus-transformed Rat Cells Research Articles

Altered processing of a major secreted phosphoprotein correlates with tumorigenicity in Rous sarcoma virus-transformed mammalian cells.

Anchorage-independent growth is highly correlated with neoplastic growth in vivo, and the retinoids (vitamin A and its analogs) inhibit this property in a wide variety of oncogenically transformed cells. We report here that retinoic acid-treated Rous sarcoma virus-transformed rat (RR1022) and vole (SR-1T) cells, which show reversible loss of anchorage-independent growth and assume nontransformed morphology, secrete a major 69-kilodalton phosphoprotein (pp69) instead of the 62-kilodalton phosphoprotein (pp62) secreted by their untreated counterparts. As determined by V8 protease mapping and by two-dimensional electrophoretic analysis, this 69-kilodalton polypeptide was indistinguishable from the pp69 released by nontransformed normal rat kidney cells. Neither retinoic acid-treated RR1022 cells nor normal rat kidney cells secreted pp62, and retinoic acid treatment did not have any significant effect on the synthesis, subcellular localization, or phosphokinase activity of pp60src. Furthermore, treatment with retinoic acid did not alter the synthesis of the transformation-specific 53-kilodalton phosphoprotein (p53) and secretion of the transforming growth factors in RR1022 cells. Our studies showed that there is a clear correlation between the release of pp69 or pp62 and the ability of cells to grow in vitro with or without anchorage. This may provide an important clue for elucidating specific biochemical events involved in anchorage regulation of growth.

A calmodulin-dependent protein kinase in Rous sarcoma virus-transformed rat cells and normal liver

A calmodulin-dependent protein kinase has been purified extensively from a Rous sarcoma virus-transformed rat cell line (RR1022) and from normal rat liver. The calmodulin-dependent protein kinase activity was manifested by in vitro phosphorylation of a single M r 57 000 endogenous phosphoprotein (pp57) present in both the virally transformed cells and normal rat liver. The calmodulin-dependent protein kinase from transformed cells fractionated with the viral src gene product, pp60 v-src, through a 650-fold purification of the oncogene product. However, purification of the calmodulin-dependent protein kinase from normal liver demonstrated that the calmodulin-dependent kinase was distinct from pp60 v-src. Phosphorylation of pp57 by the kinase purified from the transformed cell line required Ca 2+ and calmodulin, was inhibited by EDTA and was unaffected by cAMP or the heat- and acid-stable protein inhibitor of cAMP-dependent protein kinase. Troponin C did not substitute for calmodulin. A virtually identical calmodulin-dependent protein kinase activity was purified from rat liver by affinity chromatography on calmodulin-Sepharose. Phosphorylation of pp57 by the affinity-purified liver protein kinase was also observed, and required Ca 2+ and calmodulin. EGTA and trifluoroperazine inhibited pp57 phosphorylation. The calmodulin-dependent protein kinase reported here did not phosphorylate substrates of known calmodulin-dependent protein kinases in vitro (myosin light chain, phosphorylase b, glycogen synthease, microtubule-associated proteins, tubulin, α-casein). Because none of these proteins served as substrates in vitro and pp57 was the only endogenous substrate found, the properties of this enzyme appear to be different from any previously described calmodulin-dependent protein kinase.

Diverse mitogenic agents induce rapid phosphorylation of a common set of cellular proteins at tyrosine in quiescent mammalian cells.

Protein phosphorylation of quiescent human skin fibroblasts was analyzed following stimulation by epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, serum, or 12-O-tetradecanoyl-phorbol-13-acetate. In mitogen-treated cells, a markedly increased phosphorylation of two Mr = 43,000 proteins and two Mr = 41,000 proteins was always detected by two-dimensional gel electrophoresis. More acidic forms were the dominant species and contained phosphotyrosine, phosphoserine, and phosphothreonine, while the basic forms contained phosphotyrosine and phosphoserine. The two Mr = 41,000 proteins were structurally related to each other. All mitogens seemed to stimulate the phosphorylation of each protein with the same site specificity. Induction of the same set of phosphoproteins was observed in mitogen-stimulated rat and mouse fibroblasts as well. These stimulated phosphorylations occurred rapidly, were maximal 5 min after exposure of cells to mitogens, and diminished gradually after 30 min. Mitogen-induced phosphorylation of these proteins was correlated to the extent of mitogen-stimulated DNA synthesis. In addition, such increased protein phosphorylation was not observed in exponentially growing cells, nor in Rous sarcoma virus-transformed rat cells. Thus, phosphorylation of the Mr = 43,000 and 41,000 proteins, which represents a common and specific response of cells to mitogens, could constitute an early event involved in the control of cellular G0----G1 transition.

Unphosphorylated gelsolin is localized in regions of cell-substratum contact or attachment in Rous sarcoma virus-transformed rat cells.

Regions associated with cell-substratum contact or attachment in Rous sarcoma virus (RSV)-transformed rat fibroblasts (RR1022 cells) were identified by reflection-interference microscopy. Electron microscopy of such regions revealed the presence of discrete membrane-associated structures composed of a paracrystalline lattice of hexagons and pentagons to which actin filaments appear to be attached. Staining of actin by biotin-labeled heavy meromyosin showed that transformed cells, unlike normal fibroblasts, lack prominent actin fibers, and that, instead, much of the fluorescence is concentrated in loci corresponding to locations of transient association between the cell and the substratum. In stationary cells, such loci were found in rosette formation, predominantly in the region beneath the nucleus. In cells engaged in active movement, such as during migration into a wound, the actin-containing spots were concentrated in the region of the leading edge. A similar pattern of staining was observed with antibody to gelsolin, a 91,000-dalton Ca2+-dependent actin filament-shortening protein. Since the action of gelsolin on actin is reversible and dependent on physiologically relevant changes in calcium concentration, the localization of gelsolin, together with actin-bundling proteins such as alpha-actinin, in the regions containing many small microfilament bundles on the ventral side of cytoplasm suggests that gelsolin may be a component of the mechanism for the disassembly and assembly of actin during the dissolution and reformation of structures for cell-substratum contact during cell locomotion. Regulation of gelsolin activity was not dependent on protein phosphorylation, as shown by lack of 32P-incorporation into gelsolin in either transformed or normal fibroblasts.

Reduced synthesis of pp60src and expression of the transformation-related phenotype in interferon-treated Rous sarcoma virus-transformed rat cells.

Treatment of Rous sarcoma virus-transformed rat cells with rat interferon-alpha (specific activity, 10(6) U/mg of protein) for 24 h caused a 50% reduction in intracellular pp60src-associated protein kinase activity. Staphylococcus aureus V8 protease digestion of pp60src, derived from 32P-labeled monolayer cultures incubated with or without interferon, revealed no differences either in the phosphopeptide pattern or in the phosphoserine-phosphotyrosine ratio. However, [3H]leucine pulse-labeling experiments showed that the synthesis of pp60src was reduced by 42 to 48%, relative to the level of bulk protein synthesis, in the interferon-treated cultures. Rat interferon-alpha also reduced the growth rate of Rous sarcoma virus-transformed rat cells in a dose-dependent manner over a 72-h period. The decrease in growth rate was accompanied by increases in the thickness and number of actin fibers per cell and by a decline in intracellular tyrosine phosphorylation by pp60src. The results suggest that interferon can inhibit the expression of the transformation-related phenotype by selectively reducing the synthesis of the Rous sarcoma virus transforming gene product. However, the interferon effects on the cytoskeletal organization and proliferation of Rous sarcoma virus-transformed cells may be due at least in part to the predominance of interferon-induced phenotypic changes over those caused by pp60src.

Behavior of cells seeded in isolated fibronectin matrices.

Cell-free fibronectin matrix (FN-matrix) isolated from chick embryo fibroblasts was used to study cell-matrix interaction. After 24 h, most fibroblastic cells, including those without cell surface fibronectin, adopted bipolar fusiform morphology. Cells grew in parallel arrays and aligned with each other apparently along FN-matrix. Since the orientation of fibronectin fibers was determined by chick embryo fibroblasts, our results suggested that intercellular organization of "matrix-using" cell type may be influenced by "matrix-producing" cell type. Whereas the elongation and alignment effects induced by FN-matrix have been detected in fibroblasts (both normal and transformed), myoblast, aortic endothelial cells, neural cell lines (B103 and RT4D1), and cardiac muscle cells, similar effects are not detected in bone marrow hemopoietic cells, circulating lymphocytic T and B cells, and sympathetic neurons. For epithelial cells, FN-matrix has varying effects. Elongation and alignment effects are detected only in transformed epithelial cells with a great reduction in keratin expression. The morphology of normal or transformed epithelial cells with abundant keratin appears unaffected by FN-matrix. FN-matrix reduced the growth of several transformed fibroblastic lines up to 25%, but did not restore the appearance of actin stress fibers and the normal migratory activities of Rous sarcoma virus-transformed rat cells.

Frameshift and intragenic suppressor mutations in a rous sarcoma provirus suggest src encodes two proteins

We have analyzed two mutations in src responsible for phenotypic reversion and subsequent retransformation of a Rous sarcoma virus-transformed rat cell. Comparison of the nucleotide sequences of cloned proviral DNAs reveals a single base pair insertion approximately 438 bp from the 5′ end of src in the revertant line 000. This frameshift mutation accounts for an 18 kd protein as the prematurely terminated product of src. The mutation is suppressed in the retransformed line (000∗) by a 242 bp duplication that corrects the reading frame to permit synthesis of a 68 kd src protein. A 43 kd src protein with a normal carboxyl terminus is also present in 000 and 000∗ cells. To make this protein, translation must begin at an internal AUG, found just upstream from the frameshift mutation; in both lines, two src proteins appear to be initiated independently from the same mRNA species. Our results imply that a protein of 7 kd is synthesized from a second reading frame within wild-type src.

Phosphorylation of the M r = 34,000 protein in normal and Rous sarcoma virus-transformed rat fibroblasts

Antiserum was raised against the M r = 34,000 chick cell protein which may serve as a substrate for the Rous sarcoma virus transforming gene product. The antiserum specifically immunoprecipitated 2 proteins from [ 35S]methionine labeled Rous sarcoma virus-transformed rat cell extracts (a M r = 35,000 and a M r = 38,000 protein). Partial protease treatment revealed these two proteins to be very closely related. The protein of apparent M r = 38,000 was phosphorylated and the phosphate was present exclusively on tyrosine residues. The effect of epidermal growth factor on phosphorylation of the M r = 35,000 protein was examined in several normal rat fibroblast cell lines. EGF treatment had no effect on phosphorylation of the M r = 35,000 protein for any normal cell line and also failed to elevate overall levels of phosphotyrosine.

A src-related 120,000-dalton protein expressed in an avian sarcoma virus-transformed rat cell line

A Schmidt-Ruppin strain of Rous sarcoma virus-transformed rat cell line, SRYI, produced a 120,000-dalton phosphoprotein (P120) immunoprecipitated by a tumor-bearing rabbit serum in addition to the src gene product, pp60 src and the gag gene product. Antisera against viral structure components did not precipitate P120, and preabsorption of the tumor-bearing rabbit serum with disrupted virions did not affect the precipitation of P120 and pp60 src. One-dimensional peptide mapping by partial proteolysis revealed that P120 is a protein consisting of peptides common with pp60 src SRYl cells contained 28 and 21 S virus-specific RNA species and P120 was translated in vitro from 28 S polyadenylic acid-containing RNA. The expression of P120 in all subclones of SRY1 suggested that the genome coding P120 is integrated in the SRY1 cell DNA. The rescued virus from SRYl cells, however, failed to produce P120 in chick embryo cells.

Purification of a tyrosine-specific protein kinase from Rous sarcoma virus-induced rat tumor.

We have identified a tyrosine kinase activity present in tumors which were raised in rats by subcutaneous injection of Rous sarcoma virus-transformed rat cells (SR-NRK). This kinase phosphorylates tyrosine on the heavy chain of IgG from tumor-bearing rabbit (TBR) sera specific for the src gene product, pp60src. Using TBR-IgG phosphorylation as an assay, we have purified this kinase over 7200-fold. The purification procedure involves detergent extraction of tumors followed by sequential column chromatography on hydroxylapatite, DEAE-Sephacel, oligodeoxyadenosine-cellulose, an affinity column prepared from TBR-sera, and Sephacryl S-200. The IgG kinase activity behaves as a molecule of apparent Mr = 54,000 on Sephacryl S-200 molecular sieve chromatography. Analysis of the Sephacryl fractions by SDS-PAGE indicates that a major Coomassie blue-stained band with an apparent Mr = 54,000 (p54), co-elutes with the peak of kinase activity. From 600 g of tumors, approximately 200 micrograms of p54 are obtained. We have four types of evidence which show that p54 is related to pp60src. 1) Purified p54 is capable of undergoing endogenous phosphorylation in the presence of [gamma-32P]ATP producing a 32P-labeled pp54 polypeptide which is specifically immunoprecipitated by TBR-sera and contains only phosphotyrosine. 2) Purified p54 competes with 32P-labeled pp60src for binding to TBR-IgG, indicating a degree of purification over starting material which agrees very well with the results obtained by the IgG kinase assay. 3) V8 protease digestion of pp60src and p54 suggests that they share a common 26,000 fragment. 4) Antibodies to partially purified p54 specifically precipitate pp60src from Rous sarcoma virus-transformed chicken cells.

Novel localization of pp60 src 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.

Tyrosylprotein kinase and phosphatase activities in membrane vesicles from normal and Rous sarcoma virus-transformed rat cells.

Membrane vesicles; isolated from normal and Rous sarcoma virus-transformed rat cells, have an associated cyclic-AMP independent kinase that phosphorylates a Mr 37,000 protein in vesicles from normal cells and proteins of Mr 37,000, 50,000, and 67,000 in vesicles from transformed cells. Proteins in vesicles from normal and transformed cells contain 9% and 77%, respectively, of their labeled phospho amino acids as phosphotyrosine. Thus, isolation of vesicles and subsequent incubation with [gamma-32P]ATP enriches the proportion of labeled phosphotyrosine in proteins (relative to other phospho amino acids) by two orders of magnitude over that found in intact cells. The in vitro phosphorylation of each of these proteins is enhanced in the presence of 10 microM Zn2+, a phosphotyrosylprotein phosphatase inhibitor. From these studies it appears that membrane vesicles may be a valuable system for examination of transformation-specific phosphorylation of proteins.

A Rous sarcoma virus provirus is flanked by short direct repeats of a cellular DNA sequence present in only one copy prior to integration.

The Rous sarcoma virus (RSV)-transformed rat cell line RSV-NRK-2 contains a single complete RSV provirus. We have obtained recombinant lambda clones that contain both ends of the RSV provirus and the flanking rat sequences. The provirus is integrated in unique DNA and is present in only one of the two homologous chromosomes. The rat sequences into which the RSV provirus integrated were also cloned from the RSV-NRK-2 cell line. The sequences of the regions involved in the recombination event have been determined and compared. Our data suggest that, compared with the sequence of viral DNA in the large circular form of unintegrated viral DNA, the provirus lacks two base pairs at each end and that the provirus is flanked by a six-base-pair direct repeat of cellular DNA. This six-base-pair repeat was apparently created during the integration event because this sequence was present only once at the integration site before the provirus was inserted. A survey of eight other independent RSV transformed rat cell lines demonstrates that, in agreement with earlier results, the RSV proviruses have entered different segments of rat cell DNA. We have also determined the sequence of a second virus DNA-host cell DNA junction from a second RSV-transformed rat cell line (RSV-NRK-4) and find that there are no obvious similarities between the two integration sites or between the integration sites and the termini of viral DNA.

Organization of pp60src and selected cytoskeletal proteins within adhesion plaques and junctions of Rous sarcoma virus-transformed rat cells.

The localization of pp60src within adhesion structures of epithelioid rat kidney cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus was compared to the organization of actin, alpha-actinin, vinculin (a 130,000-dalton protein), tubulin, and the 58,000-dalton intermediate filament protein. The adhesion structures included both adhesion plaques and previously uncharacterized adhesive regions formed at cell-cell junctions. We have termed these latter structures "adhesion junctions." Both adhesion plaques and adhesion junctions were identified by interference-reflection microscopy and compared to the location of pp60src and the various cytoskeletal proteins by double fluorescence. The results demonstrated that the src gene product was found within both adhesion plaques and the adhesion junctions. In addition, actin, alpha-actinin, and vinculin were also localized within the same pp60src-containing adhesion structures. In contrast, tubulin and the 58,000-dalton intermediate filament protein were not associated with either adhesion plaques or adhesion junctions. Both adhesion plaques and adhesion junctions were isolated as substratum-bound structures and characterized by scanning electron microscopy. Immunofluorescence revealed that pp60src, actin, alpha-actinin, and vinculin were organized within specific regions of the adhesion junctions. Heavy accumulations of actin and alpha-actinin were found on both sides of the junctions with a narrow gap of unstained material at the midline, whereas pp60src stain was more intense in this central region. Antibody to vinculin stained double narrow lines defining the periphery of the junctional complexes but was excluded from the intervening region. In addition, the distribution of vinculin relative to pp60src within adhesion plaques suggested an inverse relationship between the presence of these two proteins. Overall, these results establish a close link between the src gene product and components of the cytoskeleton and implicate the adhesion plaques and adhesion junctions in the mechanism of Rous sarcoma virus-induced transformation.

Suppression of the transformed phenotype with retention of the viral ‘ src’ gene in cell hybrids between rous sarcoma virus-transformed rat cells and untransformed mouse cells

Hybrid cell lines between untransformed mouse 3T3TK-cells and normal rat kidney (NRK) cells transformed by the B77 strain of Rous Sarcoma Virus (RSV) express a non-transformed phenotype, as determined by anchorage-dependent growth and organization of microfilament bundles. Virus rescue experiments and genetic experiments using an RSV mutant temperature-sensitive for maintenance of the transformed phenotype demonstrate that RSV is retained in the non-transformed hybrids. The action of the viral transformation gene ‘ src’ therefore appears to be ‘suppressed’ in these hybrids. The suppressed hybrids generate variants in which the expression of the transformed phenotype and the ‘ src’ gene is regained. This system should prove to be of value in identifying cellular genes involved in the expression of virally induced transformation.

Partial purification and characterization of DNA polymerases from a rous sarcoma virus-transformed rat cell line

A new DNA polymerase was partially purified from cell-free extracts of a continuous rat cell-line (XC). The XC cells had been transformed by the Prague strain of Rous sarcoma virus but did not produce infectious virus. The molecular weight of the DNA polymerase is 70 000, as estimated by glycerol gradient centrifugation and by Sephadex gel filtration. This enzyme can be distinguished from the other cellular DNA polymerases by its elution pattern on DNA-cellulose column chromatography, its molecular weight, and its primer-template specificity. The enzyme has some characteristics of the murine leukemia virus reverse transcriptase. It is partially inhibited by immunoglobulin G purified from rabbit antiserum prepared against Rauscher leukemia virus reverse transcriptase, but is not inhibited by IgG from rat antiserum prepared against avian myeloblastosis virus reverse transcriptase. However, the XC cell enzyme can be distinguished from the murine leukemia virus reverse transcriptase by its inefficiency in copying an oligo(dG) 12 · poly(rC) primer-template.

The importance of DNA size for successful transfection of chicken embryo fibroblasts

Recovery of Rous sarcoma virus (RSV) from chicken embryo fibroblasts exposed to DNA from RSV transformed rat cells (XC cells) was successful only after shearing the large molecular weight DNA. Transfection was demonstrated after a single exposure of chicken cells to the DNA, and was achieved as well in chf-negative chicken embryo cells.

Interaction of herpes simplex virus type 1 with Rous sarcoma virus-transformed rat cells [XC and R(B77) cell lines

Two cell lines [R(B77) and XC] of Rous sarcoma virus-transformed rat cells were infected with herpes simplex virus type 1 (HSV-1) grown in BSC-1 cells. Analysis of the DNA species synthesized in XC, R

Appearance of virus-specific DNA in mammalian cells following transformation by Rous sarcoma virus

To detect Rous sarcoma virus-specific DNA in mammalian cells, we have measured the capacity of unlabeled cell DNA to accelerate the reassociation of labeled double-stranded DNA synthesized by the Rous sarcoma virus RNA directed DNA polymerase. Two populations of double-stranded polymerase products are identified by their reassociation kinetics and represent approximately 5% and 30% of the viral 70 S RNA genome. Using two strains of Rous sarcoma virus and four lines of transformed mammalian cells, we found two copies of DNA homologous to both DNA populations in Rous sarcoma virustransformed rat and mouse cells, but not in normal cells. The Rous sarcoma viruslike DNA can be demonstrated in the non-repeated fraction of transformed cell DNA and in nuclear DNA. The results are supported by evidence that the techniques employed detect the formation of extensive well-matched duplexes of cell DNA and viral polymerase products.

Reverse transcriptase-containing particles induced in rous sarcoma virus-transformed rat cells by arginine deprivation.

Incubation of rat cells transformed by Rous sarcoma virus (RSV) in an arginine-deficient medium resulted in accumulation of particles in the culture medium. Such particles did not appear when the transformed rat cells were incubated in a complete medium nor in the medium of primary rat cells which were incubated either in arginine-deficient or complete media. The particles which were released from the arginine-deprived transformed rat cells resemble C-type particles in their properties. These particles band in sucrose gradients at a density of 1.16 g/ml and contain 35S ribonucleic acid (RNA) molecules and a reverse transcriptase activity. Analysis of the cytoplasm of transformed and primary rat cells, deprived and undeprived of arginine, revealed the presence of reverse transcriptase-containing particles which banded in sucrose gradients at a density of 1.14 g/ml. These particles differed from the particles released into the medium by the arginine-deprived RSV-transformed rat cells. The deoxyribonucleic acid (DNA) molecules synthesized in vitro by the reverse transcriptase present in the particles isolated from the medium of arginine-deprived cells hybridized to RSV RNA, whereas the DNA synthesized by the cell-bound enzyme had no homology to RSV RNA.