The study of rous sarcoma virus-transformed baby hamster kidney cells using fluorescent probes

The fluorescent probes pyrene, pyrene butyric acid and $\text{N-}\text{phenyl}$ 1-naphthylamine have been used to investigate the changes that accompany in vitro transformation of a baby hamster kidney cell line using Rous sarcoma virus. The fluorescent probes which reside in the membrane were used to compare the changes in microviscosity and polarity of the membranes of normal cells with two transformed cell lines. The spectrofluorimetric data indicate that following transformation the probe $\text{N-}\text{phenyl}$ 1-naphthylamine resides in a more polar environment. However, using the probe pyrene, the yield of excimer indicates decreased mobility of this probe in the membrane of transformed cells. The data also indicate differences between the two transformed cell lines. Laser photolysis was used to study the lifetime of the pyrne probes and the quenching of the pyrene fluorescence in the membrane by several different quenching molecules. The data indicate differences between the three cell lines and suggest that transformation decreases movement within the membrane.     

Comparison of protein sulfation in control and virus-transformed baby hamster kidney cells

Protein sulfation in baby hamster kidney cells (BHK) and their polyoma virus transformants (PY-BHK) was studied comparatively. On in vivo labeling, [35S]-sulfate was incorporated into the 50K protein and proteins in the 100-180K range, represented by the 155K protein. The incorporation into both the 50K and 155K protein was elevated 2-3 fold in PY-BHK cells compared to in BHK cells. Tyrosine-O-sulfate was the only identifiable sulfated amino acid in both proteins. On in vitro labeling with [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS), at least 6 radioactive protein bands were discernible on gel electrophoresis. Of these, sulfation of the 57K and 60K proteins was elevated in PY-BHK cells compared to in BHK cells, whereas sulfation of the 39K protein was depressed in PY-BHK cells. Tyrosine-O-sulfate was the only identifiable sulfated amino acid in these proteins.     

Control and virus-transformed baby hamster kidney cells resistant to ethidium bromide. II. Membrane properties

Aspects of membrane stucture and functions were studied in ethidium bromide resistant cells. Submitochondrial particles were solubilized and electrophoresed. The gel patterns, representing mitochondiral membrane proteins, demonstrated qualitative and quantitative alterations in mitochondrial preparations derived from virus-transformed cells and ethidium bromide resistant cells as compared to the control cells. The plasma membrane glycoproteins were labelled by the sodium borohydride method. The glycoporteins were released with Triton X-100 and electrophoresed. Fluorograms of the gels demonstratred some marked differences between the ethidium bromide resistant cells and their parental strain. The observed alterations in the membrane glycoproteins did not result in altered glucose transport properties or in the elution patterns of plasma membrane glycopeptides as analyzed by Sephadex G-50 chromatography. Dye uptake and binding studies with intact parental and drug resistant cells and their isolated mitochondria demonstrated no alteration of the membrane permeability or the number of binding sites for ethidium bromide. Similar results were also obtained with a cyanine dye. This latter finding was significant in that it permitted one to exclude dye exclusion as a mechanism for ethidium bromide resistance.     

Oligosaccharides of the Hazelhurst vesicular stomatitis virus glycoprotein are more extensively processed in Rous sarcoma virus-transformed baby hamster kidney cells

Because of the extensive oligosaccharide heterogeneity of the membrane glycoprotein (G) from the Hazelhurst strain of vesicular stomatitis virus, this virus has been used as a specific intracellular probe of altered protein glycosylation in Rous sarcoma virus-transformed versus normal baby hamster kidney cells. Over 70% of G protein from virus released from the transformed cells had acidic-type oligosaccharides at both glycosylation sites, compared to less than 50% from the corresponding normal host cells. The remaining G protein contained an acidic-type oligosaccharide at one site and an endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide at the other. The major endoglycosidase-sensitive species were sialylated hybrid-type (NeuNAc-Gal-GlcNAc-Man5GlcNAc2-Asn) from the transformed and neutral-type (Man5-6GlcNAc2-Asn) from the normal host cells. The degree of branching of the acidic-type oligosaccharides was not increased in the transformed cells (approx. 80% biantennary for viral G protein from both cell types). At a reduced growth temperature (24 versus 37 degrees C), the G protein oligosaccharides were more extensively processed in both cell types (approximately 85-95% of G protein contained acidic-type structures at both sites), even though the level of viral protein synthesis and virus release was decreased. Essentially all of the minor, endoglycosidase-sensitive oligosaccharides on mature viral G protein were sialic acid-containing hybrid-type structures. At 24 degrees C the branching of the acidic-type oligosaccharides was increased in the virus released from the transformed cells versus normal cells.     

Novel glycosylation-defective baby hamster kidney cells.

The plant lectin wheat germ agglutinin (WGA) has previously been used to select more than ten different glycosylation-defective phenotypes in a variety of mammalian somatic cells. Three WGA-resistant phenotypes have now been obtained spontaneously from baby hamster kidney (BHK) cells. These mutant BHK cells exhibit a pattern of cross resistance and sensitivity to multiple plant lectins, suggesting that the cell surface carbohydrates of these cells are altered. Two WGA-resistant BHK phenotypes appear similar to WGA-resistant CHO cells that lack terminal sialic acid and galactose residues on their cell surface carbohydrates. The third WGA-resistant BHK cell phenotype has not previously been seen in WGA-resistant mammalian cells.     

Comparison of glycopeptides from control and virus-transformed baby hamster kidney fibroblasts

Glucosamine-labeled glycopeptides from control and virus-transformed BHK fibroblasts were characterized by size, lectin affinity, charge, and composition. As already demonstrated, on the basis of elution position on a column of Sephadex G-50, transformed cells contained a greater proportion of large glycopeptides than did control cells. Transformed cells also contained a larger proportion of glycopeptides which do not bind to Con A-Sepharose. By sequential chromatography on Sephadex G-50, Con A-Sepharose, and DEAE-Sephadex, approximately 40 individual peaks were partially or completely resolved. If sialic acid was removed from the glycopeptides prior to analysis by ion-exchange chromatography, 95% of the glycopeptides from control cells and 85% of the glycopeptides from transformed cells were no longer bound by DEAE-Sephadex. It was concluded that the DEAE-Sephadex elution properties of the glycopeptides are determined almost entirely by the sialic acid content of the molecules. A comparison of the profiles of control and transformed cell glycopeptides simultaneously eluting from columns of DEAE-Sephadex revealed that the differences between the two cells were largely quantitative; however, the possibility of the existence of qualitative differences as well cannot be excluded. In particular, there was one component present on the surface of transformed cells that was virtually absent in control cells. It was degraded by nitrous acid hydrolysis and heparinase and appeared to be heparan sulfate like material. After fractionation, each isolated glycopeptide population was analyzed for carbohydrate and, in some cases, amino acid content. The apparently larger glycopeptides, group A, the dominant population in transformed cells, were found to contain 3 to 4 mannose residues/glycopeptide when the sugars were normalized to sialic acid content. On the basis of the same criteria, group B glycopeptides contained 4-6 mannose residues/glycopeptide. The carbohydrate and amino acid compositions of the glycopeptides from transformed cells were, with a few exceptions, similar to those from control cells. Some isolated glycopeptides appeared to contain both O-glycosidic anad N-glycosidic linkages on the same oligopeptide.     

Vanadate-treated baby hamster kidney fibroblasts show cytoskeleton and adhesion patterns similar to their Rous sarcoma virus-transformed counterparts

Rous sarcoma virus-transformed baby hamster kidney fibroblasts (RSV/B4-BHK) adhere to a fibronectin-coated substratum by means of dot-like adhesion sites called podosomes in view of their shape and function as cellular feet (Tarone et al.: Exp Cell Res 159:141, 1985). Podosomes concentrate tyrosine-phosphorylated proteins, including pp60v-src, and appear in many cells transformed by oncogenes coding for tyrosine kinases. In this paper we used orthovanadate, an inhibitor of phosphotyrosine phosphatases, in order to increase the cellular concentration of phosphotyrosine and to study whether this treatment induced the cytoskeleton remodeling leading to the formation of podosomes. Indeed, orthovanadate (10-100 microM) induced in a time- and dose-dependent manner the redistribution of F-actin and the formation of podosomes in BHK cells. Cytoskeleton remodeling occurred along with a marked increase of tyrosine phosphorylated proteins. The vanadate effect on the cytoskeletal phenotype was enhanced by the simultaneous treatment of cells with a phorbol ester. Under the latter conditions almost all BHK cells showed podosomes. The vanadate effect was reversible insofar as podosomes and tyrosine-phosphorylated proteins disappeared. Then, vanadate treatment of normal cells induced the cascade of events leading to the cytoskeletal changes typical of transformation and suggested that the transformed cytoskeletal phenotype may be primarily induced by the tyrosine phosphorylation of unknown target(s) operated by endogenous kinases.     

Mutator genes of baby hamster kidney cells.

Two mutator genes of mammalian cells were demonstrated. One was associated with the ribonucleoside diphosphate reductase, and the other was associated with an extreme adenosine sensitivity.     

Sulfation of chondroitin sulfate secreted by baby hamster kidney cells and their polyoma virus-transformed counterparts

Sulfation of glycosaminoglycans (GAGs) secreted by baby hamster kidney (BHK) cells and the polyoma virus-transformants (PY-BHK) was investigated. It has been reported that chondroitin sulfate (CS) of cell membranes from PY-BHK cells is undersulfated compared to that from BHK cells (Cancer Res. 43, 2712-2717, 1983). In the first series of experiments of the present study, cells were incubated with [3H]glucosamine and [35S]sulfate, and GAGs isolated from the culture medium were examined. GAG composition was comparable between the BHK and PY-BHK cultures. Disaccharide analysis of the chondroitinase ACII digests of the hyaluronate lyase-resistant materials showed a high proportion (68% for BHK and 47% for PY-BHK) of delta Di-0S, with delta Di-4S (32% for BHK and 53% for PY-BHK) as the major sulfated disaccharide on the basis of 3H-radioactivities. The beta-D-xyloside treatment did not alter the degree of undersulfation of the CS of either culture. In the second series of experiments, disaccharide analysis of the chondroitinase ABC digests of unlabeled GAGs demonstrated similar disaccharide composition for the two cell types. The BHK and PY-BHK preparations showed 28 and 17% (mol percent) of delta Di-0S, 58 and 72% of delta Di-4S, and 14 and 11% of delta Di-6S, respectively. These results indicate a considerable degree of undersulfation of secretory CS from both cells, and a slightly higher degree, if any, of under-sulfation of secretory CS from BHK cells if compared between the two cell types, which is in contrast to the results reported for membrane CS.     

Hyaluronate-protein complex of rous sarcoma virus-transformed chick embryo fibroblasts

Involvement of covalently linked protein or peptide in the structure or synthesis of hyaluronate has not previously been convincingly demonstrated. We have developed conditions for double-labeling with [3H]leucine and [14C]acetate, then isolating and characterizing the cell-associated and secreted hyaluronate-protein complexes of Rous sarcoma virus-transformed chick embryo fibroblasts. The preparations were purified by Bio-Gel A-15m gel filtration and CsCl density gradient ultracentrifugation under dissociative conditions, followed by acid agarose gel electrophoresis in the presence of 0.1% Nonidet P-40. The purified hyaluronate preparations did not change their 3H:14C ratios after further sodium dodecyl sulfate or alkali treatment. The cell-derived hyaluronate-protein was resistant to pronase but susceptible to proteinase K in the presence of sodium dodecyl sulfate. After chondroitinase ABC digestion, the cell-derived 3H-labeled protein was separated from the 14C-labeled hyaluronate disaccharides, then shown to give a broad band corresponding to Mr approximately 12,000 on sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and to be susceptible to both pronase and proteinase K. The corresponding 3H-labeled peptide was prepared in the same manner from the medium hyaluronate and the [3H]leucine shown to be present in material smaller in amount and size than that from the cell. We propose from these and other published data that the cell-associated hyaluronate-protein may be bound to the cell surface and that the hyaluronate in the medium may be derived from it as a result of proteolytic scission.     

Rous sarcoma virus-transformed baby hamster kidney cells express higher levels of asparagine-linked tri- and tetraantennary glycopeptides containing [GlcNAc-beta (1,6)Man-alpha (1,6)Man] and poly-N-acetyllactosamine sequences than baby hamster kidney cells

The alterations in complex-type N-linked oligosaccharides that can occur when an animal cell line is transformed by two dissimilar viruses were examined by comparing the N-linked oligosaccharides of baby hamster kidney (BHK) cells, metabolically radiolabeled with [2-3H]mannose, to the same class of oligosaccharides from BHK cells separately transformed by Rous sarcoma virus (RS-BHK), an RNA retrovirus, and polyoma virus (PY-BHK), a DNA papovavirus. Based on experiments that utilized serial lectin affinity chromatography, glycosidase digestions, and methylation analyses, both RS-BHK and PY-BHK cells demonstrated a significant increase in the relative amounts of tri- and tetraantennary complex-type N-linked oligosaccharides containing the branching sequence, [GlcNAc-beta(1,6)Man-alpha(1,6)Man], compared to the nontransformed BHK cells. In addition, almost all of the poly-N-acetyllactosamine sequence, [GlcNAc-beta(1,3)-Gal-beta(1,4)], was expressed on the tri- and tetraantennary N-linked oligosaccharides from BHK and RS-BHK cells that contain the sequence, [GlcNAc-beta(1,6)Man-beta(1,6)Man]. The increase in the relative amounts of this latter sequence in the transformed cells, therefore, most likely results in an increase in the amount of poly-N-acetyllactosamine sequence on the N-linked glycopeptides of these cells. The analysis of the degree of sialylation of the complex-type N-linked oligosaccharides from BHK and RS-BHK cells by ion exchange chromatography revealed no apparent differences, and in both of these cell types approximately 3% of the glycopeptide fraction radiolabeled with mannose was recovered in a highly negatively charged fraction that was identified by keratanase digestion to be keratan sulfate.     

Growth stimulation of rous sarcoma virus-transformed BHK cells by biotin and serum lipids

Biotin or a serum lipid extract stimulated proliferation of G1 arrested Rous sarcoma virus-transformed BHK cells in modified Eagle's MEM (BM). The cells could be maintained continuously in BM plus biotin (BMB), but not in BM plus serum lipid extract (BM X L). Avidin inhibited growth stimulation when added to BMB, but did not inhibit growth when added to BM X L. 14C-acetate incorporation into total cellular lipids was stimulated in BMB, but not in BM. Thin-layer chromatography of the labeled cellular lipid extract indicated that relatively large amounts of 14C-acetate were incorporated into phosphatidylserine and little into the other major phospholipids. In the neutral lipids, the largest amount of incorporation was in cholesterol. G1 arrested cells multiplied rapidly in BM supplemented with dialyzed serum (BM X DS), but they did not multiply in BM with delipidized serum (BM X DLS). The addition of biotin or serum lipid extract to BM X DLS stimulated growth. Growth stimulation in BM X DLS by biotin was inhibited by avidin, but avidin had no effect on growth stimulation by serum lipid extract. Biotin stimulated additional multiplication in BM X DS and avidin inhibited this additional growth stimulation. These results suggest that growth stimulation requires lipids supplied by serum lipids or by de novo synthesis stimulated by biotin. In the absence of serum, the stimulation of the synthesis of growth factor(s) by biotin are also required for continuous multiplication.     

Control and virus-transformed baby hamster kidney cells resistant to ethidium bromide. I. Characterization and the respiratory enzymes

Cell lines resistant to ethidium bromide have been developed from cultured mammalian BHK21/C13 cells and these same cells transformed by Rous sarcoma virus (C13/B4). Cells resistant to 2 micrograms ethidium bromide per milliliter have been cloned. One clone of the control and one of the virus-transformed cell lines has been employed for characterization. The resistant cells, in the presence of 2 micrograms ethidium bromide/ml, grow at approximately the same rate as the untreated parental cells. The control cells possess a "normal" karyotype (44 chromosomes), while the corresponding ethidium bromide mutant has a reduced chromosome number of 41 and a number of translocations. The mitochondria displayed morphological alterations compared to the parental lines during the transition phase prior to the isolation of the ethidium bromide-resistant cells. The mitochondria of the ethidium bromide-resistant mutants appear somewhat enlarged with a normal morphology. The effect of ethidium bromide on selected respiratory enzymes in normal and virus-transformed ethidium bromide-resistant baby hamster kidney cells was determined. Ethidium bromide-resistant cells exhibited a depressed level of cytochrome aa3. This depression could not be reversed by growth in ethidium bromide-free media. Ethidium bromide-resistant cells possessed the same cytochrome b, c, and c1 levels per cell as their corresponding parental lines. Purified mitochondria isolated from virus-transformed ethidium bromide-resistant cells exhibited a depression in cytochrome oxidase-specific activity, while the ethidium bromide-resistant control cells did not. All cell lines studied showed a depression in NADH-ferricyanide and NADH-cytochrome c reductase-specific activities relative to their parental BHK21/C13 cells. No increase was observed in virus-transformed ethidium bromide-resistant cells. Ethidium bromide-resistant control cells exhibited a two-fold increase in oligomycin-insensitive adenosine triphosphatase activity relative to their parental cells. All of the cell lines studied possessed equivalent oligomycin-sensitive adenosine triphosphatase-specific activity except for the virus-transformed, dye-resistant mutant, whose activity was increased.     

Inhibition of protease activity in cultures of rous sarcoma virus-transformed cells: effect on the transformed phenotype.

We have examined the role of proteolytic activity in the genesis and maintenance of the transformed phenotype by growing cultures of chick embryo fibroblasts transfromed by Rous sarcoma virus either in medium containing plasminogen-free serum or in medium to which protease inhibitors were added. Alterations in morphology, adhesiveness, and hexose transport were used as markers for the transformed state. Addition of the trypsin inhibitors NPGB or Soy Bean Trypsin Inhibitor at concentrations which inhibited transformation-associated fibrinolysis restored adhesiveness and morphology to near normal, but did not affect the rate of hexose transport. Growth of Rous-infected cells in plasminogen-free medium blocked the appearance of morphological and adhesive alterations, but allowed the rate of hexose transport to increase to the transformed level. Thus we were able to separate the appearance of transformation-specific changes in morphology and adhesiveness (which apparently require fibrinolytic activity) from the increased rate of hexose transport (which is independent of fibrinolytic activity). Another trypsin inhibitor, TLCK, although it did not inhibit fibrinolysis, was very effective at restoring adhesiveness and morphology as well as hexose transport to normal. This raises the possibility that there is another, perhaps earlier, protease involved in the genesis of the transformed phenotype.     

Isolation and characterization of norleucine-resistant baby hamster kidney cells

Variants resistant to low and high levels of the methionine analogue norleucine were isolated in baby hamster kidney cells and in two clonal sublines, B1 and TG2. Clones resistant to high levels of norleucine were observed only after chemical mutagenesis, whereas clones capable of growing in low concentrations of norleucine occurred with equal frequency spontaneously and after mutagenesis. The variants were characterized with respect to uptake of ¹⁴C-norleucine and ¹⁴C-methionine. Five clones were found to be deficient in ¹⁴C-norleucine uptake, and of these, four showed reduced ¹⁴C-methionine uptake. The variants were tested also for increased activity of N₅-methyl-tetrahydrofolate: homocysteine methyltransferase, the enzyme which catalyses the terminal reaction in methionine biosynthesis. In four clones, higher levels of the methyltransferase were present than in the wild-type cells, suggesting overproduction or stabilization of this enzyme.     

Potassium fluxes and ouabain binding in growing,density-inhibited and rous sarcoma virus-transformed chicken embryo cells

Potassium fluxes, ouabain binding, and Na⁺ and K⁺ intracellular concentrations were determined for cultures of growing normal, density-inhibited and Rous sarcoma virus-transformed chicken embryo fibroblasts. No significant differences in K⁺ influx or ouabain binding were detected between growing normal cells and Rous sarcoma virus-transformed cells; however, ouabain binding and ouabain-sensitive K⁺ influx were 1.5- to 1.8-fold lower in density-inhibited cells. Thus, potassium influx in this system can be classified as a growth-related, but not transformation-specific change. As determined by both flame photometry and radioisotopic (⁴²K) equilibration, growing normal and density-inhibited cells had similar potassium contents, whereas transformed cells exhibited 1.4-fold higher potassium levels. Sodium ion levels, as measured by flame photometry, were also 2- to 4.5-fold higher in transformed than normal or density-inhibited cells. Complementary studies of potassium efflux showed a 1.3- to 1.5-fold higher rate (based on the percentage of pool exiting the cell) in growing normal versus density-inhibited or transformed fibroblasts. Because of the larger potassium pool in transformed cells, efflux based on absolute number of potassium ions is similar in normal and transformed chicken embryo fibroblasts.