Surface morphology and agglutinability with concanavalin A in normal and transformed murine fibroblasts

The surface morphology of attached and suspended normal and transformed fibroblasts has been studied with the scanning electron microscope. Normal murine fibroblasts (3T3) grow in vitro with widely extended leading lamellae. During most parts of the cell cycle the surfaces of these cells are practically free of microvilli. When the cells round up for mitosis, their cell surfaces become adorned with many microvilli. In contrast, simian virus 40-transformed fibroblasts (SV3T3) grow more compact, and their cell surfaces remain smooth throughout the life cycle. When confluent 3T3 and SV3T3 cells are suspended with ethylenediaminetetraacetic acid (EDTA) for agglutination assays, similar differences in surface morphology are found: 3T3 cells always bear many microvilli, whereas most SV3T3 cells are essentially free of microvilli. The addition of concanavalin A (Con A) does not influence the surface morphology of the suspended cells. The morphological differences described here may be important for the agglutination process of the normal and transformed 3T3 cells, because they affect the real cell surface area and thus the density of Con A-binding sites.     

The dissociation of the surface architecture described by enhanced lectin agglutinability and the transformed phenotype expressed as anchorage independence.

Using a series of cold-sensitive variants of chemically transformed BHK-21 cells, revertants to the normal phenotype derived from a dimethyl-nitrosamine transformed clone of BHK-21 as well as revertants to the normal phenotype derived from polyoma transformed BHK-21 cells we have demonstrated that the surface phenotype described by enhanced agglutinability with Con A and WGA can be dissociated from the transformed phenotype described by anchorage independence (growth in semisolid medium). Specifically we have demonstrated that the surface characteristic of enhanced agglutinability may be found in a variety of cell lines which fail to display to grow in agar. Our work clearly shows that the two phenotypes described are not concomitantly controlled and tends to suggest that the phenotype of enhanced lectin agglutinability may be dissociated from the transformed phenotype.     

Transformation of BALB/c-3T3 cells by tsA mutants of simian virus 40: temperature sensitivity of the transformed phenotype and retransofrmation by wild-type virus.

The function of the A gene of simian virus 40 (SV40) in transformation of BALB/c-3T3 cells was investigated by infecting at the permissive temperature with wild-type SV40 and with six tsA mutants whose mutation sites map at different positions in the early region of the SV40 genome. Cloned transformants were then characterized as to the temperature sensitivity of the transformed phenotype. Of 16 tsA transformants, 15 were temperature sensitive for the ability to overgrow a monolayer of normal cells, whereas three of three wild-type transformants were not. This pattern of temperature sensitivity of the transformed phenotype was also observed when selected clones were assessed for the ability to grow in soft agar and in medium containing low concentration of serum. The temperature resistance of the one exceptional tsA transformant could be attributed neither to the location of the mutation site in the transforming virus nor to transformation by a revertant virus. This temperature-resistant tsA transformant, however, was demonstrated to contain a higher intracellular concentration of SV40 T antigen than a temperature-sensitive line transformed by the same tsA mutant. A tsA transformant displaying the untransformed phenotype at the nonpermissive temperature was found to be susceptible to retransformation by wild-type virus at this temperature, demonstrating that the temperature sensitivity of the tsA transformants is due to the viral mutation and not to a cellular defect. These results indicate that continuous expression of the product of the SV40 A gene is required to maintain the transformed phenotype in BALB/c-3T3 cells.     

Cytochemical study of concanavalin A binding sites and their mobility in normal, cystic fibrosis, and SV40 transformed human fibroblasts in vitro

Concanavalin A (Con A) binding sites and their mobility were studied by peroxidase (Po) and ferritin labeling techniques in normal and SV40 transformed human fibroblasts. Binding sites were visualized either as osmium black of 3'3-diaminobenzidine (DAB) reactions or as ferritin particles. DAB reaction products were localized at the external surface of the plasma membrane and in some multivesicular bodies of fixed cells. The labeling was continuous in normal and SV40 transformed human fibroblasts. When living cells were treated with Con A-Po at 4 degrees C and incubated at 37 degrees C, both normal and transformed cells showed remarkable changes. The foci of membrane indentations (caps or patches) are formed on the cell surface. Many labeled internalized vacuoles and vesicles appeared within the cytoplasm and in close proximity to the Golgi region of all cell types. The cellular changes occurred more quickly in transformed cells than in normal cells. It is concluded that normal cells do cap under certain conditions and that the plasma membranes of transformed cells are more fluid than those of normal cells.     

SV40 transformation of mouse brain cells: critical role of gene A in maintenance of the transformed phenotype.

Brain cells derived from the NIH Swiss mouse strain have been established in tissue culture. Astrocytic neuroglial cells, identified by morphology and staining properties, predominate. The brain cell culture was successfully transformed with SV40 wild type virus and with a representative early (A239) and late (C219) mutant. When subjected to growth analysis the A239 transformant displayed selective loss of six characteristics of the transformed phenotype at the restrictive temperature (40.5 degrees C): doubling time, saturation density, ability to grow in low serum, efficiency of growth on plastic and on normal brain cell layers, and cloning in soft agar. Temperature shift experiment demonstrated the reversibility of the differences in saturation density. T-antigen was expressed at both temperatures. Alteration in uptake of 2-deoxyglucose was not a characteristic of the transformed phenotype. The cell lines may be utility in brain culture work and in studies on the mechanism of SV40 transformation.     

Isolation of a simian virus 40 T-antigen-positive, transformation-resistant cell line by indirect selection.

In an attempt to identify cellular genes that might be involved in simian virus 40 (SV40) transformation, we have set out to isolate cells which express T antigen but are not transformed. SV40 DNA and the herpes simplex virus thymidine kinase gene were cotransfected into tk- 3T3 fibroblasts. Of 72 colonies screened that were resistant to hypoxanthine-aminopterin-thymidine, 57 were T antigen positive as judged by immunofluorescence. One of these lines, A27, had a normal growth phenotype in monolayer overgrowth and soft agar assays. It contained intact SV40 sequences that could be rescued by fusion to permissive cells. This rescued virus was fully capable of transforming nonpermissive cells to the same extent as did wild-type virus. The A27 cells, however, were not transformable by infection with SV40 or by transfection of SV40 DNA. It is likely that these cells were altered in a cellular function required for the establishment of the transformed state.     

SV40-transformed cells with temperature-dependent serum requirements.

We have isolated temperature-sensitive SV40-transformed 3T3 cells which are unable to grow in low or depleted serum at the nonpermissive temperature. At 39 degrees C, these cells do not grow in 1 percent serum, but they grow if the serum concentration is raised to 10 percent. At 32 degrees they grow in both serum concentrations. This phenotype seems to be due to a cellular mutation, as the virus rescued from these cells is wild-type. We tested whether other characteristics of transformed cells were expressed in a temperature sensitive way. While high saturation density is ts in these cells, other parameters of transformation are expressed at both temperatures. In addition, when these cells are incubated in low serum at 39 degrees C, they keep synthesizing DNA and lose viability very fast, while under the same conditions normal 3T3 cells remain viable for long times and are unable to initiate DNA synthesis. These cells therefore do not appear to revert to a normal phenotype at the high temperature, and they are more likely to represent transformed cell variants with a temperature-dependent serum requirement.     

Trypanosoma rhodesiense Bloodstream Trypomastigote and Culture Procyclic Cell Surface Carbohydrates1, 2, 3

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRATat 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diaminobenzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A < PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WGA, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M. The expression of lectin binding cell surface saccharides of T. rhodesiense WRATat 1 is related to the parasite stage. Sugars resembling α-D-mannose are on the surface of bloodstream trypomastigotes and culture procyclics; n-acetyl-D-galactosamine and D-galactose residues are on bloodstream forms; and n-acetyl-D-glucosamine-like sugars are on procyclic stages.     

Sister chromatid exchange in FR3T3 rat fibroblasts transformed by Simian virus 40

The frequency of Sister Chromatid Exchange (SCE) was determined at low (33 degrees C) and high (40.5 degrees C) temperatures in cell lines derived from FR3T3 rat fibroblast cells after transformation either with Wild-Type Simian Virus 40 (SV40-WT), with an origin-defective SV40 (SV40-ori-), or with the early temperature-sensitive mutant tsA30. Of these cell lines, SV40-WT-, SV40-ori--, and one class of tsA30-transformants (A-type) express the transformed phenotype both at 33 and 40.5 degrees C. The other tsA30-transformants (N-type) revert to a normal phenotype at high temperature. As compared with normal FR3T3 cells, all transformants exhibited, at 33 degrees C, increased numbers of metaphases with high SCE rates. At 40.5 degrees C, all cell lines which expressed a transformed phenotype (SV40-WT, tsA30 type A, SV40-ori-) exhibited substantially increased SCE rates. That this increase was not related to a possible induction of viral replication by BrdU, was proven by Southern blot analysis and by SCE data on SV40-ori--transformed cells. By contrast, no such temperature-induced increase of SCE rates was observed in tsA30-transformants of type N.     

Similarities in the membrane fluidity of 3T3 and SV101-3T3 cells and its relation to concanavalin A- and wheat germ agglutinin-induced agglutination.

Intact, viable ultransformed 3T3 and transformed SV101-3T3 cells were labeled with fatty acid spin labels and with 2,2,6,6-tetramethylpiperidine-1-oxyl in order to measure the fluidity properties of membrane lipids. Both cell types were grown in regular calf serum and in a lipid-depleted serum supplemented with either oleate or elaidate. The temperature dependence of the spectra obtained revealed inflections that correlate with the temperature below which agglutination with concanavalin A is inhibited, and another inflection that correlates with the temperature below which agglutination with wheat germ agglutinin is inhibited, suggesting that (a) the lipid phase(s) in the vicinity of the receptor(s) for these two lectins differ, and (b) a fluid membrane in the vicinity of the lectin receptor(s) is necessary for agglutination with either concanavalin A or wheat germ agglutinin. Studies with a partially characterized plasma membrane fraction suggest that the plasma membrane fluidity parameters closely resemble those of the intact cell. 3T3 and SV101-3T3 cells show virtually identical fluidity profiles by all of the tests we have applied.     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.     

In vitro tumoricidal activity of macrophages against virus-transformed lines with temperature-dependent transformed phenotypic characteristics.

The susceptibility of targets to destruction by tumoricidal rat and mouse macrophages was studied with virus-transformed cell lines in which various elements of the transformed phenotype are only expressed at specific temperatures. BHK cells transformed by the ts3 mutant of polyoma virus, rat embryo 3Y1 cells transformed by a temperature-sensitive A cistron mutant of simian virus 40 (SV40) and the ts-H6-15 temperature-sensitive line of SV40-transformed mouse 3T3 cells were killed in vitro by macrophages at both the permissive (33 °C) or nonpermissive (39 °C) temperatures for expression of the transformed phenotype. 3T3, 3Y1 and BHK cells transformed by wild-type SV40 or polyoma virus were also destroyed by tumoricidal macrophages at both 33 and 39 °C, but untransformed 3T3, 3Y1, and BHK cells were not. Thus, transformed cells are killed by macrophages regardless of whether or not they express cell surface LETS protein or Forssman antigen, display surface changes which permit agglutination by low doses of plant lectins, express SV40 T antigen, have a low saturation density, or exhibit density-dependent inhibition of DNA synthesis.     

Trypanosoma rhodesiense bloodstream trypomastigote and culture procyclic cell surface carbohydrates

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRAT at 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diamino-benzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A greater than PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WAG, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Surface Alterations of Cells Carrying RNA Tumour Virus Genetic Information

CELLS transformed by the DNA tumour viruses, polyoma virus and SV40, are agglutinated by lectins such as wheat germ agglutinin¹, concanavalin A (Con A)² and soybean agglutinin³. Agglutination in these cases presumably reflects changes in the cell surface related to the transformed properties of the cell; studies with a temperature-dependent mutant of polyoma virus has shown that cell surface changes are controlled by viral genes⁴. Here we describe experiments in which we investigated the agglutinability of cells transformed by RNA tumour viruses. One recent report had suggested that cells transformed by RNA tumour viruses were not specifically agglutinated⁵, whereas a second more recent report claimed the specific agglutination of cells transformed by RSV⁶. We find that transformed rat, mouse and cat cells that replicate the sarcoma-leukaemia virus complex of murine (MSV) and feline (FeSV) origin are strongly agglutinated by Con A, but mouse and human cells that replicate the murine and feline leukaemia virus components alone are not agglutinated. The ability to agglutinate is rapidly acquired by normal mouse cells on infection with the murine sarcoma virus at a rate that parallels virus replication. In contrast to the results obtained with cells producing virus, non-virus-producing transformed hamster and mouse cells that synthesize virus-specific RNA are either not agglutinated or are agglutinated to a lesser degree. These results suggest that the cell surface alterations responsible for agglutination are not necessarily associated with the transformed state of the cell, but rather with the possession of sarcoma virus-specific information.     

Lectin-bearing liposomes: differential binding to normal and to transformed mouse fibroblasts

The binding of covalent conjugates of concanavalin A (Con A) or wheat germ agglutinin (WGA) and liposomes (lectin-liposomes) to the surface of normal and transformed mouse fibroblasts was studied. Quantitation of the binding was performed by means of microfluorometry and radioactive lipid label counting using both sparse and dense cell cultures. It was found that 2.5-3 times more lectin-conjugated liposomes are bound to L or SV3T3 cells than to the mouse embryo fibroblasts and 3T3 cells in a broad concentration range. The binding of Con A- and WGA-liposomes was inhibited up to 70% in the presence of the corresponding carbohydrate inhibitors. A decreased binding of lectin-liposomes to cells was also observed when cells were pretreated with the free lectin. Trypsinization of the cells resulted in an increase in the Con A-liposomes binding to normal fibroblasts. When free fluorescent Con A or WGA was used in binding studies no profound differences in the binding of lectin to normal or transformed cells were detected. The relation of the lectin-liposome/cell to cell/cell interactions is discussed.     

Differences in density of Concanavalin A-binding sites due to differences in surface morphology of suspended normal and transformed 3T3 fibroblasts.

Calculations of the density of Concanavalin A (Con A)-binding sites on normal and transformed fibroblasts have, as yet, been based on the unproven assumption that suspended cells are smooth spheres. We studied the surface morphology of suspended normal and transformed fibroblasts with scanning and transmission electron microscopes, and found a large difference in surface morphology between suspended normal and transformed 3T3 cells. When this difference in surface morphology was taken into account, the estimated cell surface area of normal 3T3 cells was approximately seven times larger than that of transformed 3T3 cells. Since equal numbers of 3H-Con A molecules are bound on normal and transformed cells, the density of Con A-binding sites is approximately seven times greater on transformed than on normal 3T3 cells. The difference in density of Con A-binding sites between normal and transformed fibroblasts might be sufficient to explain the difference in agglutination response, as originally suggested by Burger, and may also be the cause of the different degrees of clustering of Con A-binding sites on the plasma membrane of these cells.     

Receptor mobility and the binding of cells to lectin-coated fibers

The ability of cells to bind to nylon fibers coated with lectin molecules interspaced with varying numbers of albumin molecules has been analyzed. The cells used were lymphoma cells, normal lymphocytes, myeloid leukemia cells, and normal and transformed fibroblasts, and the fibers were coated with different densities of concanavalin A or the lectins from soybean or wheat germ. Cells fixed with glutaraldehyde did not bind to lectin-coated fibers. The number of cells bound to fibers could be increased by increasing the density of lectin molecules on the fiber, the density of specific receptors on the cell, or the mobility of the receptors. It is suggested that binding of cells to fibers involves alignment and binding of specific cell surface receptors with lectin molecules immobilized on the fibers, and that this alignment requires short-range rapid lateral mobility (RLM) of the receptors. The titration of cell binding to fibers coated with different densities of lectin and albumin has been used to measure the relative RLM of unoccupied cell surface receptors for the lectin. The results indicate a relationship of RLM to lectin-induced cell-to-cell binding. The RLM or receptors for concanavalin A (Con A) was generally found to be higher than that of receptors for the lectins from wheat germ or soybean. Receptor RLM could be decreased by use of metabolic inhibitors or by lowering the temperature. Receptors for Con A had a lower RLM on normal fibroblasts than on SV40-transformed fibroblasts, and trypsinization of normal fibroblasts increased Con A receptor RLM. Normal lymphocytes, lymphoma cells, and lines of myeloid leukemia cells that can be induced to differentiate had a high receptor RLM, whereas lines of myeloid leukemia cells that could not be induced to differentiate had a low receptor RLM. These results suggest that the RLM of Con A receptors is related to the transformation of fibroblasts and the ability of myeloid leukemia cells to undergo differentiation     

SV40 transformation of Swiss 3T3 cells can cause a stable reduction in the calcium requirement for growth

A well-characterized SV40-transformed Swiss 3T3 line, SV101, and its revertants were tested for the ability to grow in reduced Ca++ (0.01 mM). Transformants and revertants did not differ from the parent 3T3 line in their Ca++ requirements. All three classes of cells grew less well in low Ca++ than in regular Ca++ (2.0 mM). SV40 transformants were then selected for the ability to grow in reduced Ca++. This new class of transformants was found to grow in 1% serum, grow in soft agarose, have a reorganized actin cytoskeleton, and express viral T antigens, as well as grow well in low Ca++. One of the selected clones was found to be T antigen-negative, yet was transformed in the serum, anchorage, actin, and Ca++ assays. It is possible that this clone was a spontaneous transformant. However, Southern blot analysis revealed the presence of integrated SV40 DNA. In addition, this analysis revealed the absence of an intact early region fragment, which codes for the viral T antigens. One explanation of this result may be that the mechanism of viral transformation for growth in low Ca++ involves viral-host DNA interactions that may not require a fully functional T antigen. In this case SV40 integration may be acting as a nonspecific cellular mutagen.     

Fluorescence photobleaching recovery measurements of surface lateral mobilities on normal and SV40-transformed mouse fibroblasts

Lateral mobilities of fluorescent cell surface probes have been measured on normal (3T3) and transformed (SV3T3) cultured mouse fibroblasts. There is little discernible difference in the mobilities of a lipid analogue (diI), a fluorescent ganglioside derivative (GM1), and tetramethylrhodamine-labeled succinylated concanavalin A. The two cell lines showed expected differences in their abilities to grow in agar, to grow without serum, and to be agglutinated by lectins, indicating that changes of these properties in transformed cells are probably not mediated through increased overall membrane fluidity but are associated with distinct alterations in the mobilities of cell surface receptors. Both fluorescent dextran derivatives and antimouse cell surface antibodies were distinctly less mobile on SV3T3 cells, and the mobile fraction of Con A receptors was lower on SV3T3 cells.     

Epidermal growth factor induced membrane changes in 3T3 cells.

Epidermal growth factor (EGF) is a mitogen for Swiss 3T3 cells. Short incubation periods with physiological concentrations of EGF induced increased binding of Swiss 3T3 cells to Con A-coated nylon fibers. This effect was not induced in an EGF non-responsive 33 variant, in the transformed murine XC cells or in Swiss SV3T3 cells. The increase in Con A fiber-binding seems to be specific for EGF, since it was not observed in response to insulin, prostaglandin F2alpha or a higher serum concentration, which also initiate cell devision of confluent quiescent 3T3 cells. EGF also reduced Con A-mediated hemadsorption to 3T3, but had no effect on hemadsorption by the EFG non-responsive 3T3 variant. There was no change in the number of Con A-receptors on 3T3 cells after EGF treatment. Binding to WGA-coated fibers and WGA-mediated hemadsorption were not effected by preincubation with EGF.