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.     

Studies on lectin-induced agglutination of Drosophila embryonic cell lines

The agglutination responses of three Drosophila cell lines to concanavalin A and wheat germ agglutinin have been examined. Although the cell lines were originally derived from late embryonic stages of the Ore-R strain of Drosophila melanogaster, they show quantitative differences in lectin-induced agglutination. Line 1 cells were least agglutinable with both lectins. All three cell lines reached maximum agglutination with concanavalin A concentrations at 25 μg/ml, but the agglutination response to wheat germ agglutinin was biphasic such that an initial rapid increase in agglutination with concentrations up to 25 μg/ml was followed by slower agglutination above this concentration. Cells of lines 1 and 2 from ten-day old cultures exhibited greater lectin-induced agglutination than cells from three-day old cultures. Age-dependent differences were not found for line 3 cells which gave maximum agglutination responses in both young and old cultures. Cell agglutination by concanavalin A was almost completely inhibited by pretreatment of the lectin with methyl-α-d-mannopyranoside, but preincubation of wheat germ agglutinin with N-acetyl-d-glucosamine caused only partial blockage. Lectin-induced agglutination was not reversible by treatment with the monosaccharide inhibitors. These observations have been discussed with reference to the origin of the three cell lines and their cell surface properties.     

THE RELATIONSHIP OF CONCANAVALIN A BINDING TO LECTIN-INITIATED CELL AGGLUTINATION

We have investigated the relationship of concanavalin. A binding to the cell surface of normal and transformed cells and the subsequent agglutination of the transformed cells. At room temperature almost no differences could be detected in agglutinin binding between transformed and untransformed cells. At 0°C, however, where endocytosis was negligible, the transformed cells bound three times more agglutinin. However, transformed cells and trypsin-treated normal cells do not agglutinate at 0°C although the amounts of agglutinin bound at 0°C are sufficient to permit agglutination when such cells are shifted up to room temperature. Both transformed and trypsin-treated normal cells show a marked increase in agglutination at 15°C as compared to agglutination at 0°C. From this, as well as the observation that mild glutaraldehyde fixation of the cell surface inhibited agglutination but not agglutinin binding, it was concluded that concanavalin A-mediated cell agglutination requires free movement of the agglutinin receptor sites within the plane of the cell surface.     

Changes in surface architecture during murine erythroleukemia cell differentiation as detected by lectin binding and agglutination

Cell surface alterations occurred during murine erythroleukemia cell (clone 745) differentiation that were detected by both agglutination and lectin binding. Agglutination of erythroleukemia cells was produced by wheat germ agglutinin; whereas, concanavalin A, Ricin, soybean agglutinin and fucose-binding protein were either ineffective or much less efficacious. Treatment of leukemia cells with the inducer of erythroid differentiation dimethylsulfoxide (DMSO) caused a progressive accumulation of hemoglobin-containing cells in culture and a decrease in the rate of agglutination by wheat germ agglutinin, which began at 24 h after exposure to the polar solvent, reached a nadir at 48 h, and remained essentially constant thereafter. The binding of radioactive wheat germ agglutinin by untreated control erythroleukemia cells increased with time in culture, reaching a maximum value at 48 h, and decreased progressively thereafter. Although an increase in ³H-labeled wheat germ agglutinin binding also occurred in DMSO-treated cells, the level bound was significantly lower than that observed in control cells at 24–96 h. The treatment of erythroleukemia cells with various concentrations of DMSO resulted in a decrease in the number of wheat germ agglutinin receptor sites. Other inducers of differentiation (i.e., dimethylformamide, bis(acetyl)diaminopentane) also inhibited the rate of wheat germ agglutinin-induced agglutination of erythroleukemia cells while, in contrast, the inducer tetramethylurea did not. These studies indicate that membrane changes occur during differentiation and suggest that there may be more than one mechanism involved in the initiation of maturation which ultimately leads to the common pathway of erythroid development.     

Inhibition of spontaneous aggregation by wheat germ agglutinin in suspensions of BALB/c-3T3 and BHK21 tissue culture fibroblasts

A quantitative method of measuring cytoaggregation based on the Coulter electronic cell counter has been applied to the agglutination of BALB/c-3T3 and BHK21 tissue culture fibroblasts by wheat germ agglutinin. When agglutinin is added to transformed or trypsinized cell suspensions high aggregation rates are seen, and the results obtained are in close agreement with the well-known sensitivity of transformed cells to agglutination by lectins.In the absence of agglutinin, a small but reproducible amount of spontaneous aggregation can also be detected. It is related in some way to growth, since it is absent in suspension prepared from confluent (density-inhibited) cultures and is induced by either transfer to low density, additional serum, or transformation by viruses. Under conditions favouring growth, both BALB/c-3T3 and BHK21 cells show aggregation indices close to 25, corresponding to 10% of the maximum aggregation rate seen.This spontaneous aggregation is susceptible to inhibition by agglutinin. Inhibition occurs at low concentration (about 10 μg/ml) and aggregation rates thus pass through a minimum as the concentration of agglutinin is increased. Among the four different cell lines studied, sensitivity to inhibition is inversely related to agglutination. Thus 3T3 cells, which are barely agglutinated by 1 mg/ml of agglutinin, show 90% inhibition; polyoma virus-transformed BHK cells, which are agglutinated by 10 μg of agglutinin, show no inhibition at all.It is suggested that the agglutination of transformed cells is a consequence of their failure to respond to an inhibitory effect exerted by lectins upon an intrinsic adhesive property of the cell surface.     

Cholesterol levels and plasma membrane fluidity in 3T3 and SV101-3T3 cells

Polyene antibiotics such as filipin selectively inhibit wheat germ agglutinin-induced agglutination of transformed and malignant cells compared to normal cells (Hatten ME, Burger MM: Biochemistry 18:739, 1979). Since filipin binds specifically to cholesterol, we measured cholesterol levels in 3T3 cells and SV101-3T3 cells. SV101-3T3 cells contained 50-100% more cholesterol per cell than 3T3 cells. Both cell types were starved for cholesterol by growth in lipid-depleted medium plus 25-hydroxycholesterol. The cholesterol level of SV101-3T3 cells decreased by 30-50%, while the level in 3T3 cells remained constant. Filipin-stained SV101–3T3 cells revealed bright patches of filipin under fluorescence microscopy. These patches were absent in 3T3 cells and in SV101–3T3 and 3T3 cells starved for cholesterol. We selectively labeled plasma membranes of these cells with a spin label analog of phosphatidylcholine. The spin label indicated differences in plasma membrane fluidity that may be related to the different cholesterol levels in 3T3 and SV101–3T3 cells.     

Interaction of lectins with human platelets. Effects on platelet stimulation by thrombin and ristocetin.

Wheat germ agglutinin induced aggregation and secretion of fresh platelets. Aggregation, but not secretion of serotonin by platelets in plasma, by the lectin was inhibited by 5 mM EDTA. Further, the lectin-induced stimulation of fresh platelets was blocked by prostaglandin E1. Thus, this lectin stimulates platelets by a mechanism which closely mimics thrombin activation and is independent of intercellular crosslinking. Lentil lectin did not stimulate platelets. Each platelet contained about 6 . 10(-5) binding sites for the lectins with an apparent dissociation constant of 3.0 . 10(-7) M. Wheat germ agglutinin, which binds mainly to glycoprotein I (Mr 150 000), increased the subsequent binding of thrombin to fixed platelets while lentil lectin was without effect. It appears that thrombin and wheat germ agglutinin bind to independent but interacting sites. Wheat germ agglutinin, but neither thrombin nor lentil lectin, inhibited the agglutination of platelets by ristocetin. Further, rat platelets were not aggregated by either ristocetin or wheat germ agglutinin. It appears that the interaction sites of ristocetin and wheat germ agglutinin on platelets are overlapping.     

Cell surface saccharide differences in drug-susceptible and drug-resistant strains of Trichomonas vaginalis.

Surface carbohydrates of drug-resistant and drug-susceptible strains of Trichomonas vaginalis were analysed using lectins. The presence of D-GalNAc, D-Gal and mannose-like residues was detected in T. vaginalis. Marked differences in exposed surface carbohydrates were documented, e.g. wheat germ agglutinin (WGA) selectively agglutinated the drug-susceptible strain whereas drug-resistant parasites reacted preferentially with concanavalin A (Con A). In drug-resistant, but not in drug-susceptible strains, trypsinization induced the appearance of soybean agglutinin. Binding studies using fluorescein-labelled WGA and Con A essentially confirmed the agglutination experiments. Both the intense cell agglutination and the fluorescent WGA-binding displayed by a drug-susceptible strain, were completely nullified by neuraminidase treatment, suggesting the presence of an exposed sialic acid moiety on the T. vaginalis surface.     

Contact Inhibition of Transformed Cells Incompletely Restored by Dibutyryl Cyclic AMP

INCREASED levels of cyclic AMP have been found in normal cells as compared with malignant cells^1,2. Several types of malignant cells become morphologically similar to untransformed cells when incubated in media containing cyclic AMP or its derivative dibutyryl adenosine 3′:5′-cyclic monophosphate (dibutyryl cyclic AMP)^3,4. Sheppard reported that 3T3 mouse fibroblasts, transformed by polyoma virus, grew to low saturation density and became less agglutinable with wheat germ agglutinin if theophylline and dibutyryl cyclic AMP were added to the medium⁵.     

Lateral diffusion of lectin receptors in fibroblast membranes as a function of cell shape

Anchorage-dependent fibroblasts respond to biochemical growth signals only when attached to and spread on a suitable substrate surface. Attachment of fibroblasts initiates a cytoskeletal assembly process that results in the organization of long actin stress fibers and microtubules which may be required for transmembrane signal transduction. Fibroblasts maintained in suspension, however, remain spherical with no apparent stress fibers or lengthy microtubules. Because of the significant differences in cytoskeletal organisation induced by shape modification, and the resulting possible changes in organization and dynamics of membrane receptors, the technique of fluorescence redistribution after photobleaching (FRAP) was employed to examine the lateral mobility of wheat germ agglutinin (WGA) and succinylated concanavalin A (sCon A) receptors in the plasma membrane of untransformed and Kirsten murine sarcoma virus-transformed Balb/c 3T3 fibroblasts in the spread and spherical state. An examination of FITC-WGA and FITC-sCon A binding to the plasma membrane for both cell lines in a spread or spherical state demonstrated no significant differences in the number of WGA or Con A receptors as a function of shape or transformation. The primary observations from this study are (a) membrane WGA and sCon A receptors in spherical Balb/c 3T3 fibroblasts display mobility 12 times faster than in the spread state, while phospholipid mobility is similar and apparently shape independent, (b) transformed cells in the spread state have WGA and sCon A receptor mobilities similar to those of untransformed cells in the spread state, (c) flat adherent, but not unattached spherical, Balb/c 3T3 fibroblasts are subject to Con A-induced global modulation and (d) transformed cells in the spherical state contain a significant population of cells (approximately 30%) with WGA receptor mobilities faster than those observed in spherical untransformed cells. These observations are discussed in terms of a linked matrix model for membrane protein diffusion.     

Effects of wheat germ agglutinin on membrane transport

(1) Low concentrations of wheat germ agglutinin are cytotoxic toward several tissue culture lines, including Chinese hamster ovary cells, Swiss 3T3 cells, mouse L cells and baby hamster kidney cells. The LD50 ranged from 1 to 5 microgram wheat germ agglutinin per ml. Similar concentrations of the lectin inhibited the transport of the non-utilizable amino acids alpha-aminoisobutyric acid and cycloleucine and inhibited the uptake of thymidine. In contrast, 2-deoxy-D-glucose uptake was not altered and colchicine uptake was enhanced. (2) The inhibition of alpha-aminoisobutyric acid uptake occurred within minutes after lectin addition and was maximal by 1 h. Maximal inhibition ranged from 50 to 70% of control values. Studies of the kinetics of the uptake demonstrated that wheat germ agglutinin decreased the V of the uptake by 70% without affecting the apparent Km. Ovomucoid, a haptene inhibitor of wheat germ agglutinin-binding to cell surface receptors, prevented the wheat germ agglutinin-induced inhibition of alpha-aminoisobutyric acid transport. Three other lectins (Concanavalin A, Phaseolus vulgaris E-phytohemagglutinin and L-phytohemagglutinin) inhibited the uptake by 20% or less at doses up to 50 microgram/ml. (3) We propose that the cytotoxicity of wheat germ agglutinin probably results in part, if not totally, from membrane alterations which impair multiple membrane transport systems.     

Different locations of carbohydrate-containing sites in the surface membrane of normal and transformed mammalian cells

Summary A soybean agglutinin was found to agglutinate mouse, rat and human cell lines transformed by viral carcinogens, but not hamster cells transformed by viral or non-viral carcinogens. Normal cells from which the transformed cells were derived were not agglutinated by this agglutinin, but they were rendered agglutinable after short incubation with trypsin or pronase. The transformed hamster cells, on the other hand, became agglutinable only after prolonged treatment with pronase. The agglutination was specifically inhibited by N-acetyl-d-galactosamine, indicating that N-acetyl-d-galactosamine-like saccharides are part of the receptor sites for soybean agglutinin on the surface membrane. Such sites exist in a cryptic form in normal cells; they are exposed in transformed mouse, rat and human cells, but become less accessible in transformed hamster cells. The receptor sites for soybean agglutinin differ from the receptors for two other plant agglutinins (wheat germ agglutinin that interacts with N-acetyl-d-glucosamine-like sites and Concanavalin A that interacts with -d-glucopyranoside-like sites) which become exposed upon transformation of all lines tested. In normal hamster cells, the receptors for all three agglutinins become exposed after incubation with trypsin, but the exposure of N-acetyl-d-galactosamine-like sites requires the longest enzyme treatment. The results indicate a difference in the location of different carbohydrate-containing sites in the surface membrane. The differences in the exposure of carbohydrate-containing sites in the membrane could not be correlated with the levels of carbohydrate-splitting glycosidases in normal and transformed cells.     

Effect of trypsinization on lectin binding to germ cells from ICR and T/t6 mice

Flow cytometry was used to quantify the binding of fluorescein isothiocyanate (FITC)-labeled lectins to testis cells from ICR and T/t6 mice before and after trypsin treatment. Soybean agglutinin, wheat germ agglutinin, and concanavalin A bound well to testis cells of both mouse strains. Limax flavus agglutinin (LFA) bound very slightly and Ulex europeas agglutinin (UEA) did not bind at all. Trypsinization increased binding of soybean agglutinin and decreased binding of wheat germ agglutinin in both mouse strains, providing evidence for masked carbohydrate-binding sites on the surface of germ cells. It did not affect binding of the other lectins. Trypsin treatment was an attempt to increase lectin binding, particularly the binding of LFA and UEA to the reported T/t-specific carbohydrates, sialic acid, and L-fucose, respectively. These studies indicate that the T/t6 locus alleles do not alter the surface carbohydrate content of testis cells sufficiently to be detected by lectin-binding differences.     

Agglutination of Staphylococcus saprophyticus: a structural and cytochemical study

Abstract Staphylococcus saprophyticus was shown to be agglutinated by wheat germ agglutinin, wheat germ agglutinin-biotin and bovine serum albumin- p -aninophenyl- N -acetyl-β-d-glucosaminide (GlcNAc-BSA), and sheep red blood cells. In these agglutinations, filamentous or amorphous structures radiating from the surface of S. saprophyticus were demonstrated by electron microscope observation. Cytochemical analyses of the agglutination revealed the binding sites of wheat germ agglutinin in S. saprophyticus and the binding sites of GlcNAc in the sheep red blood cells and S. saprophyticus . Since GlcNAc-BSA contains N -acetylglucosamine to which wheat germ agglutinin can bind, it is most likely that an interaction between a wheat germ agglutinin-bindable substance in S. saprophyticus and an N -acetylglucosamine-bindable substance in sheep red blood cells is involved in the agglutination.     

Surface alterations in transformed epithelial and fibroblastic cells in culture: A disturbance of membrane degradation versus biosynthesis?

Fibroblasts which were transformed with oncogenic DNA or RNA viruses as well as by chemicals or spontaneously have, with very few exceptions, been shown to possess increased agglutinabilities with various plant agglutinins. This surface membrane alteration is now shown to hold true also for transformed epithelial cells and for tumors of epithelial origin as monitored by wheat germ agglutinin, Concanavalin A, phytohemagglutinin, Ricinus communis and Great Northern bean (GNB) agglutinin. An agglutinin from lentils did the opposite. It permitted the agglutination of normal liver cells but not that of hepatoma cells which led to the suggestion that some membrane glycoproteins or glycolipids may possibly be turned inside out while others are turned outside in (during the transformation process. Inhibition of protein synthesis in normal cells brought about the agglutinable membrane state in non-growing or resting cells but not in actively growing cells. Since this agglutinability could be inhibited by protease inhibitors and since proteolytic enzymes could bring about the agglutinable membrane state in growing normal cells, it was suggested that growing cells have as active a biosynthesis of membrane proteins as do non growing cells but that non-growing cells are actively catabolizing their membranes with proteases, much more so than non growing, stationary cells.     

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.     

Drosophila cell fusion induced by wheat germ agglutinin.

The effects of wheat germ agglutinin on Drosophila embryonic cell lines growing on cover-glasses was examined by scanning electron microscopy. At low concentrations of the lectin (5-10 mug/ml), cells spread against the glass surface and fused to form syncytia. At high concentration, damage to the cell surface was evidenced as extensive membrane shrivelling and loss of surface microfilaments. Fusion also occurred under these conditions. There was some indication that the morphology of cells in division remains undisturbed by wheat germ agglutinin. The coalescence of cells and morphologic disotrtion induced by wheat germ agglutinin were not inhibited by N-acetylglucosamine, the hapten inhibitor of the lectin, under the conditions utilized in this study.     

Requirement of bromodeoxyuridine for the maintenance of “transformed” characteristics in bromodeoxyuridine dependent cells

A bromodeoxyuridine (BUdr) dependent cell line, called B4, which requires BUdr not only for optimal growth but also for the maintenance of the non-contact inhibited state was described previously. We have now shown that contact inhibition in the B4 cells in the absence of BUdr is associated with a marked decrease in the percent of cells synthesizing DNA. The transition to the contact inhibited state in the absence of BUdr does not seem to be due to changes in cyclic AMP levels. It has also been shown that several but not all of the characteristics which distinguish transformed from untransformed cells also distinguish B4 cells grown in the presence of BUdr from B4 cells grown in the absence of BUdr. In addition to being contact inhibited, B4 cells grown in the absence of BUdr have a higher serum requirement, grow less well in soft agar, and are less agglutinable by wheat germ agglutinin than B4 cells grown in the presence of BUdr. Agglutinability by concanavalin A, however, is the same for B4 cells grown in the presence and absence of BUdr. Dependent cells maintained in the presence of BUdr do not form tumors and it is not yet clear how the transformed characteristics of the dependent cells are related to malignancy.