Interactions of concanavalin A with chick embryo fibroblasts transformed by Rous sarcoma virus. Study with an RSV mutant thermosensitive for transformation.

The interactions between concanavalin A and chick embryo fibroblasts, normal and infected with Rous sarcoma virus (RSV-BH) or its thermosensitive mutant RSV-BH-Ta, have been studied. Normal chick embryo cells and RSV-BH transformed cells showed at 4 and 25 degrees C a similar number of concanavalin A receptors per cell. Analysis of the binding data by the Scatchard relation showed that apparent changes in binding as a function of temperature are due to the thermodynamic properties of the process and not to endocytosis. The lectin receptors on the cell surface of normal and RSV-BH infected cells showed homogeneity in their binding properties. Chick cells infected with RSV-BH-Ta showed a lectin binding behavior that was dependent on the temperature at which the cells were grown. At the permissive temperature for transformation (37 degrees C), the binding process was similar to that observed for normal and RSV-BH infected cells. At the nonpermissive temperature (41 degrees C), the cells showed at least two sets of concanavalin A receptors. The new set of receptors on the cell surface had a lower lectin affinity than those observed in the same cells at 37 degrees C. Chick cells infected with RSV-BH showed an enhanced agglutinability by concanavalin A, as compared with normal cells. Cells infected with RSV-BH-Ta showed a reversal of the correlation between increased concanavalin A agglutinability and the transformed state. At the permissive temperature for transformation, the cells were not agglutinable, whereas at the nonpermissive temperature they presented agglutinability indexes as high as those observed with RSV-BH infected cells. This enhanced agglutinability observed with cells maintained at the nonpermissive temperature for transformation may be related to the new set of low affinity receptors present at 41 degrees C.     

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     

Studies on the iodinated surface membrane proteins and concanavalin A agglutination of transformed Syrian hamster cells.

Chemically transformed Syrian hamster cells exhibit marked agglutination in the presence of the plant lectin, concanavalin A. In this report, we describe conditions which can alter this concanavalin A agglutinability, and compare the surface proteins from transformed cells which express different degrees of agglutinability. Lactoperoxidase-catalyzed iodination of tertiary Syrian hamster cells reveals the major iodinatable protein to be approximately 220 000 daltons. The transformed Syrian hamster cells do not contain this protein in an iodinatable form. Analyses of the transformed cells grown under conditions which decrease the concanavalin A agglutinability do not demonstrate any iodination of the 220 000 mol. wt. protein. These results depict the effects of growth and dibutyryl cyclic AMP on the iodinatable cell surface proteins of transformed cells and indicate that the absence of the I-220 000 mol. wt. protein is probably not a major determinant of concanavalin A agglutination.     

Studies on the iodinated surface membrane proteins and concanavalin A agglutination of transformed Syrian hamster cells

Chemically transformed Syrian hamster cells exhibit marked agglutination in the presence of the plant lectin, concanavalin A. In this report, we describe conditions which can alter this concanavalin A agglutinability, and compare the surface proteins from transformed cells which express different degrees of agglutinability. Lactoperoxidase-catalyzed iodination of tertiary Syrian hamster cells reveals the major iodinatable protein to be approximately 220 000 daltons. The transformed Syrian hamster cells do not contain this protein in an iodinatable form. Analyses of the transformed cells grown under conditions which decrease the concanavalin A agglutinability do not demonstrate any iodination of the 220 000 mol. wt. protein. These results depict the effects of growth and dibutyryl cyclic AMP on the iodinatable cell surface proteins of transformed cells and indicate that the absence of the 1–220 000 mol. wt. protein is probably not a major determinant of concanavalin A agglutination.     

Interactions of concanavalin A with chick embryo fibroblasts transformed by rous sarcoma virus Study with an RSV mutant thermosensitive for transformation

The interactions between concanvalin A and chick embryo fibroblasts, normal and infected with Rous sarcoma virus (RSV-BH) or its thermosensitive mutant RSV-BH-Ta, have been studied. Normal chick embryo cells and RSV-BH transformed cells showed at 4 and 25 °C a similar number of concanavalin A receptors per cell. Analysis of the binding data by the Scatchard relation showed that apparent changes in binding as a function of temperature are due to the thermodynamic properties of the process and and not to endocytosis. The lectin receptors on the cell surface of normal and RSV-BH infected cells showed homogeneity in their binding properties. Chick cells infected with RSV-BH-Ta showed a lectin binding behavior that was dependent on the temperature at which the cells were grown. At the permissive temperature for transformation (37 °C), the binding process was similar to that observed for normal and RSV-BH infected cells. At the nonpermissive temperature (41 °C), the cells showed at least two sets of concanavalin A receptors. The new set of receptors on the cell surface had a lower lectin affinity than those observed in the same cells at 37 °C.Chick cells infected with RSV-BH showed an enhanced agglutinability by concanavalin A, as compared with normal cells. Cells infected with RSV-BH-Ta showed a reversal of the correlation between increased concanavalin A agglutinability and the transformed state. At the permissive temperature for transformation, the cells were not agglutinable, whereas at the nonpermissive temperature they presented agglutinability indexes as high as those observed with RSV-BH infected cells. This enhanced agglutinability observed with cells maintained at the nonpermissive temperature for transformation may be related to the new set of low affinity receptors present at 41 °C.     

Lectin-induced differentiation of transformed neuroretinal cells in vitro.

The orderly course of chick neuroretinal cell differentiation was disrupted in vitro by infection with a temperature-sensitive strain of the Rous sarcoma virus (LA29). The resulting cell culture LA29NR remained mitotically active at 42 degrees C, yet rapidly adopted a transformed phenotype upon activation of the pp60v-src oncogene product at 37 degrees C. As a further indication of metabolic state, LA29NR cells expressed the protooncogene product c-Fos, as shown by Western blot analysis. Highly proliferative LA29NR cells proved refractory to standard differentiation agents such as cAMP, and prostaglandin E1. In our novel approach, succinylated concanavalin A (SCA), a nontoxic derivative of the lectin concanavalin A, induced dramatic, reversible morphological changes in LA29NR cells, including neurite outgrowth and increased cell-to-cell adhesion. Fluoresceinated SCA appeared to localize to Golgi and lysosomal structures. Cellular response to SCA treatment included decreased growth rate, reversible decrease in the phosphorylation state of a 41-kDa phosphoprotein, and induction of neuron-specific enolase. The glial marker vimentin was also evident in these cultures. These data suggest that SCA is an effective differentiation agent for cells of neuroectodermal origin, permitting neuronal as well as glial phenotypic expression within these cell populations.     

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.     

Characterization of an SV40-transformed 3T3 cell line expressing an unusual phenotype.

A transformed variant derived as a clone from normal 3T3 cells infected with simian virus 40 (SV40) has been found to possess a phenotype intermediate between that of normal cells and that characteristic of the transformed state, yet cells of the variant still test positively for the SV40-specific nuclear T-antigen. The variant exercises growth control, although not as stringently as do normal cells. Its cell size more closely resembles that of normal cells than of transformed cells. The variant also exhibits levels of spontaneous agglutination that are in line with those characteristic of the normal cells from which it was derived, and far higher than corresponding values for cells exhibiting the fully transformed phenotype. Plasma membranes of variant cells more closely resemble those of transformed cells than of normal cells as estimated by polyacrylamide gel electrophoresis. Perhaps the most distinguishing characteristic of the transformed variant is its complete immunity to agglutination by concanavalin A (Con A), even at concentrations of the lectin as high as 500 mug/ml. Moreover, trypsinization does not render variant cells as agglutinable in the presence of Con A as are untreated fully transformed cells. By contrast the variant displays a low tolerance of Con A toxicity, as monitored by ability to grow after treatment with the lectin, and on this count resembles transformed cells. Moreover a survey of several normal cell lines has revealed that even they do not consistently show resistance to Con A toxicity. These observations indicate that Con A-mediated agglutination and inability to grow after treatment with Con A are quite independent and do not bear a cause and effect relationship.     

Selection of Revertants of Kirsten Sarcoma Virus Transformed Nonproducer BALB/3T3 Cells

Revertants of Kirsten sarcoma virus transformed nonproducer BALB/3T3 cells (KA31 cells) were isolated after exposing the transformed cells to 5-fluorodeoxyuridine at high cell density, or when suspended in methylcellulose. Revertants were also isolated by treating KA31 cells with the lectin, concanavalin A, which is manyfold more toxic to transformed cells than for normal cells. The revertants resemble BALB/3T3 cells in their morphology and growth characteristics in that they have a low saturation density, fail to grow in 1% calf serum or when suspended in methylcellulose, and cease to synthesize DNA after reaching their saturation density. Infection by murine leukemia virus rescues Kirsten sarcoma virus from only the concanavalin-A-selected variants, though all the revertants are susceptible to infection by leukemia virus. The concanavalin A revertants also become transformed after infection with murine leukemia virus. All the revertants can be transformed by Kirsten sarcoma virus but not by simian virus 40.     

Modulation of the cell cycle during reversible growth inhibition of 3T3 and SV40-3T3 cells with succinylated concanavalin A

Succinylated concanavalin A (ConA), a non-toxic, non-agglutinating derivative of the jack bean lectin ConA inhibits the growth of both normal and SV40-transformed 3T3 cells. The quantitative but not the qualitative growth inhibitory effect of succinyl-ConA can be modulated by the composition of the growth medium. Succinyl-ConA inhibited cells show a low rate of DNA synthesis and accumulate in the G0/G1 phase of the cell cycle. Upon removal of the succinyl-ConA, inhibited cells re-enter the cell cycle synchronously.     

Electrokinetic response of granulocytes to concanavalin A and succinyl-concanavalin A

Electrophoretic light scattering has been used to study the effects of concanavalin A (Con A) and succinyl-Con A on the electrophoretic mobility distribution of resident guinea-pig peritoneal eosinophils and human peripheral blood polymorphonuclear leukocytes. In both cell types, incubation with Con A (a tetrameric lectin) decreases slightly the mean mobility and increases substantially the width of the electrophoretic mobility distribution. These effects can be abolished by alpha-methyl-D-mannoside, a hapten sugar of Con A. Succinyl Con A, a dimeric derivative, was found to have no effect on the mobility distribution. These results are strikingly similar to our previous report of the response of the resident guinea-pig macrophage (19), suggesting possible parallels in the endocytic mechanisms of these cell types.     

Effects of local anesthetics on membrane properties II. Enhancement of the susceptibility of mammalian cells to agglutination by plant lectins

Treatment of untransformed mouse and hamster cells with the tertiary amine local anesthetics dibucaine, tetracaine and procaine increases their susceptibility to agglutination by low doses of the plant lectin concanavalin A. Agglutination of anesthetic-treated untransformed cells by low doses of concanavalin A is accompanied by redistribution of concanavalin A receptors on the cell surface to form patches, similar to that occurring in spontaneous agglutination of virus-transformed cells by concanavalin A. Immunofluorescence and freeze-fracture electronmicroscopic observations indicate that local anesthetics per se do not induce this redistribution of concanavalin A receptors but modify the plasma membrane so that receptor redistribution is facilitated on binding of concanavalin A to the cell surface. Fluorescence polarization measurements on the rotational freedom of the membrane-associated probe, diphenylhexatriene, indicate that local anesthetics produce a small increase in the fluidity of membrane lipids. Spontaneous agglutination of transformed cells by low doses of concanavalin A is inhibited by colchicine and vinblastine but these alkaloids have no effect on concanavalin A agglutination of anesthetic-treated cells. Evidence is presented which suggests that local anesthetics may impair membrane peripheral proteins sensitive to colchicine (microtubules) and cytochalasin-B (microfilaments). Combined treatment of untransformed 3T3 cells with colchicine and cytochalasin B mimics the effect of local anesthetics in enhancing susceptibility to agglutination by low doses of concanavalin A. A hypothesis is presented on the respective roles of colchicine-sensitive and cytochalasin B-sensitive peripheral membrane proteins in controlling the topographical distribution of lectin receptors on the cell surface.     

Effects of local anesthetics on membrane properties. II. Enhancement of the susceptibility of mammalian cells to agglutination by plant lectins.

Treatment of untransformed mouse and hamster cells with the tertiary amine local anesthetics dibucaine, tetracaine and procaine increases their susceptibility to agglutination by low doses of the plant lectin concanavalin A. Agglutination of anesthetic-treated untransformed cells by low doses of concanavalin A is accompanied by redistribution of concanavalin A receptors on the cell surface to form patches, similar to that occurring in spontaneous agglutination of virus-transformed cells by concanavalin A. Immunofluorescence and freeze-fracture electronmicroscopic observations indicate that local anesthetics per se do not induce this redistribution of concanavalin A receptors but modify the plasma membrane so that receptor redistribution is facilitated on binding of concanavalin A to the cell surface. Fluorescence polarization measurements on the rotational freedom of the membrane-associated probe, diphenylhexatriene, indicate that local anesthetics produce a small increase in the fluidity of membrane lipids. Spontaneous agglutination of transformed cells by low doses of concanavalin A is inhibited by colchicine and vinblastine but these alkaloids have no effect on concanavalin A agglutination of anesthetic-treated cells. Evidence is presented which suggests that local anesthetics may impair membrane peripheral proteins sensitive to colchicine (microtubules) and cytochalasin-B (microfilaments). Combined treatment of untransformed 3T3 cells with colchicine and cytochalasin B mimics the effect of local anesthetics in enhancing susceptibility to agglutination by low doses of concanavalin A. A hypothesis is presented on the respective roles of colchicine-sensitive and cytochalasin B-sensitive peripheral membrane proteins in controlling the topographical distribution of lectin receptors on the cell surface.     

Dynamic state of concanavalin A receptor interactions on fibroblast surfaces.

Cultured normal and transformed fibroblasts were treated "in situ" by the concanavalin A-peroxidase labelling technique. It is known that peroxidase recognizes only a fraction of the bound lectin depending on the cell type. Kinetics studies revealed that 80 to 95 percent of the peroxidase and only 10 percent of the lectin are released from the cell surface when the labelled cells were reincubated at 37 degrees C. It is shown that it is mostly the concanavalin traced by peroxidase that is released and also that the lectin and the enzyme are shed as a complex or concomitantly. Consequently, the shedding pattern of the enzyme is used to demonstrate heterogeneity in the lectin binding sites; there are two main components labelled by concanavalin and peroxidase, one which has a short period (from 6 to 16 min) and another one with a much longer one (1.3 to 3 h). It is shown that when cells are incubated at 37 degrees C after a lectin treatment, secondary binding forces occur between the lectin and cell surface components which render the lectin unavailable for inhibiting sugars. Under the same conditions, some peroxidase can still be bound and a slight agglutination can still occur.     

Dynamic state of concanavalin a receptor interactions on fibroblast surfaces

Cultured normal and transformed fibroblasts were treated “in situ” by the concanavalin A-peroxidase labelling technique. It is known that peroxidase recognizes only a fraction of the bound lect in depending on the cell type. Kinetics studies revealed that 80 to 95% of the peroxidase and only 10% of the lectin are released from the cell surface when the labelled cells were reincubated at 37 °C. It is shown that it is mostly the concanavalin traced by peroxidase that is released and also that the lectin and the enzyme are shed as a complex or concomitantly. Consequently, the shedding pattern of the enzyme is used to demonstrate heterogeneity in the lectin binding sites: there are two main components labelled by concanavalin and peroxidase, one which has a short period (from 6 to 16 min) and another one with a much longer one (1.3 to 3 h).It is shown that when cells are incubated at 37 °C after a lectin treatment, secondary binding forces occur between the lectin and cell surface components which render the lectin unavailable for inhibiting sugars. Under the same conditions, some peroxidase can still be bound and a slight agglutination can still occur.     

Modulation of endothelial cell surface glycoconjugate expression by organ-derived biomatrices

Cell surface molecules play an important role in cellular communication, migration, and adherence. Here, we show the effect of organ-derived biomatrices on endothelial cell surface glycosylation. Five different lectins (with and without neuraminidase treatment) have been used as probes in an enzyme-linked lectin assay to quantitatively detect glycoconjugates on endothelial cells (BAEC) grown on tissue culture plastic or biomatrices isolated from bovine lung, liver, and kidney. BAEC generally exhibit strong binding of concanavalin A (Con A), Ricinus communis agglutinin I (RCA-I), wheat germ agglutinin (WGA), and soybean agglutinin, and peanut agglutinin after neuraminidase pretreatment of cells (Neu-SBA and Neu-PNA), while SBA and PNA consistently bind weakly to BAEC. BAEC grown on organ-derived biomatrices exhibit significantly altered binding intensities of Con A, RCA-I, WGA, and Neu-PNA: BAEC cultured on lung- or kidney-derived biomatrices express significantly stronger binding affinities for Con A and RCA-I than BAEC grown on liver-derived biomatrix or tissue culture plastic. In contrast, BAEC binding of WGA and PNA (after treatment of cells with neuraminidase) is significantly reduced when BAEC are grown on liver- or kidney-derived biomatrix. Quantitative lectin immunogold electron microscopy reveals consistently stronger lectin binding over nuclear regions compared to junctional regions between neighboring cells. These results indicate that extracellular matrix components regulate endothelial cell surface glycoconjugate expression, which determines cellular functions, e.g., preferential adhesion of lymphocytes or metastatic tumor cells.     

Variants of hamster fibroblasts resistant to Ricinus communis toxin (ricin).

1. Variant baby-hamster kidney (BHK) cell lines were isolated that grow in the presence of high concentrations of ricin, the toxic lectin of castor beans (Ricinus communis). The variant lines were independently derived from several cultures of normal BHK cells which had been exposed to the mutagen, methyl-N-nitro-N-nitrosoguanidine, before selection by ricin. 2. The cell lines maintain a high degree of resistance to ricin after growth in lectin-free medium for prolonged periods and therefore exhibit stable phenotypes that are different from normal BHK cells. 3. A preliminary classification of the phenotypes was made. Several cell lines bind normal amounts of 125I-labelled ricin, whereas other bind the lectin poorly. 4. A loss of surface receptors for two other lectins, R. communis RCA and Axinella polyploides, which have specificities similar to ricin, was also found in some but not all of the cell lines showing decreased surface concentrations of ricin receptors. 5. The binding to the ricin-resistant cells of lectins of different sugar specificity, namely Lens culinaris lectin and concanavalin A, was similar to, or higher than, to normal BHK cells. 6. Several of the ricin-resistant cell lines were shown to be cross-resistant to the weak cytotoxicity of Phaseolus vulgaris lectin. By contrast, some cell lines were more sensitive to concanavalin A than were normal BHK cells.     

Low density cell culture of locust neurons in closed-channel microfluidic devices

Microfluidic channel systems were fabricated out of polydimethylsiloxane (PDMS) and used as culture vessels for primary culture of neurons from locust thoracic ganglia. In a biocompatibility study it was shown that cell adhesion and neuronal cell growth of locust neurons on uncoated PDMS was restricted. Coating with concanavalin A improved cell adhesion. In closed-channel microfluidic devices neurons were grown in static-bath culture conditions for more than 15 days. Cell densities of up to 20 cells/channel were not exceeded in low-density cultures but we also found optimal cell growth of single neurons inside individual channels. The first successful cultivation of insect neurons in closed-channel microfluidic devices provides a prerequisite for the development of low density neuronal networks on multi electrode arrays combined with microfluidic devices.     

Early changes in concanavalin A-stimulated lymphocytes detected by the fluorescent probe N-phenyl-1-naphthylamine

Summary The hydrophobic fluorescent cell-membrane probe N-phenyl-1-naphthylamine (NPN) is a useful investigative tool for studies of early lymphocyte activation. NPN-labelled mouse thymus cells incubated with 5 g/ml concanavalin A (Con A) for 30 min at 37° C gave a reproducible increase in mean cell-fluorescence intensity measured by microfluorimetry on 100 single cells. The dose-response curve was similar to that obtained by ³H-thymidine assay.Increased fluorescence was not observed in the presence of 10 mM -methyl mannoside, 5mM sodium azide, 10^–5 M cytochalasin B, or Ca²⁺-free culture medium.However, incubation with 10^–5 M colchicine did not alter the probe response. Fluorescence change was also shown by spleen cells from a normal mouse but not from an athymic mouse, indicating T cell dependence of the response.Comparison with other lectins showed that increased fluorescence followed incubation with phytohaemagglutinin, and the non-mitogenic wheat germ lectin, but there was no change with succinyl-Con A, and decreased fluorescence with pokeweed mitogen. Use of fluorescent-labelled lectins showed that the NPN fluorescence change did not correlate with surface receptor patching and capping. Increased phospholipid-fatty acid turnover and subsequent increased membrane fluidity with alteration of molecular polarity are suggested as likely explanations of increased NPN fluorescence.Supported by a grant from the Anti-Cancer Council of VictoriaWe are grateful to Miss R. Jenkins and Mr. R. McGready for preparations of succinyl-Con A, to Dr. H.A. Ward for helpful discussion, and to Dr. M. Hohnes of the Walter and Eliza Hall Institute for providing BALB/c.nu mice     

Metabolic stimulation of polymorphonuclear leucocytes: Effects of tetravalent and divalent concanavalin A

Summary Polymorphonuclear leucocytes (PMNL) undergo a marked activation of their oxidative metabolism upon interaction with surface-reactive soluble stimuli as well as with phagocytosable objects. To get some insight into the mechanism of this stimulation, we have compared the stimulatory activity of the tetravalent lectin concanavalin A (Con A) with that of the divalent derivative succinyl-Con A (S-Con A). Both lectins bind to the PMNL surface to the same extent. S-Con A, however, is much less efficient in stimulating the PMNL metabolism. When S-Con A-treated PMNL are further reacted with antiserum to Con A, a potentiation of the metabolic stimulation is observed. Normal serum or addition to PMNL of antiserum to Con A in the absence of lectin has no effect. Furthermore, if S-Con A is displaced from its receptors on the cell membrane with -methyl mannopyranoside, the addition of antiserum fails to cause a respiratory stimulation. These results suggest that the initial triggering of the metabolic stimulation of PMNL is in part accomplished through cross-linkage of membrane constituents.