Immunological detection of cell surface components related with aggregation of Chinese hamster and chick embryonic cells.

Previous studies suggested that Chinese hamster V79 cells possess two mechanisms for their mutual adhesion, Ca²⁺-dependent and Ca²⁺-independent ones. We could prepare cells with only the Ca²⁺-dependent mechanism intact by dispersing cell monolayers with trypsin (0.01%) containing Ca²⁺. In the present study, we found that cells dispersed with a very low concentration of trypsin (0.0001%) in the absence of Ca²⁺ retain only the Ca²⁺-independent mechanism intact. Fab fragments of antibodies directed against surface antigens of V79 cells inhibited the aggregation of V79 cells by the Ca²⁺-independent mechanism, but did not inhibit the aggregation of these cells by the Ca²⁺-dependent mechanism. These results suggest that the two mechanisms of cell adhesion are based on different cellular components. Molecules responsible for the Ca²⁺-independent adhesion mechanism are probably cell surface components, because they were released from cells by the treatment with 0.01% trypsin without losing their specific antigenicity. The presence of adhesion mechanisms similar to those in V79 cells was shown in neural retinal cells of chick embryos. It was assumed, therefore, that these mechanisms of cell adhesion are generally present among a variety of cell types.     

Differential inhibition of myoblast fusion.

The aggregation and fusion of myoblasts in the presence of either metabolic inhibitors or alterations in the incubation medium or under conditions which result in structural changes in the cells was studied using previously described assays for the intercellular interactions of myoblasts in suspension [Knudsen, K. A., and Horwitz, A. F. (1977). Develop. Biol.58, 328]. These perturbations inhibit myoblast fusion differently. For example, energy poisons, prior trypsin or glutaraldehyde treatment, and inhibitors of protein or cholesterol synthesis all inhibit the Ca²⁺-mediated myoblast aggregation. In contrast, whereas myoblasts aggregate in the presence of 20 mM Mg²⁺, these aggregates are dispersed, even after 1–2 hr, with EDTA or trypsin. Furthermore, enriching the fatty acyl chains in elaidate or prior incubation of the myoblasts in the presence of cytochalasin B or colchicine results in aggregates which, after 1–2 hr, are dispersed by trypsin but not by EDTA. Aggregates of unaltered, control myoblasts, on the other hand, begin to show resistance to dispersion by trypsin after these times. These observations support the suggestion that multinucleate cell formation results from a sequence of events. The influence of these perturbations on cellular aggregation also provides some initial, tentative insight into the molecular mechanism of myoblast fusion. Recognition (calcium-mediated aggregate formation) appears to be mediated by a protein(s) that is turning over during the period of fusion competence, while membrane union (formation of aggregates resistant to dispersion by trypsin) most likely involves the direct participation of membrane lipid.     

Effect of protease and protease inhibitors on the adhesion of Ehrlich ascites tumour cells to plastic

The adhesion of Ehrlich ascites tumour cells to plastic was inhibited by the addition to the incubation medium of either of two chemically unrelated trypsin inhibitors at non-toxic concentrations. Supplementing the medium with low concentrations of trypsin enhanced adhesion. These findings are compatible with the existence of intrinsic proteolytic activity at the surface of the tumour cell and support the view that such a protease would affect the adhesive characteristics of the cell.     

Requirements for the Ca2+-independent component in the initial intercellular adhesion of C2 myoblasts

Using a sensitive and quantitative adhesion assay, we have studied the initial stages of the intercellular adhesion of the C2 mouse myoblast line. After dissociation in low levels of trypsin in EDTA, C2 cells can rapidly reaggregate by Ca2+-independent mechanisms to form large multicellular aggregates. If cells are allowed to recover from dissociation by incubation in defined media, this adhesive system is augmented by a Ca2+-dependent mechanism with maximum recovery seen after 4 h incubation. The Ca2+-independent adhesion system is inhibited by preincubation of cell monolayers with cycloheximide before dissociation. Aggregation is also reduced after exposure to monensin, implicating a role for surface-translocated glycoproteins in this mechanism of adhesion. In coaggregation experiments using C2 myoblasts and 3T3 fibroblasts in which the Ca2+-dependent adhesion system was inactivated, no adhesive specificity between the two cell types was seen. Although synthetic peptides containing the RGD sequence are known to inhibit cell-substratum adhesion in various cell types, incubation of C2 myoblasts with the integrin-binding tetrapeptide, RGDS, greatly stimulated the Ca2+-independent aggregation of these cells while control analogs had no effect. These results show that a Ca2+- independent mechanism alone is sufficient to allow for the rapid formation of multicellular aggregates in a mouse myoblast line, and that many of the requirements and perturbants of the Ca2+-independent system of intercellular myoblast adhesion are similar to those of the Ca2+-dependent adhesion mechanisms.     

Effect of dimethyladipimidate and dimethylsuberimidate on cell-cell adhesion in rat fibroblasts

In a study performed to identify the molecular mechanisms which regulate cell to cell adhesion and contact inhibition in neoplastic and syngeneic normal cells of the rat we have observed that the adhesive capacity depends on the reagents used, either EDTA or trypsin, to release the cells from monolayer. Taking profit of this last property and of the possibility of blocking free -NH2 groups on membrane proteins with specific cross-linking reagents "in vitro", we have studied in this work the behaviour of the proteins of the cell coat involved in cell to cell adhesion of rat fibroblasts FG/2. The cross-linking reagents used were dimethyladipimidate (DMA) and dimethylsuberimidate (DMS). The cells were exposed to the reagents at 0 degrees C for 30'. Cell to cell adhesion was measured by determining the percentage of single cells labeled with 3H-leucine, adhering to a confluent monolayer at different incubation times. The inhibitory effect on cell to cell adhesion brought about by cross-linking reagents indicates that a) EDTA-released cells are more sensitive to both imides than those released with trypsin, b) DMA is more effective on trypsin-released cells and c) DMS is more effective on EDTA-released cells. Therefore, we conclude that the inhibition of adhesion by reaction with the two cross-linking reagents is more likely due to a stiffening of the molecules of the cell coat involved in the adhesion, rather than to the modification of -NH2 residues which should specifically participate to adhesive process.     

Evidence for Distinct Aggregation Factors and Receptors in Cells

SINCE the original observations by Wilson¹ that dissociated sponge cells could reassociate in vitro, cell aggregation (or reaggregation) has been widely used as an operational criterion for the study of intercellular adhesion². The introduction of rotation-mediated methods to promote cell aggregation^3,4 led to the possibility of obtaining reproducible quantitative data. In these methods, suspensions of dissociated single cells are shaken under defined conditions of speed and temperature and cell aggregation is measured by either the size of aggregates or the number of single cells. The aggregation of dissociated cells from sponges⁵, chick and mouse embryos⁴ and tissue culture cells⁶ has been investigated with this method. Cells maintained in vitro seemed particularly suitable for studying mechanisms of cell aggregation as they represent a histotypically homogeneous population.     

Two distinct adhesion mechanisms in embryonic neural retina cells. II. An immunological analysis

Antibodies were raised against neural retina cells prepared by dissociation in EGTA alone (E cells, Ca²⁺-independent aggregation), in trypsin + Ca²⁺ (TC cells, Ca²⁺-dependent aggregation), or in trypsin + EGTA (TE cells, nonadhesive). Anti-E-cell Fab selectively inhibited Ca²⁺-independent aggregation, anti-TC-cell Fab selectively inhibited Ca²⁺-dependent aggregation, and anti-TE-cell Fab inhibited neither. Fab from a fourth preparation, also raised against E cells, inhibited both Ca²⁺-independent and Ca²⁺-dependent aggregation but was separated by immunoadsorption into two fractions, one specific for each mode of aggregation. In cells which utilize both modes simultaneously (LTC cells), each was inhibited exclusively by the appropriate Fab. The immunological data presented here demonstrate the existence in the same cells of two distinct and functionally independent adhesion mechanisms, each responsible for one of the two modes of aggregation. The differing adhesive properties of retinal cells prepared by different procedures are explained by the presence, absence, or degree of activity of these two mechanisms, qualities regulated by the concentrations of trypsin and Ca²⁺ used in the tissue dissociation.     

A STUDY ON THE MECHANISM OF INTERCELLULAR ADHESION : Effects of Neuraminidase, Calcium, and Trypsin on the Aggregation of Suspended HeLa Cells

Aggregation of suspended HeLa cells is increased on removal of cell surface sialic acid. Calcium ions promote aggregation whereas magnesium ions have no effect. The calcium effect is abolished by previous treatment of the cells with neuraminidase. Trypsinization of the HeLa cells followed by thorough washing diminishes the rate of mutual cell aggregation. Subsequent incubation with neuraminidase restores the aggregation rate to the original value before trypsin treatment. Cells which had acquired a greater tendency for aggregation after removal of peripheral sialic acid lose this property when subsequently treated with trypsin. Calcium ions have no aggregative effect on trypsinized cells. In contrast to HeLa cells, aggregation of human erythrocytes was not increased after treatment with neuraminidase or on addition of calcium. The results with HeLa cells are interpreted as follows: (a) Trypsin-releasable material confers adhesiveness upon the cells. (b) The adhesive property of this material is counteracted by the presence of cell surface sialic acids. (c) Calcium ions exert their effect by attenuating the adverse effect of sialic acid.     

Cleavage stage mouse embryos share a common cell adhesion system with teratocarcinoma cells

We examined similarities in adhesive properties of mouse cleaving embryos at one- to eight-cell stages and of teratocarcinoma cells by aggregation studies. Teratocarcinoma cells and fibroblastic cells have a Ca²⁺-dependent cell-cell adhesion site (CDS), which is resistant to trypsin in the presence of Ca²⁺ but sensitive in the absence of Ca²⁺. When several embryos treated with trypsin in the presence of Ca²⁺ (TC) were kept in contact with each other, they fused into a single aggregate in the medium with Ca²⁺ but not without Ca²⁺. Embryos treated with trypsin in the absence of Ca²⁺ (TE) did not show such Ca²⁺-dependent aggregation. Aggregation of TC-treated embryos was inhibited by Fab fragments of antibody raised against TC-treated teratocarcinoma F9 cells. The aggregation-inhibitory effect of the Fab was removed by absorption with TC-treated teratocarcinoma cells, but not with TE-treated teratocarcinoma cells. This effect was not removed by absorption with fibroblasts and some other tissue cells. TC-treated embryos adhered to TC-treated teratocarcinoma cells, but not to TC-treated fibroblastic cells. These results suggest that early mouse embryos share a common CDS molecule with teratocarcinoma cells but not with fibroblastic cells.     

Calcium-dependent adhesion of Drosophila embryonic cells

Summary By using an in vitro functional assay, we have shown that Drosophila embryonic cells possess Ca²⁺-dependent adhesive sites, which resemble in many respects those described for vertebrate cells and tissues. The cells, obtained by mechanical disruption of gastrulastage embryos, form aggregates within 30 min when maintained under constant rolling. The aggregation is completely dependent on the presence of Ca²⁺ in the medium. In its absence, the cells remain dispersed but the process is reversible by readdition of Ca²⁺. In addition the aggregation is temperature-dependent. No aggregation occurs at 4° C but it can be restored by raising the temperature to 25° C. These properties are characteristic of these cells: established cell lines do not aggregate under the same conditions and mixing of cell lines and embryonic cells does not result in chimeric aggregates, thus pointing towards cell-type selectivity with respect to aggregability. Observations in electron microscopy have shown that the embryonic cells in the aggregates tightly adhere to one another and form, as early as after 30 min, maculae adherens junctions. Drosophila embryonic cells have adhesion sites that are protected from trypsin proteolysis in the presence of Ca²⁺ and sensitive in its absence. The cells' aggregation can be inhibited by a mouse antiserum directed against cell-surface components and a good correlation exists between neutralization of the inhibitory activity of the antiserum and the presence of trypsin-sensitive sites on the cells. These data are in favour of cell-cell adhesion mediated by specific adhesion proteins.     

Transport and metabolism of glucose by dissociated brain cells: Effects of trypsin

A study was carried out to determine the effect of trypsin on glucose transport into brain cells. Two suspensions of dissociated cells were prepared from the two brain hemispheres of adult rats—one using only mechanical means to dissociate the cells and one using trypsin. The use of trypsin for preparation of dissociated brain cells caused a marked reduction in the rate of transport of [1,2-³H]-2-deoxy-d-glucose compared to uptakes of this glucose analog by cells prepared without trypsin. Responses of the two cell preparations to inhibitors of glucose transport (cytochalasin B and phloretin) were similar. Rates of oxidation of [6-¹⁴C]glucose to¹⁴CO₂ by trypsin-treated cells were nearly double those in cells prepared without trypsin. Electron microscopic examination of the two preparations revealed much less preservation of structural integrity if trypsin was used to prepare the cells. The findings suggest that trypsin alters cell structure and affects receptor-regulated events in brain cells.     

The Multistep Process of Homotypic Neutrophil Aggregation: A Review of the Molecules and Effects of Hydrodynamics

Homotypic adhesion of neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes are supported by L-selectin and β₂-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand the hydrodynamic stresses. Using cone and plate viscometry to apply a uniform linear shear field to suspensions of neutrophils and flow cytometry to quantitate the size distribution of aggregates formed over the time course of formyl peptide stimulation, we conducted a detailed examination of the affect of shear rate and shear stress on the kinetics of cell aggregation. The efficiency of aggregate formation was fit from a mathematical model based on Smoluchowski's two-body collision theory. Over a range of venular shear rates (400–800 s^-1), β90% of the single cells are recruited into aggregates ranging from doublets to groupings larger than sextuplets. Adhesion efficiency fit to the kinetics of aggregation increased with shear rate from β20% at 100s^-1 to a maximum level of β80% at 400 s^-1. This increase to peak adhesion efficiency was dependent on L-selectin and β₂-integrin, and was resistant to shear stress up to β7 dyn/cm². When L-selectin was blocked with antibody, β₂-integrin (CD11a, b) supported adhesion at low shear rates (< 400 s^-1). Aggregates formed over the rapid phase of aggregation remain intact and resistant to shear up to 120 s. At the end of this plateau phase of stability, aggregates spontaneously dissociate back to singlets. The rate of cell disaggregation is linearly proportional to the applied shear rate. The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to firm but reversible adhesion.     

Removal of sialic acid from the surface of human MCF-7 mammary cancer cells abolishes E-cadherin-dependent cell-cell adhesion in an aggregation assay

Summary MCF-7 human breast cancer cells express E-cadherin and show, at least in some circumstances, E-cadherin-dependent cell-cell adhesion (Bracke et al., 1993). The MCF-7/AZ variant spontaneously displays E-cadherin-dependent fast aggregation; in the MCF-7/6 variant, E-cadherin appeared not to be spontaneously functional in the conditions of the fast aggregation assay, but function could be induced by incubation of the suspended cells in the presence of insulinlike growth factor I (IGF-I) (Bracke et al., 1993). E-cadherin from MCF-7 cells was shown to contain sialic acid. Treatment with neuraminidase was shown to remove this sialic acid, as well as most of the sialic acid present at the cell surface. Applied to MCF-7/AZ, and MCF-7/6 cells, pretreatment with neuraminidase abolished spontaneous as well as IGF-I induced, E-cadherin-dependent fast cell-cell adhesion of cells in suspension, as measured in the fast aggregation assay. Treatment with neuraminidase did not, however, inhibit the possibly different, but equally E-cadherin-mediated, process of cell-cell adhesion of MCF-7 cells on a flat plastic substrate as assessed by determining the percentage of cells remaining isolated (without contact with other cells) 24 h after plating.     

Calcium-Dependent and Calcium-Independent Adhesive Mechanisms Are Present During Initial Binding Events of Neural Retina Cells

The hypothesis that intercellular adhesion can be subdivided into two separable phenomena, an initial recognition event and a subsequent stabilization, is supported by the use of a new cell binding assay that provides a quantitative measure of intercellular binding strengths. Radioactive single cells are brought into contact with cell monolayers at 4°C in sealed compartments. The compartments are inverted and a centrifugal force is then applied tending to dislodge the probe cells from the monolayers. By varying the speed of centrifugation, the force maintaining associations between embryonic chick neural retina cells was determined to be on the order of 10^?5 dynes after incubation at 4°C. Brief incubations at 37°C resulted in significant strengthening of the intercellular bond. Using this cell binding assay, neural retina cells were shown to exhibit both a Ca⁺⁺-independent and a Ca⁺⁺-dependent mechanism in their initial binding to one another.     

STUDIES ON THE KINETICS OF INITIAL CYCLE PROGRESSION IN VITRO OF ASCITES TUMOUR CELLS SUBSEQUENT TO ISOLATION FROM ASCITES FLUID

A method is described for determining the duration of cell cycle phases traversed by cells responding to release from proliferation restraint. Experiments have been performed with arrested Yoshida ascites hepatoma cells allowed to re-enter the growing stage after transfer of cells from the late stage of ascites into an in vitro incubation system. Experimentally, this method requires information on the rate of incorporation of labelled thymidine and on the rate of increase in cell number. The rate of [¹⁴C]thymidine incorporation in vitro was shown to be directly proportional to the number of cells synthesizing DNA. This was shown by correlating data from measurements of the rate of thymidine incorporation with those from measurements of the labelling index of the cell population. Theoretically, the method is based on analysis of the region limited by two integral curves, one corresponding to the kinetics of cell entry into and the other to the kinetics of exit from the S-phase. From data on the actual rate of increase in the total number of cells and data on the S-phase duration it is possible to obtain information on the cytokinetics of growth resumption by the ascites cell population.     

Intercellular adhesion in butyrate-treated HeLa S3 cultures : Flow cytometric analysis

The application of DNA flow cytometry (FCM) for analysis of sodium butyrate-induced intercellular adhesion in human carcinoma (HeLa S3) cell cultures is described. To prepare cell suspensions for FCM, the monolayers of cells were treated with medium containing 10% serum, 0.2% non-ionic detergent Triton X-100 and 1 μg/ml DNA fluorochrome 4,6′-diamidino-2-phenylindole (DAPI). Total numbers of single cells, and aggregates containing two, three, four or more cells, were determined from DNA histograms. In cultures treated with 5 mM butyrate for 16 h, more than 80% of the cells were aggregated. Intercellular adhesion began to appear 8 h after addition of butyrate, was maximal at 16–24 h and stable in the presence of butyrate, but disappeared 24 h after its removal. Treatment with EDTA (0.2%) dissociated only 50%, whereas trypsin (0.1%) separated all cell aggregates into single cells. Actinomycin D (actD) (0.5 μg/ml) prevented cell adhesion while blocking of cells in S phase with 250 μM 5-fluorouracil or 10 μM methotrexate did not interfere with aggregation. The number of cell aggregates estimated from DNA histograms of butyrate-treated HeLa S3 cultures was the same after staining with DAPI in the presence of Triton X-100 or after vital staining with Hoechst 33342. The DNA content was used as a marker to estimate the cellular composition of aggregates in mixed cultures of HeLa S3 cells and human fibroblasts (U cells). Intercellular adhesion in these cultures was seen only between HeLa S3 cells, indicating specificity of butyrate-induced cell aggregation. FCM provides fast automatic measurement of cell aggregate formation, estimates frequency of aggregates containing different cell numbers, shows participation of cells at different cycle phases in aggregates, and allows the detection of homotypic from heterotypic cell aggregates if the interacting cells have different DNA ploidy.     

Preparation of rat liver cells : III. Enzymatic requirements for tissue dispersion

Isolated perfused rat livers were dispersed by a two-step procedure of Ca²⁺ removal (probably including the removal of a Ca²⁺-dependent adhesion factor) followed by enzymatic treatment. Collagenase, crude or purified, converted all of the parenchymal tissue to a suspension of cells which were 95% intact. Other enzymes such as hyaluronidase, lysozyme and trypsin were ineffective. Since hypoxic conditions during enzymatic treatment did not affect cellular viability, the oxygenation apparatus can be omitted to facilitate sterile preparation of cells.Purification of the parenchymal cells by differential centrifugation reduced contamination by non-parenchymal cells from 10–20% initially to 1–2% finally, with 40% of the parenchymal cells recovered and no loss in viability. The purified cells were 20% binucleated and had the same biochemical composition as intact liver tissue. The cells were active in the synthesis of RNA, protein, glycogen and various metabolites, and showed sensitivity towards steroid and polypeptide hormones. Many of the freshly isolated cells had marked constriction furrows which disappeared upon incubation.     

The interaction of a range of serine proteinase inhibitors with bovine trypsin and Costelytra zealandica trypsin

Kinetic and other properties of the interaction between two serine proteinases, bovine trypsin and Costelytra zealandica (grass grub) larval trypsin and a range of proteinaceous serine proteinase inhibitors were investigated. Twenty-six inhibitors or isoinhibitors from 10 different inhibitor families were analysed. A 1700-fold range in equilibrium dissociation constant (K_d) values was obtained for bovine trypsin and a 10⁵-fold range for grass grub trypsin. The ratios of K_d (grass grub)/K_d (bovine) also spanned a range of 10⁵-fold. Qualitative observations indicated that the second order association rate constants were high for all except two inhibitors. Two classes of first order dissociation rate constant were determined from the dissociation of trypsin-inhibitor complexes induced by substrate. While most inhibitors were cleaved by grass grub trypsin, they still inhibited larval midgut crude extracts during long incubations. We suggest using the K_d value to assess the potential for any inhibitor to act as a grass grub larval resistance factor in plants, in preference to other parameters.     

N-linked oligosaccharides are not involved in the function of a cell-cell binding glycoprotein E-cadherin

E-cadherin is a Ca2+-dependent cell-cell adhesion molecule identified as a glycoprotein with a molecular weight (MW) of 124,000. To study the role of the sugar moieties of this adhesion molecule, we tested the effect of tunicamycin on aggregation mediated by E-cadherin of teratocarcinoma cells. Immunoblot analysis using a monoclonal antibody to E-cadherin showed that in cells treated with tunicamycin this adhesion molecule is converted into two forms with MW of 118,000 and 131,000. The smaller one was exposed on the cell surface and showed a trypsin sensitivity characteristic to E-cadherin, suggesting that this is the peptide moiety of E-cadherin whose glycosylation with N-linked oligosaccharides was blocked by tunicamycin. The larger one was not removed by trypsin treatment of cells, suggesting an intracellular location. These tunicamycin-treated cells aggregated in a Ca2+-dependent manner, and the aggregation was inhibited by a monoclonal antibody to E-cadherin. These results suggested that N-linked oligosaccharides are not involved in the functional sites of this adhesion molecule.     

Two distinct adhesion mechanisms in embryonic neural retina cells. I. A kinetic analysis

The reaggregation kinetics of embryonic chick neural retina cells prepared using several different dissociation procedures were monitored through decreases in the small-angle light scattering of aggregating samples. Two distinct modes of aggregation were revealed, one Ca²⁺ independent, the other Ca²⁺ dependent, suggesting the existence of two separate adhesion mechanisms. By varying the concentrations of Ca²⁺ and trypsin in the dissociation medium, we obtained cells which exhibited both, either, or neither mode of aggregation. The Ca²⁺-independent adhesiveness is active in the absence of proteolysis, is resistant to low levels of trypsin (0.001%), but is readily inactivated at higher trypsin concentrations in either the presence or absence of Ca²⁺. It is relatively temperature independent. By contrast, the Ca²⁺-dependent adhesiveness is not detected before exposure of the cells to proteolysis. It is expressed after tryptic proteolysis in the presence of Ca²⁺ and is then highly temperature dependent. It is resistant to further digestion by trypsin in the continued presence of Ca²⁺ but is lost when Ca²⁺ is subsequently removed, apparently through the expression of tryptic cleavage incurred earlier. We suggest that its increased activity may result at least in part from the clustering of surface components into adhesive patches. A provisional model is presented correlating these data.