Inhibited Cell Attachment Research Articles

Contribution of α- d-galactopyranosyl end groups to attachment of highly and low metastatic murine fibrosarcoma cells to various substrates

There are much greater numbers of cell surface terminal, non-reducing α- d-galactorpyranosyl groups in highly malignant (metastatic) cells than are found in low malignant cells derived from the same murine fibrosarcoma. We have examined the contribution of these residues to attachment of the cells to various collagens and to plastic. Removal of these carbohydrate groups with α-galactosidase or blocking them with lectins from Griffonia simplicifolia seeds or with anti-blood group B antiserum all dramatically inhibited the attachment of both the highly malignant and the low malignant cells. Following removal with the enzyme, the α- d-galactopyranosyl end groups were rapidly resynthesized. This resynthesis was inhibited by tunicamycin, an inhibitor of de novo glycoprotein synthesis. This antibiotic also impaired cell attachment and, when used in addition to treatment with α-galactosidase, it inhibited cell attachment more than did treatment with the enzyme alone. The effects of all treatments on cell attachment were greater for the highly malignant than for the low malignant cells. With the latter cells, inhibition by lectin was seen only in the absence of serum, whereas the adhesion of highly malignant cells was affected in both the presence and the absence of serum. On their surface membrane the highly malignant cells express much more than do the low malignant cells of a glycoprotein that cross-reacts immunologically with laminin. The basement membrane glycoprotein laminin promotes cell attachment to collagen, and both glycoproteins contain terminal, non-reducing α- d-galactopyranosyl groups. Attachment of cells is a requirement for the formation of a metastasis, and thus the laminin-like molecule and the α- d-galactopyranosyl end groups (whether on the laminin-related moiety or on other cell surface molecules) may both be important for expression of the most malignant phenotype.

Synthesis and effects of basement membrane components in cultured rat Schwann cells.

Schwann cells, the myelin-forming cells of the peripheral nervous system, are surrounded by a basement membrane. Whether cultured rat Schwann cells synthesize the basement membrane-specific components, laminin and collagen type IV, and whether these components influence the adhesion, morphology, and growth of these cells have been investigated. Both laminin and collagen type IV were detected in the cytoplasm of Schwann cells by immunofluorescence. After ascorbate treatment, laminin and collagen type IV were both found in an extracellular fibrillar matrix bound to the Schwann cell surface. Laminin was further localized on the Schwann cell surface by electron microscopy using gold immunolabeling. Anti-laminin IgG-labeled gold particles were scattered over the cell surface, and linear rows of particles and small aggregates were found along the cell edges and at points of contact with other cells. When added to the culture medium, laminin acted as a potent adhesion factor, stimulating Schwann cell adhesion as much as eightfold above control levels on type IV collagen. In the presence of laminin, the cells became stellate and by 24 hr had extended long, thin processes. Laminin also stimulated cell growth in a dose-dependent manner and anti-laminin IgG completely inhibited cell attachment and growth in the absence of exogenous laminin. Thus, cultured Schwann cells synthesize laminin and collagen type IV, two major components of basement membrane, and laminin may trigger Schwann cell differentiation in vivo during early stages of axon-Schwann cell interaction before myelination.

Recognition of extracellular matrix components by neonatal and adult cardiac myocytes

Recognition of extracellular matrix (ECM) components by isolated cardiac myocytes from neonatal (4–5 days postpartum) and adult rats was determined by measuring cell attachment to substrates made of ECM components. The substrates were petri dishes coated with either fibronectin, laminin, native monomers of collagen types I, II, III, IV, and V, denatured collagen, or gels containing reconstituted collagen fibers. Adult myocytes attached efficiently to laminin and type IV collagen, weakly to fibronectin, but not at all to the other types of collagen. Neonatal myocytes attached well to all types of collagen and to fibronectin and laminin. Antibodies raised against surface membranes of neonatal myocytes, adult myocytes, or adult hepatocytes were assayed for their ability to inhibit cell attachment to the various ECM substrates. Antibodies against the surface of neonatal myocytes as well as antibodies against the hepatocyte cell surface inhibited the attachment of neonatal myocytes and hepatocytes to collagen but not to fibronectin. Antibodies against the adult myocyte cell surface did not inhibit the attachment of neonatal myocytes or hepatocytes to ECM components. These results indicate the presence of binding molecules on the surface of neonatal myocytes that are involved in the recognition of collagen at a time when collagen is being secreted and formed into a three-dimensional network that attaches to the cell surface of the myocytes. This recognition and adhesion to collagen occurs by a mechanism independent of fibronectin. The binding molecules for collagen could not be detected on normal adult myocytes isolated at a time when the formation of the collagen network has already been completed.

Fibroblast spreading and phagocytosis: similar cell responses to different-sized substrata.

Experiments were carried out to test the hypothesis that cell spreading and phagocytosis are similar cell responses to different-sized substrata. The following morphological and biochemical studies provided evidence for this supposition. Cells phagocytosed 1.09-micron and 5.7-micron latex beads, but were unable to completely ingest 15.8-micron or 25.7-micron beads. With the larger beads, the cells spread around the bead surfaces with an appearance typical of cells spread on culture dishes. Biochemical studies with cytochalasin D, azide, and iodoacetate, as well as temperature-dependence studies, demonstrated similar responses of cell spreading and phagocytosis to these treatments. Similar cell surface receptors were involved in cell spreading and phagocytosis based upon experiments using antibodies to baby hamster kidney (BHK) cell wheat germ agglutinin receptors. And finally, BHK cell variants with defective plasma fibronectin (pFN) receptors were unable to spread on pFN-coated dishes or ingest pFN-coated beads. Evidence also is presented concerning the "contact" process in cell adhesion. It was found that azide and low temperature inhibited cell attachment per se but did not block fibronectin-receptor interactions based upon cell binding of pFN-coated beads. A possible explanation for the contact process is presented based upon the resistance of cells and beads to shear forces.

Substrate adhesion of rat hepatocytes: on the mechanism of attachment to fibronectin.

We examined the mechanisms of cell attachment to fibronectin-coated substrates. Inhibition of cell attachment was obtained by species-specific antifibronectin antibodies, which presumably recognize a distinct antigenic structure in the protein located at, or in the immediate vicinity of, the cell-binding site. The inhibiting antibodies could be adsorbed on a column of Sepharose substituted with plasma fibronectin. The initial phase of cell attachment was also inhibited by addition of soluble fibronectin to the incubation medium in a reaction that exhibited specificity and concentration dependence. These data suggest that cell-binding sites are available in an active form on the surface of soluble fibronectin. However, the inhibitory effect of fibronectin was greatly enhanced by adding the protein together with heparin, heparan sulfate, collagen, or a fibronectin-binding collagen peptide (CB-7), which is consistent with an "activation" of fibronectin on binding to these matrix components. A similar activation of fibronectin was obtained by cleaving the protein with trypsin. We discuss these findings in relation to conformational rearrangements in the fibronectin molecule. Data is presented supporting a mechanism of cell attachment to fibronectin involving multiple weak interactions between cellular receptors and substrate molecules, although some steps in the attachment process appear to disobey the requirements for such a mechanism.

Liposomes inhibit intercellular attachment.

Phosphatidylcholine liposomes bound to the surface of L cells inhibit cell attachment to L-cell monolayers or to lipid films. Aggregation of L cells or of mouse embryo fibroblasts is also diminished upon treatment with liposomes. However, they neither inhibit cell attachment to glass or cellulose acetate substrata, nor diminish conA-mediated cell aggregation. It is supposed that liposome-binding sites on the cell surface described earlier are involved in cell-cell attachment.

Laminin inhibits the adhesion of a murine tumor of macrophage origin

The adhesive behavior of the M5076 reticulum cell sarcoma, a highly metastatic murine tumor of macrophage origin, was investigated in vitro both in the presence and in the absence of purified exogeneous laminin. Although laminin enhanced the adhesion of other murine cell lines to collagen-coated and plastic surfaces, it reduced the attachment to both substrates of M5076 cells, peritoneal macrophages and the macrophage cell line WEHI-3. Thus, the inhibition appeared to be related to the macrophage nature of the M5076 tumor. The effects of laminin were reversible and did not cause loss of viability or functional capacity of the M5076 cells. Laminin appeared to exert this inhibitory effect by binding to the substrates rather than binding to the cells. These studies indicate that laminin, a glycoprotein from the basement membrane, may either stimulate or inhibit cell attachment, depending on the type of cell.

Enhancement of cell attachment of a substrate coated with oral bacterial endotoxin by plasma fibronectin.

Normal human fibroblasts were inhibited from attaching to a culture surface that was coated with endotoxic lipopolysaccharide (LPS) isolated from oral strains of Bacteroides or Fusobacterlum. The LPS from Fusobacterium inhibited cell attachment to a greater degree (p < 0.001) than that of the LPS from Bacteroides, and at a concentration as low as 12.5 ng/cm2 (p < 0.001), When LPS‐coated culture dishes (12.5 ng/cm2) were incubated with concentrations of plasma fibronectin as low as 50 μg/ml for at least 15 min, the fibronectin significantly enhanced cell attachment, returning it to control levels (p < 0.001). Futhermore, when plasma fibronectin (50 μg/ml) was compared to other agents such as bile acids, citrated serum, and human plasma fraction IV1 (Cohn), which have been shown to affect the toxicity of LPS, fibronectin led to significantly more cell attachment in the presence of LPS than any other treatment (p < 0.001). Evidence is presented by means of transmission electron microscopy (TEM) that LPS may bind to fibronectin. Subsequently, the cell surface interacts with the fibronectin‐LPS and internalizes it via phagocytosis. This mechanism provides for the clearance of LPS from the culture surface.

A noncollagenous matrix for attachment of rat hepatocytes in culture.

Adult rat hepatocytes efficiently attach to intact connective tissue fibers prepared from normal rat liver; this material has been given the name of "biomatrix". The cells remain alive and differentiated for at least 4 months in culture. The liver biomatrix contains both collagen and noncollagenous glycoproteins. Heretofore, the functions of the different components of the biomatrix in facilitating cell adhesion and promoting survival and differentiation of rat hepatocytes have not been investigated. We now report on an aggregate of glycoproteins which were with dilute acetic acid from liver biomatrix and which facilitate hepatocyte adhesion to plastic dishes and collagen. One protein in the extract, with a molecular weight larger than 300,000, was selectively immunoprecipitated with an antibody prepared against the extract and immunoadsorbed to glutaraldehyde-cross-linked rat serum. The antibody did not cross-react with cold-insoluble globulin and selectively inhibited cell attachment. The protein of the molecular weight of 300,000 appears to be specific for liver, since similar extracts prepared from rat lung, kidney, and heart biomatrices did not cross-react with the antibody. Furthermore, the latter extracts did not facilitate cell attachment above values obtained with plain culture dishes. The results suggest that glycoproteins in the biomatrix, which are antigenically distinct from fibronectin, can mediate attachment of rat hepatocytes to plastic dishes and collagen.

Localization of the binding site for cell attachment in the alpha1(I) chain of collagen

Certain cells such as CHO (Chinese hamster ovary) and fibroblasts attach to a substrate of type I collagen via proteins that link the cell surface to collagen. Serum contains a glycoprotein, c-CAP (collagen cell attachment protein), that can mediate this adhesion. Previously, we established that alpha1(I)-CB7 (cyanogen bromide peptide 7), which lies within residues 552 to 822 of collagen, contains a major binding site for c-CAP(alpha1(I)-CB7 has been renumbered to account for an additional triplet recently identified at residue 613 (P. P. Fietzek and R. W. Glanville, manuscript in preparation). Now we have examined the ability of various peptides derived by proteolytic digestion of alpha1(I)-CB7 to bind to c-CAP based upon their ability to inhibit cell attachment to collagen. The binding site lies within residues 757 to 791. It is likely that this is the sole binding site in the alpha1(I) chain since cleavage of the bond between residues 775 and 776 in the alpha1(I) chain destroys cell attachment activity.

Cell attachment to a substratum and cell surface proteases

The attachment of BHK cells to a substratum in serum-containing medium was inhibited when inhibitors of proteolytic enzymes were added to the incubation medium. The inhibitors found to be effective were l-1-tosylamide-2-phenylethyl chloromethyl ketone, N-α- p-tosyl- l-lysine chloromethyl ketone HCl, and phenylmethyl sulfonyl fluoride. Soybean trypsin inhibitor had no effect on cell attachment when added to the incubation medium. Comparative studies were carried out with normal, virus-transformed and suspension-culture adapted BHK cells in which the cells were treated with various concentrations of l-1-tosylamide-2-phenylethyl chloromethyl ketone and then resuspended in fresh medium and tested for their adhesiveness. The relative efficiency of inhibition by l-1-tosylamide-2-phenylethyl chloromethyl ketone was similar with all three cell lines. The possibility was investigated that cell attachment involves proteolytic activation by cell surface proteases of serum proteins absorbed to the substratum. It was found that trypsin or chymotrypsin treatment of substrata coated with serum resulted in an enhanced rate of subsequent cell attachment to the substrata. This effect did not involve bulk removal of serum from the substrata or the presence of residual trypsin in the incubations. Moreover, inhibition of cell attachment by l-1-tosylamide-2-phenylethyl chloromethyl ketone was less pronounced when cell attachment to trypsin-activated substrata was compared with attachment to unactivated substrata. In related studies, it was observed that the spreading of BHK cells onto their substratum requires the presence of serum components in solution in the incubation medium.

Polymer exclusion, cell adhesion and membrane fusion

FROM physicochemical studies on the adhesion of fibroblasts to synthetic substrata1,2 I suggested that, in general, surface-bound polymers (for example adsorbed serum proteins) probably inhibit cell attachment because of their steric exclusion volume. This effect is well known in colloid science (Faraday used gelatin to stabilise gold sols3) but does not seem to be widely appreciated in cell biology. Thus, a recent review on the role of proteins and divalent cations in cell adhesion, viewed as a problem in colloid stability, discusses only charge repulsion and calcium bridging4. Furthermore, standard texts on the cell surface5 present the classical DLVO (Derjaguin, Landau, Verwey and Overbeek) theory of charge repulsion against dispersive attraction, but omit the later development by Overbeek and coworkers of the principle that even uncharged surfaces, if covered by adsorbed polymer molecules, may repel each other6. I now show that steric exclusion by glycocalyx polymers on the plasma membrane would explain some recent observations on fusion and attachment, by a variety of cells. The term steric exclusion3 comprises not only geometric exclusion by rigid molecules, such as rods and spheres, but also unfavourable thermodynamic parameters such as free energy, summed up over many segments, and, for random coil polymers, entropy resulting from the ability of each molecule to claim extra lebensraum by flexing its segments.

Studies on the mechanism of cell attachment to a substratum with serum in the medium: Further evidence supporting a requirement for two biochemically distinct processes

Treatment of baby hamster kidney cells with cytochalasin B or omission of divalent cations from the culture medium are conditions resulting in an inhibition of cell attachment at rest; however, these conditions do not result in inhibition of cell attachment in a centrifugal field. In marked contrast, treatment of cells with trypsin or with tranquilizers such as fluphenazine results in an inhibition of cell attachment at rest or in a centrifugal field. The evidence is interpreted to indicate that cell adhesion involves at least two biochemical processes: formation of the adhesive bond per se (inhibited by tranquilizers or trypsin) and a mechanical process of cell-to-substratum contact and/or spreading (inhibited by cytochalasin B or omission of divalent cations from the medium).

The effects of anionic polymers on cell attachment and growth behaviour, with a note on a similarity in the effect of fresh human serum

Anionic polymers in small concentrations inhibit cell attachment to a glass surface and cause cells to clump. The mechanism of these two effects seems to be similar; when the attachment is inhibited HeLa cells will stick together. Neither in protein-free medium nor in albumin-containing medium, the anionic polymers have any effect. The effect of anionic polymers is indistinguishable from the effect of fresh clumping serum. Both effects can be inhibited by thrombin. The clumping effect of fresh human serum is inhibited by anti-β-lipoprotein sera. The effect of the anionic polymers seems to be due to their electrochemical properties. The possible mechanism for the action of anionic polymers is briefly discussed. It is suggested that the anionic polymers act mainly on the cell surface increasing its negative charge, decreasing the deformability of the cell or changing the micro environment around the cell. The effect of the anionic polymers may also be attributable to their interaction with β-lipoproteins.