Early events during substrate adhesion of normal and virus-transformed mouse fibroblasts.

The relationship between attachment of Balb/c3T3 cells and their SV40 transformants to glass or plastic substrates and deposition of substrate-attached material (SAM-proteoglycans implicated in substrate adhesion) has been examined very early after inoculation of cells subcultured with ethylenebis (oxyethylenenitrilo) tetra-acetic acid (EGTA). The metabolic inhibitors cycloheximide and colchicine minimally affected the kinetics or short-term stability of attachment of cells or deposition of SAM. SAM deposition on to the substrate began immediately after inoculation of cells and was maximal prior to the highest cell attachment level (30-40 min after inoculation). At 4 degrees C, there was no attachment of cells to the substrate and no deposition of leucine- or glucosamine-radiolabelled SAM on to the substrate. 3T3 cells deposited SAM to a maximal level earlier during the attachment process than SV40-transformed cells. SVT2 cells deposited much smaller amounts of SAM (measured on a per-cell basis) to 3T3 SAM-coated substrates during attachment processes, whereas 3T3 cells and concanavalin A (con A) revertant variants of SVT2 cells, which have regained density-dependent inhibition of growth, deposited identical amounts of SAM (per-cell) on untreated or SAM-coated substrates. Serial attachment experiments with SVT2 cells indicated that all SVT2 cells reduced their deposition amounts on SAM-coated substrates, rather than there being an ability of a small proportion of cells to attach preferentially to SAM-coated substrates while being unable to deposit SAM themselves. The data are consistent with the presence of a sizeable pool of SAM-like proteoglycans being present on the surface of EGTA-removed cells whose deposition may be a requirement for, but may not necessarily be sufficient for, stable adhesion of cells to the substrate.     

Electron microscopic study of the interaction between neoplastic fibroblasts and the substrate in tissue culture]

Electron microscope study of neoplastic L fibroblasts was carried out in 15, 30, 60, and 90 min after their attachment to solid substratum. Comparative analysis of neoplastic and normal fibroblasts at the same stages was carried out. Spreading rate of L fibroblasts proved slower than that of normal fibroblasts. Primary reaction of neoplastic cells was disturbed on the contact with substratum: it was morphologically manifested in changing of structure of the cell lower surface. Bundles of microfilaments were absent from the neoplastic fibroblasts' cytoplasm. The above changes, apparently, may entail the lesser degree of spreading and the weaker attachement of neoplastic fibroblasts to the substratum.     

Activation of the small GTPase Rac is sufficient to disrupt cadherin-dependent cell-cell adhesion in normal human keratinocytes

To achieve strong adhesion to their neighbors and sustain stress and tension, epithelial cells develop many different specialized adhesive structures. Breakdown of these structures occurs during tumor progression, with the development of a fibroblastic morphology characteristic of metastatic cells. During Ras transformation, Rac-signaling pathways participate in the disruption of cadherin-dependent adhesion. We show that sustained Rac activation per se is sufficient to disassemble cadherin-mediated contacts in keratinocytes, in a concentration- and time-dependent manner. Cadherin receptors are removed from junctions before integrin receptors, suggesting that pathways activated by Rac can specifically interfere with cadherin function. We mapped an important region for disruption of junctions to the putative second effector domain of the Rac protein. Interestingly, although this region overlaps the domain necessary to induce lamellipodia, we demonstrate that the disassembly of cadherin complexes is a new Rac activity, distinct from Rac-dependent lamellipodia formation. Because Rac activity is also necessary for migration, Rac is a good candidate to coordinately regulate cell-cell and cell-substratum adhesion during tumorigenesis.     

Changes in the surface morphology of normal and transformed mouse fibroblasts following disruption of cell-substrate adhesion]

By means of scanning electron microscopy surface morphology of cultured normal mouse embryo fibroblasts (MEF) and transformed mouse fibroblasts of L strain was studied in the course of alteration of cell-substrate adhesion with proteases, EDTA and urea. The morphology of cell rounding induced by the above agents in MEF and L cells was almost independent on the type of the agent. The rounding of MEF proceeded through three stages and was accompanied by substantial changes of cell surface relief. L cells lacked the intermediate stage (formation of thick processes) during their rounding which proceeded without any changes of cell surface relief. It is suggested that the observed differences are related to the poorer development of the lamelloplasm and microfilaments bundles in the transformed cells ascompared to the normal ones.     

Autophagy of metabolically inert substances injected into fibroblasts in culture

We have examined the morphologic characteristics of fibroblasts cultured from the beige mouse, a genetic variant phenotypically similar to human Chediak-Higashi syndrome (CHS). These cultured fibroblasts are characterized by large, amorphous dense body inclusions in their cytoplasm, often as large as the cell nucleus. Using time-lapse video phase-contrast microscopy, we have observed the formation of these large dense bodies through fusion of relatively normal-appearing lysosomes in the beige mouse fibroblast. After formation of these large inclusions, cells occasionally extruded the contents of these structures through apparent fusion with the plasma membrane and rapid exocytosis. Fibroblasts cultured from normal black mice showed no evidence of fusion between lysosomes or exocytosis of lysosomes. However, the uptake of extracellular medium through macropinocytosis, subsequent actions of lysosomes on these macropinosomes through saltatory motion, cellular migration and ruffling activity appeared normal in beige mouse fibroblasts. Immunocytochemical localization of α₂-macroglobulin, a normal serum protein commonly incorporated into lysosomes in cultured fibroblasts by receptor-mediated endocytosis, showed that the large dense bodies contained α₂-macroglobulin, in keeping with their lysosomal origin. This suggested further that receptor-mediated endocytosis in these cells was relatively normal. In addition, light and electron microscopic cytochemistry showed these large inclusions to be acid-phosphatase positive, further characterizing them as lysosomal. The electron microscopic appearance of these dense inclusions was consistent with their origin through repeated fusion of lysosomes. It is suggested that a primary defect in this disease may be the ability of mature lysosomal membranes to fuse, unlike normal lysosomal membranes, indicating perhaps an alteration in some specific component of the lysosomal membranes in CHS.     

Effects of cell adhesion to the substratum on the growth of chick embryo fibroblasts

Chick embryo fibroblasts were plated on Petri dishes that had not been treated for use in tissue culture (bacteriological dishes). On these dishes the cells grow at the same exponential rate as cells plated on tissue culture dishes, but their growth becomes inhibited sooner after plating, and therefore at a lower cell number per dish. The inhibition of cell growth on bacteriological dishes is correlated with the formation of cell clumps. Clump formation is reversible by mechanical transfer of the clumps to a tissue culture dish: the cells migrate out of the clumps, form a monolayer, and cell growth resumes.Clump formation was studied by time-lapse cinematography, and was found to be due to reduced adhesion of the cells to the bacteriological dish surface. This reduced adhesiveness of the substratum is due to a lower number of negatively-charged residues on the bacteriological dish surface, which can be measured by the binding of crystal violet. The number of negatively-charged residues, and therefore the adhesiveness of the substratum can be altered by treatment of the dishes with sulfuric acid. Serum components of the medium were found to affect cell adhesion to the bacteriological dishes, consequently altering the efficiency of cell attachment, the extent of cell growth and the pattern of clump formation.The cells in clumps were compared with those in confluent monolayers on tissue culture dishes. Growth-inhibited cells on both types of dish were found to be equally viable. Cells in clumps on bacteriological dishes were found to be inhibited in the G1 phase of the cell cycle, as are cells in density-inhibited monolayers. Infection by the oncogenic virus, Rous sarcoma virus, can release the cells from growth-inhibition on both types of dish. Cell-induced alterations of the medium are not involved in the growth inhibition of cells on bacteriological dishes.     

Phospholipid composition and turnover in normal and SV40 transformed fibroblasts. Effect of cell density]

The phospholipid composition and turnover in normal and in SV 40 transformed hamster fibroblasts were studied. The amount of phospholipid phosphorus relative to protein is lower in transformed hamster fibroblasts than in normal fibroblasts. This amount decreases with increasing cell density until stationary growth is reached. The decrease is largest for the normal fibroblasts. In transformed cells, less sphingomyelin and more diphosphatidyl glycerol are found than in normal cells. The turnover of 32P in sphingomyelin is slower in transformed cells than in normal cells ; the contrary is observed with diphosphatidyl glycerol. On the other hand, in transformed cells, phosphatidyl ethanolamine has a faster turnover than phosphatidyl choline, whereas the contrary is observed in normal cells. Finally, the change to stationary growth slows down the turnover of 32P of all phospholipids, this decrease being more important in transformed cells.     

The cell adhesion protein P-selectin glycoprotein ligand-1 is a substrate for the aspartyl protease BACE1

The aspartyl protease BACE1 cleaves the amyloid precursor protein and the sialyltransferase ST6Gal I and is important in the pathogenesis of Alzheimer's disease. The normal function of BACE1 and additional physiological substrates have not been identified. Here we show that BACE1 acts on the P-selectin glycoprotein ligand 1 (PSGL-1), which mediates leukocyte adhesion in inflammatory reactions. In human monocytic U937 and human embryonic kidney 293 cells expressing endogenous or transfected BACE1, PSGL-1 was cleaved by BACE1 to generate a soluble ectodomain and a C-terminal transmembrane fragment. No evidence of the cleavage fragment was seen in primary cells derived from mice deficient in BACE1. By using deletion constructs and enzymatic deglycosylation of the C-terminal PSGL-1 fragments, the cleavage site in PSGL-1 was mapped to the juxtamembrane region within the ectodomain. In an in vitro assay BACE1 catalyzed the formation of the PSGL-1 products seen in vivo. The cleavage occurred at a Leu-Ser peptide bond as identified by mass spectrometry using a synthetic peptide. We conclude that PSGL-1 is an additional substrate for BACE1.     

Effect of hormonal and substrate backgrounds on cell membrane function in normal males

Hormonal and substrate influences on in vivo cellular membrane function were evaluated in 15 healthy male volunteers. Each subject underwent serial evaluations of membrane function in the anterior tibialis muscle, as assessed by transcutaneous measurement of resting membrane potential (Em). Group A subjects (n = 9) underwent measurement of resting Em in the basal state and again during the 10th day of intravenous feeding (IVF). Group B subjects (n = 6) underwent measurement of resting Em in the basal state during epinephrine infusion and again during epinephrine infusion on the 7th day of IVF. Percutaneous needle biopsy of the vastus lateralis muscle permitted calculation of transmembrane electrolyte distribution from the Nernst equation, using the measured Em and the chloride space method. Hospitalization with intake of a defined-formula enteral diet for 3 days resulted in depolarization (P less than 0.05) of resting Em (-75.3 +/- 1.6 mV) compared with normal (-79.8 +/- 0.9 mV). Despite 10 days of subsequent IVF, further depolarization (P less than 0.05) of resting Em (-71.2 +/- 1.2 mV) was observed. In the dual presence of IVF and exogenous epinephrine infusion, there was an increase (P less than 0.05) in intracellular potassium concentration and repolarization of resting Em (-80.6 +/- 0.8 mV) to normal levels. These data indicate that hormonal background and substrate availability contribute to the in vivo modulation of cellular membrane function in human skeletal muscle, possibly through facilitation of sodium-dependent amino acid transport across the cell membrane.     

Effect of substrate mechanics on chondrocyte adhesion to modified alginate surfaces

This study characterized the attachment of chondrocytes to RGD-functionalized alginate by examining the effect of substrate stiffness on cell attachment and morphology. Bovine chondrocytes were added to wells coated with 2% alginate or RGD-alginate. The alginate was crosslinked with divalent cations ranging from 1.25 to 62.5 mmol/g alginate. Attachment to RGD-alginate was 10-20 times higher than attachment to unmodified alginate and was significantly inhibited by antibodies to integrin subunits α₃ and β₁, cytochalasin-D, and soluble RGD peptide. The equilibrium level and rate of attachment increased with crosslink density and substrate stiffness. Substrate stiffness also regulated chondrocyte morphology, which changed from a rounded shape with nebulous actin on weaker substrates to a predominantly flat morphology with actin stress fibers on stiffer substrates. The dependence of attachment on integrins and substrate stiffness suggests that chondrocyte integrins may play a role in sensing the mechanical properties of the matrices to which they are attached.     

An antibody that inhibits fibronectin-independent adhesion of fibroblasts to extracellular matrix material

Chinese hamster ovary (CHO) fibroblasts adhere to the extracellular matrix by both fibronectin-dependent and -independent mechanisms (Harper and Juliano, 1981a,b). Previous studies have suggested that a trypsin-sensitive, 265,000-dalton membrane glycoprotein (gp265) is involved in the fibronectin-independent adhesion process. Using a polyclonal antibody against soluble products obtained from trypsin-treated CHO cells, we have been able to further analyze this involvement. This antibody immunoprecipitates a trypsin-sensitive 265,000-dalton protein from detergent-solubilized cells. Incubation of AdvF11, a variant cell line that does not utilize fibronectin for adhesion, with this antibody blocks their adhesion to extracellular matrix material (ECM). The immunoglobulin fraction will also partially block adhesion of the parental cell line to ECM particularly when the ECM is first treated with an antifibronectin antibody. Taken together these results add support for the involvement of gp265 in fibronectin-independent adhesion and provide a methodology for further characterization.     

Characterization of a membrane-associated glycoprotein complex implicated in cell adhesion to fibronectin

We have characterized a 140-kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band 1), 135,000 (band 2), and 120,000 (band 3). In two-dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band 1), pH 5.6 (band 2), and pH 5.3 (band 3). These three major bands were compared by two-dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co-migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S. Amino acid analysis revealed no detectable hydroxyproline and a composition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells.     

Effect of colcemid on the spreading of fibroblasts in culture

The effect of colcemid upon the spreading of mouse embryo fibroblast-like cells on substrates was studied with the aid of time-lapse microcinematography and scanning electron microscopy. Two types of substrates were used: flat glass and narrow strips of glass surrounded by non-adhesive lipid film; on the latter, spreading and polarization of cells proceeded simultaneously. On glass, colcemid did not prevent transition of cells into a well-attached state; however, the time required for this transition increased considerably as compared with control cultures. Similar effects were caused by two other drugs inhibiting the formation of microtubules: colchicine and vinblastine. The intermediate stages of spreading on flat glass had several abnormal features in the colcemid-containing medium: (a) the shape of cytoplasmatic outgrowths formed by the cell was altered and their distribution became less regular; (b) partial detachment of the attached parts of the cells was very frequent; (c) the spreading of various parts of the cell was not well correlated: the central part of the cell could remain unspread long after the spreading of the peripheral part. Similar effects of colcemid were observed in experiments with cells spreading on the narrow strips of the glass. In addition, colcemid prevented stabilization of the cell surface, i.e., differentiation of the cellular edge into active and stable parts. About two-thirds of the cells attached to the narrow strips of glass were completely detached from the substrate in the course of spreading in colcemid-containing medium. The possible mechanisms of the action of colcemid on spreading are discussed and it is suggested that intracellular structures sensitive to colcemid are essential for the coordination of the reactions in various parts of the cell in the course of spreading.     

Effect of cell trypsinization on nuclear proteins of WI-38 fibroblasts in culture.

When resting confluent monolayers of WI-38 fibrolasts are trypsinized and replated at a lower density they are stimulated to proliferate again with an interval of 18 hours between replating and the onset of DNA synthesis. Trypsinization of resting cells causes a 40% loss of nuclear proteins as well as of cytoplasmic proteins. The amount of nuclear proteins remains low for the first six hours after the cells have been replated and then it increases rapidly, reaching the same level of non-trypsinized resting cells by ten hours after plating. The proteins that are lost from the nucleus immediately after trypsinization are chromatin-associated proteins and most of them are non-histone chromosomal proteins, although a modest loss of histones cannot be ruled out. The loss of non-histone chromosomal proteins from cells that have been trypsinized causes changes in the structure of chromatin that can be detected by circular dichroism and by viscosity measurements. These results show that cell trypsinization causes an extensive loss of proteins from chromatin and that the loss is restored only several hours after the cells have been replated at a lower density.     

Lymphocyte adhesion to epithelia and endothelia mediated by the lymphocyte endothelial-epithelial cell adhesion molecule glycoprotein.

Upon encountering the relevant vascular bed, lymphocytes attach to endothelial adhesion molecules, transmigrate out of circulation, and localize within tissues. Lymphocytes may then be retained at microanatomic sites, as in tissues, or they may continue to migrate to the lymphatics and recirculate in the blood. Lymphocytes also interact transiently, but with high avidity, with target cells or APC that are infected with microbes or have taken up exogenous foreign Ags. This array of adhesive capabilities is mediated by the selective expression of lymphocyte adhesion molecules. Here, we developed the 6F10 mAb, which recognizes a cell surface glycoprotein designated lymphocyte endothelial-epithelial cell adhesion molecule (LEEP-CAM), that is distinct in biochemical characteristics and distribution of expression from other molecules known to play a role in lymphocyte adhesion. LEEP-CAM is expressed on particular epithelia, including the suprabasal region of the epidermis, the basal layer of bronchial and breast epithelia, and throughout the tonsillar and vaginal epithelia. Yet, it is absent from intestinal and renal epithelia. Interestingly, it is expressed also on vascular endothelium, especially high endothelial venules (HEV) in lymphoid organs, such as tonsil and appendix. The anti-LEEP-CAM mAb specifically blocked T and B lymphocyte adhesion to monolayers of epithelial cells and to vascular endothelial cells in static cell-to-cell binding assays by approximately 40-60% when compared with control mAbs. These data suggest a role for this newly identified molecule in lymphocyte binding to endothelium, as well as adhesive interactions within selected epithelia.     

Fibronectin-independent adhesion of fibroblasts to the extracellular matrix: mediation by a high molecular weight membrane glycoprotein

Fibroblastic CHO cells readily adhere to fibronectin (Fn) coated substrata. From the parental cell population we have recently selected a series of adhesion variants (ADV cells) that cannot adhere to Fn substrata (Harper and Juliano. 1980. J. Cell. Biol. 87:755-763). However, ADV cells readily adhere to substrata coated with extracellular matrix material (ECM) derived from human diploid fibroblasts by a mechanism that does not involve fibronectin (Harper and Juliano. 1981. Nature (Lond.). 290:136-138). Te Fn-dependent adhesion mechanism of parental cells (type 1 adhesion) and the ECM- dependent adhesion of ADV cells (type II adhesion) can also be discriminated on the basis of their differential sensitivity to proteolysis, with the type II mechanism being far more sensitive. In this communication we report that parental CHO cells possess both type I and type II mechanisms whereas ADV cells possess only the type II mechanism. We also identify a high molecular weight membrane glycoprotein (gp 265) that seems to play a role in type II adhesion. This component is detected by [125I]lactoperoxidase of [3H]borohydride- galactose oxidase labeling of surface proteins in WT and AD cells. Cleavage of gp 265 with low doses of proteases correlates completely with the loss of type II adhesion capacity. Thus CHO cells possess two functionally and biochemically distinct adhesion mechanisms, one involving exogenous Fn and the other mediated by the membrane component gp 265.     

Phenolic substances in the cell suspension culture ofCentaurium erythraea

The content of phenolic substances in the cell suspension culture ofCentaurium erythraea fluctuated during a 21-day-long subcultivation period in dependence on the growth phase. The total relative content of the phenolics reached its maximum at the time of transition to the exponential growth phase, similarly as the fraction of free phenolic acids, glycosides, and the fraction of phenolic acids released from the cells after alcaline hydrolysis. On the other hand, the content of phenolic acid esters decreased at this growth phase of the culture. Changes in the level of phenolic substances in the culture medium corresponded in their character to changes in the relative content of the phenolics in the cells.     

Release of antigens into the culture medium by LS fibroblasts in culture]

LS fibroblasts cultivated for 1 or more days in unsupplemented Eagle's MEM release antigens into the medium, without showing any evidence of lysis, but on the contrary continuing to proliferate. These antigens give up to three precipitation lines when tested by means of immunodiffusion and immunoelectrophoresis against specific rabbit antisera; one or two of them give identity reactions with antigens obtained by repeatedly washing the fibroblasts with balanced salt solutions. Evidence has been obtained that they are surface components, easily stripped or spontaneously shed by the cells.