Modification of the calcium-independent mechanism of cell adhesion in transformed BHK cells

The calcium-independent mechanism of cell adhesion was studied in normal and polyoma virus-transformed BHK cells. The degree of Ca2+-independent adhesion was greatly reduced in pyBHK cells, whereas CA2+-dependent adhesion occurred to the same degree as in BHK cells. This decrease was shown not to be caused by simple masking of the adhesion sites or by their altered sensitivity to trypsin. Adhesion-blocking antibodies were used to identify molecules responsible for Ca2+-independent adhesion. The antibodies precipitated surface molecules specific for adhesion-competent cells. These have tentatively been named CIDSBHK and CIDSpyBHK. Both were glycoproteins with respective apparent molecular weights of 120K and 125K. CIDSpyBHK incorporated 3H-glucosamine more than CIDSBHK did. Possible modification of the Ca2+-independent adhesion mechanism in pyBHK cells is discussed.     

Reduced contact-inhibition and substratum adhesion in epithelial cells expressing GlcNAc-transferase V

Malignant transformation of fibroblast and epithelial cells is accompanied by increased beta 1-6 N-acetylglucosaminyltransferase V (GlcNAc-TV) activity, a Golgi N-linked oligosaccharide processing enzyme. Herein, we report that expression of GlcNAc-TV in Mv1Lu cells, an immortalized lung epithelial cell line results in loss of contact- inhibition of cell growth, an effect that was blocked by swainsonine, an inhibitor of Golgi processing enzyme alpha-mannosidase II. In serum- deprived and high density monolayer cultures, the GlcNAc-TV transfectants formed foci, maintained microfilaments characteristic of proliferating cells, and also experienced accelerated cell death by apoptosis. Injection of the GlcNAc-TV transfectants into nude mice produced a 50% incidence of benign tumors, and progressively growing tumors in 2:12 mice with a latency of 6 mo, while no growth was observed in mice injected with control cells. In short term adhesion assays, the GlcNAc-TV expressing cells were less adhesive on surfaces coated with fibronectin and collagen type IV, but no changes were observed in levels of cell surface alpha 5 beta 1 or alpha v beta 3 integrins. The larger apparent molecular weights of the LAMP-2 glycoprotein and integrin glycoproteins alpha 5, alpha v and beta 1 in the transfected cells indicates that their oligosaccharide chains are substrates for GlcNAc-TV. The results suggest that beta 1-6GlcNAc branching of N-linked oligosaccharides contributes directly to relaxed growth controls and reduce substratum adhesion in premalignant epithelial cells.     

Plasma fibronectin-binding glycosaminoglycans in the substratum adhesion sites of neural tumor cells

The metabolism of high-density lipoprotein (HDL) in cells of five human cancer cell lines maintained in monolayer culture was investigated. In cells of some of the lines there was evidence of high-affinity binding sites for HDL, whereas in others this could not be demonstrated. However, in one cell line, viz., HEC-B-296 (human endometrial carcinoma), degradation of the protein component of HDL was demonstrated. The proteolytic activity was specific for HDL in so far as human serum albumin was not degraded by these cells. However, this degradative process did not involve internalization of the HDL molecule and degradation was not mediated by lysosomal proteolytic enzymes. HDL, when present in the medium, did not affect the degradation of low-density lipoprotein and low-density lipoprotein did not affect the degradation of HDL. HDL did not affect significantly cholesterol biosynthesis or cholesteryl ester biosynthesis as estimated from the activity of the regulatory enzymes, 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl-CoA:cholesterol acyltransferase. The degradation of HDL by HEC-B-296 cells was inhibited, to various degrees, when trypsin inhibitor or a protease inhibitor such as leupeptin, was present in the culture medium. It is concluded that degradation of the protein component of HDL by human neoplastic cells of the HEC-B-296 line was the result of activity of a proteolytic enzyme that is present on the external surface of the cells.     

Innate non-specific cell substratum adhesion

Adhesion of motile cells to solid surfaces is necessary to transmit forces required for propulsion. Unlike mammalian cells, Dictyostelium cells do not make integrin mediated focal adhesions. Nevertheless, they can move rapidly on both hydrophobic and hydrophilic surfaces. We have found that adhesion to such surfaces can be inhibited by addition of sugars or amino acids to the buffer. Treating whole cells with αlpha-mannosidase to cleave surface oligosaccharides also reduces adhesion. The results indicate that adhesion of these cells is mediated by van der Waals attraction of their surface glycoproteins to the underlying substratum. Since glycoproteins are prevalent components of the surface of most cells, innate adhesion may be a common cellular property that has been overlooked.     

SHAP potentiates the CD44-mediated leukocyte adhesion to the hyaluronan substratum

CD44-hyaluronan (HA) interaction is involved in diverse physiological and pathological processes. Regulation of interacting avidity is well studied on CD44 but rarely on HA. We discovered a unique covalent modification of HA with a protein, SHAP, that corresponds to the heavy chains of inter-alpha-trypsin inhibitor family molecules circulating in blood. Formation of the SHAP.HA complex is often associated with inflammation, a well known process involving the CD44-HA interaction. We therefore examined the effect of SHAP on the CD44-HA interaction-mediated lymphocyte adhesion. Under both static and flowing conditions, Hut78 cells (CD44-positive) and CD44-transfected Jurkat cells (originally CD44-negative) adhered preferentially to the immobilized SHAP.HA complex than to HA. The enhanced adhesion is exclusively mediated by the CD44-HA interaction, because it was inhibited by HA, but not IalphaI, and was completely abolished by pretreating the cells with anti-CD44 antibodies. SHAP appears to potentiate the interaction by increasing the avidity of HA to CD44 and altering their distribution on cell surfaces. Large amounts of the SHAP.HA complex accumulate in the hyperplastic synovium of rheumatoid arthritis patients. Leukocytes infiltrated to the synovium were strongly positive for HA, SHAP, and CD44 on their surfaces, suggesting a role for the adhesion-enhancing effect of SHAP in pathogenesis.     

Cooperativity of ganglioside-dependent with protein-dependent substratum adhesion and neurite extension of human neuroblastoma cells

The potential involvement of gangliosides in the adherence and neurite extension of human neuroblastoma cells (Platt and La-N1) was investigated on tissue culture substrata coated with the ganglioside GM1-binding protein, cholera toxin B (CTB) subunit, for comparison with similar processes on plasma fibronectin (pFN)-coated substrata. Cells attached with reduced efficiency on CTB substrata as compared with pFN substrata and required a much longer time to form neurite processes for a small percentage of cells on CTB. The specificity of these processes for GM1 binding was tested in a variety of ways. Supplementation of the cells with exogenous GM1, but not GD1a, identified a larger population of cells adherent on CTB (comparable to pFN-adherent cells) and dramatically increased the proportion of cells capable of forming neurites without reducing the time requirement. In ultrastructural studies using the scanning electron microscope (SEM) and immunofluorescence (IF) analyses to discriminate microtubule distributions, neurites of GM1-supplemented cells on CTB were virtually identical with pFN-adherent neurites, whereas unsupplemented cells on CTB generated processes with fine-structural differences. Treatment of cells during the GM1 supplementation period with cycloheximide completely abolished the ability of cells to generate neurites on CTB and decreased the adhesive capacity of cells as well; a similar treatment of cells had no adverse effect on adherence or neurite extension on pFN. The importance of one or more proteins in GM1-dependent processes was further confirmed by demonstrating the trypsin sensitivity of a cell surface component(s) required to achieve maximal attachment on CTB; in contrast, adherence and neurite extension on pFN were much more resistant to this treatment process. Therefore, these experiments demonstrate (a) that certain cell surface gangliosides are capable of mediating adherence and neurite outgrowth of human neuroblastoma cells on a suitable ganglioside-binding substratum; (b) this ganglioside dependence is cooperative with one or more cell surface proteins which can now be analysed. These results are discussed in light of the identification in ref. [16] (Exp cell res 169 (1987) 311) of a second ‘cell-binding’ domain on the pFN molecule competent for adherence and neurite extension of these neuroblastoma cells, as well as the potential role of pFN binding to a complex ganglioside on the surface of these neural tumor cells in these processes.     

Inhibition of cell adhesion to plastic substratum by phosphorothioate oligonucleotide.

Antisense oligonucleotides have been widely used to achieve specific inhibition of targeted gene expression. However, the mechanism of action is not well understood and in many systems sequence-independent effects occur. We have recently shown that chronic administration of an antisense c-myc phosphorothioate oligonucleotide can specifically inhibit expression of the c-myc protein and growth in human breast cancer cells. We now identify an additional effect of the same oligonucleotide on cell adhesion. Transient delivery through electroporation of 2.5 microM antisense-myc oligonucleotide to MCF-7 cells results in 85% inhibition of adhesion to plastic substratum within 24 h. Both the onset of this effect and the subsequent recovery occur without a change in cell viability, growth, or alteration of adhesion to Matrigel, collagen IV, laminin, or fibronectin. However, no parallel changes in c-myc mRNA or protein expression are detectable, suggesting that in this instance inhibition of adhesion caused by antisense-myc oligonucleotide may involve a mechanism independent of the target sequence.     

Adhesion of culture cells to their substratum

The attachment of cells to culture dishes has been investigated by replica techniques and scanning electron microscopy. Cells were removed from their substratum either in a stream of medium or by micromanipulation. In the most usual case of interphase cells one finds many small points of attachment uniformly distributed over the whole cell underside. Attachment sites are also found under the lamellipodia and at the trailing edge of the cells. A large portion of the cell underside is sometimes left behind. This is probably because the sole plate is reinforced by cytoskeletal elements, and does not necessarily indicate the presence of large adhesion points. The distal ends of the retraction fibers formed as the cells round-up in trypsin, or in the cold, represent the attachment points of the cell to the substratum. Agents which tend to stabilize microtubules greatly slow cell detachment by proteolytic agents. The primary effect of trypsin is not on the glue which holds the cells to the substratum but rather on the cell shape itself, affecting the rigidity of cytoskeletal elements.     

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.     

Proteoglycans from the substratum adhesion sites of MSV-transformed Balb/c 3T3 cells

Kirsten murine sarcoma virus-transformed Balb/c 3T3 cells (KiMSV) are highly tumorigenic and metastatic in the appropriate murine host, are loosely adherent to the tissue culture substratum, and can be readily detached from the substratum by ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid treatment leaving their adhesion sites as substratum-attached material. Both long-term culture-generated adhesion sites (L-SAM) of KiMSV cells and newly formed adhesion sites of reattaching cells (R-SAM) contain high levels of hyaluronate (HA) and chondroitin sulfate (CS) whereas the R-SAM of parental Balb/c 3T3 cells is enriched in heparan sulfate (HS). A sizable fraction of KiMSV L-SAM proteoglycans (PG) and a smaller fraction of R-SAM PG's aggregate into two size classes of supramolecular complexes, after extraction off the substratum with 4 m guanidine hydrochloride, as determined by chromatography on columns of Sepharose CL2B in several buffer systems. Isopycnic density gradient analyses under associative conditions of KiMSV L-SAM generated three classes of material—high-density G_A1 which contained some HA but principally CS and HS; intermediate-density G_A2 which contained only HA; and low-density G_A3 which contained some HA and principally glycoprotein. R-SAM gradients contained no G_A2 but a sizable amount of “low-density” HA in G_A3. When centrifuged under dissociative conditions, most of G_A1 and all of G_A2 from L-SAM shifted to the top of the gradient, whereas most of the HS-PG in R-SAM remained at the bottom of dissociative gradients. Comparison of these analyses with previous analyses of Balb/c 3T3 extracts demonstrates that (a) KiMSV cells generate adhesion sites with different PG contents than 3T3 sites; (b) the PG's of KiMSV sites have a reduced potential to aggregate into high-molecular-weight complexes but do form intermediate-size complexes not apparent in material from 3T3 sites; (c) these data support the hypothesis that HA is important in detachment of cells from extracellular matrices; and (d) HS-PG's in newly formed adhesion sites of KiMSV cells are considerably different from sites which have “matured”, indicating that there is metabolic activity in these sites during prolonged adherence and movement of transformed cells.     

Collagen synthesis in normal BHK cells and temperature-sensitive chemically transformed BHK cells

Summary Collagen synthesis in normal BHK 21/cl 13 and chemically transformed temperature-sensitive BHK 21/cl 13 cells (Me₂N4) was assessed by examination of hydroxyproline formation and collagenase-susceptible protein. The Me₂N4 cells lost their ability to synthesize collagen at both permissive and nonpermissive temperatures for transformation. These conclusions were confirmed by polyacrylamide-gel electrophoresis and CM-cellulose chromotography. Prolyl hydroxylase activity was present in both normal and transformed cells even when no collagen could be demonstrated. The production of noncollagen protein, although decreased in the transformed cell, did not change as drastically as the collagen synthesis. This paper was supported in part by a grant from the Public Health Service (AG00001), and by the Medical Research Service of the Veterans Administration.     

Coated pits and asialoglycoprotein receptors redistribute to the substratum during hepatocyte adhesion to galactoside surfaces

Rat hepatocytes bind in a sugar-specific and concentration-dependent manner to flat polyacrylamide matrices containing covalently attached galactosyl (Gal) groups. Previous studies (Weigel, P.H., J. Cell Biol. 87, 855, 1980) concluded that binding was likely mediated by the asialoglycoprotein receptor. Here we confirm that adhesion is mediated by this receptor, since cell binding is inhibited by antireceptor antibody and a threshold binding response is also observed when hepatocytes adhere to surfaces coated with asialoorosomucoid, a ligand for this receptor. Cells that had bound to a Gal surface and were then sheared from the surface left a membrane patch behind on the substratum. The cytoplasmic side of these plasma membrane patches was visualized on the substratum by indirect immunofluorescence using antireceptor antibody or anticlathrin antibody. The density of punctate coated pits, visualized with the latter antibody, was enriched in a circular membrane region of about 4 microns 2 area that mediated cell binding. This zone also contained concentrated receptors, although the staining pattern with antireceptor antibody was more uniform and less punctate. The results show that both asialoglycoprotein receptors and coated pits are redistributed at the substratum interface on hepatocytes bound to Gal surfaces.     

Locomotion and adhesion of polymorphonuclear leukocytes effects of the supporting substratum

Contact angle measurements have been used to correlate surface hydrophobicity of a supporting substratum with adhesion and locomotion of polymorphonuclear leukocytes. The binding of human serum albumin, a well-known chemokinetic substance, to hydrophilic glass slides gave rise to hydrophobic surfaces with adhesive properties conducive, to cell polarization thus allowing cell locomotion. Parallel contact angle and cell adhesion measurements suggested that albumin modified the cellsubstratum interaction by increasing the van der Waals forces of attraction and reducing the electrostatic forces. By allowing cells to adhere to a hydrophobic surface (siliconized glass), it was found that protein could be omitted from in vitro test systems for leukocyte locomotion. It is suggested that quantitatively equal cell adhesion values may, depending on the type of attraction forces working in adhesion to the substratum, result in different locomotion patterns.     

Heparan sulfate proteoglycans in the substratum adhesion sites of human neuroblastoma cells: modulation of affinity binding to fibronectin

Tissue culture substratum adhesion sites from EGTA-detached Platt human neuroblastoma cells were extracted with a buffer containing ocytlglucoside, NaCl, guanidine hydrochloride, and a variety of protease inhibitors, an extraction which resulted in quantitative solubilization of the 35SO4 = -radiolabeled proteoglycans and 3H-leucine-radiolabeled proteins. Of the sulfate-radiolabeled material, the vast majority was heparan sulfate proteoglycan (Kav = 0.15 on Sepharose C14B columns) and the remainder was chondroitin sulfate chains (no single chains of heparan sulfate were observed). This extract was then fractionated on DEAE-Sephadex columns under two different buffer elution conditions. Under DEAE-I conditions in low ionic strength acetate buffer, two major peaks of 35SO4 = -radiolabeled material (A,B) and a minor peak (C) could be resolved in the NaCl gradient; however, three-fourths of the material required 4 M guanidine hydrochloride to elute it from the column (peak D). Under DEAE-II conditions in acetate buffer supplemented with 8 M urea, the vast majority of the proteoglycan material could be eluted in the NaCl gradient as peak AB. Peak D material was shown to contain aggregated proteoglycan, along with nonproteoglycan protein, which high concentrations of urea or guanidine could dissociate, but not nonionic or zwitterionic detergents. Three different affinity chromatography systems were used to further characterize these components. Approximately 60% of peak A heparan sulfate proteoglycan from DEAE-I binds to the hydrophobic matrix, octyl-Sepharose, while 80% of the proteoglycan in DEAE-I peak D binds to this hydrophobic column. A sizable fraction of peak A proteoglycan fails to bind to plasma fibronectin but does bind to platelet factor-4 affinity columns. In contrast, peak AB proteoglycan from DEAE-II columns yields a much higher proportion of molecules which do bind to fibronectin. To examine the basis for these differences in affinity binding, nonproteoglycan protein from these adhesion sites was mixed with peak AB proteoglycan prior to affinity chromatography; proteoglycan binding to fibronectin decreased markedly while binding to platelet factor-4 was unaffected. This modulating activity involves the binding of nonproteoglycan protein in adhesion site extracts to both fibronectin on the column, as well as to heparan sulfate proteoglycan itself, and it could not be mimicked by a number of known proteins in adhesion site extracts or several other proteins. These results demonstrate selectivity and specificity in this modulation and indicate that a previously unidentified protein(s) is responsible.(ABSTRACT TRUNCATED AT 400 WORDS)     

The supply of oxygen to submerged cultures of BHK 21 cells

Oxygen solution rates were measured in 4, 30, and 100 liter culture vessels, and the oxygen demand of growing BHK 21 cells estimated. This data was used to calculate the minimal sparged air rates necessary to satisfy oxygen demand throughout the cell growth cycle, and in this way adequate oxygen was supplied without the damaging effects of excessive sparging. Comparable results were obtained when oxygen was supplied by this method and when pO₂ was controlled at 80 mmHg, but both cell growth rate and maximum cell density were reduced when pO₂ was controlled at other values.     

Collagen synthesis in normal BHK cells and temperature-sensitive chemically transformed BHK cells.

Collagen synthesis in normal BHK 21/cl 13 and chemically transformed temperature sensitive BHK 21/cl 13 cells (Me2N4) was assessed by examination of hydroxyproline formation and collagenase-susceptible protein. The Me2N4 cells lost their ability to synthesize collagen at both permissive and nonpermissive temperatures for transformation. These conclusions were confirmed by polyacrylamide-gel eletrophoresis and CM-cellulose chromatography. Prolyl hydroxylase activity was present in both normal and transformed cells even when no collagen could be demonstrated. The production of noncollagen protein, although decreased in the transformed cell, did not change as drastically as the collagen synthesis.