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.     

Effect of proteases on activation of resting chick embryo fibroblasts and on cell surface proteins.

The relationship between activation of resting chick embryo fibroblasts by proteases and proteolytic alteration of the cell surface has been investigated. Five different proteases were examined: trypsin, collagenase, plasmin, alpha-chymotrypsin, and thrombin. All of these proteases, when added to the culture medium at concentrations of 0.08-2.2 mug/ml, stimulated deoxyglucose uptake and induced cell division. The absolute levels of stimulation depended on the specific protease. Activation ranged from a doubling in cell number in 24 hr for trypsin and thrombin down to a 47% increase in cell number for alpha-chymotrypsin. Except in the case of thrombin, the stimulatory effects of these proteases correlated with breakdown of Z, a protein which is the major chick surface protein as revealed by lactoperoxidase-catalyzed iodination and which disappears upon transformation. In the case of thrombin, stimulatory concentrations brought about no detectable loss of surface components. Thus loss of Z is not a necessary condition for activation of chick fibroblasts; it may be a sufficient condition for activation of part of the cell population.     

Effects of lectins on cytoskeleton and morphology or cultured chick embryo fibroblasts

The microfilaments and microtubules of cultured chick embryo skin fibroblasts were studied in the presence of exogenous lectins by an indirect immunofluorescence technique. Lectin treatment induced modifications in the arrangement of myosin, actin and tubulin, determined depolymerization of the proteins and caused changes in cell shape and size. The results suggest that the interaction between lectins and their specific membrane receptors triggers a series of changes in the cytoskeletal pattern via transmembrana as yet unknown mechanisms and that these are responsible for the alterations in cell shape and size.     

Growth-inhibitory glycopeptides obtained from the cell surface of cultured chick embryo fibroblasts

Cell surface glycopeptides were obtained from cultured chick embryo fibroblasts (CEF) by digestion with Pronase E, and a fraction exerting growth-inhibitory activity on CEF was isolated by high performance gel permeation chromatography. The active fraction, tentatively termed cell surface glycopeptide-2 (CSGP-2), was soluble in 5% trichloroacetic acid (TCA) or 75% ethanol. It inhibited the growth of CEF reversibly at 10-20 micrograms sugar/ml, but did not inhibit BALB/c mouse 3T3, SV40-transformed 3T3, and human diploid cells at similar concentration. The growth-inhibitory activity of CSGP-2 was reduced or lost after digestion with neuraminidase or oxidation with sodium metaperiodate. Cellulose acetate electrophoresis revealed that CSGP-2 was a mixture of sialoglycopeptides. A similar growth inhibitor was also isolated from chicken embryonic tissues.     

Molecular properties of a major cell surface protein from chick embryo fibroblasts.

The molecular structure of chick embryo fibroblast cell surface protein has been investigated by ultracentrifugation, circular dichroism, and fluorescence. Most measurements were restricted to alkaline solutions because of the limited solubility of this protein at more neutral pH values. A very high frictional ratio for the protein suggests an asymmetric structure. However, there are elements of organized structure since typical thermal transition curves were found by several methods. Consequently, a model in which ordered domains are connected by flexible polypeptide chains seems to account for all the hydrodynamic and optical data.     

Involvement of cell surface phosphatidylinositol-anchored glycoproteins in cell-cell adhesion of chick embryo myoblasts

During myogenesis myoblasts fuse to form multinucleate cells that express muscle-specific proteins. A specific cell-cell adhesion process precedes lipid bilayer union during myoblast fusion (Knudsen, K. A., and A. F. Horwitz. 1977. Dev. Biol. 58:328-338) and is mediated by cell surface glycoproteins (Knudsen, K. A., 1985. J. Cell Biol. 101:891- 897). In this paper we show that myoblast adhesion and myotube formation are inhibited by treating fusion-competent myoblasts with phosphatidylinositol-specific phospholipase C (PI-PLC). The effect of PI-PLC on myoblast adhesion is dose dependent and inhibited by D-myo- inositol 1-monophosphate and the effect on myotube formation is reversible, suggesting a specific, nontoxic effect on myogenesis by the enzyme. A soluble form of adhesion-related glycoproteins is released from fusion-competent myoblasts by treatment with PI-PLC as evidenced by (a) the ability of phospholipase C (PLC)-released material to block the adhesion-perturbing activity of a polyclonal antiserum to intact myoblasts; and (b) the ability of PLC-released glycoprotein to stimulate adhesion-perturbing antisera when injected into mice. PI-PLC treatment of fusion-competent myoblasts releases an isoform of N-CAM into the supernate, suggesting that N-CAM may participate in mediating myoblast interaction during myogenesis.     

External cell surface protein phosphorylation in normal and Rous sarcoma virus transformed chick embryo fibroblasts

Normal and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts growing on plastic dishes were incubated with ATP (γ³²P) in situ to detect external cell surface protein kinase activity. Under the conditions employed, ³²P was incorporated exclusively into proteins, specifically those at the external cell surface, as radioactivity was removed by tryspin treatment of labeled whole cells. In addition, exogenous histones were phosphorylated when added to the reaction mixture. Cyclic nucleotides had virtually no effect on ³²P incorporation, suggesting that little or no cyclic nucleotide-dependent protein kinase activity was present at the external cell surface. Cell surface protein kinase activity was higher in transformed than in normal cells, and, using a temperature-sensitive RSV src mutant, this difference was shown to be transformation-specific. Several differences were observed in the cell surface proteins phosphoryllated in normal and transformed cells and at least two of these were transformation-specific. These data suggest that changes in external cell surface protein physphorylation are associated with RSV transformation and thus could play a role in the formation of the transformed cell phenotype.     

Actomyosin organisation for adhesion, spreading, growth and movement in chick fibroblasts.

Examination of the actomyosin structures and their relation to adhesion, movement and growth in the first fibroblasts migrating from chick heart explants shows striking differences with fibroblasts adapted to grow in culture. The latter have focal adhesions which seem to immobilize them for anchorage-dependent growth, rather than facilitate their movement. The fibroblasts specialized for movement from the explants, though equally well spread, make contact with substratum through extensive areas of relatively unspecialized membrane, have less well developed stress fibres and a low growth rate.     

Neuraminidase activity and cell surface sialic acid turnover in Rous sarcoma virus transformed chick embryo fibroblasts

Neuraminidase activity of Rous sarcoma virus transformed chick embryo fibroblasts (RSV-CEF) was assayed using an exogenous substrate, neuraminlactitol-[3H], and endogenous, cell surface [14C]-N]-acetyl-neuraminic acid. RSV-CEF had higher neuraminidase activity toward both substrates than did chick embryo fibroblasts (CEF) or nontransformed, Rous associated virus infected CEF (RAV-CEF). The total sialic acid content of RSV-CEF was lower than CEF or RAV-CEF, and more of the total sialic acid was accessible to extracellular Clostridium perfringens neuraminidase. Activity of the enzymes synthesizing and degrading the substrate for sialyltransferase, cytidine-5'-monophosphate-N-acetyl-neuraminic acid (CMP-AcNeu) was measured in order to determine whether control of substrate levels for sialyltransferase might contribute to the decreased levels of glycoprotein bound sialic acid. No change in activity of these enzymes was found in RSV-CEF as compared to CEF or RAV-CEF.     

Most iodinatable fibroblast surface proteins accompany the cytoplast membrane during cytochalasin B-mediated enucleation of chick embryo fibroblasts

Six different proteins are found to be reproducibly exposed on the cell surface of chicken embryo fibroblasts (CEF) by the criterion of lactoperoxidase-catalyzed iodination (250,000, 185,000, 130,000, 100,000, 87,000, and 75,000 daltons). We wondered whether cell enucleation might lead to a differential partition of these surface proteins with the karyoplast or cytoplast membrane. We found that there is a marked enrichment of most iodinatable cell surface proteins in the cytoplast after cytochalasin-mediated enucleation of cell monolayers. Nearly all the iodinatable fibronectin remains with the cytoplast. Of the six labeled proteins, the karyoplast membrane contains a small amount of the 130 kdalton protein as well as trace levels of the 100-, 85-, and 75-kdalton proteins. Proteolysis or selective shedding of membrane proteins were not significant factors in the relative exclusion of iodinatable membrane proteins from the karyoplast. The cytoplast could replace some exposed membrane proteins after removal by trypsinization; however, fibronectin was not detectable within 10 h. That the karyoplast was not capable of membrane protein synthesis and/or insertion was suggested by the lack of any change in the labeling pattern of karyoplasts up to 8-h incubation after enucleation. A variety of control studies indicated that the surface proteins identified in this report were cell-derived and not adsorbed serum components. That some of the iodinatable proteins are intrinsic membrane proteins was suggested by their resistance to removal by conditions thought to extract extrinsic membrane proteins (i.e., low salt, high salt, and NaOH washes). lack of effect of cytoskeletal disrupting agents (preliminary evidence) suggests the nonrandom partition of membrane proteins may depend on anchoring of membrane proteins by a system(s) in the cytoplast other than intact microtubules and microfilaments.     

Extracellular ATP and ADA-buffer enable chick embryo fibroblasts to grow in secondary cultures in protein-free, hormone-free, extracellular growth factor-free medium

The replacement of HEPES with ADA buffer and addition of ATP to the serum-free, protein-free Eagle's minimal essential medium (EMEM) caused stimulation of proliferation in sparse, secondary cultures of resting chick embryo fibroblasts. Better cell growth and highly reproducible results were obtained if the cells had been dispersed from primary cultures with EGTA and thereafter remained without any further contact with serum or other extracellular proteins than when trypsin was used. The frequent changes of culture medium caused no retardation of cell growth. The observed more than 12 fold increase in the cell number after stimulation of the cultures with ATP is interpreted in favour of the conceptions assuming a significant role of intracellular calcium and intracellular pH in regulation of cell proliferation.     

Neutral protease activity of Rous sarcoma (RSV) transformed chick embryo fibroblasts.

The proteolytic activities of normal, Schmidt-Ruppin Rous sarcoma virus (SR-RSV) transformed, and infected (RAV) chick embryo fibroblasts (CEF) have been measured by a highly sensitive technique using 3H-acetylated haemoglobin as a substrate.When all 3 types of CEF cells were maintained in serumless media, no differences were detected in the amount of pH 3-4 protease activity released into the media over a 24-h period, and only negligible amounts of pH 7-6 proteolytic activity were found. When normal, transformed, and infected cells were maintained in serumless media and later incubated with 3H-acetylate haemoglobin, a significant proteolysis of the haemoglobin, a 6-fold increase compared to the normal CEF cells, was associated only with plates containing SR-RSV-CEF cells. A fluorescent assay for peptides confirmed that SR-RSV-CEF cells have increased cell-associated proteolytic activity. The net surface charge of the transformed CEF cells was unchanged by maintenance in serumless media but the net surface negativity of the normal and RAV-CEF cells was significantly increased by incubation in media minus serum for 24 h. This suggests that normal CEF cells, maintained in media plus serum, have a substance masking their surface charge which is absent from the surface of transformed cells, possibly because of proteolytic degradation.     

Tunicamycin-induced alterations in the synthesis of sulfated proteoglycans and cell surface morphology in the chick embryo fibroblast

The effect of tunicamycin (TM) on the synthesis and secretion of sulfated proteoglycans and hyaluronate was examined in chick embryo fibroblasts and chondrocytes. The incorporation of the precursors [³H]glucosamine, [³H]mannose and [³⁵S]sulfate into glycoconjugates in both the cell layer and medium of cultures was determined. In the chick embryo fibroblast, but not in the chondrocyte, synthesis of sulfated proteoglycan was inhibited 60–75% by TM (5 × 10⁻⁸ M), while synthesis of hyaluronate and protein was only inhibited slightly. The inhibition of sulfate incorporation into glycosaminoglycans of the chick embryo fibroblast was overcome to a great extent by addition of β-xyloside, which provides an exogenous initiator for chondroitin sulfate synthesis. TM treatment also altered cell shape and surface morphology in chick embryo fibroblasts, as observed by phase contrast and scanning electron microscopy (SEM). Cells treated with TM became rounded, and increased numbers of microvilli and blebs appeared on the cell surface. These alterations in cell morphology were reversed by removal of TM, but not by exogenous addition of xyloside, chondroitin sulfate or the adhesive cell surface glycoprotein fibronectin. These results demonstrate that TM inhibits synthesis of sulfated proteoglycans in the chick embryo fibroblast and causes a dramatic alteration in cell shape and surface morphology.     

Mechanism of the decrease in the major cell surface protein of chick embryo fibroblasts after transformation.

The major cell surface glycoprotein of cultured chick embryo fibroblasts (CSP, a LETS protein) is substantially decreased after neoplastic transformation. We investigated the regulation of this glycoprotein by determining the kinetics of CSP biosynthesis, transit to the cell surface, and degradation before and after transformation by Rous sarcoma virus. CSP synthesis, as measured by immunoprecipitation after pulse-labeling with ¹⁴C-leucine, is decreased 3–6 fold after transformation by the Bryan high titer, Schmidt-Ruppin and temperature-sensitive ts68 and T5 strains of Rous sarcoma virus. Steady state quantities of CSP in intracellular pools are also decreased 4–5 fold after transformation. However, the rate at which newly synthesized CSP is processed and exported to the cell surface is similar before and after transformation.Degradation and release of CSP from cells were measured after labeling for 24 hr. The half-life of CSP on normal cells is 36 hr and is decreased to 16–26 hr after transformation. The absolute amount of intact CSP released into the culture medium is decreased 3 fold after transformation; these amounts, however, represent losses of approximately 20 and 40% of the total CSP synthesized by normal and transformed cells, respectively. These results indicate that the major mechanism for the decrease in CSP after transformation is reduction in its biosynthesis, although increased degradation and loss from the cell surface also contribute significantly. These changes can account for the observed 5–6 fold decreases in cell-associated CSP after transformation of chick embryo fibroblasts.     

Effects of IPO-63, Carbaryl, Foschlor and Reglone on Newcastle disease virus replication in chick embryo and chick embryo cell cultures

Cell cultures and chicken embryos were treated with maximum tolerant concentrations of different compounds and infected with Newcastle disease virus simultaneously or 24 hours after the compounds were introduced. The similar results were obtained in both cases. It was found that Reglone inhibited Carbaryl increased virus multiplication. The study on dynamics of virus multiplication indicates that only in the case of IPO and Carbaryl their stimulatory effect on virus at the final stage was preceded by its inhibition.     

Effects of colcemid and taxol on microtubules and intermediate filaments in chick embryo fibroblasts

Reports on how changes in microtubule (MT) distribution or polymerization affect the distribution of intermediate filaments (IFs) differ. Therefore, we have used cytoimmunofluorescence techniques and electron microscopy to systematically examine and compare the arrangements of MTs and IFs in cultures of chick embryo fibroblasts under the following conditions: at different times during the cell cycle, in the presence of Colcemid or of taxol, in the presence of both drugs in succession or simultaneously in varying ratios, and during recovery from treatment with Colcemid or taxol. We have found that depolymerization of MTs by 1 microM Colcemid resulted in the rapid formation of massive IF-cables, structures distinct from "collapsed IFs" or "juxtanuclear coils." Neither the rapid formation of IF-cables nor their dispersion during recovery required protein synthesis. Cells treated with 10 microM taxol rapidly formed MT-bundles, as well as aggregates of intertwining IFs, termed "IF-skeins." MT-bundles and IF-skeins displayed strikingly complementary distributions. This reciprocal distribution of packed MTs and IFs was also obvious in untreated anaphase and telophase cells. When 10 microM taxol and 1 microM Colcemid were applied simultaneously, the complementary distributions of MT-bundles and IF-skeins mimicked those in taxol alone. This ability of taxol to block Colcemid's effects was concentration dependent. Decreasing the taxol: Colcemid ratio allowed the depolymerization of MTs, which correlated with the formation of IF-cables.