Effect of fibronectin on the adhesion of an established cell line to a surface reactive biomaterial.

We have demonstrated that an established hamster cell line (NIL 8 M-2) will adhere to the bioceramic bioglass. The rate at which the NIL 8 M-2 cells assume a spread morphology on bioglass is density dependent and the morphology displayed by NIL 8 M-2 cells attached to bioglass is much more elongated than that displayed by NIL 8 M-2 cells attached to nonreactive glass. Precoating the bioglass with the plasma form of human fibronectin significantly reduces the density dependent nature of cell spreading. Coating the bioglass with fibronectin also reduces the time required for cell spreading and changes the morphology of the attached cells from an elongated to an extremely flattened shape. Our work raises the possibility that bone-implant adhesion might be improved by introducing molecules relevant to cell-substrate attachment into the biomaterial prior to implantation.

Nuclear envelope of Chinese hamster ovary cells. Re-formation of the nuclear envelope following mitosis.

We have developed a technique for isolating nuclei and nuclear envelope(s) (NE) from Chinese hamster ovary (CHO) cells which does not depend on the use of detergents to solubilize contaminating cytoplasm. In our procedure NE are prepared from purified nuclei by nuclease digestion and subsequent high salt-sucrose gradient centrifugation. The nuclei and NE fractions are free of significant contamination by other subcellular organelles as judged by electron microscopy and enzyme analysis. Examination of the peptide and glycopeptide composition of the NE fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a very complex coomassie blue staining profile with prominent bands in the 55 000-75 000 molecular weight range. Using this NE isolation technique, we have examined the breakdown and re-formation of the NE during a limited stage (late G2, M, and early G1) of the replicative cycle in synchronized populations of CHO cells. Our data demonstrate that a minimum of 60% of the early G1 NE protein and a minimum of 50% of the early G1 NE phospholipid were present in the cell during the preceding G2 phase of the cell cycle and were reutilized in the re-formation of the NE occurring during late M and early G1. Our evidence suggests that the vast majority of the newly synthesized peptides and glycopeptides of the NE which appear in the daughter NE are synthesized during the early G1 phase of the replicative cycle. Examination of the NE peptides by one-dimensional gel electrophoresis suggests that no reproducible changes in NE peptide composition can be correlated with specific phases of the cell cycle.

An analysis of concanavalin A-mediated agglutination in two Chinese hamster ovary subclones whose surface phenotypes respond to maintenance in medium supplemented with dibutyryl cyclic AMP. V. Biochemical composition of the plasma membrane.

We have used two Chinese hamster ovary subclones whose surface phenotype has been extensively investigated with regard concanavalin A-mediated cell-cell agglutination and concanavalin A-induced receptor site clustering to investigate what changes in membrane composition, if any, can be correlated with the concanavalin A-detected changes in surface phenotype. These cell clones are uniquely disposed for this purpose since maintenance of the cells under different growth conditions produces changes in agglutinability and receptor site mobility in one cell clone (H-7W) but not the other (K-1). After extensive characterization of the surface membranes of these two subclones we have been unable to identify any change in the membrane peptides, glycopeptide, cholesterol, or fatty acid composition which can be directly correlated with the concanavalin A-detected surface phenotypes. It is of particular interest to note that we have been unable to correlate the presence or absence of the large external transformation-sensitive glycoprotein with the relative mobility of the lectin receptors or with the degree of concanavalin A-mediated cell agglutination. Furthermore we have been unable, in this system, to corroborate earlier data suggesting a role for cholesterol in determining the relative mobility of the lectin receptors. Thus using a cell system consisting of genetically matched cell clones, we have been unable to identify any changes in the biochemical composition of the plasma membrane which might be associated with the surface phenotypes detected by concanavalin A.

Production of monoclonal antibodies against a cell surface concanavalin A binding glycoprotein.

Concanavalin A-binding (Con-A)-binding cell surface glycoproteins were isolated, via Con A-affinity chromatography, from Triton X-100-solubilized Chinese hamster ovary (CHO) cell plasma membranes. The Con A binding glycoproteins isolated in this manner displayed a significantly different profile on sodium dodecyl sulfate--polyacrylamide gels than did the Triton-soluble surface components, which were not retarded by the Con-A-Sepharose column. [125I]-Con A overlays of the pooled column fractions displayed on sodium dodecyl sulfate--polyacrylamide gel electro-phoresis (SDS-PAGE) demonstrated that there were virtually no Con A receptors associated with the unretarded peak released by the Con A-Sepharose column, whereas the material which was bound and specifically eluted from the Con A-Sepharose column with the sugar hapten alpha-methyl-D-mannopyranoside contained at least 15 prominent bands which bound [125I]-Con A. In order to produce monoclonal antibodies against various cell surface Con A receptors, Balb/c mice were immunized with the pooled Con A receptor fraction. Following immunization spleens were excised from the animals and single spleen cell suspensions were fused with mouse myeloma P3/X63-Ag8 cells. Numerous hybridoma clones were subsequently picked on the basis of their ability to secrete antibody which could bind to both live and glutaraldehyde-fixed CHO cells as well as to the Triton-soluble fraction isolated from the CHO plasma membrane fraction. Antibody from two of these clones was able to precipitate a single [125I]-labeled CHO surface component of approximately 265,000 daltons.

Characterization of the membrane fraction isolated by the fluorescein mercuric acetate technique of Barland and Schroeder.

Using scanning electron microscopy we have demonstrated that tha membrane fraction isolated by the fluorescein mercuric acetate technique of Barland and Schroeder (Barland, P. and Schroeder, E.A. (1975) J. Cell Biol. 45, 662-668) represents a topologically distinct membrane which circumscribes the cell nucleus. Our data suggest that not all the cells within a non-synchronized cell population release a membrane fraction after treatment according to the technique of Barland and Schroeder, but rather that the efficiency of membrane release achieved using this preparative technique is dependent on the morphology of individual cells. Our work has also demonstrated that the peptide composition of the membrane fraction isolated by the technique of Barland and Schroeder differs from the peptide composition of the plasma membrane-enriched fraction isolated by the technique of Brunette and Till (Brunette, D.M. and Till, J.E. (1971) J. Membrane Biol. 5, 215-224). This difference in peptide composition is particularly noticeable among the higher molecular weight proteins, glycoproteins and iodineateable membrane components. The data which we have accumulated suggest that the compositional differences noted between the two membrane isolates do not result from differential extraction of membrane components during the ZnCl2-fluorescein mercuric acetate treatments required in the isolation technique originally described by Barland and Schroeder. However, our data do clearly demonstrate that the membrane isolation technique of Barland and Schroeder cannot be used to study the general composition of the plasma membrane.

An analysis of Con A-mediated agglutination in a Chinese hamster ovary subclone which responds morphologically to growth in dibutyryl cyclic AMP. III. The role of microvilli in the agglutination process.

We have used the H-7w subclone of a Chinese hamster ovary cell line (K1) to investigate the role of cell surface architecture (specifically microvilli, blebs, and sheets) in determining the relative agglutinability of a cell line with Con A. Our evidence clearly demonstrates that no specific, immediately recognizable surface architecture is associated with the agglutinable or non-agglutinable phenotype. Our data suggest that the expression of microvilli on the cell surface is neither necessary to nor sufficient for the phenotype described by enhanced agglutinability with Con A. Furthermore our work demonstrates that cells covered with blebs are as agglutinable as cells covered with microvilli thereby suggesting that the intertwining of microvilli may not be an essential facet of the agglutination phenomenon.

The effect of TLCK on transcription and its role in modifying cell growth.

The synthetic protease inhibitor N-tosyl-L-lysine-chloromethyl ketone (TLCK) acts to inhibit transcription when added to cell lines growing in vitro. This inhibition of transcription is most pronounced in transformed cells where TLCK is very toxic at concentrations as low as 25 mug/ml of culture medium. Non-transformed cells are more resistant to the effect of TLCK, requiring ten times more TLCK to produce a comparable inhibition of transcription. The effect of this protease inhibitor on transcription can be prevented by preincubation of the cells in reduced glutathione or cysteine; however, the cells can not be rescued from the effect of TLCK even if glutathione or cysteine are added to the culture medium within five minutes of the addition of TLCK.

The dissociation of the surface architecture described by enhanced lectin agglutinability and the transformed phenotype expressed as anchorage independence.

Using a series of cold-sensitive variants of chemically transformed BHK-21 cells, revertants to the normal phenotype derived from a dimethyl-nitrosamine transformed clone of BHK-21 as well as revertants to the normal phenotype derived from polyoma transformed BHK-21 cells we have demonstrated that the surface phenotype described by enhanced agglutinability with Con A and WGA can be dissociated from the transformed phenotype described by anchorage independence (growth in semisolid medium). Specifically we have demonstrated that the surface characteristic of enhanced agglutinability may be found in a variety of cell lines which fail to display to grow in agar. Our work clearly shows that the two phenotypes described are not concomitantly controlled and tends to suggest that the phenotype of enhanced lectin agglutinability may be dissociated from the transformed phenotype.

The effect of SV40 transformation on the chromosomal proteins of 3T3 mouse embryo fibroblasts.

The composition and metabolism of chromosomal proteins-histones and nonhistones chromosomal proteins-were examined in normal and SV40 transformed 3T3 mouse cells. Variations were observed, many of which were similar to those previously reported for normal and SV40 transformed W138 human diploid fibroblasts. The possible implications of these viral induced changes in the protein component of the genome for the phenotypic modifications which occur in transformed cells are discussed.

An analysis of lectin-initiated cell agglutination in a series of CHO subclones which respond morphologically to growth in dibutyryl cyclic AMP.

We have investigated the molecular basis of the agglutinability of CHO subclones which respond differentially in terms of morphology and surface architecture in the presence of dB-cAMP in the medium. We have demonstrated that the agglutinability of these subclones with both wheat germ agglutinin (WGA) and concanavalin A (Con A) probably depends on the free lateral mobility of the lectin receptor sites in the plane of the membrane. The nonagglutinable surface architecture seems to depend on the presence in the membrane of a protease-labile peptide(s), which appears to be distinct from the lectin receptors, as well as on continuous protein and RNA synthesis. This dependence on continuous transcription and translation may be related to the maintenance of the protease-labile peptide(s) in such a state as to restrict mobility of the lectin receptors. The surface architecture defined as nonagglutinable also depends on the state of polymerization of the intracellular microtubules and microfilaments. It is suggested that these microskeletal elements serve to anchor the lectin receptors in such a manner as to restrict their mobility and thereby reduce the relative agglutinability of a cell line. We suggest that control of the free mobility of both the Con A and WGA receptor sites is dependent on two constraints, one applied by protease-labile ("surface") membrane components and the other by components of the intracellular microskeletal system.

Leukemia virus infection of mammalian cells: effect on two "transformation-associated" surface properties.

We demonstrated that the productive infection of three different mammalian cell lines with two separate leukemia viruses is sufficient to induce a change in surface architecture that may be detected as enhanced agglutinability with two different plant lectins. Subsequent transformation of one of these cell lines with a chemical carcinogen did not further modify the agglutinability of the cell lines. Using a polyoma virus-transformed derivative of one of the parental lines, we have demonstrated that the LETS protein (whose absence from the surface membrane has been considered a marker of the transformed phenotype) may be present in cells displaying the capacity to plate in soft agar.

Isolation and preliminary characterization of that part of the plasma membrane which is apposed to the substratum.

We have developed a membrane isolation technique which allows us to isolate that part of the plasma membrane which is apposed to the solid substratum (LPM) to which cells in vitro attach free of the remainder of the plasma membrane (UPM). Our evidence suggests that the UPM is essentially free of contaminating cellular organelles while the LPM may be somewhat contaminated with endoplasmic reticulum fixed to it during the isolation procedure. Characterization of the peptides of the UPM and LPM suggests that some membrane components are nonrandomly distributed to one or the other membrane fraction while the bulk of the membrane peptides appear to be present in both membrane fractions; Membrane components labeled with 3H-fucose or 3H-glucosamine show a 3--5-fold higher specific activity in the LPM than in the UPM. A number of iodineateable surface components, in particular the LETS protein, show a preferential localization to the LPM. The development of this membrane isolation procedure should allow us to begin investigating the molecular basis of differences in cell-substratum adhesion and cell motility, two functions assigned operationally to the LPM.

The relationship of concanavalin A binding to lectin-initiated cell agglutination.

We have investigated the relationship of concanavalin. A binding to the cell surface of normal and transformed cells and the subsequent agglutination of the transformed cells. At room temperature almost no differences could be detected in agglutinin binding between transformed and untransformed cells. At 0 degrees C, however, where endocytosis was negligible, the transformed cells bound three times more agglutinin. However, transformed cells and trypsin-treated normal cells do not agglutinate at 0 degrees C although the amounts of agglutinin bound at 0 degrees C are sufficient to permit agglutination when such cells are shifted up to room temperature. Both transformed and trypsin-treated normal cells show a marked increase in agglutination at 15 degrees C as compared to agglutination at 0 degrees C. From this, as well as the observation that mild glutaraldehyde fixation of the cell surface inhibited agglutination but not agglutinin binding, it was concluded that concanavalin A-mediated cell agglutination requires free movement of the agglutinin receptor sites within the plane of the cell surface.