A system is described for the formation of bone tissue in culture from isolated rat bone cells. The isolated bone cells were obtained from embryonic rat calvarium and periosteum or from traumatized, lifted periosteum of young rats. The cells were cultured for a period of up to 8 wk, during which time the morphological, biochemical, and functional properties of the cultures were studied. Formation of bone tissue by these isolated bone cells was shown, in that the cells demonstrated osteoblastic morphology in light and electron microscopy, the collagen formed was similar to bone collagen, there was mineralization specific for bone, and the cells reacted to the hormone calcitonin by increased calcium ion uptake. Calcification of the fine structure of the cells and the matrix is described. Three stages in the calcification process were observed by electron microscopy. It is concluded that these bone cells growing in vitro are able to function in a way similar to such cells in vivo. This tissue culture system starting from isolated bone cells is therefore suitable for studies on the structure and function of bone.

A series of stromal cell lines were studied for their growth properties, electron microscopic morphology, cytochemical profile, collagen types, production of myelopoietic factors, and modulation of leukemic cell growth. Three cell types were identified in addition to the previously described macrophages (14M and 14M1) and preadipocytes (14F). MBA-1 cells were found to be fibroblasts by their ability to synthesize collagen types I and III, while the cell line MBA-13 shared properties in common with both fibroblasts and endothelial cells (collagen types I, III, IV, V). The third cell type, represented by the stromal cell line MBA-2, produced mainly collagen types IV and V and exhibited junctional complexes between adjacent cells. All of the cell lines tested produced and secreted a macrophage-colony-stimulating factor, CSF-1. MBA-2 and to a lesser extent, MBA-13, produced an additional activity resistant to anti-CSF-1 antiserum. Trypsin extraction of outer surface components from two clones of the MBA-2 cell line (MBA-2.1 and MBA-2.4) yielded high molecular weight factor(s) that specifically inhibited the growth of a plasmacytoma cell line (MPC-11). Such inhibitory activity was not detected in other stromal cell lines. It is possible that this variability in the nature of stromal cell lines represents corresponding diversity of cell types comprising the hematopoietic microenvironment in vivo.

A glycoprotein immunologically related to plasma cold-insoluble globulin (CIG) and fetal skin fibroblast fibronectin has been purified from second-trimester human amniotic fluid. This protein (amniotic fluid fibronectin) migrated more slowly than CIG on sodium dodecyl sulfate gel electrophoresis and showed greater polydispersity which could result, at least in part, from heterogeneity in glycosylation. Cloned human amniotic fluid epithelioid and fibroblastic cells synthesized and secreted a protein with similar properties into the culture medium. Fibronectin was shown to be associated with the pericellular and extracellular matrix of cultured amniotic fluid cells by immunofluorescence, lactoperoxidase-catalyzed iodination, and labeling with ferritin-conjugated antibodies. The kinetics of secretion of the protein were consistent with its role as a matrix protein. We anticipate that amniotic fluid fibronectin will prove to be the same protein which elsewhere in the body is incorporated into connective tissues and basement membranes. Amniotic fluid could, therefore, serve as a convenient source of in vivo synthesized flbronectin for biological and structural studies. [37]) is a high molecular weight glycoprotein synthesized by many cells in culture and found both in the culture medium and in association with the cell surface (3, 16). The protein is immunologically related to cold-insoluble globulin (CIG), 1 a disulfide-bonded, dimeric 1 Abbreviation~ used in this paper: fl-APN, fl-aminopropionitrile; BSA, bovine serum albumin (fraction V); plasma glycoprotein with a subunit molecular weight of -220,000 (32, 36). However, the relation of fibronectin to CIG, at a structural level, is not known.CIG, cold-insoluble globulin; DMEM, Dulbeceo-Vogt modified Eagle's medium; DTr, dithiothreitol; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid; MalNEt, N-ethylmaleimide; PBS, calciumand magnesium-free phosphate-buffered saline; PhCH,SOzF, phenylmethanesulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Tris-saline, 0.15 M NaC1, 0.05 M Tris-HCl, pH 7.5.

Normal and virally transformed mouse (3T3) and human cells were treated with tunicamycin, an inhibitor of lipid-carrier-dependent glycosylation of proteins.Incubation of cells with tunicamycin (1 jug/ml) caused detachment and death of simian virus 40-and polyoma-transformed cells within 24 hr; these effects were not seen with nontransformed cell lines. However, the proliferation of 3T3 cells was inhibited by tunicamycin and, after a few days, a distinct change from an epithelioid to an abnormally elongated shape was observed. Both inhibition of growth and the morphological changes were reversible. A marked decrease in concanavalin A agglutinability was observed in virally transformed cells treated with tunicamycin (0.5 gg/ml), but agglutination by wheat germ agglutinin or soybean agglutinin was unaffected. Analysis of biosynthetically labeled proteins showed that a high-molecular-weight protein, presumed to be related to fibronectin, is markedly reduced in the medium of cells cultured in the presence of tunicamycin. These results suggest that tunicamycin interferes with the insertion or function of one or more cell-surface glycoproteins. Su~,h cell-surface changes could affect a number of cellular properties, including attachment, cell shape, and agglutinabilityby some lectins. Many aspects of the social behavior of cells are influenced by the composition, arrangement, and interaction of cell-surface macromolecules (1). Thus, alterations in plasma membrane composition and structure in malignant and transformed cells, compared with normal cells, appear to contribute to differences in such characteristics as cell adhesion, contact inhibition of movement, and tumorogenicity (2). Cell-surface glycoproteins, in particular, participate in a number of membrane-modulated phenomena, including responsiveness to hormones, agglutination by lectins, and recognition by antibodies (3, 4); these properties are frequently altered following transformation (5, 6).Extensive studies have been made of cell-surface glycoproteins and of the alterations that occur after transformation (5, 6), but little is known about the function of the carbohydrate moieties in these proteins or of the significance of transformation-associated changes in their structure (7-9). A prominent finding in transformed cells has been the absence or marked reduction of a high-molecular-weight cell-surface glycoprotein variously known as LETS protein, fibroblast surface antigen, or fibronectin (6).We have examined the influence of tunicamycin, an antibiotic that inhibits protein glycosylation (10-12), on the behavior of normal and virally transformed cells in culture. Tunicamycin blocks the transfer of GlcNAc from UDP-GIcNAc to a polyisoprenyl lipid carrier (13-15). Consequently, synthesis and transfer of the core oligosaccharide to asparaginyl sideThe costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore...

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