Low Density High Density

Mouse 3T3 cells and their Simian Virus 40-transformed derivatives (3T3SV) were used to assess the relationship of transformation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan (GAG). Glucosamineand galactosamine-containing G A G were labeled equivalently by [all]glucose regardless of culture type, allowing incorporation into the various G A G to be compared under all conditions studied. Three components of each culture type were examined: the cells, which contain the bulk of newly synthesized G A G and are enriched in chondroitin sulfate and heparan sulfate; cell surface materials released by trypsin, which contain predominantly hyaluronic acid; and the media, which contain predominantly hyaluronic acid and undersulfated chondroitin sulfate. Increased cell density and viral transformation reduce incorporation into G A G relative to the incorporation into other polysaccharides. Transformation, however, does not substantially alter the type or distribution of newly synthesized GAG; the relative amounts and cellular distributions were very similar in 3T3 and 3T3SV cultures growing at similar rates at low densities. On the other hand, increased cell density as well as density-dependent growth inhibition modified the type and distribution of newly synthesized GAG. At high cell densities both cell types showed reduced incorporation into hyaluronate and an increase in cellular G A G due to enhanced labeling of chondroitin sulfate and heparan sulfate. These changes were more marked in confluent 3T3 cultures which also differed in showing substantially more G A G label in the medium and in chondroitin-6-sulfate and heparan sulfate at the cell surface. Since cell density and possibly density-dependent inhibition of growth but not viral transformation are major factors controlling the cellular distribution and type of newly synthesized GAG, differences due to GAGs in the culture behavior of normal and transformed cells may occur only at high cell density. The densityinduced G A G alterations most likely involved are increased chondroitin-6-sulfate and heparan sulfate and decreased hyaluronic acid at the cell surface. 280 THE JOURNAL OF CELL BIOLOGY 9 VOLUME 71, 1976 9 pages 280-294 on A uust 8, 2017 jcb.rress.org D ow nladed fom Glycosaminoglycans are major components of the carbohydrate at the surfaces of animal cells (20). Cell surface glycosaminoglycan (GAG) has been implicated in a variety of phenomena including cell recognition and intercellular adhesion (32), cell mobility (49), embryonic induction (27), and maintenance of embryonic organ morphology (1). Several studies implicate cell surface G A G in the control of fibroblast growth in vitro. The growth rate of cells in suspension cultures is inhibited by addition of several types of sulfated G A G (24) and of dextran sulfate, a GAG-like polyanion (13, 28), which also reversibly reduces saturation densities of cells in monolayer culture (6). Viral transformation of cells to the neoplastic state alters their in vitro growth behavior, and transformation has repeatedly been shown to decrease sulfated G A G synthesis (12, 34, 36, 48), but there are conflicting reports of its effect on hyaluronic acid synthesis (15, 39). Compared to normal cells, the surfaces of transformed ceils show greater heterogeneity of glucosamine-labeled G A G (52) and a thicker layer of ruthenium red-staining material, presumably representing G A G (25). After removal of cells from the substratum with chelators, a greater proportion of sulfate-labeled G A G remains bound to the substratum in cultures with low saturation densities than in transformed cell cultures (34), although studies of the G A G remaining bound to the substratum using glucosamine labeling revealed few quantitative differences between the cell types (34). Heparan sulfate at the cell surface is selectively lost to the medium just before mitosis (22) and is more susceptible to removal by trypsin on normal cells than on transformed cells (4), suggesting that it may play a role in controlling cell growth. A possible mechanism for this control has recently been proposed (34, 48) which suggests that increased G A G at the cell surface may be involved in enhancing cell-substratum adhesion, thereby reducing saturation densities. While such studies have suggested that surface G A G influences or reflects the growth behavior of cells in culture, most of these studies have used radiosulfate as the G A G precursor, preventing assessment of hyaluronic acid and undersulfated proteoglycan production, and in no instance to our knowledge has the cellular distribution of sulfated and nonsulfated G A G been systematically examined in normal and transformed cells under varying growing conditions. In the present study, the cellular distribution of newly synthesized glucose-labeled G A G was assessed in mouse 3T3 and SV40-transformed 3T3 cells at low cell densities where the cells were growing at equivalent rates and at high cell densities where the 3T3 cells were growth inhibited. Under the labeling conditions used, G A G precursors were at the same specific activities regardless of culture type. Three components were isolated from each culture type (Fig. 1): (a) the medium, which contains G A G secreted during the labeling period; (b) the cell surface, containing the G A G removed from the cells and the substratum with crystalline trypsin; and (c) the cells, which were free of trypsin-sensitive surface materials. For each fraction, [3H]glucose incorporation into the following G A G was measured: the galactosaminoglycans, chondroitin-6-sulfate, dermatan and chondroitin4-sulfate, and chondroitin; and the glucosaminoglycans, hyaluronic acid, and heparan sulfate. This experimental design allowed an assessment of the relationship of transformation, cell density, and density-dependent growth inhibition to the cellular distribution of newly synthesized GAG. MATERIALS AND METHODS