Density-dependent regulation of growth of BSC-1 cells in cell culture: growth inhibitors formed by the cells.

Abstract

Inhibitors formed by a monkey epithelial cell line, BSC-1, play an important role in limiting growth at high cell densities. At least three inhibitors are formed: lactic acid, ammonia, and an unidentified inhibitor that may be an unstable protein. The unidentified inhibitor is destroyed by shaking the conditioned medium, by bubbling gas through the medium, or by heating or storing the medium in the absence of cells. The concentrations of lactic acid and ammonia that accumulate in conditioned medium inhibit growth when added to fresh medium. These results, together with earlier studies, indicate that density-dependent regulation of growth of BSC-1 cells results from the combined effects of (a) inhibitors formed by the cells, (b) decreased availability of receptor sites for serum growth factors as the cells become crowded, and (c) limiting concentrations of low molecular weight nutrients in the medium. In contrast, density-dependent regulation of growth in 3T3 mouse embryo fibroblasts results almost entirely from inactivation of serum factors.Evidence in the literature suggests that there are differences between the growth controls of epithelial cells and fibroblasts in cell culture (1-4). Due to these differences and to the importance of epithelial cells in the origin of tumors, we have made a thorough study of the factors that control the growth of BSC-1 cells, an epithelial cell line of African green monkey kidney origin (5). In previous papers (4, 6) we demonstrated that (a) serum factors and (b) the concentrations of low molecular weight nutrients in the medium contribute to growth regulation of this cell line. The present paper describes the regulation of growth of BSC-1 cells by the formation of inhibitory materials in the culture medium.The initial evidence for the presence of inhibitors was a slight but consistent stimulation of the labeling index in crowded cells whenever conditioned medium with 0.1% serum was replaced by fresh medium without serum. Although the labeling index along the edge of a "wound" in the cell layer often decreased after this treatment, there was a consistent increase of the labeling index in the crowded cell layer. The-increase could not be attributed to fresh serum factors, for serum was not added.Nor could it be due to an increase in the concentrations of nutrients in the fresh medium: the concentrations of nutrients in conditioned medium were found to be at least half those in fresh medium; changing the cultures to fresh serum-free medium with only half the normal concentrations of the nutrients still stimulated DNA synthesis in the cell layer.Tests of known metabolites identified lactate and ammonium ions as significant growth inhibitors in BSC-1 cell cultures. However, the inhibition observed when these ions were added to fresh medium, at the concentrations found in conditioned medium, was not adequate to explain the above results.Evidence that the cells produce an additional, unidentified inhibitor was obtained initially from experiments in which conditioned medium w...

Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth.

“…In this regard, TGF-I3 and a growth inhibitor from green monkey kidney cells (BSC-1) have been shown to have nearly identical biologic activity, and to compete for binding to the same membrane receptor (13,34). The BSC-1 cells produce the inhibitor when arrested at saturation density.…”

“…The BSC-1 cells produce the inhibitor when arrested at saturation density. The growth inhibitor has been purified from media conditioned by density-arrested cells, and has been shown to inhibit the proliferation of the same cells at a low cell density (34).…”

Transforming growth factors and the regulation of cell proliferation

“…African green monkey (BSC-1) cell-secreted growth inhibitor described by Holley et al [35] has since been shown to be essentially identical to human TGFP2 [36]. cDNA cloning from human [37, 381, porcine [37] and chicken [39] libraries have identified a gene, TGFP3, which shares similarity with TGFPl and TGFP2.…”

Enhanced jun gene expression is an early genomic response to transforming growth factor beta stimulation.

“…It was originally described as a growth inhibitor of BSC-1 monkey kidney epithelial cells (20) and was isolated as a factor that stimulated the anchorage-independent growth of normal fibroblasts alone or in combination with epidermal growth factor (EGF) or TGFa (4,10,44,55). TGF, induces the growth of AKR-2B mouse fibroblasts in soft agar and, to some extent, the agar growth of NRK-49F rat kidney cells (55).…”