Two high molecular weight growth inhibitors have been isolated from the culture medium of BSC-1 cells, epithelial cells of African green monkey kidney. The purified kidney epithelial cell growth inhibitors, at ng/ml concentrations, reversibly arrest the growth of BSC-1 cells in the GI phase of the cell cycle. Their action is selective; they are most active on BSC-1 cells, are less active as inhibitors of the growth of rat lung and human breast epithelial cells, and do not inhibit the growth of 3T3 mouse embryo fibroblasts and human skin fibroblasts in culture. Their growth inhibitory action on BSC-1 cell cultures is counteracted by epidermal growth factor or calf serum.One of the areas of ignorance that remains in our broad understanding of the control of growth of mammalian cells is the subject of endogenous growth inhibitors. The existence of a number of growth inhibitors has been postulated (1, 2), and growth-inhibitory fractions have been obtained from various mammalian cells and tissues (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Nevertheless, there are doubts as to the significance of endogenous growth inhibitors because most of the inhibitor preparations have low specific activity, the inhibitors have been difficult to purify, and with some preparations the causes of growth inhibition have, at least in part, turned out to be trivial (15-17). There is no doubt that highly active growth inhibitors exist for mammalian cells, because interferon and corticotropin are extremely active as growth inhibitors in certain cell cultures (18,19), but the inhibitory activities of these two substances could be incidental to their other biological activities. Because of the potential significance of growth inhibitors and because purified inhibitors could be very useful in the manipulation of growth of mammalian cells, we have undertaken further studies in this area.Density-dependent regulation of growth of BSC-1 cells (African green monkey kidney epithelial cells) has been found to result in part from the accumulation of growth inhibitors in the culture medium (20). The present paper describes the isolation of high molecular weight growth inhibitors from the culture medium of these cells. The growth inhibitors have very high specific activities, approximately equal to the specific activities of interferon and corticotropin in their respective cell culture assays. The new growth inhibitors appear to be selective in the types of cells on which they act. MATERIALS AND METHODSCell Cultures. BSC-1 cell stock cultures were maintained as previously described (21) in Dulbecco's modified Eagle's medium (DME medium) (22) Preparation of Conditioned Medium. Dense BSC-1 cell cultures, used for preparation of conditioned medium, were grown in 15-cm Falcon tissue culture plates in DME medium with 1% calf serum. The medium was changed once a week until the cells reached a density of approximately 1.5 X 105 cells per cm2. The cultures were then maintained in DME medium with 0.1% calf serum. To prepare conditioned medium, the cultu...

BSC-1 cells grow slowly, to high cell density, in medium with 0.1% calf serum. An increase in the serum concentration increases both the growth rate of the cells and the final cell density. The serum can be replaced to some extent by epidermal growth factor (EGF). Initiation of DNA synthesis in BSC-1 cells that have spread into a "wound" in a crowded cell layer requires the addition of a trace of serum or EGF, if the cells have previously been deprived of serum. The binding of 125I-labeled EGF to low-density and high-density BSC-1 cells has been studied. Binding is faster to low-density cells. Cells at low cell density also bindmuch more EGF per cell than cells at high cell density. The fraction of bound 12.1-labeled EGF that is present on the cell surface as intact EGF is larger at low than at high cell density. The results indicate that the number of available EGF receptors per cell decreases drastically as the cell density increases. It is suggested that a decrease in the number of available EGF receptor sites per cell, and the accompanying decrease in sensitivity of the cells to EGF, contributes to density-dependent regulation of growth of these cells. Information in the literature suggests that growth controls of epithelial cells differ from those of fibroblasts in cell culture (1-3). Epithelial cells often have a lower serum requirement, and the cell density to which they grow appears to be less dependent on the serum concentration and more dependent on the surface area of the culture dish (2). Because of these differences and because of the importance of epithelial cells in the origin of tumors, we have made a thorough study of the factors that control the growth of an epithelial cell line. The cell line, BSC-1 cells, originated from an African green monkey kidney (4). We have found that the growth of these epithelial cells is controlled by (i) the concentrations of serum factors added to the culture medium, (ii) the concentrations of low molecular weight nutrients in the medium, and (iii) the concentrations of inhibitory materials formed by the cells. The present paper summarizes the role of serum factors. MATERIALS AND METHODS

The complete amino acid sequence of the BSC-1 cell growth inhibitor, including its precursor polypeptide, is reported. The sequence was deduced from the nucleotide sequence of the cDNA. The N-terminal amino acid sequence of the mature bioactive BSC-1 cell growth inhibitor is identical with the N-terminal sequences of the factors that have been called type j32 transforming growth factor and cartilageinducing factor B, suggesting that these are identical. The complete amino acid sequence of the mature BSC-1 cell growth inhibitor differs from that of human type 13 transforming growth factor in 32 of the 112 amino acids. Polyergin is proposed as the name for the BSC-1 cell growth inhibitor.A growth inhibitor (GI) produced by BSC-1 African green monkey kidney epithelial cells has been isolated from serumfree growth medium exposed to high-density cultures of the cells (1). The GI is very active in arresting the growth of a variety of cells in culture (2) and it can inhibit the growth of a human mammary carcinoma in vivo in nude mice (2). The BSC-1 cell GI has been found to be functionally related to human platelet type 8 transforming growth factor (TGF-83) (3). TGF-p is one of a number of structurally related growth and differentiation factors (4). The present paper reports the complete amino acid sequence of BSC-1 GI, and its precursor polypeptide, as deduced from the nucleotide sequence of the cDNA.t MATERIALS AND METHODSIsolation of the BSC-1 Cell GI. High-density cultures of BSC-1 cells (5) were maintained in roller bottles (Corning, 490 cm2) in 125 ml of Dulbecco's modified Eagle's medium with 1% calf serum. To prepare conditioned medium, the culture medium was decanted from the bottles and the bottles were filled with Dulbecco's modified Eagle's medium without serum (pH 7.2-7.4, 37°C) and the bottles were incubated for 24 hr at 37°C without rolling (6). The conditioned medium was collected and was concentrated in a 2.5-liter Amicon unit with a 150-mm YM10 ultrafiltration membrane. Each group of nine roller bottles gave 12 liters of conditioned medium and the inhibitor was obtained at the conclusion of the concentration procedure in a total of 30 ml of 1 M acetic acid washes of the YM10 membrane (1, 7). Six sets of 30-ml washes were combined and concentrated to 2 ml in a 400-ml Amicon unit, using a 76-mm YM10 membrane. The concentrate was diluted with 30 ml of 1 M acetic acid and reconcentrated to 2 ml. The concentrate was combined with two 2 ml of 1 M acetic acid washes of the YM10 membrane and the combined solution was subjected to gel filtration through a column of Bio-Rad P60 (2 x 30 cm) in 1 M acetic acid (1, 8). After assay (1) the fractions with highest specific activities (fractions from 60-84 ml of the effluent, with 2.5 Ag of bovine serum albumin added per ml) were lyophilized, the residues were transferred to a single tube using small volumes of 1 M acetic acid, and the solution was relyophilized. The final residue was dissolved in 200 p.l of 0.1% trifluoroacetic acid and the solution was injec...

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...

A kidney epithelial cell growth inhibitor, isolated from BSC-1 cell-conditioned medium, has been found to be active on certain lung and mammary gland cell lines in culture. The most responsive cell observed thus far is the CCL64 mink lung cell line. With CCL64 cells, 60% inhibition of [3H]thymidine incorporation is observed at a 0.1 nanogram/ml concentration of the growth inhibitor, and approximately 95% inhibition at 1 nanogram/ml. A human mammary tumor cell line, Hs578T, shows 75% inhibition of [3H]thymidine incorporation, in cell culture. Preliminary studies indicate that injection of the kidney epithelial cell growth inhibitor in vivo into human mammary carcinomas growing in nude mice inhibits [3H]thymidine incorporation in the tumors.