Density-dependent regulation of growth of BSC-1 cells in cell culture: Control of growth by low molecular weight nutrients

Abstract

BSC-1 cells, epithelial cells of African green monkey kidney origin, show pronounced density-dependent regulation of growth in cell culture. Growth of the cells is rapid to a density of approximately 1.5 X 105 cells/per cm2 in Dulbecco-modified Eagle's medium supplemented with 10% calf serum. Above this "saturation density," growth is much slower. It has been found that the glucose concentration in the culture medium is important in determining the "saturation density." If the glucose concentration is increased 4-fold, the "saturation density" increases approximately 50%. Reduction of the "saturation density" of BSC-1 cells is also possible by decreasing the concentrations of low molecular weight nutrients in the culture medium. In medium supplemented with 0.1% calf serum, de-creasing the concentrations of all of the organic constituents of the medium, from the high levels present in Dulbecco-modified Eagle's medium to concentrations near physiological levels, decreases the "saturation density" by approximately half. The decreased "saturation density" is not the result of lowering the concentration of any single nutrient but rather results from reduction of the concentrations of several nutrients. When the growth of BSC-1 cells is limited by low concentrations of all of the nutrients, some stimulation of growth results from increasing, separately, the concentrations of individual groups of nutrients, but the best growth stimulation is obtained by increasing the concentrations of all of the nutrients. The "wound healing" phenomenon, one manifestation of density-dependent regulation of growth in cell culture, is abolished by lowering the concentration of glutamine in the medium. Density-dependent regulation of growth of BSC-1 cells in cell-culture thus appears to be a complex phenomenon that involves an interaction of nutrient concentrations with other regulatory factors.There is considerable evidence (1-4) that the growth controls of epithelial cells differ from those of fibroblasts in cell culture. Because of this 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 BSC-1 cells, an epithelial cell line of African green monkey kidney origin (5). We have found that the growth of these epithelial cells in cell culture is controlled by (a) the concentrations of serum factors added to the culture medium, (b) the concentrations of low molecular weight nutrients in the medium, and (c) the concentrations of inhibitory materials formed by the cells. The previous paper summarized the role of serum factors (4). The present paper considers growth controls that respond to the concentrations of low molecular weight nutrients in the culture medium. MATERIALS AND METHODSCell Cultures. BSC-1 cell cultures were maintained as described in the previous paper (4). Cells were detached from the culture dishes with 0.025% trypsin in calcium-and magnesium-free Tris/saline buffer (6) with 0.5 mM sodium ethylenediaminetetraacetate. The ce...

Insulin and IGFs induce apoptosis in chick embryo retinas deprived of L-glutamine

“…Recently we have observed that glutamine deprivation causes a lowering of DNA synthesis in chick embryo retinas cultured in vitro (unpublished results). This observation agrees with the considerations previously made by many authors, that the absence of L-glutamine in the medium leads to an arrest of cell growth in G1 phase (Holley and Kiernan, 1974;Holley et al, 1978). Moreover, when insulin or IGF-I were added to retinas cultured in serum-and glutamine-free medium a further relevant lowering of DNA synthesis was observed, particularly in the youngest retinas studied (7 ± 9 days) of`in ovo' development (Tesoriere et al, 1992).This paper clearly demonstrates that the addition of IGF-I, IGF-II or insulin induces, in chick embryo retinas cultured in vitro without L-glutamine, DNA fragmentation and cell death via apoptosis.…”

“…As shown in this paper, a necessary condition for insulin or IGF-I to induce apoptosis in the retinas is incubation in a medium deprived of glutamine or phosphate. It is well known that deprivation of nutrients arrests the growth of cells cultured in vitro and favours the shift between proliferative and quiescent states (Holley and Kiernan, 1974;Holley et al, 1978). The addition to serum-and glutamine-free medium of IGF-I or insulin, compounds which are known to stimulate cell growth, may produce an unresolved conflict of signals that could be lethal for retinal cells.…”

Vascular wall growth control: the role of the endothelium.

“…This is similar to other experiments showing that low concentrations of serum are sufficient to stimulate growth of sparse density cultures of 3T3 cells, while higher concentrations of serum are needed to stimulate confluent cultures. 27 In summary, there is very little known about the actual mechanisms that control movement or replication during regeneration at wound edges. In part, this may result from the diversity of systems studied.…”

Density-dependent regulation of cell growth: An example of a cell-cell recognition phenomenon

“…The interpretation given was that the drop in EGF receptor levels was a primary cause of the density-dependent growth regulation of BSC-1 cells. They have since shown that the growth of BSC-1 cells is also exquisitely sensitive to the concentrations of nutrients in the media [37]. Thus, either a reduced nutrient concentration in the media, or the cells developing an increased requirement for EGF, would be enough to inhibit cell growth.…”

“…Holley et al [37,38] have studied the growth regulation of BSC-1 cells. One of their findings was that confluent cells contained one-tenth the number of receptors for EGF than did growing cells (this is the converse of all other cases examined, where confluent cells up-regulated their mitogen receptors).…”