Serum factors modify the cellular requirement for Ca2+, K+, Mg2+, phosphate ions, and 2-oxocarboxylic acids for multiplication of normal human fibroblasts.

Abstract

The rate of multiplication of a population of cultured human lung fibroblasts is determined by the level of common nutrients and serum factors in the medium. By application of the principles of Henri-Michaelis-Menten kinetic analysis to cell growth in vitro, the relationship between the level of a macromolecular fraction of serum that contains growth factors and the concentration of individual nutrient that is required to support a half-maximal rate of cell multiplication was explored. The requirement for the majority of individual nutrients is independent of the level of serum factors in the medium. This includes all 20 amino acids, glucose, purines, pyrimidines, polyamines, choline, and inositol. In contrast, serum factors determine the cellular requirement for Ca2+, K+, Mg2+, Pi, and 2-oxocarboxylic acids. These nutrients are most likely involved in cellular processes related to the mechanism by which growth factors in serum control the cell multiplication rate.Serum-derived factors regulate the multiplication of untransformed cells in culture (1-4). Although much is known about the interaction of several purified growth factors with the cell membrane (5-11), less is understood about how the factors cause the biochemical and morphological events that culminate in cell division. Several studies suggest a relationship between regulatory factors and extracellular nutrients in the control of cell division (12-16). Observations on changes in uptake, intracellular pool sizes, and in rate of nutrient-related processes that occur parallel to changes in rate of DNA synthesis circumstantially implicate almost every major nutrient (15, 16). Of primary concern is which of these changes are involved in control of cell multiplication and which changes occur parallel to DNA synthesis but are not causal processes whose rates limit multiplication (17). Serum growth factors may control multiplication-limiting processes where nutrients are involved by control of the intracellular level of a nutrient around the Km for the nutrient in the rate-limiting process, or serum factors may alter the Km of the process for the nutrient whose normal level in the microenvironment is near the Km. Theoretically, both types of relationships could be reflected in an effect of serum factors on the extracellular requirement for a nutrient for multiplication of cells in culture, where extracellular levels of nutrients and serum factors can be closely controlled and manipulated. We have applied the principles that are used in classical enzyme kinetic analysis to simultaneously relate the level of serum factors and the level of individual nutrients in the medium directly to the multiplication rate of cells (18,19). The analysis revealed that serum factors modify the cellular requirement for a small subset of the nutrients commonly present in the extracellular environment. This subset consists of Ca2+, K+, Mg2+, Pi, and 2-oxocarboxylic acids. We propose that serum growth factors control cell proliferation by control of access to or requirement...

Phosphatidyl choline and the growth in serum‐free medium of vascular endothelial and smooth muscle cells, and corneal endothelial cells

“…Nevertheless, PC is still quite effective in replacing HDL for relatively short-term growth. McKeehan and McKeehan (1980) recently suggested that PC i s one of the extracellular nutrients most likely to be involved in the mechanism by which serum growth factors control cell multiplication. Consistent with this hypothesis are the recent reports linking membrane phospholipid synthesis, in particular that of PC, to DNA synthesis (Hirata and Axelrod, 1980) and passage through the cell cycle (Cornell and Horwitz, 1980).…”

Extracellular regulation of fibroblast multiplication: A direct kinetic approach to analysis of role of low molecular weight nutrients and serum growth factors

“…Growth factors may change multiplication rate by causing a change in the cellular requirement (So.so value) for a nutrient that is analogous to a change in the K, of an enzyme for its substrate. To examine for this, we applied the kinetic approach to relate simultaneously the level of FBSP or other growth factors and the level of individual nutrients in the medium to cell multiplication rate [19,20,23,[33][34][35][36]. Three different types of relationships were observed between FBSP and individual nutrients (Figs.…”

“…Although some lipids 1301, niacinamide 1291 and the ions of Fe and Se showed class I and I1 relationships with FBSP, a functional modification of their requirement in multiplication by FBSP could not be distinguished from their addition to deficient culture medium as a contaminant in the FBSP [35]. Table I1 summarizes the classification of all the nutrients tested.…”

“…Lechner and Kaighn also showed that EGF reduced the Ca2' requirement for multiplication of a normal prostatic epithelial cell line [20]. Fibroblast growth factor (FGF) also reduced the Ca2+ requirement for proliferation of bovine endothelial cells carboxylic acids may be closely involved in the mechanism by which growth regulatory factors exert their controls on cell multiplication [23,35]. On the one hand, if the reduced dependence on extracellular Ca", Mg2+, K', Pi, and 2-oxocarboxylic acids that is caused by growth factors reflects an increase in availability of the nutrients to key intracellular processes that control multiplication, then these 5 nutrients can be considered mediators of the primary action of serum growth factors.…”

“…SV-HLF required significantly less (P < 0.001) of these nutrients for multiplication than did N-HLF. Conclusions about class I and I1 nutrients (Table 11) were more difficult because of their complicated kinetic relationships with serum factors in both maintenance (Table 111) and multiplication [35,36] (Tables I1 and 111). However, when compared at equivalent and limiting levels of serum factors in the medium, a reduction in the requirement for Ca2', hfg", K' , Fe, and Se ions for multiplication of SV-HLF was apparent (Tables I1 and 111 specific nutrients appear quantitatively important in cell division beyond their roles in cell maintenance, and the quantitative requirement for multiplication of several (but not all) of these nutrients was reduced as a consequence of viral transformation and evolution of the transformed cell line.…”