Calcium, magnesium, and serum factors in multiplication of normal and transformed human lung fibroblasts

A spontaneously transformed clone was isolated from repeatedly passaged BALB/c 3T3 cells. The transformed cells were rounded or slender and elongated, were randomly arranged in an overlapping pattern, grew to high cell density, and had a low requirement for serum. The rates of multiplication and DNA synthesis of the nontransformed and the transformed lines were reduced for several days by drastic reduction in the Mg2+ concentration of the medium, but the rate of DNA synthesis in the Mg2'-deprived cultures increased after [6][7][8] We wished to develop a quantitative assay that would indicate whether the transformed cells had indeed adopted the more stringent growth requirements of nontransformed cells. The largest quantifiable difference between the transformed and the nontransformed cells is in the amount ofserum required for the initiation of DNA synthesis. The transformed cells require only 1% or 2% serum to reach their maximum rate ofDNA synthesis, but the nontransformed cells require 10-20% serum to reach an equivalent state. We found that a stringent serum dependency of DNA synthesis could be imposed on the transformed cells within a 3-day period by limiting the supply of Mg2' and controlling the seeding density ofthe cells. Although the Mg2+ content of the cells is only slightly reduced in Mg2+-deficient medium, the Ca2+ content triples, exceeding the level found in nontransformed cells. The Mg2+-deprived cells therefore not only look like, but behave like, and have the ionic composition of nontransformed cells. These observations are consistent with the hypothesis that Mg2+ plays an important regulatory role in cell growth (2, 3) and that a specific loss ofthis role occurs in transformation (4, 5). MATERIALS AND METHODSCells and Culture Methods. The cells used were a flat subclone of the A31 clone of BALB/c 3T3 cells obtained from J. Bartholomew. When they reached confluency, they were arranged in a systematic monolayered array, had a fixed saturation density, and did not form colonies in agar. Repeated transfer of the cells gave rise to a spontaneously transformed clone that differed markedly in appearance and behavior from the nontransformed cells (1). The transformed cells were rounded or slender and elongated, were randomly arranged and overlapped one another, grew to high cell densities, and formed large colonies in agar. The nontransformed cells were designated clone 2, and the transformed cells were designated clone 14. They were grown in MCDB 402 medium (6) Proc. Natl. Acad. Sci. USA 78 (1981) 2351 labeled nuclei by autoradiography, and protein content by the Lowry procedure were as described (2). Intracellular Mg2" and Ca2" contents were determined by the procedure of Sanui and Rubin (7). Briefly, the cultures were rinsed 5 times with CO2-free sucrose solution (pH =7) and once with C02-saturated sucrose solution (pH =4). The cells were harvested by scraping them from the dishes with a polyethylene policeman and suspended in distilled deionized water. The cell suspensions were s...

supporting

confidence: 91%

Restoration of normal appearance, growth behavior, and calcium content to transformed 3T3 cells by magnesium deprivation.

Rubin¹,

Vidair²,

Sanui³

1981

“…Cell culture minimal essential medium (ME medium) and medium 199 were from GIBCO. Medium MCDB 107 was prepared from high-quality chemicals as described (13). Bovine pituitary extract (BPE) was prepared (14) from frozen pituitaries obtained from Pel-Freez.…”

Section: Methodsmentioning

confidence: 99%

Brain- and liver cell-derived factors are required for growth of human endothelial cells in serum-free culture.

Hoshi¹,

McKeehan²

1984

A test of the mitogenic activity of >40 purified and crude sources of hormones and growth factors revealed that epidermal growth factor, high density lipoprotein, an extract of bovine pituitary, hypothalamus, or whole brain, and the medium conditioned by differentiated human hepatoma cells were (9,10), and an acid pituitary-or brain-derived factor (5,11,12) are active mitogens for endothelial cells derived from human umbilical vein (HUVE). In addition, thrombin has been reported to potentiate the action of EGF, basic fibroblast growth factor (6-8), and partially purified platelet-derived growth factor (9). Although there is no consensus on the importance of platelet mitogens (5), thrombin (7), and the basic (5, 11) and acidic (8) growth factors from brain, it appears agreed that EGF and one or more factors derived from neural tissue are mitogens for HUVE (5-8). However, in the presence of both EGF and brain-derived factors, HUVE stillThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6413have a fastidious requirement for unidentified plasma-derived factors (5-12).Our studies show that, in addition to EGF and brain-derived factors, one or more factors from the medium of differentiated human hepatoma cells is required for HUVE proliferation in the absence of serum. This provides a serum-free medium in which to study and clarify the endocrinology of growth, function, and aging of cultured human endothelial cells.

“…Clonal multiplication was estimated by densitometric measurement of the size of colonies formed from single cells as described (4,(20)(21)(22)(23) (vol/vol) perchloric acid at 100'C. Pyruvic acid and other 2-oxocarboxylic acids were assayed as described (25).…”

Section: Introductionmentioning

confidence: 99%

“…All tissue culture methods and the development and preparation of the media series, MCDB 100, and fetal bovine serum proteins (FBSP) have been described (4,(20)(21)(22)(23). FBSP was used as a complete source of factors that stimulate cell multiplication rate above that supported by the defined medium and other optimized culture conditions (4,(20)(21)(22)(23). Multiplication rate in medium MCDB 107 is half-maximal at 43.4 + 10.2 ,gg of FBSP per ml (mean + SEM of six determinations).…”

mentioning

confidence: 99%

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

McKeehan¹,

McKeehan²

1980

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...