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

Abstract

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...

Self-normalization of highly transformed 3T3 cells through maximized contact interaction.

“…Clone 14 cells grow to a high density with as little as 1% serum whereas nontransformed cells multiply to a much lower density in low serum concentrations (2). At low density, clone 14a cells reach their maximal rate of DNA synthesis in 1% serum whereas, at very high density, there is a large increase in [3H]thymidine incorporation as the serum level is increased from 1% to 30%.…”

“…They also acquire the serum dependency and Ca2+ content of nontransformed cells (2). In these particulars, Mg2+ deprivation "normalizes" the transformed cells but, in another particular, it does not appear to do so.…”

“…Effects similar to those of crowding can be produced among transformed cells at low cell densities by decreasing the external Mg2+ concentration (1,2 is involved in the response to crowding (9).…”

“…Transformed cells that have reached their saturation density under conventional culture conditions have not been shown to accumulate in the G1/GO period ta the same extent as nontransformed cells do (3)(4)(5). Although Mg2+ deprivation decreases the initial rate of multiplication oftransformed and nontransformed cells to the same extent, the former usually continue to multiply until they approach the saturation density ofcells in physiological Mg2+ concentrations (2).…”

“…2 b andf). Those transferred from the saturation-density coverslips spread and flattened out in a manner characteristic of nontransformed cells (1,2) and maintained that appearance for about a-day (Fig. 2d) erslip cultures-i.e., considerably greater than the increases in population density-it seemed a priori unlikely that limitation of cell multiplication was due to medium depletion.…”