Growth regulation, reverse transformation, and adaptability of 3T3 cells in decreased Mg ²⁺ concentration

Abstract

A nontransformed and a spontaneously transformed clone of BALB/c 3T3 cells were compared for their capacity to multiply in decreased concentrations of Mg2e. Cells of the nontransformed clone were flat, formed regularly patterned, nonoverlapping arrays, required high serum concentration for multiplication, had a low saturation density, and did not make colonies in agar. Cells of the transformed clone were slender and spiky, formed random, overlapping arrays, multiplied in low serum concentrations, and had no fixed saturation density, and 20-30% of them formed colonies in agar. The When animal cells in culture are treated with growth stimulants such as insulin or serum, various reactions in the cells are accelerated (1). This group of cellular reactions has been designated "the coordinate response" (2, 3). Many of the early reactions of the coordinate, response are independent of one another and of macromolecular synthesis (1). The only intracellular substance that has the capacity, both theoretically and experimentally, to modulate the various reactions of the coordinate response is Mg2", and I have proposed that Mg2+ acts as a second messenger for these growth stimulants (2, 3). McKeehan and coworkers concurred in this conclusion and additionally proposed that cell transformation causes a selective loss ofthe regulatory role ofMg2+ (4, 5). Their conclusions were based on measurements of the size of colonies of normal and transformed cells formed in varying concentrations ofMg2+ and in other putative growth regulatory substances.I set out to extend their findings by comparing the effects of exposure to different concentrations ofMg2+ on growth rate and saturation density of a nontransformed clone of 3T3 cells and a spontaneously occurring transformed variant of that line. I found that the differential requirements' ofthe two cell lines for Mg2+ depend on a complex set of variables which will be described elsewhere. The experiments did show a consistent relationship between the initial multiplication rate of nontransformed cells and their saturation density, which clarifies the relative contributions of growth stimulatory materials and population density in fixing limits to the size of a population. Deprivation of Mg2`caused the transformed cells to resemble nontransformed cells in appearance and growth behavior. After about 1 week in Mg2+-deficient medium, the rate of DNA synthesis began an increase which continued through the second week in both nontransformed and transformed cells, and the latter reverted to their original appearance. MATERIALS AND METHODSCell Types, Transfers, and Labeling Techniques. The original cells used in these experiments were of the A31 clone of BALB/c 3T3 cells which had been recloned by J. Bartholomew to obtain a uniform culture of flat cells. This "parental clone" was obtained in January 1979 and maintained by weekly transfers at low population density in Dulbecco's modification of Eagle's medium containing 10% calf serum in the early passages and 10% fetal calf serum lat...

Magnesium deprivation inhibits the expression of differentiation‐related phenotypes in human promyelocytic leukemia HL‐60 cells

“…It has been reported that magnesium deprivation reduces DNA, RNA, and protein synthesis in chicken fibroblasts (Rubin, 1979, and restores both normal appearances and growth regulation in spontaneously transformed BALB/c 3T3 cells (Rubin, 1981). Magnesium specifically regulates adenylate cyclase activity in numerous cells and species (Cech et al, 1980) and enhances insulin-dependent stimulation of cyclic AMP phosphodiesterase in adipocytes (Ueda et al, 1984).…”

Magnesium restriction induces granulocytic differentiation and expression of P27Kip1 in human leukemic HL-60 cells

“…As compared to their normal counterpart, several neoplastic cells may nevertheless exhibit higher levels of intracellular Mg [Rubin, 1982a]. Furthermore, it has been described that spontaneously or virus transformed fibroblasts and other cells grown under Mg restriction may rapidly assume mor-phological and functional characteristics of differentiated cells [Rubin, 1981[Rubin, , 1982bMcKeehan et al, 1978;Hoftiezer et al, 1985]. Dietary restriction of Mg and cell Mg deficiency have also been shown to accelerate aging both in vivo and in vitro [Gü nther, 1991;Costello et al, 1992;Calviello et al, 1993].…”

Mg2+‐sensitive alterations in Ca2+ regulation associated with cell transformation

“…The spontaneously transformed clone (clone 14) and the flat, nontransformed clone (clone 2) were isolated as described (Rubin, 1981). Cells were maintained in MCDB 402 medium with 10% calf serum.…”

“…The subsequent procedure for determining the rate of thymidine incorporation has been described (Rubin, 1981).…”