A spontaneously transformed clone of BALB/c 3T3 cells became more transformed after more than 90 passages as indicated by increased rounding of cells, multiplication to a higher saturation density, and increased ability to form colonies when suspended in agar. When the extracellular concentration of Mg2+ was sharply reduced, the highly transformed cells flattened, assumed the shape of nontransformed cells, and became regularly arranged in cohesive arrays. If crowded when deprived of Mg2+, they lost more intracellular Mg2+ than did nontransformed and early passage-transformed cells and remained at constant cell density for at least 10 days. The intracellular content of neither Na+ nor K+ changed consistently with Mg2+ deprivation, but the Ca2+ content increased more than 2-fold. The sensitivity of the onset of DNA synthesis to inhibition by Mg2+ deprivation increased with the extent of crowding of the cultures. This was demonstrated by varying population density within a single culture dish as well as from culture to culture. The loss of intracellular Mg2+ in low concentrations of extracellular Mg2+ increased with cell crowding as did the inhibition of DNA synthesis per fractional loss of intracellular Mg2+. Neither deprivation of K+ or Ca2+ nor addition of cyclic adenosine 3′:5′-monophosphate produced a density-dependent inhibition of DNA synthesis.

The results indicate that a reduction of the Mg2+ content of highly transformed cells restores density-dependent inhibition of the onset of DNA synthesis, which is a characteristic property of nontransformed cells. The differences in Mg2+ retentiveness with population density may reflect differences of intracellular distribution and binding of Mg+, which could in turn explain some of the regulatory effects of population density on metabolism and growth.

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Supported by Research Grants CA-15744 from the National Cancer Institute and DE-ATO3-79EV10277 from the United States Department of Energy.

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