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Effect of Magnesium Content on Density-dependent Regulation of the Onset of DNA Synthesis in Transformed 3T3 Cells¹

Department of Molecular Biology, University of California, Berkeley, California 94720

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.

¹ Supported by Research Grants CA-15744 from the National Cancer Institute and DE-ATO3-79EV10277 from the United States Department of Energy.

Received 7/27/81. Accepted 1/22/82.