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

Nontransformed and moderately and highly transformed BALB/c 3T3 cells maintained on small coverslips in a large volume of medium multiplied to 2, 3, and 4 times higher population density, respectively, than they did in conventional cultures. Deprivation of Mg2+ caused highly transformed cells on coverslips to assume the appearance of nontransformed cells, decrease their rate of multiplication, and stop further growth at a much lower saturation density than the same cells in physiological Mg2+. The latter cells reached a saturation density of 10(6)/cm2 and their rate of DNA synthesis decreased progressively with increased crowding. At saturation density, cells in physiological Mg2+ took on an appearance and arrangement similar to normal fibroblasts. They developed a high requirement for serum to initiate DNA synthesis. When transferred at low density, they flattened out on a plastic surface and maintained the appearance of nontransformed cells for approximately 1 day. Onset of DNA synthesis and multiplication in the transferred cells was delayed for periods characteristic of quiescent nontransformed cells stimulated by fresh medium or transfer. Cells from crowded coverslips were approximately 1/10th as efficient at colony formation when suspended in agar as cells from uncrowded coverslips. They also had a significantly lower Mg2+ content. The crowded cells returned to their transformed morphological and growth behavior 2 to 3 days after transfer at low density. We conclude that a very high degree of crowding causes highly transformed cells to revert to the phenotype of nontransformed cells. Other treatments such as deprivation of Mg2+ or inorganic orthophosphate can achieve similar results. It appears that a balanced reduction in rates of metabolism and multiplication can restore the normal phenotype to transformed cells, implying that they differ only quantitatively from nontransformed cells. The putative role of Mg2+ in the regulation of multiplication and in transformation of animal cells is discussed.