Solute concentration effects on the expression of cellular heterogeneity of anchorage-independent growth among spontaneously transformed BALB/c3T3 cells.

Clones were derived in culture from a tumor initiated by spontaneously transformed 3T3 cells and tested for their colony-forming efficiency in agar (CFEag). Incubation of petri dish cultures was done in subsaturation humidity to minimize mold contamination. There was great variation in CFEag between clones but also, under certain conditions, within clones. The most prominent condition that generated phenotypic diversity in CFEag was partial evaporation of the medium, which may occur during the protracted development of a mass population from a single cell. Evaporation was disproportionately great in 35-mm dishes and peripheral wells of multiwell plates. If the supraphysiological solute concentration resulting from evaporation was greater than 133% of normal, there was progressive suppression of cell growth in the succeeding transfer in agar or on plastic, even if isotonic medium was substituted 1 d before transfer. The effect of supraphysiological concentrations of all the solutes of the medium could be reproduced by simply increasing the NaCl concentration. Damaged cells were restored to their full growth potential after 3 d in isotonic medium. When nontransformed cells were chronically exposed to increased salt, irreversible increases in 2-deoxyglucose uptake were produced. With continued exposure of these cells to high salt, they became morphologically transformed, produced colonies in agar with high efficiency, and formed sarcomas when inoculated into nude mice.