Resting state depth and saturation density as modulated by tumor promoters and culture conditions: relationship to chemical transformation in C3H/10T1/2 cells.

With the use of cultures of C3H/10T1/2 fibroblasts, the relationships of saturation density and cell resting state (Go) depth to oncogenic transformation by 3-methylcholanthrene (MCA) were investigated. The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), caused a small increase in the saturation density, but saccharin (SAC), a suspected promoter, did not, Correspondingly, TPA tested positively as a promoter of transformation in a typical two-stage transformation assay with these cells while SAC did not. By altering the confluent-phase serum concentration and/or the medium renewal frequency of MCA-treated cultures, it was found that the magnitude of transformation for a given concentration of MCA was independent of the saturation density. Oscillations in this density were observed even when the medium was renewed as frequently as every 3 days, indicating that cell death and replacement occur during what is generally regarded to be a period of cellular stability. Neither TPA nor SAC blocked the cells from progressing into deeper resting states over time, as indicated by the prereplicative lag time after a medium change at 42 days. However, the serum concentration and medium renewal frequency did affect the resting state depth. Daily renewals with medium containing 10% serum blocked the resting state from deepening and kept the cells from achieving a saturation density. Under these circumstances, transformation was completely inhibited in MCA-treated cultures.