Evidence that a second event in X-ray-induced oncogenic transformation in vitro occurs during cellular proliferation

It has previously been hypothesized that radiation transformation in vitro is a two-step process; the first step is a frequent alteration occurring among a large fraction of the irradiated cells, while the second step, malignant transformation, is a rare event occurring with an approximate frequency of 10(-6) among the progeny of the irradiated cells. Data are reported here on the distributions of transformed-cell clone sizes in irradiated cultures reseeded at various times post-treatment. The results suggest that the second event in transformation occurs randomly during the growth of irradiated cultures of C3H 10T1/2 cells to confluence. When the same number of irradiated C3H 10T1/2 cells were seeded into petri dishes of different sizes [35 mm (8 cm2), 60 mm (21 cm2), 100 mm (55 cm2), 150 mm (145 cm2)], the number of foci which arose per dish was dependent on the final cell numbers at confluence in the various dish sizes, such that the number of foci/cm2 was constant. When irradiated cells and parental C3H 10T1/2 cells were mixed in different proportions at low density, the number of foci which ultimately arose was a function of the number of progeny of irradiated cells present in the culture at confluence. The results presented here confirm previous studies and give further evidence that the radiation-induced malignant transformation of cells occurs in an indirect, multistage fashion.