Incipient and overt stages of neoplastic transformation.

A subline of NIH 3T3 cells was developed that multiplies to low saturation density and produces no transformed foci in a standard assay at confluence if the cells had been kept in continuous exponential growth by passage at extremely low density. Repeated cycles of extended incubation at confluence result in the production of large dense foci with large increases in saturation density of the culture. I investigated the changes preceding overt transformation and the conditions that favor them by making a preliminary 2- or 3-week incubation of cells at high density in 2, 5, or 10% (vol/vol) calf serum (CS) followed by three consecutive assays at 2-week intervals, all in 2% CS. The cell counts on transfer of each consecutive assay constituted their saturation density, and staining of sister dishes exhibited transformed foci and other morphological evidence of transformation. To strengthen the significance of the observations, each CS category consisted of four separate but parallel lineages. The initial incubation in 2, 5, and 10% CS is referred to as the first-round (1 degree) assay. In the second-round (2 degrees) assay in 2% CS after the primary incubation for 2 weeks in different concentrations of CS, there were small but consistent increases in saturation density that were proportional to the CS concentration used in the first round. There were also large numbers of very light foci in the 2 degrees assay that increased in size as a function of the CS concentration used in the 1 degree assay but were uniform within each CS category. In the 3 degrees and 4 degrees successive assays, large dense foci appeared in a sporadic manner but these were also dependent on the CS concentration in the initial incubation. Cultures kept in the 1 degree assay in 5 and 10% CS for 3 weeks produced large dense foci at an earlier stage of the subsequent assays than did those kept for only 2 weeks in the 1 degree assay. The barely perceptible nature of the early morphological changes suggests an analogy to incipient neoplasia. Their graded nature, relatively high frequency, regularity of occurrence, and uniformity of appearance are characteristic of an epigenetic process, which Foulds [Foulds, L. (1969) Neoplastic Development, Vol. I (Academic, New York), preface, pp. 41-89] cited as the most plausible basis for incipient neoplasia [corrected]. In contrast, the low-frequency and sporadic occurrence of the large dense foci, which have the main features of overt neoplasia, are consistent with both genetic and epigenetic processes.