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Cells of the NIH 3T3 line gain the capacity to produce neoplastically transformed foci when they are maintained at high density for more than 1 week and transferred in a standard assay for focus formation. This change in cell behavior has been variously attributed to an adaptive response to the constraint of the high population density or to a spontaneous genetic change that increases in probability for a culture with the increase in the total number of cell divisions. To distinguish between these alternatives, 200 cells of the 28H subline were seeded in many culture dishes of two size classes differing 6-fold in surface area and allowed to multiply for 1, 2, and 3 weeks. At each weekly interval, 18 dishes of each class were assayed for focus formation, and two of the original dishes were stained for focus formation. The cells in the small (S) and large (L) dishes multiplied to the same extent at 1 week and produced only a few small light foci in some of the assay dishes. At 2 weeks, cells in the S dishes had become confluent and had only one-third the number of cells as those in the nonconfluent L dishes. Upon assay, 14 of the 18 S cultures produced some foci whereas only 9 of the L cultures did so. In addition, 4 of the S cultures produced large dense foci while none of the L cultures did. By 3 weeks, the L cultures were confluent and had four times as many cells as the S cultures. When assayed at this time, both sets produced dense foci in many of the cultures and light foci in the remaining ones, indicating a narrowing of the differences between the S and L cultures between 2 and 3 weeks of incubation. There were differences in the morphology of the foci produced in parallel assays from different cultures. The results showed that transformation is a diverse graded response to the growth constraint of high population density and not a spontaneous event dependent on the number of cell divisions in a cell culture. Transformation thus is basically an epigenetic process since it represents a response to physiological restraint, but the final form of response may be modulated by genetic alterations.

A critical test of the role of population density in producing transformation.