Transformation of C3H10T1/2 cells by N-ethyl-N-nitrosourea occurs as a single, low frequency, mutation-like event

C3H10T1/2 cells have been found to be relatively insensitive to transformation by N-ethyl-N-nitrosourea (ENU) and some other strongly carcinogenic, direct-acting, monofunctional alkylating agents. Because these agents are strong inducers of gene mutations we have considered the possibility that they transform 10T1/2 cells through a gene mutation. Such an event could have been missed in the standard assay, in which relatively low numbers of cells are treated. We found that the yield of ENU-induced transformed foci could be increased drastically by increasing the seeding density (up to 10(4) viable cells/cm2). The transformation frequency (foci/survivor) after ENU treatment was lower but much less dependent on cell density than after 3-methylcholanthrene. To investigate the mechanism of transformation by ENU in more detail, we used a modified transformation protocol which involved treatment of the cells at high density, followed by a period of exponential growth and reseeding at high density for focus selection. We found that ENU-induced transformation frequency is independent of the length of the growth period, thus independent of the total number of posttreatment cell divisions. This means that the focus-forming ability is completely fixed within maximally four generations. The transformation frequency does, however, depend on the number of divisions between the final reseeding and confluence. This suggests that the phenotypic expression of at least a part of the ENU-induced transformants is influenced by their clone size at confluence. Finally, we observed that the dose dependency of transformation was qualitatively and quantitatively similar to that of mutation at the Na-K-ATPase locus. Together, these experiments indicate that transformation of C3H10T1/2 cells by ENU is the result of a single, low frequency event, probably a gene mutation.