Embryo Fibroblast Cells Multistep Origin of Tumor-forming Ability in Chinese Hamster

Twenty-one anchorage-independent subclones and ten subclones with reduced serum requirements were isolated as single-step mutants spontaneously or after ethylmethanesulfonate or A/-methyl-/V'-nitro-/V-nitrosoguanidine mutagenesis of CHEF/18 diploid Chinese hamster embryo fibroblasts. An chorage-independent mutants retain the high serum require ment and nontransformed morphology typical of CHEF/18. Only four of 21 anchorage mutants have spontaneously pro duced tumors when injected at 107/site in nude mice, and these were only at a fraction of sites. Low-serum (LS) mutants acquire transformed morphology and increased anchorageindependent growth simultaneously with the loss of high-serum requirement. Only two of ten LS mutants have spontaneously produced tumors. However, when some anchorage and LS mutants were remutagenized and when mutagenized popula tions were injected into nude mice, tumors appeared at many of the injected sites. In contrast, untreated CHEF/18 cells have never given tumors (0 of 34 sites), and mutagenized CHEF/18 cells have given tumors at only three of 29 sites. These results demonstrate that malignant transformation is a multistep proc ess in the Chinese hamster embryo fibroblast system. Most one-step mutants selected for anchorage indepen dence or reduced serum requirements do not have tumorforming potentials higher than that of the parent CHEF/18. Thus, anchorage-independent and LS phenotypes per se do not account for the increase in tumor-forming potential. It is proposed that the genomic rearrangement process as well as specific mutations may contribute to tumorigenicity.