Multistep origin of tumor-forming ability in Chinese hamster embryo fibroblast cells.
Twenty-one anchorage-independent subclones and ten subclones with reduced serum requirements were isolated as single-step mutants spontaneously or after ethylmethanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis of CHEF/18 diploid Chinese hamster embryo fibroblasts. Anchorage-independent mutants retain the high serum requirement and nontransformed morphology typical of CHEF/18. Only four of 21 anchorage mutants have spontaneously produced tumors when injected at 10(7)/site in nude mice, and these were only at a fraction of sites. Low-serum (LS) mutants acquire transformed morphology and increased anchorage-independent 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 populations 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 DHEF/18 cells have given tumors at only three of 29 sites. These results demonstrate that malignant transformation is a multistep process in the Chinese hamster embryo fibroblast system. Most one-step mutants selected for anchorage independence or reduced serum requirements do not have tumor-forming 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.