Isolation of Human Diploid Cell Variants With Enhanced Colony-Forming Efficiency in Semisolid Medium After a Single-Step Chemical Mutagenesis: Brief Communication 2

To examine the role of somatic mutation in malignant transformation, we studied the induction of anchorage-independent variants from normal human diploid fibroblasts by single-step mutagenesis in vitro. Primary cell strains from normal human embryo and newborn (foreskin) tissues were treated with N-methyi-N′-nitro-N-nitrosoguanidine and plated in methylcellulose. Growing colonies were obtained at a frequency of about 10-5 per mutagenized cell. In untreated controls, the frequency of colony-forming cells was less than 10-8. In many of the colonies isolated from methylcellulose, the enhanced ability to form colonies in the semisolid medium was maintained as a stable property. These “anchorage-transformed” clones were unchanged in morphology but formed multilayered foci in confluent monolayer culture. In chromosome composition, ability to plate in 1% serum, and susceptibility to cellular senescence, they were not distinguishable from the parental cells. When injected into nude mice, the variants did not produce tumors. These results indicated that human diploid cell variants specifically transformed with respect to anchorage regulation of growth can be induced by a single-step mutagenesis in vitro, and that other transformed phenotypes are not necessarily expressed in such variant.