Characterization of a Quantitative Assay for the in Vitro Transformation of Normal Human Diploid Fibroblasts to Anchorage Independence by Chemical Carcinogens1

We have characterized an assay for the quantitative measure ment of the frequency of conversion to anchorage-independent growth of W-acetoxy-2-acetylaminofluorene-treated normal hu man diploid fibroblasts. We investigated the effects of the follow ing parameters on the absolute number and on the frequency of anchorage-independent colonies scored: (a) the number of cells seeded per dish; (b) the type of posttreatment medium; (c) the number of population doublings allowed posttreatment prior to seeding in suspension; and (d) the carcinogen dose. The assay was linear over the range of 1.9 x 103 to 3.8 x 104 cells seeded per 60-mm dish for both total colonies scored and the induced frequency of anchorage-independent growth. The medium used posttreatment affected both the frequency and the kinetics of appearance of the anchorage-independent phenotype. The number of population doublings and the number of days allowed posttreatment prior to assaying for anchorage-indepen dent growth potential also influenced the frequency of recovery of this phenotype. Under standardized conditions, the assay yielded a dose-response relationship for transformation to an chorage independence over the concentration range of 0 to 10 lÃ-M/v-acetoxy-2-acetylaminofluorene.