Characterization of a quantitative assay for the in vitro transformation of normal human diploid fibroblasts to anchorage independence by chemical carcinogens

• Published in: Cancer research (Chicago, Ill.) Vol. 43; no. 5; pp. 2176 - 2182
• Main Authors: Zimmerman, R J, Little, J B
• Format: Journal Article
• Language: English
• Published: United States 01.05.1983
• Subjects: 2-Acetylaminofluorene - analogs & derivatives; Acetoxyacetylaminofluorene - pharmacology; Cell Adhesion - drug effects; Cell Count; Cell Division - drug effects; Cells, Cultured; Culture Media; Cytological Techniques; Diploidy; Dose-Response Relationship, Drug; Fibroblasts - drug effects; Fibroblasts - ultrastructure; Humans; Infant, Newborn; Male; Phenotype; Skin
• ISSN: 0008-5472

We have characterized an assay for the quantitative measurement of the frequency of conversion to anchorage-independent growth of N-acetoxy-2-acetylaminofluorene-treated normal human diploid fibroblasts. We investigated the effects of the following 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 10(3) to 3.8 X 10(4) cells seeded per 6-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-independent growth potential also influenced the frequency of recovery of this phenotype. Under standardized conditions, the assay yielded a dose-response relationship for transformation to anchorage independence over the concentration range of 0 to 10 microM N-acetoxy-2-acetylaminofluorene.