Preneoplastic potential of morphologically distinct transformed foci induced by 3‐methylcholanthrene

Individual variability of scoring foci positive for transformation presents a difficult problem in assessing the transformation assay. In this study, an attempt was made to identify five morphologically distinct types of transformed foci based on size (2–3, 3–4, and ≥4 mm in diameter), invasiveness (smooth vs. invading margins), and other properties (piling vs. spread) induced by 3‐methylcholanthrene in Balb/c‐3T3 cells. The transformed focal cells were used in in vitro studies including anchorage‐independent analysis, focal reconstruction, gene transfection using NIH‐3T3 host cells, and Southern blotting to assess amplification of five proto‐oncogenes (K‐ras, H‐ras, c‐fos, c‐jun, c‐myc) and a tumor suppressor (p53) gene. Results showed that 1) there was a significant increase in anchorage‐independent growth of all five types of foci ranging from 7–12%; 2) all five morphological types of transformed foci showed 8–15% focal reconstruction; 3) DNA from all five types of transformed foci induced transformation in NIH‐3T3 cells at a level significantly above the control DNA; 4) gene amplification studies indicated amplification in both K‐rasand H‐rasproto‐oncogenes; however, c‐fos, c‐jun,and c‐mycdid not show DNA amplification. The tumor suppressor gene (p53) was activated and the increase was up to 3‐fold over the normal Balb/c‐3T3 DNA. These findings are consistent with our hypothesis that all five morphologically different foci have preneoplastic potential and that any foci of size ≥ 2 mm regardless of invasiveness and piling should be scored as positive during the transformation assay. Environ. Mol. Mutagen. 32:369–376, 1998 © 1998 Wiley‐Liss, Inc.