Neoplastic conversion of preneoplastic Syrian hamster cells: rate estimation by fluctuation analysis

Analysis of the role of gene mutations in the multistep process of neoplastic transformation requires that the discrete steps in carcinogenesis first be dissected. Toward this end, we have isolated and characterized preneoplastic Syrian hamster cells which exhibit in vitro a trait highly correlated with neoplastic conversion in vivo. Previous findings (J. C. Barrett, Cancer Res. 40:91-94, 1980) indicate that spontaneous neoplastic transformation of Syrian hamster cells occurs in at least two steps. An intermediate stage, characterized by an aneuploid established cell line which has a propensity to become neoplastic spontaneously upon further growth in vitro, has been described. These preneoplastic cells differ from diploid early-passage Syrian hamster cells in becoming capable of anchorage-independent growth in semisolid agar, as well as becoming neoplastic in vivo when attached to a solid substrate. Evidence presented here demonstrates that anchorage-independent conversion in vitro is a reliable marker for neoplastic conversion in this cell system. Fluctuation analyses, patterned after those described by Luria and Delbruck for microbial genetics, demonstrate that anchorage-independent variants are generated randomly from clonally derived preneoplastic cells at the rate of 10(-8) to 10(-7) variants per cell per generation. These results establish a multistep stochastic process for transformation in vitro and indicate that conversion to anchorage independence may be necessary for Syrian hamster cells to become tumorigenic. The possible role of gene mutation in this step during neoplastic progression is discussed.