The hypothesis that induction of immortalization of rodent cells follows one-hit kinetics was tested by the determination of frequencies of immortalization of Syrian hamster embryo cells after treatment with benzo(a)pyrene, X-rays, or ethylnitrosourea. Contrary to expectation, immortalization did not occur in a single step. Full immortalization appeared to be a process which required at least three steps: extension of life span (step 1), increase in cloning efficiency (step 2), and increase in growth rate (step 3). These three steps occur in this fixed sequence. The first step appears to be induced by the carcinogenic treatment, while the two other steps occur spontaneously in the progeny of cells which underwent the first step. The frequency of induction of the first step is in the order of magnitude of mutation induction, which suggests that mutation in one allele of a limited number of loci is sufficient to initiate the process of immortalization. However, the spontaneous frequency of immortalization is below 2.4 × 10-9/cell/generation, which appears to be too low for a spontaneous mutation frequency. The frequencies/cell/generation of the second and the third step are in the order of magnitude of spontaneous mutation frequencies.

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The research was supported by the Dutch Cancer Foundation, Grant IKW 87–4, by the J. A. Cohen Institute for Radiopathology and Radiation Protection, and by Contract B-16-E-141-NL and EV4V-0047-NL of the Association between the European Community with the University of Leiden.

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