Mechanisms involved in the immortalization of mammalian cells by ionizing radiation and chemical carcinogens.

Immortalization is a prerequisite for the clonal evolution and malignant transformation of normal mammalian cells in culture. In order to gain a mechanistic insight into the genetics of carcinogen-induced cellular immortality, a cell culture assay has been developed based on the use of freshly explanted Syrian hamster dermal (SHD) fibroblasts. The relative efficacies of a variety of chemical and physical carcinogens at immortalizing SHD cells (against a zero background of spontaneous immortalization) were compared. Ionizing radiation and nickel chloride appeared to be more effective as immortalizing agents than powerful point mutagens, suggesting (but not proving) that clastogenic damage may be more significant in the immortalization process than point mutation. Frequencies of induced immortality (10(-6)-10(-7)/treated cell) were arguably consistent with a direct mutational mechanism involving a single genetic target. However, detailed cytogenetic characterization of a panel of newly immortalized cell lines revealed no non-random chromosomal alterations in the cells at the level of G-banding. Furthermore, additional experiments with the SHD system have provided confirmatory evidence that immortalization can occur as an indirect consequence of carcinogen exposure following an induced high frequency change in the treated population, rather than through direct targeted mutagenesis. Previous somatic cell genetic studies have suggested the possibility that a target gene for immortalization exists on the human and Chinese hamster X chromosomes. Here we provide strong evidence that the normal SHD X chromosome displays powerful senescence-inducing properties when introduced, by microcell transfer, into newly immortalized SHD recipients. These results suggest that induction of the immortal phenotype in SHD cells by carcinogens results primarily from functional inactivation of a senescence gene which may be X-linked. One possible mechanism for senescence gene inactivation consistent with our observations is through a sub-microscopic interstitial genetic deletion. However, the considerable efficacy of nickel (a human carcinogen) as an immortalizing agent at nonmutagenic doses raises the alternative possibility that immortalization may occur through an epigenetic mechanism.