High frequency of carcinogen-induced early, preneoplastic changes in rat tracheal epithelial cells in culture

To study the mechanisms of carcinogenesis, we have developed a system that uses normal cells from an environmentally and epidemiologically relevant tissue, respiratory epithelium. The induction of preneoplastic variants of epithelial cells in culture was quantitated on a per-cell basis following exposure of rat tracheal epithelial (RTE) cells in vitro to the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Following treatment of normal RTE cells, large colonies of altered cells exhibiting an enhanced growth potential under selective culture conditions were observed, while normal RTE cells ceased proliferation after several cell doublings. After further growth in culture, these altered cells acquired the ability to grow in semisolid medium and to produce squamous cell carcinomas when injected into nude mice. The induction of enhanced growth variants of RTE cells by MNNG occurred with a high frequency (greater than or equal to 2.6%/colony-forming cell). In addition, a linear dose-response curve with a slope of approximately 1 was observed when the logarithm of MNNG-induced transformation frequency was plotted versus the logarithm of MNNG dose. These results are consistent with a one-hit mechanism for induction of preneoplastic variants of RTE cells by MNNG. Similar frequencies and kinetics of induction of preneoplastic variants in other culture systems using diploid cells have been observed, suggesting a common mechanism for this early step in carcinogenesis. The RTE cell system will be useful for mechanistic studies of early as well as late changes in the development of neoplasia by epithelial cells.