Identification and quantification of a carcinogen-induced molecular initiation event in cell transformation

All transformed foci of Balb/c 3T3 clone A31-1-1 cells induced by 7,12-dimethylbenz[a]anthracene (DMBA) (42 out of 42 examined) contained an A to T transversion at codon 61 (A182 to T) of the Ki-ras gene. The transformants induced by other carcinogens tested did not contain such a mutation, except one out of nine 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced transformed foci. Thus, we hypothesized that this mutation is a specific DMBA-induced initiating event in Balb/c 3T3 cell transformation and we have measured its frequency of induction before transformation occurs, employing our recently developed method. Such mutations can be detected in the cell population as early as 3 days after exposure to DMBA. The same mutation was also detected in the Ha-ras gene. No detectable level (< 10(-6) of these mutations was induced by other carcinogens tested. The mutation frequency of the Ha-ras gene reached a plateau after 1 week's exposure, but that of the Ki-ras gene continued to increase. These results suggest that the A182 to T mutation of the Ki-ras gene, but not that of the Ha-ras gene, contributes to morphological transformation of Balb/c 3T3 cells. We have demonstrated that the level of expression of ras genes determines the rate of recruitment of cells into transformation. Quantitative analysis of the frequencies of ras gene mutations (initiation) and of transformation suggests that about 25% of those cells with the Ki-ras mutation were recruited into the full transformation process and that, in the presence of the tumor promoter TPA, about 56% of them completed morphological cell transformation.