Increased DNA content as an early marker of transformation in carcinogen-exposed rat tracheal cell cultures

To determine if abnormal cellular DNA content, suggestive of aneuploidy, is an early indicator of transformation by chemical carcinogens, we exposed primary cultures of rat tracheal epithelial cells to N-methyl-N' -nitro-N-nitrosoguanidine (MNNG), 12-O-tetradecanoylphorbol-13-acetate (TPA), MNNG followed by TPA, or solvent as a control. After 40 and 60 days in culture suspensions of the cells were made, fixed, stained with DNA-dye Hoechst 33342, and the fluorescence per cell measured with a flow cytometer. The DNA histogram showed that freshly isolated rat tracheal epithelial cells, and control cells at days 40 and 60 had predominantly diploid DNA content values. The late primary (day 40) and early passaged (day 60) control cells commonly show increased numbers of cells with tetraploid DNA contents. TPA induced aneuploidy in few cultures by day 40, but by day 60 all samples tested had significant numbers of cells with aneuploid DNA content. In contrast a single MNNG treatment or MNNG followed by TPA regularly caused extensive aneuploidization by day 40. Multiple cycling subpopulations with aberrant DNA contents appear. These dramatic changes in cellular DNA content suggestive of drastic ploidy changes in MNNG and MNNG + TPA exposed cultures are early events, preceding other evidence of neoplastic transformation by many cell generations. Our results suggest that aneuploidy is an early event in the transformation of rat tracheal epithelial cell cultures by chemical carcinogens.