Chromosomal and c-K-ras oncogene alterations induced by a chemical carcinogen and phorbol ester in skin fibroblasts of individuals with familial polyposis coli

Skin fibroblasts derived from three patients with familial polyposiscoli (FPC) were treated in vitro with N-methyl-N’-nitro-N-nitrosoguanidine(MNNG) alone or in combination with the tumor promoter, 12-O-tetradecanoylphorbol-13-ace-tate(TPA). None of the cultures treated four times with MNNG alone(1µg/ml each) or in combination with TPA (eight applications,0.1 µg/ml each) showed either morphological transformationor anchorage-independent growth for 18 months after treatments.FPC cells treated with MNNG alone showed cell growth inhibition,breakage and loss of chromosomes, as well as the deletion of5.7 kilobase (kb) EcoRl fragment in the c-K-ras locus as detectedby Southern blot analysis with v-K-ras specific probe (cloneKBE-2). The control cells contained two EcoRl fragments of 5.7and 4.2 kb. On the other hand, cells treated with MNNG firstand then followed by treatment with TPA not only showed an increasein the rate of cell growth but also exhibited two novel EcoRlfragments of 9.4 and 12 kb. Treatment with TPA alone appearedto stimulate cell division long after application and to inducechromosomal aberrations but had no effect on c-K-ras sequences.The most likely explanation for the appearance of chromosomepulverization and hyperploidy in FPC cells treated with TPAis the induction of premature chromosome condensation of theS phase cells due to fusion with the mitotic chromosomes.