7,12-DIMETHYLBENZ[A]ANTHRACENE-INDUCED MOUSE KERATINOCYTE MALIGNANT TRANSFORMATION INDEPENDENT OF HARVEY RAS ACTIVATION

Independent clones of mouse keratinocytes initiated in vitro gave rise to tumor phenotypes typical of mouse skin multi-stage carcinogenesis and histologically similar to human tumors of the skin, and head and neck. High-molecular-weight genomic DNAs isolated from two 7,12-dimethylbenz[a]anthracene (DMBA)-initiated murine epithelial carcinoma cell lines and one papilloma cell line were examined for transforming activity by transfection into NIH3T3 cells. DNAs from each of these cell lines resulted in the formation of foci morphologically unlike foci containing an activated c-Ha-ras oncogene. Following polymerase chain reaction amplification of the c-Ha-ras gene, Xba I restriction analysis and oligonucleotide differential hybridization did not detect 61st, 12th, or 13th codon mutations. Southern and Northern analysis confirmed that the normal c-Ha-ras gene was not activated by amplification or overexpression. These results provide evidence that 7,12-dimethylbenz[a]anthracene-induced induced malignant transformation of murine keratinocytes occurred independent of point mutations associated with c-Ha-ras activation. The absence of an activated c-Ha-ras oncogene in these cell lines distinguishes our model from other mouse models of carcinogenesis and may provide a model for functional genetic changes during initiation and progression of human epithelial cancers.