c-Ha-ras gene mutation and activation precede pathological changes in DMBA-induced in vivo carcinogenesis.

We have previously reported a stage-specific and sequential overexpression of the c-Ha-ras and c-erbB genes in 7, 12-dimethylbenzanthracene (DMBA)-induced in vivo carcinogenesis in hamster buccal pouch epithelium (HBPE). In this investigation, the immunoreactive protein product of the c-Ha-ras gene (p21 protein) was identified in HBPE cells, specifically in treated tissues and cultured cells established after 3 weeks of DMBA treatment. Microscopic examination did not show any histopathological changes in these tissues. The p21 protein was detected in a few selective cells, which were dispersed away from the more densely populated basal layer. The overexpression of the c-Ha-ras gene was accompanied by a point mutation of A----T in codon 61 (CAA), inducing an amino acid substitution from the wild-type glutamine to leucine in the peptide. The concurrent molecular modifications preceded any detectable histopathological changes. The cellular morphology and orientation in treated HBPE at this early stage was indistinguishable from the control tissue. Yet the genetic alterations, such as the point mutation and overexpression of the gene, were evident at the predysplastic stage. Amplification and overexpression of the second proto-oncogene, c-erbB, and its product, epidermal growth factor receptor (EGFR), were detected in HBPE cells at the later stages of extensive cell proliferation and invasion. By using double antibodies and two immunoreporter systems, we demonstrated overexpression of both c-Ha-ras and c-erbB genes in the same HBPE cells during this chemically induced in vivo carcinogenesis.