The multistage development of chemically induced mouse skin tumors involves three stages: initiation, promotion and progression. It is hypothesized that soon (ie. within one week) after chemical initiation there are epidermal cells with different Harvey(Ha)-ras point mutations and tumor promotion by 12-0-tetradecanoyl-phorbol-13-acetate(TPA) or okadaic acid (OA) mediates clonal expansion of epidermal cells with specific Ha-ras mutations to yield benign papillomas. It is also hypothesized that OA mediates transcriptional activation through the transcription factor AP-1 acting on a TPA response element (TRE) by altered c-jun phosphorylation or through a distinct OA response element (ORE) that is activated by a jun related transcription factor. The conversion of papillomas to squamous cell carcinomas (SCCs) involves a number of phenotypic alterations including acquisition of the invasive phenotype. Evidence has been obtained implicating two AP-1 regulated genes in invasion, transin or stromelysin and plasminogen activator type (PA) urokinase. Enhanced AP-1 activity was found in malignant variants of benign papilloma producing cells. Therefore, it is hypothesized that constitutively enhanced AP-1 activity is responsible for increased expression of the two protease genes and therefore may be functionally involved in the invasive phenotype. The approach to test these hypotheses involves the use of a recently developed mutation specific polymerase chain reaction (PCR) amplification technique for the sensitive detection of a small number of epidermal cells with various Ha-ras mutations. The effects of OA on AP-1 activity will be investigated by studying altered phosphorylation of the c-jun protein and transcriptional effects on the c-jun gene. Transacting factor protein(s) that bind a distinct OA response element will be characterized. The role of constitutive AP-1 activity in regulating stromelysin gene expression and invasion will be investigated using dominant negative Jun mutant constructs. A more complete understanding of specific gene alterations that occur during the development of experimental animal SCCs will lead to better diagnosis of and intervention in human SCCs.
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