CHARACTERIZATION OF BENZO[A]PYRENE-INITIATED MOUSE SKIN PAPILLOMAS FOR HA-RAS MUTATIONS AND PROTEIN-KINASE-C LEVELS

The frequency and spectrum of Ha-ras mutations in benzo[a]pyrene ]P)-i nitiated/12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted CD-1 mous e skin papillomas were characterized by amplifying high molecular weig ht papilloma DNA using the polymerase chain reaction (PCR) followed by direct DNA sequencing. Analysis of 10 individual B[a]P-initiated earl y emergence papillomas indicated that 90% contained a Ha-ras mutation. Twenty percent of these papillomas contained a GGA --> GTA transversi on in the 12th codon, 50% contained a GGC --> GTC transversion in the 13th codon and 20% contained a CAA --> CTA transversion in the 61st co don. A characteristic of 7,12-dimethylbenz[a]anthracene (DMBA)-initiat ed papillomas, which contain an A-T mutation in the 61st codon of Ha-r as, is that they exhibit a constitutive decrease in both protein kinas e C (PKC) activity and PKCalpha and beta2 isozyme levels when compared to epidermis. In the present study we found that total PKC activity, as well as PKC alpha and beta2 isoforms, were markedly decreased in B[ a]P-initiated early emergence papillomas and that this decrease was al so accompanied by an altered subcellular distribution of PKC activity. The particulate/cytosolic (P/C) ratio of PKC activity in the epidermi s was 0.39, whereas the P/C ratio in the papillomas was 0.77. These re sults demonstrate that B[a]P-initiated/TPA-promoted papillomas exhibit a high incidence of specific ras mutations and that PKC levels are co nstitutively decreased in these papillomas, indicating that an activat ed ras gene is associated with and may contribute to the observed decr ease in PKC levels.