LOSS OF MOUSE EPIDERMAL PROTEIN-KINASE-C ISOZYME ACTIVITIES FOLLOWINGTREATMENT WITH PHORBOL ESTER AND NONPHORBOL ESTER TUMOR PROMOTERS

The present study has examined changes in activities and levels of fou r protein kinase C (PKC) isozymes (PKC alpha, PKC beta, PKC gamma and PKC delta) detectable in mouse epidermal preparations following both s ingle and multiple treatments with 12-O-tetradecanoylphorbol-13-acetat e (TPA). In addition, PKC epsilon and PKC eta protein levels were moni tored by immunoblotting following TPA application. Finally, PKC isozym e activity profiles were also examined in epidermal preparations from mice treated with single applications of two non-phorbol ester tumor p romoters: chrysarobin (CHRY) and okadaic acid (OA). Fifteen minutes fo llowing topical treatment with a tumor promoting dose of TPA (3.4 nmol ), the activities of PKC beta and PKC gamma decreased in the epidermal cytosol to 30% and 50% of control values, respectively, while these a ctivities were increased in the epidermal particulate fraction by appr oximately 50%. PKC delta activity, found predominantly in the particul ate fraction of control epidermis, was greatly diminished in both subc ellular fractions at 15 min while PKC alpha activity was translocated similar to 20% from cytosol to particulate fraction. Significant reduc tions in all four detectable PKC isozyme activities in both particulat e and cytosol fractions were observed 48 h after a single treatment wi th TPA, although particulate PKC alpha activity appeared to be less af fected at this point in time compared to the other PKC isozymes. Immun oblotting analyses of PKC isozyme protein levels after TPA treatment f ollowed the changes in activity for cytosolic PKC alpha, PKC beta and PKC gamma. However, particulate PKC delta and PKC epsilon protein leve ls remained relatively unchanged while particulate PKC eta protein lev els were significantly down-regulated after a single TPA treatment. Mu ltiple topical treatments (twice-weekly for 2 weeks) with TPA produced a pattern of loss followed by only partial recovery of total PKC acti vity. Furthermore, all four PKC isozyme activities examined by hydroxy lapatite (HA) chromatography were significantly reduced, including PKC alpha, after four applications of TPA. Cytosolic PKC alpha, PKC beta and PKC gamma protein levels as determined by Immuno-blotting again fo llowed the activity profiles; particulate PKC eta protein levels were significantly reduced, whereas particulate PKC delta and PKC epsilon l evels again appeared relatively unchanged. Fifteen minutes after topic al application of 220 nmol CHRY, an similar to 25% decrease in particu late associated with PKC alpha activity was observed while particulate activities associated with PKC beta, PMC gamma, and PKC delta were un affected by CHRY at this time point. Cytosolic PKC isozyme activities also were relatively unaffected by CHRY at this time point except that PKC alpha was increased similar to 30%. Fifteen minutes after a singl e treatment with OA (12.5 nmol) no membrane translocation was observed although there was a 50% decrease in PKC alpha, PKC beta and PKC gamm a isozyme activities in both the particulate and cytosol fractions, an d an similar to 80% decrease in PKC delta activity in the particulate fraction. By 48 h after application of both nonphorbol ester promoters , significant loss of enzyme activities associated with all four PKC i sozymes was observed. In conclusion, although the initial effects of t umor promoters on PKC isozyme activities were different, a general pat tern involving progressive loss of most PKC isozyme activities was obs erved with all three compounds. The present results suggest that loss of epidermal PKC isozyme activities may be a critical change in the re sponse to a variety of skin tumor promoting agents and that more than one mechanism may account for loss of PKC enzymatic activity.