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Loss of mouse epidermal protein kinase C isozyme activities following treatment with phorbol ester and non-phorbol ester tumor promoters*
Department of Carcinogenesis, The University of Texas MD, Anderson Cancer Center, Science Park-Research Division Smithville, TX 78957, USA
1To whom correspondence should be addressed
The present study has examined changes in activities and levels of four protein kinase C (PKC) isozymes (PKC
, PKCß, PKC
and PKC
) detectable in mouse epidermal preparations following both single and multiple treatments with 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, PKC
and PKC
protein levels were monitored by immunoblotting following TPA application. Finally, PKC isozyme activity profiles were also examined in epidermal preparations from mice treated with single applications of two non-phorbol ester tumor promoters: chrysarobin (CHRY) and okadaic acid (OA). Fifteen minutes following topical treatment with a tumor promoting dose of TPA (3.4 nmol), the activities of PKCß and PKC decreased in the epidermal cytosol to 30% and 50% of control values, respectively, while these activities were increased in the epidermal particulate fraction by approximately 50%. PKC activity, found predominantly in the particulate fraction of control epidermis, was greatly diminished in both subcellular fractions at 15 min while PKC activity was translocated {small tilde}20% from cytosol to particulate fraction. Significant reductions inall four detectable PKC isozyme activities in both particulate and cytosol fractions were observed 48 h after a single treatment with TPA, although particulate PKC activity appeared to be less affected at this point in time compared to the other PKC isozymes. Immunoblotting analyses of PKC isozyme protein levels after TPA treatment followed the changes in activity for cytosolic PKC, PKCß and PKC. However, particulate PKC and PKC; protein levels remained relatively unchanged while particulate PKC protein levels were significantly down-regulated after a single TPA treatment Multiple topical treatments (twice-weekly for 2 weeks) with TPA produced a pattern of loss followed by only partial recovery of total PKC activity. Furthermore, all four PKC isozyme activities examined by hydroxylapatite (HA) chromatography were significantly reduced, including PKC, after four applications of TPA. Cytosolic PKC, PKCß and PKC protein levels as determined by immunoblotting again followed the activity profiles; particulate PKC protein levels were significantly reduced, whereas particulate PKC and PKC levels again appeared relatively unchanged. Fifteen minutes after topical application of 220 nmol CHRY, an {small tilde}25% decrease in particulate associated with PKC activity was observed while particulate activities associated with PKCß, PKC and PKC were unaffected by CHRY at this time point. Cytosolic PKC isozyme activities also were relatively unaffected by CHRY at this time point except that PKC was increased {small tilde}30%. Fifteen minutes after a single treatment with OA (12.5 nmol) no membrane translocation was observed although there was a 50% decrease in PKC, PKCß and PKC isozyme activities in both the particulate and cytosol fractions, and an {small tilde}80% decrease in PKC 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 isozymes was observed. In conclusion, although the initial effects of tumor promoters on PKC isozyme activities were different, a general pattern involving progressive loss of most PKC isozyme activities was observed with all three compounds. The present results suggest that loss of epidermal PKC isozyme activities may be a critical change in the response to a variety of skin tumor promoting agents and that more than one mechanism may account for loss of PKC enzymatic activity.
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