Observations on the mechanism of skin tumor promotion by phorbol esters

Data from three different sorts of experiments that were designed to provide additional information on the mechanism of tumor formation are presented. The aim of the first approach was to obtain further evidence for the possible relevance of the induction of ornithine decarboxylase (ODC) activity to tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin. The irreversible inhibitor of ODC activity, alpha-difluoromethylornithine, not only prevented ODC activity in a dose-dependent manner following skin application, it also prevented skin tumor promotion by TPA. Because in tumor promotion experiments TPA must be applied repetitively in order to elicit tumors, the effect of various time intervals between two or more applications on ODC induction and polyamine levels was investigated. Double applications at intervals greater than 48 hr led to a larger induction of ODC than after a single application. In contrast, at intervals of 24 hr or less, the first application caused a refractory state; the second application did not induce ODC activity even at times after ODC activity had returned to the very low, control activity. The aim of the third approach was to more directly determine the function of TPA in the promotion process by identifying the receptor(s) for TPA and to ascertain the function of the receptor(s). A receptor was purified from the particulate protein fraction of mouse brain and it was found that divalent calcium and phosphatidylserine were essential for the maintenance of binding activity during purification. Furthermore the receptor copurified with protein kinase C; it may be that a TPA receptor is protein kinase C and that TPA activates phosphorylating activity.