Cellular and biochemical changes during multistage skin tumor promotion

Skin tumor promotion in mice which may involve a free radical mechanism can be operationally and mechanistically further divided into at least two stages. The first stage, which is partially irreversible for 4 to 6 weeks, can be accomplished by a single application of 12-O-tetradecanoylphorbol-13-acetate (TPA) or by nonpromting agents such as 4-O-methyl-TPA, calcium ionophore A23187, and hydrogen peroxide, as well as by wounding. These agents plus wounding increase the number of dark basal keratinocytes, which suggest that these cells are important in the first stage of promotion. Recent data also suggest that the dark cells may be the critical target of skin tumor initiators. Prostaglandin E2 was found to specifically enhance stage I and increase the number of dark cells induced by TPA, whereas the protease inhibitor, tosyl phenylalanine chloromethylketone, specifically inhibited stage I of promotion and counteracted the TPA-induced dark cells. We have recently found that TPA and other first stage promoters can decrease the number of epidermal glucocorticoid receptors. Fluocinolone acetamide (FA) was found to inhibit both stages but was more effective in counteracting stage I of promotion. FA also prevents the TPA-induced dark cells and the decrease in glucocorticoid receptors caused by TPA. The second stage of promotion is initially reversible but later becomes irreversible. The weak promoting agent mezerein and the nonpromoting agent 12-deoxyphorbol-13-2,4,6-decatrienoate are effective stage II promoters. Polyamines, gene amplification and epidermal cell proliferation appear to be important events in stage II of promotion. Putrescine was found to specifically enhance stage II, whereas retinoic acid, difluoromethylornithine, and butylated hydroxyanisole specifically inhibited stage II of promotion and the mezerein-induced polyamine levels but not the mezerein-induced hyperplasia. The inhibition of stage II of promotion by antioxidants gives further support for the role of free radicals in tumor promotion. Mezerein was found to be much more effective in amplifying methotrexate resistance than TPA. Although, mezerein can not significantly decrease the number of glucocorticoid receptors or increase the number of dark cells after repetitive treatment, mezerein can maintain the TPA effect in a two-stage promotion protocol.