Studies on the mechanism of skin tumor promotion: Evidence for several stages in promotion

<jats:p> The effects of nonpromoting and weakly promoting diterpenes on skin tumor promotion by 12- <jats:italic>O</jats:italic> -tetradecanoylphorbol 13-acetate (TPA) were investigated. When phorbol and phorbol 12,13-diacetate (both nonpromoting) were given simultaneously with TPA after 7,12-dimethylbenz[ <jats:italic>a</jats:italic> ]-anthracene (DMBA) initiation in female mice, they had no effect on TPA promotion. However, the nonpromoter 4- <jats:italic>O</jats:italic> -methyl-TPA and the weak promoter mezerein were found to inhibit TPA promotion in a dose-dependent manner when given simultaneously with TPA. Because mezerein was found to be an effective inhibitor of TPA promotion when given simultaneously and because it induces many biological responses similar to those to TPA, the capacity of mezerein to act as an incomplete promoter in a two-stage promotion protocol was also investigated. Twice-weekly applications of 1,2, or 5 μg of TPA for 2 weeks after DMBA initiation produced 0, 0, and 0.5 papilloma per mouse, respectively, at 20 weeks. When the twice-weekly applications of TPA for 2 weeks were followed by twice-weekly treatments with 2 μg of mezerein for 18 weeks, the number of papillomas per mouse was 2.2, 3.5, and 9.0, respectively. Twice-weekly applications of 2 μg of TPA for 2 weeks followed by twice-weekly treatments with 1, 2, or 4 μg of mezerein for 18 weeks produced 2.1, 3.5, and 6.8 papillomas per mouse, respectively, in DMBA-treated mice. Twice-weekly doses as high as 40 μg of 4- <jats:italic>O</jats:italic> -methyl-TPA were not effective in producing tumors when given after a limited treatment with TPA; however, 4- <jats:italic>O</jats:italic> -methyl-TPA had weak activity as a first-stage promoter. The results suggest that although mezerein by itself is a weak promoter and mimics TPA in many biochemical and morphological effects it is a potent second-stage promoter in a two-stage promotion regimen. </jats:p>