Mirex is a potent
tumor promoter in 7,1 2-dimethylbenz[a]
anthracene (DMBA)-initiated female CD-1 mouse skin. Like 12-O-tetradecanoylphorbol-13-acetate (TPA),
mirex promotes
papillomas that have a Ha-ras mutation; however, unlike TPA promotion,
mirex promotion does not involve a general hyperplastic response. We used
proliferating cell nuclear antigen (
PCNA) and
5-bromo-2'-deoxyuridine (
BrdU) immunohistochemical staining to further examine the proliferative capacity of
mirex. The numbers of
PCNA- and
BrdU-positive epidermal S-phase cells were highly concordant in all treatment groups. Unlike a single application of TPA, a single application of
mirex had little or no effect on the number of S-phase epidermal cells, and chronic application of
mirex to mouse skin produced only minimal increases in S-phase cells. Moreover,
mirex did not significantly alter the growth of BALB/MK-2 keratinocytes in media containing either 0.05 or 1.2 mM Ca++. These results suggest that
mirex may have highly specific effects on the proliferation of initiated cells and support the existence of a unique
mirex mechanism and/or distinct population of
mirex-promotable mutant Ha-ras epidermal cells. To begin to address this issue of a distinct population of
mirex-promotable mutant Ha-ras cells, we conducted a tandem experiment in which DMBA-initiated mice were treated twice weekly with a maximal promoting dose of
mirex. Then, when the number of
papillomas reached a plateau, these same mice were treated twice weekly with a maximal promoting dose of TPA. Mice treated with
mirex developed a maximum of 6.4
papillomas/mouse. These mice were then promoted with TPA, which produced 8.9 additional
papillomas/mouse for a total of 15.3
papillomas/mouse. The maximum
tumor yields from other groups of mice treated with only TPA or
mirex were 9.8 and 7.3
papillomas/mouse, respectively. Therefore, under these tandem conditions,
tumor yields were additive, indicating that there are at least two distinct populations of mutant Ha-ras cells: one promoted by
mirex and the other by TPA.