Cytogenetic effects caused by phorbol ester tumor promoters in primary mouse keratinocyte cultures: correlation with the convertogenic activity of TPA in multistage skin carcinogenesis.

The effects of the convertogenic ('first-stage') tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), the non-convertogenic ('second stage') tumor promoter 12-O-retinoyl-phorbol-13-acetate (RPA) and the non-promoting phorbol esters 4-O-MeTPA and 4-alpha-PDD on the chromosomes of mouse keratinocytes in primary cultures were investigated. In these target cells of tumor promotion TPA caused severe numerical and structural chromosomal aberrations, which were evident after two cell cycles and accumulated after multiple applications. Numerical aberrations were visible as hypo- and hyperdiploidy, with non-random loss or gain of specific chromosomes. The clastogenic effects were evident as simple alterations such as gaps and breaks, but more severe alterations such as tri- and quadriradial chromatid interchanges and ring chromosomes, as well as translocations could be observed. The structural aberrations were nonrandomly distributed in the genome and chromosomes no. 1, 2, 5, 6 and 18 were preferentially involved in rearrangements. In addition to the aneuploidogenic, clastogenic and recombinogenic effects induced by TPA, short treatment with this tumor promoter was efficient in producing cytogenetic equivalents of gene amplification, i.e. double minute chromosomes. The cytogenetic effects were not merely due to cytotoxicity since they occurred after a low TPA dose (10(-8) M) and did not considerably increase with a higher dose (10(-6) M). Moreover, at both dose levels cell cycle traverse of mouse keratinocytes was not drastically altered. In contrast, the non-convertogenic tumor promoter RPA and the non-promoting phorbol esters 4-alpha-PDD and 4-O-MeTPA (at the same dose level) did not cause any substantial chromosomal alterations. This discrepancy between the action of TPA and RPA suggests that effects which result in chromosomal alterations in the target cells may be critical for the conversion stage of skin tumor promotion. This conclusion is supported by experiments with substances such as antipain and eicosa-5,8,11,14-tetraynoic acid which inhibit both tumor induction in initiated skin and the cytogenetic alterations induced by TPA in cultured keratinocytes. These studies provide for the first time the possibility of differentiating between convertogenic and non-convertogenic tumor promoters in an in vitro assay using the target cells of mouse skin carcinogenesis.