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), thenon-convertogenic (‘second stage’) tumor promoter12-O-retinoylphorbol-13-acetate (RPA) and the non-promotingphorbol esters 4-O-MeTPA and 4--PDD on the chromosomes of mousekeratinocytes in primary cultures were investigated. In thesetarget cells of tumor promotion TPA caused severe numericaland structural chromosomal aberrations, which were evident aftertwo 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 clastogeniceffects were evident as simple alterations such as gaps andbreaks, but more severe alterations such as tri- and quadriradialchromatid interchanges and ring chromosomes, as well as translocationscould be observed. The structural aberrations were nonrandomlydistributed in the genome and chromosomes no. 1, 2, 5, 6 and18 were preferentially involved in rearrangements. In additionto the aneuploidogenic, clastogenic and recombinogenic effectsinduced by TPA, short treatment with this tumor promoter wasefficient in producing cytogenetic equivalents of gene amplification,i.e. double minute chromosomes. The cytogenetic effects werenot merely due to cytotoxicity since they occurred after a lowTPA dose (10^–8 M) and did not considerably increase witha higher dose (10^–6 M). Moreover, at both dose levelscell cycle traverse of mouse keratinocytes was not drasticallyaltered. In contrast, the non-convertogenic tumor promoter RPAand the non-promoting phorbol esters 4--PDD and 4-O-MeTPA (atthe same dose level) did not cause any substantial chromosomalalterations. This discrepancy between the action of TPA andRPA suggests that effects which result in chromosomal alterationsin the target cells may be critical for the conversion stageof skin tumor promotion. This conclusion is supported by experimentswith substances such as antipain and eicosa-5,8,ll,14-tetraynoicacid which inhibit both tumor induction in initiated skin andthe cytogenetic alterations induced by TPA in cultured keratinocytes.These studies provide for the first time the possibility ofdifferentiating between convertogenic and non-convertogenictumor promoters in an in vitro assay using the target cellsof mouse skin carcinogenesis.