Isolation and Characterization of Complementary DNA Clones Corresponding to Genes Induced in Mouse Epidermis in Vivo by Tumor Promoters1

Complementary DNA clones representing genes in SENCAR mouse epidermis, the expression of which is induced 4 h after one topical application of the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) were isolated. Of 56 isolated complementary DNA clones, 32 were identified to be identical to either metallothioneins (MT-I and Mi ll) or endogenous retroviral like (VL30) sequences. In situ hybridization and analysis of mRNA levels in cell fractions separated by density gradient centrifugation revealed that Ml induction was restricted to keratinocytes in the basal cell layer. Immunohistochemistry and timekinetic studies on mRNA levels in mouse epidermis showed that the increase in MT and VL30 RNAs coincide in time with a TPA-induced transient block in basal cell proliferation (3-12 h after TPA treatment). MT immunoreactivity and transcript levels had returned to control values at a time point (24 h after treatment) when epidermis is known to hyperproliferate. Treatment with other types of tumor promoters showed that MT-I and M 1-II mRNAs were coordinately induced and indicated that in-l,2-dioctanoylglycerol, 12-O-retinoylphorbol-13-acetate, and mezerein induced MT to a lesser degree than TPA. The calcium ionophore A23187 induced mRNA levels for MTs as well as VL30. VL30 and MT mRNA levels were not found to be elevated in epidermal tumors whereas the mRNA level corresponding to glyceraldehyde-3-phosphate dehydrogenase was elevated in tumors and induced by TPA with timekinetics that correlate with a TPA-induced hyperproliferation. These complementary DNA clones provide useful tools in the study of the generegulating effects of TPA in a target tissue relevant for tumor promotion.