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

Karolinska Institute, Center for BioTechnology, NOVUM, S-141 52 Huddinge, Sweden

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-O-tetradecanoylphorbol-13-acetate (TPA) were isolated. Of 56 isolated complementary DNA clones, 32 were identified to be identical to either metallothioneins (MT-I and MT-II) or endogenous retroviral like (VL30) sequences. In situ hybridization and analysis of mRNA levels in cell fractions separated by density gradient centrifugation revealed that MT induction was restricted to keratinocytes in the basal cell layer. Immunohistochemistry and time-kinetic 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 MT-II mRNAs were coordinately induced and indicated that sn-1,2-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 time-kinetics 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.

¹ The present study was supported by the Swedish Work Health Fund (Project 88-0162), the Swedish Medical Research Council (Project B90-13X-07482-05), and by Centrala Försöksdjursnämden (Project 88-01). S. B. is a recipient of a predoctoral fellowship from the Swedish Cancer Research Society.

² To whom requests for reprints should be addressed.

Received 8/ 4/89. Revised 11/ 8/89. Accepted 11/28/89.