Increased expression of the ornithine decarboxylase gene in mouse skin by ultraviolet light: detection by in situ hybridization technique

Ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine synthesis, is a well-established indicator of tumor promotion [7]. The mechanism of ODC induction has been intensively studied using Northern blot analysis, especially in 12-O-tetradecanoylphorbol-13-acetatetreated (TPA-treated) skin [4, 10]. Following ultraviolet (UV) irradiation, ODC activity is markedly induced in the skin [5]. We have previously demonstrated a UVinduced increase in ODC gene expression using Northern blotting of mRNAs extracted from the whole skin of the mouse [6]. In the present study, we examined the spatial distribution o fODC mRNA in UV-irradiated mouse skin using an in situ hybridization technique. Female Charles River CD-1 mice aged 7 to 10 weeks were used in all experiments. The dorsal skin of the mouse was shaved 3 to 4 days before the experiments. The dorsa of the mice were irradiated with 40 mJ/cm 2 UV using two fluorescent lamps [FL20SE-30, Toshiba, Japan: UV emission between 280 and 320 nm (peak, 305 nrn)]. The dorsal skin was excised 24 h after irradiation, fixed in 4% paraformaldehyde (PFA), embedded in OCT compound (Lab-Tek, Naperville, Ill., USA), frozen in liquid nitrogen and sectioned at 4 ~tm. The sections were processed for in situ hybridization using a modification of methods previously described [3, 8]. In brief, sections were treated with proteinase K, post-fixed in 4% PFA, acetylated in acetic anhydride and dehydrated in an ethanol series. The samples were pre-hybridized for 30 rain at 42~ in a solution containing 50% formamide, 0.6 M NaC1, 20% dextran sulfate, 200 ~tg/ml bovine serum albumin, 0.02% polyvinylpyrrolidone, 250 I, tg/ml yeast tRNA, 100 ~tg/ml salmon sperm DNA, 0.02% Ficoll