Phorbol ester induction of rat hepatic metallothionein in vivo and in vitro.
1. The induction of metallothionein (MT) protein by TPA (O-tetradecanoyl phorbol acetate), a protein kinase C activator, was demonstrated in vivo in rat liver and in vitro in rat hepatocytes in primary culture. In vivo half maximal induction at 25 hr was seen at 26 nmol TPA/kg body wt. Five- to seven-fold inductions were seen in vivo. De novo protein synthesis was required for this induction as demonstrated by cycloheximide inhibition of [35S]cysteine incorporation into MT protein. 2. TPA induction of MT protein in primary cultures of rat hepatocytes reached levels of 2.6-4.1-fold, as assessed by [35S]cysteine incorporation, 1.34-2.20-fold, as assessed by 109Cd binding in a metal displacement/HPLC assay, and 2.5-5-fold, as assessed by 109Cd binding in a metal displacement/Sephadex G-75 Superfine assay. 3. The induction of MT mRNA by TPA was demonstrated in vivo in rat liver and in vitro in 2 rat hepatoma cell lines, EC3 and 2M. MT mRNA was quantitated using dot blot and Northern gel assays. In vivo TPA induced hepatic MT mRNA 2.36-5.88-fold (dot blot) and 7.4-22-fold (Northern gels). In vitro TPA induced MT mRNA 1.71-15.26-fold in EC3 cells and 2.23-8.43-fold in 2M cells. MT mRNA was 0.54 kb, and alpha-tubulin mRNA was 1.62 kb in size on Northern gels. 4. TPA induction of MT protein and mRNA in vivo and in vitro is rapid and persistent and occurs at low concentrations. The 2 rat hepatoma cell lines provide a useful system in which to study MT induction in vitro without confounding secondary effects which can occur in vivo.
Garrett
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Department of Biochemistry and Molecular Biology, University of South Dakota School of Medicine, Vermillion 57069.
Xiong
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Arizono
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Brady
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D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D004790:Enzyme Induction / Q000187:drug effects; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D008297:Male; D008668:Metallothionein / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D008668:Metallothionein; D013755:Tetradecanoylphorbol Acetate