Molecular characterization of canine prostaglandin G/H synthase-2 and regulation in prostatic adenocarcinoma cells in vitro

• Published in: Endocrinology (Philadelphia) 2002-03, Vol.143 (3), p.1134-1143
• Main Authors: Boutemmine, Djamila, Bouchard, Nadine, Boerboom, Derek, Jones, Helen E, Goff, Alan K, Doré, Monique, Sirois, Jean
• Format: Article
• Language: English
• Subjects: Adenocarcinoma - enzymology; Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Cell Line; Cloning, Molecular; Dinoprostone - metabolism; Dogs; Epithelial Cells - metabolism; Gene Expression Regulation, Enzymologic - genetics; Gene Expression Regulation, Neoplastic - genetics; Immunoblotting; Immunohistochemistry; Male; Molecular Sequence Data; Prostaglandin-Endoperoxide Synthases - metabolism; Prostatic Neoplasms - enzymology; Radioimmunoassay; RNA, Messenger - biosynthesis; RNA, Messenger - genetics
• ISSN: 0013-7227
• DOI: 10.1210/en.143.3.1134

Induction of PG G/H synthase-2 (PGHS-2), a key rate-limiting enzyme in the PG biosynthetic pathway, has been implicated in prostatic adenocarcinomas in humans and dogs in vivo, but the molecular control of PGHS-2 expression in prostate cancer remains poorly understood. Using the dog model, the specific objectives of this study were to clone and characterize canine PGHS-2, and to study the regulation of its transcript, protein, and activity in a canine prostatic adenocarcinoma (CPA) cell line in vitro. The canine PGHS-2 cDNA was cloned by a combination of cDNA library screening and 5'-rapid amplification of cDNA ends, and shown to contain a 5'-untranslated region of 28 bp, an open reading frame of 1815 bp, and a 3'-untranslated region of 1655 bp. The open reading frame encodes a 604-amino acid protein that is 89% identical to the human homolog. The regulation of PGHS-2 protein and PGE(2) synthesis was studied in CPA cells cultured in the absence or presence of graded doses of phorbol 12-myristate 13-acetate (PMA), TNFalpha, and lipopolysaccharides. Results from immunoblots, immunocytochemistry, and RIAs showed that PGHS-2 protein and PGE(2) were present at low levels in control cells and were significantly induced after agonist treatment (P < 0.05), with PMA being the strongest inducer. Northern blot analyses also revealed a significant increase of PGHS-2 mRNA by PMA, TNFalpha, and lipopolysaccharides treatment (P < 0.05). Agonist-dependent induction of PGHS-2 mRNA was not dependent on new protein synthesis (coincubation with cycloheximide; 10 microg/ml) but was blocked by transcription inhibitor actinomycin D (5 microg/ml), suggesting that PGHS-2 acts an immediate early-response gene in prostatic epithelial cells. Thus, this study characterizes for the first time the structure of canine PGHS-2 and provides an in vitro model to unravel the molecular basis of PGHS-2 expression in prostatic adenocarcinomas.