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Molecular Characterization of Canine Prostaglandin G/H Synthase-2 and Regulation in Prostatic Adenocarcinoma Cells in Vitro

Centre de recherche en reproduction animale (D.B., N.B., A.K.G., J.S.) and Département de pathologie et microbiologie (M.D.), Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada J2S 7C6; and the Tenovus Center for Cancer Research (H.E.J.), Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, United Kingdom

Address all correspondence and requests for reprints to: Dr. Jean Sirois, Faculté de médecine vétérinaire, Université de Montréal, C. P. 5000, Saint-Hyacinthe, Québec, Canada J2S 7C6. E-mail: . siroisje{at}medvet.umontreal.ca

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 PGE₂ synthesis was studied in CPA cells cultured in the absence or presence of graded doses of phorbol 12-myristate 13-acetate (PMA), TNF, and lipopolysaccharides. Results from immunoblots, immunocytochemistry, and RIAs showed that PGHS-2 protein and PGE₂ 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, TNF, and lipopolysaccharides treatment (P < 0.05). Agonist-dependent induction of PGHS-2 mRNA was not dependent on new protein synthesis (coincubation with cycloheximide; 10 µg/ml) but was blocked by transcription inhibitor actinomycin D (5 µg/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.

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