Molecular characterization of canine prostaglandin G/H synthase-2 and regulation in prostatic adenocarcinoma cells in vitro.
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.
Boutemmine
Djamila
D
Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada J2S 7C6.
Bouchard
Nadine
N
Boerboom
Derek
D
Jones
Helen E
HE
Goff
Alan K
AK
Doré
Monique
M
Sirois
Jean
J
D000230:Adenocarcinoma / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D015232:Dinoprostone / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D015151:Immunoblotting; D007150:Immunohistochemistry; D008297:Male; D008969:Molecular Sequence Data; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000201:enzymology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011863:Radioimmunoassay
D012333:RNA, Messenger; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone