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Endocrinology Vol. 143, No. 3 1134-1143
Copyright © 2002 by The Endocrine Society

CANCER

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

Djamila Boutemmine, Nadine Bouchard, Derek Boerboom, Helen E. Jones, Alan K. Goff, Monique Doré and Jean Sirois

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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{alpha}, 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{alpha}, 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTATE CANCER IS the most frequent cancer in men in the Western world (1), with an estimated 198,100 new cases in the United States alone in 2001 (2). It is a locally invasive and insidious disease that metastasizes to distant tissues, preferentially to lymph nodes and bones (3). Despite its high incidence, very few risk factors have been identified and include primarily age, race, and family history (4, 5, 6). Environmental factors have also been implicated in some populations, and dietary fat has been the focus of several investigations, although its causal relationship remains questioned (7, 8). Precursor lesions of prostate cancer have been identified, with prostatic intraepithelial neoplasia being the best characterized precancerous lesion (9, 10). Different animal models have been used for studying prostatic carcinogenesis, including recent transgenic and reconstitution models in mice and experimentally induced models in rodents (11, 12, 13). However, besides man, the dog is the only species that frequently develops spontaneous prostate cancer (14). Interestingly, many aspects of the disease are common to both species. As observed in men, adenocarcinoma of the prostate in dogs affects older subjects, is associated with high-grade prostatic intraepithelial neoplasia, and is a locally invasive and malignant disease with a propensity for skeletal metastasis (15, 16).

Increased synthesis of PGs has been implicated in the development and progression of various cancers because of their role in cell proliferation, apoptosis, immune surveillance, and xenobiotic metabolism (17, 18, 19). The first rate-limiting step in the biosynthesis of all PGs from arachidonic acid is catalyzed by the enzyme PG G/H synthase (PGHS, also known as cyclooxygenase or COX) (20, 21). The enzyme has two sequential catalytic activities, a cyclooxygenase and a peroxidase activity, that ultimately produce PGH2, a common precursor for the synthesis of all PGs. Two isoforms of PGHS have been identified, PGHS-1 and PGHS-2 (20, 21). Although both isoforms share similar enzymatic functions, they differ markedly in their pattern of expression. PGHS-1 is present in a wide variety of tissues and is often referred to as the constitutive isoform involved in housekeeping functions, whereas PGHS-2 is generally undetectable in most tissues but can be induced by a variety of agonists, and is referred to as the inducible isoform (20, 21). Increased expression of PGHS-2, but not of PGHS-1, has been associated with various cancers in humans and animals (18, 22), and a cause-effect relationship between PGHS-2 expression and cancer was best supported in the Apc mouse model of human familial adenomatous polyposis in which a null mutation of the PGHS-2 gene was shown to cause an 86% reduction in the number of intestinal polyps (23).

The first evidence of an induction of PGHS-2 in prostate cancer originated from a study in dogs in which 75% of prostatic adenocarcinomas were shown to contain epithelial cells expressing PGHS-2, in contrast to normal prostates where no PGHS-2 was detected (24). Subsequently, a number of recent studies provided evidence for an increased expression of PGHS-2 in human prostate cancer (25, 26, 27, 28, 29). The implication of PGHS-2 and PGs in the development of prostate cancer is also supported by the inhibitory effects of nonsteroidal antiinflammatory drugs (NSAIDs), which block PG synthesis by inhibiting PGHS enzymes, on prostatic carcinogenesis (30, 31, 32, 33, 34). NSAIDs and particularly the new generation of PGHS-2 selective NSAIDs have recently been proposed as chemopreventive agents (18, 19). Although the precise roles of PGHS-2 and PG synthesis in prostatic carcinogenesis remain to be fully characterized, mounting evidence suggest that PGs could act by promoting cell proliferation and preventing apoptosis, by stimulating tumor cell invasiveness, and by promoting angiogenesis (35, 36). It is also thought that the oxidative activity generated by the PGHS-2 enzyme could contribute to DNA oxidation and be involved in the induction of mutations (37). Thus, although the implication and role of PGHS-2 and PG synthesis in prostate cancer are clearly emerging, the molecular control of PGHS-2 expression in prostate cancer cells remains poorly understood. The general objective of the present study was to characterize the regulation of PGHS-2 in a well-characterized epithelial cell line derived from a canine prostatic adenocarcinoma (CPA; 38). The specific objectives were to clone and describe the primary structure of canine PGHS-2, and to study the regulation of its transcript, protein, and activity in CPA cells in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant TNF-{alpha} was purchased from R&D Systems (Minneapolis, MN); phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), diethyldithiocarbamic acid (DEDTC), actinomycin D, cycloheximide, diaminobenzidine tetrahydrochloride, and octyl ß-D-glucopyranoside (octyl glucoside) were obtained from Sigma (St. Louis, MO); [125I]Protein A and Biotrans nylon membranes (0.2 µm) were purchased from ICN Pharmaceuticals, Inc. (Montréal, Québec, Canada); nitrocellulose membranes (0.45 µm) were obtained from Schleicher \|[amp ]\| Schuell, Inc. (Keene, NH); Rainbow molecular weight markers and Kodak film X-OMAT AR were purchased from Amersham Pharmacia Biotech (Baie D’Urfé, Québec, Canada); PGE2 antiserum was obtained from Assay Designs Inc. (Ann Arbor, MI); QuikHyb hybridization solution and the ExAssist/SOLR system were purchased from Stratagene Cloning Systems (La Jolla, CA); [{alpha}-32P]dCTP was obtained from Mandel Scientific-NEN Life Science Products (Mississauga, Ontario, Canada); Prime-a-Gene labeling system, 5'-rapid amplification of cDNA ends (RACE) system, and pGEM-T easy Vector System I were purchased from Promega Corp. (Fisher Scientific, Montréal, Québec, Canada); TRIzol total RNA isolation reagent, 1-kb DNA ladder, synthetic oligonucleotides, bovine fetal serum, tissue culture media, and additives were obtained from Invitrogen Canada Inc. (Burlington, Ontario, Canada); Bio-Rad Laboratories, Inc. protein assay and all electrophoretic reagents were purchased from Bio-Rad Laboratories, Inc. (Richmond, CA); Vectastain ABC kit was purchased from Vector Laboratories, Inc. (Burlingame, CA); cell scrapers, Lab-Tek II 8-chamber glass slides and all tissue culture plasticware were obtained from Corning, Inc.-Costar (Fisher Scientific).

Cell culture
The CPA 1 cell line, a previously characterized epithelial cell line derived from a canine prostatic adenocarcinoma, was used (38). Cells were cultured at 37 C in 95% air 5% CO2 in DMEM supplemented with penicillin (100 U/ml)-streptomycin (100 µg/ml), Fungizone (2.5 µg/ml), and 10% FBS, and were passaged by exposure to trypsin/EDTA (0.05/0.02% wt/vol) in PBS. CPA cells were cultured for variable lengths of time in the absence or presence of PMA (0.1–100 ng/ml), TNF{alpha} (0.1–100 ng/ml), and LPS (0.1–100 ng/ml), as described in each experiment, and coincubation with the transcription inhibitor actynomicin D (5 µg/ml) or the translation inhibitor cycloheximide (10 µg/ml) was performed in some studies. For all experiments, cells were grown to 70–80% confluency and then cultured in serum-free media for 24 h before agonist treatment. Cells were cultured in tissue culture flasks (75 cm) (2) for studies involving extraction of protein and RNA, and in 24-well culture dishes for all experiments investigating PGE2 production in vitro. At the end of the culture period, cells were recovered with a cell scraper, pelleted by centrifugation at 1,000 x g for 5 min, and stored at -70 C until used for analysis. Similarly, media samples were collected and frozen at -20 C until assayed.

Solubilized cell extracts and immunoblot analysis
Solubilized cell extracts were prepared as previously described (24). Briefly, cells were homogenized on ice in TED homogenization buffer (50 mM Tris,10 mM EDTA, 1 mM DEDTC, pH 8.0) supplemented with 2 mM octyl glucoside, and centrifuged at 30,000 x g for 1 h at 4 C. The crude pellets containing membranes, nuclei, and mitochondria were sonicated (5 sec/cycle; 4 cycles) in TED sonication buffer (20 mM Tris, 50 mM EDTA; 0.1 mM DEDTC, pH 8.0) containing 32 mM octyl glucoside. The sonicates were centrifuged at 13,000 x g for 25 min at 4 C. The supernatants (solubilized cell extracts) were stored at -70 C until immunoblot analysis. The protein concentration was determined by the method of Bradford (Bio-Rad Laboratories, Inc. protein assay) (39). Proteins (50 µg) of cell extracts were resolved by one-dimensional SDS-PAGE, and electrophoretically transferred onto nitrocellulose filters. Filters were incubated with a selective anti-PGHS-2 antibody (MF243), and 125I-labeled protein A was used to visualize immunopositive proteins as described (24). Filters were exposed to x-ray film at -70 C.

PGE2 RIA
Concentrations of PGE2 were measured directly in culture media, as previously described (40). The antiserum was purchased from Assay Designs Inc. (Ann Arbor, MI); its cross-reactivities against PGE1, PGF1{alpha} PGF2{alpha}, and 6-keto PGF1{alpha} were 70%, 1.4%, 0.7%, and 0.6%, respectively. The sensitivity of the assay was 40 pg/ml, and the intra and interassay coefficients of variation were 8.2% and 10.8%, respectively. At the end of the culture period, the total amount of protein per well was determined by the method of Bradford (Bio-Rad Laboratories, Inc. protein assay) (39), and concentrations of PGE2 were expressed as picogram per microgram of protein.

Immunocytochemistry
CPA cells were cultured in eight-chamber glass slides (Lab-Tek II) in DMEM supplemented with 10% FBS until they were 70–80% confluent, as described above. After 24 h of serum starvation, cells were cultured for 24 h in the absence or presence of PMA (10 ng/ml) or TNF{alpha} (50 ng/ml). After agonist treatment, cells were fixed with 95% ethanol/5% acetic acid, and immunocytochemical staining for PGHS enzymes was performed using the Vectastain avidin:biotin complex (ABC kit), as previously described (24). Briefly, endogenous peroxidase was quenched by incubating the slides in 0.3% hydrogen peroxide in methanol for 30 min. After rinsing in PBS for 15 min, sections were incubated with diluted normal goat serum for 20 min at room temperature. A PGHS-2 selective antibody (MF243; diluted at 1:1000) was applied, and slides were incubated overnight at 4 C. Controls were incubated with PBS alone or with a PGHS-1 selective antibody (8223; diluted at 1:100) (24). After rinsing in PBS for 10 min, a biotinylated goat antirabbit antibody (1:222 dilution) was applied, and slides were incubated for 45 min at room temperature. They were washed in PBS for 10 min and incubated with the avidin DH-biotinylated horseradish peroxidase H reagents (Vectastain ABC kit) for 45 min at room temperature. After a 10-min PBS wash, the reaction was revealed using diaminobenzidine tetrahydrochloride as the peroxidase substrate. Sections were counterstained with Gill’s hematoxylin stain and mounted.

Cloning and characterization of canine PGHS-2
The complete coding region of the canine PGHS-2 transcript was isolated using a two-step cloning strategy. A canine spleen cDNA library (Dr. R. Nash, Fred Hutchinson Cancer Research Center, Seattle, WA) was screened with a 5' 1.2-kb EcoRI fragment of the mouse PGHS-2 cDNA (41). The probe was labeled with [{alpha}-32P]deoxy-CTP using the Prime-a-Gene labeling system (Promega Corp.) to a final specific activity greater than 1 x 108 cpm/µg DNA. Approximately 300,000 phage plaques were screened, and hybridization was performed at 55 C with QuikHyb hybridization solution (Stratagene). Eight positive clones were plaque purified through secondary and tertiary screenings, and pBluescript phagemids containing the cloned DNA insert were excised in vivo with the Ex-Assist/SOLR system (Stratagene). DNA sequencing was performed using an ABI autosequencer (PE Applied Biosystems, Foster City, CA). Because the clones obtained by library screening were incomplete at the 5'-end, the 5'-RACE system, version 2.0 kit (Invitrogen) was used as directed by the manufacturer. Reverse transcription was performed using 5 µg of total RNA isolated from CPA cells treated for 6 h with PMA (10 ng/ml) and the canine PGHS-2 antisense primer 5'-CATAATTGTATTTCGCAG-3'. After terminal deoxynucleotidyl transferase tailing, the first PCR was performed with the sense abridged anchor primer (5'-RACE system, Invitrogen) and the canine PGHS-2 antisense primer 5'-AATGTTCCAGACTCCCTTGAAGTG-3', and the second nested PCR reaction was performed with the sense universal amplification primer (5'-RACE system, Invitrogen) and the canine PGHS-2 antisense primer 5'-GTATGTAGTGTACTGTATTTGGAGTG-3'. PCRs were performed with Taq DNA polymerase (Amersham Pharmacia Biotech), using 35 (first reaction) or 30 (second reaction) cycles of 94 C for 30 sec, 55 C for 1 min, and 72 C for 2 min, with a final 7-min extension step at 72 C. The largest 5'-RACE products were isolated, subcloned into pGEM-T easy (Promega Corp.) and sequenced.

RNA extraction and Northern blot
Total RNA was extracted from cells using TRIzol (Life Technologies, Inc.) according to the manufacturer’s protocol. For Northern blot analysis, RNA samples (10 µg) were denatured at 55 C for 15 min in 45% formamide, 5.4% formaldehyde, separated by electrophoresis in a 1% agarose gel, and transferred onto a nylon membrane as described (40). The membrane was first hybridized to a 1.4-kb fragment of the canine PGHS-2 cDNA using the QuikHyb solution (Stratagene). After stripping the radioactivity with 0.1% SSC-0.1% SDS for 30 min at 100 C, the same blot was hybridized with the rat elongation factor Tu (EFTu) cDNA as a control gene for RNA loading (42). Each probe was labeled with the [{alpha}-32P]deoxy-CTP using the Prime-a-Gene labeling system (Promega Corp.) to a final specific activity greater than 1 x 108 cpm/µg DNA, and membranes were exposed to film at -70 C.

Statistical analysis
Relative levels of PGHS-2 mRNA was quantified by densitometric analysis of autoradiographic bands using the ImageQuant software version 1.1 (Molecular Dynamics, Inc.). The EFTu signal was also quantified and used to normalize results, and data were expressed as ratios of PGHS-2 to EFTu. One-way ANOVA was used to test the effect of time of treatment or dose of agonist on relative levels of PGHS-2 transcripts in CPA cells and on PGE2 production in vitro. When ANOVAs indicated significant differences (P < 0.05), the Dunnett’s test was used for multiple comparisons with respective controls. Statistical analyses were performed using the computer program JMP (SAS Institute, Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of PGHS-2 protein and PGE2 synthesis in CPA cells
To determine whether PGHS-2 is expressed in CPA cells in vitro, the regulation of PGHS-2 protein and PGE2 production was studied in cultures of CPA cells stimulated with graded doses of PMA, TNF{alpha}, and LPS. Results showed that PGHS-2 protein (Mr = 72,000) was expressed at low levels, and that PGE2 was produced in vitro (2.8 ± 0.3 pg/µg protein) by CPA cells cultured in the absence of agonist (Fig. 1Go; A, 0 ng/ml PMA). An induction in PGHS-2 protein expression was observed after agonist treatment; however, the pattern of induction differed among agonists (Fig. 1Go). Levels of PGHS-2 protein were maximal with 10 ng/ml and 50 ng/ml of PMA and TNF{alpha}, respectively, whereas no marked difference was observed with the different doses of LPS tested. PGE2 production was also significantly increased after agonist treatment (P < 0.05) and results paralleled for the most part those observed for PGHS-2 protein as PMA and LPS appeared as the strongest and weakest inducers of PGE2 production, respectively (Fig. 1Go).



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  		Figure 1. Dose-dependent induction of PGHS-2 protein and PGE2 synthesis by PMA, TNF{alpha}, and LPS in CPA cells. CPA cells were cultured in the absence of agonist (A; 0 ng/ml) or presence of graded doses of PMA (A; 0.1–100 ng/ml), TNF{alpha} (B; 0.1–100 ng/ml), and LPS (C; 0.1–100 ng/ml) as described in Materials and Methods. Cell extracts were prepared from cultures of canine cells after 12 h of agonist stimulation, and proteins (50 µg/lane) were analyzed by one-dimensional SDS-PAGE and immunoblotting techniques. Results from a representative experiment (autoradiograms) are shown; the control culture (0 ng/ml) shown in A is also the no treatment control for B and C. Similar results were obtained from two other independent experiments. Markers on the right indicate migration of intact PGHS-2 protein (Mr = 72,000). Concentrations of PGE2 in culture medium were determined after 24 h of agonist stimulation. Results are presented as picogram of PGE2 per microgram of cellular protein (mean ± SEM of duplicate cultures from three independent experiments). Bars marked with an asterisk are significantly different from controls (P < 0.05).

 
To better characterize the agonist-dependent induction of PGHS-2 protein in CPA cells in vitro, cells were cultured in the absence or presence of PMA (10 ng/ml), TNF{alpha} (50 ng/ml), and LPS (10 ng/ml) for 1, 3, 6, 12, and 24 h. Results showed a progressive time-dependent increase in PGHS-2 protein in cultures stimulated with PMA and TNF{alpha}, with maximal induction observed at 12 and 24 h of stimulation (Fig. 2Go, B and C). In contrast, PGHS-2 was modestly induced after 3 h of LPS treatment, and no marked difference was noted thereafter (Fig. 2DGo). A time-dependent accumulation of PGE2 in culture media was observed with all agonists, with maximal levels observed at 24 h of culture (Fig. 2Go). Concentrations of PGE2 increased from 2.2 ± 0.5 to 393.1 ± 60.5, 0.7 ± 0.3 to 266.2 ± 43.2, and 1.4 ± 0.4 to 32.2 ± 4.6 pg/µg protein between 1 and 24 h of culture with PMA, TNF{alpha}, and LPS, respectively (P < 0.05). Levels of PGHS-2 protein remained relatively low at all time points in control cells (Fig. 2AGo) compared with agonist-stimulated cells, but a significant increase in concentrations of PGE2 also occurred between 1 and 24 h in cultures of control cells (from 1.2 ± 0.4 to 4.5 ± 0.5 pg/µg protein, respectively; P < 0.05).



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  		Figure 2. Time-dependent induction of PGHS-2 protein and PGE2 synthesis by PMA, TNF{alpha}, and LPS in CPA. CPA cells were cultured for 1, 3, 6, 12, and 24 h in absence (A) or presence of PMA (B; 10 ng/ml), TNF{alpha} (C; 50 ng/ml), and LPS (D; 10 ng/ml) as described in Materials and Methods. Cell extracts were prepared, and proteins (50 µg/lane) were analyzed by one-dimensional SDS-PAGE and immunoblotting techniques. A representative blot is shown for each agonist; similar results were obtained from two other independent experiments. Markers on the right indicate migration of intact PGHS-2 protein (Mr = 72,000). Concentrations of PGE2 in culture medium were determined by specific RIAs. Results are presented as picogram of PGE2 per microgram of cellular protein (mean ± SEM of duplicate cultures from three independent experiments). Bars marked with an asterisk are significantly different from the 0-h time point (P < 0.05).

 
To characterize the pattern of PGHS-2 protein expression in CPA cells in situ, immunocytochemistry was performed on cells cultured in the absence or presence of PMA (10 ng/ml) and TNF{alpha} (50 ng/ml) for 24 h. Immunoreactive PGHS-2 was observed in control cells (Fig. 3Go, A and B), but levels were markedly increased after PMA (Fig. 3Go, C and D) and TNF{alpha} treatment (Fig. 3FGo). Immunostaining with a PGHS-1 selective antibody revealed very low or negligible reactivity in control cells (data not shown) and no induction after PMA (Fig. 3EGo) or TNF{alpha} treatment (data not shown).



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  		Figure 3. Immunocytochemical detection of PGHS-2 in CPA cells. Immunocytochemistry was performed on CPA cells cultured for 24 h in the absence (A and B) or presence of PMA (10 ng/ml, C, D, and E) and TNF{alpha} (50 ng/ml, F), as described in Materials and Methods. Immunostaining with a PGHS-2 selective antibody (antibody 243) revealed that PGHS-2 immunoreactivity was present in control cells (A and B), and was markedly increased after PMA (C and D) and TNF{alpha} (F) stimulation. Immunostaining with a PGHS-1 selective antibody indicated the presence of low or undetectable of PGHS-1 immunoreactivity in PMA-stimulated cultures (E). Control staining with normal rabbit serum or PBS was negative (data not shown). Results are representative of three independent experiments.

 
Cloning and characterization of canine PGHS-2
To clone the canine PGHS-2 cDNA, a spleen cDNA library was screened with a 5'-fragment of the mouse PGHS-2 cDNA probe (41). A total of eight positive clones isolated from an initial screen of approximately 300,000 plaques were purified and characterized by restriction mapping and partial DNA sequencing. Two of these clones (clones 1S-4 and 2S-5) that overlapped were sequenced on both strands and shown to represent the near full-length canine PGHS-2. The missing 5'-end of the cDNA was obtained by 5'-RACE. Collectively, these results showed that the canine PGHS-2 cDNA contains a 5'-untranslated region of 28 bp, an open reading frame of 1815 bp (including the stop codon), and a long 3'-untranslated region of 1655 bp containing multiple repeats of the Shaw-Kamen sequence 5'-ATTTA-3' (n = 10; Fig. 4Go).



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  		Figure 4. Nucleotide sequence of the canine PGHS-2 cDNA. The complete nucleotide sequence was obtained by a combination of cDNA library screening and 5'-RACE, as described in Materials and Methods. The canine PGHS-2 cDNA is composed of a 5'-untranslated region of 28 bp (lowercase letters), an open reading frame of 1815 bp (uppercase letters), and a 3'-untranslated region of 1655 bp (lowercase letters). The translation initiation (ATG) and stop (TAG) codons are highlighted in bold; repeats of the Shaw-Kamen’s sequence (ATTTA) in the 3'-untranslated region are underlined; and numbers on the right refer to the last nucleotide on that line. The nucleotide sequence was submitted to GenBank (accession no. AY044905).

 
The amino acid sequence of canine PGHS-2 was deduced from the coding region of the cDNA, and compared with the human homolog (43) (Fig. 5Go). The canine PGHS-2 coding region encodes a 604-amino acid protein, which is identical in length to that of human and all other known mammalian PGHS-2 proteins except for ovine PGHS-2 that has only 603 residues (44). Comparisons between canine and human PGHS-2 revealed an 89% identity at the amino acid level, with all putative structural and functional domains implicated in PGHS-2 function conserved in the canine protein (Fig. 5Go).



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  		Figure 5. Predicted amino acid sequence of canine PGHS-2 and comparison with the human homolog. Identical residues are indicated by a printed period. The signal peptide cleavage site is indicated with an inverse triangle, and the putative transmembrane region is double underlined. The tyrosine (Tyr-371) associated with the cyclooxygenase active site is underlined, heme-coordination residues (His-295 and His-374) are overlined, and the aspirin-acetylated serine residue (Ser-516) is indicated by a number sign. Potential N-glycosylation sites are marked with an asterisk.

 
Regulation of PGHS-2 mRNA in canine prostatic adenocarcinoma cells
To investigate whether the agonist-dependent regulation of PGHS-2 protein in CPA cells was associated with the induction of transcripts, Northern blot analyses were performed on total RNA extracted from cells cultured for 6 h in the absence or presence of graded doses of PMA, TNF{alpha}, and LPS. Results showed a marked regulation of steady-state levels of PGHS-2 transcripts in CPA cells after agonist treatment (P < 0.05). PGHS-2 mRNA was not detected in cells cultured in the absence of agonist (Fig. 6AGo; 0 ng/ml), but a progressive increase in transcript levels was observed after PMA and TNF{alpha} treatment. When results from three independent experiments were expressed as ratios of PGHS-2 to EFTu (elongation factor Tu; control gene), maximal induction was obtained with 10 ng/ml of PMA and 50 ng/ml of TNF{alpha} (Fig. 6Go), in keeping with results obtained for the PGHS-2 protein (Fig. 1Go). In contrast, levels of PGHS-2 mRNA remained relatively constant after LPS treatment (Fig. 6Go), as previously observed for the PGHS-2 protein (Fig. 1Go).



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  		Figure 6. Dose-dependent induction of PGHS-2 mRNA by PMA, TNF{alpha}, and LPS in CPA cells. CPA cells were cultured for 6 h in the absence of agonist (0 ng/ml; A) or presence of graded doses of PMA (A), TNF{alpha} (B), and LPS (C), and samples of total RNA (10 µg/lane) were analyzed by Northern blotting using a 32P-labeled canine PGHS-2 cDNA probe, as described in Materials and Methods. Results from a representative experiment (autoradiograms) are shown; the control culture (0 ng/ml) shown in A is also the no treatment control for B and C. Markers on the right indicate migration of intact PGHS-2 mRNA (4.0 kb). The same blots were stripped of radioactivity and hybridized with a cDNA encoding the EFTu as a control gene for RNA loading (data not shown). For semiquantitative analyses, the PGHS-2 signal intensity was quantified by densitometric analysis, and normalized with the control gene EFTu. Results are presented as PGHS-2 mRNA levels relative to EFTu (mean ± SEM of three independent experiments). Columns marked with an asterisk are significantly different from controls (0 ng/ml; P < 0.05).

 
To provide a better characterization of PGHS-2 mRNA induction in CPA cells in vitro, cells were cultured in the absence or presence of PMA (10 ng/ml), TNF{alpha} (50 ng/ml), and LPS (10 ng/ml) for 1, 3, 6, 12, and 24 h. No PGHS-2 transcript was detected in control cells throughout the culture period (Fig. 7AGo). Levels of PGHS-2 mRNA were significantly induced by 1 h of PMA stimulation (P < 0.05) (Fig. 7BGo). Induction of PGHS-2 transcripts was also observed after 1 or 3 h of TNF{alpha} and LPS treatments, but the induction appeared more modest than with PMA and because of inherent variability among replicate experiments, significance was reached only after 3 and 12 h of LPS treatment (P < 0.05) (Fig. 7Go, C and D).



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  		Figure 7. Time-dependent induction of PGHS-2 mRNA by PMA, TNF{alpha}, and LPS in CPA cells. CPA cells were cultured for 1–24 h in presence or absence of PMA (10 ng/ml), TNF{alpha} (50 ng/ml), and LPS (10 ng/ml), and samples of total RNA (10 µg/lane) were analyzed by Northern blotting using a 32P-labeled canine PGHS-2 cDNA probe, as described in Materials and Methods. A representative blot is shown for each agonist; markers on the right indicate migration of intact PGHS-2 mRNA (4.0 kb). The same blots were stripped of radioactivity and hybridized with a cDNA encoding the EFTu as a control gene for RNA loading (data not shown). For semiquantitative analyses, the PGHS-2 signal intensity was quantified by densitometric analysis and normalized with the control gene EFTu. Results are presented as PGHS-2 mRNA levels relative to EFTu (mean ± SEM of three independent experiments). Columns marked with an asterisk are significantly different from the 0-h time point (P < 0.05).

 
Transcriptional- and translational-dependent regulation of canine PGHS-2
To determine whether the agonist-dependent induction of PGHS-2 in CPA cells was dependent on transcription and translation events, the transcription inhibitor actinomycin D and the translation inhibitor cycloheximide were added to cells cultured in the presence or absence of PMA, TNF{alpha}, and LPS. Results from immunoblot analyses showed an induction of PGHS-2 protein by all three agonists when compared with control cells, with PMA being the strongest inducer, as expected (Fig. 8AGo). However, coincubation with actinomycin D and cycloheximide blocked the protein induction by all three agonists, but had not effect on basal levels observed in control cells (Fig. 8AGo). Northern blot analyses showed that the transcription inhibitor blocked the PMA-, TNF{alpha}-, and LPS-dependent increase in PGHS-2 mRNA (Fig. 8BGo). In contrast, the translation inhibitor did not block this increase but rather caused a further rise (i.e. superinduction) in transcript levels (Fig. 8BGo). This effect was also observed in control cells in which low levels of PGHS-2 mRNA were detected in cells treated with cycloheximide, as revealed by a longer exposure of the blot presented in Fig. 8BGo (data not shown).



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  		Figure 8. Effect of transcriptional and translational inhibitors on agonist-dependent induction of PGHS-2 in CPA cells. CPA cells were cultured in absence or presence of PMA (10 ng/ml), TNF{alpha} (50 ng/ml), and LPS (10 ng/ml) with or without actinomycin D (5 µg/ml) or cycloheximide (10 µg/ml), as described in Materials and Methods. A, Cell extracts were prepared after 6 h of agonist stimulation, and proteins (50 µg/lane) were analyzed by one-dimensional SDS-PAGE and immunoblotting techniques using a PGHS-2 selective antibody. B, Total RNA was extracted from cells after 3 h of agonist stimulation and samples (10 µg/lane) were analyzed by Northern blotting using a 32P-labeled canine PGHS-2 cDNA probe. The same blots were stripped of radioactivity and hybridized with a cDNA encoding the EFTu as a control gene for RNA loading. Representative immunoblots (A) and Northern blots (B) from one experiment are shown; similar results were obtained from two other independent experiments. Markers on the right indicate migration of intact PGHS-2 protein (Mr = 72,000) and mRNA (4.0 kb), and EFTu mRNA (1.8 kb).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study characterizes for the first time the regulation of PGHS-2 and PG synthesis in an epithelial cell line derived from a canine prostatic adenocarcinoma (38). Other than man, the dog is the only known mammal to suffer from spontaneous age-dependent prostatic diseases such as benign prostatic hyperplasia and prostate cancer (15), thus providing an attractive model to study prostate carcinogenesis. Interestingly, prostate cancer in dogs bears numerous similarities to the human disease (15, 16) and, as companion animals, dogs may be susceptible to the same environmental factors that are thought to be risk factors of prostate cancer in humans. An additional similarity between dog and human prostate cancer was recently uncovered when the induction of the PGHS-2 in canine prostatic adenocarcinoma in vivo (24) was observed in humans (25, 26, 27, 28, 29). Numerous experimental and epidemiological studies have provided evidence for a role of PGHS-2 and PGs in carcinogenesis in humans and animals (17, 18, 19, 22, 23, 45); however, the molecular control of PGHS-2 expression in prostate tumor cells remains poorly characterized.

Our results demonstrate that the expression of PGHS-2 in canine prostatic carcinoma cells can be markedly up-regulated in a dose- and time-dependent manner using agonists working through different transduction pathways. The strongest induction of PGHS-2 was observed following treatment with the tumor promoter PMA, an activator of PKC, and this induction was accompanied by a significant increase in PGE2 production. PMA has been shown to enhance the transcription of PGHS-2 in normal epithelial cells such as human mammary and oral epithelial cells (46, 47) as well as in neoplastic cells such as human breast cancer cell lines (48). Interestingly, certain compounds displaying antiinflammatory and chemopreventive properties, such as resveratrol and ursolic acid, have recently been shown to suppress the activation of PGHS-2 gene expression in PMA-treated human mammary and oral epithelial cells through the inhibition of the PKC signal transduction pathway (47, 49).

The cytokine TNF{alpha} was able to significantly increase PGHS-2 expression in canine prostatic tumor cells. Suganuma et al. (50) recently demonstrated that TNF{alpha} is a critical cytokine involved in tumor promotion in mouse skin and suggested that TNF{alpha} could also play a role in human carcinogenesis. TNF{alpha} is known to stimulate immediate early genes and is recognized as a strong inducer of PGHS-2 gene expression in various cell types, including human prostatic cells (18, 51). Indeed, a recent report demonstrated that TNF{alpha} increases PGHS-2 protein levels in tumoral human prostatic cell lines (51). Interestingly, the authors characterized in the same study the level of PGHS-2 expression in three normal and three cancerous human prostatic cell lines and found very high levels of PGHS-2 mRNA and protein in one normal cell line and low levels in all other normal and neoplastic cell lines. These results are surprising considering that, in vivo, PGHS-2 is undetectable or present in very low amount in normal prostate but is highly expressed in human prostate cancer (25, 26, 27, 28, 29). In the dog, where there is also a marked induction of PGHS-2 in prostatic adenocarcinoma in vivo (24), we recently found that PGHS-2 expression in an epithelial cell line derived from a normal canine prostate (52) was higher (data not shown) than that observed in the untreated tumor cells (CPA cells in this study). Collectively, these observations suggest that, both in humans and dogs, the control of PGHS-2 expression in some prostatic epithelial cells lines may not accurately reflect the regulation observed in vivo.

Highs concentrations of PGE2 have recently been found in the tissues of canine prostatic carcinomas (45). The PMA- and TNF{alpha}-dependent induction of PGHS-2 in CPA cells was also associated with a marked increase in PGE2 production in vitro. Several pieces of evidence support a role for PGE2 at different steps of the carcinogenesis process in the prostate. Liu et al. (32, 53) recently reported that PGE2 production could be involved in tumor angiogenesis in the prostate by up-regulating the expression of vascular endothelial growth factor, a key factor in the promotion of angiogenesis in malignancy. PGE2 has also been implicated as a potential mediator in the process of degradation of the basement membrane by prostatic tumor cells since selective inhibition of PGHS-2 reduced in vitro invasiveness of prostatic cells, and this inhibition could be reversed by PGE2 (30). Additionally, PGE2 was shown to significantly stimulate the growth of human prostatic carcinoma cells, and is possibly implicated in the induction of the immediate early gene c-fos in prostate cancer cells (54, 55). PGE2 is also thought to play a role in the apoptotic process as studies have reported that selective inhibition of PGHS-2 in human prostate cancer cells induces apoptosis (33, 34).

Of the three agonists used, LPS was the weakest inducer of PGE2 production in prostatic cancer cells. LPS released from the surface of the cell membrane of Gram-negative bacteria can be found in the circulation during systemic states such as endotoxemia. Although few studies have looked at the direct effect of LPS on epithelial cells, a report by Kojima et al. (56) recently found that LPS was a potent regulator of PGHS-2 expression and PGE2 secretion in intestinal cancerous cells in vitro. Our results with canine prostatic epithelial cells suggest that the potency of LPS to regulate PG synthesis may vary depending on the origin of epithelial cells. Results from the present study also suggest that the induction of PGHS-2 mRNA by PMA, TNF{alpha}, and LPS in CPA cells is dependent on transcriptional events but not on protein synthesis, indicating that the complement of transcription factors needed for PGHS-2 gene induction is already present in cells before agonist stimulation and that the activation of these factors likely involves posttranscriptional or posttranslational modifications. The cycloheximide-dependent superinduction of PGHS-2 transcript following agonist treatment is in keeping with similar results observed in most cell types where PGHS-2 is an immediate early response-gene (57, 58). One notable exception remains the induction of PGHS-2 by gonadotropins in granulosa cells, which is clearly dependent on transcription and translation (59).

Lastly, this study is the first to report the cloning and characterization of canine PGHS-2, and comparative analyses further underscore the highly conserved nature of PGHS-2 across species. The length of the canine PGHS-2 protein, 604 amino acids, is identical to that of other cloned mammalian homologs, except for the ovine enzyme that is one residue shorter (44). The deduced amino acid sequence of the canine enzyme is very similar to that of other species, being more than 88% identical to other mammalian PGHS-2 (reviewed in Ref. 40). Importantly, all structural and functional domains putatively involved in PGHS-2 activity are present in the canine enzyme, including four N-linked glycosylation, a transmembrane domain, heme-coordinating histidines 295 and 374, the cyclooxygenase active-site tyrosine 371, and the aspirin acetylation-site serine 516 (40, 43, 44). The presence of multiple copies of the Shaw-Kamen sequence 5'-ATTTA-3', which corresponds to a 5'-AUUUA-3' motif in the transcript, in the 3'-untranslated region of the canine PGHS-2 cDNA is also a conserved structural feature in other species (40, 43, 44). This motif has been described as an instability determinant of rapidly degraded mRNAs (60) and is in keeping with the unstable nature of the PGHS-2 transcripts.

In summary, this is the first report to characterize the molecular structure of canine PGHS-2 and to demonstrate the regulation of the enzyme in a cell line derived from a canine prostatic adenocarcinoma. Based on numerous similarities observed between human and canine prostate cancer, our study provides an interesting model to further investigate the fine molecular control of PG synthesis in prostate cancer. Moreover, the dog model could provide unique advantages to test the chemopreventive or therapeutic efficacy of PG synthesis inhibitors, considering that its relatively large size, compared with rodents, enables the use of various imaging and diagnostic procedures.


   Acknowledgments
 
The authors would like to thank Dr. D.L. Simmons (Brigham Young University) for the mouse PGHS-2 cDNA, Dr. Richard Nash (Fred Hutchinson Cancer Research Center) for the canine spleen cDNA library, and Dr. R. Levine (Cornell University) for the rat EFTu cDNA.


   Footnotes
 
This study was supported in part by Morris Animal Foundation Grant D00CA-47 (to M.D. and J.S.), Canadian Institutes of Health Research (CIHR) Grant MT-13190 (to J.S.), and Natural Sciences and Engineering Research Council of Canada Grant 183964 (to M.D.). The nucleotide sequence reported in this paper has been submitted to GenBank with accession no. AY044905. D.B. was supported by a CIHR Doctoral Research Award. J.S. was supported by a CIHR Investigator Award.

Abbreviations: CPA, Canine prostatic adenocarcinoma; DEDTC, diethyldithiocarbamic acid; EFTu, rat elongation factor Tu; LPS, lipopolysaccharides; NSAIDs, nonsteroidal antiinflammatory drugs; PGHS, PG G/H synthase, also known as cyclooxygenase or COX; PMA, phorbol 12-myristate 13-acetate; RACE, rapid amplification of cDNA ends.

Received September 20, 2001.

Accepted for publication November 13, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lalani EN, Laniado ME, Abel PD 1997 Molecular and cellular biology of prostate cancer. Cancer Metastasis Rev 16:29–66[CrossRef][Medline]
  2. Greenlee RT, Hill-Harmon MB, Murray T, Thun M 2001 Cancer statistics 2001. CA Cancer J Clin 51:15–36[Abstract/Free Full Text]
  3. Jacobs SC 1983 Spread of prostatic cancer to bone. Urology 21:337–344[CrossRef][Medline]
  4. Parkin DM, Whelan SL, Ferlay J, Raymond L, Young J 1997 Cancer incidence in five continents. Vol. VII. Lyon (France): IARC Sci Publ No.143; p 316
  5. Merrill RM, Potosky AL, Feuer EJ 1996 Changing trends in U.S. prostate cancer incidence rates. J Natl Cancer Inst 88:1683–1685[Free Full Text]
  6. Steinberg GD, Carter BS, Beaty TH, Childs B, Walsh PC 1990 Family history and the risk of prostate cancer. Prostate 17:337–347[Medline]
  7. Xue L, Yang K, Newmark H, Lipkin M 1997 Induced hyperproliferation in epithelial cells of mouse prostate by a Western-style diet. Carcinogenesis 18:995–999[Abstract/Free Full Text]
  8. Kolonel LN, Nomura AMY, Cooney RV 1999 Dietary fat and prostate cancer: current status. J Natl Cancer Inst 91:414–428[Abstract/Free Full Text]
  9. Bostwick DG 1995 High grade prostatic intraepithelial neoplasia: the most likely precursor of prostate cancer. Cancer 75:1823–1836[CrossRef]
  10. Bostwick DG, Pacelli A, Lopez-Beltran A 1996 Molecular biology of prostatic intraepithelial neoplasia. Prostate 29:117–143[CrossRef][Medline]
  11. Lucia MS, Bostwick DG, Bosland M, Cockett ATK, Knapp DW, Leav I, Pollard M, Rinker-Schaeffer C, Shirai T, Watkins B 1998 Rodent models of prostate cancer. Prostate 36:49–55[CrossRef][Medline]
  12. Green JE, Greenberg NM, Ashendel CL, Barrett JC, Boone C, Getzenberg RH, Henkin J, Matusik R, Janus TJ, Scher HI 1998 Transgenic and reconstitution models of prostate cancer. Prostate 36:59–63[CrossRef][Medline]
  13. Cohen MB, Padarathsingh M, Hendrix MJC 2000 Experimental models of prostate cancer research. Am J Pathol 156:355–358[Free Full Text]
  14. Rivenson A, Silverman J 1979 The prostatic carcinoma in laboratory animals: a bibliography survey from 1900 to 1977. Invest Urol 16:468–472[Medline]
  15. Maini A, Archer C, Wang CY, Haas GP 1997 Comparative pathology of benign prostatic hyperplasia and prostate cancer. In Vivo 11:293–299[Medline]
  16. Cornell KK, Bostwick DG, Cooley DM, Hall G, Harvey HJ, Hendrick MJ, Pauli BU, Render JA, Stoica G, Sweet DC, Waters DJ 2000 Clinical and pathologic aspects of spontaneous canine prostate carcinomas: a retrospective analysis of 76 cases. Prostate 45:173–183[CrossRef][Medline]
  17. Badawi AF 2000 The role of prostaglandin synthesis in prostate cancer. Br J Urol Int 85:451–462
  18. Fosslien E 2000 Biochemistry of cyclooxygenase (COX)-2 inhibitors and molecular pathology of cancer. Crit Rev Clin Lab Sci 37:431–502[CrossRef][Medline]
  19. Subbaramaiah K, Zakim D, Weksler BB, Dannenberg AJ 1997 Inhibition of cyclooxygenase: a novel approach to cancer prevention. Proc Soc Exp Biol Med 216:201–210[Abstract]
  20. Smith WL, DeWitt DL, Garavito RM 2000 Cyclooxygenases: structural, cellular, and molecular biology. Annu Rev Biochem 69:145–182[CrossRef][Medline]
  21. Vane JR, Bakhle YS, Botting RM 1998 Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol 38:97–120[CrossRef][Medline]
  22. de Almeida EM, Piche C, Sirois J, Dore M 2001 Expression of cyclo-oxygenase-2 in naturally occurring squamous cell carcinomas in dogs. J Histochem Cytochem 49:867–876[Abstract/Free Full Text]
  23. Oshima M, Dinchuk JE, Kargman SL, Oshima H, Hancock B, Kwong E, Trzaskos JM, Evans JF, Taketo MM 1996 Suppression of intestinal polyposis in Apc {delta}716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell 87:803–809[CrossRef][Medline]
  24. Tremblay C, Dore M, Bochsler PN, Sirois J 1999 Induction of prostaglandin G/H synthase-2 in a canine model of spontaneous prostatic adenocarcinoma. J Natl Cancer Inst 91:1398–1403[Abstract/Free Full Text]
  25. Kirschenbaum A, Klausner AP, Lee R, Unger P, Yao S, Liu XH, Levine AC 2000 Expression of cyclooxygenase-1 and cyclooxygenase-2 in the human prostate. Urology 56:671–676[CrossRef][Medline]
  26. Gupta S, Srivastava M, Ahmad N, Bostwick DG, Mukhtar H 2000 Over-expression of cyclooxygenase-2 in human prostate adenocarcinoma. Prostate 42:73–78[CrossRef][Medline]
  27. Yoshimura R, Sano H, Masuda C, Kawamura M, Tsubouchi Y, Chargui J, Yoshimura N, Hla T, Wada S 2000 Expression of cyclooxygenase-2 in prostate carcinoma. Cancer 89:589–596[CrossRef][Medline]
  28. Tanji N, Kikugawa T, Yokoyama M 2000 Immunohistochemical study of cyclooxygenases in prostatic adenocarcinoma; relationship to apoptosis and Bcl-2 protein expression. Anticancer Res 20:2313–2319[Medline]
  29. Uotila P, Valve E, Martikainen P, Nevalainen M, Nurmi M, Harkonen P 2001 Increased expression of cyclooxygenase-2 and nitric oxide synthase-2 in human prostate cancer. Urol Res 29:23–28[Medline]
  30. Attiga FA, Fernandez PM, Weeraratna AT, Manyak MJ, Patierno SR 2000 Inhibitors of prostaglandin synthesis inhibit human prostate or cell invasiveness and reduce the release of matrix metalloproteinases. Cancer Res 60:4629–4637[Abstract/Free Full Text]
  31. Wechter WJ, Leipold DD, Murray Jr ED, Quiggle D, McCracken JD, Barrios RS, Greenberg NM 2000 E-7869 (R-flurbiprofen) inhibits progression of prostate cancer in the TRAMP mouse. Cancer Res 60:2203–2208[Abstract/Free Full Text]
  32. Liu XH, Kirschenbaum A, Yao S, Lee R, Holland JF, Levine AC 2000 Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo. J Urol 164:820–825[CrossRef][Medline]
  33. Liu XH, Yao S, Kirschenbaum A, Levine AC 1998 NS398, a selective cyclooxygenase-2 inhibitor, induces apoptosis and down-regulates bcl-2 expression in LNCaP cells. Cancer Res 58:4245–4249[Abstract/Free Full Text]
  34. Hsu AL, Ching TT, Wang DS, Song X, Rangnekar VM, Chen CS 2000 The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2. J Biol Chem 275:11397–11403[Abstract/Free Full Text]
  35. Tsujii M, Kawano S, DuBois RN 1997 Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc Natl Acad Sci USA 94:3336–3340[Abstract/Free Full Text]
  36. Gately S 2000 The contributions of cyclooxygenase-2 to tumor angiogenesis. Cancer Metastasis Rev 19:19–27[CrossRef][Medline]
  37. Nikolic D, van Breemen RB 2001 DNA oxidation induced by cyclooxygenase-2. Chem Res Toxicol 14:351–354[CrossRef][Medline]
  38. Eaton CL, Pierrepoint CG 1988 Growth of a spontaneous canine prostatic adenocarcinoma in vivo and in vitro: isolation and characterization of a neoplastic prostatic epithelial cell line, CPA 1. Prostate 12:129–143[Medline]
  39. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  40. Liu J, Antaya M, Goff AK, Boerboom D, Silversides DW, Lussier JG, Sirois J 2001 Molecular characterization of bovine prostaglandin G/H synthase-2 and regulation in uterine stromal cells. Biol Reprod 64:983–991[Abstract/Free Full Text]
  41. Simmons DL, Xie W, Chipman JG, Evett JE 1992 Multiple cyclooxygenases: cloning of a mitogen-inducible form: In: Martyn Bailey J, ed. Prostaglandins, leukotrienes, lipoxins and PAF. New York: Plenum Press; 67–78
  42. Levine RA, Serdy M, Guo L, Holzschu D 1993 Elongation factor Tu as a control gene for mRNA analysis of lung development and other differentiation and growth regulated systems. Acids Res 21:4426
  43. Hla T, Neilson K 1992 Human cyclooxygenase-2 cDNA. Proc Natl Acad Sci USA 89:7384–7388[Abstract/Free Full Text]
  44. Zhang V, O’Sullivan M, Hussain H, Roswit WT, Holtzman MJ 1996 Molecular cloning, functional expression, and selective regulation of ovine prostaglandin H synthase-2. Biochem Biophys Res Commun 227:499–506[CrossRef][Medline]
  45. Mohammed SI, Coffman K, Glickman NW, Hayek MG, Waters DJ, Schlittler D, DeNicola DB, Knapp DW 2001 Prostaglandin E2 concentrations in naturally occurring canine cancer. Prostaglandins Leukot Essent Fatty Acids 64:1–4[CrossRef][Medline]
  46. Mestre JR, Subbaramaiah K, Sacks PG, Schantz SP, Tanabe T, Inoue H, Dannenberg AJ 1997 Retinoids suppress phorbol ester-mediated induction of cyclooxygenase-2. Cancer Res 57:1081–1085[Abstract/Free Full Text]
  47. Subbaramaiah K, Chung WJ, Michaluart P, Telang N, Tanabe T, Inoue H, Jang M, Pezzuto JM, Dannenberg AJ 1998 Resveratol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells. J Biol Chem 273:21875–21882[Abstract/Free Full Text]
  48. Liu XH, Rose DP 1996 Differential expression and regulation of cyclooxygenase-1 and -2 in two human breast cancer cell lines. Cancer Res 56:5125–5127[Abstract/Free Full Text]
  49. Subbaramaiah K, Michaluart P, Sporn MB, Dannenberg AJ 2000 Ursolic acid inhibits cyclooxygenase-2 transcription in human mammary epithelial cells. Cancer Res 60:2399–2404[Abstract/Free Full Text]
  50. Suganuma M, Okabe S, Marino MW, Sakai A, Sueoka E, Fukjiki H 1999 Essential role of tumor necrosis factor-{alpha} (TNF-{alpha}) in tumor promotion as revealed by TNF-{alpha}-deficient mice. Cancer Res 59:4516–4518[Abstract/Free Full Text]
  51. Subbarayan V, Sabichi AL, Llansa N, Lippman SM, Menter DG 2001 Differential expression of cyclooxygenase-2 and its regulation by tumor necrosis factor-{alpha} in normal and malignant prostate cells. Cancer Res 61:2720–2726[Abstract/Free Full Text]
  52. Eaton CL, Pierrepoint CG 1982 Epithelial and fibroblastoid cell lines derived from the normal canine prostate. I. Separation and characterization of epithelial and stromalcomponents. Prostate 3:277–290[Medline]
  53. Liu XH, Kirschenbaum A, Yao S, Stearns ME, Holland JF, Claffey K, Levine AC 1999 Upregulation of vascular endothelial growth factor by cobalt chloride-simulated hypoxia is mediated by persistent induction of cyclooxygenase-2 in a metastatic human prostate cancer cell line. Clin Exp Metastasis 17:687–694[CrossRef][Medline]
  54. Chen Y, Hughes-Fulford M 2000 Prostaglandin E2 and the protein kinase A pathway mediate arachidonic acid induction of c-fos in human prostate cancer cells. Br J Cancer 82:2000–2006[CrossRef][Medline]
  55. Hughes-Fulford M, Chen Y, Tjandrawinata RR 2001 Fatty acid regulates gene expression and growth of human prostate cancer PC-3 cells. Carcinogenesis 22:701–707[Abstract/Free Full Text]
  56. Kojima M, Morisaki T, Izuhara K, Uchiyama A, Matsunari Y, Katano M, Tanaka M 2000 Lipopolysaccharide increases cyclo-oxygenase-2 expression in a colon carcinoma cell line through nuclear factor-{kappa}B activation. Oncogene 19:1225–1231[CrossRef][Medline]
  57. Herschman HR 1996 Prostaglandin synthase 2. Biochim Biophys Acta 1299:125–140[Medline]
  58. Bartlett SR, Sawdy R, Mann GE 1999 Induction of cyclooxygenase-2 expression in human myometrial smooth muscle cells by interleukin-1ß: involvement of p38 mitogen-activated protein kinase. J Physiol 520:399–406[Abstract/Free Full Text]
  59. Wong WY, DeWitt DL, Smith WL, Richards JS 1989 Rapid induction of prostaglandin endoperoxide synthase in rat preovulatory follicles by luteinizing hormone and cAMP is blocked by inhibitors of transcription and translation. Mol Endocrinol 3:1714–1723[Abstract]
  60. Chen CY, Shyu AB 1994 Selective degradation of early-response-gene mRNAs: functional analyses of sequence features of the AU-rich elements. Mol Cell Biol 14:8471–8482[Abstract/Free Full Text]



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