The Aldo-Keto Reductase AKR1C3 Is a Novel Suppressor of Cell

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[CANCER RESEARCH 63, 505–512, January 15, 2003] The Aldo-Keto Reductase AKR1C3 Is a Novel Suppressor of Cell Differentiation That Provides a Plausible Target for the Non-Cyclooxygenase-dependent Antineoplastic Actions of Nonsteroidal Anti-Inflammatory Drugs1

Julian C. Desmond, Joanne C. Mountford, Mark T. Drayson, Elizabeth A. Walker, Martin Hewison, Jonathan P. Ride, Quang T. Luong, Rachel E. Hayden, Elio F. Vanin, and Christopher M. Bunce2 Division of Medical Sciences [J. C. D., J. C. M., E. A. W., M. H.], Division of Immunity and Infection [M. T. D.], and School of Biosciences [J. P. R., Q. T. L., R. E. H., C. M. B.], University of Birmingham, Birmingham, B15 2TH United Kingdom, and Division of Experimental Hematology, St Jude Children’s Research Hospital, Memphis, Tennessee 38105 [E. F. V.]

ABSTRACT molar doses of drugs that are achieved in vivo (reviewed in Ref. 8). Consequently, the novel target has remained uncertain. However, We and others have demonstrated expression of the aldo-keto reductase micromolar concentrations of a broad spectrum of NSAIDs also AKR1C3 in myeloid leukemia cell lines and that inhibitors of the enzyme, potently inhibit the aldo-keto reductase AKR1C3 (9–12). We and including nonsteroidal anti-inflammatory drugs (NSAIDs), promote HL-60 differentiation in response to all-trans retinoic acid (ATRA) and others have shown previously that myeloid leukemia cell lines express ␣ AKR1C3 and that steroidal and NSAID inhibitors of the enzyme 1 ,25-dihydroxyvitamin D3 (D3). Here, we demonstrate that overexpres- promote their sensitivity to the physiological inducers of differentia- sion of AKR1C3 reciprocally desensitizes HL-60 cells to ATRA and D3, thus confirming the enzyme as a novel regulator of cell differentiation. tion ATRA and D3 (13–17). AKR1C3 possesses marked 11-ketoreductase activity converting prosta- In this report, we demonstrate that overexpression of AKR1C3 in glandin (PG) D2 to PGF2␣. Supplementing HL-60 cultures with PGD2 HL-60 cells generates the reciprocal phenotype, with the cells becom- mimicked treatment with AKR1C3-inhibitors by enhancing the differen- ing more resistant to ATRA and D3, thus confirming the enzyme as a tiation of the cells in response to ATRA. However, PGD2 is chemically novel regulator of nuclear receptor-regulated cell differentiation. unstable, being converted first to PGJ and then stepwise to 15-deoxy- 2 AKR1C3 is able to metabolize diverse substrates including 3␣- and ⌬12,14 ⌬ -prostaglandin J2 (15 -PGJ2), a natural ligand for the peroxisome ␤ ␥ ␥ 17 -hydroxysteroids (9, 12). However, the best substrate in terms of proliferator-activated receptor- (PPAR ). Consistent with this, PGD2 affinity (Km) and rate of turnover [Kcat/Km (specificity constant)] is was rapidly converted to PGJ2 under normal tissue culture conditions but PG D2 (or PGD2), against which the enzyme exerts 11-ketoreductase not in the presence of recombinant AKR1C3 when PGF2␣ was predomi- activity resulting in the generation of PGF ␣ (11). We also demon- nantly formed. In addition, PGJ2 but not PGF2␣ recapitulated the poten- 2 tiation of HL-60 differentiation by PGD2 and AKR1C3 inhibitors. Fur- strate that the provision of excess PGD2, but not steroid substrates of thermore, the capacity of all of these treatments to potentiate HL-60 cell AKR1C3, mimicked AKR1C3 inhibition by potentiating ATRA- and ␥ differentiation was significantly reduced in the presence of the PPAR - D3-induced HL-60 cell differentiation. PGD2 is unstable and is non- antagonist GW 9662. We conclude that AKRIC3 protects HL-60 cells ⌬ enzymatically converted to PGJ2 and, then, stepwise to 15 -PGJ2,a against ATRA and D3-induced cell differentiation by limiting the produc- natural ligand of the PPAR␥ (reviewed in Ref. 18). Importantly, we tion of natural PPAR␥ ligands via the diversion of PGD toward PGF 2 2␣ also show that the capacity of AKR1C3 inhibitors or PGD to poten- and away from PGJ . In addition, these observations identify AKR1C3 as 2 2 tiate ATRA-induced HL-60 cell differentiation was replicated by plausible target for the non-cyclooxygenase-dependent antineoplastic ac- ␥ tions of NSAIDs. PGJ2 and, furthermore, that the PPAR antagonist GW 9662 reversed the enhancement of cell differentiation conferred by all of these treatments. We, therefore, propose that AKR1C3 functions to regulate INTRODUCTION cell differentiation by the diversion of PGD2 catabolism from the generation of J-series prostanoids and toward the generation of 3 ␥ NSAIDs have enjoyed widespread clinical use and have been PGF2␣, thereby protecting PPAR from activation by endogenous implicated in chemoprevention of certain cancers. To date, the diverse ligands. The broader implications of these findings for the possible actions of these compounds have been attributed to their capacity to exploitation of AKR1C3 as a target in human malignancies and on the inhibit COXs (1–7). However, a number of studies have highlighted understanding of the antineoplastic activity of NSAIDs and dietary that these drugs may have other important targets. Although a number AKR1C3 inhibitors are also discussed. of non-COX NSAID targets have been described, most require the drugs to be present in millimolar concentrations, exceeding the micro- MATERIALS AND METHODS

Received 5/30/02; accepted 11/14/02. Cells and Cell Culture. HL-60 cells used in this study were from the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with European Cell and Culture Collection (ECACC; www.ecacc.org; Ref No: 18 U.S.C. Section 1734 solely to indicate this fact. 98070106). Stock cultures were maintained in exponential growth in RPMI 1 Supported by the Leukaemia Research Fund (LRF). C. M. B. is a LRF Bennett Senior 1640 ϩ glutamine (Life Technologies, Inc.-Invitrogen Corp., Paisley, United Research Fellow, J. C. D. was supported by a British Society for Endocrinology Prize Kingdom) supplemented with 10% v/v heat-inactivated FBS (Life Technolo- Studentship, and, at the time of the study, J. C. M. was the recipient of a United ␮ Birmingham Hospitals Barling Ratcliffe Prize Fellowship. gies, Inc.), 100 units/ml penicillin, and 100 g/ml streptomycin (Life Tech- 2 To whom requests for reprints should be addressed, at School of Biosciences, nologies, Inc.). Experimental cultures were seeded at 2.5 ϫ 105 cells/ml in University of Birmingham, Birmingham, B15 2TH United Kingdom. Phone: 44-0-121- 25-cm2 filter-capped cell culture flasks (Nucleon, Nunc Nalge International, 414-3770; Fax: 44-0-121-414-5925; E-mail: [email protected]. Paisley, United Kingdom) or at 5 ϫ 104 cells in 750 ␮l in 48-well cell culture 3 The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; COX, ␣ plates (Nuncleon), and fed appropriately to allow for cellular proliferation. All cyclooxygenase; ATRA, all-trans retinoic acid; D3,1 ,25-dihydroxyvitamin D3; PG, prostaglandin; PPAR␥, peroxisome proliferator-activated receptor-␥; FBS, fetal bovine of the cultures were incubated at 37°C in a fully humidified atmosphere with serum; MPA, medroxyprogesterone acetate; GFP, green fluorescence protein; FACS, 5% CO2. fluorescence-activated cell sorting; HSD, hydroxysteroid dehydrogenase; MSCV, murine Modulators of Cellular Proliferation and Differentiation. One mM stem cell virus; VSV-G, vesicular stomatitis virus-G; IRES, internal ribosome entry site; ⌬ ⌬12,14 ATRA (Sigma, Dorset, United Kingdom) prepared in DMSO was stored in the DHT, dihydrotestosterone; 15 -PGJ2, 15-deoxy- -prostaglandin J2; AR, androgen receptor; wt, wild type. dark at Ϫ20°C. Working stocks were prepared for each experiment by diluting 505

this stock in culture medium and were stored at 4°C. A stock solution of 4 mM Retroviral Delivery of AKR1C3. HL60 cells in exponential growth were ϫ 5 ␮ ϩ D3 in isopropanol was provided as a gift by Dr. Lisa Binderup (Leo Pharma- seeded at a concentration of 2 10 cells/ml in 500 l of RPMI 1640 10% ceuticals, Ballerup, Denmark) and stored in the dark at Ϫ20°C. Working stocks FCS and 500 ␮l of concentrated viral suspension with 6 ␮g/ml Polybrene were prepared as required by diluting this stock in culture medium and were (Sigma) and were incubated overnight. The multiplicity of infection for the stored at 4°C. Twenty mM indomethacin (Sigma) and 5 mM MPA (Sigma) were MSCV-AKR1C3 was 13 plaque-forming units/cell and for MSCV-IRES-GFP prepared in DMSO, stored at 4°C, and replaced every 4 weeks. The PPAR␥ was 100 plaque-forming units/ml. The following morning, 1 ml of fresh RPMI antagonist GW 9662 (Cayman Chemical Company, Ann Arbor, MI) was 1640 ϩ 10% was added to each culture, and the cells were cultured for an prepared as 1 mM stock in DMSO) and used at a final concentration of 1 ␮M. additional 48 h. To determine the transduction efficiency, the percentage of

Stock solutions of 20 mM PGD2 and PGJ2 (both from Affiniti Research GFP-positive cells was determined by FACS (39 and 81% GFP positive for Products, near Exeter, United Kingdom) were prepared in ethanol and stored MSCV-AKR1C3 and MSCV-IRES-GFP, respectively), these populations at Ϫ20°C and Ϫ80°C, respectively. Stocks of 5 mM 5␣-DHT (5␣-androstane- were expanded for an additional 48 h before FACS sorting for populations of 17␤-diol-3-one; Sigma) and 5 mM 3␣-androstanediol (5␣-androstane-3␣,17␤- GFP-positive cells. After sorting, cells were cultured for an additional 3 days diol; Sigma) were prepared in ethanol and stored at Ϫ20°C. to obtain sufficient numbers of cells for assays and for frozen stocks. Assessment of Cell Proliferation and Differentiation. Viable cell counts Western Blot Analysis of AKR1C3 Expression. Approximately 1 ϫ 107 were performed using a hemocytometer and phase contrast microscopy. cells were pelleted, washed in PBS, and resuspended in 200 ␮l of radioim- CD11b is a marker of both HL-60 neutrophil and monocyte differentiation that munoprecipitation assay (RIPA) buffer containing protease inhibitors (Boeh- is expressed by Ͻ5% of cells in cultures not exposed to inducers of differen- ringer Mannheim, Mann., Germany). After 30 min on ice with occasional tiation. Phycoerythrin-conjugated mouse monoclonal antibody against human shaking, samples were microfuged and the supernatant stored at Ϫ20°C. CD11b (Leu-15 Becton Dickinson; supplied by Coulter-Immunotech, Luton, Protein concentrations were determined using the Bio-Rad protein assay kit 2 United Kingdom) was used to stain cells for 30 min at 4°C. The cells were (Bio-Rad, Hertfordshire, United Kingdom) using BSA as a standard. Before washed and fixed with 100 ␮l of PBS containing 1% (v/v) formalin (Sigma) separation protein samples were diluted 1 in 2 (v/v) in sample buffer, incubated and 2% (v/v) FBS and were assayed for CD11b expression using a Becton at 100°C for 5 min, and microfuged for 10 min at 13,000 ϫ g. Proteins were Dickinson FACS-Calibur cell sorter and Becton Dickinson Cell Quest software. separated using a 10% polyacrylamide resolving gel (pH 8.8) and a 4.5% To permit morphological analysis of cell differentiation, 75–100 ␮l of a cell polyacrylamide stacking gel (pH 6.8). Five ␮l of prestained high molecular suspension at a density of 3.5–5 ϫ 105 cells/ml were spun onto an alcohol- weight rainbow markers (Amersham Pharmacia Biotech, Buckinghamshire, washed microscope slide at 500 rpm for 3 min using a cytocentrifuge (Shandon United Kingdom) were loaded in one lane of each gel. After electrophoresis, Cytospin 2). The slides were then air-dried before being fixed in methanol the proteins were transferred to an immobilon-P membrane (0.4 ␮m; Millipore (Fisher, Leicestershire, United Kingdom) for 5 min at room temperature and Corp, Bedford, MA) using a trans-blot SD semi-dry transfer cell (Bio-Rad) at were stained using a conventional Jenner Giemsa protocol. 15 V for 2 h. After transfer, the membrane was stained with Ponceau stain Production of AKR1C3 Expression Vectors. The pBluescript SKϩ vec- (Sigma), to ensure the proteins had resolved, that the transfer had been tor (Stratagene, La Jolla, CA) containing human AKR1C3 cDNA (KIAA0119; successful and to confirm equal loading of protein. Membranes were blocked Ref. 14) kindly donated by Dr. T. Nagase of the Kazusa DNA Research for 1 h with 20% (w/v) nonfat milk powder (Marvel; Premier Brands, Lin- Institute, Chiba, Japan. The cDNA fragment encoding AKR1C3 was removed colnshire, United Kingdom) in PBS 0.1% (v/v) Tween (Sigma) and probed using BamHI and Not1 restriction enzymes (Promega, Southampton, United overnight with rabbit polyclonal anti-3␣-HSD [a kind gift from Professor T. Kingdom), was gel-purified, and was ligated with similarly digested mamma- Penning, University of Pennsylvania, Philadelphia, PA, and used in our earlier lian expression vector, pcDNA3.1/Hygro (ϩ) (Invitrogen Life Technologies, studies (16)]. After washing, membranes were probed with horseradish per- Inc. Paisley, United Kingdom). Orientation and nucleotide sequence of the oxidase-conjugated donkey antirabbit immunoglobulin (Amersham Life Sci- construct pcDNA3.1-AKR1C3 were confirmed by sequencing. ences, Amersham, United Kingdom) and binding was revealed using enhanced The MSCV-AKR1C3-I-GFP retroviral vector plasmid was constructed by a chemiluminescence (Amersham Pharmacia Biotech) as instructed by the

three-way ligation in which the 1.2-kbp SalI-PshAI fragment from pcDNA3.1/ manufacturer. Hygro (ϩ)/AKR1C3 was ligated to the 1.3-kbp HpaI-NotI and the 5.3-kbp Purification of his-Tagged AKR1C3. The generation of his-tagged NotI-XhoI fragments from MSCV-I-GFP (19). AKR1C3 will be described in detail elsewhere.4 Recombinant protein was The VSV-G-pseudotyped MSCV-AKR1C3-I-GFP retroviral vector contain- purified using the Novagen (Madison, WI) Ni-NTA His•Bind Resin kits and ing supernatants were made essentially as described previously (19). 293T the manufacturer’s protocols. Eluates were subsequently analyzed using the cells (4–5 ϫ 106) were transfected by calcium phosphate coprecipitation of the Bio-Rad protein assay kit 2 (Bio-Rad) and by SDS-PAGE. Protein aliquots retroviral vector plasmid MSCV-AKR1C3-I-GFP (15 ␮g) with the plasmid were stored on ice at 4°C to avoid precipitation that occurred at Ϫ80°C. pEQ-PAM3-E (15 ␮g), which expresses the retroviral gag and pol (20), and Recombinant AKR1C3 activity assays contained 100 mM potassium phosphate the plasmid pSR␣-G (10 ␮g), which expresses the envelope protein of the buffer (Sigma; pH 7.0) along with either 2.3 mM NADPH for reduction vesicular stomatitis virus (21). The following morning, the medium was reactions or 2.3 mM NADP for oxidation reactions (both Sigma). One ␮lof changed, and 48 h later, the conditioned medium, containing the VSV-G [3H]-labeled substrate (5␣-DHT; Amersham Pharmacia Biotech), 3␣-andro-

pseudotyped retroviral vector, was harvested and concentrated by ultracentrif- stanediol (NEN Life Science, Boston, MA) or PGD2 (Amersham Pharmacia ugation, passed through a 0.45 ␮M filter, snap-frozen on dry ice, and subse- Biotech), and 2–6 ␮g of recombinant protein were added in a final reaction quently stored at Ϫ80°C until needed. The presence of active viral vectors was volume of 100 ␮l. The reactions were performed in stoppered borosilicate confirmed by titration onto 3T3 cells as described previously (21). glass culture tubes (13 ϫ 100 mm; Corning, New York) and incubated at 37°C Liposomal Delivery of AKR1C3. Five ␮l of DMRIE-C (Invitrogen Life for 1 or 2 h. Reactions were quenched by the addition of 400 ␮l ethyl acetate Technologies, Inc., Paisley, United Kingdom) reagent were added to 0.5-ml (Aldrich, Dorset, United Kingdom), and the mixtures then evaporated to aliquots of RPMI 1640 supplemented with 1% (v/v) ITSϩ (Stratech Scientific, dryness under a stream of air at room temperature. Luton, United Kingdom.) and mixed before adding 4 ␮g of pcDNA3.1: Cell Line Activity Assays. To measure AKR1C3 activity in intact cells, 1 AKR1C3 in 0.5 ml of RPMI 1640:ITSϩ and incubating at room temperature ␮lof[3H]-3␣-androstanediol was added to 4 ml cultures set at 5 ϫ 105 for 45 min. After incubation, 2 ϫ 106 cells in 200 ␮l of RPMI 1640:ITSϩ were cells/ml, and the cells were cultured overnight. To extract steroid metabolites, added to each well, and the suspension was mixed gently. The plates were then cultures were split into two 2-ml aliquots and placed in borosilicate glass ␮ incubated at 37°C with 5% CO2 for 5 h before the addition of 800 l of RPMI culture tubes (Corning); 4 ml of chloroform (Fisher Chemicals) were added, 1640, 10% FBS, and antibiotics. After 48 h, cells were exposed to 500 ␮g/ml and the mixtures were vortexed for 4 min. The reaction mixtures were G418 (Sigma) for 1 week before transfer to methylcellulose (Sigma) in the centrifuged at 2500 rpm for 10 min, and the aqueous and proteinaceous layers continued presence of G418 selection antibiotic. Clones, growing through were removed by aspiration. The sterol-containing chloroform fraction was selection after an additional 3 weeks, were isolated and cultured out of methyl cellulose in RPMI culture medium with G418, and their transfection status was 4 A. L. Lovering, J. P. Ride, C. M. Bunce, J. C. Desmond, S. M. Cummings, and S. A. confirmed by PCR for the neomycin resistance gene of pcDNA3.1 (data not White, Acetate and NSAID inhibition of human 3␣ hydroxysteroid dehydrogenase type 2 shown). (AKR1C3): crystal structures of binary and ternary complexes, manuscript in preparation. 506

then evaporated to dryness under a stream of air at 35°C. The sterols were cells in ATRA-treated HL-60AKRIC3 cultures reflected fewer cells redissolved in 40 ␮l of methanol and spotted onto Silica gel TLC plates (Fluka; undergoing terminal cell differentiation (Fig. 1b). Sigma). Elevated AKR1C3 expression was also associated with resistance For extraction of prostanoids from culture medium, the medium was split to the antiproliferative action of ATRA. For example, 5-day into four 1-ml aliquots, placed in borosilicate glass culture tubes (Corning), AKR1C3 50-nM ATRA-treated MSCV:HL-60 cultures contained and 2 ml of methanol (Fisher Chemicals) and 10 ␮l 10% (v/v) formic acid Ϯ ϫ 5 Ϯ ϫ 5 (Sigma) were added to each aliquot. After vortexing for 2 min, 3.9 ml of 7.2 0.3 10 cells as compared with 5.2 0.6 10 in parallel ϭ chloroform (Sigma) and 200 ␮l of 4.2% (w/v) KCl (Sigma) were also added, MSCV:HL-60 vector control cultures (P 0.033). Consequently, it and the samples were vortexed for an additional 4 min. The reaction mixtures was possible that similar actual numbers of maturing cells were were then centrifuged at 2500 rpm for 10 min and the resulting aqueous and produced in HL-60AKR1C3 and control cultures. In this scenario, the proteinaceous layers removed by aspiration. The prostanoid-containing organic action of AKR1C3 overexpression may have been to promote the fractions were then pooled and evaporated to dryness under a stream of air at proliferation (and/or survival) of HL-60 cells that failed to respond to ␮ room temperature. The prostanoids were redissolved in 40 l of chloroform ATRA, thereby increasing the cell number in ATRA-treated cultures and applied to Silica gel TLC plates (Fluka). and diminishing the proportion of cells expressing CD11b. However, Steroids were separated using chloroform and ethyl acetate (4:1 v/v) and as shown in Fig. 1a, combining the number of cells produced with the prostanoids using chloroform/methanol/glacial acetic acid (Sigma)/distilled H O (90:8:1:0.8 v/v/v/v). The solvents were added to the running tank and proportion of mature cells at day 5 clearly demonstrated that fewer 2 AKR1C3 allowed to equilibrate for 2 h before use. The plates were developed at room cells were differentiating in HL-60 cultures than in controls. temperature for ϳ2 h. After chromatography, the plates were removed from Cell viability in all of the cultures was recorded to be 95–98%. Thus, the running tank and allowed to dry at room temperature. The location of the elevated AKR1C3 did not alter cell survival, and, therefore, its over- radiolabelled sterols and prostanoids was then determined using a Bioscan expression most likely resulted in reduced differentiation by altering plate reader (Bioscan Inc., Washington, DC). Sterols and prostanoids were the proportion of cells entering terminal differentiation. identified by their comigration with standards the positions of which were Loss of AKR1C3 trans-Gene Expression by HL-60 Cells identified under an UV lamp. Correlated with Loss of Resistance to ATRA. trans-Gene expression by both pcDNA3.1:HL-60AKR1C3 and MSCV:HL-60AKR1C3 di- RESULTS minished on prolonged passage of the cells. At the time of their AKR1C3 Overexpression of AKR1C3 by HL-60 Cells Results in Dimin- isolation, overexpression of AKR1C3 by pcDNA3.1:HL-60 ished Sensitivity to ATRA. We used two approaches to overexpress was readily detected by both Western blot analyses and by their ␤ ␣ AKR1C3 in HL-60 cells. The first used liposomal delivery in the elevated 17 -HSD activity against 3 -androstanediol (Fig. 2a). How- mammalian expression vector pcDNA3.1 and the second, retroviral ever, after 4-weeks passage, AKR1C3 overexpression was no longer delivery using MSCV pseudotyped with VSV-G. pcDNA3.1 trans- detectable by either parameter and, concomitantly, the transfectants fected cells were selected using 500 ␮g/ml G418- and MSCV- lost their increased resistance to ATRA (Fig. 2a). In the case of AKR1C3 transduced cells by GFP expression driven independently of AKR1C3 MSCV:HL-60 cells, the progressive loss of trans-gene expres- expression, by virtue of an interstitial IRES. Although generated using sion was most readily monitored by the loss of GFP expression and different vectors and selection protocols, all AKR1C3 overexpressing was again closely associated with a progressive diminution of the HL-60 cells (HL-60AKR1C3) displayed increased resistance to ATRA cellular resistance to ATRA (Fig. 2b). However, regular resorting of (Fig. 1). For example, 5 days exposure of pcDNA3.1:HL-60AKR1C3 to GFP-bright MSCV:HL-60AKR1C3 cells sustained the AKR1C3- 50 nM ATRA resulted in 30.8 Ϯ 3.4% of cells expressing the differ- associated phenotype (data originally shown in Fig. 1). entiation marker CD11b as compared with 72.5 Ϯ 5.7% of wt-HL-60 The strong ATRA resistance of HL-60AKR1C3 cells, irrespective of cells (P ϭ 0.00035). Similarly 50 nM ATRA induced differentiation in the vector used, and the clear association of the phenotype with the 37.8 Ϯ 5.0% of MSCV:HL-60AKR1C3 cells compared 76.3 Ϯ 2.1% of level of trans-gene expression, combines with our earlier inhibitor MSCV:HL-60 vector control cells (P ϭ 0.00019). Studies of cell studies to clearly demonstrate that AKR1C3 functions as a key reg- morphology confirmed that the reduced numbers of CD11b-positive ulator of ATRA-induced differentiation. Furthermore, as predicted by

Fig. 1. AKR1C3 overexpression results in increased resistance to ATRA-induced cell differentiation. a, analyses of CD11b expression. Top panel, wt-HL-60 (F) and pcDNA3.1:HL-60AKR1C3 cells (E) were treated for 5 days with the doses of ATRA shown and were analyzed for expression of the differentiation-associated surface marker CD11b using FACS. Data are the mean Ϯ SE of three experiments. Middle panel, MSCV:HL-60 (F) and MSCV:HL-60AKR1C3 cells (E) were treated for 5 days with the doses of ATRA shown and were analyzed for expression of the differentiation-associated surface marker CD11b using FACS. Data are the mean Ϯ SE of six experiments. Bottom panel, MSCV:HL-60 (F) and MSCV:HL-60AKR1C3 cells (E) were treated for 5 days with the doses of ATRA shown, and the actual numbers of cells expressing CD11b were determined from the data from the middle panel and the mean total numbers of cells/culture (ϫ104) for the same six experiments. b, analyses of cell morphology. Jenner-Giemsa- stained cytospin preparations of MSCV:HL-60 and MSCV:HL-60AKR1C3 cells, treated in parallel for 5 days with the doses of ATRA shown. HL-60 neutrophil maturation is characterized by the development of pleiomorphic and, occasionally, fully lobed nuclei. The images show greater differentiation by ATRA-treated MSCV:HL-60 cells compared with MSCV:HL-60AKR1C3 cells.

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former inhibitor studies (15, 16), AKR1C3 overexpression similarly

bestowed HL-60 cells with increased resistance to D3. For example MSCV:HL60 cultures contained 41.2 Ϯ 2.2% CD11b-expressing Ϯ cells after 5-days exposure to 4 nM D3 as compared with 25.3 1.6% in MSCV:HL-60AKR1C3 cells (P ϭ 0.002). Analysis of cell morphology also confirmed reduced differentiation of MSCV:HL-60AKR1C3

cells in response to D3 (not shown). Thus, the capacity of AKR1C3 to suppress cell differentiation is not limited to a single cell lineage nor restricted to the blockade of ATRA-associated differentiation. ␣ ␣ PGD2 but not the AKR1C3 Substrates 5 -DHT or 3 -Andro- stanediol Potentiates ATRA-induced HL-60 Cell Differentiation. We next determined which activity of AKR1C3 might be responsible for the actions on cell differentiation. We first treated HL-60 cells with an excess (5 ␮M) of the preferred AKR1C3 3␣-hydroxysteroid and 17␤-hydroxysteroid substrates 5␣-DHT and 3␣-androstanediol,

respectively (9) and the prostanoid substrate PGD2 (11). The striking finding was that, whereas 5␣-DHT and 3␣-androstanediol had no effect on the ATRA response of HL-60 cells, both the antiproliferative

and the differentiation responses were markedly enhanced by PGD2 (Fig. 3a and b, and negative data not shown). For example, cultures ␮ treated for 5 days with either 5 M PGD2 or 1.56 nM ATRA alone, generated near identical cell numbers (19.8 Ϯ 1.8 ϫ 105 and 19.8 Ϯ 1.6 ϫ 105, respectively) that were not statistically different from those generated in control cultures (20.8 Ϯ 1.3 ϫ 105). In stark contrast, cultures treated with 1.56 nM ATRA, together with 5␮ M Fig. 2. Resistance to ATRA-induced differentiation is correlated with the level of PGD , generated significantly fewer cells (11.4 Ϯ 0.34 ϫ 105; P ϭ transgene expression. a, pcDNA3.1-transfected cells. Top panel, Western blot analyses of 2 AKR1C3 expression relative to wt-HL-60 cells in three clones of pcDNA3.1:HL- 0.001 with respect to control cells). Furthermore, cultures treated with 5 60AKR1C3 cells before and after 4 weeks continual passage in the absence of G418. Middle 6.25 nM ATRA and 5 ␮M PGD generated just 4.1 Ϯ 0.5 ϫ 10 cells ␤ ␣ 2 panel, analysis of the relative 17 -oxidation of 3 -androstanediol (an activity of AKR1C3 (ϳ20% of controls) which was similar to the maximal response to but not AKR1C4) by wt-HL-60 cells and by pcDNA3.1:HL-60AKR1C3 cells before and 5 after 4 weeks continual passage in the absence of G418. Data are the mean Ϯ SE of three ATRA alone (100 nM ATRA, 3.2 Ϯ 0.37 ϫ 10 cells; ϳ15% of experiments. Bottom panel, analysis of the relative sensitivity of wt-HL-60 cells and of AKR1C3 controls). Most noticeable was the observation that the enhanced pcDNA3.1:HL-60 cells to differentiation induced by 5 days of exposure to 50 nM ␮ ATRA, assessed by FACS analysis of CD11b staining, before and after 4 weeks continual antiproliferative effect of combined ATRA and 5 M PGD2 paralleled passage in the absence of G418. Data are the mean Ϯ SE of three experiments. b, that observed when ATRA was combined with the NSAID-AKR1C3 MSCV-transduced cells. Top panels, FACS analyses of GFP expression by MSCV: inhibitor indomethacin (Fig. 3a). Therefore, as predicted by our HL60AKR1C3 cells before and after 4 weeks continual culture. Bottom panel, changes in the relative sensitivity of MSCV:HL60AKR1C3 cells to ATRA-induced differentiation as model, the provision of an excess of the appropriate AKR1C3 sub- measured by CD11b expression at 5 days. Cells were treated with the shown ATRA doses strate mimicked the effect of inhibiting the enzyme. at one (ƒ), two (Ⅺ), three (᭛), and 4 (‚) weeks after FACS sorting for GFP-positive cells. MSCV:HL60 cells (F), sorted in parallel and in the same window of GFP expression, PGD2 also greatly enhanced ATRA-induced differentiation, were also treated with the same doses of ATRA 1 week after sorting. whereas 5␣-DHT and 3␣-androstanediol did not (Fig. 3b). After

Fig. 3. a, the AKR1C3 inhibitor indomethacin and PGD2 equally sensitize HL-60 cells to the antiproliferative actions of ATRA. HL-60 cells were treated for 5 days with the doses of ATRA shown, either alone (E) or in combination with 20 ␮ Ⅺ ␮ F M indomethacin ( )or5 M PGD2 ( and the number of cells generated by each culture determined as described in “Materials and Methods.” b, excess of the AKR1C3 substrate ␣ ␣ PGD2 but not the steroid substrates 5 -DHT or 3 -androstanediol promotes HL-60 cell differentiation. HL-60 cells were treated with the doses of ATRA shown, either alone (E)orin combination with 5 ␮M 3␣-androstanediol (‚), 5 ␮M 5␣-DHT Ⅺ ␮ F ( )or5 M PGD2 ( ) and the resultant differentiation assayed by FACS analysis of CD11b expression. c, escalation of PGD2 concentration results in cell death. The number of viable cells present in 5-day HL-60 cultures treated with the Ϯ shown doses of PGD2. Data in a and c are the mean SE (ϫ105) of three experiments. Data in b are the mean Ϯ SE of three experiments. d, morphology of PGD2-treated HL-60 cells. Jenner-Giemsa-stained cytospin preparations of HL-60 cells treated as shown.

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5 days exposure to 1.56 nM ATRA alone, only 3.9 Ϯ 1.8% of the cells tissue culture medium (ϳ0.2 nM) was able to convert ϳ70% of a 5-nM Ϯ ϳ ␮ expressed CD11b as compared with 53 12% in cultures also treated radiolabelled PGD2 solution to PGF2␣ injust2h( 5.4 pmol/ gof ␮ ϭ with 5 M PGD2 (P 0.006). Cultures treated with 1.56 nM ATRA AKR1C3/min). Thus, at least in vitro, AKR1C3 is able to inhibit the alone also lacked features consistent with ATRA-induced differenti- formation of PGJ2 from PGD2 by the efficient catalysis of its con- ␮ ation, whereas the majority of cells also treated with 5 M PGD2 version to PGF2␣. In a previous study, we demonstrated the consti- showed features characteristic of HL-60-derived neutrophils (Fig. 3c). tutive generation of PGD2 by HL-60 cells (16). The data we have ␮ Furthermore, prolonged culture of HL-60 cells in 5 M PGD2 alone shown here now suggest that, if not tightly regulated, such a pool of led to the cells acquiring features of partial differentiation and the PGD2 would lead to the endogenous generation of J-series PGs, development of weak CD11b staining by up to 25% of the cells resulting in the promotion of differentiation.

(Fig. 3d). Thus PGD2 may be in itself a weak stimulator of differen- The Ability of PGD2, PGJ2, and Steroidal and Nonsteroidal tiation. However, increasing PGD2 doses did not enhance cell differ- Inhibitors of AKR1C3 to Promote HL-60 Cell Differentiation Is entiation but rather resulted in cell death with features of apoptosis Antagonized by the PPAR␥ Antagonist GW 9662. In other in vitro

(Fig. 3, c and d), an observation consistent with the ability of PGD2 models of cell differentiation J-series PGs have been shown to pro- to induce apoptosis in other cell models (reviewed in Ref. 18). In mote differentiation via activation of PPAR␥ (reviewed in 18). To test ␥ contrast to PGD2, its AKR1C3 metabolite PGF2␣ displayed no capac- whether this was also the case in HL-60 cells, we used the PPAR ity to induce either HL-60 cell differentiation or apoptosis. Instead, antagonist GW 9662, which has been shown by others to inhibit the 20 ␮M produced a small but significant increase in cell numbers PPAR␥-dependent differentiation of osteoclasts (22). As shown in Ϯ ϫ 5 generated over 5 days [(50 0.2) 10 ] compared with parallel Fig. 5a, GW 9662 clearly diminished the capacity of either PGD2 or Ϯ ϫ 5 ϭ control cultures [(45 0.1) 10 ; P 0.05]. PGJ2 to promote the antiproliferative effects of low-dose ATRA. AKR1C3 Activity Protects against the Chemical Conversion of Similarly, GW 9662 also severely restricted the enhanced antiprolif-

PGD2 to PGJ2. Although biologically active in diverse systems, erative actions of ATRA mediated by either the NSAID AKR1C3- PGD2 is chemically unstable and is rapidly converted, nonenzymati- inhibitor indomethacin or the steroidal AKR1C3-inhibitor MPA ⌬ ␥ cally, to PGJ2 and, subsequently, to 12-PGJ2, and then to the PPAR (Fig. 5b). In addition to reversing the potentiation of HL-60 anti- ⌬ ligand 15 -PGJ2. Thus, many of the biological activities ascribed to proliferative responses by PGD2, PGJ2, and AKR1C3 inhibitors, GW PGD2 are likely to be mediated by J-series PGs. We observed that, 9226 also diminished the enhanced HL-60-cell differentiation associ- after overnight incubation of PGD2 in tissue culture conditions, only ated with these treatments. For example, as stated above, when treated Ϯ Ϯ 23.9 3.4% remained as PGD2, and the majority had converted to a with 1.56 nM ATRA alone, only 3.9 1.8% of the cells expressed ␮ metabolite that comigrated with PGJ2 in TLC assays (see also CD11b, and treatment with combined 1.56 nM ATRA and 5 M PGD2 Ϯ ϭ Fig. 4a). Furthermore, PGJ2 treatment of HL-60 cells resulted in an increased this to 53 12% (P 0.006). However, in the presence of ␮ Ϯ enhanced HL-60 response to ATRA that was similar to that observed 1.56 nM ATRA, 5 M PGD2, and GW 9662, only 4.1 0.3% of cells ϭ in response to PGD2 (Fig. 4b). Together these observations indicate became CD11b positive (P 0.016 when compared with 1.56 nM ␮ ϭ that the actions of PGD2 in enhancing HL-60 cell differentiation are ATRA and 5 M PGD2 without GW 9662, and P 0.9 when mediated downstream of its chemical conversion to PGJ2. compared with 1.56 nM ATRA alone). Thus, GW 9662 completely The chemical instability of PGD2 in the extracellular environs abrogated the ability of PGD2 to potentiate the differentiation of of HL-60 cultures precluded us from demonstrating the PGD2 11- HL-60 cells in response to this low dose of ATRA. Similarly, al- ketoreductase activity of AKR1C3 within HL-60 cells. However, as though indomethacin elevated differentiation in response to 1.56 nM shown in Fig. 4a,6␮g of human recombinant-AKRIC3 in 1 ml of ATRA (23 Ϯ 2.1% CD11b positive), this did not occur in the additional presence of GW 9662 (7.0 Ϯ 1.4% CD11b positive; P ϭ 0.009 when compared with 1.56 nM ATRA and indomethacin without GW 9662; P ϭ 0.1 when compared with 1.56 nM ATRA alone). We, therefore, conclude that the constitutive action of AKR1C3 in

reducing the sensitivity of HL-60 cells to physiological ATRA and D3 is mediated by the prevention of PPAR␥ activation via the directed

metabolism of PGD2 toward PGF2␣ and the consequent depletion of J-series PGs. The phenomenon of “prereceptor signaling” by ligand metabolizing enzymes is well established for many nuclear receptors,

including thyroid hormone and steroid, retinoid, and D3 receptors. However, ours is the first to identify an enzyme that controls PPAR␥- mediated cell activities.

DISCUSSION AKRs are expressed throughout the evolutionary spectrum from prokaryotes to flowering plants and higher mammals (23). This ex- tensive conservation strongly suggests that these enzymes have important ancestral roles in fundamental aspects of cellular behavior. Consistent with this, our study has highlighted AKR1C3, as a novel Fig. 4. PGD2 mediates its action upon HL-60 cell differentiation downstream of its chemical conversion to PGJ2. a, AKR1C3 activity prevents PGD2 degradation to PGJ2. and potentially important regulator of myeloid cell proliferation and Typical TLC traces for PGD2 (top panel), PGD2 incubated for2hat37°C in the presence differentiation. of human recombinant AKR1C3 (middle panel), and PGD2 incubated overnight at 37°C in naked tissue culture medium (bottom panel). PGJ2 and PGF2␣ were identified by Other workers have demonstrated the expression of both COX-1 comigration of “cold” standards. b, PGJ2 promotes ATRA-induced HL-60 cell differen- and COX-2 by HL-60 cells, and in an earlier study, we demonstrated tiation. HL-60 cells were treated with the doses of ATRA shown either alone (F)orin ␮ E the generation of multiple PGs by HL-60 cells (16, 24–26). We combination with 5 M PGJ2 ( ) and the resultant differentiation assayed by FACS Ϯ analysis of CD11b expression. Data are the mean SE of three experiments. further showed that indomethacin near totally diminished PGE2 pro- 509

prostate, it has been suggested that the enzyme protects the AR by sequential 3␣-reduction and 17␤-oxidation of the potent androgen 5␣-dihydrotestosterone to form the inactive androgen androsterone (9, 17). However, we and others have failed to detect AR expression by HL-60 cells, and thus, it appears likely that the capacity of AKR1C3 to regulate myeloid cell differentiation is independent of androgen action (Ref. 27 and unpublished observations).5 In contrast, the experiments that we have described here strongly

implicate PGD2 as the key AKR1C3 substrate and that the enzyme

diverts its metabolism toward PGF2␣ and away from J-series prostanoids. These findings are supported by those of others demonstrating

that PGD2 and PGJ2 inhibited the clonogenic growth not only of the HL-60 cells but also of the K562, KG1, U937, and THP1 myeloid cell lines and, importantly, normal myeloid progenitor cells CFU-GM

(28). Thus PGD2 and PGJ2 are powerful modulators of both normal and malignant myeloid cell activities. Our findings are also supported by the study of Asou et al. (29) that demonstrated the expression of PPAR␥ across a panel of myeloid leukemia cell lines and the capacity of its synthetic ligand troglitazone to potentiate the antiproliferative actions of ATRA.

Normal bone marrow is one of the most PGD2-rich tissues in the body (30). Intuitively, therefore, it appears that myeloid progenitor cells must transiently protect themselves from the antiproliferative

and prodifferentiative actions of PGD2 that are mediated downstream

of its chemical conversion to PGJ2. Although abundant in PGD2, the

extracellular environment is not rich in free PGJ2, the levels of which appear to be tightly controlled by diverse mechanisms that are as yet poorly defined (31–35). Consequently, it is endogenously generated

PGJ2 from which myeloid progenitor cells have to protect themselves.

Our study now indicates that the ability of AKR1C3 to divert PGD2

toward PGF2␣ and away from PGJ2 represents a major component in this mechanism. However, the role of AKR1C3 in regulating PPAR␥ activity may be more complex than mere regulation of ligand avail-

ability, because in the adipocyte model, PGF2␣, reciprocally, nega- tively regulates PPAR␥ activation via induction of its mitogen- activated protein kinase (MAPK)-dependent phosphorylation (36). Therefore, the level of AKR1C3 activity in cells may determine the relative activity of PPAR␥ by controlling the relative abundance of

PGF2␣ and J-series prostanoids. Fig. 5. The potentiation of HL-60 differentiation by PGD2, PGJ2, and the AKR1C3 inhibitors indomethacin and MPA is dependent on PPAR␥. a, HL-60 cells were cultured Present therapies for acute myeloid leukemia (AML) rely on com- for 4 days in the presence of 0 (F), 1.56 (Œ), 3.125 (f), and 6.25 (᭜)nM ATRA in bined chemotherapy. However, overall response rates and disease-free combination with the shown concentrations of PGD2 or PGJ2 and in either the presence or the absence of the PPAR␥ antagonist GW 9662. Data are the mean of two experiments survival remain poor. This is, in part, because the majority of patients performed in triplicate. b, HL-60 cells were cultured for 4 days in the doses of ATRA are not able to tolerate the most intensive treatments. Adjuvant ther- shown alone (E), in the presence of AKR1C3 inhibitors (ƒ), or in the presence of AKR1C3 inhibitors and PPAR␥ antagonist GW 9662 (f). Data are the mean of two apies that increase patient responses but which are themselves asso- experiments performed in triplicate. ciated with low toxicities are therefore required. A well-established paradigm for this principle is the success of combined cytotoxic therapy and ATRA in acute promyelocytic leukemia (APL). The data duction (a widely used assay for COX inhibition). Importantly, how- presented here, together with those of our previous studies and those of other workers, now indicate that inhibitors of AKR1C3 may be ever, PGD2 was not ablated by this treatment, but, in contrast, its similarly exploited. In this regard, it is important to note that, unlike AKR1C3-derived product, PGF2␣, was (16). Thus, at the doses that potentiate HL-60 differentiation, indomethacin acts as both a COX in some tumors, NSAIDs have not been reported to be beneficial in and a AKR1C3 inhibitor. It is, therefore, not possible to exclude acute myeloid leukemia. However, the lack of in vivo evidence from that the potentiation of differentiation by NSAIDs is dependent on leukemia patients is, in part, compounded by the gut-irritant actions of both activities. However, the data that we have described here are NSAIDs. Given the fact that reduced platelet counts are associated the first to clearly demonstrate the role of AKR1C3 in these with leukemia, it is rare that these patients receive NSAIDs. None- processes. Furthermore, in our previous study, we observed that a theless a number of studies have shown that NSAIDs promote hemo- spectrum of AKR1C3 inhibitors, including steroidal inhibitors that poiesis in rodents (37–40). Thus, in the particular case of leukemia, do not inhibit COX-inhibitory actions, all potentiated HL-60 cell attempts to exploit AKR1C3 inhibition therapeutically will require differentiation (16). the use of steroidal inhibitors or indeed the development of novel AKR1C3 has a broad tissue distribution and, at least in vitro, inhibitors. recognizes diverse substrates. This raises the possibility that the enzyme has separate roles in different tissues. In the case of the 5 Unpublished observations. 510

However, the above issues are less important in the context of other 15. Nagase, T., Miyajima, N., Tanaka, A., Sazuka, T. Seki, N., Sato, S., Tabata, S., tumors, it is significant that many studies have demonstrated that Ishikawa, K., Kawarabayasi, Y., Kotani, H., and Nomura, N. Prediction of the coding sequences of unidentified human genes. III. The coding sequences of 40 new genes AKRIC3 has a broad tissue distribution and that PGD2 and PGJ2 (KIAA0081–KIAA0120) deduced by analysis of cDNA clones from human cell line promote the in vitro differentiation and apoptosis of diverse cells KG-1. DNA Res., 2: 37–43, 1995. 16. Sokoloski, J. A., and Sartorelli, A. C. Induction of the differentiation of HL-60 (reviewed in Refs. 18 and 41). Thus, it remains possible that AKR1C3 promyelocytic leukemia cells by nonsteroidal anti-inflammatory agents in combina- activity may influence the progression of diverse malignancies and tion with low levels of vitamin D3. Leuk. Res., 22: 153–161, 1998. that the capacity of NSAIDs to protect against certain tumors may in 17. Bunce, C. M., Mountford, J. C., French, P. J., Mole, D. J., Durham, J., Michell, R. H., and Brown, G. Potentiation of myeloid differentiation by anti-inflammatory agents, part be mediated by its inhibition. In this regard, it has been demon- by steroids and by retinoic acid involves a single intracellular target, probably an strated that natural and synthetic PPAR␥ ligands are antiproliferative enzyme of the aldoketoreductase family. Biochem. Biophys. Acta., 1311: 189–198, against in vitro and in vivo models of colon and prostate carcinoma, 1996. ␥ 18. Straus, D. S., and Glass, C. K. Cyclopentenone prostaglandins: new insights on and the synthetic PPAR ligand troglitazone has been shown to biological activities and cellular targets. Med. Res. Rev., 21: 185–210, 2001. inhibit chemically induced colitis and aberrant crypt formation in rats 19. Persons, D. A., Mehaffey, M. G., Kaleko, M., Nienhuis, A. W., and Vanin, E. F. An and mice (42–46). Similarly, in a study of human prostate carcinoma, improved method for generating retroviral producer clones for vectors lacking a selectable marker gene. Blood Cells Mol. Dis., 24: 167–182, 1998. treatment with troglitazone resulted in an increased incidence of 20. Persons, D. A., Allay, J. A., Allay, E. R., Smeyne, R. J., Ashmun, R. A., Sorrentino, prolonged stabilization of prostate-specific antigen (PSA) levels (47). B. P., and Nienhuis, A. W. Retroviral-mediated transfer of the green fluorescent It is important to note that attempts to better exploit the antineoplastic protein gene into murine hematopoietic cells facilitates scoring and selection of transduced progenitors in vitro and identification of genetically modified cells in vivo. activities of NSAIDs has centered on the derivation of drugs that Blood, 90: 1777–1786, 1997. selectively inhibit COX-2 over COX-1 (1–4). In future studies, it will 21. Yang, Y., Vanin, E. F., Whitt, M. A., Fornerod, M., Zwart, R., Schneiderman, R. D., be important to determine whether the clinical efficacy of these drugs Grosveld, G., and Nienhuis, A. W. Inducible, high-level production of infectious murine leukemia retroviral vector particles pseudotyped with vesicular stomatitis correlates with their ability to also inhibit AKR1C3. Finally, both gut virus G envelope protein. Hum. Gene. Ther., 6; 1203–1213, 1995. and prostate tumors share a common etiology in as much as diets high 22. Bendixen, A. C., Shevde, N. K., Dienger, K. M., Willson, T. M., Funk, C. D., and in vegetable content have been demonstrated to protect against both Pike, J. W. IL-4 inhibits osteoclast formation through a direct action on osteoclast precursors via peroxisome proliferator-activated receptor ␥1. Proc. Natl. Acad. Sci. diseases (48–50). As yet it is uncertain exactly how this protection USA, 98: 2443–2448, 2001. occurs. However, a recent study has identified more than 20 dietary 23. Jez, J. M., Bennett, M. J., Schlegel, B. P., Lewis, M., and Penning, T. M. Comparative anatomy of the aldo-keto reductase superfamily. Biochem. J. 326: 625–636, 1997. plant constituents that act as inhibitors of AKR1C3 (51), and, there- 24. Honda, A., Raz, A., and Needleman, P. Induction of cyclo-oxygenase synthesis in fore, it is now possible to provide a plausible rationale for unifying the human promyelocytic leukaemia (HL-60) cells during monocytic or granulocytic protective antineoplastic actions of NSAIDs and diet in these diseases. differentiation. Biochem. J., 272: 259–262, 1990. 25. Jang, M., and Pezzuto, J. M. Cancer chemopreventative activity of resveratrol. Drugs Exp. Clin. Res., 25;65–77, 1999. REFERENCES 26. Koll, S., Goppelt-Struebe, M., Hauser, I, and Goerig, M. Monocytic-endothelial cell interaction: regulation of prostanoid synthesis in human co-culture. J. Leukoc. Biol., 1. Xu, X. C. COX-2 inhibitors in cancer treatment and prevention, a recent development. 61; 679–688, 1997. Anticancer Drugs, 13: 127–137, 2002. 27. Danel, L., Menouni, M., Cohen, J. H., Magaud, J. P., Lenoir, G., Revillard, J. P., and 2. Stack, E., and DuBois, R. N. Role of cyclooxygenase inhibitors for the prevention of Saez, S. Distribution of androgen and estrogen receptors among lymphoid and colorectal cancer. Gastroenterol. Clin. North Am., 30: 1001–1010, 2001. haemopoietic cell lines. Leuk Res., 9; 1373–1378, 1985. 3. Gupta, R. A., and Dubois, R. N. Colorectal cancer prevention and treatment by 28. Tsao, C. J., Ozawa, K., Hosoi, T., Urabe, A., and Takaku, F. In vitro effects of inhibition of cyclooxygenase-2. Nat. Rev. Cancer, 1: 11–21, 2001. antineoplastic prostaglandins on human leukemic cell growth and normal myelopoi- 4. Gro¨sch, S., Tegeder, I., Niederberger, E., Bra¨utigam, L., and Geisslinger, G. COX-2 esis. Leuk. Res., 10: 527–532, 1986. independent induction of cell cycle arrest and apoptosis in colon cancer cells by the 29. Asou, H., Verbeek, W., Williamson, E., Elstner, E., Kubota, T., Kamada, N., and selective COX-2 inhibitor celecoxib. FASEB J., 15: 2742–2744, 2001. Koeffler, H. P. Growth inhibition of myeloid leukemia cells by troglitazone, a ligand 5. Zhang, X., Morham, S. G., Langenbach, R., and Young, D. A. Malignant transfor- for peroxisome proliferator activated receptor ␥, and retinoids. Int. J. Oncol., 15: mation and antineoplastic actions of nonsteroidal anti-inflammatory drugs (NSAIDs) 1027–1031, 1999. on cyclooxygenase-null embryo fibroblasts. J. Exp. Med., 190: 451–459, 1999. 30. Ujihara, M., Urade, Y., Eguchi, N., Hayashi, H., Ikai, K., and Hayaishi, O. Prosta- 6. Wechter, W. J. Kantoci, D., Murray, E. D., Jr., Quiggle, D. D., Leipold, D. D., glandin D2 formation and characterization of its synthetases in various tissues of adult Gibson, K. M., and McCracken, J. D. R-flurbiprofen chemoprevention and treatment rats. Arch. Biochem. Biophys., 260: 521–531, 1988. of intestinal adenomas in the APC(Min)/ϩ mouse model: implications for prophy- 31. Bogaards, J. J., Venekamp, J. C., and van Bladeren, P. J. Stereoselective conjugation laxis and treatment of colon cancer. Cancer Res., 57: 4316–4324, 1997. of prostaglandin A2 and prostaglandin J2 with glutathione, catalyzed by the human 7. Wechter, W. J. Leipold, D. D., Murray, E. D., Jr., Quiggle, D., McCracken, J. D., glutathione S-transferases A1–1, A2–2, M1a–1a, and P1–1. Chem. Res. Toxicol., 10: Barrios, R. S., and Greenberg, N. M. E-7869 (R-flurbiprofen) inhibits progression of prostate cancer in the TRAMP mouse. Cancer Res., 60: 2203–2208, 2000. 310–317, 1997. 8. Tegeder, I., Pfeilschifter, J., and Geisslinger, G. Cyclooxygenase-independent actions 32. Ellis, C. K., Smigel, M. D., Oates, J. A., Oelz, O., and Sweetman, B. J. Metabolism of cyclooxygenase inhibitors. FASEB J., 15: 2057–2072, 2001. of prostaglandin D2 in the monkey. J. Biol. Chem., 254: 4152–4163, 1979. 9. Lin, H. K., Jez, J. M., Schlegel, B. P., Peehl, D. M., Pachter, J. A., and Penning, T. M. 33. Person, E. C., Waite, L. L., Taylor, R. N., and Scanlan, T. S. Albumin regulates ␥ ␥ Expression and characterization of recombinant type 2 3␣-hydroxysteroid dehydro- induction of peroxisome proliferator-activated receptor- (PPAR ) by 15-deoxy- ␦ ␥ genase (HSD) from human prostate: demonstration of bifunctional 3␣/17␤-HSD (12–14)-prostaglandin J(2) in vitro and may be an important regulator of PPAR activity and cellular distribution. Mol. Endocrinol., 11: 1971–1984, 1997. function in vivo. Endocrinology, 142: 551–556, 2001. 10. Pawlowski, J., Huizinga, M., and Penning, T. M. Isolation and partial characterization 34. Chen, Y., Morrow, J. D., and Roberts, L. J., II. Formation of reactive cyclopentenone of a full-length cDNA clone for 3␣-hydroxysteroid dehydrogenase: a potential target compounds in vivo as products of the isoprostane pathway. J. Biol. Chem., 274: enzyme for nonsteroidal anti-inflammatory drugs. Agents Actions, 34: 289–293, 10863–10868, 1999. 1991. 35. Hirata, Y., Hayashi, H., Ito, S., Kikawa, Y., Ishibashi, M., Sudo, M., Miyazaki, H., ␦ ␦ 11. Matsuura, K., Shiraishi, H., Hara, A., Sato, K., Deyashiki, Y., Ninomiya, M., and Fukushima, M., Narumiya, S., and Hayaishi, O. Occurrence of 9-deoxy- 9, 12– Sakai, S. Identification of a principal mRNA species for human 3␣-hydroxysteroid 13,14-dihydroprostaglandin D2 in human urine. J. Biol. Chem., 263: 16619–16625, dehydrogenase isoform (AKR1C3) that exhibits high prostaglandin D2 11-ketoreduc- 1988. tase activity. J. Biochem. (Tokyo), 124: 940–946, 1998. 36. Reginato, M. J., Krakow, S. L., Bailey, S. T., and Lazar, M. A. Prostaglandins 12. Penning, T. M., Burczynski, M. E., Jez, J. M., Lin, H. K., Ma, H., Moore, M., Ratnam, promote and block adipogenesis through opposing effects on peroxisome proliferator- K., and Palackal, N. Structure-function aspects and inhibitor design of type 5 activated receptor ␥. J. Biol. Chem., 273: 1855–1858, 1998. 17␤-hydroxysteroid dehydrogenase (AKR1C3). Mol. Cell. Endocrinol., 171: 137– 37. Fontagne, J., Adolphe, M., Semichon, M., Zizine, L., and Lechat, P. Effect of in vitro 149, 2001. treatment with indomethacin on mouse granulocyte-macrophage colony forming cells 13. Penning, T. M., Burczynski, M. E., Jez, J. M., Hung, C. F., Lin, H. K., Ma, H., Moore, in culture (CFUC). Possible role of prostaglandins. Exp. Hematol., 8; 1157–1164, M., Palackal, N., and Ratnam, K. Human 3␣-hydroxysteroid dehydrogenase isoforms 1980. (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and 38. Pelus, L. M. Blockade of prostaglandin biosynthesis in intact mice dramatically tissue distribution reveals roles in the inactivation and formation of male and female augments the expansion of committed myeloid progenitor cells (colony-forming sex hormones. Biochem. J., 351: 67–77, 2000. units-granulocyte, macrophage) after acute administration of recombinant human 14. Mills, K. I., Gilkes, A. F., Sweeney, M., Choudhry, M. A., Woodgate, L. J., Bunce, IL-1␣. J. Immunol., 143; 4171–4179, 1989. C. M., Brown G., and Burnett, A. K. Identification of a retinoic acid responsive 39. O’Reilly, M., and Gamelli, R., L. Indomethacin augments granulocyte-macrophage aldoketoreductase expressed in HL60 leukaemic cells. FEBS Lett. 440: 158–162, colony stimulating factor-induced hematopoiesis following 5-FU treatment. Exp. 1998. Hematol., 18; 974–978, 1990. 511

40. Gamelli, R., L., He, L., K., and Liu, H. Marrow granulocyte-macrophage progenitor heterodimer. A basis for new therapeutic strategies. J. Exp. Med., 193: 827–838, cell response to burn injury as modified by endotoxin and indomethacin. J. Trauma, 2001. 37; 339–346, 1994. 46. Tanaka, T., Kohno, H., Yoshitani, S., Takashima, S., Okumura, A., Murakami, A., 41. Sasaki, H., and Fukushima, M. Prostaglandins in the treatment of cancer. Anticancer and Hosokawa, M. Ligands for peroxisome proliferator-activated receptors ␣ and ␥ Drugs, 5: 131–138, 1994. inhibit chemically induced colitis and formation of aberrant crypt foci in rats. Cancer 42. Kubota, T., Koshizuka, K., Williamson, E. A., Asou, H., Said, J. W., Holden, S. Res., 61: 2424–2428, 2001. Miyoshi, I., and Koeffler, H. P. Ligand for peroxisome proliferator-activated receptor 47. Hisatake, J. I., Ikezoe, T., Carey, M., Holden, S., Tomoyasu, S., and Koeffler, H. P. ␥ (troglitazone) has potent antitumor effect against human prostate cancer both in Down-Regulation of prostate-specific antigen expression by ligands for peroxisome vitro and in vivo. Cancer Res., 58: 3344–3352. 1998. proliferator-activated receptor ␥ in human prostate cancer. Cancer Res., 60: 5494– 43. Butler, R., Mitchell, S. H., Tindall, D. J., and Young, C. Y. Nonapoptotic cell death 5498, 2000. associated with S-phase arrest of prostate cancer cells via the peroxisome proliferator- 48. Giovannucci, E., Rimm, E. B., Liu, Y., Stampfer, M. J., and Willett, W. C. A activated receptor gamma ligand, 15-deoxy-⌬12,14-prostaglandin J2. Cell Growth Differ., 11: 49–61, 2000. prospective study of tomato products, lycopene, and prostate cancer risk. J. Natl. 44. Sarraf, P., Mueller, E., Jones, D., King, F. J., DeAngelo, D. J., Partridge, J. B., Cancer Inst. (Bethesda), 94: 391–398, 2002. Holden, S. A., Chen, L. B., Singer, S., Fletcher, C., and Spiegelman, B. M. Differ- 49. Kennedy, A. R. The evidence for soybean products as cancer preventive agents. entiation and reversal of malignant changes in colon cancer through PPAR␥. Nat. J. Nutr., 125: 733S–743S, 1995. Med., 4: 1046–1052, 1998. 50. Messina, M. J., Persky, V., Setchell, K. D., and Barnes, S. Soy intake and cancer risk: 45. Desreumaux, P. Dubuquoy, L., Nutten, S., Peuchmaur, M., Englaro, W., Schoonjans, a review of the in vitro and in vivo data. Nutr. Cancer, 21: 113–131, 1994. K., Derijard, B., Desvergne, B., Wahli, W., Chambon, P., Leibowitz, M. D., Colo- 51. Krazeisen, A., Breitling, R., Moller, G., and Adamski, J. Phytoestrogens inhibit mbel, J. F., and Auwerx, J. Attenuation of colon inflammation through activators of human 17␤-hydroxysteroid dehydrogenase type 5. Mol. Cell. Endocrinol., 171: the retinoid X receptor (RXR)/peroxisome proliferator-activated receptor ␥ (PPAR␥) 151–162, 2001.

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Cancer Res 2003;63:505-512.

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