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Endocrinology Vol. 140, No. 10 4486-4493
Copyright © 1999 by The Endocrine Society

ARTICLES

Identification and Cloning of a Novel Androgen-Responsive Gene, Uridine Diphosphoglucose Dehydrogenase, in Human Breast Cancer Cells1

Jacques Lapointe and Claude Labrie

Laboratory of Molecular Endocrinology, Laval University Medical Research Center and Laval University, Ste-Foy, Québec, Canada G1V 4G2

Address all correspondence and requests for reprints to: Dr. Claude Labrie, Laboratory of Molecular Endocrinology, Laval University Medical Research Center, 2705 Laurier Boulevard, Ste-Foy, Québec, Canada G1V 4G2. E-mail: claude.labrie{at}crchul.ulaval.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgens inhibit the growth of breast cancer cells, but the mechanism of androgen-induced growth inhibition has not yet been elucidated, and few androgen-responsive genes have been identified. We, therefore, used differential display PCR to identify novel androgen-responsive genes in ZR-75–1 human breast cancer cells. The human UDP-glucose dehydrogenase gene (UDPGDH), which was not known to be androgen regulated, was detected and cloned by complementary DNA library screening. The UDPGDH open reading frame codes for a protein of 494 amino acids that migrates at an apparent molecular mass of approximately 54 kDa. Northern blot analysis revealed the existence of two messenger RNA species of approximately 3.5 and 2.7 kb in all of the human breast cancer cell lines examined. The major UDPGDH transcript was induced rapidly (within 6 h) by dihydrotestosterone in ZR-75–1 cells, and a maximal 13-fold induction was observed after 24 h of treatment. The increase in UDPGDH messenger RNA was completely prevented by coincubation with the pure antiandrogen hydroxyflutamide, but not by cycloheximide, indicating that UDPGDH is directly regulated by the androgen receptor. As UDPGDH is required for the production of uridine 5'-diphosphoglucuronic acid, a substrate for the steroid-conjugating uridine diphospho-glucuronosyltransferase enzymes, up-regulation of UDPGDH expression by androgens might play an important role in the control of sex steroid inactivation via glucuronidation in breast cancer cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGENS are known to inhibit human breast cancer cell proliferation in vitro (1) as well as in vivo (2), and clinical observations indicate that androgens or androgenic compounds can have beneficial effects on breast cancer growth in women (3). The most potent natural androgen is 5{alpha}-dihydrotestosterone (DHT). DHT interacts with high affinity with the androgen receptor (AR), a cellular DHT-binding transcription factor (4, 5, 6, 7). The AR is a member of the steroid hormone receptor superfamily, and the mechanism of action of DHT is similar to that of other steroid hormones (8, 9). Upon binding to DHT, the AR becomes activated and associates with the regulatory regions of DHT-responsive genes via androgen response elements. In humans, the best-characterized androgen-responsive gene is the prostate-specific antigen gene (10, 11).

The AR is expressed in androgen target tissues such as the prostate as well as in the mammary gland and a large proportion of human breast tumors (12, 13, 14, 15). However, the role of androgens in breast physiology and the mechanisms of androgen-induced growth inhibition of breast cancer cells are poorly understood. We have previously reported that DHT prolongs the duration of the cell cycle in ZR-75–1 cells in vitro (16). Part of the growth inhibitory effect of androgens may also be attributed to androgen-induced down-regulation of the estrogen receptor (17). However, the inhibitory effect of androgens on breast cancer cell growth is additive to that of antiestrogens and also occurs in the absence of estrogenic stimulation, suggesting that androgens exert a direct inhibitory effect on breast cancer cell proliferation that is independent of estrogen receptor levels (1, 2).

Identifying androgen-responsive genes in breast cancer cells will probably yield informative clues on the mechanism of the beneficial androgen action in breast cancer. Androgens are known to up-regulate GCDFP-15 (gross cystic disease breast fluid protein) and GCDFP-24 (apolipoprotein D) messenger RNA (mRNA) levels in breast cancer cells (18, 19), but the role of these proteins in androgen-induced growth inhibition is unknown. More recently, we observed that androgens down-regulate the expression of the antiapoptotic protooncogene bcl-2 in ZR-75–1 cells, suggesting that modulation of apoptosis may be involved in androgen-induced growth inhibition (20).

To better understand the mechanism of androgen action, we proceeded to identify a larger number of androgen-regulated transcripts in ZR-75–1 cells by differential display PCR. We report here the identification and cloning of UDP-glucose dehydrogenase (UDPGDH), a novel androgen-induced gene in human breast cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
17ß-Estradiol (E2) and DHT were purchased from Steraloids (Wilton, NH). The pure antiandrogen hydroxyflutamide was provided by Dr. R. Neri, Schering-Plough Corp. Research Institute (Kenilworth, NJ). Cycloheximide and actinomycin D were obtained from ICN Biomedicals, Inc. (Aurora, OH).

Cell culture
The MCF-7, ZR-75–1, T-47D, MDA-MB-231, MDA-MB-468, and BT-20 cell lines were obtained from American Type Culture Collection (Manassas, VA). MCF-7 cells were propagated in DMEM-Ham’s F-12 medium containing 5% (vol/vol) FBS supplemented with 2 mM L-glutamine, 100 IU penicillin/ml, 50 µg streptomycin/ml, and 1 nM E2. T-47D and ZR-75–1 cells were cultured in RPMI medium containing 10% FBS, glutamine, antibiotics, and 1 nM E2. MDA-MB-231, MDA-MB-468, and BT-20 cells were grown in MEM with 5% FBS, 1% nonessential amino acids, and antibiotics.

For experimental protocols, ZR-75–1 cells were plated in phenol red-free RPMI 1640 supplemented with 2 mM L-glutamine, 100 IU penicillin/ml, 50 µg streptomycin/ml, 5% (vol/vol) dextran-coated charcoal-treated FBS, and 0.1 nM E2. After 72 h, the medium was replaced with fresh medium containing either 0.1 nM E2 alone or in combination with 1 nM DHT. For the differential display experiment, ZR-75–1 cells were harvested after 48 h of incubation with E2 or E2 and DHT. Selection of the 48-h incubation period was based on the observation that pS2 mRNA levels were significantly down-regulated in DHT-treated cells at this time point (data not shown). For the time-course experiment, ZR-75–1 cells were cultured as described above and harvested after 0, 1, 6, 24, and 48 h of treatment. Experiments with the protein synthesis inhibitor cycloheximide, the RNA synthesis inhibitor actinomycin D, and the antiandrogen hydroxyflutamide were performed by adding the compounds to the cell culture medium at concentrations of 10 µg/ml, 1 µg/ml, and 3 x 10-6 M respectively, for 6 h in the presence of E2 and DHT.

Differential display PCR
Differential display PCR was performed using the Delta Differential Display Kit (CLONTECH Laboratories, Inc., Palo Alto, CA). Total RNA was isolated from ZR-75–1 human breast cancer cells using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH) and treated with ribonuclease-free deoxyribonuclease I to remove contaminating DNA. DNA-free RNA (2 µg) from control and DHT-treated cells was processed to complementary DNA (cDNA) using Moloney murine leukemia virus reverse transcriptase and an oligo(deoxythymidine) [oligo(dT)] primer. PCR reactions were performed in the presence of 50 µM deoxy (d)-NTPs, 1 µM primers, 50 nM [{alpha}-33P]dATP (2000 Ci/mmol; NEN Life Science Products, Boston, MA), and 1 x Advantage KlenTaq Polymerase Mix and reaction buffer (CLONTECH Laboratories, Inc.). Thermal cycling was performed using a RoboCycler Gradient 40 (Stratagene, La Jolla, CA) as follows: one cycle at 94 C for 5 min, 40 C for 5 min, and 68 C for 5 min, followed by two cycles at 94 C for 40 sec, 40 C for 40 sec, and 68 C for 5 min, followed by 23 cycles at 94 C for 30 sec, 60 C for 40 sec, and 68 C for 130 sec, and then 68 C for an additional 7 min. After amplification, PCR products were analyzed by electrophoresis on 6% polyacrylamide-7 M urea gels and visualized by autoradiography. Differentially expressed bands were excised from the dried gel, reamplified by PCR using the corresponding 5'- and 3'-primers used for differential display and subcloned into pBluescript-SK vector. Inserts were sequenced by dideoxynucleotide sequencing.

Northern blot analysis
For Northern blot analyses, 5 or 10 µg total RNA were electrophoresed through 1% agarose-formaldehyde gels in 1 x MOPS buffer (40 mM morpholino-propanesulfonic acid, 10 mM sodium acetate-3 H2O, and 0.1 mM EDTA). The size-fractionated RNA was transferred to nylon membranes (GeneScreen Plus, NEN Life Science Products) and cross-linked by UV light. RNA blots were prehybridized in 50% (vol/vol) formamide, 5 x Denhardt’s solution [0.1% (wt/vol) polyvinylpyrrolidone, 0.1% (wt/vol) BSA, and 0.1% (wt/vol) Ficoll 400], 5 x SSPE (750 mM NaCl, 50 mM NaH2PO4-H2O, and 5 mM EDTA), 10% (wt/vol) dextran sulfate, 1% (wt/vol) SDS, and 100 µg/ml sonicated salmon sperm DNA. Hybridizations were performed in fresh prehybridization buffer without salmon sperm DNA.

32P-Labeled UDPGDH cDNA probes were prepared using the random priming method and [{alpha}-32P]dCTP (NEN Life Science Products). The P4/T1 cDNA fragment corresponding to UDPGDH coding nucleotides 904-1037 was used to confirm that the fragment isolated by differential display corresponded to an androgen-responsive mRNA (Fig. 2Go). All other RNA blots were probed with a BamHI-EcoRI cDNA fragment derived from the UDPGDH cDNA that corresponded to coding nucleotides 1197–1409 of the 1482-bp coding region. The 593-bp AR probe (coding nucleotides 470-1083) was generated by digesting the full-length AR cDNA with PvuII. RNA blots were prehybridized for 1 h at 42 C and hybridized overnight at 42 C with 1 x 106 cpm/ml radiolabeled probe. Membranes were washed twice in 2 x SSPE at room temperature for 15 min and once in 2 x SSPE-2% SDS at 55 C for 30 min. Northern blots were exposed to Hyperfilm MP (Amersham Pharmacia Biotech, Aylesbury, UK) at -80 C using intensifying screens. Subsequently, Northern blots were probed with a 32P-labeled GAPDH cDNA as a loading control. As the GAPDH and UDPGDH mRNAs differ significantly in size, the blots were not stripped between sequential hybridizations. X-Ray films were quantitated by scanning densitometry using the Bioimage 110 S (Millipore Corp., Bedford, MA) to measure UDPGDH and GAPDH mRNA levels. The apparent mol wt of UDPGDH mRNAs was estimated using the 0.24- to 9.5-kb RNA ladder (Life Technologies, Inc., Gaithersburg, MD).



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  		Figure 2. Northern blot analysis of androgen-regulated mRNAs. Total RNA was isolated from ZR-75–1 cells grown for 48 h in the presence of estradiol (10-10 M) alone (control) or in combination with DHT (10-9 M). The differentially displayed P4/T1 cDNA fragment isolated from DHT-treated cells was used as a probe for the Northern analysis. The blot was probed for GAPDH mRNA as a control.

 
Construction and screening of an MCF-7 cDNA library
An amplified cDNA library was constructed from human MCF-7 breast cancer cell polyadenylated RNA using the Zap Express cDNA synthesis kit and the oligo(dT) linker primer according to the supplier’s protocol (Stratagene, La Jolla, CA). Approximately 1.5 x 106 recombinant phage plaques were screened using the 32P-labeled P4/T1 cDNA fragment isolated by differential display. {lambda} phage plaques were transferred to Hybond-N nylon membranes (Amersham Pharmacia Biotech). Membranes were prehybridized and hybridized in 50% (vol/vol) formamide, 5 x Denhardt’s solution, 5 x SSPE, 0.1% (wt/vol) SDS, and 100 µg/ml salmon sperm DNA. Filters were prehybridized for 3 h at 42 C and hybridized overnight at 42 C. The filters were then washed twice in 2 x SSC (300 mM NaCl, 30 mM sodium citrate dihydrate)-0.1% SDS at room temperature for 15 min and in 0.1% SSC-0.1% SDS at 50 and 55 C for 30 min. The membranes were exposed to Hyperfilm MP (Amersham Pharmacia Biotech) at -80 C with intensifying screens. Twenty-eight plaques that hybridized to the radiolabeled UDPGDH fragment were replated and subjected to a second screening using the same probe. Single clones were excised according to the instructions provided by Stratagene to yield phagemids containing the UDPGDH inserts in pBK-CMV phagemid vector.

Double stranded P4/T1 UDPGDH fragment and UDPGDH cDNA templates were sequenced using the dideoxy chain termination method with [{alpha}-35S]dATP and T7 polymerase (Amersham Pharmacia Biotech). The two longest UDPGDH cDNAs were sequenced in both orientations. The 5'- and 3'-extremities of shorter clones were also sequenced. The nucleotide sequence of the P4/T1 cDNA fragment obtained by differential display and the UDPGDH cDNA obtained by cDNA screening were compared against the National Center for Biotechnology Information databases using the BLAST Sequence Similarity Searching program.

Expression plasmids
To construct an expression vector for epitope-tagged UDPGDH, the coding region of human UDPGDH was amplified by standard PCR techniques using 5'- and 3'-primers complementary to coding nucleotides 1–24 and 1782–1758, respectively. The 5'-primer also contained a KpnI site for subcloning and a Kozak consensus sequence for optimal translation, whereas the 3'-primer contained an XhoI site for subcloning. The PCR fragment was inserted into the KpnI and XhoI sites of pcDNA3-HA (21) in-frame with sequences encoding the hemagglutinin (HA) epitope (22). Based on the confirmed sequence of plasmid pcDNA3-UDPGDH-HA, the deduced sequence of the tagged UDPGDH protein consists of UDPGDH amino acids 1–494, followed by the amino acids encoded by the vector polylinker sequences (RYLEHASRGR) and a C-terminal HA tag (YPYDVPDYASL).

Transient transfections and Western blotting
For transient expression of HA-tagged UDPGDH, MCF-7 cells were plated at 5 x 105 cells/well in six-well culture plates. The cells were transfected 24 h later with 2.5 µg pcDNA3HA or pcDNA3-UDPGDH-HA plasmid/well using 5 µl Exgen 500 reagent according to the manufacturer’s instructions (MBI Fermentas, Amherst, NY). Approximately 16 h after transfection, the cells were washed twice with PBS and lysed on ice for 30 min in 50 µl Nonidet P-40 lysis buffer [150 mM NaCl, 50 mM Tris (pH 8), and 1% (vol/vol) Nonidet P-40]. Insoluble material was removed by centrifugation. Proteins (50 µg/lane) were separated on 12% SDS-polyacrylamide gels and electroblotted to 0.2-µm nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH). The blots were blocked in 20 mM Tris and 137 mM NaCl containing 5% (wt/vol) dried milk, 0.05% (vol/vol) Tween-20, and 0.05% (vol/vol) Nonidet P-40 at room temperature for 1 h and incubated at 4 C overnight with a polyclonal antibody against the HA epitope (Berkeley Antibody Co., Richmond, CA). After a 1-h incubation with peroxidase-conjugated antirabbit antibodies (Amersham Pharmacia Biotech), proteins were detected using the SuperSignal Ultra chemiluminescent detection system (Pierce Chemical Co., Rockford, IL). The apparent mol wt of epitope-tagged UDPGDH was estimated using prestained protein mol wt standards (Life Technologies, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of UDPGDH by differential display PCR
We used differential display PCR to identify androgen-regulated mRNAs in ZR-75–1 human breast cancer cells. Estrogen-sensitive ZR-75–1 cells were grown in the presence of 0.1 nM E2 (to stimulate cell proliferation) alone or in combination with 1 nM of the androgen DHT. The mRNA expression patterns of control and DHT-treated ZR-75–1 cells were compared using primers P4 (5'-ATTAACCCTCACTAAATGCTGGTAG-3') and T1 (5'-CATTATGCTGAGTGATATCTTTTTTTTTAA-3'). A radiolabeled cDNA fragment of approximately 500 bp was detected in cDNA samples derived from total RNA of DHT-treated cells, but not in samples prepared from control ZR-75–1 cells (Fig. 1AGo). The DNA fragment was excised from the gel, reamplified by PCR using the P4 and T1 primers used for differential display, gel purified (Fig. 1BGo), and subcloned.



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  		Figure 1. Differential display PCR analysis. A, Human ZR-75–1 breast cancer cells were grown for 48 h in the presence of estradiol (10-10 M) alone (control) or in combination with DHT (10-9 M). Total RNA was isolated from control and DHT-treated cells and used for cDNA synthesis in the presence of oligo(dT) primer and Moloney murine leukemia virus reverse transcriptase. A fraction of each cDNA was amplified by PCR using P4 and T1 primers, [{alpha}-33P]dATP, and dNTP mix. The products were analyzed on a denaturing 6% acrylamide gel. The gel was dried and exposed to x-ray film. B, The differentially displayed cDNA was amplified by PCR using the P4 and T1 primers and was separated on a 1% agarose gel along with DNA size markers.

 
The 492-bp P4/T1 cDNA fragment was then radiolabeled and used to probe a Northern blot of the same pool of mRNAs used for differential display. Figure 2Go shows that the fragment hybridized to a prominent 3.5-kb transcript and a fainter 2.7-kb mRNA in ZR-75–1 cells. In agreement with the results of the differential display analysis, the two transcripts were more abundant in DHT-treated cells than in control cells, confirming that the P4/T1 cDNA fragment corresponded to an androgen-responsive gene. These results were confirmed using different pools of mRNAs from DHT-treated and control ZR-75–1 cells (data not shown).

Cloning and expression of human UDPGDH
The sequence of the P4/T1 cDNA fragment was compared with National Center for Biotechnology Information databases using the BLAST Sequence Similarity Searching program. Comparison of the potential open reading frames of this fragment suggested that it might correspond to the human homolog of the bovine UDPGDH (GenBank accession no. AF001310).

To obtain a full-length cDNA clone for putative human UDPGDH, we screened an MCF-7 breast cancer cell cDNA library using the P4/T1 cDNA fragment as a probe. The 2 longest overlapping clones were sequenced in their entirety in both orientations. The longest cDNA was 2924 bp in length and contained a 32-nucleotide 5'-untranslated region and a 3'-untranslated region of 1410 bp with three consensus polyadenylation signals (AAUAAA) located at positions 2103, 2310, and 2880 of the cDNA sequence. The most 3'-polyadenylation signal is located 21 nucleotides upstream from the polyadenylase tail. The longest uninterrupted open reading frame of 1482 bp encoded a protein of 494 residues (Fig. 3Go). Sequencing of the 5'- and 3'-ends of seven shorter cDNAs indicated that they were comprised within the longest cDNA.



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  		Figure 3. Nucleotide and deduced amino acid sequence of human UDPGDH. The numbers at the right indicate the nucleotide number. Amino acid positions are shown on the left. The termination codon is indicated by an asterisk. Nucleotide differences between the MCF-7 cDNA and the published composite sequence are underlined.

 
During the course of this work, Spicer et al. produced a composite human UDPGDH sequence by aligning expressed sequence tags to form a continuous cDNA and using RT-PCR fragments to bridge gaps in the sequence (23). The sequence of the MCF-7 UDPGDH cDNA that we isolated is identical to the published sequence (GenBank accession no. AF061016) with the exception of a few nucleic acid differences in the 3'-untranslated region. These changes are underlined in Fig. 3Go. The cDNA sequence published by Spicer et al. contains an additional 46 nucleotides of 5'-untranslated nucleotides with an in-frame termination codon located 42 bp upstream of the putative translation initiation codon, indicating that the cloned UDPGDH cDNAs encode the full-length human UDPGDH protein. Human UDPGDH shows 98% similarity with bovine UDPGDH, but differs at its C-terminus, where it contains an additional 26 amino acids (24).

The calculated molecular mass of human UDPGDH is 55 kDa. To estimate the molecular mass of human UDPGDH in vivo, the coding region of UDPGDH was subcloned into an expression vector in-frame with sequences encoding a C-terminal HA epitope tag. The resulting plasmid, pcDNA3-UDPGDH-HA, was transiently transfected into MCF-7 cells, and whole cell extracts were analyzed by immunoblotting using a polyclonal antibody directed against the HA epitope. HA-tagged UDPGDH migrated at an apparent molecular mass of approximately 54 kDa (Fig. 4Go). As the epitope-tagged protein contains 21 heterologous residues at its C-terminus, the apparent molecular mass of the native protein is estimated to be approximately 52 kDa, which corresponds to the apparent molecular mass of bovine UDPGDH (24).



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  		Figure 4. UDPGDH-HA expression in MCF-7 cells. MCF-7 cells were transiently transfected with pcDNA3-HA (lane 1) or pcDNA3-UDPGDH-HA (lane 2) expression plasmid. Whole cell extracts were prepared 16 h after transfection and analyzed by Western blotting using a polyclonal antibody directed against the HA epitope.

 
Expression of UDPGDH mRNA in breast cancer cell lines
To determine whether UDPGDH is widely expressed in human breast cancer cells, we examined the expression of UDPGDH mRNA in the estrogen-sensitive MCF-7, ZR-75–1, and T-47D human breast cancer cell lines as well as in the estrogen-insensitive MDA-MB-231, MDA-MB-468, and BT-20 cell lines. RNA was isolated from cells grown under routine culture conditions, and UDPGDH and androgen receptor expression was evaluated by Northern blot analysis. AR mRNA was expressed abundantly in ZR-75–1 and T-47D cells, whereas UDPGDH mRNA was detected in all six cell lines examined regardless of the androgen receptor status (Fig. 5Go). As in ZR-75–1 cells, the long (3.5-kb) transcript was more abundant than the short (2.7-kb) mRNA, but both mRNAs were generally coexpressed. In addition to breast cancer cells, UDPGDH mRNA was also detected in commercially available RNA samples prepared from normal human breast tissue (CLONTECH Laboratories, Inc.) as well as in a wide variety of human tissues, including the heart, placenta, liver, prostate, ovary, pancreas, and digestive tract (data not shown). The presence of two transcripts was also observed by Spicer et al. (23). It is presently unknown whether the presence of these two transcripts arises by use of different polyadenylation sites or alternative splicing.



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  		Figure 5. UDPGDH mRNA expression in breast cancer cell lines. Total RNA was isolated from MCF-7, ZR-75–1, T-47D, MDA-MB-231, MDA-MB-468, and BT-20 cells and used for Northern analysis. The blot was probed for the UDPGDH and AR mRNAs. GAPDH mRNA is shown as a loading control.

 
Regulation of UDPGDH mRNA levels by DHT in ZR-75–1 cells
We measured UDPGDH mRNA levels in ZR-75–1 cells harvested after 1–48 h of DHT treatment to determine the time course of UDPGDH mRNA induction in DHT-treated ZR-75–1 cells. Cells harvested before the addition of DHT and after an additional 48 h of incubation with E2 alone served as controls. As illustrated in Fig. 6Go, Northern blot analysis revealed that the steady state levels of UDPGDH mRNA had already increased markedly after 6 h of DHT treatment, whereas maximal levels of UDPGDH RNA were attained 24 h after the addition of DHT. Based on densitometry data averaged from independent experiments, the levels of the long UDPGDH transcript were 4, 13, and 13 times higher in cells incubated with DHT for 6, 24, and 48 h, respectively, than in control cells. The levels of the short UDPGDH mRNA were 3, 10, and 10 times higher after 6, 24, and 48 h of DHT treatment, respectively. In agreement with the presence of AR mRNA in T-47D cells, UDPGDH mRNA levels also increased after incubation of T-47D cells with DHT (data not shown).



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  		Figure 6. Time course of DHT treatment on UDPGDH mRNA levels in ZR-75–1 cells. ZR-75–1 cells were grown for 0, 1, 6, 24, and 48 h in medium containing estradiol (10-10 M) supplemented with DHT (10-9 M). Cells grown for 48 h in the absence of DHT are shown in the last lane on the right. The blot was probed for UDPGDH mRNA, and GAPDH mRNA is shown as a loading control.

 
To determine whether the induction of UDPGDH mRNA requires protein synthesis, ZR-75–1 cells were treated with DHT in combination with the protein synthesis inhibitor, cycloheximide (10 µg/ml), for 6 h. As shown in the Northern blot of Fig. 7Go, UDPGDH mRNA levels increased after a 6-h incubation with DHT. This induction was not abolished by cycloheximide (Fig. 7Go), suggesting that the increase in UDPGDH mRNA caused by DHT is independent of new protein synthesis. In contrast to cycloheximide, the RNA synthesis inhibitor actinomycin D (1 µg/ml) abolished the induction of UDPGDH mRNA in DHT-treated cells (Fig. 7Go). The pure antiandrogen hydroxyflutamide also abolished the induction of UDPGDH by DHT (Fig. 7Go), indicating that the induction of UDPGDH mRNA by DHT is an AR-dependent event.



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  		Figure 7. Northern analysis of UDPGDH mRNA levels after DHT, cycloheximide, actinomycin D, and hydroxyflutamide treatment in ZR-75–1 cells. ZR-75–1 cells were grown in medium containing estradiol (10-10 M) supplemented with 10-9 M DHT, DHT plus cycloheximide, DHT plus actinomycin D, and DHT plus hydroxyflutamide (3 x 10-6 M) for 6 h. The first lane represents control cells grown in medium containing estradiol (10-10 M) alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present data identify a new androgen-responsive gene in ZR-75–1 human breast cancer cells. We, in fact, found that UDPGDH transcripts are ubiquitously expressed in human breast cancer cells and that they are coinduced by DHT in androgen-sensitive ZR-75–1 cells. In contrast to other androgen-induced genes in mammary cells that are usually responsive to estrogens, UDPGDH appears to be specifically regulated by androgens, as antiestrogens had no effect on UDPGDH mRNA levels in ZR-75–1 breast cancer cells (data not shown). Androgens and estrogens exert opposite effects on several parameters in breast cancer cells. For example, androgens block estrogen-induced cell proliferation as well as estrogen-induced cathepsin D (18) and pS2 gene expression (20). Androgens and estrogens also exert opposite effects on GCDFP-15 and GCDFP-24 expression (18, 19).

UDPGDH converts UDP-glucose to uridine 5'-diphosphoglucuronic acid (UDPGA). This activity has been demonstrated using bovine UDPGDH (25) and cloned mouse UDPGDH (23), which show 98% amino acid identity with the cloned human enzyme (Ref. 23 and this paper). In vertebrates, uridine diphospho-glucuronosyltransferases (UGTs) catalyze the transfer of the glucuronyl group from UDPGA to endogenous lipid-soluble molecules, such as bilirubin, drugs, and steroid hormones (26, 27). The resulting glucuronidated products are more polar and more easily excreted. Therefore, up-regulation of UDPGDH in androgen-treated breast cancer cells may play an important role in modulating the actions of sex steroids in breast cancer cells by affecting sex steroid metabolism.

This detoxification mechanism is very active in the liver, but several lines of evidence indicate that sex steroid glucuronidation is an important inactivating pathway in the gonads (28, 29) as well as in peripheral target tissues such as the normal prostate (30, 31) and mammary gland (32) as well as in prostate (LNCaP) and breast (MCF-7, ZR-75–1) cancer cell lines (33, 34). In fact, UGT enzymes are widely expressed in target tissues. The isoform UGT2B15 is expressed in the mammary gland, in LNCaP prostate cancer cells, as well as in MCF-7 breast cancer cells (35), whereas UGT2B17 mRNA has been detected in the mammary gland and LNCaP cells (36). Unfortunately, the expression profile of UGT enzymes in other breast cancer cell lines has not been studied extensively.

Steroid glucuronidation in target tissues is regulated by a number of different factors. Both sex steroids and growth factors have been shown to decrease androgen glucuronidation and UGT expression in LNCaP cells, thereby potentially augmenting the useful lifetime of androgens (36). In fact, DHT itself inhibits the formation of DHT glucuronide (DHT-G), and growth factors such as epidermal growth factor as well as acidic and basic fibroblast growth factor decrease DHT-G formation in LNCaP cells (37, 38, 39). In addition, the cytokine interleukin-1{alpha} inhibits the formation of DHT-G and down-regulates UGT2B17 mRNA levels in LNCaP cells (40). Presumably, up-regulation of UDPGDH by DHT in ZR-75–1 cells may affect the inactivation of sex steroids, although it is not known whether the abundance of UDPGDH is limiting for sex steroid glucuronidation. The observation that interleukin-1ß also augments UDPGDH mRNA levels in fibroblasts supports the concept that the modulation of UDPGDH mRNA levels may be an important regulatory mechanism in various physiological processes (23).

Sex steroid inactivation via glucuronidation represents an active pathway for controlling the action of antagonistic sex steroids in target cells such as the mammary gland. In fact, as mentioned above, androgens and estrogens exert opposite effects on breast cancer cell proliferation (1), and estrogens have been shown to stimulate DHT inactivation via glucuronidation in MCF-7 breast cancer cells (41). Thus, one could assume that the up-regulation of UDPGDH in DHT-treated ZR-75–1 cells might contribute to the inactivation of growth-stimulating estrogens. In addition, up-regulation of UDPGDH may also affect as yet unidentified gene regulatory pathways in addition to those that lie downstream of the classical sex steroid receptors. For instance, DHT metabolites were thought to be devoid of biological activity until the recent discovery that some of these metabolites can modulate the activity of an orphan nuclear receptor in the mouse (42). As DHT metabolites are also eliminated via glucuronidation, this finding suggests that glucuronidation of DHT and its metabolites may be an important regulatory mechanism.

UDPGDH is expressed in several human tissues, including the mammary gland, and it is ubiquitously expressed in the breast cancer cell lines that we examined. The widespread expression of UDPGDH is not surprising, as UDPGA is essential for the biosynthesis of proteoglycans such as heparan and chondroitin sulfate that are found predominantly on the cell surface and in the extracellular matrix. In Drosophila, embryos with a mutated UDPGDH gene show embryonic cuticle phenotypes similar to those that result from loss of function mutations in genes of the Wingless signaling pathway (43, 44, 45). It has been proposed that proteoglycans may serve as low affinity coreceptors for growth factors such as fibroblast growth factor and transforming growth factor-ß and thereby modulate the ability of the growth factor or its cognate signaling receptor to produce a biological response (for review, see Ref. 46). If UDPGDH up-regulation were to affect proteoglycan formation, one could speculate that the resulting effects on growth factor action might contribute to the growth inhibitory effect of DHT in breast cancer cells.

Differential display PCR is a powerful tool for identifying hormone-responsive genes (47). Novel androgen-regulated genes, such as NKX3.1 (48) and clathrin (49), have been identified in this manner in the prostate. Unfortunately, our understanding of the mechanism of androgen action and androgen-induced growth inhibition of breast cancer cells is limited by the small number of androgen-responsive target genes that have been isolated to date. The identification of additional androgen-regulated genes, such as UDPGDH, by differential display should greatly contribute to our understanding of androgen action in breast cancer.


   Acknowledgments
 
We are grateful to F. Labrie and O. Barbier for critical reading of the manuscript. We thank A. Fournier for help in cell culture, the members of our group for helpful discussions, and the members of the Centre Hospitalier Université Laval Research Center Illustration Service for artwork.


   Footnotes
 
1 This work was supported by grants from the Canadian Breast Cancer Research Initiative (no. 6292 and 9430), a fellowship from Le Fonds de Recherche en Santé du Québec (to C.L.), and a scholarship from Le Fonds de Recherche en Santé du Québec. Additional financial support was provided by Endorecherche. Back

Received March 2, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Poulin R, Baker D, Labrie F 1988 Androgens inhibit basal and estrogen-induced cell proliferation in the ZR-75–1 human breast cancer cell line. Breast Cancer Res Treat 12:213–225[CrossRef][Medline]
  2. Dauvois S, Geng CS, Levesque C, Merand Y, Labrie F 1991 Additive inhibitory effects of an androgen and the antiestrogen EM-170 on estradiol-stimulated growth of human ZR-75–1 breast tumors in athymic mice. Cancer Res 51:3131–3135[Abstract/Free Full Text]
  3. Labrie F, Simard J, de Launoit Y, Poulin R, Theriault C, Dumont M, Dauvois S, Martel C, Li SM 1992 Androgens and breast cancer. Cancer Detect Prev 16:31–38[Medline]
  4. Chang CS, Kokontis J, Liao ST 1988 Molecular cloning of human and rat complementary DNA encoding androgen receptors. Science 240:324–326[Abstract/Free Full Text]
  5. Lubahn DB, Joseph DR, Sar M, Tan J, Higgs HN, Larson RE, French FS, Wilson EM 1988 The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate. Mol Endocrinol 2:1265–1275[Abstract]
  6. Tilley WD, Marcelli M, Wilson JD, McPhaul MJ 1989 Characterization and expression of a cDNA encoding the human androgen receptor. Proc Natl Acad Sci USA 86:327–331[Abstract/Free Full Text]
  7. Trapman J, Klaassen P, Kuiper GG, van der Korput JA, Faber PW, van Rooij HC, Geurts van Kessel A, Voorhorst MM, Mulder E, Brinkmann AO 1988 Cloning, structure and expression of a cDNA encoding the human androgen receptor. Biochem Biophys Res Commun 153:241–248[CrossRef][Medline]
  8. Beato M 1989 Gene regulation by steroid hormones. Cell 56:335–344[CrossRef][Medline]
  9. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889–895[Abstract/Free Full Text]
  10. Riegman PH, Vlietstra RJ, van der Korput JA, Brinkmann AO, Trapman J 1991 The promoter of the prostate-specific antigen gene contains a functional androgen responsive element. Mol Endocrinol 5:1921–1930[Abstract]
  11. Young CY, Montgomery BT, Andrews PE, Qui SD, Bilhartz DL, Tindall DJ 1991 Hormonal regulation of prostate-specific antigen messenger RNA in human prostatic adenocarcinoma cell line LNCaP. Cancer Res 51:3748–3752[Abstract/Free Full Text]
  12. Bryan RM, Mercer RJ, Bennett RC, Rennie GC, Lie TH, Morgan FJ 1984 Androgen receptors in breast cancer. Cancer 54:2436–2440[CrossRef][Medline]
  13. Kimura N, Mizokami A, Oonuma T, Sasano H, Nagura H 1993 Immunocytochemical localization of androgen receptor with polyclonal antibody in paraffin-embedded human tissues. J Histochem Cytochem 41:671–678[Abstract]
  14. Lea OA, Kvinnsland S, Thorsen T 1989 Improved measurement of androgen receptors in human breast cancer. Cancer Res 49:7162–7167[Medline]
  15. Ormandy CJ, Hall RE, Manning DL, Robertson JF, Blamey RW, Kelly PA, Nicholson RI, Sutherland RL 1997 Coexpression and cross-regulation of the prolactin receptor and sex steroid hormone receptors in breast cancer. J Clin Endocrinol Metab 82:3692–3699[Abstract/Free Full Text]
  16. de Launoit Y, Dauvois S, Dufour M, Simard J, Labrie F 1991 Inhibition of cell cycle kinetics and proliferation by the androgen 5{alpha}-dihydrotestosterone and antiestrogen N,N-butyl-N-methyl-11-[16'{alpha}-chloro-3',17ß-dihydroxy-estra-1',3',5'-(10')triene-7'{alpha}-yl]undecanamide in human breast cancer ZR-75–1 cells. Cancer Res 51:2797–2802[Abstract/Free Full Text]
  17. Poulin R, Simard J, Labrie C, Petitclerc L, Dumont M, Lagace L, Labrie F 1989 Down-regulation of estrogen receptors by androgens in the ZR-75–1 human breast cancer cell line. Endocrinology 125:392–399[Abstract]
  18. Simard J, Hatton AC, Labrie C, Dauvois S, Zhao HF, Haagensen DE, Labrie F 1989 Inhibitory effect of estrogens on GCDFP-15 mRNA levels and secretion in ZR-75–1 human breast cancer cells. Mol Endocrinol 3:694–702[Abstract]
  19. Simard J, Dauvois S, Haagensen DE, Levesque C, Merand Y, Labrie F 1990 Regulation of progesterone-binding breast cyst protein GCDFP-24 secretion by estrogens and androgens in human breast cancer cells: a new marker of steroid action in breast cancer. Endocrinology 126:3223–3231[Abstract]
  20. Lapointe J, Fournier A, Richard V, Labrie C 1999 Androgens down-regulate bcl-2 protooncogene expression in ZR-75–1 human breast cancer. Endocrinology 140:416–421[Abstract/Free Full Text]
  21. Lapointe J, Lachance Y, Labrie Y, Labrie C 1996 A p18 mutant defective in CDK6 binding in human breast cancer cells. Cancer Res 56:4586–4589[Abstract/Free Full Text]
  22. Field J, Nikawa J, Broek D, MacDonald B, Rodgers L, Wilson IA, Lerner RA, Wigler M 1988 Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol 8:2159–2165[Abstract/Free Full Text]
  23. Spicer AP, Kaback LA, Smith TJ, Seldin MF 1998 Molecular cloning and characterization of the human and mouse UDP-glucose dehydrogenase genes. J Biol Chem 273:25117–25124[Abstract/Free Full Text]
  24. Hempel J, Perozich J, Romovacek H, Hinich A, Kuo I, Feingold DS 1994 UDP-glucose dehydrogenase from bovine liver: primary structure and relationship to other dehydrogenases. Protein Sci 3:1074–1080[Abstract]
  25. Zalitis J, Feingold DS 1969 Purification and properties of UDPG dehydrogenase from beef liver. Arch Biochem Biophys 132:457–465[CrossRef][Medline]
  26. Burchell B, Coughtrie MW 1989 UDP-glucuronosyltransferases. Pharmacol Ther 43:261–289[CrossRef][Medline]
  27. Tephly TR, Burchell B 1990 UDP-glucuronosyltransferases: a family of detoxifying enzymes. Trends Pharmacol Sci 11:276–279[CrossRef][Medline]
  28. Prevost J, Brochu M, Belanger A, Lambert R 1987 Conjugated and unconjugated C-21, C-19 and C-18 steroid concentrations in human follicular fluid from hyperstimulated follicles. Gynecol Endocrinol 1:331–338[Medline]
  29. Chen F, Ritter JK, Wang MG, McBride OW, Lubet RA, Owens IS 1993 Characterization of a cloned human dihydrotestosterone/androstanediol UDP-glucuronosyltransferase and its comparison to other steroid isoforms. Biochemistry 32:10648–10657[CrossRef][Medline]
  30. Belanger A, Brochu M, Lacoste D, Noel C, Labrie F, Dupont A, Cusan L, Caron S, Couture J 1991 Steroid glucuronides: human circulatory levels and formation by LNCaP cells. J Steroid Biochem Mol Biol 40:593–598[CrossRef][Medline]
  31. Belanger G, Beaulieu M, Marcotte B, Levesque E, Guillemette C, Hum DW, Belanger A 1995 Expression of transcripts encoding steroid UDP-glucuronosyltransferases in human prostate hyperplastic tissue and the LNCaP cell line. Mol Cell Endocrinol 113:165–173[CrossRef][Medline]
  32. Belanger A, Caron S, Labrie F, Naldoni C, Dogliotti L, Angeli A 1990 Levels of eighteen non-conjugated and conjugated steroids in human breast cyst fluid: relationships with cyst type. Eur J Cancer 26:277–281
  33. Adams JB, Phillips NS, Young CE 1989 Formation of glucuronides of estradiol-17ß by human mammary cancer cells. J Steroid Biochem 33:1023–1025[CrossRef][Medline]
  34. Guillemette C, Hum DW, Belanger A 1995 Specificity of glucuronosyltransferase activity in the human cancer cell line LNCaP, evidence for the presence of at least two glucuronosyltransferase enzymes. J Steroid Biochem Mol Biol 55:355–362[CrossRef][Medline]
  35. Levesque E, Beaulieu M, Green MD, Tephly TR, Belanger A, Hum DW 1997 Isolation and characterization of UGT2B15(Y85): a UDP-glucuronosyltransferase encoded by a polymorphic gene. Pharmacogenetics 7:317–325[Medline]
  36. Beaulieu M, Levesque E, Hum DW, Belanger A 1996 Isolation and characterization of a novel cDNA encoding a human UDP-glucuronosyltransferase active on C19 steroids. J Biol Chem 271:22855–22862[Abstract/Free Full Text]
  37. Belanger A, Hum DW, Beaulieu M, Levesque E, Guillemette C, Tchernof A, Belanger G, Turgeon D, Dubois S 1998 Characterization and regulation of UDP-glucuronosyltransferases in steroid target tissues. J Steroid Biochem Mol Biol 65:301–310[CrossRef][Medline]
  38. Guillemette C, Levesque E, Beaulieu M, Turgeon D, Hum DW, Belanger A 1997 Differential regulation of two uridine diphospho-glucuronosyltransferases, UGT2B15 and UGT2B17, in human prostate LNCaP cells. Endocrinology 138:2998–3005[Abstract/Free Full Text]
  39. Levesque E, Beaulieu M, Guillemette C, Hum DW, Belanger A 1998 Effect of fibroblastic growth factors (FGF) on steroid UDP-glucuronosyltransferase expression and activity in the LNCaP cell line. J Steroid Biochem Mol Biol 64:43–48[CrossRef][Medline]
  40. Levesque E, Beaulieu M, Guillemette C, Hum DW, Belanger A 1998 Effect of interleukins on UGT2B15 and UGT2B17 steroid uridine diphosphate-glucuronosyltransferase expression and activity in the LNCaP cell line. Endocrinology 139:2375–2381[Abstract/Free Full Text]
  41. Roy R, Dauvois S, Labrie F, Belanger A 1992 Estrogen-stimulated glucuronidation of dihydrotestosterone in MCF-7 human breast cancer cells. J Steroid Biochem Mol Biol 41:579–582[CrossRef][Medline]
  42. Forman BM, Tzameli I, Choi HS, Chen J, Simha D, Seol W, Evans RM, Moore DD 1998 Androstane metabolites bind to and deactivate the nuclear receptor CAR-ß. Nature 395:612–615[CrossRef][Medline]
  43. Binari RC, Staveley BE, Johnson WA, Godavarti R, Sasisekharan R, Manoukian AS 1997 Genetic evidence that heparin-like glycosaminoglycans are involved in wingless signaling. Development 124:2623–2632[Abstract]
  44. Hacker U, Lin X, Perrimon N 1997 The Drosophila sugarless gene modulates Wingless signaling and encodes an enzyme involved in polysaccharide biosynthesis. Development 124:3565–3573[Abstract]
  45. Haerry TE, Heslip TR, Marsh JL, O’Connor MB 1997 Defects in glucuronate biosynthesis disrupt Wingless signaling in Drosophila. Development 124:3055–3064[Abstract]
  46. Schlessinger J, Lax I, Lemmon M 1995 Regulation of growth factor activation by proteoglycans: what is the role of the low affinity receptors? Cell 83:357–360[CrossRef][Medline]
  47. Liang P, Pardee AB 1992 Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967–971[Abstract/Free Full Text]
  48. Prescott JL, Blok L, Tindall DJ 1998 Isolation and androgen regulation of the human homeobox cDNA, NKX3.1. Prostate 35:71–80[CrossRef][Medline]
  49. Prescott JL, Tindall DJ 1998 Clathrin gene expression is androgen regulated in the prostate. Endocrinology 139:2111–2119[Abstract/Free Full Text]



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