From the Laboratory for Experimental Medicine and
Endocrinology and the ¶ Division of Biochemistry, Faculty of
Medicine, Onderwijs en Navorsing, Gasthuisberg, Catholic University of
Leuven, B-3000 Leuven, Belgium
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ABSTRACT |
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Diazepam-binding
inhibitor/acyl-CoA-binding protein (DBI/ACBP), a highly conserved
10-kDa polypeptide, has been implicated in various physiological
processes including -aminobutyric acid type A receptor binding,
acyl-CoA binding and transport, steroidogenesis, and peptide hormone
release. Both in LNCaP prostate cancer cells and 3T3-L1 preadipocytes,
the expression of DBI/ACBP is stimulated under conditions that promote
lipogenesis (treatment with androgens and insulin, respectively) and
that involve the activation of sterol regulatory element-binding
proteins (SREBPs). Accordingly, we investigated whether DBI/ACBP
expression is under the direct control of SREBPs. Analysis of the human
and rat DBI/ACBP promoter revealed the presence of a conserved sterol
regulatory element (SRE)-like sequence. Gel shift analysis confirmed
that this sequence is able to bind SREBPs. In support of the
functionality of SREBP binding, coexpression of SREBP-1a with a
DBI/ACBP promoter-reporter gene resulted in a 50-fold increase in
transcriptional activity in LNCaP cells. Disruption of the SRE
decreased basal expression and abolished SREBP-1a-induced
transcriptional activation. In agreement with the requirement of a
co-regulator for SREBP function, transcriptional activation by SREBP-1a
overexpression was severely diminished when a neighboring NF-Y site was
mutated. Cholesterol depletion or androgen treatment, conditions that
activate SREBP function in LNCaP cells, led to an increase in DBI/ACBP
mRNA expression and SRE-dependent transcriptional activation.
These findings indicate that the promoter for DBI/ACBP contains a
functional SRE that allows DBI/ACBP to be coregulated with other genes
involved in lipid metabolism.
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INTRODUCTION |
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Diazepam-binding inhibitor
(DBI)1 is a highly conserved
10-kDa polypeptide that is expressed in a wide variety of species
ranging from yeast to mammals. It is found in various tissues and
organs and was identified independently in various different
experimental settings. DBI was first isolated from rat brain based on
its ability to displace diazepam (Valium) from the -aminobutyric
acid A receptor (1). When injected intraventricularly in rats, it
produces conflict behavior, an action that can be blocked by the
benzodiazepine antagonist flumazenil (2, 3). Independently, a 10-kDa
peptide was isolated from bovine liver by virtue of its ability to bind and induce the synthesis of medium-chain acyl-CoA esters. This peptide
was designated acyl-CoA-binding protein (ACBP) (4). Sequencing revealed
that it was the bovine homologue of rat DBI (5, 6). ACBP has been shown
to bind acyl-CoA esters with high affinity, to protect them from
hydrolysis and to attenuate acyl-CoA inhibition of enzymatic activities
such as acetyl-CoA carboxylation (7, 8). In addition, ACBP has been
shown to extract acyl-CoA from phosphatidylcholine membranes and donate it for
oxidation or glycerolipid synthesis (8). Based on these
findings, together with the observation that overexpression of
recombinant bovine ACBP in yeast leads to a substantial increase in
cellular acyl-CoA content (9), it has been proposed that ACBP functions
as a pool former and transporter of acyl-CoA. In another experimental
setting, a protein able to stimulate mitochondrial steroid synthesis
was isolated from adrenal cortex and found to be identical to DBI
(10-12). Furthermore, in Leydig and in glial cells, DBI/ACBP has been
shown to stimulate steroidogenesis by facilitating cholesterol
translocation to the inner mitochondrial membrane. This process is
mediated by a peripheral-type benzodiazepine receptor (see Refs. 13 and
14 for review). A protein with sequences identical to DBI was also
isolated from porcine intestine and found to inhibit both early and
late phases of glucose-induced insulin release from isolated perfused
rat pancreas (15). This finding has been confirmed by several other
investigators and may be of physiological significance (16-19). In
fact, DBI-like immunoreactivity was found in cells of pancreatic islets
(16-17). In yet another study, a trypsin-sensitive peptide that is
secreted intraduodenally and that functions as a potent
cholecystokinin-releasing peptide in the intestine was found to be
identical to DBI (20). Furthermore, peptides with sequences identical
to DBI have been shown to have antibacterial properties (21) and to
function as paracrine or autocrine modulators of cell proliferation and cell function (22, 23).
In agreement with its expression in various tissues and cell types and its postulated roles in many different biological processes, the promoter of the DBI/ACBP gene displays all the hallmarks of a typical housekeeping gene (24, 25) but may also allow controlled activation related to specific regulatory pathways, including hormonal stimulation (26). Hormones that have been shown to stimulate the expression of DBI/ACBP include insulin and androgens. Insulin regulation of DBI/ACBP expression has been observed in 3T3-L1 preadipocytes (27). Regulation by androgens has been found in various male accessory sex organs (28) and in the human prostate cancer cell line LNCaP (25, 29). Interestingly, both conditions promote lipogenesis and involve the activation of sterol regulatory element-binding proteins (SREBPs) (30-36). SREBPs are cholesterol-regulated transcription factors that are synthesized as inactive membrane-bound precursors (see Ref. 36 for review). Proteolytic activation results in release and translocation to the nucleus. In the nucleus, SREBPs bind to specific sterol-responsive elements (SREs) and in cooperation with the generic transcription factors SP-1 or NF-Y (37-46), they coordinately modulate the transcription of a wide array of genes involved in cholesterol and fatty acid metabolism (36).
In view of the postulated role of DBI/ACBP in fatty acid and cholesterol metabolism and its induction under conditions that involve SREBP activation, we explored whether DBI/ACBP is directly controlled by SREBPs. Both LNCaP and HepG2 cells were used as experimental paradigm.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- LNCaP and HepG2 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and were cultured as described previously (32, 47). To assess the impact of cholesterol on gene expression, cells were incubated in media supplemented with 5% lipoprotein-deficient serum (LPDS) (Perimmune, Rockville, MD) in the absence or presence of 10 µg/ml cholesterol and 1 µg/ml 25-hydroxycholesterol added from stock solutions in ethanol. Control cultures received similar amounts of ethanol only. In experiments assessing the effects of steroids, fetal calf serum was pretreated with dextran-coated charcoal (CT-FCS) to reduce the background levels of steroids. The synthetic androgen R1881 (methyltrienolone), purchased from DuPont New England Nuclear (Dreiech, Germany), was dissolved in absolute ethanol and added to the cultures. Final ethanol concentrations did not exceed 0.2%. All experiments involving LNCaP cells were carried out with cells of passages 30 to 75.
Electrophoretic Mobility Shift Assay--
Recombinant
6×His-tagged SREBP-la (amino acids 1-490) was expressed in
Escherichia coli BL21(DE3)pLysS (Stratagene, La Jolla, CA)
from plasmid pRSA (kindly provided by Dr. T. Osborne, Department of
Molecular Biology and Biochemistry, University of California, Irvine,
USA) (37) and was purified by means of TALON metal affinity chromatography (CLONTECH). Complementary
single-stranded oligonucleotides corresponding to nucleotides 137 to
113 of the published human DBI promoter sequence (25) were
end-labeled with [
-32P]ATP and T4 polynucleotide
kinase and annealed. The probe (50,000 cpm) was incubated with
recombinant SREBP-1a in a solution containing 10 mM
Tris-HCl, pH 7.6, 50 mM NaCl, 0.05 mM EDTA, 2.5 mM MgCl2, 8.5% glycerol, l mM
dithiothreitol, 0.5 µg/ml poly(dI-dC), 0.1% Triton X-100, and 2.5 mg/ml nonfat milk for 20 min on ice. Unlabeled wild type or mutated
double-stranded oligonucleotides were added as indicated in the figure
legends. In the oligonucleotides designated "mutSRE," the SRE-like
sequence CTCGCCCGAG was replaced by CTACAAAATG. Oligonucleotides
designated "delSRE" encompassed bases
145 to
105, but lacked
the SRE-like sequence. Where indicated, antibody K-10 against SREBP-la
(Santa Cruz Biotechnology, Santa Cruz, CA) was added and incubation on
ice was continued for another 20 min. The DNA-protein complexes were
resolved on a 5% nondenaturing polyacrylamide gel in 0.25×
TBE and 0.02% Triton X-100 at room temperature. Gels were vacuum-dried
and exposed to Kodak X-Omat AR film.
Plasmids--
A cDNA fragment encoding amino acids 1-460 of
human SREBP-la was generated by polymerase chain reaction and inserted
into the eukaryotic expression vector pIRES1neo
(CLONTECH). The resulting plasmid was designated
pSREBP-la1-460. Plasmid pDBI-264luc encompassing bases
264 to
13 of the human DBI/ACBP gene linked to a luciferase
reporter gene has been described previously (25) and is here referred
to as "wt." Mutations or deletions within the promoter were created
using the Quikchange mutagenesis kit (Stratagene, La Jolla, CA). The
constructs mutSRE and delSRE contain the same mutation and deletion,
respectively, as the oligonucleotides described under
"Electrophoretic Mobility Shift Assay." In construct "mutNF-Y,"
the A at position
142, which is part of a putative NF-Y site, was
replaced by a C. In construct +4A, 4 A residues were inserted at
position
131, which is located between the NF-Y and the SRE-like
sequence. Plasmid pFASluc, a fatty acid synthase promoter-reporter
construct, and plasmid pSV-AR0 expressing the androgen
receptor, have been described previously (32, 48). Plasmid pPA-7, a
luciferase-based plasmid with the prostate-specific antigen (PSA)
promoter (49), was kindly provided by Dr. J. Trapman (Erasmus
University, Rotterdam, The Netherlands).
Transient Transfections and Reporter Gene Assays--
LNCaP
cells were seeded in 6-cm dishes in Dulbecco's modified Eagle's
medium containing 10% fetal calf serum at a density of 7 × 105 cells. On the next day, the medium was replaced with
Dulbecco's modified Eagle's medium with 2% CT-FCS. Cells were
transfected with 5 µg of the indicated luciferase reporter
constructs, the indicated amounts of pIRES1neo or
pSREBP-1a1-460, and a plasmid encoding -galactosidase.
After 4 h of exposure to DNA, cells were subjected to a glycerol
shock and washed with phosphate-buffered saline. To explore potential
effects of androgens, cells were cotransfected with an androgen
receptor expression vector (pSV-ARo) (48) and incubated with
10
8 M R1881 or with ethanol vehicle in medium
containing 5% CT-FCS. In experiments assessing the effects of sterols,
cells were incubated in medium containing 5% LPDS in the absence or in
the presence of 10 µg/ml cholesterol and 1 µg/ml
25-hydroxycholesterol, or ethanol vehicle. One day after treatment,
cells were washed with phosphate-buffered saline and harvested in 500 µl of reporter lysis buffer (Promega, Madison, WI). Aliquots of 10 µl of cleared lysate were assayed for luciferase activity using a
luciferase reporter assay kit from Promega and a Berthold Microlumat LB
96P luminometer. The activity of
-galactosidase was used to
normalize for transfection efficiencies.
Northern Blot Analysis-- Total RNA was prepared using a modified guanidinium/CsCl ultracentrifugation method as described previously (29). Equal aliquots of total RNA (20 µg) were denatured and subjected to electrophoresis in a 1% agarose gel containing formaldehyde. The RNA was transferred to Biotrans + membranes (ICN Pharmaceuticals, Inc., Costa Mesa, CA), prehybridized and hybridized with DBI, FAS, PSA, and 18 S probes as described before (29, 31). Blots were autoradiographed by exposure to Amersham Hyperfilm-MP or to Kodak Biomax film (Amersham International, Buckinghamshire, UK). Hybridization signals were quantitated using PhosphorImager screens (Molecular Dynamics, Sunnyvale, CA) and normalized for differences in RNA loading.
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RESULTS |
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Identification of a SREBP-binding Site in the DBI/ACBP
Promoter--
Examination of the nucleotide sequences of the human
DBI/ACBP promoter for potential SREBP sites revealed a sequence
CTCGCCCGAG at positions 127 to
118 of the published sequence (25),
resembling the 10-base pair SREs found in other SREBP-regulated genes
(Fig. 1). This sequence is most
homologous to the SRE of the farnesyl-diphosphate synthase gene (57)
and differs only at two positions. In agreement with the requirement
for a coregulator for SREBP function (SP-1 or NF-Y) (37-46), the
putative SRE is closely positioned to a reverse CCAAT box, which is a
potential binding site for the generic heterotrimeric transcription factor NF-Y. Interestingly, both SRE and NF-Y sites are
perfectly conserved in the otherwise less homologous rat DBI/ACBP promoter (Fig. 1A).
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Transcriptional Regulation of DBI/ACBP Promoter-Reporter Genes by
Coexpressed SREBP--
In order to determine whether SREBP binding is
functional, we transiently transfected LNCaP cells with a DBI/ACBP
promoter-reporter construct harboring the SRE site (pDBI-264luc, here
referred to as wt), together with a plasmid encoding -galactosidase
and increasing amounts of pSREBP-1a1-460, a plasmid
encoding transcriptionally active SREBP-1a. Two days after the
transfection, the luciferase activity was measured and the values were
corrected for any differences in transfection efficiency, as determined
from the
-galactosidase assay. As Fig.
3A shows, the transcriptional
activity of the DBI/ACBP promoter was elevated with increasing amounts
of co-transfected pSREBP-1a1-460. Maximal effects were
reached at 20 ng of pSREBP-1a1-460. In order to
demonstrate that the stimulation of transcriptional activity by
SREBP-la overexpression is mediated by the SRE-like site, we generated
DBI/ACBP promoter-reporter constructs in which the SRE site is mutated
(mutSRE) or deleted (delSRE) (Fig. 3B). Transfection
experiments were carried out as described above with maximally
effective amounts of pSREBP-1a1-460 (20-50 ng). In
support of the involvement of the SRE site in SREBP-induced transcriptional activation, stimulation of luciferase activity was
severely decreased when the SRE was deleted or mutated (Fig. 3C). Additionally, the basal transcriptional activity of the
mut and del constructs was 5-10-fold lower than that of the wild type construct, demonstrating the importance of the SRE site for the transcriptional activity of the DBI/ACBP gene (Fig. 3D).
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Requirement of NF-Y Binding for SREBP Activation of DBI/ACBP Transcription-- SREBPs are weak activators of transcription in isolation and are known to function more efficiently when a co-regulatory factor (SP-1 or NF-Y) binds to a neighboring site (37-46). The SRE in the DBI/ACBP promoter is preceded by a potential NF-Y site. To test whether this latter site is important for SREBP-induced activation of DBI/ACBP transcription, we generated a DBI/ACBP promoter-reporter construct in which the NF-Y site is mutated (Fig. 4A). In another construct, we increased the distance between the NF-Y and the SRE sites by insertion of four deoxyadenosine residues. Transient co-transfection of these constructs with pSREBPla1-460 revealed that the stimulatory effect of SREBP overexpression as observed with the wild type construct is severely diminished when the NF-Y site is mutated or when the distance between the NF-Y and the SRE sites is modified (Fig. 4B). Basal transcriptional activities, however, remained high. Mutation of the NF-Y site even led to a 3-4-fold increase in basal transcription. Insertion of four bases between the NF-Y and the SRE site caused a 2-fold decrease in luciferase activity (Fig. 4C).
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Regulation of DBI/ACBP Expression by Sterols-- Having demonstrated that overexpression of SREBPs leads to SRE-dependent activation of the transcriptional activity of DBI/ACBP promoter-reporter genes, we examined whether these constructs are also responsive to physiological changes in endogenous SREBP levels. One of the main and universal physiological signals that trigger SREBP processing resulting in increased nuclear levels of SREBPs is cholesterol depletion. Fig. 5A shows that the luciferase activity of LNCaP cells that were transiently transfected with DBI/ACBP promoter-reporter constructs was 2-fold higher in cells that were deprived of cholesterol as compared with sterol-treated cells. Similar results were obtained with a luciferase reporter construct harboring the promoter of the fatty acid synthase gene (31), a well known sterol-regulated gene (38, 54, 60). The promoter activity of a reporter construct containing a promoter fragment of the PSA gene (a gene encoding a prostate-secreted protein that is not directly related to cholesterol and fatty acid metabolism) (49), was not affected by sterols, indicating that the effects of cholesterol were specific. In support of the involvement of SREBPs in the effects of cholesterol depletion on DBI/ACBP gene transcription, no effects of sterols were observed when LNCaP cells were transfected with DBI/ACBP constructs in which the SRE-site was mutated or deleted (Fig. 5A).
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Involvement of SREBPs in the Regulation of DBI/ACBP Expression by
Androgen--
Based on our recent findings that androgens coordinately
stimulate the expression of lipogenic genes in LNCaP cells and that these effects are (at least in part) mediated by an androgen-induced increase in nuclear SREBP levels (32), we determined whether the
previously reported effects of androgens on DBI/ACBP expression (25,
29) are also mediated by SREBPs. To this end, we transfected LNCaP
cells with DBI/ACBP promoter-reporter constructs together with a
-galactosidase encoding plasmid and a plasmid encoding the androgen
receptor. This latter plasmid was included since the wild type DBI/ACBP
promoter-reporter construct is poorly responsive to androgens in
standard transient transfection experiments in LNCaP cells (25).
Cotransfection with an androgen receptor plasmid has been shown to
improve the androgen responsiveness of promoter-reporter constructs
from several other genes including prototypical androgen-responsive genes such as PSA (61, 62). Under these conditions androgens consistently stimulated the transcriptional activity of the wild type
DBI/ACBP construct (Fig. 6). In support
of the involvement of SREBPs in the effects of androgens, stimulatory
effects of androgens were severely reduced when the SRE site was
mutated or deleted.
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DISCUSSION |
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The current experiments provide evidence that DBI/ACBP is a SREBP-responsive gene. A SRE-like sequence was found in the human DBI/ACBP promoter by visually scanning the DNA sequence. This site resembles the SREs of other SREBP-responsive genes and is present also in the promoter of the rat homologue. The SRE-like site is functional; it binds purified SREBP-1a and mediates transcriptional activation by overexpressed SREBP-1a in cotransfection experiments. Like the genes encoding farnesyl diphosphate synthase, HMG-CoA synthase, squalene synthase, SREBP-2, and glycerol-3-phosphate acyltransferase (41-46), regulation by SREBPs requires a neighboring binding site for the generic transcription factor NF-Y. Furthermore, physiological conditions that are known to activate SREBP processing and stimulate their nuclear translocation (such as cholesterol depletion or androgen treatment of LNCaP cells) increase the expression of DBI/ACBP promoter-reporter genes and of the endogenous gene. Consistent with this finding is the report by Hansen et al. (27) that DBI/ACBP expression is stimulated during insulin-induced lipogenesis in 3T3-L1 preadipocytes, another physiological condition that involves changes in SREBP levels (35). Together with our finding that a functional SRE is important also for basal DBI/ACBP gene transcription, the current experiments illustrate the importance of SREBPs in the regulation of DBI/ACBP gene expression.
Regulation of DBI/ACBP by SREBPs is consistent with the postulated role for DBI/ACBP in fatty acid metabolism and allows DBI/ACBP to be coregulated with other proteins and enzymes involved in lipid metabolism. Our finding that DBI/ACBP is under the control of cholesterol may be of special interest in view of the role of DBI/ACBP in cholesterol translocation across mitochondrial membranes, a rate-limiting step in the biochemical synthesis of steroids.
Our observation that the here identified SRE also plays a role in the
androgen regulation of DBI/ACBP transcription is consistent with our
previous finding that androgens enhance the nuclear accumulation of
SREBP-l in LNCaP cells, and is reminiscent of the involvement of SREBPs
in the androgen regulation of FAS gene expression (32). Furthermore,
the involvement of SREBPs in the androgen regulation of DBI/ACBP
expression may fit with our previous observation that androgen
regulation of DBI/ACBP expression may be indirect (29). It is unlikely,
however, that the entire effect of androgens on DBI/ACBP expression is
mediated by the SRE. (i) The effects of androgens on the 264 DBI/ACBP
promoter-reporter construct are observed only under conditions
(cotransfection with an androgen receptor-expressing vector) that
enhance androgen responsiveness. (ii) The effects of androgens on
steady state mRNA for DBI/ACBP are more pronounced than the effects
on transcriptional activation of the DBI/ACBP luciferase reporter
constructs in transient transfection experiments. (iii) Another
androgen-responsive region was found in the DBI/ACBP promoter sequence
upstream of the SRE site (25).
Issues that remain to be addressed include the question whether different SREBPs (SREBP-1a, SREBP-1c, SREBP-2) are equally effective in regulating DBI/ACBP expression, and whether changes in SREBP-mediated DBI/ACBP expression may also affect other functions of DBI/ACBP not directly related to lipid metabolism.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. Osborne of the University of California, Irvine for providing the pRSA plasmid encoding recombinant SREBP-1a, and Dr. J. Trapman and Dr. A. Brinkmann, Erasmus University, Rotterdam, the Netherlands, for providing the PSA promoter-reporter plasmid and the androgen receptor expression plasmid. We also acknowledge Frank Vanderhoydonc and Bart Maes for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by a grant from "Geconcerteerde Onderzoeksactie van de Vlaamse Gemeenschap," by research grants from the Fund for Scientific Research-Flanders (Belgium) (to J. V. S. and to G. V.), by the "Schenking Rimaux-Bartier" (to J. V. S.), by a grant from the "Vereniging voor Kankerbestrijding," and by a grant from the "Interuniversity Poles of Attraction Program-Belgian State, Prime Minister's Office, Federal Office for Scientific, Technical and Cultural Affairs."The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X94563.
§ Senior Research Assistant of the Fund for Scientific Research-Flanders (Belgium). To whom correspondence should be addressed: LEGENDO, Onderwijs en Navorsing, Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32-16-34-59; Fax: 32-16-34-59-34; E-mail: johan.swinnen{at}med.kuleuven.ac.be.
Supported by a scholarship of the "Vlaams Instituut voor de
Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de
Industrie."
The abbreviations used are: DBI, diazepam-binding inhibitor; ACBP, acyl-CoA-binding protein; SRE, sterol regulatory element; SREBP, sterol regulatory element-binding protein; FAS, fatty acid synthase; HMG, 3-hydroxy-3-methylglutaryl; LPDS, lipoprotein-deficient serum; CT-FCS, charcoal-treated fetal calf serum; PSA, prostate-specific antigen; wt, wild type; HMG, hydroxymethylglutaryl.
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REFERENCES |
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