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Endocrinology Vol. 144, No. 8 3433-3440
Copyright © 2003 by The Endocrine Society

Characterization of the Androgen-Regulated Prostate-Specific T Cell Receptor {gamma}-Chain Alternate Reading Frame Protein (TARP) Promoter

Wing-Shing Cheng, Valeria Giandomenico, Ira Pastan and Magnus Essand

Clinical Immunology (W.-S.C., V.G., M.E.), Rudbeck Laboratory, Uppsala University, SE-75185 Uppsala, Sweden; and Laboratory of Molecular Biology (I.P.), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Magnus Essand, Clinical Immunology, Rudbeck Laboratory, Uppsala University, SE-75185 Uppsala, Sweden. E-mail: magnus.essand{at}klinimm.uu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TARP (T cell receptor {gamma}-chain alternate reading frame protein) is uniquely expressed in males in prostate epithelial cells and prostate cancer cells. Here we demonstrate that TARP expression is regulated by testosterone at the transcriptional level through specific binding of androgen receptor to an androgen response element in the proximal TARP promoter. We further demonstrate that the promoter specifically initiates reporter gene expression in TARP-positive prostate cancer cell lines. To develop a regulatory sequence for prostate-specific gene expression, we constructed a chimeric sequence consisting of the TARP promoter and the prostate-specific antigen (PSA) enhancer. We found that in the prostatic adenocarcinoma cell line LNCaP, the transcriptional activity of the regulatory sequence consisting of a TARP promoter and PSA enhancer is 20 times higher than the activity of a regulatory sequence consisting of the PSA promoter and PSA enhancer. Thus, our studies define a regulatory sequence that may be used to restrict expression of therapeutic genes to prostate cancer cells and may therefore play a role in prostate cancer gene therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IDENTIFICATION OF prostate-specific gene products and prostate-specific gene promoters is of outmost importance for the development of immunotherapy and gene therapy of prostate cancer. Proteins that are uniquely expressed in normal and neoplastic prostate cells may serve as vaccines for the activation of host immune responses against cancer cells. Promoters that are uniquely active in prostate tissues can be used to restrict the expression of therapeutic genes to prostate cancer cells.

Many genes with unique or preferential expression in human prostate tissues have been characterized (1), including three genes from the human kallikrein (KLK) family (2). Genes that are specifically expressed in human prostate tissues are often regulated by testosterone at the transcriptional level. Testosterone and the more potent agonist dihydrotestosterone (DHT) bind to the androgen receptor (AR). Upon ligand activation the AR is phosphorylated and forms a homodimer that is transported to the nucleus in which it activates transcription by binding to androgen-response elements (AREs) in promoter and enhancer regions of target genes. The best-characterized androgen-responsive gene in the human prostate gland is KLK-3, the gene coding for prostate-specific antigen (PSA). KLK-3 transcription is regulated through at least two AREs, one in the proximal promoter and one in an upstream enhancer sequence (3). The ARE consensus sequence is a 15-nucleotide long imperfect palindromic sequence consisting of two 6-bp half-sites that are separated by a three-nucleotide long spacer (4). However, natural AREs often contain one nearly canonical half-site of TGTTCT and one half-site of considerable deviation from this sequence. The ARE consensus sequence is recognized by the androgen receptor as well as the glucocorticoid receptor and the progesterone receptor (5). The specificity mediated by the AR can arise from coregulators, such as ARA-70 that bridges the AR to the preinitiation complex or from proximal transcription factors, such as prostate-derived Ets factor and Oct-1 that interacts with the AR to activate transcription (6, 7, 8). In addition, the natural sequence variants of the consensus ARE have been shown to be important for the differential responses of the androgen, glucocorticoid and progesterone receptors (9).

We have previously shown that human prostate epithelium expresses a unique transcript from a portion of the nonrearranged T cell receptor {gamma}-chain (TCR {gamma}) locus (10). The transcript encodes a small protein designated TARP (T cell receptor {gamma}-chain alternate reading frame protein) (11). TARP mRNA expression is retained in hyperplastic and neoplastic cells of the prostate and breast as well as in the prostate adenocarcinoma cell line LNCaP. The level of TARP mRNA is increased when LNCaP is cultured in growth media containing DHT (12).

In this article we describe the cloning and characterization of the proximal TARP promoter. We show that TARP mRNA expression is directly up-regulated by testosterone at the transcriptional level and that the AR can bind an ARE in the proximal TARP promoter sequence. To develop a regulatory sequence for prostate-specific gene expression, we constructed a chimeric sequence consisting of the TARP promoter and the PSA enhancer. The activity of the chimeric TARP/PSA sequence is highly prostate specific and strictly controlled by testosterone. Our results suggest that this regulatory sequence may be used to restrict expression of therapeutic genes to prostate cancer cells and may therefore play a role in gene therapy of prostate cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines
The prostate adenocarcinomas LNCaP, PC346-C, and PC-3; the breast carcinomas MCF 7, ZR-75-1, and Hs 578T; the bladder carcinoma T24; the colon carcinoma HT-29; the pancreatic carcinoid Bon-1; the glioma U343; the chronic myeloid leukemia K562; and the umbilical vein endothelial cells HUVEC were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES, and 1 mM sodium-pyruvate (all cell culture reagents from Invitrogen, Carlsbad, CA). The cervix adenocarcinoma HeLa, embryonic kidney 293, and embryonic retinoblast 911 were cultured in DMEM supplemented with 5% FBS, and 2 mM L-glutamine. Steroid-depleted cell culture medium was RPMI 1640 supplemented with 5% charcoal/dextran-treated FBS (HyClone, Logan, UT), 2 mM L-glutamine, 10 mM HEPES, and 1 mM sodium-pyruvate.

Northern blot hybridization
LNCaP cells were grown in steroid-depleted culture medium for 48 h and treated for 12 h in steroid-depleted culture medium with or without 10 nM of the synthetic androgen, R1881 (NEN Life Science Products, Boston, MA). PolyA mRNA isolations and Northern blot hybridizations were performed as described previously (10).

RT-PCR
LNCaP cells were grown in steroid-depleted culture medium for 48 h and treated for 12 h in steroid-depleted culture medium with or without 10 nM of R1881. During R1881 treatment, actinomycin-D (ActD, Sigma, St. Louis, MO) was added at 1 µg/ml to block RNA synthesis and cyclohexamide (CHX, Sigma) was added at 10 µg/ml to block protein synthesis. RT-PCR was performed using conditions and primers described previously (11, 12).

Promoter sequence analysis
Genomic DNA was extracted from LNCaP cells by using a DNA extraction kit (QIAGEN, Valencia, CA). A 2.7-kb genomic sequence between the TCR J{gamma}1.1 and J{gamma}1.2 gene segments was amplified by PCR using a proofreading polymerase enzyme (Roche, Indianapolis, IN), sequenced (BigDye terminator, Perkin-Elmer, Norwalk, CT), and deposited into GenBank (AF151104). Additional sequence from the TCR{gamma} locus was obtained from GenBank (AF159056). Nine thousand nucleotides of genomic DNA sequence, upstream of the TARP transcription initiation site, was analyzed for regulatory sequence elements by using the MacVector 6.5 program (Oxford Molecular Group, Oxford, UK) with the addition of the ARE consensus sequence (4), NKX3.1-binding motif (13), and prostate-derived Ets factor-binding motif (7).

Luciferase reporter construct
Various portions of the genomic 5'-flanking sequence of TARP were amplified by PCR from human genomic DNA (Roche) and MluI/XmaI directionally cloned into the pGL3-Basic luciferase reporter gene plasmid (Promega Corp., Madison, WI). The names given to reporter constructs indicate the first and last nucleotides of included genomic sequence, relative to the TARP transcription initiation site. The -6715 to -6233 sequence was KpnI/MluI directionally cloned into T(-201, +45) to create T(-201, +45) + T(-6715, -6233). PSA73luc (3) containing the PSA promoter, -632 to +12, and the PSA enhancer, -4758 to -3884, were obtained from Dr. J. Trapman (Erasmus University, Rotterdam, The Netherlands). The PSA enhancer sequence was removed from PSA73 luc to create the PSA promoter luciferase reporter construct, P(-632, +12). Excised PSA enhancer sequence was subcloned into T(-201, +45) to create the chimeric T(-201, +45) + P(-4758, -3884) reporter construct. PSA73luc is designated P(-632, +12) + P(-4758, -3884) in this article. Inserted sequences were sequenced (BigDye terminator, Perkin-Elmer).

Transient transfections and luciferase assay
Cells were transiently transfected by using Lipofectamine plus (Invitrogen) with reporter constructs described above together with a CMV/ß-gal plasmid (Stratagene, La Jolla, CA) used as an internal control of transfection efficiency. The pGL3-Basic (Promega) was used for background luciferase activity. After transfection, cells were grown either in fresh culture medium or fresh steroid-depleted culture medium in the presence or absence of 10 nM R1881. After 36 h cells were lysed with lysis buffer (PharMingen, San Diego, CA). Luciferase activities were determined in duplicate samples as suggested by the manufacturer (PharMingen). Luciferase activities were calculated by dividing the relative light unit value with the ß-galactosidase value.

EMSA
Double-stranded oligonucleotides constituting either the wild-type or the mutated ARE at -186, relative to the TARP transcription initiation site, were 5'end labeled with [{gamma}32P]dATP using T4 polynucleotide kinase (Amersham Bioscience, Piscataway, NJ). The wild-type oligonucleotide sequence is underlined in Fig. 2AGo, with mutated nucleotides indicated above. Full-length human AR cDNA was subcloned from pSVAR0 (14) to pBluescript II SK+ (Stratagene). In vitro-translated human AR was produced by transcription-coupled translation by using T7 RNA polymerase and wheat germ extract (Promega). Translated AR was incubated with either wild-type ARE(-186) probe or mutated ARE(-186) probe in a buffer containing 20 mM HEPES (pH 7.5), 30 mM KCl, 2 mM MgCl2, 10 µM ZnCl2, 1 mM EDTA, 1 mM dithioerythritol, 1 mM phenylmethylsulfonyl fluoride, 10 nM R1881, 15% glycerol, 1 µg poly(dI-dC)poly(dI-dC), and 1 µg BSA. Samples were resolved by electrophoresis on 5% Tris-borate EDTA/polyacrylamide gels. The gels were dried and subjected to autoradiography.



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  		FIG. 2. Characterization of the proximal TARP promoter. A, Genomic DNA sequence upstream of the TARP gene with putative regulatory motifs in bold. The names of the motifs are given below the sequence. The TARP transcription initiation site is marked +1. B, Comparison between putative AREs identified upstream of the TARP gene, the ARE consensus sequence, and natural AREs in promoters and enhancers of the human KLK 2 (hK2), PSA, rat probasin, and mouse aldose reductase-like protein genes. C, LNCaP cells were transiently transfected with luciferase reporter constructs containing different portions of the genomic 5' flanking sequence of TARP and cultured in normal culture medium. Luciferase activities were expressed in relation to the background activity of pGL3-Basic. D, LNCaP cells were transiently transfected with luciferase reporter constructs and cultured in steroid-depleted medium with 10 nM R1881. Luciferase activities were expressed in relation to activities from transfected cells cultured without R1881. Average activities with SD from three independent experiments with duplicate samples are shown.

 
DNA precipitation (DNAP) assay
Double-stranded, biotinylated oligonucleotides were prepared for ARE(-186) and mutated ARE(-186) as indicated in Fig. 2AGo. LNCaP total extract was prepared in radioimmunoprecipitation assay buffer [10 mM Tris (pH 7.5), 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 1 mM Pefabloc SC (Roche)]. Total extract was precleared with streptavidin-agarose (Amersham Biosciences) and incubated with biotinylated probes for 1 h at 4 C. DNA-bound proteins were precipitated with streptavidin-agarose for 30 min at 4 C, washed three times with radioimmunoprecipitation assay buffer, resolved by 4–20% SDS-PAGE (Bio-Rad) and detected by Western blot analysis using a mouse monoclonal anti-AR antibody (AR441, Santa Cruz Biotechnology, Santa Cruz, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TARP is regulated by testosterone at the transcriptional level
We have previously shown that TARP mRNA is expressed in normal and neoplastic prostate epithelium and the prostate adenocarcinoma cell line LNCaP (10). We have further shown that the amount of TARP mRNA is increased when LNCaP is cultured in the presence of DHT (12). To determine whether TARP transcription is regulated by testosterone at the transcriptional level, we performed Northern blot analyses and RT-PCR analyses. TARP mRNA expression increased when LNCaP was cultured in medium supplemented with synthetic testosterone, R1881 (Fig. 1AGo). The increment in mRNA level caused by R1881 was completely abolished by the RNA synthesis inhibitor ActD, indicating that R1881 increases TARP expression through synthesis of new mRNA (Fig. 1BGo). The increase in mRNA expression was not altered by the addition of the protein synthesis inhibitor CHX, indicating that R1881 acts directly on TARP mRNA synthesis, without requirement of newly synthesized second messenger proteins (Fig. 1BGo). Taken together, the results demonstrate that testosterone regulates TARP expression at the transcriptional level.



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  		FIG. 1. TARP is regulated by testosterone at the transcriptional level. A, Northern blot analyses were performed using polyA mRNA isolated from LNCaP cells starved of testosterone by culture in steroid-depleted medium for 48 h and then treated in steroid-depleted medium with 10 nM of R1881 for 12 h. B, RT-PCR analyses were performed using polyA mRNA isolated from LNCaP cells cultured for 48 h in steroid-depleted medium and treated for 12 h in steroid-depleted medium without (-) R1881; with (+) 10 nM R1881; with 10 nM R1881 and 1 µg/ml of ActD; and with 10 nM R1881 and 10 µg/ml of CHX. ß-Actin was used as mRNA control.

 
Characterization of the proximal TARP promoter
To further investigate the transcriptional regulation of TARP, we cloned the genomic sequence of the TARP gene. Several sequence motifs with potential role in transcriptional regulation were identified upstream of the transcription initiation site, +1 (Fig. 2AGo). These include a TATA-box (6/7 nucleotide matched sequence) located at -26; a CAAT-box (8/9) at -103; and three c-Jun-binding sites at -111 (4/4, antisense), -120 (4/4), and -132 (4/4). Sequences with homology to the consensus ARE were found at several locations. The homologies of putative AREs at -186 and -6301, the ARE consensus sequence, and natural AREs in promoters and enhancers of androgen responsive genes are shown in Fig. 2BGo. A potential transcription response region was identified at -1100, relative to the transcription initiation site, with a putative cAMP response element (CRE) (8/8) and a putative activator protein 1 (AP-1) response element (7/7).

To identify the proximal TARP promoter, we generated luciferase reporter constructs, containing various portions of the genomic 5'-flanking sequence of TARP, and transiently transfected LNCaP cells under normal culture conditions. The T(-201, +45) construct yielded a luciferase activity of 7 times above background level (pGL3-Basic), and shorter constructs yielded activities of 2 times or less above background level (Fig. 2CGo). The highest activity was observed for T(-2646, +45) at approximately 18 times above background, and the activity for T(-6767, +45) was significantly lower, indicating the presence of a silencing region upstream of -2646. We did not observe significant difference in activity between T(-1042, +45) and T(-1168, +45), even though the latter construct contains putative CRE- and AP-1-binding sites.

To investigate the regulatory role of testosterone on TARP transcription, LNCaP cells were transiently transfected with reporter constructs and grown in steroid-depleted culture medium with or without 10 nM R1881 (Fig. 2DGo). The synthetic androgen caused a 4-fold induction of activity for T(-201, +45), but no induction was observed for T(-181, +45), indicating the presence of a functional ARE in T(-201, +45) that is not present in T(-181, +45). Therefore, the ARE-like sequence at -186, relative to the transcription initiation site, appears to be important for testosterone-dependent expression of TARP. The T(-6767, +45) construct yielded only 2-fold induction, further indicating the presence of a silencing region upstream of -2646. A reporter construct with the T(-201, +45) sequence and an upstream region (-6715 to -6233) containing a putative ARE at -6301 yielded slightly higher induction by testosterone than T(-201, +45). Taken together, the ARE at -186 appears to be pivotal for testosterone-dependent transcriptional regulation of TARP, but the ARE-like sequence at -6301 appears to be of lesser importance.

The androgen receptor binds the ARE in the proximal TARP promoter
To determine whether the AR is able to bind the ARE sequence at -186, we performed EMSA using in vitro-translated AR. We observed a DNA-protein complex that is specific for ARE(-186), (Fig. 3AGo, lane 2), because formation of this complex was completely abolished by a 100-fold molar excess of nonlabeled ARE(-186) competitor (Fig. 3AGo, lane 3). Furthermore, in vitro-translated AR was not able to bind a mutated ARE sequence because the specific DNA-protein complex observed with the wild-type ARE(-186) probe, (Fig. 3AGo, lane 5) was not detected with the mutated ARE(-186) probe (Fig. 3AGo, lane 7). Anti-AR antibodies blocked DNA binding without generating a visible supershifted complex (data not shown). To further address the AR binding to ARE(-186), total extract was prepared from LNCaP cells and subjected to DNAP assays, by using biotinylated oligonucleotides for wild-type and mutated ARE(-186). The wild-type ARE(-186) oligonucleotide was able to precipitate AR from the total extract, as detected by Western blot using an anti-AR antibody (Fig. 3BGo). On the contrary, the mutated ARE(-186) oligonucleotide was not able to precipitate AR. These results demonstrate that AR can specifically bind the ARE at position -186, relative to the TARP transcription initiation site.



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  		FIG. 3. The AR specifically binds the ARE sequence at -186 in the TARP promoter. A, In vitro-translated AR was used in EMSA with a wild-type 32P-labeled ARE(-186) probe derived from the TARP promoter and a mutated 32P-labeled ARE(-186) probe. The wild-type ARE(-186) sequence is underlined in Fig. 2AGo with mutated nucleotides in the mutated probe indicated above. A specific AR-DNA complex is detected with the wild-type ARE(-186) probe but not with the mutated ARE(-186) probe. The AR-DNA complex specifically formed with the wild-type probe is completely abolished by a 100 molar excess (x100) of nonlabeled wild-type ARE(-186) competitor. B, Total extract was prepared from LNCaP cells and used in DNAP assays with either a biotinylated wild-type ARE(-186) probe or biotinylated mutated ARE(-186) probe. AR from the cell lysate in complex with the probe was precipitated with streptavidin-agarose and detected by Western blot using an anti-AR antibody.

 
High and prostate-specific activity of a chimeric TARP/PSA regulatory sequence
LNCaP and PC346-C cells were transiently transfected with luciferase reporter constructs containing either the minimal PSA promoter P(-632, +12) or the minimal TARP promoter T(-201, +45) (Fig. 4AGo). Transfected cells were grown in steroid-depleted culture medium in the presence (10 nM R1881) or absence (no stimuli) of testosterone. Transcriptional activities were expressed in relation to pGL3-Basic. We did not detect significant activity for the minimal PSA promoter in LNCaP cells neither with nor without testosterone. Under steroid-depleted conditions (no stimuli), the TARP promoter yielded an activity of 2.1 and with testosterone (10 nM R1881) the relative activity was 9.9. The transcriptional activity of the TARP promoter was higher than the activity of the PSA promoter also in PC346-C (Fig. 4AGo). We next analyzed luciferase reporter constructs containing either the PSA promoter and the PSA enhancer P(-632, +12) + P(-4758, -3884) or the TARP promoter and the PSA enhancer T(-201, +45) + P(-4758, -3884) (Fig. 4BGo). The PSA promoter/PSA enhancer sequence showed very low basic transcriptional activity in LNCaP under steroid-depleted conditions (no stimuli), although when cells were grown with testosterone (10 nM R1881), the transcriptional activity was 58 times above background. The chimeric TARP promoter/PSA enhancer showed a transcriptional activity of 2.9 under steroid-depleted conditions (no stimuli) and an activity of 1188 in the presence of testosterone (10 nM R1881). Therefore, the induction of the TARP promoter/PSA enhancer caused by testosterone is 400-fold, and under those conditions the transcriptional activity of the TARP promoter/PSA enhancer is 20 times higher than the activity of the PSA promoter/PSA enhancer. Because LNCaP expresses a mutated AR, we also examined the activities in PC346-C, a prostate cancer cell line expressing a normal androgen receptor (Trapman, J., unpublished results). The transcriptional activity of the TARP/PSA chimeric regulatory sequence was higher than the activity of the PSA/PSA regulatory sequence also in PC346-C cells, although the difference was less pronounced than in LNCaP.



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  		FIG. 4. The chimeric TARP/PSA regulatory sequence yields high transcriptional activity. LNCaP and PC346-C cells were transiently transfected with luciferase reporter constructs and cultured in steroid-depleted medium either with or without 10 nM R1881 for 36 h before analysis. A, Transcriptional activities of the PSA promoter, P(-632, +12), and the TARP promoter, T(-201, +45). B, Transcriptional activities of the PSA promoter with PSA enhancer, P(-632, +12) + P(-4758, -3884), and the TARP promoter with PSA enhancer, T(-201, +45) + P(-4758, -3884). Luciferase activities were expressed in relation to background activity of pGL3-Basic. Average activities with SD from three independent experiments with duplicate samples are shown.

 
The prostate specificity of the TARP/PSA chimeric regulatory sequence was confirmed in luciferase reporter gene assays by screening cancer cell lines of different origin (Fig. 5Go). Transfected cells were grown in the presence of 10 nM R1881, and the transcriptional activities were expressed in relation to pGL3-Basic. The highest transcriptional activity was found in LNCaP. Significant activity was also found in PC346-C, but the activities were virtually at background level in the other tested cell lines, including the prostate adenocarcinoma cell line PC-3, which does not express an AR and does not express TARP.



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  		FIG. 5. The chimeric TARP/PSA regulatory sequence yields prostate-specific activity. Various cancer cell lines were transiently transfected with a reporter construct in which the chimeric TARP promoter/PSA enhancer sequence controls luciferase gene expression. Transfected cells were cultured in steroid-depleted medium supplemented with 10 nM R1881 for 36 h before analysis. Luciferase activities were expressed in relation to background activity of pGL3-Basic. Average activities with SD from three independent experiments with duplicate samples are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously reported that a prostate-specific mRNA is transcribed from the nonrearranged TCR{gamma} locus and that the transcript encodes for TARP, an alternative reading frame protein, compared with the TCR{gamma} chain protein expressed on {gamma}{delta}-T lymphocytes. Here we present evidence that TARP mRNA is directly up-regulated by testosterone at the transcriptional level. We further describe the TARP promoter sequence and show that it contains DNA motifs for general transcription factors involved in initiation and regulation (15, 16) and an ARE. The significance of the ARE in the TARP promoter sequence was confirmed in luciferase reporter gene assays, EMSAs and DNA precipitation assays. We concluded that the sequence up to -201 is the proximal TARP promoter, containing a TATA box, a CAAT box, three c-Jun binding sites, and a functional ARE.

The ARE consensus sequence consists of two asymmetric 6-bp half-site elements that are separated by a three-nucleotide-long spacer (4). Natural AREs often have high homology to the consensus sequence on at least one half-site and the other half-site can deviate significantly. For example, the rat probasin element displays strong androgenicity in the context of its native promoter. It contains two AREs, both with high homology on one half-site and considerably less homology on the other half-site (17). The mouse aldose reductase-like protein has absolute homology with the ARE consensus sequence on the right half-site but only two nucleotides homology on the left half-site, yet it is a fully functional ARE (18). The ARE at position -186 in the TARP promoter has high homology on the right half-site, 5/6 nucleotides match with the ARE consensus sequence and the left half-site diverges in positions -1, -2, and -3. In contrast, AREs in the promoters and enhancers of KLK-2 (hK2) and KLK-3 (PSA) have high overall homology with the ARE consensus sequence, with complete match on positions ±2, ±3, and ±5 on both half-sites (19, 20).

It is known that a single ARE and TATA box can be sufficient for androgen induction and that two AREs can result in a synergistic or cooperative increase of transcription in response to androgens. Cleutjens et al. (3) have shown strong testosterone-dependent induction for a reporter gene controlled by the minimal PSA promoter together with an upstream PSA enhancer. They also showed that the ARE in the enhancer has a higher impact on the induction process of PSA than the ARE in the promoter. In the current article, we describe a functional ARE in the proximal TARP promoter. We also demonstrate that testosterone induces TARP promoter activity, in our hands, more significantly than PSA promoter activity.

AREs are found in promoters of genes expressed in different tissues including prostate, brain, kidney, liver, and testis. Therefore, it is unlikely that the AR alone regulates the cell-type-specific expression of TARP. AR-dependent transcription occurs through the attenuation of the function of other transcription factors, such as proteins interacting with AP-1 and CRE-binding protein. For example, it has been shown that androgen induction of PSA is repressed by protein-protein interaction between the AR and AP-1/c-Jun in LNCaP cells (21). Conversely, it was shown that the AR can inhibit AP-1 activity (21). It has also been shown that CRE-binding protein can work as a coactivator for AR and mediate cross-talk with AP-1 to reduce its inhibiting activity on AR (22). The T(-1168, +45) construct contains a putative AP-1-binding sites that is not present in the T(-1042, +45) construct, but both constructs contain the functional ARE at -186. Preliminary results on transfected LNCaP cells treated with the phorbol ester phorbol-12-myristate-13-acetate with or without simultaneous R1881 treatment did not reveal a significant difference in reporter gene expression between the two constructs. Further studies are therefore needed to identify the potential role of the AP-1, CRE region in the TARP promoter.

There is an increasing interest in using tumor- and/or tissue-specific regulatory sequences for therapeutic gene expression. Because the prostate is an organ that is not essential for life, regulatory sequences that are prostate specific may be used to restrict therapeutic gene expression to prostate and prostate cancer cells. Yu et al. (20) produced an attenuated replication competent adenovirus for prostate cancer therapy. These adenoviral constructs use promoter and enhancer domains from the PSA and hK2 genes. Other groups have developed similar therapeutic strategies based on prostate-specific transcriptional regulation of genes encoding therapeutic molecules (23, 24, 25). We constructed a prostate-specific chimeric regulatory sequence consisting of the proximal TARP promoter and the PSA enhancer. We found that the transcriptional activity of the TARP promoter/PSA enhancer is 20 times higher than the activity of the PSA promoter/PSA enhancer in LNCaP cells grown in culture medium with addition of testosterone. Because the AR is mutated in LNCaP, we analyzed the activity of the various constructs on PC346-C cells, which has a normal AR (J. Trapman, unpublished results). In PC346-C the transcriptional activity of the TARP promoter/PSA enhancer is approximately three times higher than the activity of the PSA promoter/PSA enhancer. The superior activity of the chimeric regulatory sequence, observed both in LNCaP and PC346-C, might be due to that TARP expression is primarily regulated by the ARE in the proximal promoter, and PSA expression is primarily regulated be the ARE in the upstream enhancer region. Therefore, a chimeric transcriptional regulatory sequence with a potent and specific promoter from one gene and a potent and specific enhancer from another gene may be advantageous.

In summary, our studies show that TARP expression is regulated by testosterone at the transcriptional level through AR recognition of the ARE sequence at -186 in the proximal TARP promoter. We further show that it is possible to use a chimeric regulatory sequence consisting of the proximal TARP promoter and the upstream PSA enhancer to control prostate-specific gene expression. This chimeric regulatory sequence yields both higher transcriptional activity and higher induction by testosterone than a regulatory sequence consisting of the PSA promoter and PSA enhancer. Therefore, we believe that the chimeric sequence may be used to restrict expression of therapeutic genes to prostate cancer cells. It will be of outmost importance to use our findings to develop prostate-specific vectors for prostate cancer gene therapy.


   Acknowledgments
 
We thank Dr. Jan Trapman (Erasmus University, Rotterdam, The Netherlands) and Dr. Johan Ericsson (Ludwig Institute for Cancer Research, Uppsala Branch, Uppsala, Sweden) for fruitful discussions and suggestions.


   Footnotes
 
This work was supported by the Swedish Cancer Society, 4419-B01-02XBB.

Abbreviations: ActD, Actinomycin-D; AP-1, activator protein-1; AR, androgen receptor; ARE, androgen response element; CHX, cyclohexamide; CRE, cAMP response element; DHT, dihydrotestosterone; DNAP, DNA precipitation; FBS, fetal bovine serum; hK2, human kallikrein 2; KLK, kallikrein; PSA, prostate specific antigen; TARP, TCR{gamma} alternate reading frame protein; TCR{gamma}, T cell receptor {gamma}-chain.

Received January 27, 2003.

Accepted for publication April 22, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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