[CANCER RESEARCH 60, 1348–1352, March 1, 2000]

Prostate-specific Transcription Factor hPSE Is Translated Only in Normal ProstateEpithelial Cells

Masahiro Nozawa, Kentaro Yomogida, Nobufumi Kanno, Norio Nonomura, Tsuneharu Miki, Akihiko Okuyama,Yoshitake Nishimune, and Masami Nozaki1

Department of Urology, Osaka University Medical School [M. Nozaw., N. K., N. N., T. M., A. O.] and Department of Science for Laboratory Animal Experimentation, ResearchInstitute for Microbial Diseases [M. Nozaw., K. Y., Y. N., M. Nozak.], Osaka University, Osaka 565-0871, Japan

ABSTRACT

We recently cloned a novel transcription factor gene,hPSE, whichbelongs to the Ets gene family.hPSEmRNA was expressed specifically inprostate glandular epithelial cells and also in the human prostate carci-noma cell lines PC-3 and LNCaP. On the other hand, on immunoblotanalysis with anti-hPSE antiserum, hPSE protein was detected only inhuman prostate tissue samples and not in PC-3 or LNCaP culture cells.Immunohistochemistry and in situ hybridization analysis revealed thathPSE protein was translated in normal prostate glandular epithelial cells,but not in carcinoma cells with hPSE transcripts. These findings suggestthat expression ofhPSE is regulated translationally in prostate epithelialcells and that hPSE protein is a candidate for a marker distinguishingnormal cells from cancer cells in the prostate. It appeared that the 5*- and3*-untranslated regions ofhPSE transcripts might be necessary for trans-lational control of hPSE, on the basis of results of transfection analysis innon-prostate lineage cells (HEK-293) using some deletion mutants ofhPSE cDNA.

INTRODUCTION

Prostate cancer is a significant health problem in advanced nations.It is the most common cancer diagnosed and is the second leadingcause of cancer death among males in the United States (1). Surgeryis one of the most effective radical therapies for this disease. Cur-rently, there is no curative therapy for advanced prostate cancer. Onthe other hand, it has been revealed by autopsy that as many as 73%of men over the age of 75 years have identifiable prostate carcinomaswithout clinical symptoms (2). It is possible that some patients haveclinically nonsignificant prostate cancer that has little effect on theirlives. If this is true, surgery may be overtreatment for such patients,given its risk of postoperative incontinence and impotence. At present,there is no clinical marker that distinguishes those prostatic carcino-mas that are potentially aggressive from those that are unlikely tocause advanced prostatic cancer (3). Clarification of the molecularmechanisms of prostate carcinogenesis and of invasion and metastasisis an urgent task.

Alterations in normal control of cellular pathways that regulate cellgrowth and differentiation can lead to cancer. Dysregulation of tran-scription factor proto-oncogene expression results in the developmentof several neoplasias (4). We recently cloned a novel Ets family gene,hPSE, from the PC-3 human prostate carcinoma cell line.2 Membersof the Ets family are transcription factors involved in the transcrip-tional control of genes associated with development, angiogenesis,cell cycle control, and cell proliferation. All Ets family members

contain a conserved DNA binding domain of about 85 amino acidsthat recognizes purine-rich sequences containing a GGAA/T core(5–7). We have shown thathPSEmRNA expression is restricted tohuman prostate epithelial cells. hPSE might play an important role indifferentiation and growth of prostate epithelial cells.hPSEmRNAexpression is also observed in human prostate carcinoma cell lines.However, it is necessary to investigatehPSEexpression at the proteinlevel.

In this study, we demonstrated thathPSEtranscripts were translatedonly in normal prostate glandular epithelial cells, and not in malignantones. Furthermore, we showed that the 59- and 39-UTRs3 of hPSEtranscripts are necessary for translational control ofhPSE. Our find-ings suggest that the expression ofhPSEis regulated translationally inprostate epithelium. Clarification of the regulation ofhPSEexpressionmay aid understanding of prostatic carcinogenesis.

MATERIALS AND METHODS

Cell Lines and Tissue Samples.Human embryonic kidney epithelial cellline HEK-293 and human prostate carcinoma cell lines PC-3 and DU145 weregrown in DMEM (Life Technologies, Inc., Grand Island, NY), and humanprostate carcinoma cell line LNCaP was grown in RPMI 1640 (Life Technol-ogies, Inc.) supplemented with 10% fetal bovine serum, 200 units/ml penicil-lin, 200mg/ml streptomycin, and 0.5mg/ml amphotericin B (Sigma, St. Louis,MO) at 37°C and 5% CO2.

Human prostate tissue samples were obtained through surgeries such as totalcystectomy for bladder cancer, radical prostatectomy for prostate cancer, andretropubic prostatectomy or transurethral resection of the prostate for benignprostate hyperplasia.

Polyclonal Antiserum Preparation. The GST-fusion hPSE proteins wereproduced inEscherichia coliusing the pGEX-2T vector system (Pharmacia,Piscataway, NJ). Polyclonal antisera were obtained by injection of the GST-fusion hPSE proteins into two New Zealand White rabbits. Antisera (anti-PSE1and anti-PSE2) were reacted with the GST-fusion proteins and the T7-taggedprotein (see “Constructs forhPSEExpression”).

Immunoblot Analysis. Cultured cell lines on the dishes were collected inradioimmunoprecipitation buffer [10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1mM EDTA, 1% NP40, 0.1% sodium deoxycholate, 0.1% SDS, 10 mg/mlaprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM DTT]. Humanprostate tissue pieces were homogenized in radioimmunoprecipitation buffer,and the supernatant was collected by centrifugation. The supernatant wasboiled in SDS/DTT buffer and separated by 10% SDS-PAGE. The proteins inthe gel were transferred electrophoretically onto polyvinylidene difluoridemembranes (Millipore, Saint Quentin en Yvelines, France). The membraneswere blocked with 5% skimmed milk in TBS-T [50 mM Tris-HCl (pH 7.5), 150mM NaCl, and 0.05% Tween 20]. Anti-PSE1 antiserum was used for detectionof hPSE. After incubation with primary antibodies, the membranes werewashed and incubated with peroxidase-conjugated antirabbit IgG antibodies(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). After exten-sive washing in TBS-T, reactive bands were detected by development withdiaminobenzidine in 50 mM Tris-HCl (pH 7.5) plus 0.3% H2O2. For detectionof the T7-tagged protein, we used an anti-T7 monoclonal antibody (CNBiosciences, Inc., Darmstadt, Germany) as a primary antibody and peroxidase-

Received 8/31/99; accepted 12/28/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Science forLaboratory Animal Experimentation, Research Institute for Microbial Diseases, OsakaUniversity, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-8338; Fax:81-6-6879-8339; E-mail: mnozaki@biken.osaka-u.ac.jp.

2 M. Nozaki, N. Kanno, N. Yamada, S. Amekawa, K. Yomogida, M. Nozawa, H.Miyamoto, T. Fujiwara, N. Nonomura, T. Miki, A. Okuyama, and Y. Nishimune. PSE: anEts-related transcription factor is preferentially expressed in the human prostate epithe-lium, submitted for publication.

3 The abbreviations used are: UTR, untranslated region; GST, glutathioneS-transfer-ase; ORF, open reading frame; IRE, iron-responsive element.

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conjugated antimouse IgG antibody (Jackson ImmunoResearch Laboratories,Inc.) as a secondary antibody.

Immunohistochemistry. Human prostate tissue samples were frozen inOCT embedding compound (Tissue-Tek; Sakura Finetechnical Co., Ltd., To-kyo, Japan), and cryosections (7mm) were placed on silane-coated slides(Matsunami Glass Inc., Ltd., Osaka, Japan). The sections were air-dried andfixed in 4% paraformaldehyde. Specimens were blocked with 2% normaldonkey serum in PBS and incubated with anti-PSE1 antiserum (1:100 dilution)in PBS. The slides were rinsed with PBS and incubated with antirabbitimmunoglobulin antibody conjugated with horseradish peroxidase (1:200;Jackson ImmunoResearch Laboratories, Inc.) in PBS. A positive reaction wasdetected by incubating with 3,39-diaminobenzidine and counterstaining with1% methyl green (Muto Pure Chemicals, Ltd., Tokyo, Japan).

Northern Blot Analysis. Total RNA was prepared from cultured cells andfrozen tissue samples using the Trizol reagent (Life Technologies, Inc.). RNA(15 mg) was resolved on a 1% agarose gel containing 6.7% formaldehyde,transferred to a Zeta-probe blotting membrane (Bio-Rad Laboratories, Hercu-les, CA), and hybridized to a [a-32P]dCTP-labeledhPSEcDNA probe.

In Situ Hybridization. In situ hybridization was performed with thedigoxigenin-labeled Riboprobe system (Boehringer Mannheim, Mannheim,Germany) as described previously (8, 9).hPSEprobes were generated from a1.0-kbEcoRI/BamHI fragment cloned into pBluescript II SK1. After hybrid-ization, the bound probe was detected with anti-digoxigenin-Fab fragmentsconjugated with alkaline phosphatase (Boehringer Mannheim). Sections werecounterstained with 1% methyl green (Muto Pure Chemicals, Ltd.).

Constructs for hPSE Expression. The T7-tagged hPSE protein was ex-pressed in HEK-293 cells with the pPSE-T7 expression construct. pPSE-T7was constructed by inserting thehPSE coding region and double T7 tag(MASMTGGQQMGAAMASMTGGQQMG) into pRCCMV (Invitrogen, Carls-bad, CA). pRCCMV-T7, which contained a double-T7 tag, was used as acontrol vector. We recently demonstrated transcriptional activity of hPSE withthe expression vector.2 To examine control of the expression ofhPSE, weprepared several expression constructs (Fig. 4A). We first subcloned a 59-UTR-deleted clone (E17) and full-lengthhPSEcDNA into pSG5 (Stratagene, LaJolla, CA) as an expression vector (designated vectors g and f, respectively).The construct, designated construct d, contained only the coding region ofhPSE, which was generated by PCR with primers 59-GGGAATTCCAGCG-GCATGGGCAGCGCCAGC-39 (forward) and 59-CGGGATCCTCAGAT-GGGGTGCACGAACTGG-39(reverse) with construct g as a template. Theconstructs consisting of the coding region with the full-length 59-UTR or withthe deleted 59-UTR (192 bp), named constructs a and c, respectively, weregenerated by recombination of construct d with vector g or f, using theXhoIrestriction site. Construct e was generated by recombination of construct d with

vector f. Construct b was obtained by digestion of construct a withTth111Irestriction enzyme.

Transfection Analysis. Cultured cells were transfected with each expres-sion vector using LipofectAMINE Plus Reagent (Life Technologies, Inc.)according to the manufacturer’s recommendations. The levels of expression ofmRNA and protein were evaluated with the Microcomputer Imaging DeviceMCID/mcid image analyzing system (Imaging Research Inc., St. Catharines,Ontario, Canada). The protein expression level was standardized with themRNA expression level. All of the results shown in this study were obtainedfrom at least four independent experiments.

RESULTS

Identification of hPSE Protein. To examine the expression ofhPSE protein, we generated hPSE-specific antisera (anti-PSE1 and anti-PSE2) using the GST-fusion protein. Each antiserum detected the GST-fusion protein on immunoblot analysis (data not shown). To confirm thespecificity of these antisera, the T7-tagged hPSE was expressed in HEK-293 cells with the pPSE-T7 expression construct. On immunoblot anal-ysis, anti-PSE1 and anti-PSE2 recognized two protein bands (Mr 56,000and Mr 47,000), which were identical to those detected by anti-T7antibody, against total lysates of HEK-293 cells transfected withpPSE-T7 (Fig. 1,A andB). Each antiserum detected no band against totallysates of HEK-293 cells transfected with an expression vector containingonly the T7 tag (Fig. 1B). These findings indicate that both anti-PSE1 andanti-PSE2 specifically recognize hPSE protein. Immunohistochemistrywith anti-PSE1 demonstrated that hPSE protein was expressed in normal

Fig. 1. Identification of hPSE protein by anti-PSE1 antiserum. Immunoblot analysisusing anti-T7 monoclonal antibody (A) and anti-PSE1 antiserum (B).Lanes representlysates of HEK-293 cells transfected with pPSE-T7 (AandB, Lane 1) or the control vectorpRCCMV-T7 (B,Lane 2). The T7-tagged hPSE protein was detected as a major band ofapproximatelyMr 56,000 and as an additional minor band of approximatelyMr 47,000 byanti-PSE1 antiserum and anti-T7 antibody.MW, approximate molecular weight in thou-sands.C and D, results of immunohistochemistry in a normal human prostate tissuespecimen using preimmunized serum (C) or anti-PSE1 (D). hPSE protein is specificallyexpressed in prostate epithelial cells.C andD, 3100 (bars, 100mm).

Fig. 2. Expression ofhPSEin human prostate carcinoma cell lines and human prostatetissue samples.A, Northern blot analysis. A 1.9-kb fragment was detected in both humanprostate carcinoma cell lines and human prostate tissue samples, but not in DU145 cells.B, immunoblot analysis using anti-PSE1 antiserum. hPSE protein was detected in allhuman prostate tissue samples (Mr 39,000) but was not detected in any human prostatecarcinoma cell line.Patients 1and2, pathologically benign prostate hyperplasias.Patients3 and4, poorly differentiated adenocarcinoma and moderately differentiated adenocarci-noma, respectively.Patients 5and6, normal human prostate tissues obtained from totalcystectomy for bladder cancer.MW, approximate molecular weight in thousands.

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prostate epithelial cells, especially in the nuclei (Fig. 1,C andD). Thelocalization of hPSE protein suggested that hPSE might be a prostate-specific transcription factor.

hPSE Transcripts Were Not Translated in Human ProstateCarcinoma Cell Lines. hPSE mRNA was strongly expressed inhuman prostate carcinoma cell lines PC-3 and LNCaP (Fig. 2A).However, hPSE protein expression was detected in neither PC-3 nor

LNCaP cells on immunoblot analysis (Fig. 2B). In another humanprostate carcinoma cell line, DU145,hPSEexpression was not de-tectable by Northern blot analysis or by immunoblot analysis (Fig. 2,A andB).

hPSE Transcripts Were Translated Only in Normal ProstateCells. To investigate the expression ofhPSEmRNA and protein inhuman prostate tissues, we obtained specimens from 12 patients: (a)

Fig. 3. Histological analysis ofhPSEexpression in prostate cancer tissues.A andB, in situ hybridization of poorly differentiated adenocarcinoma of the prostate with the antisenseprobe and sense probe, respectively.hPSEmRNA was expressed in prostate carcinoma cells.C andD, immunohistochemistry using anti-PSE1 antiserum. The same portion of tissueas shown for thein situ hybridization above (C) and the normal prostate glands of the same specimen (D) were used. hPSE protein was observed in the normal glandular cells, butnot in the carcinoma cells.E, H&E staining of the same portion as shown inA. F, H&E staining of moderately differentiated adenocarcinoma of the prostate. Thearrowheadsindicatethe carcinomatous portion.G andH, immunohistochemistry of the same portion as shown inF using anti-PSE1 antiserum.A, C–E, andH, 3100.B, F, andG, 350 (bars, 100mm).

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5 transurethral resections and 3 retropubic prostatectomies from pa-tients with benign prostate hyperplasia; (b) 2 normal prostate tissuesamples from total cystectomies for bladder cancer; and (c) 2 clini-cally localized prostate cancer specimens from radical prostatectomies(pathologically, a moderately differentiated adenocarcinoma and apoorly differentiated adenocarcinoma).hPSE expression was ob-served at the levels of transcription and translation in all humanprostate tissue samples (Fig. 2,A and B). We hypothesized that theefficacy of translation ofhPSEwas much lower in prostate cancercells than in normal prostate epithelial cells, on the basis of the findingthat hPSE protein was not detected in prostate carcinoma cell linesPC-3 and LNCaP. To test this hypothesis, we performedin situhybridization and immunohistochemistry to check the expression ofhPSEat the cellular level in human prostate tissues.In situ hybrid-ization revealed thathPSE was specifically expressed in prostateepithelial cells in normal glands including those with hyperplasia(data not shown) and in cancer cells (Fig. 3,A andB). Immunohis-tochemistry demonstrated that hPSE protein was expressed only innormal prostate epithelial cells and not in prostate cancer cells (Fig. 3,C, D, G,andH). These findings suggest that transcripts ofhPSEaretranslated only in normal prostate epithelial cells.

Identification of the Region Controlling hPSETranslation. Wefound that hPSE protein was expressed only in normal prostateepithelial cells (Fig. 3,C, D, G, and H). It is possible that theexpression ofhPSEis regulated at the translational level. To study theregions controlling translation ofhPSEtranscripts, we produced sev-eral deletion mutants, as indicated in Fig. 4A, and transfected each ofthem into HEK-293 cells. Northern blot analysis revealed nearly equallevels of expression of mRNA of each mutant (Fig. 4B). However, thedegree of expression of hPSE protein differed according to the lengthof the 59-UTR and the presence of the 39-UTR (Fig. 4, C and D).Deletion of 221 bp from the 59-end of the 59-UTR and deletion of the39-UTR significantly enhanced translation ofhPSE, indicating thatthese portions might be concerned with the translational regulation ofhPSEin prostate epithelial cells.

DISCUSSION

hPSEis a novel Ets family gene that is specifically transcribed inprostate epithelial cells. Ets family members are transcription factorsthat regulate the transcription of genes involved in development,differentiation, and cell proliferation (10, 11). Dysregulation of suchgenes should cause cancerous changes in normal cells. A relationshipbetween Ets family genes and human cancer has been demonstratedfor leukemias and solid tumors (5–7). ETS2 has previously beenreported as a member of the Ets family involved in prostate cancer(12, 13). ETS2 was shown to be expressed at elevated levels inprostate cancer (12) and was shown to be required for maintenance ofthe transformed state in prostate cancer cells (13). In the present study,we demonstrated that transcripts ofhPSE were translated only innormal prostate epithelial cells and not in prostate cancer cells. hPSEappears to play roles in determining the properties of normal prostateepithelial cells, given its specificity of expression. Interestingly, theexpression ofhPSEis suppressed at the translational level in PC-3 andLNCaP cells and at the transcriptional level in DU145 cells. It hasbeen reported that DU145 and PC-3 cells are invasive, whereasLNCaP cells are not, as measured by migration through Matrigel-coated membranes (14). There may be a correlation between themalignancy of prostate cancer cells and the level of regulation ofhPSEexpression.

Several genes containing Ets elements in their regulatory region areup-regulated in prostate cancer. For example, c-met, the receptor forhepatocyte growth factor/scatter factor, is regulated by Ets (15), and

the presence of met protein is associated with higher-grade adenocar-cinomas (16). Mitogenic signaling through the ErbB/neu receptor ismediated through Ets (17, 18), and elevated neu expression is asso-ciated with metastatic conversion of prostate cancer (19, 20). Maspin,a tumor-suppressing protease inhibitor that is expressed in normalprostate epithelial cells but not in prostate cancer cell lines, is alsotranscriptionally regulated by Ets (21). Because the pattern of expres-sion of hPSE protein is identical with that of maspin, hPSE might beinvolved in the regulation of transcription ofmaspin. hPSE may be acandidate for a negative maker of prostate cancer.

Our transfection analysis of HEK-293 cells with several expressionconstructs suggested that the 59- and 39-UTRs ofhPSE transcriptsshould contain translational regulatory regions. A system that over-comes translational suppression should function in normal prostateepithelial cells. The process of translation can be divided into threedistinct stages: (a) initiation; (b) elongation; and (c) termination (22,23). The primary target for translational control is the initiation step.Control of translation initiation on individual mRNAs is determinedprimarily by the structural properties of the mRNA, particularly the59-UTR (24). IREs are a well-characterized case in which the second-ary structure located within the 59-UTR plays a role in translational

Fig. 4. A, schema of constructs for transfection. Deletion mutants (a–f) of full-lengthhPSEcDNA (g) were prepared as described in “Materials and Methods.”M, the ORF;f,the UTR.a, full-length 59-UTR (413 bp) and the ORF.b, 360 bp of the deleted 59-UTRand the ORF.c, 192 bp of the deleted 59-UTR and the ORF.d, the ORF alone.e, the ORFand full-length 39-UTR.f, 192 bp of the deleted 59-UTR, the ORF, and full-length39-UTR. These constructs were subcloned into vector pSG5. Thearrowheadindicates theposition of theXhoI restriction site.B, expression of mutant mRNAs in transientlytransfected HEK-293 cells. The intensity of each mRNA was nearly the same.C,immunoblot analysis using anti-PSE1 for hPSE protein expression 24 h after transfection.hPSE protein was expressed most strongly in HEK-293 cells transfected with the expres-sion vector including construct c.MW, approximate molecular weight in thousands.D,evaluation of efficiency of translation by densitometry. The expression level againstmRNA of hPSE in HEK-293 cells transfected with the expression vector includingconstruct d was arbitrarily assigned a value of 100%. Each band density in the immunoblotwas standardized with the level of mRNA expression in the Northern blot. Resultsrepresent the mean of four independent experiments.Bars, SD.a–g in B–D correspond toa–g in A.

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regulation.Ferritin mRNA contains an IRE in its 59-UTR to which theIRE-binding protein binds when intracellular concentrations of ironare low and inhibits translation offerritin mRNA (25). It is becomingclear that 39-UTRs of some mRNAs can play important roles intranslation of these mRNAs. A domain within the pseudoknot in the39-UTR of the tobacco mosaic virus RNA regulates its translation (26,27). A pyrimidine-rich motif in the 39-UTR of 15-lipoxygenasemRNA interacts with a RNA-binding protein (28). An AU-rich se-quence within the 39-UTR of human cytokine mRNA inhibits itstranslation (29). The selenocysteine insertion sequence within the39-UTR of a number of eukaryotic mRNAs directs insertion of sel-enocysteine at in-frame UGA codons (30). In addition, mammalianhistone mRNAs terminate in stem-loop structures that are functionallysimilar to the poly(A) tail (31). The direct molecular communicationbetween the 59- and 39-ends of mRNAs is thought to play an importantrole in regulation of their translation. For example, mRNAs with basecomplementarity between the 59- and 39-UTRs and that form a stablesecondary structure are translated poorly, if at all, in COS cells (32).To our knowledge, there is no consensus motif in the 59- and 39-UTRsof hPSEcDNA.

In future, if we can express hPSE protein in prostatic cancer cells,these cancer cells might regain properties of normal prostate cells.Clarification of the mechanism of regulation ofhPSEexpression inprostate lineage cells will aid in understanding of human prostaticcarcinogenesis and in developing new methods for the diagnosis andtherapy of prostate cancer.

ACKNOWLEDGMENTS

We thank Dr. Masaru Shin for advice on pathological examination.

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TRANSLATIONAL CONTROL OF hPSE

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2000;60:1348-1352. Cancer Res   Masahiro Nozawa, Kentaro Yomogida, Nobufumi Kanno, et al.   in Normal Prostate Epithelial CellsProstate-specific Transcription Factor hPSE Is Translated Only

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