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[Cancer Research 63, 329-336, January 15, 2003]
© 2003 American Association for Cancer Research

Carcinogenesis

A Novel Human Prostate-specific Gene-1 (HPG-1)

Molecular Cloning, Sequencing, and Its Potential Involvement in Prostate Carcinogenesis1

Elizabeth A. Herness and Rajesh K. Naz2

Division of Research, Department of Obstetrics and Gynecology, Medical College of Ohio, Toledo, Ohio 43614


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostate-specific genes that have a role in normal and abnormal prostate growth are needed for early and specific diagnosis and treatment of prostate cancer. In the present study, the differential display-PCR technique was used to obtain a prostate-specific ~339-bp cDNA fragment. On screening the human-prostate {lambda}gt10 library with this fragment, a full-length 1468-bp human prostate-specific gene (HPG-1) with an open reading frame of 127 amino acids (aa) was retrieved. Extensive database search revealed that the HPG-1 has novel nucleotide/aa sequences. It was localized on Homo sapiens 3q26 chromosomal locus, a region that has been shown to be involved in prostate carcinoma. The computer-generated translated protein has a calculated molecular mass of 14.8 kDa with several potential glycosylation and phosphorylation sites including two N-linked glycosylation, one tyrosine phosphorylation, and one N-myristoylation sites. The in vitro transcription and translation procedures using HPG-1 cDNA yielded a protein of similar molecular mass of ~15 kDa. Hydrophilicity analysis of the deduced aa sequence indicated that HPG-1 is a membrane-anchored/attached protein. Analysis for tissue specificity by using the Northern blot and reverse transcription-PCR-Southern blot procedures using 19 different human tissues revealed that HPG-1 is expressed specifically only in prostate tissue. To examine its involvement in prostate carcinogenesis, three prostate cancer epithelial cell lines, one androgen-responsive (LNCaP) and two androgen-nonresponsive (DU-145 and PC-3), were examined for the expression of HPG-1. Using the Northern blot and quantitative reverse transcription-PCR procedures it was found that LNCaP and DU-145 cells and not the PC-3 cells have HPG-1 expression, with LNCaP cells having approximately 2–3-fold higher levels of HPG-1 mRNA transcripts compared with DU-145 cells. In vitro culture of LNCaP cell with antisense and not the sense oligonucleotide decreased the HPG-1 mRNA levels and inhibited the cell growth in a concentration-dependent manner; at 72 h there was an 86% inhibition of cell growth. HPG-1 mRNA expression in LNCaP cells was found to be responsive to androgen. Thus, the novel androgen-responsive HPG-1, which has prostate-specific expression and seems to be involved in carcinogenesis, may have applications in the specific diagnosis and treatment of prostate cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostate adenocarcinoma is the most frequently diagnosed cancer and is second only to lung adenocarcinoma as having the highest cancer mortality (1 , 2) . Numerous factors are attributed to the increase in prostate cancer incidence. Two predominant factors include: (a) the explosion in the aging population demographic; age is the single most significant risk factor for development of prostate cancer (3) ; and (b) the availability of recent technologies (digital rectal examination and the prostate-specific antigen test) for the early detection of prostate cancer (4) . These clinical diagnostic tests lack sensitivity and specificity in appropriately distinguishing between prostate cancer and benign abnormalities such as BPH3 and prostatitis (5 , 6) . Additional prostate-specific molecules need to be identified to design more sensitive/specific tests for early disease diagnosis and to develop more effective therapies.

Numerous laboratories, including ours, are actively searching for genes that are prostate-specific and have a role in carcinogenesis (7) . Several genes have been identified using various approaches, such as, RNA DD analysis (8, 9, 10) , expressed sequence tag database analysis (11, 12, 13, 14, 15) , exon trapping (16) , representational difference analysis (17) , subtractive cDNA libraries (18 , 19) , suppression subtractive hybridization (20) , microarray technology (21 , 22) , serial analysis of gene expression (23) , and yeast two hybrid system analysis (24) . The genes identified using these techniques include the cell surface antigens, namely, prostatic carcinoma tumor inducing gene-1, prostate stem cell antigen, prostate-specific G-coupled receptor, six-transmembrane epithelial antigen of the prostate, and prostein (8 , 13 , 17 , 20 , 22 , 24) . Other genes reported are noncoding riboregulators, DD3 and PCGEM1 (9 , 10) , serine protease, TMPRSS2 (16) , small nuclear proteins, T-cell receptor {gamma}-chain alternate reading frame protein, and prostate/rectum/colon gene (14 , 15) , tumor suppressor genes, NKX3.1 and prostate androgen-regulated transcript-1 (11 , 21) , coregulator of the androgen receptor, PDEF (12) , androgen-regulated gene, PMEPA-1 (23) , and the calcium channel protein, transient receptor potential-8 (19) . Most of the genes have not been extensively examined for their prostate-specificity and/or for their role in carcinogenesis. Thus, their application in early diagnosis or immunotherapy of prostate cancer and BPH remains unclear. Only three, namely, prostate-specific antigen, prostatic acid phosphatase, and prostate-specific membrane antigen have been investigated for prostate-specificity and for their utility in diagnosis of human prostate disease (25, 26, 27) . Additional molecules need to be identified that are prostate-specific and have a role in prostate carcinogenesis, and can thus be used for early diagnosis and for developing specific immunotherapeutic modalities for treatment of prostate abnormalities (cancer and BPH).

The objective of the present study was to identify genes expressed specifically in normal human prostate. To identify genes that are specifically expressed in human prostate, we used the DD-PCR technology. Herein, we describe the identification, molecular cloning, and sequencing of a novel HPG-1 that is expressed only in the prostate. The expression of HPG-1 is up-regulated in androgen-responsive LNCaP cells compared with androgen-nonresponsive DU-145 and PC-3 cells, and the expression in LNCaP cells is enhanced with androgens. Treatment of LNCaP cells with antisense oligonucleotide decreases cell growth in a concentration-dependent manner by decreasing the level of specific mRNA transcripts, indicating its role in prostate carcinogenesis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Prostate-specific cDNA Fragment(s) by DD-PCR Procedure.
The Delta RNA DD method (Clontech Laboratories, Inc., Palo Alto, CA) was used to obtain prostate-specific cDNAs, and the procedure was carried out according to the manufacturer’s instructions (28) . Briefly, first-strand cDNA was synthesized from total RNAs from normal human prostate, liver, and spleen (Clontech), and then reversed transcribed 42°C for 1 h. Each DD primer-pair fingerprint, consisting of two dilutions (1:10 and 1:40) of a cDNA template, 20 ng of starting RNA, and the appropriate buffer and water controls, were subjected to PCR amplification in the presence of 50 pmol of a selected P and T primer pair, 10x reaction buffer, 5 mM dNTP, 10 µCi [{alpha}35]dATP, and Taq polymerase; 12 different combinations of primers were used in various experiments. The bands were resolved on a 6% urea denaturing gel, the gel was dried, and exposed to X-ray film. On autoradiography, mRNAs that appeared to be differentially expressed in prostate tissue were selected, and the cDNA fragments were recovered, cloned into pBluescript II SK+ at the SmaI site, and sequenced using the Sequenase 2.0 (USB Corporation, Cleveland, OH). The nucleotide sequence was subjected to a sequence homology search in the GenBank database, and only those cDNA fragments that showed no homology with any existing gene in the database were selected for additional studies. The DD-PCR procedure furnished a novel 339-bp cDNA fragment, designated as P17, which was expressed only in the prostate and not in liver and spleen, the tissues tested.

Library Screening, Isolation of Full-length cDNA, and Sequence Analysis.
The human prostate 5'-STRETCH {lambda}gt10 cDNA library (Clontech) was screened with the P17 cDNA fragment to obtain the full-length cDNA. The partial cDNA fragment was cut from pBluescript II SK+ by digestion with PstI and BamHI and labeled with [{alpha}-32P]dCTP. The library was plated at a density of ~10 x 103 plaque-forming units/100-mm Petri dish with Escherichia coli C600 Hfl as the host bacterium. After growth at 37°C for 5.5 h, the plaques were lifted onto nitrocellulose membranes, denatured, neutralized, and UV cross-linked. The membranes were blocked with ExpressHyb solution (Clontech) for 15 min, and then probed with the 32P-labeled P17 probe for 2 h. The filters were washed with 0.1% SDS in 2x SSC solution (room temperature, 5 min), 0.1% SDS in 1x SSC (room temperature, 5 min), and then with 0.1% SDS in 1x SSC (55°C; 10 min). The putative positive clones were subjected to secondary and tertiary screening, and the cDNA insert of the selected clone was subcloned into pBluescript II SK+ at the EcoRI site, and sequenced. The nt and aa sequence search were performed using BLASTN and BLASTX algorithms (29) using the National Center for Biotechnology information web server.4 The sequence was analyzed for putative ORF and plotted for hydrophobicity according to the Kyte and Doolittle (30) , and Engelman et al. (31) scales using the Vector Nti Suite software program (InforMax Inc., North Bethesda, MD). Potential phosphorylation sites, protein motifs, and glycosylation sites were analyzed using the GCG program (University of Wisconsin, Madison, WI). The full-length cDNA was designated as HPG-1.

Northern Blot Procedure.
The human multiple tissue Northern blots were obtained from Clontech, and the Northern blots of normal prostate tissue and cultured prostate cancer cells were prepared in the laboratory (28) . Total RNA from cultured prostate cancer cells (LNCaP, DU-145, and PC-3) that had reached 75–80% confluence was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. After electrophoresis on a denaturing gel, all of the RNAs were found to have distinct 28S and 18S ribosomal bands with intensity ratio of at least 1.5:1. The gel containing ~2 µg of poly(A)+ RNA per lane from normal human prostate (Clontech) or cultured prostate cancer cells was subjected to electrophoresis, blotted onto nitrocellulose membrane, and UV cross-linked. Blots (multiple tissue Northerns, prostate, and prostate cancer cells) were prehybridized (60°C; 10 min) in ExpressHyb solution and then hybridized (65°C; 2 h) with [{alpha}-32P]dCTP-labeled cDNA probe incorporating the HPG-1 ORF. The cDNA probe (ORF) was prepared by PCR amplification of the HPG-1 cDNA, using forward (5'-CGGGATCCATGATAAAAAAAAATCTT-3') incorporating the BamHI site and reverse primer (5'-CGGAATTCTCATTGTGAACAAAATGC-3') incorporating the EcoRI site. These primers correspond with the start and termination codons, respectively, of ORF of HPG-1 cDNA. PCR amplification cycles involved initial denaturation at 94°C for 10 min, and 30 cycles: 94°C for 1 min, 55°C for 2 min, and 72°C for 2 min, followed by a final extension at 72°C for 15 min. The final volume of the PCR reaction mixture was 50 µl consisting of 20 ng cDNA, 50 pmol of the respective forward and reverse primers, 1x PCR buffer, 1.5 mM MgCl2, and Taq polymerase. The PCR-amplified and gel-purified HPG-1 cDNA (ORF) was labeled with [{alpha}-32P]dCTP by using the random hexamer method (Life Technologies, Inc., Rockville, MD). The filters were washed (room temperature, 15 min) with 2xx SSC/0.1% SDS and then with 1x SSC/0.2% SDS. The blots were exposed to X-ray film at -70°C. The filters were stripped and rehybridized (60°C, 1 h) with [{alpha}-32P]dCTP-labeled ß-actin to examine the integrity and equal loading of RNA.

Cell Culture.
The human prostate cancer cell lines LNCaP, DU-145, and PC-3 were a gift from Dr. Parmender Mehta (University of Nebraska Medical Center, Omaha, NE). All three of the cell lines were grown in RPMI 1640 supplemented with 2 mM L-glutamine, 10% FCS, and 50 µg/ml 1entamicin, and maintained in an atmosphere of 5% CO2/95% air at 37°C.

RT-PCR and Southern Blot Analysis.
The expression of HPG-1 in normal human tissues and prostate cancer cells was also examined using the RT-PCR/Southern blot procedure (28 , 32) . The total RNA from 10 normal human tissues, namely, brain, heart, kidney, liver, lung, mammary gland, prostate, skeletal muscle, testis, and trachea were purchased from Clontech. Total RNA from LNCaP, DU-145, and PC-3 cells was extracted as described above.

To prepare cDNA, the RNA was heated to 70°C for 10 min, cooled on ice for 5 min, and then reversed transcribed by using 5x buffer [250 mM Tris-Hcl (pH 8.3), 375 mM KCl, and 15 mM MgCl2], 0.5 µg oligo(dT)15 primer, 0.5 µl of rRNasin RNase inhibitor (40 units/µl), 2 µl of 10 mM MgCl2, 1 µl of 10 mM dNTPs, and 1 µl of Moloney murine leukemia virus reverse transcriptase (2.5 units/µl). Integrity of the cDNA was confirmed by the amplification of ß-actin transcripts in a 30-cycle PCR reaction with specific forward and reverse 29-mer primers (Clontech) that were based on the conserved regions between rat and human ß-actin cDNAs sequences. This primer set is expected to amplify a 256-bp fragment. Specific forward and reverse 21-mer HPG-1 cDNA primers (5'-GATTATTCCAATTGATGCTGA-3' and 5'-GATAGGGATGACATTGAATCT-3') were designed to incorporate the ORF and are expected to amplify a 692-bp fragment.

To examine the specificity of the amplified PCR products the Southern blot procedure was performed. The amplified products on agarose gel were transferred to nitrocellulose membrane, and the membrane was prehybridized (60°C, 10 min) with ExpressHyb solution and then hybridized (65°C, 1 h) with [{alpha}-32P]dCTP-labeled HPG-1 cDNA or ß-actin cDNA probe. The membranes were washed and exposed to X-ray film.

In Vitro Transcription and Translation Procedures.
The recombinant HPG-1 clone in pBluescript II SK+ plasmid was restricted with XhoI or NotI to linearize for the runoff transcription procedure using T3 and T7 RNA polymerase (Promega, Madison, WI; Ref. 28 ). The restricted linear DNA was incubated (37°C, 30 min) with proteinase K, followed by phenol-chloroform extraction, and ethanol precipitation. One µg of the restricted and proteinase K-treated DNA template was incubated (37°C, 30 min) with 5 µl of 5x transcription buffer containing 2 µl of 100 mM DTT, 20 units of RNasin RNase inhibitor, 1 µl of 10 mM dNTP, and 10 units of T3 or T7 polymerase with or without [{alpha}-32P]dCTP in a final volume of 20 µl. Template RNA from the transcription reaction was digested at 37°C for 15 min with RNase-free DNase (1 unit/µg template DNA), followed by phenol-chloroform extraction and ethanol precipitation. The transcribed RNA was dissolved in diethylpyrocarbonate-treated water and used for the in vitro translation procedure (Promega). The labeled RNA was subjected to agarose gel electrophoresis followed by autoradiography to visualize the transcript. For the in vitro translation procedure, the template mRNA was heated at 67°C for 10 min followed by immediate cooling on ice. The translated mRNA (100 ng/µl) was incubated (30°C, 60 min) in a total volume of 50 µl containing 20 µl of rabbit reticulocyte lysate-nuclease, 1 µl of 1 mM amino acid mixture without methionine, 1 µl of RNasin RNase inhibitor (40 units/µl), and 4 µl of L-[35S]methionine (10 mCi/ml). Luciferase RNA was used as a control. After the reaction was completed, a 20-µl aliquot was analyzed in the reducing SDS-PAGE (5–10%; Ref. 33 ), followed by autoradiography.

Quantitative RT-PCR Using Standardized CTs.
Quantitative RT-PCR was used to examine the relative quantities of HPG-1 mRNA transcripts in cancer cells. The primers for HPG-1 and the housekeeping ß-actin gene were designed as described by Celi et al. (34) . They were designed to ensure the absence of stable duplex formation and false priming sites, and were synthesized by MWG Biotech (High Point, NC). The NT forward primer is a conventional PCR primer 28 nt in length that corresponds to the target sequence, and the 28 nt reverse primer corresponds to its complimentary strand. The CT 56-nt primer was a hybrid with 28 nt in the 5'-end identical to the reverse primer and 28 nt in the 3'-end corresponding the complimentary strand of HPG-1 cDNA located in a region approximately 100–120 bp upstream of the original reverse primer. The 28 bp at the 3'-end of the CT primer allows it to bind to its complimentary sequence on the target gene, but it cannot be extended with Taq polymerase. The primer sequences, nt position, and the PCR product length for both the NT and CT of HPG-1 and ß-actin are described in Table 1Citation .


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  		Table 1 RT-PCR primers for HPG-1 and ß-actin native and competitive templates

 
Synthesis and quantification of CT standards were performed as described by Willey et al. (35) . The forward NT primers and CT primers of HPG-1 and ß-actin, were used to amplify CT standards from cDNAs of LNCaP, DU-145, and PC-3 cells. CT standards were synthesized in a 50-µl reaction mixture that contained 5 µl of 10x PCR buffer, 50 pmol CT primer or primers, 50 pmol forward NT primer, 20 ng of cDNA, 1.5 µl of 50 mM MgCl2, 0.5 µl of 10 mM dNTPs, and 0.2 µl of Taq polymerase (5 units/µl). PCR amplification cycles involved initial denaturation at 94°C for 10 min and then 30 cycles: 94°C for 1 min, 58°C for 2 min, and 72°C for 2 min followed by a final extension at 72°C for 15 min. HPG-1 and ß-actin CTs were checked by electrophoresis for predicted sizes, and then cut from the gel, eluted, and purified. The concentrations of gel-purified CTs were determined by electrophoresis on a 2% agarose gel containing 0.5 µg/ml EtBr using a known amount of HindIII-digested {lambda} DNA markers. The bands were visualized using a Foto/Eclipse Investigator image capture system (Fotodyne, Hartland, WI) and quantified using the GelPro densitometric scan software program (Fotodyne). CT molarity was calculated from the pixel quantity and compared with that of a known amount of a fragment from the HindIII-digested {lambda} DNA marker. CTs were diluted in 10 mM Tris and 1 mM ethylenediaminetetraacetic acid buffer (pH 8.0), so that the forward and reverse primers yielded CT and NT PCR products of approximately equal intensity in EtBr-stained electrophoretic gels (31) . CT mixtures and dilutions containing known amounts of both HPG-1 and ß-actin CTs were prepared from the same original stock solutions (31) . CT mixtures were then diluted to contain ß-actin (A) and HPG-1 (P) at the following molar (M) concentration: A-13/P-13, A-13/P-14, A-13/P-15, A-13/P-16, A-13/P-17, A-14/P-15, A-14/P-16, A-14/P-17, and A-14/P-18. Each quantitative competitive RT-PCR reaction (50 µl of total volume) required 5 µl of the CT mixture, and thus, the final CT molarity was 10-fold lower than the above concentrations. The appropriate CT mixture was selected based on the relative cDNA concentration in prostate cancer cells and on the relative expression of HPG-1 to ß-actin. In our study, 1 µl of cDNA was found to compete equally with 105–106 molecules of ß-actin CT and 102–106 molecules of HPG-1 CT in PCR. Primer pair for each gene was expected to amplify a single band from the CT mixture when no native cDNA was present.

The quantitative competitive RT-PCR was carried out as described elsewhere (35) . Briefly, a 50-µl reaction mixture containing 50 pmol of ß-actin forward primer, 50 pmol of ß-actin reverse primer, 50 pmol of HPG-1 forward primer, 50 pmol of HPG-1 reverse primer, 5 µl of CT mixture, 2 µl of cDNA, 5 µl of 10x PCR buffer, 1.5 µl of 50 mM MgCl2, 0.5 µl of 10 mM dNTPs, and 0.2 µl Taq polymerase, was subjected to PCR, and the products were quantified and analyzed after the conditions described above. The intensity of the EtBr-stained band of NT and CT products was mathematically corrected for size by the after equation as described by Allen et al. (36) . iN = initial number of native molecules; ND = native band density; CTD = CT band density; CTs = CT size in bp; Ns = native size in bp; and iCT is the initial number of CT molecules:

The number of initial native molecules in a sample was calculated based on the amount of CT standard added, and the ratio of native gene product and CT gene products. Results were expressed as the number of molecules of HPG-1 mRNA per 100 molecules of ß-actin.

Effect of Antisense Oligonucleotide on mRNA Transcript Levels in LNCaP Cells.
The human prostate cancer LNCaP cells were grown in RPMI 1640, supplemented with 2 mM L-glutamine, 10% FCS, 50 µg/ml 1entamicin, and maintained in an atmosphere of 5% CO2/95% air at 37°C. Exponentially growing LNCaP cells were seeded into 2 ml of RPMI 1640 with 10% charcoal/dextran-stripped, delipidated, heat-inactivated, FCS (Sigma, St. Louis, MO) in culture dishes at a concentration of 2 x 105 cells/ml. After 24 h, the cells were washed with HBSS (Life Technologies, Inc., Grand Island, NY), and resuspended in fresh RPMI 1640/charcoal-stripped calf serum medium and 2.5–20 µM (final concentration) of sense/antisense phosphorothioate-conjugated oligonucleotides. The sense (5'-ATGATAAAAAAAAAT-3') and antisense (5'-ATTTTTTTTTATCAT-3') oligonucleotides were based on the translation initiation region (nt sequence 318–332) of the HPG-1 cDNA sequence, had 6.7% G/C content, and were synthesized at the MWG-Biotech (High Point, NC). LNCaP cells were incubated with the oligonucleotides for 24, 48, and 72 h, washed, and total RNA was extracted and examined for integrity as described above. Approximately 2 µg of poly(A)+ RNA per lane from LNCaP cells cultured with various concentrations of sense or antisense phosphorothioate oligonucleotides were subjected to gel electrophoresis, blotted onto nitrocellulose membrane, and UV cross-linked for Northern blot analysis. Blots were prehybridized (60°C, 10 min) in ExpressHyb solution, hybridized (65°C, 2 h) with [{alpha}-32P]dCTP-labeled cDNA probe incorporating HPG-1 ORF, washed, and exposed to X-ray film. The bands were visualized and quantified as described above. HPG-1 transcript intensity in each lane was normalized to the ß-actin band intensity.

Effect of Antisense Oligonucleotide on Growth of LNCaP Cells.
Exponentially growing LNCaP cells were seeded into 2 ml of RPMI 1640 with 10% FCS in tissue culture dishes at the concentration of 2 x 105 cells/ml. After 24 h, cells were washed with HBSS and resuspended in 2 ml of fresh RPMI 1640/charcoal-stripped calf serum medium containing 2.5–20 µM of sense or antisense phosphorothioate oligonucleotide. LNCaP cells were incubated with the various concentrations of phosphorothioate oligonucleotides for 24, 48, and 72 h, trypsinized, and total cell counts were determined by hemocytometer. Cell viability was assayed using the trypan blue dye exclusion assay (Sigma). Five independent experiments were performed on different days using different passages of LNCaP cells. Significance of differences between sense- and antisense-treated and the control groups was analyzed by the one-way ANOVA test. Post-hoc analysis was performed by using the Bonferroni test. A P of <0.05 was considered significant.

Effect of Androgens on HPG-1 Expression in LNCaP Cells.
LNCaP cells were cultured in phenol red-free RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD) supplemented with charcoal-stripped calf serum, and the experiments were performed on cells cultured between passages 12 and 16. Cells were incubated with (5{alpha}-DHT; Sigma) at 10-7 M, 10-8 M, 10-9 M, 10-10 M, and 10-11 M concentrations for 6 h, washed with PBS, and the RNA was extracted and examined for integrity as described above. Approximately 2 µg of poly (A)+ RNA per lane from the LNCaP cells treated with the various concentrations of 5{alpha}-DHT were subjected to electrophoresis, blotted onto nitrocellulose membrane, and UV cross-linked for Northern blot analysis. Blots were prehybridized (60°C, 10 min) in ExpressHyb solution, hybridized (65°C, 2 h) with [{alpha}-32P]dCTP-labeled cDNA probe incorporating HPG-1 ORF, washed, and exposed to X-ray film. HPG-1 mRNA levels were visualized and quantified as described above. LNCaP cells treated identically without 5{alpha}-DHT served as a control.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HPG-1 Full-Length Clone.
To identify novel genes that are expressed exclusively in the normal prostate, we compared mRNA expression patterns of normal human prostate, liver, and spleen by the DD-PCR technique. A total of 5 cDNA fragments were found that were differentially expressed in the prostate. These fragments were subcloned into pBluescript II SK+, sequenced, and analyzed for nt and aa homology in the GenBank database, and for prostate-specific expression in the Northern blot analysis. Of these, a 339-bp cDNA fragment, designated as P17, did not show a significant homology with any nt/aa sequence in the database and demonstrated prostate-specific expression, and thus was selected for additional studies. Using P17 as a probe, the full-length 1,468-bp cDNA, designated as novel HPG-1, was obtained after screening 873,000 clones from the normal human prostate {lambda}gt10 cDNA library.

HPG-1 Sequence Analysis.
HPG-1 cDNA was found to be a novel sequence without any homology with any known nt/aa sequence in the database. HPG-1 cDNA was found to be located on human 3q26 chromosome locus. Analysis of HPG-1 cDNA using the DNA analysis program revealed that it has an ORF starting with an ATG initiation codon at nt 318–320 with a termination codon at nt 306–308 (Fig. 1A)Citation . It has a 5'-UTR of 317 bp and a 3-UTR of 767 bp. The canonical 3' polyadenylation signal, AATAAA, is located at nt positions 720–725.



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  		Fig. 1. nt and aa sequences of HPG-1 cDNA. A, HPG-1 contains a 317-bp 5' UTR, a 384-bp ORF, and a 767-bp 3' UTR. Inframe termination (TER) codons are located at nt positions 306–308 and at nt positions 699–701. HPG-1 encodes a 127 aa protein with putative post-translational modification sites especially two N-glycosylation sites at aa 35–38 and 97–100 ({square}), respectively and one tyrosine phosphorylation site at aa 8–19 (__). The canonical 3'-polyadenylation signal is double underlined. B, hydrophilicity plot of the deduced aa sequence of HPG-1 protein. It was computed by using methods of Kyte and Doolittle (30) , and Engelman et al. (31) .

 
Analysis of the predicted peptide using Vector NTI DNA analysis software program revealed that the predicted peptide of HPG-1 has 127 amino acid residues with a calculated molecular mass of 14,810 Da and a isoelectric point of 9.42. The motif search of the predicted peptide showed two potential N-linked glycosylation sites on the hydrophilic portions of the peptide at 35–38 and 97–100 aa, respectively, one Asn-myristoylation site at 10–15 aa, two casein kinase II phosphorylation sites at 42–45 and 75–78 aa, respectively, and six protein kinase C phosphorylation sites at 37–39, 63–65, 75–77, 99–101, 100–102, and 113–115 aa residues, respectively. HPG-1 has one potential tyrosine phosphorylation site at aa positions 8–19, two threonine phosphorylation sites at aa positions 59–67 and 71–79, and six serine phosphorylation sites at aa positions 25–33, 33–41, 39–47, 58–66, 95–103, and 109–117, respectively. The encoded protein contains ~15% serine/threonine residues, which are potential sites for O-linked glycosylation. The aa sequence has 52.8% hydrophilic and 47.2% hydrophobic amino acid residues, respectively. It seems that the HPG-1 has a slightly more hydrophilic characteristic. Analysis of the hydrophilicity profile of the deduced amino acid sequence of HPG-1 (ORF; Fig. 1BCitation ) indicated that this peptide has a hydrophobic NH2-terminal region, a central core with several hydrophilic and hydrophobic domains, and a tendency toward hydrophility at the COOH terminus. Both the Kyte and Doolittle (30) and Engelman et al. (31) computations indicated similar patterns.

Tissue-specific Expression of HPG-1 cDNA.
The tissue-specific expression of HPG-1 cDNA was examined by the Northern blot and RT-PCR-Southern blot analyses. Human multiple northern blots containing ~2 µg of poly (A)+ RNA per lane from 16 human tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, adrenal medulla, thyroid, adrenal cortex, testis, thymus, small intestine, stomach, and prostate) were probed with HPG-1 cDNA (ORF). Northern blot analysis showed that the specific transcript of HPG-1 mRNA (~1.5-kb) was observed only in the prostate lane (Fig. 2A)Citation . The estimated transcript size of HPG-1 on human Northern blots corresponded to the size of the insert cDNA. The HPG-1 (ORF) cDNA probe did not hybridize with any other tissue even when the membrane was exposed for 2 weeks. Fig. 2ACitation , bottom row, shows hybridization of all of the tissues to the control human ß-actin cDNA probe revealing a positive signal of equal intensity of the ~2.0-kb transcript (Fig. 2B)Citation .



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  		Fig. 2. Prostate-specific expression of HPG-1 in Northern blots. A, two human multiple tissue blots and prostate blot were prehybridized with ExpressHyb solution, then hybridized with [{alpha}-32P]dCTP-labeled HPG-1 (ORF) cDNA probe, washed, and exposed to X-ray film for 24 h to 3 weeks. The HPG-1 transcript of ~1.5-kb was detected only in the prostate (arrowhead) and not in any of the other 15 tissues tested. B, the blots were stripped off the HPG-1 cDNA probe and rehybridized with [{alpha}-32P]dCTP-labeled ß-actin probe that showed a band of ~2.0-kb of equal intensity in all lanes.

 
The tissue-specific expression of HPG-1 cDNA (ORF) was additionally confirmed by RT-PCR-Southern blot analysis. The total RNA from 10 various human tissues was examined using specific primers based on HPG-1 or ß-actin cDNA. The HPG-1-specific primers amplified the expected 692-bp fragment only in the prostate RNA (Fig. 3A)Citation . The Southern blot hybridization additionally confirmed the specificity of the amplified 692-bp fragment (Fig. 3B)Citation . The ß-actin-specific primers amplified the expected 256-bp fragment in all of the tissues by RT-PCR (Fig. 3C)Citation that was specifically hybridized with the ß-actin cDNA probe in the Southern blot procedure (Fig. 3D)Citation .



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  		Fig. 3. RT-PCR-Southern blot pattern of HPG-1 in various normal human tissue RNAs. A, specific primers based on HPG-1 cDNA incorporating ORF amplified a specific 692-bp fragment only in the prostate tissue RNA, as seen in the EtBr-stained agarose gel. B, the 692-bp amplified fragment was specifically recognized by the HPG-1 cDNA in the Southern blot procedure. C, ß-actin primers amplified a specific 256-bp fragment in all 10 tissue RNAs that was specifically recognized by the ß-actin cDNA probe in the Southern blot procedure (D).

 
In Vitro Transcription and Translation Products.
In vitro transcription and translation procedures were performed to examine the apparent molecular mass of the translated polypeptide. The subcloned HPG-1 cDNA in pBluescript II SK+ vector was analyzed for orientation of mRNA-like strand (sense strand) sequence. To obtain the flanking insert sequence with either T3 or T7 RNA polymerase, the XhoI- or NotI-restricted recombinant plasmid was used for the in vitro transcription. T7 and T3 RNA promoter polymerase-generated run-off transcripts (Fig. 4A)Citation were subjected to the in vitro translation procedure by using the rabbit reticulocyte lysate system. The translated products were resolved in the SDS-PAGE and subjected to autoradiography. Autoradiography of the gel showed a single band of ~15 kDa translated product when the NotI-restricted recombinant plasmid with T7 RNA polymerase was used. The run-off transcript generated by T7 RNA polymerase using NotI-restricted recombinant plasmid indicated that it was a sense strand (Fig. 4B)Citation . The run-off transcript generated with T3 RNA polymerase using XhoI-restricted plasmid did not yield any translated product, indicating it to be an antisense strand (Fig. 4B)Citation .



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  		Fig. 4. Analysis of in vitro transcription and translation products of HPG-1 sense (S) and antisense (AS) strands. A, in the in vitro transcription procedure, NotI (Lane a) and XhoI (Lane b)-restricted recombinant plasmids revealed ~ 1.5-kb transcript (arrowhead). B, autoradiography of the dried 5–10% SDS-PAGE gel of the in vitro translation products of the various run-off transcripts. The translation product of the NotI-restricted recombinant plasmid using T7 is shown in Lane a. The translation product of the XhI-restricted recombinant plasmid using T3 is shown in Lane b. The translation product of the NotI-restricted (S strand) recombinant plasmid is Mr ~15,000 (arrowhead). No translation product was observed in the XhoI-restricted recombinant plasmid (AS strand).

 
HPG-1 Expression in Prostate Cancer Cells.
To investigate the role of HPG-1 in carcinogenesis, its expression was examined in the androgen-responsive (LNCaP) and androgen-nonresponsive (DU-145 and PC-3) cells by the Northern blot analysis. HPG-1 mRNA transcript of ~1.5-kb was detected in the LNCaP and DU-145 cells and not in PC-3 cells; the LNCaP transcript band was stronger (~2-fold) than that of DU-145 cells (Fig. 5A)Citation . The control ß-actin transcript of ~2.0-kb was present in equal amounts in all of the three lanes indicating equal loading and RNA integrity.



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  		Fig. 5. Expression pattern of HPG-1 in prostate cancer cells. A, 45 µg of total RNA from three prostate cell lines were separated on a denaturing gel and then transferred to nitrocellulose membrane for Northern blot analysis. The blot was prehybridized with ExpressHyb solution, then hybridized with [{alpha}-32P]dCTP-labeled HPG-1 cDNA probe, washed, and exposed to X-ray film for 24 h to 3 weeks. The HPG-1 transcript of ~1.5-kb was detected in LNCaP and DU-145 cells and not in PC-3 cells with the LNCaP cell showing the strongest band. The blots were stripped off the HPG-1 cDNA probe and rehybridized with [{alpha}-32P]dCTP-labeled ß-actin probe that showed a band of ~2.0-kb of equal intensity in all lanes. B, in the quantitative RT-PCR procedure, the LNCaP cells showed significantly (P < 0.05) higher amount of HPG-1 mRNA transcripts as compared with DU-145 and PC-3 cells. PC-3 cells demonstrated no HPG-1 mRNA transcript but only ß-actin mRNA transcripts. The results are expressed as mean (n = 9 experiments) of HPG-1 transcripts/100 ß-actin molecules; bars, ±SE.

 
The quantitative measurements of HPG-1 transcript levels in the cultured prostate cancer cell lines were determined by the quantitative RT-PCR (Fig. 5B)Citation . LNCaP cells showed significantly higher levels of HPG-1 transcripts per 100 ß-actin molecules (~2.3 HPG-1 transcripts/100 ß-actin molecules) as compared with DU-145 cells (~1 HPG-1 transcript/100 ß-actin molecules). There was no expression observed in the androgen-nonresponsive PC-3 cells (Fig. 5B)Citation .

Effect of Antisense Oligonucleotide on HPG-1 mRNA Transcript Levels in LNCaP Cells.
The overexpression of HPG-1 in LNCaP cells implied that it may play a critical role in the cellular processes in the androgen-responsive cells. We proceeded to examine whether treatment of LNCaP cells with 2.5, 5, 10, or 20 µM of antisense oligonucleotide would reduce HPG-1 transcript levels. Sense and antisense oligonucleotides based on the initiation codon and the following four codons were selected for the study. These oligonucleotides were chemically modified with a phosphorothioate backbone that enabled it to be resistant to cellular nucleases, while maintaining the ability for the oligonucleotide to bind to the target mRNA and recruit RNaseH. Because of the cellular toxicity observed at higher concentration (20 µM) and no effect observed at lower concentration (2 µM) of antisense oligonucleotide in the pilot experiments, the 5 µM and 10 µM concentrations were selected for these studies. Treatment of LNCaP cells with 10 µM of antisense oligonucleotide resulted in a 54% decrease (P = 0.023) at 24 h and a 59% decrease (P = 0.028) in HPG-1 mRNA levels at 48 h as compared with control (Fig. 6A)Citation . The sense oligonucleotide did not significantly (P > 0.05) affect HPG-1 mRNA transcript level at these time points. There was no significant effect of sense or antisense oligonucleotide on HPG-1 mRNA levels at 72 h.



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  		Fig. 6. Effect of antisense oligonucleotide on HGP-1 mRNA transcript levels in LNCaP cells. LNCaP cells (2 x 106 cells/dish) were incubated with a single treatment of 5 or 10 µM of HPG-1 sense (S) or antisense (AS) phosphorothioate oligonucleotide or vehicle control for 24 h. After incubation, the cells were removed from the plates, washed, and the total RNA was extracted by using TRIzol reagent. RNA (25 µg/lane) was run in gel electrophoresis, the bands transferred to nitrocellulose membrane, and the Northern blot was probed with [{alpha}-32P]dCTP-labeled HPG-1 or ß-actin cDNA to detect specific RNA bands by autoradiography. The intensity of various bands was quantitated by Gel Pro analyzer software program. The HPG-1 mRNA band intensity was normalized with ß-actin bands and expressed as percentage of control mRNA expression (A). The pictures of actual autoradiograms of representative Northern blots showing HPG-1 and ß-actin mRNA signals for each treatment are included in B. The incubation of LNCaP cell with antisense and not the sense oligonucleotide significantly decreased HPG-1 mRNA levels and not the ß-actin transcript levels at day 1 and day 2; bars, ±SE.

 
Effect of Antisense Oligonucleotide on Growth of LNCaP Cells.
The effect of antisense oligonucleotide on LNCaP cells was also determined. The incubation of LNCaP cells with 10 µM of antisense oligonucleotide significantly (P < 0.05) decreased cell growth at all of the time points tested (days 1, 2 and 3; Fig. 7Citation ). The incubation of cells with 5 µM of antisense oligonucleotide significantly decreased cell growth at days 2 and 3. At day 3, there was a 63% reduction (P = 0.026) with 5 µM and an 86% reduction (P = 0.013) with 10 µM concentration of antisense oligonucleotide compared with control. The sense oligonucleotide did not significantly (P > 0.05) affect LNCaP cell growth.



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  		Fig. 7. Effect of HPG-1 antisense oligonucleotide on LNCaP cell growth. LNCaP cells (2 x 106 cells/dish) were incubated with a single treatment of 5 or 10 µM HPG-1 sense (S) or antisense (AS) phosphorothioate oligonucleotide or medium control, and for 24, 48, or 72 h. Total cell counts were determined at the various time points by hemocytometer. Inclusion of antisense oligonucleotide in the medium significantly (P = 0.013–0.026) inhibited cell growth as compared with controls. The sense oligonucleotide did not significantly (P > 0.05) affect the cell growth; bars, ±SE.

 
Effect of Androgens on HPG-1 Expression in LNCaP Cells.
LNCaP cells express high-affinity androgen receptors with the ability to respond to androgens. To examine the effect of androgen on HPG-1 mRNA levels, various concentrations of 5{alpha}-DHT were added to LNCaP cells cultured in charcoal-stripped serum and phenol red-free medium. HPG-1 mRNA levels were determined by using the Northern blot analysis, and their levels were quantified and normalized to ß-actin mRNA levels. HPG-1 mRNA levels increased with increasing concentrations of 5{alpha}-DHT (Fig. 8)Citation . Significant increases in HPG-1 mRNA level occurred when LNCaP cells were treated with 10-10 M (P = 0.044), 10-9 M (P = 0.031), 10-8 M (P = 0.012), and 10-7 M (P = 0.003), concentrations of 5{alpha}-DHT as compared with controls.



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  		Fig. 8. Effect of 5{alpha}-DHT on HPG-1 mRNA transcripts in LNCaP cells. LNCaP cells were grown in phenol red-free RPMI 1640 supplemented with 10% charcoal/dextran-stripped calf serum for 24 h and then incubated with various concentrations (10–11-10-7 M) of 5{alpha}-DHT or vehicle control. After incubation, the cells were washed, total RNA extracted, run in a gel, the bands transferred to nitrocellulose, and the Northern blot was probed with [{alpha}-32P]dCTP-labeled HPG-1 or ß-actin cDNA to detect specific RNA bands by autoradiography. The intensity of various bands was quantitated by Gel Pro analyzer software program. The HPG-1 mRNA bands intensity was normalized with ß-actin bands and expressed as percentage of control mRNA expression (mean, n = 3). The picture of an autoradiogram of a representative Northern blot showing mRNA signals (arrowhead) at various concentrations of 5{alpha}-DHT is also included (inset). The HPG-1 mRNA levels of cells grown in the presence of 10% FCS has been included for comparison (Lane A). The incubation of LNCaP cells with 5 {alpha}-DHT caused a concentration-dependent increase in the levels of HPG-1 mRNA transcripts; bars, ±SE.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The aim of the present study was to obtain a cDNA that is prostate-specific and is involved in carcinogenesis. The DD-PCR technique was used for this purpose. Using this technique, five cDNA fragments expressed only in the prostate and not in the other tissues tested were obtained. All were subcloned, sequenced, searched for homology with known genes in the database, and analyzed for prostate-specific expression in the Northern blot analysis involving RNAs from a limited number of tissues. Of these five, the P17 cDNA fragment did not reveal homology with any known gene in the database and also indicated prostate specificity in the Northern blot analysis. Screening the human prostate 5'-stretch {lambda}gt-10 cDNA library with this prostate-specific 339-bp cDNA fragment yielded a full-length HPG-1 cDNA comprised of 1468 nt having an ORF encoding for a 127 aa peptide. Both nt and aa sequences did not show homology with any gene reported previously in the database, indicating it to be a novel cDNA. The presence of a termination codon at the 5' end before the ATG start codon, and a polyadenylation signal at the 3' end, indicate it to be a full-length cDNA. It has a 5'-UTR of 317-bp and a 3'-UTR of 767-bp. The UTRs may be involved in stabilizing mRNA for translation regulation (37) . Most eukaryotic mRNAs possess short 5' UTRs of 20–100 nucleotides that enable efficient cap-dependent ribosome scanning (38) . Long 5' UTRs are found in proto-oncogenes, growth factors, growth factor receptors, and homeodomain proteins, and mutations in this region have been implicated in cancer progression (39) .

The deduced aa of the HPG-1 ORF translated to a polypeptide of 14.8 kDa. The in vitro transcription and translation procedures confirmed the molecular mass of the HPG-1 protein to be ~15 kDa. The deduced aa of HPG-1 indicated two potential N-linked and several O-linked glycosylation sites; one tyrosine, two threonine, and six serine phosphorylation sites; six kinase C and two casein kinase II phosphorylation sites; and one N-myristoylation site. Hydrophilicity analysis of the deduced protein indicated a hydrophobic NH2-terminal region, a central core of several hydrophilic and hydrophobic domains, and a tendency toward hydrophilicity at the COOH-terminal region. This hydrophilicity profile indicates HPG-1 to be a membrane-anchored protein. The presence of potential N-myristoylation site close to the NH2-terminus also indicates that HPG-1 protein may have a propensity to bind to the plasma membrane of the cell (40) . However, there is no signal peptide sequence at the NH2-terminus, and several O-linked and two N-linked glycosylation sites. It is possible that HPG-1 protein is present in both a membrane-attached/anchored and secreted forms. The presence of multiple potential phosphorylation sites renders the molecule with signal transducing ability that may have a role in cellular growth, differentiation, and proliferation (41 , 42) .

The HPG-1 has a prostate-specific expression as indicated by the Northern blot and RT-PCR/Southern blot procedures using 19 different tissues. The prostate-specific expression suggests a unique role of this molecule in normal and possibly abnormal prostate function and growth. The HPG-1 transcripts were present in LNCaP and DU-145 cells, and absent in PC-3 cells as indicated by the Northern blot and quantitative RT-PCR procedures. LNCaP is an androgen-responsive cancer cell line, and the DU-145 and PC-3 are androgen-nonresponsive cancer cell lines. There was a 2–3-fold higher HPG-1 mRNA expression in the androgen-responsive (LNCaP) cells compared with androgen-nonresponsive (DU-145 and PC-3) cells as observed in both procedures. These findings indicate that HPG-1 may play a role in regulating androgen-dependent responses in prostate adenocarcinomas. Indeed, the in vitro incubation with antisense and not the sense oligonucleotide caused a significant, concentration- and time-dependent inhibition in LNCaP cells; at 72 h there was an 86% inhibition of cellular growth. These findings indicate a role of HPG-1 in prostate carcinogenesis. The growth-inhibitory effects of antisense oligonucleotides were specific because they specifically reduced the levels of HPG-1 mRNA transcripts (and not the ß-actin mRNA levels) that correlated with the inhibition of LNCaP cellular growth. The incubation of LNCaP cells with the androgen, 5 {alpha}-DHT, that cannot be metabolized to estrogen, caused an up-regulation of HPG-1 mRNA levels in a concentration-dependent manner confirming that HPG-1 is an androgen-responsive gene.

The molecular mechanisms involved in the growth inhibition of LNCaP cells require additional study. It is possible that the HPG-1 is involved in a signal transduction cascade through phosphorylation of serine/threonine/tyrosine residues (41, 42, 43, 44) . Many peptide growth factors, such as epidermal growth factor, fibroblast growth factor, insulin-like growth factor, and transforming growth factor-ß, and phosphorylation events have been shown to participate in normal and abnormal prostate growth (45) .

The computer-based genomic analysis mapped HPG-1 cDNA to Homo sapiens chromosome 3q26 locus. Studies by Sattler et al. (46) showed that 3q25-q27 chromosomal locus, including interleukin 12A, myelodysplasia syndrome 1, solute carrier family 2 member 2, and sex determining region-Y box 2 genes, is amplified in human prostate carcinoma. Because HPG-1 is localized in this chromosomal locus, it may be one of the genes amplified in this cluster.

The utility of an antigen in the specific diagnosis and treatment of prostate cancer is contingent on its prostate specificity and involvement in carcinogenesis. The novel androgen-responsive HPG-1 seems to fulfill both these criteria, and thus may provide an interesting molecular marker for the specific diagnosis of prostate cancer. The involvement of HPG-1 in prostate carcinogenesis is indicated by its prostate-specific expression, inhibition of cancer cell growth by antisense oligonucleotides, and its localization at the 3q26 chromosome locus that has been shown to be involved in prostate cancer. It appears to be a membrane-anchored/attached protein; however, it needs to be determined whether or not it is also secreted in blood and/or semen. The immunotherapeutic strategies involving passive immunization with HPG-1 antibodies and a vaccine could be developed against this molecule for the treatment of prostate abnormal growth (47) . The HPG-1 expression and its role in different tumor size and grade, in indolent and aggressive cancers, in androgen-independent cancer growth, and in BPH need additional investigation.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by NIH Grant HD24425 (to R. K. N.). Back

2 To whom requests for reprints should be addressed, at Division of Research, Health Education Building, Room 211, Medical College of Ohio, 3055 Arlington Avenue, Toledo, OH 43614-5806. Phone: (419) 383-3502; Fax: (419) 383-4473; E-mail: Rnaz{at}mco.edu Back

3 The abbreviations used are: BPH, benign prostatic hyperplasia; DD, differential display; HPG-1, human prostate gene-1; dNTP, deoxynucleotide triphosphate; nt, nucleotide; aa, amino acid; ORF, open reading frame; RT-PCR, reverse transcription-PCR; NT, native template; CT, competitive template; EtBr, ethidium bromide; 5{alpha}-DHT, 5{alpha}-androstan-17ß-ol-3-one; UTR, untranslated region. Back

4 Internet address: http://www.ncbi.nlm.nih.gov/BLAST/. Back

Received 8/ 6/02. Accepted 11/13/02.


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 MATERIALS AND METHODS
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 DISCUSSION
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