Molecular Cloning and Expression of ps20 Growth Inhibitor
A NOVEL WAP-TYPE "FOUR-DISULFIDE CORE" DOMAIN PROTEIN EXPRESSED IN SMOOTH MUSCLE*

Melinda LarsenDagger , Steven J. Ressler§, Bing Lu§, Michael J. Gerdes§, Lauren McBride§, Truong D. Dang§, and David R. RowleyDagger §

From the § Department of Cell Biology and the Dagger  Cell and Molecular Biology Program, Baylor College of Medicine, Houston, Texas 77030

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

We previously reported the purification of ps20 (Rowley, D. R., Dang, T. D., Larsen, M., Gerdes, M. J., McBride, L., and Lu, B. (1995) J. Biol. Chem. 270, 22058-22065), a urogenital sinus mesenchymal cell secreted protein having growth-inhibitory properties. We report here cloning of the 1.03-kilobase rat ps20 cDNA clone from the PS-1 (adult rat prostate smooth muscle) cDNA library. Partial clones were obtained by nested polymerase chain reaction with degenerate primers, and full-length ps20 cDNA clones were isolated by plaque hybridization. Sequence analysis revealed that ps20 protein contains a WAP-type "four-disulfide core" motif and is a novel member of the WAP signature protein family composed primarily of secreted serine protease inhibitors. Native ps20 immunoprecipitated from smooth muscle cells and recombinant ps20 both resolved on SDS-polyacrylamide gel electrophoresis with apparent molecular mass of 27-29 kDa under reducing conditions and 21-23 kDa under non-reducing conditions, respectively. Stable ps20-transfectant COS-7 cell lines secreted ps20 and were growth-inhibited relative to mock transfectants. In addition, COS-7 and prostate carcinoma PC-3 cells were growth-inhibited by bacterially expressed ps20. Northern analysis indicated differential expression by tissue with highest expression in the heart. Immunohistochemical localization of ps20 protein showed cell-specific expression by both visceral and vascular smooth muscle in all tissues, including the prostate gland. These results indicate ps20 is a novel growth-regulatory member of the WAP signature family expressed by smooth muscle cells.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Signaling between stromal and epithelial cells directs specific growth and differentiation patterns in many tissues, including the lung (1, 2), gastrointestinal tract (3), liver (4), skin (5), the mammary gland (6), and the prostate gland (7-10). Growth factors comprise a key component of stromal-epithelial interactions. Growth factors implicated in prostate gland biology include: the fibroblast growth factor family (11, 12), the transforming growth factor-beta family (13), transforming growth factor-alpha (14), the insulin-like growth factor family (15), epidermal growth factor (16, 17), heparin-binding epidermal growth factor-like growth factor (18), tumor necrosis factor-alpha (19), and heparin growth factor-scatter factor (20). Each of these growth factors are bound to the extracellular matrix, cell membrane, or other binding proteins in an inactive or sequestered form and their activity regulated by secreted proteases and protease inhibitors. The actions of proteases and protease inhibitors are fundamental to tissue homeostasis through the regulation of both growth factor bioavailability and cell interaction with extracellular matrix. Alterations in protease and protease inhibitor actions affect tissue development patterns, angiogenesis, cell motility, and tumor invasion (21-24).

In previous studies to identify stromally derived paracrine-acting factors which regulate prostatic epithelial cell proliferation and differentiation, we identified a urogenital sinus mesenchymal secreted growth-inhibitory activity (UGIF1 activity), which altered epithelial cell proliferation, morphology, and protein secretion in several epithelial cell lines (25, 26). A novel mesenchymally secreted protein, termed ps20 (20-kDa prostate stromal protein), responsible for UGIF activity was purified to homogeneity and its amino acid sequence determined (27). Purified ps20 inhibited PC-3 prostatic epithelial carcinoma cell proliferation in a dose-dependent and saturable manner, altered phenotypic morphology, and stimulated protein synthesis and secretion in vitro (27). Specific mechanisms of ps20 action have not yet been determined.

We report here the cloning of full-length cDNA encoding ps20, expression of biologically active recombinant ps20, development of a ps20 antibody, and analysis of in vivo expression patterns. The ps20 protein is a novel member of the whey acidic protein (WAP)-type "four-disulfide core" domain protein family (28, 29). This family is composed of small, secreted serine protease inhibitors, which exhibit a variety of growth- and differentiation-regulatory functions and affect extracellular matrix remodeling and carcinoma progression. We demonstrate here that recombinant ps20 inhibits the proliferation of prostate carcinoma PC-3 cells and COS-7 cells. In addition, we show that ps20 is expressed specifically in the smooth cell type of the prostate gland and other tissues including vascular smooth muscle. These studies suggest that ps20 may function as a mediator of local growth and differentiation mechanisms.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Preparation of the PS-1 (Adult Rat Prostate Smooth Muscle Cell Line) ZAP Express (PS-1_Bfslambda ZAP) cDNA Library-- An oligo(dT) reverse transcribed cDNA library of 109 to 1012 plaque-forming units/ml was prepared using the ZAP Express cDNA synthesis kit (Stratagene, La Jolla, CA). PS-1 cells (adult rat prostate smooth muscle-like cell line) (30), were grown to post-confluence in Bfs medium (90% Dulbecco's modified Eagle's medium (DMEM), 5% fetal calf serum, 5% NuSerum (Becton Dickinson, Bedford, MA), 5 µg/ml insulin, 100 units/ml penicillin, and 100 µg/ml streptomycin) supplemented with androgens (0.5 µg/ml testosterone) (26). RNA was prepared using the RNAgents total RNA isolation system and poly(A) RNA selected with the PolyATtract® system (both from Promega, Madison, WI). RNA was size-fractionated on a Sephacryl column prior to ligation with vector arms.

ps20 Cloning-- The amino-terminal amino acid sequence of ps20 was determined and previously reported (27): N-Thr-Trp-Glu-Ala-Met-Leu-Pro-Val-Arg-Leu-Ala-Glu-Lys-Ser-X-X-X-X-Val-Ala-Ala-(Thr)-Gly-X-Arg-Gln-Pro-(His)-C. Degenerate ps20 primers were designed from this sequence: primer P34 (256-fold degenerate, amino acids 1-7), primer P36 (16-fold degenerate, amino acids 1-7), primer P37 (64-fold degenerate, amino acids 7-13), and primer P38 (16, 384-fold degenerate, amino acids 27-19). Inosines were incorporated at sites of complete degeneracy (31) in primers P34 and P38, whereas optimal mammalian codon usage was assumed in the design of primers P36 and P37. The ps20-specific primers P40 and P42 were designed from clone 3438-1pCRII sequence and ps20-specific primers P45 and P46 from clone 42T7pCRII sequence. Primer sequences (IUPAC nomenclature) are identified in terms of rat ps20 full-length cDNA sequence (Fig. 2): P34 (nt 130-150), 5'-ACI TGG GAR GCI ATG YTI CC-3'; P36 (nt 130-150), 5'-ACM TGG GAR GCY ATG CTS CC-3'; P37 (nt 148-168), 5'-CCY GTS MGR CTG GCY GAR AA-3'; P38 (nt 209-183), 5'-GGC TGY CKI IIS CCK GTK GCR GCN AC-3'; P40 (nt 169-151), 5'-TCA GCT TGG GAC TTC TCA G-3'; P42 (nt 153-171), 5'-GAG AAG TCC CAA GCT GAA G-3'; P45 (nt 425-444), 5'-GGA GGA AGT GTT ACA AGC AG-3'; and P46 (nt 1011-992), 5'-GGT GGG CAG ATT TAT TCG GG-3. Primers P34-P42 were synthesized and purified using high performance liquid chromatography by Keystone Labs, Inc. (Menlo Park, CA). Primers P45 and P46 were synthesized by Genosys (The Woodlands, TX).

Primary PCR amplifications were performed using a ps20 degenerate primer and a vector primer (T3 or T7) with 1 µl of the PS-1_Bfslambda ZAP express cDNA library phage lysate as template under the following conditions: 94 °C for 1 min, 54 °C for 2 min, 72 °C for 30 s to 2 min, for 35 cycles, followed by 72 °C for an additional 7 min. Secondary PCR reactions were performed under identical conditions using a 1-µl aliquot of a primary amplification reaction as template and were resolved by 20% polyacrylamide gels or 3% low melt agarose gels for small (<100 bp) fragments and by 0.7% low melt agarose gels for larger fragments. To amplify specific bands for cloning into the pCRII vector (Invitrogen, San Diego, CA), tertiary PCR reactions were performed repeating the conditions used for secondary PCR reactions but using products of secondary reactions resolved on low melt agarose gels and excised as template. Standard conditions for 20-µl PCR reactions were: 0.5 unit of Taq polymerase (storage buffer B), 1.5 mM MgCl, 0.2 mM each dNTP (all Promega), and 20 pmol of each primer.

Full-length cDNA clones were isolated by plaque hybridization of 42T7-1pCRII insert with the PS-1_Bfslambda ZAP express library. The complexity of plaques to be screened was reduced by using ps20-specific primers P45 and P46 (see Fig. 1D) to screen pools of PS-1_Bfslambda ZAP library lysate for the presence of ps20 clones by detection of the diagnostic 586-bp fragment. Initially, 24 pools of 50,000 plaques were screened with a positive signal detected in each pool. One positive pool was further diluted 1:50 into pools of 1000 plaques for rescreening. Of 10 pools, 5 scored positive. One of these pools was diluted 1:10 into pools of 100 plaques. Of 12 pools, 5 scored positive and 3 of these were screened by plaque hybridization. Plaques were transferred to Nytran 0.45-µm membranes (Schleicher & Schuell), DNA cross-linked by the Stratalinker UV cross-linker (Stratagene), and membranes prehybridized for 2 h at 42 °C in hybridization solution (50% formamide, 2 × PIPES buffer, 0.5% (w/v) SDS, 100 µg/ml sonicated salmon sperm DNA). Clone 42T7pCRII insert was purified from an agarose gel slice by Spin-X columns (Corning Costar Corp., Cambridge, MA), 150 ng labeled with [alpha -32P]dCTP (Amersham Corp.) by random priming with Klenow enzyme (labeling grade, Boehringer Mannheim). Unincorporated nucleotides were removed by NucTrap probe purification column (Stratagene), and denatured probe hybridized with filters at 1-3 × 106 counts/ml overnight, at 42 °C in hybridization mixture. Filters were washed three times for 10 min each at room temperature in 2 × SSC, 0.1% SDS; two times for 20 min each at 55 °C in 0.2 × SSC, 0.1% SDS; and exposed to X-OMAT AR film (Eastman Kodak Co.). Positive plaques were screened by PCR, phagemids excised from lambda  arms by the Rapid Excision Kit (Stratagene), and DNA prepared either by mini alkaline lysis (32) or by Qiagen tip-500 (Qiagen, Chatsworth, CA).

The full-length ps20 cDNA clone, ps20-2bpBKCMV, was confirmed by sequencing. For expression in mammalian cells, the lacZ ATG and prokaryotic 5'-untranslated sequences were removed from the vector of the ps20-2bpBKCMV clone by NheI and SpeI digestion and re-ligation to generate the ps20-2bpBKCMV-lac construct.

Sequencing-- Multiple pass DNA sequencing was performed on an Applied Biosystems model 377 Sequencer version 2.1.1 (University of Texas Houston Molecular Genetics Core Facility) using Ampli-Taq polymerase, 3 pmol of primer (pBKT3-1-, pBKT7-1-, M13-20-, and ps20-specific primers), and 1 µg of double-stranded DNA per reaction, or by manual sequencing with Sequenase version 2.0 DNA sequencing kit (U. S. Biochemica Corp./Amersham Life Science) (33). Sequences were assembled using MacVector version 4.1 and AssemblyLign version 1.0 (Kodak).

Sequence Analysis-- Nucleotide sequence searches were performed on all available data bases using the BLASTN and TBLASTN (blast enhanced alignment utility) algorithms (34). Secondary structure predictions of deduced amino acid sequence were made by SOPMA (Self Optimized Prediction Method from Alignment) (35) and SBASE (36) and confirmed by BLASTX and TBLASTX (34) searches of the available data bases (37). Secretory peptide prediction was confirmed by PSORT version 6.3.

Northern Analysis-- Total RNA was isolated from adult male Sprague-Dawley rat tissues (intact, castrate 2 weeks, and castrate 6 weeks), adult female rat tissues (kindly provided by J. S. Richards, Baylor College of Medicine) and from U4F cells grown to post-confluence in Bfs medium. Total RNA was prepared using RNA Stat-60 total RNA isolation reagent (Tel-Test "B," Inc., Friendswood, TX). RNA was electrophoresed through 1% agarose gels (containing 1 × MOPS and 5.1% of a 37% formaldehyde solution), which were transferred to neutral charged nylon membranes (Schleicher & Schuell) by downward capillary blotting. Inserts from T340pCRII and 42T7pCRII were excised, gel-purified, labeled by random priming with [alpha -32P]dCTP, purified, and denatured as described above. Blots were UV-cross-linked and exposed to probe at 1-5 × 106 counts/ml, overnight, at 42 °C; washed with multiple 20 min washes at 60 °C in 0.1% SDS containing 2 to 0.1 × SSC; and exposed to Kodak XAR film for 24 h to 2 weeks, as necessary.

Preparation of ps20 Antisera and Affinity Purification-- The amino-terminal sequence of native ps20 was determined as described previously (27). A unique 14-amino acid synthetic peptide, ps20 peptide-(1-14), corresponding to the amino terminus of purified ps20 (see Fig. 2), was synthesized on an Applied Biosystems model 430A peptide synthesizer: N-Thr-Trp-Glu-Ala-Met-Leu-Pro-Val-Arg-Leu-Ala-Glu-Lys-Ser-C (Baylor College of Medicine Protein Core Facility). For initial immunization, ps20 peptide was solubilized in sterile, tissue culture grade H2O (400 µg/ml), mixed with Freund's complete adjuvant (1:1 ratio), and injected in 500-µl (100-µg) aliquots at multiple subcutaneous and intramuscular sites of three female New Zealand rabbits. At 3 weeks following immunization, sera samples were analyzed by solid phase enzyme-linked immunoabsorbent assay (ELISA). Each rabbit received a booster of 100 µg of peptide in 500 µl of Freund's incomplete adjuvant injected subcutaneously and intramuscularly at 2 weeks and 5 weeks. Serum samples were tested for ps20-specific antibody every 2 weeks and immunoglobulin subtype determined by ELISA analysis. High titer antiserum (reactive at 1:1 × 106 dilution) was pooled, and the IgG class was precipitated by ammonium sulfate (50% saturation), resolubilized in 1 × PBS, and dialyzed overnight against 1 × PBS at 4 °C. This antibody preparation is referred to as ps20ab-1.

ps20ab-1 was affinity-purified by peptide column chromatography. ps20 peptide-(1-14) (10 mg) was coupled to 1 g of cyanogen bromide-activated Sepharose 4B (Pharmacia Biotech Inc.) (38) and poured into a 2-ml Poly-Prep column (Bio-Rad). ps20ab-1 was diluted in BBS buffer (200 mM sodium borate, 160 mM sodium chloride, pH 8.0) and chromatographed through the column at 4 °C. The column was washed extensively in BBS (10-15 column volumes) and bound antibodies eluted with glycine-Cl buffer (0.05 M glycine, 0.15 M NaCl, pH 2.28). Fractions (2 ml) were eluted and collected directly in tubes containing 0.5 ml of neutralizing buffer (0.5 M phosphate, pH 7.7). Fractions were assayed for protein content, and peak fractions were pooled and assayed for specific immunoreactivity by ELISA.

Cell Culture-- The U4F urogenital sinus mesenchymal cell line (26) was derived from a urogenital sinus organ explant (25) and the PS-1 cell line (30) derived from an adult rat prostate organ explant. U4F cells and PS-1 cells were maintained in medium Bfs as described previously (26, 27, 30). PC-3 cells (ATCC CRL-1435, human prostate adenocarcinoma) were maintained as recommended by the ATCC and as described previously (27). The COS-7 cell line (ATCC CRL-1651, SV40 transformed African green monkey kidney fibroblast-like) was maintained in 90% DMEM, 10% fetal calf serum (DMEM-10) (BioWhittaker, Walkersville, MD), 100 units/ml penicillin, and 100 µg/ml streptomycin. Cultures were tested for mycoplasma every 6 months.

COS-7 Cell Transient Transfection-- COS-7 cells were seeded at 1.7 × 106 cells/ml in 100-mm culture dishes in 90% DMEM, 10% fetal calf serum (DMEM-10) with no antibiotics 24 h prior to transfection. Cells were transfected at 80% confluence with 4.1 µg of ps20-2bpBKCMV-lac DNA or pBKCMV-lac control plasmid DNA (prepared by Qiagen tip-500) and 41.0 µl of LipofectAMINE reagent (Life Technologies, Inc.), following recommendations of the manufacturer.

COS-7 Cell Stable Transfectant Cell Lines-- To select for stably transfected COS-7 cell lines, 48 h following transfection with either ps20-2bpBKCMV-lac DNA or pBKCMV-lac control plasmid DNA cells were split 1:15 and replated in COS-7 cell medium containing 600 µg/ml G418 (Sigma). Cells were grown in G418-containing medium for 3 weeks when pools of G418-resistant colonies were collected. Clonal cell lines were obtained by serial dilution in 96-well plates and assayed for ps20 expression by Western analysis of the conditioned medium. Once established, stable cell lines were maintained in 400 µg/ml G418-containing medium (DMEM-10/G418).

Crystal Violet Staining-- Cells were grown to desired density and then incubated with a solution of crystal violet (0.5% (w/v) crystal violet, 3.2% formaldehyde, 0.17% (w/v) NaCl, 22% ethanol overnight, washed three times with H2O, allowed to dry, and photographed.

[3H]Thymidine Incorporation Assays-- Cells were seeded in a 150-µl volume in 96-well plates at a density of 5.0 × 104 cells/ml (PC-3) and 2.5 × 104 cells/ml (COS-7-derived cell lines), and allowed to attach overnight. Sample was added in triplicate in a 50-µl volume (if applicable) and allowed to incubate an additional 24 h. Cultures were pulsed with 10 µl of 2 µCi/ml [3H]thymidine (Amersham) during the final 3 h of incubation, fixed, and processed as described previously (27). To assay for ps20 activity in the conditioned medium (CM) of transfectant lines, cultures were seeded in a 5-ml volume in 25-mm2 flasks at 1 × 105 cells/ml in standard medium, CM collected after 24 or 48 h, and 100 or 200 µl of CM added to pBKCMV-4 cells and cultures assayed as described above. All assays were performed in triplicate and each experiment repeated multiple times (n >=  3). Data are presented as mean ± S.E. (error bars) and significance determined by Student's t test.

Immunoprecipitation-- 75-mm2 flasks of PS-1 and U4F cells, grown in Bfs medium, were allowed to reach post-confluence. Cells were then incubated in M medium for 6 h then for 24 h in 4 ml of medium M made with DMEM lacking Cys, Met, and glutamine (ICN, Costa Mesa, CA), 2 mM L-glutamine, and 0.6 mCi of 35S (ICN, Boston, MA). 300 µl of 35S-labeled medium was incubated overnight with an equal amount of RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 8.0) and 10 µl of ps20ab-1 or 50 µl preimmune sera followed by a 1-h incubation with 50 µl of Protein A-Sepharose (Pharmacia). Beads were washed four times in 500 µl of RIPA buffer, resuspended in SDS sample buffer with or without 100 mM dithiothreitol, and resolved by 12% SDS-PAGE. The gel was fixed for 30 min in 30% methanol with 10% acetic acid, incubated for 20 min in Enlightning (NEN Life Science Products), dried, and exposed to X-OMAT AR film (Kodak) for 12-48 h.

Expression and Purification of rps20-His-- PCR primers, P69 and P60, were used to synthesize a modified 585-nt fragment of the ps20 coding region for cloning into the bacterial expression vector, pET23c (Novagen, Madison, WI), a vector that allows incorporation of a His tag (six consecutive histidine residues) at the carboxyl terminus. At the amino terminus, the secretory peptide (amino acids 1-26) was replaced with a Met-Ala and the fifth codon was modified to optimize for bacterial expression. Primer P69 (nt 117-153; 5'-C TCT cat atg GCT ACT TGG GAA GCA ATG TTG CCG GTC-3') includes an NdeI site (lowercase) containing an internal ATG start site. Nucleotides modified from ps20 cDNA sequence are underlined. Primer P60 (nt 672-697; 5'-C TCC gaa ttc TGG AAA GTG CCT CTG T-3'), contains an EcoRI site (lowercase) immediately following the final amino acid for cloning into pET23c. Because the EcoRI site in the pET23c vector is out of frame with the His tag, the vector was modified by deleting the four-nucleotide SacI 3' overhang downstream of the EcoRI site; the entire construct was digested with SacI, digested with T4 polymerase in the presence of 100 µg/ml dNTPs at 37 for 10 min, re-ligated, and confirmed by sequencing. The ps20 cDNA clone ps20-2bpBKCMV (1 ng) was used as template using standard PCR procedures with primers P69 and P60 to amplify a PCR product that was cloned into the T-tailed vector, pCRII. The insert from one clone, rps20pCRII, confirmed by automated sequencing, was excised by a NdeI/EcoRI double digest and ligated with NcoI/EcoRI-digested pET23c. An insert-containing clone, rps20-1pET23c, was confirmed by sequencing and transformed into the BL21(DE3)pLysS bacterial expression cell line (Novagen). 500-ml cultures were grown to OD600 = 0.6, protein expression induced by 0.4 mM isopropyl-1-thio-beta -D-galactopyranoside for 3 h, and soluble cell extracts generated by sonication in SB buffer (50 mM phosphate, 300 mM NaCl, pH 8.0) on ice followed by centrifugation at 15,000 × g for 20 min. Soluble recombinant rat ps20 His-tagged protein (rps20-His) was purified on a nickel-charged resin (Ni+-NTA, Qiagen). The column was washed and eluted with an imidazole gradient, as recommended by the manufacturer. Fractions containing rps20-His with apparent >95% purity were pooled and dialyzed to remove imidazole.

Cloning, Expression, and Purification of GST-rps20-- The rps20pCRII clone was modified by cloning the oligonucleotide 5'-CATAcGGATCCCTCGAGCATATG-3' into the NdeI site providing an artificial ATG prior to nucleotide 129 (amino acid 26) of the rps20 cDNA sequence. The lowercase nucleotide represents a mutation in the 5' NdeI site, and the underlined nucleotides represent BamHI and XhoI sites, respectively. This clone, rps20BXpCRII, was digested with BamHI and EcoRI and the 647-base pair fragment purified on a 0.8% agarose gel by the following procedure. The 647-base pair fragment was run onto DE-81 paper (Whatman) and incubated overnight at 25 °C in 500 µl of 0.5 × TBE, 1.25 M NaCl. The paper was then filtered through a 0.45-µm filter, and the DNA was ethanol-precipitated and resuspended in dH2O. This rps20 fragment was then cloned into the BamHI and EcoRI sites in the pGEX2TK vector (Pharmacia) (39), containing a thrombin cleavage and cAMP-dependent protein kinase phosphorylation site, creating the vector rps20pGEX2TK.

To purify GST-rps20 or GST protein preparations, a 1-ml overnight bacterial culture of rps20pGEX2TK or pGEX2TK in XA90 bacteria was used to inoculate 100 ml of fresh LB plus ampicillin (50 µg/ml) at 37 °C. When the culture reached an OD600 = 0.5 M, 1 mM isopropyl-1-thio-beta -D-galactopyranoside was added and the culture allowed to grow for 2.25 h. The culture was centrifuged at 1800 × g at 4 °C for 15 min. The pellet was resuspended in 10 ml of cold CHAPSO buffer (0.5% CHAPSO, 300 mM NaCl, 1 mM EDTA, 50 mM Tris, pH 8.0, 10 mM beta -mercaptoethanol) and sonicated with three 10-s pulses. Cell debris was pelleted at 17,000 × g for 25 min, 1 ml of glycerol added to the supernatant, and the lysate stored at -80 °C. 1 ml of pGST-2TK-rps20 or 100 µl of pGST-2TK lysate was incubated with 40 µl of glutathione-Sepharose beads for 15 min, rocking, at room temperature. The beads were centrifuged at 1800 × g and washed twice in CHAPSO buffer and twice in the same buffer without CHAPSO. The protein was eluted for 10 min at room temperature with 400 µl of elution buffer (40 mM reduced glutathione, 100 mM Tris pH 8.0, 120 mM NaCl). The beads were centrifuged at 1800 × g in a microcentrifuge and the supernatant dialyzed against 2 × DMEM overnight at 4 °C.

For thrombin cleavage of GST-rps20, three tubes of 1 ml each of rps20pGEX2TK lysate were allowed to bind separately to 40 µl of glutathione-Sepharose beads, as described previously except the beads were further washed twice in PBS. The beads from the three tubes were combined and resuspended in 76 µl of PBS. For cleavage of GST, 200 µl of pGEX2TK lysate were allowed to bind to 40 µl of glutathione-Sepharose beads and processed as described above. 8 µl of thrombin (Pharmacia, 1 unit/µl) was added to either GST or GST-rps20 bound to glutathione-Sepharose for 3 h at room temperature. The beads were centrifuged at 1800 × g in a microcentrifuge, and the supernatant dialyzed against 2 × DMEM overnight at 4 °C. Following cleavage of the GST-rps20 fusion, the resulting protein (rps20) contained an additional 11 amino acids; N-Gly-Ser-Arg-Arg-Ala-Ser-Val-Gly-Leu-Glu-His-C were added to the amino terminus of rps20.

Western Analysis-- CM from transfected COS-7 cells and from U4F cells were prepared identically for Western analysis. U4F cells were seeded in Bfs medium and COS-7 stable transfectants seeded in DMEM-10/G418. Both were switched to serum-free, fully defined medium M (MCDB-110, ITS, 0.5 µg/ml testosterone, 0.1 µg/ml epidermal growth factor, 100 units/ml penicillin, and 100 µg/ml streptomycin) (40). CM-M was collected every 48-72 h, clarified by centrifugation, and stored at -20 °C until use. Proteins from CM were precipitated with ammonium sulfate (0-45% saturation) for 3 h at 4 °C, centrifuged at 9.5 × g for 20 min at 4 °C, and pellets resuspended in 0.5 ml of 1 M acetic acid. Samples were dialyzed 12-16 h against 1 M acetic acid in Spectra/Por Mr 3500 dialysis tubing (Spectrum Medical Industries, Inc., Houston, TX), dried by Speed Vac (Savant Instruments), and resolubilized in 1 × SDS Laemmli sample buffer containing (700 mM) beta -mercaptoethanol at 37 °C. Bacterial cell extracts and purified protein were mixed with 3 × SDS-sample buffer with or without 700 mM beta -mercaptoethanol or 100 mM dithiothreitol. Samples were boiled at 95 °C for 10 min, and resolved by 0.75-mm or 1.5-mm 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) following the procedures of Laemmli (41), as described previously (27).

Gels were transferred to 0.45-µm nitrocellulose membranes (Bio-Rad). Membranes were blocked in 5% (w/v) powdered milk (Carnation) in 10 mM Tris, 150 mM NaCl, pH 7.5 (1 × TBS) at 4 °C, overnight. Primary and secondary antibodies were diluted in 1 × TBS-T (1 × TBS containing 0.05% Tween 20) (Sigma) containing 1% milk and incubated with blots 1 h rocking at room temperature. Antibody concentrations were determined empirically and used at the following dilutions: ps20ab-1, 1:2000; ps20ab-1 preabsorbed against bacterial extract, 1:400; preimmune sera, 1:378; anti-His (COOH-terminal) antibody, 1:5000 (Invitrogen); and horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody (Amersham), 1:1000; goat anti-mouse secondary antibody, 1:1000 (Sigma); and ExtrAvidin-conjugated peroxidase, 1:1000 (Sigma). For preabsorbed negative control, 10 µl of ps20ab-1 was preincubated at 37 °C for 30 min with 800 µg of ps20 peptide-(1-14) and centrifuged at 16,000 × g to pellet precipitate. Following each incubation, blots were washed three times for 10 min in 1 × TBS-T. Blots were visualized with a chemiluminescent detection system (ECL, Amersham), and exposed to ECL hyperfilm (Amersham) for 1 s to 30 min, as necessary.

Immunohistochemistry-- Two-month-old and 6-month-old male Sprague-Dawley rats were sacrificed (intact and 2-week and 8-week castrates), and tissues removed and fixed in 10% neutral buffered formalin overnight. Fixed tissues were dehydrated in a graded series of ethanol washes, embedded in paraffin, cut into 5-µm-thick sections, applied to poly-L-lysine-coated slides, and baked at 37 °C prior to staining. Tissue sections were deparaffinized by immersion in Hemo D (Fisher) twice for 10 min each; rehydrated by 5-min graded washes in 100%, 95%, and 70% ethanol; permeabilized by immersion in 1 × PBS, 0.1% Triton X-100 for 5 min; and treated 5 min with 3% hydrogen peroxide (H2O2) to neutralize endogenous peroxidase activity. Conditions for primary antibody incubations were determined empirically. Affinity-purified ps20ab-1 was incubated with tissue sections at a 1:4 dilution overnight at room temperature in a humid chamber. Negative controls included affinity-purified ps20ab-1 preabsorbed with peptide, preimmune sera (1:18.9), and secondary antibody alone. Mouse monoclonal antibody against the smooth muscle-specific isoform of alpha -actin (clone asm-1, Boehringer Mannheim) was incubated at a 1:4 dilution for 1 h at room temperature. Immunoreactivity was visualized by a 45-min incubation with either biotinylated goat anti-rabbit or goat anti-mouse secondary antibodies (Sigma), diluted 1:15; followed by a 30-min incubation with ExtrAvidin-conjugated peroxidase (Sigma), diluted 1:15; and concluding with a 7-min incubation with diaminobenzadine and mounting on a glass slide with Gel Mount.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Cloning of ps20 cDNA-- The amino-terminal amino acid sequence of ps20 was previously determined and reported (27) (see "Experimental Procedures"). Degenerate ps20 primers, P34 (corresponding to amino acids 1-9) and P38 (corresponding to amino acids 19-27) (primer sequences given under "Experimental Procedures"), were designed from this amino acid sequence and used in a nested PCR strategy in combination with vector primers to amplify a fragment of ps20 cDNA from the PS-1_Bfslambda ZAP cDNA library, a cDNA library developed from the PS-1 adult rat prostate smooth muscle cell line (30). Degenerate ps20 primer P38 was used in a primary PCR amplification with vector primer T7 to amplify a product from PS-1 cDNA library phage lysate. This primary amplification reaction was used as template for a secondary PCR amplification with primer P38 and degenerate ps20 primer P34 to yield a single 81-bp reaction product (Fig. 1A). To confirm that the 81-bp fragment was derived from ps20 sequence, two additional ps20 degenerate primers, P36 (amino acids 1-7) and P37 (amino acids 7-13), were generated and used independently in combination with P38 to amplify expected 81-bp and 63-bp fragments, respectively, in analogous nested PCR reactions. The 81-bp fragment produced by P34 and P38 was cloned into the pCRII T-tailed vector (clone 3438pCRII), and sequenced (Fig. 1A). Clone 3438pCRII sequence (primer sequence omitted) was: 5'-GGTCAGGCTGGCTGAGAAGTCCCAAGCTGAAGAG-3'. The deduced amino acid sequence of clone 3438pCRII matched directly with native ps20 amino acid sequence (27). The clone 3438pCRII was therefore assumed to be a fragment of the DNA sequence encoding the amino terminus of mature ps20 protein.


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Fig. 1.   ps20 nested PCR cloning strategy. A nested PCR strategy was used to isolate ps20 PCR clones from the PS-1_Bfslambda ZAP cDNA library lysate. In this diagram, PCR primers (see "Experimental Procedures" for sequences) are shown as arrows, vector sequence as bold lines, and partial ps20 PCR clones as single lines. Two ps20 degenerate primers, P34 and P38, were generated based on ps20 amino acid sequence. A, for nested PCR, primers T3 and P38 were used in a primary PCR amplification, which was sequentially used as template for a secondary amplification with primers P34 and P38. The 81-bp fragment was cloned into vector pCRII (3438pCRII) and sequenced. The deduced amino acid sequence corresponded completely with purified ps20 amino acid sequence. From 3438pCRII sequence, ps20 primers P40 and P42 were designed. B, primers T3 and P40 were used to clone the ps20 5' end (184-bp clone T340pCRII) in a secondary nested reaction following initial amplification with primers T3 and P38. C, primers P42 and T7 were used to clone the ps20 3' end (868-bp clone 42T7pCRII) following a primary PCR reaction with P34 and T7. D, primers P45 and P46 were used to screen plates for the presence of ps20 clones prior to the isolation of full-length ps20 cDNA by plaque hybridization with 32P-labeled 42T7pCRII insert as probe.

From clone 3438pCRII sequence, non-degenerate ps20-specific primers P40 (amino acids 17-11) and P42 (amino acids 12-18) were developed and used in a similar nested PCR strategy in combination with vector primers T3 and T7 to clone the 5' and 3' ends of ps20 cDNA. Primers T3 and P38 were used in a primary PCR amplification of PS-1_Bfslambda ZAP library lysate. An aliquot of the primary amplification reaction was used as template for a secondary amplification with primers T3 and P40 to generate additional 5' sequence. A consistent 182-bp PCR reaction product was observed, cloned (T340pCRII), and sequenced (Fig. 1B). The 182-bp T340pCRII clone was identified as the 5' terminus of ps20 since the in-frame (based on the first ATG) derived amino acid sequence corresponded precisely with amino acids 1-17 of native ps20 sequence. The ps20 3' cDNA sequence was cloned by a similar nested PCR strategy using primers P34, P42, and T7. The final product (864 bp) obtained by PCR with primer P42 and primer T7 was cloned (42T7pCRII) and sequenced (Fig. 1C). Since the 3' nucleotide sequence terminated with a poly(A) sequence, the 5' nucleotide sequence overlapped with clone T340pCRII sequence, and the deduced amino acid sequence corresponded directly with native ps20 sequence amino acids 12-28, clone 42T7pCRII was determined to contain the 3' nucleotide sequence of full-length ps20 cDNA.

Using the ps20 clone 42T7pCRII as probe, multiple full-length ps20 cDNA clones were isolated from the PS-1_Bfslambda ZAP cDNA library by plaque hybridization. Pools of phage lysate were screened for the presence of ps20 clones by PCR and enriched for ps20 cDNA clones through three sequential rounds of screening. Three separate pools enriched for ps20 clones were screened by plaque hybridization with 32P-labeled 42T7 insert as a probe. Of six positive clones obtained, two were sequenced by multipass sequencing and found to be identical. The sequence of the full-length ps20 cDNA clone ps20-2bpBKCMV was identical to the sequences of PCR clones 3438pCRII, T340pCRII, and 42T7pCRII and terminated with a poly(A) sequence. The 5' ends of ps20-2bpBKCMV and of the 5' partial clone, T340pCRII, were identical in sequence and length, indicating ps20-2bpBKCMV represented full-length cDNA. Shown in Fig. 2 is the nucleotide and deduced amino acid sequence of the ps20 full-length cDNA clone ps20-2bpBKCMV.


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Fig. 2.   Nucleotide sequence and deduced amino acid sequence of rat ps20 full-length cDNA. The 1029-bp ps20 cDNA contains a start codon at nt 52-54, a stop codon at nt 688-690 (asterisk), and a polyadenylation signal at nt 1000-1002 (thick underline). This open reading frame predicts expression of a 212-amino acid protein with a predicted signal peptide cleavage site immediately following Gly26 (arrow). The deduced protein sequence is numbered beginning with the first amino acid of the signal peptide. Thr27-His54 (underlined) directly correspond in sequence with Thr1-His28 of native ps20. The sequence of peptide-(1-14) used to generate ps20ab-1 is underlined (dotted line). The WAP-type four-disulfide core domain is shaded, and the eight cysteines composing the domain are underlined. Potential protein modifications are indicated: casein kinase II phosphorylation sites at Ser40, Ser77, Ser137, Thr138, and Ser150 (dot), and an O-linked glycosylation site at Thr48 (dagger).

To confirm clone ps20-2bpBKCMV represented full-length ps20 message, total RNA was extracted from U4F mesenchymal cells and probed by Northern analysis with the ps20 clone 42T7pCRII insert, as shown in Fig. 3. A single transcript of approximately 1.1 kb was recognized in agreement with the 1029-bp size of ps20-2bpBKCMV (Fig. 3, lane 1). Identical results were obtained using clone T340 as probe (data not shown). To examine ps20 transcript expression in vivo, Northern analysis was performed on total RNA isolated from rat dorsal prostate and multiple other tissues (Fig. 3). A single 1.1-kb transcript identical in size to the transcript detected in U4F cells was recognized, indicating an identical size ps20 message was expressed in vivo. Of multiple tissues surveyed, the highest levels of ps20 message expression was observed in the heart. It was detectable in female reproductive tissues and, hence, is not a male-specific product. A minor, diffuse region of hybridization (2.0-3.0 kb) was also differentially observed, the significance of which is unknown.


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Fig. 3.   Northern analysis of ps20 mRNA expression in U4F cells and in adult rat tissues. Total RNA (15.0 µg/lane) isolated from the U4F cell line and from adult rat tissues was analyzed by Northern analysis with 32P-labeled clone 42T7pCRII insert (868 bp) as probe. A single mRNA species of 1.1 kb was recognized in both U4F cells and in several rat prostate tissues. The highest expression was observed in the heart with detectable message observed in dorsal-lateral prostate, vas deferens, testis, uterus, and ovary. The size of RNA markers is indicated at left. The gel was stained with ethidium bromide to visualize ribosomal RNA as a loading control prior to transfer (lower panel).

The full-length ps20 cDNA is 1029 bp long and contains a 636-bp predicted open reading frame initiating at the first ATG (nt 52-54) and terminating at the first TGA stop codon (nt 688-690) (see Fig. 2). The predicted translated product is a 212-amino acid protein containing an amino-terminal hydrophobic signal peptide with a perfect predicted signal peptidase cleavage site following Gly26, as determined by the rules of Von Heijne (42). The amino acid sequence of Thr27-His54 of the deduced protein corresponds directly with Thr1-His28 of native purified ps20 amino-terminal sequence, as reported previously (27), consistent with signal peptide cleavage following Gly26. The mature secreted protein is predicted to be 186 amino acids with a molecular mass of 20.7 kDa, in complete agreement with the 20-21-kDa size of native, biologically active ps20 purified from U4F conditioned medium under non-reducing conditions (27).

Sequence homology searches of all available data bases revealed no identity or significant similarity of ps20 cDNA nucleotide sequence with known sequences. Analysis of ps20 amino acid sequence revealed the presence of a single potential functional domain, a 44-amino acid WAP-type four-disulfide core domain, or WAP signature motif (28), named for whey acidic protein (WAP) (29). As noted in Fig. 2, the WAP signature domain consists of a conserved pattern of eight cysteine residues, which form four intradisulfide bonds (29) shown to be important for folding and function (43). The ps20 WAP signature motif shares a 27-40% sequence identity and 40-70% sequence similarity with other members of the WAP protein family, as shown in Fig. 4. Members of the WAP signature motif protein family are small, secreted proteins. The best characterized WAP family proteins, elafin (ELAF) (44), antileukoproteinase (ALK) (45), and chelonianin (IBP) (46), are secreted serine protease inhibitors, in which the protease inhibitor activity has been mapped to the WAP domain. Several other WAP family members have specific functions that have not yet been determined, including: mammary gland whey acidic protein (WAP) (29), WDNM1 (47), HE4 (48), and the Kallmann syndrome protein (49), although all have been hypothesized to act as protease inhibitors.


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Fig. 4.   Amino acid sequence comparison of the WAP-type four-disulfide core domains of ps20 and other family members. The ps20 WAP-type four-disulfide core domain amino acid sequence was aligned with the WAP domains of other family members. Conserved residues are in reversed contrast, and conservative substitutions are shaded. Within the entire WAP-type four-disulfide core domain, ps20 is 39.4% identical to chelonianin (IBP), 35.4% identical to antileukoproteinase 1 (ALK1), 35.3% identical to whey acidic protein (WAP), 33.3% identical to WDNM1, 33.3% identical to HE4, 31.2% identical to Kallmann syndrome protein (KALM), 29.2% identical to elafin (ELAF), and 27.1% identical to caltrin-like protein II (CALU). The multiple sequence alignment was assembled using the PILEUP program (Genetics Computing Group, Madison, WI), spaces introduced to optimize alignment, and presented using BOXSHADE (ISREC). Alignment scores were calculated by pairwise alignments using ALIGN (EERIE) with default settings.

Analysis of Endogenous ps20 Secreted by U4F and PS-1 Cells-- Prior to expression of recombinant ps20 protein, CM from U4F cells (the cell line from which ps20 was identified) (25-27) was analyzed by Western analysis using ps20ab-1. CM from U4F cells was collected, partially purified (as described under "Experimental Procedures"), and analyzed by Western analysis under reducing conditions with ps20ab-1. Two specific bands of apparent molecular mass 21 and 27 kDa were specifically recognized by ps20ab-1 (Fig. 5A, lane 1), relative to preabsorbed control (Fig. 5A, lane 2).


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Fig. 5.   Analysis of native and recombinant ps20 protein by SDS-PAGE. A, concentrated U4F CM was analyzed by Western analysis under reducing conditions and probed with ps20ab-1 (lane 1) and ps20ab-1 preabsorbed with ps20 peptide-(1-14) (lane 2). ps20 was detected as both 21- and 27-kDa bands. B, ps20 was immunoprecipitated from PS-1 CM with ps20ab-1 (lane 1) or preimmune sera (lane 2) or from U4F CM with ps20ab-1 (lane 3) or preimmune sera (lane 4) and resolved by 12% SDS-PAGE gels under reducing conditions. Identical samples were resolved under non-reducing conditions: lane 5, PS-1 CM immunoprecipitated with ps20ab-1; lane 6, PS-1 CM immunoprecipitated with preimmune sera; lane 7, U4F CM immunoprecipitated with ps20ab-1; lane 8, U4F immunopreciptated with preimmune sera. From both PS-1 and U4F cell CM, 27- and 21-kDa bands are specifically detected under reducing conditions when immunoprecipitated with ps20ab-1 (lanes 1 and 3) and a 21-kDa protein detected under non-reducing conditions (lanes 5 and 7). C, ps20 was expressed as a His-tagged fusion protein, rps20-His, in the BL21(DE3)pLysS bacterial cell line. Crude extracts of cells prior to induction (lane 1) and following induction (lane 2) were resolved by reducing SDS-PAGE and visualized by Coomassie Blue stain. Western analysis with ps20ab-1 (lane 3) demonstrated an intact amino terminus, and Western analysis with anti-His (COOH-terminal) antibody (lane 4) confirmed the protein was full-length. Soluble protein extracts were purified by nickel chromatography, resolved by SDS-PAGE analysis under reducing (lane 5) and non-reducing (lane 6) conditions, and visualized by silver staining. The 29-kDa rps20-His protein observed under reducing conditions (lane 5) migrated with an apparent mass of approximately 23 kDa under non-reducing conditions (lane 6). The apparent migration rate (molecular mass) of protein markers (in kDa) is indicated at left.

Because detection of ps20 by Western analysis required partial purification of CM, U4F, and PS-1 cells were 35S-labeled, and CM analyzed by immunoprecipitation with ps20ab-1 to confirm the ps20 forms in crude CM. Immunoprecipitation of native ps20 from PS-1 cell CM detected 21- and 27-kDa bands under reducing conditions (Fig. 5B, lane 1). When analyzed in the absence of reducing agents, only the 20-22-kDa band was detected (Fig. 5B, lane 5). The apparent change in native ps20 migration rate under reducing and non-reducing conditions was also observed with ps20 immunoprecipitated from U4F cell CM (Fig. 5B, lanes 3 and 7, respectively). Immunoprecipitations using preimmune sera confirmed specificity of interaction with both the 21-kDa and 27-kDa bands (Fig. 5B, lanes 2, 4, 6, and 8). Together, these data suggest ps20 is expressed and secreted as a protein resolvable by SDS-PAGE analysis as 20-22 kDa under non-reducing conditions and as 27 kDa under reducing conditions. These data are consistent with previous studies in which ps20 was purified and reported as a protein of 20-22 kDa apparent molecular mass under non-reducing conditions (27).

Expression of Recombinant ps20 Protein-- To further clarify the molecular forms of ps20, the ps20 open reading frame without signal peptide was subcloned into a bacterial expression vector and expressed as a carboxyl-terminal histidine (His)-tagged fusion protein (rps20-His) in bacteria. A protein of 29 kDa was observed by SDS-PAGE under reducing conditions in induced cells (Fig. 5C, lane 2). Western analysis with ps20ab-1 (ps20, amino-terminal) and anti-His antibody (His tag, carboxyl-terminal), demonstrated specific immunoreactivity of both antibodies with the 29-kDa protein (Fig. 5C, lanes 3 and 4, respectively), confirming intact amino and carboxyl termini. rps20-His protein was purified by nickel affinity chromatography, resolved by 15% SDS-PAGE under both reducing and non-reducing conditions, and visualized by silver staining. Consistent with immunoprecipitation of endogenous protein (Fig. 5A), rps20-His migrated at an apparent molecular mass of 29 kDa under reducing conditions (Fig. 5C, lane 5) and at 22-24 kDa (Fig. 5C, lane 6) under non-reducing conditions. These data confirm the apparent molecular mass of ps20 resolved by SDS-PAGE is sensitive to reducing agents. In addition, these data suggest that the bacterial and mammalian expressed forms of ps20 are equivalent in their structural properties as resolved by SDS-PAGE. Given the presence of the WAP four-disulfide core domain, it is not surprising that ps20 in its folded, non-reduced (21-23 kDa) form exhibits a more rapid migration in SDS-PAGE as compared with the reduced (27-29 kDa) form.

To demonstrate the expression of the full-length ps20 cDNA clone in eukaryotic cells and secretion of the recombinant protein, COS-7 cells were transfected with ps20 cDNA or vector control. Both transiently transfected cultures and stable transfectant clonal lines were established. CM from both transiently and stably transfected COS-7 cells was collected, processed, resolved by reducing SDS-PAGE, and analyzed by Western analysis with ps20ab-1. As expected under reducing conditions, the 27-kDa protein was detected in the CM of COS-7 cells transfected with ps20 cDNA (Fig. 5D, lane 2) but not in CM of mock-transfected COS-7 cells (Fig. 5D, lane 1). All ps20 stable transfectant line CM was screened for ps20 secretion with identical results (data not shown). These data confirm that COS-7 cells transfected with ps20 cDNA express and secrete an immunoreactive protein of molecular mass consistent with endogenous ps20 secreted by U4F and PS-1 cells (Fig. 5, A and B).

Biological Activity of Recombinant ps20 Protein-- As reported previously for ps20/UGIF-treated cells (25-27), growth inhibition of the ps20 transfectant cell lines was measured using [3H]thymidine incorporation assays. Four ps20 stable transfectant COS-7 cell lines (rps20-1, rps20-3, rps20-4, and rps20-6pBKCMV COS-7), one mock-transfected cell line (pBKCMV-4 COS-7), and the parent cell line (COS-7) were seeded at identical densities, and the rate of [3H]thymidine incorporation by each cell line measured daily over a 4-day time course (Fig. 6A). The rate of [3H]thymidine incorporation by the ps20 transfectant lines was significantly reduced (p < 0.01) relative to the mock transfectant line and the COS-7 parent line at each time point assayed. Consistent with previous data indicating that ps20-induced inhibition of [3H]thymidine incorporation correlated directly with cell number (27), direct cell counting of these cultures indicated 14-45% fewer cells in cultures of the ps20 transfectant cell lines relative to the mock transfectant line when counted 24-96 h after seeding (data not shown). To demonstrate that the growth inhibition of the ps20 transfectant COS-7 cell lines was due to an activity secreted into the growth medium, mock transfectant cells were incubated with CM from ps20 transfectant or mock cell lines and assayed for [3H]thymidine incorporation. Incorporation of [3H]thymidine in cultures incubated with CM from ps20 transfectant cell lines was reduced (p < 0.01) relative to cultures incubated with CM from mock transfectant cells (Fig. 6B).


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Fig. 6.   Inhibition of [3H]thymidine uptake by ps20 stable transfectant COS-7 cell lines. A, the stable transfectant COS-7 cell lines rps20-1, rps20-3, rps20-4, and rps20-6pBKCMV COS-7; the mock stable transfectant cell line pBKCMV-4; and COS-7 cells were seeded in 96-well plates at identical densities and assayed for [3H]thymidine incorporation. Incorporated [3H]thymidine (cpm) was plotted at 24-h intervals. B, pBKCMV-4 cells were seeded in 96 well plates, incubated with increasing amounts of CM from ps20 transfectant cell lines and the mock transfectant cell line, and assayed for [3H]thymidine incorporation. Each experiment was repeated three times. At all time points, samples were assayed in triplicate ± S.E. (error bars) and significance determined by Student's t test (p < 0.01 for all samples at all time points shown). C, COS-7, pBKCMV-4, and rps20-4 were exposed to crystal violet stain/fixative and photographed.

In addition to an inhibition of [3H]thymidine incorporation, the morphology of ps20 transfectant COS-7 cell lines was altered as compared with mock or parental lines. Whereas COS-7 cells are polygon-shaped, closely packed cells (Fig. 6C), ps20 stable transfectant COS-7 cells exhibited a morphology similar to ps20-treated PC-3 cells in that they were larger and more spread out (27), and similar to ps20-treated NBT-II cells (25) in that they exhibited an extensive array of filopodia and lamellipodia. Shown in Fig. 6C is the ps20 transfectant cell line (rps20-4); the other ps20 stable transfectant cell lines exhibited an identical morphology (data not shown). The mock transfectant cell line (pBKCMV-4) exhibited a morphology similar to COS-7 cells. Together, these data indicate that ps20 transfectant COS-7 cells exhibit properties consistent with cell lines treated with either crude or purified native rat ps20 reported in previous studies (25-27).

To confirm that inhibition of ps20 transfectant COS-7 cell lines was due to soluble ps20 protein, purified bacterially expressed ps20 protein was assayed for activity. Analysis of the biological properties of rps20-His was impeded by a propensity of the purified fusion protein to aggregate and remain insoluble. In an attempt to generate soluble recombinant protein in a bacterial expression system, ps20 was expressed as a GST fusion protein (32). GST and GST-rps20 were purified by glutathione-Sepharose chromatography and analyzed by Western analysis (Fig. 7A). As expected, GST-rps20 (lane 2) was immunoreactive with ps20ab-1 and expressed as fusion protein of expected mass 50 kDa. Because prostate carcinoma PC-3 cells are a target cell type previously shown to be sensitive to native ps20 activity (27), PC-3 cells and COS-7 cells were assayed for GST-rps20-induced inhibition of [3H]thymidine incorporation. GST-rsp20 induced only a minor inhibition of [3H]thymidine incorporation in both COS-7 (15% inhibition) and PC-3 cells (9.6% inhibition) relative to GST as shown in Fig. 7B. To determine if rps20 activity could be enhanced by removal of the GST moiety, GST-rps20 was cleaved with thrombin and released rps20 was analyzed by Western under reducing conditions. Following cleavage with thrombin, rps20 was detectable as two immunoreactive protein forms of the predicted 23-kDa and 29-kDa sizes (Fig. 7A, lane 3). The rps20 exerted a 61% inhibition in COS-7 cells and a 47% inhibition in PC-3 cells (Fig. 7B) relative to thrombin-cleaved GST. These data support results shown in Fig. 6 with transfectant COS-7 cell lines and indicate that COS-7 cells are directly inhibited by ps20. In addition, these data show that bacterially expressed ps20 inhibits PC-3 prostatic carcinoma cells, a target cell type previously reported to be growth-inhibited by purified native rat ps20 (27).


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Fig. 7.   Inhibition of COS-7 and PC-3 cell [3H]thymidine uptake by bacterial produced recombinant ps20. A, GST (lane 1), GST-rps20 (lane 2), and rps20 (lane 3) were resolved by reducing SDS-PAGE and analyzed by Western analysis with ps20ab-1. GST-rps20 was expressed as a 50-kDa immunoreactive protein and rps20 as two migratory forms of 23 and 29 kDa. Molecular masses of standard protein markers are shown at left. B, COS-7 cells were seeded at 2.5 × 104 cells/ml in 96 well plates and incubated with: thrombin-treated GST (1, labeled GST), GST-rps20 (2), or thrombin-cleaved GST-rps20 (3, labeled rps20) for 24 h. PC-3 cells were seeded at 5 × 104 cells/ml and incubated with GST (4), GST-rps20 (5), or rps20 (6) for 24 h and assayed for [3H]thymidine incorporation (counts/min). GST-rps20 inhibited PC-3 and COS-7 cells by 9.6% and 15%, and rps20 inhibited by 47% and 61%, respectively. The 70-kDa mildly cross-reactive band is apparently a bacterial protein previously reported to co-purify with GST fusion proteins (56). Each experiment was repeated three times. At all time points, samples were assayed in triplicate ± S.E. (error bars).

Localization of ps20 Protein in Vivo-- Immunohistochemical analysis with affinity-purified ps20ab-1 and adult rat tissues was performed to assess ps20 protein expression patterns in specific cell types. In adult rat prostate, specific immunoreactivity was apparent only in the periacinar smooth muscle cells of the stroma (Fig. 8c), as determined by comparison of a serial section probed with a monoclonal antibody recognizing the smooth muscle-specific isoform of alpha -actin (SM alpha -actin), a smooth muscle cell marker (Fig. 8d). The smooth muscle staining pattern was eliminated by preabsorption of ps20ab-1 with ps20 peptide-(1-14) (Fig. 8b). No immunoreactivity was observed in the absence of primary antibody, and only nonspecific background reactivity was observed with preimmune sera (data not shown). No immunoreactivity was observed in the stromal fibroblast population. A weak immunoreactivity, noted in some epithelial cells, was eliminated by preabsorption with ps20 peptide. ps20 was detected in smooth muscle of both normal and castrate animals, with an apparent moderate increase of immunoreactivity noted in the smooth muscle of castrate animals (data not shown).


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Fig. 8.   ps20 immunolocalization in rat prostate smooth muscle and vascular smooth muscle. Affinity-purified ps20ab-1 was used to immunolocalize ps20 protein expression in adult rat prostate tissue: a, hematoxylin and eosin counterstain; b, affinity-purified ps20ab-1 preabsorbed with ps20 peptide-(1-14) (1:4); c, affinity-purified ps20ab-1 (1:4); and d, SM alpha -actin (1:4) (magnification, ×520). Immunoreactivity with ps20ab-1 (c) corresponded with SM alpha -actin immunolocalization in smooth muscle (d) adjacent to acinar epithelial cells. Affinity-purified ps20ab-1 immunolocalized ps20 protein expression in vascular smooth muscle: e, hematoxylin and eosin counterstain of a typical artery (labeled A) and a typical vein (labeled V) embedded in parathyroid tissue; f, affinity-purified ps20ab-1 (1:4) in parathyroid tissue (magnification, ×235); g, hematoxylin and eosin counterstain of aorta; and h, affinity-purified ps20ab-1 (1:4) in aorta (magnification, ×470). ps20ab-1 shows specific immunoreactivity in the tunica media of arteries and veins and in the tunica media (labeled TM) of aorta and not in the other tissue layers, including the tunica adventitia (TA) and tunica intima (TI) composed of fibroblasts and endothelial cells, respectively).

Differential immunoreactivity was observed in the visceral smooth muscle of all tissues analyzed that contain smooth muscle including: prostate, anterior prostate, seminal vesicle, vas deferens, abdominal esophagus, stomach, colon, bladder, and skin. A consistent immunoreactivity was detected in vascular smooth muscle, particularly in the large elastic arteries (aorta) and in all muscular arteries. Fig. 8h shows specific ps20 staining in the tunica media (smooth muscle) of the aorta. Fig. 8f shows characteristic specific ps20 staining of the tunica media of both an artery and a vein. Excluding detection in vascular smooth muscle, no specific immunoreactivity with ps20 antibody was seen in tissues that do not contain visceral smooth muscle, which include: kidney, liver, testis, lung, brain, heart, and salivary gland. No cross-reactivity was observed in other cell types including: skeletal muscle, cardiac muscle, neurons, glial cells, endothelial cells, or fibroblasts. Immunoreactivity with ps20 antibody did not always correlate with expression of SM alpha -actin; myoepithelial cells, found in several tissues including the salivary gland, were not immunoreactive with ps20ab-1, but were reactive with SM alpha -actin (data not shown).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We report here the cloning of full-length cDNA encoding ps20, expression of biologically active recombinant ps20 protein, and characterization of ps20 mRNA and protein expression patterns in vivo. The cloning of ps20 from the PS-1 (prostate smooth muscle-like cell line) cDNA library and immunolocalization of ps20 expression in smooth muscle in vivo is consistent with the previous characterization of ps20 secretion by prostatic mesenchymal and smooth muscle cell lines in culture (27). We have demonstrated here that both bacterial and mammalian expressed recombinant ps20 protein exhibit growth-inhibitory effects on mammalian cells in culture, consistent with properties reported for UGIF/native ps20 protein (25-27). The ps20 amino acid sequence contains a whey acidic protein (WAP)-type four-disulfide core motif (29), which places ps20 in this family of small secreted proteins.

Biological activity of ps20 is consistent with the biological activity of other WAP family proteins, the majority of which have protease inhibitor activity. Protease inhibitors have been reported previously to have growth-inhibitory effects both in vitro and in vivo. Pefabloc, a synthetic serine protease inhibitor, inhibited prostatic PC-3 carcinoma cell proliferation through inhibition of an IGFBP-3 protease that regulated availability of active insulin-like growth factor-II (12). Other serine protease inhibitors, including maspin (50), plasminogen activator inhibitors (51), and tissue inhibitors of matrix metalloproteinases (52), have exhibited growth-inhibitory activities. The WAP family member elafin inhibited abnormal vascular smooth muscle proliferation in an in vivo rabbit arteriopathy model of coronary neoinitimization (12). In vitro, the mechanism for elafin-induced inhibition of pulmonary artery smooth muscle cell proliferation involves inhibition of elastase-mediated proteolytic release of mitogenic biologically active FGF-2 from extracellular matrix (12).

Members of the WAP signature motif family exhibit fundamental roles in growth control, differentiation, and tissue remodeling in development and maintenance of homeostasis in adult tissue. The Kallmann syndrome protein is a peripheral membrane protein, which is proteolytically released in soluble form and may be a neuronal axon chemoattractant (53). Mutations or deletions in the Kallmann syndrome protein lead to defective embryonic migration and differentiation of olfactory and gonadotrophin-releasing hormone-synthesizing neurons in development, resulting in the hypogonadotropic hypogonadism and anosmia of Kallmann syndrome (49). Expression of elafin is down-regulated in poorly differentiated squamous cell carcinoma (48) and in mammary carcinoma cells (54), and its loss of expression has been postulated to facilitate tumor progression in these tissues. WDNM1 was identified in a subtractive hybridization screen to identify potential tumor suppressor genes in mammary cancer (47, 55) as a transcript down-regulated in metastatic breast cancer cell lines. Although the significance of ps20 expression in vivo is not yet known, alterations in the expression of other WAP family members clearly influence growth control in vivo.

We initially identified ps20 protein as the major protein component of the mesenchymally produced UGIF activity, which has a growth-inhibitory effect on epithelial cell lines in vitro (25-27). We now report that ps20 localizes to the periacinar ring of smooth muscle in immediate contact with the secretory epithelial acini in the prostate gland. Whether or not the weak immunoreactivity observed in epithelial cells represents a low level of expression by these cells or stromally produced ps20 incorporated by these cells is not yet clear. The in vivo localization of ps20 in smooth muscle supports a putative role for ps20 in stromal-epithelial interactions in the prostate gland.

The significance of the apparent differential expression of ps20 by different types of smooth muscle cells is unknown, but is evident at both the mRNA and protein levels. Expression of ps20 message and protein was relatively low in many organs containing visceral smooth muscle, including the stomach and small and large intestines, which have thick walls of smooth muscle layers. Of interest, the staining intensity of ps20 in vascular smooth muscle, particularly in both larger elastic arteries and small muscular arteries, was consistently high and similar to or higher than in prostate smooth muscle. The elevated mRNA expression observed in heart tissue by Northern analysis is not likely due to expression by cardiac muscle cells, which were not immunoreactive, but is likely due to high expression by coronary artery and large elastic artery smooth muscle cells, which were highly immunoreactive with ps20ab-1. Vascular diseases are typified by medial hypertrophy and neointimal proliferation. The localization of ps20 in vascular smooth muscle suggests ps20 may play an autocrine role in regulation of smooth muscle proliferation and maintenance of homeostasis in the vasculature.

The specific mechanisms of ps20 biological action and functional role in vivo are not yet known. ps20 could potentially inhibit cell proliferation through alterations of growth factor bioavailability, extracellular matrix remodeling, or alterations of cell adhesion. Based on our data to date, no specific ps20 mechanism can be ruled in or ruled out. Definitive characterization of ps20 mechanisms of biological action awaits a more complete functional characterization of activity in vitro and in vivo.

    ACKNOWLEDGEMENTS

We thank Dr. Jeff Rosen for helpful discussions and Liz Hopkins for excellent technical assistance in preparation of tissue sections for immunohistochemical analysis.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA58093, DK45909, and SPORE CA58204 and by a grant from Sheffield Medical Technologies.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF037272.

To whom requests for reprints should be addressed: Dept. of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-6220; Fax: 713-790-1275; E-mail: drowley{at}bcm.tmc.edu.

1 The abbreviations used are: UGIF, urogenital sinus mesenchymal secreted growth-inhibitory factor; kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; CM, conditioned medium; TBS, Tris-buffered saline; ELISA, enzyme-linked immunosorbent assay; DMEM, Dulbecco's modified Eagle's medium; SM, smooth muscle; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

  1. Smith, B. T., and Sabry, K. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1951-1954[Abstract]
  2. Smith, B. T. (1979) Science 208, 1094-1095
  3. Lacroix, B., Kedinger, M., Simon-Assmann, P. M., Haffen, K. (1984) Differentiation 28, 129-135[Medline] [Order article via Infotrieve]
  4. Dow, K. E., Sabry, K., and Smith, B. T. (1983) Cell Tissue Res. 231, 83-91[Medline] [Order article via Infotrieve]
  5. Sawyer, R. H., O'Guin, W. M., and Knapp, L. W. (1984) Dev. Biol. 101, 8-18[Medline] [Order article via Infotrieve]
  6. Haslam, S. Z., and Counterman, L. J. (1992) Endocrinology 129, 2017-2023[Abstract]
  7. Cunha, G. R. (1972) Anat. Rec. 172, 179-196[Medline] [Order article via Infotrieve]
  8. Cunha, G. R., Reese, B. A., and Sekkingstad, M. (1980) Endocrinology 107, 1767-1770[Abstract]
  9. Cunha, G. R., Fujii, H., Neubauer, B. L., Shannon, J. M., Sawyer, L., Reese, B. A. (1983) J. Cell Biol. 96, 1662-1670[Abstract]
  10. Neubauer, B. L., Chung, L. W. K., McCormick, K. A., Taguchi, O., Thompson, T. C., Cunha, G. R. (1983) J. Cell Biol. 96, 1671-1676[Abstract]
  11. Saksela, O., and Rifkin, D. B. (1990) J. Cell Biol. 110, 767-775[Abstract]
  12. Thompson, K., and Rabinovitch, M. (1996) J. Cell. Physiol. 166, 495-505[CrossRef][Medline] [Order article via Infotrieve]
  13. Taipale, J., Koli, K., and Keski-Oja, J. (1992) J. Biol. Chem. 267, 25378-25384[Abstract/Free Full Text]
  14. Bringman, T. S., Lindquist, P. B., and Derynck, R. (1987) Cell 48, 429-440[Medline] [Order article via Infotrieve]
  15. Angelloz-Nicoud, P., and Binoux, M. (1995) Endocrinology 136, 5485-5492[Abstract]
  16. Frey, P., Forand, R., Maciag, T., and Shooter, E. M. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 6294-6298[Abstract]
  17. Jahnke, G. D., Chao, J., Walker, M. P., Diaugustine, R. P. (1994) Endocrinology 135, 2022-2029[Abstract]
  18. Goishi, K., Higashiyama, S., Klagsbrun, M., Nakano, N., Umata, T., Ishikawa, M., Mekada, E., and Taniguchi, N. (1995) Mol. Biol. Cell 6, 967-980[Abstract]
  19. Kriegler, M., Perez, C., DeFay, K., Albert, I., and Lu, S. D. (1988) Cell 53, 45-53[Medline] [Order article via Infotrieve]
  20. Taipale, J., and Keski-Oja, J. (1997) FASEB J. 11, 51-59[Abstract/Free Full Text]
  21. Liotta, L. A., Steeg, P. S., and Stetler-Stevenson, W. G. (1991) Cell 64, 327-336[Medline] [Order article via Infotrieve]
  22. Sympson, C. J., Talhouk, R. S., Alexander, C. M., Chin, J. R., Clift, S. M., Bissell, M. J., Werb, Z. (1994) J. Cell Biol. 125, 681-693[Abstract]
  23. Koolwijk, P., van Erck, M. G. M., de Vree, W. J. A., Vermeer, M. A., Weich, H. A., Hanemaaijer, R., van Hinsbergh, V. W. M. (1996) J. Cell Biol. 132, 1177-1188[Abstract]
  24. Wilson, M. J. (1995) Microsc. Res. Tech. 30, 305-318[Medline] [Order article via Infotrieve]
  25. Rowley, D. R., and Tindall, D. J. (1987) Cancer Res. 47, 2955-2960[Abstract]
  26. Rowley, D. R. (1992) In Vitro Cell. Dev. Biol. 28, 29-38
  27. Rowley, D. R., Dang, T. D., Larsen, M., Gerdes, M. J., McBride, L., Lu, B. (1995) J. Biol. Chem. 270, 22058-22065[Abstract/Free Full Text]
  28. Bairoch, A. (1991) Nucleic Acids Res. 19, 2241-2245[Medline] [Order article via Infotrieve]
  29. Hennighausen, L. G., and Sippel, A. E. (1982) Nucleic Acids Res. 10, 2677-2684[Abstract]
  30. Gerdes, M. J., Dang, T. D., Lu, B., Larsen, M., McBride, L., and Rowley, D. R. (1996) Endocrinology 137, 864-872[Abstract]
  31. Knoth, K., Roberds, S., Poteet, C., and Tamkum, M. (1988) Nucleic Acids Res. 16, 109-132
  32. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. (1996) Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York
  33. Kyprianou, N., and Isaacs, J. T. (1988) Endocrinology 123, 2124-2131[Abstract]
  34. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410[CrossRef][Medline] [Order article via Infotrieve]
  35. Geourjon, C., and Deleage, G. (1993) CABIOS 9, 87-91 [Abstract]
  36. Pongor, S., Hatsagi, Z., Degtyarenko, K., Fabian, P., Skerl, V., Hegyi, H., Murvai, J., and Bevilacqua, V. (1994) Nucleic Acids Res. 22, 3610-3615[Abstract]
  37. Kuroki, J., Hosoya, T., Itakura, M., Hirose, S., Tamechika, I., Yoshimoto, T., Ghoneim, M. A., Nara, K., Kato, A., Suzuki, Y., Furukawa, M., Tachibana, S. (1995) J. Biol. Chem. 270, 22428-22433[Abstract/Free Full Text]
  38. Post, M., and Smith, B. T. (1984) Biochim. Biophys. Acta 793, 297-299[Medline] [Order article via Infotrieve]
  39. Okada, A., Belloq, J., Chenard, M., Rio, M., Chambon, P., and Basset, P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2730-2734[Abstract]
  40. Gerdes, M. J., Larsen, M., McBride, L., Dang, T. D., Lu, B., and Rowley, D. R. (1998) J. Histochem. Cytochem., in press
  41. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  42. von Heijne, G. (1984) J. Mol. Biol. 173, 243-251[Medline] [Order article via Infotrieve]
  43. Tsunemi, M., Matsuura, Y., Sakakibara, S., and Katsube, Y. (1996) Biochemistry 35, 11570-11576[CrossRef][Medline] [Order article via Infotrieve]
  44. Wiedow, O., Schroder, J.-M., Gregory, H., Young, J. A., Christophers, E. (1990) J. Biol. Chem. 265, 14791-14795[Abstract/Free Full Text]
  45. Heinzel, R., Appelhans, H., Gassen, G., Seemuller, U., Machleidt, W., Fritz, H., and Steffens, G. (1986) Eur. J. Biochem. 160, 61-67[Abstract]
  46. Kato, I., and Tominaga, N. (1979) Fed. Proc. 38, 832
  47. Dear, T. N., Ramshaw, I. A., and Kefford, R. F. (1988) Cancer Res. 48, 5203-5209[Abstract]
  48. Kirchhoff, C., Habben, I., Ivell, R., and Krull, N. (1991) Biol. Reprod. 45, 350-357[Abstract]
  49. Legouis, R., Hardelin, J.-P., Levilliers, J., Claverle, J.-M., Compain, S., Wunderle, V., Millasseau, P., Le Paslier, D., Cohen, D., Caterina, D., Bougueleret, L., Delemarre-Van de Waal, H., Lutfalla, G., Weissenbach, J., Petit, C. (1991) Cell 67, 423-435[Medline] [Order article via Infotrieve]
  50. Sheng, S., Carey, J., Seftor, E. A., Dias, L., Hendrix, M. J. C., Sager, R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11669-11674[Abstract/Free Full Text]
  51. Thorsen, S., and Philips, M. (1987) in Fundamental and Clinical Fibrinolysis (Samama, M. M., and Takata, A., eds), pp. 83-98, Elservier, Amsterdam
  52. Alexander, C. M., and Werb, Z. (1992) J. Cell Biol. 118, 727-739[Abstract]
  53. Rugarli, E. I., Ghezzi, C., Valsecchi, V., and Ballabio, A. (1996) Hum. Mol. Genet. 5, 1109-1115[Abstract/Free Full Text]
  54. Zhang, M., Zou, Z., Maass, N., and Sager, R. (1995) Cancer Res. 55, 2537-2541[Abstract]
  55. Dear, T. N., and Kefford, R. F. (1991) Biochem. Biophys. Res. Commun. 176, 247-254[Medline] [Order article via Infotrieve]
  56. Majmudar, G., Nelson, B. R., Jensen, T. C., Johnson, T. M. (1994) Mol. Carcinog. 11, 29-33[Medline] [Order article via Infotrieve]


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