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[Cancer Research 61, 2390-2394, March 15, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

Activated in Prostate Cancer

A PDZ Domain-containing Protein Highly Expressed in Human Primary Prostate Tumors1

Hassan Chaib, Mark A. Rubin, Neil R. Mucci, Lang Li, Jeremy M. G. Taylor, Mark L. Day, Johng S. Rhim and Jill A. Macoska2

The Departments of Surgery, Section of Urology [H. C., M. A. R., M. L. D., J. A. M.], Pathology [M. A. R., N. R. M.], and Biostatistics [L. L., J. M. G. T.], and the Comprehensive Cancer Center [M. A. R., J. M. G. T., M. L. D., J. A. M.], The University of Michigan, Ann Arbor, Michigan 48109-0946, and The Center for Prostate Disease Research, The Uniformed Services University of the Health Sciences, Rockville, Maryland 20852 [J. S. R.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 
Critical events in prostate tumorigenesis and metastasis likely include the abnormal activation and expression of specific genes. Using RNA expression profiling techniques, we have identified a transcript originating from the activated in prostate cancer (AIPC) gene, the expression of which is preferentially up-regulated in several cultured prostate tumor cell lines and human primary prostate tumors. Sequence analysis revealed that the AIPC protein encodes six PDZ domains, which are protein-protein binding domains likely involved in protein clustering and scaffolding. Immunohistochemical analysis of a tissue microarray comprising 158 tumor, 18 high-grade prostatic intraepithelial neoplasia, and 91 normal prostate specimens with an anti-AIPC antibody demonstrated abundant AIPC protein expression in 75% of tumors, 83% of prostatic intraepithelial neoplasia lesions, and 3% of normal tissues (P < 0.0001). These data suggest that the accumulation of AIPC protein may be closely associated with the initiation or early promotion of prostate tumorigenesis.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
The activation of proto-oncogenes and inactivation of tumor suppressor genes are critical events in tumorigenesis and metastasis. However, the identification of such genes has been hampered in the analysis of prostate tumors by factors such as the histological heterogeneity of the disease and the failure of conventional genetic analysis techniques to identify amplified or overexpressed genes. To overcome some of these limiting factors, we used an RNA profiling technique, differential display, to identify genes the transcription of which is either induced or repressed in primary prostate tumor-derived cell lines and primary prostate tumor tissues (1 , 2) . This strategy successfully identified 25 differentially expressed genes. Twenty of the 25 genes demonstrated transcriptional up-regulation in the tumor-derived cell lines, suggesting that the inappropriate transcriptional activation of several genes may contribute to prostate tumorigenesis. One of the 20 genes, AIPC,3 is specifically up-regulated at the RNA and protein levels in human prostate tumors and tumor-derived cell lines. Moreover, the protein structure of AIPC includes six PDZ motifs, which are protein-protein binding domains likely involved in protein clustering and scaffolding. These studies suggest that the inappropriate up-regulation of novel proteins with the ability to recruit, bind, and coordinate the activities of other proteins may contribute to tumorigenesis in the human prostate.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
Cell Lines and Culture
The 1535N, 1532T, 1535T, and 1542T cell lines were produced through immortalization of normal (N) or malignant (T) prostate cells by transduction with a recombinant retrovirus encoding the E6 and E7 genes of human papillomavirus type 16, as described previously (3) . The 267B1 cells were produced through transfection of neonatal prostate cells with the pRSV-T plasmid containing SV40 early region genes (4) . 1535N and 267B1 cells are genotypically and phenotypically normal (4, 5, 6) , whereas 1532T, 1535T, and 1542T possess karyotypic alterations consistent with prostate cancer and express the malignant phenotype (5 , 6) . All cell lines were grown in defined keratinocyte-SFM (Life Technologies, Inc.), 5% FBS, and 1% penicillin/streptomycin/fungizone antibiotic mixture (BioWhittaker) in a humidified incubator at 37°C with 5% CO2.

RNA Profiling and Transcript Characterization
RNA was prepared from cell lines using TRIzol reagent (Life Technologies, Inc., Rockville, MD) according to the manufacturer’s directions. Differential display reactions were modified from the original description of this technique to allow for visualization by agarose gel electrophoresis (2) . A total of 14 oligonucleotide primer sets (A-H, M-P, R, and S), each comprising 20 10-mer oligonucleotide primers, were used in the RT-PCR reaction assays (Operon Technologies, Inc., Alameda, CA). All reactions were performed in 96-well microtiter plates, which facilitated the ability to perform a large number of reactions at one time. Transcripts were categorized as overexpressed if they appeared in reactions using RNA from tumor but not normal cells and as underexpressed if they appeared in reactions using RNA from normal but not tumor cells. Differentially expressed transcripts were subcloned into pGEM-T Easy vectors (Promega Corp., Madison, WI) and sequenced. Database searches using the BLAST algorithm were then conducted to identify sequence homologies at the RNA and protein levels (7 , 8) . Transcript expression patterns detected through differential display were verified by Northern blot analysis with poly(A)+ RNA purified using the PolyATract mRNA magnetic bead-based isolation system (Promega) from the same cell lines used in the differential display experiments. Conventional autoradiography or phosphorimaging was used to visualize homologous sequences in cell line RNA. 5' RACE was performed using sequence-specific oligonucleotide primers, RNA from the 1542T cell line, and the FirstChoice RLM-RACE kit according to the manufacturer’s recommendations (Ambion, Austin, TX). Clone sequences were compared with cDNA and genomic DNA sequences using Sequencher Software (Gene Codes Corp., Ann Arbor, MI) to define the ATG start site and the entire AIPC open reading frame. The entire AIPC coding sequence, 7926 bp in length, is available through GenBank (accession no. AF338650).

Protein Profiling
Determination of Protein Structure.
The predicted 2642-residue AIPC protein sequence was examined for the presence of functional motifs through a ProfileScan search of the protein profile entries in PROSITE, Pfam, and MultAlin accessed through the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (9 , 10) .4

Antibody Production.
A peptide comprising amino acid residues 1742–1752 of AIPC was synthesized on a branching lysine core through a single solid-phase synthesis to construct a MAP, which was then used to immunize two rabbits (11) . The anti-AIPC antibody was subsequently affinity purified from immunized rabbit serum (Bethyl Laboratories, Inc.).

Western Blot Analysis.
To verify that the AIPC antibody specifically recognized the AIPC protein, oligonucleotide primers P1 (5'-ACGTACGTGGATCCCCTCCTCAGCCGAAAACAAACCT-3')containing a BamHI restriction site and P2 (5'-ACGTACGTGAATTCGCGCCCTGCCCTCTGCTCTACA-3') containing an EcoRI site were used in a PCR reaction to amplify a portion of the AIPC sequence spanning nucleotides 4959–5490 (amino acid residues 1653–1830) inclusive of the antigenic site. The resulting PCR product was subcloned into a pGEX-2T plasmid, sequence verified, and expressed in Escherichia coli DH5 {alpha} cells as a GST fusion protein. Cell lysates were incubated with glutathionine-Sepharose 4B beads, and the AIPC-GST fusion protein was eluted with glutathionine elution buffer according to the manufacturer’s protocols (Amersham Pharmacia Biotech, Piscataway, NJ). Twenty-five mg of protein were separated by electrophoresis through a 10% Tris-Glycine Laemmli gel (Novex, San Diego, CA). The AIPC-GST fusion protein was detected using the anti-AIPC primary antibody at 1:1000 dilution and a donkey-antirabbit secondary antibody (Amersham Pharmacia Biotech) and detected with the anti-GST primary antibody at 1:1000 dilution (Amersham Pharmacia Biotech) and a rabbit-antigoat secondary antibody (Sigma-Aldrich, St. Louis, MO) using an ECL detection system (Amersham Pharmacia Biotech).

To quantitate AIPC protein in mammalian cells, cells were lysed for 50 min on ice in lysis buffer [50 µM Tris-HCl (pH 8), 120 µM NaCl, 0.5% NP40, 40 µM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 200 µM sodium orthovanadate]. Lysates were centrifuged, the supernatants were collected, and protein was quantitated using a Bradford assay. Forty mg of protein/cell line were separated by electrophoresis through a 6% Tris-Glycine Laemmli gel (Novex, San Diego, CA) and electroblotted. AIPC protein was detected using the anti-AIPC primary antibody at 1:1000 dilution with a donkey-antirabbit secondary antibody and visualized using an ECL detection system.

Tissue Microarray Construction and Immunohistochemical Analysis.
A high-density tissue microarray was constructed using fixed archival prostate tissue samples collected retrospectively from patients who had undergone radical retropubic prostatectomy at the University of Michigan Medical Center (Ann Arbor, MI). The array was assembled using the manual tissue puncher/array (Beecher Instruments, Silver Spring, MD) to produce 432 tissue cores ~0.6 mm in diameter, which were inserted into a 45 x 20 x 12-mm recipient block and spaced at a distance of 0.7 mm apart, as described previously (12) . Sections taken from the donor block were stained with H&E for pathological evaluation or subjected to standard indirect immunoperoxidase procedures for protein detection. Slides were pretreated by microwaving at 100°C in Tris-EDTA buffer for antigen retrieval, and incubation in primary AIPC antibody was carried out for 1 h at 1:800 dilution. Digital images of tissue microarray elements were captured for telepathological review using the x20 objective of an Olympus BX50 microscope, Sony Digital camera, and IP-Lab Imaging Software. Images were archived on a JAZ disc and presented for evaluation of immunohistochemical staining in I-View Multimedia on an Apple G3 workstation. H&E-stained images were paired with matching AIPC-stained array elements. Immunohistochemical staining of epithelial cytoplasm was graded into four groups: absent (0), weak (1), moderate (2), or strong (3) staining. All data were compiled in a customized relational database (Microsoft Access) that maintained patient/tissue diagnosis, array location, and histology results.

Statistical Analysis
A cumulative logistic regression mixed model for ordinal responses with repeated measures was used to describe the relationship between the immunohistochemical staining results and tissue type or clinical variables (predictors) using MIXOR (version 2; Ref. 13 ). Univariate analysis was used to determine significance levels.


   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
The AIPC Transcript Is Up-Regulated in Human Prostate Tumor Cells.
The 1535N normal and 1532T, 1535T, and 1542T primary tumor-derived prostate cell lines were completely characterized by differential display analysis using 14 Operon primer sets. At 20 primers/set, these experiments entailed 280 PCR reactions, in duplicate, for a total of 560 PCR reactions/cell line. These experiments identified 25 transcripts, suggesting that ~9% (25 of 280) of all transcripts examined by these methods were differentially expressed. One of the differentially expressed transcripts comprised a 1.4-kb PCR product that was substantially up-regulated in RNA purified from the 1532T and 1542T prostate primary tumor-derived cell lines compared with the normal tissue-derived 1535N cell line (Fig. 1A)Citation . This PCR product was subcloned and sequenced. A BLAST search showed that it was 100% homologous at the nucleotide level to a unique, 7621-bp unidentified human transcript, KIAA0300 (GenBank accession no. AB002298) and 64% homologous to the Rattus norvegicus PAPIN gene (GenBank accession no. AF169411; Ref. 14 , 15 ). However, Northern blot analysis using the subcloned 1.4-kb PCR product as a probe demonstrated a single 10-kb transcript, suggesting that the KIAA0300 cDNA clone was 5' truncated (Fig. 1B)Citation . Therefore, 5' RACE assays were undertaken using RNA purified from 1542T cells to identify the ATG initiation codon. These assays identified a total of 26 overlapping clones that were used in conjunction with the KIAA0300, PAPIN, and Homo sapiens chromosome 5 working draft sequence segment, NT_006455, sequences to assemble the complete AIPC coding sequence. The complete AIPC transcribed sequence is 10,718 bp in length with a 7,926-bp open reading frame comprising 3,097 bp of sequence identified in this study and 4,829 bp from the known KIAA0300 sequence, as well as a 2,792 bp 3' untranslated region (Fig. 2A)Citation . The first 483 bp of AIPC are virtually identical to the first 483 bp of the PAPIN and include the ATG initiation codon. The entire 7,926 complete coding region for the AIPC gene has been deposited in GenBank (accession no. AF338650). Because both the RT-PCR RNA profiling and Northern blot experiments demonstrated abundant expression of this transcript exclusively in the prostate tumor-derived cell lines, we designated it AIPC.



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  		Fig. 1. RNA profiling of AIPC expression. A, differential display analysis. Total cellular RNA from 1535N (Lane 1), 1532T (Lane 2), 1542T (Lane 3), and 1535T (Lane 4) cells was reverse-transcribed and subjected to PCR using the B7 primer set (Operon). The 1.4-kb AIPC transcript (arrow) is abundantly expressed in the 1532T and 1542T cells, expressed less abundantly in 1535T cells, and absent in 1535N cells. Lane 5, 100-bp molecular weight marker (Life Technologies, Inc.). B, Northern blot Analysis. One µg of poly(A)+ RNA purified from 1535N (Lane 1), 1542T (Lane 2), and 1535T (Lane 3) cells was electrophoresed through a 1.0% agarose/formaldehyde gel, blotted onto nylon, and hybridized to the radiolabeled 1.4-kb AIPC probe. The 10-kb AIPC transcript is abundantly expressed in 1542T and 1535T cells but absent from 1535N cells (top panel). The same membrane was stripped and rehybridized with a radiolabeled probe specific for ß-actin as a loading control (bottom panel).

 


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  		Fig. 2. AIPC gene sequence and Western blot analysis. A, AIPC gene. The nucleotide structure of the 7926-bp AIPC open reading frame is shown schematically. Functional motifs shown here include the ATP/GTP-binding site motif A (P-loop; cross-hatched box) and the six PDZ domains (light gray boxes); the nucleotide positions of motif boundaries, initiation codon, and stop codon sequences, are indicated. The sequence subcloned to test the specificity of the anti-AIPC antibody is bracketed (nucleotides 4959-5490). *, sequence used to construct the MAP peptide. The 2792-bp 3' UTR is shown by the dark gray box. The entire AIPC sequence may be accessed through GenBank (accession no. AF338650). B, the anti-AIPC antibody is specific for the AIPC sequence. Twenty-five mg of protein from cell lysates of E. coli DH5 {alpha} cells expressing an AIPC-GST fusion protein was separated in duplicate by electrophoresis through 10% Tris-Glycine Laemmli gels and then incubated with 1:1000 dilutions of anti-AIPC primary or anti-GST primary antibodies (as described in the text). Both antibodies detected the same Mr 58,000 fusion protein, demonstrating the specificity of the anti-AIPC antibody for the AIPC sequence. C, Western blot analysis of AIPC protein expression in human cultured prostate cells. Forty µg of protein prepared from 267B1 (Lane 2), 1535N (Lane 3), 1535T (Lane 4), 1532T, (Lane 5), and 1542T (Lane 6) were electrophoresed through 6% Tris-Glycine Laemmli gel, electroblotted onto nitrocellulose, and incubated with the AIPC primary antibody and antirabbit secondary antibody. Proteins were visualized using standard ECL methods. AIPC protein expression is abundant in all three tumor-derived cell lines, less abundant in 1535N cells, and absent from 267B1 cells. A Mr 220,000 protein molecular weight marker is shown (Lane 1).

 
AIPC Encodes a High Molecular Weight Protein Containing Six PDZ Domains.
Translation of the 7,926-bp AIPC open reading frame results in a protein sequence of 2642 amino acid residues with a theoretical protein weight of Mr 280,000 (determined using the Compute pI/Mw tool accessed through the ExPASy proteomics server). ProfileScan analysis of the AIPC ORF revealed the presence of an ATP/GTP-binding site motif A (P-loop) at amino acid residues 1983–1990 (nucleotides 5949–5970). It also revealed the presence of six PDZ domains at amino acid residues 94–160, 335–376, 406–492, 548–618, 2424–2496, and 2552–2637; the corresponding nucleotide positions of these domains are shown in Fig. 2ACitation . Consistent with other PDZ domains described in the literature, each of the AIPC PDZ domains are 80–90 amino acid residues in length and contain a signature GLGF/L sequence (16 , 17) .

AIPC Protein Expression Is Up-Regulated in Human Prostate Tumor Cell Lines and Tissues.
We next produced an antibody to the AIPC protein to examine its expression in human prostate cell lines and tissues. A hydrophilicity and antigenicity profile revealed a potentially antigenic site at amino acid residues 1742–1753, and a MAP peptide of these residues proved immunogenic in rabbits. To test the specificity of this antibody, a portion of the AIPC sequence between nucleotides 4959 and 5490 and inclusive of the antigenic site was subcloned as part of a GST fusion protein construct. Immunoblots against E. coli protein lysates using antibodies against either AIPC or GST identified the same Mr 58,000 fusion protein, demonstrating that the anti-AIPC antibody was specific for the antigenic sequence (Fig. 2B)Citation .

AIPC Protein Expression Is Up-Regulated in Cultured Human Prostate Tumor Cells.
Western blot analysis using affinity-purified AIPC-specific antibody demonstrated a single predominant band in cultured prostate cells of high molecular weight (Mr >220,000), consistent with a theoretical protein weight of Mr 280,000. Moreover, this protein was abundantly expressed in 1532T, 1535T, and 1542T human prostate tumor-derived cultured cells and was more variable in normal human prostate cultured cells, with a low level of expression evident in 1535N cells and virtually no expression in 267B1 cells (Fig. 2C)Citation . These results were consistent with those observed by RT-PCR and Northern blot analysis and suggested that AIPC was abundantly expressed at both the RNA and protein levels in prostate tumor cells.

AIPC Protein Expression Is Up-Regulated in Human Prostate Tumors.
Expression of the AIPC protein was next evaluated in normal and neoplastic human prostate tissues by immunohistochemical analysis of a tissue microarray. The tissue microarray comprised 313 tissue elements from 42 separate prostate glands representing 158 primary tumor, 18 high-grade PIN, 46 normal atrophic, and 91 normal benign prostate specimens. Staining intensity was evaluated for each tissue element on a scale of 0–3, corresponding to absent (0), weak (1), moderate (2), or strong (3) staining. A portion of the array is shown in Fig. 3Citation . This analysis demonstrated that the AIPC protein was highly expressed (at a staining intensity of 2 or 3) in 75% of tumors, 83% of PIN lesions, 18% of atrophic, and 3% of normal benign tissues (P < 0.0001; Fig. 4Citation ). These results showed that the AIPC protein is abundantly expressed in high-grade PIN and human prostate tumors. The observation of abundant AIPC protein expression in high-grade PIN, a putative premalignant lesion of the prostate, and tumor glands suggests that the activation of AIPC expression is an early event in human prostate tumorigenesis.



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  		Fig. 3. AIPC evaluated on high-density tissue microarray of human prostate tissues. A–C, composition of the tissue microarray. The H&E-stained, high-density tissue microarray was composed of benign dysplastic (high-grade PIN) and malignant prostate tissue (A, x5; B–C, x200). All tissue samples were confirmed histologically as either benign, high-grade PIN, or prostate carcinoma (B and C, x200). D, benign prostate tissues. AIPC protein expression is seen to stain the basal cell layer in benign prostate but not the overlying secretory cells (D, x200; inset, x1000). E and F, malignant prostate tissues. Infiltrating prostate carcinoma demonstrates strong cytoplasmic staining of the AIPC protein (E–F, x200; inset, x1000).

 


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  		Fig. 4. Correlation of AIPC protein expression with clinical and pathological variables. A, summary of AIPC expression in human prostate tissues. Immunohistochemical analysis of AIPC protein expression as absent or weak (0, 1) versus moderate or strong (2, 3) is shown correlated with tissue type, tumor stage, tumor grade, and patient preoperative serum prostate-specific antigen. Statistical significance is indicated as P values. B, histogram of AIPC staining pattern in human prostate tissues. The percentage of each tissue type expressing AIPC protein at absent or weak (0, 1; light gray) levels versus moderate or strong (2, 3; dark gray) levels is shown. Both high-grade PIN and tumor specimens demonstrated significantly more abundant AIPC protein expression than normal or atrophic tissues.

 
AIPC staining was cytoplasmic in all positive cases, consistent with the absence of any transmembrane domain-associated residues in the amino acid sequence (Fig. 3)Citation . Of the 42 malignant prostate glands present on the array, 39 were represented by 2 or more tissue elements for a total of 158 tumor tissue elements. Malignant tissues from 27 of 42 glands demonstrated no heterogeneity for AIPC staining, whereas those from 15 of 42 glands demonstrated heterogeneity for AIPC staining spanning 2 or more units on a scale of 0–3. However, 11 of the 15 heterogeneously-staining glands demonstrated a median staining intensity of 2.0–3.0, indicating that the majority of tissue elements associated with these glands expressed AIPC abundantly at the protein level. Thus, AIPC staining was largely uniform and intense for the majority of the 158 tumor elements examined.

The AIPC protein was highly expressed in the majority of prostate tumors and did not associate with the specific diagnostic and prognostic variables of tumor pathological stage or grade and patient preoperative prostate-specific antigen level (Fig. 4A)Citation .

Possible Role(s) for the AIPC Protein in Prostate Tumorigenesis.
The six PDZ domains within the AIPC protein sequence may mediate binding interactions between AIPC and other cellular proteins. PDZ domains may occur in multiple copies/protein, with each PDZ domain having similar or different binding specificities. PDZ domain-mediated protein-protein interactions have been implicated in the organization of protein complexes in signal transduction cascades, in coupling channels and transmembrane receptors to downstream signaling elements, in clustering transmembrane receptors and channels, in recruiting cytosolic proteins to membrane complexes, in organizing large two-dimensional complexes like cell junctions and plasma membrane domains, and in interactions with the cortical cytoskeleton (16 , 17) . Some of these PDZ-associated functions, particularly those involving signal transduction, clustering, and scaffolding, may have important functions in tumorigenesis. One recent study reported up-regulation of a PDZ domain-containing protein, PCD1, in several human tumor types, including malignant prostate tissues (18) . Another study demonstrated that the APC protein binds to the second of five PDZ domains of the protein tyrosine phosphatase protein PTP-BL and suggested that this binding interaction might modulate the phosphorylation of associated proteins and thereby play a role in several cellular activities that are dysfunctional in cancer cells, including cell division, migration, and adhesion (19) . Although the binding specificities of the six PDZ domains within the AIPC protein are unknown, future studies will identify which cellular proteins bind the AIPC PDZ domains and how these interactions may contribute to tumorigenesis in the prostate.

Another intriguing feature of AIPC protein expression is its localization to the basal cell layer in normal prostatic glands and its ubiquitous expression in PIN and malignant glands. A current hypothesis is that prostatic stem cells reside in the basal cell compartment of prostatic epithelium and may actually comprise the majority of basal cells (20) . AIPC staining was absent from secretory cells but was observed in the basal cell layer in normal prostatic glands and was both uniform and intense in neoplastic (PIN and tumor) glands. If stem cells actually give rise to differentiated secretory cells and undifferentiated neoplastic cells in the prostate, it is possible that AIPC protein expression is down-regulated in differentiated secretory cells but is up-regulated in proliferative stem cells and neoplastic cells. Moreover, the observed abundant expression of AIPC protein in both high-grade PIN and tumor cells is consistent with a role for the AIPC protein in the initiation or early promotion of malignant transformation in the prostate. Future studies will test this hypothesis and will focus on the possible contribution of the AIPC protein and its protein binding partners to prostate tumorigenesis.


   ACKNOWLEDGMENTS
 
We thank Drs. Kim Taussig-Orth, Marco Muda, and Jack E. Dixon for valuable input and helpful discussions.


   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 by NIH/National Cancer Institute Grant 1P50 CA69568 Specialized Program of Research Excellence in Prostate Cancer (to J. A. M., M. A. R., and J. M. G. T.). Back

2 To whom requests for reprints should be addressed, at Department of Surgery, Section of Urology, The University of Michigan, 7306 CCGC, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0946. Phone: (734) 647-8121; Fax: (734) 647-9480; E-mail: jcoska{at}umich.edu Back

3 The abbreviations used are: AIPC, activated in prostate cancer; RT-PCR, reverse transcription-PCR; RACE, rapid amplification of cDNA ends; MAP, multiple antigenic peptide; GST, glutathione S-transferase; PIN, prostatic intraepithelial neoplasia. Back

4 Internet address: http://www.expasy.ch. Back

Received 5/23/00. Accepted 1/31/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
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