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PrLZ, a Novel Prostate-Specific and Androgen-Responsive Gene of the TPD52 Family, Amplified in Chromosome 8q21.1 and Overexpressed in Human Prostate Cancer

¹ Molecular Urology and Therapeutics, Department of Urology, ² Department of Pathology, and ³ Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia; ⁴ Laboratory of Cancer Genetics, Institute of Medical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland; ⁵ Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia; and ⁶ Division of Human Biology, Fred Hutchinson Cancer Research Center, University of Washington, Seattle, Washington

	ABSTRACT

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We report a previously unrecognized prostate-specific protein, PrLZ (prostate leucine zipper), a new member of the Tumor Protein D52 (TPD52) family. The gene for PrLZ was localized at chromosome 8q21.1, a locus most frequently amplified in human prostate cancer. Multiple tissue analyses demonstrated PrLZ predominantly in the prostate gland. Although its expression was enhanced by androgens in androgen receptor-expressing cells, PrLZ was detected in all of the human prostate cancer cell lines, regardless of androgen receptor status. Monoclonal anti-PrLZ antibodies were produced and intense immunohistochemical staining of PrLZ was observed in prostate epithelial cells in intraepithelial neoplasia and prostate cancer, whereas lower-level staining was detected in normal and benign epithelial components of the prostate gland. As the only prostate-specific gene identified in the most frequently amplified genomic region in prostate cancer, PrLZ may be the link between chromosome 8q amplification and malignant transformation of the prostate epithelia.

	Introduction

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Metastasis of prostate cancer (PCa) results from the malignant progression of localized tumor (known as prostatic intraepithelial neoplasia; PIN), and is the main cause of the morbidity and mortality of this disease. The mechanism for the development and progression of PCa has yet to be elucidated. To study the development and progression of PCa with a lineage-related cell model, we established a series of LNCaP sublines, including the C4, C4–2, and C4–2B cell lines, through tumor-stroma interaction and xenograft selection (1) . The LNCaP/C4–2 PCa model mimics the progression of clinical PCa. Whereas growth of the LNCaP is androgen-dependent, proliferating only in intact but not in castrated male hosts, its derivative C4–2 and C4–2B sublines are androgen-independent, capable of growing in castrated male hosts. These sublines express androgen receptor (AR), secrete prostate specific antigen (PSA) in the absence of androgenic hormones, and metastasize to lymph node and bone. Furthermore, these cells exhibit bone-like properties, expressing osteoblastic factors and secreting bone matrix proteins (2 , 3) .

We studied the LNCaP/C4–2 progression model for clues to genetic and expressional changes, which may cause the transition from androgen-dependent to androgen-independent status. For this report, we isolated and characterized a novel prostate-specific transcript, PrLZ, based on its differentiated expression between LNCaP and lineage-related C4–2 cells. Our data suggest that overexpression of PrLZ is associated with PCa progression. PrLZ may function to promote prostatic epithelial proliferation and transformation.

	Materials and Methods

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Cell Lines and Reagents.
PCa cell lines PC3 and DU145 were obtained from American Type Culture Collection (Manassas, VA). C4–2 and C4–2B cells were derived from chimeric LNCaP cell tumors (1) . ARCaP was from a PCa metastasis (4) . Cells were maintained in T-medium (Invitrogen, Carlsbad, CA) with 10% of fetal bovine serum and antibiotics (penicillin, 100 unit/ml and streptomycin, 100 µg/ml) at 37°C with 5% CO₂ in a humidified incubator. To study the effect of androgen, cells were plated on a 10-cm dish at 50% confluence, subjected to androgen starvation in phenol red-free RPMI 1640 for 48 h, and treated with a synthetic androgen, methyltrienolone (R1881; Perkin-Elmer Life and Analytical Sciences, Boston, MA), in fresh phenol red-free RPMI 1640 with 10% dextran/charcoal absorbed fetal bovine serum for 12 h.

Microarray Expression Analysis.
Total RNA samples from LNCaP and C4–2 cells were used for synthesizing fluorescence-labeled cDNA probes to screen an array of 1500 unique sequences derived from the prostate expression database (5) . The detailed protocol used for analyzing prostate gene expression has been described previously (5) . Two hybridizations were performed for each sample, and for each arrayed gene four data points were collected for statistical comparison.

DNA Cloning and Sequencing.
A cDNA library of the C4–2 cells was constructed into the ZAPExpress phage (Stratagene, La Jolla, CA) by the manufacturer’s recommended protocol. A 330-bp insert from the original expressed sequence tag clone, identified through cDNA microarray, was used as a probe to screen 2 x 10⁵ plaque-forming units of the library. Positive clones rescued into pBK-CMV phagemid were subjected to restriction mapping and DNA sequencing. A human genomic library in bacterial artificial chromosome was screened by PCR with 5'-GCCTGAACTGTTTGTACC TCTG-3' and 5'-GAGTAGGTGATCCGGGTGGAGATG-3' as primers. Restriction fragments were subjected to nested deletion with the Erase-A-Base kit (Promega, Madison, WI). Manual DNA sequencing was performed with the Taq Sequenase II kit (USB, Cleveland, OH), and automated sequencing was on an ABI sequencer (Applied Biosystems, Foster City, CA).

Expression and Multiple Tissue Expression Assays.
The PrLZ-specific fragment was cloned to pGEM-T easy (Promega) after amplification of the PrLZ cDNA with primers: 5'-GCCTGAACTGTTTGTACCTCTG-3' and 5'-GAGTAGGTGATCCGGGTGGAGATG-3'. Similarly, coding sequences of the PSA and prostate-specific membrane antigen (PSMA) were cloned with primers 5'-ATGTGGGTCCCGGTTGTCTTCCTCACCCTGTC-3' and 5'-TCAGGGGTTGGCCACGATGGTGTCCTTGATC-3' (PSA); and 5'-ATAGGATCCATGTGGAATCTCCTTCACGAAACC-3' and 5'-CATAAGCTTTTAGGCTACTTCACTCAAAGTCTC-3' (PSMA) from prostate RNA (Clontech, Palo Alto, CA). Manufacturer-recommended protocols were used in hybridizations to the Multiple Tissue Expression array, the Multiple Tumor Expression array, and the Human Total RNA Master Panel (Clontech).

Immunohistochemical Staining.
Polyclonal antibodies were produced by immunizing mice with synthetic peptides, followed by establishing and screening hybridoma cells for monoclonal antibody production (University of Virginia Hybridoma Core Facility). Antibody specificity was confirmed by Western blot and immunoprecipitation. Preimmune sera or control hybridoma fluid were used as negative control.

Multiple tumor arrays prepared from 100 radical prostatectomies performed at Emory University were subjected to immunohistochemical staining with the anti-PrLZ antibody. The data were evaluated by two pathologists (S. D. L. and M. A.). PrLZ staining was set as low if 0–15% of the cells were stained stronger than the normal level, and high if >15% cells were strongly stained. The data were subjected to Fischer’s exact test for cross-table analysis, with P < 0.05 considered statistically significant.

PCR Analyses.
All of the PCR reactions were initiated with incubation at 94°C for 2 min, followed by 30 cycles of 94°C, 30 s; 55°C, 30 s; and 72°C, 2 min. Reactions were finished with a 72°C, 7-min extension. Primers used were: 5'-CCTGAACTGTTTGTACCTCTGGGCCATATTGC-3' and 5'-CAAATTTCTGAAGAGTAGGTGATCCGGGTGGAG-3' for exon 1 of the PrLZgene; 5'-TCTAAAGTAGGGGGAACCAAGCCTGCTGGTGGTG-3' and 5'-ACTGATAGATGGAATTTATTAAGCTTTTCACATG-3' for exon 7 of the PrLZgene; 5'-CAAGTTAACTGAGCTTTTTCTTAATTTCATTC C-3' and 5'-GTTGGTACCTCCACAGAAGATGTTTATTTGATGTAAC-3' for 3' untranslated region of the HIF1A gene. Primers for amplification of the mitochondrial DNA were 5'-AGTCAATAGAAGCCGGCG-3' and 5'-GGGGATTTAGAGGGTTCTGT-3'. Comparison of the expression of PrLZand TPD52 was done with gene-specific primer pairs of 5'-ATGGATTGTAGAGAGATGGACTTATATGAGG-3, 5'-TCACAGGCTCTCCTGTGTCTTT TCTGGAAGAGG-3' (PrLZ), and 5'-ATGGACCGCGGCGAGCAA-3', 5'-TCACAGGCTCTCCTGTGTCTTTTCTGGAAGAGG-3' (TPD52).

Fluorescence in Situ Hybridization.
The PrLZ-specific probe was labeled with either digoxigenin (Roche Diagnostics, Mannheim, Germany) or Alexa Fluor 594-dUTP (Molecular Probes, Eugene, OR) by nick-translation, and hybridized together with FITC-dUTP (DuPont, Boston, MA) -labeled chromosome 8 centromeric probe (pJM128) to metaphase chromosomes as described (6) . Slides were counterstained with 0.1 µM 4,6-diaminido-2-phehylindole in an antifade solution. Assignment of the gene to chromosome was based on 4,6-diaminido-2-phehylindole banding pattern.

Fluorescence in situ hybridization was used to determine PrLZ gene amplification in PCa specimens. Forty formalin-fixed, paraffin-embedded, locally recurrent, hormone-refractory specimens (transurethral resection of prostate) from Tampere University Hospital were analyzed (6) . PrLZ amplification was classified into three groups, no amplification (no increase in PrLZsignal number), low-level amplification (3–4 copies of signals per cell), and high-level amplification (5 copies of signals or clusters of signals per cell).

	Results

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PrLZ Isolation.
We studied 1500 arrayed genes for their expression in LNCaP and C4–2 cells. Comparative analysis and subsequent Northern blot hybridization identified 8 clones with >2-fold higher expression in C4–2 than in LNCaP (Fig. 1A) . One of the clones showed a 9-fold higher expression in C4–2 (PrLZ; Fig. 1A ). Using this expressed sequence tag clone (probe P; Fig. 1B ) as probe, we screened a cDNA library of C4–2 cells and isolated 24 positive clones. DNA sequencing analyses revealed that these clones were from two transcripts that shared a homologous sequence in their 3' half, but contained distinctive 5' regions (Fig. 1B) . Homology search through the GenBank revealed that one represented a previously reported TPD52 (7) , except the new isolate had a longer 5' untranslated region and a shorter 3' untranslated region. The other transcript contained a unique 502-bp sequence in its 5' region, completely divergent from that of TPD52. It encoded a polypeptide with a 41-residue unique NH₂ terminus, 35 residues longer than the TPD52 protein (Fig. 1B ; GenBank accession no. AF202897). We named this new isolate PrLZ, and characterized its transcript and protein product.

View larger version (45K): [in this window] [in a new window]   Fig. 1. PrLZ isolation. A, Northern blot hybridization to confirm differentiated gene expression. After microarray screening, insert from selected clones was used as a probe to confirm differentiated expression in LNCaP and C4–2 cells. In each panel, the specific hybridization of the gene being studied is shown at the top. At the bottom, the same membrane was then hybridized to ß-actin probe to determine equal loading. Except for PrLZ, other candidate genes are indicated with their array coordinates. B, schematic of the relationship between PrLZ and TPD52. Shaded area indicates coding region and slashed area shares identical sequence among cDNA clones. Probe (P) was used to screen the cDNA library. PrLZ-502 is a fragment containing the PrLZ-specific region and was used as probe in hybridizations. C, prostate-specific expression of PrLZ as determined on Multiple Tissue Expression array. PrLZ-502 was used to detect specific PrLZ expression. Predominant PrLZ expression was seen in prostate (coordinate E8), with minimal expression seen in the gastrointestinal system, including stomach (B5), duodenum (C5), jejunum (D5), ileum (E5), ilocecum (F5), appendix (G5), ascending colon (H5), transverse colon (A6), descending colon (B6), and rectum (C6). Still lower levels of expression were seen in kidney (A7), pituitary (D3), salivary (E9), and mammary gland (F9). G12 and H12 represent 100 ng and 500 ng control human DNA, respectively. Examined on the array were 64 normal adult human tissues, 8 embryonic tissues, and a series of cell lines. A list of these samples can be found elsewhere.7 The same membrane was hybridized with ß-tubulin probe provided by the manufacturer. Ubiquitous and uniform signals were seen (data not shown).

Prostate Specificity of PrLZ.
The first 502 bp of the PrLZ cDNA, determined as unique and PrLZ-specific (probe PrLZ-502; Fig. 1B ), was used as a probe to study the expression of PrLZ in various tissue and cell types. PrLZ was expressed predominantly in human prostate (Fig. 1C) , with only minimal expression in the gastrointestinal tract and a few other glandular tissues with secretory functions (i.e., pituitary, salivary, and mammary glands, pancreas, and kidney). The expression in prostate was at least 12-fold higher than in any other tissues. Importantly, PrLZ was not detected in the rest of the 52 different embryonic and adult human tissues, nor in a list of human cell lines of extraprostatic origin. Similar prostate-specific expression of PrLZ was found in additional studies with the Multiple Tumor Expression array and with the Human Total RNA Master Panel (data not shown). In contrast, TPD52 was expressed in numerous human tissues without evidence of tissue specificity.

Chromosomal Localization and Amplification of the PrLZ Gene.
Taking advantage of the gene specificity of the 502-bp fragment, we localized the PrLZ gene at human chromosome 8q21.1 (Fig. 2A) . The PrLZgene was isolated in a 180-kb genomic fragment in a bacterial artificial chromosome clone. Exons were mapped and corresponding restriction fragments subcloned for DNA sequencing. The PrLZ gene was also contained in the draft sequence of 8q21.1 (GenBank accession no. NT_023700). PrLZ messenger was transcribed from 7 exons, which were scattered in a 45-kb region (Fig. 2B) . Similar to other members of the TPD52 family, there were alternatively spliced messengers of the PrLZ gene. Distinct from other genes of the TPD52 family, we found that alternative splicing events were from unique locations of the PrLZgene, producing novel polypeptides (data not shown).

View larger version (58K): [in this window] [in a new window]   Fig. 2. PrLZgene amplification at chromosome 8q21.1. A, localization of PrLZgene. Shown is a representative micrograph of the fluorescence in situ hybridization analysis (FISH), with red signals defining PrLZ at chromosomal 8q21.1 and green signals chromosome 8 centromeres. Inset is a magnification of the chromosome 8 and chromosome 8 ideogram. B, structure of the PrLZgene. Genomic structure of PrLZis shown at the top, with solid bars indicating exons. The corresponding region in cDNA is indicated in the lower portion. C, amplification of PrLZgene in prostate cancer (PCa) specimens as detected by PCR. In this study, paired genomic DNA samples isolated from cancer cells (T), and unaffected glandular cells (N) of the same PCa specimen (depicted with case numbers), were used to detect the first and last exons of the PrLZ gene. Equal amounts (100 pg) of DNA were used in each sample. In the control lane (C), 10 ng of normal human genomic DNA (Clontech) was used. PrLZ gene was amplified in four of the seven cases tested (266T, 316T, 285T, and 320T). The equal amount of genomic DNA was controlled by PCR of 3' untranslated region of the hypoxia inducible factor 1 (HIF1A), which is rarely amplified in PCa (21) . Mitochondrial DNA (MtDNA) was amplified for similar purposes. D, amplification of PrLZ gene in PCa specimens as detected by FISH. In contrast to the signal in normal prostate epithelial cells (top left panel), nuclei of PCa cases showed low-level amplification (top right panel) and high-level amplification of the PrLZ gene. Amplification is seen as scattered (bottom left panel) or clustered (bottom right panel) hybridization signals. Green signals outside the nuclei were due to autofluorescence, typical for FISH analysis with tissue sections.

Regulated Expression of PrLZ.
Under regular culture conditions (T-medium with 10% fetal bovine serum), PrLZ was expressed in all of the tested PCa cell lines, independent of the AR status (Fig. 3A) . This was in sharp contrast with PSA and PSMA, which were seen only in the AR-expressing LNCaP and its lineage derivatives. To investigate the regulation of PrLZ by androgens, we treated the PCa cells with an androgen analog, R1881 (1 nM), after androgen deprivation. After culturing the PCa cell lines under this condition (phenol red-free RPMI 1640, fetal bovine serum-free, for 48 h), the PrLZ signal markedly declined. Nonetheless, androgen promoted the expression of PrLZ in LNCaP and its lineage-derivative cells (Fig. 3B) . Thus, PrLZ expression could be regulated by serum and androgen. In contrast, TPD52 was uniformly expressed (Fig. 3A) , its expression neither affected by androgen deprivation nor by androgen stimulation (Fig. 3B) . Compared with its regulation of PSA, androgen treatment seemed to have distinguishable effect on PrLZ expression. The effect of R1881 on PSA expression was seen only in LNCaP cells, whereas the constitutive expression in C4–2 and C4–2B was largely unaffected (Fig. 3B) . In contrast, androgen-enhanced PrLZ expression was seen in both parental and LNCaP-lineaged cells, with the highest up-regulation seen in C4–2B cells. The transition from androgen-dependent to androgen-independent state seemed a heterologous process: whereas LNCaP sublines lost the control for PSA expression, these cells still maintain the capability of controlling some other androgen responsive genes, such as PrLZ.

View larger version (76K): [in this window] [in a new window]   Fig. 3. Expression of the PrLZ in prostate cancer cell lines. A, detection of PrLZ in prostate cancer cell lines by reverse transcription-PCR. In this study, the 502-bp gene-specific sequence in 5' of the PrLZ cDNA, and the full coding sequences of TPD52 and prostate-specific antigen were subjected to PCR amplification. The experiment was repeated at least three times; a representative result is shown. B, PrLZ expression is enhanced by androgen, as detected by Northern blot hybridization with PrLZ as probe (top panel) and reverse transcription-PCR detecting the 502-bp gene-specific sequence in 5' of the PrLZ cDNA (bottom panel). Expression of prostate-specific antigen (positively regulated by androgen) and PSMA (negatively regulated by androgen) was used to determine the effect of androgen. Full coding sequence of the TPD52 was amplified to show the distinctive response of PrLZ to androgen. The experiment was repeated three times; a representative result is shown. C, detection of PrLZ protein by Western blotting. In the top panel, control vector (lane 1), FLAG-tagged PrLZ (lane 2), and FLAG-tagged TPD52 (lane 3) were stably overexpressed in HEK293 cells, which did not express PrLZ. Whole cell lysates were subjected to immunoblotting with anti-FLAG (left) or anti-PrLZ antibody (right). Anti-PrLZ antibody specifically recognized the PrLZ protein (right). The bottom panel shows a representative result in which the anti-PrLZ antibody was used in immunoblotting to detect endogenous PrLZ expression in LNCaP and C4–2 cell lines in the presence (+) or absence (-) of androgen (R1881; 1 nM).

PrLZ Expression in PCa Specimens.
Using the specific antibodies, we determined the expression of PrLZ in association with clinical PCa. Specimens from 100 PCa cases were used. PrLZ was generally low in unaffected secretory epithelia (75.3% being low) and in benign prostatic hyperplasia (BPH) (78.2% being low; Fig. 4B ). It was highly expressed in high-grade prostatic intraepithelial neoplasia (PIN) (84.5% being high) and PCa (75% being high; Fig. 4B ). Gleason grade 4 tumors stained more than Gleason grade 3 tumors. Remarkably, intense PrLZ staining was always limited to malignant cells, with the neighboring unaffected epithelial cells stained at the normal level (Fig. 4B) . Enhanced expression of PrLZ was, therefore, tumor cell specific.

View larger version (163K): [in this window] [in a new window]   Fig. 4. Abnormally enhanced PrLZ expression in prostate cancer (PCa) specimens. Representative data from immunohistochemical studies of 100 PCa specimens are shown. A, PrLZ level in a normal, healthy, 42-year-old prostate, with low but discernible staining seen in glandular epithelia. B, PrLZ expression in PCa nodules with adjacent BPH nodular region. Whereas the PCa nodules were heavily stained, BPH in their vicinity was low for PrLZ, comparable with the normal level. C, PrLZ in high-grade PIN with adjacent normal glands. Epithelia of the high-grade PIN exhibited a general up-regulation of PrLZ; adjacent normal or unaffected epithelial cells displayed low level staining. D, PrLZ expression in PCa nodules with neighboring normal or unaffected glands. In contrast to intense PrLZ staining in PCa cells, normal or unaffected glands in the immediate neighborhood showed low level staining. E, uniformly high expression of PrLZ in PCa cells of Gleason score 3 tumor. F, representative specimens showed prevalently high PrLZ expression in PCa of typical Gleason score 4 tumors, with higher PrLZ staining than Gleason score 3 tumors.

	Discussion

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PrLZ appears to be a new member of the TPD52 family, a group of homologous proteins identified due to overexpression of D52 in tumors (7 , 9 , 10) . Distinctively, PrLZ expression is prostate-specific and androgen-responsive, whereas TPD52 could be detected in many tissue and cell types (10) , and is not affected by androgen (Fig. 1C and Fig. 3B ). Using a gene-specific probe, we localized the PrLZ gene to chromosome 8q21.1 (Fig. 2A) and determined the gene structure by studying a bacterial artificial chromosome clone (Fig. 2B) . Previously, other laboratories used the full cDNA as a probe to localize the TPD52 gene on 8q, proximate to the locus in which the PrLZ gene was localized (7 , 10) . We have not been able to determine whether the transcripts for PrLZ and TPD52 are derived from the same gene by selective usage of promoters or arise from separate genes in close vicinity. We determined the genomic organization of the PrLZ by partially sequencing a bacterial artificial chromosome clone, which contained all of the exons of PrLZwithin a 50-kb span. We did not find TPD52-specific exon in the PrLZ gene or within a 12-kb 5' flanking region. In the draft sequences of the human genome, a TPD52-specific exon is located 107 kb upstream from the second exon of the PrLZ gene, raising the possibility that PrLZ and TPD52 are splicing variants of one single gene. Both PrLZ and TPD52 are abnormally expressed in malignant tissues, with PrLZ seen specifically in PCa and TPD52 in many tumors. Studying PrLZ may elucidate the biological and pathophysiological function of the TPD52 family.

Prostate specificity and enhanced expression in high-grade PIN and PCa make PrLZ an attractive diagnostic marker. Androgen-independent, tumorigenic, and bone metastatic C4–2 expressed higher PrLZ levels than LNCaP in the LNCaP/C4–2 PCa progression model (Fig. 1A ; Fig. 2, B and C ; and Fig. 3B ). Although it can be activated through liganded AR, PrLZ expression does not rely on AR presence (Fig. 2, B and C) . Thus, enhanced PrLZ expression in PCa is correlated to malignancy rather than to AR activity.

Oncogenic Property of PrLZ.
The biological function of PrLZ is unclear. Besides its unique NH₂-terminal structure, PrLZ contains conserved domains of the TPD52 family. Members of the TPD52 family are involved in protein-protein interaction (11, 12, 13) . Annexin VI (14) , MAL2 (13) , syntaxin I, and VAMP2 (15) have been identified as interacting partners of TPD52 proteins. 14–3-3, a crucial player in Ras signaling, vesicular transport, and cytoskeletal organization, was revealed recently as another interacting partner (11) . Also, TPD52 proteins could be modified post-translationally with phosphorylation, N-glycosylation, or protease digestion. In a separate study we found that PrLZ was highly expressed in the developing prostate gland (data not shown) with a spatial and temporal expression suggesting that PrLZ may help regulate growth, morphogenesis, and cytodifferentiation of glandular ducts during development (16) .

The role of PrLZ in PCa development and progression has yet to be elucidated. The coding sequence of the PrLZ in PCa cell lines contained no mutations. PrLZ cDNA, when transfected to ARCaP cells, markedly stimulated their growth in immune-deficient mice (data not shown). Other TPD52 family members have been found to be associated with cancers in multiple tissues and organs (7 , 10 , 17) . In addition, retroviral integration in avian TPD52 (R10) was accompanied by neuroepithelial proliferation (18) . In a leukemic (HL-60 and K-562) cell differentiation model, expression of TPD52 members was associated with cell proliferation (9) .

PrLZ gene is located in 8q21. Amplification of this gene in PCa is independent from 8q23–24 amplification (19) . Within the 8q21 amplicon, we found previously that TCEB1 gene was 6 mb pairs centromeric to the PrLZ(20) . Fluorescence in situ hybridization analyses revealed that PrLZ and TCEB1 genes were coamplified in PCa specimens (data not shown). Additional investigation would clarify whether both of these genes are involved in malignancies of the prostate gland.

In summary, we have identified a prostate-specific and androgen-inducible gene, PrLZ, located at the most frequently amplified 8q chromosome region in human PCa. PrLZ was seen in the majority of the high-grade PIN specimens. Possibly up-regulation of PrLZ is an early sign of malignant transformation. PrLZ expression persisted in locally recurrent, hormone-refractory, and metastatic PCa. Additional elucidation of the function of PrLZ in PCa cells could provide insight into the malignant progression of PCa cells.

	ACKNOWLEDGMENTS

 
We thank Drs. Lara Harik, Fan Yeung, Yuanyuan Cui, and Zhihui Xie for technical assistance. We also acknowledge the technical support provided by Dr. Jay Fox of the Core Facilities at the University of Virginia for the chemical synthesis of PrLZ peptides.

	FOOTNOTES

 
Grant support: United States Department of Defense (DAMD 17–03-2–0033; L. W. K. Chung, H. E. Zhau, R. Wang; and DAMD PC991274; P. S. Nelson) and NIH (CA098912 and CA76620; L. W. K. Chung; CA82739, H. E. Zhau; and CA96994, J. A. Petros).

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.

Requests for reprints: Ruoxiang Wang, Department of Urology, Emory University School of Medicine, 1365B Clifton Road, Suite B5204, Atlanta, GA 30322. Phone: (404) 778-5116; Fax: (404) 778-3965; E-mail: rwang2{at}emory.edu

⁷ Internet address: http://www.clontech.com.

Received 10/22/03. Revised 1/15/04. Accepted 1/19/04.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 

1. Thalmann G. N., Anezinis P. E., Chang S. M., Zhau H. E., Kim E. E., Hopwood V. L., Pathak S., von Eschenbach A. C., Chung L. W. Androgen-independent cancer progression and bone metastasis in the LNCaP model of human prostate cancer. Cancer Res., 54: 2577-2581, 1994.[Abstract/Free Full Text]
2. Keller E. T. The role of osteoclastic activity in prostate cancer skeletal metastases. Drugs Today (Barc.), 38: 91-102, 2002.
3. Taichman R. S., Cooper C., Keller E. T., Pienta K. J., Taichman N. S., McCauley L. K. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone. Cancer Res., 62: 1832-1837, 2002.[Abstract/Free Full Text]
4. Zhau H. Y., Chang S. M., Chen B. Q., Wang Y., Zhang H., Kao C., Sang Q. A., Pathak S. J., Chung L. W. Androgen-repressed phenotype in human prostate cancer. Proc. Natl. Acad. Sci. USA, 93: 15152-15157, 1996.[Abstract/Free Full Text]
5. Lin B., White J. T., Ferguson C., Bumgarner R., Friedman C., Trask B., Ellis W., Lange P., Hood L., Nelson P. S. PART-1: a novel human prostate-specific, androgen-regulated gene that maps to chromosome 5q12. Cancer Res., 60: 858-863, 2000.[Abstract/Free Full Text]
6. Saramaki O., Willi N., Bratt O., Gasser T. C., Koivisto P., Nupponen N. N., Bubendorf L., Visakorpi T. Amplification of EIF3S3 gene is associated with advanced stage in prostate cancer. Am. J. Pathol., 159: 2089-2094, 2001.[Abstract/Free Full Text]
7. Byrne J. A., Tomasetto C., Garnier J. M., Rouyer N., Mattei M. G., Bellocq J. P., Rio M. C., Basset P. A screening method to identify genes commonly overexpressed in carcinomas and the identification of a novel complementary DNA sequence. Cancer Res., 55: 2896-2903, 1995.[Abstract/Free Full Text]
8. Visakorpi T., Kallioniemi A. H., Syvanen A. C., Hyytinen E. R., Karhu R., Tammela T., Isola J. J., Kallioniemi O. P. Genetic changes in primary and recurrent prostate cancer by comparative genomic hybridization. Cancer Res., 55: 342-347, 1995.[Abstract/Free Full Text]
9. Byrne J. A., Mattei M. G., Basset P. Definition of the tumor protein D52 (TPD52) gene family through cloning of D52 homologues in human (hD53) and mouse (mD52). Genomics, 35: 523-532, 1996.[CrossRef][Medline]
10. Chen S. L., Maroulakou I. G., Green J. E., Romano-Spica V., Modi W., Lautenberger J., Bhat N. K. Isolation and characterization of a novel gene expressed in multiple cancers. Oncogene, 12: 741-751, 1996.[Medline]
11. Boutros R., Bailey A. M., Wilson S. H., Byrne J. A. Alternative splicing as a mechanism for regulating 14–3-3 binding: interactions between hD53 (TPD52L1) and 14–3-3 proteins. J. Mol. Biol., 332: 675-687, 2003.[CrossRef][Medline]
12. Sathasivam P., Bailey A. M., Crossley M., Byrne J. A. The role of the coiled-coil motif in interactions mediated by TPD52. Biochem. Biophys. Res. Commun., 288: 56-61, 2001.[CrossRef][Medline]
13. Wilson S. H., Bailey A. M., Nourse C. R., Mattei M. G., Byrne J. A. Identification of MAL2, a novel member of the mal proteolipid family, though interactions with TPD52-like proteins in the yeast two-hybrid system. Genomics, 76: 81-88, 2001.[CrossRef][Medline]
14. Thomas D. D., Kaspar K. M., Taft W. B., Weng N., Rodenkirch L. A., Groblewski G. E. Identification of annexin VI as a Ca2+-sensitive CRHSP-28-binding protein in pancreatic acinar cells. J. Biol. Chem., 277: 35496-35502, 2002.[Abstract/Free Full Text]
15. Proux-Gillardeaux V., Galli T., Callebaut I., Mikhailik A., Calothy G., Marx M. D53 is a novel endosomal SNARE-binding protein that enhances interaction of syntaxin 1 with the synaptobrevin 2 complex in vitro. Biochem. J., 370: 213-221, 2003.[CrossRef][Medline]
16. Davies J. A. Do different branching epithelia use a conserved developmental mechanism?. Bioessays, 24: 937-948, 2002.[CrossRef][Medline]
17. Balleine R. L., Fejzo M. S., Sathasivam P., Basset P., Clarke C. L., Byrne J. A. The hD52 (TPD52) gene is a candidate target gene for events resulting in increased 8q21 copy number in human breast carcinoma. Genes Chromosomes Cancer, 29: 48-57, 2000.[CrossRef][Medline]
18. Proux V., Provot S., Felder-Schmittbuhl M. P., Laugier D., Calothy G., Marx M. Characterization of a leucine zipper-containing protein identified by retroviral insertion in avian neuroretina cells. J. Biol. Chem., 271: 30790-30797, 1996.[Abstract/Free Full Text]
19. Nupponen N. N., Kakkola L., Koivisto P., Visakorpi T. Genetic alterations in hormone-refractory recurrent prostate carcinomas. Am. J. Pathol., 153: 141-148, 1998.[Abstract/Free Full Text]
20. Porkka K., Saramaki O., Tanner M., Visakorpi T. Amplification and overexpression of Elongin C gene discovered in prostate cancer by cDNA microarrays. Lab. Invest., 82: 629-637, 2002.[CrossRef][Medline]
21. Saramaki O. R., Savinainen K. J., Nupponen N. N., Bratt O., Visakorpi T. Amplification of hypoxia-inducible factor 1 gene in prostate cancer. Cancer Genet. Cytogenet., 128: 31-34, 2001.[CrossRef][Medline]

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