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Endocrinology Vol. 143, No. 12 4788-4796
Copyright © 2002 by The Endocrine Society

ARTICLE

Identification and Characterization of a Novel Testosterone-Regulated Prominin-Like Gene in the Rat Ventral Prostate

Qiuheng Zhang, Riffat Haleem, Xiaoyan Cai and Zhou Wang

Departments of Urology (Q.Z., R.H., X.C., Z.W.) and Molecular Pharmacology and Biological Chemistry (Z.W.), The Robert H. Lurie Comprehensive Cancer Center (Z.W.), The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: Zhou Wang, Ph.D., Department of Urology, Tarry 11-715, Feinberg Medical School of Northwestern University, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: wangz{at}northwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
More than two dozen androgen-responsive genes were identified from the castrated rat ventral prostate on the basis of their induction by exogenous testosterone. One of the identified genes encodes a novel 886-amino-acid protein that was named prominin-like protein 2 (PROML2) because it shares 32% identity to prominin and prominin-like protein 1, a family of important plasma membrane proteins with five transmembrane domains. The rat PROML2 gene is expressed abundantly in the glandular epithelial cells of the rat ventral prostate. The PROML2 gene is expressed in the human prostate and human prostate cancer cell lines with the highest level in less aggressive LNCaP cells and low expression in highly aggressive PC3 and DU145 cells, suggesting a correlation between PROML2 down-regulation and aggressiveness of prostate cancer cells. Transient transfection of green fluorescent protein-tagged rat PROML2 expression vector into prostate cancer cell lines showed that PROML2 protein is localized to the nuclear envelope and perinuclear region and induces cell death in all of the transfected prostate cancer cells. Taken together, our results argue that PROML2 is a novel proapoptotic membrane protein and its down-regulation may associate with aggressive phenotype of prostate cancer cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGENS ARE REQUIRED for the maintenance of structural and functional integrity of the prostate (1). Androgen ablation by castration induces dramatic prostatic regression via apoptosis (2, 3), and androgen replacement stimulates the regrowth of a regressed prostate (4). The regrowth stops once the prostate reaches a normal size. These observations have led Bruchovsky et al. (1) to postulate that androgens induce not only mitogenic factors but also nullifiers that terminate growth once the number of prostatic cells is restored to the normal level. The regrowth and maintenance of prostatic structural and functional integrity require a coordinated interplay between mitogens, nullifiers, and other factors.

Androgens play an important role in prostate cancer progression (5, 6, 7). Most, if not all, prostate cancers are androgen dependent in the early phase of their progression. Androgen ablation is the standard therapy for patients with metastatic prostate cancer. Unfortunately, prostate cancer patients eventually relapse with androgen-refractory prostate tumors. The mechanisms responsible for prostate tumor transition from an androgen-dependent state to a lethal androgen-independent state are poorly understood. Elucidation of the basic mechanism of androgen action in the prostate will facilitate the investigation of androgen-refractory prostate cancers.

Androgen regulation of cell proliferation, differentiation, and apoptosis is mediated through the androgen receptor, a ligand-dependent transcription factor that controls the expression of androgen-responsive genes (8, 9). Identification and characterization of prostatic androgen-responsive genes will undoubtedly provide important insights into the mechanism of androgen action and the role of androgens in prostate cancer progression. To elucidate the mechanism of androgen action, we have conducted an extensive search for androgen-responsive genes on the basis of their induction by androgen replacement in 7-d castrated rat ventral prostate using gene expression screen, a PCR-based cDNA subtraction method (10, 11). Our effort has led to the identification of more than 20 prostatic androgen-responsive genes. One of the identified genes (previously named U20) encodes a novel protein with about 32% identity to prominin and prominin-like protein 1 (PROML1). Thus, we renamed U20 prominin-like protein 2 (PROML2).

Prominin is a transmembrane protein initially identified from mouse neuroepithelium by Weigmann et al. (12). Mouse prominin and human prominin-like 1 (also called AC133 antigen or CD133) share about 61% identity in protein sequence and are thought to be the murine and human orthologues (13, 14, 15, 16). This emerging family of the multispan membrane proteins is predicted to have five transmembrane domains and multiple glycosylation sites. Prominin is expected to exhibit a novel topology. This family of proteins is conserved evolutionarily and distributed widely throughout the animal kingdom. Prominins are primarily localized to microvilli and other plasma membrane protrusions via a novel cholesterol-based lipid microdomain, suggesting a role for prominin in plasma membrane protrusion morphogenesis. A frame shift mutation in prominin-like 1 in human was reported to be responsible for retinal degeneration, probably due to defects in generation of the evaginations and/or conversion of the evaginations to disks (15). The above information indicates that prominins are functionally important.

Identification of PROML2 adds a new member to the prominin family. Investigating the expression and function of PROML2 will enhance our understanding of this family of pentatransmembrane proteins and provide insights into the role of PROML2 in the prostate. This paper describes the characterization of PROML2 expression, intracellular localization, and potential functions in androgen action and prostate cancer progression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Young adult male Harlan Sprague Dawley rats (250–300 g) were purchased from Harlan, Inc. (Indianapolis, IN). The rats were castrated by removing testes, fat pads, and epididymis in a room dedicated for animal manipulation according to a protocol approved by the Northwestern University Animal Care and Use Committee (Chicago, IL). The castrated animals were maintained in the Northwestern University animal facility. Androgen replacement of the castrated rats was carried out 7 d after castration by daily sc injections of testosterone propionate at 2 mg/rat. For each experimental condition, at least three rats were killed by decapitation after anesthesia. The ventral prostate lobes were removed, weighed, and immediately frozen in liquid nitrogen before RNA isolation. Additional tissues including the liver, kidney, heart, brain, muscle, seminal vesicles, and testis were also collected for tissue specificity studies.

Cell lines
PC3, DU145, and LNCaP human prostate cancer cell lines were obtained from American Type Culture Collection (ATCC Manassas, VA). These cell lines were maintained in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal calf serum, 1% glutamine, and 1% penicillin-streptomycin (Invitrogen Carlsbad, CA) at 37 C in the presence of 5% CO2 in a humidified incubator.

Cloning and sequencing of full-length rat PROML2 (rPROML2) cDNA
A full-length cDNA clone encoding for the rPROML2 was isolated from a {lambda}-ZAP phage cDNA library derived from the normal rat ventral prostate. In vivo excision of the {lambda}-ZAP phage resulted in a plasmid pBluescript II SK with the rPROML2 cDNA inserted at the EcoRI and XhoI sites. To sequence the PROML2 cDNA, 5' and 3' nested deletion mutants were generated using the Erase-a-Base kit (Promega Corp. Madison, WI). Both strands of the cDNA were sequenced using ALFExpress automated sequencing machine (Amersham Biosciences Piscataway, NJ). Sequence assembly and analysis were performed using the DNASIS program (Hitachi Software South San Francisco, CA).

RNA isolation and Northern blot analysis
Total RNA was isolated using the guanidinium/CsCl gradient method. Purified RNA samples were fractionated in a 1% agarose-formaldehyde gel. Ten micrograms of total RNA sample were loaded in each lane. After electrophoresis, RNA was transferred to a nylon membrane by capillary blotting and then cross-linked to the membrane by UV irradiation. Northern blot hybridization of the membrane was carried out at 42 C overnight in a buffer containing 5x SSPE, 2x Denhardt’s solution, 0.1% sodium dodecyl sulfate (SDS), 100 µg/ml denatured salmon sperm DNA, and 50% formamide in the presence of DNA probes labeled by random priming. The membrane was then washed at room temperature with 1x SSC and 0.1% SDS for 20 min followed by three 20-min washes at 65 C with 0.2x SSC and 0.1% SDS. Full-length rPROML2 cDNA was used in the Northern blot analysis of rat RNA samples. The RT-PCR product of hPROML2 was used in the Northern blot analysis of human RNA samples.

In situ hybridization
Digoxigenin (DIG)-labeled rPROML2 sense and antisense RNA probes for in situ hybridization were synthesized using linearized, proteinase K-treated plasmid DNA templates. A full-length rPROML2 cDNA, inserted at the multiple cloning sites of pBluescript II SK plasmid, was used in template preparation. Synthesis of sense and antisense RNA probes was carried out by in vitro transcription using DIG RNA labeling mix of nucleotides (Roche Molecular Biochemicals Indianapolis, IN) with either T3 or T7 RNA polymerase (Promega Corp). In situ hybridization analysis of rPROML2 expression in the rat ventral prostate was carried out the same as described previously (17).

RT-PCR
RT-PCR was performed to evaluate hPROML2 expression in human prostate cancer cell lines using the One-Step-RT-PCR kit (Invitrogen Carlsbad, CA). Two hPROML2 gene specific primers were designed based on the human homolog sequence (GenBank accession no. NM_144707). The primer sequences are 5'-GTGGTGAAGACCAGCATGGAGC-3' (hPROML2 sense from nucleotides 2021 to 2042) and 5'-GATGTGGAAGAGCTGAGTCTC-3' (hPROML2 antisense from nucleotides 2591 to 2611). The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was also assayed as a control in the RT-PCR. The primers for GAPDH gene are 5'- ACCACAGTCCATGC CATCAC-3' (GAPDH sense) and 5'-TACAGCAACAGGGTGGT GGA-3' (GAPDH antisense).

The cDNA synthesis was performed using 50 ng of total RNA as template at 50 C for 30 min. The PCR amplification program consisted of denaturation at 94 C for 30 sec, annealing at 50 C for 30 sec, and extension at 72 C for 90 sec. After 20 cycles of amplification, the RT-PCR products were analyzed on a 1% agarose gel. The 591-bp RT-PCR product was sequenced and verified that it is derived from hPROML2.

Cloning of rPROML2 expression vector and cell transfection
The rPROML2 coding region was generated by PCR with anchor primers (I) 5'-ATTTCCTCGAGGCCACCATGGTGCCTCTGCTGGGC-3', (II) 5'-GAATTCCCGCGGTCACGAGCAGAAAGAGTA-3'. Underlined sequence within primer I is a BglII site and in primer II a SacII site. Boldface ATG is the initiation codon, and boldface TCA is the stop codon in antisense orientation. The rPROML2 coding region was cloned into pEGFP-C1 and pEGF-N1 (CLONTECH Laboratories, Inc., Palo Alto, CA), which are the green fluorescent protein (GFP) expression vectors for creating fusion proteins with GFP at the N or C terminus. The PCR-amplified rPROML2 coding regions were digested with BglII and SacII and then inserted into the BglII and SacII sites of the pEGFP-C1 and pEGFP-N1 vectors. The pEGFP-C1-PROML2 and pEGFP-N1-PROML2 constructs were confirmed by restriction digestion and sequencing. The plasmid DNAs for transient transfection were prepared using double-CsCl gradient banding.

The pEGFP-C1-PROML2 or pEGFP-N1-PROML2 construct was then transfected into PC3, DU145 and LNCaP cells by using FuGENE 6 (Roche Molecular Biochemicals). Transfection was carried out according to the manufacturer’s instruction. PC3, DU145, or LNCaP cells were plated in six-well plates and allowed to grow to 20% confluence on the day of transfection. For each well of cells, 4 µg of DNA and 10 µl of the FuGENE 6 transfection reagent were used. The DNA and transfection reagent were diluted separately, each in 100 µl of Opti-MEM reduced serum medium. The diluted DNA and FuGENE 6 were mixed and allowed to form DNA-lipid complexes at room temperature for 20 min before addition to cultured cells in 2 ml serum-free Roswell Park Memorial Institute 1640 medium. The GFP was visualized via fluorescent microscopy 16 h after transfection.

Confocal microscopy
Cells were cultured on Bio-coat cover slips (Fisher ScientificPittsburgh, PA) placed in six-well plates. The cells were then fixed in 4% paraformaldehyde for 30 min at room temperature and visualized under Zeiss LSM510 Laser Scanning Confocal Microscope (Zeiss, Jena, Germany) at the Cell Imaging Facility at Northwestern University.

Hoechst staining
Cell death was studied morphologically by staining the nuclei with Hoechst 33342 (Molecular Probes Eugene, OR). Cultured cells were stained directly with 10 µM Hoechst 33342 for 10 min and then analyzed under a fluorescence microscope (Leica Corp. Wetzlar, Germany).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sequence analysis of rPROML2 cDNA
One of the androgen-responsive genes, initially called up-regulated gene 20 (U20) (11), encodes a protein related to prominin, sharing 32% identity with prominin and prominin-like 1 (PROML1) (Fig. 1Go). Thus, we renamed U20 prominin-like protein 2 (PROML2) (accession no. AF486828). The rPROML2 cDNA we cloned consists of a 2658-bp open reading frame, a 111-bp 5' untranslated region, and a 1522-bp 3' untranslated region. Hydropathy plot analysis (Protean, DNAStar, Madison, WI) of rPROML2 amino acid sequence revealed five hydrophobic regions that constitute putative transmembrane segments (Fig. 1Go). A database search with rPROML2 cDNA sequence identified a mouse prominin-like protein (accession no. AF128113) that shares 90% homology to the rPROML2 cDNA. This mouse prominin-like protein is likely to be the mouse counterpart of the rPROML2 (mPROML2). Human also appears to have a counterpart for the rPROML2. Our database search found a human genomic DNA fragment (accession no. AC009238.3) in chromosomal region 2q11.1 containing putative exons that share about 90% identity with the rPROML2 cDNA at the nucleotide level. These observations indicate that PROML2 sequence is conserved, at least in mammals.



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  		Figure 1. Predicted amino acid sequences of rat PROML2 (accession no. AF486828), mouse prominin (accession no. NM-008935), and human PROML1 (accession no. NM-006017). Underlines represent the five putative transmembrane domains, and asterisks (*) under prominin sequence indicate putative N-glycosylation sites. Broken lines (- -) represent gaps in amino acid sequences.

 
PROML2 lacks putative N-glycosylation sites (Fig. 1Go). Prominin and PROML1 are N-glycosylated (14, 18). Prominin has been predicted to have 8 putative N-glycosylation sites in its extracellular domains (18). However, these putative N-glycosylation sites found in prominin are not present in PROML2, suggesting that PROML2 may not undergo N-glycosylation.

Androgen regulation of rPROML2 in the rat ventral prostate
PROML2 was identified initially as an androgen-responsive gene. To further characterize androgen regulation of PROML2 expression, we have carried out Northern blot analysis of total RNA samples isolated from ventral prostates of the 7-d castrated rats and the castrated rats treated with testosterone replacement (Fig. 2Go). The induction of rPROML2 mRNA was initially observed within 6.5 h and reached maximum 36 h after the androgen replacement. rPROML2 induction is similar to the induction of early androgen-responsive genes, including prostatein C3, adrenomedullin, and calreticulin, which also occurred within 6.5 h and peaked 24 h after the androgen replacement (11). Therefore, rPROML2 is likely to be an early androgen-responsive gene.



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  		Figure 2. Northern blot analysis of total RNA from the ventral prostate of 7-d castrated rats treated with testosterone propionate for indicated number of hours. The amount and quality of total RNA loaded in the gels were examined by staining the transferred nylon membrane with methylene blue. Full-length rPROML2 cDNA was used as the template in probe synthesis. The experiment was reproduced once.

 
There are three mRNA bands, with sizes at about 5.5 kb, 4.3 kb, and 3.6 kb (Fig. 2Go). The rPROML2 cDNA clone contains an insert of 4.3 kb, which is probably derived from the 4.3 kb rPROML2 mRNA. Different sizes of rPROML2 mRNA are likely resulted from alternative splicing or incomplete mRNA processing. However, we cannot rule out the possibility of partial mRNA degradation.

Localization of rPROML2 mRNA expression in the rat ventral prostate
To further characterize the expression of rPROML2 gene, we performed in situ hybridization analysis using the intact rat ventral prostate. The result in Fig. 3Go showed that rPROML2 mRNA is abundantly expressed in the glandular epithelial cells. No staining was observed in prostatic stromal cells despite the strong staining in the epithelial cells, suggesting that PROML2 expression is epithelial specific in the prostate.



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  		Figure 3. rPROML2 mRNA localization in the rat ventral prostate. In situ hybridization analysis was conducted using antisense (top panel) and sense (bottom panel) rPROML2 RNA probes labeled with DIG. The hybridized probes were visualized with alkaline phosphate conjugated with an anti-DIG antibody. The glandular epithelial cells (E) and stromal cells (S) were indicated. The experiment was reproduced once.

 
Tissue specificity of PROML2 mRNA expression
To test whether PROML2 is regulated by androgens in the prostate specifically, we have carried out Northern blot analysis of total RNA isolated from the liver, kidney, heart, brain, muscle, prostate, and seminal vesicles of the testis-intact rats, castrated rats, and castrated rats followed by 2 d of androgen replacement. The testis RNA was also included in the Northern blot analysis. Our total RNA Northern blot analysis did not detect rPROML2 expression in the liver, kidney, heart, brain, muscle, seminal vesicles, and testis (Fig. 4AGo). The result shown in Fig. 4AGo indicates that rPROML2 mRNA is expressed abundantly and regulated by androgen manipulation in the rat ventral prostate. Because we have only examined eight different rat tissues, it is possible that rPROML2 is expressed in untested tissues.



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  		Figure 4. Northern blot analysis of tissue specificity of PROML2 expression. A, Northern blot analysis of rPROML2 mRNA of different tissues during androgen manipulation in rat. N, Tissue from the testis-intact rats; -, tissue from 7-d castrated rats; +, tissue from the rats castrated for 7 d followed by androgen replacement for an additional 2 d. The amount and quality of total RNA loaded in the gels were examined by staining the transferred membrane with methylene blue. The rPROML2 cDNA probe was used in the Northern blot analysis. The experiment was reproduced once. B, Northern blot analysis of PROML2 expression in different human tissues. Multiple-tissue poly A+ RNA membranes (catalog nos. 7780-1 and 7784-1; CLONTECH Laboratories, Inc.) were hybridized with a labeled hPROML2 cDNA fragment. The ß-actin expression was used as a control.

 
To determine whether PROML2 is also abundantly expressed in the human prostate, we performed Northern analysis on two CLONTECH Laboratories, Inc. filters containing poly A+ RNA samples isolated from 24 different human tissues (Fig. 4BGo). Abundant hPROML2 expression was observed in the prostate and several other tissues including the placenta, liver, kidney, trachea, thyroid, stomach, spinal cord, mammary gland, and adrenal gland. The above-described observation indicates that the tissue specificity of PROML2 expression in human is different from that in the rat.

Northern blot analysis (Fig. 4BGo) detected several bands in the human poly A+ RNA samples. Similar to the rPROML2 transcripts, the hPROML2 also contains the transcripts with 5.5 kb, 4.3 kb, and 3.6 kb in size. The 4.3-kb transcripts is present in every tissue that expresses PROML2. In addition to the 5.5-, 4.3-, and 3.6-kb transcripts, smaller size bands were also observed in the Northern blot analysis (Fig. 4BGo). It is not clear whether these small RNA bands were derived from alternative splicing or partial degradation of hPROML2 mRNA.

Expression of PROML2 in human prostate cancer cell lines
Because prostate tumors are derived from glandular epithelial cells that express PROML2, we studied the expression of PROML2 in prostate cancer cell lines. hPROML2 mRNA expression is very low in prostate cancer cells and difficult to be detected by Northern blot analysis (data not shown). RT-PCR analysis was carried out to compare the expression of hPROML2 in LNCaP (19), PC3 (20), and DU145 (21) human prostate cancer cell lines. Figure 5Go showed that the hPROML2 expression level in LNCaP cells is significantly higher than that in PC3 and DU145 cells.



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  		Figure 5. Expression of hPROML2 in LNCaP, PC3, and DU145 prostate cancer cell lines. GAPDH was used as an internal control in the RT-PCR. The experiment was reproduced twice. RT-PCR was carried out as described in Materials and Methods.

 
Subcellular localization of rPROML2-GFP fusion proteins
Prominin and PROML1 proteins are localized to plasma membrane (14, 22). To determine whether PROML2 is also localized to plasma membrane, we cloned the rPROML2 cDNA into pEGFP-C1 and pEGFP-N1 expression vectors that express rPROML2 fusion protein with GFP tagged either at the N terminus or C terminus of rPROML2 protein. Transfection of rPROML2 fusion protein expression vectors into PC3 prostate cancer cell line showed that rPROML2 fusion proteins are localized to the nuclear envelope and perinuclear region (Fig. 6Go). The localization of rPROML2 with GFP tagged at N terminus is indistinguishable from that of rPROML2 with GFP tagged at C terminus, indicating that the position of GFP tagging did not affect the localization of rPROML2. Additional transient transfection experiments showed that rPROML2 fusion proteins are also localized to the nuclear envelope and perinuclear region in DU145 and LNCaP cells (results not shown), suggesting that PROML2 intracellular localization is independent of prostate cell line models.



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  		Figure 6. Subcellular localization of rPROML2 GFP fusion proteins in PC3 cells. Cultured PC3 cells were transiently transfected with a GFP expression vector (left) or a rPROML2 fusion protein expression vector with GFP tagged either at N terminus (center) or at C terminus (right) of PROML2. The localization was visualized by a confocal microscope. The experiment was reproduced twice.

 
Overexpression of rPROML2 in prostate cancer cells induces cell death
One interesting phenomenon we observed after transfection of rPROML2-GFP fusion protein expression vectors into prostate cancer cells was that cells expressing rPROML2-GFP fusion protein were unable to survive. The transfected cells exhibited condensed nuclei and membrane blebbing, suggesting that rPROML2 overexpression induced apoptosis. To provide further evidence that apoptosis was induced by rPROML2 overexpression, we have performed Hoechst staining. As expected, Hoechst staining revealed the condensed and fragmented nuclei in the transfected cells (Fig. 7AGo). Quantitative analysis (Fig. 7BGo) showed that cell death induced by rPROML2-GFP fusion protein was very efficient, with 57% of the transfected cells dead by d 1, 87% by d 2, and 99% by d 3. In contrast, about 5% of the GFP expression vector transfected and parental PC3 cells stained positive with Hoechst dye.



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  		Figure 7. Induction of apoptosis by rPROML2 overexpression in prostate cancer cell lines. A, Hoechst staining of transfected PC3 cells. Top panels show the expression of GFP or rPROML2-GFP fusion protein visualized under green fluorescent light. Bottom panels show the Hoechst staining of the nuclei of the same fields. rPROML2-GFP-positive cells are indicated by arrows. B, Induction of cell death by rPROML2-GFP overexpression in PC3 cells. C, Induction of cell death by rPROML2-GFP overexpression in DU145 cells. D, Induction of cell death by rPROML2-GFP overexpression in LNCaP cells. Quantitative analysis of cell death was carried out by counting the percent of dead cells d 1, 2, and 3 after the transfection of a rPROML2-GFP expression vector. At least 150 transfected cells in each dish were counted and three dishes were used in each experimental condition. The cells that are detached or exhibit fragmented nuclei are considered dead. The results were based on four independent experiments.

 
To rule out the possibility that rPROML2 induced cell death is limited to PC3 cells, we transfected the rPROML2-GFP expression vector into DU145 cells and LNCaP cell lines. In transfected DU145 cells, rPROML2-GFP expression also induced very efficient cell death, with 53% by d 1, 81% by d 2, and 99.5% by d 3. Only 5% of GFP transfected or parental DU145 was positive with Hoechst staining. The effect of rPROML2-GFP expression on LNCaP cells was similar to that observed with PC3 and DU145 cells, with 48% by d 1, 73.5% by d 2, and 98.8% by d 3 (Fig. 7DGo). These observations showed that overexpression of rPROML2-GFP fusion proteins induced cell death in all of the assayed prostate cancer cell lines.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This paper describes characterization of PROML2, a novel prominin-like protein encoded by an androgen-responsive gene, in the prostate and prostate cancer cell lines. Our studies suggest that PROML2 is distinct from other members in the prominin family. We have also obtained results suggesting that PROML2 may be proapoptotic and associated with growth suppression in androgen action and prostate cancer progression.

Our studies showed that rPROML2 gene is expressed abundantly in the rat ventral prostate (Fig. 4AGo), suggesting that rPROML2 may play an important role in androgen action in the prostate. Northern blot analysis of rPROML2 expression in different rat tissues showed that its expression in the ventral prostate is much more abundant than any other assayed tissues. Human tissue-specific Northern blot analysis showed that hPROML2 is also abundantly expressed in the prostate (Fig. 4BGo). hPROML2 mRNA is also abundantly expressed in many other human tissues including placenta, liver, kidney, trachea, thyroid, stomach, spinal cord, mammary gland, and adrenal gland, suggesting that hPROML2 may play important role in many different tissues.

Our in situ hybridization analysis showed that rPROML2 mRNA is localized to the glandular epithelial cells in the rat ventral prostate (Fig. 3Go). It is likely that PROML2 is also expressed in epithelial cells of other tissues. Similarly, prominin and PROML1 proteins are known to be expressed in epithelial cells (14, 22).

PROML2 appears to be a distinct member of the prominin family. rPROML2 and prominins have only about 30% amino acid sequence identity, whereas both have 5 transmembrane domains (Fig. 1Go). The homology between rPROML2 and prominins is scattered over the entire peptide sequences. Prominin is predicted to have eight N-glycosylation sites in its extracellular domains (18, 22). In contrast, rPROML2 does not have these putative glycosylation sites (Fig. 1Go). N-Glycosylation status may influence intracellular localization of these penta-transmembrane proteins. rPROML2, which lacks putative N-glycosylation sites, is localized to the nuclear envelope and perinuclear region. In contrast, prominin, which is N-glycosylated, is localized to the plasma membrane. The functional significance of N-glycosylation or lack of it in this family of proteins remains unclear.

Our transient transfection experiments showed that overexpression of rPROML2 induced cell death in prostate cancer cells (Fig. 7Go). We have transfected rPROML2 cDNA expression vector into PC3, DU145, and LNCaP prostate cancer cell lines. The induction of cell death was detected within 1 d after transient transfection. rPROML2 induces cell death probably via an apoptotic process because the dead cells exhibited condensed and fragmented nuclei with Hoechst staining. It is remarkable that virtually all of the transfected cells undergo cell death, suggesting that apoptosis induction by PROML2 overexpression is very effective. In contrast, overexpression of prominin has not been reported to induce cell death. Our studies suggest that PROML2 is a distinct proapoptotic penta-transmemberane protein. Because proteins with proapoptotic activities are often growth suppressive, PROML2 is likely to play a growth suppressive role in the normal and cancerous prostate.

PROML2 down-regulation appears to associate with the aggressiveness of human prostate cancer cells. The level of hPROML2 mRNA in LNCaP cells is much higher than that in PC3 and DU145 cells (Fig. 5Go). LNCaP cell line is much less aggressive relative to PC3 and DU145 cell lines. These observations suggest that PROML2 down-regulation may be associated with aggressive phenotype in prostate cancers, which is consistent with the proapoptotic activity of PROML2.

As a proapoptotic protein, PROML2 may be involved in growth suppression in androgen action in the prostate. Androgen-induced prostate growth is complex. Bruchovsky et al. (1) pointed out that exogenous androgens stimulate growth only in the regressed prostate but not in the fully grown prostate. The regrowth of the prostate stops once the number of prostatic cells reaches the normal level. Prostatic regrowth requires coordinated actions of mitogenic factors that induce proliferation and nullifiers that terminate proliferation.

Endogenous PROML2 in the normal prostate in the presence of androgens is unlikely to induce apoptosis because the apoptotic activity is very low in the normal prostate. On the other hand, efficient cell death induction by PROML2 overexpression was observed in cultured prostate cancer cells. The differential effect between endogenous PROML2 in the normal prostate and transfected PROML2 in cultured cancer cells may reflect the differences between in vivo and in vitro conditions. Alternatively, PROML2 may be apoptotic in prostate cancer cells but not in the normal prostatic epithelial cells.

In conclusion, our studies showed that PROML2 mRNA is expressed abundantly and regulated by androgens in prostatic epithelial cells in vivo. PROML2 is a novel penta-transmembrane protein localized predominantly in nuclear envelope and perinuclear region. PROML2 expression is down-regulated in aggressive prostate cancer cell lines and transient transfection of PROML2 expression vectors induced apoptosis in cultured prostate cancer cells, suggesting a tumor suppressive role for PROML2. PROML2 expression is likely to be involved in growth suppression in the prostate. Down-regulation of PROML2 may disrupt normal prostatic homeostasis and lead to uncontrolled prostate growth.


   Acknowledgments
 
We thank Sui Huang for help in assessing the intracellular localization of rPROML2-GFP fusion proteins and Jomol Cyriac, Zehra Dincer, and Feng Jiang for critical reading of the manuscript.


   Footnotes
 
This work was supported by NIH Grant R01-DK-51193 and Prostate Cancer SPORE P50 CA-90386.

Abbreviations: DIG, Digoxigenin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; PROML1, prominin-like protein 1; PROML2, prominin-like protein 2; rPROML2, rat PROML2; SDS, sodium dodecyl sulfate.

Received May 17, 2002.

Accepted for publication August 2, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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 Stem CellsHome page
C. A. Fargeas, D. Corbeil, and W. B. Huttner
AC133 Antigen, CD133, Prominin-1, Prominin-2, Etc.: Prominin Family Gene Products in Need of a Rational Nomenclature
Stem Cells, July 1, 2003; 21(4): 506 - 508.
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C. A. Fargeas, M. Florek, W. B. Huttner, and D. Corbeil
Characterization of Prominin-2, a New Member of the Prominin Family of Pentaspan Membrane Glycoproteins
J. Biol. Chem., February 28, 2003; 278(10): 8586 - 8596.
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