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Endocrinology, doi:10.1210/en.2003-0415
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Endocrinology Vol. 144, No. 11 4955-4966
Copyright © 2003 by The Endocrine Society

Gene Expression Analysis of Prostate Hyperplasia in Mice Overexpressing the Prolactin Gene Specifically in the Prostate

Karin Dillner, Jon Kindblom, Amilcar Flores-Morales, Ruijin Shao, Jan Törnell, Gunnar Norstedt and Håkan Wennbo

Department of Pharmacology and Physiology, Göteborg University (K.D., J.K., R.S., J.T.), 405 30 Göteborg, Sweden; Department of Molecular Medicine, Karolinska Institute (K.D., A.F.-M., G.N.), 171 76 Stockholm, Sweden; and AstraZeneca Transgenic Center (J.T.) and Integrative Pharmacology (H.W.), AstraZeneca R&D, 431 83 Mölndal, Sweden

Address all correspondence and requests for reprints to: Dr. Karin Dillner, Department of Molecular Medicine, Karolinska Institute, CMM L8:01, Karolinska Hospital, 171 76 Stockholm, Sweden. E-mail: karin.dillner{at}cmm.ki.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The probasin (Pb)-PRL transgenic mice that overexpress the rat PRL gene specifically in the prostate develop a dramatic enlargement of the prostate gland. The objective of this study was to characterize the molecular mechanisms involved in the prostate hyperplasia seen in the Pb-PRL transgenic mice. cDNA microarray analysis was used to identify differentially expressed transcripts in the hyperplastic prostates of 6-month-old transgenic mice compared with age-matched controls. We report the identification of 266 genes (175 up-regulated and 91 down-regulated) that were differentially expressed in the enlarged transgenic prostates compared with controls. Subsequential real-time RT-PCR was used to verify a set of differentially regulated transcripts. The hyperplastic prostates of Pb-PRL transgenic mice demonstrate a molecular pattern supporting the importance of reduced degree of apoptosis for the development of the phenotype. Immunohistochemical analysis of apoptotic activity using two different markers of apoptosis (single-stranded DNA and activated caspase-3) were performed, and the results showed diminished apoptosis activity in the prostate of Pb-PRL transgenic mice compared with control prostates. The increased stromal/epithelial ratio of the Pb-PRL transgenic prostate together with up-regulation of a significant fraction of genes involved in tissue remodeling activity, including the synthesis and degradation of the extracellular matrix and changes in protease activity, suggest that activation of the stroma is involved in the development of prostate hyperplasia. Overall, the differentially expressed transcripts identified in this study show many molecular similarities between the prostate hyperplasia of PRL-transgenic mice and human prostate pathology, including both benign prostatic hyperplasia and prostate cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BENIGN PROSTATIC hyperplasia (BPH) is one of the most common conditions associated with ageing in the male population (1). BPH is characterized as a slow, progressive enlargement of the prostate gland, which eventually causes obstruction and subsequent problems with urination. The altered endocrine status of aging men is likely to be of importance for development of the disease. Testosterone and GH levels decrease with age, whereas estrogen levels increase. Conflicting data exist on changes in PRL levels with increasing age (2, 3, 4, 5).

Despite the obvious importance of BPH as a major health problem, little is known in terms of the biological processes that contribute to the pathogenesis of BPH. The BPH progression is characterized by hyperplasia of both the stromal and epithelial compartments. Although the exact etiology of BPH is unknown, it is thought to arise as a result of epithelial-stromal interactions in a certain hormonal milieu (6). Tissue growth depends upon a complex balance between the rates of cell proliferation and cell death (apoptosis). Alterations in the molecular mechanisms regulating these two processes may underlie the abnormal growth of the gland, leading to BPH and even prostate carcinoma. Quantitative analyses, comparing BPH and normal prostatic tissues, have revealed that the total increase in both stromal and epithelial cells is a result of reduced apoptotic activity in parallel with increased cell proliferation (7, 8). However, other studies comparing normal and BPH epithelium observed increased cell proliferation but similar levels of apoptosis (9). When calculating the ratio of stromal to epithelial compartments in human BPH tissue, clinical reports have firmly established a dominance of the stromal component in BPH tissue, which is in contrast to the predominance of the epithelial compartments in normal prostate (10, 11, 12, 13). Furthermore, in symptomatic BPH patients the stromal/epithelial ratio has been reported to be significantly higher than in asymptomatic patients (11). Together these observations support the long-held contention that BPH arises from changes in the fibromuscular stroma (14). This proposition was based on BPH histopathology features from which McNeal concluded that the prostate stroma undergoes an "embryonic reawakening," resulting in inductive effects of the local stroma possess, which, in turn, induces hyperplastic changes in the epithelium through stromal-epithelial interactions.

Studies in different species have demonstrated the action of PRL, both independently and in synergy with androgens, in the regulation of development, growth, as well as secretory and metabolic functions of the prostate gland (15, 16). We have previously demonstrated that ubiquitous transgenic expression of the rat PRL gene in male mice (Mt-PRL) leads to a dramatic prostate enlargement with parallel chronic hyperprolactinemia and elevated serum androgen levels (17, 18). Several other in vivo studies in rodents, have demonstrated the growth-promoting effects of PRL on the prostate (19, 20, 21). Moreover, PRL has been shown to stimulate growth and significantly increase the cell proliferation rate in human BPH organ cultures, human primary prostate epithelium, and the androgen refractory human prostate cancer cell lines PC-3 and DU145 (22, 23, 24, 25). Along with inducing proliferation, PRL has been shown to decrease epithelial apoptosis induced by androgen deprivation (26, 27).

To evaluate the role of local PRL action in the prostate under physiological androgen levels, we recently generated a new transgenic mouse model using the prostate-specific rat probasin (Pb) promoter to drive expression of the rat PRL gene. The male mice expressing the transgene (Pb-PRL) developed a significant enlargement of both the dorsolateral prostate lobe (DLP) and ventral prostate lobe (VP), evident from 10 wk of age, and the prostate enlargement progress throughout adulthood (28). Histologically, the Pb-PRL prostate displays a significant stromal hyperplasia, ductal dilation, and focal areas of epithelial dysplasia, resulting in a major increase in the stromal/epithelial ratio, similar to observations made in human BPH tissue (10, 11, 12, 13). The glandular dysplastic lesions demonstrated in the Pb-PRL prostate resemble the low grade prostatic intraepithelial neoplasia (PIN) described in the recently proposed classification of PIN in genetically engineered mice (29), and those observations may indicate a potential for malignant disease. However, no progression to high grade PIN or prostate tumor formation has yet been detected in Pb-PRL transgenic prostate. From this study we concluded that abnormal growth of the Pb-PRL prostate gland occurs primarily at the postpubertal stage and in a setting of normal serum androgen levels, thereby resembling the situation in the adult human prostate.

The objective of this study was to characterize the molecular mechanisms involved in prostate hyperplasia seen in Pb-PRL transgenic mice. Using cDNA microarray technology, we identified differential expression of genes involved in proliferation, apoptosis, and tissue remodeling in the enlarged prostate of Pb-PRL transgenic mice compared with controls. Furthermore, we examined the apoptosis activity in the different prostate lobes of transgenic and control animals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Pb-PRL transgenic mice
Transgenic mice were generated in C7BL/6JxCBA-f2 embryos by standard microinjection procedures as previously described (28). All experiments were performed on heterozygous transgenic animals, and nontransgenic littermates served as controls. All animal experimentation was conducted in accord with accepted standards of humane animal care and approved by the local ethical committee at Goteborg University.

Prostate samples
The genital tract was removed en bloc, and the individual prostate lobes (DLP and VP) were carefully dissected and separated under a stereomicroscope. For tissues that later were used for RNA preparation, dissections were carried out in RNA later (Qiagen, Chatsworth, CA), subsequently snap-frozen in liquid nitrogen, and stored at -70 C until RNA preparation. Prostate used for immunohistochemistry analysis were dissected in calcium- and magnesium-free Hanks’ solution.

Generation of cDNA microarrays
Approximately 6250 cDNA clones were selected from the TIGR Rat Gene Index (www.tigr.org), Research Genetics, and our own collection of rat and mouse cDNA libraries (30, 31). Procedures for array fabrication, including quality controls and sequence verification, have been described previously (32, 33).

RNA preparation, cDNA labeling, purification, and hybridization
Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The quality of all RNA samples was ascertained on a denaturing agarose gel. Animals at 6 months of age were used. cDNA generated from each experimental group was synthesized from 20 µg pooled total RNA (control, n = 5; transgenic, n = 4) using a cDNA synthesis kit (Promega Corp., Madison, WI). The protocol employed for probe labeling and purification was essentially that described previously (34). Labeled cDNA was produced by oligo-deoxythymidine-primed RT reaction using SuperScript II (Life Technologies, Inc., Grand Island, NY). Oligo-deoxythymidine primers and cyanine (Cy)-labeled nucleotides were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). Cy3- and Cy5-labeled cDNA probes were combined and purified using Microcon 30 (Millipore Corp., Bedford, MA). The labeled and purified cDNA was added to the array at a final volume of 25 µl in hybridization buffer [0.75 M NaCl, 75 mM sodium citrate, 0.2% sodium dodecyl sulfate, 10 µg poly(A) RNA, and 10 µg yeast tRNA]. The array was covered by a plastic 22 x 40-mm coverslip (Grace Biolabs, Bend, OR) and put in a sealed hybridization chamber (Corning, Inc., Corning, NY). After hybridization, which took place at 65 C for 15–18 h, the array was washed (34) and dried. Three independent experiments were performed for DLP and VP, respectively.

Image analysis, data acquisition, and statistical evaluation
The array was scanned using a GMS 418 scanner (Affymetrix, Santa Clara, CA). Image analysis was performed using GenePix Pro software (Axon Instruments, Foster City, CA). Automatic flagging was used to localize absent or weak spots (<=2 times above background), which were excluded from analysis. The value of the signal from each spot was calculated as the average intensity minus the background. To allow for interarray comparisons, each array was normalized using the Pin-wise LOWESS normalization method in the SMA (Statistics of Microarrays Analysis) package (35, 36) as described in detail previously (31, 37). The variability of the repeated experiments was estimated using SAM (Significance Analysis of Microarray) software (38). SAM is a statistical technique for finding significantly regulated genes in a set of microarray experiments. Briefly, SAM assigns a score to each transcript on the basis of changes in gene expression relative to the SD of multiple independent measurements. Thereby, SAM allows us to select differentially regulated genes based on estimation of the percentage of genes identified as differentially regulated by chance, the so-called false discovery rate (FDR). To each of the genes in the array a q value is assigned. This value is similar to the familiar p value and measures the lowest FDR at which the gene is called significant. Only genes were expression ratios could be measured in all replicates where used in the analysis. According to the SAM analysis, the list of differentially expressed genes (see the supplemental data published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org/) has an associated FDR of less than 5%, meaning that of the 266 genes identified, less than 14 are expected to be falsely classified as differentially regulated. To the statistically based criteria we have added a further requirement based on the absolute changes in expression ratios. Only genes with average changes of more than 70% were counted as differentially expressed. Although lower levels of changes in gene expression may have important biological consequences, insufficient information exists regarding its reproducibility by independent methodologies. As most of the array elements are rat cDNA clones, and the RNAs analyzed were of mouse origin, we performed a homology search where rat cDNA sequences were compared with the mouse established sequence tag division of the GenBank database using the BLAST program (39). The identity of the mouse clone with the highest sequence homology to the corresponding rat clone is shown in the supplemental data together with the e-value and the percentage of identity. For each of the mouse genes reported here as being regulated based on a rat probe (array element), a highly homologous mouse clone could be identified (e-value <= e-10). Hence, the high level of homology between rat and mouse genes used in this study justifies the use of rat cDNA clones to measure expression in tissues of mouse origin. According to the BLAST results, the possibility of misidentifying a mouse gene as being differentially regulated based on an orthologous rat probe is very small and is comparable to the likelihood of cross-hybridization between two distinct genes within the same species (rat). Moreover, significant cross-hybridization with homologies above 80% has been previously reported using a similar technology (40), which further supports the use rat probes to measure orthologous mouse genes. All values are expressed as the mean ± SEM.

Real-time RT-PCR verification
Ten different rat cDNA clones were selected for real-time RT-PCR verification. The verification was performed using mouse-specific primers for the orthologous mouse genes. Gene-specific real-time RT-PCR primers were designed in the coding sequence for respective mouse transcripts using the PrimerExpress software (PerkinElmer/PE Applied Biosystems, Foster City, CA). These sequences can be seen in Table 2Go. Total RNA was extracted from control and transgenic animals, respectively (controls, n = 5; transgenics, n = 4) as described above. Pooled total RNA was treated with deoxyribonuclease I (DNA-free; Ambion, Austin, TX), followed by cDNA synthesis using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. The analysis was performed using the NM_007475 Mus musculus acidic ribosomal phosphoprotein PO (Arbp; forward, 5'-GAG GAA TCA GAT GAG GAT ATG GGA-3'; reverse, 5'-AAG CAG GCT GAC TTG GTT GC-3') as an internal standard. A PCR Mastermix was prepared using SYBR Green PCR Core Reagents (PerkinElmer/PE Applied Biosystems) and was aliquoted into microplate wells together with 5 µl template and 10 pmol of each primer for a final volume of 20 µl/reaction. The ABI PRISM 7700 Sequence Detection System (PerkinElmer/PE Applied Biosystems) was used for PCR and detection of the fluorescent signal. For data evaluation, the comparative threshold cycle (CT) method was used according to the manufacturer’s instructions, and the level of significance was set to a 1.7-fold relative difference between samples (CT < 0.6 or CT > 1.7). The analysis was performed at the lobe level (DLP and VP, respectively) in three replicates.


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  		TABLE 2. List of designed primers for the real-time RT-PCR analysis

 
Immunohistochemistry analysis
New sets of transgenic (n = 5) and wild-type (n = 5) prostate lobes (DP, LP and VP) were removed as described and thereafter fixed in 4% paraformaldehyde in PBS (pH 7.4) for 6 h, processed, and embedded in paraffin blocks for sectioning. Six-month-old animals were used. Fixation times were identical in transgenic and control groups. All tissues from transgenic and control mice were processed in the same assay to minimize discrepancies due to variability in staining intensity. Analysis was performed blind to genotype. Images were digitally obtained as described in the following paragraphs. Fields were selected by a systematic random sampling scheme, and genotype information was not recorded, so that the subsequent analysis was blind to genotype.

Fluorescence immunohistochemical detection of activated caspase-3
Embedded tissue was sectioned at 5 µm, mounted onto poly-L-lysine-coated slides, deparaffinized in xylene, and rehydrated through a graded series of ethanol. After washing with Tris-buffered saline (TBS) three times, nonspecific binding was blocked by incubating sections in 3% BSA and 10% normal goat serum in TBS with 0.5% Tween 20 for 1 h at room temperature. The activated caspase-3 primary antibody (BD PharMingen, San Diego, CA) was diluted 1:250 in TBS containing 3% BSA and incubated overnight at 4 C in a humidified chamber. After washing with TBS, immunodetection was accomplished using a biotin-conjugated antirabbit antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc.). Sections were washed and mounted with Fluorescent Mounting Media (DAKO, Carpinteria, CA). Slides were viewed on a Nikon E-1000 microscope (Tokyo, Japan) and photomicrographed using Easy Image 1 (Bergström Instrument AB, Solna, Sweden). The specificity of activated caspase-3 primary antibody was monitored in the separate control sections and confirmed by processing with TBS containing 1% BSA and normal rabbit serum (DAKO) with equivalent concentrations in place of primary antibodies. Additional control sections were processed as described above but with the same volume of distilled water substituted for omission of primary antibodies. All controls gave negative results.

Visualization of apoptosis
Immunohistochemistry was used for detection of denatured DNA with a monoclonal antibody against single-stranded DNA (ssDNA) to identify specific apoptotic cells as described previously (41). Deparaffinized sections were subsequently treated with PBS containing 0.2 mg/ml saponin (Sigma-Aldrich Corp., St. Louis, MO) and 20 µl/ml proteinase K (Roche, Mannheim, Germany) for 20 min at room temperature. Sections were then washed with distilled water and heated in 50% formamide prewarmed to 56 C for 20 min. After rinsing three times for 5 min each time in ice-cold PBS, sections were treated to remove endogenous peroxidase activity with 3% hydrogen peroxide in PBS for 10 min, blocked for nonspecific binding by 1% BSA and 3% nonfat dry milk for 30 min, and then incubated with anti-ssDNA primary antibody (monoclonal antibody F7–26, Alexis Biochemicals, San Diego, CA) with a dilution of 10 µg/ml in PBS containing 1% nonfat dry milk for 1 h at 4 C in a humidified chamber. After washing with PBS, sections were stained using the avidin-biotinylated-peroxidase complex detection system (ABC kit, Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s instructions. Immunostaining was then visualized using 3,3-diaminobenzidine tetrahydrochloride (0.5 mg/ml in PBS and 0.01% H2O2, pH 7.6) for 2 min. Sections were consecutively counterstained with Richardson’s modified methylene blue for 1 min, dehydrated, and coverslipped using Histo Mounting Medium (Mountex, Histolab, Sweden). Slides were viewed on a Nikon E-1000 microscope under brightfield optics and were photomicrographed using Easy Image 1 (Bergström Instrument AB). Negative controls were processed in an identical manner, except anti-ssDNA primary antibody was omitted, incubation was performed with either normal mouse IgG (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) or 100 U/ml S1 nuclease (Sigma-Aldrich Corp.) in acetate buffer after rinsing in ice-cold PBS or PBS with 1% nonfat dry milk, and the controls yielded no reaction product in parallel experiments (data not shown).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Global analysis of gene expression in the enlarged prostate lobes of prostate-specific PRL transgenic mice
cDNA microarray analysis was used to examine the molecular mechanisms involved in the prostate hyperplasia of Pb-PRL transgenic mice overexpressing the PRL gene specifically in the prostate. Global changes in gene expression were analyzed using a cDNA microarray chip containing about 6250 cDNAs of rat or mouse origin. The gene expression analysis was performed in the DLP and VP, separately. We have chosen to denote genes as differentially regulated if their level of expression was changed by 70% (corresponding to a fold change of 1.7) or more in a statistically significant fashion. This level of change has previously been shown to be valid compared with other direct methods, such as Northern blot, ribonuclease protection assay, and RT-PCR (32, 33, 37, 42) (see also Table 3Go). The identities of genes showing variation by a factor of 1.7 or more are shown in Table 1GoGo (the complete list of differentially expressed genes can be found in the supplemental data published on The Endocrine Society’s Journals Online web site, http://endo.endojournals.org/). Of the 6250 cDNA clones analyzed, 2344 showed hybridization in all independent determinations (DLP, 2003; VP, 1962). Of those, 266 nonredundant transcripts were found to be differentially expressed (175 up-regulated and 91 down-regulated) in the enlarged prostate of PRL transgenic mice compared with controls in at least 1 of the lobes analyzed. Figure 1AGo is a diagram that schematically illustrates the total number of differentially expressed transcripts in each lobe as well as the number of transcripts that were commonly differentially expressed in both lobes. One hundred and fifty-nine were differentially expressed in the DL lobe (111 up and 48 down) and 224 in the VP (159 up and 65 down). Of those, 117 transcripts were commonly differentially expressed in both DLP and VP (95 up and 22 down). As many as 84 of the 266 nonredundant differentially expressed transcripts were transcripts with unknown function, identified as established sequence tags. The differences between the gene expressions of VP and DLP can reflect biological differences and/or be a consequence of the limited detection sensitivity associated with the technique of cDNA microarray. Consequently, we have not paid specific attention to these differences.


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  		TABLE 3. Confirmation of altered expression levels in the prostates of Pb-PRL transgenic mice compared to controls with real-time RT-PCR

 

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  		TABLE 1. Differentially expressed transcripts in the Pb-PRL transgenic prostates compared to controls

 

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  		TABLE 1A. Continued

 


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  		FIG. 1. A, The total number of differentially expressed genes, determined by DNA microarray technology, DLP and VP of Pb-PRL transgenic compared with wild-type mice. All animals used were 6–7 months of age. RNA was separately isolated from DLP and VP of Pb-PRL transgenic (n = 4) and age-matched control (n = 5) mice. Analysis was performed as detailed in Materials and Methods. Genes were denoted as differentially regulated if their level of expression was changed by 70% or more in a statistically significant fashion. The left circle represents the DLP, and the right circle represents the VP. Overlapping regions indicate the differentially expressed genes that were commonly regulated in both lobes. The number of genes in the areas that do not overlap indicates the total number of differentially regulated genes in respective lobe. B, Functional classification of differentially expressed transcript, defined as more than 70% variation, in DLP and VP of Pb-PRL transgenic compared with control mice. The length of the bars represent the number of differentially regulated genes in each functional category. Within each functional category, different colors indicate the number of genes that were up-regulated (red) and down-regulated (green), respectively. Solid colors indicate the number of genes differentially expressed in both DLP and VP. Patterns indicate the number of genes differentially expressed specifically in one of the lobes. Transcripts with unknown function are not shown.

 
Functional classification of the differentially expressed genes
The differentially expressed transcripts were categorized based on their known functions. As each gene may have diverse physiological and cellular functions, this functional grouping primarily serves to facilitate data visualization. Effects were seen in virtually all cellular processes (Fig. 1BGo). The two largest functionally categorized groups, of both the DLP and VP are those of signal transduction and cell tissue structure. The majority of the genes in these two categories were highly expressed in the hyperplastic relative to the control prostate lobes, suggesting that prostate hyperplasia is closely associated with up-regulation of genes in these two categories. Several immune system-associated transcripts were found to be up-regulated, which is in line with previous observations of areas of mild to moderate chronic inflammation, exhibiting stromal mononuclear (primarily lymphocytes and macrophage) infiltrate, in both ventral and lateral Pb-PRL prostate lobes (28).

Validation of differential gene expression by quantitative real-time RT-PCR
Ten transcripts found to be differentially expressed using cDNA microarray analysis were subjected to real-time RT-PCR verification using mouse-specific primers for the orthologous mouse genes (see Table 2Go). The same pool of total RNA that was used in the cDNA microarray experiments was used in the real-time RT-PCR reactions (see Materials and Methods). As in the cDNA microarray experiments, we chose to denote genes as differentially regulated if their level of expression was changed by 70% or more (CT < 0.6 or CT > 1.7). The analysis was performed in triplicate at the lobe level. The real-time RT-PCR results showed that almost all of the differentially expressed transcripts [TIMP-1, secreted apoptosis-related protein 1 (SARP-1), cathepsin B, cathepsin D, clusterin, decorin, nuclear protein 1 (p8), BOK, castration-induced prostatic apoptosis-related protein-1 (CIPAR-1), and epidermal growth factor] could be confirmed to be regulated in the same direction and to a similar degree as the fold changes obtained from the cDNA microarray experiments (Table 3Go). Moreover, the verification using mouse-specific primers for the orthologous mouse genes to the corresponding cDNA clone of rat origin on the cDNA microarray further supports the use of rat probes to measure orthologous mouse genes.

Assessment of apoptosis activity in Pb-PRL transgenic and control prostates
To assess possible differences in apoptotic activity in the Pb-PRL transgenic and control prostates, two established apoptosis markers were used. Activation of caspase-3 is considered an early apoptotic event and has been shown to play a central role in cellular commitment to apoptosis in response to a variety of stimuli (43). In contrast, detection of ssDNA signifies the downstream event of DNA fragmentation. DNA fragmentation may be detected in both histologically defined apoptotic cells and morphologically intact apoptotic cells. The immunohistochemical method involving an antibody specific against ssDNA in cells allows accurate assessment of apoptosis (44, 45). Furthermore, detection of ssDNA is considered more apoptosis-specific than the widely used terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling method for detection of DNA fragmentation and also detects apoptotic cells at an earlier stage than terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (41, 46). In Pb-PRL transgenic prostate, no activation of caspase-3 was detected using immunofluorescence (Fig. 2AGo). In control prostate, distinct clusters of apoptotic epithelial cells were infrequently detected in distal regions of the ductal system (Fig. 2AGo). Furthermore, detectable levels of ssDNA were absent in all lobes of Pb-PRL transgenic prostate using immunohistochemistry (Fig. 2BGo). In contrast, control littermate prostate focally displayed distinct nuclear ssDNA immunoreactivity in a small portion of epithelial cells, located almost exclusively in the distal ductal regions in all lobe types (DP, LP, and VP; Fig. 2BGo). Apoptotic bodies reactive with ssDNA antibody were often detected as small round dots on nuclei of epithelial cells; in other cells a more uniform staining of the nucleus was observed. Taken together, the ssDNA and caspase-3 immunohistochemistry results clearly indicate an overall diminished apoptotic activity in all prostate lobes of the Pb-PRL transgenic mice compared with controls.



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  		FIG. 2. A, Representative immunofluorescence photomicrographs showing the distribution of activated caspase-3-immunoreactive cells in control and Pb-PRL transgenic prostate lobes at 6 months of age. Transgenic prostate did not display any sign of caspase-3 immunoreactivity (left panel). In contrast, distal regions of both DP and VP of nontransgenic littermates exhibited areas of immunoreactivity (right panel). The caspase-3 immunosignal was principally present in the cytoplasm of ductal epithelial cells. B, Representative photomicrographs demonstrating ssDNA immunochemical staining in normal and Pb-PRL transgenic prostate lobes at 6 months of age. A complete lack of ssDNA immunoreactivity is seen in the DP, LP, and VP of transgenic males (left panel). In contrast, positive ssDNA nuclear immunostaining was frequently demonstrated in distal ductal regions of all lobe types in controls (right panel). Magnification: DL and VP, x40; and LP, x20.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have used cDNA microarray analysis to characterize the molecular mechanisms involved in the development of prostate hyperplasia in Pb-PRL transgenic mice. We identified several differentially expressed genes related to apoptosis and tissue remodeling in the enlarged prostate of Pb-PRL transgenic mice. Real-time RT-PCR was used to verify 10 of the differentially expressed transcripts. Furthermore, we demonstrated a lower apoptotic activity in the enlarged prostates of Pb-PRL transgenic mice compared with controls.

In the present investigation we used two different apoptotic markers to identify reduced apoptotic activity in the hyperplastic prostates of Pb-PRL transgenic mice. Diminished apoptosis activity has also been seen in human BPH tissue, and a shift in the proliferative/apoptotic balance is considered a key element in the etiology of BPH (7, 8). Interestingly, several apoptosis-related transcripts were found to be differentially expressed in the Pb-PRL transgenic prostate compared with controls. For example, the proapoptotic member of the Bcl-2 family, Bok (Bcl-2-related ovarian killer protein) (47), was showed to be down-regulated in Pb-PRL transgenic prostate. Together with the ssDNA and the caspase-3 immunoassay results, this expression pattern supports a role of Bok as a proapoptosis protein as previously shown in ovarian cancers (47). Apoptosis involves two essential steps: decision, which involves the interaction of pro- and antiapoptotic proteins (for example, Bcl-2 family members), and execution, which involves proteolytic enzymes such as the apoptotic caspases. The differential expression of this proapoptotic protein may indicate that the regulation of the apoptotic activity in Pb-PRL transgenic prostate occurs at the decision step of apoptosis. Two additional transcripts associated with proapoptotic activity, CIPAR-1 (also known as PARM-1) (48) and nuclear protein 1 (8), were found to be down-regulated in the Pb-PRL transgenic mice compared with controls. Furthermore, two transcripts with antiapoptotic activity, clusterin (also known as testosterone-repressed prostate message-2 or sulfated glycoprotein-2) and SARP-1, were shown to be up-regulated in Pb-PRL transgenic prostate. Clusterin is a ubiquitous protein that is expressed in almost all cells (49). Compared with normal prostate, its expression is known to be highly up-regulated in both hyperplastic and malignant rat prostate after sex hormone treatment (50). Moreover, a powerful survival/antiapoptotic activity of clusterin has been noted in vitro in human prostate cancer cells (51). SARP-1, also referred to as secreted frizzled-related protein 2, is a protein that appears to act as a soluble modulator of Wnt signaling by competing with membrane-bound frizzled receptors for the binding of Wnt ligands. SARP-1 has also been shown to possess antiapoptotic activity (52). Interestingly, two other transcripts in the Wnt signaling pathway were found to be differentially regulated in Pb-PRL transgenic mice compared with controls [Dvl1 disheveled 1 and Drosophila polarity gene (frizzled) homolog, both up-regulated]. The autonomous expression of all three Wnt-signaling molecules suggests that the Wnt-signaling pathway is involved in the regulation of prostate hyperplasia formation in our model.

Taken together, the reduced apoptosis activity, assessed by immunohistochemical analysis, in combination with down-regulation of transcripts with proapoptotic activity and up-regulation of transcripts with antiapoptotic activity supports the importance of reduced apoptosis activity in the pathogenesis of prostate hyperplasia in Pb-PRL transgenic mice. Furthermore, this allows a molecular insight into how the apoptosis activity might be regulated in BPH. An alternative explanation is that the decreased epithelial apoptosis activity in Pb-PRL transgenic mice is related to PIN formation rather than hyperplasia formation.

In parallel with reduced apoptosis activity, the prostate lobes of Pb-PRL transgenic mice display a significant increase in primarily the stromal compartment, resulting in an increased stroma/epithelium ratio (28). Furthermore, the epithelial dysplastic lesions in the enlarged prostate resemble low grade PIN, described in the recently proposed classification of PIN in genetically engineered mice, and are considered murine counterparts of the premalignant changes seen in human prostate (29). Several studies have proved the importance of epithelial-stromal interactions in both normal prostate development as well as the influence of abnormal reciprocal interaction between epithelial cells and the embryonic mesenchyme or adult stroma in the progression of neoplastic growth in the prostate gland (53). Although the exact mechanisms of such tissue interactions are still not fully understood, there is growing evidence that they may operate through cell-extracellular matrix (ECM) interactions, remodeling of ECM, and auto-/paracrine growth factors (54). Interestingly, several of the differentially expressed transcripts, between the enlarged prostates of Pb-PRL transgenic and control mice, are transcripts associated with tissue remodeling, including cell tissue structure (cytoskeleton and the ECM proteins) and proteases. Furthermore, many of the differentially expressed transcripts are directly associated with stromal cells and the ECM proteins that are secreted from stromal cells, including fibronectin, vimentin, laminin, osteonectin, and collagens. This may indicate that the activity of the prostatic stromal cells has been changed in our model. In addition to their structural role, ECM proteins have a pronounced influence on tissue remodeling regulating cell growth, differentiation, communication, and migration (54). Moreover, degradation of the ECM, mediated by a variety of proteolytic enzymes, such as matrix metalloproteinases (MMPs) and other proteases, have significant roles in normal and pathological tissue remodeling, including wound repair and tumorigenesis (55). Several transcripts related to protease activity and regulation, including the membrane-type MT1-MMP (known as MMP-14 in mouse), tissue inhibitors of MMPs (TIMP-1 and TIMP-2), and several members of the cathepsin family, were found to be up-regulated in the Pb-PRL transgenic prostate compared with controls. TIMP-1 and TIMP-2 have previously been demonstrated to be expressed in prostatic stroma and epithelial cells (56). Interestingly, TIMPs also possess proliferative and antiapoptotic features that may contribute to the observed prostate phenotype in Pb-PRL transgenic mice (57, 58). Several members of the cathepsin family (cathepsins A, B, D, H, L, and S) were found to be up-regulated in the Pb-PRL transgenic prostate compared with controls. Besides the role of cathepsins as proteases that degrade ECM, several studies suggest that some cathepsins also have important proliferative activity. Cathepsin B has previously been demonstrated in both benign and neoplastic prostatic cells, where the expression was suggested to be involved in tumor progression, angiogenesis, and stromal invasion (59, 60). Moreover, cathepsin B has been found to be frequently coexpressed with cathepsin S in early development of prostatic neoplasms (61). Up-regulation of cathepsin D (or procathepsin D) has been observed in prostate and breast cancer cells, where the protease was found to possess growth factor activity (62). Moreover, the expression of cathepsin D has been demonstrated to correlate to alterations in the expression of ECM proteins (fibronectin, laminin, and collagen) in breast tumors (63), resembling our own findings in the Pb-PRL transgenic prostate.

The phenomenon of tumorigenesis promotion by an activated stroma (generation of a so-called reactive stroma) has previously been associated with prostate pathology and other human cancers (64). The reactive stroma is characterized by ECM remodeling, elevated protease activity, increased angiogenesis and an influx of inflammatory cells. The list of differentially regulated transcripts in the Pb-PRL transgenic prostate has much in common with the processes involved in what is in the literature described as characteristics of the reactive stroma (reviewed in Ref.64). Up-regulation of vimentin together with down-regulation of desmin suggest a myofibroblastic-like nature of stroma cells, in line with the described phenotype of reactive stroma. In addition, reactive stroma cells typically express high levels of ECM components, such as collagen type I and III, fibronectin, and proteoglycans, as well as proteases that degrade the ECM, observations in accordance with our present results. Furthermore, the matricellular glycoprotein SPARC (secreted protein, acidic, rich in cysteine, also known as osteonectin and BM-40), which modulates the cellular interaction with ECM, is known to be expressed in tissues undergoing remodeling. SPARC was also found to be highly up-regulated in Pb-PRL transgenic prostate, in agreement with the dramatic up-regulation of SPARC in the reactive stroma associated with human epithelial ovarian cancer (65).

One hypothesis is that PRL might influence the initial induction of prostatic hyperplasia by modulating the stromal-epithelial interaction that, in one way or the other, results in activation of stroma with phenotypical features of reactive stroma. Stromal expansion is the dominant feature of human BPH, as numerous morphometric studies have demonstrated (10, 12, 13).

In the Mt-PRL transgenic mouse model, also generated by us, the PRL gene was ubiquitously expressed in all tissues, which resulted in a histologically similar prostate hyperplasia as that in the Pb-PRL transgenic mice, but with elevated serum androgen levels (17, 18). Molecular characterization of Mt-PRL transgenic mice has previously been performed using cDNA representational difference analysis, and in this study we also found differential regulation of apoptosis-associated transcripts (31). Furthermore, three other transcripts were found to be differentially regulated in a similar fashion in Mt-PRL and Pb-PRL transgenic prostates. In both studies vimentin was found to be up-regulated, and mRNA for the RIL protein as well as the established sequence tag clone IMAGE: 4988271 were both found to be down-regulated. The differential expression of the transcript of the RIL protein is exciting because this protein is speculated to be a candidate tumor suppressor. As expected, these results indicate molecular similarities between the two different prostate hyperplasia models, in parallel with the earlier reported histological similarities (18).

Although one might have expected some previously described PRL-regulated transcripts to be differentially regulated in the prostate of Pb-PRL transgenic mice, this was not the case. There can be several explanations for this, but we believe that the most important one is that our mouse model is a prostate hyperplasia model with chronically elevated PRL levels in the prostate, rather than a model to study acute PRL effects, as in most previous experiments. By this means we do not discount the idea that PRL has several important proliferative effects on the prostate. However, further functional studies of differentially expressed transcripts are needed before we denote any genes as being directly regulated by PRL or decide they are more a consequence of prostate hyperplasia.

In conclusion, the hyperplastic prostate of Pb-PRL transgenic mice demonstrates a reduced apoptotic activity and has a molecular pattern supporting the importance of a reduced degree of apoptosis for the development of the phenotype. The increased stromal/epithelial ratio of the Pb-PRL transgenic prostate together with up-regulation of a significant fraction of genes involved in tissue remodeling activity, including synthesis and degradation of the ECM and changes in protease activity suggest that activation of the stroma is involved in the development of prostate hyperplasia. Overall, the differentially expressed transcripts identified in this study show many molecular similarities between the prostate hyperplasia of PRL-transgenic mice and human prostate pathology, including both BPH and prostate cancer.


   Acknowledgments
 
We thank Mia Umaerus and Kåre Hultén for excellent technical assistance.


   Footnotes
 
This work was supported by grants from the Swedish Society for Medical Research, the Assar Gabrielsson’s Fund, the King Gustav V Jubilee Clinic Cancer Research Foundation (Sahlgrenska University Hospital), the Konrad and Helfrid Johansson’s Foundation, as well as the Foundation Clas Groschinsky’s Memorial Fund.

Abbreviations: BPH, Benign prostatic hyperplasia; CIPAR-1, castration-induced prostatic apoptosis-related protein-1; CT, comparative threshold cycle; Cy, cyanine; DLP, dorsolateral prostate lobe; ECM, extracellular matrix; FDR, false discovery rate; MMP, matrix metalloproteinase; Pb, probasin; PIN, prostatic intraepithelial neoplasia; SARP-1, secreted apoptosis-related protein 1; SPARC, secreted protein, acidic, rich in cysteine; ssDNA, single-stranded DNA; TBS, Tris-buffered saline; TIMP, tissue inhibitor of matrix metalloproteinase; VP, ventral prostate lobe.

Received April 3, 2003.

Accepted for publication July 25, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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