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,
,
From the Departments of * Urology and
Pathology, and
University of Pittsburgh Cancer Institute,
University of Pittsburgh, Pittsburgh, Pennsylvania; and
Gene Logic Inc, Gaithersburg, Maryland.
| Correspondence to: Dr Robert H. Getzenberg, Department of Urology Research Laboratories, University of Pittsburgh, Shadyside Medical Center G-40, 5200 Centre Avenue, Pittsburgh, PA 15232. |
| Received for publication September 23, 2003; accepted for publication February 4, 2004. |
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Abstract |
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Key words: Benign prostatic hyperplasia, prostate cancer, genes, gene expression
During development, androgens and the androgen receptor (AR) regulate several key events in the development and differentiation of major target tissues such as the prostate, seminal vesicles, and epididymis (Marker et al, 2003). Furthermore, it is generally held that androgens are not only required for normal prostate function, but they also have been implicated in prostate disease as well. Thus, identifying target genes that are androgen regulated may help to better understand the molecular basis of prostate physiology during health and disease.
Our earlier microarray studies using prostate samples from different groups of patients revealed that the JM-27 transcript is up-regulated by as much as 17-fold in patients with symptomatic BPH over that of normal patients with asymptomatic BPH, and in the neoplastic prostate (Prakash et al, 2002). Furthermore, our studies indicated that JM-27 expression was remarkably tissue specific; JM-27 messenger RNA (mRNA) was detected only in the prostate in males, and in uterine tissue in females.
Given the highly restricted expression pattern of JM-27 and its confinement to the stromal compartment, we continued our interests and undertook a detailed study of the functional aspects of this gene. In this study we evaluated a possible function for JM-27 by investigating its androgen regulation by utilizing a rat castration model.
Unlike the human prostate, numerous studies have described the rat prostate being divided not only anatomically, but also functionally. The rat prostate is composed of 2 anatomical regions, designated as the ventral prostate (VP) and the dorsolateral prostate (DLP). VP and DLP are known to exhibit cytological, biochemical, and functional differences that include differential susceptibilities to cancer-inducing substances (Wubah et al, 2002). Prior studies have shown that the DLP is more sensitive to hyperplasia and to cancer induced by sex hormones, and is more susceptible to prostate cancer (Banerjee PP et al, 1998; Thompson et al, 2002). The present study evaluates expression of JM-27 in both VP and the DLP in the rat.
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Materials and Methods |
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Rats were killed by CO2 asphyxiation, and the prostate glands were removed and dissected into ventral and dorsolateral lobes. The seminal vesicles were also dissected. All tissue was placed in phosphate-buffered saline (PBS) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and stored at -80°C until the study was completed.
The tissues were processed for total lysates following previously published procedures with minor modifications. Frozen tissue was cut into fine pieces using scissors, transferred to Eppendorf tubes containing 200–500 µL of high-salt buffer (10 mM NaPO4 pH 7.0, 150 mM NaCl, 2 mM EDTA, 1% sodium deoxycholate, 1% NP-40, 0.1% sodium dodecyl sulfate [SDS], 50 mM NaF, 200 mM Na3VO4, 0.1% ß-mercaptoethanol, 1 mM PMSF, 4 µg/mL aprotinin, and 2 µg/mL leupeptin [Sigma, St Louis, Mo]) and homogenized at 4°C until a slurry formed. The mixture was freeze-thawed 3 times repeatedly, and placed on a shaker for 30 minutes before centrifugation at 20 800 x g for 20 minutes at 4°C. The clarified lysates were recovered, aliquoted, and stored at -80°C. An aliquot was used for protein estimation via the Pierce Assay according to the manufacturer's specifications.
Cell Culture
Primary human prostate stromal cells were obtained from the tissue and cell
culturing facility at the University of Pittsburgh Department of Pathology and
plated in T-25 culture flasks. Cells were maintained in RPMI 1640 culture
media without phenol red, supplemented with 10% fetal bovine calf serum, 1%
glutamine, and 1% penicillin/streptomycin. Cells were subcultured when they
were 75% confluent and replenished with fresh media every other day. The SV2+
(Invitrogen, Carlsbad, Calif) expression vector containing the JM-27 encoding
the complementary DNA (cDNA) sequence in the sense orientation was constructed
by standard cloning techniques between the BamHI and PstI
restriction enzyme sites. Plated cells at 75% confluency were transfected with
the SV2+ vector using the Lipofectamine protocol (Gibco BRL, Gaithersburg,
Md). Control cells were transfected with expression vector only. All cells
were plated on 25-cm2 culture flasks and treated with 3 µg of
vector and 3 µL of Lipofectamine in 2 mL of serum-free medium. The reaction
was allowed to incubate for 3 hours on the respective cells, after which
serum-free medium was added to the mixture, bringing the volume up to 5 mL.
Twenty-four hours after transfection, the media were replaced with complete
medium containing serum. Cells were grown for 3 days and then lysed using
high-salt buffer. Transient transfection efficiency was evaluated by measuring
beta-galactosidase–specific activity in both JM-27 and
control-vector-only transfected cell populations as per the SV2+ vector
instructions (Invitrogen). Specific activity in nontransfected cells was
evaluated and subtracted from the values obtained from the transfected
cells.
Antibodies
An antipeptide antibody against the JM-27 protein was generated against a
synthetic peptide encoded in the sequence of the JM-27 gene. A synthetic
peptide of human JM-27, CPGQEREGTPPIEERKVE (amino acid residues 44–60),
was used to produce polyclonal antibodies in rabbits. Before they were used,
the antibodies were affinity-purified using the synthetic peptide. The
prostatein antibody (Accurate Chemical, Westbury, NY) reacts with the C3
subunit of the protein that was derived from the prostate. Beta-actin C-11
antibody is a polyclonal antisera obtained from goats (Santa Cruz
Biotechnologies, Santa Cruz, Calif).
Immunoblot
Total cell or tissue lysates were obtained, and 30 µg of protein in
6x loading buffer was loaded onto a 12% acrylamide gel and allowed to
run for 1 hour at 150 volts. Once the dye reached the bottom of the gel, it
was removed and equilibrated in transfer buffer with a pre-wet Immobilon-P
membrane for 30 minutes. Proteins were transferred to the membrane on a
Transblot-SD semidry transfer cell, where they remained for 40 minutes. The
membrane was then blocked overnight in 4% PBS-Tween milk. The membrane was
incubated on a shaker at room temperature with 2% PBS-Tween milk for 2 hours
with primary antibody for JM-27 at a dilution of 1:500. The membrane was
washed 3 times for 15 minutes with PBS-Tween. The membrane was then incubated
for 1 hour at room temperature with secondary antibody, horseradish peroxidase
(HRP)-conjugated anti-rabbit, at a dilution of 1:15 000. The membrane was
again washed 3 times for 15 minutes in 2% PBS-Tween milk. The membrane was
then sealed and exposed to an x-ray to visualize JM-27 protein expression. The
following day, the membrane was evaluated for actin expression as a measure of
equal loading using a primary antibody for actin (goat anti-donkey) at a
dilution of 1:500, and a secondary antibody, HRP-conjugated-anti-donkey, at a
dilution of 1:15 000.
Immunohistochemistry
Immunohistochemistry was performed on formalin-fixed paraffin-embedded
sections of tissue obtained from the same samples that were previously used
for our microarray analysis (Prakash et
al, 2002). Immunohistochemical examination was performed with the
standard avidin-biotin technique and a protease pretreatment step was
included.
Immunohistochemical analysis was performed using the Dako Envision+ System (DAKO, Carpenteria, Calif), based on an HRP-labeled polymer, which is conjugated to a secondary antibody. The labeled polymer does not contain avidin or biotin. The chromagen we used was diaminobenzidine, which elicited a brown color. The intensity of staining was subjectively graded from zero to 3+. Zero was determined as the absence of staining, whereas a 3+ grading was given to intense expression of the protein. Based on the Kruskal-Wallis test, which is the nonparametric analog to the one-way analysis of variance, the 4 groups were statistically different (P = .001). Pair-wise differences were then tested using the Wilcoxon rank-sum test (the nonparametric analog to the unpaired t test). The donor group was not significantly different from either the RP or other BPH subjects without symptoms (P = .60 and P = .12, respectively), but donors did differ significantly from the symptomatic BPH group (P = .001). The RP and other BPH group without symptoms were not significantly different from each other (P = .27), but both groups were statistically different from the symptomatic BPH group (P = .003 for RP and P = .02 for others without symptoms).
Identification of Androgen Response Element Motifs in JM-27 Protein
The representative nucleotide sequence for JM-27, NM_007003, was retrieved
from the RefSeq database at the National Center for Biotechnology Information
(NCBI). This mRNA sequence was mapped to assembled genomic sequence at Xp11.23
on chromosome X with a cDNA to a genomic DNA mapping tool, SIM4
(Florea et al, 1998). Five
kilobases of genomic sequence immediately upstream of the first exon of
NM_007003 were extracted and used for the androgen response element (ARE)
motif search. The FindPatterns program in the Wisconsin Package by Accelrys
(San Diego, Calif) was used to search for both experimentally-verified ARE
motif A (Nelson et al, 2002)
and putative ARE motif B.
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Results |
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Androgen Regulation of JM-27
The effect of androgen depletion via castration was examined on JM-27 in
the rat prostate. JM-27 expression in both the ventral and dorsolateral lobes
is significantly decreased in castrated animals, and the decrease appears to
be time-dependent, showing the greatest decrease in JM-27 protein expression
(89% ± 3.2%) within 1 week. Thus, while a dramatic loss of JM-27
protein expression is seen after 1 week of castration, its levels decreased
only slightly after 15 hours (31% ± 4.6%) and 24 hours (26% ±
6.1%) postcastration (Figure
2). To verify that the loss of JM-27 expression following
castration was indeed an effect attributed to loss of androgens, castrated
rats were treated with time-released testosterone pellets (15 mg) immediately
following castration and throughout the time period of each study. As
expected, exogenous administration of testosterone after castration was able
to maintain JM-27 expression in these animals
(Figure 3).
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Taken together these data suggest a formal dependence of JM-27 expression on androgen. However, the data do not distinguish between transcriptional and posttranscriptional regulation by androgen. We examined the nucleotide sequence of the human JM-27 gene located on the X-chromosome for the presence of putative AREs. Recently, Nelson et al (2002) identified a number of androgen-regulated genes in neoplastic prostate epithelium. A motif called the ARE motif A with the sequence 5'-AGAACAnnnAGTACT-3' was identified as the consensus, based on similar ARE motifs in promoter regions of androgen-responsive genes in humans. Furthermore, these were all experimentally verified to be responsive to androgen. We searched the JM-27 gene sequence for the ARE motif A element. As shown in Table 1, several putative AREs located on the JM-27 promoter region were readily identifiable between nucleotide positions -25 and -2186 from the translation start site, providing at least circumstantial evidence that regulation of this gene could be at the transcriptional level. Nelson et al (2002) also identified a second consensus ARE motif (motif B) based on several genes they identified but that did not experimentally show a response to androgen (Nelson et al, 2002). When we used this consensus motif B to compare it to the individual ARE-like motifs found in the JM-27 sequence, we found a high degree of identity (83%) to at least one ARE motif B, which was located at -2186 (Table 2). These observations strongly suggest that JM-27 may indeed be regulated by androgen in vivo. Additional experiments to examine the role of these putative AREs are currently under way in our laboratory.
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JM-27 and the Androgen Receptor
Given the intriguing expression patterns and preliminary evidence that
JM-27 may be directly regulated by androgens, we hypothesized that a putative
function for JM-27 is its involvement in rendering the tumor cell insensitive
to androgens. To test this hypothesis, primary human stromal prostate cultures
were transfected with an expression vector containing the cDNA sequence
encoding human JM-27. Measurement of JM-27 protein expression levels via
immunoblot analysis revealed that JM-27 transfection resulted in increased
expression of the 27-kd isoform of the JM-27 protein. The transfected cells
were then evaluated for AR expression using an AR-specific antibody, and
revealed that cells that express greater amounts of JM-27 had decreased levels
of AR (by 65.3% + 5.4% as measured by phosphor-image analysis)
(Figure 4). These results
provide strong evidence that JM-27 may be involved in the down-regulation of
the AR, and suggest a negative feedback loop between JM-27 and the AR
expression.
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Expression of JM-27 in the Diseased Prostate
Using representative samples from 3 of 4 patient groups studied, we had
earlier shown that JM-27 expression correlates with its transcript expression
in that, like the JM-27 mRNA, the protein is highly expressed only in
symptomatic BPH but not in normal or asymptomatic BPH. Furthermore, within the
prostate tissue, JM-27 protein expression is confined to the stromal component
of the gland. However, its histologic expression in patients with BPH and
prostate cancer was only preliminarily determined in this study
(Prakash et al, 2002). In the
present study, we not only extended the immunohistochemical analysis using 31
patient samples, but also quantitated JM-27 protein expression. In prostate
samples from young (N1) and old (N2) "normal" donors, JM-27
protein expression was virtually undetectable and received a score of 1.5
arbitrary units. In contrast, in samples from patients with symptomatic BPH,
JM-27 protein is significantly overexpressed, as was seen by the intense
staining pattern, and this group was assigned a score of 2.5. Samples from
patients with asymptomatic BPH and those from patients with histologic BPH and
prostate cancer, interestingly, show barely detectable levels of protein
expression, and hence received an intermediate score of 1.75 arbitrary units
(Table 3).
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Discussion |
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Numerous studies have described the rat prostate as being not only anatomically divided, but also functionally divided. The rat prostate is composed of two anatomically defined regions: ventral prostate and dorsolateral prostate. VP and DLP are known to exhibit cytological, biochemical, and functional differences that include differential susceptibilities to cancer-inducing substances (Wubah et al, 2002). Prior studies have shown that the DLP is more sensitive to hyperplasia and to cancer induced by sex hormones (Banerjee et al, 1998; Thompson et al, 2002). These lobe-specific responses to disease onset may be partially accounted for by JM-27 expression. Further investigation is ongoing in our laboratory to clarify such a mechanism. Because our findings show that patients with BPH as well as patients with prostate cancer show an up-regulation of the JM-27 protein, we have postulated that this gene may be involved in growth disregulation in the diseased prostate. The role of androgens in growth is well documented. In order to test this hypothesis, we evaluated the androgen regulation of JM-27.
The importance of androgens in proper functioning and growth of the prostate gland is substantially supported in the literature (Jiang and Wang, 2003). Presently, we investigated whether JM-27 is androgen-regulated by utilizing a castrated rat model. Our data revealed that JM-27 significantly decreased 1 week after castration and that administration of exogenous androgens maintains JM-27 expression in castrated rats. Furthermore, the observation that several ARE-like sequence motifs are present strengthens this argument. Further evaluation of JM-27 overexpressing cells reveals that AR expression decreases in these cells. These findings show that, not only does JM-27 expression depend on androgen stimulation, but that increased JM-27 expression decreases AR expression. A possible feedback loop may be involved in this protein product and in the AR. The AR has been found to play a significant role in prostate cancer, specifically in invasion. Studies have shown that a functional AR contributes to cell differentiation as well as decreased invasiveness of cancer (Baldi et al, 2003). In regards to our findings, the JM-27 protein, when up-regulated, decreases AR expression. Our data suggest that the cells are becoming less responsive to androgen, and therefore, more progressed in the disease state. Because the importance of androgens on prostate growth and development, and the mechanism by which androgens act on proper functioning of the prostate gland remain inconclusive, it is especially important to evaluate genes such as JM-27 and to determine how it correlates with androgen action on prostate cell types.
A number of studies indicate that both normal and diseased prostates are dependent on androgens for growth and differentiation (Lu et al, 1997). Studies in rodents with nonfunctional ARs during fetal life never develop a prostate gland (Marker et al, 2003). Testosterone or dihydrotestosterone act via the AR to induce a cascade of intracellular signaling events that in turn act on the cell cycle as well as other cell functions. Studies to investigate the role of androgens in growth control in the prostate have shown that, at least in the initial stages, prostate cancer is androgen-dependent for growth, and, in many cases, it progresses to an androgen-insensitive stage (Mobbs et al, 1974). The JM-27 protein may be involved in rendering the prostate to become less androgen sensitive. By evaluating androgen regulation of JM-27 in the rat prostate as well as quantitating the immunohistochemical analysis of JM-27 expression in normal versus diseased prostatic tissue, we have clarified one aspect of the JM-27 gene, which is androgen regulation of its protein product. Our data suggesting that JM-27 is involved in the AR-ligand cascade further support the complexity of androgen action on the prostate gland. To our knowledge, this is the first investigation to examine androgen regulation of this unique prostate-associated gene.
Previous studies in our laboratory have shown that JM-27 is isolated to the stromal component of the prostate gland in humans (Prakash et al, 2002). Because the prostate gland is made up of a dynamic environment of both stromal and epithelial components that interact via paracrine, endocrine, and autocrine regulation, it is possible that the JM-27 gene and protein exerts its effects via one of these mechanisms. The diseased prostate has been extensively studied, and it is known that changes occur within the heterogeneous cell types, leading to loss of growth control, loss of function, and, ultimately, considerable discomfort to the patient. In prostate cancer, this balance is abnormally disrupted. There is thus a critical need to resolve whether the role of stromal cells is the same in androgen-regulated normal tissue as it is in malignant prostatic tissue. The JM-27 gene may, potentially, be a crucial stromal-expressing component that will elucidate the role of stromal interaction in the diseased prostate. In conclusion, it is important that we investigate the precise function of genes such as JM-27, which potentially play a significant role in growth disregulation in both normal and diseased prostates.
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Footnotes |
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References |
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