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Original Studies |
-Reductase1
University Departments of Surgery (C.W.B., F.K.H.), Pathology (F.D.), Medicine (K.C.), and Urology (P.B.), Western General Hospital, Edinburgh; and the Department of Urology, Glasgow Royal Infirmary (C.B.), Glasgow, Scotland
Address all correspondence and requests for reprints to: Dr. Fouad K. Habib, University Department of Surgery, Western General Hospital, Edinburgh, Scotland EH4 2XU. E-mail: fkh{at}srvo.med.ed.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-reductase (type I and type II) as well as maintained
expression of androgen receptors and prostate-specific antigen.
Furthermore, levels of prostate-specific antigen secreted by cocultured
epithelial cells were increased by treatment with androgens, mimicking
the situation in the human gland. This contrasted with conventionally
cultured fibroblasts or epithelial cells, which failed to express
5-reductase type II and rapidly lost expression of androgen
receptors and androgen sensitivity upon being placed into culture.
Electron microscopy demonstrated intracellular structures indicative of
the differentiated state of the cocultured cell types, including round
nuclei, tonofibrils, and microvilli in epithelial cells and elongated
nuclei; large amounts of Golgi and cilia; along with immature collagen
fibers in fibroblasts. The present study demonstrates that the
coculture model reflects more closely the in vivo system
for human BPH and is thus a far more suitable model for investigating
the molecular and cellular events that underlie BPH than current
in vitro systems. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reductase syndrome (4). Even so the precise
role for androgens in the pathogenesis of BPH is unclear, and there may
be other putative factors involved in the regulation of prostate
growth.
The investigation of the events that lead to BPH has been hampered by
the lack of suitable in vitro model systems. Although
primary cells derived from prostate can be maintained in culture
in vitro, and a number of prostate derived cell lines have
been established (5, 6, 7, 8, 9, 10), all of these systems have many limitations
compared to human BPH in particular with regard to their androgen
sensitivity/hormone responsiveness. Androgen receptors are rapidly lost
upon primary culture of prostate cells and are either absent or present
at low levels in established cell lines (11), making it difficult to
investigate the role of androgens in prostate growth and
differentiation. Furthermore, prostate epithelium and stroma interact
during the normal development and function of the gland (12), but in a
conventional culture system, epithelial cells are grown in isolation
and, as a result, behave abnormally. This is particularly evident with
immortalized cell lines that suffer from clonogenicity and
phenotypic/genotypic alterations (13, 14). These alterations are
highlighted by the inability of cultured epithelial cells to secrete
prostate-specific antigen (PSA) (11, 15) and to express 5-reductase
type II (5-RII) (15).
To improve our understanding of the processes involved in the development of BPH, there is a need for a model that maintains the in vivo characteristics of fibroblast and epithelial cells. Here we describe an in vitro model for BPH that maintains many of the characteristics of the in vivo state and does not suffer from the same shortcomings as earlier models.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Establishment of primary cultures. BPH tissue was obtained from men undergoing transurethral resection of the prostate. The histological status of the tissue was checked by an independent pathologist. Primary cultures of separated fibroblast and epithelial cells were established as previously detailed (5, 16). In brief, the prostate tissue was washed with phosphate-buffered saline to remove all traces of blood before being diced into approximately 1-mm3 pieces using forceps and scissors. The diced tissue was then incubated for 20 h at 37 C in a collagenase solution. After digestion with collagenase, the epithelial acini and fibroblast cells were separated by centrifugation. The epithelial acini were resuspended in WAJC 404 (Kyoto Pharmaceutical Co., Tokyo, Japan) supplemented with 0.5% FCS, 2.5 µg/L fungizone, 100,000 IU/mL penicillin, 100,000 µg/mL streptomycin, and 0.5 µg/L. The fibroblast cells were resuspended in RPMI 1640 (Life Technologies, Paisley, Scotland) supplemented with 200 mmol/L L-glutamine, 100,000 IU/L penicillin, 100,000 µg/mL streptomycin, 2.5 µg/L fungizone, and 10% FCS. The separated cells were then incubated at 37 C in 5% CO2. The identity and purity of the separated cultures were confirmed by immunohistochemistry and phase contrast microscopy as previously described (5, 16).
Establishment of cocultures. Confluent cultures of epithelial cells were harvested by trypsinization and seeded onto six-well plates at a density of 10,000 cells/well in 2 mL of a 50:50 (vol/vol) mix of epithelial cell growth medium [WAJC 404 (Kyoto Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 0.5% FCS, 2.5 µg/L fungizone, 100,000 IU/mL penicillin, 100,000 µg/mL streptomycin, and 0.5 µg/L insulin] and fibroblast cell growth medium [RPMI 1640 (Life Technologies, Paisley, Scotland, UK) supplemented with 200 mmol/L L-glutamine, 100,000 IU/L penicillin, 100,000 µg/mL streptomycin, 2.5 µg/L fungizone, and 10% FCS]. The epithelial cells were then incubated at 37 C in 5% CO2 for 2 h to allow attachment. Fibroblasts harvested after initially being placed in culture were subsequently seeded at a density of 2,000 cells/well in 2 mL of the same medium onto microporous membrane inserts (Millicell, Sigma Chemical Co., Poole, UK) that were placed into separate 6-well plates and left for 2 h to allow cell to attach. The inserts containing fibroblast were then removed using sterile forceps and placed into the wells containing the epithelial cells, thus allowing for interpopulation communication by means of diffusible elements but without any direct contact between epithelial cells and fibroblasts. Cocultured cells were maintained for up to 8 days. Cells were examined by electron microscopy for typical intracellular structures characteristic of epithelial cells and fibroblasts. This also served to determine the purity of the cultures.
Measurement of functional 5-reductase activity
5-Reductase activity was assayed as described previously
(17). Immediately before the assay, 120,000 cells were harvested by
trypsinization, pelleted by centrifugation, resuspended in RPMI 1640,
and counted. For assays, cells were then recentrifuged and resuspended
in 200 µL sodium phosphate buffer (4 mmol/L) at either pH 7.5
(5-RI) or pH 5.5 (5-RII) containing 0.32 mol/L sucrose and 1
mmol/L dithiothreitol. Cell suspensions were added to glass tubes
containing 1 µCi [3H]testosterone
([1,2,6,7-3H]testosterone; SA, 105 Ci/mmol; Amersham
International, Aylesbury, UK) and an NADPH-generating system consisting
of 0.5 mmol/L NADP, 0.1 U/mL glucose-6-phosphate, and 5 mmol/L
glucose-6-phosphate dehydrogenase, and the final assay volume was
adjusted to 1 mL by the addition of the appropriate pH buffer.
Reactions were agitated at 37 C for 30 min in a water bath. Reactions
were stopped by the addition of an equal volume of diethylether
containing 500 cpm [14C]dihydrotestosterone
([14C]DHT; SA, 50 mCi/mmol; Amersham International) and
25 µg each of unlabeled 3-androstanediol, 3ß-androstanediol,
testosterone, DHT, and androstenedione (all from Sigma). After
vortexing, steroids were extracted by evaporating to dryness in a
vacuum oven at 40 C. The residue was resuspended in 50 µL ethanol,
and steroids were separated on TLC plates (Gelman Sciences, Ann Arbor,
MI) in dichloromethane-diethylether (9:1, vol/vol). Steroids were
visualized by spraying with phosphomolybdic acid reagent spray, and the
radioactive metabolites were quantified using a Tri-Carb liquid
scintillation counter (Canberra Packard, Panbourne, Berks, UK) as
previously described (5, 18). Conversion of
[3H]testosterone was linear for at least 60 min for both
5-RI and 5-RII under the conditions used in these experiments. At
both pH 7.5 (5-RI assay) and pH 5.5 (5-RII assay), DHT was the
major metabolite and accounted for approximately 70% and 60% of the
total metabolites formed by the cocultured fibroblast and epithelial
cells after a 30-min incubation.
Ribonucleic acid (RNA) preparation and reverse transcription-PCR
(RT-PCR) amplification of 5-reductase isoenzyme expression
Total cellular RNA was extracted by the method of Chomczynski
and Sacchi (19). The presence of messenger RNA (mRNA) encoding 5-RI
and 5-RII was then determined by RT-PCR. One microgram of total RNA
was reverse transcribed using a commercial RT kit (Promega,
Southampton, UK) following the manufacturer’s protocol. Twenty
microliters of the RT reaction were then used for PCR analysis in a
reaction volume of 100 µL containing 8 µL 10 x assay buffer
(pH 9.0; Promega), 0.2 µL Taq polymerase (5000 U/mL), 2
µL deoxy-NTP mix (1.25 mmol/L), and 10 µL intron-spanning primers
(50 µg/mL) as follows.
The 5-RI primers used were 5'-TGCTGATGACTGGGTAACAG and
5'-GTTGGCTGCAGTTACGTATTC amplifying a 171-bp DNA fragment between
nucleotides 453–624; for 5-RII, the primers used were
5'-CCTTGTACGTCGCGAAGC and 5'-CCACCCATCAGGGTATTCAG amplifying a
350-bp fragment between nucleotides 98–447. PCR reactions were carried
out in a Hybaid Thermal reactor (Hybaid, Teddington, UK). Conditions
for 5-RI were 30 cycles of 96 C for 1.5 min, 52 C for 1 min, and 72
C for 1.5 min. Conditions for 5-RII amplification were 35 cycles of
96 C for 1.5 min, 56 C for 1 min, and 72 C for 1.5 min. PCR products
were separated on 1% agarose and visualized by ethidium bromide
staining under UV transillumination. Control reactions from which
complementary DNA (cDNA) or RT had been omitted were included in each
set of PCR reactions. The authenticity of all PCR products was verified
by restriction analysis as previously described (20).
PAGE and Western blot analysis for androgen receptor
Cells to be analyzed by PAGE were lysed directly in 1 mL loading buffer [100 mmol/L Tris-HCl (pH 6.8), 200 mmol/L dithiothreitol, 4% SDS, 0.4% bromophenol blue, and 20% glycerol]. One hundred-microliter samples (25 µg protein/mL) were electrophoresed on 6% SDS-polyacrylamide gels following the method of Laemmli (21). Gels were electroblotted onto nitrocellulose and incubated with a rabbit polyclonal antibody to human androgen receptor (Novocastra Laboratories, Newcastle, UK). Protein (54 µg/mL) from LNCaP cells (a prostate cancer cell line known to express androgen receptors) was included as a posi-tive control. Primary antibodies were detected using an ECL kit (Amersham).
Cell proliferation and response to androgens
Cellular responsiveness to androgens was determined by replacing normal growth medium with medium supplemented with 10% FCS stripped of endogenous steroids by treatment with dextran-coated charcoal. After a 24-h incubation period, the cells were supplied with fresh stripped medium containing 100 nmol/L testosterone (Sigma) and maintained for a further 4 days. Cells were subsequently removed by trypsinization, their densities were estimated with a hemocytometer, and counts are given as the number of cells per well. Controls that received no testosterone were similarly analyzed. All results are reported as the mean ± SEM of three replicates.
PSA measurement and expression
Immunoenzymetric assay. Cells were maintained in the same medium for 4 days, after which conditioned medium from six wells was collected then dialyzed against H2O for 24 h in membranes with a 6000–8000 mol wt cut-off. The dialysate was lyophilized and reconstituted in 100 µL sterile H2O, and PSA was measured using a Tandem-MP assay kit (Hybritech, Liege, Belgium) according to the manufacturer’s protocol. Results were expressed as the mean (nanograms per mL) ± SD of three replicates.
Immunohistochemistry of PSA. Epithelial cells grown under normal or coculture conditions were fixed directly in six-well plates in 1% formaldehyde in phosphate-buffered saline. After washing twice in Tris-buffered saline [TBS; 60 mmol/L Tris-HCl (pH 7.4) and 137 mmol/L NaCl], endogenous peroxidase activity was blocked with 3% H2O2. Nonspecific binding of antibodies was blocked with 20% sheep serum for 20 min at room temperature. Incubation was carried out overnight at 4 C with anti-PSA monoclonal antibody (Bionostics, Wyboston, UK) diluted in TBS (1:50,000). Cells were given two 5-min washes with TBS, then incubated in biotinylated sheep antimouse antibody (Dako, High Wycombe, UK) for 30 min at room temperature, followed by two five-min washes in TBS. After a 30-min incubation with horseradish peroxidase-conjugated streptavidin, cells were washed twice with TBS, and the peroxidase enzyme was visualized by immersion in 0.05% (wt/vol) diaminobenzidine for 10 min. Cells were counterstained with hematoxylin for 1 min. For each staining experiment, a negative control with the primary antibody omitted from the staining procedure and a positive control (LNCaP cell line) were included.
Electron microscopy
Separated primary and cocultured epithelial and fibroblast cells were harvested by trypsinization, pelleted by centrifugation, and fixed with 3% glutaraldehyde in 0.1 mol/L cacodylate-HCl buffer at 4 C overnight. Secondary fixation of samples was carried out with osmium tetroxide in deionized water for 45 min at room temperature. Samples were impregnated with EMIX resin and cut into 90-nm sections, which were mounted on 300-µm mesh copper grids before staining using the uranyl acetate-lead citrate method (22). After processing, the samples were visualized using a JEOL 100CXII transmission electron microscope (JEOL, Peabody, MA) operating at 60 kV.
Statistical analysis
Statistical significance was determined using two-tailed Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells grown in coculture were examined by electron microscopy for
specific intracellular structures characteristic of prostate fibroblast
and epithelial cells. The morphology of these cells was also compared
to that of separated primary culture cells maintained on plastic.
Cocultured epithelial cells (Fig. 1A
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exhibited an abundance of tonofibrils, maintained a complex microvilli
structure, and possessed a large number of secretory vesicles. They
also showed greater differentiation than separated primary epithelial
cells, which grew in monolayer and exhibited very few microvilli (Fig. 1B). The primary and cocultured epithelial cells were also stained for
specific markers of basal epithelial cells using the mouse antihuman
high mol wt cytokeratin antibody from Dako, which reacts with
cytokeratins 1, 5, 10, and 14. This demonstrated good staining for the
primary cultured epithelial cells, highlighting the basal nature of the
cells. The cocultured cells also expressed these basal epithelial cell
markers, indicating that the epithelial cells grown in coculture are
also basal in nature (results not shown).
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), on the
other hand, were characterized by the presence of cilia, which are
known to be specific to this type of cell and the formation of
collagen. The difference between primary and cocultured fibroblast
cells is demonstrated in Fig. 2, A–C. The cocultured fibroblast cells
shown in Fig. 2A demonstrate the characteristic shape of a fibroblast
cell, abundant cilia, Golgi, and collagen production (Fig. 2B). The
primary fibroblast cells shown in Fig. 2C are rounder in appearance and
demonstrate little cilia and no collagen production, but have large
amounts of Golgi. There is no evidence of smooth muscle cells in either
the cocultured epithelial or fibroblast.
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Expression of androgen receptors. Androgen receptor expression
in fibroblast and epithelial cells at passage 1 (P1) was examined by
Western blot analysis and compared to expression in the same cell types
in coculture (Fig. 3). Cocultured
epithelial and fibroblast cells contained a single immunoreactive band
at 110 kDa corresponding to the protein for human androgen receptor and
coincident with the 110-kD immunoreactive protein in LNCaP cells, an
androgen receptor-expressing prostate cell line (11). No similar bands
were seen in separated primary cultured fibroblast or epithelial cells
(Fig. 3).
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Immunostaining employing a specific antibody to the PSA demonstrated
positive PSA staining in cocultured epithelial cells (Fig. 4b), in contrast to separately cultured
epithelial cells, which showed no PSA staining (Fig. 4c). No PSA
immunoreactivity was seen in fibroblasts either separately cultured or
cocultured.
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Two-day treatment of fibroblast and epithelial cells grown in
coculture with DHT at 100 nmol/L produced no significant increase
(P < 0.05) in cell numbers compared to those of
control cells (Fig. 5) in the absence of
androgens. However, longer exposure to the androgens (4 days) yielded a
significant increase (P < 0.05) in number of
cocultured epithelial and fibroblast cells compared to that in control
cocultures (Fig. 5). Noteworthy are our earlier studies on separated
primary cultures of fibroblast and epithelial cells that demonstrated
no significant increase in cell numbers after exposure to exogenous DHT
compared with controls (11).
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-RI and 5-RII
Examination of total RNA isolated from co-cultured and
separated primary epithelial and fibroblast cells demonstrated striking
differences in the extent of 5-RI and 5-RII gene expression as
assessed by RT-PCR (Figs. 6 and 7). Whereas separately cultured
fibroblast and epithelial cells derived from BPH specimens clearly
contained a 5-RI mRNA easily detectable by RT-PCR (Fig. 6), no
amplification of 5-RII cDNA was seen. In contrast, RT-PCR carried
out on total RNA isolated from cocultured fibroblast or epithelial
cells (from the same tissue sample) generated a very strong 5-RI and
5-RII signal in the RT-PCR reaction (Fig. 7). This demonstrates that
cocultures of fibroblast and epithelial prostate cells maintained the
expression of 5-RII and 5-RI seen in intact prostate (20) in
contrast to separately cultured cells that expressed the type I
isoenzyme only.
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-RI and 5-RII mRNA expressed in
cocultured cells were translated into functional enzyme, we compared
the activities of the two 5-reductase isoenzymes in cocultured and
separately cultured epithelial cells and fibroblasts. The results
confirmed the data generated from the mRNA analyses. Cocultured
fibroblasts and epithelial cell suspensions (
60,000 cells/mL)
exhibited high levels of 5-RI and 5-RII activities, as measured
by the conversion of [3H]testosterone to
[3H]DHT at pH 7.5 (5-R1) and pH 5.5 (5-RII; Table
1). In contrast, the separately cultured
fibroblast and epithelial cells showed much lower levels (30-fold)
of 5-RI activity, and 5-RII activity was undetectable (Table 1).
We also noted that whereas cocultured epithelial cells expressed
similar levels of 5-RI and 5-RII activities, cocultured
fibroblasts contained 6 times more 5-RII activity than 5-RI
enzyme activity (Table 1). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To overcome the manifested limitations of earlier models, we have
herein described an in vitro cell model for investigating
prostate cell function and differentiation. Cocultured fibroblast and
epithelial cells maintained high levels of 5-RI and 5-RII
expression, expressed PSA, and were responsive to androgens, thus
mimicking the in vivo situation. Furthermore, the coculture
system allowed separation of the two cell types at the end of the
culture period, permitting separate analysis to be performed on the two
cell types. This model has very significant advantages over other
in vitro models described, all of which have limitations in
their use and validity (5, 6, 7, 8, 11, 12, 13, 14, 24, 25, 26, 27, 28, 29). In particular, the
growth of the prostate gland is dependent on androgens; none of the
in vitro models currently in use, with the notable exception
of the LNCaP cell line, contains functional androgen receptors, and
therefore, they do not exhibit androgen-sensitive cell growth and gene
expression. In contrast, our cocultured prostate cells both expressed
androgen receptors and showed androgen-induced alterations in cell
properties, and the growth of both the fibroblast and epithelial
components of the coculture was stimulated by the presence of exogenous
androgens. To the best of our knowledge, this is the first example
where androgen has been noted to stimulate the proliferation of
fibroblasts in prostate tissue.
Our studies also showed that the cultivation of prostate epithelial cells on standard tissue culture flasks reduced their capacity to secrete PSA and promoted a dedifferentiated morphology. However, once the epithelial cells were cocultured with fibroblasts, they regained many of the in vivo characteristics of the differentiated human prostate gland, including round nuclei as well as the presence of tonofibrils and microvilli, whereas the fibroblasts showed elongated nuclei and the presence of Golgi and cilia. In addition, we observed enhanced PSA secretory activity, which was increased significantly after androgen stimulation. The importance of the fibroblast to the clonal growth of prostate epithelial cells had been demonstrated previously (31), and a subsequent report Fong et al. (9) demonstrated that human prostate epithelial cells grown on basement membrane promoted PSA secretion, which was potentiated by the presence of androgens. In that study the prostate epithelium was in contact with the basement membrane, whereas in our own coculture system the two cell populations were separated by a microporous membrane, thus allowing for epithelial/fibroblast cross-talk by means of diffusible elements. It is evident from these studies that cell to cell contact is not required for PSA expression, but the results suggest that the promotion of PSA secretion is influenced by secondary products from the fibroblast; these factors remain to be identified.
The present report also showed that the coculture
microenvironment was crucial to the maintenance of 5-reductase
activity in both fibroblasts and epithelial cells. Expression of the
isoenzyme types I and II was observed in both cell types, and this was
further confirmed by assays of functional activities at pH 5.5
(5-RII) and 7.5 (5-RI), thus reflecting the patterns seen in
human BPH (20). The loss of 5-RII expression in separated primary
cultures highlights the importance of epithelial/fibroblast cross-talk
to maintain the activity of this isoenzyme. It is likely that a
diffusible factor(s) produced by one or both cell types is responsible
for the expression of this isoenzyme. In the absence of the factor(s),
as in primary cultures of fibroblasts or epithelial cells grown in
isolation, the responsible mechanism(s) is switched off, and no
5-RII is detected. We are at present attempting to elucidate the
nature of this mechanism(s).
Collectively, the findings reported in this study demonstrated that many of the properties of differentiated epithelium and fibroblast are preserved in the coculture system described here. This model allows for a better analysis of any possible in vivo effects of a test compound on the prostate than was previously possible using the earlier model systems. It also opens up opportunities for the investigation of fibroblast/epithelial cell interactions that may be important in understanding the processes involved in the development of not only BPH but also prostate cancer.
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Acknowledgments |
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Footnotes |
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Received May 29, 1997.
Revised September 10, 1997.
Accepted September 25, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reductase deficiency. J Clin Endocrinol
Metab. 75:1022–1026.[Abstract]
-reductase (type I)
detected in DU145 cells and expressed in most cells. J Steroid Biochem
Mol Biol. 48:347–352.[CrossRef][Medline]
-Reductase expression by prostate cancer cell lines
and benign prostatic hyperplasia. J Clin Endocrinol Metab81
:1361–1366.
-reductase. J Steroid Biochem. 22:461–467.[Medline]
-reductase type I and
type II mRNA in human hyperplastic prostate and in prostate primary
cultures. J Endocrinol. In press.
-reductase inhibitor, finasteride. J Steroid Biochem Mol Biol. 37:375–378.[CrossRef][Medline]
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