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Endocrinology |
and ER-ß in Normal and Malignant Prostatic Epithelial Cells: Regulation by Methylation and Involvement in Growth Regulation1
Division of Urology, Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts 01655 [K-M. L., S-M. H.], Department of Biology, Tufts University, Medford, Massachusetts 02155 [K-M. L., M. L.] and Urological Clinic, New England Medical Center, Boston, Massachusetts 02111 [J. L.]
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ABSTRACT |
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and those of ER-ß were
expressed in our normal PrEC primary cultures, in a commercially
available PrEC preparation (PrEC; Clontech), in an immortalized PrEC
line established from a benign prostatic hyperplasia specimen (BPH-1),
and in three prostatic cancer cell lines (LNCaP, PC-3, and DU145).
Expression levels of ER- and ER-ß transcripts were related to
those of two estrogen-responsive genes [progesterone receptor (PR) and
pS2], at the message levels, to gain insights into the functionality
of the ER subtypes in PrECs. Interestingly, only transcripts of ER-ß,
but not those of ER-, were found in our primary cultures of normal
PrECs, along with both PR and pS2 mRNA. These data strongly suggest
that estrogen action was signaled exclusively via ER-ß in normal
human PrECs. In contrast, PrEC (Clontech) and BPH-1 cells expressed
both ER- and ER-ß transcripts and no PR nor pS2 mRNA in PrEC and
only a minimal level of PR mRNA in BPH-1. Among the three prostate
cancer cell lines, LNCaP expressed ER-ß mRNA along with transcripts
of PR and pS2, DU145 expressed messages of ER-ß and PR, and PC-3
cells exhibited ER-, ER-ß, and pS2 mRNA. Thus, unlike normal
PrECs, expression patterns of these genes in malignant PrECs are more
variable.
Treatment of prostate cancer cells with demethylation agents
effectively reactivated the expression of ER- mRNA in LNCaP and
DU145 and that of pS2 message in DU145. These findings provide
experimental evidence that ER- gene silencing
in prostate cancer cells, and perhaps also in normal PrECs, are caused
by DNA hypermethylation.
To evaluate the potential of using antiestrogens as prostate cancer therapies, we have assessed the growth-inhibitory action of estrogens (estradiol and diethylstilbestrol) and antiestrogens (4-hydroxy-tamoxifen and ICI-182,780) on PC-3 and DU-145 cells. In PC-3 cells, which express both ER subtypes, estrogens as well as antiestrogens are effective inhibitors. In contrast, in DU145 cells, which express only ER-ß, antiestrogens, but not estrogens, are growth inhibitors. By comparison, ICI 182,780 is the more effective cell growth inhibitor. Importantly, the ICI 182,780-induced antiproliferative effects were reversed by cotreatment of DU145 cells with an ER-ß antisense oligonucleotide, hence lending additional support to a central role played by ER-ß in mediating growth-inhibitory action of antiestrogens.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), the two ER subtypes may regulate
different sets of cellular functions (18
, 24)
. Recent
findings from an ER-ß knockout mouse suggest that ER-ß may suppress
proliferation and prevent hyperplasia in the rodent prostate
(25)
. Taken together, these new findings raise an
intriguing possibility that ER-ß is expressed in normal and/or
malignant human PrECs and plays a role in mediating estrogen action in
these cell types.
Knowledge of the distribution of ER-ß in normal and malignant human
PrECs is limited at this time. A recent study reported a lack of ER-ß
expression in human prostate tissues (26)
, whereas several
preliminary reports noted expression of this receptor subtype in basal
epithelial cells of the human (27, 28, 29)
. In this study, we
reported the development of an effective method to obtain and culture
"pure" or highly enriched populations of normal PrECs from needle
biopsies of the peripheral zone of the human prostate. Expression
levels of ER- and ER-ß transcripts in our primary cultures of
normal PrECs were compared with those found in a PrEC preparation
obtained from a commercial source (PrEC; Clontech), in an immortalized
PrEC cell line established from a BPH specimen (BPH-1; Ref.
30
), and in three prostatic cancer cell lines (DU145,
PC-3, and LNCaP). Expression levels of ER- and ER-ß in normal and
malignant PrECs were then related to the transcript expression levels
of two estrogen-responsive genes (PR and pS2) to gain insights into the
functionality of the ER subtypes. Additionally, we have compared the
efficacy of two estrogens (DES and E2) to those
of two antiestrogens (4OH-TAM and ICI) in inhibiting cell growth in
PC-3 and DU145 cells. An ER-ß antisense ODN was then used to
demonstrate that the antiestrogen-induced growth-inhibitory effects on
prostate cancer cells were mediated via an ER-ß signaling mechanism.
Finally, we provided the first experimental evidence in support of DNA
methylation-mediated transcriptional inactivation of gene expression as
the mechanism of ER- silencing in PrECs.
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MATERIALS AND METHODS |
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Each harvested tissue specimen was then washed three times with HBSS
and cut into five to seven smaller pieces. The pieces were suspended in
2 ml of freshly prepared growth medium (see below) and transferred to a
60-mm Falcon culture dish (Becton Dickinson, Lincoln Park, NJ) coated
with type I rat tail collagen (Collaborative Biomedical Products,
Bedford, MA). An epithelial cell selection medium (the growth medium),
reported previously (31)
, consisted of keratinocyte
serum-free medium with 25 µg/ml bovine pituitary extract, 5 ng/ml
epidermal growth factor, 2 mM L-glutamine, 10
mM HEPES buffer, P/S (100 units/ml penicillin and 100
µg/ml streptomycin), 5.5 µl/ml fungizone, 20 ng/ml cholera toxin,
and 1% heat-inactivated FBS was used to obtain enriched populations of
PrECs. All culture reagents were obtained from Life Technologies, Inc.
(Grand Island, NY) except for FBS, which was purchased from Sigma
Chemical Co. (St. Louis, MO). The culture was incubated at 37°C in a
5% CO2 atmosphere without disturbance for 7 days
to allow epithelial cells to grow out of the tissue pieces. Culture
medium was then routinely replaced every 4 days until cell culture
reached 
80% confluence. The cells were split once before they were
used for RNA extraction.
In addition to primary cultures obtained from biopsy explants, a batch of normal human PrECs were purchased from Clonetics Co. (San Diego, CA). The PrEC cells were cultured in the PrEGM medium (Clontech) supplemented with SingleQuots (Clonetics Co.) according to the manufacturer’s recommended protocol.
Maintenance of Established Prostatic Cell Lines.
All culture reagents were obtained from Life Technologies and FCS from
Sigma (St. Louis, MO), except otherwise specified. BPH-1
(30)
, a nontumorigenic, SV40-immortalized, highly
differentiated human prostate epithelial cell line, was provided as a
gift by Dr. Simon Hayward at the University of California (San
Francisco, CA). This cell line was maintained in RPMI 1640 with 10
mM HEPES, 1 mM sodium pyruvate, 2
mM L-glutamine, 4.5 g glucose/l, and
1.5 g of sodium bicarbonate/L (American Type Culture Collection,
Rockville, MD) plus 5% heat-inactivated FBS, ITS
(insulin-transferrin-selenium mixture; Collaborative Biomedical
Research, Bedford, MA), and P/S. Three human prostate cancer cell lines
(DU145, PC-3, and LNCaP) were purchased from American Type Culture
Collection. For routine maintenance, DU145 and PC-3 cells were grown in
DMEM/F-12 supplemented with heat-inactivated FBS, 2
mML-glutamine, 1 mM sodium
pyruvate, 0.1 M nonessential amino acids, P/S, 0.05
mM ß-mercaptoethanol (Sigma), and 1% ITS+. LNCaP cells
were maintained in the same medium used for BPH-1 except that ITS+ was
left out from the medium. All cell cultures were incubated at 37°C
under a 5% CO2 atmosphere.
RNA Isolation and RT-PCR.
Total cellular RNA was isolated using RNA Stat-60 reagent (Tel-Test,
Inc., Friendswood, TX) according to protocols provided by the
manufacturer. The quality of each total RNA sample was checked and
controlled by the following steps: (a) measurement of
absorbance; (b) running of a denaturing RNA gel capable of
detecting possible RNA degradation, as judged by the integrity and
intensity of the 18S and the 28S rRNA signals; and (c)
conducting a semiquantitative RT-PCR for the 18S rRNA at low cycle
numbers. One µg of total cellular RNA was reverse transcribed using
the GeneAmp RNA PCR kit (Perkin-Elmer, Norwalk, CT), and 2 µl
of the resulting cDNA were used in each PCR.
Intron-spanning primers were either obtained from published literature
or designed using the Primer3 Output
program.5
Primer sequences for GAPDH, ER- (primer set #1, exon 1–3), ER-ß,
PR, and AR are given in Table 1
. All PCR conditions were optimized for quantification of relative
message contents under nonsaturating conditions. Preliminary
experiments were conducted to ensure linearity for all semiquantitative
procedures. Hot-start PCR using AmpliTaq Gold DNA polymerase
(Perkin-Elmer) was used in all amplification reactions. The enzyme was
activated by preheating the reaction mixtures at 95°C for 6 min prior
to PCR. This protocol was chosen to minimize nonspecific product
amplification. The routine PCR program was 30 cycles of 1 min at
94°C, 1 min at 60°C (annealing temperature), and 1 min at 72°C
with the following modifications: (a) amplification for
ER-ß cDNA used an annealing temperature of 58°C; (b)
amplifications of ER- cDNA and AR cDNA were carried out at an
annealing temperature of 55°C; (c) cycle number for ER-
cDNA amplification was set at 35; and (d) GAPDH cDNA was
amplified at 26 cycles. GAPDH cDNA levels served as a loading control.
Amplification of the correct sequence was verified by direct DNA
sequencing of each PCR product from at least two different samples.
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mRNA (see
"Results"), we used two additional pairs of primers (primer sets #2
and #3, Table 1
) to amplify regions downstream of the coding sequence
of ER- (668 bp of exon 3–6 and 710 bp of exon 5–8, respectively).
Together, these three pairs of pairs of ER- primers (Table 1)
covered most of the length of the coding sequence. They had been used
successfully in our recent study on the expression of ER- mRNA in
ovarian cancer (32)
. If PCR at high cycle number failed to
yield products from all three pairs of primers, we considered the
sample to be devoid of ER- cDNA.
Treatment of DU145, PC-3, and LNCaP Cells with Demethylating
Agents.
The three prostatic cancer cell lines were seeded at a density of
104 cells/ml medium in
25-cm2 culture flasks, allowed to attach during a
24-h period, and exposed to two demethylating agents separately. The
demethylating agents were added daily in aqueous solution.
5'-Azacytidine was added at final concentrations of 2.5 and 5
µM and 5'-aza-2'-deoxycytidine at 0.5 and 0.75
µM, respectively. Culture medium was changed every 4
days, and cells were subjected to a total of 8 days of demethylating
agent treatment. At the end of the treatment period, the medium was
removed, and cellular RNA was extracted for RT-PCR.
Treatment of DU145 and PC-3 Cells with Estrogens/Antiestrogens.
Cells were seeded at a density of 5 x
103 per ml into 24-well plates (Falcon; Becton
Dickinson Labware, Lincoln Park, NJ) in a final volume of 1 ml of
culture medium with 5% charcoal-stripped FBS. Twenty-four h after
seeding, triplicate wells of cells were treated in with 1, 10, and 100
µM of E2, DES, 4OH-TAM, or ICI.
E2, DES, and 4OH-TAM were purchased from Sigma,
and ICI was a generous gift from Zeneca Pharmaceuticals (Macclesfield,
United Kingdom). Estrogens and antiestrogens were dissolved in absolute
ethanol (Sigma) and added to the media daily. Cell cultures that were
not treated with estrogenic compounds received absolute ethanol as a
vehicle control. Total additive ethanol concentrations never exceeded
0.2% throughout the culture period. The cells were refed with freshly
prepared medium every other day. At the end of a 4-day treatment
period, cells in each well were trypsinized, and cell count was
determined by direct counting using the trypan blue exclusion method.
All treatment experiments were repeated at least three times to
generate statistically relevant data.
Treatment of DU145 Cells with ICI and ER-ß Antisense ODN.
DU145 cells (5 x 103 cells/well)
were plated in 24-well plates (Falcon; Becton Dickinson Labware). After
allowing 24 h for cell attachment, cell cultures were treated in
triplicate with 1 µM of ICI in the presence of 2.5
µM ER-ß antisense, sense, or mismatch ODNs for 4 days.
The ER-ß antisense ODN, an 18-mer, was designed to recognize the
first translation start site on the ER-ß mRNA and its immediate 5'
flanking region (Table 2)
. The nucleotide sequence of sense ODN is complementary to those of
ER-ß antisense ODN (Table 2)
. On the basis of the sequence of ER-ß
antisense ODN, five nucleotides were scrambled to generate a mismatch
ODN that retains the same GC ratio of the ER-ß antisense ODN (Table 2)
. Both the sense and the mismatch ODNs served as controls for the
antisense ODN. In all three ODNs, the first and the last three
nucleotides were phosphorothioate modified to increase their stability
in cellulo. The number of viable cells in each well was
determined by direct counting using the trypan blue exclusion method
after a 4-day treatment period. At least three individual experiments
were performed to obtained statistically relevant data.
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RESULTS |
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Transcripts, in Normal PrECs in Primary Cultures.
) demonstrated that our normal PrEC cultures retained high levels of AR
mRNA expression, which usually disappeared in late-passage normal PrEC
primary cultures or in established PrECs (33)
.
Interestingly, all five cultures of normal PrECs expressed uniform
levels of ER-ß RNA and transcripts of the estrogen-responsive genes,
PR and pS2. In contrast, expression of ER-
mRNA was noticeably absent in all five cultures, even when high cycle
number PCR (>42 cycles) was used to amplify the cDNA. Interestingly,
PrEC (Clonetics Co.), a commercially prepared normal PrEC culture, and
BPH-1, a SV40 immortalized prostatic epithelial cell line, expressed
both ER- and ER-ß, but no PR or pS2 transcripts in PrEC
(Clontech), and only a minimal level of PR mRNA in BPH-1.
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PR, and pS2 mRNA in Prostatic Cancer
Cell Lines.
). In contrast,
ER- mRNA was expressed only in the PC-3 cells. Interestingly, PR
transcripts were detected only in DU145 and LNCaP cells and not in PC-3
cells. Messages of pS2 were found in PC-3 and LNCaP cells but not in
DU145 cells. In accordance with reports in the literature, AR mRNA
expression was only noted in LNCaP cells.
Expression of ER- Variant in Prostate Cell Lines.
When RT-PCR analyses were conducted for ER- mRNA semiquantification
in PrEC (Clonetics Co.), BPH-1, or PC-3 cells, we noticed that, in
addition to the expected PCR product, a smaller PCR product was
coamplified (Fig. 1, a and b
). Sequencing
analysis (data not shown) revealed that this smaller PCR product was
derived from an ER- mRNA variant that had the entire exon 2 deleted.
We reported recently the coexistence of this ER- mRNA variant with
wild-type transcripts in normal and malignant human ovarian surface
epithelial cells (33)
.
Demethylation Reactivates ER- and pS2 mRNA Expressions in DU145
Cells and ER- Expression in LNCaP Cells.
Prior to exposure to demethylating agents, ER- and pS2 transcripts
were not detected in RNA samples prepared from DU145 cells (Fig. 1, a and c
, and Fig. 2
). After the 8-day treatment with 5'-aza-cytidine (2.5 and 5
µM) or 5'-aza-2'deoxycytidine (0.5 and 0.75
µM), DU145 cells regained expression of both
transcripts (Fig. 2
). Interestingly, the absence of AR mRNA expression
in DU145 cells was not reversed by treatment with demethylating agents.
Exposure of LNCaP cells to demethylating agents also reactivated ER-
mRNA expression (data not shown).
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). A dose-dependent inhibition of cell
numbers was observed in cultures exposed to ICI for 4 days when
compared with control cultures treated with vehicle (absolute ethanol).
A 40% reduction (P < 0.001) in the cell
numbers was achieved with an ICI dose of 1 µM.
A similar growth-inhibitory response was observed when DU145 cells were
treated with 4OH-TAM. However, cell number reduction achieved with 1
µM 4OH-TAM was only 25%
(P < 0.001). In contrast, exposure of DU145
cells to 1 µM estrogens
(E2 and DES) did not affect cell growth in 4-day
exposure experiments (Fig. 3, C and D
). However,
in a separate series of experiments, DU145 cells were treated with 10
µM DES, and we observed a significant reduction
in cell number (data not shown). Furthermore, we had conducted
competition experiments to determine whether E2
could reverse the growth-inhibitory effects induced by ICI and 4OH-TAM.
DU145 cells were treated with 10-6
M ICI or 4OH-TAM in the absence or presence of
10-8, 10-7,
10-6 M
E2. E2 at all three
concentrations was unable to reverse the ICI- or 4OH-TAM-induced cell
growth inhibition (data not shown).
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). Furthermore, exposure of PC-3 cells to
E2 at 1 or 10 µM
concentrations also induced inhibition of cell growth
(P < 0.05 and P <
0.01, respectively; Fig. 4C
). Interestingly, treatment with
DES at the various concentrations up to 10-6
M did not elicit statistically significant cell
growth inhibition in PC-3 cells (Fig. 4D
). However, in a
separate experiment, PC-3 cells were treated with 10
µM DES, and we observed a significant reduction
in cell number (data not shown). Additionally, we had conducted
competition experiments to determine whether DES could reverse the
growth-inhibitory effects induced by ICI and 4OH-TAM. PC-3 cells were
treated with 10-6 M ICI or
4OH-TAM in the absence or presence of 10-8,
10-7, or 10-6
M of DES. DES was able to reverse the
ICI-induced, but not 4OH-TAM-induced, cell growth inhibition in a dose
dependent manner (data not shown).
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). Cotreatment of DU145 cells with
ICI and an ER-ß antisense ODN led to restoration of cell number
(P < 0.001; Fig. 5
) whereas cotreatments with an ER-ß sense ODN or a mismatch ODN (Table 2)
did not reverse the ICI-induced effects. These data support the
notion that the ICI-induced cell growth inhibition in DU145 cells is
mediated via an ER-ß signaling mechanism.
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DISCUSSION |
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Traditionally, the actions of estrogens/antiestrogens are thought to be
mediated via the classical ER, the subtype, which has been
localized to the stromal compartment and basal epithelial cells of
human and rodent prostates (22
, 26
, 45, 46, 47, 48)
. Because
ER- is not expressed in the normal glandular epithelium of rat or
human prostate (4
, 21
, 26
, 46, 47, 48, 49)
, it is widely believed
that the action of estrogen/antiestrogen on normal PrECs is indirect,
likely mediated via estrogen-induced stromal factors. However, after
the discovery of ER-ß (19)
and its localization to the
epithelial compartment of rodent prostates (19
, 21
, 50)
, a
distinct possibility has been raised that estrogen/antiestrogen could
influence PrEC function via an ER-ß signaling pathway. However, at
present, information on ER-ß in human PrECs is limited. Only one
recent study (26)
has evaluated the expression pattern of
ER-ß transcripts and proteins in human prostatic tissues and found
nondetectable levels in both normal and diseased tissues. In contrast,
the present study unequivocally demonstrated expression of ER-ß mRNA
in highly enriched or pure human PrEC cultures established from
peripheral zone biopsies. Furthermore, because ER- message was
undetectable but transcripts of two estrogen-dependent genes,
PR and pS2, were expressed in these cultures,
these data strongly suggest that ER-ß is the cellular mediator of
estrogen action in normal human PrECs. Of interest to note is that both
ER- and ER-ß mRNA, but not PR or pS2 transcripts, were expressed
in a PrEC preparation purchased from a commercial source
(PrEC; Clontech) and in the immortalized PrEC line, BPH-1, with only a
minimal level of PR transcripts. The discrepancies between ER subtype,
PR, and pS2 expression in our primary PrEC cultures and those
observed in PrEC (Clontech) and BPH-1 could be attributable to
the tissue of origin of these cell cultures/lines. In this regard, PrEC
cultures (Clontech) are routinely prepared from whole prostates, and
BPH-1 was derived from a benign hyperplastic specimen
(30)
, whereas our primary cultures were established from
ultrasound-guided peripheral zone biopsies.
Issues relating to whether ER, and which subtype, is expressed in
cancerous PrECs remain unsettled. Several investigators (51
, 52) observed ER- expression in human prostate cancer cell
lines, including LNCaP, PC-3, and DU-145, whereas others
(53)
did not. Similarly, observations on ER- expression
in prostate cancer specimens were equally controversial. Bonkhoff
et al. (26)
reported recently that ER-
expression was infrequent in low-to-moderate grade adenocarcinoma but
common in high-grade and metastatic cancers. Conversely, Konishi
et al. (54)
noted the presence of ER-
immunopositivity in well-differentiated adenocarcinomas but not in
poorly differentiated specimen. The latter observation was supported by
two additional studies that reported no ER- expression in lymph node
and distant metastases (53
, 55)
. These issues become more
convoluted when the expression pattern of ER-ß is taken into
consideration. A lack of ER-ß expression in human prostate tissues
was reported by Bonkhoff et al. (26)
, whereas
several preliminary reports noted expression of this receptor subtype
in dysplastic and cancerous tissues (27, 28, 29)
. Findings in
the present study revealed that ER-ß mRNA was expressed in all three
cancer cell lines (PC-3, DU145, and LNCaP), although in accordance with
the literature, the ER- message was only detected in PC-3 cells
(51)
. Unlike primary PrEC cultures, which uniformly
expressed both PR and pS2 transcripts, PR mRNA expression was only
detected in DU145 and LNCaP cells whereas pS2 transcripts were found in
PC-3 and LNCaP cells. Hence, despite uniform expression of ER-ß, the
expression patterns of ER-, PR, and pS2 among these prostatic cancer
cell lines was variable. Because transcription of PR and pS2 is well
recognized to be regulated by estrogen (56, 57, 58)
, the loss
of expression of these two genes in some prostatic cancer cell lines
suggests a deregulation of estrogen signaling in these cells. A similar
phenomenon has been observed in ovarian cancer cell lines that express
both ER subtype but no PR (33)
.
Until now, it remains unknown as to why prostatic epithelium expresses
only ER-ß and not ER-. In the present study, we demonstrated that
treatment of DU145 and LNCaP cells with demethylating agents
reactivated ER- expression in these cells. These data provide the
first experimental evidence in support of DNA methylation-mediated gene
silencing (59
, 60)
as a mechanism of ER- inactivation
in PrECs. In breast cancers, it had been shown that hypermethylation of
the promoter region of ER- was associated with loss of expression of
this receptor subtype in hormone-refractory cancers
(61, 62, 63, 64)
. Because of the fragile nature of PrECs in
primary culture, we had not subjected them to demethylating agent
treatment; it is reasonable to assume that the same mechanism
transcriptional inactivation operates in ER- silencing in the normal
prostatic epithelium. Interestingly, loss of pS2 expression in DU145
cells might also be linked to hypermethylation-mediated transcriptional
inactivation because exposure of this cell line to demethylating agents
revived pS2 expression. The pS2 gene encodes an 84-amino
acid, cystein-rich, secretary protein, which is widely expressed in
estrogen-sensitive tissues (57)
. Its expression in
prostate cancer specimens has been shown to be associated with
premalignant changes and neuroendocrine differentiation (65
, 66)
.
Different variants of ER- transcripts are often found to coexist
with the wild-type transcript in normal and malignant tissues
(67
, 68) . These variants, produced by alternative
splicing, are whole exon deletion variants that may have "outlaw
functions." In PC-3 cells, a previous study has demonstrated the
expression of an exon 4 deletion variant (51)
. In the
current study, we found an exon 2 deletion variant in PC-3, BPH-1, and
PrEC (Clontech) cells. Whether ER transcript variants have functional
or regulatory roles in prostatic cells is a topic of future
investigation.
Therapies for metastatic prostate cancers are limited. In addition to androgen ablation therapies, estrogens/antiestrogens have been used, singularly or in combination with other modalities, for treatment of the disease. DES is an effective treatment therapy; however, its estrogenicity induces significant adverse effects in patients and have resulted in termination of its use (7) . In contrast, TAM, a nonsteroidal antiestrogen, is better tolerated but produces little objective responses in multiple trials (8, 9, 10, 11, 12) . With the discovery of ER-ß as a new estrogen signaling pathway and the availability of pure antiestrogen such as ICIs (13) , it becomes appropriate to address the issue of whether pure antiestrogens could be considered in the treatment of prostate cancer. In this study, we have compared the efficacy of two estrogens (E2 and DES) an two antiestrogens (4OH-TAM and ICI) in inhibiting cell growth in two androgen-refractory prostate cancer cell lines, PC-3 cells that we found express both ER subtypes, and DU145 cells that express only ER-ß. Past studies have shown that DES, when given at high concentrations (10–30 µM range), exerts colchicine-like action, inhibits tubulin assembly, and induces apoptosis in prostate cancer cells (69 , 70) . Because the focus of the current study is to elucidate receptor-mediated action of DES, we have chosen to use concentrations not higher than 10-6 M. Our results demonstrated all four estrogenic/antiestrogenic compounds, at relatively low doses, exerted antiproliferative effects on PC-3 cells, with antiestrogens exhibiting greater potencies. Furthermore, competition experiments showed that DES could reverse the effect of ICI, thus suggesting that estrogens and antiestrogens may share similar cellular mediators in this cell line. In contrast, DU145 cells responded only to antiestrogens with regard to cell growth inhibition. In both cases, ICI was found to be more potent than 4OH-TAM as a growth inhibitor. Furthermore, competition experiments demonstrated that E2 did not reverse the ICI-induced or 4OH-TAM-induced growth inhibition. This finding suggests that estrogens have little impact on the antiestrogenic action in DU145 cells. Importantly, the antiestrogen-induced growth-inhibitory response in DU145 cells was reversible by cotreatment with an ER-ß antisense ODN. Taken together, these findings raise several significant implications: (a) it is apparent that the estrogen/antiestrogen-induced antiproliferative action on prostatic cancer cells is ER subtype dependent. It supports the prediction for the antiproliferative action of ER-ß as previously discussed (71) ; (b) because ICI consistently expresses a higher potency, it may be better suited to be used in prostate cancer treatment. In clinical trails for breast cancer treatment, this compound has demonstrated high efficacy and low toxicity (72) ; and (c) our data have provided the first demonstration that estrogen/antiestrogen action in prostatic cancer cells could signal via an ER-ß pathway. Because ER-ß selective ligands have been reported recently (73) , this development raises the likelihood of using receptor subtype ligands as cancer therapeutics in the future.
In summary, this study has demonstrated that human normal PrECs express
exclusively ER-ß and likely signal via this receptor subtype for
estrogen/antiestrogen action. On the contrary, prostatic cancer cells
exhibit a more variable pattern of ER subtype expression, and their
responses to individual estrogen or antiestrogen will depend on the ER
subtype(s) expressed in the cells. Significantly, we provide the first
experimental evidence that ER- gene silencing in prostate
cancer cells, and perhaps also in normal cells, may be caused by DNA
hypermethylation. Overall, data from this study lend support to the
notion that ER-ß plays a central role in estrogen/antiestrogen
signaling in normal and malignant human PrECs.
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FOOTNOTES |
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1 Research was supported in part by United States
Army Grant DAMD17-98-1-8606 and NIH Grants CA15776 (to S-M. H.). 
2 Present address: New England College of
Osteopathy, Biddeford, ME 04005.
3 To whom requests for reprints should be
addressed, at Division of Urology, Department of Surgery, Rm S4-746,
University of Massachusetts Medical School, 55 Lake Avenue North,
Worcester, MA 01655. E-mail: shuk-mei.ho{at}umassmed.edu
4 The abbreviations used are: DES,
diethylstilbestrol; PCa, prostatic adenocarcinoma; TAM, tamoxifen; ER,
estrogen receptor; E2, 17ß-estradiol; PrEC, prostatic
epithelial cell; BPH, benign prostatic hyperplasia; PR, progesterone
receptor; 4OH-TAM, 4-hydroxy-TAM; ICI, ICI-182,780; ODN,
oligodeoxynucleotide; FBS, fetal bovine serum; P/S,
penicillin/streptomycin; RT-PCR, reverse transcription-PCR; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; AR, androgen receptor.
5 Internet address:
http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi.
Received 11/24/99. Accepted 4/13/00.
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REFERENCES |
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and ß by both the antiestrogens ICI 164,384 and ICI 182–780 leading to opposite effects on receptor stability. Biochem. Biophys. Res. Commun., 261: 1-5, 1999.[Medline]
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and -ß, progesterone receptor, and androgen receptor mRNA in normal and malignant ovarian epithelial cells. Proc. Natl. Acad. Sci. USA, 96: 5722-5727, 1999. (ER) and estrogen receptor-ß (ERß) messenger ribonucleic acid in the wild-type and ER-knockout mouse. Endocrinology, 138: 4613-4621, 1997.This article has been cited by other articles:
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 P. Li, H. Lee, S. Guo, T. G. Unterman, G. Jenster, and W. Bai AKT-Independent Protection of Prostate Cancer Cells from Apoptosis Mediated through Complex Formation between the Androgen Receptor and FKHR Mol. Cell. Biol., January 1, 2003; 23(1): 104 - 118. [Abstract] [Full Text]
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S. Huerta, J. R. Arteaga, R. W. Irwin, T. Ikezoe, D. Heber, and H. P. Koeffler PC-SPES Inhibits Colon Cancer Growth in Vitro and in Vivo Cancer Res., September 15, 2002; 62(18): 5204 - 5209. [Abstract] [Full Text] [PDF]
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I. Y. Kim, B.-C. Kim, D. H. Seong, D. K. Lee, J.-M. Seo, Y. J. Hong, H.-T. Kim, R. A. Morton, and S.-J. Kim Raloxifene, a Mixed Estrogen Agonist/Antagonist, Induces Apoptosis in Androgen-independent Human Prostate Cancer Cell Lines Cancer Res., September 15, 2002; 62(18): 5365 - 5369. [Abstract] [Full Text] [PDF]
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H. T. Kim, B. C. Kim, I. Y. Kim, M. Mamura, D. H. Seong, J.-J. Jang, and S.-J. Kim Raloxifene, a Mixed Estrogen Agonist/Antagonist, Induces Apoptosis through Cleavage of BAD in TSU-PR1 Human Cancer Cells J. Biol. Chem., August 30, 2002; 277(36): 32510 - 32515. [Abstract] [Full Text] [PDF]
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I. Y. Kim, D. H. Seong, B.-C. Kim, D. K. Lee, A. T. Remaley, F. Leach, R. A. Morton, and S.-J. Kim Raloxifene, a Selective Estrogen Receptor Modulator, Induces Apoptosis in Androgen-responsive Human Prostate Cancer Cell Line LNCaP through an Androgen-independent Pathway Cancer Res., July 1, 2002; 62(13): 3649 - 3653. [Abstract] [Full Text] [PDF]
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N. Kuwajerwala, E. Cifuentes, S. Gautam, M. Menon, E. R. Barrack, and G. P. V. Reddy Resveratrol Induces Prostate Cancer Cell Entry into S Phase and Inhibits DNA Synthesis Cancer Res., May 1, 2002; 62(9): 2488 - 2492. [Abstract] [Full Text] [PDF]
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E. Corey, J. E. Quinn, M. J. Emond, K. R. Buhler, L. G. Brown, and R. L. Vessella Inhibition of Androgen-independent Growth of Prostate Cancer Xenografts by 17{beta}-Estradiol Clin. Cancer Res., April 1, 2002; 8(4): 1003 - 1007. [Abstract] [Full Text] [PDF]
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 J. F. Schmitt, D. S. Millar, J. S. Pedersen, S. L. Clark, D. J. Venter, M. Frydenberg, P. L. Molloy, and G. P. Risbridger Hypermethylation of the Inhibin {alpha}-Subunit Gene in Prostate Carcinoma Mol. Endocrinol., February 1, 2002; 16(2): 213 - 220. [Abstract] [Full Text] [PDF]
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M. E. Taplin and S.-M. Ho The Endocrinology of Prostate Cancer J. Clin. Endocrinol. Metab., August 1, 2001; 86(8): 3467 - 3477. [Full Text] [PDF]
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L. G. Horvath, S. M. Henshall, C-S. Lee, D. R. Head, D. I. Quinn, S. Makela, W. Delprado, D. Golovsky, P. C. Brenner, G. O'Neill, et al. Frequent Loss of Estrogen Receptor-{beta} Expression in Prostate Cancer Cancer Res., July 1, 2001; 61(14): 5331 - 5335. [Abstract] [Full Text] [PDF]
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S. Signoretti and M. Loda Estrogen Receptor {beta} in Prostate Cancer : Brake Pedal or Accelerator? Am. J. Pathol., July 1, 2001; 159(1): 13 - 16. [Full Text] [PDF]
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I. Leav, K.-M. Lau, J. Y. Adams, J. E. McNeal, M.-E. Taplin, J. Wang, H. Singh, and S.-M. Ho Comparative Studies of the Estrogen Receptors {beta} and {{alpha}} and the Androgen Receptor in Normal Human Prostate Glands, Dysplasia, and in Primary and Metastatic Carcinoma Am. J. Pathol., July 1, 2001; 159(1): 79 - 92. [Abstract] [Full Text] [PDF]
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D. Pasquali, V. Rossi, D. Esposito, C. Abbondanza, G. A. Puca, A. Bellastella, and A. A. Sinisi Loss of Estrogen Receptor {beta} Expression in Malignant Human Prostate Cells in Primary Cultures and in Prostate Cancer Tissues J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 2051 - 2055. [Abstract] [Full Text]
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A. Latil, I. Bièche, D. Vidaud, R. Lidereau, P. Berthon, O. Cussenot, and M. Vidaud Evaluation of Androgen, Estrogen (ER{{alpha}} and ER{beta}), and Progesterone Receptor Expression in Human Prostate Cancer by Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction Assays Cancer Res., March 1, 2001; 61(5): 1919 - 1926. [Abstract] [Full Text]
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2) in PC-3 cells.
c, a summary of the data from RT-PCR analyses performed
in three individual experiments. +, detectable level of
expression; -, undetectable level of expression.
).
