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- Original Article -
Expression of human AR cDNA driven by its own promoter
results in mild promotion, but not suppression, of growth in
human prostate cancer PC-3 cells
Saleh Altuwaijri1, 2, Cheng-Chia
Wu1, Yuan-Jie Niu1, Atsushi
Mizokami1,3, Hong-Chiang
Chang1,4, Chawnshang Chang1
1Departments of Pathology and Urology, and the Cancer Center, University of Rochester Medical Center, Rochester,
New York 14642, USA
2King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia
3Department of Urology, Karazawu University, Karazawu City, Japan
4Department of Urology, National Taiwan University, Taipei, Taiwan, China
Abstract
Aim:
Keywords:
Correspondence to: Dr Chawnshang Chang, George H. Whipple Laboratory for Cancer Research-Pathology and Urology, 601 Elmwood
Ave., Box 626, Rochester, New York 14642, USA.
Tel: +1-585-275-9994 Fax: +1-585-756-4133
E-mail: chang@URMC.rochester.edu
Received 2006-09-09 Accepted 2006-10-24
DOI: 10.1111/j.1745-7262.2007.00258.x
1 Introduction
Prostate cancer is the commonest cancer in men and
is the second leading cause of cancer related deaths in
men in the USA [1]. Like normal prostate tissues,
prostate cancer proliferation and maintenance of tissue
differentiation depends on androgen signaling; this
property has been targeted therapeutically by androgen
deprivation treatment in prostate cancer [2]. Although
androgen ablation is effective at slowing initial progression of
tumor growth, it is commonly known that long-term
treatment eventually results in loss of efficacy as a result of
the development of androgen-independent prostate
cancer (AIPC) [3]. Although AIPC cells lose their
dependence on androgen as a proliferative signal,
immunohistochemical data suggest that cells from many
androgen-independent tumors continue to express the androgen
receptor (AR) [4, 5]. Thus, the role of AR in AIPC
remains unclear.
PC-3 is an androgen insensitive human prostate
cancer cell line derived from a bone metastasis that has been
used to study AIPC for several years [6]. These cells
lack expression of AR, despite having normal AR gene.
Commonly, the PC-3 cell line has been used to study
AIPC in vitro or in vivo, and furthermore the use of
PC-3 has increased in studying the role of the AR in AIPC
through transfection of functional AR cDNA. However,
results remain controversial. Marcelli
et al. [6] reported that the transfection of a constitutively active AR driven
by the cytomegalovirus (CMV) promoter resulted in growth inhibition in PC-3 cells. In support of this, Heisler
et al. [5] also created a series of AR expressing PC-3
clones stably transfected with the CMV promoter-driven
AR expressed at various levels; from one-quarter to twice
the AR expression concentration of AR-positive LNCaP
prostate cancer cell line. Even with the wide range of
AR expression levels, dihydrotestosterone (DHT)
treatment resulted in an inhibition of cellular growth in PC-3
clones with low-level (PC-3[AR]13), moderate-level
(PC-3[AR]2) and high-level (PC-3[AR]10) AR expression [5].
In addition to the use of the CMV promoter to drive the
AR expression, AR expressing PC-3 clones were also
created with the pSG5-AR vector in which expression
of the AR was controlled by the SV40 promoter. Similarly, the expression of the AR in this PC-3 clone
caused androgen-sensitivity resulting in a suppression of
the cell growth [7]. With these results, however, the AR
expression within different PC-3 cell lines is artificially
controlled by viral promoters and might not represent
the control of the AR function under physiological and
cellular conditions. Observations of the AR function in
AIPC PC-3 cell lines with continual AR expression might
be insufficient for showing the physiological
contributions of androgen stimulated AR signaling for cell growth
and proliferation.
In the present study, we generated PC-3(AR)9, a
PC-3 cell line that expresses the AR under the control of
the intrinsic AR promoter, and compared the
proliferation of this cell line to that of PC-3(AR)2, which
consistently expresses high levels of the AR under androgen
treatment. Because the intrinsic AR promoter is
regulated by various intracellular responses [8, 9], we hope
to examine the AR function in AIPC PC-3 cell expressed
under physiological control. We now report that DHT
induces cell cycle arrest and inhibits the proliferation of
PC-3(AR)2. In contrast, growth of PC-3(AR)9 cells is
increased mildly or does not change when exposed to
physiological conditions (1 nmol/L) of DHT
[10].
2 Materials and methods
2.1 Cell culture, plasmids and reagents
PC-3(AR)2 was a generous gift from Dr T. J. Brown
[5]. Human prostate cancer cell lines PC-3(pIRES)
(a mammalian expression vector, internal ribosome entry
site), PC-3(AR)2 (AR expression driven by human CMV),
and PC-3(AR)9 (AR expression driven by its own
natural promoter) were maintained in RPMI 1640 media
containing 10% fetal calf serum (FCS), 25 U/mL penicillin
and 25 µg/mL streptomycin. DHT and hydroxyflu-tamide
(HF) were purchased from Sigma (St. Louis, MO, USA).
Anti-AR polyclonal antibody, NH27, was produced as
previously described [11, 12]. An anti-actin monoclonal
antibody was purchased from Amersham Biosciences (Piscataway, NJ, USA). The natural promoter driven
AR plasmid was constructed by inserting a 3.6-kDa hAR
promoter, the entire hAR 5'-UTR, and the full-length AR
cDNA into the pIRES plasmid. Expression of the AR
was placed under the control of the 3.6 kDa proximal
AR promoter region cloned into pIRES. Neomycin resistant cells were selected by incubation with
500 mg G418/mL.
2.2 Transient transfections and reporter gene assays
Transfections were carried out using the calcium
phosphate precipitation method [13]. Luciferase (Luc)
assays were carried out as described previously [17].
Briefly, 1_4 × 105 cells were plated on 35- or 60-mm
dishes 24 h before adding the DNA precipitation mix
containing either mouse mammay tumor virus (MMTV)-Luc or androgen response element 4-Luc (ARE[4]-Luc)
reporter plasmid DNA. The medium was changed to
phenol-red-free RPMI 1640 with 10% charcoal-dextran
treated FCS (CD-FCS) 1 h before transfection. In each
experiment, the total amount of transfected
DNA/dish was equalized by the addition of
empty expression vector (pCMV) to make a total of 10 µg/60-mm dish. After
24-h transfection, the medium was changed, the cells
were treated with various concentrations of DHT, harvested,
and whole cell extracts were used for Luc assay. Luc
activity was determined using a
Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI,
USA) and a luminometer.
2.3 Western blot analysis
PC-3(pIRES), PC-3(AR)2 and PC-3(AR)9 cells were
treated with ethanol or 1 nmol/L DHT CD-FCS for 24 h.
The medium was removed and the attached cells were
washed with phosphate buffered saline (PBS). Protein
extraction was accomplished by cell lysis with sodium
dodecyl sulfate (SDS) [14]. Protein concentrations were
measured using bicinchoninic acid protein (BCA)
protein reagent (Pierce Chemical, Rock-ford, IL, USA).
Equal amounts of total protein (50 µg) were loaded onto
an SDS-polyacrylamide gel with a Tris/glycine running
buffer system and subjected to electrophoresis.
Proteins were then transferred to a 0.2-µm polyvinylidene
difluoride membrane (PVDF) in a mini-electrotransfer unit
(Bio-Rad, Hercules, CA, USA). Membranes were probed
with anti-AR and anti-actin antibodies. Immunoblot
analysis was carried out with horseradish
peroxidase-conjugated anti-rabbit and anti-mouse IgG using
enhanced chemiluminescence Western blotting detection
reagents purchased from Amersham Biosciences (Piscataway, NJ, USA).
2.4 Thiazolyl blue assay
Thiazolyl blue (MTT) assay is a quantitative
colorimetric assay for mammalian cell survival and proliferation.
Briefly, 5 × 104 PC3-3(pIRES), PC-3(AR)2 and
PC-3(AR)9 cells were seeded in 24-well plates in RPMI 1640 with
10% CD-FCS. After 24 h, cells were treated with either
vehicle or selected concentrations of DHT for 9 days.
Daily cell growth was determined by adding 50 µL of
thiazolyl blue (5 mg/mL MTT; Sigma, St. Louis, MO,
USA) into each well of a plate with 500 µL of medium
for 2 h. After incubation, 400 µL of 0.04 mol/L HCL in
isopropanol was added to each well. Absorbency was
read at a test wavelength of 590 nm.
2.5 Cell cycle analysis (flow cytometry assay)
PC-3(pIRES), PC-3(AR)2 and PC-3(AR)9 cells grown in 100-mm dishes were treated with either
ethanol or 1 nmol/L DHT in CD-FCS for the indicated times.
At the end of each time period, the cells were digested
by trypsin_EDTA. As many as
1 × 106 cells were harvested and fixed in 70% ethanol at 4ºC. After 12 h, cells
were centrifuged (1 000 × g, 7 min, 4ºC),
resuspended in PBS containing 0.05 mg/mL RNase A (Sigma, St.
Louis, MO, USA), and then incubated at room temperature for 30 min. After washing, the cells were
stained with 10 mg/mL propidium iodide, filtered
through a 60-mm mesh, and 10 000 cells per treatment
were analyzed by flow cytometry (FACSCalibur; BD Company, San Jose, CA, USA) [15] with MODFIT
software (Verity Software House, Topsham, MA, USA).
3 Results
3.1 Expression of AR in PC-3 cells stably transfected
with a full length AR cDNA under the control of the
natural promoter (p-3.6 hAR)
Cell lines expressing AR under the control of the
natural AR promoter were generated by transfecting PC-3
cells with p-IRES-3.6hAR. The plasmid was constructed
using a 3.6-kDa proximal human AR promoter region to
drive expression of a full-length human AR cDNA (Figure
1A). Several G418-resistant colonies were selected from
the original transfected pool using cloning cylinders,
followed by expansion and an assay for the AR expression.
A rabbit anti-human AR polyclonal antibody was used to
probe Western blots of the protein extracts from each
cell line. A protein band with a molecular weight of 110
kDa was present in several transfected lines designated as
PC-3(AR)7, PC-3(AR)8 (data not shown) and
PC-3(AR)9. No AR protein band was detected in PC-3(pIRES) cells.
The 110 kDa proteins detected in the natural promoter
transfected PC-3(AR)9 cell were the same size as those
from the CMV promoter derived cell line PC-3(AR)2 (Figure 1B, C). Furthermore, the level of AR protein
detected in PC-3(AR)9 cells using the natural AR
promoter was consistently less then that detected in the CMV
promoter-driven cell line in the presence or absence of
1 nmol/L DHT and the levels of AR expression in
PC-3(AR)9 varied between vehicle or 1 nmol/L DHT
treatment (Figure 1B, C).
3.2 The functional assay of AR in PC-3(AR)9 compared
with AR in PC-3(AR)2
Androgen responsiveness was tested using transient
transfection assays. Cell lines PC-3(AR)2 and
PC-3(AR)9 were transfected with a Luc reporter plasmid driven by
the MMTV promoter, treated with various concentrations of DHT, and assayed for Luc activity (Figure 2A).
Both PC-3(AR)2 and PC-3(AR)9 showed an increase in
relative Luc activity with the addition of DHT from
0.0001 nmol/L to 10 nmol/L. The addition of 1 nmol/L
DHT resulted in a maximal 20-fold induction of Luc
reporter gene in PC-3(AR)2, whereas PC-3(AR)9 showed
a 5-fold induction. This is consistent with the higher
level of the AR protein expressed in the CMV
promoter-driven cell line PC-3(AR)2 (Figure 1B). The level of the
AR protein expressed correlates with the degree of AR
transactivation observed using both MMTV promoter driven reporter plasmid and ARE reporter plasmid. Finally,
the transactivation activity was inhibited by HF in both
the PC-3(AR)2 and PC-3(AR)9 cell lines, indicating that
expression of luciferase activity from the reporter
plasmid is androgen/AR-mediated (Figure 2B, C).
3.3 Differential modulation of cell culture growth in
PC-3(AR)2 and PC-3(AR)9 cells
A MTT assay in PC-3(pIRES), PC-3(AR)2 and PC-3(AR)9 cell lines were used to determine the effect of
androgen treatment on cell viability. Cells were plated in
24-well tissue culture plates
(5 × 104 cells/well) and the
number of viable cells was determined after 9 days. In
PC-3(AR)2, DHT inhibits cell proliferation at all tested
concentrations of DHT (Figure 3B, E). This is
consistent with a previous report in which DHT was shown to
induce cell cycle arrest and inhibit cell proliferation in
PC-3(AR)2 cells [5]. In contrast, PC-3(AR)9 cells
showed a slight increase or no change in cell
proliferation when treated with 1 nmol/L DHT (Figure 3C, F).
3.4 The high level of AR expression inducing cell cycle
arrest
It has been proposed that androgen inhibits cell
proliferation in PC-3(AR)2 cells by inducing cell cycle arrest
[7]. Using flow cytometry, we examined the effect of
DHT on cell cycle phase distribution. After incubating
cells for 9 days in medium containing
1 nmol/L DHT, flow cytometry analysis with propidium iodide staining showed
that 1 nmol/L DHT treatment increased
the proportion of PC-3(AR)2 cells in the
G0_G1 phase from 39% to 62%
(P < 0.05; Figure 4C). This was completely prevented
by the addition of HF (data not shown). However, in
PC-3(pIRES) and PC-3(AR)9 cells, DHT had little effect on the cell cycle (Figure 4A, B), suggesting that
DHT-induced G1 cell cycle arrest is dependent on the
expressed level of the AR protein.
4 Discussion
In contrast to experimental results in which the
commonly used AIPC cell lines DU145 and PC-3 were
AR-negative, more than 90% of prostatic cancers obtained
directly from patients failing androgen ablation actually
overexpress the AR; 30% of this overexpression is to the
result of genetic amplification [16]. Despite this high
percentage of AR expression in AIPC, the role of the AR
in AIPC is unknown. In the present study, using the
AIPC cell line PC-3, we created PC-3(AR)9 stable cell
line transfected with the normal human AR natural
promoter driven AR vector in order to examine the AR role
in AIPC under physiological conditions.
Numerous reports have shown that androgen/AR can
modulate AIPC cell growth [13, 16]. However, the
precise role of androgen/AR remains unclear. By disrupting
AR in two different androgen-refractory sublines of
LNCaP (LNCaP-Rf and LNCaP-C4), Zegarra-Moro
et al. [3] and Xu et al. [17] showed the dependence of AIPC
on the AR for proliferation. In contrast, using AR
restoration strategies in the PC-3 AR negative AIPC cell line,
multiple groups have shown that an overexpression of
AR resulted in AIPC growth inhibition [5_7]. Therefore,
whether the AR stimulates or inhibits cell growth is a
question that remains controversial. In related studies, it
has been reported that estrogen insensitive breast cancer
cells transfected with the estrogen receptor (ER) cDNA
showed a similar controversial cell growth inhibition. Like
the AR, the ER is a DNA-binding protein that can
interact with general transcription factors. Thus, the growth
inhibitory effects seen in the transfected breast cancer
cells might be the result of non-specific effects caused
by overexpression of the ER [5, 18]. This might also be
the case in the AR transfected PC-3 related studies in
which the strong viral promoter-driven AR vectors (CMV
or SV40 promoters) were used [5]. Therefore, in order
to mimic the physiological levels of androgen and allow
for intracellular modifications of the AR expression, we
constructed a natural promoter driven AR plasmid for
transfection into PC-3 cells. This was carried out in
order to establish a stable PC-3(AR)9 clone in which
androgen/AR mediates cell proliferation and other
functions including cell cycle and cell invasion.
Having established the PC-3(AR)9 clone, we first
compared the AR protein level as well as AR transactivation
to that of the previously established PC-3(AR)2 cell line
[5]. Levels of AR in PC-3(AR)9 were consistently less
than that of PC-3(AR)2. In addition, the AR protein
levels of PC-3(AR)9 with 1 nmol/L DHT treatment showed
an increase compared to that of vehicle treatment. Lin
et al. [19] reported that testosterone and DHT under
normal conditions act as moderate up-regulators of AR
mRNA expression in smooth muscle cells
in vitro. It has been reported that various stimuli might also modify
the CMV promoter driven expression levels [20]; thus,
this change in the AR protein level might be an artificial
cause of the CMV promoter-modification in
PC-3(AR)2. In order to characterize the function of AR in
PC-3(AR)9, we used the MMTV promoter-driven Luc reporter gene
to show the AR transactivation after DHT treatment.
Treatment of PC-3(AR)2 with 1 nmol/L DHT resulted in
a maximal 20-fold increase in Luc activity as compared
with PC-3(AR)9, in which DHT treatment induced a
5-fold increase. This correlates with the difference in the
AR protein levels detected in the two cell lines. In order
to characterize the AR role in PC-3 AIPC cell proliferation,
an MTT assay was carried out. In agreement with
previous publications, PC-3(AR)2 showed a decrease in cell
growth when treated with various concentrations of DHT
[5]. In contrast, after treating PC-3(AR)9 with
physiological levels (1 nmol/L) of DHT, we observed a slight
increase in cell growth in comparison with vehicle
treatment. Furthermore, in comparing cell cycle
analysis using flow cytometry, treating PC-3(AR)2 cells with
1 nmol/L of DHT resulted in an increase in G0/G1 cell
cycle arrest. This phenomenon was also observed
previously by Heisler et al. [5]. As for PC-3(AR)9, little
change in cell cycle was observed.
The rationale for the use of a natural AR promoter in
creating a PC-3 AR positive cell line is to allow cellular
modification of AR expression according to its
physiological needs. We observed that under the control of
the normal AR promoter, PC-3(AR)9 AR expression was
significantly less than that in the PC-3(AR)2 cell line.
Furthermore, DHT treatment of PC-3(AR)9 showed that
androgen/AR increased cell proliferation or did not change
in PC-3, in contrast to the inhibition reported previously
by groups that used viral promoter driven AR expression.
Whether this difference in the role of androgen/AR in
AIPC cell proliferation is a result of the AR protein level
is still debatable. Heisler et al. [5] created a series of
PC-3(AR) stable clones that expressed the AR at levels
one-quarter (PC-3[AR]13) to twice that of LNCaP endogenous AR. At various concentrations of DHT
treatment, the investigators observed a consistent
inhibition of PC-3(AR) growth through the androgen/AR. The
AR expression level of the PC-3(AR)13 cell line exceeded
the necessary threshold for inducing
androgen/AR-mediated increase in cell proliferation. This is possible
because the CMV promoter drives its AR expression,
however, further investigation is needed. We believe that
PC-3(AR)9 will be an excellent model that most closely
resembles the physiological function of the AR in AIPC.
This will give us further insight into the AR function in
the PC-3 AIPC cell line, and has introduced greater
considerations and criteria for the use of AR overexpression
strategies.
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