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INTRODUCTION |
Androgens are responsible for the development, maintenance, and
regulation of male phenotype and reproductive physiology. These
activities are mediated through the
AR1 (1-4), which belongs to
the steroid/thyroid receptor superfamily, a group of ligand-regulated
transcription factors (5). Like other members of the family, the AR
protein is modular in nature and composed of an amino-terminal A/B
region, a DNA-binding domain and a "hinge" region in the middle,
and a hormone-binding domain at the carboxyl terminus. Whereas the
amino-terminal A/B region contains the major transcriptional activation
function, a weaker activation function in the hormone-binding domain
also contributes to the total transcriptional activity. The
hormone-binding domain represses the activation functions until
androgens bind to it and relieve the repression by inducing the
formation of a conformation suitable for the interaction with
transcriptional cofactors (6-8). In addition, the polymeric stretches
within the amino-terminal region (9) and receptor phosphorylation (10,
11) also contribute to the AR transcriptional activities.
Besides their established physiological functions, androgens are
implicated in multiple pathological processes including prostate cancer
(PCa), which is the most commonly diagnosed malignancy and second only
to lung cancer in the mortality rate of American males (12).
Chronically maintained androgen level sustains the total prostate cell
number by both stimulating the rate of proliferation and inhibiting the
rate of death of prostate epithelial cells (13); both lead to an
increase in the cell number. To maintain the homeostasis of prostate
epithelium, there must exist a "brake" system that opposes these
effects of androgens.
PTEN tumor suppressor is a 403-amino acid
phosphoprotein/phospholipid dual-specificity phosphatase (14, 15).
Somatic mutation of PTEN is a common event in diverse human cancers
including PCa (14, 15), and heterozygous deletion of PTEN in mice leads to neoplasm in multiple tissues including the prostate (16, 17). The
significance of the phosphoprotein phosphatase in tumor suppression is
largely unknown, although one known substrate is focal adhesion kinase
(18). In contrast, multiple findings support a role of the lipid
phosphatase activity in PTEN-mediated tumor suppression (19-23). PTEN
lipid phosphatase catalyzes the dephosphorylation of
phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3)
(24), resulting in inactivation of the downstream protein kinase B, also named AKT, activity (21, 22). Ectopic PTEN expression in PTEN-null
PCa cells induced cell cycle arrest and apoptosis (22, 25); both
activities are opposite to those of androgens, suggesting that PTEN may
provide the brake to balance the functions of AR in prostate cells.
In the current study, we provide experimental evidence that the
activities of PTEN and AR are antagonistic in PCa cells. Interestingly, PTEN and AR do not simply shut down each others activity but
differentially antagonize and selectively dominate each other in PCa
cell apoptosis and proliferation.
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EXPERIMENTAL PROCEDURES |
Plasmids--
AREe1bLuc (26), pCMVhAR (27), pLEN
gal (28),
PSALuc (29), GalLuc (30), and Gal-VP16 (6), pSG5L-HA-PTEN:WT (22), pSG5L-HA-PTEN:G129R (22), PLNCX-HA-myr-AKT (22), and
PLNCX-HA-myr-AKT179 M (22) have already been described.
pCMV
and pLNCE are from CLONTECH (Palo Alto,
CA). pLENhAR was constructed by replacing
-galactosidase (
-gal)
cDNA of pLEN
gal with human AR cDNA. Partial human AR
cDNA was excised from pCMVhAR with BamHI and
EagI. Because EagI cuts the AR cDNA at an
internal position close to the initiator ATG, the digestion yield a
partial AR cDNA fragment. The partial AR cDNA was ligated to a
synthetic linker that extended the AR cDNA sequence from the
internal EagI site to a designed BamHI site
immediately upstream of the initiator ATG. The full-length AR cDNA
was inserted into the BamHI site of pLEN vector in the correct orientation for mammalian expression.
Transfections and Transcriptional Assays--
PC3 cells were
plated in Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum (FBS) at 2 × 105 cells/well in 6-well
plates and LNCaP cells were plated in RPMI 1640 at 4 × 105 cells/well. One day after plating, cells were
transfected with a LipofectAMINE plus-mediated transfection procedure
following the protocol from Life Technologies, Inc. Transfected cells
were treated with synthetic androgens or phosphoinositide 3-kinase (PI3K) inhibitors in medium containing 1% charcoal-stripped FBS for
24 h and washed with phosphate-buffered saline (PBS). Cell lysate
was prepared by directly adding lysis buffer (25 mM
Tris-phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N',N',N',N'-tetraacetic acid,
10% glycerol, 0.2% Triton X-100) to the cells on ice. Luciferase activity was determined using luciferase assay systems from Promega Corporation (Madison, WI) following the company's protocol.
-gal activity was determined as previously described (31).
ELISA and Colorimetric MTT Assay--
LNCaP cells were plated in
RPMI 1640 containing 10% FBS at 1 × 105 cells/well
in 96-well plates for MTT assays. After cells attached, they were
starved in RPMI 1640 with 1% charcoal-stripped FBS for 48 h and
treated with synthetic androgens or PI3K inhibitors. The medium was
collected and PSA levels were determined using the Tandem-E PSA
ImmunoEnzyMetric Assay Kit (Hybritech Inc., San Diego, CA) following
manufacturer's protocols. The absorbance at 405 nm was measured using
a UV spectrophotometer. PSA concentration (ng/ml) was determined based
on a standard curve generated with PSA controls of known concentrations
provided by the kit. MTT assays were performed as described (32). The
plates were read on a MRX microplate reader (DYNEX Technologies,
Chantilly, VA) using a test wavelength of 595 nm.
Apoptotic Assays--
Transfected cells were washed with PBS and
fixed in 3.7% formaldehyde/PBS at room temperature for 10 min. The
viability of transfected cells in each well was determined by counting
the total number of green cells in each well under a fluorescence microscope. For the demonstration of apoptotic cells, fixed cells were
stained at room temperature for 15 min with
4',6'-diamidino-2-phenylindole (DAPI) at a concentration of 1.5 mg/ml
in the VECTASHIELD Mounting Medium (Vector laboratories, Burlingame,
CA). Green and blue fluorescence were observed with a Leitz Orthoplan 2 Microscope. Representative micrographs were captured by a CCD camera
with the Smart Capture Program (Vysis, Downers Grove, IL). The
apoptotic index of GFP-positive cells was determined by scoring 300 GFP-positive cells for chromatin condensation and apoptotic body formation.
In Vitro Immunocomplex Kinase Assays--
LNCaP cells were
transfected as described above for transcriptional assays, washed with
ice-cold PBS, and lysed in 20 mM Tris, pH. 7.6, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 5 mM
-glycerophosphate, 100 µM
Na3VO4, 0.5% Nonidet P-40, and protease
inhibitor mixture (1 tablet/10 ml). After centrifugation, half of the
supernatant was immunoprecipitated with the 12CA5 anti-HA monoclonal
antibody (Roche Molecular Biochemicals Corp., Indianapolis, IN). Kinase reactions were performed at 30 °C in 30 µl of buffer containing 20 mM HEPES, pH 7.4, 10 mM
p-nitrophenyl phenylphosphonate, 20 mM
MgCl2, 2 mM EDTA, 2 mM EGTA, 100 µM Na3VO4, 10 µM
ATP, 10 µCi [
-32P]ATP with 3 µg of H2B as
substrate. After incubation for 30 min, reactions were terminated by
adding 2× SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample
buffer, analyzed by SDS-PAGE, and visualized by autoradiography.
Immunoblotting Analysis--
To determine the expression of AR,
duplicate wells of PC3 cells transfected in parallel to the cells
prepared for transcriptional assays were pooled and lysed in the same
buffer as described above for kinase assays. The lysate was mixed with
one-sixth of 6× SDS-PAGE sample buffer, heated for 10 min at
100 °C, and separated on 8% SDS-PAGE. To determine the expression
of HA-AKT, half of the cell lysate prepared for kinase assays was
processed similarly as for AR and separated on 10% SDS-PAGE. After
separation on SDS-PAGE, samples were transferred to a nitrocellulose
membrane, probed with the PG-21 anti-AR (Upstate Biotechnology, Lake
Placid, NY) or the 12CA5 anti-HA antibodies, and visualized using ECL
as described (28).
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RESULTS |
Repression of AR Transcriptional Activity by PTEN and a PI3K
Inhibitor and Relief of the Repression by a Dominantly Active
AKT--
To test whether PTEN inhibits AR transcriptional activity, we
transfected into AR-negative, PTEN-null PC3 cells (22) an AR expression
vector, pLENhAR, together with a PTEN expression vector or a control
vector expressing a phosphatase-inactive PTEN. The transcriptional
activity of AR was measured by assaying the luciferase activity from a
co-transfected AR reporter, AREe1bLuc, in which luciferase expression
is under the control of synthetic androgen response elements (AREs)
placed in front of the simple adenovirus E1b promoter. The
effect of PTEN on the AR activity was measured by comparing the AR
activity in cells transfected with PTEN to those transfected with the
control vector. Because of the concern that co-transfected PTEN may
alter the expression of AR by affecting the activity of the promoter or
the enhancer of the AR expression vector, the reporter activity was
normalized with
-gal activity from co-transfected pLEN
gal vector
in which
-gal expression is under the control of human
metallothionin-II promoter and SV40 enhancer, the same regulatory
sequences used by the AR vector. As shown in Fig.
1A, AR basal activity was low but the activity was significantly induced by treatment with R1881, a
stable synthetic androgen agonist. Whereas the basal activity was not
affected, R1881-induced AR activity was repressed, but not abolished,
by PTEN in a dosage-dependent manner.

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Fig. 1.
PTEN repressed the transcriptional activity
of the AR. A, dosage-dependent inhibition
of AR activity by PTEN in PC3 cells. Cells were transfected with 0.1 µg of pLENhAR, 0.5 µg of pLEN gal, 0.5 µg of AREe1bLuc, and the
indicated amounts of pSG5L-HA-PTEN:WT (WT) or
pSG5L-HA-PTEN:G129R (MT). Transfected cells were treated for
24 h with 10 8 M R1881 or ethanol
(EOH) as vehicle controls. All samples were normalized with
-gal activity and expressed as relative luciferase unit
(RLU). Duplicate samples were analyzed for each single data
point, and the data have been reproduced three times. B,
inhibition of AR activity by PTEN was not caused by decreased AR
protein levels. PC3 cells were transfected with 0.1 µg of pCMVhAR,
0.5 µg of pCMV , 0.5 µg of AREe1bLuc, and 0.1 µg of
pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R. The level of AR protein was
detected by Western blotting.
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Because both AR and
-gal were expressed using the same vector, the
way we normalize the reporter activity should have eliminated the
potential interference caused by either variation in transfection efficiency or PTEN-induced alterations in the promoter activity of the
AR expression vector. However, the lower AR activity in the presence of
PTEN could arguably be because of lower level of AR protein as a result
of decreased protein stability or reduced efficiency of protein
translation. To test whether the level of AR protein was altered by
PTEN, PC3 cells were transfected with pCMVhAR and pCMV
, which
express the receptor and
-gal, respectively, under the control of
the stronger cytomegalovirus (CMV) promoter, permitting the analysis of
AR protein level and transcriptional activity in the same experiment.
The AR activity from pCMVhAR was decreased by PTEN to the same degree
as demonstrated for pLENhAR (Fig. 1A). Parallel Western
blotting demonstrated that PTEN, in the presence of R1881, did not
decrease the AR protein level (Fig. 1B). These data
demonstrate that PTEN repressed the transcriptional activity of the AR
per molecule. In the absence of R1881, PTEN expression caused a
decrease in the level of AR protein. The decrease, based on later
studies, was evidently because of the death of transfected cells that
expressed PTEN to high levels.
To further substantiate the AR repression by PTEN, we performed a time
course study as shown in Fig. 2. In cells
transfected with mutant PTEN, R1881-induced a dramatic AR activation at
times as early as 12 h post-transfection and the continuous
treatment proportionally increased the AR activity over time. Whereas
the basal AR activity in the absence of R1881 was not affected, the proportional increase in AR activity as a result of prolonged R1881
treatment was almost eliminated by PTEN.

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Fig. 2.
PTEN repressed the androgen-induced increase
in AR activity over time. PC3 cells were transfected with 0.1 µg
of pCMVhAR, 0.5 µg of pCMV , 0.5 µg of AREe1bLuc, and 0.1 µg of
pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R. Transfected cells were treated
for indicated times with 10 8 M R1881 or
ethanol (EOH) as vehicle controls. AR activity was assayed
and expressed as described in the legend to Fig. 1.
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In theory, the AR repression could be mediated through either the
protein or lipid phosphatase activity of PTEN. Because the lipid
phosphatase activity has a well established role in tumor suppression,
we decided to first investigate the role of the lipid phosphatase
activity in the AR repression. PTEN lipid phosphatase is known to
decrease the level of PI(3,4,5)P3 by catalyzing its dephosphorylation. Thus, we examined whether the decrease of
PI(3,4,5)P3 by other means such as inhibition of its
synthesis will have the same effect on AR activity as PTEN expression.
AR was transfected into PC3 cells and treated with either R1881 alone
or co-treated with LY294002, a synthetic inhibitor for PI3K. As shown
in Fig. 3A, LY294002 repressed
the AR activity in a dosage-dependent manner. A time course
study showed that the percentage of AR repression by LY294002 was best
observed at the shortest time point (Fig. 3B). This is
different from the PTEN data (Fig. 2) and probably reflects the fact
that LY294002 exerts its effect through PI3K-mediated signaling
pathways (which usually takes just minutes), and the effectiveness of
synthetic drugs decreases over time because of the issue of stability
and active clearance by cells. These studies indicate that the
PTEN-induced AR repression is most likely mediated through the
PI(3,4,5)P3 phosphatase activity of PTEN.

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Fig. 3.
A chemical inhibitor of PI3K also repressed
AR activity. A, dosage-dependent inhibition
of AR by LY294002. PC3 cells were transfected with 0.1 µg of pLENhAR,
0.5 of µg AREe1bLuc, 0.5 µg of pLEN Gal and treated for 24 h
with or without 10 8 M R1881 and LY294002
(LY) at indicated concentrations. B, repression
of AR activity by LY294002 over time. PC3 cells were transfected as
described in A and treated for indicated times with or
without 10 8 M R1881 and 20 µM
LY294002. LY294002 was dissolved in Me2SO, and the absolute
amount of Me2SO was the same for all samples.
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Because AKT is the major downstream target of PI(3,4,5)P3,
we next examined the involvement of AKT in PTEN-induced AR repression. PC3 cells were transfected with PTEN, AR, and a dominantly active AKT
of which the activity cannot be repressed by PTEN or a kinase-inactive AKT. The AR repression by PTEN was analyzed as shown in Fig.
4. Whereas PTEN-induced AR repression
occurred in the presence of the kinase-inactive AKT, co-expression of
the dominantly active AKT blocked the AR repression by PTEN. These data
suggest that the down-regulation of PI(3,4,5)P3 by PTEN
lipid phosphatase, and the subsequent inactivation of AKT kinase
mediated the AR repression, establishing the role of the PTEN lipid
phosphatase in AR repression.

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Fig. 4.
A dominantly active AKT blocked PTEN-induced
AR repression. PC3 cells were transfected with 0.1 µg of
pLENhAR, 0.5 µg of AREe1bLuc, 0.5 µg of pLEN Gal, 0.1 µg of
pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R and 0.1 µg of
PLNCX-HA-myr-Akt (Active) or PLNCX-HA-myr-AKTK179M
(Inactive). AR activity was assayed and expressed as
described in the legend to Fig. 1.
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Impairment of the Biological Activities of Endogenous AR by PTEN
and the PI3K Inhibitor--
So far, we have demonstrated an inhibitory
effect of PTEN and the PI3K inhibitor on the activity of transiently
transfected AR with a reporter constructed with synthetic AREs. To
determine whether PTEN also represses endogenous AR activity with
natural promoters, we transfected PSALuc into PTEN-null but
AR-positive LNCaP cells (22) and examined the effect of PTEN and the
PI3K inhibitor on endogenous AR activity. PSALuc is an AR reporter in
which luciferase expression is under the control of the promoter of
human PSA, a PCa marker, which is transcriptionally regulated by AR
through complex AREs (29). As shown in Fig.
5A, the transcriptional activity of the endogenous AR in cells transfected with the PSALuc was
increased by R1881, and R1881 induction was blocked by PTEN. This
demonstrates that PTEN represses endogenous AR activity on natural
promoters and thus is not limited to ectopic AR or synthetic AREs.

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Fig. 5.
PTEN repressed the transcriptional activity
of endogenous AR in LNCaP cells. A, PTEN repression of
endogenous AR activity on PSA promoter in LNCaP cells. Cells were
transfected with 0.5 µg of PSALuc, 0.5 µg of pLEN Gal, and 0.1 µg of pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R. Transfected cells were
treated with or without 10 8 M R1881, and AR
activity was assayed and expressed as described in the legend to Fig.
1. B, lack of Gal-VP16 repression by PTEN in LNCaP cells.
Cells were transfected with 0.5 µg of GalLuc, 0.5 µg of pCMV ,
0.1 µg of pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R and with or without
0.1 µg of Gal-VP16. Transfected cells were treated with
10 8 M R1881. Transcriptional activity of VP16
was assayed and expressed as described in the legend to Fig. 1.
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To rule out the possibility that the AR repression was caused by
nonspecific repression of LNCaP cell transcription by PTEN, we
transfected into LNCaP cells a Gal-VP16 expression vector and a Gal4
reporter and measured the activity of the fusion activator in the
presence or absence of PTEN. As shown in Fig. 5B, the
reporter was inactive in the absence of Gal-VP16, and the co-expression of the fusion activator induced the activity dramatically. More importantly, the Gal-VP16 activity was not inhibited by PTEN. These
data demonstrate that the AR repression was not caused by a nonspecific
effect of PTEN on LNCaP cell transcription.
To determine the biological consequence of the observed repression of
AR transcriptional activity, the effect of PI3K inhibitor on
androgen-induced PSA production and cell proliferation was examined in
LNCaP cells. LNCaP cells were treated with either R1881 alone or
co-treated with LY294002 for indicated times. Culture medium from the
treated cells was collected and analyzed for PSA level by ELISA. As
shown in Fig. 6A, R1881
treatment stimulated PSA production and the stimulation was inhibited
by the co-treatment with LY294002. Similar to PSA production, R1881
induced an increase in cell number as measured by MTT assays, and this
increase was blocked by co-treatment with LY294002 (Fig.
6B). LY294002 did not consistently decrease the basal PSA
level or LNCaP cell numbers in the absence of R1881. These analyses
demonstrate that the repression of AR transcriptional activity by the
PI3K inhibitor impaired the biological functions of endogenous AR.

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Fig. 6.
PI3K inhibitor impaired the biological
activities of the AR. A, repression of androgen-induced
PSA production in LNCaP cells by LY294002. LNCaP cells were starved and
treated with vehicle, 10 8 M R1881 or
10 8 M R1881 plus 20 µM LY294002
for the indicated times. Absolute amounts of Me2SO and
ethanol were the same for all samples. PSA levels were quantified by
ELISA. Duplicate samples were analyzed for each data points, and the
experiment was repeated three times. B, repression of
androgen-induced LNCaP cell proliferation by LY294002. LNCaP cells were
starved and treated as in A. Cell numbers were determined by
the MTT colorimetric assays. Eight samples were analyzed for each data
point, and the data were reproduced three times.
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AR-dependent Protection of PTEN-induced Apoptosis by
Androgens in PCa Cells--
Our data so far have established a
functional relationship between PTEN and AR by showing that PTEN or a
PI3K inhibitor opposed AR function in PCa cells. We next investigated
whether androgen also opposes the function of PTEN. Because the
expression of PTEN in LNCaP cells induced apoptosis (25), we examined
the effect of androgens on PTEN-induced apoptosis. LNCaP cells were
transfected with a green fluorescence protein (GFP) expression vector
and the PTEN expression vector or the control vector expressing
the mutant PTEN. The transfected cells were then treated with or
without R1881, and the viability of transfected cells was determined. As shown in Fig. 7A, the
viability of PTEN-transfected cells in the absence of R1881 was only
about 15% of the cells transfected with the control vector. Treatment
with R1881 restored the viability of PTEN-transfected cells to the
level of controls. This decrease in the viability of PTEN-transfected
cells as well as its blockage by R1881 was more directly shown by the
number of GFP-positive cells in representative micrographic fields
(Fig. 7B, panels 1-3).

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Fig. 7.
Androgen protected LNCaP cells from
PTEN-induced apoptosis. A, androgen effect on the
viability of PTEN-transfected LNCaP cells. LNCaP cells were transfected
with 0.5 µg of pLNCE, a GFP expression vector and 0.1 µg of
pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R. Transfection cells were
treated with 10 8 M R1881 or ethanol for
24 h, and the viability of transfected cells in each well was
determined by counting the total number of green cells.
Triplicate samples were analyzed for each data point, and the
data were reproduced three times. B, representative
micrographs of PTEN-transfected cells in the presence or absence of
androgen. Cells were transfected and fixed as in A. Fixed
cells were stained with DAPI, and representative micrographs were
captured by a CCD camera attached to the fluorescence microscope using
objective lens of × 10 (panels 1-3) or × 100 (panels 4-6). C, androgen effect on PTEN-induced
increase in apoptotic index of LNCaP cells. Cells were processed as in
B, and the apoptotic index of GFP-positive cells was
determined by scoring 300 GFP-positive cells for chromatin
condensation. Triplicate samples were analyzed per data point, and the
graph represents three independent experiments.
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To confirm that the decreased viability of PTEN-transfected cells is
the result of cell apoptosis, cells were fixed after transfection and
stained with DAPI, and the nuclear morphology of transfected cells was
examined for features of apoptosis under a fluorescence microscope that
permits the simultaneous visualization of both blue and green
fluorescence. As shown in Fig. 7B, panels 4-6,
as representative micrographs, cells transfected with the control
vector displayed a normal morphology similar to surrounding non-transfected cells (Fig. 7B, panel 4). Cells transfected
with PTEN without R1881 treatment frequently displayed an apoptotic morphology (Fig. 7B, panel 5). Similar to controls, most
cells transfected with PTEN but treated with R1881 showed a normal
morphology (Fig. 7B, panel 6). Apoptotic index, as
determined by counting apoptotic cells in 300 green cells per sample
24 h after treatment, was 5% for controls, 20% for cells
transfected with PTEN without R1881 treatment, and 5% for cells
transfected with PTEN but treated with R1881 (Fig. 7C).
These analyses show that PTEN induced apoptosis in LNCaP cells, and
this PTEN function was blocked by androgen treatment.
Because of the presence of endogenous AR in LNCaP cells, LNCaP cell
experiments did not show that the androgen protection of PTEN-induced
apoptosis is mediated through the AR. To determine whether the androgen
effect on apoptosis is AR-dependent, AR-negative PC3 cells
were transfected with or without AR, and the effect of androgen on
PTEN-induced apoptosis was examined as in LNCaP cells. As shown in
Fig. 8A, R1881 had no effect
on the viability of PTEN-transfected cells in the absence of ectopic AR
expression. After co-transfection with AR, both PTEN-induced decreases
in cell viability (Fig. 8A) and increases in apoptotic index
(Fig. 8B) were blocked by R1881, demonstrating that the
androgen protection of PTEN-induced apoptosis was mediated through the
AR.

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Fig. 8.
Androgen effect on PTEN-induced apoptosis was
AR-dependent. A, AR-dependent
androgen effect on PTEN-induced decrease in PC3 cell viability. PC3
cells were transfected with 0.5 µg of pLNCE, 0.1 µg of
pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R and with or without 0.1 µg of
pLENhAR. Transfected cells were treated and processed for cell
viability determination as described in the legend to Fig. 7.
B, effect of androgen on PTEN-induced increase in apoptotic
index in AR-transfected PC3 cells. Cells were transfected with AR and
PTEN expression vectors and processed as in A. Apoptotic
index was scored as described in the legend to Fig. 7. C,
androgen protection of PTEN-induced PC3 cell death over time. Cells
were transfected with AR and PTEN expression vectors as in A
and treated with 10 8 M R1881 or ethanol for
indicated times. The viability of transfected cells in each well was
determined as described in the legend to Fig. 7.
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To further analyze the anti-apoptotic function of AR, a time course
study was performed (Fig. 8C). In this study, the number of
transfected (green) cells peaked at 24 h post-transfection, and
R1881 protected PC3 cells from PTEN-induced death similarly at all
three tested time points, presumably because of the synchronized expression of AR and PTEN proteins in co-transfected cells.
Lack of an Androgen Effect on AKT Activities in PCa Cells--
Our
data in Fig. 1 showed that the PTEN repression of AR depended on the
down-regulation of AKT activity. In addition, co-expression of the
dominantly active AKT blocked PTEN-induced apoptosis in LNCaP cells
(Fig. 9A), demonstrating that
PTEN-induced apoptosis was mediated through AKT down-regulation. So we
investigated the possibility that androgens might protect apoptosis by
regulating AKT activity. Western blotting with
anti-[phospho-Ser437]AKT antibody did not detect an
androgen effect on endogenous AKT activity in LNCaP cells (data not
shown).

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Fig. 9.
Androgen protection of PTEN-induced apoptosis
in LNCaP cells occurred independent of AKT activity. A,
inhibition of PTEN-induced apoptosis by dominantly active AKT. PC3
cells were transfected with 0.5 µg of pLNCE, 0.1 µg of
pSG5L-HA-PTEN:WT or pSG5L-HA-PTEN:G129R and 0.5 µg of
PLNCX-HA-myr-AKT or PLNCX-HA-myr-AKTK179M. The viability of transfected
cells was determined as described in the legend to Fig. 7.
B, lack of androgen effect on PTEN-induced down-regulation
of AKT activity. LNCaP cells were transfected with 0.5 µg of
PLNCX-HA-AKT (wtAKT) and 0.1 µg of pSG5L-HA-PTEN:WT or
pSG5L-HA-PTEN:G129R. Transfected cells were treated with
10 8 M R1881 or vehicle for 24 h. Both
AKT kinase activity (top panel) and level of expression
(bottom panel) were analyzed by parallel immunocomplex
kinase assays and Western blotting using the 12CA5 anti-HA monoclonal
antibody. Phosphorylated histone H2b (P-H2b) was resolved on
a 15% SDS-PAGE gel and visualized by autoradiography.
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LNCaP cells are PTEN-null and contain high levels of endogenous AKT
activity. Although androgens do not have a direct effect on endogenous
AKT activity, it may affect the negative regulation of AKT by PTEN. So
we next examined whether androgen treatment blocked PTEN-induced AKT
down-regulation. We transfected a HA-tagged wild-type AKT into LNCaP
cells with the PTEN expression vector and analyzed the AKT activity by
in vitro immunocomplex kinase assays. As shown in Fig.
9B, in either the presence or absence of R1881, the kinase
activity of AKT was dramatically decreased by PTEN expression whereas
the level of AKT protein was slightly reduced (Fig. 9B). The
data demonstrate that R1881 did not block the down-regulation of AKT
activity by PTEN in LNCaP cells.
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DISCUSSION |
Our studies demonstrated an antagonistic interaction and selective
dominance between PTEN and AR in the proliferation and apoptosis of PCa
cells as well as a differential involvement of AKT in the interaction.
The lack of an androgen effect on both AKT activity and its
down-regulation by PTEN in PCa cells suggests that androgens protect
PTEN-induced apoptosis either by activating a survival pathway that is
independent of AKT as suggested by a previous study (33) or by
activating the same survival pathway at steps downstream of AKT.
It appears paradoxical that PTEN-or LY294002-induced repression of AR
transcriptional activity was sufficient to block androgen-induced proliferation and PSA production but unable to override the protective effect of androgens on apoptosis. One possibility is that
AR-dependent protection of apoptosis by androgens might be
mediated through non-genomic effects of the receptor. Our data clearly
showed that the androgen effect on PTEN-induced apoptosis is
AR-dependent. Consistent with our data, reported studies
demonstrated that the "decoy" of ARE triggered apoptosis in LNCaP
cells (34), suggesting that the anti-apoptotic effect of androgens is
mediated through the genomic effect of the AR. Because the AR
transcriptional activity was repressed but not abolished by PTEN or
LY294002 in our experiments, it is likely that the androgen induction
of genes involved in promoting cell proliferation, and those in
apoptosis protection require different amounts of AR transcriptional activity.
Because our studies indicate that androgen target genes involved in
proliferation and apoptosis protection may have a differential sensitivity to cellular status of PI(3,4,5)P3 signal
pathway, it would be interesting to determine whether AR could still
mediate the induction of anti-apoptotic genes under conditions when
androgen-induced cell proliferation is blocked by the suppression of
PI(3,4,5)P3 signaling. Unfortunately, genes mediating the
anti-apoptotic effect of androgens in PCa cells remain to be
identified. Under the same conditions when PTEN-induced apoptosis of
LNCaP cells was blocked by androgens, we did not detect any androgen
effect on Bcl-2 expression (data not shown), although Bcl-2
up-regulation by androgens in LNCaP cells had been described in
previous studies (35).
Phosphorylation is known to regulate the transcriptional activity of
steroid receptors including AR (10, 11, 36). Based on the consensus
sequence for AKT phosphorylation (37), there are two potential AKT
phosphorylation sites in AR: Ser212 in the A/B region and
Ser782 in the ligand-binding domain. Therefore, it is
conceivable that AR activation may need the phosphorylation of these
sites by AKT and that AR repression by PTEN might be caused by the
inhibition of the AKT phosphorylation. However, transfection of the
dominantly active AKT into DU145 cells that contain wild type PTEN and
low endogenous AKT did not cause either ligand-independent activation nor enhanced the androgen-induced activity of co-transfected AR (data
not shown), suggesting that PTEN repression of AR activity may involve
more complex processes than a simple inhibition of AR phosphorylation
at the potential AKT sites.
Our data are the first to clearly demonstrate a mutual antagonism
between PTEN and AR in PCa cells. The mutual antagonism implies that
the balance between the function of PTEN and AR may maintain the
homeostasis of prostate epithelium in adult males. The
demonstration that AR is more active in the absence of functional PTEN
suggests that the loss of PTEN function may induce prostatic tumorigenesis by exposing prostatic epithelial cells to unopposed AR
activity. Similarly, excessive androgens may induce prostatic tumorigeneis by blocking the apoptosis-promoting function of PTEN. The
induction of apoptosis by the restored PTEN expression in LNCaP cells
only occurred in the absence of androgen, implying that PTEN mutation
or decreased expression may contribute to the resistance of PCa to
androgen ablation and that the combinational inhibition of both
PI3K/AKT and androgen signals could be an effective approach for the
treatment of AR-positive PCa.