Androgen-Driven Prostate Epithelial Cell Proliferation and Differentiation in Vivo Involve the Regulation of p27
David Waltregny,
Irwin Leav,
Sabina Signoretti,
Peggy Soung,
Douglas Lin,
Frederick Merk,
Jason Y. Adams,
Nandita Bhattacharya,
Nicola Cirenei and
Massimo Loda
Department of Adult Oncology (D.W., S.S., P.S., D.L., N.B., N.C.,
M.L.) Dana-Farber Cancer Institute, and Department of Pathology
(S.S., M.L.) Brigham and Womens Hospital Harvard Medical
School Boston, Massachusetts 02115
Department of
Pathology (I.L., F.M., J.Y.A.) Medicine and Veterinary
School Tufts University Boston, Massachusetts 02111
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ABSTRACT
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Androgens control both growth and differentiation
of the normal prostate gland. However, the mechanisms by which
androgens act upon the cell cycle machinery to regulate these two
fundamental processes are largely unknown. The cyclin-dependent kinase
(cdk) inhibitor p27 is a negative cell cycle regulator involved in
differentiation-associated growth arrest. Here, we investigate the role
and regulation of p27 in the testosterone proprionate (TP)-stimulated
regeneration of the ventral prostate (VP) of castrated rats. Continuous
TP administration to castrated rats triggered epithelial cell
proliferation, which peaked at 72 h, and then declined despite
further treatment. Castration-induced atrophy of the VP was
associated with a significant increase in p27 expression as compared
with the VP of intact animals. Twelve hours after the initiation of
androgen treatment, total p27 levels as well as its fraction bound to
cdk2, its main target, significantly dropped in the VP of castrated
rats. Thereafter, concomitantly to the induction of epithelial cell
proliferation, the glandular morphology of VP was progressively
restored at 4896 h of TP treatment. During this period of the
regenerative process, whereas both proliferating basal and secretory
epithelial cells did not express p27, the protein was selectively
up-regulated in the nonproliferating secretory epithelial compartment.
This up-regulation of p27 expression was coincident with an increase in
its association with, and presumably inhibition of, cdk2.
At each time point of TP treatment, p27 abundance in the VP was
inversely correlated with the level of its proteasome-dependent
degradation activity measured in vitro in VP lysates,
whereas only slight changes in the amount of p27 transcripts were
detected. In addition, the antiandrogen flutamide blocked maximal
TP-induced p27 degradation completely. Finally, the expression of skp2,
the ubiquitin ligase that targets p27 for degradation, was seen to
increase with androgen administration, preceding maximal proliferation
and concomitantly to augmented p27 degradation activity.
Taken together, our data indicate that androgens mediate both
proliferation and differentiation signals in normal prostate epithelial
cells in vivo, through regulation of p27.
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INTRODUCTION
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Cell proliferation and differentiation are controlled by
extracellular signals that impinge upon the cell cycle machinery and
modulate the expression/activity of key cell cycle regulators. In the
normal prostate gland, these two fundamental processes are regulated to
a large extent by androgens (1). Like its human counterpart, the
epithelial compartment of the rat prostate is composed of two types of
cells: basal and secretory cells. Secretory cells compose approximately
85% of the total cells in the ventral prostate (VP) of a sexually
mature male rat. Basal cells are numerically less abundant, and their
function is still poorly understood, although they are believed to be
precursors of secretory cells (2, 3). After castration, the rat
prostate rapidly involutes as a result of a major loss of secretory
cells (60%70% within 7 days of androgen deprivation), which
chronically require physiological levels of androgens for their
maintenance (3, 4). Testosterone stimulation can dramatically
accelerate the proliferation rate of prostate epithelial cells in a
sexually immature rat, yet once the organ has attained adult size
additional androgen has little influence on proliferation (5).
Likewise, sustained androgen administration to mature castrated rats
can trigger the regrowth of the prostate gland, which will eventually
return to its original size (5, 6, 7). This regenerative process is timely
regulated since proliferation rates decline to the baseline levels
found in intact animals despite further androgen treatment (6, 8).
Concomitantly with their mitogenic activity, androgens induce
histological and biochemical changes characteristic of glandular
differentiation in the prostate of castrated rats (5, 6, 7, 9, 10). For
example, testosterone replenishment in castrated rats results in the
reconstitution of the normal secretory cell compartment simultaneous
with a significant reduction in VP expression levels of basal
cell-associated cytokeratins (7, 9). In addition, the mRNA levels of
the C3 gene, which encodes for a major subunit of a prostate secretory
protein (prostatic steroid-binding protein) exclusively expressed in
secretory cells, are dramatically induced in the regenerating VP of
castrated rats (11, 12).
The regulation of mammalian cell proliferation by growth-stimulatory
and growth- inhibitory extracellular signals occurs during the first
gap (G1) phase of the cell cycle. The enzymes
that regulate G1 phase progression include the
cyclin-dependent kinases cdk4 and cdk6, which can be activated through
their association with D-type cyclins, and cdk2, which forms active
complexes with cyclins E and A (for reviews, see Refs. 13, 14, 15). The
activity of these cdk/cyclin complexes is negatively regulated by
cyclin-dependent kinase inhibitors, which belong to two known families
(16). The INK4A family comprises p16, p15, p18, and p19, which bind to
and specifically inhibit cdk4 and cdk6. The CIP/KIP family includes
p21, p27, and p57, which preferentially target cdk2 complexes
(16, 17, 18).
The cdk inhibitor p27 was initially identified as an inhibitor of
cdk2/cyclin E complex activity in transforming growth
factor-ß-treated or contact-inhibited mink lung epithelial cells
(19), and it was found to induce cell cycle arrest when overexpressed
in cultured cells (20, 21). p27 has since been shown to play a pivotal
role in mediating G1 arrest in some normal and
neoplastic cells in response to a variety of antiproliferative signals,
including growth in suspension (22), cAMP agonists (23), interferon-ß
(24) and -
(25), interleukin-6 (26), and rapamycin (27). In contrast
to p21, p27 expression is highest in quiescent cells (28, 29) and
declines as cells are stimulated to reenter the cell cycle (27, 28).
p27 abundance is regulated primarily at the posttranslational level.
Once activated, cdk2/cyclin E complexes phosphorylate p27 at
Threonine-187 (30, 31, 32, 33), thereby signaling for its degradation by the
ubiquitin-proteasome system through the ubiquitin-ligase SCF
(Skp1/Cul1/F-box)-skp2 complex (34, 35). Hence, maximum degradation of
p27 by the ubiquitin-proteasome pathway is detected in extracts
prepared from cells in S-phase (36). Accumulation of p27, however, can
also be regulated by other mechanisms, including transcriptional (26, 37, 38, 39) and translational control (40, 41).
Several lines of evidence from studies in p27 knock-out mice indicate a
direct involvement of p27 in differentiation-associated cell cycle
arrest. Homozygous p27-deficient female mice display infertility with
both impaired release of eggs during the estrus cycle and deficient
implantation of embryos (42, 43, 44). In this instance, the absence of p27
prevents the coupling of differentiation with growth arrest in
granulosa cells in response to LH (45, 46). Other studies using
p27-/- mice have highlighted an important role
for this protein in regulating the differentiation of oligodendrocytes
(47) and osteoblasts (48). In addition, p27 expression increases during
the differentiation of various normal and neoplastic cell types, both
in vivo and in vitro (49). For example, treatment
of LNCaP human prostate cancer cells with interleukin-6 (50) or the
flavanoid antioxidant silibinin (51) results in increased p27
expression associated with G1 arrest and
neuroendocrine differentiation.
Prior data have suggested that androgens regulate the expression
of p27 in both normal and neoplastic prostate epithelial cells, and
that p27 levels may mediate the androgen-driven proliferative and
differentiation signals in such cells (7, 52, 53, 54, 55). However, the
mechanisms by which androgens modulate the expression of p27 are
unknown. Testosterone-induced regeneration of the castrated rat VP
follows a highly reproducible time course, thus making this system an
excellent model for scrutinizing the androgen-mediated regulation of
proteins involved in the cell cycle. In this study, we have used this
model to investigate the regulation of p27 by testosterone proprionate
(TP). We herein provide convincing evidence that in this system 1) p27
plays an important role in the control of TP-stimulated proliferation
and differentiation of normal prostate epithelial cells and 2) TP
regulates the expression of p27 mainly through modulation of its
specific degradation by the ubiquitin-proteasome proteolytic
system.
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RESULTS
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Epithelial Cell Proliferation in the VP of Castrated Rats after
Androgen Treatment
As expected, TP administration (6.6 mg/kg, once daily) to
the castrated rats caused a dramatic regrowth of their VP, which
returned to a size almost comparable to that of age-matched
noncastrated rats after 4 days of treatment (data not shown). In
situ, proliferating epithelial cells were recognized by
immunohistochemical detection of BrdU. Figure 1A
shows representative examples of
anti-BrdU immunostaining of VP epithelial cells at each time point of
TP treatment as well as those from intact animals. Virtually no
detectable anti-BrdU nuclear staining was identified in epithelial
cells from untreated castrated or intact rats. The induction of cell
proliferation became apparent 48 h after the initiation of TP
treatment, and BrdU incorporation was detected in both basal and
secretory cell subsets (Fig. 1A
, inset). The proportion of
DNA-synthesizing basal and secretory cells reached a peak at 72 h
(11.7% ± 0.2%), and then declined in both cell types despite further
androgen administration (Fig. 1B
). Interestingly, at peak
proliferation, the percentage of BrdU-positive basal cells (22.9% ±
0.6%) was almost 2.5 times higher than the percentage of BrdU-positive
secretory cells (9.2% ± 0.5%) (paired t test,
P < 0.0001).

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Figure 1. Epithelial Cell Proliferation in the VP of
Castrated Rats Treated by TP
A, Castrated rats were treated by TP (6.6 mg/kg, once daily) for 4
days. At 0, 12, 24, 48, and 96 h after the initiation of the
treatment, rats (seven per time point) were injected with BrdU (10
mM) and killed 30 min later. Seven untreated noncastrated
age-matched male rats were also included as control animals. Tissue
sections were cut from each paraffin-embedded VP sample, immunostained
with either anti-p63 monoclonal antibody or anti-BrdU monoclonal
antibody, and counterstained with methyl green and hematoxylin,
respectively as described in Materials and Methods. For
recognition of basal vs. secretory cells,
immunohistochemistry for p63 is shown in the first
panel. p63 positive basal cells show nuclear immunoreactivity.
Representative examples of BrdU immunodetection are shown for each
group of seven rats. Positive staining was completely abolished when
the primary antibody was omitted from the staining procedure (data not
shown). Original magnification: x200. Inset, Detection of anti-BrdU immunoreactivity in the nucleus of both
secretory and basal cells. Arrow, Identification of a
BrdU-positive epithelial cell. B, Epithelial cell proliferation rates
in the VP of castrated rats treated by TP. The percentage of
proliferating epithelial cells in the VP of each animal was calculated
by dividing the number of BrdU-positive epithelial cells by the total
number of epithelial cells counted ( 1,000 cells). Assessment of the
percentage of BrdU-positive epithelial cells was also done in the VP of
seven intact rats. Values are expressed as the mean ±
SE (error bar) for each group of seven
animals.
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Expression of G1-Related cdks,
Cyclins, and cdk Inhibitors in the VP of Androgen-Replenished Castrated
Rats
To understand the relationship between androgen stimulus and the
proliferation/differentiation response of regenerating VPs, we next
investigated the temporal changes in expression levels of p27 and other
key regulators involved in the G1 phase
progression of the cell cycle. Changes in androgen receptor (AR)
expression were also examined. Western blot analysis of AR, cdk2, cdk4,
cdk6, cyclin D1, cyclin E, p21, and p27 expression was performed by
using total VP protein lysates from rats killed at each time point of
androgen treatment (Fig. 2
).

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Figure 2. Expression of G1 Phase-Related
Cyclin-Dependent Kinases, Cyclins, and Cyclin-Dependent Kinase
Inhibitors in the VP of Testosterone-Treated Castrated Rats
Castrated rats (7 per time point) were killed 0, 12, 24, 48, 72, and
96 h after the beginning of androgen replenishment (6.6 mg/kg TP,
once daily). The VP of each animal was harvested. VP specimens from
seven age-matched intact animals were also used. Tissue sample
duplicates for each group of seven rats were obtained by pooling
separately the VP specimens from three and four animals. Total proteins
were extracted from the pooled samples. Protein lysates (100 µg per
sample) were subjected to Western blot analysis of AR, cdk2, cdk4,
cdk6, cyclin D1, cyclin E, p21, and p27 expression, as described in
Materials and Methods. Ponceau S staining of the
membranes showed equal protein sample loading and transferring (data
not shown). Each Western blot experiment was performed at least twice
with both sets of pooled samples and consistent results were observed.
Fold increases (+) or decreases (-) in p27 protein levels quantified
by the ImageQuant program are indicated under the
Western blot.
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Although the AR protein was expressed as early as 12 h after the
initiation of TP treatment, the levels of cdks and their associated
cyclins remained unchanged at this time point. The expression levels of
these cell cycle proteins gradually increased at 24 h of treatment
and peaked during the next 3 days. p21 protein was virtually
undetectable in the VP of untreated castrated rats and during the first
24 h of TP replenishment. Thereafter, its expression gradually
increased. The highest level of p21 expression was detected in the VP
of intact rats.
p27 expression levels followed more complex temporal changes. The
protein levels were highest in the VP of untreated castrated rats. TP
administration induced an early (12 h) substantial decrease in the
amount of p27 protein. Subsequently, p27 levels increased and peaked at
72 h (concomitantly with maximal proliferation rate) and decreased
again at 96 h of treatment. The protein abundance was lowest in
the VP of intact animals.
In Situ Expression of p27 in the Regenerating VP of
Castrated Rats
Since our Western blot experiments were performed with the
use of total protein extracts, it was possible that the changes in p27
expression levels detected with this technique may not reflect those
that take place in the epithelial cells. Thus, to rule out this
potential bias as well as to assess p27 expression in the two subsets
of VP epithelial cells, we used immunohistochemistry. Figure 3A
shows representative examples of
anti-p27 immunostaining in VP glands during the first 96 h of TP
administration to castrated rats as well as in the VP of intact
animals. p27 expression was scored separately in the basal and
secretory cells according to the percentage of positive cells (Fig. 3B
).

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Figure 3. In Situ Expression of p27 in the
Regenerating VP of Castrated Rats
A, Immunodetection of p27 in the VP of castrated rats treated by TP.
After treatment by TP (6.6 mg/kg, once daily) for the indicated times,
castrated rats (7 per time point) were killed and the VP from each
animal was harvested. Seven intact age-matched male rats were also
used. Tissue sections from each paraffin-embedded VP were cut,
immunostained with either anti-p63 monoclonal antibody or anti-p27
monoclonal antibody, and counterstained with methyl green and
hematoxylin, respectively, as described in Materials and
Methods. For recognition of basal vs. secretory
cells, immunohistochemistry for p63 is shown in the first panel.
p63-positive basal cells show nuclear immunoreactivity. Representative
examples of p27 staining are shown for each group of seven animals.
Substitution of the primary antibody with PBS did not yield any
specific staining for either antibody (data not shown). Original
magnification: x400. B, Levels of p27 expression in the VP of
castrated rats treated by TP, as determined by immunohistochemistry.
Scoring of p27 immunostaining in the VP of each rat was done according
to the percentage of epithelial cells ( 500 cells counted) exhibiting
nuclear anti-p27 reactivity. p27 scoring was also done in the VP of seven intact rats. p27 scores were determined in the
basal (closed circles) and secretory cell (open
diamonds) subsets separately. All scoring values are expressed as
the mean ± SE (error bar) for each group
of seven rats. The paired t test was used to assess
whether there were significant differences in p27 expression levels
between basal and secretory cells at each treatment time point.
Statistically significant differences are indicated as
asterisks (*, P < 0.05; **,
P < 0.01; ***, P < 0.005).
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p27 immunoreactivity in the epithelial cells was mainly nuclear (Fig. 3A
). Overall, temporal changes in p27 expression levels were similar in
basal and secretory cell compartments and strictly paralleled those
detected by Western blot in the corresponding total protein extracts
(compare Fig. 3B
and Fig. 2
). The percentages of p27-expressing
epithelial cells were high in the VP of untreated castrated rats and,
after 12 and 24 h of TP treatment, fell significantly in both
secretory (unpaired t test, P = 0.03) and
basal cells (unpaired t test, P = 0.05).
Subsequently, p27 expression levels increased in both cell types and
peaked at 72 h of treatment. However, this increase in p27 levels
was significant only in the secretory cell subset (unpaired
t test, P = 0.2 and P <
0.0001 for basal and secretory cells, respectively). p27 levels then
slightly dropped in both subsets of cells at 96 h of treatment
(unpaired t test, P = 0.62 and P =
0.47 for secretory and basal cells, respectively). The lowest
level of p27 expression was found in the VP epithelial cells of intact
animals.
In the VP of untreated castrated rats, a significantly higher
percentage of p27-positive cells was observed in the basal compartment
(64.7% ± 12.8%) than in the secretory one (45.6% ± 9.3%) (paired
t test, P = 0.005) (Fig. 3B
). After 24
h of TP treatment, the level of p27 expression was similar in both cell
types (paired t test, P = 0.38).
Subsequently, at 4896 h of treatment, p27 expression levels were
significantly higher in secretory than in basal cells (paired
t test, P < 0.05). Also, a significant
difference in the percentages of basal (4.6% ± 2%) and secretory
(7% ± 2.8%) p27-positive cells was observed in the VP of intact
animals (paired t test, P < 0.05).
Thus, both our immunohistochemical and immunoblot data unexpectedly
indicated that the expression levels of the cell cycle inhibitor were
high in the regenerating VP at peak proliferation (72 h). To understand
this paradox, we performed double immunofluorescence experiments for
BrdU and p27 using VP tissue sections from animals killed at 72 h
of TP treatment. Remarkably, all epithelial cells in S-phase
(BrdU-positive) exhibited no detectable p27 expression (Fig. 4
, AC). Thus, during the proliferation
phase, two main subsets of epithelial cells could be distinguished:
cells in S phase (BrdU positive/p27 negative) and cells likely to be
arrested in G1 phase (BrdU negative/p27
positive). In addition, a small proportion of basal and secretory cells
were double negative for both antibodies.

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Figure 4. Absence of p27 Expression in Proliferating
Epithelial Cells from the Regenerating VP of Castrated Rats
Double immunofluorescence staining for p27 and BrdU was performed in
paraffin-embedded VP specimens from seven castrated rats that had been
killed after 3 days of continuous TP treatment (6.6 mg/kg, once daily).
Tissue sections from each VP were immunostained with an anti-p27
antibody, and detection was performed utilizing FITC-conjugated
streptavidin (green staining). Subsequently, the same
sections were immunostained with anti-BrdU monoclonal antibody, and
detection was done using Texas red-conjugated streptavidin
(red staining). Substitution of the primary antibodies
with PBS did not yield any specific staining (data not shown).
Photomicrographs of double immunofluorescence staining for p27 and BrdU
in representative prostate glands were taken under an UV microscope
using appropriate filters for detecting FITC/p27 (A, x200; D, x400;
G, x1,000), Texas red/BrdU (B, x200; E, x400; H, x1,000), and both
FITC/p27 and Texas Red/BrdU (C, x200; F, x400; I, x1,000).
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Association of p27 with cdk2/Cyclin Complexes in the Regenerating
VP of Castrated Rats
To further substantiate our results, we determined whether the
increased levels of p27 expression detected at 72 h of TP
replenishment would translate into an increased association of p27 with
cdk2. We performed coimmunoprecipitation experiments in which cdk2 was
pulled down from the VP protein lysates at the different time points of
TP treatment with the use of an anti-cdk2 antibody. The
immunoprecipitates were subjected to SDS-PAGE and transferred to
nitrocellulose membranes that were blotted with an anti-p27 antibody
(Fig. 5A
).

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Figure 5. Association of p27 with cdk2 in the Regenerating
Prostate of Castrated Rats
After treatment by TP (6.6 mg/kg, once daily) for the indicated times,
castrated rats (7 per time point) were killed, and the VP from each
animal was harvested. The VP from seven intact age-matched male rats
was also collected. The VP specimens from each group of seven rats were
randomly pooled in duplicates containing three and four VP specimens,
each. Total proteins were extracted and used in cdk2/p27
coimmunoprecipitation experiments. A, Protein lysates (500 µg per
sample) were immunoprecipitated with 1.5 µg of anti-cdk2 antibody, as
detailed in Materials and Methods. The
immunoprecipitates were subjected to SDS-PAGE and transferred to
nitrocellulose membranes, which were blotted with an anti-p27 antibody.
Immunoprecipitates of VP lysates (500 µg) from rats treated by TP for
72 h with 1.5 µg of rabbit IgG were used as negative controls.
One hundred micrograms of nonimmunoprecipitated intact VP protein
lysates were also used in the Western blotting procedure.
Coimmunoprecipitation experiments were performed with both sets of
pooled samples and yielded reproducible results. B, p27-probed
membranes were subsequently stripped and reprobed with an anti-cdk2
antibody to examine the amount of immunoprecipitated cdk2 in the
different samples.
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Temporal changes in the association of p27 with cdk2 matched those in
endogenous p27 protein expression (compare Fig. 5A
and Fig. 2
). Indeed,
as compared with the VP of untreated castrated rats, the amount of
cdk2/p27 complexes was substantially decreased and almost undetectable
in the VP of castrated rats treated by TP for 12 or 24 h. The
abundance of p27 bound to cdk2 increased significantly at 48 h of
TP treatment, peaked at 72 h, and then declined at 96 h
despite further TP treatment. The lowest amount of cdk2- bound p27 was
found in the VP of intact animals. These coimmunoprecipitation
experiments were performed using high amounts of protein lysates and
low amounts of anti-cdk2 antibody to saturate anti-cdk2 antibodies with
cdk2 in each condition. Thus, since the abundance of immunoprecipitated
cdk2 was similar in all samples (Fig. 5B
), the increased association of
p27 with cdk2 at 4896 h of TP treatment was not merely due to an
increased amount of cdk2 protein in the lysates.
Evidence for Androgen Regulation of p27 Expression by Degradation
via the Ubiquitin-Proteasome Pathway
We next searched for the mechanisms responsible for the regulation
of p27 expression by TP. We first tested for levels of p27
ubiquitin-proteasome-mediated degradation activity in the different VP
lysates by using a previously described assay (36, 56, 57) in which
purified recombinant his6-tagged p27 serves as a
substrate to the lysates. Kinetic profiles of p27 degradation obtained
by using the samples from each time point of TP treatment clearly
showed that VP protein lysates were able to degrade exogenous p27 and
that this ability to degrade the protein was modulated in time, as
prostates were replenished with TP (Fig. 6A
). p27 was
degraded in a proteasome-dependent manner since the addition of a
specific proteasome inhibitor (MG-132) to the VP protein lysates (which
contained the nonproteosomal protease inhibitors
phenylmethylsulfonylfluoride , aprotinin, and leupeptin) completely
nullified p27 degradation (Fig. 6C
).

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Figure 6. Kinetics of p27 and p21 Degradation in the
Regenerating VP of Castrated Rats
After treatment by TP (6.6 mg/kg, im, once daily) for the indicated
times, castrated rats (7 per time point) were killed and the VP from
each animal was harvested. The VP from seven intact age-matched male
rats was also collected. The VP specimens from each group of seven rats
were randomly pooled in duplicates containing three and four VP
specimens each. Total proteins were isolated from each pooled sample.
Purified human recombinant his6-tagged p27 (A) and p21 (B)
proteins (300 ng) were incubated for the indicated times at 37 C in the
presence of a degradation mix containing 100 µg of each VP protein
lysate, as described in Materials and Methods. The
degradation of the his6-tagged proteins by the lysates was
analyzed by immunoblotting with an antihistidine antibody. Each
degradation assay was performed at least twice with both sets of pooled
samples and consistent results were observed. Control experiments
included the omission of the lysates from the degradation mix. No
specific signal was obtained when the tagged proteins were not added to
the degradation mix (data not shown). C, Protein lysates (100 µg)
from the VP of intact animals were assayed for his6-p27
degradation activity in the presence or absence of the proteasome
inhibitor MG-132 (100 µM) in the degradation mixture.
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To measure p27 degradation activity levels in the different samples and
to correlate these levels with the expression levels of the
endogenous protein in the corresponding samples, the relative amounts
of undegraded his6-p27 after 0 and 4 h of
incubation in the presence of the lysates were analyzed by Western
blotting and quantified by densitometry. p27 abundance was inversely
correlated with the level of degradation activity for the protein
during the course of TP treatment (Fig. 7A
). Importantly, the highest level of
p27 degradation activity was observed in the intact VP, while
castration abolished this activity almost completely.

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Figure 7. Androgen Regulation of p27 and p21 Expression in
the Regenerating Prostate of Castrated Rats
After treatment by TP (6.6 mg/kg, once daily) for the indicated times,
castrated rats (7 per time point) were killed and the VP from each
animal was harvested. The VP from seven intact age-matched male rats
was also harvested. The VP specimens from each group of seven rats were
randomly pooled in duplicates containing three and four VP specimens
each. A, The degradation activity of the VP lysates for
his6-tagged p21 and p27 proteins was analyzed by
immunoblotting with an antihistidine antibody, as described in
Materials and Methods. Quantification of degradation
activity for the exogenous proteins was done by densitometric analysis
of the bands at ±22 kDa and ±28 kDa corresponding to undegraded
his6-tagged p21 and p27 proteins, respectively. Relative
degradation activity of the samples at each time point after TP
injections (closed circles) was calculated by dividing
the densitometric value of the band obtained with the sample without
incubation (0 h) by the one obtained with the sample incubated for
4 h in the degradation mix. Relative levels of p27 and p21
degradation activity were compared with the endogenous levels of p27
and p21 proteins (open diamonds) in the VP at each time
point (see Fig. 2 ). All values are normalized to those found in the VP
of untreated castrated rats. B, Total RNA was extracted from each VP
tissue duplicate. One microgram of total RNA per sample was
reverse-transcribed and a one-twentieth of each RT reaction was
subjected to Taqman Real-Time PCR amplification, as described in
Materials and Methods. The specific rat p27 and p21
primers and probes used in the PCR reactions are shown in Table 1 . The
housekeeping GAPDH gene was used as endogenous control. The relative
amounts of p27 and p21 transcripts in each sample were determined using
the standard curve method and were normalized to GAPDH mRNA expression
levels. All values represent the mean of triplicates and are normalized
to those found in the VP of untreated castrated rats. Error
bars stand for SDs. Taqman PCR experiments were
performed with both sets of pooled samples, and consistent results were
observed. PCR reactions with samples in which the reverse transcriptase
or the target RNA was omitted from the RT reaction did not yield any
significant amplification (data not shown).
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Some degradation of p21, which is also a target of the
ubiquitin-proteasome pathway (58, 59, 60, 61), was noted in all the samples
tested (Fig. 6B
). However, in contrast to p27, the levels of p21
degradation activity remained stable (Fig. 7A
).
We then determined whether Flutamide (F) blocked TP-induced degradation
of p27 as further proof that degradation of p27 was under androgen
control and that this effect was AR-mediated. As shown in Fig. 8
, top panel, F completely
blocked degradation induced by TP in castrate rats. A Western blot of
VP lysates probed with probasin antibody (bottom panel), a
marker of testosterone activity in mouse and rat prostate (56), showed
that castration abolished its expression. Probasin expression was
partially restored by 24 h of TP treatment, and the TP-induced
probasin expression was almost completely blocked by F.

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Figure 8. Effects of Flutamide (F) on TP-Mediated Degradation
of p27
Fourteen days after surgical castration, rats were injected sc with 10
mg of F , twice a day, for 48 h. At 24 h from the initial F
injection, rats were injected with 6.6 mg/kg of TP im, once a day, for
24 h. The animals were then killed. Castrated rats injected with
TP only and killed 24 h after injection served as controls. The
VPs of castrates (first three lanes, top panel),
castrates treated with TP alone (middle three lanes, top
panel) or castrates treated with both F and TP (last three
lanes, top panel) were harvested, degradation assays for
p27 were performed, and immunoblots were probed with antihistidine
( -his) antibodies as described. Zero-, 1-, and 4-h time points are
shown for all treatment groups. Antiprobasin Western blot performed on
lysates of castrates, TP-treated castrates, and TP- and F-treated
castrates is shown in the lower panel.
|
|
Expression of p27 transcripts during the Androgen-Induced
Regeneration of Castrated Rat VP
We also investigated whether p27 and p21 mRNA levels were
modulated in the regenerating VP of castrated rats using Taqman
Real-Time RT-PCR (PE Applied Biosystems, Foster City, CA). After
inducing a drop in the level of p27 transcripts at 12 h of
treatment (2.6-fold decrease), sustained TP administration resulted in
a slight but consistent increase in p27 mRNA levels, which peaked at
96 h of treatment (2.3-fold induction) (Fig. 7B
). The highest
level of p27 transcripts was detected in the VP from intact rats. On
the other hand, p21 mRNA levels were gradually and substantially
up-regulated, with maximal levels attained at 96 h of TP treatment
(5.7-fold induction). Also, the level of p21 transcripts was almost 3
times higher in the VP of intact rats than that in the VP of untreated
castrated animals. Thus, our results strongly suggest that testosterone
regulates p27 expression mainly through its
ubiquitin-proteasome-mediated degradation while it transcriptionally
induces p21 expression in the regenerating rat prostate.
Expression of skp2, the Ubiquitin Ligase for p27, in the
Regenerating Rat Prostate
To further elucidate the mechanism(s) by which the
proteasome-mediated degradation of p27 is restored by readministration
of TP to castrate animals, we assessed expression of skp2, the p27
targeting F-box protein in the ubiquitin ligase for p27, at the various
time points of the treatment. Whereas no change was noted between VP
lysates from intact and those from castrate rats, a substantial
increase in skp2 levels, which preceded times of peak proliferative
rates and paralleled p27 degradation activity, was observed starting as
early as 12 h after TP administration and consistently decreasing
thereafter (Fig. 9
). These results
suggest that, when androgen-driven proliferation is induced, increased
skp2 may be responsible for the enhanced degradation of p27.

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Figure 9. Expression of skp2 in the VP of
Testosterone-Treated Castrated Rats
Castrated rats (7 per time point) were killed 0, 12, 24, 48, 72, and
96 h after the beginning of androgen replenishment (6.6 mg/kg TP,
once daily). The VP of each animal was harvested. VP specimens from
seven age-matched intact animals were also used. Tissue sample
duplicates for each group of seven rats were obtained by pooling
separately the VP specimens from three and four animals. Total proteins
were extracted from the pooled samples. Protein lysates (100 µg per
sample) were subjected to Western blot analysis of skp2 expression, as
described in Materials and Methods. Ponceau S staining
of the membranes showed equal protein sample loading and transferring
(data not shown). Each Western blot experiment was performed at least
twice with both sets of pooled samples and consistent results were
observed. Fold increases (+) or decreases (-) in skp2 protein levels
quantified by the ImageQuant program are indicated under the Western
blot.
|
|
 |
DISCUSSION
|
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In the present study, we have used a castration-regeneration
rat prostate model for investigating the in vivo regulation
of p27 expression by androgens. In agreement with other reports, our
results demonstrate that sustained TP administration to castrated rats
stimulates VP epithelial cell proliferation, whose rate peaks at
72 h of androgen replenishment and then declines despite further
treatment (1, 3, 5, 62, 63, 64).
Using Western blot experiments, we show that the castration-induced
atrophy of the VP is associated with a significant up-regulation of p27
expression as compared with the VP of intact animals. In addition, TP
administration to castrated rats induces an early (12 h) and
substantial drop in p27 levels while the expression levels of several
G1-associated cdks and cyclins remains unchanged.
This significant decrease in p27 expression levels is also detected by
immunohistochemistry in both basal and secretory cells. Furthermore,
the amount of p27 bound to cdk2, its main target, declines markedly in
the early phase of TP replenishment, suggesting a release in the
inhibition of cdk2 activity. Together, these results thus indicate that
the initial down-regulation of p27 in the androgen-induced regeneration
of VP in castrated rats may represent a primary cell cycle event,
thereby allowing VP epithelial cells to proliferate.
Unexpectedly, the level of p27 expression detected by Western blot
peaks at the same time as the rate of epithelial cell proliferation and
the levels of G1-related cdks and cyclins are
maximal. Indeed, this observation is in apparent contradiction with the
fact that p27 expression is known to be down-regulated in proliferating
cells (36). Findings from our immunohistochemical experiments have
helped clarify this paradoxical observation. First, the levels of p27
in prostate epithelial cells parallel those detected by Western blot in
the corresponding total protein extracts during the course of TP
treatment, indicating that the changes in p27 expression detected in
the crude homogenates are representative of those occurring in the VP
epithelial compartment. Second, when the rate of cell proliferation
reaches its maximum, the majority of epithelial cells express p27
whereas a minority of them proliferate. Third, the detection of BrdU
and the expression of p27 in epithelial cells are mutually exclusive.
In addition, the increased levels of p27 expression detected at peak
TP-induced proliferation are coincident with an increased association
of p27 with, and presumably inhibition of, cdk2. Since cells expressing
p27 are BrdU-negative, our results indicate that in this model, p27 may
play a significant role in controlling the regrowth and restoring the
glandular structure of the VP by limiting the proliferation of
epithelial cells and favoring their postmitotic differentiation.
Further evidence is provided by our observation that, concomitant with
the androgen-stimulated enhancement of cell proliferation and
progressive reconstitution of VP morphology, p27 expression is
significantly induced only in nonproliferating secretory cells. It is
also noteworthy that, in the VP of intact rats, the percentage of
p27-positive cells is also significantly higher in the secretory
compartment than in the basal one. This is in keeping with the wide
expression of p27 in human prostate secretory cells as compared with
the more restricted expression of this protein in basal cells (65) and
the lack of its expression in the putative, transiently proliferating
intermediate cell compartment (52). Altogether, our results thus add
weight to the body of data indicating an important role for p27 in the
switch from proliferation to differentiation of precursor/progenitor
cells from different lineages (41, 42, 43, 44, 45, 46, 47, 48, 66).
In the VP of untreated castrated rats, p27 expression levels are
significantly higher in basal than in secretory cells. We have observed
a similar although more striking pattern of selective distribution of
p27 expression in the VP gland of rats that had been castrated for 4
months. In those rats, a high level of anti-p27 nuclear reactivity is
detected in virtually all basal cells while residual secretory cells
are consistently devoid of any staining (M. Loda, I. Leav, and D.
Waltregny, unpublished data). Since basal cells do not suffer a
substantial loss in number after castration (3), it is tempting to
hypothesize that the sustained, high expression of p27 observed in
basal cells after castration may be related to the suggested role of
this protein in the prevention of cell death. Recent studies on
p27-/- mice have revealed an antiapoptotic role
of p27 in growth factor-deprived mesangial cells and fibroblasts (67).
p27 also protects cells from cyclohexamide-induced apoptosis (71) and
has been shown to inhibit apoptosis after inflammatory injuries in
renal glomerular and tubular cells (68). In a human leukemia cell line,
overexpression of p27 confers resistance to induction of apoptosis by
several cytotoxic agents (69). Thus, although our hypothesis that p27
overexpression may safeguard basal cells from androgen
deprivation-induced apoptosis remains at this time highly speculative,
it certainly deserves further investigation.
It is known that p27 expression can be regulated at multiple levels
(49), but the relative contribution of transcriptional, translational,
or proteolytic control to p27 regulation in various physiological and
pathological contexts in the prostate gland remains largely unknown.
Our results provide the first demonstration that androgens regulate p27
expression in an animal model. Using lysates from VPs at the various
time points, we determined that p27 is regulated primarily by
ubiquitin-proteasome-mediated degradation. In fact, a maximal level of
p27 degradation activity is observed in the VP of intact animals,
whereas castration abrogates this activity almost completely.
Progressive restoration of p27 degradation activity is obtained with TP
replenishment and blocked by the antiandrogen flutamide. In contrast to
the p21 gene, to date, no androgen response element has been found in
the cloned portion of the p27 gene promoter (70). Our results reveal
that TP treatment slightly modulates p27 transcription in the VP of
castrated rats. However, in view of the kinetics and magnitude of
changes in p27 mRNA expression, androgen-mediated p27 transcriptional
regulation, alone, is insufficient to explain the levels of p27 protein
expression detected during the course of the treatment. Furthermore,
since the highest level of p27 transcripts is found in the VP of intact
rats while the protein level is the lowest, we conclude that in the VP
of sexually mature rats TP induces a high p27 protein turnover by
augmenting its proteasome-mediated degradation.
The specific mechanism by which androgens regulate p27 degradation is
unknown. It has been recently shown that the F-box protein skp2 is the
targeting subunit of the ubiquitin ligase complex specific for p27 (34, 35, 71). Skp2 levels are highest in S-phase when p27 degradation is
also maximal (34, 35, 71, 72, 73). We therefore assessed levels of this
protein in the various experimental conditions. We provide evidence
that the amount of skp2 parallels the level of p27 degradation activity
in the regenerating (proliferating) prostate and, importantly, its
increased expression precedes peak proliferation. Although the
mechanism regulating p27 degradation activity in the nonproliferative
state, i.e. the intact prostate, remains unknown, our data
suggest that skp2 is induced by testosterone and may mediate this
process when prostate epithelial cells are stimulated to
proliferate.
The regulation of p27 proteasome-mediated proteolytic activity in the
regenerating prostate may be specific for this protein. Indeed,
the levels of p21 degradation activity, another target of the
ubiquitin-proteasome pathway (58, 59, 60, 61), remain unchanged during the
course of TP treatment and thus cannot account for the gradual increase
in expression levels of this protein observed during TP treatment. In
fact, it has been shown that the p21 gene can be transcriptionally
activated by androgens in vitro through binding of AR with
an androgen response element present in its proximal promoter (74). In
this report, we substantiate these findings in an in vivo
model by demonstrating that the levels of p21 transcripts are
significantly up-regulated by androgens.
In summary, the results from the present study convincingly suggest
that p27 plays an important role in the control of
testosterone-stimulated proliferation and differentiation of normal
prostate epithelial cells. In addition, these results support the
hypothesis that the regulation of p27 levels by androgens in these
cells is dependent on the AR and achieved through modulation of its
specific degradation by the ubiquitin-proteasome proteolytic
system.
 |
MATERIALS AND METHODS
|
---|
Animals and Tissues
All animals were maintained in accordance with the NIH Guide for
the Care and Use of Laboratory Animals, and the specific protocol used
in this study was approved by the Dana-Farber Cancer Institute Animal
Care and Use Committee. Twelve week-old male Noble rats were purchased
from Charles River Laboratories, Inc. (Wilmington, MA).
Fourteen days after surgical castration, rats were injected im with 6.6
mg/kg TP (Sigma, St. Louis, MO) in tocopherol-stripped
corn oil (ICN Biomedicals, Inc., Aurora, OH), once a day,
for 4 days. At 0, 12, 24, 48, 72, and 96 h after the beginning of
TP injections, animals (seven rats per time point) were injected ip
with 5 ml of PBS containing 10 mM BrdU (Roche Molecular Biochemicals, Mannheim, Germany) and killed 30
min later by CO2 asphyxiation. The ventral
prostate (VP) was then harvested, freed of fat, and two-thirds of each
VP were immediately flash-frozen in liquid nitrogen and then stored at
-80 C for subsequent RNA and protein isolation. The remaining third of
each VP was fixed in 10% phosphate buffered formalin overnight,
dehydrated in graded alcohols, and paraffin embedded for
immunohistochemical procedures. Seven noncastrated untreated
age-matched male rats were also included as control animals.
Castrated rats were also injected sc with 10 mg of the anti-androgen
flutamide (F) (Sigma, St. Louis, MO), twice a day, for
48 h. At 24 h from the initial F injection, rats were
injected im with 6.6 mg/kg of TP (Sigma), once a day, for
24 h. The animals were then killed and the VPs were harvested and
processed as described above. Castrated rats injected with TP only and
killed 24 h after injection served as controls.
Every 20 mg of flutamide was dissolved in 75 µl of methanol, 75 µl
of butanol, and 850 µl of tocopherol-stripped corn oil (ICN Biomedicals, Inc., Aurora, OH).
Protein and RNA Extraction
The frozen VP specimens from each group of seven rats were
randomly pooled in two separate sample duplicates containing three and
four VP specimens, each. All the experiments described in this study
were performed with both sets of pooled samples and yielded
reproducible results (data not shown). The pooled frozen VP samples
were homogenized by pulverization using a Mikro-Dismembrator S (B.
Braun Biotech Intl. GmbH, Melsungen, Germany) to generate a
tissue powder that was immediately processed for protein and RNA
extraction. Total RNA was extracted from 1020 mg of each tissue
homogenate by using the RNeasy mini kit (QIAGEN, Valencia,
CA), according to the manufacturers protocol. The remaining tissue
powder was lysed in 3.5 vol/wt of lysis buffer [10% sucrose, 1%
Nonidet P-40, 20 mM Tris (pH 8.0), 137 mM NaCl,
10% glycerol, 2 mM EDTA, 10 mM NaF, 1
mM Na3VO4]
containing phenylmethylsulfonyl fluoride (1 mM), soybean
trypsin inhibitor (10 µg/ml), leupeptin (1 µg/ml), and aprotinin (1
µg/ml). Protein lysates were placed in ice for 30 min, vortexed every
10 min, and then cleared by centrifugation at 12,000 x
g for 20 min at 4 C. The supernatants were retrieved and
frozen at -80 C until use in immunoblot, coimmunoprecipitation, and
degradation assays. The protein concentration was measured using the
Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc.
Hercules, CA).
Western Blot Analysis
Protein lysates (100 µg from each sample) were resolved by
size on 12% or 16% SDS-polyacrylamide gels and transferred onto
nitrocellulose membranes (Protran, Schleicher & Schuell, Inc., Keene, NH), which were stained with Ponceau S
(Sigma) to examine the equal protein sample loading and
transferring (data not shown). The membranes were blocked with 5%
nonfat dry milk in Tris-buffered saline [20 mM Tris base
(pH 7.6), 150 mM NaCl] containing 0.1% Tween-20 (TBS-T),
and probed with the following antibodies: anti-cdk2 (M2) (0.4 µg/ml),
anti-cdk4 (C-22) (0.4 µg/ml), anti-cdk6 (C-21) (0.4 µg/ml),
anticyclin E (M-20) (0.4 µg/ml), anti-p21 (C-19) (0.4 µg/ml),
anti-skp2 (H-435) (0.4 µg/ml) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-AR (PG-21) (1.5 µg/ml)
(Upstate Biotechnology, Inc., Lake Placid, NY), anticyclin
D1 (CC12) (0.8 µg/ml) (Calbiochem, Cambridge, MA), anti-p27
(0.1 µg/ml) (Transduction Laboratories, Inc., Lexington,
KY) and antiprobasin (1:3,000) (56) (kind gift of Dr. Norman Greenberg,
Baylor College of Medicine, Houston, TX) antibodies. After washing in
TBS-T, membranes were incubated with horseradish peroxidase
(HRP)-conjugated secondary antibodies (Bio-Rad Laboratories, Inc. Hercules, CA) and developed using an enhanced
chemiluminescence detection system (ECL detection kit; Amersham Pharmacia Biotech, Arlington Heights, IL) according to the
instructions of the manufacturer. Membranes were exposed to X-Omat AR
film (Eastman Kodak Co., Rochester, NY). The immunoblots
were quantitated by densitometric analysis using ImageQuant software
(Molecular Dynamics, Inc., Sunnyvale, CA).
Coimmunoprecipitation Assay
To study the association of p27 with cdk2/cyclin complexes,
lysates containing 500 µg of total proteins normalized to a 1 ml
volume in lysis buffer were precleared by incubation with 100 µl of a
1:1 (vol/vol) slurry of protein A-sepharose beads (Sigma)
for 45 min on a rotator at 4 C. Protein complexes were
immunoprecipitated from the precleared lysates by addition of 1.5 µg
of anti-cdk2 antibodies overnight at 4 C with constant rotation,
followed by the addition of 50 µl of a 50% slurry of beads for 45
min at 4 C with mild agitation. After three washes with 1 ml cold lysis
buffer, pelleted beads were quenched in 20 µl of 2x Laemmli sample
buffer and boiled. The mixtures were spun down at 10,000 x
g for 30 sec and 20 µl of each supernatant were retrieved
and analyzed by immunoblot using anti-p27 antibody as described above.
The filters were subsequently stripped and reprobed with an anti-cdk2
antibody to examine the amount of immunoprecipitated cdk2 in the
different samples tested. Immunoprecipitations with 1.5 µg of rabbit
IgG (Vector Laboratories, Inc. Burlingame, CA) instead of
anti-cdk2 antibodies were used as negative control experiments.
Degradation Assay
Degradation assay experiments were performed as previously
described (36, 57, 75) with minor modifications. Three hundred
nanograms of human hexahistidine(his6)-tagged p27
and p21 proteins (bacterially expressed and purified as in Ref. 36)
were incubated at 37 C for 0, 1, 4, and 15 h in 60 µl of
degradation mix containing 100 µg of protein homogenates, 50
mM Tris-HCl, pH 8.0, 5 mM
MgCl2, 1 mM dithiothreitol, 2
mM ATP, 60 µg/ml creatine phosphokinase, 10
mM creatine phosphate, and 5 µM ubiquitin.
The reactions were carried out in a PCR instrument and stopped at the
different time points by addition of 2x Laemmli sample buffer. The
degradation of his6-tagged p27 and p21 proteins
by the rat VP protein lysates was analyzed by immunoblotting with an
antihistidine monoclonal antibody (0.1 µg/ml) (QIAGEN,
Valencia, CA), as detailed above. To ensure that endogenous p27 present
in the VP lysates was not significantly competing for proteasome
degradation with his6-tagged p27, we had
previously checked by Western blot using an anti-p27 antibody that the
amount of exogenous p27 added to the degradation mixtures was in large
excess to that of endogenous p27 (data not shown). Control degradation
experiments included the omission of the
his6-tagged protein as well as the omission of
the lysate from the degradation mix. To demonstrate that p27
degradation activity was dependent on the proteasome proteolytic
activity of the lysates, degradation assays were performed, in which
either the proteasome inhibitor MG-132
(carbobenzoxyl-L-leucyl-L-leucyl-L-leucinal,
Calbiochem-Novabiochem Corp., La Jolla, CA) dissolved in
dimethylsulfoxide (DMSO) (100 µM final concentration) or
DMSO alone was added to the degradation mixture. Quantification of
degradation activity was done by densitometric analysis of the bands of
slowest electrophoretic migration, in extenso the bands at
±22 kDa and ±28 kDa corresponding to undegraded
his6-tagged p21 and p27 proteins, respectively.
Relative degradation activity of the samples at each time point after
TP injections was then calculated by dividing the densitometric
assessment of the band intensity obtained without incubation (0 h) by
the one obtained after 4 h of incubation in the degradation
mix.
Real-Time RT-PCR (Taqman RT-PCR)
Reverse Transcription.
For cDNA synthesis, 1 µg of total RNA was reverse-transcribed in a 20
µl reaction mixture containing 250 µM of each
deoxynucleoside triphosphate (dNTP), 20 U of RNase inhibitor, 50
U of MuLV Reverse Transcriptase (RT), 2.5 µM random
hexamers, and 1x buffer (1.5 mM
MgCl2) (all reagents purchased from PE Applied Biosystems, Foster City, CA). The reaction mix was
incubated at 42 C for 45 min and then denatured at 99 C for 5 min.
Reactions not containing the RT or omitting the target RNA were used as
controls.
Primers and Probes.
Specific primers and probes for rat p27 and p21 genes (Table 1
) were designed from sequences available
in the GenBank database, using the Primer Express 1.0 Software
(PE Applied Biosystems). The housekeeping
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (Taqman rodent
GAPDH control reagents kit, PE Applied Biosystems, Foster
City, CA) was used as endogenous control to normalize the amount of p27
and p21 transcripts in each reaction. The choice of this reference gene
was guided by results from a previous study showing that GAPDH mRNA
levels were not significantly altered in the regenerating prostate of
castrated rats treated by TP (12). All sets of primers and probe were
selected to work under identical cycling conditions. cDNA amplification
products with the different primer pairs had been previously checked to
yield a single band of the expected size after electrophoretic
migration in a 2% agarose gel stained with ethidium bromide (data not
shown). Probes were synthesized by PE Applied Biosystems.
Real-Time PCR.
Taqman PCR was performed on the cDNA samples using an ABI PRISM 7700
Sequence Detector (PE Applied Biosystems). The Taqman PCR
Core Reagent kit (PE Applied Biosystems) was used
according to the manufacturers directions with the following
modifications: dUTP was replaced by dTTP at the same concentration, and
incubation with AmpErase was omitted. For each sample tested, PCR
reaction was carried out in a 50 µl volume containing 1 µl of cDNA
reaction (equivalent to 50 ng of template RNA) and 2.5 U of AmpliTaq
Gold (PE Applied Biosystems). Oligonucleotide primers and
fluorogenic probes were added to a final concentration of 100
nM each. After activation of AmpliTaq Gold for 10 min at 94
C, the amplification step consisted of 60 cycles of 94 C for 45 sec, 58
C for 45 sec, and 64 C for 1 min.
In each experiment, seven additional reactions with serial dilutions
(500x magnitude) of intact VP cDNA as template were performed with
each set of primers and probe in the same 96-well plate to generate
standard curves relating the threshold cycle
(CT) to the log input amount of template.
All samples were run in triplicate. The relative amounts of p27 and p21
transcripts in each sample were determined using the standard curve
method and were normalized to GAPDH mRNA expression levels, as
described in detail in ABI PRISM Sequence Detection System User
Bulletin 2 (PE Applied Biosystems) and elsewhere (76).
Immunohistochemistry and Immunofluorescence
Antibodies and Immunodetection of p63, p27, and BrdU.
Immunostaining experiments for p27 and BrdU were performed in all
paraffin-embedded VP tissue specimens with anti-p63 (4A4, gift of Frank
McKeon, Harvard Medical School), anti-p27 (Transduction Laboratories, Inc., Lexington, KY), and anti-BrdU monoclonal
antibodies (Becton Dickinson and Co., Mansfield, MA),
respectively. Double immunofluorescence staining for p27 and BrdU was
performed in the paraffin-embedded VP specimens from seven castrated
rats that had been killed at 72 h of TP treatment.
Five-micrometer sections were deparaffinized in xylene, and then
rehydrated and subjected to microwaving in 10 mM citrate
buffer, pH 6.0 (BioGenex Laboratories, Inc. San Ramon, CA)
in a 750 W oven for 15 min. Slides were allowed to cool at room
temperature for 30 min. Immunohistochemistry was performed by an
automated processor (Optimax Plus 2.0 bc, BioGenex Laboratories, Inc. San Ramon, CA). After quenching of the endogenous
peroxidase activity with 3% hydrogen peroxide in methanol for 10 min,
slides were incubated for 10 min with a buffered casein solution (Power
Block Reagent, BioGenex Laboratories, Inc.) to block the
nonspecific binding sites. Antibodies (anti-p63, 1:50 dilution,
anti-p27, 1:3,000 dilution; anti-BrdU, 1:100 dilution) were applied at
room temperature for 2 h in the automated stainer. Detection steps
were performed by the instrument utilizing the MultiLink-HRP kit
(BioGenex Laboratories, Inc.). Standardized development
times with the chromogenic substrate [3,3'-diaminobenzidine
tetrahydrochloride (DAB) (for anti-p27 staining) ± nickel
chloride (DAB-NC) (for anti-BrdU staining)] allowed accurate
comparison of all samples. Sections were counterstained with
hematoxylin, rehydrated, and mounted for microscopic examination.
For double immunofluorescence staining experiments, antigen retrieval
and blocking steps were performed as described above. Anti-p27 antibody
(dilution 1:100) was applied at room temperature for 1 h. Sections
were then incubated with secondary biotinylated antibody (MultiLink,
BioGenex Laboratories, Inc.), followed by detection with
fluorescein isothiocyanate (FITC)-conjugated streptavidin (Vector Laboratories, Inc. Burlingame, CA). Sections were then blocked
with, successively, FITC-conjugated antimouse immunoglobulin antibody
(Vector Laboratories, Inc.), a blocking solution (Power
Block, BioGenex Laboratories, Inc.), avidin, and biotin
(both from BioGenex Laboratories, Inc.), for 10 min each.
Subsequently, anti-BrdU antibody (dilution 1:10) was applied for 1
h at room temperature. Then, sections were incubated with a secondary
antibody (MultiLink, BioGenex Laboratories, Inc.) and
detection was performed by addition of Texas red-conjugated
streptavidin (Vector Laboratories, Inc.). Slides were
mounted with an antifading fluorescent medium for microscopic
examination and photomicrography under an BX50 microscope
(Olympus Corp., Lake Success, NY) equipped with
appropriate filters.
Substitution of the primary antibodies with PBS served as negative
staining control. For p27, strong immunoreactivity in small
lymphocytes, which invariably infiltrated the prostate stroma, was used
as an internal positive control.
Evaluation of Immunohistochemical Staining.
The percentage of BrdU-positive epithelial cells in each VP was
calculated by dividing the number of epithelial cells with
BrdU-positive nucleus by the total number of epithelial cells counted
(
1000 cells). BrdU counts were done separately in basal cells and
secretory cells from the VP of castrated rats treated by TP for 72
h. Scoring of p27 immunostaining was done according to the percentage
of epithelial cells (
500 cells counted) exhibiting detectable
nuclear anti-p27 reactivity, as previously described (57, 77). p27
scores were determined in the basal and secretory cell subsets
separately in all VP specimens. The distinction between basal and
secretory cells was done according to the location and morphology of
the nucleus of these epithelial cells, as determined by microscopic
examination of both hematoxylin-eosin-stained and anti-p27
immunostained sections. In addition, to confirm the morphological
assessment of basal cells, immunohistochemistry for the prostate basal
cell marker p63 was performed in serial sections from each VP, as
previously detailed (78). All scoring values are expressed as the
mean ± SE for each group of seven rats.
Statistical Analysis
The paired t test was used to assess whether there
were significant differences in p27 expression (percentage of positive
cells) between basal and secretory cells within the same VP specimen at
each time point of TP treatment. The same test was performed to examine
the statistical significance of differences in the percentages of
BrdU-positive cells between the basal and secretory compartments in the
VP of rats treated by TP for 72 h. The unpaired t test
was used to determine whether there were significant differences in the
percentages of p27-positive basal or secretory cells between rats
treated by TP for different times. The tests were two-tailed and
P < 0.05 was considered statistically significant. The
analyses were performed with a statistics software package (Statview
4.02, Abacus Concepts Inc., Berkeley, CA).
 |
ACKNOWLEDGMENTS
|
---|
The authors thank William Sellers, Myles Brown, and Michele
Pagano for critical review of the manuscript and Jane Hayward for
assistance with photography.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Massimo Loda, M.D., Department of Adult Oncology, Dana Building 740B, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115. E-mail:
Massimo_Loda{at}dfci.harvard.edu
This work was supported by Department of Defense Grant PC970273
and National Cancer Institute Grant 5RO1CA-8175503 (to M.L.) and by
the National Fund for Scientific Research (Belgium), and the Léon
Frédérick Foundation (Liège, Belgium) (to D.W.).
Received for publication July 14, 2000.
Revision received February 12, 2001.
Accepted for publication February 16, 2001.
 |
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