Androgen Regulation of the Cyclin-Dependent Kinase Inhibitor p21 Gene through an Androgen Response Element in the Proximal Promoter
Shan Lu,
Min Liu,
Daniel E. Epner,
Sophia Y. Tsai and
Ming-Jer Tsai
Department of Cell Biology (S.L., M.L., S.Y.T., M.-J.T.) and
Department of Medicine (D.E.E., M.-J.T.) Baylor College of
Medicine Houston, Texas 77030
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ABSTRACT
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Androgen is essential for the physiological
maintenance of the integrity of prostatic epithelial cells, and
castration causes the cells to undergo apoptosis. To study the
molecular mechanism of androgen-dependent cell growth, we
showed that androgen up-regulates the expression of the
cyclin-dependent kinase inhibitor p21 (WAF1, CIP1, SDI1, CAP20)
gene at both the mRNA and protein levels. Nuclear run-on assays
demonstrated that androgen stimulates endogenous p21 gene expression at
the transcriptional level. Transient transfection experiments showed
that androgen can enhance the activity of a 2.4-kb promoter of the p21
gene linked to a luciferase reporter. These results suggested that a
putative androgen response element (ARE), which mediates androgen
response to enhance the p21 transcription, is included in the 2.4-kb
promoter fragment. Deletion analysis of the promoter revealed a
functional ARE (AGCACGCGAGGTTCC) located at -200 bp of the p21 gene
proximal to the promoter region. Electrophoretic mobility shift
assay further demonstrated that the androgen receptor
specifically binds to this element. Wild-type ARE, but not mutant ARE,
confers androgen responsiveness to a heterologous promoter. The
up-regulation of p21 gene expression by androgen suggests that p21 may
have an antiapoptotic function in prostatic epithelial cells. However,
this hypothesis will need to be tested in future experiments.
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INTRODUCTION
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Regulation of the cell cycle is essential for a cell to determine
whether it will undergo proliferation, differentiation, or cell death
(1). The cell cycle is controlled by the sequential activation of
cyclin-dependent kinases (CDKs) upon association with their
partner cyclins and the subsequent phosphorylation and
dephosphorylation of the CDKs. Checkpoint regulation of the cell cycle
is effected by CDK inhibitors (CKIs), of which two classes have
recently been identified. The first class of CDK inhibitors comprises
p16 (also called MTS1, INK4a) (2), p15 (also called MTS2, INK4b) (3),
p18 (4), and p19 (5). Each of these genes encodes a protein with
ankyrin-like repeats that specifically inhibits CDK4 and CDK6.
Mutations and deletions of p16 and p15 genes have been found to be
associated with tumors, suggesting a tumor suppressor function for this
class of genes (6, 7, 8, 9). The second class of CDK inhibitors consists of
p21 (also called WAF1, CIP1, SDI1, CAP20) (10, 11, 12), p27 (also called
KIP1) (13, 14), and p57 (also called KIP2) (15, 16), which possess
considerable sequence similarity and can inhibit all known CDK
subtypes. This class of genes is mainly involved in development and
differentiation (17, 18).
Androgens, acting through their receptors (ARs), are essential for the
maintenance of prostatic epithelial cell proliferation and
differentiation during development (19). The rates of epithelial cell
growth and death in adult prostate glands are in equilibrium, such that
there is no net change in cell growth. In an adult rat, the glandular
epithelial cells constitute approximately 80% of the total cells in
the ventral prostate, and approximately 70% of these cells die by 7
days postcastration (20, 21). The molecular mechanisms of the
androgen-mediated maintenance of the integrity of prostatic epithelial
cells have not been elucidated and remains an area of active
research.
There are many androgen-regulated genes (22). However, only a few of
the genes, such as PSA and KLK-2 (23), C(3) protein (24), Slp (25), and
probasin (26), have been well characterized, and androgen response
elements (AREs) in these genes were identified. Whether these
characterized genes are involved in mitogenic signaling of androgen is
still unknown. In studying the molecular mechanisms of androgen action
on cell growth, we were the first to demonstrate that androgen
stimulates the expression of cell cycle genes CDK2 and CDK4 and
represses the expression of CDK inhibitor p16 gene, resulting in
increased CDK kinase activities (27). Those studies revealed a
new class of androgen target genes. In addition, those observations
suggested a possible signaling pathway by which androgen stimulates
prostate cell growth. In the current study, we identified the CDK
inhibitor, p21, as a target gene of androgen. Androgen induces the
expression of the p21 gene in prostate cancer cells through an ARE in
the proximal p21 promoter.
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RESULTS
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Androgen Up-Regulates the Expression of the CDK Inhibitor p21 in
LNCaP-FGC Cells
To investigate the molecular mechanisms of
androgen-dependent growth of prostatic epithelial cells, we used an
androgen-dependent prostatic carcinoma cell line, LNCaP-FGC, as an
in vitro model (27). We observed that androgen can
up-regulate the expression of the CDK inhibitor p21. After culturing
the cells for a week in medium containing 10% stripped FBS, the
expression levels of the p21 gene were examined in response to androgen
stimulation. As shown in Fig. 1A
, the
level of p21 mRNA in LNCaP-FGC cells was increased after 2 h of
treatment with the AR agonist R1881 (10-8 M)
and continued to increase thereafter up to 48 h. In contrast, no
detectable p21 mRNA was observed in androgen-independent PC-3 cells.
The enhancement of p21 expression is specific, since the expressions of
CDK inhibitor p27 (KIP 1) is not altered in response to androgen
stimulation in both LNCaP-FGC and PC-3 cells. We also found that p21
protein levels in LNCaP-FGC cells, as measured by Western blot
analysis, increased in response to R1881 within 8 h (Fig. 1B
).

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Figure 1. Up-Regulation of the p21 Gene by Androgen
A, After LNCaP-FGC and PC-3 cells were cultured in medium containing
10% stripped FBS for 1 week, R1881, at a concentration of
10-8 M, was added to these cells and the cells
were incubated for 0, 2, 8, 24, and 48 h. Subsequently, the total
RNAs were isolated, and 20 µg of RNA per sample were used in Northern
analysis as described in Materials and Methods. The same
filter was used for hybridization with p21, p27, and GAPDH cDNA probes,
respectively. Fold increases in the p21 mRNA levels quantified by
ScanAnalysis program were indicated under the Northern blot. B,
LNCaP-FGC cells were cultured in medium containing 10% stripped FBS
for 1 week. Subsequently, R1881 (10-8 M) was
added for 2, 8, and 24 h. Total cellular proteins were isolated
and subjected to Western blot analysis using 50 µg protein per
sample. Ponceau S staining was used as protein loading control. Fold
increases of p21 protein levels were indicated under each Western blot.
Each Northern or Western analysis experiment has been repeated more
than three times with sample collected at different times, and
consistent results were observed. C, After LNCaP-FGC cells were
cultured in medium containing 10% stripped FBS for 1 week,
cycloheximide (CHX, 50 uM) was added to the cells for
48 h and R1881 (10-8 M) was added to
these cells for 0, 2, 8, 24, and 48 h. Subsequently, the total
RNAs were isolated and 20 µg of RNA per sample was used in Northern
analysis. D, After LNCaP-FGC cells were cultured in medium containing
10% stripped FBS for 1 week, R1881 was added to the cells overnight,
and nuclei were isolated from both control and R1881-treated cells.
Subsequently, run-on transcription was performed for 30 min followed by
RNA isolation. -Probe strips containing 500 ng of either p21 cDNA or
GAPDH cDNA on each dot slot were used for hybridization probed by
labeled RNA from both control and R1881-treated cells.
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Androgen Up-Regulates the p21 Gene Expression at the
Transcriptional Level
To investigate whether androgen-dependent up-regulation of
p21 is dependent on protein synthesis, LNCaP-FGC cells were treated
with protein synthesis inhibitor, cycloheximide, for 48 h with
R1881 stimulation for various times. Northern blot analysis showed that
cycloheximide superinduces the expression of p21 gene with or without
R1881 treatment, and the enhanced expression of p21 gene in response to
androgen stimulation was not observed in the presence of cycloheximide
(Fig. 1C
). It is possible that this phenomenon is due to an enhanced
p21 mRNA stability stimulated by cycloheximide by an unknown mechanism.
We next did nuclear run-on assays to determine whether androgen
directly stimulates p21 gene transcription. As shown in Fig. 1D
, we
found an enhanced mRNA synthesis in the RNA preparation isolated from
R1881-treated nuclei as compared with the RNA isolated from control
nuclei. As a control, glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) expressions are similar between control and R1881-treated
sample.
To examine whether androgen directly up-regulates the expression
of the p21 gene, transient cotransfection experiments were performed.
An AR expression vector (CMV-AR) and a reporter construct
(p21(-2400)-Luc) containing 2.4 kb of the p21 promoter fused to a
luciferase reporter were cotransfected into COS 1 cells. Subsequently,
the reporter luciferase activity in response to androgen stimulation
was determined. AR agonist R1881 (10-8 M)
induces an approximately 2-fold increase in luciferase activity in COS
1 cells cotransfected with the p21(-2400)-Luc and cytomegalovirus
(CMV)-AR vectors (Fig. 2A
). There is no
increase in luciferase activity upon androgen stimulation in COS 1
cells transfected with p21(-2400)-Luc vector without the AR expression
vector. This result suggests that the COS 1 cells contain no, or low
levels of, endogenous AR. As a control, a reporter construct,
RARE-tk-Luc, which contains two copies of retinoic acid response
element (RARE) fused upstream of the minimal thymidine kinase (tk)
promoter, fails to respond to R1881 when cotransfected with AR
expression vector in COS 1 cells. Since these experiments were carried
out in the COS 1 cell line, which is monkey kidney cells, the results
may not reflect the physiological condition. Thus, these experiments
were also carried out in prostatic carcinoma LNCaP-FGC cells, which
contain a high expression level of a gain-of-function mutated
form of endogenous AR. Figure 2
showed that an increased expression of
luciferase activity was also observed in LNCaP-FGC cells transfected
with p21(-2400)-Luc vector alone in response to R1881 (Fig. 2B
). These
results indicate that androgen-dependent induction of luciferase
reporter gene expression is specifically mediated by AR, and a
functional ARE is likely to reside in the 2.4-kb fragment of the
p21 promoter.

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Figure 2. Determination of Androgen Responsiveness of the p21
Promoter by Transient Transfection Assay
A, COS 1 cells (105) were seeded in six-well tissue culture
plates. The next day, the cells were cotransfected with 0.25 µg of
p21(-2400)-Luc or RARE-tk-Luc vector with or without 0.25 µg CMV-AR
expression vector. The cells were stimulated with 10-8
M R1881 for 48 h. Cell extracts were prepared
according to an in vitro luciferase assay kit followed
by luciferase assay as described in Materials and
Methods. B, LNCaP-FGC cells (2 x 105) were
seeded in six-well tissue culture plates. The next day, the cells were
transfected with 0.25 µg p21(-2400)-Luc vector for the transient
transfection assay. The error bar represents the average
of duplicate samples of each experiment. Each transient transfection
assay has been repeated more than five times.
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Characterization of a Functional ARE in the p21 Promoter
To identify the ARE(s) in the p21 promoter, a series of
progressive 5'-promoter deletion mutants were generated (Fig. 3A
). COS 1 cells were used for transient
transfection experiments by cotransfecting these p21 deletion
constructs and CMV-AR expression vector. As shown in Fig. 3A
, the
constructs p21(-2400)-Luc, p21(-1800)-Luc, p21(-600)-Luc, and
p21(-215)-Luc, which include 2.4 kb, 1.8 kb, 0.6 kb, and 0.215 kb of
the p21 promoter fragment, respectively, showed an increased promoter
activity upon androgen stimulation. In contrast, the construct
p21(-60)-Luc, which contains only 60 bp of the p21 gene promoter,
fails to respond to androgen. These results strongly suggested that the
p21 promoter spanning from nucleotides -215 to -60 contains a
functional ARE. A dose-dependent induction of the expression of
luciferase reporter gene driven by 215 bp of the p21 promoter fragment
by varying concentrations of R1881 showed that a concentration as low
as 10-11 M can stimulate the reporter gene
expression (Fig. 3B
). The dose-response curve reached a plateau at
concentrations higher than 10-10 M of R1881,
implying that all binding sites have been saturated. A similar
dose-response curve was obtained by transfecting only the reporter
construct p21(-215)-Luc into LNCaP-FGC cells with R1881
EC50 at approximately 5 x 10-11
M (data not shown).

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Figure 3. Localization of ARE in the p21 Promoter by Deletion
Analysis
A, The recombinant luciferase reporter constructs containing deleted
p21 promoter fragments were shown on the left panel.
Luciferase activities were determined by transient cotransfection
assay. COS 1 cells were cotransfected with 0.25 µg reporter construct
with 0.25 µg CMV-AR expression vector for 48 h in the presence
and absence of R1881 (10-8 M), followed by
luciferase assay as described in Materials and Methods.
B, Dose-dependent up-regulation of p21 promoter activity by androgen.
COS 1 cells were cotransfected with 0.25 µg of p21(-215)-Luc with
0.25 µg of CMV-AR expression vector for 48 h in the presence of
increasing concentrations of R1881.
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Sequence analysis of the p21 promoter spanning the regions between
-215 to -60 bp revealed a likely ARE sequence (AGCACGCGAGGTTCC)
located at the -200 bp position of the promoter. This putative ARE is
homologous to the AREs found in the promoters of the hKLK2, C(3), PSA,
and Slp genes (Fig. 4A
). Therefore, a
double-stranded oligonucleotide containing the putative ARE sequence
corresponding to the region from nucleotides -215 to -186 of the p21
gene promoter was used in an electrophoretic mobility shift assay
(EMSA) to determine whether AR can bind to this sequence. Figure 4B
showed that the binding of AR to the oligo probe can be supershifted by
anti-AR antibody but not by a nonspecific rabbit IgG, suggesting that
AR binds specifically to the oligo probe. Unlabeled wild-type
oligonucleotide duplexes including AREs from the p21 gene and C(3) gene
could compete efficiently for the binding of AR to the ARE in the p21
gene, indicating that binding is specific. To further support that the
binding is specific, multiple ARE mutants were generated by changing
the conserved nucleotides of the p21 gene ARE as shown in Fig. 4A
.
EMSAs showed that there is no specific binding of AR to mutant-2 ARE
and a drastic decrease in AR binding to mutant-3 ARE as compared with
the efficient binding of AR to the wild-type ARE (Fig. 5A
). Similarly, the specific binding of
AR to the wild-type ARE can be competed for the unlabeled wild-type ARE
oligo probe of the p21 gene but not by the unlabeled mutant-2 ARE (Fig. 5B
). These results collectively indicate that mutation of more than one
conserved nucleotide in the ARE decreases or abolishes the binding by
AR.

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Figure 4. Binding of AR to the ARE in p21 Promoter
A, Sequence comparison of the ARE in the p21 gene with the well
characterized AREs in hKLK2, C(3 ), PSA, and Slp genes. Mutated AREs for
EMSA are indicated as ARE-mut1, ARE-mut2, and ARE-mut3. B, Cell nuclear
extract prepared from LNCaP-FGC cells treated with R1881 was analyzed
for AR complex formation with the ARE in the p21 gene in a EMSA. The
end-labeled oligonucleotide containing the wild-type ARE in the p21
gene was used as probe. Twenty-fold excess of the unlabeled ARE
oligonucleotide probe either from the p21 gene or from the C(3 ) gene
was used for binding competition.
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Figure 5. Mutational Analysis of Specific Binding of AR to
the ARE from the p21 gene in EMSA
A, The end-labeled wild-type and mutated ARE oligonucleotides as
indicated in Fig. 4A were used as probe for EMSA. B, Dose-dependent
competition of AR binding to the wild-type ARE by the unlabeled
wild-type ARE oligonucleotide and ARE-mut2 oligonucleotide; 20-, 10-,
5-, 2.5-, and 1.2-fold of unlabeled wild-type or mutated
oligonucleotides were added in each binding reaction, respectively.
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To further determine the physiological relevance of the ARE of the p21
gene, we investigated whether this ARE can confer androgen
responsiveness to a heterologous promoter. An oligo containing two
wild-type or two mutated ARE sequences separated by six nucleotides
from the p21 gene was subcloned into the pXP2-
NF1-Luc vector
containing the human tissue transglutaminase gene minimal promoter
(-32
+5 nucleotides). The resulting constructs are
ARE-TATA-Luc and AREmut-TATA-Luc vectors, respectively (Fig. 6
) (28). Transient transfection of these
reporter vectors into LNCaP-FGC cells in the presence or absence of
R1881 demonstrated that only the wild-type ARE, but not the mutated
ARE, can confer the androgen responsiveness to the minimal promoter of
the human tissue transglutaminase gene (Fig. 6
). In conclusion, these
results further support the notion that the ARE found in the p21 gene
is authentic.

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Figure 6. The Wild-Type but Not Mutated Form of the ARE in
the p21 Gene Confers Androgen Responsiveness to a Heterologous Promoter
Two copies of the wild-type or mutated AREs spaced by six nucleotides
were subcloned upstream of the human tissue transglutaminase minimal
promoter fused with luciferase reporter to generate ARE-TATA-Luc and
AREmut-TATA-Luc constructs. LNCaP-FGC cells (2 x 105)
were seeded in six-well tissue culture plates. Next day, the cells were
transfected with ARE-TATA-Luc or AREmut-TATA-Luc (0.5 µg) for 48
h in the presence and absence of R1881. Cell extracts were prepared
according to an in vitro luciferase assay kit followed
by luciferase assay as described in Materials and
Methods. The error bar represents an average of
duplicate samples of each experiment. Each transient transfection assay
has been repeated more than five times.
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DISCUSSION
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Multiple factors have been demonstrated to regulate the expression
of the p21 gene. In stimulating myelomonocytic cell differentiation,
vitamin D transcriptionally activates the expression of the p21 gene,
and a vitamin D response element (VDRE) was identified at the promoter
sequence from -788 to -756 bp (29). The multifunctional growth factor
transforming growth factor-ß (TGFß) can cause cell growth arrest by
the up-regulation of the expression of the p21 gene through a
TGFß-response element (RE) located at -84
-74 bp position
of the p21 promoter (30). By inhibiting cell growth in response to
epidermal growth factor and interferon-
treatment, the p21
gene can be up-regulated through STAT1 (signal transducers and
activators of transcription). Three STAT-inducible elements (SIEs) in
the promoter of the p21 gene were identified, which are located at
-640, -2540, and -4183 bp (31). A functional p53-RE was also located
at approximately 2.4 kb upstream of the p21 gene-coding region (11). It
has also been demonstrated that the E2A gene products, E12 and E47, can
up-regulate the p21 gene expression through two E boxes in the proximal
p21 promoter to suppress cell growth (32). Here we demonstrated that
androgen transcriptionally regulates the expression of the p21 gene.
Our studies thus reveal a cross-talk of cell cycle control and androgen
action at the molecular level.
Determination of the biological functions ascribed to p21 suggests that
p21 is a multifunctional protein playing a key role in the cell cycle
control, DNA repair, and antiapoptosis (33, 34, 35). When one p21 molecule
binds to one cyclin-CDK complex, the resulting complex is active and
can phosphorylate Rb to allow cell cycle to progression. When two or
more p21 molecules bind per cyclin-CDK complex, kinase activities are
inhibited, and cell cycle progression is blocked. p21 is known to
inhibit proliferating-cell nuclear antigen in DNA replication
but not in DNA repair, leading to the inactivation of chromosomal
replication while allowing DNA damage-responsive repair (36). In
addition, it has been shown that during terminal differentiation of
myocytes, p21 induction is correlated with the acquisition of an
apoptotic-resistant phenotype (34). p21 Is also required for the
survival of differentiating neuroblastoma cells (37). More direct
evidence is that p21 protects against p53-mediated apoptosis of human
melanoma cells (35). Furthermore, a transcriptionally incompetent p53
can induce apoptosis, but not growth arrest, whereas induction of p21,
which is a major transcriptional target of p53, can induce growth
arrest but not apoptosis (38). Therefore, this evidence suggests that
p21 plays a major role in cell proliferation, differentiation, and
apoptosis.
To investigate the molecular mechanisms in the development of
androgen-independent growth of prostate cancer, we have established an
androgen-independent prostatic carcinoma cell line LNCaP-AI by
culturing the parental androgen-dependent LNCaP-FGC cell line in medium
containing stripped serum. It is interesting to find that there is a
drastic increase in the basal expression of the p21 gene in this
androgen-independent cell line, although the cells can grow very well
in the absence of androgen (S. Lu, S. Tsai, and M.-J. Tsai, unpublished
observation). Androgen is essential for the maintenance of the
integrity of the prostatic epithelium, and androgen withdrawal results
in massive apoptosis of the prostatic epithelial cells and prostate
evolution. bcl-2 Was demonstrated to be involved in the development of
androgen-independent growth (39, 40). In normal prostatic secretory
epithelial cells, there are no detectable levels of bcl-2 expression.
The correlation between the up-regulation of the p21 gene and
acquisition of androgen-independent cell growth led us to speculate
that in the normal prostatic epithelial cells, up-regulation of p21
gene by androgen may play an antiapoptotic role in an
androgen-dependent manner but not an inhibitory role in cell cycle
progression.
p21 Is the fourth androgen-regulated cell cycle gene we have
identified. To investigate the molecular mechanisms of androgen
induction of p21 expression, we have performed detailed functional
analyses on the p21 promoter. By deletion and mutation analyses, we
have defined a consensus ARE sequence located at -200 bp of the p21
promoter that is required for androgen-activated transcription. EMSA
with the wild-type and mutated ARE sequences of the p21 gene using
nuclear extracts from LNCaP-FGC cells revealed one specific band of
retarded mobility, which can be supershifted by anti-AR antibody. The
presence of this retarded band is correlated functionally with the
ability of the ARE to drive androgen-mediated transcription to a
heterologous promoter.
Recent studies have demonstrated that a glucocorticoid response element
(GRE)- or progesterone response element (PRE)-like element with
consensus sequence 5'-GGA/TACAnnnTGTTCT-3' functions as ARE, which may
be due to the homologous properties between AR, GR, and PR. However,
AREs in Slp and probasin genes exhibit preference for AR (25, 26).
According to the characteristics of the identified ARE, some of them
are simple ARE, which typically consists of an imperfect palindrome
sequence with a 3-bp spacer between the two half-sites. The examples
are the AREs in PSA and hKLK2 genes (23, 41). There are also complex
AREs in which multiple elements and the binding of multiple proteins to
these elements are required for full androgen-induced activity. These
are exemplified in AREs in Slp and probasin genes (25, 26). For the ARE
in the p21 gene, it appears to be a simple ARE element.
In summary, our studies show convincingly that androgen acts on the p21
gene. This finding points to an interesting possibility that the CDK
inhibitor p21 may be involved in the androgen-mediated antiapoptotic
function in the prostatic epithelial cell. Further studies are
currently underway to investigate this hypothesis.
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MATERIALS AND METHODS
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Cell Culture
Human metastatic prostate adenocarcinoma cell line LNCaP-FGC
[American Type Culture Collection (ATCC), Manassas, VA] was
maintained in RPMI-1640 (Life Technologies, Inc., Gaithersburg, MD)
supplemented with either 10% FBS or 10% charcoal/dextran-treated
(stripped) FBS (HyClone Laboratories, Logan, UT) at 37 C in 5%
CO2. Human prostate adenocarcinoma cell line PC-3 (ATCC)
was cultured in DME/F12 medium (Life Technologies, Inc.) supplemented
with either 10% FBS or stripped FBS.
Reagents
R1881 was purchased from Dupont Biotechnology Systems (Boston,
MA). Human p27 and p21 cDNA probes were kindly provided by Dr. Wade
Harper (Department of Biochemistry, Baylor College of Medicine). The
vector RARE-tk-Luc was a gift from Dr. Richard Heyman (Ligand, La
Jolla, CA). Anti-AR antibody was a gift from Dr. Nancy Weigel
(Department of Cell Biology, Baylor College of Medicine).
Northern Blot Analysis
Total cellular RNAs from control and R1881-treated samples were
isolated using Ultraspec RNA isolation reagent (Biotecx Laboratories,
Inc., Houston, TX). Total RNA (20 µg/sample) was fractionated on a
1% formaldehyde agarose gel and transferred onto a nylon filter
(Hybond-N, Amersham Life Science, Arlington Heights, IL). Northern
hybridization was performed using Quikhyb hybridization solution
according to the manufactures recommendations (Stratagene, La Jolla,
CA). Quantitation of each band was performed using ScanAnalysis
computer program (BIOSOFT, Ferguson, MO).
Western Blot Analysis
Aliquots of samples with same amount of protein,
determined using the Bradford assay (Bio-Rad, Hercules, CA), were mixed
with loading buffer [final concentrations of 62.5 mM
Tris-HCl (pH 6.8), 2.3% SDS, 100 mM dithiothreitol, and
0.005% bromophenol blue], boiled, fractionated in a 15% SDS-PAGE,
and transferred onto a 0.45-µm nitrocellulose membrane (Bio-Rad). The
filters were blocked with 2% fat-free milk in PBS and probed with
anti-CDK2 and anti-CDK4 antibodies (0.05 µg/ml IgG) (Santa Cruz
Biotechnology, Santa Cruz, CA) in PBS containing 0.1% Tween 20 (PBST)
and 1% fat-free milk. The membranes were then washed once in PBST and
incubated with horseradish peroxidase-conjugated F(ab')2 of
goat antirabbit secondary antibody (Bio-Rad) in PBST containing 1%
fat-free milk. After washing four times in PBST, the membranes were
visualized using the enhanced chemiluminescence (ECL) Western blotting
detection system (Amersham Life Science). Quantitation of each band was
performed using ScanAnalysis computer program (BIOSOFT).
Nuclear Run-on Assay
LNCaP-FGC cells were cultured in medium containing 10% stripped
serum for 1 week. Nuclei were isolated from approximately 5 x
107 of either control cells or cells treated overnight with
R1881. Cells were lysed twice in NP-40 lysis buffer [10 mM
Tris-Cl (pH 7.4), 10 mM NaCl, 3 mM
MgCl2, and 0.5% NP-40] by incubating on ice for 5 min
each time, and obtained nuclei were frozen at -80 C in 100 µl of
glycerol storage buffer [50 mM Tris-Cl (pH 8.3), 40%
(vol/vol) glycerol, 5 mM MgCl2, 0.1
mM EDTA]. To perform nuclear run-on transcription, the
frozen nuclei were thawed at room temperature. One hundred microliters
of 2x reaction buffer with nucleotides [10 mM Tris-Cl (pH
8.0), 5 mM MgCl2, 0.3 M KCl, 1
mM ATP, 1 mM CTP, 1 mM GTP, and 5
mM DTT] plus 10 µl of 10 mCi/ml
[
-32P]UTP were added. The reaction was incubated 30
min at 30 C with shaking. The nuclei were spun 5 min at 500 x
g and 4 C. RNAs were isolated using Ultraspec RNA isolation
reagent (Biotecx Laboratories, Inc.). For hybridization, 500 ng of
denatured p21 or GAPDH cDNA was used for each dot slot of Zeta-probe
membrane (Bio-Rad). The dot-slot strips were prehybridized in Quick-hyb
hybridization solution (Stratagene) for 3 h at 68 C. Subsequently,
2 x 106 cpm/ml denatured RNA probe were added and
hybridization was performed for 24 h at 68 C. The strips were
washed twice in 2x saline sodium citrate (SSC) and 0.1% SDS at
room temperature for 15 min and once in 0.2x SSC and 0.1% SDS at 50 C
for 15 min followed by x-ray exposure.
Transient Transfection Assay
COS 1 cells (105) or 2 x 105
LNCaP-FGC cells were seeded in six- well tissue culture plates. Next
day, lipofectin-mediated transfection was used for the transient
transfection assays according to the protocol provided by Life
Technologies, Inc.. Cell extracts were prepared according to in
vitro luciferase assay kit (Promega, Madison, WI). Luciferase
assays were performed in a Monolight 2010 Luminometer (Analytical
Luminescence Laboratory, San Diego, CA). For each assay, cell extract
(20 µl) was added into a cuvette, and the reaction was started by
injection of 100 µl luciferase substrate. Each reaction was measured
for 10 sec in the Luminometer. Luciferase activity was defined as light
units/mg protein.
EMSA
EMSA was carried out as described in a bandshift assay
system (Promega). Nuclear extract isolated from LNCaP-FGC cells treated
with R1881 was used. The oligo probe containing p21-ARE is
AAGCTTAGTACGTGATGTTCTAAGCTT. The probes
containing mutated AREs are shown in Fig. 4A
.
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ACKNOWLEDGMENTS
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We thank Miss Naomi Lee for assistance in manuscript
preparation.
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FOOTNOTES
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Address requests for reprints to: Address requests for reprints to: Dr. Ming-Jer Tsai, Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: mtsai{at}bcm.tmc.edu
This work was supported by Baylor Specialized Program of Research
Excellence in prostate cancer (CA-58204).
Received for publication June 25, 1998.
Revision received October 28, 1998.
Accepted for publication November 30, 1998.
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