Progression of LNCaP Prostate Tumor Cells during Androgen Deprivation: Hormone-Independent Growth, Repression of Proliferation by Androgen, and Role for p27Kip1 in Androgen-Induced Cell Cycle Arrest
John M. Kokontis,
Nissim Hay1 and
Shutsung Liao
The Ben May Institute for Cancer Research (J.M.K., N.H., S.L.)
The Departments of Pharmacology and Physiology (N.H.)
and Biochemistry and Molecular Biology (S.L.) The University of
Chicago Chicago, Illinois 60637
 |
ABSTRACT
|
---|
The molecular mechanism of androgen-independent
growth of prostate cancer after androgen ablation was explored in LNCaP
cells. An androgen-dependent clonal subline of the LNCaP human prostate
carcinoma cell line, LNCaP 104-S, progressed to a slow growing stage
(104-R1) and then to a faster growing stage (104-R2) during more than 2
yr of continuous culture in the absence of androgen. Androgen-induced
proliferation of 104-S cells is inhibited by the antiandrogen Casodex,
while proliferation of 104-R1 and 104-R2 cells is unaffected by
Casodex. This indicates that proliferation of 104-R1 and 104-R2 cells
is not supported by low levels of androgen in the culture medium.
Compared with LNCaP 104-S cells, both 104-R1 and 104-R2 cells express
higher basal levels of androgen receptor (AR), and proliferation of
these two cell lines is paradoxically repressed by androgen. After
continuous passage in androgen-containing medium, 104-R1 cells reverted
back to an androgen-dependent phenotype. The mechanism of androgenic
repression of 104-R1 and 104-R2 sublines was further evaluated by
examining the role of critical regulatory factors involved in the
control of cell cycle progression. At concentrations that repressed
growth, androgen transiently induced the expression of the
cyclin-dependent kinase (cdk) inhibitor
p21waf1/cip1 in 104-R1 cells, while expression
of the cdk inhibitor p27Kip1 was persistently
induced by androgen in both 104-R1 and 104-R2 cells. Induced expression
of murine p27Kip1 in 104-R2 cells resulted in
G1 arrest. Specific immunoprecipitates of Cdk2 but not Cdk4 from
androgen-treated 104-R1 cells contained both
p21waf1/cip1 and
p27Kip1. This observation was confirmed by
in vitro assay of histone H1 and Rb (retinoblastoma
protein) phosphorylation by the proteins associated with the immune
complex. Furthermore, inhibition of Cdk2 activity correlated with the
accumulation of p27Kip1 and not
p21waf1/cip1. From these results we conclude
that androgenic repression of LNCaP 104-R1 and 104-R2 cell
proliferation is due to the induction of
p27Kip1, which in turn inhibits Cdk2, a factor
critical for cell cycle progression and proliferation.
 |
INTRODUCTION
|
---|
The resistance to androgen ablation therapy (1) acquired by
prostate tumor cells during androgen deprivation remains a severe
obstacle to the effective treatment of metastatic prostate cancer (2).
Tumor cells that were formerly sensitive to antiandrogen or androgen
ablation strategies almost always emerge as androgen-independent
tumors after 13 yr of treatment (3). Understanding the mechanism(s)
by which hormonally sensitive tumor cells lose hormone dependency or
otherwise acquire the ability to defeat hormone deprivation-based
therapies has therefore been an important objective in the study of
prostate cancer. To examine this phenomenon, investigators have derived
androgen-independent cells from the Dunning tumor (4), the Shionogi
mouse mammary carcinoma 115 (5, 6, 7), a mouse prostate carcinoma system
(8), and the LNCaP cell line (9), among others.
We reported previously that adaptation of an androgen-dependent clonal
isolate of the LNCaP human prostate carcinoma cell line, LNCaP 104-S,
to androgen deprivation in vitro after 4060 passages was
accompanied by increased androgen receptor (AR) expression and activity
(10). Cells with heightened sensitivity to androgen, called 104-R
cells, proliferated at a much faster rate than 104-S cells in medium
deficient in androgen, and their proliferation rate was, in fact,
severely repressed by androgen even at low concentrations. This
repression by androgen was accompanied by reduced c-myc
expression and was blocked by ectopic overexpression of
c-myc. We also found that tumors derived from LNCaP 104-R
cells grown in athymic mice are repressed by testosterone and that this
growth repression could be blocked by the 5
-reductase inhibitor
finasteride (11). Others have also studied androgen-independent LNCaP
cells arising as tumors after the inoculation of androgen-dependent
cells into castrated nude mice (12, 13, 14, 15) or arising spontaneously
in vitro (16, 17, 18, 19, 20). The LNCaP cell line, therefore, provides
a good model for changes that occur in prostate tumor cells as the
cells are subjected to androgen deprivation both in vitro
and in vivo. In this paper we report on the continued
adaptation of LNCaP 104-R cells, now designated 104-R1 cells, to
androgen withdrawal and the progression of 104-R1 cells to more rapidly
proliferating cells designated 104-R2. We also report on the ability of
LNCaP 104-R1 cells to adapt to repressive levels of androgen and to
reacquire positive proliferative responsiveness to androgen.
To study the mechanism of androgen repression, we examined the
expression of several cyclin-dependent kinase inhibitors, including
p21waf-1/cip-1 and p27Kip1, in LNCaP 104-S,
104-R1, and 104-R2 cells after androgen treatment. The cyclin-dependent
kinase (cdk) inhibitors p21waf-1/cip-1 and
p27Kip1 are members of a structurally related family of
proteins that can mediate G1 cell cycle arrest (for review, see Ref.
21). Cell cycle arrest mediated by p21waf1/cip1 or
p27Kip1 is thought to occur because these proteins inhibit
the phosphorylation by cdks of members of the retinoblastoma protein
(Rb) family of regulators (Rb, p107, and p130). Hypophosphorylated Rb
family members form complexes with members of the E2F family (E2F-1
through 5) of transcription factors and the DP (DP-1, DP-2) family of
associated proteins (22). Phosphorylation of Rb by cdks results in
release of active E2F/DP complexes that transactivate genes required
for G1 transit and progression through S phase, or in release of Rb/E2F
complexes that can repress transcription of such genes. Results
described in this paper show that, although androgen induces a
transient elevation of p21waf1/cip1 in 104-R1 cells, it is
the persistent androgen-induced accumulation of p27Kip1 in
both 104-R1 and 104-R2 cells that correlates with the inhibition of
Cdk2 activity and G1 cell cycle arrest.
 |
RESULTS
|
---|
Androgen-Dependent and -Independent Proliferation of LNCaP 104
Sublines
Previously we had shown that LNCaP 104-S cells adapt to androgen
withdrawal after about 40 passages (PA- 40) in
androgen-deficient medium and progress to 104-R1 cells (formally called
104-R; Ref. 10). This adaptation is manifested as a higher
proliferation rate of 104-R1 cells in androgen-deficient medium as
compared with 104-S cells and is accompanied by increases in AR
expression and activity. However, the proliferation of LNCaP 104-R1
cells is repressed by androgen at concentrations that are optimal for
LNCaP 104-S growth. After about 60 additional passages in
androgen-deficient medium, 104-R1 cells attained proliferation rates in
the absence of androgen equivalent to or higher than 104-S cells
maximally stimulated by 0.1 nM
17ß-hydroxy-17-methylestra-4,9,11-trien-3-one (R1881) (Fig. 1
). LNCaP 104 cells at this stage
(>PA- 100) are designated 104-R2 to distinguish them from
104-R1 cells, which proliferate at a lower rate. The acquisition of
higher proliferation rates occurred gradually so the boundary between
104-R1 and 104-R2 cells is somewhat arbitrary. LNCaP 104-R2 cells are
somewhat less sensitive than 104-R1 cells to androgenic repression of
growth.

View larger version (24K):
[in this window]
[in a new window]
|
Figure 1. Proliferation of Various LNCaP Sublines in the
Presence of R1881
For testing the effect of androgen on cell proliferation, LNCaP cells
were plated in triplicate wells (3 x 104 cells per
well) in 2 ml of DMEM supplemented with 10% CS-FBS and the indicated
concentration of R1881 in a 12-well dish. After 6 days, cells were
trypsinized and counted with a hemocytometer. PA- 80 and
PA- 221 refer to the number of passages grown in the
absence of androgen at the time of testing.
|
|
Proliferation of LNCaP cells, which express a mutant form of AR (23, 24), is stimulated by antiandrogens such as hydroxyflutamide and
cyproterone acetate (23, 24, 25) but is inhibited by Casodex (26). Casodex
at a concentration of 5 µM severely repressed LNCaP 104-S
cell proliferation induced by 0.1 nM R1881, as expected,
but had little or no effect on LNCaP 104-R1 or 104-R2 cell
proliferation in the absence of R1881, indicating that the
proliferation of these cells is not dependent on residual androgen that
may be present in the medium (Fig. 2
). On
the other hand, Casodex efficiently blocked the repressive effect of
R1881 on 104-R1 and 104-R2 cell proliferation. This shows that androgen
repression of the proliferation of 104-R1 and 104-R2 cells is mediated
through AR.

View larger version (44K):
[in this window]
[in a new window]
|
Figure 2. Effect of R1881 and Casodex on Cell Proliferation
of LNCaP 104-S, 104-R1, and 104-R2 Cells
LNCaP cells were plated in triplicate wells (3 x 104
cells per well) in a 12-well dish in 2 ml of DMEM supplemented with
10% CS-FBS and ethanol vehicle (control), 0.1 nM R1881, 5
µM Casodex, or both 0.1 nM R1881 and 5
µM Casodex, as indicated. After 6 days, cells were
trypsinized and counted with a hemocytometer.
|
|
While the proliferation of LNCaP 104-R1 cells is severely inhibited by
R1881 at concentrations of 0.1 nM and higher, these cells
exhibited an ability to adapt over time and to grow in high
concentrations of R1881. Starting at PA- 55, 104-R1 cells
were grown continuously in DMEM with 10% dextran-coated,
charcoal-stripped (CS)-FBS supplemented with 20 nM R1881.
During the first 510 passages, the proliferation rate was low.
Gradually, the proliferation rate climbed to levels similar to 104-R1
cells grown in the absence of androgen. When these adapted cells,
termed 104-R1Ad, were grown over a range of R1881 concentrations, the
optimal concentration of R1881 for growth was now 110 nM
(Fig. 3
). Casodex, at 5 µM,
repressed R1881-induced proliferation, similar to its effect on 104-S
cells. R1881 at 100 nM appeared to overcome the inhibition
by Casodex. These observations indicate that 104-R1 cells can reacquire
positive proliferative sensitivity to previously repressive levels of
androgen after they are subjected to long-term exposure. The
proliferative responses of the various LNCaP 104 sublines to androgen
and Casodex are summarized in Table 1
.

View larger version (20K):
[in this window]
[in a new window]
|
Figure 3. Effect of R1881 and Casodex on Cell Proliferation
of LNCaP 104-R1Ad Cells
LNCaP 104-R1 cells were cultured for 30 passages in 20 nM
R1881 to generate 104-R1Ad cells. 104-R1Ad cells were passaged twice in
medium supplemented with 20 nM DHT before testing to clear
the cells of R1881. DHT was used because, unlike R1881, it is rapidly
metabolized in LNCaP cells (49 ) and therefore is efficiently removed
from cells before the proliferation assays. LNCaP 104-R1Ad cells were
plated in triplicate wells (3 x 104 cells per well)
in 2 ml of DMEM supplemented with 10% CS-FBS, the indicated
concentration of R1881, and 5 µM Casodex (open
circles) or ethanol vehicle (filled circles) in
a 12-well dish. After 6 days, cells were trypsinized and counted with a
hemocytometer.
|
|
Expression of AR and Prostate-Specific Antigen (PSA) in LNCaP
104-S, 104-R1, and 104-R2 Cells
AR expression in 104-R1 and 104-R2 cells was about 20-fold higher
compared with 104-S cells as quantified by densitometric scans of AR
immunoblots (Fig. 4
). AR level in
104-R1Ad cells, however, was dramatically lower than the level of AR in
104-R1 cells and even lower than the level found in 104-S cells. If
cells were cultured for 4 days in medium containing 0.1 or 10
nM R1881, AR levels in 104-S and 104-R1Ad cells were
enhanced 2- to 3-fold but were still lower than AR levels in 104-R1 or
104-R2 cells. The increased amount of AR in 104-S and 104-R1Ad cells
treated for 4 days with R1881 probably reflects stabilization of the AR
protein by androgen (27), since AR mRNA is down-regulated by androgen
in LNCaP cells (10, 28, 29). Probing of HindIII-digested
genomic DNA from 104-S, 104-R1, and 104-R2 cells with an amino-terminal
AR-specific cDNA probe revealed that the AR gene had not undergone
amplification in these sublines (data not shown).

View larger version (36K):
[in this window]
[in a new window]
|
Figure 4. AR Immunoblot of Total Cellular Proteins Extracted
from LNCaP 104-S, 104-R1, 104-R2, or 104-R1Ad Cells after 4 Days of
Growth in R1881 at 0, 0.1, and 10 nM R1881
Cells from 6-cm plates were lysed in 0.5 ml 2x Laemmli gel loading
buffer lacking bromophenol blue. After quantification of protein
concentration using the Bradford reagent, 40-µg aliquots were run on
10% SDS-PAGE gels. Blots were incubated with rabbit polyclonal
antibody AN-21 raised against the N-terminal 21 amino acids of hAR
(10 ), and bound antibody was visualized using peroxidase-conjugated
goat antirabbit IgG (Stratagene) and an enhanced chemiluminescence
procedure (15-sec exposure) AR migrated as a 110-kDa protein.
|
|
The ability of AR to stimulate transcription in these sublines was
examined by measuring induction of PSA mRNA by androgen. The
5'-regulatory region of the PSA gene contains multiple androgen
response elements (30). Induction of PSA mRNA by androgen was higher in
104-R1 cells and 104-R2 cells than in 104-S cells (Fig. 5
). The level of PSA mRNA in LNCaP 104-R1
and 104-R2 cells increased 10- to 15-fold within 48 h after
treatment with 1 nM R1881, while the PSA mRNA level in
104-S cells increased only about 5-fold. Therefore, AR in LNCaP 104-R1
and 104-R2 cells is functional and transcriptionally activated by
androgen, even though these cells are proliferatively repressed by
androgen.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 5. Induction of PSA mRNA by Androgen in LNCaP 104
Sublines
RNAse protection assay using 32P-labeled antisense PSA and
ß2-microglobulin (ß2-MG) RNA probes (10 )
was employed to measure the induction of PSA mRNA expression by R1881
in LNCaP 104-S, 104-R1 (PA- 68), and 104-R2
(PA- 210) cells. Total RNA was isolated from LNCaP 104-S,
104-R1, and 104-R2 cells grown in DMEM supplemented with 10% CS-FBS in
the presence or absence of 1 nM R1881 for 48 h.
Ordinate values were calculated by dividing the amount of radioactivity
present in PSA mRNA bands by the amount of radioactivity present in
ß2-MG mRNA bands and setting the ratio of one of the
untreated 104-S points to an arbitrary value of 1.
|
|
Androgen Induction of G1 Arrest in 104-R1 and 104-R2 Cells
Flow cytometric analysis of LNCaP 104-S, 104-R1, and 104-R2 cells
grown in the presence of R1881 demonstrated that androgen induces cell
cycle arrest at the G1 phase in 104-R1 and 104-R2 cells, but not in
104-S or 104-R1Ad cells (Fig. 6
, A and
B). Flow cytometric analysis of the time course of G1 arrest induced by
R1881 in 104-R1 cells showed that arrest was first apparent 24 h
after addition of hormone (Fig. 6C
). The percentage of cells in S phase
declined to the minimum by 72 h after addition of hormone. The
decline in the percentage of untreated control cells in S phase over
the last 2 days of the 4-day culture period can be attributed to the
gradual overcrowding of cells in the culture dishes, even though medium
was changed on day 2.

View larger version (39K):
[in this window]
[in a new window]
|
Figure 6. Androgen Induction of G1 Arrest in 104-R1 and
104-R2 Cells
A, Representative histograms of 104-R1 cells incubated for 4 days in
the absence or presence of 10 nM R1881. B, Flow cytometry
of 104-S, 104-R1, 104-R2, and 104-R1 cells adapted to growth in 20
nM R1881 after 4 days of growth in 0, 0.1, and 10
nM R1881. Cells (3 x 105) were plated on
6-cm plates. The next day, medium supplemented with 10% CS-FBS was
replaced with fresh medium containing the indicated concentration of
R1881, and the cells were incubated for 4 days before trypsinization
and fixation for flow cytometric analysis. C, Flow cytometric analysis
of the time course of G1 arrest in 104-R1 cells treated with 10
nM R1881 for the indicated periods. Data in panels B and C
represent the mean ± SE of three independent
experiments.
|
|
Androgen Induction of Accumulation of the cdk Inhibitors
p21waf1/cip1 and
p27Kip1 in 104-R1 Cells and
p27Kip1 in 104-R2 Cells
Because androgen specifically induced a G1 cell cycle arrest in
104-R1 and 104-R2 cells, we surveyed the expression of several cdk
inhibitors that are known to elicit G1 arrest when overexpressed. From
our time course studies of androgen-induced G1 arrest in 104-R1 cells,
we knew that increased expression of pertinent cdk inhibitors should be
apparent within 24 h after androgen treatment. Therefore we first
examined the expression of cdk inhibitors in 104-R1 cells grown for
increasing lengths of time in 10 nM R1881. Expression of
p21waf1/cip1 was induced 2- to 3-fold 12 h and 24
h after addition of R1881, but declined to the basal level at the 48-
and 72-h timepoints and was almost undetectable by 96 h (Fig. 7A
). Expression of p27Kip1
was induced 2- to 3-fold in 104-R1 cells after 24 h of incubation
in 10 nM R1881 and appeared to peak at 4872 h. Unlike
p21waf1/cip1, the level of p27Kip1 persisted at
a high level up to 96 h of incubation with androgen. Expression of
cdk inhibitors p57Kip2, p16INK4A, and
p15INK4B were not affected significantly by androgen in any
of the cell types (data not shown).

View larger version (38K):
[in this window]
[in a new window]
|
Figure 7. Effect of Androgen on Accumulation of
p21waf1/cip1 and p27Kip1 in 104-R1 and 104-R2
Cells
A, Time course analysis of p21waf1/cip1 and
p27Kip1 protein expression in 104-R1 cells treated with 10
nM R1881 for the indicated periods. B, Western blot of
p21waf1/cip1 and p27Kip1 expression in 104-S,
104-R1, 104-R2, and 104-R1Ad cells after 24 h
(p21waf1/cip1) or 48 h (p27Kip1) days of
growth in R1881 at 0, 0.1, 1, and 10 nM R1881. Cells from
6-cm plates were lysed in 0.5 ml 2x Laemmli gel loading buffer lacking
bromophenol blue. After quantification of protein concentration using
the Bradford reagent, 40-µg (p21waf1/cip1) or 20-µg
(p27Kip1) aliquots were run on 12% SDS-PAGE gels. Blots
shown are representative of three independent experiments.
|
|
Next we examined the effect of a range of R1881 concentrations on
p21waf1/cip1 and p27Kip1 expression in 104-S,
104-R1, 104-R2, and 104-R1Ad cells at 24 h
(p21waf1/cip1) and 48 h (p27Kip1) of
incubation (Fig. 7B
). R1881 at 0.1 nM induced only
p27Kip1 and not p21waf1/cip1 in 104-R1 cells,
suggesting that p27Kip1 is more sensitive to
androgen stimulation than p21waf1/cip1. In 104-R2
cells, R1881 induced p21waf1/cip1 slightly only
at the highest concentration. To check the possibility that the
kinetics of p21waf1/cip1 induction are delayed in 104-R2
cells compared with 104-R1 cells, a time course experiment similar to
that shown in Fig. 6A
was performed with 104-R2 cells. It revealed that
p21waf1/cip1 does not accumulate in 104-R2 cells at any
time within 96 h (data not shown). The basal level of
p27Kip1 expression in these cells was higher than in 104-R1
cells, and accumulation of p27Kip1 appeared to be less
sensitive to R1881. In 104-S and 104-R1Ad cells, R1881 did not induce
accumulation of p21waf1/cip1 or p27Kip1.
Rather, R1881, at the proliferatively optimal concentration of 0.1
nM, reduced the expression of both p21waf1/cip1
and p27Kip1 in 104-S cells. In 104-R1Ad cells, R1881
reduced the level of p21waf1/cip1 in a dose-dependent
manner, while R1881 at all concentrations only slightly reduced the
level of p27Kip1. These observations suggest that 1) in
104-R1 cells, p27Kip1 appears to be more sensitive to low
concentrations of R1881, but accumulates with slower kinetics than
p21waf1/cip1 and is persistently expressed; 2) in 104-R2
cells, accumulation of p27Kip1 appears to be less sensitive
to androgen than in 104-R1 cells, and accumulation of
p21waf1/cip1 is insensitive to androgen except at the
highest dose used, consistent with the generally lower sensitivity of
104-R2 cells to androgen-induced G1 arrest (Fig. 6B
); and 3) in
androgen-dependent 104-S cells and in 104-R1Ad cells, accumulation
of p21waf1/cip1 and p27Kip1 may have a role in
growth arrest after androgen deprivation, opposite to the effect of
androgen in 104-R1 and 104-R2 cells.
We then measured the levels of p21waf1/cip1 and
p27Kip1 mRNA in 104-R1 cells incubated in 10 nM
R1881 over a time course spanning 96 h. The message levels of each
were increased slightly after 12 h of incubation in 10
nM R1881, but this increase was not significant (Fig. 8
) and did not correlate with the protein
levels of p21waf1/cip1 or p27Kip1,
suggesting that the induction of p21waf1/cip1 and
p27Kip1 proteins by androgen occurred mainly by a
posttranscriptional mechanism.

View larger version (73K):
[in this window]
[in a new window]
|
Figure 8. RNase Protection Analysis Using
32P-Labeled 419-Base and 355-Base Antisense RNA Probes for
p21waf1/cip1 and p27Kip1 mRNA Expression,
Respectively, in 104-R1 Cells Grown for 24 h
(p21waf1/cip1) or 48 h (p27Kip1) in the
Presence of 10 nM R1881
A 109-base antisense GAPDH probe was used as internal control for the
total amount of RNA present. The lower panel represents
the means of normalized values from two independent experiments. The
values were calculated by dividing the amount of radioactivity present
in protected p21waf1/cip1 or p27Kip1 mRNA bands
by the amount of radioactivity present in GAPDH mRNA bands and setting
the ratio of one of the untreated points to an arbitrary value of 1.
The same probe preparations were used for each experiment.
|
|
Induced Expression of Exogenous
p27kip1 Results in G1 Arrest
To demonstrate that induced expression of
p27Kip1 can elicit G1 cell cycle arrest independently of
androgen, a clone of 104-R2 cells expressing a modified lac repressor
protein (31) was infected with LNXRO2 retrovirus (32) containing a cDNA
encoding murine p27Kip1. LNCaP 104-R1 cells were not used
because we were unable to obtain 104-R1 clones that stably expressed
the lac repressor protein. Expression of p27Kip1 was
induced in three selected clones by incubation in 4 mM
isopropyl ß-D-thiogalactoside (IPTG) for 48 h (Fig. 9A
). Murine p27Kip1 had a
faster mobility on Western blots than endogenous human
p27Kip1 and was induced at three different levels in the
three clones, with the lowest level corresponding roughly to the
induction of endogenous p27Kip1 by androgen. Interestingly,
endogenous human p27Kip1 was induced in IPTG-treated clones
as well as the retroviral mouse p27Kip1. Flow cytometry of
retrovirally infected cells incubated in the presence or absence of
IPTG showed that expression of p27Kip1 was sufficient to
arrest cells in the G1 stage, and the extent of G1 arrest correlated
with the level of p27Kip1 expression in the three clones
(Fig. 9B
). IPTG had no effect on parental 104-R2 cells expressing only
the lac repressor. A higher level of murine p27Kip1 was
required to elicit the same degree of G1 arrest observed by androgen
induction of endogenous p27Kip1. This suggests that murine
p27Kip1, which has 87% amino acid identity to human
p27Kip1, is not as active in LNCaP cells as human
p27Kip1. However, the observation that endogenous
p27Kip1 is elevated after IPTG treatment may mean that
murine p27Kip1 acts to cause G1 arrest indirectly by
raising endogenous human p27Kip1 levels through an unknown
mechanism. Since p27Kip1 is thought to be degraded through
the ubiquitin/proteosome pathway (33), it is possible that
overexpressed murine p27Kip1 competes with human
p27Kip1 at steps along this pathway.

View larger version (47K):
[in this window]
[in a new window]
|
Figure 9. Induction of G1 Arrest by Exogenous Expression of
p27Kip1
A, IPTG induces expression of murine p27Kip1 in three
104-R2 clones (5 11 16 ) infected with LNXRO2-murine
p27Kip1 retrovirus. Parental 104-R2 cells not infected with
the LNXRO2-murine p27Kip1 retrovirus are designated as
3'SS. Cells (5 x 105) were plated in 6-cm plates and
grown for 48 h. Medium was then replaced with control medium or
medium containing 4 mM IPTG and cells were incubated for an
additional 48 h. Cells were then lysed by the addition of 0.5 ml
2x Laemmli gel loading buffer lacking bromophenol blue. Aliquots
containing 20 µg of protein were run on 12% SDS-PAGE gels. B, IPTG
induces G1 arrest in 104-R2 clones infected with LNXRO2-murine
p27Kip1 retrovirus. Flow cytometric analysis was performed
on cells treated with IPTG as described in panel A. Data represent the
mean ± SE of three independent experiments.
|
|
p21waf1/cip1 and
p27Kip1 Inhibit Cdk2 but Not Cdk4
Activity in 104-R1 Cells Arrested by Androgen
To identify the targets of p21waf1/cip1 and
p27Kip1 in androgen-repressed LNCaP cells, 104-R1 cells
were incubated for 24 or 48 h in the absence or presence of R1881,
and whole cell extracts were immunoprecipitated with either Cdk2 or
Cdk4 polyclonal antibody that had been preadsorbed to Protein A-agarose
beads. Washed immunoprecipitates were separated by SDS-PAGE,
transferred to nitrocellulose membranes, and probed with
p21waf1/cip1 or p27Kip1 antibodies. Both
p21waf1/cip1 and p27Kip1 proteins
coimmunoprecipitated with Cdk2 but not Cdk4, and androgen increased the
amount of p21waf1/cip1 and p27Kip1 complexed
with Cdk2 by roughly the same extent as was seen in total cell extracts
(Fig. 10A
). Under identical conditions,
the Cdk4 antibody was able to coimmunoprecipitate
p21waf-1/cip-1 and Cdk4 in PC-3 prostate carcinoma cells
overexpressing p21waf1/cip1 (data not shown). The total
amount of Cdk2 and Cdk4 in cell lysates was not altered by androgen
(Fig. 10B
). Cdk2 and Cdk4 immunoprecipitates were used in in
vitro assays of kinase activity using histone H1 and a
glutathione-S-transferase (GST)-Rb fusion protein (Rb
residues 769921) as substrates. Cdk2 activity in immunoprecipitates
from R1881-treated 104-R1 cells was reduced more than 95% compared
with untreated cells, but had no effect on Cdk4 activity (Fig. 10C
). In
a time course study, Cdk2 activity, as measured by histone H1
phosphorylation, was not reduced by androgen treatment of cells until
24 h after addition of androgen and was almost complete by 48
h (Fig. 10D
). This result mirrors the onset of G1 arrest and the
accumulation of p27Kip1 but not the induction of
p21waf1/cip1 (Figs. 6C
and 7A
). Therefore, in LNCaP 104-R1
cells, although androgen transiently induces the accumulation of
p21waf1/cip1 before that of p27Kip1, it is the
accumulation of p27Kip1 and not p21waf1/cip1
that correlates directly with inhibition of Cdk2 activity and onset of
G1 arrest.

View larger version (47K):
[in this window]
[in a new window]
|
Figure 10. Effect of Androgen on Cdk2 and Cdk4 Activity in
104-R1 Cells
A, Androgen induces association of p21waf1/cip1 and
p27Kip1 with Cdk2 but not Cdk4. 104-R1 cells were grown for
24 h (p21waf1/cip1) or 48 h (p27Kip1)
in the absence or presence of 10 nM R1881. Whole cell
lysates were made, and Cdk2 and Cdk4 were immunoprecipitated from
lysate (2 mg protein) with Protein A-agarose preloaded with anti-Cdk2
or anti-Cdk4 antibody. After washing, immunoprecipitated protein was
eluted by boiling in Laemmli SDS-PAGE loading buffer, separated on 12%
SDS-PAGE gels, blotted onto nitrocellulose, and probed with
anti-p21waf1/cip1 or anti-p27Kip1 antibody. B,
Immunoblot showing that the total amount of Cdk2 and Cdk4 in 104-R1
cells is unaffected by R1881. C, Inhibition of Cdk2 activity by
androgen in 104-R1 cells. Cdk2 and Cdk4 immunoprecipitates from 104-R1
cells grown in the absence or presence of 10 nM R1881 for
48 h were used in histone H1 and GST-Rb phosphorylation reactions.
D, Time course assay of Cdk2 activity, using histone H1 as substrate,
from Cdk2 immunoprecipitates obtained from 104-R1 cells treated with 10
nM R1881 for the indicated periods. Blots and
autoradiograms are representative of three independent experiments.
|
|
 |
DISCUSSION
|
---|
The findings reported in this paper support the contention that
androgen-dependent prostate tumor cells can adapt to an
androgen-depleted environment and give rise to androgen-independent
cells that can grow unchecked by antiandrogen strategies. These
hormone-independent cells instead are paradoxically repressed by
androgen through a mechanism involving induction of the cdk inhibitor
p27Kip1. Because the 104-S cell line is clonally derived,
the possibility that preexistent androgen-independent cells emerge by
selection (34) is excluded. LNCaP 104-S cells, while not strictly
androgen-dependent in vitro, are extremely slow growing in
the absence of androgen and must undergo more than 2 yr of passage in
androgen-depleted medium during the progression to first the 104-R1
stage and then to the 104-R2 stage. 104-R2 cells proliferate in
androgen-depleted medium at a rate equivalent to 104-S cells grown in
an optimal concentration of androgen. LNCaP 104-S tumors grown in male
athymic mice rapidly regress after castration, suggesting that 104-S
cells are strictly androgen-dependent in vivo (11). The
insensitivity of 104-R1 and 104-R2 cells to Casodex inhibition shown in
this report indicates that 104-R1 and 104-R2 cells do not scavenge
residual androgen from the culture medium but are truly proliferatively
independent of androgen. If an extremely low concentration of androgen
was supporting 104-R1 and 104-R2 cell proliferation, then a many
log-fold excess of Casodex should have repressed proliferation even
more effectively than in 104-S cells growing in 0.1 nM
R1881. Hypersensitivity to estradiol was observed in MCF-7 human breast
cancer cells after hormone deprivation, although the growth of
hypersensitive cells was not repressed by estradiol (35). The change
was characterized as an adaptive response to low levels of hormone.
Significantly, LNCaP 104-R1 and 104-R2 cells, although proliferatively
independent of androgen, continue to express high levels of functional
AR. LNCaP 104-R1 and 104-R2 cells may therefore be classified as both
androgen-independent and androgen-sensitive. This is consistent with
the finding that most androgen-independent metastatic prostate tumors
retain AR expression. When the AR status of distant
androgen-independent metastatic prostatic tumors in 18 patients was
analyzed, all were found to express AR (36). Earlier studies had also
found that the majority of prostate tumors from patients experiencing
relapse exhibited widespread AR expression (37, 38). Additionally,
amplification of the AR gene was observed in 30% of metastatic
prostate tumors after androgen ablation (39, 40). In the Shionogi
mammary carcinoma, progression to androgen independence does not
correlate with loss of AR expression or functionality (41, 42). It is
possible that functional AR is required for the proliferation of
androgen-independent cells if AR is activated independently of ligand,
e.g. through peptide growth factors (43, 44), or through
activation of a protein kinase A-signaling pathway (45). However,
activation of AR by insulin-like growth factor-I or through activation
of protein kinase A is inhibited by Casodex (43, 45), while
proliferation of LNCaP 104-R1 and 104-R2 cells is clearly insensitive
to Casodex in the absence of androgen. Additionally, LNCaP 104-R1 and
104-R2 cells do not exhibit androgen-independent PSA expression, which
might be expected if AR could activate transcription in the absence of
androgen. Androgen-dependent PSA expression in LNCaP 104-R1 and 104-R2
cells stands in contrast to the results of Gleave et al.
(46) and Sato et al. (14), who found that LNCaP tumors grown
in castrated athymic mice can acquire androgen-independent growth and
androgen-independent PSA production. The recently established human
prostate tumor cell line, ARCaP, is repressed by androgen (47). Unlike
LNCaP 104-R1 and 104-R2 cells, however, ARCaP cells exhibit
androgen-repressible PSA expression, which coincides with the
repressive effect of androgen on ARCaP cell proliferation.
LNCaP 104-R1 and 104-R2 cells appear to be hypersensitive to the
repressive effects of 0.1 nM R1881 on proliferation, which
can be seen in 104-S cells (grown in medium supplemented with untreated
FBS; Ref. 10) and most other LNCaP cells (48, 49, 50, 51) only at higher
concentrations of R1881 or other androgens. Soto et al. (20)
observed similar hypersensitivity with LNCaP-LNO cells. The heightened
sensitivity in 104-R1 and 104-R2 cells is probably due to heightened AR
expression as compared with 104-S cells. Consistent with this is the
recent observation that 1
,25-dihydroxyvitamin D3
up-regulates AR expression and synergizes with 5
-dihydrotestosterone
(DHT) in the repression of LNCaP cell proliferation (52). LNCaP 104-R1
and 104-R2 cells, however, can also adapt to high concentrations of
androgen so that previously repressive concentrations are required for
optimal proliferation rate. This adaptation is accompanied by reduced
AR expression, providing further evidence that the level of AR
expression may be related to whether a positive or a negative
androgenic effect on proliferation is seen. Langeler et al.
(19) and Soto et al. (20) had found previously that
LNCaP-FGC and LNCaP-LNO cells can also adapt to repressively high
androgen concentration.
Androgen treatment of 104-R1 cells results in the transient elevation
of p21waf1/cip1 levels. The significance of this transient
androgen-induced elevation is not clear at the present time. This
elevation does not correlate with the kinetics of G1 arrest or
inhibition of Cdk2 activity. Forty-eight hours after addition of
androgen, the level of p21waf1/cip1 has returned to basal
levels, while Cdk2 activity is maximally repressed. Further,
p21waf1/cip1 is induced only minimally by androgen at a 10
nM concentration in 104-R2 cells, but these cells are
substantially arrested at both 0.1 and 10 nM R1881
concentrations. Unlike p21waf1/cip1, elevated
p27Kip1 levels are sustained for prolonged periods in both
104-R1 and 104-R2 cells treated with androgen. This sustained level of
p27Kip1 expression coincides with the repression of Cdk2.
Moreover, expression of exogenous p27Kip1 induced by IPTG
clearly elicited G1 arrest in 104-R2 cells in the absence of androgen,
suggesting that p27Kip1 accumulation by itself can account
for all of the repressive effects of androgen. Therefore, we conclude
that androgen-induced G1 arrest of 104-R1 and 104-R2 cells is mediated
by the accumulation of p27Kip1. We cannot, however, rule
out the participation of cdk inhibitors for which we have not screened.
Both p21waf1/cip1 and p27Kip1 have recently
been implicated in progesterone-induced growth arrest of T47D breast
cancer cells (53). These investigators also found that
p21waf1/cip1 induction precedes induction of
p27Kip1 and is more transient in duration.
The observation that p21waf1/cip1 or p27Kip1
steady state mRNA levels are not significantly affected by androgen
leads to the conclusion that elevation of these cdk
inhibitors occurs by a translational or posttranslational mechanism.
While regulation of p21waf1/cip1 is known to occur
principally at the transcriptional level, interference with protein
degradation rate has been reported to induce its accumulation (54).
Posttranscriptional control is consistent with previous findings that
p27Kip1 is regulated at the level of rate of translation
and protein turnover (33, 55, 56). Recently, Peng, et al.
(57) reported that the antiproliferative activity of an antibody
directed against epidermal growth factor receptor in DU145 prostate
carcinoma cells coincided with elevation of both p27Kip1
mRNA and protein. As in the present study, Cdk2 and not Cdk4 (nor Cdk6)
was found to be inhibited by p27Kip1 in DU145 cells.
It is possible that androgen regulates p27Kip1 through a
mechanism involving c-myc. Previous studies by others have
shown that c-Myc overexpression can block p27Kip1-induced
cell cycle arrest (58), and c-Myc activation or overexpression leads to
the loss of p27Kip1 protein in Rat1 fibroblasts (59) and in
rat embryo fibroblasts when co-expressed with activated H-ras (60).
Expression of c-myc is repressed by androgen in 104-R1 cells
(10), and others have reported that androgen represses c-myc
expression in LNCaP cells through a rapid transcriptional mechanism
(61). Therefore, it is possible that p27Kip1 level rises as
a result of a drop in c-myc expression. The observation that
constitutive retroviral c-myc overexpression in 104-R1 cells
blocks the repression of cell proliferation by androgen (10) is
consistent with this mechanism. Preliminary data show that
c-myc overexpression blocks androgen-induced G1 arrest in
104-R1 cells and reduces the accumulation of p27Kip1 in
104-R1 cells after androgen treatment (our unpublished
observations).
LNCaP 104-R1Ad cells that have adapted to growth in androgen no longer
accumulate high levels of either p21waf1/cip1 or
p27Kip1 in response to androgen, suggesting that the
androgen-signaling pathway leading to the accumulation of these
inhibitors in these cells has somehow become altered. It is possible
that a second, distinct proliferation pathway (or set of pathways),
that is activated in 104-S cells growing in 0.1 nM R1881
and is masked in hypersensitive 104-R1 and 104-R2 cells by the
repressive p27Kip1 pathway, is now revealed in these cells.
It is unclear, however, why 104-R1Ad cells lose some of their
previously acquired ability to grow independently of androgen. This
particular phenomenon may be related to the observation that prostate
tumor cells subjected to intermittent androgen deprivation remain
androgen-dependent for a longer time than cells subjected to continuous
androgen deprivation (14, 62, 63).
Recently, expression of the cdk inhibitor p16INK4a was
reported to be down-regulated in LNCaP-FGC cells induced to proliferate
by androgen (64). In LNCaP 104-S and 104-R1Ad cells, down-regulation of
both p21waf1/cip1 and p27Kip1 was much stronger
than the slight down-regulation of p16INK4a we observed
after androgen treatment. In addition, we did not observe changes in
Cdk2 or Cdk4 expression after androgen treatment, in contrast to the
report of Lu et al. (64). This variability among LNCaP
sublines suggests that cdk and cdk inhibitor expression may be far
downstream of the primary sites of androgen action in the induction of
cell proliferation. The mechanism(s) by which androgen induces cell
proliferation in 104-S cells is probably fundamentally more complex
than the mechanism by which androgen induces G1 arrest in 104-R1 and
104-R2 cells. However, both mechanisms appear to be subject to
reversible, epigenetic adaptation driven by changes in the hormonal
environment. Further identification of gene products and pathways that
mediate androgen-dependent growth, androgen-independent growth, and
androgen repression of growth in LNCaP cells in different hormonal
environments may lead to a better general understanding of how
hormone-dependent prostate tumor cells become independent and
ultimately, how to better treat hormone-independent prostate
cancer.
 |
MATERIALS AND METHODS
|
---|
Materials
The LNCaP 104-S and 104-R1 (formerly called 104-R) cell lines
were described previously (10). [
-32P]UTP (800
Ci/mmol), [
-32P]dCTP (3000 Ci/mmol), and
[
-32P]ATP (5000 Ci/mmol) was purchased from Amersham
(Arlington Heights, IL). A Super-Signal chemiluminescence detection kit
was from Pierce Chemical Co. (Rockford, IL). Restriction endonucleases
and other enzymes were purchased from Boehringer Mannheim
(Indianapolis, IN), New England Biolabs (Beverly, MA), Stratagene (La
Jolla, CA), and Life Technologies (Gaithersburg, MD). AN-21, a
polyclonal rabbit antibody raised against a 21-residue peptide
corresponding to the first 21 amino acids of human and rat AR was
described previously (10). Polyclonal antibodies against
p21waf-1/cip-1 (C-20), p27Kip1 (C-19),
p57Kip2 (C-20), p16INK4a (C-20) and
p15INK4b (C-20), Cdk2 (M2), and Cdk4 (C-22) and a GST-Rb
fusion protein (residues 769921) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). The 3'SS vector and anti-lac repressor
antibody were purchased from Stratagene, and the LNXRO2 vector was
generously provided by I. Roninson at the University of Illinois at
Chicago. Histone H1 from calf thymus was purchased from Boehringer
Mannheim (Indianapolis, IN). R1881 was from New England Nuclear
(Boston, MA). Casodex (ICI 176, 334;
(2RS)-4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3'-(trifluoromethyl)-propionanilide;
bicalutamide) was a generous gift from Zeneca Pharmaceuticals
(Wilmington, DE).
Cell Culture and Stable Retroviral Infection
LNCaP 104-S, 104-R1, and 104-R2 cells were passaged and
maintained as described previously (10). One passage consisted of cells
split 1:10 and grown for 5 days with a medium change on day 3. Cells
were infected with amphotropic pMV7 control retrovirus and pMV7 c-myc
retrovirus generated in the packaging cell line PA317 as described
previously (10) and selected by growth in 400 µg/ml G418 (Geneticin,
Life Technologies). One hundred to 200 colonies were pooled and
maintained in 400 µg/ml G418. For proliferation assays, cells were
trypsinized and resuspended in DMEM supplemented with 10%
dextran-coated CS-FBS (65) (Summit Biotechnology, Ft. Collins, CO).
Cells (3 x 104 cells per well) were added to wells in
a 12-well dish containing 2 ml DMEM supplemented with 10% CS-FBS and
the appropriate concentration of R1881 and/or Casodex. The amount of
ethanol added as vehicle never exceeded 0.1% of the total volume.
RNA Analysis
Isolation of total RNA and RNAse protection analysis of PSA mRNA
were carried out as described previously (10). For RNAse protection
assay of p21waf1/cip1 mRNA, an antisense
32P-labeled riboprobe that protects a 419-base
p21waf1/cip1 mRNA fragment was synthesized by using T7 RNA
polymerase and PstI-digested p21waf1/cip1 cDNA
(66) inserted into pBluescript SK+ (pBS; Stratagene).
Template for a 355-base human p27Kip1 antisense mRNA was
isolated by PCR amplification from an LNCaP 104-R1 UniZAP cDNA library
(10) using Pfu polymerase (Stratagene) and the primers
5'-ACCACGAAGAGTTAACCCGG and 5'-GGTCGCTTCCTTATTCCTGC, which correspond
to nucleotides 110129 and 464445 of human p27Kip1 (67).
Template for a 109-base glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) antisense mRNA probe was isolated by PCR amplification from the
same library using the primers 5'-CCATGGGGAAGGTGAAGGTCGGAG and
5'-GGGTCATTGATGGCAACAATA-TCC, which correspond to nucleotides 5982
and 167144 of human GAPDH (68). The amplification products were
ligated into EcoRV-digested pBS. For generation of antisense
probes, pBS-p27Kip1 was linearized with HindIII
and transcribed with T3 RNA polymerase; pBS-GAPDH was linearized with
EcoRI and transcribed with T7 RNA polymerase. The amount of
radioactivity in protected bands was quantified by scanning dried gels
in an AMBIS radioanalytic imaging system (AMBIS Systems, San Diego,
CA).
Immunoblot Analysis
Extraction of cellular proteins and immunoblot analysis with
polyclonal anti-AR (AN-21) antibodies were performed essentially as
described previously (10). Prestained molecular weight markers were
from BRL Life Technologies. For analysis of p21waf1/cip1
and p27Kip1 protein levels by SDS-PAGE, 12% polyacrylamide
gels were used. Protein concentration in cell lysates was determined
with the Bradford reagent using BSA standards. All antibodies were used
at a concentration of 0.5 µg/ml.
Flow Cytometric Analysis
LNCaP 104 cell lines and retrovirally infected LNCaP 104-R1
cells were plated (5 x 105 cells per 6-cm dish) in
DMEM supplemented with 10% CS-FBS and were incubated overnight. The
next day, medium was replaced with fresh medium containing R1881 at
various concentrations or ethanol vehicle, and cells were grown for
either 4 days (with a change of medium on day 2) or for varying lengths
of time in the time course experiment. Cells were trypsinized,
resuspended in complete medium, pelleted, and fixed in 70%
ethanol/30% PBS overnight at -20 C. Cells were then washed in PBS,
incubated for 30 min with 100 µg/ml RNAse A, and stained with 50
µg/ml propidium iodide in PBS. Cell cycle profiles and distributions
were determined by flow cytometric analysis of 104 cells
using the LysisII program on a FACScan flow cytometer
(Becton-Dickinson, San Jose, CA). Clumped cells were excluded from cell
cycle distribution analysis by gating.
Induced Expression of Exogenous
p27Kip1
LNCaP 104-R2 cells were transfected with the 3'SS vector
(Stratagene) encoding a modified lac repressor protein (31). Clones
expressing large amounts of repressor were selected in hygromycin B and
screened with anti-lac repressor antibody (Stratagene). One clone,
clone 8, was infected with retrovirus generated as described above from
the vector LNXRO2 (32) containing a cDNA encoding mouse
p27Kip1, and clones were selected in G418. The LNXRO2
vector contains an Rous sarcoma virus (RSV) promoter with two lac
operator sequences upstream of the cDNA insert site. Three clones, 5,
11 and 17, exhibited induced expression of p27Kip1 after
48 h of incubation in 4 mM IPTG and were used in
subsequent cell cycle analysis.
Immunoprecipitation and Kinase Assays
LNCaP 104-R1 cells were plated and treated with R1881 as
described above. After 48 h of incubation in the presence or
absence of R1881, cells were washed twice in PBS, lysed, and scraped
from the dishes in 1 ml of buffer containing 50 mM Tris HCl
(pH 8.0), 100 mM NaCl, 0.5% NP-40, 0.5 mM
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml
leupeptin. Lysate was agitated for 15 min at 4 C and centrifuged for 10
min at 4 C to pellet debris. Protein concentration of supernatant was
determined with Bradford reagent, and an aliquot of the supernatant (2
mg of protein) was preincubated for 1 h at 4 C with 1 µg rabbit
IgG and Protein A-agarose beads (25 µl packed volume; Santa Cruz
Biotechnology) equilibrated with lysis buffer. Beads were removed by
centrifugation, and lysates were incubated for 1 h at 4 C with
Protein A-agarose beads (25 µl packed volume) loaded with 1 µg
rabbit anti-Cdk2 or anti-Cdk4 polyclonal antibody. Beads were washed
three times with lysis buffer, and immunocomplexes were eluted by
boiling in 25 µl 2x Laemmli gel loading buffer. After
electrophoresis and transfer, cdk inhibitors were detected using goat
anti-p21waf1/cip1 or anti-p27Kip1 antibody and
horseradish peroxidase-conjugated donkey anti-goat IgG (Santa Cruz
Biotechnology). In vitro cdk phosphorylation assays were
performed according to Serrano et al. (69). Cdk2 and Cdk4
were immunoprecipitated as described above. Protein A-agarose beads
were washed twice in kinase buffer (20 mM Tris-HCl, 10
mM MgCl2, 1 mM dithiothreitol, 1
mM EDTA, pH 8.0) after which beads were resuspended in 25
µl kinase buffer containing 10 µCi [
-32P]ATP, 1
µM unlabeled ATP, and 2.5 µg of either histone H1 or
GST-Rb fusion protein. After 20 min at room temperature, reactions were
terminated by adding 0.33 volumes of 4x Laemmli gel loading buffer and
boiling. Aliquots were run on 10% SDS-PAGE gels, which were dried and
analyzed by autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Dr. Richard Hiipakka for helpful comments on
the manuscript and Dr. Igor Roninson (University of Illinois at
Chicago) for the LNXRO2 vector.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Shutsung Liao, The Ben May Institute for Cancer Research, Box MC6027, The University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637.
This work was supported by NIH Grants CA-58073 and DK-41670 to S.L. and
by NIH Grant CA-71874 and American Chemical Society Grant CB-133 to
N.H.
Results described in this paper were presented at the Schilling
Research Conference, Hormones and Cancer in Santa Cruz, CA, September
1821, 1997.
1 Present address: Department of Molecular Genetics, The University of
Illinois College of Medicine at Chicago, 900 South Ashland Avenue,
Chicago, Illinois 60607. 
Received for publication January 28, 1998.
Revision received March 17, 1998.
Accepted for publication March 25, 1998.
 |
REFERENCES
|
---|
-
Huggins C, Stevens RE, Hodges CV 1941 Studies on
prostatic cancer. II. The effects of castration on advanced carcinoma
of the prostate gland. Arch Surg 43:209223
-
Catalona WJ 1994 Management of cancer of the prostate. N
Engl J Med 331:9961004[Free Full Text]
-
Crawford ED, Eisenberger MA, McLeod DG, Spaulding JT, Benson
R, Dorr FA, Blumenstein BA, Davis MA, Goodman PJ 1989 A controlled
trial of leuprolide with and without flutamide in prostatic carcinoma.
(published erratum appears in N Engl J Med 1989, 321:1420)
N Engl J Med 321:419424
-
Isaacs JT, Isaacs WB, Feitz WFJ, Scheres J 1986 Establishment
and characterization of seven Dunning rat prostatic cancer cell lines
and their use in developing methods for predicting metastatic abilities
of prostatic cancers. Prostate 9:261281[Medline]
-
Bruchovsky N, Rennie PS, Coldman AJ, Goldenberg SL, To M,
Lawson D 1990 Effects of androgen withdrawal on the stem cell
composition of the Shionogi carcinoma. Cancer Res 50:22752282[Abstract]
-
Sato N, Watabe Y, Suzuki H, Shimazaki J 1993 Progression of
androgen-sensitive mouse tumor (Shionogi carcinoma 115) to
androgen-insensitive tumor after long-term removal of testosterone. Jpn
J Cancer Res 84:13001308[Medline]
-
Koga M, Kasayama S, Matsumoto K, Sato B 1995 Molecular
mechanism of androgen-dependent growth in transformed cells. Pathway
from basic science to clinical application. J Steroid Biochem Mol Biol 54:16[CrossRef][Medline]
-
Baley PA, Yoshida K, Qian W, Sehgal I, Thompson TC 1995 Progression to androgen insensitivity in a novel in vitro
mouse model for prostate cancer. J Steroid Biochem Mol Biol 52:403413[CrossRef][Medline]
-
Horoszewicz JS, Leong SS, Kawinski E, Karr J, Rosenthal H,
Chu TM, Mirand EA, Murphy GP 1983 LNCaP model of human prostatic
carcinoma. Cancer Res 43:18091818[Abstract]
-
Kokontis J, Takakura K, Hay N, Liao S 1994 Increased androgen
receptor activity and altered c-myc expression in prostate
cancer cells after long-term androgen deprivation. Cancer Res 54:15661573[Abstract]
-
Umekita Y, Hiipakka RA, Kokontis JM, Liao S 1996 Human
prostate tumor growth in athymic mice: inhibition by androgens and
stimuation by a 5
-reductase inhibitor. Proc Natl Acad Sci USA 93:1180211807[Abstract/Free Full Text]
-
Thalmann GN, Anezinis PE, Chang S-M, Zhau HE, Kim EE, Hopwood
VL, Pathak S, von Eschenbach AC, Chung LWK 1994 Androgen-independent
cancer progression and bone metastasis in the LNCaP model of human
prostate cancer. Cancer Res 54:25772581[Abstract]
-
Wu H-C, Hsieh J-T, Gleave ME, Brown NM, Pathak S, Chung LWK 1994 Derivation of androgen-independent human LNCaP prostatic cancer
cell sublines: role of bone stromal cells. Int J Cancer 57:406412[Medline]
-
Sato N, Gleave ME, Bruchovsky N, Rennie PS, Goldenberg SL,
Lange PH, Sullivan LD 1996 Intermittent androgen suppression delays
progression to androgen-independent regulation of prostate-specific
antigen gene in the LNCaP prostate tumour model. J Steroid Biochem Mol
Biol 58:139146[CrossRef][Medline]
-
Hyytinen ER, Thalmann GN, Zhau HE, Karhu R, Kallioniemi OP,
Chung LW, Visakorpi T 1997 Genetic changes associated with the
acquisition of androgen-independent growth, tumorigenicity and
metastatic potential in a prostate cancer model. Br J Cancer 75:190195[Medline]
-
Hasenson M, Hartley-Asp B, Kihifors C, Lundin A, Gustafsson
J-Å, Pousette Å 1985 Effect of hormones on growth and ATP content of
a human prostatic carcinoma cell line, LNCaP-r. Prostate 7:183194[Medline]
-
van Steenbrugge GJ, Groen M, van Dongen JW, Bolt J, van der
Korput H, Trapman J, Hasenson M, Horoszewicz JS 1989 The human
prostatic carcinoma cell line LNCaP and its derivatives. Urol Res 17:7177[Medline]
-
van Steenbrugge GJ, van Uffelen CJC, Bolt J, Schröder FH 1991 The human prostatic cancer cell line LNCaP and its derived
sublines: an in vitro model for the study of androgen
sensitivity. J Steroid Biochem Mol Biol 40:207214[CrossRef][Medline]
-
Langeler EG, van Uffelen CJC, Blankenstein MA, van Steenbrugge
GJ, Mulder E 1993 Effect of culture conditions on androgen sensitivity
of the human prostatic cancer cell line LNCaP. Prostate 23:213223[Medline]
-
Soto AM, Lin T-M, Sakabe K, Olea N, Damassa DA, Sonnenschein C 1995 Variants of the human prostate LNCaP carcinoma cell line as tools
to study discrete components of the androgen-mediated proliferative
response. Oncol Res 7:545558[Medline]
-
Sherr CJ, Roberts JM 1995 Inhibitors of mammalian
G1 cyclin-dependent kinases. Genes Dev 9:11491163[CrossRef][Medline]
-
Weinberg RA 1995 The retinoblastoma protein and cell cycle
control. Cell 81:323330[Medline]
-
Veldscholte J, Ris-Stalpers C, Kuiper GGJM, Jenster G,
Berrevoets C, Claassen E, van Rooij HCJ, Trapman J, Brinkmann AO,
Mulder E 1990 A mutation in the ligand binding domain of the androgen
receptor of human LNCaP cells affects steroid binding
characteristics and response to anti-androgens. Biochem Biophys Res
Commun 173:534540[Medline]
-
Kokontis J, Ito K, Hiipakka RA, Liao S 1991 Expression and
function of normal and LNCaP androgen receptors in androgen-insensitive
human prostatic cancer cells: altered hormone and antihormone
specificity in gene transactivation. Receptor 1:271279[Medline]
-
Olea N, Sakabe K, Soto AM, Sonnenschein C 1990 The
proliferative effect of "anti-androgens" on the androgen-sensitive
human prostate tumor cell line LNCAP. Endocrinology 126:14571463[Abstract]
-
Veldscholte J, Berrevoets CA, Brinkmann AO, Grootegoed JA,
Mulder E 1992 Anti-androgens and the mutated androgen receptor of the
LNCaP cells: differential effects on binding affinity, heat-shock
protein interaction, and transcription activation. Biochemistry 31:23932399[Medline]
-
Kemppainen JA, Lane MV, Sar M, Wilson EM 1992 Androgen
receptor phosphorylation, turnover, nuclear transport, and
transcriptional activation. J Biol Chem 267:968974[Abstract/Free Full Text]
-
Quarmby VE, Yarbrough WG, Lubahn DB, French FS, Wilson EM 1990 Autologous down-regulation of androgen receptor messenger ribonucleic
acid. Mol Endocrinol 4:2228[Abstract]
-
Krongrad A, Wilson C, Wilson J, Allman D, McPhaul M 1991 Androgen increases androgen receptor protein while decreasing receptor
mRNA in LNCaP cells. Mol Cell Endocrinol 76:7988[CrossRef][Medline]
-
Cleutjens KB, van der Korput HA, van Eekelen CC, van Rooij HC,
Faber PW, Trapman J 1997 An androgen response element in a far upstream
enhancer region is essential for high, androgen-regulated activity of
the prostate-specific antigen promoter. Mol Endocrinol 11:148161[Abstract/Free Full Text]
-
Fieck A, Wyborski DL, Short JM 1992 Modifications of the E.
coli Lac repressor for expression in eukaryotic cells: effects of
nuclear signal sequences on protein activity and nuclear accumulation.
Nucleic Acids Res 20:17851791[Abstract]
-
Chang B-D, Roninson I 1996 Inducible retroviral vectors
regulated by lac repressor in mammalian cells. Gene 183:137142[CrossRef][Medline]
-
Pagano M, Tam SW, Theodoras AM, Beer-Romero P, Del Sal G, Chau
V, Yew PR, Draeta GF, Rolfe M 1995 Role of the ubiquitin-proteosome
pathway in regulating abundance of the cyclin-dependent kinase
inhibitor p27. Science 269:682685[Medline]
-
Isaacs JT, Coffey DS 1981 Adaptation vs. selection
as the mechanism responsible for the relapse of prostate cancer to
androgen ablation therapy as studied in the Dunning R-3327-H
adenocarcinoma. Cancer Res 41:50705075[Abstract]
-
Masamura S, Santner SJ, Heitjan DF, Santen RJ 1995 Estrogen
deprivation causes estradiol hypersensitivity in human breast cancer
cells. J Clin Endocrinol Metab 80:29182925[Abstract]
-
Hobisch A, Culig Z, Radmeyer C, Bartsch G, Klocker H, Hittmair
A 1995 Distant metastases from prostatic carcinoma express androgen
receptor protein. Cancer Res 55:30683072[Abstract]
-
van der Kwast TH, Schalken J, Ruizeveld de Winter JA, van
Vroonhoven CCJ, Mulder E, Boersma W, Trapman J 1991 Androgen receptors
in endocrine-therapy-resistant human prostate cancer. Int J Cancer 48:189193[Medline]
-
Ruizeveld de Winter JA, Janssen PJA, Sleddens HMEB,
Verleun-Mooijman MCT, Trapman J, Brinkmann AO, Santerse AB,
Schröder FH, Van der Kwast TH 1994 Androgen receptor status in
localized and locally progressive hormone refractory human prostate
cancer. Am J Pathol 144:735746[Abstract]
-
Visakorpi T, Hyytinen ER, Koivisto P, Tanner M, Keinänen
R, Palmberg C, Palotie A, Tammela T, Isola JJ, Kallioniemi O-P 1995 In vivo amplification of the androgen receptor gene and
progression of human prostate cancer. Nat Genet 9:401406[Medline]
-
Koivisto P, Kononen J, Palmberg C, Tammela T, Hytinen E, Isola
J, Trapman J, Cleutjens K, Noorrdzij A, Visakorpi T, Kallioniemi O-P 1997 Androgen receptor gene amplification: a possible molecular
mechanism for androgen deprivation therapy failure in prostate cancer.
Cancer Res 57:314319[Abstract]
-
Darbre PD, King RJB 1987 Progression to steroid insensitivity
can occur irrespective of the presence of functional steroid receptors.
Cell 51:521528[Medline]
-
Furuya Y, Shirasawa H, Sato N, Watabe Y, Simizu B, Shimazaki J 1992 Loss of androgen dependency with preservation of functional
androgen receptors in androgen-dependent mouse tumour (Shionogi
carcinoma 115). J Steroid Biochem Mol Biol 42:569574[CrossRef][Medline]
-
Culig Z, Hobisch A, Cronauer MV, Radmayr C, Trapman J,
Hittmair A, Bartsch G, Klocker H 1994 Androgen receptor activation in
prostate tumor cell lines by insulin-like growth factor-I, keratinocyte
growth factor, and epidermal growth factor. Cancer Res 54:54745478[Abstract]
-
Reinikainen P, Palvimo JJ, Jänne O 1996 Effects of
mitogens on androgen receptor-mediated transactivation. Endocrinology 137:43514357[Abstract]
-
Nazareth LV, Weigel NL 1996 Activation of the human androgen
receptor through a protein kinase A signaling pathway. J Biol Chem 271:1990019907[Abstract/Free Full Text]
-
Gleave ME, Hsieh J-T, Wu H-C, von Eschenbach AC, Chung LWK 1992 Serum prostate specific antigen levels in mice bearing human
prostate LNCaP tumors are determined by tumor volume and endocrine and
growth factors. Cancer Res 52:15981605[Abstract]
-
Zhau HY, Chang SM, Chen BQ, Wang Y, Zhang H, Kao C, Sang QA,
Pathak SJ, Chung LW 1996 Androgen-repressed phenotype in human prostate
cancer. Proc Natl Acad Sci USA 93:1515215157[Abstract/Free Full Text]
-
Wolf DA, Schulz P, Fittler F 1991 Synthetic androgens suppress
the transformed phenotype in the human prostate carcinoma cell line
LNCaP. Br J Cancer 64:4753[Medline]
-
Smith CM, Ballard SA, Wyllie MG, Masters JRW 1994 Comparison
of testosterone metabolism in benign prostatic hyperplasia and human
prostate cancer cell lines in vitro. J Steroid Biochem Mol
Biol 50:151159[CrossRef][Medline]
-
Joly-Pharaboz M-O, Soave M-C, Nicolas B, Mebarki F, Renaud M,
Foury O, Morel Y, Andre JG 1995 Androgens inhibit the proliferation of
a variant of the human prostate cancer cell line LNCaP. J Steroid
Biochem Mol Biol 55:6776[CrossRef][Medline]
-
Kim IY, Kim J-H, Zelner DJ, Ahn H-J, Sensibar JA, Lee C 1996 Transforming growth factor-ß1 is a mediator of androgen-regulated
growth arrest in an androgen-responsive prostatic cancer cell line,
LNCaP. Endocrinology 137:991999[Abstract]
-
Zhao X-Y, Ly LH, Peehl DM, Feldman D 1997 1
,25-dihydroxyvitamin D3 actions in LNCaP human prostate
cancer cells are androgen-dependent. Endocrinology 138:32903298[Abstract/Free Full Text]
-
Groshong SD, Owen GI, Grimison B, Schaueer IE, Todd MC, Langan
TA, Sclafani RA, Lange CA, Horwitz KB 1997 Biphasic regulation of
breast cancer cell growth by progesterone: role of cyclin-dependent
kinase inhibitors, p21 and p27Kip1. Mol Endocrinol 11:15931607[Abstract/Free Full Text]
-
Blagosklonny MV, Wu GS, Omura S, el-Deiry WS 1996 Proteasome-dependent regulation of p21WAF1/CIP1 expression. Biochem
Biophys Res Commun 227:564569[CrossRef][Medline]
-
Hengst L, Reed SI 1996 Translational control of
p27Kip1 accumulation during the cell cycle. Science 271:18611864[Abstract]
-
Millard SS, Yan JS, Nguyen H, Pagano M, Kiyokawa H, Koff A 1997 Enhanced ribosomal association of p27Kip1 mRNA is a
mechanism contributing to accumulation during growth arrest. J
Biol Chem 272:70937098[Abstract/Free Full Text]
-
Peng D, Fan Z, Lu Y, DeBlasio T, Scher H, Mendelsohn J 1996 Anti-epidermal growth factor receptor monoclonal antibody 225
up-regulates p27KIP1 and induces G1 arrest in prostatic
cancer cell line DU145. Cancer Res 56:36663669[Abstract]
-
Vlach J, Hennecke S, Alevizopoulos K, Conti D, Amati B 1996 Growth arrest by the cyclin-dependent kinase inhibitor
p27Kip1 is abrogated by c-Myc. EMBO J 15:65956604[Abstract]
-
Steiner P, Philipp A, Lukas J, Godden-Kent D, Pagano M,
Mittnacht S, Bartek J, Eilers M 1995 Identification of a Myc-dependent
step during the formation of active G1 cyclin-cdk
complexes. EMBO J 14:48144826[Abstract]
-
Leone G, DeGregori J, Sears R, Jakoi L, Nevins JR 1997 Myc and
Ras collaborate in inducing accumulation of active cyclin E/Cdk2 and
E2F. Nature 387:422426[CrossRef][Medline]
-
Wolf DA, Kohlhuber F, Schulz P, Fittler F, Eick D 1992 Transcriptional down-regulation of c-myc in human prostate
carcinoma cells by the synthetic androgen mibolerone. Br J Cancer 65:376382[Medline]
-
Akakura K, Bruchovsky N, Goldenberg SL, Rennie PS, Buckley AR,
Sullivan LD 1993 Effects of intermittent androgen suppression on
androgen-dependent tumors. Cancer 71:27822790[Medline]
-
Goldenberg SL, Bruchovsky N, Gleave ME, Sullivan LD, Akakura K 1995 Intermittent androgen suppression in the treatment of prostate
cancer: a preliminary report. Urology 45:839845[CrossRef][Medline]
-
Lu S, Tsai SY, Tsai MJ 1997 Regulation of androgen-dependent
prostatic cancer cell growth: androgen regulation of CDK2, CDK4, and
CKI p16 genes. Cancer Res 57:45114516[Abstract]
-
Horwitz KB, McGuire WL 1978 Estrogen control of progesterone
receptor in human breast cancer. J Biol Chem 253:22232228[Medline]
-
El-Deiry WS, Tokino T, Velculesco VE, Levy DB, Parsons R,
Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B 1993 WAF1, a potential mediator of p53 tumor suppression. Cell 75:817825[Medline]
-
Polyak K, Lee M-H, Erdjument-Bromage H, Koff A, Roberts JM,
Tempst P, Massagué J 1994 Cloning of p27kip1, a
cyclin-dependent kinase inhibitor and a potential mediator of
extracellular antimitotic signals. Cell 78:5966[Medline]
-
Tokunaga K, Nakamura Y, Sakata K, Fujimori K, Ohkubo M, Sawada
K, Sakiyama S 1987 Enhanced expression of a glyceraldehyde-3-phosphate
dehydrogenase gene in human lung cancers. Cancer Res 47:56165619[Abstract]
-
Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW 1997 Oncogenic ras provokes premature cell senescence associated
with accumulation of p53 and p16INK4a. Cell 88:593602[CrossRef][Medline]