Antiprogestin Inhibition of Cell Cycle Progression in T-47D Breast Cancer Cells Is Accompanied by Induction of the Cyclin-Dependent Kinase Inhibitor p21
Elizabeth A. Musgrove,
Christine S. L. Lee,
Ann L. Cornish,
Alex Swarbrick and
Robert L. Sutherland
Cancer Research Program Garvan Institute of Medical
Research St. Vincents Hospital Sydney, New South Wales 2010,
Australia
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ABSTRACT
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Progestin antagonists inhibit the
proliferation of progesterone receptor-positive cells, including breast
cancer cells, by G1 phase-specific actions, but
the molecular targets involved are not defined. Reduced phosphorylation
of pRB, a substrate for G1 cyclin-dependent
kinases (CDKs) in vivo, was apparent after 9 h
treatment of T-47D breast cancer cells with the antiprogestins RU 486
or ORG 31710, accompanying changes in S phase fraction. Although the
abundance of cyclin D1, Cdk4, and Cdk6 did not decrease, cyclin
D1-associated kinase activity was reduced by
50% at 918 h.
Similarly, cyclin E-associated kinase activity decreased by
60% at
1224 h in the absence of significant changes in the abundance of
cyclin E and Cdk2. The CDK inhibitor p21 increased in mRNA and protein
abundance and was present at increased levels in cyclin D1 and cyclin E
complexes at times when their kinase activity was decreased. Increased
p21 protein abundance was observed in another antiprogestin-sensitive
cell line, BT 474, but not in two breast cancer cell lines insensitive
to antiprogestins. These data suggest increased p21 abundance and
concurrent inhibition of CDK activity as a mechanism for antiprogestin
induction of growth arrest. Antiprogestin effects on proliferation
were markedly reduced after ectopic expression of cyclin D1, indicating
that inhibition of cyclin D1 function is a critical element in
antiprogestin inhibition of proliferation. However, these data
also implicate regulation of cyclin E function in antiprogestin
regulation of cell cycle progression.
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INTRODUCTION
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Cancers of steroid hormone target tissues, i.e. breast,
prostate, endometrium, and ovary, account for a third of newly
diagnosed cancers (1). Many of these cancers retain steroid
responsiveness, leading to the development of synthetic steroid
antagonists as potential therapeutic agents for hormone-dependent
cancers. The success of this strategy is illustrated by the
nonsteroidal antiestrogen tamoxifen, which is among the most effective
specific therapies for breast cancer (2). In animal models of mammary
cancer the antiprogestin RU 486 is as effective as tamoxifen in
inhibition of tumor growth (3). Preliminary clinical trials of RU 486
in patients with metastatic breast cancer demonstrated some efficacy
(4, 5), but frequent side effects, attributed to the potent
antiglucocorticoid activity of RU 486, were observed (5). This problem
may be minimized by more recently developed antiprogestins that display
little antiglucocorticoid activity (6). Thus, antiprogestins may be of
use in the treatment of breast cancer, in addition to their obstetric
and gynecological uses (7), and their optimal clinical use is likely to
be aided by a more detailed understanding of their molecular modes of
action as anticancer agents.
Studies using breast cancer cells in vitro and rodent
mammary tumors in vivo suggest that the antitumor activity
of antiprogestins is mediated by inhibition of proliferation (8). Like
other steroid or retinoid receptor ligands, antiprogestins have cell
cycle phase-specific effects on cell proliferation (9, 10).
Antiprogestin treatment leads to accumulation of breast cancer cells in
G1 phase at the expense of cells in S, G2, and
M phases (11, 12, 13), but the molecular targets involved in mediating this
effect have not been defined. However, recent studies implicate
regulation of cyclin/cyclin-dependent kinase (CDK) activity,
particularly cyclin D-associated kinase activity, in steroidal
regulation of proliferation. Cyclin D1 appears to be necessary for the
progesterone-dependent development and differentiation of the mammary
gland, because cyclin D1-deficient mice fail to develop lobular alveoli
during pregnancy (14, 15), and this phenotype is shared by progesterone
receptor-deficient mice (16). Furthermore, antiestrogen and retinoid
inhibition of breast cancer cell cycle progression is accompanied by
decreased cyclin D1 function (17, 18, 19), whereas decreased cyclin D3
function contributes to glucocorticoid inhibition of lymphoma cell
proliferation (20).
Sequential activation of cyclin/CDK enzyme complexes regulates progress
through the cell cycle. These enzymes are regulated at multiple levels,
providing a variety of possible means by which overall activity of the
complex, and hence the rate of cell cycle progression, might be
modulated. The catalytic activity of CDKs depends not only on cyclin
association but also on appropriate phosphorylation of the CDK subunit
(21). The CDK-activating kinase (CAK) is itself a cyclin/CDK complex
(cyclin H/Cdk7) regulated by phosphorylation. However, regulation of
cyclin abundance governs much of the regulation of CDK activity during
cell cycle progression and is a frequent response to treatment either
by mitogens or inhibitors of cell proliferation (22, 23). Alteration of
cyclin abundance is sufficient to alter the rate of cell cycle
progression because overexpression of cyclins D or E accelerates cells
through G1 and, conversely, inhibition of their function by
antibody microinjection prevents entry into S phase (23).
A further means of regulating cyclin/CDK function is provided by
endogenous low molecular weight proteins that physically associate with
the cyclins, CDKs, or their complexes and inhibit CDK activity (24). A
growing family of such inhibitors, for which p16INK4 is the
prototype, selectively targets Cdk4 and Cdk6 (24). A second family of
inhibitors, including p21 (WAF1, Cip1, Sdi1) and p27 (Kip1), are active
against a wider range of cyclin/CDK complexes (24). The balance between
levels of inhibitor and cyclin/CDK complexes is thought to set a
threshold for activation of the kinase. The mechanism for inhibition of
CDK activity has not been defined, but p21 immunoprecipitates display
kinase activity indicating that association per se is not
sufficient for inactivation (25, 26). Increased p21 or p27 abundance
accompanies inhibition of proliferation during quiescence, senescence,
and differentiation and contributes to inhibition of growth by
transforming growth factor-ß (24, 27).
The retinoblastoma tumor suppressor protein, pRB, is a critical
substrate for the G1 CDKs. pRB is hypophosphorylated during
early G1 and in this form is growth-inhibitory. The pRB
hyperphosphorylation that relieves this growth inhibition is first
apparent in late G1 phase and continues during the
remainder of the cell cycle (28). Cells without functional pRB lose
dependence on cyclin D1 for G1 progression but demonstrate
an absolute requirement for cyclin D1 upon reintroduction of pRB (29, 30). These data provide compelling evidence that pRB is a critical
physiological target for cyclin D1. However, it is likely that cyclin
E/Cdk2 also phosphorylates pRB in vivo, perhaps contributing
to the further phosphorylation of pRB as cells progress into S phase
(28).
In breast cancer cells cyclin D1 is both necessary and sufficient for
G1 phase progression (31, 32). Furthermore, cyclin D1
abundance is rate-limiting in these cells as well as in fibroblasts
(32, 33, 34). Initial studies demonstrated that neither cyclin D1 nor Cdk4
mRNA decreased in abundance after antiprogestin treatment, despite
inhibition of proliferation (12, 19). However, as outlined above, other
mechanisms for regulation of cyclin function exist and, in view of
increasing evidence for regulation of cyclin function after treatment
with steroids, steroid antagonists, and retinoids, the possibility that
antiprogestins might regulate cyclin function in T-47D human breast
cancer cells was investigated. Both RU 486, which is the prototypic
progestin antagonist but also has glucocorticoid antagonist activity,
and ORG 31710, which is representative of newer progestin antagonists
with little antiglucocorticoid activity, were used. These studies
demonstrate that antiprogestins induce p21 and regulate G1
cyclin function and indicate that this is likely to account for their
inhibition of breast cancer cell proliferation.
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RESULTS
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T-47D human breast cancer cells were cultured under conditions
leading to optimal growth rates, i.e. in medium supplemented
with insulin and FCS, to maximize sensitivity to growth inhibition.
Initial experiments sought to define the effects of progestin
antagonists on cell cycle progression under these conditions. Both RU
486 and ORG 31710 led to a concentration-dependent decrease in relative
cell number (Fig. 1A
). ORG 31710 was more potent than RU
486 such that the response to ORG 31710 was maximal at 1
nM, compared with 10 nM after RU 486 treatment
(Fig. 1A
). The decrease in cell number was associated with a sustained
decrease in the proportion of cells in S phase over the same
concentration range, apparent within 1 day of treatment and at
concentrations of
1 nM, maintained until the conclusion
of the experiment at 5 days (Fig. 1B
). Maximally effective
concentrations of RU 486 (100 nM) or ORG 31710 (10
nM) were chosen on the basis of these data and used for
further experiments.

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Figure 1. Effect of Antiprogestins on T-47D Cell
Proliferation
A, Exponentially proliferating T-47D human breast cancer cells were
treated with RU 486 ( ) or ORG 31710 () at the indicated
concentrations. Proliferation was assayed after 67 days treatment
using a colorimetric assay, and absorbance of the treated wells is
presented relative to that in vehicle-treated control wells, as
relative cell number. Data points indicate mean ± SEM
of values from two or three replicate experiments each performed in
quadruplicate or (points without error bars) mean of quadruplicates in
a single experiment. B, Exponentially proliferating T-47D human breast
cancer cells were treated with ethanol vehicle or the indicated
concentrations of RU 486 or ORG 31710. Individual flasks were harvested
and stained for DNA analysis by flow cytometry after 1 ( ), 3 ( ),
or 5 days ( ) treatment. Data represent mean ± SEM
or range of results from two to four experiments. The S phase of
control cells was similar over the entire experiment and, therefore,
data from days 1, 3, and 5 have been pooled and are shown as mean
± SEM (C, ). C, Exponentially proliferating T-47D human
breast cancer cells were
treated with ethanol vehicle (x) RU 486 (100 nM,
), or ORG 31710 (10 nM, ). Individual flasks were
then harvested and stained for DNA analysis by flow cytometry. Data
represent mean ± SEM, where this is greater than the
size of the symbol used, of at least three points from a total of seven
experiments (RU 486) or five experiments (ORG 31710). The S phase
fraction is significantly reduced compared with time-matched controls
after 924 h treatment: P < 0.04 at 9 h for
both compounds, P < 0.0001 for 1224 h RU 486
treatment, and P < 0.001 for 1224 h ORG 31710.
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To characterize the initial decrease in S phase in more detail, cell
cycle phase distribution was determined at intervals after
antiprogestin treatment. The proportion of cells in S phase remained
near control levels for 6 h but began to decline at 9 h and
reached a minimum after 1218 h (Fig. 1C
). Although the time courses
for the two compounds were similar, ORG 31710 was slightly more
effective. The effects of both antiprogestins on S phase fraction
decreased between 1824 h exposure but were thereafter maintained
(Fig. 1
, B and C).
Since pRB is a physiological substrate for the G1 phase
CDKs, pRB phosphorylation in vivo was examined to determine
whether changes in CDK activity might accompany antiprogestin
treatment. In untreated exponentially proliferating cells pRB was
predominantly in the hyperphosphorylated form (ppRB, Fig. 2
). After 9 h or more of antiprogestin treatment
the abundance of hypophosphorylated pRB increased, reaching a more than
2-fold increase after 18 h ORG 31710 treatment, and there was a
concomitant decrease in the abundance of ppRB (Fig. 2
and data not
shown). On average, the ppRB/pRB ratio was reduced to
50% of
control at 1224 h (Fig. 2B
).

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Figure 2. Effect of Antiprogestins on pRB Phosphorylation
Exponentially proliferating T-47D human breast cancer cells were
treated with RU 486 (100 nM) or ORG 31710 (10
nM), and whole cell lysates were prepared at intervals.
Equal amounts of protein were separated by SDS-PAGE, transferred to
nitrocellulose, and blotted using a monoclonal antibody specific for
pRB. A, Representative Western blot. Hyperphosphorylated pRB (ppRB)
exhibits reduced electrophoretic mobility compared with
hypophosphorylated pRB (pRB). B, The ratio between the optical
densities of the ppRB and pRB bands is presented relative to the
average of vehicle-treated controls. Data represent mean ±
SEM from a total of six experiments. Control (X); RU 486
( ); ORG 31710 (). Ratio is significantly less than control;
P < 0.02 for 924 h RU 486 treatment;
P = 0.05 for 9 h ORG 31710 treatment; and
P < 0.01 for 12 or 18 h ORG 31710
treatment.
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To identify changes in CDK activity that might be responsible for the
decreased pRB phosphorylation, immunoprecipitates from cells treated
with antiprogestin were used in kinase assays in vitro.
Cyclin D1-associated kinase activity was measured using a pRB fusion
protein substrate. The specificity of this assay has been demonstrated
as follows: kinase activity was inhibited by addition of either
recombinant GST-p16INK4 or GST-p21, was directed toward pRB
fusion proteins but not histone H1, and was increased after ectopic
cyclin D1 expression (Ref. 35 and B. Sarcevic, unpublished data).
Kinase activity decreased significantly to reach a minimum of
50%
after 918 h ORG 31710 treatment (Fig. 3A
). This
decrease, while modest, was statistically significant at 9 and 12
h. Cyclin E-associated kinase activity was measured using histone H1
substrate and was decreased by
60% from 1224 h ORG 31710
treatment (Fig. 3B
). RU 486 treatment led to similar effects on kinase
activity (not shown). These data demonstrated a decrease in
G1 cyclin-associated kinase activity which paralleled
decreased pRB phosphorylation.

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Figure 3. ORG 31710 Effects on Cyclin D1-Associated and
Cyclin E-Associated Kinase Activity
Exponentially proliferating T-47D human breast cancer cells were
treated with ORG 31710 (10 nM), and cell lysates were
prepared at intervals. A, Kinase activity of cyclin D1
immunoprecipitates was measured using a pRB fusion protein substrate. A
representative autoradiogram is shown. Graph shows mean ±
SEM of data pooled from three experiments after background
subtraction as described in the Materials and Methods.
Hatched bar indicates SEM of pooled control
data. Activity is significantly less than control at 9 h
(P = 0.0075) and 12 h (P =
0.046). B, Kinase activity of cyclin E immunoprecipitates was measured
using histone H1 substrate. A representative autoradiogram is shown.
Graph shows mean of data pooled from duplicate
experiments. Range is shown where greater than the size of the symbol
used. Hatched bar indicates SEM of pooled
control data. Activity is significantly less than control from 624 h
(P < 0.05 at 12 and 18 h;
P < 0.0025 at 6 and 24 h).
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Cyclin and CDK abundance were examined by Western blotting to identify
possible mechanisms underlying the changes in CDK activity. Cyclin D1
protein abundance did not decrease after antiprogestin treatment but
rather was either unchanged or slightly increased in abundance (Fig. 4A
), consistent with previous examination of cyclin D1
mRNA after RU486 treatment (12, 19). No change in the abundance of
cyclin E, Cdk4, or Cdk6 was detected (Fig. 4
), and there was at most a
minor decrease in Cdk2 abundance (not shown). Since regulation of the
abundance of these cyclins and CDKs did not appear to contribute to
decreased kinase activity CDK inhibitor expression was next examined.
No change in the abundance of p27 was detected, but p21 levels
increased markedly after 12 h or more antiprogestin treatment
(Fig. 4B
). Quantification of data from multiple experiments showed an
average 4- to 5-fold increase after ORG 31710 treatment and a slightly
smaller increase of >3-fold after RU 486 treatment (Fig. 4C
). To
determine the basis for the increase in p21 abundance Northern analysis
was performed after ORG 31710 treatment. Within 6 h treatment p21
mRNA increased by
3-fold and the increase was maintained at 18
h (Fig. 5
), indicating that antiprogestin regulation of
p21 mRNA levels is one cause of the increase in p21 protein levels.

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Figure 4. Antiprogestin Effects on Cyclin, CDK, and CDK
Inhibitor Abundance
Exponentially proliferating T-47D human breast cancer cells were
treated with RU 486 (100 nM) or ORG 31710 (10
nM), and cell lysates were prepared at intervals. Equal
amounts of protein were separated by SDS-PAGE and transferred to
nitrocellulose (A, B). Data in panel B were obtained by sequential
incubation of the same blots with the indicated primary antibodies. C,
Graph presents densitometric analysis of Western blots as the mean ±
SEM of three to five experiments. RU 486 ( ); ORG 31710
(). The abundance of p21 is significantly increased from 624 h RU
486 treatment (P < 0.05) and from 1224 h ORG 31710
treatment (P < 0.02).
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Figure 5. ORG 31710 Effects on p21 mRNA Expression
Exponentially proliferating T-47D human breast cancer cells were
treated with ORG 31710 (10 nM) for the indicated times and
total RNA was extracted for Northern analysis.
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To ascertain whether the abundance of p21 in cyclin immunoprecipitates
increased as its abundance increased, cyclin D1 and cyclin E
immunoprecipitates were examined. Cyclin D1 immunoprecipitates from
cells treated with antiprogestin for up to 24 h contained cyclin
D1, Cdk4, and p27 in amounts similar to those in control cells (data
not shown). The amount of p21 coimmunoprecipitating with cyclin D1 was
increased after
12 treatment (Fig. 6A
), to an average
of
3-fold relative to control. Cyclin E immunoprecipitates contained
similar amounts of cyclin E and Cdk2 in control and
antiprogestin-treated cells, but the amount of coimmunoprecipitated p21
increased by 2- to 3.3-fold after 1224 h ORG 31710 treatment (Fig. 6B
), when the kinase activity was decreased (Fig. 3B
). Overall, these
data are consistent with the interpretation that increased p21
abundance contributes to the reduction in cyclin-associated kinase
activity after antiprogestin treatment.

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Figure 6. ORG 31710 Effects on Cyclin D1 and Cyclin E Complex
Composition
Exponentially proliferating T-47D human breast cancer cells were
treated with ORG 31710 (10 nM) and cell lysates were
prepared. A, Cyclin D1 immunoprecipitates immunoblotted for p21. B,
Cyclin E immunoprecipitates were immunoblotted sequentially for p21 and
Cdk2.
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To determine whether antiprogestin induction of p21 was always
associated with inhibition of proliferation the effects of
antiprogestin treatment were examined in several breast cancer cell
lines expressing a range of progesterone receptor levels. MDA-MB-231
cells are progesterone receptor negative, while MCF-7 cells express
9.1 x 104 sites per cell and BT 474 cells express
4.5 x 105 sites per cell, in comparison with T-47D,
which express 2.3 x 106 sites per cell (36). In MCF-7
or MDA-MB-231 cells no significant effect of antiprogestin was observed
on the S phase fraction after 18 h treatment, when maximal effects
were observed in T-47D cells (Fig. 7A
). Similarly, there
was no effect on cyclin D1, p21, or p27 abundance, nor was there any
alteration in the amount of p21 coimmunoprecipitating with cyclin D1
(Fig. 7
, B and C). In BT 474 no effect on S phase was observed after
15 h treatment but a significant, albeit small, effect was
observed after
30 h (Fig. 7A
). This delay in action is likely a
reflection of the longer doubling time of these cells,
3 days
compared with 11.5 days for the other three cell lines. Concomitant
with the decrease in S phase fraction, there was an approximately
3-fold increase in p21 abundance but no change in the abundance of
cyclin D1 or p27 (Fig. 7B
). Cyclin D1 immunoprecipitates indicated
increased p21 bound to cyclin D1 after ORG 31710 treatment of BT 474
(Fig. 7C
). However, the level of p21 in cyclin D1 immunoprecipitates
from treated BT 474 cells was still markedly lower than that in
immunoprecipitates of a similar amount of cyclin D1 from T-47D cells.
These data indicate an association between antiprogestin induction of
p21 and inhibition of cell proliferation.

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Figure 7. ORG 31710 Effects on MCF-7, MDA-MB-231, and BT 474
Cells
Exponentially proliferating MCF-7, MDA-MB-231, and BT 474 human breast
cancer cells were treated with vehicle or ORG 31710 (100
nM) for 18 h (MCF-7, MDA-MB-231) or 30 h (BT 474)
unless otherwise indicated. A, S phase fraction was determined by flow
cytometry. BT 474 data represent mean ± SEM of flasks
harvested after 2439 h treatment in two independent experiments. B,
Cell lysates were prepared for immunoblotting. For each cell line, data
were obtained by sequential incubation of a single filter with the
indicated primary antibodies. C, Cyclin D1 immunoprecipitates
immunoblotted for cyclin D1, p21, and Cdk4. Exponentially growing T-47D
cells were immunoprecipitated and blotted in parallel for comparison.
The amount of cellular protein immunoprecipitated was adjusted for each
cell line to yield comparable amounts of immunoprecipitated cyclin D1.
Data were obtained by sequential incubation of one filter with the
indicated primary antibodies.
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The data presented above suggest that decreased CDK activity
after antiprogestin treatment might result from induction of p21 and
subsequent association with cyclin/CDK complexes. However, the effect
of the observed increase in p21 on kinase activity is likely to depend
on the initial p21 occupancy of the cyclin/CDK complexes.
Immunoblotting of cyclin D1 immunoprecipitates in parallel with cyclin
D1-depleted lysate and mock-depleted lysate revealed that approximately
a third of the total cellular p21 was associated with cyclin D1 in
T-47D cells (Fig. 8A
), suggesting that the cyclin D1
complexes in control cells contained significant amounts of p21. To
assess the degree of saturation of the complexes, the effect of adding
recombinant GST-p21 fusion protein to a constant amount of cyclin D1
immunoprecipitate from exponentially proliferating cells was
determined. Recruitment of GST-p21 into the cyclin D1 complexes
increased in proportion to the amount of GST-p21 added until the total
p21 (the sum of the endogenous p21 and GST-p21) reached approximately 3
times the initial abundance (Fig. 8
, B and C). However, increasing the
amount of added GST-p21 by 10-fold led to a relatively modest further
increase in the total p21 bound (Fig. 8
, B and C). Thus the cyclin D1
complexes became saturated with p21 upon a 4-fold increase in
associated p21. These data indicate that the
3-fold relative
increase in cyclin D1-associated p21 after antiprogestin treatment is
likely to be sufficient to account for the observed decrease in kinase
activity.

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Figure 8. Association of p21 with Cyclin D1 Complexes
A, Precleared lysates of exponentially proliferating T-47D cells were
immunoprecipitated using either cyclin D1 antiserum linked to protein A
beads (D1) or protein A beads alone (Con). Aliquots of the supernatants
(Sup.) or immunoprecipitated protein (Pellet) were immunoblotted. Each
lane contains protein derived from an equal amount of cellular protein.
B and C, Increasing amounts of recombinant GST-p21 fusion protein were
added to cyclin D1 immunoprecipitates of 750 µg lysate from
exponentially proliferating T-47D cells. After 1 h at 30 C to
allow association, the resulting complexes were immunoblotted (B). The
intensities of the endogenous and exogenous p21 bands were added to
yield total cyclin D1-associated p21, which is presented relative to
the amount of endogenous p21 (C).
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Since the activity of both cyclin D1- and cyclin E-associated
CDKs was decreased, further experiments were designed to test the
relative contributions of these responses to growth inhibition. T-47D
cells transfected with a metal-responsive cyclin D1 construct, T-47D
MTcycD1-3 (32, 35), were used to examine the effects of increased
cyclin D1 abundance on the response to antiprogestins. After zinc
treatment of these cells cyclin D1 protein abundance increases within
3 h and reaches maximum levels by 69 h (32). The resulting
acceleration of cells through G1 phase leads to an increase
in the proportion of cells in S phase, which is maximal at 1524 h
(32). Since zinc induction of cyclin D1 and antiprogestin induction of
p21 occur over a similar time course, the cyclin D1/p21 ratio would be
expected to remain relatively constant after simultaneous treatment
with ZnSO4 and antiprogestin. The increase in cyclin D1
abundance after zinc treatment was unaffected by simultaneous treatment
with ORG 31710 (Fig. 9A
). ORG 31710 treatment reduced
the S phase fraction relative to untreated control cells when cyclin D1
levels were increased by up to
2-fold (i.e. zinc
concentrations of 30 µM or below) (Fig. 9B
). A 2.5-fold
increase in cyclin D1 abundance after treatment with 40
µM zinc prevented ORG 31710 treatment from decreasing the
S phase fraction relative to untreated control cells, while a further
increase in cyclin D1 abundance led to increased S phase despite the
presence of antiprogestin (Fig. 9B
). In the same experimental design,
treatment of parental T-47D cells with up to 50 µM zinc
did not significantly increase either cyclin D1 abundance or S phase
fraction.
The S phase fraction of T-47D
MTcycD1-3 cells treated with zinc
together with antiprogestin was consistently lower than that of cells
treated with zinc in the absence of antiprogestin treatment, despite
equivalent cyclin D1 levels (Fig. 9
, A and B). However, consistent with
previous data (32), cyclin D1 abundance and S phase fraction were
linearly related in both cases (Fig. 9C
). Lines of best fit generated
by linear regression were essentially parallel, with slopes of 1.52 and
1.59 for control and ORG 31710, respectively (Fig. 9C
), i.e.
in either the presence or absence of antiprogestin a given absolute
increase in cyclin D1 abundance led to the same absolute increase in S
phase. Thus, the effect of antiprogestin treatment was to increase the
amount of cyclin D1 necessary to achieve a particular S phase value, as
would be expected if the threshold for cyclin D1 function had been
increased as a result of p21 induction after antiprogestin
treatment.
 |
DISCUSSION
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The cell cycle-phase specific effects of steroids and steroid
antagonists (10) suggest that their molecular targets include genes
involved in the regulation of cell cycle progression through
G1. Cyclin D1 is steroid-regulated (Refs. 17, 19, 38, and
39 and O. W. J. Prall, B. Sarcevic, E. A. Musgrove, C. K. W. Watts, and
R. L. Sutherland, submitted), and recent data argue that activation of
other G1 cyclins also contributes to steroid-induced
mitogenesis (Ref. 39 and O. W. J. Prall, B. Sarcevic, E. A. Musgrove,
C. K. W. Watts, and R. L. Sutherland, submitted). However, decreased
cyclin D1 expression does not always accompany inhibition of breast
cancer cell proliferation (18, 19). This manuscript further
investigated the effects of antiprogestins and demonstrates that while
regulation of cyclin/CDK function is likely to account for their
inhibition of cell cycle progression, this is not mediated by
regulation of cyclin abundance.
Accumulation of underphosphorylated pRB was coincident with the
decrease in S phase fraction. Both were first evident at 9 h and
reached a minimum after 18 h ORG 31710 treatment. These responses
were preceded slightly by decreased cyclin D1- and cyclin E-associated
kinase activity, which reached a minimum by 12 h. Since pRB is a
key substrate for G1 cyclin-associated kinases, these data
suggest that decreased pRB phosphorylation results from decreased CDK
activity and that it is the presence of growth-inhibitory,
underphosphorylated, pRB that ultimately mediates G1
arrest. Investigation of possible mechanisms underlying the decrease in
CDK activity revealed that the abundance of the CDK inhibitor p21 was
increased by 3- to 4-fold after 1224 h treatment, when decreased
kinase activity was observed. Antiprogestin induction of p21 mRNA was
also observed, suggesting that the increased protein abundance was, at
least in part, a result of either increased transcription or
stabilization of p21 mRNA. Induction of p21 was not observed in either
a progesterone receptor-negative cell line (MDA-MB-231) or a
progesterone receptor-positive MCF-7 variant that is insensitive to
antiprogestins. However, it was observed in BT 474 cells accompanying
an antiprogestin-induced decrease in S phase fraction, indicating a
close correspondence between p21 induction and growth inhibition.
Regulation of the abundance of cyclin D1, Cdk4, Cdk6, or p27 did not
appear to contribute to the decreased kinase activity, nor did the
relative Cdk4 abundance in cyclin D1 immunoprecipitates alter after
antiprogestin treatment (our unpublished data). The latter observation
argues against the induction of p16INK4 or a related CDK
inhibitor after antiprogestin treatment because this would be expected
to displace Cdk4 from the complexes (24). Furthermore, preliminary
examination of cyclin D1 immunoprecipitates from
35S-methionine-labeled T-47D cells did not provide evidence
for alterations in complex composition other than increased p21
association after antiprogestin treatment (our unpublished data).
Similarly, decreases in the abundance of cyclin E or Cdk2 sufficient to
account for the decrease in cyclin E-associated kinase activity were
not observed, but the relative abundance of p21 increased in cyclin E
immunoprecipitates at times when kinase activity was reduced.
In a variety of cell types, ectopic expression of p21 leads to arrest
in G1 phase (26, 40, 41), consistent with the hypothesis
that induction of p21 might be responsible for the G1 phase
arrest after antiprogestin treatment. Further experiments indicated
that the observed increase in p21 abundance was likely to be sufficient
to account for the decrease in cyclin D1-associated kinase activity.
Although addition of p21 to recombinant cyclin D1/Cdk4 complexes
inhibits kinase activity in a concentration-dependent fashion, the
complexes retain near-maximal kinase activity in the presence of p21
levels representing 2550% of the level required to completely
inhibit kinase activity (42). Although the precise details of the p21
binding required for inhibition remain undefined, it is apparent that
kinase activity is likely to decrease as the complexes approach
saturation. In untreated T-47D cells a third of the total cellular p21
coimmunoprecipitated with cyclin D1 (Fig. 8
), consistent with data from
other cell types (e.g. Ref.43). This observation suggested
that the cyclin D1 complexes contained p21 at a level near that
required for inhibition of kinase activity. Addition of recombinant
GST-p21 to cyclin D1 immunoprecipitates indicated that saturation of
the cyclin D1 complexes with p21 would occur after an approximately
4-fold increase in bound p21 (Fig. 8
) and thus that the observed
3-fold increase in p21 coimmunoprecipitating with cyclin D1 was
likely to be sufficient to decrease cyclin D1-associated kinase
activity. The observation that the threshold for cyclin D1 function was
increased after antiprogestin treatment (Fig. 9
) is consistent with
this interpretation since the abundance of CDK inhibitors is thought to
set this threshold (24). Although the degree of induction of p21 in BT
474 was similar to that in T-47D, there was more Cdk4 and less p21
coimmunoprecipitated with cyclin D1 in BT 474, even after antiprogestin
induction of p21 (Fig. 7C
), suggesting a greater proportion of active
complexes. This could then contribute to the modest level of the
decrease in BT 474 S phase fraction after ORG 31710 treatment.
Because the activity of both cyclin D1 and cyclin E is required for
progress into S phase (44, 45), inhibition of either could account for
growth inhibition after antiprogestin treatment. However, despite the
antiprogestin-induced decrease in both cyclin D1-associated and cyclin
E-associated kinase activity, a 2.5-fold increase in cyclin D1
abundance alone prevented antiprogestin inhibition of cell cycle
progression, apparently overriding effects on cyclin E-associated
kinase activity. Thus, inhibition of cyclin D1-associated kinase
activity appears to be a critical element in antiprogestin inhibition
of cell cycle progression. An implication of this conclusion is that
sensitivity to inhibition by antiprogestins may, in part, depend on the
abundance of cyclin D1 and hence that the significant fraction,
3050%, of breast cancers that overexpress cyclin D1 (31, 46, 47, 48, 49) may
display altered sensitivity to antiprogestin therapy.
Occupancy of cyclin E complexes by p21 was not investigated in such
detail as that of cyclin D1 complexes, but the correspondence between
increased p21 binding and decreased kinase activity is consistent with
increased p21 abundance contributing to the inhibition of cyclin
E-associated kinase activity. However, it does not exclude other
mechanisms, including alterations in the level or activity of the
kinases and phosphatases controlling the level of Cdk2 phosphorylation
and hence its activity. Furthermore, there is increasing evidence for
growth-inhibitory effects of p21 not mediated via direct inhibition of
CDK activity but rather by interaction with other cell cycle-regulatory
pathways. For example, the activity of E2F, a transcription factor with
a central role in cell proliferation, is repressed by p21 (50, 51). A
possible mechanism is suggested by p21 disruption of the interaction
between Cdk2, E2F, and the pRB-related proteins p107 and p130 (50, 51, 52),
and this has been suggested to play a role in the inhibitory function
of p21 (51). In addition, p21 binds the proliferating cell nuclear
antigen (PCNA), blocking its ability to activate DNA polymerase
,
and the PCNA-binding domain alone is capable of blocking cell cycle
progression (24). Since the PCNA-binding domain is a less effective
growth inhibitor than the CDK-inhibitory domain (24), it is unlikely
that inhibition of DNA replication by this mechanism makes a major
contribution to antiprogestin inhibition of proliferation. Finally, p21
has recently been shown to inhibit the activity of kinases other than
CDKs, i.e. the stress-activated protein kinases (also known
as the c-Jun amino-terminal kinases) and protein kinase CK2 (53, 54),
and it is conceivable that such inhibition also contributes to its
growth-inhibitory effects.
The effects of antiprogestin on breast cancer cells are not limited to
inhibition of cell proliferation. Other responses including induction
of differentiation and apoptosis are apparent after several days
antiprogestin treatment (55, 56). Although both differentiation and
apoptosis can occur in the absence of p21 induction (24), increased p21
has been associated with differentiation in a number of cellular
systems both in vivo and in vitro (24) and with
retinoid induction of apoptosis in breast cancer cells (57).
Overexpression of p21 in human melanocytes led to morphological changes
characteristic of differentiation and increased melanin production, in
some cases followed by cell death (41), while overexpression in MCF-7
and T-47D breast cancer cells led to apoptosis (58). These data suggest
that p21 induction could contribute to antiprogestin effects other than
growth arrest alone.
In summary, the data presented in this manuscript suggest a model for
the effects of antiprogestin treatment on proliferation in which
decreased activity of both cyclin D1/CDK and cyclin E/CDK and
consequent decreased pRB phosphorylation result in inhibition of entry
into S phase. Decreased kinase activity does not result from regulation
of cyclin abundance but induction of the CDK inhibitor p21 accompanies
these changes and is thus a likely mediator of the effects of
antiprogestins on cell proliferation.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
The human breast cancer cells were obtained from the following
sources: T-47D and MDA-MB-231, EG & G Mason Research Institute
(Worcester, MA); MCF-7, Michigan Cancer Foundation (Detroit, MI); BT
474, American Type Culture Collection (Rockville, MD). T-47D
MTcycD1-3 are a clonal derivative of T-47D expressing ectopic cyclin
D1 under the control of a metal-inducible truncated human
metallothionein IIA promoter lacking steroid-responsive sequences (32, 35). RPMI 1640 medium was supplemented with 10 µg/ml human insulin
(Actrapid, CSL-Novo, North Rocks, NSW, Australia), HEPES (20
mM), sodium bicarbonate (14 mM), and
L-glutamine (6 mM). Stock cultures were
maintained as previously described (36), in medium supplemented with
10% FCS and without antibiotics. Experiments used cells cultured in
medium supplemented with 5% FCS. RU 486 (generously provided by Dr J-P
Raynaud of Roussel-Uclaf, Romainville, France) and ORG 31710
(generously provided by Dr W. Schoonen, Organon, Oss, The Netherlands)
were dissolved in ethanol at 1000- or 2000-fold final concentration and
added to cells in exponential growth. Control cultures received ethanol
to the same final concentration. Data presented are representative of
at least two experiments. Cell cycle phase distribution was determined
by flow cytometry (59).
Antiprogestin effects on cell number were measured using a colorimetric
cell proliferation assay (CellTiter, Promega, Madison, WI). Cells
(103) were seeded into 96-well plates, and the next day
antiprogestin or vehicle was added to quadruplicate wells for each
treatment. Plates were assayed at intervals and relative absorbances,
i.e. relative cell numbers, were determined near the end of
exponential growth for control cultures, after 67 days exposure to
antiprogestin.
Recombinant p21
The coding region of p21 was amplified by PCR using pZL.WAF1
(60) as a template and p21N (TAC ATG GAT CCA TGT CAG AAC CGG CTG GGG A)
and p21C (AGA CTG AAT TCT TAG GGC TTC CTC TTG GAG A) as primers. The
resulting fragment was cloned into the
BamHI/EcoRI sites of the expression vector
pGEX-2t (Pharmacia, Uppsala, Sweden) to yield pGEX-p21. To prepare
GST-p21, Eschericia coli were transformed with pGEX-p21, and
expression of the protein was induced by incubation with 0.1
mM isopropylthioglycoside) (2.5 h, room temperature).
Frozen cell pellets were lysed by sonication at 4 C after resuspension
in PBS with protease inhibitors (1 mM
phenylmethylsulfonylfluoride, 0.5 M EDTA, 0.05% (vol/vol)
ß-mercaptoethanol, 10 µg/µl aprotonin, 10 µg/µl leupeptin).
After addition of 0.5% Triton X-100, the lysate was centifuged at 4 C.
The supernatant was then incubated with gentle rotation at 4 C for
1 h with 0.5 ml of a 50% (vol/vol) suspension of
glutathione-agarose (Sigma, St.Louis, MO). The resin was washed once
with ice-cold PBS/0.05% ß-mercaptoethanol/0.5% Triton and twice
with ice-cold PBS/0.05% ß-mercaptoethanol and finally resuspended in
0.5 ml ice-cold PBS/0.05% ß-mercaptoethanol/0.1% azide. The fusion
protein was then eluted with 50 mM Tris-Cl, pH 8.0, 15
mM reduced glutathionine, 0.05% ß-mercaptoethanol, and
the purity of fusion protein was assessed by PAGE followed by Coomassie
blue staining.
Cell Lysis, Western Blot Analysis, and Immunoprecipitation
Cells were lysed either as described below for cyclin
D1-associated kinase assays or as previously described, using lysis
buffer consisting of 50 mM HEPES (pH 7.5), 150
mM NaCl, 10% (vol/vol) glycerol, 1% Triton X-100, 1.5
mM MgCl2, 1 mM EGTA, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, 200
µM sodium orthovanadate, 10 mM sodium
pyrophosphate, and 100 mM NaF (32, 61). Similar results
were obtained from Western blotting or immunoprecipitation using either
lysis technique.
Cell lysates were precleared by incubation with protein A-Sepharose
beads (Zymed, San Francisco, CA) (1 h, 4 C) then immunoprecipitated by
incubation (3 h, 4 C) with protein A-Sepharose beads that had been
conjugated with either an anti-cyclin E antibody (C-19, Santa Cruz
Biotechnology, Santa Cruz, CA) or rabbit polyclonal anti-cyclin D1
serum [raised against a human cyclin D1-GST fusion protein (35)]. In
some experiments the antibodies were chemically cross-linked to the
protein A-Sepharose beads by incubation in 5 mg/ml dimethyl
pimelimidate/0.2 M sodium tetraborate (pH 9.0) for 30 min
at room temperature, essentially as described (62). The
immunoprecipitated proteins were then washed as previously described
(35). In the experiments presented in Fig. 8
, recombinant GST-p21 was
added to the immunoprecipitates, and the samples were incubated at 30 C
for 1 h with vortexing every 10 min. The beads were then washed
with 50 mM HEPES (pH 7.5), 1 mM dithiothreitol
before resuspension in SDS-PAGE sample buffer.
Samples of immunoprecipitated or total protein in SDS-PAGE sample
buffer were heated to 95 C for 3 min, then separated by SDS-PAGE and
transferred to nitrocellulose. Specific proteins were visualized by
chemiluminescence (Dupont NEN, Boston, MA) after incubation (24 h at
room temperature or overnight at 4 C) with the following primary
antibodies: p21 antiserum kindly provided by Dr David Beach (Cold
Spring Harbor, NY); cyclin E (HE12), Cdk2 (M2), Cdk4 (C-22), and Cdk6
(C-21) antibodies from Santa Cruz Biotechnology; cyclin D1 antibody
(DCS6) from Novocastra, Newcastle-upon-Tyne, U.K.; pRB (14001A)
antibodies from Pharmingen (San Diego, CA); p21 (C24420) and p27
(K25020) antibodies from Transduction Laboratories (Lexington, KY).
Relative abundance was quantitated using a Molecular Dynamics
(Sunnyvale, CA) densitometer and IP LabGel analysis software (Signal
Analytics, Vienna, VA).
Kinase Assays
The histone H1 kinase activity of cyclin E immunoprecipitates
was measured as previously described for Cdk2 assays (35) using 10 µg
histone H1 as substrate. For cyclin D1-associated kinase assays, cells
were harvested and lysed as previously described using kinase lysis
buffer [50 mM HEPES (pH 7.5), 1 mM
dithiothreitol, 150 mM NaCl, 1 mM EDTA, 2.5
mM EGTA, 0.1% Tween-20, 10% glycerol, 10 mM
ß-glycerophosphate, 1 mM NaF, 0.1 mM sodium
orthovanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.1
mM PMSF] (35, 63). Kinase activity of cyclin D1
immunoprecipitates of these lysates was measured using either a
pRB(379928)-maltose binding protein fusion protein, GST-pRB(769921)
fusion protein substrate (Santa Cruz) or GST-pRB(773928) (64) as
previously described (35). After termination of kinase reactions,
samples were incubated at 90 C for 2 min in SDS sample buffer and
separated using 10% SDS-PAGE. Relative intensities were quantitated
using a Molecular Dynamics PhosphorImager Scanner (model 445 SI)
followed by analysis using IP LabGel analysis software (Signal
Analytics) or in some cases after exposure to x-ray film as decribed
above for Western analysis. The degree of background phosphorylation in
cyclin D1-associated kinase assays was estimated from parallel control
samples either immunoprecipitated using preimmune serum or assayed
after incubation of immunoprecipitates with an excess of GST-p21 (30
min, 30 C) and has been subtracted in the data presented in Fig. 3A
.
RNA Isolation and Northern Analysis
Total RNA was extracted (using a guanidinium
isothiocyanate-cesium chloride procedure) and blotted as previously
described, using 20 µg total RNA/lane (46). The membranes were
hybridized overnight at 50 C in 50% (vol/vol) formamide, 2x SSPE (0.3
M NaCl, 20 mM NaH2PO4,
2 mM EDTA, pH 7.4), 1% (wt/vol) SDS, 0.5% (wt/vol) low
fat skim milk (Diploma, St Kilda, Victoria, Australia), 10% (wt/vol)
dextran sulfate (Mr 500,000), 200 µg/ml yeast RNA, 40
µg/ml polyadenylic acid (5'), 500 µg/ml salmon sperm DNA. The
2.1-kb p21 cDNA from pZL.WAF1 (60) was labeled with
[
-32P]dCTP (Amersham Australia, North Ryde, New South
Wales, Australia; specific activity
3000 Ci/mmol) to a specific
activity of approximately 1 x 109 cpm/µg DNA using
the Multiprime DNA labeling kit (Amersham Australia) then added to the
hybridization mix at a final concentration of
10 ng/ml. The
membranes were washed at a highest stringency of 0.2 x SSC (30
mM NaCl, 3 mM sodium citrate, pH 7.0), 1% SDS
at 65 C and exposed to Kodak X-OMAT film at -70 C. Equivalent RNA
loading was verified as previously described (46) by hybridizing
membranes with a [
-32P]ATP end-labeled oligonucleotide
complementary to 18S rRNA.
Statistical Analysis
Data pooled from multiple experiments were analyzed using
Statview II software (Abacus Concepts, Inc, Berkeley, CA). The
significance of differences from control was determined using a
one-tailed t test.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Drs David Beach and Jiri Lukas for supplying
antibodies.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Elizabeth A. Musgrove, Cancer Research Program, Garvan Institute of Medical Research, St. Vincents Hospital, Sydney, New South Wales 2010 Australia.
This study was supported by research grants from the National Health
and Medical Research Council of Australia and the New South Wales State
Cancer Council.
Received for publication September 16, 1996.
Accepted for publication October 3, 1996.
 |
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