CDK2 Is a Target for Retinoic Acid-Mediated Growth Inhibition in MCF-7 Human Breast Cancer Cells
Christine Teixeira and
M. A. Christine Pratt
Department of Pharmacology University of Ottawa Ottawa,
Ontario, Canada, K1H 8M5
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ABSTRACT
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Retinoic acid (RA) inhibition of breast cancer
cell growth is associated with an accumulation of cells in G1 phase of
the cell cycle. We have investigated the effects of RA on the
expression and activity of cell cycle-regulatory proteins in MCF-7
human breast cancer cells. Flow cytometry analysis of MCF-7 cells
treated with RA revealed a decrease in the percentage of cells in S
phase by 48 h, which was maximal by 72 h. Phosphorylation of
the retinoblastoma protein (pRb) was partially reduced in RA-treated
cells accompanied by a decrease in the level of retinoblastoma protein.
Expression of the cyclin D1 transcript was reduced by 48 h and
cyclin-dependent kinase 2 (cdk2) mRNA levels declined within
8 h posttreatment followed by a decrease in cyclin D1 and cdk2
protein levels. Message and protein levels of cdk4 and cdc2 were
not affected by RA. While cdk4 activity was similar in control and
RA-treated cells, cdk2 activity began to decrease within 48 h of
exposure to RA and was profoundly reduced after 72 h. This reduced
activity was associated with decreased phosphorylation of cdk2. The
decrease in cdk2 activity occurred in the absence of RA-mediated
increases in the levels of the cdk inhibitors p21and p27. However,
assays of cdk2 from pooled lysates from RA-treated and control cells
showed that RA-treated cells contain a cdk2-inhibitory activity. Our
results show that RA inhibits cell cycle progression of MCF-7 cells by
inhibiting cdk2 mRNA and protein production and by decreasing cdk2
activity.
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INTRODUCTION
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The two retinoid isomers, all-trans-retinoic acid (RA)
and 9-cis-RA signal through nuclear receptors [retinoic
acid receptors (RARs) and retinoid X receptors] that act as
ligand-inducible transcription factors on target genes containing
RA-response elements in their regulatory regions (1, 2). Retinoids
influence growth and differentiation in a wide variety of cell types
(3, 4). Among those cells that are growth inhibited by RA are a subset
of breast cancer cells that are estrogen receptor (ER) positive. In
most instances, ER-positive breast cancer cells such as MCF-7 cells
also express RARs whereas ER-negative cells do not (5, 6). We (7) and
others (8, 9) have shown that RA inhibits activation of the ER in
breast cancer cells. Using transient transfection of MCF-7 cells, we
have shown that this inhibition involves transcriptional interference
through the RAR AF-2 activation domain (7). However, this is unlikely
to be the sole mechanism by which RA inhibits the growth of breast
cancer cells since this interference is incomplete, and ER-negative
cells transfected with the RAR become sensitive to RA-induced growth
inhibition (10, 11).
Past studies have shown that RA causes breast cancer cells to
accumulate in G1 of the cell cycle (12). Cyclins and cyclin-dependent
kinases (cdks) play a central role in regulation of the cell cycle in
eukaryotic cells (13, 14). Cyclins are positive regulatory subunits for
cdks, and together they form active complexes that phosphorylate
substrates involved in cell cycle progression. The D-type cyclins and
cyclin E, in association with cdk4, cdk6, and cdk2, govern progression
through G1 (15, 16). The activity of cyclin:cdk complexes is subject to
positive and negative regulation. Both cyclin binding and
phosphorylation by the cdk-activating kinase (cak) are required for
activation whereas inhibitory phosphates are removed by members of the
cdc25 phosphatase family (17, 18, 19). Recently, several new inhibitor
proteins have been identified that bind to and negatively regulate
either cyclin D-cdk4 complexes (p15,p16,p18,p19) (20, 21, 22, 23) or
inhibit both cdk2 and cdk4-cyclin complexes
(p27Kip1,p21,p57) (24, 25, 26, 27, 28, 29, 30, 31). A major function of cdk4
complexed with D cyclins is the phosphorylation of the tumor suppressor
protein, retinoblastoma (pRb) (32, 33, 34, 35). Phosphorylation of pRb prevents
the binding of members of the E2F/DP family of transcription factors
(36, 37, 38, 39). Free E2F/DP proteins act by promoting the transcription of
genes whose products facilitate progression through G1 (40). Cdk2 has
also been implicated in the phosphorylation of pRb (41) and is required
for the G1 to S phase transition (42) and DNA synthesis (43).
In this report we demonstrate that RA induces accumulation of MCF-7
cells in G1 of the cell cycle. These cell cycle effects are accompanied
by a decrease in pRb phosphorylation. Both cdk2 mRNA and protein levels
were decreased by RA treatment of MCF-7 cells associated with a
profound decrease in cdk2 activity. This occurred in the absence of
alterations in p27 and p21 levels although cdk2-inhibitory activity was
present in extracts from RA-treated cells. We conclude that RA induces
accumulation of breast cancer cells in G1 phase, at least in part, as a
result of decreased expression and inhibition of cdk2 activity.
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RESULTS
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Effect of RA on Cell Cycle Phase Distribution
While numerous studies have shown that RA inhibits the growth of
MCF-7 cells (44, 45), it has been suggested that there is some clonal
variation in responsiveness (9, 46). To establish the effects of RA on
our MCF-7 cells, flow cytometric analysis was performed on control and
RA-treated populations. The cells were partially synchronized by
contact inhibition to help clarify RA effects on specific phases of the
cell cycle. After release from contact inhibition, cells were plated in
the presence or absence of 1 µM RA and harvested at the
indicated intervals. Table 1
shows that almost 70% of
contact-inhibited (CI) cells were in G0/G1 phase. Both RA-treated and
control cells showed similar phase distribution over the initial 24-h
period after release. However, by 48 h the percentage of
RA-treated cells in S phase began to decline compared with control
cells accompanied by a proportional transient increase in G2/M phase
cells. At 72 h after release there was a clear accumulation of
RA-treated cells in G0/G1 accompanied by a decline in the S phase
population, which persisted to the 96-h time point.
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Table 1. Cell Cycle Distribution of CI MCF-7 Cells and
Cells Released from Contact Inhibition in the Presence or Absence of 1
µM RA
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RA Decreases Rb Phosphorylation and Protein Levels in MCF-7
Cells
Since the Rb protein is central to regulation of the cell
cycle we examined the effects of RA on pRb phosphorylation (Fig. 1A
). pRb was present in CI cells in various states of
phosphorylation. At 24 h following release, no difference was
observed between control and RA-treated cells; however, by 48 h,
hypophosphorylated pRb was observed in the RA-treated cells. By 72
h hypophosphorylated pRb was present in both populations, which may
indicate that a subpopulation of untreated cells were leaving M phase
and entering G1. At 96 h post-RA, no hyperphosphorylated forms of
pRb were evident in contrast to control cells, which only contained
hyperphosphorylated forms of pRb. Additionally, there appeared to be
less total pRb at the final time point compared with earlier times. To
determine whether RA acted to simply prevent the semi-synchronized
cells from exiting G1, we performed a similar analysis on asynchronous
cultures of both MCF-7 and T47-D cells. Like MCF-7 cells, this breast
cancer cell line also expresses the ER. The results in Fig. 1B
show a
profound reduction in hyperphosphorylated pRb after RA treatment in
both cell lines accompanied by a decrease in total pRb levels. The
kinetics of this reduction in phosphorylated forms of pRb were more
rapid than in the semi-synchronized cells. Reactivity of the blots with
-actin is shown below to control for total protein levels.

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Figure 1. RA Decreases pRb Phosphorylation and Protein
Levels
A, Western blot analysis of pRb was performed on lysates from CI cells
and at the indicated times after release in the presence or absence of
RA. Immunoreactive protein was detected by chemiluminescence as
described in Materials and Methods. Multiple
phosphorylated species of pRb are indicated by the open
box, and hypophosphorylated pRb is indicated by the
arrow. B, Western blot analysis of pRb was performed on
exponentially growing MCF-7 and T47-D cells after treatment with RA.
Phosphorylated forms of pRb are indicated. Immunoreactivity with an
-actin antibody was used to control for loading of protein.
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RA Decreases Cyclin D1 and cdk2 Gene Expression
To determine whether changes in the gene
expression for cell cycle proteins might account for the RA-induced
changes in cell cycle progression, we performed Northern blot analysis
of RNA from MCF-7 cells released from contact inhibition in the
presence or absence of RA. Figure 2A
shows that cyclin A
mRNA was present in CI cells, increased transiently in control cells,
was reduced at 24 h, and underwent a second increase at 48 h,
which persisted through 96 h. The transient decrease in cyclin A
appeared earlier in RA-treated cells (8 h), and the second increase was
also accelerated, beginning at 24 h. This early decrease in cyclin
A appeared to be without major effects on cell growth since both
control and RA-treated cells were equally distributed in the cell cycle
by 24 h after release. However, the oscillation in cyclin A levels
in both control and RA cells may indicate a differential cell cycle
phase distribution of the cells. Consistent with this, cyclin A levels
in RA and control cells were again similar by 48 h, but by 96 h,
cyclin A was again lower in RA-treated MCF-7 cells. Cyclin D1 mRNA
levels were high in CI cells and were similar in control and RA-treated
cells until 48 h, at which point there was a reduction in the
4.5-kb transcript in the RA-treated cells compared with the control.
The lower (1.5 kb) transcript did not decrease significantly in these
cells.

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Figure 2. Effect of RA on Expression of mRNA for Cyclins and
cdks
Twenty micrograms of total RNA from CI cells and cells released from
contact inhibition in the presence or absence of RA were
electrophoresed and transferred to nylon as described in
Materials and Methods. Two separate RNA blots were
hybridized with cDNA probes for: cyclin A and cyclin D1 (A); cdk2 and
cdk4 (B). C, Twenty micrograms of RNA from exponentially growing MCF-7
cells treated for the indicated times with RA were subjected to
Northern blot analysis of cdk2 mRNA as described above. All blots were
reacted with GAPDH to control for equivalency of loading. D,
Densitometric scan of cdk2 mRNA. The graph depicts the ratio of the
relative density of bands in RA-treated samples in panel B to the GAPDH
control for the same time point.
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Northern blot analysis of the expression of the G1 kinases, cdk2
and cdk4, in Fig. 2B
indicated that while cdk4 transcript levels did
not change over the course of the cell cycle in either control or
RA-treated MCF-7 cells, a decrease in the expression of cdk2 mRNA was
evident within 8 h after RA addition, which persisted over the
course of culture. A densitometric analysis of cdk2 mRNA levels in
treated vs. control cells showed initial oscillations in
mRNA levels that fell to 20% of control levels by 48 h (Fig. 2C
).
To ensure that the effect of RA on cdk2 mRNA was not due to prevention
of exit from G1, we performed Northern blot analysis of RNA from
exponentially growing MCF-7 cells treated with RA for various times.
The result in Fig. 2D
shows that RA produces a similar rapid decrease
in cdk2 mRNA under these conditions. RA had no effect on cyclin E or D3
mRNA expression in MCF-7 cells (data not shown).
RA Decreases Levels of Cyclin D1 and cdk2 Protein in MCF-7
Cells
Since we observed decreases in the mRNA levels for both cyclin D1
and cdk2 in RA-treated cells, we looked for associated changes in
protein levels. Cyclin D1 protein levels correlated well with
expression of the 4.5-kb transcript decreasing within 48 h after
release from contact inhibition in the presence of RA (Fig. 3A
). The p33cdk2 protein levels remained stable for
several hours after RA treatment of MCF-7 cells, but by 48 h the
protein levels decreased by 30% and were further reduced by 80% at
72 h and 96 h after treatment with RA (Fig. 3B
), as
determined densitometrically. By contrast, RA did not alter cdk4
protein levels (Fig. 3C
).

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Figure 3. RA Decreases cdk2 and Cyclin D1 Protein Levels
Fifty micrograms of MCF-7 cell extract from CI cells and cells released
in the presence or absence of RA as described in Materials and
Methods were used for Western blot analysis of cyclin D1 (A);
Western blot analysis of cdk2 using 5 µg cell extract (B); Western
blot analysis of cdk4 using 5 µg cell lysate (C). An antibody to
-actin was used to control for loading equivalency.
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RA Decreases the Phosphorylation of cdk2
To determine whether RA affects the phosphorylation of cdk2, we
metabolically labeled cdk2 with 32Pi in the
presence or absence of RA and analyzed the cdk2 immunoprecpitates by
electrophoresis followed by transfer to a nylon membrane and
autoradiography. Figure 4
is an autoradiogram of
metabolically labeled cdk2, which shows a marked decrease in
phosphorylation of p33cdk2 after a 72-h exposure to RA. Western blot
analysis with cdk2 antibody (shown below) confirmed that the difference
in labeling was not due to differential amounts of immunoprecipitated
cdk2 protein in the treated and untreated lanes since the RA-treated
lysate has been overloaded with respect to the control. Two other
labeled bands, one at 34 kDa and the other at 30 kDa, that
coimmunoprecipitated with cdk2 are evident in the immunoprecipitate.
The 30-kDa band also cross-reacts with the cdk2 antibody on Western
blot analysis.

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Figure 4. Metabolic Labeling of cdk2 with
32Pi
Extracts were prepared from MCF-7 cells 72 h after release from
contact inhibition in the presence or absence of RA. The cells were
metabolically labeled for the last 16 h of culture in the presence
of 32Pi, and the extracts were
immunoprecipitated with a cdk2 antibody, subjected to SDS-PAGE, and
transferred to a PVDF membrane as described in Materials and
Methods. The membrane was exposed to film to detect
32P-labeled cdk2 then subjected to Western blot analysis
with cdk2 antibody to determine total cdk2 protein in the
immunoprecipitate.
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Cdk2 Activity but Not cdk4 Activity Is Inhibited by RA
The decrease in cdk2 protein and phosphorylation predicted that
the activity of cdk2 would be decreased by RA. Therefore, cultures of
CI MCF-7 cells were released in the presence or absence of RA. The
results in Fig. 5A
show that CI MCF-7 cells contain high
levels of cdk2 activity measured by histone H1 phosphorylation.
Twenty-four hours after release from CI, cdk2 activity decreased in
both control and RA-treated cells. By 48 h both control and
RA-treated cells contained increased cdk2 activity, although this
activity was significantly lower in RA-treated cells. Within 72 h,
cdk2 activity was inhibited by 40% in RA-treated cells and dropped to
less than 10% of control levels after 96 h. The decrease in
cyclin D1 protein levels in RA-treated MCF-7 cells suggested that RA
might also inhibit cdk4 activity. We therefore assayed cdk4 activity in
control and RA-treated cells using a glutathione-S-transferase (GST)-Rb
fusion protein as substrate. The lower panel in Fig. 5A
shows the results of this assay and indicates that there was no change
in cdk4 activity in RA-treated cells compared with control cells over
the 96-h treatment period. Figure 5B
is a graph of the densitometric
analysis of the ratio of phosphorylated histone H1 in RA-treated cells
to that in control cells.

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Figure 5. RA Inhibits cdk2 Activity in MCF-7 Cells
A, MCF-7 cell extracts from CI cells or after release for the indicated
times in the presence or absence of RA were prepared as described in
Materials and Methods. For assays of cdk2 and cdk4
kinase activities, cdk2 and cdk4 immunoprecipitates were mixed with
histone H1 or a GST-Rb fusion protein, respectively, in the presence of
-32P-ATP as described in Materials and
Methods. Kinase reactions were subjected to SDS-PAGE followed
by autoradiography. Upper panel, cdk2 phosphorylation of
H1 histone; lower panel, cdk4 phosphorylation of GST-Rb.
B, Densitometric scan of histone H1 phosphorylation expressed as a
ratio of activity in RA-treated to untreated control lysate at each
time point.
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RA Does Not Decrease Levels or Activity of the cdk-Activating
Kinase, cak
To investigate whether the profound decrease in levels of
phosphorylated cdk2 is due to RA modulation of cak (cdk7), the kinase
responsible for the activating phosphorylation of cdk2 (47), we
assessed cak protein levels. Figure 6A
shows that cak
exists primarily as a 37 kDa protein in CI MCF-7 cells, and increasing
amounts of a faster migrating species appeared after release in both
control and RA-treated cells. We then wished to determine whether
decreased cdk2 activity might be due to reduced cak activity in
RA-treated cells. We therefore assayed cak activity in anti-cak
immunoprecipitates using GST-cdk2 fusion protein as a substrate. Figure 6B
shows that CI MCF-7 and released cells grown in the absence or
presence of RA all contained equivalent levels of cak activity. Thus, a
decrease in cak protein levels or activity is not responsible for the
decreased levels of cdk2 phosphorylation in RA-treated cells.

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Figure 6. Effect of RA on cak Protein and Activity
A, Western blot analysis of cak. Extracts from CI MCF-7 cells and after
release in the presence or absence of RA for the indicated times were
used for Western blot analysis of cak. Two immunoreactive bands are
evident in all samples except in CI cells. Equivalency of loading was
assessed by reactivity with an -actin antibody. B, Assay of cak
activity. Extracts from MCF-7 cells cultured as in panel A were
immunoprecipitated with an anti-cak antibody. Immunoprecipitates were
mixed with a GST-cdk2 fusion protein in the presence of
-32P-ATP as described in Materials and
Methods and reactions subjected to SDS-PAGE followed by
autoradiography.
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RA Does Not Alter Levels of Cyclin Inhibitor Proteins
Analysis of TGFß-treated mink lung epithelial cells has shown
that cdk inhibitors play a role in mediating growth inhibition induced
by this factor (48). We have studied the effects of RA on two of these
inhibitors, p21 and p27Kip1, in MCF-7 cells by Western blot
analysis of CI and released cells (Fig. 7
, A and B). p21
and p27Kip1 were both present in CI MCF-7 cells. While the
levels of p27Kip1 did not change in either control or
RA-treated released cells over the 6-day study period, p21 protein
levels were reduced compared with control by 96 h of treatment.
Next we determined whether p21 or p27 levels were altered in
asynchronous cells treated with RA. The results in Fig. 7C
show that
neither p21 nor p27 were increased after exposure to RA. Both p21 and
p27Kip1 can form complexes with cdks to inhibit their
activity. To determine whether RA inhibits cdk2 activity by increasing
the level of these inhibitors complexed with cdk2, we
immunoprecipitated cdk2 protein and performed Western blot analysis of
p21 and p27Kip1 in the immunoprecipitate. The results in
Fig. 7D
indicate that cdk2-associated p21 does not increase but instead
decreases by 72 h in RA compared with control cells, while
p27Kip1 levels remain relatively constant in control and
RA-treated cells. Since a decrease in cdk2-associated cyclin E could
also have an impact on cdk2 activity, we also performed Western blot
analysis of cdk2-associated cyclin E. The third panel in Fig. 7D
shows
that cyclin E levels are unchanged in RA-treated cells compared with
controls. Western blot analysis of immunoprecipitated cdk2 was
performed to control for cdk2 levels in the complexes. The decreased
level of cdk2 immunoprecipitated after 96 h likely reflects the
overall decrease in cdk2.

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Figure 7. Western Blot Analysis of cdk Inhibitors
Extracts from semi-synchronized MCF-7 cells were subjected to Western
blot analysis as described in Materials and Methods.
Blots were incubated with anti-p21 antibody (A) and
anti-p27Kip1 antibody (B). Extracts from exponentially
growing MCF-7 cells were subjected to Western blot analysis of p21 and
p27Kip1 (C). All blots were reacted with an anti- actin
antibody to control for loading equivalency. Western blot analysis of
cdk2-associated p21, p27, and cyclin E (D). Semi-synchronized MCF-7
cells were released in the presence or absence of RA. At the indicated
times, extracts were immunoprecipitated with cdk2 antibody and the
complexes subjected to SDS-PAGE followed by immunoblot analysis with
anti-p21, anti-p27, and anti-cyclin E. The blot was reacted with
anti-cdk2 to control for efficiency of the immunoprecipitation.
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RA-Treated MCF-7 Cells Contain a cdk2-Inhibitory Activity
Cdk-inhibitory activity induced by TGFß has been characterized
by the ability of treated cell lysates to inhibit cdk kinase activity
in control lysates (49, 50). We have thus performed a similar
experiment using RA-treated cell lysates to determine whether an
inhibitor of cdk2 activity is present in these cells. Figure 8
shows that immunoprecipitation of cdk2 from two pooled
control lysates (100 µg each) yielded a high level of histone H1
kinase activity while immunoprecipitation of 100 µg of protein from a
single control lysate produced about half of this activity. As
expected, the activity in RA-treated lysates was much reduced compared
with the control lysate. Strikingly, rather than producing an additive
effect, the mixing of control lysate with RA-treated lysate resulted in
levels of kinase activity similar to those in the RA lysate consistent
with the presence of a cdk2-inhibitory activity in the RA-treated
cells.

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Figure 8. RA-Treated MCF-7 Cells Contain a cdk2-Inhibitory
Activity
Control (C) or RA-treated MCF-7 cell lysates (RA) were
immunoprecipitated with cdk2 antibody (C or RA) or premixed for 1
h at 37 C before immunoprecipitation with cdk2 antibody (C+C or C+RA).
Immunoprecipitates were used to phosphorylate histone H1 in the
presence of 32Pi as described in
Materials and Methods. The reaction mixture was then
subjected to PAGE after which the gel was dried and subjected to
autoradiography.
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DISCUSSION
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Studies of factors that either promote or inhibit cell growth have
shown that individual agents may target specific components of the cell
cycle-regulatory apparatus. Several years ago it was shown that
treatment of MCF-7 cells with RA results in an accumulation of these
cells in G1 of the cell cycle (12). In this study we have attempted to
identify the cell cycle-regulatory targets associated with RA-induced
changes in cell cycle progression in MCF-7 breast cancer cells. RA
treatment of MCF-7 cells results in a decrease in the S phase
population after 48 h, which continues to decrease accompanied by
an increase in the G1 population. Thus the first cell cycle alterations
associated with RA exposure begin at 48 h after treatment and peak
by 72 h. Of the cell cycle components studied, levels of cdk2 and
cyclin D1 mRNA and protein were reduced in this time frame. In
addition, cdk2 activity decreased in RA-treated cells within 48 h
following RA treatment coincident with the initial decrease in the S
phase cell population. These results contrast with the effects of an
antiestrogen on MCF-7 cells, which causes a decrease in the S phase
fraction associated with repression of cdk4 activity (51). The relative
lack of effect on cdk4 activity was somewhat surprising given that
cyclin D1, a G1 partner for cdk4 (52), was reduced in RA-treated cells.
However, cyclin D3 was abundant in both RA-treated and control cells,
and it is possible that it might substitute for cyclin D1 in an active
kinase complex. To this end, cyclin D1 and D3 have both been shown to
elevate cdk4 activity (35).
The observation that RA results in accumulation of underphosphorylated
pRb in both synchronous and asynchronous cells suggests that RA is not
acting to simply prevent semi-synchronized cells from exiting G0/G1 but
rather to cause the accumulation of cells in G1. Furthermore, the
profound effects of RA on pRb phosphorylation in T47-D cells supports
the notion that this is not a cell line-specific effect of RA. There do
appear, however, to be some differences between the cell lines. Wilcken
et al. (53) showed that RA treatment of T47-D cells results
in an inhibition of cdk4 activity and a small decrease in pRb
phosphorylation that was not associated with alterations of cyclin D1
or cdk enzyme levels. The kinetics of RA reduction of the S phase
population in these cells were faster than what we observed in MCF-7
cells with decreases in S phase being evident by 24 h after RA
treatment compared with 48 h in this study. The reasons for the
differences between responses to RA in T47-D and MCF-7 cells are not
clear but may reflect cell-specific responses to RA.
Unlike what has been observed for prostaglandin A2 (54), an agent that
reduces G1 cdk activity in MCF-7 breast cancer cells without alteration
of pRb levels, both antiestrogen (51) and RA treatment (this study)
cause a decrease in pRb that may reflect a lack of requirement for the
Rb protein in maintaining growth inhibition.
The recent discovery of several cdk inhibitors has established another
level of regulation of cdk activity. These proteins have been shown to
function as targets of certain growth inhibitors. For instance
TGFß-treated Mv1Lu mink lung epithelial cells exhibit decreased
phosphorylation and activity of cdk2-cyclin E complexes (49) mediated
by the heat-stable inhibitor, p27Kip1 (50). TGFß appears
to act by increasing the levels of the cdk4 inhibitor,
p15Ink4B, resulting in the release of p27Kip1,
thereby freeing it to bind to cdk2 and inhibit its activity (48). The
response varies with cell type since keratinocytes respond to TGFß by
increasing both p15 and p21Cip1 (51). Although these
inhibitors can prevent cdk activation by cak, they do not inhibit the
activity of cak (30). Kato et al. (55) have shown that
p27Kip1 association with cdk4-cyclin D complexes prevents
cak from phosphorylating cdk4 to activate the enzyme complex in
macrophages arrested in G1 after treatment with cAMP. Extracts from
quiescent mouse mammary epithelial cells also contain
p27Kip1 inhibitory activity (56) while prostaglandin A2-
treated MCF-7 cells undergo growth arrest associated with marked
increases in p21 levels (54). Since p21 can block the phosphorylation
of cdks by cak (57), it was also a possible mediator of RA effects on
cdk2. Surprisingly, RA did not increase levels of either p21 or
p27Kip1 in MCF-7 cells. These effects are unlike those of
an antiestrogen wherein both p21 and p27Kip1 are weakly
increased in MCF-7 cells (51, 58) in apparent antagonism of the effects
of estrogen, which decreases the level of p27Kip1 in MCF-7
cells (58). In contrast, we observed a decrease in p21 and
cdk2-associated p21 in RA-treated cells.
Our data show that a major downstream target for RA inhibition in MCF-7
breast cancer cells is the cell cycle protein, cdk2, through a decrease
in mRNA and protein levels that apparently does not involve increases
in the levels of cdk2-associated cell cycle inhibitors, p21 and
p27Kip1. It is not clear whether RA acts directly or
indirectly to decrease cdk2 mRNA levels. The cdk2 promoter has recently
been cloned and contains regulatory sequences corresponding to sites of
interaction for E2F, AP-1, and several other factors (59).
Interestingly the ligand-bound RA receptor has been shown to interfere
with transcription from AP-1 sites through binding of a common
transcriptional coactivator protein, the cAMP response element binding
protein (CBP) (60). Future studies will determine the mechanism of
retinoid inhibition of this gene.
Results of metabolic labeling in the presence of RA showed that the
phosphorylation of cdk2 was decreased, suggesting that RA may induce
the activity of a phosphatase. Several phosphatases, including cdc25
(61) and protein phosphatases 1 and 2a [PP-1 and PP2A (62)], are
known to play a role in regulation of cdk activity. While cdc25 removes
the inhibitory phosphates at residues Y15 and T14 (61), PP1 and PP2A
have been implicated in the inactivation of a cdk (cdc2) through the
removal of an activating phosphate on T160 (62). Since RA treatment
results in decreased total phosphorylation of cdk2, it is possible that
both types of phosphatase activity are increased by RA. The other
possibility is that the phosphorylation of cdk2 by cak is inhibited by
RA. Although our results show that the intrinsic activity of cak has
not been decreased by RA treatment, it is possible that the
phosphorylation of cdk2 is blocked in RA-treated cells.
The results of the cdk2-inhibitory assay show that a putative
RA-inducible inhibitor can block cdk2 activity in control lysates
containing preactivated cdk2. This may result from one or more
mechanistic possibilities. The first is that the decrease in cdk2
protein in RA-treated cells in the absence of a change in cellular
levels of p27Kip1 results in an increase in free
p27Kip1 relative to cdk2. The free p27Kip1
would then be available to interact with cdk2 complexes from control
lysates to inhibit their activity. Alternatively, a novel inhibitor may
be induced by RA in these cells, resulting in a decrease in cdk2
activity.
In combination with cyclin E, cdk2 is necessary for the G1 to S
phase transition (42). The cyclin A-cdk2 complex binds to E2F-1 (63, 64) and inactivates E2F-1 DNA-binding activity by phosphorylation (65),
an activity that is necessary for orderly progression through S phase
(66). In addition, recent evidence shows that cdk2 activity is also
necessary for entry into mitosis since it activates the mitotic
cyclin-cdc2 kinase activity (43). In light of the expansive role
of cdk2 in cell cycle regulation, RA-mediated decreases in cdk2
levels and activity are likely to have profound effects on several
aspects of proliferation.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
MCF-7 ER-positive breast cancer cells (obtained from Dr. Leigh
Murphy, Manitoba) and T47-D ER-positive breast cancer cells (ATCC,
Rockville, MD) were maintained in DMEM (GIBCO BRL, Burlington, Ontario)
containing phenol red and supplemented with nonessential amino acids,
5% FBS (GIBCO BRL), 0.3% glucose, and 2 µg/ml gentamicin sulfate at
37 C/5%CO2. In some experiments cells were
semi-synchronized by contact inhibition achieved by maintaining the
cells at confluence for 48 h without a change of medium. This
routinely resulted in a blockage of approximately 70% of the cells in
G1 of the cell cycle. Cells were released by trypsinization and plated
at low density in the presence or absence of 1 µM RA
(Sigma Chemical Co., St. Louis, MO) added from a 1 mM stock
in ethanol.
Flow Cytometry
Cells were fixed in 100% ethanol for 1 h at 4 C and washed
twice with PBS before staining with 32 µM propidium
iodide containing 500 µg/ml RNase A. DNA content was analyzed on a
Coulter Epics V FACscan (Coulter Corp., Hialeah, FL), and cell cycle
phase distributions were estimated by computer fit using the Multicycle
analysis program.
RNA Isolation and Northern Blot Analysis
Total RNA was isolated from LiCl-urea as described (67). Twenty
micrograms of total RNA were electrophoresed in denaturing agarose gels
and transferred to Hybond N (Amersham, Oakville, Ontario). Membranes
were hybridized with multiprime-labeled probes, washed, and
autoradiographed. Equivalency of RNA loading was monitored by
hybridization of the blot with a glyceraldehyde phosphate dehydrogenase
(GAPDH) probe whose level is not altered in RA-treated MCF-7 cells. The
cDNAs encoding human cyclins and cdks were obtained from Dr. Paul Hamel
(Toronto, Ontario, Canada).
Western Blot Analysis
Protein extracts were prepared from cultures washed twice with
PBS and lysed with 1 ml per 107 cells of RIPA buffer (1%
NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml
phenymethylsulfonyl fluoride (PMSF), 3 µg/ml aprotinin, 10 mg/ml
sodium orthvanadate in PBS). Insoluble material was removed by
centrifugation at 4000 rpm for 15 min at 4 C. Protein concentrations
were determined using a Bio-Rad (Mississauga, Ontario) protein assay
kit. Samples were resolved on 7.5% (for detection of pRb) or 12% (for
all other proteins) SDS porous polyacrylamide gels (68) or the Laemmli
gel system (cdk2 only) and transferred electrophoretically to PVDF
polyscreen membranes (DuPont NEN, Boston, MA). Blots were incubated
with primary and secondary antibodies according to the suppliers
directions. Antibodies were obtained from the following sources: pRb
(Pharmingen, Cedarlane Labs, Hornby, Ontario); cyclin A, cyclin D1,
cyclin E, cdk2, cdk4, cak, p21, and p27 (Santa Cruz Biotechnology,
Santa Cruz, CA);
-actin (Sigma); goat anti-mouse IgG-horseradish
peroxidase (Jackson Labs, BioCan Sci, Mississauga, Ontario). Bound
antibodies were detected using the ECL chemiluminescence system (Dupont
NEN). In some experiments densitometry was performed on Northern and
Western blots using the microcomputer imaging densitometry (MCID)
software system (Imaging Research, Brock University, Ontario,
Canada).
In Vitro Kinase Assays
For cdk2 and cak assays, 400 µg of cell lysed as described
(69) were precleared by incubation with protein A Sepharose (Pharmacia,
Baie dOr, Quebec) and incubated with antibody to cdk2 or cak adsorbed
to protein A Sepharose at 4 C overnight. Immune complexes were washed
four times with RIPA buffer. Cdk2 activity was assayed by resuspending
the beads in 40 mM Tris (pH 7.5), 10 mM
MgCl2, 5 µM ATP, 0.5 mM
dithiothreitol (DTT), 0.5 mM EGTA, 400 µg/ml histone H1
(Sigma), 50 µCi [
-32P]ATP (Amersham) and incubation
at room temperature for 20 min. To assay cak activity, beads were
resuspended in 50 mM HEPES (pH 7.5), 10 mM
MgCl2, 4 mM ATP, 1 mM DTT, 400
µg/ml of a glutathione-S-transferase-cdk2 fusion protein (GST-cdk2)
and 10 µCi [
32P]ATP and incubated for 30 min at 30
C. To assay cdk4 activity, cells were lysed by sonication at 4 C in 50
mM HEPES (pH 7.5), 150 mM NaCl, 1
mM EDTA, 1 mM DTT, 0.1% Tween 20 containing
10% glycerol, 0.1 mM PMSF, 10 mM
ß-glycerophosphate, 1 mM NaF, 0.1 mM sodium
orthovandate, and 3 µg/ml aprotinin as described (35). Lysates were
cleared by centrifugation, precleared with protein A-Sepharose, and
incubated for 2 h at 4 C with protein A-Sepharose precoated with
anti-cdk4. Immunoprecipitates were washed four times with lysis buffer
and twice with 50 mM HEPES (pH 7.5), 1 mM DTT.
Beads were resuspended in 50 mM HEPES (pH 7.5), 1
mM NaF, 2.5 mM EGTA, 1 mM DTT, 10
mM ß-glycerophosphate, 0.1 mM sodium
orthovanadate, 10 mM MgCl2, 20 µM
ATP, and 10 µCi[
-32P]ATP and incubated for 30 min at
30 C with 400 µg/ml GST-pRB fusion protein. All kinase reactions were
resolved by SDS-PAGE and subjected to autoradiography. GST-pRb was
obtained from Dr. Paul Hamel (Toronto, Ontario, Canada); GST-cdk2 was
obtained from Dr. Tim Hunt (Oxford, U.K.).
Metabolic Labeling
MCF-7 cells released from contact inhibition were plated in the
presence or absence of 1 µM RA. After 56 h, cells
were washed twice with DMEM without phosphate then preincubated in the
same medium for 30 min. Cells were then cultured for 16 h in 3 ml
phosphate-free DMEM containing 1 mCi of 32Pi
(Amersham). Cells were lysed in RIPA buffer and immunoprecipitated with
cdk2 antibody as described above. Precipitated complexes were subjected
to SDS-PAGE and transferred to a PVDF membrane for autoradiography
followed by Western blot analysis with cdk2 antibody.
Cdk2 Inhibitor Assay
Cells incubated with or without 1 µM RA for
96 h were lysed in a buffer containing 20 mM Tris, pH
7.5, 250 mM NaCl, 0.1% NP-40, 1 mM Na
orthovanadate, 10 mM NaF, and 1 mM PMSF. One
hundred micrograms of lysates were mixed where indicated for 1 h,
preadsorbed with protein A Sepharose, and incubated with antibody
against cdk2 overnight at 4 C. Immunoprecipitates were formed after
incubation with protein A Sepharose for 1 h at 4 C, pelleted,
washed four times with lysis buffer, and assayed for cdk2 kinase
activity as described above. The reaction was allowed to proceed for 20
min at room temperature, stopped by the addition of SDS-PAGE sample
buffer, boiled, and loaded on a 10% SDS polyacrylamide gel. The fixed,
dried gel was subjected to autoradiography at -80 C.
 |
FOOTNOTES
|
---|
Address requests for reprints to: M. A. Christine Pratt, Department of Pharmacology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada, K1H 8M5.
This work was supported by grants from the Medical Research Council of
Canada and the American Institute for Cancer Research (No. 96A020) to
M.A.C.P.
Received for publication August 2, 1996.
Revision received December 23, 1996. Revision received April 30, 1997.
Accepted for publication May 14, 1997.
 |
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