Inhibitory Effects of 1
,25-Dihydroxyvitamin D3 on the G1S Phase-Controlling Machinery
Simon Skjøde Jensen,
Mogens Winkel Madsen,
Jiri Lukas,
Lise Binderup and
Jiri Bartek
Institute of Cancer Biology (S.S.J., J.L., J.B.) The Danish Cancer
Society, DK-2100 Copenhagen, Denmark; and LEO Pharmaceutical
Products (S.S.J., M.W.M., L.B.), DK-21002750 Ballerup, Denmark
Address all correspondence and request for reprints to: Dr. Simon Skjode Jensen, Department of Molecular Biology and Biochemistry, Leo Pharmaceutical Products, Industriparken 55, Ballerup, Denmark 2750.
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ABSTRACT
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The nuclear hormone 1
,25-dihydroxyvitamin D3 induces
cell cycle arrest, differentiation, or apoptosis depending on target
cell type and state. Although the antiproliferative effect of
1
,25-dihydroxyvitamin D3 has been known for years, the
molecular basis of the cell cycle blockade by 1
,25-dihydroxyvitamin
D3 remains largely unknown. Here we have investigated the
mechanisms underlying the G1 arrest induced upon
1
,25-dihydroxyvitamin D3 treatment of the human breast
cancer cell line MCF-7. Twenty-four-hour exposure of exponentially
growing MCF-7 cells to 1
,25-dihydroxyvitamin D3 impeded
proliferation by preventing S phase entry, an effect that correlated
with appearance of the growth-suppressing, hypophosphorylated form of
the retinoblastoma protein (pRb), and modulation of cyclin-dependent
kinase (cdk) activities of cdk-4, -6, and -2. Time course
immunochemical and biochemical analyses of the cellular and molecular
effects of 1
,25-dihydroxyvitamin D3 treatment for up to
6 d revealed a dynamic chain of events, preventing activation of cyclin
D1/cdk4, and loss of cyclin D3, which collectively lead to repression
of the E2F transcription factors and thus negatively affected cyclin A
protein expression.
While the observed 10-fold inhibition of cyclin D1/cdk 4-associated
kinase activity appeared independent of cdk inhibitors, the activity of
cdk 2 decreased about 20-fold, reflecting joint effects of the lower
abundance of its cyclin partners and a significant increase of the cdk
inhibitor p21CIP1/WAF1, which blocked the remaining cyclin
A(E)/cdk 2 complexes.
Together with a rapid down-modulation of the c-Myc oncoprotein in
response to 1
,25-dihydroxyvitamin D3, these results
demonstrate that 1
,25-dihydroxyvitamin D3 inhibits cell
proliferation by targeting several key regulators governing the
G1/S transition.
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INTRODUCTION
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1
,25-DIHYDROXYVITAMIN
D3 (1,25-VD3) and its
analogs represent candidate compounds for treatment of
hyperproliferative diseases including psoriasis and diverse types of
cancer. A major advantage of these reagents lies in the ability not
only to halt proliferation, but also to induce differentiation or cell
death [reviewed in (1, 2)].
1,25-VD3 is the physiologically active
ligand to the VDR. The VDR forms stable receptor complexes, preferably
as heterodimers with the RXR. The receptor dimers regulate
transcription in either a negative or positive fashion
(3, 4, 5). The VDR-RXR dimer binds specific palindromic
vitamin D response elements, located in the promoters of
1,25-VD3-regulated genes (6, 7). The
1,25-VD3-regulated genes are numerous and range
from genes involved in bone mineralization and proliferation to
transcription factors, interleukins, and structural proteins (reviewed
in Ref. 8).
Over the last decade, many antiproliferative agents have been shown to
interfere with the cell cycle machinery and arrest cells in
G1 phase of the cell cycle. Cell cycle
transitions are largely governed by a family of cyclin-dependent
kinases (cdks), which target critical substrates such as pRb, the
inactivation of which by cdk-mediated phosphorylation in mid-to-late
G1 phase is a prerequisite for entry into S phase
(reviewed in Refs. 9 and 10).
cdk Activity is regulated by association with its activating cyclin
partner, physical interaction with cdk inhibitors, and by both positive
and negative regulatory phosphorylations (11, 12). cdk
Inhibitors (cdki) include first the INK4 family:
p15INK4B, p16INK4A,
p18INK4C, and p19INK4D,
which bind and inactivate the major G1 phase
kinases, cdk4 and cdk6, by forming inactive dimeric complexes
(13, 14, 15). Another family of cdkis include
p21CIP1/WAF1, p27KIP1, and
p57KIP2, which inhibit a broader range of cdks,
including cdk1, -2, -4, and -6 (16, 17, 18). Regulation at the
level of phosphorylation is best characterized for the cyclin B-cdk1
complex, but the mechanisms are conserved in other cdk complexes acting
in G1 and S phase. Stimulatory phosphorylations
occur at Thr 160 in cdk2 and Thr 172 in cdk4. This activating
phosphorylation is carried out by the cdk activating kinase (CAK), a
complex consisting of cdk7, cyclin H, and the MAT1 assembly factor
(19, 20). Cdk4/6 and cdk2 contribute to pRb
phosphorylation in G1 and S phase, respectively,
resulting in derepression/activation of the pRb-regulated E2F
transcription factors required for further cell cycle progression
(21, 22, 23).
The cell cycle arrest induced by 1,25-VD3 and its
analogs has been investigated in tumor cells of leukemic
(24, 25, 26, 27), prostate (28, 29, 30), pancreatic
(31), and breast cancer origin (32, 33, 34) as
well as in normal keratinocytes (35, 36). The consensus
view emerging from these studies identifies G1
phase as the major target of the observed cell cycle blockade and
points to the p21CIP1/WAF1 or
p27KIP1 cdkis as candidate mediators of these
cell cycle effects.
In MCF-7 breast cancer cells, the 1,25-VD3 analog
EB1089 up-regulates p21CIP1/WAF1, which then
targets and inactivates cdk2 complexes (32). BT-20 and
ZR75 breast cancer cells respond in a similar manner, possibly
including induction of p27KIP1 (32).
The finding of a vitamin D response element in the
p21CIP1/WAF1 promoter and the induction of
p21CIP1/WAF1 transcript within 2 h after
1,25-VD3 addition suggested that
p21CIP1/WAF1 represents an early mediator of the
1,25-VD3-induced cell cycle arrest
(24).
The aim of this study was to further elucidate the mechanisms by which
1,25-VD3 exerts its growth-inhibitory activities.
We chose the human breast cancer cell line MCF-7 as a model, which
expresses VDR and responds to 1,25-VD3 in a
growth-inhibitory manner, to investigate the effects of
1,25-VD3 on key cell cycle regulators, the
cyclin-cdk complexes controlling the mammalian
G1S phase transition, and their cognate
inhibitors.
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RESULTS
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1,25-VD3-Treated MCF-7 Cells Display Decreased
Proliferation and Accumulation in G1 Phase
To determine the proliferation-inhibitory effect of
1,25-VD3 on the MCF-7 cells, proliferation assays
with concentrations of 1,25-VD3 at
10-7 M were performed; these
concentrations were shown by others to have antiproliferative,
but not toxic effects, in several cell lines including MCF-7 (32, 37, 38). As seen in Fig. 1A
, proliferation of exponentially growing MCF-7 cells was inhibited after
only 2 d of 1,25-VD3 treatment, and the
cell number did not increase significantly after day 4. To determine
the cell cycle phase in which MCF-7 cells arrest when exposed to
1,25-VD3, we analyzed the DNA content of the
cells by flow cytometry. After 24 h treatment, the
G1 phase population increased significantly from
49 to 57% of the total cell population, correlating with a concomitant
S phase decrease (Fig. 1B
). The G1 phase
accumulation in 1,25-VD3-treated cells remained
approximately 2030% above control throughout the time course. After
6 d of treatment, approximately 80% of the treated cells were in
G1 phase, indicating that the MCF-7 cell
population does not respond with a complete G1
phase blockade. This incomplete response was caused by heterogeneity in
the MCF-7 cell line showing a subpopulation of nearly
1,25-VD3-resistant cells (data not shown).
Control cells increased in the G1 population at
later time points, reflecting the characteristic growth of MCF-7 cells
in "islands," in the center of which the cells become increasingly
contact inhibited during the time course, eventually arresting in
G1 phase in a
p27KIP1-dependent manner (39). Taken
together, these data show that MCF-7 cells respond to
1,25-VD3 and accumulate in
G1 phase of the cell cycle after only 24 h
of treatment.

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Figure 1. Antiproliferative Effect of
1,25-VD3 on MCF-7 Cells
A, A summary of growth curves from three experiments, in which day 0
corresponds to the time when 1,25-VD3 (10-7
M) was added (24 h after plating the cells). Error
bars represent SD. B, Summary of analysis of cell
cycle phase distribution of cells treated as in panel A, analyzed by
flow cytometry over a 6-d period. The change in G1 phase
cells was significant from 24 h treatment, and the decrease in S
phase population was significant from 48 h treatment
(P < 0.05, analyzed by Students t
test, n = 35).
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1,25-VD3-Treated MCF-7 Cells Exhibit Hypophosphorylated
pRb and Impaired Expression of Cyclin A and E mRNA and Protein
The 1,25-VD3-induced accumulation of cells
in G1 phase indicated a potential effect on pRb,
whose growth-suppressive, hypophosphorylated form is characteristic for
cells in early G1 (40, 41). In a 4-d
time course experiment, we analyzed pRb in total cell lysates
by gel electrophoresis and immunoblotting (Fig. 2A
). At day 0, the bulk of pRb was in the
slower migrating, hyperphosphorylated form (marked
pRbpp) (40), but after only 2436 h
treatment, the hypophosphorylated form started to accumulate when
compared with control cells (Fig. 2
, A and D). Accumulation of the
hypophosphorylated form increased throughout the time course, whereas
the hyperphosphorylated form gradually decreased in
1,25-VD3 treated cells. The middle
panel shows a blot of less separated pRb, which indicates decrease
of pRb in lovastatin-arrested cells and in MCF-7 cells treated for 72
and 96 h, relative to loading control (cdk7-blot). Given the
ability of the hypophosphorylated pRb to repress the E2F transcription
factors (23, 42), we next examined mRNA and protein levels
of cyclin A and E, both known E2F targets, in
1,25-VD3 treated cells. Expression of cyclin A
and E were modulated at both transcript and protein levels by
1,25-VD3 treatment. Cyclin A and E mRNA in
1,25-VD3-treated cells remained at low levels as
seen at the zero time point, whereas the control cells increased
transcript levels as the cells entered an exponential growth phase and
reached a 2-fold higher level after 4 days of treatment (Fig. 2B
).
Western blot of total cell extracts showed increased cyclin A protein
levels in control cells during the time course with highest levels
after 7296 h, whereas 1,25-VD3 treatment
prevented this increase and retained cyclin A protein close to the
initial low levels at time zero. These data indicate that
1,25-VD3 prevents entry into exponential growth
phase by targeting the pRb pathway, resulting in hypophosphorylated,
active pRb, and consequently preclude E2F activation and transcription
of E2Fregulated genes such as cyclin A.

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Figure 2. Effects of 1,25-VD3 on Cell Cycle
Regulators
A, Western blot analysis of pRb in total cell extracts from mock (-)-
or 1,25-VD3 (+)-treated MCF-7 cells at the indicated time
points. The upper panel allowed good separation of the
pRb species in an 8% gel separated overnight; pRbpp
marks the hyperphosphorylated form of pRb above the hypophosphorylated
pRb, for which cells treated with lovastatin for 48 h served as
control (marked L). The hypophosphorylated pRb was quantitated from
several experiments and plotted as fold to control ±
SEM in panel D (upper graph). The
middle panel (A) shows a less separated blot of pRb,
allowing quantitative interpretation of pRb amounts in each lysate. B,
Northern blot analysis of cyclin A and cyclin E expression using 10
µg of total RNA from MCF-7 cells at the indicated times. The 36B4
probe was used as a loading control. C, Time course Western blot
analysis of the indicated cell cycle-regulatory proteins in MCF-7 cell
extracts, with antibodies specified in Materials and
Methods. Statistical analyses of multiple experiments were
performed. Protein levels of cyclin A, cyclin D3, cdk2, and the faster
migrating CAK-activated cdk2 species marked Cdk2CAK were
all significantly reduced from 48 h treatment. c-Myc was
significantly reduced from 24 h treatment and cdk6 was reduced
from 72 h treatment. p21 was significantly increased from 72
h treatment, whereas protein levels of p27, cyclin D1, and cdk4 were
not significantly altered. Cdk7 served as a loading control. D,
Representative diagram showing statistical analysis of selected
proteins; bars indicate SEM (data evaluated
using Students t test, n = 36,
P < 0.05).
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1,25-VD3 Modulates Protein Levels of Important
G1S Phase Regulators
Western blots performed with total cell extracts showed altered
protein levels of cyclin and cdk regulators, controlling
G1S phase progression. Protein levels of cdk2 and the
activated cdk2CAK migrating slightly faster than
the inactive cdk2 increased in control cells as they entered
exponential growth, whereas 1,25-VD3 treatment
retained cdk2 at the low initial levels, with only a minor appearance
of the activated cdk2CAK (Fig. 2C
). Cdk6
displayed a similar pattern, with significant difference between
control and treated cells from 72 h 1,25-VD3
treatment, whereas cdk4 was not significantly decreased.
Cyclin D1, which primarily activates cdk4 in MCF-7 cells, showed
only a minor change after 4 d of 1,25-VD3
treatment, whereas cyclin D3 was significantly decreased from 48 h
treatment (Fig. 2C
). The cdk inhibitor
p21CIP1/WAF1 was reduced in control cells at 72
and 96 h of treatment, but remained at high levels in
1,25-VD3-treated cells, correlating with the
difference in growth properties previously identified in Fig. 1
.
Analysis of p27KIP1 and the INK4 cdki proteins
revealed no significant change upon 1,25-VD3
treatment (data not shown).
Interestingly, protein levels of the c-Myc decreased significantly from
24 h of 1,25-VD3 treatment relative to the
zero time point, but also relative to control cells, which
showed increased c-myc protein levels, correlating to
the increased proliferation state (Fig. 1
).
These data show that 1,25-VD3 prevents the cells
from entering the exponential growth phase, as seen in control cells,
by uncoupling expression of proteins required for cell proliferation,
e.g. c-Myc, cdk, and cyclin proteins. Simultaneous high
levels of the cdk inhibitor p21CIP1/WAF1 further
sustain growth retardation.
The Effects of 1,25-VD3 on cdk Activity
The observed G1 phase arrest and the lack of
pRb phosphorylation strongly suggested that the activities of pRb
kinases, including cyclin D/cdk4 (6) and cyclin E(A)/cdk2,
may be affected upon treatment of cells with
1,25-VD3. Investigation of the cdk complexes
responsible for pRb phosphorylation in G1 and S
phase of the cell cycle showed a general inhibition of cdk activity,
when assayed by immunoprecipitation (ip) of the complexes followed by
in vitro kinase assays using
glutathione-S-transferase (GST)-pRb as substrate. The kinase
activity of cyclin D1-cdk4, cyclin D3-cdk4/6, and cdk6-cyclin D1/3 were
all strongly inhibited in MCF-7 cells treated with
1,25-VD3 for 4 d (Fig. 3A
). The most abundant
G1 phase complex cyclin D1-cdk4 is inhibited more
than 6-fold, and the less abundant complexes containing cyclin D3
and/or cdk6 show somewhat weaker activity and are significantly
inhibited 2- to 3-fold relative to vehicle- treated controls. Cdk2 is
activated by cyclin E at G1S phase transition
and in S phase by cyclin A (43, 44). To examine whether
1,25-VD3 leads to inhibition of cdk2 associated
with both cyclin E and A, we performed kinase assays with complexes
immunoprecipitated with antibodies to cyclin E, cyclin A, or cdk2. As
shown in Fig. 3B
, 4
-d exposure to 1,25-VD3 leads
to a 3-fold reduction of cyclin E-associated kinase activity, while
cyclin A-associated kinase activity was reduced 17-fold and cdk2-cyclin
E(A) activity was reduced 25-fold, showing an overall dramatic
inhibition of cdk2. Examination of the dynamics of the cdk2
(4) regulation showed increased cdk activity in control
cells as exponential growth progressed, but with cdk activity in
1,25-VD3-treated cells remaining at low levels
throughout the time course (Fig. 3
, C and 3D). Surprisingly, activity
of both cdk2 and cdk4 kinase was weakly but significantly increased
after 8 h of treatment, effects that could be related to
nongenomic fast responses mediated by 1,25-VD3
(45).

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Figure 3. 1,25-VD3 Treatment Modulates
G1 and S Phase cdk Activities
A, Kinase activities of the indicated cyclin D-associated complexes
toward GST-pRb substrate, assayed after 4 d of mock (-) or
1,25-VD3 (+) treatment in immunoprecipitates from MCF-7
cell extracts obtained with antibodies DCS6 (recognizing only cdk-free
cyclin D1; negative control), 5D4 (recognizing cyclin D1/cdk4 complex),
DCS28 (recognizing cyclin D3/cdk4(6 )), and DCS130 (against the C
terminus of cdk6). B, Kinase activities assayed in cells treated as in
panel A, in immunoprecipitates obtained with antibodies HE12
(recognizing only cdk-free cyclin E; negative control), HE172
(recognizing cyclin E complexed to cdk2), sc-751 (cyclin A/cdk
complexes), and sc-163 (recognizing cdk2 complexed with cyclins E or
A). All complexes were significantly inhibited relative to control
and mean ± SEM indicated as percent of control
below the panels (n = 23, P
< 0.05). Panels C and D, Four to 6-d time course analysis of cdk4
(C)- and cdk2 (D)-associated kinase activities in extracts of mock
(-)- or 1,25-VD3-treated (+) MCF-7 cells. The
diagrams below the panels show a summary of three to
five independent kinase assays indicating mean value ±
SEM, evaluated by a one-sided, unpaired t
test. After 8 h of treatment there was a significant increase in
cdk2 (4 ) kinase activity relative to control
(P < 0.05). After 24 h and throughout the
time course, cdk2 and cdk4 were significantly inhibited relative to
control (P < 0.005). AD, The phosphorylation
signals in GST-pRb were quantitated using a PhosphorImager
(Molecular Dynamics, Inc.).
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Figure 4. 1,25-VD3-Induced
p21CIP1/WAF1 Targets cdk2 but Not cdk4
A, Time course Northern blot analysis of p21CIP1/WAF1 mRNA
in mock (-)- and 1,25-VD3-treated (+) MCF-7 cells
(36B4 = loading control). B and C, MCF-7 cells treated for 96
h with 1,25-VD3 (+) or vehicle (-) were analyzed by ip of
equal amount of lysates with antibodies against the proteins shown on
the top, followed by Western blotting with antibodies
against the proteins indicated on the left. B, WCE
(whole-cell extract) marks direct immunoblots on WCEs; and
cdk2CAK indicates the CAK-activated form of cdk2.
Below the blots, analyses of independent experiments are
shown as mean ± SEM and indicated as decrease in
total cdk2/increase in associated p21 relative to control (n =
25, P < 0.05). C, Analysis of independent
experiments of cdk4(6 )-cyclin D-p21-p27 complexes revealed
no significant stoichiometric alteration in these complexes, except
that the cdk4-cyclin D3 complex was significantly reduced to 46 ±
14% of untreated controls, after 96 h treatment (n = 4,
P < 0.05).
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The M phase-specific cdk1-cyclin B1 complex showed a 3-fold
inhibition of kinase activity toward the histone H1 substrate after
4 d of 1,25-VD3 treatment (data not shown).
Taken together, these data show that the known cdk complexes essential
for G1 and S phase progression are not activated
in 1,25-VD3-treated MCF-7 cells, which are
consequently precluded from entering a productive exponential growth
phase.
Inhibition of cdk2 Correlates with Increased Targeting of
p21CIP1/WAF1, Decreased Cyclin A and E Association, and
Lack of CAK-Mediated Phosphorylation
Since cyclin A was affected at the level of total protein (as
described above, Fig. 2C
), lack of cdk-cyclin association could
contribute to failure of cdk2 activation. In addition, members of the
p21CIP1/WAF1/p27KIP1
family of cdk inhibitors were identified as important
mediators of glucocorticoid-, retinoic acid-, and
1,25-VD3-induced cdk inhibition (24, 32, 46, 47) and would therefore also be good candidates for
mediating the effects on cdk2 kinase activity in our model system.
Northern blot analysis showed an increase of
p21CIP1/WAF1 transcript in MCF-7 cells after
only 8 h of 1,25-VD3 treatment
(Fig. 4A
). The increased
p21CIP1/WAF1 mRNA levels were seen throughout
the time course, with a maximal 3-fold induction after
72 h. Coimmunoprecipitation experiments from cells treated for
96 h revealed that the increase in
p21CIP1/WAF1 protein correlated with increased
binding of p21CIP1/WAF1 with cyclin A(E)-cdk2
complexes (Fig. 4B
, lower panel, lanes 510). In whole-cell
extracts (Fig. 4B
, lanes 12), cdk2 was largely shifted to the slower
migrating, inactive form, known to reflect the lack of CAK
activation/phosphorylation of cdk2 (see also Fig. 2C
). When
p21CIP1/WAF1 ips were analyzed, both active and
inactive cdk2 were seen in treated cells, showing that
p21CIP1/WAF1 preferentially targets the active
cdk2 complexed to cyclins, despite the low amounts of active cdk2 after
4 days of treatment (Fig. 4B
, upper panel, lanes 34). In
immunoprecipitates of cyclin E and A, mainly containing the
CAK-phosphorylated active cdk2, an overall significant decrease in the
levels of cyclin-associated cdk2 was seen after 4 days of treatment,
possibly reflecting the lower levels of cyclin A protein identified in
total cell extract (Fig. 4B
, upper panel, lanes 58). These
data indicate that after 4 d of 1,25-VD3
treatment, cdk2 kinase activity is deregulated by a combination of
increased association with the cdk inhibitor
p21CIP1/WAF1 and decreased association/activation
with both cyclin A and cyclin E. Consequently, the overall proportion
of the active form of cdk2, phosphorylated at Thr 160 by CAK, is
reduced after 1,25-VD3 treatment.
Regulation of G1 Phase Complexes Independently of
p21CIP1/WAF1
The G1 phase complexes cyclin D1(3)-cdk4(6)
and their cognate inhibitors were also examined. Ips with antibodies
against p21CIP1/WAF1,
p27KIP1, cdk4, cdk6, cyclin D1, and cyclin D3 did
not show any dramatic increase in either
p21CIP1/WAF1 or p27KIP upon
1,25-VD3 treatment in any of these complexes
(Fig. 4C
). The lack of cdk4-cyclin D1 activation in treated cells, as
seen in control cells in Fig. 3C
, could not be explained by disruption
of the complex, since the stoichiometry between cdk4-cyclin D1 remained
largely preserved upon treatment (Fig. 4C
, lanes 56 and 910). In
contrast to cdk4-cyclin D1, complexes containing cyclin D3 were
disrupted (Fig. 4C
, lanes 1112), showing decreased levels of cdk4
associated with cyclin D3 upon 1,25-VD3
treatment. This effect was probably due to decreased protein levels of
cyclin D3 following 4 d of 1,25-VD3
treatment (Fig. 2C
). Furthermore, these ips show that cyclin D1
primarily associates with cdk4, rather than cdk6, strongly indicating
that the kinase complex assayed by the anticyclin D1 antibody in Fig. 3A
, lanes 2 and 3, and in Fig. 3C
, is mainly cyclin D1-cdk4.
These data suggest that 1,25-VD3 treatment
prevents activation of the major G1 phase
complex, cdk4-cyclin D1, by mechanisms distinct from targeting by cdkis
and dissociation of the cyclin-cdk complex.
Cdk2 Kinase Activation Is Prevented by p21CIP1/WAF1
Targeting and Accompanied by Decreased Cyclin A Activation
As proliferation of the MCF-7 cells was affected by
1,25-VD3 after only 24 h treatment
(Fig. 1B
), we next analyzed the dynamics of cdk2 activity in an attempt
to identify the events that may initiate the effects on cdk2 kinase
activity. cdk2 Was immunoprecipitated every 6 h between 18 and
48 h of 1,25-VD3 treatment, and the
complexes were analyzed for associated cyclin A and
p21CIP1/WAF1 proteins. Analysis of
cdk2-coprecipitated proteins (Fig. 5A
)
showed a weak reduction in precipitated cdk2 relative to control from
24 h treatment, correlating to the different cdk2 protein levels
seen in total cell extract (Fig. 2C
). When the coprecipitated
p21CIP1/WAF was quantitated relative to the
decreased levels of cdk2, the p21CIP1/WAF1-cdk2
ratio was significantly increased from 36 h treatment (Fig. 5
, A
and C). Analysis of coprecipitated cyclin A revealed a slightly lower
cyclin A level in treated vs. control cells from 24 h,
but since cdk2 was reduced in a similar manner, the cdk2-cyclin A ratio
was not significantly reduced until after 48 h of treatment. The
cdk2-cyclin E ratio was affected even later in the time course (not
shown). Immunoblotting of p21CIP1/WAF1 in total
cell extracts prepared as above showed a significant increase in
p21CIP1/WAF1 protein levels relative to cdk7
(loading control) from 30 h of treatment and throughout the time
course (Fig. 5
, B and D).

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Figure 5. Dynamics of 1,25-VD3-Induced
Modulation on cdk2 Activity
A, cdk2-Associated proteins (coprecipitated with antibody sc-163) were
analyzed at the indicated time points by Western blotting using
antibodies to cyclin A, cdk2, and p21CIP1/WAF1. B, Detailed
time course analysis of p21CIP1/WAF1 protein abundance by
direct immunoblotting of total cell extracts. cdk7 served as loading
control. C and D, Six independent experiments were evaluated by
unpaired, one-sided t test, with the following values *,
0.05 > P > 0.025; **,
0.025 > P > 0.01; and ***,
P < 0.01. Protein levels of
p21CIP1/WAF1 were quantitated by the ECF system
(Amersham Pharmacia Biotech) and correlated to the amount
of cdk2 (A), or cdk7 (B) in each lane, and finally to mock (-)-treated
cells standardized to 1. Bars indicate
SEM.
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These data suggest that the initial events responsible for
deficient cdk2 kinase activation in
1,25-VD3-treated MCF-7 cells include increased
association between cdk2 and p21CIP1/WAF1,
accompanied by a lack of cdk2 activation by cyclin A association after
48 h of treatment.
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DISCUSSION
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Our current data show that 1,25-VD3
treatment of MCF-7 cells prevented activation of
G1 phase complexes cdk4(6)-cyclin D1(3), and the
G1-S governing complexes of cdk2-cyclin A(E).
Based on these results we propose a model (Fig. 6
), of 1,25-VD3
targets and effects that lead to cell cycle block in
G1 phase. 1) cdk4(6)-cyclin D1(3) kinase activity
is deregulated starting from 24 h after
1,25-VD3 treatment, preventing pRb
phosphorylation/inactivation concomitant with sequestration and
inactivation of E2F. Continued silencing of E2F by active pRb prevents
cyclin A and E protein expression (21, 22, 23), resulting in
deficient cdk2 activation. 2) Induction of
p21CIP1/WAF1 leads to failure of cdk2 activation
after 2436 h. 3) c-Myc deregulation could possibly contribute to
silencing of cdk2 activity, since c-Myc has been shown to induce
cdk2 activity and concomitant S phase entry in a cyclin E- and
Cdc25A-dependent manner (48).

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Figure 6. Schematic Model of 1,25-VD3
Effects on Diverse Pathways Controlling G1/S Transition
1,25-VD3 affects at least three pathways of the cell cycle
machinery: 1) Inhibition of cyclin D-dependent kinases leading to
activation of pRb and thereby repression of E2F; 2) Down-regulation of
c-Myc abundance; and 3) Induction of p21CIP1/WAF1; all of
which contribute to regulation of cdk2 kinase activity and
G1 phase blockade.
|
|
Deregulation of G1 phase complexes including
cdk6 and cyclin D3 can be explained by lack of protein induction and/or
decreased protein levels of cdk6 and cyclin D3 from approximately
48 h of 1,25-VD3 treatment. At present, we
have no mechanistic explanation for the observed effects on cdk4-cyclin
D1 kinase activity after 24 h treatment.
The cdk4-cyclin D1 complex stoichiometry was not affected, despite a
7-fold difference in kinase activity after 96 h of treatment. This
indicates that no INK4 proteins are involved, since this would result
in disruption of the cdk4-cyclin D1 complex and sequestration of cdk4
in a dimeric complex with the INK4 proteins (17, 49, 50, 51).
Sequestration of cdk4 into HSP90/cdc37 complexes is another known
mechanism for cdk4 inactivation (52, 53, 54), but since this
process apparently involves monomeric cdk4 only, it would also require
cdk4-cyclin D1 disassembly and is therefore unlikely to play a role in
our model system. A plausible mechanism for deregulation of cdk4 by
1,25-VD3 could therefore reflect regulation at
the level of phosphorylation. Cdk4 was reported to be phosphorylated at
the inhibitory Tyr 17 upon entry into quiescence induced by either
contact inhibition or serum starvation in rat kidney fibroblasts
(55, 56), or after TGFß treatment of MCF-10A cells, in
the latter case due to decreased activity of the Cdc25A phosphatase.
Thus, Cdc25A could be a good candidate for an upstream regulator of
both cdk2 and cdk4 activity in the
1,25-VD3-mediated cell cycle arrest (48, 57).
In this context, 1,25-VD3-mediated regulation of
cdc25A activity could account for the difference in timing
between significant inhibition of cdk2 kinase activity
(Fig. 3D
), at 24 h (60% of control) until
p21CIP1/WAF1 could be significantly identified in
cdk2 complexes at 36 h (Fig. 5
, A and C).
Consistent with others we found induction of
p21CIP1/WAF1 transcript within 8 h of
1,25-VD3 treatment (24, 28, 33).
Induction of the p21CIP1/WAF1 transcript occurs
very likely by a VDR-dependent mechanism, since a VDR response element
has been identified in the p21CIP1/WAF1 promoter
(24). These data suggest that
p21CIP1/WAF1 induction is an event upstream of
cdk2 inhibition, independent of the pathways leading to cdk4
inhibition and c-Myc down-regulation. Surprisingly, the early
increase in p21CIP1/WAF1 transcript does not
directly correspond to an increase in
p21CIP1/WAF1 protein, which first occurs after
30 h treatment, where we identified a significant increase in
p21CIP1/WAF1 protein, suggesting regulation at
the level of p21CIP1/WAF1 translation or
stability of p21CIP1/WAF1 mRNA or protein. The
increased p21CIP1/WAF1 protein contributed to the
failure of cdk2 activation and possibly prevented CAK activation of
cdk2, by analogy with the effects of PGA2-induced
p21CIP1/WAF1 (58). A similar
phenomenon was reported for p27KIP1 in inhibition
of cdk4 in response to contact inhibition (59).
The pathways of 1,25-VD3 signaling, leading
either through VDR or nongenomic signaling through possible
1,25-VD3 membrane receptors, are poorly
understood. Candidate molecules on the pathway leading to the
1,25-VD3-mediated cell cycle arrest remain
elusive, but possibly transcription factors such as c-jun
and c-Myc could play important roles, since they are affected early by
1,25-VD3 treatment (60, 61, 62, 63). AP1
activation and c-jun induction have been shown to occur
within 3060 min after 1,25-VD3 addition, in a
process possibly involving PKC, JNK1, and ERK2 (63, 64, 65).
Other studies report an increase in intracellular
Ca2+, which possibly activates
Ca2+-dependent PKC isoforms (63, 66). Collectively, 1,25-VD3 signaling
through VDR-independent mechanisms involves activation of MAPK
pathways, leading to activation of transcriptional complexes such as
AP1. Since c-Myc is a downstream target of ERK2 (67),
activation of this pathway could possibly play a role in the observed
down-regulation of c-Myc in our model system.
Our data show that the strong antiproliferative effect of
1,25-VD3 in human breast cancer cells reflects
targeting of several key cell cycle regulators and warrants further
research and attempts to develop 1,25-VD3 analogs
suitable for cancer treatment. One of the most promising analogs of
1,25-VD3 in terms of cancer treatment is EB1089,
for which significant anticancer effects were reported from both
in vitro and in vivo experiments [reviewed in
(68, 69)]. The antiproliferative potency of this compound
is greatly improved with an IC50 value 50200
times below that of 1,25-VD3. In vivo,
EB1089 causes significant inhibition of tumor progression in both rats
(70) and mice (71, 72), and it lacks the
serious hypercalcemic side effects characteristic for
1,25-VD3.
In conclusion, our data show that cdk2 and cdk4 kinase activities are
deregulated within 24 h of 1,25-VD3
treatment. Prevention of cdk4 activation likely contributes to the
G1 phase arrest by decreased phosphorylation of
pRb, leading to sustained E2F sequestration, and thereby decreased
cyclin A expression and subsequent repression of cdk2 kinase activity,
which is essential for G1/S phase transition. The
mechanism of initial cdk2 deregulation likely includes a combination of
p21CIP1/WAF1 increase, followed by lack of cyclin
A association and activation. Lack of cdk2 activation is further
promoted via low cdk2 protein levels and decreased cdk2-cyclin E
association.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
The human breast cancer cell line MCF-7 (obtained from DkFZ,
TZB610030) was cultured in DMEM without phenol red (Life Technologies, Inc., Gaithersburg, MD, catalog no. 11880)
supplemented with 5% FCS, 2 mM glutamine, penicillin (10
U/ml), and streptomycin (10 U/ml). The cells were routinely passaged
every week, and media were changed every second to third day. The cell
line was regularly tested for mycoplasma infection (PCR-primer set,
Stratagene, La Jolla, CA), and found to be mycoplasma
negative. Twenty-four hours before 1,25-VD3 addition, the
cells were passaged with 1 mM EDTA, spun down, and plated
as single cells at a density of 35 x 103
cells/cm2. 1
,25(OH)2vitamin D3
was synthesized in the Department of Chemical Research, Leo Pharmaceuticals Products, where purity and concentration were
determined. 1,25-VD3 was diluted in media from an
isopropanol stock at 4 x 10-4 M to a
final concentration of 10-7 M. In experiments
the media was changed every 24 h with fresh prewarmed
CO2-equilibrated media including either vehicle or
1,25-VD3. Vehicle concentration was kept below
0.0025%.
Antibodies
Western blot against pRb was performed with a monoclonal mouse
antibody (mMAb); G3245 from PharMingen (San Diego, CA),
against p27KIP1 with a rabbit polyclonal antibody
(PC55), was from Calbiochem (La Jolla, CA). mMAbs against
cyclin D1 (DCS6), cyclin D3 (DCS28), cdk6 (DCS130), and cdk7 (MO-1.1)
were produced and used as either hybridoma supernatant or ascites. 5D4
was against cyclin D1/2 and donated by M. Sato; HE12 and HE172 are
mMAbs against cyclin E (73). Rabbit antisera against
p21CIP1/WAF1 (sc-397),
p27KIP1 (sc-776), cyclin A (sc-751), cdk2
(sc-163), cdk4 (sc-601), and cdk6 (sc-177) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Cell Cycle Analysis and Proliferation Assay
Progression through the cell cycle was determined by flow
cytometry analysis of DNA content of cell populations stained with
propidium iodide as described previously (74).
Proliferation assays were done in six-well plates with initially 1
x 103 cells/cm2. Every
counting was done in triplicate as the average of two countings in a
Coulter counter. Before counting the cells were passaged through a
syringe to prevent formation of cell aggregates.
Kinase Assays
cdk2, cdk4, and cdk6 kinase assays were performed as described
previously (75, 76, 77), using a short C-terminal GST-pRb
substrate (amino acids 773928) and the antibodies indicated in each
figure. The incubations were at 30 C for 30 min, and the kinase
reaction was stopped by adding 8 µl 4x Laemmli sample buffer
including 10 mM EDTA. The kinase reactions were separated
by SDS-PAGE, and the proteins were transferred to nitrocellulose
membranes by the semidry method. Membranes were exposed on a
phosphorimage screen to measure incorporation of
32P
ATP into the substrate using the STORM
analyzer from Molecular Dynamics, Inc. (Sunnyvale,
CA).
Immunochemical Analysis
Extraction of protein for ip, estimation of protein content, and
ip procedures were as described previously (76, 78). For
each ip in Figs. 4
and 5
the protein content of each lysate was
measured using the BCA protein assay reagent (Pierce Chemical Co., Rockford, IL) ensuring ip from equal amounts of lysate.
Cross-linking of cdk2 antibody was done as described previously
(79). Western blot was performed as in (80),
except for secondary antibodies (PI1000 and PI2000), which were from
Vector Laboratories, Inc. (Burlingame, CA). For
quantitation in Fig. 5
, the ECF system was used from
Amersham Pharmacia Biotech (Arlington Heights, IL), on a
Storm analyzer from Molecular Dynamics, Inc.
Northern Blot
Total cellular RNA was extracted and purified using the
RNAqueous kit from Ambion, Inc. (Austin, TX, catalog no.
1912) according to the manufacturers instructions. Ten micrograms of
RNA were electrophoresed on a 1% agarose formaldehyde gel and
transferred onto a nylon membrane. Expression of the
p21CIP1/WAF1, cyclin A, and cyclin E transcripts
were monitored using the full-length cDNA from each gene as probes, cut
out from appropriate plasmids. The 36B4 cDNA probe encoding acidic
ribosomal phosphoprotein PO was used in parallel to control for
balanced loading.
 |
ACKNOWLEDGMENTS
|
---|
We thank M. Sato, S. I. Reed, and E. Harlow for important
reagents, and D. Hansen for help with fluorescence-activated cell
sorting analysis.
 |
FOOTNOTES
|
---|
This work was supported by the Danish Cancer Society and the Danish
Medical Research Council.
Abbreviations: CAK, cyclin-dependent kinase-activating kinase;
cdk, cyclin-dependent kinase; cdki, cyclin-dependent kinase inhibitor;
GST, glutathione-S-transferase; ip, immunoprecipitation;
mMAb, monoclonal mouse antibody; pRb, retinoblastoma protein;
1,25-VD3,1
,25-dihydroxyvitamin D3.
Received for publication June 22, 2000.
Accepted for publication April 11, 2000.
 |
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