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Mol Cell Biol, April 1998, p. 1812-1825, Vol. 18, No. 4
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Mechanisms of Cyclin-Dependent Kinase
Inactivation by Progestins
Elizabeth A.
Musgrove,*
Alexander
Swarbrick,
Christine S. L.
Lee,
Ann L.
Cornish, and
Robert L.
Sutherland
Cancer Research Program, Garvan Institute of
Medical Research, St Vincent's Hospital, Sydney, New South Wales
2010, Australia
Received 15 August 1997/Returned for modification 6 October
1997/Accepted 22 December 1997
 |
ABSTRACT |
The steroid hormone progesterone regulates proliferation and
differentiation in the mammary gland and uterus by cell cycle phase-specific actions. In breast cancer cells the predominant effect
of synthetic progestins is long-term growth inhibition and arrest in
G1 phase. Progestin-mediated growth arrest of T-47D breast
cancer cells was preceded by inhibition of cyclin D1-Cdk4, cyclin
D3-Cdk4, and cyclin E-Cdk2 kinase activities in vitro and reduced
phosphorylation of pRB and p107. This was accompanied by decreases in
the expression of cyclins D1, D3, and E, decreased abundance of cyclin
D1- and cyclin D3-Cdk4 complexes, increased association of the
cyclin-dependent kinase (CDK) inhibitor p27 with the remaining Cdk4
complexes, and changes in the molecular masses and compositions of
cyclin E complexes. In control cells cyclin E eluted from Superdex 200 as two peaks of ~120 and ~200 kDa, with the 120-kDa peak displaying
greater cyclin E-associated kinase activity. Following progestin
treatment, almost all of the cyclin E was in the 200-kDa, low-activity
form, which was associated with the CDK inhibitors p21 and p27; this
change preceded the inhibition of cell cycle progression. These data
suggest preferential formation of this higher-molecular-weight, CDK
inhibitor-bound form and a reduced number of cyclin E-Cdk2 complexes as
mechanisms for the decreased cyclin E-associated kinase activity
following progestin treatment. Ectopic expression of cyclin D1 in
progestin-inhibited cells led to the reappearance of the 120-kDa active
form of cyclin E-Cdk2 preceding the resumption of cell cycle
progression. Thus, decreased cyclin expression and consequent increased
CDK inhibitor association are likely to mediate the decreases in CDK
activity accompanying progestin-mediated growth inhibition.
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INTRODUCTION |
Steroid hormones regulate cellular
proliferation and differentiation by cell cycle phase-specific actions
(40). Estrogen, acting in concert with other hormones and
growth factors, appears to be the main drive to proliferation in the
female reproductive tract and mammary gland. In contrast with the
proliferative effects of estrogen, progesterone acts as the
differentiating female sex steroid. In this role it can either
stimulate or inhibit proliferation in a cell type- and tissue-specific
manner (5). For example, the primary function of
progesterone in the uterus is to facilitate implantation, and in this
organ progesterone acts synergistically with estrogen to stimulate
proliferation of stromal cells but inhibits estrogen-induced mitosis in
the epithelium. In the mammary gland progesterone stimulates
proliferation and development of alveoli, a requirement for subsequent
lactation. In breast cancer cells, a widely used model for studies of
the effects of steroids on cell proliferation, treatment with synthetic
progestins results in a biphasic change in the rate of cell cycle
progression, consisting of an initial transient acceleration through
G1 phase and a subsequent increase in the S phase fraction,
followed by cell cycle arrest and growth inhibition accompanied by a
decrease in the S phase fraction (23, 25, 38, 55, 61). Thus,
two distinct, opposing effects of progestins on cell cycle progression
can be observed within the one cell type, emphasizing the complexity of
progestin effects on cell proliferation. Data from both breast cancer
cells in tissue culture and in vivo studies of the uterus and mammary gland demonstrate that sensitivity to both stimulation and inhibition is present only during G1 phase (5, 38, 55).
Since endogenous hormones play a key role in the development of
hormone-dependent cancers, exposure to exogenous steroid hormones through the use of oral contraceptives and hormone replacement therapies might influence the risk of developing such cancers. Combined
oral contraceptives or hormone replacement therapies containing both an
estrogen and progestin confer protection from endometrial cancer, while
treatment with estrogen alone leads to an increase in risk
(46). In contrast, while the effect of hormonal therapies on
breast cancer risk has been controversial, there appears to be a slight
increase in risk in recent or current users (7, 8), and in
postmenopausal women the risk of breast cancer associated with estrogen
use does not appear to be reduced by the addition of progestin
(6). Thus, progestins appear to be protective against
endometrial cancer but not breast cancer. Nevertheless, synthetic
progestins have an established role in the therapy of both breast and
endometrial cancers (46, 49, 60). The mechanism for the
antitumor action of progestins is unknown, but inhibition of breast
cancer cell proliferation is a likely contributor. Despite these issues
and the role of progesterone in normal mammary development and
differentiation, the effects of progesterone and synthetic progestins
on cell proliferation have not been widely studied from a mechanistic
viewpoint, and mechanisms for progestin inhibition of proliferation
remain unidentified at a molecular level. However, the demonstration of
steroidal control of cell cycle progression at defined points within
G1 phase (40) suggests that these agents and
their antagonists act via their respective receptors to regulate,
directly or indirectly, the expression of genes with a central role in
the control of cell cycle progression through G1 phase.
Research aimed towards identification of these molecular targets has
focused on the roles of c-Myc and cyclins and the associated
cyclin-dependent kinases (CDKs) (1, 10, 11, 35, 37, 38, 44,
64).
The sequential activation of CDKs and consequent phosphorylation of
specific substrates govern progress through the cell cycle. Key
substrates of the CDKs with G1 phase-specific actions
include the retinoblastoma gene product, pRB, and the related protein p107, although it is likely that other substrates remain to be identified: unlike cyclin D1, cyclin E is required for G1
progression in the absence of functional pRB (reviewed in references
24, 50, and 51). CDK activity is
subject to multiple levels of regulation. Since CDKs are inactive in
the absence of cyclin binding, cyclin abundance is a major determinant
of cyclin-CDK activity (24, 32, 50, 51). Each cyclin is thus
typically present for only a restricted portion of the cell cycle.
Alteration of cyclin abundance is sufficient to alter the rate of cell
cycle progression, since overexpression of either of the principal
G1 cyclins, cyclins D and E, accelerates cells through
G1 and, conversely, inhibition of their function by
antibody microinjection prevents entry into S phase (24, 50,
51). CDK activity is also regulated by a network of kinases and
phosphatases so that cyclin binding is sufficient only for partial
activation (32). The CDK-activating kinase (CAK)
phosphorylates a conserved threonine residue in the T-loop of the
kinase, stabilizing the cyclin-CDK interaction and altering the
conformation of the substrate binding site (33); this
phosphorylation is necessary for full activity. Even in the presence of
activating phosphorylation at this threonine residue and cyclin
binding, CDKs can be inhibited by phosphorylation of threonine and
tyrosine residues within the catalytic cleft. The dual-specificity
Cdc25 phosphatases activate CDKs by dephosphorylating these inhibitory
residues (32). In vertebrate cells three such phosphatases
have been identified: Cdc25A, -B, and -C. Cdc25A appears to target CDKs
active at the G1-S transition, since microinjection of
anti-Cdc25A antibodies blocked progression from G1 into S
phase (22, 27), while anti-Cdc25C antibodies led to arrest
in G2 phase (31). Overexpression of dominant
negative Cdc25B mutants indicates that Cdc25B regulates
G2-M transit (13).
A further level of control results from the actions of two families of
specific CDK-inhibitory proteins. One family, for which the prototype
is p16INK4, specifically targets the kinases which
associate with the D-type cyclins, Cdk4 and Cdk6. Their inhibitory
activity appears to be largely due to competition with the cyclin for
CDK binding, although they can also interact with cyclin D-Cdk4 and
cyclin D-Cdk6 complexes (21, 45, 51). Members of the other
family, of which p21 (WAF1, Cip1, sdi1) and p27 (Kip1) are the best
studied, interact with a broader range of CDKs, including Cdk2 as well
as Cdk4 and Cdk6. Recent structural studies of p27 bound to cyclin
A-Cdk2 have indicated that p27 interacts with both cyclin A and Cdk2, occluding the catalytic cleft and causing multiple structural changes
around it (47). Sequence conservation of the residues involved in cyclin and CDK interaction suggests that other members of
this inhibitor family probably bind in a similar fashion
(47). Both CAK and Cdc25 activation of the kinase are also
prevented by inhibitor binding, probably by steric hindrance (2,
48). Despite these multiple modes of inhibition of CDK activity,
not all cyclin-CDK complexes containing p21 or p27 are inactive.
Histone H1 kinase activity, most likely due to Cdk2, is present in p21 immunoprecipitates (66), perhaps representing an alternate
mode of interaction where either the cyclin or CDK, but not both,
interacts with p21 (12). Histone H1 activity is not present
in p27 immunoprecipitates, although they display pRB kinase activity,
apparently due to Cdk4 and Cdk6 (53). Recent data indicate
that p21 and p27 as well as a related inhibitor, p57Kip2,
stabilize cyclin D-Cdk4 and cyclin D-Cdk6 complexes in vitro (30). High-stoichiometry p21 or p27 binding appears to be
required for inhibition of Cdk4 activity (3, 30).
Consequently, these molecules appear to have functions in addition to
CDK inhibition, perhaps as adapters which not only promote assembly of
the cyclin-CDK complexes but also target these complexes to specific
intracellular compartments or substrates.
To identify the sequence of molecular events linking progestin action
with inhibition of cell cycle progression, we have examined the effects
of progestin treatment of T-47D human breast cancer cells on the
abundance and activity of cyclin D1-Cdk4, cyclin D3-Cdk4, and cyclin
E-Cdk2, the major G1 phase CDK complexes in these cells.
These experiments demonstrate that progestins not only regulate cyclin
abundance but also lead to increased association of the CDK inhibitor
p27 with G1 cyclin-CDK complexes, providing a mechanism for
their growth-inhibitory effects in breast cancer cells.
 |
MATERIALS AND METHODS |
Cell culture.
T-47D human breast cancer cells (obtained from
the EG & G Mason Research Institute, Worcester, Mass.) were cultured in
RPMI 1640 medium supplemented with 5% fetal calf serum, insulin (10 µg/ml), and gentamicin (20 µg/ml). ORG 2058 (16
-ethoxy-21-hydroxy-19-norpregn-4-en-3,20-dione; Amersham
Australia, Castle Hill, New South Wales, Australia) was dissolved in
ethanol at a 1,000- or 2,000-fold final concentration and added to
cells in exponential growth. Control cultures received ethanol to the
same final concentration. In some experiments untreated control
cultures were harvested at the time of ORG 2058 or vehicle addition to
the remainder of the replicate cultures.
A clonal derivative of T-47D cells expressing cyclin D1 under control
of the metal-responsive metallothionein promoter, T-47D
MTcycD1-3,
was used for the experiments presented in Fig. 12. The derivation and
characteristics of this cell line have been previously described
(36, 39); these cells retain progestin sensitivity similar
to that of the parent cell line. T-47D
MTcycD1-3 cells were treated
with 10 nM ORG 2058 or vehicle as described above, and 24 to 48 h
later either zinc (50 to 75 µM ZnSO4) or water (vehicle)
was added.
Generation of recombinant proteins.
The pRB fusion protein
substrate for the Cdk4 activity assay was a glutathione
S-transferase (GST)-pRB construct encoding amino acids 773 to 928 (supplied by Ed Harlow, Massachusetts General Hospital Cancer
Center, Charlestown). A restriction fragment of pVL1392
His6-Cdk2 (44) encompassing the Cdk2 open
reading frame was cloned into the expression vector pGEX-2T (Pharmacia,
Uppsala, Sweden) to yield a construct encoding the GST-Cdk2 fusion
protein used as a substrate for measurement of CAK activity. GST-Cdc25A was generated by using a construct provided by David Beach, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y. Escherichia coli
transformed with the appropriate expression vectors was induced by the
addition of 0.2 to 0.4 mM isopropylthiogalactopyranoside and incubated
for 3 h at room temperature. The bacterial pellets were lysed by
sonication, and the fusion proteins were purified by affinity
chromatography on glutathione-agarose beads and then eluted with 15 mM
reduced glutathione.
Western blot analysis and immunoprecipitation.
Cells were
lysed as follows. Cell monolayers were washed twice with ice-cold
phosphate-buffered saline and then scraped into ice-cold lysis buffer
(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 of aprotinin
per ml, 10 µg of leupeptin per ml, 1 mM phenylmethylsulfonyl fluoride, 200 µM sodium orthovanadate, 10 mM sodium pyrophosphate, 20 mM NaF) or ice-cold kinase lysis buffer (50 mM HEPES [pH 7.5], 1 mM
dithiothreitol, 150 mM NaCl, 10% [vol/vol] glycerol, 0.1% Tween 20, 1 mM EDTA, 2.5 mM EGTA, 10 mM
-glycerophosphate, 10 µg of
aprotinin per ml, 10 µg of leupeptin per ml, 0.1 mM
phenylmethylsulfonyl fluoride, 0.1 mM sodium orthovanadate, 1 mM NaF).
At selected time points an aliquot of this suspension was diluted in
RPMI 1640-5% fetal calf serum and stained for later flow cytometric DNA analysis by addition of ethidium bromide (50 µg/ml) and Triton X-100 (0.2%). Lysates in lysis buffer were incubated for 5 min on ice,
and the cellular debris was cleared by centrifugation (15,000 × g, 5 min, 4°C). Lysates in kinase lysis buffer were snap
frozen in liquid nitrogen and later thawed on ice, vortexed every 10 min for 60 min, and finally cleared by centrifugation (15,000 × g, 5 min, 4°C). The cleared lysates were aliquoted and stored at
80°C. Similar results were obtained from Western blotting or immunoprecipitation by either lysis technique.
For immunoprecipitation, whole-cell lysates (typically 500 µg) or
lysates fractionated by gel filtration chromatography were precleared
by incubation with protein A-Sepharose beads (Zymed, San Francisco,
Calif.) (1 h, 4°C) and then immunoprecipitated by incubation (1 to
2 h, 4°C) with either anti-cyclin E (C19) or anti-Cdk4 (H22)
antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.)
followed by incubation with protein A-Sepharose beads (0.5 to 1 h,
4°C). In some experiments the antibodies were chemically cross-linked
to the protein A-Sepharose beads by incubation in 5 mg of dimethyl
pimelimidate per ml-0.2 M sodium tetraborate (pH 9.0) for 30 min at
room temperature, essentially as described previously (17).
The immunoprecipitated proteins were washed with lysis buffer and
resuspended in sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) sample buffer (63 mM Tris-HCl [pH 6.8],
10% [vol/vol] glycerol, 2% SDS, 5%
-mercaptoethanol).
Samples of immunoprecipitated or total protein (40 to 100 µg) in
SDS-PAGE sample buffer were heated to 95°C for 3 min and then
separated by SDS-PAGE and transferred to nitrocellulose. Following
transfer, the nitrocellulose membrane was stained with 0.5% Ponceau S
in 10% acetic acid to monitor protein loading; blots with more than
one lane not displaying equivalent loading were discarded, and the
procedure was repeated. The membranes were incubated (1 to 2 h at
room temperature or overnight at 4°C) with the following primary
antibodies: p107 (C-18), cyclin D3 (C-16), cyclin E (HE12), Cdk2 (M2),
and Cdk4 (C-22) antibodies from Santa Cruz Biotechnology; cyclin D1
antibody (DCS6) from Novocastra, Newcastle-upon-Tyne, United Kingdom;
pRB (14001A) antibody from Pharmingen, San Diego, Calif.; p21 (C24420)
and p27 (K25020) antibodies from Transduction Laboratories, Lexington, Ky.; rabbit polyclonal antibody raised against a pRB peptide
phosphorylated on the site corresponding to Ser780, a residue
preferentially phosphorylated by cyclin D1-Cdk4 (29),
generously provided by Yoichi Taya, National Cancer Center Research
Institute, Tokyo, Japan; and affinity-purified rabbit polyclonal Cdc25A
antibody, generously provided by Ingrid Hoffmann, Deutsches
Krebforschungszentrum, Heidelberg, Germany. Following incubation (1 h
at room temperature) with horseradish peroxidase-conjugated sheep
anti-mouse or donkey anti-rabbit secondary antibody (Amersham
Australia), specific proteins were visualized by chemiluminescence
(Dupont NEN, Boston, Mass.). Where the proteins of interest were of
sufficiently different mobilities, membranes were incubated either
sequentially or simultaneously with several primary antibodies. Before
incubation with primary antibodies requiring a different secondary
antibody, the horseradish peroxidase on the initial secondary antibody
was inactivated by incubation (overnight, 4°C) in 10 mM Tris (pH
7.4)-150 mM NaCl-0.05% Triton X-100-5% skim milk powder-0.2%
sodium azide. Where proteins of similar mobility were examined by using
the same membrane, it was stripped by incubation (20 min, 50°C) in
62.5 mM Tris-HCl (pH 6.7)-2% SDS-100 mM
-mercaptoethanol.
Relative abundances were quantitated with a Molecular Dynamics
(Sunnyvale, Calif.) densitometer and IP LabGel analysis software (Signal Analytics, Vienna, Va.). Quantitation of protein levels by this
method was linear over the range of intensities measured. Within each
experiment data were available from multiple control samples (typically
four or five vehicle-treated controls harvested in parallel with the
ORG 2058-treated samples and, in some experiments, an additional
untreated control harvested at the time of treatment). Protein
abundances in treated cultures were calculated relative to the means
for these controls, which typically had a standard error of the mean
(SEM) of ~10%.
Kinase assays.
The histone H1 kinase activity of cyclin E
immunoprecipitates (100 µg of cell lysate) was measured as previously
described (37, 44) with 10 µg of histone H1 as the
substrate. There was little or no detectable phosphorylation in samples
immunoprecipitated by using beads without antibody or following
blocking of specific antibody binding with the appropriate antigenic
peptide. For Cdk4 assays, cells were harvested and lysed by using
kinase lysis buffer as described above. The kinase activity of Cdk4
immunoprecipitates of >400 µg of these lysates was measured by using
10 µg of GST-pRB773-928 fusion protein substrate as
previously described (44). The degree of background
phosphorylation in each pRB phosphorylation assay was estimated from
parallel control samples immunoprecipitated either by using beads
without antibody or following blocking of specific antibody binding
with the appropriate antigenic peptide. The two controls yielded
similar amounts of background phosphorylation.
Cdk7 (06-377; Upstate Biotechnology, Lake Placid, N.Y.)
immunoprecipitates from whole-cell lysates were assayed for CAK
activity by using GST-Cdk2 as the substrate. To determine the degree of background phosphorylation, control lysate was immunoprecipitated with
antibody which had been preincubated in the presence of competing antigenic peptide at 30°C for 30 min. The kinase reaction was initiated by resuspending each immunoprecipitate in 30 µl of 50 mM
HEPES (pH 7.5)-30 mM MgCl2-1 mM dithiothreitol-90 µM
ATP containing 10 µCi of [
-32P]ATP and 10 µg of
GST-Cdk2. After incubation for 20 min at 30°C, the reaction was
terminated by the addition of SDS-PAGE sample buffer.
Following termination of kinase reactions, samples were incubated at
95°C for 2 min in SDS sample buffer and separated by SDS-10% PAGE.
Substrate phosphorylation was quantitated with a Molecular Dynamics
PhosphorImager Scanner (model 445 SI) followed by analysis with IP
LabGel analysis software (Signal Analytics), and relative activity was
calculated as described above for Western blots.
RNA isolation and Northern analysis.
Total RNA was extracted
(by a guanidinium isothiocyanate-cesium chloride procedure) and blotted
as previously described, using 20 µg of total RNA/lane
(4). Filters were hybridized (overnight, 50°C) with cDNA
probes radioactively labelled with [
-32P]dCTP to a
specific activity of approximately 109 cpm/µg of DNA by
using the Prime-A-Gene labelling kit (Promega Australia, Annandale, New
South Wales, Australia). Restriction enzyme digestion was used to
provide the listed fragments from plasmids supplied by the following
investigators: cyclin D1, a 1.3-kb EcoRI fragment
encompassing the entire open reading frame (David Beach, Cold Spring
Harbor Laboratory); cyclin E, a 2.5-kb EcoRI fragment
encompassing the entire open reading frame (Steven Reed, Scripps
Research Institute, La Jolla, Calif.); Cdc25A, a 2.3-kb
EcoRI fragment encompassing the entire open reading frame (David Beach); c-myc, a 0.45-kb PstI fragment
corresponding to the second exon (Geoff Symonds, RW Johnson
Pharmaceutical Research Institute, Sydney, New South Wales, Australia);
and 36B4, a 0.22-kb PstI fragment (Pierre Chambon, Institut
de Genetique et de Biologie Moleculaire et Cellulaire, Strasbourg,
France).
Cdc25A activation assay.
Cyclin E or Cdk4 immunoprecipitates
were washed twice in lysis buffer or kinase lysis buffer, respectively,
without phosphatase inhibitors and twice in phosphatase buffer
containing 50 mM HEPES (pH 8.2), 15 mM MgCl2, 1 mM
dithiothreitol, and 1 mM ATP. The immunoprecipitates were incubated
with GST-Cdc25A in phosphatase buffer for at least 1 h at 30°C
and then washed twice in lysis buffer and twice in 10 mM HEPES (pH
7.5)-1 mM dithiothreitol. Kinase activity was assayed as described
above.
Gel filtration.
Cell lysates prepared in kinase lysis buffer
were passed through a 0.22-µm-pore-size MILLEX-GV4 filter (Millipore,
Lane Cove, New South Wales, Australia) and fractionated on a Hiload
16/60 Superdex 200 column-fast protein liquid chromatography system (Pharmacia Biotech, Uppsala, Sweden) with 20 mM HEPES (pH 7.5)-250 mM
NaCl-1 mM EDTA-0.1% (vol/vol)
-mercaptoethanol-0.01% Tween 20 at a flow rate of 1.2 ml/min. The void volume of the column was 45 ml.
Either 2- or 3-ml fractions were collected between elution volumes of
55 and 95 ml. Column calibration was performed under the same
conditions with ferritin (440 kDa), aldolase (158 kDa), and ovalbumin
(43 kDa). Protein was concentrated prior to Western blotting: 500 µl
of each fraction was placed at
70°C for 3 h with 2.5 ml of
acetone and 10 µg of carrier bovine serum albumin protein, and
pellets were collected by centrifugation (15,000 × g,
10 min, 4°C) and then resuspended by boiling for 4 min in 30 µl of
SDS sample buffer.
 |
RESULTS |
Previous experiments in this laboratory characterized the effects
of synthetic progestins on the cell proliferation kinetics of human
breast cancer cell lines, particularly T-47D, and defined a biphasic
response consisting of initial acceleration of cell cycle progression
and subsequent inhibition of proliferation (38, 55). The
relative magnitudes of the two components of the response are dependent
in part on the growth rate and hence on the culture conditions. To
emphasize the growth-inhibitory response, for the present series of
experiments cells were cultured under conditions leading to optimal
growth rates, i.e., in medium supplemented with insulin and fetal calf
serum. Consistent with previous data (38, 55), treatment
with a maximally effective concentration of the progestin ORG 2058 (10 nM) led to an initial small increase in the S phase fraction, followed
by arrest in G1 phase and consequent accumulation of
G1 phase cells at the expense of the other phases of the
cell cycle (Fig. 1). Inhibition of entry
into S phase was first apparent after 18 h of treatment, as
indicated by a relative lack of cells in early S phase (Fig. 1A),
although the S phase fraction had not yet decreased significantly below
that of control cells (Fig. 1B). The decrease in the S phase fraction
was maximal at 24 h (Fig. 1B), and the G2-plus-M
fraction subsequently decreased (Fig. 1A) such that by 30 h up to
86% of the cells were in G1 phase.

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FIG. 1.
Progestin inhibition of cell cycle progression.
Exponentially proliferating T-47D breast cancer cells were treated with
the synthetic progestin ORG 2058 (10 nM) or vehicle (Control). At
intervals thereafter, cells were lysed and an aliquot was stained for
flow cytometric measurement of DNA content. (A) Representative DNA
histograms. (B) Percentages of control cells ( ) or ORG 2058-treated
cells ( ) in S phase. Data from a total of six experiments have been
pooled and are shown as means ± SEMs, where the SEM exceeds the
size of the symbol used.
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Inhibition of G1 CDK activity and reduced
phosphorylation of pRB and p107 following progestin treatment.
Initial experiments examined the phosphorylation of pRB and p107, in
vivo substrates of G1 CDKs. In control exponentially proliferating cells both phosphorylated and underphosphorylated pRB
were apparent, although the underphosphorylated form comprised only
~20% of the total (Fig. 2A and B).
Little change in the proportions of phosphorylated and
underphosphorylated pRB was apparent at 6 or 12 h after progestin
treatment (Fig. 2A and B), although there was a slight, <50%,
increase in the total pRB abundance and a consequent increase in the
absolute amount of underphosphorylated pRB at 12 h (Fig. 2C). By
18 h the proportion of pRB in the underphosphorylated form had
begun to increase, and after 24 h or more, 70 to 80% of pRB was
in this form (Fig. 2A and B). Although the total pRB abundance
decreased by ~50% at 24 to 30 h, the large increase in the
proportion of underphosphorylated pRB resulted in an ~2-fold increase
in the abundance of the underphosphorylated form from 18 to 30 h
(Fig. 2C). Phosphorylated pRB, detected by antibodies recognizing
either the whole molecule or a site specifically phosphorylated by
cyclin D1-Cdk4 (RB-P-Ser780 [29]), began to decrease
in abundance by 18 h and was markedly reduced by 24 h (Fig.
2). The effects of progestin treatment on p107 phosphorylation and
abundance paralleled those on pRB: at 18 h more
underphosphorylated p107 was apparent, and after 24 h or more,
p107 was largely unphosphorylated and reduced in total abundance (Fig.
2A). Comparison of the data in Fig. 1B and 2C indicated that the
accumulation of underphosphorylated pRB reached its maximum by 18 h, a time when little change in the S phase fraction was detected, and
thus accumulation of the inhibitory form of pRB preceded the inhibition
of entry into S phase by up to 6 h.

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FIG. 2.
Progestin effects on pRB and p107. Exponentially
proliferating T-47D breast cancer cells were treated with the synthetic
progestin ORG 2058 (10 nM) or vehicle. At intervals thereafter
whole-cell lysates were immunoblotted with antibodies raised against
pRB, p107, or a pRB-derived phosphopeptide that contains a cyclin
D1-Cdk4-specific target (RB-P-Ser780). (A) Representative blots from
one of five experiments (pRB) or two experiments (p107 and
RB-P-Ser780). (B) The slower-mobility (hyperphosphorylated) ( and
) and faster-mobility (hypophosphorylated) ( and ) forms of
pRB in the blot shown in panel A were quantitated by densitometry and
are presented as the proportion of total pRB for both control cells
(open symbols) and ORG 2058-treated cells (closed symbols). (C) Raw
densitometric measurements of pRB abundance after ORG 2058 treatment
for the representative blot shown in panel A. For clarity, data from
control lanes have been pooled and are presented (means ± SEMs)
as the 0-h points. , total; , hyperphosphorylated; ,
hypophosphorylated. arb., arbitrary.
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Since decreased CDK activity was a probable mechanism for the decreased
phosphorylation of pRB and p107, the activities of CDK complexes active
in G1 phase were measured by using in vitro kinase assays.
T-47D cells express both cyclin D1 and cyclin D3, but cyclin D2 is
essentially undetectable (4, 56). Both Cdk4 and Cdk6 are
expressed, but cyclin D1 immunoprecipitates contain little detectable
Cdk6 under conditions where Cdk6 can be readily detected in parallel
immunoprecipitates from other cell lines (56). Thus,
measurement of the activities of Cdk4 immunoprecipitates (comprising
both cyclin D1-Cdk4 and cyclin D3-Cdk4) and cyclin E immunoprecipitates
allows estimation of activity of the major G1 phase CDK
complexes in these cells. Cdk4 immunoprecipitates from control cells
phosphorylated a truncated pRB fusion protein, and this kinase activity
was substantially reduced in the presence of a competing peptide or in
the absence of primary antibody (Fig. 3A
and data not shown), confirming the specificity of the assay. No
significant change in Cdk4 activity was apparent in the first 12 h
following progestin treatment (Fig. 3). However, Cdk4 activity declined
after
18 h of progestin treatment (Fig. 3A), such that the specific
Cdk4 activity (after background subtraction) was reduced to 40% of the
control value by 18 h and to 10% of the control value by 24 h (Fig. 3B). A slight increase in cyclin E-associated kinase activity
was apparent at 6 h, followed by a reduction to ~40% of the
control value by 18 h (Fig. 3). The activity of this kinase
reached a minimum of 23% at 24 h and remained below 50% of the
control value at 30 h, although partial recovery of kinase activity was observed at 30 h (Fig. 3B). Since a marked decrease in the activities of both Cdk4 and cyclin E-associated kinases preceded
a significant decrease in the S phase fraction by up to 6 h
(compare Fig. 1 and 3), the decrease in CDK activity is implicated in
the accompanying inhibition of pRB and p107 phosphorylation and hence
arrest in G1 phase following progestin treatment.

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FIG. 3.
Progestin inhibition of CDK activity. Exponentially
proliferating T-47D breast cancer cells were treated with the synthetic
progestin ORG 2058 (10 nM) or vehicle. At intervals thereafter whole
cell-lysates were prepared and immunoprecipitated with antibodies to
Cdk4 or cyclin E. Either GST-pRB773-928 (Cdk4 activity) or
histone H1 (cyclin E-Cdk2 activity) was used as a substrate. (A) The
upper panel shows phosphorylated GST-pRB773-928 from a
representative experiment, visualized by SDS-PAGE and autoradiography.
The first lane (+ pep) contains a sample from untreated cells harvested
at the time of ORG 2058 or vehicle addition, in which the
immunoprecipitation was performed in the presence of competing
antigenic peptide. The lower panel shows phosphorylated histone H1 from
a representative experiment visualized by SDS-PAGE and autoradiography.
(B) Phosphorylated substrates were detected with
PhosphorImager. Points represent the means ± SEMs from
two to four observations, relative to the average control value for
each experiment. For Cdk4 activity the background in each assay was
estimated from samples immunoprecipitated in the presence of competing
peptide or in the absence of antibody and has been subtracted from the
individual data points. , Cdk4 activity; , cyclin E-Cdk2
activity.
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Decreased expression of cyclins D1, D3, and E and increased
expression of the CDK inhibitors p21 and p27 following progestin
treatment.
To investigate possible causes of the decreased kinase
activity, the expression of components of the G1 cyclin
complexes was examined. Cyclin D1 protein initially slightly increased
in abundance (Fig. 4). However, this was
transient, and at 18 to 24 h cyclin D1 levels in progestin-treated
cells were ~50% of those in control cells (Fig. 4). Northern
analysis showed that, similarly, cyclin D1 mRNA was initially induced
but subsequently declined to ~60% of control at 18 h, remaining
below control levels at 24 h (Fig. 4C). A slight decrease in
cyclin E protein expression was apparent as early as 12 h, and the
decline continued until cyclin E reached a minimum of 25% of the
control value at 24 h (Fig. 4). Consistent with the partial
recovery of cyclin E-associated kinase activity observed at 30 h
(Fig. 3), cyclin E expression partially recovered between 24 and
30 h to reach 50% of the control value (Fig. 4). The cyclin E
mRNA abundance was reduced following 18 to 24 h of progestin
treatment, to ~50% of the control value or less (Fig. 4C). The
changes in cyclin D1 and E mRNAs were similar in timing and magnitude
to the changes in protein abundance, suggesting that progestin
treatment regulated cyclin D1 and E expression in large part via
transcriptional mechanisms. No significant changes in cyclin D3, Cdk2,
or Cdk4 protein expression were apparent until 24 h after
progestin treatment, when decreases of 40 to 50% were observed; the
decreases were maintained at 30 h (Fig. 4). The abundance of p21
protein did not change until 18 h, when it increased ~2-fold;
the increase was maintained at 24 h but was of smaller magnitude
thereafter (Fig. 5). Upon more extended
progestin treatment, p21 levels fell below control values (see Fig.
12). The abundance of p27 began to increase slightly at 18 to 24 h
and had increased approximately twofold at 30 h (Fig. 5). Thus, of
the proteins examined, only cyclin D1, cyclin E, and p21 displayed
alterations in abundance of
2-fold at 18 h, when marked
decreases in Cdk4 activity and cyclin E-associated kinase activity were
observed.

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FIG. 4.
Cyclin and CDK expression following progestin treatment.
Exponentially proliferating T-47D breast cancer cells were treated with
the synthetic progestin ORG 2058 (10 nM) or vehicle, and at intervals
thereafter whole-cell lysates or total cellular RNA were prepared. (A)
Representative Western blots for cyclin D1, cyclin D3, cyclin E, Cdk2,
and Cdk4. UT, untreated control harvested at the time of treatment. (B)
Data representing the means ± SEMs from three to five
observations (cyclin D1), three or four observations (cyclin D3), or
two to four observations (cyclin E) are expressed relative to the
average control value in each experiment. (C) Representative Northern
blots for cyclin D1, cyclin E, and, as a control for RNA loading on the
cyclin E blot, 36B4. The loading control for the cyclin D1 blot is
shown in Fig. 6.
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FIG. 5.
CDK inhibitor expression following progestin treatment.
Exponentially proliferating T-47D breast cancer cells were treated with
the synthetic progestin ORG 2058 (10 nM) or vehicle, and at intervals
thereafter whole-cell lysates were prepared. (A) Representative Western
blots for p21 and p27. (B) Data representing the means ± SEMs
from three to six (p21) or four or five (p27) observations are
expressed relative to the average control value in each experiment.
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Changes in Cdc25 or CAK activity do not account for
progestin-induced decreases in CDK activity.
Further experiments
examined the possibility that altered CDK phosphorylation might
contribute to the decrease in activity following progestin treatment.
First, regulation of phosphorylation at inhibitory residues within the
catalytic cleft was addressed, since such inhibition is dominant over
activation resulting from either cyclin binding or CAK activation (see
the introduction). Of the family of phosphatases responsible for
relieving this inhibition, Cdc25A appears to have specificity for
G1 CDKs in vivo, and therefore its expression was examined.
Following ORG 2058 treatment a small increase of <50% was apparent at
6 h, but thereafter Cdc25A mRNA decreased in abundance to reach a
minimum of ~30% of the control value at 18 to 24 h (Fig.
6A). In view of evidence for
c-myc regulation of Cdc25A (14), c-myc
expression was also examined. Consistent with previous data (38,
65), progestin treatment increased c-myc expression
after 1 h, but thereafter c-myc expression decreased to
<50% of the control value (Fig. 6A). The decrease in c-myc expression was apparent within 6 h, thus preceding the decline in
Cdc25A expression by >12 h. Measurement of Cdc25A protein levels revealed only an ~50% decrease in abundance at 18 to 24 h (Fig. 6B).

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FIG. 6.
Cdc25A and c-myc expression following
progestin treatment. Exponentially proliferating T-47D breast cancer
cells were treated with the synthetic progestin ORG 2058 (10 nM) or
vehicle (Control), and at intervals thereafter total cellular RNA or
whole-cell lysates were prepared. (A) Representative Northern blot
sequentially probed with Cdc25A, c-myc, and 36B4 (as a
control for RNA loading). (B) Representative Western blot for Cdc25A.
UT, untreated cells harvested at the time of treatment.
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To determine whether a lack of activating phosphatase activity might
contribute to the reduction in CDK activity following progestin
treatment, attempts were made to reactivate kinase complexes from ORG
2058-treated cells by using bacterially expressed recombinant GST-Cdc25A. Incubation of GST-Cdc25A with cyclin E immunoprecipitates from exponentially proliferating T-47D cells resulted in activation of
the cyclin E-associated kinase (Fig. 7A).
The activation was abolished in the presence of the phosphatase
inhibitors sodium vanadate and sodium fluoride, and incubation with
recombinant GST alone had no effect on cyclin E-associated kinase
activity (Fig. 7A), demonstrating that the activation of the kinase
resulted from Cdc25A phosphatase activity. The effect of Cdc25A on the CDK activity of cyclin E immunoprecipitates from control or
progestin-treated cells was then examined. These experiments
demonstrated activation of cyclin E-Cdk2 from both control and
progestin-treated cells (Fig. 7B). However, since the degree of
activation was similar in both cases, following Cdc25A activation the
cyclin E-Cdk2 activity from progestin-treated lysates remained at
~20% of that observed in Cdc25A-activated immunoprecipitates from
control cells. This difference could not be accounted for by a lack of
Cdk2 in the immunoprecipitates, since in parallel cyclin E
immunoprecipitates the amount of Cdk2 was reduced by <40% (Fig. 7B).
Thus, a lack of Cdc25A activity did not appear to contribute to the
decreased cyclin E-Cdk2 activity following progestin treatment. Similar experiments failed to demonstrate Cdc25A activation of Cdk4
immunoprecipitates from either control or progestin-treated cells,
despite 15-fold increases in the amount of added Cdc25A (Fig. 7C).
These data argue against decreased Cdc25A expression contributing to
the decrease in Cdk4 activity following progestin treatment. Since the
presence of inhibitory phosphorylation did not contribute to the
decrease in CDK activity following progestin treatment, the possibility
that CAK activity might be regulated by progestins was next examined.
Bacterially expressed GST-Cdk2 was used as a substrate for Cdk7
immunoprecipitates, since in eukaryotes complexes containing Cdk7
(MO15) appear to be responsible for CAK activity (32).
However, these experiments did not demonstrate a decrease in CAK
activity following progestin treatment (Fig. 7D).

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FIG. 7.
Regulation of Cdc25 or CAK activity does not account for
decreased CDK activity following progestin treatment. (A) Replicate
cyclin E immunoprecipitates from 250 µg of lysate prepared from
exponentially proliferating T-47D cells were incubated with increasing
amounts of GST-Cdc25A (170, 340, 680, and 1,360 µg) or GST (360 and
1,440 µg) or without further addition ( ) at 30°C for 1 h and
washed, and then kinase activity was measured with a histone H1
substrate. One sample (Van) was incubated with 680 µg of GST-Cdc25A
in the presence of the phosphatase inhibitors vanadate (10 mM) and NaF
(50 mM). (B) T-47D breast cancer cells were treated with the synthetic
progestin ORG 2058 (10 nM) or vehicle for 24 h. Cyclin E
immunoprecipitates were incubated with or without Cdc25A as indicated,
and cyclin E-Cdk2 activity was measured with a histone H1 substrate.
Parallel immunoprecipitates were Western blotted with Cdk2 antibodies.
(C) Cdk4 immunoprecipitates from control or ORG 2058-treated (10 nM,
30 h) cells were incubated with GST-Cdc25A, and Cdk4 activity was
measured with GST-pRB773-928. (D) The kinase activity of
Cdk7 immunoprecipitates from control or ORG 2058-treated T-47D cells
was measured with a GST-Cdk2 substrate. The first lane (+ pep) contains
a sample from untreated cells in which the immunoprecipitation was
performed in the presence of competing antigenic peptide.
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Progestin treatment alters the molecular mass of cyclin E complexes
but not cyclin D complexes.
The data presented in Fig. 7 argued
that changes in CDK phosphorylation were unlikely to contribute to the
decreases in Cdk4 and cyclin E-associated kinase activities. This
suggested that alterations in either the abundance or composition of
the CDK complexes were probable mechanisms for the decreased kinase
activity. Since recent data have indicated that CDK complexes with
different compositions and activities can be resolved by gel filtration chromatography (for example, see references 44 and
54), lysates from control and progestin-treated
cells were fractionated to determine whether progestin treatment
affected the formation or composition of these complexes. Aliquots of
protein eluted from a Superdex 200 column were acetone precipitated and
Western blotted. In control lysates cyclin D1 typically eluted in a
single major broad peak with an apparent molecular mass of ~160 kDa
(Fig. 8A), consistent with previous data
from Superose 12 fractionation of Swiss 3T3 cells (42); in
some experiments (e.g., that shown in Fig. 8), a proportion also eluted
at a lower apparent molecular mass (Fig. 8A). The majority of cyclin D3
coeluted with cyclin D1 at ~160 kDa, but a form with a lower apparent
molecular mass was consistently observed. Since the
lower-molecular-mass forms of cyclin D1 and D3 coeluted with cyclin D1
produced by in vitro transcription and translation in reticulocyte
lysate (data not shown), it is likely that these represent free cyclin
D. Cyclin E eluted over a broad range of apparent molecular mass,
indicating the presence of two overlapping peaks with apparent
molecular masses of ~120 and ~200 kDa (Fig. 8B and
9; see also Fig. 12).

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FIG. 8.
Elution profiles of cyclins, CDKs, and CDK inhibitors in
control and progestin-treated lysates. Exponentially proliferating
T-47D breast cancer cells were treated with the synthetic progestin ORG
2058 (10 nM) or vehicle (Control) for 24 h. Whole-cell lysates
were fractionated on a Superdex 200 gel filtration column (void volume,
45 ml). The elution volumes of markers with known molecular masses
(ferritin, 440 kDa; aldolase, 158 kDa) are indicated; ovalbumin (43 kDa) eluted at 85 ml, corresponding to fraction 15. Fractions (2 ml)
corresponding to elution volumes of 57 to 82 ml were acetone
precipitated and then Western blotted for the proteins indicated.
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FIG. 9.
Time course of changes in elution profiles of cyclin E,
p21, and p27 following progestin treatment. Exponentially proliferating
T-47D breast cancer cells treated with the synthetic progestin ORG 2058 (10 nM) or vehicle (Control) in the same experiment as presented in
Fig. 8 were harvested after 12 or 18 h. Whole-cell lysates were
fractionated on a Superdex 200 gel filtration column (void volume, 45 ml). The elution volumes of markers with known molecular masses
(ferritin, 440 kDa; aldolase, 158 kDa) are indicated; ovalbumin (43 kDa) eluted at 85 ml, corresponding to fraction 15. (A) Fractions (2 ml) corresponding to elution volumes of 57 to 80 ml were acetone
precipitated and then Western blotted for the proteins indicated. (B)
Western blots of 2-ml fractions were quantitated by densitometry and
are presented as percentages of the total signal observed for each
protein. Data from three control samples (12, 18, and 24 h of
vehicle treatment) have been pooled and are presented as means ± SEMs, where the SEM exceeds the size of the symbol. , control; ,
ORG 2058.
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Both Cdk2 and Cdk4 eluted in two major peaks. One peak, representing
about 50% of the Cdk4 and the majority of the Cdk2, eluted at an
apparent molecular mass of <50 kDa (data not shown), close to the
expected position of monomeric CDK. For Cdk4 the other peak coeluted
with the major peaks of cyclins D1 and D3 at ~160 kDa (Fig. 8A). The
higher-apparent-molecular-mass form of Cdk2 encompassed the fractions
containing both the 120- and 200-kDa forms of cyclin E. A small
proportion of cyclins D1, D3, and E, Cdk2, and Cdk4 could be detected
at very high apparent molecular masses, >400 kDa (Fig. 8), suggesting
the existence of a small fraction of cyclin-CDK complexes associated
with other intracellular proteins. Examination of the elution profiles
of p21 and p27 suggested that these CDK inhibitors were all associated
with cyclin-CDK complexes, since no p21 or p27 with an apparent
mobility of below ~120 kDa was detected and both coeluted with cyclin
D1, cyclin D3, and cyclin E (Fig. 8). Overall, the mobilities of p27
and p21 were similar, although there was a tendency for more p21 to elute at a higher apparent molecular weight (Fig. 8A and 9).
In lysates prepared following 24 h of progestin treatment, several
significant differences in the elution profiles of the cyclins, CDKs,
and CDK inhibitors were apparent. The most marked of these was loss of
the ~120-kDa peak of cyclin E, such that essentially all the
detectable cyclin E coeluted with the higher-molecular-weight form of
cyclin E present in control cells (Fig. 8B and 9). There was a
corresponding shift in the elution profile of Cdk2, such that less Cdk2
was present in fractions corresponding to the 120-kDa peak of cyclin E
protein (Fig. 8B). In contrast with the marked changes in the elution
profile of cyclin E, there was no apparent change in the position of
the major peak of cyclin D1 or D3 (Fig. 8A). While a significant
fraction of p21 and p27 coeluted with cyclins D1 and D3 in lysates from
either control or progestin-treated cells, there was a clear increase
in the amount of p27 and a less marked increase in the amount of p21
eluting at apparent molecular masses of
200 kDa following progestin
treatment (Fig. 8A and 9). These higher-apparent-molecular-mass
fractions contained cyclin E, suggesting increased association between
cyclin E-Cdk2 complexes and these inhibitors following progestin
treatment.
The changes in the elution profiles of cyclin E, p27, and p21 were
further examined to determine the temporal relationship between these
changes and the inhibition of proliferation. After 12 h of
progestin treatment, before any inhibition of entry into S phase could
be detected, no change in the elution profile of any of these proteins
was detected (Fig. 9). However, after 18 h of progestin treatment,
when inhibition of entry into S phase was just commencing (Fig. 1), the
proportion of cyclin E in the 120-kDa form was markedly reduced, and
there was a corresponding increase in the proportion in the 200-kDa
form (Fig. 9). Similarly, after 18 h of progestin treatment, the
amounts of p21 and p27 eluting at >200 kDa (fractions 3 to 5 in Figure
9) were increased compared with the control values. The changes in the
elution profiles of cyclin E and p27 were more pronounced after 24 h than after 18 h of treatment (Fig. 9). Thus, these changes
paralleled the decrease in cyclin E-Cdk2 activity and preceded the
decrease in the S phase fraction by several hours, suggesting that they
were a cause rather a consequence of the inhibition of proliferation.
Increased association of p27 but not p21 with cyclin D-Cdk4
complexes following progestin treatment.
Since the data presented
in Fig. 8 indicated that the majority of cyclin-CDK complexes might
also contain CDK inhibitors, in further experiments immunoprecipitates
of fractionated lysates were used in in vitro kinase assays to
determine which fractions contained active CDK. In view of the limited
sensitivity of Cdk4 assays, Cdk4 activity was measured in 3-ml
fractions rather than in 2-ml fractions. Cdk4 activity from control
lysates displayed an elution profile similar to that of the
high-molecular-mass form of Cdk4, reaching a maximum at 150 to 200 kDa
(compare Fig. 8A and 10). However,
consistent with data obtained with whole-cell lysates (Fig. 3),
progestin treatment resulted in a marked decrease in Cdk4 activity
(Fig. 10A). Western blotting of Cdk4 immunoprecipitates from
fractionated control lysates indicated that the active fractions contained significant amounts of coimmunoprecipitating cyclin D1,
cyclin D3, Cdk4, p21, and p27 (Fig. 10B). The same fractions from
progestin-treated lysates contained less Cdk4 and significantly less
cyclin D1 and cyclin D3, consistent with the decrease in total
abundance of these proteins (Fig. 10B). However, no increase in p21 was
observed, and in some experiments the abundance of p21 appeared to
decrease relative to the abundance of other components of the complex
following progestin treatment (Fig. 10B). In contrast, the decrease in
the amount of cyclin D1 and cyclin D3 in the Cdk4 immunoprecipitates
was more marked than the decrease in the amount of associated p27 (Fig.
10B). In fraction 3, the abundance of cyclin D3 decreased by ~60%
while the abundance of p27 changed little, such that the p27/cyclin D3
ratio increased 2.3-fold. Similarly, in fraction 5, the abundance of
cyclin D1 decreased by >50% and there was a smaller decrease in the
amount of p27, such that the p27/cyclin D1 ratio increased ~2-fold.
These data suggest that the decreased abundance of cyclins D1 and D3 is
a major factor in the decrease in Cdk4 activity but that increased p27
association also likely contributes to the loss of activity.

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FIG. 10.
Cdk4 activity and complex composition of fractionated
T-47D lysates. Exponentially proliferating T-47D breast cancer cells
were treated with the synthetic progestin ORG 2058 (10 nM) or vehicle
(Control) for 24 h. Whole-cell lysates were fractionated on a Superdex
200 gel filtration column, and 3-ml fractions corresponding to elution
volumes of 58 to 81 ml were then immunoprecipitated with a Cdk4
antibody. (A) Kinase activities of the immunoprecipitates were measured
with a GST-pRB773-928 substrate. (B) Immunoprecipitates of
the fractions indicated were Western blotted with the antibodies
indicated.
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The low-activity form of cyclin E is associated with CDK
inhibitors.
Measurement of kinase activity in cyclin E
immunoprecipitates from gel filtration fractions revealed a peak of
activity associated with the 120-kDa form of cyclin E in control cells
(fractions 8 to 10) but much less activity in fractions 4 to 7, associated with the ~200-kDa form (Fig.
11A), although similar amounts of cyclin E were present in the two forms (Fig. 8B and 9). Some activity, comprising up to ~20% of the total, was present in the fractions with very high apparent molecular masses (>400 kDa). Following progestin treatment, near-background levels of kinase activity were
observed in fractions 8 to 11, consistent with the lack of cyclin E in
these fractions, and little activity could be detected associated with
the remaining major ~200-kDa peak of cyclin E protein (Fig. 11A). The
peak of activity in fraction 4 of progestin-treated lysate in Fig. 11A
was not consistently observed. Thus, while kinase activity was reduced
across the elution profile, this was most marked in the fractions
corresponding to the 120-kDa form of cyclin E.

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FIG. 11.
Cyclin E-Cdk2 activity and complex composition of
fractionated T-47D lysates. Exponentially proliferating T-47D breast
cancer cells were treated with the synthetic progestin ORG 2058 (10 nM)
or vehicle (Control) for 24h. Whole-cell lysates were fractionated on a
Superdex 200 gel filtration column, and fractions were then
immunoprecipitated with a cyclin E antibody. (A) Kinase activities of
immunoprecipitates corresponding to elution volumes of 59 to 61 (fractions 2 and 3), and 63 to 78 ml in 2-ml increments (fractions 4 to
11) were measured with a histone H1 substrate. (B) Immunoprecipitates
from fractions encompassing the peak of cyclin E protein were
resuspended in 25 µl of SDS sample buffer, and various volumes were
Western blotted in parallel with the antibodies indicated. (C) Cyclin E
immunoprecipitates corresponding to elution volumes of 65 to 68 ml
(fractions 5 and 6) and 72 to 74 ml (fractions 8 and 9) were Western
blotted with a Cdk2 antibody.
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Fractions encompassing the major peaks of cyclin E protein were
immunoprecipitated to examine whether differences in composition were
associated with the differences in activity. To facilitate comparison
between fractions containing different amounts of cyclin E, various
amounts of each fraction were Western blotted in parallel. These
experiments revealed clearly detectable p21 and p27 in fraction 6, corresponding to the ~200-kDa form of cyclin E, in control cells but
little in the remaining fractions (Fig. 11B). Thus, the ~120-kDa form
was relatively deficient in p21 and p27. Cdk2 displays a characteristic
increase in electrophoretic mobility upon activation via CAK
phosphorylation (15), and higher-resolution Western blotting
of cyclin E immunoprecipitates from control cells demonstrated that all
the detectable Cdk2 in the lower-molecular-weight complexes was
apparently CAK phosphorylated, while the higher-molecular-weight complexes contained Cdk2 of both electrophoretic mobilities (Fig. 11C).
Following progestin treatment, fractions encompassing the peak of
cyclin E protein at 200 kDa (fractions 4, 6, and 8), contained a 2.0- to 2.5-fold increase in the relative abundance of cyclin E-associated
p27 compared with the same fraction from control lysates (Fig. 11B).
Although in the experiment presented in Fig. 11B the relative abundance
of p21 also increased in fraction 6 following ORG 2058 treatment, this
was not a consistent observation. In progestin-treated cells, a smaller
proportion of cyclin E-associated Cdk2 was CAK phosphorylated (Fig.
11C). The mobility shift of the Cdk2 in these fractions following ORG
2058 treatment suggested that the reduced specific activity of the
complexes might in part be due to a lack of CAK activation, perhaps
resulting from increased p27 association.
Cyclin D1 induction in progestin-inhibited cells leads to a
reinitiation of cell cycle progression and altered molecular mass of
cyclin E complexes.
To determine to what degree reduced cyclin
expression contributed to the reduction in CDK activity and inhibition
of cell cycle progression following progestin treatment, a clonal
derivative of T-47D cells expressing cyclin D1 under the control of the
heavy-metal-responsive metallothionein promoter, T-47D
MTcycD1-3,
was treated with progestin for 24 to 48 h to inhibit cell cycle
progression and then with zinc to induce cyclin D1 expression.
Induction of cyclin D1 was sufficient to stimulate a cohort of
progestin-inhibited cells to resume cell cycle progression, such that
the S phase fraction, 10% after progestin pretreatment, increased to a
maximum of >40% after zinc treatment (Fig.
12A). Zinc treatment of cells
pretreated with vehicle rather than progestin increased the proportion
of cells in S phase to a similar maximum level, although the relative increase was smaller due to the higher S phase fraction in the absence
of progestin pretreatment (Fig. 12A). Western blots of cells harvested
after zinc treatment of either vehicle- or progestin-pretreated cells
demonstrated marked induction of cyclin D1 in progestin-pretreated cells and more modest induction of cyclin D1 in vehicle-pretreated cells (Fig. 12B). In progestin-pretreated cells cyclin D1 induction restored cyclin E expression to a level similar to that observed in
cells not treated with progestin, consistent with the observation that
cyclin D1 induction of cell cycle progression in these cells leads to
induction of cyclin E (39), but had little effect on the
abundances of p21 and p27. Induction of cyclin D1 in
progestin-pretreated cells also increased Cdk4 activity and cyclin
E-Cdk2 activity to levels similar to that following cyclin D1 induction
in vehicle-pretreated cells (Fig. 12B).

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FIG. 12.
Resumption of cell cycle progression after cyclin D1
induction in progestin-pretreated cells. Exponentially proliferating
T-47D MTcycD1-3 breast cancer cells, expressing cyclin D1 under the
control of a zinc-inducible promoter, were pretreated with the
synthetic progestin ORG 2058 (10 nM) or vehicle (ethanol [EtOH]) and
then treated with either zinc (ZnSO4) or vehicle (water).
(A) Percentages of cells in S phase after 24 h of ORG 2058 or EtOH
pretreatment followed by 15 h of zinc (50 µM) or water
treatment. (B) Lysates harvested from cells pretreated for 30 h
with either ORG 2058 or EtOH followed by 15 h of zinc (75 µM) or
water treatment were Western blotted for the indicated proteins or
immunoprecipitated with either Cdk4 or cyclin E antibodies for
measurement of kinase activity as described in the legend to Fig. 3.
(C) Whole-cell lysates from the experiment presented in panel B were
fractionated on a Superdex 200 gel filtration column (void volume, 45 ml). Fractions (2 ml) corresponding to elution volumes of 57 to 82 ml
were acetone precipitated and then Western blotted for the proteins
indicated. The elution volumes of markers with known molecular masses
(ferritin, 440 kDa; aldolase, 158 kDa) are indicated; ovalbumin (43 kDa) eluted at 85 ml, corresponding to fraction 15. (D) The Western
blots presented in panel C were quantitated by densitometry and are
presented as percentages of the total signal observed for each
protein.
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To determine whether the restoration of CDK activity and resumption of
cell cycle progression following cyclin D1 induction was accompanied by
alteration in the molecular mass of the cyclin-CDK complexes, lysates
from zinc-treated cells were separated by gel filtration
chromatography. Despite the partial synchronization following cyclin D1
induction in vehicle-pretreated cells, the elution profiles of cyclin
D1, cyclin E, p27, and p21 were similar to those observed in the
absence of zinc treatment (compare Fig. 12C and D with Fig. 8 and 9).
As expected, the elution profiles of cyclins D1 and E, p21, and p27 in
progestin-treated T-47D
MTcycD1-3 cells were similar to those
observed in progestin-treated T-47D cells. Interestingly, some p27
eluted at a low molecular mass (~70 to 90 kDa) following progestin
treatment (ORG 2058/water in Fig. 12C and D); this likely represents
monomeric p27 (42). However, following cyclin D1 induction
in progestin-pretreated cells, the elution profiles of cyclin E and p27
were altered compared with those from cells treated with progestin but
not zinc, such that they closely resembled the profiles obtained from
control cells. Thus, the 120-kDa form of cyclin E was clearly apparent following cyclin D1 induction in progestin-pretreated cells, while the
amount of p27 present in high-molecular-mass (>200-kDa) complexes decreased (Fig. 12C and D). These changes in elution profile preceded entry into S phase, since the lysates used for the gel filtration chromatography were harvested from cells just beginning to enter S
phase following cyclin D1 induction; i.e., the S phase fraction in
progestin-pretreated cells had not increased to more than 20%. These
data suggest that altered cyclin abundance is a major determinant of
the altered cyclin-CDK complex distribution following progestin treatment.
 |
DISCUSSION |
This study has focused on regulation of CDK activity accompanying
growth inhibition by the progestin ORG 2058 as a step towards dissecting the mechanisms that mediate progestin effects on normal physiology and on breast cancer. Marked decreases in both Cdk4 activity
and cyclin E-Cdk2 activity, with consequent decreases in the
phosphorylation of pRB and p107, preceded inhibition of entry into S
phase. Several different molecular events potentially contributing to
the decrease in CDK activity were observed: initial alterations in the
abundances of cyclin D1 and cyclin E, later changes in the abundances
of cyclin D3 and p27, and increased relative p27 association with
cyclin D-Cdk4 and cyclin E-Cdk2 complexes. These events were all
preceded by an early decline in c-myc expression, first
apparent within 3 to 6 h of progestin treatment.
Since both Cdk4 activity and cyclin E-Cdk2 activity are required for
progress from G1 into S phase (24, 50, 51), the observed decrease in these activities (Fig. 3) could account for progestin-mediated inhibition of proliferation. Substrates for these
CDKs, including pRB and p107, can thus be seen as the eventual targets
for this aspect of progestin action. Both pRB and p107 decreased in
total abundance as well as phosphorylation, such that very little of
the phosphorylated form of either protein was present following 24 h or more of progestin treatment. However, despite the decrease in
total abundance, the underphosphorylated form was increased in
abundance, reaching a maximum by 18 h, which was maintained until
at least 30 h. Thus, the accumulation of the growth-inhibitory
forms of these proteins preceded the inhibition of entry into S phase,
which was first apparent at 18 h. The partial recovery of cyclin
E-associated kinase activity between 24 and 30 h was not
accompanied by a detectable increase in pRB phosphorylation. Since at
that time little Cdk4 activity was present, this observation is
consistent with the view that cyclin E-Cdk2 phosphorylation of pRB is
dependent on prior pRB phosphorylation by cyclin D-associated kinases
(19). However, it is also possible that the recovery of
cyclin E-Cdk2 activity is sufficient to overcome the lack of Cdk4
activity in a fraction of the cell population, thus contributing to the
slow resumption of proliferation following extended progestin treatment
(55). Induction of differentiation is a major physiological role of progestins (5), but the decrease in pRB abundance
following progestin treatment contrasts with the frequent observation
of increased pRB expression accompanying differentiation in diverse cell types (9, 52, 57). However, since the mammary gland differs from most other tissues in that differentiation is followed by
involution, some differences in control of differentiation may be
expected. In this context it is interesting that pRB deficiency is
associated with increased apoptosis (63).
Examination of potential mechanisms for the decrease in CDK activity
accompanying progestin inhibition of proliferation revealed decreased
expression of mRNA encoding the CDK-activating phosphatase Cdc25A.
Reduced Cdc25A expression and increased tyrosine phosphorylation of
Cdk2 and Cdk4 have been implicated in growth arrest by different agents, including UV irradiation, alpha interferon, and transforming growth factor
(TGF-
) (26, 58, 59). In addition, since cyclin E-Cdk2 activates Cdc25A by phosphorylation (22),
decreased Cdc25A expression in combination with decreased cyclin E-Cdk2 activity might result in marked decreases in Cdc25A activity. Thus,
lack of Cdc25A activity provided an attractive mechanism contributing
to reduced CDK activity following progestin treatment. However, the
decrease in Cdc25A protein levels was less marked than the decrease in
the corresponding mRNA, consistent with the suggestion that the protein
may have a long half-life (26). Furthermore, treatment with
recombinant Cdc25A failed to restore the activity of cyclin
E-associated kinases to a level consistent with the level of Cdk2
present in the complexes, and no Cdc25A activation of Cdk4 from either
control or progestin-treated lysates was observed. Although the
possibility that the amount of Cdc25A used was insufficient to activate
Cdk4 cannot be excluded, this result is consistent with the lack of
tyrosine phosphorylation on Cdk4 and Cdk6 (but not Cdk2) in
exponentially proliferating MCF10A mammary epithelial cells
(26). These data indicate that regulation of Cdc25A does not
make a major contribution to progestin inhibition of proliferation.
The initial progestin-mediated decreases in Cdk4 and cyclin E-Cdk2
activity were accompanied by alterations in the abundances of several
of the components of these complexes, i.e., cyclin D1, cyclin E, and
p21. Separation of whole-cell lysates by gel filtration chromatography
indicated that cyclin E and Cdk2 displayed a distinct alteration in
elution profile following progestin treatment. In control cells the
major peak of cyclin E-associated kinase activity eluted at ~120 kDa,
although some activity, ~20% of the total, was present in complexes
of very high molecular mass. A significant fraction of the cyclin E in
control cells eluted at ~200 kDa and displayed relatively little
associated kinase activity, likely due to p21 and p27 association. This
observation is consistent with other data indicating that cyclin E is
present in a largely inactive ~250-kDa form and a more active
~120-kDa form (54) and that a minority of cyclin E
accounts for the majority of the associated kinase activity (41,
44). Following progestin treatment, almost all of the cyclin E
was in a low-activity, p21- and p27-bound form eluting at ~200 kDa.
This peak of cyclin E also contained less CAK-activated Cdk2 than the
corresponding fractions from a control lysate, consistent with data
demonstrating that these CDK inhibitors interfere with activation by
CAK (2). Thus, the reduced number of cyclin E-Cdk2 complexes
present following progestin treatment preferentially assemble into the
less active CDK-inhibitor bound form at the expense of the more active
120-kDa form. This redistribution preceded the inhibition of entry into S phase and coincided with the inhibition of cyclin E-Cdk2 activity, implicating it as a cause of the inhibition of kinase activity. Further
evidence for a causative link between the redistribution of cyclin
E-Cdk2 complexes and their activity is provided by the observation that
following cyclin D1 induction in progestin-pretreated cells, the more
active 120-kDa form of cyclin E-Cdk2 reappeared and kinase activity was
restored at a time when the majority of the stimulated population had
yet to reenter S phase. These data also suggest that alterations in
cyclin abundance, especially cyclin D1 abundance, are sufficient to
lead to changes in the compositions as well as the relative abundances
of various cyclin-CDK complexes.
Cyclin D1 and cyclin D3 differed from cyclin E in that the cyclin-CDK
complexes and the associated kinase activity displayed the same elution
profile, peaking at ~160 kDa, although this peak clearly contained
p21- and p27-bound CDK (Fig. 10). This is consistent with data
indicating that p21 and p27 association with Cdk4 does not necessarily
inhibit kinase activity (3, 30, 53), and indeed, some pRB
kinase activity was present in p27 immunoprecipitates from
exponentially proliferating T-47D cells, equivalent to approximately a
third of the Cdk4 activity (data not shown). Following progestin treatment, although major shifts in the elution profile of cyclin D1,
cyclin D3, or Cdk4 were not observed, Cdk4 immunoprecipitates of
fractionated lysates contained less cyclin D1 and cyclin D3 and had an
increased p27/cyclin D ratio (Fig. 10). Overall, these data suggest
that a major factor in the decrease in Cdk4 activity following
progestin treatment is a decrease in the abundance of the cyclin
D1-Cdk4 and cyclin D3-Cdk4 complexes and that increased p27 association
with these complexes may contribute to their decreased kinase activity.
The ability of ectopic cyclin D1 induction to reinitate cell cycle
progression following progestin pretreatment is consistent with this
conclusion.
The data presented above indicate a redistribution of p27 between
different cyclin-CDK complexes at times when there was little alteration in p27 abundance. Furthermore, although the p21 abundance increased ~2-fold following progestin treatment, there appeared to be
no corresponding recruitment of p21 into either cyclin E-Cdk2 complexes
or Cdk4 complexes. One explanation for the latter observation might be
that following progestin treatment p21 was in excess over the number of
available binding sites, but gel filtration indicated that neither p21
nor p27 was present in monomeric form except after extended progestin
treatment. Furthermore, immunoprecipitation studies indicated that the
majority of p21 and p27 is associated with cyclin D1 and Cdk4, in
either the presence or absence of progestin treatment (reference
37 and data not shown). In contrast, while a
significant proportion of cyclin E is associated with p21 and p27, this
accounts for a small fraction (<10%) of the total pool of these CDK
inhibitors.
One important factor in the formation of complexes between
these proteins is the relative affinities of the CDK
inhibitors for different cyclin-CDK complexes, and this,
combined with the reduction in the abundance of several cyclin-CDK
complexes, suggests a possible explanation for the redistribution of
these CDK inhibitors. Experiments using purified components have
indicated that the affinity of p27 for cyclin E-Cdk2 is ~10-fold
higher than its affinity for cyclin D2-Cdk4, while in contrast, p21 has
a higher affinity for cyclin D2-Cdk4 than for cyclin E-Cdk2
(18). If cyclin D1-Cdk4 and cyclin D3-Cdk4 have affinities
for p27 similar to that of cyclin D2-Cdk4, cyclin D-Cdk4 complexes are
likely to act as high-capacity, low-affinity binding sites for p27,
consistent with the idea that they represent a sink for p27 (24,
50, 51). Thus, the data are consistent with the interpretation
that a relatively small decrease in the abundance of cyclin D-Cdk4 complexes makes sufficient p27 available for cyclin E-Cdk2 binding to
account for its redistribution following progestin treatment, and
conversely, induction of cyclin D1 sequesters p27 in cyclin D1-Cdk4
complexes, favoring the formation of the more active, p27-depleted form
of cyclin E-Cdk2. Thus, increased p27 association with cyclin E-Cdk2
following progestin treatment may be a consequence of the decreased
abundances of cyclins D1 and D3. In contrast with p27, the lower
relative affinity of p21 for cyclin E-Cdk2 suggests that upon reduction
in cyclin D-Cdk4 complex numbers, it would compete poorly with p27 for
the available cyclin E-Cdk2 and in consequence associate with other
cyclin-CDK complexes, perhaps cyclin A-Cdk2, for which it displays an
affinity similar to its affinity for cyclin D2-Cdk4 (18).
This suggestion is consistent with observations that following
progestin treatment, an increased proportion of p21 was present in the
fractions containing cyclin A-Cdk2 (~300 kDa) and that Cdk2
immunoprecipitates contained increased amounts of p21 (data not shown).
While it is likely that the composition of cyclin-CDK complexes is
largely determined by the relative abundances and affinities of the
cyclins, CDKs, and CDK inhibitors involved, it is also possible that
interactions with other intracellular proteins contribute to the
availability or association of these molecules. For example, a
Myc-regulated heat-labile inhibitor of p21 has been described (20). By an apparently similar mechanism, in cells which
express abundant p27 but little p21, Myc induction inhibits the ability of p27 to bind cyclin E-Cdk2, most likely by causing the p27 to be
sequestered (34, 41, 62). While cyclin D-Cdk4 or cyclin D-Cdk6 complexes provide an attractive possibility for the
sequestration of p27 or p21, increased association between these
proteins and p27 was not observed following c-Myc induction
(62). Similarly, data from this laboratory suggest that
following c-Myc induction in human breast cancer cells, p21 and p27 are
not sequestered by cyclin D-Cdk4 (43). Recent data suggest
that the sequestered p27 is targeted for degradation, raising the
possibility that the sequestering proteins are involved in the
degradation process (34). It is possible that the decrease
in c-myc expression following progestin treatment results in
decreased expression of p21- and p27-sequestering molecules,
contributing to increased availability of p21 and p27 for cyclin-CDK
binding. However, the ability of cyclin D1 induction to restore CDK
activity and cell cycle progression in progestin-pretreated cells
suggests a more major role for cyclin expression than for
c-myc expression in the redistribution of cyclin E-Cdk2
complexes.
Decreased CDK activity preceded inhibition of entry into S phase and
thus provides a mechanism for inhibition of proliferation by
progestins. However, no decreases in cyclin expression or CDK activity
were apparent after less than 12 h of progestin treatment, indicating that while these are unlikely to be consequences of inhibition of proliferation, they are likely to be indirect effects of
progestin action. Cell cycle kinetic studies of progestin inhibition in
T-47D cells led