MOLECULAR AND CELLULAR BIOLOGY,
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Feb. 2001, p. 794–810Vol. 21, No. 3
Multifaceted Regulation of Cell Cycle Progression by Estrogen:
Regulation of Cdk Inhibitors and Cdc25A Independent of
Cyclin D1-Cdk4 Function
JAMES S. FOSTER,1DONALD C. HENLEY,1ANTONIN BUKOVSKY,1PREM SETH,2
AND JAY WIMALASENA1*
Department of Obstetrics and Gynecology, Graduate School of Medicine, University of Tennessee Medical Center,
Knoxville, Tennessee 37920,1The Medicine Branch, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland 208922
Received 18 July 2000/Returned for modification 25 August 2000/Accepted 9 November 2000
Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating
G1/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks),
and phosphorylation of the retinoblastoma protein (pRb). We have utilized blockade of cyclin D1-Cdk4
complex formation through adenovirus-mediated expression of p16INK4ato demonstrate that estrogen regu-
lates Cdk inhibitor expression and expression of the Cdk-activating phosphatase Cdc25A independent of cyclin
D1-Cdk4 function and cell cycle progression. Expression of p16INK4ainhibited G1/S transition induced in
MCF-7 cells by 17-?-estradiol (E2) with associated inhibition of both Cdk4- and Cdk2-associated kinase
activities. Inhibition of Cdk2 activity was associated with delayed removal of Cdk-inhibitory activity in early G1
and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21Cip1and p27Kip1was
decreased, however, in both control and p16INK4a-expressing cells 20 h after estrogen treatment. Expression of
Cdc25A mRNA and protein was induced by E2in control and p16INK4a-expressing MCF-7 cells; however,
functional activity of Cdc25A was inhibited in cells expressing p16INK4a. Inhibition of Cdc25A activity in
p16INK4a-expressing cells was associated with depressed Cdk2 activity and was reversed in vivo and in vitro by
active Cdk2. Transfection of MCF-7 cells with a dominant-negative Cdk2 construct inhibited the E2-dependent
activation of ectopic Cdc25A. Supporting a role for Cdc25A in estrogen action, antisense CDC25A oligonucle-
otides inhibited estrogen-induced Cdk2 activation and DNA synthesis. In addition, inactive cyclin E-Cdk2
complexes from p16INK4a-expressing, estrogen-treated cells were activated in vitro by treatment with recom-
binant Cdc25A and in vivo in cells overexpressing Cdc25A. The results demonstrate that functional association
of cyclin D1-Cdk4 complexes is required for Cdk2 activation in MCF-7 cells and that Cdk2 activity is, in turn,
required for the in vivo activation of Cdc25A. These studies establish Cdc25A as a growth-promoting target of
estrogen action and further indicate that estrogens independently regulate multiple components of the cell
cycle machinery, including expression of p21Cip1and p27Kip1.
Estrogenic steroids, including 17-?-estradiol (E2), regulate
cellular function in a wide variety of tissues and influence
proliferation in the female reproductive tract and mammary
gland (31). A role for estrogens in breast cancer etiology is well
established and clearly relates to their growth-stimulatory ac-
tion (35). Estrogens elicit proliferative responses in breast can-
cer cells in vivo (85) and in vitro (43) and are essential for
initiation and progression of breast cancer in animal models
(35). Studies of estrogen receptor (ER)-positive breast cancer
cell lines indicate that estrogens (41) and antiestrogens (86) act
on sensitive populations of cells in early to mid-G1phase.
G1/S transition is under the control of cyclin-dependent ki-
nases (Cdks) activated by specific complex formation with reg-
ulatory cyclins. Cdk4 and Cdk6 are activated by binding to
D-type cyclins and act early in G1phase, while Cdk2 kinase
functions in conjunction with cyclins E and A and is necessary
for progression through late G1and entry into S phase (81, 83,
92, 98). A primary target of Cdk action in G1phase is the
retinoblastoma susceptibility gene product (pRb), which me-
diates G1arrest through sequestration of transcriptional fac-
tors of the E2F-DP family. Phosphorylation of pRb and other
members of the pocket protein family (p107 and p130) by
active cyclin-Cdk complexes leads to release of E2F and DP
transcription factors and transcription of requisite genes for
S-phase entry (98). Recently a parallel, Cdk2-driven pathway
promoting the G1/S transition independent of D cyclin-Cdk4
activation, pRb phosphorylation, and E2F release has been
described in model systems utilizing cooperative Ras-Myc ac-
tivation (40), and overexpression of cyclin E (45, 74).
Cdk activation depends upon removal of inhibitory Thr/Tyr
phosphorylation by members of the Cdc25 phosphatase family
(17, 21, 25, 77). Cdc25 phosphatases are candidate oncogenes
and are overexpressed in a wide variety of tumors, including
roughly 30% of breast carcinomas (20). Cdc25A expression is
required for S-phase entry (17, 27, 33) and is induced in G1(3,
27, 33) by Myc (18, 74) and E2F (7, 19, 30, 93). Cdc25A is
active from mid-G1through S phase and participates in acti-
vation of Cdk2 (3, 27, 33). Overexpression of Cdc25A is suffi-
cient for transformation of Rb?/?fibroblasts and cooperates
with Ras in causing tumors in mice (20). Coexpression of
* Corresponding author. Mailing address: Department of Obstetrics
and Gynecology, University of Tennessee Medical Center, 1924 Alcoa
Highway, Knoxville, TN 37920. Phone: (865) 544-8960. Fax: (423)
544-6863. E-mail: firstname.lastname@example.org.
Cdc25A and cyclin E elicits G1/S transition in fibroblasts (93)
and in U2-OS cells independent of pRb inactivation (74).
D-type cyclins play an essential role in recognition of extra-
cellular growth stimuli and initiation of G1transit (71, 80), and
several lines of evidence have linked estrogen regulation of
cellular proliferation to cyclin D1 expression. Estrogen-in-
duced proliferation of normal uterine and breast epithelium in
vivo is associated with increased expression of cyclin D1
mRNA and protein (2, 23, 73, 90). Cyclin D1?/?knockout
mice exhibit normal development of reproductive tissues and
mammary gland ductal epithelium, yet estrogen-dependent de-
velopment of lobular-alveolar structures in mammary epithe-
lium during pregnancy is disrupted (14, 84). Expression of
cyclin D1 in breast tumor isolates correlates with ER-positive
status (28, 52, 59). MCF-7 breast cancer cells treated with
estrogen exhibit increased expression of cyclin D1 mRNA and
protein, formation of active cyclin D1-Cdk4 complexes, and
phosphorylation of pRb leading to G1/S transition (1, 15, 64,
69). Estrogen-induced S-phase entry in these cells is inhibited
by microinjection of antibodies to cyclin D1 (44). Ectopic ex-
pression of cyclin D1 regulates exit from G0in MCF-7 cells
(102) and is sufficient for Cdk activation and S-phase entry in
MCF-7 and T47D breast cancer cells (56, 68). Antiestrogen-
induced growth arrest of ER-positive breast cancer cells is
associated with decreased cyclin D1 expression (97). Collec-
tively, these studies are consistent with a model of estrogen
action in which receptor activation induces increased cyclin D1
expression, Cdk4 activation, and cell cycle progression. An
upstream role for cyclin D1 has been suggested by recent
reports describing direct physical interactions between cyclin
D1 and the ER, leading to recruitment of steroid receptor
coactivators and activation of ER-dependent transcription.
This occurs in the absence of hormone and is independent of
D cyclin association with Cdk4 (49, 57, 101, 103).
Constraint upon Cdk activity and G1progression is provided
by the universal Cdk inhibitors of the Cip-Kip family, including
p21Cip1and p27Kip1, and the specific Cdk4 and Cdk6 inhibitors
of the INK4 family, typified by p16INK4a(26, 39, 65, 82, 91).
The p16INK4agene product inhibits formation of active D
cyclin-Cdk complexes through specific binding interactions
with Cdk4 or Cdk6 that prevent D cyclin-Cdk association (46,
50, 63). Overexpression of p16INK4ain cells with functional
pRb results in inhibition of both Cdk4- and Cdk6-associated
kinase activity and pRb phosphorylation, with subsequent cell
cycle arrest (46, 50). In addition, inhibition of D cyclin-Cdk4
complex formation by p16INK4aprevents sequestration of
p21Cip1and p27Kip1by these complexes in early G1, leading to
suppression of cyclin E-Cdk2 activity (32, 48, 53).
Adenoviral transduction of p16INK4ainto MCF-7 cells leads
to G1arrest associated with inhibited Cdk activity (8, 9). Pre-
vious studies in our laboratory indicated that cell cycle pro-
gression induced by estradiol requires action of the steroid
through mid-G1, well beyond the point of cyclin D1-Cdk4
activation (15). In this study, we utilized adenovirus-mediated
overexpression of p16INK4ato examine in detail the role of
cyclin D1-Cdk4 complexes in cell cycle progression induced in
these cells by estrogen. This approach allowed for the study of
estrogen action independent of D cyclin-Cdk4 complex forma-
tion and cell cycle progression. Our results demonstrate that
functional association of cyclin D1-Cdk4 is required for estro-
gen-induced Cdk2 activation and G1/S transition and that es-
trogen regulates expression of p21Cip1, p27Kip1, and Cdc25A
independent of D cyclin-Cdk4 function. The results further
demonstrate a requirement for in vivo activation of Cdc25A by
MATERIALS AND METHODS
Reagents and antibodies. Cell culture media and antibiotics, E2, insulin, epi-
dermal growth factor (EGF), histones, glutathione-agarose beads, RNase A,
propidium iodide, and other chemicals were from Sigma Chemical Co. (St.
Louis, Mo.) unless otherwise noted. ICI 182,780 was kindly supplied by Alan
Wakeling at Zeneca Pharmaceuticals (Alderly Park, Cheshire, United King-
dom). Recombinant glutathione S-transferase (GST)-pRb, protein A/G beads,
GST-agarose beads, affinity-purified antibodies to Cdk2 (M2), Cdk4 (C22), pRb
(C15), p27Kip1(C19), p21Cip1(C19), Cdc25A (N15), Raf-1 (C12), Pim-1 (C20),
and cyclin D1 (H295), as well as monoclonal antibodies to cyclin E (HE12 and
HE111) and cyclin A (BF683), were from Santa Cruz Biotechnology (Santa Cruz,
Calif.). Monoclonal anti-Cdc25A (Ab-3) and anti-E2F-1 (Ab-7) antibodies were
from Labvision (Freemont, Calif.), and both monoclonal antiactin and anti-
hemagglutinin (HA) (12CA5) antibodies were from Boehringer Mannheim (In-
dianapolis, Ind.) [?-32P]ATP, Tran35S label, and [methyl-3H]thymidine were
from ICN (Irvine, Calif.). Fetal bovine serum (FBS) was from Summit Biotech-
nology (Fort Collins, Colo.). Horseradish peroxidase-conjugated protein A/G
and secondary antibodies were from Jackson Immunoresearch (West Grove,
Pa.). MG132, geldanamycin, PD98059, and roscovitine were from Calbiochem
(La Jolla, Calif.). Flavopiridol was acquired from the National Cancer Institute
(Bethesda, Md.). Antisense and control oligonucleotides for c-myc and CDC25A
were based upon published sequences and were obtained from Genosys (Hous-
ton, Tex.) in partially phosphothiorated form (three linkages at 3? and 5? ends).
Oligonucleotide sequences were as follows: antisense CDC25A, 5?-GGGCTCG
GGCCCAGTTCCAT-3?; antisense c-myc, 5?-AAGCTAACGTTGAGGGGCA
T-3?; and nonsense control oligonucleotide, 5?-AGATAGCTTAGTGCGGACG
Cell culture and transfections. MCF-7 cells were a kind gift from R. P. Shiu
(13) and were maintained in Dulbecco’s minimal essential medium (DMEM)
supplemented with antibiotics and 5% FBS. MCF-7/tTA cells stably expressing
the Tet transactivator protein were derived by transfection with pTet-off (Clon-
tech, Palo Alto, Calif.) and selection in geneticin. For experiments requiring
growth arrest, cells were plated in 60- or 100-mm-diameter dishes and grown to
50 to 60% confluency. Cells were growth arrested by 2 to 3 days of culture in
phenol red-free DMEM–0.1% FBS with 10 nM ICI 182,780. This protocol
results in arrest of greater than 90% of the cells in G0/G1as described previously
(15, 69). Stock solutions of E2and ICI 182,780 were prepared in ethanol and
added to growth-arrested cultures as indicated in the text. Control cultures
received equal amounts of ethanol or dimethyl sulfoxide (DMSO) as vehicle
controls where appropriate. MCF-7/MVLN cells (66) were maintained and
treated for experiments in the same fashion as MCF-7 cells. MCF-7/MVLN cells
are derived from MCF-7 cells and contain a stably integrated vitellogenin A2-
luciferase reporter of estrogen-induced transcriptional activity. Plasmid vectors
for p16 (pBPSTRI-p16) and cyclin E (pMTcyclin E) were provided by G. Peters
(48) and J. M. Roberts (83), respectively. Transfections were carried out on the
day of growth arrest with Superfect transfection reagent (Qiagen, Valencia,
Viral vectors and infection of MCF-7 cells. A replication-defective adenoviral
vector for expression of p16INK4a(Ad.p16) was constructed in the laboratory of
P.S. (8) by homologous recombination in human embryonic kidney 293 cells.
Adenoviral backbone (Ad type 5, 9.24-100mu) was cotransfected by calcium
phosphate precipitation with shuttle vector pCC2, which contains a p16INK4a
expression cassette. Control adenovirus (Ad.Con) and adenoviral vectors ex-
pressing p27Kip1(Ad.p27) and p21Cip1(Ad.p21) were derived similarly (9).
Adenoviral vectors for expression of cyclin E (Ad.cycE), Cdk2 (Ad.Cdk2), the
constitutively active caaX mutant of Raf-1 (Ad.Raf-1caaX), and dominant-nega-
tive Ras (Ad.RasN17) were kindly provided by J. Nevins (40, 76). Adenoviruses
were propagated in 293 cells (American Type Culture Collection), and titers of
viral lysates for use in experiments were determined by a standard plaque assay.
For experiments with MCF-7 cells, cultures undergoing growth arrest were
infected with adenoviral vectors at appropriate multiplicities of infection (MOIs)
in phenol red-free DMEM–0.1% FBS. Unless otherwise noted, MOIs were 50
PFU/cell. After infection, the medium was replaced with phenol red-free
DMEM–0.1% FBS plus ICI 182,780, and the cultures were incubated for an
additional 48 to 72 h before treatment and harvest. A plasmid retroviral vector
VOL. 21, 2001 MULTIPLE MODES OF CELL CYCLE REGULATION BY ESTROGEN795
for Cdc25A was obtained from M. Roussel (78). The retroviral vector was
produced by transfection of PT67 cells (Clontech) and selection of producer
pools in geneticin. MCF-7 cells were infected with retroviral supernatants in the
presence of 4 ?g of Polybrene per ml selected in 0.5 mg of geneticin per ml, and
surviving colonies were pooled for experiments.
Flow cytometric analysis and thymidine incorporation. MCF-7 cells were
growth arrested and treated as described above. For flow cytometric analysis,
cells were harvested in saline-EDTA, fixed in cold 70% ethanol, and stored at
?20°C. Fixed cells were subsequently washed, treated with 100 ?g of RNase A
per ml, and stained with 50 ?g of propidium iodide per ml. Analysis of DNA
content was performed in a Becton-Dickinson FACScan with a minimum of
15,000 events collected for analysis with Becton-Dickinson Cell Quest software.
For flow cytometric analysis of transfected cultures, MCF-7 cells were trans-
fected with pEGFPN1 (Clontech) along with appropriate plasmids (1:5 mass
ratio) and treated as for other experiments. Harvested cells were fixed in 0.5%
formalin followed by ethanol fixation and propidium iodide staining. At least
30,000 events were collected for analysis of DNA content in the cell fraction
exhibiting green fluorescence. Thymidine uptake was performed as described
previously (15). Briefly, growth-arrested MCF-7 cultures at 20 to 30% confluency
in 24-well plates were treated as described in the text, and 1 ?Ci of [methyl-
3H]thymidine per well (60 Ci/mmol) was added 18 to 20 h after treatment. After
allowing 8 to 12 h for uptake, the monolayer was washed twice with ice-cold 5%
trichloroacetic acid, solubilized with 0.2 N NaOH, and counted in aqueous
scintillant with a Packard ?-scintillation counter (Packard Instrument Co.,
Downers Grove, Ill.).
Western blot analysis. Cells were lysed as described previously (15). Briefly,
following treatment, cell monolayers were washed in ice-cold phosphate-buffered
saline and lysed by addition of ice-cold NP-40 lysis buffer (20 mM Tris [pH 7.5],
250 mM NaCl, 0.5% NP-40, 0.1 mM EDTA, 1 mM NaOV4, 10 mM NaF, 10 ?g
of aprotinin per ml, 10 ?g of leupeptin per ml, 1 mM phenylmethyl sulfonyl
fluoride). Cells were scraped off of the plates, briefly sonicated, and centrifuged
at 15,000 ? g for 10 min at 4°C to remove cellular debris. The supernatant was
aliquoted and frozen at ?80°C for later use. For Western blots, equal amounts
of protein (50 to 100 ?g) were separated by sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose, and
membranes were incubated in blocking buffer (20 mM Tris [pH 7.5], 150 mM
NaCl, 0.1% Tween 20, 0.5% casein) for 10 to 15 min. Membranes were incubated
with primary antibodies in blocking buffer 2 h at room temperature or overnight
at 4°C. All primary antibodies were used at 0.5 ?g/ml. Bound antibodies were
detected with horseradish peroxidase-conjugated secondary antibodies and
chemiluminescent immunodetection (ECL; Amersham). Equal protein loading
was verified for all lysate blots by reprobing for actin expression. For Western
blots of immunoprecipitated proteins, proteins were immunoprecipitated from
0.5 to 1 mg of cell lysate, and horseradish peroxidase-conjugated protein A/G was
used for detection to minimize interference by the precipitating antibodies.
Immune complex kinase assays. Histone kinase assays were performed essen-
tially as described previously (15). Equal amounts of lysate proteins prepared as
described above (100 to 200 ?g) were precleared and immunoprecipitated with
1 ?g of anti-Cdk2 (M2), anti-Pim-1 (C20), or monoclonal anti-cyclin E (HE111)
antibodies, along with protein A/G agarose beads for 4 to 18 h in NP-40 lysis
buffer. The immunoprecipitates were washed three times in lysis buffer, washed
twice in kinase buffer (20 mM HEPES [pH 7.5], 20 mM MgCl2), and resus-
pended in kinase buffer supplemented with 400 ?g of histones per ml (type
III-SS), 10 ?M ATP, 1 mM dithiothreitol, 0.5 mM EGTA, 0.2 mM NaOV4, and
5 ?Ci of [?-32P]ATP (7,000 Ci/mmol). Kinase reaction mixtures were incubated
for 1 h at room temperature. For pRb kinase assays, cell lysates were prepared
in Tween 20 lysis buffer as described previously (47), and immunoprecipitations
were carried out with anti-Cdk4 (C22) antibodies. Precipitates were washed in
Tween 20 lysis buffer, and the kinase reaction mixtures contained 0.2 ?g of
GST-pRb fusion protein per sample (amino acids 769 to 928). All kinase assays
were stopped with 25 ?l of 2? SDS-PAGE sample buffer. Reaction products
were separated on SDS-PAGE gels, followed by autoradiography with Kodak
XAR film and quantification by video densitometry.
Northern blot analysis and luciferase assay. Total cellular RNA was extracted
from 100-mm-diameter dishes by a standard guanidinium isothiocynate proce-
dure. Analysis of mRNA expression was performed following separation of 10 ?g
of total RNA per lane and transfer to nylon membranes. Membranes were
hybridized at high stringency with cDNA probes labeled with [?-32P]dCTP by
random primer extension (Decaprime, Ambion, Austin, Tex.). Relative mRNA
expression was assessed by densitometry of autoradiograms normalized to ?-ac-
tin or 18S rRNA expression. For luciferase assay of estrogen-induced transcrip-
tional activity, MCF-7/MVLN cell lysates were prepared and assayed for enzy-
matic activity with a commercial assay kit (Promega, Madison, Wis.) and
measured with a Packard scintillation counter in the single-photon counting
mode. Results were obtained from measurements in triplicate and equalized for
protein content. E2F-induced transcriptional activity was measured by cotrans-
fection of MCF-7 cultures with a cyclin A promoter-luciferase construct (100)
along with pSV-?-gal (Promega) for normalization of transfection efficiency.
Cultures were infected with adenoviral vectors, growth arrested, and treated with
E2. Lysates were prepared after 20 h and assayed for luciferase and ?-galacto-
sidase activity with a commercial assay (Promega).
Assay of Cdk inhibitors, Cdc25A treatment of cyclin E complexes, and Cdc25A
assay. For assay of Cdk inhibitors, active Cdk2 was immunoprecipitated from
lysates of E2-treated MCF-7 cells (2 mg of cell lysate) by using 500 ng of
anti-Cdk2 antibody and protein A/G agarose beads. Cdk2 precipitates were
washed, divided (50 ng of bead-bound antibody per aliquot), and incubated for
1 h at room temperature with lysates (0.2 mg) of cells treated as indicated in the
text. After incubation, complexes were washed and assayed for histone kinase
activity as described above. Treatment of cyclin E-Cdk2 complexes with purified
GST-Cdc25A was carried out by incubating cyclin E immunoprecipitates, pre-
pared from 100 ?g of cell lysate, with 2 ?g of GST-Cdc25A for 30 min at 30°C
in phosphatase buffer (50 mM Tris [pH 8], 150 mM NaCl, 2 mM dithiothreitol,
2.5 mM EDTA). Phosphatase-treated complexes were washed and assayed for
histone kinase activity. GST-Cdc25A was purified by affinity chromatography of
crude bacterial lysates on glutathione-agarose beads as described previously (17).
The assay of endogenous Cdc25A activity in MCF-7 cell lysates was adapted
from the method of Galaktionov and Beach (17). Cdc25A was immunoprecipi-
tated from MCF-7 cell lysates (1 to 2 mg) with anti-Cdc25A (N15) and eluted
from the washed beads with 0.1 M glycine (pH 2.5). Tyrosine-phosphorylated
cyclin B1-Cdc2 complexes precipitated from lysates of hydroxyurea-treated
MCF-7 cells were washed and incubated with the eluted Cdc25A for 1 h at 30°C
in phosphatase buffer followed by two washes and a standard histone kinase
assay. For assay of activity of exogenously expressed Cdc25A, MCF-7/tTA cells
were transfected with pBI-HACdc25A (74) along with plasmid constructs for
p16INK4aor dominant-negative Cdk2 (92) at a mass ratio of 1:5, followed by
growth arrest and treatment. The control plasmid was pcDNA3 (Invitrogen).
Cdc25A activity was assayed as described above in anti-HA immunoprecipitates
from 100 ?g of lysate. For pull-down assays of Cdc25A-associated kinase activity
(72), cell lysates (200 ?g) were precleared with GST-agarose beads followed by
incubation with 8 ?g of GST-Cdc25A on glutathione-agarose beads for 2 h at
4°C. The beads were washed and assayed for histone kinase activity as described
above. Immunodepletion of lysates before assay was carried out by incubation
with 1 ?g of the specified antibodies or control immunoglobulin G (IgG) along
with protein A/G beads. In vitro treatment of Cdc25A with active Cdk2 was
carried out by incubating Cdc25A immunoprecipitates with active, soluble cyclin
A-Cdk2 complexes in kinase buffer with 0.1 mM ATP. Cyclin A-Cdk2 was
purified on glutathione-agarose beads from lysates MCF-7 cells transfected with
a plasmid encoding GST-cyclin A (72) and treated for 24 h with hydroxyurea.
Metabolic labeling and immunoprecipitation. MCF-7 cells in six-well plates
were growth arrested and treated with E2as required, and at appropriate times,
proteins were labeled with [35S]Met/Cys as described previously (15). Cells were
starved in phenol red-free, Met/Cys-free DMEM for 20 min at 37°C, followed by
labeling in Met/Cys-free medium plus 50 ?Ci of Tran35S label (1,000 Ci/mmol)
for 1 to 2 h at 37°C. Monolayers were washed twice in ice-cold phosphate-
buffered saline, and lysates were prepared in NP-40 lysis buffer as described
above for Western blots. For immunoprecipitation analysis, lysates were equal-
ized based on protein concentration and precleared with protein A/G agarose
beads followed by incubation with 1 ?g of the appropriate antibodies (anti-cyclin
D1 [HD11], anti-Cdk4 [C22], and anti-Cdc25A [Ab-3]) along with protein A/G
agarose beads. After 18 h at 4°C, immunoprecipitates were washed in lysis buffer,
suspended in SDS-PAGE sample buffer, boiled, and separated on SDS-PAGE
gels. Gels were soaked in 1 M salicylate before drying and fluorography.
Adenoviral transduction of p16INK4ablocks E2-induced
G1/S transition, formation of cyclin D1-Cdk4 complexes, and
pRb phosphorylation. Estrogen treatment of growth-arrested
MCF-7 cells elicits cyclin D1 expression, Cdk activation, pRb
phosphorylation, and ultimately the onset of DNA synthesis (1,
15, 64, 69). Cell cycle progression in these cells is regulated by
cyclin D1 (44, 61, 68, 102), and adenoviral transduction of the
Cdk inhibitors p16INK4a, p21Cip1, and p27Kip1into MCF-7 cells
elicits G1arrest (8, 9). To determine the effects of Cdk inhib-
796FOSTER ET AL.MOL. CELL. BIOL.
itor transduction on E2-induced S-phase entry in MCF-7 cells,
cultures were infected with control adenovirus or vectors ex-
pressing the appropriate Cdk inhibitors, and growth was ar-
rested by serum withdrawal and antiestrogens. Assays of thy-
midine uptake 20 h after growth arrest and treatment with 10
nM E2(Fig. 1A, left panel) demonstrated that DNA synthesis
induced by estrogen treatment was completely inhibited in
MCF-7 cells transduced with vectors for p16INK4a, p21Cip1, or
p27Kip1. Flow cytometric analysis of DNA content confirmed
the specific inhibition of E2-induced G1/S transition by trans-
duction of Cdk inhibitors (data not shown). Recent studies
have shown that ER-cyclin D1 interactions elicit ER-depen-
dent transcription in ER-positive cells, independent of ligand
binding and independent of interactions between cyclin D1
and Cdk4 (57, 103). Transduction of MCF-7/MVLN cells with
Ad.p16, Ad.p21, or Ad.p27 had no effect on upon E2-induced
transcription from an ERE-luciferase reporter (Fig. 1A, right
panel). Thus, while ectopic expression of p16INK4a, p21Cip1, or
p27Kip1provided an effective blockade of G1/S transition in-
duced by estrogen, ligand-induced transcription mediated by
the ER was unaffected by Cdk inhibitor expression.
Estrogen treatment elicits formation of active cyclin D1-
Cdk4 complexes in MCF-7 cells (1, 15, 64, 69). Specific binding
interactions of p16INK4awith Cdk4 and Cdk6 in vivo inhibit
complex formation with D-type cyclins and prevent kinase ac-
tivation (46, 50, 63). We analyzed cyclin D1 synthesis and cyclin
D1-Cdk4 complex formation in MCF-7 cells expressing
p16INK4a. MCF-7 cells were infected with Ad.Con or Ad.p16
FIG. 1. Blockade of estrogen-induced G1/S transition in MCF-7 cells by p16INK4ais associated with inhibition of cyclin D1-Cdk4 complex
formation and pRb kinase activity. (A, left panel) Cdk inhibitor effects on E2-induced DNA synthesis. MCF-7 cells were infected with adenoviral
vectors for p16INK4a, p21Cip1, p27Kip1, or with control adenovirus (MOI ? 50), growth arrested, and treated with 10 nM E2as indicated. Thymidine
incorporation was measured from 20 to 32 h after treatment with E2and is given as the mean ? standard deviation from four replicates. (A, right
panel) Analysis of estrogen-induced transcriptional activity in MCF-7/MVLN cells. MCF-7/MVLN cells were infected with the indicated adenoviral
vectors (MOI ? 50), growth arrested, and treated with 10 nM E2for 24 h. Results from the luciferase assay are given as relative light units (mean ?
standard deviation from three replicates) based on an arbitrary designation. (B) Analysis of de novo-synthesized cyclin D1 and Cdk4. MCF-7 cells
were infected with Ad.Con or Ad.p16 (MOI ? 50) followed by growth arrest and treatment with 10 nM E2as indicated. Cultures were labeled
for 2 h with Tran35S label, 4 h after estrogen treatment. Immunoprecipitations (IP) were performed with antibodies to cyclin D1 (upper panel)
and Cdk4 (lower panel). Two different exposures are given for Cdk4 precipitates. (C) Assay of Cdk4-associated kinase activity. MCF-7 cells were
infected with Ad.p16 and Ad.Con (MOI ? 50), growth arrested, and treated for 6 h with 10 nM E2as indicated. Lysates were assayed for
Cdk4-associated pRb kinase activity in an immunocomplex assay with GST-pRb as the substrate. (C, lower panel) Western blotting analysis of total
pRb phosphorylation: MCF-7 cultures were infected as indicated and treated for 20 h with E2. Western blot analysis of pRb was performed with
whole-cell lysates. Slowly migrating, phosphorylated pRb forms are indicated (pRb-P). Nonspecific bands on Western blots are indicated by an
asterisk. (D) Analysis of p21Cip1-p27Kip1association with Cdk4. MCF-7 cells were infected as indicated, followed by growth arrest and a 10-h
treatment with 10 nM E2. Proteins in Cdk4 immunoprecipitates were analyzed by Western blotting (WB) analysis for p16INK4a, cyclin D1, p21Cip1,
p27Kip1, and Cdk4.
VOL. 21, 2001 MULTIPLE MODES OF CELL CYCLE REGULATION BY ESTROGEN797
and growth arrested, and de novo-synthesized proteins were
labeled from 4 to 6 h after E2treatment. Estrogen treatment
increased cyclin D1 synthesis as described previously (15), and
this increase was independent of p16INK4aexpression (Fig.
1B). Complex formation between de novo-synthesized cyclin
D1 and Cdk4 was increased by E2in Ad.Con-infected cells, but
was markedly inhibited in p16INK4a-expressing cells irrespec-
tive of estrogen treatment (Fig. 1B, lower panels). Cdk4 im-
munoprecipitates from Ad.p16-infected cells also contained
detectable p16INK4aon long film exposures (data not shown).
Western blotting studies confirmed the equivalent expression
of cyclin D1 in lysates of E2-treated MCF-7 and MCF-7/
MVLN cells, whether infected with Ad.Con or Ad.p16 (data
Immunocomplex assays of Cdk4-associated kinase activity in
Ad.Con- and Ad.p16-infected MCF-7 cultures demonstrated
that estrogen treatment increased Cdk4-associated pRb kinase
activity in Ad.Con-infected cells measured 6 h after E2treat-
ment, while expression of p16INK4ablocked induction of kinase
activity (Fig. 1C). Phosphorylation of pRb in vivo was assessed
by Western blotting of lysates from cultures treated for 20 h
with E2. Estrogen treatment elicited pRb phosphorylation, as
demonstrated by the appearance of slower-migrating forms in
Ad.Con-infected cells, while phosphorylated forms of pRb
were not evident in E2-treated MCF-7 cells expressing
p16INK4a(Fig. 1C, lower panel). Cdk4-specific phosphorylation
of Ser 780 of pRb (37) was also inhibited by p16INK4a, as shown
when blots were reprobed with phosphospecific antibodies to
this residue (data not shown).
Binding of p21Cip1and p27Kip1to newly formed, intact cyclin
D1-Cdk4 complexes leads to sequestration of Cdk-inhibitory
activity and is a critical element of cell cycle transit in early G1
(65, 67, 82, 83). Since p16INK4aeffectively inhibited formation
of cyclin D1-Cdk4 complexes in the studies shown above, the
association of p21Cip1and p27Kip1with Cdk4 was evaluated by
Western blot analysis of Cdk4 immunoprecipitates from con-
trol and p16INK4a-expressing MCF-7 cells treated with estro-
gen. Association of both p21Cip1and p27Kip1with Cdk4 was
markedly decreased in cells expressing p16INK4awhen com-
pared to controls (Fig. 1D), while association of p16INK4awith
Cdk4 was readily evident. In these experiments, transduction
of MCF-7 cells with p16INK4aagain inhibited the association of
cyclin D1 with Cdk4.
Expression of p16INK4ainhibits Cdk2 activation in estrogen-
treated MCF-7 cells. In normal cells, activation of Cdk2 in
mid-to-late G1phase depends upon prior formation of active
D cyclin-Cdk4 or -Cdk6 complexes (82, 83). To evaluate cyclin
E-Cdk2 activation in p16INK4a-expressing MCF-7 cells, cul-
tures of Ad.Con- and Ad.p16-infected MCF-7 cells were
treated with E2for 10 h and cyclin E immunoprecipitates were
assayed for kinase activity. Estrogen treatment activated cyclin
E-Cdk2 in Ad.Con-infected cells (Fig. 2A), as has been previ-
ously demonstrated (1, 15, 64, 69), while cyclin E-Cdk2 activity
was completely inhibited in MCF-7 cells expressing p16INK4a.
Cyclin E expression is limiting for G1-phase Cdk2 activation in
MCF-7 cells (87) and is regulated by E2F (22). Estrogen treat-
ment does not, however, elicit increased cyclin E expression in
MCF-7 cells despite pRb inactivation and E2F release (15, 69).
In multiple experiments, expression of endogenous cyclin E
was not affected by estrogen treatment or by p16INK4aexpres-
sion (Fig. 2A) (data not shown). We further tested the extent
to which p16INK4ainhibition of cyclin E-Cdk2 activity relates to
limiting expression of cyclin E in these cells. Ad.Con- or
Ad.p16-infected MCF-7 cultures were coinfected with adeno-
viral vector for cyclin E. Cyclin E expression and associated
kinase activity were elevated in Ad.cycE-transduced cells
treated with estrogen (Fig. 2A). Cyclin E-associated kinase
activity was again suppressed by coexpression of p16INK4a, yet
exceded that in E2-treated control cultures. The data demon-
strate that overexpression of cyclin E facilitates formation of
active cyclin E-Cdk2 complexes in the context of p16INK4a-
mediated blockade of cyclin D1-Cdk4 function.
Cyclin E-Cdk2 activity in MCF-7 cells peaks 10 to 12 h after
release of growth arrest (15, 69), and Cdk2 activity is associ-
ated more predominantly with cyclin A as MCF-7 cells ap-
proach the G1/S border (69). Kinase activity in Cdk2 immuno-
precipitates was assayed from MCF-7 cultures transduced with
Ad.Con or Ad.p16 and treated for 20 h with E2, and again,
Cdk2 activation was inhibited by p16INK4aexpression (Fig. 2B,
left panel). Cyclin A expression in late G1is regulated by E2F
(10, 75) and is increased by estrogen treatment of growth-
arrested MCF-7 cells (69). Expression of cyclin A is inhibited
by p16INK4ain U2-OS osteosarcoma cells (48, 53), likely
through repression of the cyclin A promoter, which represents
one proposed mechanism of cell cycle arrest mediated by ac-
tive pRb (38). Western blot analysis indicated that cyclin A
expression in response to estrogen was inhibited by Ad.p16
transduction of MCF-7 cells (Fig. 2B, right panel). The results
in Fig. 2 demonstrate that Cdk2 activation elicited by estrogen
treatment of MCF-7 cells requires functional association of
cyclin D1-Cdk4 and further suggest that p16INK4ainhibition of
Cdk2 activity relates to limiting cyclin expression, particularly
that of cyclin A at the G1/S border.
Given the reversal of Cdk2 inhibition evident when p16INK4a-
expressing MCF-7 cells were cotransduced with Ad.cycE, we
evaluated whether G1/S transition was facilitated in these cells
when cyclin E was overexpressed. Overexpression of cyclin E
or cyclin A provides relief of p16INK4a-mediated G1arrest in
U2-OS cells (53). For these experiments, MCF-7 cells were
transfected with plasmid vector for p16INK4aalong with either
control plasmid, or a plasmid vector for cyclin E. G1/S transi-
tion induced in these cells by E2was again inhibited by
p16INK4aexpression, and this inhibition was almost fully re-
versed upon transfection of cells with vector for cyclin E (Fig.
2C). In separate experiments, transfection of MCF-7 cells with
plasmid cyclin E vector fully reversed p16INK4asuppression of
activity associated with cotransfected, exogenous HA-Cdk2
(data not shown).
Cdk-inhibitory activity and p21Cip1and p27Kip1expression
are downregulated late in G1independent of p16INK4aexpres-
sion. Activation of Cdk2, and G1transit requires elimination of
Cdk-inhibitory activity associated with p21Cip1and p27Kip1(39,
65, 82, 83). Previous studies indicate that Cdk-inhibitory activ-
ity in MCF-7 cells is predominantly attributable to p21Cip1(64,
69) and that estrogen treatment leads to removal of Cdk inhi-
bition in part through redistribution of p21Cip1into cyclin D1-
Cdk4 complexes (64). Expression of p16INK4ain U2-OS cells
prevents Cdk inhibitor sequestration by D cyclin-Cdk4 com-
plexes leading to inhibition of Cdk2 (32, 48, 53). Since the
experiments described above demonstrated that cyclin D1-
798FOSTER ET AL.MOL. CELL. BIOL.
Cdk4 complex formation and Cdk2 activation were effectively
inhibited by transduction with Ad.p16, we determined whether
inhibition of Cdk2 activity in p16INK4a-expressing MCF-7 cells
relates to a failure in elimination of Cdk inhibitors. We per-
formed functional assays of Cdk-inhibitory activity in whole-
cell lysates from control and Ad.p16-infected cultures treated
with estrogen for a range of times (0 to 20 h). Our results
indicated that Cdk-inhibitory activity in MCF-7 lysates was
effectively eliminated by 20-h estrogen treatment in both
p16INK4a-expressing and nonexpressing cells (Fig. 3A). Down-
regulation of this activity was delayed, however, in cells ex-
pressing p16INK4a, with the majority of Cdk-inhibitory activity
still present at 7.5 to 10 h after treatment (Fig. 3A). At these
times, Cdk-inhibitory activity was absent, and substantial cyclin
E-Cdk2 activity was detected in control MCF-7 cells treated
with estrogen (Fig. 3A) (data not shown). The results indicate
that 20-h estrogen treatment leads to elimination of Cdk-in-
hibitory activity in MCF-7 cells independent of sequestration
by D cyclin-Cdk4 complexes, while removal of Cdk inhibition
at earlier times is effectively prevented by p16INK4a. Thus, in
early G1and during the normal time frame of cyclin E-Cdk2
activation, p16INK4a-mediated blockade of D cyclin-Cdk4 com-
plex formation maintains Cdk-inhibitory activity and prevents
Estrogen treatment of growth-arrested MCF-7 cells leads to
decreased expression of both p21Cip1and p27Kip1and forma-
tion of cyclin E-Cdk2 complexes depleted in content of either
Cdk inhibitor (15, 69). We determined whether regulation of
p21Cip1and p27Kip1expression by E2is dependent upon cyclin
D1-Cdk4 complex formation. Western blotting analysis indi-
cated that steady-state protein levels of p21Cip1and p27Kip1
declined 50 to 60% in MCF-7 cells treated for 20 h with E2,
independent of p16INK4a-induced G1blockade (Fig. 3B, left
panels). In agreement with earlier studies (69), we did not
observe any decline in levels of p21Cip1and p27Kip1on Western
blots until at least 8 h after estrogen treatment, and at 12 h
after E2treatment, 70 to 75% of p21Cip1and p27Kip1remained
(J. Wimalasena and S. Ahamed, submitted for publication).
Cyclin E immunoprecipitates from both Ad.Con- and Ad.p16-
infected cultures were depleted of p21Cip1and p27Kip1follow-
ing E2treatment (Fig. 3B, right panels), and Cdk2 in these
complexes was phosphorylated in response to estrogen, as
demonstrated by the appearance of faster-migrating forms of
the enzyme (25).
In mammalian cells undergoing G1transit, p27Kip1protein is
subject to ubiquitin-targeted degradation in the proteasome,
leading to a shortened half-life for the protein and decreased
overall abundance (62, 82). Expression of p21Cip1is regulated
by the proteasome as well (6). Treatment of MCF-7 cells with
the specific proteasome inhibitor MG132 along with E2pre-
FIG. 2. Expression of p16INK4aprevents Cdk2 activation in E2-treated MCF-7 cells. (A) For assay of E2-induced cyclin E-Cdk2 activity, MCF-7
cells were infected with Ad.Con or Ad.p16 (MOI ? 50). Additional cultures were coinfected with Ad.cycE as indicated (all MOIs ? 50). Cultures
were growth arrested and treated with 10 nM E2for 12 h, and cyclin E-associated histone kinase activity was determined with equal amounts of
lysate. The histone band (HH1) is indicated. Relative kinase activities based on densitometry are indicated under the appropriate lanes. Western
blot analysis of cyclin E expression in the same lysates is given in the lower panel with identical arrangement of lanes. (B, left panel) Effects of
p16INK4aexpression on Cdk2-associated kinase activity in MCF-7 cells. MCF-7 cells were infected with the indicated adenoviral vectors, growth
arrested, and treated for 20 h with E2. Histone kinase activity was determined with Cdk2 immunoprecipitates. Numbers represent relative kinase
activities. (B, right panel) Cyclin A expression in control and p16INK4a-expressing MCF-7 cells was determined by Western blotting of whole-cell
lysates from MCF-7 cells infected as indicated and treated for 20 h with E2. (C) Reversal of p16INK4a-mediated G1arrest by cyclin E. MCF-7/tTA
cells were transfected with pEGFPN1 along with control vector (pcDNA3), pBPSTRI-p16 (p16), or pMTcyclin E (cycE). The cultures were growth
arrested and treated for 24 h with E2, and the proliferative fraction (S?G2/M) was determined by DNA content analysis of the transfected
population as given in Materials and Methods. The results are given as the mean ? standard deviation from three independent experiments.
VOL. 21, 2001 MULTIPLE MODES OF CELL CYCLE REGULATION BY ESTROGEN 799
vented downregulation of both p21Cip1and p27Kip1, leading to
a 15-fold increase in p21Cip1levels and an 8-fold increase in
p27Kip1(Fig. 3C). The control protease inhibitor E64 had no
effect on levels of p21Cip1or p27Kip1in MCF-7 cells (data not
shown). Inhibition of proteasome function by MG132 also led
to increased p21Cip1in cyclin E-Cdk2 complexes (Fig. 3C,
lower panels), while levels of p27Kip1in these complexes were
unchanged. As described previously (64, 69), we found that
Cdk-inhibitory activity in whole-cell lysates of MCF-7 cells was
eliminated by immunodepletion with p21Cip1antibodies (data
not shown). Given this apparent intracellular excess of p21Cip1
and the greater extent of p21Cip1accumulation, a predominance
of p21Cip1in cyclin E-Cdk2 complexes might be expected when
proteasome function is inhibited. In further support of proteo-
lytic mechanisms of Cdk inhibitor downregulation, estrogen
treatment also decreased expression of ectopic p21Cip1and
p27Kip1expressed from a cytomegalovirus promoter in MCF-7
cells transduced with Ad.p21 or Ad.p27, as observed for the
endogenous proteins (Fig. 3D) (data not shown).
Downregulation of p27Kip1requires Cdk2-mediated phos-
phorylation of the protein on Thr 187 (55, 79, 83, 89). Trans-
fection of MCF-7 cells with the epitope-tagged T187A mutant
of p27Kip1and Western blotting analysis of expression of the
protein demonstrated that phosphorylation of this residue was
required for downregulation of the protein subsequent to es-
trogen stimulation (Fig. 3E). Epitope-tagged wild-type p27Kip1
FIG. 3. Estradiol downregulates Cdk-inhibitory activity and decreases expression of p21Cip1and p27Kip1proteins in MCF-7 cells arrested in G1
by p16INK4aexpression. (A) Effects of p16INK4aexpression on downregulation of Cdk-inhibitory activity in MCF-7 cells. Cdk-inhibitory activity in
Ad.Con- and Ad.p16-infected MCF-7 cells was assayed after treatment of growth-arrested cells with 10 nM E2for the indicated times. Whole-cell
lysates were mixed with Cdk2 immunoprecipitates followed by assay of histone kinase activity. Results are presented in graphic form based on
densitometric measurements. (B, left panels) Expression of p21Cip1and p27Kip1proteins in MCF-7 cells. Cultures were infected with Ad.p16 and
Ad.Con (MOI ? 50), growth arrested, and treated with 10 nM E2for 20 h. Lysates were assayed by Western blotting (WB) for expression of
p27Kip1, p21Cip1, p16INK4a, and actin as indicated. (B, right panels) Levels of Cdk2, p21Cip1, and p27Kip1proteins in cyclin E immunoprecipitates
(IP) from cultures treated in the same fashion were analyzed by Western blotting. (C) Proteasomal regulation of Cdk inhibitor levels. Ad.p16-
infected MCF-7 cells were growth arrested and treated with 10 nM E2and the specific proteasome inhibitor MG132 (10 ?M) as indicated. Lysates
prepared after 20 h were analyzed by Western blotting for p21Cip1, p27Kip1, and actin (upper three panels). In the lower panel, cyclin E
immunoprecipitates from cultures treated in the same fashion were analyzed for p27Kip1and p21Cip1content by Western blotting. (D) Estrogen
regulation of ectopic p27Kip1expression. MCF-7 cells were infected with Ad.p27, growth arrested, and treated for 20 h with E2. Expression of
p27Kip1was determined by Western blotting of whole-cell lysates. Lysates of uninfected, growth-arrested MCF-7 cells (NV) were included for
comparison. (E) Western blot analysis of p27Kip1(T187A) expression in MCF-7 cells. MCF-7 cells were transfected with the HA-p27-T187A vector,
growth arrested, and treated with E2for 20 h. Expression of the p27Kip1mutant was determined by Western blot analysis of lysates with anti-HA
800FOSTER ET AL.MOL. CELL. BIOL.
expressed in MCF-7 cells was downregulated by estrogen treat-
ment, as observed above with endogenous and ectopic p27Kip1
(data not shown). The T187A mutant of p27Kip1accumulated
in cells treated with MG132, indicating that steady-state levels
of the protein are nonetheless regulated to some extent by
proteasomal degradation. Together the results in Fig. 2 and 3
suggest that estrogen treatment of MCF-7 cells leads to elim-
ination of Cdk-inhibitory activity in two phases. The early
phase (0 to 8 h) is dependent upon sequestration of the Cdk
inhibitors by D cyclin-Cdk4 complexes and mediates rapid
removal of Cdk-inhibitory activity allowing activation of cyclin
E-Cdk2 in mid-G1. At later times following estrogen adminis-
tration (8 to 20 h), Cdk inhibitors are subject to degradation
mediated via the proteasome, a mechanism of Cdk inhibitor
removal independent of cyclin D1-Cdk4 function. Further-
more, downregulation of p27Kip1in MCF-7 cells requires phos-
phorylation at Thr 187, although this occurs under conditions
of only minimal Cdk2 activation.
Estrogen induces Cdc25A expression in MCF-7 cells.
Cdc25A is required for S-phase entry and activates Cdk2 in
vitro through removal of inhibitory phosphorylation (25, 27,
33). Our studies indicated that Cdk2 was phosphorylated after
estrogen treatment in p16INK4a-expressing MCF-7 cells, yet
remained inactive (Fig. 3). Hence, we wished to ascertain the
extent of Cdc25A expression in estrogen-treated cells. Cdc25A
is a downstream target of Myc-induced transactivation (18, 74).
Transcription of c-myc is rapidly induced by E2in MCF-7 cells
(11, 12), and increased expression of Cdc25A protein in estro-
gen-treated MCF-7 cells has been reported (96). In prelimi-
nary studies, estrogen induced c-myc mRNA expression in
both Ad.Con- and Ad.p16-infected cells, with peak expression
1 h after treatment (Fig. 4A). Estrogen treatment also in-
FIG. 4. Estradiol induces expression of Cdc25A mRNA and protein in MCF-7 cells. (A) MCF-7 cells were infected with Ad.p16 or Ad.Con
(MOI ? 50), growth arrested, and treated for the indicated times with 10 nM E2. Total RNA was analyzed for expression of c-myc and Cdc25A
mRNA by Northern blotting. Membranes were reprobed for 18S rRNA as a loading control. (B) Cdc25A protein synthesis. Ad.p16- and
Ad.Con-infected MCF-7 cultures were growth arrested and treated with 10 nM E2or 10 nM E2plus 500 nM ICI 182,780 as indicated. Cultures
were labeled with Tran35S-label from 10 to 12 h after treatment. Control (Con.), antisense CDC25A, or c-myc antisense (AS) oligonucleotides
(oligo) (10 ?M) were added to the culture medium 18 h before treatment and remained in the culture until labeling. Equal amounts of protein
lysates were immunoprecipitated with monoclonal anti-Cdc25A (Ab-3) antibodies and analyzed by SDS-PAGE. (C) Western blot analysis of
Cdc25A expression. MCF-7 cultures were infected with Ad.Con or Ad.p16, growth arrested, and treated for 20 h with E2as indicated. Cdc25A was
immunoprecipitated with monoclonal anti-Cdc25A (Ab-3) and analyzed by Western blotting with anti-Cdc25A (N15). (D, left panel) E2F-
dependent transcriptional activity was measured in lysates of Ad.Con- and Ad.p16-infected cells 30 h after treatment with E2as given in Materials
and Methods. Values are given as relative light units (mean ? standard deviation from three replicates). Values given represent estrogen-induced
transcription, with activity in untreated cells subtracted. (D, right side panels) Western blot analysis of E2F-1 and actin expression in whole-cell
lysates of MCF-7 cells treated as described above in panel C.
VOL. 21, 2001 MULTIPLE MODES OF CELL CYCLE REGULATION BY ESTROGEN801
creased the expression of Cdc25A mRNA in both control and
p16INK4a-expressing MCF-7 cells, with maximal expression by
10 to 12 h (Fig. 4A). Metabolic labeling of de novo-synthesized
proteins from 10 to 12 h after E2treatment demonstrated
increased synthesis of Cdc25A (Fig. 4B), which was completely
inhibited by cotreatment of cultures with the steroidal anties-
trogen ICI 182,780. Prior treatment of MCF-7 cultures with
antisense DNA oligonucleotides directed to the translation-
initiation regions of either c-myc or CDC25A also inhibited
E2-induced Cdc25A protein synthesis, while control oligonu-
cleotides and p16INK4atransduction had no effect (Fig. 4B).
Western blot analysis of Cdc25A immunoprecipitates demon-
strated an increase in total Cdc25A protein expression induced
in MCF-7 cells by E2irrespective of p16INK4a-expression (Fig.
4C). In addition to Myc, Cdc25A expression is also regulated
by transcription factors of the E2F family through transactiva-
tion by free E2F-1 and repression or derepression mediated by
E2F-pocket protein complexes (7, 19, 30, 93). Functional anal-
ysis of E2F transcriptional activity in estrogen-treated MCF-7
cells demonstrated that E2F activity generated following es-
trogen treatment is largely inhibited by p16INK4aexpression
(Fig. 4D, left panel). In addition to eliciting E2F activity
through Cdk activation and pRb phosphorylation, estrogen
treatment of MCF-7 cells induces E2F-1 expression (95).
Western blot analysis confirmed that E2treatment increased
E2F-1 protein expression (fourfold) in control MCF-7 cells
(Fig. 4D, right panels); however, transduction with p16INK4a
yielded marked suppression of E2F-1 expression. The results
in Fig. 4 demonstrate that Cdc25A is expressed in response to
E2in control and p16INK4a-expressing MCF-7 cells and would
further indicate that Cdc25A induction in cells transduced with
p16INK4aoccurs in the presence of minimal E2F activity.
Cdc25A regulates estrogen-induced Cdk2 activation and
G1/S transit in vivo, but is inactive in p16INK4a-expressing
MCF-7 cells. Cdc25A is rate limiting for G1transit (3, 78), and
microinjection of anti-Cdc25A antibodies inhibits S-phase en-
try in vivo (17, 27, 33). In experiments to clarify the in vivo role
of Cdc25A in MCF-7 cells, antisense CDC25A oligonucleo-
tides, which effectively inhibit synthesis of the protein (Fig.
4B), were found to inhibit estrogen-induced DNA synthesis,
while control oligonucleotides had no inhibitory effect (Fig.
5A, upper panel). In agreement with earlier studies (68), the
Cdk2 inhibitor roscovitine also inhibited DNA synthesis in-
duced by estrogen. Cdc25A activates Cdk2 in vitro (25) and
promotes G1transit in vivo through Cdk2 activation as well (3,
27, 33). In direct support of an in vivo role for Cdc25A in the
activation of Cdk2 in MCF-7 cells, cyclin E-dependent Cdk2
activity measured 12 h after estrogen treatment of growth-
arrested MCF-7 cells was specifically inhibited by antisense
CDC25A oligonucleotides (Fig. 5A, lower panel).
Previous studies suggest that Cdc25A is phosphorylated and
activated in vivo by cyclin E-Cdk2 in late G1(27). To deter-
mine if Cdc25A expressed in estrogen-treated MCF-7 cells is
fully active, Cdc25A was immunoprecipitated from lysates of
estrogen-treated control and p16INK4a-expressing cells and as-
sayed for activation of cyclin B1-Cdc2 complexes (3, 17). In
multiple experiments, Cdc25A activity was markedly lower in
p16INK4a-expressing cells than in Ad.Con-infected cells (Fig.
5B). Equal amounts of Cdc25A were precipitated from E2-
treated cells irrespective of p16INK4aexpression (Fig. 4C) (data
not shown). In control experiments, Cdc2-activating activity in
Cdc25A immunoprecipitates was inhibited by sodium or-
thovanadate and by blocking peptide included in the immuno-
precipitation reaction (data not shown). As a further control,
purified recombinant GST-Cdc25A was found to readily acti-
vate cyclin B1-Cdc2 complexes in the assay (Fig. 5B). In agree-
ment with previous studies (3, 27), Cdc25A activity was largely
absent in growth-arrested cultures (Fig. 5B, lower panel).
These results would indicate that Cdc25A is largely inactive in
MCF-7 cells transduced with p16INK4a, despite increased ex-
pression of the protein in response to estrogen, and might
suggest a failure to activate Cdc25A in vivo in these cells.
Inactive cyclin E-Cdk2 complexes from p16INK4a-expressing
cells are activated in vitro by Cdc25A. Given our results in Fig.
5 indicating that Cdc25A is inactive in p16INK4a-expressing
cells, we determined if cyclin E-Cdk2 complexes in p16INK4a-
expressing MCF-7 cells were inactive due to inhibitory phos-
phorylation and, as such, could be activated in vitro by Cdc25A
phosphatase (64). Cyclin E immunoprecipitates from control
and p16INK4a-expressing MCF-7 cells were incubated with pu-
rified, recombinant GST-Cdc25A before assay of kinase activ-
ity. Cyclin E-Cdk2 from Ad.p16-infected, E2-treated cells was
activated by treatment with Cdc25A (Fig. 6A), yielding histone
kinase activity similar to that in complexes from estrogen-
treated control cultures (relative activity of 4.0 versus 4.3).
Active cyclin E-Cdk2 complexes from E2-treated, Ad.Con-in-
fected cultures were further activated by treatment with
Cdc25A (activity of 10.2). As reported previously (64), Cdc25A
phosphatase did not activate cyclin E-Cdk2 complexes from
untreated MCF-7 cultures, irrespective of p16INK4aexpression,
perhaps owing to the presence of Cdk inhibitors in the com-
plexes and/or a lack of activating phosphorylation. Inclusion of
sodium orthovandate in the in vitro phosphatase reaction com-
pletely inhibited activation of cyclin E-Cdk2 complexes by
GST-Cdc25A (data not shown). The results indicate that a
substantial fraction of cyclin E-Cdk2 complexes elicited in both
control and p16INK4a-expressing MCF-7 cells remain inactive
due to inhibitory phosphorylation. Following treatment in vitro
with Cdc25A, the active fraction of Cdk2 is increased irrespec-
tive of in vivo p16INK4aexpression, yet remains greater when
derived from cells not expressing p16INK4a.
In vivo overexpression of Cdc25A leads to early activation of
Cdk2 and accelerated S-phase entry in HeLa cells and NIH
3T3 fibroblasts (3, 78). MCF-7 cells transduced with retroviral
Cdc25A expression vector overexpressed Cdc25A protein 8- to
10-fold (Fig. 6B). These cells exhibited elevated functional
Cdc25A activity, yet did not exhibit accelerated G1/S transition
or early activation of cyclin E-Cdk2 complexes, which would
indicate that following estrogen stimulation, Cdc25A activity is
not limiting in MCF-7 cells (data not shown). When the effects
of p16INK4aexpression on Cdk2 activation were assessed in
these cells, transduction with Ad.p16 inhibited Cdk2 activation
in parental MCF-7 cells as before, but inhibited that in
Cdc25A-overexpressing cells to a lesser degree (Fig. 6B). Cdk2
activity was higher overall in Cdc25A-overexpressing cells. In
flow cytometric assays of S-phase entry after estrogen treat-
ment, however, in vivo overexpression of Cdc25A provided no
relief from the G1/S blockade afforded by p16INK4atransduc-
tion (Fig. 6C). The results indicate that despite the increase in
Cdk2 activity provided by Cdc25A overexpression, the degree
802 FOSTER ET AL.MOL. CELL. BIOL.
of activation achieved is nonetheless insufficient for G1/S tran-
sition. Together the results in Fig. 5 and 6 lend support to an
in vivo role for Cdc25A in Cdk2 activation and G1/S transit
induced by estrogen in MCF-7 cells. The results also indicate
that a population of Cdk2 in p16INK4a-expressing MCF-7 cells
can be activated in vitro and in vivo by Cdc25A. Cdk2 inhibi-
tion and cell cycle arrest in these cells are, however, a result of
mechanisms of which Cdc25A is only a part.
Cdc25A activity is regulated in vivo by p27Kip1expression
and Cdk2 activity. Cdc25A interacts with cyclin E-Cdk2 com-
plexes through a cyclin-binding domain similar to that in Cip
and Kip Cdk inhibitors and competes with p21Cip1for cyclin-
Cdk2 binding (72). Activation of Cdc25A has been related to
this interaction with cyclin-Cdk complexes in vitro and in vivo
(17, 27). To further investigate the mechanisms of Cdc25A
activation in MCF-7 cells, we sought to determine whether
p27Kip1overexpression had any effect on Cdc25A activation.
As shown in Fig. 7A, Cdc25A activity was inhibited in MCF-7
cells transduced with Ad.p27, similar to effects seen in Ad.p16-
infected cells. Assay of Cdk2 activity in the same lysates con-
firmed that p27Kip1overexpression effectively inhibited Cdk2
in these cells, similar to the effects seen with Ad.p16 transduc-
tion (Fig. 7A). MG132, which effectively inhibited downregu-
lation of p21Cip1and p27Kip1(Fig. 3), inhibited Cdc25A acti-
vation as well (data not shown). These results would indicate
that deregulated expression of Cdk inhibitors inhibits Cdc25A
activation in MCF-7 cells, whether occurring as a result of
ectopic overexpression, through inhibition of proteasome func-
tion, or as observed in cells where cyclin D1-Cdk4 function is
inhibited by overexpression of p16INK4a.
Catalytic activity of Cdc25A is increased in vitro upon phos-
phorylation by cyclin E-Cdk2 (27), Raf-1 (19), and Pim-1 (54).
Recent studies indicate that E2activates the Ras-Raf-Erk
pathway (5, 70), and our own studies have demonstrated that
adenoviral transduction of a dominant-negative Ras mutant
(Ad.RasN17) inhibits E2-induced Cdk2 activation and G1/S
transition in MCF-7 cells (J.W., unpublished data). We exam-
ined the effects of Ad.RasN17 transduction on Cdc25A acti-
vation in vivo, and as depicted in Fig. 7A, dominant-negative
Ras inhibited Cdc25A activity without influencing expression
of the protein. Cdk2 activity was inhibited by Ad.RasN17 trans-
duction as well. Ras activity is required for p27Kip1downregu-
lation (34, 88), and dominant-negative Ras expression led to an
accumulation of p27Kip1in both control and estrogen-treated
cultures (Fig. 7B). Previous studies and our own unpublished
results indicate that growth factor treatment of MCF-7 cells
leads to more prolonged and pronounced activation of the
Ras-Raf-Erk pathway than the transient activation observed
with estrogen treatment alone (44, 70). Cdc25A activity was
FIG. 5. Cdc25A is required for estrogen-induced Cdk2 activation
and DNA synthesis in MCF-7 cells and is inactive in cells expressing
p16INK4a. (A, upper panel) Inhibition of S-phase entry by antisense
(AS) CDC25A oligonucleotides (Oligo). DNA synthesis in E2-treated
MCF-7 cells was assayed as given above in Fig. 1. Antisense oligonu-
cleotides were added to the culture medium at the indicated concen-
trations 18 h before estrogen treatment and remained in the culture
throughout the assay. Values represent percent thymidine incorpora-
tion relative to that in estrogen-treated control (Con.) cultures and are
given as the mean ? standard deviation from four replicates. (A, lower
panel) Inhibition of Cdk2 activation by antisense CDC25A. MCF-7
cells were treated with antisense CDC25A or control oligonucleotides
as described above, and lysates prepared 12 h after E2treatment were
assayed for cyclin E-associated histone kinase activity. (B) Assay of
endogenous Cdc25A activity. Cdc25A immunoprecipitates were pre-
pared from MCF-7 cultures treated as indicated, and Cdc25A was
eluted from the beads as described in Materials and Methods. The
function of eluted Cdc25A was assayed as activation of cyclin B1-Cdc2
complexes measured in a standard histone kinase assay. In lane 4,
maximal activation is demonstrated by treatment of complexes with
purified recombinant GST-Cdc25A. Results from a separate experi-
ment are presented graphically in the lower portion of panel B, with
activity in lysates of untreated, Ad.Con-infected cells taken as 1.
VOL. 21, 2001 MULTIPLE MODES OF CELL CYCLE REGULATION BY ESTROGEN803
inhibited by p16INK4a, however, when MCF-7 cells were
treated with estrogen and growth factors in combination, as
was observed in cultures treated only with estrogen (data not
shown). Our studies thus suggest some role for the Ras–Raf-1
pathway in estrogen-induced activation of Cdc25A. While this
potentially involves direct activation of Cdc25A by interaction
with active Raf-1, the results may reflect a requirement for Ras
in p27Kip1downregulation and subsequent activation of Cdk2.
Pim-1 kinase is expressed at the G1/S border in a wide
variety of cells (42) and activates Cdc25A in vitro (54). We
examined whether Cdc25A activity in MCF-7 cells was related
to expression and/or activity of Pim-1 in control and p16INK4a-
expressing MCF-7 cells. Estrogen treatment of growth-ar-
rested cells elicited increased Pim-1-associated histone kinase
activity, and this was associated with increased expression of
the protein (Fig. 7C). Increased Pim-1 expression and kinase
activity were evident in both control and p16INK4a-expressing
cells, which would not suggest any relationship between Pim-1
FIG. 6. Cyclin E-Cdk2 complexes from p16INK4a-expressing MCF-7
cells are activated in vitro and in vivo by Cdc25A. (A) Activation of
cyclin E-Cdk2 complexes by Cdc25A in vitro. MCF-7 cells were in-
fected with Ad.Con or Ad.p16, growth arrested, and treated for 20 h
with E2as indicated. Cyclin E immunoprecipitates were incubated with
GST-Cdc25A and assayed for histone kinase activity. Relative kinase
activities from densitometric measurements are given beneath the fig-
ure. (B) Effect of in vivo overexpression of Cdc25A on Cdk2 activity in
MCF-7 cells. (Left panel) Cdc25A expression was assayed in parental
MCF-7 cells and in cells transduced with retroviral vector for Cdc25A.
Western blot analysis of Cdc25A immunoprecipitates from proliferat-
ing parental and Cdc25A-overexpressing cells is shown. (Right panel)
Cdk2-associated kinase activity was assayed in lysates of parental and
Cdc25A-overexpressing MCF-7 cells infected with Ad.Con or Ad.p16.
Cultures were growth arrested and treated for 20 h with 10 nM E2as
indicated. Relative activity based on densitometry is provided beneath
each lane. (C) Flow cytometric analysis of estrogen-induced S-phase
entry in parental and Cdc25A-overexpressing MCF-7 cells is given.
Parental and Cdc25A-overexpressing MCF-7 cells were infected with
Ad.Con or Ad.p16 as indicated and growth arrested, and the prolifer-
ative fraction (S?G2/M) was determined 24 h after E2treatment by
flow cytometric analysis of DNA content.
FIG. 7. Cdc25A activation in vivo is inhibited by p27Kip1and by
dominant-negative Ras. (A, top panel) Effects of transduction with
Ad.p16, Ad.p27, and Ad.RasN17 on generation of Cdc25A activity in
MCF-7 cells. MCF-7 cells were infected with Ad.Con, Ad.p16, Ad.
RasN17, or Ad.p27 as indicated, and Cdc25A activity was assayed
following growth arrest and a 20-h estrogen treatment. Results are
presented in graph form with input cyclin B1-Cdc2 activity taken as a
value of 1. (A, lower panel) Western blotting (WB) analysis of Cdc25A
expression in MCF-7 cells. Cdc25A was immunoprecipitated from 600
?g of the same lysates assayed above. Relative Cdk2 activities assayed
in the same lysates are given below the panel based on densitometric
analysis. (B) Expression of p27Kip1was assayed by Western blotting of
lysates from MCF-7 cells infected with Ad.Con and Ad.rasN17 vectors
following growth arrest and 20-h treatment with estrogen as indicated.
(C) Pim-1-associated histone kinase activity (upper panel) was deter-
mined as given in Materials and Methods with lysates of control and
Ad.p16-infected MCF-7 cells 20 h after E2treatment. Western blot
analysis of Pim-1 protein expression in the same lysates is given in the
804 FOSTER ET AL.MOL. CELL. BIOL.
activity and the relative inactivity of Cdc25A in cells trans-
duced with p16INK4a.
Cdk2 activates Cdc25A in vivo and in vitro. Active cyclin
E-Cdk2 complexes associate with Cdc25A in vitro and with
ectopic Cdc25A in vivo (72). To our knowledge, no study has,
as yet, demonstrated in vivo association of endogenous
Cdc25A and cyclin-Cdk complexes. To assess interactions of
Cdc25A and potential in vivo activators in MCF-7 cells, kinase
activity associated with Cdc25A in vitro was measured in pull-
down kinase assays using beads coated with recombinant
Cdc25A and lysates from control and p16INK4a-transduced
MCF-7 cells treated for 12 h with E2(mid-G1). Histone kinase
activity bound to Cdc25A was readily demonstrable in lysates
of estrogen-treated cells, but was greatly diminished in cells
expressing p16INK4a(Fig. 8A, top panel). No kinase activity
was associated with beads coated only with GST (data not
shown). Kinase activity associated with Cdc25A was almost
completely abolished by addition of flavopiridol and roscovi-
tine to the in vitro assay (Fig. 8A, middle panel). As controls,
PD98059, a specific MEK inhibitor, and geldanamycin, an in-
hibitor of tyrosine kinases, were found to have no effect on
Cdc25A-associated kinase activity in vitro. Immunodepletion
of MCF-7 lysates with specific antibodies to Cdk2 and Pim-1
decreased kinase activity associated with Cdc25A, while anti-
bodies to Raf-1 had no effect (Fig. 8A, bottom panel). These
results indicate that the active Cdk2 and Pim-1 elicited in
estrogen-treated MCF-7 cells can interact with Cdc25A in vitro
and would support a potential role for these regulators in
Cdc25A activation in vivo.
Cdc25A is inactive in MCF-7 cells where Cdk2 is rendered
inactive by transduction with p16INK4a, p27Kip1, or dominant-
negative Ras (Fig. 5 and 7). In light of these results and since
active Cdk2 from E2-treated MCF-7 cells associates with
Cdc25A in vitro (Fig. 8A), we determined if Cdc25A activity in
FIG. 8. Cdc25A is activated in vivo by Cdk2. (A) Analysis of Cdc25A-associated kinase activity. (A, top panel) Control-infected and Ad.p16-
infected MCF-7 cells were growth arrested and treated for 12 h with E2. Lysates were precleared and incubated with GST-Cdc25A-coated beads,
and histone kinase activity associating with Cdc25A was measured as given in Materials and Methods. (A, middle panel) The in vitro assay of
Cdc25A-associated kinase activity was carried out with the addition of flavopiridol, roscovitine, PD9059, or geldanamycin (all 2.5 ?M). DMSO was
used as a solvent control. Lysates were from estrogen-treated MCF-7 cells. (A, bottom panel) Immunodepletion analysis of Cdc25A-associated
kinase activity is shown. Lysates of estrogen-treated MCF-7 cells were subjected to immunodepletion with the indicated specific antibodies or
control goat IgG before incubation with Cdc25A beads and assay. (B) Reversal of Cdc25A inhibition in vivo. MCF-7 cells were infected with
Ad.Con and Ad.p16 vectors along with Ad.Con, Ad.cycE, or Ad.Raf-1caaxas indicated. Cdc25A activity was assayed following growth arrest and
a 20-h treatment with E2. (C) Reactivation of Cdc25A in vitro. Cdc25A immunoprecipitates from lysates of estrogen-treated Ad.p16-infected
MCF-7 cells prepared as described above were incubated in vitro with soluble, active cyclin A-Cdk2 complexes as described in Materials and
Methods and assayed for activation of cyclin B1-Cdc2 complexes. Activity in Cdc25A immunoprecipitates from Ad.Con-infected cells is given for
comparison. In panels C and D, relative activity based on densitometry is given above the respective lanes. (D) Activation and inhibition of ectopic
Cdc25A activity. MCF-7/tTA cells were transfected with pBI-HACdc25A along with control plasmid (pcDNA3), pBPSTRI-p16 (p16), or
dominant-negative Cdk2 vector (DNCdk2). Following growth arrest and E2treatment (20 h), Cdc25A activity was assayed in anti-HA immuno-
precipitates as given in Materials and Methods. Relative activity is given under each lane. In the lower panel, equal expression of HA-Cdc25A was
verified by anti-Cdc25A immunoblot analysis of anti-HA immunoprecipitates prepared from the same lysates.
VOL. 21, 2001 MULTIPLE MODES OF CELL CYCLE REGULATION BY ESTROGEN805
p16INK4a-expressing MCF-7 cells could be restored in vivo or
in vitro by Cdk2. To examine this, MCF-7 cells were infected
with Ad.p16 along with Ad.Con, Ad.cycE, or adenoviral vector
expressing constitutively active Raf-1caax, and Cdc25A activity
was assayed after growth arrest and E2treatment. Ad.cycE
transduction increases Cdk2 activity in p16INK4a-expressing
MCF-7 cells (Fig. 2) and led to generation of Cdc25A activity
in these cells comparable to that in Ad.Con-infected cells (Fig.
8B). Expression of the Raf-1 mutant also activated Cdc25A in
p16INK4a-expressing cells. In vitro treatment of Cdc25A immu-
noprecipitates from p16INK4a-expressing cells with soluble, ac-
tive cyclin A-Cdk2 also increased activity to levels similar to
those in immunoprecipitates from Ad.Con-transduced cells
To test directly whether in vivo inhibition of Cdk2 activity
leads to suppression of Cdc25A activity, MCF-7 cells were
transfected with HA-tagged Cdc25A along with vectors for
p16INK4aor a kinase-inactive, dominant-negative Cdk2 mu-
tant. Assays of Cdc25A activity in HA immunoprecipitates
demonstrated that activity of the exogenous enzyme was in-
creased following E2treatment (Fig. 8D) and furthermore
demonstrated that Cdc25A activation was inhibited by domi-
nant-negative Cdk2. Transfection with p16INK4ainhibited ac-
tivation of ectopic Cdc25A as well. Our results in Fig. 7 and 8
indicate that the inability of estrogen to generate full Cdc25A
activity in MCF-7 cells under conditions of G1blockade en-
forced by p16INK4aexpression stems from low levels of Cdk2
activity. Cdk2 inhibition is, in turn, associated with inhibition of
p21Cip1-p27Kip1sequestration in p16INK4a-expressing cells, as
well as a lack of cyclin A expression late in G1. Full activity of
Cdc25A in vivo thus requires activation of cyclin-Cdk com-
plexes and the availability of these complexes for interaction
Our studies demonstrate that estrogens promote cell cycle
progression in MCF-7 cells at multiple points within the ma-
chinery governing G1/S transition (see the schematic in Fig. 9).
Using adenoviral transduction of p16INK4ato provide G1
blockade, we have shown that estrogen regulates expression of
Cdk inhibitors and induces expression of Cdc25A and that
regulation at this level is independent of D cyclin-Cdk4 func-
tion. The data show that formation of ternary complexes be-
tween cyclin D1-Cdk4 and p21Cip1and p27Kip1is an essential
aspect of estrogen action in G1, since expression of p16INK4ain
MCF-7 cells completely inhibited S-phase entry induced by E2
treatment (Fig. 1). Blockade of cyclin D1-Cdk4 association by
p16INK4aprevented sequestration of p21Cip1and p27Kip1, in-
hibited cyclin A induction by preventing pRb inactivation and
E2F release, and led to abolition of Cdk2 activity (Fig. 2). As
a consequence of Cdk2 inhibition, activation of Cdc25A in vivo
was inhibited as well (Fig. 5, 7, and 8).
Three previous studies in U2-OS osteosarcoma cells associ-
ated Cdk2 inhibition and G1arrest by p16INK4awith inhibition
of D cyclin-Cdk4 complex formation, redistribution of Cdk
FIG. 9. Schematic model of estrogen-mediated promotion of S-phase entry. Transcriptional activation of Myc and cyclin D1 expression in early
G1(dark arrows) facilitates cyclin E-Cdk2 activation in mid-to-late G1- and S-phase entry. Expression of cyclin D1 and complex formation with
Cdk4 leads to sequestration of p21Cip1and p27Kip1Cdk inhibitors and initiates phosphorylation and inactivation of pocket proteins, including pRb.
Conversely, expression of p16INK4aprevents cyclin D1-Cdk4 association, delays removal of Cdk-inhibitory activity, and effectively inhibits pocket
protein phosphorylation and release of E2F transcription factors. Estrogen downregulates expression of both p21Cip1and p27Kip1independent of
cyclin D1-Cdk4 function and at least in part through the proteasome. It is not yet clear to what extent this is related to estrogen-induced Myc
expression, Ras activation, or induction of as-yet-unidentified mediators. Myc further participates in cyclin E-Cdk2 activation by eliciting Cdc25A
expression. As suggested in earlier studies, full activation of both Cdc25A and Cdk2 hinges upon interaction and mutual activation between these
two regulators. Ultimately, active cyclin E-Cdk2 likely elicits S-phase entry both through contribution to pocket protein phosphorylation and E2F
release and through phosphorylation of additional, unknown mediators of S-phase entry. Upon inactivation of pocket proteins, derepression at
E2F-dependent promoters and consequent induction of cyclin A, Cdc25A, and E2F-1 provides further reinforcement for G1/S transition and
progression through the S phase.
806 FOSTER ET AL.MOL. CELL. BIOL.
inhibitors from D cyclin-Cdk4 into cyclin E-Cdk2 complexes,
and cyclin A repression (29, 32, 53). In our studies, ectopic
p16INK4awas found in association with Cdk4, inhibited forma-
tion of complexes between Cdk4 and cyclin D1, and prevented
association of p21Cip1and p27Kip1proteins with these com-
plexes (Fig. 1). Our studies essentially agree with those with
U2-OS cells in that p16INK4aexpression in MCF-7 cells caused
delayed removal of Cdk-inhibitory activity in early G1(0 to 8 h
after E2treatment, Fig. 3), leading to inhibition of cyclin E-
Cdk2 activation. In contrast, our studies show that downregu-
lation of Cdk-inhibitory activity was evident in both control
and p16INK4a-expressing cells 20 h after estrogen administra-
tion. Cdk inhibitor downregulation at this time was associated
with decreased expression of p21Cip1and p27Kip1, required
proteasomal action, and was reflected in decreased p21Cip1and
p27Kip1content of cyclin E-Cdk2 complexes (Fig. 3). This sug-
gests a functional dissociation of Cdk inhibitor sequestration in
early G1and downregulation through protein degradation in
the proteasome in mid to late G1. It is not clear at this time to
what extent these particular observations are specific to MCF-7
cells. Studies with U2-OS cells have utilized asynchronous cell
populations (29, 32, 53), which might not allow for discrimina-
tion of Cdk inhibitor regulation in early and late G1. The
relative contributions of complex formation and sequestration
and protein degradation to removal of the Cdk-inhibitory
threshold associated with p21Cip1and p27Kip1are not known at
this time. As with p16INK4a-transduced cells, MCF-7 cells
treated with MG132 do not exhibit active Cdk2, nor do they
enter S phase (Fig. 1 and 2) (data not shown). Thus, Cdk
inhibitor regulation at the level of protein degradation would
appear to be necessary, though insufficient, for G1/S transition
in MCF-7 cells. Our results would suggest that sequestration
and degradation of Cdk inhibitors are requisite and comple-
mentary mechanisms facilitating Cdk2 activation in MCF-7
Our observation that inactive cyclin E-Cdk2 complexes in
p16INK4a-expressing MCF-7 cells were phosphorylated (Fig. 3)
led us to investigate Cdc25A expression and function in MCF-7
cells. Expression of Cdc25A is transcriptionally regulated by
Myc (18, 74) and E2F-1 (7, 19, 30, 93), both of which are
expressed in MCF-7 cells in response to estrogen (11, 12, 95).
Estrogen treatment of MCF-7 cells induced Cdc25A expres-
sion independent of p16INK4a-induced G1blockade (i.e., where
pRb remained in the hypophosphorylated state and both
E2F-1 expression and functional E2F activity were minimal)
(Fig. 1 and 4). Synthesis of Cdc25A protein induced by E2was
inhibited by c-myc antisense oligonucleotides (Fig. 4); thus,
Cdc25A expression in these cells would appear to be a down-
stream effect of estrogen-induced Myc expression.
The activity of cyclin E-Cdk2 complexes from p16INK4a-
expressing MCF-7 cells was increased by in vitro treatment
with recombinant Cdc25A, although the levels of activity did
not equal that of enzyme-treated complexes derived from con-
trol cultures (Fig. 6). This indicates that at least some portion
of cyclin E-Cdk2 complexes in p16INK4a-expressing cells were
inactive due to inhibitory phosphorylation. In vivo overexpres-
sion of Cdc25A partially restored Cdk2 activation in p16INK4a-
expressing cells as well, although no relief of the p16INK4a-
mediated blockade of G1/S transition was provided (Fig. 6B
and C). Thus, both in vivo and in vitro, Cdc25A failed to
restore full activation of Cdk2 derived from cells expressing
p16INK4a. The data would indicate that in MCF-7 cells, the role
of Cdc25A in Cdk2 activation is secondary to the requirement
for D cyclin-Cdk4 complex formation and Cdk inhibitor se-
questration in early G1.
Our studies do, however, support an in vivo role for Cdc25A
in G1/S transition induced in MCF-7 cells by estrogen, since
antisense CDC25A oligonucleotides inhibited expression of
Cdc25A, inhibited Cdk2 activation, and prevented the onset of
DNA synthesis (Fig. 4 and 5). In conjunction with increased
Cdc25A expression, estrogen treatment increased measurable
in vivo Cdc25A activity in MCF-7 cells, but this activity was
largely absent when these cells were transduced with p16INK4a
(Fig. 4, 5, 7, and 8). Previous studies demonstrated that in
HeLa cells, expression and activity of Cdc25A increase as cells
approach the G1/S border (3, 27). In these cells Cdc25A was
phosphorylated in vivo at the G1/S border, and in vitro phos-
phorylation or activation of Cdc25A was associated with cyclin
E-Cdk2 in cell lysates (27). Our studies provide a direct dem-
onstration of the separate regulation of Cdc25A expression
and activity in MCF-7 cells and thus lend in vivo support to
earlier studies suggesting that full enzymatic activity of
Cdc25A requires activation in vivo through phosphorylation
(17, 19, 27, 54). Cdc25A synthesis in MCF-7 cells occurs in
mid-G1phase, and activation of the enzyme in vivo correlates
with Cdk2 activity. The relative inactivity of Cdc25A in
p16INK4a-expressing cells is associated with a lack of cyclin
E-Cdk2 kinase activity and may directly relate to the disruption
of p21Cip1-p27Kip1sequestration by p16INK4a. Overexpression
of p27Kip1also led to inhibition of Cdc25A activity (Fig. 7). The
evidence at this time indicates that deregulated expression of
p27Kip1-p21Cip1, cyclin A repression, and the consequent inhi-
bition of Cdk2 leads to Cdc25A inhibition in p16INK4a-express-
ing cells. Accordingly, in vivo overexpression of cyclin E and
incubation in vitro with active Cdk2 restored activity of
Cdc25A derived from p16INK4a-transduced cells (Fig. 8B and
C). In addition, active Cdk2 in MCF-7 cell lysates associated
with Cdc25A in vitro (Fig. 8A). Chemical Cdk inhibitors fla-
vopiridol and roscovitine (51) also inhibited kinase activity
associating with Cdc25A in vitro (Fig. 8A). To underscore the
independence of Cdc25A expression and in vivo activation,
ectopic Cdc25A was activated following estrogen treatment of
growth-arrested MCF-7 cells (Fig. 8D). Furthermore, activa-
tion of exogenous Cdc25A was prevented by expression of
p16INK4aand a dominant-negative Cdk2 mutant (Fig. 8D).
Cdc25A is phosphorylated and activated in vitro by cyclin
E-Cdk2 (27), Raf-1 (19), and Pim-1 (54), although no previous
study has conclusively demonstrated whether these constitute
mediators of Cdc25A phosphorylation or activation in vivo.
Estrogen elicits activation of cyclin E-Cdk2 (1, 15, 64, 69),
Raf-1 (5, 70), and Pim-1 (Fig. 7) (this study) in MCF-7 cells, so,
potentially, any of these mediators might participate in
Cdc25A activation in vivo. The inhibition of Cdc25A activity
we observed in MCF-7 cells transduced with dominant-nega-
tive Ras might suggest a requirement for the Ras–Raf-1 path-
way in Cdc25A activation, yet this was also associated with
p27Kip1accumulation and depressed Cdk2 activity (Fig. 7).
Raf-1 activation by E2is transient (5, 70) and may not coincide
with the time frame of Cdc25A activation in mid-G1. A con-
stitutively active Raf-1 mutant activated Cdc25A in vivo in
VOL. 21, 2001MULTIPLE MODES OF CELL CYCLE REGULATION BY ESTROGEN 807
MCF-7 cells expressing p16INK4a(Fig. 8B), which would sug-
gest that Raf-1–Cdc25A interactions are nonetheless func-
tional in vivo. Recent studies have suggested, however, that
these interactions serve to regulate phosphorylation and activ-
ity of Raf-1 (99) and thus may lie outside the realm of any
direct participation in G1transit. Our studies cannot at this
time support a role for Pim-1 in Cdc25A activation, since
Cdc25A remained inactive in p16INK4a-expressing cells where
Pim-1 was activated (Fig. 7). Active Pim-1 kinase in lysates of
E2-treated MCF-7 cells, however, was found to associate with
Cdc25A in vitro (Fig. 8), as described previously (54).
In conclusion, a wide range of studies have suggested roles
for cyclin D1 and p16INK4ain control of growth and senescence
of normal mammary epithelial cells (4, 16, 73) and in mam-
mary carcinogenesis (36, 59, 94). Estrogen induces expression
of cyclin D1 mRNA and protein in normal uterine and mam-
mary gland epithelium and in MCF-7 cells (1, 2, 15, 23, 69, 73,
90), and our studies and the others cited herein would suggest
that cyclin D1 is a requisite downstream target of estrogen
action with respect to growth promotion. Our studies cannot
support recent investigations suggesting that direct cyclin
D1-ER interactions serve to promote growth in a Cdk4-inde-
pendent fashion (49, 57, 101, 103). Rather, the studies de-
scribed herein would suggest that in MCF-7 cells, growth pro-
motion by cyclin D1 is mediated largely through interactions
with Cdk4. These studies further demonstrate that estrogen
independently regulates multiple components of the cell cycle
machinery in addition to cyclin D1, including expression of
p21Cip1-p27Kip1and Cdc25A (Fig. 9). Our studies thus indicate
that estrogen exerts a regulatory influence upon Cdk2 activa-
tion in MCF-7 cells independent of cyclin D1-Cdk4 function
and identify Cdc25A as a growth-promoting target for estrogen
action. Recent investigations demonstrate that cyclin E and
Cdc25A function cooperatively along with Myc to generate
active Cdk2 and elicit G1/S transition independent of pRb
inactivation (74). Cyclin E and Cdc25A are overexpressed in a
substantive portion of breast carcinomas (20, 24, 36, 58, 59)
and cyclin E expression, cyclin E-Cdk2 kinase activity, and
p27Kip1expression may hold predictive value with respect to
the proliferative rate and severity of the disease (58–60). Es-
trogens modulate expression and function of a variety of genes
across a range of target cell types, and our studies fit this
paradigm of multifaceted regulation. Elucidation of the mul-
tiple sites of estrogen action may be of considerable impor-
tance to our understanding of the normal biology of estrogen
target tissues and our understanding of breast cancer etiology.
We thank Eva Bukovska for technical assistance and Richard An-
drews for performing the flow cytometric analysis. We are also grateful
to Joseph Nevins for providing adenoviral vectors for dominant-neg-
ative Ras, Raf-1caax, and cyclin E. Plasmid vectors used for Northern
blotting analysis of Cdc25A, c-myc, and cyclin D1 were kindly provided
by Yue Xiong and David Beach (Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.). MCF-7/MVLN cells were provided by Michel
Pons (INSERM, Strasbourg, France). The retroviral expression vector
for Cdc25A was kindly provided by M. Roussel (St. Jude’s Children’s
Research Hospital, Memphis, Tenn.). The GST-cyclin A expression
vector was provided by Bruce Mayer (Harvard Medical School, Bos-
ton, Mass.). Plasmid vectors for p16INK4a, cyclin E, and dominant-
negative Cdk2 were kindly provided by G. Peters, J. Roberts, and E.
Harlow, respectively. The p27Kip1wild-type and T187A vectors were
provided by J.-Y. Kato at the Nora Institute of Science and Technol-
ogy, Japan. We also thank J. Lukas and E. Santoni-Rugiu (Institute of
Cancer Biology, Danish Cancer Society) for the HA-Cdc25A expres-
This work was supported by National Institutes of Health grant
CA-68538 and by CA-84048 (J.W.).
While this paper was in the review process, a related study
was published (4a) that provides support for a role for Cdc25A
in breast cancer and in proliferation of MCF-7 cells in vitro.
This study demonstrates that Cdc25A is overexpressed in up to
50% of breast carcinomas and that Cdc25A overexpression was
associated with poor patient survival. Cdc25A overexpression
also correlated with elevated Cdk2 activity in tumor tissues. In
addition, antisense CDC25A oligonucleotides were shown to
specifically inhibit Cdk2 activity and proliferation in asynchro-
nous cultures of MCF-7 cells.
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