[CANCER RESEARCH 54, 1077 10~4. February 15, t994]
Regulation of Cyclin B1 by Estradiol and Polyamines in MCF-7
Breast Cancer Cells 1
T h r e s i a T h o m a s 2 a n d T. J. T h o m a s
Department of Environmental and Community Medicine, Environmental and Occupational Health Sciences Institute IT. T.], and the Program in Clinical Pharmacology, Clinical
Research Center [T. J. T.], University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854
ABSTRACT
Recent studies have identified a family of proteins called cyclins that
control cell cycle. Among these proteins, cyclin B synthesis and degradation are necessary and sufficient to cause a Xenopus egg cell-free system to
oscillate between S and M. To understand the link between hormonal
regulation of cell growth and the expression of B-type cyclins, we studied
the effect of estradiol on cyclin BI mRNA in a hormone-responsive breast
cancer cell line, MCF-7. Cells were synchronized at GI by isoleucine
starvation, and estradioi was added along with the removal of cell cycle
block. Flow cytometric analysis showed 81 + 7% cells in G~ after 30 h of
isoleucine starvation. Significant population of cells progressed to S by 16
h after the addition ofestradiol, whereas a comparable transition occurred
in control cells by 36 h only. In cells progressing from Gj~S--*Gz--*M
under the influence of estradioi, there was a significant increase in cyclin
B1 mRNA at 30 and 36 h, consistent with the accumulation of this cyclin
in Gz/M. In addition, we found that cyclin BI mRNA degradation occurred early in G~, and this process was accelerated by estradiol. At 2 h
after removal of the isoleucine block, there was a 40% reduction in the
level of cyclin B1 mRNA in estradiol-treated cells compared to untreated
controls. Cyclin B1 protein degradation followed a similar pattern, as
determined by Western blots using a monoclonal anti-cyclin BI antibody.
Since previous studies suggested a polyamine pathway in the mechanism
of action of estradiol, we questioned whether polyamines are important in
controlling the level of cyclin B1 mRNA. Treatment of synchronized cells
with the polyamine biosynthetic inhibitor, difluoromethylornithine attenuated cyclin B1 mRNA degradation in the presence of estradiol. This process was mostly reversed by exogenous putrescine and spermidine but not
by putrescine homologues. Collectively, these data suggest that the mechanism of cell growth regulation by estradiol in MCF-7 cells includes alterations in cyclin B1 mRNA. Our data also indicate molecular pathways for
the action of polyamines in estrogenic control of cell cycle.
INTRODUCTION
Estradiol exerts pleiotropic growth stimulatory effects on breast
epithelial cells and other sex accessory tissues as part of their normal
development (1-3). Estradiol is also involved in the induction and
progression of breast cancer, and a subset of breast cancers regresses
by ablation of estradiol. Estrogen receptor, a protein that mediates the
action of estradiol, is present in the majority of breast tumors, and its
presence correlates with responsiveness of the tumor to antiestrogen
therapy (4-6). Several pathways have been proposed for estrogenic
regulation of breast cancer cells (7-10): (a) direct gene regulatory
effects mediated by estrogen receptor facilitate the expression of
growth-related genes such as c-los, c-myc, and ODC, 3 resulting in
DNA synthesis and cell division; (b) estradiol exerts its effects on cell
Received 6/25/92; accepted 12/14/93.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported, in part, by the NIH Grant CA 42439 (T. T.) and by the
National Institute of Environmental Health Sciences Center of Excellence Grant ES05022.
2 To whom requests for reprints should be addressed, at Department of Environmental
and Community Medicine, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes
Lane, Piscataway, NJ 08854.
3 The abbreviations used are: ODC, ornithine decarboxylase; MPF, M phase promoting
factor; DFMO, difluoromethylornithine; C3, 1,3-diaminopropane; C4, 1,4-diaminobutane
(putrescine); C5, 1,5-diaminopentane; C6, 1,6-diaminohexane; DCC, dextran-coated charcoal; SDS, sodium dodecyl sulfate; cDNA, complementary DNA; PBS, phosphate-buffered saline; HPLC, high-performance liquid chromatography.
growth by stimulating the production of growth factors, particularly
epidermal growth factor and insulin-like growth factor I (8, 9); (c)
estradiol may release the growth-inhibitory effects of certain serumderived factors and thereby enhance cell growth (10). Despite extensive work on the involvement of estradiol in breast cancer, the control
of the cell cycle and cell proliferation by this hormone is not fully
understood.
Several of the molecules that control cell cycle have been recently
identified (11-13). Two major regulatory points in eukaryotic cell
cycle are at G j and Ga/M transitions. In the Ge/M transition, the
protein complex, MPF, drives interphase cells into mitosis (14-16). A
component of MPF, cyclin was originally named as a result of its
unusual pattern of protein expression during early embryonic cycles of
sea urchins and clams. The protein kinase activity of MPF is activated
only after its association with cyclins. Pines and Hunter (17, 18)
characterized human cyclins A and B1 as the mitotic cyclins because
these proteins induced maximal activation of associated protein kinase
in G2/M. Another group of cyclins including C, DI, D2, D3, and E
were described later as Gt cyclins (19, 20). In spite of this classification, mutant yeast that were deficient in G1 cyclins could be rescued
by mitotic cyclins, suggesting that the functional domains of these
proteins might be acting in a similar manner. Recent studies also
suggest that cyclin B protein has multiple functions including the
activation of a specific tyrosine phosphatase (21). Thus regulation of
G~ as well as mitotic cyclins might be important in the control of
breast cancer cell growth. In this report, we describe the effect of
estradiol on the control of cyclin B1 mRNA and protein in synchronized MCF-7 cells.
Early studies on the action of estrogens in target tissues showed that
this hormone stimulates ODC activity (22). ODC is a key enzyme in
the biosynthesis of the naturally occurring polyamines, putrescine
[H2N(CH2)4NH2] , spermidine [H2N(CH2)3NH(CH2)4)NH2 ], and
spermine [HeN(CH2)3NH(CH2)4NH(CH2)3NH2; Refs. 22-24]. Recent studies suggest that the polyamine pathway is interlinked with
estrogenic regulation of cell growth in breast cancer cells (25-28). The
inhibitory effect of antiestrogens on breast cancer cells could be
reversed by the addition of polyamines (25, 27). Furthermore, polyamines are capable of altering the structural organization and DNA
binding of estrogen and other steroid receptors (28-30). Therefore, we
questioned whether estrogenic control of cell cycle and the expression
of cyclin B1 mRNA were related to the accumulation of polyamines
in these cells. For these studies, we used an inhibitor of ODC, DFMO,
for polyamine depletion and exogenous putrescine, spermidine, or
putrescine homologues for polyamine repletion.
Our results indicate that estradiol accelerates the down-regulation
of cyclin BI mRNA in early Gt and its accumulation in S and G2/M.
Our data further indicate an important role for polyamines in the rapid
degradation of cyclin B1 mRNA in early G1.
MATERIALS AND
METHODS
Chemicals and Reagents. DFMO was a gift from Peter E McCann
of the Marion Merrell Dow Research Institute, Cincinnati, OH. C4,
spermidine-3HCl, spermine.4HC1, and aminoguanidine were obtained from
Sigma Chemical Co. (St. Louis, MO). Putrescine analogues, C3, C5, and C6,
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REGUI.ATION OF CYCLIN I31 BY ESTRADIOL AND POLYAMINES
were obtained in the form of their dihydrochlorides from Aldrich Chemical Co.
(Milwaukee, WI). Cyclin B1 cDNA probe was a gift from Drs. Pines and
Hunter of the Salk Institute, La Jolla, CA. Monoclonal anti-cyclin B1 antibody
was obtained from Pharmingen (San Diego, CA). Chemiluminescence based a
Western blot detection system was obtained from Amersham (Arlington
Heights, IL).
Cell Culture. MCF-7 cells were obtained from the American Type Culture
Collection. Cells were maintained in Dulbecco's modified Eagles' medium
supplemented with 100 units/ml penicillin, 100/xg/ml streptomycin, 40/xg/ml
gentamicin, 2 /.r
insulin, and 10% fetal calf serum (Hyclone, Salt Lake
City, UT; Ref. 27). Culture medium, trypsin, antibiotics, and fetal bovine serum
were purchased from Gibco Laboratories, Grand Island, NY. One week before
each experiment, MCF-7 cells were grown in phenol red-free Dulbecco's
modified Eagle's medium supplemented with fetal calf serum which was
pretreated with DCC. For this purpose, we used a stock DCC suspension
consisting of 0.5% Norit A, 0.05% Dextran T-70, 10 mM Tris-HC1 (pH, 7.5), 1
mM EDTA, and 0.25 M sucrose. This suspension (200 ml) was centrifuged at
5000 • g for 10 min and decanted. Then, 200 ml of serum was added to the
pellet and stirred at 4~ for 30 min. The mixture was centrifuged at 5000 • g
for 10 min. The serum was decanted and sterilized by filtration through a 0.22
/zm membrane. We conducted all experiments with MCF-7 cells in phenol
red-free medium and DCC-treated serum to avoid estrogenic effect of phenol
red (31).
In experiments with DFMO, a 200-mM stock solution of this compound was
made in double distilled water, pH adjusted to 7.4, and small volumes were
added to the culture medium. In polyamine repletion experiments, cells were
treated with polyamines along with 1 mM aminoguanidine in order to inhibit
serum-derived amine oxidase from degrading exogenous polyamines.
Flow Cytometry. Cells were grown in 100-mm dishes, washed with PBS,
and covered with a buffer containing 40 mM sodium citrate, 250 mM sucrose,
and 5% dimethylsulfoxide; then cells were frozen at -70~ (32, 33). On the
day of DNA analysis, cells were thawed, and the citrate buffer was removed.
Cells were then trypsinized for 10 min and further treated with a solution
containing trypsin inhibitor and RNase for 10 min. Cells were then stained by
adding propidium iodide solution in sodium citrate buffer and analyzed by a
Coulter flow cytometer.
RNA Extraction. RNA was extracted using the guanidium isothiocyanate
procedure (34). Cells (2 • 106) were seeded in 100-mm culture dishes. After
48 h, medium was removed, and cells were treated for 30 h with isoleucine-free
medium. Estradiol or ethanol vehicle was then added for specific time periods.
Estradiol was dissolved in ethanol as a concentrated stock solution to maintain
ethanol concentrations below 0.1%. The cells were trypsinized (0.25% trypsin
in 2.5 mM EDTA), washed once in cold PBS (pH 7.2), and centrifuged at 800
• g for 5 min. The pellet was resuspended in 3 ml of a solution containing 5
M guanidium isothiocyanate, 50 mM Tris-HCl (pH 7.6), and 5% 13-mercaptoethanol. This cell lysate was immediately layered on a 5.7-M CsCI cushion and
centrifuged at 114,000 • g for 18 h at 20~ The RNA pellet was resuspended
in 1 ml of a buffer containing 10 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 1%
SDS. The RNA solution was extracted with a 4:1 mixture of chloroform and
butanol, precipitated with absolute ethanol, and resuspended in sterile water.
The RNA concentration was determined by measuring the optical density at
260 nm.
Northern Blot Hybridization. RNA prepared from MCF-7 cells was separated by agarose (1%) gel electrophoresis under denaturing conditions (35).
Ethidium bromide (50/xg/ml) was added to samples immediately before loading. The gel and running buffer contained 2.2 M formaldehyde, 50 mM morpholino-propanesulfonic acid, 10 mM sodium acetate, and 1 mM EDTA (pH
7.0). 18S and 28S rRNA from calf thymus (P. L. Biochemicals, Piscataway, NJ)
was used as molecular size markers. The integrity of the 18S and 28S bands of
the extracted RNA indicated that RNA molecules have not degraded during the
extraction process. RNA was transferred onto a nylon membrane by a modified
version of the Maniatis procedure (35) for the Probtech 2 automated transfer
system (Oncor, Gaithersberg, MD). Slot blots were prepared by applying
purified RNA at 0.5 and 1 /xg levels on nylon membrane using a slot blot
apparatus obtained from Bethesda Research Laboratories, Bethesda, MD.
The plasmid (pGEM 4Z) harboring human cyclin B1 cDNA sequences was
cleaved by BamHI, and the insert was purified by a Gene Clean kit from Bio
101, La Jolla, CA. The DNA was recovered by ethanol precipitation and
resuspended at a concentration of approximately 0.1 /xg/ttl in a buffer con-
taining 10 mM Tris-HC1 (pH 7.5), 0.1 mM EDTA, and 20 mM NaC1. The DNA
was labeled with 32p using a nick translation reagent kit from Oncor.
The RNA blots were hybridized with 32p-labeled cyclin B1 cDNA. Hybridization conditions and washing procedures were optimized using standard
protocols (36). The specific activity of the cDNA probe was in the range of
5-10 • 107 cpm//zg DNA. Prehybridization was for 1 h using Hybrisol I
(Oncor) containing 50% formamide and other salts adapted from Maniatis et
aL (35). Hybridization was conducted in the same buffer at 45~ for 20 h.
Filters were washed two times with buffer containing 0.1X sodium saline
citrate and 0.1% SDS at 22~ for 15 min. Subsequently, filters were washed for
1 h at 52~ in the same washing buffer. Washed filters were blotted on a paper
towel and then exposed to Kodak X-Omat AR film for 24 to 72 h at -70~
before development. The radioactive cyclin B1 probe was removed by boiling
the filter in RNase-free water, and the filter was rehybridized using 32p-labeled
/3-actin probe (Oncor).
Western Blots for Cyclin B1 Protein. Experiments on the effects of estradiol on cyclin B1 protein accumulation/degradation were conducted as
described above for RNA preparation. Cell pellets were lysed in 100/tl of 50
mM Tris-HC1 (pH 8.0) containing 0.5% SDS and 1 mM dithiothreitol. Samples
were boiled for 5 min and then diluted with 400/.tl RIPA buffer [10 mM sodium
phosphate (pH 7.0), 150 mM NaCI, 1% Nonidet P-40, 1% sodium deoxycholate, 2 mM (ethylenedinitrilo)-tetra acetic acid, 50 mM sodium fluoride, 100/ZM
sodium vanadate; and 1% aprotinin; Ref 37]. Samples were centrifuged at
29,000 x g for 1 h, and the supernatant was used for the analysis. Proteins (20
p~g) from the cellular extract were separated by 10% polyacrylamide gel.
Electrophoresis was performed using a Bio-Rad minigel system. The proteins
were transferred to PVDF immobilon membrane (Bedford, MA) and probed
with a purified monoclonal mouse anti-human cyclin B1 protein antibody at 1:
500 dilution. Cyclin proteins were then visualized using horseradish peroxidase-labeled anti-mouse secondary antibody (1:5000 dilution) with a chemiluminescence-based detection system.
Measurement of Intracellular Polyamines. Approximately 3 • 106 cells
were harvested from culture, washed with PBS, and pelletized. The cell pellet
was treated with 300/xl of 8% sulfosalicylic acid and sonicated for 15 s in ice.
The solution was incubated on ice for 1 h and centrifuged at 900 x g for 5 min
to remove the precipitated protein. Intracellular polyamine levels were determined by HPLC after derivatization to their dansyl derivatives as described by
Singh et al. (38). C6 was used as an internal standard. HPLC was performed
on a Perkin-Elmer system using Binary LC Pump 250 and a Fluorescence
Detector LS 40.
Statistical Analysis. Statistical significance of difference between control
and treatment groups was determined by the Student t test using the computer
program Statview.
RESULTS
Synchronized M C F - 7 cells w e r e seeded in phenol red-free m e d i u m
containing charcoal-treated serum for 1 w e e k prior to the experiment.
Growth of M C F - 7 cells under these conditions was necessary to
obtain maximal sensitivity to estradiol and to avoid the estrogenic
effects o f phenol red (27, 31). Two days after the experimental seeding, cells were fed with isoleucine-free m e d i u m for 30 h in order to
arrest cells in G1. Estradiol was added along with the removal of the
cell cycle block with isoleucine containing medium. Table 1 shows the
results o f cell cycle analysis by flow c y t o m e t r y as a percentage o f cells
in different phases o f cell cycle. Eighty one % o f the cells w e r e in G1
after isoleucine starvation. There was no significant change in the
distribution o f cells 12 h after the release o f cell cycle block. Marked
changes between the percentages o f G1 and S cells w e r e observed at
16 h after the addition o f estradiol. Significant difference ( P < 0.05;
n = 4) from control and estradiol-treated cultures was also observed
at 16 h after treatment. In contrast, a marked change from GI to S
appeared to occur in control cells after 36 h.
In order to d o c u m e n t the general pattern o f cyclin B1 m R N A
accumulation in estradiol-treated M C F - 7 cells traversing through
G1--~S----~M, we extracted total R N A at 0, 12, 16, 24, 30, and 36 h after
estradiol addition using the guanidium isothiocyanate procedure (34).
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REGUI.ATION OF CYCLIN B1 BY ESTRADIOL AND POLYAMINES
Table 1 Distribution of MCF-7 cells in cell cycle phases as measured by flow cytometpy
MCF-7 cells grown in phenol red-free medium were treated with isoleucine free medium for 30 h. Estradiol was added along with change to medium containing isoleucine. Controls
received equivalent amount of ethanol vehicle.
Percentage of cells in different phases of cell cycle
G1
Time after the removal of G1 block (h)
0
12
16
24
36
S
G2/M
Control
E2
Control
Ez
81 + 6
87•
83 • 4
80 • 2
74•
81•
73•
63 • 6"
31 • 5"
54•
14_+2
11 •
16 -+ 3
20 • 3
22---2
14•
13•
35 • 4 a
68 • 4 a
67•
'~
Control
4 -+
2 -+
1•
0.5 •
5 •
E2
1
1
0.5
0.5
1
4• 1
3• 1
2 _+ 1
1 --- 1
5•
a Significantly different from controls (P < 0.05).
RNA was analyzed by Northern blotting and hybridization was conducted with a 32p-labeled cyclin B1 cDNA probe. Fig. 1 shows
changes in cyclin B1 mRNA levels during cell cycle progression from
G1 to M. Results shown are from two experiments, one for 12 to 24
h and the other for 30 and 36 h time points. A major species of mRNA
was observed at about 1.7 kilobases, but a second, larger mRNA was
found in overexposed autoradiograms. Densitometer scanning of hybridization signal (after correction for variations in RNA levels)
showed that estradiol induced a 10 to 20% increase in cyclin B1
mRNA levels at 12 to 24 h time points and a significant 37 and 56%
increase (P < 0.05; n = 4, including slot blots) at 30 and 36 h time
points. The latter time points mark the transition of MCF-7 cells to
G2/M, although cells tend to lose synchrony by this time. Thus, by 36
h, an increase in the percentage of G~ cells is observed as cells reenter
G1 after the first cell division in the presence of estradiol. These
results would suggest that estradiol-induced facilitation of cell cycle
involves an increase in cyclin B1 mRNA at GJM.
Previous studies (17) on HeLa cells have shown that cyclin B1
mRNA degradation occurred in early G]. In order to examine whether
this degradation is affected by estradiol, we examined the effects of
estradiol on cyclin B1 mRNA in MCF-7 cells in early G1. For this
purpose, total RNA was extracted from synchronized cells treated
with estradiol at 2, 4, and 8 h after its addition. Control cells that were
not exposed to estradiol were also harvested at these time points. Fig.
2A represents an autoradiogram from Northern blot analysis of cyclin
B1 mRNA. There was a decrease in the intensity of cyclin B1 mRNA
signal in estradiol-treated cells compared to controls. Fig. 2B shows
the quantification of this decrease by a scanning densitometer. There
was a significant 40% (P < 0.05; n = 3) decrease in cyclin B1 mRNA
intensity in estradiol-treated cells compared to controls at 2 h after the
removal of the cell cycle block. The decrease in cyclin B1 mRNA
intensity continued for up to 8 h after the removal of the G] block, and
estradiol accelerated this process.
In the next set of experiments, we examined whether estradiolinduced alterations in cyclin B1 mRNA could be detected at the
protein level. Cells harvested as in previous experiments were used for
the analysis of cyclin B1 protein by Western blots using a monoclonal
anti-cyclin B1 antibody. As shown in Fig. 3, two proteins were rec-
ognized by this cyclin B1 antibody; the upper band coincided with the
reported molecular weight of 62,000 of cyclin B1 protein. The lower
band appears to be a degradation product of the Mr 62,000 protein
because we observed an increase in the intensity of this band at the 6-h
time point as the intensity of the upper band decreased. The observation of immunoreactive cyclin B1 protein in these experiments is
consistent with our finding of relatively high levels of the mRNA in
G~. Estradiol accelerated the degradation of this protein as in the case
of the mRNA. Quantification of the cyclin B1 protein (Fig. 3) showed
that its degradation continued up to 12 h after the addition of estradiol,
reaching 31 ___8% of the control at 2 h. This decrease was statistically
significant (P < 0.02; n = 3). Additional three experiments using
immunoprecipitated cyclin B1 protein provided a similar pattern of
cyclin B1 degradation (results not shown).
A
1
2
3
4
5
6
9
--
~
1 1 ~
B
m
e-
e-
lOO
~0
m
0
G,
0o
m
Z
n,
.
1
2
.
3
.
4
.
5
.
6
.
7
.
8
9
10
E
--18S
I0
~
0
2
4
$
Time, h
Fig. 1. Accumulation of cyclin B1 mRNA in ceils progressing from GI. Cyclin B1
mRNA was detected in cells treated with estradiol for 0 (Lane 1), 12 (Lane 2), 16 (Lane
3), and 24 (Lane 4) h after treatment with estradiol by Northern blot analysis. Lane 5,
RNA from cells cycling in the absence of estradiol for 24 h. Lanes 6-10, results of a
separate experiment. Lanes 6, 7, and 9, controls (no estradiol) at 0, 30, and 36 h; Lanes
8 and 10, estradiol-treated cells at 30 and 36 h. Arrow, cyclin B1 mRNA (1.7 kilobase
pairs).
Fig. 2. A, estradiol-induced down-regulation of cyclin B1 mRNA in early G1 by
Northern blot analysis. Lanes 1, 3, and 5, controls (no estradiol) at 2, 4, and 8 h,
respectively; Lanes 2, 4, and 6, estradiol-treated cells at 2, 4, and 8 h. B, quantitation of
cyclin B1 mRNA signals using Hoefer GS 300 scanning densitometer from three Northern
blot experiments representing control ([5) and estradiol-treated ([]) cells. Hybridization
signals were normalized to the amount of RNA using negatives of gel pictures after
ethidium bromide staining. Data are the mean • SD of three separate experiments. Signals
from estradiol-treated cells were significantly different from those of control cells at all
time points tested (P < 0.05; n = 3).
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REGULATION OF CYCLIN t31 BY ESTRAI)IO[. AND I'OLYAMINES
p62 Cyclin B1
1 2
BJ
3
4
5
~
i
m a y be related to the release of cells from G1 block and growth
stimulation. Previous studies have s h o w n an increase in the expression
of other structural genes such as ot-tubulin in growth-stimulated cells
6
(37).
To investigate the role of p o l y a m i n e s in estrogenic regulation of
cyclin B1 m R N A , we used D F M O for depleting p o l y a m i n e s (39).
Dose- and t i m e - d e p e n d e n t effects of D F M O on p o l y a m i n e levels in
isoleucine-starved M C F - 7 cells were first d o c u m e n t e d for this purpose. Results s h o w n in Fig. 5A d e m o n s t r a t e d that m a x i m a l reduction
of putrescine occurred with 1 mM D F M O treatment for 48 h, even
though significant reduction was observed at the 24-h time point ( P <
0.05; n = 4). At 48 h, 1 mM D F M O reduced putrescine concentrations
to 10% of that in the control cells at the same time point. There were
no significant differences in the reduction of putrescine b e t w e e n 1 and
2 mM concentrations of D F M O . The effect of D F M O on spermidine
concentration was also time dependent, the m a x i m a l effect occurring
only at 7 2 - 9 6 h (Fig. 5B). At the 48-h time point, 1 mM D F M O
treatment reduced spermidine concentration to 25% of that in the
control cells at the same point. In contrast to a significant reduction
>' 120
=-
100
o.
~
60
o.
c
20
O
0
2
6
12
Time, h
Fig. 3. Estradiol-induced down-regulation of cyclin B1 protein in G]. Proteins were
detected by Western blots using a chemiluminescence detection system. Lanes I, 3, and
5, controls; Lanes 2, 4, and 6, estradiol-treated samples at 2, 6, and 12 h, respectively, after
the addition of estradiol and removal of the cell cycle block. Columns, the intensity of the
protein bands quantitated by densitometer for control (IS]) and estradiol-treated (I~)
samples. Bars, + SD in 3 separate experiments.
[]
OFMO:0 i~1
Q t~MO:S00~
[]
OFMO:1000FM
[] oF~o:z~oo~M
c-
i
0
o
.
.
.
.
1oo
=
4
0
,
0
,
10
,
,
,
20
Time,
,
30
,
/
o
40
:~
h
~:
Fig. 4. Changes in cyclin B1 and/3-actin mRNA intensity as a function of cell cycle
progression. Membranes used for probing with cyclin B1 eDNA (e) were boiled to
remove the radioactive probe and then hybridized with a/3-actin probe (C]). Autoradiograms from a 24-h exposure were used for densitometric scanning. Points, mean of four
separate experiments, two Northern blots, and two slot blots.
We also analyzed the a c c u m u l a t i o n of cyclin B1 protein at 30 and
36 h time points w h i c h mark G ~ M (results not shown). There was a
4-fold increase in the intensity o f the protein band at 36 h c o m p a r e d
to that at 12 h. However, the increase in intensity of the protein band
at these time points in estradiol-treated cells was not statistically
significant w h e n c o m p a r e d to that of control cells at the same time
points.
To c o m p a r e the oscillation of cyclin B1 m R N A to the expression of
a structural gene, each blot was boiled to r e m o v e cyclin B1 c D N A and
then reprobed with 32p-labeled ~O-actin probe. Densitometric scanning
f r o m autoradiograms that were e x p o s e d 24 h was used for this purpose. Quantification o f autoradiograms (Fig. 4) s h o w e d a slow increase in /3-actin gene expression f r o m 0 to 36 h, while cyclin B1
decreased in early G1 and then increased in S. This result was reproducible in four separate experiments. T h e up-regulation of /3-actin
0
~
"~
"
"
.
.
0
1
2
a
4
DAYS OF TREATMENT
Fig. 5. Dose-and time-dependent effect of DFMO on putrescine (A), spermidine (B),
and spermine (C) levels in G1 synchronized MCF-7 cells. Isoleucine-starved cells were
treated with 0, 0.5, 1, and 2 mM concentrations of DFMO for different time points. Cells
were harvested, and polyamine levels were analyzed using a previously described HPLC
method (38).
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REGULATION OF CYCLIN B1 BY ESqRADIOL AND POLYAMINES
(P < 0.001; n = 4) of putrescine and spermidine, DFMO had no
significant effect on spermine levels in these cells (Fig. 5C).
To study the effect of polyamine depletion on cyclin B1 mRNA,
cells were synchronized by 30-h treatment of isoleucine-free medium
and then treated with 0, 0.5, 1, and 2 mM concentrations of DFMO for
48 h. Cells were then changed to regular medium containing the
appropriate concentrations of DFMO as well as 4 nM estradiol. Cells
were harvested 2 h after the addition of estradiol and the change to
regular medium. Total RNA was extracted and analyzed for cyclin B1
m R N A levels as in the previous experiments. Fig. 6 shows that the
intensity of cyclin B1 m R N A signal was up-regulated in DFMOtreated cells at all concentrations tested. Densitometric scanning of the
autoradiograms with normalization for RNA levels in the gel indicated
a 2- to 3-fold increase in cyclin B1 m R N A in the presence of DFMO
compared to that of untreated cells. These increases were statistically
significant (P < 0.01; n = 3). In separate experiments, we found that
0.1 mM DFMO had no significant effect on cyclin B1 m R N A levels
(results not shown). Thus, optimal effect of DFMO on cyclin B1
m R N A was achieved at 0.5 to 1 mM concentration.
In the next set of experiments, we determined the effect of exogenous putrescine and spermidine on DFMO-mediated increase in cyclin B1 m R N A in G1 of MCF-7 cells in the presence of estradiol. Fig.
7 shows our results on the effect of polyamine repletion on cyclin B1
mRNA. Both compounds reversed the effect of DFMO on cyclin B1
m R N A stability and accelerated its degradation in a dose-dependent
manner. At 100/XM putrescine, the cyclin B1 m R N A level was 10% of
that in DFMO-treated cells. There was no significant change in the
level of cyclin B1 m R N A at 250, 500, and 1000 ttM concentrations of
putrescine, compared to that at 100 tiM. Spermidine at 25 /XM did not
affect cyclin B1 m R N A levels significantly, but at 50, 100, and 250
tXM concentrations, cyclin B1 m R N A was rapidly degraded. These
experiments were conducted in the presence of 1 mM aminoguanidine
to prevent oxidative metabolism of polyamines by serum-derived
amine oxidases (40). Treatment with aminoguanidine alone had no
effect on cyclin B1 m R N A levels (results not shown). These results
were reproducible in three separate experiments and confirm our
hypothesis that polyamines play a major regulatory role in controlling
the levels of cyclin B1 mRNA. Measurement of polyamine levels of
isoleucine-starved and DFMO-treated cells showed that exogenous
putrescine and spermidine were transported into the cells within 30
min of its addition to the medium, and they remained high for at least
8 h in these cells (41).
In another set of experiments, we examined the structural specificity of polyamines in cyclin B1 m R N A stability using homologues of
putrescine. Isoleucine-starved cells were treated with 1 mM DFMO for
48 h and then with 100 /~M C3, C4 (putrescine), C5, or C6 in the
presence of aminoguanidine for 2 h. Cell cycle was then initiated by
changing to regular medium containing 1 mM DFMO, 100 tim putrescine, or its homologues, 1 mM aminoguanidine and 4 nM estradiol.
A
1
2
3
4
5
:'
-18S
B
1
2
3
4
5
-18S
Fig. 7. Reversal of DFMO-mediatedstabilization of cyclin B1 mRNA by putrescine
(A) and spermidine (B). Ceils were synchronized by treatment with isoleucine-free
mediumand then treatedwith 1 mMDFMOfor 48 h in isoleucine-freemedium.Cellswere
then treatedfor 2 h with 1 mMDFMO, 1 mMaminoguanidine,and differentconcentrations
of putrescine or spermidine in isoleucine-freemediumfor 2 h. Ceils were then changed
to regular mediumcontainingDFMO, putrescine/spermidine, 1 mMaminoguanidine,and
4 nMestradiol. Cells were harvested2 h after the estradiol addition,and cyclin B1 mRNA
was determined.(A) Lane 1, 0; Lane 2, 100; Lane 3, 250; Lane 4, 500; and Lane 5, 1000
tZMputrescine. (B) Lane 1, 0; Lane 2, 25; Lane 3, 50; Lane 4, 100; and Lane 5, 250/~M
spermidine.
Cells were harvested 2 h after change to this medium; RNA was
extracted and analyzed by Northern blots. Putrescine was the most
effective diamine in reversing the effect of DFMO on cyclin B1
m R N A (80% reversal; P < 0.05; n = 3), whereas C3, C5, and C6 had
no significant effect (10-25% reversal; P > 0.1). HPLC analysis
showed that all diamines were internalized into MCF-7 cells. Thus,
polyamine-induced effects on cyclin B1 m R N A are unlikely to be due
to ionic effects of these molecules and indicate structure-specific
interactions of polyamines in facilitating the degradation of cyclin B1
mRNA.
We also examined the effects of DFMO on cyclin B1 m R N A in the
presence and absence of estradiol. Cells were treated with DFMO and
4 nM estradiol, and cyclin B1 m R N A was analyzed and quantified by
slot blot experiments (autoradiogram not shown). Quantitation of slot
blot signals (Fig. 8) showed a significant 3-fold increase (P < 0.01; n
= 3) in cyclin B1 m R N A levels with 1 m u DFMO, both in the
presence and absence of estradiol.
DISCUSSION
1
2
3
4
. . . .
18S
Our results demonstrate that estradiol plays an important role in the
regulation of cyclin B1 gene expression in MCF-7 cells during the cell
cycle traverse through G1----~S----~G2---~M. In both control and estradioltreated cells, down-regulation of cyclin B1 m R N A appears to occur
immediately after the removal of the G1 cell cycle block. Within 2 h
of estradiol addition, there was a 40% decrease in cyclin B1 m R N A
level compared to that of untreated control cells. The cyclin B1 m R N A
level in the control cultures at 8 h after the release of cell cycle block
was comparable to that of estradiol-treated cells at 2 h. A similar effect
of estradiol was observed on the degradation of cyclin B1 protein in
G~. DFMO inhibited the degradation of cyclin B1, while the addition
of exogenous putrescine and spermidine accelerated the degradation
process when these compounds were added after DFMO-treatment.
Fig. 6. Effect of DFMO on cyclin B1 mRNA levels. Cells were synchronizedby
treatment with isoleucine-freemediumand then treatedwith (1) 0; (2) 0.5, (3) 1, and (4)
2 mM DFMO for 48 h. Cells were then allowed to progress in isoleucine-containing
medium in the presenceof estradiol and were harvested 2 h later. Cyclin B1 mRNAwas
analyzed by Northern blot hybridization.
1081
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Research.
REGULATION
OF ( ' Y ( ' I . I N
131
BY
ESTI,~AI)IOI. A N D
I'OI.YAMINES
to be conducive to the requirements of GI transit, as cells move very
slowly through G, in the absence of estradiol. Estradiol-induced eno
hancement of cyclin B1 degradation appears to be one of the factors
facilitating cell cycle transit through G1.
el
The induction of cyclin B1 mRNA at Gz/M may involve transcrip300
tional regulation. In addition to the direct effects of estrogen receptor,
these later events may include those mediated by transcription factors
and DNA binding proteins induced at the early stage of estradiol
action. Estradiol induction of cyclin B1 mRNA accumulation in Gz/M
tin
200
may also make a contribution from the fact that 24 h after the addition
of estradiol, treated and untreated cultures are at different stages of
Z
cell cycle progression. In contrast, effects of estradiol and DFMO at
n"
the early stages of cell cycle progression could be attributed to the
100
E
direct action of these agents. Accelerated degradation of cyclin B1
mRNA
may contribute to estradiol-induced decrease in the duration of
r
MCF-7
cell cycle. For example, the duration of G1 of MCF-7 cells
r~
growing in the absence of phenol red and estradiol was 27 h as
1
2
3
4
o
determined by the BrdUrd labeling method, and it was reduced to 16
Treatment
O
h in the presence of estradiol (46, 47). Estradiol-induced decrease in
Fig. 8. Quantificationof the effectof DFMOon cyclinBI mRNAlevelsin the presence the duration of G~ was also reported for another estrogen-dependent
and absence of estradiol. SynchronizedMCF-7 cells were treated with 1 mMDFMO for breast cancer cell line, CAMA-1 (48). The 81% cell synchrony that we
48 h followedby estradiol for 2 h, and RNAwas extracted. Treatmentswere: (1) control
without DFMOand estradiol; (2) 4 nMestradiol; (3) 1 mMDFMO;and (4) 1 rnMDFMO obtained by isoleucine starvation is comparable to other studies using
+ 4 nM estradiol. Data are the mean of three slot-blot experiments. Bars, +__SD. One purified mitotic cells. For example, Taylor et al. (49) observed 85%
hundred % represents the intensityof cyclinB1 mRNAlevel after estradiol treatment.
synchrony after mitotic selection and found that about 15% of MCF-7
cells could be categorized as noncycling cells.
The interaction between estrogen receptor and cyclin B1 mRNA
might be important in its destabilization. This could be a result of the
Our results indicate that critical concentrations of polyamines might
be necessary for the degradation of cyclin B1 and for cell cycle reorganization of macromolecular interactions with the ligand-induced changes in the conformation of estrogen receptor, the dissociaprogression through G~. These results are consistent with previous
tion of heat shock proteins from the receptor, and the recruitment of
studies from our laboratory and others that suggest a polyamine pathtranscription factors (1-3). Our previous studies have shown that
way in estrogenic regulation of breast cancer cell growth (25-27).
polyamines increase the stability of ligand-receptor and estrogen reOur observation of cyclin B1 mRNA in Ga is comparable to other
ceptor-DNA interactions in vitro (28-30). Thus, polyamines might be
reports in the literature (17, 39). Pines and Hunter (17) conducted
experiments on HeLa cells to determine if cyclin B1 mRNA levels fell important cellular components that facilitate estrogenic action and the
during M or early Ga by blocking cells in pseudo-metaphase state regulation of cyclin B1 accumulation and destruction during different
using a microtubule polymerization inhibitor, nocadazole. Results of phases of cell cycle. Since DFMO-induced increase in cyclin B1
mRNA occurred even in the absence of estradiol, polyamines may
those experiments indicated that cyclin B1 mRNA level remained
also have a direct role in the degradation of cyclin B1 mRNA.
constant until the end of M and then decreased during the initial part
Our finding that polyamine depletion in MCF-7 cells blocked esof G1. A recent study on the expression of cyclins during liver regeneration, however, did not show mRNA for B-type cyclins in the early tradiol-induced degradation of cyclin B1 mRNA indicates that cyclin
stage of stimulation (37). On the other hand, studies on HL-60 cells B1 could be a target site at which the effect of estradiol is linked to
cellular polyamine concentrations. DFMO exerted a time- and doseshow significant levels of cyclin B1 mRNA in purified GI cells (42).
These results raise the possibility of cell type-specific regulation of dependent effect in depleting putrescine and spermidine, and the effect
of DFMO on cyclin BI mRNA was significant only after substantial
this mRNA. More recently, while our work was in progress, Keyomarsi and Pardee (43) reported that human breast cancer cell lines reduction of these polyamine levels was achieved. Exogenous putrescine and spermidine reversed the effect of DFMO by a relatively
show abnormal expression of cyclins A and B1 in G,, whereas such
expression was not detected in immortalized normal breast epithelial short-term exposure of the cells to these compounds. The effect of
cells. This result suggests a correlation of the overexpression of mi- polyamines appear to be structure-specific since putrescine homototic cyclins in G, and the expression of the malignant phenotype. It logues differing by 1 or 2 methylene groups in their chemical structure
is also important to note that estradiol, a hormone that stimulates the had no effect on the degradation of cyclin B1. The polyamine strucproliferation of MCF-7 cells, accelerated the degradation of this tural specificity that we observed in this study is comparable to
mRNA, while DFMO, a growth inhibitory agent that arrests cells in polyamine effects on DNA conformational transitions and the ability
of polyamine homologs to support cell growth (50-52).
GI, attenuated its degradation.
We also characterized estradiol-induction of ODC activity and
Cyclin B1 protein is degraded in M in well-characterized embrypolyamine levels in cells synchronized by isoleucine starvation (41).
onic model systems such as X e n o p u s oocytes and sea urchin eggs (44,
45). Introduction of deletion mutants of cyclin B1 protein defective in The peak of ODC activity was broad starting from 8 to 16 h after
degradation leads to growth arrest in M. Thus, overexpression or lack addition of estradiol. Polyamine levels peaked at the 8- to 12-h time
period. Thus, an increase in polyamine levels might be required for the
of degradation of cyclin B1 protein is not consistent with abnormal
proliferation based on the current knowledge of these events in lower dissociation and regrouping of protein-DNA interactions that are necorganisms. However, detection of cyclin B1 protein in GI may indi- essary during a rapid phase of transcription and protein synthesis that
should precede DNA replication. Furthermore, polyamines are likely
cate an alternate pathway by which these proteins activate cdc2to be involved in the interactions between transcription factors and
related kinases in G1, bypassing the regulation of this kinase in normal
specific DNA sequences and in the control of mRNA stability. Our
cells. Nevertheless, the presence of cyclin B1 protein does not appear
1082
ti,-.
~
v
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Research.
REGULATION OF CYCLIN BI BY ESTRADIOL AND I'OIYAMINES
results on the effects of DFMO and polyamines on cyclin B1 mRNA
indicate that the pool of polyamines available in early G~ of the cell
cycle might have an important effect in driving the cell cycle by
accelerating the degradation of mitotic cyclins, such as B1, that are
aberrantly expressed in this phase.
Phosphorylation levels of cyclin B1 and p34 cdc2 are other important
factors that determine the activation of cdc2 kinase activity (53, 54).
Kinase inhibitors have been reported to suppress estrogen-stimulated
growth of MCF-7 cells (55). Thus, a complete characterization of the
role of cyclin B1 on estradiol-induced regulation of cell cycle will
require the determination of concentrations of cyclin B1 mRNA,
protein, its phosphorylation status, and its association with p34 cdc2.
Recent studies demonstrate that cdc2 and homologous kinases are
critical to the control of cell cycle, not only at the initiation of M but
also at the onset of S (56). Estradiol may affect these processes at
many different levels.
In summary, our results show a differential effect of estradiol on
cyclin B1 mRNA levels during different phases of cell cycle. During
early G1, estradiol facilitates the down-regulation of cyclin B1
mRNA, whereas during Gz/M, it enhances the accumulation of cyclin
B1 mRNA. Effect of estradiol on cyclin B1 mRNA in G1 was correlated to that on its protein. Polyamines appear to be necessary for the
degradation of cyclin B1 mRNA both in the presence and absence of
estradiol. The effect of estradiol on cyclin B1 in G1 may constitute a
part of estrogenic action in shortening the duration of MCF-7 cell
cycle and thus contribute to the hormonal regulation of cell growth.
ACKNOWLEDGMENTS
We thank Chia-Ching Chao and Carol A. Faaland for technical assistance
and Dr. E d w a r d Yurkov for flow cytometry. We thank Drs. Jonathon Pines and
Tony Hunter of the Salk Institute (La Jolla, C A ) for the cyclin B1 c D N A probe
and Dr. Pines for a critical reading of the manuscript. We also a c k n o w l e d g e
Marion Merrell D o w (Cincinnati, Ohio) for the supply of D F M O .
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Regulation of Cyclin B1 by Estradiol and Polyamines in MCF-7
Breast Cancer Cells
Thresia Thomas and T. J. Thomas
Cancer Res 1994;54:1077-1084.
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