[cancer research 54, 1077 10~4. february 15, t994 ... · control cell cycle. among these proteins,...

[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-buff- ered 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 serum- derived factors and thereby enhance cell growth (10). Despite exten- sive 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 classifi- cation, 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]. Re- cent 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 A N D M E T H O D S

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-mercapto- ethanol. 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 load- ing. The gel and running buffer contained 2.2 M formaldehyde, 50 mM mor- pholino-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. Hybrid- ization 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 deoxycho- late, 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 peroxi- dase-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.

R E S U L T S

Synchronized MCF-7 cells were seeded in phenol red-free med ium

containing charcoal- treated serum for 1 week prior to the experiment.

Growth of MCF-7 cells under these conditions was necessary to

obtain maximal sensitivity to estradiol and to avoid the estrogenic

effects of phenol red (27, 31). Two days after the experimental seed-

ing, cells were fed with isoleucine-free med ium 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 f low cytometry as a percentage o f cells

in different phases of cell cycle. Eighty one % of the cells were in G1

after isoleucine starvation. There was no significant change in the

distribution of cells 12 h after the release of cell cycle block. Marked

changes between the percentages o f G1 and S cells were observed at

16 h after the addition of 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 f rom GI to S

appeared to occur in control cells after 36 h.

In order to document the general pattern of cyclin B1 m R N A

accumulat ion in estradiol-treated MCF-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).

1078



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.

Time after the removal of G1 block (h) Control

Percentage of cells in different phases of cell cycle

G1 S G2/M

E 2 Control Ez Control E 2

0 81 + 6 8 1 • 14_+2 1 4 • 4 - + 1 4 • 1 12 8 7 • 7 3 • 11 • 1 3 • 2 - + 1 3 • 1 16 83 • 4 63 • 6" 16 -+ 3 35 • 4 a 1 • 0.5 2 _+ 1 24 80 • 2 31 • 5" 20 • 3 68 • 4 a 0.5 • 0.5 1 --- 1 36 7 4 • 5 4 • 22---2 6 7 • '~ 5 • 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 estradiol- induced 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-

1 2 3 4 5 6 7 8 9 10 . . . . . . . - - 1 8 S

4 $

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).

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

~ m 0

0 G, 0o

Z n,

E

I0

~ 0

m

2

Time, h 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).

1079



REGULATION OF CYCLIN t31 BY ESTRAI)IO[. AND I'OLYAMINES

1 2 3 4 5 6 p62 Cyclin B1 B J i ~

>' 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.

may be related to the release of cells f rom G1 block and growth

st imulat ion. Previous studies have shown an increase in the expression

of other structural genes such as ot-tubulin in growth-s t imula ted cells

(37). To invest igate the role of po lyamines in es t rogenic regulat ion of

cyclin B1 m R N A , we used D F M O for deple t ing po lyamines (39).

Dose- and t ime-dependen t effects of D F M O on po lyamine levels in

isoleucine-s tarved MCF-7 cells were first documen ted for this pur-

pose. Results shown in Fig. 5A demons t ra ted that max imal reduct ion

of putrescine occurred with 1 mM D F M O treatment for 48 h, even

though significant reduct ion was observed at the 24-h t ime point (P <

0.05; n = 4). At 48 h, 1 mM D F M O reduced putrescine concentra t ions

to 10% of that in the control cells at the same t ime point. There were

no significant differences in the reduct ion of putrescine be tween 1 and

2 mM concentra t ions of DFMO. The effect of D F M O on spermidine

concentra t ion was also t ime dependent , the maximal effect occurr ing only at 72 -96 h (Fig. 5B). At the 48-h t ime point, 1 mM D F M O

treatment reduced spermidine concentra t ion to 25% of that in the

control cells at the same point. In contrast to a s ignif icant reduct ion

[ ] OFMO: 0 i~1 Q t~MO: S00 ~ [ ] OFMO: 1000 FM [] oF~o: z~oo ~M

c-

i 0 o . . . .

1oo

= 4

0 , , , , , , , / o

0 10 20 30 4 0 :~

Time, h ~: Fig. 4. Changes in cyclin B1 and/3-actin mRNA intensity as a function of cell cycle 0

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]). Autoradio- ~ " " grams 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 accumula t ion of cycl in B1 protein at 30 and 36 h t ime points which mark G ~ M (results not shown). There was a

4-fold increase in the intensity o f the protein band at 36 h compared

to that at 12 h. However , the increase in intensity of the protein band

at these t ime points in estradiol- treated cells was not statistically

significant when compared to that of control cells at the same t ime points.

To compare the oscil lat ion of cycl in B1 m R N A to the express ion of

a structural gene, each blot was boi led to r emove cycl in B1 c D N A and . .

then reprobed with 32p-labeled ~O-actin probe. Dens i tomet r ic scanning 0 1 2 a 4

f rom autorad iograms that were exposed 24 h was used for this pur- DAYS OF TREATMENT pose. Quant i f icat ion of au toradiograms (Fig. 4) showed a s low in- Fig. 5. Dose-and time-dependent effect of DFMO on putrescine (A), spermidine (B), crease in /3-actin gene expression f rom 0 to 36 h, whi le cycl in B1 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 decreased in early G1 and then increased in S. This result was repro- were harvested, and polyamine levels were analyzed using a previously described HPLC ducible in four separate exper iments . The up-regula t ion of /3-actin method (38).

1080



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

mRNA levels as in the previous experiments. Fig. 6 shows that the intensity of cyclin B1 mRNA signal was up-regulated in DFMO-

treated 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 mRNA 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 mRNA levels (results not shown). Thus, optimal effect of DFMO on cyclin B1

mRNA was achieved at 0.5 to 1 mM concentration. In the next set of experiments, we determined the effect of exog-

enous putrescine and spermidine on DFMO-mediated increase in cy-

clin B1 mRNA 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 mRNA stability and accelerated its degradation in a dose-dependent manner. At 100/XM putrescine, the cyclin B1 mRNA level was 10% of that in DFMO-treated cells. There was no significant change in the

level of cyclin B1 mRNA at 250, 500, and 1000 ttM concentrations of putrescine, compared to that at 100 tiM. Spermidine at 25 /XM did not affect cyclin B1 mRNA levels significantly, but at 50, 100, and 250 tXM concentrations, cyclin B1 mRNA 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 mRNA 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 mRNA 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.

B

1 2 A

3 4 5 : ' -18S

1 2 3 4 5 - 1 8 S

Fig. 7. Reversal of DFMO-mediated stabilization of cyclin B1 mRNA by putrescine (A) and spermidine (B). Ceils were synchronized by treatment with isoleucine-free medium and then treated with 1 mM DFMO for 48 h in isoleucine-free medium. Cells were then treated for 2 h with 1 mM DFMO, 1 mM aminoguanidine, and different concentrations of putrescine or spermidine in isoleucine-free medium for 2 h. Ceils were then changed to regular medium containing DFMO, putrescine/spermidine, 1 mM aminoguanidine, and 4 nM estradiol. Cells were harvested 2 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 tZM putrescine. (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 mRNA (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 mRNA 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 mRNA in the

presence and absence of estradiol. Cells were treated with DFMO and 4 nM estradiol, and cyclin B1 mRNA 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 mRNA levels with 1 mu DFMO, both in the

presence and absence of estradiol.

DISCUSSION

1 2 3 4 . . . . 18S

Fig. 6. Effect of DFMO on cyclin B1 mRNA levels. Cells were synchronized by treatment with isoleucine-free medium and then treated with (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 presence of estradiol and were harvested 2 h later. Cyclin B1 mRNA was analyzed by Northern blot hybridization.

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 estradiol- treated cells, down-regulation of cyclin B1 mRNA 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 mRNA level compared to that of untreated control cells. The cyclin B1 mRNA 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.

1081



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200

100

R E G U L A T I O N OF ( 'Y( ' I . IN 131 BY ESTI,~AI)IOI. AND I 'OI .YAMINES

Fig. 8. Quantification of the effect of DFMO on cyclin BI mRNA levels in the presence and absence of estradiol. Synchronized MCF-7 cells were treated with 1 mM DFMO for 48 h followed by estradiol for 2 h, and RNA was extracted. Treatments were: (1) control without DFMO and estradiol; (2) 4 nM estradiol; (3) 1 mM DFMO; and (4) 1 rnM DFMO + 4 nM estradiol. Data are the mean of three slot-blot experiments. Bars, +__ SD. One hundred % represents the intensity of cyclin B1 mRNA level after estradiol treatment.

Our results indicate that critical concentrations of polyamines might be necessary for the degradation of cyclin B1 and for cell cycle progression through G~. These results are consistent with previous studies from our laboratory and others that suggest a polyamine pathway in estrogenic regulation of breast cancer cell growth (25-27).

Our observation of cyclin B1 mRNA in Ga is comparable to other reports in the literature (17, 39). Pines and Hunter (17) conducted experiments on HeLa cells to determine if cyclin B1 mRNA levels fell during M or early Ga by blocking cells in pseudo-metaphase state using a microtubule polymerization inhibitor, nocadazole. Results of those experiments indicated that cyclin B1 mRNA level remained constant until the end of M and then decreased during the initial part of G1. A recent study on the expression of cyclins during liver regeneration, however, did not show mRNA for B-type cyclins in the early stage of stimulation (37). On the other hand, studies on HL-60 cells show significant levels of cyclin B1 mRNA in purified GI cells (42). These results raise the possibility of cell type-specific regulation of this mRNA. More recently, while our work was in progress, Keyo- marsi and Pardee (43) reported that human breast cancer cell lines show abnormal expression of cyclins A and B1 in G,, whereas such expression was not detected in immortalized normal breast epithelial cells. This result suggests a correlation of the overexpression of mitotic cyclins in G, and the expression of the malignant phenotype. It is also important to note that estradiol, a hormone that stimulates the proliferation of MCF-7 cells, accelerated the degradation of this mRNA, while DFMO, a growth inhibitory agent that arrests cells in GI, attenuated its degradation.

Cyclin B1 protein is degraded in M in well-characterized embryonic model systems such as Xenopus oocytes and sea urchin eggs (44, 45). Introduction of deletion mutants of cyclin B1 protein defective in degradation leads to growth arrest in M. Thus, overexpression or lack of degradation of cyclin B1 protein is not consistent with abnormal proliferation based on the current knowledge of these events in lower organisms. However, detection of cyclin B1 protein in GI may indicate an alternate pathway by which these proteins activate cdc2- related kinases in G1, bypassing the regulation of this kinase in normal cells. Nevertheless, the presence of cyclin B1 protein does not appear

to be conducive to the requirements of GI transit, as cells move very slowly through G, in the absence of estradiol. Estradiol-induced en- hancement of cyclin B1 degradation appears to be one of the factors facilitating cell cycle transit through G1.

The induction of cyclin B1 mRNA at Gz/M may involve transcriptional 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 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 cell cycle progression. In contrast, effects of estradiol and DFMO at the early stages of cell cycle progression could be attributed to the direct action of these agents. Accelerated degradation of cyclin B1 mRNA may contribute to estradiol-induced decrease in the duration of MCF-7 cell cycle. For example, the duration of G1 of MCF-7 cells growing in the absence of phenol red and estradiol was 27 h as determined by the BrdUrd labeling method, and it was reduced to 16 h in the presence of estradiol (46, 47). Estradiol-induced decrease in the duration of G~ was also reported for another estrogen-dependent breast cancer cell line, CAMA-1 (48). The 81% cell synchrony that we obtained by isoleucine starvation is comparable to other studies using purified mitotic cells. For example, Taylor et al. (49) observed 85% 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 reorganization of macromolecular interactions with the ligand-induced changes in the conformation of estrogen receptor, the dissociation of heat shock proteins from the receptor, and the recruitment of transcription factors (1-3). Our previous studies have shown that polyamines increase the stability of ligand-receptor and estrogen receptor-DNA interactions in vitro (28-30). Thus, polyamines might be important cellular components that facilitate estrogenic action and the regulation of cyclin B1 accumulation and destruction during different phases of cell cycle. Since DFMO-induced increase in cyclin B1 mRNA occurred even in the absence of estradiol, polyamines may also have a direct role in the degradation of cyclin B1 mRNA.

Our finding that polyamine depletion in MCF-7 cells blocked estradiol-induced degradation of cyclin B1 mRNA indicates that cyclin B1 could be a target site at which the effect of estradiol is linked to cellular polyamine concentrations. DFMO exerted a time- and dose- dependent effect in depleting putrescine and spermidine, and the effect of DFMO on cyclin BI mRNA was significant only after substantial reduction of these polyamine levels was achieved. Exogenous putrescine and spermidine reversed the effect of DFMO by a relatively short-term exposure of the cells to these compounds. The effect of polyamines appear to be structure-specific since putrescine homologues differing by 1 or 2 methylene groups in their chemical structure had no effect on the degradation of cyclin B1. The polyamine structural specificity that we observed in this study is comparable to polyamine effects on DNA conformational transitions and the ability of polyamine homologs to support cell growth (50-52).

We also characterized estradiol-induction of ODC activity and polyamine levels in cells synchronized by isoleucine starvation (41). The peak of ODC activity was broad starting from 8 to 16 h after 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 dissociation and regrouping of protein-DNA interactions that are necessary during a rapid phase of transcription and protein synthesis that should precede DNA replication. Furthermore, polyamines are likely to be involved in the interactions between transcription factors and specific DNA sequences and in the control of mRNA stability. Our

1082



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 corre- lated 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. Edward Yurkov for f low cytometry . We thank Drs. Jonathon Pines and

Tony Hunter o f the Salk Institute (La Jolla, CA) for the cycl in B1 c D N A probe

and Dr. Pines for a critical reading of the manuscript . We also acknowledge

Mar ion Merrel l D o w (Cincinnati , Ohio) for the supply of D F M O .

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1994;54:1077-1084. Cancer Res Thresia Thomas and T. J. Thomas Breast Cancer CellsRegulation of Cyclin B1 by Estradiol and Polyamines in MCF-7

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