human breast cancer cell cycle synchronization by...

[CANCER RESEARCH 44, 1433-1439, April 1984]

Human Breast Cancer Cell Cycle Synchronization by Estrogens andAntiestrogens in Culture1

C. Kent Osborne,2 David H. Boldt, and Phillip Estrada

Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78284

ABSTRACT

The mechanisms by which estrogens and antiestrogens modulate breast cancer growth have not been totally defined. Wehave examined the cell cycle kinetic effects of estrogens andantiestrogens in cultured human breast cancer cell lines. In aprevious study, we showed that tamoxifen induces a transitiondelay in early to mid-d phase of the cell cycle. In the present

study, we show that this cell kinetic alteration by tamoxifen isdose dependent and that other antiestrogens have identicaleffects. As little as 0.01 to 0.1 ^M tamoxifen reduces the S andG2+M fractions and increases the d fraction of MCF-7 cellsgrowing in medium with 5% charcoal-stripped bovine serum.

More than 90% of cells are in G, 72 to 96 hr after the additionof 1 MMtamoxifen, a concentration achieved in patients treatedwith the drug. Nafoxidine and trioxifene have identical activity.Partial reversal of tamoxifen growth inhibition is observed with asimple change to tamoxifen-free medium. Complete reversal ofthe tamoxifen effect is observed with the addition of 170-estra-

diol. By 24 hr after the addition of estrogen, 60 to 70% oftamoxifen-inhibited cells have progressed through d and into Sphase, indicating that tamoxifen-treated cells remain viable. Thisestrogen "rescue" effect is observed even in the absence of a

change to tamoxifen-free medium. A 100-fold-lower concentra

tion of estradici can totally reverse the inhibitory effects of 1.0UMtamoxifen. Stimulation of the progression of GÃ¬cells to enterS phase is also observed when estradici is added to cellsmaintained for four days in medium with stripped serum, even inthe absence of tamoxifen. Similar effects are observed in theestrogen receptor-positive ZR75-1 breast cancer cells. No effects of antiestrogens or estrogens are observed in the receptor-negative MDA-231 cells, suggesting that these effects are me

diated through the estrogen receptor. In summary, antiestrogensand estrogens have prominent effects on the cell cycle kineticsof endocrine-dependent human breast cancer cells. Antiestro

gens cause an accumulation of cells in GÃ¬phase. Estrogenreverses this block with a synchronous cohort of cells progressing through the cell cycle. These data have important implicationsfor the design of rational clinical trials of combined chemoendo-

crine therapy.

INTRODUCTION

Antiestrogens are widely used for palliation of women withhormone-responsive human breast cancer. More recently, an-

tiestrogen treatment has been combined with cytotoxic chemotherapy in both patients with metastatic disease and thoseundergoing adjuvant therapy following mastectomy with thehope of achieving additive antitumor activity. Although prelimi-

1This work was supported by NIH Grant CA 30251.2To whom requests for reprints should be addressed.

Received July 22,1983; accepted January 3,1984.

nary results suggest that certain subgroups of patients mightbenefit from the combined approach (12,27), other studies reportno significant response or survival improvement (4-7), or even a

deleterious effect of combining the antiestrogen tamoxifen withcytotoxic agents (12). The lack of the expected additive effectssuggests that tamoxifen may have an antagonistic interactionwith the cytotoxic agents or with the host (5, 20).

The mechanisms of antiestrogen action are not well defined.Antiestrogens can bind to and translocate cytoplasmic estrogenreceptor to the nucleus, where presumably they inhibit estrogen-mediated events leading to tumor growth (13). Several estrogen-

sensitive processes are inhibited by exposure of breast cancercells to antiestrogens (10, 14-16), but the net effect of these

biochemical events on cell proliferation and specifically on cellcycle kinetics is not clear.

Recent reports suggest, however, that antiestrogens haveprofound effects on breast cancer cell cycle kinetics that couldpartially explain the possible antagonism observed in the clinicaltrials of chemoendocrine therapy. Incubation of human breastcancer cells with concentrations of tamoxifen achieved in vivoresults in accumulation of cells in early d phase (21, 25, 26),where they may be less susceptible to the lethal effects of cycle-

active cytotoxic drugs. We now extend these initial observationsand show that (a) other antiestrogens, in addition to tamoxifen,block cell cycle progression in d ; (b) the cell cycle block can bereversed by incubation with estrogen, resulting in a synchronizedcohort of cells progressing through S phase; and (c) the cellcycle effects are not observed in estrogen receptor-negative

cells, suggesting that the effects are mediated via the estrogenreceptor.

MATERIALS AND METHODS

Breast Cancer Cells and Culture Techniques. The human andmammary origins of the MCF-7, ZR75-1, and MDA-231 breast cancercell lines have been described previously in detail (18). The MCF-7 andZR75-1 cells contain estrogen receptor, whereas the MDA-231 cells do

not. The cell culture techniques used for the maintenance of these cellshave been reported previously (23).

Hormones. Tamoxifen citrate was generously provided by StuartPharmaceuticals, and nafoxidine, by The Upjohn Co. Trioxifene (LY133314) was the generous gift of Lilly Research Laboratories. 17/3-

Estradiol was purchased from Calbiochem (San Diego, CA).Cell Kinetic Studies. Breast cancer cells were plated into 100-mm

culture dishes (Corning Glass Works, Coming, NY) in complete culturemedium (Richter's Improved Eagle's Minimal Essential Medium-ZO;

Grand Island Biological Co., Grand Island, NY) supplemented with insulin(1.0 nw) and 5% bovine serum (MCF-7 and ZR75-1 cells) or 10% fetalbovine serum (MDA-231). After the cells had firmly attached to the dish

(24 hr), complete medium was replaced with identical medium supplemented only with 5% bovine serum stripped with dextran-coated char

coal to reduce the endogenous estrogen concentration. After an additional 24 hr, hormones or ethanol (vehicle control, final concentration =

APRIL 1984 1433

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C. K. Osborne et al.

0.1%) was added directly to the culture dishes.The TLI3 was performed by a modification of the method of Livingston

ef al. (19). Cells were incubated for a 30 min pulse with tritiated thymidine(47 Ci/mmol; Amersham) at a final concentration of 0.3 /iCi/ml, washed3 times, suspended in PBS with 0.02% EDTA, and centrifuged (600 rpmfor 2 min). Cells were resuspended in PBS (2.5 x 105 cells/ml), and 0.2

ml of the suspension was cytocentrifuged for 5 min at 1000 rpm. Afterdrying, slides were fixed, developed, and stained as described previously(19). A total of 500 cells was counted for each point. The TLI (labeled/total cells) was calculated by counting the fraction of intact cells containing 8 or more grains over the nucleus.

Cell cycle distributions were obtained by computer analysis of DMAhistograms obtained by flow cytometry of mithramycin-stained cells (1,

9, 21). Cell monolayers were suspended in 0.02% EDTA in PBS andincubated for 10 min, and then a single-cell suspension was prepared bypassing the cells through 22-, 25-, and 27-gauge needles. Cells were

pelleted, resuspended in PBS, and fixed in 70% ethanol (final concentration) while vortexing. The fixed cells were pelleted, washed once in PBS,and stained overnight in the dark at 4Â°with mithramycin as described

previously (1, 21). Prior to flow cytometry, the cell suspension was againpassed through a 27-gauge needle and through a 60-/im nylon mesh

screen to remove clumps. Doublets constituted less than 3% of the totalcell suspension as determined by light microscopy. Histograms wereobtained by analyzing 1 x 104 cells on a Coulter Model TPS-1 cell sorter

equipped with a 2-watt argon ion laser tuned to 457 nw. The coefficient

of variation of the d peak varied from 4 to 8% using this method.

RESULTS

Cell Cycle Effects of Antiestrogens. Initially, the TLI wasused to estimate the fraction of cells actively synthesizing DMAafter incubation with tamoxifen. After 96 hr with 1 //M tamoxifen,a concentration readily achieved in patients administered standard doses (11), the TLI was reduced in MCF-7 cells growing inserum-free medium or in medium supplemented with 5% stripped

serum (Table 1). Although the effects of tamoxifen were slightlymore pronounced under serum-free conditions, all subsequent

experiments were conducted with cells maintained in mediumcontaining stripped serum, conditions in which near-optimal cell

proliferation is maintained.It has been reported previously that the reduction in the S-

phase fraction of MCF-7 cells with tamoxifen is accompanied by

an increase in the GÃ¬fraction (21, 25, 26). Flow cytometry ofcells stained with the DNA fluorochrome mithramycin revealedthat this antiestrogen effect is dose dependent (Chart 1, A andB). Concentrations of tamoxifen between 0.01 and 1.0 MM resulted in a stepwise reduction in the fraction of cells in S andG2+M phases and a concomitant accumulation of cells in GÃ¬.Fewer than 10% of cells were in S phase, whereas greater than90% were in GÃ¬after exposure to 1.0 /tw tamoxifen.

This antiestrogenic effect was also time dependent and wasobserved with other antiestrogens as well as tamoxifen (Chart2). After a 96-hr exposure of MCF-7 cells to tamoxifen, nafoxi-

dine, or trioxifene, more than 90% of cells were in GÃ¬phasecompared to 65% in control dishes. There was a concomitantreciprocal reduction in the fraction of cells in the S and G2+Mphases observed with the antiestrogens.

Effect of Estrogen. An important question is whether or notthe cells accumulating in G, phase after antiestrogen exposureare destined to die. Previous reports have demonstrated that

Table1Effect of tamoxifen on the TLI of MCF-7 cells

MCF-7 cells were cultured in serum-free medium or medium containing 5%dextran-coated charcoal-stripped bovine serum for 96 hr in the absence or presenceof tamoxifen (1 JIM). The TLI was determined as an estimate of the fraction of S-phase cells.

TU at 96 hrCondition

Control, stripped serumControl, serum freeTamoxifen, stripped serumTamoxifen, serum free

251753

3The abbreviations used are: TLI, tritiated thymidine labeling index; PBS,Dulbecco's phosphate-buffered saline without calcium or magnesium.

cell proliferation can be restored by the addition of 17/3-estradiolto tamoxifen-treated cultures (8, 15, 16). We observed a dose-dependent ability of 17/3-estradiol to recruit tamoxifen-treatedMCF-7 cells into S phase (Chart 3). After 72 hr in tamoxifen, the

TLI (S fraction) was less than 10%. At that time, medium waschanged to fresh medium (with 5% stripped bovine serum)supplemented with increasing concentrations of estradici. Asimple change to tamoxifen-free medium resulted in a significant

increase in the TLI in 24 hr. This stimulation of cells into S phasewas probably related to replenishment of essential nutrients orother growth factors, rather than removal of tamoxifen, since achange to fresh medium containing tamoxifen resulted in a similarpattern (see below). With the addition of estradici to the tamox-ifen-inhibited cells, a further increase in the fraction of S-phase

cells at 24 hr was evident. Even 0.1 HM estradici caused amodest increase in the TLI above that observed with a changein medium alone. Maximal effects were observed with 10 nwestradici; 65% of cells were in S phase.

To study the effects of estrogen replenishment in more detail,we studied the cell cycle phase distributions of tamoxifen-inhib-

ited cells supplemented with estradici and analyzed by flowcytometry (Charts 4 and 5). DNA histograms (Chart 4) demonstrate that a synchronous wave of cells entered and passedthrough the S phase between 12 and 24 hr after adding estradici.Chart 5 displays in more detail the time course of the effects ofestrogen on control and tamoxifen-inhibited cells analyzed by

flow cytometry. A sharp drop in the S fraction with a concomitantincrease in the GÃ¬fraction was observed with a 96-hr exposure

to tamoxifen. The S fraction also declined somewhat in controlcultures, probably secondary to nutrient depletion. At 96 hr,medium was changed to fresh medium alone (control) or mediumcontaining estradici or tamoxifen. In both control and tamoxifen-

inhibited cultures, a simple change in medium, even mediumcontaining tamoxifen, resulted in a significant fraction of cellsleaving d phase and passing through the S compartment. Theaddition of estradici to tamoxifen-treated or control cells caused

a greater increase in the S fraction which peaked at about 70%.The S fraction in control cells treated with a medium changealone or with estradiol had already reached the maximal increaseby the first time point examined (12 hr). The lag time was longerin tamoxifen-inhibited cells rescued by estradiol; the maximal

increase in S fraction in these cells required 18 hr. This differencein the timing of the estrogen "rescue" effect on control or

tamoxifen-pretreated cells was consistent in several experi

ments.Control or tamoxifen-treated MCF-7 cells can be stimulated to

enter S phase by estrogen without a change to fresh culturemedium (Chart 6). In this experiment, cells were incubated in theabsence of hormone (controls) or with tamoxifen for 72 hr.

1434 CANCER RESEARCH VOL. 44



Synchronization of Human Breast Cancer

Control

C, * 7l 7%

S ' 19 I %

TAM lOnMG,' 781%

~Ã•28

0)JD

S >Â«3%

G]M < 12 5%

TAMlOnMG,'787%S -183%

G,MÂ«30%

TAM IMM

G, â€¢919%

S >BI%

GtM â€¢00%

Relative Fluorescence Intensity(Channel Number)

50

IO'7 IO"

Tamoxifen (M)

Chart 1. Effect of tamoxifen (TAM) concentration on DMA histograms obtained by flow cytometry of mithramycin-stained MCF-7 cells. Cells were grown in 5%charcoal-stripped bovine serum with increasing concentration of tamoxifen for 96 hr. A single-cell suspension was prepared, fixed, stained, and analyzed as described in"Materials and Methods." A. histograms obtained from analysis of 1 x 10* cells. The coefficient of variation of the G, peaks varied from 4 to 8%. B. G, and S fractions

computed from the histograms and plotted in chart form.

IOO-

90-

80-

70

6050-

L

O 40-|

03Ã›. 30-

20

lOi

20-1

48

Time (hours)Chart 2. Time course of the effect of antiestrogens on MCF-7 celt cycle kinetics.

MCF-7 cells were cultured in control medium (â€¢)or medium containing 1 Â¡Mtamoxifen (O), nafoxidine (D). or tnoxifene (A). Cell cycle phase distributions weredetermined as described in "Materials and Methods."

70

60

BOS'Â»0-

3 30-

20-

10-

0-

TAM

t

24 48 72 96Time (hours)

Chart 3. Effect of estrogen (Â£2)on the TLI of tamoxifen-treated cells. MCF-7cells were incubated with tamoxifen (1 UM. â€¢)for 72 hr. Spent medium wasexchanged for fresh medium supplemented with 5% charcoal-stripped serum andincreasing concentrations of I7.j-estradio! (O). After 24 hr, the TLI (S phase) wasdetermined.

Estradici was then added directly to the cultures. Cells left G,phase and traversed S phase in a sequence similar to that notedabove, with tamoxifen-pretreated cells demonstrating a longer

lag time. Since cells could be stimulated by estradiol to enter Sphase after growing for several days in medium with charcoal-

stripped bovine serum, we also examined the effect of adding17|8-estradiol to cells at the beginning of the experiment (time =0 hr). Interestingly, initial exposure of MCF-7 cells to 10 nw

estradiol resulted in a modest decline in the S fraction and an

APRIL 1984 1435



C. K.Osborne et al.

Ohr

O).o

O)O

G, =88%

S '7%

GjM'5%

12hr

I2fl 128

18 hr 24 hr

128 128

Relative Fluorescence Intensity (Channel Number)Chart 4. Effect of tamoxifen followed by estrogen on MCF-7 cell cycle kinetics. Cells were incubated with tamoxifen (1 Â¿IM)for 96 hr. The medium was then changed

to fresh medium containing estradiol (10 PM). DNA histograms were plotted at 0,12, 18, and 24 hr after estrogen addition.

increase in the GÃ¬fraction by 72 hr. Additional estrogen at thatpoint had no effect on the cell cycle distribution, as the majorityof cells remained in GÃ¬phase.

Effect of Estrogen and Antiestrogen on Other Breast Cancer Cell Lines. We next examined the effects of tamoxifen and17/8-estradiol on the cell cycle kinetics of 2 other human breastcancer cell lines, the ZR75-1 and the MDA-231. The ZR75-1cells contain estrogen and progesterone receptors; the MDA-

231 cells contain glucocorticoid receptors but lack receptors forestrogen and progesterone (18). The cell kinetic effects of estrogen on the ZR75-1 cells were similar to those described abovefor the MCF-7 cells (Chart 7). These cells proliferate more slowlythan do MCF-7 cells, and only about 10% of cells were in Sphase at time 0. A 96-hr exposure to tamoxifen did not signifi

cantly alter the cell cycle distribution of these cells. However, aswith the MCF-7 cells, estrogen added to controls or to tamoxifen-treated ZR75-1 cells resulted in an increase in the S compartment

and a decrease in the d compartment at 24 hr. A mediumchange alone had no effect on the cell cycle phase distribution.

No significant cell kinetic effects of either tamoxifen, estrogen,or a change to fresh medium were observed in the MDA-231cells (Chart 8). These cells proliferate more rapidly than do MCF-7 or ZR75-1 cells, and a relatively low proportion were in d at

the beginning of the experiment. The fraction in d graduallyincreased with time as the cells neared confluence.

DISCUSSION

Previous studies have shown that tamoxifen slows macro-molecular synthesis and proliferation of MCF-7 human breastcancer cells growing in medium with charcoal-stripped serum (8,

15, 16). Growth could be restored by the addition of 170-estradiol. The timing of this estrogen "rescue" effect was inter

preted to indicate that tamoxifen might arrest cells in a uniformposition within the cell cycle. The present data extend theprevious work reported by Sutherland and coworkers (25, 26)and by our own laboratory (21), demonstrating that tamoxifen-treated MCF-7 cells accumulate in the d phase of the cell cycle.

This effect is not restricted to tamoxifen but is also observedwith other antiestrogens, including nafoxidine and trioxifene.Furthermore, concentrations of tamoxifen observed in breastcancer patients (11) are effective, supporting the notion that thismechanism of inhibition of cell proliferation may be operative invivo.

Our data demonstrate that tamoxifen inhibition can be reversed by the addition of 17/3-estradiol at a 100-fold-lower concentration. Estrogen-treated cells progress through d and into

S phase. Furthermore, transit through the cell cycle can berestored by estrogen even when the antiestrogen remains in theculture medium. This is not surprising, since 17/3-estradiol hasmore than 100-fold-greater affinity for the estrogen receptor than

does tamoxifen (8, 16) and since estradiol can compete withtamoxifen and nafoxidine for the nuclear estrogen receptor withrestoration of receptor processing and other estrogen events(14). Effects of estrogen are also observed in the estrogenreceptor-positive ZR75-1 cells but not in the receptor-negativeMDA-231 cells, further suggesting that these cell cycle effects

are mediated through the estrogen receptor.We consistently observed that the lag time between estrogen

addition and the maximal accumulation of cells in S phase islonger for tamoxifen-treated cells than for controls. It is doubtful

that this increased lag time is due to the time required for





90-

96 6 12 18 24 30 36

Ti me (hours)Chart 5. Time course of the effect of tamoxlfen and estrogen on MCF-7 cell

cyde kinetics. Cells were incubated in control medium (â€¢)or with tamoxifen (1 tint,O) for 96 hr. Medium was then changed to fresh control medium (â€¢,O), mediumcontaining 1 UMtamoxifen (â€¢.O), or medium containing 10 nw estradici (A, A). Cellcycle phase analysis was performed at the times shown.

estradici to enter cells, replace tamoxifen on the nuclear estrogenreceptor, and initiate transcription. These events require only afew min (14). However, this observation is consistent with ourprevious report demonstrating that MCF-7 cells treated withtamoxifen accumulate in early to mid-d phase compared to lateGÃ¬for control cells (21). Tamoxifen-inhibited cells released by

the addition of estrogen would then require more time to traverseGT phase before entering the S compartment.

We also observed that the antiestrogen block could be partiallyovercome by changing to fresh medium without or even withtamoxifen. This indicates that other factors present in mediumand/or serum (nutrients, growth factors, etc.) are potent growthregulators that can modulate the inhibitory properties of anties-

trogens. It is also possible that conjugated estrogens present in"stripped" serum account for the observed effect. This seems

unlikely, however, since the effect of fresh serum is observedeven when tamoxifen is added at concentrations that shouldblock the effects of low concentrations of estrogen. A morepronounced rescue effect, however, is noted when estradiol ispresent in the medium. Clearly, the effect of estrogen does notrequire fresh medium or serum factors, since the addition ofestrogen stimulated progression of tamoxifen-blocked cells into

90-,

80-

70

60-

50-

4030-

O 60-03O- 50-

40-

30-

20-

10-

T ime (hours)Chart 6. Effect of tamoxifen and estrogen without a change in culture medium

on MCF-7 cell cycle kinetics. Cells were grown for 72 hr in control medium (â€¢),

medium with 10 nM estradiol (A), or medium with 1 /JMtamoxifen (â€¢)Then, 10 nmestradiol (O, A, G) was added directly to all wells without a change to fresh medium.Cell cycle phase analysis was performed at the times shown.

90-1

80 -

70-

60-

50-

co>o1_CD

CL

30-,

Time (hours)Chart 7. Effect of tamoxifen and estrogen on the cell cycle kinetics of ZR75-1

breast cancer cells. Cells were treated as described in Chart 6 in control (â€¢)or(amoxifen-supplemented medium (O). After 96 hr, medium was changed to freshcontrol medium (â€¢,O), medium with estradiol (A, A), or tamoxifen {â€¢.O).

S phase even in the absence of a medium change.It is interesting that no stimulation of cells to enter S phase is

observed when estradiol is added to cells shortly after plating.In fact, there is a slight reduction in the S fraction after several

APRIL 1984 1437



O.K. Osbomeetal.

80-,

96 12 24

Time (hours)36

Chart 8. Effect of tamoxifen and estrogen on the cell cycle kinetics of MDA-231cells. Cells were processed as described in Chart 7. Symbols are identical.

days. Estrogen stimulation is only observed in cells maintainedfor several days in charcoal-stripped serum or in cells blockedby antiestrogen. Conflicting reports on the ability of estrogen tostimulate proliferation of MCF-7 cells have been published previously (10, 16, 24). One might postulate that cells growing inmedium supplemented with bovine or fetal calf serum are alreadyproliferating at a maximal rate due to the estrogens (or perhapsother growth factors) present in serum. Additional estrogenwould then have little, if any, effect. A recent report, suggestingthat a serum "factor" may actually be required for the growth

response of MCF-7 cells to estradici, clouds this hypothesis (24).The disparate results might also be explained by genetic differences among the MCF-7 cell lines used in various laboratories.In any event, it is clear that estradici has potent growth-promoting activity in antiestrogen-treated cells or in cells growing inestrogen-depleted medium.

Whether antiestrogens in pharmacological concentrations arecytocidal for breast cancer cells is not clear. Certainly, lethaleffects would not be required for tumor regression to occur invivo. Ongoing cell shedding and/or death mediated by hostdefense mechanismswould result in slow tumor regressionwhencell proliferation has been inhibited by antiestrogen therapy.Although cell death might result from prolonged GÃ¬delay, wepostulate that inhibition of cell proliferation by antiestrogens isthe major mechanism involved in tumor regression for severalreasons, (a) In short-term (1 to 2 weeks) in vitro growth experiments, antiestrogens do not cause a reduction in cell numberbut rather a gradual slowing in the rate of proliferation (8,15,16,21, 26). Very high tamoxifen concentrations (>5.0 UM)or greatlyreduced serum concentrations are required for lethal effects (16,26). (o) Cultured cells exposed to antiestrogens remain viableand can be recruited back into the proliferative pool by the

addition of estrogen, (c) Our preliminary studies of MCF-7 cellsgrowing as s.c. tumors in immune-deficient athymic nude miceshow that prolonged treatment with estrogen depletion and/orantiestrogen results in a block of tumor growth but not in tumorregression (22). Furthermore, tumor growth resumes when estrogen is replenished, even after several weeks of estrogendeprivation or tamoxifen treatment. Tumor regression in patientstreated with tamoxifen may result from a block of cell proliferationcombined with a functioning host defense system.

Our results have potentially important implications for thetreatment of patients with breast cancer, especially concerningthe use of antiestrogen therapy combined with cytotoxic agents.The ability to synchronize the endocrine-sensitive cells in G,phase with tamoxifen or in S phase with short-term estrogentherapy could be used to design more rational treatment strategies. Cytotoxicity of drugs effective in G! phase might be enhanced by combined therapy with tamoxifen. Alternatively, theactivity of S-phase-specific drugs might be synergistically enhanced by synchronization of tumor cells into S phase withestrogen. Preliminaryreports using laboratory models and clinicaltrials of patients with breast cancer support these hypotheses(2,3,17,28).

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APRIL 1984 1439



1984;44:1433-1439. Cancer Res C. Kent Osborne, David H. Boldt and Phillip Estrada and Antiestrogens in CultureHuman Breast Cancer Cell Cycle Synchronization by Estrogens

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