Download Effects of Tamoxifen on Human Breast Cancer

[CANCER RESEARCH 43. 3583-3585,
August 1983]
Effects of Tamoxifen on Human Breast Cancer Cell Cycle Kinetics:
Accumulation of Cells in Early GìPhase1
C. Kent Osborne,2 David H. Boldt, Gary M. Clark, and Jeffrey M. Trent3
Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78284 [C. K. O., D. H. B., G. M. C.], and the University of Arizona Health Science
Center, Tucson, Arizona 85724 [J. M. T.]
ABSTRACT
We have studied the effects of tamoxifen on the cell cycle
kinetics of the endocrine-responsive MCF-7 human breast cancer
cells. Tamoxifen inhibits proliferation of MCF-7 cells. The tritiated
thymidine labeling index is markedly reduced by tamoxifen, in
dicating a reduction in the fraction of cells in S phase. Flow
cytometry of mithramycin-stained cells reveals that cells accu
mulate in Gìphase, with a concomitant depletion of S- and G2M-phase cells with tamoxifen. Mapping of G1-phase cells by
morphology of prematurely condensed chromosomes demon
strated that tamoxifen-treated
cells accumulate in early G,.
These studies indicate that tamoxifen inhibits proliferation of
MCF-7 human breast cancer cells by invoking a transition delay
early in the d phase of the cell cycle.
INTRODUCTION
The antiestrogen tamoxifen is now widely used for the treat
ment of patients with breast cancer. However, its mechanism of
action is not totally defined. Although specific binding sites for
tamoxifen have been identified recently in certain tissues (15),
tamoxifen, like other antiestrogens, is thought to exert its effects
by binding to estrogen receptor and then translocating it to the
nucleus, where steps leading to estrogen-regulated events such
as tumor growth are inhibited (6). Tamoxifen has been shown to
inhibit breast tumor growth in experimental animals (8) and to
inhibit a variety of biochemical events in cultured human breast
cancer cells including nuclear processing of estrogen receptor
(7), the activity of key enzymes such as DNA polymerase (3),
and cell proliferation (10). Little is known about the effects of
tamoxifen on cell cycle kinetics. Previous studies using [3H]thymidine incorporation techniques suggest that tamoxifen may
block cells in a uniform phase of the cell cycle (9), possibly in G,
(12, 16, 17). Here, we demonstrate that tamoxifen causes a
transition delay early in the d phase of the cell cycle.
MATERIALS AND METHODS
Cell Proliferation. The tissue culture methodology used for propaga
tion of the MCF-7 human breast cancer cells has been described previ
ously (14). For determination of the effects of tamoxifen on cell prolifer
ation, MCF-7 cells were replicately plated into multimeli culture dishes in
tissue culture medium (Richter's Improved Eagle's Minimal Essential
Medium ZO; Grand Island Biological Co., Grand Island, N. Y.) supple
mented with insulin (1.0 HM) and 5% bovine serum. After 24 hr, plating
medium was replaced with medium supplemented with 5% dextrancoated charcoal-stripped bovine serum to reduce endogenous estro' This work was supported by NIH Grant CA 30251 and by an institutional grant
from the American Cancer Society (IN-116C).
1 To whom requests for reprints should be addressed.
3 Recipient of National Cancer Institute Grants CA 29476 and CA 23074.
Received December 9,1982; accepted May 4,1983.
AUGUST
gens. After an additional 24 hr, tamoxifen (1 »M)or ethanol (final concen
tration, 0.1%) was added to the dishes. At the indicated times, cells were
suspended and counted in a hemocytometer.
Cell Kinetic Studies. Cells were plated and cultured as described
above. At the indicated times, cells were pulsed with tritiated thymidine
(0.3 fiCi/ml) and washed twice with PBS,4 and a cell suspension was
prepared. Uhe TLI was determined autoradiographically as described
previously (11, 14). The cell cycle distribution of MCF-7 cells was
determined by flow cytometry of mithramycin-stained cells (1). Cells were
washed once and, then, suspended in 0.02% EDTA in PBS. After a 5to 10-min incubation, a single-cell suspension was prepared mechanically
by passing the cells through progressively smaller gauge needles (22,
25, and 27 gauge). The cells were pelleted by centrifugaron (800 rpm
for 2 min) and resuspended in 0.4 ml of PBS. The cells were then fixed
by adding ethanol (final concentration, 70%) while vortexing and stored
at 4°.Fixed cells were pelleted by centrifugation, and the ethanol was
discarded. Cells were washed once with PBS and, then, resuspended in
staining solution (1.0 mg mithramycin:0.87 g NaCI:0.15 ml 1 M MgCb:9.85
ml distilled H2O) to a concentration of 5 x 105 cells/ml. The cells were
protected from light at 4°overnight prior to flow cytometry. DNA histo
grams were obtained by analyzing a total of 1 x 104 cells on a Coulter
Model TPS-1 cell sorter equipped with a 2-watt argon-ion laser which
was tuned to 457.9 nM. Histograms were analyzed by the computerassisted method of Dean and Jett (2). The coefficient of variation of the
d peak varied from 4 to 8% using this method.
Distribution of GìCells. MCF-7 cells were cultured as described
above and then incubated with either ethanol (controls) or tamoxifen (1
¿¿M)
for 72 hr. G, cells were mapped by the method of Hittelman and
coworkers (4, 5, 18), which analyzes the morphology of PCC. Briefly,
mitotic HeLa cells were obtained following a 24-hr exposure to thymidine
(2.5 rtiM), followed by a 15-hr exposure to colchicine (0.1 MM). This
procedure routinely provides a 97% pure population of mitotic cells (as
distinguished by light microscopy following acetoorcein staining). Equal
numbers of mitotic HeLa cells and MCF-7 cells were mixed in serumfree medium and pelleted (150 x g for 2 min). Cells were resuspended
in 0.5 ml of PBS containing 2000 hemagglutinating units of UV-inactivated
Sendai virus (a generous gift of Dr. W. Hittelman, Houston, Texas). Cells
were then incubated at 4°for 15 min after the addition of 0.05 ml of 20
rnw MgCl2 and Colcemid (5 ng/mi). Samples were warmed to 37°and
incubated for an additional 45 min. Cell pellets were obtained by centrif
ugation (150 x g, 5 min), and 7 ml of 0.075 M KCI (prewarmed to 37°)
were added for 10 min. Cells were then recentrifuged, the supernatant
was removed, and 7 ml of fresh cold fixative (3:1 methanol:glacial acetic
acid) were added. Air-dried slides were then prepared and stained with
4% Giemsa (Gurr's R-66). G, PCC were graded on a scale of 1 through
6, depending on the degree of condensation. PCC given values of 1 to
3 (highly condensed) were considered to be in early G,, whereas those
given values of 4 to 6 (less condensed) were judged to be in late Gì(4).
RESULTS
When cells from the well-characterized MCF-7 estrogen recep4The abbreviations
used are: PBS, phosphate-buffered
saline [NaCI (8 g/
Bter):KCI (0.2 g/liter):Na2HPO4 (1.15 g/liter):KH2PO, (0.2 g/liter); TLI, tritiated thy
midine labeling index; PCC, prematurely condensed chromosomes.
1983
Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1983 American Association for Cancer Research.
3583
C. K. Osborne et al.
tor-positive human breast cancer cell line were incubated with
tamoxifen (Chart 1), inhibition of cell proliferation similar to that
observed in previous reports (9, 17) was observed. In 5% char
coal-stripped serum, control cultures proliferated rapidly and had
reached confluence by Day 6. Tamoxifen (1 fiM) slowed the rate
of proliferation slightly during the first 48 hr. Thereafter, prolifer
ation was markedly reduced, cell number nearly plateaued, and
the monolayers remained at a subconfluent density. These data
suggest that more than one passage through the cell cycle is
required for expression of maximal effects of tamoxifen at these
concentrations. When MCF-7 cells were incubated with tamoxi
fen for longer durations or were incubated with tamoxifen in
serum-free medium, cells would begin to detach from the monolayer and appear as floaters. It is important to emphasize,
however, that, under the experimental conditions described in
Chart 1, cell number did not decline with tamoxifen. These data
suggest that tamoxifen slowed proliferation but did not have an
immediate cytocidal effect on the MCF-7 cells.
We next examined the effect of tamoxifen on the distribution
of MCF-7 cells in the cell cycle. The TLI was used to estimate
the fraction of cells in the S phase of the cell cycle (Chart 2). The
TLI of control cells growing in medium supplemented with dextran-coated charcoal-stripped calf serum was 35% at the begin
ning of the experiment. The TLI decreased slightly over 96 hr in
control cultures, probably reflecting depletion of nutrients from
the medium and/or density-dependent growth inhibition as the
monolayer neared confluence. Tamoxifen resulted in a marked
reduction in the TLI, and by 72 to 96 hr, less than 10% of the
cell population was in S phase.
To determine the effects of tamoxifen on the distribution of
MCF-7 cells in the other cell cycle phases, DMA histograms were
obtained by flow cytometry of mithramycin-stained cells (Chart
3). After 72 hr in control medium, 72% of cells were in d as
revealed by computer analysis of DNA histograms, 19% were in
S phase (similar to the TLI result), and 9% were in G2-M phases.
Tamoxifen treatment resulted in a marked accumulation of cells
in Gì
(92%), concomitant with a depletion of cells from the S and
Û2-M compartments. When experiments were carried out for
longer time periods (6 days), cells exposed to tamoxifen re
mained in Gì,indicating that the block in G, phase was not
temporary.
It has been reported that serum-deprived or plateau-phase
40 n
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24
72
48
96
Time (hours)
Chart 2. Effect of tamoxifen on the TLI of MCF-7 cells.
Control
G, = 7I.7%
S =19.1 %
G2M = 9.2%
0)
-Q
128
TAM \fj.M
G, = 91.9%
S =81%
G2M = 0.0%
128
Relative Fluorescence Intensity
(Channel Number)
Chart 3. Effect of tamoxifen (TAM) on the cell cycle distribution of MCF-7 cells.
A, control cells; B, tamoxifen for 72 hr. Hatched area, computer estimate of the Sphase fraction of cells.
normal (nontransformed) cells accumulate in early Gì,whereas
malignant or transformed cell populations accumulate in late Gì
(4). Furthermore, certain metabolic inhibitors, such as actinomycin D or cycloheximide, block cells in early Gìin contrast to
inhibitors of DNA synthesis, such as thymidine or hydroxyurea,
which block cell cycle progression at the d-S boundary (4).
Chart 1. Effect of tamoxifen on proliferation of MCF-7 cells. Cells growing in
medium supplemented with 5% dextran-coated charcoal-stripped bovine serum
were incubated in the absence (•)or presence (O) of tamoxifen (1 ,,M). Fresh
medium and tamoxifen were exchanged for spent medium on Day 4. Points, mean
of triplicate determinations; bars, S.D.
3584
Thus, we were interested in determining where in the d phase
the tamoxifen block occurred. Utilizing the morphology of PCC,
cells can be classified as occurring early or late in the d phase
based upon the structural criteria of Hittleman and Rao (4). This
is possible because the chromosomes from early d PCC are
highly condensed, whereas those from late G, PCC are long and
extended. Morphological assessment of PCC (Table 1) revealed
that 62% of GìPCC in control cultures were in late Gì.In
contrast, only 35% of tamoxifen-treated d PCC were present
in late G, (p = 0.001). The majority (65%) of tamoxifen-treated
cells had accumulated early in d. Furthermore, exact chromo
some counts of d PCC revealed that both control and tamoxi
fen-treated d cells had similar chromosome numbers (70), indiCANCER
RESEARCH
VOL. 43
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Tamoxifen in Human Breast Cancer
Table 1
Gìdistribution of control or tamoxifen-treated
eating that accumulation of d cells with tamoxifen (as deter
mined by analysis of DNA histograms) was not an artifact due
to chromosome loss.
for primary breast cancer with adjuvant antiestrogens may have
to be continued indefinitely or at least long enough for host
defense or normal cell attrition to eradicate residual tumor cells.
Premature discontinuation of therapy would lead to tumor regrowth, (o) The combined use of antiestrogens with cytotoxic
chemotherapy in an attempt to obtain an additive or synergistic
effect will require careful evaluation. Tamoxifen might enhance
the killing effect of drugs most active in Gìcells, whereas the
cytotoxic effect of drugs acting specifically during DNA synthesis
(S phase) might be diminished. This hypothesis is currently being
investigated.
DISCUSSION
ACKNOWLEDGMENTS
We have demonstrated that the antiestrogen, tamoxifen, inhib
its proliferation of estrogen receptor-positive human breast can
We would like to acknowledge the technical assistance of Philip Estrada, Sharon
Olson, and Kathy Mosly.
Earty G, (Stages 1-3)
Control
Tamoxifen
31 (38)"
49 (65)
MCF-7 cells
Late G, (Stages (4-6)
51 (62)
26 (35)
p = 0.001
Numbers in parentheses, percentage of cells.
cer cells. These results are similar to those reported by Lippman
ef al. (9) and Sutherland ef al. (17) who found that, at concentra
tions of tamoxifen observed in women treated for breast cancer
(<1.0 UM), the antiestrogen slowed proliferation but was not
lethal to MCF-7 cells in short-term culture. At suprapharmacological concentrations (10 MM), a cytocidal effect was observed
(17), although the relevance of this effect to in vivo drug mech
anism of action is speculative. The slight differences observed in
the effects of tamoxifen in these studies may be related to
differences in the type (charcoal stripped or not) and concentra
tion of serum used.
Our data suggest that tamoxifen inhibits cell proliferation by
invoking a transition delay or block in early to mid-d phase of
the cell cycle. This delay may require more than one complete
passage of some cells through the cell cycle, since cell number
more than doubled before plateau growth was observed, and
since 72 to 96 hr (2 to 3 generation times) were required for
maximal accumulation of cells in Gì.A small percentage of cells
was refractory to tamoxifen and remained in the proliferative
pool (5 to 10% in S phase). The mechanism of resistance of
these cells to tamoxifen will require further study. We have
shown recently that the tamoxifen effect can be reversed by the
addition of 170-estradiol and that tamoxifen has no effect on the
cell cycle kinetics of the receptor-negative MDA-MB-231 cells,
suggesting that the antiestrogen effect is mediated through the
estrogen receptor (13). Furthermore, these inhibitory effects are
not restricted to tamoxifen. In our preliminary studies, the anties
trogens nafoxidine and trioxifene have identical effects on breast
cancer cell kinetics.
These data are consistent with the hypothesis that antiestro
gens, and perhaps other forms of endocrine therapy, induce
regression of breast cancer in vivo by simply blocking progres
sion of estrogen-dependent cells through the cell cycle rather
than by a direct cytotoxic effect. With cell replication inhibited,
tumors might then regress because of ongoing cell shedding or
death or because of interaction with normal host defenses. This
hypothesis would account for the very slow rate of tumor regres
sion observed in many patients undergoing endocrine therapy.
These results may have important clinical implications for the
treatment of breast cancer, (a) If antiestrogens are simply putting
endocrine-responsive breast cancer cells into a "resting" phase
of the cell cycle (G0-Gi), then treatment of patients after surgery
AUGUST
1983
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3585
Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1983 American Association for Cancer Research.
Effects of Tamoxifen on Human Breast Cancer Cell Cycle
Kinetics: Accumulation of Cells in Early G 1 Phase
C. Kent Osborne, David H. Boldt, Gary M. Clark, et al.
Cancer Res 1983;43:3583-3585.
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