[CANCER RESEARCH 46, 2526-2531. May 1986]
Estrogen- and Tamoxifen-induced Rearrangement of Cytoskeletal and Adhesion
Structures in Breast Cancer MCF-7 Cells1
Anna Sapino, Francesca Pietribiasi, Gianni Bussolati,2 and Pier Carlo Marchisio
Section of Pathological Anatomy and Histology; Department of BiomédicalSciences and Oncology, University of Turin, School of Medicine, Via Santena 7,
10126 Torino, Italy (A. S., F. P., G. B.J and Section of Histology and Embryology, Department of BiomédicalSciences and Oncology, University of Turin, School of
Medicine, Corso M. D'Azeglio 52, 10126 Torino, Italy [P. C. M.J
ABSTRACT
The cytoskeleton, the shape, and the adhesion complexes of MCF-7
breast carcinoma cells have been studied by fluorescence, phase contrast,
and interference reflection microscopy. Cells have been grown in media
containing different concentrations of estrogen and with or without the
addition of the antiestrogen tamoxifen. The pattern of actin microfilaments and keratin intermediate filaments (tonofilaments) and the distri
bution of adhesion areas change as a function of the estrogen concentra
tion.
When cells are cultured in estrogen-deprived medium, they appear
roundish and flattened and adhere firm!) to the substratum, with multiple
vinculin-positive adhesion plaques at their ventral surface. Upon stimu
lation with estrogen, these cells display pseudopodial cytoplasmic protru
sions and ruffling membranes; in interference reflection microscopy the
adhesion areas are mostly localized in these projections. A rearrangement
of microfilamcnts and of tonofilaments in the cell projections and the
formation of a dense network of keratin Tibers takes place. Tamoxifen
affects cellular shape and Cytoskeletal arrangement in a way similar to
that induced by estrogen.
An effect of estrogen-receptor stimulation on the adhesion structures
and on the rearrangement of intermediate and actin filaments (and
accordingly of the shape and internal structure of breast cancer cells) can
be suggested. Such an effect might be direct or mediated through unknown
mechanisms; it seems, however, to be independent of the well known
estrogenic effect on cell proliferation.
INTRODUCTION
Estrogen stimulation specifically affects metabolism and
structure of breast cancer cells (1). This has been extensively
investigated by "in vitro" cultures; the estrogen receptor positive
MCF-7 human breast carcinoma cell line responds to estrogens
by an increase in mitotic index (2, 3), metabolic activity and
synthesis of progesterone receptors (4, 5), plasminogen activa
tor (6, 7), and other proteins the function and significance of
which have not yet been defined (8-10).
Scanning and transmission electron microscopy have been
used to show structural changes in the MCF-7 cell line; physi
ological concentrations of estradiol have been observed to in
duce a secretory phenotype and an increase in the number and
length of microvilli. A parallel change of the overall cell shape
occurs, since they become more globular and less tightly at
tached to culture dishes (1).
These findings suggest that estrogens might induce, either
directly or indirectly, a rearrangement of the cytoskeleton,
resulting in a modification of cell shape. Since each Cytoskeletal
component has a different significance and controls different
cellular functions, it is interesting to know whether Cytoskeletal
patterns are under hormonal control. In order to study this
possibility experimentally we have cultured MCF-7 cells in the
Received 12/3/84; revised 7/5/85, 1/3/86: accepted 1/29/86.
' Supported by grants from the M.P.I., Rome, the C.N.R. Progetto Finalizzato
"Oncologia" (grants n. 84.00478.44 and n. 84.00658.44), and the A.I.R.C., Milan,
Italy.
2 To whom requests for reprints should be addressed, at Department of
BiomédicalSciences and Oncology, Section of Pathological Anatomy and His
tology, University of Turin, Via Santena 7. 10126 Torino. Italy.
presence of different concentrations of 17/3-estradiol and ana
lyzed Cytoskeletal patterns by immunofluorescence microscopy
using specific antibodies to vinculin, keratin, vimentin, actin,
and tubulin.
MATERIALS AND METHODS
The MCF-7 cell line, originally obtained by a pleural effusion (11)
and serially cultured in Dr. Lippmann's laboratory (National Cancer
Institute, Bethesda, MD), was kindly supplied by Prof. I. Nenci (Insti
tute of Pathological Anatomy, University of Ferrara, Italy). The pres
ence of estrogen receptors in this same cell line was confirmed by
biochemical and cytochemical procedures (12, 13).
Cells were routinely cultured in RPMI (Grand Island Biological Co.,
Grand Island, NY) medium supplemented with glutamine (0.6 g/liter),
penicillin (200 units/ml), streptomycin (200 Mg/ml) (Eurobio), and 10%
PCS3 (Grand Island Biological Co., Grand Island, NY) at 37°Cin a
water-saturated atmosphere containing 5% CO2. The content in 170estradiol of the PCS was measured by radioimmunoassay and found to
be about 41 pg/ml. In order to remove the hormone, the PCS was
adsorbed with dextran coated charcoal suspension according to a pro
cedure outlined by Heyns et al. (14): the adsorbed serum was found to
have a reduced estrogen content of about 0.1 pg/ml. An alcoholic
solution of 17fi-estradiol (Merck, Darmstadt, Federal Republic of Ger
many) was used to supplement estrogen in the culture medium.
MCF-7 cells have been cultured for 3 and 6 days in the following
media:
(A) Maintenance medium composed of RPMI medium supplemented
with 10% PCS, having a final estrogen concentration of about
10-' M.
(B)
Estrogen-deprived medium, composed of RPMI medium with
10% of charcoal adsorbed PCS, with a final estrogen concentra
tion of approximately 10"12M.
(C)
Estrogen-supplemented medium composed as above (B) but sup
plemented with 170-estradiol in order to obtain a final concen
tration of 10"8M.
(D) Estrogen-deprived, tamoxifen-supplemented medium, composed
as medium B, but supplemented with tamoxifen (trans-P-dimethylaminoethoxyphenyl-l-l,2-diphenylbut-l-en,
ICI 46474)
dissolved in ethanol to reach a final concentration of lO^M.
In order to obtain a growth curve, cells were seeded at a density of
about 125,000 cells in 60-mm Petri dishes. Hemocytometer cell counts
from triplicate culture dishes were performed every 3 days. The medium
was changed every 2 days.
The cells, grown serially in flasks, were trypsinized and transferred
for the experiments onto glass coverslips in 60-mm Petri dishes (5 x
10s cells/dish).
Coverslip-attached cells were processed for fluorescence microscopy
essentially as described (15, 16). Briefly, coverslips were fixed for 10
min at room temperature in 3.7% formaldehyde (from paraformaldehyde) in PBS, pH 7.6, containing 2% sucrose. Permeabilization was
obtained either by methanol and acetone at -20°C (5 min and 5 s,
respectively) or by exposure to 0.1% Triton XI 00 for 2 to 5 min at
0°C.For staining microtubules, the methanol-acetone procedure was
used (15), while for staining keratin intermediate filaments formalde3The abbreviations used are: FCS. fetal calf serum; PBS, phosphate-buffered
saline (43.85 g NaCI, 1.10 g NaH2PO4 H2O. and 11.95 g Na2HPO, 12H3O
dissolved in 5 liters of water, pH 7.2; 1RM, interference reflection microscopy;
F-PHD, fluorescein-isothiocyanate phalloidin: R-PHD, rhodamine-isothiocyanate phalloidin.
2526
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CYTOSKELETON IN BREAST CANCER CELLS
•
17BE2 io"8 M
•
17Q-E2 10"12M
hyde fixation prior to methanol-acetone permeabilization was omitted
(17). After rinsing, coverslips were incubated sequentially in primary
and secondary antibodies for 30 min at 37°C,rinsed, and mounted in
Mowiol as described (15). In double-labeling experiments, cells were
incubated previously with vinculin monoclonal antibodies, followed by
rinsing and subsequent incubation in rhodamine isothiocyanate-labeled
secondary antibody and F-PHD. When cells were simultaneously ex
amined in phase contrast and in IRM, coverslips were mounted in 50%
glycerol in PBS (16).
In order to stain actin microfilaments, either immunofluorescence
using actin antibodies (see below) or direct staining using F-PHD or
R-PHD (kindly supplied by Prof. T. Wieland, Max Planck Institute for
Experimental Medicine, Heidelberg, Federal Republic of Germany)
was adopted. The latter reagent, which specifically binds to F-actin,
was used according to the method of Wulf et al. (18) at a concentration
of2-10Mg/mI in PBS.
The following antibodies have been used in this investigation. Rabbit
anti-calf brain tubulin prepared according to the method of Weber et
al. (19) or mouse monoclonal anti-a or .i-tubulin (Amersham Interna
tional, U.K.) was used. Mouse anti-vimentin monoclonal antibody was
used also. The specificity of this antibody was tested against a large
variety of different cell lines and found to meet the criteria (20) adopted
for identifying intermediate filament types.4 Rabbit anti-keratin serum,
prepared as described (21), was a kind gift of Dr. M. Osborn, Max
Planck Institute for Biophysical Chemistry, Goettingen, Federal Re
public of Germany. Rabbit anti-actin from Dyctiostelium discoideum
was prepared, affinity-purified, and kindly donated by Dr. P. Cappuccinelli, Institute of Microbiology, Sassari, Italy. Mouse anti-vinculin
monoclonal antibodies were obtained from Bio Yeda, Rehovot, Israel.
Fluorescein- or rhodamine-labeled secondary antibodies were pur
chased from Kirkegaard and Perry, Gaithersburg, MD.
Stained coverslips were observed in a Leitz Dialux microscope
equipped with epifluorescence illumination. Photographic recording
was obtained as described previously ( 16).
RESULTS
The MCF-7 cell line used in the present study grows as
reported (2) and shows a proliferation curve dependent on the
estradiol concentration of the culture medium. The addition of
tamoxifen to the estrogen-deprived medium at the concentra
tion reported by Vic et al. (1) results in the reduction of cell
growth at day 3 and 6 as compared to tamoxifen-free controls
(Fig. 1).
Cytoskeletal Structures in Estrogen-deprived Cells
MCF-7 cells cultured for 3 or 6 days in estrogen-deprived
medium appear roundish and grow either scattered or in clumps
in which occasional intercellular contacts are observed (Fig.
2a). These cells show bundles of microfilaments (stained either
by actin antibodies or by R-PHD) located primarily at their
peripheral rim (Fig. 2b). The edges of these peripheral microfilament bundles (e.g., at arrows in Fig. 2b) end at punctate or
elongated areas which represent sites of vinculin enrichment
(e.g., at arrows in Fig. 2c). The latter correspond to intensely
dark areas (e.g., at arrows in Fig. 2d) in IRM. Since this optical
mean shows the distance between the ventral membrane of a
tissue culture cell and the adhesion substratum (22, 23) it can
be assumed that these dark areas represent focal contacts of the
"adhesion plaque" type in which the cell-substratum distance
is in the order of 10 nm. Then, MCF-7 cells grown in estrogendeprived medium appear to adhere firmly to the substratum.
Under the same culture conditions, MCF-7 cells after 6 days
of culture show occasional actin-rich membrane ruffles predom
inantly located on the dorsal surface (e.g., at arrows in Fig. le).
4 M. Prat, G. C. Corbascio, P. M. Comoglio, and P. C. Marchisio, submitted
for publication.
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DAYS OF CULTURE
12
Fig. 1. Effects of hormones on the proliferation of MCF-7 cells. Cells were
seeded at a density of about 125,000 cells/dish and cultured in three different
media: •.estrogen (!7ß-E2)
deprived medium (10~12]«);•,
estrogen supplemented
medium (10~8 M); A, estrogen deprived tamoxifen (TAM) supplemented medium
(10~12M; tamoxifen = IO"4M). Hemocytometer cell counts from triplicate culture
dishes were performed every 3 days. Standard deviations were less than 5%.
Intermediate filaments of the keratin type appear as wavy
slender fibers distributed in the cytoplasm (Fig. 2f), occasionally
forming perinuclear rings or aiming at cell-to-cell contacts. A
looser network of intermediate filaments of the vimentin type
is also shown by a specific monoclonal antibody. Such a network
is much less dense than that formed by keratin intermediate
filaments.
Polyclonal antibodies to tubulin stain a network of microtubules mostly located in the paranuclear area (Fig. 2g). Microtubule distribution does not display any peculiar feature as
compared to other cultured epithelial cells (17).
Cytoskeletal Structures in MCF-7 Cells Cultured in Estrogensupplemented Medium
Estrogen treatment stimulates cell proliferation (Fig. 1) and
leads to the appearance of cell clusters showing irregular glandlike features. Some cells, notably those located at the periphery
of clusters, display pseudopodial cytoplasmic protrusions (e.g.,
at arrow in Fig. 3a) which are rich in F-actin (e.g., at arrows in
Fig. 3¿>).
When stained with vinculin antibodies, the same cells
show rare stained streaks at the tips of pseudopodial protrusions
(e.g., at arrows in Fig. 3c) which correspond to tight adhesion
areas in IRM (e.g., at arrows in Fig. 3rf).
A major feature of estrogen-stimulated MCF-7 cells is the
wealth of actin-rich microfilamentous structures associated with
polymorphous peripheral protrusions sprouting from the whole
peripheral rim (Fig. 3e). These cells acquire sometimes a fibroblast-like appearance which, together with presence of actin
containing ruffles, appears even more marked after 6 days of
exposure to estrogen (Fig. 3/).
Furthermore estrogen stimulation induces an apparent in
crease of keratin filamentous network which occupies also
peripheral protrusions (Fig. 3g). The distribution of vimentin
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CYTOSKELETON IN BREAST CANCER CELLS
Fig. 2. Cytoskeletal and adhesion struc
tures in estrogen deprived cells, a to d, the
same clump of cells after 3 days of culture. In
phase contrast the cells appear roundish and
flattened over the dish (a). Double-labeled flu
orescence for actin microfilaments (by F-PHD)
(e.g., at arrows; b) and for vinculin (by a specific
monoclonal antibody) (e.g.. at arrows; c) shows
that the microfilaments bundles termini over
lap with vinculin positive dots. In d, IRM black
adhesion areas correspond to the sites of vin
culin enrichment (e.g., at arrows), a and d: bar.
13 „in:h and <: bar. 11 „m.In e. staining for
F-actin by R-PHD after 6 days of culture
shows occasional membrane ruffles (e.g., at
arrows). Bar, 8 »im.Inf. wavy slender fibers of
intermediate filaments, revealed by keratin an
tibodies, are characteristically distributed in
the cytoplasm. Bar, 8 urn. In g, microtubules.
here revealed by a rabbit anti-tubulin serum,
form a dense network mostly located in the
paranuclear areas. Bar. 8 urn.
filaments (not shown) and microtubules (Fig. 3h) is similar to
that of cells cultured in estrogen-deprived medium.
Cytoskeleton of MCF-7 Cells Grown in Estrogen-deprived Tamoxifen-supplemented Medium
The addition of tamoxifen to the culture medium further
reduces the rate of cell proliferation below the values obtained
with estrogen-deprived medium (Fig. 1) and results in marked
changes of cell structure.
The morphology of cells is similar to that obtained in estro
gen-supplemented medium, and irregular gland-like patterns
appear in cell clusters. Pseudopodial cytoplasmic protrusions
(Fig. 4a) contain prominent microfilament bundles (e.g., at
arrows in Fig. 46) which end at vinculin-rich streaks (e.g., at
arrows in Fig. 4c) corresponding to IRM dark areas (e.g., at
arrows in Fig. 4d). The cell periphery is strongly positive for
actin, which is often located in dorsal ruffles, and in some areas,
microfilaments tend to form straight bundles of the stress fiber
type (Fig. 4e). The distribution patterns of keratin filaments
(Fig. 4/) and of microtubules (Fig. 4g) in cells cultured in
tamoxifen-supplemented
medium are overall similar to those
of estrogen-treated cells.
DISCUSSION
Breast cancer cell lines have been investigated mainly to get
information on metabolic and mitotic responses to different
culture conditions.
Several studies on the breast carcinoma MCF-7 cell line have
been reported (11). The interest in this cell line stems from its
well proven estrogen dependency; receptors for 170-estradiol
have been demonstrated ( 12, 13), and addition of estrogen in
the culture medium induces an increase in mitotic index and in
protein turnover (2, 3, 8-10, 24).
We have investigated these cells in order to show whether
their overall response to estrogen stimulation is also matched
by changes in their cytoskeletal architecture and, in general,
whether the cell shape, which is of obvious importance in
cytopathological diagnosis, represents a hormone-dependent
phenomenon.
Previous ultrastructural investigations (1) showed that estro
gen stimulation of MCF-7 cells induces an increase in number
and length of microvilli at the cell surface; this effect precedes
cell detachment. In agreement with these investigations, we
have shown that a marked change in cell shape and in cellsubstratum relationship is induced by estradici. This is evident
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CYTOSKELETON IN BREAST CANCER CELLS
Fig. 3. Cytoskeletal and adhesion struc
tures in MCF-7 cells cultured in estrogen sup
plemented medium, a to d: A cluster of cells
(cultured for 3 days) display, in phase contrast
(e.g., at arrow, a), long cytoplasmic projec
tions, which are rich in F-actin (e.g., at arrows;
b), here stained with F-PHD. The same cells
stained with a monoclonal antibody against
vinculin (e.g., at arrows; c) show a few small
patches at the tips of the pseudopodial protru
sions. These patches correspond to adhesion
areas in IRM (e.g., at arrows; d). a and d: bar,
13 urn; b and c: bar, 11 ¿im.In e and/ staining
of F-actin by R-PHD shows bundles of microfilaments mostly associated with the cell pe
ripheral rim and along irregular cytoplasmic
projections. Straight bundles of the stress fiber
type are evident in cells grown for 3 days (i),
while in cells grown for 6 days {/) dorsal mem
brane ruffling (e.g., at arrows) is quite promi
nent. Bar, 8 »mi.In g, staining for keratin
reveals a rich network of intermediate fila
ments, diffuse in the cytoplasm and present
also in the irregular projections. Bar, 8 urn. In
h, microtubules revealed by a rabbit anti-tubulin serum are mainly localized in a parami
clear area, with a distribution similar to that
observed in cells cultured in estrogen-deprived
medium (see Fig. lg). Bar, 8 ><m.
on phase contrast and IRM; long cytoplasmic projections
sprout from the cell body, and there is a reduction of adhesion
areas which appear located just at the tips of these projections,
so that the cell body becomes relatively detached from the
substratum. Cells grown in estrogen-deprived medium appear
instead markedly flattened over the dish, with adhesion plaques
developed all around the cell periphery, as shown also by
vinculin staining. Loss of adhesion and the appearance of
similar projections have also been observed in Shionogi 115
mouse mammary tumor cells stimulated by testosterone (25)
and might therefore correspond to a general specific response
to steroid stimulation in hormone dependent tumors. Other
studies on these androgen-dependent cells (26) showed steroidmodulated changes of microfilaments and microtubules, while
intermediate filaments were not investigated.
The data reported above suggest that structural changes
might either follow or precede the specific metabolic and proliferative response of endocrine-dependent breast cancer cells.
Changes of cell shape and of different cytoskeletal structures in
response to endocrine stimulation have not been reported pre
viously on MCF-7 or on other estrogen-dependent breast car
cinoma cell lines. Our experiments show that some classes of
cytoskeletal filaments (e.g., microtubules and vimentin inter
mediate filaments) do not appear significantly affected by hor
mone conditioning; conversely, a rearrangement of keratin fil
aments and the formation of a rich network in the cell body
and mainly along cytoplasmic projections represent instead the
main effect of estrogen treatment. Microfilaments also undergo
a marked redistribution and appear in a thick meshwork at the
cell periphery in connection with the apparent increase of
surface activity. Microfilament bundle termini appear to match
the adhesion areas at the tip of cytoplasmic projections; in
addition, microfilaments tend to form in some cells straight
bundles of the stress fiber type (27).
Another notable difference was the appearance, in cells grown
for 6 days in estrogen-supplemented medium, of a prominent
membrane ruffling, as shown by actin staining. A change in
microfilament distribution between cells grown for 3 or 6 days
was less marked but was observed also in cell grown in estrogen
deprived medium; such a change appears to be a general phe
nomenon, possibly related to membrane activity.
These changes may not be directly related to the increase in
mitotic index and metabolism; conversely they might be a
primary effect of estrogen stimulation mediated through the
specific high affinity sites for estradiol receptors. These struc
tures have been suggested recently (28) to be bound to cyto-
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CYTOSKELETON IN BREAST CANCER CELLS
Fig. 4. Cytoskeleton and adhesion struc
tures of MCF-7 cells grown in estrogen-delMIi ni tamoxifen supplemented medium, a to
</: In phase contrast MCF-7 cells, treated for
6 days with tamoxifen. appear irregularly
shaped (a), vim microfilaments are stained
with F-PHD (e.g.. at arrows: b). The ends of
these microfilament bundles match with vinculin-rich streaks (e.g.. at arrows: c) and cor
respond to IRM dark areas (e.g.. at arrows: d).
a and ¡I:
bar, 13 ,.m: b and c: bar. 11 ,.m. In e.
a fìbroblast-likeshape and the presence at the
periphery of straight bundles of the stress fibre
type is present in cells cultured for 3 days and
stained for actin with F-PHD. Bar, 8 /im. In/
staining with keratin antibodies reveals a nest
of fibers filling the protrusions sprouting from
the cell body. Bar, 8 ,;m. In <;.the distribution
of microtubules, revealed by o- and , inimlin
monoclonal antibodies, is not modified by ta
moxifen treatment, being similar to that pre
sented in Figs. \g and 2/r. Bar, 8 ^m.
skeletal components. To clarify this point, we undertook exper
iments on MCF-7 cells grown on estrogen-deprived tamoxifensupplemented medium. The addition of this anti-estrogen to
the medium is known to cause a marked decrease in mitotic
index and protein turnover (29), an effect apparently related to
a specific binding to estrogen receptors and to interference with
the trophic effects of estrogen (2).
Tamoxifen treatment induced the appearance of fibroblastlike phenotypes, with a cytoskeletal organization overall similar
to that of estrogen treated cells. However, cells treated with
tamoxifen show a good adhesion to the substratum, as shown
also by the appearance of abundant vinculin containing streaks
corresponding to adhesion plaques. In long term estrogentreated cells there is a clear reduction of this characteristic.
The effect of tamoxifen on the cytoskeleton might possibly
be related to the induced growth arrest. However, we think that
the discrepancy which we have recorded between the proliferative and metabolic effects of tamoxifen and the structural rear
rangement of the cytoskeletal components is in line with our
hypothesis that cell shape in cancer might be independently
affected. It seems in keeping with this hypothesis that the
tamoxifen, in addition to the inhibitory effects on cell prolifer
ation, is known to display "in vivo" a partial agonistic activity
(30).
On the basis of these results we propose that a target for
hormonal signals may be the direct relationship of neoplastic
cells with their environment and notably with other cells. Such
a phenomenon, which involves the distribution of cytoskeletal
structures, seems either totally independent or loosely related
to the general metabolic and proliferative control of cancer
cells.
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1986 American Association for Cancer Research.
Estrogen- and Tamoxifen-induced Rearrangement of
Cytoskeletal and Adhesion Structures in Breast Cancer MCF-7
Cells
Anna Sapino, Francesca Pietribiasi, Gianni Bussolati, et al.
Cancer Res 1986;46:2526-2531.
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