Progesterone Induces Focal Adhesion in Breast Cancer Cells MDA-MB-231 Transfected with Progesterone Receptor Complementary DNA
Valerie C.-L. Lin,
Eng Hen Ng,
Swee Eng Aw,
Michelle G.-K. Tan,
Esther H.-L. Ng and
Boon Huat Bay
Department of Clinical Research (V.C.-L.L., S.E.A., M.G.-K.T.)
Department of General Surgery (E.H.N., E.H.-L.N) Singapore General
Hospital Republic of Singapore 169608
Department of
Anatomy (B.H.B.) National University of Singapore Republic of
Singapore 169608
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ABSTRACT
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Since the effects of progesterone are mediated
mainly via estrogen-dependent progesterone receptor (PR), the
expression of the effects of progesterone may be masked or overridden
by the influence of estrogen under conditions in which priming with
estrogens is required. We have established a PR-positive but estrogen
receptor-
(ER-
) negative breast cancer cell model by transfecting
PR cDNA into ER-
- and PR-negative MDA-MB-231 cells in order that the
functions of progesterone can be studied independently of estrogens. We
have demonstrated using this model that progesterone markedly inhibited
cell growth. We have also discovered that progesterone induced
remarkable changes in cell morphology and specific adhesion structures.
Progesterone-treated cells became considerably more flattened and well
spread than vehicle-treated control cells. This was associated with a
striking increase of stress fibers, both in number and diameter, and
increased focal contacts as shown by the staining of focal adhesion
proteins paxillin and talin. There were also distinct increases in
tyrosine phosphorylation of focal adhesion protein paxillin and focal
adhesion kinase in association with increased focal adhesion.
The staining of tyrosine-phosphorylated proteins was concentrated at
focal adhesions in progesterone-treated cells. More
interestingly, monoclonal antibody (Ab) to ß1 integrin was able to
inhibit progesterone-induced cell spreading and formation of actin
cytoskeleton. To our knowledge, this is the first report describing a
direct effect of progesterone in inducing spreading and adhesion of
breast cancer cells, and ß1-integrin appeared to play an essential
role in the effect. It is known that the initial step of tumor
metastasis is the breakaway of tumor cells from primary tumor mass when
they lose the ability to attach. Hence, progesterone-induced cell
spreading and adhesion may have significant implications in tumor
metastasis.
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INTRODUCTION
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Progesterone is critically involved in the growth, development,
and differentiation of the breast (1, 2, 3), and its effects are mostly
mediated via progesterone receptors (PRs) (2, 4). Mice lacking PR
displayed incomplete mammary ductal branching and failure of
lobular-alveolar development (5). Although PRs are regulated by a
number of hormones and growth factors, they are estrogen receptor
(ER)-dependent gene products (6, 7, 8, 9). Hence, target cells usually need
to be primed by estrogenic compounds before the effects of progesterone
can be studied. It is conceivable that the effects of estrogen will be
present for almost as long as the effects of progesterone can last.
Indeed, Otto (10) has shown that a pulse of 1 nM estrogen
for 1 min was sufficient to partially stimulate cell proliferation for
5 days. As a result, it is often difficult to distinguish the specific
effects of progesterone from that of estrogen as progesterone-induced
response may be overshadowed by those of estrogens.
Since T47D breast cancer cells were found to constituitively express
high levels of PR independent of estrogens (11), the cell line and its
variants have been the major models by which to study the functions of
progesterone in the regulation of cell growth and other cellular
processes (12, 13). We have established ER-independent expression of PR
by stably transfecting PR cDNA into ER-
- and PR-negative breast
cancer cell line MDA-MB-231, which has recently been reported to
express ER-ß mRNA (14). The resulting PR-positive but ER-
-negative
cell model allows us to assess PR-mediated progesterone-regulated
cellular processes independent of estrogen and ER. We have reported
that progestins markedly inhibit cell proliferation of these PR
transfectants (15). The findings are similar to what was reported for
the growth-inhibitory effects of progestins in T47D cells (1) and in a
PR-negative subline T47D-Y transfected with either the B or A isoforms
of PR (T47D-YB and T47D-YA) (13). In this report, the novel finding
that progesterone induced remarkable cell spreading and focal adhesion
in PR-transfected MDA-MB-231 cells is described. Progesterone-induced
cell adhesion was associated with increased tyrosine phosphorylation of
focal adhesion protein paxillin and focal adhesion kinase (FAK) and
inhibited by Ab to ß1-integrin.
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RESULTS
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Effect of Progesterone on Cell Spreading and Attachment
ER- and PR-negative breast cancer cells MDA-MB-231-CL2 were
transfected with PR expression vectors hPR1 and hPR2 as reported in
detail previously (15). Vector pBK-CMV was cotransfected with PR
expression vectors as it contains neomycin-resistant gene as selection
marker for initial screening. Eight transfectant clones expressing both
PR-A and PR-B were generated. The ratios of the two isoforms expressed
differ among the transfectants. It is to be noted that
progesterone-mediated effects described below are similar in these
clones. For simplicity of interpretation, effects of progesterone are
only described for clone ABC28, which expressed PR of 660 fmol/mg
protein. Clone CTC15 transfected with both pSG5 and pBK-CMV plasmids
were used as transfectant control. No PR was detected in CTC15 by
either enzyme immunoassay or Western blot analysis.
A comprehensive report on the effects of progesterone on the growth of
PR-transfected MDA-MB-231 cells (ABC28) has been previously published
(15). We have described in that report that progesterone markedly
inhibited the growth of ABC28 cells in a concentration-dependent
fashion. Maximal inhibition of cell growth was observed with
10-9 M of progesterone, and the cell
number was reduced by a maximum of 70% as compared with the
vehicle-treated controls.
Although progesterone markedly inhibited the growth of PR-transfected
MDA-MB-231 cells, progesterone-treated cells did not exhibit any sign
of apoptotic or necrotic death as shown in Fig. 1
. Instead, progesterone induced
remarkable changes in cell morphology and specific adhesion structures
in these cells. The majority of PR-transfectant cells in control medium
appeared rounded in shape and attached to the substratum poorly (Fig. 1
, a and c). In contrast, progesterone-treated cells became
considerably flattened and more spread with much larger cell surface
than the vehicle-treated control (Fig. 1
, b and d). The effect on cell
spreading began to be visible after 8 h of progesterone treatment
as more cells were flattened and adhered to the substratum (Fig. 1b
).
The cell spreading and flattening were very prominent after 48 h
treatment (Fig. 1d
). The flattened and well-spread morphology of
progesterone-treated cells is best illustrated in the micrograph (Fig. 2b
), as compared with the much more
rounded control cells (Fig. 2a
). Progesterone had no detectable effect
on vector-transfected control cells CTC15 or the parental cell line
MDA-MB-231-CL2 cells (not shown).

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Figure 1. Effect of Progesterone on the Morphology of
PR-Transfected MDA-MB-231 Cell Clone ABC28 Cells
Cells grown in six-well plates were treated with either 1
nM progesterone in 0.1% ethanol or 0.1% ethanol only for
the required period of time before they were viewed and photographed
under a Carl Zeiss AXIOVERT 35 phase contrast microscope.
After 8 h (a and b) and 48 h (c and d) of treatment,
progesterone-treated ABC28 cells (b and d) are more flattened and
spread than the vehicle-treated controls (a and c). Cells in panel e
are treated with monoclonal Ab to integrin ß1 at 24 h after
progesterone treatment and photographed after a further 24-h incubation
(bar, 100 µm). Photos with + signs show cells treated
with progesterone, and photos with - signs show cells not treated
with progesterone.
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Figure 2. Micrographs of Scanning Electron Microscopy of
Progesterone-Treated and Control ABC28 Cells
Cells were processed for scanning electron microscopy analysis as
described in Materials and Methods. Progesterone-treated
cells (b) are more flattened and spread out than the vehicle-treated
controls (a) (bar, 10 µm).
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Progesterone Stimulated the Formation of Stress Fibers and Focal
Adhesions
To determine whether the flattened and well spread morphology
induced by progesterone was associated with the formation of new stress
fibers, filamentous actin was visualized by the use of fluorescein
isothiocyante (FITC)-phalloidin after 48 h treatment.
Vehicle-treated cells contained few stress fibers (Fig. 3a
). In contrast, progesterone-treated
cells were characterized by a very well developed stress fiber system,
and the new stress fibers appeared to be increased both in number and
diameter (Fig. 3b
). To confirm that progesterone induced specific focal
adhesion, cells were stained with Ab to focal adhesion proteins, talin
and paxillin, after 48 h of treatment. Progesterone-treated cells
(Fig. 4b
for talin and Fig. 4d
for
paxillin) showed increased focal contacts as evidenced by the much more
prominent staining of focal adhesion proteins than that in
vehicle-treated controls (Fig. 4a
for talin and Fig. 4c
for paxillin).
The nuclei regions of the cells showed strong staining by the Abs, and
we are not sure of the significance of this staining. Figure 4
, e and
f, showed cells stained with Ab to talin (red) and paxillin
(red), respectively, in conjunction with
FITC-phalloidin, the probe to filamentous actin. It can be seen that
focal adhesion proteins, talin and paxillin, colocalized with the ends
of stress fibers, thus playing the role of anchoring the cytoskeleton
to the extracellular matrix and hence enabling the cells to adhere and
spread.

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Figure 3. Effect of Progesterone on the Development of Stress
Fibers in ABC28 Cells
ABC28 cells were treated with control vehicle (a) or 1 nM
progesterone (b) for 48 h before they were stained with
FITC-conjugated phalloidin. There are marked increases in stress fibers
in the cells both in number and size after progesterone treatment.
Cells in panel c were treated with integrin ß1 Ab (10 µg/ml) at
24 h after progesterone treatment and photographed after a further
24-h incubation (bar, 25 µm).
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Figure 4. Immunostaining of Focal Adhesion Proteins Talin and
Paxillin Alone (ad) or in Combination with Either F-Actin (e and f)
or Phosphotyrosine (g and h) in ABC28 Cells
Cells grown on glass coverslips were treated with 1 nM
progesterone in 0.1% ethanol or 0.1% ethanol only for 48 h
before they were fixed and permeabilized. The cells were then incubated
with mouse monoclonal Ab to paxillin and talin and rabbit polyclonal Ab
to phosphotyrosine either alone or in combinations (for colocalization)
overnight at 4 C, followed by incubation with FITC- or
rhodamine-conjugated sheep antimouse, antirat, or antirabbit IgG
at room temperature for 1 h. For colocalization of F-actin with
talin and paxillin, 10 µg/ml FITC-phalloidin were added together with
the secondary Ab. Stained cells were viewed and photographed using the
model LSM 510 Carl Zeiss confocal laser scanning
microscope. There were notable increases in focal contacts in
progesterone-treated cells (panel b for talin and panel d for paxillin)
as compared with vehicle-treated controls cells (panel a for talin and
panel c for paxillin). Talin and paxillin was localized at the tips of
the stress fibers as shown by costaining of F-actin
(green) with talin (panel e, red) and
paxillin (panel f, red) in progesterone-treated cells.
The staining of phosphotyrosine (panel g, red) was found
to be concentrated at focal adhesions as shown by its
immunocolocalization with focal adhesion protein paxillin (4 h,
green) in progesterone-treated cells
(bar, 10 µm).
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It is clear that progesterone induces cell spreading and focal
adhesions in PR cDNA-transfected MDA-MB-231 cells. We further
investigated signaling pathways that are associated with
progesterone-induced cell adhesion. Tyrosine phosphorylation of
paxillin and FAK are thought to be involved in the assembly of focal
adhesion complexes (16, 17). Therefore, focal adhesion protein paxillin
and FAK were immunoprecipitated and analyzed by Western blotting for
tyrosine phosphorylation in response to progesterone treatment. The
assay was performed twice and the results showed similar trends. FAK
and paxillin were identified at molecular masses 125 kDa and 6570
kDa, respectively (Fig. 5a
). Lanes in
panel A were probed with antityrosine Ab, and lanes in panel B were
probed with Ab to FAK and paxillin, respectively, to reveal the amount
of immunoprecipitated protein analyzed. There were increased tyrosine
phosphorylations of FAK and paxillin in progesterone-treated
cells (+ lanes, Fig. 5
, panel A) as compared with
the vehicle-treated controls (-lanes, Fig. 5a
, panel A) after
normalization with the amount of immunoprecipitated protein analyzed
(panel B). Densitometry data of two experiments revealed that there was
an average of 62% increase in tyrosine phosphorylation of FAK after
8 h and 24 h of 1 nM progesterone treatment, and
100% and 240% increases, respectively, in tyrosine phosphorylation of
paxillin after 8 h and 24 h of 1 nM progesterone
treatment. It is to be noted that cell spreading began to be visible
after 8 h of progesterone treatment and by 24 h the
phenomenon was well established. Hence, increased tyrosine
phosphorylation by progesterone accompanies the function of induction
of focal adhesion by progesterone. This observation is consistent with
the idea that tyrosine phosphorylation of focal adhesion proteins is
involved in the development of new focal adhesions. Indeed, the
staining of Ab to phosphotyrosine in progesterone-treated cells was
concentrated at focal contacts as shown by colocalization of the
staining of Ab to phosphotyrosine (Fig. 4g
) with that of the focal
adhesion protein paxillin (Fig. 4h
).

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Figure 5. Tyrosine Phosphorylation of FAK and Paxillin in
Progesterone-Treated ABC28 Cells
Cell lysates were collected after 8 h and 24 h of treatment.
A, FAK and paxillin were immunoprecipitated with their specific Ab and
analyzed by Western Blotting analysis. After probing with
antiphosphotyrosine Ab PY20 (panel A), the membranes were stripped and
reprobed with the specific Ab to FAK or paxillin to determine the
relative amounts of each protein expressed (panel B). There were
increased phosphorylations of FAK and paxillin in progesterone-treated
cells (+ lanes) as compared with vehicle-treated controls (-
lanes). B, Densitometry analysis of tyrosine phosphorylation of FAK and
paxillin in progesterone-treated ABC28 cells. The results are the means
of two experiments and are each normalized with the total amount of
immunoprecipitated proteins. , Ethanol-treated controls; , 1
nM progesterone-treated group.
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Involvement of ß1-Integrin in Progesterone-Induced Cell Spreading
and Adhesion
Integrins are major adhesion molecules that mediate interactions
between cytoskeleton and extracellular matrix proteins (18). To
determine whether progesterone-induced cell spreading and focal
adhesion are integrin-mediated, cells were tested for blocking effects
of monoclonal Ab to integrin subunits ß1, ß4,
2,
3,
4,
5, and
6. It was revealed that monoclonal Ab to ß1-integrin
dramatically reversed progesterone-induced cell spreading and adhesion
when it was incubated for 24 h with the cells that had been
treated with progesterone for 24 h when the focal adhesions had
already been established. Similar inhibition of progesterone-induced
spreading and adhesion by ß1-integrin Ab was demonstrated when the Ab
was added at the same time as progesterone. Cells treated with Ab to
ß1-integrin became smaller and less protracted (Fig. 1e
) as compared
with progesterone-treated cells without ß1-integrin Ab (Fig. 1d
).
There was also considerable reduction in the amount of stress fibers in
ß1-integrin Ab-treated cells (Fig. 3c
) as compared with the cells
treated with progesterone alone (Fig. 3b
).
Quantitative assessment of reversing effect of ß1-integrin Ab on
progesterone-induced cell spreading is shown in Table 1
. Images of light micrographs of 76115
cells were acquired via a charge-coupled device (CCD) camera,
and the surface area of individual cells was analyzed by KS 400
software (Kontron Instruments Ltd.). Progesterone-treated
cells were, on average, 3.4 times larger in area than the
ethanol-treated controls (P < 0.00001). ß1-Integrin
Ab at 1:100 was able to reduce progesterone-induced spreading by nearly
40% after 24 h treatment (P < 0.0005). These
observations suggest that the ß1-integrin subunit is critically
involved in progesterone-induced cell spreading and adhesion. This is
in line with the belief that integrins function to establish bridges
between extracellular matrix proteins and cytoskeleton at focal
adhesion sites.
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Table 1. Quantitative Measurement of the Reversing
Effect of ß1-Integrin Ab on Progesterone-Induced Cell
Spreading
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DISCUSSION
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One unique feature in the classical model for the mechanism of
action of progesterone is that the hormone acts via estrogen-dependent
PR (2, 4, 6, 7, 8, 9). According to this model, progesterone can only exert
its effect in the cell that has already been stimulated by estrogens.
Our goal was to establish a PR-positive but ER-negative cell model that
would enable us to separate the functions of progesterone from
the influence of estrogen and its receptor. We have stably transfected
PR cDNA into ER and PR-negative MDA-MB-231 cells. These PR
transfectants express both A and B receptor isoforms with
varying A:B ratio. Using PR transfectant clone ABC28, we have
discovered that progesterone induced remarkable cell spreading,
formation of new stress fibers, and focal adhesion assemblies in the
PR-transfected MDA-MB-231 cells. This is the first report describing
such a direct effect of progesterone on cell adhesion, demonstrating
the usefulness of the PR-positive but ER-
-negative model in
revealing the function of progesterone.
There has been recent evidence suggesting the involvement of
progesterone in cell-cell adhesion. Estrogen was shown to suppress the
expression of E-cadherin and
- and ß-actenin in Ishikawa cells of
well differentiated endometrial cancer cells, which could lead to
decreased cell-to-cell adhesion. The use of progestins reversed the
suppressions induced by estrogen (19). In T47D cells, progestins
induced the expression of desmoplakins (3), which are essential
intracellular attachment proteins that connect intermediate filaments
with the desmosomes, which in turn interact with transmembrane linker
proteins to hold the adjacent membranes together. These findings
suggest promoting mechanisms of progesterone for cell-cell interaction.
However, these T-47D cells displayed much more rounded morphology after
progestin treatment, which is opposite to what we have found in
PR-transfected MDA-MB-231 cells. Another recent finding was that in
transgenic mice carrying an imbalance in the native ratio of A to B
forms of PR, the mammary glands exhibited decreased cell-cell adhesion
(20). This is an in vivo demonstration of the role of
progesterone in cell-cell adhesion that is mediated by a balanced
effort of A to B forms of PR. It remains to be determined whether the
function of progesterone in inducing focal adhesion reported here
reflects a normal physiological function and whether it can also be
reproduced in other PR-positive but ER-negative cell models.
Progesterone-induced cell adhesion was associated with increased
tyrosine phosphorylation of FAK and focal adhesion protein paxillin
(16, 17). FAK was first identified to be concentrated at focal
adhesions in 1992 (21). Substantial evidence suggests that FAK is
capable of autophosphorylation in response to integrin clustering
(21, 22, 23, 24). Upon activation, FAK can itself phosphorylate paxillin, which
will then serve to recruit additional signaling molecules to focal
contacts and hence to catalyze the formation of focal adhesion
assemblies and to initiate signals that may direct the activation of
other cellular signaling pathways. In PR-transfected MDA-MB-231 cells,
ß1-integrin was shown to play an essential role in
progesterone-induced cell spreading and formation of actin
cytoskeleton as Ab to ß1-integrin inhibited progesterone-induced cell
adhesion. Our preliminary results also revealed that the ß1-integrin
Ab was able to reverse progesterone-induced tyrosine phosphorylation of
FAK and paxillin. Hence, ß1-integrin may be responsible for the
activation of FAK, which will then trigger the generation of other
signaling molecules such as the phosphorylation of paxillin in the
focal contacts as is generally proposed for the model of signal
transduction in focal adhesion (22, 23).
It is to be noted that the magnitude of ß1-integrin Ab-mediated
reduction of progesterone-induced cell spreading was about 40%. The
inability of ß1-integrin Ab to completely reverse
progesterone-induced focal adhesion may be because the functional
efficiency of ß1-integrin Ab is not 100% or there may be other
mechanisms working in parallel with ß1-integrin to promote cell
adhesion.
At present, we can only speculate on the mechanisms of integrin
activation by progesterone. Progestins have been shown to increase the
expression of laminin receptor mRNA in T47-D cells (24), and ß1
integrin-associated heterodimers are well known laminin receptors (25, 26). However, our study revealed no progesterone-induced changes in the
expression of ß1-integrin or in tyrosine phosphorylation of
ß1-integrin in these PR-transfected MDA-MB-231 cells that are known
to express high level of ß1-integrin (26, 27). We hypothesize that
progesterone induces focal adhesion by activating ß1-integrin via
intermediate signaling molecules, notably the growth factor-mediated
signaling pathways. This hypothesis is supported by several lines of
evidence. First, progesterone is known to mediate the expression of a
number of growth factors and their receptors. These include fibroblast
growth factor (28), epidermal growth factor (29), transforming growth
factor (30), and insulin-like growth factors (IGF) and IGF binding
proteins (IGFBPs) (31, 32, 33, 34). Second, several reports suggest that growth
factors are involved in cell adhesion. IGF-I has been reported to
stimulate the formation of adhesion structures. In SH-SY5Y
neuroblastoma cells, IGF-I induces lamellapodia extension and formation
of stress fibers, and this is associated with increased tyrosine
phosphorylation of FAK and paxillin (35). IGF-I also stimulates
chemotaxis of breast cancer cells lines MCF-7 and MDA-MB-231 in which
specific types of integrins are required for the IGF-I-mediated
response (36). Furthermore, direct interactions between IGFBP-I and
integrins have been reported both in vitro (37) and in
vivo (38). Accordingly, recent evidence also highlighted the
importance of interactions between integrins and classical growth
factor signaling pathways, with several reports showing integration of
integrin and growth factor signal transduction pathways (39, 40, 41).
Direct evidence of progesterone acting as a mediating factor of growth
factor signaling in breast tissue has emerged recently. Progesterone
was shown to up-regulate type I growth factor receptors, and
selectively amplify downstream MAPK cascade (42). Progesterone also
primes breast cells for growth factors action. For example, T47D
cells primed by progestins for approximately 48 h become highly
sensitive to the proliferative effect of epidermal growth factor (EGF)
that is not mitogenic in these cells in the absence of progesterone
(42, 43). In bovine mammary tissue transplanted to nude mice,
progesterone significantly augmented the mitogenic effect of EGF (44).
In agreement with published results, preliminary results from our
laboratory also suggested progesterone-dependent effect of EGF on cell
spreading in the PR-transfected MDA-MB-231 cells. EGF-mediated
activation of ß1-integrin by progesterone is currently under
investigation in our laboratory.
Cell adhesion is a key process in the establishment of tissue structure
and differentiation. Complex and coordinated reductions and increases
in adhesion have been proposed to be necessary for tumor invasion and
metastasis. The findings that progesterone induced cell-extracellular
matrix adhesion suggest that this hormone may play a significant role
in the process of tumor invasion and metastasis and the effect may be
mediated by integrins. Integrins are generally believed to promote the
cell-substrate adhesion. Ab to ß1-integrin significantly inhibited
the adhesion of MDA-MB-231 cells to extracellular matrix, bone matrix,
and to human umbilical vein endothelial cells (27, 28, 45).
ß1-Integrin Ab also inhibited the attachment of rat mammary tumor
cells on the lymph node stroma (46). The integrin-promoted attachment
can be positively or negatively related to metastasis, depending on
whether the integrin functions to adhere the tumor cells to basement
membrane surrounding the primary tumor or whether it functions to aid
in adhesion at a secondary site. Since the initial step of metastasis
is believed to be the detachment of tumor cells from the primary tumor
mass when the cells lose the ability to attach (47), increased
adherence of tumor cells to basement membrane may prevent the tumor to
metastasize to a secondary site. Experiments are underway to study the
effect of progesterone on cell invasion in ABC28 cells both in
vitro and in vivo.
It is also interesting to note that morphogenesis of human mammary
cells in collagen gel was prevented by ß1-integrin Ab (48, 49, 50) and
progesterone is well known to be involved in mammary morphogenesis (2).
It would be interesting to determine whether in vivo
development of lobule-alveolar structure of the mammary gland requires
the activation of ß1-integrin by progesterone.
In conclusion, we have demonstrated that progesterone induces
remarkable focal adhesion in PR-transfected MDA-MB-231 cells. In
association with progesterone-induced focal adhesion was the increased
tyrosine phosphorylation of focal adhesion protein paxillin and FAK. Ab
to ß1-integrin distinctively inhibited progesterone-induced focal
adhesion and tyrosine phosphorylation of FAK and paxillin, suggesting
that ß1-integrin plays an important role in progesterone-induced
focal adhesion. This is the first report describing a direct effect of
progesterone on focal adhesion. The results provide new directions to
which the therapeutic potential of progesterone in breast cancer can be
explored. It remains to be studied whether progesterone also induces
focal adhesion in physiological situation in which both PR and ER are
naturally present.
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MATERIALS AND METHODS
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Materials
MDA-MB-231 cells were obtained from American Tissue Culture
Collection (ATCC, Manassas, VA) in 1995 at passages 28.
They were cloned using a 96-well plate by the method of single-cell
dilution, and clone 2 (MDA-MB-231-CL2) was used for the study described
herein. FITC-phalloidin and mouse monoclonal antitalin Abs were
obtained from Sigma (St. Louis, MO). Mouse monoclonal
antipaxillin Ab and rabbit polyclonal antiphosphotyrosine Ab were from
Transduction Laboratories, Inc. (Lexington, KY).
Antiintegrin ß1, ß4, and
2-
6 were from Becton Dickinson and Co. (San Jose, CA) Mouse monoclonal
antiphosphotyrosine (PY20) Ab was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All fluorescence-conjugated
secondary Abs were purchased from Roche Molecular Biochemicals
(Indianapolis, IN). All tissue culture reagents were obtained
from Life Technologies (Gaithersburg, MD). Tissue culture
plastic wares were from Corning, Inc. (Corning, NY).
Cell Culture
All cells were routinely maintained in phenol red containing
DMEM supplemented with 5% FCS, 2 mM glutamine, and 40
mg/liter gentamicin. For all experiments, cells were grown in phenol
red-free DMEM supplemented with 5% dextran charcoal-treated FCS
to remove the endogenous steroid hormones that might interfere with the
analysis. Cells were treated with progesterone from 1000-fold stock in
ethanol. This gave a final concentration of ethanol of 0.1%. Treatment
controls received 0.1% ethanol only.
Transfection
PR expression vectors hPR1 and hPR2 were generous gifts of
Professor P. Chambon (Institute of Genetics and Molecular and Cellular
Biology, Strasbourg, France). Vectors hPR1 and hPR2 contained
human PR cDNA coding for PR isoform B and isoform A, respectively, in
pSG5 plasmid (51). Vector pBK-CMV (Stratagene, La Jolla,
CA) containing the neomycin-resistant gene was cotransfected with hPR1
and hPR2 into MDA-MB-231-CL2 cells using Lipofectin reagent (Life Technologies, Inc.). Neomycin-resistant clones selected in
medium containing G418 were screened for vector pSG5 sequence by PCR
and for PR using the PR enzyme immunoassay kit from Abbott Laboratories (North Chicago, IL). Eight PR-positive clones
expressing both PR isoforms A and B were isolated and characterized.
They showed similar responses to progesterone treatment. For simplicity
of interpretation, the effects of progesterone on clone ABC28 that
expressed approximately 660 fmol PR per mg protein were described in
this report. The cells at passage numbers 530 were used. Cells stably
transfected with both vectors pBK-CMV and pSG5 were used as
transfection controls.
Light Microscopy
Cells were grown in six-well plates and treated with either
progesterone in 0.1% ethanol or 0.1% ethanol for the required period
of time before they were viewed and photographed under an AXIOVERT 35
phase contrast microscope (Carl Zeiss, Thornwood, NY).
Quantitative measurement of cell sizes was according to that described
by Bay and Tay (52). Light micrographs containing 76115 cells taken
at magnifications of 100 times were analyzed. Image acquisition was
performed via a Variocam CCD camera mounted on a copy stand and
analyzed with KS 400 software (Kontron Instruments Ltd.,
Eching, Germany).
Immunofluorescence Microscopy
Cells were grown on glass coverslips in six-well plates and
treated with progesterone in 0.1% ethanol or 0.1% ethanol for 48
h. After rinsing with PBS, the cells were fixed in 4% paraformaldehyde
for 10 min and permeabilized with 0.2%Triton X-100 for 10 min. This
was followed by incubation with 2% normal horse serum in PBS for
1 h to block nonspecific binding. All the subsequent incubations
with Ab were carried out in PBS containing 2% normal horse serum. Ab
to paxillin, talin, and phosphotyrosine alone or in combinations (for
colocalization) were incubated with the cells overnight at 4 C,
followed by incubation with FITC- or rhodamine-conjugated sheep
antimouse, antirat, or antirabbit IgG at room temperature for 1 h.
For F-actin staining, the fixed and permeabilized cells were incubated
with 10 µg/ml FITC-phalloidin in PBS for 1 h at room
temperature. After washing in PBS, the coverslips were mounted on
slides with fluorescence mounting media from DAKO Corp.
(Carpinteria, CA). Stained cells were viewed and photographed using the
model LSM 510 Carl Zeiss confocal laser scanning
microscope.
Immunoprecipitation
Cells (15 x 106) grown on 100-mm
petri dishes were lysed with 200 µl cold lysis buffer (50
mM Tris, 150 mM NaCl, 2 mM EDTA,
0.5 mM EGTA, 1 mM sodium vanadate, 0.1% sodium
deoxycholate, 0.5% Triton X-100, and a cocktail of protease inhibitors
for serine, cysteine, and metalloproteases, pH 7.5) at 4 C for 30 min
before they were scraped and harvested. The protein supernatants were
collected by centrifugation at 30,000 x g for 30 min
and the protein concentrations in the lysates were determined using a
protein assay kit (Bio-Rad Laboratories, Inc.). Protein
(400 µg) was incubated with Ab against paxillin, FAK, or integrin
ß1 in lysis buffer overnight at 4 C. The Ab-bound proteins were
precipitated with protein A/G Sepharose. The protein A/G Sepharose
beads were then boiled for 5 min in sample buffer, and the supernatants
containing the protein of interest were analyzed by Western blotting
using ECL kit (Amersham Pharmacia Biotech, Arlington
Heights, IL). After probing with antiphosphotyrosine Ab, the membrane
was stripped in buffer containing 62.5 mM
Tris-HCl, pH 6.7, 2% SDS, and 100 mM
ß-mercaptoethanol for 30 min at 55 C. The membrane was reprobed with
the respective Ab of interest to determine the relative amounts of each
protein expressed.
Antibody Inhibition
Cells grown on six-well plates were treated with 1
nM progesterone or 0.1% ethanol for 48 h. Ab to
integrins
2,
3,
4,
5,
6, ß1, or ß4 were added to
cells at 24 h after progesterone treatment and were incubated for
a further 24 h. Effects of these integrin Ab to
progesterone-induced cell spreading and focal adhesion were viewed and
photo- graphed.
Scanning Electron Microscopy
Cells on glass coverslips were fixed in 100 mM
cacodylate buffer containing 5% glutaraldehyde, pH 7.2, for 20 min.
After extensive washing with cacodylate buffer, coverslips were
incubated for 15 min in 1% osmium tetroxide in cacodylate buffer and
then dehydrated by successive 5-min incubations in 50%, 75%, and 95%
ethanol. The coverslips were incubated 3 times for 5 min each in 100%
ethanol, and then were dried in a Balzers CPD 030 critical point
dryer using liquefied carbon dioxide. Coverslips were sputter coated
with 20 nM gold before viewing in a scanning electron
microscope.
Statistical Analysis
Differences between treatments were tested by ANOVA. When
significant differences were detected by ANOVA, multiple comparisons
among means were performed by the least significant difference test.
Correlation analysis was performed using the program in
Excel.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to express their sincere thanks to Professor
Pierre Chambon of Institute of Genetics and Molecular and Cellular
Biology, Strasbourg, France, for kindly providing the progesterone
receptor expression vectors hPR1 and hPR2. We would also like to thank
Dr. I. A. Forsyth of Babraham Institute, Cambridge, U.K., for the
discussion of this manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Valerie C-L Lin, Department of Clinical Research, Singapore General Hospital, Block 6, level 6, Room B22, Outram Road, Singapore 169608.
This work was funded by the National Medical Research Council, Republic
of Singapore.
Received for publication March 3, 1999.
Revision received October 25, 1999.
Accepted for publication November 22, 1999.
 |
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