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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-{alpha} (ER-{alpha}) negative breast cancer cell model by transfecting PR cDNA into ER-{alpha}- 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-{alpha}- 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-{alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go. 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. 1Go, 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. 1Go, 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. 1bGo). The cell spreading and flattening were very prominent after 48 h treatment (Fig. 1dGo). The flattened and well-spread morphology of progesterone-treated cells is best illustrated in the micrograph (Fig. 2bGo), as compared with the much more rounded control cells (Fig. 2aGo). 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).

 
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. 3aGo). 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. 3bGo). 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. 4bGo for talin and Fig. 4dGo 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. 4aGo for talin and Fig. 4cGo 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 4Go, 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 (a–d) 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).

 
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 65–70 kDa, respectively (Fig. 5aGo). 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. 5Go, panel A) as compared with the vehicle-treated controls (-lanes, Fig. 5aGo, 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. 4gGo) with that of the focal adhesion protein paxillin (Fig. 4hGo).



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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. {square}, Ethanol-treated controls; {blacksquare}, 1 nM progesterone-treated group.

 
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, {alpha}2, {alpha}3, {alpha}4, {alpha}5, and {alpha}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. 1eGo) as compared with progesterone-treated cells without ß1-integrin Ab (Fig. 1dGo). There was also considerable reduction in the amount of stress fibers in ß1-integrin Ab-treated cells (Fig. 3cGo) as compared with the cells treated with progesterone alone (Fig. 3bGo).

Quantitative assessment of reversing effect of ß1-integrin Ab on progesterone-induced cell spreading is shown in Table 1Go. Images of light micrographs of 76–115 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

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-{alpha}-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 {alpha}- 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}2-{alpha}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 5–30 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 76–115 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 (1–5 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 {alpha}2, {alpha}3, {alpha}4, {alpha}5, {alpha}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 Balzer’s 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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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