Bcl-2 expression decreases cadherin-mediated cell-cell adhesion

Laiji Li, Jody Backer, Annisa S. K. Wong, Erin L. Schwanke, Brian G. Stewart and Manijeh Pasdar*

Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G2H7, Canada

* Author for correspondence (e-mail: mpasdar{at}ualberta.ca)

Accepted 1 May 2003


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 Summary
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 Materials and Methods
 Results
 Discussion
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Bcl-2, a member of the apoptosis-regulating family of proteins confers a survival advantage on cells by inhibiting apoptosis. Bcl-2 expression is estrogen-responsive and high in various tumors. Overexpression of Bcl-2 has been associated with the loss of contact inhibition, unregulated growth and foci formation in culture.

In this study, we have examined the effects of bcl-2 overexpression and expression on cell-cell adhesion in MCF-7 and MDCK epithelial cell lines respectively. Overexpression of Bcl-2 in estrogen receptor-positive MCF-7 mammary carcinoma cells led to decreased cell surface E-cadherin and the disruption of junctional complexes concurrent with intracellular redistribution of their components. Particularly noticeable, was the partial nuclear localization of the tight junction-associated protein ZO-1 which coincided with upregulation of ErbB2. The expression of this EGF co-receptor is regulated by the ZO-1-associated transcription factor ZONAB. Growth in estrogen-depleted media led to downregulation of Bcl-2 expression and upregulation and membrane localization of all junctional proteins. Similar disruption in junctions, accompanied by decreased transepithelial resistance, was observed when Bcl-2 was expressed in MDCK cells.

These results strongly suggest that Bcl-2 expression decreases the level of functional E-cadherin thereby interfering with junction formation. The inhibition of junction formation decreases cell-cell adhesion leading to the loss of contact inhibition, which, in vivo, can lead to unregulated growth and tumorigenesis.

Key words: E-cadherin, Catenin, Bcl-2, Junction, Cell-cell adhesion


    Introduction
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 Introduction
 Materials and Methods
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Cadherins are calcium-dependent transmembrane glycoproteins that mediate cell-cell adhesion and play essential roles in development, morphogenesis and cell polarity (Takeichi, 1991Go). E-cadherin (E-cad), the prototypic member of the classic cadherin family, mediates cell-cell adhesion in epithelia and plays critical roles in histogenesis and organogenesis (Marrs and Nelson, 1996Go; Huber et al., 1996Go; Larue et al., 1994Go). Cadherin molecules on adjacent cells form homophilic interactions in a zipper-like fashion, binding cells together (Nagar et al., 1996Go). To function, the cytoplasmic domain of cadherins must bind to the actin cytoskeleton via proteins called catenins (Aberle et al., 1996Go; Gumbiner, 2000Go). Alpha-catenin ({alpha}-cat) is a vinculin-related molecule, whereas ß-catenin (ß-cat), {gamma}-catenin [plakoglobin (Pg)] and p120 belong to the Armadillo family of proteins (Peifer et al., 1994Go). Beta-catenin and Pg interact with E-cad in a mutually exclusive manner and connect it to {alpha}-cat, which then interacts with the actin cytoskeleton (Adams and Nelson, 1998Go; Ben-Ze'ev and Geiger, 1998Go; Imamura et al., 1999Go). p120 strengthens the E-cad adhesive complexes (Thoreson et al., 2000Go).

E-cad expression is necessary for the formation of tight, adherens and gap junctions and desmosomes (Gumbiner et al., 1988Go). The formation of normal junctions is essential for the integrity and function of epithelial tissues (Yeaman et al., 1999Go). Loss of E-cad expression leads to epithelial tumorigenesis (Perl et al.,1998Go) and disruption of the cadherincatenin complex because decreased E-cad expression is often observed in many advanced, poorly differentiated carcinomas (Berx and Van Roy, 2001Go; Hajra and Fearon, 2002Go).

The regulated balance between cell proliferation and programmed cell death or apoptosis is essential for normal tissue homeostasis and development. Bcl-2, the prototypic member of the apoptosis-regulating family of proteins (Adams and Cory, 2001Go), confers a survival advantage on cells by inhibiting apoptosis. In epithelial cells, Bcl-2 expression is associated with proliferation, tissue development and morphogenesis (Lu et al., 1996Go). High levels or aberrant patterns of Bcl-2 expression occur in various carcinomas (Reed, 1999Go; Reed et al., 1996Go).

Bcl-2 expression increases in response to estrogen, and is correlated with estrogen receptor (ER) positivity in breast carcinomas (Schorr et al., 1999Go; Gee et al., 1994Go). Bcl-2+, ER+ breast tumors respond favorably to estrogen withdrawal (Leek et al., 1994Go). Estrogens regulate gene expression via estrogen receptors and their cognate response elements within the promoter of estrogen-responsive genes. Estrogen deprivation may inhibit the growth of ER+ breast tumors, in part by downregulating Bcl-2 and inducing apoptosis (Gompel et al., 2000Go; Wolf and Davidson, 2001). Supporting this notion, Perillo et al. (Perillo et al., 2000Go) showed that two estrogen-responsive elements in the coding sequence of Bcl-2 can act as enhancers in cells that express estrogen receptors. Furthermore, estrogen can upregulate Bcl-2 expression in the ER+-MCF-7 breast carcinoma cells (Pratt et al.1998Go).

E-cad expression is also modulated by estrogen (Blaschuk et al., 1994Go; Meng et al., 2000Go; Habermann et al., 2001Go; Malaguti and Rossini, 2002Go). In breast carcinoma cells, estrogen withdrawal or estrogen antagonists led to increased E-cad levels (DePasquale, 1999Go; Meng et al., 2000Go). However, this effect may be indirect since estrogen response elements have not been found in the E-cad promoter. Interestingly, Bcl-2 overexpression in MCF-7 cells can promote an epidermal to mesenchymal transition along with the loss of E-cad (Lu et al., 1995Go).

Here, we have examined the effects of Bcl-2 expression on cell-cell adhesion in MCF-7 breast carcinoma and in Madin-Darby canine kidney (MDCK) epithelial cell lines. We show that Bcl-2 expression leads to the disruption of junctional components. Overexpression of Bcl-2 in MCF-7 mammary carcinoma cells grown in normal, estrogen-containing media led to decreased cell surface E-cad levels and disappearance of tight junctions, adherens junctions and desmosomes. The disappearance of the junctions coincided with intracellular redistribution of their components. Particularly noticeable was the partial nuclear localization the tight junction-associated protein ZO-1, which coincided with upregulation of ErbB2, an EGF co-receptor that is normally expressed at very low levels in the parental MCF-7 cells. ErbB2 expression is regulated by the ZO-1-associated transcription factor ZONAB [ZO-1-associated nucleic acid-binding protein (Balda and Matter, 2000Go)]. Growth in estrogen-depleted media led to downregulation of Bcl-2 expression and upregulation and membrane localization of all junctional proteins.

Bcl-2 expression had similar effects in MDCK cells. Junction formation was inhibited, along with the redistribution and increased solubility of junctional proteins. Interestingly, the soluble ZO-1 in these cells was codistributed with Bcl-2. This codistribution was only detected for ZO-1 among all junctional markers. The disruption of junctions in Bcl-2-expressing MDCK and MCF-7 cells also coincided with a reduction in their transepithelial resistance (TER). Together, these results suggest that Bcl-2 expression leads to decreased membrane-associated E-cad and, consequently, inhibition of junction formation. The absence of junctions results in decreased cell-cell adhesion and loss of contact inhibition which, in vivo, can lead to unregulated growth and tumorigenesis.


    Materials and Methods
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 Materials and Methods
 Results
 Discussion
 References
 
Cell lines and culture conditions
Human mammary carcinoma cell lines MCF-7 and Bcl-2-overexpressing MCF-7 (hereafter referred to as MCF-7B) cells were provided by Dr David Andrews (McMaster University, Hamilton, Ontario, Canada). MCF-7B cells were generated by transfecting MCF-7 cells with the plasmid pRc/CMV-Bcl-2 encoding wild-type Bcl-2 and selection of G418-resistant clones (Thangaraju et al., 1999Go). MCF-7 and 7B cells were grown in {alpha}-minimal essential medium ({alpha}-MEM, Invitrogen). Breast cancer cell line SKBR-3 and MDCK cells were grown in MEM and DMEM (Invitrogen) respectively. All media were supplemented with 10% FBS and 1% antibiotic. For estrogen-free experiments, phenol-red-free-{alpha}-MEM was supplemented with charcoal-filtered FBS.

Antibodies and reagents
All reagents were purchased from Sigma unless stated otherwise. Table 1 outlines a list of the primary and secondary antibodies used in this study. The source of each antibody and their respective dilution in each assay are also indicated.


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Table 1. Antibodies and their respective dilutions in specific assays

 

Plasmids and transfection
The expression vector BMG-Neo (Karasuyama and Melchers, 1988Go) and expression plasmid BMG-Bcl-2 (under the control of a metallothionein promoter) were provided by Dr Michele Barry, University of Alberta. MDCK cells were grown to 80% confluence and transferred into low calcium (5 µM)-containing DMEM (LCM) for 5 hours. For transfections, 10 µg of BMG-Neo (control) or BMGBcl-2 plasmids were used with LipofectAMINE 2000 (Invitrogen) reagent according to the manufacturer's instruction. Forty-eight hours later, the medium were changed to DMEM with 600 µg/ml G418. Bcl-2 expression in G418-resistant colonies was induced by addition of 5 µM ZnCl2 and verified by immunofluorescence (IF) and immunoblotting (IB). Two independent transfections were performed using the BMG-Bcl-2 plasmid that produced transfectants, MDCKB1 and MDCK-B2, with different levels of Bcl-2 expression.

Cell fractionation, cell surface biotinylation and immunoblotting
Total cell extracts (TCE) were prepared by solubilizing cells in hot SDS-sample buffer followed by boiling for 10 minutes. For cell fractionation, cells were extracted with cytoskeleton extraction buffer [CSK: 300 mM sucrose, 10 mM Pipes, pH 6.8, 50 mM NaCl, 3 mM MgCl2, 0.5% (v/v) Triton X-100, 1.2 mM PMSF, 0.1 mg/ml DNase, and 0.1 mg/ml RNase (Pasdar and Nelson, 1988aGo)] for 10 minutes. CSK separates the cytoskeleton-associated (insoluble; P) pool of junctional proteins from the cytoplasmic (soluble; S) pool. The CSK extracts were centrifuged at 48,000 g for 10 minutes and the resulting S and P fractions were denatured using 10x and 1x hot SDS sample buffer respectively.

Protein concentrations were determined by BCA assay (Sigma) according to the manufacturer's instructions. Equal amounts of protein from each fraction were resolved on SDS/7.5% or 10% gels and transferred to nitrocellulose membranes. Membranes were incubated with antibodies at dilutions indicated in Table 1 and developed using ECL (Amersham Pharmacia).

Cell surface biotinylation was done using EZ-Link-Sulfo-NHS-LC-biotin (Pierce). Biotin reagent was dissolved in Hepes-saline (280 mM NaCl, 50 mM Hepes and 1.5 mM Na2HPO4) and added to confluent cultures at 500 µg/ml for 30 minutes at room temperature. After rinsing, cells were solubilized in SDS buffer without DTT (1% SDS, 10 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.5 mM PMSF), vortexed and boiled for 10 minutes. SDS was then diluted to 0.1% and extracts were processed for avidin-agarose pull down.

Biotinylated proteins were precipitated by incubating 250 µg of biotinylated cell lysates with 50 µl avidin-agarose beads. After 3 hours of incubation on a rocker rotator, lysates were centrifuged and beads washed 3 times, once with wash buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl and 0.5% NP-40), once with high salt buffer (wash buffer + 1 M LiCl) followed by a final wash with the wash buffer. Biotinylated proteins were eluted from the beads with SDS sample buffer containing 100 mM DTT, separated by gel electrophoresis and transferred to nitrocellulose membranes. Membranes were processed for IB with E-cad antibodies and developed by ECL.

Protein bands were quantitated using NIH-imager software and normalized to the amount of tubulin present in the same extract. All experiments were reproduced 3-5 times and results of a typical experiment are shown.

Immunofluorescence (IF) and electron microscopy (EM)
For indirect IF staining, cells were grown to confluence on uncoated (MCF-7/7B) or collagen-coated (MDCK) glass coverslips (Pasdar and Nelson, 1988bGo). Cells were rinsed with PBS and either fixed in 3.7% (v/v) paraformaldehyde for 15 minutes and permeabilized with CSK buffer for 2-5 minutes on ice, or extracted with CSK buffer before fixation. Coverslips were blocked in PBS with 3% (v/v) goat serum and 50 mM NH4Cl for 30 minutes and incubated for 1 hour with various primary antibodies (Table 1), followed by 20 minutes incubation with the appropriate secondary antibodies. Coverslips were mounted in elvanol containing 0.2% (w/v) paraphenylene diamine (PPD, pH 8.6) and viewed with a 100x objective using a BX50 Olympus IF microscope. Images were captured using the Advanced Spot software.

For electron microscopy (EM), confluent cells grown on carbon- (MCF-7/7B) or carbon and collagen (MDCK)-coated glass coverslips and were fixed in 1.25% (v/v) glutaraldehyde, 1% (v/v) OsO4 in 50 mM Pipes, pH 7.2 for 5 minutes at room temperature, processed as described (Pasdar et al., 1995aGo) and examined using a Philips 410 transmission electron microscope.

Transepithelial resistance (TER) measurements
TER was measured as described previously (Stevenson and Begg, 1994Go) using a Millicell-ERS apparatus (Millipore Corporation, Bedford, MA). Cultures of MDCK-Neo, -B1, -B2, MCF-7 and MCF-7B cells were grown to confluence on uncoated (MCF-7/7B) or collagen-coated (MDCK) 24 mm transwell filters (0.4 µm pore size; Costar Corp.) in medium containing 1.8 mM calcium (HCM). In each experiment, duplicate MDCK-Neo cultures were transferred to LCM 3-4 hours before TER measurement. TER ({Omega}xcm2) were calculated by subtracting the resistance of a blank filter multiplied by the area of the monolayer. Results are shown as the mean ± s.e.m. of three independent measurements for each cell line.


    Results
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 References
 
MCF-7B cells overexpressing Bcl-2 do not assemble well-formed junctions
Upon confluence, MCF-7 cells formed a uniform monolayer of tightly associated cells in which cellular boundaries were obscured (Fig. 1A). In contrast, MCF-7B cells formed a less uniform monolayer and the entire culture could never be focused upon in one plane. In MCF-7B cultures, the cellular boundaries were always distinct (Fig. 1B).



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Fig. 1. Phase contrast image of confluent cultures (A,B) and electron micrograph images showing cell structure (C,D) of MCF-7 (A,C) and MCF-7B (B,D) cells. TJ, tight junction; AJ, adherens junction; Des, desmosome. (A,B) Magnification, 250x. Scale bars: (C) 0.42 µm; (D), 0.21 µm

 

Ultrastructural examination of MCF-7 cells showed tightly opposed lateral membranes between cells and well-formed tight and adherens junctions and desmosomes (Fig. 1C). MCF-7B cells however, showed wide intercellular spaces with numerous interdigitations, and lacked well-formed junctions (Fig. 1D). Most striking was the lack of membrane fusion at the tight junction and the integral membrane domains in adherens junctions and desmosomes (Fig. 1D).

Subcellular distribution of junctional proteins in MCF-7 and MCF-7B cells
Stable junctions are formed when integral membrane proteins interact with the cytoskeleton via the cytoplasmic plaque proteins. Since EM studies showed a complete lack of integral membrane domains, we compared the distribution of junctional proteins in the parental MCF-7 and MCF-7B transfectants.

MCF-7 cells expressed low levels of endogenous Bcl-2 and the the cadherin-catenin complex (E-cad, {alpha}-cat, ß-cat and p120) was primarily membrane-associated (Fig. 2). Similarly, the staining for desmoplakins (DPs) and desmoglein (Dsg) showed punctate peripheral staining typical of desmosomes. The distribution of Pg, a common component of desmosomes and adherens junctions, was also primarily peripheral. Tight junction-associated proteins, ZO-1 and occludin were also localized to the cell periphery. Together, the ultrastructure and IF analysis of MCF-7 cells showed stable junctions, as seen previously (van Deurs et al., 1987Go; Sommers et al., 1994Go; Mauro et al., 2001Go; Zhu et al., 2001Go; Vizirianakis et al., 2002Go).



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Fig. 2. Subcellular localization of Bcl-2 and components of the adherens junction, tight junction and desmosomes in MCF-7 and MCF-7B cells. Cultures were grown on glass coverslips to confluence, formaldehyde fixed, CSK permeabilized and stained with antibodies to Bcl-2, and the indicated junctional proteins at concentrations listed in Table 1. Primary antibodies were detected by FITC- or rhodamine-conjugated species-specific secondary antibodies and viewed with a 100x objective using a BX50 Olympus microscope. Images were captured using Advance Spot software. Scale bar: 50 µm.

 

In MCF-7B cells, the staining for Bcl-2 was strong and distributed throughout the cells, consistent with its mitochondrial and endoplasmic reticulum distribution (Fig. 2). Compared to MCF-7 cells, MCF-7B cells showed less intense staining for E-cad particularly at the membrane (Fig. 2). Consistent with decreased E-cad membrane staining, MCF-7B cells showed considerable intracellular staining for {alpha}- and ß-cat (Fig. 2). Similarly, p120 showed both membrane and intracellular staining (Fig. 2). For Pg, the staining was primarily intracellular and much more intense than that observed in MCF-7 cells (Fig. 2). This redistribution was also observed for the other desmosomal proteins, DPs and Dsg (Fig. 2). The most pronounced differences between MCF-7 and MCF-7B cells were those observed in the distribution of the tight junction-associated proteins ZO-1 and occludin. The continuous peripheral ZO-1 ring observed in MCF-7 cells was completely disrupted in MCF-7B cells. In addition, a small pool of this protein appeared to localize to the nucleus (Fig. 2). As well, we detected high levels of cytoplasmic occludin in contrast to its exclusive membrane association in MCF-7 cells (Fig. 2). The disrupted ZO-1 staining, the decreased membrane staining for E-cad, Dsg and occludin, and intracellular redistribution of the components of the three junctions further support the absence of well-formed junctions in MCF-7B that was apparent by EM.

Spatial reorganization of the junctional proteins in MCF-7 and MCF-7B cells upon estrogen withdrawal
MCF-7 cells express very little Bcl-2 and have estrogen receptors (Leek et al., 1994Go). It is well known that estrogen can regulate Bcl-2 expression (Teixeira et al., 1995Go). After 8 days growth in estrogen-depleted medium, MCF-7 cells showed decreased staining for endogenous Bcl-2 (Fig. 3). These cells developed numerous processes, showed decreased cell-cell contact and were more motile. Consistent with this morphology, junctional proteins were redistributed from the periphery into the cytoplasm. The staining for E-cad showed a more or less homogeneous cytoplasmic distribution with a higher concentration in the processes. Similar staining patterns were observed for {alpha}-cat, ß-cat, p120, Pg, DPs and occludin. The peripheral ZO-1 staining (Fig. 2) was disrupted concurrent with lightly stained nuclei. This pattern of ZO-1 staining resembles that seen in MDCK cells at low confluence or upon the removal of calcium from the medium (Gottardi et al., 1996Go). The decreased cell to cell contact in MCF-7 cells in estrogen-depleted medium also coincided with increased motility and cell death.



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Fig. 3. Reorganization of the junctional proteins in MCF-7 and MCF-7B cells grown in the absence of estrogen. Cells were grown to confluence on glass coverslips, transferred to estrogen-depleted medium and maintained for another 8 days. Cells were fixed and processed for indirect IF as outlined in the legend of Fig. 2. Scale bar: 50 µm.

 

The effects of estrogen removal in MCF-7B were completely different. Eight days after estrogen removal, the staining pattern for Bcl-2 did not change (compare MCF-7B, Bcl-2 in Figs 2 and 3). Unfortunately, the highly expressed exogenous Bcl-2 obscured the detection of any decrease in the endogenous protein. Regardless, estrogen removal from MCF-7B cells led to increased cell-cell contact with partial redistribution of junctional proteins to the peripheries. The staining for E-cad was more intense at the membrane (MCF-7B, E-cad in Figs 2 and 3). Similarly, more intense peripheral staining was seen for {alpha}-cat, ß-cat, p120, Pg and DPs. ZO-1 staining remained disrupted at the periphery but increased intracellularly (compare MCF-7B, ZO-1 in Figs 2 and 3). Likewise, the exclusively intracellular distribution of occludin was modified as some membranous aggregates were also detected in these cells (compare MCF-7B, occludin in Figs 2 and 3). Whereas MCF-7 cells became more motile and underwent cell death without estrogen, neither of these changes occurred in MCF-7B cells. There was, however, a clear drop in their rate of proliferation (data not shown). Together, estrogen removal appeared to cause MCF-7 cells to differentiate/die and MCF-7B to become more adhesive. The latter was supported by the downregulation of Bcl-2 and upregulation and redistribution of junctional proteins in subpopulations of MCF-7B cells deprived of estrogen for longer duration, up to 2.5 weeks (Fig. 4A, 4B).




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Fig. 4 (A,B) Membrane redistribution of junctional proteins in MCF-7B cells upon long-term absence of estrogen. Coverslip-grown confluent cultures were transferred into estrogen-deprived medium for 16 days. IF localization of Bcl-2 and various junctional proteins was carried out as outlined in the legend of Fig. 2. Double staining was performed by detecting two different primary antibodies with FITC- or rhodamine-conjugated species-specific secondary antibodies. Merged images (right column, A: bottom row, B) were constructed using the Adobe Photoshop software. CK, cytokeratins; Pg, plakoglobin. Arrows in A indicate the absence of Bcl-2 expression or membrane localization of junctional proteins. Scale bars: (A) 150 µm; (B) 75 µm

 

In Fig. 4A,B, confluent cultures of MCF-7B cells grown on coverslips in normal medium were transferred to estrogen-free medium and allowed to grow for another 16 days. Replicate samples were then processed for double staining with Bcl-2, and various junctional proteins in the indicated combinations.

After 16 days without estrogen, MCF-7B cultures developed islands of cells in which Bcl-2 was undetectable (Fig. 4A,B). Double staining of these cultures with cell adhesion protein antibodies detected exclusively peripheral distributions in cells without Bcl-2 (e.g., Bcl-2, p120 and Bcl-2/p120, Fig. 4A,B). In contrast, cells that maintained Bcl-2 expression continued to display a primarily cytoplasmic distribution for these proteins as observed previously (Fig. 3). In cells in which Bcl-2 was undetectable, E-cad was localized exclusively to the membrane and overlapped in the apical region with ZO-1 and occludin (Fig. 4A,B). Bcl-2-negative cells also showed organized distributions of cytokeratins in contrast to Bcl-2-positive cells where cytokeratins remained disorganized (Fig. 4A). Similar peripheral distributions were also detected for {alpha}-cat, ß-cat, p120, Pg and occludin (Fig. 4A,B). Together, these results suggest a direct correlation between downregulation of Bcl-2 expression and relocation of the junctional proteins from cytoplasm to the membrane.

Bcl-2 overexpression changes the steady state levels of {alpha}-catenin and p120 and decreases the amount of membrane-associated E-cadherin
To determine if membrane redistribution of junctional proteins was associated with changes in their steady state levels, MCF-7 and MCF-7B cells were grown to confluence and replicate cultures deprived of estrogen for 2, 5, 8 and 16 days. TCEs of control and estrogen-deprived cultures were processed for IB using Bcl-2, junctional proteins and tubulin antibodies.

In MCF-7 cells, a decrease in the endogenous Bcl-2 levels was detected after 5 days without estrogen (Fig. 5A). The amounts of E-cad, {alpha}-cat, ß-cat, Pg, p120 and ZO-1 remained essentially unchanged between control and estrogen depleted cultures of MCF-7 cells (Fig. 5A). However, proteolytic fragments of ß-cat, Pg and p120 were detected after estrogen removal. The size of the ß-cat and Pg fragments are similar to those seen in epithelial cells undergoing apoptosis (Brancolini et al., 1997Go; Brancolini et al., 1998Go; Schmeiser et al., 1998Go; Fukuda, 1999Go; Steinhusen et al., 2000Go; Ling et al., 2001Go). The quantities of these fragments were highest at day 16, when cell numbers were the lowest.



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Fig. 5. Bcl-2 over expression modifies the level of junctional proteins and decreases the surface E-cad. (A) MCF-7 and MCF-7B cells were grown to confluence in normal medium (day 0). Replicate cultures were transferred into estrogen-free medium for 2 to 16 days. Equal amounts of TCEs from control and estrogen-depleted cultures was resolved on 10% (Bcl-2 and tubulin) or 6% (E-cad, {alpha}-cat, ß-cat, Pg, p120 and ZO-1) gels and processed for IB with various antibodies at concentrations listed in Table 1. (B) MCF-7 and MCF-7B were grown in normal medium and duplicate cultures were transferred into estrogen-deprived media for 12 days. Cultures were biotinylated as described in Materials and Methods and TCEs prepared. From each line, 250 µg of TCE was precipitated with streptavidin-agarose beads. Biotinlylated proteins were eluted from the beads and, together with 50 µg of TCEs, processed for IB with E-cad antibodies. To verify comparable loading the TCE blot was reprobed for tubulin. (C) Confluent MCF-7 and MCF-7B cells were either fixed and then permeabilized (Total) or CSK-extracted and then fixed (Surface) and processed for staining with an E-cad antibody (Transduction Laboratories). Scale bar: 50 µm.

 

In estrogen-depleted MCF-7B cultures, Bcl-2 levels increased up to 8 days with little or no subsequent changes. Although we do not know the exact cause of this increase we suspect that it may be due to the death of the cells that do not overexpress Bcl-2 within the first week. In these cultures, only E-cad level was increased after 12 days (Fig. 5A, days 12 and 16) and estrogen removal had no effect on the amounts of {alpha}-cat, ß-cat, Pg, p120 and ZO-1 (Fig. 5A). However, MCF-7B cells generally appeared to have less {alpha}-cat and p120 than MCF-7 cells. In addition, smaller isoforms of p120 were detected in MCF-7B cells after 12 days of estrogen depletion (Fig. 5A), concurrent with increased E-cad levels.

IF of MCF-7 and MCF-7B cells showed less surface E-cad in MCF-7B cells. Furthermore, increased E-cad membrane staining was detected in Bcl-2 negative colonies that appeared within estrogen-depleted MCF-7B cultures. Therefore, we used cell surface biotinylation to assess the amount of membrane-associated E-cad between the two cultures.

In Fig. 5B, control and 12-day estrogen-depleted cultures were biotinylated and TCEs prepared. Biotinylated proteins were isolated by streptavidin-agarose beads and processed for IB with E-cad antibodies. As shown in Fig. 5B, whereas MCF-7 cells showed a slight decrease in the cell surface E-cad upon estrogen removal, more biotinylated E-cad was detected in estrogen-depleted MCF-7B cells. This was further confirmed by IF staining of E-cad in MCF-7 and MCF-7B cultures after differential extraction/fixation procedure. Coverslip grown cultures were either fixed and then permeabilized, to determine the total E-cad (Fig. 5C, Total), or CSK-extracted first and then fixed, to determine the surface E-cad (Fig. 5C, Surface). Very little difference was detected between total and surface E-cad staining in MCF-7 cells whereas significantly more E-cad staining was detected in MCF-7B cells before extraction (Fig. 5C). Together, these results suggest that decreased Bcl-2 levels upon estrogen removal leads to increased cell surface E-cad in MCF-7B cells.

Increased ErbB2 protein levels in MCF-7B cells
IF staining showed disruption of peripheral ZO-1 staining concurrent with its partial redistribution into the nucleus. To assess the significance of this redistribution, we examined the levels of the EGF co-receptor ErbB2 the expression of which is known to be modulated by ZO-1 and it's partner transcription factor ZONAB (Balda and Matter, 2000Go).

In Fig. 6A, MCF-7 and MCF-7B cells were processed for IF with ErbB2 and ZO-1 antibodies. MCF-7 cells with little ErbB2 showed a typical peripheral ZO-1 staining. In contrast, significantly more ErbB2 was detected in MCF-7B cells which showed disrupted ZO-1 peripheral distribution concurrent with its increased nuclear staining. The increase in ErbB2 levels in MCF-7B cells was confirmed by IB of the TCEs of MCF-7, MCF-7B and SKBR-3 (an ErbB2-positive breast carcinoma cell line) cells (Fig. 6B).



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Fig. 6. Disruption and intracellular redistribution of ZO-1 coincide with increased levels of ErbB2 in MCF-7B cells. (A) Cultures were established on glass coverslips, formaldehyde-fixed, CSK permeabilized and processed for IF staining with ErbB2 and ZO-1 antibodies. Scale bar: 50 µm. (B) TCEs were prepared for confluent MCF-7, MCF-7B and SKBR-3 cultures and 50 µg from each line processed for IB with ErbB2 antibodies.

 

Bcl-2 expression in MDCK cells also inhibits the formation of junctional structures
To show that the effect of Bcl-2 on E-cad and junction formation was not cell-type specific, we transfected MDCK cells with the inducible BMG-Neo empty vector or BMG-Neo encoding Bcl-2 and isolated stable, independent transfectants with various levels of Bcl-2 expression. For all experiments described here, controls included either MDCK-Neo or MDCK-Bcl-2 cells maintained in normal medium (without Zn+2). Identical results were obtained with both controls and one of them is included in various experiments. Cultures were grown to confluence with or without Zn+2 and processed for ultrastructural examination (Fig. 7A) and IF staining with antibodies to Bcl-2 and various junctional markers. Fig. 7B,C,D shows the results obtained for ZO-1 and p120.



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Fig. 7. Bcl-2 expressed in MDCK cells inhibits junction formation and co-distributes with the soluble ZO-1. (A) Ultrastructural organization of uninduced and Zn2+-induced Bcl-2-expressing MDCK cells. MDCK-Bcl-2 transfectants were grown on collagen- and carbon-coated glass coverslips without (left) or with (right) Zn2+ and processed for electron microscopy. (B) Confluent Zn2+-induced, MDCK-Bcl-2 transfectants grown on coverslips were processed for double IF with a mixture of Bcl-2 and ZO-1 antibodies. Primary antibodies were detected with species-specific secondary antibodies and viewed with a 63x objective. (C) Duplicate cultures from B were either formaldehyde fixed, CSK-permeabilized (Fixed) or CSK extracted first and then fixed (Extracted) and processed for Bcl-2 and ZO-1 staining. (D) Duplicate cultures from B were formaldehyde fixed, CSK-permeabilized and processed for double IF staining with Bcl-2 and p120 antibodies. Scale bars: 1.25 µm (A); 85 µm (B); 40 µm (C); 50 µm (D).

 

BMG-Neo or BMG-Bcl-2 transfectants grown without Zn2+showed closely opposed lateral membranes and well-formed junctions (Fig. 7A, left). In contrast, BMG-Bcl-2 transfectants grown with Zn2+ showed wide intercellular spaces and lacked well-formed junctions (Fig. 7A, right). These observations were very similar to those obtained with MCF-7B cells.

MDCK-Neo or MDCK-Bcl-2 cells grown without Zn2+ were negative for Bcl-2 staining. In contrast, strong Bcl-2 staining was detected in subpopulations of MDCK-Bcl-2 cultures when Zn2+ was present (Fig. 7B, Bcl-2). The staining pattern for Bcl-2 was consistent with its localization to both mitochondria and endoplasmic reticulum. In Bcl-2-negative cells, all junctional proteins were primarily, if not exclusively, localized to the cell periphery in the areas of cell-cell contact (Fig. 7B, ZO-1, and data not shown). In contrast, in Bcl-2-expressing cells all junctional markers showed partial intracellular redistribution (Fig. 7B, ZO-1, and data not shown). Most interestingly, ZO-1 was detected in two distinct distributions, a typical peripheral ring similar to the Bcl-2-negative cells and an intracellular pool (Fig. 7B, ZO-1). Moreover, the intracellular ZO-1 was co-distributed with Bcl-2 (Fig. 7B, ZO-1/Bcl-2). Differential fixation/extraction was used to assess the solubility of these two pools of ZO-1. Fig. 7C, fixed, shows the total cellular ZO-1 and Bcl-2 and their co-distribution in the cytoplasm. Upon extraction (Fig. 7C, Extracted), most of the intracellular ZO-1 was removed. These cells also showed reduced cytosolic Bcl-2 staining. Only ZO-1-and Bcl-2 showed this co-distribution as the other junctional proteins were distributed independently of Bcl-2 (e.g., p120, Fig. 7D).

Increased soluble cadherin and catenins and decreased cell surface E-cadherin in MDCK-Bcl-2 cells
Since junction formation in MDCK cells is associated with increased insolubility of the junctional proteins, we assessed the solubility of these proteins in control and Bcl-2-expressing cultures. Two independent Zn2+-induced MDCK-Bcl-2 transfectants (Fig. 8A, B1, B2) were compared with control cultures. B1 and B2 are the parental transfectants derived from two independent transfections and their difference lies in the number of colonies (within each line) that express Bcl-2. Confluent cultures from each transfectant were CSK extracted to separate the soluble (S) proteins from the insoluble (P) and processed for IB of various junctional markers (Fig. 8A). Although the amount of Bcl-2 was very similar in the Bcl-2-expressing cells of both transfectants, B1 transfectants had fewer positive colonies, an observation that was also reflected in the amount of Bcl-2 detected by IB (Fig. 8A, Bcl-2).



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Fig. 8. Bcl-2 expression in MDCK cells decreases the cytoskeleton-associated pool of the junctional proteins and the surface E-cad. (A) Confluent, vector transfected (Neo) and two Zn2+-induced independent MDCK-Bcl-2 transfectants (B1, B2) were extracted with CSK buffer and the soluble (S) and insoluble (P) fractions separated. Fractions were resolved on 10% (Bcl-2 and tubulin) or 6% (E-cad, {alpha}-cat, ß-cat, Pg, and ZO-1) gels and processed for IB with various antibodies. This experiment was repeated 5 times producing similar results with very little variability among the experiments. (B) The protein bands in A were scanned and quantitated using the NIH imager software. The value obtained for each protein/cell line was normalized to the value obtained for tubulin in the same lysate/cell line and the ratio of the soluble to insoluble calculated. (C) Confluent MDCK-Neo and Zn2+-induced MDCK-B1 and MDCK-B2 cultures were biotinylated and equivalent amounts of total proteins precipitated by streptavidinagarose beads. Biotinylated complexes were eluted, separated on SDS/6% gels and together with 50 µg of the TCE from each line were processed for IB with E-cad antibodies. To confirm equal loading, the TCE blots were reprobed with tubulin antibodies. (D) Confluent MDCK-Neo and Zn2+-induced MDCK-Bcl-2 cells were either fixed and then permeabilized (Total) or CSK-extracted and then fixed (Surface) and processed for staining with an E-cad antibody (3G8). Scale bar: 50 µm.

 

Fig. 8A shows a decrease in the pool of insoluble proteins for all junctional markers in Bcl-2 expressing MDCK cells relative to the MDCK-Neo. The degree of reduction was correlated with the level of Bcl-2 for all proteins. The lowest level of insoluble E-cad, {alpha}-catenin, ß-catenin, Pg and ZO-1 was found in the MDCK-B2 transfectants that had the most Bcl-2 protein. The ratio of S/P for each protein is presented in Fig. 8B. The increased soluble pool of junctional proteins was also supported by the decreased membrane-associated E-cad. MDCK-Neo and Zn2+-induced MDCK-Bcl-2 cells were surface biotinylated and TCEs prepared (Fig. 8C). Biotinylated proteins were isolated with streptavidin-agarose beads and processed for IB with a monoclonal anti-E-cad. As shown in Fig. 8C, there was less biotinylated E-cad in the TCEs prepared from the B2 transfectants. These results were also supported by decreased IF staining of E-cad in extracted Bcl-2-expressing MDCK cells relative to that of MDCK-Neo cells (Fig. 8D).

Bcl-2-expressing cells have lower transepithelial resistance (TER)
Junction integrity and tightness of the monolayers was also determined by measuring the TER of control and Bcl-2-expressing cultures. Cultures were established on transwell filters until confluent. As a control, MDCK-Neo cultures were transferred into LCM medium 3 hours prior to the TER measurement. LCM leads to junction disassembly in MDCK cells (Pasdar et al., 1995aGo). The TER of the MDCKNeo cells grown in normal medium (HCM, 1.8 mM calcium) was ~94 {Omega}xcm2 (Fig. 9). Transfer to LCM (5 µM calcium) reduced the TER to ~21{Omega}xcm2 (Fig. 9 MDCK-Neo-LCM). The TER of MDCK-B1 and B2 transfectants was 62 and 49 {Omega}xcm2 respectively (Fig. 9). These values were inversely correlated with the number of Bcl-2-expressing colonies in each transfectant. We also measured TER of filter-grown confluent MCF-7 and MCF-7B cells. Similar to MDCK transfectants, the Bcl-2-overexpressing MCF-7B cells had lower TER than MCF-7 cells (26 and 40 {Omega}xcm2 respectively, Fig. 9).



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Fig. 9. Decreased transepithelial resistance (TER) in Bcl-2 expressing MDCK and MCF-7B cultures. Confluent MDCK Neo, Zn2+-induced MDCK-B1 and MDCK-B2, MCF-7 and MCF-7B cells were grown on filters in normal medium (1.8 mM calcium, HCM) as described in Materials and Methods. Duplicate MDCK Neo cultures were transferred into LCM for 3 hours and TER measurements were performed on all filters. Histograms represent the mean±s.e.m. of three independent measurements for each cell line. The absence of error bars indicates the small differences in the three measurements.

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We show here that Bcl-2 expression leads to decreased cell surface E-cad and inhibition of junction assembly. Junctions are essential for the integrity and function of epithelial tissues and their absence is a major contributing factor to loss of contact inhibition of growth and tumorigenesis (Perl et al., 1998Go; Hajra and Fearon, 2002Go).

MCF-7 is a breast carcinoma cell line that has typical epithelioid morphology, is ER+, expresses low levels of Bcl-2, and is poorly invasive. In cultures, MCF-7 cells form tight monolayers and have well-formed junctions. IF staining of various junctional proteins in these cells showed membrane localization for all junctional proteins. In contrast, Bcl-2-overexpressing MCF-7B cells did not have well-formed junctions. These cells showed decreased cell surface E-cad and a disruption in the membrane localization of all junctional markers that coincided with their intracellular redistribution. Cell surface biotinylation confirmed the decreased cell surface E-cad in MCF-7B cells. This decrease explains the absence of well-formed junctions and intracellular redistribution of junctional proteins in these cells. Lu et al. (Lu et al., 1995Go) also reported a similar reduction in E-cad levels in another Bcl-2-overexpressing mammary epithelial cell line. In the case of ZO-1, the disruption in the membrane organization coincided with its partial nuclear localization. The nuclear localization of ZO-1 and other cytoplasmic markers of the tight junction has been shown in subconfluent cultures of epithelial cells or in cells maintained in LCM, conditions under which tight junctions can not form (Gottardi et al., 1996Go; Islas et al., 2002Go; Laura et al., 2002Go). ZO-1 is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins (Gonzalez-Mariscal et al., 2000Go). Members of this family interact with transcription factors and regulate gene expression (Hsueh et al., 2000Go; Balda and Matter, 2000Go). ZO-1 interacts with an Y-box transcription factor, ZONAB. The ZO-1-ZONAB complex can regulate the expression of ErbB2 in a cell-density-dependent manner (Balda and Matter, 2000Go). ErbB2/her2/neu is a transmembrane tyrosine kinase co-receptor of the EGFR family that is important for epithelial differentiation and morphogenesis and is often over-expressed in breast carcinomas (Hynes and Stern, 1994Go; Alroy and Yarden, 1997Go; Harari and Yarden, 2000Go; Olayioye et al., 2000Go). Over expression of ErbB2 in mammary carcinomas inhibits transcription of the E-cad gene (D'Souza and Taylor-Papadimitriou, 1994Go). In addition, in certain carcinoma cell lines ErbB2 has been shown to interact with the cadherincatenin complex via ß-cat or Pg (Hoschuetzky et al., 1994Go; Ochiai et al., 1994Go; Kanai et al., 1995Go; Ougolkov et al., 2000Go). ErbB2 can phosphorylate ß-cat/Pg and destabilize the cadherin-catenin complex leading to decreased intercellular adhesion (Jawhari et al., 1999Go). We also detected an increase in ErbB2 levels in MCF-7B cells along with reduced surface E-cad and partial redistribution of ZO-1 to the nuclei.

In mammary epithelial cells, estrogens control cell proliferation by regulating the expression of genes involved in cell cycle progression (Altucci et al., 1996Go). However, there is cross talk between factors that regulate cell proliferation and the pathways that control apoptosis (White, 1996Go; White, 2001Go). Bcl-2, the prototype of the prosurvival proteins that inhibit apoptosis is expressed in mammary epithelium at various stages of differentiation and is present in 80% of breast cancers (Kumar et al., 2000Go). Bcl-2 expression is regulated by estrogen (Pratt et al., 1998Go; Perillo et al., 2000Go; Alkayed et al., 2001Go) and its presence in breast cancers reliably predicts a favorable response to hormone therapy (Gee et al., 1994Go; Silvestrini et al., 1996Go; Zhang et al., 1999Go). Estrogen can downregulate E-cad expression (Blaschuk et al., 1994Go; Meng et al., 2000Go; Eger et al., 2000Go; Habermann et al., 2001Go; Malaguti and Rossini, 2002Go) whereas estrogen antagonists restore E-cad expression and function (Bracke et al., 1994Go; Mbalaviele et al., 1996Go; Vermeulen et al., 1995Go; Meng et al., 2000Go). Here, in MCF-7 cells deprived of estrogen for 8 days, Bcl-2 levels were decreased, cells became larger, developed numerous processes, exhibited decreased cell-cell adhesion and finally died.

In MCF-7B cells, 8 days without estrogen produced little change in the cell morphology or Bcl-2 level. We detected only a slight increase in membrane-localized E-cad, catenins, Pg and DPs, but not ZO-1 and occludin. However, when these cells were deprived of estrogen for longer periods (>16 days), small islands of cells lacking Bcl-2 appeared within the cultures. In these islands, E-cad staining was increased and was almost exclusively peripheral. Similarly, all junctional makers were redistributed to the membrane, typical for tight and adherens junctions and desmosomes. Consistent with this result we found an increase in the level of E-cad and its surface distribution by IB and biotinylation. These results are consistent with increased E-cad levels found in breast carcinoma cell lines treated with estrogen antagonists (Bracke et al., 1994Go; Meng et al., 2000Go). Further, they suggest that the downregulation of Bcl-2 expression results in a restoration of junctional components.

Our observations with MCF-7 cells are not cell-type specific because we also found similar results with MDCK cells. MDCK cells are well characterized in terms of junctional protein expression and have been used as a model system to study the regulation of junction assembly and function (Gonzalez-Mariscal et al., 1985Go; Pasdar and Nelson, 1988aGo; Pasdar and Nelson, 1989b; Gumbiner et al., 1988Go; Shore and Nelson, 1991Go; Nathke et al., 1994Go; Hinck et al., 1994Go). Bcl-2-expressing MDCK cells showed wide intercellular spaces and lacked well-formed junctions. The organization of lateral membranes in these cells was similar to MDCK cells maintained in LCM, a condition under which junctions cannot form (Pasdar et al., 1995aGo). Bcl-2-negative MDCK cells showed membrane localization for various junctional proteins. In contrast, we found increased intracellular staining for almost all of these proteins in the Bcl-2 expressing cells, similar to that of MCF-7B cells. The one exception was the difference in the distribution of ZO-1. Unlike MCF-7B cells in which the membrane distribution of ZO-1 was disrupted, Bcl-2-expressing MDCK cells showed a continuous ring of peripheral staining. However, there was also a large intracellular extractable pool of ZO-1 in these cells that overlapped with Bcl-2. This codistribution was not seen for any of the other junctional markers. It is not known if ZO-1 and Bcl-2 interact, however, interactions between Bcl-2 and focal adhesion proteins paxillin and FAK have been reported during epithelial morphogenesis (Sorenson and Sheibani, 1999Go).

The absence of junctions in MDCK cells has been associated with an increased intracellular soluble pool of junctional proteins. Consistent with this observation, we found increased intracellular and extractable pools of junctional proteins in MDCK-Bcl-2 cells. In addition, similar to MCF-7B cells, Bcl-2-expressing MDCK cells had less membrane-associated E-cad. Furthermore, the decreased TER of MDCK-Bcl-2 and MCF-7B cells relative to the controls further supported the lack of tight junctions in these transfectants.

Together, the results of these studies in two independent cell lines show that Bcl-2 expression leads to decreased cell-cell adhesion. Many tumors express Bcl-2 and show reduced cell-cell adhesiveness. Disruption in cell-cell adhesion can lead to unregulated growth and increased motility and invasiveness. Although the molecular mechanism by which Bcl-2 inhibits junction formation is not yet known, the following is a possible hypothesis based on our results and those of previous studies. Initially, Bcl-2 expression inhibits membrane association of ZO-1, leading to its intracellular accumulation. The intracellular pool of ZO-1 can now bind to its cognate transcription factor ZONAB, translocate into the nucleus and upregulate the expression of ErbB2. Increased ErbB2 expression can decrease E-cad levels either directly (D'Souza and Taylor-Papadimitriou, 1994Go) and/or by phosphorylating and destabilizing the cadherincatenin complex (Jawhari et al., 1999Go). Disruption of this complex increases the level of intracellular catenins promoting their interactions with transcription factors. ß-cat-Lef/Tcf complexes have been shown to downregulate E-cad (Huber et al., 1996Go) and upregulate the expression of genes involved in proliferation [Myc, cyclin D1 (He et al., 1998Go; Shtutman et al., 1999Go)] and invasion [matrilysin (MMP-7) (Crawford et al., 1999Go)]. MMP-7 has been shown to induce cell-cell dissociation by cleaving the extracellular domain of E-cad (Davies et al., 2001Go). The decreased cell surface E-cad can ultimately lead to the inhibition of junction formation

In conclusion, there are several integrative pathways that regulate E-cad expression and function. Many tumors expressing high levels of Bcl-2 have decreased cell-cell adhesion. Bcl-2 expression in culture can also lead to unregulated growth and disruption of contact inhibition (Lu et al., 1995Go; Hakimelahi et al., 2000Go). We have presented data that show the expression of Bcl-2, an estrogen-responsive gene, leads to decreased cell-cell adhesion by reducing the level of functional E-cad. Our results provide a molecular basis for the interplay between pathways regulating cell adhesion and cell death. Furthermore, they may explain why ER+, Bcl-2-expressing breast tumors have a better prognosis because of a more favorable response to hormone withdrawal.


    Acknowledgments
 
We thank Drs David Andrews, Michele Barry and Bruce Stevenson for providing reagents and Honey Chan for assistance in electron microscopy. We are grateful to Drs Ellen Shibuya and Walter Dixon for their critical review of the manuscript. This work was supported by the Canadian Institutes of Health Research (CIHR) and Cancer Research Society


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 Materials and Methods
 Results
 Discussion
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