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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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
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Introduction |
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E-cad expression is necessary for the formation of tight, adherens and gap
junctions and desmosomes (Gumbiner et al.,
1988). The formation of normal junctions is essential for the
integrity and function of epithelial tissues
(Yeaman et al., 1999
). Loss of
E-cad expression leads to epithelial tumorigenesis
(Perl et al.,1998
) and
disruption of the cadherincatenin complex because decreased E-cad expression
is often observed in many advanced, poorly differentiated carcinomas
(Berx and Van Roy, 2001
;
Hajra and Fearon, 2002
).
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, 2001), 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.,
1996
). High levels or aberrant patterns of Bcl-2 expression occur
in various carcinomas (Reed,
1999
; Reed et al.,
1996
).
Bcl-2 expression increases in response to estrogen, and is correlated with
estrogen receptor (ER) positivity in breast carcinomas
(Schorr et al., 1999;
Gee et al., 1994
).
Bcl-2+, ER+ breast tumors respond favorably to estrogen
withdrawal (Leek et al.,
1994
). 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., 2000
; Wolf and
Davidson, 2001). Supporting this notion, Perillo et al.
(Perillo et al., 2000
) 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.1998
).
E-cad expression is also modulated by estrogen
(Blaschuk et al., 1994;
Meng et al., 2000
;
Habermann et al., 2001
;
Malaguti and Rossini, 2002
).
In breast carcinoma cells, estrogen withdrawal or estrogen antagonists led to
increased E-cad levels (DePasquale,
1999
; Meng et al.,
2000
). 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., 1995
).
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, 2000)]. 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.
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Materials and Methods |
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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.
|
Plasmids and transfection
The expression vector BMG-Neo
(Karasuyama and Melchers,
1988) 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, 1988a)]
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, 1988b).
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., 1995a) and
examined using a Philips 410 transmission electron microscope.
Transepithelial resistance (TER) measurements
TER was measured as described previously
(Stevenson and Begg, 1994)
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 (
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.
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Results |
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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, -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.,
1987
; Sommers et al.,
1994
; Mauro et al.,
2001
; Zhu et al.,
2001
; Vizirianakis et al.,
2002
).
|
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 - 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., 1994). It is
well known that estrogen can regulate Bcl-2 expression
(Teixeira et al., 1995
). 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
-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.,
1996
). The decreased cell to cell contact in MCF-7 cells in
estrogen-depleted medium also coincided with increased motility and cell
death.
|
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 -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).
|
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 -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
-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, -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., 1997
;
Brancolini et al., 1998
;
Schmeiser et al., 1998
;
Fukuda, 1999
;
Steinhusen et al., 2000
;
Ling et al., 2001
). The
quantities of these fragments were highest at day 16, when cell numbers were
the lowest.
|
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 -cat, ß-cat, Pg, p120 and ZO-1
(Fig. 5A). However, MCF-7B
cells generally appeared to have less
-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, 2000).
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).
|
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.
|
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).
|
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,
-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., 1995a). The
TER of the MDCKNeo cells grown in normal medium (HCM, 1.8 mM calcium) was
94
xcm2 (Fig.
9). Transfer to LCM (5 µM calcium) reduced the TER to
21
xcm2 (Fig.
9 MDCK-Neo-LCM). The TER of MDCK-B1 and B2 transfectants was 62
and 49
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
xcm2
respectively, Fig. 9).
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Discussion |
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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.,
1995) 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.,
1996
; Islas et al.,
2002
; Laura et al.,
2002
). ZO-1 is a member of the membrane-associated guanylate
kinase (MAGUK) family of proteins
(Gonzalez-Mariscal et al.,
2000
). Members of this family interact with transcription factors
and regulate gene expression (Hsueh et
al., 2000
; Balda and Matter,
2000
). 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, 2000
). 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,
1994
; Alroy and Yarden,
1997
; Harari and Yarden,
2000
; Olayioye et al.,
2000
). Over expression of ErbB2 in mammary carcinomas inhibits
transcription of the E-cad gene (D'Souza
and Taylor-Papadimitriou, 1994
). 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.,
1994
; Ochiai et al.,
1994
; Kanai et al.,
1995
; Ougolkov et al.,
2000
). ErbB2 can phosphorylate ß-cat/Pg and destabilize the
cadherin-catenin complex leading to decreased intercellular adhesion
(Jawhari et al., 1999
). 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., 1996).
However, there is cross talk between factors that regulate cell proliferation
and the pathways that control apoptosis
(White, 1996
;
White, 2001
). 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.,
2000
). Bcl-2 expression is regulated by estrogen
(Pratt et al., 1998
;
Perillo et al., 2000
;
Alkayed et al., 2001
) and its
presence in breast cancers reliably predicts a favorable response to hormone
therapy (Gee et al., 1994
;
Silvestrini et al., 1996
;
Zhang et al., 1999
). Estrogen
can downregulate E-cad expression
(Blaschuk et al., 1994
;
Meng et al., 2000
;
Eger et al., 2000
;
Habermann et al., 2001
;
Malaguti and Rossini, 2002
)
whereas estrogen antagonists restore E-cad expression and function
(Bracke et al., 1994
;
Mbalaviele et al., 1996
;
Vermeulen et al., 1995
;
Meng et al., 2000
). 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.,
1994; Meng et al.,
2000
). 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.,
1985; Pasdar and Nelson,
1988a
; Pasdar and Nelson, 1989b;
Gumbiner et al., 1988
;
Shore and Nelson, 1991
;
Nathke et al., 1994
;
Hinck et al., 1994
).
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.,
1995a
). 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,
1999
).
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, 1994) and/or by phosphorylating and
destabilizing the cadherincatenin complex
(Jawhari et al., 1999
).
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., 1996
) and
upregulate the expression of genes involved in proliferation [Myc, cyclin D1
(He et al., 1998
;
Shtutman et al., 1999
)] and
invasion [matrilysin (MMP-7) (Crawford et
al., 1999
)]. MMP-7 has been shown to induce cell-cell dissociation
by cleaving the extracellular domain of E-cad
(Davies et al., 2001
). 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., 1995;
Hakimelahi et al., 2000
). 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.
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