From the Department of Surgery, Section of Urology,
University of Michigan and the University of Michigan Comprehensive
Cancer Center, Ann Arbor, Michigan 48109 and the ¶ Memorial
Sloan-Kettering Cancer Center, New York, New York 10021
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ABSTRACT |
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E-cadherin and the retinoblastoma tumor
suppressor (Rb) are traditionally associated with diverse regulatory
aspects of cell growth and differentiation. However, we have discovered
new evidence, which suggests that these proteins are functionally
linked in a physiologic pathway required for cell survival and
programmed cell death. Pharmacological activation of protein kinase C
(PKC) or inducible overexpression and activation of the Tissue development and homeostasis are dependent on a complex
microenvironment regulated by extracellular adhesion mechanisms as well
as soluble growth regulatory molecules. The
PKC1 family of
serine/threonine protein kinases represents one of the most prominent
signal transduction mechanisms activated by these environmental stimuli
(1). Historically, PKC activity has been associated with the regulation
of cell growth and differentiation; however, recent studies have
demonstrated that specific isozymes of the PKC family may regulate
apoptotic programs as well (2-4). Although PKC has become the focus of
more extensive research in the apoptosis field, a specific mechanism
has not been elucidated. A potential target of PKC action, with
important ramifications in differentiation and development, is the
cadherin family of transmembrane adhesion proteins. This hypothesis is
supported by studies in which the PKC activator,
12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to
induce redistribution of E-cadherin to membranes and premature
compaction in the mouse embryo (5). Other studies using activators and
inhibitors of PKC have also demonstrated a functional association
between PKC and cadherins in a variety of cell types (6-9); however,
the identity of PKC isozymes and their regulation of specific cadherin
targets have not been reported.
In the prostate gland and mammary gland, inter-epithelial membrane
adhesion is dependent on the homotypic interaction of E-cadherin (10,
11). Such homophilic cell-cell adhesion results in the formation of
desmosomes and adherens junctions that are required for tissue
morphogenesis and the maintenance of the differentiated phenotype (10).
The intracellular domain of E-cadherin is linked to the actin
cytoskeleton through its interaction with the cytoplasmic adapter
proteins Homeostasis of the prostate and mammary glands is dependent upon
androgenic and estrogenic steroids, respectively. Depletion of these
hormones activates a series of poorly defined molecular events leading
to involution and remodeling of the tissue resulting from apoptotic
death of the luminal epithelium. Although critical for tissue
homeostasis, steroid hormones may not be exclusive to the regulation of
cell survival since suppression of epithelial apoptosis also depends on
their homotypic placement with adjacent epithelial cells and their
attachment to the extracellular matrix. The first visible stage of
prostate and mammary involution is the disruption of intercellular
adhesion and anchorage prior to the onset of apoptosis (17). Thus,
disruption of basal and lateral adhesion is often associated with
apoptosis in involuting tissues (17, 18). Previous studies have
demonstrated that anchorage-dependent epithelium will
undergo apoptosis following loss of integrin contact with the
extracellular matrix (19, 20) or inhibition of integrin-mediated organoid formation (21). It is now believed that just as integrins function to mediate cell-extracellular matrix interactions in anchorage-dependent survival, cadherins may also act in
such a capacity, possessing a functional role in the regulation of
intercellular adhesion-dependent survival. Several studies
have reported the association between N-cadherin-mediated intercellular
adhesion and survival of gut and ovarian epithelium (22, 23), and
recently an association between E-cadherin-mediated aggregation and
survival of oral squamous carcinoma cells has been reported (24). These studies suggested that homophilic binding of cadherin molecules on
adjacent cells could transduce apoptotic suppressive signals; however,
the actual mechanism by which cadherins mediate these signals is not known.
We have previously demonstrated that integrin-mediated survival of
prostate epithelial cells was uniquely regulated through the Rb cell
cycle control pathway (25). We have also investigated the functional
role of PKC in the regulation of epithelial apoptosis, and we have
demonstrated that PKC signal transduction, like integrin-regulated mechanisms, is capable of recruiting a Rb-regulated apoptotic pathway
in prostate epithelial cells (3). In many cell types, apoptosis is
thought to be a default pathway occurring where opposing or conflicting
cell proliferation signals arise. For example, in the absence of serum
growth factors, overexpression of c-Myc can initiate such a conflict by
promoting the aberrant entry of cells into cycle (26). We have found
that such a conflict arises when Rb growth-suppressive activity opposes
mitogenic signals in proliferating cells. In support of this
possibility, we found that transient expression of a constitutively
active form of Rb induced apoptosis and that the inhibition of Rb
function, by transfection of the Rb inhibitory oncogene, E1a,
suppressed apoptosis (3, 25). These findings are in stark contrast with
studies in which homozygous deletion of the RB1 gene
resulted in increased apoptosis in mouse cells, suggesting an
apoptotic-suppressive role for Rb (27-30). A possible explanation for
this discrepancy is that Rb may influence both outcomes and participate
in both cell survival and cell death.
The adhesion molecules that mediate intercellular and extracellular
matrix contact (31) largely regulate the normal growth of cells. Thus,
an inadequate cell adhesion system, in which cells can easily
dissociate from the primary tumor, may be a key determinant in the
metastatic progression of certain cancers. In tumors of epithelial
origin, the disruption of cellular adhesion appears to arise through
alterations in the E-cadherin/ Herein, we describe a novel cell survival mechanism initiated by PKC in
prostate and mammary epithelial cells in which E-cadherin-mediated aggregation resulted in Rb activation and G1 arrest
necessary for survival. However, non-aggregated cells, which do not
arrest in G1, undergo apoptosis, which we believe results
from a fatal cell cycle conflict that occurs in S phase cells which
contain hypophosphorylated Rb. Additionally, these findings are
supported by the observation that the loss of membrane E-cadherin and
subsequent hypophosphorylation of Rb in luminal epithelial cells
occur in the involuting prostate gland.
Cell Culture Reagents and Transfections--
The cell line LNCaP
(ATCC) was propagated in RPMI 1640 medium supplemented with 10% fetal
calf serum, 2 mM L-glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). The
cells were kept at 37 °C in a humidified atmosphere of 5%
CO2 and subcultured weekly. The LN E-cadherin Blocking Experiments--
For the E-cadherin blocking
antibody studies, LNCaP cells were plated on 5 µg/ml
fibronectin-coated tissue culture plates, and the SUM185 cells were
plated on poly-D-lysine-coated tissue culture plates
(Collaborative Biomedical Products), and all cells were allowed to
attach for 24-48 h prior to experimentation. LNCaP and SUM185 cells
were identically plated, and the cultures were 50% confluent prior to
treatment. For both cell lines, the E-cadherin blocking antibody
(Zymed Laboratories Inc., catalog number 13-5700) and
the IgG2a antibody isotype control (PharMingen, catalog number 03020D)
were added to the cultures 1-h prior to the addition of TPA. Both
antibodies were used at a concentration of 60 µg/ml in culture. Cell
viability was followed by trypan blue exclusion (Life Technologies,
Inc.) or by the colorimetric
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay (Promega).
Experimental Cell Culture Plating and Density--
LNCaP,
LN Western Blot and Protein Analysis--
Expression of E-cadherin
and Rb proteins was determined by Western blot analysis using the
following antibodies: anti-human E-cadherin (Zymed
Laboratories Inc., catalog number 13-1700), anti-rat E-cadherin
(Transduction Laboratories, catalog number C20820), anti-Rb
(PharMingen, catalog number 14001A), and anti-human E1a (Calbiochem,
catalog number DP11). The donkey anti-mouse peroxidase-conjugated IgG
(Amresco, catalog number E974) was used as a secondary antibody for all
monoclonal antibodies described here. For protein extraction, tissue
culture cells were lysed in 50 mM Tris at pH 7.5, 120 mM NaCl, 0.5% Nonidet P-40 with the following protease
inhibitors: 40 µM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 200 µM
orthovanadate. The cells were allowed to lyse on ice for 1 h; the
resulting lysates were centrifuged, and the supernatants were collected
and quantitated. Rat prostate lysates were prepared from frozen tissue
as described below in a buffer containing 1% Nonidet P-40, 0.5%
sodium deoxycholate, 0.1% SDS in phosphate-buffered saline with the
following protease inhibitors: 40 µM phenylmethylsulfonyl
fluoride, 5 µg/ml leupeptin, 50 µg/ml aprotinin, and 200 µM orthovanadate. Protein lysates were quantitated using
a Bradford assay, separated by 6% Tris/glycine precast Novex gels, and
analyzed under denaturing conditions using the Novex and ECL (Amersham
Pharmacia Biotech) detection systems as described previously (3).
Flow Cytometry Analysis--
LNCaP cells (log phase cells) were
grown and PKC-activated as previously reported (3). Cell cycle analysis
of asynchronous LNCaP cultures treated with TPA over a 48-h time course
was accomplished utilizing propidium iodide staining and multicycle
analysis. Cultures were harvested, fixed with 70% EtOH, stained in a
solution containing 5 µg/ml propidium iodide, 50 µg/ml Range A,
phosphate-buffered saline, and analyzed by flow cytometry with
multicycle (see below). For experiments involving growth arrest through
high density contact inhibition, LNCaP cells were plated at higher
density and retained in culture for 5 days. For experiments
manipulating late G1/S cell cycle arrest, subconfluent
LNCaP cultures (2 days after plating) were treated for 3 days with 2 mM hydroxyurea (Sigma). Hydroxyurea cultures were released
from the block with hydroxyurea-free medium 5 h prior to PKC
activation. LNCaP cells were prepared at the times indicated and
stained with propidium iodide and Hoechst for flow cytometry as
described (37, 38). Flow cytometry was done at the University of
Michigan flow cytometry facility using a Coulter ELITE cell sorter
(Beckman-Couler, Miami, FL) equipped with a 5-watt UV-capable laser.
Analysis regions were set to include viable, dead, and apoptotic cell
populations, based on untreated control samples. Data, acquired to 105 events per sample, were analyzed using Coulter's ELITE software. DNA
histograms resulting from the H342 staining were analyzed using
algorithms available in MultiCycle software (Phoenix Flow Systems, San
Diego, CA) to estimate G1, S, and
G2M compartment percentages.
Rat Castration and Histology--
250-g male Sprague-Dawley rats
were castrated at the indicated times, and the ventral prostate was
excised and immediately frozen in liquid nitrogen. Immunohistochemical
studies were performed on 3-µm serial frozen sections fixed in 100%
methanol and incubated with the rat anti-E-cadherin antibody
(Transduction Laboratories, catalog number C20820) diluted 1:40,000.
The secondary antibody was a biotinylated horse anti-mouse (Vector
Laboratories, catalog number BA-2001) conjugated with peroxidase and
was used at a 1:200 dilution. The sections were counterstained with
hematoxylin, dehydrated, and mounted. For direct comparison of
E-cadherin expression and TUNEL analysis, the TUNEL assay was performed
as described by the manufacturer (Apoptag kit, Oncor) in the subsequent
sections. In this fashion, alternating sections were used for
immunohistochemistry and TUNEL analysis. The sections were examined
at × 1000 magnification.
Activation of PKC Results in Apoptosis and Aggregation-associated
Survival of Prostate and Breast Epithelial Cells--
We have
previously shown that prolonged activation of PKC recruited a novel
apoptotic pathway in prostate epithelial cells (2, 39). We examined PKC
isozyme specificity of this mechanism in detail using a
tetracycline-regulated expression system in which the E-cadherin Mediates Aggregation-dependent Survival of
Prostate and Mammary Epithelial Cells--
The observation that cell
survival was closely associated with increased aggregation suggested a
regulatory role for E-cadherin in survival of prostate and mammary
epithelial cells. To begin to evaluate the functional role of
E-cadherin in epithelial aggregation and survival, we examined the
expression of E-cadherin protein in aggregated and non-aggregated
populations of both prostate and mammary epithelial cells following
activation of PKC. Protein extracts from the aggregated population of
LNCaP cells and SUM185 cells were analyzed for E-cadherin expression
using an E-cadherin-specific monoclonal antibody (Fig.
3). Immunoblotting revealed strong, yet
transient induction of E-cadherin protein post-PKC activation in both
cell lines. Induction of E-cadherin was first detected by 6 h,
which coincided precisely with the onset of cellular aggregation. This
finding suggested that E-cadherin-mediated aggregation is necessary for
survival following apoptotic stimuli. We postulated, however, that a
perturbation to E-cadherin-mediated adhesion must occur in the
non-aggregated population as dissociated or individual cells were very
rarely observed in treated cultures. Therefore, we examined the
expression of E-cadherin in the non-aggregated population. Due to
decreased basal adhesion, the non-aggregated cells were separated from
aggregated cells as suspended cells. In this non-aggregated population,
we found very little expression of E-cadherin at the 48-h time point,
which correlated with a dramatic reduction in cell viability (Fig. 3).
In comparison, E-cadherin expression continued to increase in the
aggregated populations through 48 h without loss of viability.
Increased aggregation concurrent with E-cadherin induction strongly
suggested that E-cadherin was mediating the survival of prostate and
breast epithelial cells. To establish a functional role for E-cadherin
in intercellular adhesion and cell survival, we attempted to disrupt
PKC-induced aggregation in prostate and mammary epithelial cells with
an E-cadherin-blocking antibody. In the presence of the blocking
antibody, aggregation was completely inhibited, and the cells underwent
synergistic cell death with more than a 3-fold increase in apoptosis as
compared with minus antibody and isotype controls (Fig.
4, A and B).
Although more diffuse and somewhat rounded by treatment with the
blocking antibody, the LNCaP and SUM185 cells remained viable and
continued to proliferate.
The observation that PKC activation resulted in morphologic changes, in
addition to E-cadherin accumulation, suggested that the surviving,
aggregated cells had undergone dramatic phenotypic alterations.
However, the fact that PKC activation also resulted in apoptosis of
non-aggregated cells was supportive evidence that PKC recruits a
pathway, which leads to either survival in an aggregated state or leads
to apoptosis in non-aggregated cells. To confirm that these events were
regulated through a common PKC mechanism, we examined the ability of
staurosporine, a potent catalytic inhibitor of serine/threonine
kinases, including PKC, to inhibit these changes. Not only did
staurosporine prevent the changes in cell morphology and aggregation
(Fig. 2, c and f), it also inhibited E-cadherin induction (Fig. 3B) and apoptosis (Figs. 1 and 2,
c and f).
E-cadherin-mediated Aggregation Results in G1 Growth
Arrest and Survival--
We had previously demonstrated that the Rb
cell cycle control mechanism plays a crucial role in regulating
apoptosis induced by anchorage disruption (25). In the current study,
we have shown that the survival of mammary and prostate epithelial
cells following PKC activation appears to be dependent on cellular
aggregation. In the presence of the E-cadherin-blocking antibody,
aggregation of prostate and mammary epithelial cells was inhibited, and
the cells were hypersensitive to PKC-induced apoptosis. We postulated that E-cadherin-dependent aggregation, similar to
integrin-mediated anchorage, might mediate survival by influencing Rb
activity and cell cycle regulation as well. To determine if a
functional link existed between E-cadherin and Rb, we first examined
the cell cycle profile of LNCaP cells at the onset of PKC activation (0 h) as well as cell viability and Rb phosphorylation in four TPA-treated populations during a 48-h TPA time course (Fig.
5). TPA treatment of subconfluent, log
phase LNCaP cells resulted in complete Rb hypophosphorylation within
24 h and 53% apoptosis by 48 h. However, TPA treatment of
log phase cultures results in two distinct populations as follows:
aggregated surviving cells and non-aggregated apoptotic cells, both of
which contained hypophosphorylated Rb. How could such divergent
effects, survival of aggregated cells and apoptosis of dissociated
cells, be explained by Rb activation? When we specifically examined the
aggregated population at the 48-h time point, we found that these cells
expressed only hypophosphorylated Rb and had accumulated in
G1 but were completely viable, suggesting that contact-induced G1 arrest facilitated survival. This was
confirmed by the finding that when a subconfluent culture was grown to
confluence and arrested in G1 prior to PKC activation, the
culture exhibited only a 3% reduction in viability following 48 h
of TPA exposure (Fig. 5). In support of this observation, we found that
cells cultured at subconfluent densities and arrested in G1
by serum starvation also contained the hypophosphorylated form of Rb
prior to PKC activation and were resistant to apoptosis (Fig. 5). The finding that aggregated cells were growth-arrested suggested that aggregation-mediated survival may depend on the ability of cells to
initiate Rb-dependent G1 arrest. To help
confirm the role of E-cadherin-mediated adhesion in Rb and cell cycle
regulation, we examined cell cycle profile at the time of PKC
activation (0 h). We also determined cell viability and Rb
phosphorylation in cultures plated at the same density as the confluent
cultures but were pretreated with the E-cadherin-blocking antibody
(Fig. 5). The blocking antibody inhibited PKC-induced aggregation when added to subconfluent cultures. Immunoblot and flow cytometric analysis
revealed that these dissociated cells contained the inactive, hyperphosphorylated form of Rb and were in log phase growth prior to TPA treatment (0 h). Addition of TPA to non-aggregated cells resulted in the conversion of Rb to the hypophosphorylated form within
24 h and resulted in 87% apoptosis at 48 h. This result was
in complete contrast to the slight apoptosis (3%) observed in the
aggregated population at the same time.
Apoptosis Results from a Fatal Growth Signal Conflict in S Phase
Cells--
Our results demonstrated that aggregated,
G1-arrested cells were resistant to TPA-induced apoptosis,
whereas cycling cells remained highly sensitive. We next attempted to
determine what phase of the cell cycle that TPA-treated cells were
susceptible to apoptosis. Propidium iodide staining of asynchronous
LNCaP cells treated with TPA resulted in a 3-fold reduction in S phase (Fig. 6A). To confirm that
apoptosis was actually occurring in S phase, we examined the
susceptibility of synchronized S phase cultures to TPA-induced cell
death utilizing Hoechst and propidium iodide staining and flow
cytometry (Fig. 6B). When PKC was activated in cells
synchronized at the G1/S border and released into S phase for 5 h, we observed an extremely rapid decline in cell viability that was reduced to 41% by only 8 h and 13 and 6% at 24 and
48 h, respectively (Fig. 6B and data not shown). We
have previously postulated that PKC-induced apoptosis resulted from a
conflict between Rb growth-suppressive signals in opposition to
growth-promoting signals in proliferating epithelial cells (3).
However, mechanistic details of this conflict were not elucidated.
Because of the dramatic apoptosis that is induced in S phase cultures,
we speculated that the hypophosphorylation of Rb as cells enter S phase
might manifest such an apoptotic conflict. Surprisingly, TPA treatment
not only resulted in Rb hypophosphorylation in the S phase cells, but
it occurred with kinetics correlating precisely with cell death. Following PKC activation, approximately 50% of Rb protein was dephosphorylated by 8 h, which correlated with 59% apoptosis at this time. Total conversion to the active conformation was complete by
24 h which correlated to 87% reduction in viability, which was
2-fold greater than the levels of apoptosis seen in asynchronous cells
(Fig. 5).
To determine whether functional inhibition of endogenous Rb could
suppress S phase apoptosis, we overexpressed the Rb inhibitory oncogene, E1a, in LNCaP cells. As shown in Fig.
6C, E1a-expressing LNCaP cells did not undergo
apoptosis after addition of TPA. Because E1a has cellular
targets in addition to Rb, we used a control E1a expression
vector with a mutation at nucleotide 928 that blocks interaction with
Rb without disrupting interactions with the other proteins. In contrast
to wild type E1a, the Rb-specific E1a point mutant (E1a-928) did not block apoptosis in TPA-treated
LNCaP cells, suggesting that the S phase anti-apoptotic activity of E1a results from inhibition of Rb function alone.
Loss of E-cadherin and Rb Activation Are Early Events of Prostate
Involution--
To determine if E-cadherin plays a role in
contact-dependent survival of epithelial cells in
vivo, we examined the expression of E-cadherin in the rat ventral
prostate following castration. The glandular epithelium constitutes
approximately 85% of the cells in the ventral prostate. Nearly 80% of
these cells undergo apoptosis within 7 days following castration (40,
41). After castration, apoptotic cells begin to appear at 3 days, with
the maximum number of apoptotic cells being observed by day 4 (25). This apoptotic phase, during prostate involution, is complete by day 7. Immunoblot analysis of E-cadherin protein in the rat ventral prostate
following castration revealed a dramatic reduction of E-cadherin
expression between 24 and 48 h which was followed by the
appearance of apoptotic cells at 72 h (Fig.
7). Immunohistochemical analysis
demonstrated that E-cadherin was localized exclusively to the
junctional membranes of the luminal epithelium of intact animals. By
comparison, the 72-h castrates exhibited a dramatic reduction in
membrane staining to nearly undetectable levels. TUNEL analysis of
serial sections revealed that apoptotic cells were not observed in the
intact animal but were prominent throughout the luminal epithelium in
the 72-h castrates. TUNEL staining was not observed in cells exhibiting
strong membrane E-cadherin immunoreactivity in the castrates. We have
previously reported increased expression of Rb immunoreactivity and
mRNA exclusively in the glandular epithelium during prostate
involution (25, 42); however, the accumulation of Rb mRNA and
protein is only suggestive of Rb activity in this process. To further
associate Rb activity with prostate involution, we examined the
phosphorylation state of Rb following castration. Immunoblot analysis
revealed that the Rb protein immunopurified from the 48-h castrate
co-migrated with the control 105-kDa hypophosphorylated Rb isolated
from cycling LNCaP cells. The appearance of hypophosphorylated Rb was
concurrent with the loss of membrane E-cadherin expression at 48 h.
E-cadherin is essential for the formation of intercellular
junctional complexes and the establishment of cell polarity, and its
role in epithelial differentiation and maintenance of tissue integrity
is well established (10, 43). In the current study, we have
demonstrated that E-cadherin functions in a novel
adhesion-dependent survival pathway that can suppress
apoptosis of prostate and mammary epithelial cells. Our observation
that PKC activation resulted in the rapid accumulation of E-cadherin
and aggregation suggested that PKC initiated a phenotypic program in
surviving prostate and mammary epithelial cells. Regardless of the
phenotypic outcome, the observation that staurosporine inhibited all
morphologic and molecular changes induced by TPA suggested that the
fate of these cells, whether survival in an aggregated state or death
of non-aggregated cells, had vastly divergent outcomes originating from
a common PKC signal.
Following PKC activation we found that the surviving population of
aggregated cells contained hypophosphorylated Rb, were growth-arrested,
and were resistant to apoptosis. These results strongly suggested that
E-cadherin-mediated survival is achieved through Rb-mediated
G1 arrest and precluded apoptosis. By inhibiting aggregation with an E-cadherin-blocking antibody, we found that the
cells contained hyperphosphorylated Rb and remained in logarithmic growth at the time of PKC activation. Following activation of PKC,
these non-aggregated cells rapidly converted Rb to the active, hypophosphorylated form that preceded a 3.5-fold increase in apoptosis. This result suggested that Rb activity was involved in signaling apoptosis in cycling cells. If aggregated, G1-arrested
cells are able to suppress apoptosis, then in what phase of the cycle
are cells susceptible to apoptotic signals? We determined that
PKC-induced apoptosis occurred in the S phase of the cell cycle. This
was demonstrated in two experiments in which we observed a dramatic reduction in viable S phase cells from asynchronous cultures and almost
complete loss of cell viability in synchronized S phase cultures.
The functional role of Rb is traditionally associated with cell cycle
regulation; however, recent studies have accumulated significant data
linking Rb activity with programmed cell death (44-46). We have
previously demonstrated a functional role for Rb in signaling apoptosis
of epithelial cells induced by anchorage disruption or inducible
overexpression and activation of PKC Short term PKC activity is necessary for the regulation of cell growth
and differentiation (1). However, sustained activation of PKC is
thought to have oncogenic activity in various types of adenocarcinomas
(47). E-cadherin, whose function is critical for epithelial
differentiation, has also been implicated in tumorigenesis, specifically as a metastasis suppressor protein (48). Supporting this
hypothesis is the observation that the loss of E-cadherin is a common
event in advanced adenocarcinoma of the prostate (34). In the mammary
gland the same trend is prevalent; however, late stage or metastatic
tumors exhibit some dependence on intercellular adhesion suggesting
that the loss or gain of cell-cell adhesion during tumor progression is
a regulated and dynamic process (20, 36). Some of the genetic events
that occur in prostate tumorigenesis involve alterations in cell death
programs present in the normal, androgen-dependent prostate
epithelium. One of the most common genetic alterations associated with
prostate cancer is allelic loss at the RB locus as reported in 27-67%
of prostate tumors examined (49-51); however, the pathobiological role
of Rb in this disease has not been elucidated.
Tumor suppression is achieved not only by cell cycle arrest but by the
initiation of cell death programs as well. The role of the p53 tumor
suppressor gene product in apoptosis has been extensively documented in
cells that are responding to DNA-damaging agents, chemotherapeutic
agents, or in cells that have a deregulated cell cycle (reviewed in
Ref. 1). Although Rb has been shown to inhibit multiple
p53-dependent apoptotic pathways (1, 23), accumulation of
hypophosphorylated Rb leading to G1 arrest and apoptosis
occurs in multiple p53-independent pathways as well (36, 37). Two such
p53-independent pathways are induced by release of the lipid second
messenger ceramide (38, 39) or by DNA-damaging agents (40), both of
which result in the accumulation of hypophosphorylated Rb,
G1 arrest, and apoptosis. We have examined the contribution
of p53 to apoptosis in the LNCaP model and have found that although p53
protein is rapidly induced 30-40-fold over basal expression following
UV irradiation, there is absolutely no induction of p53 following PKC
activation.3 Coupled to the
observation that apoptosis occurs normally in androgen-dependent prostate epithelium of p53( Hormone ablation, by surgical or pharmacological castration, induces
rapid involution of the prostate gland. This process involves extensive
remodeling of the ductal architecture that regresses to an atrophic or
underdeveloped state. The predominance of apoptotic cells observed in
the luminal compartment 3-4 days following castration indicates that
this regression results from programmed cell death of the luminal
epithelium. The first visible stage of individual cell death is the
loss of intercellular adhesion as the desmosomal contacts between the
epithelial cells are lost prior to cytoplasmic and nuclear condensation
(17). We investigated whether an E-cadherin/Rb-regulated pathway was
activated in the prostate gland following castration. Examination of
E-cadherin expression and its cellular distribution following
castration revealed a dramatic reduction in the junctional membranes
prior to apoptosis. Previous studies have reported that the levels
of prostatic cyclin D1 and E are reduced following castration (40), suggesting that G1 cyclin-dependent kinase
activity is diminished and that Rb is likely in the hypophosphorylated
form. This observation that Rb was hypophosphorylated subsequent to the
loss of E-cadherin and coincidental to the onset of apoptosis
during involution of the prostate gland suggested that androgen
ablation signals the same apoptotic pathway in epithelial cells as was
observed in vitro.
Considering the coordinated regulation between proliferation and cell
death that is required for normal growth, it is not surprising that
apoptosis is a fundamental component of numerous developmental programs
(52). Therefore, it is feasible that cell survival and cell death could
be regulated through common pathways. However, a mechanism that
controls such divergent outcomes has not previously been described.
Here, we present evidence that prostate and mammary epithelial cells
utilize a mechanism in which E-cadherin and Rb are linked in a novel,
regulatory pathway that is required for cell survival following PKC
activation and in resting prostate tissue. In vitro,
PKC-induced hypophosphorylation of Rb occurs in cells that are randomly
proceeding through cycle and, although providing a survival signal in
cells that are able to aggregate and arrest in G1, also
signals an apoptotic conflict in non-aggregated, S phase cells (Fig.
8).
isozyme of PKC (PKC
) resulted in approximately 60% apoptosis of mammary and
prostate epithelial cells. Interestingly, the surviving cells had
undergone dramatic aggregation concurrent with increased E-cadherin expression. When aggregation was inhibited by the addition of an
E-cadherin-blocking antibody, apoptosis increased synergistically. We
hypothesized that survival of the aggregated population was associated
with contact-inhibited growth and that apoptosis might result
from aberrant growth regulatory signals in non-aggregated, cycling cells. This hypothesis was confirmed by experiments that demonstrated that E-cadherin-dependent aggregation resulted in Rb-mediated G1 arrest and survival. Immunoblot
analysis and flow cytometry revealed that hypophosphorylated Rb was
present in non-aggregated, S phase cultures concurrent with synergistic
cell death. We have also determined that the loss of membrane
E-cadherin and subsequent hypophosphorylation of Rb in luminal
epithelial cells preceded apoptosis induced by castration. These
findings provide compelling evidence that suggests that
E-cadherin-mediated aggregation results in Rb activation and
G1 arrest that is critical for survival of prostate and
mammary epithelial cells. These data also indicate that Rb can initiate
a fatal growth signal conflict in non-aggregated, cycling cells when
the protein is hypophosphorylated as these epithelial cells enter S phase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-,
-, and
-catenin/plakoglobin (12-14), which is
essential for intercellular adhesion. Although central to the cell
adhesion mechanism, E-cadherin has also been implicated in physiologic
roles beyond the mere mechanical interconnection of cells. More recent
evidence suggests that E-cadherin may also be associated with
regulatory pathways involved in various aspects of cell fate including
developmental decisions, cellular differentiation, and possibly cell
survival (15, 16).
,
-catenin/adenomatous polyposis
coli system, and as such, the loss of growth control may not be due to
growth-promoting mutations but by the escape from apoptotic mechanisms
(31, 32). Accordingly, E-cadherin expression is often reduced in more
advanced prostate tumors and lobular mammary tumors, supporting growing
speculation about the role of E-cadherin as a metastasis-suppressor
protein in these cancers (33-36).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
17, a clone of LNCaP transfected with PKC
under control of the tetracycline-repressible promoter, was propagated as described (3). The human epithelial breast
cancer cell line, SUM185 was grown in Ham's F-12 supplemented with 5%
fetal bovine serum, 5 µg/ml insulin, 1 mg/ml hydrocortisone, gentamycin, and fungizone. SUM185 PE is one of 12 breast cancer cell
lines developed in the laboratory of Dr. Stephen
Ethier.2 Bryostatin 1, staurosporine, and TPA (LC Laboratories, catalog numbers B-6697,
S-8451, and P-1680, respectively) were dissolved in 100% ethanol,
aliquoted, and stored at
20 °C. Bryostatin 1 and TPA were used at
a final concentration of 10 nM. Staurosporine was used at
50 nM concentration as a pretreatment 90 min prior to PKC activation.
17, and SUM185 log phase cells were plated and allowed to attach
to the tissue culture flask for 48 h, at which time the cells were
at 50% confluence (subconfluent) prior to PKC activation for all
experiments unless otherwise described. For confluent cultures and
subconfluent plus E-cadherin-blocking antibody cultures (Fig. 6), log
phase cells were plated out at a higher density so that 48 h
post-plating the cells were at 70% confluence. At this point one
culture was given the blocking antibody and the other allowed to grow
to 100% confluence. Both cultures were then treated with 10 nM TPA and harvested for viability and protein extraction
48 h later.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
member of the
PKC family (PKC
) was expressed 40-fold over basal levels in the
parental prostate epithelial line, LNCaP (3). In this earlier study, we
demonstrated that activation of exogenous PKC
in the transfected
clones resulted in dramatic morphological changes and subsequent
apoptosis. Identical results were obtained in this study by using the
PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), not only in the parental LNCaP cells but also in the human breast epithelial cell line, SUM185. Treatment of LNCaP and SUM185 cells with TPA resulted in approximately half of the cells undergoing apoptosis. Interestingly, the extent of cell survival correlated with
increasing cell density such that high density cultures were more
resistant to PKC-induced apoptosis (Fig.
1). This density-associated survival
could possibly be explained by the observation that all surviving cells
had undergone dramatic aggregation (Fig.
2, a, b, and
d, e). The association of survival with high
density cultures suggested that cells, which were in close proximity at
the time of PKC activation, were more likely to aggregate and
survive.
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Fig. 1.
Effect of culture density on cell survival
following PKC activation. LNCaP cells were plated in 96-well
microtiter dishes at the indicated densities. Cells were treated with
10 nM TPA ( ) or pretreated for 90 min with 50 nM staurosporine prior to TPA treatment
(STS/TPA,
), and viability was obtained 48 h later
by the
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt assay. The data are presented as an average and S.D.
of quadruplicate counts expressed as a percentage of untreated
control.
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Fig. 2.
PKC activation results in cellular
aggregation of prostate and breast epithelial cells.
Photomicrographs were taken of LNCaP prostate cells (a-c)
and of SUM185 breast cells (d-f) as follows: untreated
(a and d), treated with 10 nM TPA for
6 h (b and e), or pretreated for 90 min with
50 nM staurosporine (c and f) prior
to TPA treatment, and viability was obtained 48 h later by
the
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt assay. The data were presented as an average and S.D.
of quadruplicate counts expressed as a percentage of untreated control.
Magnification × 200.
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Fig. 3.
Accumulation of E-cadherin is associated with
aggregation of prostate and breast epithelial cells. Protein
extracts were analyzed by immunoblot analysis employing an
E-cadherin-specific monoclonal antibody. A, protein extracts
were obtained at the indicated times from aggregated or non-aggregated
PKC -expressing LN
17 prostate cells treated with 10 nM
bryostatin 1 and from SUM185 breast cells treated with 10 nM TPA (collectively labeled as PKC).
B, identical cultures were also pretreated for 90 min with
50 nM staurosporine prior to PKC activation
(STS/PKC). The percent viable cell number was measured by
trypan blue exclusion at 24 and 48 h post-PKC activation and is
presented as an average of triplicate cell counts.
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Fig. 4.
An E-cadherin -blocking antibody inhibits
PKC-induced aggregation and results in synergistic apoptosis of
prostate and breast epithelial cells. Prior to PKC activation
LNCaP prostate (A) and SUM185 breast cells (B)
were not treated with antibody ( Ab), were pretreated for
1 h with 60 µg/ml of an anti-E-cadherin antibody
(
E), or were pretreated with 60 µg/ml IgG2a isotype
control (
2a) in untreated (
TPA) LNCaP cells
(a, c, and e) and SUM185 cells
(a, c, and e) or in TPA-treated
(+TPA) LNCaP cells (b, d, and
f) and SUM185 cells (b, d, and
f). 48 h later viable cell number was obtained by
trypan blue exclusion and represents the average and S.D. of duplicate
or triplicate cell counts. Magnification × 200.
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Fig. 5.
Apoptosis occurs in subconfluent, cycling
cells but is suppressed in contact-inhibited and serum-starved
G1-arrested cells. Cell cycle analysis of
G1, S, and G2/M was performed using
propidium iodide/Hoechst staining and multicycle analysis of cells just
prior to PKC activation (cell cycle 0 h) in four types of cultures
plated at the same density (70% confluence 24 h after plating in
serum-containing medium). The cells were then treated with 10 nM TPA as a subconfluent culture (Subconfluent)
or treated with 10 nM TPA following growth to 100%
confluence (Contact-inhibited) or treated with 10 nM TPA following 5 days in serum-free medium
(Serum-starved) or pretreated as a subconfluent culture with
the E-cadherin-blocking antibody prior to treatment with 10 nM TPA ( -E-cadherin). Whole cell protein
extracts were prepared at the indicated times following PKC activation
and were analyzed by immunoblotting employing an Rb-specific monoclonal
antibody. Viable cell number was measured by trypan blue exclusion
expressed as a percentage of untreated control.
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Fig. 6.
PKC-induced apoptosis occurs in S phase and
is associated with rapid hypophosphorylation of Rb. A,
multicycle analysis of asynchronous untreated ( , control) and
TPA-treated (
, TPA) LNCaP cells at the indicated times.
B, LNCaP cells were released from hydroxyurea-induced
G1/S block for 5 h prior to TPA treatment at the
indicated times. Cell cycle was analyzed by multicycle analysis
of propidium iodide/Hoechst-stained cells just prior to PKC
activation (Cell cycle 0 h). Protein extracts were prepared
from whole cultures at the indicated times following PKC activation and
analyzed by immunoblotting employing an Rb-specific monoclonal
antibody. C, TPA-induced apoptosis is blocked through the
functional inhibition of Rb by transfection of E1a into
LNCaP cells. For each, 1 × 106 cells were plated for
48 h and then treated with 10 nM TPA. Cultures of
untransfected control (LNCaP Parental) or transfected,
nonfunctional E1a (928 E1a) or transfected,
functional E1a (wt E1a) were harvested 48 h
post-TPA treatment. Protein lysates from whole cultures were prepared,
quantitated, and subjected to Western analysis employing an
E1a-specific antibody. Viable cell number was measured by
trypan blue exclusion and represents the average and S.D. of triplicate
cell counts as compared with parallel untreated controls.
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Fig. 7.
Loss of membrane E-cadherin
correlates with Rb hypophosphorylation and apoptosis in the luminal
epithelium of the prostate following castration. A,
whole prostate protein extracts were prepared from an intact animal and
from castrates at the indicated times and analyzed by immunoblot
employing a rat E-cadherin-specific monoclonal antibody
(E-cad) or by immunoprecipitation and immunoblotting using
Rb-specific antibodies (Rb). The immunoprecipitation control
distinguishes the hyperphosphorylated (110 kDa) and the
hypophosphorylated (105 kDa) species from cycling LNCaP
cells for molecular weight comparison. B, 3 µm serial
sections of rat ventral prostate gland were obtained from intact
animals (a and b) and 72-h castrates
(c and d). Immunohistochemical analysis was
performed on a single section from the intact (a) and 72-h
castrate (c) using a rat E-cadherin-specific monoclonal
antibody. The succeeding 3-µm sections from the intact animal
(b) and from the 72-h castrate (d) were subjected
to TUNEL analysis. Arrows indicate individual apoptotic
cells (d), and the corresponding E-cadherin membrane
staining from the previous 3-µm section (c).
Magnification × 1000.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(3, 25). In the latter study,
we demonstrated that Rb-signaled apoptosis was executed through the
caspase family of cysteine proteases (3); however, the exact mechanism
by which Rb initiates this apoptotic response was unclear. Since a
variety of regulatory signals from the extracellular environment are
critically important in determining cell fate, we and others (26) have
hypothesized that opposing signals can arise in some cells, such as
forced c-Myc expression in fibroblasts in the absence of serum growth factors, resulting in dysfunctional cell cycle and apoptosis. A similar
conflict occurs in our model, one in which Rb activation provides a
growth inhibitory signal in direct opposition to growth-promoting signals in proliferating cells. In support of this possibility we found
previously that when this conflict was prevented by eliminating Rb
function in Rb
/
cells or by transfection with the Rb inhibitory oncogene, E1a, apoptosis did not occur (see Ref. 25 and Fig. 6C). Although many aspects of PKC regulation of cellular
differentiation and development are known, the mechanism by which PKC
induces apoptosis in epithelial cells is not completely understood. The theory that defective developmental programs may precipitate some forms
of apoptosis substantiates our observations that the rapid hypophosphorylation of Rb in S phase cells does not result in G1 arrest but leads to a fatal conflict of growth
regulatory signals. These findings may begin to address
inconsistencies surrounding the functional role of Rb in the
regulation of programmed cell death. One view, strongly supported by RB
knock-out studies, suggests that Rb may play a protective role in
developing tissue by suppressing apoptotic programs (27-30). We
believe that Rb also has the capacity to initiate an apoptotic program,
which results from conflicting growth regulatory signals in the cell.
The results of this study may provide an explanation as to how Rb can
regulate cell survival through the induction of G1 arrest.
Alternatively, our results also suggest that Rb can initiate a cell
death program when it is hypophosphorylated as cells enter S phase.
/
) mice
following castration (41) indicated that apoptosis in prostate
epithelium is a p53-independent process. The ability of epithelial
cells to survive and proliferate in the absence of extracellular
contact is critical for tumor progression. We postulate that E-cadherin functions to monitor extracellular contact, which during
differentiation or tumorigenesis may control contact-independent growth
of autonomous cells by inducing apoptosis. Thus, the inactivation of
the E-cadherin/Rb apoptotic pathway may be a critical regulatory
control that is lost during the metastatic progression of epithelial tumors.
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Fig. 8.
Schematic representation of
aggregation-mediated survival and apoptosis during TPA-induced
aggregation and apoptosis. Prostate and mammary epithelial cells
undergo dramatic aggregation following activation of PKC. All cells
appear to hypophosphorylate Rb in response to PKC activation. Cells
that undergo aggregation will arrest in G1 and survive;
however, cells that are in S phase when Rb is activated undergo
apoptosis.
In conclusion, our data suggest that E-cadherin may play a role in the
regulation of homeostasis through a pathway that coordinates cell
survival and cell death. Many extracellular growth regulatory pathways
terminate with the modulation of Rb activity and the regulation of
G1 transit. Although we do maintain that the primary role
of Rb is to signal G1 arrest, we also suggest that
coordinated communications between the extracellular and intracellular
environment dictate whether an epithelial cell will survive in a
quiescent or differentiated state or die in response to this Rb signal.
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ACKNOWLEDGEMENTS |
---|
We thank Mark A. KuKuruga and Stephen Dewey for skillful technical support. We also thank Dr. Stephen Ethier for the SUM185 mammary cell line and scientific comments and advice.
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FOOTNOTES |
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* This study was supported by the Specialized Project of Research Excellence (SPORE) in Prostate Cancer P50 CA69568 (to M. L. D.) and DK/CA47650 (to C. T. P.) from the National Institutes of Health and by Grants JFRA-531 and TPRN-98-111-01 CSM from the American Cancer Society (to M. L. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Box 0944, Rm. 6219 CGC, 1500 East Medical Center Dr., Ann Arbor, MI 48109. Tel.: 734-647-8121; Fax: 734-647-9271; E-mail: mday{at}umich.edu.
2 On-line address is as follows: http://www.cancer.med.umich.edu/umbnydb.html.
3 M. L. Day, X. Zhao, C. J. Vallorosi, C. T. Powell, C. Lin, and K. C. Day, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PKC, protein kinase C; Rb, retinoblastoma; TPA, 12-O-tetradecanoylphorbol-13-acetate; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP biotin nick end labeling.
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