Oncogene (2002) 21, 1989 ± 1999
ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00
www.nature.com/onc
Gap junction intercellular communication propagates cell death in
cancerous cells
Vladimir A Krutovskikh*,1, Colette Piccoli2 and Horashi Yamasaki3
1
Unit of Gene-Environment Interactions, International Agency for Research on Cancer, Lyon, 69372, France; 2Unit of GeneEnvironment Interactions, International Agency for Research on Cancer, Lyon, 69372, France; 3Kwansei Gakuin University,
1-1-155 Uegahara, Nishinomiya, 662-8501 Japan
Gap junction intercellular communication (GJIC) or cell
coupling has an important function in maintaining tissue
homeostasis and is thus a critical factor in the life and death
balance of cells. While the role of GJIC in cell growth
regulation has been much studied, its involvement in
apoptosis remains unclear. In this study we elucidated the
possibility that cell death is propagated via gap junctions,
employing the rat bladder carcinoma cell line BC31. BC31
cells proliferate quickly, are tumorigenic, and are wellcoupled via gap junctions that contain the gap junction
protein Connexin43 (Cx43). In addition, these cells are
predisposed to spontaneous death by apoptosis, particularly
upon achieving con¯uency. We found that many dying
BC31 cells express Cx43 just as their non-apoptotic
counterparts do. Furthermore, Cx43 in apoptotic cells
could be functionally competent, supporting coupling of
these cells with their non-apoptotic neighbors, and as a
result, clusters of coordinately dying cells were observed.
The role of Cx43 and GJIC in propagating cell death was
shown by analysing clones of BC31 cells expressing a
mutant of Cx43 that is a dominant negative inhibitor of
GJIC, and by using b-glycyrrhetinic acid to inhibit intrinsic
cell coupling in BC31 cells: in both cases the formation of
clusters of dying cells was abrogated, and the intensity of
cell death was considerably decreased. These results suggest
that GJIC spreads cell-killing signals initially generated by
a single cell that spontaneously initiates apoptosis, into
healthy surrounding cells, thus increasing the level of cell
death. Treatment of BC31 cells with the sleep-inducing lipid
Oleamide, which selectively restricts gap junction permeability to Ca2+ ions, did not abrogate coordinated cell death
by clusters, indicating that Ca2+ ions are the most probable
cell-killing signals spread through gap junctions.
Oncogene (2002) 21, 1989 ± 1999. DOI: 10.1038/sj/
onc/1205187
Keywords: gap junctions; apoptosis; Connexin43
Introduction
Gap junctions are tiny aqueous channels which connect
the cytoplasms of contiguous cells, enabling cells to share
directly small metabolites by the process called gap
junction intercellular communication (GJIC) or cell
coupling. GJIC has a role in maintaining tissue homeostasis or its variant morphostasis, which is largely
achieved by regulating the balance between cell gain and
cell loss. There is a large body of evidence that GJIC are
implicated in the regulation of cell growth, mostly by
negative control of cell proliferation (Loewenstein and
Rose, 1992). Correspondingly, disorders of GJIC are
strongly associated with aberrant cell growth diseases,
including cancer (Yamasaki, 1990; Krutovskikh and
Yamasaki, 1997). Due to their frequent functional
alteration in tumors, the genes of gap junction proteins
± Connexins (Cx) ± have been classi®ed as tumor
suppressors (Chipman, 1995; Yamasaki and Naus, 1996).
In cancer, an increase in cell number is often a result
of suppressed cell loss (apoptosis) rather than increased
cell proliferation (McDonnell, 1993). In view of this,
and the ubiquitous role of cell coupling in the
maintenance of tissue homeostasis, its implication in
regulation of apoptosis was postulated.
Previous experiments have found that cell coupling
could propagate cell injury in cultured astrocytes (Cortina
et al., 1998). Furthermore, cell ± cell communication via
gap junctions increases the volume of brain infarct, and gap
junction blockers e€ectively inhibit this spreading (Rawanduzy et al., 1997). However, until now there has been no
experimental evidence that cell coupling could play a
signi®cant role in regulating cell death in cancerous cells.
Here we report experimental evidence that due to cell
coupling, individual dying bladder cancer cells can spread
cell death signals into adjacent cells which then also die by
apoptosis, and that the messenger molecules which pass
through gap junctions to kill cells are very likely Ca2+ ions.
Results
*Correspondence: V Krutovskikh; E-mail: [email protected]
Received 8 May 2001; revised 26 November 2001; accepted 22
January 2002
BC31 cells as a model to study the role of cell coupling in
propagation of cell death
The BC31 rat bladder carcinoma cell line has a high
coupling capacity due to the expression of only one
Gap junctions propagate cell death
VA Krutovskikh et al
1990
type of connexin protein, Cx43 (Asamoto et al., 1994;
Krutovskikh et al., 1998). Another characteristic of
these cells is their propensity to die spontaneously. The
combination of these features o€ered an opportunity to
employ these cells as a model for investigating the
possible role of gap junction intercellular communication in regulating cell death.
BC31 cells die by apoptosis
Examination of the morphology of con¯uent monolayer of BC31 cells reveals the frequent presence of
either individual cells, or small clusters of cells with
obvious signs of death; i.e. they are round in shape,
often with a `blebbed' membrane, and are poorly
attached to the surface of culture dishes (Figure 1a). In
situ staining for apoptotic markers using Annexin V
and Propidium Iodide detected substantial number of
individual cells positive either on Annexin V alone or
for both these markers of apoptosis, indicating therefore, on apoptotic origin of cell death (Figure 1b).
In addition, agarose gel electrophoresis of DNA
prepared from the fraction of dying cells collected by
mechanical exfoliation from cell culture, as described in
`Materials and methods', revealed a `ladder' pattern of
DNA degradation, providing, thus, additional evidence
that individual BC31 cells die by apoptosis (Figure 1c).
Expression of Cx43 by apoptotic BC31 cells
Using this same method of separating dying from
healthy cells, we were able to collect cells in sucient
quantity to examine, by Western blot analysis, whether
apoptotic cells express the gap junction protein
Connexin43 (Cx43), as their non-apoptotic counterparts do (Asamoto et al., 1994; Krutovskikh et al.,
1998). The immunoblotting study used an antiserum
raised against a Cx43 peptide to test for the presence of
Cx43 in protein prepared from the fraction of dying
cells, and revealed levels of Cx43 comparable to those
in the fraction of non-apoptotic BC31 cells. The only
di€erence between apoptotic and healthy cells revealed
by the immunoblotting analysis of Cx43 expression,
was a less neat electrophoretic separation of individual
bands corresponding to the di€erently phosphorylated
species of Cx43 i.e. P0, P1, P2, in a sample from the
fraction of dying BC31 cells (Figure 2a).
Immunostaining also detected Cx43 in some apoptotic cells, localizing its expression to parts of lateral
plasma membrane that were engaged in intercellular
contacts with non-apoptotic neighbors in plaque-like
forms (Figure 2b). This ®nding posed the question
whether these gap junction-like structures between
apoptotic and non-apoptotic cells could be functionally
competent. Functional heterologous coupling between
apoptotic and non-apoptotic cells could be visualized
by selectively loading tracers speci®cally permeable for
gap junctions, e.g. Lucifer yellow (LY) dye into the
cytoplasm of apoptotic cells, and observing the transfer
of dye into adjacent non-apoptotic cells in the
monolayer. The initial selective loading of dye into
apoptotic cells was readily achieved because, as
previously reported, metabolically inhibited, in other
words dying cells, increase the permeability of their
plasma membranes and therefore, unlike healthy cells,
become permeable to Lucifer yellow dye from outside
of cells (Li et al., 1996; John et al., 1999). We
con®rmed the validity of this for BC31 cells by brie¯y
Figure 1 BC31 cells frequently die by apoptosis. (a) Numerous individual dying BC31 (indicated by arrows) are morphologically
distinguishable and poorly attached to the cell monolayer (bar 100 mm). (b) In situ Annexin V/Propidium iodide staining of non-®xed
BC31 cells reveals multiple individual cells which are positive for these markers of apoptosis (bar 200 mm). (c) Agarose gel
electrophoresis of DNA samples isolated either from healthy, ®rmly attached cells in the monolayer (left) or from dying cells, which are
poorly attached to the monolayer (right). The `ladder' pattern of DNA degradation that is typical of apoptosis is seen for right sample.
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exposing them to medium containing LY and found
that LY was indeed selectively taken up from the
extracellular medium by numerous individual cells with
morphological signs of apoptosis, such as `blebbing' of
the plasma membrane (Figure 3). There were no signs
of direct LY uptake by healthy non-apoptotic cells in
monolayer.
GJIC between apoptotic and non-apoptotic cells in
monolayer of BC31 cells
However, microscopic examination of a monolayer of
BC31 cells after exposure to LY occasionally revealed,
in addition to numerous individual apoptotic cells,
groups or clusters of non-apoptotic cells that were
positive for LY. It was noteworthy that in the middle
of each group of such LY-positive non-apoptotic cells
there was a single bright cell, or a few particularly
bright cells, with morphological features of apoptosis
(Figure 4). The higher concentration of LY in such
cells indicated that most probably they are the source
from which LY spread into adjacent non-apoptotic
cells in the monolayer. The gradual decrease in
intensity of LY staining of the cytoplasms of normal
cells, as one moves from the center of LY-positive
focus towards its periphery, resembles the pattern of
LY spreading in a cell monolayer after its microinjection into a single cell, and therefore indirectly
1991
Figure 2 Expression of Connexin43 in healthy and apoptotic BC31 cells. (a) Western blotting detected comparable levels of Cx43
protein in both healthy (1) and dying (2) BC31 cells. However, sample from dying cells displayed less neatly separated
phosphorylated species of Cx43 (P0, P1 and P2). (b) Immunostaining reveals Cx43 plaque-like structures in parts of the lateral
plasma membranes involved in cell-to-cell contacts between a single apoptotic cell (in the middle) and several non-apoptotic cells
that surround it (shown by arrows). Propidium iodide counterstaining reveals the nuclear fragmentation that is typical of apoptosis,
in a cell in the middle of the ®eld (bar 20 mm)
Figure 3 Spontaneous uptake of Lucifer yellow (LY) dye by apoptotic cells from extracellular medium (see Materials and
methods). Several individual bright cells of round shape are seen (bar 100 mm). Insertion: An individual cell that has taken up LY
shows the membrane blebbing that is typical of apoptosis (bar 100 mm)
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Gap junctions propagate cell death
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Figure 4 Functional GJIC between apoptotic and non-apoptotic BC31 cells as revealed by LY uptake. Groups of cells, ranging in
size from a very few (a) to several (b) cells contain LY dye, as the apoptotic cells which were permeable to the LY added to the
medium, spread this dye into their non-apoptotic neighbors. Note a gradient of intensity LY from the center of such coupled foci
towards their periphery. (bars 100 mm)
corroborates the suggestion that LY was initially taken
up by an apoptotic cell in the center of such focus and
then spread into the cell monolayer. It is noteworthy
that Propidium Iodide (PI), which we used to stain the
nuclei of cells to reveal the DNA fragmentation
characteristic of apoptosis, is known to be another
dye to which gap junctions are speci®cally permeable.
Double vital staining of BC31 cell monolayer by
Annexin V and Propidium Iodide, showed that sometimes a single apoptotic cell that stains positive with
both Annexin V and Propidium Iodide is surrounded
by several non-apoptotic cells positive for Propidium
Iodide only (Figure 5). Again we assume that PI could
get into the cytoplasms of healthy cells only through a
single Annexin V-positive apoptotic cell in the middle
of such focus.
Taken together, we consider that our observations
obtained by the spontaneous LY uptake assay and by
the Annexin V/ Propidium Iodide staining experiments
can be interpreted as an evidence of GJIC between
apoptotic and non-apoptotic cells. The limited number
of apoptotic cells found coupled with non-apoptotic
counterparts, compared to the total number of
individual apoptotic cells permeable for LY, suggested
that GJIC between apoptotic and healthy cells in
monolayer is a very short-term event during apoptosis.
Consecutive brief exposures of BC31 cell monolayer to
®rst Propidium Iodide and then LY, showed that with
a 20-min interval between the two exposures there are
no cells positive for both these dyes.
Cell death due to spreading of killing messengers from
apoptotic cells to non-apoptotic neighbors
Observation of BC31 cell culture during its exponential
phase of growth revealed the progressive formation of
morphologically distinguishable clusters of dying cells
(Figure 6a). Subsequently, when con¯uency was
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achieved, particularly big clusters of dying cells
detached from the cell monolayer simultaneously,
which eventually resulted in the formation of defects
in the cell monolayer (Figure 6b). At that stage of
culture a signi®cant decrease in the number of cells in
monolayer was detected (Figure 6c). This formation of
particularly big clusters of dying cells and/or defects in
cell monolayer coincided with the highest coupling
capacity of BC31 which is observed soon after
con¯uence, prompting us to associate these events.
Inhibition of intrinsic GJIC in BC31 cells by
dominant-negative mutant of Cx43 decreases the level of
cell death
In order to verify whether cell coupling is indeed a means
of propagating cell death in a cell monolayer, we
measured the e€ect of abrogating the intrinsic GJIC
between BC31 cells, on the total level of cell death at
di€erent stages of cell growth. Previously we demonstrated that several Cx mutants are strong dominant
negative inhibitors of GJIC in di€erent cell lines (Du¯otDancer et al., 1997; Krutovskikh et al., 1998, 2000).
Speci®cally, we found that Cx43 lacking seven residues
from the internal loop at positions 130 ± 136, e€ectively
abrogated GJIC in BC31 cells (Krutovskikh et al., 1998)
by occluding the channels of established gap junctions in
the lateral plasma membrane of cells. Clones of BC31
cells de®cient in cell coupling due to expression of this
Cx43D mutant were used in this study.
In situ staining of apoptosis markers using Annexin
V and Propidium Iodide did not reveal an obvious
di€erence in the number of individual apoptotic cells
between communication-de®cient clones of BC31 and
the well-coupled parental cell line. This indicates that
inhibiting intrinsic GJIC has no apparent impact on
the frequency with which `primary' apoptosis spontaneously occurs in individual BC31 cells.
Gap junctions propagate cell death
VA Krutovskikh et al
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Figure 5 GJIC between apoptotic and non-apoptotic BC31 cells shown by double Annexin V/Propidium iodide staining of live
cells. A single apoptotic cell, positive on Annexin V and permeable for Propidium iodide dye, is shown in the middle of ®gure (a)
and outlined by a dotted line on ®gure (b). Propidium iodide has spread into the cytoplasms of non-apoptotic Annexin V-negative
neighbors (b). Note the normal shape of the nuclei stained by Propidium iodide in the non-apoptotic cells (b). Pictures A and B have
been taken from the same ®eld with di€erent ®lters (bar 100 mm)
Figure 6 Coordinated death by clusters in communication-pro®cient BC31 cells. (a). Morphologically-distinguishable clusters of
dying cells (bar 500 mm) (b). Defect in monolayer as a result of cell death by clusters (bar 500 mm)
The Lucifer yellow uptake assay demonstrated that
expression of Cx43D dominant negative mutant in
BC31 cells did not prevent uptake of this dye from
extracellular medium by single apoptotic cells.
Meanwhile, as we expected, there was no spreading of LY from individual apoptotic cells into
contiguous non-apoptotic counterparts in the monolayer of communication-de®cient clones of BC31 cells
which expressed the dominant negative mutant of
Cx43D. Furthermore, no clusters of dying cells were
observed in non-coupling clones of BC31 cells at
di€erent stages of cell culture (Figure 7a). As a
result, the intensity of cell loss after con¯uency in
communication-de®cient clones of BC31 cells was
considerably less than in the well-coupled control
(Figure 7b). A similar selective inhibitory e€ect, on
only `bystander', or secondary cell death, was
observed when intrinsic GJIC in BC31 cells was
suppressed by another dominant-negative mutant of
Cx43 (E166G) (not shown). Unlike Cx43D, the
E166G mutant abrogates intrinsic GJIC by retaining
of Cx43 complexes intracytoplasmically (Krutovskikh
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Gap junctions propagate cell death
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et al., 2000). Thus we conclude that the protection
a€orded against cell death by dominant negative
mutants of Cx43 should be attributed to their
capacity to inhibit cell ± cell coupling.
The inhibition of GJIC by the blocking agent
18b-glycyrrhetinic acid also decreases the level of cell
death
In order to verify our interpretation of the
experiments using dominant-negative Cx43 mutants
to abrogate cell coupling, and to exclude any
possible e€ects of enforced expression of Cx43
protein on cell growth per se, we conducted
experiments using the GJIC inhibitor 18b-glycyrrhetinic acid (bGA). This compound was shown to
block cell coupling fully (Davidson et al., 1986),
presumably by disassembling gap junctional channels
(Guan et al., 1996); the inhibitory e€ect on GJIC
lasts for many hours and apparently has no direct
e€ect on cell proliferation.
Unlike the well-coupled control BC31 cells, BC31
cells treated with bGA at a dose (70 mM), which was
not toxic, but e€ective in disrupting cell coupling,
did not form either clusters of dying cells, or defects
in the cell monolayer (Figure 8a,b). A quantitative
analysis revealed that the bGA-treated cells remained
attached to the monolayer after con¯uency a lot
better than non-treated control cells (Figure 8c).
The nature of cell death signals spread through gap
junctions
We then addressed the critical question what is/are the
killing messenger(s) that are generated in individual
apoptotic cells and then spread through gap junctions
into the cytoplasms of healthy adjacent cells in the
monolayer. The permeability of gap junctions (about
1 kD) limits the list of putative candidates to Ca2+,
potassium, IP3, and ATP. Previous studies indicate
that, at least in neural tissue, Ca2+ ions are the most
likely messengers of death (Badd and Lipton, 1998).
In order to verify whether Ca2+ is the most likely
cell-killing signal in BC31 cells, rather than other
molecules of bigger size that spread through gap
junctions, we studied the e€ect of selectively restricting
GJIC permeability in BC31 cells using the sleepinducing agent cis-9-Octadecenamide (Oleamide). This
neuropeptide con®nes the permeability of gap junctions to calcium waves (Guan et al., 1997). Our
experiments showed that treatment of BC31 cells with
100 mM of Oleamide e€ectively blocks the spreading of
LY and that this e€ect lasts at least 24 h. Further
studies showed, however, that treatment of BC31 cells
by Oleamide at this dose did not prevent the
formation of either clusters of dying cells or defects
in the cell monolayer. Furthermore, estimation of the
number of cells remaining in the cell monolayer did
not reveal any signi®cant di€erence between Oleamide-
Figure 7 Expression of the dominant-negative Cx43 mutation abrogates cell coupling and a€ects BC31 cell growth and death. The
cell-coupling capacity of parental BC31 cells (a) is shown by the spread of LY dye after microinjection of LY into a single cell (see
insertion, bar 500 mm). By contrast, clones of the BC31 cell line that express a dominant-negative mutant of Cx43 (b) show a lack of
cell coupling after LY microinjection into a single cell (see insertion, bar 500 mm). After con¯uency, defects arise in the monolayer
of parental BC31 cells due to intensive coordinated cell loss by clusters (a). Such defects are not seen after con¯uency in a culture of
BC31 cells that express the dominant-negative mutant of Cx43 (b). A quantitative assessment of cell growth (c, see Materials and
methods) shows that cell loss of parental BC31 cells occurs from day 4 to day 6 of cell culture; the cells shown in (a) and (b)
correspond to day 6 of the experiment shown in (c). (bars 500 mm)
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Figure 8 18 b-glycyrrhetinic acid abrogates cell coupling and reduces cell loss in post-con¯uent cultures of BC31 cells. Cultures of
non-treated control BC31 cells (a) and BC31 cells treated with b-glycyrrhetinic acid (bGA) (b) are shown after 6 days of culture, 2 ±
3 days after reaching con¯uency (bars 500 mm). The insertions illustrate the inhibitory e€ect of bGA on cell coupling, as revealed by
LY injection (bar 500 mm); the main panels show that bGA treatment during exponential phase of growth and after con¯uency
(days 2 ± 6 of experiment) abolishes the formation of the defects in the cell monolayer that are seen everywhere in communication
pro®cient non-treated control BC31 cells. Growth curves (c) show that bGA treatment did not a€ect the rate of cell growth during
the exponential phase, but decreased both the level of con¯uency (days 3 and 4 of culture) and intensity of cell loss afterwards (days
4 ± 6 of culture). (Vertical arrows indicate changes of medium to ensure a supply of active bGA)
treated cells and non-treated coupled BC31 cells
(Figure 9).
Discussion
In this study we obtained several lines of experimental evidence that gap junction intercellular
communication in cancer cells is a means of
propagating cell death. Speci®cally, using the rat
bladder carcinoma BC31 cell line, we found that
individual cells, which spontaneously commit to die,
could be coupled with non-apoptotic counterparts.
Apparently early apoptotic events do not alter gap
junctions; Cx43, which is the only connexin to
provide coupling in these cells (Asamoto et al.,
1994; Krutovskikh et al., 1998), was found in
apoptotic cells and was neither degraded nor
translocated from its normal location in the lateral
plasma membrane into the cytoplasm. Furthermore,
the spontaneous loading of apoptotic cells by Lucifer
yellow dye con®rmed that dying cells could be
functionally coupled with their healthy neighbors.
Apparently, communication between apoptotic and
non-apoptotic cells is a short-lived event, as only a
few of the numerous individual dying cells, which
were able to take up LY from the medium during
5 min of exposure to this dye, could then spread it
into the normal cells surrounding them. It is
noteworthy that coupling between apoptotic and
non-apoptotic cells is a two-way process: after
microinjecting LY into an individual healthy cell that
was situated next to a morphologically-distinguishable
single apoptotic cell in the monolayer, we found that
LY could spread into the apoptotic cell (not shown).
Apparently, a transient functional coupling between
dying and healthy cells nevertheless suces to trigger
irreversible processes, which eventually result in the
death of these adjacent, previously healthy, cells. Our
and others' (Cortina et al., 1998) observation that only
communicating cells die in culture by clusters,
corroborates this suggestion. It also appears, however,
that the spread of killing signals through gap junctions
is somehow limited by protective mechanisms which
determine the size of the clusters of dying cells: even in
very well-coupled cells, the propagation of death never
became totally generalized.
Ca2+ has a role in both the initiation and regulation
of apoptosis (McConkey and Orrenius, 1997). Very
high concentrations of Ca2+, e.g. due to its in¯ux from
extracellular space as a result of metabolic inhibition or
oxidative insult, or after treatment of cells with Ca2+
ionophores (Martikainen and Isaacs, 1990), may
trigger apoptosis. Thus, it is very likely that the
spontaneous apoptosis of individual BC31 cells is
associated with a large increase in [Ca2+]i concentration. Due to coupling with neighboring cells, a single
cell that spontaneously dies by apoptosis, could spread
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1996
Figure 9 Partial inhibition of GJIC of BC31 cells by Oleamide treatment does not prevent cell loss after con¯uency. Oleamide
treatment from days 2 ± 6 of BC31 cell culture e€ectively inhibited cell coupling for Lucifer yellow (b, insertion, bar 500 mm), but did
not eliminate the formation of defects in cell monolayer (b), or modulate the rate of cell growth during days 1 ± 4 of culture or
prevent cell loss after achieving con¯uency (days 4 ± 6) (c). (bars 500 mm)
excess Ca2+ into the cell monolayer, i.e. generate a
Ca2+ wave. The possibility that Ca2+ waves spread
through gap junctions may kill cells has been shown
before in a study on the development of neural tissue
(Walszon et al., 1994). It is likely that the limitation of
the size of the focus of dying cells is, at least partially,
due to the relatively slow speed with which Ca2+ waves
spread through gap junctions (Jorgensen et al., 1997).
In addition, the passage of a Ca2+ wave through gap
junctions does not provoke Ca2+ release from
intracellular stores. Therefore, one may suggest that
after passing through several cells the intensity of a
Ca2+ wave is greatly decreased. In addition, it is
known that gap junction gating is Ca2+ dependent
(Pfahnl and Dahl, 1999). Thus cells that are a few cells
away from the original, spontaneously-apoptosing cell,
may receive an attenuated Ca2+ wave and have time to
respond by closing their gap junctions before receiving
enough Ca2+ to trigger their own apoptosis. It should
be noted, however, that cell coupling has been shown
to have a role in protecting against cell injury, by
reducing the concentrations of harmful molecules in
cytosol or by the maintenance of [Ca2+]i homeostasis
(Blanc et al., 1998).
A direct demonstration of the cell killing power of
Ca2+ would be to show that introducing a high
concentration of these ions into the cytoplasm of a
single healthy cell indeed kills it. Unfortunately, all our
attempts to demonstrate this by microinjecting a highly
concentrated solution of Ca2+ have been, so far,
unsuccessful, probably due to the limitations of
microinjection manipulation, which does not allow
the introduction of Ca2+ in amounts optimal to kill the
recipient cell.
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However, our data from the experiments using the
sleep-inducing lipid Oleamide, show that restricting gap
junction permeability to molecules of the size of Ca2+
ions, does not prevent the spreading of cell death
signals through gap junctions, thus indirectly implicating Ca2+ ions in this process. These data also exclude
the implication of IP3 in mediating of spreading of
Ca2+ waves, which kill cells.
Apparently gap junctions are not the only means of
propagating cell death in cell cultures. Thus, gap
junction-independent clustering of apoptotic cells, due
to the release of toxic compounds from an initially
single dying cell into the culture medium, has been
described, and hydrogen peroxide was suggested as the
death signal (Reznikov et al., 2000).
The capacity of cells to kill each other through gap
junctions has been shown before in co-culture experiments and was named `bystander death'. For instance,
glioma cells that resisted apoptosis due to the enforced
expression of Bcl2 could, nevertheless, be killed by
di€erent injuries (metabolic inhibition, oxidative
stress), when they were coupled via gap junctions with
vulnerable non-transfected counterparts (Lin et al.,
1998). This phenomenon is being pursued for its
potential bene®t in cancer therapy. Despite a low
eciency of transfecting cancer cells with the herpes
simplex virus thymidine kinase (HSV-tk)-gene, which
converts the anti-tumor compound ganciclovir from a
prototoxin into an active form, it has been shown that
the therapeutic e€ect of this compound could be
essentially exacerbated by cell coupling, as the toxic
metabolite of ganciclovir produced in individual HSVtk gene-bearing cells spreads via gap junctions into
numerous cells which themselves lack the HSV-tk gene,
Gap junctions propagate cell death
VA Krutovskikh et al
and eventually kills them (Mesnil et al., 1996; Estin et
al., 1999).
Similar `bystander cell death' or `fratricide' phenomena have been observed during the development of foci
of ischemia-induced brain damage, when cell death was
found to spread slowly away from the core of infarct
(Cortina et al., 1998), presumably by means of gap
junction intercellular communication, but again, up to
certain limits (Badd and Lipton, 1998). Furthermore,
inhibiting GJIC by administrating the gap junction
inhibitors, octanol and hepthane, signi®cantly reduced
infarct volume during experimental focal brain stroke
(Rawanduzy et al., 1997).
There is a large body of evidence that gap junction
intercellular communication plays an essential role in
tumor suppression. Until now, the tumor suppressive
e€ect of the gap junction proteins known as Connexins
was attributed to their capacity to inhibit cell growth,
which in some cases directly correlates with cell ± cell
communication (Zhang et al., 1998), or is, at least,
contingent upon the formation of gap junctions (Mehta
et al., 1999). Now we show that in cancer cells
intercellular communication via gap junctions propagates cell death and thereby contributes to the tumor
suppression mediated by Connexins, particularly when
their direct inhibitory e€ect on cell proliferation is not
evident.
1997
Immunostaining
After removing the culture medium, cells were brie¯y washed
with Ca2+ Hanks solution, ®xed in 4% paraformaldehyde on
PBS for 10 min at room temperature, washed with PBS
several times and permeabilized with 0.5% Triton X-100
solution for 10 min. After incubation with primer antibodies
against Cx43, immunoreactivity was revealed using the TSA
(Tyramid signal ampli®cation)-direct technique from NEN1
Life Science Products, according to the manufacturer's
instructions. Counterstaining with Propidium Iodide helped
to reveal cells with morphological alterations of their nuclei
that are typical of apoptosis.
Immunoblotting
Apoptotic and non-apoptotic cells, collected as described
above, were lysed in a bu€er containing sodium dodecyl
sulfate, 2%; dithiothreitol, 0.1 M; Tris HCl, 60 mM, 6.8 pH;
phenylmethylsulfonyl ¯uoride, 1 mM and glycerol, 10%. The
samples were sonicated for 30 s, centrifuged, resolved by
electrophoresis on a 10% polyacrylamide gel and blotted
onto a nitrocellulose membrane. Preincubation of the
membrane and its subsequent exposure to the primary and
secondary antibodies and washing were done with bu€er
containing 0.1% Tween 20 in 100 mM Tris, 0.9% sodium
chloride, pH 7.5. A positive reaction with antibody was
revealed by NiCl2-DAB staining after incubation with
peroxidase-conjugated goat anti-rabbit antiserum (Sigma
Co., St. Louis, MO, USA).
Microinjection dye transfer assay
Materials and methods
BC31 cells culture
The rat BC31 cell line, established from a primary
chemically-induced rat bladder transitional cell carcinoma
(Asamoto et al., 1994), was maintained in culture in
Dulbecco's modi®ed Eagle's medium supplemented with
10% fetal bovine serum on plastic under 5% CO2/95% air
at 378C in a humidi®ed incubator.
Preparation of fractions of apoptotic and non-apoptotic cells
It was possible to collect selectively the fraction of dying cells
from cultures of BC31 cells because one of the features of
these apoptotic cells is their poor attachment to the surface of
the culture dish, and they could be detached from the
monolayer of healthy cells by intensive pipetting. Microscopic
examination revealed that intensive pipetting e€ectively
removed only those cells which were poorly attached to the
surface of the culture, and did not a€ect the cellular
monolayer. The cells left attached to the culture dishes after
this manipulation represented a healthy population of BC31
cells and were collected separately by mechanical scraping.
DNA and protein samples were extracted from each of these
cell fractions and were analysed by electrophoresis.
Antibodies
Polyclonal primary anti-Cx43 antibodies, raised in a rabbit
against a synthetic peptide corresponding to residues 363 ±
382 of native Cx43, were provided by Dr E Rivedal, Oslo,
Norway.
In order to estimate cell coupling quantitatively, microinjections of 5% Lucifer yellow CH solution in 0.33 M lithium
chloride were performed essentially as described previously
(Fitzgerald et al., 1983).
Functional assay to reveal heterologous coupling between
apoptotic and non-apoptotic BC31 cells in monolayer
To estimate the possibility of functional heterologous
coupling between individual apoptotic and surrounding
non-apoptotic BC31 cells, the former must be speci®cally
loaded by a tracer dye speci®c for gap junctions such as
Lucifer Yellow. We achieved this by exposing the monolayer
of BC31 cells to PBS with 5% of Lucifer yellow dye for
5 min and then washing the cell monolayer three times in
PBS. The increased permeability of the lateral membranes of
dying cells allowed LY to get into their cytoplasms; by
contrast no signs of spontaneous penetration of LY into the
cytoplasms of healthy cells in the monolayer were seen.
Immediately after this exposure the cell monolayer was
examined by inverted ¯uorescence microscope.
Detection of apoptosis
± by in situ staining Non-®xed BC31 cells were stained in
situ by AnnexinV/Propidium Iodide using the Annexin-VFLUOS Staining kit (Boehringer Mannheim GmbH, Germany) according to the manufacturer's protocol. Immediately
after this procedure the stained cells were examined under an
inverted microscope with epi-¯uorescent equipment.
±by the pattern of DNA degradation DNA samples from
di€erent cell fractions, collected from cell culture as described
before, were resolved in agarose gel and stained by Ethidium
Oncogene
Gap junctions propagate cell death
VA Krutovskikh et al
1998
Bromide to reveal whether the DNA was degraded in the
ladder pattern typical of apoptotic cells.
Inhibition of GJIC
± by dominant negative mutant of Cx43 As we described
previously (Du¯ot-Dancer et al., 1997, Krutovskikh et al.,
1998, 2000), several mutations of Cx proteins have been
characterized as dominant negative because on being coexpressed with wild type Cx these communication-defective
Cx mutants are able to interfere with the functional normal
Cx protein and deactivate its function. One such Cx43
mutant with a strong dominant negative property has seven
residues deleted from its cytoplasmic loop at positions 130 ±
136 (Krutovskikh et al., 1998). Communication-de®cient
clones of BC31 cells stably transfected with this Cx43 mutant
were used in the study.
± by 18 b-glycyrrhetinic acid Bearing in mind that enforced
expression of Cx protein could have an e€ect on cell growth
per se, independent of its modulation of cell coupling
capacity, we also inhibited intrinsic GJIC in BC31 cells using
the known gap junction blocking agent ± 18 b-glycyrrhetinic
acid (Davidson et al., 1986). An initial experiment showed
that a 70 mM concentration of 18 b-glycyrrhetinic acid
e€ectively inhibits GJIC between BC31 cells, without being
cytotoxic. We also found, that exposure of BC31 cells to the
medium supplied with 70 mM of 18 b-glycyrrhetinic acid
e€ectively inhibited their coupling for approximately 24 h.
In order to achieve inhibition of cell coupling throughout the
several days required to assess the e€ects on growth rate, the
medium was replaced with fresh medium containing 18 bglycyrrhetinic acid on a daily basis.
± by cis-9-Octadecenamide (Oleamide) The sleep-inducing
lipid cis-9-Octadecenamide (Oleamide) has been described as
a compound which restricts the permeability of gap junctions
to Ca2+ ions (Guan et al., 1997). To obtain this e€ect on
BC31 cells, Oleamide was dissolved in DMSO and supplied
to the culture medium at a ®nal concentration of 100 mM; in
an initial experiment this concentration of Oleamide was
found to be non-toxic for BC31 cells, but e€ective in blocking
the intercellular passage of Lucifer yellow dye at least for
24 h. To obtain stable long-lasting inhibition of cell ± cell
communication the medium with freshly supplied Oleamide
was changed daily.
Growth rates of cells in vitro
Cells (56104) were seeded on a 35-mm culture dish with
supplemented medium, as described before, at day 0 of the
experiment. Cells from three dishes were trypsinized and
counted independently every next day of experiment.
Statistical analyses
All in vitro experiments were repeated three times. Statistical
analyses were performed using a commercial statistical
analysis package (GraphPad Prizm 3.0).
Acknowledgments
We thank Dr J Cheney for editing this manuscript.
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