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Disruption of Gap Junctions in Toxicity and Carcinogenicity
J. Kevin Chipman,1
Angela Mally, and Gareth O. Edwards
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
Received October 11, 2002; accepted November 16, 2002
Although the specific role of connexin-mediated gap junctional
intercellular communication in the control of cell homeostasis,
proliferation, and death are still not clear, several lines of evidence
support these roles. The disturbance of this communication,
through multiple mechanisms, may in the short term be a protec-tive mechanism to limit the spread of toxicity in a tissue following
chemical or radiation damage. However, sustained downregula-
tion confers a loss of tumor-suppressive action. Consequently,
connexin dysfunction has been associated with both the action of
many carcinogens and being a feature of cancerper se. Connexins
offer not only a target for cancer chemoprevention but also for
exploitation in chemotherapy through the bystander effect.
Key Words: connexin, gap junction, carcinogenesis, bystander,
intercellular communication.
Structure and Function of Gap Junctions
Gap junctions are intercellular membrane channels that al-low the direct exchange of small molecules (1.2 kD) between
adjacent cells. They are comprised of two hemichannels (con-
nexons), which are in turn formed by the oligomerization of six
protein subunits, termed connexins (Cxs). To date, at least 21
members of this multigene family have been discovered and
cloned (Willecke et al., 2002). Cxs consist of four highly
conserved membrane-spanning domains, two extracellular
loops, a cytoplasmic loop, and cytoplasmic N- and C-terminal
regions. While the extracellular loops are thought to be re-
quired for connexon docking, the cytoplasmic domains appear
to play a role in channel gating and intracellular signaling, and
they vary between members of the Cx family. Small ions such
as water, Ca2
, cAMP, and inositol triphosphate can passthrough the gap junctions from one cell to another, while larger
molecules such as proteins, complex lipids, and polysaccha-
rides are retained within the cell (Saez et al., 1989). This
process of exchange of molecules between neighboring cells
via gap junctions is termed gap junction intercellular commu-
nication (GJIC). A major physiological role of GJIC is the
maintenance of tissue homeostasis and proliferation, the regu-
lation of embryonic development and differentiation, and elec-
tric coupling of electrically excitable cells such as cardiomy-
ocytes (Loewenstein, 1981). However, more recently, evidence
for an intracellular signaling role of connexins in the control ofcell proliferation has also emerged (see below).
Mechanisms of Regulation of GJIC
Gap junction-mediated intercellular communication can be
regulated at several levels, including transcription, mRNA
processing, protein synthesis, and post-translational modifica-
tion, assembly, trafficking, connexon docking, and gating of
the gap junction channel (see Fig. 1).
Several promoter response elements and transcription fac-
tors that are involved in the transcriptional regulation of con-
nexins have been identified. For example, the promoter of the
rat cx32 gene contains several GC-boxes, and Sp1, HNF-1,
YY-1, and NF-1 binding sites have been recognized (Bai et al.,
1995; Piechocki et al, 2000). In the human, rat and, mouse
cx43 promoters, several sites, including AP1, cAMP and es-
trogen response elements have been identified (Chen et al.,
1995a; Yu et al., 1994). The expression of cx genes can be
controlled in a tissue-specific manner. For example, the liver
cell-specific expression of cx32 has been shown to be depen-
dent (at least in part) on expression of HNF-1 (Piechocki et al.,
2000). Additional promoters have been identified that control
the transcription of cx32 in neuronal tissue (Neuhaus et al.,
1995). The methylation of CpG islands of promoter regions
can lead to gene silencing and methylation of the Cx43 pro-
moter has been reported in MH1C1 rat hepatoma cells, whichexpress Cx32 but not Cx43, while the cx32 promoter was
methylated in WB-F344 rat liver epithelial cells, which express
Cx43 but not Cx32 (Piechocki et al., 1999).
It appears that connexin mRNA stability also plays an im-
portant role in controlling GJIC. Decreased Cx32 mRNA half-
life has been observed in regenerating liver (Kren et al., 1993),
while sequences located within the 3 untranslated region of
the Cx43 mRNA appear to stabilize this transcript (Lefebvre et
al.,1995).
1 To whom correspondence should be addressed. Fax: 44 121 414 5925.
E-mail: [email protected].
TOXICOLOGICAL SCIENCES71, 146153 (2003)
Copyright 2003 by the Society of Toxicology
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The activity of GJ is also regulated by post-translational
modification of connexin proteins. Regulation can be by volt-
age, calcium and various kinases that act on the intracellular
domains (Fig. 1; Holder et al., 1993). Phosphorylation of the
C-terminal domain of the connexins (with the exception of
Cx26) influences Cx trafficking, channel gating and turnover
(Saezet al., 1998). Inhibition of communication through Cx43
hyperphosphorylation has been brought about both chemically
(phorbol ester, via MAP kinase and possibly protein kinase C)
and by epidermal growth factor through the action of MAP
kinase (Rivedal and Opsahl, 2001). Hepatic Cx32 appears also
to be inactivated by PKC activation (Elcock et al., 2000).
Furthermore a number of oncogene products mediate inhibitionof GJIC, such as through the tyrosine phoshorylation of Cx43
by v-src (reviewed by Yamasaki et al.,1999). However, there
are multiple phosphorylation sites on different Cx molecules
and the balance of the phosphorylation status determines func-
tion. It is predictable, therefore, that phosphorylation is medi-
ated by various signal transduction pathways.
Another important mechanism involves the trafficking of
connexins from intracellular pools to the plasma membrane.
Following protein synthesis a number of the connexin polypep-
tides have been shown to be inserted into the membrane of the
ER and subsequently the Golgi where they oligomerize to form
hemichannels. Little is known about what initiates and medi-
ates the transport of connexons to the plasma membrane and
the formation of the complete gap junction channel, but cell
cell adhesion appears to be especially important. There is a
requirement for E-cadherin expression for Cx43 transport to
the cell membrane and for communication to be recovered in a
mouse skin papilloma cell line (Hernandez-Blazquez et al.,
2001). It is also known that Cxs can associate with other
proteins in the membrane, specifically ZO-1 with Cx43 and
Cx45 (Defamie et al.,2001; Kausalya et al.,2001). Clustering
of gap junction channels into plaques is required for functionalcell coupling (Bukauskas et al., 2000). Finally, several mech-
anisms have been implicated in GJ degradation, including
lysosomal and proteasomal pathways.
It is therefore not surprising to find that there are multiple
ways by which GJIC can be regulated and disrupted (Saez et
al.,1993). Aliphatic alcohols (octanol, heptanol) and anesthet-
ics such as halothane impair gap junction activity by interfering
with membrane fluidity that leads to contraction of intercell
channels. Other compounds have been reported to physically
FIG. 1. Summary of the multiple regulatory points that control GJIC. Specificity of regulation and function are dependent on the connexin forms.
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block GJs. Chemical gating is regulated by pH, calcium ions
and free radicals (Saez et al., 1993). Thus, toxicant-induced
changes in cellular calcium levels and redox state can lead to
inhibition of GJIC at least in part through mechanisms that
involve disturbance of phophorylation status (Trosko et al.,
1998).
GJIC and Control of Cell Proliferation and Apoptosis:
Multiple Signaling Roles
GJIC has long been considered to play an important role in
the regulation of cell growth and cell death. For example, in the
course of liver regeneration following partial hepatectomy, GJ
expression is temporarily decreased until liver mass and lobu-
lar conformation are restored. Similar effects on GJIC are seen
during acute liver injury and tissue restoration after treatment
with toxic chemicals such as thioacetamide (Kojima et al.,
1994). Studies on liver regeneration after partial hepatectomy,
where synchronous initiation of DNA synthesis was preceded
by a decrease in the expression of Cx proteins, suggest thatdownregulation of GJs may be involved as a trigger for cell
proliferation (Dermietzelet al.,1987). However, Temme et al.
(2000) reported that Cx32-deficient mice showed normal re-
generative response following two-thirds hepatectomy, although
the extent of initiation and termination of DNA synthesis was
slightly altered. Furthermore, hepatocytes completely devoid
of gap junctional communication are able to enter the cell cycle
upon mitogenic stimulation (Fladmarket al., 1997). Neveu et
al. (1990) demonstrated that a decrease of Cx32 in livers of
phenobarbitone-treated rats was not associated with increased
hepatocyte proliferation. Similarly, we have recently shown
that downregulation of Cxs by a number of nongenotoxic
carcinogens does not necessarily correlate with induction of
cell proliferation (Mally and Chipman, 2002). Although a
certain stage of mitosis has been associated with decreased
levels of Cxs (Dermietzel et al., 1987), loss of GJIC per se
does not appear to provide a signal for cells to divide but rather
permits cell-cycle progression in the presence of mitotic stim-
uli (Trosko and Ruch 1998). It has been suggested that down-
regulation of GJs during mitosis may serve to maintain sepa-
rate cellular pools of signaling molecules (Fladmark et al.,
1997). This would ensure that during critical stages of cell
division, proliferating cells remain isolated from potentially
damaging signals mediated by neighboring cells.
Just how gap junctions regulate cell growth is poorly under-stood. Forced expression of Cx43 and Cx32 in neoplastic lung
and liver cells restored G1 growth control and was associated
with increased p27 (kip-1) and decreased cyclin D1 expression
(Koffler et al., 2000). Similarly, overexpression of Cx43 in
osteosarcoma cells inhibited cell cycle transition from G1 to S
phase by increasing the expression of p27 (Zhanget al., 2001).
Chen et al. (1995b) reported alterations in the expression of
genes involved in cell cycle control after transfection of Cx43
into a transformed kidney cell line. However, it remains to be
established whether the observed decrease in cyclin A, D1 and
D2 and the cyclin dependent kinases CDK5 and CDK6 were a
direct effect of Cx43 expression or a result of the accompanied
density-dependent inhibition of proliferation and prolongation
of the G1 and S phases of the cell cycle.
It is becoming increasingly apparent that gap junctions play
an important role in intracellular signaling in relation to pro-liferation in addition to junctional functionality (Duflot-Dancer
et al., 1997). Moorby and Patel (2001) reported opposing
effects on cell growth in three clones transfected with different
Cx43 mutants when grown under noncoupling conditions. In
addition, they were able to show that the cytoplasmic carboxyl
domain of Cx43, which includes various phosphorylation sites
and has no channel activity, is as effective as wild-type Cx43
in blocking cell growth. These studies suggest that Cxs may
regulate cell growth independent of intercell communication.
As mitosis and apoptosis share some common regulatory
features (Kasten and Giordano, 1998), it is not surprising that
GJIC appears to play a role in both processes. Wilson et al.
(2000) reported relatively high levels of GJIC during earlystages of apoptosis and mitosis, which then decreased as each
process progressed. During both processes, activation of signal
transduction pathways involving Cdc2/cyclin B kinase is an
important event (King and Cidlowski, 1995; Pandey and
Wang, 1995) and the phosphorylation of Cx43 in vitro is
dependent on Cdc2 kinase (Lampe et al.,1998). However, the
question remains whether phosphorylation of Cx43 influences
apoptosis and mitosis or whether initiation of these pathways
results in Cx phosphorylation and inhibition of GJIC.
Since several tumor promoters and oncogenes are known to
inhibit both GJIC and apoptosis, while several antitumor pro-
moters enhance GJIC and apoptosis, Trosko and Goodman
(1994) hypothesized that a death signal may be mediated
through gap junctions. The ability of nongenotoxic carcinogens
such as the peroxisome proliferators and phorbol esters to
disrupt GJIC and at the same time suppress apoptosis suggests
a strong link between GJ and apoptosis in homeostatic control
of solid tissue. However, although GJIC may be one of the
mechanisms contributing to apoptosis signaling, there is little
evidence to indicate that functional gap junctions are (gener-
ally) required for apoptosis. We have recently investigated the
effects of a number of nongenotoxic carcinogens on gap junc-
tions in relation to apoptosis in target tissues in the rat. In this
study, the peroxisome proliferator Wy-14,643 decreased both
the number of hepatocyte gap junction plaques containingCx32 and the rate of hepatocyte apoptosis, while 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) appeared to downregu-
late Cx32 without effects on apoptosis. Most importantly,
treatment with methapyrilene led to a dramatic loss of connex-
ins and increased rate of apoptosis (Mally and Chipman, 2002).
Similarly, propylthiouracil and low-iodine diet decreased GJIC
and at the same time enhanced apoptosis in rat thyroid (Kolaja
et al.,2000b). These data clearly show that apoptosis can occur
in the absence of functional GJs, despite the fact that with
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certain other compounds a correlation between reduced GJ
function, elevated mitogenesis, and reduced apoptosis exists
(Kolaja et al., 2000a). It may be that functional GJIC is
required for the initial signaling in apoptosis but that this
communication is lost at a later stage. In summary, it appears
that rather than suppressing growth or signaling a cell to
undergo apoptosis, gap junctions may serve to maintain opti-mal rates of cell growth and cell death. Disruption of GJIC per
se may not result in alterations of the rates of proliferation or
apoptosis but require the presence of additional stimuli (Trosko
and Ruch, 1998). Such a stimulus may include the activation of
a cellular oncogene or inactivation of a tumor suppressor in
initiated or preneoplastic cells (Watanabe and Tonosaki, 1995).
Downregulation of GJIC as an Adaptive,
Protective Response to Toxicity
While GJIC can be important in tissue homeostasis and
integrated tissue responses, it follows that GJs have the poten-
tial to propagate toxic responses also through the transfer ofmolecules such as superoxide and calcium ions, if elevated in
the process of cell toxicity (e.g., in cerebral ischemia; Lin et
al.,1998). It is likely, therefore, that the rapid, temporary, and
reversible closure of GJ during cellular stress may be an
important protective means for preventing the spread of cellu-
lar toxicity (Peracchia et al., 2000; Saez et al. 1989). Closure
of gap junctions is thought to prevent the spread of apoptotic
and inflammatory signals between cells, and furthermore, heart
and brain infarction sizes are reduced when cells are uncoupled
(reviewed in Brosnan et al., 2001). Studies have demonstrated
that when cells are irradiated with 0.31.0 cGy doses of-par-
ticles (Azzamet al., 2001; Zhou et al., 2000), stress responses
(mutations, micronucleus formation, p53 phosphorylation,p21waf1 induction) are observed in more cells than were directly
exposed. Upon incubation of cells with GJIC blockers such as
lindane, the number of responding cells was significantly re-
duced. However, when communicating cells were irradiated
with soft x-rays at sublethal doses in the range of 1 to 5 Gy,
GJIC was reduced in a dose-dependent manner (Edwardset al.,
2002). Thus, depending on dose, the inhibition of GJIC can
limit the spread of stress-inducing molecules through this very
same system. This is of importance when considering the dose
dependency of effects of exposure to ionizing radiation.
Sustained Aberrant Localization or Downregulation
of Cxs Provides a Tumor-Promoting Stimulus
While deliberate, temporary closure of gap junctions serves
to protect healthy cells by preventing the spread of toxic
molecules and the loss of metabolically important substances,
sustained inhibition of GJIC may contribute to carcinogenesis
or conditions such as reproductive, neurological, and cardio-
vascular dysfunction through loss of homeostatic control. Un-
timely inhibition of GJIC during critical stages of development
may result in embryonic toxicity or teratogenesis. Indeed,
many chemicals known to be tumor promoters, teratogens, or
neurotoxins modulate GJIC (Elmore et al., 1987; Rosenkranz
et al., 1988; Trosko et al., 2002).
There is substantial evidence to suggest that sustained down-
regulation of GJIC provides a tumor-promoting stimulus. Nor-
mal, contact-inhibited cells exhibit functional GJIC, while
transformed cells and many tumors are characterized by re-duced expression of GJs involving aberrant localization or
downregulation of Cxs and nonfunctional homologous or het-
erologous GJIC (Loewenstein and Rose, 1992; Trosko and
Ruch, 1998; Yamasaki, 1990). Activation of some cellular
oncogenes inhibits GJIC and induces malignant transformation
(Jou et al., 1995; reviewed in Yamasaki et al., 1999), and
tumor-promoting conditions such as wounding and necrosis are
associated with decreased GJIC. Moreover, compelling evi-
dence that links loss of GJ function to carcinogenesis has come
from the observation that many nongenotoxic carcinogens and
tumor-promoting agents inhibit GJIC. A range of nongenotoxic
carcinogens has tested positive for inhibitory effect on GJICin
vitro. These include the pesticides DDT, DDE, lindane, hep-
tachlor, and dieldrin, the peroxisome proliferators nafenopin,
Wy-14,643, clofibrate, and di-2-ethylhexyl-phthalate, and sev-
eral other classical tumor promoters in rodents, such as phe-
nobarbitone, the phorbol ester 12-O-tetradecanoylphorbol 13-
acetate, TCDD, and polychlorinated biphenyls (Swierenga and
Yamasaki, 1992). Importantly, inhibition of GJIC by tumor-
promoting agents has been demonstrated in several in vivoand
ex vivo studies (Ito et al., 1998; Kolaja et al., 2000a; Kru-
tovskikhet al., 1995; Mally and Chipman, 2002; Tateno et al.,
1994). A very important aspect that has received insufficient
attention is the need to distinguish between specific inhibition
of GJIC and the inhibition that may result secondarily to, orcoincidentally with, cell death. In the latter case, such as is seen
with carbon tetrachloride in rat liver (Cowles et al., 2000),
compensatory cell proliferation is likely to be a more important
tumor-promoting influence than the inhibition of gap junctions.
However, in many cases, the window between the doses re-
quired for GJ disruption and loss of cell viability is substantial
both in vitro and in vivo, and the disruption has often been
shown to be reversible (e.g., Elcock et al., 2000; Mally and
Chipman, 2002; Rivedal and Opsahl, 2001; Swierenga and
Yamasaki, 1992).
It is thought that chronic disruption of GJIC may release
potentially initiated cells from growth constraints imposed by
normal neighboring cells, resulting in clonal expansion and
ultimately tumor formation (Fig. 2). Following withdrawal of
the tumor-promoting stimulus, GJ function is restored and is
associated with reduced size of chemically induced preneoplas-
tic foci (Neveu et al., 1990). Reduced expression of GJs
following treatment with nongenotoxic carcinogens appears to
be a target organ-specific effect (Mally and Chipman, 2002;
Mesnil et al., 1988). Furthermore, the ability of some com-
pounds to modulate GJIC correlates well with sex- and species-
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specific tumor incidence (Klaunig et al., 1989; Plante et al.,
2002).Many nongenotoxic carcinogens have been shown to cause
gene hypermethylation, and this may be important in the re-
duced expression of certain tumor-suppressive genes (Sug-
imura and Ushijima, 2000). It is known that methylation is one
important mechanism for the control of Cx expression (see
above); and, in particular, the Cx26 gene promoter is found to
be hypermethylated (notably at the SP-1 site) in certain breast
cancer cell lines (Tan et al., 2002), and treatment with the
demethylating agent 5-aza-2-deoxycytidine resulted in re-ex-
pression of Cx26.
Connexin Genes and Tumor Suppression
Although cx gene mutations in tumors are rare, there is a
wealth of evidence to suggest that Cxs function as tumor
suppressors. As indicated above, the loss of GJIC is a common
feature of tumors and appears to be important in the mecha-
nism of action of many nongenotoxic carcinogens. Dominant
negative mutants of cx32 and cx26 genes also caused loss of
growth control, although this effect in some instances may be
independent of GJIC per se (Duflot-Dancer et al., 1997).
Numerous studies demonstrate that transfection of Cxs into
communication deficient tumor cells can restore growth control
in vitro and in vivo (Chen et al., 1995b; Eghbali et al., 1991;
Mehta et al., 1991; Omori and Yamasaki 1999; Rae et al.,
1998; Yano et al., 2001). Reduction of the malignant pheno-type in some of these studies was associated with reduced
saturation density, increased doubling times, reduced growth
capacity in soft agar, and delayed onset of tumor formation in
nude mice. Similarly, treatment of BALB/c 3T3 cells with
Cx43 antisense oligonucleotides resulted in inhibition of GJIC
and increased saturation density (Ruch et al.,1995). Dominant
negative inhibition of GJIC by mutant Cx43 enhanced tumor-
igenicity of a rat bladder carcinoma cell line (Krutovskikh et
al., 1998). In a coculture experiment Esinduy et al. (1995)
demonstrated inhibition of growth of neoplastically trans-
formed, communication-deficient cells by cocultured, highlycommunicating, nontransformed cells. Not only do these stud-
ies show potential for the use of connexins in gene therapy but
the upregulation of GJIC by drugs or dietary factors may also
provide chemoprevention (Trosko and Chang, 2001). Most
important will be attention to the relative expression of differ-
ent Cxs, the balance of Cx43:Cx32 being important in the GJIC
of human malignant prostate epithelial cells (Carruba et al.,
2002). A clear reduction of malignant potential of human
hepatoma cells was indicated by an inhibition of dedifferenti-
ation, reappearance of cell adhesion complex, and reduced
proliferation coupled with enhanced apoptosis through trans-
fection of Cx26 cDNA (Maramatsu et al., 2002; Yano and
Yamasaki, 2001).Additional evidence for the role of Cxs as tumor suppressors
has emerged from studies on transgenic mice lacking Cx genes.
Temmeet al. (1997) reported increased susceptibility of Cx32
knockout mice for the development of spontaneous and chem-
ically induced liver tumors. Interestingly, phenobarbitone treat-
ment did not further increase the incidence of liver tumors in
Cx32-null animals, supporting the role of Cx32 as a crucial
target in tumor promotion (Moennikeset al., 2000). Connexin
expression is also inversely correlated to metastatic potential.
The loss of cooperation between cells when GJ are disrupted
may lead to heterogeneity and cell dissociation, thus support-
ing metastasis (Carystinos et al., 2001). More specifically,
Cx26 expression can reduce invasiveness of a human hepatoma
cell line through E-cadherin expression and suppression of
matrix metalloproteinase 9 (Yano and Yamasaki, 2001). It needs
to be recognized, however, that different Cxs appear to have
contrasting effects on metastagenicity (Carystinoset al.,2001).
Role of GJIC in Bystander Effect in Tumor Therapy
GJIC may also play an important role in cancer treatment in
both radiotherapy and gene therapy. When radiotherapy is
FIG. 2. A possible mechanism whereby inhibition of GJIC may contribute to tumor development.
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carried out, the possibility arises that positive effects may be
seen in unirradiated areas of organs that may involve bystander
effects mediated both by GJIC as well as through the release of
signaling compounds into medium (Mothersill and Seymour,
2001).
Furthermore, transfection of a small number of tumor cells
with the herpes simplex virus thymidine kinase gene (HSV-TK) followed by treatment with the nucleotide analogue gan-
ciclovir (GCV) leads to phosphorylation of the latter and
incorporation into the cells DNA, thus terminating DNA po-
lymerization and leading to cell death. Tumors can enter re-
gression even when only a small number of cells are treated,
demonstrating bystander killing. This is thought to be GJIC-
mediated, as it is believed that phosphorylated GCV may pass
through gap junctions as with natural nucleotides. Such by-
stander killing of tumor cells may be enhanced by transfection
with connexin genes (Tanaka et al., 2001) provided that the
function of their products can be maintained. Another use of
connexins in gene therapy is focused on human glioblastoma.
When cultured in vitro and transformed with cx43 (Huang etal., 2001) such cells become sensitive to chemotherapeutic
drugs that induce apoptosis (i.e., etoposide, paditaxel, and
doxorubicin).
Conclusion
The apparent role of the connexin family of proteins in such
a wide range of cell functions relating to differentiation, pro-
liferation, cell death, and migration, and the multitude of
potential mechanisms of disruption, not surprisingly leads to
the implication of these molecules in the processes of carcino-
genesis and renders them potential useful targets in cancer
therapy. A major future direction to aid understanding will bethrough targeted mutagenesis (Plum et al., 2000), dominant
negative mutants (Yamasaki et al., 1999), and knock-in of
specific cx genes in mice.
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