toxicol. sci. 2003 chipman 146 53

8/12/2019 Toxicol. Sci. 2003 Chipman 146 53

1/8

FORUM

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

146


2/8

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.

147FORUM


3/8

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

148 FORUM


4/8

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-

149FORUM


5/8

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.

150 FORUM


6/8

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.

REFERENCES

Azzam, E. I., de Toledo, S. M., and Little, J. B. (2001). Direct evidence for the

participation of gap junction-mediated intercellular communication in the

transmission of damage signals from -particle irradiated to nonirradiated

cells. Proc. Natl. Acad. Sci. U.S.A. 98, 473478.

Bai, S., Schoenfeld, A., Pietrangelo, A., and Burk, R. D. (1995). Basal

promoter of the rat connexin 32 gene: Identification and characterization of

an essential element and its DNA-binding protein. Mol. Cell. Biol. 15,14391445.

Brosnan, C. F., Scemes, E., and Spray, D. C. (2001). Cytokine regulation of

gap-junction connectivity: An open-and-shut case or changing partners at

the nexus? Am. J. Path. 158, 15651569.

Bukauskas, F. F., Jordan, K., Bukauskiene, A., Bennet, M. V. L., Lampe, P. D.,

Laird, D. W., and Verselis, V. K. (2000). Clustering of connexin 43-

enhanced green fluorescent protein gap junction channels and functional

coupling in living cells. Proc. Natl. Acad. Sci. U.S.A. 97, 25562561.

Carruba, G., Stefano, R., Cocciadiferro, L., Saladino, F., Di Christina, A.,

Tokar, E., Quader, S. T., Webber, M. M., and Castagnetta, L. (2002).

Intercellular communication and human prostate carcinogenesis. Ann. N.Y.

Acad. Sci. 963, 156168.

Carystinos, G. D., Bier, A., and Batist, G. (2001). The role of connexin-

mediated cellcell communication in breast cancer metastasis.J. Mammary

Gland Biol. Neoplasia 6, 431440.

Chen, S. C., Pelletier, D. B., Ao, P., and Boynton, A. L. (1995b). Connexin43

reverses the phenotype of transformed cells and alters their expression of

cyclin/cyclin-dependent kinases.Cell Growth Differ. 6, 681690.

Chen, Z.-Q., Lefebvre, D., Bai, X.-H., Reaume, A., Rossant, J., and Lye, S. J.

(1995a). Identification of two regulatory elements within the promoter

region of the mouse connexin 43 gene. J. Biol. Chem. 270, 38633868.

Cowles, C., Lee, A. J., and Chipman, J. K. (2000). Carbon tetrachloride

reduces connexin 32 expression in rat liver in vivo only in association with

hepatotoxicity.Toxicology 148, 64 (Abstract).

Defamie, N., Mograbi, B., Roger, C., Cronier, L., Malassine, A., Brucker-

Davis, F., Fenichel, P., Segretain, D., and Pointis, G. (2001). Disruption of

gap junctional intercellular communication by lindane is associated with

aberrant localization of connexin43 and zonula occludens-1 in 42GPA9

Sertoli cells. Carcinogenesis 22, 15371542.

Dermietzel, R., Yancey, S. B., Traub, O., Willecke, K., and Revel, J. P. (1987).

Major loss of the 28-kD protein of gap junction in proliferating hepatocytes.

J. Cell Biol.105,

19251934.Duflot-Dancer, A., Mesnil, M., and Yamasaki, H. (1997). Dominant-negative

abrogation of connexin-mediated cell growth control by mutant connexin

genes.Oncogene 15, 21512158.

Edwards, G. O., Meldrum, R., Wharton, C. W., Botchway, S. W., and Chip-

man, J. K. (in press). Assessing the effect of ionizing radiation on a gap

junction-mediated component of the bystander effect. Proceedings British

Toxicology Society Meeting, April 2002. Toxicology.

Eghbali, B., Kessler, J. A., Reid, L. M., Roy, C., and Spray, D. C. (1991).

Involvement of gap junctions in tumorigenesis: Transfection of tumor cells

with connexin 32 cDNA retards growthin vivo.Proc. Natl. Acad. Sci U.S.A.

88, 1070110705.

Elcock, F. J., Deag, E., Roberts, R. A., and Chipman, J. K. (2000). Nafenopin

causes protein kinase C-mediated serine phosphorylation and loss of func-

tion of connexin 32 protein in rat hepatocytes without aberrant expression or

localisation. Toxicol. Sci. 56, 8694.

Elmore, E., Milman, H., and Wyatt, G. (1987). Applications of the Chinese

hamster V79 metabolic cooperation assay in toxicology.Adv. Mod. Environ.

Toxicol. 14, 265292.

Esinduy, C. B, Chang, C. C., Trosko, J. E., and Ruch, R. J. (1995). In vitro

growth inhibition of neoplastically transformed cells by non-transformed

cells: Requirement for gap junctional intercellular communication. Carci-

nogenesis16, 915921.

Fladmark, K. E., Gjertsen, B. T., Molven, A., Mellgren, G., Vintermyr, O. K.,

and Doskeland, S. O. (1997). Gap junctions and growth control in liver

regeneration and in isolated rat hepatocytes. Hepatology 25, 847855.

Hernandez-Blazquez, F. J., Joazeiro, P. P., Omori, Y., and Yamasaki, H.

(2001). Control of intracellular movement of connexins by E-cadherin in

murine skin papilloma cells. Exp. Cell Res. 270, 235247.

Holder, J. W., Elmore, E., and Barrett, J. C. (1993). Gap junction function andcancer. Cancer Res. 53, 34753485.

Huang, R. P., Hossain, M. Z., Huang, R., Gano, J., Fan, Y., and Boynton

(2001). Connexin 43 (Cx43) enhances chemotherapy-induced apoptosis in

human glioblastoma cells. Int. J. Cancer92, 130138.

Ito, S., Tsuda, M., Yoshitake, A., Yanai,T., and Masegi, T. (1998). Effect of

phenobarbital on hepatic gap junctional intercellular communication in rats.

Toxicol. Pathol. 26, 253259.

Jou, Y. S., Layhe, B., Matesic, D. F., Chang, C. C., de Feijter, A. W.,

Lockwood, L., Welsh, C. W., Klaunig, J. E., and Trosko, J. E. (1995).

Inhibition of gap junctional intercellular communication and malignant

151FORUM


7/8

transformation of rat liver epithelial cells by neu oncogene. Carcinogenesis

16,311317.

Kasten, M. M., and Giordano, A. (1998). pRb and the cdks in apoptosis and the

cell cycle. Cell Death Differ. 5, 132140.

Kausalya, P. J., Reichert, M., and Hunziker, W. (2001). Connexin45 directly

binds to ZO-1 and localizes to the tight junction region in epithelial MDCK

cells. FEBS Lett. 505, 9296.

King, L. K., and Cidlowski, J. A. (1995). Cell cycle and apoptosis: Common

pathways of life and death. J. Cell. Biochem. 58, 175180.

Klaunig, J. E., Ruch, R. J., and Lin, E. L. (1989). Effects of trichloroethylene

and its metabolites on rodent hepatocyte intercellular communication. Toxi-

col. Appl. Pharmacol. 99, 454465.

Koffler, L., Roshong, S., Kyu Park, I., Cesen-Cummings, K., Thompson, D. C.,

Dwyer-Nield, L. D., Rice, P., Mamay, C., Malkinson, A. M., and Ruch, R. J.

(2000). Growth inhibition in G(1) and altered expression of cyclin D1 and

p27(kip-1) after forced connexin expression in lung and liver carcinoma

cells. J. Cell. Biochem. 79, 347354.

Kojima, T., Sawada, N., Zhong, Y., Oyamada, M., and Mori, M. (1994).

Sequential changes in intercellular junctions between hepatocytes during the

course of acute liver injury and restoration after thioacetamide treatment.

Virchows Arch. 425, 407412.

Kolaja, K. L., Engelken, D. T., and Klaassen, C. D. (2000a). Inhibition of

gap-junctional communication in intact rat liver by nongenotoxic hepato-

carcinogens.Toxicology 146, 1522.

Kolaja, K. L., Petrick, J. S., and Klaassen, C. D. (2000b). Inhibition of

gap-junctional-intercellular communication in thyroid-follicular cells by

propylthiouracil and low iodine diet. Toxicology 143, 195202.

Kren, B. T., Kumar, N. M., Wang, S.-Q., Gilula, N. B., and Steer, C. J. (1993).

Differential regulation of multiple gap junction transcripts and proteins

during liver regeneration. J. Cell. Biol. 123, 707718.

Krutovskikh, V. A., Mesnil, M., Mazzoleni, G., and Yamasaki, H. (1995).

Inhibition of rat liver gap junction intercellular communication by tumor-

promoting agentsin vivo. Association with aberrant localization of connexin

proteins. Lab. Invest. 72, 571577.

Krutovskikh, V. A., Yamasaki, H., Tusda, T., and Asamoto, M. (1998).

Inhibition of intrinsic gap junction intercellular communication and en-

hancement of tumorigenicity of the rat bladder carcinoma cell line BC31 by

a dominant negative connexin 43 mutant. Mol. Carcinog. 23, 254261.

Lampe, P. D., Kurata, W. E., Warn-Cramer, B. J., and Lau, A. F. (1998).

Formation of a distinct connexin43 phosphoisoform in mitotic cells is

dependent upon p34cdc2 kinase. J. Cell Sci. 111, 833841.

Lefebvre, D. L., Piersanti, M., Bai, X.-H., Chen, Z.-Q., and Lye, S. J. (1995).

Myometrial transcriptional regulation of the gap junction gene, connexin-43.

Reprod. Fertil. Dev. 7, 603611.

Lin, J. H., Weigel, H., Cotrina, M. L., Liu, S., Bueno, E., Hansen, A. J., Hansen

Goldman, S., and Nedergaard, M. (1998). Gap junction-mediated propaga-

tion and amplification of cell injury. Nat. Neurosci. 1, 494500.

Loewenstein, W. R. (1981). Junctional intercellular communication: The cell-

to-cell membrane channel. Physiol. Rev. 61, 829913.

Loewenstein, W. R., and Rose, B. (1992). The cellcell channel in the control

of growth. Semin. Cell Biol. 3, 5979.

Mally, A., and Chipman, J. K. (2002). Non-genotoxic carcinogens: Early

effects on gap junctions, cell proliferation, and apoptosis in the rat. Toxi-

cology180, 233248.

Maramatsu, A., Iwai, M., Morikawa, T., Tanaka, S., Mori, T., Harad, Y., and

Okanoue, T. (2002). Influence of transfection with connexin 26 gene on

malignant potential of human hepatoma cells. Carcinogenesis23, 351358.

Mehta, P. P., Hotz-Wagenblatt, A., Rose, B., Shalloway, D., and Loewenstein,

W. R. (1991). Incorporation of the gene for a cell-cell channel protein into

transformed cells leads to normalization of growth. J. Membr. Biol. 124,

207225.

Mesnil, M., Fitzgerald, D. J., and Yamasaki, H. (1988). Phenobarbital specif-

ically reduces gap junction protein mRNA level in rat liver. Mol. Carcinog.

1, 7981.

Moennikes, O., Buchmann, A., Romualdi, A., Ott, T., Werringloer, J., Wil-

lecke, K., and Schwarz, M. (2000). Lack of phenobarbital-mediated promo-

tion of hepatocarcinogenesis in connexin32-null mice. Cancer Res. 60,50875091.

Moorby, C., and Patel, M. (2001). Dual functions for connexins: Cx43 regu-

lates cell growth independently of gap junction formation. Exp. Cell Res.

271, 238248.

Mothersill, C., and Seymour, C. (2001). Radiation-induced bystander effects:

Past history and future directions. Radiation Res. 155, 759767.

Neuhaus, I. M., Dahl, G., and Werner, (1995). Use of alternate promoters for

tissue-specific expression of the gene coding for connexin32. Gene 158,

257262.

Neveu, M. J., Hully, J. R., Paul, D. L., and Pitot, H. C. (1990). Reversible

alteration in the expression of the gap junction protein Connexin 32 during

tumor promotion in rat liver and its role during cell proliferation. Cancer

Commun.2, 2131.

Omori, Y., and Yamasaki, H. (1999). Gap-junction proteins connexin32 andconnexin43 partially acquire growth-suppressive function in HeLa cells by

deletion of their C-terminal tails. Carcinogenesis 20, 19131918.

Pandey, S., and Wang, E. (1995). Cells en route to apoptosis are characterized

by the upregulation of c-fos, c-myc, c-jun, cdc2, and RB phosphorylation,

resembling events of early cell-cycle traverse.J. Cell. Biochem58,135150.

Peracchia, C., Wang, X. G., and Peracchia L. L. (2000). Chemical gating of

gap junction channels. Methods 20, 188195.

Piechocki, M. P., Burk, R. D., and Ruch, R. J. (1999). Regulation of con-

nexin32 and connexin 43 gene expressions by DNA methylation in rat liver

cells.Carcinogenesis 20, 401406.

Piechocki, M. P, Toti, R. M., Fernstrom, M. J., Burk, R. D., and Ruch, R. J.

(2000). Liver cell-specific transcriptional regulation of connexin32. Bio-

chim. Biophys. Acta 149, 107122.

Plante, I., Charbonneau, M., and Cyr, D. G. (2002). Decreased gap junctional

intercellular communication in hexachlorobenzene-induced, gender-specific

hepatic tumor formation in the rat. Carcinogenesis 23, 12431249.

Plum, A., Hallas, G., Magin, T., Dombrowski, F., Hagendorff, A., Schuma-

cher, B., Wolpert, C., Kim, J., Lamers, W. H., Evert, M., Meda, P., Traub,

O., and Willecke, K. (2000). Unique and shared functions of different

connexins in mice. Curr. Biol. 10, 10831091.

Rae, R. S., Mehta, P. P., Chang, C. C., Trosko, J. E., and Ruch, R. J. (1998).

Neoplastic phenotype of gap-junctional intercellular communication-defi-

cient WB rat liver epithelial cells and its reversal by forced expression of

connexin 32. Mol. Carcinog. 22, 120127.

Rivedal, E., and Opsahl, H. (2001). Role of PKC and MAP kinase in EGF- and

TPA-induced connexin43 phosphorylation and inhibition of gap junction

intercellular communication in rat liver epithelial cells. Carcinogenesis 22,

15431550.

Rosenkranz, H. S., Pollack, N., and Cunningham, A. R. (1988). Nongenotoxicmechanisms in carcinogenesis: Role of inhibited intercellular communica-

tion. InBanbury Report31.Carcinogen Risk Assessment: New Directions in

the Quantitative and Qualitative Assessment Aspects (R. W. Hart and F. D.

Hoerger, Eds.), pp 139170. Cold Spring Harbor Laboratory Press, Cold

Spring Harbor, New York.

Ruch, R. J., Guan, X., and Siegler, K. (1995). Inhibition of gap junctional

intercellular communication and enhancement of growth in BALB/c 3T3

cells treated with connexin43 antisense oligonucleotides.Mol. Carcinog.14,

269274.

Saez, J. C., Berthoud, V. M., Moreno, A. P. and Spray, D. C. (1993). Gap

152 FORUM


8/8

junctions: Multiplicity of controls in differentiated and undifferentiated cells

and possible functional implications. Adv. Second Messenger Phosphopro-

tein Res. 27, 163168.

Saez, J. C., Connor, J. A., Spray, D. C., and Bennett, M. V. (1989). Hepatocyte

gap junctions are permeable to the second messenger, inositol 1,4,5-

trisphosphate, and to calcium ions. Proc. Natl. Acad. Sci. U.S.A. 86, 2708

2712.

Saez, J. C., Martinez, A. D., Branes, M. C., and Gonzalez, H. E. (1998).Regulation of gap junctions by protein phosphorylation. Braz. J. Med. Biol.

Res. 31, 593600.

Sugimura, T., and Ushijima, T. (2000). Genetic and epigenetic alterations in

carcinogenesis.Mutat. Res. 462, 235246.

Swierenga, S. H., and Yamasaki, H. (1992). Performance of tests for cell

transformation and gap-junction intercellular communication for detecting

nongenotoxic carcinogenic activity. IARC Sci. Publ. 1992 (116), 165193.

Tan, L.-W., Bianco, T., and Dubrovic, A. (2002). Variable promoter region

CpG island methylation of the putative tumor suppressor gene Connexin 26

in breast cancer. Carcinogenesis 23, 231236.

Tanaka, T., Yamasaki, H., and Mesnil, M. (2001). Stimulation of intercellular

communication of poor-communicating cells by gap-junction-competent

cells enhances the HSV-TK/GCV bystander effect in vitro. Int. J. Cancer

91,538542.

Tateno, C., Ito, S., Tanaka, M., Oyamada, M., and Yoshitake, A. (1994). Effect

of DDT on hepatic gap junctional intercellular communication in rats.

Carcinogenesis15, 517521.

Temme, A., Buchmann, A., Gabriel, H. D., Nelles, E., Schwarz, M., and

Willecke, K. (1997). High incidence of spontaneous and chemically induced

liver tumors in mice deficient for connexin 32. Curr. Biol. 7, 713716.

Temme, A., Ott, T., Dombrowski, F., and Willecke, K. (2000). The extent of

synchronous initiation and termination of DNA synthesis in regenerating

mouse liver is dependent on connexin32 expressing gap junctions.J. Hepa-

tology 32, 627635.

Trosko, J. E., and Chang, C.C. (2001) Mechanism of up-regulated gap junc-

tional intercellular communication during chemoprevention and chemother-

apy of cancer. Mutat. Res. 480481,219229.

Trosko, J. E., Chang, C. C., and Upham, B. (2002). Modulation of gap

junctional communication by epigenetic toxicants: A shared mechanism

in teratogenesis, carcinogenesis, atherogenesis, immunomodulation, repro-

ductive- and neurotoxicities. In Biomarkers of Environmental Associated

Disease(S. H. Wilson and W. A. Suk, Eds.), pp. 445454. Lewis Publish-

ers, Boca Raton, FL.

Trosko, J. E., Chang, C. C., Upham, B., and Wilson, M. R. (1998). Epigenetic

toxicology as toxicant-induced changes in intracellular signaling leading to

altered gap junctional intercellular communication.Toxicol. Lett. 102103,

7178.

Trosko, J. E., and Goodman, J. I. (1994). Intercellular communication may

facilitate apoptosis: Implications for tumor promotion. Mol. Carcinog. 11,

812.

Trosko, J. E., and Ruch, R. J. (1998). Role of cellcell communication in

carcinogenesis.Front. Biosci. 3, D208236.

Watanabe, H., and Tonosaki, A. (1995). Gap junctions in apoptosis. In

Progress in Cell Research(Y. Kanno, K. Kataoka, Y. Shiba, Y. Shibata, and

T. Shimazu, Eds.), Vol 4. Elsevier, Amsterdam.

Willecke, E., Eiberger, J., Degen, J., Eckardt, D., Romualdi, A., Guldenagel,

M., Deutsch, U., and Sohl, G. (2002). Structural and functional diversity of

connexin genes in the mouse and human genome. Biol. Chem. 383, 752

737.

Wilson, R. M., Close, T. W., and Trosko, J. E. (2000). Cell population

dynamics (apoptosis, mitosis, and cellcell communication) during disrup-

tion of homeostasis. Exp. Cell Res. 254, 257268

Yamasaki H. (1990). Gap junctional intercellular communication and carcino-

genesis.Carcinogenesis, 11, 10511058.

Yamasaki, H., Krutovskikh, V., Mesnil, M., Tanaka, T., Zaidan-Dagli, M. L.,

and Omori, Y. (1999). Role of connexin (gap junction) genes in cell growth

control and carcinogenesis. C. R. Acad. Sci. III322, 151159.

Yano, T., Hernandez-Blazquez, F. J., Omori, Y., and Yamasaki, H. (2001).

Reduction of malignant phenotype of HEPG2 cell is associated with the

expression of connexin 26 but not connexin 32. Carcinogenesis 22, 1593

1600.

Yano, T., and Yamasaki, H. (2001). Regulation of cellular invasion and matrix

metalloproteinase activity in HepG2 cell by connexin 26 transfection. Mol.

Carcinog. 3, 101109.

Yu, W., Dahl, G., and Werner, R. (1994) The connexin43 gene is responsive

to oestrogen. Proc. R. Soc. Lond. [Biol.] 255, 125132.

Zhang, Y. W., Morita, I., Ikeda, M., Ma, K. W., and Murota, S. (2001).

Connexin43 suppresses proliferation of osteosarcoma U2OS cells throughpost-transcriptional regulation of p27. Oncogene 20, 41384149.

Zhou, H., Randers-Pehrson, G., Waldren, C. A., Vannais, D., Hall, E. J., and

Hei, T. K. (2000). Induction of a bystander mutagenic effect of -particles

in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 97, 20992104.

153FORUM

toxicol. sci. 2003 chipman 146 53

Documents