TOXICOLOGICAL SCIENCES 71, 146 153 (2003) Copyright 2003 by the Society of Toxicology

FORUM Disruption of Gap Junctions in Toxicity and Carcinogenicity

J. Kevin Chipman, 1 Angela Mally, and Gareth O. Edwards
Downloaded from http://toxsci.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on March 24, 2014 School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom Received October 11, 2002; accepted November 16, 2002

Although the specic 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 protective mechanism to limit the spread of toxicity in a tissue following chemical or radiation damage. However, sustained downregulation 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 cancer per 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.

maintenance of tissue homeostasis and proliferation, the regulation of embryonic development and differentiation, and electric coupling of electrically excitable cells such as cardiomyocytes (Loewenstein, 1981). However, more recently, evidence for an intracellular signaling role of connexins in the control of cell 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 modication, assembly, trafcking, connexon docking, and gating of the gap junction channel (see Fig. 1). Several promoter response elements and transcription factors that are involved in the transcriptional regulation of connexins have been identied. 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 estrogen response elements have been identied (Chen et al., 1995a; Yu et al., 1994). The expression of cx genes can be controlled in a tissue-specic manner. For example, the liver cell-specic expression of cx32 has been shown to be dependent (at least in part) on expression of HNF-1 (Piechocki et al., 2000). Additional promoters have been identied 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 promoter has been reported in MH 1C 1 rat hepatoma cells, which express 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 important role in controlling GJIC. Decreased Cx32 mRNA halflife 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).

Structure and Function of Gap Junctions Gap junctions are intercellular membrane channels that allow the direct exchange of small molecules (1.2 kD) between adjacent cells. They are comprised of two hemichannels (connexons), 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 required 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, Ca 2, cAMP, and inositol triphosphate can pass through the gap junctions from one cell to another, while larger molecules such as proteins, complex lipids, and polysaccharides 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 communication (GJIC). A major physiological role of GJIC is the
1 To whom correspondence should be addressed. Fax: 44 121 414 5925. E-mail: j.k.chipman@bham.ac.uk.

146

FORUM

147

Downloaded from http://toxsci.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on March 24, 2014

FIG. 1.

Summary of the multiple regulatory points that control GJIC. Specicity of regulation and function are dependent on the connexin forms.

The activity of GJ is also regulated by post-translational modication of connexin proteins. Regulation can be by voltage, 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) inuences Cx trafcking, channel gating and turnover (Saez et 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 inhibition of 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 function. It is predictable, therefore, that phosphorylation is mediated by various signal transduction pathways. Another important mechanism involves the trafcking 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 mediates 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, specically ZO-1 with Cx43 and Cx45 (Defamie et al., 2001; Kausalya et al., 2001). Clustering of gap junction channels into plaques is required for functional cell coupling (Bukauskas et al., 2000). Finally, several mechanisms have been implicated in GJ degradation, including lysosomal and proteasomal pathways. It is therefore not surprising to nd that there are multiple ways by which GJIC can be regulated and disrupted (Saez et al., 1993). Aliphatic alcohols (octanol, heptanol) and anesthetics such as halothane impair gap junction activity by interfering with membrane uidity that leads to contraction of intercell channels. Other compounds have been reported to physically

148

FORUM

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 lobular 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 that downregulation of GJs may be involved as a trigger for cell proliferation (Dermietzel et al., 1987). However, Temme et al. (2000) reported that Cx32-decient mice showed normal regenerative 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 (Fladmark et 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 stimuli (Trosko and Ruch 1998). It has been suggested that downregulation of GJs during mitosis may serve to maintain separate 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 understood. 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 (Kofer 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 (Zhang et 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 proliferation in addition to junctional functionality (Duot-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 early stages 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 inuences 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 promoters 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 (generally) required for apoptosis. We have recently investigated the effects of a number of nongenotoxic carcinogens on gap junctions 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 containing Cx32 and the rate of hepatocyte apoptosis, while 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) appeared to downregulate Cx32 without effects on apoptosis. Most importantly, treatment with methapyrilene led to a dramatic loss of connexins 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

Downloaded from http://toxsci.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on March 24, 2014

FORUM

149

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 optimal 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 potential to propagate toxic responses also through the transfer of molecules 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 cellular toxicity (Peracchia et al., 2000; Saez et al. 1989). Closure of gap junctions is thought to prevent the spread of apoptotic and inammatory 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 -particles (Azzam et al., 2001; Zhou et al., 2000), stress responses (mutations, micronucleus formation, p53 phosphorylation, p21 waf1 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 signicantly reduced. 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 (Edwards et 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 cardiovascular dysfunction through loss of homeostatic control. Untimely 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 downregulation of GJIC provides a tumor-promoting stimulus. Normal, contact-inhibited cells exhibit functional GJIC, while transformed cells and many tumors are characterized by reduced expression of GJs involving aberrant localization or downregulation of Cxs and nonfunctional homologous or heterologous 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 evidence 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 GJIC in vitro. These include the pesticides DDT, DDE, lindane, heptachlor, and dieldrin, the peroxisome proliferators nafenopin, Wy-14,643, clobrate, and di-2-ethylhexyl-phthalate, and several other classical tumor promoters in rodents, such as phenobarbitone, the phorbol ester 12-O-tetradecanoylphorbol 13acetate, TCDD, and polychlorinated biphenyls (Swierenga and Yamasaki, 1992). Importantly, inhibition of GJIC by tumorpromoting agents has been demonstrated in several in vivo and ex vivo studies (Ito et al., 1998; Kolaja et al., 2000a; Krutovskikh et al., 1995; Mally and Chipman, 2002; Tateno et al., 1994). A very important aspect that has received insufcient attention is the need to distinguish between specic inhibition of GJIC and the inhibition that may result secondarily to, or coincidentally 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 inuence than the inhibition of gap junctions. However, in many cases, the window between the doses required 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 preneoplastic foci (Neveu et al., 1990). Reduced expression of GJs following treatment with nongenotoxic carcinogens appears to be a target organ-specic effect (Mally and Chipman, 2002; Mesnil et al., 1988). Furthermore, the ability of some compounds to modulate GJIC correlates well with sex- and species-

Downloaded from http://toxsci.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on March 24, 2014

150

FORUM

Downloaded from http://toxsci.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on March 24, 2014

FIG. 2.

A possible mechanism whereby inhibition of GJIC may contribute to tumor development.

specic 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 reduced expression of certain tumor-suppressive genes (Sugimura 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-expression 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 mechanism 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 (Duot-Dancer et al., 1997). Numerous studies demonstrate that transfection of Cxs into communication decient 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 phenotype 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 tumorigenicity 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 transformed, communication-decient cells by cocultured, highly communicating, nontransformed cells. Not only do these studies 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 different 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 dedifferentiation, reappearance of cell adhesion complex, and reduced proliferation coupled with enhanced apoptosis through transfection 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. Temme et al. (1997) reported increased susceptibility of Cx32 knockout mice for the development of spontaneous and chemically induced liver tumors. Interestingly, phenobarbitone treatment 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 (Moennikes et 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 supporting metastasis (Carystinos et al., 2001). More specically, 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 (Carystinos et 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

FORUM

151

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 (HSVTK) followed by treatment with the nucleotide analogue ganciclovir (GCV) leads to phosphorylation of the latter and incorporation into the cells DNA, thus terminating DNA polymerization and leading to cell death. Tumors can enter regression even when only a small number of cells are treated, demonstrating bystander killing. This is thought to be GJICmediated, as it is believed that phosphorylated GCV may pass through gap junctions as with natural nucleotides. Such bystander 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 et al., 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, proliferation, 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 carcinogenesis and renders them potential useful targets in cancer therapy. A major future direction to aid understanding will be through targeted mutagenesis (Plum et al., 2000), dominant negative mutants (Yamasaki et al., 1999), and knock-in of specic 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, 473 478. Bai, S., Schoenfeld, A., Pietrangelo, A., and Burk, R. D. (1995). Basal promoter of the rat connexin 32 gene: Identication and characterization of an essential element and its DNA-binding protein. Mol. Cell. Biol. 15, 1439 1445. 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 43enhanced green uorescent protein gap junction channels and functional coupling in living cells. Proc. Natl. Acad. Sci. U.S.A. 97, 2556 2561. 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, 156 168. Carystinos, G. D., Bier, A., and Batist, G. (2001). The role of connexinmediated cell cell communication in breast cancer metastasis. J. Mammary Gland Biol. Neoplasia 6, 431 440. 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, 681 690. Chen, Z.-Q., Lefebvre, D., Bai, X.-H., Reaume, A., Rossant, J., and Lye, S. J. (1995a). Identication 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., BruckerDavis, 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. Duot-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 Chipman, 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 growth in 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 function of connexin 32 protein in rat hepatocytes without aberrant expression or localisation. Toxicol. Sci. 56, 86 94. 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. Carcinogenesis 16, 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, 847 855. 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 and cancer. 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. Cancer 92, 130 138. 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 Downloaded from http://toxsci.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on March 24, 2014

152

FORUM transformed cells leads to normalization of growth. J. Membr. Biol. 124, 207225. Mesnil, M., Fitzgerald, D. J., and Yamasaki, H. (1988). Phenobarbital specifically reduces gap junction protein mRNA level in rat liver. Mol. Carcinog. 1, 79 81. Moennikes, O., Buchmann, A., Romualdi, A., Ott, T., Werringloer, J., Willecke, K., and Schwarz, M. (2000). Lack of phenobarbital-mediated promotion of hepatocarcinogenesis in connexin32-null mice. Cancer Res. 60, 50875091. Moorby, C., and Patel, M. (2001). Dual functions for connexins: Cx43 regulates cell growth independently of gap junction formation. Exp. Cell Res. 271, 238 248. Mothersill, C., and Seymour, C. (2001). Radiation-induced bystander effects: Past history and future directions. Radiation Res. 155, 759 767. Neuhaus, I. M., Dahl, G., and Werner, (1995). Use of alternate promoters for tissue-specic 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 and connexin43 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. Biochem 58, 135150. Peracchia, C., Wang, X. G., and Peracchia L. L. (2000). Chemical gating of gap junction channels. Methods 20, 188 195. Piechocki, M. P., Burk, R. D., and Ruch, R. J. (1999). Regulation of connexin32 and connexin 43 gene expressions by DNA methylation in rat liver cells. Carcinogenesis 20, 401 406. Piechocki, M. P, Toti, R. M., Fernstrom, M. J., Burk, R. D., and Ruch, R. J. (2000). Liver cell-specic transcriptional regulation of connexin32. Biochim. Biophys. Acta 149, 107122. Plante, I., Charbonneau, M., and Cyr, D. G. (2002). Decreased gap junctional intercellular communication in hexachlorobenzene-induced, gender-specic hepatic tumor formation in the rat. Carcinogenesis 23, 12431249. Plum, A., Hallas, G., Magin, T., Dombrowski, F., Hagendorff, A., Schumacher, 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-decient WB rat liver epithelial cells and its reversal by forced expression of connexin 32. Mol. Carcinog. 22, 120 127. 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). Nongenotoxic mechanisms in carcinogenesis: Role of inhibited intercellular communication. In Banbury Report 31. Carcinogen Risk Assessment: New Directions in the Quantitative and Qualitative Assessment Aspects (R. W. Hart and F. D. Hoerger, Eds.), pp 139 170. 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, 269 274. Saez, J. C., Berthoud, V. M., Moreno, A. P. and Spray, D. C. (1993). Gap

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. Toxicol. Appl. Pharmacol. 99, 454 465. Kofer, 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, 407 412. Kolaja, K. L., Engelken, D. T., and Klaassen, C. D. (2000a). Inhibition of gap-junctional communication in intact rat liver by nongenotoxic hepatocarcinogens. 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 tumorpromoting agents in 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 enhancement of tumorigenicity of the rat bladder carcinoma cell line BC31 by a dominant negative connexin 43 mutant. Mol. Carcinog. 23, 254 261. 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, 833 841. 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, 603 611. 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 propagation and amplication of cell injury. Nat. Neurosci. 1, 494 500. Loewenstein, W. R. (1981). Junctional intercellular communication: The cellto-cell membrane channel. Physiol. Rev. 61, 829 913. Loewenstein, W. R., and Rose, B. (1992). The cell cell channel in the control of growth. Semin. Cell Biol. 3, 59 79. Mally, A., and Chipman, J. K. (2002). Non-genotoxic carcinogens: Early effects on gap junctions, cell proliferation, and apoptosis in the rat. Toxicology 180, 233248. Maramatsu, A., Iwai, M., Morikawa, T., Tanaka, S., Mori, T., Harad, Y., and Okanoue, T. (2002). Inuence of transfection with connexin 26 gene on malignant potential of human hepatoma cells. Carcinogenesis 23, 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

Downloaded from http://toxsci.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on March 24, 2014

FORUM junctions: Multiplicity of controls in differentiated and undifferentiated cells and possible functional implications. Adv. Second Messenger Phosphoprotein 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 1,4,5trisphosphate, 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, 593 600. 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, 538 542. Tateno, C., Ito, S., Tanaka, M., Oyamada, M., and Yoshitake, A. (1994). Effect of DDT on hepatic gap junctional intercellular communication in rats. Carcinogenesis 15, 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 decient 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. Hepatology 32, 627 635. Trosko, J. E., and Chang, C.C. (2001) Mechanism of up-regulated gap junctional intercellular communication during chemoprevention and chemotherapy of cancer. Mutat. Res. 480481, 219 229. 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, reproductive- and neurotoxicities. In Biomarkers of Environmental Associated Disease (S. H. Wilson and W. A. Suk, Eds.), pp. 445 454. Lewis Publishers, Boca Raton, FL.

153

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, 8 12. Trosko, J. E., and Ruch, R. J. (1998). Role of cell cell communication in carcinogenesis. Front. Biosci. 3, D208 236. 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 cell cell communication) during disruption of homeostasis. Exp. Cell Res. 254, 257268 Yamasaki H. (1990). Gap junctional intercellular communication and carcinogenesis. 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. III 322, 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 through post-transcriptional regulation of p27. Oncogene 20, 4138 4149. 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, 2099 2104.

Downloaded from http://toxsci.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on March 24, 2014