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PubMed2003

Supplemental role of the Ames mutation assay and gap junction intercellular communication in studies of possible carcinogenic compounds from diesel exhaust particles.

This study presents a new strategy for the carcinogenic evaluation of complex chemical mixtures based on genotoxic and nongenotoxic assays. We studied the ability of organic extracts of diesel exhaust particles (DEP) to induce point mutations in five different Salmonella typhimurium strains (Ames test) and to inhibit gap junction intercellular communication (GJIC) in rat liver epithelial cell lines. A crude extract of DEP was prepared by extraction with dichloromethane (DCM), and fractionated according to polarity into five fractions: aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAH), nitro-PAH, dinitro-PAH, and polar compounds. Statistical experimental design, multivariate data analysis, and modeling were used to quantify the mutagenicity of individual and combined DEP fractions in the Ames assay. Quantitative determination of GJIC was carried out using a recently described combination of scrape loading and digital image analysis. Both assays responded to the DEP extract, but the responses were due to different fractions. The nitro-PAH fraction showed the strongest mutagenic potential, followed by the dinitro-PAH fraction. The effect on GJIC was due to the fraction containing the polar components, followed by the dinitro-PAH fraction. The extract was found to induce both basepair substitutions and frameshift mutations, through activation by bacterial nitroreductases. Hyperphosphorylation of connexin43, the major connexin in the epithelial cell lines, was less evident for DEP extract than for other communication inhibitors such as phorbol esters and growth factors, and consequently inhibitors of the protein kinase C (PKC) and mitogen-activated protein (MAP) kinase pathway were unable to counteract the inhibition by DEP extract. Since the Ames test is a well accepted method to screen for substances with genotoxic activity while inhibition of GJIC is associated with effect of tumor promoters and nongenotoxic carcinogens, it is not surprising but encouraging and interesting that the present data indicate that the two endpoints supplement each other as screening tests and in the evaluation of hazardous compounds in complex mixtures.

Animals↗
PubMedDate not supplied

The preventive effect of green tea on the gap junction intercellular communication in renal epithelial cells treated with a renal carcinogen.

OBJECTIVE: Clinical studies imply that (-)-epigallocatechin-3-gallate (EGCG), a main ingredient of green tea catechins, has a chemopreventive action against cancers and suppresses the proliferation of cancer cells. However, there is no report about its chemopreventive effect for renal cancer. We previously determined that renal carcinogens suppressed the gap junction intercellular communication (GJIC) of renal epithelial cells. In this study, we investigated the effect of EGCG on the GJIC of renal epithelial cells treated with a renal carcinogen. MATERIALS AND METHODS: Mardin-Darby canine kidney (MDCK) cells were used to determine the protective effects of EGCG on dimethylnitrosamine-induced alteration of GJIC and connexin 43 (Cx 43). The maximum concentration of EGCG was determined by the lactate dehydrogenase assay method. The scrape-loading dye transfer method was used to assess the expression and cellular localization of Cx 43. The phosphorylation status of Cx 43 was determined by Western blot analysis. RESULTS: The optimal noncytotoxic concentration of EGCG was determined to be 10 microg/ml. The levels of GJIC and Cx 43 expression were markedly decreased in MDCK cells exposed to dimethylnitrosamine. A 12-h pretreatment with EGCG greatly ameliorated the GJIC-inhibitory effects of dimethylnitrosamine. CONCLUSION: These results suggest that the preservation of GJIC may indicate the chemopreventive effect of green tea on renal epithelial cells treated with a renal carcinogen in vitro.

Animals↗
PubMed1996

Gap junctional intercellular communication of bovine luteal cells from several stages of the estrous cycle: effects of cyclic adenosine 3',5'-monophosphate.

Cellular interactions mediated by both contact-dependent and contact-independent mechanisms are probably important to maintain luteal function. The objective of the present study was to evaluate the role of cAMP in regulation of contact-dependent gap junctional intercellular communication (GJIC) of bovine luteal cells from several stages of luteal development. In experiment 1, corpora lutea (n = 5) from the mid-luteal phase of the estrous cycle were dissociated with collagenase, and cells were preincubated in a medium with serum. Then the medium was changed to serum-free media containing a cAMP agonist (dbcAMP; 1 mM) or antagonist (Rp-cAMPS; 0, 3, 10, 30, or 100 microM). In experiment 2, corpora lutea from the early (n = 7), mid- (n = 6), and late (n = 6) luteal phases of the estrous cycle were dissociated and preincubated as in experiment 1, and luteal cells were then incubated with no treatment, LH (100 ng/ml), dbcAMP (1 mM), forskolin (1 microM), Rp-cAMPS (100 microM), or LH+Rp-cAMPS. After incubation of luteal cells with treatments for 18-24 h, media were collected for determination of progesterone and cAMP concentrations. Then the rate of GJIC was evaluated for selected cells (small luteal cells in contact with small luteal cells, and large luteal cells in contact with small luteal cells) by using the fluorescence recovery after photo-bleaching technique and laser cytometry. In experiment 1, dbcAMP increased (p < 0.01) but Rp-cAMPS (p < 0.05) decreased GJIC between small luteal cells and between large and small luteal cells. In addition, dbcAMP stimulated (p < 0.01) but Rp-cAMPS did not affect progesterone secretion. In experiment 2, treatments affected (p < 0.05) GJIC and progesterone production of luteal cells from the mid- and late but not from the early luteal phase of the estrous cycle. GJIC between small luteal cells was increased (p < 0.01) by LH, dbcAMP, and forskolin. GJIC between large and small luteal cells was increased (p < 0.05) by dbcAMP and forskolin. Rp-cAMPS decreased (p < 0.01) GJIC between small luteal cells (mid-luteal phase) and between large and small luteal cells (mid- and late luteal phases). In addition, Rp-cAMPS inhibited (p < 0.05) the stimulatory effects of LH on GJIC between small luteal cells from the mid- and late luteal phases of the estrous cycle. For luteal cells from the mid- and late luteal phases, progesterone production was increased (p < 0.05) by LH, dbcAMP, forskolin, and LH+Rp-cAMPS, but was not affected by Rp-cAMPS. Across all stages of the estrous cycle, cyclic AMP accumulation in media was greater (p < 0.05) in LH- and forskolin-treated cultures than in control cultures and was greater (p < 0.01) in forskolin-treated than in LH-treated cultures. These data demonstrate that small and large luteal cells communicate with each other and that the rate of GJIC is modulated by LH and cAMP, as has been shown previously for other cell types. Thus, cAMP appears to be involved in the regulation of GJIC within the bovine corpus luteum, which probably is an important mechanism for coordinating luteal cell function.

Animals↗
PubMed2003

Cells heterozygous for the ApcMin mutation have decreased gap junctional intercellular communication and connexin43 level, and reduced microtubule polymerization.

Mutations in the tumour suppressor gene adenomatous polyposis coli (Apc) are early and critical events in the development of colon cancer. In the absence of functional Apc, beta-catenin is not degraded in the cytoplasm and can be transported to the nucleus and turn on transcription of several genes, including the gap junction protein connexin43. Apc also stabilizes microtubules and regulates microtubule polymerization. Changes in Wnt signalling and microtubule function are reported to affect the connexin level. To study the effect of heterozygous Apc mutation we examined gap junctional intercellular communication (GJIC) in IMCE (Immorto-Min colonic epithelium) cells with one mutated Apc allele and in YAMC (Young adult mouse colon) cells with normal Apc function. IMCE cells had only half the GJIC level compared with YAMC cells. RT-PCR showed that both YAMC and IMCE cells express a common complement of seven connexin genes (Cx26, Cx31, Cx39, Cx40, Cx43, Cx45 and Cx50), with an additional Cx29 gene expression in YAMC cells. We found that the Cx43 level was correspondingly lower in IMCE cells as detected by western blotting and immunofluorescence. There were no differences in the level or localization of beta-catenin and the downstream gene E-cadherin between the cells, indicating no activation of the Wnt-signalling pathway in cells with one mutated Apc allele. We also examined the microtubule polymerization rate, and IMCE cells had markedly slower microtubule polymerization than YAMC cells. Hence, it appears that mutation in one Apc allele is sufficient to affect microtubule function, while inactivation of both wild-type Apc alleles may be necessary for activation of Wnt signalling. Reduction in GJIC and Cx43 level in IMCE cells may be caused by reduced Cx43 transport as a result of alterations in microtubule function.

Animals↗
PubMed1999

The effect of acrylonitrile on gap junctional intercellular communication in rat astrocytes.

Rats chronically exposed to acrylonitrile (ACN) have shown a dose-dependent increase in the incidence of astrocytomas in the brain. The mechanism(s) by which ACN induces cancer in rodents has not been established. ACN does not appear to be directly genotoxic in the brain and thus a nongenotoxic mode of action has been proposed. Inhibition of gap junctional intercellular communication (GJIC) has been shown to be a property of many nongenotoxic carcinogens. The present study examined the effects of ACN on GJIC in a rat astrocyte transformed cell line, DI TNC1 cells (a target cell for ACN carcinogenicity) and primary cultured hepatocytes (a nontarget cell for ACN carcinogenicity). ACN inhibited GJIC in rat astrocytes in a dose-dependent manner. Inhibition of GJIC was observed following 2 h treatment with 0.10 mmol/L and 1.00 mmol/L ACN. However, in primary cultured hepatocytes, ACN exposed did not result in inhibition of GJIC even after 48 h of continued treatment. In the astrocytes, GJIC inhibition plateaued after 4 h of treatment and remained blocked throughout the entire experimental period examined. Inhibition of GJIC in DI TNC1 cells was reversed by removal of ACN from the culture medium after 4 or 24 h of treatment. Cotreatment of astrocytes with vitamin E reduced the effect of ACN-induced inhibition of GJIC. Similarly, inhibition of GJIC was prevented by treatment with 2-oxothiazolidine-4-carboxylic acid (OTC), a precursor of glutathione synthesis. Decreasing cellular glutathione by treatment with buthionine sulfoxamine alone (without ACN) did not affect GJIC in astrocytes. Collectively, these results demonstrate that treatment with ACN caused a selective inhibition of GJIC in rat DI TNC1 astrocytes (the target cell type), but not in rat hepatocytes (a nontarget tissue). Inhibition of GJIC in astrocytes was reversed by treatment with antioxidants and suggests a potential role for oxidative stress in ACN-induced carcinogenesis.

Acrylonitrile↗
PubMed1998

Inhibition of gap junctional intercellular communication by perfluorinated fatty acids is dependent on the chain length of the fluorinated tail.

Perfluorinated fatty acids (PFFAs), such as perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA), are known peroxisome proliferators and hepatocarcinogens. A causal link between an increase in the oxidative stress by peroxisomes and tumor promotion has been proposed to explain the hepatocarcinogenicity of PFOA and PFDA. However, the down-regulation of gap junctional intercellular communication (GJIC) has also been linked to the tumor-promoting properties of many carcinogens. Therefore, the effect of PFFAs on GJIC in WB-rat liver epithelial cells was determined. The chain length of the PFFAs tested for an effect on GJIC ranged from 2 to 10, 16 and 18 carbons. Carbon lengths of 7 to 10 inhibited GJIC in a dose-response fashion, whereas carbon lengths of 2 to 5, 16 and 18 did not appreciably inhibit GJIC. Inhibition occurred within 15 min and was reversible, with total recovery from inhibition occurring within 30 min after the removal of the compound from the growth medium. This short time of inhibition suggests that GJIC was modified at the post-translational level. Also, this short time period was not long enough for peroxisome proliferation. The post-translational modification of the gap junction proteins was not a consequence of altered phosphorylation as determined by Western blot analysis. Perfluorooctanesulfonic acid also inhibited GJIC in a dose-response fashion similar to PFDA, indicating that the determining factor of inhibition was probably the fluorinated tail, which required 7-10 carbons. Our results suggest that PFFAs could potentially act as hepatocarcinogens at the level of gap junctions in addition to or instead of through peroxisome proliferation.

Animals↗
PubMed1996

Gap junctional intercellular communication of bovine luteal cells from several stages of the estrous cycle: effects of prostaglandin F2 alpha, protein kinase C and calcium.

Cellular interactions mediated by both contact-dependent and contact-independent mechanisms are probably important to maintain luteal function. The present studies were performed to evaluate the effects of luteotropic and luteolytic hormones, and also intracellular regulators, on contact-dependent gap junctional intercellular communication (GJIC) of bovine luteal cells from several stages of luteal development. Bovine corpora lutea (CL) from the early, mid and late luteal phases of the estrous cycle were dispersed with collagenase and incubated with no treatment, LH, PGF or LH + PGF (Experiment 1), or with no treatment, or agonists or antagonists of protein kinase C (TPA or H-7) or calcium (A23187 or EGTA; Experiment 2). After incubation, media were collected for determination of progester-one concentrations. Then the rate of GJIC was evaluated for small luteal cells in contact with small luteal cells, and large luteal cells in contact with small luteal cells by using the fluorescence recovery after photobleaching technique and laser cytometry. Luteal cells from each stage of the estrous cycle exhibited GJIC, but the rate of GJIC was least (P < 0.05) for luteal cells from the late luteal phase. LH increased (P < 0.05) GJIC between small luteal cells from the mid and late but not the early luteal phase. PGF increased (P < 0.05) GJIC between small luteal cells from the mid luteal phase and diminished (P < 0.05) LH-stimulatory effects on GJIC between small luteal cells from the late luteal phase. Throughout the estrous cycle, TPA decreased (P < 0.05) the rate of GJIC between large and small, and between small luteal cells, and A23187 decreased (P < 0.05) the rate of GJIC between large and small luteal cells. LH and LH + PGF, but not PGF alone increased (P < 0.05) progesterone secretion by luteal cells from the mid and late luteal phases. Agonists or antagonists of PKC or calcium did not affect progesterone secretion by luteal cells. These data demonstrate that both luteal cell types communicate with small luteal cells, and the rate of communication depends on the stage of luteal development. LH and PGF affect GJIC between small luteal cells during the fully differentiated (mid-luteal) and regressing (late luteal) stages of the estrous cycle. In contrast, at all stages of luteal development, activation of PKC decreases GJIC between small and between large and small luteal cells, whereas calcium ionophore decreases GJIC only between large and small luteal cells. Luteotropic and luteolytic hormones, and intracellular regulators, may be involved in regulation of cellular interactions within bovine CL which likely is an important mechanism for coordination of luteal function.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2003

Diallyl disulfide (DADS) enhances gap-junctional intercellular communication by both direct and indirect mechanisms in rat liver cells.

Diallyl disulfide (DADS), a sulfur compound from garlic has been shown to exert many biological effects: induction of carcinogen detoxication, inhibition of tumor cell proliferation, etc. These effects are consistent with its anticarcinogenic properties in animal models and could account for garlic protective effects in humans. Our study demonstrates that DADS can improve gap-junctional intercellular communication (GJIC) in vitro. In rat liver epithelial cells (REL cells), using the dye transfer assay, we observe a time-dependent stimulation of GJIC by DADS at non-cytotoxic concentrations. In addition, incubation of cells with DADS for 1 h prevents the inhibition of GJIC induced by 3,5-di-tertio-butyl-4-hydroxytoluene (BHT). We have studied the direct effects of DADS on the regulation of GJIC, and especially on the expression and localization of the connexin expressed in these cells (Cx43): the enhancement of dye transfer (x1.6) by DADS from 1 to 50 micro M is associated with an increase (x1.3-1.8) in the amount of Cx43 protein (western blotting) with no alteration of its localization in the cell-cell contact regions of the plasma membrane (immunofluorescence analysis). We have also explored the possibility that DADS might act indirectly on GJIC. On one hand, DADS does not change the amount of E-cadherin, the adhesion molecule expressed in epithelial cells. On the other hand, it induces rapid inhibition of protein glycosylation. The data suggest that DADS could reduce local constraints imposed by glycoproteins, thus facilitating dye transfer. In conclusion, DADS can be included with other plant microconstituents, which have been demonstrated to improve GJIC. Its effect on REL cells can be explained by its ability to enhance the amount of Cx43 and also to diminish the level of glycosylated proteins.

Allyl Compounds↗
PubMed1995

The gap junctional intercellular communication is no prerequisite for the stabilization of xenobiotic metabolizing enzyme activities in primary rat liver parenchymal cells in vitro.

In primary monocultures of adult rat liver parenchymal cells (PC), the activities of the xenobiotic metabolizing enzymes microsomal epoxide hydrolase (mEHb), soluble epoxide hydrolase (sEH), glutathione S-transferases (GST), and phenolsulfotransferase (ST) were reduced after 7 d to values below 33% of the initial activities. Furthermore, the gap junctional intercellular communication (GJIC), measured after microinjection by dye transfer, decreased from 90% on Day 1 to undetectable values after 5 d in monoculture. Co-culture of PC with nonparenchymal rat liver epithelial cells (NEC) increased (98% on Day 1) and stabilized (82% on Day 7) the homotypic GJIC of PC. Additionally, most of the measured xenobiotic metabolizing enzyme activities were well stabilized over 1 wk in co-culture. Because GJIC is one of several mechanisms playing an important role in cell differentiation, the importance of GJIC for the stabilization of xenobiotic metabolizing enzymes in PC was investigated. PC in monoculture were, therefore, treated with 2% dimethyl sulfoxide (DMSO), a differentiation promoting factor, and 1,1,1-trichloro-2,2,-bis (p-chlorophenyl) ethane (DDT) (10 micrograms/ml), a liver tumor promotor and inhibitor of GJIC, was given to co-cultures of PC with NEC. DMSO significantly stabilized (68% on Day 7), while DDT significantly inhibited (8% on Day 7) homotypic GJIC of PC in the respective culture systems. In contrast, the activities of mEHb, sEH, GST, and ST were not affected in the presence of DMSO or DDT. These results lead to the assumption that the differentiation parameters measured in this study (i.e., homotypic GJIC and the activities of xenobiotic metabolizing enzymes) are independently regulated in adult rat liver PC.

Animals↗
PubMed2005

Metabolites of benzene are potent inhibitors of gap-junction intercellular communication.

Chronic exposure to benzene has been shown to lead to bone marrow depression and the development of leukemia. The mechanism underlying the carcinogenicity of benzene is unknown, although a number of genetic changes including chromosomal aberrations have been associated with benzene toxicity. Metabolism of benzene is required for the induced toxicological effects. We have investigated the effect of trans,trans-muconaldehyde (MUC), hydroquinone (HQ), and four MUC metabolites on gap-junction intercellular communication (GJIC). Inhibition of GJIC has been considered a possible predictor of tumor promoters and non-genotoxic carcinogens, and shown to result in perturbation of hematopoiesis. MUC was found to be a strong inhibitor of GJIC (EC50=12 micromol L(-1)) in rat liver epithelial cells IAR20, with potency similar to that of chlordane (EC50=7 micromol L(-1)). HQ inhibited GJIC with an EC50 of 25 micromolmol L(-1), and the metabolite OH/CHO with an EC50 of 58 micromol L(-1). The other MUC metabolites tested, CHO/COOH and OH/COOH were weak inhibitors of GJIC whereas COOH/COOH had no effect. Benzene itself had no effect on GJIC when tested in concentrations up to 20 micromol L(-1). The relative potency observed for the metabolites on GJIC is similar to their hematotoxic effects. The effect of MUC on GJIC was observed to take place concordant with a dramatic loss of connexin 43 (Cx43) from the cells as visualized by Western blotting. Substances with the ability to inhibit Cx43-dependent GJIC have previously been observed to interfere with normal hematopoietic development. The ability of benzene metabolites to interfere with gap-junction functionality, and especially the dramatic loss of Cx43 induced by MUC, should therefore be considered as a possible mechanism for benzene-induced hematotoxicity and development of leukemia.

Animals↗
PubMed1999

Polycyclic aromatic hydrocarbons with bay-like regions inhibited gap junctional intercellular communication and stimulated MAPK activity.

Many polycyclic aromatic hydrocarbons (PAHs) are known carcinogens. A considerable amount of research has been devoted to predicting the genotoxic, tumor-initiating potential of PAHs based on chemical structure. However, information on the correlation of structure with the non-genetoxic, epigenetic events of tumor promotion is sparse. PAHs containing a bay or bay-like region were shown to be potent inhibitors of gap-junctional intercellular communication (GJIC), an epigenetic event involved in the removal of an initiated cell from growth suppression. We tested the epigenetic toxicity of PAHs containing bay-like regions by comparing the effects of methylated vs. chlorinated isomers of anthracene on the temporal activation of mitogen-activated protein kinase (MAPK) and the regulation of GJIC. Specifically, we used anthracene, 1-methylanthracene, 2-methylanthracene, 9-methylanthracene, 9,10-dimethylanthracene, 1-chloroanthracene, 2-chloroanthracene, and 9-chloroanthracene. We determined the effect of these compounds on GJIC and on the activation of extracellular receptor kinase (ERK 1 and 2), a MAPK, in F344 rat liver epithelial cells. Results showed that bay or bay-like regions, formed by either chlorine or a methyl group, reversibly inhibited GJIC at the same doses, time, and time of recovery, whereas the linear-planar isomers had no effect on GJIC. Similarly, the GJIC-inhibitory isomers also induced the phosphorylation of ERK 1 and ERK 2, while the non-inhibitory isomers had no effect on the activation of these MAPKs. MAPK activation occurred 10-20 min after the inhibition of GJIC, which indicates that MAPK is not involved in the initial regulation of GJIC; instead altered GJIC may be affecting MAPK activation. The present study revealed that there are structural determinants of PAHs, which clearly affect epigenetic events known to be involved in the non-genetoxic steps of tumor promotion. These events are the release of a cell from growth suppression involving the reduction of GJIC, followed by the activation of intracellular mitogenic events.

Animals↗
PubMedDate not supplied

TAC-101, a novel retinobenzoic-acid derivative, enhances gap junctional intercellular communication among renal epithelial cells treated with renal carcinogens.

[4-3,5-Bis(trimethylsilyl)benzamido] benzoic acido] (TAC-101), which exhibits an anti-tumor effect, can bind to retinoic acid receptors (RARs). It has retinoid-like properties, such as chemopreventive action against cancer cells. The up-regulation of connexin (Cx) expression by retinoids is well known in various epithelial cells. In this study, we investigated whether TAC-101 up-regulates gap junctional intercellular communication (GJIC) in renal epithelial cells exposed to the renal carcinogens. Madin Darby canine kidney (MDCK) cells were incubated with TAC-101 for 3 days, then briefly exposed to renal carcinogens potassium bromate (KBrO3) or dimethylnitrosamine (DMN). TAC-101 increased the expression of connexin 43 protein without affecting Cx43 phosphorylation and prevented inadequate Cx43 localisation caused by KBrO3 or DMN. Consequently, TAC-101 prevented the disruption of GJIC in MDCK cells. These data suggested that TAC-101 enhanced GJIC by up-regulating Cx43 expression and that TAC-101 might be useful for the prevention of renal cell carcinoma.

Animals↗
PubMed2005

Altered expression of connexin43 in the inhibition of gap junctional intercellular communication by chlorohydroxyfuranones in WB-F344 rat liver epithelial cells.

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA), and 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) promote foci formation in the two-stage cell transformation assay in vitro. These chlorohydroxyfuranones (CHFs) and their structural congener 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF) inhibit gap junctional intercellular communication (GJIC) in Balb/c 3T3 mouse fibroblast cells. In the present study, the effects of MX, MCA, CMCF, and MCF on GJIC were evaluated in liver cells (WB-F344 rat liver epithelial cells), the target cells of MX-induced carcinogenicity, using the scrape-loading dye transfer technique. The CHFs inhibited GJIC after 1 h exposure in a concentration-dependent fashion. The order of potency was MX>CMCF approximately MCA>MCF. In terms of the lowest observed effective concentrations, the difference in the potency was about 27-fold (MX 1.875 microM, MCF 50 microM). After a prolonged exposure period (12 h), the inhibition of GJIC by MX and CMCF remained stable, but MCA and MCF exhibited increasing inhibitory effects. After removal of the CHFs, the GJIC slowly recovered. At the transcriptional level, CHFs caused essentially no change in the level of connexin43 (Cx43) mRNA. Preincubation of cells with the protein kinase C (PKC) inhibitor did not modify the response, but the specific MEK 1 inhibitor PD98059 decreased substantially the inhibition of GJIC by all four CHFs. Activation of the mitogen-activated protein kinases (MAPKs) signaling pathway was necessary for inhibition of GJIC. CHFs did not increase the basal phosphorylation state of the Cx43 protein, but all CHFs caused a concentration-dependent degradation of the Cx43 protein. The results indicate that all the studied CHFs inhibit GJIC in WB-F344 cells by altering Cx43 expression.

Animals↗
PubMed2003

Effects of multipurpose solutions (MPS) for hydrogel contact lenses on gap-junctional intercellular communication (GJIC) in rabbit corneal keratocytes.

To ensure the effects of multipurpose solutions (MPS) for hydrogel contact lenses on the cornea, the inhibitory activity of three types of MPS on corneal cells has been evaluated with the use of scrape loading and dye transfer assay (SLDT assay) and Western blotting on rabbit corneal keratocytes (RC4). In SLDT assay, MPS-A and poloxamine showed dose-dependent inhibitory activity, suggesting the inhibitory action of MPS-A and poloxamine to gap junctional intercellular communication (GJIC) in the tested cells. Moreover, after treatment with MPS-A, the GJIC was initially inhibited within 4 h, and thereafter gradually turned to an approximately 60% level of the initial value. When MPS-A was removed from the incubation media after exposure of the cells, the recovery of GJIC was time dependent and returned to approximately initial levels at 8 h. Complete recovery was established after approximately 24 h. These findings suggested that the inhibitory action of MPS-A on corneal keratocytes was reversible. This inhibition was accompanied by a decrease in the quantity of connexin-43, which is a major protein constituting the gap junctional channel of these cells, and its change in the phosphorylation state. Taken together, it was suggested that MPS-A interacts with connexin-43, inducing an inhibitory action on GJIC.

Animals↗
PubMed1995

Redox-active chalcogen-containing glutathione peroxidase mimetics and antioxidants inhibit tumour promoter-induced downregulation of gap junctional intercellular communication between WB-F344 liver epithelial cells.

Evidence is mounting supporting a role for oxidative stress in the mechanism of tumour promotion in response to agents such as 12-O-tetradecanoylphorbol-13-acetate (TPA). In this paper we demonstrate that glutathione peroxidase-mimetic xenobiotics, ebselen, ebselen-glutathione, alpha-(phenylselenenyl) acetophenone and bis-(4-aminophenyl) telluride (at concentrations between 10 microM and 50 microM) all demonstrate protective effects on TPA-induced downregulation of gap-junctional intercellular communication (GJIC) between WB-F344 rat liver epithelial cells. These effects were, in each case, diminished if the cells were depleted of their intracellular glutathione, and potentiated if glutathione was supplemented into the incubations. Additionally, bis-(4-aminophenyl) selenide and several N-substituted analogues, possessing potent antioxidant activity but being devoid of GSH peroxidase-mimetic activity, demonstrated remedial activity against TPA-induced downregulation of GJIC. Structure-activity relationships between these molecules showed a strong correlation to the oxidation potential of the selenium atom in the compound as the bis-(4-nitrophenyl)- and bis-(4-cyanophenyl)- derivatives, which possess poor antioxidant capacity and a half-wave redox potential well above +1.0 V, did not affect TPA-induced effects on GJIC. Examination of the mechanism of action of these redox-active compounds demonstrated correlations between their abilities to (i) prevent TPA-induced downregulation of GJIC, (ii) abolish the accumulation of intracellular oxidants and (iii) prevent the hyper-phosphorylation and internalization of connexin 43 in the cells. The active compounds were also able to prevent the rapid, TPA-induced translocation of protein kinase C to the particulate fraction of the cells, without affecting phorbol ester binding. These data support a synergistic role for oxidants and other TPA-dependent responses within the cell in mediating the downregulation of GJIC. Such oxidative metabolism may play a role in the control of translocation of protein kinase C from the cytosol to membranes in response to TPA within these cells. Despite the nature of the in vitro test system studied, the data also clarify the molecular basis for a potential anti-tumour promotive effect of antioxidants, based on established redox chemistries of several series of structurally-related molecules.

Animals↗
PubMed1995

Non-genotoxic mechanisms of carcinogenesis: studies of cell transformation and gap junctional intercellular communication.

It is widely accepted that a series of genetic changes accumulate during carcinogenesis. In addition, it is likely that various non-genotoxic mechanisms also operate at different stages of carcinogenesis. It is even possible that non-genotoxic mechanisms indirectly generate genetic changes, e.g., through induction of cell proliferation, active oxygen species or cytosine methylation. This may partially explain why many carcinogens are devoid of activity when tested in the usual genetic toxicology assays. In vitro cell transformation mimics certain stages of in vivo carcinogenesis. It has therefore been proposed that both genotoxic and non-genotoxic aspects of carcinogenesis can be studied in cell transformation systems, with tumor formation by transformed cells in syngenic animals or nude mice as the endpoint. Many genotoxic as well as non-genotoxic carcinogens induce transformation of Syrian hamster embryo, murine Balb/c 3T3 and murine C3H10T1/2 cells; interaction of genotoxic and non-genotoxic mechanisms can be clearly seen in 2-stage cell transformation studies in which a genotoxic initiating agent and a non-genotoxic promoting agent act synergistically to induce transformation of rodent cells. Aberrant control of gap junctional intercellular communication (GJIC) in cell transformation and carcinogenesis is well documented. Possible genotoxic as well as non-genotoxic mechanisms involved in abnormal gap junction communication control in multistage carcinogenesis are discussed.

Animals↗
PubMed2000

Gap junction intercellular communication and cytotoxicity in normal human cells after exposure to smoke condensates from cigarettes that burn or primarily heat tobacco.

Heating tobacco, rather than burning it, reduces tobacco combustion and pyrolysis products. This study tested the hypothesis that the simplified smoke chemistry of a cigarette which primarily heats tobacco (TOB-HT) significantly reduces the potential to alter the structure or function of cellular plasma membranes relative to low "tar" 1R4F and ultra low "tar" lR5F Kentucky reference cigarettes which burn tobacco. Gap junction intercellular communication (GJIC) and lactate dehydrogenase release (LDH) were used to quantify functional and structural changes to the plasma membrane, respectively. Cigarette smoke condensate (CSC) from the mainstream smoke of TOB-HT, lR4F and 1R5F cigarettes were compared in the GJIC and LDH release assays following a 1-hr exposure in vitro. Human bronchial/tracheal epithelial cells, coronary artery endothelial cells, coronary artery smooth muscle cells, foreskin keratinocytes and the WB-344 rat liver epithelial cell line were studied. TOB-HT did not inhibit GJIC in any of the human cell types tested (P0.05) at concentrations where 1R4F and lR5F did inhibit GJIC (P<0.05). TOB-HT did not elevate LDH release (P0.05) when tested at concentrations where lR4F and lR5F did elevate LDH release (P<0.05). Our results suggest that CSC from TOB-HT cigarettes is less damaging to the structure or function of the cellular plasma membranes of a variety of human cell lines than CSC from 1R4F and 1R5F tobacco burning reference cigarettes.

Adult↗
PubMed2005

Effects of the histone deacetylases inhibitors sodium butyrate and trichostatin A on the inhibition of gap junctional intercellular communication by H2O2- and 12-O-tetradecanoylphorbol-13-acetate in rat liver epithelial cells.

The histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and sodium butyrate (NaBu) are considered as potent therapeutic agents for cancer treatment presenting therapeutic benefits with less risk of side effects. The microbial metabolite, TSA is a potent reversible and highly specific inhibitor of mammalian histone deacetylases. NaBu causes hyperacetylation of core histones with effects similar to TSA but it is not a specific inhibitor of HDACs. The gap junction is a channel in the plasma membrane of most cell types which allows direct communication (gap junctional intercellular communication; GJIC) of small molecules and ions. Modulation of GJIC is a known cellular event associated with tumor promotion. The effects of NaBu and TSA on the H(2)O(2)- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced GJIC inhibition of WB cells and the mechanisms involved in the process were assessed. TSA and NaBu exerted differential preventive effects on the H(2)O(2) and TPA-induced inhibition of GJIC as well as hyperphosphorylation of connexin43 (Cx43) in WB-F344 rat liver epithelial cells (WB cells). NaBu prevented the TPA-induced GJIC inhibition via ERK1/2 inactivation whilst TSA restored the H(2)O(2)-induced GJIC inhibition and Cx43 hyperphosphorylation by preventing p38 MAP kinase. The inhibition of tyrosine phosphorylation and down-regulation of src protein observed may also contribute to Connexin 43 dephosphorylation and GJIC restoration by TSA and NaBu partly through depletion of src protein pool. Thus, TSA and NaBu exert differential effects on chemically induced GJIC inhibition via modulation of MAP kinases and partly, tyrosine kinases.

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