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PubMed2001

Stimulation of gap junctional intercellular communication by thalidomide and thalidomide analogs in human fetal skin fibroblasts (HFFF2) and in rat liver epithelial cells (WB-F344).

Gap junction channels maintain cell-cell communication and are essential for the coordination of tissues, playing a pivotal role in embryonal development. Gap junctional intercellular communication (GJIC), studied here in human fetal skin fibroblasts (HFFF2) and in rat liver epithelial cells (WB-F344), was almost doubled upon exposure to thalidomide (10 microM) in the presence of NADH or NADPH (20 microM). Neither in HFFF2 nor in WB-F344 cells did any detectable alteration in GJIC occur with the thalidomide analog EM 16 (10 microM), known as a non-teratogenic compound. The thalidomide analog EM 364 (10 microM) increased GJIC without prior metabolic activation. It is suggested that GJIC modification may be related to the pharmacological and toxicological properties of thalidomide.

Angiogenesis Inhibitors↗
PubMed1996

The use of initiated cells as a test system for the detection of inhibitors of gap junctional intercellular communication.

The effects of five non-mutagenic carcinogens--Aroclor 1260, benzoyl peroxide (BP), phenobarbital (PB), 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and 1,1'-(2,2,2-trichloroethylidene)bis[4-chlorobenzene] (DDT)--on gap junctional intercellular communication (GJIC) were tested in a cell line consisting of initiated cells (3PC). Four agents suspected of tumor promotion activity--o-anisidine, clofibrate, L-ethionone and d-limonene--were also tested for their effects on GJIC. Finally sodium fluoride (NaF), whose carcinogenic property is still unclear, was tested for its effects on GJIC in the 3PC cell line. Four of the five selected tumor promoters (Aroclor 1260, BP, DDT and TPA) decreased GJIC between these initiated epidermal cells. The four non-mutagenic carcinogens with tumor-promoting activity in vivo (o-anisidine, clofibrate, L-ethionine and d-limonene) all inhibited GJIC, whereas NaF had no effect. Seven compounds (o-anisidine, Aroclor 1260, BP, DDT, L-ethionine, d-limonene and TPA) had a dose-dependent as well as time-dependent inhibitory effect on GJIC. Under the experimental conditions used, clofibrate showed only a dose-related inhibition of GJIC. PB showed no inhibitory effect on GJIC in the 3PC cell line. In order to determine the role of biotransformation in the tumor-promoting activity of PB, its effect on GJIC was also examined in the presence of an Aroclor 1254-induced rat liver homogenate (S9 mix) and in the hepatoma cell line HepG2. In the presence of rat liver homogenate PB decreased GJIC in the 3PC cell line, whereas in the HepG2 cells PB showed a time- and dose-dependent inhibitory effect. To study the potential differences in susceptibility of cells representing different stages in the process of tumor formation, the effect of the selected tumor promoters on GJIC was also investigated in primary mouse keratinocytes and in a mouse skin carcinoma-derived cell line (CA3/7). Primary keratinocytes were sometimes more (BP and clofibrate) and sometimes less sensitive (ethionine and limonene) for inhibitory effects on GJIC compared to the effects in the cell line 3PC. Except for TPA and anisidin, GJIC between the CA3/7 cells was less affected by the selected agents compared to the 3PC cell line. These results show that, during the process of tumor formation the susceptibility of cells to inhibition of GJIC by tumor promoters is variable. Overall the CA3/7 cells are less sensitive compared to 3PC cells. The susceptibility of primary keratinocytes is variable compared to 3PC cells, depending on the agent used. These results also show that GJIC is a valid parameter for testing the tumor-promoting activity of compounds. Finally, this study demonstrates that mouse keratinocyte cell lines could serve as an in vitro model for the detection of non-mutagenic carcinogens with diverse target organs in vivo. For this use the cell line consisting of initiated cells (3PC) is more sensitive than the carcinoma-derived cell line CA3/7.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2001

Stimulation of intercellular communication of poor-communicating cells by gap-junction-competent cells enhances the HSV-TK/GCV bystander effect in vitro.

We have previously shown that gap-junctional intercellular communication (GJIC) appears to play a role in the bystander effect that is observed in anticancer suicide gene therapy mediated by herpes simplex virus (HSV) thymidine kinase (tk) and ganciclovir (GCV). We now report that when connexin-expressing (Cx+) cells are present within a noncommunicating population of cells (Cx-), there is GJIC between the Cx+ and Cx- cells and that due to this stimulation of GJIC, the bystander effect also occurs when the 2 cell types are mixed. We transfected HeLa cells, which do not express any detectable level of connexin, with Cx43. The Cx+ and Cx- HeLa cells were further transfected with the tk gene, giving 4 phenotypes: Cx+tk-, Cx+tk+, Cx-tk+ and Cx-tk-. We observed GJIC between Cx+ and Cx- cells, but not between Cx- and Cx- cells, regardless of the tk genotype. Similarly, we observed the HSV-tk/GCV bystander effect in Cx+tk-/Cx-tk+ and Cx+tk+/Cx-tk- cocultures. The extent of the bystander effect in cocultures of Cx+tk- and Cx-tk+ cells was stronger than in cocultures of Cx+tk+ and Cx-tk- cells when each mixture had the same ratio of Cx+ and tk+ cells. These results suggest that Cx-expressing HeLa cells stimulate GJIC capacity between them and non-Cx-expressing HeLa cells, which mediates the bystander effect in mixtures of Cx+ cells and Cx- cells in vitro. Thus, Cx expression even in only a limited fraction of tumor cells may enhance the efficacy of the HSV-tk/GCV strategy by inducing a bystander effect.

Antiviral Agents↗
PubMed2002

Retinoids inhibit squamous cell carcinoma growth and intercellular communication.

BACKGROUND: Retinoids have been shown to inhibit the growth of squamous cell carcinoma and other malignancies. They have also been shown to alter gap junctional intercellular communication (GJIC) and the expression of connexins, the protein subunits of gap junctions. We report in this study that the alteration of GJIC by retinoids may be directly related to inhibitory effects on cell growth. MATERIALS AND METHODS: SCC-13 cells were treated with all-trans retinoic acid (tRA) and 13-cis retinoic acid (cRA) at 10(-7) and 10(-6) M concentrations in culture. No treatment and ethanol vehicle controls were included for each experiment. Serial cell counts of parallel cultures were performed to determine cell growth. The parachute technique was performed in combination with fluorescence activated cell sorting (FACS) analysis to determine GJIC. Northern and Western blot analysis were performed to assess connexin mRNA and protein expression. RESULTS: The growth rate was inhibited for cells treated with tRA (10(-6) M) (P < 0.05) and cRA (10(-6) M) (P = 0.068) vs. vehicle control. GJIC was significantly inhibited with both tRA (10(-7) and 10(-6) M) (P < 0.001) and cRA (10(-7) and 10(-6) M) (P < 0.001) at 24, 48, and 96 h as determined by FACS analysis. To correlate GJIC with cell growth, we studied the effect of glycyrrhetinic acid, a known inhibitor of GJIC. Glycyrrhetinic acid also significantly inhibited cell growth (P < 0.05) vs. control. Connexin 26 and connexin 43 mRNA and protein expression were not significantly altered after retinoid treatment. CONCLUSION: Retinoic acids inhibit both cell growth and GJIC in SCC-13 cells. Retinoids may inhibit cell growth through alteration of GJIC in SCC-13 cells.

Blotting, Northern↗
PubMed2001

Chronic effects of endothelin 1 and angiotensin II on gap junctions and intercellular communication in cardiac cells.

Endothelin-1 (ET-1) and angiotensin-II (AT-II) participate in the pathophysiology of cardiovascular diseases. Regulation of gap junctional intercellular communication may influence heart function and its response to cardiac injury. In this study, we examined the effects of ET-1 and AT-II on connexin43 (Cx43) and connexin40 (Cx40) in cultured neonatal rat ventricular cardiomyocytes (NRCs) and the role of mitogen-activated protein kinase signaling in the ET-1- and AT-II-induced responses. NRCs were incubated for 24 h with either ET-1 or AT-II (each at concentrations ranging from 10 to 1000 nM), and Cx43 expression and phosphorylation increased with increasing concentrations of both. ET-1 effects were significantly blocked by ETA (BQ123), but not by ETB (BQ788), receptor antagonists. AT-II-induced Cx43 induction could be completely inhibited by the AT1 receptor antagonist losartan. In contrast to Cx43, Cx40 expression did not change in either ET-1- or in AT-II-treated NRCs. Thus, these two connexins were differentially regulated. ET-1 and AT-II increased the gap junctional conductance between the cardiomyocytes in culture as measured using a dual-cell voltage clamp. Mitogen-activated protein kinase inhibition revealed that ERK1/2 was critical for up-regulation of Cx43 in response to ET-1, whereas both ERK and p38 signal pathways were involved in the regulation of Cx43 by AT-II. Thus, stimulation of the ERK and p38 signal pathways via ETA and AT1 receptors may partcipate in the regulation of cardiac gap junctions under (patho)physiological conditions.

Angiotensin II↗
PubMed2003

[Research on the gap junctional intercellular communication of CHL cells and human cancer cells induced by c9, t11-conjugated linoleic acid].

In order to determine the effects on the gap junctional intercellular communication(GJIC) of CHL cells and human cancer cells induced by c9, t11-CLA and its mechanism. GJIC was observed by using scrape loading and dye transfer(SLDT) on CHL cells blocked by TPA and cancer cells(SGC-7901 and MCF-7) which were treated with various c9, t11-conjugated linoleic acid (CLA) concentrations (25, 50, 100 and 200 mumol/L) of c9, t11-CLA for 24 and 48h, with a negative control. The results showed that c9, t11-CLA can recover GJIC of CHL cell blocked by TPA and similar to negative control at 200 mumol/L 48 h of c9, t11-CLA; c9, t11-CLA also can recover cancer cells(SGC-7901 and MCF-7) at doses of 24 h 100 mumol/L for MCF-7 cells and 24 h 200 mumol/L, 48 h 100 mumol/L, 200 mumol/L for SGC-7901 and MCF-7 cells. It was suggested that c9, t11-CLA can recover GJIC of CHL cells blocked by TPA and SGC-7901 and MCF-7 cells.

Adenocarcinoma↗
PubMed2005

Connexin43 synthesis, phosphorylation, and degradation in regulation of transient inhibition of gap junction intercellular communication by the phorbol ester TPA in rat liver epithelial cells.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces transient inhibition of gap junction intercellular communication (GJIC) in several cell types. The initial block in GJIC has been attributed to protein kinase C (PKC) mediated phosphorylation of connexin gap junction proteins, including connexin43 (Cx43). Restoration of GJIC, associated with normalization of the Cx43 phosphorylation status, has been ascribed to different events, including dephosphorylation of Cx43 and de novo synthesis of Cx43 or other, non-gap junctional, proteins. The data presented suggest that restoration of GJIC during continuous TPA exposure in normal and transformed rat liver epithelial cells is dependent on synthesis of Cx43 protein, as well as the transport of already synthesized Cx43 from intracellular pools to the plasma membrane. Reactivation of inactivated Cx43 by dephosphorylation does not appear to be involved in the recovery of GJIC. Both PKC and MAP kinase is involved in TPA-induced degradation of Cx43 and inhibition of GJIC. We show that coincubation of TPA with the protein synthesis inhibitor cycloheximide or the transcription inhibitor actinomycin D results in synergistic enhancement of the level of activated ERK1/2. Together, the present data highlight Cx43 degradation and synthesis as critical determinants in TPA-induced modifications of cell-cell communication via gap junctions.

Animals↗
PubMed1998

Effect of airborne particles from selected indoor and outdoor environments on gap-junctional intercellular communication.

The effect of airborne particles from diesel exhaust, rubber and metal industry, urban air and biological sources (poultry, pig farming, compost industry) on gap-junctional intercellular communication (GJIC) were compared, using HEPA1c1c7 cells. Particles as such were compared with aqueous and organic extracts. Significant inhibition of GJIC by particle suspensions was only observed for the diesel and rubber samples, and for one biological sample (compost). Up to 83% of the inhibition of the whole suspension could be attributed to the particles as such. Washing the particles with organic solvents (aceton, methanol, hexane) did not result in a significant loss of activity from the particles, although the organic fractions showed a significant activity towards GJIC. More active organics was eluted from the rubber industry particles than from the diesel particles by the organic solvent. It is suggested that cancer promoting potential as measured by inhibition of GJIC may vary widely depending on the particle source, and that this effect may be exerted by the particles as such and/or by means of tightly bound bio-active material to the surface.

Agriculture↗
PubMed2000

Prevention of the down-regulation of gap junctional intercellular communication by green tea in the liver of mice fed pentachlorophenol.

Much evidence has been documented supporting the hypothesis that the down-regulation of gap junctional intercellular communication (GJIC) is a cellular event underlying the tumor promotion process and that treatment to prevent the down-regulation or to up-regulate GJIC is important in preventing tumor promotion. We explored the potential preventive effects of green tea against the promoting action of pentachlorophenol (PCP) in mouse hepatocarcinogenesis, examining whether drinking green tea prevents the down-regulation of GJIC inhibition in the liver caused by tumorigenic doses of PCP. We used a modified in vivo GJIC assay, the incision loading/dye transfer method. Male B6C3F1 mice were given a green tea infusion for 1 week and then PCP was fed at a dose of 300 or 600 p.p.m. in the diet for the following 2 weeks, along with green tea treatment. A dose-related inhibition of GJIC in the hepatocytes was evident in the mice treated with PCP alone that was associated with a reduction in connexin32 (Cx32) plaques in the plasma membrane and an increase in the cell proliferation index. Drinking green tea significantly protected mice against GJIC inhibition, the reduction in Cx32 and the elevation of the labeling index. These findings suggest that green tea might act as an anti-promoter against PCP-induced mouse hepatocarcinogenesis via its ability to prevent down-regulation of GJIC.

Animals↗
PubMed2002

[Downregulation of gap junctional intercellular communication induced by silicon dioxide in the pulmonary alveolar epithelial cell].

OBJECTIVE: To explore the effect of silica dioxide(SiO2) on proliferation and downregulation of gap junctional intercellular communication (GJIC) in pulmonary alveolar epithelial cells (CCL-64 cells). METHODS: The pulmonary alveolar macrophages(PAMs) were incubated in the serum-free RPMI 1640 containing the various concentration of SiO2 for 24 hours. The supernatants were prepared and added 5% (V/V) into 2% (V/V) NBS RPMI 1640 to stimulate the proliferation of CCL-64 cells for 24 hours. A set of "blank control", run in parallel, contained RPMI 1640 + 2% (V/V) NBS alone. The proliferation of CCL-64 cells was detected using MTT assay(to show as the absorbency, A570nm). GJIC function was measured using the fluorescence redistribution after photobleaching(FRAP) assay [to express as the transfer rate of the fluorescence, K (x 10(-3)/s)], with a laser scanning confocal microscope(LSCM, Leica TCS SP). RESULTS: The silica-exposed PAM supernatants could induce both the proliferation(F = 9.679, P < 0.01) and downregulation of GJIC(F = 20.587, P < 0.01) of CCL-64 cells. In the range of 50-500 micrograms/ml SiO2 concentrations, the proliferation (A570nm values) and GJIC(the transfer rate, K) were fitted well in a dose-dependent manner(proliferation: r = 0.891, P < 0.05; GJIC: r = -0.943, P < 0.05). CONCLUSION: By way of stimulating the PAM, SiO2 could inhibit GJIC function in lung alveolar epithelial cells, and induce epithelial cell proliferation. In the pathogenesis of silicosis, the downregulation of GJIC of the pulmonary epithelial cells may play an important role in silica-mediated alveolar epithelial cell injury.

Cell Communication↗
PubMed1993

Inhibition of intercellular communication in rat hepatocytes by phenobarbital, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and gamma-hexachlorocyclohexane (lindane): modification by antioxidants and inhibitors of cyclo-oxygenase.

Several tumour promoting chemicals have been shown to inhibit intercellular communication (IC) through gap junctions in cell cultures. In the present investigation we studied the effect of the hepatic tumour promoters phenobarbital (PB), 1,1,1-trichloro-2,2-(p-chlorophenyl)ethane (DDT) and gamma-hexachlorocyclohexane (lindane) on IC in rat hepatocyte cultures. IC was evaluated by microinjection of fluorescent Lucifer Yellow CH dye and visualization of dye spread to adjacent hepatocytes. Incubation of hepatocytes with PB (2 mM), DDT (30 microM) and lindane (25 microM) decreased dye-coupling of the cells by about 30%, 42% and 35%, respectively; dye-coupling in untreated cultures was 88.1 +/- 0.7%. Inhibition of IC was reversible when the xenobiotics were removed from the medium. The antioxidant vitamin E (100 microM) prevented inhibition of dye-coupling by PB and lindane and partially that by DDT. Superoxide dismutase (100 units/microliters) counteracted the effect on dye-coupling by PB, but not that by the insecticides. Similarly, the cyclo-oxygenase inhibitors indomethacin and aspirin only reversed the effect of PB on IC, but not that of DDT or lindane. As indicated by further experiments, prevention by non-steroidal anti-inflammatory agents of PB-induced inhibition of IC is most likely not mediated by inhibition of cyclo-oxygenase. The results indicate significant differences in the action of PB, DDT and lindane on IC in hepatocyte cultures. This is suggested by the differential effects of superoxide dismutase and non-steroidal anti-inflammatory agents on the action of the three tumour promoting chemicals. Whereas superoxide radicals may be involved in the inhibition of dye-coupling by PB, radical intermediates of the insecticides may be responsible for the decrease in dye-coupling by DDT and lindane.

Animals↗
PubMed1995

Cardiac intercellular communication: consequences of connexin distribution and diversity.

Gap junctions contain channels which allow the exchange of ions and small molecules between adjacent cells. In the heart, these channels are crucial for normal intercellular current flow and the propagation of action potentials throughout the myocardium. Molecular cloning studies have demonstrated that these channels are formed by members of a family of related proteins called connexins each containing conserved and unique regions. There are several consequences of this multiplicity of connexins. Multiple connexins are expressed in differing, but sometimes overlapping, distributions within cardiovascular and other tissues. Connexin40, connexin43, and connexin45 are all found in cardiac myocytes, but their abundance differs in specialized cardiac regions with disparate conductive properties. Individual connexins form channels with differing voltage-dependence, conductance, and permeability properties, as demonstrated by functional expression of the cloned sequences. Connexins differ in their modification by phosphorylation, which may contribute to physiological regulation of intercellular communication. Expression of multiple connexins may lead to the formation of multiple channel types in a single tissue or cell and potentially allows mixing to form heterotypic and/or heteromeric channels. Thus, multiple connexins may contribute to the differences in intercellular resistance in cardiac regions with differing conductive properties and possibly may allow differences in the signalling molecules that pass between cells.

Animals↗
PubMed2006

Noise magnetic fields abolish the gap junction intercellular communication suppression induced by 50 hz magnetic fields.

Previously, we have reported that exposure to 50 Hz coherent sinusoidal magnetic fields (MF) for 24 h inhibits gap junction intercellular communication (GJIC) in mammalian cells at an intensity of 0.4 mT and enhances the inhibition effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) at 0.2 mT. In the present study, we further explored the effects of incoherent noise MF on MF-induced GJIC inhibition. GJIC was determined by fluorescence recovery after photobleaching (FRAP) with a laser-scanning confocal microscope. The rate of fluorescence recovery (R) at 10 min after photobleaching was adopted as the functional index of GJIC. The R-value of NIH3T3 cells exposed to 50 Hz sinusoidal MF at 0.4 mT for 24 h was 30.85 +/- 14.70%, while the cells in sham exposure group had an R-value of 46.36 +/- 20.68%, demonstrating that the GJIC of NIH3T3 cells was significantly inhibited by MF exposure (P < .05). However, there were no significant differences in the R-values of the sham exposure, MF-plus-noise MF exposure (R: 49.58 +/- 19.38%), and noise MF exposure groups (R: 46.74 +/- 21.14%) (P > .05), indicating that the superposition of a noise MF alleviated the suppression of GJIC induced by the 50 Hz MF. In addition, although MF at an intensity of 0.2 mT synergistically enhanced TPA-induced GJIC inhibition (R: 24.90 +/- 13.50% vs. 35.82 +/- 17.18%, P < .05), further imposition of a noise MF abolished the synergistic effect of coherent MF (R: 32.51 +/- 18.37%). Overall, the present data clearly showed that although noise MF itself had no effect on GJIC of NIH3T3 cells, its superposition onto a coherent sinusoidal MF at the same intensity abolished MF-induced GJIC suppression. This is the first report showing that noise MF neutralizes 50 Hz MF-induced biological effect by using a signaling component as the test endpoint.

Animals↗
PubMed2004

Inhibition of gap junction intercellular communications in cell culture by polycyclic aromatic hydrocarbons (PAH) in the absence of PAH metabolism.

We have studied the effect of polycyclic aromatic hydrocarbons (PAH) on gap junction intercellular communications (GJIC) in culture of hepatoma cells Hep G2 and G27. Carcinogenic PAH inhibited GJIC in both cultures in contrast to non-carcinogenic PAH. We showed that both constitutive and inducible expressions of mRNAs of Ah receptor and cytochrome P4501A1 (the main isoform involved in PAH metabolism) were absent in hepatoma G27 cells. We concluded that the initial, non-metabolized molecules of carcinogenic PAH are responsible for changes in GJIC through interaction with an unknown factor in the cellular membrane.

Base Sequence↗
PubMed2004

Anisomycin downregulates gap-junctional intercellular communication via the p38 MAP-kinase pathway.

Phosphorylation of connexin 43 (Cx43) molecules (e.g. by extracellular signal-regulated kinase) leads to reductions in gap-junctional intercellular communication (GJIC). GJIC levels also appear to be lower in the presence of p38 mitogen-activated protein (MAP) kinase, for unknown reasons. In this study, we used assays of the recovery of fluorescence by photobleached WB-F344 cells to demonstrate that GJIC levels are decreased by anisomycin [a protein synthesis inhibitor as well as an activator of p38 MAP kinase and c-Jun N-terminal kinases (JNK)] as a result of time-dependent depletion of the phosphorylated forms of Cx43. Using immunohistochemistry, we also detected far less of the Cx43 proteins at cell borders. These findings agree with the photobleaching assay results. Moreover, prior treatment with SB203580 (a specific inhibitor of p38 MAP kinase) appeared to be effective in preventing the loss of phosphorylated forms of Cx43 and the loss of Cx43 proteins at cell borders. Total protein labelling with [(35)S]-methionine and [(32)P]-orthophosphates labelling of Cx43 showed that anisomycin enhanced the phosphorylation level of Cx43 along with inhibition of protein synthesis. SB203580 prevented the former but not the latter. The effect of anisomycin on GJIC was not dependent on the inhibition of protein synthesis because the addition of SB203580 completely maintained the level of GJIC without restoring protein synthesis. The Cx43 phosphorylation level increased by anisomycin treatment, whereas the amount of phosphorylated forms of Cx43 decreased, suggesting that activation of Cx43 phosphorylation might lead to the loss of Cx43. These results suggest that activation of p38 MAP kinase leads to reduction in the levels of phosphorylated forms of Cx43, possibly owing to accelerated degradation, and that these losses might be responsible for the reduction in numbers of gap junctions and in GJIC.

Animals↗
PubMed1994

Inhibition of gap junctional intercellular communication in Syrian hamster embryo cells by TPA, retinoic acid and DDT.

12-O-Tetradecanoylphorbol-13-acetate (TPA), trans-retinoic acid (RA) and DDT inhibit gap junctional intercellular communication in Syrian hamster embryo (SHE) cells. The inhibition is rapid and takes place within minutes. Northern blot analysis shows that SHE cells express connexin 43 and that exposure to these compounds for up to 20 h has no effect on connexin 43 mRNA level. Immune cytochemistry shows that the connexon structures in SHE cells are scattered over the cell, and not confined to the cell-cell boundaries as is the case in the rat liver epithelial cell line IAR20. RA and TPA induce the disappearance of the connexon structures in parallel to the induced inhibition of communication in SHE cells. The disappearance of the connexon spots takes place with no apparent effect on the cellular content of connexin protein measured by immunoblotting, and is probably caused by disaggregation of the connexon structures rather than disappearance or degradation of the connexin protein. DDT shows little or no apparent effect on connexin immunostaining in SHE cells, indicating a different mechanism of action. In the IAR20 cells, exposure to TPA and RA also results in loss of immunostainable connexon structures while exposure to DDT results in relocalization of the connexons away from the cell-cell borders. Immunoblotting of connexin 43 in SHE cells results in three major bands with apparent mol. wts of 40-50 kDa where the two higher mol. wt bands represent phosphorylated connexin 43 protein. Exposure of the cells to the communication inhibiting compounds results in reduction or loss of the highest mol. wt phosphorylated band, indicating a relation between a specific connexin phosphorylation and aggregation of connexin 43 protein to functional communicating gap junctions. The results suggest the presence of various post-transcriptional control mechanisms in the regulation of connexin function which are vulnerable to exogenous stimuli.

Animals↗
PubMed1993

Phosphatase inhibitors, gap junctional intercellular communication and [125I]-EGF binding in hamster fibroblasts.

A number of phosphatase inhibitors (okadaic acid, calyculin A, aluminium fluoride, sodium molybdate, sodium orthovanadate, pervanadate and vanadyl sulphate) were investigated for their effects on gap junctional intercellular communication (GJIC) and [125I]-epidermal growth factor (EGF) binding in early passage Syrian hamster embryo cells (mainly fibroblast-like cells) and in V79 Chinese hamster lung fibroblasts. Only pervanadate decreased GJIC significantly. After the initial pervanadate-induced decrease the GJIC recovered rapidly. Only pervanadate was able to change the band pattern of the gap junction protein connexin43 (cx43) in Western blots. Together this may indicate either that there is a low turnover of phosphate groups in cx43 under basal conditions or that the putative phosphatases are not sensitive to most of the phosphatase inhibitors applied. In contrast, pervanadate, orthovanadate and molybdate decreased [125I]-EGF binding. 12-O-Tetradecanoylphorbol-13-acetate (TPA) is able to induce the phosphorylation of both cx43 and the EGF receptor, concomitantly with a decrease in GJIC and [125I]-EGF binding. These effects are reversible after removal of TPA. It could be imagined that other phosphatases would act on cx43 and the EGF receptor after the forced phosphorylation of the two molecules. Thus TPA was used to downregulate GJIC and [125I]-EGF binding and phosphatase inhibitors were applied in the upregulation phase. Only pervanadate affected the upregulation of GJIC, and pervanadate, orthovanadate and molybdate affected the upregulation of [125I]-EGF binding. Thus it is not an identical complement of phosphatases that act on cx43 and the EGF receptor. All the downregulating agents are assumed to be phosphotyrosine phosphatase inhibitors.

Aluminum Compounds↗
PubMed2001

Upregulation of gap junctional intercellular communication and connexin 43 expression by cyclic-AMP and all-trans-retinoic acid is associated with glutathione depletion and chemosensitivity in neuroblastoma cells.

PURPOSE: Downregulation of gap junctional intercellular communication (GJIC) has been implicated in carcinogenesis. This is a result of altered expression of connexins, the proteins that mediate GJIC, including connexin 43 (Cx43). Our aim was to evaluate the effect of known inducers of Cx43 on the chemosensitivity of the human neuroblastoma cell line IMR-32 to chemotherapeutic agents. METHODS: We examined the effect of dibutyryl-cyclic AMP (db-cAMP) and all-trans-retinoic acid (tRA) on Cx43 and GJIC, glutathione (GSH) and gamma-glutamyl-cysteine-synthetase (gamma-GCS) levels, and glutathione S-transferase (GST) activity. Finally, we performed cell survival assays to measure the response of IMR-32 cells to the chemotherapeutic drugs doxorubicin, melphalan and bis-chloronitrosourea (BCNU), after treatment with db-cAMP and/or tRA. RESULTS: Exposure to db-cAMP led to the upregulation of GJIC and Cx43 expression and phosphorylation. On the other hand, exposure to tRA led to the upregulation of GJIC but Cx43 expression and phosphorylation were not greatly affected. The combination of both agents was more potent in inducing GJIC in comparison to treatment with db-cAMP or tRA alone. Treatment with db-cAMP, but not with tRA, was associated with a significant increase in the cytotoxic effects of the anticancer drugs doxorubicin, melphalan and BCNU as shown by a decrease in their IC50 values. Concomitant exposure to db-cAMP and tRA, however, had a more pronounced effect on cell sensitization to chemotherapy drugs (particularly doxorubicin) than exposure to db-cAMP or tRA alone. Under the db-cAMP and tRA treatment conditions (which upregulate GJIC and modulate drug response), GSH levels were significantly reduced while the levels of GST and gamma-GCS activities remained unchanged. CONCLUSIONS: This study suggests that GJIC plays a role in cellular drug resistance, and highlights the potential use of GJIC modulators in combination with chemotherapy. Also, this is the first study exploring the ability of both db-cAMP and tRA to enhance cell chemosensitivity.

Bucladesine↗