Resveratrol counteracts gallic acid-induced down-regulation of gap-junction intercellular communication

Since reactive oxygen species (ROS) play a key role in carcinogenesis, many studies have focused on the chemopreventive activities of naturally occurring antioxidants. However, the possibility that different antioxidants in food exert opposing effects on carcinogenesis has not been adequately investigated. Gap-junction intercellular communication (GJIC), which is strongly related to carcinogenesis (particularly the tumor promotion stage), may be a suitable model for investigating the tumor-promoting and antitumor-promoting effects of phytochemicals. The present study investigated the possible combined effects of resveratrol and gallic acid (GA), which are major antioxidants in red wine, on GJIC in WB-F344 rat liver epithelial (RLE) cells. GA at 100 microM, but not resveratrol, inhibited GJIC and generated hydrogen peroxide. The GA-induced inhibition of GJIC was recovered by resveratrol, but only partially recovered by catalase. Resveratrol did not attenuate GA-induced generation of hydrogen peroxide, but it did block GA-induced phosphorylation of connexin 43 (Cx43), a key modulator of GJIC. Furthermore, resveratrol down-regulated GA-induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2, one of the critical regulators of Cx43. However, catalase partially blocked the GA-induced phosphorylation of Cx43 and ERK1/2. Collectively, these findings suggest that the combined effects of red wine phenolic phytochemicals on GJIC and antioxidants differ in ROS-mediated carcinogenesis depending on their dosages and structures.     

The chemopreventive role of dietary phytochemicals through gap junctional intercellular communication

Dietary phytochemicals offer protection from oxidative damages and lower the risks of chronic diseases, by complementary and overlapping action mechanisms. These include antioxidant activity, regulation of gene expression and cell cycle, stimulation of the immune and hormonal systems and modulation of cell–cell communication. Gap-junction intercellular communication (GJIC) plays an important role in maintaining tissue homeostasis by allowing the intercellular exchange of ions and regulatory molecules associated with cell proliferation, differentiation and apoptosis, and by contributing to intracellular signaling. This mechanism is strictly regulated and abnormal GJIC can result in several pathological conditions. GJIC is deregulated in cancer cells and reversible GJIC inhibition is strongly related to the promotion phase of carcinogenesis, likely mediated by reactive oxygen species. Whereas, the reversible inhibition of GJIC is related to the promotion phase of carcinogenicity, enhancers of GJIC are expected to prevent cancer. Several dietary plant compounds demonstrated the ability to control GJIC at the epigenetic levels and to prevent GJIC down-regulation by tumor promoting compounds, thus preventing cancers. In this Commentary, a number of reported studies on several phytochemicals in dietary and medicinal plants, which were able to affect GJIC and their structural proteins, i.e., connexins, in different in vivo and in vitro systems, were examined. The growing evidence, on the involvement of plant-derived molecules in the modulation of GJIC and in understanding of the specific action mechanisms, might offer a new perspective of the protective and/or preventive effects of dietary phytochemicals, in addition to possible chemotherapeutic use.     

Subcytotoxic mercury chloride inhibits gap junction intercellular communication by a redox- and phosphorylation-mediated mechanism

Gap junctions play a central role in coordinating intercellular signal-transduction pathways to control tissue homeostasis. Deregulation of gap junctional intercellular communication is a common phenotype of cancer cells and supports its involvement in the carcinogenesis process. Many carcinogens, like environmental heavy-metal chemical pollutants, are known to activate various signal transduction mechanisms and modulate GJIC. They act as tumor promoters on preexisting "initiated" cells, rather than as genotoxic initiators, albeit their mode of action is often unknown. In this study we investigated the effect of Hg(II) (HgCl(2)) on GJIC in cultured human keratinocytes. It is shown that subcytotoxic concentrations of HgCl(2) as low as 10 nM cause inhibition of the GJIC, assessed by dye transfer assay, despite enhanced expression of connexins. In addition, HgCl(2)-treated keratinocytes exhibited a decrease of free thiols and accumulation of mitochondria-derived reactive oxygen species, albeit no effect on the respiratory chain activity was observed. Treatment of HgCl(2)-exposed keratinocytes with the PKC inhibitor calphostin C and with all-trans retinoic acid resulted in rescue of the mitochondrial ROS overproduction and full recovery of the GJIC. Similar results were obtained with the PKA activator db-cAMP. Overall, the presented results support a cross-talk between the altered intracellular redox tone and PKA- and PKC-mediated signaling in HgCl(2)-challenged keratinocytes. These events, although not cytotoxic, lead to inhibition of GJIC and possibly to carcinogenic priming.     

Intercellular communication and carcinogenesis

Two types of intercellular communication (humoral and cell contact-mediated) are involved in control of cellular function in multicellular organisms, both of them mediated by membrane-embedded proteins. Involvement of aberrant humoral communication in carcinogenesis has been well documented and genes coding for some growth factors and their receptors have been classified as oncogenes. More recently, cell contact-mediated communication has been found to have an important role in carcinogenesis, and some genes coding for proteins involved in this type of communication appear to form a family of tumor-suppressor genes. Both homologous (among normal or (pre-)cancerous cells) as well as heterologous (between normal and (pre)cancerous cells) communications appear to play important roles in cell growth control. Gap junctional intercellular communication (GJIC) is the only means by which multicellular organisms can exchange low molecular weight signals directly from within one cell to the interior of neighboring cells. GJIC is altered by many tumor-promoting agents and in many human and rodent tumors. We have recently shown that liver tumor-promoting agents inhibit GJIC in the rat liver in vivo. Molecular mechanisms which could lead to aberrant GJIC include: (1) mutation of connexin genes; (2) reduced and/or aberrant expression of connexin mRNA; (3) aberrant localization of connexin proteins, i.e., intracytoplasmic rather than in the cytoplasmic membrane; and (4) modulation of connexin functions by other proteins, such as those involved in extracellular matrix and cell adhesion. Whilst mutations of the cx 32 gene appear to be rare in tumors, cx 37 gene mutations have been reported in a mouse lung tumor cell line. Our results suggest that aberrant connexin localization is rather common in cancer cells and that possible molecular mechanisms include aberrant phosphorylation of connexin proteins and lack of cell adhesion molecules. Studies on transfection of connexin genes into tumor cells suggest that certain connexin genes (e.g., cx 26, cx 43 and cx 32) act as tumor-suppressor genes.     

Loss of gap junctional intercellular communication in rat lung epithelial cells exposed to quartz particles

Chronic inhalation of quartz particles has been implicated in lung diseases including silicosis and cancer. The aim of this study was to investigate whether quartz particles affect gap junctional intercellular communication (GJIC) in rat lung epithelial cells (RLE-6TN). Here, we demonstrate that exposure of RLE-6TN cells to subtoxic doses of DQ12 standard quartz resulted in an up to 55% reduction of GJIC, as determined in a dye transfer assay. We show that connexin-43 (Cx43) is the major connexin responsible for intercellular communication in these lung epithelial cells and that exposure to quartz particles induces a significant internalization of Cx43. Downregulation of GJIC was attenuated by N-acetyl cysteine, suggesting the involvement of reactive oxygen species and/or cellular thiol homeostasis in the regulation of GJIC. Furthermore, an inhibitor of activation of extracellular signal-regulated kinases prevented the loss of GJIC in cells exposed to DQ12 quartz, although no direct phosphorylation of Cx43 upon exposure to DQ12 was detected.     

Role of gap junctional intercellular communication in radiation-induced bystander effects in human fibroblasts

Involvement of gap junctional intercellular communication (GJIC) in bystander responses of confluent human fibroblasts irradiated with a carbon-ion beam was investigated. It was found that the lower the radiation dose, the higher the yield of radiation-induced micronuclei per nuclear traversal, suggesting the existence of bystander effects. This low-dose sensitivity was increased when GJIC was enhanced by treating cells with 8-Br-cAMP, but it was partly reduced by treating cells with DMSO, an effective scavenger of reactive oxygen species (ROS). Moreover, no low-dose sensitivity was observed when cells were treated with 100 micro M lindane, an inhibitor of GJIC. The survival of irradiated cells was increased by DMSO but was not influenced significantly by cAMP or lindane. On the other hand, G(1)-phase arrest was detected in the irradiated cells, and it was enhanced by cAMP. In contrast, this arrest was reduced or almost eliminated by DMSO or lindane, respectively, even when cells were irradiated with such a high dose that each cell received five nuclear traversals on average. Thus the bystander responses occurred after both low-dose and relatively high-dose irradiation. Our results indicated that both GJIC and ROS contributed to the radiation-induced bystander effect, but gap junctional channels might play an essential role by modulating the release of radiation-induced signaling factors.     

Pathological Significance of Intracytoplasmic Connexin Proteins: Implication in Tumor Progression

A considerable amount of evidence has established that gap junctional intercellular communication (GJIC) suppresses tumor development by halting the stage of tumor promotion. Consistently, GJIC is downregulated in tumors. The downregulation of GJIC is caused by not only the reduced expression level of connexin proteins but also their aberrant cytoplasmic localization. Although it has long been thought that cytoplasmic localization of connexin proteins is merely one of the mechanisms of the downregulation of GJIC, careful studies with human tumor samples have indicated that the expression level of intracytoplasmic connexin proteins correlates well with the grade of malignancy and the progression stage of tumors. Hypothesizing that intracytoplasmic connexin proteins should have their proper functions and that their increase should facilitate tumor progression such as cell migration, invasion and metastasis, we examined the effects of overexpressed connexin32 (Cx32) protein on the phenotype of human HuH7 hepatoma cells, which express a basal level of endogenous Cx32 only in cytoplasm. The cells were retrovirally transduced with the Tet-off Cx32 construct so that withdrawal of doxycycline from the culture medium could induce overexpression of Cx32 protein in cytoplasm. Even when overexpressed, Cx32 protein was retained in cytoplasm, i.e., Golgi apparatuses, and did not induce GJIC. However, overexpression of Cx32 protein in cytoplasm enhanced both the motility and the invasiveness of HuH7 cells and induced metastasis when the cells were xenografted into SCID mice. Taken together, cytoplasmic accumulation of connexin proteins may exert effects favorable for tumor progression.     

Modulation of cell-cell communication in the cause and chemoprevention/chemotherapy of cancer

Chemopreventive or chemotherapeutic agents have been those that either kill cancer cells to a differential degree over the non-cancer cells or those chemicals that either block the induction of tumors in carcinogen-treated animals or retard transplanted tumors in animals. Carcinogenesis is a multi-stage, multi-mechanism process, involving the irreversible alteration of a stem cell ("initiation"), followed by the clonal proliferation of the initiated cell ("promotion"). To develop a strategy for intervention with chemoprevention/chemotherapeutic chemicals, the basic mechanism(s) of carcinogenesis must be understood. Gap junction intercellular communication (GJIC) regulates cell growth, differentiation, apoptosis and adaptive functions of differentiated cells. Normal cells have functional GJIC while cancer cells do not. Tumor promoters and oncogenes inhibit GJIC, while anti-tumor promoter and anti-oncogene drugs can reverse the down-regulation of GJIC. Transfection of gap junction genes (connexins) has been shown to reverse the tumorigenic phenotype. If prevention/treatment of cancer is to occur, prevention of the chronic down regulation of GJIC by tumor promoters in non-tumorigenic but initiated cells or the up-regulation of GJIC in stably down-regulated GJIC in tumor cells must occur to prevent or to treat cancers.     

Oxidative protein cross-linking reactions involving L-tyrosine in transforming growth factor-beta1-stimulated fibroblasts

The mechanisms by which ligand-stimulated generation of reactive oxygen species in nonphagocytic cells mediate biologic effects are largely unknown. The profibrotic cytokine, transforming growth factor-beta1 (TGF-beta1), generates extracellular hydrogen peroxide (H2O2) in contrast to intracellular reactive oxygen species production by certain mitogenic growth factors in human lung fibroblasts. To determine whether tyrosine residues in fibroblast-derived extracellular matrix (ECM) proteins may be targets of H2O2-mediated dityrosine-dependent cross-linking reactions in response to TGF-beta1, we utilized fluorophore-labeled tyramide, a structurally related phenolic compound that forms dimers with tyrosine, as a probe to detect such reactions under dynamic cell culture conditions. With this approach, a distinct pattern of fluorescent labeling that seems to target ECM proteins preferentially was observed in TGF-beta1-treated cells but not in control cells. This reaction required the presence of a heme peroxidase and was inhibited by catalase or diphenyliodonium (a flavoenzyme inhibitor), similar to the effect on TGF-beta1-induced dityrosine formation. Exogenous addition of H2O2 to control cells that do not release extracellular H2O2 produced a similar fluorescent labeling reaction. These results support the concept that, in the presence of heme peroxidases in vivo, TGF-beta1-induced H2O2 production by fibroblasts may mediate oxidative dityrosine-dependent cross-linking of ECM protein(s). This effect may be important in the pathogenesis of human fibrotic diseases characterized by overexpression/activation of TGF-beta1.     

The role of human adult stem cells and cell-cell communication in cancer chemoprevention and chemotherapy strategies

Since carcinogenesis is a multi-stage, multi-mechanism process, involving mutagenic, cell death and epigenetic mechanisms, during the "initiation/promotion/and progression" phases, chemoprevention must be based on understanding the underlying mechanism(s) of each phase, In principle, prevention of each of these phases could reduce the risk to cancer. However, because reducing the mutagenic/initiation phase to a zero level is impossible, the most efficacious intervention would be at the promotion phase that requires a sustained exposure to promoting conditions/agents. In addition, assuming the "target" cells for carcinogenesis are the pluri-potent stem cells and their early progenitor or transit cells, chemoprevention strategies for inhibiting the promotion of these two types of pre-malignant "initiated" cells will require different kinds of agents. A hypothesis will be proposed that involves adult stem cells, which express Oct-4 gene and lack gap junctional intercellular communication (GJIC-) or the early progenitor cells which express GJIC+ and are partially-differentiated, if initiated, will be promoted by agents that either inhibit secreted negative growth regulators or by inhibitors of GJIC. Consequently, anti-tumor promoting chemopreventing agents to each of these two types of initiated cells must have different mechanisms of action and work on different target cells. Assuming stem cells are target cells for carcinogenesis, an alternative method of chemoprevention would be to reduce the stem cell pool. Many classes of anti-tumor promoter chemopreventive agents, such as green tea components, resveratrol, caffeic acid phenethylene ester, either up-regulate GJIC in stem cells or prevent the down regulation of GJIC by tumor promoters in early progenitor cells.     

Serial passage of MC3T3-E1 cells alters osteoblastic function and responsiveness to transforming growth factor-beta1 and bone morphogenetic protein-2.

The murine-derived clonal MC3T3-E1 cell is a well-studied osteoblast-like cell line. To understand the effects of serial passages on its cellular function, we examined changes in cell morphology, gap junctional intercellular communication (GJIC), proliferation, and osteoblastic function between early passage (<20) and late passage (>65) cells. MC3T3-E1 cells developed an elongated, spindle shape after multiple passages. Intercellular communication decreased significantly (33%) in late vs. early passage cells. Transforming growth factor-beta1 (TGF-beta1) stimulated cell proliferation in early passage cells and induced c-fos expression, while it inhibited proliferation in late passage cells. Using alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion as markers for osteoblastic function and differentiation, we demonstrated that both markers were significantly reduced after multiple cell passages. Bone morphogenetic protein-2 (BMP-2) significantly enhanced ALP activity and OC secretion in early passage cells while TGF-beta1 exerted an opposite effect. Both BMP-2 and TGF-beta1 had minimal effects on late passage cells. We conclude that serial passage alters MC3T3-E1 cell morphology, and significantly diminishes GJIC, osteoblastic function, TGF-beta1-mediated cell proliferation, and responsiveness to TGF-beta1 and BMP-2. Cell passage numbers should be clearly defined in functional studies involving MC3T3-E1 cells.     

Interaction of MSC with tumor cells

Tumor development and tumor progression is not only determined by the corresponding tumor cells but also by the tumor microenvironment. This includes an orchestrated network of interacting cell types (e.g. immune cells, endothelial cells, fibroblasts, and mesenchymal stroma/stem cells (MSC)) via the extracellular matrix and soluble factors such as cytokines, chemokines, growth factors and various metabolites. Cell populations of the tumor microenvironment can interact directly and indirectly with cancer cells by mutually altering properties and functions of the involved partners. Particularly, mesenchymal stroma/stem cells (MSC) play an important role during carcinogenesis exhibiting different types of intercellular communication. Accordingly, this work focusses on diverse mechanisms of interaction between MSC and cancer cells. Moreover, some functional changes and consequences for both cell types are summarized which can eventually result in the establishment of a carcinoma stem cell niche (CSCN) or the generation of new tumor cell populations by MSC-tumor cell fusion.     

Increased gap junctional intercellular communication is directly related to the anti-tumor effect of all-trans-retinoic acid plus tamoxifen in a human mammary cancer cell line

Additive effects against tumor cells might be achieved by combining anti-neoplastic agents directed against one or more altered mechanisms in cancer. We investigated the participation of gap junctional intercellular communication (GJIC), which is commonly dysfunctional in tumor cells as a possible mediating mechanism of the effect of all-trans-retinoic acid (RA) and tamoxifen (Tx) in MCF-7 human breast cancer cell lines. The combination of RA + Tx stimulated GJIC in approximately 53 +/- 3% of MCF-7 cells as early as after 6 h of treatment remaining communicated through 144 h of culture. The GJIC enhancement occurred along with immunolocalization of Cx26 and 43 at the membrane of contacting cells and correlated with higher protein levels. Cx40 immunoreactive plaques were detected at cell-to-cell contacts during 48 h of RA + Tx treatment that did not involve higher protein expression, to the contrary, a downregulation occurred after 72 h of treatment. Cell proliferation inhibition upon RA + Tx exposure was observed with optimal effects at 96-120 h of culture with an accumulation of cells primarily in G2/M and G0/G1 cell cycle boundaries. An enhancement of the pre-existing E-cadherin levels was observed after drug exposure along with a downregulation of Bcl-2 and C-myc protein levels and a reduction of telomerase activity, suggesting partial tumor phenotype reversion. Blockage of the RA + Tx-induced GJIC with 18-beta-glycyrrhetinic acid (beta-Gly) prevented in 34% the inhibition of MCF-7 proliferation and the E-cadherin increment in 30% at 96 h of culture. GJIC blockage did not alter the downregulation of Bcl-2, c-Myc, or telomerase activity induced by RA + Tx. Our results showed the participation of GJIC as a mediator mechanism of the combined action of RA and Tx in MCF-7 cells. The chemopreventive modulation of GJIC might represent an approachable alternative for the improvement of cancer therapy.     

Induction of tenascin-C by cyclic tensile strain versus growth factors: distinct contributions by Rho/ROCK and MAPK signaling pathways

Expression of the extracellular matrix (ECM) protein tenascin-C is induced in fibroblasts by growth factors as well as by tensile strain. Mechanical stress can act on gene regulation directly, or indirectly via the paracrine release of soluble factors by the stimulated cells. To distinguish between these possibilities for tenascin-C, we asked whether cyclic tensile strain and soluble factors, respectively, induced its mRNA via related or separate mechanisms. When cyclic strain was applied to chick embryo fibroblasts cultured on silicone membranes, tenascin-C mRNA and protein levels were increased twofold within 6 h compared to the resting control. Medium conditioned by strained cells did not stimulate tenascin-C mRNA in resting cells. Tenascin-C mRNA in resting cells was increased by serum; however, cyclic strain still caused an additional induction. Likewise, the effect of TGF-beta1 or PDGF-BB was additive to that of cyclic strain, whereas IL-4 or H2O2 (a reactive oxygen species, ROS) did not change tenascin-C mRNA levels. Antagonists for distinct mitogen-activated protein kinases (MAPK) inhibited tenascin-C induction by TGF-beta1 and PDGF-BB, but not by cyclic strain. Conversely, a specific inhibitor of Rho-dependent kinase strongly attenuated the response of tenascin-C mRNA to cyclic strain, but had limited effect on induction by growth factors. The data suggest that regulation of tenascin-C in fibroblasts by cyclic strain occurs independently from soluble mediators and MAPK pathways; however, it requires Rho/ROCK signaling.     

Ascorbic acid 6-palmitate suppresses gap-junctional intercellular communication through phosphorylation of connexin 43 via activation of the MEK–ERK pathway

Although the health benefits of dietary antioxidants have been extensively studied, their potential negative effects remain unclear. L-Ascorbic acid 6-palmitate (AAP), a synthetic derivative of ascorbic acid (AA), is widely used as an antioxidant and preservative in foods, vitamins, drugs, and cosmetics. Previously, we found that AA exerted an antitumor effect by protecting inhibition of gap-junctional intercellular communication (GJIC), which is closely associated with tumor progression. In this study, we examined whether AAP, an amphipathic derivative of AA, has chemopreventive effects using a GJIC model. AAP and AA exhibited dose-dependent free radical-scavenging activities and inhibited hydrogen peroxide (H₂O₂)-induced intracellular reactive oxygen species (ROS) production in normal rat liver epithelial cells. Unexpectedly, however, AAP did not protect against the inhibition of GJIC induced by H₂O₂; instead, it inhibited GJIC synergistically with H₂O₂. AAP inhibited GJIC in a dose-dependent and reversible manner. This inhibitory effect was not due to the conjugated lipid structure of AAP, as treatment with palmitic acid alone failed to inhibit GJIC under the same conditions. The inhibition of GJIC by AAP was restored in the presence of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126, but not in the presence of other signal inhibitors and antioxidant (PKC inhibitors, EGFR inhibitor, NADPH oxidase inhibitor, catalase, vitamin E, or AA), indicating the critical involvement of MEK signaling in the GJIC inhibitory activity of AAP. Phosphorylation of ERK and connexin 43 (Cx43) was observed following AAP treatment, and this was reversed by U0126. These results suggest that the AAP-induced inhibition of GJIC is mediated by the phosphorylation of Cx43 via activation of the MEK–ERK pathway. Taken together, our results indicate that AAP has a potent carcinogenic effect, and that the influence of dietary antioxidants on carcinogenesis may be paradoxical.     

Modulatory effects of connexin-43 expression on gap junction intercellular communications with mast cells and fibroblasts

The influence of mast cells upon aberrant wound repair and excessive fibrosis has supportive evidence, but the mechanism for these mast cell activities is unclear. It is proposed that heterocellular gap junction intercellular communication (GJIC) between fibroblasts and mast cells directs some fibroblast activities. An in vitro model was used employing a rodent derived peritoneal mast cell line (RMC-1) and human dermal derived fibroblasts. The influence of the expression of the gap junction channel structural protein, connexin 43 (Cx-43) on heterocellular GJIC, the expression of microtubule β-tubulin and microfilament α smooth muscle actin (SMA) were investigated. The knockdown of Cx-43 by siRNA in RMC-1 cells completely blocked GJIC between RMC-1 cells. SiRNA knockdown of Cx-43 within fibroblasts only dampened GJIC between fibroblasts. It appears Cx-43 is the only expressed connexin (Cx) in RMC-1 cells. Fibroblasts express other Cxs that participate in GJIC between fibroblasts in the absence of Cx-43 expression. Heterocellular GJIC between RMC-1 cells and fibroblasts transformed fibroblasts into myofibroblasts, expressing α SMA within cytoplasmic stress fibers. The knockdown of Cx-43 in RMC-1 cells increased β-tubulin expression, but its knockdown in fibroblasts reduced β-tubulin expression. Knocking down the expression of Cx-43 in fibroblasts limited αSMA expression. Cx-43 participation is critical for heterocellular GJIC between mast cells and fibroblasts, which may herald a novel direction for controlling fibrosis.     

Advantages and limitations of commonly used methods to assay the molecular permeability of gap junctional intercellular communication

The role of gap junctional intercellular communication (GJIC) in regulation of normal growth and differentiation is becoming increasingly recognized as a major cellular function. GJIC consists of intercellular exchange of low molecular weight molecules, and is the only means for direct contact between cytoplasms of adjacent animal cells. Disturbances of GJIC have been associated with many pathological conditions, such as carcinogenesis or hereditary illness. Reliable and accurate methods for the determination of GJIC are therefore important in cell biology studies. There are several methods used successfully in numerous laboratories to measure GJIC both in vitro and in vivo. This review comments on techniques currently used to study cell-to-cell communication, either by measuring dye transfer, as in methods like microinjection, scrape loading, gap-fluorescence recovery after photobleaching (gap-FRAP), the preloading assay, and local activation of a molecular fluorescent probe (LAMP), or by measuring electrical conductance and metabolic cooperation. As we will discuss in this review, these techniques are not equivalent but instead provide complementary information. We will focus on their main advantages and limitations. Although biological applications guide the choice of techniques we describe, we also review points that must be taken into consideration before using a methodology, such as the number of cells to analyze.     

Peroxynitrite diminishes gap junctional communication: protection by selenite supplementation

Loss of intercellular communication via gap junctions has been correlated with progression of cells to a malignant phenotype. Here, we show that peroxynitrite, a mediator of toxicity in inflammatory processes, diminishes gap junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells, assayed by the scrapeloading dye-transfer technique as well as by microinjection of a fluorescent dye into single cells. Exposure of cultured cells to a steady-state concentration of peroxynitrite of 1.6 microM for 4 min or to 3-morpholinosydnonimine (SIN-1) at 0.5 mM strongly diminished GJIC. These concentrations of peroxynitrite or SIN-1 were not cytotoxic. When cells were grown in a medium supplemented with sodium selenite (0.1-1 microM) for 72 h, substantial protection was afforded against the decrease in GJIC by peroxynitrite. Thus, peroxynitrite can disrupt GJIC, and selenium-containing proteins protect.