A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.  Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.     

Limitations of the scrape-loading/dye transfer technique to quantify inhibition of gap junctional intercellular communication

Gap junctional intercellular communication (GJIC) is recognized as playing an important role in normal cell proliferation and development. Chemically induced alteration of GJIC has been proposed to be associated with abnormal cellular growth and/or tumor promotion. Several in vitro assays are currently used to determine the effects of chemicals on GJIC between cultured mammalian cells. One of these assays, the scrape-loading dye transfer (SLIDT) technique, is based on monitoring the transfer of the fluorescent dye Lucifer yellow from one cell into adjacent cells via functional gap junctions. The objective of our study was to evaluate and compare various approaches for quantifying results obtained with the SL/DT technique. Confluent cultures of either WB rat liver epithelial cells or LC-540 rat leydig cells were exposed to the animal tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), solvent (0.1% ethanol), or culture medium for one hour at 37° C prior to analysis of GJIC. Inhibition of dye transfer was clearly evident following TPA exposure. Quantification of this dye transfer was assessed via four approaches: manually counting the number of labeled cells; measuring the distance of dye travel from the scrape line; quantifying the amount of cellular dye uptake; and determining the distribution of dye away from the scrape line. Our results suggest that while the SL/DT technique can be effectively used as a tool to determine the qualitative presence or absence of GJIC, its use in quantifying changes in GJIC following chemical exposure is limited. Since concentration-dependent responses are critical in chemical testing, application of the SLIDT method should be restricted to a screening assay for qualitatively assessing the presence or absence of GJIC. Another assay (e.g., electrical coupling, microinjection, metabolic cooperation, radioactive metabolite transfer, or fluorescence redistribution after photobleaching) should be considered to quantify changes in GJIC and construct chemical concentration-response curves.Abbreviations FBS, fetal bovine serum - GJIC, gap junctional intercellular communication - HBSS, Hank's balanced saline solution - SL/DT, scrape-loading/dye transfer - TPA, 12-O-tetradecanoylphorbol-13-acetate.     

Quantitative determination of gap junction intercellular communication using flow cytometric measurement of fluorescent dye transfer

Gap junction intercellular communication (GJIC) is involved in several aspects of normal cell behaviour, and disturbances in this type of communication have been associated with many pathological conditions. Reliable and accurate methods for the determination of GJIC are therefore important in studies of cell biology. (Tomasetto, C., Neveu, M.J., Daley, J., Horan, P.K. and Sager, R. (1993) Journal of Cell Biology, 122, 157-167) reported some years ago the use of flow cytometer to determine transfer between cells of a mobile dye, calcein, as a measure of cell communication through gap junctions. In spite of this being a method with potential for quantitative and reliable determination of GJIC, it has been modestly used, possibly due to technical difficulties. In the present work we have illustrated several ways to use flow cytometric data to express cell communication through gap junctions. The recipient cells were pre-stained with the permanent lipophilic dye PKH26, and the donor cell population were loaded with the gap junction permeable dye, calcein. We show that the method may be used to measure the effect of chemicals on GJIC, and that the information is reliable, objective and reproducible due to the large number of cells studied. The data may give additional information to that obtained with other methods, since the effect observed will be on the establishment of cell communication as compared to what is observed for microinjection or scrape loading, where the effect is on already established communication. This is probably the reason for the more potent effects of DMSO on GJIC measured by the present method than on already existing GJIC measured by microinjection or quantitative scrape loading. We also show that the problem related to the mobile dye calcein not being fixable with aldehydes will not affect the results as long as the cells are kept on ice in the dark and analysed by flow cytometer within the first hours after formalin cell fixation.     

Quantitative Determination of Gap Junction Intercellular Communication Using Flow Cytometric Measurement of Fluorescent Dye Transfer

Gap junction intercellular communication (GJIC) is involved in several aspects of normal cell behaviour, and disturbances in this type of communication have been associated with many pathological conditions. Reliable and accurate methods for the determination of GJIC are therefore important in studies of cell biology. (Tomasetto, C., Neveu, M.J., Daley, J., Horan, P.K. and Sager, R.(1993) Journal of Cell Biology, 122, 157–167) reported some years ago the use of flow cytometer to determine transfer between cells of a mobile dye, calcein, as a measure of cell communication through gap junctions. In spite of this being a method with potential for quantitative and reliable determination of GJIC, it has been modestly used, possibly due to technical difficulties. In the present work we have illustrated several ways to use flow cytometric data to express cell communication through gap junctions. The recipient cells were pre-stained with the permanent lipophilic dye PKH26, and the donor cell population were loaded with the gap junction permeable dye, calcein. We show that the method may be used to measure the effect of chemicals on GJIC, and that the information is reliable, objective and reproducible due to the large number of cells studied. The data may give additional information to that obtained with other methods, since the effect observed will be on the establishment of cell communication as compared to what is observed for microinjection or scrape loading, where the effect is on already established communication. This is probably the reason for the more potent effects of DMSO on GJIC measured by the present method than on already existing GJIC measured by microinjection or quantitative scrape loading. We also show that the problem related to the mobile dye calcein not being fixable with aldehydes will not affect the results as long as the cells are kept on ice in the dark and analysed by flow cytometer within the first hours after formalin cell fixation.     

Effect of cadmium on gap junctional intercellular communication in primary cultures of rat renal proximal tubular cells.

Cadmium mainly accumulates in the kidney and causes renal injury. To clarify the mechanism of Cd nephrotoxicity, we investigated the effects of this element on intercellular communication through gap junction channels in primary cultures of rat renal proximal tubular cells. Sixty minutes after exposure to 100 microM Cd, dye coupling experiments showed that gap junctional intercellular communication (GJIC) was significantly inhibited. This inhibition occurred before the appearance of cytotoxicity. Intracellular calcium concentrations [Ca2+]i, which modulate the function of gap junctions, gradually increased after exposure to Cd and reached a maximum after 60 minutes. These results suggest that the inhibition of GJIC as a result of Cd exposure is related to an increase in [Ca2+]i, and that GJIC inhibition may be an indicator of nephrotoxicity.     

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.     

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.     

Gap junctional intercellular communication capacity by gap-FRAP technique: a comparative study

Gap junctions play an important role in vital functions, including the regulation of cell growth and cell differentiation. Connexins 43 (Cx43) are the most widely expressed gap junction proteins. Cellular localization of phosphorylated Cx43 has been implicated in the capacity of gap junctional intercellular communication (GJIC). To follow the functionality of GJIC of different cell types, in monolayer cultures, characterized by different patterns of phosphorylated Cx43, we used a fluorescence recovery after photobleaching (FRAP) technique, and compared two tracers, 5(6)-carboxyfluorescein diacetate (CFDA) and calcein acetoxymethylester (AM). The GJIC capacity was quantified by estimating fluorescence redistribution parameters. The functionality of GJIC was in relation with the staining localization of phosphorylated Cx43 to the cell-cell contact areas, corresponding to gap junctions between contacting cells. GJIC involvement in fluorescence restitution after photobleaching was checked by a gap junction channel inhibition assay. We demonstrated that the choice of the dye did not significantly influence the fluorescence recovery percentages despite a cell line-dependent CFDA release, whereas it had an important impact on fluorescence kinetic profiles. This study reinforces the interest of the gap-FRAP approach to quantify modifications in the functionality of gap junctions and, above all, argues about the limits of CFDA for 3-D future approaches.     

Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.     

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.     

Intercellular communication of notochord cells during their differentiation in Cynops orientalis

Intercellular communication of notochord cells during their differentiation was studied by microinjection of a fluorescent dye.Lucifer Yellow,Close correlation existed between the incidences of dye coupling and quantitative evaluation of gap junctions.high incidences of dye coupling and of gap junctions occurred at a stage when notochord cells were active in the change of cell shape and cell arrangement.With the subsidence of cell movements,both dye coupling and gap junctions were reduced to lower levels.It was,therefore,Suggested that intercellular communication via gap junctions played an important role in the coordination of notochord cell movements.Gap Junctions of altered configuration occurred in notochord cells in late taibud stage.The comparison of incidences of dye coupling at this stage with those at other stages strongly suggested that the gap junctions of altered configuration functioned just as those of generalized type.     

Cell-to-cell communication competence in simian virus 40-transfected rat ovarian cells is reduced following tumor selection

Summary A pSV3neo-transfected rat ovarian cell line (SV-GC) was developed from a primary granulosa culture (GC) to study gap junctional intercellular communication (GJIC) during Simian virus 40 (SV40) transformation. SV-GC expressed SV40 large T-antigen (T-ag), grew indefinitely in culture without luteinization, was anchorage independent, and formed tumors in nude mice. Ultrastructural analysis identified abundant gap junctional membrane and suggested that SV-GC was arrested at an early stage of differentiation. Functional GJIC, measured by a dye transfer technique (gap FRAP), was comparable to that observed in normal granulosa cells, suggesting that the expression of T-ag alone was insufficient to reduce GJIC. However, there was approximately a 50% loss in the rate of GJIC in the nude mouse SV-GC-tumor derived and G418 selected cell line (T-SV-GC). SV-GC→T-SV-GC also resulted in a transition from migration of cells as an epithelial sheet to the dissociation of individual fibroblastoid cells. Tumor cell detachment was also seen in migrating malignant human (A2780 and 547) and rat (DC3) ovarian cell lines. Co-culture combinations of normal (GC)→transformed (SV-GC) → tumor-derived (T-SV-GC) cells indicated that the rate of heterologous GJIC was characteristic of the least communicating partner. Taken together, these data suggested that SV-GC → T-SV-GC represented progression toward metastasis with concomitant reduction of GJIC and adhesiveness. These sequentially derived cell lines may be a useful in vitro model system for studies focusing on the mechanisms involved in the detachment of cells during the progression of ovarian cancer.     

Gap junctions in myometrial cell cultures: evidence for modulation by cyclic adenosine 3':5'-monophosphate.

Primary cultures of myometrial cells from juvenile rats, continuous cultures maintained by serial passage, and a pSV3neotransfected myometrial cell line were established and utilized for the study of development and modulation of gap junctional intercellular communication (GJIC) in vitro. The smooth muscle origin and homogeneity of the cultures were verified by immunofluorescence staining of alpha-smooth muscle actin and cellular desmin. Although gap junctions were not detected in thin sections of juvenile and adult myometrial tissues by transmission electron microscopy, they were detected in cultured myometrial cells derived from juvenile and adult animals. The presence of GJIC in cultured cells was confirmed using a fluorescence recovery after photo-bleaching assay. Administration of exogenous estradiol-17 beta (10(-7) M) resulted in an increase in GJIC in primary and passage 9 myometrial cultures, whereas pSV3neo-transfected myometrial cells were not significantly different from untreated controls. The lack of estrogen responsiveness in pSV3neo-transfected cultures correlated with lower levels of estrogen receptors than in primary cultures. Addition of 1 mM 8-bromo-cAMP resulted in rapid (within 2 min) increases in dye transfer in both control and estradiol-17 beta-primed primary cultures. Uncoupling of cells by treatment with 1 mM 1-octanol, followed by addition of 1 mM 8-bromo-cAMP, resulted in increased GJIC in control and estradiol-17 beta-primed cultures, although up-regulation of GJIC in estradiol-17 beta-primed cultures was much greater than in control cultures. Comparative experiments carried out on a spontaneously immortalized rat granulosa cell line (SIGC), which expresses the same connexin43 species as myometrial cells, exhibited similar responses to exogenous 8-bromo-cAMP following uncoupling of gap junctions with octanol. While the results of these investigations may not be extrapolated to myometrium in vivo, they suggest that myometrial cell culture may offer additional opportunities to explore the temporal expression and modulation of GJIC in myometrium.     

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.     

Regulation of Lens Gap Junctions by Transforming Growth Factor Beta

Gap junction–mediated intercellular communication (GJIC) is essential for the proper function of many organs, including the lens. GJIC in lens epithelial cells is increased by FGF in a concentration-dependent process that has been linked to the intralenticular gradient of GJIC required for lens transparency. Unlike FGF, elevated levels of TGF-β are associated with lens dysfunction. We show that TGF–β1 or -2 up-regulates dye coupling in serum-free primary cultures of chick lens epithelial cells (dissociated cell-derived monolayer cultures [DCDMLs]) via a mechanism distinct from that utilized by other growth factors. Remarkably, the ability of TGF-β and of FGF to up-regulate GJIC is abolished if DCDMLs are simultaneously exposed to both factors despite undiminished cell–cell contact. This reduction in dye coupling is attributable to an inhibition of gap junction assembly. Connexin 45.6, 43, and 56–containing gap junctions are restored, and intercellular dye coupling is increased, if the activity of p38 kinase is blocked. Our data reveal a new type of cross-talk between the FGF and TGF-β pathways, as well as a novel role for TGF-β and p38 kinase in the regulation of GJIC. They also provide an explanation for how pathologically increased TGF-β signaling could contribute to cataract formation.     

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.