The bystander effect in photodynamic inactivation of cells

Treatment of MDCK II cells with the lipophilic photosensitizer tetra(3-hydroxyphenyl)porphyrin and light was found to induce a rapid apoptotic response in a large fraction of the cells. Furthermore, the distribution of apoptotic cells in microcolonies of eight cells was found to be different from the binomial distribution, indicating that the cells are not inactivated independently, but that a bystander effect is involved in cell killing by photodynamic treatment. The observation of a bystander effect disagrees with the common view that cells are inactivated only by direct damage and indicates that communication between cells in a colony plays a role in photosensitized induction of apoptosis. The degree of bystander effect was higher for cells dying by necrosis than for cell dying by apoptosis.     

Radiation-induced bystander effects in malignant trophoblast cells are independent from gap junctional communication

It is controversially discussed that irradiation induces bystander effects via gap junction channels and/or diffusible cellular factors such as nitric oxide or cytokines excreted from the cells into the environment. But up to now the molecular mechanism leading to a bystander response is not well understood. To discriminate between both mechanisms of bystander response, (i) mediated by gap junctional communication and/or (ii) mediated by diffusible molecules, we used non-communicating Jeg3 malignant trophoblast cells transfected with inducible gap junction proteins, connexin43 and connexin26, respectively, based on the Tet-On system. We co-cultivated X-ray irradiated and non-irradiated bystander Jeg3 cells for 4 h, separated both cell populations by flow cytometry and evaluated the expression of activated p53 by Western blot analysis. The experimental design was proven with communicating versus non-communicating Jeg3 cells. Interestingly, our results revealed a bystander effect which was independent from gap junctional communication properties and the connexin isoform expressed. Therefore, it seems more likely that the bystander effect is not mediated via gap junction channels but rather by paracrine mechanisms via excreted molecules in Jeg3 cells.     

Reversible Inhibition of Gap Junctional Intercellular Communication, Synchronous Contraction, and Synchronism of Intracellular Ca Fluctuation in Cultured Neonatal Rat Cardiac Myocytes by Heptanol

We analyzed by Fotonic Sensor, a fiber-optic displacement measurement instrument, the effects of heptanol on synchronized contraction of primary neonatal rat cardiac myocytes cultured at confluent density. We also examined the effect of heptanol on the changes in gap junctional intercellular communication by using the microinjection dye transfer method, and on intercellular Ca²⁺ fluctuation by confocal laser scanning microscopy of myocytes loaded with the fluorescent Ca²⁺ indicator fluo 3. In addition, we studied expression, phosphorylation, and localization of the major cardiac gap junction protein connexin 43 (Cx43) using immunofluorescence and Western blotting. At Day 6 of culture, numerous myocytes exhibited spontaneous, synchronous contractions, excellent dye coupling, and synchronized intracellular Ca²⁺ fluctuations. We treated the cells with 1.5, 2.0, 2.5, and 3.0 mmol/liter heptanol. With 1.5 mmol/liter heptanol, we could not observe significant effects on spontaneous contraction of myocytes. At 3.0 mmol/liter, the highest concentration used in the current experiment, heptanol inhibited synchronous contractions and even after washing out of heptanol, synchronous contraction was not rapidly recovered. On the other hand, at the intermediate concentrations of 2.0 and 2.5 mmol/liter, heptanol reversely inhibited synchronized contraction, gap junctional intercellular communication, and synchronization of intracellular Ca²⁺ fluctuations in the myocytes without preventing contraction and changes of intracellular Ca²⁺ in individual cells. Brief exposure (5-20 min) to heptanol (2.0 mmol/liter) did not cause detectable changes in the expression, phosphorylation, or localization of Cx43, despite strong inhibition of gap junctional intercellular communication. These results suggest that gap junctional intercellular communication plays an important role in synchronous intracellular Ca²⁺ fluctuations, which facilitate synchronized contraction of cardiac myocytes.     

The role of gap junction communication and oxidative stress in the propagation of toxic effects among high-dose α-particle-irradiated human cells

We investigated the roles of gap junction communication and oxidative stress in modulating potentially lethal damage repair in human fibroblast cultures exposed to doses of α particles or γ rays that targeted all cells in the cultures. As expected, α particles were more effective than γ rays at inducing cell killing; further, holding γ-irradiated cells in the confluent state for several hours after irradiation promoted increased survival and decreased chromosomal damage. However, maintaining α-particle-irradiated cells in the confluent state for various times prior to subculture resulted in increased rather than decreased lethality and was associated with persistent DNA damage and increased protein oxidation and lipid peroxidation. Inhibiting gap junction communication with 18-α-glycyrrhetinic acid or by knockdown of connexin43, a constitutive protein of junctional channels in these cells, protected against the toxic effects in α-particle-irradiated cell cultures during confluent holding. Upregulation of antioxidant defense by ectopic overexpression of glutathione peroxidase protected against cell killing by α particles when cells were analyzed shortly after exposure. However, it did not attenuate the decrease in survival during confluent holding. Together, these findings indicate that the damaging effect of α particles results in oxidative stress, and the toxic effects in the hours after irradiation are amplified by intercellular communication, but the communicated molecule(s) is unlikely to be a substrate of glutathione peroxidase.     

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.     

Tentative novel mechanism of the bystander effect in glioma gene therapy with HSV-TK/GCV system.

Although many works support gap junctional intercellular communication (GJIC) having a close relation to bystander cell killing in herpes simplex virus thymidine kinase (HSV-TK) gene and ganciclovir (GCV) treatment, our previous work suggested that other factors involved in bystander effect besides GJIC exist. To confirm our primary work, we evaluated the mode of the bystander cell (C6) co-cultured with TK-positive cells (TF10.2) in our designed "insert plates" in which two cell lines could be separated but share the same medium. Another method that we used was adding the supernatant from the medium of GCV-treated TF10.2 cells to the wild type C6. Growth inhibition of the bystander cells was observed despite the absence of GJIC. In addition, apoptotic cell death of TK+ cells and bystander cells was obvious. These studies suggested that other pathways besides cell-cell contacts may play a role in bystander cell killing; the factors released from TK-positive cells could induce apoptosis of bystander cells.     

Connexins,gap junctional intercellular communication and kinases

A number of kinases and signal transduction pathways are known to affect gap junctional intercellular communication and/or phosphorylation of connexins. Most of the information is available for protein kinase A, protein kinase C, mitogen-activated protein kinase, and the tyrosine kinase Src. Much less is known for protein kinase G, Ca(2+)-calmodulin dependent protein kinase, and casein kinase. However, the present lack of knowledge is not necessarily synonymous with lack of importance in the regulation of intercellular communication and phosphorylation of connexins. Kinases and the phosphorylation of connexins may be involved in the regulation of gap junctional intercellular communication at all levels ranging from the expression of connexin genes to the degradation of the gap junction channels. The exact role of the phosphorylation depends both on the kinase and the connexin involved, as well as the cellular context.     

Tanshinone IIA Increases the Bystander Effect of Herpes Simplex Virus Thymidine Kinase/Ganciclovir Gene Therapy via Enhanced Gap Junctional Intercellular Communication

The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV). This effect is reported to be mediated by gap junctional intercellular communication (GJIC), and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA), a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy.     

Connexins and their channels in cell growth and cell death

Direct communication between cells, mediated by gap junctions, is nowadays considered as an indispensable mechanism in the maintenance of cellular homeostasis. In fact, gap junctional intercellular communication is actively involved in virtually all aspects of the cellular life cycle, ranging from cell growth to cell death. For a long time, it was believed that this was merely a result of the capacity of gap junctions to control the direct intercellular exchange of essential cellular messengers. However, recent data show that the picture is more complicated than initially thought, as structural precursors of gap junctions, connexins and gap junction hemichannels, can affect the cellular homeostatic balance independently of gap junctional intercellular communication. In this paper, we summarize the current knowledge concerning the roles of connexins and their channels in the control of cellular homeostasis, with the emphasis on cell growth and cell death. We also briefly discuss the role of gap junctional intercellular communication in carcinogenesis and the potential use of connexins as tools for cancer therapy.     

Connexins and cancer

The hypothesis, that gap junctional intercellular communication plays a key role in carcinogenesis and more generally in growth control was formulated nearly 40 years ago. From this time, data accumulated, showing that this type of communication is frequently decreased or absent in cells treated with tumor promoting agents, among transformed cells or between transformed/tumor cells and normal cells. This observation has been made on various cell types and whatever their tissue and species origins, by using in vitro and in vivo models. It led to the general assumption that the inhibition of gap junctional intercellular communication may play a role in carcinogenesis at two levels: (1) during tumor promotion by favoring the clonal expansion of initiated cells and (2) after the phenotypic transformation of cells by preventing the diffusion of putative "normalizing" factors between tumor cells and surrounding normal cells. During the past decade, the discovery that gap junction proteins, the connexins (Cx), may act as tumour suppressors, by reverting the phenotype of transformed cells confirmed the idea that their lack of function would be actively involved in carcinogenesis. However, we still do not know precisely what are the molecular processes that gap junctional intercellular communication may regulate and still do have very few data concerning the gap junction situation in human cancers. All these aspects are presented from an historical point of view and discussed below.     

Free radical-initiated and gap junction-mediated bystander effect due to nonuniform distribution of incorporated radioactivity in a three-dimensional tissue culture model

To investigate the biological effects of nonuniform distribution of radioactivity in mammalian cells, we have developed a novel three-dimensional tissue culture model. Chinese hamster V79 cells were labeled with tritiated thymidine and mixed with unlabeled cells, and multicellular clusters (approximately 1.6 mm in diameter) were formed by gentle centrifugation. The short-range beta particles emitted by (3)H impart only self-irradiation of labeled cells without significant cross-irradiation of unlabeled bystander cells. The clusters were assembled in the absence or presence of 10% dimethyl sulfoxide (DMSO) and/or 100 microM lindane. DMSO is a hydroxyl radical scavenger, whereas lindane is an inhibitor of gap junctional intercellular communication. The clusters were maintained at 10.5 degrees C for 72 h to allow (3)H decays to accumulate and then dismantled, and the cells were plated for colony formation. When 100% of the cells were labeled, the surviving fraction was exponentially dependent on the mean level of radioactivity per labeled cell. A two-component exponential response was observed when either 50 or 10% of the cells were labeled. Though both DMSO and lindane significantly protected the unlabeled or bystander cells when 50 or 10% of the cells were labeled, the effect of lindane was greater than that of DMSO. In both cases, the combined treatment (DMSO + lindane) elicited maximum protection of the bystander cells. These results suggest that the bystander effects caused by nonuniform distributions of radioactivity are affected by the fraction of cells that are labeled. Furthermore, at least a part of these bystander effects are initiated by free radicals and are likely to be mediated by gap junctional intercellular communication.     

Suicide gene therapy with Herpes simplex virus thymidine kinase and ganciclovir is enhanced with connexins to improve gap junctions and bystander effects

Connexins are proteins that form gap junctions between cells in various mammalian tissues. Because of their role in intercellular communication, connexins are important in the bystander cell death seen in Herpes simplex virus-thymidine kinase (HSV-TK) gene therapy for brain tumors. A selective review of connexin transduction/transfection studies with particular emphasis to central nervous system tumor cells is presented. In addition, specific references to studies with cell types that demonstrate low gap junction intercellular communication are presented. Data are included with the HT-29 colorectal tumor cell line to support the concept that enhancing gap junction protein expression in otherwise low gap junction communicating HT-29 cells increases bystander cell death and reduces tumor burden beyond what might be expected from HSV-TK and ganciclovir (GCV) treatment alone. Maximum in vitro bystander cell death was always produced when GCV treated co-cultures of TK-transduced and non-TK-transduced HT-29 cell lines were also transduced with connexin-43. When connexin was present in only one group of cells in the co-culture, there was more bystander cell death observed with connexin transduced into the non-TK-transduced cells, rather than the TK-transduced cells. The data presented reinforces conclusions made from earlier findings from cell line mixing experiments in which the non-TK-transduced cell population determined the level of bystander cell death (Burrows et al., 2002).     

Differential regulation of junctional complex assembly in renal epithelial cell lines

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and -catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner. calpeptin; tight junctions; adherens junctions; Rho; cadherin; p120ctn     

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the ras oncogene modulate expression and phosphorylation of gap junction proteins.

Gap junctional intercellular communication is inhibited in response to tumor promoters and oncogene transformation, suggesting that loss of this function is an important step in tumor formation. To elucidate the molecular mechanisms responsible for this inhibition, we examined the expression of gap junction proteins and mRNA in mouse primary keratinocytes after treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or ras transformation. During normal cell growth, keratinocytes expression the alpha 1 (connexin 43) and beta 2 (connexin 26) proteins. Within 5 min of TPA treatment, the alpha 1 protein became rapidly phosphorylated on serine residues and its expression was dramatically reduced by 24 h. The beta 2 protein, after an initial increase in expression, was also significantly reduced 24 h after treatment with TPA. ras transformation caused changes similar to those induced by TPA. The alpha 1 protein underwent an increase in serine phosphorylation, although its expression declined only slightly, while beta 2 expression was greatly reduced. The effects of TPA and ras on alpha 1 expression were additive; treatment of ras-transformed cells with TPA resulted in increased alpha 1 phosphorylation, with greatly decreased protein levels, much lower than those generated by either agent alone. These data provide a likely explanation for the similar and synergistic inhibition of gap junctional intercellular communication by phorbol esters and ras.     

Quantitative analysis of gap-junctional intercellular communication in precision-cut mouse liver slices

Direct intercellular communication through gap junction channels is involved in the maintenance of tissue homeostasis and suppression of carcinogenesis. Gap-junctional communication is often altered in tumor cells but it can also be modulated in response to tumor promotors or inflammatory signals. In order to evaluate the effect of nongenotoxic compounds, suggested to be involved in tumor promotion, on gap junctional intercellular communication in the liver, we have developed a direct dye transfer method. The fluorescent dye Alexa Fluor 488 was iontophoretically injected into hepatocytes of freshly prepared, precision-cut mouse liver slices (250 microm). The area of dye spreading was monitored and quantified by microscopy. Comparison of dye spreading in connexin-32-deficient versus wild-type liver revealed a 96% decrease in connexin-32-deficient tissue. Induction of an acute phase response in connexin-32-deficient mice by intraperitoneal injection of lipopolysaccharide increased dye coupling by 33%, probably due to upregulation of connexin-26-containing gap junction channels.     

Protein phosphorylation and hydrogen ions modulate calcium-induced closure of gap junction channels.

The regulation of the cell-to-cell pathway formed by gap junctions seems to involve the interaction of the junctional channels with either calcium or hydrogen ions, as well as protein phosphorylation and calmodulin. These mechanisms of junctional regulation have been considered to act independently on specific sites of the gap junction protein; however, the possibility that they may be interrelated has not been adequately explored mainly due to the difficulties involved in simultaneous measurement of intracellular cations and protein phosphorylation. To further understanding of mechanisms regulating gap junctions, we have internally perfused coupled lateral axons from crayfish with solutions containing different calcium and hydrogen concentrations under conditions favoring phosphorylation, while monitoring the junctional conductance. We found that calcium ions regulate cell communication probably through a direct interaction with the channel protein. Phosphorylation and low pH do not alter junctional conductance themselves, but appear only to modulate the effects of calcium, possibly by altering the affinity of the channel for calcium. We propose that a combination of free intracellular calcium and protein phosphorylation form an important physiological mechanism regulating intercellular communication.     

Connexin Over-Expression Differentially Suppresses Glioma Growth and Contributes to the Bystander Effect Following HSV-Thymidine Kinase Gene Therapy

Neoplastic transformation is frequently associated with a loss of gap junctional intercellular communication and reduced expression of connexins. The introduction of connexin genes into tumor cells reverses the proliferative characteristics of such cells. However, there is very little comparative information on the effects of different connexins on cancer cell growth. We hypothesized that Cx26, Cx32, or Cx43 would display differential growth suppression of C6 glioma cells and uniquely modulate the bystander effect following transduction of C6 cells with HSVtk followed by suicide gene therapy. The bystander phenomenon is the death of a greater number of tumor cells than are expressing the HSVtk gene, presumably due to the passage of toxic molecules through gap junction channels. To test this hypothesis, we used retroviral vectors to infect C6 glioma cells producing connexin-expressing and HSVtk-expressing cell lines. All three connexin-expressing cell lines grew significantly slower than GFP-infected or native C6 cells. Cx32 and Cx26 were significantly more effective at mediating the bystander effect in cocultures of C6-connexin cells with C6-HSVtk cells. These studies indicate that connexins have unique properties that contribute to their tumor suppressive function.     

HDACi phenylbutyrate increases bystander killing of HSV-tk transfected glioma cells

Malignant glioma patients have a dismal prognosis with an urgent need of new treatment modalities. Previously developed gene therapies for brain tumors showed promising results in experimental animal models, but failed in clinical trials due to low transfection rates and insufficient expression of the transgene in tumor cells, as well as low bystander killing effects. We have previously shown that the histone deacetylase inhibitor 4-phenylbutyrate (4-PB) enhances gap junction communication between glioma cells in culture. In this study, we demonstrate an activation of recombinant HSV-tk gene expression, and a dramatic enhancement of gap junction-mediated bystander killing effect by administration of the HSV-tk prodrug ganciclovir together with 4-PB. These findings that 4-PB potentiates "suicide gene" expression as well as enhances gap junctional communication and bystander killing of tumor cells justify further testing of this paradigm as an adjunct to suicide gene therapy of malignant gliomas.     

Connexin over-expression differentially suppresses glioma growth and contributes to the bystander effect following HSV-thymidine kinase gene therapy

Neoplastic transformation is frequently associated with a loss of gap junctional intercellular communication and reduced expression of connexins. The introduction of connexin genes into tumor cells reverses the proliferative characteristics of such cells. However, there is very little comparative information on the effects of different connexins on cancer cell growth. We hypothesized that Cx26, Cx32, or Cx43 would display differential growth suppression of C6 glioma cells and uniquely modulate the bystander effect following transduction of C6 cells with HSVtk followed by suicide gene therapy. The bystander phenomenon is the death of a greater number of tumor cells than are expressing the HSVtk gene, presumably due to the passage of toxic molecules through gap junction channels. To test this hypothesis, we used retroviral vectors to infect C6 glioma cells producing connexin-expressing and HSVtk-expressing cell lines. All three connexin-expressing cell lines grew significantly slower than GFP-infected or native C6 cells. Cx32 and Cx26 were significantly more effective at mediating the bystander effect in cocultures of C6-connexin cells with C6-HSVtk cells. These studies indicate that connexins have unique properties that contribute to their tumor suppressive function.     

The Sleep-inducing Lipid Oleamide Deconvolutes Gap Junction Communication and Calcium Wave Transmission in Glial Cells

Oleamide is a sleep-inducing lipid originally isolated from the cerebrospinal fluid of sleep-deprived cats. Oleamide was found to potently and selectively inactivate gap junction–mediated communication between rat glial cells. In contrast, oleamide had no effect on mechanically stimulated calcium wave transmission in this same cell type. Other chemical compounds traditionally used as inhibitors of gap junctional communication, like heptanol and 18β-glycyrrhetinic acid, blocked not only gap junctional communication but also intercellular calcium signaling. Given the central role for intercellular small molecule and electrical signaling in central nervous system function, oleamide- induced inactivation of glial cell gap junction channels may serve to regulate communication between brain cells, and in doing so, may influence higher order neuronal events like sleep induction.