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.     

Effect of gap junctional intercellular communication on radiation responses in neoplastic human cells

Gap junctional intercellular communication (GJIC) is an important function of metazoan cells and is believed to have beneficial effects in anti-tumor therapy. In this study, we found that, when neoplastic human salivary gland (HSG) cells were irradiated with a 100 keV/microm carbon-ion beam, micronuclei, G(2)/M-phase arrest, and cell killing were induced and that their induction increased with dose. Treatment of confluent HSG cells with 8-Br-cAMP increased GJIC between cells. After release from this treatment, the cell cycle progress and the formation of binucleated cells were still similar to those of untreated cells. However, radiation-induced cellular damage, including micronucleus (MN) formation and G(2)/M-phase arrest of that cAMP-treated population, was less than that of the untreated population and that the surviving fraction was slightly enhanced by cAMP treatment, suggesting that increased GJIC protects HSG cells from lethal radiation damage. Moreover, when confluent HSG cells were treated with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), a scavenger of nitric oxide (NO) free radical, MN induction and cell killing in the irradiated population were increased. Our results indicate that NO may be involved in GJIC-mediated radioprotection of HSG cells, which may have implications for radiotherapy.     

Calcium fluxes modulate the radiation-induced bystander responses in targeted glioma and fibroblast cells

Bystander responses have been reported to be a major determinant of the response of cells to radiation exposure at low doses, including those of relevance to therapy. This study investigated the role of changes in calcium levels in bystander responses leading to chromosomal damage in nonirradiated T98G glioma cells and AG01522 fibroblasts that had been either exposed to conditioned medium from irradiated cells or co-cultured with a population where a fraction of cells were individually targeted through the nucleus or cytoplasm with a precise number of microbeam helium-3 particles. After the recipient cells were treated with conditioned medium from T98G or AG01522 cells that had been irradiated through either nucleus or cytoplasm, rapid calcium fluxes were monitored in the nonirradiated recipient cells. Their characteristics were dependent on the source of the conditioned medium but had no dependence on radiation dose. When recipient cells were co-cultured with an irradiated population of either T98G or AG01522 cells, micronuclei were induced in the nonirradiated cells, but this response was eliminated by treating the cells with calcicludine (CaC), a potent blocker of Ca(2+) channels. Moreover, both the calcium fluxes and the bystander effect were inhibited when the irradiated T98G cells were treated with aminoguanidine, an inhibitor of nitric oxide synthase (NOS), and when the irradiated AG01522 cells were treated with DMSO, a scavenger of reactive oxygen species (ROS), which indicates that NO and ROS were involved in the bystander responses generated from irradiated T98G and AG01522 cells, respectively. Our findings indicate that calcium signaling may be an early response in radiation-induced bystander effects leading to chromosome damage.     

Mitochondrial reactive oxygen species-mediated genomic instability in low-dose irradiated human cells through nuclear retention of cyclin D1

Mitochondria are associated with various radiation responses, including adaptive responses, mitophagy, the bystander effect, genomic instability, and apoptosis. We recently identified a unique radiation response in the mitochondria of human cells exposed to low-dose long-term fractionated radiation (FR). Such repeated radiation exposure inflicts chronic oxidative stresses on irradiated cells via the continuous release of mitochondrial reactive oxygen species (ROS) and decrease in cellular levels of the antioxidant glutathione. ROS-induced oxidative mitochondrial DNA (mtDNA) damage generates mutations upon DNA replication. Therefore, mtDNA mutation and dysfunction can be used as markers to assess the effects of low-dose radiation. In this study, we present an overview of the link between mitochondrial ROS and cell cycle perturbation associated with the genomic instability of low-dose irradiated cells. Excess mitochondrial ROS perturb AKT/cyclin D1 cell cycle signaling via oxidative inactivation of protein phosphatase 2A after low-dose long-term FR. The resulting abnormal nuclear accumulation of cyclin D1 induces genomic instability in low-dose irradiated cells.     

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.     

Refinement of the dichlorofluorescein assay for flow cytometric measurement of reactive oxygen species in irradiated and bystander cell populations

Reactive oxygen species (ROS) have been implicated in many ionizing radiation-related phenomena, including bystander effects. The oxidation of 2'7'-dichlorofluorescin (DCFH) to fluorescent 2'7'-dichlorofluorescein (DCF) is commonly used for the detection of radiation-induced ROS. The DCF assay was adapted for efficient, systematic flow cytometry quantification of low-linear energy transfer (LET) gamma-radiation-induced ROS in vitro in Chinese hamster ovary (CHO) cells. This method is optimized for increased sensitivity to radiation-induced ROS and to discriminate against measurement of extracellular ROS. This method can detect a significant increase in ROS in cells exposed to gamma radiation at doses as low as 10 cGy. The antioxidants N-acetyl-cysteine and ascorbic acid (vitamin C) significantly reduced the amount of ROS measured in cells exposed to 5 Gy ionizing radiation. This method was used to measure the intracellular ROS in unirradiated CHO bystander cells co-cultured with low-LET-irradiated cells. No increase in ROS was measured in bystander cell populations co-cultured with the irradiated cells beginning 9 s after radiation exposure.     

Bystander normal human fibroblasts reduce damage response in radiation targeted cancer cells through intercellular ROS level modulation

The radiation-induced bystander effect is a well-established phenomenon which results in damage in non-irradiated cells in response to signaling from irradiated cells. Since communication between irradiated and bystander cells could be reciprocal, we examined the mutual bystander response between irradiated cells and co-cultured with them non-irradiated recipients. Using a transwell culture system, irradiated human melanoma (Me45) cells were co-cultured with non-irradiated Me45 cells or normal human dermal fibroblasts (NHDF) and vice versa. The frequency of micronuclei and of apoptosis, ROS level, and mitochondrial membrane potential were used as the endpoints. Irradiated Me45 and NHDF cells induced conventional bystander effects detected as modest increases of the frequency of micronuclei and apoptosis in both recipient neighbors; the increase of apoptosis was especially high in NHDF cells co-cultured with irradiated Me45 cells. However, the frequencies of micronuclei and apoptosis in irradiated Me45 cells co-cultured with NHDF cells were significantly reduced in comparison with those cultured alone. This protective effect was not observed when irradiated melanomas were co-cultured with non-irradiated cells of the same line, or when irradiated NHDF fibroblasts were co-cultured with bystander melanomas. The increase of micronuclei and apoptosis in irradiated Me45 cells was paralleled by an increase in the level of intracellular reactive oxygen species (ROS), which was reduced significantly when they were co-cultured for 24h with NHDF cells. A small but significant elevation of ROS level in NHDF cells shortly after irradiation was also reduced by co-culture with non-irradiated NHDF cells. We propose that in response to signals from irradiated cells, non-irradiated NHDF cells trigger rescue signals, whose nature remains to be elucidated, which modify the redox status in irradiated cells. This inverse bystander effect may potentially have implications in clinical radiotherapy.     

Inhibitory effects of eicosapentaenoic acid (EPA) on the hypoxia/reoxygenation-induced tyrosine kinase activation in cultured human umbilical vein endothelial cells

We have previously reported that the n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) inhibited the abnormal gap junctional intercellular communication (GJIC) induced by hypoxia/reoxygenation (H/R) via suppressing tyrosine kinase (TK) activation (Zhang et al., Prostaglandins Leukot Essent Fatty Acids, 1999; 61: 33-40). However, the mechanisms by which EPA-inhibited TK activation remained unidentified. In this study we investigated whether reactive oxygen species (ROS) and growth factor-receptor systems would contribute to the H/R-induced TK activation or not. The results showed that H/R-induced ROS production, which reached the peak after 30 min of reoxygenation. Pretreatment with 10 microM EPA significantly inhibited this ROS production. However, the TK inhibitor genistein (10 microM) failed to inhibit the generation of ROS, although it completely inhibited TK activation. On the other hand, the ROS inhibitor DMSO (0.5% v/v) showed little effect on TK activation while it significantly blocked ROS production. Further EPA and genistein, but not DMSO and superoxide dismutase (SOD, 300 U/ml), prevented cells from GJIC injury induced by H/R. Moreover, EPA protected against VEGF-induced reduction in GJIC and phosphorylation of connexin 43. These data suggest that growth factor, but not ROS, might be involved in the EPA-inhibited TK activation induced by H/R.     

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.     

Neoplastic transformation in vitro after exposure to low doses of mammographic-energy X rays: quantitative and mechanistic aspects

The induction of neoplastic transformation in vitro after exposure of HeLa x skin fibroblast hybrid cells to low doses of mammography-energy (28 kVp) X rays has been studied. The data indicate no evidence of an increase in transformation frequency over the range 0.05 to 22 cGy, and doses in the range 0.05 to 1.1 cGy may result in suppression of transformation frequencies to levels below that seen spontaneously. This finding is not consistent with a linear, no-threshold dose- response curve. The dose range at which possible suppression is evident includes doses typically experienced in mammographic examination of the human breast. Experiments are described that attempt to elucidate any possible role of bystander effects in modulating this low-dose radiation response. Not unexpectedly, inhibition of gap junction intercellular communication (GJIC) with the inhibitor lindane did not result in any significant alteration of transformation frequencies seen at doses of 0.27 or 5.4 cGy in these subconfluent cultures. Furthermore, no evidence of a bystander effect associated with factors secreted into the extracellular medium was seen in medium transfer experiments. Thus, in this system and under the experimental conditions used, bystander effects would not appear to be playing a major role in modulating the shape of the dose-response curve.     

DNA double-strand breaks induced by very low X-ray doses are largely due to bystander effects

Ojima, M., Ban, N. and Kai, M. DNA Double-Strand Breaks Induced by Very Low X-Ray Doses are Largely due to Bystander Effects. Radiat. Res. 170, 365-371 (2008).Phosphorylated ATM immunofluorescence staining was used to investigate the dose-response relationship for the number of DNA double-strand breaks (DSBs) induced in primary normal human fibroblasts irradiated with doses from 1.2 to 200 mGy. The induction of DSBs showed a supralinear dose-response relationship. Radiation-induced bystander effects may explain these findings. To test this hypothesis, the number of DSBs in cells treated with lindane, an inhibitor of radiation-induced bystander effects, prior to X irradiation was assessed; a supralinear dose-response relationship was not observed. Moreover, the number of DSBs obtained by subtracting the number of phosphorylated ATM foci in lindane-treated cells from the number of phosphorylated ATM foci in untreated cells was proportional to the dose at low doses (1.2-5 mGy) and was saturated at doses from 10-200 mGy. Thus the increase in the number of DSBs in the range of 1.2-5 mGy was largely due to radiation-induced bystander effects, while at doses >10 mGy, the DSBs may be induced mainly by dose-dependent direct radiation effects and partly by dose-independent radiation-induced bystander effects. The findings in our present study provide direct evidence of the dose-response relationship for radiation-induced bystander effects from broad-beam X rays.     

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.     

Radiation-induced bystander effect in healthy G(o) human lymphocytes: biological and clinical significance

To study the bystander effects, G(0) human peripheral blood lymphocytes were X-irradiated with 0.1, 0.5 and 3 Gy. After 24h, cell-free conditioned media from irradiated cultures were transferred to unexposed lymphocytes. Following 48 h of medium transfer, viability, induction of apoptosis, telomere shortening, reactive oxygen species (ROS) levels and micronuclei (after stimulation) were analyzed. A statistically significant decrement in cell viability, concomitant with the loss of mitochondrial membrane potential, telomere shortening, increases in hydrogen peroxide (H(2)O(2)) and superoxide anion (O(2)(-)) with depletion of intracellular glutathione (GSH) level, and higher frequencies of micronuclei, were observed in bystander lymphocytes incubated with medium from 0.5 and 3 Gy irradiated samples, compared to lymphocytes unexposed. Furthermore, no statistically significant difference between the response to 0.5 and 3 Gy of irradiation in bystander lymphocytes, was found. However, when lymphocytes were irradiated with 0.1 Gy, no bystander effect with regard to viability, apoptosis, telomere length, and micronuclei was observed, although a high production of ROS level persisted. Radiation in the presence of the radical scavenger dimethyl sulfoxide (DMSO) suppressed oxidative stress induced by 3 Gy of X-rays with the effective elimination of bystander effects, suggesting a correlation between ROS and bystander signal formation in irradiated cells. The data propose that bystander effect might be mostly due to the reactions of radiation induced free radicals on DNA, with the existence of a threshold at which the bystander signal is not operative (0.1 Gy dose of X-rays). Our results may have clinical implications for health risk associated with radiation exposure.     

Detection of chromosomal instability in alpha-irradiated and bystander human fibroblasts

There is increasing evidence biological responses to ionizing radiation are not confined to those cells that are directly hit, but may be seen in the progeny at subsequent generations (genomic instability) and in non-irradiated neighbors of irradiated cells (bystander effects). These so called non-targeted phenomena would have significant contributions to radiation-induced carcinogenesis, especially at low doses where only a limited number of cells in a population are directed hit. Here we present data using a co-culturing protocol examining chromosomal instability in alpha-irradiated and bystander human fibroblasts BJ1-htert. At the first cell division following exposure to 0.1 and 1Gy alpha-particles, irradiated populations demonstrated a dose dependent increase in chromosome-type aberrations. At this time bystander BJ1-htert populations demonstrated elevated chromatid-type aberrations when compared to controls. Irradiated and bystander populations were also analyzed for chromosomal aberrations as a function of time post-irradiation. When considered over 25 doublings, all irradiated and bystander populations had significantly higher frequencies of chromatid aberrations when compared to controls (2-3-fold over controls) and were not dependent on dose. The results presented here support the link between the radiation-induced phenomena of genomic instability and the bystander effect.     

Gap junctions mediate STAT5-independent β-casein expression in CID-9 mammary epithelial cells

Crosstalk between gap junction intracellular communication (GJIC), STAT5 and OCT-1 in gap junction (GJ)-dependent β-casein expression was investigated. CID-9 mammary cells plated with prolactin on non-adherent substratum (poly-HEMA) expressed β-casein independent of STAT5 only in the presence of the GJIC inducer, cAMP. Nuclear STAT5 levels were not detectable. By contrast, cells on EHS-drip expressed β-casein in a STAT5-dependent manner and nuclear STAT5 levels were up-regulated. A 75 kDa OCT-1 isoform was detected in conditions that induced β-casein expression regardless of substratum. Interestingly, 40 and 28 kDa OCT-1 isoforms were induced in cells on polyHEMA with cAMP. Electrophoretic mobility shift assays (EMSA) for OCT-1 revealed two band shifts in cells on polyHEMA with cAMP and on EHS-drip, which were repressed by the GJIC inhibitor, 18α-GA. These studies demonstrated that mammary cells on polyHEMA expressed β-casein in response to prolactin in a pathway that involves GJIC and OCT-1 and is independent of STAT5 nuclear translocation.     

Gap junctions mediate STAT5-independent β-casein expression in CID-9 mammary epithelial cells

Crosstalk between gap junction intracellular communication (GJIC), STAT5 and OCT-1 in gap junction (GJ)-dependent β-casein expression was investigated. CID-9 mammary cells plated with prolactin on non-adherent substratum (poly-HEMA) expressed β-casein independent of STAT5 only in the presence of the GJIC inducer, cAMP. Nuclear STAT5 levels were not detectable. By contrast, cells on EHS-drip expressed β-casein in a STAT5-dependent manner and nuclear STAT5 levels were up-regulated. A 75 kDa OCT-1 isoform was detected in conditions that induced β-casein expression regardless of substratum. Interestingly, 40 and 28 kDa OCT-1 isoforms were induced in cells on polyHEMA with cAMP. Electrophoretic mobility shift assays (EMSA) for OCT-1 revealed two band shifts in cells on polyHEMA with cAMP and on EHS-drip, which were repressed by the GJIC inhibitor, 18α-GA. These studies demonstrated that mammary cells on polyHEMA expressed β-casein in response to prolactin in a pathway that involves GJIC and OCT-1 and is independent of STAT5 nuclear translocation.     

Persistence of DNA double-strand breaks in normal human cells induced by radiation-induced bystander effect

Our previous study suggested that the DNA double-strand breaks (DSBs) induced by very low X-ray doses are largely due to bystander effects. The aim of this study was to verify whether DSBs created by radiation-induced bystander effects are likely to be repaired. We examined the generation of DSBs in cells by enumeration of phosphorylated ataxia telangiectasia mutated (ATM) foci, which are correlated with DSB repair, in normal human fibroblast cells (MRC-5) after X irradiation at doses ranging from 1 to 1000 mGy. At 24 h after irradiation, 100% (1.2 mGy), 58% (20 mGy), 12% (200 mGy) and 8.5% (1000 mGy) of the initial number of phosphorylated ATM foci were detected. The number of phosphorylated ATM foci in MRC-5 cells treated with lindane, an inhibitor of radiation-induced bystander effects, prior to X irradiation was assessed; phosphorylated ATM foci were not observed at 5 h (20 mGy) or 24 h (200 mGy) postirradiation. We also counted the number of phosphorylated ATM foci in MRC-5 cells cocultured with MRC-5 cells irradiated with 20 mGy. After 48 h of coculture, 81% of the initial numbers of phosphorylated ATM foci remained. These findings suggest that DSBs induced by the radiation-induced bystander effect persist for long periods, whereas DSBs induced by direct radiation effects are repaired relatively quickly.     

Effects of pyrethroid insecticides on gap junctional intercellular communications in Balb/c3T3 cells by dye-transfer assay

The effects of fenvalerate, esfenvalerate, permethrin, cypermethrin, deltamethrin, p-chlorophenylisovaleric acid (CPIA, major metabolite of fenvalerate) and DDT, a liver tumor promoter, on gap junctional intercellular communication (GJIC) were examined in Balb/c3T3 cells by dye-transfer assay. Separate groups of Balb/c3T3 cells were exposed to the chemicals for 1 day. On the following day, GJIC was measured by counting the number of dye-transferring cells per injection of Lucifer Yellow under a fluorescent microscope. Fenvalerate, esfenvalerate, permethrin, cypermethrin, deltamethrin and DDT inhibited GJIC at noncytotoxic concentrations, while CPIA did not inhibit GJIC even, at a cytotoxic concentration. It is concluded that the examined pyrethroid insecticides, but not a metabolite, have inhibitory effects on GJIC in Balb/c3T3 cells.Abbreviations DDT 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane - DMSO dimethyl sulfoxide