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(The FASEB Journal. 2003;17:1422-1427.)
© 2003 FASEB

Bystander effect induced by counted high-LET particles in confluent human fibroblasts: a mechanistic study

CHUNLIN SHAO*,1, YOSHIYA FURUSAWA*,2, YASUHIKO KOBAYASHI{dagger}, TOMOO FUNAYAMA{dagger} and SEIICHI WADA{dagger}

* Heavy-Ion Radiobiology Research Group, National Institute of Radiological Sciences, Inage, Chiba 263-8555, Japan; and
{dagger} Biotechnology Laboratory, Takasaki Radiation Chemistry Establishment, Japan Atomic Energy Research Institute, Takasaki Gunma 370-1201, Japan

2Correspondence: National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage, Chiba 263-8555, Japan. E-mail: Furusawa{at}nirs.go.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The possible mechanism of a radiation-induced bystander response was investigated by using a high-LET heavy particle microbeam, which allows selected cells to be individually hit with precise numbered particles. Even when only a single cell within the confluent culture was hit by one particle of 40Ar (~1260 keV/µm) or 20Ne (~380 keV/µ m), a 1.4-fold increase of micronuclei (MN) was detected demonstrating a bystander response. When the number of targeted cells increased, the number of MN biphasically increased; however, the efficiency of MN induction per targeted cell markedly decreased. When 49 cells in the culture were individually hit by 1 to 4 particles, the production of MN in the irradiated cultures were ~2-fold higher than control levels but independent of the number and LET of the particles. MN induction in the irradiated-culture was partly reduced by treatment with DMSO, a scavenger of reactive oxygen species (ROS), and was almost fully suppressed by the mixture of DMSO and PMA, an inhibitor of gap junctional intercellular communication (GJIC). Accordingly, both ROS and GJIC contribute to the above-mentioned bystander response and GJIC may play an essential role by mediating the release of soluble biochemical factors from targeted cells.—Shao, C., Furusawa, Y., Kobayashi, Y., Funayama, T., Wada, S. Bystander effect induced by counted high-LET particles in confluent human fibroblasts: a mechanistic study.


Key Words: microbeam • micronucleus • gap junction • reactive oxygen species


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IT HAS BEEN COMMONLY ACCEPTED for a long time that damage to DNA, either by direct ionization or by the production of reactive free radicals in water molecules, is required to induce heritable damage in cells. Numerous microdosimetric models impose the assumption that the traversals of {alpha}-particles through the nuclei of cells within the lung alone cause the induction of cancer (1) . However, considerable evidence has recently been accumulated in support of the existence of a "bystander effect," in which cells having received no irradiation show biological consequences from their neighboring irradiated cells (2 3 4) . A characteristic of bystander responses is that the proportion of cells showing biological damage, such as sister chromatid exchange (SCE) in fibroblast cells, greatly exceeds the statistical number of the cells having sustained a particle traversal. This phenomenon was first reported by Nagasawa and Little (5) and then demonstrated by other studies with various biological end points, including cell killing, micronucleus (MN) induction, mutation induction, and changes in cell growth (6 7 8 9 10) . These observations suggest that reconsideration may be in order of the current cancer risk assessments for low doses of radiation, especially after high-LET exposure.

It is believed that there are two pathways involved in radiation-induced bystander responses. Some medium-derived soluble factors such as short-lived reactive oxygen species (ROS) (11) , nitric oxide (NO) (12 13 14) , and long-lived cell line dependent transforming growth factor ß1 (TGF-ß1) (15 , 16) could be released from irradiated cells, then further induce a series of damage in the nonirradiated neighbor cells. On the other hand, gap junctional intercellular communication (GJIC) was found to be relevant to the molecular events leading to the modulation of p53 and p21 gene expression in nonirradiated bystander cells within a confluent human fibroblast population exposed to {alpha}-particles at a very low dose, and these expressions were reduced when GJIC was inhibited by lindane (17 18 19) . With a 3-dimensional tissue culture model, it was also found that GJIC played a role in enhancing cell damage and thus reducing the survival of nonirradiated neighboring V79 cells (20 , 21) .

Single-cell microbeam facilities in which cells are individually irradiated by a predefined exact number of particles allow the effects of individual particle traversals to be assessed, and these methods have become a useful tool in the study of bystander responses (22 23 24 25) . A bystander mutagenic effect has been found in nontraversed cells when a proportion of mammalian cells have suffered a precise number of nuclear traversals by {alpha}-particles (26 , 27) . Belyakov et al. (28) reported that when a single cell within a population was targeted by an {alpha}-particle, typically an additional ~100 damaged cells were observed in the surrounding population. The microbeam technique offers the possibility of cytoplasmic irradiation, and this has been found to be effective in causing cellular killing and mutation (29) .

{alpha}-Particles, helium-3 ions, and protons are currently used to study the microbeam irradiation-induced bioresponses. In this work, primary human fibroblast cells within a confluent population were individually hit by a high-LET heavy particle microbeam of 40Ar or 20Ne with LET values of ~1260 keV/µ m and 380 keV/µm, respectively. A clear bystander response, independent of these LETs and the number of charged particles delivered to the targeted cells, was observed and its possible formation pathway was further investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and treatments
AG1522 normal human diploid skin fibroblasts purchased from Coriell Cell Repositories were cultured at 37°C in humidified atmosphere of 95% air and 5% CO2 with Eagle’s minimum essential medium (Sigma M5650) supplemented with 2.0 mM L-glutamine and 18% fetal bovine serum (Lenexa) plus 100 µg/mL streptomycin and 100 U/mL penicillin. Cells destined for heavy-ion irradiation were seeded in specially made 35 mm diameter dishes with 8 µm-thick replaceable Kapton® polyimide film bottoms at a density of 2x105 cells per dish 4 days before irradiation. The medium was changed at day 2 so that at the time of irradiation the cells were in full confluence (~7x105 cells/dish). In some dishes, the cell culture medium was replaced with medium containing 1 nM PMA (4ß, 9{alpha} , 12ß, 13{alpha}, 20-pentahydro-xytiglia-1, 6-dien-3-one 12ß -myristate 13-acetate, 1 µM prepared in DMSO) 1 h before irradiation in order to prohibit GJIC; in this case, 14 mM DMSO was contained in the culture medium. In the following, we refer to this process simply as PMA treatment. Parallel to the PMA treatment, in some of other dishes the culture medium was replaced with one containing 14 mM DMSO 1 h before irradiation.

Just before irradiation, a Kapton® film was floated on the medium surface and the medium was removed. This process kept the cells fully hydrated during irradiation, which was <30 min. After irradiation, 2 mL medium was immediately supplied to each dish and cells were subsequently cultivated for 15 h until they were harvested for the MN assay. During this process, with respect to PMA pretreated culture, PMA with a reduced concentration of 0.5 nM in case of any toxic effect was added in the medium to maintain the inhibition level of GJIC. Correspondingly, for the DMSO pretreated culture, 7 mM DMSO was contained in the culture medium after irradiation. Nonirradiated control dishes were treated with the same protocol except for irradiation. Chemical treatment of PMA or DMSO did not show a significant toxic effect on cells.

Cell irradiation
A charged particle microbeam apparatus installed below a vertical beam line of the cyclotron at TIARA (Takasaki Ion Accelerators for Advanced Radiation Application) of JAERI (Japan Atomic Energy Research Institute) was used to deliver precise number of heavy ions of 260 MeV 20Ne and 460 MeV 40Ar with calculated LET values of ~380 keV/µ m and 1260 keV/µ m, respectively, for cell irradiation. Particles traversing through horizontal film dish and cells were detected by plastic scintillator and photomultiplier tube (PMT). The spatial resolution achieved by this collimated facility was ±5 µm; this was substantially smaller than the measured average cell size of 1370 µm2, so that in the case of multiparticle irradiation, the particles have a possibility of >98% to traverse through in the same single cell rather than through different cells.

Two inverted optical microscopes (Nikon, TMD-300) were operated to locate cell position. One microscope mounted with the PMT was installed below the vertical beam line as an "on-line microscope" for on-line cell observing and cell targeting. The other microscope was used as an "off-line microscope" in the preparation room for cell finding before irradiation. A local network connected the microscope control systems allowing the database of the cells selected at the off-line microscope to be used by the cell targeting system. The cells in confluent culture were irradiated in two ways; 1 to 121 cells with a matrix distribution of 11x11 mm2 in the culture center were individually hit by 1 particle; on the other hand, 49 matrix-distributed cells within a confluent culture were individually hit by particles of numbers from 1 to 4. Because it has been shown that both nuclear and cytoplasmic traversals are able to generate cellular responses (29 , 30) , we did not specially target the cell nucleus or cytoplasm. This consideration also enabled us to avoid using any chemical staining treatment to locate the nucleus or cytoplasm, as has been used in other studies.

Dye transfer assay
GJIC situation in confluent culture was tested by the scrape loading and dye transfer technique (31) Briefly, 2 mL of 0.05% Lucifer yellow (Sigma) in PBS was added in 35 mm cell dish where the confluent cell population had been washed by PBS twice. Then, the cell layer was scraped with a scalpel blade and kept in an incubator at 37°C for 3 min. After three rinses with fresh PBS to remove the dye solution, the cell layer was observed with a fluorescence microscope.

MN assay
The formation of MN was assayed by using the cytokinesis block technique developed by Fenech and Morley (32) . The harvested cells were incubated in the presence of 2.5 µg/mL cytochalasin-B. After 48 h, the cells were collected, suspended in a 0.075 M KCl hypotonic solution at 37°C for 10 min, and fixed by methanol at -30°C overnight. A portion of the fixed cells was placed on a glass slide and stained with 10 µg/mL acridine orange for 5 min. After this cytochalasin-B treatment, ~20% of cells became binucleated (BN) cells. MN in BN cells was checked by fluorescence microscopy and morphologically identified by the criteria method (33) . At least 1000 BN cells were scored at each dose point for the MN measurement in three separate experiments.

Statistical analysis
Data from three independent experiments, each run in duplicates, were presented as mean ± SE. Significance levels were assessed using the Student’s t test; P<0.01 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Influence of PMA on GJIC
The intact AG1522 monolayer was impermeable to Lucifer yellow, but when scraped, Lucifer yellow of a low molecular mass (457.2 Da) permeated into the damaged cells and subsequently migrated readily to neighboring cells through gap junctions. A competent transfer of Lucifer yellow in the cultures with a scraped row was observed in the cell layer without any chemical treatment (Fig. 1 A), which indicates that good cell–cell communication exists in this confluent cell population. However, the dye transfer profile in Fig. 1B provided evidence that GJIC was inhibited by the treatment of 1 nM PMA since the ability of Lucifer yellow transfer was reduced extensively. This inhibition was maintained when the culture was subsequently treated by 0.5 nM PMA after microbeam irradiation. However, DMSO treatment did not influence the dye transfer situation in the cell layer (data not show).



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  		Figure 1. Transfer of the fluorescent dye Lucifer yellow through gap junctions in AG1522 confluent culture without any treatment (A) or with treatment of 1 nM PMA for 1 h (B).

Response of MN induction to the number of targeted cells
When a few selected cells within a population in the dish were individually hit by a precisely numbered particle and maintained in culture, additional MN was significantly produced in the surviving BN cells generated by the cytokinesis block technique. Most micronucleated BN cells had one MN and a few contained two or three MN. Figure 2 illustrates the yield of MN induction (i.e., the ratio of the number of MN to the number of the observed BN cells) of the culture where 1 to 121 cells are individually targeted by one 40Ar particle or 20Ne particle. Even when only a single cell within the population was targeted with one particle, the level of MN was increased ~1.4-fold above that of the unirradiated control. Based on the observed yield of MN and the frequency of BN formation, it was calculated that additional MN could be produced in ~3000 BN cells when only one cell was actually hit in the confluent culture. This additional MN induction must result from some factors other than a direct radiation effect, i.e., a bystander response.



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  		Figure 2. Response of the yield of MN to the number of targeted cells. Cells with a matrix distribution within a confluent culture were individually hit by one 40Ar particle (A) or one 20Ne particle (B) and then were incubated for 15 h before MN assay. The culture was treated by PMA ({square}), DMSO ({blacktriangleup}), or was not treated (•) before and after irradiation (see text).

To investigate the possible pathways of the bystander effect, we treated the cultures with PMA or DMSO before and after irradiation as described above. Neither treatment itself significantly changed the MN background. It is seen from Fig. 2 that the yield of MN of the PMA-treated culture where selected cells were individually hit by one particle of 40Ar or 20Ne was reduced to a very low level, comparable to the control without any irradiation, and the MN yield was partly (around half) reduced by the treatment of DMSO. These results indicate that both GJIC and ROS contribute to the microbeam irradiation-induced bystander effect.

Although the LET values of 40Ar and 20Ne particles were quite different, the yields of MN induced by these two kinds of irradiations had no significant difference. For instance, when 49 cells within the population were individually hit by one particle, the yield of MN was 0.074 for 40Ar irradiation and 0.072 for 20Ne irradiation. Thus, the MN induction was independent of the LET of these particles delivered to the selected cells within a confluent culture.

With respect to both 40Ar and 20Ne irradiated cultures, Fig. 2 shows that the yields of MN have biphasic responses to the number of targeted cells. When the number of targeted cells increased from 1 to 4, the yield of MN increased with a slope larger than when the number of targeted cells increased from 16 to 121. This biphasic response is clearly shown in Fig. 3 by replotting the data in Fig. 2 using the efficiency of MN induction: the yield of MN per targeted cell as a function of the number of targeted cells. The highest efficiency of MN induction was generated in the case of a single cell irradiated by one particle. When the number of targeted cells increased, the efficiency of MN induction drastically decreased.



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  		Figure 3. Response of the yield of MN per targeted cell to the number of targeted cells. The data were calculated from that in Fig. 2 . Cells without any treatment were hit by 40Ar particle and 20Ne particle, respectively.

Response of MN induction to the particle number
We further studied the dose effect of MN production induced by counted 40Ar and 20Ne particle irradiation. For this purpose, 49 cells within the confluent culture were hit by 1 to 4 particles. Figure 4 shows that the yields of MN in the irradiated cultures are ~twofold higher than the unirradiated control level, which means that additional MN are produced in ~4500 BN cells since a bystander response ensued even though only 49 cells are actually irradiated. Moreover, it was found that MN induction in those cultures where 49 selected cells were irradiated by 40Ar or 20Ne particles of different numbers was not significantly influenced by the number of particle traversals, but was partly reduced or almost totally inhibited by the treatment of DMSO or PMA, respectively. In general, the higher the LET and dose of radiation, the more serious the cellular damage. However, the results in Fig. 4 indicate that the level of bystander-induced MN does not depend on the amount of direct damage induced in the targeted cells. Thus the bystander phenomenon is quite different from broad beam irradiation-induced cellular damage, which has a positive relationship to radiation dose and LET (34) , and illustrates that the bystander effect is dominated by signaling type of behavior whereas direct damage is governed by stochastic mechanisms.



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  		Figure 4. Response of the yield of MN to the number of particles delivered to 49 cells with a matrix distribution within a confluent culture. Cells were individually hit by 40Ar particles (A) or 20Ne particles (B), then incubated for 15 h before MN assay. The culture was treated by PMA (blank bar) or DMSO (hatched bar) (see text) or was not treated (black bar). *P < 0.01 compared with control without irradiation and to the DMSO- or PMA-treated cultures, **P < 0.01 compared with control without irradiation and to the PMA-treated culture.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, it was found that single cell targeted by one particle was enough to trigger a bystander response throughout a cell population, producing MN in thousands of nontargeted neighboring cells (see Fig. 2 ). This observation of the irradiation-induced bystander effect may be significant in terms of cancer risk of low-dose high-LET radiations such as occur in space flight and with indoor radon contamination. Considering our previous findings that a cell proliferation enhancement effect accompanied by DNA damage in a tumor cell population could be induced by irradiated bystander cells (13) , an additional cancer risk may arise in neighboring nontargeted normal tissues cells if a bystander response occurs during a radiation therapy.

On the other hand, when the number of cells individually targeted by one particle increased from 1 to 121, the efficiency of MN induction per targeted cell decreased markedly, although the overall number of MN increased biphasically (see Fig. 3 ). Thus, the number of targeted cells appears to be a nonessential factor in the induction of bystander responses. Provided the targeted cell received one or more particle traversals, the bystander response was triggered and caused neighboring nonirradiated cells to be damaged. The newly damaged cells will possibly cause other cellular damage. With a series of cascade responses, the original radiation-induced cellular damage will be significantly amplified.

To investigate the factors involved in the bystander responses, we treated cells with DMSO so as to scavenge radiation-induced ROS. This treatment partly reduced MN induction (see Fig. 2 ), indicating that ROS contributes to the above-mentioned bystander response. Once the original radiation-induced ROS is scavenged, any further subsequent cascade cellular damage responses will certainly be halted. Therefore, it can be expected that DMSO with a low concentration will be enough to reduce ROS-induced bystander effect. Here the applied concentration of DMSO of 14 mM gives a scavenging capacity of 9.3 x 107 s-1 to •OH free radical (K•OH=6.6x109 M-1s-1) (35) . The amount of DMSO molecules could be enough to react with ROS released by one or a few targeted cells and diffused in the culture medium. It has been reported that this scavenging capacity effectively suppresses radiation-induced DNA damage in vitro (36) . On the other hand, even though the concentration of DMSO increased to as high as 5% in experiments using a very low dose of broad heavy-ion irradiation, our unpublished data demonstrated that even this treatment only partly reduced radiation-induced bystander MN formation in a confluent AG1522 population. Therefore, additional factors that cannot be scavenged by DMSO appear to contribute to the microbeam irradiation-induced bystander effect. It has been found that, in association with a decreased TP53/CDKN1A bystander response, TGF-ß was released from irradiated fibroblast cells so that the growth of bystander cells was altered (16) . It has been reported that nitric oxide is released from heavy-ion irradiated human salivary gland neoplastic cells to further induce damage in other nonirradiated cells(10) .

We further treated cells with PMA containing DMSO solvent. This treatment is most effective in decreasing the MN induction in the cell population irradiated by precise numbered charged particles (see Figs. 2 and 4 ). Therefore, GJIC is another key factor in the induction of the bystander response in a confluent culture. The GJIC-mediated pathway and the medium-derived pathway most likely are not separate pathways but are interrelated. One possibility is that GJIC regulates the release of irradiation-induced diffusible biochemical factors. When the gap junctional channels are closed by treatment with PMA, most of the biochemical factors will be inhibited from being released by damaged cells. However, when the culture is treated with the mixture of PMA and DMSO, the MN induction is still not fully decreased to the background level. The direct irradiation effect could be one source of this MN induction. Another possible source might be other bystander signal factors that cannot be mediated by GJIC. For instance, it has been reported that membrane signaling such as the second messenger ceramide is involved in the irradiation-induced bystander mutation effect (37) . But GJIC may have poor control over release of the cell surface receptor involved in signaling molecules.

Based on the present and other findings, we hypothesize that the bystander effect induced by the counted particle irradiation may have the following mechanism. When a cell within a confluent culture is traversed by heavy particles, it can be expected to release some biochemical factors, including ROS and NO, during the processing of cell damage repair or during programmed cell death (38) . These chemical factors will react with the neighboring nontargeted cells; the damaged neighboring cells will release the biochemical factors again and downstream cause other cells to be damage. With this kind of cascade reaction, the damage generated in the original targeted cell is amplified so that a detectable bystander response is produced. The release of the biochemical factor may be mediated by the functionality of cellular gap junctions.


   ACKNOWLEDGMENTS
 
We are grateful to Professor Barry D. Michael, Gray Cancer Institute of U.K., for his valuable suggestions.


   FOOTNOTES
 
1 Present address: Gray Cancer Institute, P.O. Box 100, Mount Vernon Hospital, Northwood, Middlesex, HA6 2JR, UK. E-mail: Shao{at}gci.ac.uk

Received for publication November 19, 2002. Accepted for publication March 28, 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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