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[Cancer Research 62, 5436-5442, October 1, 2002]
© 2002 American Association for Cancer Research

Biochemistry and Biophysics

Oxidative Metabolism Modulates Signal Transduction and Micronucleus Formation in Bystander Cells from {alpha}-Particle-irradiated Normal Human Fibroblast Cultures1

Edouard I. Azzam, Sonia M. de Toledo, Douglas R. Spitz and John B. Little2

Department of Cancer Cell Biology, Laboratory of Radiobiology, Harvard School of Public Health, Boston, Massachusetts 02115 [E. I. A., S. M. d. T., J. B. L.]; Department of Radiology, New Jersey Medical School, Newark, New Jersey 07103 [E. I. A., S. M. d. T.]; and Free Radical and Radiation Biology Program, Department of Radiation Oncology, University of Iowa, Iowa City, Iowa 52242 [D. R. S.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of oxidative metabolism in the up-regulation/activation of stress-induciblesignaling pathways as well as induction of micronucleus formation in bystander cells was investigated. By immunoblotting and in situ immunofluorescence, active Cu-Zn superoxide dismutase (SOD) enzyme and active catalase enzyme were shown to inhibit the up-regulation of p21Waf1 as well as the induction of micronucleus formation in bystander cells from confluent cultures of normal human diploid fibroblasts irradiated with 0.3–3 cGy of {alpha}-particles. Enzyme activity assays indicated that exogenous SOD became significantly associated with the cells. Reactive oxygen species apparently derived from a flavin-containing oxidase enzyme [presumably an NAD(P)H-oxidase] appeared to be major contributors to the bystander-induced up-regulation of p53 and p21Waf1 as well as micronucleus formation, as evidenced by the inhibition of these effects with diphenyliodonium. Rapid activation of nuclear factor {kappa}B, Raf-1, extracellular signal-regulated kinase 1/2, c-Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase and their downstream effectors activator protein 1, ELK-1, p90RSK, and activating transcription factor 2 was also observed in cultures exposed to very low fluences of {alpha}-particles. Significant attenuation in the activation of these kinases and transcription factors occurred in irradiated cultures treated with either SOD or catalase. Overall, these results support the hypothesis that superoxide and hydrogen peroxide produced by flavin-containing oxidase enzymes mediate the activation of several stress-inducible signaling pathways as well as micronucleus formation in bystander cells from cultures of human cells exposed to low fluences of {alpha}-particles.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increasing experimental evidence indicates that traversal of the nucleus by an ionizing particle or photon is not a prerequisite to elicit a radiation response. Nontraversed bystander cells that are in the vicinity of irradiated cells (1 , 2) or are recipients of growth medium from radiation-exposed cultures (3) have been shown to exhibit responses similar to those of irradiated cells. Changes in gene expression (4 , 5) , induction of genetic changes including mutations (6 , 7) , DNA damage (8 , 9) , lethality (10) , and transformation to the neoplastic state (11) have been observed in bystander cells of various types, suggesting that these cells contribute to the risk of exposure to ionizing radiation.

Whereas evidence for radiation-induced effects in nontargeted cells has been well established, a clear understanding of the basic mechanisms by which damaging effects are transmitted from irradiated to nonirradiated cells is only beginning to emerge. {alpha}-Particles have been previously shown to initiate the biological production of ROS3 (superoxide anions and hydrogen peroxide) in human cells (12 , 13) . In other studies, the antioxidant DMSO inhibited the induction of mutations by cytoplasmic irradiation of human-hamster hybrid cells (14) . Oxidative stress has also been implicated in toxic effects observed in bystander cells in which {gamma}- or ß-particle radiations were used (15, 16, 17) .

Whereas radiation-induced ROS are known to participate in damage to various cellular components (reviewed in Refs. 18 and 19 ) and produce double-strand breaks in addition to base damage and single-strand breaks (reviewed in Refs. 20, 21, 22 ), the involvement of {alpha}-particle-induced metabolic ROS production in the activation of signaling pathways in bystander cells is not well characterized. In the present study, immunoblot analyses and in situ immunodetection techniques were used to examine the role of ROS formed by metabolic processes in the induction of stress-inducible proteins (in the p53 and MAPK pathways) in bystander cells from confluent cultures of human diploid fibroblasts exposed to fluences of {alpha}-particles that resulted in only a very small fraction of cells being traversed by a particle. The contribution of flavin-containing oxidase enzymes to the production of ROS [presumably NAD(P)H-oxidase(s)] and the involvement of ROS in the induction of micronuclei formation in bystander cells were also evaluated.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The AG1522, GM6419, and GM5758 human diploid skin fibroblast cell strains were obtained from the Genetic Cell Repository at the Coriell Institute for Medical Research (Camden, NJ). Cells for {alpha}-particle irradiation were grown in stainless steel dishes with 1.5-µm-thick replaceable mylar bottoms (23) at a seeding density of about 1.2 x 105 cells/dish. The cells were subsequently fed on days 5, 7, and 9 with Eagle’s MEM supplemented with 15% (v/v) heat-inactivated FCS, 50 units/ml penicillin, and 50 µg/ml streptomycin. Experiments were started 48 h after the last feeding. At that time, 95–98% of the cells were in G0-G1 as determined by labeling with [3 H]thymidine and/or flow cytometry (9) . Because cellular radiation sensitivity changes at different phases of the cell cycle (24 , 25) , the cells were synchronized in G0-G1 by confluent, density inhibition of growth to eliminate complications in the interpretation of the results. Passage 10 or 11 cells maintained in a 37°C humidified incubator (atmosphere = 5% CO2 in air) were used. Control cells were sham-treated and handled in parallel with the test cells.

Antioxidants and Enzyme Assays.
Lyophilized bovine liver Cu-ZnSOD (Oxis International) was dissolved in PBS and added to cell cultures at a concentration (100 µg/ml, 300 units/ml) that had been shown to provide significant protection against the lethal effects of superoxide radicals generated photochemically or by X-rays (26 , 27) . Catalase (Oxis International) in PBS was added to the cells to a final concentration of 20 µg/ml (103 units/ml). DPI (Sigma), an inhibitor of flavoprotein oxidases, was dissolved in DMSO and added to the cells to a final concentration of 0.2 or 2 µM. SOD and catalase were added to the cultures in 20-µl quantities, whereas DPI was added in 2-µl quantities to a final concentration of 0.1% DMSO. Sham-treated cultures received the same volume of PBS or DMSO diluents. For experiments in which inactive SOD or catalase was used, solutions of either enzyme were placed in a boiling water bath for 1–2 h. Care was taken to ensure resuspension of the inactive enzymes in solution before addition to cell cultures. The inactivation of enzymes as well as enzymatic activity in homogenates from cultures washed three times with PBS was determined using a previously described spectrophotometric activity assay (28 , 29) . Briefly, this assay is a competitive inhibition assay using xanthine-xanthine oxidase-generated superoxide to reduce NBT at a constant rate, which is monitored spectrophotometrically. Increasing amounts of purified SOD or cellular homogenates containing SOD activity progressively inhibit this rate of reduction of NBT. The amount of protein that inhibits the NBT reduction 50% of maximal inhibition is defined as 1 unit of SOD activity (6–9 ng = 1 unit of activity for purified CuZnSOD from Oxis International). Inhibition of CuZnSOD by cyanide is used to differentiate between CuZnSOD and MnSOD activities. CuZnSOD activity is determined by subtracting MnSOD activity from total SOD activity. Protein concentration is determined by the method of Lowry et al. (30) , and enzymatic activity is expressed in units/mg protein.

{alpha}-Particle Irradiation,
Cells were exposed to {alpha}-particles from a 238Pu-collimated source at a dose rate of 9.9 cGy/min as described previously (23) . Irradiation was carried out from below with {alpha}-particles of 3.65 MeV average energy at the cell layer. The fraction of cells whose nucleus was traversed by a {alpha}-particle was derived from Poisson statistics and estimates involving cell geometry, {alpha}-particle fluence, and energy loss.

Western Analysis.
After irradiation, cell cultures were held at 37°C in 5% CO2 atmosphere for various time intervals before harvesting for analysis. The cells were lysed in chilled radioimmunoprecipitation assay buffer supplemented with phosphatase inhibitors as described previously (9) . Anti-p21Waf1 (Ab-1), anti-p53 (Ab-6), and anti-{alpha}-tubulin (Ab-1), which were used to verify equal loading of the samples, were obtained from Oncogene Science. Antibodies against the active (phosphorylated) forms of Raf1, p38MAPK, JNK, ERK1/2, ELK-1, p90RSK and ATF2 were from New England Biolabs. Antimouse or antirabbit IgG secondary antibody conjugated with horseradish peroxidase was used to detect the various proteins by chemiluminescence.

Immunofluorescence.
Cell cultures were rinsed in PBS+ (PBS supplemented with 1 mM MgCl2 and 0.1 mM CaCl2) and fixed in 3% paraformaldehyde in PBS+. Permeabilization, reaction to anti-p21Waf1, and detection with a FITC-conjugated goat antimouse IgG secondary antibody (Sigma) were performed as described previously (9) . Microscopy of coded samples was carried out using a Leica TCSNT scanning confocal microscope equipped with an argon laser (excitation at 488 nm).

DNA Binding Assay.
DNA binding activity of NF-{kappa}B, ATF2, and AP-1 was assessed using the electrophoretic mobility shift assay system from Promega. Briefly, nuclear extracts from AG1522 cells (10 µg) were annealed with end-labeled oligonucleotides, electrophoresed in a 2% agarose gel, and submitted for autoradiography according to the manufacturer’s instructions.

Micronucleus Assay.
The frequency of micronucleus formation was measured by the cytokinesis block technique (31) . After treatments, cultures were dissociated by trypsinization, and approximately 3 x 104 cells were seeded in chamber flaskettes (Nunc) in the presence of 2 µg/ml cytochalasin B (Sigma) and incubated at 37°C. After 72 h, the cells were rinsed in PBS+, fixed in cold methanol, stained with Hoechst 33342 solution (1 µg/ml), and viewed under a fluorescence microscope (9) . At least 500 cells were examined, and only micronuclei in binucleate cells were considered for analysis. At the concentration used, cytochalasin B was nontoxic to AG1522 cells. Binomial statistics were applied in the analysis of the data, whereby a certain number of cells were found to be micronucleated in a population of binucleate cells. The frequency of micronucleus formation (r0) was calculated as: r0 = a/b, where a is the total number of micronucleated cells scored, and b is the total of binucleate cells examined. The error associated with r0 is given by the following formula: {Delta}r0 = [(a/b) (1 - a/b)]1/2. {chi}2 analysis was used to determine whether the frequency of micronucleus formation observed in the presence of antioxidants was different from that observed in their absence.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Attenuation of the Bystander Induction of p21Waf1 by SOD.
By means of gene expression end points (increased levels of p21Waf1, a p53 downstream effector that is highly sensitive to stress) and induction of DNA damage (micronucleus formation), our previous studies had shown that nonirradiated bystander cells participate in the overall response of confluent, monolayer cultures of human and rodent cells exposed to very low fluences of {alpha}-particles (5 , 9 , 32) . To investigate the contribution of ROS to the expression of bystander effects in such cultures, the ability of SOD to alter the induction of the stress-inducible p53 signaling pathway in density-inhibited normal human fibroblasts was determined. CuZnSOD (100 µg/ml) was added to the cell culture medium 30 min before irradiation (mean doses in the range of 0–10 cGy) and remained for 3 h thereafter until cells were harvested for Western blot analysis or fixed using paraformaldehyde for immunostaining.

The representative immunoblot shown in the top panel of Fig. 1ACitation demonstrated a 2-fold increase (by scanning densitometry) in p21Waf1 expression levels in control GM6419 cultures exposed to a mean dose of 1 cGy but showed only a 4-fold increase in cultures exposed to 10 cGy. Previously reported dose-response studies (9) extending the dose to 85 cGy indicated that the p21Waf1 band at 10 cGy is not at the saturation level. At a mean dose of 10 cGy, about 8-fold more cells in the population would experience a nuclear traversal by one or more {alpha}-particles than at 1 cGy (54% traversed at 10 cGy versus 7% at 1 cGy). The data in Fig. 1ACitation are therefore consistent with the previous finding that at low fluences of {alpha}-particles, a greater number of cells than those traversed are responding to radiation. The addition of CuZnSOD to the irradiated cultures inhibited detectable increases in p21Waf1 immunoreactive protein at mean doses in the range of 0.3–1 cGy (Fig. 1A)Citation (one-tailed t test, paired, P < 0.001; n = 5). At doses greater than 1 cGy, p21Waf1 levels were induced, but at an attenuated level as compared with irradiated cells not incubated with SOD. The suppression of the effect by SOD at low mean doses supports the hypothesis that superoxide radicals are involved in mediating the bystander response. The lack of total inhibition of p21Waf1 induction at high mean doses (5–10 cGy) suggests that at these doses, the effect is predominantly a consequence of direct cellular traversal by an {alpha}-particle. Similar results were obtained with the normal human fibroblast strains GM5758 (data not shown; n = 1) and AG1522 (Fig. 1ACitation , bottom panel; P < 0.015; n = 5). The representative data in Fig. 1ACitation , bottom panel, indicate a 2.1- and 2.7-fold increase in p21Waf1 levels at 0.6 and 2 cGy, respectively, in control irradiated cells, but no increase was detected in irradiated cells incubated with SOD. The data in Fig. 1BCitation obtained with AG1522 cells confirm the SOD effect observed in Fig. 1ACitation . These data also show that SOD activity was necessary to cause a reduction in accumulation of p21Waf1 immunoreactive protein in irradiated cultures, as evidenced by the results obtained with inactive enzyme. Densitometric analyses indicated that the expression levels of p21Waf1 in 1 cGy-irradiated/SOD-incubated cultures were significantly different from those of 1 cGy-irradiated/inactive SOD-incubated cultures (P < 0.009; n = 3); however, the data obtained with 1 cGy-irradiated control and 1 cGy-irradiated/inactive SOD-incubated cultures were not significantly different (P < 0.37; n = 3).



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  		Fig. 1. A, Western blot analysis of p53 and p21Waf1 expression levels in {alpha}-particle-irradiated GM6419 and AG1522 human fibroblasts in the presence or absence of SOD (100 µg/ml). Confluent, density-inhibited cultures were exposed to {alpha}-particles at mean doses ranging from 0.3 to 10 cGy and held at 37°C for 3 h. Cell lysates from irradiated and control sham-treated cultures were prepared and examined. B, Western analyses of p53 and p21Waf1 in AG1522 cultures exposed to {alpha}-particles in the presence of active or inactive ({Delta}) SOD (100 µg/ml). C, in situ immunofluorescence detection of p21Waf1 (colored cells on blue background) in control and irradiated (0.3 cGy) cultures exposed to {alpha}-particles in the presence or absence of SOD. In irradiated control cultures, p21Waf1 induction is seen to occur in aggregates of cells (about 1 cell in 50 would be traversed by an {alpha}-particle). SOD inhibits this pattern of induction.

 
To examine the role of superoxide in the bystander response on a cell by cell basis, p21Waf1 expression in individual cells was analyzed by in situ immunodetection. As observed previously (5 , 9) , the data in Fig. 1CCitation indicate that up-regulation of p21Waf1 expression occurred in clusters of AG1522 cells in close proximity to each other after exposure to 0.3 cGy. The fraction of cells demonstrating enhanced p21Waf1 expression greatly exceeded the expected 2% actually traversed by an {alpha}-particle (P < 0.001; n = 4). Microscopic examination of pits etched in CR-39 plastic after a 1-min exposure to the {alpha}-particles delivered by our 238Pu source showed a uniform distribution down to the 2500 µm2 level (23) . Consistent with the previous data showing that SOD inhibited the radiation-induced effects in bystander cells (Fig. 1, A and B)Citation , the in situ immunofluorescence data in Fig. 1CCitation indicate that SOD inhibits p21Waf1 accumulation in aggregates of neighboring cells (compared with irradiated/control cultures, about 6-fold fewer cells accumulated p21Waf1 in irradiated/SOD-incubated cultures). The immunostaining pattern for p21Waf1 in control cells treated with SOD was similar to that seen in sham-treated, nonirradiated control cultures (data not shown). The enzyme activity analyses shown in Table 1Citation (representing five repeat experiments) also indicate that the exogenously added SOD enzymatic activity becomes significantly associated with the cells (P < 0.016). Whether this association is limited to the plasma membrane or is internalized by the cells remains to be established. Overall, the in situ immunofluorescence data in Fig. 1CCitation indicate that SOD inhibits p21Waf1 induction in bystander cells, consistent with the results shown in Fig. 1, A and BCitation .


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  		Table 1 Cell-associated SOD (in units of activity/mg protein) activity measured in control and SOD-incubated (3.5 h) AG1522 cultures

 
The Effect of Catalase on Bystander Effects.
The effect of SOD on the bystander-induced accumulation of p21Waf1 is presumably attributed to the rapid dismutation of O2 to hydrogen peroxide and oxygen (33 , 34) . The H2O2 formed from the dismutation reaction may undergo further reactions catalyzed by reduced metal ions (i.e., Fe2+ or Cu+) with the net result of conversion of O2 to the more reactive hydroxyl radical (·OH) via the Fenton reaction (35) . However, H2O2 levels can be controlled through the action of enzymes such as catalase, glutathione peroxidase, and thioredoxin peroxidase (18 , 36) that convert H2O2 into water and molecular oxygen. Nevertheless, studies in irradiated yeast mutants and mammalian cells have shown protection by SOD in the absence of catalase (26 , 37) . To further examine the involvement of ROS in the observed bystander responses, the effect of catalase on the induction of p21Waf1 by low fluences of {alpha}-particles delivered to confluent AG1522 cultures was determined. The Western blot shown in Fig. 2Citation indicates that, similar to the findings with SOD, only active catalase enzyme (20 µg/ml, 103 units/ml) was capable of suppressing the increase in p21Waf1 levels observed after irradiation with a mean dose of 2 cGy. Densitometric analyses indicated that p21Waf1 expression in irradiated/sham-manipulated cultures was significantly different from that in irradiated/catalase-treated cultures (P < 0.0006; n = 3); however, the data with irradiated control cultures were not significantly different from those with irradiated/inactive catalase-incubated cultures (P < 0.21; n = 3). These results support the hypothesis that H2O2 participates in the bystander effects seen in this system.



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  		Fig. 2. Western blot analysis of p21Waf1 expression in {alpha}-particle-irradiated AG1522 confluent fibroblast cultures in the presence or absence of active or inactive ({Delta}) catalase (20 µg/ml).

 
Activation of NF-{kappa}B and Its Inhibition by SOD in Irradiated Cultures.
Increasing experimental evidence has indicated that ROS act as second messengers regulating gene expression at the level of alterations in signal transduction, leading to the activation of several redox-sensitive transcription factors including NF-{kappa}B and AP-1 in a cell type-dependent manner (38, 39, 40, 41, 42, 43, 44) . Consistent with a role for radiation-induced alterations in redox-sensitive transcription factor activation in bystander cells after exposure to low fluences of {alpha}-particles, increases in the DNA binding activity of NF-{kappa}B were observed in AG1522 cultures exposed to doses as low as 0.3 cGy (Fig. 3)Citation . A similar level of increased DNA binding activity was observed at 0.6 and 3 cGy, although a 5-fold greater fraction of cell nuclei are traversed at 3 cGy than at 0.6 cGy. Importantly, the data in Fig. 3Citation show that when SOD (100 µg/ml, 300 units/ml) was added to the culture 30 min before irradiation, the increase in NF-{kappa}B DNA binding activity was inhibited, further supporting the role of O2 in the bystander response of AG1522 cells.



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  		Fig. 3. Electrophoretic mobility shift assay in {alpha}-particle-irradiated AG1522 fibroblast cultures indicates activation of NF-{kappa}B DNA binding by low mean doses at 30 min after exposure.

 
Attenuation of Micronuclei Formation by SOD or Catalase in Low Fluence {alpha}-Particle-irradiated Cultures.
Our previous analyses in AG1522 cultures exposed to low fluences of {alpha}-particles showed that micronuclei were induced in bystander cells (9) . The ability of SOD to inhibit this type of DNA damage after exposure of confluent AG1522 cultures to low fluences of {alpha}-particles is shown in Table 2Citation . A 3–4-fold increase in the fraction of cells with micronuclei was observed in cultures exposed to mean doses of 1 or 2 cGy, whereas only an about 5-fold increase occurred after exposure to 10 cGy. With a mean dose of 10 cGy, 7–8-fold more cells in the population experience one or more nuclear traversals by an {alpha}-particle than after 1 cGy. Incubation with SOD led to a significant reduction (50%; P < 0.01 at 1 cGy) in the production of micronuclei by low mean doses of {alpha}-particles (Table 2)Citation . Similar results were observed when irradiated AG1522 cells were incubated with catalase (70% reduction at 1 cGy; P < 0.01) and when irradiated GM6419 cells were incubated with either SOD or catalase (data not shown). Whereas, compared with sham-irradiated and nontreated AG1522 cells, a significant increase in micronucleus frequency was observed in sham-irradiated and catalase-treated cells, such an increase was not observed when GM6419 cells were used in experiments.


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  		Table 2 Micronucleus formation in AG1522 cell cultures exposed to {alpha}-particles

Percentage of binucleate cells containing micronuclei over total binucleate cells in sham-manipulated control, SOD (100 µg/ml)-, catalase (100 µg/ml)-, or DPI (0.2 µM)-treated cultures exposed to mean doses of 0, 1, 2, or 10 cGy, subcultured, and plated for micronucleus formation 3 h after irradiation.

 
The Potential Role of Flavin-containing NAD(P)H-oxidase Enzyme(s) in Triggering Increases in the Accumulation of p53 and p21waf1 as well as Micronucleus Formation.
NAD(P)H-oxidase enzymes are known to produce ROS in quantities capable of stimulating signaling pathways, and these enzymes are rapidly activated by a variety of soluble mediators and engagement of cell surface receptors (45) . Previous results have also suggested that NAD(P)H-oxidase-mediated ROS production may be implicated in the formation of increased numbers of sister chromatid exchanges in bystander cells from cultures exposed to low fluences of {alpha}-particles (12) . This hypothesis was confirmed and extended when the enhanced accumulation of p53 and p21Waf1 immunoreactive protein occurring after mean doses of 1–3 cGy was significantly reduced by the presence of the flavoprotein oxidase inhibitor DPI, which has also been shown to inhibit ROS production by NAD(P)H-oxidase enzymes (Fig. 4)Citation . Interestingly, 2 µM DPI also appeared to inhibit the transactivating function of p53 as detected by the lack of induction of its downstream effector p21Waf1 in cultures exposed to 10 cGy. However, 0.2 µM DPI was only able to inhibit the increased accumulation of p53 and p21Waf1 at fluences less than or equal to 3 cGy. These results support the hypothesis that a DPI-sensitive flavin-containing oxidase activity [presumably NAD(P)H-oxidase(s)] may represent a significant source of ROS production in AG1522 human fibroblast cultures exposed to low fluences of {alpha}-particles. These results also suggest that ROS production originating from NAD(P)H-oxidase(s) may trigger the signaling pathway leading to the accumulation of p21Waf1 and p53 after exposure to these doses of radiation. In addition, incubation of AG1522 or GM6419 cells with 0.2 µM DPI completely inhibited the 3–4-fold increase in micronucleus formation observed after exposure to {alpha}-particles (1 or 2 cGy; Table 2Citation ). Overall, based on several biological end points as well as treatment with inhibitors of ROS production and scavengers of ROS, Figs. 1Citation 2Citation 3Citation 4Citation and Tables 1Citation and 2Citation strongly support the hypothesis that ROS mediate the bystander response observed in confluent cultures of human diploid fibroblasts exposed to low fluences of {alpha}-particles.



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  		Fig. 4. Western blot analyses of p53 and p21Waf1 in {alpha}-particle-irradiated control and DPI-treated AG1522 fibroblast cultures. Cultures were held at 37°C for 3 h before harvest.

 
Rapid Modulation of Oxidative Stress-responsive Signaling Pathways in Low-fluence {alpha}-Particle-exposed Human Fibroblast Cultures.
The sequence of molecular signaling events leading to expression of biological effects in {alpha}-particle-irradiated cells is unknown. The MAPK pathway, like the NF-{kappa}B pathway, contains groups of kinases that are sensitive to ROS produced in response to environmental stresses (46 , 47) . Therefore, the accumulation of the phosphorylated (active) forms of key proteins in the ERK, JNK, and p38MAPK subgroups was determined in control and {alpha}-particle-irradiated confluent AG1522 cultures.

The immunoblot in Fig. 5ACitation shows the accumulation of the phosphorylated (activated) forms of ERK1/2, JNK, p38, and the upstream Raf-1 kinase (48) within 1 min after exposure to a mean dose of 5 cGy. Activation of these kinase pathways is further supported by the rapid accumulation of the phosphorylated (activated) forms of their downstream effecters p90RSK, ATF2, and ELK-1 [Fig. 5ACitation , right panel (39 , 49) ]. Consistent with the occurrence of DNA damage in the irradiated cultures, detectable accumulation of p53 was noted by 15 min after irradiation and an increase in phosphorylation of Ser15 on p53 was also observed by 1 min postirradiation (Fig. 5A)Citation . Fig. 5BCitation indicates that longer time intervals (15 min) were needed to observe detectable increases in the levels of ERK1/2 and JNK after 1 cGy. By 30 min after irradiation, the level of activation in cultures exposed to either 1 or 10 cGy was similar, suggesting that at the low mean dose a significantly greater number of cells participated in the response than expected and that time was required to propagate the stress-induced signal in these cells. The activation of these kinases persisted for at least 60 min after exposure. The electrophoretic mobility shift assays in Fig. 5CCitation indicate that low mean doses (0.6 cGy) significantly increased the DNA binding activity of the AP-1 and ATF2 transcription factors that was inhibited by SOD. Consistent with a role for oxidative metabolism in the regulation of the MAPK signaling pathway (39 , 50) , the data in Fig. 5DCitation also indicate that activation of ERK1/2 and JNK was attenuated by SOD or catalase added 30 min before irradiation.



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  		Fig. 5. A, Western blot analysis of active Raf-1, ERK1/2, JNK, and p38MAPK and p90RSK, ATF2, ELK-1, p53, and p53 Ser15 in AG1522 cultures irradiated with 5 cGy of {alpha}-particles. B, expression of active ERK1/2 and JNK in AG1522 cultures exposed to 1 or 10 cGy as a function of time after irradiation. C, electrophoretic mobility shift assay of AP-1 and ATF2 DNA binding in AG1522 cultures 30 min after exposure to {alpha}-particles in the presence or absence of SOD. D, expression levels of active ERK1/2 and JNK in control, SOD-treated, or catalase-treated AG1522 cultures 30 min after irradiation.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The activation of cellular signal transduction pathways by DNA-damaging agents has been a topic of intensive research for the past decade. The data presented in this study indicate significant activation of several signaling pathways in normal human fibroblast cultures in which a very small fraction of the cells have been irradiated. In addition to up-regulation of p21Waf1 in bystander cells (Fig. 1C)Citation , the current study reveals the increased DNA binding activity of NF-{kappa}B and the activation of the MAPK group of signaling pathways by very low fluences of {alpha}-particles (Fig. 5)Citation .

The in situ immunostaining (Fig. 1C)Citation for p21Waf1 in cultures irradiated with a mean dose of 0.3 cGy confirms our previous findings (5 , 9) and clearly indicates the up-regulation of this stress-inducible protein in a significantly greater fraction of cells than those expected to be traversed by an {alpha}-particle. The affected cells occurred in aggregates of neighboring cells presumably including irradiated and bystander cells (Fig. 1C)Citation . Furthermore, the magnitude of the modulation of various oxidative stress-responsive proteins, including NF-{kappa}B, Raf1 kinase, JNK, p38MAPK, ERK1/2, and their downstream effectors ATF2, ELK-1, and AP-1 (Figs. 3Citation and 5Citation ) in AG1522 cultures irradiated with low mean doses (0.3–1 cGy) provides further supporting evidence for the occurrence of bystander effects in low fluence {alpha}-particle-irradiated cell cultures. This is further highlighted by the kinetics of induction of proteins in the MAPK group of pathways. Whereas JNK and ERK1/2 were rapidly activated (by 1 min) in cultures exposed to mean doses of 5 cGy (where a significant fraction of the cells are irradiated), longer time periods (15–30 min) were needed for activation after exposure to 1 cGy, where the effect would be primarily occurring in bystander cells (Fig. 5B)Citation .

Most importantly, this study shows that addition of the superoxide-scavenging enzyme, CuZnSOD, before and 3 h after irradiation with very low fluences of {alpha}-particles inhibited the induction of p21Waf1 in aggregates of neighboring cells (Fig. 1C)Citation . Consistent with the notion that this finding represents inhibition of radiation effects in bystander cells, SOD-treated cultures demonstrated p21Waf1 induction mainly in single isolated cells. These results suggest that superoxide, generated by metabolic processes in irradiated cells, is an important intermediate in inducing biological effects in bystander cells. The involvement of ROS (superoxide and hydrogen peroxide) in the induction of several stress-related signaling pathways as well as micronucleus formation in bystander cells was further confirmed in cultures treated with active SOD or catalase before exposure to low mean doses of {alpha}-particles (Figs. 1Citation 2Citation 3Citation 4Citation 5Citation , Table 2Citation ).

The role of ROS in the indirect effects of ionizing radiation as well as the oxygen effect associated with ionizing radiation is well documented (51) . However, the involvement of metabolically produced ROS in mediating the expression of radiation effects in bystander cells is not well characterized. Previous studies examining the effects of SOD have suggested that the protective effects may extend several hours postirradiation (18) . Consistent with the notion that increased levels of ROS are released by cells after irradiation, it has been suggested that ROS mediate the effects observed in cells treated with growth medium taken from {gamma}-irradiated cultures (52) . Furthermore, molecular analyses of HPRT mutations induced in hamster fibroblasts exposed to very low fluences of {alpha}-particle have suggested the involvement of ROS in producing mutations in bystander cells after radiation exposure (53) . Whereas deletions were the predominant type of mutation observed in the directly irradiated cells, point mutations typical of cells exposed to ROS were the most common mutations found in bystander cells (53) . These previous results, taken together with the current results, support the hypothesis that SOD may act in irradiated cells by scavenging the short-lived superoxide radicals produced at the time of irradiation as well as those produced as a result of oxidative metabolism after exposure.

Consistent with our previous studies (9) , the data in Table 2Citation indicate a greater frequency of micronucleus formation than expected in cultures exposed to mean doses of 1 or 2 cGy. A similar frequency of micronuclei formation was observed in cultures exposed to 1, 2, or 10 cGy. The apparent induction of similar levels of DNA damage at all doses suggests that damage occurs in bystander cells after low mean dose exposures. Significantly, the frequency of micronuclei in cultures exposed to the low mean doses (1–2 cGy) was reduced (P < 0.013) by 40–50% when SOD was added to the cultures before irradiation. SOD had no significant effect on cultures exposed to 10 cGy. These studies suggest a role for superoxide radicals in mediating the production of micronuclei in bystander cells. The lack of a SOD effect at 10 cGy indicates that at this dose most cells are damaged through a mechanism acting directly in the irradiated cell, as shown previously for {alpha}-particles from 210Po (54) .

The data in Table 1Citation indicate a significant association of exogenous added SOD activity with AG1522 cells. Whether the exogenous enzyme is internalized (55, 56, 57) or simply remains associated with the cell membrane, it has an opportunity to scavenge superoxide produced at or near the cell membrane, inhibiting the formation of micronuclei in bystander cells. Superoxide anion has been shown to permeate into lipid bilayers (58 , 59) , where it can contribute to lipid peroxidation and alter the activity of membrane-bound enzymes and/or kinases. Membrane-associated SOD can effectively intercept the reactive species before they can induce changes in membrane originating signaling pathways. Clearly, fractionation of the cell homogenates would determine the level at which exogenous SOD associates with AG1522 cells.

Consistent with ROS from a source external to the bystander cells participating in micronucleus formation, catalase was also found to inhibit micronucleus formation by up to 70% in cultures irradiated with low mean doses (1–2 cGy) of {alpha}-particles (Table 2)Citation . The fact that neither SOD nor catalase was capable of completely inhibiting induction of micronucleus formation in response to low mean fluences of {alpha}-particles suggests that both superoxide and hydrogen peroxide were participating in causing this effect. Because superoxide is converted to hydrogen peroxide by the action of SOD, these results also suggest that the superoxide-mediated reactions leading to micronuclei formation in bystander cells are not identical to those of hydrogen peroxide. Because superoxide is a rather weak oxidant but an excellent reductant, one could speculate that the superoxide-mediated reduction of metal ions from their relatively nonreactive oxidized forms (i.e., Fe3+ and Cu2+) to their highly reactive reduced forms (i.e., Fe2+ and Cu+) could represent such a reaction. The reduced forms of these metals could then initiate the direct oxidation of membrane components such as polyunsaturated fatty acids, forming cytotoxic and mutagenic byproducts of lipid peroxidation (i.e., hydroperoxides and aldehydes), or participate in the decomposition of hydroperoxides, such as H2O2 or organic hydroperoxides, to form highly reactive hydroxyl or alkoxyl radicals. In this way, SOD could protect by inhibiting the superoxide-mediated reduction of metal ions, and catalase could protect by scavenging hydrogen peroxide. This hypothesis would also explain why partial protection against the induction of micronucleus formation in bystander cells could be obtained with either enzyme.

Cell membrane involvement in the bystander response to low fluences of {alpha}-particles is further emphasized by the inhibition of radiation-induced p21Waf1 accumulation and micronucleus formation in cultures pretreated with DPI (Fig. 4)Citation . DPI is an inhibitor of flavin-containing oxidase enzymes (i.e., nonphagocytic NAD(P)H-oxidase enzymes) associated with the plasma membrane and found in a variety of cells including fibroblasts (60 , 61) . Activation of NAD(P)H-oxidase enzymes in leukocytes is thought to involve phosphorylation and rapid assembly of constitutive subunits in response to signals originating from outside the cell (reviewed in Refs. 45 and 62 ). The data presented in this study indicate significant activation, in irradiated cultures, of several members of the MAPK family (Fig. 5)Citation . Whether these kinases participate in the activation of NAD(P)H-oxidase in {alpha}-particle-irradiated AG1522 cultures remains to be examined. In neutrophils, NAD(P)H-oxidase activation was prevented upon inhibition of MAPK kinase and p38 by chemical inhibitors (63 , 64) . Of further interest is the recent finding that NAD(P)H-oxidase enzymes can be activated in fibroblasts by H2O2 exposure (65) , suggesting a feed-forward mechanism by which H2O2 production in response to low mean doses of radiation may amplify NAD(P)H-oxidase activation and propagation of ROS-mediated bystander effects in unirradiated cells.

The current study also suggests the activation of Raf-1 kinase by {alpha}-particles. Raf-1 is upstream in the Ras/Rac signaling pathway (48) and is also an integral component of the pathway leading to the activation of NAD(P)H-oxidase. Hence the data in Fig. 5ACitation showing activation of Raf-1 by {alpha}-particles suggest that this may be a signaling pathway by which NAD(P)H-oxidase activity is induced. The data in Fig. 4Citation indicating total suppression of p21Waf1 induction in low-fluence irradiated cultures that were pretreated with DPI, while the response in high fluence irradiated cultures was preserved, again points to an important role for NAD(P)H-oxidase activation in the bystander response observed in {alpha}-particle-irradiated cell cultures.

Our previous studies showed that gap-junction intercellular communication is an important mechanism mediating transmission of stress effects from irradiated to nonirradiated cells (9) . Whether this occurs cooperatively or independently of oxidative metabolism is not yet clear. ROS-activated kinase(s) [e.g., member(s) of the MAPK superfamily] or phosphatase(s) in directly irradiated cells may activate gap-junction proteins, enhancing the selective passage of small molecules from irradiated to bystander cells. Binding sites for AP-1 and NF-{kappa}B, which are activated by low fluences of {alpha}-particles (Figs. 3Citation and 5CCitation ), have been shown to exist in the connexin43 gene promoter region (66) .

In summary, the data reported in this study indicate that irradiation of confluent human diploid fibroblast cultures under conditions in which a small fraction of cells are traversed by an {alpha}-particle results in activation of p53-dependent pathways as well as several members of the MAPK superfamily in nonirradiated bystander cells. Presently, it is not known whether the activated proteins (p53, ERK1/2, JNK, and their downstream effectors) are a consequence of the response in bystander cells, or whether they are active participants in communicating stress effects to bystander cells. In this regard, these data support the hypothesis that ROS (superoxide and hydrogen peroxide) are potentially important modulators of redox-sensitive signaling pathways in bystander cells from irradiated cultures, which correlates with redox-sensitive induction of micronuclei formation in bystander cells.


   ACKNOWLEDGMENTS
 
We thank Drs. Bruce Demple and Roger Howell for helpful discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Research Grant FG02–98ER62685 from the United States Department of Energy; Center Grant ES-00002 from the National Institute of Environmental Health Sciences; Grants RO1-HL51469, P01-CA66081, and 1RO1-CA92262-01A1 from the NIH; and Grant 02-1081-CCR-S2 from the New Jersey Commission on Cancer Research. Back

2 To whom requests for reprints should be addressed, at Department of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115. Back

3 The abbreviations used are: ROS, reactive oxygen species; SOD, superoxide dismutase; DPI, diphenyliodonium; NF-{kappa}B, nuclear factor {kappa}B; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; ATF2, activating transcription factor 2; AP-1, activator protein 1; NBT, nitroblue tetrazolium. Back

Received 4/ 9/02. Accepted 8/ 1/02.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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