J. Radiat. Res., 48, 407–415 (2007)

Regular Paper

Low-dose of Ionizing Radiation Enhances Cell Proliferation Via Transient ERK1/2 and p38 Activation in Normal Human Lung Fibroblasts Cha Soon KIM1,2, Jin-Mo KIM1,3, Seon Young NAM1, Kwang Hee YANG1, Meeseon JEONG1, Hee Sun KIM1, Young-Khi LIM1, Chong Soon KIM1, Young-Woo JIN1* and Joon KIM2† Low-dose of ionizing radiation/ERK1/2/p38/Cell proliferation/Normal human diploid cells. This study shows the human cellular responses and the mechanism of low-dose ionizing radiation in CCD 18 Lu cells, which are derived from normal human lung fibroblasts. Cell proliferation and viability assay were measured for the cells following γ-irradiation using trypan blue, BrdU incorporation, and Wst1 assay. We also examined genotoxicity using a micronuclei formation assay. The activation of the MAPKs pathway was determined by Western blot analysis, and the siRNA system was used to inhibit the expression of ERK1/2 and p38. We found that 0.05 Gy of ionizing radiation stimulated cell proliferation and did not change Micronuclei frequencies. In addition, 0.05 Gy of ionizing radiation activated ERK1/2 and p38, but did not activate JNK1/2 in cells. A specific ERK1/2 inhibitor, U0126, decreased the phosphorylation of ERK1/2 proteins induced by 0.05 Gy of ionizing radiation, and a similar suppressive effect was observed with a p38 inhibitor, PD169316. Suppression of ERK1/2 and p38 phosphorylation with these inhibitors decreased cell proliferation, which was stimulated by 0.05 Gy of ionizing radiation. Furthermore, downregulation of ERK1/2 and p38 expression using siRNA blocked the cell proliferation that had been increased by 0.05 Gy of ionizing radiation. These results suggest that 0.05 Gy of ionizing radiation enhances cell proliferation through the activation of ERK1/2 and p38 in normal human lung fibroblasts.

INTRODUCTION Ionizing radiation (IR) is an invaluable diagnostic and therapeutic tool that is widely used in medicine. Human beings are constantly exposed to low levels of natural background radiations. In addition, exposure to low-dose ionizing radiation (LDIR) from medical and industrial sources is becoming a worldwide problem. The biological effects of LDIR have attracted attention for nearly two decades.1) Despite considerable worldwide effort, the biological effects of LDIR remain unclear. Therefore, there is growing interest *Corresponding author: Phone: +82-2-3499-6660, Fax: +82-2-3499-6669, E-mail: [email protected] † Corresponding author: E-mail: [email protected] 1 Radiation Health Research Institute, Korea Hydro & Nuclear Power Co., LTD, Seoul 132-703, Republic of Korea; 2School of Life Sciences & Biotechnology, Korea University, Seoul 136-701, Republic of Korea; 3Inha University College of Medicine, Department of Physiology, Jungsoek building 6th floor, Shinheung-dong 3-ga, Junggu Inchoen, 400-712, Republic of Korea. doi:10.1269/jrr.07032

in quantification of the biological effects of LDIR. Several investigations have recently reported that LDIR causes contradictory phenomena such as hypersensitivity or cell proliferation. Exposure of many cells exposed to LDIR caused low-dose hypersensitivity, which is an unexpectedly high cell death compared with high dose of ionizing radiation (HDIR).2) There are currently data available on the lowdose response of human tumor cell lines, of which 80% exhibit low-dose hypersensitivity.3,4) There is a significant body of evidence addressing this phenomenon in cell line experiments, animal models, and normal human tissues. However, some evidence supports the theory, while some does not. In another phenomenon of cell proliferation, exposure of cells to LDIR has been found to stimulate the activity of cell metabolism and enhancing cell growth.5) Irradiation at low doses of between 0.02 and 0.05 Gy caused stimulated proliferation of normal human diploid cells.6) Exposure to 0.5 Gy of IR stimulated induction of cell proliferation in mouse hematopoietic cells.7) In general, the cell signal pathway of cell proliferation is the MAPK pathway. MAPKs are a family of serine/threonine kinases that regulate a diversity of cellular activities. They are important mediators of signal transduction from

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the cell surface to the nucleus.8) Three major classes have been described: ERKs, JNKs and p38.9) JNKs and p38 mediate signals in response to cytokines and environmental stress, whereas the ERK subtypes are classically recognized as key transducers in the signaling cascade that mediates cell proliferation in response to growth factors such as plateletderived growth factor and endothelial growth factor. It is becoming increasingly clear that the ERK pathway, like those of p38 and JNK, is activated by environmental stresses, including reactive oxygen species such as H2O2.10) MAPK is also seen with the activation of stress-responsive pathways after IR.11) Several studies have reported that HDIR activates ERK 1/2 through the stimulation of tyrosine kinase associated with the membrane,12) which results in the activation of several transduction factors that regulate genes involved in cell growth. Thus, MAPK mediates the transduction of the signal from the membrane to the nucleus, and it may alleviate the lethal effects of radiation. Recently, a study reported that exposure to 1 Gy of IR caused greater alternations in the activities of ERK 1/2 than that to 6 Gy of IR.13) On the other hand, it was also reported that LDIR (0.01 Gy) caused activation of SAPK/JNK and p38 in mice.14) LDIR (0.02–0.06 Gy) also activated ERK 1/2 in normal human diploid cells.6) However, molecular mechanisms to LDIR remain unresolved. In the present study, we examined the effect of LDIR on proliferation in normal human diploid cells and the activation of the MAPK pathway, which can explain the proliferation of cells exposed to LDIR.

MATERIALS AND METHODS

Antibodies and chemical inhibitors Antibodies such as anti-ERK1/2 antibody, anti-phosphoJNK1/2 antibody, anti-JNK1/2 antibody, anti-phospho-p38 antibody, anti-p38 antibody, anti-phospho-Elk1 antibody, anti-Elk1 antibody, anti-phospho-p90RSK, and anti-phosphoATF2 antibody (Cell Signaling Technology), and anti-phospho-ERK1/2 antibody (Santacruz) were purchased from Cell Signaling Technology and Santa Cruz Biotechnolgy, Inc.. A specific MEK1 inhibitor, U0126, and p38 inhibitor, PD169316, were obtained from Cell Signaling Technology and Calbiochem, respectively.

Cells culture CCD 18Lu, human lung fibroblast cells were purchased from the American Type Culture Collection (Gaithersburg, MD). Cells were grown in minimal essential medium (MEM) (Gibco). Cells were cultured at 37°C in a humidified incubator at 5% CO2. Cells were harvested from the T75 flask by trypsinization, and 1 × 106 cells were seeded into a 60mm culture dish.

Irradiation

All samples were exposed to doses of γ-irradiation rang-

ing from 0.015 Gy to 2 Gy at room temperature using a 137Cs γ-irradiator (IBL 437 N, CIS International Co.) which is 0.8 Gy/min with a 5 mm plumbum filter. The controls were treated similarly except for irradiation.

Proliferation and viability assay Three methods were used to measure cell proliferation and viability. For the first method, cells were irradiated after 18–20 hours of seeding at 5 × 104 cells/dish. At 24 hour intervals, cells were harvested by trypsinization and seeded in 96-well culture dishes. Viable cells were detected using a BrdU incorporation kit (cell proliferation ELISA system) according to the protocols of the manufacturer (Roche, USA, Cat# 1 647 229). Twenty-five μl of 1M H2SO4 was added to each well, and plates were shaken for 1 min at 300 rpm. The absorbances of the samples were measured in an ELISA reader (Lab system, Finland) at 450 nm (reference wavelength: 690 nm). Measurement was carried out within 5 min after the addition of stop solution. For the second method, cells were irradiated after 18–20 hours of seeding at 5 × 104 cells/dish. At 24 hour intervals, cells were harvested by trypsinization and seeded in 96-well culture dishes. Viable cells were detected using a Wst-1 assay kit for cell proliferation according to the protocols of the manufacturer (Roche, USA, Cat# 1 644 807). Twenty μl/well of Wst-1 were added, and the incubation was continued for an additional 1–2 hours. After shaking for 1 min at 300 rpm, absorbance was measured in an ELISA reader (Lab system, Finland) at 450 nm (reference wavelength: 690 nm). For the third method, cells were harvested from the T75 flask by trypsinization, and cells (1 × 106 or 5 × 104) were seeded into 60mm or 30mm culture dishes and incubated for 24 hours. Then, cells were irradiated with γ-irradiation as indicated for an additional 24, 48, or 72 hours. After trypsinization, living cells were counted based on dye exclusion after 0.4% trypan blue staining in a hemacytometer.

Micronuclei formation assay

Cells were plated at 5 × 104 cells/dish, and were cultured overnight. After irradiation, cells were incubated with 3 μg/ ml of cytochalasin B (Sigma) for 24 hours before fixation. The optimal conditions of cytochalasin concentration and the incubation period used were determined in this system (data not shown). Initially, hypotonic treatment of 1% sodium citrate (w/v) was added for 5 min to swell cells. An equivalent volume of 3:1 methanol : glacial acetic acid (v/v) was then added, and cells were incubated for 5 min. The mixture was removed and replaced 3:1 with fresh methanol : glacial acetic acid (v/v) for 10 min. All solutions and incubations were at room temperature. The chambers were removed and the slides air-dried. Three slides were prepared per point. Slides were cooled and stored at –20°C. Just before scoring, cells were stained with 1:1 4’,6-diamidino-2-phenylindole (DAPI) : propidium iodide (both Oncor, Gaithesburg, MD)

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LDIR induces cell growth via MAPKs

using a fluorescence microscope. Micronucleus formation was estimated in binucleate cells. For the data shown, a minimum of 1000 binucleate cells were scored in each experiment.

Western blot analysis Cells were lysed in mammalian protein extraction reagent (Pierce) as described in the instructions of the manufacturer. The cell lysate was cleared by centrifugation at 14,000 rpm for 20 min at 4°C, and the supernatant was used as total cellular protein. The protein concentration was determined by a Bradford dye binding assay using BSA as a standard.15) Protein samples (50 μg) were electroporated on a SDSpolyacrylamide gel. The proteins were electrophoretically transferred to a polyvinyldene difluoride membrane in a transfer buffer (100mM Tris, 192mM glycine). After an overnight incubation in TBST buffer (20mM Tris-HCl, pH7.6, 137mM NaCl, 0.1% Tween 20) plus 5% skim milk, the membrane was incubated with primary antibodies. Bands were developed by an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech) using secondary antibodies coupled with horseradish peroxidase (Cell Signaling Technology).

Transfection of siRNAs siRNAs to p38 and ERK2 (p38 and ERK2 SMARTpool® siRNA reagents) and nonspecific control SMARTpool® siRNA reagents were obtained from Upstate Biotechnology, Inc. Cells were plated in a 35mm dish (5 × 104 cells). One day later, medium was removed and 1ml of serum-free MEM containing 100nM siRNA was added to each dish. MEM with 10% FBS was changed after 4 hours of siRNA treatment, and cells were incubated for 24 hours. After irradiation with 0.05 Gy, cells were incubated for an additional 24 hours.

Statistical analysis Each experiment was repeated three times with triplicate samples. All results were expressed as means ± standard deviation (SD). Statistical comparison between the control and irradiated groups was performed using the Student’s t test or one-way analysis of variance followed by the Dunnett test for multiple comparison. Differences with a p-value < 0.01 were considered statistically significant. **p < 0.01.

RESULTS

Effect of ionizing radiation on cell growth The effects of cell survival include both delayed cell death and chronically increased doubling times, resulting in reduced and less uniform colony size even after doses below 0.5 Gy.16) Thus, the use of cell proliferation methods instead of the colony counting method would be a simple solution to this problem because the detection of subtle differences

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in colony formation after LDIR is a very difficult matter. The effects of LDIR on cell proliferation were investigated by BrdU incorporation and Wst-1 assay. As shown in Fig. 1A and B, cell proliferation was significantly enhanced by lowdose irradiation at 0.05 Gy (p < 0.01 by Dunnett tests). The BrdU incorporation assay system, illustrated in Fig. 1A, also showed a significantly enhanced cell proliferation for 0.015 Gy irradiated cells (p < 0.01 by Dunnett tests). In contrast, cells irradiated with a dose of between 0.5 and 2 Gy showed significantly decreased live cells compared with control cells. In addition, Fig. 1C shows the growth kinetics of cells in the presence and absence of irradiation with 0.05 Gy. It was found that 0.05 Gy irradiated cells showed a significant increase in cell numbers compared to control cells at 24 and 48 hours after irradiation (p < 0.01 by the Student’s t test). However, at 72 hours, 0.05 Gy irradiated cells did not increase cell numbers significantly compared to control cells. Therefore, this increase of cell proliferation was also confirmed in cell growth kinetics (Fig. 1C). Ionizing radiation induces DNA damage and mutagenetic effects. Therefore, we also examined whether 0.05 Gy irradiated cells have a genotoxic effect. Cells irradiated with 2 Gy were used as a positive control, because cells irradiated with between 0.5 and 2 Gy showed decreased cell proliferation (Figs. 1A and B). A micronucleus (MN) assay was used for 0.05 or 2 Gy irradiated cells (Table 1). The effect of 0.05 Gy irradiation was similar to that of the control, but 2 Gy irradiated cells showed a stronger effect than control cells. Therefore, these results suggest that cell proliferation is promoted without genotoxicity at 0.05 Gy of IR.

Transient activation of p38 and ERK1/2 by 0.05 Gy of ionizing radiation In general, since the cell signaling pathway of cell proliferation is related to MAPKs, we examined the phoshporylation of MAPK pathways after irradiation. To examine the different time courses of MAPKs phoshporylation after low and high doses of ionizing radiation, we performed Western blot analysis of CCD-18 Lu cells irradiated with 0.05 and 2 Gy (Fig. 2A). Cells irradiated with 0.05 Gy induced transient phoshporylation of ERK1/2 and p38. This phosphorylation peaked at 6 hours, which then returned to the basal level at 10 hours. However, JNK phoshporylation was not altered during these periods. We also observed the phoshporylation of MAPKs in CCD-18 Lu cells irradiated with 2 Gy. Cells irradiated with 2 Gy resulted in the prolonged phoshporylation of ERK1/2, p38, and JNK1/2 from 2 hours to 10 hours. While CCD-18 Lu cells were irradiated for the designated amount of time, none of the total proteins of JNK1/2, ERK1/2, and p38 were changed.

Activation of transcriptional factors by 0.05 Gy of ionizing radiation Because 0.05 Gy irradiation stimulated the phosphoryla-

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Fig. 1. Cell proliferation is enhanced by low-dose of ionizing radiation in CCD 18Lu cells. A, Cell proliferation was determined by BrdU incorporation. B, Cell proliferation was determined by Wst-1 assay. C, Growth curves of cells irradiated with Control (●) and 0.05 Gy (▲). Live cells were counted based on dye exclusion after trypan blue staining in a hemacytometer. (** < 0.01 by Student’s t test or Dunnett tests).

Table 1. Analysis of genetic instability by micronuclei assay on CCD 18Lu cells irradiated with 0.05 or 2 Gy of ionizing radiation Dose (Gy)

Cells (No.)

Total MN (No.)

YMN

0

1071

11

0.010

0.05

2573

21

0.008

2

1189

238

0.200

tion of ERK1/2 and p38, we determined whether the downstream effectors such as Elk-1, p90RSK and ATF-2 were activated. We found that transcriptional factors and downstream effectors such as Elk-1, p90RSK, and ATF-2 were also activated by 0.05 Gy of IR (Fig. 2B). Phosphorylation of transcriptional factors, Elk-1, p90RSK, and ATF-2, was observed at 6 hours. This result confirms the status of ERK1/ 2 and p38 activation (Fig. 2B).

YMN ; Number of micronuclei / cell

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Fig. 2. Low-dose of ionizing radiation causes activation of ERK1/2, p38, and various transcription factors. A, Phosphorylation of ERK 1/2, p38, and JNK was determined by Western blot analysis at the indicated time points after irradiation at 0.05 and 2 Gy. B, Phosphorylation of p90RSK, Elk, and ATF-2 was determined by Western blot analysis.

Fig. 3. Suppressive effects of the U0126 and PD169316 on enhanced cell proliferation by low-dose of ionizing radiation. A, Cells were pretreated with or without 1, 5, and 10 μM of U0126 for 60min and then irradiated with 0.05 Gy. Phosphorylation of ERK 1/2 was determined by Western blot analysis. B, Cells were pretreated with or without 5, 10, and 15 μM of PD169316 for 60min and then irradiated with 0.05Gy. Phosphorylation of p38 was determined by Western blot analysis. C, Exponentially-growing cells were pretreated with 5 μM of U0126 and 10 μM PD169316 for 60min, and were then irradiated with 0.05 Gy. Viable cells were detected using a BrdU incorporation kit (** < 0.01 by Student’s t test). J. Radiat. Res., Vol. 48, No. 5 (2007); http://jrr.jstage.jst.go.jp

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Effects of inhibitors on activation of p38, ERK1/2 phosphorylation and enhanced cell proliferation The role of ERK1/2 activation was examined using U0126, a specific inhibitor of MEK, which is an upstream kinase of ERK (Fig. 3). We found that the administration of 5 μM of U0126 inhibited the phosphorylation of ERK1/2 in cells irradiated with 0.05 Gy (Fig. 3). The effects of an inhibitor for p38, PD169316, on p38 phosphorylation were

also examined. Treatment of the cells with 10 or 15 μM of PD169316 suppressed the phosphorylation of p38 in cells irradiated with 0.05 Gy. The suppressive effect of the inhibitors on enhanced cell proliferation by 0.05 Gy of IR is shown in Fig. 3. All 5 μM of U0126 and 10 μM of PD169316 diminished the stimulation of cell proliferation. These results suggest that the proliferative signal that was induced by 0.05 Gy of IR can pass through ERK1/2 and p38.

Fig. 4. Suppressive effects of enhanced cell proliferation and expression of MAPKs by low-dose of ionizing radiation in ERK2 and p38 siRNA-transfected cells. Exponentially-growing cells were treated with ERK2 and p38 siRNAs for 35min. Then, MEM with 10% FBS was changed after 4 hours of siRNA treatment, and cells were incubated for 24 hours. After irradiation with 0.05 Gy, cells were incubated for an additional 24hours. ERK2 and p38 expression and cell viability were analyzed by Western blot analysis with ERK2, p38, P-p90rsk, P-Elk1, P-ATF2, or β-actin antibody (A and B), and viable cells were detected using a BrdU incorporation kit (C) (** < 0.01 by Student’s t test). J. Radiat. Res., Vol. 48, No. 5 (2007); http://jrr.jstage.jst.go.jp

LDIR induces cell growth via MAPKs

Effects of p38 and ERK2 siRNA on enhanced cell proliferation In order to make sure that 0.05 Gy of IR influences the activation of ERK1/2 and p38, we performed a small interfering RNA (siRNA) knockdown strategy to silence the expression of ERK2 and p38 mRNAs in CCD 18 Lu cells. We first found that transfection of cells using ERK2 and p38 siRNA resulted in more than 50% knockdown of ERK2 and 90% knockdown of p38, respectively, but non-specific control (negative control) alone had no effect on ERK2 and p38 expression (Figs. 4A and B) and enhanced the proliferation of cells induced by low-dose irradiation. Phosphorylation of transcriptional factors, Elk-1, p90RSK, and ATF-2 also decreased in each transfected cells using ERK2 and p38 siRNA (Figs. 4A and B). Furthermore, each cell treated with p38 and ERK2 siRNAs completely abolished the stimulation of cell proliferation by 0.05 Gy of IR (Fig. 4C).

DISCUSSION The biological effects of LDIR in different biological systems have been extensively investigated and characterized.16) In this study, we demonstrated that LDIR stimulates a proliferation of normal human diploid cells. It has been reported that this effect was observed in low-dose irradiated cells such as Raji lymphoma, Chinese hamster fibroblasts, and normal human diploid cells.5,6) We also observed the MN induction frequencies because IR induces DNA damage and mutagenetic effects. In our data, MN induction was not observed in the LDIR-irradiated cells, which shows that LDIR does not have genotoxic effects. LDIR has been shown to modulate cellular signal transduction by altering the expressions of various genes and proteins.17,18) However, the biological and molecular mechanisms that underlie the cellular response to LDIR are not fully understood. Because we observed that LDIR induces a stimulation of cell proliferation, it appears to activate certain signal transduction pathways. In general, MAP kinases are enzymes that are activated by phosphorylation in response to a wide variety of extracellular stimuli.19,20) However, the activation of various MAP kinases in response to LDIR is unclear. Therefore, we used antibodies specifically recognizing the activated form of MAP kinases, in which specific threonine and tyrosine are doubly phosphorylated. Although detectable levels of all three of the MAP kinases were observed in normal human diploid cells, 0.05 Gy of IR stimulated transient phosphorylation of ERK1/2 and p38, but it did not affect the phosphorylation of JNK1/2. Time course experiments showed that phosphorylation of ERK1/2 and p38 peaked at 6 hours after irradiation (Fig. 2A), and the prolonged activation of ERK1/2, p38, and JNK1/2 peaked from 2 to 10 hours after 2 Gy irradiation. However, since it was reported that prolonged activation of MAP kinases induces cell cycle arrest and cell death,21) and that the acti-

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vation of JNK induces apoptosis and cell cycle arrest,22,30) we also observed that 2 Gy irradiation induced apoptosis and cell cycle arrest, but 0.05 Gy irradiation did not show these phenomena (data not shown). IR is known to activate both the cytotoxic stress-activated kinases (JNKs, p38) and the cytoprotective MAPKs (ERKs), which send divergent signals to the nucleus. All of these MAPKs were activated, and downstream effectors such as Elk-1, CREB, c-Jun, and ATF-2 proteins were phosphorylated, indicating that the three pathways remain intact in these cells.24–26) Recent studies have shown that ERK1/2 and p38 phosphorylate not only transcription factors, but also other protein kinases and downstream mediators such as p90RSK. We also examined whether the downstream effectors, Elk-1, p90RSK, and ATF-2, were activated, because 0.05 Gy of IR stimulated ERK1/2 and p38 phosphorylation. We found that transcriptional factors such as Elk-1, p90RSK, and ATF-2 were also activated by 0.05 Gy of IR (Fig. 2B). Phosphorylation of the transcriptional factors, Elk-1, p90RSK, and ATF-2, was observed at 6 hours. The present results indicated that the same mechanism may be involved in ERK1/2 and p38 activation by LDIR. As shown in Fig. 3A and B, both 5 or 10 μM of U0126 and 10 or 15 μM of PD169316 mM inhibited the activation of ERK1/2 and p38 by 0.05 Gy of IR. Suppressive effects of U0126 and PD169316 were observed in the enhancement of cell proliferation by 0.05 Gy of IR (Fig. 3C). These results indicated that 0.05 Gy of IR transduces the signal to ERK1/ 2 and p38. Activated ERK1/2 and p38 phosphorylated Elk1 and ATF2, which are involved in the induction of the growth-related genes, which suggests that the stimulating effects of LDIR were mediated by ERK1/2 and p38 activation. Involvement of ERK1/2 and p38 activation in growth stimulation by LDIR was confirmed by the results presented in Fig 3. First, two kinds of inhibitors, U0126 and PD169316, were used, and they suppressed the phosphorylation of ERK1/2 and p38, and also increased cell proliferation. Second, phosphorylation of ERK and p38 by LDIR potentiated a stimulatory effect on cell proliferation. And Fig. 2A showed that expression levels did not change by irradiation. While the expression of ERK2 or p38 was downregulated by siRNA for those, which completely abrogated the cell proliferation that had been enhanced by LDIR (Fig. 4). A recent study also showed that inhibition of MAPK potentiated the cell killing by IR.20) It seems that radiationinduced activation of MAPK plays a protective role against cell death. Thus, our results indicate that both of ERK1/2 and p38 pathway by LDIR showed the increased proliferation of cells through the phosphorylation and activation of such growth-related factors. It can be concluded that LDIR transiently stimulates ERK1/ 2 and p38, and enhances cell proliferation, whereas a higher dose of IR induces prolonged activation of ERK1/2, p38, and JNK1/2, which antagonizes the proliferative effect and results

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in cell cycle arrest. Several studies have shown that transient activation of ERK1/2 and p38 leads to an induction of the genes involved in cell proliferation, which suggests that the activation of ERK1/2 and p38 has cytoprotective effects. Although the mechanism of the cytoprotective effect of ERK1/2 is unknown, a recent study has shown that ERK1/2 phosphorylates Histone H3, the phosphorylation of which is hypothesized to be involved in the transcriptional activation of immediate-early genes through chromatin remodeling.27,28) In addition, p38 promotes cell proliferation through ATF-2mediated upregulation of cyclin D1 and controls of cyclin A, and mediates the regulation of cell cycle genes.29,30) Therefore, it is highly likely that the activation of ERK1/ 2 and p38 by LDIR may induce gene expression related to cell survival by remodeling the chromatin structure and regulating the cell cycle. Thus, present results indicate that both of ERK1/2 and p38 pathway may be the mechanism by which the proliferation of cells exposed to LDIR can be explained in normal human cells.

ACKNOWLEDGMENTS We thank Dr. Sungkwan Ahn for helpful comments during the preparation of this manuscript. This work was supported in part by R01-2005-000-10798-0 from the Ministry of Science and Technology, and by R-2006-1-043 Grant from KHNP and MOCIE, Republic of Korea. REFERENCES 1. Luckey, T. D. (1982) Physiological benefits from low levels of ionizing radiation. Health Phys. 43: 771–789. 2. Joiner, M. C., Marples, B., Lambin, P., Short, S. C. and Turesson, I. (2001) Low dose hypersensitivity: Current status and possible mechanisms. Int. J. Radiat. Oncol. Biol. Phys. 49: 379–389. 3. Beauchesne, P. D., Bertrand, S., Branche, R., Linke, S. P., Revel, R., Dore, J. E. and Pedeux, R. M. (2003) Human malignant glioma cell lines are sensitive to low radiation doses. Int J Cancer. 105: 33–40. 4. Wouters, B. G., Sy, A. M. and Skarsgard, L. D. (1996) Lowdose hypersensitivity and increased radioresistance in a panel of human tumor cell lines with different sensitivity. Radiat. Res. 146: 399–413. 5. Korystov, Yu. N., Eliseeva, N. A., Kublik, L. N. and Narimanov, A. A. (1996) The Effect of Low-Dose Irradiation on Proliferation of Mammalian Cells In Vitro. Radiat. Res. 146: 329–332. 6. Suzuki, K., Kodama, S. and Watanabe, M. (2001) Extremely Low-Dose Ionizing Radiation Causes Activation of Mitogenactivated Protein Kinase Pathway and Enhances Proliferation of Normal Human Diploid Cells. Cancer res. 61: 5396–5401. 7. Wang, G. J. and Cai, L. (2000) Induction of Cell-Proliferation Hormesis and Cell-Survival Adaptive Response in Mouse Hematopoietic Cells by Whole-Body Low-Dose Radiation. Toxicol Sci. 53: 369–376.

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