X-Radiation-Induced Down-Regulation of the EGF Receptor ... - BioOne

RADIATION RESEARCH

173, 620–628 (2010)

0033-7587/10 $15.00 g 2010 by Radiation Research Society. All rights of reproduction in any form reserved. DOI: 10.1667/RR1793.1

X-Radiation-Induced Down-Regulation of the EGF Receptor in Primary Cultured Rat Hepatocytes Akiko Maruko,a Yosuke Ohtake,a,1 Masasumi Kawaguchi,a Tomonori Kobayashi,a Taisuke Baba,b Yoshikazu Kuwahara,c Hironobu Nakagawa,d Tsutomu Shimura,c Manabu Fukumotoc and Yasuhito Ohkuboa Department of Radiopharmacy, Tohoku Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan; b Molecular Delivery System Laboratory, Department of Biochemical Engineering, Graduate School of Biomedical Engineering, Tohoku University, 1-1, Seiryomachi, Aoba-ku, Sendai, Miyagi 980-8574, Japan; c Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, 4-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan; and d Division of Oral and Maxillofacial Surgery, Department of Oro-Maxillofacial Surgical Science, Tohoku University Graduate School of Dentistry, 4-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan

a

within a week after surgery (1, 2). This regenerative growth proceeded in a characteristic pattern and resulted in several sequential waves of DNA synthesis and mitosis starting in the periportal area and ending in the perivenous area. Ionizing radiation affects proliferative activity during liver regeneration. High-dose ionizing radiation suppresses liver regeneration after partial hepatectomy, but the same dose applied to an intact liver has no effect (3). Although the effects of proliferation induced by partial hepatectomy on the response of the liver to ionizing radiation have been studied, the molecular basis for ionizing radiation-induced changes in cell proliferation after hepatectomy is not well understood. Many growth factors and cytokines appear to play important roles in the process of liver regeneration. Periportal hepatocytes (PPH; portal area) and perivenous hepatocytes (PVH; venous area) show different responses to growth factors such as EGF and hepatocyte growth factor (HGF) (4–6). Our previous studies demonstrated that zonal differences in cell growth between PPH and PVH were caused by the zonal specificity of EGF receptor (EGFR) activity (7, 8). Thus EGFR is thought to play an important role in the proliferation of hepatocytes. Irradiation of the cellular membrane leads to a breakdown of the membrane-associated signaling pathways including EGFR (9, 10). In several tumor cells, ionizing radiation has been shown to activate the phosphorylation of tyrosine residues on the EGFR and ERK, which are important steps for normal cell proliferation (11). Therefore, it is possible that ionizing radiation affects the proliferative activity of hepatocytes through the modification of EGFR activation. EGFR is a member of the ErbB family of receptor tyrosine kinases. In response to stimulation by ligands such as EGF or transforming growth factor a, the receptor homodimerizes or heterodimerizes with other ErbB family members and undergoes autophosphoryla-

Maruko, A., Ohtake, Y., Kawaguchi, M., Kobayashi, T., Baba, T., Kuwahara, Y., Nakagawa, H., Shimura, T., Fukumoto, M. and Ohkubo, Y. X-Radiation-Induced DownRegulation of the EGF Receptor in Primary Cultured Rat Hepatocytes. Radiat. Res. 173, 620–628 (2010). Exposure to X radiation is associated with a decline in the proliferative activity of the liver, but the molecular mechanism(s) is not well understood. We investigated whether exposure to X radiation is involved in functional changes in the epidermal growth factor (EGF) receptor (EGFR), thereby causing a reduction of EGF-induced DNA synthesis using periportal hepatocytes (PPH) and perivenous hepatocytes (PVH), which differ in their proliferative activity. X radiation dose-dependently decreased DNA synthesis in both subpopulations. The rate of decline in the DNA synthesis was greater in PPH than in PVH, but the zonal difference disappeared after exposure to 10 Gy X radiation. [125I]EGF binding studies indicated that highaffinity EGFRs in both subpopulations were down-regulated after X irradiation. Furthermore, EGF-induced EGFR dimerization and phosphorylation at Y1173 in both subpopulations were down-regulated after X irradiation, and the rate of decline was greater in PPH than in PVH. In contrast, phosphorylation at Y845 after EGF treatment was dose-dependently upregulated after X irradiation in both subpopulations. These results suggest that the X-radiation-related decline in EGFinduced DNA synthesis is caused at least partly by the modification of EGFR function. g 2010 by Radiation Research Society

INTRODUCTION

The liver is the central organ for metabolism, and it has a large regenerative capability. In an experimental model, after 70% hepatectomy, the remaining liver tissue underwent rapid regeneration and regained its lost mass 1 Address for correspondence: Department of Radiopharmacy, Tohoku Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan; e-mail: [email protected]. jp.

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tion at a number of tyrosine residues, including 974, 992, 1045, 1068, 1086, 1148 and 1173 (12). Alternatively, the Src nonreceptor kinase can phosphorylate EGFR at tyrosine residues 845 and 1101 (13, 14). Phosphorylation of EGFR activates the downstream signaling pathways involving STAT, AKT, extracellular signal-regulated kinases (ERK), and protein kinase C, which induce cellular responses such as proliferation, oncogenesis, survival and transformation. In the present study, we investigated the effect of X radiation on the amount of EGFR, its affinity for its ligand, and the dimerization and phosphorylation of EGFR in normal rat hepatocytes using hepatocytes isolated in the periportal and perivenous regions of the liver, which differ in proliferative capacity and EGFR characteristics. MATERIALS AND METHODS Animals and Materials Male Wistar rats weighing 200–230 g (SLC, Hamamatsu, Japan) were kept at a controlled temperature (23 ± 1uC) under a 12-h lightdark cycle and were maintained on a standard diet and water. All animal experiments were performed in strict accordance with our institutional animal committee’s criteria for the care and use of laboratory animals. [methyl-3H]Thymidine and [125I]EGF were purchased from Perkin-Elmer Life Sciences, Inc. (Boston, MA). Collagenase was obtained from Nitta Gelatin (Osaka, Japan). Mouse EGF was obtained from Biomedical Technologies, Inc. (Stoughton, MA). Anti-actin antibody was obtained from Sigma-Aldrich (St. Louis, MO). Anti-EGFR polyclonal antibody, PY20 (phosphotyrosine-specific) antibody and anti-phospho-EGFR antibody specific to residue Y1173 were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-EGFR (phospho Y845) was obtained from Abcam (Cambridge, UK). All other reagents were readily available commercial products of analytical grade and were used without further purification.

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Inc., Boston, MA) incorporated into cells was measured using a Beckman LS 6500 liquid scintillation counter (Beckman Coulter, Fullerton, CA). Protein concentrations were determined according to the method of Bradford (17) using bovine serum albumin (BSA) as a standard. DNA Fragmentation Assay DNA fragmentation was assessed by agarose gel electrophoresis. DNA from cultured hepatocytes was prepared using Isogen (Nippon Gene Co., Ltd., Tokyo, Japan) according to the manufacturer’s instructions. Precipitated DNA was dissolved in a gel loading buffer and then analyzed on a 2% agarose gel containing 0.1 mg/ml of ethidium bromide for visualization of DNA ladders. Assay for Binding of [125I]EGF to Cultured Hepatocytes After irradiation, hepatocytes were incubated for 1 h at 37uC in serum-free medium. The culture medium was then replaced with a binding buffer containing 50 mM Hepes (pH 7.4), 128 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 5 mM MgSO4 and 0.5% BSA. Hepatocytes were then incubated for 4 h at 4uC with a binding buffer containing [125I]EGF. Ligand binding affinities were determined using a fixed concentration of [125I]EGF in competition with unlabeled EGF. Nonspecific binding was determined in the presence of a 1,000-fold concentration of unlabeled EGF and amounted to less than 10% of the total binding. The radioactivity of [125I]EGF (PerkinElmer Life Sciences) bound to hepatocytes was measured using a cray counter (Aloca ARC-370M, Tokyo, Japan). Crosslinking of EGFRs To determine the EGFR dimerization, EGFR crosslinking was carried out as follows. Hepatocytes were plated in 35-mm dishes. After irradiation, hepatocytes were incubated for 1 h at 37uC in serum-free medium and then were treated with EGF at intervals of 5, 30 and 90 min. The hepatocytes were washed twice in PBS and were incubated for 1 h at 4uC with the crosslinker bis (sulfosuccinimidyl) suberate (BS3) (Pierce) dissolved to a final concentration of 2 mM in PBS. The quench solution (0.5 M Tris-HCl buffer, pH 7.4) was then added to a final concentration of 0.25 M and incubated for 10 min at 4uC. The hepatocytes were then washed twice in PBS and were subjected to hepatic membrane fractionation.

Isolation and Culture of PPH and PVH PPH and PVH subpopulations were isolated using the digitonin/ collagenase perfusion technique described by Quistroff (15) with modifications as described by Imai et al. (16) as described previously (5). PPH and PVH were placed at a density of 0.8 3 105 cells/cm2 in WE medium containing 10% fetal bovine serum (FBS), 1029 M insulin, 1029 M dexamethasone and 1% (v/v) antibiotics. After 3 h incubation at 37uC in an atmosphere of 95% air and 5% CO2 at 100% relative humidity, the medium was replaced with serum-free medium.

Hepatic Membrane Fractionation Cultured hepatocytes on 35-mm dishes were homogenized in 50 mM Tris-HCl buffer containing 1 mM EDTA and 10% glycerol supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich) and 1% phosphatase inhibitor cocktail (Sigma-Aldrich) with 30 strokes on a Dounce homogenizer. The homogenates were then centrifuged at 500g for 5 min. The supernatant was collected and centrifuged at 100,000g for 30 min. The pellet containing hepatic membranes was subjected to Western blot analysis.

X Radiation Irradiation was performed 24 h after the hepatocytes were plated. Hepatocytes were irradiated with 1, 5 and 10 Gy at a dose rate of 1 Gy/min using an MRB-1520 X-ray machine (Hitachi). All irradiations were carried out at room temperature. The irradiated hepatocytes were kept at 37uC for 1 h, and then the culture medium was immediately replaced with fresh medium. Measurement of DNA Synthesis DNA synthesis was measured with and without 1028 M EGF, a concentration that induced the maximum proliferative effect. The radioactivity of [methyl-3H]thymidine (Perkin-Elmer Life Sciences,

Western Blot Analysis Samples were mixed with sample buffer for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis containing 62.5 mM TrisHCl buffer (pH 6.8), 10% glycerol, 2% SDS, 5% b-mercaptoethanol and 0.025% bromophenol blue. The mixture was boiled at 100uC for 3 min and was size-separated by SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide gels (Bio-Rad). The separated membrane or lysate proteins were transferred onto polyvinylidene difluoride membranes (GE Healthcare), and immunoblot analysis was carried out using the appropriate antibodies. Immunoreactive bands were detected with ECL Western blotting detection reagents (GE healthcare) and exposed with X-ray film (Fujifilm).

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Statistical Analysis The Student’s t test and one-way ANOVA were used for the statistical analysis of [methyl-3H]thymidine incorporation. For the binding assay of [125I]EGF, the Kd values of PPH and PVH were compared as correlation coefficients.

RESULTS

Effect of X Radiation on DNA Synthesis in Primary Cultured PPH and PVH The dose-dependent effects of X radiation on EGFinduced DNA synthesis in primary cultured PPH and PVH are shown in Fig. 1. The effect of EGF on DNA synthesis was greater in PPH than in PVH. There was no significant effect of 1 Gy X rays on DNA synthesis in either subpopulation. After 5 and 10 Gy irradiation, DNA synthesis decreased in a dose-dependent manner in both subpopulations; the decreases were greater in PPH than in PVH. Effect of Radiation on DNA Fragmentation in Primary Cultured PPH and PVH Ionizing radiation causes lesions in DNA, including DNA single- and double-strand breaks, which may result in cell death or growth arrest. We examined whether X-radiation-induced cell growth arrest was caused by DNA breaks. Figure 2 shows the results of the DNA fragmentation assay by agarose gel electrophoresis. DNA fragmentation was not observed at 6 and 24 h after X irradiation in either subpopulation. Moreover, X radiation had no apparent effect on cell viability (data not shown). Scatchard Analysis of EGF Binding to Primary Cultured PPH and PVH We investigated the dose effect of different doses of X radiation on [125I]EGF binding to hepatocytes. Figure 3 shows a Scatchard plot of bound/free [125I]EGF and the [125I]EGF binding rate to the receptor. The binding data showed a curvilinear relationship and yielded two apparent dissociation constants (Kd), indicating highand low-affinity binding sites. The Kd values and the numbers of binding sites (Bmax) in PPH and PVH after various doses of X radiation are shown in Table 1. When cultures were not irradiated, PPH had a greater affinity than PVH for EGF, but there was no significant difference in the Bmax values. X radiation dose-dependently decreased the high-affinity EGFRs but did not affect the low-affinity EGFRs in either subpopulation. After 10 Gy, the Kd values of the PPH and PVH increased markedly to approximately five and 1.7 times the control levels at 0 Gy, respectively. The decrease in the affinity of EGFR to EGF was greater in PPH than in PVH.

FIG. 1. Effect of X radiation on DNA synthesis in primary cultured PPH and PVH. DNA synthesis induced by 1028 M EGF in primary cultured (panel a) PPH and (panel b) PVH was measured by the incorporation of [methyl-3H]thymidine. EGF was added at 1 h after X irradiation [methyl-3H]thymidine was added 24 h later, and cells were incubated for an additional 24 h. Values are means ± SEM. *P , 0.05 compared to respective EGF-untreated group by unpaired two-tailed Student’s t test. #P , 0.05 compared to EGF alone control by one-way ANOVA.

Effect of X Radiation on EGFR Dimerization Induced by EGF Treatment of Primary Cultured PPH and PVH We investigated the effect of X radiation on EGFR dimerization. The addition of EGF induced a redistribution of the receptor from the 170–175-kDa band to the 340–350-kDa band corresponding to the monomeric and dimeric forms of the EGFR, respectively. Hepatic membranes without EGF treatment were used as the controls for EGFR activation. As shown in Fig. 4, at 0 Gy, the dimerization of EGFRs in PPH and PVH increased greatly in response to EGF stimulation. Peak dimerization was attained within 5 min of the EGF treatment and decreased thereafter. The dimerization of EGFRs after


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FIG. 2. Effect of X radiation on DNA fragmentation in primary cultured PPH and PVH. PPH and PVH were irradiated and incubated for 6 or 24 h at 37uC, and DNA was prepared for the DNA fragmentation assay. Results are from representative experiments that were repeated at least three times.

EGF treatment was greater in PPH than in PVH after 0 Gy. X radiation decreased the dimerization of EGFRs in both subpopulations. After 1 and 5 Gy, dimerization of EGFR in PPH and PVH was decreased by approximately 48% and 32%, respectively, but dimerization was similar in the two subpopulations after 10 Gy. Effect of X Radiation on EGFR Phosphorylation in Primary Cultured PPH and PVH We examined effects of X radiation on phosphorylation of EGFR after EGF treatment (Fig. 5). Hepatic membranes from cultured hepatocytes without EGF treatment were used as a control. The level of phosphotyrosine was analyzed by Western blotting using the anti-phosphotyrosine monoclonal antibody PY20. Exposure to X radiation dose-dependently increased the phosphotyrosine at 170 kDa after EGF treatment in both subpopulations. The peak amount of EGFR phosphorylated at Y845 was similarly enhanced, while that of EGFR phosphorylated at Y1173 was reduced in a dose-dependent manner. Elevation of EGFR phosphorylation at Y845 was greater in PVH

FIG. 3. Scatchard analysis of EGF binding to primary cultured PPH and PVH. Ligand binding affinities for (panel a) PPH and (panel b) PVH were determined at a fixed concentration of [125I]EGF by competition with unlabeled EGF. All determinations were done in duplicate and experiments were repeated a minimum of three times. Scatchard plots of ligand binding affinities and receptor numbers were calculated using GraphPad Prism 5.00.

than in PPH. The EGFR phosphorylation at Y1173 in PPH and PVH after various doses of X radiation correlated with dimerization of EGFR. DISCUSSION

The molecular basis for ionizing radiation-induced changes in the proliferation of normal cells is not well understood. To understand this phenomenon, we focused on EGFR, which plays an important role in the proliferation of normal cells and is influenced by ionizing radiation.

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TABLE 1 Effect of X Radiation on EGFR in Primary Cultured PPH and PVH PPH

PVH Bmaxb (pmol/33105 cells)

Kda (pM) Dose (Gy)

High

c

d

c

d

Low

High

Low

0 1 5 10

8.58 10.08 34.2 42.6

107 97 105 112

0.89 0.98 1.97 2.31

7.04 7.24 6.85 6.16

Bmaxb (pmol/33105 cells)

Kda (pM) c

High

Low

26.9 29.9 35.6 46.9

212 198 201 224

d

Highc

Lowd

1.41 1.65 1.81 1.57

6.59 6.39 6.15 6.29

Notes. Estimates of EGF binding affinities and EGFR numbers were calculated using GraphPad Prism 5.00. A Scatchard plot of the binding data showed a curvilinear relationship and yielded two apparent dissociation constants (Kd) of high- and low-affinity binding sites. Scatchard plots are shown in Fig. 3. a Dissociation constants. b Number of binding sites of EGFR. c High-affinity binding sites of EGFR to its ligand. d Low-affinity binding sites of EGFR to its ligand.

PPH and PVH showed different responses to X radiation. EGF-induced DNA synthesis decreased with increasing radiation dose and was greater in PPH than in PVH (Fig. 1). Low-LET radiations, including X and c rays, act mainly by generating reactive oxygen species (ROS) (18). Highly reactive oxygen radicals produced by ionizing radiation cause lesions in DNA (19), including DNA single- and double-strand breaks (20), which may result in cell death or apoptosis (21). However, in the present study, exposure to X radiation did not decrease cell viability or induce DNA fragmentation during apoptosis in either subpopulation (Fig. 2). We also examined the effect of the indirect ionization that is induced by the generation of free radicals in the medium; no significant effect on DNA synthesis was observed in either subpopulation (data not shown). Therefore, the radiation-induced decrease in DNA synthesis may be caused by direct ionization but not by indirect ionization. Plasma membranes are another target for oxidative damage in cells. The amount of membrane lipid peroxidation (LPO) increases with increasing radiation dose in mitochondria and microsome in rat liver (22). If overproduction of LPO occurs, oxidative damage can lead to radiation-induced cytotoxicity (23). However, our present results indicate that thiobarbituric acid reactive substances (TBARS) as measured by lipid peroxidation did not change in X-irradiated hepatocytes (data not shown). Others have also reported that no membrane changes were found, as measured by LPO in Chinese hamster fibroblasts (24). We recently reported that zonal differences in cell growth between PPH and PVH are at least partly caused by down-regulation of EGFR activity (7). In the present study, the zonal differences in the two subpopulations were abolished by exposure to X radiation. Therefore, drastic change in DNA synthesis in both subpopulations after X radiation must be due to changes in the characteristics of EGFR. Scatchard analysis using [125I]EGF was used to examine the changes in the binding affinity of EGF to

EGFR after X irradiation (Fig. 3). The affinity of EGF to EGFR decreased with increasing radiation dose. EGFR on PPH responded to radiation exposure with greater sensitivity than that on PVH, resulting in the complete disappearance of any difference in highaffinity EGFR in PPH and PVH after 10 Gy irradiation (Fig. 3). However, in a subclass of low-affinity EGFR on PPH and PVH, there were no significant difference in Kd and Bmax values after irradiation. These results suggest that a subclass of high-affinity but not lowaffinity EGFR is down-regulated by X radiation. EGFR normally has two subpopulations: a majority of low-affinity receptors (95–98%) and a minority of high-affinity receptors (2–5%) (25). Activation of the EGFR signal transduction cascade was reported to occur completely through the binding of EGF to a subclass of high-affinity EGFR (26, 27). We confirmed that high-affinity EGFR is more highly expressed in PPH than in PVH (7). Considering the results of previous studies in addition to the present one, it is possible that down-regulation of a subclass of highaffinity EGFR by X radiation causes a radiationrelated decrease in DNA synthesis of hepatocytes in both subpopulations. The binding of EGF to the high-affinity EGFR causes dimerization of EGFRs, and its downstream signaling pathways, including the phosphorylation of tyrosine residues on the receptors, are activated (28, 29). In the present study, EGF-induced dimerization of its receptors in both subpopulations reduced by X radiation, and the rate of decrease in the dimerization of EGFR was greater in PPH than in PVH (Fig. 4). The present data suggest that this X radiation-induced down-regulation of dimerization of EGFRs is related to down-regulation of a subclass of high-affinity EGFRs. EGF binding induces dimerization of the receptor, rapidly leading to trans-autophosphorylation on the intracellular tyrosine kinase domain and subsequent activation of the downstream signaling pathways (30, 31). The present data


FIG. 4. Effect of X radiation on EGFR dimerization induced by EGF treatment in primary cultured PPH and PVH. Protein samples from lysed hepatocytes were immunoblotted with anti-EGFR antibodies, and the bands were quantified with National Institutes of Health Image software. Panel a: Gels from a typical experiment. Panel b: Bars are means ± SEM of three independent experiments, expressed relative to the peak of EGFR dimer in PPH at 5 min after EGF treatment.

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FIG. 5. Effect of X radiation on EGFR phosphorylation induced by EGF treatment in primary cultured PPH and PVH. Cell membranes were lysed and the protein samples were immunoblotted and probed with antibodies to phosphotyrosine (PY20), EGFR phosphorylated at Y845, Y1173 and EGFR. To determine specific tyrosine phosphorylation of the EGFR, PVDF membranes proved with several antibodies were stripped with a solution of 0.1 M glycine (pH 2.1) and reprobed with anti-EGFR antibody. Quantification of the bands was performed with National Institutes of Health Image software. Panel a: Blots of EGFR phosphorylation and EGFR from a typical experiment. Panel b: Bars are means ± SEM from three independent experiments.

indicate that the pattern of EGFR phosphorylated at Y1173 was nearly parallel to the pattern of dimerization of its receptor in both subpopulations of X-irradiated hepatocytes (Figs. 4 and 5). After activation of EGFR kinase and phosphorylation of EGFR at Y1173, activation of the mitogen-activated protein kinase (MAPK) pathway and phosphatidylinositol 3-kinase (PI3K) are induced (32). This MAPK and PI3K activation is essential for EGF-induced hepatocyte proliferation in the primary culture system. Therefore, we think that EGFR phosphorylated at Y1173 reflects

hepatocyte proliferative capacity. On the other hand, phosphorylation of EGFR at Y845 after EGF treatment was dose-dependently up-regulated after X irradiation in both subpopulations. The phosphorylation of EGFR at Y845 may be independent of EGFR dimerization. Y845 of EGFR has been identified as specifically phosphorylated by c-Src, cytosolic tyrosine kinase (13, 33). The phosphorylation of Y845 is involved in the modulation of EGFR function as well as in tumor progression (13). Kitagawa et al. reported that ultraviolet-radiationinduced ERK activation, which provides a survival


signal against stress-induced apoptosis, is mediated through Src-dependent Tyr phosphorylation of EGFR (34). Therefore, we think that the cooperative action of X radiation and EGF induces the phosphorylation of EGFR at Y845, resulting in the induction of a survival signal against apoptosis. The present results suggest that Y845 and Y1173 of EGFR may have different functions and that multiple phosphorylation sites are required to fully assess EGFR signaling. Moreover, zonal differences in proliferative activity between PPH and PVH in response to X radiation may be caused by heterogeneous phosphorylaton of Y845 and Y1173. Ionizing radiation has been shown to activate EGFR and ERK (35–37). Activation of EGFR by ionizing radiation initiates the Ras/Raf/ERK signaling cascade, resulting in cell proliferation in several tumors. However, in the present study, ionizing radiation-induced EGFR activation resulting in hepatocyte proliferation without EGF treatment was not observed. A possible reason for this discrepancy is the different cell types and conditions used. In the present study, hepatocytes in each population were cultured at a subconfluent cell density (8 3 104 cells/cm2 at plating), at which there is modest cell-cell contact. Proliferation depending on exogenous growth factors in hepatocytes in the normal intact liver is regulated by cell-cell contact (38, 39). When hepatocytes are cultured at low density, the cells undergo DNA synthesis in response to growth factors. However, when the cells are confluent, DNA synthesis is largely inhibited even in the presence of growth factors. Growth factors such as EGF and HGF play pivotal roles in hepatocyte proliferation through the specific receptor tyrosine kinase. It has been proposed that protein-tyrosine phosphatase activities are involved in regulating the balance of tyrosine phosphorylation states of receptor tyrosine kinase (40). Machide et al. reported that protein-tyrosine phosphatase plays a definitive role in inactivation, i.e. tyrosine dephosphorylation of HGF receptor through physical interactions, which specifically occurs in hepatocytes under confluent conditions (41). Therefore, we think that at subconfluent cell densities, EGFR and the downstream signaling molecules may have difficulty activating through the specific tyrosine kinase that is stimulated only by ionizing radiation. EGFR, a member of ErbB family (ErbB1–4), is a transmembrane tyrosine kinase receptor and is widely expressed in human and animal tissues. It is possible that other ErbB members are involved in X-radiationinduced hepatocyte growth arrest. Carver et al. reported that only ErbB1 (EGFR) and ErbB3 were detected in adult rat hepatocytes (42). Moreover, EGF binds exclusively to EGFR and induces EGFR phosphorylation but not ErbB3 (42). Therefore, we think that other forms of the ErbB family do not complement EGFR function.

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Our present data support the possibility that the ionizing radiation-induced decrease in the proliferative activity of hepatocytes is at least partly caused by down-regulation of EGFR activity. These results are explained by consideration of the zonal differences between PPH and PVH, which differed in proliferative activity. The present data provide useful information for further studies of the ionizing radiation-induced decrease in proliferative activity in the liver. ACKNOWLEDGMENT The authors received no outside funding for this study. Received: March 17, 2009; accepted: November 5, 2009; published online: February 12, 2010

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