Toxicological Sciences 55, 97-106 (2000)
Copyright © 2000 by the Society of Toxicology
Immunotoxicology |
Low-Dose Whole-Body Irradiation (LD-WBI) Changes Protein Expression of Mouse Thymocytes: Effect of a LD-WBI-Enhanced Protein RIP10 on Cell Proliferation and Spontaneous or Radiation-Induced Thymocyte Apoptosis
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* Institute of Radiation Medicine, Norman Bethune University of Medical Sciences, Changchun, People's Republic of China; and
Department of Pathology, The University of Western Ontario, London, Ontario, Canada
Received October 29, 1999; accepted January 10, 2000
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Low-dose radiation (LDR) can potentiate cellular metabolic activities or immune functions in vivo (hormesis), and can render cells resistant to DNA or chromosome damage caused by subsequent high-dose radiation (adaptive response). Protein synthesis was required for these cellular responses to LDR. In the present study, the early expression of proteins by thymocytes in response to low-dose whole-body irradiation (LD-WBI) was investigated. The expression of novel and previously existing proteins was found in the nucleus, cytoplasm, and extracellular fluid of thymocytes at 4 hours after WBI with 75-mGy X-rays. A 10 kD protein (RIP10) was seen in the cytoplasm of thymocytes after LD-WBI was further investigated. The fraction containing RIP10 separated by Sephadex G 100 gel filtration potentiated spontaneous thymocyte, and mitogen-induced splenocyte proliferation. Western blotting demonstrated that an anti-RIP10 antibody could react with a 10-kD cytoplasm protein and also with a 13-kD nuclear protein in thymocytes at 4 h after LD-WBI. Immunocytochemical staining showed the existence of RIP10 in several immune tissues including thymus, spleen, and lymph node. RIP10 expression, as determined by immunocytochemical staining and flow cytometry, was enhanced at 4–8 h after LD-WBI. Cell-cycle arrest (G0/G1 block with decreased percentage of S-phase cells), and increased levels of spontaneous or radiation-induced apoptosis were observed in thymocytes incubated with RIP10 antibody in vitro for 4 h or 24 h. These results directly demonstrated the role of RIP10 in modulating cell proliferation and apoptosis. This finding is important to understand the mechanisms underlying LDR-induced hormesis and adaptive response.
Key Words: low-dose X-rays; whole-body irradiation; thymocytes; protein expression; RIP10.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The biological effects of low-dose radiation (LDR) have attracted attention for nearly two decades (Cai 1999
; Luckey 1980; Wolff and Olivieri, 1996). LDR stimulates cellular metabolic activities such as protein synthesis, DNA repair, antioxidant activities; a phenomenon termed radiation hormesis (Liu, 1989, 1996; Luckey, 1980). Cells exposed to LDR in vitro or in vivo can develop high resistance to subsequently high-dose radiation-induced gene mutation, DNA damage, and cell death. This phenomenon is termed radiation adaptive response (Cai and Liu, 1990, 1992; Olivieri et al., 1984; Wolff 1992; Yoshida et al., 1993). The molecular mechanisms underlying these phenomena are poorly understood, although several mechanisms have been proposed such as the stimulation of DNA repair and antioxidant activity, as well as the formation of protective proteins (Cai 1999; Cai and Jiang, 1995; Cai and Wang, 1995).
Studies on gene expression and signal transduction have attempted to understand the mechanisms of radiation hormesis and adaptive response. For example, Nogami et al. (1993) and Melkonyan et al. (1995) demonstrated the enhanced expression of HSP and mdr by chronic or multiple LDR. Genes including E-selectin (Hallahan et al., 1995), 
IIbß3 Integrin (Onoda et al., 1992), c-jun, c-fos, c-myc, c-Ha-ras (Martin et al., 1995; Prasad et al., 1995), TGF-ß1 (Ehrhart et al., 1997) and thioredoxin (Hoshi et al., 1997) are up-regulated in cells following LDR. These investigations are very important to the understanding of the molecular mechanisms of cells in response to LDR. However, those studies only considered the changes of a few known genes and paid no attention to the expression of novel genes and proteins.
Two approaches are used to screen and explore novel genes or proteins. At the transcription level, differential display and differential hybridization are used to screen and identify novel genes induced by LDR (Boothman et al. 1993, 1996; Wolff 1996; Woloschak et al., 1994). In U1-Mel cells, Boothman et al. (1993) discovered several novel genes in response to 10 cGy X-rays, and named them as XIP-1, XIP-2, XIP-3, XIP-4, XIP-5, XIP-6, XIP-9, XIP-11, XIP-12 and XIP-13. XIP-3, XIP-6, and XIP-11 were identified as DT diaphorase, t-PA (tissue-type plasminogen activator), and thymidine kinase, respectively. At the translation level, 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is used to investigate changes of protein expression in cells following LDR in vitro (Wolff et al., 1988). Since a limited amount of information was presented, investigation of protein expression in organs of animals exposed to LDR-WBI is desired.
Apoptosis is an important regulator of tissue kinetics under normal and neoplastic conditions (Kerr and Harmon, 1991). The effects of oxygen-free radicals, including DNA and cell membrane damage, are associated with the occurrence of apoptosis. Ionizing radiation induces apoptosis at certain dose levels. A dose-dependent increase in thymocyte apoptosis was found in vitro and in vivo with doses of 0.25-Gy or higher X-rays (Liu et al., 1996; Mori et al., 1992). However, the rate of apoptosis was lower in thymocytes exposed to LDR (0.2 Gy or less) in vitro or in vivo than that in controls (Liu et al., 1996; Shaposhnikova and Korystov, 1995). Pre-exposure to LDR decreased apoptotic cell death caused by secondary high-dose radiation in mouse splenocytes in vivo (Yoshida et al., 1993) or in normal and neoplastic cells in vitro (Kim et al., 1996, 1997; Park et al., 1999). LDR-induced adaptive response (reducing the amount of DNA damage and apoptosis) was prevented by protein synthesis inhibition (Cai and Liu, 1992; Kim et al., 1996, 1997; Youngblom et al., 1991), suggesting the importance of protein synthesis in LDR-induced hormesis and cytogenetic or cell-survival adaptive response.
In the present study, the 2D-PAGE technique was used to screen changes of LD-WBI-induced protein expression. In subsequent experiments, a LD-WBI-induced protein RIP10 was selected to investigate its effect on cell proliferation and apoptotic cell death both spontaneous or induced by radiation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and irradiation.
Male Kunming mice, weighing 20 ± 2 g, were used in accordance with guidelines approved by the University's Animal Care Committee. A Phillips therapeutic X-ray machine operated at 200 kVp and 10 mA in the presence of 1 mm Al, and 0.5-mm Cu filter plates were used for X-irradiation. The dose rates were 0.05 Gy/min for a dose of 75 mGy and 0.28 Gy/min for a dose of 2 Gy.
Preparation of mouse thymocyte suspensions and protein extracts.
Mice in control and LD-WBI groups were sacrificed to collect thymus and spleen tissues. Cell suspensions were prepared as described by Chen et al. (1999). After determining the cell number with a hemocytometer, the cell suspensions were centrifuged at 500 x g for 5 min and the supernatant was collected as extracellular fluid. The cell pellets were washed twice with PBS. A whole-cell extract was obtained using a lysis buffer [50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% Nonidet P-40; 0.1% SDS; 1 mM phenylmethylsulfonyl fluoride (PMSF); and 1 µg/ml aprotinin]. The cytoplasmic and nuclear protein extracts were prepared based on the method described by Dignam et al. (1983). Protein concentrations for nuclei, cytoplasm, extracellular fluid, and whole-cell extracts were determined using Bradford's method (Bradford, 1976). The extracts were stored at –70°C.
2D-PAGE and silver staining.
Nuclei, cytoplasm, and extracellular fluid thymocyte extracts from control and LD-WBI mice were analyzed using 2D-PAGE, and the gel was stained using the sliver-staining method previously reported by Chen et al. (1999).
Sephadex G-100 Gel filtration.
Four ml (60 mg) of whole protein thymocyte extract from mice irradiated and sham-irradiated was subjected to a Sephadex G-100 gel-filtration column (1 x 50 cm) with 150 mM phosphate-buffered saline, pH 7.4, containing 0.01% sodium azide at 4°C. A flow rate of 0.06 ml/min was controlled by a low-pressure chromatography system (Bio-Rad, U.S.) and protein concentration was determined using Bradford's method. Five ml of elution was collected for each fraction.
Preparation of polyclonal anti-p10 rabbit serum.
Fraction 12 of protein extracts in LD-WBI group via Sephadex G-100 gel filtration was run on a 10% SDS-PAGE gel. A 10-kD single band of gel was cut containing approximately 50 µg of protein. The gel was broken into small fragments by freezing and thawing and passage through a narrow-gauge hypodermic needle. The gel fragments were emulsified in an equal volume of Freund's complete adjuvant and injected intradermally into a rabbit. A booster injection was administered after 4 weeks. Ten days after booster injection, blood was drawn and the serum was tested for the presence of RIP10 antibody (1:50) by ELISA (St. Clair et al., 1988), and the samples were stored at –20°C.
Immunocytochemical stain for RIP10.
Immunocytochemical localization for RIP10 was performed on thymus and spleen from irradiated mice at 0–24 h after WBI with 75-mGy X-rays. Briefly, 5-µm paraffin-embedded tissue sections were deparaffinized and dehydrated, then immersed in 2% H2O2 with methanol for 30 min to remove the endogenous peroxidase activity. Sections were further incubated with 10% normal rabbit serum for 30 min and washed with Tris-HCl buffer (0.05 M, pH 7.6). The sections were sequentially incubated overnight with 1:50 dilute rabbit polyclonal serum at 4°C. The sections were treated with a biotinylated secondary anti-rabbit antibody (1:1000) at 37°C for 1 h and incubated with streptavidin-linked peroxidase (Vector Laboratories, CA). The slides were developed by immersion in 0.05% diaminobenzidine with 0.03% H2O2 and counterstained with hematoxylin and eosin.
Western blotting.
Thymocytes were lysed and whole-cell, nuclear, and cytoplasmic protein extracts were run on a 12% SDS-PAGE gel and transferred to a cellulose membrane as described previously (Chen and Liu, 1997). RIP10 was detected using anti-RIP10 rabbit polyclonal serum (1:50). The membrane was incubated at room temperature with anti-RIP10 for 2 h, followed by incubation with biotinylated anti-rabbit IgG (1:1000; Vector, U.S.) for 2 h, and a final incubation with APK-streptavidin (1:1000; Vector, U.S.) for 2 h. Color was developed using a solution made up of 100-mM Tris-HCl, pH 9.5; 100 mM NaCl; 5 mM MgCl2; 165-µg BCIP/ml; and 330 µg NBT/ml.
Proliferating response of thymocytes and splenocytes to LD-WBI.
Thymocyte proliferation was monitored with 3H-TdR incorporation. Thymocytes were incubated in RPMI 1640 medium with 10% fetal bovine serum. Each well of a 96-well microplate contained 1 x 106 cells and 0.5 µCi 3H-TdR. The protein concentration of each fraction was measured as described above. Fifteen µg of protein from the RIP10 fraction of Sephadex G-100 gel filtration from WBI-irradiated mice or from sham-irradiated mice was added to each well. After incubation for 6 h at 37°C with 5% CO2, samples were collected on to a glass cellulose filter. The amount of 3H-TdR incorporation was measured using a scintillation counter.
To determine the splenocyte-proliferating response, 3H-TdR incorporation was measured by the procedure identical to that for thymocyte proliferation, with the addition of Con A (5 µg/ml) to each well for 66 h prior to the addition of the RIP10 fraction component and 3H-TdR for an extra 6-h incubation.
Flow cytometry (FCM).
Flow cytometry was used to measure (1) RIP10 expression in thymocytes of WBI-irradiated and control mice; (2) the cell cycle of thymocytes in response to RIP10 antibody in vitro; and (3) the amount of spontaneous and radiation-induced apoptosis in thymocytes after reaction with RIP10 antibody in vitro.
The RIP-10 expression of thymocytes from irradiated and sham-irradiated mice at 4 and 24 h after WBI with 75-mGy X-rays was analyzed with FACScan (Becton-Dickinson, U.S.) using anti-RIP10 rabbit polyclonal serum (1:50). Briefly, cells were incubated with anti-RIP10 serum for 45 min at 4°C. Goat anti-rabbit IgG-FITC (1:1000, Jackson, U.S.) was used as the secondary antibody. Ten thousand cells were counted using the laser excitation wavelength of 488 nm.
Thymocytes, at 4 h after sham-irradiation and WBI-irradiation with 2-Gy X-rays, were collected and incubated with anti-RIP10 serum (1:50) at 4 and 24 h in vitro, respectively. To analyze the DNA content, cells were treated with RNase A for 30 min and stained with propidium iodide. Each sample was measured for fluorescence by counting 10,000 cells, cell cycles were analyzed by Cellfit software, and apoptotic bodies were analyzed with Lysys Software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In previous studies, 75-mGy X-ray was determined to be an optimal dose to enhance thymocyte viability and immunological function (Liu et al., 1987
, 1994, 1996). Doses of X-rays at 50–100-mGy could also induce the maximum magnitude of the adaptive response (Cai and Liu, 1990, 1992; Yoshida et al., 1993). A 4–6-h period of post-irradiation was required for the induction of an adaptive response by LDR (Cai and Liu, 1990). Therefore, in the present study, protein expression in mouse thymocytes was investigated at 4-h post exposure to 75-mGy WBI.
General Alteration of Protein Expression in Thymocytes after LD-WBI
Thymocytes collected 4 h after exposure to LD-WBI and extracts of nucleus, cytoplasm, and extracellular fluid were subjected to 2D-PAGE. In the nuclear fraction, 27 proteins, ranging in size from 10 to 62 kD, with pI of 5.0–8.2, were differentially displayed in the irradiated group as compared to the sham-irradiated group (Table 1). Fourteen novel proteins, 3 up-regulated and 1 down-regulated, were observed in the irradiated group compared to the sham-irradiated group, and 9 proteins disappeared in the WBI-irradiated group.
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In the cytoplasmic fraction of irradiated mouse thymocytes, alterations of 14 proteins, ranging in size from 10 to 53 kD with a pI of 5.0–8.2, were displayed (Table 1
). Seven proteins were novel, 2 were up-regulated, 2 were down-regulated, and 3 proteins were missed in the irradiated group as compared to the control group. Figure 1 is a representative gel of cytoplasmic extracts from sham-irradiated and LD-WBI groups. It is interesting to note that, compared to controls (Fig. 1A), a 10 kD protein with pI 8.2 was significantly seen in the LD-WBI group (Fig. 1B).
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The extracellular-fluid extract of irradiated mouse thymocytes displayed only 6 proteins, ranging in size from13 to 69 kD with a pI of 5.0–8.2 difference from control (Table 1
). One protein was novel, one was up-regulated and one was down-regulated. The other 3 proteins disappeared in the irradiated group.
Certain proteins exchanged position between the extracellular fluid and the cytoplasm (Table 1). For example, a 67-kD/5.8 (MW/pI) protein, which was found in the cytoplasm of sham-irradiated thymocytes, appeared in the extracellular fluid of thymocytes in response to LDR. A 53-kD/5.0 protein observed in the sham-irradiated thymocyte cytoplasm was decreased in WBI-irradiated cytoplasm, but markedly increased in the WBI-irradiated extracellular fluid. Two proteins, 32 kD/5.0 and 13 kD/5.0 observed in the sham-irradiated extracellular fluid were found in the irradiated thymocyte cytoplasm.
Identification of 28 kD and 10 kD Proteins, and the Stimulating Effect of the 10 kD Protein on Cell Proliferation
Thymocytes were collected at 4 h after LD-WBI to extract proteins from both the cytoplasm and the nucleus and then filtered with the Sephadex G-100 gel filtration. Fractions were separated with a 10% SDS–PAGE gel. Two peaks at UV 280 absorption were found (Fig. 2A). Two enhanced proteins, 28 kD (Fig. 2B) and 10 kD (Fig. 2C), were found in fractions 7 and 12 and were named RIP28 and RIP10, respectively.
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RIP10 was found in thymocyte nucleus (Table 1
) and cytoplasm (Table 1 and Fig. 1B), as well as splenocyte cytoplasm and nucleus (Chen et al., 1999). Therefore, in future experiments, the RIP10-containing fractions from control and LD-WBI mouse thymocytes were added to thymocyte cultures to observe the effect of RIP10 on thymocyte spontaneous proliferation. The rate of 3H-TdR incorporation into thymocytes was enhanced by the presence of RIP10 (Fig. 3). An enhanced mitogen-induced proliferation response of splenocytes to Con A in the presence of RIP10 for 72 h was also observed (Fig. 3).
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Evaluation of RIP10 through the Biological Influence of Anti-RIP10 Antibody
Estimation and localization of RIP10 in cells.
First, whole cell extracts from sham-irradiated mouse thymocytes and cytoplasmic extracts or nuclear extracts from LDR-irradiated mouse thymocytes were run on a 14% SDS–PAGE gel. The gel was transferred to a cellulose membrane, which was blotted with anti-RIP10 antibody. An undetectable band of 10 kD was found in the thymocyte extract from sham-irradiated mice, while two intense bands for a 10-kD protein in the cytoplasmic extract and a 13-kD protein in the nuclear extract from LDR-irradiated mice were observed (Fig. 4A
). There was no other detectable band in any areas but the 10 and 13 kD locations, suggesting the specific reaction of RIP10 antibody with RIP10.
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Immunocytochemical staining with the RIP10 antibody showed a wide distribution of RIP10-positive cells (microphages, interdigitating cells, and large lymphocytes) throughout the thymus (Fig. 4B
), spleen, and lymph node (data not shown). The stain was localized in the cytoplasm of these cells and reached a maximum level at 4–8 h after LD-WBI (Fig. 4B). To quantify RIP10 expression, thymocytes were collected at 4 and 24 h after LDR and incubated with the RIP10 antibody for 45 min at 4°C in vitro. FCM analysis (Fig. 4C) showed a high level of RIP10 at the early stage (4 h) as compared to that at late stage (24 h), which was consistent with immunochemical data (Fig. 4B). These results indicated that the LD-WBI might cause an early and transiently enhanced expression of RIP10.
Influence of blocking RIP10 by RIP10 antibody on cell cycle and apoptotic cell death.
The effect of RIP10 antibody on the cell cycle of thymocytes incubated for 4 and 24 h in vitro was examined using FCM. Since the RIP10 antibodies were made from rabbit, one group of cultures with normal rabbit serum was included as a negative control. No effect of the normal rabbit serum on cell cycle was observed (Table 2). However, the cell cycle blocking effect of the RIP10 antibodies, as shown by the increase in the number of Go/G1 cells and decrease in the number of S-phase cells, was observed statistically independent of incubating time (Table 2). These results demonstrated a positive regulatory action of RIP10 on cell proliferation.
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The effect of the RIP10 antibody on thymocyte apoptosis in vitro was investigated with sham-irradiated and 2-Gy-WBI mouse thymocytes incubated with or without RIP10 antibody for 4 and 24 h in vitro at 4 h after WBI. Again, normal rabbit serum was used as a negative control. Unexpectedly, normal rabbit serum inhibited apoptotic cell death of thymocytes incubated in vitro for 4 h to a below normal level (Fig. 5A
). This was likely due to the nutritional effect of serum since the effect disappeared with a longer incubation period of 24-h (Fig. 5A). The RIP10 antibody could eliminate the inhibitory effect of normal rabbit serum on thymocyte apoptosis incubated for 4 h (Fig. 5A). Since normal rabbit serum inhibited the apoptotic cell death of thymocytes (Fig. 5A), the effect of RIP10 antibody on the amount of radiation-induced apoptosis was compared to that with normal serum (Fig. 5B). Incubation with RIP10 antibodies, either for 4 h or 24 h, enhanced 2-Gy WBI-induced thymocyte apoptosis (Fig. 5B). These results suggested that RIP10 is required for normal cell proliferation. If RIP10 was inhibited by antibody, spontaneous and radiation-induced apoptosis were enhanced.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ionizing radiation has been shown to modulate gene and protein expression as well as cellular signal transduction. However, the biochemical and molecular mechanisms that underlie the cellular response to radiation are not fully understood. After exposure of cells in vitro to high-dose radiation, proteins were enhanced and suppressed when compared to control (Boothman et al., 1989
; Ramsamooj et al., 1995; Rand and Thomas, 1994). Wolff et al. (1988) reported novel and enhanced protein expressions after cells were exposed to LDR in vitro. For the first time, we have used high-resolution 2D-PAGE and silver staining to analyze these patterns in tissue after mice were exposed to LD-WBI. Seventeen nuclear proteins, 9 cytoplasmic proteins and 2 extracellular proteins were found to be novel or enhanced in thymocytes in response to LD-WBI. Ten nuclear proteins, 5 cytoplasmic proteins, and 4 extracellular fluid proteins disappeared after LD-WBI. We assumed that LD-WBI-induced alterations of these proteins might play an important role in the molecular radiation response.
The Importance of Altered Protein Expression in the Induction of Hormesis and Adaptive Response by LDR
Protein synthesis was required for the induction of hormesis and adaptive response after cells in vitro or tissues in vivo were exposed to LDR (Cai and Liu, 1992; Kim et al., 1997; Yoshida et al., 1993; Youngblom et al., 1989). Human lymphocytes exposed in vitro to an LDR effective for inducing cytogenetic adaptive response showed some novel or enhanced protein expressions (Wolff et al., 1988). Splenocytes of mice chronically exposed to LD-WBI showed elevated levels of HSP70 mRNA, HSC70 and HSP72 as well as an increased level of proliferation (Nogami et al., 1993). Since hyperthermia also induced a radio-adaptive response, a common mechanism may underlie both hyperthermia and LDR-induced radio-adaptive response or hormesis. We showed a radio-adaptive response induced by pretreatment with zinc through induction of metallothionein (MT), and with a known IFN inducer (Cai and Cherian, 1996; Cong et al., 1998). MT as a free radical scavenger or antioxidant showed a significant radio-protective action (Cai et al., 1999). All these actions suggest that LDR could enhance the expression of some existing or novel proteins. Among these proteins, antioxidants or protective proteins may protect against radiation-induced DNA or cell membrane damage, resulting in the adaptive response, while other proteins may stimulate the metabolic action resulting in cellular radiation hormesis.
LDR induces novel or enhanced protein expression, but it also inhibits certain protein expressions. In our early study, cycloheximide (CHM) alone could reduce the rate of spontaneous or radiation-induced chromosome aberrations (i.e., radio-adaptation) when added to cell culture at 4 or 6 h prior to high-dose radiation exposure (Cai and Liu, 1992). This suggests that in normal cells there are genes and proteins responsible for ROS formation resulting in spontaneous chromosome aberration. LDR-induced adaptive response may be partially due to the inhibition of these genes and proteins leading to a decrease in spontaneous chromosome damage. More and Barouki (1999) demonstrated that oxidative stress-induced adaptive response is not just via activation of an antioxidant defense system, but also due to depression of certain genes and proteins which may facilitate the formation of ROS. Down regulation of certain proteins in thymocytes of mice exposed to LD-WBI in the present study may be related to this factor. Using mouse splenocytes, we recently reported that a fraction of the splenocyte extract at 4 h after exposure to LD-WBI could protect human lymphocytes in vitro from radiation-induced chromosome aberration (Chen et al., 1999). When this fraction was subjected to HPLC analysis, two proteins (17 kD and 18.5 kD) were missed and one protein (50.7 kD) was up-regulated. These results suggested the importance of up-regulated and down-regulated proteins in LDR-induced hormesis and adaptive response. Therefore, we should not ignore the negative results when studying specific gene or protein expression to illustrate the molecular response to LDR.
RIP10, as a Factor to Modulate Cell Proliferation and Apoptotic Cell Death, May Play an Important Role in LDR-induced Hormesis and Adaptive Response
To date, LDR-induced hormesis and adaptive response have been found in various organs and tissues such as thymus, spleen, bone marrow and spermatogenic cells (Cai and Cherian, 1996; Cai and Jiang, 1995; Cai and Liu, 1990, 1992; Cai and Wang, 1995; Hyun et al., 1997; Kim et al., 1996; Liu et al., 1987, 1994, 1996; Olivieri et al., 1984; Yoshida et al., 1993). Mitogen-stimulated proliferation of mouse splenocytes could be enhanced by LDR, and this enhancement could be prevented by inhibition of protein synthesis (Hyun et al., 1997). In the present study, low expression of RIP10 was found in normal tissues, but enhanced expression could be induced by LD-WBI at 4–8 h in several immune tissues such as thymus, spleen, and lymph nodes (Fig. 4). Direct addition of RIP10 resulted in stimulating spontaneous thymocyte and mitogen-induced splenocyte proliferation in vitro (Fig. 4). If RIP10 was blocked functionally by its specific antibody, the cell cycle of these thymocytes incubated for 4 or 24 h in vitro was arrested (Table 2). This further supports the important role of RIP10 in LDR-induced radiation hormesis and adaptive response.
Apoptosis is important in regulating tissue kinetics in normal and abnormal conditions (Kerr and Harmon, 1991). Yoshida et al. (1993) and Kim et al. (1996) have found that pre-exposure of cells to LDR could reduce high-dose radiation-induced apoptosis, as a cell survival adaptive response. A significant reduction of apoptosis was found in thymocytes exposed in vivo or in vitro to 0.2-Gy X-rays or lower, compared to control thymocytes (Liu et al., 1996; Shaposhnikova and Korystov, 1995). In contrast, a dose-dependent increase in apoptosis was demonstrated when thymocytes were exposed in vivo or in vitro to 0.25-Gy X-rays, or higher (Liu et al., 1996; Mori et al., 1992). Therefore, the variations of cellular responses to different doses of radiation may result from different protein expressions. The molecular sizes of the enhanced proteins after exposure to high-dose radiation were much larger (ranging in size from 126 to 275 kD, Boothman et al. 1989) than those after exposure to LD-WBI (ranging in size from 10 to 69 kD, present study). RIP10 was assumed to be required for reducing spontaneous and radiation-induced apoptosis, based on the following facts. (1) Protein synthesis inhibition could prevent the induction of cell-survival adaptive response induced by LDR (Hyun et al., 1997; Kim et al., 1997). (2) RIP10 antibodies could enhance the spontaneous and radiation-induced apoptosis of thymocytes incubated in vitro for both 4 and 24 h (Fig. 5). It should be mentioned that the addition of RIP10 to cultures offered a significant increase in 3H-TdR incorporation (Fig. 3); however, the addition of RIP10 antibody only produced a slight increase in both cell-cycle arrest (Table 2) and spontaneous or radiation-induced apoptosis (Fig. 5). As mentioned above, Western blot did not show a detectable RIP10 protein in normal thymocytes. One could not expect a large increase in cell-cycle arrest, and spontaneous or radiation-induced apoptotic cell death in normal thymocyte cultures where limited RIP10 protein was neutralized by specific antibody. However, one can expect a significant effect by directly adding excess RIP10 in cultures. This further confirms the direct effect of RIP10 on cell proliferation and against apoptotic cell death.
We do not currently know the identity of RIP10. Only a few proteins with a molecular size of 10 kD have been documented. Apoptotic signals are transmitted through a few common pathways that include the target steps of the death-driving interleukin-1ß-converting enzyme (ICE)-family proteases and anti-cell death protein Bcl-2 in the cytoplasm. The signals must be transferred between the cytoplasm and the nucleus. It is known that small signal molecules may diffuse across nuclear pores, but larger molecules are transported by active mechanisms. Protein import across the nuclear pore complex is mediated by at least four soluble factors. One of them is p10 (a 10 kD protein), which appears to coordinate the Ran-dependent association and disassociation reactions underlying nuclear import. Mutant cells that lack p10 do not survive (Nehrbass and Blobel, 1996), and excess amounts of p10 protein could prevent Fas-induced apoptotic cell death (Yasuhara et al., 1997). Furthermore, p10 was inducible by a variety of environments such as metals, LPS, and cytokines (Lanier et al., 1997; Neville et al., 1997). Interestingly, in the present study, we found that anti-RIP10 antibody reacted with a 13-kD protein in WBI-irradiated thymocyte nucleus, and we assumed that both 10-kD and 13-kD proteins might be the same protein since certain proteins shift between the nucleus and cytoplasm with a small modification (modification of molecular size) after LDR exposure. This finding suggests that RIP10 may play a role in the signal transduction between the cytoplasm and nucleus, and may act as a factor required for the induction of hormesis or adaptive response by LDR. In addition, active ICE is composed of 20-kD and 10-kD polypeptides (p20 and p10) (Yamin et al., 1997). Alnemri et al. (1995) cloned four isoforms of ICE. One of them, ICE
cDNA, codes for a protein that corresponds to the p10 subunit of ICE. They found that ICE could not induce cell death, but does protect from or delay apoptotic cell death. They also found that the complex of ICE/p20 did not induce apoptosis, suggesting that ICE could form a heterodimer with the p20 subunit to inhibit ICE activity (Alnemri et al., 1995).
In the present study, we do not know the sequence of RIP10 or the relationship of RIP10 with the 2 p10 proteins mentioned above. However, we cannot ignore the fact that RIP10 can be induced by LD-WBI, and it can stimulate cell proliferation and prevent spontaneous cell-cycle arrest and apoptosis, or radiation-induced apoptosis.
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NOTES |
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1 To whom correspondence should be addressed at 511 South Floyd Street, MDR Building, Gastro Lab (Room 531), Louisville, Kentucky 40202. Fax: (502) 852-6004. E-mail: lcai1{at}hotmail.com.
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Alnemri, E. S., Fernandes-Alnemri, T., and Litwack, G. (1995). Cloning and expression of four novel isoforms of human interleukin-1ß-converting enzymes with different apoptotic activity. J. Biol. Chem. 270, 4312–4317.
Boothman, D. A., Bouvard, L., and Hughes, E. N. (1989). Identification and characterization of X-ray-induced proteins in human cells. Cancer Res. 49, 2871–2878.
Boothman, D. A., Fukunage, N., Meyers, M., and Lee, S. W. (1993). Isolation of X-ray-inducible transcripts from radio-resistant human melanoma cells. PNAS 90, 7200–7204.
Boothman, D. A., Meyers, M., Odegaard, E., and Wang, M. (1996). Altered G1 checkpoint control determines adaptive survival responses to ionizing radiation. Mutat. Res. 358, 143–154.[ISI][Medline]
Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–252.[ISI][Medline]
Cai, L. (1999). Research of the adaptive response induced by low-dose radiation: where have we been and where should we go? Hum. Exp. Toxicol. 18, 419–425.
Cai, L., and Cherian, M. G. (1996). Adaptive response to ionizing radiation-induced chromosome aberrations in rabbit lymphocytes: effect of pre-exposure to zinc, and copper salts. Mutat. Res. 369, 233–241.[ISI][Medline]
Cai, L., and Jiang, L. (1995). Mild hyperthermia can induce adaptation to cytogenetic damage caused by subsequent x-irradiation. Radiat. Res. 134, 26–33.
Cai, L., and Liu, S. Z. (1990). Induction of cytogenetic adaptive response of somatic and germ cells in vitro and in vivo by low dose X-irradiation. Int. J. Radiat. Biol. 58, 187–196.[ISI][Medline]
Cai, L., and Liu, S. Z. (1992). Effect of cycloheximide on adaptive response induced by low-dose radiation. Biomed. Environ. Sci. 5, 46–52.[Medline]
Cai, L., Satoh, M., Tohyama, C., and Cherian, M. G. (1999). Metallothionein in radiation exposure: its induction and protective role. Toxicology 132, 85–98.[ISI][Medline]
Cai, L., and Wang, P. (1995). Induction of a cytogenetic adaptive response in germ cells of irradiated mice with very low-dose rate of chronic gamma-irradiation and its biological influence on radiation-induced DNA or chromosomal damage and cell killing in their male offspring. Mutagenesis 10, 95–100.
Chen, S. L., Cai, L., Li, X. K., and Liu, S. Z. (1999). Low-dose whole-body irradiation induces alteration of protein expression in mouse splenocytes. Toxicol. Lett. 105, 141–152.[ISI][Medline]
Chen, S. L., and Liu, S. Z. (1997). Tyrosine phosphorylation of proteins in mouse thymocytes: a comparison between immunoprecipitation and Western blotting. Journal of Norman Bethune University of Medical Sciences 23, 678–682 (in Chinese with English abstract).
Cong, X. L., Wang, X. L., Su, Q., and Cai, L. (1998). Protective effects of extracted human-liver RNA, a known interferon inducer, against radiation-induced cytogenetic damage in male mice. Toxicol. Lett. 94, 189–198.[ISI][Medline]
Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983). Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nuclear Acids Res. 11, 1475–1489.
Ehrhart, E. J., Segarini, P., Tsang, M. L. S., Carroll, A. G., and Barcellos-Hoff, M. H. (1997). Latent transforming growth factor ß1 activation in situ: quantitative and functional evidence after low-dose 
-irradiation. FASEB J. 11, 991-1002.[Abstract]
Hallahan, D., Clark, E. J., Kochibhotla, J., Gewertz, B. L., and Collins, T. (1995). E-selectin gene induction by ionizing radiation is independent of cytokine induction. Biochem. Biophys. Res. Commun. 217, 784–795.[ISI][Medline]
Hoshi, Y., Tanooka, H., Miyazaki, K., and Wakasugi, H. (1997). Induction of thioredoxin in human lymphocytes with low-dose ionizing radiation. Biochem. Biophy. Acta 1359, 65–70.
Hyun, S. J., Yoon, M. Y., Kim, T. H., and Kim, J. H. (1997). Enhancement of mitogen-stimulated proliferation of low dose radiation-induced mouse splenocytes. Anticancer Res. 17(1A), 225–229.[ISI][Medline]
Kerr, J. F. R., and Harmon, B. V. (1991). Definition and incidence of apoptosis: an historical perspective. In Apoptosis: The Molecular Bases of Cell Death (L. D. Tomei and F. O. Cope, Eds.), pp. 5–29. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Kim, J. H., Hahm, K. H., Cho, C. K., and Yoo, S. Y. (1996). Protein biosynthesis in low dose ionizing radiation-adapted human melanoma cells. J. Radiat. Res. 37, 161–169.
Kim, J. H., Hyun, S. J., Yoon, M. Y., Ji, Y. H., Cho, C. K., and Yoo, S. Y. (1997). Pretreatment of low-dose radiation reduces radiation-induced apoptosis in mouse lymphoma (EL4) cells. Arch. Pharmacol. Res. 20, 212–217.
Lanier, L. M., Storm, K., Shafaie, A., and Volkman, L. E. (1997). Copper treatment increases recombinant baculovirus production and polyhedrin and p10 expression. Biotechniques 23, 728–735.[ISI][Medline]
Liu, S. Z. (1989). Radiation hormesis: a new concept in radiological science. Chin. Med. J. 102, 750–756.[Medline]
Liu, S. Z. (1994). Editoral: Low-dose radiation effect. Chin. Med. J. 107, 403–404.[Medline]
Liu, S. Z. (1996). Radiation Hormesis with Low Level Exposures. Science Press, Beijing.
Liu, S. Z., Liu, W. H., and Sun, J. B. (1987). Radiation hormesis: its expression in the immune system. Health Phys. 52, 579–583.[ISI][Medline]
Liu, S. Z., Zhang, Y. C., Mu, Y., Su, X., and Liu, J. X. (1996). Thymocyte apoptosis in response to low-dose radiation. Mutat. Res. 358, 185–191.[ISI][Medline]
Luckey, T. D. (1980). Hormesis with Ionizing Radiation. CRC Press, Boca Raton, FL.
Martin, M., Crechet, F., Ramounet, B., and Lefaix, J. L. (1995). Activation of c-fos by low-dose radiation: a mechanism of the adaptive response in skin cells. Radiat. Res. 141, 118–119.
Melkonyan, H. S., Ushakova, T. E., and Umansky, S. R. (1995). Hsp 70 gene express in mouse lung cells upon chronic -irradiation. Int. J. Radiat. Biol. 68, 277–280.[ISI][Medline]
Morel, Y., and Barouki, R. (1999). Repression of gene expression by oxidative stress. Biochem. J. 342, 481–496.
Mori, N., Okumoto, M., Morimoto, J., Imai, S., Matsuyama, T., Takamori, Y., and Yagasaki, O. (1992). Genetic analysis of susceptibility to radiation-induced apoptosis of thymocytes in mice. Int. J. Radiat. Biol. 62, 153–159.[ISI][Medline]
Nehrbass, U., and Blobel, G. (1996). Role of the nuclear transport factor p10 in nuclear import. Science 272, 120–123.[Abstract]
Neville, L. F., Mathiak, G., and Bagasra, O. (1997). The immunobiology of interferon-gamma inducible protein 10kD (IP-10): a novel, pleiotropic member of the C-X-C chemokine superfamily. Cytokine Growth Factor Review 8, 207–219.
Nogami, M., Huang, J. T., James, S. J., Lubinski, J. M., Nakamura, L. T., and Makinodan, T. (1993). Mice chronically exposed to low-dose ionizing radiation possess splenocytes with elevated HSP70 mRNA, HSC70, and HSP72 and with an increased capacity to proliferate. Int. J. Radiat. Biol. 63, 775–783.[ISI][Medline]
Olivieri, G., Bodycote, J., and Wolff, S. (1994). Adaptive response of human lymphocytes to low concentrations of radioactive thymidine. Science 223, 594–597.
Onoda, J. M., Piechocki, M. P., and Honn, K. V. (1992). Radiation-induced increase in expression of the IIbß3 Integrin in melanoma cells: effects on metastatic potential. Radiat. Res. 130, 281–288.[ISI][Medline]
Park, S. H., Lee, Y., Jeong, K., Yoo, S. Y., Cho, C. K., and Lee, Y. S. (1999). Different induction of adaptive response to ionizing radiation in normal and neoplastic cells. Cell Biol. Toxicol. 15, 111–119.[ISI][Medline]
Prasad, A. V., Mohan, N., Chandrasekar, B., and Meltz, M. L. (1995). Induction of transcription of "immediate early genes" by low-dose ionizing radiation. Radiat. Res. 143, 263–272.[ISI][Medline]
Ramsamooj, P., Notario, V., and Dritschilo, A. (1995). Enhanced expression of calreticulin in the nucleus of radioresistant squamous carcinoma cells in response to ionizing radiation. Cancer Res. 55, 3016–3021.
Rand, A., and Thomas, M. K. (1994). Coordinate regulation of proteins associated with radiation resistance in cultured insect cells. Radiat. Res. 138, S13–S16.[ISI][Medline]
Shaposhnikova, V. V., and Korystov, Y. N. (1995). Thymocyte proliferation and apoptosis induced by ionizing radiation. Scan. Micros. 9, 1203–1206.
St. Clair, E. W., Pisetsky, D. S., Reich, C. F., Chambers, J. C., and Keene, J. D. (1988). Quantitative immunoassay of anti-La antibodies using purified recombinant La antigen. Arthritis Rheum. 31, 506–514.[ISI][Medline]
Wolff, S. (1992). Is radiation all bad? The search for adaptation. Radiat. Res. 131, 117–123.[ISI][Medline]
Wolff, S. (1996). Aspects of the adaptive response to very low doses of radiation and other agents. Mutat. Res. 358, 135–142.[ISI][Medline]
Wolff, S., and Olivieri, G. (1996). The adaptive response to very low doses of ionizing radiation. Mutat. Res. 358(Special Issue), 1–244.[ISI][Medline]
Wolff, S., and Wiencke, J. K. (1988). The induction of chromosomal repair enzymes by 1 cGy (1 rad) of X-rays to human lymphocytes. Abstracts of the 19th Annual Meeting of EMS. Environ. Mol. Mutag. 2(Suppl. 11), 114.
Woloschak, G. E., Chang-Liu, C. M., Panozzo, J., and Libertin, C. R. (1994). Low doses of neutrons induce changes in gene expression. Radiat. Res. 138(Suppl 1), 56–59.
Yamin, T. T., Ayala, J. M., and Miller, D. K. (1997). Activation of the native 45-kD precursor form of interleukin-1-converting enzyme. J. Biol. Chem. 271, 13273–13282.
Yasuhara, N., Eguchi, Y., Tachibana, T., Imamoto, N., Yoneda, Y., and Tsujimoto, Y. (1997). Essential role of active nuclear transport in apoptosis. Genes Cells 2, 55–64.[Abstract]
Yoshida, N., Imada, H., Kunugita, N., and Norimura, T. (1993). Low-dose radiation-induced adaptive survival response in mouse spleen T-lymphocytes in vivo. J. Radiat. Res. 34, 269–276.
Youngblom, J. H., Wiencke, J. K., and Wolff, S. (1989). Inhibition of the adaptive response of human lymphocytes to very low doses of ionizing radiation by the protein synthesis inhibitor, cycloheximide. Mutat. Res. 227, 257–261.[ISI][Medline]
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