3.1 BIO-MEDICAL SCIENCE Biochemistry and Biophysics


3.1 BIO-MEDICAL SCIENCE
 Biochemistry and Biophysics

Studies on Neon Beam Induced DNA Double-Strand Breaks and Break Features which Depend on Chromatin Structure
The Role of DNA Repair on Cell Killing by Neutrons
Low Dose Irradiation-Induced Protective Function of Drug Metabolizing Enzyme System in Rat Liver Microsomes against Higher Doses of Radiation
A Mechanism of Radiation-induced Activation of Protein Kinase C in Primary Cultured Rat Hepatocytes
Discontinuous Translation and mRNA Secondary Structure: Globin
Formation of New Nitroxyl Radicals due to Chlorine Dioxide (ClO２) Radical and Various Spin-Traps in Aqueous Solutions
Effects of Hydroxyl Radicals on the Ion Permeability of Planar Lipid Bilayers through Incorporated Purified Cardiac Ryanodine Receptor
Expression of IL-1β mRNA in Mice after Whole Body X-Irradiation
Immediate-early, Transient Induction of the Interleukin-1β Gene in Mouse Spleen Macrophages by Ionizing Radiation
Molecular Dynamics (MD) Simulation on Normal and Damaged DNA
Radiolytic Products of Bromodeoxyuridine in Solids Produced by Co-60 Gamma-Rays and Monoenergetic Soft X-Rays at the K-Absorption Edge of Bromine
Dependence of RBE on LET and Particle Species

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1.Studies on Neon Beam Induced DNA Double-Strand Breaks and Break Features which Depend on Chromatin Structure

Masahiro Murakami, Kiyomi Eguchi-Kasai, Koki Sato, Shinichi Minohara, Fumio Yatagai■ and Tatsuaki Kanai (■RIKEN)

Keywords:DNA double-strand break, heavy ions, chromatin structure

The nature of DNA breaks induced by heavy ion beams with high LET may differ from those induced with X-rays. The main biological effects of heavy-ion radiation are thought to be mediated by DNA damage. Heavy-ion radiation deposits an energy more densely along its track than X-rays and this may cause unequal distribution of ionization in a cell. On the other hand, the distribution of DNA filaments in a cell changes as chromatin structure changes. We hypothesized that the quantity of DNA double-strand breaks induced by heavy-ion radiation may vary with the chromatin structure. DNA double-strand breaks induced by X-ray or neon beam irradiation in an ionizing radiation sensitive mouse mutant cell line (SL3-147) were examined before or after chromatin proteolysis. Because SL3-147 cells show DNA double-strand break repair deficiency, the DNA double-strand breaks were expected to remain unrepaired. 　Cells were exposed to X-rays from a Shimazu model Shinai X-ray Irradiator at 200kVp, 20mA, with 0.5mm aluminum and 0.5mm copper filtration, at NIRS, or they were exposed to neon beams from a cyclotron at the initial energy of 135 MeV】u, at RIKEN. Doses for the cell irradiation with X-rays were 30, 60 and 80 Gy. Doses with the neon beams were 30, 60 and 80 Gy and each dose was applied at the LET of 62.8 keV】μm or 205.5 keV】μm. The amount of DNA double-strand breaks for both beam irradiations was estimated by pulsed-field gel electrophoresis. There were no obvious differences in the size distribution of DNA fragments, based on electrophoresis analysis after the irradiations. One band (about 5.7 Mbp in size) and some smear bands occurred between 1 Mbp and 5.7 Mbp. The increase in the number of DNA double-strand breaks was dose dependent for both beam irradiations. 　We also analyzed the relation between chromatin structure and generation of DNA double-strand breaks by comparing the intact cell with the proteolyzed chromatin (naked DNA) irradiated with X-rays or neon beams. Both beam irradiations showed remarkable dose responses for the DNA double-strand breaks on naked DNA irradiation. The number of DNA double-strand breaks induced by X-ray or neon beam irradiations after proteinase K treatment was greater than that for non-treated cells. A decrease in the 5.7 Mbp DNA band was observed in concurrence with the increase in the relatively smaller smear bands between 5.7 Mbp and 1 Mbp. 　These results strongly suggest that destruction of the chromatin structure accounts for the hypersensitivity to X-rays and heavy-ionradiation after proteolysis treatment.

[Publications]Murakami, M., Eguchi-Kasai, K., Sato, K., Minohara, S., Yatagai, F. and Kanai, T.: J. Radiat. Res., 36, 258-264, 1995

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2. The Role of DNA Repair on Cell Killing by Neutrons

Kiyomi Eguchi-Kasai, Masahiro Murakami, Hiromi Itsukaichi, Kumiko Fukutsu, Koki Sato and Hiroshi Ohara■ (■Okayama Univ.)

Keywords :Keywords: neutrons, DNA double-strand break, radiosensitive mutants

It has been suggested that initial double-strand break (dsb) correlates well with cell death induced by conventional radiations. However for high linear energy transfer (LET) radiations, values of relative biological effectiveness (RBE) for dsb induction were about 1 for particles with LET below 300 keV】μm and even smaller than unity for very high LET ions, which were far lower than those for cell killing. Therefore it is not simply dsb, but the non-repairable breaks that are associated with high biological effectiveness in the cell killing effect for high LET radiation. Here, we have examined the effectiveness of high LET neutrons on cell death in 19 mammalian cell lines including radiosensitive mutants. 　Fast neutrons were generated by the cyclotron at NIRS. The contribution of γ-rays was less than 5◇ of the total dose. The dose rate was approximately 0.7 Gy】min. Cells were irradiated through a 5 mm plastic plate to obtain full build up. X-rays of 200 kVp filtered with 0.5 mm Cu and 0.5 mm Al were used for reference. Some of the radiosensitive lines were deficient in DNA dsb repair such as LX830, M10, V3, SX9, SX10, and L5178Y-S cells. They showed lower values of RBE for fast neutron beams if compared with their parent cell lines, L5178Y, CHO-AA8, and SR-1. The average RBE value of dsb repair deficient cells was 1.23 ∞ 0.15 (mean ∞ standard deviation) in contrast to that of their parent cell group (normal group) of 1.86 ∞ 0.16. The other lines of human ataxia telangiectasia fibroblasts, irs 1, irs 2, irs 3 and irs1SF cells, which were also radiosensitive but known to be proficient in dsb repair (AT type group), showed a moderate RBE of 1.68 ∞ 0.15. The averages of these 3 groups were significantly different (p°0.05) from each other, and the RBE value for dsb repair deficient cells was much lower than values for the other 2 groups (p°0.0005). 　AT type cell lines are hypersensitive to X-rays, but behave like normal cells towards high LET neutrons. This suggests that the repair system which the AT type cell lines lack does not have a major role in causing the strong cell killing effects of neutrons. On the other hand, cell lines lacking dsb repair ability behave similarly towards X-rays and neutrons. Therefore dsb seems to be a key determining the cell killing effect of high LET radiations. We hypothesize that the main cause of cell death induced by high LET radiations is non-repairable dsb, which is produced at a higher rate than in low LET radiations.

[Publications]Eguchi-Kasai, K., Murakami, M., Itsukaichi, H., Fukutsu, K., Kanai, T., Furusawa, Y., Sato, K., Ohara, H. and Yatagai, F.: Adv. in Space Res., 18, 109-118, 1996.

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3.Low Dose Irradiation-Induced Protective Function of Drug Metabolizing Enzyme System in Rat Liver Microsomes against Higher Doses of Radiation

Osami Yukawa and Tetsuo Nakajima

Keywords:low dose radiation, free radical, radical scavenger, drug metabolizing enzyme system, biological membrane.

　As one of the mechanisms of adaptive responses induced by low dose irradiation, an increase in cellular free radical scavengers is postulated from the viewpoint that biological effects of low LET radiation are mainly due to free radicals generated following radiation. We have previously reported that whole body irradiation of male Wistar rats with 5cGy of X-rays caused an increase in liver cytosolic radical scavenging activity and a prior irradiation with 5cGy protected the subsequent 7.1Gy irradiation-induced decrease in microsomal drug metabolizing enzyme activity using hexobarbital as a substrate. Our present study dealt with changes in the components of the microsomal drug metabolizing enzyme system after 7.1Gy irradiation combined with 5cGy; we wanted to clarify the protective mechanism for this membrane-bound enzyme system by low dose irradiation against action of higher doses of radiation. 　Whole body irradiation of rats with 5cGy was carried out 1 day before irradiation with 7.1Gy and changes in drug metabolizing enzymes in liver microsomes were determined 5 days after the second irradiation. The microsomal drug metabolizing enzyme system is composed of NADPH-cytochrome P-450 reductase and cytochrome P-450 and the metabolizing activity is strongly dependent on microsomal phospholipids. Significant decreases in activities of NADPH oxidation and NADPH-cytochrome P-450 reductase, in the content of cytochrome P-450, and in the binding capacity of cytochrome P-450 for hexobarbital were observed after irradiation with 7.1Gy of X-rays alone. Irradiation with 5cGy prior to the 7.1Gy irradiation caused a marked protection of the decreases in activities of NADPH oxidation and of NADPH-cytochrome P-450 reductase and a complete protection of damages to the content and hexobarbital binding capacity of cytochrome P-450. Our previous study demonstrated that this enzyme system was inactivated by radiation-induced microsomal lipid peroxidation which was suppressed by liver cytosol. Therefore, the present study suggests that the substantial protection of the components of the drug metabolizing enzyme system, especially of cytochrome P-450 which is easily inactivated by lipid peroxidation, is due to the suppression of radiation-induced lipid peroxidation through the increase in radical scavenging activity following low dose irradiation. Analysis of inducible antioxidative substances present in microsomal membranes is in progress, in relation to the protection of membrane-bound enzymes.

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4. A Mechanism of Radiation-induced Activation of Protein Kinase C in Primary Cultured Rat Hepatocytes

Tetsuo Nakajima and Osami Yukawa

Keywords:rat hepatocytes, signal transduction , protein kinase C, lipid peroxidation, trolox

　We have already demonstrated that active oxygens produced by radiation induce lipid peroxidation in primary cultured rat hepatocytes. In addition, we observed that protein kinase C (PKC) was activated following irradiation and that the activation was due to translocation of PKC molecules from cytosol to membranes. In the present experiment, we investigated the time course of the PKC activation and the relationship between PKC activation and lipid peroxidation after irradiation of cultured hepatocytes to elucidate further details of the activation mechanism and its cellular function. 　The time course of PKC activation was measured as an increase of PKC molecules in the membrane fraction of hepatocytes after irradiation with 50Gy of γ-rays. Phorbol -12,13-dibutyrate(PdBu) binding assay was used to determine the number of PKC molecules. PdBu binding activity in the membrane fraction began to increase 15 min after irradiation of the cells, reached a maximum 30 min after irradiation, and decreased 90 min after irradiation, indicating a rapid and transient activation of PKC following irradiation. The effect of trolox, a water soluble vitamin E derivative that has radical scavenging properties, was also investigated in hepatocytes to clarify the relationship between radiation-induced lipid peroxidation of hepatocytes and translocation of PKC. More than 85◇ of radiation-induced lipid peroxidation was suppressed by the addition of trolox(5-10mM) to the cells. Furthermore, trolox (10mM) treatment showed a simultaneous suppression of the PKC activation to the level observed in the non-irradiated controls without trolox. These results suggested that the translocation of PKC from the cytosol to the membrane is inhibited by the prevention of radiation- induced lipid peroxidation of the membrane. 　From these results, we concluded that the rapid and transient activation of PKC in hepatocytes is closely related to membrane lipid peroxidation induced by radiation. Further studies to determine PKC isoforms activated in the membrane and intracellular activators are in progress.

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5. Discontinuous Translation and mRNA Secondary Structure: Globin

Mitsuo Zama

Keywords: Discontinuous translation, mRNA secondary structure, globin

　It is known for several proteins that growing polypeptide chains, which are being translated from mRNA, transiently accumulate as discrete size classes.This is due to discontinuities or pauses in the translation process, which may occur at specific sites in mRNA templates. In a series of studies for such proteins, silk fibroin, chicken type ⅲ collagen, colicin A and chloroplast photosystem ⅲ reaction center protein D1, we have so far presented evidence to support the view that the mRNA secondary structure of the proteinⅲcoding region is responsible for the discontinuous translation. Continuing from this now we have focused our attention on hemoglobin. 　It was shown in earlier studies by other authors that the rate of translation of hemoglobin mRNA molecules from rabbit reticulocytes varies during the synthesis of the globin chains in a manner which suggests that slow steps are present during synthesis of specific regions of the globin molecules. We obtained the stability map of the local secondary structure of the mRNA for each of the rabbit reticulocyte α and β globin genes, by plotting the free energy of the optimal secondary structure of the local segment of the mRNA against the segment position along the base sequence of the mRNA. We found that the positions of the minima or lower free energy in the map for the α globin mRNA are approximately located at the regions where slow steps or pauses during synthesis of globin molecules are observed. This suggested that the synthesis pauses of globin chains arise from the mRNA secondary structure of the α globin gene. Clarification of the physiological significance of the discontinuous translation and its relation to the structure of the proteinⅲcoding region of mRNA might give us information on the origins and evolution of the nucleotide sequences of proteinⅲcoding genes.

[Publications]Zama, M: Nucleic Acids Res. Symp. Ser., 35, 293-294, 1996.

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6.Formation of New Nitroxyl Radicals due to Chlorine Dioxide (ClO２) Radical and Various Spin-Traps in Aqueous Solutions

Toshihiko Ozawa, Yuri Miura and Jun-ichi Ueda

Keywords:chlorine dioxide, ESR, spin trapping, Ti３≤, KClO３, free radicals, oxidation

　The reactivities of chlorine dioxide (ClO２), which is a stable free radical, towards some water-soluble spin-traps were investigated in aqueous solutions by electron spin resonance (ESR) spectroscopy. The ClO２ radical was generated from the redox reaction of Ti３≤ with potassium chlorate (KClO３) in aqueous solutions. When one of the spin-traps, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), was included in the Ti３≤-KClO３ reaction system, the ESR spectrum due to the ClO２ radical completely disappeared and a new ESR spectrum [aＮ(1)♂0.72 mT, aＨ(2)♂0.41 mT], which differed from that of DMPO-ClO２ adduct, was observed. The ESR parameters of this new ESR signal were identical to those of 5,5-dimethylpyrrolidone-(2)-oxyl-(1) (DMPOX) (Fig.12), suggesting the radical species giving the new ESR spectrum was assignable to DMPOX. A similar ESR spectrum consisting of a triplet [aＮ(1)♂0.69 mT] was observed when the derivative of DMPO, 3,3,5,5-tetramethyl-1-pyrroline N-oxide (M４PO), was included in the Ti３≤-KClO３ reaction system. This radical species was attributed to the oxidation product of M４PO, 3,3,5,5-tetramethylpyrrolidone-(2)-oxyl-(1) (M４POX) (Fig.1). When another nitrone spin-trap, α-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN) was used, the ESR signal intensity due to the ClO２ radical decreased and a new ESR signal consisting of a triplet [aＮ(1)♂0.76 mT] was observed. A similar ESR spectrum was observed when N-t-butyl-α-nitrone (PBN) was used as a spin-trap. This ESR parameter [aＮ(1)♂0.85 mT] was identical to that of the oxidation product of PBN, PBNX (Fig.12). Thus, the new ESR signal observed from POBN could be assigned to the oxidation product of POBN, POBNX (Fig.12). These results suggested that the ClO２ radical did not form stable spin adducts with nitrone spin-traps, but oxidized these spin-traps to give the corresponding nitroxyl radicals. On the other hand, the nitroso spin-traps, 5,5-dibromo-4-nitrosobenzenesulfonate (DBNBS), and 2-methyl-2-nitrosopropane (MNP), did not trap the ClO２ radical. This result indicated that the unpaired electron of the ClO２ radical was localized on an oxygen atom, because nitroso spin-traps cannot form a stable spin adduct with an oxygen-centered radical. 　

[Publications]Ozawa, T., Miura, Y., and Ueda, J.: Free Radic. Med. Biol. 20, 837-841, 1996.

Fig.1 The structures of oxidized nitrone spin-traps:           (1) DMPOX  (2) M４POX  (3) PBNX
 (4) POBNX.

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7.Effects of Hydroxyl Radicals on the Ion Permeability of Planar Lipid Bilayers through Incorporated Purified Cardiac Ryanodine Receptor

Kazunori Anzai, Kunitaka Ogawa１, Toshihiko Ozawa, Haruhiko Yamamoto１, Akihiko Kuniyasu２ and Hitoshi Nakayama２ (１Kanagawa Univ., ２Kumamoto Univ.)

Keywords:ion channel, ryanodine receptor, planar lipid bilayer, hydroxyl radical

Ryanodine receptor (RyR), a Ca2≤-release channel, is involved in physiological Ca2≤-release from sarcoplasmic reticulum (SR) in both skeletal and cardiac muscles. Various oxidative stresses have been shown to alter the Ca2≤ permeability of the SR membranes. Reconstitution studies of the SR vesicles to planar bilayers have revealed that ionic current through the RyR channel is modified by oxidative stress [Stoyanovsky et al. (1994) Arch. Biochem. Biophys., 308, 214-221; Boraso and Williams (1994) Am. J. Physiol., 267, H1010-H1016]. In these reports, it is not clear if hydroxyl radicals are involved as the crucial oxidant and if the oxidative stress directly alters the ryanodine receptor molecule or indirectly affects the receptor through modification of lipids or accessory proteins which affect the RyR. In the present study, we have measured the effects of hydroxyl radicals on the ion permeability through purified cardiac RyR incorporated into the planar bilayer membrane. 　The effects of the hydroxyl radicals on the permeability of the bilayer without incorporation of the purified RyR were also measured. The cardiac RyR, purified from pig heart and reconstituted with asolectin as proteoliposome, was incorporated into a planar bilayer membrane. The RyR showed a single channel activity with a conductance of 731 pS in 900】200 mM (cis】trans) KCl and ion selectivity of PK:PCl♂1:0.03. These characteristics are similar to those reported for the RyR incorporated into planar bilayer membranes through SR vesicles. Hydroxyl radicals chemically generated by the reaction of H２O２ and Cu(ethylenediamine)２ at the cis compartment increased the open provability of the channel from Po♂0.27 to 0.94 after a lag time of about 2 min. On the other hand, no permeability change occurred in the lipid bilayer without incorporation of the purified RyR even at higher concentrations of hydroxyl radical generating system. The results indicate that the hydroxyl radicals directly modify the RyR channel protein and increase the Ca２≤-release through it. 　Pretreatment of the RyR with SH reagent, 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), quenched the reactivity of hydroxyl radicals with RyR. One or several SH groups, when blocked with DTNB, prevented hydroxyl radicals from enhancing the increase in ion permeability. These findings demonstrate that hydroxyl radicals react with some SH groups of the RyR channel protein and increase the Ca２≤ release from SR through RyR, which may physiologically cause harmful effects on the cardiac muscle cells. 　

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8. Expression of IL-1β mRNA in Mice after Whole Body X-Irradiation

Hiroshi Ishihara, Kumie Nemoto-Nojima, Izumi Tanaka, Gen Suzuki, Kazuko Tsuneoka, Kazuko Yoshida and Hiroshi Ohtsu

Keywords:interleukin-1β, radiation damage, myeloid cells, quantitative Northern hybridization, in situ hybridization

Ionizing radiation stimulates various kinds of biological reactions in mammalian cells. A gene for the interleukin(IL)-1β encoding radioprotector is induced by x-irradiation in normal mice cells. There are multiple profiles for increase in IL-1β mRNA after irradiation. Immediately after whole body irradiation at a high dose (20 Gy) of x-rays, monocytes】macrophages in spleen accumulate mRNA for IL-1β. The increased level of the message returns to the normal level within 120 min after the irradiation. This probably reflects an early stage of one of the signal transduction mechanisms triggered by x-ray-induced damages. 　In contrast, late response for induction of IL-1β gene is moderate and constitutive. When mice were irradiated with a sublethal dose (3-6 Gy) of x-rays, accumulation of IL-1β mRNA is observed several days after irradiation in spleen cells. Since the dose of 3Gy x-rays to C3H】He inbred mice induces myeloid leukemia at the highest incidence (20-30◇ of individuals), we analyzed the relationship between IL-1β expression and population change in spleen and bone marrow of irradiated mice. 　Normal mouse spleen has a number of cell types such as myeloid and lymphoid cells at various stages of differentiation. Spleens from 3 mice were isolated from whole body-irradiated C3H】He mice at 1, 3, 5, 7, 9, 11, 13, 15 and 17 days after irradiation. They were simultaneously analyzed by histochemical staining, flow cytometric analyses, CFU-GM assay and quantitative Northern blot hybridization. To avoid any effect of variation between mice groups, the similar experiments were done using three different lots of C3H】He mice. In the Northern analyses, the amount of IL-1β mRNA in the same amount of cells was measured. Increase in IL-1β mRNA was observed 5-9 days after irradiation. Fig.1A shows experimental results with the message peak 7 to 9 days after irradiation. Total cell number and CFU-GM amount decreased 1 day after irradiation and recovered to the normal level 15-17 days after irradiation. Between 5 to 9 days after irradiation, cell number in spleen partially recovered with an increase in contents of monocytes】macrophages. The time of the partial recovery was identical to that of the peak in IL-1β message level. 　Differentiation of monocytes】macrophages results in increased expression of IL-1β mRNA. If monocytes】macrophages are not differentiated, an increase in their content at 5-9 days leads to detection of accumulation of IL-1β mRNA in spleen by Northern analysis. We analyzed the heterologous spleen cells by in situ hybridization analysis (Fig.1B). Although monocytes】macrophages in spleen express a small amount of IL-1β mRNA before irradiation, a small number of them produce a large amount of the message. 　Results of the study imply that an increase in the relative ratio of monocytes】macrophages with differentiation is due to removal of lethally damaged cells in spleen and it is reflected as an increase in IL-1β mRNA level. Since IL-1β stimulates a wide variety of myeloid and lymphoid cells to produce other cytokines, the finding probably reflects an early stage of recovering by hematopoietic cells after radiation damage. 　

[Publications]Nemoto, K., Ishihara, H., Tanaka, I., Suzuki, G., Tsuneoka, K., Yoshida, K. and Ohtsu, H.: J. Radiat. Res., 36, 125-133, 1995. 
Fig.1. (A) Each lane on the Northern blots contained 10μⅲ of total spleen RNA from 3 mice sacrificed before (0 day) or on various days as indicated after irradiation.  
(B) In situ hybridization probing IL-1β cRNA was performed using spleen cells before or after indicated days following irradiation.  Each pair of microphotographs consists of light-field (left) and dark-field (right) views of microautoradiographs of the same area.

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9.Immediate-early, Transient Induction of the Interleukin-1β Gene in Mouse Spleen Macrophages by Ionizing Radiation

Hiroshi Ishihara, Izumi Tanaka, Kumie Nemoto-Nojima, Kazuko Tsuneoka, Cheeratana Cheeramakara■, Kazuko Yoshida and Hiroshi Ohtsu (■Visiting Scientist from Mahidol Univ.,Thailand)

Keywords:interleukin-1β, early response gene, transcriptional regulation, mRNA stability, in situ hybridization

Interleukin(IL)-1β is a multifunctional cytokine and has important roles in a variety of biological reactions. IL-1β is known as a radioprotector, since the survival rate for mice which have been irradiated rises after injection of the protein. We previously found that IL-1β gene is induced by x-irradiation in normal mouse spleen cells. The fact that the gene encoding radioprotector is responsive to exposure of ionizing radiation suggests the presence of radiation-responsive mechanisms to minimize damages by radiation. 　IL-1β mRNA level increases immediately after irradiation and decreases within 120 min in isolated spleen cells from normal mice. The immediate-early, transient induction reflects a signal transduction mechanism directed by the ionizing radiation. Therefore, we focused on the early regulation mechanisms, although IL-1β is also induced several hours after irradiation in a late response to radiation. 　Lymphoid and myeloid cells are found at various stages of differentiation in normal spleen. We determined the type of cells which causes the immediate-early induction of IL-1β gene after x-irradiation. Normal spleen cells were bound to one of the following primary antibodies; MOMA2 and F4】80 for monocytes】macrophages, anti-gra1 for granulocytes, anti-B2】20 for B lymphocytes, anti-CD3 for T lymphocytes. After mixing them with the secondary antibodies which were bound to DynabeadsTM, different types of cells were successfully separated using a magnet. After removal of the antibodies by preincubation, the cells were x-irradiated at 20 Gy. The irradiated cells were fixed on a slide glass and analyzed by in situ hybridization probed with 35S-labeled antisence IL-1β RNA. As shown in Fig. A, an increased level of mRNA for IL-1β was observed after irradiation in the cells which bound to MOMA2 and F4】80. In contrast, cells which had gra-1, B2】20 or CD3 did not express IL-1β messages before and after irradiation. The results show that monocytes】macrophages in the normal spleen express IL-1β gene immediately after x-irradiation. 　To the further molecular studies, we used a macrophage cell line, L-8704, derived from C3H】He mice. An increase in mRNA for IL-1β was observed immediately after x-irradiation, similar with normal spleen cells. When the cells were irradiated in the presence of protein kinase inhibitors, x-ray responsive induction of IL-1β disappeared. This shows that x-ray-responsive immediate expression of IL-1β is controlled by signal transduction mechanisms including protein kinase networks at the transcriptional level. To confirm the contribution of transcriptional regulation, the nuclear run-on assay was performed. Isolated nuclei from non-irradiated and irradiated L-8704 cells were incubated with α-32P-UTP to label transcripts. The radioactive RNAs were hybridized with IL-1β cDNA which binds nitrocellulose. As shown in Fig. B, IL-1β-related transcripts were synthesized when nuclei from irradiated cells were used. This shows that the immediate-early induction of IL-1β is regulated at the transcriptional level. 　As described above, x-ray-responsive immediate-early induction of gene for IL-1β which encodes a radioprotector is transcriptionally regulated in monocytes】macrophages. This probably reflects the fact that x-irradiation stimulates radioprotective mechanisms, including IL-1β expression, via signal transduction. To clarify the mechanisms, further analysis of the events from irradiation to initiating transcription is necessary. 　

[Publications]Ishihara, H., Tanaka, I., Nemoto, K., Tsuneoka, K., Cheeramakara, C., Yoshida, K. and Ohtsu, H.: J. Radiat. Res., 36, 112-124, 1995. 
Fig.1. (A)  Spleen cells were separated using the indicated surface antibodies.  Before and immediately after irradiation of 20 Gy x-rays, they were analyzed by in situ hybridization probed with antisence IL-1β RNA.  Each pair of microphotographs shows the light-field (left) and dark-field (right) views of microautoradiographs of the same area.  (B)  Spots on the nitrocellulose filter containing 10 μⅲ of bacterial plasmid (pBSII) as a negative control, β-actin DNA as a positive control and IL-1β cDNA.  Nulei of non-irradiated and irradiated L-8704 cells were used to synthesize the probe.

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