The PC12 cell is a cell line from rat pheochromocytoma which alters in morphology to neuron-like cells with neurite extensions in response to nerve growth factor (NGF). Therefore, the PC12 cell is considered a useful model to study neuronal cell differentiation at the cellular level. Some reports indicate that the cell differentiation can be induced with conventional γ-ray irradiations. Radiation effects in PC12 cells have been interested for studies of the mechanism of neuron differentiation.
We have investigated the radiation-induced neuronal differentiation and neurite extension of the single PC12 cell with X-ray microbeams of 10 μm in diameter. The X-ray microbeam system was composed of micro focus X-ray tube and glass capillary. Targeted PC12 cells were irradiated at the dose rate of 0.05 Gy/s and the control of neuron cell differentiation have been investigated.

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Recently, Copper(II)-diacetyl-bis(N4-methyothiosemicarbazone), Cu-ATSM, has been reported to concentrate in hypoxic tissues and cells. In this study, the chromosomal aberrations were investigated in Cu-ATSM treated cultured cells irradiated with monochromatic X rays at around K-shell absorption edge of the Cu atom. Exponentially growing human cells (GM05389, HSG) were incubated with 3000nM Cu-ATSM at both hypoxic and normoxic conditions for 10 hours. After Cu-ATSM treatment, cells were irradiated with monochromatic X rays having energy of 9.027 (CuK-H) and 8.973 keV (CuK-L) (corresponding energies to the slightly above and below the Cu K-shell absorption edge). Soon after the irradiation, the chromosomal aberrations were analyzed by premature chromosomal condensation (PCC) method using calyculin A. Under hypoxic conditions, cells took up Cu-ATSM significantly a lot compared with those treated under normoxic conditions, and the chromosomal aberrations induced by the irradiation with Cu-K edge X rays were much greater than those without Cu-ATSM. Also, chromosomal aberrations of Cu-ATSM treated cells irradiated with CuK-H X rays were greater than those irradiated with CuK-L X rays. This study shows clearly the enhancement of chromosomal aberrations of hypoxic cultured cells with Cu-ATSM by through ionization or excitation of Cu atoms in cells.

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This study aims to investigate the medium-mediated bystander effect and the role of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in the bystander effect. The induction of chromosomal aberrations was observed in recipient normal human fibroblast cells treated with conditioned medium that was harvested from donor fibroblasts exposed to low-linear energy transfer (LET) X-rays and high-LET heavy ions. The recipient cells were treated for 12 h with conditioned medium, which were harvested from donor cells at 24 h after exposure to 10 Gy of 30 kVp soft X-rays and 20Ne ions (437 keV/micro-m), followed by analyses of chromosome aberrations in recipient cells with premature chromosome condensation methods. To examine the role of DNA-PKcs and nitric oxide (NO), cells were treated with its inhibitor LY294002 (LY) and its scavenger (c-PTIO), respectively. Increased frequency of chromosomal aberrations in recipient cells treated with conditioned medium from irradiated but not from un-irradiated donor cells was observed which was independent of radiation type. Bystander induction of chromosomal aberrations in recipient cells was mitigated when donor cells were treated with LY before irradiation and with c-PTIO after irradiation, and was enhanced when recipient cells were treated with LY before treatment of recipient cells with conditioned medium from irradiated donor cells. Irradiated normal human cells secrete NO and other molecules which in turn transmit radiation signals to un-irradiated bystander cells.

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K-shell photoabsorption of DNA constituent atoms is predicted to induce the critical DNA damage because the constituent atom can be selectively ionized by the K-shell photoabsorption, and ejected secondary low-energy electron tracks cause local multiple damage sites to DNA molecules. To investigate the K-shell photoabsorption effect (photoelectric effect and subsequent secondary electron effect) of DNA damage, dry plasmid DNA (pUC18) films were irradiated with synchrotron monochromatic ultrasoft X-rays. Four photon energies, 270, 380, 435, and 560eV, a value below the carbon K-edge, below and above the nitrogen K-edge, and above the oxygen K-edge, respectively, were chosen for the irradiation experiments. The experiments were performed at the beamline BL23SU in the SPring-8. Irradiated plasmid DNA was analyzed by agarose gel electrophoresis and the yields of strand breaks were determined by measuring the band intensity of the separated closed circular, open circular and linear forms of the plasmid DNA. The yields of base lesions were determined by the post-irradiation-treatment of the DNA with enzymatic probes (Fpg and Nth) which excise base lesion. The obtained yields of strand breaks (1∼2×10^-10ssb/Gy/Da) were almost the same as that obtained by the irradiation with soft X-rays (2keV), which were investigated by previous reports. The role of ionization effect of carbon, nitrogen and oxygen will be discussed in terms of their efficiency to induce strand breaks, base lesions, and clustered damage sites.

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Gap junction (GJ) mediated X-ray bystander effect has been examined by using lindane (γ-BHC), an inhibitor of the GJ function. X-ray microbeam with a diameter of 100 micrometer was available from a micro-capillary tube installed in a table-top X-ray fluorescence analyzer for local specimen area (XGT-2700, HORIBA Ltd.). First, the concentration and the timing of the administration of lindane were determined using human normal skin fibroblast NB1RGB cells. The GJ function restraint by lindane was detected with Lucifer Yellow Transfer method. The results showed that 1) the GJ function was completely restrained by 2 hr incubation with 100 μM of lindane, and 2) the restraint by lindane begins to release immediately after removal of the reagent and fully recovered after 4 hrs. These results suggest that lindane is a useful tool for the examination of time-dependent progression of the bystander effects via GJ. Second, a bystander effect was estimated by p53 expression area which was observed using the fluorescent antibody technique after 6hr incubation from irradiation. The expression area was limited to the beam diameter by the addition of 100 μM lindane. The result indicated the important role of GJ in the bystander effect. The progression of the bystander effect after irradiation is under examination by varying the administration timing or the removal timing of lindane.

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Ultraviolet-B radiation (UVB; 280-320 nm) can damage DNA by causing formation of cyclobutane pyrimidine dimer (CPD). Photoreactivation mediated by an enzyme, photolyase, is the major pathway for repairing UVB-induced CPD in plants. Plant cell has different genome in each nuclei, chloroplast and mitochondria. It has been reported that CPD photoreactivation was involved in nuclei, but in the other two organelles in Arabidopsis. We previously reported at the meeting (the 48th Annual Meeting of The Japan Radiation Research Society, 2005) that the levels of CPD induced by UVB radiation on not only nuclei but also chloroplast and mitochondrial genome decreased dependently by exposure of blue irradiation. These results suggested that CPD photolyase, which is encoded by a single-copy gene in nuclei, could function in nuclei, chloroplast and mitochondria in rice. In this study, in order to verify this suggestion, we compared the CPD photorepair capacity in each organelle among two rice strains (Sasanishiki and Surjamkhi) and antisense CPD photolyase gene transgenic plant: Surjamkhi was more deficient in CPD photolyase activity compared with Sasanishiki, and the antisense transgenic plant has little CPD photolyase activity. As a result, we found that there was a significant difference in CPD photorepair capacity in each organelle among these plants: the CPD photorepair capacity in each organelle was faster in Sasanishiki than that in Surjamkhi, and was not detected in antisense transgenic plant. Thus, CPD photolyase functions in nuclei, chloroplast and mitochondria in rice plant.

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Ultraviolet-B radiation (UV-B, 280~320nm) can damage DNA by causing formation of cyclobutane pyrimidine dimer (CPD). Photoreactivation mediated by an enzyme, photolyase, is the major pathway for repairing UVB-induced CPD in plants. Sensitivity to UVB varies widely among rice cultivars. We previously indicated that a difference in UVB sensitivity among rice cultivars is tightly linked with an alteration of function of photolyase resulting from spontaneously occurring mutations in the CPD photolyase gene. Here, in order to demonstrate directly that CPD photolyase activity is a crucial factor for determining UVB sensitivity in rice, we constructed transgenic rice plants with five to thirty-fold higher CPD photolyase activity or very low activity in comparison with UV-resistant rice cultivar Sasanishiki (wild type). Rice plants with higher CPD photolyase activity, by overexpressing CPD photolyase, showed strong resistance to UVB-caused growth inhibition, whereas rice plants with very low activity, by insertion ob antisense CPD photolyase gene, was severely damaged. These results emphasize that CPD photolyase activity plays an important role in preventing UVB-caused growth inhibition, and the increases in CPD photolyase activity can lead to increasing resistance to UVB radiation.

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