Kaoru Takakura

Repair of DNA Damage Induced by Accelerated Heavy Ions in Mammalian Cells Proficient and Deficient in the Non-homologous End-Joining Pathway

Abstract Okayasu, R., Okada, M., Okabe, A., Noguchi, M., Takakura, K. and Takahashi, S. Repair of DNA Damage Induced by Accelerated Heavy Ions in Mammalian Cells Proficient and Deficient in the Non-homologous End-Joining Pathway. Radiat. Res. 165, 59–67 (2006). Human and rodent cells proficient and deficient in non-homologous end joining (NHEJ) were irradiated with X rays, 70 keV/μm carbon ions, and 200 keV/μm iron ions, and the biological effects on these cells were compared. For wild-type CHO and normal human fibroblast (HFL III) cells, exposure to iron ions yielded the lowest cell survival, followed by carbon ions and then X rays. NHEJ-deficient xrs6 (a Ku80 mutant of CHO) and 180BR human fibroblast (DNA ligase IV mutant) cells showed similar cell survival for X and carbon-ion irradiation (RBE = ∼1.0). This phenotype is likely to result from a defective NHEJ protein because xrs6-hamKu80 cells (xrs6 cells corrected with the wild-type KU80 gene) exhibited the wild-type response. At doses higher than 1 Gy, NHEJ-defective cells showed a lower level of survival with iron ions than with carbon ions or X rays, possibly due to inactivation of a radioresistant subpopulation. The G1 premature chromosome condensation (PCC) assay with HFL III cells revealed LET-dependent impairment of repair of chromosome breaks. Additionally, iron-ion radiation induced non-repairable chromosome breaks not observed with carbon ions or X rays. PCC studies with 180BR cells indicated that the repair kinetics after exposure to carbon and iron ions behaved similarly for the first 6 h, but after 24 h the curve for carbon ions approached that for X rays, while the curve for iron ions remained high. These chromosome data reflect the existence of a slow NHEJ repair phase and severe biological damage induced by iron ions. The auto-phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs), an essential NHEJ step, was delayed significantly by high-LET carbon- and iron-ion radiation compared to X rays. This delay was further emphasized in NHEJ-defective 180BR cells. Our results indicate that high-LET radiation induces complex DNA damage that is not easily repaired or is not repaired by NHEJ even at low radiation doses such as 2 Gy.

Simple biodosimetry method for use in cases of high-dose radiation exposure that scores the chromosome number of Giemsa-stained drug-induced prematurely condensed chromosomes (PCC)

There is a need for quick dose estimation by a simple method in radiation accidents. This study develops a simple and rapid dose estimation protocol for victims of such accidents, in particular those involving high radiation doses. Human peripheral blood lymphocytes (PBL) were γ-irradiated in vitro at several dose points up to 60 Gy, and were stimulated with phytohaemagglutinin-P (PHA-P) for 2 days to obtain dividing cells. PBL were then forced to condense prematurely, using 50 nM calyculin A, and the obtained chromosome spreads were Giemsa stained. The G2-PCC (prematurely condensed chromosomes) index and chromosome number for each radiation dose point were scored. G2-PCC were stably induced using calyculin A within 24 h delays in stimulation of PBL with PHA-P. The chromosome number of G2-PCC increased steeply with radiation doses up to 30 Gy at a rate of 0.31 Gy − 1 and then decreased at 0.30 Gy − 1 up to 40 Gy. More than 10% of G2-PCC index remained up to a 15 Gy dose. Even after 40 Gy irradiation, about 2% PCC index was obtained, and this value was enough to score a sufficient number of chromosome spreads for analysis. Therefore, the combined use of chromosome number and G2-PCC index allows biodosimetry to be done easily and rapidly. If PCC are not induced using calyculin A, it is strongly suggested that the radiation dose is over 50 Gy. A rapid and easy dose estimation for large dose exposure whole-body was realized by combined analysis of Giemsa-stained chromosome number of G2-PCC and PCC index using calyculin A. This simple method will be of use for rapid decision making of therapy for radiation accident victims. This method also has potential for use as a biodosimeter for partial-body exposure accidents.

Role of DNA-PKcs in the bystander effect after low- or high-LET irradiation

Purpose: To investigate the role of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in the medium-mediated bystander effect for chromosomal aberrations induced by low-linear energy transfer (LET) X-rays and high-LET heavy ions in normal human fibroblast cells. Materials and methods: The recipient cells were treated for 12 h with conditioned medium, which was harvested from donor cells at 24 h after exposure to 10 Gy of soft X-rays (5 keV/µm) and 20Ne ions (437 keV/µm), 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 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (c-PTIO), respectively. Results: Increased frequency of chromosome 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 chromosome 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. Conclusion: Irradiated normal human cells secrete NO and other molecules which in turn transmit radiation signals to un-irradiated bystander cells, leading to the induction of bystander chromosome aberrations partially repairable by DNA-PKcs-mediated DNA damage repair machinery, such as non-homologous end-joining repair pathways.

SINGLE‐STRAND BREAKS IN SUPERCOILED DNA INDUCED BY VACUUM‐UV RADIATION IN AQUEOUS SOLUTION

We studied the induction of single‐strand breaks in the DNA of plasmid pBR 322 by vacuum‐UV radiation above 145 nm in aqueous solutions in relation to the production of OH‐radicals in water. The similarity and dissimilarity were examined of the wavelength dependence between the two effects. The maximum of single strand breaks at 150 nm could be explained by the action of OH‐radicals derived from direct water photolysis: the maximum at 180 nm remains unexplained. There was no indication that the direct absorption of photon by the DNA molecule plays an important role in the production of single‐strand breaks.

Action spectrum for the induction of single-strand breaks in DNA in buffered aqueous solution in the wavelength range from 150 to 272 nm: dual mechanism.

The induction of single-strand breaks by vacuum-u.v. and far-u.v. radiations (150 nm-272 nm) in plasmid Col E1 DNA in buffered aqueous solution was studied by agarose gel electrophoresis. The results indicated that the mechanisms of DNA strand-break induction are different in the two u.v. regions: above 210 nm the direct excitation of DNA seemed to be the main cause; below 180 nm the photolysis of solvent water seemed to be essential. The demonstration of a systematic protection at 160 nm and not at 209 nm by OH scavengers, such as ethyl alcohol, potassium iodide and D-mannitol, supports the involvement of OH radicals in the vacuum-u.v. region.

Auger effects on bromo-deoxyuridine-monophosphate irradiated with monochromatic X-rays around bromine K-absorption edge

SummaryIn order to study the enhanced effect by Auger cascade, samples of bromo-deoxyuridine-monophosphate (Br-dUMP) in aqueous solutions were irradiated with monochromatic X-rays at 13.49 keV and 13.43 keV, just above and below the K-absorption edge of bromine, using synchrotron radiation as a source. Radiolytic products such as deoxyuridine-monophosphate (dUMP), uracil and bromo-uracil (Br-uracil) were isolated using high performance liquid chromatography. Their amounts were quantitatively analysed as a function of the absorbed dose in the solutions containing Br-dUMP for the energy of the X-rays.G values for these products were calculated on the basis of the absorbed energy. As the results, the ratios of G values of radiolytic products from Br-dUMP between X-rays of 13.49 keV and 13.43 keV were 2.2 for dUMP, 1.02 for Br-uracil and 1.23 for uracil, suggesting clearly the energy dependent enhancement. On the other hand, little significant difference between X-rays of 13.49 keV and 13.43 keV was observed for theG values of uracil released from dUMP irradiated in aqueous solutions. It seemed to confirm that the Auger electrons from K-shell of bromine atoms might play the main role for energy-dependent enhancement at induction of these radiolytic products.

Development of photon microbeam irradiation system for radiobiology

Abstract In order to evaluate the risk of low dose or low-dose rate irradiation, a microbeam irradiation system using synchrotron radiation has been proposed, which overcomes the difficulty of the Poisson distribution of hit cells in the majority of the non-hit cell population. Up to now, we succeeded in developing a system having the following specifications. The smallest X-ray beam size obtained is 2 μm, practically 10 μm with X-ray energy of 5.35 keV. Cells grown and irradiated on specially designed dishes can be identified, after various treatments, for detection of irradiated response. Presently, the system works only in “point and shoot” mode. Automatic recognition and irradiation of cells, which enables the irradiation of 1000 cells/h, will soon be operative.

Double-strand breaks in DNA induced by the K-shell ionization of calcium atoms

A calcium (Ca)-DNA complex in a buffered aqueous solution was irradiated with monochromatic x-rays at around the K-shell absorption edge of the Ca atom. The energies were 4.030 and 4.071 keV, which correspond to slightly below and above the K-shell absorption edge of Ca (4.047 keV). The number of double-strand breaks (DSB) and single-strand breaks (SSB) per one molecule of Col E1 DNA was calculated using Povirks equation after the electrophoretic separation of three forms of plasmid DNA (forms I, II and III, which are supercoiled closed circular, open circular and linear, respectively). In a solution with Ca ions, the DSB yield in DNA irradiated with 4.071 keV x-rays is 1.27 times higher than that with 4.030 keV x-rays. In contrast, in the absence of Ca ions, the DSB yield is the same at both the x-ray energies. The SSB yields are also independent of the x-ray energy in the presence and absence of Ca ions. A reference experiment regarding irradiation with 60Co gamma-rays shows that the ratio of the DSB yield to the SSB yield upon irradiation with 4.071 keV x-rays is higher (factor of 3.2) than that with 60Co gamma-rays.

Chromosomal Aberrations in Normal Human Cells Induced by the Auger Effect via Ca Atoms

Purpose: To quantify the Auger effect on chromosomal aberrations via Ca atoms in human cells. Material and Methods: Exponentially growing human normal fibroblasts (GM05389) were irradiated with 4.047 (CaK‐P), 4.026 (CaK‐L) and 4.067 (CaK‐H) keV X‐rays (corresponding to the resonance absorption edge of the Ca K‐shell and slightly below and slightly above the edge, respectively) using synchrotron radiation at the photon factory (PF) of the High Energy Accelerator Organization located in Tsukuba. Chromosomal aberrations induced by the irradiation were analyzed by the premature chromosome condensation (PCC) method using calyculin A. The dependency of the chromosomal aberrations on the incubation time post 2 Gy irradiation was observed for each energy. Irradiation using 200 kVp conventional X‐rays was also examined as a reference to CaK irradiation. Results: 1. Soon after irradiation with 2 Gy, the enhancement ratios of CaK‐H X‐rays to CaK‐L X‐rays were 1.21, 1.51 and 2.70 for breaks/gaps, isochromatid breaks and exchanges, respectively. The enhancement ratios of CaK‐P X‐rays to CaK‐L X‐rays were 1.82, 0.98 and 6.30, for breaks/gaps, isochromatid breaks and exchanges, respectively. 2, After a 6‐hr incubation treatment post 2 Gy irradiation, the enhancement ratios of CaK‐H X‐rays to CaK‐L X‐rays were 1.59, 2.03 and 2.14 for breaks/gaps, isochromatid breaks and exchanges, respectively. The enhancement ratios of CaK‐P X‐rays to CaK‐L X‐rays were 1.69, 1.66 and 2.00 for breaks/gaps, isochromatid breaks and exchanges, respectively. 3, Soon after irradiation, the ratios of the efficiencies of CaK‐P X‐rays to those of 200 kVp X‐rays were 1.74, 1.29 and 2.51 for breaks/gaps, isochromatid breaks and exchanges, respectively. And after a 6‐hr incubation treatment, the ratios were 5.50, 1.93 and 1.81 for breaks/gaps, isochromatid breaks and exchanges, respectively. Conclusions: An effective enhancement of chromosomal aberrations, such as breaks/gaps, isochromatid breaks and exchanges, was caused by Ca K‐shell ionization or excitation. Auger electrons emitted by Ca atoms in irradiated cells appear to have an important role in causing this enhancement. Comparing these efficiencies of chromosomal aberrations with those produced by 200 kVp conventional X‐rays suggests un‐repaired and complicated damage is induced by the X‐rays around the Ca K‐shell resonance absorption edge.

Comparison of the Kinetics of Radiation-Induced Apoptosis in DT40 Cells Irradiated with Low and High Doses of X Rays

Abstract In this study, we attempted to clarify the influence of the DNA repair genes RAD54 and KU70, components of the homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways, respectively, on apoptosis induced by 1 Gy (low-dose) and 5 Gy (high-dose) irradiation. All experiments were performed using chicken B-lymphocyte DT40 cells and the DNA repair-deficient cell lines KU70−/−, RAD54−/− and KU70−/−/RAD54−/−. Morphological changes were detected by fluorescence methods, and the sub-G1 fraction and the activated caspases in DT40 cells were analyzed by flow cytometry. Irradiation with 1 Gy significantly increased the level of apoptosis in cells with the defective DNA repair genes, with the maximum apoptosis occurring in double mutant cells, KU70−/−/RAD54−/−, demonstrating that 1 Gy is enough to induce apoptosis in DNA repair-deficient DT40 cells, and that KU70 and RAD54 must have almost the same role in low-dose radiation-induced apoptosis. After 5 Gy, fast induction of apoptosis, within 2 h, was seen in both wild-type cells and RAD54−/− cells, indicating that functional KU70 must be important for the rescue of the cells from the induction of fast apoptosis.