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/microm) and 20Ne ions (437 keV/microm), 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 unirradiated 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.

Repair of DNA damage induced by accelerated heavy ions in mammalian cells proficient and deficient in the non-homologous end-joining pathway.

Human and rodent cells proficient and deficient in non-homologous end joining (NHEJ) were irradiated with X rays, 70 keV/microm carbon ions, and 200 keV/microm 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 = approximately 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 G(1) 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.

Enhancement of chromosomal aberrations in tumor cells with a non-labeled Cu-PTSM and irradiation with Cu K-shell monochromatic X rays.

This study aims to clarify the enhancement effect on chromosomal aberrations via selective energy absorption by Cu atoms in cultured bone cancer cells with non-labelled Cu-pyruvaldehyde-bis(N(4)-methylsemicarbazone) (Cu-PTSM) and monochromatic X rays. The X rays having the energy of the Cu K-shell absorption edge arising from synchrotron radiation (KEK-PF) was used as radiation source. Cu-PTSM labelled with copper radionuclides has been developed for medical imaging using positron emission tomography (PET). Evidence clearly showed that the absorption of X rays in the Cu K-shell caused this enhancement. After cells were treated with 1000 nM Cu-PTSM, the enhancement ratio of the initial yield of isochromatid breaks caused by CuK-H X-ray irradiation to that by 200 kV(p) X rays was approximately 2.8. About 5.6 times of the remaining isochromatid breaks were observed at 4 h in Cu-PTSM treated samples irradiated with CuK-H X rays comparing to that with 200 kV(p) X rays. In this study, uniqueness in physical property such as Cu atom K-shell ionisation was applied for the enhancement of biological effects in cancer cells.

EPR studies of 5-bromouracil crystal after irradiation with X rays in the bromine K-edge region.

Radicals induced in a single crystal of 5-bromouracil (BrUra) by synchrotron soft X rays in the bromine K-edge region (13.461-13.482 keV) were investigated using the X-band EPR method. The crystal was irradiated at three peak energies of the absorption spectrum at room temperature or at 80 K. A hydrogen abstraction radical derived from N1 of the pyrimidine ring was commonly observed for all of the energies used, though with some variation in quantity. Similar characteristics were also observed in the EPR signal for the off-K-edge low-energy (13.42 keV) and (60)Co gamma rays used for comparison. When irradiated at 80 K, a much larger exposure (roughly 10 times) of soft X rays was needed to obtain the same signal intensity as that observed at room temperature. EPR signals were not detectable with gamma irradiation at liquid nitrogen temperature.

Radiosensitization of normal human cells by LY294002: cell killing and the rejoining of DNA and interphase chromosome breaks.

The radiosensitizing effect of a phosphoinositide-3 kinase (PI3K) inhibitor, wortmannin, has been studied rather extensively, but there have been few studies on the radiosensitizing effect of another PI3K inhibitor, LY294002. In this report, we present the radiosensitizing effect of LY294002 using normal human cells. Clonogenic cell survival indicated that LY294002 enhanced the killing effect of gamma-rays in a dose-dependent manner, although this drug by itself did not affect the cell killing. To obtain a 10% cell survival, about one half of the radiation dose was needed when cells were treated with 50 microM LY294002 as compared to cells without the drug. A mild inhibition of repair of DNA double strand breaks (DSBs) was observed in irradiated normal human cells pre-treated with LY294002 (50 microM). At the interphase chromosome level, we also observed an increase in the number of residual breaks when irradiated cells were pre-treated with this drug (about 2-fold at 5 Gy). These results suggest that the inhibition of DSB repair mediated the radiosensitizing effect of LY294002 at the dose level that we used.

Comparison of the kinetics of radiation-induced apoptosis in DT40 cells irradiated with low and high doses of X rays.

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-G(1) 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.

Electronic structure of DNA nucleobases and their dinucleotides explored by soft X-ray spectroscopy.

The electronic structures of a series of DNA nucleobases and their dinucleotides were investigated by N 1s X-ray absorption, X-ray photoemission, and resonant X-ray emission spectroscopy. Resonant X-ray emission spectra of the guanine base and its dinucleotide indicate that it has a weak structure at the lowest binding energy; at this energy, it isolates from the main valence band and forms the HOMO state. This indicates that the HOMO state is localized in the guanine base, as claimed by valence and core photoemissions and expected from theoretical predictions. In addition, the XAS and XES profiles of the guanine dinucleotide indicate that disruption of the aromatic character of the six-membered ring results in the localization of the pi state at the imine (-N=) site of the guanine base; this may favor charge transfer among stacked guanine bases and further influence the conductivity of DNA.