Tomoo Funayama

Bystander effect induced by counted high-LET particles in confluent human fibroblasts: a mechanistic study

The possible mechanism of a radiation‐induced bystander response was investigated by using a high‐LET heavy particle microbeam, which allows selected cells to be individually hit with precise numbered particles. Even when only a single cell within the confluent culture was hit by one particle of 40Ar (∼1260 keV/µm) or 20Ne (∼380 keV/µ m), a 1.4‐fold increase of micronuclei (MN) was detected demonstrating a bystander response. When the number of targeted cells increased, the number of MN biphasically increased; however, the efficiency of MN induction per targeted cell markedly decreased. When 49 cells in the culture were individually hit by 1 to 4 particles, the production of MN in the irradiated cultures were ∼2‐fold higher than control levels but independent of the number and LET of the particles. MN induction in the irradiated‐culture was partly reduced by treatment with DMSO, a scavenger of reactive oxygen species (ROS), and was almost fully suppressed by the mixture of DMSO and PMA, an inhibitor of gap junctional intercellular communication (GJIC). Accordingly, both ROS and GJIC contribute to the above‐mentioned bystander response and GJIC may play an essential role by mediating the release of soluble biochemical factors from targeted cells.—Shao, C., Furusawa, Y., Kobayashi, Y., Funayama, T., Wada, S. Bystander effect induced by counted high‐LET particles in confluent human fibroblasts: a mechanistic study. FASEB J. 17, 1422–1427 (2003)

PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation.

The extraordinary radiation resistance of Deinococcus radiodurans results from the efficient capacity of the bacterium to repair DNA double‐strand breaks. By analysing the DNA damage repair‐deficient mutant, KH311, a unique radiation‐inducible gene (designated pprA) responsible for loss of radiation resistance was identified. Investigations in vitro showed that the gene product of pprA (PprA) preferentially bound to double‐stranded DNA carrying strand breaks, inhibited Escherichia coli exonuclease III activity, and stimulated the DNA end‐joining reaction catalysed by ATP‐dependent and NAD‐dependent DNA ligases. These results suggest that D. radiodurans has a radiation‐induced non‐homologous end‐joining repair mechanism in which PprA plays a critical role.

Identification and disruption analysis of the recN gene in the extremely radioresistant bacterium Deinococcus radiodurans.

We isolated a radiosensitive mutant strain, KR4128, from a wild-type strain of Deinococcus radiodurans, which is known as a extremely radioresistant bacterium. The gene that restore the defect of the mutant in DNA repair was cloned, and it turned out to be the homolog of the recN gene of Escherichia coli. The recN gene encoded a protein of 58 kDa, and, in its N-terminal region, a potential ATP binding domain was conserved as expected for a prokaryotic RecN protein. An analysis of sequence of the mutant recN gene revealed a G:C to T:A transversion near the 3 end of the coding region. This alteration causes an ochre mutation, and results in the truncation of 47 amino acids from the C-terminal region of the RecN protein. The null mutant of recN gene was constructed by insertional mutagenesis, and it showed substantial sensitivities to various types of DNA damaging agents, indicating that a single defect in the recN gene can directly affect the DNA damage resistant phenotype in D. radiodurans. The recN locus of KR4128 was also disrupted and the disruptant indicated the sensitivity that was indistinguishable from its progenitor. The result indicate that the transversion in the recN gene of KR4128 cells causes a complete loss of function of the RecN protein and thus the C-terminal region of the RecN protein includes domain essential to its function.

Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30

Deinococcus radiodurans strain rec30, which is a DNA damage repair-deficient mutant, has been estimated to be defective in the deinococcal recA gene. To identify the mutation site of strain rec30 and obtain information about the region flanking the gene, a 4.4-kb fragment carrying the wild-type recA gene was sequenced. It was revealed that the recA locus forms a polycistronic operon with the preceding cistrons (orf105a and orf105b). Predicted amino acid sequences of orf105a and orf105b showed substantial similarity to the competence-damage inducible protein (cinA gene product) from Streptococcus pneumoniae and the 2-5 RNA ligase from Escherichia coli, respectively. By analyzing polymerase chain reaction (PCR) fragments derived from the genomic DNA of strain rec30, the mutation site in the strain was identified as a single G:C to A:T transition which causes an amino acid substitution at position 224 (Gly to Ser) of the deinococcal RecA protein. Furthermore, we succeeded in expressing both the wild-type and mutant recA genes of D. radiodurans in E. coli without any obvious toxicity or death. The gamma-ray resistance of an E. coli recA1 strain was fully restored by the expression of the wild-type recA gene of D. radiodurans that was cloned in an E. coli vector plasmid. This result is consistent with evidence that RecA proteins from many bacterial species can functionally complement E. coli recA mutants. In contrast with the wild-type gene, the mutant recA gene derived from strain rec30 did not complement E. coli recA1, suggesting that the mutant RecA protein lacks functional activity for recombinational repair.

Irradiation of single mammalian cells with a precise number of energetic heavy ions-Applications of microbeams for studying cellular radiation response

Abstract A single cell irradiation system has been developed for targeting cells individually with a precise number of high-LET heavy ions to elucidate radiobiological effects of single heavy ions and to investigate the interaction of damages produced by separate events. The system has been installed at a high-energy collimated heavy-ion microbeam apparatus under a vertical beam line of the JAERI-Takasaki AVF-cyclotron. Using the heavy ion microbeam apparatus, mammalian cells are irradiated in the atmosphere with a single or precise number of heavy ions, 13.0 MeV/u 20 Ne or 11.5 MeV/u 40 Ar. Positional data of the individual cells are obtained at an off-line microscope before irradiation, then the cells are targeted and irradiated semi-automatically using the on-line microscope of the microbeam apparatus according to the obtained data. The number of ions penetrating the cells attached on the ion track detector CR-39 were counted with a plastic scintillator-photomultiplier tube assembly and a constant fraction discriminator. Immediately after the irradiation, the position and the number of ion tracks traversed the cell was detected with etching of CR-39 from the opposite side of the cell with alkaline-ethanol solution at 37 °C. The growths of the cells were observed individually up to 60 h after irradiation. The continuous observation of the individual cell growth indicated that a single ion traversal of a cell nucleus resulted in complete growth inhibition of the irradiated cells.

Radiosurgery using heavy ion microbeams for biological study: Fate mapping of the cellular blastoderm-stage egg of the silkworm, Bombyx mori

We investigated the effects of heavy ions on embryogenesis of the silkworm, Bombyx mori using a collimated heavy ion microbeam from the vertical beam line of an AVF-cyclotron. Eggs were exposed to carbon ions at the cellular blastoderm stage. Microbeams were found to be extremely useful for radio-microsurgical inactivation of nuclei or cells in the target site. Spot irradiation caused abnormal embryos, which showed localized defects such as deletion, duplication and fusion, depending on dose, beam size and site of irradiation. The location and frequency of defects on the resultant embryos were closely correlated to the irradiation site. Based on this correlation, a fate map was established for the Bombyx egg at the cellular blastoderm stage.

Evaluation of the Resistance of Euglena gracilis to Ion Beam Radiation

Abstract The resistance of Euglena (E.) gracilis to ionizing radiation was investigated using seven kinds of ion beams each with different energy characteristics. The minimum effective dose of the most lethal ion beams was 40 Gy. Given its substantially high resistance to heavy ion beams, E. gracilis possesses great potential in acting as an effective support system to produce food and regenerate oxygen in a space station. The lethal effect of ionizing radiation was dependent on the linear energy transfer value of the heavy ion beams, and reached a maximum at 196 keV/μm. This value was different from those obtained by previous irradiation experiments using mammalian and plant cells, suggesting that the radiation response of E. gracilis is distinct from that of mammalian and plant cells.

Detection of DNA damage induced by heavy ion irradiation in the individual cells with comet assay

Investigating the biological effects of high-LET heavy ion irradiation at low fluence is important to evaluate the risk of charged particles. Especially it is important to detect radiation damage induced by the precise number of heavy ions in the individual cells. Thus we studied the relationship between the number of ions traversing the cell and DNA damage produced by the ion irradiation. We applied comet assay to measure the DNA damage in the individual cells. Cells attached on the ion track detector CR-39 were irradiated with ion beams at TIARA, JAERI-Takasaki. After irradiation, the cells were stained with ethidium bromide and the opposite side of the CR-39 was etched. We observed that the heavy ions with higher LET values induced the heavier DNA damage. The result indicated that the amount of DNA damage induced by one particle increased with the LET values of the heavy ions.

Cloning of Structural Gene of Deinococcus radiodurans UV-Endonuclease β

To characterize its enzymic property we cloned and sequenced the gene of Deinococcus radiodurans encoding UV-endonuclease β, an alternative enzyme to UvrABC repairing damaged DNA. Amino acid substitutions were found in UV-sensitive mutants. The putative amino acid sequence had some similarity with those of eukaryotic UV-endonucleases and with a sequence found in a protein data base of Bacillus subtilis.