Effects of the mutation on X-ray-induced deletions in the brain and spleen of delta mice.

Background: DNA-dependent protein kinase (DNA-PK), consisting of a Ku heterodimer (Ku70/80) and a large catalytic subunit (DNA-PKcs), plays an important role in the repair of DNA double-strand breaks via non-homologous end-joining (NHEJ) in mammalian cells. Severe combined immunodeficient () mice carry a mutation in the gene encoding DNA-PKcs and are sensitive to ionizing radiation. To examine the roles of DNA-PKcs in the generation of deletion mutations in vivo, we crossed mice with delta transgenic mice for detecting mutations.

Biological effects of space environmental factors: A possible interaction between space radiation and microgravity.

In the mid-1980s, space experiments began to examine if microgravity could alter the biological effects of space radiation. In the late 1990s, repair of DNA strand breaks was reported to not be influenced by microgravity using the pre-irradiated cells, because the exposure doses of space radiation were few due to the short spaceflight. There were, however, conflicting reports depending on the biological endpoints used in various systems.

Frozen human cells can record radiation damage accumulated during space flight: mutation induction and radioadaptation.

To estimate the space-radiation effects separately from other space-environmental effects such as microgravity, frozen human lymphoblastoid TK6 cells were sent to the "Kibo" module of the International Space Station (ISS), preserved under frozen condition during the mission and finally recovered to Earth (after a total of 134 days flight, 72 mSv). Biological assays were performed on the cells recovered to Earth. We observed a tendency of increase (2.

An approach to estimate radioadaptation from DSB repair efficiency.

In this review, we would like to introduce a unique approach for the estimation of radioadaptation. Recently, we proposed a new methodology for evaluating the repair efficiency of DNA double-strand breaks (DSB) using a model system. The model system can trace the fate of a single DSB, which is introduced within intron 4 of the TK gene on chromosome 17 in human lymphoblastoid TK6 cells by the expression of restriction enzyme I-SceI.

Contributions of direct and indirect actions in cell killing by high-LET radiations.

The biological effects of radiation originate principally in damages to DNA. DNA damages by X rays as well as heavy ions are induced by a combination of direct and indirect actions. The contribution of indirect action in cell killing can be estimated from the maximum degree of protection by dimethylsulfoxide (DMSO), which suppresses indirect action without affecting direct action.

Transcriptional infidelity promotes heritable phenotypic change in a bistable gene network.

Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate.

Repair of I-SceI induced DSB at a specific site of chromosome in human cells: influence of low-dose, low-dose-rate gamma-rays.

We investigated the influence of low-dose, low-dose-rate gamma-ray irradiation on DNA double strand break (DSB) repair in human lymphoblastoid TK6 cells. A single DSB was introduced at intron 4 of the TK+ allele (chromosome 17) by transfection with the I-SceI expression vector pCBASce. We assessed for DSB repair due to non-homologous end-joining (NHEJ) by determining the generation of TK-deficient mutants in the TK6 derivative TSCE5 (TK +/-) carrying an I-SceI recognition site.

Mutagenic radioadaptation in a human lymphoblastoid cell line.

We investigated the mutagenic radioadaptive response of human lymphoblastoid TK6 cells by pretreating them with a low dose (5 cGy) of X-rays followed by a high (2 Gy) dose 6h later. Pretreatment reduced the 2-Gy-induced mutation frequency (MF) of the thymidine kinase (TK) gene (18.3 x 10(-6)) to 62% of the original level (11.

Mutation induction in cultured human cells after low-dose and low-dose-rate gamma-ray irradiation: detection by LOH analysis.

To study the genetic effects of low-doses and low-dose-rate ionizing radiation (IR), human lymphoblastoid TK6 cells were exposed to 30 mGy of gamma-rays at a dose-rate of 1.2 mGy/hr. The frequency of early mutations (EMs) in the thymidine kinase (TK) gene locus was determined to be 1.

Cellular and molecular effects for mutation induction in normal human cells irradiated with accelerated neon ions.

We investigated the linear energy transfer (LET) dependence of mutation induction on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in normal human fibroblast-like cells irradiated with accelerated neon-ion beams. The cells were irradiated with neon-ion beams at various LETs ranging from 63 to 335 keV/microm. Neon-ion beams were accelerated by the Riken Ring Cyclotron at the Institute of Physical and Chemical Research in Japan.

Exploration of "over kill effect" of high-LET Ar- and Fe-ions by evaluating the fraction of non-hit cell and interphase death.

The reason why RBE for cell killing fell to less than unity (1.0) with very high-LET heavy-ions ((40)Ar: 1,640 keV/microm; (56)Fe: 780, 1,200, 2,000 keV/microm) was explored by evaluating the fraction of non-hit cell (time-lapse observation) and cells undergoing interphase death (calculation based on our previous data). CHO cells were exposed to 4 Gy (30% survival dose) of Ar (1,640 keV/microm) or Fe-ions (2,000 keV/microm).

Tumor induction in mice locally irradiated with carbon ions: a retrospective analysis.

Tumor induction in mice legs that were locally irradiated with carbon ions was compared to tumor induction by gamma rays after single and fractionated irradiation. A total of 250 tumors were induced in 1104 mice that received carbon-ion doses of 5 through 65 Gy. A total of 77 tumors were induced in 371 mice that received gamma-ray doses of 45 through 95 Gy.

Mutations induced by heavy charged particles.

The relative biological-effectiveness of radiation is increased when cells or tissue are exposed to densely ionizing (high-LET) radiation. A large number of studies focus on the following aspects of the biological effects of high-LET radiation: (i) basic understanding of radiation damage and repair; (ii) developing radiotherapy protocols for accelerated charged particles; and (iii) estimation of human risks from exposure to high-LET heavy charged particles. The increased lethal effectiveness (cell inactivation) of high-LET radiation contributes to new methods for using radiation therapy, but it is also necessary to study the enhanced mutagenic effect of high LET radiation, because higher frequencies of mutation can be expected to provide higher rates of carcinogenicity with human exposure.

Detection of adaptive response at chromosome level.

We have just started the basic study to detect the genetic alterations at chromosome level as a result of radioadaptive response. The assay system is based upon the analysis of loss of heterozygosity (LOH) induced in human lymphoblastoid cell TK6, which were pre-irradiated with low-doses of ionizing radiation (IR) before the challenging irradiation. In our previous study, this analysis was shown to be very sensitive to IR because the radiation-specific hemizygous LOHs (interstitial deletions) were observed after 10 cGy of IR (X-rays or accelerated carbon-ion beam).

Radiosensitization by hyperthermia in the chicken B-lymphocyte cell line DT40 and its derivatives lacking nonhomologous end joining and/or homologous recombination pathways of DNA double-strand break repair.

Hyperthermia has a radiosensitizing effect, which is one of the most important biological bases for its use in cancer therapy with radiation. Although the mechanism of this effect has not been clarified in molecular terms, possible involvement of either one or both of two major DNA double-strand break (DSB) repair pathways, i.e.

Further characterization of loss of heterozygosity enhanced by p53 abrogation in human lymphoblastoid TK6 cells: disappearance of endpoint hotspots.

Loss of heterozygosity (LOH) is the predominant mechanism of spontaneous mutagenesis at the heterozygous thymindine kinase locus (tk) in TK6 cells. LOH events detected in spontaneous TK(-) mutants (110 clones from p53 wild-type cells TK6-20C and 117 clones from p53-abrogated cells TK6-E6) were analyzed using 13 microsatellite markers spanning the whole of chromosome 17. Our analysis indicated an approximately 60-fold higher frequency of terminal deletions in p53-abrogated cells TK6-E6 compared to p53 wild-type cells TK6-20C whereas frequencies of point mutations (non-LOH events), interstitial deletions, and crossing over events were found to increase only less than twofold by such p53 abrogation.

Qualitative and quantitative difference in mutation induction between carbon- and neon-ion beams in normal human cells.

We investigated the difference in cell-killing effect and mutation induction between carbon- and neon-ion beams in normal human cells. Carbon- and neon-ion beams were accelerated by the Riken Ring Cyclotron (RRC) at the Institute of Physical and Chemical Research in Japan. Cell-killing effect was measured as the reproductive cell death using the colony formation assay.

Localization of human Mcm10 is spatially and temporally regulated during the S phase.

Mcm10 (Dna43) is an essential protein for the initiation of DNA replication in Saccharomyces cerevisiae. Recently, we identified a human Mcm10 homolog and found that it is regulated by proteolysis and phosphorylation in a cell cycle-dependent manner and that it binds chromatin exclusively during the S phase of the cell cycle. However, the precise roles that Mcm10 plays are still unknown.

Deletion, rearrangement, and gene conversion; genetic consequences of chromosomal double-strand breaks in human cells.

Chromosomal double-strand breaks (DSBs) in mammalian cells are usually repaired through either of two pathways: end-joining (EJ) or homologous recombination (HR). To clarify the relative contribution of each pathway and the ensuing genetic changes, we developed a system to trace the fate of DSBs that occur in an endogenous single-copy human gene. Lymphoblastoid cell lines TSCE5 and TSCER2 are heterozygous (+/-) or compound heterozygous (-/-), respectively, for the thymidine kinase gene (TK), and we introduced an I-SceI endonuclease site into the gene.

Sensitive detection of LOH events in a human cell line after C-ion beam exposure.

A molecular analysis of the loss of heterozygosity (LOH) events in human cells after low-dose heavy-ion exposure could contribute to the sensitive detection of the genetic influences caused by high-LET radiation. We exposed human lymphoblastoid TK6-20C cells to 10 cGy of an accelerated C-ion (22 keV/microm) beam, and observed a 3.1-fold increase in the mutation frequency (MF) at the heterozygous thymidine kinase (TK) locus over the background level.

Mutation induction by heavy ion irradiation of gpt delta transgenic mice.

Using the new transgenic mice produced by mating gpt delta with p53 knockout, mutation induction by heavy-ion irradiation and the effect of p53 background on such induction were studied. After the whole body irradiation with 10 Gy of 135 MeV/u carbon-ion beam, the genomic DNA was isolated from the different organs and the lambda DNA was rescued as a lambda phage. Mutations in the transgene on the lambda DNA were determined by the spi(-) selection (deletion assay).

Delayed cell cycle progression in human lymphoblastoid cells after exposure to high-LET radiation correlates with extremely localized DNA damage.

To compare the genotoxic effects of high-LET ionizing radiation to those of low-LET radiation, we investigated the responses of human lymphoblastoid cells to DNA damage TK6 after treatment with either low-LET X rays or high-LET iron ions (1000 keV/microm). A highly localized distribution of gammaH2AX/RAD51 foci was observed in the nuclei of cells irradiated with iron ions, in sharp contrast to cells exposed to X rays, where the distribution of foci was much more uniform. This implied the occurrence of a relatively high frequency of closely spaced double-strand breaks, i.

Heavy-ion-induced mutations in the gpt delta transgenic mouse: effect of p53 gene knockout.

The influence of the loss of p53 gene on heavy-ion-induced mutations was examined by constructing a new line of transgenic mice, p53 knockout (p53(-/-)) gpt delta. In this mouse model, deletions in lambda DNA integrated into the mouse genome are preferentially selected as Spi(-) phages, which can then be subjected to molecular analysis. Mice were exposed to 10 Gy of whole-body carbon-ion irradiation.