Emiko Sekine

Single extreme low dose/low dose rate irradiation causes alteration in lifespan and genome instability in primary human cells

To investigate the long-term biological effect of extreme low dose ionising radiation, we irradiated normal human fibroblasts (HFLIII) with carbon ions (290 MeV u−1, 70 keV μm−1) and γ-rays at 1 mGy (total dose) once at a low dose rate (1 mGy 6–8 h−1), and observed the cell growth kinetics up to 5 months by continuous culturing. The growth of carbon-irradiated cells started to slow down considerably sooner than that of non-irradiated cells before reaching senescence. In contrast, cells irradiated with γ-rays under similar conditions did not show significant deviation from the non-irradiated cells. A DNA double strand break (DSB) marker, γ-H2AX foci, and a DSB repair marker, phosphorylated DNA-PKcs foci, increased in number when non-irradiated cells reached several passages before senescence. A single low dose/low dose rate carbon ion exposure further raised the numbers of these markers. Furthermore, the numbers of foci for these two markers were significantly reduced after the cells became fully senescent. Our results indicate that high linear energy transfer (LET) radiation (carbon ions) causes different effects than low LET radiation (γ-rays) even at very low doses and that a single low dose of heavy ion irradiation can affect the stability of the genome many generations after irradiation.

High LET heavy ion radiation induces lower numbers of initial chromosome breaks with minimal repair than low LET radiation in normal human cells

We investigated the earliest possible chromosome break and repair process in normal human fibroblasts irradiated with low and high LET (linear energy transfer) heavy ion radiation using the modified premature chromosome condensation (PCC) technique utilizing wortmannin (WM) during the fusion incubation period [M. Okada, S. Saito, R. Okayasu, Facilitated detection of chromosome break and repair at low levels of ionizing radiation by addition of wortmannin to G1-type PCC fusion incubation, Mutat. Res., 562 (2004) 11-17]. The initial numbers of breaks were approximately 10/cell/Gy in X-irradiated samples, followed by carbon (LET: 70 keV/microm), neon, and the number was around 5/cell/Gy in silicon (LET: 70 and 200 keV/microm) and iron (LET: 200 keV/microm) samples. If WM was not used, the initial numbers of breaks with silicon and iron were higher than those of X-rays. To quantify these data, we used initial repair ratio (IRR) defined as the number of G1 PCC breaks with WM divided by the number of breaks without WM. X-irradiation gave the maximum IRR ( approximately 2.0), while iron as well as silicon irradiation showed the minimum IRR ( approximately 1.0), suggesting almost no rejoining at the initial stage. Although there is a comparatively good correlation between the IRR value and the cell survival, the survival fraction with the repair data at 2 or 6h correlates better statistically. Our data indicate that high LET heavy ion irradiation induces a lower number of initial chromosome breaks with minimal repair when compared with low LET irradiation. These results at the chromosome level substantiate and extend the notion that high LET radiation produces complex-type DNA double strand breaks (DSBs).

Down regulation of BRCA2 causes radio‐sensitization of human tumor cells in vitro and in vivo

In order to study the role of BRCA2 protein in homologous recombination repair and radio‐sensitization, we utilized RNA interference strategy in vitro and in vivo with human tumor cells. HeLa cells transfected with small‐interfering BRCA2 NA (BRCA2 siRNA) (Qiagen) as well as negative‐control siRNA for 48 h were irradiated, and several critical end points were examined. The radiation cell survival level was significantly reduced in HeLa cells with BRCA2 siRNA when compared with mock‐ or negative‐control siRNA transfected cells. DNA double strand break repair as measured by constant field gel‐electrophoresis showed a clear inhibition in cells with BRCA2 siRNA, while little inhibition was observed in cells with negative control siRNA. Our immuno‐staining experiments revealed a significant delay in Rad51 foci formation in cells with BRCA2 siRNA when compared with the control populations. However, none of the non‐homologous end joining proteins nor the phosphorylation of DNA‐dependent protein kinase catalytic subunit was affected in cells transfected with BRCA2 siRNA. In addition, the combined treatment with radiation and BRCA2 siRNA in xenograft model with HeLa cells showed an efficient inhibition of in vivo tumor growth. Our results demonstrate down‐regulation of BRCA2 leads to radio‐sensitization mainly through the inhibition of homologous recombination repair type double‐strand break repair; a possibility of using BRCA2 siRNA as an effective radiosensitizer in tumor radiotherapy may arise. (Cancer Sci 2008; 99: 810–815)

Ionizing radiation downregulates ASPM, a gene responsible for microcephaly in humans

Microcephaly is a malformation associated with in utero exposed atomic bomb survivors and can be induced in mice by fetal exposure to ionizing radiation (IR). The pathogenesis of IR-induced microcephaly, however, has not been fully understood. Our analyses of high-coverage expression profiling (HiCEP) demonstrated that the abnormal spindle-like microcephaly associated gene (ASPM) was down-regulated in irradiated human diploid fibroblasts. ASPM was recently reported as the causative gene for MCPH-5, the most common type of congenital microcephaly in humans. Here, we show that the expression of the Aspm gene was significantly reduced by IR in various human and murine cells. Additionally, Aspm was found downregulated in the irradiated fetal mouse brain, particularly in the ventricular zones. A similar suppression was observed in the irradiated neurosphere cultures. This is the first report suggesting that the suppression of Aspm by IR could be the initial molecular target leading to the future microcephaly formation.

Amplification of glutathione-mediated oxidative stress by catalase in an aqueous solution at hyperthermal temperatures

The glutathione (GSH)-mediated superoxide (O2•−) generation in an aqueous solution and relation of hydrogen peroxide (H2O2) and effect of catalase were investigated. GSH-induced O2•− generation in hyperthermal temperatures was measured by the nitroblue tetrazolium (NBT) mehod. Heating an aqueous solution containing GSH caused superoxide from dissolved O2. H2O2 was generated simultaneously in this reaction mixture probably from the hydroperoxy radical (HO2•), which is equilibrated with O2•− in an aqueous condition, and then H2O2 consumed O2•−. Coexisting catalase in the reaction mixture, as a result, could increase O2•− generation. The catalase-exaggerated extracellular O2•− generation could give a harmful effect to living cells. This GSH-induced oxidative stress can be a part of mechanisms of hyperthermia therapy.