Ryuichi Okayasu

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.

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.

Cytotoxicity of cigarette smoke condensate is not due to DNA double strand breaks: Comparative studies using radiosensitive mutant and wild-type CHO cells

Purpose: To determine whether cigarette smoke condensate (CSC) without metabolic activation induces direct DNA double strand breaks (DSB) in the G1 phase of various radiosensitive mutants of CHO cells and whether these breaks display collateral hypersensitivity to CSC with respect to cell killing. Materials & methods: We treated the G1-phase cultures of wild-type and DNA repair deficient mutants of CHO cells with various concentrations of CSC and examined the cell survival by colony formation assay and the induction of DNA double strand breaks by constant field gel electrophoresis as well as the phophorylated histone H2-A variant X (γ-H2AX) assay. Results: Gel analysis and γ-H2AX focus assay showed significantly fewer, but still detectable levels of DSB per cell after CSC treatment compared to ionizing radiation (IR) exposures, even when equitoxic radiation exposures were delivered at a low dose rate over the same 8-hour exposure used for CSC treatments. None of the three non-homologous end joining (NHEJ) deficient mutants were remarkably hypersensitive to CSC compared to wild-type cells. In contrast, UV-1 cells that are hypersensitive to several base damage and cross-linking agents showed a higher sensitivity to CSC compared to the other CHO cell lines. Conclusions: DNA DSB produced directly by CSC are not principally responsible for its cytotoxicity. Further, the present study does not rule out the possibility that some of these lesions may secondarily result in DSB, such as may occur during impeded DNA replication and whose repair may require systems other than NHEJ.

DNA topoisomerase inhibitor, etoposide, enhances GC-box-dependent promoter activity via Sp1 phosphorylation.

Modification of transcription factors by anticancer agents plays an important role in both apoptotic and survival signaling. Here we report that both DNA topoisomerase I and II inhibitors such as SN‐38 and etoposide, but not cisplatin, 5‐fluorouracil or actinomycin D, can induce phosphorylation of the transcription factor Sp1. Furthermore, DNA topoisomerase inhibitors were shown to transactivate GC‐box‐dependent promoters such as the SV40 and vascular endothelial growth factor promoters. The phosphorylated form of Sp1 was detectable within 30 min of etoposide treatment and was greatly diminished by the presence of the PI3K inhibitor wortmannin and by DNA‐dependent protein kinase (DNA‐PK) knockdown. We also confirmed that the phosphorylated form of DNA‐PK was increased by treatment with both etoposide and SN‐38. Taken together, these findings demonstrate a novel genomic response to anticancer agents that induce Sp1 phosphorylation, and might contribute to tumor progression and drug resistance. (Cancer Sci 2007; 98: 858–863)