Defective repair of radiation-induced DNA damage is complemented by a CHORI-230-65K18 BAC clone on rat chromosome 4

AbstractThe Long Evans cinnamon (LEC) rat is highly susceptible to X-irradiation due to defective DNA repair and is thus a model for hepatocellular carcinogenesis. We constructed a bacterial artificial chromosome (BAC) contig of rat chromosome 4 completely covering the region associated with radiation susceptibility. We used transient and stable transfections to demonstrate that defective DNA repair in LEC cells is fully complemented by a 200-kb BAC, CHORI-230-65K18. Further analysis showed that the region associated with radiation susceptibility is located in a 128,543-bp region of 65K18 that includes the known gene Rpn1. However, neither knockdown nor overexpression of Rpn1 indicated that this gene is associated with radiation susceptibility. We also mapped three ESTs (TC523872, TC533727, and CB607546) in the 128,543-bp region, suggesting that 65K18 contains an unknown gene associated with X-ray susceptibility in the LEC rat

gammaH2AX foci formation in x-irradiated mammalian cell lines with different radio-sensitivities

DNA double-strand breaks (DSBs) induced by ionizing radiation represent the most serious damage in cells. Immediately after the formation of DSB, Ser-139 of histone H2AX is phosphorylated at the site of DSB. The phosphorylated H2AX (gammaH2AX) foci can be observed with an immunohistochemical staining using anti-gammaH2AX antibody. It is also known that each gammaH2AX focus corresponds to one DSB and disappears when the DSB is rejoined. Using this method, we compared the amount of unrejoined DSBs among cell lines with different radiation sensitivities after X irradiation. At 30 min following 2 Gy of X-rays, the number of foci observed is similar in all cell lines studied. However, the disappearance of foci in radioresiatant colon cancer cell lines (SW-480 and CaCo-2) was faster than that in radiosensitive ones (SW-48 and LoVo). At 12 hr after X-irradiation, few foci were observed in SW-480 and CaCo-2 cells, while many foci were remained in SW-48 and LoVo cells. Furthermore, the number of focus disappeared much slower in irradiated radiosensitive AT and 180BR cells as compared to normal human fibroblasts. Similar results were obtained when cells from radiosensitive LEC rat were compared with cells from Fisher rat. Our data demonstrate that the rejoining kinetics of DSBs measured by gammaH2AX foci formation correlates with the cellular radiosensitivities.The 12th International Congress of Radiation Research(ICRR

ZDHHC8 knockdown enhances radiosensitivity and suppresses tumor growth in a mesothelioma mouse model.

Mesothelioma is an aggressive tumor caused by asbestos exposure, the incidence of which is predicted to increase globally. The prognosis of patients with mesothelioma undergoing conventional therapy is poor. Radiation therapy for mesothelioma is of limited use because of the intrinsic radioresistance of tumor cells compared with surrounding normal tissue. Thus, a novel molecular-targeted radiosensitizing agent that enhances the radiosensitivity of mesothelioma cells is required to improve the therapeutic efficacy of radiation therapy. ZDHHC8 knockdown reduces cell survival and induces an impaired G2/M checkpoint after X-irradiation in HEK293 cells. In the present study, we further analyzed the effect of the combination of ZDHHC8 knockdown and X-irradiation and assessed its therapeutic efficacy in mesothelioma models. SiRNA-induced ZDHHC8 knockdown in 211H and H2052 mesothelioma cells significantly reduced cell survival after X-irradiation. In 211H cells treated with ZDHHC8 siRNA and X-irradiation, the G2/M checkpoint was impaired and there was an increase in the number of cells with micronuclei, as well as apoptotic cells, in vitro. In 211H tumor-bearing mice, ZDHHC8 siRNA and X-irradiation significantly suppressed tumor growth, whereas ZDHHC8 siRNA alone did not. Immunohistochemical analysis showed decreased cell proliferation and induction of apoptosis in tumors treated with ZDHHC8 siRNA and X-irradiation, but not with ZDHHC8 siRNA alone. These results suggest that ZDHHC8 knockdown with X-irradiation induces chromosomal instability and apoptosis through the impaired G2/M checkpoint. In conclusion, the combination of ZDHHC8 siRNA and X-irradiation has the potential to improve the therapeutic efficacy of radiation therapy for malignant mesothelioma

Correlation with residual rH2AX and radiation susceptibility after irradiation

本研究では、細胞の放射線感受性と放射線照射の一定時間経過後に再結合されないDNA二本鎖切断（DSBｓ）の存在との間に関連があることを、γH2AXを指標として確認した。放射線照射によって生じたDSBｓ近傍のヒストンH2AXの139位のセリンがATMやATRキナーゼによってリン酸化される。リン酸化されたH2AX（γH2AX)は免疫組織染色することでフォーカスとして観察できる。放射線に対して高感受性あるいは抵抗性を示す種々のヒトがん細胞株（大腸癌、乳癌、膵臓癌由来）および正常繊維芽細胞に2GyのX線を照射し、経時的にγH2AXのフォーカス数の増減を観察することで、一定時間経過後の再結合されないDSBsの存在を調べた。実験に用いた全ての細胞株において放射線照射直後からフォーカス形成が認められ、30−60分後にその数はピークに達した。その後、放射線抵抗性細胞におけるフォーカスは速やかに消失し、12時間後にはほとんど観察されなかった。しかし、放射線高感受性細胞においては照射後12時間目においても多数のフォーカスが残存していたことから、放射線抵抗性細胞に比べてDSBsの再結合能が低下していることが示唆された。すなわち、放射線照射後の残存γH2AXの量が細胞の放射線感受性の指標として有用であることが示された。しかしながらγH2AXの定量は困難であるため、現在自動定量化を検討している。日本放射線影響学会第４６回大