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

Radiosynthesis and preclinical characterization of a 11C-labeled aminolevulinicacid

"Radiosynthesis and preclinical characterization of a 11C-labeled aminolevulinic acid derivative as an imaging probe to estimate tumor protoporphyrin IX accumulation induced by exogenous aminolevulinic acid"Objective: Fluorescence-guided resection, photodynamic therapy, and sonodynamic therapy for tumors using tumor-specific accumulation of protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) administration are promising therapeutic strategies. Pretreatment noninvasive imaging to estimate the quantity and spatial distribution of ALA induced PpIX accumulation in tumors is expected to improve the efficacy of ALA-based tumor therapies. PpIX synthesis from exogenous ALA has been reported to be regulated by ALA influx and/or ALA dehydratase (ALAD) activity which catalyzes the first step of the synthesis. In this study, we radiosynthesized a 11C-labeled ALA analog, 5-amino-4-oxo-[6-11C]hexanoic acid ([11C]MALA) and characterized its in vitro and in vivo properties as a positron emission tomography (PET) probe to estimate PpIX accumulation.Materials and methods: [11C]MALA was synthesized via 11C-methylation of a Schiff-base-activated precursor in the presence of tetrabutylammonium fluoride, followed by the hydrolysis of ester and imine groups. In vitro uptake of [11C]MALA and [3H]ALA were determined in five tumor cell lines (AsPC-1, Sk-Br-3, U-87 MG, BxPC-3, and MIA PaCa-2) after 10 min incubation with each tracer at 37°C. The expression levels of ALAD were determined by western blot analysis. Tumor-bearing mice were intravenously injected with the mixture of [11C]MALA and [3H]ALA, and 11C and 3H radioactivity in tissues were measured. Dynamic PET scans were performed for 90 min just after injection of [11C]MALA. In vitro and in vivo accumulation of ALA-induced PpIX was determined by measuring fluorescence in extracts of cells or tumors. Results and conclusions: [11C]MALA was obtained in a radiochemical yield (decay uncorrected, referred to [11C]carbon dioxide) of 4.4 ± 1.7% using remote-controlled synthesis module within 35 min from the end of bombardment. In vitro uptake of [11C]MALA in five tumor cell lines was correlated with ALAD expression levels and PpIX accumulation. In vivo biodistribution and dynamic PET studies showed that [11C]MALA was rapidly incorporated into tumors and the tumor-to-muscle ratio of [11C]MALA at 1 min after injection was significantly correlated with that of [3H]ALA. [11C]MALA in tumors was continuously decreased thereafter, and the elimination rate of [11C]MALA from AsPC-1 tumors with the highest ALAD expression level was slower compared with those from other tumors with lower expression levels. These results suggest that the influx and intracellular retention of [11C]MALA reflect ALA influx and ALAD expression levels, respectively. Tumor accumulation of [11C]MALA at 60 min post-injection was strongly correlated with PpIX accumulation in tumor tissues. In conclusion, PET imaging with [11C]MALA has the potential to noninvasively estimate the quantitative and spatial accumulation of exogenous ALA-induced PpIX.11th Congress of the World Federation of Nuclear Medicine and Biology

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

A Loss of Function Screening for Radiation Susceptibility Genes

　Carcinogenesis is thought to be a multistep process that occurs through the accumulation of mutations in multiple genes required for the maintenance of normal growth control. Genomic instability is considered the earliest cellular event in the process of carcinogenesis. Although genomic instability is induced by ionizing radiation, the molecular mechanisms underlying this process are poorly understood. Cell cycle checkpoints play a key role in cell survival following DNA damage. The failure of DNA repair and cell cycle regulation in response to DNA damage are thought to be important factors in the early stages of genomic instability. Several known genes are reportedly associated not only with DNA damage repair and cell cycle regulation but also with radiation susceptibility. Thus, the identification of novel radiation susceptibility genes will likely help elucidate the molecular mechanisms underlying DNA damage–induced genomic instability.　By the large-scale expression profiling of 15 human cell lines and three mouse strains having varying degrees of susceptibility to ionizing radiation, we identified 200 genes correlated with radiation susceptibility. We constructed the shRNA library of these 200 genes and screened this library using a 96-well format and measuring the cell survivals, as determined by a sulforhodamine B–based cell proliferation assay, after X-irradiation. We identified 12 genes involved in cellular proliferation after irradiation. Three genes, ATM, ATR and CDKN1A, were known to be radiation susceptibility genes and other nine genes have not been reported to be associated with radiation susceptibility. Eight of the 12 genes were reported to directly or potentially regulate cell cycle, and the remaining four genes have not been reported biological functions. We then performed cell cycle analysis of cells transfected with shRNA vectors against thee four genes following X-irradiation, and found that knockdown cells of one gene did not accumulate at G2/M phase. This result suggests that one gene is associated with G2/M checkpoint after DNA damage. Further study of these 12 genes would help elucidate the molecular mechanism for genomic instability and carcinogenesis, and develop novel drugs for effective tumor radiotherapy and early diagnosis.１３ｔｈ International Congress of Radiation Reserc

A loss of function screen identifies nine new radiation susceptibility genes

Genomic instability is considered a hallmark of carcinogenesis, and dysfunction of DNA repair and cell cycle regulation in response to DNA damage caused by ionizing radiation are thought to be important factors in the early stages of genomic instability. We performed cell-based functional screening using an RNA interference library targeting 200 genes in human cells. We identified three known and nine new radiation susceptibility genes, eight of which are linked directly or potentially with cell cycle progression. Cell cycle analysis on four of the genes not previously linked to cell cycle progression demonstrated that one, ZDHHC8, was associated with the G(2)/M checkpoint in response to DNA damage. Further study of the 12 radiation susceptibility genes identified in this screen may help to elucidate the molecular mechanisms of cell cycle progression, DNA repair, cell death, cell growth and genomic instability, and to develop new radiation sensitizing agents for radiotherapy

Down regulation of ZDHHC8 enhances radiosensitivity of human mesothelioma in vitro and in vivo

Malignant mesothelioma is highly lethal and the prognosis following therapy is very poor. The effect of radiation monotherapy for mesothelioma is reported to be minimal. To improve the efficacy of radiotherapy, a novel molecular-targeted radiosensitizing agent is needed to increase radiosensitivity of mesothelioma cells. We have previously identified ZDHHC8 as a novel radiation susceptibility gene based on the functional screening in human cells. In this study, we analyzed the effect of ZDHHC8 knockdown with radiation on mesothelioma cells and assessed the therapeutic efficacy in mouse model. In mesothelioma cells, knockdown of ZDHHC8 by siRNA significantly reduced cell survival after irradiation, induced the impairment of G2/M checkpoint, and increased the frequency of cells with micronuclei and apoptosis. In the mouse model, the treatment with ZDHHC8 siRNA and irradiation significantly suppressed tumor growth and increased apoptosis, whereas ZDHHC8 siRNA alone did not. These results suggest that ZDHHC8 knockdown with X-irradiation induced chromosomal instability and cell death including apoptosis through the defects of G2/M checkpoint, and the combination of ZDHHC8 depletion and irradiation has the potential to be effective therapy for malignant mesothelioma.14th International Congress of Radiation Research(ICRR’2011