M-FISH Analysis of Chromosome Aberrations in Human Fibroblast Cells After In Vitro Exposure to Low- and High-LET Radiation

The recently commercialized multiplex fluorescence in situ hybridization (m-FISH) technique, which allows human chromosomes to be painted in 24 different colors, was used to analyze chromosome aberrations in diploid human fibroblast cells after in vitro radiation exposure. Confluent flasks of a normal primary fibroblast cell line (AG 1522) were irradiated at high dose rates with either gamma rays or 200 MeV/nucleon Fe ions (LET = 440 keV/micron), incubated at 37 C for 24 hours after exposure, and subsequently subcultured. A chemically induced premature chromosome condensation technique was used to collect chromosome samples 32 hours after subculture. Results showed that the fraction of exchanges which were identified as complex, i.e. involving misrejoining of three or more DSB, were higher in the Fe-irradiated samples compared with the gamma-irradiated samples, as has been shown previously using FISH with one or two painted chromosomes . The ratios of complex/simple type exchanges were similar for samples irradiated with 0.7 Gy and 3 Gy of Fe ions, although exchanges involving five or more breaks were found only in 3 Gy irradiated samples. The fraction of incomplete exchanges was also higher in Fe- than gamma-irradiated samples. Data on the distribution of individual chromosome involvement in interchromosomal exchanges will be presented

Analysis of Heavy Ion-Induced Chromosome Aberrations in Human Fibroblast Cells Using In Situ Hybridization

Confluent human fibroblast cells (AG1522) were irradiated with gamma rays, 490 MeV/nucleon Si, or with Fe ions at either 200 or 500 MeV/nucleon. The cells were allowed to repair at 37 0 C for 24 hours after exposure, and a chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Unrejoined chromosomal breaks and complex exchanges were analyzed in the irradiated samples. In order to verify that chromosomal breaks were truly unrejoined, chromosome aberrations were analyzed using a combination of whole chromosome specific probes and probes specific for the telomere region of the chromosome. Results showed that the frequency of unrejoined chromosome breaks was higher after high-LET radiation, and consequently, the ratio of incomplete to complete exchanges increased steadily with LET up to 440 keV/micron, the highest LET value in the present study. For samples exposed to 200 MeV/nucleon Fe ions, chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique that allows identification of both complex and truly incomplete exchanges. Results of the mFISH study showed that 0.7 and 3 Gy dose of the Fe ions produced similar ratios of complex to simple exchanges and incomplete to complete exchanges, values for which were higher than those obtained after a 6 Gy gamma exposure. After 0.7 Gy of Fe ions, most complex aberrations were found to involve three or four chromosomes, indicating the maximum number of chromosome domains traversed by a single Fe ion track.

Equivalency of the quality of sublethal lesions after photons and high-linear energy transfer ion beams

The quality of the sublethal damage (SLD) after irradiation with high–linear energy transfer (LET) ion beams was investigated with low-LET photons. Chinese hamster V79 cells and human squamous carcinoma SAS cells were first exposed to a priming dose of different ion beams at different LETs at the Heavy Ion Medical Accelerator in the Chiba facility. The cells were kept at room temperature and then exposed to a secondary test dose of X-rays. Based on the repair kinetics study, the surviving fraction of cells quickly increased with the repair time, and reached a plateau in 2–3 h, even when cells had received priming monoenergetic high-LET beams or spread-out Bragg peak beams as well as X-ray irradiation. The shapes of the cell survival curves from the secondary test X-rays, after repair of the damage caused by the high-LET irradiation, were similar to those obtained from cells exposed to primary X-rays only. Complete SLD repairs were observed, even when the LET of the primary ion beams was very high. These results suggest that the SLD caused by high-LET irradiation was repaired well, and likewise, the damage caused by the X-rays. In cells where the ion beam had made a direct hit in the core region in an ion track, lethal damage to the domain was produced, resulting in cell death. On the other hand, in domains that had received a glancing hit in the low-LET penumbra region, the SLD produced was completely repaired

Induction of Chromatin Damage and Distribution of Isochromatid Breaks in Human Fibroblast Cells Exposed to Heavy Ions

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p53 変異型ヒト口腔がん細胞における高LET 放射線によるp53 非依存Akt 生存シグナルの抑制

Although mutations and deletions in the p53 tumor suppressor gene lead to resistance to low linear energy transfer (LET) radiation, high-LET radiation efficiently induces cell lethality and apoptosis regardless of the p53 gene status in cancer cells. Recently, it has been suggested that the induction of p53-independent apoptosis takes place through the activation of Caspase-9 which results in the cleavage of Caspase-3 and poly (ADP-ribose) polymerase (PARP). This study was designed to examine if high-LET radiation depresses serine/threonine protein kinase B (PKB, also known as Akt) and Akt-related proteins. Human gingival cancer cells (Ca9-22 cells) harboring a mutated p53 (mp53) gene were irradiated with 2 Gy of X-rays or Fe-ion beams. The cellular contents of Akt-related proteins participating in cell survival signaling were analyzed with Western Blotting 1, 2, 3 and 6h after irradiation. Cell cycle distributions after irradiation were assayed with flow cytometric analysis. Akt-related protein levels decreased when cells were irradiated with high-LET radiation. High-LET radiation increased G(2)/M phase arrests and suppressed the progression of the cell cycle much more efficiently when compared to low-LET radiation. These results suggest that high-LET radiation enhances apoptosis through the activation of Caspase-3 and Caspase-9, and suppresses cell growth by suppressing Akt-related signaling, even in mp53 bearing cancer cells.博士（医学）・甲第598号・平成25年3月15日Copyright © 2012 Elsevier Inc. All rights reserve

Relative biological effectiveness (RBE) and potential leathal damage repair (PLDR) of heavy-ion beam

150KV X線,中性子線及び炭素(LET13, 20, 50, 90, 140, 150, 153, 200keV/μm)を照射したマウスNIH3T3細胞の生存率曲線のLD(10)から(60)Coγ線に対する生物学的効果比(RBE)を求めた｡RBEは150KV X線では1.26,中性子線では2.44,炭素線(LET13, 20, 50, 90, 140, 150, 153, 200keV/μm)ではそれぞれ1.41, 1.47, 2.22, 2.61, 1.61, 2.05, 1.57であった｡LETとRBEの関係では100keV/μm付近にピークを認めた｡150KVX線のLETは13keV/μm,中性子線のLETは70keVμmに相当した｡(60)Co γ線の潜在性致死損傷からの回復(PLDR)は大きかった｡炭素線(13keV/μm)照射でもPLDRが観察されるがLETが大きくなるとPLDRは減少したが,LET90keV/μmの炭素線でもPLDRが認められた｡照射時の細胞状態の検討では増殖期の細胞の感受性は定常期細胞に比し僅かに高かった｡Relative biological effectiveness (RBE) and repair of potential lethal damage (PLDR) of NIH3T3 cells against heavy-ion radiation were studied. RBE of 150 KV X-rays and neutron estimated from LD(10) dose of dose response survival curves compared to (60)Co γ-ray were 1.26 and 2.44, respectively. RBE of 13, 20, 50, 90, 140, 150, 153, 200 keV/μm of LET of carbon beam were 1.41, 1.47, 2.22, 2.61, 2.61, 1.61, 2.05 and 1.57, respectively. Potential lethal damage repair (PLDR) after exposure to carbon beam was observed. The magnitude of PLDR of (60)Co γ-ray was the biggest. As for the carbon beam of LET of 13 keV/μm as well, PLDR were observed. PLDR decreased when LET of carbon beam grew big

Biological background of carbon ion radiation therapy

NIRS-KI Joint Symposium on Carbon Ion Therap