Temporally distinct response of irradiated normal human fibroblasts and their bystander cells to energetic heavy ions

ヒト正常線維芽細胞への重粒子線照射効果とバイスタンダー効果

浜田 信行*; Ni, M.*; 舟山 知夫; 坂下 哲哉; 小林 泰彦

Hamada, Nobuyuki*; Ni, M.*; Funayama, Tomoo; Sakashita, Tetsuya; Kobayashi, Yasuhiko

ヒト正常線維芽細胞における重粒子線照射後誘導蛋白質を定量的ウェスタンブロット法によって解析した。Cイオン(220MeV)又はNeイオン(260MeV)をD相当線量、すなわちCでは1.3Gy(7.610p/cm)、Neでは1.9Gy(2.710p/cm)をブロードビームで照射した細胞では、p53蛋白質Ser15部位でのリン酸化レベルが、照射後2時間と6時間とで変化せず高いレベルを保持していた。一方、高密度培養した細胞シャーレ内の125か所に1か所あたり10粒子照射した場合の、細胞シャーレ内の圧倒的多数をしめるバイスタンダー細胞では、照射後2時間よりも6時間で上昇することがわかった。この結果は、直接の重イオン照射細胞と非照射のバイスタンダー細胞における分子応答機構の違いを示唆している。

To characterize the energetic heavy ion-induced bystander response, we employed precise microbeams of C and Ne ions for targeting only a very small fraction of cells in confluent fibroblast cultures. Conventional broad field irradiation was conducted in parallel to see the effects in irradiated cells. Exposure of 0.00026% of cells led to nearly 10% reductions in the clonogenic survival and twofold rises in the apoptotic incidence regardless of ion species. Whilst apoptotic frequency increased with time up to 72 h post irradiation in irradiated cells, its frequency escalated up to 24 h post irradiation but declined at 48 h post irradiation in bystander cells, indicating that bystander cells exhibit transient commitment to apoptosis. C and Ne-ion microbeam irradiation similarly caused almost twofold increments in the levels of serine 15-phosphorylated p53 proteins, irrespective of whether 0.00026, 0.0013 or 0.0066% of cells were targeted. Whereas the levels of phosphorylated p53 were elevated and remained unchanged at 2 and 6 h post irradiation in irradiated cells, its levels rose at 6 h post irradiation but not at 2 h post irradiation in bystander cells, suggesting that bystander cells manifest delayed p53 phosphorylation.