It is thought that heavy-ion radiation therapy is a promising modality of cancer therapy that can deliver higher biological effects on tumors than conventional X-ray radiation therapy, and may also conserve the function of normal organs because of its excellent dose distribution.(1) Recently, excellent clinical results were reported in the treatment of lung cancer, prostate cancer, cancer of the head and neck, and malignant tumors of bone and soft tissue.(2) Heavy-ion has high linear energy transfer (LET) with increased relative biological effectiveness (RBE). However, the possible mechanism of increasing cell killing in heavyion therapy is not fully understood. Previous studies have demonstrated that the induction of cell killing of non-irradiated cells by adjacent cells that are directly irradiated by heavy-ions is considered to play an important role in radiation-induced cell death, not only in tumors but also in normal organs. This phenomenon is termed the "bystander effect", and was reported by Nagasawa and Little in 1992.(3) They reported that increased sister chromatid exchanges were observed in more than 30% of cells when less than 1% of the nuclei of cultured cells were actually traversed by an α-particle in Chinese hamster ovary cells. It has been proposed that the mediating mechanism involves secreted soluble factors, (4)(5)(6) an increase in oxidative stress,plasma membrane-bound lipid rafts, (8) and gap junctional intracellular communication (GJIC). (4,7,(9)(10)(11)(12)(13) Gap junctions are membrane channels that connect the cytoplasm of cells and allow direct exchange of small molecules and ions between adjacent cells. GJIC is essential for the maintenance of tissue homeostasis and proliferation and the regulation of embryonic development and differentiation, and it plays a key role in induction of the bystander effect. (14)(15)(16)(17) Here, bystander killing of cells by heavy-ion irradiation was investigated, focusing on the involvement of GJIC on cell survival, to clarify the underlying mechanisms of the bystander effect in a human lung cancer cell line in vitro.
Materials and MethodsCell cultures. A549 (a human lung cancer cell line with wild-type p53) was obtained from the Cell Resource Center for Biomedical Research at the Institute of Development, Aging and Cancer, Tohoku University, Japan. Cells were cultured in RPMI-1640 medium (Immuno-Biological Laboratories, Takasaki, Japan) supplemented with 10% fetal calf serum (PAA Laboratories, Pasching, Austria). Two days prior to irradiation experiments, 5 × 10 5 and 5 × 10 4 cells were inoculated into a specially designed 35-mm microbeam dish that was constructed as previously described, (18) to prepare confluent cultures and sparsely populated cultures, respectively. Unless otherwise described, all cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO 2 in air. The size of the cell nucleus and the whole cells was determined to be 14.6 ± 4.3 and 106 ± 24.9 μm 2 , respectively. Drug treatment. Prior to irradiation, cells were p...
