Gammacell 3000 elan irradiator

The human breast cancer MCF-7 cells, the human promyelocytic leukemia HL-60 cells and the human erythroleukemic K562 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Biowest, Riverside, MO, USA), 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate (Invitrogen, Carlsbad, CA, USA). All cells were maintained in a humidified incubator containing 5% CO₂ at 37°C. To induce DNA double strand breaks, exponentially growing cells were irradiated from ¹³⁷Cs source (Gamma cell 3000 Elan irradiator, Best Theratronics, Ottawa, Canada) at different doses depending on the types of experiments and allowed to recover at 37°C. For terminal differentiation, MCF-7 cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA: Sigma), final concentration of 100 nM for 3 days. To induce macrophage and megakaryotes differentiation, HL-60 or K562 cells were treated with 32 nM TPA or 16 nM TPA for 2 days, respectively. Another differentiation reagent, Retinoic Acids (RA, Sigma) was treated to induce differentiation. MCF-7 cells were treated with 10 μM RA for 3 days, and HL60 or K562 cells were treated with 0.1 μM for 3 days or 3 μM for 4 days, respectively.

The human cervix adenocarcinoma HeLa cells, the human bone osteocarcinoma U2OS cells, the human embryonic kidney HEK293T cells, the human breast cancer BT549, MCF7, MDA-MB 231, MDA-MB 453 cells, and the human prostate cancer DU145 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL, Grand Island, NY, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Cambrex Corp., East Rutherford, NJ, USA), 100 units/mL penicillin, and 100 µg/mL streptomycin sulfate (Invitrogen, Carlsbad, CA, USA). All cells were maintained in a humidified incubator containing 5% CO₂ at 37 °C. To induce DNA double strand breaks, exponentially growing cells were irradiated from ¹³⁷Cs source (Gamma cell 3000 Elan irradiator, Best Theratronics, Ottawa, ON, Canada) at different doses depending on the types of experiments and allowed to recover at 37 °C. To inhibit Akt1 function, HeLa cells were treated with 50 μM perifosine (Sigma, St. Louis, MO, USA) for 24 h.

Human hepatoma cells (Huh7, Hep3B, SK-hep1 and PLC/PRF-5) were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 100 μg/mL penicillin and 0.25 μg/mL streptomycin (Invitrogen) and maintained in a humidified incubator at 37°C with 5% CO₂. Cells were preincubated with serum-free DMEM and irradiated at 15- Gy using a cesium-137 source delivering 3.1 Gy/min (Gammacell 3000 Elan irradiator; Best Theratronics, Ottawa, Canada). After 15- Gy irradiation, the cells were plated at the same density for the in vitro study.