Figure 3

The effects of S44563 on tumor cell radiosensitivity. (a) Effect of S44563 on SCLC cells radiosensitivity. The SCLC cells were seeded for colony formation in 35 mm dishes containing methylcellulose-based medium at 500–10 000 cells/dish with various doses of S44563 for 21–28 days period. Radiation was given 2 h after incubating cells in methylcellulose-based medium containing S44563. Upon intervals of 21–28 days, colonies were manually counted using microscopy. All colonies of 50 cells or more were then counted. The survival fraction (SF) was estimated according to the formula: SF=number of colonies formed/number of cells seeded plating efficiency of the control group. Experiments were performed in triplicate (points, mean; bars, standard deviation, *P<0.05 as compared with unirradiated cells). The figure is representative of four independent experiments. (b) Effect of S44563 on radiation-induced sub-G1 fraction. Radiation was given at a dose of 6 Gy 2 h after giving S44563 at a dose of 100 nmol/l for H146 cells. Cells were harvested after 72 h exposure to S44563 and stained with a propidium iodide solution for 24 h. Stained nuclei were analyzed for DNA-propidium iodide fluorescence using a Becton Dickinson FACScan flow cytometer. Resulting DNA distributions were analyzed by Modfit (Verity Software House, Inc.) for the proportion of cells in sub-G₀, G₁, and G₂-M phases of the cell cycle (columns, mean; bars, standard deviation, *P<0.05 as compared with untreated cells). Results from two experiments were combined. (c) The H146 cells were treated as experiment 3B and immunoblotting for cleaved caspase 3 was performed. The ratio of cleaved caspase 3/GAPDH was measured using dedicated software. The combination of radiation and S44563 results in 2.5-fold enhancement of cleaved caspase 3/GAPDH ratio