Purpose Radiation-induced autophagy has been shown to play two different roles, in malignant glioma (MG) cells, cytocidal or cytoprotective. to 5 after irradiation. Apoptosis measured by annexin-V staining and Western blotting of cleaved poly(ADP-ribose) polymerase exhibited relatively late appearance 3 days after irradiation that increased for up to 7 days. Blocking of pan-caspase (Z-VAD-FMK) did not affect apoptosis after irradiation, but silencing of Atg5 effectively reduced radiation-induced autophagy, which decreased apoptosis significantly. Inhibition of autophagy in Atg5 knockdown cells was shown to be beneficial for cell survival. Stable transfection of GFP-LC3 cells was observed after irradiation. Annexin-V was localized in cells bearing GFP-LC3 punctuated spots, indicating autophagy in immunofluorescence. Some of these punctuated GFP-LC3 bearing cells formed conglomerated spots and died in final phase. Conclusion These findings suggest that autophagy appears earlier than apoptosis after irradiation and that a portion of the apoptotic populace that appears later is usually autophagy-dependent. Thus, autophagy is usually a pathway to cell death after irradiation of MG cells. (circle) on 1 day after irradiation (upper, right panel). (B) Malignant glioma cells were Teriflunomide irradiated with different doses of radiation and incubated for the indicated time. Radiation-induced G2/M arrest was minimal or recovered without a significant increase of the subG1 populace in response to sublethal dose (2 Gy for all those 3 cell lines and 5 Gy for U373 and U87), but prominent G2/M arrest decreased from 1 day after irradiation and was transformed into subG1 accumulation from 3 to 7 days after irradiation with lethal dose (5 Gy or more for LN229 and 10 Gy or more for U373 and U87). These data suggest that G2/M arrest is an earlier event after irradiation than subG1 accumulation, which is usually wellknown to be suggestive of apoptosis. The decrease in G2/M transition occurred concurrently with accumulation of the subG1 populace at lethal doses, while it did not occur at 2 Gy and was repaired or accumulated as the subG1 populace at 5 Gy. 3. Radiation-induced autophagy occurs early and is dose-dependent We measured autophagy after irradiation by counting AVO made up of cells in given time frames and dose ranges (Fig. 2A). Increases in autophagy from 1 to 7 days after irradiation were exhibited at 10 and 20 Gy and a dosedependent increase in autophagy after irradiation was Teriflunomide confirmed at each designated time of measurement in all three cell lines. At 2 Gy, significant increases in AVO made up of cells were not observed any of Teriflunomide the cell lines. At 5 Gy, a discernible increase in AVO made up of cells in proportion to days after irradiation was only observed in LN229 cells. Open in a separate windows Fig. 2. Analysis of autophagy after radiation according to time and radiation dose. (A) Glioma cells were irradiated with different doses of radiation and Rabbit polyclonal to CBL.Cbl an adapter protein that functions as a negative regulator of many signaling pathways that start from receptors at the cell surface. then incubated for the indicated occasions. To measure acidic vesicular organelle (AVO), which is usually indicative of autophagy formation, cells were stained with acridine orange and analyzed quantitatively by flow cytometry. Data from three impartial experiments were combined. The error bars indicate standard error of mean. (B) Western blot analysis of LC3 according to time and radiation dose. Western blotting of LC3 generated two individual bands (an upper one of 18-kDa for LC3-bI and a lower one of 16-kDa for LC3-bII). An increase in the lower band accompanied by an increase in the ratio of the lower to the upper band is usually indicative of autophagy. Each membrane was stripped and re-blotted with -actin to confirm equal loading. (C) U373 cells stably expressing GFP-LC3 revealed an increase of LC3 dots from 5 to 10 Gy and from 3 to 5 5 days (200). Cells bearing GFP-LC3 puncta, which were quantified and averaged from 10 high-power fields, showed a dose-dependent increase at 3 days after irradiation (D) and an increase from 3 to 5 5 days after irradiation at 10 Gy (E). We checked the expression of LC3 protein, which is known to be a standard indicator of autophagy. Among two individual bands observed upon Western blot (an upper one of 18-kDa for LC3-bI and a lower one of 16-kDa for LC3-bII), the lower band representing fusion of the autophagosome to lysosome was increased according to the time after irradiation at 10 Gy (Fig. 2B, left column) and in proportion to the radiation dose given at 5 days (Fig. 2B, right column). We also checked the amount of autophagy after irradiation using stable GFP-LC3 expression Teriflunomide in U373 Teriflunomide cells. The punctuated pattern of GFP-LC3 granules was identified as an indicator of autophagy after irradiation. The number of autophagic cells, which was counted under a fluorescence microscope, increased from 3 to 5 5 days and showed a dosedependent increase at 3 days after irradiation.