FIGURE 1. PI3K/Akt pathway inhibition radiosensitizes U251MG glioblastoma cells.

A, clonogenic survival assays indicate radiosensitization of U251MG after pharmacologic inhibition of PI3K/Akt. U251MG cells were seeded as single cell suspensions. Three hours after seeding, LY294002 (10 μM) or vehicle only was added and then followed 1 h later by mock-irradiation or exposure to ionizing radiation at the indicated doses. After 24 h of exposure, the LY294002 was removed and the cells left to grow undisturbed for 7–10 days; the resultant colonies were stained and counted. B, induction of wild-type or mutant PTEN. U251MG cells engineered to be inducible for either wild-type PTEN or phosphatase-dead PTEN (C124S) were exposed to doxycycline (1 μg/ml). After 24 h, cells from each group were harvested and the resultant lysates immunoblotted for the proteins indicated. Doxycycline-induced PTEN protein diminishes activated Akt as indicated by reduced levels of Akt phosphorylated at serine 473. C, radiosensitization of U251MG after induction of wild-type PTEN. Clonogenic assays performed on U251-PTEN in the presence or absence of doxycycline. Cells were exposed to doxycycline or vehicle for 24 h, followed by reseeding onto fresh plates and mock-irradiation or irradiation with the indicated doses. D, lack of radiosensitization with induction of mutant PTEN. Clonogenic assays performed on U251 cells engineered to be inducible for catalytically inactive PTENC124S in the presence or absence of doxycycline. Cells were exposed to doxycycline or vehicle for 24 h, followed by reseeding onto fresh plates and mock-irradiation or irradiation with the indicated doses. After 4 h to allow for attachment, they were irradiated. Doxycycline was removed after 24 h. Survival fraction is plotted on the y-axis versus dose of radiation on the x-axis. All surviving fractions with radiation were normalized to these base-line plating efficiencies.