As we had previously shown that EGFR activation was required for IR induced Akt activation, we next tested to whether EGFR signaling modulated radioresist ance sellckchem in U87 cells. To do this, we pre treated U87MG cells with the EGFR Inhibitors,Modulators,Libraries inhibitor AG1478, then treated them with various doses of IR and performed clonogenic survival assays. As depicted in Fig. 3C, inhibition of EGFR had the expected effect of enhancing the radiosensitivity of U87 cells, consistent with its effect on IR induced Akt activa tion. Genetic inhibition of PI3K signaling enhances the radiosensitivity of U87MG cells In addition to abolishing PI3K activity, LY294002 has been reported to inhibit other PI3K like kinases, such as mTOR, DNK, and ATM. These kinases play impor tant roles in IR induced DNA damage repair, and mTOR regulates the PI3K Akt signaling pathway at multi ple levels.

As such, it remained possible that the effect of LY294002 on radiosensitivity was independent of its effect on PI3K signaling. Therefore, a genetic approach was used to specifically modulate PI3K Akt activation and determine the effect on radiosensitivity. U87MG cells have mutant PTEN genes, leading to a high Inhibitors,Modulators,Libraries level of Akt phosphorylation. To modulate Akt acti vation in these cells, genetically modified versions of U87MG cells harboring tetracycline inducible wild type or mutant PTEN transgenes were studied. In the absence of doxycycline treatment, both cell lines expressed little PTEN protein and high levels of phospho Akt.

Treatment for 24 hr with doxycycline induced robust expression of Inhibitors,Modulators,Libraries both wild type and mutant PTEN, and only the induction of wild type Inhibitors,Modulators,Libraries PTEN led to a significant decrease in Akt phosphorylation, confirming that functional PTEN is required for inhibiting Akt activation in GBM. Next, these U87MG clones were treated with or without doxycycline for Inhibitors,Modulators,Libraries 24 hr, followed by radiation treatment. 4 hr after IR, the cells were trypsinized and subjected to clo nogenic survival assays. As shown in Fig. 4B, expression of wild type but not mutant PTEN enhanced the radiosensi tivity of U87MG cells. This result is consistent with the results from LY294002 as well as reports from Jiang et al, and confirms that IR induced Akt activation contrib utes to the radioresistance of U87MG cells. Pharmacological inhibition of Akt enhances the radiosensitivity of U87MG cells Next, we used Akt inhibitors to directly inhibit IR induced find FAQ Akt activation, and assessed the effect on radiosensitivity. Two different Akt inhibitors, SH 5 and MK 2206 were tested. InhibitioncellsPI3K Akt signaling with PI3K inhibitor LY294002 or EGFR inhibitor AG1478 increases the radiosensitivity of Inhibition of PI3K Akt signaling with PI3K inhibitor LY294002 or EGFR inhibitor AG1478 increases the radio sensitivity of U87MG cells. A.