As shown in Figure 6A, ABT 263 increased the phosphorylation of GSK 3B

Further more, activation of NF κB by expression of constitutively ac tive IKK and IKKB reduced the cleavage of caspase 3 and PARP augmented by GsQL, indicating in hibition of NF κB activity augments radiation induced apoptosis. Next, the effect of radiation and Gs on NF κB activation was examined. Radiation increased nu clear translocation of NF kB p50 and p65 with a peak at 2 h ARQ 197 availability after irradiation, and the expression of GsQL reduced the radiation induced translocation of p50 and p65. Then, the effect on NF κB dependent promoter activity was analyzed. Radiation slightly increased NF κB dependent promoter activity, and the expression of GsQL reduced the promoter ac tivity until 24 h after irradiation. Next, the role of ATM in NF κB activation was assessed.<br><br> Inhibition of ATM activation by treatment with an ATM inhibitor, KU55933, or by knockdown with siRNA reduced the NF κB dependent promoter activity before and 2 h after irradi ation. Activation of ATM by pretreatment with chloroquine abolished the reducing effect of GsQL on NF κB dependent promoter activity. The ex pression AZD0530 ic50 of GsQL also reduced the NF κB activity before and after irradiation in A549 lung cancer cells. These results suggest that Gs augments radiation induced apoptosis by reducing ATM dependent activa tion of NF κB in lung cancer cells. To probe the mechanism how ATM activate NF kB after irradiation, we determined the effect of Gs on the level of phosphorylated ATM in the cytosol, where IκB is located and degraded following phosphorylation.<br><br> Al though most of the phosphorylated ATM is localized in the nucleus, a small amount of phosphorylated ATM in the cytosol could be visualized after ray irradiation by exposing blots to the gel documentation system for AMN-107 641571-10-0 a longer period of time. Ray irradiation increased the amount of phosphorylated ATM in the cytosol, and GsQL expression decreased the amount of phosphorylated ATM in the cytosol following irradiation. This result indicates that Gs reduced the translocation of phosphory lated ATM into the cytosol, which might decrease phos phorylation and degradation IκB protein and reduce activation of NF κB in H1299 lung cancer cells. Prostaglandin E2 and isoproterenol affected ATM activation and apoptosis similarly to Gs To confirm the effects observed upon GsQL expres sion, we analyzed the effects of prostaglandin E2 and isoproterenol, two agonists for Gs coupled receptors.<br><br> Pretreatment with prostaglandin E2 and isoproterenol increased the phosphorylation of PP2A B56 and de creased ATM phosphorylation following ray irradi ation. Pretreatment with prostaglandin E2 decreased NF κB luciferase activity 12 h after irradiation and the activity was not recovered until 24 h after irradi ation. Isoproterenol treatment showed a similar inhibitory effect on radiation induced NF κB dependent promoter activity. The inhibitory effect of prostaglandin E2 and isoproterenol on ATM phosphorylation was abol ished by treatment with a PKA inhibitor, H 89. Prostaglandin E2 or isoproterenol treat ments also enhanced the cleavage of caspase 3 and PARP and increased the proportion of early apoptotic H1299 cells. Moreover, treatment with prostaglandin E2 significantly decreased survival of the irradiated cells.