Th AZD6244 revealed no evidence of redistribution in the radiosensitive phases of the cell cycle. Treatment with AZD6244 has entered Born a smaller percentage AS-1404 DMXAA of cells in G2 / M cell cycle compared to cells treated with vehicle alone. Another m Possible source of radiosensitization is the abolition of the G2 checkpoint-induced cell death as compared to radiation protection. The analysis by flow cytometry of phosphorylated histone H3 in the 4N cell population at different times after irradiation was used to cells in the G2 and M phases to distinguish the cell cycle. This test provides a measure for the progression of cells in the G2 and M and the activation of the G2 checkpoint. As shown in Figure 3B, irradiation has entered Born a rapid reduction in the mitotic index reached a maximum decrease of 3 hours, which the early G2 checkpoint activation.
AZD6244 treatment prevents the decrease in mitotic index after irradiation, suggesting that AZD6244 treatment of the monitored station canceled The early G2. No difference in the mitotic index was known in A549 cells at 24 and 48 h after irradiation with 4 Gy Chk1 way that judges are involved in G2 checkpoint activation and radiation response. We observed an abolition of G2 arrest after irradiation in cells treated with AZD6244. Therefore, we evaluated the phosphorylation of Chk1 in treated cells irradiated with me Trise vehicle or AZD6244. Treatment with AZD6244 has entered Born eingeschr Nkter Chk1 phosphorylation after irradiation to that in cells with vehicle-treated patients observed in the comparison.
Additionally, treatment with AZD6244 reduces the expression of the entire Chk1 protein in non-irradiated cells as compared to with Tr hunter non-treated irradiated cells. Davies et al. an increase of caspase 3 is activated, one of the main effector of apoptosis in a xenograft model after treatment with AZD6244. To assess the contribution of apoptosis to the AZD6244-mediated radiosensitization of cancer cells, membrane Ver Changes were in early stages of apoptosis in cells destined to be defined at 24, 48 and 72 hours after irradiation. As shown in Figures 5A and B, there was a significant h Higher apoptosis in both the radiation and the treatment are compared with AZD6244 to untreated controls, was the degree of apoptosis in measured AZD6244 and the combination less than additive RT in both cell lines A549 and MiaPaCa2.
Thus shows enhance the combination of AZD6244 and RT-radiation-induced death in 1 had no effect on the rate of cell death by apoptosis. These data show that AZD6244-mediated radiosensitization of A549 cells does not imply a significantly elevated Hte sensitivity to apoptosis. The observation that cells not treated with AZD6244 in G2 arrest after irradiation, suggesting that mitotic catastrophe may be a mechanism for increased Hte cell death after treatment with radiation and AZD6244 have. To test if mitotic catastrophe to be responsible k Can for the decreased clonogenic survival in A549 cells with AZD6244 and RT, the number of cells with abnormal nuclei as a function of time was treated after the irradiation achieved. Cells that differentiate a mitotic catastrophe clearly on the individual treatment of IR and AZD6244, and the combination. As in