Inhibition Expression Of ATM By Wortmannin Enhances The Radiation Sensitivity Of Human Malignant Glioma Cells

Brain glioma is one of the most common intracranial tumor. Glioma has the features of invasive growth, no definite border from the normal brain tissue, a tendency to recur after operation, and cannot totally remove by operations. So postoperative radiotherapy is being a major therapeutic method to it. However, the total therapeutic efficacy is not satisfactory because of the radio-resistant ability of brain glioma and limit of the normal brain tissue tolerance dose of radiation. Therefore, it is imminent to find a new way to increase the radiation sensitivity of glioma. Recently, gene therapy combined with radiosensitization has become one of focus researches, and its aim is to increase the sensitivity of tumor cells to X- rays by up-regulation or inhibition expression of target genes. Now, it has been reported that ATM gene is close correlate with the radiation sensitivity, so our study is to observe the change of radiation sensitivity in human malignant glioma cells(U251)by altering the expression of ATM.ATM is an important member of the PI-3K family, participates function regulation of many genes associated with tumors, such as p53 and p21, and correlates with cell cycle and apoptosis. WT as the specific inhibitor of the PI-3K family can inhibit the expression and activity of ATM. So our study used WT of higher concentration (≥10μmol/L) to treat U251 cells and then detected the content of ATM by immunofluorescence staining and RT-PCR. After this treatment of WT, U251 cells were exposed to X-irradiation and cell cycle was detected. The proliferations of U251 cells were evalued by colony-forming assay and PCNA immunofluorescence staining, apoptosis and necrosis were investigated by Hoechst 33258 fluoroassay kit and Annexin V-EGFP apoptosis kit, the morphous of U251 cells apoptosis was observed by transmission electron microscope, the changes of cytoskeleton were viewed byβ-actin immunofluorescence staining, and caspase-3 as a gene associated with apoptosis was detected by immunocytochemical stain. From above assays, we evalued the change of radiation sensitivity of U251 cells.The results were as follow:①The immunofluorescence assay showed that the ATM expression was weak in control group, the ATM expression of U251 cells was increased when cells were exposed to X-irradiation and there was significant differences between this two groups (P<0.05). After treatment with WT, ATM expression was lower than that in alone irradiated group. However, The ATM expression of U251 cells after treatement with WT and without irradiation was similar to that of control group. Our data suggested that WT could not regulated of ATM expression alone, and inhibited ATM expression only when ATM was activated. The changes of ATM mRNA detected by RT-PCR were in accordance with the results above.②The results of cell cycle showed that U251cells in WT plus irradiation group were more arrested in G2/M phase, and cells in S phase were increased obviously than the control group(P<0.05).③In the colony forming assay, we found that the clone forming rates of WT (10μmol/L,20μmol/L) plus irradiation groups were lower than that of control group and the alone irradiation group(P<0.05). The clone forming rate of 20μmol/L WT combined with irradiation (10Gy) was only 6.63%. The reaults illustrated that WT inhibited ATM activity of U251 cells, and obviously increased the lethal effect of X-irradiation to cells.④The results of PCNA immunofluorescence assay showed that the PCNA expression was strong in control group, and weaken in alone irradiation group, and lowest in WT (10μmol/L,20μmol/L) plus irradiation groups. There was significant differences among the four groups (P<0.05), except the difference between 10μmol/L WT and 20μmol/L WT plus irradiation group. This mean that WT could decreas ATM expression and enhance the inhibitory effect of X-irradiation to proliferation of U251 cells.⑤In the apoptosis assay, we investaged that the numbers of apoptosis and necrosis cells were increased significantly in WT plus irradiation groups, and were increased to about 1.5 times as compared with the alone irradiation group. These data indicated that down-regulation of ATM expression by WT could increase radiation sensitivity of U251 cells and apoptosis and necrosis growed in number obviously. There were more U251 cells that showed typical ultrastructure morphous characteristic of apoptosis and necrosis in WT plus irradiation groups by transmission electron microscope.⑥The cytoskeleton staining byβ-actin showed amoebocytes in WT plus irradiation groups were increased, microfilaments arranged more disorderly, fluorescence enhanced in perinuclear and cell membrane of U251 cells and parts of cells became aggregate. That demonstrated in WT plus irradiation groups, microfilaments of U251 cells were depolymerized, cytoskeleton changed obviously, and cells would appear functional impairment and apoptosis would happened at last.⑦The results of caspase-3 immunocytochemical stain showed that the caspase-3 expression was weaken in control group, increased in alone irradiation group, and was most strong in WT plus irradiation group. The results manifested that more caspase-3 in more U251 cells were activated when cells were irradiated after treatment by WT and then caspase-3 priminged the process of apoptosis by many ways.In summary, the expression of ATM could be activated by X-irradiation in human malignant glioma cell(sU251). However, WT as the specific inhibitor of the PI3K family can inhibit the activity of ATM. Therefore, when U251 cells were exposed to X-irradiation, WT could lead to the change of cell cycle, the inhibition of cells proliferation, the increasing of apoptosis, and enhancement of radiation sensitivity by down-regulation of ATM expression in U251 cells The study on relation of ATM gene and radiation sensitivity is expected to provide a new target and theoretical foundation for the clinical therapy of glioma.