Tichý Ales, Záskodová Darina, Rezácová Martina, Vávrová Jirina, Vokurková Doris, Pejchal Jaroslav, Vilasová Zdena, Cerman Jaroslav, Osterreicher Jan
Department of Radiobiology, Faculty of Military Health Sciences in Hradec Králové, University of Defence in Brno, Czech Republic.
Acta Biochim Pol. 2007;54(2):281-7. Epub 2007 Jun 12.
ATM kinase (ATM) is essential for activation of cell cycle check points and DNA repair in response to ionizing radiation (IR). In this work we studied the molecular mechanisms regulating DNA repair and cell death in human T-lymphocyte leukemic cells, MOLT-4. Apoptosis was evaluated by flow-cytometric detection of annexin V. Early apoptotic cells were determined as sub-G1 cells and late apoptotic cells were determined as APO2.7-positive ones. Proteins involved in ATM signalling pathway were analysed by Western-blotting. We observed a rapid (0.5 h) phosphorylation of ATM declining after 6 h after irradiation by all the doses studied (1.5, 3.0, and 7.5 Gy). Checkpoint kinase-2 (Chk-2) was also phosphorylated after 0.5 h but its phosphorylated form persisted 4, 2, and 1 h after the doses of 1.5, 3.0, and 7.5 Gy, respectively. The amount of p53 protein and its form phosphorylated on Ser-392 increased 1 h after irradiation (1-10 Gy). The lethal dose of 7.5 Gy caused an immediate induction and phosphorylation of p53 after 0.5 h post-irradiation. At the time of phosphorylation of p53, we found simultaneous phosphorylation of the oncoprotein Mdm2 on Ser-166. Neither ATM nor its downstream targets showed a dose-dependent response after 1 h when irradiated by the doses of 1-10 Gy. MOLT-4 cells were very sensitive to the effect of IR. Even low doses, such as 1.5 Gy, induced apoptosis 16 h after irradiation (evaluated according to the cleavage of nuclear lamin B to a 48-kDa fragment). IR-induced molecular signalling after exposure to all the tested doses was triggered by rapid phosphorylation of ATM and Chk-2. Subsequent induction of p53 protein and its phosphorylation was accompanied by concomitant phosphorylation of its negative regulator, oncoprotein Mdm2, and followed by induction of apoptosis.
ATM激酶（ATM）对于响应电离辐射（IR）激活细胞周期检查点和DNA修复至关重要。在这项工作中，我们研究了调节人T淋巴细胞白血病细胞MOLT-4中DNA修复和细胞死亡的分子机制。通过膜联蛋白V的流式细胞术检测评估细胞凋亡。早期凋亡细胞被确定为亚G1期细胞，晚期凋亡细胞被确定为APO2.7阳性细胞。通过蛋白质印迹分析ATM信号通路中涉及的蛋白质。我们观察到，在所研究的所有剂量（1.5、3.0和7.5 Gy）照射后，ATM迅速（0.5小时）磷酸化，并在6小时后下降。检查点激酶2（Chk-2）在0.5小时后也被磷酸化，但其磷酸化形式在1.5、3.0和7.5 Gy剂量照射后分别持续4、2和1小时。p53蛋白的量及其在Ser-392位点磷酸化的形式在照射后1小时（1-10 Gy）增加。7.5 Gy的致死剂量在照射后0.5小时导致p53立即诱导和磷酸化。在p53磷酸化时，我们发现癌蛋白Mdm2在Ser-166位点同时磷酸化。当用1-10 Gy剂量照射1小时后，ATM及其下游靶点均未显示出剂量依赖性反应。MOLT-4细胞对IR的作用非常敏感。即使是低剂量，如1.5 Gy，也会在照射后16小时诱导细胞凋亡（根据核纤层蛋白B裂解为48 kDa片段进行评估）。暴露于所有测试剂量后，IR诱导的分子信号由ATM和Chk-2的快速磷酸化触发。随后p53蛋白的诱导及其磷酸化伴随着其负调节因子癌蛋白Mdm2的磷酸化，随后诱导细胞凋亡。
