Objective: Activation of ATM-kinase (ATM) is involved in cell cycle arrest and reparation of ionizing radiation (IR)-induced DNA damage. Caffeine, a nonspecific inhibitor of ATM, shortens IR-induced G2 arrest. It was shown to inhibit ATM/Chk-2/p53 signalling pathway and to diminish DNA reparation of IRinduced double strand breaks. In this work we studied effect of ATM inhibition on activation of other proteins involved in induction of apoptosis and cell cycle control in human T-lymphocyte

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... mic MOLT-4 cells (p53 wild type). We evaluated expression of tumour-suppressor p53 (itself and phosphorylated at Ser-15 and Ser-392) and p21 (a cell cycle regulator). Methods: We added 2mM caffeine 30 min prior irradiation ( 60 Co) and washed out after 24 h. The cells were irradiated by the doses of 1 or 3 Gy, lysed and the proteins were separated by SDS-PAGE and detected by Western-blotting. Apoptosis (annexin V detection) and DNA content were determined by flow-cytometry. Results: Protein p53 was up-regulated 2 h after irradiation by the doses of 1 and 3 Gy with maximum after 16 h. After 24 h its amount decreased. Both doses of IR induced phosphorylation at both Ser-15 and Ser-392 after 2 h with maximum after 4 h. Adding caffeine significantly inhibited both p53 phosphorylations. Similarly, p21 was up-regulated 4 h after irradiation by the doses of 1 and 3 Gy with maximum after 24 h. Caffeine caused a substantial decrease of p21 in combination with both doses of IR. Three days after irradiation caffeine significantly potentiated induction of apoptosis. Conclusion: Caffeine significantly inhibited ATM/Chk-2/p53 signalling pathway in MOLT-4 cells, which via decreased expression of p21 led to inhibition of cyclin-dependent kinases. This resulted in shortened cell cycle arrest (necessary for effective DNA reparation) and induction of apoptosis. Therefore we conclude that ATM activity inhibitors such as caffeine have a radio-sensitising effect and could be exploited in radio-therapy as radio-sensibilisators. Acknowledgement: The authors acknowledge the financial support of Ministry of Education of Czech Republic (project no. MSM 0021620820) and Ministry of Defence of Czech Republic (project no. MO0FVZ0000501).