J Appl Biomed 9:49-56, 2011 | DOI: 10.2478/v10136-009-0031-7

Caffeine-suppressed ATM pathway leads to decreased p53 phosphorylation and increased programmed cell death in gamma-irradiated leukaemic molt-4 cells

Aleš Tichý^1,3,*, Darina Muthná³, Jiřina Vávrová¹, Jaroslav Pejchal², Zuzana Šinkorová¹, Lenka Zárybnická¹, Martina Řezáčová³

¹ Department of Radiobiology, Faculty of Military Health Sciences in Hradec Králové, University of Defence in Brno, Czech Republic

² Centre of Advanced Studies, Faculty of Military Health Sciences in Hradec Králové, University of Defence in Brno, Czech Republic

³ Department of Medical Biochemistry, Faculty of Medicine in Hradec Králové, Charles University in Prague, Czech Republic

Ionising radiation (IR) is one of the main treatment modalities in oncology. However, we still search for substances which can radio-sensitize tumour cells. In this study we used caffeine, a non-specific ataxia-telangiectasia mutated kinase (ATM) inhibitor, and studied its effect on the activation of the proteins involved in cell cycle control and the induction of apoptosis in human T-lymphocyte leukaemic MOLT-4 cells (p53 wt). We evaluated the expression of the tumour-suppressor p53 (itself and phosphorylated on Ser¹⁵ and Ser³⁹²), the cell cycle regulator p21, and the anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1). After treatment with 2 mM caffeine, the cells were irradiated by 1 or 3 Gy, lysed and the proteins detected by Western-blotting. Apoptosis was determined by flow-cytometric annexin V/propidium iodine detection. Irradiation by 1 or 3 Gy induced p53 phosphorylation at Ser¹⁵ and Ser³⁹² after 2 h with maximum after 4 h. Adding caffeine significantly inhibited Ser¹⁵ phosphorylation, which is ATM-dependent but surprisingly also Ser³⁹² phosphorylation, which is ATM-independent, suggesting that caffeine might have another cellular target (protein kinase). Similarly, caffeine caused a substantial decrease in p21 in combination with both doses of IR and also Mcl-1 was down-regulated. Three days after irradiation, caffeine significantly increased induction of apoptosis. The ATM/p53 pathway was suppressed by caffeine, which led to increased apoptosis accompanied by a p53-independent decrease in Mcl-1. It also caused down-regulation of p21, which possibly contributed to the shortened cell cycle arrest necessary for effective DNA repair and thus impeded radio-resistance. Caffeine promotes the cytotoxic effect of ionising radiation and provides a possible platform for the development of new anti-cancer therapeutics known as radio-sensitizers.

Keywords: ATM; p53; p21; Mcl-1; caffeine; ionising radiation; MOLT-4

Received: April 20, 2010; Revised: June 9, 2010; Published: March 31, 2011  Show citation