ISSN 1214-0287 (on-line), ISSN 1214-021X (printed)
J Appl Biomed
Volume 9 (2011), No 1, p 49-56
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

Ales Tichy, Darina Muthna, Jirina Vavrova, Jaroslav Pejchal, Zuzana Sinkorova, Lenka Zarybnicka, Martina Rezacova

Address: Ales Tichy, Department of Radiobiology, Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic
tichy@pmfhk.cz

Received 20th April 2010.
Revised 9th June 2010.
Published online 5th November 2010.

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SUMMARY
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.

KEY WORDS
ATM; p53; p21; Mcl-1; caffeine; ionising radiation; MOLT-4

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CITED

Novotna E, Tichy A, Pejchal J, Lukasova E, Salovska B, Vavrova J. DNA-dependent protein kinase and its inhibition in support of radiotherapy. Int J Rad Biol. 89: 416-423, 2013.

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