The ATM kinase signaling induced by the low-energy β-particles emitted by 33P is essential for the suppression of chromosome aberrations and is greater than that induced by the energetic β-particles emitted by 32P

Abstract

Ataxia-telangiectasia mutated (ATM) encodes a nuclear serine/threonine protein kinase whose activity is increased in cells exposed to low doses of ionizing radiation (IR). Here we examine ATM kinase activation in cells exposed to either 32P- or 33P-orthophosphate under conditions typically employed in metabolic labelling experiments. We calculate that the absorbed dose of IR delivered to a 5 cm × 5 cm monolayer of cells incubated in 2 ml media containing 1 mCi of the high-energy (1.70 MeV) β-particle emitter 32P-orthophosphate for 30 min is ∼1 Gy IR. The absorbed dose of IR following an otherwise identical exposure to the low-energy (0.24 MeV) β-particle emitter 33P-orthophosphate is ∼0.18 Gy IR. We show that low-energy β-particles emitted by 33P induce a greater number of ionizing radiation-induced foci (IRIF) and greater ATM kinase signaling than energetic β-particles emitted by 32P. Hence, we demonstrate that it is inappropriate to use 33P-orthophosphate as a negative control for 32P-orthophosphate in experiments investigating DNA damage responses to DNA double-strand breaks (DSBs). Significantly, we show that ATM accumulates in the chromatin fraction when ATM kinase activity is inhibited during exposure to either radionuclide. Finally, we also show that chromosome aberrations accumulate in cells when ATM kinase activity is inhibited during exposure to ∼0.36 Gy β-particles emitted by 33P. We therefore propose that direct cellular exposure to 33P-orthophosphate is an excellent means to induce and label the IR-induced, ATM kinase-dependent phosphoproteome.