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

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 (32)P- or (33)P-orthophosphate under conditions typically employed in metabolic labelling experiments. We calculate that the absorbed dose of IR delivered to a 5cm×5cm monolayer of cells incubated in 2ml media containing 1mCi of the high-energy (1.70MeV) β-particle emitter (32)P-orthophosphate for 30min is ∼1Gy IR. The absorbed dose of IR following an otherwise identical exposure to the low-energy (0.24MeV) β-particle emitter (33)P-orthophosphate is ∼0.18Gy IR. We show that low-energy β-particles emitted by (33)P induce a greater number of ionizing radiation-induced foci (IRIF) and greater ATM kinase signaling than energetic β-particles emitted by (32)P. Hence, we demonstrate that it is inappropriate to use (33)P-orthophosphate as a negative control for (32)P-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 ∼0.36Gy β-particles emitted by (33)P. We therefore propose that direct cellular exposure to (33)P-orthophosphate is an excellent means to induce and label the IR-induced, ATM kinase-dependent phosphoproteome.