Ionizing radiation induces a transient increase in cytosolic free [Ca2+] in human epithelial tumor cells

Free cytosolic [Ca2+] ([Ca2+]f) was measured at the single cell level using digitized video-intensified fluorescence microscopy and the fluorescent Ca2+ indicator, fura-2. Cells were irradiated at 1-4 Gy (dose rate of 6 Gy/min) with a 90Sr eye applicator mounted on the microscope. HeLa cells responding to ionizing radiation exhibited a maximal 3-4-fold transient rise in cytosolic and nuclear [Ca2+]f immediately upon irradiation, which persisted for at least 5 min before returning to baseline in most but not all cells. The radiation-induced rise in [Ca2+]f was blocked by 1 mM La3+, 100 nM nifedipine, or membrane potential depolarization, suggesting that HeLa cells possess a voltage-dependent Ca2+ channel that mediates the response to radiation. Experiments with Mn2+, a paramagnetic probe for extracellular Ca2+, showed that radiation stimulated an increase in Mn2+ influx, as witnessed by loss of fura-2 fluorescence. Thapsigargin and ryanodine, inhibitors of intracellular Ca2+ mobilization, also completely blocked the radiation effect, implying a linkage between the radiation-induced influx and mobilization of internal Ca2+ stores. Not all cells in an asynchronous culture responded identically to radiation. Upon synchronization with thymidine/aphidicolin, cells in the S- and G2/M-phase exhibited radiation-induced changes in [Ca2+]f, whereas G1-phase cells did not. In addition, the increased [Ca2+]f of irradiated G2/M phase cells did not fully return to pretreatment levels. Further studies utilizing MDA-MB-231 and MCF-7 human breast and HT-29 human colon cancer cell lines indicate that radiation can alter Ca2+ homeostasis in other epithelial cell types. In the case of MDA-MB-231 and HT-29 cells, oscillations in cytosolic [Ca2+]f levels were observed that persisted for up to 50 min. The kinetics and inhibitor sensitivities differed from HeLa cells, indicating a different type of mechanism for the radiation effects on cell [Ca2+]f. Survival studies with HeLa and MDA-MB-231 cells did not reveal a connection between the radiation effects on cellular Ca2+ homeostasis and cell survival.