IONIZING-RADIATION INDUCES A TRANSIENT INCREASE IN CYTOSOLIC-FREE [CA2-CELLS(] IN HUMAN EPITHELIAL TUMOR)

Free cytosolic [Ca2+] ([Ca2+](f)) mas measured at the single cell leve l using digitized video-intensified fluorescence microscopy and the fl uorescent Ca2+ indicator, fura-2. Cells were irradiated at 1-4 Gy (dos e rate of 6 Gy/min) with a Sr-90 eye applicator mounted on the microsc ope. HeLa cells responding to ionizing radiation exhibited a maximal 3 -4-fold transient rise in cytosolic and nuclear [Ca2+](r) 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+](r) was blocked by 1 mM La3+, 100 nM nifedipine, or membrane pote ntial depolarization, suggesting that HeLa cells possess a voltage-dep endent Ca2+ channel that mediates the response to radiation. Experimen ts 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 int racellular Ca2+ mobilization, also completely blocked the radiation ef fect, implging a linkage between the radiation-induced influx and mobi lization of internal Ca2+ stores. Not all cells in an asynchronous cul ture responded identically to radiation. Upon synchronization with thy midine/aphidicolin, cells in the S- and G(2)/M-phase exhibited radiati on-induced changes in [Ca2+](r), whereas G(1)-phase cells did not In a ddition, the increased [Ca2+](f) of irradiated G(2)/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 epithelia l 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 5 0 min. The kinetics and inhibitor sensitivities differed from HeLa cel ls, indicating a different type of mechanism for the radiation effects on cell [Ca2+](f). Survival studies with Beta and MDA-MB-231 cells di d not reveal a connection between the radiation effects on cellular Ca 2+ homeostasis and cell survival.