Influx of extracellular calcium participates in rituximab-enhanced ionizing radiation-induced apoptosis in Raji cells

We have previously shown that rituximab has a significant radiosensitizing effect on Raji cells in vitro. To investigate whether calcium signals participate in rituximab- and radiation-induced cell death in Raji cells, confocal laser scanning microscopy was used to detect kinetic changes in intracellular free calcium concentration ([Ca2+]i). Cell survival, the rates of apoptosis in Raji cells and the kinetics of γ-H2AX foci induction and loss were also evaluated. X-irradiation of Raji cells induced an initial increase of [Ca2+]i in both the presence and absence of extracellular calcium, followed by a decrease in [Ca2+]i over time. Rituximab enhanced both the amplitude and the duration of intracellular calcium signals in the irradiated cells. EGTA significantly inhibited radiation- or radiation/rituximab combination treatment-induced apoptosis. However, the calcium chelators EGTA and BAPTA/AM conferred no survival advantage on the irradiated cells. Furthermore, although no significant difference was seen after 1 h, the treatment of cells with a combination of irradiation and rituxiamb caused an increase of γ-H2AX foci when compared with irradiated cells after 8 h. Both EGTA and BAPTA/AM suppressed the number of γ-H2AX foci induced by either radiation or radiation combined with rituximab. Our results suggest that rituximab increases the level of [Ca2+]i in irradiated Raji cells. The entry of calcium from the extracellular space plays an essential role in [Ca2+]i-dependent radiation-induced apoptosis in Raji cells. The calcium chelators inhibited the formation of γ-H2AX foci, which are thought to prevent the activation of Ca2+/Mg2+-dependent endonucleases and subsequent DNA fragmentation. The calcium chelators most likely modulate only particular features of apoptosis and fail to change the fates of cells that are already committed to die.

► Rituximab enhanced intracellular calcium signaling in irradiated Raji cells.
► [Ca2+]i entry from extracellular space plays an essential role in [Ca2+]i-dependent radiation-induced apoptosis.
► [Ca2+]i is related to the formation of radiation-induced γ-H2AX foci.