Targeting TRPM2 Channels Impairs Radiation-Induced Cell Cycle Arrest and Fosters Cell Death of T Cell Leukemia Cells in a Bcl-2-Dependent Manner

Figure 6

Ca²⁺-signaling via ACA-sensitive Ca²⁺ entry contributes to IR-induced G₂/M cell cycle arrest and decreases IR-induced cell death of Jurkat cells. (a) Immunoblots from whole lysates of irradiated (0 or 5 Gy, 4 h after IR) Jurkat-vector and Jurkat-Bcl-2 cells probed against phospho-CaMKII and total CaMKII isoforms, against phospho-cdc2, and against β-actin. Cells were irradiated and postincubated in the presence of ACA (0 or 20 μM). (b, d) Histograms showing the DNA-specific PI fluorescence intensity of irradiated (5 Gy, 24 h after IR) Jurkat-vector cells pre- (0.25 h) and postincubated (24 h) with 0 μM (black) or 20 μM ACA (red) in (b) or 0 μM (black) or 20 μM clotrimazole (CLT, red) in (d). (c, e) Mean (± SE, ) percentage of irradiated (0 Gy, open bars, or 5 Gy, closed bars) and ACA- in (d) or CLZ- in (e) (both 0 or 20 μM) cotreated Jurkat-vector and Jurkat-Bcl-2 cells arrested in G₂ phase of cell cycle (upper line) or belonging to the dead cells accumulating in the subG₁ population (lower line). (f) Knock-down of TRPM2 by RNA interference mimics the effect of ACA. Electroporation with TRPM2-specific siRNA decreases the TRPM2 protein abundance in Jurkat-vector cells to about a half of that of nontargeting RNA- (nt RNA-) transfected control cells as analyzed by TRPM2 and β-actin immunoblots, 48 h after electroporation (insert, mock: electroporation without RNA). Lower line: mean (± SE, ) percentage of irradiated (0 Gy, open bars, or 5 Gy, closed bars, 24 h after IR) in G₂/M arrest (left) or in subG₁ population as analyzed by PI staining and flow cytometry as shown in (b, d). Cells were either mock-electroporated or transfected with nt RNA or TRPM2-specific siRNA. Mock and nt RNA data did not differ and were pooled. and indicate and , ANOVA, respectively.

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