hERG K+ Channels Promote Survival of Irradiated Leukemia Cells

Figures

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  Figure 1. Irradiation modifies voltage-dependent hERG channels in K562 cells. (A) Current tracings recorded from a control (0 Gy, upper line) and a 5 Gy-irradiated cell (lower line). Records were obtained with K-d-gluconate pipette and bath solution during a hyperpolarizing voltage step to −100 mV delivered from pre-pulses ranging from −100 to +100 mV (5 mV increments, see insert). Deactivating inward tail currents were recorded before (control), during (E4031) and after (wash-out) of the hERG1 inhibitor E4031 (1 µM). Red line indicates zero current. (B) Relationships between mean (± SE, n = 9–16) peak current density of the deactivating tail currents at −100 mV clamp voltage and the pre-pulse voltage in control (0 Gy, left) and 5 Gy-irradiated (right) K562 cells recorded as in (A) before (open circles) and during administration of E4031 (closed triangles). (C) Relationships between mean (± SE, n = 6–9) E4031-sensitive fractions of peak current density and activating pre-pulses in control (0 Gy, black circles) and 5 Gy-irradiated (red circles) as recorded in (A). * indicates p ≤ 0.05, two-tailed (Welch-corrected) t-test.

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  Figure 2. Radiation augments in K562 cells the modulation of cytosolic free Ca2+ concentration (c[Ca2+]free) by the hERG1 inhibitor E4031 and the Kv3.4 blocker tetraethylammonium (TEA): effect of E4031 on Ca2+-regulated signaling. (A) Time course of the mean (± SE, n = 7–10) ratio between the 340 nm- and the 380 nm-excited fura-2 fluorescence as a measure of c[Ca2+]free. Ratios were recorded 2–3 h post-irradiation with 0 Gy (upper line, n = 27) and 5 Gy-irradiated (lower line) K562 cells before, during, and after administration of E4031 (1 µM, closed triangles) or TEA (open circles). (B) Mean (± SE) change in fura-2 340/380 nm ratio (Δ ratio) elicited in 0 Gy (open bars) and 5 Gy (closed bars)-irradiated cells by E4031 (n = 90–111, left) and TEA (n = 57–59, right) application as recorded in (A). (C) Immunoblots showing the abundance of phosphorylated (Thr286, first panel) and total CaMKII isoforms (second panel), phospho-(Tyr15)-cdc2 (third panel), or ß-actin (fourth panel) as loading control in total lysates of K562 cells cropped 2 h after irradiation with 0 and 5 Gy. Cells were irradiated and post-incubated in the absence or presence of the hERG-inhibitor E4031 (1 µM). (D) Mean (± SE, n…

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  Figure 3. Inhibition of hERG channels stimulates release from S phase arrest. (A) Flow cytometry dot plots showing DNA incorporation of the base analogon bromodeoxyuridine (BrdU) and DNA amount by propidium iodide staining during 8 h of incubation following irradiation with 0 Gy (1st and 2nd plot) and 5 Gy (3rd and 4th plot). Irradiation (0 and 5 Gy) and post-incubation (8 h) were carried out either in the absence (1st and 3rd plot) or presence (2nd and 4th plot) of the hERG channel inhibitor E4031 (3 µM). Cells which did not accumulate BrdU and which resided in G1 and G2 phase of cell cycle are outlined in violet and yellow, respectively. BrdU-positive cells which resided in G1, S and G2 phase of cell cycle are outlined in red, turquoise, and gray, respectively. (B, C) Mean (± SE, n = 8–9) normalized percentage (B) and radiation (5 Gy)-induced cell cycle re-distribution (mean percent points ± SE, n = 8–9) of K562 cells (C) incubated in the absence (open bars, vehicle) and presence of E4031 (closed bars) residing in the G2 low BrdU, G1 low BrdU, S, G2 high BrdU, or G1 high BrdU population as defined in (A). * indicates p ≤ 0.05, ANOVA.

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  Figure 4. hERG channels delay recovery from radiation-induced release from S and G2/M arrest in K562 cells. (A) Flow cytometry histograms depicting the propidium iodide fluorescence (Nicoletti staining) of K562 cells irradiated (0 or 5 Gy) and post-incubated (48 h) with vehicle (black) or hERG inhibitor E4031 (3 µM). (B) Time course of radiation (5Gy)-induced changes in cell cycle progression in the absence (vehicle, open circles) and presence of E4031 (3 µM, closed triangles). Shown are mean (± SE, n = 21) percentages of cells residing in G1 (left), S (middle), or G2 (right) phase of cell cycle 0, 24, 48, and 72 h after irradiation. (C) Mean percentage (± SE, n = 27) of irradiated (0 or 5 Gy) K562 cells residing 48 h after irradiation in G1 (left), S (middle), or G2 (right) phase of cell cycle. Cells were pre- and post-incubated with vehicle (open bars) or E4031 (3 µM, closed bars). * indicates p ≤ 0.05, two-tailed t-test in (B) and ANOVA in (C).

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  Figure 5. hERG channels confer radioresistance to K562 cells without promoting DNA repair. (A) Flow cytometry histograms highlighting the dead population of irradiated (0 Gy in black and 5 Gy in red) K562 cells with low (subG1) propidium iodide fluorescence (Nicoletti staining). K562 cells were irradiated (0 or 5 Gy) and post-incubated (24 h) in the absence (vehicle, left) or presence of hERG inhibitor E4031 (1 µM, right). (B) Mean (± SE, n = 9) percentage of dead (subG1) K562 cells 24 h after irradiation with 0 (left) or 5 Gy (right) and co-incubation with vehicle alone (open bars) or E4031 (1 µM, closed bars). (C) Mean (± SE, n = 12–24) survival fraction of irradiated (0, 2, 4, 6 Gy) K562 cells as determined by pre-plating colony formation assay. Cells were irradiated and post-incubated in the absence (vehicle, black) or presence of E4031 (1 µM, red). (D) Fluorescence micrographs of 0 Gy- (left) and 4 Gy-irradiated (closed symbols) K562 cells immunostained against γH2AX (green) and counterstained against DNA with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Cells were irradiated and 24 h post-incubated in the absence or presence of E4031 (3 µM) before analysis. (E, F) Mean (± SE, n =…

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  Figure 6. Primary chronic myeloid leukemia (primary CML) cells express radiation-induced hERG currents: involvement in G2/M cell cycle arrest. (A) Whole-cell deactivating tail current tracings from a control (0 Gy, left) and an irradiated primary CML cell (5 Gy, right). Records were obtained with K-D-gluconate pipette and Na-D-gluconate bath solution (insert). Square pulses to −80 mV (insert) were either delivered from a holding potential of +40 mV (black tracings) or −50 mV (blue tracings) as indicated. The current tracing in red color was recorded during administration of E4031 (3 µM). Gray lines indicate zero current. (B, C) Peak current densities of the deactivating tail current after stepping the clamp voltage to −80 mV either from a holding potential of −50 or +40 mV (B) and holding potential-dependent peak current density fractions of the deactivating tail current (C) of control (0 Gy, open symbols) or 5 Gy-irradiated primary (closed symbols) CML cells. Symbols and red lines indicate individual values (n = 7–9) and medians, respectively. (D) Flow cytometry histograms showing the propidium iodide fluorescence (Nicoletti staining) of primary CML cells that were irradiated (5 Gy) and…