Non-selective cation channel-mediated Ca2+-entry and activation of Ca2+/calmodulin-dependent kinase II contribute to G2/M cell cycle arrest and survival of irradiated leukemia cells.
Genotoxic stress induces cell cycle arrest and DNA repair which may enable tumor cells to survive radiation therapy. Here, we defined the role of Ca(2+) signaling in the cell cycle control and survival of chronic myeloid leukemia (CML) cells subjected to ionizing radiation (IR). To this end, K562 erythroid leukemia cells were irradiated (0-10 Gy). Tumor survival was analyzed by clonogenic survival assay and cell cycle progression via flow cytometry. Plasma membrane cation conductance was assessed by patch-clamp whole-cell recording and the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) was measured by fura-2 Ca(2+) imaging. Nuclear activity of Ca(2+)/calmodulin-dependent kinase II (CaMKII) was defined by Western blotting. In addition, the effect of IR (5 Gy) on the cation conductance of primary CML cells was determined. The results indicated that IR (10 Gy) induced a G(2)/M cell cycle arrest of K562 cells within 24 h post-irradiation (p.i.) and decreased the clonogenic survival to 0.5 % of that of the control cells. In K562 cells, G(2)/M cell cycle arrest was preceded by activation of TRPV5/6-like nonselective cation channels in the plasma membrane 1-5 h p.i., resulting in an elevated Ca(2+) entry as evident from fura-2 Ca(2+) imaging. Similarly, IR stimulated a Ca(2+)-permeable nonselective cation conductance in primary CML cells within 2-4 h p.i.. Ca(2+) entry, into K562 cells was paralleled by an IR-induced activation of nuclear CaMKII. The IR-stimulated accumulation in G(2) phase was delayed upon buffering [Ca(2+)](i) with the Ca(2+) chelator BAPTA-AM or inhibiting CaMKII with KN93 (1 nM). In addition, KN93 decreased the clonogenic survival of irradiated cells but not of control cells. In conclusion, the data suggest that IR-stimulated cation channel activation, Ca(2+) entry and CaMKII activity participate in control of cell cycle progression and survival of irradiated CML cells.
Department of Physiology, University of Tübingen, Tübingen, Germany.
Nicole
N
Heise
Daniela
D
Palme
Milan
M
Misovic
Saisudha
S
Koka
Justine
J
Rudner
Florian
F
Lang
Helmut R
HR
Salih
Stephan M
SM
Huber
Guido
G
Henke
D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002455:Cell Division; D002614:Chelating Agents / Q000737:chemistry / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D016257:Fura-2 / Q000737:chemistry / Q000494:pharmacology; D016195:G2 Phase; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000201:enzymology / Q000378:metabolism* / Q000532:radiotherapy; D018408:Patch-Clamp Techniques; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011836:Radiation Tolerance; D011839:Radiation, Ionizing; D050916:TRPV Cation Channels / Q000378:metabolism*
D015220:Calcium Channels; D002614:Chelating Agents; D047428:Protein Kinase Inhibitors; D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; C422827:TRPV6 protein, human; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium; D016257:Fura-2