No abstract

Genotoxic stress induces cell cycle arrest and DNA repair which may enable tumor cells to survive radiation therapy. Here, we defined the role of Ca²⁺ 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 Ca²⁺ concentration ([Ca²⁺]_i) was measured by fura-2 Ca²⁺ imaging. Nuclear activity of Ca²⁺/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 G₂/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, G₂/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 Ca²⁺ entry as evident from fura-2 Ca²⁺ imaging. Similarly, IR stimulated a Ca²⁺-permeable nonselective cation conductance in primary CML cells within 2-4 h p.i.. Ca²⁺ entry, into K562 cells was paralleled by an IR-induced activation of nuclear CaMKII. The IR-stimulated accumulation in G₂ phase was delayed upon buffering [Ca²⁺]_i with the Ca²⁺ 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, Ca²⁺ entry and CaMKII activity participate in control of cell cycle progression and survival of irradiated CML cells.

Aberrant ion channel expression in the plasma membrane is characteristic for many tumor entities and has been attributed to neoplastic transformation, tumor progression, metastasis, and therapy resistance. The present study aimed to define the function of these "oncogenic" channels for radioresistance of leukemia cells. Chronic myeloid leukemia cells were irradiated (0-6 Gy X ray), ion channel expression and activity, Ca(2+)- and protein signaling, cell cycle progression, and cell survival were assessed by quantitative reverse transcriptase-polymerase chain reaction, patch-clamp recording, fura-2 Ca(2+)-imaging, immunoblotting, flow cytometry, and clonogenic survival assays, respectively. Ionizing radiation-induced G2/M arrest was preceded by activation of Kv3.4-like voltage-gated potassium channels. Channel activation in turn resulted in enhanced Ca(2+) entry and subsequent activation of Ca(2+)/calmodulin-dependent kinase-II, and inactivation of the phosphatase cdc25B and the cyclin-dependent kinase cdc2. Accordingly, channel inhibition by tetraethylammonium and blood-depressing substance-1 and substance-2 or downregulation by RNA interference led to release from radiation-induced G2/M arrest, increased apoptosis, and decreased clonogenic survival. Together, these findings indicate the functional significance of voltage-gated K(+) channels for the radioresistance of myeloid leukemia cells.

Many tumor cells express highly elevated activities of voltage-gated K + channels in the plasma membrane which are indispensable for tumor growth. To test for K + channel function during DNA damage response, we subjected human chronic myeloid leukemia (CML) cells to sub-lethal doses of ionizing radiation (0-8 Gy, 6 MV photons) and determined K + channel activity, K + channel-dependent Ca 2+ signaling, cell cycle progression, DNA repair, and clonogenic survival by whole-cell patch clamp recording, fura-2 Ca 2+ imaging, Western blotting, flow cytometry, immunofluorescence microscopy, and pre-plating colony formation assay, respectively. As a result, the human erythroid CML cell line K562 and primary human CML cells functionally expressed hERG1. Irradiation stimulated in both cell types an increase in the activity of hERG1 K + channels which became apparent 1-2 h post-irradiation. This increase in K + channel activity was paralleled by an accumulation in S phase of cell cycle followed by a G 2 /M cell cycle arrest as analyzed between 8 and 72 h post-irradiation. Attenuating the K + channel function by applying the hERG1 channel inhibitor E4031 modulated Ca 2+ signaling, impaired inhibition of the mitosis promoting subunit cdc2, overrode cell cycle arrest, and decreased clonogenic survival of the irradiated cells but did not affect repair of DNA double strand breaks suggesting a critical role of the hERG1 K + channels for the Ca 2+ signaling and the cell cycle control during DNA damage response.