Abstract
To understand the impact of ionizing irradiation from diagnostics and radiotherapy on cells, we examined K+ channel activity before and immediately after exposing cells to X-rays. Already, low dose in the cGy range caused in adenocarcinoma A549 cells within minutes a hyperpolarization following activation of the human intermediate-conductance Ca2+-activated K+ channel (hIK). The response was specific for cells, which functionally expressed hIK channels and in which hIK activity was low before irradiation. HEK293 cells, which do not respond to X-ray irradiation, accordingly develop a sensitivity to this stress after heterologous expression of hIK channels. The data suggest that hIK activation involves a Ca2+-mediated signaling cascade because channel activation is suppressed by a strong cytosolic Ca2+ buffer. The finding that an elevation of H2O2 causes an increase in the concentration of cytosolic Ca2+ suggests that radicals, which emerge early in response to irradiation, trigger this Ca2+ signaling cascade. Inhibition of hIK channels by specific blockers clotrimazole and TRAM-34 slowed cell proliferation and migration in “wound” scratch assays; ionizing irradiation, in turn, stimulated the latter process presumably via its activation of the hIK channels. These data stress an indirect radiosensitivity of hIK channels with an impact on cell differentiation.
Similar content being viewed by others
References
Andersson B, Janson V, Behnam-Motlagh P, Henriksson R, Grankvist K (2006) Induction of apoptosis by intracellular potassium ion depletion: using the fluorescent dye PBF1 in a 96-well plate method in cultured lung cancer cells. Toxicol In Vitro 20:986–994
Ashcroft FM (2000) Ion Channels and Disease. Academic, Orlando
Bertrand R, Solary E, O’Connor P, Kohn KW, Pommier Y (1994) Induction of a common pathway of apoptosis by staurosporine. Exp Cell Res 211:314–321
Bortner CD, Cidlowski JA (2014) Ion channels and apoptosis in cancer. Philos Trans R Soc Lond B Biol Sci 3;369(1638):20130104
Brugnara C, Gee B, Armsby CC, Kurth S, Sakamoto M, Rifai N, Alper SL, Platt OS (1996) Therapy with oral clotrimazole induces inhibition of the Gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease. J Clin Invest 5:1227–1234
Catacuzzeno L, Fioretti B, Franciolini F (2012) Expression and role of the intermediate-conductance calcium-activated potassium channel KCa3.1 in Glioblastoma. J Signal Transduct 2012:421564
Corre I, Niaudet C, Paris F (2010) Plasma membrane signaling induced by ionizing radiation. Mutat Res 704:61–67. doi:10.1016/j.mrrev.2010.01.014
Dallaporta B, Hirsch T, Susin SA, Zamzami N, Larochette N, Brenner C, Marzo I, Kroemer G (1998) Potassium leakgage during the apoptotic degradation phase. J Immunol 160:5605–5615
Elliott JI, Higgins CF (2003) IKCa1 activity is required for cell shrinkage, phosphatidylserine translocation and death in T lymphocyte apoptosis. EMBO Rep 4:189–194
Facompré M, Wattez N, Kluza J, Lansiaux A, Bailly C (2000) Relationship between cell cycle changes and variations of the mitochondrial membrane potential induced by etoposide. Mol Cell Biol Res Commun 4:37–42
Fanger CM, Ghanshani S, Logsdon NJ et al (1999) Calmodulin mediates calcium-dependent activation of the intermediate conductance KCa channel, IKCa1. J Biol Chem 274:5746–5754
Fertig N, Blick RH, Behrends JC (2002) Whole cell patch clamp recording performed on a planar glass chip. Biophys J 82:3056–3062
Fournier C, Winter M, Zahnreich S, Nasonova E, Melnikova L, Ritter S (2007) Interrelation amongst differentiation, senescence and genetic instability in long-term cultures of fibroblasts exposed to different radiation qualities. Radiother Oncol 83:277–282
Gerlach AC, Gangopadhyay NN, Devor DC (2000) Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1. J Biol Chem 275:585–598
Ghanshani S, Wulff H, Miller MJ, Rohm H, Neben A, Gutman GA, Cahalan MD, Chandy KG (2000) Up-regulation of the IKCa1 potassium channel during T-cell activation. Molecular mechanism and functional consequences. J Biol Chem 275:37137–37149
Goeckenjan G et al (2011) Prevention, diagnosis, therapy and follow-up of lung cancer: interdisciplinary guideline of the German Respiratory Society and the German Cancer Society. Pneumologie 65:39–59
Grgic I, Eichler I, Heinau P, Si H, Brakemeier S, Hoyer J, Köhler R (2005) Selective blockade of the intermediate-conductance Ca2+-activated K+ channel suppresses proliferation of microvascular and macrovascular endothelial cells and angiogenesis in vivo. Arterioscler Thromb Vasc Biol 25:704–709
Grissmer S, Nguyen AN, Cahalan MD (1993) Calcium-activated potassium channels in resting and activated human T lymphocytes. Expression levels, calcium dependence, ion selectivity, and pharmacology. J Gen Physiol 102:601–630
Grupe M, Myers G, Penner R, Fleig A (2010) Activation of store-operated I(CRAC) by hydrogen peroxide. Cell Calcium 48:1–9. doi:10.1016/j.ceca.2010.05.005
Hammill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch clamp technique for high-resolution recording from cells and cell-free membrane patches. Pflugers Arch 391:85–100
Hille B (2001) Ion channels of excitable membranes, 3rd edn. Sinauer, Sunderland
Hulkower KI, Herber RL (2011) Cell migration and invasion assays as tools for drug discovery. Pharmaceutics 3:107–124
Iftinca M, Waldron GJ, Triggle CR, Cole WC (2001) State-dependent block of rabbit vascular smooth muscle delayed rectifier and Kv1.5 channels by inhibitors of cytochrome P450-dependent enzymes. J Pharmacol Exp Ther 298:718–728
Jang SH, Choi SY, Ryu PD, Lee SY (2011) Anti-proliferative effect of Kv1.3 blockers in A549 human lung adenocarcinoma in vitro and in vitro. Eur J Pharmacol 651:26–32
Jehle J, Schweizer PA, Katus HA, Thomas D (2011) Novel roles for hERG K+ channels in cell proliferation and apoptosis. Cell Death Dis 2:e193. doi:10.1038/cddis.2011.77
Jensen BS, Strøbaek D, Olesen SP, Christophersen P (2001) The Ca2+-activated K+ channel of intermediate conductance: a molecular target for novel treatments? Curr Drug Targets 2:401–422
Jung J-W, Hwang SY, Hwang JS, Oh ES, Park S, Han IO (2007) Ionising radiation induces changes associated with epithelial-mesenchymal transdifferentiation and increased cell motility of A549 lung epithelial cells. Eur J Cancer 437:1214–1224
Kolesnick R, Fuks Z (2003) Radiation and ceramide-induced apoptosis. Oncogene 22:5897–5906
Kunzelmann K (2005) Ion channels and cancer. J Membr Biol 205:159–173
Kuo SS, Saad AH, Koong AC, Hahn GM, Giaccia AJ (1993) Potassium-channel activation in response to low doses of gamma-irradiation involves reactive oxygen intermediates in nonexcitatory cells. Proc Natl Acad Sci U S A 90:908–912
Kuras Z, Yun YH, Chimote AA, Neumeier L, Conforti L (2012) KCa3.1 and TRPM7 channels at the uropod regulate migration of activated human T cells. PLoS ONE 7:e43859
Lang F, Stournara C (2014) Ion channels in cancer: future perspectives and clinical potential. Philos Trans R Soc Lond B Biol Sci (1638):20120108. doi:10.1098/rstb.2013.0108
Lee EL, Hasegawa Y, Shimizu T, Okada Y (2008) IK1 channel activity contributes to cisplatin sensitivity of human epidermoid cancer cells. Am J Physiol Cell Physiol 294:C1398–C1406
Li X, Ishihara S, Yasuda M, Nishioka T, Mizutani T, Ishikawa M, Kawabata K, Shirato H, Haga H (2013) Lung cancer cells that survive ionizing radiation show increased integrin α2β1- and EGFR-dependent invasiveness. PLoS ONE 8:e70905. doi:10.1371/journal.pone.0070905
McFerrin MB, Turner KL, Cuddapah VA, Sontheimer H (2012) Differential role of IK and BK potassium channels as mediatoirs of intrinsic and extrinsic apoptotic cell death. Am J Physiol Cell Physiol 294:C1070–C1078
Nagai T, Yamada S, Tominaga T, Ichikawa M, Miyawak A (2004) Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci U S A 101:10554–10559
Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279
Ouadid-Ahidouch H, Roudbaraki M, Delcourt P, Ahidouch A, Joury N, Prevarskaya N (2004) Functional and molecular identification of intermediate-conductance Ca2+-activated K+ channels in breast cancer cells: association with cell cycle progression. Am J Physiol Cell Physiol 287:C125–C134
Panyi G, Varga Z, Gáspár R (2004) Ion channels and lymphocyte activation. Immunol Lett 92:55–66
Pardo LA, Stühmer W (2014) The roles of K+ channels in cancer. Nat Rev Cancer 14:39–48. doi:10.1038/nrc3635
Pedersen KA, Schrøder RL, Skaaning-Jensen B, Strøbaek D, Olesen SP, Christophersen P (1999) Activation of the human intermediate-conductance Ca2+-activated K+ channel by 1-ethyl-2-benzimidazolinone is strongly Ca2+-dependent. Biochim Biophys Acta 1420:231–240
Pouget JP, Mather SJ (2001) General aspects of the cellular response to low- and high-LET radiation. Eur J Nucl Med 28:541–561
Remillard CV, Yuan JX (2004) Activation of K+ channels: an essential pathway in programmed cell death. Am J Physiol Lung Cell Mol Physiol 286:49–67
Saad AH, Zhou LY, Lambe EK, Hahn GM (1994) Mutagenesis in mammalian cells can be modulated by radiation-induced voltage-dependent potassium channels. Mut Res 324:171–176
Schmidt-Ullrich RK, Mikkelsen RB, Dent P, Todd DG, Valerie K, Kavanagh BD, Contessa JN, Rorrer WK, Chen PB (1997) Radiation-induced proliferation of the human A431 squamous carcinoma cells is dependent on EGFR tyrosine phosphorylation. Oncogene 15:1191–1197
Schwab A, Fabian A, Hanley PJ, Stock C (2012) Role of ion channels and transporters in cell migration. Physiol Rev 92:1865–1913. doi:10.1152/physrev.00018.2011
Shao C, Folkard M, Michael BD, Prise KM (2004) Targeted cytoplasmic irradiation induces bystander responses. Proc Natl Acad Sci U S A 101:13495–13500
Singleton KR, Will DS, Schotanus MP, Haarsma LD, Koetje LR, Bardolph SL, Ubels JL (2009) Elevated extracellular K+ inhibits apoptosis of corneal epithelial cells exposed to UV-B radiation. Exp Eye Res 89:140–151. doi:10.1016/j.exer.2009.02.023
Szabo I, Zoratti M, Gulbins E (2010) Contribution of voltage-gated potassium channels to the regulation of apoptosis. FEBS Lett 584:2049–2056
Tharp DL, Bowles DK (2009) The intermediate-conductance Ca2+ -activated K+ channel (KCa3.1) in vascular disease. Cardiovasc Hematol Agents Med Chem 7:1–11
Todd DG, Mikkelsen RB (1994) Ionizing radiation induces a transient increase in cytosolic free Ca2+ in human epithelial tumor cells. Cancer Res 54:5224–5230
Valencia-Cruz GI, Shabala L, Delgado-Enciso I, Shabala S, Bonales-Alatorre E, Pottosin II, Dobrovinskaya OR (2009) Kbg and Kv1.3 channels mediate potassium efflux in the early phase of apoptosis in Jurkat T lymphocytes. Am J Physiol Cell Physiol 297:C1544–C1553. doi:10.1152/ajpcell.00064.2009
Vermes I, Haanen C, Steffens-Nakken H, Reutellingsperger C (1995) A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immun Meth 184:39–51
Wang S, Melkoumian Z, Woodfork K, Cather C, Davidson AG, Wonderlin WF, Strobl JS (1998) Evidence for an early G1 ionic event necessary for cell cycle progression and survival in the MCF-7 human breast carcinoma cell line. J Cell Physiol 176:456–464
Wang Y, Yang H, Liu H, Huang J, Song X (2008) Effect of staurosporine on the mobility and invasivness of lung adenocarcinoma A549 cells: an in vitro study. BMC Cancer 9:174
Wild-Bode C, Weller M, Rimner A, Dichgans J, Wick W (2001) Sublethal irradiation promotes migration and invasiveness of glioma Cells: implications for radiotherapy of human glioblastoma. Cancer Res 61:2744–2750
Wonderlin W, Strobl JS (1996) Potassium channels, proliferation and G1 progression. J Membr Biol 154:91–107
Wonderlin WF, Woodfork KA, Strobel JS (1995) Changes in membrane potential during the progression of MCF-7 human mammary tumor cells through the cell cycle. J Cell Physiol 165:177–185
Wulff H, Knaus HG, Pennington M, Chandy KG (2004) K+ channel expression during B cell differentiation: implications for immunomodulation and autoimmunity. J Immunol 173:776–786
Wulff H, Miller MJ, Hansel W, Grissmer S, Cahalan MD, Chandy KG (2000) Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant. Proc Natl Acad Sci U S A 97:8151–8156
Zhou YC, Liu JY, Li J et al (2011) Ionizing radiation promotes migration and invasion of cancer cells through transforming growth factor-beta-mediated epithelial-mesenchymal transition. Int J Radiat Oncol Biol Phys 81:1530–1537. doi:10.1016/j.ijrobp.2011.06.1956
Acknowledgments
This work was supported, in part, by the HIC-for-Fair project, the ESA-IBER project, the BMBF (project 02NUK017B), and the GRK 1657. We thank Dr. Atsushi Miyawaki (RIKEN Brain Science Institute) for calcium sensor YC3.60 and Sandro Conrad and Markus Löbrich (TU Darmstadt) for technical support during X-ray experiments and Franz Rödel (Uni. Frankfurt) and Marco Durante (GSI) for useful discussions. We are also grateful to continuous support from the NanIon team.
Author information
Authors and Affiliations
Corresponding author
Supplemental material
Below is the link to the electronic supplementary material.
ESM 1
Fig. S1 shows a transcription analysis of K+ channel genes in different cell lines. Fig. S2 shows that a sham-irradiation of A549 cells has no effect on membrane conductance (a). During long recordings A549 cells show a run down (b). Fig. S3 shows that challenging A549 cells with μM concentrations of H2O2 increases the hIK conductance in A549 cells. Fig. S4 shows the typical distribution of cell cycle states of A549 cells used in this study (a,b). Data in c and d show that ionizing irradiation (1Gy) has no impact on apoptosis of A549 cells. (PDF 604 kb)
Rights and permissions
About this article
Cite this article
Roth, B., Gibhardt, C.S., Becker, P. et al. Low-dose photon irradiation alters cell differentiation via activation of hIK channels. Pflugers Arch - Eur J Physiol 467, 1835–1849 (2015). https://doi.org/10.1007/s00424-014-1601-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00424-014-1601-4