No abstract

Messenger RNA data of lymphohematopoietic cancer lines suggest a correlation between expression of the cation channel TRPM2 and the antiapoptotic protein Bcl-2. The latter is overexpressed in various tumor entities and mediates therapy resistance. Here, we analyzed the crosstalk between Bcl-2 and TRPM2 channels in T cell leukemia cells during oxidative stress as conferred by ionizing radiation (IR). To this end, the effects of TRPM2 inhibition or knock-down on plasma membrane currents, Ca2+ signaling, mitochondrial superoxide anion formation, and cell cycle progression were compared between irradiated (0–10 Gy) Bcl-2-overexpressing and empty vector-transfected Jurkat cells. As a result, IR stimulated a TRPM2-mediated Ca2+-entry, which was higher in Bcl-2-overexpressing than in control cells and which contributed to IR-induced G2/M cell cycle arrest. TRPM2 inhibition induced a release from G2/M arrest resulting in cell death. Collectively, this data suggests a pivotal function of TRPM2 in the DNA damage response of T cell leukemia cells. Apoptosis-resistant Bcl-2-overexpressing cells even can afford higher TRPM2 activity without risking a hazardous Ca2+-overload-induced mitochondrial superoxide anion formation.

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.

K channels crosstalk with biochemical signaling cascades and regulate virtually all cellular processes by adjusting the intracellular K concentration, generating the membrane potential, mediating cell volume changes, contributing to Ca signaling, and directly interacting within molecular complexes with membrane receptors and downstream effectors. Tumor cells exhibit aberrant expression and activity patterns of K channels. The upregulation of highly "oncogenic" K channels such as the Ca-activated IK channel may drive the neoplastic transformation, malignant progression, metastasis, or therapy resistance of tumor cells. In particular, ionizing radiation in doses used for fractionated radiotherapy in the clinic has been shown to activate K channels. Radiogenic K channel activity, in turn, contributes to the DNA damage response and promotes survival of the irradiated tumor cells. Tumor-specific overexpression of certain K channel types together with the fact that pharmacological K channel modulators are already in clinical use or well tolerated in clinical trials suggests that K channel targeting alone or in combination with radiotherapy might become a promising new strategy of anti-cancer therapy. The present article aims to review our current knowledge on K channel signaling in irradiated tumor cells. Moreover, it provides new data on molecular mechanisms of radiogenic K channel activation and downstream signaling events.

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.

No abstract

Ultraviolet radiation (UV) induces a series of morphological and ultrastructural alterations in human epidermis. Alterations observed in irradiated keratinocytes in morphological studies done before were cell retraction with loss of intercellular interactions, surface blebbing, and eventually cell death by apoptosis. The aim of this study was to investigate effect of UV-A, UV-B, and UV-C irradiation on the cytoskeleton of human keratinocytes. Keratinocytes were obtained by exfoliative scrubbing procedure from buccal mucosa of healthy individuals, and treated with UV-A, UV-B, and UV-C radiation. Afterward, treated cell were labeled with anti-alfa-tubulin and anti-human-cytokeratin and analyzed by light and confocal microscopy. The intensity of the cytokeratin labeling was found to be much higher in all irradiated cells than in control cells as observed by light microscope and measured with the Image J program. This measurement showed that with the decrease in the wavelengths of UV irradiation the intensity of the labeling of cells increases. Although the authors expected to find the collapse of microtubules toward the cell nucleus or their rearrangement in UV-treated cells, these alterations were not verified on cell smears labeled with anti-alfa tubulin observed by confocal microscope. When they used electron microscopy to examine in more detail the morphology of irradiated cells, they did not find apoptotic cells, but found features of autophagy in UV-treated keratinocytes. The authors assume that autophagy found as a result of UV radiation of human keratinocytes acts as a cytoprotective mechanism against UV-induced apoptosis.

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.