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Gliomas are morbid brain tumors that are extremely resistant to available chemotherapy and radiology treatments. Some studies have suggested that calcium-activated potassium channels contribute to the high proliferative potential of tumor cells, including gliomas. However, other publications demonstrated no role for these channels or even assigned them antitumorogenic properties. In this work we characterized the expression and functional contribution to proliferation of Ca2+-activated K+ channels in human glioblastoma cells. Quantitative RT-PCR detected transcripts for the big conductance (BK), intermediate conductance (IK1), and small conductance (SK2) K+ channels in two glioblastoma-derived cell lines and a surgical sample of glioblastoma multiforme. Functional expression of BK and IK1 in U251 and U87 glioma cell lines and primary glioma cultures was verified using whole-cell electrophysiological recordings. Inhibitors of BK (paxilline and penitrem A) and IK1 channels (clotrimazole and TRAM-34) reduced U251 and U87 proliferation in an additive fashion, while the selective blocker of SK channels UCL1848 had no effect. However, the antiproliferative properties of BK and IK1 inhibitors were seen at concentrations that were higher than those necessary to inhibit channel activity. To verify specificity of pharmacological agents, we downregulated BK and IK1 channels in U251 cells using gene-specific siRNAs. Although siRNA knockdowns caused strong reductions in the BK and IK1 current densities, neither single nor double gene silencing significantly affected rates of proliferation. Taken together, these results suggest that Ca2+-activated K+ channels do not play a critical role in proliferation of glioma cells and that the effects of pharmacological inhibitors occur through their off-target actions.
Citation: Abdullaev IF, Rudkouskaya A, Mongin AA, Kuo Y-H (2010) Calcium-Activated Potassium Channels BK and IK1 Are Functionally Expressed in Human Gliomas but Do Not Regulate Cell Proliferation. PLoS ONE 5(8): e12304. doi:10.1371/journal.pone.0012304
Editor: Masato Asanuma, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan
Received: May 21, 2010; Accepted: July 25, 2010; Published: August 20, 2010
Copyright: © 2010 Abdullaev et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by AMC grant 206-465247 (to AAM and YHK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Gliomas are primary brain tumors that arise from glial cells. They represent 30 to 60% of CNS primary tumors, with the incidence of 2 to 3 new cases per 100,000 persons annually [1], [2]. Based on their histopathological characteristics, gliomas are classified as grades I through IV, with higher grades being more de-differentiated and malignant [3], [2]. Grade IV gliomas, also known as glioblastoma multiforme (GBM), have a very high proliferative potential, are invasive and resistant to currently available therapies [3], [4]. Only 30% of GBM patients survive one year, and the average life expectancy remains approximately 14–18 months even with maximal therapy including gross total surgical resection followed by chemotherapy and radiation therapy [5], [6]. Despite medical advances over the past 30 years, there has not been a significant impact on GBM patient survival [4]. Therefore, a better understanding GBM biology is needed in order to develop novel therapeutic treatments for this disease.
There is extensive literature suggesting that proliferation of normal and transformed animal cells requires activity of certain potassium (K+) channels (see for example [7], [8], [9], [10]). K+ channels are thought to facilitate progression through cell cycle checkpoints, likely via modulation of membrane potential (reviewed in [11], [12]). Particularly, progression through the G1/S checkpoint in many cell types is associated with increased K+ channel activity and transient hyperpolarization [12]. Conversely, G2/M transition may involve depolarization and be associated with increased Cl− currents [12]. On the other hand, a number of studies suggest that concomitant modulation of K+ and Cl− channel activity may lead to cell cycle-dependent changes in cell volume (reviewed in [13], [14]).
One group of K+ channels that may be relevant to proliferation of both malignant and non-malignant cells are the Ca2+-activated K+ channels ([14], see also references below). These channels are subdivided into big conductance (large or maxi-) K+ channels (BK), intermediate conductance K+ channels (IK1), and small conductance K+ channels (SK) [15]. Each of these sub-classes can be discriminated based on their biophysical and pharmacological properties. BK channels, also known as SLO1, are broadly expressed in various tissues but have particular functional significance for membrane potential regulation in excitable and exocrine cells [15], [16]. The pore-forming subunit of BK is encoded by the KCNMA1 gene. BKs are voltage-gated channels that are activated at high positive potentials with a large unitary conductance of 100–300 pS. Increases in the intracellular Ca2+ concentration to the micromolar levels strongly shift the voltage-dependence of BK to more negative potentials [16]. Paxilline, penitrem A, iberiotoxin, and low concentrations of tetraethyl ammonium are potent and specific inhibitors of BK [16]. IK1 channels, also known as KCa 3.1 or SK4, are the product of the KCNN4 gene. They are expressed in a variety of tissues and play diverse physiological roles [15], [17]. Unlike BK, IK1 channels are strictly Ca2+ dependent as their channel-forming subunit is constitutively associated with calmodulin [18]. Once activated by cytosolic Ca2+, they show an intermediate single channel conductance of 33–42 pS [17]. Clotrimazole and its derivative TRAM-34 are potent inhibitors of IK1, discriminating it from other Ca2+-activated K+ channels [19], [17].
Prior studies have implicated BK channels in the proliferation and migration of glioblastoma cells [20], [21], [22]. BK has also been associated with growth control in cervical, ovarian, prostate, and breast cancer derived cell lines [23], [24], [25]. However, a number of other studies contradict these findings and suggest that BK channels are not required for proliferation or even have antitumorogenic properties, including in glioma cells [26], [27], [28], [29]. In addition to BK, intermediate conductance Ca2+-activated K+ channels (IK1) have also been proposed to regulate growth rate in numerous types of malignant and non-malignant mammalian cells [30], [31], [32], [33], [34], [35]. More importantly, the inhibitor of the IK1 clotrimazole strongly reduces tumor load and metastases in vivo in several cancer types including GBM [36], [37], [38]. It should be noted, however, that some studies question the functional expression of IK1 in GBM cells [22].
Because of the uncertain functional significance of BK and IK channels in glioblastoma proliferation, in the present study we used a combination of pharmacological and molecular biology tools to explore their functional significance. We found that pharmacological inhibitors of both BK and IK1 strongly suppress glioma cell growth in an additive fashion. However, low concentration of the same blockers that were sufficient to inhibit channel activity had no effect on cell proliferation. To address this discrepancy, we downregulated BK and IK1 channels using gene-specific siRNAs. siRNA transfections caused strong reductions in K+ current densities but no changes in cell growth. These data argue against a critical role for BK and IK1 in GBM proliferation.
Materials and Methods
Clotrimazole, paxilline, penitrem A, 8,14-Diaza-1,7(1,4)-diquinolinacyclotetradecaphane trifluoroacetate salt (UCL1848), and all other salts and chemicals were from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted. 1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) was purchased from Tocris Biosciences (Ellisville, MO, USA). Stock solutions of paxilline (20 mM), penitrem A (20 mM), clotrimazole (30 mM), and TRAM-34 (30 mM) were prepared in DMSO and stored at −30°C. UCL1848 was diluted in water at 20 mM.
Cell cultures
Two established astrocytoma/glioblastoma cell lines, U251 MG and U87 MG, were used in this study. U251 cells were a gift of Dr. M.G. Kaplitt (Weill-Cornell Medical Center, New York, USA); their exact passage is unknown. U87 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and used at passages 78–90. Both U251 and U87 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin, and 50 mg/ml streptomycin at 37°C in a humidified atmosphere containing 95% air and 5% CO2. All cell culture reagents are from Invitrogen-Gibco (Carlsbad, CA, USA). Culture media was changed twice a week and cells were passaged using recombinant protease TrypLE when 90–95% confluency was reached.
Primary glioblastoma cells were prepared from a surgical sample of glioblastoma multiforme. Tissue sample was obtained with written consent under a protocol approved by Albany Medical Center Institutional Review Board. Tumor tissue (∼200–300 mg) was washed twice with ice-cold Ca2+-, Mg2+-free phosphate-buffered saline (PBS, pH 7.4), minced to small pieces and treated with solution of 0.125% Trypsin/0.015% EDTA in PBS containing 250 µg/ml DNAse I. After brief digestion, tissue fragments were triturated using fire-polished glass Pasteur pipette, and the resulting cell suspension was filtered through a Nylon cell strainer (70 µm, BD Falcon, Bedford, MA, USA). Cell were grown in T75 cell culture flasks in DMEM plus 20% FBS supplemented with 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in a humidified atmosphere containing 95% air and 5% CO2.
Analysis of gene expression in tumor samples and cultured cells
Expression of various Ca2+-activated K+ channels in cultured cells and the surgical GBM tissue sample was analyzed using quantitative RT-PCR. Cellular or tissue mRNA was isolated using the RNAqueous-4PCR kit (Applied Biosystems-Ambion, Austin, Texas, USA) according to the manufacturer's instructions. Cultured cells were grown in a 60-mm dish to 60–80% confluency and lyzed in 700 µl of lysis buffer. The GBM tumor sample (∼100–150 mg) was homogenized in 1 ml lysis buffer provided with the RNAqueous-4PCR kit. Concentration of mRNA in resulting samples was quantified using NanoDrop 1000 (ThermoFisher Scientific, Wilmington, DE, USA). mRNA was converted to cDNA using the iScript cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's instructions. One µg of mRNA was used in each 20-µl cDNA synthesis reaction mix.
Gene expression was quantified by a real-time PCR using the CFX96 Real-Time PCR Detection System (Bio-Rad) and iTaq SYBR Green Supermix kit (Bio-Rad) according to the manufacturer's instructions. One µl of cDNA product prepared as described above was used for each qPCR reaction. Gene expression was analyzed with the gene-specific QuantiTect Primer Assays (Qiagen, Hilden, Germany; see Table 1 for the list of primers). Two housekeeping genes, GAPDH and RPL13A, were used as internal reference.
Western blot analysis
Protein expression of the IK1 channel was assessed by the Western blot analysis using polyclonal antibody raised against synthetic peptide corresponding to a region of human IK1 (LYDLQQNLSSSHRALEKQIDTLAGKLDALTELLSTALGPRQLPEPSQQSK, Sigma-Aldrich; cat.# AV35098). Whole cell lysates were diluted with a reducing Laemmli buffer. Proteins were separated on 10% polyacrylamide gel followed by transfer onto an Immun-Blot PDVF membrane (Bio-Rad). The membrane was blocked for 1 hr with 5% nonfat milk in Tris-Phosphate buffer containing 0.05% Tween 20 (TBS-T). It was further incubated overnight at 4°C with primary antibody (1∶500 dilution). After five washes for 5 min with TBS-T, membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare/Amersham Biosciences, Piscataway, NJ; 1∶10,000 dilution), followed by four TBS-T washes. The horseradish peroxidase signal was detected and digitized using a chemiluminescence ECLplus reagent (GE Healthcare/Amersham Biosciences) and a luminescent image analyzer LAS-4000 (FujiFilm Medical Systems, Stamford, CT).
BK and IK1 currents were recorded employing conventional whole-cell patch clamp technique [39]. Ion currents were recorded using an Axopatch-200B amplifier (Molecular Devices, Sunnyvale, CA, USA). Patch pipettes were prepared from borosilicate glass (Warner Instruments, Hamden, CT, USA) using a P-97 micropipette puller (Sutter Instruments, Novato, CA, USA). Pipette resistances were 2.5–4 MOm when filled with a solution containing (in mM): 135 KCl, 2 MgSO4, 10 HEPES, 2.5 ATP-Na, 0.1 EGTA (pH = 7.2, adjusted with KOH). Bath solution contained (in mM): 140 NaCl, 5 KCl, 3 CaCl2, 1.2 MgSO4, 10 HEPES, 10 glucose (pH = 7.4, adjusted with NaOH). In experiments to detect IK1 currents, calcium concentration in pipette solution was increased to 750 nM by adding CaCl2. Free Ca2+ levels were calculated using CaBuf software (G. Droogmans, KU Leuven, Leuven, Belgium). In all experiments series resistance was <15 MOm.
BK currents were recorded from a holding potential of −80 mV in response to step pulses of −80 mV to +140 mV in 20 mV increments. BK current development and inhibition was monitored by applying repetitive (every 3 sec) depolarization ramps from −120 mV to +140 mV. Because the pipette solution is nominally Ca2+-free, other Ca2+ activated K+ channels, such as IK1 and SK1-3 are silent under these experimental conditions.
IK1 currents were recorded from a holding potential of −80 mV in response to step pulses from −120 mV to +80 mV in 20 mV increments. Development and inhibition of the Ca2+-dependent IK1 currents were monitored by applying repetitive (every 3 sec) depolarization ramps from −120 mV to +80 mV. Since these conditions also favor activation of BK currents, 2 µM paxilline was added to bath solution to isolate the IK1 currents.
Cell proliferation assays
Cell proliferation rates were quantified using two approaches: Coulter counter technique and colorimetric MTT proliferation assay. U251 and U87 cells were plated in 24-well cell culture plates (TPP, Trasadingen, Switzerland, Europe) at the density of 10,000 cells per well and were allowed to grow overnight. The next day, culture media was replaced with serum-containing or serum-free media including the various pharmacological inhibitors or vehicle controls. Initial cell density was quantified at this time in a separate "baseline" plate prepared in the same fashion. We used two types of culture media in pharmacological experiments: serum-containing (DMEM plus 10% FBS), or serum-free medium prepared using OptiMEM with B27 supplement (Invitrogen; 1∶50 dilution). To preclude the potential degradation of inhibitors, we replaced inhibitor-containing media with freshly prepared every 24 hrs. 48 hrs after first application of inhibitors, extracellular media were removed and cells were processed for proliferation assays.
For MTT assays, cells were briefly washed with basal solution containing (in mM): 135 NaCl, 3.8 KCl, 1.2 MgSO4, 1.3 CaCl2, 1.2 KH2PO4, 10 HEPES, and 10 glucose (pH = 7.4, adjusted with NaOH). Aliquots of basal solution containing 0.5 mg/ml thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich) were then added to each well. After 30-min incubation at 37°C MTT solution was removed, and cells were solubilized in acidified isopropanol to dissolve newly formed MTT-formazan particles. 250-µl aliquots from each well were transferred into a 96-well plate. Absorbances at 562 nm were measured using ELx800 Absorbance microplate reader (BioTek Instruments, Winooski, VT, USA).
For Coulter counter assays, cells were detached from substrate using recombinant protease TrypLE and counted using a Z1 Series Coulter Counter (Beckman Coulter, Miami, FL, USA).
siRNA transfections
To downregulate BK and IK1, U251 cells were transfected with gene-specific siRNAs using a Nucleofector II (Lonza, Cologne, Germany) and the Amaxa Nucleofection kit T according to the manufacturer's protocol. Cells were removed from the substrate with TrypLE, counted, centrifuged at 300 g for 10 min, and mixed with nucleofection suspension buffer containing 0.5 µg of GFP cDNA (as a control of transfection efficacy) and 1 µM siRNA of choice. Cells were electroporated using program T-20 and then plated on either 24-well plates for proliferation assays or glass coverslips for patch clamp experiments. We used two to three siRNA constructs per gene that were obtained from Qiagen or Ambion. Target sequences for each siRNA are listed in Table 2. As a negative control we used nonsense AllStars negative control siRNA (Qiagen) or siRNA to an unrelated K+ channel TREK.
Statistical analysis
All data presented as mean values ±SE. Number of independent experiments per group is indicated in each figure legend. Statistical significance was determined by one-way analysis of variance (ANOVA) and a priori Newman-Keuls test for multiple comparisons. p<0.05 was accepted as significant. In those experiments where experimental values were normalized to controls, statistical difference from controls was calculated with unpaired Student's t-test. Origin 6.0 (Origin Labs, Northampton, MA, USA) and Prism 5 (GraphPad Software, San Diego, CA, USA) were used for statistical analysis.
Expression of mRNAs encoding the Ca2+-activated K+ channels BK, IK1 and SK1-3 in glioma cell lines and in a surgical sample of glioblastoma multiforme
We initially evaluated the expression of Ca2+-activated K+ channels in glioma cell lines at the mRNA level. U251 and U87 are both derived from human gliomas, but demonstrate different morphological and growth characteristics as well as substantial difference in gene expression profiles [40], [41]. For example, U87 cell express wild type p53, while U251 carry its mutated form [40]. We found expression of BK, IK1, and SK2 transcripts in U251 and U87 cells. The levels of transcripts for each of these channels were quantitatively similar, representing ∼0.5–2% of the mRNA expression levels for the housekeeping gene GAPDH (Fig. 1). SK1 and SK3 expression levels were negligible. In order to determine if glioma cells in situ have a similar Ca2+-activated K+ channel expression profile, we isolated mRNA from a surgical sample that was histopathologically identified as a GBM. In the GBM sample, BK and IK1 mRNA expression levels were comparable to those in U251 and U87 cells (Fig. 1). However, GBM SK2 transcript levels were substantially higher than in glioma cell lines. Additionally, we found the expression of SK1 and SK3 in the surgical sample that was not present in the glioma cell lines (Fig. 1).
Relative levels of mRNA expression for BK, IK1 and SK1-3 channels were quantified in U251, U87, and a GBM tissue sample using reverse transcription quantitative PCR. Expression levels were normalized to the expression of two housekeeping genes, GAPDH (shown in graph) and ribosomal protein RPL13A. Mean values ±SE of gene expression in three independent cell preparations for U251 and U81, and three independent measurements for GBM are shown.
Functional expression of BK and IK1 in glioma cell lines and in primary GBM cells
Expression of mRNA does not necessarily result in production of functional plasmalemmal channels. For example, Weaver et al. found high levels of the IK1 mRNA but no IK1 immunoreactive signal in Western blot analysis or functional IK1 currents in electrophysiological experiments in D54 and U251 glioma cell lines [22]. Therefore, we assessed the functional expression of Ca2+-activated K+ channels using conventional patch clamp technique.
When U251 cells were dialyzed with a pipette solution containing no Ca2+, we registered large macroscopic K+ currents that were activated in response to depolarizing step pulses >40 mV (Fig. 2A, D). This electrophysiological profile is characteristic of BK channels [16]. Under these Ca2+-free recording conditions, the addition of specific BK channel blockers paxilline (2 µM, Fig. 2B and C) or penitrem A (1 µM, Fig. 2E and F) nearly completely suppressed transmembrane K+ currents. Paxilline and penitrem A data were corroborated with the BK peptide inhibitor charybdotoxin (Fig. S1). Average BK current density, calculated as the paxilline-sensitive component of macroscopic K+ currents at +140 mV, was 67±9 pA/pF (n = 11). U87 cells also exhibited BK currents of similar amplitude (data not shown).
(A) Representative recordings of the whole-cell BK currents elicited by depolarization step pulses from −80 mV to +140 mV in 20 mV intervals. To prevent concomitant activation of IK1 and SK channels, no Ca2+ was added into pipette solution. (B) In the same cell shown in (A), whole-cell K+ currents were potently suppressed by the specific inhibitor of BK channels paxilline (2 µM). (C) Whole cell BK currents in response to depolarization ramps from −80 mV to +140 mV. Representative traces in the absence or presence of paxilline are shown. (D–F) Results of similar experiments employing another selective BK blocker penitrem A (1 µM).
To record IK1 currents, we increased [Ca2+] in the pipette to 750 nM and applied step pulses from –120 mV to +80 mV in 20 mV increments. To prevent concomitant activation of the BK currents, 2 µM paxilline was included in the bath solution. Under these recording conditions, we observed macroscopic K+ currents that were saturated at voltages ≥40 mV, consistent with the electrophysiological profile of IK1 channels (Fig. 2A and C) [35]. The specific blockers of IK1 channels 2 µM clotrimazole (Fig. 2B and C), and 1 µM TRAM-34 (Fig. 3E and F) inhibited the K+ currents, indicating that they are indeed mediated by the IK1 channels. In U251 cells, average IK1 current density, calculated as the clotrimazole-sensitive component, was 25±6 pA/pF at 0 mV (n = 6). Similar IK1 currents were recorded in U87 cells (Fig. S2).
(A) Representative recordings of the whole-cell IK1 currents elicited by depolarization step pulses from −120 mV to +80 mV in 20 mV intervals. Whole cell IK1 currents were activated by clamping pipette [Ca2+] at 750 nM. To prevent concomitant activation of BK currents, 2 µM paxilline was added into bath solution. (B) In the same cell shown in (A), the whole cell IK1 currents were inhibited by the potent IK1 blocker clotrimazole (2 µM). (C) Whole cell currents in response to depolarization ramps from −120 mV to +80 mV in the absence or presence of clotrimazole. (D–F) Results of similar experiments employing the selective IK1 blocker TRAM-34 (1 µM).
Since immortalized cell lines frequently have different gene expression profile from cells in situ and primary cell cultures, we performed recordings in primary glioma cultures prepared from a human surgical sample of GBM. As shown in Fig. 4, primary GBM cells also exhibited large paxilline-sensitive BK currents under Ca2+-free conditions (Fig. 4A–C). Five out of five recorded cells had BK currents, with average current density 80±33 pA/pF at +140 mV. We also observed the clotrimazole-sensitive IK1 currents when [Ca2+]i was clamped at 750 nM and 2 µM paxilline was added to the bath solution (Fig. 4D–F). Three out of six recorded GBM cells showed IK1 currents with an average current density of 13±6 pA/pF at 0 mV. The reasons for heterogeneity of IK1 current expression are not clear. However, when observed, GBM IK1 current densities were not statistically different from those found in U251 cells.
(A) Representative recordings of macroscopic BK currents in primary cells cultured from surgical sample of glioblastoma multiforme. Currents were elicited by step pulses from −80 mV to +140 mV. (B) In the same cell shown in (A), the BK blocker paxilline (2 µM) potently inhibited macroscopic K+ currents. (C) Representative traces of K+ currents elicited in response to depolarization ramps from −80 mV to +140 mV in the absence and presence of paxilline. (D) Representative recordings of the whole-cell IK1 currents activated by step pulses from −120 to +60 mV. To isolate IK1 currents, [Ca2+]pipette was clamped at 750 nM and 2 µM paxilline was added into bath solution. (E) The specific IK1 inhibitor clotrimazole (2 µM) potently suppressed macroscopic K+ currents. (F) Representative traces of K+ currents elicited in response to depolarization ramps −120 mV to +60 mV in the absence and presence of clotrimazole. For additional experimental details, see legend to Fig. 2 and Results section.
Our finding of the functional IK1 currents in all glial cell lines was in conflict with the previous observations of Weaver et al. [22]. Therefore, we performed additional Western Blot analysis of the IK1 protein expression in U251, U87 and GBM cells. Using commercially available polyclonal IK1 antibody, we identified a weak immunopositive band of ∼46–48 kDa in protein lysates prepared from all three cell types (Fig. S3). This molecular weight is very close to the predicted molecular weight of the IK1 protein (45 kDa).
Effects of BK and IK1 channel blockers on proliferation of U251 and U87 cells
To explore functional involvement of BK and IK1 channels in proliferation, we tested the effects of pharmacological inhibitors of BK and IK1 using two different techniques, Coulter counter and MTT proliferation assays. Using two alternative approaches was important because MTT conversion into light-absorbing formazan is mediated by mitochondrial dehydrogenases and may be affected without changes in cell numbers or viability [42]. In the FBS-containing media, the BK inhibitor paxilline (10 µM) had no effect on cell growth, while the IK1 blocker clotrimazole (10 µM) moderately suppressed cell proliferation of U251 (Fig. 5). The combination of these two drugs caused a significant reduction of cell proliferation as measured by both MTT and Coulter counter assays (Fig. 5 and Fig. S4A). To test whether serum components may reduce the inhibitory effects of channel blockers, we performed additional proliferation assays under serum-free conditions. In OptiMEM medium supplemented with serum substitute B27, U251 proliferation rates were similar to those in cells cultured in DMEM plus 10% FBS. In both media we observed approximately six-fold increases in cell numbers over 48 hrs (data of Coulter counter assays). Under serum-free conditions the efficacy of paxilline was dramatically increased (Fig. 5 and Fig. S4B). Furthermore, the combination of paxilline and clotrimazole produced strong and additive inhibition of cell growth, reducing it by approximately 70% (Fig. 5, Fig. S4B). The combination of drugs also induced strong changes in cell morphology (see Fig. 6A–D). Qualitatively similar data were obtained in U87 cells, although the latter cells were less sensitive to the actions of clotrimazole (see Fig. S5 and micrographs in Fig. 6E–H). To test if drug bio-availability was reduced by the major serum component albumin, we supplemented serum-free media (OptiMEM+B27) with 2 mg/ml bovine serum albumin. Addition of albumin reverted the inhibitory properties of paxilline and clotrimazole to the levels seen in serum-containing media, rendering paxilline completely ineffective (Fig. 5).
Paxilline (10 µM) and clotrimazole (10 µM) were added to culture media alone or in combination, and rates of cell proliferation were determined 48 hrs later using an MTT proliferation assay. Proliferation assays were performed in standard cell culture medium (DMEM +10% FBS), serum-free OptiMEM medium supplemented with serum substitute B27, or in OptiMEM+B27 additionally containing bovine serum albumin (2 mg/ml). Quantitatively similar data were obtained using Coulter counter assay (see Fig. S2). *, p<0.05, **p<0.01, ***p<0.001 vs. control; ##p<0.01, combination of drugs vs. paxilline or clotrimazole alone.
Cells were grown in the serum-free OptiMEM media supplemented with serum substitute B27. The BK blocker paxilline (10 µM) and the IK1 inhibitor clotrimazole (10 µM) were added as indicated. Images of the cells were captured ∼48 hrs after addition of channel blockers using Hoffman modulation contrast optics in Olympus IX71 microscope at 10×10 magnification.
We next tested the dose dependency of the effects of paxilline and clotrimazole on U251 cell proliferation and compared their efficacy to the specific BK inhibitor penitrem A [43] and the selective IK1 blocker TRAM-34 [19]. Since high levels of SK2 message were found in both immortalized cell lines and primary cells, we also tested UCL1848, an inhibitor of SK1, SK2, and SK3 [44]. The BK blockers, paxilline and penitrem A, inhibited U251 proliferation in a dose dependent fashion (Fig. 7). At the highest tested concentration (30 µM), both agents were cytotoxic, i.e. reduced cell numbers below seeding densities (Fig. 7A). The IC50 values for both inhibitors were substantially higher than those reported for inhibition of BK currents. In our hands, paxilline blocked cell proliferation by 50% at ∼13 µM (Fig. 7A), whereas the reported IC50 for inhibition of the whole-cell currents is ∼30 nM [45]. Penitrem A was somewhat more effective and inhibited proliferation with the IC50 of ∼3.5 µM (Fig. 7A), but this value greatly exceeded ∼10 nM concentration required for half-maximal inhibition of the BK activity [45]. The IK1 inhibitors clotrimazole and TRAM-34 reduced cell proliferation by 50% at the concentrations of ∼6 and 14 µM, respectively (Fig. 7B). These values were drastically higher than the IC50 of 70 nM (clotrimazole) and 20 nM (TRAM-34) that were obtained in electrophysiological experiments [19]. The specific blocker of the SK1-3 channels UCL1848 showed no effect on U251 cell proliferation (Fig. 7C).
(A) Dose response curves for the effects of the BK channel inhibitors, paxilline and penitrem A, on proliferation of U251 cells. Arrow indicates concentration of paxilline that was used to block K+ currents in the electrophysiology experiments presented in (D). (B) Dose response curves for the effects of the IK1 channel inhibitors, clotrimazole and TRAM-34, on proliferation of U251 cells. Arrow indicates concentration of clotrimazole that was used to block K+ currents in the electrophysiological experiments presented in (E). (C) Dose response curves for the effects of the selective SK1-3 channel blocker UCL1848 on proliferation of U251 cells. (D) Effect of the "aged" paxilline on the whole-cell BK currents. Cell proliferation medium containing 10 µM paxilline was collected after 24-hr incubation in cell proliferation assays, diluted 10-fold with electrophysiological bath solution, and used for inhibiting BK currents. Nominal concentration of paxilline was 1 µM (indicated by arrow in (A), to compare proliferation and electrophysiology data). Representative of three experiments. (E) Effect of the "aged" clotrimazole on the whole-cell IK1 currents. Cell proliferation medium containing 10 µM clotrimazole was collected after 24-hr incubation in cell proliferation assays, diluted 10-fold, and used for inhibiting IK1 currents. Nominal concentration of clotrimazole was 1 µM (indicated by arrow in (B)). Representative of three experiments. (F) Comparison of normalized effects of the "aged" paxilline and clotrimazole on the whole cell K+ currents to their effects on cell proliferation. Inhibition of BK and IK1 currents was calculated as average inhibition of the whole K+ cell currents at +140 and 0 mV for BK and IK1, respectively.
The disparity between the IC50 values for inhibition of proliferation and channel activity may be explained by decomposition of tested compounds in culture media, or by nonspecific absorption of the drugs during long incubation. To address this possibility, we tested the inhibitory properties of culture media containing nominally 10 µM of either paxilline or clotrimazole after 24-hr incubation in cell proliferation assays. We diluted aliquots of the inhibitor-containing culture media 10-fold with electrophysiological bath solution and performed whole-cell patch recordings. Such dilution was necessary to minimize differences between cell culture media and bath solution. The final nominal concentrations of paxilline and clotrimazole were 1 µM. As seen in Fig. 7D, E, "aged" paxilline and clotrimazole solutions inhibited BK and IK1 currents by >90% and >70%, respectively. Notably, at the nominal concentration of 1 µM, neither paxilline nor clotrimazole affected cell proliferation (compare to the values indicated by the block arrows in Fig. 7A and B). Thus, the effects of paxilline and clotrimazole (as well as the other inhibitors) on cell proliferation are unlikely related to their actions on BK and IK1 activities.
Effects of gene-specific siRNAs on BK and IK1 channel activity and cell proliferation
We decided to further test the functional involvement of BK and IK1 in glioma cell proliferation using an alternative approach. For this purpose, we downregulated BK and IK1 expression using an siRNA technique. At 48 hrs after transfection, the two BK-specific siRNAs significantly downregulated BK mRNA expression levels by 62% and 58% (Fig. 8A, p<0.01). These values likely underestimate the degree of mRNA downregulation in individual cells because only 65–80% of the cells were successfully transfected, as routinely verified by co-transfection with GFP (see Fig. S6). U251 cells transfected with siRNA targeting the unrelated K+ channel TREK-1 (KCNK2) did not affect levels of BK mRNA expression (n = 3, data not shown). The BK siRNA#2 was used to test for functional suppression of BK channels. As seen in Fig. 8B, C, the BK siRNA#2 induced a 52% decrease in the BK current density 72 hrs after transfection (29±5 pA/pF for BK siRNA vs. 60±8 pA/pF for negative control siRNA, p = 0.002). While the BK siRNA#2 successfully downregulated the BK current densities, neither siRNA#2 nor siRNA#1 affected U251 cell proliferation (Fig. 8D, cell counts performed 72 hrs after transfection).
(A) Effect of the BK-specific siRNAs on BK mRNA expression levels. U251 cells were transfected with negative control siRNA (NC) or two different siRNAs targeting BK (si1-BK, and si2-BK). 48 hrs post transfection, mRNA levels were quantified and normalized to housekeeping genes GAPDH and RPL13A using reverse transcription qPCR. Data are the mean values ±SE of 3-5 independent transfections. (B) Representative traces of whole-cell BK currents measured at 72 hrs after transfection with negative control siRNA (NC) or siRNA targeting BK (si2-BK). (C) Average BK current densities in U251 cells transfected with negative control (NC) or si2-BK siRNAs, as compared to non-transfected cells (WT). BK current density was measured as paxilline-sensitive K+ currents at +140 mV. Data are the mean values ±SE from 11-17 cells per group. **p<0.01, vs. negative control and non-transfected cells. (D) Effect of transfection with negative control (NC) or BK siRNAs (si1-BK, si2-BK) on proliferation of U251 measured 72 hrs post transfection using Coulter counter assay. Data are the mean values ±SE of 3 independent cell transfections. (E) Effect of the IK1-specific siRNAs on IK1 mRNA expression levels. mRNA levels were quantified and normalized to housekeeping genes GAPDH and RPL13A 48 hrs after transfection with negative control siRNA (NC), and three different siRNAs targeting IK1 (si5-IK1, and si6-IK1, si7-IK1. Data are the mean values ±SE in 3 independent transfections. (F) Representative whole-cell K+ currents measured 72 hrs after transfection with negative control siRNA (NC) or IK1siRNA (si7-IK1). (G) Average IK1 current densities in cells transfected with negative control (NC) or si7-IK1 siRNAs, as compared to non-transfected cells (WT). IK1 current density was measured as clotrimazole-sensitive K+ currents at 0 mV with 2 µM paxilline added to bath solution. Data are the mean values ±SE from 14-15 cells per experimental group, and n = 6 in WT. **p<0.01, vs. negative control and non-transfected cells. (H) Effect of nucleofection with negative control siRNA (NC), or IK1-siRNA (si5-IK1, si6-IK1, si7-IK1) on cell proliferation measured 72 hrs post nucleofection using MTT assay. Data are the mean values ±SE of 3 independent cell transfections.
Two out of three IK1-specific siRNAs tested by us downregulated IK1 mRNA levels by 52% and 70% 48 hrs after transfection, while the third construct proved ineffective (Fig. 8E). The most effective IK1 siRNA construct (siIK1#7) was further tested in electrophysiological functional assays. This siRNA reduced the IK1 current density by 67% 72 hours after transfection (7±2 pA/pF for IK siRNA vs. 21±4 pA/pF in negative control transfections, p = 0.003). As in the case of BK downregulation, the IK1 knockdowns had no effect on proliferation of U251 cells (Fig. 8H).
Because paxilline and clotrimazole reduced cell proliferation in an additive fashion, we explored the effects of simultaneous BK and IK1 downregualtion on rate of U251 cell growth. As in the case of individual channel knockdowns, a combination of the BK siRNA#2 and the IK1 siRNA#7 (two constructs that effectively suppressed BK and IK1 current densities) was ineffective in inhibiting cell growth (Fig. S7).
The main finding of this study is the lack of functional involvement of the Ca2+-activated K+ channels in proliferation of U251 and U87 glioma cell lines and primary GBM cells. This conclusion is at odds with some previous reports that also used pharmacological agents to assay for functional contribution of BK and/or IK1 to proliferation. Although all the inhibitors of BK and IK1 that we tested in this study suppressed glioma cell proliferation, their effects occurred at concentrations significantly exceeding those needed to block channel function. Downregulation of BK and IK1 activity using siRNA did not cause changes in cell growth. Therefore, the antiproliferative properties of the BK and IK1 channel blockers are likely mediated by off target actions.
BK channels are highly expressed in glioma cell lines and in human glioma tissue [46], [47], [48], [22]. The BK channel expression correlates with glioma malignancy, with higher levels of the BK protein seen in more malignant glioma biopsy samples [48]. BK channels have been implicated in regulation of cell proliferation in a number of malignant cell lines, such as HeLa and A2780 carcinomas, MCF-7 breast cancer, PC-3 prostate cancer, as well as in 1321N1 and D54-MG human glioma lines [20], [21], [23], [24], [25]. These proliferation studies relied on pharmacological inhibitors of BK, such as TEA and iberiotoxin. However, more recent publications, that employed molecular biology techniques and/or BK channel openers, put forward the opposite idea that BK channels may have antiproliferative and antitumorogenic properties. For example, Cambien et al. [29] found that silencing of BK channels in human osteosarcoma cells strongly increases tumor load in vivo. In A2780 ovarian cancer cells, the BK opener NS1619 produced potent inhibition of cell proliferation [28]. Our own results strongly suggest that BK channels do not play a role in growth of glioma cells in vitro. While the BK blockers paxilline and penitrem A inhibited proliferation of U251 cells in a dose-dependent manner, their inhibitory effects were seen at concentrations greatly exceeding those necessary for complete channel block. This points to possible off-target effects of pharmacological agents. Such conclusion was supported by the results of siRNA experiments, in which downregulation of BK channel expression had no impact on U251 proliferation rates. The discrepancy between our BK data and several other reports in glioma cell lines may be explained by different conditions employed in cell proliferation assays. Positive results implicating BK in control of glioma cell growth have been collected using specific cell growth conditions, such as elevated extracellular [K+] [20] or serum deprivation [21]. In other malignant cell types, the effects of BK blockers could be seen only when cell growth was stimulated with certain factors such as estradiol [25], [24]. Hence, even if BK may contribute to cell proliferation under specific conditions, its activity is not a prerequisite for cell cycle progression in glioma cells.
A somewhat surprising finding of our work was the detection of functional IK1 channels in two established glioma lines, U251and U87, as well as in primary GBM cells. Although IK1 mRNA is consistently detected in immortalized glioma cell lines and human GBM samples, Weaver et al. found no evidence for the IK1 protein expression in numerous glioma samples and were unable to detect whole-cell IK1 currents in U251 cells [22]. Nevertheless, Fioretti et al. recently reported functional expression of the IK1 channels in GL-15 and U251 lines and in primary GBM cells [49], [50], [51], similar to findings in the present work. The disparity between these findings and the results of Weaver et al. may be due to differences in conditions employed in electrophysiological experiments. In order to isolate the IK1 currents, we and Fioretti et al. suppressed the concomitant BK currents by adding BK blockers into bath solutions. Furthermore, we recorded IK1 currents with ATP present in the pipette solution, which may strongly enhance IK1 activity via a PKC- and/or PKA-dependent mechanism [52], [53]. Unlike Weaver et al., we also found weak immunopositive protein bands at the molecular weight that was very close to the predicted molecular weight for the IK1 protein. This band was detected in cell lysates prepared from U251, U87, and primary GBM cells. Such difference in Western blots results may be due to the higher sensitivity of antibody that was used in our study.
The detection of the IK1 subtype of Ca2+-activated K+ channels in glioma cells may be of substantial importance. These channels have been implicated in tumorogenesis and regulation of cell proliferation in a number of cell types, including pancreatic and endometrial cancer cells, non-malignant endothelial and smooth muscle cells, and bone marrow mesenchymal cells [31], [33], [54], [35]. In in vivo experiments, systemic application of the IK1 inhibitor clotrimazole was reported to decrease tumor load and prolong survival in animal models of glioma and melanoma [36], [38]. In our hands, the specific IK1 blockers clotrimazole and TRAM-34 strongly inhibited proliferation of U251 cells. However, as in the case of BK blockers, their effective concentrations strongly exceeded those that were necessary for complete inhibition of the IK1 currents. Moreover, siRNA downregulation of IK1 did not affect cell proliferation. Recently, Sciaccaluga et al. discovered a critical role for the IK1 channels in chemotactic motility of glioblastoma cells [51]. In this latter study, downregulation of IK1 expression using shRNA completely inhibited cell migration in response to the chemokine ligand CXCL12 in several glioblastoma cell lines and primary GBM cells, suggesting a possible role of IK1 in glioblastoma invasiveness [51].
Overall, our data provide strong evidence against a requirement for the Ca2+-activated K+ channels BK and IK1 for proliferation of U251 cells, and likely other glioma cells. Further work is needed to define the mechanisms responsible for the antiproliferative effects of pharmacological inhibitors of BK and IK1, such as paxilline, penitrem A, clotrimazole, and TRAM-34. Our results suggest that strong caution should be taken when interpreting positive results obtained with pharmacological ion channel blockers and that such experiments should be supplemented with molecular biology studies targeting expression of proteins of interest.
Supporting Information
Figure S1.
The BK blocker charybdotoxin (100 nM) potently suppresses whole-cell K+ currents in U251 glioma cells. For methodological details see Methods and legend to Fig. 2 in the main text.
Figure S2.
U87 glioma cells express typical IK1 currents that were potently suppressed by the IK1 blocker clotrimazole (2 µM). For methodological details see Methods and legend to Fig. 3 in the main text.
Figure S3.
Western blot analysis of the IK1 protein expression in glioma cell lines and primary glioblastoma cells. Protein lysates (20 µg/lane) of primary glioblastoma (GBM), U87, and U251 cells were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to the PVDF membrane and probed with polyclonal anti-IK1 antibody as described in Materials and Methods. Immunopositive signal was detected and digitized using chemiluminescence ECLplus reagent kit (GE Healthcare/Amersham Biosciences) and luminescent image analyzer LAS-4000 (FujiFilm Medical Systems, Stamford, CT). Bars and numbers on the left indicate positions of molecular weight standards (MW). Weak immunopositive band indicated by asterisk was detected at the molecular weight of ∼46–47 kDa that is close to the predicted molecular weight of the IK1 protein (45 kDa).
Figure S4.
Effects of paxilline and clotrimazole on proliferation of U251 cells in serum-containing and serum-free media measured using two alternative cell proliferation assays. (A) Effects of paxilline (10 µM) and clotrimazole (10 µM), or their combination on proliferation of U251 in media containing 10% FBS. Cells were treated with pharmacological inhibitors for 48 hrs and rates of proliferation were quantitatively assessed using Coulter Counter (white bars) and MTT (grey bars) assay. Plating cell density is indicated by dashed line. (B) Effects of paxilline (10 µM) and clotrimazole (10 µM) or their combination on proliferation of U251 cells in serum-free media supplemented with serum substitute B27. Cell proliferation was quantified using Coulter Counter (open bars) or MTT assay (grey bars). (C) Effects of paxilline (10 µM) and clotrimazole (10 µM) or their combination on proliferation of U251 cells in serum-free media supplemented with serum substitute B27 and bovine serum albumin. Proliferation rates were measured using MTT proliferation assay. Data are the mean values ±SE of proliferation normalized to controls in the same experiments. **p<0.01 vs. control; **p<0.05 vs control;***p<0.001 vs. control.
Figure S5.
Effects of paxilline and clotrimazole on proliferation of U87 cells in serum-containing and serum-free media. (A) Effects of paxilline (10 µM) and clotrimazole (10 µM) or their combination on proliferation of U87 in media containing 10% FBS. Cells were treated with pharmacological inhibitors for 48 hrs and rates of proliferation were quantitatively assessed using MTT assay. Data are the mean values of proliferation ±SE normalized to controls in the same experiments. (B) Effects of paxilline (10 µM) and clotrimazole (10 µM) or their combination on proliferation of U87 cells in serum-free media supplemented with serum substitute B27. Data are the mean values of proliferation ±SE normalized to controls in the same experiments. **p<0.01 vs. control; ***p<0.001 vs. control.
Figure S6.
Representative images of U251 cells transfected with the GFP-expressing plasmid using amaxa nucleofection protocol. Images of the same field were acquired using Olympus IX71 Hoffman modulation contrast optics (A) and GFP fluorescence (B) 72 hrs after nucleofection at 10×10 magnification. Transfection efficacy varied between 60–90% in various cell preparations.
Figure S7.
Double knockdown of BK and IK1 K+ channels does not affect proliferation of U251 glioma cells. Effect of transfection with negative control (NC), BK-specific siRNA #2 (siBK), IK1-specific siRNA#7 (siIK1) or combination of BK- and IK1-specific siRNAs (siBK+siIK1) on proliferation of U251 measured 72 hrs post transfection using MTT proliferation assay. Data are the mean values ±SE of 3 independent cell transfections.
We thank Dr. Michael G. Kaplitt for the gift of U251 cells, Drs. James A. Bennet and Thomas T. Andersen for providing U87 cells, Dr. Fassil B. Mesfin for help in preparation of GBM cells, and Dr. Mohamed Trebak for critical reading of the manuscript. We greatly appreciate the technical support of Maria C. Hyzinski.
Author Contributions
Conceived and designed the experiments: IFA AR AAM YHK. Performed the experiments: IFA AR. Analyzed the data: IFA AR AAM YHK. Contributed reagents/materials/analysis tools: YHK. Wrote the paper: IFA AAM YHK.
- 1. Wrensch M, Minn Y, Chew T, Bondy M, Berger MS (2002) Epidemiology of primary brain tumors: current concepts and review of the literature. Neuro Oncol 4: 278–299.
- 2. Schwartzbaum JA, Fisher JL, Aldape KD, Wrensch M (2006) Epidemiology and molecular pathology of glioma. Nat Clin Pract Neurol 2: 494–503.
- 3. Kleihues P, Burger PC, Scheithauer BW (1993) The new WHO classification of brain tumours. Brain Pathol 3: 255–268.
- 4. Barnholtz-Sloan JS, Sloan AE, Schwartz AG (2003) Relative survival rates and patterns of diagnosis analyzed by time period for individuals with primary malignant brain tumor, 1973-1997. J Neurosurg 99: 458–466.
- 5. Sanai N, Berger MS (2008) Glioma extent of resection and its impact on patient outcome. Neurosurgery 62: 753–764.
- 6. Bondy ML, Scheurer ME, Malmer B, Barnholtz-Sloan JS, Davis FG, et al. (2008) Brain tumor epidemiology: consensus from the Brain Tumor Epidemiology Consortium. Cancer 113: 1953–1968.
- 7. Amigorena S, Choquet D, Teillaud JL, Korn H, Fridman WH (1990) Ion channel blockers inhibit B cell activation at a precise stage of the G1 phase of the cell cycle. Possible involvement of K+ channels. J Immunol 144: 2038–2045.
- 8. Nilius B, Wohlrab W (1992) Potassium channels and regulation of proliferation of human melanoma cells. J Physiol 445: 537–548.
- 9. Woodfork KA, Wonderlin WF, Peterson VA, Strobl JS (1995) Inhibition of ATP-sensitive potassium channels causes reversible cell-cycle arrest of human breast cancer cells in tissue culture. J Cell Physiol 162: 163–171.
- 10. Bianchi L, Wible B, Arcangeli A, Taglialatela M, Morra F, et al. (1998) herg encodes a K+ current highly conserved in tumors of different histogenesis: a selective advantage for cancer cells? Cancer Res 58: 815–822.
- 11. Wonderlin WF, Strobl JS (1996) Potassium channels, proliferation and G1 progression. J Membr Biol 154: 91–107.
- 12. Blackiston DJ, McLaughlin KA, Levin M (2009) Bioelectric controls of cell proliferation: ion channels, membrane voltage and the cell cycle. Cell Cycle 8: 3519–3528.
- 13. Lang F, Ritter M, Gamper N, Huber S, Fillon S, et al. (2000) Cell volume in the regulation of cell proliferation and apoptotic cell death. Cell Physiol Biochem 10: 417–428.
- 14. Wang Z (2004) Roles of K+ channels in regulating tumour cell proliferation and apoptosis. Pflugers Arch 448: 274–286.
- 15. Vergara C, Latorre R, Marrion NV, Adelman JP (1998) Calcium-activated potassium channels. Curr Opin Neurobiol 8: 321–329.
- 16. Salkoff L, Butler A, Ferreira G, Santi C, Wei A (2006) High-conductance potassium channels of the SLO family. Nat Rev Neurosci 7: 921–931.
- 17. Pedarzani P, Stocker M (2008) Molecular and cellular basis of small- and intermediate-conductance, calcium-activated potassium channel function in the brain. Cell Mol Life Sci 65: 3196–3217.
- 18. Xia XM, Fakler B, Rivard A, Wayman G, Johnson-Pais T, et al. (1998) Mechanism of calcium gating in small-conductance calcium-activated potassium channels. Nature 395: 503–507.
- 19. Wulff H, Miller MJ, Hansel W, Grissmer S, Cahalan MD, et al. (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.
- 20. Basrai D, Kraft R, Bollensdorff C, Liebmann L, Benndorf K, et al. (2002) BK channel blockers inhibit potassium-induced proliferation of human astrocytoma cells. Neuroreport 13: 403–407.
- 21. Weaver AK, Liu X, Sontheimer H (2004) Role for calcium-activated potassium channels (BK) in growth control of human malignant glioma cells. J Neurosci Res 78: 224–234.
- 22. Weaver AK, Bomben VC, Sontheimer H (2006) Expression and function of calcium-activated potassium channels in human glioma cells. Glia 54: 223–233.
- 23. Han X, Wang F, Yao W, Xing H, Weng D, et al. (2007) Heat shock proteins and p53 play a critical role in K+ channel-mediated tumor cell proliferation and apoptosis. Apoptosis 12: 1837–1846.
- 24. Coiret G, Borowiec AS, Mariot P, Ouadid-Ahidouch H, Matifat F (2007) The antiestrogen tamoxifen activates BK channels and stimulates proliferation of MCF-7 breast cancer cells. Mol Pharmacol 71: 843–851.
- 25. Bloch M, Ousingsawat J, Simon R, Schraml P, Gasser TC, et al. (2007) KCNMA1 gene amplification promotes tumor cell proliferation in human prostate cancer. Oncogene 26: 2525–2534.
- 26. Chin LS, Park CC, Zitnay KM, Sinha M, DiPatri AJ Jr, et al. (1997) 4-Aminopyridine causes apoptosis and blocks an outward rectifier K+ channel in malignant astrocytoma cell lines. J Neurosci Res 48: 122–127.
- 27. Ouadid-Ahidouch H, Roudbaraki M, Ahidouch A, Delcourt P, Prevarskaya N (2004) Cell-cycle-dependent expression of the large Ca2+-activated K+ channels in breast cancer cells. Biochem Biophys Res Commun 316: 244–251.
- 28. Han X, Xi L, Wang H, Huang X, Ma X, et al. (2008) The potassium ion channel opener NS1619 inhibits proliferation and induces apoptosis in A2780 ovarian cancer cells. Biochem Biophys Res Commun 375: 205–209.
- 29. Cambien B, Rezzonico R, Vitale S, Rouzaire-Dubois B, Dubois JM, et al. (2008) Silencing of hSlo potassium channels in human osteosarcoma cells promotes tumorigenesis. Int J Cancer 123: 365–371.
- 30. Parihar AS, Coghlan MJ, Gopalakrishnan M, Shieh CC (2003) Effects of intermediate-conductance Ca2+-activated K+ channel modulators on human prostate cancer cell proliferation. Eur J Pharmacol 471: 157–164.
- 31. Jager H, Dreker T, Buck A, Giehl K, Gress T, et al. (2004) Blockage of intermediate-conductance Ca2+-activated K+ channels inhibit human pancreatic cancer cell growth in vitro. Mol Pharmacol 65: 630–638.
- 32. Ouadid-Ahidouch H, Roudbaraki M, Delcourt P, Ahidouch A, Joury N, et al. (2004) Functional and molecular identification of intermediate-conductance Ca(2+)-activated K(+) channels in breast cancer cells: association with cell cycle progression. Am J Physiol Cell Physiol 287: C125–C134.
- 33. Grgic I, Eichler I, Heinau P, Si H, Brakemeier S, et al. (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.
- 34. Wang ZH, Shen B, Yao HL, Jia YC, Ren J, et al. (2007) Blockage of intermediate-conductance-Ca(2+) -activated K(+) channels inhibits progression of human endometrial cancer. Oncogene 26: 5107–5114.
- 35. Tao R, Lau CP, Tse HF, Li GR (2008) Regulation of cell proliferation by intermediate-conductance Ca2+-activated potassium and volume-sensitive chloride channels in mouse mesenchymal stem cells. Am J Physiol Cell Physiol 295: C1409–C1416.
- 36. Benzaquen LR, Brugnara C, Byers HR, Gatton-Celli S, Halperin JA (1995) Clotrimazole inhibits cell proliferation in vitro and in vivo. Nat Med 1: 534–540.
- 37. Khalid MH, Shibata S, Hiura T (1999) Effects of clotrimazole on the growth, morphological characteristics, and cisplatin sensitivity of human glioblastoma cells in vitro. J Neurosurg 90: 918–927.
- 38. Khalid MH, Tokunaga Y, Caputy AJ, Walters E (2005) Inhibition of tumor growth and prolonged survival of rats with intracranial gliomas following administration of clotrimazole. J Neurosurg 103: 79–86.
- 39. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391: 85–100.
- 40. Ishii N, Maier D, Merlo A, Tada M, Sawamura Y, et al. (1999) Frequent co-alterations of TP53, p16/CDKN2A, p14ARF, PTEN tumor suppressor genes in human glioma cell lines. Brain Pathol 9: 469–479.
- 41. Camphausen K, Purow B, Sproull M, Scott T, Ozawa T, et al. (2005) Influence of in vivo growth on human glioma cell line gene expression: convergent profiles under orthotopic conditions. Proc Natl Acad Sci U S A 102: 8287–8292.
- 42. Akimova OA, Mongin AA, Hamet P, Orlov SN (2006) The rapid decline of MTT reduction is not a marker of death signaling in ouabain-treated cells. Cell Mol Biol 52: 71–77.
- 43. Knaus HG, McManus OB, Lee SH, Schmalhofer WA, Garcia-Calvo M, et al. (1994) Tremorgenic indole alkaloids potently inhibit smooth muscle high-conductance calcium-activated potassium channels. Biochemistry 33: 5819–5828.
- 44. Chen JQ, Galanakis D, Ganellin CR, Dunn PM, Jenkinson DH (2000) bis-Quinolinium cyclophanes: 8,14-diaza-1,7(1, 4)-diquinolinacyclotetradecaphane (UCL 1848), a highly potent and selective, nonpeptidic blocker of the apamin-sensitive Ca(2+)-activated K(+) channel. J Med Chem 43: 3478–3481.
- 45. Gessner G, Schonherr K, Soom M, Hansel A, Asim M, et al. (2005) BKCa channels activating at resting potential without calcium in LNCaP prostate cancer cells. J Membr Biol 208: 229–240.
- 46. Brismar T, Collins VP (1989) Potassium and sodium channels in human malignant glioma cells. Brain Res 480: 259–267.
- 47. Brismar T (1995) Physiology of transformed glial cells. Glia 15: 231–243.
- 48. Liu X, Chang Y, Reinhart PH, Sontheimer H, Chang Y (2002) Cloning and characterization of glioma BK, a novel BK channel isoform highly expressed in human glioma cells. J Neurosci 22: 1840–1849.
- 49. Fioretti B, Castigli E, Calzuola I, Harper AA, Franciolini F, et al. (2004) NPPB block of the intermediate-conductance Ca2+-activated K+ channel. Eur J Pharmacol 497: 1–6.
- 50. Fioretti B, Castigli E, Micheli MR, Bova R, Sciaccaluga M, et al. (2006) Expression and modulation of the intermediate- conductance Ca2+-activated K+ channel in glioblastoma GL-15 cells. Cell Physiol Biochem 18: 47–56.
- 51. Sciaccaluga M, Fioretti B, Catacuzzeno L, Pagani F, Bertollini C, et al. (2010) CXCL12-induced glioblastoma cell migration requires intermediate conductance Ca2+-activated K+ channel activity. Am J Physiol Cell Physiol 299: C175–C184.
- 52. Wulf A, Schwab A (2002) Regulation of a calcium-sensitive K+ channel (cIK1) by protein kinase C. J Membr Biol 187: 71–79.
- 53. Hayashi M, Kunii C, Takahata T, Ishikawa T (2004) ATP-dependent regulation of SK4/IK1-like currents in rat submandibular acinar cells: possible role of cAMP-dependent protein kinase. Am J Physiol Cell Physiol 286: C635–C646.
- 54. Si H, Grgic I, Heyken WT, Maier T, Hoyer J, et al. (2006) Mitogenic modulation of Ca2+ -activated K+ channels in proliferating A7r5 vascular smooth muscle cells. Br J Pharmacol 148: 909–917.


text:
PPIx::EditorTools::RenameVariable - Lexically replace a variable name in Perl code
version 0.11
my $munged = PPIx::EditorTools::RenameVariable->new->rename( code => $code, line => 15, column => 13, replacement => 'stuff', ); my $code_as_strig = $munged->code; my $code_as_ppi = $munged->ppi; my $location = $munged->element->location;
This module will lexically replace a variable name.
Constructor. Generally shouldn't be called with any arguments.
Accepts either a PPI::Document
to process or a string containing the code (which will be converted into a PPI::Document
) to process. Renames the variable found at line, column with that supplied in the replacement
parameter and returns a PPIx::EditorTools::ReturnObject
with the new code available via the ppi
or code
accessors, as a PPI::Document
or string
, respectively. The PPI::Token
found at line, column is available via the element
Instead of specifying an explicit replacement variable name, you may choose to use the to_camel_case
or from_camel_case
options that automatically convert to/from camelCase. In that mode, the ucfirst
option will force uppercasing of the first letter.
You can not specify a replacement name and use the to/from_camel_case
Croaks with a "no token" exception if no token is found at the location. Croaks with a "no declaration" exception if unable to find the declaration.
This class inherits from PPIx::EditorTools
. Also see App::EditorTools, Padre, and PPI.
This software is copyright (c) 2010 by The Padre development team as listed in Padre.pm.
This is free software; you can redistribute it and/or modify it under the same terms as the Perl 5 programming language system itself.

subset: commoncrawl


text:
Artist description:
There is only snow beyond the window, so it's only logical to draw something like this :)
Java Draw Created Jan 05, 2013 640 x 460px Unless noted Copyright 2013 Predelnik.
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text:
Five Anglican bishops have announced their resignation from the Church of England with the intention to set up an English Ordinariate in communion with the Pope. Archbishop of Canterbury, Rowan Williams, regretfully accepted their decision and wished them well.
The bishops
include Andrew Burnham, Bishop of Ebbsfleet, Keith Newton, Bishop of
Richborough, John Broadhurt, Bishop of Fulham, Edwin Barnes, ermeritus
Bishop of Richborough, and David Silk, an emeritus assistant bishop of
In a statement released by the bishops
their reason for leaving the Church was over "developments they believed
to be incompatible with the historic vocation of Anglicanism in faith
and order".
This comes after a change in the Anglican church to allow the ordination of female bishops and other liberal innovations.
Archbishop of Canterbury, Rowan Williams, regretfully accepted their decision and wished them well.
Catholic liaison officer, Bishop Alan Hopes, welcomed the bishops to
the Ordinariate for England, which will be soon be established.


text:
No Room for Error
The Story Behind the USAF Special Tactics Unit
Col. John T. Carney & Benjamin F. Schemmer
This book is available for download with iBooks on your Mac or iOS device, and with iTunes on your computer. Books can be read with iBooks on your Mac or iOS device.
"John Carney is one of the few heroes I have."
–LT. COL. L. H. "BUCKY" BURRUSS, USA (Ret.)
Founding member and Deputy Commander of Delta Force
When the U.S. Air Force decided to create an elite "special tactics" team in the late 1970s to work in conjunction with special-operations forces combating terrorists and hijackers and defusing explosive international emergencies, John T. Carney was the man they turned to. Since then Carney and the U.S. Air Force Special Tactical units have circled the world on sensitive clandestine missions. They have operated behind enemy lines gathering vital intelligence. They have combated terrorists and overthrown dangerous dictators. They have suffered many times the casualty rate of America's conventional forces. But they have gotten the job done–most recently in stunning victories in the war on terrorism in Afghanistan, which Carney calls "America's first special-operations war." Now, for the first time, Colonel Carney lifts the veil of secrecy and reveals what really goes on inside the special-operations forces that are at the forefront of contemporary warfare.
Part memoir, part military history, No Room for Error reveals how Carney, after a decade of military service, was handpicked to organize a small, under-funded, classified ad hoc unit known as Brand X, which even his boss knew very little about. Here Carney recounts the challenging missions: the secret reconnaissance in the desert of north-central Iran during the hostage crisis; the simple rescue operation in Grenada that turned into a prolonged bloody struggle. With Operation Just Cause in Panama, the Special Tactical units scored a major success, as they took down the corrupt regime of General Noriega with lightning speed. Desert Storm was another triumph, with Carney's team carrying out vital search-and-rescue missions as well as helping to hunt down mobile Scud missiles deep inside Iraq.
Now with the war on terrorism in Afghanistan, special operations have come into their own, and Carney includes a chapter detailing exactly how the Air Force Special Tactics d.c. units have spearheaded the successful campaign against the Taliban and Al Qaeda.
Gripping in its battle scenes, eye-opening in its revelations, No Room for Error is the first insider's account of how special operations are changing the way modern wars are fought. Col. John T. Carney is an airman America can be proud of, and he has written an absolutely superb book.
From the Hardcover edition.
- 8,49 €
- Category: Military
- Published: 12 November 2002
- Publisher: Random House Publishing Group
- Print Length: 368 Pages
- Language: English


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Urgent. Make sure you ask if the Jacket is fused or fully canvassed. Apparently unless you specify fully canvassed, its fused. Having said that had a recent wander in their and I found them to be quite friendly!!!! Very pleasantly surprised. I might go back there again. Let me know about the jacket. He's got some great fabrics just sitting there ready to go.
I always make sure beforehand to insist on full canvassing, in our email discussions and when I'm there in person.
Originally Posted by Journeyman
Just a gentle suggestion, but perhaps you should wait until your suit is finished first so as to ensure that you're happy with it, before you order a second item.
They did so well with the first fitting already that I'm happy to move forward with another commission from them. Second fitting for my dark grey PoW suit this coming week.


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(KSA 1) Water Resource Management
Population growth, urbanisation, development, and climate change are creating growing challenges for the water supplies on which South Africa's families, businesses, farms, industry and natural environment rely. In many areas of the country, such as the arid North, dwindling water supplies, lengthening droughts, and rising demand for water are forcing communities, stakeholders, and governments to explore new ideas and find new solutions that will help ensure stable, secure water supplies for future generations.
Some major urban centres such as City of Cape Town already face periodic water shortages compounded by water pollution occurrence, the latter often originating from water-dependent and water-impacting agricultural and industrial activities. This Key strategic area focuses on questions relating to water resources management and usage optimisation across many economic and social sectors, including agriculture, industry, urban development, energy, environment, tourism; etc. The White paper and the National Water Act number 36 of 1998.contain the principles and concepts used to manage, control, protect, water resources and its use.
Lack of a holistic perspective regarding water has led to dispersed and sometimes disorganised systems of water management. Responsibilities for the management of the resource in areas such as water monitoring, reserve determination, water quality compliance and enforcement, are often allocated or shared between a variety of different administrative departments. Water-related activities and their management are also present within a wide range of user sectors, are subsequently managed by that sector's institution, and result in often uncoordinated management. As the water resource is finite and its utilisation needs to be equitable, efficient and planned, the challenge will be to bring all sector strands of management together within agreed on management frameworks.
Water's special character of being essential to development and health as well as a key component in social and economic activities, has resulted in a special cultural status and consequently a special position in public policy. Freshwater resources have traditionally been regarded as something to which all members of the human community have rights to access for domestic as well as productive purposes. Over and above, it is viewed as pertinent that water users have active role to play in the decentralised management of water resources through representative water management institutions. The success of managing water resources is often perceived as a government or agency responsibility whereas water policies carve a space for robust stakeholder involvement in all matters relating to monitoring, use and enforcement of rules. This KSA will have a special focus to this area.
The use of water in the various social and economic contexts has typically been at most very low-cost; probably compromising the viability of decentralised institutions. There are important implications of subsidies and cost recovery in an era of water stress, among which are water profligacy and wasteful practices, or mismanaged water services and infrastructures. In the face of water shortages and environmental concerns, discussions in some international fora have called for water to be regarded as a social and public good and not to be available for the marketplace. This is particularly relevant in the South African socio-economic context where inequitable access to water remains rive. The recognition of existing lawful users from the past laws has reinforced this reality. However, the NWA provides for a number of mechanisms to make sure that there is a rectification of this situation through compulsory licensing, schedule 1 allowance and General Authorisations. The legal issues pertaining to property rights and the level of enforcement of reallocating water in the public interest for either environmental concerns or equity targets will require special attention.
The need to examine collectively the entire range of uses to which freshwater is put, and to design management which neither squander precious resources nor fail to respect other competing and complementary water needs, was translated into a policy and programme principle and strategy known as Integrated Water Resources Management (IWRM). This principle is the response to the growing pressure on water resources resulting from growing population and socio-economic developments. It comprises a holistic approach that makes the management and protection of water resources compatible with the development of systems serving all types of consumers. It is a vital part of the challenge facing water-related development cooperation today. IWRM contributes to the quantitative and qualitative sustainable management of interlinked surface waters, groundwater and aquifers and coastal waters, thus ensuring the social and economic development that is also dependent on vitally important ecosystems.
However, a change in strategic direction on water is underway. Given the complexities implied in the implementation of IWRM, the concept of IWRM has incorporated the concept of 'Water for Growth and Development, adaptive management, water, food and energy nexus, etc. This concept re-emphasises that water cannot be dealt with in isolation, but requires a high degree of collaboration and engagement between the ministry responsible for water ministries and the ministries responsible for driving social and economic development, such as ministries of infrastructure, energy, mining, agriculture and trade. Water is therefore seen more as a horizontal cross-cutting issue within many facets of development, rather than as a stand-alone 'sector'.
Fundamental global challenges affect KSA 1 such as climate change, population growth and urbanisation. The results of these drivers are clear pressure manifestations such as demand far exceeding available freshwater resources, increased competition between sectors and deteriorating water quality.
The current situation in which there is, perpetually growing water demands and competition is leading to deepened water insecurity in certain locations. The ability and preparedness of sectors to engage on water issues has improved with the enhanced comprehension of water shortages yet water management is not as high as it should in the national political agenda. Water quality remains to be a concern where causes and management options are well researched but the need to implement control and/or incentive measures require additional work. Deteriorating water quality has overall compromised water resource integrity and its resilience in adapting to natural as well as man induced impacts to become sustainable and be able to support national as well as regional sustainable development.
Decisions around allocations and de-allocations, water tenure and trade-offs in satisfying demands for food security, energy and sustained environmental services will become much more heightened focusing largely on understanding the barriers to policy implementation at the different levels. The democratisation of the management of water resources through decentralised management will need to amplify the developmental dimension for ensuring equitable access to water and its use and economic benefits.
The KSA will support the knowledge base in addressing the above impacts through 5 focus areas as defined in the diagram below from institutional arrangements, reform and governance, catchment assessment and planning, water quality management, water resource protection, to water resources and climate.
Because of this broad scope, all the above WRC Knowledge Tree goals are strongly, directly, and sometimes indirectly, served by this KSA.
Previous research will inform the research agenda for the next 5 years. Integration, transdisciplinarity, synthesis, adaptive management absorptive capacity and requisite level of simplicity will be the principles that will guide the future research approach. Emerging research areas are around water security and trade-offs between water, food, energy and the environment, the need for an equity framework and revised tenure systems for water, complete value chain assessments from water resources to raw water supply to tap water supply to wastewater treatment and back to freshwater systems or the coast. This should create stronger links with KSA3, 2 and 4 through most of the defined WRC flagship programmes; the water sensitive Urban Design and the wastewater as a resource, the green village/model catchment as well as the water, energy food nexus.
Video Clip: http://www.youtube.com/watch?v=iuEbPYIu0YE
Overview and description of thrusts and programmes
Scope: This thrust focuses on articulating the thinking for the new roles and responsibilities of the various stakeholders, based on catchment and water management area boundaries. The marked shift from central management of resources to a more localised scale is critical to the main founding concepts of IWRM. The defined management boundary based on watershed boundaries is another fundamental provision in IWRM as a concept. This thrust will support the suitable implementation of IWRM in South Africa. The further articulation of the NWA for the benefit of all South Africans and the fulfilment of the developmental role of the state within the water resource limitations will be investigated. Lessons learnt and evaluations of the IWRM applications in South Africa to date will be part of this portfolio, focusing on home-grown approaches and experiences in water resource management.
Programme 1:
Water governance and institutional reforms
Scope: The principle of subsidiarity, or, as sometimes referred to, democratisation of water resource management, has brought about challenges, both conceptually and in terms of application. Although current reforms in South Africa are based on sound IWRM principles, to date the implementation thereof continues to break new ground, proving that institutional engineering cannot provide a one-size-fits-all solution to the new management paradigm. Further understanding and research are hence needed to learn and to decide on best practice as defined in the South African or similar socio-economic settings.
Programme 2:
Compliance and enforcement
Scope: For the implementation of state-of-the-art legislation like the NWA, a matching enforcement and compliance regime needs to be in place to ensure effective implementation. The regulatory environment in the South African water sector is in its infancy and requires substantial support from research in creating the understanding and knowledge for informed decision making. Benchmarking and best practice are crucial here to accelerate learning.
Programme 3:
Pricing and financing WRM
Scope: The issues of financial sustainability, affordability of charges by users, transparency and corporate governance are becoming central in the decentralisation era. The new infrastructure agency responsible for new developments and maintaining national assets provides good ground-breaking research opportunities, especially to assess if water tariffs can indeed pay for managing and sustaining water resources. Does pricing water and introducing the water resource charge exclude the poor and will it further cripple local government from delivering services? The waste discharge charge is another serious introduction to the water sector fraught with considerable challenges. This programme can project and assess such issues.
Programme 4:
Transboundary water resources
Scope: This programme will provide tools and guidelines for resolving potential water-centred conflicts for the management of shared international rivers and transboundary aquifer systems, including development of appropriate institutional forms and functions, development and harmonisation of policy and regulation in shared river basins, strategies for knowledge-sharing and joint management of shared river basins. A need has been identified to define the roles and interrelationships between local WRM institutions and international basin organisations.
Programme 5:
Future scenarios
Scope: This activity has been assigned a separate programme to ensure that local South African expertise is qualified to explore future scenarios and answer the 'what if' questions in support of reflection and evaluation of national policy applications. Projecting the water resource management and development institutional arrangements landscape 10 or 15 years from now would be of interest to decision makers to define policy reviews and enhance decision making. This is considered as one of the tools for assisting in learning and allowing for dialogue to take place around options. Other tools exist which will also be explored in due course such as Game Theory especially in support of water allocation options.
Thrust 2: WATER RESOURCE ASSESSMENT AND PLANNING
Scope: This thrust focuses on developing a scientific understanding of the hydrological cycle (and inter-linkages) in order to promote systematic water assessment and planning. The thrust will promote better understanding of the variability of the quantity and quality of water available for use and development in South Africa. Recent changes in national water resource infrastructure management, the awareness of the poor state of water resource infrastructure and increased knowledge of water resource planning needs are expected to receive attention, through the support of competent and sustainable solutions. Sound water resource assessment and planning can only be achieved with reasonably accurate and consistently recorded and processed data and information.
Programme 1: Catchment data, information systems and predictive tools
Scope: This programme will support the provisions of Chapter 14 of the National Water Act, especially Part 2: National Information Systems on Water Resources. This programme is focused on supporting the national initiative for improving the available water resource information, better management of the information and improved information dissemination to stakeholders. It will establish direct linkages to the national information systems as well as identifying and resolving water resource information gaps. In this programme researched water resource information will be integrated into the national information system that is being established by DWA. The programme will also support the process of decentralising identified water resource data and information from broader national perspectives to detailed and highly-resolved local and catchment scales. The need to prepare the country to cope with global climate change and regional climate variability is of paramount and strategic importance. Taking the view that water is South Africa's key resource implies the need to adapt water resource management progressively as global climate change progresses, in order to maintain optimal levels of both resource protection and beneficial use of water for society. The development of coping strategies will require the development of informed, quantitative scenarios of potential impacts, at regional and catchment level, on rainfall regimes and rainfall variability, hydrological and geohydrological regimes, water availability and reliability, water quality, ecosystem structure and functions and ecological processes. This programme will therefore focus on the following key issues: select and use GCM-generated scenarios of global climate change of appropriate confidence level as a basis for development of model projections; improve techniques for downscaling of scenarios from global (GCMs) to regional and catchment scales to enable or support management at higher resolution scale and to ensure high level of reliability and robustness; improve on detection and attribution of anthropogenic impacts of climate change in the Southern African context in order to distinguish those from natural climate variability and change-related impacts. The programme will also deal with: the choice of relevant and appropriate climate indicators and variables as well as monitoring systems that need to be in place in this regard; determination of the frequency and magnitude of resultant extreme rainfall and flow events; use of existing conceptual and numerical models to utilise global change-related, downscaled, hydro-climatic information effectively, to provide information regarding likely inter-related land-use, ecosystem, hydrological (including geohydrological), water yield and water quality changes at regional/catchment level; modification of existing management strategies and tools for adaptation purposes; determining the likely socio-economic impacts for a given structure of society in Southern Africa; and appropriate technological, social and political coping strategies. Other areas that will be attended to include: improving understanding of and forecasting of the variability of rainfall, flow and groundwater recharge, as the ability to forecast at very short time scales would greatly benefit flood management and disaster mitigation and adaptation activities; and improving the understanding of global climate change impacts and vulnerability for the purposes of better informing the nation on permanent changes of the climate which require long-term solutions and adaptation actions. Through this programme, support will be provided for weather and climate disaster mitigation programmes at various levels which will include regional, national and provincial as well as other, more localised, scales.
Programme 2: Surface water / groundwater hydrology
Scope: This programme focuses on developing and utilising integrated hydrological approaches in surface water and groundwater assessments, water resource explorations, planning and management. It will take advantage of gains made in improved understanding of groundwater and surface water hydrological processes as well as the availability of better hydrological data, especially the various forms of more accurate remotely-sensed data with better coverage. Through this programme, strategic partnerships with international expertise in both groundwater and surface water hydrological research will be encouraged to flourish. Hydrological tools that have been developed in the past are expected to be upgraded, redeveloped or replaced by tools that are more suited to the current data availability, the improved knowledge and the recent technological advances in hydrological modelling. In this programme, the continued deterioration of hydrological gauging processes and other installed earth measurement devices will be addressed through the intensive use of new data sources from remote sensing coupled with the limited earth-based measurements.
Programme 3: Water resource planning flood and drought management
Scope: This programme will address water resource planning for the purposes of improved water allocation, better management of water use activities and to ensure secure, sustainable and adequate national water resources. It is also focused on the development of tools that will address planning gaps such as the absence of reliable information in ungauged areas and the persistent record gaps which exist in present data sets. The programme will promote a deliberate shift towards the development of water system plans that will benefit from real-time, historic and stochastic data on a countrywide basis. Impacts of climate change on water resources and the planning processes will be accounted for so as to ensure a proactive approach and allow for national preparedness. Integration will also be achieved through aligning this programme to wider national water resource planning needs as expressed in the objectives of Water for Growth and Development as well as through accounting for other factors, which include poverty alleviation, economic benefit, empowerment and the importance of meeting the Millennium Development Goals. Research on the planning of water resources will also address the information gaps in the understanding and subsequent utilisation of seawater in building water resource security. Saline water, brackish water, and other water bodies that can be purified and made available for regular water uses will be investigated and included as part of future water resource plans.
Flooding and drought are major natural hazards to human society and have important influences on social and economic development. The most vulnerable communities are often those who are poorly resourced since they barely have means to cope, and also often live in informal settlements notorious for being drought- or flood-prone with poor infrastructure. This programme focuses on research that will result in the development and implementation of integrated institutional frameworks and technological tools to reduce and combat floods and their negative effects, while enhancing positive flooding patterns that are important to the natural ecosystem. Research related to drought management will focus on integrated tools and strategies for early identification and mitigation of the social and economic impacts of drought, with the aim of supporting collaborative, multi-institutional processes and programmes.
Programme 4: Water resource infrastructure
Scope: There is an increasing need to develop systems for the efficient maintenance of the aging water infrastructure as the demand for the development of new and expensive water resource infrastructure is increasing due to the growing economy and population growth. This programme will seek to develop strategies and priorities for water resource infrastructure development and management to address the uncertainties and risks associated with climate change. While built infrastructure development such as dams, reservoirs, irrigation and flood barriers, are important options for addressing these issues, this programme will also explore the potential use of natural infrastructure such as wetlands, floodplains, artificial recharge (to aquifers), etc., to complement built infrastructure (but with an added advantage of healthy ecosystems).
Scope: Secure and sustainable access to water is essential for a wide range of critical uses such as human health, economic growth, food security, etc. However, in semi-arid environments such as South Africa, conventional water sources are not sufficient to meet the ever growing demand. Therefore, the understanding and assessments of alternative sources of water such as fog water, desalination, water transfers, etc is essential. The programme will also promote research on the transboundary water issues (wrt water quantity and quality) to ensure water security for South Africa. Other issues to be researched include cooperation on the shared surface water and groundwater resources, as well as the integration of social, economic, and environmental considerations as key components of sustainable water resource development. Climate change risk management seeks to promote sustainable development by reducing vulnerability associated with climate risks. The approach involves a range of actions including reduction of vulnerabilities or enhancement of resilience amongst people and societies, protection of ecosystem goods and services, early response systems, strategic diversification, and improved institutional capacities. Climate adaptation refers to the ability of the system to adjust to climate change, variability or extreme to moderate potential damage or to cope with the consequences. This programme is aimed at reducing vulnerabilities among communities and people through development or implementation of systems, tools, approaches and strategies (some of which would have been developed under Programme 1, such as modification of structures or implementation of early preparedness programme for extreme events). Protection or restoration of ecosystem goods and services that are vulnerable to climate variability and change as well as strengthening capacity of people and institutions are some of the techniques that will be investigated under this programme. Climate risk management strategies to be developed under this programme also aim to maximise opportunities in climate-sensitive economic sectors, even under uncertain climatic conditions of high variability. The programme could also deal with implementation of capacity building and awareness programmes including sharing of climate information as part of a broader adaptation programme.
Scope: This thrust acknowledges the significant water quality problems in our natural water resources. Water quality is generally reflected in concentrations of substances and microorganisms, physico-chemical attributes, radioactivity, as well as biological responses to these. These concentrations will accumulate over a long-term to result in impacts that pose a risk to the human health, environment (ecology) as well as the economy. Within each of the programmes in this thrust, research will focus on three broad fronts, namely, (1) consolidation and knowledge transfer; (2) alertness to emerging issues and (3) risk to human health, environment and the economy.
Consolidation is necessary of the vast amount of existing water quality-related research outputs in priority domains. The primary aim will be to distil effective decision support for management of our water quality problems. Emphasis will be more on formulating solutions (development of monitoring tools, methods, reporting systems including real-time and early warning/ prediction) than on formulating problems. By actively sharing knowledge with decision makers and other stakeholders (Community of Practice, Local Government, Departments of Health, Environment, Agriculture etc.), and working closely with them, the decision support must explicitly address their absorptive capacity in its broadest sense. On the one hand, solutions need to be based on a thorough holistic and realistic examination of likely consequences of implementation of those solutions. This must create confidence that risks of unintended consequences will be minimised. However, on the other hand, solutions must cater for the inherent complexity (and hence uncertainty) of both the institutional and natural environment. Research will also be encouraged that heightens awareness, and/or recommends management approaches, specifically to important emerging issues, i.e., those potential or recognised concerns that are either not addressed, or are only partly addressed, in current water quality management practice and research. High priority issues include those of national concern, those for which the frequency or probability of adverse conditions occurring is high, and the consequences are severe, and so on. Water quality necessarily cuts across various KSAs as well as thrusts within this KSA. The scope of this particular thrust focuses primarily on water quality of inland surface waters, its management and the risks associated with the water quality impacts.
Water quality monitoring
Scope: Monitoring data is crucial to sustainable management because it provides information on the current status and trends and can be useful in the prediction of the future. Creative yet soundly-scientific approaches to monitoring are required that optimise information, minimise costs (inclusion of screening techniques that are less costly and detailed monitoring at problem areas) and that can be useful across all spheres of authority (including local/ catchment, provincial/ regional, national and cross boundary).
Water quality modelling
Scope: The programme will focus on the development of the community of practice (researchers, model developers, water resource quality managers) in order to collectively identify areas of support that can be addressed through research. This will however, not limit research into new models for the prediction of the future. The work in this programme is aimed at encouraging a move to open-source modelling platforms that benefit individual model developers, while allowing effective interfacing with other modelling modules in a way that provides integrated, scientifically-defensible water quality information. Business models of such platforms must be as much in the interests of users of such information (e.g. catchment management agencies) as the service providers and modellers.
Impacts on and of water quality
Scope: This programme will focus on identifying, characterising, and understanding (1) the changes (current, long-term and climate related) in the state of water quality in our water resources (use available data to support the efforts of annual reporting on the health of the water resources as required by the NDP, NWA etc.) associated with either point or non-point pollution sources, and (2) the associated impacts (human health, environment and economy) of such compromised water quality.
Thrust 4: WATER RESOURCE PROTECTION
Scope: Reliable supply of good quality water is required for the health, environmental, social and economic wellbeing of the country. The National Water Act of 1998 recognises that protection in relation to a water resource means: (1) maintenance of the quality of the water resource to the extent that the water resource may be used in an ecological sustainable way; (2) prevention of the degradation of the water resource, and (3) the rehabilitation of the water resource. There are significant gaps in our knowledge on how to protect our water resources in an integrated manner. While Thrust 3 will look mainly at the quality of the water within our systems this thrust focuses on protecting the water resources, by reducing the quantity of harmful materials reaching the water resources, within a broader framework for all uses. Broadly, research in this thrust focuses on the generation of knowledge and understanding of the catchment processes and land use activities that influence the quality and quantity, negatively or positively, of the water resources. Scientific, technological and institutional approaches that will help to characterise and address these problems include: (1) assessment, monitoring and prediction; (2) tools and control strategies; (3) innovation to assist with prediction and control; and (4) implementation and technology transfer options. The following programmes support this thrust:
Source water protection
Scope: Source water protection refers to protecting source water (water from dams, wetlands, rivers, aquifers, etc.) from contamination and overuse. Specific driving forces, or a combination thereof, which have an impact on water resources will be researched. Integrated protection strategies and approaches will be researched and tested. The development of source water planning, control and response strategies, to minimise adverse impacts on source waters by reducing pollution risks and securing water availability, is a key component of this programme. The source water protection approach will look at, among others, land use (see Programme 2 below), vulnerability assessments and catchment plans and strategies (for both surface and groundwater).
Land-water linkages
Scope: This programme will enhance our knowledge on the interaction of water and land at various scales. This programme will focus on the driving forces (new developments, emergency spills, erosion, leaks, soil enhancements, etc.) that can impact water resources from land-based activities. The aim is also to research, evaluate and develop common regulatory tools to overcome the challenge of different technical and procedural approaches for water resource and land use management, in order to enhance our water resource protection capabilities. Techniques to delineate, protect and remediate areas, and/or the activities occurring within these areas, will be researched. Research will also be bi-directional where potential impacts on water resources from land-based activities or processes are investigated as well as the impact of water resources on land-based activities (e.g. floods and droughts).


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Subject: Re: Order of paying off? | Date: 10/22/2013 9:58 AM | |
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AJ, this is gold! Thanks. Everything AJ contributes to this board is gold. She is a genius. So is JAFO. You got two of the best contributors responding to your issue. It was a well presented question and fantastic working through of the options. I hope this thread gets read by tons of people in the future. It's the kind of short and sweet thread that opens the eyes and minds of anybody keen enough to see the value therein. xtn |
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