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J. Biol. Chem., Vol. 283, Issue 12, 7421-7428, March 21, 2008

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Eag1 and Bestrophin 1 Are Up-regulated in Fast-growing Colonic Cancer Cells^*

From the Institut für Physiologie, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany

Received for publication, May 7, 2007 , and in revised form, December 31, 2007.

	ABSTRACT

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Ion channels like voltage-gated ether-á-go-go (Eag1) K⁺ channels or Ca²⁺-activated Cl^– channels have been shown to support cell proliferation. Bestrophin 1 (Best1) has been proposed to form Ca²⁺-activated Cl^– channels in epithelial cells. Here we show that original T₈₄ colonic carcinoma cells grow slowly (T₈₄-slow) and express low amounts of Eag1 and Best1, whereas spontaneously transformed T₈₄ cells grow fast (T₈₄-fast) and express high levels of both proteins. Both Eag1 and Best1 currents are up-regulated in T₈₄-fast cells. Eag1 currents were cell cycle-dependent with up-regulation during G₁/S transition. T₈₄-slow, but not T₈₄-fast, cells formed tight monolayers when grown on permeable supports. RNA interference inhibition of Eag1 and Best1 reduced proliferation of T₈₄-fast cells, whereas overexpression of Best1 turned T₈₄-slow into fast-growing cells. Eag1 and Best1 improve intracellular Ca²⁺ signaling and cell volume regulation. These results establish a novel role for bestrophins in cell proliferation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
An increasing number of studies demonstrate proliferative effects of membrane ion channels (1). Voltage-gated K⁺ channels and other types of K⁺ channels are expressed in numerous types of tumors, where they may serve as diagnostic and prognostic markers and potential drug targets (2–4). Eag1 channels are probably necessary for progression through the G₁ phase and G₀/G₁ transition of the cell cycle (5). A recent study demonstrates the hyperpolarizing effects of Eag1 and other Kv channels on the membrane voltage of T₈₄ cells, which supports intracellular pH regulation and Ca²⁺ increase necessary for proliferation (6).

Much less is known about the role of Ca²⁺-activated Cl^– channels in cell proliferation (7). This may be due to the ongoing controversy regarding the molecular nature of Ca²⁺-activated Cl^– channels (8). A family of putative Ca²⁺-activated Cl^– channels has been identified that also controls cell-cell adhesion, apoptosis, and the cell cycle. However, the structure and biophysical properties of these channels are poorly understood (9, 10). Recent studies defined bestrophin proteins as bona fide Ca²⁺-activated Cl^– channels. The Cl^– currents generated upon expression of bestrophin show many of the properties found for native Ca²⁺-activated Cl^– currents (11–13). However, bestrophins have also been proposed to function as regulators of voltage-gated L-type Ca²⁺ channels (14). Our own ongoing work in epithelial cells supports both concepts, in that bestrophins may form part of a Cl^– channel complex or may couple intracellular Ca²⁺ signals to Cl^– channels of unknown molecular identity (15).

In the present report we demonstrate that both voltage-gated Eag1 K⁺ channels and bestrophin 1 (Best1) Cl^– channels support proliferation of fast-growing T₈₄ colonic carcinoma cells. The fast-growing T₈₄ cell clone was obtained through spontaneous transformation of slow-growing T₈₄ cells. In contrast to slow-growing T₈₄ cells, transformed cells do not form polarized monolayers and show a remarkable up-regulation of Eag1 and Best1 expression. We demonstrate that both currents are in charge of enhanced cell proliferation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture and Proliferation Studies—Human colorectal carcinoma epithelial T₈₄ cells (ATCC, Manassas, VA) were grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium (1:1) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen, Karlsruhe, Germany) at 5% CO₂ and 37 °C. Cells were seeded on fibronectin (Invitrogen)/collagen (Cellon)-coated glass coverslips or permeable supports (Snapwell, Costar). Typically these cells grow slowly as polarized monolayers (T₈₄-slow). Because of spontaneous transformation, a T₈₄ cell line was selected that grew remarkably faster (T₈₄-fast). For proliferation assays, cells were plated at a density of 2000 cells/0.35 cm² and incubated 2 days later with either niflumic acid (0.01–100 mM) or astemizole (0.5–5000 nM). Cell proliferation was assessed by 5-bromo-2'-deoxyuridine (BrdUrd)³ incorporation using an enzyme-linked immunosorbent assay kit (Roche Diagnostics, Penzberg, Germany) and cell counting. The cell number was assessed after fixation in 3.7% formaldehyde and 0.5% Triton X-100 for 30 min at room temperature and after staining with Mayer's hemalaun (Merck, Darmstadt, Germany) for 5 min. Digitized microscopic images were taken (Fluovert FS, Leitz), and nuclei were counted using imaging software (TINA version 2.09g). Toxicity of the blockers was assessed using trypan blue (Sigma). Each experiment was performed at least in triplicate.

Cell Cycle, FACS Analysis, Caspase Assay, and RT-PCR—Cells were synchronized into early G₁ by 24-h serum starvation. Incubation in thymidine (2 mM) (Sigma) halted the cells at G₁/S transition. 36-h treatment with demecolcine (0.05 µg/ml) (Sigma) synchronized cells into M phase. Synchronization was verified by FACS (COULTER EPICS® XL-MCL, Beckman) using propidium iodide staining of the DNA (Sigma). Apoptosis was analyzed after 8- and 24-h incubation with the protein kinase C inhibitor staurosporine (1 µM) (Sigma) and with detection of cleaved caspase-3 in Western blots using rabbit anti-human caspase-3 antibody (1:1000) (Cell Signaling Technology, Inc., Danvers, MA). For RT-PCR, total RNA was isolated using NucleoSpin RNA II columns (Macherey-Nagel, Düren, Germany). 1 µg of total RNA was reverse-transcribed for 1 h at 37 °C using random primer and RT (Moloney murine leukemia virus reverse transcriptase, Promega, Mannheim, Germany). For PCR, the following primers were used: hEag1 (KCNH1, GenBank^TM accession number NM_002238 [GenBank] ), 5'-CGCATGAACTACCTGAAGACG-3' (sense) and 5'-TCTGTGGATGGGGCGATGTTC-3' (antisense), 560 bp; and hBest1 (VMD2, GenBank^TM accession number NM_004183 [GenBank] ), 5'-CTGCTCTGCTACTACATCATC-3' (sense) and 5'-GTGTCCACACTGAGTACGC-3' (antisense), 552 bp. The conditions were 94 °C for 2 min and 35 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min. PCR products were visualized by loading on 2% agarose gels and verified by sequencing.

Down-regulation of Best1 and Eag1 Expression by RNAi—Three different batches (A, B, and C) of duplexes of 21-nucleotide RNAi with a 3'-overhanging TT were purchased from Invitrogen. The sense strands of the RNAi used to silence the Best1 gene were 5'-AAUUCCUGUCGACAAUCCAGUUGGU-3' (A), 5'-AUCUCAUCCACAGCCAACAGGGACA-3' (B), and 5'-UAAAUAAAGCGGAUGAUGUAGUAGC-3' (C). RNAi sequences for Eag1 are shown in Ref. 6. Fluorophore-labeled RNAi and exposure to the transfection reagent Lipofectamine 2000 (Invitrogen) served as controls. After 48 h, cells were further processed in proliferation assays for Western blotting.

Detection of Eag1 and Best1 by Western Blotting—T₈₄ cells were homogenized in lysis buffer (150 mmol/liter NaCl, 50 mmol/liter Tris, 100 mmol/liter dithiothreitol, 1% Nonidet P-40, and 1% protease inhibitor mixture) (Sigma). Equal amounts of total protein (50 µg) were separated by 7% SDS-PAGE, transferred to Hybond-P (Amersham Biosciences, Freiburg, Germany), and incubated with either rabbit anti-human Kv10.1 (Eag1) (Alomone Labs, Jerusalem, Israel) or rabbit anti-human VMD2 (Best1) (15) antibodies. Proteins were visualized using goat anti-rabbit IgG conjugated to horseradish peroxidase (Acris Antibodies, Hiddenhausen, Germany) and ECL (Amersham Biosciences). Signals were detected by a Fluor-STM MultiImager (Bio-Rad).

Measurement of the Intracellular Ca²⁺ Concentration and Cell Volume—T₈₄ cells were loaded with 2 µM Fura-2/AM (Molecular Probes, Eugene, OR) in Opti-MEM I medium (Invitrogen) with 2.5 mM probenecid for 1 h at room temperature. Fluorescence was detected in cells perfused with Ringer's solution containing 2.5 mM probenecid (Sigma) at 37 °C using an inverted microscope (IMT-2, Olympus, Nürnberg, Germany) and a high speed polychromator system (VisiChrome, Puchheim, Germany). Fura-2 was excited at 340/380 nm, and emission was recorded between 470 and 550 nm using a charge-coupled device camera (CoolSnap HQ, Visitron). [Ca²⁺]_i was calculated from the 340/380 nm fluorescence ratio after background subtraction. The formula used to calculate [Ca²⁺]_i was [Ca²⁺]_i = K_d x (R–R_min)(R_max–R) x (S_f2/S_b2), where R is the observed fluorescence ratio. The values R_max and R_min (maximum and minimum ratios) and the constant S_f2/S_b2 (fluorescence of free and Ca²⁺-bound Fura-2 at 380 nm) were calculated using 2 µmol/liter ionomycin (Calbiochem), 5 µmol/liter nigericin, 10 µmol/liter monensin (Sigma), and 5 mmol/liter EGTA to equilibrate intracellular and extracellular Ca²⁺ in intact Fura-2-loaded cells. The dissociation constant for the Fura-2·Ca²⁺ complex was taken as 224 nmol/liter.

Patch Clamping—Cell culture dishes were mounted on the stage of an inverted microscope (IM35, Zeiss) and perfused continuously (37 °C) with Ringer's solution. Patch clamp experiments were performed in the fast and slow whole cell configuration. Patch pipettes had an input resistance of 2–4 megaohms when filled with a solution containing 30 mM KCl, 95 mM potassium gluconate, 1.2 mM NaH₂PO₄, 4.8 mM Na₂HPO₄, 1 mM EGTA, 0.758 mM calcium gluconate, 1.034 mM MgCl₂, 5 mM D-glucose, and 3 mM ATP. pH was adjusted to 7.2; Ca²⁺ activity was 0.1 µM. The access conductance was monitored continuously and was 60–120 nanosiemens. Currents (voltage clamp) and voltages (current clamp) were recorded using an EPC7 amplifier (HEKA, Darmstadt, Germany), an LH1600 interface and PULSE software (HEKA), and Chart software (ADInstruments, Spechbach, Germany). In intervals, membrane voltages were clamped in steps of 10 mV from–50 to +50 mV relative to resting potential. Membrane conductance G_m was calculated from the measured current (I) and clamped membrane voltage values according to Ohm's law.

Cell volume was measured directly by a Zeiss Axiovert 200M/ApoTome microscope using Axiovision software or was assessed by fluorescence measurements in calcein (2 µM; Molecular Probes)-loaded cells at an excitation of 500 nm and emission of 520–550 nm. The experiments were done in the presence of 2.5 mM probenecid. The control isotonic solution (290 mosM) was prepared by adding 120 mM mannitol. The hypotonic (170 mosM) and control isotonic solutions contained 85 mM NaCl.

Materials and Statistical Analysis—All compounds used were of the highest purity grade available. Astemizole, niflumic acid (NFA), DIDS, tetrapentylammonium, carbachol, and ATP were all from Sigma. Student's t test (for paired or unpaired samples as appropriate) and analysis of variance (ANOVA) were used for statistical analysis. p < 0.05 was accepted as significant.

	RESULTS

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Fast-growing Colonic Carcinoma Cells (T₈₄-fast) Express Eag1 and Best1—T₈₄ cells typically grow in slowly expanding patches (T₈₄-slow). At passage number 73, the cell line spontaneously changed its growth pattern, i.e. the cells grew remarkably faster (T₈₄-fast) as single cells, non-polarized and above each other (Fig. 1, A and B). T₈₄-slow cells formed tight mono-layers with a transepithelial resistance of 2.2 ± 0.3 kiloohms x cm² (n = 25) when grown on permeable supports, whereas T₈₄-fast cells did not (0.12 ± 0.07 kiloohms x cm²; n = 23). Thus, growth pattern or phenotype was not changed by the way the cells were grown. We examined apoptosis in both cell lines by Western blotting and analyzed uncleaved and cleaved caspase-3. Small amounts of cleaved caspase-3 were detected in T₈₄-slow, but not in T₈₄-fast, cells after treatment with the protein kinase C inhibitor staurosporine (1 µM) (Fig. 1C).

View larger version (52K): [in this window] [in a new window]   FIGURE 1. Slow- and fast-growing T84 cells. A, typical growth patterns of T84-slow cells as obtained from ATCC and spontaneously transformed T84-fast cells. Scale bars indicate 50µm. B, proliferation curves for T84-slow and T84-fast cells as obtained by cell counting (n = 4). C, Western blot for uncleaved and cleaved caspase-3 in T84-slow and T84-fast cells after 8- and 24-h incubation with 1 µM staurosporine (stauro). D, Western blot analysis of the expression of Eag1 and Best1 in slow- and fast-growing T84 cells. Actin indicates equal loading of the gel. The bar graph indicates large up-regulation of ion channel expression in T84-fast cells. E, whole cell currents measured in slow- and fast-growing T84 cells under control (con) conditions (upper panels) and corresponding current/voltage relationships (n = 8 for both cell types; lower panels). Replacement of extracellular Cl– by gluconate (5Cl) shifted the I/V curve to more depolarized clamp voltages only in fast-growing T84 cells, indicating the presence of a base-line Cl– conductance. RT-PCR and Western blotting were performed at least in triplicate. Vc, clamped membrane voltages.

Eag1 Controls Proliferation of Fast- but Not Slow-growing T₈₄ Cells—Eag1 has been demonstrated to support proliferation of several different cell types (6, 16). We therefore examined if high expression levels of Eag1 correlate with increased proliferation of T₈₄-fast cells. To that end, T₈₄-slow and T₈₄-fast cells were treated with three different batches (A–C) of siRNA for Eag1. Incubation of the cells with either fluorescently labeled scrambled oligos or lipid (transfection reagent) and nontreated cells served as controls. Measurement of BrdUrd incorporation clearly indicates inhibition of proliferation of T₈₄-fast cells after treatment with siRNA for Eag1 (Fig. 2A). siRNA treatment of T₈₄-slow cells did not affect cell proliferation, suggesting a proliferative function of Eag1 only in T₈₄-fast cells. Notably, Eag1 expression was significantly up-regulated by the readdition of 10% fetal calf serum in serum-starved cells (data not shown). Using the inhibitor astemizole (5 µM), we examined the contribution of Eag1 to whole cell currents measured in T₈₄-slow and T₈₄-fast cells. Astemizole inhibited whole cell currents in both cell types; however, the effect was more pronounced in T₈₄-fast cells, and thus astemizole-sensitive whole cell currents were significantly larger in T₈₄-fast cells (Fig. 2, B and C). The normalized conductance/voltage relationship for Eag1 (astemizole-sensitive whole cell conductances) was not different for T₈₄-fast (solid line) and T₈₄-slow (dashed line) cells (Fig. 2D).

A previous report (6) supplied evidence that hyperpolarized Eag1 currents assist in the increase of intracellular Ca²⁺ ([Ca²⁺]_i) when cells are stimulated with secretagogues (such as ATP and carbachol) or mitogens and may thereby support proliferation. Both Ca²⁺ release from endoplasmic reticulum stores and Ca²⁺ influx through store-operated Ca²⁺ channels depend on Kv channel function as reported earlier (6). We compared the increase in [Ca²⁺]_i in Fura-2-loaded T₈₄-slow and T₈₄-fast cells upon stimulation with ATP (100 µM). ATP binds to purinergic P2Y₂ receptors and thereby induces a peak and plateau [Ca²⁺]_i increase in both cell lines (Fig. 2E). The peak [Ca²⁺]_i increase was doubled in T₈₄-fast cells when compared with T₈₄-slow cells, which suggests a role of Eag1 in Ca²⁺ signaling in T₈₄-fast cells (Fig. 2, E and F). Enhanced Ca²⁺ signaling is not due to an increased expression of the abundant purinergic receptor P2Y₂ in T₈₄-fast cells. In contrast, P2Y₂ expression was slightly higher in T₈₄-slow cells (Fig. 2G).

Eag1 Activity in T₈₄-fast Cells Is Cell Cycle-dependent—In other cell types Eag1 activity varies during the cell cycle (16). Therefore we examined cell cycle dependence of Eag1 in T₈₄-fast cells. Cells were synchronized in early G₁ (eG₁), G₁/S, or M phase (see "Materials and Methods"), and synchronization was verified by FACS analysis (Fig. 3A). We found that astemizole-sensitive whole cell currents were augmented in cells synchronized in G₁/S when compared with eG₁ or M phase (Fig. 3B). T₈₄ cells also express other voltage-gated K⁺ channels such as Kv1.5 and Kv3.4, which are blocked by the inhibitor tetrapentylammonium (6). However, in contrast to Eag1, tetrapentylammonium-sensitive whole cell currents were not cell cycle-dependent (data not shown). We further compared the increase in [Ca²⁺]_i in T₈₄-fast cells synchronized in eG₁ and G₁/S phase. Both peak and plateau [Ca²⁺]_i increases were significantly enhanced in G₁/S cells. Moreover, the Eag1 blocker astemizole inhibited peak and plateau [Ca²⁺]_i increases in both cell cycle phases, but the effect of astemizole on plateau [Ca²⁺]_i was augmented in the G₁/S phase (Fig. 3C). These results supply evidence for cell cycle-regulated Eag1 currents in T₈₄-fast cells and cell cycle-dependent effects of Eag1 on Ca²⁺ signaling.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. Eag1 supports proliferation of fast-growing T84 cells. A, proliferation of slow- and fast-growing T84 cells as measured by BrdUrd (BrdU) incorporation. Cells were treated with three different batches (A–C) of siRNA for Eag1. Incubation of the cells with fluorescently labeled scrambled oligos or transfection reagent only (Lipids) and non-treated cells (Control) served as controls. Assays were performed at least in triplicate. *, significant inhibition of proliferation by RNAi in T84-fast cells (ANOVA). B, whole cell currents in T84-slow and T84-fast cells and the effect of the Eag1 inhibitor astemizole (Aste; 5 µM). con, control. C, summary of the calculated astemizole-sensitive whole cell conductances (GAste) in T84-slow (n = 8) and T84-fast (n = 9) cells. GAste was significant in both cell lines and was enhanced in T84-fast cells (unpaired t test). nS, nanosiemens. D, normalized conductance/voltage relationship for Eag1 (i.e. astemizole-sensitive whole cell conductance) measured in T84-fast (solid line) and T84-slow (dashed line) cells. Vc, clamped membrane voltages. E, increase of [Ca2+]i by stimulation with ATP (100 µM) in T84-slow and T84-fast cells. F, summary of the ATP-induced peak and plateau increases in [Ca2+]i in slow-growing (n = 59) and fast-growing (n = 69) T84 cells. G, RT-PCR analysis of mRNA expression for P2Y2 receptors (764 bp) in T84-slow and T84-fast cells. When compared with the internal β-actin standard, expression of P2Y2 receptors was slightly enhanced in T84-slow. H2O served as the control. For PCR conditions see "Materials and Methods." *, significant difference in peak [Ca2+]i increase in T84-fast cells. hP2Y2, human P2Y2.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. Eag1 operates as a cell cycle-regulated channel in fast-growing T84 cells. A, FACS analysis of fast-growing T84 cells synchronized in eG1, G1/S transition, and M phase (see "Materials and Methods"). Experiments were performed at least in triplicate. B, astemizole-sensitive whole cell conductances (GAste) measured in T84-fast cells synchronized in eG1 (n = 14), G1/S (n = 9), and M (n = 9) phase. GAste was significant during all cell cycle phases but was enhanced during G1/S (ANOVA). nS, nanosiemens. C, upper panels, summary of the peak and plateau [Ca2+]i increases induced by carbachol in T84-fast cells synchronized into eG1 and the effects of 0.5 µM astemizole (Aste; n = 73); lower panels, summary of the peak and plateau [Ca2+]i increases induced by carbachol in T84-fast cells synchronized into G1/S and the effects of 0.5 µM astemizole (n = 64). The effect of astemizole was significant in all series (paired t test) and was enhanced for the plateau [Ca2+]i increase in G1/S when compared with eG1 (unpaired t test). con, control. *, significant difference when compared with control; #, significant difference when compared with eG1.

View larger version (43K): [in this window] [in a new window]   FIGURE 4. Best1 supports proliferation of fast-growing T84 cells. A, Western blot analysis of Best1 in T84-slow and T84-fast cells treated with three different batches (A–C) of siRNA for Best1 (siBest1). Incubation of the cells with fluorescently labeled scrambled oligos or non-treated cells (control) served as controls. actin indicates equal loading of the gels. B, densitometric analysis of Best1 expression and the ratio between Best1 and actin expression. hBest1, human Best1. C, proliferation of slow- and fast-growing T84 cells as measured by BrdUrd (BrdU) incorporation. Assays were performed at least in triplicate. *, significant inhibition of proliferation by RNAi in T84-fast cells (ANOVA).

View larger version (26K): [in this window] [in a new window]   FIGURE 5. T84-fast, but not T84-slow, cells have a Cl– conductance. A, summaries for the base-line whole cell conductances (G) and the effects of the inhibitors of Ca2+-activated Cl– channels, NFA (10 µM) and DIDS (100 µM), in slow- and fast-growing T84 cells (n = 8–11). *, significant effects of NFA and DIDS (paired t test). µS, microsiemens. B, whole cell currents measured in T84-slow and T84-fast cells. Stimulation of the cells with ATP (100 µM) activated a whole cell conductance only in T84-slow, but not T84-fast, cells. con, control. C, summaries for the whole cell conductances (upper panels) and membrane voltages (lower panels) measured in T84-slow (n = 13) and T84-fast (n = 14) cells and the effects of ATP (100 µM). *, significant effects on whole cell conductance and membrane voltages (paired t test). D, summaries for the whole cell conductances measured in T84-slow (n = 9) and T84-fast (n = 8) cells and the effect of replacement of extracellular Cl– by gluconate (5Cl) and ATP (100 µM). *, significant activation of conductance by ATP in T84-slow cells and significant inhibition by gluconate (5Cl) (paired t test). nS, nanosiemens.

View larger version (32K): [in this window] [in a new window]   FIGURE 6. Expression of Best1 in T84-slow cells enhances Cl– currents and proliferation. A, Western blot analysis of human Best1 (hBest1) expression in T84-slow cells after transfection of 50 or 100 ng of Best1 cDNA or after mock transfection (50 and 100 ng) and comparison with the level of Best1 expression in T84-fast cells and non-transfected T84-slow cells. actin indicates equal loading of the gel. B, growth pattern of mock-transfected (–Best1) and Best1-transfected (+Best1) T84-slow cells. Scale bars indicate 50 µm. C, upper panels, whole cell currents measured in mock-transfected and Best1-transfected T84-slow cells and the effect of DIDS (100 µM); lower panels, summaries of the whole cell conductances and the effects of DIDS on mock-transfected (n = 7) and Best1-transfected (n = 7) T84-fast cells. *, significant inhibition of whole cell conductance by DIDS (paired t test). con, control; nS, nanosiemens. D, proliferation of mock-transfected (50 and 100 ng) and Best1-transfected (50 and 100 ng) cells. Non-transfected cells (Control) and cells exposed to the transfection reagent only (Lipids) served as controls. *, significant increase in proliferation (ANOVA). BrdU, BrdUrd.

View larger version (35K): [in this window] [in a new window]   FIGURE 7. Eag1 and Best1 support intracellular Ca2+ signaling and volume regulation. A, summary of intracellular peak (left) and plateau (right) Ca2+ increases in T84-fast cells upon stimulation with ATP (100 µM). Cells were treated with scrambled siRNA (scrbld), Eag1 siRNA (siEag1), or Best1 siRNA (siBest1)(n = 24–57). B, hypotonic (170 mosmol) cell swelling assessed by volume measurement in T84-slow and T84-fast cells (upper trace) and in T84-fast cells treated with Best1 siRNA or control transfection (green fluorescent protein (GFP)) (lower trace). n = 5 for each time point. C, change in calcein fluorescence due to hypotonic cell swelling. D and E, summaries for maximal change in fluorescence intensity and slope of recovery (from swelling) of T84-slow and T84-fast cells (n = 12). *, significant difference (paired t test).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Transformation of Colonic Epithelial Cells Increases Proliferation and Ion Conductances—T₈₄ colonic carcinoma cells are well established (17, 18). They grow slowly in patches and form tight and polarized monolayers when cultured on permeable supports. T₈₄ cells were used for numerous electrophysiological studies and resemble a model for electrolyte transport in the colonic epithelium (17). They show many aspects of native epithelial cells such as relatively hyperpolarized membrane voltage and Cl^– secretion by cAMP-dependent cystic fibrosis transmembrane conductance regulator channels. When stimulated by secretagogues such as ATP and carbachol that increase the intracellular Ca²⁺ concentration, predominantly K⁺ channels are activated, hyperpolarizing the membrane voltage. This is comparable with colonic crypt cells, which also activate Ca²⁺-activated K⁺ channels upon stimulation of basolateral muscarinic M3 and apical purinergic P2Y₂ receptors (18, 19). In contrast, T₈₄ cells that had undergone spontaneous transformation were remarkably different: they proliferated much faster, were unable to polarize or form tight monolayers when grown on permeable supports, showed typical features of malignancy, and had different membrane conductances.

Anderson and Welsh (20) as well as Morris and Frizzell (21) reported earlier that expression of ion channels in epithelial cells is significantly affected by the underlying substrate. Thus Ca²⁺-activated Cl^– channels were prominent in non-differentiated HT₂₉ colonic carcinoma cells but disappeared with polarization (22). Although T₈₄-fast cells did not polarize when grown on permeable supports, a change in Best1 expression upon differentiation of filter-grown HT₂₉ cells may explain earlier results (22).

A change in membrane ion conductance has also been reported for spontaneously transformed Madin-Darby canine kidney cells. These fast-growing cells express Ca²⁺-activated K⁺ channels that largely enhance cell migration (23). The fast-growing T₈₄ cells described in the present study showed a strong increase in the expression of the Best1 Cl^– channel along with Eag1 potassium channels. Eag1 currents were detected despite a relatively high (100 nM) Ca²⁺ concentration in resting cells. However, up to 1 µM [Ca²⁺]_i is required for complete inhibition of Eag1, and thus a fraction (40%) of the Eag1 conductance is still detectable in T₈₄-fast cells (24). Notably, the Cl^– channels were already active in non-stimulated cells, i.e. without purinergic stimulation. Additional increase of intracellular Ca²⁺ by ATP or carbachol did not further increase Cl^– conductance. A similar result was obtained when Best1 was overexpressed in T₈₄-slow cells and has also been observed when Best1 was expressed in HEK293 cells (8). This suggests that additional components may be required for Ca²⁺ regulation of Best1 currents, which may not be present in cancer cells. The presence of these DIDS-sensitive Cl^– currents along with astemizole-inhibited Eag1 K⁺ currents clearly augmented cell proliferation.

Ion Channels Induce Malignancy and Metastatic Cell Growth—Clonal selection of fast proliferating T₈₄ cells (T84SF) has been reported previously (25). These cells demonstrate invasive and metastatic cell growth when transferred into nude mice. Basal tyrosine phosphorylation and expression of Src kinase were enhanced in these T84SF cells (25). Along this line, the Src inhibitor PP2 reduced cell growth, invasion, and cell adhesion of T84SF cells (26). In Jurkat T lymphocytes, Src kinase controls voltage-dependent Kv1.3 K⁺ channels, which also affect cell proliferation (27). Src kinase may also be up-regulated in fast-growing T₈₄ cells and may be responsible for changes in membrane conductance. It will be interesting to examine in subsequent studies the impact of Eag1 and Best1 on cell migration and tissue invasion since we found previously that genomic amplification of Eag1 in human colorectal carcinoma is an independent marker of adverse prognosis (28).

How Do Ion Channels Determine Malignancy?—K⁺ and Cl^– channels are essential for cell migration and metastasis of cancer (29). It has been shown that enhanced intracellular Ca²⁺ activates Kv channels during intestinal wound healing (30). Cell migration and formation of tumor metastasis are due to fluctuations in the activity of membrane transporters and ion channels because they cause localized cell swelling and shrinkage (29). These changes in cell volume appear to be a prerequisite for cell migration and malignant invasion. There are only a few studies that have investigated the role of Cl^– channels in cell migration. Cl^– channels are probably necessary for cell movement because K⁺ transport needs to be accompanied by a counterion (23). Notably, bestrophin has been shown to operate as a volume-sensitive Cl^– channel (31).

A Novel Function of Bestrophin for Cell Proliferation?—Cl^– currents induced by expression of bestrophins share many of the properties attributed to Ca²⁺-activated Cl^– channels, such as anion selectivity of I^– > Cl^– and inhibition by NFA and DIDS (11–13, 15). However, there is an ongoing controversy regarding whether bestrophins are actually channel-forming proteins or, rather, regulators of ion channels. Moreover, other proteins have also been proposed as molecular candidates for the Ca²⁺-activated Cl^– channel (reviewed in Ref. 8). The present results would support the role of Best1 as a Cl^– channel. As known from previous studies, Ca²⁺ and volume-regulated Cl^– channels support cell proliferation, and bestrophin is activated by an increase in intracellular Ca²⁺ as well as cell swelling (7, 32). In an ongoing study with M1-collecting duct cells expressing high levels of Best1, we found inhibition of proliferation by the Cl^– channel blockers DIDS and NFA.⁴ Thus bestrophins may provide the molecular basis for an understanding of the role of Ca²⁺ and volume-regulated Cl^– channels in cell proliferation.

	FOOTNOTES

 
^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB699 A7 and the Else-Kröner-Fresenius-Stiftung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 49-954-4302; Fax: 49-941-4315; E-mail: uqkkunze{at}mailbox.uq.edu.

³ The abbreviations used are: BrdUrd, 5-bromo-2'-deoxyuridine; RNAi, RNA interference; siRNA, small interfering RNA; FACS, fluorescence-activated cell sorter; RT, reverse transcription; NFA, niflumic acid; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; ANOVA, analysis of variance; eG₁, early G₁ phase.

⁴ M. Spitzner, J. R. Martins, R. Barro Soria, J. Ousingsawat, K. Scheidt, R. Schreiber, and K. Kunzelmann, unpublished results.

	ACKNOWLEDGMENTS

 
We acknowledge the expert technical assistance by E. Tartler and A. Paech.

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