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CELLULAR AND MOLECULAR

Inhibition of Tumor Cell Proliferation by Ligands Is Associated with K⁺ Channel Inhibition and p27^kip1 Accumulation

University of Nice Sophia-Antipolis Centre National de la Recherche Scientifique Unité Mixte Recherche, Laboratoire de Physiologie des Membranes Cellulaires, Bt. Jean Maetz, La Darse, Chemin du Lazaret, Villefranche-sur-Mer, France (A.R., V.W., J.E., O.S.); and Institut National de la Santé et de la Recherche Médicale U385, Biology and Physiology of the Skin, Faculté de Médecine, Nice Cedex, France (A.-A.C.)

Received June 9, 2004; accepted July 26, 2004.

	Abstract

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Previous studies have shown that receptors are overexpressed in tumor cells. However, the role of receptors remains enigmatic. Recently, we and others have demonstrated that -1 receptor modulates K⁺ channels in pituitary. In the present report, patch-clamp and Western blot assays were used in small cell lung cancer (SCLC, NCI-H209, and NCI-H146) and leukemic (Jurkat) cell lines to investigate the effects of ligands on voltage-gated K⁺ channels and cell proliferation. The ligands (+)-pentazocine, igmesine, and 1,3-di(2-tolyl)guanidine (DTG) all reversibly inhibited voltage-activated K⁺ currents in both cell lines. The potency of ligand-induced inhibition (10 µM) was igmesine = (+)-pentazocine > DTG, pointing to the involvement of -1 receptors. Addition of the K⁺ channel blockers tetraethylammonium (TEA) and 4-aminopyridin or one of cited ligands in the culture media reversibly inhibited Jurkat cell growth. Interestingly, K⁺ channel blockers and ligands caused an accumulation of the cyclin-dependent kinase inhibitor p27^kip1 and a decrease in cyclin A expression in Jurkat and SCLC cells, whereas no effect could be detected on p21^cip1. Moreover, ligands and TEA had no effect on caspase 3 activity. Accordingly, incubation of cells with ligands did not provoke DNA laddering. These data demonstrate that ligands and voltage-dependent channel blockers inhibit cell growth through a cell cycle arrest in the G₁ phase but not via an apoptotic mechanism. Altogether, these results indicate that the -1 receptor-induced inhibition of the cell cycle is, at least in part, the consequence of the inhibition of K⁺ channels.

Receptors were first postulated as being a subtype of opioid receptors but subsequent studies have revealed that receptors form a class of proteins unrelated to other receptors, distributed in the nervous, endocrine and immune systems, and in organs such as liver and kidney (for review, see Bowen, 2000; Su and Hayashi, 2003). Receptors bind heterolog classes of exogenous compounds such as (+)-benzomorphans, guanidines, alcaloid deriveds, and neuroleptics (Bowen, 2000). Neurosteroids have been proposed as the endogenous ligands on the basis of in vivo and in vitro functional studies showing that progesterone, pregnenolone, and D-hydroepiandrosterone interact with receptors in the central nervous system (Monnet et al., 1995; Su et al., 1998). However, because neurosteroids are low-affinity receptor ligands, they are not fully accepted as the natural receptor ligands. Two subtypes of receptors have been characterized, namely, -1 and -2 receptors, on the basis of pharmacological, functional, and biochemical studies (Bowen, 2000). The -1 receptors were cloned in 1996 and the sequence yielded a 24-kDa protein with one or two predicted membrane spanning domains (Hanner et al., 1996; Aydar et al., 2002). This protein is unrelated to other mammalian proteins but shares 66.4% of similarity with a yeast C8-C7 sterol isomerase (Hanner et al., 1996). At the cellular level, -1 receptors are distributed in the endoplasmic reticulum and the plasma membrane (Hanner et al., 1996; Aydar et al., 2002). By contrast, the -2 receptor has not been isolated so far.

Receptors have been involved in memory, hormone secretion, synaptic activity, and cell electrical activity (Monnet et al., 1995; Maurice et al., 1998; Soriani et al., 1998; Lupardus et al., 2000). However, receptors may play a major role in cancer: a number of studies have demonstrated the presence of high densities of receptors in tumor cells, up to 10 times more than in normal organs or quiescent cells (Bowen, 2000). Interestingly, it has also been demonstrated that expression of receptors is positively correlated with the proliferating status of tumor cells (Al-Nabulsi et al., 1999). These results have led to the development of various ligands as tumor markers for photon emission tomography and single-photon emission-computed tomography analysis in vivo (John et al., 1999). Moreover, it has recently been reported that the -1 receptor gene is a target of the oncogene c-Myc (Fernandez et al., 2003), suggesting that this receptor is involved in tumor genesis. Besides, several studies have indicated that receptor ligands inhibit the growth of various tumor cell types, including lung, prostate, colon, and breast cancer cells (John et al., 1999; Moody et al., 2000; Berthois et al., 2003). These results indicate that receptors are potential targets for therapeutic agents. However, the mechanisms leading to the ligand-induced cell growth arrest remain poorly understood.

Recently, we and others have demonstrated that one of the major functions of -1 receptors is the modulation of VOK channels in pituitary and brain cells through G protein coupling or protein-protein interactions (Soriani et al., 1998, 1999a,b; Lupardus et al., 2000; Aydar et al., 2002).

Therefore, in the present study, we have used both SCLC (NCI-H209 and NCI-H146) and T-leukemic (Jurkat) cell lines to characterize the effects of ligands on VOK channels and cell growth. NCI-H146 and H209 are commonly used cell models for the study of SCLC (Gazdar et al., 1985). Moreover, it has been demonstrated that both cell types express -1 receptors (Moody et al., 2000). The Jurkat cell line is a widely accepted model of leukemia from which the -1 receptor has been isolated previously (Ganapathy et al., 1999). In this study, three different ligands were used i.e., igmesine, (+)-pentazocine, and DTG. Igmesine and (+)-pentazocine are highly selective -1 receptor ligands, whereas DTG is a mixed -1/-2 receptor ligand (Bowen, 2000; Su and Hayashi, 2003). Our results demonstrate for the first time that ligands induce a cell cycle arrest in the G₁ phase through the accumulation of the cell cycle inhibitor p27^kip1, but not apoptosis. In addition, the present findings indicate that this growth inhibition is the consequence of a down-regulation of voltage-dependent K⁺ channels.

	Materials and Methods

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Cell Culture. The SCLC cell lines NCI-H209 and H146 were obtained from CLS (Heidelberg, Germany). The T-leukemic cell line Jurkat was a gift from Dr. P. Auberger (Institut National de la Sante et de la Recherche Medicale U526, Nice, France). All cell lines were grown at 37°C with 5% of CO₂ in RPMI 1640 medium supplemented with L-glutamine (2 mM), sodium-pyruvate (1 mM), penicillin/streptomycin (100 U/ml), and fetal-bovine serum (10% for SCLC and 5% for Jurkat). Medium was routinely changed three times a week. For SCLC, dead cells were excluded by a Ficoll gradient separation technique one time a week (lymphocyte separation medium; Cambrex Bio Science Verviers S.p.r.l., Verviers, Belgium).

Drugs and Reagents. (+)-Pentazocine, DTG, TEA, 4-AP, apamin, and poly-D-lysine were purchased from Sigma-Aldrich (St. Quentin Fallavier, France). Igmesine is a generous gift from Dr. F. Roman (Pfizer, Fresne, France). Ac-Asp-Glu-Val-Asp-pNA (DEVD-pNA) and DEVD-CHO were from Alexis (Coger, Paris, France). Anti-actin (A2066), anti-cyclin A (C4710), and secondary anti-mouse horseradish peroxidase-coupled antibodies were obtained from Sigma-Aldrich. Santa Cruz anti-p27^kip1 (C-19) and anti-p21^Cip1 (C-19) antibodies were from TEBU International (Le Perray-en-Yvelines, France). Secondary anti-rabbit horseradish peroxidase-coupled antibodies (11-035-144; Jackson ImmunoResearch Laboratories, West Grove, PA) were purchased from Interchim (Montlucon, France).

Electrophysiolology. For whole-cell patch-clamp recordings, cells were plated on glass coverslips coated with poly-D-lysine (10 nM) and incubated for 2 to 4 h in RPMI 1640 medium. NCI-H209 and H146 cells need to be mechanically dissociated before being plated. Patch-clamp experiments were made at room temperature with an external solution of the following composition: 2.8 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂, 140 mM N-methyl-D-gluconate-Cl, and 10 mM Hepes (pH adjusted to 7.4 with HCl, 304.6 mOsM/l). Soft glass patch electrodes (borosilicate glass capillaries GC150TF-7.5; Harvard Apparatus, Edenbridge, Kent, UK) were made on a horizontal pipette puller (P-97; Sutter Instrument Company, Novato, CA) to achieve a final resistance ranging from 3 to 5 M. The internal solution was of the following composition: 129 mM KCl, 2 mM MgCl₂, 5 mM NaCl, 1 mM CaCl₂, 11 mM EGTA, and 10 mM Hepes (pH adjusted to 7.2 with KOH, 298 mOsM/l). ATP (2 mM) and GTP (100 µM) were extemporaneously added to the internal solution. Electric signals were amplified with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) and acquired on an IBM-compatible personal computer with a DIGIDATA 1200 interface and pCLAMP 8 software (Axon Instruments). K⁺ currents were recorded at a 5-kHz-sampling frequency and filtered at 2 kHz.

DTG was dissolved in methanol [final concentration of methanol <0.1% (v/v)]. (+)-Pentazocine was dissolved in methanol/acid [one-half methanol + one-half HCl 0.1 M (v/v), final concentration of methanol <0.1% (v/v)]. Solvent alone had no effect on K⁺ currents at this concentration. Igmesine was dissolved in water. The ligand solutions were administered in the vicinity of the cell under study through the use of a gravity-feed system (rate 2 ml/mn). The excess of bathing solution was continuously aspired via a suction needle.

Current amplitudes were determined with the pCLAMP 8 analysis software (Clampfit). Current/voltage and current/time relationships were fitted by using Microcal Origin analysis software (Sega, Paris, France). Quantitative data are expressed as mean ± S.E.

Cell Growth Analysis. To assess cell growth, cells were seeded at day 0 at a density of 0.25 x 10⁶ cells/ml^-1 and counted 24, 48, and 72 h after incubation with TEA or igmesine. For cell density evaluation, an aliquot of 25 µl of cell suspension was mixed with 25 µl of trypan blue, and the number of cells was counted using a Malassez chamber. Only viable cells (which excluded trypan blue) were counted. This enabled us to differentiate easily between a reduced cell proliferation rate and cell death. Percentage of cell growth inhibition was calculated after 3 days in culture as follows: Inhibition = [1 - (IN₃ - IN₀)/(Ctl₃ - Ctl₀)] x 100, were IN₀ and IN₃ are the cell densities at day 0 and 3 for cells incubated with either ligands or TEA, respectively, Ctl₃ and Ctl₀ are the cell densities at day 0 and 3 for cells in control conditions.

Igmesine was dissolved in methanol [final concentration of methanol <0.1% (v/v)]. Solvent alone had no effect on cell proliferation at this concentration. TEA was directly dissolved in the culture medium.

DEVD-pNA Cleavage Assay. Caspase activity was measured using a kinetic colorimetric assay according to the supplier specification. In brief, control cells or cells incubated with a proapoptotic agent (staurosporine) or ligands were washed in phosphate-buffered saline and then lysed 30 min at 4°C in lysis buffer (50 mM Hepes, 150 mM NaCl, 20 mM EDTA, 0.2% Triton X-100, 20 µg/ml aprotinin, 10 µM leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Lysates were then centrifuged 15 min at 15,000g, and 50 µg of cell extract was incubated with 5 mM dithiothreitol and 200 µM Ac-Asp-Glu-Val-Asp-pNA (DEVD-pNA) preferentially cleaved by members of the CPP32 family of cysteine proteases. Liberation of pNA was monitored continuously at 37°C by using an excitation wavelength of 405 nm. Measurements were recorded over the linear range of assay, and caspase activity was controlled by adding in the cell extract an apopain/CPP32 inhibitor (DEVD-CHO). Substrates without lysates served as negative control.

DNA Fragmentation. Jurkat and NCI-H209 cells (1 x 10⁶ cells) were harvested and centrifugated at 1000g for 5 min. Cell pellets were lysed by incubation in 10 mM Tris (pH 8.0) containing 1 mM EDTA, 0.2% Triton X-100, and 100 µg/ml RNase A for 30 min at 37°C. Proteinase K was then added to give a final concentration of 100 µg/ml, and the reaction was continued for an additional 30 min. The lysate was extracted with 10% 5 M NaCl and 1 volume of isopropanol, incubated 2 h at -20°C, and then centrifuged at 15,000g for 15 min at 4°C. The supernatant was washed with 70% ethanol and centrifuged. The pellets were dried and suspended in Tris-EDTA buffer (10 mM Tris, pH 8, and 1 mM EDTA). DNA was incubated at 55°C for 30 min, and electrophoresis was performed in 1.5% agarose gel containing 0.5 µg/ml ethidium bromide at 50 V for 50 min. DNA was visualized by UV illumination and photographed.

Western Blot. After 3 days of incubation with igmesine, (+)-pentazocine, DTG, or TEA, cells were washed in phosphate-buffered saline and then lysed under agitation in ice-cold lysis buffer (50 mM Tris, pH 7.4, 200 mM NaCl, 1 mM EDTA, 0.2% Nonidet P-40, 10 mM NaF, 0.1 mM -glycerophosphate, 1 mM NaVO₄, and a protease inhibitor cocktail, Complete; Roche, Meylan, France). The lysate was then centrifuged (11,000g, 15 min, 4°C), and the resulting supernatants were analyzed by immunoblotting. Total protein concentration was determined with a Bio-Rad protein assay (Bio-Rad, Munich, Germany) with bovine serum albumin as the standard. Proteins (50 µg/lane) were resolved on a 13% acrylamide gel by SDS-polyacrylamide gel electrophoresis, electrotransferred to nitrocellulose, blocked in 5% nonfat milk, and incubated overnight at 4°C with a primary antibody directed against either p27^kip1 (1:200), p21^Cip1 (1/200), cyclin A (1:750), or actin (1:200) human proteins. Blots were incubated with horseradish peroxidase-conjugated secondary antibody (anti-rabbit, 1:15,000; anti-mouse, 1:50,000) for 1 h at room temperature. Labeled proteins were visualized by enhanced chemiluminescence (Pierce SuperSignal West Pico chemiluminescent; Interchim) using Kodak Bio-Max MR film (Sigma-Aldrich).

	Results

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Ligands Inhibit Voltage-Operated K⁺ Currents in Tumor Cells. We first have studied the potential modulatory effects of ligands on VOK currents expressed in both SCLC and Jurkat cells. Currents were recorded by using the patch-clamp technique in the voltage-clamp mode and the standard whole-cell configuration. Internal and external solutions were designed to isolate K⁺ currents (see Materials and Methods).

In SCLC cells, K⁺ currents were evoked by successive 200-ms pulses from -80 to +20 mV in 20-mV intervals from a holding potential of -50 mV (Fig. 1A). In all tested NCI-H209 cells (n = 117), voltage pulses positive to -40 mV elicited a TEA- and 4-AP-sensitive, noninactivating outward current corresponding to the delayed-rectifier K⁺ current (I_K) previously described in SCLC (Pancrazio et al., 1993) (Fig. 1). The effects of ligands on I_K were studied in a total of 66 cells. In 89% of the tested cells, applications of either igmesine, (+)-pentazocine, or DTG induced dose-dependent and reversible inhibitions of the current evoked by pulses positive to -40 mV (10 nM-10 µM; Fig. 2). The rank order potency at 10 µM was igmesine (+)-pentazocine > DTG, which corresponds to the pharmacological profile of the -1-receptor (Fig. 2D). In addition, the EC₅₀ of the DTG-induced inhibition of the current was similar to the EC₅₀ value obtained in a previous study dealing with the characterization of the -1 receptor using binding methods in NCI-H209 cells (206 and 90 nM, respectively; Moody et al., 2000; Fig. 2E). In the present work, the inhibitory effects induced by ligands on I_K were also observed in NCI-H146 cells (not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Electrophysiological and pharmacological characterization of the VOK current recorded in NCI-H209 cells in the whole-cell configuration and the voltage-clamp mode. A, family of currents recorded in response to pulses ranging from -80 to 20 mV in 20-mV increments (Vh, -50 mV) as shown by the voltage protocol represented underneath. B, corresponding current-voltage plot. C, time course of the TEA (10 mM)-induced inhibition of the current elicited by voltage pulses from -50 to 20 mV (200 ms). D, histogram showing the inhibitory effects induced by TEA (10 mM) and 4-AP (1 mM) on the K+ current. Experiments with TEA and 4-AP were performed on naive cells coming from different culture batches. Same protocol as in C.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Effects of ligands on the VOK current recorded in NCI-H209 cells. A, two families of currents obtained from a single cell in response to pulses ranging from -80 to 40 mV in 20-mV increments (Vh, -50 mV) as shown by the voltage protocol represented underneath. These currents were recorded in the absence (left) or the presence (right) of (+)-pentazocine (Ptz; 10 µM). B, current-voltage plots deduced from the currents presented in A. C, time course of the igmesine (10 µM)-induced inhibition of the current. Same protocol as in Fig. 1C. D, histogram showing the inhibitory effects induced by igmesine (10 µM), (+)-pentazocine (10 and 0.1 µM), and DTG (10 µM) on the K+ current. Same protocol as in Fig. 1C. E, dose-response curve of the DTG-induced inhibition of the K+ current. Values are mean ± S.E. Experiments with (+)-pentazocine, igmesine, or DTG were performed on naive cells coming from different culture batches. Same protocol as in Fig. 1C.

In Jurkat cells, membrane was clamped at -80 mV between the depolarizing steps to avoid any voltage-dependent inactivation of the current. Voltage pulses (200 ms) ranging between -100 and +80 mV in 20-mV intervals applied to 28 Jurkat cells gave rise to an outward current for potentials positive to -40 mV. The current presented a fast activation and a slow inactivation over time. These kinetic characteristics correspond to the Kv1.3 channel previously described as the main VOK current occurring in this cell line (Panyi and Deutsch, 1996; Fig. 3, A and B). Application of TEA (10 mM; n = 3) or 4-AP (2.5 mM; n = 3) induced a clear-cut reduction in current amplitude, in a good agreement with the pharmacological profile of the Kv 1.3 channel (Fig. 3C).

View larger version (20K): [in this window] [in a new window]   Fig. 3. Electrophysiological and pharmacological characterization of the VOK current recorded in Jurkat cells in the whole-cell configuration and the voltage-clamp mode. A, family of currents elicited by voltage pulses from -100 to 80 mV in 20-mV increments (Vh, -80 mV) as shown by the voltage protocol represented underneath. B, corresponding current-voltage plot. C, histogram representing the inhibitory effect of 4-AP (1 mM) and TEA (10 mM) on the K+ current. Currents were monitored by iterative applications of pulses from - 80 to 50 mV. Experiments with TEA and 4-AP were performed on naive cells coming from different culture batches. Values are mean ± S.E.

External application of (+)-pentazocine (10 µM) induced a strong inhibition of the current for voltage pulses positive to -40 mV (Fig. 4, A and B). This effect was mimicked by igmesine (10 µM) and was reversible after cell washing. The inhibitory effect of ligands occurred in 81% of tested cells (n = 16; Fig. 4, C and D). These results demonstrate that ligands inhibit VOK currents expressed in both SCLC and Jurkat leukemic cells.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Effects of ligands on the VOK current recorded in Jurkat cells in the whole-cell configuration and the voltage-clamp mode. A, two family of currents elicited by voltage pulses from -100 to 80 mV in 20-mV increments (Vh, -80 mV) as shown by the voltage protocol represented underneath. The currents were recorded in the absence (left) or the presence (right) of (+)-pentazocine (Ptz; 10 µM). B, corresponding I/V plots. C, time course of the (+)-pentazocine (10 µM)-induced inhibition of the K+ current. The currents were evoked by pulses from -80 to 50 mV (300 ms). The inset shows superimposed currents recorded before (1) and during (2) (+)-pentazocine application. D, histogram representing the inhibitory effect of (+)-pentazocine and igmesine (10 µM each). Same protocol as in Fig. 3C. Values are mean ± S.E. Experiments with (+)-pentazocine and igmesine were performed on naive cells coming from different culture batches.

Igmesine and VOK Blockers Inhibit Jurkat Cell Growth. To determine whether ligands and K⁺ channel blockers had an effect on the proliferation rate of Jurkat cells, we next studied the effects of igmesine and Kv 1.3 channel blockers on Jurkat cell growth over 3 days. Cell density was measured by direct counting of living cells 24, 48, and 72 h after seeding. In control experiments, the number of cells increased exponentially to reach a cell density of 1.24 ± 0.07 x 10⁶ cells/ml after 3 days (n = 12). Incubations of Jurkat cells with igmesine (10 or 30 µM), TEA (10 mM), or 4-AP (2.5 mM) provoked a significant slow down in cell growth (Fig. 5, A-D). After 3 days in culture, igmesine significantly reduced the cell density by 23.9 ± 4.6 and 82.8 ± 3.1% at 10 and 30 µM, respectively (n = 5 each; Fig. 3, A and B). Similarly, a 3-day incubation of cells with TEA (10 mM) induce a 60.6 ± 10.6% decrease in cell density (n = 3; Fig. 5C). Jurkat cells express two main K⁺ conductances, i.e., Kv 1.3 and the small Ca²⁺-activated K⁺ channel hSK2 (Panyi and Deutsch, 1996; Fanger et al., 2001). Because TEA blocks both voltage-dependent and Ca²⁺-activated K⁺ channels, we also tested 4-AP (2.5 mM), which specifically acts on voltage-dependent K⁺ channels. Similar to TEA, 4-AP induced a 75.7 ± 9.3% decrease in cell density (n = 3; Fig. 3D). By contrast, a high concentration of apamin (20 nM), a specific blocker of small Ca²⁺-activated K⁺ channels, had no effect on Jurkat cell growth (Fig. 5E), indicating that hSK2 channels are not involved in the control of Jurkat cell growth. Finally, incubation of cells with igmesine (30 µM) together with TEA (10 mM) did not provoke a significantly different inhibition of cell growth after 3 days than igmesine alone (Fig. 5F). This result indicates that there is no addition of the effects of VOK blockers and ligands.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Effects of igmesine, TEA, 4-AP, and apamin on Jurkat cell growth. A, plots showing the growth evolution over 3 days in the absence and in the presence of igmesine, 10 µM (black squares and black circles, respectively; n = 5). B, plots showing the growth evolution over 3 days in the absence and in the presence of igmesine, 30 µM (black squares and black circles, respectively; n = 6). C, plots showing the growth evolution over 3 days in the absence and in the presence of TEA, 10 mM (black squares and black circles, respectively; n = 4). D, plots showing the growth evolution over 3 days in the absence and in the presence of 4-AP, 1 mM (black squares and black circles, respectively; n = 3). E, plots showing the growth evolution over 3 days in the absence and in the presence of apamin, 20 nM (black squares and black circles, respectively; n = 3). F, histogram comparing the number of cells after 3 days in culture (DIC) in the absence (Ctl) and the presence of TEA (10 mM), igmesine (Igm; 10 µM), or igmesine + TEA (10 µM and 10 mM, respectively). Values are mean ± S.E. * P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0005; N.S., nonsignificant (paired Student's t test). Note that none of the solvents used to prepare ligands solutions (DMSO or methanol) had any effect on cell growth (data not shown).

Together, these data indicate that incubation of Jurkat cells with a ligand induce an inhibition in cell growth. Interestingly, this effect is mimicked by the K⁺ channel blockers TEA and 4-AP, but not apamin, demonstrating that -1 receptors modulate cell proliferation through the inhibition of Kv 1.3 channels.

Ligands Stimulate p27^kip1 and Inhibit Cyclin A Expressions in Tumor Cells but Have No Effect on p21^cip1. A further characterization of the inhibitory effects induced by ligands and K⁺ channel blockers was necessary to understand whether these compounds reduce cell growth via a cell cycle arrest or by inducing cell death. Therefore, we next examined their effects on the expression of p27^kip1, p21^cip1, and cyclin A, three key proteins involved in the cell cycle progression. Cyclin A is up-regulated in growing cells at the G₁/S transition and is involved in the S-phase progression. Its stimulation results from the sequential activation of cyclinD/CDK4 and cyclinE/CDK2 and can be regulated by CDKs inhibitors such as p27^kip1 and p21^cip1 (Sherr and Roberts, 1995; Ekholm and Reed, 2000). Jurkat cells were harvested after 3 days in culture in the presence of ligands, TEA, 4-AP, apamine, or the carrier alone. After cell lysis, proteins were subjected to immunoblotting with antibodies directed against human p27^kip1, p21^cip1, or cyclin A. Actin was used as an internal loading control. We observed a net increase in p27^kip1 level in cells challenged with igmesine, DTG, or (+)-pentazocine (30 µM each) compared with control cells (Fig. 4A, top). The expression level of p27^kip1 was significantly enhanced after 24 h of incubation with igmesine (10 and 30 µM) and gradually increased to reach a maximal level after 3 days (not shown). Interestingly, incubation of cells with TEA or 4-AP (10 and 2.5 mM, respectively) led to a p27^kip1 level increase, whereas apamin had no effect (Fig. 6A, bottom). A previous report has indicated that the growth of SCLC cells was altered by incubation with ligands (Moody et al., 2000). It was thus interesting to understand whether this alteration could be explained by a modulation of cyclin inhibitors. Similar to Jurkat cells, higher levels of p27^kip1 were detected after treatment of SCLC cells with igmesine, (+)-pentazocine, or DTG (30 µM; 3 days each) compared with control cells (Fig. 6B, top). The same results were observed with NCI-H209 cells incubated with either TEA or 4-AP (10 and 2.5 mM, respectively; Fig. 6B, bottom). By contrast, apamin (20 nM) had no effect on p27^kip1, ruling out any involvement of Ca²⁺-dependent K⁺ channels in cell cycle progression. However, neither DTG, igmesine or (+)-pentazocine (30 µM each), nor TEA (10 mM) had any effect on the level of the CDK inhibitor p21^cip1 expressed in both SCLC and Jurkat cells (Fig. 7).

View larger version (34K): [in this window] [in a new window]   Fig. 6. Effects of ligands and K+ channel blockers on the level of p27kip1 in Jurkat and NCI-H209 cells. p27kip1 expression was assessed by Western blotting. A, p27kip1 expression in Jurkat cells in the absence (Control; Ctl) and the presence of igmesine (Igm), (+)-pentazocine (Ptz), DTG, TEA, 4-AP, or apamin. B, p27kip1 expression in NCI-H209 cells in the absence (Ctl) and the presence of Igm, Ptz, DTG, TEA, 4-AP, or apamin. Cells were treated with each drug for 3 days. Actin levels were used as controls in each experiment. The immunoblottings show typical examples from three to eight independent experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 7. Effects of ligands and TEA on the level of p21Cip1 in Jurkat and NCI-H209 cells. p21Cip1 expression was assessed by Western blotting. A, p21Cip1 expression in Jurkat cells in the absence (Control; Ctl) and the presence of (+)-pentazocine (Ptz), DTG, or TEA. B, p21Cip1 expression in NCI-H209 cells in the absence (Ctl) and in the presence of igmesine (Igm), DTG or TEA. Cells were treated with each drug for 3 days. Actin levels were used as controls in each experiment. The immunoblottings show typical examples from three to eight independent experiments.

The effects of ligands on the expression level of cyclin A were next examined in the two cell types. Igmesine, (+)-pentazocine, or DTG (30 µM; 3 days each) clearly decreased cyclin A level in cell treated (Fig. 8). A similar result was observed with cells challenged with TEA (10 mM; Fig. 8).

View larger version (29K): [in this window] [in a new window]   Fig. 8. Effects of ligands and TEA on the level of cyclin A in Jurkat and NCI-H209 cells. Cyclin A expression was assessed by Western blotting. A, cyclin A expression in Jurkat cells in the absence (Control; Ctl) and the presence of igmesine (Igm), (+)-pentazocine (Ptz), DTG, or TEA. B, cyclin A expression in NCI-H209 cells in the absence (Ctl) and the presence of Igm, Ptz, DTG, or TEA. Cells were treated with each drug for 3 days. Actin levels were used as controls in each experiment. The immunoblottings show typical examples from three to eight independent experiments.

Altogether, these results demonstrate that the activation of -1 receptors leads to the same effects as the pharmacological inhibition of voltage-dependent K⁺ channels, i.e., p27^kip1 accumulation and a decrease in cyclin A likely underlying a G₁ cell cycle arrest in Jurkat and SCLC cells.

Ligands and TEA Do Not Induce Apoptosis in Tumor Cells. To rule out the possibility of a cell death-induced growth arrest, we have tested whether ligands had any proapoptotic effect. Caspases are a family of proteases that are executioners of apoptotic signals (Cohen, 1997). Among the different caspases involved in this process, caspase 3 has been shown to be a crucial effector of various stimuli triggering apoptosis, including DNA laddering (Porter and Janicke, 1999). The activity of caspase 3 was quantified by monitoring the release of pNA, resulting from the cleavage of DEVD-pNA, a synthetic substrate of the protease. In both NCI-H209 and Jurkat cells, incubation with the proapoptotic agent staurosporine (1 µM; 4 or 15 h) gave rise to a dramatic increase in caspase 3 activity (Fig. 9). By contrast, neither igmesine nor DTG (30 µM each; 24 h) was able to induce any enhancement of caspase 3 activity (Fig. 9), even after extending incubation time with ligands up to 48 h (not shown). Similarly, incubation of cells with TEA (10 mM; 24 h) failed to provoke any activation of caspase 3 activity in both cell types (Fig. 9). In a good agreement with these observations, no DNA laddering occurred in Jurkat or SCLC cells treated with DTG (30 µM; 24 h) compared with the staurosporine treatment (1 µM; 15 h; Fig. 10).

View larger version (12K): [in this window] [in a new window]   Fig. 9. Effects of ligands and TEA on caspase 3 level in Jurkat and NCI-H209 cells. Specific caspase 3 activity is expressed in nanomoles of pNA per minute per milligram. A, caspase activity of Jurkat cells in the presence of staurosporine (Stauro; 1 µM; 4 h), in control conditions (Ctl, 4 and 24 h after cell seeding), and in the presence of igmesine (Igm; 30 µM; 24 h), DTG (30 µM; 24 h), or TEA (10 mM; 24 h). B, caspase 3 specific activity of NCI-H209 cells incubated with Stauro (1 µM; 15 h), Igm (30 µM; 24 h), DTG (30 µM; 24 h), or TEA (10 mM; 24 h). Values are mean ± S.E. (n = 3).

View larger version (66K): [in this window] [in a new window]   Fig. 10. DNA fragmentation assay after exposure to staurosporine and DTG in SCLC and Jurkat cells. Left, DNA fragmentation of SCLC cells in control condition (Ctl; 15 h) and in the presence of stauroporine (Stauro; 1 µM; 15 h) or DTG (30 µM; 15 h). Right, DNA fragmentation of Jurkat cells in Ctl condition (4 h) and in the presence of Stauro (1 µM; 4 h) or DTG (30 µM; 4 h). The DNA electrophoresis are typical examples from three independent experiments.

Cell death (both necrosis and apoptosis) is a nonreversible process, thus the reversibility of the igmesine-induced arrest in Jurkat cell growth was also examined. After a 3-day incubation of cells with igmesine (30 µM), cells were diluted (250,000 cells/ml) and split into two flasks containing igmesine (30 µM) or the carrier alone. After three further days in culture, the cells grown in fresh culture media alone had recovered a normal rate of growth, whereas cells still challenged with igmesine presented an inhibited profile of proliferation (Fig. 11). The cell density observed after 3 days post-washing (1.02 ± 0.08 x 10⁶ cells/ml; n = 3) was not significantly different from the value obtained in control conditions for naive cells (1.24 ± 0.07 x 10⁶ cells/ml; n = 8; P > 0.09), indicating that the cell growth inhibition due to igmesine is a reversible mechanism.

View larger version (10K): [in this window] [in a new window]   Fig. 11. Reversibility of the igmesine-induced inhibition of Jurkat cell growth. Plots showing the growth evolution over 6 days in the presence of igmesine or after washing. Cells were treated with igmesine (30 µM; n = 3) for 3 days (black squares, continuous line), and then diluted to a cell density of 0.25 x 106 /ml and split in two populations. The two populations of cells were allowed to grow three more days in fresh medium alone (empty circles; n = 3) or in the presence of igmesine (30 µM; black squares, dashed line; n = 3). Values are mean ± S.E. (n = 3; ***, P < 0.005; ****, P < 0.0005, paired Student's t test).

These results demonstrate that pharmacological activation of -1 receptors has no proapoptotic effect and further confirm that ligands inhibit cell proliferation through a cell cycle arrest.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Recently, we and others have demonstrated in pituitary and brain cell models that one of the major role of -1 receptors is the modulation of K⁺ channels (Soriani et al., 1998, 1999a,b; Aydar et al., 2002). The aim of our study was to explore the link between cell growth arrest and the modulation of VOK by receptors in tumor cells. We demonstrate for the first time that activation of -1 receptors induce a tumor cell cycle arrest in the G₁ phase through a modulation of p27^KIP1 and cyclin A levels. Moreover, our findings demonstrate that this effect is the consequence of the blockade of VOK channels by -1 receptors.

Patch-clamp experiments performed in NCI-H209 cells elicited an outward current presenting the characteristics of the delayed rectifier K⁺ current, e.g., a slow activation for potentials positive to -40 mV, a weak inactivation over time and a strong sensitivity to TEA and 4-AP, as described previously in SCLC cells (Pancrazio et al., 1993). The current was inhibited by three ligands, igmesine, (+)-pentazocine, and DTG (Bowen, 2000). The rank order potency of this inhibition in NCI-H209 cells was consistent with the pharmacological profile of -1 receptors, pointing out the involvement of this receptor in our model (Bowen, 2000; Su and Hayashi, 2003). This result is in agreement with previous reports demonstrating the functional link between -1 receptors and VOK currents in pituitary (Soriani et al., 1998, 1999a,b). We next investigated the effects of ligands on the VOK current expressed in Jurkat cells. The current recorded presented the characteristics of the Kv1.3 channel that was cloned and identified as the main VOK channel in this cell type (Panyi and Deutsch, 1996). We demonstrate herein that ligands inhibit K⁺ currents in lymphocytes. Previous studies have shown that -1 receptors are tightly coupled to Kv1.4 and Kv1.5 channel subunits expressed in Xenopus oocytes (Aydar et al., 2002). Thus, the present results indicate that receptors down modulate Kv1.3, another member of the Kv channel family, showing for the first time that -1 receptors modulate membrane electrical properties in lymphocytes.

Recently, several studies have demonstrated the involvement of channels of the Kv family, including channels underlying the delayed-rectifier current, in cell cycle events such as apoptosis and cell division in various cell types. For example, Kv1.5 and 1.3 channels are up-regulated in proliferative rat oligodendrocyte progenitor cells, whereas antagonists of Kv1.3-induced currents inhibit the S-phase entry (Chittajallu et al., 2002). In the same cells, [³H]thymidine incorporation is decreased by Kv channel blockers (Attali et al., 1997). In addition, inhibition of delayed-rectifier channels of the Kv protein family by antagonists such as TEA or 4-AP alters cell growth in various tumor cell types (Pancrazio et al., 1993; Rouzaire-Dubois and Dubois, 1998; Rybalchenko et al., 2001). Consistent with these observations, we show here that TEA and 4-AP, but not the hSK channel blocker apamin, inhibit both the VOK current and cell growth in Jurkat cells. It can be concluded from these experiments that Kv1.3 channel activity is strongly involved in cell growth. More interestingly, the similar effects induced by the -1 receptor activation and Kv channel blockers on both channel activity and cell proliferation raise the following hypothesis: ligands may block tumor cell growth through a -1 receptor-dependent modulation of VOK. Accordingly, our results clearly show that TEA does not increase the response induced by igmesine, demonstrating that ligands inhibit cell growth through K⁺ channel inhibition.

In this perspective, both channels blockers and ligands should trigger the same cellular pathways. However, the mechanism by which the activation of -1 receptors alters tumor cell growth has not been described so far. At least two different cellular events may underlie a diminution in growth rate: a cell cycle arrest or cell death. Consequently, the effects of -1 ligands and pharmacological K⁺ channels blockers were examined on cell cycle. We focused our study on p27^kip1, p21^cip1, and cyclin A, three key proteins involved in cell cycle progression (Sherr and Roberts, 1995). p27^kip1 and p21^cip1 are known as inhibitors of cyclin D-cdk 4/6 and cyclin E-cdk 2 complexes, both responsible for pRb phosphorylation. The phosphorylation of pRb is a critical step for G₁-to-S transition through the liberation of transcription factors necessary for the activation of S-phase genes such as cyclin A (for review, see Sherr and Roberts, 1995; Ekholm and Reed, 2000). In pRb-deficient cells lines such as SCLC cells, p27^kip1 has also been shown to block cyclin E-dependant transactivation of cyclin A (Zerfass-Thome et al., 1997). The findings presented here demonstrate for the first time, in both Jurkat and SCLC cells, that ligands induce a cell cycle arrest in the G₁ phase through the accumulation of the cell cycle inhibitor p27^kip1 and the concomitant decrease in cyclin A. However, ligands had no effect on the expression of p21^cip1, indicating that -1 receptors specifically modulate cell cycle through a p27^kip1-dependent pathway. Incubation of both SCLC and Jurkat cells with TEA or 4-AP led to a similar modulation profile of protein level, i.e., p27^kip1 level increase, cyclin A inhibition but no effect on p21^cip1. It can be concluded that the pharmacological inhibition of Kv channels leads to the modulation of the same cell cycle regulators as those triggered by ligands. Altogether, these findings are in a good agreement with the model of a -1 receptor-induced cell cycle arrest in the G₁ phase through voltage-dependent K⁺ channel inhibition. Recently, Crawford and Bowen (2002) have shown that -2 receptor ligands were responsible for apoptosis induction in breast tumor cell lines. Thus, one might speculate that micromolar doses of -1 ligands may interact with -2 receptors and consequently alter cell proliferation through apoptosis. However, the antiproliferative effect of ligands we report here likely differs from apoptosis when one consider that 1) the effects of ligands on Jurkat cell growth were reversible after washing, and 2) the treatment of Jurkat and SCLC cells with ligands neither stimulated caspase 3 activity levels nor provoked DNA laddering. These data clearly indicate that unlike -2 receptors, which induce apoptosis, -1 receptors activation specifically alters cell division. Accordingly, TEA had no effect on caspase 3 activity, indicating that inhibition of K⁺ channels do not induce apoptosis. Consistent with this concept, it is known that the inhibition of cell division is associated with either an inhibition of voltage-dependent K⁺ conductances or a decrease in related channel expression. By contrast, apoptosis induction has been shown to be dependent of an enhancement of K⁺ currents (Storey et al., 2003). In conclusion, our results demonstrate for the first time that ligands induce a cell cycle arrest in the G₁ phase and that this inhibition is a consequence of the down-modulation of VOK by -1 receptors.

A question arises from this model: by what mechanism can the inhibition of VOK channels arrest the cell cycle? First, a large hyperpolarization of the cell membrane is required for the G₁-to-S phase progression (Wang et al., 1998). This hyperpolarization would influence calcium-dependent events involved in the division process. Thus, the depolarization that can be expected from -1 receptor activation may alter Ca²⁺ homeostasis (Soriani et al., 1999a) and consequently inhibit proliferation. Alternatively, Dubois at al. (1998) have proposed that the inhibition of K⁺ channels impairs the RVD, which represents a critical event for the G₁-to-S phase progression. Cell volume changes may alter the concentration of cellular components involved in the expression or activity of cell cycle-regulating proteins. Moreover, cytoskeleton rearrangements due to cell volume changes may affect the protein kinases or phosphatases responsible for the control of cell cycle progression (Huang and Ingber, 1999). Experiments recently performed in our laboratory showing that ligands delay RVD in Jurkat cells seem to be in a good agreement with this latter hypothesis (our unpublished data). Although the molecular mechanisms linking ion channels to cell cycle remain elusive, the critical importance of K⁺ channels in cell proliferation make of these proteins potent targets for pharmacological tumor treatments. However, VOK channels are also widely expressed in normal organs such as central nervous system and muscles, rendering a potential use of classical K⁺ current blockers hazardous. Therefore, because 1) -1 receptors are biological markers of tumors (Bowen, 2000); 2) the concentrations of ligands required to produce a 50% inhibition of K⁺ currents are about 10 times higher in normal cells (Soriani et al., 1998; Lupardus et al., 2000) than in malignant cell models studied herein, suggesting that tumor cells may be more sensitive to ligands that nonproliferative cells; and 3) the perfusion of ligands reduces SCLC tumor xenografts in mice without altering animal viability (Moody et al., 2000), the model presented here indicates that new pharmacological tools using -1 receptor ligands may be developed to specifically target in vivo VOK that regulate cancer cell proliferation.

	Acknowledgements

 
We thank Dr. François Roman (Pfizer) for the gracious gift of igmesine and Prof. Patrick Auberger (Institut National de la Sante et de la Recherche Medicale U526) for kindly providing Jurkat cells. We also thank Dr. Beatrice Rayet, Dr. Sandra Schmieder, and Prof. Franck Delaunay (Centre National de la Recherche Scientifique Unité Mixte Recherche 6078, Villefranche-sur-Mer, France) for helpful discussions.

	Footnotes

 
This work was supported by the Centre National de la Recherche Scientifique, the Association pour la Recherche sur le Cancer, the University of Nice Sophia-Antipolis. A.R. and A.A.C. are research fellows of the Ministère de la Recherche et de l'Enseignement Supérieur.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.072413.

ABBREVIATIONS: VOK, voltage-operated K⁺ channel; SCLC, small cell lung carcinoma; DTG, 1,3-di(2-tolyl)guanidine; TEA, tetraethylammonium; I_K, delayed-rectifier K⁺ current; CDK, cyclin-dependent kinase; pRb, retinoblastoma protein; 4-AP, 4-aminopyridin.

Address correspondence to: Dr. Olivier Soriani, UNSA Centre National de la Recherche Scientifique UMR 6078, Laboratoire de Physiologie des Membranes Cellulaires, Bt. Jean Maetz, La Darse, 284, Chemin du Lazaret, 06230 Villefranche-sur-Mer, France. E-mail: soriani{at}obs-vlfr.fr

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