1Laboratoire de Physiologie Cellulaire, Institut National de la Santé et de la Recherche Médicale EMI 0228, SN3, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cédex; 2Laboratoire de Physiologie Cellulaire et Moléculaire, Université Picardie Jules Verne, Faculté des Sciences, 80000 Amiens; 4Institut Fédératif de Recherche IFR22: "Biologie et Pathologie des Régulations Cellulaires," IRCL, 59045 Lille Cédex, France; and 3Laboratoire de Physiologie Animale, Université Ibnou Zohr, Faculté des Sciences, Agadir, Morocco BP2815 80000
Submitted 5 November 2003 ; accepted in final form 20 February 2004
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ABSTRACT |
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breast cancer; calcium-activated potassium channels; proliferation
At the present time, the mechanisms responsible for the hyperpolarization of membrane potential through the G1 and S phases have yet to be clearly identified.
Changes in the cytosolic calcium concentration ([Ca2+]i) may also provide important regulatory signals during the cell cycle. Ca2+ has been observed to be required for progression through G1 and for the G1/S transition in several cell types, including human embryonic lung fibroblasts (20, 48) and L1210 leukemic cells (7). A link between [Ca2+]i and membrane potential was first reported in melanoma cells, where membrane hyperpolarization increased [Ca2+]i simply by controlling the electrochemical gradient for Ca2+ entry into the cell (37). This increase in [Ca2+]i may, in turn, induce the activation of Ca2+-activated K+ (KCa) channels. In breast cells, two types of Ca2+-activated K+ channels, studied using the patch-clamp recording technique, were shown to be involved in some responses to EGF and insulin stimulation (47). A Ca2+-activated K+ channel has been characterized in MCF-7 cells, and its incidence was observed to increase during insulin-stimulated logarithmic growth compared with cells in the plateau phase. The greater incidence of these channels was associated with cell populations exhibiting a more hyperpolarized membrane potential (50). Moreover, in MCF-7 cells, ATP acting extracellularly through the purinergic receptor, P2Y2, raised intracellular Ca2+ levels and increased proliferation (49). In mouse mammary epithelial cells, the enhancement of Ca2+ mobilization and capacitative Ca2+ entry by EGF induced the proliferating stage (21).
The goal of this research is to define the pathway leading to the hyperpolarization of membrane potential during the cell cycle. We combined electrophysiological and molecular methods to demonstrate, for the first time, that both hEAG and hIK1-like K+ channels were responsible for the progression of the cell cycle. An initial hyperpolarization, produced by the activation of hEAG K+ channels, permitted the cell to enter the G1 phase and the activation of hIK1 contributed to its progression through G1, to G1/S transition and on, through the S phase.
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MATERIALS AND METHODS |
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Cell synchronization. The cells were plated in 35-mm dishes in EMEM containing 10% FCS. After 24 h, the cells were rinsed twice with PBS and incubated with serum-free EMEM for 24 h to synchronize them in early G1. To obtain cells progressing through the G1 phase, we replaced the serum free-medium by EMEM with 10% FCS for 710 h. To synchronize cells on the G1/S boundary (end G1), the cells were incubated with 2 mM thymidine in EMEM with 10% FCS for 24 h.
To obtain cells accumulated in the S phase, the cells incubated with thymidine for 24 h were rinsed twice with PBS and incubated with EMEM, without thymidine, with 10% serum for 79 h. Cell-cycle distribution patterns were identified by measuring cellular DNA content using flow cytometry.
Cell proliferation assay. Cells were seeded in 24-well plates in EMEM with 5% FCS. After 24 h, the cells were treated with various agents. The medium was changed every other day. After 4 days of treatment, the cell number was determined by a colorimetric method (CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay).
Flow cytometry. Flow cytometry assays were performed on cell populations cultured in triplicate 25-cm2 flasks. Approximately 106 cells were fixed with 1 ml ice-cold 70% methanol for 30 min. After fixing, cells were pelleted by centrifugation to remove the fixatives, washed three times with phosphate-buffered saline (PBS) at 4°C, resuspended in 100 µl PBS, treated with 100 µl RNase (1 mg/ml, Sigma), and stained with propidium iodide (PI, Sigma) at a final concentration of 50 µg/ml. The stained cells were stored at 4°C in the dark and analyzed within 2 h. The stained samples were measured on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Data were acquired for 7,000 events with a variation coefficient of <5%, and red fluorescence was measured using a fluorescence detector 3 (FL3) on the x-axis. The data were stored and analyzed using CellQuest software to assess cell cycle distribution patterns (G0/G1, S, and G2/M phases).
Electrophysiology.
For electrophysiological analysis, the cells were cultured in 35-mm petri dishes at a density of 6,000 cells/cm2. Currents and membrane potential were recorded in voltage-clamp or current-clamp mode, using an Axopatch 200 B patch-clamp amplifier (Axon Instruments, Burlingame, CA) and Labmaster hardware (Digidata 2000, Axon Instrument). pClamp software (ver. 6.03, Axon Instruments) was used to control voltage, as well as to acquire and analyze data. The whole cell mode of the patch-clamp technique was used with 3- to 5-M resistance borosilicate fire-polished pipettes (A-M systems, Everett, WA). Seal resistance was typically in the 10- to 20-G
range. The maximum uncompensated series resistance was <10 M
during whole cell recordings, so the voltage error was <5 mV for a current amplitude of 500 pA. Recordings where series resistance resulted in errors >5 mV in voltage commands were discarded. Whole cell currents were allowed to stabilize for 5 min before K+ currents were measured. The capacitance of the membrane was measured by voltage clamp with a voltage pulse after completion of a whole cell patch-clamp procedure and the compensation of the electrode capacitance with electronic circuits built into the patch-clamp amplifier. Results were expressed using current densities instead of current amplitude. The cell surface of the MCF-7 cells was thus estimated by measuring their membrane capacitance (32 ± 4 pF, n = 90). The voltage-dependent outward currents were recorded using the whole cell patch-clamp technique during ramps from 120 to +60 mV, applied from a holding potential of 40 mV for 250 ms. The hIK1 current value was measured at the end of the ramp protocol. To eliminate a possible involvement of Kv1.1 K+ channels, all experiments were conducted in the presence of 10 nM
-dendrotoxin (
-DTX).
Cells were allowed to settle in petri dishes placed at the opening of a 250-µm-inner diameter capillary for extracellular perfusions (MSC-200, Manual solution changer, Bio-Logic Instruments, France). The cell under investigation was continuously superfused with control or test solutions. All electrophysiological experiments were performed at room temperature.
Solutions.
External and internal solutions had the following compositions (in mM): external, 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES at pH 7.4 (NaOH), osmolarity 300 mosM; internal, 150 KCl, 10 HEPES, 0.1 EGTA, 2 MgCl2, at pH 7.2 (KOH), osmolarity 292 mosM (measured with a freezing-point depression osmometer). AST and clotrimazole (CLT; Sigma) were made in DMSO. Final concentrations were obtained by appropriate dilution in an external control solution. The final concentration of DMSO was <0.1%. -DTX and r-IbTX (Latoxan) were made up in 1% BSA, HEPES 5 mM (pH 7.2). Final concentrations were obtained by appropriate dilution in an external control solution.
Free Ca2+ concentrations for the solution applied from the inner side of the membrane were buffered with 10 mM EGTA and calculated using Maxc Software (from Chris Patton, Hopkins Marine Station, Stanford University). For example, to produce 1 µM of free Ca2+, solutions with (in mM) 8.3 CaCl2, 1 MgCl2 and 10 EGTA were used (pH 7.2).
Statistical analysis. Results were expressed as means ± SD. Experiments were repeated at least three times. Statistical analysis was performed using GraphPad InStat Software. The Student's t-test for multiple comparisons modified by the Tukey-Kramer honestly significant difference method was used to compare treatment means with control means, and one-sided t-tests were used to test the significance of drug block with P < 0.05.
Ca2+ measurements. MCF-7 cells were grown on glass coverslips for Ca2+ imaging experiments. The [Ca2+]i was measured using fura 2-loaded cells. MCF-7 cells were loaded for 1 h at room temperature with 2 µM fura 2-AM prepared in saline solution and subsequently washed three times with the same dye-free solution. The coverslip was then transferred onto a perfusion chamber on an Olympus IX70 microscope equipped for fluorescence. Fluorescence was excited at 340 and 380 nm alternately, using a monochromator (Polychrome IV, TILL Photonics, Planegg, Germany), and captured by a MicroMax 5-MHz CCD camera (Princeton Instruments, Evry, France) after filtration through a long-pass filter (510 nm). Metafluor 4.5 software (Universal Imaging, West Chester, PA) was used for acquisition and analysis. The [Ca2+]i was derived from the ratio of fluorescence intensities for each of the excitation wavelengths (F340/F380) and from the equation of Grynkiewicz et al. (18). All recordings were carried out at room temperature. The cells were continuously perfused with the saline solution, and chemicals were added via the perfusion system. The flow rate of the whole chamber perfusion system was set to 1 ml/min, and the chamber volume was 500 µl.
RNA extraction and RT-PCR. Total RNA was isolated from MCF-7 unsynchronized cells, synchronized cells in the early G1 phase, cells arrested at the end of the G1 phase, and cells accumulated in the S phase, using the guanidium thiocyanate-phenol-chloroform extraction procedure (6). After a DNase I (Life Technologies) treatment to eliminate genomic DNA, 2.5 µg of total RNA were reverse transcribed into cDNA at 42°C, using random hexamer primers (Perkin Elmer) and MuLV reverse transcriptase (Perkin Elmer) in a 20-µl final volume, followed by PCR. PCR was carried out on the RT-generated cDNA using a GeneAmp PCR System 2400 thermal cycler (Perkin Elmer). To detect hIK cDNAs, PCR was performed by adding 1 µl (125 ng RNA equivalents) of RT template to the following mixture (final concentrations): 50 mM KCl, 10 mM Tris·HCl (pH 8.3), 2.5 mM MgCl2, 200 µM of each dNTP, 1 µM of sense and antisense primers, and 1 U AmpliTaq Gold (Perkin Elmer), giving a final volume of 25 µl. To control genomic DNA amplification, PCRs were also performed on the non-reverse-transcribed RNA (where the reverse transcriptase was omitted in the RT mix) of each sample. The DNA amplification conditions included an initial 10-min denaturation step at 95°C (which also activates the Gold variant of the Taq polymerase), and 40 cycles of 30 s at 95°C, 30 s at 60°C, 1 min at 72°C, and finally 7 min at 72°C. Half of the PCR samples were analyzed by electrophoresis in 2% agarose gel and stained with ethidium bromide (0.5 µg/ml). The PCR primers used to amplify the RT-generated hIK1 cDNAs were designed on the basis of established GenBank sequences. The primers were synthesized by Life Technologies. The primers for hIK1 cDNA were 5'-TCAATCAAGTCCGCTTCCG-3' (nucleotides 677695, GenBank accession AF033021 [GenBank] ) and 5'-TCAATCAAGTCCGCTTCCG-3' (nucleotides 12541275). The expected DNA length was 599 bp. To confirm the identity of the amplified products, restriction analysis was carried out on each PCR product using specific restriction enzymes.
Semiquantitative multiplex PCR.
To estimate the rate of the hIK1 mRNA in different phases of the cell cycle, hIK1 cDNA was amplified along with -actin cDNA as an internal control. The following (+) and () strand oligonucleotide primers were used to amplify
-actin cDNA (227 bp): 5'-CAGAGCAAGAGAGGCATCCT-3' and 5'-ACGTACATGGCTGGGGTGTTGAA-3'. The duplex PCR was performed with 10-fold-diluted cDNA of different MCF-7 samples. The conditions were 10 min at 95°C, then 28 cycles of 1 min extension at 72°C each, and a final 7-min extension step. The reaction products were separated by electrophoresis, using a 2% agarose gel in Tris-borate-EDTA buffer. The gel was stained with ethidium bromide and viewed by Gel Doc 1000 (Bio-Rad, Hercules, CA). The quantity of each PCR product was determined using Molecular Analyst software (Bio-Rad). The image density of the hIK1 PCR product was compared with the density of coamplified
-actin to determine the hIK1 mRNA expression ratio in unsynchronized and synchronized cells. All relative values of hIK1 expression in synchronized cells were compared and expressed as a percentage of that of unsynchronized cells. Control experiments using MCF-7 cell cDNA showed that the amount of each amplimer obtained in a multiplex PCR was independent of the presence of the other primers (cross-correlation analysis), thereby excluding the possibility of strong interference between primers. The number of cycles and the final reaction conditions were then adjusted to correspond to the exponential phase of the amplification of each product. Finally, the amount of each PCR product in a multiplex reaction was found to increase linearly with the amount of starting cDNA (550 ng RNA equivalents), ensuring that changes in the ratio of PCR product to control gene product truly reflect a change in the gene's mRNA abundance of the gene.
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RESULTS |
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Taken together, the results suggested that other types of K+ channels may be involved in the G1 phase, in G1/S transition, and in the progress of cells through the S phase.
Pharmacological profile of the Ca2+-activated K+ channels in hyperpolarized MCF-7 cells. It was previously suggested that the predominant K+ channel type in MCF-7 cells was a Ca2+-activated K+ channel (50). In the hyperpolarized MCF-7 cells (membrane potential = 69 mV), we recorded, under control conditions, an outward current of 120 to +60 mV for 250 ms with 150 mM K+ in the pipette (n = 60, Fig. 2A), while no current was activated when the pipette solution was 150 mM Cs (n = 15, Fig. 2A). The outward currents were K+ selective, as seen from the intersection of their current vs. the voltage-ramp curve [mean reversal potential (Erev) = 80 ± 3 mV (n = 25); Nernst potential of K (EK) = 86 mV]. Moreover, in symmetrical K+ solutions, the K+ current exhibited an inward rectification and Erev = 0 mV (n = 6, data not shown). Then the cell currents were pharmacologically characterized with several Ca2+-activated K+ channel-selective peptide toxins. Voltage ramps (120 to 40 mV, 250-ms duration) were applied every 30 s. The addition of apamin (1 µM), a SK2 and a SK3 channel blocker, had no effect on the K+ current (Fig. 2B). Moreover, the outward current was insensitive to D-tubocurarine (250 µM), a blocker of a small-conductance KCa channels (data not shown). CLT, a hIK1 channel blocker, blocked the K+ current by 25, 51, and 90% when used at 1, 2, and 5 µM, respectively (Fig. 2C). In the control conditions, ramp voltage from 120 to 40 mV rapidly elicited whole cell currents with an Erev (80 ± 3 mV, n = 14) close to the EK for K (86 mV); the 5 µM CLT perfusion essentially blocked a linear component without affecting the reversal potential (Fig. 2D). Moreover, the linear component was also blocked by 100 nM TRAM-34 (n = 10, data not shown). Finally, the perfusion of r-IbTX, a large-conductance Ca2+-activated K+ (BK) channel blocker, reduced the voltage-activated K+ current by 52 ± 5% (n = 15), when used at 50 nM (Fig. 2E). Furthermore, the effect of r-IbTX and CLT were additive (Fig. 2E).
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Thus the sensitivity to blocking by CLT and r-IbTX as well as insensitivity toward the selective SK2 and SK3 channel blocker apamin is consistent with the current being conducted by Ca2+-activated K+ channels closely related to the human hIK1 and BK channels.
To eliminate a possible involvement of Kv1.1 K+ channels, all experiments were conducted in the presence of 10 nM -dendrotoxin (
-DTX).
Ca2+ activates hIK1 and induces hyperpolarization in MCF-7 cells arrested in the early G1 phase. Figure 3A illustrates the activation of hIK1 currents with Ca2+ ionophore ionomycin (1 µM) in MCF-7 cells arrested in early G1 and preincubated with tetraethylammonium (TEA) (5 mM). The ionomycin-activated current was sensitive to 5 µM CLT (Fig. 3A). Moreover, Fig. 3B shows the outward currents that were recorded from MCF-7 synchronized in the early G1 with pipette solutions containing 1 µM free Ca2+ concentrations in the absence or presence of 5 mM TEA alone or combined with CLT 5 µM.
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The result indicated that activation of mainly hIK1 mediates hyperpolarization in the response to [Ca2+]i increase, while the activation of BK seems to play a minor or no role in the hyperpolarization.
Expression of hIK1 channels during the cell cycle. To determine the contribution of hIK1 channels during the cell cycle, whole cell patch-clamp measurements were carried out on cells arrested in the early G1, late G1, and S phases and the current density values for hIK1 K+ conductance were calculated. hIK1 current density was determined by dividing the CLT-sensitive current by membrane capacitance. hIK1 current density increased significantly in cells synchronized at the end of G1 phases (20 ± 1.8 pA/pF, n = 20, P < 0.001) or S (27 ± 5 pA/pF, n = 25, P < 0.001) compared with those in the early G1 phase (3 ± 2 pA/pF, n = 10).
These results suggest that hIK1 K+ conductance was only activated in cells arrested at the end of G1 and during S phases.
Comparison of the mRNA expression of hIK1 channels during the cell cycle.
Figure 4A shows the results of RT-PCR analysis of the hIK1 expression in total RNA extracts from unsynchronized MCF-7 cells. The primers designed for hIK1 amplified the fragments of the expected size of 599 bp for hIK1, thereby showing the expression of the hIK1 potassium channel mRNA in MCF-7 cells. To study the variation in hIK1 mRNA expression during the cell cycle, we used the semiquantitative RT-PCR as described in MATERIALS AND METHODS. The relative abundance of the major hIK1 mRNA expressed in MCF-7 cells (hIK1) in different phases of the cell cycle was determined by comparing the amount of the amplification product for hIK1 with the amplification of -actin mRNA expressed in the same sample. These studies established the linearity of the amplification rate under these experimental conditions. Figure 4B shows that the level of hIK1 mRNA expression increased in cells synchronized in the early G1 (229 ± 12%), reached a maximum in cells synchronized at the end of the G1 phase (282 ± 19%), and increased in cells accumulated in the S phase (402 ± 24%) compared with unsynchronized cells (100%, Fig. 4B). These experiments clearly show a strong variation in hIK1 K+ channel mRNA expression during cell-cycle progression.
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Cell-cycle analysis. We predicted that the growth inhibition produced by CLT was likely to be accompanied by the arrest of the cells in a specific cell-cycle phase. Cells were analyzed using flow cytometry after 4 days in culture in the presence of CLT (5 µM), econazole (10 µM), and ketoconazole (10 µM). Exponentially growing cells had a distribution of 75 ± 3.1% in G1, 16 ± 3.7% in S, and 9.2 ± 0.8% in G2/M (n = 6). In CLT-treated cells, the proportion of G1 cells increased significantly, to 85.9 ± 0.57% (P < 0.001, n = 6), while there was a significant decrease in S-phase cells (4.4 ± 0.79%, P < 0.001, n = 6). In econazole-treated cells, the proportion of G1 cells also increased, to 82 ± 0.27% (P < 0.001, n = 6), and a significant decrease in S-phase cells was also observed (5 ± 0.4%, P < 0.001, n = 6). However, in cells treated with ketokonazole, no accumulation in the G1 phase or decrease in the S phase was observed. These results show that hIK1 K+ channel inhibition by CLT and econazole leads to cell-cycle arrest and growth inhibition. Moreover, in cells treated with both AST and CLT, we observed a significant increase in the number of cells arrested in the G1 phase (99.5 ± 0.1%, P < 0.001, n = 6, Fig. 7D) compared with CLT alone as well as a drastic decrease in the S phase (Fig. 7D).
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DISCUSSION |
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In this study, we demonstrate, for the first time, that in addition to the initial hyperpolarization produced by the activation of hEAG K+ channels in MCF-7 cells, the activation of hIK1-like channels also contributes to the progression through the G1 phase and in the G1/S transition.
Our experiments show that SK4/hIK1-like channels mediate some of the native currents in MCF-7 cells. These channels (hIK1-like) expressed in MCF-7 cells are not the typical Ca2+-dependent IK channels. The channel reported in this paper, which we termed hIK1-like, is sensitive to CLT at 1 µM, TRAM-34, and to Ba2+, but not to charybdotoxin (ChTX). The lacking effect of ChTX on hIK1-like is an unexpected finding, since ChTX is known to be a classical Gardos channel blocker. Moreover, the perfusion of hIK1 activators CZ and ZOX activated the outward current recorded in MCF-7 cells and induced a hyperpolarization as expected for K+ channel openers. However, the prolonged exposure (3 days) of MCF-7 cells to CZ or ZOX inhibited cell proliferation. The results are in agreement with the work of Koegel et al. (29), who reported a downregulation of both hIK1 mRNA levels and channel activity after 3 days of treatment of HaCaT keratinocytes with hIK1 openers, i.e., 1-EBIO, CZ, and ZOX. The downregulation of hIK1 was accompanied by a loss of mitogenic activity and a strong increase in cell size (29). They suggested that 1-EBIO induces a conformational change in the channel protein that leads to more rapid internalization and/or degradation. The inhibition of hIK1 channel activity leads also to inhibition of cell proliferation. However, the block of hIK1 channels induces a membrane depolarization that is sufficient to inhibit cell proliferation by elimination of the inward force for Ca2+ and then a decrease in [Ca2+]i.
A number of native channels with characteristics different from those of the erythrocyte-type IK channel have traditionally been classified as IK channels. Thus the IK channel group is quite heterogeneous, comprising channels that differ widely in single-channel conductances, degree of rectification, pharmacology, Ca2+ sensitivity, and calmodulin (CaM) regulation (19, 31, 10). Thus, besides methodological differences, these controversial findings could be due to species differences. However, one possibility is that hIK1-like channel expressed in MCF-7 cells must interact with another K+ channel subunit to reconstitute a functional heterotetramer but not a homotetramer IK channel.
We also showed a large BK conductance inhibited by r-IbTX and ChTX. Moreover, the lack of sensitivity of the outward current to the bee venom peptide toxin apamin or the plant alkaloid D-tubocurarine indicated the absence of small conductance channels (SK2, SK3) in MCF-7 cells.
Using the RT-PCR method, Jensen et al. (23) have already reported the expression of the hIK1 transcript in the mammary gland. In MCF-7 cells, the expression of hIK1-like channels is not constant throughout the cell cycle. The hIK1 current density was not detected in cells arrested in early G1 but increased in cells arrested at the end of G1 and S phases. These changes are consistent with previous RT-PCR studies showing an increase in hIK1 mRNA levels in cells arrested at the end of G1. Thus it is likely that hIK1 channels are involved in cell cycle progression. Furthermore, hIK1 expression has been shown to regulate mitogenic cell growth and proliferation in other cells, such as human fibroblasts (33), B and T lymphocytes (15, 17, 43), as well as in prostate cancer cells (41, 44). Moreover, hIK1 channels are also important in regulating the membrane potential (30) and appears to be coupled to Cl secretion in epithelial cells in response to agonists that elevate intracellular Ca2+ (12).
In human breast cancer cells, the electrophysiological and molecular studies have shown that the MCF-7 cell line expresses a plethora of distinct ionic channels (1, 28, 36, 38, 39, 50). The first K+ channel characterized was a KCa channel (50). Moreover, Wonderlin et al. (52) suggested that KCa channels were expressed in MCF-7 cells and postulated that these channels were involved in cell cycle regulation. Our results show that hIK1-like channel expression is dramatically enhanced in cells at the end of G1 and those entering the S phase, in parallel with a strong hyperpolarization and an enhanced [Ca2+]i. Pharmacological blocking of the hIK1-like channel has been shown to depolarize and reduce [Ca2+]i, suggesting that hIK1-mediated hyperpolarization increases the electrochemical driving force for Ca2+ influx. Indeed, an important physiological role of the IK channel is to help maintain sharp electrical gradients for the sustained transport of ions such as the Ca2+ influx that controls cell proliferation (for review, see Ref. 24).
The contribution of hIK1-like channels to MCF-7 cell proliferation and cell cycle progression is still poorly understood. The treatment of MCF-7 cells with hIK1-inhibitors (CLT, econazole) reduced cell proliferation in a dose-dependent manner, suggesting that hIK1-like channels were involved in regulating MCF-7 cell proliferation. Moreover, the growth inhibition produced by both CLT and econazole was accompanied by an accumulation of cells in the G1 phase and a reduction in the number of cells in the S phase.
To distinguish between the contributions of hEAG and hIK1-like channels to MCF-7 cell proliferation, we used AST to block hEAG and CLT to block hIK1. As expected, blocking hIK1 inhibited proliferation of MCF-7 less effectively, i.e, by 25% compared with 60% when hEAG was blocked. Blocking both channels (AST + CLT) was additive, reducing MCF-7 proliferation by 80 ± 2%. Furthermore, the growth inhibition produced by AST and CLT, alone or combined, was accompanied by an accumulation of cells in the G1 phase. However, blocking hEAG meant that greater numbers of cells accumulated in the G1 phase compared with the hIK1-like block. When used together, 99.5 ± 0.1% of cells were synchronized in the G1 phase.
Ca2+ is an important messenger involved in several phases of the cell cycle, and increased Ca2+ activity and/or Ca2+ levels have been associated with increased proliferation in most cell types. As expected, both AST and CLT reduce the basal [Ca2+]i in the MCF-7 cell. However, the effect of AST on cytosolic [Ca2+] is observed in the cells arrested in the early G1, while CLT induced a greater decrease in [Ca2+]i in cells arrested at the end of G1 and throughout S phases. To further clarify the respective roles of Ca2+, membrane changes and the inhibition of MCF-7 proliferation by AST, CLT, or both, we measured the membrane potential and the basal [Ca2+]i after the prolonged exposure of MCF-7 cells to these substances. Our results show a direct correlation between the membrane depolarization and the decrease in the basal [Ca2+]i. Thus inhibition of K+ channels and the decrease in [Ca2+]i represents a possible means of specifically inhibiting MCF-7 cell proliferation.
How might both hEAG and hIK1-like channels contribute to MCF-7 cell proliferation and progression through the cell cycle? The ability of the hEAG blocker to inhibit proliferation and to arrest the cell cycle indicates that this type of K+ channel may play a critical regulatory role in the growth of human breast cancer cells. However, as hEAG expression is transient and the membrane potential continued to hyperpolarize through the G1 and S phases (Fig. 1), we propose a model that links the activity of these two classes of K+ channels. In early G1, the membrane potential is depolarized (approximately 20 mV) with little or no activation of hIK1-like, due perhaps to a low resting [Ca2+]i. hEAG is voltage gated, activated by depolarization, and its steady-state activity is not null at 20 mV. A neo-expression of hEAG channels [increase in mRNA levels (39)] induces an increase in the current density, thence hyperpolarizing the membrane potential and increasing Ca2+ entry (Fig. 6A). Gating of the hIK1 channel is voltage independent but exquisitely sensitive to increases in internal Ca2. Thus the initial Ca2+ entry during G1 is regeneratively amplified by the activation of hIK1-like channels, resulting in the strong hyperpolarization of the membrane potential during progression through G1 and into the S phase.
Several studies have reported that hIK1 activity may be dynamically regulated by phosphorylation (16, 26, 42, 45). Interestingly, both hEAG and hIK1 channels are regulated by intracellular Ca2+ and CaM (26, 46) in reverse, i.e., [Ca2+]i decreases hEAG and increases hIK1 channel activity. This apparent paradox is explained by the system's different regulatory mechanisms: hEAG channels are closed by the binding of only one CaM molecule (46), whereas it has been reported that four Ca2+-loaded CaM molecules are required to activate hIK1 channels (14, 25). This reverse regulation can take place in the same cell, for example, in human melanoma, where both hEAG and hIK1 channels have been identified (35). Based on these observations, we speculate that the phosphorylation-dependent modulation of hEAG and hIK1 plays a critical role in modulating the progression of cells through G1 and into the S phase. In breast cells, it has been reported that Ca2+ is involved in controlling cell growth through its interaction with CaM (13). Furthermore, MCF-7 cells require CaM to traverse the G1/S boundary (2, 34, 47). Treating MCF-7 cells with CaM antagonists (calmidazolium and W12) inhibited proliferation and caused an increase in the percentage of cells in the G1 phase, accompanied by a decrease in the percentage in the S phase (47).
In summary, our results provide new evidence that the hIK1-like K+ channel is important for progression in the G1 phase and through the G1/S transition and imply that blockers for this potassium channel may have a therapeutic potential for the treatment of cancers.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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2. Bachs O, Agell N, and Carafoli E. Calcium and calmodulin function in the cell nucleus. Biochim Biophys Acta 1113: 259270, 1992.[ISI][Medline]
3. Boonstra G, Mummery CL, Tertoolen LGJ, Van der Saag P, and De Laat SW. Cation transport and growth regulation in neuroblastoma cells. Modulations of K+ transport and electrical membrane properties during the cell cycle. J Cell Physiol 107: 7583, 1981.[ISI][Medline]
4. Brüggemann A, Stühmer W, and Pardo LA. Mitosis-promoting factor-mediated suppression of a cloned delayed rectifier K+ channel expressed in Xenopus oocytes. Proc Natl Acad Sci USA 94: 537542, 1997.
5. Chittajallu R, Chen Y, Wang H, Yuan X, Ghiani CA, Heckman T, McBain CJ, and Gallo V. Regulation of Kv1 subunit expression in oligodendrocyte progenitor cells and their role in G1/S phase progression of the cell cycle. Proc Natl Acad Sci USA 99: 23502355, 2002.
6. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156159, 1987.[CrossRef][ISI][Medline]
7. Cory JG, Carter GL, and Karl RC. Calcium ion-dependent proliferation of L1210 cells in culture. Biochem Biophys Res Commun 145: 556562, 1987.[ISI][Medline]
8. Day ML, Johnson MH, and Cook DI. A cytoplasmic cell cycle controls the activity of a K+ channel in pre-implantation mouse embryos. EMBO J 17: 19521960, 1998.
9. Day ML, Pickering SJ, Johnson MH, and Cook DI. Cell-cycle control of a large-conductance K+ channel in mouse early embryos. Nature 365: 560562, 1993.[CrossRef][ISI][Medline]
10. Del Carlo B, Pellegrini M, and Pellegrini M. Calmodulin antagonists do not inhibit IK(Ca) channel in human erythrocytes. Biochim Biophys Acta 1558: 133141, 2002.[ISI][Medline]
11. Deutsch C, Price M, Lee S, King VF, and Garcia ML. Characterization of high affinity binding sites for charybdotoxin in human T lymphocytes. Evidence for association with the voltage-gated K+ channel. J Biol Chem 266: 36683674, 1991.
12. Devor DC, Singh AK, Gerlach AC, Frizzell RA, and Bridges RJ. Inhibition of intestinal Cl-secretion by clotrimazole effect on basolateral membrane K+ channels. Am J Physiol Cell Physiol 273: C531C540, 1997.
13. Etindi R and Manni A. TGF- and IGF-I effects on calcium ion transients in human breast cancer cells. Cancer Lett 66: 131137, 1992.[CrossRef][ISI][Medline]
14. Fanger CM, Ghanshani S, Logsdon NJ, Rauer H, Kalman K, Zhou J, Beckingham K, Chandy KG, Cahalan MD, and Aiyar J. Calmodulin mediates calcium-dependent activation of the intermediate conductance KCa channel, IKCa1. J Biol Chem 274: 57465754, 1999.
15. Fanger CM, Neben AL, and Cahalan MD. Differential Ca2+ influx, KCa channel activity, and Ca2+ clearance distinguish Th1 and Th2 lymphocytes. J Immunol 164: 11531160, 2000.
16. Gerlach AC, Gangopadhyay NN, and Devor DC. Kinase-dependent regulation of the intermediate conductance, calcium-activated potassium channel, hIK1. J Biol Chem 275: 585598, 2000.
17. Ghanshani S, Wulff H, Miller MJ, Rohm H, Neben A, Gutman GA, Cahalan MD, and Chandy G. Upregulation of the IKCa1 potassium channel during T-cell activation. J Biol Chem 275: 3713737149, 2000.
18. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 34403450, 1985.[Abstract]
19. Hay M and Kunze DL. An intermediate conductance calcium-activated potassium channel in rat visceral sensory afferent neurons. Neurosci Lett 167, 179182, 1994.[ISI][Medline]
20. Hazelton B, Mitchell B, and Tupper J. Calcium, magnesium, and growth control in the WI-38 human fibroblast cell. J Cell Biol 83: 487498, 1979.[Abstract]
21. Ichikawa J, Furuya K, Miyata S, Nakashima T, and Kiyohara T. EGF enhances Ca2+ mobilization and capacitative Ca2+ entry in mouse mammary epithelial cells. Cell Biochem Funct 18: 215225, 2000.[CrossRef][ISI][Medline]
22. Iftinca M, Waldron GJ, Triggle CR, and Cole WC. State-dependent block of rabbit vascular smooth muscle delayed rectifier and Kv1.5 channels by inhibitors of cytochrome P450-dependent enzymes. J Pharmacol Exp Ther 298: 718728, 2001.
23. Jensen BS, Strobaek D, Christophersen P, Jorgensen TD, Hansen C, Silahtaroglu A, Olesen SP, and Ahring PK. Characterization of the cloned human intermediate conductance Ca2+-activated K+ channel. Am J Physiol Cell Physiol 275: C848C856, 1998.[Abstract]
24. Jensen BS, Strobaek D, Olesen SP, and Christophersen P. The Ca2+-activated K+ channel of intermediate conductance: a molecular target for novel treatments? Curr Drug Targets 2: 401422, 2001.[ISI][Medline]
25. Keen JE, Khawaled R, Farrens DL, Neelands T, Rivard A, Bond CT, Janowsky A, Fakler B, Adelman JP, and Maylie J. Domains responsible for constitutive and Ca2+ dependent interactions between calmodulin and small conductance Ca2+-activated potassium channels. J Neurosci 19: 88308838, 1999.
26. Khanna R, Chang MC, Joiner WJ, Kaczmarek LK, and Schlichter LC. hSK4/hIK1, a calmodulin-binding KCa channels in human T lymphocytes. J Biol Chem 274: 1483814849, 1999.
27. Kiefer H, Blume AJ, and Ebert JD. Membrane potential changes during mitogenic stimulation of mouse spleen lymphocytes. Proc Natl Acad Sci USA 77: 22002204, 1980.[Abstract]
28. Klimatcheva E and Wonderlin WF. An ATP-sensitive K+ current that regulates progression through early G1 phase of the cell cycle in MCF-7 human breast cancer cells. J Membr Biol 171: 3546, 1999.[CrossRef][ISI][Medline]
29. Koegel H, Kaesler S, Burgstahler R, Werner S, and Alzheimer C. Unexpected down-regulation of the hIK1 Ca2+-activated K+ channel by its opener 1-ethyl-2-benzimidazolinone in HaCaT keratinocytes. Inverse effects on cell growth and proliferation. J Biol Chem 278: 33233330, 2003.
30. Kohler R, Degenhardt C, Kuhn M, Runkel N, Paul M, and Hoyer J. Expression and function of endothelial Ca2+-activated K+ channels in human mesenteric artery: a single-cell reverse transcriptase-polymerase chain reaction and electrophysiological study in situ. Circ Res 87: 496503, 2000.
31. Latorre R, Oberhauser A, Labarca P, and Alvarez O. Varieties of calcium-activated potassium channels. Annu Rev Physiol 51: 385399, 1989.[CrossRef][ISI][Medline]
32. Lepple-Wienhues A, Berweck S, Böhmig M, Leo CP, Meyling B, Garbe C, and Wiederholt M. K+ channels and the intracellular calcium signal in human melanoma cell proliferation. J Membr Biol 151: 149157, 1996.[CrossRef][ISI][Medline]
33. Logsdon NJ, Kang J, Togo JA, Christian EP, and Aiyar J. A novel gene, hSK4, encodes the calcium-activated potassium channel in human T lymphocytes. J Biol Chem 272: 3272332726, 1997.
34. Lu KP and Means AR. Regulation of the cell cycle by calcium and calmodulin. Endocr Rev 14: 4058, 1993.[ISI][Medline]
35. Meyer R, Schonherr R, Gavrilova-Ruch O, Wohlrab W, and Heinemann SH. Identification of ether à go-go and calcium-activated potassium channels in human melanoma cells. J Membr Biol 171: 107115, 1999.[CrossRef][ISI][Medline]
36. Mu D, Chen L, Zhang X, See LH, Koch CM, Yen C, Tong JJ, Spiegel L, Nguyen KCQ, Servoss A, Peng Y, Pei L, Marks JR, Lowe S, Hoey T, Jan LY, McCombie WR, Wigler MH, and Powers S. Genomic amplification and oncogenic properties of the KCNK9 potassium channel gene. Cancer Cell 3: 297302, 2003.[ISI][Medline]
37. Nilius B and Wohlrab W. Potassium channels and regulation of proliferation of human melanoma cells. J Physiol 445: 537548, 1992.[Abstract]
38. Ouadid-Ahidouch H, Chaussade F, Roudbaraki M, Slomianny C, Dewailly E, Delcourt P, Prevarskaya N. Kv1.1 K+ channels identification in human breast carcinoma cells: involvement in cell proliferation. Biochem Biophys Res Commun 278: 272277, 2000.[CrossRef][ISI][Medline]
39. Ouadid-Ahidouch H, Le Bourhis X, Roudbaraki M, Toillon RA, Delcourt P, and Prevarskaya N. Changes in the K+ current-density of MCF-7 cells during progression through the cell cycle: possible involvement of a h.ether-à-gogo K+ channel. Receptors Channels 7: 345356, 2001.[ISI][Medline]
40. Pardo LA, Brüggemann A, Camacho J, and Stühmer W. Cell cycle-related changes in the conducting properties of r-eag K+ channels. J Cell Physiol 143: 767775, 1998.
41. Parihar AS, Coghlan MJ, Gopalakrishnan M, and Shieh CC. Effects of intermediate-conductance Ca2+-activated K+ channel modulators on human prostate cancer cell proliferation. Eur J Pharmacol 471: 157164, 2003.[CrossRef][ISI][Medline]
42. Pellegrino M and Pellgrini M. Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous cAMP-dependent protein kinase. Pflügers Arch 436: 749756, 1998.[CrossRef][ISI][Medline]
43. Pena TL and Rane SG. The fibroblast intermediate conductance KCa channel, FIK, as prototype for the cell growth regulatory function of the IK channel family. J Membr Chem 172: 249257, 1999.
44. Rane SG. The growth regulatory fibroblast IK channel is the prominent electrophysiological feature of rat prostatic cancer cells. Biochem Biophys Res Commun 269: 457463, 2000.[CrossRef][ISI][Medline]
45. Roch B, Baro I, Hongre AS, and Escande D. ATP-sensitive K+ channels regulated by intracellular Ca2+ and phosphorylation in normal (T84) and cystic fibrosis (CFPAC-1) epithelial cells. Pflügers Arch 429: 355363, 1995.[CrossRef][ISI][Medline]
46. Schrönherr R, Lober K, and Heinemann H. Inhibition of human ether à go-go potassium channels by Ca2+/calmodulin. EMBO J 19: 33633271, 2000.
47. Strobl JS, Wonderlin WF, and Flynn DC. Mitogenic signal transduction in human breast cancer cells. Gen Pharmacol 26: 16431649, 1995.[CrossRef][Medline]
48. Tupper JT, Kaufman L, and Bodine PV. Related effects of calcium and serum on the G1 phase of the human W138 fibroblast. J Cell Physiol 104: 97103, 1980.[ISI][Medline]
49. Wagstaff SC, Bowler WB, Gallagher JA, and Hipkind RA. Extracellular ATP activates multiple signaling pathways and potentiates growth factor-induced c-fos gene expression in MCF-7 breast cancer cells. Carcinogenesis 21: 21752181, 2000.
50. Wegman EA, Young JA, and Cook DI. A 23-pS calcium-activated potassium channel in MCF-7 human breast carcinoma cells: an apparent correlation of channel incidence with the rate of cell proliferation. Pflügers Arch 417: 562570, 1991.[ISI][Medline]
51. Wonderlin WF and Strobl JS. Potassium channels, proliferation and G1 progression. J Membr Biol 154: 91107, 1996.[CrossRef][ISI][Medline]
52. Wonderlin WF, Woodfork KA, and Strobl JS. Changes in membrane potential during the progression of MCF-7 human mammary tumor cells through the cell cycle. J Cell Physiol 165: 77185, 1995.[ISI][Medline]
53. Woodfork KA, Wonderlin WF, and Strobl JS. Inhibition of ATP-sensitive potassium channels causes reversible cell-cycle arrest of human breast cancer cells in tissue culture. J Cell Physiol 162: 163171, 1995.[ISI][Medline]