Ca2+-activated K+ channels in human melanoma cells are up-regulated by hypoxia involving hypoxia-inducible factor-1 and the von Hi
1 Institute of Molecular Cell Biology, Research Unit Molecular and Cellular Biophysics, Friedrich Schiller University Jena, Drackendorfer Str. 1, D-07747 Jena, Germany
2 Clinics for Dermatology, Friedrich Schiller University Jena, Erfurter Str. 35, D-07740 Jena, Germany
3 Department of Neurosurgery, Yokohama City, University Graduate School of Medicine, Yokohama 236-0004, Japan
Abstract
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Under chronic hypoxia, tumour cells undergo adaptive changes involving hypoxia-inducible factors (HIFs). Here we report that ion currents mediated by Ca2+-activated K+ (KCa) channels in human melanoma IGR1 cells are increased by chronic hypoxia (3% O2), as well as by hypoxia mimetics. This increase involves the HIF system as confirmed by overexpression of HIF-1 or the von Hippel-Lindau tumour suppressor gene. Under normoxic conditions the KCa channels in IGR1 cells showed pharmacological characteristics of intermediate conductance KCa subtype IK channels, whereas the subtype SK2 channels were up-regulated under hypoxia, shown with pharmacological tools and with mRNA analysis. Hypoxia increased cell proliferation, but the KCa channel blockers apamin and charybdotoxin slowed down cell growth, particularly under hypoxic conditions. Similar results were obtained for the cell line IGR39 and for acutely isolated cells from a biopsy of a melanoma metastasis. Thus, up-regulation of KCa channels may be a novel mechanism by which HIFs can contribute to the malignant phenotype of human tumour cells.
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Introduction
Many human tumours develop hypoxic microenvironments as a result of insufficient and uncoordinated growth of blood vessels (Brown & Giaccia, 1998). In this milieu, tumours proliferate and gradually acquire aggressive phenotypic traits. Consequently, oxygen-deprived regions in tumours were found to indicate a poorer prognosis and reduced survival rates (Hckel et al. 1996; Brizel et al. 1996). The various mechanisms by which hypoxia can increase tumour malignancy include: (1) an increased expression of angiogenic cytokines (Shannon et al. 2003); (2) selection for tumour cells with diminished sensitivity to radiotherapy or chemotherapy (Graeber et al. 1996; Shannon et al. 2003); and (3) the promotion of migration and metastatic spread (Brizel et al. 1996; Rofstad & Halsr, 2002). In the last few years, hypoxia-inducible factors (HIFs) have been recognized as key regulators of these processes, controlling the expression of responsive genes including those encoding erythropoietin, vascular endothelial growth factor, tyrosin hydroxylase and various glycolytic enzymes (Jelkmann, 1992; Czyzyk-Krzeska et al. 1992; Goldberg & Schneider, 1994; Semenza et al. 1994; Semenza, 2004).
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HIF is a heterodimeric complex of an oxygen-sensitive HIF- protein and a stable HIF- subunit. Under normoxic conditions, HIF-1 is a target of oxygen-dependent proline hydroxylases, which catalyse hydroxylation at two conserved residues (Pro-402 and Pro-564) (Jaakkola et al. 2001; Ivan et al. 2001) and thereby allow binding of the von Hippel-Lindau protein (pVHL) to HIF-1 (Maxwell et al. 1999; Ohh et al. 2000). The tumour suppressor protein pVHL forms part of a ubiquitin ligase complex, which adds ubiquitin to HIF-1 and targets it for proteasomal destruction (Ohh et al. 2000; Tanimoto et al. 2000).
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There is increasing evidence that membrane ion channels play an important role in cell proliferation and cell cycle control (Wonderlin & Strobl, 1996; Wang, 2004; Pardo, 2004). The relationship between K+ channels and cell cycle has been studied for more than a decade, because the most obvious function of K+ channels in non-excitable cells, including tumour cells, is the control of the resting membrane potential (Wonderlin & Strobl, 1996; Lepple-Wienhues et al. 1996; Gavrilova-Ruch et al. 2002; Ouadid-Ahidouch et al. 2004). Several studies have shown that membrane hyperpolarization is required for the passage of cells through the G1/S phase transition of the cell cycle and this hyperpolarization is mediated by the opening of K+ channels (Kiefer et al. 1980; Chittajallu et al. 2002). Moreover, it has been shown that blockade of K+ channel activity leads to membrane depolarization and an arrest in the early G1 phase (Lepple-Wienhues et al. 1996; Ouadid-Ahidouch et al. 2001). We have previously reported that the human (h) ether à go-go (hEAG) K+ channels in human melanoma cells contribute to cellular proliferation and may provide initial hyperpolarization for the progression into the G1 phase of the cell cycle (Gavrilova-Ruch et al. 2002). In addition, it has been shown that not only hEAG but also Ca2+-activated K+ channels (KCa channels) are responsible for the progression of the cell cycle in human breast cancer cells (Ouadid-Ahidouch et al. 2001).
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In the human melanoma cell line IGR1, hEAG1 and KCa channels constitute the major groups of K+ channels (Meyer et al. 1999; Gavrilova-Ruch et al. 2002). KCa channels are further classified into two types, intermediate-conductance human (hIK or hSK4) and small-conductance (hSK1–3) K+ channels. In IGR1, the pharmacological profile indicated a predominance of hIK channels, being blocked by 100 nM charybdotoxin (ChTX) but not by 100 nM apamin (Meyer et al. 1999). Unlike hSK channels, hIK channels are widely expressed in peripheral tissues, with highest expression in smooth muscle cells (Joiner et al. 1997; Ishii et al. 1997); however, hIK has also been observed in melanoma cells (Meyer et al. 1999). hIK channels have been implicated in the regulation of secretion, in cellular migration and in the proliferation of mitogenically active cells (Mauro et al. 1993; Schwab et al. 1999). However, the role and regulation of the KCa channels in melanoma cells are poorly understood under normoxic as well as under hypoxic conditions.
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Here we report that KCa channels are up-regulated by hypoxia and that the HIF sensory system is responsible for this process. Hypoxia-induced up-regulation was accompanied by increased sensitivity of the currents to apamin, indicating an increased activity of SK-type KCa channels. The levels of mRNA coding for IK and SK2 channels were transiently increased upon hypoxia. Furthermore, we found that cell proliferation was enhanced by chronic hypoxia. Application of the KCa channel blockers ChTX and, with lower potency, apamin prevented this effect on hypoxic cell growth. Thus, up-regulation of apamin-sensitive KCa channels by an HIF-dependent mechanism is linked to enhanced proliferation of tumour cells under hypoxia. These findings contribute to the understanding of the pathophysiology of malignant melanoma cells and shed light on a new mechanism by which HIF-1 proteins can lead to malignant tumour progression. This is particularly relevant as we also found KCa channels with identical regulation by hypoxia in an acutely dissociated metastasis of a melanoma patient.
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Methods
Recombinant DNA
The following constructs were used in this study: von Hippel-Lindau (VHL) cDNA (Gnarra et al. 1994) encoding residues 54–213 (pVHL19) subcloned into pIRES2-EGFP (pVHL-EGFP; Clontech, Heidelberg, Germany), pVHL19 in pcDNA3.1 (Invitrogen, Karlsruhe, Germany), pVHL- (pVHL1987-130), HIF-1 (in pBOS), HIF-1-P564A (in pBOS), HIF-1-N (HIF-1-1–59 in pBOS), HIF-1-N-P564A, rat (r) SK2 (in pcDNA3), pCD8, pEGFP-N1 (Clontech). PCR-based site-directed mutagenesis was performed using the QuikChange kit (Stratagene, La Jolla, CA, USA) to produce the point mutant P564A of HIF-1.
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Cell culture and transfections
The human melanoma cell line IGR1 was maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing 10% fetal calf serum (FCS) at 37°C in a humidified 10% CO2 atmosphere (normoxic conditions). IGR39 cells were cultivated in Minimum Essential Medium (Invitrogen) supplemented with 5% FCS at 37°C in a 5% CO2. HEK 293 cells were maintained in 5% CO2 and DMEM/F-12 (1 : 1) medium with 10% FCS. Cells were transiently transfected with the PolyFect transfection reagent (Qiagen, Hilden, Germany) using a total amount of 2 μg plasmid DNA. To allow identification of transfected cells, either pEGFP-N1 or pCD8 were cotransfected in a 4 : 1 ratio of target plasmid versus the EGFP or CD8 plasmid. For stable transfection, pVHL-EGFP-transfected IGR1 cells were selected with 1 mg ml–1 G418. All transfectants were assayed under normoxic conditions.
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For hypoxic treatments, cells were plated at a density of 4 x 104 cells ml–1. Cells were transferred to an incubator that maintained an atmosphere of 3% O2–10% CO2 balanced with N2 1 day after plating. Alternatively, inhibitors of prolyl hydroxylases (desferrioxamine (DFO) or cobalt chloride (CoCl2)) were added to the culture medium at a final concentration of 100 μM. Cells were kept under these conditions for between 3 and 72 h.
Isolation of primary cells from melanoma metastases
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Biopsies from melanoma metastases (5 mm3) were obtained from patients of the Clinics for Dermatology, University of Jena with informed consent of the patients and approval by the local ethics committee. The biopsy was immediately placed in DMEM (high glucose, Invitrogen). Within 1 h after surgery, the tissue was dissected into small pieces in cold Hanks' balanced salt solution (HBSS; Invitrogen), trypsinized and passed several times through Pasteur pipettes of decreasing diameters. The dissociated cells were then cultivated in DMEM (high glucose, 10% FCS) in 24-well dishes at 37°C (10% CO2). The melanoma markers melan-A and HMB45 were used for immunohistochemical verification of isolated cells.
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Quantitative RT-PCR
Total RNA from melanoma cells was isolated from 5 x 106 to 1 x 107 cells using the RNeasy Mini kit (Qiagen) following the manufacturers instructions. RNA (1.5 μg) was used for cDNA synthesis using oligo(dT) primer and SuperScriptII reverse transcriptase (Invitrogen, SuperScript First Strand Synthesis System for RT-PCR). A Light Cycler rapid thermal cycler system and the Light Cycler Fast Start DNA MasterPlus SYBR Green I-kit were used (Roche, Mannheim, Germany) for hot start amplification and quantification of cDNA. Each run was followed by a melting curve to 95°C to exclude primer dimer formation as a source of fluorescence. Amplification of -actin mRNA was used to normalize determinations from individual RNA samples. The sequences of sense and antisense primers for IK (KCNN4, NM_002250), SK1 (KCNN1, NM_002248) and SK3 (KCNN3, NM_002249) are described by Meyer et al. (1999). For SK2 (KCNN2, NM_021614) they were: 5'-GGT ACC ATG ATC AAC AGG ATG TTA CTA GC-3' and 5'-TCA TTC AGT TTC CTC TGC TCC A-3'; for actin (ACTB, NM_001101): 5'-CCA AGG CCA ACC GCG AGA AGA TGA C-3' and 5'-AGG GTA CAT GGT GGT GCC GCC AGA C-3'.
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Western blot analysis
Cultured cells from one 75-cm2 flask were scraped into 5 ml ice cold PBS and sedimented at 2000 g. Pellets were resuspended in 100 μl ice-cold lysis buffer containing (mM): NaCl 150 , EDTA, EGTA 1, Tris 50 and 1% Triton-X 100; pH 7.5 with Complete Protease Inhibitor Mix (Roche). The lysates were centrifuged at 10 000 g for 10 min at 4°C and supernatants were used for further analysis. Protein concentrations were determined using the Bradford reagent and 40 μg per sample was resolved by 9% SDS-PAGE, followed by electro-transfer to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). Membranes were probed with mouse monoclonal anti-HIF-1 antibodies at 1 : 500 dilution (BD Transduction Laboratories, Heidelberg, Germany), or with mouse monoclonal anti- actin antibodies at 1 : 2000 dilution (Sigma, Taufkirchen, Germany). Signals were detected using the enhanced chemiluminescence method (ECL plus, Amersham, Freiburg, Germany).
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Proliferation assays
Cells were trypsinized and plated at a density of 2–4 x 104 cells ml–1 in a final volume of 100 μl well–1 in 96-well plates. Drugs were added 24 h after plating. For determination of the cell numbers, cells were cultured in 24-well plates (500 μl well–1) and counted in a cell counter (CASY Cell Counter Model DT, Schrfe System, Reutlingen, Germany). After 48 h, proliferation and metabolism were estimated using a colorimetric bromodeoxyuridine assay (Brd U, Roche) and a colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid assay (MTT, Roche), respectively. The assays were performed according to manufacturers' instructions and absorbances were measured at 450 nm (reference wavelength, 690 nm) and 570 nm, respectively.
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Electrophysiological recordings
KCa-mediated currents were recorded in the whole-cell configuration using an EPC9 patch-clamp amplifier (HEKA Elektronik, Lambrecht, Germany). Pulse protocol generation and data acquisition were controlled with Pulse or PatchMaster software (HEKA Elektronik). Series-resistance errors were compensated in the range of 70–80%. Data were filtered at 5 kHz. Patch pipettes were fabricated from Kimax-51 glass (Kimble Glass, Vineland, NJ, USA) with resistance values in the range of 1–3 M. Unless otherwise noted, holding potential was –80 mV. Off-line analysis of data was performed using FitMaster (HEKA Elektronik) and IgorPro (WaveMetrics, Lake Oswego, OR, USA). All experiments were performed at room temperature (20–22°C). For whole-cell recordings, the internal solution contained (mM): KCl 130, MgCl2 2, EGTA 10, Hepes 10 and CaCl2 9.3, and was added to yield 800 nM free Ca2+; pH was adjusted to 7.4 with KOH. The standard bath solution contained (mM): KCl 5, NaCl 135, CaCl2 2 and Hepes 10; pH was adjusted to 7.4 with NaOH. Chemicals for electrophysiological solutions were from Sigma.
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Statistical analysis
Data were analysed using Student's t test for unpaired observations. Values are presented as means ±S.E.M (n= number of cells); in some cases we also specify the number of cell batches (N) used to compile the data. P < 0.05 was regarded as statistically significant. In the Figures, P-values are indicated by asterisks: P < 0.05, P < 0.01, P < 0.001.
Results
Ca2+-activated K+ channels in IGR1 human melanoma cells
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To investigate the expression level of KCa channels in IGR1 human melanoma cells, we measured the currents in the whole-cell configuration as response to linear voltage ramps for 1.5 s from –100 to +50 mV. Figure 1A shows representative examples of current responses with and without 800 nM free Ca2+ in the patch pipette. In the absence of intracellular Ca2+, there are only small currents detectable. A few cells show larger outward current, but only above about 0 mV. Such currents are activated by voltage and are mainly carried by EAG channels (Meyer et al. 1999). In the presence of elevated Ca2+, however, large currents that reverse at the K+ equilibrium potential of about –80 mV were measured in all IGR1 cells tested. The mean current amplitude at +50 mV in this batch of cells is compared in Fig. 1C (left panels). We used cell capacitances as a measure of the cell size and normalized current amplitudes to derive current densities (Fig. 1B, centre panels). Other batches showed considerable variations in current magnitude. Therefore, in the rest of the experiments we performed individual control experiments on untreated IGR1 cells for all experiments shown. In addition, in all cases we report the fractional change (r) in current density and cell capacitance.
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A, each six examples of current responses to voltage-ramp stimulation (from –100 to +50 mV) in the absence of Ca2+ (left) and with 800 nM free Ca2+ in the pipette solution (right). Note that the reversal potential is close to the K+ equilibrium potential of about –80 mV, indicating almost no contribution of non-specific leak currents. B, box plot for the distribution of resting membrane potential of the cells described in A. C, influence of intracellular Ca2+ on the current amplitude at +50 mV, the current density and the cell capacitance. The panels on the right show the ratios of the respective parameters with indication of the statistical significance (P < 0.001); the dashed line marks a ratio of one, i.e. no change. The numbers in parentheses in B and C indicate the number of cells (n) measured.
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As it has been shown that K+ efflux could provide the membrane hyperpolarization necessary for cell cycle progression, resting membrane potential was measured in the current-clamp mode. Activation of KCa channels resulted in a hyperpolarization of the membrane. With Ca2+ in the patch pipette, the mean resting membrane potential was –77.0 ± 1.1 mV (n= 44) while it was only –22.3 ± 2.7 mV (n= 40) in the absence of intracellular free Ca2+ (Fig. 1B).
Hypoxia increases KCa currents in IGR1 cells
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To determine whether mild chronic hypoxia influences KCa channels, we performed electrophysiological experiments on IGR1 cells exposed to low ambient oxygen tension (3% O2) or under normoxic conditions. The mean KCa currents were approximately 2- to 3-fold larger in cells exposed to hypoxic conditions compared with those under normoxic conditions. For hypoxic conditions, the mean currents measured at +50 mV were 2560 ± 324 (n= 43), 3130 ± 445 (n= 26) and 2210 ± 344 pA (n= 21) for 24, 48 and 72 h, respectively (Fig. 2A). For normoxic conditions, the mean currents were 985 ± 128 (n= 45), 1210 ± 204 (n= 21) and 1150 ± 145 pA (n= 20) for 24, 48 and 72 h, respectively (Fig. 2A). The cell size, measured as electrical membrane capacitance (Cm), significantly increased under hypoxia by about 20–30%. The current density (IKCa/Cm) also increased by about a factor of two (Fig. 2A). Thus, exposure of cells to hypoxia for 24 h has the strongest effect on KCa currents, while a longer period of hypoxia results in a return towards control levels. In contrast, cell capacitance keeps increasing under long-term hypoxia. Membrane resting potential did not change (control, –76.1 ± 2.2 mV, n= 26; after 24 h hypoxia, –76.0 ± 1.2 mV, n= 27) because under control conditions and in Ca2+-containing solutions the resting potential was already close to the theoretical K+ equilibrium potential.
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A, IGR1 cells were grown for 24, 48 and 72 h in normoxia (20% oxygen, ) or hypoxia (3% O2, ). Summarized data showing mean amplitude of the macroscopic KCa currents at +50 mV. Also shown are the ratios of the current, the current density, and the cell capacitance. The numbers in parentheses indicate the number of cells (n) measured. B, similar experiments as in A, but IGR1 cells were treated for 24 h with 100 μM CoCl2 or 100 μM desferrioxamine (DFO) to mimic the effect of hypoxia (controls, ; treated cells, ). C, Western blot analysis of HIF-1 in IGR1 cells exposed for 4 and 24 h to hypoxia or treated with 100 μM CoCl2. After indicated times, cell lysates were prepared, and lysates containing equal amounts of protein were used for Western blot analysis. Blots for -actin are shown as a gel-loading control.
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Hypoxia-mimetic stimuli increase KCa currents
Iron chelators such as DFO and iron competitors such as CoCl2 exert hypoxia-mimetic effects due to their inhibition of prolyl hydroxylases that regulate the proteasomal degradation of HIF-1 under normoxic conditions (Semenza, 2004). As shown in Fig. 2B, treatment of IGR1 melanoma cells for 24 h with 100 μM CoCl2 resulted in an increase of the KCa currents to a degree similar to that produced by hypoxic stimulation. The same holds true for the effects on cell capacitance and the current density. Upon 24-h treatment with 100 μM DFO, the current amplitude increased almost 6-fold. The cell capacitance increased by about 60% and the current density by a factor of 3.5. Acute application of CoCl2 or DFO did not affect KCa currents (100 μM CoCl2, 104.0 ± 2.4%; 100 μM DFO, 102.7 ± 2.6%; n= 5 for both).
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Thus, mild hypoxia and treatment of IGR1 cells with CoCl2 and DFO result in an increase in KCa currents indicating that the HIF-1 system might be involved in the regulation. We therefore performed Western blot analysis of IGR1 cells subjected to hypoxia and treatment with CoCl2 using an antibody specific for HIF-1. As shown in Fig. 2C, both interventions resulted in an increase in the HIF-1 protein content suggesting that the functional expression of the KCa channels might be regulated by HIF-1.
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HIF-1 up-regulates KCa currents
As simultaneous up-regulation of HIF-1 and KCa channels could be a mere coincidence, we overexpressed HIF-1 in IGR1 cells and analysed them for KCa currents. HIF-1-transfected cells were identified by coexpression of EGFP; KCa currents were compared with cells cotransfected with an empty vector and EGFP. HIF-1 overexpression increased the KCa currents by about a factor of 2.8 (Fig. 3A). Also cell capacitance and current density were significantly increased. As strong overexpression of signalling proteins can have many deleterious effects on the cells under investigation, it would be important to know whether a reduction of the endogenous HIF-1 level has an effect opposite to an overexpression of HIF-1. Therefore, we overexpressed in IGR1 cells a dominant-negative HIF-1 mutant (HIF-1-N), which lacks the DNA-binding domain (Depping et al. 2004). As shown in Fig. 3A, HIF-1-N significantly reduced KCa currents and current density. In addition, it had no significant effect on the cell capacitance. These experiments clearly show that endogenous HIF-1 takes part in the regulation of KCa channels.
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A, IGR1 melanoma cells were transiently transfected with plasmids coding for HIF-1 or HIF-1-N. Either EGFP or CD8 were cotransfected as a marker. The histogram bars show the effects of HIF transfection on the KCa current amplitude (top). The lower panels show the ratios (overexpressed versus control) of the current amplitude, the current density and the cell capacitance. B, IGR1 melanoma cells were transfected with plasmids coding for VHL-IRES (transient expression), VHL- (transient expression), and VHL19 (stable expression). EGFP was used (either as IRES construct or separately) as a marker. C, IGR1-VHL19 stable cells were transiently transfected with plasmids coding for HIF-1 or HIF-1-P564A. The panels in B and C are the same as in A.
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pVHL down-regulates KCa currents
As pVHL is known to antagonize HIF-1, we examined the effect of pVHL transfection on KCa channels. Transient as well as stable transfection of pVHL strongly reduced KCa currents and current density while there was no significant effect on cell capacitance (Fig. 3B). pVHL lacking this domain may not be able to influence KCa currents because pVHL binds directly to HIF-1 through its -domain. To test this possibility, we transiently introduced a VHL -domain deletion mutant (VHL-) into IGR1 cells and found an increase of KCa currents (Fig. 3B). These results further indicate that the functional expression of KCa channels is regulated by pVHL through interaction with HIF-1.
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It has been previously shown that pVHL only binds to HIF-1 after the latter is enzymatically hydroxylated on conserved prolyl residues (Pro-402 and Pro-564) within a region of HIF-1 called the oxygen-dependent degradation domain. If this mechanism is involved in the regulation of KCa channels in IGR1, an HIF-1 mutant that cannot be hydroxylated might show even stronger effects on KCa channels than the wild-type (wt). HIF-1 and the mutant HIF-1-P564A were thus transiently introduced into the stable, pVHL-overexpressing IGR1 cell line. As shown in Fig. 3C, both wt-HIF-1 and the mutant increased current and current density of KCa channels, but the mutant was far more effective. In contrast, in wt-IGR1 cells, no increased activity of the mutant HIF-1-P564A over the wild-type could be observed (not shown) suggesting that overexpression of HIF-1 is saturating the system in a way that endogenous pVHL is insufficient for ubiquitinating the heterologously expressed HIF protein.
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KCa channel blockers reduce proliferation of IGR1 cells
Regulation of KCa channels in IGR1 cells by hypoxia might not be relevant for cell function and, in particular, for the growth of melanoma cells in general. We thus assayed proliferation of IGR1 cells in the presence of KCa channel blockers, using standard assays for DNA synthesis (BrdU assay) or metabolic activity (MTT assay). Figure 4A summarizes the results of seven individual experiments each normalized to the proliferation rate of normoxic cells in the absence of any blocker. The SK channel blocker apamin slightly reduced the cell proliferation, but more pronounced effects were induced by the IK channel blocker ChTX or by the combined application of both blockers. It is interesting that in the absence of blockers, hypoxia increased the proliferation rates during the 48-h experiments (Fig. 4A, ), compared to the normoxic controls. Under these conditions, the antiproliferative effects of channel blockers were even more pronounced, with ChTX being more potent than apamin. While the mean values show a clear effect of KCa channels on cell proliferation, it is worth noting that in two of the seven individual experiments even combined application of apamin and ChTX did not significantly inhibit the proliferation. This implies that, depending on the status of the cell culture, additional factors might become growth limiting, regardless of the KCa channel activity.
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A, proliferation of IGR1 cells under normoxic () or hypoxic conditions () during a growth period of 48 h. Proliferation rates were determined as BrdU incorporation (number of cell batches, N= 3) or MTT conversion (N= 4) and normalized to the respective normoxic controls. Mean values for all experiments are shown for the following conditions: control (–), presence of 100 nM apamin (A), 100 nM ChTX (C) or 100 nM of each blocker (AC). Hypoxia (3% O2) increased the proliferation rate by about 10%. B, up-regulation of apamin-sensitive channels by hypoxia. Mean current amplitudes of KCa currents in HEK293 cells expressing rSK2 channels and in IGR1 cells with and without 24-h incubation in 100 μM CoCl2. The open histogram bars represent the control currents and the shaded bars the currents after application of 100 nM apamin. This procedure blocks almost all of the heterologously expressed rSK2 channels. IGR1 cells under control conditions do not contain apamin-sensitive KCa channels. Treatment with CoCl2 increases KCa current magnitude and part of this up-regulated conductance is blocked by apamin. The individual ratios of the apamin effects are: rSK2, 0.12 ± 0.03; IGR1, 1.01 ± 0.02; IGR1 + CoCl2, 0.71 ± 0.04. C, RT-PCR analysis for IK and SK channel subtypes in IGR1 cells. mRNA levels detected with RT-PCR under control conditions for SK1, SK2, SK3 and IK transcripts. D, relative changes in the concentration of mRNA coding for SK2 and IK as a function of incubation time in 3% O2 (n= 4–6). Straight lines connect the data points.
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Identification of channel types underlying the regulated KCa currents
Only one or more IK/SK channel types may mediate the hypoxia-regulated KCa currents. As previously shown (Meyer et al. 1999), resting IGR1 cells harbour transcripts for IK and at least two SK channel -subunits (SK1 and SK2). Given the strength of the RT-PCR signals and the pharmacological profile of the KCa currents, Meyer et al. (1999) concluded that IGR1 cells mainly express functional IK channels, characterized by insensitivity to apamin. However, the proliferation experiments shown above indicate that apamin-sensitive channels may play a role in IGR1 cells. Thus, we used apamin to test whether there is a difference in the pharmacological profile of the KCa current before and after exposure to hypoxic stimuli. To evaluate the quality of apamin and the method of toxin application, we expressed rSK2 channels in HEK293 cells and applied 100 nM apamin. As shown in Fig. 4B, apamin blocked rSK2 currents almost completely. The KCa currents in IGR1 cells, cultivated under normoxic conditions, were not significantly affected by 100 nM apamin. Exposure to 100 μM CoCl2 for 24 h resulted in an increase in current magnitude. In addition, a part of this up-regulated current became sensitive to apamin (Fig. 4B). This result indicates that at least part of the up-regulated current must be of the SK1–3 type.
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High levels of SK2 and IK mRNA were found, using quantitative RT-PCR analysis of mRNA extracted from IGR1 cells under normoxic conditions, whereas the concentration of mRNA coding for SK1 and SK3 were at the border of detection level (Fig. 4C). High expression of IK channels is in accordance with the electrophysiological finding of channels sensitive to ChTX, but insensitive to apamin (Meyer et al. 1999). However, the message of SK2 channels does not seem to fully result in functional SK2 channels in normoxic IGR1 cells.
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If KCa currents are regulated via a transcriptional mechanism, one might be able to detect alterations in the mRNA levels of the relevant genes upon hypoxic stimulation of the IGR1 cells. RT-PCR analyses of IGR1 cells grown under hypoxic conditions were performed for SK2 and IK and normalized to -actin levels. As shown in Fig. 4D, both types of mRNA were increased after hypoxia for 3 h, but returned towards control levels after 24 h.
KCa channels in IGR39 cells and in human melanoma metastases
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To test whether the described regulation of KCa channels is limited to IGR1 cells, we also tested the melanoma cell line IGR39. We previously showed that this line, originally derived from a primary melanoma, differs from IGR1, as it does not express EAG channels (Meyer et al. 1999), which may also have oncogenic properties (Pardo et al. 1999). As in IGR1 cells, KCa currents in IGR39 cells were strongly increased by treatment of the cells for 24 h with 100 μM CoCl2 (control, 2.22 ± 0.16 nA, n= 93; 100 μM CoCl2, 5.25 ± 0.37 nA, n= 88; P < 0.001) and cell capacitance was also increased (Fig. 5A). Regulation of KCa channels by hypoxia may have a clinical relevance if such channels are also present in native human tumour material. We therefore analysed freshly isolated biopsies of melanoma metastases from three individual patients. mRNA from all three samples was positive for hIK expression in RT-PCR experiments (not shown). From one of the analysed biopsies we derived a primary cell culture, growing under standard culture conditions, that allowed further investigation. Using patch-clamp analysis, functional KCa currents with similar amplitudes as in IGR1 cells could be detected in these tumour cells (Fig. 5A). The currents were already detectable at the earliest stage of the culture (two passages) and persisted during cultivation. In addition, exposure of the primary cells to CoCl2 (100 μM) also increased the mean current amplitudes in these cells, clearly indicating that the phenomena described above might be of general importance for human melanoma (Fig. 5A).
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A, mean KCa currents, ratio of currents, current densities and cell capacitances for IGR39 cells (number of cell batches, N= 5) and primary human melanoma metastasis cells (N= 2). The open bars indicate control measurements and the grey bars indicate measurements after 24-h exposure to 100 μM CoCl2. B, proliferation of IGR39 cells under normoxic () or hypoxic conditions () during a period of 48 h. Proliferation rates were determined as BrdU incorporation (N= 1) or MTT conversion (N= 4) and normalized to the respective normoxic controls. Mean values of all experiments are shown for the following conditions: control (–), presence of 100 nM apamin (A), 100 nM ChTX (C) or 100 nM of each blocker (AC). C, mean proliferation rates of primary human melanoma cells measured by BrdU incorporation (N= 3). All symbols are the same as in B.
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Proliferation of IGR39 and freshly isolated melanoma cells
The two major findings from the proliferation studies on IGR1 are the increased proliferation rate in hypoxia and inhibition of growth by apamin and, more prominently, by ChTX. Analogous proliferation studies were performed with IGR39 cells and with the primary cell culture of melanoma biopsy material. IGR39 cell proliferation during the 48-h period was increased by almost 40% under hypoxia. In the presence of ChTX, the proliferation of hypoxic cells was reduced to the level of untreated normoxic cells. Both, under normoxic and hypoxic conditions, an inhibition by apamin was measurable, but less effective than growth inhibition by ChTX (Fig. 5B). A similar pattern was found for the primary cell culture with 40% increase in proliferation under hypoxia. In these cells the antiproliferative effect of ChTX was even more prominent than in IGR1 and IGR39 cells, reducing the proliferation rate to about 60% of the control value, both under normoxic and hypoxic conditions (Fig. 5C).
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Discussion
For the last decade, research on cellular adaptation to hypoxia meditated by HIFs has rapidly progressed. Particularly, the fact that HIF-1 is controlled by pVHL has advanced the knowledge of VHL-related disease. HIF-dependent pathways regulate the expression of hypoxia-inducible genes, thereby contributing to cell proliferation, angiogenesis, metabolism, malignant progression and metastasis formation (Harris, 2002). The regulation of ion channels via the HIF pathway and their implication in malignant growth under hypoxia is currently emerging as one important mechanism in cancer formation. In this context the HIF-1–VHL regulation system may serve an alternative function.
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Specific ion channels respond to acute hypoxia and provide physiological signals for oxygen sensing (e.g. López-Barneo et al. 2004). Chronic hypoxia has been shown to down-regulate the mRNA and protein levels of GABA-A receptors in NT2 cells (Gao et al. 2004) and of Kv channels in pulmonary arterial myocytes (Wang et al. 1997; Platoshyn et al. 2001). In contrast to those reports, chronic hypoxia has been reported to increase the Kv channel gene expression in PC12 cells (Conforti & Millhorn, 1997). Recently, it was demonstrated that the density of functional T-type Ca2+ channels is increased by lowering oxygen tension in PC12 cells; furthermore, the gene expression of 1H T-type Ca2+ channels was induced by HIF-2 (del Toro et al. 2003). KCa channels are also subject to regulation by hypoxia: in arterial myocytes chronic hypoxia down-regulates the 1 subunit of big-conductance KCa channels (Navarro-Antolin et al. 2005).
, 百拇医药
In the present study, we investigated human IGR1 cells, which are a model for highly malignant melanoma. We demonstrated that KCa currents in IGR1 cells are increased upon exposure to 3% O2 or hypoxia mimetics. More importantly, we demonstrated that hypoxia up-regulates KCa channels through activation of the HIF-dependent pathway. In addition, KCa channels are down-regulated by pVHL. We have previously reported that pVHL transforms neuroblastoma cells into neurone-like cells and this process is accompanied by alterations in the expression of two types of K+ channels: up-regulation of Kv and down-regulation of hEAG1 channels (Murata et al. 2000). VHL gene transduction into rodent neural progenitor cells induces neuronal differentiation (Kanno et al. 2000; Yamada et al. 2003). Given this differentiating potential of pVHL, one might assume that differentiation effects, independent of HIF-1, could be responsible for the influence of pVHL on KCa channels. However, our finding that expression of the HIF-1 P564A mutant was sufficient to restore KCa currents in a stable VHL-transfected cell line strongly suggests that down-regulation of KCa by pVHL relies on its capacity to degrade HIF-1.
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HIF-1 most probably regulates KCa channels in IGR1 via transcriptional control. Quantitative analysis of mRNA coding for SK2 and IK KCa channel genes in IGR1 cells under hypoxia and normoxia showed that both genes are transiently up-regulated. It is interesting that up-regulation of SK2 was stronger than that of IK, which is compatible with the pharmacological profile of the up-regulated KCa. The transient nature of the regulation is also reflected by a decreasing current magnitude after long exposure of IGR1 cells to hypoxia (Fig. 2A). Our finding of a modest increase of IK mRNA under hypoxia agrees well with a recent report by Ganfornina et al. (2005); they observed a 2-fold increase of IK (Kcnn4) mRNA in cells of the carotid body in animals kept under hypoxic conditions.
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What is the pathophysiological significance of an up-regulation of KCa channels induced by activation of the HIF-dependent pathway under hypoxia We have clearly shown that KCa channels in the tested cellular melanoma models are involved in the control of cell proliferation, as pharmacological channel blockade resulted in reduced proliferation rates. Hence, reduced cell proliferation by blockade of K+ channels appears to be a general feature in cancer cells of different origins (Wang, 2004; Pardo, 2004; Schnherr, 2005). However, the underlying mechanisms linking K+ channels and cell cycle are still under discussion. Proposed mechanisms include changes in cell volume which might affect cell-cycle check points, and increased intracellular Ca2+ concentrations, caused by membrane hyperpolarization and a stronger driving force for Ca2+ entry (Wang, 2004; Pardo, 2004). We did not address the above question directly, but in this context it is interesting to note that hypoxia and HIF-1 transfection not only affected channel activities, but also significantly increased cell size.
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A common finding for the cell systems examined here was an increased proliferation under conditions of reduced oxygen pressure. This may be surprising, as mitochondrial energy production through oxidative phosphorylation depends on the availability of O2. However, 3% O2, as used in our experiments, can be considered a mild form of hypoxia, sufficient to induce HIF-1 stabilization, but not imposing severe metabolic stress. Oxygen levels in solid tumours can drop well below 1%, requiring an enhanced glycolytic activity to support energy production (Vaupel, 2004; Hpfl et al. 2004). In all cell types tested here, the relative growth inhibition imposed by KCa channel blockers was more pronounced under hypoxia than under normoxic conditions. This implies that the up-regulation of KCa channels by hypoxia may be one of the factors causing enhanced proliferation at 3% O2. At present, we cannot exclude the possibility that the transient nature of KCa regulation by mild hypoxia is an adaptive response of the cells that might not be present at lower oxygen pressures. In general, the cellular responses to hypoxia strongly depend on the type of cell tested. Schmaltz et al. (1998) observed hypoxia-induced growth arrest in the G0/G1 phase for murine fibroblasts. In contrast, transformed fibroblasts were able to override this arrest and showed enhanced viability and growth under hypoxic conditions (Schmaltz et al. 1998).
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The identification of HIF-1/1 as a central switch for responses to hypoxia in cancer cells makes it a very attractive candidate for pharmacological intervention and strong efforts are underway to identify appropriate compounds that interfere with HIFs (Giacca et al. 2003). However, the central role of HIF-1/1 and the huge variety of HIF-responsive cellular pathways impose the risk of severe side effects for any strategy that directly targets HIF-1/1. Hence, the identification of specific downstream effectors of HIF-1/1 activity in tumour cells could provide an alternative and promising therapeutic strategy. With regard to KCa channels in melanoma cells, future work is needed to elucidate how general the expression and hypoxia regulation of these channels is in melanoma and whether anti-KCa interventions are beneficial in vivo. It is interesting that ChTX-sensitive K+ channels in melanoma cells are not only relevant for cell proliferation, but also for melanoma cell migration, one of the phenotypes required for the formation of tumour metastases (Schwab et al. 1999). Thus, HIF–VHL-dependent regulation of KCa channels may be an important criterion for melanoma progression. Expression of hypoxia-regulated KCa channels in acutely dissociated cells from a human melanoma metastasis corroborates this conclusion.
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2 Clinics for Dermatology, Friedrich Schiller University Jena, Erfurter Str. 35, D-07740 Jena, Germany
3 Department of Neurosurgery, Yokohama City, University Graduate School of Medicine, Yokohama 236-0004, Japan
Abstract
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Under chronic hypoxia, tumour cells undergo adaptive changes involving hypoxia-inducible factors (HIFs). Here we report that ion currents mediated by Ca2+-activated K+ (KCa) channels in human melanoma IGR1 cells are increased by chronic hypoxia (3% O2), as well as by hypoxia mimetics. This increase involves the HIF system as confirmed by overexpression of HIF-1 or the von Hippel-Lindau tumour suppressor gene. Under normoxic conditions the KCa channels in IGR1 cells showed pharmacological characteristics of intermediate conductance KCa subtype IK channels, whereas the subtype SK2 channels were up-regulated under hypoxia, shown with pharmacological tools and with mRNA analysis. Hypoxia increased cell proliferation, but the KCa channel blockers apamin and charybdotoxin slowed down cell growth, particularly under hypoxic conditions. Similar results were obtained for the cell line IGR39 and for acutely isolated cells from a biopsy of a melanoma metastasis. Thus, up-regulation of KCa channels may be a novel mechanism by which HIFs can contribute to the malignant phenotype of human tumour cells.
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Introduction
Many human tumours develop hypoxic microenvironments as a result of insufficient and uncoordinated growth of blood vessels (Brown & Giaccia, 1998). In this milieu, tumours proliferate and gradually acquire aggressive phenotypic traits. Consequently, oxygen-deprived regions in tumours were found to indicate a poorer prognosis and reduced survival rates (Hckel et al. 1996; Brizel et al. 1996). The various mechanisms by which hypoxia can increase tumour malignancy include: (1) an increased expression of angiogenic cytokines (Shannon et al. 2003); (2) selection for tumour cells with diminished sensitivity to radiotherapy or chemotherapy (Graeber et al. 1996; Shannon et al. 2003); and (3) the promotion of migration and metastatic spread (Brizel et al. 1996; Rofstad & Halsr, 2002). In the last few years, hypoxia-inducible factors (HIFs) have been recognized as key regulators of these processes, controlling the expression of responsive genes including those encoding erythropoietin, vascular endothelial growth factor, tyrosin hydroxylase and various glycolytic enzymes (Jelkmann, 1992; Czyzyk-Krzeska et al. 1992; Goldberg & Schneider, 1994; Semenza et al. 1994; Semenza, 2004).
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HIF is a heterodimeric complex of an oxygen-sensitive HIF- protein and a stable HIF- subunit. Under normoxic conditions, HIF-1 is a target of oxygen-dependent proline hydroxylases, which catalyse hydroxylation at two conserved residues (Pro-402 and Pro-564) (Jaakkola et al. 2001; Ivan et al. 2001) and thereby allow binding of the von Hippel-Lindau protein (pVHL) to HIF-1 (Maxwell et al. 1999; Ohh et al. 2000). The tumour suppressor protein pVHL forms part of a ubiquitin ligase complex, which adds ubiquitin to HIF-1 and targets it for proteasomal destruction (Ohh et al. 2000; Tanimoto et al. 2000).
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There is increasing evidence that membrane ion channels play an important role in cell proliferation and cell cycle control (Wonderlin & Strobl, 1996; Wang, 2004; Pardo, 2004). The relationship between K+ channels and cell cycle has been studied for more than a decade, because the most obvious function of K+ channels in non-excitable cells, including tumour cells, is the control of the resting membrane potential (Wonderlin & Strobl, 1996; Lepple-Wienhues et al. 1996; Gavrilova-Ruch et al. 2002; Ouadid-Ahidouch et al. 2004). Several studies have shown that membrane hyperpolarization is required for the passage of cells through the G1/S phase transition of the cell cycle and this hyperpolarization is mediated by the opening of K+ channels (Kiefer et al. 1980; Chittajallu et al. 2002). Moreover, it has been shown that blockade of K+ channel activity leads to membrane depolarization and an arrest in the early G1 phase (Lepple-Wienhues et al. 1996; Ouadid-Ahidouch et al. 2001). We have previously reported that the human (h) ether à go-go (hEAG) K+ channels in human melanoma cells contribute to cellular proliferation and may provide initial hyperpolarization for the progression into the G1 phase of the cell cycle (Gavrilova-Ruch et al. 2002). In addition, it has been shown that not only hEAG but also Ca2+-activated K+ channels (KCa channels) are responsible for the progression of the cell cycle in human breast cancer cells (Ouadid-Ahidouch et al. 2001).
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In the human melanoma cell line IGR1, hEAG1 and KCa channels constitute the major groups of K+ channels (Meyer et al. 1999; Gavrilova-Ruch et al. 2002). KCa channels are further classified into two types, intermediate-conductance human (hIK or hSK4) and small-conductance (hSK1–3) K+ channels. In IGR1, the pharmacological profile indicated a predominance of hIK channels, being blocked by 100 nM charybdotoxin (ChTX) but not by 100 nM apamin (Meyer et al. 1999). Unlike hSK channels, hIK channels are widely expressed in peripheral tissues, with highest expression in smooth muscle cells (Joiner et al. 1997; Ishii et al. 1997); however, hIK has also been observed in melanoma cells (Meyer et al. 1999). hIK channels have been implicated in the regulation of secretion, in cellular migration and in the proliferation of mitogenically active cells (Mauro et al. 1993; Schwab et al. 1999). However, the role and regulation of the KCa channels in melanoma cells are poorly understood under normoxic as well as under hypoxic conditions.
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Here we report that KCa channels are up-regulated by hypoxia and that the HIF sensory system is responsible for this process. Hypoxia-induced up-regulation was accompanied by increased sensitivity of the currents to apamin, indicating an increased activity of SK-type KCa channels. The levels of mRNA coding for IK and SK2 channels were transiently increased upon hypoxia. Furthermore, we found that cell proliferation was enhanced by chronic hypoxia. Application of the KCa channel blockers ChTX and, with lower potency, apamin prevented this effect on hypoxic cell growth. Thus, up-regulation of apamin-sensitive KCa channels by an HIF-dependent mechanism is linked to enhanced proliferation of tumour cells under hypoxia. These findings contribute to the understanding of the pathophysiology of malignant melanoma cells and shed light on a new mechanism by which HIF-1 proteins can lead to malignant tumour progression. This is particularly relevant as we also found KCa channels with identical regulation by hypoxia in an acutely dissociated metastasis of a melanoma patient.
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Methods
Recombinant DNA
The following constructs were used in this study: von Hippel-Lindau (VHL) cDNA (Gnarra et al. 1994) encoding residues 54–213 (pVHL19) subcloned into pIRES2-EGFP (pVHL-EGFP; Clontech, Heidelberg, Germany), pVHL19 in pcDNA3.1 (Invitrogen, Karlsruhe, Germany), pVHL- (pVHL1987-130), HIF-1 (in pBOS), HIF-1-P564A (in pBOS), HIF-1-N (HIF-1-1–59 in pBOS), HIF-1-N-P564A, rat (r) SK2 (in pcDNA3), pCD8, pEGFP-N1 (Clontech). PCR-based site-directed mutagenesis was performed using the QuikChange kit (Stratagene, La Jolla, CA, USA) to produce the point mutant P564A of HIF-1.
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Cell culture and transfections
The human melanoma cell line IGR1 was maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing 10% fetal calf serum (FCS) at 37°C in a humidified 10% CO2 atmosphere (normoxic conditions). IGR39 cells were cultivated in Minimum Essential Medium (Invitrogen) supplemented with 5% FCS at 37°C in a 5% CO2. HEK 293 cells were maintained in 5% CO2 and DMEM/F-12 (1 : 1) medium with 10% FCS. Cells were transiently transfected with the PolyFect transfection reagent (Qiagen, Hilden, Germany) using a total amount of 2 μg plasmid DNA. To allow identification of transfected cells, either pEGFP-N1 or pCD8 were cotransfected in a 4 : 1 ratio of target plasmid versus the EGFP or CD8 plasmid. For stable transfection, pVHL-EGFP-transfected IGR1 cells were selected with 1 mg ml–1 G418. All transfectants were assayed under normoxic conditions.
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For hypoxic treatments, cells were plated at a density of 4 x 104 cells ml–1. Cells were transferred to an incubator that maintained an atmosphere of 3% O2–10% CO2 balanced with N2 1 day after plating. Alternatively, inhibitors of prolyl hydroxylases (desferrioxamine (DFO) or cobalt chloride (CoCl2)) were added to the culture medium at a final concentration of 100 μM. Cells were kept under these conditions for between 3 and 72 h.
Isolation of primary cells from melanoma metastases
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Biopsies from melanoma metastases (5 mm3) were obtained from patients of the Clinics for Dermatology, University of Jena with informed consent of the patients and approval by the local ethics committee. The biopsy was immediately placed in DMEM (high glucose, Invitrogen). Within 1 h after surgery, the tissue was dissected into small pieces in cold Hanks' balanced salt solution (HBSS; Invitrogen), trypsinized and passed several times through Pasteur pipettes of decreasing diameters. The dissociated cells were then cultivated in DMEM (high glucose, 10% FCS) in 24-well dishes at 37°C (10% CO2). The melanoma markers melan-A and HMB45 were used for immunohistochemical verification of isolated cells.
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Quantitative RT-PCR
Total RNA from melanoma cells was isolated from 5 x 106 to 1 x 107 cells using the RNeasy Mini kit (Qiagen) following the manufacturers instructions. RNA (1.5 μg) was used for cDNA synthesis using oligo(dT) primer and SuperScriptII reverse transcriptase (Invitrogen, SuperScript First Strand Synthesis System for RT-PCR). A Light Cycler rapid thermal cycler system and the Light Cycler Fast Start DNA MasterPlus SYBR Green I-kit were used (Roche, Mannheim, Germany) for hot start amplification and quantification of cDNA. Each run was followed by a melting curve to 95°C to exclude primer dimer formation as a source of fluorescence. Amplification of -actin mRNA was used to normalize determinations from individual RNA samples. The sequences of sense and antisense primers for IK (KCNN4, NM_002250), SK1 (KCNN1, NM_002248) and SK3 (KCNN3, NM_002249) are described by Meyer et al. (1999). For SK2 (KCNN2, NM_021614) they were: 5'-GGT ACC ATG ATC AAC AGG ATG TTA CTA GC-3' and 5'-TCA TTC AGT TTC CTC TGC TCC A-3'; for actin (ACTB, NM_001101): 5'-CCA AGG CCA ACC GCG AGA AGA TGA C-3' and 5'-AGG GTA CAT GGT GGT GCC GCC AGA C-3'.
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Western blot analysis
Cultured cells from one 75-cm2 flask were scraped into 5 ml ice cold PBS and sedimented at 2000 g. Pellets were resuspended in 100 μl ice-cold lysis buffer containing (mM): NaCl 150 , EDTA, EGTA 1, Tris 50 and 1% Triton-X 100; pH 7.5 with Complete Protease Inhibitor Mix (Roche). The lysates were centrifuged at 10 000 g for 10 min at 4°C and supernatants were used for further analysis. Protein concentrations were determined using the Bradford reagent and 40 μg per sample was resolved by 9% SDS-PAGE, followed by electro-transfer to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). Membranes were probed with mouse monoclonal anti-HIF-1 antibodies at 1 : 500 dilution (BD Transduction Laboratories, Heidelberg, Germany), or with mouse monoclonal anti- actin antibodies at 1 : 2000 dilution (Sigma, Taufkirchen, Germany). Signals were detected using the enhanced chemiluminescence method (ECL plus, Amersham, Freiburg, Germany).
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Proliferation assays
Cells were trypsinized and plated at a density of 2–4 x 104 cells ml–1 in a final volume of 100 μl well–1 in 96-well plates. Drugs were added 24 h after plating. For determination of the cell numbers, cells were cultured in 24-well plates (500 μl well–1) and counted in a cell counter (CASY Cell Counter Model DT, Schrfe System, Reutlingen, Germany). After 48 h, proliferation and metabolism were estimated using a colorimetric bromodeoxyuridine assay (Brd U, Roche) and a colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid assay (MTT, Roche), respectively. The assays were performed according to manufacturers' instructions and absorbances were measured at 450 nm (reference wavelength, 690 nm) and 570 nm, respectively.
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Electrophysiological recordings
KCa-mediated currents were recorded in the whole-cell configuration using an EPC9 patch-clamp amplifier (HEKA Elektronik, Lambrecht, Germany). Pulse protocol generation and data acquisition were controlled with Pulse or PatchMaster software (HEKA Elektronik). Series-resistance errors were compensated in the range of 70–80%. Data were filtered at 5 kHz. Patch pipettes were fabricated from Kimax-51 glass (Kimble Glass, Vineland, NJ, USA) with resistance values in the range of 1–3 M. Unless otherwise noted, holding potential was –80 mV. Off-line analysis of data was performed using FitMaster (HEKA Elektronik) and IgorPro (WaveMetrics, Lake Oswego, OR, USA). All experiments were performed at room temperature (20–22°C). For whole-cell recordings, the internal solution contained (mM): KCl 130, MgCl2 2, EGTA 10, Hepes 10 and CaCl2 9.3, and was added to yield 800 nM free Ca2+; pH was adjusted to 7.4 with KOH. The standard bath solution contained (mM): KCl 5, NaCl 135, CaCl2 2 and Hepes 10; pH was adjusted to 7.4 with NaOH. Chemicals for electrophysiological solutions were from Sigma.
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Statistical analysis
Data were analysed using Student's t test for unpaired observations. Values are presented as means ±S.E.M (n= number of cells); in some cases we also specify the number of cell batches (N) used to compile the data. P < 0.05 was regarded as statistically significant. In the Figures, P-values are indicated by asterisks: P < 0.05, P < 0.01, P < 0.001.
Results
Ca2+-activated K+ channels in IGR1 human melanoma cells
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To investigate the expression level of KCa channels in IGR1 human melanoma cells, we measured the currents in the whole-cell configuration as response to linear voltage ramps for 1.5 s from –100 to +50 mV. Figure 1A shows representative examples of current responses with and without 800 nM free Ca2+ in the patch pipette. In the absence of intracellular Ca2+, there are only small currents detectable. A few cells show larger outward current, but only above about 0 mV. Such currents are activated by voltage and are mainly carried by EAG channels (Meyer et al. 1999). In the presence of elevated Ca2+, however, large currents that reverse at the K+ equilibrium potential of about –80 mV were measured in all IGR1 cells tested. The mean current amplitude at +50 mV in this batch of cells is compared in Fig. 1C (left panels). We used cell capacitances as a measure of the cell size and normalized current amplitudes to derive current densities (Fig. 1B, centre panels). Other batches showed considerable variations in current magnitude. Therefore, in the rest of the experiments we performed individual control experiments on untreated IGR1 cells for all experiments shown. In addition, in all cases we report the fractional change (r) in current density and cell capacitance.
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A, each six examples of current responses to voltage-ramp stimulation (from –100 to +50 mV) in the absence of Ca2+ (left) and with 800 nM free Ca2+ in the pipette solution (right). Note that the reversal potential is close to the K+ equilibrium potential of about –80 mV, indicating almost no contribution of non-specific leak currents. B, box plot for the distribution of resting membrane potential of the cells described in A. C, influence of intracellular Ca2+ on the current amplitude at +50 mV, the current density and the cell capacitance. The panels on the right show the ratios of the respective parameters with indication of the statistical significance (P < 0.001); the dashed line marks a ratio of one, i.e. no change. The numbers in parentheses in B and C indicate the number of cells (n) measured.
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As it has been shown that K+ efflux could provide the membrane hyperpolarization necessary for cell cycle progression, resting membrane potential was measured in the current-clamp mode. Activation of KCa channels resulted in a hyperpolarization of the membrane. With Ca2+ in the patch pipette, the mean resting membrane potential was –77.0 ± 1.1 mV (n= 44) while it was only –22.3 ± 2.7 mV (n= 40) in the absence of intracellular free Ca2+ (Fig. 1B).
Hypoxia increases KCa currents in IGR1 cells
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To determine whether mild chronic hypoxia influences KCa channels, we performed electrophysiological experiments on IGR1 cells exposed to low ambient oxygen tension (3% O2) or under normoxic conditions. The mean KCa currents were approximately 2- to 3-fold larger in cells exposed to hypoxic conditions compared with those under normoxic conditions. For hypoxic conditions, the mean currents measured at +50 mV were 2560 ± 324 (n= 43), 3130 ± 445 (n= 26) and 2210 ± 344 pA (n= 21) for 24, 48 and 72 h, respectively (Fig. 2A). For normoxic conditions, the mean currents were 985 ± 128 (n= 45), 1210 ± 204 (n= 21) and 1150 ± 145 pA (n= 20) for 24, 48 and 72 h, respectively (Fig. 2A). The cell size, measured as electrical membrane capacitance (Cm), significantly increased under hypoxia by about 20–30%. The current density (IKCa/Cm) also increased by about a factor of two (Fig. 2A). Thus, exposure of cells to hypoxia for 24 h has the strongest effect on KCa currents, while a longer period of hypoxia results in a return towards control levels. In contrast, cell capacitance keeps increasing under long-term hypoxia. Membrane resting potential did not change (control, –76.1 ± 2.2 mV, n= 26; after 24 h hypoxia, –76.0 ± 1.2 mV, n= 27) because under control conditions and in Ca2+-containing solutions the resting potential was already close to the theoretical K+ equilibrium potential.
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A, IGR1 cells were grown for 24, 48 and 72 h in normoxia (20% oxygen, ) or hypoxia (3% O2, ). Summarized data showing mean amplitude of the macroscopic KCa currents at +50 mV. Also shown are the ratios of the current, the current density, and the cell capacitance. The numbers in parentheses indicate the number of cells (n) measured. B, similar experiments as in A, but IGR1 cells were treated for 24 h with 100 μM CoCl2 or 100 μM desferrioxamine (DFO) to mimic the effect of hypoxia (controls, ; treated cells, ). C, Western blot analysis of HIF-1 in IGR1 cells exposed for 4 and 24 h to hypoxia or treated with 100 μM CoCl2. After indicated times, cell lysates were prepared, and lysates containing equal amounts of protein were used for Western blot analysis. Blots for -actin are shown as a gel-loading control.
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Hypoxia-mimetic stimuli increase KCa currents
Iron chelators such as DFO and iron competitors such as CoCl2 exert hypoxia-mimetic effects due to their inhibition of prolyl hydroxylases that regulate the proteasomal degradation of HIF-1 under normoxic conditions (Semenza, 2004). As shown in Fig. 2B, treatment of IGR1 melanoma cells for 24 h with 100 μM CoCl2 resulted in an increase of the KCa currents to a degree similar to that produced by hypoxic stimulation. The same holds true for the effects on cell capacitance and the current density. Upon 24-h treatment with 100 μM DFO, the current amplitude increased almost 6-fold. The cell capacitance increased by about 60% and the current density by a factor of 3.5. Acute application of CoCl2 or DFO did not affect KCa currents (100 μM CoCl2, 104.0 ± 2.4%; 100 μM DFO, 102.7 ± 2.6%; n= 5 for both).
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Thus, mild hypoxia and treatment of IGR1 cells with CoCl2 and DFO result in an increase in KCa currents indicating that the HIF-1 system might be involved in the regulation. We therefore performed Western blot analysis of IGR1 cells subjected to hypoxia and treatment with CoCl2 using an antibody specific for HIF-1. As shown in Fig. 2C, both interventions resulted in an increase in the HIF-1 protein content suggesting that the functional expression of the KCa channels might be regulated by HIF-1.
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HIF-1 up-regulates KCa currents
As simultaneous up-regulation of HIF-1 and KCa channels could be a mere coincidence, we overexpressed HIF-1 in IGR1 cells and analysed them for KCa currents. HIF-1-transfected cells were identified by coexpression of EGFP; KCa currents were compared with cells cotransfected with an empty vector and EGFP. HIF-1 overexpression increased the KCa currents by about a factor of 2.8 (Fig. 3A). Also cell capacitance and current density were significantly increased. As strong overexpression of signalling proteins can have many deleterious effects on the cells under investigation, it would be important to know whether a reduction of the endogenous HIF-1 level has an effect opposite to an overexpression of HIF-1. Therefore, we overexpressed in IGR1 cells a dominant-negative HIF-1 mutant (HIF-1-N), which lacks the DNA-binding domain (Depping et al. 2004). As shown in Fig. 3A, HIF-1-N significantly reduced KCa currents and current density. In addition, it had no significant effect on the cell capacitance. These experiments clearly show that endogenous HIF-1 takes part in the regulation of KCa channels.
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A, IGR1 melanoma cells were transiently transfected with plasmids coding for HIF-1 or HIF-1-N. Either EGFP or CD8 were cotransfected as a marker. The histogram bars show the effects of HIF transfection on the KCa current amplitude (top). The lower panels show the ratios (overexpressed versus control) of the current amplitude, the current density and the cell capacitance. B, IGR1 melanoma cells were transfected with plasmids coding for VHL-IRES (transient expression), VHL- (transient expression), and VHL19 (stable expression). EGFP was used (either as IRES construct or separately) as a marker. C, IGR1-VHL19 stable cells were transiently transfected with plasmids coding for HIF-1 or HIF-1-P564A. The panels in B and C are the same as in A.
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pVHL down-regulates KCa currents
As pVHL is known to antagonize HIF-1, we examined the effect of pVHL transfection on KCa channels. Transient as well as stable transfection of pVHL strongly reduced KCa currents and current density while there was no significant effect on cell capacitance (Fig. 3B). pVHL lacking this domain may not be able to influence KCa currents because pVHL binds directly to HIF-1 through its -domain. To test this possibility, we transiently introduced a VHL -domain deletion mutant (VHL-) into IGR1 cells and found an increase of KCa currents (Fig. 3B). These results further indicate that the functional expression of KCa channels is regulated by pVHL through interaction with HIF-1.
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It has been previously shown that pVHL only binds to HIF-1 after the latter is enzymatically hydroxylated on conserved prolyl residues (Pro-402 and Pro-564) within a region of HIF-1 called the oxygen-dependent degradation domain. If this mechanism is involved in the regulation of KCa channels in IGR1, an HIF-1 mutant that cannot be hydroxylated might show even stronger effects on KCa channels than the wild-type (wt). HIF-1 and the mutant HIF-1-P564A were thus transiently introduced into the stable, pVHL-overexpressing IGR1 cell line. As shown in Fig. 3C, both wt-HIF-1 and the mutant increased current and current density of KCa channels, but the mutant was far more effective. In contrast, in wt-IGR1 cells, no increased activity of the mutant HIF-1-P564A over the wild-type could be observed (not shown) suggesting that overexpression of HIF-1 is saturating the system in a way that endogenous pVHL is insufficient for ubiquitinating the heterologously expressed HIF protein.
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KCa channel blockers reduce proliferation of IGR1 cells
Regulation of KCa channels in IGR1 cells by hypoxia might not be relevant for cell function and, in particular, for the growth of melanoma cells in general. We thus assayed proliferation of IGR1 cells in the presence of KCa channel blockers, using standard assays for DNA synthesis (BrdU assay) or metabolic activity (MTT assay). Figure 4A summarizes the results of seven individual experiments each normalized to the proliferation rate of normoxic cells in the absence of any blocker. The SK channel blocker apamin slightly reduced the cell proliferation, but more pronounced effects were induced by the IK channel blocker ChTX or by the combined application of both blockers. It is interesting that in the absence of blockers, hypoxia increased the proliferation rates during the 48-h experiments (Fig. 4A, ), compared to the normoxic controls. Under these conditions, the antiproliferative effects of channel blockers were even more pronounced, with ChTX being more potent than apamin. While the mean values show a clear effect of KCa channels on cell proliferation, it is worth noting that in two of the seven individual experiments even combined application of apamin and ChTX did not significantly inhibit the proliferation. This implies that, depending on the status of the cell culture, additional factors might become growth limiting, regardless of the KCa channel activity.
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A, proliferation of IGR1 cells under normoxic () or hypoxic conditions () during a growth period of 48 h. Proliferation rates were determined as BrdU incorporation (number of cell batches, N= 3) or MTT conversion (N= 4) and normalized to the respective normoxic controls. Mean values for all experiments are shown for the following conditions: control (–), presence of 100 nM apamin (A), 100 nM ChTX (C) or 100 nM of each blocker (AC). Hypoxia (3% O2) increased the proliferation rate by about 10%. B, up-regulation of apamin-sensitive channels by hypoxia. Mean current amplitudes of KCa currents in HEK293 cells expressing rSK2 channels and in IGR1 cells with and without 24-h incubation in 100 μM CoCl2. The open histogram bars represent the control currents and the shaded bars the currents after application of 100 nM apamin. This procedure blocks almost all of the heterologously expressed rSK2 channels. IGR1 cells under control conditions do not contain apamin-sensitive KCa channels. Treatment with CoCl2 increases KCa current magnitude and part of this up-regulated conductance is blocked by apamin. The individual ratios of the apamin effects are: rSK2, 0.12 ± 0.03; IGR1, 1.01 ± 0.02; IGR1 + CoCl2, 0.71 ± 0.04. C, RT-PCR analysis for IK and SK channel subtypes in IGR1 cells. mRNA levels detected with RT-PCR under control conditions for SK1, SK2, SK3 and IK transcripts. D, relative changes in the concentration of mRNA coding for SK2 and IK as a function of incubation time in 3% O2 (n= 4–6). Straight lines connect the data points.
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Identification of channel types underlying the regulated KCa currents
Only one or more IK/SK channel types may mediate the hypoxia-regulated KCa currents. As previously shown (Meyer et al. 1999), resting IGR1 cells harbour transcripts for IK and at least two SK channel -subunits (SK1 and SK2). Given the strength of the RT-PCR signals and the pharmacological profile of the KCa currents, Meyer et al. (1999) concluded that IGR1 cells mainly express functional IK channels, characterized by insensitivity to apamin. However, the proliferation experiments shown above indicate that apamin-sensitive channels may play a role in IGR1 cells. Thus, we used apamin to test whether there is a difference in the pharmacological profile of the KCa current before and after exposure to hypoxic stimuli. To evaluate the quality of apamin and the method of toxin application, we expressed rSK2 channels in HEK293 cells and applied 100 nM apamin. As shown in Fig. 4B, apamin blocked rSK2 currents almost completely. The KCa currents in IGR1 cells, cultivated under normoxic conditions, were not significantly affected by 100 nM apamin. Exposure to 100 μM CoCl2 for 24 h resulted in an increase in current magnitude. In addition, a part of this up-regulated current became sensitive to apamin (Fig. 4B). This result indicates that at least part of the up-regulated current must be of the SK1–3 type.
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High levels of SK2 and IK mRNA were found, using quantitative RT-PCR analysis of mRNA extracted from IGR1 cells under normoxic conditions, whereas the concentration of mRNA coding for SK1 and SK3 were at the border of detection level (Fig. 4C). High expression of IK channels is in accordance with the electrophysiological finding of channels sensitive to ChTX, but insensitive to apamin (Meyer et al. 1999). However, the message of SK2 channels does not seem to fully result in functional SK2 channels in normoxic IGR1 cells.
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If KCa currents are regulated via a transcriptional mechanism, one might be able to detect alterations in the mRNA levels of the relevant genes upon hypoxic stimulation of the IGR1 cells. RT-PCR analyses of IGR1 cells grown under hypoxic conditions were performed for SK2 and IK and normalized to -actin levels. As shown in Fig. 4D, both types of mRNA were increased after hypoxia for 3 h, but returned towards control levels after 24 h.
KCa channels in IGR39 cells and in human melanoma metastases
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To test whether the described regulation of KCa channels is limited to IGR1 cells, we also tested the melanoma cell line IGR39. We previously showed that this line, originally derived from a primary melanoma, differs from IGR1, as it does not express EAG channels (Meyer et al. 1999), which may also have oncogenic properties (Pardo et al. 1999). As in IGR1 cells, KCa currents in IGR39 cells were strongly increased by treatment of the cells for 24 h with 100 μM CoCl2 (control, 2.22 ± 0.16 nA, n= 93; 100 μM CoCl2, 5.25 ± 0.37 nA, n= 88; P < 0.001) and cell capacitance was also increased (Fig. 5A). Regulation of KCa channels by hypoxia may have a clinical relevance if such channels are also present in native human tumour material. We therefore analysed freshly isolated biopsies of melanoma metastases from three individual patients. mRNA from all three samples was positive for hIK expression in RT-PCR experiments (not shown). From one of the analysed biopsies we derived a primary cell culture, growing under standard culture conditions, that allowed further investigation. Using patch-clamp analysis, functional KCa currents with similar amplitudes as in IGR1 cells could be detected in these tumour cells (Fig. 5A). The currents were already detectable at the earliest stage of the culture (two passages) and persisted during cultivation. In addition, exposure of the primary cells to CoCl2 (100 μM) also increased the mean current amplitudes in these cells, clearly indicating that the phenomena described above might be of general importance for human melanoma (Fig. 5A).
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A, mean KCa currents, ratio of currents, current densities and cell capacitances for IGR39 cells (number of cell batches, N= 5) and primary human melanoma metastasis cells (N= 2). The open bars indicate control measurements and the grey bars indicate measurements after 24-h exposure to 100 μM CoCl2. B, proliferation of IGR39 cells under normoxic () or hypoxic conditions () during a period of 48 h. Proliferation rates were determined as BrdU incorporation (N= 1) or MTT conversion (N= 4) and normalized to the respective normoxic controls. Mean values of all experiments are shown for the following conditions: control (–), presence of 100 nM apamin (A), 100 nM ChTX (C) or 100 nM of each blocker (AC). C, mean proliferation rates of primary human melanoma cells measured by BrdU incorporation (N= 3). All symbols are the same as in B.
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Proliferation of IGR39 and freshly isolated melanoma cells
The two major findings from the proliferation studies on IGR1 are the increased proliferation rate in hypoxia and inhibition of growth by apamin and, more prominently, by ChTX. Analogous proliferation studies were performed with IGR39 cells and with the primary cell culture of melanoma biopsy material. IGR39 cell proliferation during the 48-h period was increased by almost 40% under hypoxia. In the presence of ChTX, the proliferation of hypoxic cells was reduced to the level of untreated normoxic cells. Both, under normoxic and hypoxic conditions, an inhibition by apamin was measurable, but less effective than growth inhibition by ChTX (Fig. 5B). A similar pattern was found for the primary cell culture with 40% increase in proliferation under hypoxia. In these cells the antiproliferative effect of ChTX was even more prominent than in IGR1 and IGR39 cells, reducing the proliferation rate to about 60% of the control value, both under normoxic and hypoxic conditions (Fig. 5C).
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Discussion
For the last decade, research on cellular adaptation to hypoxia meditated by HIFs has rapidly progressed. Particularly, the fact that HIF-1 is controlled by pVHL has advanced the knowledge of VHL-related disease. HIF-dependent pathways regulate the expression of hypoxia-inducible genes, thereby contributing to cell proliferation, angiogenesis, metabolism, malignant progression and metastasis formation (Harris, 2002). The regulation of ion channels via the HIF pathway and their implication in malignant growth under hypoxia is currently emerging as one important mechanism in cancer formation. In this context the HIF-1–VHL regulation system may serve an alternative function.
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Specific ion channels respond to acute hypoxia and provide physiological signals for oxygen sensing (e.g. López-Barneo et al. 2004). Chronic hypoxia has been shown to down-regulate the mRNA and protein levels of GABA-A receptors in NT2 cells (Gao et al. 2004) and of Kv channels in pulmonary arterial myocytes (Wang et al. 1997; Platoshyn et al. 2001). In contrast to those reports, chronic hypoxia has been reported to increase the Kv channel gene expression in PC12 cells (Conforti & Millhorn, 1997). Recently, it was demonstrated that the density of functional T-type Ca2+ channels is increased by lowering oxygen tension in PC12 cells; furthermore, the gene expression of 1H T-type Ca2+ channels was induced by HIF-2 (del Toro et al. 2003). KCa channels are also subject to regulation by hypoxia: in arterial myocytes chronic hypoxia down-regulates the 1 subunit of big-conductance KCa channels (Navarro-Antolin et al. 2005).
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In the present study, we investigated human IGR1 cells, which are a model for highly malignant melanoma. We demonstrated that KCa currents in IGR1 cells are increased upon exposure to 3% O2 or hypoxia mimetics. More importantly, we demonstrated that hypoxia up-regulates KCa channels through activation of the HIF-dependent pathway. In addition, KCa channels are down-regulated by pVHL. We have previously reported that pVHL transforms neuroblastoma cells into neurone-like cells and this process is accompanied by alterations in the expression of two types of K+ channels: up-regulation of Kv and down-regulation of hEAG1 channels (Murata et al. 2000). VHL gene transduction into rodent neural progenitor cells induces neuronal differentiation (Kanno et al. 2000; Yamada et al. 2003). Given this differentiating potential of pVHL, one might assume that differentiation effects, independent of HIF-1, could be responsible for the influence of pVHL on KCa channels. However, our finding that expression of the HIF-1 P564A mutant was sufficient to restore KCa currents in a stable VHL-transfected cell line strongly suggests that down-regulation of KCa by pVHL relies on its capacity to degrade HIF-1.
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HIF-1 most probably regulates KCa channels in IGR1 via transcriptional control. Quantitative analysis of mRNA coding for SK2 and IK KCa channel genes in IGR1 cells under hypoxia and normoxia showed that both genes are transiently up-regulated. It is interesting that up-regulation of SK2 was stronger than that of IK, which is compatible with the pharmacological profile of the up-regulated KCa. The transient nature of the regulation is also reflected by a decreasing current magnitude after long exposure of IGR1 cells to hypoxia (Fig. 2A). Our finding of a modest increase of IK mRNA under hypoxia agrees well with a recent report by Ganfornina et al. (2005); they observed a 2-fold increase of IK (Kcnn4) mRNA in cells of the carotid body in animals kept under hypoxic conditions.
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What is the pathophysiological significance of an up-regulation of KCa channels induced by activation of the HIF-dependent pathway under hypoxia We have clearly shown that KCa channels in the tested cellular melanoma models are involved in the control of cell proliferation, as pharmacological channel blockade resulted in reduced proliferation rates. Hence, reduced cell proliferation by blockade of K+ channels appears to be a general feature in cancer cells of different origins (Wang, 2004; Pardo, 2004; Schnherr, 2005). However, the underlying mechanisms linking K+ channels and cell cycle are still under discussion. Proposed mechanisms include changes in cell volume which might affect cell-cycle check points, and increased intracellular Ca2+ concentrations, caused by membrane hyperpolarization and a stronger driving force for Ca2+ entry (Wang, 2004; Pardo, 2004). We did not address the above question directly, but in this context it is interesting to note that hypoxia and HIF-1 transfection not only affected channel activities, but also significantly increased cell size.
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A common finding for the cell systems examined here was an increased proliferation under conditions of reduced oxygen pressure. This may be surprising, as mitochondrial energy production through oxidative phosphorylation depends on the availability of O2. However, 3% O2, as used in our experiments, can be considered a mild form of hypoxia, sufficient to induce HIF-1 stabilization, but not imposing severe metabolic stress. Oxygen levels in solid tumours can drop well below 1%, requiring an enhanced glycolytic activity to support energy production (Vaupel, 2004; Hpfl et al. 2004). In all cell types tested here, the relative growth inhibition imposed by KCa channel blockers was more pronounced under hypoxia than under normoxic conditions. This implies that the up-regulation of KCa channels by hypoxia may be one of the factors causing enhanced proliferation at 3% O2. At present, we cannot exclude the possibility that the transient nature of KCa regulation by mild hypoxia is an adaptive response of the cells that might not be present at lower oxygen pressures. In general, the cellular responses to hypoxia strongly depend on the type of cell tested. Schmaltz et al. (1998) observed hypoxia-induced growth arrest in the G0/G1 phase for murine fibroblasts. In contrast, transformed fibroblasts were able to override this arrest and showed enhanced viability and growth under hypoxic conditions (Schmaltz et al. 1998).
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The identification of HIF-1/1 as a central switch for responses to hypoxia in cancer cells makes it a very attractive candidate for pharmacological intervention and strong efforts are underway to identify appropriate compounds that interfere with HIFs (Giacca et al. 2003). However, the central role of HIF-1/1 and the huge variety of HIF-responsive cellular pathways impose the risk of severe side effects for any strategy that directly targets HIF-1/1. Hence, the identification of specific downstream effectors of HIF-1/1 activity in tumour cells could provide an alternative and promising therapeutic strategy. With regard to KCa channels in melanoma cells, future work is needed to elucidate how general the expression and hypoxia regulation of these channels is in melanoma and whether anti-KCa interventions are beneficial in vivo. It is interesting that ChTX-sensitive K+ channels in melanoma cells are not only relevant for cell proliferation, but also for melanoma cell migration, one of the phenotypes required for the formation of tumour metastases (Schwab et al. 1999). Thus, HIF–VHL-dependent regulation of KCa channels may be an important criterion for melanoma progression. Expression of hypoxia-regulated KCa channels in acutely dissociated cells from a human melanoma metastasis corroborates this conclusion.
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