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KCNMA1 gene amplification promotes tumor cell proliferation in human prostate cancer

Abstract

Molecular mechanisms of prostate cancer progression are poorly understood. Here, we studied gene amplification of the large conductance calcium-activated potassium channel alpha subunit (KCNMA1), which is located at the chromosomal region 10q22. Fluorescence in situ hybridization (FISH) revealed KCNMA1 amplification in 16% of 119 late-stage human prostate cancers and in the hormone-insensitive prostate cancer cell line PC-3. In contrast, KCNMA1 amplification was absent in 33 benign controls, 32 precursor lesions and in 105 clinically organ-confined prostate cancers. Amplification was associated with mRNA and protein overexpression as well as increased density of BK channel protein and β-estradiol-insensitive BK currents in PC-3 cells as compared to non-amplified control cell lines. Specific blockade of BK channels by iberiotoxin or RNAi significantly inhibited K+ currents and growth of PC-3 cells. The data demonstrate that 10q22 amplification drives KCNMA1 expression and cell proliferation. Thus, KCNMA1 qualifies as a promising diagnostic and therapeutic target in patients with prostate cancer.

Introduction

Prostate cancer is the most frequent malignant tumor among males in developed countries and the second leading cause for cancer-related death (Hsing et al., 2000). Androgen withdrawal is an effective therapy for patients with advanced prostate cancer, but progression to hormone independence occurs ultimately in almost all patients after a few months or years (Petrylak et al., 2004). Although numerous non-hormonal agents have been evaluated in patients with hormone-refractory prostate cancer, these agents have limited antitumor activity with modest or no effect on survival (Petrylak et al., 2004). Therefore, identification and selected inhibition of molecular targets that mediate progression of prostate cancer are highly warranted.

The molecular mechanisms underlying progression to metastatic and hormone-refractory prostate cancer are poorly understood. High-level amplifications can pinpoint to genomic sites containing activated oncogenes with major biologic and therapeutic importance (Albertson et al., 2003). We have recently identified an amplification at 10q22 prevailing in approximately 15% of hormone-refractory prostate cancers and in the hormone-insensitive prostate cancer cell line PC-3 (Bernardino et al., 1997; El Gedaily et al., 2001). Amplification of 10q22 appears to be absent or exceedingly rare in other tumor types, suggesting that this chromosomal region harbors genes that specifically drive progression of prostate cancer (Knuutila et al., 2000).

The KCNMA1 gene is located at 10q22 and encodes the pore-forming α-subunit of the large-conductance Ca2+-activated K+ channel (also known as BK-channel, Maxi K+ channel, slowpoke) that can tetramerize to a fully active BK channel (Tseng-Crank et al., 1994; Quirk and Reinhart, 2001). In mammalian tissues, KCNMA1 is almost ubiquitously expressed along with four regulatory β-subunits KCNMB1-4 (Rebhan et al., 1997; Orio et al., 2002).

Numerous functions have been attributed to BK channels, including modulation of smooth muscle tone of blood vessels and uterus (Zhou et al., 2000; Amberg et al., 2003), synaptic neurotransmitter release (Robitaille and Charlton, 1992) and epithelial transport (Kunzelmann and Mall, 2002). Most recently, attention has been drawn to the BK channel as a critical player in blood pressure regulation (Amberg and Santana, 2003) and in the neurotropic effect of ethanol (Davies et al., 2003). Hence, potassium channel-modulating agents have been suggested as therapeutic agents in neurological and cardiovascular disorders (Calderone, 2002).

Apart from this, growing evidence exists that K+ channels may also be involved in oncogenesis. For example, BK channel activation has been shown to drive tumor cell proliferation in astrocytoma (Basrai et al., 2002). Moreover, a previous report has suggested a role of potassium channels for the proliferation of prostate cancer (Van Coppenolle et al., 2004). Here, we demonstrate that KCNMA1 is amplified in a subset of late-stage prostate cancers in vivo, and that BK channel activity alters growth of human prostate cancer in vitro.

Results

KCNMA1 amplification and overexpression of BK channels

Human prostate cell lines were analysed for KCNMA1 amplification by fluorescence in situ hybridization (FISH). Amplification, defined as a signal ratio of the KCNMA1 gene/centromere of chromosome 102 and at least five gene signals, was present in the hormone-insensitive prostate cancer cell line PC-3 but not in the hormone-sensitive prostate cell lines BPH-1 or LNCaP (Figure 1). To explore whether this amplification also prevails in vivo, a series of 298 formalin-fixed and paraffin-embedded human prostate specimens was analysed on a tissue microarray. We found amplification in 16.0% of 119 advanced prostate cancers including untreated locally aggressive tumors (20%), distant metastases (13%) and local recurrences (22%) from hormone-refractory tumors but not in benign prostatic hyperplasia, precursor lesions (high-grade prostatic intraepithelial neoplasia) and clinically localized prostate cancers (Table 1). These results suggest that amplification of KCNMA1 is associated with progression to late-stage, metastatic and hormone-refractory human prostate cancer. The FISH results of KCNMA1 were compared with PLAU, a previously suggested candidate target gene at 10q22 (Helenius et al., 2001). Comparable to KCNMA1, PLAU amplification was present in PC-3 but not in BPH-1 and LNCaP. Analysis of 133 specimens revealed a similar prevalence and distribution of PLAU amplification among the different tumor stages as KCNMA1 amplification (Table 1). PLAU amplification was absent in specimens of benign prostatic hyperplasia and in clinically localized prostate cancers but present in untreated locally aggressive tumors (14%), lymph node metastases (13%), distant metastases (13%) and in hormone-refractory local recurrences (12%). The difference in the prevalence of amplification of these two genes in the different subgroups of tumors stages was statistically not significant.

Figure 1
figure1

FISH with KCNMA1-specific BAC RP11-428P16. (a) Metaphase control: Green KCNMA1 signals and red centromere signals on chromosome 10. (b) BPH-1, transformed benign prostate hyperplasia cell line, showing two signals for BAC probe and centromeric probe, each. (c) PC-3, tumourigenic prostate cancer cell line with KCNMA1 amplification at 10q22: two clusters of green probe signals and two red signals of centromere 10 (arrows). (d) Tumor cells from clinical prostate cancer with normal probe copy number. (e) and (f) Prostate cancers with amplification. Dense clusters of green probe signals but only rare red centromeric signals (arrows). Blue channel: DAPI. Green channel: FITC (BAC RP11-428P16). Red channel: Spectrum Orange (centromere 10). Magnification × 1000.

Table 1 Prevalence of KCNMA1 and PLAU amplification in prostate cancer

To study whether amplification of KCNMA1 results in overexpression, we compared the mRNA levels of KCNMA1 in the amplified PC-3 cell line with the non-amplified cell lines BPH-1 and LNCaP using semiquantitative reverse transcriptase–polymerase chain reaction (RT–PCR). Expression of KCNMA1 was significantly higher in PC-3 as compared to LNCaP and BPH-1, suggesting that KCNMA1 amplification drives overexpression of the channel protein (Figure 2a). In fact, increased levels of KCNMA1 protein in PC-3 as opposed to the control cell lines were documented by immunofluorescence (Figure 2b). The fluorescence intensity (mean of optical density±error of the mean) was significantly higher in PC-3 (27 759±2061; 10 cells) than in LNCaP (10 806±259; 12 cells) and BPH-1 (317±19.2; 13 cells; P<0.0001; stains were repeated twice). Immunofluorescence on human prostatic tissue revealed strong BK expression in smooth muscle cells of the fibromuscular stroma. This is in line with previous reports showing strong expression in smooth muscle of other sites such as blood vessels and colonic wall (McCobb et al., 1995; Sausbier et al., 2006). BK expression also prevailed in all six prostatic adenocarcinomas and the intensity varied from weak to moderate (Figure 3). None of the tumors met the criteria for KCNMA1 gene amplification by FISH. One tumor had increased gene copy number of KCNMA1 (5–7 gene signals and 4–5 reference signals) and showed moderate BK expression (Figure 3a). However, expression at a similar level was also found in tumors with a normal gene copy number (Figure 3f). Benign prostatic glands showed weak expression of BK.

Figure 2
figure2

Expression of KCNMA1. (a) mRNA expression of KCNMA1 was significantly enhanced in the amplified prostate cancer cell line PC-3 as compared to the non-amplified cell lines LNCaP (P=0.0002) and BPH-1 (P=0.0001). Figure is drawn to be relative to the expression in PC-3. (b) Immunofluorescence of the prostate cancer cell lines PC-3, LNCaP and BPH-1. Protein expression of KCNMA1 was strong in PC-3 cells, weak in LNCaP and indiscernible in BPH-1 (P<0.0001). Magnification × 1000. Stains were repeated twice; (n)=number of cells measured.

Figure 3
figure3

Immunofluorescence of anti-BK channel antibody on human prostate cancer tissue. Antibody labeled in green (FITC), nuclei counterstained in blue (DAPI). (a) Well-detectable BK channel staining in the cytoplasm of hormone-refractory prostate cancer (long arrow), and strong staining in surrounding smooth muscle cells (short arrow). This tumor showed increased KCNMA1 gene copy number by FISH. (b) Negative control on the same specimen as (a). Absence of staining indicates specificity of the antibody. (c) Bundle of smooth muscle cells in the same specimen as (a). Note the very intense expression of the BK channel at the cellular membrane (short arrow). (d) Hormone-refractory prostate cancer infiltrating between smooth muscle cells. Note the intense staining of the smooth muscle cells (short arrow) and the very weak staining of the tumor cells (long arrow). This tumor had normal KCNMA1 gene copy number by FISH. (e) Benign prostatic gland. Weak cytoplasmic immunofluorescence (long arrow). The bright spots in the cytoplasm represent autofluorescent lipofuscin granules that are typically present in benign prostatic glands (arrowhead). (f) Hormone-insensitive prostate cancer infiltrating between smooth muscle cells. Well-detectable cytoplasmic and membranous immunofluorescence in tumor cells (long arrows) and strong positivity in adjacent smooth muscle cells (short arrow). This tumor had normal KCNMA1 gene copy number by FISH. Magnifications: (a)–(d) × 1000, (e)–(f) × 630.

BK channel currents in prostate cancer cells

The prostate cell lines PC-3 and BPH-1 were used as in vitro models to study the influence of KCNMA1 currents for cell proliferation. Functional expression of BK channels in PC-3 and BPH-1 cells was examined in patch clamp experiments. Iberiotoxin (IBTX), a well-known and highly specific inhibitor of the BK channel (Galvez et al., 1990), reversibly inhibited whole-cell current (Im) and conductance (Gm) in PC-3 (Figure 4a and b). Inhibition of whole-cell currents was paralleled by a depolarization of the membrane voltage (Vm). Other than in PC-3 cells, IBTX had only minor effects on the whole-cell current of LNCaP and the benign BPH-1 cells and did not depolarize the membrane voltage (Figure 4c). Whole-cell patch clamp experiments were performed in the same media as proliferation assays. Under these ‘proliferative’ conditions, the membrane voltage (Vm) of both BPH-1 (−18±1.8 mV, n=13) and PC-3 (−23±2.0 mV, n=17) cells was depolarized. Although depolarization of Vm allows for activation of BK channels, IBTX inhibited membrane conductance was only found in PC-3 cells but not in BPH-1 cells. In Ringer solution both PC-3 and BPH-1 cells were hyperpolarized to −58±2.1 mV (n=14) and −59±3.8 mV (n=17) and IBTX-sensitive membrane conductance was reduced (data not shown). Other Ca2+-activated K+ channels such as small and intermediate conductance channels showed negligible contribution to the whole-cell conductance of PC-3 and BPH-1 cells (data not shown).

Figure 4
figure4

BK currents in prostate cancer cells. (a) Continuous recording of the whole-cell current (Im, upper trace) and membrane voltage (Vm, lower trace) in a fast whole-cell recording of a PC-3 cell. Symbol on top indicates configuration and concentrations of K+. IBTX (20 nmol/l) reduced the outward current and depolarized Vm. (b) Concentration dependence of the inhibitory effect of IBTX on the whole-cell conductance (Gm) of PC-3 cells. (c) Summary of the whole-cell conductances (Gm) of PC-3, BPH-1 and LNCaP cells and effects of IBTX (20 nmol/l) on Gm as compared to negative control (con). IBTX significantly reduced Gm in PC-3 and LNCaP (P<0.001 and P<0.02, respectively). (d) Summary of the whole-cell conductances (Gm) of PC-3 and BPH-1 cells and effects of 17β-estradiol (E, 10 μmol/l). 17β-estradiol lead to a significantly increased Gm in BPH-1 (P<0.01). (e) Single BK channel currents in inside out membrane patches at different clamp voltages. Channel openings are indicated by brief downward current deflections, while - - -c indicates the closed state of the channel. Symbols indicate configuration and concentrations of K+. *Significance (paired t-test); (n)=number of experiments.

The whole-cell K+ conductance could not be augmented by the BK channel activator 17β-estradiol in PC-3 (Valverde et al., 1999), but was further enhanced along with a hyperpolarization of the membrane voltage in BPH-1 (Figure 4d). The results suggest a high activity of BK channels in PC-3 that cannot be further increased by 17β-estradiol. These findings were supported by inside/out excision of membrane patches from PC-3 cells into a bath solution containing high Ca2+ concentration. Under these conditions, large conductance BK channels were regularly detected in PC-3 (Figure 4e), but only occasionally found in membrane patches of BPH-1. The open probability (Po) of the single channel was enhanced at depolarized membrane voltages, and the single channel conductance was about 220 pS at symmetrical K+ concentrations of 150 mM, which is characteristic for BK channels.

Impact of BK channel activity on proliferation of prostate cancer cells

We examined the response of PC-3, BPH-1 and LNCaP to modulation of BK channel activity (Figure 5). The basal growth rate was higher in PC-3 than in BPH-1 and was significantly inhibited by the BK channel inhibitor IBTX (P=0.015). This indicates a central role of BK channels for proliferation of PC-3 under normal growth conditions (Figure 5a). In contrast, there was no significant effect of IBTX on the growth of BPH-1 and LNCaP (P>0.05, Figure 5b and c). 17β-estradiol significantly stimulated growth of BPH-1 (P=0.0021) and LNCaP (P=0.0039) but not PC-3 cells (Figure 5a–c). The growth effect of 17β-estradiol was abolished by IBTX both in BPH-1 and LNCaP. This finding suggests that the steroid exerts its stimulatory effects on cell proliferation in BPH-1 and LNCaP through activation of BK channels. In contrast, rapidly growing PC-3 cells proliferate independently of 17β-estradiol, as the abundantly expressed BK channels operate already at high activity.

Figure 5
figure5

Growth rates of PC-3, BPH-1 and LNCaP cells in response to IBTX and 17β-estradiol (E). (a) Growth of PC-3 cells was reduced in response to IBTX (P=0.015) and was not significantly influenced by 17β-estradiol. (b) BPH-1 cells showed increased growth in response to 17β-estradiol (P=0.0021), which was prevented by IBTX (P=0.0175). (c) LNCaP cells showed increased growth in response to 17β-estradiol (P=0.0039), which was prevented by IBTX (P=0.026). Con=untreated control, IBTX=iberiotoxin, E=17β-estradiol, E+IBTX=17β-estradiol+iberiotoxin. y-axis: growth rate k as N(t)=N0 * ekt. N(t): number of cells at time t, N0=number of cells at time 0, t in days.

The role of BK channels for proliferation of prostate cancer cells was further substantiated by gene-specific mRNA silencing using RNA interference (RNAi). Four different types of anti-KCNMA1 small interference RNA molecules (siRNA K1–K4) were transfected into PC-3 cells, which all significantly reduced KCNMA1 mRNA expression and cell proliferation. The most potent KCNMA1 siRNA (K2) inhibited mRNA expression (Figure 6a) and cell proliferation (Figure 6b) by 80–90%, when compared to non-transfected cells (c-si) or cells transfected with scrambled sequence siRNA (scr). Expression of KCNMA1 protein was largely reduced in siRNA K2-treated cells, when compared to non-transfected cells or cells transfected with scrambled sequence siRNA (Figure 6c). In whole-cell patch clamp experiments, inhibition of the membrane conductance and depolarization of the membrane voltage by IBTX was significantly reduced in RNAi-treated cells (Figure 7a). The BK-inhibitor paxillin rather than IBTX was used for analysis of excised inside out membrane patches as paxillin is applicable to the cytosolic side of the membrane. Analysis of excised inside out membrane patches of RNAi-treated cells revealed largely attenuated BK channel activity (nPo) and a reduced effect of the BK channel blocker paxillin (Figure 7b). Inhibition of BK channel activity by paxillin was much reduced in RNAi-treated PC-3 cells and was not detectable in BPH-1 cells (Figure 7c). Taken together, the present data indicate a crucial role of KCNMA1 for proliferation of advanced and hormone-refractory prostate cancer cells.

Figure 6
figure6

Inhibition of KCNMA1 expression by RNAi in PC-3. (a) RNAi reduced KCNMA1 mRNA expression to 15–50% compared to non-transfected cells (c-si) or cells transfected with scrambled sequence RNAi (scr) (P=0.005 or P=0.0021, respectively). Figure is drawn to be relative to the expression in the control (c-si). (b) KCNMA1-RNAi using all four active siRNA K1-4 had a pronounced inhibitory effect on PC-3 cell growth. y-axis shows total number of cells per well. (c) Western blot analysis of non-transfected (c-si) PC-3 cells and PC-3 cells which have been transfected with scrambled sequence (scr) and KCNMA1-RNAi (K2).

Figure 7
figure7

Inhibition of KCNMA1 currents by RNAi. (a) Summary of the whole-cell conductances (Gm) and membrane voltages (Vm) of non-transfected (c-si) PC-3 cells and PC-3 cells which have been transfected with anti KCNMA1-RNAi. GIBTX=Gm under inhibition with IBTX, VIBTX=Vm under inhibition with IBTX (P<0.001 and P<0.0007, respectively). (b) Multiple BK channels in inside out membrane patches at 0 mV clamp voltage. - - -c indicates current level when all channels are closed. BK channels are much more abundant in non-transfected (c-si) when compared with RNAi-transfected PC-3 cells. The BK channel inhibitor paxillin had a more pronounced effect in non-transfected (c-si) cells. (c) Product of open probability and number of channels (nPo) in non-transfected (c-si) and RNAi-transfected PC-3 cells as well as BPH-1 cells. The Po was significantly reduced by paxillin (Pax) in non-transfected (c-si) PC-3 cells and in PC-3 cells treated by RNAi (P<0.001 and P<0.02, respectively). The effect of paxillin was significantly greater in non-transfected PC-3 cells (c-si) than in RNAi-transfected PC-3 cells (#; P<0.03). *Significance (paired t-test); (n)=number of experiments. con=untreated controls.

Discussion

There is mounting evidence that K+ channels play a crucial role in oncogenesis, and their potential as therapeutic targets in cancer has been recognized (Pardo et al., 1999; Pei et al., 2003; Conti, 2004; Wang, 2004). Here, we demonstrate that the large-conductance Ca2+-activated potassium channel (KCNMA1) is amplified at 10q22 in 16% of late-stage, metastatic and hormone-refractory human prostate cancers. In the cell line PC-3, amplification of KCNMA1 is associated with overexpression of mRNA and protein, and a high density of BK channels in the cellular membrane as opposed to non-amplified cell lines. Importantly, growth inhibition by specific blockade of KCNMA1 activity using IBTX and RNAi in the amplified prostate cancer cell line PC-3 strongly argues for an oncogenic effect of KCNMA1 amplification and functional overexpression. Expression of BK channel protein is not restricted to cell lines in vitro but also present in human prostate cancers in vivo. As well-detectable BK expression was found in tumors without KCNMA1 amplification, factors other than increased gene dosage must also be involved in the regulation of BK expression in prostate cancer.

The oncogenic role of KCNMA1 found in this study is in line with the previously reported growth inhibition of human astrocytoma by IBTX (Basrai et al., 2002; Weaver et al., 2004). Interestingly, the intermediate conductance K+ channel (IK) rather than KCNMA1 has been proposed to be important for PC-3 and LNCaP cells by Parihar et al. (2003). Parihar et al. performed the efflux experiments under non-proliferative conditions, that is, in Ringer solution rather than in growth medium. In Ringer solution, however, the cells were hyperpolarized and thus BK channels and other voltage-gated K+ channels show little activity (Pardo, 2004; O’Grady and Lee, 2005). The present data suggest that analysis of ion channels relevant to proliferation should be conducted under proliferative conditions, that is, in the presence of serum-containing culture media. Serum and glucocorticoid-inducible kinase, phosphoinositide-3 kinase and the mitogen-activated protein kinase pathway are activated by serum and have been shown to stimulate BK channels (Henke et al., 2004; O’Malley and Harvey, 2004).

The role of estrogens in prostate cancer is controversial and is also challenged by the present results. Despite the traditional use of estrogens for the treatment of prostate cancer, it is now known that estrogens can also induce neoplastic transformation in the prostate (Ho, 2004). In addition, 17β-estradiol directly activates BK channels by binding to the β1-regulatory subunit, as shown in vascular smooth muscle cells (Valverde et al., 1999). The present data provide evidence that 17β-estradiol stimulates growth of prostate cells through activation of BK channels. This growth stimulation by 17β-estradiol was pronounced in BPH-1 and LNCaP, but was absent in PC-3 cells. Missing activation of BK and lack of stimulation of cell proliferation by 17β-estradiol in PC-3 cells suggests that BK channels operate at maximal activity in these cells owing to overexpression driven by amplification. Interestingly, it has recently been shown that a proliferative effect of 17β-estradiol in the breast cancer cell line MCF-7 is also mediated by BK channels independently of estrogen receptor (Coiret et al., 2005). PC-3 and BPH-1 are known to express estrogen receptor alpha and beta, whereas LNCaP only expresses the estrogen receptor beta (Lau et al., 2000). Thus, 17β-estradiol might stimulate growth of BPH-1 and LNCaP by binding to the estrogen receptors. Alternatively, one could hypothesize that the growth effect of 17β-estradiol in LNCaP is due to altered ligand binding of the mutated androgen receptor (Veldscholte et al., 1992). However, the prevention of the growth stimulation of estradiol by IBTX suggests that the BK channel activity plays a central role in this growth stimulation. This is also in accordance with the previously shown direct activation of BK channels by 17β-estradiol (Valverde et al., 1999). Taken together, activation of BK channel might be a major target of 17β-estradiol in prostate cancer in the absence of KCNMA1 amplification and overexpression. However, it is likely that BK channels are modulated by a complex network of different molecules and molecular pathways (O’Malley and Harvey, 2004; Olsen et al., 2005).

There is increasing evidence that gene amplification leads to growth advantage of tumor cells, as a set of multiple genes rather than a single gene is overexpressed (Reiter et al., 2000; Kauraniemi et al., 2001). Along this line the urokinase-type plasminogen activator gene (uPA; syn. PLAU) has previously been suggested as amplification target at 10q22 in human prostate cancer (Helenius et al., 2001; Helenius et al., 2006). As expected, PLAU amplification preferentially occurred in advanced prostate cancer at a similar prevalence as amplification of KCNMA1 in our study. Prosaposin (PSAP) at 10q22, which has growth- and invasion-promoting activities, is another gene that has recently been found to be amplified in PC-3 and a few prostate cancer xenografts and lymph node metastases by single nucleotide polymorphism array and quantitative real-time PCR analysis (Koochekpour et al., 2005). Notably, PLAU stimulates Ca2+-activated K+ channels in the human promyelocytic cell line U937 (Christow et al., 1999), suggesting synergistic effects of co-amplification of KCNMA1 and PLAU. This challenges new promising studies on the interaction between KCNMA1 and other genes within the amplified chromosomal region at 10q22.

In summary, the data presented in this study unmask KCNMA1 as an amplified target gene at 10q22 that confers growth advantage to prostate cancer cells. Thus, BK channel may be a suitable pharmacological target in patients with late-stage prostate cancer that show amplification and overexpression of KCNMA1.

Materials and methods

FISH probes

The bacterial artificial chromosome (BAC) RP11-428p16, which contains major parts of the KCNMA1 gene sequence, and the BAC RP11-417o11, which contains the whole genomic sequence of PLAU, were obtained from the Wellcome Trust Sanger Institute (Cambridge, UK). Growth conditions and DNA extraction were as recommended by the supplier. One microgram of purified plasmid DNA was labeled using a modified Bio Nick kit (Invitrogen, Carlsbad, CA, USA). The nucleotide solution of the nick kit was replaced by 0.2 mM each dCTP, dGTP, dTTP, 0.1 mM dATP, 500 mM Tris-HCl (pH 7.8), 50 mM MgCl2, 100 mM-mercaptoethanol, 100 μg/ml nuclease-free bovine serum albumin (BSA), 1 μl Digoxigenin (Roche Diagnostics, Basel, CH), and 1 μl DNA Polymerase 1 (Invitrogen) was added. Nick translation was performed at 16°C for 90 min. The labeled FISH probes were purified by precipitation and re-dissolved in 50 μl H2O.

Clinical specimens

The 298 formalin-fixed and paraffin-embedded clinical specimens on the prostate tissue microarray were from the archive of the Institute for Pathology, University Hospital Basel, Switzerland. The tissue microarray was constructed as described previously (Kononen et al., 1998). The size of each tissue spot was 0.6 mm. In addition, sections from eight frozen prostate tissue specimens were used for immunofluorescence. They were from four patients with hormone-refractory local recurrences treated by transurethral resection, two patients with clinically organ-confined disease treated by radical prostatectomy and from two patients with benign prostatic hyperplasia treated by transurethral resections. Specimens were kept anonymous, and the experiments were according to the guidelines of the ethical committee of the University of Basel.

Fluorescence in situ hybridization

Paraffin removal (3 × 5 min in Xylene followed by 2 × 2 min ethanol 95% and air-drying) and enzymatic tissue pretreatment (Vysis pretreatment solution, 80°C, 15 min) of tissue sections mounted on glass slides were performed in a VP2000 Processor device (Vysis, Downers Grove, IL, USA). Slides were rinsed in water for 2 min, incubated in protease K solution (Vysis) at 37°C for 150 min and rinsed in water again. Subsequently, slides were dehydrated in an ascending ethanol series (70, 80, 95%) and air-dried. Then, a premixed hybridization cocktail containing 0.5 μl centromere 10 probe (CEP 10 Spectrum orange labeled; Vysis), 1.5 μl KCNMA1 probe (digoxigenin labeled), 1 μl Cot DNA (Invitrogen) and 7 μl hybridization buffer (Vysis) was added to each slide. For probe and target DNA denaturation, slides were heated to 72°C for 10 min. Probes were allowed to hybridize overnight at 37°C in a humidified chamber. The next day, slides were washed in 2 × standard sodium citrate, 0.3%NP40, ph 7–7.5 at 72°C for 2 min. The KCNMA1 probe was detected using the Dig detection kit (Roche Diagnostics). Amplification was defined as a signal ratio of gene probe/centromere 102 and at least five gene signals.

Cell lines and cell culture

We used the cell lines PC-3, LNCaP and BPH-1. PC-3 was originally established from a lumbar vertebral metastasis of a 62-year-old Caucasian man after treatment with diethylstilbestrol and bilateral orchiectomy (Kaighn et al., 1979). LNCaP cells were isolated from a needle aspiration biopsy of the left supraclavicular lymph node metastasis of a 50-year-old Caucasian man (Horoszewicz et al., 1980). BPH-1 originates from a 68-year-old patient with transurethral resection of the prostate for benign prostatic hyperplasia. The donor had not undergone hormonal therapy and did not have malignant disease. The cells were immortalized with a virus carrying the SV40T antigen and selected for resistance to geneticin. This cell line does not express androgen receptor and prostate specific antigen (Hayward et al., 1995).

For the growth assays, 6 × 25 000 cells were plated on a six-well plate (Falcon, Becton-Dickinson, Franklin Lakes, NJ, USA). PC-3 and BPH-1 were grown in 3 ml OPTI-MEM (Invitrogen)+10% fetal calf serum (FCS, BioConcept, Allschwil, CH). After 1 day, the medium was changed to OPTI-MEM+1% FCS. At day 2, the medium was changed to OPTI-MEM+0.4% Albumax (Invitrogen) containing IBTX (Sigma, Fluka Production GmbH, St Louis, MO, USA) or 17 β-estradiol (Sigma, Fluka Production GmbH). IBTX was diluted in phosphate-buffered saline (PBS) to a final concentration of 20 nM. 17 β-estradiol was diluted in 70% ethanol to a final concentration of 1 nM. In contrast to PC-3 and BPH-1, LNCaP did not grow in Optimem+0.4% Albumax. Thus, the effects of estradiol and IBTX on growth of LNCaP were assayed in Optimem+5% FCS+0.2% Albumax. This medium contains reduced serum and still allows growth of LNCaP. After two days of growth, cell numbers were counted using a Neubauer Chamber. The growth rate k was calculated as N(t)=N0* ekt, t=time in days.

Western blot and immunofluorescence

Cells were lysed in sample buffer containing 1% sodium dodecyl sulfate (SDS) and 100 mmol/l dichlorodiphenyl-trichloroethane. Protein concentrations were determined according to a modified Laury method. Lysates of PC-3 cells were resolved by 7% SDS–PAGE, transferred to Hybond-P (Amersham Pharmacia Biotech, Freiburg, BRD) and incubated with anti-KCNMA1 polyclonal antibody (generous gift by Professor Dr P Ruth, Institute of Pharmacy, University of Tübingen, Germany). Proteins were visualized using a sheep anti rabbit immunoglobulin G, conjugated to horse radish peroxidase and ECL Advance Detection Kit (Amersham Pharmacia Biotech). For immunocytochemistry, PC-3, BPH-1 and LNCaP cells were grown on glass cover slips, washed three times in PBS and fixed in methanol at −20°C. After washing in PBS, cells were incubated for 10 min in blocking buffer containing 10% BSA and 10% fish skin gelatine (both from Sigma). Cells were incubated overnight at 4°C in blocking solution, containing the primary antibody (1:1000 dilution). Subsequently, cells were washed in PBS and incubated with the fluorescein isothiocynate (FITC)-linked secondary antibody for 1 h at 37°C. Tissues were counter stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution (Sigma) and embedded in Mowiol (Sigma). Immunofluorescence was detected using an Axiovert 200 microscope and MetaFluor imaging software. Immunofluorescence on frozen tissue sections was performed as described previously (Sausbier et al., 2006).

RNA interference (RNAi)

Transfection was performed following the siRNA transfection protocol for Lipofectamine 2000 (Qiagen, KJ Venlo, NL, USA). Briefly, 50 000 cells were plated in six-well plates (Falcon, Becton-Dickinson) one day before transfection. The transfection was performed in OPTI-MEM (Invitrogen) for 4 h. The transfection mixture contained 5 μl of siRNA and Lipofectamine, each, in totally 3 ml OPTI-MEM. After 4 h, the transfection mixture was replaced by OPTI-MEM +10% FCS. The siRNA sequences were: K1 IndexTermgactggcagagtcctggttgt, K2 IndexTermgtgggtctgtccttccctact, K3 IndexTermgaccgtcctgagtggccatgt, K4 IndexTermacgcccttagaggtggctaca.

Semiquantitative RT–PCR

Cells washed with PBS were lysed in 350 μl RLT buffer (RNAeasy minikit, Qiagen). RNA extraction was done according to the protocol of Qiagen. cDNA synthesis was performed with 0.25 μg of total RNA using the superscript2 cDNA synthesis kit (Invitrogen). RT–PCR was performed using the LightCycler-system and the LightCycler FastStart DNA Master Hybridization Probes kit (Roche Diagnostics). Primers for G6PD and KCNMA1 hybridization products were obtained from TIB MolBiol (Berlin, BRD). PCR primers were: KCNMA1: KCNMA1_s: IndexTermcctggcctcctccatggt, KCNMA1_a: IndexTermttctgggcctccttcgtct, G6PD: G6ex7,8 R IndexTermttctgcatcacgtcccgga, G6ex6 S IndexTermaccactacctgggcaaggag. Hybridization probes were: KCNMA1: KCNMA1_fl: IndexTermagcgtccgccagagcaagat, KCNMA1_lc: IndexTermatgaagaggcccccgaagaaagt, G6PD: G6ex_FL: IndexTermcagatggggccgaagatcctgtt, G6ex_LC: IndexTermcaaatctcagcaccatgaggttctgcac. PCR conditions were: activation: 10 min 95°C PCR cycle: 5 s at 95°C, annealing 5 s at 57°C, elongation 15 s at 72°C, 40 cycles. Raw data for KCNMA1 expression were corrected by comparing to the related G6PD controls. Then these corrected values were compared to each other to obtain relative expression.

Patch clamp experiments

Cell culture dishes were mounted on the stage of an inverted microscope (IM35, Carl Zeiss AG, Oberkochen, BRD) and kept at 37°C. Patch clamp experiments were performed in the fast whole-cell and cell excised inside/out configuration. The patch pipettes had an input resistance of 2–4 MΩ when filled with a solution containing (mmol/l) KCl 30, K-gluconate 95, NaH2PO4 1.2, Na2HPO4 4.8, ethyleneglycol tetraacetate 1, CaCl2 0.726, MgCl2 1.034, D-glucose 5, adenosine triphosphate 1 (32 Cl). The pH was adjusted to 7.2, the Ca2+ activity was either 0.1 or 1 μmol/l. The access conductance was between 30 and 120 nS. In regular intervals, membrane voltages (Vm) were clamped in steps of 10 mV from −50 to +50 mV and whole-cell conductances were calculated according to Ohm's law. Currents (voltage clamp) and voltages (current clamp) were recorded using a patch clamp amplifier (EPC 7, List Medical Electronic, Darmstadt, Germany), the LIH1600 interface and PULSE software (HEKA) as well as Chart software (AD-Instruments). Data were analysed using PULSE and Origin software. In membrane patches containing several BK channels, channel activity was determined by multiplying single channel Po with the number of active channels in the membrane patch. NS1619, IBTX, paxillin and β-estradiol were from Sigma.

Statistical analysis

Statistical significance was verified in the Figures 2, 4 and 5 with an analysis of variance test using JMP-IN software (release 5.1, SAS Institute GmbH, Heidelberg, BRD). Statistical significance was assumed when P<0.05; n=3 for these figures. For all patch clamp experiments, Student's t-test P-values <0.05 were accepted to indicate statistical significance (*).

References

  1. Albertson DG, Collins C, McCormick F, Gray JW . (2003). Chromosome aberrations in solid tumors. Nat Genet 34: 369–376.

    CAS  Article  Google Scholar 

  2. Amberg GC, Bonev AD, Rossow CF, Nelson MT, Santana LF . (2003). Modulation of the molecular composition of large conductance, Ca(2+) activated K(+) channels in vascular smooth muscle during hypertension. J Clin Invest 112: 717–724.

    CAS  Article  Google Scholar 

  3. Amberg GC, Santana LF . (2003). Downregulation of the BK channel beta1 subunit in genetic hypertension. Circ Res 93: 965–971.

    CAS  Article  Google Scholar 

  4. Basrai D, Kraft R, Bollensdorff C, Liebmann L, Benndorf K, Patt S . (2002). BK channel blockers inhibit potassium-induced proliferation of human astrocytoma cells. Neuroreport 13: 403–407.

    CAS  Article  Google Scholar 

  5. Bernardino J, Bourgeois CA, Muleris M, Dutrillaux AM, Malfoy B, Dutrillaux B . (1997). Characterization of chromosome changes in two human prostatic carcinoma cell lines (PC-3 and DU145) using chromosome painting and comparative genomic hybridization. Cancer Genet Cytogenet 96: 123–128.

    CAS  Article  Google Scholar 

  6. Calderone V . (2002). Large-conductance, Ca(2+)-activated K(+) channels: function, pharmacology and drugs. Curr Med Chem 9: 1385–1395.

    CAS  Article  Google Scholar 

  7. Christow SP, Bychkov R, Schroeder C, Dietz R, Haller H, Dumler I et al. (1999). Urokinase activates calcium-dependent potassium channels in U937 cells via calcium release from intracellular stores. Eur J Biochem 265: 264–272.

    CAS  Article  Google Scholar 

  8. Coiret G, Matifat F, Hague F, Ouadid-Ahidouch H . (2005). 17-Beta-estradiol activates maxi-K channels through a non-genomic pathway in human breast cancer cells. FEBS Lett 579: 2995–3000.

    CAS  Article  Google Scholar 

  9. Conti M . (2004). Targeting K+ channels for cancer therapy. J Exp Ther Oncol 4: 161–166.

    CAS  PubMed  Google Scholar 

  10. Davies AG, Pierce-Shimomura JT, Kim H, VanHoven MK, Thiele TR, Bonci A et al. (2003). A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans. Cell 115: 655–666.

    CAS  Article  Google Scholar 

  11. El Gedaily A, Bubendorf L, Willi N, Fu W, Richter J, Moch H et al. (2001). Discovery of new amplification loci in prostate cancer by comparative genomic hybridization. Prostate 46: 184–190.

    CAS  Article  Google Scholar 

  12. Galvez A, Gimenez-Gallego G, Reuben JP, Roy-Contancin L, Feigenbaum P, Kaczorowski GJ et al. (1990). Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus. J Biol Chem 265: 11083–11090.

    CAS  PubMed  Google Scholar 

  13. Hayward SW, Dahiya R, Cunha GR, Bartek J, Deshpande N, Narayan P . (1995). Establishment and characterization of an immortalized but non-transformed human prostate epithelial cell line: BPH-1. In vitro Cell Dev Biol Anim 31: 14–24.

    CAS  Article  Google Scholar 

  14. Helenius MA, Saramaki OR, Linja MJ, Tammela TL, Visakorpi T . (2001). Amplification of urokinase gene in prostate cancer. Cancer Res 61: 5340–5344.

    CAS  PubMed  Google Scholar 

  15. Helenius MA, Savinainen KJ, Bova GS, Visakorpi T . (2006). Amplification of the urokinase gene and the sensitivity of prostate cancer cells to urokinase inhibitors. BJU Int 97: 404–409.

    CAS  Article  Google Scholar 

  16. Henke G, Maier G, Wallisch S, Boehmer C, Lang F . (2004). Regulation of the voltage gated K+ channel Kv1.3 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid inducible kinase SGK1. J Cell Physiol 199: 194–199.

    CAS  Article  Google Scholar 

  17. Ho SM . (2004). Estrogens and anti-estrogens: key mediators of prostate carcinogenesis and new therapeutic candidates. J Cell Biochem 91: 491–503.

    CAS  Article  Google Scholar 

  18. Horoszewicz JS, Leong SS, Chu TM, Wajsman ZL, Friedman M, Papsidero L et al. (1980). The LNCaP cell line – a new model for studies on human prostatic carcinoma. Prog Clin Biol Res 37: 115–132.

    CAS  PubMed  Google Scholar 

  19. Hsing AW, Tsao L, Devesa SS . (2000). International trends and patterns of prostate cancer incidence and mortality. Int J Cancer 85: 60–67.

    CAS  Article  Google Scholar 

  20. Kaighn ME, Narayan KS, Ohnuki Y, Lechner JF, Jones LW . (1979). Establishment and characterization of a human prostatic carcinoma cell line (PC-3). Invest Urol 17: 16–23.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Kauraniemi P, Barlund M, Monni O, Kallioniemi A . (2001). New amplified and highly expressed genes discovered in the ERBB2 amplicon in breast cancer by cDNA microarrays. Cancer Res 61: 8235–8240.

    CAS  Google Scholar 

  22. Knuutila S, Autio K, Aalto Y . (2000). Online access to CGH data of DNA sequence copy number changes. Am J Pathol 157: 689.

    CAS  Article  Google Scholar 

  23. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S et al. (1998). Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 4: 844–847.

    CAS  Article  Google Scholar 

  24. Koochekpour S, Zhuang YJ, Beroukhim R, Hsieh CL, Hofer MD, Zhau HE et al. (2005). Amplification and overexpression of prosaposin in prostate cancer. Genes Chromosomes Cancer 44: 351–364.

    CAS  Article  Google Scholar 

  25. Kunzelmann K, Mall M . (2002). Electrolyte transport in the mammalian colon: mechanisms and implications for disease. Physiol Rev 82: 245–289.

    CAS  Article  Google Scholar 

  26. Lau KM, LaSpina M, Long J, Ho SM . (2000). Expression of estrogen receptor (ER)-alpha and ER-beta in normal and malignant prostatic epithelial cells: regulation by methylation and involvement in growth regulation. Cancer Res 60: 3175–3182.

    CAS  PubMed  Google Scholar 

  27. McCobb DP, Fowler NL, Featherstone T, Lingle CJ, Saito M, Krause JE et al. (1995). A human calcium-activated potassium channel gene expressed in vascular smooth muscle. Am J Physiol 269: H767–H777.

    CAS  PubMed  Google Scholar 

  28. O’Grady SM, Lee SY . (2005). Molecular diversity and function of voltage-gated (Kv) potassium channels in epithelial cells. Int J Biochem Cell Biol 37: 1578–1594.

    Article  Google Scholar 

  29. Olsen ML, Weaver AK, Ritch PS, Sontheimer H . (2005). Modulation of glioma BK channels via erbB2. J Neurosci Res 81: 179–189.

    CAS  Article  Google Scholar 

  30. O’Malley D, Harvey J . (2004). Insulin activates native and recombinant large conductance Ca(2+)-activated potassium channels via a mitogen-activated protein kinase-dependent process. Mol Pharmacol 65: 1352–1363.

    Article  Google Scholar 

  31. Orio P, Rojas P, Ferreira G, Latorre R . (2002). New disguises for an old channel: MaxiK channel beta-subunits. News Physiol Sci 17: 156–161.

    CAS  Google Scholar 

  32. Pardo LA . (2004). Voltage-gated potassium channels in cell proliferation. Physiology (Bethesda) 19: 285–292.

    CAS  Google Scholar 

  33. Pardo LA, del Camino D, Sanchez A, Alves F, Bruggemann A, Beckh S et al. (1999). Oncogenic potential of EAG K(+) channels. EMBOJ 18: 5540–5547.

    CAS  Article  Google Scholar 

  34. Parihar AS, Coghlan MJ, Gopalakrishnan M, Shieh CC . (2003). Effects of intermediate-conductance Ca2+-activated K+ channel modulators on human prostate cancer cell proliferation. Eur J Pharmacol 471: 157–164.

    CAS  Article  Google Scholar 

  35. Pei L, Wiser O, Slavin A, Mu D, Powers S, Jan LY et al. (2003). Oncogenic potential of TASK3 (Kcnk9) depends on K+ channel function. Proc Natl Acad Sci USA 100: 7803–7807.

    CAS  Article  Google Scholar 

  36. Petrylak DP, Tangen CM, Hussain MH, Lara Jr PN, Jones JA, Taplin ME et al. (2004). Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer. N Engl J Med 351: 1513–1520.

    CAS  Article  Google Scholar 

  37. Quirk JC, Reinhart PH . (2001). Identification of a novel tetramerization domain in large conductance K(ca) channels. Neuron 32: 13–23.

    CAS  Article  Google Scholar 

  38. Rebhan M, Chalifa-Caspi V, Prilusky J, Lancet D . (1997). GeneCards: integrating information about genes, proteins and diseases. Trends Genet 13: 163.

    CAS  Article  Google Scholar 

  39. Reiter RE, Sato I, Thomas G, Qian J, Gu Z, Watabe T et al. (2000). Coamplification of prostate stem cell antigen (PSCA) and MYC in locally advanced prostate cancer. Genes Chromosomes Cancer 27: 95–103.

    CAS  Article  Google Scholar 

  40. Robitaille R, Charlton MP . (1992). Presynaptic calcium signals and transmitter release are modulated by calcium-activated potassium channels. J Neurosci 12: 297–305.

    CAS  Article  Google Scholar 

  41. Sausbier M, Matos JE, Sausbier U, Beranek G, Arntz C, Neuhuber W et al. (2006). Distal colonic K(+) secretion occurs via BK channels. J Am Soc Nephrol 17: 1275–1282.

    CAS  Article  Google Scholar 

  42. Tseng-Crank J, Foster CD, Krause JD, Mertz R, Godinot N, DiChiara TJ et al. (1994). Cloning, expression, and distribution of functionally distinct Ca(2+)-activated K+ channel isoforms from human brain. Neuron 13: 1315–1330.

    CAS  Article  Google Scholar 

  43. Valverde MA, Rojas P, Amigo J, Cosmelli D, Orio P, Bahamonde MI et al. (1999). Acute activation of Maxi-K channels (hSlo) by estradiol binding to the beta subunit. Science 285: 1929–1931.

    CAS  Article  Google Scholar 

  44. Van Coppenolle F, Skryma R, Ouadid-Ahidouch H, Slomianny C, Roudbaraki M, Delcourt P et al. (2004). Prolactin stimulates cell proliferation through a long form of prolactin receptor and K+ channel activation. Biochem J 377: 569–578.

    CAS  Article  Google Scholar 

  45. Veldscholte J, Berrevoets CA, Ris-Stalpers C, Kuiper GG, Jenster G, Trapman J et al. (1992). The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens. J Steroid Biochem Mol Biol 41: 665–669.

    CAS  Article  Google Scholar 

  46. Wang Z . (2004). Roles of K+ channels in regulating tumour cell proliferation and apoptosis. Pflugers Arch 448: 274–286.

    CAS  Article  Google Scholar 

  47. Weaver AK, Liu X, Sontheimer H . (2004). Role for calcium-activated potassium channels (BK) in growth control of human malignant glioma cells. J Neurosci Res 78: 224–234.

    CAS  Article  Google Scholar 

  48. Zhou XB, Wang GX, Ruth P, Huneke B, Korth M . (2000). BK(Ca) channel activation by membrane-associated cGMP kinase may contribute to uterine quiescence in pregnancy. Am J Physiol Cell Physiol 279: C1751–C1759.

    CAS  Article  Google Scholar 

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Acknowledgements

We are grateful to Martina Mirlacher, Alex Rufle Hedvika Novotny, Ursula Dürrmüller and Dr Heide Tullberg from the Institute for Pathology, University Hospital Basel, and Dr Rainer Schreiber, Professor Dr Richard Warth and Melanie Spitzner at the University of Regensburg for their expert technical advice. We also thank Professor Dr Peter Ruth, Dr Matthias Sausbier and Ulrike Sausbier from the Institute of Pharmacy, University of Tübingen, Germany for generously providing the BK channel antibody.

The PC-3 cell line was kindly provided by Spyro Mousses, PhD (Translational Genomics Research Institute, Gaithersburg, MD, USA), LNCaP was from Meera Srivastava, PhD (Uniformed Services University of the Health Sciences, Bethesda, MD, USA) and BPH-1 was from Holger Rumpold, MD (Institute for Biomedical Ageing, Innsbruck, Austria).

This study was supported by the Stammbach Foundation/SWISS BRIDGE (KLS 01114-02-2001), by the Swiss National Science Foundation (Grant No. 3100A0-105413) and by the Deutsche Forschungsgemeinschaft DFG SCHR 752/2-1.

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Correspondence to L Bubendorf.

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Bloch, M., Ousingsawat, J., Simon, R. et al. KCNMA1 gene amplification promotes tumor cell proliferation in human prostate cancer. Oncogene 26, 2525–2534 (2007). https://doi.org/10.1038/sj.onc.1210036

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Keywords

  • prostate cancer
  • progression
  • amplification
  • BK channel
  • KCNMA1

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