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Advances in Brief |
Department of Medicine, Montreal Heart Institute, University of Montreal, Montreal, Quebec, H1T 1C8 Canada
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ABSTRACT |
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). Immunostaining and immunocoprecipitation reveal coexpression of HERG and TNF receptor 1 on the cytoplasmic membrane, which is correlated with greater activities of nuclear transcription factor, nuclear factor-
B, in HERG-expressing tumor cells. Our data suggest that HERG K+ channel is a regulator of tumor cell proliferation and apoptosis and provide a potential new target for cancer therapy. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Materials and Methods |
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Whole Cell Patch-Clamp Recordings.
The techniques have been described in detail elsewhere (11)
. Borosilicate glass electrodes (1-mm absorbance) had tip resistances of 1–3 M
when filled with pipette solution (0.1 mM GTP, 110 mM potassium aspartate, 20 mM KCl, 1 mM MgCl2, 5 mM Mg-ATP, and 10 mM HEPES, pH 7.3). Junction potentials were zeroed before formation of the membrane-pipette seal in Tyrode’s solution (135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, and 10 mM glucose, pH 7.4). For SK-Br-3, SH-SY5Y, A549, and SK-Mel-28 cells, the external KCl and NaCl were 40 and 100 mM, respectively. Experiments were conducted at 36°C.
Measurement of DNA Fragmentation.
The methods for ELISA and TUNEL quantification of DNA fragmentation and Annexin V detection of early apoptosis have been described in detail previously (12)
. To induce apoptosis, H2O2 was added to the culture medium to a final concentration of 400 µM and incubated with cells for 5 h. For experiments involving Dof, cells had been pretreated with the drug (1 µM) for 30 min before H2O2 was added.
Determination of PDT.
Cell proliferation was assessed by characterizing the log phase growth with PDT calculated by the equation: 1/(3.32 x (logNH - logNI)/(t2 - t1), where NH is the number of cells harvested at the end of the growth period (t2) and NI is the number of cells at 5 h (t1) after seeded. Cells were counted by a flow cytometer (EPICS XL; Beckman Coulter Canada, Inc.), and the number obtained at 5 h (t1) after seeding was taken as an initial cell number (NI), and the number at 48 h (t2) after seeding was taken as an endpoint number (NH).
Immunocytochemical Analysis.
For immunostaining of wild-type and S633A (negative dominant mutant) HERG K+ channels, HEK293 cells transfected with wild-type or S633A mutant HERG channels were fixed with freshly prepared 3% paraformaldehyde in PBS for 20 min at 4°C. After washes with PBS, cells were permeabilized in 1% Triton for 5 min and blocked in 1% BSA. The cells were then incubated overnight at 4°C with primary antibody (anti-HERG residues 1106–1159; Alomone Labs, Jerusalem, Israel) diluted in 1% BSA, followed by incubation with donkey antirabbit FITC-conjugated secondary antibody (Jackson ImmunoResearch, Baltimore, MD) at room temperature for 2 h. The coverslip was mounted on a slide with 10 µl of anti-fading solution, and the cells were examined under a confocal microscope. For double staining of HERG and TNFR1, anti-HERG and anti-TNFR1 (Research Diagnostics, Flanders, NJ) antibodies were mixed and added to the cells in 1% BSA. Donkey antirabbit, FITC-conjugated antibodies and donkey antigoat, FITC-conjugated antibodies were used as secondary antibodies for HERG and TNFR1, respectively. Rabbit anti-cleaved caspase-3 antibody was purchased from Cell Signaling Technology (Beverly, MA) and used for analyzing caspase activity. The active form of NF-B activity was analyzed with mouse anti-NF-B p65 subunit monoclonal antibody (Chemicon International). Simultaneous PI staining was performed for identifying the nuclei caspase-3 and NF-B experiments.
Immunocoprecipitation for HERG and TNFR1.
Cells were rinsed with ice-cold PBS and scraped off into the lysis buffer (200 mM NaCl, 33 mM NaF, 10 mM EDTA, and 50 mM HEPES, pH 7.4) plus protease inhibitor cocktail (100 µM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, and 4 µl/ml aprotinin; Sigma). The cells were sonicated and spun at 500 x g for 10 min. The membrane fractions were pelleted from the low-speed supernatants by centrifugation at 60,000 rpm for 1 h at 4°C and resuspended in 50 mM Tris-HCl, 15 mM ß-mercaptoethanol, and 1% SDS. For immunoprecipitation, a protein sample (60 µg) was incubated with anti-TNFR1 or anti-HERG antibody for 1 h at 4°C, followed by the addition of 1:1 slurry of protein G-Sepharose beads (Sigma) and incubated overnight at 4°C. The beads were washed with extraction buffer, and bound proteins were eluted with SDS-PAGE sample buffer and boiled for 5 min. Samples were subjected to SDS-PAGE and immunoblotting (11
, 12) with anti-HERG or anti-TNFR1.
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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and immunostaining using antibody raised against the COOH-terminal sequence of HERG (Fig. 4)
. The cells grown in the normal culture medium to 80% confluence were incubated with H2O2 (400 µM) for 5 h and then examined under a light microscope for morphological alterations to identify the apoptotic cells (shrinking, blebbing, detached, and round up; Ref. 12
). As shown in Fig. 1
, H2O2 caused considerable cell death in cells expressing endogenous IHERG relative to cells devoid of IHERG (Fig. 1A)
. To confirm that the observed cell death was caused by apoptosis, ELISA (12)
and TUNEL (12)
methods were used to detect DNA/chromosomal fragmentation, a biochemical hallmark of apoptosis, and the results are presented in Fig. 1, B and C
. Consistent with the morphological data, DNA fragmentation is substantially higher in HERG-expressing cells than in cells lacking HERG channel conductance. In addition, Annexin V and PI double staining was performed to identify the cells in the early stage of apoptosis (12)
. Our data showed clearly that after exposure to H2O2 for 2 h, a majority of cells expressing HERG, but not the cells lacking HERG, displayed positive Annexin V staining (Fig. 1D
, green).
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, Dof prevented cell death and DNA fragmentation in cells with endogenous IHERG but did not affect the apoptosis in cells lacking IHERG.
Also noteworthy is that the concentration of H2O2 required to reach the same degree of apoptosis was much higher in IHERG-lacking cells relative to IHERG-expressing cells. For example, 400 µM H2O2 induced 
50% cell death in all IHERG-expressing tumor cells tested, but only 5% in the IHERG-lacking cells, and to obtain 50% cell death in these IHERG-lacking cells, H2O2 concentration had to be increased to >1.2 mM.
To further assess the role of IHERG in regulating H2O2-induced apoptosis, we performed similar experiments in HEK293 cells stably transfected with HERG (HERG-HEK; Ref. 10
). Our data demonstrated that H2O2 (400 µM) caused minimal apoptosis in nontransfected or mock-transfected HEK cells. By comparison, substantial apoptosis was consistently seen in HERG-HEK cells, an effect effectively prevented by pretreatment with Dof (Fig. 1)
. To investigate whether the apoptosis promotion in HERG-expressing cells was ascribed to HERG channel expression per se or was attributable to HERG channel conductance, we used the HEK cells stably transfected with the negative dominant construct of HERG channel (S633A mutant). Expression of S633A HERG proteins in the cytoplasmic membrane was verified by immunostaining, and the absence of HERG conductance was confirmed by whole-cell patch-clamp recording (Fig. 2B)
. As shown in Fig. 1D
, the HEK cells transfected with S633A lost the ability to enhance H2O2-induced apoptosis. Our data thus suggest that HERG conductance or IHERG promotes apoptosis induced by H2O2 insult.
Proapoptotic effect of other K+ currents has been recognized recently. The elegant studies reported by several groups including Yu et al. (13
, 14)
, Bortner and Cidlowski (15)
, Vu et al. (16)
, and Maeno et al. (17)
open up a new era in the field of K+ channel research. Their data, together with some other studies, provide convincing evidence that decreased intracellular K+ content attributable to K+ efflux through K+ channels results in activation of apoptotic signaling pathways. Loss of intracellular K+ can lead to cell shrinkage because K+ is a major determinant of intracellular osmolarity (17)
and can also mean a withdrawal of inhibitory factor for caspase activation (executioners of apoptosis; Refs. 15
, 16
). We performed experiments to assess the effects of H2O2 on IHERG in the tumor cells. As illustrated in Fig. 2A
, H2O2 (400 µM) significantly increased outward IHERG in various cells tested and shifted the HERG activation to more negative potentials. For example, in HERG-HEK cells, minimal IHERG was seen at potentials negative to -40 mV under control conditions. However, 10 min after H2O2, ample activation of IHERG was observed starting from -60 mV. Particularly noticeable is that the outward tail currents that were virtually absent under control conditions became prominent in the presence of H2O2 in HERG-expressing tumor cells. Dof (1 µM) effectively inhibited IHERG in the various cells tested.
To study whether HERG conductance was correlated with caspase activation, immunostaining analysis of caspase-3 activity using antibody directed against the cleaved form of the enzyme was performed. Our data show clearly that H2O2 produced more pronounced activation of caspase-3 in HERG-expressing cells than in cells lacking HERG or in HEK cells transfected with S633A. Preincubation of cells with Dof significantly prevented caspase-3 from activating in HERG-expressing cells (Fig. 2C)
.
Moreover, promotion of apoptosis by HERG is also observed when 10 ng/ml TNF- was used to induce apoptosis in both HERG-expressing and HERG-lacking cells, an effect effectively abrogated by Dof only in the former but not in the latter. And similar to the results obtained with H2O2, TNF- consistently caused more apoptotic cell death (Fig. 3A)
and more pronounced activation of caspase-3 (data not shown) in HERG-expressing cells than in HERG-lacking cells.
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was also seen and became more obvious when the apoptosis was less prominent with reduced TNF- concentrations to 1 and 0.1 ng/ml. This growth-facilitating effect of TNF- was significantly more pronounced in the HERG-expressing cells than in the HERG-lacking tumor cells. PDT was determined to quantify the cell growth. PDT was markedly shortened by TNF- (0.1 or 1 ng/ml) in HERG-expressing cells. For example, TNF- shortened PDT by 60% in SK-Br-3 cells and by 70% in HL-1 cells (Fig. 3B)
. A similar proliferation-facilitating effect of TNF- was also seen in HEK293 cells expressing the dominant-negative HERG (S633A), although HERG conductance or function was lost in this construct. Noticeably, Dof failed to affect the TNF--induced cell proliferation.
The fact that TNF- produced greater effects on growth and apoptosis in HERG-expressing cells than in HERG-lacking cells suggests a possibility that TNFR is expressed more abundantly in the former than in the latter cells. To test this notion, double staining with antibodies directed against HERG and TNFR1 was carried out, and the results are shown in Fig. 4A
. Clearly, cells that express HERG channels (green staining) demonstrated strong TNFR1 staining (red), with the antibody targeting the COOH-terminal sequence of the protein. By comparison, cells lacking HERG (SK-Mel-28 and A549) did not show positive HERG green staining, and only weak TNFR1 red staining was seen (Fig. 4A)
. Similar results were obtained when another anti-TNFR1 antibody targeting the NH2-terminal region of the protein was used (data not shown). These data indicated that HERG channels could somehow recruit TNFR1 to the cytoplasmic membrane, and there might exist physical interactions between HERG and TNFR1 proteins. To examine the notion, immunocoprecipitation was performed with membrane protein samples extracted from various cells. Anti-HERG antibody recognized a single discrete band of Mr 155,000 size in anti-TNFR1 pull-down samples, as well as in the samples without precipitation steps, from HERG-expressing cells. In contrast, anti-TNFR1 antibody identified a Mr 55,000 band in the protein samples immunoprecipitated with anti-HERG antibody. Pretreatment of the antibodies with their respective antigens abolished the bands. The data are displayed in Fig. 4B
, and only examples from A549, SK-Br-3, and HERG-HEK cells are shown in the figure. Similar results were consistently observed in other HERG-expressing and HERG-lacking cells, respectively. It is unclear what the structural components are for the interactions of two completely different categories of membrane proteins.
No significant differences of immunoreactivity to anti-TNFR2 or anti-Fas were found between cells with and without HERG expression (data not shown).
TNFR1 is known to be critical for regulating cell proliferation and apoptosis in many cells (18
, 19)
. Overexpression of TNFR1 has indeed been found in many malignant tumor cells (19
, 20)
. TNFR1 induction of apoptosis is fulfilled by sequential activation of caspase-2 and caspase-3, whereas TNFR1 induction of cell proliferation may be mediated by NF-B. NF-B is involved in the control of numerous cellular functions, particularly regulation of survival and proliferation (21
, 22)
. Translocation from cytoplasm to the nucleus is an event essential for NF-B activation. An increase in constitutive NF-B activity has been observed in a variety of malignant tumors, and it may have an important role in tumorigenesis and chemotherapy resistance because it facilitates cell proliferation and antagonizes apoptosis (21
, 22) . Here, NF-B activity was analyzed by immunostaining with the antibody raised against the active form of NF-B in various cells. The data consistently showed higher immunoreactivity of basal active NF-B in the nuclei of the cells expressing HERG than in those of the cells that do not express HERG. After treatment with TNF- (0.1 ng/ml), the immunostaining was further enhanced (reflecting the inducible activation of NF-B), and the increase was more in the HERG-expressing cells relative to the cells without HERG (Fig. 4C)
. These results, together with the data on TNFR1 expression, might explain the more pronounced increase in proliferation of HERG-expressing cells in response to TNF- stimulation (Fig. 3B)
.
We show here that H2O2 induced substantial apoptosis in cells expressing endogenous or cloned HERG channels, and the concentration of H2O2 required to induce a similar degree of apoptosis was approximately three times higher in two lines of tumor cells that do not express HERG channels. H2O2-induced apoptosis was significantly prevented by HERG blockade. Similarly, higher concentrations of TNF- (1–10 ng/ml) also induced apoptosis, which was greatly prevented by Dof. On the other hand, lower concentrations of TNF- (0.1–1 ng/ml) promoted cell proliferation in tumor cells expressing HERG channels but not in those without HERG expression, an effect unaffected by HERG blockade. Immunostaining and immunocoprecipitation experiments revealed coexpression of HERG and TNFR1 on the surface membrane, and TNFR1 expression was markedly higher in cells expressing HERG channel proteins than in cells lacking HERG. It appears from our data that HERG channel conductance promotes H2O2 (or TNF-)-induced apoptosis, and HERG protein expression seems to recruit TNFR1 to the membrane, which facilitates TNF--induced tumor cell growth. Therefore, HERG expression may represent an advantage for tumor cell growth and cancer development in the absence of apoptotic inducers such as H2O2 and higher concentrations of TNF-, indicating that HERG K+ channels might contribute to tumorigenesis. Yet, promotion of apoptosis by IHERG suggests that manipulating HERG conductance to enhance apoptotic tumor cell death may be a novel strategy for cancer therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by the Canadian Institute of Health Research, the Heart and Stroke Foundation of Quebec, and the Fonds de la Recherche de l’Institut de Cardiologie de Montreal (to Z. W.). H. W. is a research fellow of the Canadian Institute of Health Research. H. H. is a research fellow of the Heart and Stroke Foundation of Canada. Z. W. is a research scholar of the Fonds de la Recherche en Sante du Quebec. 
2 To whom requests for reprints should be addressed, at Research Center, Montreal Heart Institute, 5000 Belanger East, Montreal, PQ H1T 1C8 Canada. E-mail: wzmail{at}canada.com
3 The abbreviations used are: HERG, human ether-a-go-go related gene; TUNEL, terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling; Dof, dofetilide; PDT, population doubling time; NF-B, nuclear transcription factor-B; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor type 1; PI, propidium iodide.
Received 4/23/02. Accepted 7/16/02.
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H. Chapman, C. Ramstrom, L. Korhonen, M. Laine, K. T. Wann, D. Lindholm, M. Pasternack, and K. Tornquist Downregulation of the HERG (KCNH2) K+ channel by ceramide: evidence for ubiquitin-mediated lysosomal degradation J. Cell Sci., November 15, 2005; 118(22): 5325 - 5334. [Abstract] [Full Text] [PDF]
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, control (Ctl); -60 mV; 
, H2O2 (400 µM, bath application), -60 mV; 
, control, -40 mV; 
, H2O2, -40 mV. n 
3 cells for each group. B, immunostaining of HERG protein and whole-cell patch-clamp recording in HEK cells transfected with wild-type or S633A mutant HERG. Note that the antibody recognizes the S633A mutant in the cytoplasmic membrane, but the construct fails to carry K+ currents (bottom panel). C, immunostaining of cleaved (activated) caspase-3. Caspase-3 staining is in green (cytoplasm), and PI staining is in red (nucleus).
