1Health Science Center, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, and 2Department of Medical Genetics, Shanghai Second Medical University, Shanghai, China; 3Department of Cell Biology Neurobiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio; 4Division of Molecular Medicine, Harbor-UCLA Medical Center, David Geffen School of Medicine, University of California-Los Angeles, Torrance, California; and 5Division of Molecular Carcinogenesis, Department of Medicine, New York Medical College, Valhalla, New York
Submitted 11 January 2005 ; accepted in final form 21 March 2005
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ABSTRACT |
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growth factors; myeloblastic cells; signaling; ion channel blocker
K+ channels, ubiquitous in the cell membrane, are essential for basic and physiological functions (20). Alternation of K+ channel activity can mediate functional adaptations to a variety of chemical and physical stimulations by stabilizing membrane potential, maintaining salt and water balance, and regulating cell volume (2, 13). In addition, mounting evidence now shows that voltage-gated K+-channel activity also plays an important role in growth factor- or cytokine-stimulated activation and proliferation of T and B lymphocytes (27), corneal epithelial cells (11), and a number of tumor cell lines (18). In line with this, K+ channel antagonists usually inhibit cell proliferation (27). Consistently, mitogenic signaling mechanisms can often upregulate K+ channel expression and activity (2, 4). Cell cycle studies have indicated that voltage-gated K+ channels are required for progression through the G1 phase (27, 28). However, the underlying mechanisms remain unknown.
In our previous studies, we described (8, 23, 28) a voltage-gated delayed-rectifier K+ channel in human myeloblastic leukemic ML-1 cells. K+ channel activity, strongly activated by growth factors contained in serum, is particularly sensitive to 4-aminopyridine (4-AP), a specific blocker for voltage-gated K+ channels (8, 23, 28). K+ channel activity is high in proliferating cells and subdued in quiescent cells (23). Suppression of K+ channel activity with K+ channel blockers inhibited serum-stimulated ML-1 proliferation by inducing G1 arrest (28). Recently, it was demonstrated that EGF could evoke a rapid 4-AP-sensitive K+ current in ML-1 cells and that the EGF-stimulated ERK pathway was blunted by K+ channel blockade (29).
In the present work, we extend our finding by demonstrating that insulin-stimulated mitogenesis of serum starvation-synchronized ML-1 cells is dependent on 4-AP-sensitive K+ channel activity. K+ channel activity appears to be an upstream modulator of growth factor-mediated ERK and Akt pathways. However, the Akt pathway, but not the ERK pathway, appears to play a major role in regulating ML-1 cell proliferation. These results may further our understanding of the importance of K+ channel activity in regulating cell proliferation by a variety of mitogenic signals in general.
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MATERIALS AND METHODS |
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Cell proliferation assay. A CellTiter 96 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay was performed to determine cell growth as a proliferation index according to the instruction manual provided by the manufacturer (Promega, Madison, WI). Briefly, after serum starvation, ML-1 cells were cultured in 96-well plates (BD Biosciences, Franklin Lakes, NJ) at a density of 104 cells·100 µl medium1·well1, with each well containing the appropriate treatment as indicated. After 60-h incubation, a dye was added to each well and incubated for 4 h at 37°C. A stop solution was then added to solubilize the formazan crystals at 37°C. Cell proliferation was measured by absorbance at 570 nm with a Sunrise 96-well plate reader (Tecan Group, Maennedorf, Switzerland). Each experiment was done in triplicates, and the entire experiment was repeated three times.
Cell cycle analysis. Serum-starved ML-1 cells were either collected or treated with 0.1 µM insulin or 7.5% serum in RPMI 1640 medium for 24 h before collection. Five million cells were spun at 1,000 g for 10 min at 4°C and washed once in cold PBS. Cells were resuspended and fixed by addition of 0.3 ml of ice-cold PBS and 0.7 ml of ethanol. After incubation on ice for 1 h, cells were collected by centrifugation and washed with cold PBS. After being washed, cell pellets were resuspended in 0.5 ml of PBS staining solution containing 50 µg/ml propidium iodide (Sigma) and 0.1 mg/ml RNase A (Sigma). The cell suspension was kept at 4°C for 1 h in the dark. Analysis was done on a FACSCalibur cytometer (BD Biosciences) with CellQuest and ModFit software, with appropriate gating on the FL2-A and FL2-W channels to exclude cell aggregates. At least 10,000 events were analyzed per sample.
Western blot analysis.
ML-1 cells treated with various agents were collected for immunoblotting at the indicated times. Cells seeded at a density of 3 x 105 cells/ml were washed once with ice-cold PBS before lysis, at a ratio of 106 cells/40 µl buffer, with a lysis buffer [in mM: 20 Tris·HCl (pH 7.5), 137 NaCl, 1.5 MgCl2, 2 EDTA, 10 sodium pyrophosphate, 25 -glycerophosphate (Sigma), 1 sodium orthovanadate (Calbiochem, San Diego, CA), and 1 phenylmethylsulfonyl fluoride (Sigma), with 10% glycerol, 1% Triton X-100 (Sigma), 10 µM okadaic acid (Calbiochem), and protease inhibitor cocktail (Sigma)]. All chemicals were purchased from Shanghai Chemical Reagent (Shanghai, China) unless otherwise specified. After incubation on ice for 30 min, cell lysates were cleared by centrifugation at 13,000 g for 25 min and denatured by boiling in 2x Laemmli buffer for 5 min. Equal amounts of protein samples (the equivalent of 1.5 x 105 cells) were subjected to 11% SDS-PAGE followed by electrotransfer to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h and subsequently incubated overnight at 4°C with primary antibodies diluted in TBST containing 5% nonfat dry milk. Rabbit polyclonal antibodies against total and phosphorylated (activated) ERK1/2 and Akt were diluted to 1:1,000 (Cell Signaling Technology, Beverly, MA). Mouse monoclonal antibody against
-actin (Sigma) was diluted to 1:4,000. After incubation, the membranes were washed in TBST for 3 x 5 min and then incubated for 1 h at room temperature with either horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (Amersham Biosciences, Piscataway, NJ) diluted to 1:2,000 in TBST containing 5% nonfat dry milk or HRP-conjugated goat anti-mouse IgG (1:6,000; Santa Cruz Biotechnology, Santa Cruz, CA). After an additional wash with TBST (3 x 5 min), signals were developed with a Supersignal West Pico kit (Pierce, Rockford, IL) and captured on Kodak X-ray films. In some experiments, results were scanned digitally and optical density (OD) was quantified by Quantity One software (Bio-Rad). Relative OD was calculated by normalizing the signal of the phosphorylated protein against that of the corresponding total protein.
Statistics. Data are shown either as original values or, in the case of OD absorbance values, as means ± SE. Statistical differences between the control group and treatment groups were determined by Students t-test at a significance level of P < 0.05.
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RESULTS |
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Insulin treatment also resulted in significantly elevated phosphorylation of Akt, which was again blocked by 4-AP with a pattern similar to that of the ERK pathway (Fig. 5, C and D). Quantitative analysis revealed that insulin stimulated Akt phosphorylation about four times over the unstimulated control level, and this stimulatory effect was progressively diminished by increasing concentrations of 4-AP (Fig. 5D). Neither insulin nor 4-AP caused a significant change in total protein levels of ERK and Akt.
The specific effect of suppressing K+ channel activity on EGF- and insulin-induced cell proliferation was further studied by applications of various K+ channel blockers. These blockers have previously demonstrated effectiveness in blockade of K+ channel activity in our patch-clamp studies in ML-1 cells (23, 24). Three different K+ channel blockers including 4-AP, Ba2+, and tetraethylammonium were used in G1 phase-synchronized ML-1 cells (Fig. 6). These channel blockers significantly inhibited ML-1 cell proliferation detected by MTT assays (n = 6, P < 0.05). The results indicate that the effect of these channel blockers on inhibition of K+ channel activity is rather specific, resulting in suppression of growth factor-induced proliferation in ML-1 cells.
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DISCUSSION |
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In early literature describing ML-1 cells, bovine insulin along with human transferrin was used to supplement serum-free liquid medium to support ML-1 growth (19). By using insulin in our experiments, we were able to show that 4-AP suppressed both ML-1 proliferation and Akt and ERK phosphorylation in a concentration-dependent manner. It is noted that insulin is less effective in stimulating cell proliferation than 7.5% serum, suggesting that a combined effect of additional factors or pathways is still involved. Myeloblastic ML-1 cells are dependent on appropriate cytokines to proliferate and differentiate. One potential candidate is the JAK/STAT pathway and the cytokines associated with it (1, 17). Whether the JAK/STAT pathway plays a role in ML-1 proliferation and whether it is modulated by K+ channel activity remain to be determined.
Increased K+ channel activities result in K+ efflux and membrane hyperpolarization and secondarily lead to transient Ca2+ influx and cell swelling. These events have been associated with cell cycle progression through G1 and S phases (6, 27, 28). Stimulation of mitogens increases expression and activity of K+ channels, which has been amply documented. For example, it has been reported that in HEK293 cells, IGF-I can upregulate the expression of several Kv channels as part of its mitogenic effect (4). Consistent with other reports on rabbit corneal epithelial cells (11), our own results point to an early involvement of K+ channels in the initial signaling events at the membrane level and suggest that K+ channel modulation of mitogenic/growth factor-mediated signaling may be a more general phenomenon. From a mechanistic point of view, activation of K+ channels can lead to changes of the membrane event including receptor clustering, facilitating signaling (12). On the other hand, it is conceivable that posttranslational modification of K+ channel protein subunits and protein-protein interaction with upstream signaling transduction components may be involved in the process.
K+ channel activation has been implicated as an early event during apoptosis of hematopoietic (5, 24), corneal epithelial (7, 22), and neuronal (25, 31) cells, as well as several other cell types (10, 30). Involvement of K+ channel activity, however, appears to depend on the type of stress exerted on the cell in question. For example, UV irradiation and osmotic shock are potent activators of 4-AP-sensitive K+ channels (22, 24), but they appear to be spared in oxidative stress-induced apoptosis (5). In our experiments, we observed no loss of cell viability during serum starvation or subsequent growth factor and 4-AP challenges by Trypan blue staining (not shown). During UV-induced ML-1 apoptosis, hyperactivity of K+ channels activates the JNK pathway, whereas K+ channel activity affects activation of the ERK signaling pathway during growth factor stimulation. Exactly how K+ channel activity selectively affects signaling pathways in response to specific stresses merits further study. It is conceivable that such delineation has implications for chemical intervention in cancer therapy.
The molecular identity of the 4-AP-sensitive K+ channel in ML-1 cells remains an open question, but a similar channel was found to be a member of the Kv3.4 family in corneal epithelial cells (12). Although 4-AP is a commonly used antagonist in K+ channel studies, like all pharmacological agents, there may be nonspecific actions associated with it. It is now apparent that deciphering the precise mechanism will have to await further studies to define the channel type in ML-1 cells. Electrophysiologically, the 4-AP-sensitive channel is a voltage-gated delayed-rectifier type that has a conductance of 31 pS (8, 11, 28). 4-AP binds to the pore region of the channel to inhibit K+ fluxes and has an IC50 of 80 µM (8). Recently, Wang et al. (21) reported the identification in corneal epithelial cells of a Kv3.4 channel whose characteristics fit the biochemical and pharmacological profiles of the voltage-gated 4-AP-sensitive K+ channel. Expression studies in a heterologous system will be needed to confirm this. Given the available RNA interference technology, one can test the role of the various Kv channels in ML-1 cells. However, recent reports suggest that 4-AP may affect DNA synthesis in chondrocytes (26). In addition, there is a report that 4-AP decreases cell proliferation in GH3 cells without altering membrane potential (3). To define the specific effect of K+ channel activity on growth factor-induced cell proliferation, we used three different K+ channel blockers in the present study (Fig. 6). In fact, all of these three K+ channel blockers suppress EGF- and insulin-induced cell proliferation.
In summary, our results highlight the significance of K+ channels in cell proliferation through regulation of multiple growth factor-mediated signaling pathways. They raise the intriguing prospect of using drugs targeting specific K+ channels as a therapeutic intervention in the treatment of cancer. Investigations of the detailed molecular ordering and interplay and identification of the K+ channel responsible will be crucial to unraveling the connection between K+ channel physiology and cancer biology.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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2. Cahalan MD and Lewis RS. Functional roles of ion channels in lymphocytes. Semin Immunol 2: 107117, 1990.[Medline]
3. Czarnecki A, Dufy-Barbe L, Huet S, Odessa MF, and Bresson-Bepoldin L. Potassium channel expression level is dependent on the proliferation state in the GH3 pituitary cell line. Am J Physiol Cell Physiol 284: C1054C1064, 2003.
4. Gamper N., Fillon S., Huber SM, Feng Y, Kobayashi T, Cohen P, and Lang F. IGF-1 up-regulates K+ channels via PI3-kinase, PDK1 and SGK1. Pflügers Arch 443: 625634, 2002.[CrossRef][ISI][Medline]
5. Gao J, Wu D, Guo TB, Ruan Q, Li T, Lu Z, Xu M, Dai W, and Lu L. K+ channel activity and redox status are differentially required for JNK activation by UV and reactive oxygen species. Exp Cell Res 297: 461471, 2004.[CrossRef][ISI][Medline]
6. Lang F, Ritter M, Volkl H, and Haussinger D. The biological significance of cell volume. Renal Physiol Biochem 16: 4865, 1993.[ISI][Medline]
7. Lu L, Wang L, and Shell B. UV-induced signaling pathways associated with corneal epithelial cell apoptosis. Invest Ophthalmol Vis Sci 44: 51025109, 2003.
8. Lu L, Yang T, Markakis D, Guggino WB, and Craig RW. Alterations in a voltage-gated K+ current during the differentiation of ML-1 human myeloblastic leukemia cells. J Membr Biol 132: 267274, 1993.[ISI][Medline]
9. Reddy KB, Nabha SM, and Atanaskova N. Role of MAP kinase in tumor progression and invasion. Cancer Metastasis Rev 22: 395403, 2003.[CrossRef][ISI][Medline]
10. Remillard CV and Yuan JX. Activation of K+ channels: an essential pathway in programmed cell death. Am J Physiol Lung Cell Mol Physiol 286: L49L67, 2004.
11. Roderick C, Reinach PS, Wang L, and Lu L. Modulation of rabbit corneal epithelial cell proliferation by growth factor-regulated K+ channel activity. J Membr Biol 196: 4150, 2003.[CrossRef][ISI][Medline]
12. Rosette C and Karin M. Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors. Science 274: 11941197, 1996.
13. Sarkadi B and Parker JC. Activation of ion transport pathways by changes in cell volume. Biochim Biophys Acta 1071: 407427, 1991.[ISI][Medline]
14. Sebolt-Leopold JS. MEK inhibitors: a therapeutic approach to targeting the Ras-MAP kinase pathway in tumors. Curr Pharm Des 10: 19071914, 2004.[CrossRef][ISI][Medline]
15. Shelton JG, Steelman LS, White ER, and McCubrey JA. Synergy between PI3K/Akt and Raf/MEK/ERK pathways in IGF-1R mediated cell cycle progression and prevention of apoptosis in hematopoietic cells. Cell Cycle 3: 372379, 2004.[ISI][Medline]
16. Sordella R, Bell DW, Haber DA, and Settleman J. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 305: 11631167, 2004.
17. Steelman LS, Pohnert SC, Shelton JG, Franklin RA, Bertrand FE, and McCubrey JA. JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis. Leukemia 18: 189218, 2004.[CrossRef][ISI][Medline]
18. Strobl JS, Wonderlin WF, and Flynn DC. Mitogenic signal transduction in human breast cancer cells. Gen Pharmacol 26: 16431649, 1995.[CrossRef][Medline]
19. Taketazu F, Kubota K, Kajigaya S, Shionoya S, Motoyoshi K, Saito M, Takaku F, and Miura Y. Clonal growth of human acute myeloid leukemia cells (ML-1 and HL-60) in serum-free agar medium. Cancer Res 44: 531535, 1984.[Abstract]
20. Tang XD, Santarelli LC, Heinemann SH, and Hoshi T. Metabolic regulation of potassium channels. Annu Rev Physiol 66: 131159, 2004.[CrossRef][ISI][Medline]
21. Wang L, Fyffe RE, and Lu L. Identification of a Kv3.4 channel in corneal epithelial cells. Invest Ophthalmol Vis Sci 45: 17961803, 2004.
22. Wang L, Li T, and Lu L. UV-induced corneal epithelial cell death by activation of potassium channels. Invest Ophthalmol Vis Sci 44: 50955101, 2003.
23. Wang L, Xu B, White RE, and Lu L. Growth factor-mediated K+ channel activity associated with human myeloblastic ML-1 cell proliferation. Am J Physiol Cell Physiol 273: C1657C1665, 1997.
24. Wang L, Xu D, Dai W, and Lu L. An ultraviolet-activated K+ channel mediates apoptosis of myeloblastic leukemia cells. J Biol Chem 274: 36783685, 1999.
25. Wei L, Yu SP, Gottron F, Snider BJ, Zipfel GJ, and Choi DW. Potassium channel blockers attenuate hypoxia- and ischemia-induced neuronal death in vitro and in vivo. Stroke 34: 12811286, 2003.
26. Wohlrab D, Lebek S, Kruger T, and Reichel H. Influence of ion channels on the proliferation of human chondrocytes. Biorheology 39: 5561, 2002.[ISI][Medline]
27. Wonderlin WF and Strobl JS. Potassium channels, proliferation and G1 progression. J Membr Biol 154: 91107, 1996.[CrossRef][ISI][Medline]
28. Xu B, Wilson BA, and Lu L. Induction of human myeloblastic ML-1 cell G1 arrest by suppression of K+ channel activity. Am J Physiol Cell Physiol 271: C2037C2044, 1996.
29. Xu D, Wang L, Dai W, and Lu L. A requirement for K+-channel activity in growth factor-mediated extracellular signal-regulated kinase activation in human myeloblastic leukemia ML-1 cells. Blood 94: 139145, 1999.
30. Yu SP. Regulation and critical role of potassium homeostasis in apoptosis. Prog Neurobiol 70: 363386, 2003.[CrossRef][ISI][Medline]
31. Yu SP, Yeh CH, Sensi SL, Gwag BJ, Canzoniero LM, Farhangrazi ZS, Ying HS, Tian M, Dugan LL, and Choi DW. Mediation of neuronal apoptosis by enhancement of outward potassium current. Science 278: 114117, 1997.
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