Insulin-activated, K+-channel-sensitive Akt pathway is primary mediator of ML-1 cell proliferation

Taylor B. Guo,1,2 Jiawei Lu,1 Tie Li,4 Zhenyu Lu,2 Guotong Xu,1 Ming Xu,1,2,3 Luo Lu,1,2,4 and Wei Dai1,2,5

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


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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Voltage-gated K+ channel activities are involved in regulating growth factor-stimulated cell proliferation in a variety of cell types. Here we report that suppression of a voltage-gated K+ channel with 4-aminopyridine (4-AP), barium, and tetraethylammonium inhibited both EGF- and insulin-stimulated myeloblastic leukemia ML-1 cell proliferation in a concentration-dependent manner. Both MAPK/ERK and Akt pathways are known to mediate cell proliferative signals of a variety of growth factors including insulin. In serum-starved ML-1 cells, insulin rapidly stimulated phosphorylation of ERK1/2 and Akt, and the phosphorylation levels peaked ~30 min after treatment. Pretreatment of ML-1 cells with 4-AP potently and dose-dependently prevented phosphorylation of ERK1/2 and Akt. However, insulin-induced activation of the Akt pathway also played a role in promoting ML-1 cell proliferation. Flow cytometry analysis revealed that although ML-1 cells were primarily arrested at G1 phase by serum starvation for 36 h, they reentered the cell cycle after treatment with serum or insulin for 24 h. However, concomitant 4-AP treatment was able to attenuate cell cycle progression in synchronized ML-1 cells stimulated with growth factors. Our results strongly suggest that a 4-AP-sensitive K+ channel activity plays an important role in controlling proliferation of ML-1 cells by affecting the activation of multiple signal transduction processes induced by insulin.

growth factors; myeloblastic cells; signaling; ion channel blocker


GROWTH FACTORS contained in serum, such as insulin, IGF, and EGF, stimulate cell proliferation in various types of cells. These growth factors transmit mitogenic signals through binding to and activation of their cognate receptors. The signals, in the form of reversible phosphorylation, are transduced and amplified through downstream kinase cascades, leading to cell survival, growth, differentiation, and/or metabolic changes. The Raf-MEK-ERK axis is one of these major growth factor-mediated signaling pathways (14). ERK1 and 2, the terminal kinases in this module, regulate a myriad of substrates important for growth and differentiation (9). Extensive studies in the past have shown that the Raf-MEK-ERK pathway is intricately integrated into the cell signaling network. For example, it can both collaborate and cross talk with the phosphatidylinositol 3-kinase/Akt signaling pathway and the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway (15, 16), both of which are also essential for cell survival and growth.

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.


    MATERIALS AND METHODS
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 MATERIALS AND METHODS
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Cell cultures. ML-1 cells were cultured in RPMI 1640 supplemented with 7.5% heat-inactivated FBS, 100 µg/ml penicillin, and 50 µg/ml streptomycin sulfate in a humidified incubator supplied with 5% CO2 at 37°C. All culture reagents were from PAA Laboratories (Linz, Austria). Cells were passed at a seeding density of 3 x 105 cells/ml. Serum starvation was achieved by maintaining the cells in a medium containing 0.3% FBS for 36 h. Growth factors at the indicated concentrations or serum was added to the culture to reinitiate the cell cycle. For K+ channel blockade, varying concentrations of 4-AP (Sigma, St. Louis, MO) were added to the medium 30 min before growth factor or serum supplementation. EGF was purchased from R&D Systems (Minneapolis, MN), and insulin (Novolin R) was from Novo Nordisk.

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 medium–1·well–1, 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 {alpha}-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 {beta}-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 Student’s t-test at a significance level of P < 0.05.


    RESULTS
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We showed previously (28) that ML-1 cell proliferation stimulated by serum can be inhibited by K+ channel blockade with specific antagonists. Because the interaction of EGF with its cognate receptor results in activation of the Raf-MEK-ERK pathway in a variety of cells and because EGF-stimulated ERK phosphorylation is strongly inhibited by 4-AP (29), we tested whether EGF is a primary growth factor in the serum that was responsible for ML-1 cell proliferation. To that end, ML-1 cells that were serum starved for 36 h were released into the cell cycle by addition of EGF or serum. EGF at a concentration of 10 ng/ml induced a transient phosphorylation activation of ERK1/2, and EGF-induced phosphorylation activation of ERKs peaked at 15 min (Fig. 1A). EGF applied in synchronized cells at different concentrations elicited significant growth responses in proliferation assays (Fig. 1B). We next examined whether insulin, another major growth factor found in serum, could stimulate ML-1 proliferation. As shown in Fig. 1C, 5 µg/ml insulin significantly stimulated ML-1 cell proliferation. Compared with that of control cells, insulin stimulated 50% more cell growth, whereas the cell growth rate of ML-1 cells after 7.5% serum refeeding was 100% higher. These observations suggest that EGF was capable of activating ERK1/2 signal inputs to induce cell proliferation and insulin was also capable of activating cell proliferation, perhaps from different pathway(s) that involve sustaining cell proliferation. These results thus suggest that both EGF and insulin are the principal components in the serum that stimulate ML-1 cell proliferation.



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Fig. 1. Insulin- but not EGF-stimulated ML-1 cell proliferation. A: effect of EGF on ERK activation. Serum-starved ML-1 cells were stimulated with EGF (10 ng/ml), and at the indicated time points cells were harvested for Western blot analysis of phosphorylated (p) and total ERK1/2 levels. B: dose-dependent stimulation of ML-1 cell proliferation by EGF. C: dose-dependent stimulation of ML-1 cell proliferation by insulin. ML-1 cells were synchronized by serum starvation (in RPMI 1640 supplemented with 0.3% serum) for 36 h before being treated with different concentrations of EGF or insulin. Control cells received no treatment. Cell proliferation was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Absorbance data (optical density at 570 nm) are presented as means ± SE from a representative of 3 independent experiments. *Significant statistical difference from the control (P < 0.05).

 


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Fig. 5. Dose-dependent effect of suppressing K+ channel activity with 4-AP on insulin-induced ERK and Akt phosphorylation/activation. ML-1 cells were serum starved for 24 h before various concentrations of 4-AP were added to the medium as indicated. Half an hour later, EGF at 10 ng/ml (A) or insulin at 10 µg/ml (B and C) was added to the medium for 20 min before cells were harvested for Western blotting. Control samples were not treated with either growth factors or 4-AP. D: statistical analysis of 4-AP inhibition of insulin-stimulated Akt phosphorylation as shown in C. Data are presented as means ± SE (n = 3, P < 0.05).

 
To examine whether this insulin-stimulated proliferation is associated with activation of ERK and Akt pathways in ML-1 cells, we examined the time course of ERK1/2 and Akt phosphorylation stimulated by insulin. Western blotting revealed that insulin elicited significant difference in phosphorylation levels of ERKs 5–30 min after treatment (n = 3, P < 0.05) and that the phosphorylation lasted for at least 20 min before returning to the pretreatment level (Fig. 2, A and B). This time-dependent ERK phosphorylation by insulin is reminiscent of EGF-induced ERK activation (29). Moreover, insulin was able to increase Akt phosphorylation, the peak of which was reached ~15 min after insulin treatment (Fig. 2C). Therefore, insulin is capable of rapid activation of both ERK and Akt pathways.



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Fig. 2. Activation of ERK and Akt signaling pathways by insulin stimulation. A: time-dependent ERK phosphorylation in response to insulin stimulation. After serum starvation, ML-1 cells were treated with 1 µM insulin for the indicated times. Levels of phosphorylated (activated) and total ERK1/2 were determined by Western blot analysis. B: statistical analysis of time course of insulin-induced ERK phosphorylation. Densitometric measurement of ERK phosphorylation over time in response to insulin treatment is shown as normalized values against total ERK levels, further converted to relative values by comparing them to the value at time 0, set as 1; values are means ± SE of 2 independent experiments. *Significant statistical difference from the control (P < 0.05). C: time course of Akt phosphorylation/activation after insulin stimulation. After serum starvation, ML-1 cells treated with insulin for the indicated times were collected. Equal amounts of cell lysates were blotted for Akt and total Akt.

 
To determine whether inhibition of K+ channel activity would suppress ML-1 cell proliferation, the K+ channel blocker 4-AP was added to the cell culture 30 min before stimulation with FBS or insulin. Indeed, 4-AP inhibited both insulin- and serum-stimulated ML-1 cell proliferation in a concentration-dependent manner, from 0.1 to 2.0 mM (Fig. 3). Although insulin was consistently less potent than serum in stimulating growth, the growth inhibition with both treatments was essentially parallel. It is interesting to note that the addition of 1 mM 4-AP attenuated growth to the level of serum-starved cells when they were maintained in 0.3% serum for 36 h (Figs. 1B and 3). These results indicate that K+ channel activity is required for growth factor-induced increases in ML-1 cell proliferation.



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Fig. 3. Effect of suppressing K+ channel activity on insulin-induced ML-1 cell proliferation. MTT assay shows that 4-aminopyridine (4-AP), given 30 min before growth factor treatment, inhibits growth factor-stimulated ML-1 proliferation in a concentration-dependent manner. Values are means ± SE. {bullet}, 5 µg/ml insulin treatment; {circ}, 7.5% serum treatment. Shown is 1 representative set of data from 3 independent experiments.

 
To determine which cell cycle phase(s) was affected by inhibition of K+ channel activity, we analyzed the cell cycle status of ML-1 cells that were treated with or without 4-AP by flow cytometry. We demonstrated that in control cells after 36-h serum starvation, two-thirds of the cells remained in G1 phase; a high concentration of 4-AP (2 mM) was used because it was expected to produce the maximal effect of suppressing K+ channel activity on cell cycle progression. In our previous studies (23, 29), ML-1 cell viability was not affected by 2 mM 4-AP. At this dose, inhibition of K+ channel activity led to a 70% population of cells being accumulated in G1 phase (Fig. 4). Treatment of starved cells with insulin and serum drove the cells into the cell cycle, resulting in increases in cell populations in S phase of ~25% and ~40%, respectively (Fig. 4). When starved ML-1 cells were cultured in the presence of insulin, 4-AP treatment reduced the number of cells in M phase, with a reciprocal increase in the number of cells in G1 phase. On the other hand, when serum-starved cells were refed with serum, 4-AP treatment further suppressed accumulation of the S-phase cell population. Together, these results suggest that K+ channel blockade profoundly affects cell cycle progression.



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Fig. 4. Effect of suppressing K+ channel activity on serum- and insulin-induced cell growth analyzed by cell cycle profiling. Left: After serum starvation, ML-1 cells were treated with or without 5 µg/ml insulin or 7.5% serum in the absence (control, CNTL) or presence of 4-AP. After 24 h, they were processed for propidium iodide (PI) staining and flow cytometry. Shown are cell cycle histograms (cell counts vs. DNA content) of control cells (no 4-AP treatment) and cells treated with 2 mM 4-AP. Tall gray peaks represent the G1 population of cells, small peaks on right represent the G2/M population of cells, and hatched areas represent the S phase population of cells. Right: data were analyzed by ModFit software, and the resultant relative %cell counts were plotted against various 4-AP concentrations.

 
Having shown that K+ channel activity is required for insulin-stimulated cell proliferation, we went on to investigate the effect of K+ channel activity on insulin-stimulated ERK and Akt pathways. Serum-starved ML-1 cells were pretreated with increasing concentrations of 4-AP for 30 min, after which they were stimulated with insulin for 20 min, the time point at which both ERK and Akt were expected to be maximally phosphorylated (Fig. 2). Immunoblot analysis revealed that, similar to the inhibition of EGF-stimulated ERK1/2 phosphorylation by 4-AP (Fig. 5A), insulin-stimulated ERK1/2 phosphorylation was also severely attenuated by the K+ channel blocker (Fig. 5B). It is interesting to note that 4-AP was more effective in inhibition of EGF-stimulated phosphorylation of ERKs than insulin-stimulated phosphorylation (Fig. 5, A and B). The basal level of ERK phosphorylation was also inhibited by 4-AP treatment. Therefore, K+ channel activity is required for both insulin- and EGF-stimulated ERK activation.

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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Fig. 6. Effects of suppressing K+ channel activity on EGF- and insulin-induced proliferation. EGF- and insulin-induced proliferation were inhibited by suppression of K+ channel activity with 1 mM 4-AP (A), 2 mM Ba2+ (B), or 10 mM tetraethylammonium (TEA; C) in ML-1 cells. Data were collected from 6 independent experiments for each group. *Significant differences within the group (P < 0.05).

 

    DISCUSSION
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 MATERIALS AND METHODS
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We have provided new evidence that K+ channel activity is required for growth factor-stimulated cell proliferation through regulation of Akt and ERK pathways. Although previous experiments show that serum-stimulated proliferation can be blocked by K+ channel antagonists (28), the growth factors in the serum responsible for this effect have yet to be identified. Because EGF stimulates K+ channel activity and activation of the ERK signaling pathway (23, 28), and because the ERK signaling pathway was blocked by 4-AP, we attempted to show the effect of suppressing K+ channel activity by 4-AP on EGF-stimulated ML-1 proliferation. In fact, EGF is one of the primary mitogens for cells of the epithelial lineage; it may not be the only effective factor for hematopoietic cells. In ML-1 cells, EGF receptor expression was found to be very low, which could contribute to the weak effect in eliciting a growth response, despite the finding that EGF is able to stimulate K+ channel activity and induces activation of ERK signaling pathway, both of which were done over short periods of time immediately after EGF stimulation.

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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This work was supported in part by core support from the Chinese Academy of Sciences (CAS), a One Hundred Talent Grant from CAS (M. Xu), a National Key Program Grant (973) (NO2002CB512805), and funds from Jiaotong University (2003hzjj001) and E Research Institute of Shanghai Second Medical University.


    ACKNOWLEDGMENTS
 
We thank the members of the joint laboratory for helpful discussions.


    FOOTNOTES
 

Addresses for reprint requests and other correspondence: W. Dai, L. Lu, and Ming Xu (e-mail: wei_dai{at}NYMC.edu; lluou{at}ucla.edu; ming.xu{at}uc.edu)

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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