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[Cancer Research 60, 4358-4361, August 15, 2000]
© 2000 American Association for Cancer Research

Advances in Brief

Bcl-2 Overexpression Results in Enhanced Capacitative Calcium Entry and Resistance to SKF-96395-induced Apoptosis1

Scott S. Williams, John N. French, Mark Gilbert, Arun A. Rangaswami, Jan Walleczek and Susan J. Knox2

Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 94305-5302 [S. S. W., J. N. F., M. G., J. W., S. J. K.], and Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of California at Davis Medical Center, Sacramento, California 95817 [A. A. R.]


   ABSTRACT
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 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Although there is evidence that changes in cellular ionic concentrations are important early events in apoptosis, the regulation of ion fluxes across the plasma membrane during this process is poorly understood. We report here that Bcl-2 overexpression results in up-regulation of capacitative Ca2+ entry (CCE) and that SKF-96365, an inhibitor of CCE, is a potent inducer of apoptosis. Cells that overexpress Bcl-2 are resistant to SKF-96365-mediated apoptosis and to its inhibition of CCE. Enhanced CCE can be reversed with ouabain, suggesting that Bcl-2-associated plasma membrane hyperpolarization plays a role in up-regulating CCE and may partially explain the antiapoptotic effect of Bcl-2.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Bcl-2 is one of the most widely studied oncogenes. Despite this fact, its precise mechanism of action has not been clearly elucidated. Overexpression of Bcl-2 is known to convey resistance to apoptosis induced by many different agents, including radiation (1) . Results from our laboratory have shown that Bcl-2 has profound effects on plasma membrane functions. Specifically, we have reported that Bcl-2 overexpression is associated with hyperpolarization of the resting plasma membrane potential (2) . Furthermore, Bcl-2-overexpressing cells have a higher level of Na+/K+-ATPase pump activity (the major contributor to plasma membrane potential) than control cells. Indeed, inhibition of the Na+/K+-ATPase pump by ouabain essentially negates Bcl-2-associated radioresistance, suggesting the presence of a relationship between Bcl-2, Na+/K+-ATPase pump activity, and radiation-induced apoptosis (3) . It was therefore of interest to determine how Bcl-2-associated hyperpolarization affects other plasma membrane functions involved in apoptotic signaling pathways, such as the regulation of the [Ca2+]i.3

CCE is the specific gating of Ca2+ entry across the plasma membrane in response to depletion of intracellular stores during Ca2+ signaling and can be triggered by Tg, an irreversible inhibitor of the ER Ca2+-ATPase (4) . CCE is essential for maintaining [Ca2+]i homeostasis and may therefore be an important regulator of the induction and execution phases of apoptosis, both of which contain Ca2+-dependent components. The results of the experiments described here demonstrate that Bcl-2 overexpression is associated with enhanced CCE, further demonstrating the importance of CCE in the maintenance of cell viability and providing insight into the antiapoptotic effect of Bcl-2.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Lines.
HL60, a human promyeloid leukemia cell line, was transfected via retroviral gene transfer with the cos MSV-tk-Neor-hBcl-2 vector to produce Bcl-2-overexpressing transfectants as described previously (5) . PW is a human B-cell lymphoma line that was similarly transfected. The level of Bcl-2 overexpression and associated relative radioresistance has been reported previously (2) . Jurkat T-cell leukemia cells were transfected using pREP4 episomal mammalian expression vector and show a 3–5-fold increase in Bcl-2 expression and a 4-fold reduction in apoptosis after 10 Gy of radiation (data not shown).

Measurement of Apoptosis.
Flow cytometry was used to determine the sub-G1-G0 fraction in fixed cells stained with propidium iodide as described previously (6) . Fixed cells were stored at 4°C until they were stained and analyzed on a FACStar flow cytometer (Becton Dickinson, San Jose, CA).

Measurement of [Ca2+]i.
Parental cells or Bcl-2-overexpressing HL60, PW, and Jurkat cells were loaded with fura-2/AM (2.5 µM) in HBSS containing 0.5% BSA at 7.0–9.0 x 105 cells/ml for 45 min. at 37°C. Cells were washed and resuspended in HBSS/BSA containing 225 nM sulfinpyrazone. Final resuspension buffers also contained SKF-96365 at 10–40 µM or ouabain at 50 µM as indicated. [Ca2+]i was measured after stimulation with Tg (400 nM) using a Hitachi F2000 fluorescence spectrophotometer with dual excitation of fura-2/AM at 340 and 380 nm and detection of fluorescent emissions at 500 nm. [Ca2+]i was calculated using the ratio of fluorescence at 340 nm:fluorescence at 380 nm with Ca2+ analysis software (Hitachi Instruments, Tokyo, Japan), using the method described by Hitachi. For specific measurement of Ca2+ influx, the final wash and resuspension was done in calcium- and magnesium-free HBSS/BSA, and extracellular Ca2+ (2 mM CaCl2) was added 45 s after stimulation with Tg. Cells were incubated in Ca2+-free media for 10–30 min before analysis. The resultant rise in [Ca2+]i was indicative of CCE (7) . In prior studies, vector-transfected control cells did not differ significantly from parental cells in any of the measured end points studied.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Enhanced CCE Results from Bcl-2 Overexpression.
HL60 and PW control and Bcl-2-transfected cell lines were analyzed to determine the level of CCE after stimulation with Tg. [Ca2+]i was measured in fura-2/AM-loaded cells in the absence of extracellular Ca2+ (Fig. 1ACitation ). Tg, a well-established inducer of CCE, was added at 20 s to trigger the opening of the plasma membrane calcium release-activated calcium channels. Generally, after the addition of Tg, Ca2+ is released immediately from intracellular stores, resulting in an elevation of [Ca2+]i. However, in this experiment, cells were incubated in Ca2+-free media long enough to deplete intracellular Ca2+ stores; therefore, no immediate Ca2+ release was detected. Only after extracellular Ca2+ is replenished 45 s later does [Ca2+]i rise as the ion crosses the plasma membrane. In both HL60 and PW cell lines, the Bcl-2-overexpressing cells had significantly higher levels of [Ca2+]i than the control cells (Fig. 1BCitation ), with a 41% and 60% increase in CCE compared with parental control cells, respectively (P < 0.005). Furthermore, Bcl-2-overexpressing cells also had significantly elevated rates of calcium influx compared with similarly treated control cells (Fig. 1CCitation ). The rate of Ca2+ influx in the Bcl-2-transfected cells was 102% and 32% greater in HL60 and PW transfectants (P < 0.005 and P < 0.03), respectively, compared with parental cells.



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  		Fig. 1. Bcl-2 overexpression is associated with enhanced CCE in HL60 and PW cells. A, representative tracings of [Ca2+]i induced by Tg stimulation in Ca2+-free media followed by replenishment of extracellular Ca2+. Short-dashed tracings are controls without Tg addition. B, measurement of maximum [Ca2+]i after Tg-induced CCE. C, measurement of the slope of calcium influx. , parental cell lines, , Bcl-2 transfectants. *, P < 0.005; **, P < 0.03. HL60, n = 6; PW, n = 4. Error bars, mean ± SD.

 
Bcl-2 Transfectants Are Partially Resistant to the Effects of SKF-96365 on CCE.
Tg causes a strong Ca2+ signal by releasing ER Ca2+ stores, which is followed by compensatory CCE from the extracellular space. In HL60 parental cells, Tg results in the expected elevation of [Ca2+]i, which is suppressed in a dose-dependent fashion by SKF-96365, an inhibitor of CCE (Fig. 2ACitation ). In contrast, Bcl-2-transfected HL60 cells are resistant to the inhibitory effect of SKF-96365 on CCE (Fig. 2BCitation ). It is important to note that the rise in [Ca2+]i seen in this experiment (Fig. 2Citation ) results from both CCE and the release of intracellular Ca2+ stores. This may explain the somewhat reduced [Ca2+]i level observed in Bcl-2-overexpressing cells compared with control cells in the absence of SKF-96365, (see "Discussion"). SKF-96365, at concentrations of 10–20 µM, generally resulted in less than 11% inhibition of the Tg-induced Ca2+ signal in the Bcl-2 transfectants as compared with up to 54% inhibition in similarly treated HL60 parental cells (P < 0.0005; Fig. 2CCitation ). As can be seen even at 40 µM SKF-96365, Bcl-2-overexpressing cells showed significantly less SKF-96365-mediated inhibition of CCE than control cells (P < 0.03).



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  		Fig. 2. Bcl-2-overexpressing cells are resistant to SKF-96365-mediated inhibition of the Tg-induced [Ca2+]i signaling. A and B, representative tracings showing the effect of increasing concentrations of SKF-96365 on Tg-induced [Ca2+]i elevations in HL60 parental and Bcl-2-overexpressing cells, respectively. Tracings labeled 1–4 are in the presence of 0, 10, 20, and 40 µM SKF-96365, respectively. Cells were analyzed in Ca2+-containing HBSS; Tg was added at 20 s. Baseline [Ca2+]i values are normalized to zero. C, pooled analysis, expressed as the percentage of inhibition of maximal [Ca2+]i, showing that Bcl-2-overexpressing cells are resistant to the inhibitory effects of SKF-96365. 10 µM SKF-96365, n = 11, P < 0.0005. 20 µM SKF-96365, n = 12, P < 0.0005. 40 µM SKF-96365, n = 6, P < 0.03. Error bars, SD.

 
Inhibition of CCE with SKF-96365 Results in Apoptosis That Is Partially Inhibited by Bcl-2 Overexpression.
Next, the effect of blocking CCE on cellular viability was determined. Cells were incubated in increasing concentrations of SKF-96365 for 24 h before quantification of apoptosis (Fig. 3Citation ). A subset of similarly treated cells was also irradiated with 10 Gy before determining the apoptotic fraction. At all concentrations of SKF-96365, as in irradiated cells, the Bcl-2-overexpressing cells underwent significantly less (P < 0.05) apoptosis than similarly treated HL60 control cells. These results demonstrate the importance of CCE as a determinant of cell viability, the effects of which are modulated by Bcl-2 (Fig. 2Citation ). These results suggest that Bcl-2-mediated protection from SKF-96365-induced apoptosis may be due, in part, to the enhancement of CCE in Bcl-2-transfected cells (Fig. 1Citation ).



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  		Fig. 3. SKF-96365 induces apoptosis in HL60 cells, and Bcl-2 protects cells from SKF-96365-induced apoptosis. Cells were treated with increasing concentrations of SKF-96365 or with 10 Gy of radiation, as indicated. Apoptosis was measured at 24 h as the fraction of cells containing sub-G0-G1 DNA content. P < 0.05. Error bars, SD.

 
The Enhanced CCE Associated with Bcl-2 Is Reversed with Ouabain.
Because ouabain negates the effect of Bcl-2 on plasma membrane hyperpolarization and radioresistance, it was used to determine the effect of plasma membrane depolarization on CCE. In all three Bcl-2-overexpressing cell lines studied, the level of calcium influx in the presence of ouabain was similar to that of parental cells (Fig. 4Citation ). Ouabain, at this concentration, was previously shown to depolarize the plasma membrane potential of Bcl-2-overexpressing cells to the level of parental control cells (2) . Depolarization of parental cell lines also resulted in partial inhibition of calcium influx. This observation suggests that up-regulation of CCE is a consequence of Bcl-2-associated plasma membrane hyperpolarization, which decreases the susceptibility of cells to apoptosis.



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  		Fig. 4. Depolarization with ouabain reverses Bcl-2-associated enhancement of CCE. Fura-2/AM-loaded cells were analyzed in calcium-free HBSS, Tg was added at 20 s, and extracellular Ca2+ was replenished 45 s later. Dashed tracings were performed in 50 µM ouabain. Tracings 1 and 3 represent Bcl-2 transfectants, tracings 2 and 4 represent parental cell lines. Tracings are representative of eight, six, and two tracings in A–C, respectively.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
CCE is essential for the maintenance of Ca2+ homeostasis in nonexcitable cells (8) . Cells use two sources of Ca2+ for generating signals: (a) Ca2+ release from intracellular stores; and (b) CCE, which plays a central role in many aspects of cell signaling and is inducible by a wide range of stimuli (4) . It is depletion of ER Ca2+ stores that initiates CCE to potentiate cytoplasmic Ca2+ signals and refill intracellular Ca2+ reserves through the action of the ER Ca2+-ATPase pump. Maintenance of intracellular Ca2+ stores appears to be crucial to the well-being of cells because prolonged depletion of these stores can induce apoptosis (9) . Therefore, cells with efficient mechanisms for replenishing intracellular stores, such as enhanced CCE, may be less likely to undergo apoptosis and may have a significant survival advantage after exposure to apoptotic stimuli.

Recent data also suggest that normal [Ca2+]i homeostasis plays a fundamental role in both the generation and execution of the apoptotic pathway. For example, Ca2+ is required for endonuclease activity that results in DNA fragmentation, and alterations in mitochondrial Ca2+ homeostasis result in membrane depolarization, cytochrome c release, and caspase activation. Depletion of the ER Ca2+ stores appears to be an important apoptotic signal. Interestingly, others have reported that Bcl-2-overexpressing cells retain higher intralumenal Ca2+ levels than control cells, making Ca2+ store depletion more difficult. Increased retention of ER Ca2+ by Bcl-2-overexpressing cells may be due to overexpression of Ca2+-ATPase, the Ca2+ pump that sequesters Ca2+ in the ER (10) . These findings, however, fail to explain the results presented in Fig. 1Citation , in which cells incubated in Ca2+-free media are depleted of intracellular Ca2+ stores as indicated by the failure of Tg to elicit an immediate Ca2+ release. In the presence of Tg, addition of Ca2+ to the media resulted in the rapid accumulation of Ca2+ in the cytosol. Because Tg inhibits Ca2+-ATPase, refilling of the ER stores is not possible. Therefore, the rise in [Ca2+]i observed after the addition of Ca2+ to the media is a reflection of CCE across the plasma membrane that is independent of ER Ca2+-ATPase function. Both the magnitude and rate of CCE were greater in cells overexpressing Bcl-2 as compared with parental control cells. This observation is further substantiated by the relative insensitivity of Bcl-2-overexpressing cells to SKF-96365 (Fig. 2Citation ).

It is notable that the baseline [Ca2+]i response to Tg in Fig. 2Citation is somewhat reduced in Bcl-2 transfectants as compared with similarly treated control cells. This observation is in agreement with the findings of Lam et al. (11) , who reported that Bcl-2 was associated with reduced efflux of Ca2+ from Tg-sensitive ER stores. They suggested that it was possible that inhibition of ER Ca2+ store depletion may interfere with the signal to trigger CCE and thereby result in reduced Ca2+ influx. This interpretation leads to an apparent discrepancy in comparison to our findings, which can be explained by differences in the experimental technique used. We analyzed cells that had been incubated in Ca2+-free media before the addition of Ca2+ at a specified time point. This resulted in a more specific measurement of Ca2+ influx and revealed enhanced CCE in Bcl-2-transfected cells (Fig. 1Citation ). The results presented here as well as the observation by Lam et al. (11) that Bcl-2 transfectants have enhanced uptake of Ca2+ into the ER support the hypothesis that the antiapoptotic effect of Bcl-2 is closely associated with alterations in Ca2+ homeostasis that protect intracellular Ca2+ stores (i.e., enhanced CCE, reduced depletion of ER Ca2+ stores, and a greater ability to refill these stores).

The importance of CCE in maintaining cell viability is seen in the induction of apoptosis by SKF-96365 in HL60 cells (Fig. 3Citation ). These results are consistent with those of others who observed that SKF-96365 induced DNA fragmentation in Syrian hamster embryo cells (12) . Whereas SKF-96365 has activity on voltage-gated ion channels in excitable cells, it has been widely used and accepted as an inhibitor of CCE in nonexcitable cell types (12, 13, 14) . Nevertheless, at higher concentrations, SKF-96365 may have other, less well understood effects and should be used with caution (15 , 16) . The observation that Bcl-2-overexpressing cells are resistant to SKF-96365-induced apoptosis is novel and adds support to the idea that the protective effect of Bcl-2 is related to its modulation of Ca2+ homeostasis. This hypothesis is further supported by the observation of He et al. (9) that the antiapoptotic effect of Bcl-2 is lost at low concentrations of extracellular Ca2+, further suggesting that an adequate supply of extracellular Ca2+ is required for the antiapoptotic function of Bcl-2.

Our findings, as well as those reported by others, suggest that gating of ions across the plasma membrane is critically important to the regulation of apoptosis. Killoran and Walleczek (17) have recently shown that CCE levels are suppressed within minutes after irradiating Jurkat cells and normal human peripheral blood leukocytes with 10 Gy, further emphasizing the importance of CCE in maintaining cell viability. In addition, others have established a fundamental role for K+ ions in apoptosis (18) . Cell shrinkage, which is a very early event in the apoptotic process, can only occur after K+ efflux. Normal intracellular levels of K+ inhibit both apoptotic DNA fragmentation and caspase-3 protease activation, suggesting that intracellular K+ loss must occur early during apoptosis. In addition, there seems to be a tight coupling between cell shrinkage, K+ efflux, and changes in mitochondrial membrane potential that are independent of DNA degradation and can be largely caspase independent, depending on the particular signal transduction pathway used. Therefore, the control of K+ gating across the plasma membrane may play an integral role in the initiation of signal transduction pathways involved in apoptosis. We have reported previously that Bcl-2 overexpression is associated with a higher level of Na+/K+-ATPase activity, which could block the loss of K+ from the cell via either (a) compensation for K+ loss by pumping more K+ back into the cell or (b) establishment of a hyperpolarized membrane potential that would inhibit K+ efflux.

It is known that the influx of Ca2+ across the plasma membrane is facilitated by hyperpolarization (19) and inhibited by depolarization (20) , suggesting that the plasma membrane potential plays a crucial role in [Ca2+]i homeostasis. Therefore, plasma membrane hyperpolarization, by providing an electrochemical gradient that favors Ca2+ influx, may also help to explain the up-regulation of CCE in Bcl-2-overexpressing cells. Evidence for this is presented in Fig. 4Citation , where plasma membrane depolarization by ouabain, an inhibitor of Na+/K+-ATPase, resulted in a marked reduction in the influx of extracellular Ca2+ in both parental and Bcl-2-overexpressing cells.

The results reported here show that Bcl-2 overexpression is associated with enhanced CCE, a process that is essential for Ca2+ store refilling and cell viability. The previously reported increased Na+/K+-ATPase activity and resultant plasma membrane hyperpolarization associated with Bcl-2 may provide a mechanism of action to explain the observed enhancement in CCE. These data further establish the plasma membrane as a regulator of early signaling events in apoptotic pathways, particularly in the control of the intracellular ionic environment. These results suggest that altered membrane potential, Na+/K+-ATPase activity, and/or CCE may be important mediators of the enhanced radioresistance of Bcl-2-overexpressing tumor cells and have implications for the development of novel strategies for sensitizing these cells to radiation therapy.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by National Cancer Institute Contract Grant CA68149. Back

2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5302. Phone: (650) 723-5832; Fax: (650) 725-8231; E-mail: knox{at}reyes.stanford.edu Back

3 The abbreviations used are: [Ca2+]i, intracellular calcium concentration; CCE, capacitative Ca2+ entry; Tg, thapsigargin; ER, endoplasmic reticulum. Back

Received 2/21/00. Accepted 6/29/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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