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Originally published In Press as doi:10.1074/jbc.M201604200 on March 21, 2002
J. Biol. Chem., Vol. 277, Issue 23, 20301-20308, June 7, 2002
Bax-mediated Ca2+ Mobilization Promotes Cytochrome
c Release during Apoptosis*
Leta K.
Nutt §,
Joya
Chandra¶,
Abujiang
Pataer ,
Bingliang
Fang ,
Jack A.
Roth ,
Stephen G.
Swisher ,
Roger G.
O'Neil**, and
David J.
McConkey 
From the Departments of Cancer Biology and
¶ Pediatrics and Section of Thoracic and Molecular
Oncology, Department of Thoracic and Cardiovascular Surgery,
University of Texas M. D. Anderson Cancer Center, Houston, Texas
77030 and ** Department of Integrative Biology and
Pharmacology, University of Texas Houston Medical School, Houston,
Texas 77030
Received for publication, February 15, 2002, and in revised form, March 19, 2002
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ABSTRACT |
Previous studies have demonstrated that
Ca2+ is released from the endoplasmic reticulum (ER)
in some models of apoptosis, but the mechanisms involved and the
functional significance remain obscure. We confirmed that apoptosis
induced by some (but not all) proapoptotic stimuli was associated with
caspase-independent, BCL-2-sensitive emptying of the ER
Ca2+ pool in human PC-3 prostate cancer cells. This
mobilization of ER Ca2+ was associated with a concomitant
increase in mitochondrial Ca2+ levels, and neither ER
Ca2+ mobilization nor mitochondrial Ca2+ uptake
occurred in Bax-null DU-145 cells. Importantly, restoration of DU-145
Bax expression via adenoviral gene transfer restored ER
Ca2+ release and mitochondrial Ca2+ uptake and
dramatically accelerated the kinetics of staurosporine-induced cytochrome c release, demonstrating a requirement for Bax
expression in this model system. In addition, an inhibitor of the
mitochondrial Ca2+ uniporter (RU-360) attenuated
mitochondrial Ca2+ uptake, cytochrome c
release, and DNA fragmentation, directly implicating the mitochondrial
Ca2+ changes in cell death. Together, our data
demonstrate that Bax-mediated alterations in ER and mitochondrial
Ca2+ levels serve as important upstream signals for
cytochrome c release in some examples of apoptosis.
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INTRODUCTION |
Commitment to cell death via apoptosis appears to require the
activation of members of a family of aspartate-directed cysteine proteases known as caspases. Caspase activation is controlled in part
by changes within mitochondria that lead to the release of polypeptide
factors, including cytochrome c (1) and Smac/Diablo (2, 3),
which promote caspase activation by independent and complementary
mechanisms. Antiapoptotic members of the BCL-2 family can prevent the
mitochondrial alterations leading to factor release and caspase
activation (4-6), whereas proapoptotic members of the family promote
factor release (7-10). A subfamily of proapoptotic BCL-2 family
polypeptides may in fact be required for cytochrome c
release because mouse embryonic fibroblasts derived from
Bax / Bak / mice fail to display
cytochrome c release or downstream biochemical features of
apoptosis after exposure to a variety of death stimuli in
vitro (11). However, the biochemical mechanisms underlying the
effects of BCL-2 family polypeptides on mitochondria are still unclear.
Another large body of evidence implicates alterations in intracellular
Ca2+ homeostasis in the control of apoptosis. We
demonstrated in early studies that endogenous endonuclease activation
proceeds via a Ca2+-dependent mechanism in
thymocytes and certain other cell types exposed to a wide range of
stimuli (12). However, more recent studies indicate that alterations in
subcellular Ca2+ compartmentalization can be detected in
apoptotic cells that do not display global increases in cytosolic
Ca2+ concentration. Specifically, work by Lam et
al. (13) demonstrated that glucocorticoid-induced apoptosis was
associated with early emptying of the endoplasmic reticular
Ca2+ pool in W7MG1 mouse lymphoma cells (13). Parallel,
independent efforts by Baffy et al. (14) showed that the
same was true in interleukin-3-dependent 32D hematopoietic
progenitor cells after withdrawal of IL-3 (14). Importantly,
ER1 Ca2+ pool
depletion was not observed in cells transfected with BCL-2 (14-18),
suggesting that part of BCL-2's antiapoptotic activity involves
maintenance of the ER Ca2+ store. However, precisely how ER
Ca2+ pool depletion might contribute to the regulation of
apoptosis remains unclear.
Given the central role of mitochondria in the commitment to apoptosis,
it is possible that ER Ca2+ pool depletion triggers
secondary changes in mitochondrial Ca2+ levels that
contribute to cytochrome c release and cell death. Close
contacts exist between mitochondria and sites of ER Ca2+
release (19), such that ER Ca2+ release leads to rapid
Ca2+ accumulation within mitochondria (20, 21).
Furthermore, Hajnoczky and co-workers (22, 23) have shown that
1,4,5-inositol trisphosphate-mediated ER Ca2+ release
results in mitochondrial Ca2+ increases that play an
important role in promoting mitochondrial permeability transition and
cytochrome c release in cells undergoing apoptosis in
response to staurosporine treatment. We recently used adenoviral gene
transfer to demonstrate that apoptosis induced by overexpression of Bax
(and to a lesser extent, Bak) was associated with alterations in
the ER and mitochondrial Ca2+ stores in human PC-3 prostate
adenocarcinoma cells (24). Inhibition of mitochondrial Ca2+
uptake attenuated Bax-induced cytochrome c release and DNA
fragmentation (24), strongly suggesting that the Ca2+
fluxes participated directly in caspase activation and cell death. Whereas our study provided some of the first evidence that Bax and Bak
can affect intracellular Ca2+ stores, it did not provide
direct evidence that the effects were important for "endogenous"
signals for apoptosis, such as staurosporine, Fas engagement, or
exposure to DNA-damaging agents.
Here we characterized the effects of several well-known proapoptotic
stimuli on ER and mitochondrial Ca2+ fluxes in human PC-3
prostate adenocarcinoma cells and investigated whether or not these
fluxes played a role in promoting cytochrome c release. In
addition, we used another, Bax-deficient human prostate cancer cell
line (DU-145) to determine whether or not these Ca2+ fluxes
were dependent on cellular Bax expression. The results confirm and
extend our previous observations and demonstrate that at least two
mechanistically distinct pathways for cytochrome c release
can exist within a given cell type, one that is dependent on
ER-to-mitochondrial Ca2+ fluxes and another that is not.
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EXPERIMENTAL PROCEDURES |
Materials--
The esterified peptide caspase inhibitor Z-VAD
(O-Me) was purchased from Enzyme System Products, Inc. (Dublin, CA).
MG-132 and RU-360 were from Calbiochem. Anti-Fas antibody (CH-11; IgM) was from Kamiya Biomedical (Seattle, WA). Staurosporine, thapsigargin, and all other chemicals were obtained from Sigma.
Cell Lines and Tissue Culture--
The PC-3 human prostatic
adenocarcinoma line was obtained from American Type Culture Collection
(Manassas, VA) and maintained in RPMI 1640 medium supplemented with
10% fetal calf serum antibiotics, sodium pyruvate, and 10 mM Hepes (pH 7.4). Stable transfectants overexpressing
human BCL-2 were generated as described previously (24) and maintained
in RPMI 1640 medium supplemented with 1 mg/ml Geneticin (G418;
Invitrogen). Expression of BCL-2 was confirmed by immunoblotting with a
monoclonal anti-BCL-2 antibody (6C8; generously provided by Dr. Timothy
McDonnell, M. D. Anderson Cancer Center). Resistance to apoptosis was
also verified in cells incubated for 24-48 h with staurosporine or thapsigargin.
Adenovirus-mediated Transduction--
Construction of the
Ad/GT-Bax, Ad/GT-LacZ, and Ad/GV16 vectors was reported previously
(25). The Ad/GT-Bax vector was constructed by placing Bax cDNA
downstream of the GAL4/TATA promoter (GT) to generate the shuttle
plasmid pAd/GT-Bax. This plasmid was cotransfected into 293 cells along
with a 35-kb ClaI fragment purified from human adenoviral
type 5 to generate the Ad/GT-Bax vector. Bak or Bax gene expression can
then be induced in target tissues by coadministration of the Ad/GT-Bax
vector with the second adenoviral vector in our system, Ad/GV16 (which
produces the GAL4/GV16 fusion protein). Purified Ad/GT-Bax was obtained
by expanding the virus in 293 cells, harvesting the supernatant
of those cells, and then subjecting the supernatant to
ultracentrifugation on a cesium chloride gradient. Virus titers were
determined by optical absorbance at A260 (1 A260 unit = 1012 viral
particles/ml). The transduction efficiencies of adenoviral
vectors in various cancer cell lines were determined by infecting cells
with Ad/GT-LacZ and then determining the titers needed to transduce at
least 80% of the cells. These levels were achieved in PC-3 cells
after treatment with Ad/GT-Bax (2000 viral particles) and Ad/GV16 (1000 viral particles) and treatment with Ad/GT-LacZ (2000 viral particles)
and Ad/GV16 (1000 viral particles).
DNA Fragmentation Analysis--
We measured DNA fragmentation by
propidium iodide staining and FACS analysis as described previously
(26). Cells were harvested, pelleted by centrifugation, and resuspended
in phosphate-buffered saline containing 50 µg/ml propidium iodide,
0.1% Triton X-100, and 0.1% sodium citrate. Samples were stored at
4 °C for 16 h and vortexed before FACS analysis (BD PharMingen
FACScan; FL-3 channel).
Spectrofluorometric Caspase-3 Quantification--
Cells were
lysed in caspase-3 buffer (100 mM Hepes (pH 7.5), 10%
sucrose, 0.1% CHAPS, and 1 mM EDTA) with freshly
added 10 mM dithiothreitol, 2 mM
phenylmethylsulfonyl fluoride, and a complete mini-tablet (Roche
Molecular Biochemicals) for 1 h at 4 °C. Lysates were incubated
at 37 °C for 1 h in a total of 2 ml of caspase-3 buffer
containing 25 µM aspartate-glutamate-valine-aspartate
aminomethyl coumarin (Bachem, King of Prussia, PA). Samples were
excited at 380 nm and read at 460 nm in an RF-1501 spectrofluorometer
(Shimadzu Scientific Instruments, Columbia MD). Relative fluorescence
was calculated by subtracting the blank fluorescence (buffer plus substrate only) from the sample fluorescence and dividing by the protein content of the sample (sample blank/protein;
fluorescence units/µg protein).
Cytochrome c Release Measurements--
Release of cytochrome
c from mitochondria was measured by immunoblotting as
described previously (27). Cells were harvested by centrifugation and
gently lysed for 5 min in an ice-cold buffer containing 25 mM Tris and 5 mM MgCl2 (pH 7.4).
Lysates were centrifuged for 5 min at 16,000 × g,
supernatants were mixed with 1× Laemmli's reducing SDS-PAGE sample
buffer, and extracts from equal numbers of cells (10-20 × 106) were resolved by 15% SDS-PAGE. Polypeptides were
transferred to nitrocellulose membranes (0.2 µM;
Schleicher & Schuell), and cytochrome c was detected by
immunoblotting with the monoclonal antibody clone 7H8.2C12 (BD PharMingen).
Quantification of Intracellular Ca2+--
Cells
plated on 22 × 30-mm glass coverslips were loaded with fura-2
acetoxymethyl ester (AM) (10 µM; Molecular Probes) for 1 h at 37 °C with humidified air (5% CO2). The
coverslips were washed thoroughly with phosphate-buffered saline and
mounted on a 1.5-ml-volume chamber (with the cells facing upward). The
chamber was placed on a epifluorescence/phase-contrast microscope for Ca2+ imaging and quantification. Cells were bathed in 1 ml
of phosphate-buffered saline without Ca2+ at room
temperature. Identical results were obtained when cells were bathed in
Hanks' balanced salt solution containing 0.5 mM EGTA.
After a baseline cytosolic Ca2+ concentration
([Ca2+]i) was established, cells were then
treated with thapsigargin (5 µM) to empty
[Ca2+]ER stores. An INCA work station
(Intracellular Imaging, Inc.) was used to quantify
[Ca2+]i levels based on fura-2
fluorescence. The INCA software allowed for subtraction of background
fluorescence. Fluorescence was monitored using a ×20 fluorescence
objective. Cells were illuminated at alternating excitation
wavelengths of 340 and 380 nm using a xenon arc lamp. The
emitted fluorescence was monitored at 511 nm with a video camera, and
the calculated free [Ca2+]i was determined using
the cell-free calibration curve. The data were collected with INCA
software (Win 3.1 version).
Spectrofluorometric Analysis of Mitochondrial
Ca2+--
Cells were pelleted and resuspended in 5 ml of
RPMI 1640 medium containing 0.2% fetal bovine serum. Rhod-2 AM (50 µg; Molecular Probes) was diluted to 0.5 µg/ml in
Me2SO. Cells were loaded with Rhod-2 AM for 45 min, and the
washed cells were analyzed in a spectrofluorometer (PerkinElmer Life
Sciences model LS 50 B) at 550 nm excitation and 578 nm emission.
Analysis of washed, MitoTracker-counterstained cells by confocal
microscopy confirmed that the vast majority of Rhod-2 fluorescence was
associated with mitochondria. Furthermore, preincubation with the
mitochondrial uncoupler CCCP reduced fluorescence levels to baseline
and completely blocked the increases in fluorescence normally observed
after stimulation with thapsigargin. To obtain fluorescence maxima and
minima, cells were sequentially incubated with detergent and EGTA (10 mM, final concentration) in the presence of saturating
concentrations of extracellular Ca2+. No differences in
minimum Ca2+-dependent fluorescence were
observed when CCCP was substituted for EGTA in these studies.
Intramitochondrial Ca2+ concentrations were calculated by
the following formula: [Ca2+] = (F Fmin/Fmax - F) × Kd (Rhod-2) where F = fluorescence.
Statistical Analyses--
Values are the means ± S.E. The
number of experiments is shown in the legend of each figure.
Statistical analysis was performed by analysis of variance with
Neuman-Keuls post hoc comparison.
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RESULTS AND DISCUSSION |
Previous work has shown that certain proapoptotic stimuli cause
depletion of the endoplasmic reticular Ca2+ store (13-16).
To determine the prevalence and kinetics of this phenomenon, we treated
human PC-3 prostate adenocarcinoma cells with several common
death-inducing agents and measured their effects on ER Ca2+
levels. Cells were preincubated with various death-promoting stimuli
and loaded with the Ca2+-sensitive fluorescent dye fura-2
AM, and ER Ca2+ content was measured indirectly by
releasing the pool with the ER Ca2+ ATPase inhibitor
thapsigargin. Representative fluorescence traces obtained using this
technique in cells treated with staurosporine or the cancer
chemotherapeutic agent Adriamycin (doxorubicin) are presented in Fig.
1A, and quantitative data
obtained in several experiments are presented in Fig. 1B.
Both staurosporine and Adriamycin promoted ER Ca2+ release
several hours before cytochrome c release was first
detected, but the proteasome inhibitor MG-132 and the anti-Fas antibody CH-11 did not (Fig. 1B). Thus, ER Ca2+ release
is associated with apoptosis induced by some (but not all) proapoptotic
stimuli.

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Fig. 1.
Depletion of the ER Ca2+ store is
associated with apoptosis induced by some stimuli. A,
quantification of ER Ca2+ in PC-3 cells exposed to
staurosporine or Adriamycin. Cells were incubated with 1 µM staurosporine or 1 µg/ml Adriamycin for the times
indicated. One h before analysis, cells were loaded with 5 µM fura-2 AM and transferred to Ca2+-free
medium, and thapsigargin-induced Ca2+ increases were used
as an indirect measure of ER Ca2+ content as described
previously (13, 52). Representative traces (10-20 cells/trace) are
shown. The arrow indicates the time point of thapsigargin
(TG) addition. B, quantification of ER
Ca2+ content in cells exposed to various stimuli. Cells
were incubated for the times indicated with 1 µM
staurosporine, 1 µg/ml doxorubicin, 10 µM MG-132, or 1 µg/ml anti-Fas antibody (CH-11), and ER Ca2+ content was
measured as described above. Mean ± S.E.; n = 3-13. Significant ER Ca2+ depletion (*,
p < 0.001) was observed in staurosporine-treated cells
after 2 h and in Adriamycin-treated cells after 8 h.
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The commitment phase of apoptosis involves activation of the caspase
family of cysteine proteases. To determine whether or not ER
Ca2+ pool depletion was a consequence of caspase
activation, we measured staurosporine-induced pool depletion in cells
preincubated with the pan-caspase inhibitor zVADfmk (20 µM). The inhibitor had no effect on ER Ca2+
release (Fig. 2B), although
control experiments confirmed that it completely blocked caspase
activation (measured using a fluorogenic dye; Fig. 2B).
These results confirm that ER Ca2+ release occurs upstream
of caspase activation.

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Fig. 2.
Effects of the caspase inhibitor zVADfmk on
staurosporine-induced changes in ER Ca2+ and caspase
activation. A, effects on ER Ca2+ pool
depletion. PC-3 cells were exposed to 1 µM staurosporine
with or without 20 µM zVADfmk for 4 h. Cells were
then loaded with fura-2 AM, and ER Ca2+ pool content was
measured by exposing cells to thapsigargin (5 µM) in
Ca2+-free medium. Mean ± S.D.; n = 3. *, p < 0.001 versus control.
B, effects of zVADfmk on staurosporine-induced caspase
activation. Cells were exposed to 1 µM staurosporine
without ( ) or with ( ) 20 µM zVADfmk. At the times
indicated, cytosolic extracts were prepared (1 × 106
cell equivalents), and caspase-3-like activity was quantified by
monitoring hydrolysis of a peptide substrate
(aspartate-glutamate-valine-aspartate aminomethyl coumarin) in a
spectrofluorometer. Mean ± S.E.; n = 3. *,
p < 0.01 versus control.
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As described above, previous studies have shown that BCL-2 localizes to
the ER, where it may regulate ER Ca2+ homeostasis. To test
the effects of BCL-2 in our model system, we stably transfected the
PC-3 cells with BCL-2 and characterized their responses to
staurosporine. Immunoblotting confirmed that the transfectants
expressed high levels of BCL-2 (Fig.
3A), and functional studies
demonstrated that staurosporine-induced DNA fragmentation was reduced
in the transfectants (Fig. 3B). Consistent with some of the
earlier studies, staurosporine failed to induce ER Ca2+
pool depletion in the BCL-2 transfectants (Fig. 3C), an
effect that was associated with reduced staurosporine-stimulated
cytochrome c release from mitochondria (Fig.
3D).

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Fig. 3.
Effects of BCL-2 on staurosporine-induced
mobilization of ER Ca2+. A, expression
of BCL-2 in two representative stable transfectants (BCL-2.3 and
BCL-2.9). BCL-2 expression was determined by immunoblotting (triplicate
experiments). B, effects of BCL-2 on
staurosporine-induced DNA fragmentation. Cells were exposed to 1 µM staurosporine for the indicated times, and DNA
fragmentation was quantified by propidium iodide staining and FACS
analysis. Mean ± S.E.; n = 3 (*,
p < 0.01). C, effects of BCL-2 on
staurosporine-induced ER Ca2+ release. ER Ca2+
content was estimated by thapsigargin release in cells incubated in
Ca2+-free medium as described in Fig. 1. Mean ± S.E.;
n = 3-13 (*, p < 0.001).
D, effects of BCL-2 on staurosporine-induced cytochrome
c release. Cells were incubated with 1 µM
staurosporine for the times indicated, and cytochrome c was
measured in cytosolic extracts by immunoblotting. The results shown are
from one experiment that was representative of four independent
replicates.
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Recent studies have shown that pseudosynaptic contacts exist between
the ER and mitochondrial Ca2+ pools (19), such that release
of Ca2+ from ER stores can result in rapid Ca2+
uptake by mitochondria (20, 21). We therefore investigated the effects
of staurosporine on mitochondrial Ca2+ levels in PC-3 cells
loaded with the mitochondrial Ca2+ probe Rhod-2 AM;
representative fluorescence traces obtained using this technique are
presented in Fig. 4A.
Ca2+-independent (background) fluorescence was measured by
treating cells with the protonophore CCCP, which completely releases
the mitochondrial Ca2+ pool, and levels of this background
fluorescence were nearly indistinguishable in the cells under all
conditions examined (Fig. 4A). Control experiments also
confirmed that all of the Rhod-2 was confined to mitochondria. Exposure
of PC-3 cells to staurosporine resulted in a significant increase in
mitochondrial Ca2+ concentration that was completely
abolished by overexpression of BCL-2 (Fig. 4B). To
investigate the relationship between mitochondrial Ca2+
uptake and cytochrome c release, we compared the levels of
cytochrome c release observed in staurosporine-treated cells
in the absence or presence of a chemical inhibitor of the mitochondrial
Ca2+ uniporter (RU-360). A representative immunoblot
obtained in these experiments is shown in Fig. 4C, and
quantitative results obtained in multiple experiments are presented in
Fig. 4D. RU-360 significantly reduced staurosporine-induced
cytochrome c release (Fig. 4, C and D)
and DNA fragmentation (Fig. 4F), but it had no effect on Fas-mediated cytochrome c release (Fig. 4E).
Together, these results demonstrate that mitochondrial Ca2+
uptake is important for staurosporine-induced apoptosis and confirm that Fas-mediated apoptosis occurs via Ca2+-independent
mechanisms, at least in the PC-3 model system.

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Fig. 4.
Effects of staurosporine on mitochondrial
Ca2+ levels. A, representative fluorescence
traces. Cells were exposed to 1 µM staurosporine
(ST) for 4 h in the absence or presence of 10 µM RU-360. Cells were then loaded with Rhod-2 AM, and
mitochondrial Ca2+ was estimated based on Rhod-2
fluorescence as described under "Experimental Procedures." After
baseline fluorescence levels were obtained, cells were exposed to CCCP
to empty mitochondrial Ca2+ stores. RFU,
relative fluorescence intensity (arbitrary units). B,
effects of BCL-2 on staurosporine-induced increases in mitochondrial
Ca2+. Parental PC-3 cells or BCL-2 transfectants were
incubated with 1 µM staurosporine for 4 h. Cells
were loaded with Rhod-2 AM, and mitochondrial Ca2+ was
measured in a spectrofluorometer. Once baseline fluorescence values
were obtained, cells were exposed to CCCP to obtain a fluorescence
minimum and then lysed in the presence of extracellular
Ca2+ to obtain a fluorescence maximum. Raw fluorescence
values were then used to derive Ca2+ concentrations using
the Rhod-2 Ca2+ dissociation constant essentially as
described previously (24). Mean ± S.E.; n = 4. C and D, RU-360 attenuates
staurosporine-induced but not anti-Fas-induced cytochrome c
release. Cells were preincubated with the indicated concentrations of
RU-360 for 30 min. Cells were then incubated with or without 1 µM staurosporine for 8 h, and cytosolic cytochrome
c was analyzed by immunoblotting. Results of densitometric
quantification of cytochrome c release in cells treated with
1 µM staurosporine (D) or 1 µg/ml anti-Fas
antibody (CH-11) (E) with or without RU-360 are shown. Note
that 10 µM RU-360 inhibited thapsigargin-induced
mitochondrial Ca2+ uptake by 50% in separate experiments
(Fig. 6). Mean ± S.D.; n = 3. F,
inhibition of DNA fragmentation by RU-360. PC-3 cells were preincubated
with 10 µM RU-360 for 30 min before exposure to 1 µM staurosporine, and DNA fragmentation was quantified by
propidium iodide staining and FACS analysis at 12 h. Mean ± S.E.; n = 3. *, p < 0.01 versus control.
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We recently showed that overexpression of Bax via adenoviral gene
transfer resulted in early, caspase-independent depletion of the ER
Ca2+ pool and apoptosis (24). These observations suggested
to us that staurosporine's effects on the ER and mitochondrial
Ca2+ pools might be mediated by Bax. To directly test this
hypothesis, we characterized the effects of staurosporine on ER (Fig.
5A) and mitochondrial (Fig.
5B) Ca2+ pools in the Bax-deficient human
prostate cancer cell line DU-145. Consistent with the hypothesis,
staurosporine failed to induce significant ER Ca2+ pool
depletion or mitochondrial Ca2+ uptake in the parental
DU-145 cells. Importantly, reconstitution of Bax expression via
adenoviral gene transfer restored staurosporine's effects on the ER
(Fig. 5A, right bars) and mitochondrial (Fig. 5B, right bars) Ca2+ pools and
dramatically accelerated the kinetics of staurosporine-induced cytochrome c release (Fig. 5C). Because
mitochondrial Ca2+ uptake appears to be involved in
promoting cytochrome c release and downstream features of
apoptosis, these Bax-mediated Ca2+ changes appeared to
directly promote apoptotic cell death.

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Fig. 5.
A, staurosporine-induced ER
Ca2+ depletion is Bax-dependent. Left
bars, control DU-145 cells were incubated in medium alone or
with 1 µM staurosporine for 8 h, and ER
Ca2+ levels were measured by thapsigargin release as
described in Fig. 1. Mean ± S.E; n = 4-13.
Right bars, DU-145 cells were transduced with
adenoviral -galactosidase (Ad- -Gal) or adenoviral Bax (Ad-Bax)
for 10 h as outlined under "Experimental Procedures." Cells
were then exposed to 1 µM staurosporine for 1 h,
and ER Ca2+ content was measured by thapsigargin release as
described in Fig. 1. Mean ± S.E.; n = 4-13. *,
p < 0.001 versus control. Although ER
Ca2+ levels were similar to those of controls at 10 h,
note that prolonged (18 h) accumulation of Bax results in ER
Ca2+ pool depletion, consistent with our earlier
observations (24). An adenoviral control ( -galactosidase) had no
effect on staurosporine-induced Ca2+ fluxes.
B, Bax is required for staurosporine-induced
mitochondrial Ca2+ increases. Left bars,
Bax / DU-145 cells were exposed to staurosporine for
4 h, and mitochondrial Ca2+ concentrations were
quantified using Rhod-2 AM as described in Fig. 2. Mean ± S.E.;
n = 4-13. Right bars, transduction
with Bax restores staurosporine-induced mitochondrial Ca2+
fluxes. DU-145 cells were transduced with a control vector
(Ad- -Gal) or Ad-Bax for 10 h. Cells were then exposed to
staurosporine for 1 h, and mitochondrial Ca2+ levels
were quantified using Rhod-2 as described above. A statistically
significant increase in mitochondrial Ca2+ (*,
p < 0.001 versus controls) was observed
only in the staurosporine-treated DU-145 cells transduced with Bax.
Note that Bax itself induced increases in mitochondrial
Ca2+ at 12 h (*, p < 0.01),
consistent with our previous findings (24). C, Bax
accelerates staurosporine-induced cytochrome c release
in DU-145 cells. Cells transduced with Bax (10 h after infection) were
exposed to staurosporine for 45 min, and cytochrome c
release, Bax expression, and an internal loading standard (actin) were
measured by immunoblotting. The results shown are from one experiment
that was representative of three replicates.
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Finally, we turned our attention to the paradoxical observation that
staurosporine stimulated apoptosis more rapidly than the
Ca2+-mobilizing agent thapsigargin, even though the effects
of thapsigargin on intracellular Ca2+ fluxes were more
rapid than those of staurosporine. Specifically, thapsigargin caused
immediate ER Ca2+ release and subsequent RU-360-sensitive
mitochondrial Ca2+ uptake in PC-3 cells (Fig.
6A), but it did not promote
cytochrome c release (Fig. 6C) or DNA
fragmentation (Fig. 6B) until much later (>18 h after
exposure). These kinetics of cytochrome c release and DNA
fragmentation are consistent with previous findings (11, 28-30). These
data suggested to us that staurosporine's effects on mitochondrial
Ca2+ might cooperate with other effects (i.e.
pore formation and interaction with the permeability transition pore
complex) to promote cytochrome c release. To directly test
this possibility, we measured thapsigargin-induced cytochrome
c release in PC-3 cells that had been pretreated with staurosporine for 4 h. At this time point,
staurosporine-induced ER Ca2+ release was submaximal (Fig.
1, A and B), and no cytochrome c release had occurred (Fig. 6D). Thapsigargin induced a
nearly immediate release of cytochrome c (within 5-15 min)
in the staurosporine-pretreated cells (Fig. 6D), confirming
that staurosporine sensitized mitochondria to efflux of
Ca2+ from the ER. The mechanisms probably involved direct
effects of Bax and/or other proapoptotic members of the BCL-2 family
because previous work has established that Bax translocates to
mitochondria in response to staurosporine exposure (31-33). In
addition, exogenous Bax sensitizes mitochondria to
Ca2+-mediated cytochrome c release, but
Ca2+-independent effects of Bax are also involved (24,
34).

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Fig. 6.
Thapsigargin-induced apoptosis is slower than
staurosporine-induced apoptosis in PC-3 cells. A,
effects of thapsigargin on mitochondrial Ca2+ levels. PC-3
cells were loaded with Rhod-2 AM as described above. After baseline
fluorescence levels were obtained, cells were exposed to 1 µM thapsigargin in the absence or presence of 10 µM RU-360. The results demonstrated that thapsigargin
causes an immediate increase in mitochondrial Ca2+ level
that is inhibited by the mitochondrial Ca2+ uniporter
antagonist (RU-360). B, time course of
thapsigargin-induced DNA fragmentation. PC-3 cells were exposed to 1 µM thapsigargin for the times indicated, and DNA
fragmentation was quantified by propidium iodide staining and FACS
analysis. Mean ± S.E.; n = 3. *,
p < 0.001 versus controls.
C, time course of thapsigargin-induced cytochrome
c release. PC-3 cells were exposed to thapsigargin for the
times indicated. Cytosolic extracts were then prepared, and cytochrome
c content was determined in these extracts by
immunoblotting. The results shown are from one experiment that was
representative of three independent replicates. D,
staurosporine sensitizes PC-3 cells to Ca2+-induced
cytochrome c release. PC-3 cells were preincubated with 1 µM staurosporine for 4 h. Cells were then pulsed
with 1 µM thapsigargin for the times indicated, and
cytosolic cytochrome c was measured by immunoblotting. Cells
exposed to thapsigargin for 4 h in the absence of staurosporine
served as controls. The results shown are from one experiment that was
representative of three independent replicates.
|
|
Whereas our results establish a novel role for Bax-mediated
Ca2+ changes in apoptosis, they do not identify the
biochemical mechanisms involved in the effects of Bax on
Ca2+. Previous studies have shown that Bax can form
transmembrane ion channels in planar lipid bilayers (Refs. 35-38;
reviewed in Ref. 39) and tetramerize in response to proapoptotic
signals to form larger pores capable of directly accommodating
cytochrome c release (40, 41). Thus, one simple explanation
for our data would be that Bax forms transmembrane cation
(i.e. Ca2+) channels that allow for
Ca2+ to flow down its concentration gradient from the ER
into mitochondria. However, previous studies have shown that the
channels initially formed by Bax in lipid bilayers are selective for
monovalent anions (Cl ) rather than divalent cations such
as Ca2+ (39). Thus, it is possible that Bax must assemble
into higher order structures to exert its effects on ER and
mitochondrial Ca2+ and that its
Ca2+-independent effects on mitochondria involve some other
activity of the protein. The latter may be explained by previous
studies that demonstrated that proapoptotic BCL-2 family members
interact with components of the permeability transition pore (8, 9), which is a Ca2+-sensitive, mitochondrial polypeptide
complex that has been implicated in some pathways of cytochrome
c release by other investigators (42).
Recent work by others has demonstrated that Bax and Bak play redundant
roles in promoting cytochrome c release in mouse embryonic fibroblasts (11), and our own previous findings demonstrated that both
Bax and Bak can promote ER Ca2+ pool emptying and
mitochondrial Ca2+ uptake (24). We were therefore surprised
that staurosporine failed to cause alterations in the ER or
mitochondrial Ca2+ pools in DU-145 cells because these
cells retain wild-type Bak. We did confirm that staurosporine-induced
Ca2+ fluxes were attenuated in mouse embryonic fibroblasts
derived from Bax / mice (data not shown). However, it
was difficult to obtain satisfactory Ca2+ dye loading in
the cells, and our failure to detect residual Bak-mediated
Ca2+ changes may have been limited by the lower sensitivity
of our methods in the cells. We have no reason to expect that other
"multidomain" proapoptotic BCL-2 family members are incapable of
mediating effects on Ca2+ similar to those described here,
although our experience with Bax and Bak in PC-3 cells suggests that
Bax is significantly more potent (24). It is possible that the
involvement of Ca2+ fluxes in cytochrome c
release varies not only with the proapoptotic stimulus in question but
also with cellular background because human prostate cancer cells are
notoriously sensitive to Ca2+-mediated apoptosis.
Although BCL-2 did not affect steady-state levels of Ca2+
in the ER or mitochondria in the PC-3 cells, it did inhibit the effects of staurosporine on ER Ca2+ release and subsequent
mitochondrial Ca2+ uptake. These observations suggest that
BCL-2 acts primarily to antagonize the effects of proapoptotic stimuli
in our cells and does not exert direct effects of its own on either
Ca2+ pool. It should be stressed, however, that other
laboratories have reported different effects of BCL-2 on intracellular
Ca2+ pools. For example, at least three groups have shown
that BCL-2 lowers the steady-state level of Ca2+ within the
ER, which they argued inhibits apoptosis by reducing Ca2+
efflux across the ER membrane (15, 18, 43). In contrast, another group
showed that overexpression of BCL-2 increased steady-state levels of ER
Ca2+ (44). On the other hand, Murphy et al. (17)
concurred that BCL-2 had no significant effect on steady-state
Ca2+ levels in their cells, but they found that it
potentiated Ca2+ uptake by mitochondria, an observation
that stands in opposition to our results. Although we cannot explain
these contrasting findings at present, they are probably related to the
use of different cellular model systems; hematopoietic cells and
excitable cells (neurons) are likely to regulate intracellular
Ca2+ compartmentalization very differently from epithelial
cells. Furthermore, recent studies have demonstrated that high and low levels of BCL-2 can exert different effects on cells (45, 46). It is
therefore possible that we would have observed direct effects of BCL-2
on ER and/or mitochondrial Ca2+ pools in PC-3 cells
expressing different levels of BCL-2 protein. BCL-2 is capable of
interacting with a variety of different proteins, some of which are
involved in Ca2+-associated signal transduction
(i.e. the Ca2+-dependent protein
phosphatase calcineurin) (47), and BCL-2's activity is regulated by
phosphorylation (48-51). It therefore appears likely that cellular
context dictates precisely how BCL-2 will influence intracellular
Ca2+ pools. Additional efforts are required to more
precisely define the biochemical mechanisms involved in the regulation
of intracellular Ca2+ compartmentalization by the different
members of the BCL-2 family.
 |
FOOTNOTES |
*
This work was supported in part by grants from the National
Institutes of Health/National Cancer Institute (Grant CA69676 to
D. J. M., Grant PO1-CA78778-O1A1 to J. A. R. and S. G. S., and
Grant 2P50-CA70970-04 to J. A. R.). Additional funding was provided
by a grant from the Tobacco Settlement Funds as appropriated by the
Texas State Legislature (Project B) and The W. M. Keck Foundation.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.
§
Supported by National Institutes of Health/National Cancer
Institute Cancer Immunobiology Training Grant CA09598.

To whom correspondence should be addressed: Dept. of Cancer
Biology (173), 1515 Holcombe Blvd., Houston, TX 77030. Tel.:
713-792-8591; Fax: 713-792-8747; E-mail:
dmcconke@mdanderson.org.
Published, JBC Papers in Press, March 21, 2002, DOI 10.1074/jbc.M201604200
 |
ABBREVIATIONS |
The abbreviations used are:
ER, endoplasmic
reticulum;
FACS, fluorescence-activated cell-sorting;
AM, acetoxymethyl
ester;
CCCP, carbonyl cyanide m-chlorophenyl hydrazone;
zVADfmk, z-valine-alanine-aspartate fluoromethyl ketone;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid.
 |
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Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
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