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RAPID COMMUNICATION
Blood Cells With Reduced Mitochondrial Membrane Potential
and Cytosolic Cytochrome C Can Survive and Maintain Clonogenicity
Given Appropriate Signals to Suppress Apoptosis
By Quan Chen, Naoshi Takeyama, Ged Brady, Alastair J.M. Watson, and Caroline Dive
Reduction of mitochondrial membrane potential (Cm) and
release of cytochrome c from mitochondria appear to be key
events during apoptosis. Apoptosis was induced in IC.DP
premast cells by the withdrawal of interleukin-3 (IL-3). Cm
decreased by 12 hours and cytochrome c was detected in the
cytosol at 18 hours. Despite these changes in the mitochondria after 18 hours of IL-3 deprivation, clonogenicity was
unaffected when IL-3 was replenished at 18 hours. Activation
of v-Abl tyrosine kinase (v-Abl TK) in IC.DP cells before IL-3
depletion led to increased levels of Bcl-XL, prevented reduction of Cm and the release of mitochondrial cytochrome c,
and suppressed apoptosis. Activation of v-Abl TK 18 hours
after withdrawal of IL-3 when I10% of the cells had died
restored Cm in the remaining cells. More than 40% of cells
thus rescued by v-Abl TK between 18 and 42 hours could
subsequently form colonies in the presence of IL-3. These
data suggest that reduction in Cm precedes loss of mitochondrial cytochrome c in IC.DP cells; that v-Abl TK activation,
probably via upregulation of Bcl-XL, prevents loss of Cm and
blocks the release of cytochrome c from mitochondria; and
that neither of these mitochondrial events is sufficient for
commitment to apoptosis.
r 1998 by The American Society of Hematology.
A
through the mitochondrial intermembrane space remains undefined. A proposed common mechanism for Bcl-2 and other
antiapoptotic family members is that they either block the efflux
pathway of cytochrome c from mitochondria or its binding to
apaf-1 or both.8 In this context, it is worthwhile noting that,
under special conditions, Bcl-XL can form pores in artificial
lipid bilayers13,14; however, the relevance of this to its antiapoptotic function remains to be formally demonstrated.
Another mitochondrial event that has been demonstrated to
be an early event in apoptosis in a large number of cell systems
is loss of electrical potential across the inner mitochondrial
membrane (Cm).3,15 The reduction in Cm is thought to be due to
the opening of a Ca 21-activated, ADP-inhibited, and voltagedependent megachannel in mitochondria giving rise to the
mitochondrial permeability transition (PT) that facilitates mitochondrial swelling. The functional relevance of the induction of
PT to apoptosis is implied by observations that apoptosis can be
inhibited by cyclosporin A or bongrekic acid, compounds that
inhibit PT. The relationship between loss of mitochondrial
cytochrome c and loss of Cm is currently unclear. In HL60
myeloid leukemia cells, cytochrome c is released before a
reduction in Cm,5 whereas the reverse kinetics have been
observed in the liver.16 Moreover, during Fas-driven apoptosis
of Jurkat T cells, cytochrome c appears to be inactivated but not
lost from mitochondria.17 Furthermore, Brunet et al18 showed in
POPTOSIS IS A GENETICALLY regulated cell death
process, initially defined by distinctive morphological
criteria1 and subsequently characterized by the involvement of a
number of cell death-associated genes.2 Although induced by a
wide range of disparate stimuli, the final apoptotic response of
the cell is relatively stereotypic involving cell shrinkage,
chromatin condensation, nonrandom DNA fragmentation, and
selective proteolysis by a series of cysteine proteases collectively named caspases. This strongly implies that a common
biochemical effector mechanism mediates the latter stages of
apoptosis regardless of type of stimulus. An important question
that we address here is the identity of the commitment event(s)
that engages the execution machinery. Because mitochondria
are believed to play an important role in the induction of
apoptosis,3 we wish to examine whether changes in mitochondria exemplified by the release of cytochrome c from mitochondria and a reduction in Cm are sufficient for commitment to
death.
After exposure to a variety of stimuli that induce apoptosis,
the electron transport protein cytochrome c is released from the
mitochondria into the cytosol,4,5 where it binds apoptosis
protease activating factor-1 (apaf-1), a newly characterized
130-kD protein that shares homology with CED-4, the Caenorhabditis elegans death gene.6,7 As well as acting as an adaptor
protein for cytochrome c (also termed apaf-2), it is suggested
that apaf-1 may also bind to dATP and a so-far uncharacterized
protein called apaf-3 (reviewed in Vaux8). This putative molecular complex is thought to trigger the cleavage of the inactive
precursor of caspase 3 yielding active caspase 3, which in turn
activates a cascade of other caspases and other molecules,
including the DNA fragmentation factor DFF,9,10 the executionary machinery of apoptotic cell death.
Bcl-2 suppresses apoptosis and can block the release of
cytochrome c from mitochondria and prevent the activation of
caspase-3.5,11 The Bcl-2 homolog Bcl-XL also prevents the
accumulation of cytosolic cytochrome c, possibly by binding
directly to it.12 Bcl-2 and certain other Bcl-2 family members,
including Bcl-XL, are tethered to the outer mitochondrial
membrane, as well as to the endoplasmic reticulum (ER) and
nuclear membranes.2 The efflux pathway of cytochrome c from
its loose connection to the inner mitochondrial membrane
Blood, Vol 92, No 12 (December 15), 1998: pp 4545-4553
From the School of Biological Sciences and the Department of
Medicine, Victoria University of Manchester, Manchester, UK.
Submitted July 27, 1998; accepted October 6, 1998.
Supported by a Medical Research Grant to C.D. and A.J.M.W. C.D. is
a Lister Institute Research Fellow. N.T. is an Honorary Visiting
Research Fellow from Kansai Medical University (Osaka, Japan).
Address reprint requests to Caroline Dive, PhD, School of Biological
Sciences, Stopford Building G38, Victoria University of Manchester,
Oxford Road, Manchester M13 9PT, UK; e-mail: [email protected].
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9212-0047$3.00/0
4545
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4546
CHEN ET AL
a human lymphoblastic cell line (CEM C7A) that, during
dexamethasone-induced apoptosis, a reduction in Cm is downstream of commitment to apoptosis. Taken together, these data
suggest that the precise order of a reduction in Cm, the release of
cytochrome c from mitochondria, and commitment to apoptosis
can vary with cell type and apoptotic stimulus.
We address here the question of whether loss of Cm and
release of cytochrome c from mitochondria are reversible or
irreversible events in terms of commitment to apoptosis in
IC.DP pre-mast cells deprived of interleukin-3 (IL-3). In
particular, we sought to determine whether cells receiving an
apoptotic stimulus and that had reduced their Cm and contained
cytosolic cytochrome c could subsequently clone given appropriate survival stimuli. To approach these questions, we have
exploited the IL-3–dependent murine pre-mast cell line IC.DP
that contains a temperature-sensitive mutant of v-Abl tyrosine
kinase (v-Abl TK).19 Activation of v-Abl TK does not promote
cell proliferation, but results in the upregulation of Bcl-XL20 and
suppresses apoptosis induced by IL-3 withdrawal.21
MATERIALS AND METHODS
Unless stated, all materials were from Sigma (Poole, UK). Sucrose,
KCl, MgCl2, and HEPES were from Boehringer Mannheim BDH
Laboratories (Mannheim, Germany). Murine cytochrome c monoclonal
antibody was from Pharmingen (San Diego, CA). Cytochrome a
monoclonal antibody, DiOC(6)3, nonyl acridine orange (NAO), and
carbonyl cyanide m-chlorophenyl-hydrazone (mCCCP) were from
Molecular Probes, Inc (Eugene, OR). Annexin V apoptosis detection kit
is from R&D Systems (Oxford, UK). Murine IgG2 anti-Bcl-XL antibody
was from Transduction Laboratories (Lexington, KY).
Flow Cytometry
All studies were performed using a Becton Dickinson FacsVantage
with a Enterprise laser (Becton Dickinson [BD], Palo Alto, CA).
Excitation was at 250 mW using the 488 nm laser line. Cells were
examined at a flow rate of 200 to 300 events per second, and 10,000
events were analyzed per sample. Cellular debris and, in certain
instances, cells already undergoing apoptosis (with reduced forward and
increased orthogonal light scatter) were excluded from the analysis. All
data were analyzed using Lysis II software (BD).
Apoptosis. Apoptotic cells expose phosphatidyl serine at their
plasma membrane and this can be detected using fluorescein isothiocyanate (FITC)-conjugated antibodies to annexin V. Apoptosis was measured using the annexin V-based R&D Systems detection kit as directed
by the manufacturer. Green fluorescence of annexin V staining was
collected at 530 6 30 nm and red fluorescence due to DNA bound
propidium was collected at 630 6 22 nm.
Mitochondrial membrane potential. The Cm indicator DiOC(6)3 (2
µL of 2 µmol/L stock solution in dimethyl sulfoxide [DMSO]) was
added by hamilton syringe to 0.4 mL IC.DP cell suspension (4 3 105
cells/mL) in fresh Fischer’s medium (pH 7.2) and incubated at room
temperature for 5 minutes. A change in DiOC(6)3 fluorescence indicates
a change that could represent a change in Cm and/or a change in
mitochondrial mass. Therefore, parallel experiments were performed
using mitochondrial-specific dye NAO to determine mitochondrial
mass. NAO (100 nmol/L, final concentration) was added to cell samples
as described above. In both types of assay, propidium iodide (PI; 4 µL of
1 mg/mL stock) was added 30 seconds before analysis. DiOC(6)3
fluorescence or NAO fluorescence was collected at 530 6 30 nm.
DiOC(6)3 data were validated by addition of 20 µmol/L mCCCP after 5
minutes of DiOC(6)3 loading. Median values of green fluorescence from
the subpopulation of cells that were negative for red fluorescence were
determined. Comparative experiments were performed on the same day
and the data were normalized against the 0-hour time point.
Clonogenic Assay
Cell Culture
The hematopoietic cell line IC2.9 is an IL-3–dependent murine
pre-mast cell line that has been stably transfected with a temperaturesensitive mutant of v-Abl TK to generate the IC.DP subclone.19 v-Abl
TK is active at 32°C but inactive at 39°C. IC.DP and IC2.9 cells were
cultured in Fischer’s medium (Life Technology, Paisley, Scotland)
supplemented with 10% horse serum and 3% X60-Ag-653 cellconditioned medium containing IL-3.22 IL-3 withdrawal-mediated
induction of apoptosis was performed as described previously.21 Briefly,
cells were incubated for 18 hours at 39°C to ensure that v-Abl TK was
inactivated. Cells were then incubated for 2 hours at 32°C to activate
v-Abl TK or were maintained at 39°C for 2 hours with inactive v-Abl
TK. After extensive washing in Fischer’s medium containing only
glutamine and antibiotics, cells were resuspended at 1 3 106/mL in this
medium and reincubated at 32°C or 39°C. This time point, after IL-3
withdrawal, is referred to as 0 hours in the text and figures. IC2.9 cells
were treated using an identical protocol and were included to control for
temperature effects. None of the results presented was due to temperature, but rather due to the activation status of v-Abl TK.
Clonogenic assays were performed to determine whether v-Abl TK
activity could protect cells from apoptosis and that cells rescued in this
way could subsequently proliferate if a mitogenic stimulus were
subsequently applied. Cellular clonogenicity was measured as previously described.25 Briefly, cells were incubated for various periods of
time in the absence of IL-3 and serum with v-Abl TK either active or
inactive. Subsequently, single-cell suspensions were serially diluted to 1
or 2 cells per well in fresh Fischer’s medium supplemented with 10%
horse serum and 3% IL-3 containing medium plus glutamine and
antibiotics. This cell suspension was then aliquotted into a U-shaped
96-well plate (Costar, Cambridge, UK), with each well containing 100
µL of medium. Cell colonies were counted 14 days after initial plating
and the percentage of cells that were clonogenic was calculated by
ratioing the theoretical Poisson density for the number of negative wells
observed, against the initial cell plating density, that is, % Clonogenicity
5 (ln 3 [96/NegWells])/(Plate Density) 3 100, where NegWells is the
number of wells that have failed to grow colonies and Plate Density is
the original cell plating density per well. IL-3–replete IC.DP cells have
a absolute cloning efficiency of 40%.
Cell Fractionation
Measurement of Cell Death
The mode of cell death upon depletion of IL-3 has been extensively
studied in IC.DP cells and confirmed as being apoptosis using various
techniques.21,23,24 In this study, we routinely examined cell viability by
trypan blue exclusion. In selected studies, flow cytometric analysis of
Annexin V staining simultaneously with uptake of propidium to detect
increased plasma membrane permeability was used (see flow cytometry
section below).
Cells (1 3 107) were washed once with Fischer’s Medium and then
suspended in ice-cold mitochondria isolation buffer containing 20
mmol/L HEPES-KOH, 100 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L
EGTA, 250 mmol/L sucrose plus protease inhibitors phenylmethyl
sulfonyl fluoride (PMSF; 1 mmol/L), aprotinin (10 µg/mL), leupeptin
(10 µg/mL), pepstatin (10 µg/mL), and dithiothreitol (1 mmol/L). Five
volumes of this buffer was added to the cell pellet and left on ice for 20
minutes. The cell suspension was then homogenized with a dounce
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MITOCHONDRIAL EVENTS AND COMMITMENT TO APOPTOSIS
homogenizer (15 to 25 strokes) and the homogenate was centrifuged at
750g for 5 minutes. The pellet containing any remaining intact cells and
nuclei was washed once with the isolation buffer and then discarded.
The supernatant was pooled and then centrifuged at 10,000g for 15
minutes. The resultant pellet containing mitochondria (designated as
P10) was used for further experiments. The supernatant (termed S10)
was subjected to further ultracentrifugation at 100,000g. The resultant
supernatant was the cytosolic fraction (designated as S100) and the
pellet contained cytoplasmic membranes (termed P100). Cytochrome
oxidase assays were routinely performed (as described in Storrie and
Madden26), and these assays demonstrated that P10 contained mitochondria and that mitochondria were not detectable in the S100 fraction
containing cytosol. Cell fractions were also examined by Western
blotting (see below) for the presence of cytochrome a and cytochrome c,
which confirmed the cytochrome oxidase assay results.
Protein Analysis by Western Blotting
At specific time points after temperature switch to activate or
inactivate v-Abl TK, 1 3 107 cells were harvested, washed in
phosphate-buffered saline (PBS; pH 7.4), and lysed in buffer containing
Tris-HCl (50 mmol/L, pH 7.4), NP-40 (1% vol/vol), sodium deoxycholate (0.25% wt/vol), NaCl (150 mmol/L), EGTA (1 mmol/L), EDTA
(1 mmol/L), PMSF (1 mmol/L), aprotinin (10 µg/mL), leupeptin (10
µg/mL), pepstatin (10 µg/mL), and NaF (50 mmol/L). Protein content
was determined using the standard Bio-Rad reagents (Bio-Rad, Hemel
Hempstead, UK). Forty micrograms of S100 cytosolic or P10 mitochondria enriched fraction was loaded per sample and cellular proteins were
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 15% wt/vol gel before transfer onto a
nitrocellulous membrane. The membranes were then probed using
mouse, anti–Bcl-XL, anti-cytochrome c, or anti-cytochrome a antibody
overnight at 4°C, follwed by antimouse secondary antibody for 2 hours
Fig 1. Induction of apoptosis by withdrawal of
IL-3 from IC.DP and IC2.9 cells. (A) Kinetics of cell
death of IC2.9 cells (triangles) and IC.DP cells
(squares) after withdrawal of IL-3 measured by trypan
blue exclusion. Cells were maintained at either 32°C
(open symbols) or 39°C (solid symbols). Data points
are the mean value 6 SEM of three repeated experiments. (B) Flow cytometric analysis of apoptosis in
IC.DP and IC2.9 cells maintained at either 32°C or
39°C for 18 hours after withdrawal of IL-3 determined by binding of annexin V and uptake of propidium iodide. Results are representative of three
repeated experiments.
4547
at room temperature. Equal protein loading was confirmed by staining
the filters with Ponceau S and/or reprobing for actin using mouse IgG2a
anti-actin monoclonal antibody (Sigma). Immunreactive bands were
detected using the ECL system (Amersham Life Science, Amersham,
UK).
RESULTS
Kinetics of Apoptosis After Withdrawal of IL-3
in the Presence or Absence of v-Abl TK Activity
Figure 1A shows the rate of cell death in IC.DP (expressing ts
v-Abl) and IC2.9 (no v-Abl) cells cultured at 32°C or 39°C,
confirming the results of our previous studies.21,23 Note that
there is approximately 10% cell death in each cell sample as
determined by trypan blue uptake at the 18-hour time point.
Figure 1B demonstrates that the mode of cell death in IL-3–
depleted IC.DP and IC2.9 cells is apoptosis, identified by
positive staining for annexin V, and shows that, at 18 hours, less
than 10% cells take up propidium. In the absence of v-Abl TK
activity, the percentage of cells staining positive for annexin V
at 18 hours was always slightly greater than the percentage of
cells staining positive for trypan blue or the percentage that take
up propidium (Fig 1B, right-hand panel).
Withdrawal of IL-3 From IC.DP Cells Results in a Reduction
in Cm That Is Prevented and Reversed by Activated v-Abl TK
A reduction in Cm is believed to be an early step during the
induction of apoptosis in several cell types (eg, Decaudin et
al27). We investigated whether this was also true in IL-3–
depleted IC.DP cells. Figure 2A shows the measurement of Cm
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4548
CHEN ET AL
Fig 2. Activation of v-Abl TK prevents reduction
in Cm after withdrawal of IL-3. (A [i]) Viable cells
were defined in region R1 by exclusion of propidium
iodide. (A [ii]) Example of the measurement of Cm in
IC.DP cells (0 hours) using DiOC(6)3 (solid histogram)
and collapse of Cm with 10 mmol/L mCCCP (open
histogram). (B) Kinetics of changes in Cm in IC.DP
(squares) and IC2.9 (triangles) cells after withdrawal
of IL-3. Cells were maintained at either 32°C (open
symbols) or 39°C (solid symbols). Data were normalized against the mean DiOC(6)3 fluorescence intensity for each sample at 0 hours. Data points are the
mean 6 SEM of three duplicate experiments.
using DiOC(6)3 and propidium iodide. Cells excluding propidium were gated (panel i) and the green DiO(6)3 fluorescence
histogram was generated. A collapse of Cm was observed after
treatment with the proton ionophore mCCCP that corresponded
on average to a 2.3-fold decrease in DiOC(6)3 staining (panel ii).
Removal of IL-3 from IC.DP cells with v-Abl TK inactive
resulted in a reduction of Cm that was first detected at 12 hours
(Fig 2B) and at 18 hours (the nadir of Cm) corresponded to a
1.4-fold reduction in DiOC(6)3. Activation of v-Abl TK from 0
to 18 hours completely prevented this loss in Cm (Fig 2B).
These effects in IC.DP cells were not merely due to temperature
changes, because IC2.9 cells exhibited a similar reduction in
Cm at 32°C or 39°C as that observed for IC.DP cells at 39°C
(Fig 2B). Parallel experiments performed using the mitochondrial-selective probe NAO demonstrated that the observed
changes in Cm measured by DiOC(6)3 in IC.DP cells were not
attributable to changes in mitochondrial mass (data not shown).
We asked whether this reduction of Cm that occurred in the
absence of IL-3 and v-Abl TK activity could be reversed if the
survival stimulus provided by v-Abl TK were applied at 18
hours. IC.DP cells (106/mL) were deprived of IL-3 with v-Abl
active (32°C) or inactive (39°C) for 18 hours. Cells were then
either maintained at these temperatures for a further 24 hours or
had their culture temperature switched from either from 32°C to
39°C (to inactivate v-Abl TK) or from 39°C to 32°C (to activate
v-Abl TK) for the next 24 hours (see Fig 3, a schematic of the
experimental protocol). Total cell number, Cm, and the percentage of dead cells (uptake of propidium) were examined at 0, 18,
and 42 hours for each of these conditions. Figure 4 shows
typical results from such an experiment. Figure 4A shows the
percentage of viable cells in each sample from which the mean
values of Cm were generated. Activation of v-Abl TK at 18
hours rescued all but 9% cells when cell viability was assessed
at 42 hours (compare the bottom two dot plots of Fig 4A).
Conversely, inactivation of v-Abl TK at 18 hours resulted in the
further loss of 26% of cells between 18 and 42 hours (compare
the top two dot plots of Fig 4A). The total cell number
(including the trypan blue-positive corpses) remained constant
throughout the 42-hour period congruent with the lack of
proliferation that occurs in the absence of IL-3 with v-Abl
active or inactive.21 The data for cell viability and Cm from
three repeat experiments are summarized in Table 1. Figure 4B
shows the changes in Cm that occurred at 18 and 42 hours after
the v-Abl TK activating and inactivating temperature switches.
The downward arrows indicate the cell population mean value
for Cm at 0 hours. Figure 4B (i) shows that rescue of IC.DP cells
by v-Abl TK activation at 18 hours resulted in an elevation of
Cm from the point of rescue by temperature switch (open
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MITOCHONDRIAL EVENTS AND COMMITMENT TO APOPTOSIS
4549
Fig 3. Schematic of the experimental protocol.
histogram) to that observed at 42 hours (solid histogram).
Conversely, inactivation of v-Abl TK at 18 hours resulted in a
lowering of Cm (from the solid histogram to the open histogram), as expected for cells that are destined to die by apoptosis
(Figure 4B [ii]). Figure 4B (i) shows that there is minimal
overlap (5% cells) between the solid and open histograms,
implying that 95% of the propidium negative cells in the
population increase their Cm after the applied temperature
switch to activate v-Abl TK (Fig 4A). Taken together, these data
show that the reduction in Cm elicited by IL-3 withdrawal for 18
hours can be reversed by a survival stimulus such as activation
of v-Abl TK.
Release of Cytochrome c From Mitochondria Elicited
by IL-3 Withdrawal Is Prevented by v-Abl TK Activation
Figure 5 shows Western blots of a cytosolic extract of IC.DP
(Fig 5A) and IC2.9 cells (Fig 5B) probed for cytochrome c. No
cytosolic cytochrome c is detectable in IC.DP or IC2.9 cells at
the start of the experiment (0 hours). At 39°C in the absence of
v-Abl TK activity, the cytosol of IC.DP cells deprived of IL-3
for 18 hours contained cytochrome c. In contrast, at 32°C in the
presence of v-Abl TK activity, cytosolic cytochrome c was not
detectable in IC.DP cells deprived of IL-3 for 18 hours. This
effect was not a merely a reflection of cell culture temperature,
because cytosolic cytochrome c was detected at 18 hours after
IL-3 withdrawal in the cytosol of IC2.9 cells (which do not
contain v-Abl TK) at both temperatures. Maximal levels of
detectable cytosolic cytochrome c were seen at 18 hours. At
later time points (up to 42 hours) with v-Abl TK inactive, the
percentage of cells undergoing apoptosis increased and cytosolic cytochrome c remained detectable but did not increase in
level (data not shown). Cytosolic cytochrome a was undetectable in IL-3–deprived IC.DP and IC2.9 cells throughout the
experimental time course (data not shown), ruling out the
possibility that the fractionation procedure itself disrupted
mitochondria and caused general spillage of mitochondrial
components into the cytosolic fraction. When v-Abl TK was
activated 18 hours after IL-3 withdrawal and the cytosolic
cytochrome c level was examined 24 hours later (at 42 hours), it
was detectable but reduced compared with that observed at the
time of v-Abl TK activation (Fig 5).
Figure 6 shows the levels of cytochrome c, cytochrome a, and
Bcl-XL in the p10 intracellular membrane fraction of IL-3–
deprived IC.DP cells. Using a previously reported procedure,28
P10 fractions from IC.DP were assessed for cytochrome c and
cytochrome a levels at 0, 18, and 42 hours after the removal of
IL-3 in the presence or absence of v-Abl TK activity and in cell
populations in which v-Abl TK had been activated or inactivated at 18 hours to 42 hours. Cytochrome a remained constant
in all samples. Cytochrome c remained at a constant level from
0 to 42 hours with v-Abl TK active (lanes 1, 2, and 3). In
contrast, when v-Abl TK was inactive, the levels of cytochrome
c in the p10 were decreased at 18 hours (lane 5) and barely
detectable at 42 hours (lane 6). At 42 hours, despite a massive
loss of cytochrome c from the p10 fraction, we did not detect a
corresponding increase in the cytosolic s100 fraction, perhaps
suggesting degradation of cytosolic cytochrome c during the
apoptotic process. Inactivation of v-Abl TK at 18 hours resulted
in a decrease in mitochondrial cytochrome c (lane 4) and,
conversely, rescue of IL-3–deprived cells by activation of v-Abl
TK between 18 and 42 hours resulted in cytochrome c levels
equivalent to that seen at the start of the experiment (lane 7). We
have previously shown that v-Abl TK activity results in the
upregulation of Bcl-XL protein levels in whole cell lysates by 18
hours.22 Western blots of the p10 fraction were reprobed for
Bcl-XL to correlate the levels of this suppressor of apoptosis
with changes in the mitochondrial content of cytochrome c. As
predicted, Bcl-XL levels were elevated 18 and 42 hours after
v-Abl TK activation (lanes 2 and 3) and were dramatically
reduced if v-ABL TK was inactivated between 18 and 42 hours
(lane 4). Conversely, when IL-3–deprived cells were not
protected by v-Abl TK activity, Bcl-XL levels decreased (lanes
5 and 6), but were reestablished in cells that were rescued by
v-Abl TK activation between 18 and 42 hours (lane 7). Thus, the
levels of Bcl-xL and cytochrome c in the p10 fraction were
positively correlated.
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4550
IC.DP Cell Populations That Have Reduced Their Cm
and Contain Cytosolic Cytochrome c Retain Clonogenicity
When Provided With IL-3
Clonogenic assays were performed to determine whether
cells that appeared viable at the conclusion of the reversal
procedure described above (and shown in Fig 3) would survive
in the long term and were capable of proliferation upon
readdition of IL-3. The absolute cloning efficiency of IC.DP
cells at 0 hours was 40% 6 8%, and the data described below
CHEN ET AL
Table 1. Summary of Results
Time After
IL-3 Withdrawal
(h)
0
18
18
18-42
18-42
42
42
v-ABL
TK Status
Active
Inactive
Inactivated
at 18 h
Activated
at 18 h
Active
Inactive
Cm (fluorescence
units
normalized
to 0 h)
Cytosolic
Cyto C
Clonogenicity
(% normalized
to 0 h)
2
5
8
100
148
64
22
22
11
100
163
100
33
88
ND
10
17
10
49
148
1
ND
11
40
100
0
% Dead
(trypan
blue
positive)
Results are the average of at least three independent experiments.
Abbreviation: ND, not determined.
and in Table 1 have been normalized, taking the 0-hour value as
100%. The most striking results was observed when cells were
deprived of IL-3 for 18 hours with inactive v-Abl TK and then
assessed for their ability to form colonies when IL-3 was
readded; the clonogenicity remained 100%. Thus, long-term
survival as assessed by clonogenicity was completely unaffected in a cell population in which 95% cells had reduced Cm
(Fig 4B) and that contained cells that we demonstrated to have
cytosolic cytochrome c. There were no colonies formed when
cells were left without IL-3 or v-Abl TK activity for 42 hours.
When IL-3 was removed for 42 hours but cells were protected
for the last 24 hours of this period by v-Abl TK (Fig 3), at least
40% of cells were viable as judged by their ability to form
colonies. In this cell population, Cm had decreased in the first 18
hours and had then been restored by v-Abl TK action in the
subsequent 24-hour rescue period. Moreover, cells rescued by
v-Abl TK activity from 18 to 42 hours had a reduced amount of
cytosolic cytochrome c (Fig 5). When v-Abl TK was switched
off between 18 and 42 hours after IL-3 removal and the decrease
in Cm was not restored, only 10% cells subsequently formed
colonies.
DISCUSSION
Fig 4. Activation of v-Abl TK reverses the loss of Cm. IL-3 was
withdrawn from IC.DP cells, which were then maintained at 32°C or
39°C for 18 hours and then switched to the other temperature, ie,
from 39°C to 32°C or from 32°C to 39°C for a further 24 hours. (A) The
percentage of cells that exclude PI at each time point. The upper two
dot plots show the increase in cell death after inactivation of v-Abl TK
at 18 hours compared with that observed when v-Abl TK was
activated at 18 hours (lower two dot plots). (B) The changes in Cm
occurring before and after activation (Bi) and inactivation (Bii) of
v-Abl TK. The arrow indicates the Cm at 0 hours. Results are
representative of three independent experiments.
Many studies of the induction of apoptosis demonstrate that
there are several phases of the process, namely initiation,
commitment, and execution.18 If the promise of therapeutic
intervention along the pathway(s) leading to apoptosis in
pathological conditions is to be fulfilled, the molecular events
defining these phases need to be established. Notably, those
events that commit a cell to irreversibly engage the execution
machinery might provide useful drug targets.
Recent interest in the regulation of apoptosis has been
focussed on events occurring within mitochondria.3 In particular, the release of apoptogenic substances from mitochondria
such as cytochrome c and apoptosis-inducing factor (AIF) is
linked to the activation of caspases and thus may be an
irreversible commitment event. We asked whether a reduction
in Cm and the release of cytochrome c were irreversible events
in a well-characterized model of IL-3 withdrawal-mediated
induction of apoptosis.
In contrast to other reports in different cell types,11,29 IL-3
withdrawal from IC.DP cells resulted in a reduction in Cm 6
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
MITOCHONDRIAL EVENTS AND COMMITMENT TO APOPTOSIS
4551
Fig 5. Protein levels of cytochrome c in the
cytosolic fraction (s100) of IC.DP and IC2.9 cells
after IL-3 deprivation in the presence and absence
of v-Abl TK activation. (A) IC.DP cells. (B) IC2.9 cells.
Results shown are representative of three repeated
experiments.
hours before detectable cytochrome c in cytosolic fractions and
the appearance of cells with apoptotic morphology (Figs 1
through 5).20,21
Activation of v-Abl TK both at the point of IL-3 depletion
(from 0 hours) and, more importantly, after a reduction in Cm
and appearance of cytosolic cytochrome c had occurred (after
18 hours in the absence of IL-3 and v-Abl TK activity), resulted
in a cell population 24 hours later in which the vast majority of
the cells had intact plasma membranes and appeared viable
(Table 1). When Cm was reexamined 24 hours after v-Abl TK
activation, it was restored. Intriguingly, we noted that v-Abl TK
activity for more than 12 hours resulted in an increase in Cm
above that observed at 0 hours (Fig 2B), and this is not a
reflection of increased mitochondrial mass. We have not
investigated this phenomenon further but speculate that it may
be due to the increased availability of metabolic substrates,
because v-Abl TK upregulates glucose uptake.30 When put into
the clonogenic assay with IL-3, 40% of these cells that had been
rescued (by v-Abl TK activation for 24 hours) formed colonies
(Table 1). Notably, readdition of IL-3 at 18 hours to IL-3–
deprived cells that had not been protected by v-Abl TK and that
contained cytosolic cytochrome c resulted in 100% clonogenicity (Table 1). We argue that, taken together, these experiments
suggest that both mitochondrial events are reversible. There are
Fig 6. Protein levels of cytochrome c, cytochrome a, and Bcl-XL in
the mitochondria-enriched subcellular fraction (p10) of IC.DP cells
after IL-3 deprivation in the presence and absence of v-Abl TK
activation. Lane 1, 0 hours; lane 2, 18 hours, v-Abl TK active; lane 3, 42
hours, v-Abl TK active; lane 4, 18 hours, v-Abl TK active, and then 24
hours, v-Abl TK inactive; lane 5, 18 hours, v-Abl TK inactive; lane 6, 42
hours, v-Abl TK inactive; lane 7, 18 hours, v-Abl TK inactive, and then
24 hours, v-Abl TK active. Data are representative of three separate
experiments.
several pieces of evidence to support our argument. First, there
is only a small fraction of cells dying during the v-Abl
TK–mediated rescue period (typically ,10%; compare bottom
panels of Fig 4A), and in any case, measurements of Cm are
made after the cells already dead are excluded. Second, there
has been minimal if any cell division during the course of the
experiment, so we are not comparing different populations of
cells. Thirdly, the histograms for Cm shown in Fig 4B(i) show
single populations before and after rescue, and there is minimal
overlap in the solid and open histograms. Fourth, and shown for
the first time, cells that had reduced Cm and had released
cytochrome c from their mitochondria that were subsequently
prevented from undergoing apoptosis by v-Abl TK or readdition
of IL-3 at 18 hours retain the capacity for proliferation in a
clonogenic assay upon readdition of IL-3 (Table 1).
Enforced overexpression of Bcl-XL in U937 cells prevented
the accumulation of cytochrome c in the cytosol and suppressed
apoptosis induced by DNA damage.12 Our data also demonstrate that v-Abl TK, which upregulates the level of Bcl-XL and
potently suppresses apoptosis induced by cytokine depletion or
DNA damage (Fig 1),31 also prevents the release of cytochrome
c from mitochondria. Our preliminary data suggest that the
upregulation of Bcl-XL by v-Abl TK is of functional significance with respect to the suppression of apoptosis. Application
of antisense Bcl-XL oligonucleotide but not sense oligonucleotide reduces the level of Bcl-XL with v-Abl TK active and
restored an apoptotic response to the withdrawal of IL-3 (data
not shown). A putative protein protein interaction pertinent to
events occurring in IL-3–deprived IC.DP cells is that between
Bcl-XL and cytochrome c. This protein partnership was demonstrated by immunoprecipitation of whole U937 cell lysates.12
The mechanism by which Bcl-xL prevents release of cytochrome c remains unresolved. Cytochrome c is located in the
intermitochondrial membrane space loosely associated with the
inner mitochondrial membrane, whereas Bcl-XL is thought to be
tethered to the outer mitochondrial membrane, with the majority
of the molecule facing outwards into the cytoplasm.3 Studies of
apoptosis in Jurkat cells suggest that physical disruption of the
outer mitochondrial membrane early in apoptosis provides an
efflux pathway for cytochrome C.28 However, in contrast to
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
4552
these studies, the proapoptotic protein Bax can release cytochrome c from the mitochondria without inducing a PT or
physical disruption to the outer mitochondrial membrane.32
Another potential efflux pathway might be the PT pore complex, which forms in the outer mitochondrial membrane early in
apoptosis. However, reconstitution experiments of the PT in
vesicles containing cytochrome c suggest that PT pore opening
does not permit passage of cytochrome c.33
The data presented here suggest that the release of cytochrome c from mitochondria by whatever mechanism, observed
after IL-3 depletion from IC.DP cells at least, is a recoverable
position. This invokes a model whereby an important level of
regulation of apoptosis occurs within the cytosol, preventing
immediate irreversible engagement of apoptosis as soon as
cytochrome c leaves the mitochondria. Other studies also
suggest that release of cytochrome c does not inevitably lead to
caspase activation and cell death, at least in short-term assays.34,35 Bcl-2 can prevent Bax-induced release of cytochrome
c but can also prevent activation of caspases in cells in cells
containing cytosolic cytochrome c.34 Overexpressed Bcl-2
delayed cell death induced by microinjection of cytochrome c
(20 µmol/L).35 However, neither study examined the survival of
cell in the long term with analysis of clonogenicity. Given our
data that does show that cells with cytosolic cytochrome c can
clone, either the death promoting cytosolic partner(s) for
cytochrome c are not immediately available or active or
cytochrome c has to be modified to fulfil a lethal function. The
recent identification of apaf-1,6 which in the presence of dATP
is a death-promoting partner for cytochrome c coupling its
release into the cytosol to the activation of caspases, invites the
speculation that the availability of the apaf-1 binding site for
cytosolic cytochrome c might be the regulationary step leading
to an elusive commitment point for the engagement of apoptosis.
We acknowledge that these findings are based on a single cell
line and it should be noted that IL-3–dependent cell lines tend to
use anerobic glycolysis for ATP generation.36 This has not been
assessed in our experiments but could explain, at least in part,
why IL-3–deprived IC.DP cells can tolerate the presence of
cytosolic cytochrome c for a period of time and still be rescued
by v-Abl TK or readdition of IL-3. Moreover, we do not yet
know whether IC.DP cells harbor a defect in the cell death
pathway downstream of cytochrome c release. If they do, such a
defect might serve to delay the onset of apoptosis after IL-3
withdrawal (or exposure to chemotherapy) after cytochrome c
release from mitochondria, but cannot prevent its occurrence.
Studies are underway to examine the expression of Apaf-1 and
cellular inhibitor of apoptosis (c-IAP)37 and the activation of
caspase 3 in IC.DP cells and to determine whether these
molecules are regulated by v-Abl TK and/or IL-3. Despite these
caveats, our conclusions drawn from studies of IC.DP premast
cells are that release of cytochrome c per se does not commit a
cell to death. These conclusions are consistent with those
obtained for fibroblasts and melanoma cells in which overexpressed Bcl-2 can attenuate Bax killing downstream of cytochrome c release.34
Most recently, it was reported that constitutive Bcr-Abl TK
activity prevented the accumulation of cytochrome c in the
S100 cytosolic fraction of HL60 and K562 hematopoietic cells
CHEN ET AL
treated with cytotoxic drugs or sphingoid bases.38 Our data
confirm that a related oncogenic Abl tyrosine kinase (v-Abl TK)
can also prevent release of cytochrome c in another cellular
context. In addition, we provide data to suggest that v-Abl TK
or IL-3 can act downstream of a reduction in Cm and cytochrome c release from mitochondria to promote long-term cell
survival.
ACKNOWLEDGMENT
The authors thank Sukbinder Heer for excellent technical support of
all the flow cytometry experiments. We thank John A. Hickman for his
constructive critique of this manuscript.
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1998 92: 4545-4553
Blood Cells With Reduced Mitochondrial Membrane Potential and Cytosolic
Cytochrome C Can Survive and Maintain Clonogenicity Given Appropriate
Signals to Suppress Apoptosis
Quan Chen, Naoshi Takeyama, Ged Brady, Alastair J.M. Watson and Caroline Dive
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