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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4545-4553
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
From the School of Biological Sciences and the Department of
Medicine, Victoria University of Manchester, Manchester, UK.
 |
ABSTRACT |
Reduction of mitochondrial membrane potential ( m) 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). m 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 m and the release of mitochondrial
cytochrome c, and suppressed apoptosis. Activation of v-Abl TK 18 hours
after withdrawal of IL-3 when  10% of the cells had died restored
m 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 m precedes loss of mitochondrial cytochrome c in IC.DP
cells; that v-Abl TK activation, probably via upregulation of
Bcl-XL, prevents loss of m and blocks the
release of cytochrome c from mitochondria; and that neither of these
mitochondrial events is sufficient for commitment to apoptosis.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
APOPTOSIS 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 m 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 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
(m).3,15 The reduction in m
is thought to be due to the opening of a Ca 2+-activated,
ADP-inhibited, and voltage-dependent 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
m is currently unclear. In HL60 myeloid leukemia cells,
cytochrome c is released before a reduction in
m,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 a human lymphoblastic cell line (CEM C7A)
that, during dexamethasone-induced apoptosis, a reduction in
m is downstream of commitment to apoptosis. Taken
together, these data suggest that the precise order of a reduction in
m, 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 m 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 m 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).
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 temperature-sensitive 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
cell-conditioned 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 × 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.
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).
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 ± 30 nm and red fluorescence due to DNA bound propidium was collected at 630 ± 22 nm.
Mitochondrial membrane potential.
The m 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 × 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 m 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 ± 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
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 = (ln × [96/NegWells])/(Plate
Density) × 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
Cells (1 × 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 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 × 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 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).
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| 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 ± 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.
|
|
Withdrawal of IL-3 From IC.DP Cells Results in a Reduction in
m That Is Prevented and Reversed by
Activated v-Abl TK
A reduction in m 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 m 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 m 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
m that was first detected at 12 hours (Fig 2B) and at 18 hours (the nadir of m) 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 m (Fig 2B).
These effects in IC.DP cells were not merely due to temperature
changes, because IC2.9 cells exhibited a similar reduction in
m 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 m measured by DiOC(6)3 in IC.DP
cells were not attributable to changes in mitochondrial mass (data not
shown).
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| Fig 2.
Activation of v-Abl TK prevents reduction in
m 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 m in IC.DP cells (0 hours)
using DiOC(6)3 (solid histogram) and collapse of
m with 10 µmol/L mCCCP (open histogram). (B) Kinetics
of changes in m 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 ± SEM of three
duplicate experiments.
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|
We asked whether this reduction of m 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,
m, 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 m 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
m from three repeat experiments are summarized in
Table 1. Figure 4B shows the changes in
m 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 m 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 m
from the point of rescue by temperature switch (open histogram) to that
observed at 42 hours (solid histogram). Conversely, inactivation of
v-Abl TK at 18 hours resulted in a lowering of m (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 m after the applied
temperature switch to activate v-Abl TK (Fig 4A). Taken together, these
data show that the reduction in m elicited by IL-3
withdrawal for 18 hours can be reversed by a survival stimulus such as
activation of v-Abl TK.
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| Fig 4.
Activation of v-Abl TK reverses the loss of
m. 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 m occurring
before and after activation (Bi) and inactivation (Bii) of v-Abl TK.
The arrow indicates the m at 0 hours. Results are
representative of three independent experiments.
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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).
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| 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.
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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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| 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.
|
|
IC.DP Cell Populations That Have Reduced Their
m 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% ± 8%, and the data
described below 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 m (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, m 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 m was not
restored, only 10% cells subsequently formed colonies.
| |
DISCUSSION |
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 m 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 m 6 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 m 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 m 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 m 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 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 m 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 m 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 m 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 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 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
m 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.
| |
FOOTNOTES |
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).
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.
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: cdive{at}man.ac.uk.
| |
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Abstract
Introduction