CANCER RESEARCH 57. 2446-2451. June IS. 19971
Relationships between the Mitochondrial Permeability Transition and Oxidative
Stress during ara-C Toxicity
Karen L. Backway, Ernest A. McCulloch, Sue Chow, and David W. Hedley'
Department of Pathology. Ontario Cancer institute/Princess Margaret Hospital [K. L B.. S. C. D. W. H.], and Department ofMedical
D. W. H.], 610 University Avenue, Toronto, Ontario, M5G 2M9 C'anada
ABSTRACT
Later, there was an increase in intracellular calcium ([Ca2@]@),fol
lowed by a loss of surface membrane integrity. Cells that overex
pressed the Bcl-2 protein were protected by being held in the high
ROI/high GSH reducing state (3). This orderly sequence of events is
consistent with the suggestion that oxidative stress is an important
mechanism of drug-induced apoptosis and that the protection afforded
by Bcl-2 is related to an increased capacity to withstand oxidative
stress (6).
Recently, Kroemer and coworkers have shown that mitochondrial
changes play a pivotal role during early apoptosis (7—10).The mito
chondrial permeability transition results from the opening of large
protein pore complexes within the inner mitochondrial membrane (9,
11). An active process, the permeability transition can occur in re
sponse to a wide range of factors, including calcium signaling and
oxidative stress (I 1—15).It produces loss of the MMP, thereby un
coupling oxidative phosphorylation. Increases in ROl generation then
occur and are likely to be the result of a feedback increase in
respiratory chain activity due to the loss of ATP generation (16, 17).
The mitochondrial permeability transition could therefore be both a
cause and a consequence of oxidative stress.
In addition to mitochondrial membrane changes, there is recent
interest in alterations in the surface membrane that take place in early
apoptosis. These involve the translocation to the cell surface of
phosphatidylserine, an aminophospholipid that is normally confined
to the inner leaflet of the lipid bilayer ( 18—20).This is an active
process that allows the recognition of apoptotic cells by phagocytes.
It is likely that the various physiological events taking place during
apoptosis are interrelated and controlled by genes that determine
whether drug treatment results in cell death or survival. These pro
cesses are potential targets for novel treatment strategies. Therefore,
we developed multilaser flow cytometry methods to map the sequence
of changes in cellular physiology after ara-C treatment and identify
the critical events associated with drug resistance. Using these tech
niques, we have confirmed that the loss of MMP is an early event that
coincides with phosphatidylserine exposure and precedes high ROI
generation and the loss of ionized calcium regulation. However, these
changes seem to take place downstream of an earlier depletion in the
reduced cellular GSH content.
The mitochondrial permeability transition and oxidative stress seem to
be critical alterations in cellular physiology that take place during pro
grammed cell death. Failure to undergo apoptosis is associated with drug
resistance in acute myeloid leukemia and other cancers. Therefore, it is
important to establish causal relationships between the physiological
changes that take place in apoptosis, because these are potential targets
for novel treatment strategies to overcome this form of drug resistance.
We describe the use of multilaser flow cytometry methods to make cor
related measurements of mitochondrial membrane potential (MMP), the
generation of reactive oxygen intermediates, the cellular content of re
duced glutathione (GSH), intracellular calcium, and exposure of phos
phatidylserine on the cell surface. Using these combined methods, we have
mapped
a “death sequence― that occurs
after
treatment
of leukemic
blasts
with clinically relevant concentrations of l-@-D-arabinofuranosylcytosine
(nra-C). Dual labeling of MMP and cellular glutathione content showed
that loss of MMP, indicative of the permeability transition, took place in
cells that
were
depleted
phosphatidylsenne
of glutathione.
exposure
The loss of MMP
and preceded
a state
coincided
of high reactive
with
oxygen
generation. Finally, there was an increase in intracellular calcium. These
results
demonstrate
that
the mitochondrial
permeability
transition
takes
place during ara-C toxicity but suggest that this occurs downstream of the
loss of GSH. Thus, oxidative stress after am-C-induced toxicity seems to
be a biphasic phenomenon, with the permeability transition occurring
after
a depletion
of GSH
and
preceding
a state
of high
reactive
oxygen
generation.
INTRODUCTION
The lethal effects of ara-C2 result from incorporation of ara-CTP
into
DNA,
where
it acts
as a chain
terminator
( I ). After
initial
DNA
damage, cells eventually undergo DNA fragmentation that is charac
teristic
of apoptosis
(2). We have previously
shown
that the loss of
calcium ion regulation occurs during ara-C toxicity, suggesting that
DNA fragmentation is caused by the activation of calcium-dependent
endonucleases (3). Using multiparametric flow cytometry with probes
for the cellular redox state, we mapped a sequence of events occurring
upstream of the increase in intracellular calcium. The earliest detect
able change was a modest increase in the rate of reactive oxygen
generation, accompanied by an approximately 2-fold increase in the
cellular content of GSH. This finding is consistent with previous
reports that oxidative stress is an early event in programmed cell death
(4, 5); the source
of the ROIs
is uncertain.
The
increase
Biophysics, University of Toronto fE. A. M.,
MATERIALS
AND METHODS
in GSH
observed is likely to be a cellular response to the increased ROI that
protects the normal reducing cellular redox environment. With longer
ara-C treatment, we observed a transition to a state of very high ROl
generation and marked GSH depletion, indicating collapse of cellular
antioxidant defenses and a shift in redox balance to an oxidizing state.
Cell Culture. The OCIJAML-2cells used in these experiments are a
continuous
line of acute myelogenous
leukemia
a patient at Ontario Cancer Institute/Princess
grow independently
of added
growth
factors
blasts originally
isolated
from
Margaret Hospital. These cells
and were maintained
at 37°C in
10% FCS (Sigma, St. Louis, MO) with a-MEM (Life Technologies, Inc.,
Burlington, Canada) and subcultured routinely every 2—3days.
ara-C Treatment ara-C(UpjohnCo., Don Mills, Canada)was dissolved
Received I2/I 7/96; accepted 4/20/97.
in PBS buffer
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement
18 U.S.C. Section 1734 solely to indicate this fact.
I To
whom
requests
for
reprints
should
be addressed.
Phone:
appropriate
in accordance with
(416)
946-2262;
Fax:
2 The
abbreviations
used
are:
ara-C,
I -@3-D-arabinofuranosylcytosine;
GSH,
reduced
with 7 @tMara-C for the
and then resuspended
at I X 106 cells/mi
of medium for flow cytometric analysis. This concentration of ara-C produces
an approximately 90% loss of clonogenic survival after 24 h of exposure.
(416) 946-6546; E-mail: [email protected].
glutathione; ROl, reactive oxygen intermediate; MMP, mitochondrial membrane poten
tial; MBBr, monobromobimane; DHR-123, dihydrorhodamine 123; lCa2 @l,.intracellular
ionized cytosolic calcium.
(pH 6.5) at 10 msi. Cells were treated
length of time, counted,
ROl Generation. Generationof ROI was measuredusing DHR-l23 (Mo
lecular Probes, Eugene, OR). This is weakly fluorescent but is oxidized by ROl
to the highly fluorescent rhodamine 123. DHR-123 was made up as a 1 mist
stock solution
in 100% ethanol
and stored
in 1-mi aliquots
under
nitrogen.
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PHYSIOLOGICALCHANGES DURING ara-C TOXICITY
GSH. Thenonfluorescent
sulfydrylprobeMBBr(MolecularProbes)was
used as described previously (21). This compound binds avidly to -SH groups,
producing brightly fluorescent conjugates. Under the staining conditions used,
fluorescence is highly correlated with the cellular content of GSH (22). MBBr
was made up as a 4 mMstock solution in 100% ethanol.
MMP. This was measured using the cyanine dye DiIC6(5), obtained from
Molecular Probes. This is a structural analogue of the widely used MMP probe
RESULTS
Relationships
between Cellular Redox State and MMP. Dead
cells that had lost the capacity
to exclude
propidium
iodide
showed
low levels of MBBr and DHR- I23 fluorescence. These represented
<20%
of the total cell population
and were gated
out from further
analyses. As previously found (3), there was striking heterogeneity in
DiOC6(3) but is capable of excitation using a 633 nm HeNe laser. Because of
ROI generation and GSH content after ara-C treatment. A subpopu
their cationic and amphipathic nature, these cyanine dyes are concentrated in lation appeared with an increased rate of ROI production, whereas the
energized
mitochondria.
Dual staining of cells undergoing
apoptosis
using
distribution of individual cell GSH content became much broader,
DiOC6(3) and DiIC6(5) gave identical patterns, confirming that DiIC6(5) is a
with the appearance of cells with greater and lower values than those
suitable red light-excited MMP probe. DiIC6(5) was made up as a 40 @tM
seen in untreated control populations. Correlated two-parameter plots
working dilution in 100% ethanol.
of GSH content versus reactive oxygen show that the low GSH cells
Phosphatidylserine Expression. This was measured using a fluorescein
conjugate of the natural ligand annexin V, obtained from R&D Systems correspond to the high ROl population (Fig. I). Using DiIC6(5) to
measure MMP, a distinct population of low fluorescent cells was seen
(Minneapolis, MN). Cells were labeled with a final concentration of 5 pi/ml
after
ara-C treatment. The data are shown in Fig. 2 as correlated
annexin V-FITC for 5 mm at room temperature.
Ionized Calcium. This was done using the fluorescent calcium indicator two-parameter contour plots of glutathione content (top) and reactive
oxygen generation (bottom) plotted against MMP. The low MMP cells
indo-l , loaded into cells as the membrane-permeant ester indo-1AM (Molec
ular Probes).
appearing after drug exposure showed decreased GSH (top right
Surface Membrane Integrity. The membrane-impermeantDNA stain panel), and no cells were seen that had high levels of GSH and
propidium iodide (10 j.@g/ml)
was added before running the samples. Cells that depolarized mitochondria. The subpopulation of low MMP cells
had lost surface membrane integrity were identified by their bright nuclear showing high rates of ROI generation (bottom right panel) mapped to
fluorescence.
the low MMPIIow GSH cluster (top right panel).
Flow Cytometry. Flow cytometry was done using an Epics Elite cell sorter
Relationships between MMP, [Ca@@]1,and Phosphatidylserine
(Coulter Corp., Miami, FL) equipped with argon, HeCd, and HeNe lasers
Exposure. Using annexin V-FITC labeling, a subpopulation of cells
emitting at 488, 325, and 633 am, respectively. The argon laser was used as the
that expressed phosphatidylserine but excluded propidium iodide
reference beam, with the HeCd and HeNe running collinearly with a 40-ms
could
be clearly identified after ara-C treatment. As shown in Fig. 3,
time delay. The signals were processed using a gated amplifier. The following
annexin
V positivity was strongly correlated with low MMP, and very
excitation lasers and bandpass filters were used. Annexin V-FITC and DHR
few cells were seen in the transition from high MMP/annexin V
123 were excited by the argon laser with fluorescence collected at 525 nm, and
negative to low MMP/annexin V positive. These findings indicate that
propidium iodide was excited by the argon laser with fluorescence
collected at
640 nm. The HeCd laser was used to excite MBBr, and fluorescence was the loss of MMP and the exposure of phosphatidylserine on the cell
collected at 450 nm. Indo-l was also excited using the HeCd laser, and [Ca2 @l, surface take place rapidly and within minutes of each other. Fig. 4
was obtained from electronic ratio measurements of the calcium-bound (405 shows correlated two-parameter dot plots of ionized calcium versus
nm) and calcium-free (525 nm) emissions. The 633 nm HeNe laser was used
MMP (top) and annexin V binding (bottom). After ara-C treatment, a
to excite DiIC6(5), and emission was collected at 675 nm.
population of cells appeared with very high [Ca2@]@values. All of
Combined Labeling Methods. To identify relationshipsbetween various these high [Ca2@]@cells showed reduced MMP and were annexin V
the changes in cell function that take place during apoptosis, two quadruple
positive, whereas approximately 50% of the low MMP/annexin V
labeling methods were used, each using triple-laser excitation. Method 1,
positive
cells
showed
normal
calcium
values,
indicating
that the
designed to monitor changes in cellular redox state, involved staining with 1
increase in [Ca2@]@occurred later than the mitochondrial and surface
p.M DHR-123 and 40 nM DiIC6(5) for 25 mm at 37°C, after which cells were
membrane changes.
resuspended
in fresh medium containing
40 @tMMBBr and 10 @g/ml pro
pidium
iodide and incubated
for an additional
5 mm at 37°C. Method
Time Course of ara-C Treatment. The single 24-h time point
2, which
examines events downstream of the loss of GSH, consisted of 3 @LM
indo-lAM
and 40 nMDiIC6(5) for 25 mm at 37°Cfor 25 mm, followed by resuspension
in fresh medium containing annexin V-FITC and propidium iodide for S mm
at room temperature.
experiments suggested that a sequence of events was occurring during
ara-C toxicity. This was further investigated by making flow cytom
etry measurements at 2-h intervals during continuous ara-C treatment
for 26 h. The findings are summarized in Fig. 5, which for clarity
CONTROL
ARA-C
w
z
0
Fig. 1. Correlated contour maps of reactive oxygen generation
(log scale) versus cellular glutathione content (linear scale),
showing increased heterogeneity after 24 h of treatment with 7
@LMara-C
(right
panel).
I
I—
F-I
0
j
LOG REACTIVEOXYGEN
2447
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PHYSIOLOGICAL CHANGES DURING ara-C TOXICITY
@
@CONTROL
uJ
z
0
I
F—
F--J
0
Fig. 2. Correlated contour maps of MMP versus glutathione
z
w
content (top) and reactive oxygen generation (bottom). All cells
withloss of MMPafterara-Ctreatmentshowlow glutathione,
and a proportion of these show high reactive oxygen generation
0
(right panels).
mmCONTROL
liii
ARA-C
r-i-rn@
>.
><
0
w
>
F-0
0
w
0
0@
-J
MITOCHONDRIAL MEMBRANE POTENTIAL
apoptosis after a wide range of physiological and nonphysiological
initiating factors (7—10,12, 23, 24). It is an active process that
involves the opening of complex pore structures in the mitochondrial
inner membrane, resulting in the loss of MMP and the uncoupling of
oxidative phosphorylation (17). High levels of ROI generation may
then occur, as found in the present paper, and are probably due to the
loss of feedback inhibition of the respiratory chain after depletion of
cellular ATP. Although these events are by themselves injurious to
cells, there is evidence that the nuclear changes associated with
apoptosis are due to the release of a death effector from the perme
abilized mitochondria (8, 9). The identity of this effector is uncertain,
although it is apparently not the interleukin 1(3 converting enzyme
like protease CPP-32 that has recently been implicated in ara-C
induced apoptosis (25).
The detailed composition of the permeability transition pore com
shows only the 16-, 20-, and 24-h time points. The percentage of cells
showing loss of MMP increased with time of ara-C exposure. The
lower panels in Fig. 5 show GSH content, annexin V binding, and
[Ca2@}1plotted against MMP. Note that the basic patterns, such as the
loss of MMP in low GSH cells and the strong correlation between
MMP and annexin V labeling, were maintained throughout the time
course. Thus, the heterogeneity observed at 24 h using the live cell
function assays is explained on the basis of the cells going through a
sequence of discrete stages in an asynchronous manner. This asyn
chrony is probably due to the fact that ara-C incorporation into DNA
only takes place during S-phase of the cell cycle.
DISCUSSION
Considerable interest has been generated recently by the proposal
that the mitochondrial permeability transition plays a key role in
CONTROL
ARA-C
0
I—.
UFig. 3. Correlated dot plots of MMP versus annexin V-FITC
labeling after 24 h of nra-C treatment (right panel) gated to exclude
propidium iodide-positive cells.
>
z
w
z
z
:4,
.3.
1
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ass
tees
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is
see
MITOCHONDRIALMEMBRANEPOTENTIAL
2448
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a...
PHYSIOLOGICALCHANGES DURING ara-C TOXICITY
CONTROL
ARA-C
F--
0
0@
w
a
F—
‘.4
Fig. 4. Correlated dot plots of ionized calcium concentration
versus
MMP(top)andannexinV-FITClabeling(bottom)gatedto exclude
propidium iodide-positive cells.
a
64
0
64
S
64
S
64
0
F—
>
z
x
z
z
w
IONIZED CALCIUM
@
plex is unknown. However, there is evidence that it is associated with
the loss of MMP and phosphatidylserine exposure on the cell surface.
other inner membrane proteins such as the ADP/ATP translocase and
Phosphatidylserine is an aminophospholipid that is normally confined
a peripheral benzodiazepine receptor (24). The Bcl-2 protein might
to the inner leaflet of the cell membrane. Translocation to the outer
also be physically linked and might inhibit apoptosis by a direct action
leaflet is an active process that allows phagocytes to recognize apop
on the pore complex (9). Although not fully characterized, the per
totic cells in vivo (18—20).Costedo ci al. found that phosphatidyl
meability transition seems to be ubiquitous in mammalian cells. It can
serine exposure occurred approximately I h after the mitochondrial
be initiated by cell signaling ( 12), glucocorticoids ( 10), and also by
permeability transition in dexamethasone-treated
thymocytes (10).
oxidative processes such as disulfide bonding or the generation of
However, in our experiments, almost all propidium iodide-excluding
lipid peroxidation breakdown products within the matrix space (13— cells were either annexin V positive/low MMP or annexin V negative/
15, 23). Thus, the permeability transition may cause oxidative stress
high MMP. Furthermore, as seen in Figs. 3 and 5, the proportion of
due to increased respiratory chain activity and may also be the
cells that were in the transition between these two states was very
consequence of oxidative stress.
small. This indicates that the loss of MMP and the exposure of
Using multiparametric flow cytometry, we have mapped out a phosphatidylserine on the outer membrane take place rapidly and
sequence of events occurring during ara-C toxicity that is defined in occur within a few minutes of each other.
physiological terms. As previously reported (3), the earliest events
High levels of ROl generation were seen in some cells that had lost
detected were an increase in the cellular content of glutathione and a MMP, as has been reported by others ( 16, 17). ROIs such as super
modest increase in ROI generation. These early changes are probably
oxide anion are normally generated as byproducts of mitochondrial
due to an as-yet-uncharacterized
oxidative stress that occurs after
respiration (3 1). Because respiration is feedback-inhibited by cellular
DNA damage by ara-C. Oxidative stress has been extensively docu
ATP levels, it is likely that the high ROI generation seen after loss of
mented after a wide variety of cellular insults, such as hyperthermia
MMP was due to the uncoupling of oxidative phosphorylation, with
(26), UV irradiation (27), glucocorticoid treatment (6), or transient
increased respiratory chain activity occurring as AlP levels decrease
hypoxia (27, 28). An increase in GSH commonly occurs in response
(16,17).
to oxidative stress, probably as an antioxidant defense mechanism (28,
The final event observed before the loss of surface membrane
29). With increasing time of ara-C treatment, we later observed a integrity was a large increase in intracellular ionized calcium. Low
decrease in cellular GSH that coincided with the loss of MMP. No
calcium values were found in approximately half of the cells that had
cells with high GSH values showed loss of MMP, excluding the
lost MMP, indicating that a significant time interval occurs between
possibility that the permeability transition is the cause of the GSH
the permeability transition and the loss of calcium ion regulation. The
depletion. More likely, the permeability transition is due to failure of
most likely explanations for the increase in [Ca2 1 are the failure of
antioxidant mechanisms within the mitochondrial matrix space, as has
AlP-dependent calcium pumps or lipid peroxidation damage to mem
brane sites of calcium regulation (32). Internucleosomal fragmenta
been reported previously (13, 14, 30).
tion of DNA by the activation of calcium-dependent endonucleases is
At all time points examined, there was a strong correlation between
2449
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PHYSIOLOGICAL
CONTROL
CHANGES
DURING
ara-C
TOXICITY
2OHR
16HR
24HR
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MITOCHONDRIAL
ieee
MEMBRANE
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POTENTIAL
Fig. 5. Time course of ara-C toxicity. Cells were treated with 7 @sM
ara-C continuously for 16, 20, and 24 h. Top panels, single-parameter
histograms of MMP. The other rows are
correlated dot plots of MMP versus glutathione content (second row), annexin V-FITC labeling (third row), and ionized calcium (bottom row) gated to exclude propidium iodide-positive
cells.
considered to be a major late event during apoptosis (32), although its
relationship with more recently described death effectors, such as the
interleukin 1 3 converting enzyme-like proteases, remains to be estab
lished.
A large number of genes and their products are implicated in the
regulation of apoptosis, but there are apparent contradictions in the
published literature, and it is difficult to fit all of the potential
effector mechanisms into a unifying scheme (33). In this paper, we
have taken the alternative approach of describing ara-C-induced
apoptosis in terms of the relationships between the important
physiological
events that occur during apoptosis,
including
changes in cellular redox state, calcium ion regulation, and the
mitochondrial permeability transition. This physiological approach
provides a framework in which specific genetically controlled
events such as Bcl-2 alterations could be fitted. In addition, it may
give insights into potential new types of cancer treatment that are
designed to overcome drug resistance caused by the failure to
undergo apoptosis. For example, in the present study, we have
shown that an early event after ara-C treatment is an increase in
GSH content, which may inhibit the mitochondrial permeability
transition. Clinical trials of the GSH-depleting agent buthionine
sulfoximine have shown that this can produce up to 90% loss of
peripheral blood leukocyte GSH with minimal toxicity (34, 35).
Although buthionine sulfoximine is currently under consideration
2450
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PHYSIOLOGICALCHANGES DURING ara-C TOXICITY
as a modulator of drug resistance to alkylating agent, our results
suggest that it may also have an adjunctive role in ara-C-containing
regimens.
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19. Martin, S. J., Reutelingsperger, C. P. M., McGahon, A. J., Rader, J. A., van Schie,
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1997 American Association for Cancer Research.
Relationships between the Mitochondrial Permeability
Transition and Oxidative Stress during ara-C Toxicity
Karen L. Backway, Ernest A. McCulloch, Sue Chow, et al.
Cancer Res 1997;57:2446-2451.
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