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Centre National de Recherche Scientifique, Villejuif, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Attempts to order the sequence of apoptosis-associated events are
mainly based on multiparameter cytofluorometric analyses, the only
methodology that allows for the determination of several parameters on
a per-cell basis. Using this approach on thymocytes, primary
lymphocytes, or lymphoid cell lines (CEM-C7, 2B4.11, etc.), as well as
cancer cell lines, a common, death stimulus-independent sequence of
apoptosis-associated events has been established (20, 21, 22, 23, 24, 25, 26, 27). After
initial mitochondrial transmembrane potential (
m)
disruption (stage 1), cells reduce their content in nonoxidized GSH and
aberrantly expose phosphatidylserine (PS) residues on the plasma
membrane (stage 2). Subsequently, cells hyperproduce ROS, which leads
to cardiolipin oxidation, and undergo massive Ca2+ influx
(stage 3) shortly before the plasma membrane permeability is lost.
Commitment to cell death has already occurred at stage 1. DNA
fragmentation is initiated during stage 2 (20, 21, 22, 23, 24, 25, 26).
We and others have investigated the subcellular distribution of Ca2+ and protons during the apoptotic process (15, 20, 21, 22, 23, 24, 25, 26, 28, 29, 30, 31). Moreover, it has recently been shown that K+ extrudes from the cell during the apoptotic process (32, 33). Whereas Ca2+ is much more abundant in extracellular fluid than in the cytosol, K+ concentrates in the cytosol (physiologic concentration in mammalian cells is 100–140 mM) as compared with the extracellular medium (4–5 mM). The asymmetric distribution of K+ is vital for the maintenance of plasma membrane integrity and function. Manipulations of cytosolic K+ concentrations causing a decrease in K+, such as extracellular K+ depletion or the addition of K+-specific ionophores, can cause apoptosis (34, 35). In contrast, an extra supply of external K+ can prevent apoptosis, at least in neuronal cell lines (36, 37, 38).
Based on these premises, we decided to determine the role of K+ in several models of apoptosis. As shown in this work, a loss of cytosolic K+ occurs during programmed cell death and is located downstream of mitochondrial PT and caspase activation. Although this K+ loss becomes detectable relatively late during apoptosis, it appears crucial for the activation of endonucleases as suggested by data obtained in intact cells and cell-free systems. Collectively, our data establish that the disruption of K+ homeostasis constitutes an important step in the apoptotic degradation phase after commitment to death has occurred.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Thymocytes from female 4- to 8-wk-old BALB/c mice were cultured at 37°C with 5% CO2 in RPMI 1640 supplemented with 10% FCS, L-glutamine, antibiotics, and ß-mercaptoethanol (50 µM, Sigma, St. Louis, MO). Murine 2B4.11 T cell hybridoma cell lines that had been stably transfected with the SFFV.neo vector containing the human bcl-2 gene or the neomycin resistance gene only were a gift from J. Ashwell (National Institutes of Health, Bethesda, MD).
Induction and inhibition of apoptosis
The following cell death inducers were employed: the glucocorticoid receptor agonist dexamethasone (DEX) (at a final concentration 1 µM; Sigma), etoposide (10 µM), or an Ab specific for CD95/Fas (clone 154000D, 500 ng/ml; PharMingen, San Diego, CA). The following apoptosis inhibitors were tested: the antioxidant N-acetylcysteine (50 mM), the protein synthesis inhibitor cycloheximide (35 µM; Sigma), the IL-1ß-converting enzyme-like caspase inhibitor acetyl-Tyr-Val-Ala-Asp-chloromethylketone (100 µM), the broad spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) (at a final concentration 100 µM; stocks in 10 mM DMSO; Bachem, Basel, Switzerland), the ligand of the adenine nucleotide translocator bongkrekic acid (BA) (at a final concentration of 50 µM; kindly provided by Dr. Duine, Delft University, Delft, The Netherlands), or cyclosporin A (CsA) (1 µM; Novartis, Basel, Switzerland).
Cytofluorometric determination of mitochondrial parameters, ROS, GSH levels, and cytosolic Ca2+ or K+
For the determination of the m,
3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) (at a final
concentration of 40 nM; stock 40 µM in ethanol; excitation wave
length of 488 nm, emission 529 nm; Molecular Probes, Eugene, OR) was
used (39). The generation of ROS was monitored with hydroethidine (HE)
(at a final concentration of 5 µM; stock 10 mM in DMSO; excitation
wave length of 488 nM, emission 620 nM; Molecular Probes); the content
in nonoxidized GSH was determined using monochlorobimane (at a final
concentration of 50 µM; stock 100 mM in ethanol; excitation wave
length of 488 nM; emission 620 nM). Ca2+ levels were
measured using Fluo-3 acetoxymethyl ester (AM) (at a final
concentration of 250 nM; stock 1 mM in DMSO; excitation wave length of
488 nM, emission 525 nM; Sigma) in calcium-free HBSS (Eurobio, Paris,
France). After incubation in the presence of the indicated fluorochrome
(15–30 min, 37°C), cells were kept on ice (60-min maximum) until
analysis. For the determination of intracellular K+ levels,
cells were loaded during the 15 to 30 min with cell-permeant
potassium-binding benzofuran isophthalate (PBFI)-AM (at a final
concentration of 2.5 µM; stock 500 µM in dimethylformamide)
(40, 41, 42). The resulting PBFI fluorescence was elicited at 360 nM and
measured at 485 ± 20 nm.
Cytofluorometric analyses
The frequency of hypoploid cells was determined by ethanol fixation followed by staining with propidium iodine (PI) as previously described (43) using an EPICS Profile II Analyzer (Coulter, Hialeah, FL). All other stainings were analyzed using a FacsVantage cytofluorometer (Becton Dickinson). Data were analyzed in duplicate, and results were recorded for 10,000 cells while gating was performed either on cells exhibiting normal forward scatter (FSC) and side scatter (SSC) characteristics or on the whole cell population (normal cells plus shrunken cells) as indicated. In control experiments, cells were labeled in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) (at a final concentration of 100 µM; intermediate stock 10 mM in ethanol), the prooxidant ter-butylhydroperoxide (200 µM), the redox-cycling agent menadione (which causes the generation of superoxide anions; 1 mM; stock 1 M in water), the calcium ionophore A23187 (which allows for the influx of extracellular Ca2+; 500 nM; stock 500 µM in DMSO), or a combination of valinomycin (0.5 µM)/nigericin (2.5 µM) (which allows for the free distribution of K+) plus different concentrations of KCl/NaCl (with a final osmolarity of 300 mM) and 5 mM HEPES buffer (pH 7.4). Cell sorting was performed using an Elite II cytofluorometer (Coulter, Miami, FL).
Cytofluorometric separation of cells and determination of cellular K+ levels
Thymocytes stimulated for 4 h with DEX (1 µM) were
simultaneously stained with DiOC6(3) and PBFI as
described above and separated in a FACS (Coulter Elite II or Becton
Dickinson FACS Vantage). Cells were sorted into complete culture medium
that was kept on ice and analyzed within a maximum of 30 min (for
potassium quantitation) or 120 min after separation (for reculture and
reanalysis). After cytofluorometric separation, cells
(
106/tube) were washed three times (600 x
g, 4°C, 10 min) in 140 mM NaCl plus 10 mM HEPES (pH =
7.4) and stored as a pellet at -20°C. Cells were resuspended in 100
µl of H2O, subjected to three freeze-thaw cycles,
centrifuged (5000 x g, 4°C, 15 min) and injected
into an IL-243 flame spectrophotometer (Laboratory Instrumentation,
Lexington, MA) according to standard procedures.
Cell-free system of nuclear apoptosis
Nuclei from HeLa cells were purified on a sucrose gradient (44)
and conserved at -20°C in HNB freezing buffer (80 mM KCl, 20 mM
NaCl, 250 mM sucrose, 5 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and 10 mM
PIPES, pH 7.4) plus 50% glycerol for up to 15 days. Mouse liver
mitochondria (10 mg/ml protein) were incubated with atractyloside (5
mM) for 10 min at room temperature to induce PT and the release of AIF
and cytochrome c and then ultracentrifuged (1.5 x
105 g, 1 h, 4°C) as previously described
(12, 45). Nuclei (103 nuclei/µl) were cultured in the
presence of mitochondrial supernatants (200 ng protein/µl) for 90 min
at 37°C in cell-free system buffer (220 mM mannitol, 68 mM sucrose, 2
mM NaCl, 2.5 mM PO4H2K, 0.5 mM EGTA, 2 mM
MgCl2, 5 mM pyruvate, 0.1 mM PMSF, 1 mM dithiothreitol, and
10 mM HEPES-NaOH; pH 7.2) supplemented with variable amounts of KCl.
Nuclei were stained with PI (10 µg/ml; Sigma) for 
5 min at room
temperature), and cytofluorometric analysis was subsequently performed
in an EPICS Profile II Analyzer (Coulter) while gating the FSCs and
SSCs on single-nucleus events (46). Alternatively, nuclei were stained
with 4',6-diamidino-2-phenylindone or subjected to agarose gel
electrophoresis as previously described (12). In control experiments,
the
N-benzyloxycarbonyl-Val-Ala-Asp-7-amino-4-methyltrifluoromethylcoumarin
(Z-VAD.afc) (30 µM in CFS buffer at 37°C, 30 min; Enzyme Systems,
Livermore, CA) cleaving activity of mitochondrial supernatants was
assessed. Z-VAD.fmk, an inhibitor of AIF (45, 46), was used for control
purposes at a final concentration of 100 µM.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Upon loading with the K+-sensitive dye PBFI
(40, 41, 42), thymocytes that undergo apoptosis in response to DEX exhibit
a lower fluorescence as compared with untreated cells (Fig. 1
). Control experiments revealed that the
difference in PBFI-dependent fluorescence between viable (PI-excluding)
DEX-treated cells and viable control thymocytes disappeared in the
presence of a combination of the potassium ionophore valinomycin and
the K+/H+ exchanger nigericin (Fig. 1A), which together allow for the free distribution
of K+ between the cell and the extracellular environment
(41). Thus, the difference in PBFI fluorescence is due to a difference
in K+ concentration rather than in dye uptake. Calibration
of K+-permeant cells with variable doses of extracellular
K+ (Fig. 1, A and B) revealed
that viable, PI-excluding cells, which retain PBFI, exhibit a low
(0.6–0.8 logs) although consistent (n = 5)
K+-dependent variation of fluorescence. Although this
relatively low signal/background ratio would render this dye
inappropriate for the quantitation of apoptosis in single parameter
studies, it is possible to assess the loss of PBFI fluorescence in
combination with additional fluorochromes (see below, Figs. 2-4). In
the presence of K+ ionophores and external K+,
all cells, including the fraction of DEX-treated thymocytes with an
originally low PBFI-dependent fluorescence (PBFIlow cells),
acquire a PBFIhigh phenotype. The reduction of
K+-dependent PBFI fluorescence is detectable in a fraction
of normal-sized cells before major volume loss occurs, at least
according to the criterium of the FSC (Fig. 1C), and
is also detectable before cells become permeant to PI (Fig. 1A). A gas spectroscopic method for K+
determination confirmed that K+ loss occurred during
apoptosis (see below, Fig. 5). In
conclusion, DEX-induced thymocyte apoptosis is accompanied by both a
loss of intracellular K+ that occurs before cell shrinkage
and a complete loss of plasma membrane barrier function.
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In most cell types, including thymocytes, the first
cytofluorometrically detectable sign of apoptosis is a loss of
m and the subsequent hyperproduction of ROS on
the uncoupled respiratory chain (21, 22, 26, 28, 47). To determine the
temporal relationship between these signs of apoptosis and
K+ loss, we performed a multiparameter analysis taking
advantage of the m-sensitive dye DiOC6(3)
(green fluorescence), the ROS-dependent conversion of hydroethidine
into ethidium (Eth) (red fluorescence), and PBFI as a
K+-sensitive marker (blue fluorescence). As shown in Figure 2
, phenotypically normal (DiOC6(3)high
HE
Ethlow) cells are homogeneously PBFIhigh,
whereas DiOC6(3)low HEEthhigh
cells are PBFIlow. In contrast, cells of the intermediate
DiOC6(3)low HEEthlow stage split
into PBFIhigh and mostly PBFIlow cells,
indicating that K+ loss occurs after m
dissipation but before the acquisition of the HEEthhigh
phenotype. Kinetic multiparameter analyses confirmed this
interpretation. Cells undergoing apoptosis manifest the
m disruption before K+ leakage. Of note,
DiOC6(3)low but not
DiOC6(3)intermediate cells emit low
K+-dependent fluorescence (Fig. 3A).
K+ loss exhibits a strong correlation with PS exposure, as
determined with an FITC-annexin V conjugate. All PBFIhigh
cells are FITC-annexin Vlow (Fig. 3B),
suggesting that K+ loss occurs together with or shortly
after PS exposure. In contrast, the PBFI fluorescence correlates to a
lower degree with ROS generation and Ca2+ influx.
PBFIlow cells are distributed between the
HEEthhigh/Fluo-3high populations (among
which all cells are PBFIlow) and a fraction of cells that
still have a normal HEEthlow phenotype (Fig. 3C) and have not yet undergone the Fluo-3-detectable
terminal Ca2+ influx (Fig. 3D). In
conclusion, K+ loss occurs after the initial dissipation of
the m and after PS exposure, but before cells
hyperproduce ROS and accumulate Ca2+.
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) and the topoisomerase type
II inhibitor etoposide, and is also seen as a result of crosslinking of
the Fas/Apo-1/CD95 Ag (Fig. 4). Whenever an inhibitor interrupts the
proapoptotic signaling cascade, K+ loss is prevented. Thus,
inhibiting protein synthesis with cycloheximide or with the addition of
the thiol antioxidant N-acetylcysteine prevents DEX- and
etoposide-induced m disruption, nuclear apoptosis,
and K+ outflow (Fig. 4). However these agents have no
effect on Fas-induced apoptosis in any of its manifestations at the
levels of mitochondria, nuclei, and K+. In contrast,
acetyl-Tyr-Val-Ala-Asp-chloromethylketone (an inhibitor of caspase-1,
formerly called IL-1ß-converting enzyme) prevents caspase-1-dependent
Fas-induced apoptosis and K+ loss but has no effect on DEX-
and etoposide-induced apoptosis (Fig. 4). These data support the
conclusion that apoptotic m disruption, nuclear
apoptosis, and K+ outflow are functionally associated with
each other.
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As shown in Figures 2 through 5, K+ loss is
always secondary to m disruption. No K+
loss is observed in cells whose m remains stable.
Cytofluorometric separation of DiOC6(3)/PBFI-stained cells
and flame spectroscopic determination of K+ revealed that
DEX-treated DiOC6(3)highPBFIhigh
cells contained 10.9 ± 3.6 nmol K+/106
cells (meon ± SEM, n = 10). This value is
comparable with that of nontreated control cells. In contrast,
DiOC6(3)lowPBFIlow cells contain
only 3.1 ± 1.1 nmol K+/106 cells
(n = 10; p < 0.001, paired
Student’s t test) (Fig. 5, A and
B). Since this determination has been performed on
cells with a normal volume (i.e., cells with normal FSC and SSC
parameters), it appears that the K+ concentration drops to
less than one-third of the control value.
To further investigate the relationship between mitochondrial PT and
K+ loss, cells were treated with two inhibitors of the
mitochondrial megachannel, BA and CsA. BA is a ligand of the adenine
nucleotide translocator that prevents m disruption in
most models of apoptosis (12, 24, 27, 48) but that fails to protect
mitochondria against Fas-elicited caspase-1 (46). This agent stabilizes
the m of DEX- or etoposide-treated cells and
simultaneously prevents the loss of K+-dependent PBFI
fluorescence (Fig. 4, lower panels), suggesting that
K+ loss is secondary to mitochondrial PT.
To confirm this hypothesis, cells were stained with
DiOC6(3) and PBFI and then cytofluorometrically separated
into DiOC6(3)highPBFIhigh,
DiOC6(3)intermediatePBFIhigh,
and DiOC6(3)lowPBFIlow cells (Fig. 5A). Upon culture at 37°C, a fraction of
DiOC6(3)high cells became
DiOC6(3)intermediate and a significant
percentage of DiOC6(3)intermediate cells
became DiOC6(3)low and concomitantly reduced
PBFI fluorescence (Fig. 5C), thus corroborating the
precursor product relationship
(DiOC6(3)highPBFIhigh
DiOC6(3)intermediatePBFIhigh
DiOC6(3)lowPBFIlow) that
was suggested by the kinetic analyses (Fig. 3). CsA, a transient
inhibitor of mitochondrial PT which acts on matrix cyclophilin D (16, 49), was employed as a probe to assess the role of mitochondrial
dysfunction in this system. CsA (1–10 µM, Fig. 5 and data not shown)
prevents the initial transition
(DiOC6(3)highPBFIhigh
DiOC6(3)intermediatePBFIhigh) but
not the second one
(DiOC6(3)intermediatePBFIhigh
DiOC6(3)lowPBFIlow), suggesting
that the CsA-suppressible PT is only responsible for the first phase of
m disruption (Fig. 5C). The
transfection-enforced overexpression of Bcl-2, an endogenous inhibitor
of PT (12, 45, 50), also prevents both m dissipation
and the extrusion of K+ ions (Fig. 6).
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Functional relationship between K+ loss and caspase activation
Since caspases play a major role in apoptosis, we evaluated the
effect of caspase inhibition on cytofluorometrically detectable
apoptotic changes. Z-VAD.fmk, a broad spectrum inhibitor of caspases
and other proteases including AIF (45, 46), completely prevents
Fas-induced apoptosis at the DiOC6(3)high
stage, thus confirming the involvement of caspase cascades early during
Fas-induced apoptosis (46, 51, 52). In contrast, Z-VAD.fmk arrests DEX-
and etoposide-treated cells at a later stage of the process, namely the
DiOC6(3)intermediate stage (Fig. 4). Thus, in
support of previous results (30, 48, 53), caspase inhibitors fail to
prevent mitochondrial PT. Instead, they arrest the apoptotic process in
a postmitochondrial phase of the apoptotic process. Cells that have
been exposed to DEX plus Z-VAD.fmk (or etoposide plus Z-VAD.fmk) tend
to accumulate at the DiOC6(3)intermediate stage
(Figs. 4 and 7). At this
Z-VAD.fmk-arrested stage, cells still maintain high concentrations of
nonoxidized intracellular GSH (as detected by monochlorobimane), lack
PS exposure, and remain PBFIhigh (Fig. 7).
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m disruption,
GSH depletion/oxidation, PS exposure, and K+ loss) requires
the action of Z-VAD.fmk-inhibitable proteases. Moreover, these data
imply that caspase activation occurs before the K+ loss.
Accordingly, physiologic concentrations of K+ (100–140 mM)
fail to inhibit the acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl
coumarin-cleaving protease activity that accumulates in the cytosol of
DEX-treated thymocytes. Similarly, in the presence of 140 mM
K+, recombinant human caspases-3, -6, and -7 retain 60 to
80% of their enzymatic activity (100% value at 140 mM NaCl) (data not
shown) (32). Thus, caspases can act upstream of K+ loss in
cells with a normal K+. K+ loss is a prerequisite for endonuclease activation
In several models of apoptosis induction and inhibition, the
K+ loss exhibits a strong correlation with nuclear DNA
loss (Fig. 4). This correlation has been confirmed by cytofluorometric
purification of
DiOC6(3)highPBFIhigh,
DiOC6(3)intermediatePBFIhigh, and
DiOC6(3)lowPBFIlow cells (as Fig. 5A) and by the subsequent determination of the
frequency of hypoploid cells. Only
DiOC6(3)lowPBFIlow cells
exhibit advanced DNA fragmentation (Fig. 8), suggesting the possibility that
K+ may act as an endogenous regulator of apoptotic
degradation. Since manipulations of potassium have pleiotropic and
toxic effects on intact cells (Fig. 1) (54), we tested the effect of
variable K+ concentrations on a cell-free system of
apoptosis in which isolated HeLa nuclei are exposed to supernatants
from mitochondria that have been treated with atractyloside, an inducer
of PT leading to the release of AIF (12) and cytochrome c
(55). Physiologic concentrations of KCl corresponding to the normal
cytosolic K+ concentration (100–140 mM) inhibit chromatin
condensation and DNA fragmentation (as measured on gels or as nuclear
DNA loss) to background levels (Fig. 9,
A and B). In contrast, K+ has
a far less spectacular inhibitory effect on the Z-VAD.afc cleavage
mediated by the mitochondrial supernatant (Fig. 9C).
Thus, K+ inhibits endonuclease activation much more
efficiently than proteolytic activity, causing Z-VAD.afc cleavage (Fig. 9, B and C). Lowering of the KCl
concentration itself is not sufficient to activate endonucleases (Fig. 10B). However, it
does facilitate their activation by mitochondrial supernatants. The
apoptosis-inhibitory effect of KCl is dose dependent, with an
approximate ED50 of 80 mM (Fig. 9B). As a
consequence, K+ loses most of its suppressive effect on
endonuclease activation at the concentration that is found in the
viable PBFIlow population, about one-third of the
physiologic concentration. K+ outflow is a prerequisite for
apoptotic endonuclease activation.
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The data reported in this work are compatible with a step-wise
sequence of the common phase of apoptosis. The cytofluorometric
manifestations of this sequence are summarized in Figure 10. It may be
assumed that various proapoptotic signal transduction or damage
pathways converge at the level of the mitochondrion, the organelle
which determines the commencement of the common pathway of apoptosis
(3, 4, 5, 12, 13, 14). One of the first events of the common phase of
apoptosis is the disruption of mitochondrial membrane integrity, with
the dissipation of the m and/or the release of
proteins that are normally sequestered in the intermembrane space
(cytochrome c, AIF) in a sequence that remains to be
determined and that may be cell type- and/or inducer-specific (13, 14, 15, 20, 21, 22, 23, 24, 25, 26, 29, 30, 31, 46). Thus, the sequence of molecular and cellular
events leading to apoptosis, as described here, applies to thymocytes
and lymphoid cells but may be different in other cell types.
The dissipation of the inner membrane proton gradient (stage 1) is
mediated by PT, a process that is inhibited by CsA, BA, and Bcl-2
(
Figs. 4–6) (3, 15, 16, 21, 24, 45, 49). After initial
m disruption, cells are irreversibly committed to
death and consequently have entered the effector phase of apoptosis
(20, 30, 53). However, early after m disruption,
cells still maintain a normal morphology (20) and lack major changes in
K+/Ca2+ homeostasis (
Figs. 1–3), redox
potentials (Figs. 2, 3, and 7), or plasma membrane structure (Figs. 1, 3, and 7) (stage 1, Fig. 10).
The activation of Z-VAD.fmk-inhibitable proteases, which are triggered
as a consequence of mitochondrial changes (5, 12, 13, 14, 24, 26, 31, 45, 46), is rate-limiting for the manifestation of a subsequent block of
alterations (stage 2, Fig. 10), namely PS exposure on the cell surface,
loss of nonoxidized GSH, and K+ leakage (Figs. 4 and 7).
The functional relationship among these events is unknown. As a
possibility, the rearrangement of those plasma membrane lipids that are
linked to PS exposure might favor the extrusion of GSH and
K+ (56). Alternatively, the inhibition of Na/K adenosine
triphosphatase and/or the opening of K+ channels may be
connected to redox regulation and/or plasma membrane function (37, 57).
Thus, perturbation of redox metabolism by protonophores or menadione
favors rapid K+ efflux (Fig. 3). However, the exact
molecular mechanism of K+ loss that occurs during apoptosis
remains elusive. The cytosolic accumulation of those apoptogenic
activities (AIF, cytochrome, etc.) that are normally confined to
mitochondria (12, 45, 46, 58) stimulates nuclear endonucleases that can
only be activated in the context of a low K+ concentration
(Fig. 9).
The progressive exhaustion of antioxidant defense subsequently causes
the manifest (HE-detectable) hyperproduction of ROS, which entails
massive Ca2+ influx into the cytosol (stage 3, Fig. 10)
shortly before cells shrink, form apoptotic bodies, and lyse (
Figs. 1–3) (25).
As a caveat, it would be erroneous to assume that the pleiotropic molecules that are involved in the common pathway of apoptosis such as K+, Ca2+, ROS, or caspases only participate in the degradation phase of apoptosis. Indeed, it has been established that elevations in cytosolic Ca2+ and ROS can serve as facultative transmitters in particular (private) proapoptotic signal transduction pathways (reviewed in 3 . Similarly, caspases can be activated at the premitochondrial stage of apoptosis, for instance after Fas crosslinking (46, 51, 52), although this is not a general feature of apoptosis. By analogy, it is possible that the K+ efflux can participate in special cases of apoptosis initiation. Indeed, a reversible K+ loss has been observed early after IL-3 withdrawal in a murine hematopoietic cell line (59) and an outward K+ current may be decisive for neuronal apoptosis that is induced by staurosporine or serum deprivation (38). However, at least in the models that we have studied in this work, K+ release occurs at a relatively late stage of apoptosis, after commitment to cell death.
After submission of this work, several studies reporting the
efflux of K+ from cells undergoing apoptosis have been
published (32, 33, 60). These studies clearly establish the role of the
K+ (and Na+) efflux as a mechanism leading to
cytosolic H2O loss and consequent volume loss (32, 33, 60).
Thus, as shown here (Fig. 1), all cells with a shrunken phenotype
exhibit low K+-dependent PBFI fluorescence, and only a
minor fraction of cells that still have a normal FSC demonstrate a
PBFIlow phenotype (33). Moreover, in agreement with our
data, a rise in the concentration of extracellular K+
preventing the drop in cytosolic K+ can prevent nuclear DNA
fragmentation (32, 33). However, in contrast to our observations,
Cidlowski and coworkers (32, 33) suggest that K+ is an
endogenous inhibitor of caspase activation and has a primary role in
the regulation of cell death. This interpretation is based on the
observations that 1) the cytochrome c/deoxy ATP-driven
activation of caspase-3 is inhibited by K+ (32), and that
2) exogenous K+ can prevent endonuclease activation both in
cells and in cell-free systems of apoptosis. As shown here, the action
of a Z-VAD.afc-specific enzyme that is thought to be responsible for
caspase-3 activation (46) is not inhibited by K+ (Fig. 9).
This may imply that during early apoptosis, when K+ is
still high, caspase activation is triggered by factors other than
cytochrome c that are present in mitochondrial supernatants
(46). Clearly, in our cell-free system of nuclear apoptosis, in which
isolated nuclei are mixed with mitochondrial supernatant in the absence
of cytosol (46, 61), cytochrome c and deoxy ATP are
inactive, because they require the addition of cytosolic proteins for
caspase-3 and endonuclease activation (62). In intact cells, the
inhibition of caspase activation by Z-VAD.fmk prevents the
K+ efflux, suggesting that caspase activation occurs
upstream of this efflux (Fig. 7). Since the inhibition of caspases by
Z-VAD.fmk fails to prevent the initial m dissipation
(Fig. 7) and the subsequent cytolysis (53), it appears that the
commitment point which seals the fate of the cell is located upstream
of caspase activation and consequently upstream of K+
efflux. Therefore, although K+ outflow is critical for the
acquisition of several hallmarks of apoptotic morphology (cell
shrinkage and DNA fragmentation), it is probably not important for the
life/death/ decision, which is made before K+ drops and
before caspases and endonucleases are activated (53). This observation
applies to the models of thymocyte apoptosis studied in this work. At
present we cannot exclude the possibility that K+ levels
may play a role during the initiation or effector stages of apoptosis
in other cell types or in response to other apoptosis inducers.
In conclusion, we show in this paper that a loss of cytosolic K+ occurs as a common event in the apoptotic degradation phase of thymocytes, after mitochondrial PT and caspase activation but before cell shrinkage and before the plasma membrane becomes permeable. K+ leakage is closely associated with the loss of plasma membrane asymmetry and facilitates endonuclease-mediated chromatin degradation. These findings emphasize the functional impact of ion gradient dissipation on the apoptotic process.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Guido Kroemer, CNRS-UPR420, 19 rue Guy Môquet, BP8, F-94801 Villejuif, France. E-mail address:
3 Abbreviations used in this paper: AIF, apoptosis-inducing factor; FSC, forward scatter; SSC, side scatter; BA, bongkrekic acid; DEX, dexamethasone; PS, phosphatidylserine; DiOC6(3), 3,3'-)dihexyloxacarbocyanine iodide; m, AM, acetoxymethyl ester; mitochondrial transmembrane potential; CsA, cyclosporin A; Eth, ethidium; GSH, glutathione; HE, hydroethidine; PI, propidium iodine; CCCP, carbonyl cyanide m-chlorophenylhydrazone; PBFI, potassium-binding; benzofuran isophthalate; PT, permeability transition; ROS, reactive oxygen species; Z-VAD.afc, N-benzyloxycarbonyl-Val-Ala-Asp-7-amino-4-methyltrifluoromethylcoumarin; Z-VAD.fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone.
Received for publication November 24, 1997. Accepted for publication February 3, 1998.
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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  R. Franco, W. I. DeHaven, M. I. Sifre, C. D. Bortner, and J. A. Cidlowski Glutathione Depletion and Disruption of Intracellular Ionic Homeostasis Regulate Lymphoid Cell Apoptosis J. Biol. Chem., December 26, 2008; 283(52): 36071 - 36087. [Abstract] [Full Text] [PDF]
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 M. T. Andres, M. Viejo-Diaz, and J. F. Fierro Human Lactoferrin Induces Apoptosis-Like Cell Death in Candida albicans: Critical Role of K+-Channel-Mediated K+ Efflux Antimicrob. Agents Chemother., November 1, 2008; 52(11): 4081 - 4088. [Abstract] [Full Text] [PDF]
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 E. J. Coulson, L. M. May, S. L. Osborne, K. Reid, C. K. Underwood, F. A. Meunier, P. F. Bartlett, and P. Sah p75 Neurotrophin Receptor Mediates Neuronal Cell Death by Activating GIRK Channels through Phosphatidylinositol 4,5-Bisphosphate J. Neurosci., January 2, 2008; 28(1): 315 - 324. [Abstract] [Full Text] [PDF]
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 M. Foller, R. S. Kasinathan, S. Koka, S. M. Huber, B. Schuler, J. Vogel, M. Gassmann, and F. Lang Enhanced susceptibility to suicidal death of erythrocytes from transgenic mice overexpressing erythropoietin Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R1127 - R1134. [Abstract] [Full Text] [PDF]
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 A. J. Fay, X. Qian, Y. N. Jan, and L. Y. Jan SK channels mediate NADPH oxidase-independent reactive oxygen species production and apoptosis in granulocytes PNAS, November 14, 2006; 103(46): 17548 - 17553. [Abstract] [Full Text] [PDF]
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 F. Arrebola, E. Fernandez-Segura, A. Campos, P. V. Crespo, J. N. Skepper, and A. Warley Changes in intracellular electrolyte concentrations during apoptosis induced by UV irradiation of human myeloblastic cells Am J Physiol Cell Physiol, February 1, 2006; 290(2): C638 - C649. [Abstract] [Full Text] [PDF]
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 M. B. Friis, C. R. Friborg, L. Schneider, M.-B. Nielsen, I. H. Lambert, S. T. Christensen, and E. K. Hoffmann Cell shrinkage as a signal to apoptosis in NIH 3T3 fibroblasts J. Physiol., September 1, 2005; 567(2): 427 - 443. [Abstract] [Full Text] [PDF]
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E. E. Brevnova, O. Platoshyn, S. Zhang, and J. X.-J. Yuan Overexpression of human KCNA5 increases IK(V) and enhances apoptosis Am J Physiol Cell Physiol, September 1, 2004; 287(3): C715 - C722. [Abstract] [Full Text] [PDF]
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T. Shahar, S. B. House, and H. Gainer Neural Activity Protects Hypothalamic Magnocellular Neurons against Axotomy-Induced Programmed Cell Death J. Neurosci., July 21, 2004; 24(29): 6553 - 6562. [Abstract] [Full Text] [PDF]
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E. M. Jablonski, A. N. Webb, N. A. McConnell, M. C. Riley, and F. M. Hughes Jr. Plasma membrane aquaporin activity can affect the rate of apoptosis but is inhibited after apoptotic volume decrease Am J Physiol Cell Physiol, April 1, 2004; 286(4): C975 - C985. [Abstract] [Full Text] [PDF]
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 C. V. Remillard and J. X.-J. Yuan Activation of K+ channels: an essential pathway in programmed cell death Am J Physiol Lung Cell Mol Physiol, January 1, 2004; 286(1): L49 - L67. [Abstract] [Full Text] [PDF]
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 L. Wang, T. Li, and L. Lu UV-Induced Corneal Epithelial Cell Death by Activation of Potassium Channels Invest. Ophthalmol. Vis. Sci., December 1, 2003; 44(12): 5095 - 5101. [Abstract] [Full Text] [PDF]
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C. D. Bortner and J. A. Cidlowski Uncoupling Cell Shrinkage from Apoptosis Reveals That Na+ Influx Is Required for Volume Loss during Programmed Cell Death J. Biol. Chem., October 3, 2003; 278(40): 39176 - 39184. [Abstract] [Full Text] [PDF]
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T. Ichinose, S. Yu, X. Q. Wang, and S. P. Yu Ca2+-independent, but voltage- and activity-dependent regulation of the NMDA receptor outward K+ current in mouse cortical neurons J. Physiol., September 1, 2003; 551(2): 403 - 417. [Abstract] [Full Text] [PDF]
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S. Pal, K. A. Hartnett, J. M. Nerbonne, E. S. Levitan, and E. Aizenman Mediation of Neuronal Apoptosis by Kv2.1-Encoded Potassium Channels J. Neurosci., June 15, 2003; 23(12): 4798 - 4802. [Abstract] [Full Text] [PDF]
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D. Ekhterae, O. Platoshyn, S. Zhang, C. V. Remillard, and J. X.-J. Yuan Apoptosis repressor with caspase domain inhibits cardiomyocyte apoptosis by reducing K+ currents Am J Physiol Cell Physiol, June 1, 2003; 284(6): C1405 - C1410. [Abstract] [Full Text] [PDF]
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 X. Q. Wang, A. Y. Xiao, C. Sheline, K. Hyrc, A. Yang, M. P. Goldberg, D. W. Choi, and S. Ping Yu Apoptotic insults impair Na+, K+-ATPase activity as a mechanism of neuronal death mediated by concurrent ATP deficiency and oxidant stress J. Cell Sci., May 15, 2003; 116(10): 2099 - 2110. [Abstract] [Full Text] [PDF]
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 X. Q. Wang, A. Y. Xiao, A. Yang, L. LaRose, L. Wei, and S. P. Yu Block of Na+,K+-ATPase and Induction of Hybrid Death by 4-Aminopyridine in Cultured Cortical Neurons J. Pharmacol. Exp. Ther., May 1, 2003; 305(2): 502 - 506. [Abstract] [Full Text] [PDF]
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 L. Wei, S. P. Yu, F. Gottron, B. J. Snider, G. J. Zipfel, and D. W. Choi Potassium Channel Blockers Attenuate Hypoxia- and Ischemia-Induced Neuronal Death In Vitro and In Vivo Stroke, May 1, 2003; 34(5): 1281 - 1286. [Abstract] [Full Text] [PDF]
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O. Platoshyn, S. Zhang, S. S. McDaniel, and J. X.-J. Yuan Cytochrome c activates K+ channels before inducing apoptosis Am J Physiol Cell Physiol, October 1, 2002; 283(4): C1298 - C1305. [Abstract] [Full Text] [PDF]
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 V. Mateo, E. J. Brown, G. Biron, M. Rubio, A. Fischer, F. L. Deist, and M. Sarfati Mechanisms of CD47-induced caspase-independent cell death in normal and leukemic cells: link between phosphatidylserine exposure and cytoskeleton organization Blood, September 26, 2002; 100(8): 2882 - 2890. [Abstract] [Full Text] [PDF]
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 R. Filomenko, F. Poirson-Bichat, C. Billerey, J.-P. Belon, C. Garrido, E. Solary, and A. Bettaieb Atypical Protein Kinase C {zeta} as a Target for Chemosensitization of Tumor Cells Cancer Res., March 1, 2002; 62(6): 1815 - 1821. [Abstract] [Full Text] [PDF]
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A. Y. Xiao, L. Wei, S. Xia, S. Rothman, and S. P. Yu Ionic Mechanism of Ouabain-Induced Concurrent Apoptosis and Necrosis in Individual Cultured Cortical Neurons J. Neurosci., February 15, 2002; 22(4): 1350 - 1362. [Abstract] [Full Text] [PDF]
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S. Xia, P. A. Lampe, M. Deshmukh, A. Yang, B. S. Brown, S. M. Rothman, E. M. Johnson Jr, and S. P. Yu Multiple Channel Interactions Explain the Protection of Sympathetic Neurons from Apoptosis Induced by Nerve Growth Factor Deprivation J. Neurosci., January 1, 2002; 22(1): 114 - 122. [Abstract] [Full Text] [PDF]
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 S. Krick, O. Platoshyn, M. Sweeney, S. S. McDaniel, S. Zhang, L. J. Rubin, and J. X.-J. Yuan Nitric oxide induces apoptosis by activating K+ channels in pulmonary vascular smooth muscle cells Am J Physiol Heart Circ Physiol, January 1, 2002; 282(1): H184 - H193. [Abstract] [Full Text] [PDF]
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S. Krick, O. Platoshyn, S. S. McDaniel, L. J. Rubin, and J. X.-J. Yuan Augmented K+ currents and mitochondrial membrane depolarization in pulmonary artery myocyte apoptosis Am J Physiol Lung Cell Mol Physiol, October 1, 2001; 281(4): L887 - L894. [Abstract] [Full Text] [PDF]
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D. Ekhterae, O. Platoshyn, S. Krick, Y. Yu, S. S. McDaniel, and J. X.-J. Yuan Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells Am J Physiol Cell Physiol, July 1, 2001; 281(1): C157 - C165. [Abstract] [Full Text] [PDF]
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B. McLaughlin, S. Pal, M. P. Tran, A. A. Parsons, F. C. Barone, J. A. Erhardt, and E. Aizenman p38 Activation Is Required Upstream of Potassium Current Enhancement and Caspase Cleavage in Thiol Oxidant-Induced Neuronal Apoptosis J. Neurosci., May 15, 2001; 21(10): 3303 - 3311. [Abstract] [Full Text] [PDF]
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S. P. Yu and D. W. Choi Ions, cell volume, and apoptosis PNAS, August 15, 2000; 97(17): 9360 - 9362. [Full Text] [PDF]
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C. Dumont, A. Durrbach, N. Bidere, M. Rouleau, G. Kroemer, G. Bernard, F. Hirsch, B. Charpentier, S. A. Susin, and A. Senik Caspase-independent commitment phase to apoptosis in activated blood T lymphocytes: reversibility at low apoptotic insult Blood, August 1, 2000; 96(3): 1030 - 1038. [Abstract] [Full Text] [PDF]
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 S. N. Orlov, S. Taurin, N. Thorin-Trescases, N. O. Dulin, J. Tremblay, and P. Hamet Inversion of the Intracellular Na+/K+ Ratio Blocks Apoptosis in Vascular Smooth Muscle Cells by Induction of RNA Synthesis Hypertension, May 1, 2000; 35(5): 1062 - 1068. [Abstract] [Full Text] [PDF]
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 A. RASOLA, D. FARAHI FAR, P. HOFMAN, and B. ROSSI Lack of internucleosomal DNA fragmentation is related to Cl- efflux impairment in hematopoietic cell apoptosis FASEB J, October 1, 1999; 13(13): 1711 - 1723. [Abstract] [Full Text]
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H. Hida, S. Nagano, M. Takeda, and B. Soliven Regulation of Mitogen-Activated Protein Kinases by Sphingolipid Products in Oligodendrocytes J. Neurosci., September 1, 1999; 19(17): 7458 - 7467. [Abstract] [Full Text] [PDF]
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C. D. Bortner and J. A. Cidlowski Caspase Independent/Dependent Regulation of K+, Cell Shrinkage, and Mitochondrial Membrane Potential during Lymphocyte Apoptosis J. Biol. Chem., July 30, 1999; 274(31): 21953 - 21962. [Abstract] [Full Text] [PDF]
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 B. Dallaporta, P. Marchetti, M. A. de Pablo, C. Maisse, H.-T. Duc, D. Metivier, N. Zamzami, M. Geuskens, and G. Kroemer Plasma Membrane Potential in Thymocyte Apoptosis J. Immunol., June 1, 1999; 162(11): 6534 - 6542. [Abstract] [Full Text] [PDF]
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 S. P. Yu, C. Yeh, U. Strasser, M. Tian, and D. W. Choi NMDA Receptor-Mediated K+ Efflux and Neuronal Apoptosis Science, April 9, 1999; 284(5412): 336 - 339. [Abstract] [Full Text]
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W. Yu, K. Israel, Q. Y. Liao, C. M. Aldaz, B. G. Sanders, and K. Kline Vitamin E Succinate (VES) Induces Fas Sensitivity in Human Breast Cancer Cells: Role for Mr 43,000 Fas in VES-triggered Apoptosis Cancer Res., February 1, 1999; 59(4): 953 - 961. [Abstract] [Full Text] [PDF]
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C. D. Bortner, M. Gomez-Angelats, and J. A. Cidlowski Plasma Membrane Depolarization without Repolarization Is an Early Molecular Event in Anti-Fas-induced Apoptosis J. Biol. Chem., February 2, 2001; 276(6): 4304 - 4314. [Abstract] [Full Text] [PDF]
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C. C. Q. Vu, C. D. Bortner, and J. A. Cidlowski Differential Involvement of Initiator Caspases in Apoptotic Volume Decrease and Potassium Efflux during Fas- and UV-induced Cell Death J. Biol. Chem., September 28, 2001; 276(40): 37602 - 37611. [Abstract] [Full Text] [PDF]
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