Oncogene (2003) 22, 3927–3936
& 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00
www.nature.com/onc
Mitochondrial membrane permeabilization is a critical step of
lysosome-initiated apoptosis induced by hydroxychloroquine
Patricia Boya,1 Rosa-Ana Gonzalez-Polo,1 Delphine Poncet,1 Karine Andreau,1
Helena LA Vieira,1 Thomas Roumier,1 Jean-Luc Perfettini1 and Guido Kroemer*,1
1
Centre National de la Recherche Scientifique, UMR 8125, Institut Gustave Roussy, Pavillon de Recherche 1, 39 rue
Camille-Desmoulins, F-94805 Villejuif, France
Hydroxychloroquine (HCQ) is a lysosomotropic amine
with cytotoxic properties. Here, we show that HCQ
induces signs of lysosomal membrane permeabilization
(LMP), such as the decrease in the lysosomal pH gradient
and the release of cathepsin B from the lysosomal
lumen, followed by signs of apoptosis including caspase
activation, phosphatidylserine exposure, and chromatin
condensation with DNA loss. HCQ also induces mitochondrial membrane permeabilization (MMP), as indicated by the insertion of Bax into mitochondrial
membranes, the conformational activation of Bax within
mitochondria, the release of cytochrome c from mitochondria, and the loss of the mitochondrial transmembrane
potential. To determine the molecular order among these
events, we introduced inhibitors of LMP (bafilomycin A1),
MMP (Bcl-XL, wild-type Bcl-2, mitochondrion-targeted
Bcl-2, or viral mitochondrial inhibitor of apoptosis from
cytomegalovirus), and caspases (Z-VAD.fmk) into the
system. Our data indicate that caspase-independent
MMP is rate-limiting for LMP-mediated caspase activation. Mouse embryonic fibroblasts lacking the expression
of both Bax and Bak are resistant against hydroxychloroquine-induced apoptosis. Such Bax/ Bak/ cells
manifest normal LMP, yet fail to undergo MMP and
subsequent cell death. The data reported herein indicate
that LMP does not suffice to trigger caspase activation
and that Bax/Bak-dependent MMP is a critical step of
LMP-induced cell death.
Oncogene (2003) 22, 3927–3936. doi:10.1038/sj.onc.1206622
Keywords: Bax; Bcl-2; cell death; lysosomes; mitochondria
Introduction
Apoptosis can be induced by stresses acting on specific
organelles including nuclei, mitochondria, the endoplasmic reticulum, or lysosomes. In spite of the
heterogeneity of potential cell death inducers, apoptosis
is characterized by common morphological and biochemical alterations. This suggests the existence of
*Correspondence: Guido Kroemer; E-mail: [email protected]
Received 17 January 2003; revised 24 March 2003; accepted 15 March
2003
interorganellar crosstalk (Ferri and Kroemer, 2001). It
is an open conundrum, however, whether apoptotic cell
death involves an obligate final pathway. Caspases have
been suggested to be universal apoptotic executioners
(Nicholson and Thornberry, 1997), but recent data
describing caspase-independent death and noncaspase
death effectors argue against this possibility (Mathiasen
and Jaattela, 2002; Ravagnan et al., 2002). Alternatively, it has been proposed that mitochondrial membrane permeabilization (MMP) might constitute a
common phenomenon that would mark the point of
integration as well as the point-of-non-return of the
lethal signal transducing cascade (Green and Reed,
1998; Kroemer and Reed, 2000; Debatin et al., 2002). In
favor of this hypothesis, it appears that overexpression
of Bcl-2-like proteins (which inhibit MMP) or knockout
of the proapoptotic genes Bax and Bak (whose products
induce MMP) confers a broad cytoprotection including
against nuclear DNA damage and ER stress (Zamzami
et al., 1998; Vogelstein et al., 2000; Ferri and Kroemer,
2001; Wei et al., 2001; Boya et al., 2002; Reed, 2002;
Zamzami and Kroemer, 2003). However, the importance of mitochondria for the regulation of apoptosis is
a matter of controversy, and some investigators suggest
that they play no central role in cell death control
(Lassus et al., 2002; Marsden et al., 2002).
One of the organelles that participates in the control
of caspase-independent cell death is the lysosome, as
indicated by several lines of evidence. First, in the course
of death, lysosomal proteases from the cathepsin family
frequently translocate from the lysosomal lumen to the
cytosol (Levy-Strumpf and Kimchi, 1998; Foghsgaard
et al., 2001; Roberg, 2001; Mathiasen and Jaattela, 2002;
Yuan et al., 2002). Second, genetic and pharmacological
studies demonstrate that either cathepsin B (CB) and D
are rate limiting for death induced by interferon-g,
tumor necrosis factor-a, p53, or pro-oxidants, at least in
some cell types (Levy-Strumpf and Kimchi, 1998;
Foghsgaard et al., 2001; Roberg, 2001; Mathiasen and
Jaattela, 2002; Yuan et al., 2002). Third, overexpression
of oncogenic Ras, DAP-kinase, and DRP-1 can induce
caspase-independent cell death with increased autophagy, indicating that some lethal signal-transducing
systems can elicit lysosome-dependent cell death (Cohen
et al., 2002; Inbal et al., 2002; Kitanaka et al., 2002).
Fourth, in a model of caspase-independent neuronal cell
Lysosomes and mitochondria in apoptosis
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death, increased macroautophagy, which requires the
contribution of lysosomes, causes the self-elimination of
atrophic cells (Xue et al., 2001).
Stimulated by these findings, we decided to address
the hitherto unresolved question as to whether a
primary damage affecting lysosomes induces cell death
through activation of the mitochondrial apoptotic
pathway. To address this issue, we took advantage of
the lysosomotropic amine hydroxychloroquine, which is
clinically used for the treatment of malaria, rheumatoid
arthritis, and systemic lupus erythematosus. As do other
immunosuppressive drugs, this agent exerts potent
cytotoxic effects (Lai et al., 2001), which in part may
explain unwarranted effects on the nervous system and
the retina (Zaidi et al., 2001; Marmor et al., 2002). Here
we report that hydroxychloroquine (HCQ) induces cell
death apoptosis through a lysosomal mechanism. Our
data indicate that Bax/Bak-mediated MMP is a rate
limiting step of cell death, primarily triggered at the
lysosomal level. Mitochondrial alterations thus determine lysosome-initiated cell death induced by HCQ.
Results and discussion
HCQ triggers selective CB translocation from lysosomes
HCQ (which is fluorescent, Figure 1a) enriches in
cytoplasmic organelles as well as nucleoli (Figure 1a).
When cells are treated with bafilomycin A1 (Baf A1), a
specific inhibitor of the lysosomal proton pump
(vacuolar H þ ATPase) (Moriyama and Nelson, 1989),
the cytoplasmic organellar distribution of HCQ is lost.
Staining of cells with the acidophilic lysosomal probe
LysoTracker Red (LTR) revealed that HCQ caused an
increase in lysosomal volume. Concomitantly, HCQ
Figure 1 LMP induced by HCQ. (a) Subcellular localization of HCQ.
The low level of autofluorescence of HCQ (excitation 480740 nm,
emission 527730 nm) was detected in HCQ-treated HeLa cells (10 mg/
ml, 15 min), as well as in cells pretreated with Baf A1 (0.1 mm, 1 h before
HCQ). Note that Baf A1 prevents the appearance of a cytoplasmic
granular pattern of fluorescence, yet has no effect on the occasional
nuclear accumulation of HCQ. (b) Effect of HCQ and Baf A1 on the
staining with the acidophilic dye LTR and the marker of autophagic
vacuoles monodansylcadaverine (MDC). Doses are as in (a). Note that
HCQ transiently increases the volume and the frequency of
cytoplasmic granules staining with LTR or MDC. (c) Long-term
effect of HCQ on lysosomal AO staining. Cells were exposed to HCQ
(30 mg/ml) and then stained with AO, a metachromatic fluorochrome
exhibiting red fluorescence when highly concentrated in lysosomes and
a green fluorescence at low concentrations. Note that the frequency of
highly fluorescence red cytoplasmic granules declines over time. Cells
without red fluorescence lack lysosomes capable of accumulating AO.
(d) Cytofluorometric quantitation of AO staining. Cell treated and
stained as in (c) were subjected to FACS analysis. Values indicate the
percentage of cells manifesting an abnormally low AO fluorescence. (e)
CB translocation induced by HCQ. HeLa cells were stained for
immunofluorescence detection of the lysosomal membrane marker
LAMP and CB. Note that in control cells CB colocalizes with LAMP,
as demonstrated by the blend of the red and green fluorescence
(yellow). HCQ (30 mg/ml, 15 h) induced the translocation of CB to the
cytosol as well as to the nucleus. Pretreatment with Baf A1 greatly
reduced the frequency of cells manifesting the lysosomal release of CB.
Results are representative of at least three independent experiments
yielding similar results
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triggered an increase in the frequency of cytoplasmic
organelles staining with monodansylcadaverine, a dye
that specifically stains autophagic vacuoles (Biederbick
et al., 1995; Munafo and Colombo, 2001) (Figure 1b).
These effects were suppressed when cells were pretreated
with Baf A1. HCQ also caused a progressive decline in
the red staining of lysosomes with acridine orange (AO),
as determined at the single-cell level (Figure 1c) or by
cytofluorometry (Figure 1d). Finally, HCQ triggered the
release of CB from lysosomes (where it colocalizes
normally with the sessile lysosomal marker Lamp-1) to
the cytosol and the nucleus. This translocation of CB is
Lysosomes and mitochondria in apoptosis
P Boya et al
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inhibited by Baf A1. Altogether, these data suggest that
HCQ causes selective, Baf A1-inhibitable lysosomal
membrane permeabilization (LMP).
HCQ triggers apoptosis and MMP through a lysosomal
pathway
HCQ treatment resulted in marked cytoplasmic vacuolization and apoptosis with cellular shrinkage and
pathognonomic chromatin condensation. Importantly,
vacuolization became manifest before marked chromatin condensation and was accompanied by signs of
increased macroautophagy. These changes could be
detected by electron microscopy (Figure 2a), as well as
by Giemsa staining (Figure 2b). HCQ also caused
phosphatidylserine (PS) exposure on the outer leaflet of
the plasma membrane, which can manifest before
plasma membrane permeabilization (PMP) (Figure 2c).
Both PS exposure and PMP could be inhibited by
pretreating cells with Baf A1, indicating that they must
be secondary to LMP. HCQ did also trigger signs of
MMP. First, HCQ induced a mitochondrial transmembrane potential (DCm) loss, as detectable with two
different DCm-sensitive dyes, namely 3,30 -dihexyloxacarbocyanine iodide (DiOC6(3)) (which is amenable to
cytofluorometric analysis, Figure 3a) and JC-1 (which is
useful for microscopic observation, Figure 3b). Again,
these HCQ effects were fully blocked by BafA1
(Figure 3a), indicating that HCQ did indeed trigger
apoptosis by acting on lysosomes rather than on other
organelles such as mitochondria or nuclei. HCQ also
provoked the mitochondrial release of cytochrome c and
the concomitant activation of caspase-3 (Figure 3c), as
detected with an antibody specific for the large subunit
of caspase-3 (Casp-3a). To determine the temporal
order between LMP and MMP, HCQ-treated cells
were double-stained for the simultaneous detection of
CB and cytochrome c. While a fraction of cells
manifested a diffuse CB staining with a punctate
cytochrome c distribution (indicating LMP without
MMP), we found no cells in which cytochrome c would
be diffuse, yet CB was retained in lysosomes (Figure 3d).
Thus, LMP occurs clearly upstream of MMP in this
model.
HCQ-induced apoptosis involves the activation of
caspases and of proapoptotic Bcl-2 family members
To determine the relative contribution of cathepsines
and caspases to HCQ-induced apoptosis, cells were
pretreated with a panel of protease inhibitors. While
all tested cathepsin inhibitors failed to affect the death
of HCQ-treated cells, we found that the pan-caspase
inhibitor N-benzyloxycarbonyl-Phe-Ala-fluoromethylketone (Z-VAD.fmk) significantly reduced the HCQinduced killing (Figure 4a, b). Z-VAD.fmk fully blocked
Casp-3 activation (Figure 4c) and prevented the
HCQ-induced chromatin condensation and DNA
loss (Figure 4d). In many paradigms of apoptosis
induction, Bax translocates to mitochondria, where it
inserts into the outer membrane, exposes its N-terminus,
Figure 2 HCQ induces morphological hallmarks of autophagic
and apoptotic cell death. (a) Ultrastructure of HCQ-treated HeLa
cells. Cells were either left untreated (Co.) or treated with HCQ
(30 mg/ml, 15 h). Note that cells can manifest a notable degree
of cytoplasmic vacuolization and autophagic sequestration of
organelles before they undergo major signs of chromatin condensation. Such autophagic vacuoles are characterized by double
membranes. Later, signs of apoptotic chromatinolysis with
homogenous chromatin condensation are observed. (b) Cytoplasmic vacuolization as an early trait of HCQ-induced cell death.
Representative Giemsa stainings of control HeLa cells and HCQtreated cells showing cytoplasmic vacuolization without nuclear
apoptosis (at 6 h) or vacuolization with apoptosis (at 15 h) are
shown. Note that cells never manifest nuclear apoptosis without
prior vacuolization. Similar morphological changes have been
obtained in MEF and Rat-1 cells. (c) PS exposure determined by
annexin-V–FITC staining. HCQ-treated cells (30 mg/ml, 24 h)
optionally pretreated with Baf A1 were subjected to simultaneous
staining with Annexin V–FITC and PI, followed by cytofluorometric analysis
oligomerizes and coalesces into large complexes (Goping
et al., 1998; Gross et al., 1998; Griffiths et al., 1999).
HCQ was found to induce the ‘apoptotic conformation’
of Bax (Figure 5a), as detectable with specific monoclonal antibody recognizing the exposed N-terminus
of activated Bax. These alterations were not affected
by Z-VAD.fmk (Figure 5b), yet were inhibited by Baf
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Figure 3 HCQ induces hallmarks of MMP, downstream of LMP. (a) HCQ induces a drop in the DCm, as determined by FACS
analysis. Cells treated with HCQ and/or Baf A1 (as in Figure 2c) were simultaneously stained with the DCm-sensitive dye DiOC6(3) and
the vital dye PI. (b) HCQ induces a loss in the DCm, as determined by in situ fluorescence. HCQ-treated cells were stained with the
DCm-sensitive dye JC-1 (which indicates a DCm loss by a spectral red-green shift) and the number of green cells were determined. (c)
HCQ triggers the release of cytochrome c from mitochondria and concomitant caspase-3 activation. Cells were triple stained for the
detection of chromatin (Hoechst 33324, blue fluorescence), cytochrome c (green punctate staining in control cells, diffuse
cytosolic þ nuclear staining) and active caspase-3 (red fluorescence only detectable in cells manifesting a diffuse cytochrome c staining
pattern). (d) Cytochrome c release occurs after cathepsin B release. Control cells manifest cytoplasmic, punctate, nonoverlapping CB
and cytochrome c staining patterns. After HCQ treatment (30 mg/ml, 15 h), B30% of the cells manifest a diffuse staining pattern, both
for CB and cytochrome c, while 95% of cells show diffuse CB and punctate cytochrome c staining patterns (arrow). The opposite case
(punctate CB staining and diffuse cytochrome c staining) is not induced by HCQ, although it can be found among spontaneously
apoptotic (o2%) control cells (not shown)
A1 (not shown). Moreover, Z-VAD.fmk failed to inhibit
the release of both CB and cytochrome c from
lysosomes and mitochondria, respectively. These data
indicate that caspases operate downstream of LMP and
MMP.
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MMP is a rate limiting step of LMP-triggered apoptosis
To investigate the importance of MMP for apoptosis
induction by HCQ, we took advantage of a series of
cell lines in which MMP is blocked by genetic
Lysosomes and mitochondria in apoptosis
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Figure 5 Activation of Bax in HCQ-induced apoptosis. (a)
Detection of Bax activation by immunofluorescence. Control cells
or cells treated with HCQ (30 mg/ml, 15 h) were subjected to
immunostaining with antibodies specific for the N-terminus of Bax
(a) and the resident mitochondrial matrix protein Hsp60 and
counterstained with Hoechst 33324. Note that HCQ causes cells to
manifest mitochondrial staining of Bax with antibodies known to
react only with Bax in their apoptotic conformation. (b) Quantitation of lysosomal and mitochondrial alterations induced by HCQ
in the presence or absence of Z-VAD.fmk. Cells were treated with
HCQ as in (a) or (b), in the presence or absence of 100 mm ZVAD.fmk, followed by immunostaining to determine the percentage of cells manifesting a diffuse CB staining (determined as in
Figure 3d), a diffuse cytochrome c staining (determined as in
Figure 3c) and activation of Bax (determined as in Figure 5b).
Results are means of three determinations7s.d.
Figure 4 Involvement of caspases in HCQ-induced apoptosis. (a,
b) Inhibitory spectrum of cathepsin and caspase inhibitors on the
DCm loss (a) and the cell death (b) induced by HCQ. Cells were
treated with the indicated dose of HCQ for 15 h, in the presence or
absence of the indicated inhibitors of cathepsins (Z-FA.fmk, ZFF.fmk, Ca-074-Me, pepstatin 1) and caspases (Z-VAD.fmk), as
indicated in Materials and methods. Results (X7s.d., n ¼ 3) were
quantified by cytofluorometry after staining with DiOC6(3) and PI.
(c) Caspase-3 activation induced HCQ. Cells were treated for the
indicated period with HCQ (30 mg/ml), followed by immunoblotting for the detection of the active cleavage product of caspase-3
(Casp-3a). Note that no caspase activation occurs in the presence
of Z-VAD.fmk. Equal loading was controlled by immunodetection
of GAPDH. (d) Chromatinolysis and chromatin condensation
induced by HCQ. Cells treated for up to 60 h with HCQ (30 mg/
ml)7Z-VAD.fmk (100 mm) were subjected to ethanol fixation and
staining with DAPI followed by FACS quantification of DNA loss.
Insets demonstrate representative fluorescence micrographs. Numbers indicate the percentage of cells found in the corresponding
gate
manipulations. As shown in Figure 6a, HCQ killed
vector-only-transfected HeLa cells, yet was less efficient
in inducing MMP and PMP in HeLa cells stably
transfected with the MMP inhibitors Bcl-2 or Bcl-XL.
Similarly, HeLa cells expressing the MMP inhibitor
vMIA (viral mitochondrial inhibitor of apoptosis) (from
cytomegalovirus) (Goldmacher et al., 1999; Vieira et al.,
2001) were partially protected against the HCQ-driven
MMP and PMP (Figure 6a). These findings could be
substantiated in different cell lines, including BJAB Bcell leukemia and Jurkat T-lymphoma cells, in which
Bcl-2 or vMIA inhibited HCQ-induced killing (Figure
6b, c). Rat-1 fibrosarcoid cells were also partially
protected against HCQ, when engineered to overexpress
wild-type Bcl-2. When Bcl-2 was specifically targeted to
the endoplasmic reticulum (Bcl-Cb5) (Zhu et al., 1996),
it lost its cytoprotective action vis-à-vis of HCQ. In
contrast, Bcl-2 targeted to mitochondria (Bcl-ActA)
inhibited HCQ-mediated killing as efficiently as wildtype Bcl-2 (Figure 6c). Mouse embryonic fibroblasts
(MEF) lacking both Bax and Bak (Wei et al., 2001) were
completely resistant against HCQ-induced MMP and
PMP (Figure 7a). Partial inhibition against HCQ killing
was also obtained by knockout of Bax alone (Figure 7b).
To investigate the relative importance of Bax/Bak for
LMP and MMP, we double-stained control MEF and
Bax/Bak/ MEF cells with antibodies specific for
cytochrome c and CB. In wild-type MEF, HCQ induced
the release of both proteins from their respective
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Figure 6 MMP-inhibitory proteins inhibit HCQ-induced cell death. (a) Effect of Bcl-2, Bcl-XL, and vMIA on HCQ-induced killing in
HeLa cells. HeLa cell lines stably transfected with Bcl-2, Bcl-XL, or vMIA (UL37 from cytomegalovirus) or control cells (Neo) were
exposed to the indicated dose of HCQ during 24 h, followed by staining with DiOC6(3) and PI to assess the frequency of cells
manifesting a low DCm or plasma membrane permeabilization, respectively. (b) Effects of Bcl-XL and vMIA on HCQ-induced death in
BJAB B-cell leukemia cells. Cells were treated with HCQ (60 mg/ml, 16 h), and the frequency of DCmlow cells was determined as in (a).
(c) Effect of Bcl-2 on HCQ-induced death in Jurkat T-cell lymphoma cells. Control (Neo)- or Bcl-2-transfected Jurkat cells were
exposed to HCQ (60 mg/ml, 16 h), followed by DiOC6(3)/PI staining. (d) Effect of Bcl-2 targeted to mitochondria or to the endoplasmic
reticulum (ER) on HCQ-induced killing. Rat-1 cells engineered to express wild-type Bcl-2 or Bcl-2 specifically targeted to mitochondria
(Bcl-2 ActA) or to the ER (Bcl2 Cb5) were exposed to HCQ (60 mg/ml, 16 h) and the percentage of cells with a low DCm (DiOC6(3)low)
or a permeable plasma membrane (PI þ ) was determined as in (a) Results are representative of a minimum of three independent
determinations
organelles. In Bax/ Bak/ MEF, HCQ did induce the
redistribution of CB (Figure 7c), yet failed to induce the
mitochondrial release of cytochrome c (not shown),
loss of the DCm and cell death (Figure 6a). Altogether
these data indicate that Bax/Bak-mediated MMP, downstream of LMP, is an obligate step of LMP-triggered
apoptosis.
Concluding remarks
Based on the data accumulating in this paper, HCQ
induces a precise sequence of subcellular alterations
culminating into cell death. This sequence involves (i)
lysosomal accumulation resulting into selective LMP
with release of lysosomal enzymes such as CB, (ii)
activation of Bax with consequent MMP, and (iii)
caspase activation and apoptosis. This hierarchy
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(LMP-MMP-caspase activation) is demonstrated
by the facts that (i) Baf A1, an inhibitor of the lysosomal
accumulation of HCQ (Figure 1a), prevents all signs of
HCQ-induced LMP (Figure 1b, e), MMP (Figure 3a),
and apoptosis (Figure 2c); that (ii) inhibitors of MMP
such as Bcl-2, Bcl-XL, vMIA or the absence of Bax and/
or Bak largely prevent HCQ-induced MMP and
apoptosis but not LMP (Figures 6 and 7), and (iii) that
Z-VAD.fmk stops caspase activation and retards cell
death, yet has no effect on HCQ-induced LMP and a
minor inhibitory effect on HCQ-induced MMP (Figures
4 and 5). These data thus place MMP as an obligate link
between LMP, on the one hand, and caspase activation,
on the other hand. Although it is possible that long-term
LMP might cause cell death in the absence of MMP and
caspase activation, our data clearly indicate that
inhibition of MMP and caspases retards cell death for
a considerable period, that is several days (Figure 4d).
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Figure 7 Requirement of proapoptotic Bcl-2 family members for the induction of apoptosis by HCQ. (a) Effect of the combined
knockout of Bax and Bak on HCQ-induced killing. MEF lacking both Bax and Bak and wild-type controls were exposed to 20 or
40 mm HCQ (18 h) and then subjected to the assessment of the frequency of cells with a low DiOC6(3) incorporation, positive Annexin
V staining or PMP. (b) Effect of the knockout of Bax or Bak on HCQ-triggered cell death. MEF with the designated genotypes were
exposed to HCQ and the indicated parameters were assessed. (c) Failure of the double knockout or Bax and Bak to inhibit the HCQinduced lysosomal release of CB. Wild-type or Bax/ Bak/ MEF were exposed to HCQ (30 mg/ml, 18 h), and the frequency of cells
manifesting a diffuse CB staining (exemplified in the left panel) and diffuse cytochrome c release was assessed by immunofluorescence
staining. Results are mean values7s.d. of a minimum of three independent experiments
The data presented here are incompatible with the
previous studies performed in cell-free systems, which
suggested a direct link between lysosomal activation/
permeabilization and caspase activation. Thus, it has
been suggested that CB would directly activate caspase11 (Schotte et al., 1998) and/or trigger apoptotic
chromatin condensation and nuclear DNA loss (Vancompernolle et al., 1998). However, the presence of CB in
the nucleus is not sufficient to induce apoptosis, as shown
by the fact that Bax/Bak/ cells can release cathepsin
from lysosomes, yet manifest a normal chromatin
structure and fail to translocate cytochrome c (Figure 7).
It has been suggested that cathepsin L released from
lysosomes might proteolytically activate Bid, which in
turn would act on mitochondria to cause MMP and
subsequent caspase activation (Zamzami et al., 2000;
Stoka et al., 2001). However, Bid/ cells were found to
manifest MMP at least as efficiently as control cells (not
shown), indicating that Bid is not the major factor
linking LMP to MMP. In contrast, either Bax or Bak
are rate limiting for the LMP-induced MMP, as
demonstrated by the fact that the knockout of either
Bax or Bak (but in particular the knockout of both Bax
and Bak) greatly reduces HCQ-induced MMP and subsequent cell death (Figure 7). Bax is activated in an
orthodox fashion subsequent to HCQ-induced LMP, as
indicated by the finding that Bax manifests a mitochondrial staining pattern while exposing its N-terminus
(Figure 5a, b). This conformational change of Bax is
commonly observed in different models of apoptosis
induction (Goping et al., 1998; Gross et al., 1998;
Griffiths et al., 1999; Nechushtan et al., 2001). However,
how Bax is activated in response to LMP remains an ongoing conundrum. Thus, a variety of cathepsin-specific
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inhibitors failed to prevent MMP (Figure 4a), and cells
engineered to lack the expression of the lysosomal
cathepsins B, D, L, or S were at least as sensitive as wildtype control cells to HCQ-induced killing (not shown),
suggesting that other lysosomal enzymes thus far not
involved in apoptosis control and/or indirect changes in
cellular physiology may account for the HCQ-induced
Bax/Bak activation and MMP.
The data presented in this paper reinforce the general
idea that MMP is rate limiting for cell death induction
(Boya et al., 2003), even when apoptosis has initially
been triggered by permeabilizing the ‘suicide bags’, as
lysosomes have been initially nick-named (De Duve and
Wattiaux, 1966; Lockshin and Zakeri, 2001). This
finding has far reaching implications for our current
understanding of ‘type I’ (apoptotic) versus ‘type II’
(autophagic/lysosomal) cell death. Indeed HCQ-induced
cell death is preceded by signs of ‘type II cell death’,
namely increased autophagy (Figure 1b), organellar
sequestration in autophagosomes (Figure 2a) and cytoplasmic vacuolization (Figure 2a, b), followed by later
signs of ‘type I cell death’, as indicated by chromatin
condensation, caspase activation, DNA loss and shrinkage (Figures 1 and 2). Future studies will confirm whether
cellular demise initiated as ‘type II cell death’ always
requires a mitochondrial contribution or whether some
physiological stimuli, lysosomal activators or cell types
may bypass the stringent requirement for mitochondria.
Importantly, it appears that MMP-inhibitory, Bcl-2-like
molecules have an unsuspectedly wide range of cytoprotective action, thereby further emphasizing why these
oncoproteins are that important for cancer development.
Materials and Methods
Cell lines and culture conditions
HeLa and BJAB cells transfected with pcDNA3.1 control
vector (Neo), human Bcl-2 or the cytomegalovirus UL37 exon
1 gene coding for vMIA (were a gift by Dr V Goldmacher)
(Goldmacher et al., 1999). Jurkat cells transfected with Neo
or Bcl-2 were kindly provided by Nicole Israel (Aillet et al.,
1998). Rat-1/Myc fibroblasts (kindly provided by Dr David
Andrews) were stably transfected with pRc/CMV-based
plasmids (Neo) and engineered to express human wild-type
Bcl-2, ER-targeted Bcl-2-Cb5, or mitochondrion-targeted Bcl2 ActA (Zhu et al., 1996; Annis et al., 2001). These cells were
cultured in DMEM medium supplemented with 10% FCS,
1 mm pyruvate, 10 mm HEPES and 100 U/ml penicillin/
streptomycin at 371C in 5% CO2. SV40-transformed MEF
whose genotype was either wild-type (WT), Bax/, Bak/,
Bax/Bak/ double knockout (Wei et al., 2001) were a gift by
Dr Stanley Korsmeyer. MEF were cultured in IMDM (Life
Technologies) with 20% FCS, 1 NEAA (Sigma), and 100 U/
ml pencillin/streptomycin. Cells were cultured in the presence
of the indicated dose of HCQ (Sanofi-Synthélabo, diluted
from a stock solution of 30 mg/ml). Baf A1 (Baf A1,
Sigma, 0.1 mm), N-benzyloxycarbonyl-Phe-Ala-fluoromethylketone (z-FA-fmk, 100 mm; Bachem), N-benzyloxycarbonyl-ValAla-Asp-fluoromethylketone (z-VAD-fmk; 100 mm; Bachem),
N-benzyloxycarbonyl-Phe-Phe-fluoromethylketone (z-FF-fmk;
100 mm; Enzyme System Products), CA-074-Me (10 mm, Peptides International), or pepstatin A (100 mm, Sigma) were
added 1 h before the addition of HCQ.
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Quantification of apoptosis-associated changes by
cytofluorometry
To determine the intracellular localization of HCQ, cells
cultured on coverslips were treated with 10 mg/ml of HCQ for
15 min and subjected to fluorescence microscopy. To label
lysosomes, cells were stained with either 0.5 mm LysoTracker
Red (Molecular Probes) or 5 mm AO (Sigma) for 15–30 min at
371C and then analysed by fluorescence microscopy or flow
cytometry (Zhao et al., 2001) using a FACSs Vantage (Becton
Dickinson). DiOC6(3) (40 nm) was employed for cytofluorometric DCm quantification, propidium iodide (PI, 1 mg/ml) for
determination of cell viability, and Annexin V labeled with
fluorescein isothiocyanate (FITC) (Bender Medsystems) for
the assessment of PS exposure (Zamzami et al., 1995; Castedo
et al., 1996, 2002). Cells were trypsinized and labeled with the
fluorochromes at 371C followed by cytofluorometric analysis.
Quantification of DNA content was performed on ethanolfixed stained with 40 ,6-diamidino-2-phenylindole, dihydrochloride (DAPI, 2.5 mg/ml, Molecular Probes) for 30 min
at 371C. For the assessment of the DCm in situ, cells grown on
cover slips were incubated with 5,50 ,6,60 -tetrachloro-1,10 ,3,30 tetraethylbenzimidazolylcarbocyanine iodide (JC-1, 3 mm, Molecular Probes) and Hoechst 33342 (2 mm, Sigma) (Castedo
et al., 2001; Ferri et al., 2000).
Immunofluorescence, electron microscopy, Giemsa staining, and
immunochemistry
For immunofluorescence staining, cells were fixed with
paraformaldehyde (4% w : v) and picric acid (0.19% v : v)
(Daugas et al., 2000) and stained for the detection of
cytochrome c (mAb 6H2.B4 from Pharmingen), activated
Bax (mAb 6A7, Pharmingen), CB (Calbiochem), a polyclonal
rabbit antibody recognizing activated caspase-3 (Casp-3a,
Cell Signaling Technology), or Lamp1 (mAb from BD
Transduction laboratories), all detected by a goat anti-mouse
or goat anti-rabbit IgG conjugated with Alexas fluorochromes
(Molecular Probes). Bars represent 10 mm. For electron
microscopy, cells were fixed for 1 h at 41C in 2.5%
glutaraldehyde in PBS (pH 7.4), washed and fixed again in
2% osmium tetroxide, before embedding in Epon. Electron
microscopy was performed with an Leo 902 electron microscope, at 80 kv, on ultrathin sections (80 nm) stained with
uranyl acetate and lead citrate. Bars represent 5 mm. Giemsa
stainings were performed using a kit from Sigma. To confirm
caspase-3 activation, cells were lysed for 15 min in 50 mm
HEPES, 150 mm NaCl, 5 mm EDTA, 0.1% NP-40, supplemented with protease inhibitor cocktail (Roche), 1 mm DTT
and 1 mm PMSF, and then centrifuged at 13 000 g for 10 min
to remove cell debris. Protein (40 mg) was loaded on a 15%
SDS–PAGE. Anti-glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) (Chemicon) was used to control equal loading, and
an antiserum recognizing Casp3a (Cell Signaling Technology)
was used for the detection of caspase activation.
Abbreviations
AO, acridine orange; Baf A1, bafilomycin A1; Casp-3a,
activated caspase-3; CB, cathepsin B; DCm, mitochondrial
transmembrane potential; DAPI, 40 ,6-diamidino-2-phenylindoledihydrochloride; DiOC6(3), 3,30 -dihexyloxacarbocyanine
iodide, DN,dominant negative; GAPDH, glyceraldehyde-3phosphate dehydrogenase; JC-1,5,50 ,6,60 -tetrachloro-1,10 ,3,
30 -tetraethylbenzimidazolylcarbocyanine iodide; HCQ, hydroxychloroquine; LMP, lysosomal membrane permeabilization;
MMP, mitochondrial membrane permeabilization; PMP,
plasma membrane permeabilization; PS, phosphatidylserine;
Lysosomes and mitochondria in apoptosis
P Boya et al
3935
z-FA-fmk, N-benzyloxycarbonyl-Phe-Ala-fluoromethylketone;
Z-VAD.fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; vMIA, viral mitochondrial inhibitor of apoptosis.
Acknowledgements
We thank Drs Victor Goldmacher (ImmunoGen, Cambridge,
MA, USA) for cell lines, David Andrews (Hamilton University,
Ontario, Canada), Nicole Israel (Pasteur Institute, Paris,
France), Nathanael Larochette, and Didier Métivier (CNRS,
Villejuif, France) for assistance, Dominique Coulaud (CNRS,
UMR5826, Villejuif, France), and the NIH AIDS reagents
program (Bethesda, MD) for cell lines.This work has been
supported by a special grant from LNC, as well as grants from
ANRS, FRM, and European Commission (QLG1-CT-199900739) (to GK). PB receives a fellowship from the European
Commission (MCFI-2000-00943).
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