Oncogene (2003) 22, 3927–3936
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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
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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 Oncogene
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
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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 Oncogene
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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. Oncogene
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
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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-a`-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 Oncogene
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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 Oncogene
(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 Oncogene
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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-Synthe´labo, 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. Oncogene
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;
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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 Me´tivier (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).
References Aillet F, Masutani H, Elbim C, Raoul H, Chene L, Nugeyre MT, Paya C, Barre-Sinoussi F, Gougerot-Pocidalo MA and Israel N. (1998). J. Virol., 72, 9698–9705. Annis MG, Zamzami N, Zhu W, Penn LZ, Kroemer G, Leber B and Andrews DW. (2001). Oncogene, 20, 1939–1952. Biederbick A, Kern HF and Elsasser HP. (1995). Eur. J. Cell Biol., 66, 3–14. Boya P, Cohen I, Zamzami N, Vieira HLA and Kroemer G. (2002). Cell Death Differ., 9, 465–467. Boya P, Andreau K, Poncet D, Zamzami N, Perfettini JL, Metivier D, Ojcius DM, Ja¨a¨ttela¨ M and Kroemer G. (in press). Castedo M, Ferri KF, Blanco J, Roumier T, Larochette N, Barretina J, Amendola A, Nardacci R, Metivier D, Este JA, Piacentini M and Kroemer G. (2001). J. Exp. Med., 194, 1097–1110. Castedo M, Ferri K, Roumier T, Metivier D, Zamzami N and Kroemer G. (2002). J. Immunol. Methods, 265, 39–47. Castedo M, Hirsch T, Susin SA, Zamzami N, Marchetti P, Macho A and Kroemer G. (1996). J. Immunol., 157, 512–521. Cohen I, Castedo M and Kroemer G. (2002). Trends Cell Biol., 12, 293–295. Daugas E, Susin SA, Zamzami N, Ferri K, Irinopoulos T, Larochette N, Prevost MC, Leber B, Andrews D, Penninger J and Kroemer G. (2000). FASEB J., 14, 729–739. De Duve C and Wattiaux R. (1966). Annu. Rev. Physiol., 28, 435–492. Debatin KM, Poncet D and Kroemer G. (2002). Oncogene, 21, 8786–8803. Ferri KF, Jacotot E, Blanco J, Este´ JA, Zamzami A, Susin SA, Brothers G, Reed JC, Penninger JM and Kroemer G. (2000). J. Exp. Med., 192, 1081–1092. Ferri KF and Kroemer GK. (2001). Nat. Cell Biol., 3, E255–E263. Foghsgaard M, Wissing D, Mauch D, Lademann U, Bastholm L, Boes M, Elling F, Leist M and Ja¨a¨ttela¨ M. (2001). J. Cell Biol., 153, 999–1009. Goldmacher VS, Bartle LM, Skletskaya S, Dionne CA, Kedersha NL, Vater CA, Han JW, Lutz RJ, Watanabe S, McFarland EDC, Kieff ED, Mocarski ES and Chittenden T. (1999). Proc. Natl. Acad. Sci. USA, 96, 12536–12541. Goping IS, Gross A, Lavoie JN, Nguyen M, Jemmerson R, Roth K, Korsmeyer SJ and Shore GC. (1998). J. Cell Biol., 143, 207–215. Green DR and Reed JC. (1998). Science, 281, 1309–1312. Griffiths GJ, Dubrez L, Morgan CP, Jones NA, Whitehouse J, Corfe BM, Dive C and Hickman JA. (1999). J. Cell Biol., 144, 903–914. Gross A, Jockel J, Wei MC and Korsmeyer SJ. (1998). EMBO J., 17, 3878–3885. Inbal B, Bialik S, Sabanay I, Shani G and Kimchi A. (2002). J. Cell Biol., 157, 455–468.
Kitanaka C, Kato K, Ijiri R, Sakurada K, Tomiyama A, Noguchi K, Nagashima Y, Nakagawara A, Momoi T, Toyoda Y, Kigasawa H, Nishi T, Shirouzu M, Yokoyama S, Tanaka Y and Kuchino Y. (2002). J. Natl. Cancer Inst., 94, 358–368. Kroemer G and Reed JC. (2000). Nat. Med., 6, 513–519. Lai JH, Ho L-J, Lu K-C, Chang D-M, Shaio M-F and Han SH. (2001). J. Immunol., 166, 6914–6924. Lassus P, Opitz-Araya X and Lazebnik Y. (2002). Science, 297, 1352–1354. Levy-Strumpf N and Kimchi A. (1998). Oncogene, 17, 3331–3340. Lockshin RA and Zakeri Z. (2001). Nat. Rev. Mol. Cell Biol., 2, 545–550. Marmor MF, Carr RE, Easterbrook M, Farjo AA and Mieler WF. (2002). Ophthalmology, 109, 1377–1382. Marsden VS, O’Connor L, O’Reilly LA, Silke J, Metcalf D, Ekert PG, Huang DC, Cecconi F, Kuida K, Tomaselli KJ, Roy S, Nicholson DW, Vaux DL, Bouillet P, Adams JM and Strasser A. (2002). Nature, 419, 634–637. Mathiasen IS and Jaattela M. (2002). Trends Mol. Med., 8, 212–220. Moriyama Y and Nelson N. (1989). J. Biol. Chem., 264, 18445–18450. Munafo DB and Colombo MI. (2001). J. Cell Sci., 114, 3619–3629. Nechushtan A, Smith CL, Lamensdorf I, Yoon SH and Youle RJ. (2001). J. Cell Biol., 153, 1265–1276. Nicholson DW and Thornberry NA. (1997). Trends Biochem. Sci., 22, 299–306. Ravagnan L, Roumier T and Kroemer G. (2002). J. Cell. Physiol., 192, 131–137. Reed JC. (2002). Nat. Rev. Drug Discov., 1, 111–121. Roberg K. (2001). Lab. Invest., 81, 149–158. Schotte P, Van Criekinge W, Van de Craen M, Van Loo G, Desmedt M, Grooten J, Cornelissen M, de Ridder L, Vandkerckhove J, Fiers W, Vandenabeele P and Beyaert R. (1998). Biochem. Biophys. Res. Commun., 251, 379–387. Stoka V, Turk B, Schendel SL, Kil TH, Cirman T, Snipas SJ, Ellerby LM, Bredesen D, Freeze H, Abrahamson M, Bromme D, Krajewski S, Reed JC, Yin XM, Turk V and Salvesen GS. (2001). J. Biol. Chem., 276, 3149–3157. Vancompernolle K, Van Herrewghe F, Pynaert G, Van de Craen M, De Vos K, Totty N, Sterling A, Fiers W, Vandenabeele P and Grooten J. (1998). FEBS Lett., 6, 150–158. Vieira HL, Belzacq A-S, Haouzi D, Bernassola F, Cohen I, Jacotot E, Ferri KF, Hamel EH, Bartle LM, Melino G, Brenner C, Goldmacher V and Kroemer G. (2001). Oncogene, 20, 4305–4316. Vogelstein B, Lane D and Levine AJ. (2000). Nature, 408, 307–310. Wei MC, Zong W-X, Cheng EH-Y, Lindsten T, Panoutsakopoulou V, Ross AJ, Roth KA, MacGregor GR, Thompson CB and Korsmeyer SJ. (2001). Science, 292, 727–730. Oncogene
Lysosomes and mitochondria in apoptosis P Boya et al
3936 Xue L, Fletcher GC and Tolkovsky AM. (2001). Curr. Biol., 6, 361–365. Yuan XM, Li W, Dalen H, Lotem J, Kama R, Sachs L and Brunk UT. (2002). Proc. Natl. Acad. Sci. USA, 99, 6286–6291. Zaidi AU, McDonough JS, Klocke BJ, Latham CB, Korsmeyer SJ, Flavell RA, Schmidt RE and Roth KA. (2001). J. Neuropathol. Exp. Neurol., 60, 937–945. Zamzami N, Brenner C, Marzo I, Susin SA and Kroemer G. (1998). Oncogene, 16, 2265–2282.
Oncogene
Zamzami N, El Hamel C, Mun˜oz C, Brenner C, Belzacq A-S, Costantini P, Molle G and Kroemer G. (2000). Oncogene, 19, 6342–6350. Zamzami N and Kroemer G. (2003). Curr. Biol., 13, R71–R73. Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T, Susin SA, Petit PX, Mignotte B and Kroemer G. (1995). J. Exp. Med., 182, 367–377. Zhao M, Eaton JW and Brunk UT. (2001). FEBS Lett., 509, 405–412. Zhu W, Cowie A, Wasfy GW, Penn LZ, Leber B and Andrews DW. (1996). EMBO J., 15, 4130–4141.
