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. 2003 May 19;197(10):1323-34.
doi: 10.1084/jem.20021952.

Lysosomal membrane permeabilization induces cell death in a mitochondrion-dependent fashion

Patricia Boya  1 , Karine Andreau, Delphine Poncet, Naoufal Zamzami, Jean-Luc Perfettini, Didier Metivier, David M Ojcius, Marja Jäättelä, Guido Kroemer
Affiliations

Lysosomal membrane permeabilization induces cell death in a mitochondrion-dependent fashion

Patricia Boya et al. J Exp Med. .

Abstract

A number of diseases are due to lysosomal destabilization, which results in damaging cell loss. To investigate the mechanisms of lysosomal cell death, we characterized the cytotoxic action of two widely used quinolone antibiotics: ciprofloxacin (CPX) or norfloxacin (NFX). CPX or NFX plus UV light (NFX*) induce lysosomal membrane permeabilization (LMP), as detected by the release of cathepsins from lysosomes. Inhibition of the lysosomal accumulation of CPX or NFX suppresses their capacity to induce LMP and to kill cells. CPX- or NFX-triggered LMP results in caspase-independent cell death, with hallmarks of apoptosis such as chromatin condensation and phosphatidylserine exposure on the plasma membrane. LMP triggers mitochondrial membrane permeabilization (MMP), as detected by the release of cytochrome c. Both CPX and NFX* cause Bax and Bak to adopt their apoptotic conformation and to insert into mitochondrial membranes. Bax-/- Bak-/- double knockout cells fail to undergo MMP and cell death in response to CPX- or NFX-induced LMP. The single knockout of Bax or Bak (but not Bid) or the transfection-enforced expression of mitochondrion-targeted (but not endoplasmic reticulum-targeted) Bcl-2 conferred protection against CPX (but not NFX*)-induced MMP and death. Altogether, our data indicate that mitochondria are indispensable for cell death initiated by lysosomal destabilization.

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Figures

Figure 1.
Figure 1.
Specific lysosomal destabilization by NFX* and CPX. (A) Lysosomal localization of NFX and CPX. HeLa cells were incubated with 10 μg/ml NFX or CPX, and the CPX- or NFX-mediated fluorescence was determined in control cells or in cells pretreated with 100 nM Baf A1. The percentage of cells exhibiting a clear cytoplasmic punctate (lysosomal) or a nucleolar staining was determined. (B and C) NFX*- and CPX-mediated disruption of acidic vacuoles. Cells were stained with LysoTracker red (B) or AO (C) after treatment with UV, NFX alone, NFX*, or CPX, followed by fluorescence microscopy (B) or cytofluorometric analysis of the 630 ± 11-nm fluorescence (C). Numbers indicate the percentage of cells with decreased AO red fluorescence. (D and E) Translocation of cathepsins B and D from lysosomes. Cells treated for 8 h were fixed, permeabilized, and stained for the immunofluorescence of cathepsin B (D) and D (E). Results are representative of three to five independent determinations.
Figure 2.
Figure 2.
Caspase-independent cell death induction by NFX* and CPX. (A) PS exposure resulting from lysosomal destabilization. After the indicated treatment, cells were stained for PS exposure (with annexin V–FITC) or plasma membrane permeabilization (with PI) and subjected to cytofluorometric analysis. Numbers in each quadrant refer to the percentage of cells. (B) Quantitation of data obtained in A. (C) DNA hypodiploidy induced by NFX* and CPX. After 24 h, the nuclear DNA content was determined with DAPI by cytofluorometric analysis in fixed-permeabilized cells. Numbers indicate the percentage of cells exhibiting a subdiploid (apoptotic) DNA content. (D) Immunoblot determination of caspase-3 maturation. Cell extracts obtained after the indicated treatment and interval were subjected to immunoblotting with antibodies specific for cleaved caspase-3 (17-kD fragment) and glyceraldehyde-3-phosphate dehydrogenase (as a loading control). (E) Giemsa staining of HeLa cells treated with NFX*, CPX, or αCD95, in the absence or presence of 100 μM z-VAD-fmk. (F) Chromatin structure of living HeLa cells stably expressing a histone H2B-GFP fusion protein after treatment with NFX* or CPX. Representative micrographs of the GFP fluorescence and phase-contrast pictures are shown. (G) Ultrastructure of NFX*- or CPX-treated cells exhibiting nuclear chromatin condensation and vacuolization, as compared with apoptosis induced by αCD95. (H) Chromatin condensation induced by NFX* or CPX, both in the absence or presence of 100 μM z-VAD-fmk, as determined in F. (I) Failure of z-VAD-fmk (100 μM) to inhibit PS exposure in NFX*- and CPX-treated cells. Data are shown as mean values ± SD of three independent experiments.
Figure 3.
Figure 3.
MMP induced by NFX* and CPX. (A) Dissipation of the mitochondrial transmembrane potential (ΔΨm). After NFX* (or UV and NFX alone) or CPX treatment, cells were stained simultaneously with DiOC6(3), which incorporates into cells driven by the ΔΨm, and the vital marker propidium iodide (PI). (B) Dose response of the ΔΨm dissipation and viability loss induced by NFX* and CPX, determined as in A. (C) In situ determination of ΔΨm loss, as indicated by the spectral red–green shift of JC-1–stained cells. (D) In situ evidence for outer mitochondrial membrane permeabilization. After an 8-h treatment and fixation, cells were stained for the immunodetection of cytochrome c (green fluorescence) and counterstained with Hoechst 33324. (E) Caspase independence of mitochondrial changes. Cells were pretreated (60 min) with subtoxic doses of z-VAD-fmk (100 μM), exposed overnight to NFX* or CPX, followed by detection of ΔΨm reduction. (F) Cytochrome c release occurs after cathepsin B translocation. Cells were treated for 8 h with NFX* or CPX and subjected to simultaneous immunofluorescence staining for cytochrome c (red) and cathepsin B (green). Note that some cells (arrows) have a diffuse cathepsin B distribution, yet still retain cytochrome c in a punctate pattern.
Figure 4.
Figure 4.
NFX* and CPX exert their mitochondriotoxic and cytotoxic effect via lysosomes. (A and B) Effect of the vacuolar ATPase inhibitor Baf A1 on ΔΨm loss and cell death. Cells were pretreated for 1 h with Baf A1 and exposed overnight to NFX* or CPX, followed by staining with DiOC6(3) (A) and PI (B). (C and D) Baf A1–mediated protection against lysosomal and mitochondrial permeabilization. Cells treated as in A and B were subjected to immunofluorescence detection of cytochrome c (C) or cathepsin B (D). Representative cells showing that Baf A1 pretreatment maintains cytochrome c and cathepsin B in cytoplasmic organelles are shown.
Figure 5.
Figure 5.
Activation of Bax and Bak by NFX* and CPX through a p53-independent mechanism. (A) Conformational change of Bax and Bak. Cells were stained with monoclonal antibodies recognizing the apoptotic conformation of Bax (6A7) and Bak (Ab-1) 8 h after treatment with NFX* or CPX. (B) Time course of lysosomal and mitochondrial permeabilization induced by NFX* and CPX, as determined by staining with LysoTracker Red and DiOC6(3) in unfixed cells. (C) Kinetics of NFX*- and CPX-induced lysosomal cathepsin B release, Bax activation (determined as in A), mitochondrial cytochrome c release, and caspase-3 activation, as determined by immunofluorescence of fixed and permeabilized cells. Results are representative of three experiments. (D) Failure of NFX* and CPX to activate p53-dependent transcription. Cells were transfected with a p53-inducible GFP reporter construct, and the percentage of cells expressing GFP was determined by FACS® analysis. DNA damage by etoposide served as positive control. (E) Failure of pifithrin-α to inhibit NFX*- and CPX-induced cell death. Cells were pretreated for 60 min with the p53 inhibitor pifithrin-α, and the frequency of formula image (DiOC6(3)low) and death (PIhigh) was determined by cytofluorometry.
Figure 6.
Figure 6.
Requirement of Bax and/or Bak for NFX*- or CPX-induced cell death. (A) SV40-transformed WT MEF or Bax−/− Bak−/− DKO cells were exposed to NFX* and CPX for the indicated period, followed by determination of the frequency of DiOC6(3)low and annexin V–positive cells. (B) MEF lacking Bax, Bak, Bax and Bak (DKO), or Bid were exposed to either NFX* or CPX and the indicated parameters were measured. (C) NFX*- and CPX-induced cathepsin B release in control and Bax−/− Bak−/− DKO cells, as determined by immunostaining. The percentage of cells exhibiting diffuse cathepsin B staining as well as those with diffuse cytochrome c staining has been quantified. (D) Failure of chemical cathepsin inhibition to prevent NFX*- or CPX-induced cell death. HeLa cells were pretreated with the indicated cathepsin inhibitors, followed by determination of the frequency of DiOC6(3)low and PIhigh cells. (E) Cathepsins are dispensable for NFX*- or CPX-induced cell death. MEF with the indicated genotypes were treated with NFX* or CPX, and apoptotic parameters were measured as in D. Results are means ± SD of three independent determinations.
Figure 7.
Figure 7.
Mitochondrial membrane stabilization by Bcl-2 or vMIA prevent CPX-induced cell death. (A) HeLa cells stably transfected with vector only (Neo), or cDNAs coding for human Bcl-2 or cytomegalovirus-derived vMIA were treated with NFX* or CPX, and the frequency of DiOC6(3)low or PIhigh cells was assessed. Note that Bcl-2– and vMIA-expressing cells are more susceptible to cell death induction by NFX*, yet relatively resistant to CPX. (B) Mitochondrial, not ER-localized, Bcl-2 prevents CPX-induced cell death. Rat1 cells stably transfected with vector only (Neo), WT Bcl-2, mitochondrion-targeted Bcl-2 (Bcl-2 acta), and ER-targeted Bcl-2 (Bcl-2 cathepsin B5), were subjected to the indicated treatment, and the loss of mitochondrial and plasma membrane integrity was assessed with DiOC6(3) and PI, respectively. (C) Failure of Bcl-2 or vMIA to confer lysosomal stabilization. HeLa cells expressing Neo, Bcl-2, or vMIA were treated with NFX* or CPX, fixed, and immunostained for cathepsin B. Note that all cells exhibit a diffuse cathepsin B staining, although Bcl-2 and vMIA do inhibit CPX-induced MMP (A) and cell death (B) as an internal control of their efficacy.
Figure 8.
Figure 8.
Differential involvement of ROS in CPX- and NFX-induced cell death. (A) Relationship between ROS production and ΔΨm loss triggered by NFX* and CPX. HeLa cells were exposed to NFX* or CPX for the indicated interval, and stained simultaneously with the ΔΨm-sensitive dye DiOC6(3) and the ROS-sensitive probe hydroethidine (HE), whose oxidation by superoxide anions yields the red fluorescent product ethidium (Eth). Note that NFX*treatment causes ROS production before the ΔΨm drops, whereas after CPX treatment, ROS production is only observed in a subpopulation of DiOC6(3)low cells. (B–E) Effects of antioxidants on CPX- and NFX-induced cell death. HeLa cells were pretreated (60 min) with the cell-permeable GSH ethyl ester (10 mM) or the superoxide dismutase analogue MnTBAP (100 μM), followed by overnight treatment with NFX* or CPX and determination of the frequency of formula image, ROS-overproducing (B), PS-exposing, dead (C) cells, and cells exhibiting Bax in the apoptotic conformation (D, determined with the conformation-specific 6A7 mAB as in Fig. 5 A) and diffuse cathepsin B staining (E, determined as in Fig. 1 D).
Figure 9.
Figure 9.
Effect of the Bax/Bak DKO on apoptosis induced by two different lysomal stimuli. (A) Chloroquine-induced ΔΨm loss and cell death is inhibited by Baf A1 as well as by ablation of Bax and Bak. WT MEF or DKO MEF were exposed to 30 μg/ml chloroquine (24 h), followed by determination of DiOC6(3)/PI staining and flow cytometric analysis. (B) Chloroquine-induced cathepsin B translocation, cytochrome c release, and chromatin condensation are inhibited in DKO cells. Cells, treated as in A, were subjected to immunofluorescence analysis (anti–cathepsin B, anti–cytochrome c, and Hoechst 33324), and the cells exhibiting the indicated phenotype were determined. (C) DAP kinase-induced cell death and ΔΨm loss in WT and DKO MEF. Cells were transfected with the constitutively active DAP kinase construct ΔCaM or controls. After 72 h, the cells were stained with DiOC6(3) and DAPI, and the percentage of formula image (DiOC6(3)low) or dead (DAPI+) cells was measured by cytofluorometry.
Figure 10.
Figure 10.
Hypothetical mechanisms through which CPX and NFX* kill cells. CPX causes death through lysosomal membrane permeabilization (LMP), upstream of MMP, caspase-3 activation, and activation of caspase-independent death effectors including ROS. NFX* causes death through ROS-mediated phototoxic effects, upstream of LMP, and MMP. Perhaps as a result of the primary effect on cellular redox potentials, MMP occurs in a Bcl-2–independent fashion and does not result in caspase-3 activation.
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