Skip to main content
NCBI home page
International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2005 Oct;86(5):309–321. doi: 10.1111/j.0959-9673.2005.00442.x

Lysosomal membrane permeabilization during apoptosis – involvement of Bax?

Katarina Kågedal *, Ann-Charlotte Johansson *, Uno Johansson *, Gerd Heimlich †, Karin Roberg ‡, Nancy S Wang §, Juliane M Jürgensmeier †, Karin Öllinger *
PMCID: PMC2517437  PMID: 16191103

Abstract

Bcl-2 family members have long been known to control permeabilization of the mitochondrial membrane during apoptosis, but involvement of these proteins in lysosomal membrane permeabilization (LMP) was not considered until recently. The aim of this study was to investigate the mechanism underlying the release of lysosomal proteases to the cytosol seen during apoptosis, with special emphasis on the role of Bax. In human fibroblasts, exposed to the apoptosis-inducing drug staurosporine (STS), the release of the lysosomal protease cathepsin D to the cytosol was observed by immunocytochemistry. In response to STS treatment, there was a shift in Bax immunostaining from a diffuse to a punctate pattern. Confocal microscopy showed co-localization of Bax with both lysosomes and mitochondria in dying cells. Presence of Bax at the lysosomal membrane was confirmed by immuno-electron microscopy. Furthermore, when recombinant Bax was incubated with pure lysosomal fractions, Bax inserted into the lysosomal membrane and induced the release of lysosomal enzymes. Thus, we suggest that Bax is a mediator of LMP, possibly promoting the release of lysosomal enzymes to the cytosol during apoptosis.

Keywords: Bax, cathepsins, lysosomes, lysosomal membrane permeabilization, mitochondria

Mitochondria are considered key players in the regulation of apoptotic cell death, as they coordinate caspase activation through the release of cytochrome c. During recent years, however, we have gained increased knowledge about the complexity of programmed cell death, and many new components thereof have been discovered. The lysosome, which is an acidic degradative organelle, has recently been implicated in the regulation of cell death. A feature common to lysosomes and mitochondria is their increased membrane permeability, resulting in release of their contents, in the early phase of apoptosis (Mathiasen & Jäättelä 2002). Lysosomal membrane permeabilization (LMP) and translocation of enzymes from the lysosomal compartment to the cytosol has been reported following apoptosis induction by varying stimuli such as the synthetic retinoid CD437 (Zang et al. 2001), oxidative stress (Roberg & Öllinger 1998; Kågedal et al. 2001), staurosporine (STS) (Johansson et al. in press), TNF-α (Guicciardi et al. 2000; Foghsgaard et al. 2001) and p53 (Yuan et al. 2002).

Lysosomes contain a large number of hydrolytic enzymes that, when released from the lysosomes, can come into contact with cytosolic targets and contribute to apoptotic cell death. Agents that directly target lysosomes and permeabilize the lysosomal membrane have been used to enlighten the importance of the release of lysosomal proteases to the cytosol during the onset of apoptosis. Treatment of cells with the lysosomotropic detergent O-methylserine dodecylamide hydrochloride results in rapid permeabilization of the lysosomal membrane and ensuing apoptosis (Li et al. 2000). The same phenomenon has been reported for other lysosomotropic drugs such as the quinolone antibiotics ciprofloxacin and norfloxacin and the amine hydroxychloroquine (Boya, Andreau et al. 2003; Boya, Gonzalez-Polo et al. 2003).

It remains elusive how the lysosomal enzymes escape into the cytosol during apoptosis, but several mechanisms have been proposed. In oxidative stress-induced apoptosis, release of cathepsin D from lysosomes and induction of apoptosis could be prevented by the antioxidant α-tocopherol (Roberg & Öllinger 1998), suggesting the involvement of oxidative events. Reactive oxygen species, produced via intralysosomal iron-catalysed oxidative processes, may in some cases be responsible for lysosomal membrane destabilization (Antunes et al. 2001; Persson et al. 2003). It has also been suggested that intracellular sphingosine, which accumulates inside lysosomes, can permeabilize the membrane via a detergent mechanism (Kågedal et al. 2001). There is some evidence that permeabilization of lysosomal and mitochondrial membranes occurs by similar mechanisms. The adenine nucleotide translocator (ANT) agonist atractyloside, which triggers opening of the mitochondrial permeability transition pore and release of mitochondrial cytochrome c, has been shown to also cause release of cathepsin B from isolated lysosomes (Vancompernolle et al. 1998). Conceivably, the lysosomal membrane may, just like the mitochondrial membrane, consist of ANT-like proteins that might regulate its permeability. Other data suggest the involvement of Bcl-2 family members in both mitochondrial membrane permeabilization (MMP) and LMP. It is well known that caspase-8 cleaves the cytosolic Bcl-2 family member Bid, which upon cleavage translocates to mitochondria and promotes the release of cytochrome c via activation of Bax (Li et al. 1998; Luo et al. 1998; Desagher et al. 1999). Active caspase-8 has, however, also been reported to induce release of cathepsin B from isolated lysosomes (Guicciardi et al. 2000). Furthermore, tBid caused the release of cathepsin B to the cytosol, and such release was markedly reduced in Bid knockout cells as compared with that in wild-type cells (Werneburg et al. 2004). Collectively, these data suggest that caspase-8 and Bid can together promote the permeabilization not only of mitochondrial but also of lysosomal membranes. Furthermore, lysosomes and mitochondria seem to share mechanisms that prevent membrane permeabilization. This was demonstrated in a recent publication by Zhao and colleagues in which overexpression of Bcl-2, which is known to inhibit mitochondrial release of cytochrome c (Yang et al. 1997), was found to prevent lysosomal destabilization (Zhao et al. 2000).

Bax, which is a pro-apoptotic member of the Bcl-2 family, is central in the regulation of MMP, and its action is counteracted by Bcl-2. When the three-dimensional structure of Bcl-XL was revealed, showing a striking similarity to the pore-forming domains of certain bacterial toxins (e.g., colicins and diphtheria toxins) (Muchmore et al. 1996), it was hypothesized that members of the Bcl-2 family could mediate release of cytochrome c from mitochondria through pore formation. More recently, it was demonstrated that Bax shows the same general structure as Bcl-XL (Suzuki et al. 2000), displays channel-forming activity in liposomes (Antonsson et al. 1997) and induces release of cytochrome c from isolated mitochondria (Eskes et al. 1998; Jürgensmeier et al. 1998). Interestingly, the diphtheria toxin is known to create pores in the lysosomal membrane, allowing transport of the ADP-ribosylating A-subunit of the toxin across the membrane (Donovan et al. 1982).

We, thus, hypothesized that Bax, upon apoptosis induction, could be targeted to lysosomes and promote release of lysosomal proteases to the cytosol. By using highly purified rat liver lysosomes, and a human fibroblast cell system in which apoptosis was induced by the protein kinase inhibitor STS, Bax was identified as a mediator of LMP.

Materials and methods

Cells and culture conditions

Human foreskin fibroblasts of the AG-1518 line (passages 12–20; Coriell Institute Camden, NJ, USA) were cultured in Eagle's minimum essential medium supplemented with 2 mm glutamine, 50 IU/ml penicillin-G, 50 µg/ml streptomycin and 10% foetal bovine serum (all from Gibco, Paisley, UK). Cells were incubated in humidified air with 5% CO2 at 37 °C. Prior to experiments, cells were trypsinized and seeded in 35-mm dishes (Costar, Cambridge, MA, USA) at a density of 10,000 cells/cm2. STS, 0.1 µm (Sigma, St. Louis, MO, USA), was dissolved in dimethyl sulfoxide (DMSO) (0.2 mm stock solution), added to cultures in prewarmed serum-free medium, and the cells were incubated at 37 °C.

Detection of apoptosis

Nuclear fragmentation was analysed in paraformaldehyde-fixed cells using Vectashield® mounting medium with DAPI (1.5 µg/ml; Vector Laboratories, Burlingame, CA, USA). Cells were examined and photographed in a Nikon photomicroscope (Nikon, Tokyo, Japan), equipped with a digital colour video camera (Hamamatzu Orca 100, Hamamatzu, Japan), using UV excitation light and a blue barrier filter.

Immunofluorescence detection of cathepsin D, Bax and lysosome-associated membrane protein-2

Fibroblasts grown on coverslips were fixed in 4% paraformaldehyde for 20 min at 4 °C and then processed for immunocytochemistry (Brunk et al. 1997). Briefly, cells were incubated with a polyclonal rabbit anti-human cathepsin D antibody (1:100; Dako, Glostrup, Denmark) followed by a goat anti-rabbit IgG-Texas Red® conjugate (1:200; Vector Laboratories) or a polyclonal rabbit anti-Bax antibody (1:200; Upstate Biotechnology, Lake Placid, NY, USA) followed by a goat anti-rabbit Alexa Fluor 488® conjugate (1:400; Molecular Probes, Eugene, OR, USA) or with a monoclonal mouse anti-human lysosome-associated membrane protein-2 (LAMP-2) antibody (1:200; Southern Biotechnology Associates, Inc, Burmingham, AL, USA) followed by a goat anti-mouse IgG-Texas Red® conjugate (1:50; Vector Laboratories). Finally, the cells were rinsed in phosphate-buffered saline (PBS) and distilled water and mounted in Vectashield® Mounting Medium. Cells were examined and photographed in a fluorescence microscope using green excitation light and a red barrier filter or blue excitation light and a green barrier filter. Negative controls, incubated without the primary antibodies, showed no staining.

Fluorescence microscopical analysis of Bax localization

The subcellular localization of Bax was analysed by confocal microscopy. Lysosomes were visualized by immunocytochemical detection of endogenous LAMP-2 or by endocytosis of 37.5 µg/ml dextran-Texas Red® 40,000 Da (Sigma) for 3 days before drug exposure. Mitochondria were labelled by incubation of cells with 100 nm MitoTracker® Red (molecular probes) for 30 min at 37 °C. Images were obtained using an argon 488 (10 mW) laser or a helium neon 543 (0.1 mW) laser in a Nikon Eclipse E600W confocal microscope. Blue and green dual excitation light was used, and split images were obtained.

Immuno-electron microscopical analysis of Bax localization

Antibodies coupled to ultra-small gold particles were used to visualize Bax (Roberg & Öllinger 1998). In short, fibroblasts were fixed for 20 min at 4 °C in a solution consisting of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.15 m sodium cacodylate buffer (pH 7.6). The cultures were then immersed in a freshly prepared solution of 0.05% saponin and 1% glycine in PBS. Next, the cells were incubated with a monoclonal mouse anti-Bax antibody that recognizes Bax only in its active conformation (clone 6A7, dilution 1:200; Zymed, San Francisco, CA, USA) overnight at 4 °C. Thereafter, the cells were rinsed and incubated with goat anti-mouse F(ab′) tagged with 0.8 nm gold particles (dilution 1:100, Aurion, Wageningen, the Netherlands) over night at 4 °C. The immunogold-labelled cells were fixed for 20 min in 2.5% glutaraldehyde (Agar scientific, Essex, UK) in PBS, rinsed in PBS at room temperature and silver-enhanced for 8 min at 26 °C, and finally postfixed for 15 min at 4 °C in 0.2% osmium tetraoxide (Johnson Matthey Chemicals, Roystone, UK). Cultures were embedded in Epon-812 (Fluka AG, Buchs, Switzerland) and thin sections cut with a diamond knife (DIATOME, Bienne, Switzerland). Cells were examined and photographed in a JEM 1230 electron microscope (JEOL, Tokyo, Japan) at 100 kV.

Expression and purification of Bax(-TM) and wtBax

Recombinant C-terminally truncated Bax protein [Bax(-TM)] was prepared according to Vyssokikh et al. (2002). Bax(-TM) was concentrated in the presence of 10 mm Tris-HCl (pH 7.5) and 100 mm NaCl.

Cloning and fermentation, wtBax

Recombinant wild-type Bax protein (wtBax) was purified according to Montessuit et al. (1999) with some modifications. In short, the coding DNA sequence for mouse Bax α was cloned with the aid of a 5′-terminal Bgl II and a 3′-terminal Hind III restriction side into the prokaryotic expression vector pBad/His B (Invitrogen, San Diego, CA, USA). This cloning strategy introduces an N-terminal His-tag for affinity chromatography, an express epitope and an EK site. The resulting plasmid was checked by sequencing and retransformed into the Escherichia coli strain LMG 194 (Invitrogen). Bacteria were grown with the aid of a fermenter in RM-medium (1 × M9 salts, 2% casamino acids, 1 mm MgCl2, 0.4% glucose and 100 µg/ml ampicillin). Protein expression was performed at 30 °C for 4 h after addition of L-arabinose (1 g/l). The yield was 60-70 g bacteria (wet weight).

Purification, wtBax

Bacteria (35 g) were resuspended in 100 ml lysis buffer [100 mm HEPES-NaOH (pH 8.0), 100 mm NaCl, 1 mm MgCl2, 1% Triton X-100, 1 mm phenylmethylsulphonyl fluoride (PMSF), 1 mm benzamidine, 0.1% 2-mercaptoethanol, Protease Inhibitor Cocktail EDTA-free Complete (Boehringer-Mannheim, Mannheim, Germany), 30 µg/ml Dnase I, 50 µg/ml Rnase A and 50 µg/ml lysozyme]. The cells were broken by sonication. After centrifugation (35,000 × g, 30 min), the resulting supernatant was applied for 16 h at 4 °C to a 5 ml Nickel-NTA column (Amersham Pharmacia Biotech, Uppsala, Sweden) and equilibrated with buffer A [50 mm HEPES-NaOH (pH 8.0), 100 mm NaCl, 1 mm MgCl2, 20 mm imidazole, 2% octyl-glycoside, 0.1 mm PMSF, 0.1 mm benzamidine and 0.1% 2-mercaptoethanol]. The protein-loaded column was washed with 15-column volumes buffer A. The protein was eluted in one step with 20 ml buffer A containing 200 mm imidazole.

The wtBax containing fractions were pooled and diluted 1:4 with buffer B [50 mm HEPES-NaOH (pH 8.0), 2% octyl-glycoside, 0.1 mm PMSF, 0.1 mm benzamidine and 0.1% 2-mercaptoethanol]. This sample was applied to an anion-exchanger column (6 ml Resource Q, Amersham Pharmacia Biotech) equilibrated with buffer B. The column was washed with 40 ml buffer B. The wtBax protein was eluted with a linear gradient of 10 ml (0–40% buffer B containing 500 mm NaCl) followed by a linear gradient of 15 ml (40–100% buffer B containing 500 mm NaCl). The fractions containing wtBax protein were concentrated in the presence of 20 mm sodium phosphate (pH 7.5), 5% glycerol and 0.5% octyl glycoside by ultrafiltration (VIVA-Spin6 Concentrator, 5 kDa cut-off, VIVA Science, Göttingen, Germany) to a final concentration of 200 µg protein/ml. The resulting protein was stored at –70 °C.

Isolation of rat liver lysosomes

Sprague-Dawley rat liver lysosomes were isolated essentially as described earlier for mouse liver lysosomes (Stoka et al. 2001). The experiments were approved by the Linköping University ethical committee. Integrity of lysosomes was studied by measuring the activity of the lysosomal enzymes N-acetyl-β-glucoseaminidase (NAG) and cathepsins B and L in the absence or presence of 0.2% Triton X-100. Cross-contamination of mitochondria was investigated by measuring the activity of the mitochondrial enzyme succinic p-iodonitrotetrazolium reductase or by immunoblotting of the mitochondrial membrane protein cytochrome c oxidase (as described below). The presence of these proteins in the lysosomal fraction was compared to their presence in the initial homogenate. The purity of the lysosomal fractions was also studied using electron microscopy (methods are described below).

Activity measurement of lysosomal and mitochondrial marker enzymes

Activity of NAG was measured using 4-methyl-umbelliferyl-2-acetoamido-2-deoxy-β-D-glucopyranoside (Sigma) as substrate. Samples were incubated at 37 °C for 30 min in 0.2 m citrate buffer containing 0.8 mm substrate. The relative amount of the fluorescent product, 4-methylumbelliferone, was determined at λex 356/ λem 444 nm in a Victor plate reader (EG & G Wallac, Upplands Väsby, Sweden).

Activity of cathepsins B and L was measured using z-Phe-Arg-AMC (Bachem, Bubendorf, Switzerland) as substrate. Samples were incubated in 250 µl of 340 mm sodium acetate buffer (pH 5.5) containing 60 mm acetic acid, 4 mm EDTA, 8 mm dithiothreitol and 30 µm substrate and incubated for 15 min at 37 °C. The reaction was stopped by adding 500 µl of a buffer consisting of 100 mm sodium monochloric acetate, 30 mm sodium acetate and 70 mm acetic acid, pH 4.3. The amount of liberated fluorescent AMC was analysed at λex 380 nm/λem 460 nm in a Shimadzu RF-540 spectroflourophotometer.

Succinic p-iodonitrotetrazolium reductase was used as a mitochondrial marker, and the enzymatic activity was analysed using p-iodonitrotetrazolium violet as substrate. Samples were incubated with 2 mmp-iodonitrotetrazolium violet in 55 mm potassium dihydrogen phosphate, 55 mm succinic acid and 250 mm sucrose at pH 6.0 for 30 min at 37 °C. The reaction was stopped by addition of 10% trichloro acetic acid. After centrifugation (5 min at 16,000 × g), the pellets were resuspended in ethanol, and supernatant was finally cleared by centrifugation for 5 min at 16,000 × g and analysed at 495 nm in a Victor plate reader.

Electron microscopical analysis of lysosomal fractions

Lysosomal fractions were fixed by addition of 2% glutaraldehyde in 0.1 m sucrose/sodium-cacodylate-HCl buffer (Sigma), pH 7.2, and postfixed in osmium tetraoxide. Lysosomes were pelleted in 2% agar prior to dehydration, staining with uranyl acetate, dehydration and embedding in Epon-812. Thin sections were cut with a diamond knife, stained with lead-citrate, examined and photographed in a JEOL 1200-EX electron microscope at 100 kV.

Insertion of Bax into lysosomal membranes

Lysosomes (50 µg protein) were incubated with 0.2–1.0 µm wtBax, 0.3–1.6 µm Bax(-TM) or diluents in 50 µl sucrose/Pipes buffer (250 mm sucrose, 20 mm Pipes, pH 7.2) at 30 °C for 1 h. In a control experiment, 2 µm of either glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cytochrome c (Sigma), full-length Bid or caspase-8-cleaved Bid (kind gifts from Dr Antonsson, Serono Pharmaceutical Research Institute, Geneva, Switzerland) were incubated with lysosomes. Lysosomes were washed four times in sucrose/Pipes and pelleted by centrifugation at 18,000 × g for 10 min. Proteins loosely attached to the lysosomal membrane were removed by incubation of the pellet in 0.1 m Na2CO3 in sucrose/Pipes buffer on ice for 20 min, as described earlier for mitochondria (Antonsson et al. 2001). After centrifugation for 1 h at 1,00,000 × g, the supernatants, containing proteins attached to the membrane, and the pellets, containing proteins inserted in the membrane, were analysed by immunoblotting of Bax (as described below).

Lysosomal protease release assay

Lysosomes (50 µg protein) were incubated with 0.2–1.0 µm wtBax, 0.3–1.6 µm Bax(-TM), 0.2–1.0 µm full-length Bid, 0.013–13.0 µm active caspase-8, 2–20 mm atractyloside or diluents in 50 µl sucrose/Pipes buffer (250 mm sucrose, 20 mm Pipes, pH 7.2) at 30 °C for 1 h. Lysosomes were pelleted by centrifugation at 18,000 ×g for 10 min, and the supernatants were analysed for presence of lysosomal enzymes by activity measurements of NAG (as described above) or immunoblotting of cathepsins B and D (as described below).

Western blotting

Gel electrophoresis and Western blotting were performed as described earlier (Kågedal et al. 2001). The blots were probed with the following antibodies, a rabbit anti-Bax antibody (1:1000; Upstate Biotechnology) was used. For detection of cathepsins D and B, blots were incubated with a mouse anti-cathepsin D antibody (1:1000, Oncogene, San Diego, CA, USA) or a rabbit anti-cathepsin B antibody (1:1000, Athens Research and Technology, Athens, GA, USA). A mouse anti-cytochrome c oxidase antibody (1:1000, Molecular Probes) was used for staining of mitochondrial cytochrome c oxidase. Blots were probed with a mouse anti-GAPDH antibody (1:400; Biogenesis, Poole, UK), a mouse anti-cytochrome c antibody (1:400, Becton-Dickinson, Mountain View, CA, USA) and a rabbit anti-Bid antibody (1:1000, Cell Signalling Technology, Beverly, MA, USA) for visualization of GAPDH, cytochrome c and Bid, respectively. Next, the membranes were incubated with horse radish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies (1:1500, Dako). Protein bands were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech).

Haemoglobin release assay

Erythrocytes were prepared from fresh heparinized human blood by dilution of 0.5 ml whole blood in 40 ml of PBS (pH 7.4) and centrifugation at 5000 ×g for 10 min at 4 °C. Erythrocytes (50 µg) were incubated with 1.0 µm wtBax, 1.6 µm Bax(-TM) or diluents in 50 µl sucrose/Pipes buffer at 30 °C for 1 h. Samples were centrifuged at 5000 ×g for 5 min, the supernatant was diluted in sucrose/Pipes (1:1), and the absorbance at 405 nm was determined using a Victor plate reader.

Statistics

Experiments were repeated three times, and statistical significance was determined using the Kruskal-Wallis multiple comparison test. Differences were considered significant when P ≤ 0.05.

Results

STS induces release of lysosomal proteases to the cytosol

Release of lysosomal proteases was studied using cathepsin D as a lysosomal marker enzyme. In control human fibroblasts (AG-1518), cathepsin D was detected in granular, lysosome-like structures (Figure 1a). In STS-exposed cells, there was a diffuse staining of cathepsin D, indicating LMP with release of lysosomal enzymes to the cytosol (Figure 1b). This was earlier shown by Western blot analysis of cytosol in the same experimental system (Johansson et al. in press) and is consistent with our previous findings that cathepsins B, D and L are released to the cytosol in response to mild oxidative stress (Kågedal et al. 2001). The STS-induced release of cathepsin D was earlier demonstrated to be an important event, as inhibition of cathepsin D with pepstatin A delayed the apoptosis process (Johansson et al. 2003).

Figure 1.

Figure 1

Open in a new tab

Staurosporine (STS) exposure induces release of lysosomal enzymes. (a) Immunofluorescence of cathepsin D in control human fibroblasts (AG-1518) and (b) fibroblasts exposed to STS for 1 h. A rabbit anti-human cathepsin D antibody (Dako) and a Texas Red-conjugated secondary antibody were used.

Caspase-8 and ANT-like proteins are not involved in the release of lysosomal proteases

To test whether active caspase-8 could cause LMP with the release of lysosomal proteases, highly purified rat liver lysosomes were used. The lysosomal fractions were found to be free from mitochondrial contamination as measured by the activity of the mitochondrial marker enzyme p-iodonitrotetrazolium reductase and immunoblotting of the mitochondrial membrane protein cytochrome c oxidase (Figures 2a, b). Furthermore, examination of the isolated lysosomal fractions by electron microscopy did not reveal any contamination by mitochondria (Figure 2c). Even at the relatively high concentration of 13 µm of recombinant active caspase-8, there was only a weak tendency of LMP detected as release of NAG and cathepsin B from isolated lysosomes (Figures 3a, b). In addition, no additive effect was seen when combining caspase-8 with recombinant full-length Bid, suggesting that truncated Bid alone does not mediate the caspase-8-induced LMP earlier described (Guicciardi et al. 2000; Werneburg et al. 2004).

Figure 2.

Figure 2

Open in a new tab

Purity of isolated rat liver lysosomes. (a) Activity measurement of the mitochondrial marker enzyme p-iodonitrotetrazolium reductase and the lysosomal marker enzyme N-acetyl-β-glucoseaminidase. The enzyme activities in the lysosomal fractions were compared to activities in the initial homogenates. Values are means ± standard deviation, n = 3. (b) Western blotting of the mitochondrial membrane protein cytochrome c oxidase. A mouse anti-cytochrome c oxidase antibody (molecular probes) and a horseradish peroxidase-conjugated secondary antibody were used. The presence of cytochrome c oxidase in the lysosomal fraction was compared to its presence in the initial homogenate. (c) Electron micrograph of isolated rat liver lysosomes. Arrowheads indicate lysosomes of different sizes. Bar = 1 µm.

Figure 3.

Figure 3

Open in a new tab

Caspase-8 and adenine nucleotide translocator-like proteins are not involved in the release of lysosomal proteases. Rat liver lysosomes were incubated with various concentrations of recombinant active caspase-8, recombinant active caspase-8 combined with Bid, atractyloside or diluent for 1 h at 30 °C. Triton X-100 (0.2% v/v) was used as positive control. Lysosomes were pelleted by centrifugation, and the resulting supernatants were subjected to analysis. (a and c) Enzymatic activity of N-acetyl-β-glucoseaminidase (NAG) was measured using the fluorescent substrate 4-methyl-umbelliferyl-2-acetoamido-2-deoxy-b-D-glucopyranoside and is expressed as percentage of the Triton X-100-positive control. Values are means ± standard deviation, n = 3. (b and d) Cathepsin B was detected by Western blotting using a rabbit anti-cathepsin B antibody (Athens Research) and a horseradish peroxidase-conjugated secondary antibody. A representative blot, out of three, is shown.

An alternative mechanism of LMP is based on the possible presence of ANT-like proteins in the lysosomal membrane (Vancompernolle et al. 1998). This was investigated by incubation of isolated lysosomes with the ANT agonist atractyloside. Only at the relatively high concentrations of 15 and 20 mm of atractyloside, a weak, statistically nonsignificant increase of NAG and cathepsin B release was detected (Figures 3c, d).

Bax translocates from the cytosol to both lysosomes and mitochondria during STS-induced apoptosis

To elucidate whether Bax could be a mediator of LMP, the subcellular location of Bax was studied using immunofluorescence microscopy. Prior to apoptosis induction, cells showed a diffuse cytosolic staining of Bax. After exposure to STS, the number of cells with punctate staining of Bax increased with time, suggesting association of Bax with mitochondria and possibly lysosomes (Figures 4a, b). As, earlier, demonstrated by Desagher et al. (1999), the number of cells showing punctate staining of Bax correlated well with the number of cells displaying apoptotic fragmented nuclei (Figure 4a). To investigate whether the punctate staining of Bax during apoptosis could partially represent a lysosomal location, fibroblasts were examined by laser confocal microscopy. Lysosomes were labelled by immunostaining of the lysosomal membrane protein LAMP-2 (Figures 5b, e). Prior to apoptosis induction, cells showed a diffuse cytosolic immunostaining of Bax (Figure 5a), and no co-localization of Bax with lysosomes was detected after overlay of images (Figures 5a-c). In STS-exposed cells, there was a punctate distribution of Bax that in part co-localized with lysosomes as detected by yellow staining in overlay pictures (Figures 5d-f). The same result was obtained when lysosomes were labelled by endocytotic uptake of Texas Red®-conjugated dextran and compared with the localization of immunostained Bax (data not shown). These results suggest that Bax translocates from the cytosol to lysosomes during apoptosis.

Figure 4.

Figure 4

Open in a new tab

Staurosporine (STS) exposure induces punctate staining of Bax [▪] and nuclear fragmentation (•). Bax was detected by immunofluorescence in human fibroblasts (AG-1518) exposed to STS. A rabbit anti-Bax antibody (Upstate Biotechnology) and an Alexa Fluor 488®-conjugated secondary antibody were used. Nuclear fragmentation after exposure to STS was studied in DAPI-stained cells. (a) The percentage of cells showing punctate staining of Bax and fragmented nuclei, respectively, was determined by examining 200 cells per sample. Values are means ± standard deviation, n = 3. (b) Images of cells immunolabelled for Bax or DAPI stained for nuclear morphology.

Figure 5.

Figure 5

Open in a new tab

Bax is localized to lysosomes and mitochondria during apoptosis induced by staurosporine (STS). Control fibroblasts (AG-1518) and fibroblasts exposed to STS for 4 h were examined by laser fluorescence confocal microscopy. Bax was visualized by immunostaining (a, d, g and j) using a rabbit anti-Bax antibody (Upstate Biotechnology) and an Alexa Fluor 488-conjugated secondary antibody. Lysosomes were labelled by immunostaining (b and e) using a mouse anti-human lysosome-associated membrane protein-2 (LAMP-2) antibody (Southern Biotechnology Associates), and mitochondria were visualized with Mito Tracker® Red (h and k). Co-localization (yellow staining) of Bax and lysosomes (c and f) and of Bax and mitochondria (i and l) were detected by overlay of images. Insets in c, f, i and l show higher magnification views. Images are representative of three experiments.

Because it is well documented that Bax is localized to mitochondria during apoptosis, we compared the co-localization of Bax and lysosomes with that of Bax and mitochondria. After labelling of mitochondria with MitoTracker® Red, long vermiform mitochondria were detected in control cells (Figure 5h). During apoptosis induced by STS, cells displayed punctiform mitochondria, which co-localized with immunostaining of Bax (Figures 5j-l). In control experiments, there were no co-localization of lysosomes and mitochondria, ruling out the possibility that the observed lysosomal location of Bax is due to overlap of these organelles in the shrunken apoptotic cell. When comparing overlay images showing co-localization of Bax with lysosomes and mitochondria, respectively, it is evident that a majority of Bax is localized to mitochondria during apoptosis. There is, however, also a small fraction of Bax that localizes to lysosomes, where it may promote release of lysosomal proteases.

Finally, the subcellular localization of Bax was studied by immuno-electron microscopy, where Bax was visualized using antibodies coupled to ultra-small gold particles. As seen in Figure 6(b), gold particles are observed in close vicinity of the lysosomal membrane in cells exposed to STS, indicating the presence of Bax in the lysosomal membrane. In control cells, however, there was no lysosomal staining of Bax (Figure 6a).

Figure 6.

Figure 6

Open in a new tab

Bax is present at the lysosomal membrane during apoptosis induced by staurosporine (STS). Human fibroblasts (AG-1518) exposed to STS for 6 h were examined by immuno-electron microscopy. Bax was labelled using a mouse anti-Bax antibody (Zymed) and goat anti-mouse F(ab′) coupled to ultra-small gold particles (Aurion). (a) Control cell. (b) Cell exposed to STS. Arrows indicate Bax located at the lysosomal membrane.

Recombinant Bax is inserted into membranes of isolated rat liver lysosomes

To test whether Bax could be inserted into the lysosomal membrane, purified oligomeric recombinant Bax was added to lysosomes isolated from rat liver. A truncated form of Bax [Bax(-TM)], lacking the C-terminal 20 amino acids containing the membrane-anchoring domain, has often been used in studies on mitochondria. In this study, both the full-length wtBax and the truncated form were used. Both Bax proteins were purified in the presence of detergents favouring conformational change and oligomerization. After incubation of lysosomes with either 1 µm wtBax, 1 µm Bax(-TM) or diluent for 1 h, lysosomes were pelleted and suspended in a Na2CO3 solution (0.1 m). This will remove proteins attached to the membrane, while proteins integrated into the membrane remain in the membrane fraction. The presence of attached and inserted Bax was analysed using Western blotting. While wtBax was only found inserted into the membranes of the lysosomal fraction, Bax(-TM) was either inserted or loosely attached (Figure 7). GAPDH, cytochrome c, full-length Bid or caspase-8-cleaved Bid, which were used as negative controls, did not insert into the membranes, which demonstrate that the lysosomal membrane is not adhesive to proteins in general (data not shown). Results from this part of the study support the idea that Bax can be targeted to lysosomes.

Figure 7.

Figure 7

Open in a new tab

Recombinant Bax is inserted into membranes of isolated lysosomes. Rat liver lysosomes were incubated with 1.0 µm recombinant wild-type Bax (wtBax) or 1.0 µm recombinant Bax lacking the transmembrane domain [Bax(-TM)] for 1 h at 30 °C. Lysosomes were treated with 0.1 m Na2CO3, washed and centrifuged at 1,00,000 × g for 1 h. The supernatant containing loosely attached proteins and the alkali-resistant pellet containing the inserted proteins were analysed by Western blotting using a rabbit anti-Bax antibody (Upstate Biotechnology) and a HRP-conjugated secondary antibody. One blot representative for three experiments is shown.

Recombinant Bax induces release of lysosomal proteases from isolated rat liver lysosomes

Next, we investigated whether insertion of Bax into lysosomal membranes could mediate the release of lysosomal proteases. After incubation of rat liver lysosomes with either recombinant wtBax, Bax(-TM) or diluent for 1 h, the lysosomes were pelleted, and the presence of lysosomal proteases in the supernatants was analysed by the measurement of enzymatic activity of NAG and immunoblotting of cathepsins B and D. As shown in Figures 8(a-c), the activity of NAG and the amount of cathepsins B and D increased in the supernatants from wtBax treated lysosomes as compared with the diluent control. The wtBax-induced release of lysosomal proteases was concentration dependent. Compared with Triton X-100-treated positive controls, wtBax (1.0 µm) induced the release of about 70% of the total amount of lysosomal proteases. Bax(-TM) was not as efficient, inducing only a weak tendency of release of lysosomal proteases with increasing concentrations (Figure 8a). This suggests that the transmembrane C-terminal domain of Bax somehow facilitates the release of enzymes from lysosomes.

Figure 8.

Figure 8

Open in a new tab

Recombinant Bax induces release of proteases from isolated lysosomes. Rat liver lysosomes were incubated with various concentrations of recombinant wild-type Bax (wtBax), recombinant Bax lacking the transmembrane domain [Bax(-TM)], recombinant Bax diluent or sucrose/Pipes buffer (control) for 1 h at 30 °C. Triton X-100 (0.2% v/v) was used as positive control. The lysosomes were pelleted and the presence of lysosomal proteases in the supernatant was analysed. (a) Enzymatic activity of N-acetyl-β-glucoseaminidase (NAG) was measured using the fluorescent substrate 4-methyl-umbelliferyl-2-acetoamido-2-deoxy-b-D-glucopyranoside and is expressed as percentage of the positive control. Values are means ± standard deviation, n = 3. (b) Western blotting of cathepsin D using a mouse anti-cathepsin D antibody (Oncogene) and a HRP-conjugated secondary antibody. (c) Western blotting of cathepsin B using a rabbit anti-cathepsin B antibody (Athens Research) and a HRP-conjugated secondary antibody. Data in B and C are representative for three experiments.

To study the membrane specificity of recombinant Bax, erythrocytes isolated from human blood were incubated with wtBax and Bax(-TM). The highest concentrations of recombinant wtBax and Bax(-TM) used above were not able to induce release of haemoglobin from erythrocytes (Figure 9). These results demonstrate that the Bax-induced LMP cannot be explained by nonspecific targeting of recombinant Bax to membranes in general.

Figure 9.

Figure 9

Open in a new tab

Recombinant Bax does not induces release of haemoglobin from intact erythrocytes. Erythrocytes were incubated with 1.0 µm recombinant wild-type Bax (wtBax), 1.6 µm recombinant Bax lacking the transmembrane domain [Bax(-TM)], recombinant Bax diluent or sucrose/Pipes buffer (control) for 1 h at 30 °C. Triton X-100 (0.2% v/v) was used as positive control. The erythrocytes were pelleted, and the presence of haemoglobin in the supernatant was analysed. Release of haemoglobin from erythrocytes is expressed as percentage of the positive control. Values are means ± standard deviation, n = 2.

Discussion

Several recent studies indicate that lysosomal destabilization and lysosomal enzymes are also involved in regulation of cell death (Mathiasen & Jäättelä 2002; Turk et al. 2002). While massive lysosomal damage leads to necrosis (Kågedal et al. 2001), a controlled, yet undefined, mechanism causing a limited release of lysosomal enzymes to the cytosol results in apoptosis. It has been suggested that there exist similar mechanisms for release of mitochondrial and lysosomal proteins to the cytosol during apoptosis (Vancompernolle et al. 1998; Guicciardi et al. 2000; Zhao et al. 2001).

One proposed mechanism for the release of cytochrome c is the opening of the mitochondrial permeability transition pore. The components of this pore include the voltage-dependent anion channel and ANT, where ANT is located in the inner mitochondrial membrane and functions as an ATP/ADP carrier (Marzo et al. 1998; Narita et al. 1998). Opening of the permeability transition pore can be induced by atractyloside, an agonistic ligand to ANT (Kroemer et al. 1997). Interestingly, atractyloside was reported to cause LMP and release of cathepsin B to the cytosol (Vancompernolle et al. 1998). Atractyloside-induced release of lysosomal enzymes was not observed in the present study. Only when using relatively high concentrations of atractyloside, a weak tendency of release of lysosomal enzymes was detected. Thus, it is not likely that ANT is responsible for destabilization of the lysosomal membrane during apoptosis.

Another proposed mechanism for mitochondrial release is caspase-8-mediated activation of Bid (Li et al. 1998; Luo et al. 1998). It was recently shown that active caspase-8 alone can induce the release of cathepsin B from isolated lysosomes (Guicciardi et al. 2000). By combining caspase-8 with cytosol, this release was enhanced, and it was suggested that cytosolic Bid could be involved. This was supported by results from an additional study in which truncated Bid was found to translocate to lysosomes during apoptosis (Werneburg et al. 2004). In addition, the release of lysosomal cathepsin B was markedly reduced in Bid knockout cells. In the present study, when isolated lysosomes were incubated with caspase-8, at concentrations up to a 1000-fold higher than that earlier reported to cause LMP, there was only a very small amount of lysosomal proteases released. Moreover, no additive effect was observed when combining caspase-8 with full-length Bid. Thus, a combination of caspase-8 and Bid is not sufficient to trigger the release of proteases from isolated lysosomes.

Because it is well established that Bax associates with mitochondria and is involved in MMP during apoptosis (Wolter et al. 1997), we investigated the possibility that Bax also associates with lysosomes and promotes LMP and release of lysosomal proteases during STS-induced apoptosis. As detected in our system, Bax is translocated to punctate structures in apoptotic cells. In each individual experiment, showing Bax translocation to punctate structures, mitochondria and lysosomes were targeted by Bax, although Bax targeted lysosomes to a lesser extent. This finding is supported by our earlier published results demonstrating concurrent release of both cathepsin D and cytochrome c during STS exposure of fibroblasts (Johansson et al. 2003). The subcellular localization of Bax was also studied by immuno-electron microscopy, which confirmed that Bax is present in the lysosomal membrane. Moreover, this finding was verified in isolated rat liver lysosomes, where recombinant Bax was inserted into the membrane and caused the release of lysosomal proteases. Noteworthy, Bax was unable to mediate the release of haemoglobin from isolated erythrocytes, showing that the effect of wtBax and Bax(-TM) on lysosomal integrity is not due to nonspecific targeting to membranes in general.

The hydrophobic C-terminal domain of Bax is involved in anchoring of the proteins to intracellular membranes. For solubility reasons, most studies with recombinant Bax have been performed using a C-terminally truncated form of Bax. In the present study, we used the wild-type form of recombinant Bax, in parallel with the C-terminally truncated form to be able to compare our results to previous ones. Full-length Bax was approximately 3.5-fold more active in releasing NAG from isolated lysosomes as compared with Bax(-TM). This is in agreement with earlier studies, where full-length Bax has been found to be more efficient in releasing carboxyfluorescein from liposomes (Montessuit et al. 1999) and cytochrome c from isolated mitochondria (Eskes et al. 1998), as compared with the truncated form. It was recently demonstrated, using these two forms of recombinant Bax, that the TM domain of the protein is important for targeting to membranes. Insertion of Bax into membranes, however, was found to be mediated by helix 5/6 (Heimlich et al. 2004; Cartron et al. 2005). In lysosomes, the TM C-terminal domain seems to be of importance for the pore-forming capacity of Bax.

During apoptosis, Bax translocates to mitochondria (Hsu et al. 1997; Wolter et al. 1997), undergoes conformational change (Goping et al. 1998; Desagher et al. 1999), dimerizes or oligomerizes and inserts into membranes (Zha et al. 1996; Gross et al. 1998). The mechanism for recruitment of Bax to intracellular organelles is, however, not yet fully understood. Recent results indicate that permeabilization of the mitochondrial membrane by Bax requires interaction with a BH3-domain-only protein, such as Bid, and with the negatively charged phospholipid cardiolipin (Epand et al. 2002; Kuwana et al. 2002). The question is, could these requirements be met in the lysosomal membrane? Cardiolipin, which is abundant in the inner mitochondrial membrane, is present only in small amounts in lysosomal membranes. These membranes are, however, rich in the structurally related phospholipid bis-(monoacylglyceryl) phosphate (also called lysobisphosphatidic acid) (Wherrett & Huterer 1972). Interestingly, Bid was recently found to associate with lysosomes during apoptosis (Werneburg et al. 2004) and may, thus, promote Bax-mediated LMP. In the fibroblast-model system used in the present study, no Bid cleavage can be detected until several hours after appearance of punctate staining of Bax (Johansson et al. 2003). Other BH3-domain-only proteins may, thus, trigger insertion of Bax into lysosomal and mitochondrial membranes in this experimental system. Taken together, it is possible that Bax is involved in LMP during apoptosis.

Within the complex apoptosis machinery, the decision whether a signal should result in apoptosis or cell survival is controlled at multiple levels, among which release of lysosomal proteases to the cytosol is one. In the present study, we sought to unravel the mechanism behind LMP and found that Bax was localized not only to mitochondria but to lysosomes as well during STS-induced apoptosis. Furthermore, recombinant Bax inserted into membranes of isolated lysosomes and promoted the release of lysosomal proteases. Thus, our report provides a possible mechanism underlying LMP and release of lysosomal proteases to the cytosol during apoptosis.

Acknowledgments

We thank Dr Jiri Neuzil for fruitful discussions. This study was supported by grants from the Swedish Cancer Foundation, the Swedish Research Council, the Swedish Society for Medical Research, Östergötlands läns landsting, the Deutsche Forschungsgemeinschaft (JU 319/3-1 to JMJ) and the foundations of Lennander at Uppsala University, Tore Nilsson, Hedberg and Lars Hierta.

References

  1. Antonsson B, Conti F, Ciavatta A, et al. Inhibition of Bax channel-forming activity by Bcl-2. Science. 1997;277:370–372. doi: 10.1126/science.277.5324.370. [DOI] [PubMed] [Google Scholar]
  2. Antonsson B, Montessuit S, SancheZ B, Martinou JC. Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J Biol Chem. 2001;276:11615–11623. doi: 10.1074/jbc.M010810200. [DOI] [PubMed] [Google Scholar]
  3. Antunes F, Cadenas E, Brunk UT. Apoptosis induced by exposure to a low steady-state concentration of H2O2 is a consequence of lysosomal rupture. Biochem J. 2001;356:549–555. doi: 10.1042/0264-6021:3560549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boya P, Andreau K, Poncet D, et al. Lysosomal membrane permeabilization induces cell death in a mitochondrion-dependent fashion. J Exp Med. 2003;197:1323–1334. doi: 10.1084/jem.20021952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boya P, Gonzalez-Polo RA, Poncet D, et al. Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine. Oncogene. 2003;22:3927–3936. doi: 10.1038/sj.onc.1206622. [DOI] [PubMed] [Google Scholar]
  6. Brunk UT, Dalen H, Roberg K, Hellquist HB. Photo-oxidative disruption of lysosomal membranes causes apoptosis of cultured human fibroblasts. Free Radic Biol Med. 1997;23:616–626. doi: 10.1016/s0891-5849(97)00007-5. [DOI] [PubMed] [Google Scholar]
  7. Cartron PF, Arokium H, Oliver L, Meflah K, Manon S, Vallette FM. Distinct domains control the addressing and the insertion of Bax into mitochondria. J Biol Chem. 2005;280:10587–10598. doi: 10.1074/jbc.M409714200. [DOI] [PubMed] [Google Scholar]
  8. Desagher S, Osen-Sand A, Nichols A, et al. Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol. 1999;144:891–901. doi: 10.1083/jcb.144.5.891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Donovan JJ, Simon MI, Montal M. Insertion of diphtheria toxin into and across membranes: role of phosphoinositide asymmetry. Nature. 1982;298:669–672. doi: 10.1038/298669a0. [DOI] [PubMed] [Google Scholar]
  10. Epand RF, Martinou JC, Fornallaz-Mulhauser M, Hughes DW, Epand RM. The apoptotic protein tBid promotes leakage by altering membrane curvature. J Biol Chem. 2002;277:32632–32639. doi: 10.1074/jbc.M202396200. [DOI] [PubMed] [Google Scholar]
  11. Eskes R, Antonsson B, Osen-Sand A, et al. Bax-induced cytochrome c release from mitochondria is independent of the permeability transition pore but highly dependent on Mg2 + ions. J Cell Biol. 1998;143:217–224. doi: 10.1083/jcb.143.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Foghsgaard L, Wissing D, Mauch D, et al. Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J Cell Biol. 2001;153:999–1010. doi: 10.1083/jcb.153.5.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goping IS, Gross A, Lavoie JN, et al. Regulated targeting of BAX to mitochondria. J Cell Biol. 1998;143:207–215. doi: 10.1083/jcb.143.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gross A, Jockel J, Wei MC, Korsmeyer SJ. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J. 1998;17:3878–3885. doi: 10.1093/emboj/17.14.3878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guicciardi ME, Deussing J, Miyoshi H, et al. Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest. 2000;106:1127–1137. doi: 10.1172/JCI9914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heimlich G, McKinnon AD, Bernardo K, et al. Bax-induced cytochrome c release from mitochondria depends on alpha-helices-5. and -6. Biochem J. 2004;378:247–255. doi: 10.1042/BJ20031152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hsu YT, Wolter KG, Youle RJ. Cytosol-to-membrane redistribution of Bax and Bcl-X(L) during apoptosis. Proc Natl Acad Sci USA. 1997;94:3668–3672. doi: 10.1073/pnas.94.8.3668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Johansson AC, Steen H, Öllinger K, Roberg K. Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine. Cell Death Differ. 2003. pp. 1253–1259. [DOI] [PubMed]
  19. Jürgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D, Reed JC. Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA. 1998;95:4997–5002. doi: 10.1073/pnas.95.9.4997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kågedal K, Johansson U, Öllinger K. The lysosomal protease cathepsin D mediates apoptosis induced by oxidative stress. FASEB J. 2001;15:1592–1594. doi: 10.1096/fj.00-0708fje. [DOI] [PubMed] [Google Scholar]
  21. Kågedal K, Zhao M, Svensson I, Brunk U. Sphingosine-induced apoptosis is dependent on lysosomal proteases. Biochem J. 2001;359:335–343. doi: 10.1042/0264-6021:3590335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kroemer G, Zamzami N, Susin SA. Mitochondrial control of apoptosis. Immunol Today. 1997;18:44–51. doi: 10.1016/s0167-5699(97)80014-x. [DOI] [PubMed] [Google Scholar]
  23. Kuwana T, Mackey MR, Perkins G, et al. Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell. 2002;111:331–342. doi: 10.1016/s0092-8674(02)01036-x. [DOI] [PubMed] [Google Scholar]
  24. Li W, Yuan X, Nordgren G, et al. Induction of cell death by the lysosomotropic detergent MSDH. FEBS Lett. 2000;470:35–39. doi: 10.1016/s0014-5793(00)01286-2. [DOI] [PubMed] [Google Scholar]
  25. Li H, Zhu H, Xu CJ, Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell. 1998;94:491–501. doi: 10.1016/s0092-8674(00)81590-1. [DOI] [PubMed] [Google Scholar]
  26. Luo X, Budihardjo I, Zou H, Slaughter C, Wang X. Bid, a Bcl-2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell. 1998;94:481–490. doi: 10.1016/s0092-8674(00)81589-5. [DOI] [PubMed] [Google Scholar]
  27. Marzo I, Brenner C, Zamzami N, et al. Bax and adenine nucleotide translocator cooperate in the mitochondrial control of Apoptosis. Science. 1998;281:2027–2031. doi: 10.1126/science.281.5385.2027. [DOI] [PubMed] [Google Scholar]
  28. Mathiasen IS, Jäättelä M. Triggering caspase-independent cell death to combat cancer. Trends Mol Med. 2002;8:212–220. doi: 10.1016/s1471-4914(02)02328-6. [DOI] [PubMed] [Google Scholar]
  29. Montessuit S, Mazzei G, Magnenat E, Antonsson B. Expression and purification of full-length human Bax alpha. Protein Expr Purif. 1999;15:202–206. doi: 10.1006/prep.1998.1010. [DOI] [PubMed] [Google Scholar]
  30. Muchmore SW, Sattler M, Liang H, et al. X-ray and NMR structure of human Bcl-XL, an inhibitor of programmed cell death. Nature. 1996;381:335–341. doi: 10.1038/381335a0. [DOI] [PubMed] [Google Scholar]
  31. Narita M, Shimizu S, Ito T, et al. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc Natl Acad Sci USA. 1998;95:14681–14686. doi: 10.1073/pnas.95.25.14681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Persson HL, Yu Z, Tirosh O, Eaton JW, Brunk UT. Prevention of oxidant-induced cell death by lysosomotropic iron chelators. Free Rad Biol Med. 2003;34:1295–1305. doi: 10.1016/s0891-5849(03)00106-0. [DOI] [PubMed] [Google Scholar]
  33. Roberg K, Öllinger K. Oxidative stress causes relocation of the lysosomal enzyme cathepsin D with ensuing apoptosis in neonatal rat cardiomyocytes. Am J Pathol. 1998;152:1151–1156. [PMC free article] [PubMed] [Google Scholar]
  34. Roberg K, Öllinger K. A pre-embedding technique for immunocytochemical visualization of cathepsin D in cultured cells subjected to oxidative stress. J Histochem Cytochem. 1998;46:411–418. doi: 10.1177/002215549804600316. [DOI] [PubMed] [Google Scholar]
  35. Stoka V, Turk B, Schendel SL, et al. Lysosomal protease pathways to apoptosis. Cleavage of Bid, not pro-caspases, is the most likely route. J Biol Chem. 2001;276:3149–3157. doi: 10.1074/jbc.M008944200. [DOI] [PubMed] [Google Scholar]
  36. Suzuki M, Youle RJ, Tjandra N. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell. 2000;103:645–654. doi: 10.1016/s0092-8674(00)00167-7. [DOI] [PubMed] [Google Scholar]
  37. Turk B, Stoka V, Rozman-Pungercar J, Cirman T, Droga-Mazovec G, Oreic K. Apoptotic pathways: involvement of lysosomal proteases. Biol Chem. 2002;383:1035–1044. doi: 10.1515/BC.2002.112. [DOI] [PubMed] [Google Scholar]
  38. Vancompernolle K, Van Herreweghe F, Pynaert G, et al. Atractyloside-induced release of cathepsin B, a protease with caspase-processing activity. FEBS Lett. 1998;438:150–158. doi: 10.1016/s0014-5793(98)01275-7. [DOI] [PubMed] [Google Scholar]
  39. Vyssokikh M, Zorova L, Zorov D, Heimlich G, Jürgensmeier JM, Brdiczka D. Localisation of cytochrome c in a complex of porin and adenine nucleotide translocator: Bax targeted cytochrome c release depends on the functional state of the complex. Mol Biol Rep. 2002;29:93–96. doi: 10.1023/a:1020383108620. [DOI] [PubMed] [Google Scholar]
  40. Werneburg N, Guicciardi ME, Yin XM, Gores GJ. TNF-alpha-mediated lysosomal permeabilization is FAN and caspase 8/Bid dependent. Am J Physiol Gastrointest Liver Physiol. 2004;287:G436–G443. doi: 10.1152/ajpgi.00019.2004. [DOI] [PubMed] [Google Scholar]
  41. Wherrett JR, Huterer S. Enrichment of bis-(monoacylglyceryl) phosphate in lysosomes from rat liver. J Biol Chem. 1972;247:4114–4120. [PubMed] [Google Scholar]
  42. Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG, Youle RJ. Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol. 1997;139:1281–1292. doi: 10.1083/jcb.139.5.1281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 1997;275:1129–1132. doi: 10.1126/science.275.5303.1129. [DOI] [PubMed] [Google Scholar]
  44. Yuan XM, Li W, Dalen H, et al. Lysosomal destabilization in p53-induced apoptosis. Proc Natl Acad Sci USA. 2002;99:6286–6291. doi: 10.1073/pnas.092135599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zang Y, Beard RL, Chandraratna RA, Kang JX. Evidence of a lysosomal pathway for apoptosis induced by the synthetic retinoid CD437 in human leukemia HL-60 cells. Cell Death Differ. 2001;8:477–485. doi: 10.1038/sj.cdd.4400843. [DOI] [PubMed] [Google Scholar]
  46. Zha H, Fisk HA, Yaffe MP, Mahajan N, Herman B, Reed JC. Structure-function comparisons of the proapoptotic protein Bax in yeast and mammalian cells. Mol Cell Biol. 1996;16:6494–6508. doi: 10.1128/mcb.16.11.6494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zhao M, Eaton JW, Brunk UT. Protection against oxidant-mediated lysosomal rupture: a new anti-apoptotic activity of bcl-2? FEBS Lett. 2000;485:104–108. doi: 10.1016/s0014-5793(00)02195-5. [DOI] [PubMed] [Google Scholar]
  48. Zhao M, Eaton JW, Brunk UT. Bcl-2 phosphorylation is required for inhibition of oxidative stress-induced lysosomal leak and ensuing apoptosis. FEBS Lett. 2001;509:405–412. doi: 10.1016/s0014-5793(01)03185-4. [DOI] [PubMed] [Google Scholar]

Articles from International Journal of Experimental Pathology are provided here courtesy of Wiley

RESOURCES