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Apoptosis Supercedes Necrosis in Mitochondrial DNA-Depleted Jurkat Cells by Cleavage of Receptor-Interacting Protein and Inhibition of Lysosomal Cathepsin¹

Department of Internal Medicine (Section 4), Sapporo Medical University, School of Medicine, Sapporo, Japan

	Abstract

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In the present study, we used mitochondrial DNA-depleted Jurkat subclones (⁰ cells) to demonstrate that Fas agonistic Ab (CH-11), at the concentrations that evoke apoptotic death of the parental Jurkat cells, induced necrosis mainly through generation of excess reactive oxygen species, lysosomal rupture, and sequential activation of cathepsins B and D, and in minor part through activation of receptor-interacting protein (RIP). In the ⁰ cells treated with CH-11, ATP supplementation converted necrosis into apoptosis by the formation of the apoptosome and subsequent activation of procaspase-3. In these ATP-supplemented ⁰ cells (ATP-⁰), generation of excess ROS and lysosomal rupture were still seen, yet cathepsins B and D were inactivated and RIP was degraded. The conversion of necrosis to apoptosis, RIP degradation, and cathepsin inactivation in ATP- ⁰ cells were blocked by caspase-3 inhibitors. Activities of cathepsins B and D in the lysate of necrotic ⁰ cells were inhibited by the addition of apoptotic parental Jurkat cell lysate. Thus, apoptosis may supercede necrosis.

	Introduction

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Apoptosis and necrosis are two major forms of cell death and are generally not related to one another, neither under in vitro experimental conditions nor in in vivo tissues (1). Apoptosis is known to play a critical role in physiological elimination of cells that are no longer needed during morphogenesis or development (2). It is also linked to pathogeneses of some diseases such as T cell depletion in AIDS and premature death of neurons in Alzheimer’s disease and Parkinson’s disease, in addition to therapeutic effects of anticancer drugs.

In contrast, necrosis is a rather pathological type of tissue destruction, such as cardiac (3) or cerebral injury following ischemia-reperfusion (4), cyclosporin A-induced endothelial damage (5), radiation hazard (6), and certain congenital mitochondrial diseases (7). However, recently, investigators claimed that necrotic cell death was implicated in a certain type of physiological cell elimination (8).

The molecular basis of apoptosis has been well elucidated (9). A large amount of active caspase-8 is generated after the stimulation of death receptors, such as Fas, TNF- receptor type 1, and TRAIL receptors, leading to alterations in downstream mitochondria function and the subsequent caspase cascade.

Independent of the caspase-signaling cascade, reactive oxygen species (ROS)³ has been reported to be involved in apoptosis based on the findings that apoptosis was prevented by various antioxidants, including N-acetyl-L-cysteine (NAC) (10, 11), manganese III tetrakis, a nonpeptidyl mimic of superoxide dismutase (SOD) (12), catalase (13), and overexpression of copper/zinc SOD (14). We have linked the role of ROS to that of caspases by demonstrating that the treatment of Jurkat cells with NAC or SOD blocked the formation of an apoptosome and the subsequent caspase-3 activation pathway induced by Fas ligand (15).

Regarding necrosis, however, the molecular events underlying cell death are relatively less understood. Although involvement of ROS (16, 17), lysosomal enzymes (5), mitochondrial-derived proteins including endonuclease G, Omi/HtrA2, and apoptosis-inducing factor, and, most recently, receptor-interacting protein (RIP) (18) have been reported, the mechanisms of necrotic cell death induced by each of these factors and their relationship to one another are unclear.

Furthermore, despite the fact that apoptosis and necrosis are distinct from each other, curiously, they are interchangeable under certain conditions. Transition from a Fas-induced apoptotic state into a necrotic state was accomplished by depletion of ATP with an ATPase inhibitor, and reversal of necrosis into apoptosis was achieved by ATP supplementation (19). In Jurkat cells treated with a pan caspase inhibitor (18) or in a caspase-8-deficient subline of Jurkat cells (16), strong Fas stimulation that transduces an apoptotic signal in normal Jurkat cells instead induced necrosis.

In murine L929 fibrosarcoma cells modified to overexpress Fas, stimulation with Fas ligand rapidly led to apoptosis. When this apoptotic pathway was blocked by a pan caspase inhibitor, necrosis was evoked with the same ligand (17). Similarly, redirection of cell death toward necrosis by blocking apoptotic machinery with cyclosporin A in arterial endothelial cells was also reported (5). The molecular mechanisms underlying this switching phenomenon between necrosis and apoptosis are unclear.

In the present study, we established a subline (⁰ cell) of human Jurkat cells in which mitochondrial DNA (mtDNA) was selectively depleted by long-term exposure to ethidium bromide. Using these ⁰ cells, we showed that Fas ligand clearly induced necrosis without using a pan caspase inhibitor. This necrotic cell death was ascribed to the sequential events of release of excess ROS from mitochondria, rupture of lysosomes, and activation of cathepsins B and D in parallel with RIP activation. Finally, we showed that supplementation of ⁰ cells with ATP restored the formation of the apoptosome and the subsequent activation of caspase-3, which in turn inhibited necrotic cell death by blocking the activities of cathepsins B and D and by degrading RIP. Thus, Fas-triggered RIP activation and the parallel cellular events of ROS release from mitochondria/lysosomal rupture/activation of cathepsins to induce necrosis were found to be superceded by apoptosis.

	Materials and Methods

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Reagents

Agonistic anti-human Fas mAb, clone CH-11, and caspase-3-specific inhibitory peptide N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK) were purchased from Medical and Biological Laboratories. NAC, ATP, 1-antichymotrypsin, epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester (E-64d), chloroquine, bafilomycin cyclosporin A, and pepstatin A were from Sigma-Aldrich. Bongkrekic acid was from Calbiochem. 3'-O-acetyl-2',7'-bis(carboxyethyl)-4 or 5-carboxyfluorescein, diacetoxymethyl ester was from DOJINDO.

Cell culture

Jurkat cells (human T cell leukemia) were supplied by American Type Culture Collection and maintained in RPMI 1640 (Life Technologies) with 10% heat-inactivated FBS (Sigma-Aldrich). To deplete mtDNA in Jurkat cells, long-term culture >70 passages was conducted in medium containing 800 ng/ml ethidium bromide (Sigma-Aldrich), as described previously (20, 21). The parental Jurkat cells and a mtDNA-depleted subline were conventionally designated as ⁺ and ⁰ cells, respectively.

Transduction of the manganese superoxide dismutase (MnSOD) gene

MnSOD expression plasmid pRc/CMV-MnSOD was provided by N. Taniguchi (Department of Biochemistry, Osaka University, Osaka, Japan). Briefly, ⁰ cells were transfected with pRc/CMV-MnSOD using cationic lipid DMRIE-C reagent (Life Technologies), following the manufacturer’s instructions. Three days after the transfection, 800 µg/ml G418 sulfate (Calbiochem) was added to the culture medium to eliminate nontransfected cells. Isolated colonies were obtained by the standard limiting dilution method. Transduction of the MnSOD gene was confirmed by the PCR method using the following set of primers: 5'-GCC AGC ACT AGC AGC ATG TTG AGC C-3' (forward) and 5'-CTT TGG GTT CTC CAC CAC CGT TAG G-3' (reverse). These primers detect a part of MnSOD cDNA (358-bp DNA fragment). Then the MnSOD transfectants and control clones, which were transfected with pRc/CMV, were designated as ⁰-MnSOD and ⁰-neo, respectively.

Southern blotting

Genomic DNA was extracted from 1 x 10⁷ cells by the standard phenol/chloroform method, digested with XbaI (Takara), and loaded onto a 0.8% agarose gel for electrophoresis. After hybridization to GeneScreen membrane (NEN-DuPont), blotting was performed with a mtDNA probe radiolabeled with [-³²P]dCTP (NEN-DuPont) using the BcaBEST labeling kit (Takara). The mtDNA probe mentioned above, which was a full-length cDNA of human cytochrome oxidase subunit (COX) 3, had been amplified beforehand by the PCR method from genomic DNA of normal human monocytes using the following set of primers: 5'-ATG ACC CAC CAA TCA CAT GCC TAT-3' (forward) and 5'-AGA CCC TCA TCA ATA GAT GGA GAC-3' (reverse). The PCR product was subjected to agarose gel electrophoresis, and COX3 cDNA was excised and purified using the GeneClean kit (Bio 101).

Western blotting

Cells (5 x 10⁶) were lysed in a buffer containing 1% SDS, 20 mM Tris-HCl (pH 7.4), 5 µg/ml pepstatin A, 10 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM PMSF, and then boiled for 5 min. After passage through a 20-gauge needle 10 times and centrifugation at 15,000 rpm for 30 min, the aliquot was boiled in a standard reducing sample buffer for 3 min and subjected to SDS-polyacrylamide gel for electrophoresis. It was followed by transfer to Immobilon-P membrane (Millipore) and hybridization with mouse anti-COX2 mAb (clone 12C4; Molecular Probes), mouse anti-cathepsin B mAb (clone CA10; Calbiochem), rabbit anti-cathepsin D polyclonal Ab (JM-3191; Medical and Biological Laboratories), rabbit anti-β-actin polyclonal Ab (4967; Cell Signaling Technology), or rabbit anti-RIP Ab (ab10472; Abcam). Proteins were visualized by ECL (Amersham Biosciences).

Analysis of cell death

Cells (1 x 10⁶) were cultured with anti-Fas Ab, harvested, centrifuged, resuspended in 100 µl of PBS containing 2.5 µg/ml propidium iodide (PI; Sigma-Aldrich), and incubated for 15 min. These cells were analyzed on a FACScan flow cytometer (BD Biosciences). Cell debris was electronically gated out, based on forward and side light scatter. FL2 fluorescence-positive cells were identified as nonviable.

To distinguish necrotic cell death from apoptotic cell death, the cells stained with PI were inspected using confocal fluorescence microscopy, LSM-GB200 (Olympus). As described previously (22), fragmented nuclei or round ones were identified as apoptotic or necrotic, respectively. At least 300 cells were examined in each experiment. The percentages of apoptotic and necrotic cell death within nonviable cells are presented. For electron microscopic analysis, Hitachi H-7000 (Hitachi) was used after fixation of the cells with glutaraldehyde and OsO4. Fragmented DNA in apoptotic cells was amplified by the PCR method using the ApoAlert LM-PCR ladder assay kit (BD Clontech), according to the manufacturer’s instructions.

ATP content

Intracellular ATP content was estimated using the ATP assay kit (Calbiochem). Briefly, 1 x 10⁶ cells were Fas stimulated, washed, and resuspended in 200 µl of PBS. Then 200 µl of provided releasing reagent was added to facilitate ATP release. The luciferin/luciferase reaction was initiated by adding 100 µl of provided luciferase solution, of which the peak height was recorded by a luminometer, ML2200 (Dynatech Laboratories). Luminescence of ATP content was presented relative to control ⁺ cells, which had not been Fas stimulated.

ROS measurement

The production of ROS was quantified fluorometrically using the ROS-sensitive fluorescent probe hydroethidine (HE; Polysciences). Following the stimulation of Fas, cells were harvested, centrifuged, resuspended in 200 µl of PBS with 3 µM HE, and incubated for 30 min on ice. Then the fluorescence was analyzed on a FACScan flow cytometer. Dead cells and debris were excluded by electronic gating of forward and side light scatter. Mean channel fluorescence of ROS production was presented relative to control ⁺ cells, which had not been Fas stimulated.

Lipid peroxidation

Lipid peroxidation was assayed using a Bioxytech LPO-586 kit (OXIS). Briefly, 5 x 10⁷ cells were Fas stimulated, washed, and resuspended in 1.0 ml of PBS, and then Dounce homogenized using a type B (loose) pestle. After adding 10 µl of 0.5 M butylated hydroxytoluene (Sigma-Aldrich), homogenate was centrifuged at 3000 rpm for 10 min. Then 200 µl of the aliquot was mixed with 650 µl of provided reagent R1, N-methyl-2-phenylindole, and 150 µl of 12 N HCl (Sigma-Aldrich). Following the incubation at 45°C for 60 min, malonaldehyde, which results from peroxidation of polyunsaturated fatty acids, was detected spectrophotometrically by ImmunonMini NJ-2300 (InterMed) at 586 nm. Absorbance of lipid peroxidation was presented relative to control ⁺ cells, which had not been Fas stimulated.

Caspase activity

The activity of caspase-3 and -9 was measured using a colorimetric protease assay kit (Medical and Biological Laboratories). Briefly, after Fas stimulation, 1 x 10⁶ cells were collected and suspended in 50 µl of the provided lysis buffer. After incubation for 10 min on ice, cells were centrifuged at 15,000 rpm for 15 min. The supernatant was mixed with 50 µl of provided 2x reaction buffer and 200 µM p-nitroanilide (pNA)-conjugated colorimetric substrate of caspase-3 or -9. Following incubation for 45 min at 37°C, released pNA was detected spectrophotometrically at 405 nm.

For further confirmation, procaspase-9, procaspase-3, and their cleaved fragments were detected by Western blotting. Briefly, 5 x 10⁶ cells were Fas stimulated, collected, and suspended in ice-cold isosmotic buffer, 200 mM mannitol, 70 mM sucrose, 1 mM EGTA, 10 mM HEPES-KOH (pH 7.4), 1 µg/ml pepstatin A, 1 µg/ml leupeptin, and 1 mM pefabloc SC. Then it was Dounce homogenized with a type B (loose) pestle and centrifuged at 1,000 x g for 10 min to separate nuclei and unbroken cells, of which the supernatant was centrifuged at 8,000 x g for 10 min to pellet heavy membranes (mitochondrial fraction). The aliquot was further centrifuged at 100,000 x g to obtain a cytosolic fraction (S-100). After boiling in a standard reducing sample buffer for 3 min, the S-100 fraction was subjected to SDS-PAGE. It was followed by transfer to nitrocellulose membrane and hybridization with mouse anti-caspase-9 Ab (clone 5B4; Medical and Biological Laboratories) or mouse anti-CPP32 Ab (clone 19; BD Transduction Laboratories), which can detect both proenzyme and their cleaved forms.

Chromatographic detection of apoptosome formation

A HiPrep 16/60 S-300 Sephacryl high-resolution column (Amersham Biosciences) was equilibrated with 5% (w/v) sucrose, 0.1% (w/v) CHAPS, and 20 mM HEPES/NaOH (pH 7.0), and calibrated with a Gel Filtration Protein Standards kit (Amersham Biosciences). Then the cytosolic fraction (S-100) of 2 x 10⁸ cells was applied to and eluted from the column at a flow rate of 0.2 ml/min and fractionated every 20 min. All procedures were conducted at 4°C. Each fraction was concentrated by Centricon YM-3 (Millipore) and analyzed by Western blot with the anti-caspase-9 mouse mAb mentioned above, anti-apaf-1 mouse mAb (clone 94408.11; R&D Systems), or anti-cytochrome c mouse mAb (clone 7H8.2C12; BD Pharmingen).

Analysis of cytochrome c release

A total of 1 x 10⁶ cells was harvested and washed twice with PBS. The cytosolic fraction (S-100) was prepared, as described above. The pellet of mitochondrial fraction was resuspended in 10 mM Tris-HCl (pH 7.4) containing 1% SDS. Western blot analysis was performed with the anti-cytochrome c mouse mAb mentioned above.

Measurement of lysosomal rupture

Lysosomal rupture was analyzed using the lysosomotropic weak base acridine orange (AO; Molecular Probes). This vital dye accumulates in intact lysosomes and exhibits red fluorescence. Thus, the rupture of lysosomes can be monitored as a decrease in red fluorescence. Briefly, following the stimulation of Fas, cells were harvested, centrifuged, suspended in 200 µl of PBS with 5 µg/ml AO, and incubated for 20 min. Then FL2 fluorescence was analyzed on a FACScan flow cytometer. Dead cells and debris were excluded by electronic gating of forward and side light scatter. The percentage of FL3-negative cells was presented as lysosomal rupture relative to control ⁺ cells, which had not been Fas stimulated.

Short interfering RNA (siRNA)

Cathepsin B siRNA (100 pmol; Santa Cruz Biotechnology), cathepsin D siRNA (Santa Cruz Biotechnology), RIP siRNA (Santa Cruz Biotechnology), or control siRNA-A (Santa Cruz Biotechnology) diluted with siRNA transfection medium (Santa Cruz Biotechnology) was introduced into 1 x 10⁶ cells using siRNA transfection reagent (Santa Cruz Biotechnology), according to the manufacturer’s instructions. Six hours posttransfection, transfection medium containing transfection reagent and siRNA was exchanged to normal growth medium with FBS. Then the cells were cultured for another 48 h.

Measurement of cytosolic cathepsin activities

The activity of cathepsin B or cathepsin D was measured using Z-Arg-Arg-pNA (Calbiochem) or Bz-Arg-Gly-Phe-Phe-Pro-4MbNA (Calbiochem), respectively. Briefly, after Fas stimulation, 1 x 10⁶ cells were collected and lysed without disruption of lysosomes in 50 µl of lysis buffer (25 µg/ml digitonin, 250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, and 1 mM pefabloc (pH 7.5)). After incubation for 10 min on ice, the cells were centrifuged at 15,000 rpm for 15 min. The supernatant was diluted by reaction buffer (50 mM sodium acetate, 4 mM EDTA, 8 mM DTT, and 1 mM pefabloc (pH 6.0)) supplemented with 50 µM substrates of cathepsin B or D mentioned above. The dilution ratio was optimized in each experiment. Following incubation for 45 min at 37°C, released pNA or 4MbNA was detected spectrophotometrically at 405 nm or fluorometrically at emission 410 nm and excitation 335 nm, respectively.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Depletion of mtDNA

To deplete mtDNA selectively, Jurkat cells were exposed to ethidium bromide, as described previously (20, 21). The mtDNA was substantially depleted in ethidium bromide-treated Jurkat cells (⁰) compared with that of parental Jurkat cells (⁺), as revealed by both Southern blotting using a probe to COX3 gene and Western blotting using a specific Ab to COX2 (Fig. 1A). There were no differences in growth rates after 3 days of incubation, in morphologic characters, or in Fas expression, as analyzed by flow cytometry between ⁺ and ⁰ cells (data not shown). To validate that mtDNA-depleted cells were impaired in production of ATP, intracellular ATP content was assessed by a luciferin/luciferase method and results from ⁰ cells were compared with that of ⁺ cells. The ATP concentration in ⁰ cells was almost one-third of ⁺ cells (Fig. 1B). When the ⁰ and ⁺ cells were stimulated with anti-Fas Ab (CH-11), ATP concentrations gradually decreased, possibly due to the consumption of ATP during the process of cell death (Fig. 1B).

View larger version (41K): [in this window] [in a new window]   FIGURE 1. Fas stimulation induced necrotic cell death on 0 cells. A, Quantification of mtDNA by Southern blot analysis of COX3 (upper) and Western blot analysis of COX2 (lower) in + (left) and 0 (right) cells was performed. B and D, + () and 0 (•) cells were incubated with 100 ng/ml CH-11 for the indicated periods of time. C, + () and 0 (•) cells were incubated with the indicated concentrations of CH-11 for 12 h. E, + (left) and 0 (right) cells were incubated with 100 ng/ml CH-11 for 12 h. The cells positively stained with PI were quantified by flow cytometry as nonviable cells (C and D). ATP content (B) and DNA fragmentation (E) were also examined. + (left) and 0 (right) cells were incubated with 100 ng/ml CH-11 for 12 h. Morphological features were examined by inverted microscope (F) and electron transmission microscope (G). Based on the analysis of nuclei staining with PI by confocal fluorescence microscope (H), nonviable cells were classified as necrotic or apoptotic (I). All of the procedures are detailed in Materials and Methods. A representative result of three independent experiments is shown in A, E, F, G, and H. The data represent the means (bars, SD; n = 5) in B, C, D, and I.

Fas-induced necrosis of ⁰ cells

After incubation for 12 h with various concentrations of CH-11, ⁺ and ⁰ cells were analyzed by flow cytometry with the fluorescent probe PI, which penetrates nuclei of both apoptotic and necrotic cells and exhibits red fluorescence. The percentage of PI-positive fractions in both cells increased dose dependently and reached a plateau at 100 ng/ml CH-11 (Fig. 1C). At this concentration, 56% of ⁺ cells stained positively, whereas ⁰ cells were more sensitive to Fas stimulation, exhibiting 92% PI-positive cells (Fig. 1C).

Using 100 ng/ml CH-11, we then monitored cell death of both 0 and ⁺ over a time course (Fig. 1D). The occurrence of cell death of ⁰ lagged that of ⁺ by 1 h, but its magnitude was significantly higher, findings compatible with Fig. 1C. The nonviable cell number for both ⁰ and ⁺ reached a plateau after 12 h of CH-11 treatment. When DNA fragmentation was examined, it was obvious in ⁺ cells, but not in ⁰ cells (Fig. 1E).

Furthermore, inverted microscopic analysis revealed that Fas stimulation for 12 h induced morphologically characteristic changes in ⁰ cells, which were readily distinguished from ⁺ cells (Fig. 1F). Examination by electron transmission microscopy revealed that ⁰ showed typical necrotic death with vacuole formation, maintenance of nuclear membrane integrity, and high electron translucency of the cytoplasm (Fig. 1G). In contrast, the death of ⁺ was characterized by cytoplasmic shrinkage, condensation of chromatin, and formation of apoptotic bodies (Fig. 1G), features consistent with apoptotic death. Confocal fluorescence microscopy with PI staining (22) demonstrated that 95% of ⁺ cells exhibited apoptotic nuclei with DNA fragmentation and that 92% of ⁰ cells contained shrunken nuclei characteristic of necrosis (Fig. 1, H and I).

Besides, neither wortmanin nor 3-methyladenine, which were known to suppress autophagy, inhibited Fas-induced cell death of ⁰ (data not shown). Also, the knockdown of Beclin1 or Atg5, which were both essential signal transducers of autophagy, did not inhibit Fas-induced cell death of ⁰ (data not shown). LC3-II, into which LC3-I was converted in autophagic process, was not increased when ⁰ cells were stimulated by Fas (data not shown). These results indicated that Fas-induced cell death of ⁰ was not autophagy.

Activities of caspase-3 and -9 in ⁰ treated with Fas

To ensure that the apoptotic caspase cascade was not playing a crucial role in necrotic death of ⁰, we measured the activities of both caspase-3 and -9 induced by Fas stimulation using a colorimetric assay. The activities of these enzymes in ⁺ cells started to increase at 3 h and peaked at 6 h after Fas stimulation, whereas there was little activation in ⁰ cells (Fig. 2A). Likewise, when cleavage of procaspases-9 and -3 were examined by Western blotting, cleaved forms (active forms) of these proenzymes were clearly detected in ⁺ cells, but were undetectable in ⁰ cells (Fig. 2B). Otherwise, caspase-8 was activated in ⁰ cells as well as in ⁺ cells by the Fas stimulation (data not shown).

View larger version (43K): [in this window] [in a new window]   FIGURE 2. Caspase activity was reduced in necrotic 0 cells. + and 0 cells were incubated with 100 ng/ml CH-11 for the indicated periods of time (A and B). or •, Indicate + or 0 cells, respectively (A). Activity of caspase-9 and -3 was estimated by both colorimetric assay (A) and Western blot analysis (B), as described in Materials and Methods. The data represent the means (bars, SD; n = 5) in A. A representative result of three independent experiments is shown in B.

Impaired formation of apoptosome in Fas-stimulated ⁰

To confirm the above observation, we next examined by the chromatographic fractionation method (15) the formation of the apoptosome, the ATP/dATP-dependent process (23) of assembling apaf-1, procaspase-9, and cytochrome c into a complex. As shown in Fig. 3, A and C, without Fas stimulation, apaf-1, procaspase-9, and cytochrome c in both ⁺ and ⁰ cells were detected in accordance with their respective molecular weights in fractions 4–9, 12–15, and 17–19, respectively. After stimulation of Fas, each component in ⁺ cells was detected in the high m.w. fractions 1 and 2, indicating the formation of the apoptosome (Fig. 3B). In contrast, the components in ⁰ cells all remained in the low m.w. fractions regardless of Fas stimulation (Fig. 3D), indicating no apoptosome formation in these cells even after Fas stimulation.

View larger version (55K): [in this window] [in a new window]   FIGURE 3. Apoptosome formation was inhibited in necrotic 0 cells. + (A and B) and 0 (C–E) cells were incubated with 100 ng/ml CH-11 (B, D, and E) in the presence (E) or absence (A–D) of 10 mM ATP for 3 h. Chromatographic detection of apoptosome formation was performed, as described in Materials and Methods. A representative result of three independent experiments is shown.

Dependency of Fas-induced necrosis on ROS in ⁰

We next explored the implication of ROS in necrotic cell death by using flow cytometric analysis with the ROS-sensitive fluorescent probe HE. In ⁰ cells, intracellular ROS increased soon after Fas stimulation (1 h) and peaked after 6 h of stimulation (5-fold elevation). Generation of ROS in ⁺ cells was only 2-fold higher after 6 h (Fig. 4A).

View larger version (28K): [in this window] [in a new window]   FIGURE 4. ROS production was enhanced in necrotic 0 cells. A, + () and 0 (• and closed rhombuses) cells were incubated with 100 ng/ml CH-11 in the presence (rhombuses) or absence ( and •) of 10 mM ATP for the indicated periods of time. B and C, + () and 0 () cells were incubated with 100 ng/ml CH-11 in the presence of the indicated concentrations of NAC for 6 (B) or 12 (C) h. ROS production (A and B) and nonviable cells (C) were estimated, as described in Materials and Methods. The data represent the means (bars, SD; n = 5). D and E, 0–neo (left) and 0–MnSOD (right) cells were incubated with () or without () 100 ng/ml CH-11 for 6 (D) or 12 (E) h. ROS production (D) and nonviable cells (E) were estimated, as described in Materials and Methods. The data represent the means (bars, SD; n = 5). F, + () and 0 (• and closed rhombuses) cells were incubated with 100 ng/ml CH-11 in the presence (rhombuses) or absence ( and •) of 10 mM ATP for the indicated periods of time. Lipid peroxidation was estimated, as described in Materials and Methods. The data represent the means (bars, SD; n = 5).

As for the mechanisms by which large excess of ROS was induced in ⁰ cells by Fas stimulation (Fig. 4A), we focused on the possible involvement of the induction of mitochondrial permeability transition (MPT) and resultant cytochrome c release. By using cyclosporin A or bongkrekic acid, which suppressed the induction of MPT and resultant cytochrome c release in ⁰ cells induced by Fas stimulation (Fig. 5A), Fas-induced ROS production in ⁰ cells was effectively inhibited (Fig. 5B). Furthermore, the basal level of ROS in ⁰ cells was not higher than that in parental Jurkat, ⁺ cells (Fig. 5C). We surmise that by the trigger of Fas stimulation, the excessive ROS production is induced in ⁰ cells due to the insufficiency of respiratory chain, which usually consumes oxygen.

View larger version (16K): [in this window] [in a new window]   FIGURE 5. ROS production was induced by MPT in necrotic 0 cells. A and B, 0 cells were incubated with or without 100 ng/ml CH-11 for 3 (A) or 6 h (B) in the presence or absence of 5 µM cyclosporine A or 100 µM bongkrekic acid. C, + (left) and 0 (right) cells were incubated with no additional regents. Released cytochrome c (A) was detected, and ROS production (B and C) was estimated, as described in Materials and Methods. A representative result of three independent experiments is shown in A and C. The data represent the means (bars, SD; n = 5) in B.

Increased lipid peroxidation in Fas-stimulated ⁰

Because ROS is known to damage subcellular structure by lipid peroxidation of membranes, we measured the relative levels of lipid peroxidation by the malonaldehyde method in ⁰ cells. One hour after Fas stimulation, lipid peroxidation became evident, although at a lower level (Fig. 4F) relative to the induction of ROS (Fig. 4A). Lipid peroxidation levels gradually increased to >3-fold after 6 h of stimulation, reaching a plateau at 12 h. In ⁺ cells, no appreciable amount of lipid peroxidation was detected after 1 and 3 h of Fas stimulation, but a 1.5-fold increase was observed after 6 h. These results indicate that lipid peroxidation occurred more frequently in ⁰ cells than in ⁺ cells, although its onset appeared to be late compared with that of ROS generation.

ROS-induced lysosomal rupture in ⁰ cell

Based on our experiments and those using an ischemia-reperfusion model (24, 25, 26) to examine the involvement of lysosomal proteases in necrotic cell death, we were prompted to examine whether ROS generated by Fas stimulation caused lysosomal rupture in ⁰ cells. Lysosomal rupture in ⁰ cells as monitored by FACS using an AO indicator occurred rather slowly after Fas stimulation (Fig. 6A) compared with the rapid rise of ROS (Fig. 4A) and caspases (Fig. 2), but showed a quite similar curve to that of lipid peroxidation (Fig. 4F). However, the percentage of lysosomal rupture in ⁰ after 6 and 12 h of stimulation was significantly higher (60%) than in ⁺ cells (10%). Treatment with NAC and MnSOD transfection clearly inhibited lysosomal rupture in ⁰ cells (Fig. 6, B and C), suggesting the involvement of ROS in lysosomal rupture. When ⁰ cells were treated with CH-11 in the presence of lysosomal stabilizers chloroquine (Fig. 6D) and bafilomycin (Fig. 6E), the nonviable cell number significantly decreased, indicating lysosomal rupture in necrosis.

View larger version (23K): [in this window] [in a new window]   FIGURE 6. Lysosomal rupture was enhanced in necrotic 0 cells. A, + () and 0 (• and closed rhombuses) cells were incubated with 100 ng/ml CH-11 in the presence (rhombuses) or absence ( and •) of 10 mM ATP for the indicated periods of time. B, 0 cells were incubated with ( and ) or without () 100 ng/ml CH-11 in the presence () or absence ( and ) of 25 mM NAC for 6 h. C, 0–neo (left) and 0–MnSOD (right) cells were incubated with () or without () 100 ng/ml CH-11 for 6 h. D and E, 0 cells were incubated with or without 100 ng/ml CH-11 in the presence of the indicated concentrations of chloroquine (D) or bafilomycin (E) for 12 h. Lysosomal rupture (A–C) and nonviable cells (D and E) were estimated, as described in Materials and Methods. The data represent the means (bars, SD; n = 5).

Role of cathepsins B and D in necrotic cell death of ⁰

To identify the molecules whose activations are associated with lysosomal rupture, we examined the effect of various protease inhibitors on necrosis of ⁰. A combination of inhibitors for lysosomal proteases, cysteine protease inhibitor E-64d and aspartic protease inhibitor pepstatin A, but not serine protease inhibitor 1-antichymotrypsin, substantially suppressed necrotic death of ⁰ cells (Fig. 7A). This indicates the role of proteases after release from ruptured lysosomes in necrotic death of ⁰.

View larger version (36K): [in this window] [in a new window]   FIGURE 7. Cathepsin inhibitors suppressed necrosis of 0 cells. A, 0 cells were incubated with or without () 100 ng/ml CH-11 for 12 h in the presence () or absence () of 10 µM E-64d, 1 µM pepstatin A (Pep-A), and/or 10 µg/ml 1-antichymotrypsin (1-ACM). B and C, 0 (left) and + (right) cells were incubated with () or without () 100 ng/ml CH-11 for 6 h. D, 0 cells were incubated with or without 100 pmol of control siRNA, cathepsin B siRNA, and/or cathepsin D siRNA for 48 h. E, After the incubation with or without () 100 pmol of control siRNA, cathepsin B siRNA, and/or cathepsin D siRNA for 48 h, 0 cells were further incubated with 100 ng/ml CH-11 for 12 h ( and ). Nonviable cells (A and E) and cathepsin activities (B and C) were estimated, and Western blot analysis (D) was performed, as described in Materials and Methods. A representative result of three independent experiments is shown in D. The data represent the means (bars, SD; n = 5) in A, B, C, and E.

Role of RIP in necrotic cell death of ⁰ induced by CH-11

RIP has been previously shown to be involved in necrosis of Jurkat cells, which were treated with high doses of Fas ligand in the presence of a pan caspase inhibitor (18). We, therefore, knocked down RIP using specific siRNA (Fig. 10A) and examined the sensitivity of RIP knocked down (RIPKO) ⁰ cells to CH-11. As shown in Fig. 10B, although the difference was small, the number of nonviable RIPKO⁰ cells was significantly lower than the number of nonviable ⁰ cells. When RIPKO⁰ cells were treated with NAC, almost complete inhibition of CH-11-induced cell death was observed. These results indicate that, in addition to lysosomal cathepsins, RIP is also involved in necrosis of ⁰, although the latter involvement is a rather minor event.

View larger version (33K): [in this window] [in a new window]   FIGURE 10. Apoptotic lysate of + cells suppressed cathepsin activities in necrotic lysate of 0 cells. A, 0 cells were incubated with or without 100 pmol of control siRNA or RIP siRNA for 48 h. B, After the incubation with 100 pmol of control siRNA or RIP siRNA for 48 h, 0 cells were further incubated with or without 100 ng/ml CH-11 in the presence or absence of 25 mM NAC for 12 h. Western blot analysis (A) was performed, and nonviable cells (B) were estimated, as described in Materials and Methods. A representative result of three independent experiments is shown in A. The data represent the means (bars, SD; n = 5) in B. C and D, 0 and + cells were incubated with 100 ng/ml CH-11 for 6 h. Then the lysates of 0 and + cells were mixed in the indicated proportions. Cathepsin activity in the mixtures was estimated, as described in Materials and Methods. The data represent the means (bars, SD; n = 5). The broken lines were the expected decreases in cathepsin activity due to dilution effects. The statistical significance between expected and actual cathepsin activities was calculated using statistical software SPSS. E, After incubation with or without 20 µM Z-DEVD-FMK for 2 h, 0 cells were further incubated with or without 100 ng/ml CH-11 in the presence or absence of 10 mM ATP for 3 h. Western blot analysis was performed, as described in Materials and Methods. A representative result of three independent experiments is shown. F, Schematic overview of Fas-induced necrosis of 0 cells.

Conversion of necrosis to apoptosis in ⁰ by ATP supplementation

To elucidate whether supplementation of ATP to ⁰ cells converts necrotic death to apoptosis, we added ATP to culture medium of ⁰ cells and stimulated them with CH-11. As shown in Fig. 8A, in ATP-supplemented ⁰ cells, cell death occurred immediately after CH-11 stimulation with no lag time in a similar manner as ⁺ cells that underwent apoptosis upon CH-11 stimulation (Fig. 1D). The ATP-supplemented ⁰ cells showed dose-dependent morphological conversion from necrosis to apoptosis as ATP concentrations increased. At 10 mM ATP, almost complete conversion to apoptosis was achieved (Fig. 8B). This dose-dependent morphological conversion was consistent with the observation of DNA laddering, increasing the appearance of DNA fragmentation as the ATP concentration increased (Fig. 8C). The activation of both caspase-3 and -9 detected by colorimetric analysis (Fig. 8D) as well as by Western blotting (Fig. 8E) was also almost completely recovered by ATP treatment. Furthermore, apoptosome formation in ⁰ cells by addition of ATP was clearly confirmed (Fig. 3E).

View larger version (43K): [in this window] [in a new window]   FIGURE 8. Apoptosis was restored in necrotic 0 cells by ATP supplementation. A, 0 cells were incubated with 100 ng/ml CH-11 in the presence of 10 mM ATP for the indicated periods of time. Nonviable cells were estimated, as described in Materials and Methods. B and C, 0 cells were incubated with 100 ng/ml CH-11 in the presence of the indicated concentrations of ATP for 12 h. Microscopic counting of apoptotic () or necrotic () cells (B) and detection of DNA fragmentation by the PCR method (C) are detailed in Materials and Methods. D and E, 0 cells were incubated with 100 ng/ml CH-11 in the presence ( in D and the second lanes in E) or absence ( in D and the first lanes in E) of 10 mM ATP for 6 h. + cells were also stimulated by CH-11 without ATP as a control ( in D and the third lanes in E). Activity of caspase-9 (left) and -3 (right) was estimated by both colorimetric assay (D) and Western blot analysis (E), as described in Materials and Methods. The data represent the means in A, B, and D (bars, SD; n = 5). A representative result of three independent experiments is shown in C and E.

View larger version (26K): [in this window] [in a new window]   FIGURE 9. Caspase-3 inhibited cathepsin activities in ATP-supplemented apoptotic 0 cells. A and B, 0 cells were incubated with or without 100 ng/ml CH-11 in the presence of the indicated concentration of ATP for 6 h. C–E, After incubation with or without 20 µM Z-DEVD-FMK for 2 h, 0 cells were further incubated with 100 ng/ml CH-11 in the presence or absence of 10 mM ATP for 6 (D and E) or 12 (C) h. Cathepsin activities (A, B, D, and E) and nonviable cells (C) were estimated, as described in Materials and Methods. The data represent the means (bars, SD; n = 5).

To examine whether the caspase cascade negatively regulated necrosis through inhibition of cathepsins, we treated ATP-supplemented ⁰ cells with an inhibitor of caspase-3, an apoptosis promoter. As shown in Fig. 9, C–E, the caspase-3 inhibitor Z-DEVD-FMK induced necrosis in ATP-supplemented ⁰ cells with full recovery of cathepsin B and D activities.

The mechanisms underlying the inhibitory action of caspase-3 on cathepsins were then investigated. The possibility that caspase-3 directly cleaved these cathepsins is unlikely because there is no target sequence, DEVD, for caspase-3 in these enzymes. Furthermore, no apparent cleavage bands of cathepsins were observed in ATP-supplemented ⁰ cells treated with CH-11 (data not shown). Another possibility was that some unknown cofactor(s) that binds to cathepsins to augment their activity was cleaved by caspase-3. However, there was no apparent difference in silver staining patterns of precipitates with anti-cathepsin B and D Abs between ⁰ and ATP-supplemented ⁰ cells (data not shown). Interference of cathepsin activity by shifting the cytosolic pH away from the optimal range as the result of digestion of various target molecules in the cytosol by caspase-3 was disproved by the observation that there was no significant difference in cytosolic pH of ⁰ cells and ATP-supplemented ⁰ cells (data not shown). When we mixed cytosol of CH-11-treated ⁰ cells with CH-11-treated ⁺ cells in different proportions, as indicated in Fig. 10, C and D, the actual activities of cathepsins B and D were significantly lower than those theoretically predicted, indicating inhibition of cathepsins in ⁰ cells by cytosolic components of apoptotic cells. Besides, the activities of caspase-9 and -3 in Fas-stimulated apoptotic ⁺ cell lysate were decreased by the adding of Fas-stimulated necrotic ⁰ cell lysate with just a dilution effect of mixing (data not shown).

Degradation of RIP by caspase-3

To examine the possible effect of caspase on RIP, we performed Western blotting of RIP from ATP-supplemented ⁰ cells in the presence or absence of Z-DEVD-FMK. As shown in Fig. 10E, RIP completely disappeared in ATP-supplemented ⁰ cells concomitantly with the appearance of lower m.w. RIP species. Treatment of ATP-supplemented ⁰ with Z-DEVD-FMK restored the original RIP band, compatible with the notion that RIP, which has the caspase-3-sensitive motif DEID at aa 314–317, is being degraded by caspases. A postulated schematic overview of necrotic signaling in ⁰ cells was shown in Fig. 10F.

	Discussion

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In the present investigation, we established a mtDNA-depleted subline of Jurkat cells (⁰) to study the molecular mechanism of necrosis induced by Fas signal stimulation on the basis of previous reports that various mitochondrial diseases are associated with tissue damage characteristic of necrotic cell death (7). Furthermore, in mtDNA-depleted syndromes, necrosis of hepatocytes is generally associated (27) and cyclosporin A caused deep mitochondrial damage, followed by necrosis of endothelial cells (5).

Results obtained indeed indicate that ⁰ underwent necrosis upon stimulation with agonistic Fas Ab (CH-11), suggesting that in mitochondrial diseases (7) or mtDNA-depleted syndromes (27), chronic exposure of tissues (cells) to endogenous death triggers (such as Fas ligand and possibly TNF) by circulation might be the cause of tissue damage by necrosis. The molecules responsible for necrosis in ⁰ were found to be ROS, lysosomal enzymes (particularly cathepsins B and D), and RIP.

The role of ROS in necrosis has been noted in various experimental models and cell lines (1, 16, 17). However, the mechanisms inducing necrosis by ROS are not fully understood at present (1), and, with the exception of cyclosporin A, evoked necrotic death of endothelial cells in which ROS caused lysosomal rupture and release of cathepsins, promoters of necrosis.

In the present study, we describe a similar necrosis pathway involving ROS/lipid peroxidation/lysosomal rupture/cathepsin activation in Fas-triggered ⁰ cells, suggesting that these sequential events might be a general scheme of ROS-related necrosis, irrespective of cell types or triggering agents. Incidentally, we have previously shown that ROS is also involved in apoptotic cell death of CH-11-treated Jurkat cells (⁺) by facilitating apoptosome formation (15). Thus, ROS is considered to be a common and key factor for both apoptosis and necrosis, and, therefore, scavenging ROS may be a reasonable means of preventing tissue damage, irrespective of the type of cell death.

Although ROS-related cellular events were primarily responsible for necrotic death in ⁰ cells, RIP was also found to participate in necrosis, but as a minor player in the present study. The RIP pathway has been identified as a major causative component for necrosis in Jurkat cells treated with soluble Fas ligand with cross-linking Abs, an extremely strong stimulator of Fas, in the presence of a pan caspase inhibitor (18). The reason for the apparently less pivotal role of RIP in necrosis of ⁰ cells than in Jurkat cells may be that we did not use a pan caspase inhibitor and used relatively weak Fas stimulation (CH-11 at a concentration of 100 ng/ml compared with 1000 ng/ml in a previous report (18) with no pan caspase inhibitor).

Reversal of necrosis by addition of ATP was clearly seen in ⁰ cells in accordance with the previously reported fact that Jurkat cells, which underwent necrosis by depletion of ATP from culture medium, showed an apoptotic type of death when ATP was supplemented into the medium (19). This indicates that in ⁰ cells, ATP deficiency, not generation of excess ROS, determines necrotic cell fate. In other words, ⁰ cells never undergo necrosis even in the presence of excess ROS if ATP is sufficiently supplied, suggesting that apoptosis may be the primary type of cell death, whereas necrosis occurs only when the apoptosis cascade is not functioning.

This dominance of apoptosis over necrosis was proven by the subsequent observation that treatment of ATP-supplemented ⁰ cells with caspase-3 inhibitor resulted in necrosis of the cells (Fig. 9C).

Similar results have been repeatedly reported in various cell types, such as Fas-transfected murine fibroblast L929 cells (17), murine thymocytes (28), caspase-8-deficient Jurkat cells (16), ATP-depleted Jurkat cells (19), Jurkat cells stimulated by soluble Fas ligand with cross-linking Abs (18), and cyclosporin A-treated endothelial cells (5), of which the caspase cascade was either blocked by inhibitors or genetically impaired. However, in these previous reports, the molecular mechanism for the regulation of necrotic cell death by the caspase cascade was not elucidated. The biological implication of apoptosis dominance over necrosis is merely a matter of speculation, but could be a reflection of ontogeny, meaning that necrosis is simply an older phenomenon in ontogenical sequence than apoptosis.

In the present study, we determined that the activity of cathepsins, which appeared to be the main executor of necrosis, was inhibited by the addition of cytosol from Jurkat cells (⁺ cells) stimulated with CH-11 in the absence of a caspase inhibitor. The most plausible explanation for this inhibitory effect of ⁺ apoptotic cytosol on cathepsins from ⁰ necrotic cytosol is that the small peptides produced by cleavage of intracellular caspase-3 target proteins work as the cathepsin inhibitors. In fact, there are several proteins, such as Fodrin, DNA-dependent protein kinase catalytic subunit, protein kinase C, and plasma membrane calcium-transporting ATPase 4, which have both target sequences for caspase-3 cleavage (DEVD/DEID) and inhibitory sequences for cathepsin B (LVK) (29). However, identification of every one of these peptides is extremely laborious and remains as a future task. Incidentally, other possibilities, such as that caspase-3 altered intracellular pH from the optimal range for cathepsins and that caspase-3 cleaved cathepsins B and D, are not likely to occur (data not shown).

We also describe an inhibitory effect of caspase-3 on RIP activity, a minor executor of necrosis in ⁰ cells, as evidenced by the fact that degradation of RIP in ATP-supplemented ⁰ cells upon CH-11 stimulation was inhibited by caspase-3 (Fig. 10E). Because there is a caspase-3 target sequence (DEID) at the C-terminal end of RIP (30) and the C-terminal cleaved RIP protein is calculated to have a molecular mass of 11 kDa, our observation of a 65-kDa band in lysates of ATP-supplemented ⁰ cells is reasonable. Thus, both major and minor pathways of necrosis in ⁰ cells were considered to be under the control of caspase-3. Incidentally, it has been surmised that caspase-8, not caspase-3, is responsible for the cleavage of RIP upon Fas stimulation (18), based on the fact that caspase-8-deficient Jurkat cells underwent necrosis (16, 18) and that caspase-8 is a potent proteolytic enzyme for RIP (31). However, the present result suggesting that caspase-3 is the key controller of necrosis-apoptosis conversion does not contradict these findings because caspase-3 is downstream of caspase-8 and was shown to be capable of similarly cleaving RIP in vitro (30).

In the previous literature (17), L929 cells died Fas-induced necrotic cell death in the complete absence of all caspase activities with the induction of excessive ROS production, whereas caspase-8 is actually activated in the absence of ATP and required for the downstream events in ⁰ cells. Caspase-8 can be activated and evoke MPT in the ATP-depleted conditions of rho0 cells because ATP-dependent step of caspase cascade is downstream of MPT, that is to say apoptosome formation (32). Taking together these findings, the essential event of necrotic cell death is an excessive ROS production, irrespective of how ROS production is induced. Additionally, it is therefore not surprising that RIP is not much involved in this process, because it has been reported for RIP to lead directly to the production of ROS through activation of NADPH oxidase in a caspase-independent manner, and apparently independent of mitochondria (33).

In conclusion, we used ⁰ cells stimulated with agonistic anti-Fas Ab to demonstrate that excess ROS, which caused lysosomal rupture and release of cathepsins, is a major pathway mediating necrosis, whereas activation of RIP was a minor contributor. Reactivation of the caspase cascade by ATP supplementation almost completely shut down these necrotic events through inactivation of cathepsins and RIP by caspase-3, suggesting dominance of caspase-induced apoptosis over necrosis. Based on these results and the fact that ROS plays an essential role in both apoptosis (15) and necrosis, we suggest using antioxidative agents rather than caspase inhibitors, ATP supplementation, or cathepsin inhibitors to treat tissue damage, irrespective of necrotic or apoptotic cell death.

	Acknowledgments

 
We thank Dr. Naoyuki Taniguchi (Department of Biochemistry, Osaka University, Osaka, Japan) for the gift of the MnSOD expression plasmid pRc/CMV-MnSOD.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported in part by the Japan Society for the Promotion of Science.

² Address correspondence and reprint requests to Dr. Yoshiro Niitsu, Department of Internal Medicine (Section 4), Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo 060-8543, Japan. E-mail address: niitsu{at}sapmed.ac.jp

³ Abbreviations used in this paper: ROS, reactive oxygen species; AO, acridine orange; COX, cytochrome oxidase subunit; E-64d, epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester; HE, hydroethidine; MnSOD, manganese superoxide dismutase; MPT, mitochondrial permeability transition; mtDNA, mitochondrial DNA; NAC, N-acetyl-L-cysteine; PI, propidium iodide; pNA, p-nitroanilide; RIP, receptor-interacting protein; RIPKO, RIP knocked down; siRNA, short interfering RNA; SOD, superoxide dismutase; Z-DEVD-FMK, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone.

Received for publication December 18, 2007. Accepted for publication April 30, 2008.

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