Proteolytic Loss of bcl-xL in FL5.12 Cells Undergoing Apoptosis Induced by MK886

Toxicology and Applied Pharmacology 174, 273–281 (2001) doi:10.1006/taap.2001.9220, available online at http://www.idealibrary.com on

Proteolytic Loss of bcl-x L in FL5.12 Cells Undergoing Apoptosis Induced by MK886 Kaushik Datta, Julie C. Kern, Shyam S. Biswal, and James P. Kehrer 1 Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas, Austin, Texas 78712 Received January 15, 2001; accepted May 22, 2001

Proteolytic Loss of bcl-x L in FL5.12 Cells Undergoing Apoptosis Induced by MK886. Datta, K., Kern, J. C., Biswal, S. S., and Kehrer, J. P. (2001). Toxicol. Appl. Pharmacol. 174, 273–281. Apoptosis induced in the IL3-dependent murine pro-B lymphocytic (FL5.12) cell line by the 5-lipoxygenase activating protein inhibitor MK886 is accompanied by the rapid loss of the antiapoptotic bcl-x L and bcl-2, but not the proapoptotic bax proteins (Datta et al., J. Biol. Chem. 273, 28163–28169, 1998). Since several reports indicate important roles for noncaspase proteases in apoptosis, the participation of lysosomes, as well as serine, cysteine, or aspartic acid proteases, in the effects of MK886 were investigated. Consistent with the involvement of various proteases, lysosomal degranulation was evident, as observed by a decrease in acridine orange fluorescence at 2 h and an increase in cytosolic ␤– hexosaminidase activity at 4 h after treating FL5.12 cells with 10 ␮M MK886. The disappearance of bcl-x L from FL5.12 cells upon MK886 treatment was prevented in a dose-dependent manner by pretreatment with leupeptin, pepstatin, phenylmethylsulfonyl fluoride, or the broad-spectrum caspase inhibitor Boc-D-FMK. Each of the noncaspase protease inhibitors partially inhibited MK886induced apoptosis as measured by phosphatidylserine externalization and DNA fragmentation. The noncaspase inhibitors also blocked about half of the increase in caspase-3-like activity. Boc-D-FMK completely inhibited this enzyme and prevented apoptosis. None of the inhibitors were able to directly inhibit activated caspase-3 in cell lysates, suggesting their effects were upstream of caspase activation. These observations suggest the involvement of various proteases, possibly originating from lysosomes, upstream of active caspase-3, in the loss of bcl-x L protein and in the signaling pathway of MK886induced apoptosis in FL5.12 cells. This pathway may be unique to MK886 since these same protease inhibitors had only minimal effects on etoposide-induced apoptosis and the accompanying moderate loss of bcl-x L in FL5.12 cells. © 2001 Academic Press Key Words: apoptosis; proteases; caspase; bcl-2; bcl-x L; 5-lipoxygenase activating protein; MK886; lysosome.

DNA fragmentation was initially emphasized as the major marker of apoptotic cell death. More recent literature has highlighted the importance of proteolytic events in the effector 1

To whom correspondence should be addressed. Fax: (512) 471-5002. E-mail: [email protected].

phase of apoptosis due to the identification of several proteases that induce the morphological and biochemical features of this form of cell death. Since apoptosis can be initiated in the absence of a nucleus (Jacobson et al., 1994), there may be a “cytoplasmic regulator” of this process, such as a protease or a complex of various proteases. The family of cysteine-aspartate proteases, known as caspases, plays a vital role in both initiation and execution stages of apoptotic cell death (Cohen, 1997). The apoptosismodifying BCL-2 family of proteins may modulate the activation of caspases by controlling cytochrome c release required for stress-induced caspase activation via the apoptosome (Bratton et al., 2000). The antiapoptotic bcl-2 and bcl-x L proteins are degraded following caspase activation induced by IL-2 2 or IL-3 withdrawal (Fujita et al., 1998; Cheng et al., 1997), or treatment with TGF-␤ (Saltzman et al., 1998), sphingosine (Shirahama et al., 1997), or etoposide (Fadeel et al., 1999), suggesting this may be an important permissive step in the apoptotic process. Nevertheless, the loss of the antiapoptotic bcl proteins is not required for apoptosis, since this does not occur in all model systems, including the withdrawal of IL-3 from FL5.12 cells (Datta et al., 1998). There is growing evidence that supports the involvement of a variety of intracellular noncaspase proteases and multisubunit protease complexes in apoptosis (Kutsyi et al., 1999; Solary et al., 1998; Schmitt et al., 1997). For example, Kagaya et al. (1997) demonstrated that exposing rat fibroblasts to 4-(2-aminoethyl)benzenesulfonyl fluoride, a serine protease inhibitor, significantly blocked c-myc-mediated apoptosis and the appearance of caspase-3-like activity in vivo. In addition, pepstatin and leupeptin, potent inhibitors of noncaspase aspartic acid and serine/cysteine proteases with trypsin-like specificity, respectively, inhibited 6-hydroxydopamine-induced apoptosis in neuronal PC12 cells and primary cultured rat microglia (Takai et al., 1998). Other data show that the serine protease inhibitors tosyl-L-phenylalanine chloromethyl ketone (TPCK) and tosylL-lysine chloromethyl ketone (TLCK) inhibited chromatin deg2

Abbreviations used: AMC, 7-amino-4-methyl coumarin; Boc-D-FMK, Boc-Asp(OMe)-CH 2F; FLAP, 5-lipoxygenase activating protein; IL, interleukin; PMSF, phenylmethyl sulfonyl fluoride; TLCK, tosyl-L-lysine chloromethyl ketone; TPCK, tosyl-L-phenylalanine chloromethyl ketone.

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radation during glucocorticoid-induced apoptosis in rat thymocytes (Hughes et al., 1998). A similar inhibition of anticancer drug-induced DNA fragmentation, but not apoptosis, was seen in glioma cells treated with TPCK, TLCK, PMSF, E64, leupeptin, or pepstatin (Eitel et al., 1999). A role for lysosomal proteases in apoptosis has been supported by several studies. Lysosomes contain numerous enzymes that degrade proteins as well as most other cellular components. The lysosomal cysteine protease cathepsin B can activate procaspases (Vancompernolle et al., 1998). In addition, in cultured human fibroblasts, proteases and endonucleases released from lysosomes into the cytosol may induce apoptosis, and this release precedes the release of cytochrome c and the loss of ⌬␺ m during apoptosis induced by oxidative stress (Brunk et al., 1997; Roberg et al., 1999). Free radicals, especially, reactive oxygen species, appear to mediate apoptosis in various mammalian cell lines. Although the mechanism by which these species initiate apoptosis is unclear, it seems that several effectors are generated, including active proteases. An association between oxidized metabolites of polyunsaturated fatty acids and apoptosis has supported a role for lipoxygenase in this process (Tang et al., 1996; Tang and Honn, 1997). However, this role can be questioned since we have found that lipoxygenase inhibitors can induce apoptosis in cell lines lacking these enzymes (Biswal et al., 2000; Datta et al., 1998). The involvement of 5-lipoxygenase activating protein (FLAP) in apoptosis can similarly be questioned, since the purportedly specific FLAP inhibitor MK886 induces apoptosis independently of FLAP (Datta et al., 1999). Thus, the mechanism by which MK886 induces apoptosis in chronic myelogenous leukemia (Anderson et al., 1996) and pro B-lymphocytic FL5.12 (Datta et al., 1998) cell lines remains unclear. We earlier reported that bcl-x L and bcl-2 proteins were rapidly lost in FL5.12 cells during MK886-mediated apoptosis and that this disappearance was only partially affected by the caspase inhibitors zVAD-FMK and Ac-DEVD-CHO (Datta et al., 1998). The present study examined the mechanism of the loss of bcl-x L in more detail. MK886 rapidly induced lysosomal degranulation. Inhibitors of noncaspase serine, cysteine, and aspartate proteases effectively blocked the disappearance of bcl-x L in cells treated with MK886 and partially prevented apoptosis. Pretreatment with these inhibitors also reduced the activation of caspase-3 after MK886, although none had direct inhibitory effects on this enzyme. Only a general caspase inhibitor could completely block caspase-3 activity and prevent apoptosis. These data indicate that various proteases, perhaps originating from lysosomes, may be involved upstream of caspase-3 during MK886-induced apoptosis. MATERIALS AND METHODS Cell Culture and Treatments An IL-3-dependent murine prolymphoid progenitor cell line (FL5.12), obtained from Dr. Gabriel Nun˜ez (University of Michigan, Ann Arbor, MI)

(Simonian et al., 1996), was used for these studies. Cells were maintained in RPMI-1640 media (Gibco BRL, Grand Island, NY) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Summit Biotechnology, Ft. Collins, CO), penicillin (100 U/ml), streptomycin (100 ␮g/␮l), and 10% (v/v) WEHI3B-conditioned medium in a 5% CO 2:95% air atmosphere at 37°C. WEHI-3B cells were grown under similar conditions to produce IL-3-containing medium as described previously (Lee et al., 1982). Cultures were passaged on alternate days with fresh medium and cell counts were determined with a hemocytometer. Cells were treated with DMSO (0.1% as vehicle control), MK886 (Biomol Research, Plymouth Meeting, PA), or etoposide (Sigma, St. Louis, MO). In protease inhibitor experiments, cells were treated for 2 h with Boc-D-FMK (Enzyme Systems, Livermore, CA), PMSF, (Sigma), pepstatin, or leupeptin (Boehringer Mannheim, Indianapolis, IN) prior to MK886 or etoposide treatment. Western Blot Assay SDS–PAGE and Western blots for bcl-x L protein were done using established procedures (Datta et al., 1998). Briefly, cells (5 ⫻ 10 6) were lysed with 150 ␮l RIPA buffer (10 mM sodium phosphate, 150 mM NaCl (pH 7.4), 0.5% sodium deoxycholate, 0.1% SDS, 100 ␮g/ml PMSF, 30 ␮l/ml aprotinin, and 1 mM sodium orthovanadate) by repeatedly pipeting the cell suspension and incubating for 15 min at 4°C. The lysed cells were centrifuged at 14,000g for 10 min and the protein content of the supernatants was determined by the method of Bradford (1976) using bovine serum albumin as a standard. Supernatants were run on 15% reducing SDS polyacrylamide gels (buffer composition: 20% glycerol, 4% ␤-mercaptoethanol, 4% SDS, 0.2 M Tris–HCl (pH 6.8), 0.02% bromophenol blue). Protein was transferred onto polyvinylidene fluoride membranes and blocked for 1 h. The membrane was then incubated with anti-bcl-x L antibody (1:1500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). After membrane washing, horseradish peroxidase-conjugated antirabbit secondary antibodies were used (1:3000 dilution; Amersham, Arlington Heights, IL). Bound antibodies were detected using enhanced chemiluminescence with a kit from Amersham. Relative band densities were determined using a scanned image and UN-SCAN-IT software (Silk Scientific, Orem, UT). Lysosomal Degranulation The procedures used were based on the assay established by Brunk et al. (1997). Following treatment with 10 ␮M MK886, cells (1 ⫻ 10 6/ml) were pelleted, resuspended in medium containing acridine orange (5 ␮g/ml), and incubated for 15 min at 37°C. Cells were then pelleted, resuspended in fresh medium, and kept at room temperature for 10 min. Upon centrifugation, cell pellets were washed with and resuspended in 100-␮l cold phosphate-buffered saline. Cell suspensions (10 ␮l) were viewed under an Olympus IX70 confocal laser scanning microscope (Melville, NY) with an argon laser exciting at 488 and 514 nm. The lysosomal red emitting fluorescence was separated from green fluorescence by using a 610-nm barrier filter in the emission pathway. Images were visualized with a PlanApo 60X/1.4 oil immersion lens and analyzed using the software program FLUOVIEW 2.1.39. Quantitative changes in lysosomal red fluorescence were determined by taking the same cell suspension in cold phosphate-buffered saline and measuring 20,000 cells in a Coulter Epics-XL flow cytometer (Miami, FL) with an argon laser excitation of 488 nm and detection of red lysosomal emission at 620 nm. Lysosomal Enzymatic Activity The separation of intact lysosomes from cytosol and the measurement of ␤– hexosaminidase activity as a marker of lysosomal enzymes was determined in MK886-treated and control cells based on methods described by Stoka et al. (2001) and Storrie and Madden (1990). Twenty million cells were collected and rinsed once with 1 ml PBS and once with 1 ml 20 mM Hepes/250 mM sucrose buffer (pH 7.5) at 4°C. After centrifugation, cells were resuspended in 1 ml Hepes/sucrose buffer and homogenized with a Thomas homogenizer. The homogenate was centrifuged at 2000g for 5 min at 4°C, rinsed once with 100 ␮l buffer, and the combined supernatants were then centrifuged at 16,000g for 5 min at 4°C to separate the lysosome-containing pellet from the cytosol.

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FIG. 1. Lysosomal degranulation following treatment with 10 ␮M MK886. At 2 h after MK886, cells (1 ⫻ 10 6/ml) were pelleted, resuspended in medium containing acridine orange (5 ␮g/ml), and incubated for 15 min at 37°C. Cells were then pelleted, resuspended in fresh medium, and kept at room temperature for 10 min. Upon centrifugation, cell pellets were washed and resuspended in 100 ␮l cold PBS. The cells were viewed under an Olympus IX70 confocal laser scanning microscope using an argon laser to excite at 488 and 514 nm. The acidic lysosomal red emitting fluorescence was separated from green fluorescence by using a 610-nm barrier filter in the emission pathway. The cells shown are representative of those seen. (A) Control; (B) MK886. The number of fluorescing lysosomes was diminished in MK886-treated cells.

␤-Hexosaminidase activity was measured spectrophotometrically by its ability to convert p-nitrophenyl-n-acetyl ␤-D-glucosaminide to n-acetyl-p-nitrophenol. Fractions were resuspended in Hepes/sucrose buffer and 100-␮l samples were mixed with 400 ␮l of warmed substrate solution ⫾ Triton X-100 (1.5 ml 0.4 M sodium acetate (pH 4.4), 0.5 ml freshly made 16 mM p-nitrophenyln-acetyl ␤-D-glucosaminide, 0.4 ml 2.5 M sucrose or 10% Triton X-100, and 1.6 ml H 2O) and incubated at 37°C for 1 h. Lysosomal and nuclear fractions were also added to a Triton (1%)-containing substrate solution to determine latency (organelle intactness) and efficiency of homogenization, respectively. The reaction was stopped by addition of 1 ml of a 0.5 M glycine/0.5 M carbonate (pH 10) solution. A 410 of n-acetyl-p-nitrophenol was determined immediately, normalized to protein concentrations, and quantitated using an extinction coefficient of 18 mM ⫺ 1. Measurements of Apoptosis Flow cytometry. Phosphatidylserine externalization on the plasma membrane was measured by binding of Annexin V-FITC to phosphatidylserine in the presence of Ca 2⫹ (Immunotech, Miami, FL). Cells were also stained with propidium iodide (100 ␮g/ml) to distinguish between necrotic and nonnecrotic cell populations. The cells were analyzed with a Coulter EPICS-XL flow cytometer equipped with an argon laser. The fluorescence for both Annexin V-FITC and propidium iodide was measured for 20,000 cells to determine the percentage of both early and late apoptotic cells. ELISA. A characteristic event in apoptosis is DNA fragmentation and release of nucleosomes into the cytoplasm that can be detected and are quite specific for apoptosis relative to necrosis (Salgame et al., 1997). Cells are first washed to remove histone-associated DNA released by necrotic cells. Cell membranes are then gently lysed, releasing nucleosomes from apoptotic cells. A one-step ELISA kit using streptavidin-coated microplates, biotinylated antihistone, and anti-DNA completes the assay (Roche Molecular, Indianapolis, IN). Results are determined as the ⌬A 405– 490 relative to control cells, which indicates the enrichment of nucleosomes in the cytoplasm. Measurement of Caspase Activities Cells (2 ⫻ 10 6) were pelleted and lysed in 300 ␮l of 50 mM Tris (pH 7.5) containing 150 mM NaCl, 0.5 mM EDTA, and 0.5% v/v NP-40. Aliquots (50 ␮l) of the lysate were incubated with tetrapeptide substrate (Ac-DEVD-AMC for caspase-3, Ac-IEHC-AMC for caspase-8, and Ac-LEHD-AMC for

caspase-9; final concentrations 40 ␮M), 10 mM Hepes (pH 7.5), 50 mM NaCl, and 2.5 mM DTT in a final volume of 200 ␮l for 2 h at 37°C. The fluorescence of the released 7-amino-4-methylcoumarin (AMC) was measured using an excitation wavelength of 360 nm and emission wavelength of 450 nm and normalized to lysate protein concentrations. Fluorogenic caspase substrates were obtained from Alexis Biochemicals (San Diego, CA). Statistics Data are expressed as means ⫾ SE. Comparisons between groups were done with a one-way analysis of variance followed by Student Newman–Kuels test, or a Student’s t test when only two groups were being compared. A p value of less than 0.05 was considered to be significant.

RESULTS

The possibility that MK886 was affecting lysosomes was examined by confocal microscopy with cells stained with acridine orange that, due to its cationic nature, is accumulated within lysosomes. Control cells exhibited a granular orange fluorescence, indicative of the numerous lysosomes present in these cells (Fig. 1). Within 2 h after treatment with 10 ␮M MK886, the number of lysosomes within most cells was severely depressed. A similar effect was evident 4 h after MK886 (data not shown). A more quantitative assessment of lysosomal content was obtained using flow cytometric analyses of the acridine orange-stained cells. This revealed a clear shift toward a lower intensity of fluorescence 2 h after cells were treated with MK886 (Fig. 2). Analyses for a lysosomal marker enzyme, ␤-hexosaminidase, showed a small but statistically significant increase in cytosolic ␤-hexosaminidase at 4 h after 10 ␮M MK886 (1.12 ⫾ 0.23 vs 1.41 ⫾ 0.19 mmol n-acetyl-pnitrophenol released/h/mg protein; n ⫽ 3), in agreement with the flow cytometry and confocal microscopy data. Inhibitors with relative specificity toward serine, cysteine,

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FIG. 2. Quantitative analyses of lysosomal degranulation following treatment with 10 ␮M MK886. At 2 h after MK886, cells (1 ⫻ 10 6/ml) were pelleted, resuspended in medium containing acridine orange (5 ␮g/ml), and incubated for 15 min at 37°C. Cells were then pelleted, resuspended in fresh medium, and kept at room temperature for 10 min. Upon centrifugation, cell pellets were washed and resuspended in 500 ␮l cold PBS. Twenty thousand cells were analyzed in a Coulter flow cytometer with excitation and emission settings at 488 and 620 nm, respectively. The average and peak fluorescence intensity was decreased in MK886-treated cells.

and aspartic acid proteases, and a broad spectrum caspase inhibitor, were used to assess the role of proteolysis both in apoptosis and the disappearance of bcl-x L following the treatment of FL5.12 cells with MK886. Treatment of FL5.12 cells with 10 ␮M MK886 for 8 h caused ⬃41% apoptosis compared to ⬃9% in vehicle-treated control cells, as determined by phosphatidylserine externalization using flow cytometry (Table 1). Although not strictly comparable, a similar 4.8-fold increase in apoptosis was seen with an ELISA that measured histone-associated DNA fragments (Table 1). Subtracting out basal levels of apoptosis, a 2-h pretreatment with PMSF, pepstatin, or leupeptin (50 ␮M each) reduced apoptosis 8 h after MK886 by ⬃50%. The caspase inhibitor Boc-D-FMK was more effective, decreasing the number of apoptotic cells by

80% as shown by flow cytometry and nearly 100% using the ELISA. Extending the observation period after protease inhibitor treatment to 24 h showed that there was a time-dependent increase in viability and decrease in apoptosis in all groups (Fig. 3). The percentage of necrotic cells remained below 10% up to 24 h. This percentage increased at times beyond 24 h as apoptotic cells underwent secondary necrosis, but overall viability continued to increase (data not shown). These data indicate that the protection seen at early time points is followed by recovery rather than any delayed cell death. The effects of these various protease inhibitors, at a dose of 50 ␮M, on apoptosis (Table 1) correlated with caspase-3 like activity in MK886-treated cells (Table 2). There was, however, no increase in the activities of caspases-8 or -9 at 4 h after cells were treated with MK886 (data not shown). As expected, Boc-D-FMK pretreatment completely blocked caspase-3 activation in cells treated with MK886 (Table 2). PMSF, pepstatin, and leupeptin pretreatments diminished, but did not prevent, caspase-3 activation after MK886 (Table 2). In contrast to Boc-D-FMK, none of these inhibitors was capable of directly inhibiting active caspase-3 when added to cell lysates from MK886-treated cells (Table 3). The apoptosis and caspase-3 data suggest that PMSF, pepstatin, and leupeptin do not directly inhibit caspase-3-like activity but rather inhibit events upstream of caspase-3 activation. The role of serine, cysteine, and aspartic acid proteases, and caspases, in the disappearance of bcl-x L 8 h following MK886induced apoptosis was assessed in cells pretreated for 2 h with PMSF, pepstatin, leupeptin, or Boc-D-FMK. There was an 80 to 90% loss of bcl-x L 8 h after MK886 (Figs. 4A and 4B). This loss was diminished in a dose-dependent manner with all inhibitors tested. At a dose of 10 ␮M inhibition was ⬃18% (except for Boc-D-FMK, which inhibited the loss by 55%), increasing to ⬃38% at 25 ␮M and 82% at 50 ␮M (Fig. 4).

TABLE 1 Effect of Protease Inhibitors on Cell Viability and Apoptosis after MK886 Annexin V a

Treatments Vehicle MK886 MK886 MK886 MK886 MK886

control ⫹ ⫹ ⫹ ⫹

Boc-D-FMK PMSF pepstatin leupeptin

ELISA Assay b

Live cells (% of total)

Apoptotic cells (% of total)

⌬A 405–490

Enrichment factor (⌬A treated/⌬A control)

90.3 58.4 79.6 70.3 70.8 69.1

8.8 41.1 14.4 23.0 22.4 23.9

0.076 ⫾ 0.006 0.368 ⫾ 0.041* 0.088 ⫾ 0.010 0.125 ⫾ 0.020 0.115 ⫾ 0.018 0.105 ⫾ 0.019

1.0 4.8 1.1 1.7 1.5 1.4

Note. Cells (10 6) were pretreated with either Boc-D-FMK, PMSF, pepstatin, or leupeptin (50 ␮M each) for 2 h followed by 10 ␮M MK886 for 8 h at 37°C. Boc-D-FMK, PMSF, pepstatin, or leupeptin treatment alone did not cause apoptosis. a Cells were stained with annexin V-FITC and propidium iodide and analyzed by flow cytometry as described under Materials and Methods. b Results were determined as the mean ⌬A 405– 490 ⫾ SE relative to control cells, which indicates the enrichment of nucleosomes in the cytoplasm (n ⫽ 3). * Significantly different from all other groups ( p ⬍ 0.05).

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FIG. 3. Effect of protease inhibitors on apoptosis 12 to 24 h after MK886. Cells (10 6) were pretreated with 50 ␮M Boc-D-FMK or a mixture of 50 ␮M each PMSF, pepstatin, and leupeptin for 2 h prior to treatment with 10 ␮M MK886 for 12, 18, or 24 h. Cells were stained with annexin V-FITC and propidium iodide and analyzed by flow cytometry as described under Materials and Methods. Results are expressed as a mean percentage of 20,000 cells from three independent experiments.

In contrast to the effect of MK886 on bcl-x L protein levels, no change was evident when apoptosis was induced in FL5.12 cells by withdrawal of IL-3 (Datta et al., 1998). Inducing apoptosis with etoposide at a dose sufficient to cause ⬃40% apoptosis at 12 h (Table 4) affected bcl-x L less than did MK886 despite similar levels of apoptosis, although there was still an ⬃50% loss of this protooncogene product (Fig. 4C). This loss was unaffected by pretreatment with Boc-D-FMK, PMSF, pepstatin, or leupeptin. Furthermore, none of the protease inhibitors tested had much of an effect on etoposide-induced apoptosis (Table 4). Boc-D-FMK was the most effective, blocking ⬃40% of the phosphatidylserine externalization, while the others blocked it by 20 to 30%. Unlike MK886, etoposide-induced caspase-3 activation was not prevented by pretreatment with different protease inhibitors except Boc-DFMK (Table 4). TABLE 2 Caspase-3 Activity in Protease Inhibitor-Treated Cells

Treatments

DISCUSSION

Numerous reports have described the activation of caspases as the key proteolytic events associated with apoptosis and also suggested that this family of proteases align in a cascade that amplifies a death signal while destroying structural proteins and functional systems (Thornberry and Lazebnik, 1998). However, caspase-independent apoptosis has been identified and is receiving increasing attention. It has been suggested that, while apoptosis-associated nuclear events may require caspases, certain cytoplasmic characteristics may be triggered by other enzymes (Borner and Monney, 1999). The nature of such “other enzymes” is unclear, but it seems likely that several types of proteases are activated during apoptosis. The activities of several noncaspase proteases (Solary et al., 1998), including certain serine and cysteine proteases, have TABLE 3 Direct Effect of Various Protease Inhibitors on Caspase-3 Activity in Cell Lysates

Caspase-3 activity (% of vehicle) Treatments

Vehicle MK886 MK886 MK886 MK886 MK886

control ⫹ ⫹ ⫹ ⫹

Boc-D-FMK PMSF pepstatin leupeptin

100 751 ⫾ 93* 30 ⫾ 4.5 304 ⫾ 44† 250 ⫾ 35† 231 ⫾ 39†

Note. Cells (10 6) were treated with Boc-D-FMK, PMSF, pepstatin, or leupeptin (50 ␮M each) for 2 h before MK886 treatment (10 ␮M) for 4 h. Caspase-3 activity was measured at the end of the MK886 exposure in the cell lysate as described under Materials and Methods. Results are expressed as mean percentages ⫾ SE (n ⫽ 3). * Significantly different from other treatments (p ⬍ 0.05). † Significantly different from vehicle control and MK886 alone (p ⬍ 0.05).

Vehicle MK886 MK886 MK886 MK886 MK886

control ⫹ ⫹ ⫹ ⫹

Boc-D-FMK PMSF pepstatin leupeptin

Caspase-3 activity (% of vehicle) 100* 729 ⫾ 98 48 ⫾ 5* 731 ⫾ 89 738 ⫾ 90 725 ⫾ 99

Note. Following MK886 (10 ␮M) treatment for 8 h, cells (10 6) were lysed and lysate was further incubated with either Boc-D-FMK, PMSF, pepstatin, or leupeptin (50 ␮M each) for 30 min. Caspase-3 activity was then measured in the cell lysates as described under Materials and Methods. Results were expressed as mean percentages ⫾ SE (n ⫽ 3). * Significantly different from all other groups ( p ⬍ 0.05).

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FIG. 4. Effect of different protease inhibitors on bcl-x L expression in FL5.12 cells treated to induce apoptosis with MK886 or etoposide. Cells (5 ⫻ 10 6) were treated with DMSO (final concentration ⱕ 0.2%) and Boc-D-FMK, PMSF, pepstatin, or leupeptin for 2 h. This was followed by 10 ␮M MK886 or 20 ␮M etoposide. Cells were lysed after 8 h and the lysate was used to perform SDS–PAGE as described under Materials and Methods. Each lane was loaded with 25 ␮g total protein. (A) MK886 ⫹ 50 ␮M each inhibitor; (B) MK886 ⫹ 10 or 25 ␮M each inhibitor; (C) etoposide ⫹ 50 ␮M each inhibitor. All inhibitors prevented the loss of bcl-x L after MK886 in a dose–response fashion. No protection was evident against the smaller loss of bcl-x L after etoposide.

been linked to apoptosis (Ishisaka et al., 1998; Schmitt et al., 1997; Hara et al., 1996). Walker and Sikorska (1993) found that apoptosis could be prevented in thymocytes by serine protease inhibitors, PMSF, and dichloroisocoumarin. Likewise, another serine protease inhibitor, TPCK, prevents the death of target cells attacked by cytotoxic T lymphocytes (Chang and Eisen, 1980). Ceramide-induced apoptosis, at least in some cell types, is inhibited by leupeptin but not by a caspase inhibitor, while the reverse was true of fas-triggered cell death (Belaud-Rotureau et al., 1999). On the other hand, calpain inhibitors I or II, PMSF, TLCK, TPCK, E64, leupeptin, or pepstatin do not inhibit apoptosis induced by a variety of cytotoxic agents in glioma cells, although they do block DNA fragmentation (Eitel et al., 1999). This, along with other work (Lotem and Sachs, 1996), suggests that the specific proteases activated in response to a toxicant may exhibit some cell type and/or inducer specificity, particularly in terms of the downstream lytic pathways that are affected. Lysosomal protease(s) are capable of activating caspase-3like protease (Vancompernolle et al., 1998), and it has been suggested that leakage of such protease(s) into the cytosolic

compartment might be involved in activating this apoptosis effector caspase (Ishisaka et al., 1998). Other studies have also implicated lysosomes in the oxidative stress-induced apoptosis (Brunk et al., 1997; Roberg et al., 1999; Yuan et al., 2000). In addition, it has been suggested that bcl-2 directly blocks the effects of released lysosomal enzymes, thereby preventing the activation of downstream events (Zhao et al., 2000). Since MK886 induces a rapid loss of bcl-2 and bcl-x L (Datta et al., 1998) that is accompanied by lysosomal degranulation, and MK886 appears to enhance the overall production of oxidative species in FL5.12 cells (Datta et al., 1999), it is possible that a similar pathway is involved in the observed apoptosis. PMSF, leupeptin, and pepstatin and serine, cysteine, and aspartate protease inhibitors, were able to somewhat decrease the extent of apoptosis in FL5.12 cells induced by MK886. The same inhibitors also decreased the activation of caspase-3 mediated by MK886. The general caspase inhibitor Boc-DFMK almost completely prevented caspase-3 activation, phosphatidylserine externalization, and DNA fragmentation caused by the same stimulus. These data suggest that MK886-induced apoptosis is caspase-3 dependent and raises the question of whether PMSF-, leupeptin-, and pepstatin-inhibitable proteases are involved in the caspase cascade or affect apoptosis independently. Though PMSF, leupeptin, and pepstatin pretreatment decreased caspase-3 activation, unlike true caspase inhibitors these agents had no direct effect on caspase-3-like activity, even at very high concentrations. This finding suggests that certain serine, cysteine, or aspartate proteases may play a functional role, upstream of caspase-3 activation, in MK886induced apoptosis. This contention is supported by the ability of the serine protease inhibitor aminoethyl benzenesulfonyl TABLE 4 Effect of Various Protease Inhibitors on Etoposide-Induced Apoptosis and Caspase-3 Activation

Treatments

Live cell (% of total cells)

Apoptotic cell (% of total cells)

Caspase-3 activity (% of vehicle)

Vehicle control Etoposide Etoposide ⫹ Boc-D-FMK Etoposide ⫹ PMSF Etoposide ⫹ pepstatin Etoposide ⫹ leupeptin

90.3 59.1 69.5 67.7 67.0 69.4

8.8 39.0 24.3 29.3 30.8 28.1

100 814 ⫾ 95† 49 ⫾ 7* 733 ⫾ 82† 772 ⫾ 77† 621 ⫾ 66†

Note. Cells (10 6) were pretreated with either Boc-D-FMK, PMSF, pepstatin, or leupeptin (50 ␮M each) for 2 h followed by 10 ␮M etoposide. Apoptosis was measured after 12 h in cells stained with annexin V-FITC and propidium iodide and analyzed by flow cytometry. Caspase-3 activity was measured in cell lysates after 8 h as described under Materials and Methods. Data are expressed as means ⫾ SE (n ⫽ 3). Boc-D-FMK, PMSF, pepstatin, or leupeptin treatment alone did not cause apoptosis. * Significantly different from all other treatments ( p ⬍ 0.05). † Significantly different from vehicle control ( p ⬍ 0.05).

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fluoride to hinder c-myc-induced apoptosis and caspase-3 activation in vivo, although it has no direct inhibitory effect on this caspase in vitro (Kagaya et al., 1997). In further support, Hishita et al. (2001) suggest that lysosomal enzymes may be involved in the activation of caspase-3 in P39 cells treated with etoposide. Dong et al. (2000) demonstrates that various chymotryptic and serine protease inhibitors suppressed caspase activation and apoptosis caused by hypoxia/reoxygenation in kidney proximal tubule cells. Interestingly, the inhibitors effective in the present study were ineffective in other studies. These authors speculated that serine proteases may be involved in postmitochondrial apoptotic events that lead to caspase-9 activation. Some reports indicate that serine protease inhibitors such as TPCK and dichloroisocoumarin suppress fas- or TNFR-mediated apoptosis, although it was not shown whether the serine proteases sensitive to these inhibitors perform upstream or downstream of caspase-3 activation (Chow et al., 1995; Higuchi et al., 1995). The possibility that noncaspase protease activation is of general importance in apoptosis was explored by examining the effect of PMSF, leupeptin, and pepstatin on etoposide-induced apoptosis in FL5.12 cells. None of these protease inhibitors had a significant effect on etoposide-induced apoptosis, and they were only moderately effective in preventing changes in bcl-x L after etoposide treatment. This suggests that PMSF-, leupeptin-, and pepstatin-sensitive steps might be more related to the pathways activated by MK886. The inability of low concentrations of PMSF (0.1 ␮M) to block the activation of caspase-3-like protease mediated by lysosomal enzymes (Ishisaka et al., 1998) also suggests that the effects observed in the current study with much higher concentrations are related to inhibition of a wider array of enzymes. The importance of members of the BCL-2 family of protooncogenes in apoptosis has been well established (Chao and Korsmeyer, 1998). Several reports in the literature have suggested that the degradation of the antiapoptotic bcl-2 and bcl-x L proteins during apoptosis is important in enhancing the death process. However, a report using TNF-␣–induced apoptosis in FL5.12 cells suggests this cleavage, which was quite minor, is not essential (Johnson and Boise, 1999). A recent manuscript by Stoka et al. (2001) investigated the role of Bid, a proapoptotic BCL-2 protein, in the induction of apoptosis by lysosomal degranulation. Bid was cleaved directly by contact with a lysosomal extract. When added to intact mitochondria, the cleaved Bid resulted in cytochrome c release. Thus, although lysosomal proteases cannot directly activate caspases (Stoka et al., 2001), the release of cytochrome c subsequent to cleavage of Bid may be a pathway by which lysosomal proteases can be involved. The enzyme(s) responsible for degrading the BCL-2 proteins may involve caspase activities. For example, both IL-3 withdrawal and fas ligation-induced apoptosis stimulated the degradation of bcl-2 (Cheng et al., 1997) and bcl-x L (Clem et al.,

1998) by caspase-3-like proteases. More directly related to our work, caspases appear to mediate the degradation of bcl-2 during etoposide-induced apoptosis in human leukemia cells (Fadeel et al., 1999) and TNF-␣-induced apoptosis in FL5.12 cells (Johnson and Boise, 1999). Our earlier study (Datta et al., 1998) suggested that the rapid disappearance of bcl-x L following MK886 treatment in FL5.12 cells was unrelated to changes in bcl-x L mRNA levels and was only minimally affected by a specific caspase-3 inhibitor. It is likely, therefore, that other caspases and/or noncaspase protease(s) are involved in this process. Interestingly, pretreatment with the broad-spectrum caspase inhibitor Boc-D-FMK prevented bcl-x L disappearance prompted by MK886, implying the involvement of caspase(s) other than caspase-3. Moreover, leupeptin-, pepstatin-, and PMSF-sensitive proteases may be connected to this protein degradation, most probably via the activation of the caspase cascade. Based on the ability of noncaspase protease inhibitors to prevent the loss of bcl-x L in MK886-treated cells and the absence of a more general proteolysis of proteins (Datta et al., 1998), it is apparent that some selective proteolysis takes place in the presence of apoptotic stimuli. Although it is not clear that the breakdown of bcl-x L is essential for the observed apoptosis, it is clear that various noncaspase proteases are involved, since their inhibition modifies the extent of cell death. Overall, it is also apparent that proteolytic events during apoptosis are not only dictated by the caspase cascade but also are associated with serine, cysteine, and/or aspartyl proteases upstream of caspase-3 activation that may originate from lysosomes. ACKNOWLEDGMENTS This work was supported by NIH Grants HL51005 and CA83701. Support from NIEHS Center Grant ES07784 is also acknowledged. S. S. B. was supported by F32 ES05896. J. P. K. is the Gustavus and Louise Pfeiffer Professor of Toxicology.

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