Overexpression of manganese superoxide dismutase protects against mitochondrial-initiated poly(ADPribose) polymerase-mediated cell death K. K. KININGHAM,* T. D. OBERLEY,† S.-M. LIN,* C. A. MATTINGLY,* AND D. K. ST. CLAIR*,1 *Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536, USA; and † Department of Pathology and Laboratory Medicine Service, Veterans Administration Hospital, University of Wisconsin Medical School, Madison, Wisconsin 55705, USA ABSTRACT Mitochondria have recently been shown to serve a central role in programmed cell death. In addition, reactive oxygen species (ROS) have been implicated in cell death pathways upon treatment with a variety of agents; however, the specific cellular source of the ROS generation is unknown. We hypothesize that mitochondria-derived free radicals play a critical role in apoptotic cell death. To directly test this hypothesis, we treated murine fibrosarcoma cell lines, which expressed a range of mitochondrial manganese superoxide dismutase (MnSOD) activities, with respiratory chain inhibitors. Apoptosis was confirmed by DNA fragmentation analysis and electron microscopy. MnSOD overexpression specifically protected against cell death upon treatment with rotenone or antimycin. We examined bcl-xL, p53 and poly(ADP-ribose) polymerase (PARP) to identify specific cellular pathways that might contribute to the mitochondrial-initiated ROS-mediated cell death. Cells overexpressing MnSOD contained less bcl-xL within the mitochondria compared to control (NEO) cells, therefore excluding the role of bcl-xL. p53 was undetectable by Western analysis and examination of the proapoptotic protein bax, a p53 target gene, did not increase with treatment. Activation of caspase-3 (CPP32) occurred in the NEO cells independent of cytochrome c release from the mitochondria. PARP, a target protein of CPP-32 activity, was cleaved to a 64 kDa fragment in the NEO cells prior to generation of nucleosomal fragments. Taken together, these findings suggest that mitochondrial-mediated ROS generation is a key event by which inhibition of respiration causes cell death, and identifies CPP-32 and the PARP-linked pathway as targets of mitochondrial-derived ROS-induced cell death.—Kiningham, K. K., Oberley, T. D., Lin, S.-M., Mattingly, C. A., St. Clair, D. K. Overexpression of manganese superoxide dismutase protects against mitochondrial-initiated poly(ADP-ribose) polymerase-mediated cell death. FASEB J. 13, 1601–1610 (1999)
0892-6638/99/0013-1601/$02.25 © FASEB
Key Words: apoptosis z reactive oxygen species z caspase 3 z antimycin
Physiological and biochemical alterations occur in the mitochondria prior to cytoplasmic or nuclear events associated with apoptosis, including disruption of mitochondrial transmembrane potential, permeability transition, calcium mobilization, generation of superoxide radicals, and release of apoptogenic factors including cytochrome c (1). Release of cytochrome c from mitochondria has been shown to be an important factor in staurosporine, 1-b-arabinofuranosylcytosine, and ionizing radiation-induced apoptosis (2– 4). Several lines of evidence suggest that the release of apoptotic factors from the mitochondria may be a result of respiratory inhibition, reactive oxygen species (ROS)2 formation, and membrane permeability transition (1, 5–7). Overexpression of bcl-2, a protein found within the mitochondrial membrane, has been shown to prevent tumor necrosis factor a (TNF-a), staurosporine, ionizing radiation, and adriamycin- or thapsigargin-induced apoptosis, suggesting an important role for this organelle in apoptosis (8 –11). Recently, bcl-xL, a member of the bcl-2 family of proteins, was shown to block apoptosis by binding directly with cytochrome c and preventing its release from the mitochondria (10). Generation of ROS has been implicated in apoptotic 1 Correspondence: Graduate Center for Toxicology, 360 Health Sciences Research Bldg., University of Kentucky, Lexington, KY 40536-0305, USA. E-mail:
[email protected] 2 Abbreviations: AFC, fluorophore 7-amino-4-trifluoromethyl coumarin; DCF, 29,79-dichlorofluorescein; DCFH-DA, 29,79-dichlorofluorescin diacetate; MnSOD, manganese superoxide dismutase; NEO, neomycin-transfected cells; PARP, poly(ADP-ribose); PBS, phosphate-buffered saline; ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; SOD-H, high levels of MnSOD activity; SOD-L, low levels of MnSOD activity; TBS, 10 mM Tris-HCl and 150 mM NaCl; TE, Tris-EDTA; TNF, tumor necrosis factor.
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cell death pathways upon treatment with a variety of agents including TNF-a (12), ceramide (13), glutamate (14), amyloid b-peptide (15, 16), alkaline conditions (17), and nerve growth factor withdrawal (18, 19). With many of the apoptotic inducers, the specific subcellular source of the ROS generation is unknown. Some studies using TNF, ceramide, or glutamate have implicated the mitochondria as the source of ROS generation (13, 20). This is not surprising since mitochondria constitute a major cellular source of ROS, primarily as byproducts of aerobic respiration. Components of the electron transport chain such as the NADH–coenzyme Q reductase complex and the reduced form of coenzyme Q leak electrons onto oxygen, producing a univalent reduction to generate superoxide radicals (21). Within the mitochondrial matrix, manganese superoxide dismutase (MnSOD) is an essential antioxidant enzyme that catalyzes the conversion of superoxide radical to hydrogen peroxide and molecular oxygen. In addition, generation of cytosolic superoxide radicals can be scavenged by the mitochondria via a polarized inner mitochondrial membrane. It has been hypothesized that superoxide radicals can diffuse into the mitochondrial intermembranous space by presentation of a localized proton-rich environment. Within the intermembranous space, superoxide radicals can be protonated, followed by diffusion into the matrix where dismutation by MnSOD could occur (22). Previous studies have shown that overexpression of MnSOD in various cell lines can prevent TNF-a (23), alkaline (17), or peroxynitrite-mediated apoptosis (24). Thus, it has been hypothesized that upon treatment with these agents, radicals may be generated within one or more cellular compartments. Therefore, to ‘directly’ test the role of mitochondria derivedsuperoxide radicals in apoptotic cell death, we treated a murine fibrosarcoma cell line (FSa) with specific respiratory chain inhibitors that are known to increase superoxide levels within that organelle. Here we demonstrate that overexpression of MnSOD in murine FSa cell lines specifically protected against cell death upon treatment with either complex I or complex III, but not complex IV, inhibitors. Our results demonstrate that MnSOD protects against antimycin-induced caspase-3 activation, nuclear condensation, DNA fragmentation, and PARP cleavage independent of bcl-xL and cytochrome c release. Taken together, these findings demonstrate that mitochondrial-mediated ROS can directly signal the execution of cell death through activation of cellular proteases.
supplemented with 10% fetal bovine serum and 1% antibiotics at 37°C in a humidified atmosphere containing 5% CO2 (25). The FSa-II cells were transfected with p2V2-NEO plasmid or cotransfected with the sense MnSOD expression plasmid (pHbAPR-1), as described previously (26). After 48 h, the cells were exposed to 400 mg/ml of geneticin. Colonies originating from a single transfected cell were selected. Two clones that stably expressed different levels of MnSOD activity [i.e., low (SOD-L) and (SOD-H)] were selected together with a control clone transfected with only the pSV2-NEO plasmid (NEO). Determination of cell doubling time On the basis of the plating efficiencies of the cell lines (27), 1 3 105 cells for the NEO line and 5 3 104 cells for the MnSOD-transfected lines were plated in 10 –14 dishes. After 24 h, media was removed and replaced with fresh medium containing either antimycin (2.5–25 mM), rotenone (10 –100 nM), or sodium cyanide (10 –50 mM). Two dishes from each group were trypsinized every day, and the cells were counted using a hemacytometer. Cell doubling times were obtained by linear regression analysis after plotting the logarithm of the number of cells vs. hours after plating. Colony survival assay Single cells suspended in supplemented McCoy’s medium were plated onto 60 mm dishes in triplicate for each clone examined. Cells (500/dish for NEO and 300/dish for SOD-L and SOD-H each) were plated. After 48 h, media were removed and replaced with medium containing either antimycin (25 nM) or rotenone (10 nM). Cells were kept for 12 days in the incubator to allow for colony formation. The colonies were fixed and stained in 0.1% crystal violet and 2.1% citric acid. Colonies containing more than 50 cells were counted using a dissecting microscope. All experiments were performed in triplicate. The surviving fraction was calculated as follows: SF 5
Number of colonies formed Number of cells plated 3 plating efficiency
DNA fragmentation
MATERIALS AND METHODS
Cells were plated (63105 NEO; 33105 SOD-H) in 100 mm dishes and treated with 0 –100 mM antimycin for 0 – 48 h. Cells were washed twice with phosphate-buffered saline (PBS), resuspended in lysis buffer [50 mM Tris-HCl (pH 8.0), 20 mM EDTA, 0.5% sodium dodecyl sulfate (SDS), 200 mg/ml proteinase K], and incubated at 37°C overnight. Crude DNA preparations were extracted with phenol:chloroform:isoamylalcohol (25:24:1) and precipitated with 0.1 volume of 3 M sodium acetate and 2.0 volumes of 100% ethanol. The DNA pellet was air-dried and resuspended in Tris-EDTA (TE) buffer containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 20 mg/ml DNase-free RNase A. After an incubation for 1 h at 37°C, the concentration of nucleic acid was determined by UV absorbance at 260 nm. The same amount of nucleic acid from each sample was resolved by electrophoresis on a 1% agarose gel and visualized by UV fluorescence after staining with ethidium bromide.
Cell lines
Electron microscopy
A mouse fibrosarcoma cell line (FSa-II) that arose in a C3Hf/Sed mouse was maintained in McCoy’s medium 5a
Seventy-two hours after plating in 100 mm dishes, cells were treated with 100 mM antimycin for 0 – 48 h, rinsed with PBS,
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and fixed with 2.5% glutaraldehyde in Sorensen’s phosphate buffer at 4°C. Cells were postfixed in Caulfield’s osmium tetroxide, rinsed with water, dehydrated in a graded ethanol series with 100% propylene oxide as a transitional solvent, and embedded in Epon 812. Random areas (1 mm square) were cut and processed. Sections were cut using an LKB ultramicrotome and transferred to copper grids that were stained with lead citrate and uranyl acetate. Using a Hitachi H-300 electron microscope, 20 cells per treatment group and time period were photographed and described in a blinded fashion by a pathologist. Flow cytometry Cells were plated and treated as described above. Twenty-four to 48 h after treatment with antimycin, cells were incubated with 10 mM 29,79-dichlorofluorescin diacetate (Molecular Probes Inc., Eugene, Oreg.) for 30 min at 37°C in the dark. Cells were washed with PBS and then incubated for 6 min at 37°C with cell dissociation solution (Sigma, St. Louis, Mo.). Cells were filtered through 35 mM nylon mesh (Small Parts Inc., Miami, Fla.), counted using a hemacytometer, diluted to a final concentration of 1 3 106 cells/ml, and analyzed immediately on a FACScan flow cytometer using excitation at 488 nm and emission at 525 nm. Nuclear extract preparation The cell lines were seeded at a density of either 6.0 3 105 cells/100 mm dish (NEO) or 3.0 3 105 cells/100 mm dish (SOD-H). During the exponential growth phase, nuclear extracts were isolated as described by Dignam and Roeder (28) with the inclusion of 35% glycerol and protease inhibitors (pepstatin, leupeptin, aprotinin) at 1 mg/ml in the extraction buffer. Protein concentration was determined by a colorimetric assay (Bio-Rad Laboratories, Richmond, Calif.). Subcellular fractionation The mitochondrial fraction of FSa cells was prepared by washing the cells twice in ice-cold PBS, followed by resuspension in 5 ml of 0.25 M sucrose, 1 mM EGTA, and 10 mM Tris-HCl (pH 7.4) and centrifuging at 500 3 g for 2 min at 5°C. The supernatant was discarded and the cells were resuspended in 5 ml of the same buffer. The cells were homogenized in a glass Teflon homogenizer using 10 up-anddown strokes at 500 rpm. The homogenate was centrifuged at 1500 3 g for 10 min at 5°C. The crude mitochondrial pellet was resuspended in 100 ml of buffer. The supernatant was recentrifuged at 100,000 3 g (4°C, 1 h) to generate the S-100 fraction. Protein concentration of both fractions was determined by a colorimetric assay (Bio-Rad Laboratories).
protein sequence was used. After two washings in TBS (10 mM Tris-HCl and 150 mM NaCl)-0.05% Tween-20, the blot was incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (Cappel) at a 1:3,000 dilution in Blotto for 1 h at room temperature. The blot was washed three times with TBS-0.05% Tween-20 and once with TBS. Protein bands were visualized using the enhanced chemiluminescence detection system (ECL, Amersham, Little Chalfont, U.K.). Cytochrome c and Bcl-xL Mitochondrial proteins (35–50 mg/lane) were electrophoresed in a 12.5% gel, transferred to nitrocellulose, and blocked as described previously. For bcl-xL analysis, an affinity-purified rabbit anti-bcl-xL antibody (1:1000) purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.) was used. For cytochrome c analysis, a monoclonal antibody purchased from PharMingen (San Diego, Calif.) was used at a final concentration of 1 mg/ml. Bax Cells were washed in PBS and solubilized in lysis buffer (50 mM Tris-HCl pH 7.4, 125 mM NaCl, 0.1% SDS, 5 mM NaF, 1 mM PMSF, 1 ng/ml leupeptin, 1 ng/ml aprotinin, 1 ng/ml pepstatin) for 1 h on ice. Lysates were centrifuged at 2500 3 g for 5 min. The protein concentration in the supernatants was determined as described previously. One hundred micrograms of total cell lysate was resolved on a 12.5% SDS-PAGE gel, transferred to nitrocellulose, and probed with an affinitypurified rabbit anti-bax antibody (1 mg/ml; Santa Cruz Biotechnology). The antibody was raised against a peptide corresponding to amino acids 43– 61, mapping at the amino terminus of the murine bax protein. PARP cleavage products Antimycin-induced apoptosis was examined by proteolytic cleavage of PARP (29). Briefly, cells were treated with 100 mM antimycin for 0 – 48 h at 37°C, then washed with PBS and sonicated (10% power-60 s continuous) on ice. Protein concentration was determined by a colorimetric assay (BioRad Laboratories). Next, 40 ml of sample buffer (50 mM Tris, pH 6.8, 6 M urea, 6% b-mercaptoethanol, 0.003% bromphenol blue, and 3% SDS) was added to the samples (200 mg) and heated at 80°C for 5 min. Samples were loaded and run on an 8% SDS-PAGE gel according to the method of Laemmli (30). Transfer and blocking of the membrane were performed as described previously. The membrane was incubated with a rabbit anti-PARP antibody (1:2,000; Boehringer Mannheim, Mannheim, Germany) in Blotto at 4°C overnight. Protein bands were visualized using the enhanced chemiluminescence detection system (Amersham).
Western analysis Caspase-3 protease activity p53 One hundred micrograms of nuclear extract proteins was resolved on a 12.5% SDS-PAGE (SDS-polyacrylamide gel electrophoresis) and transferred to nitrocellulose. Transfer onto nitrocellulose membrane was assessed by incubating with 0.1% Ponceau. The membrane was washed with distilled water to remove the excess stain and blocked in Blotto [5% milk, 10 mM Tris-HCl, 150 mM NaCl (pH 8.0), and 0.05% Tween-20] for 3 h at room temperature. An affinity-purified rabbit-polyclonal antibody (1:1,000, Geneka Biotechnology) raised against amino acid residues 367–381 of the human p53 MnSOD PROTECTS AGAINST ANTIMYCIN-MEDIATED CELL DEATH
Activation of caspase-3 (CPP-32) was determined using the ApoAlert assay kit (Clontech Laboratories, Palo Alto, Calif.), which fluorometrically detects CPP-32 activity using proteolytic cleavage of the fluorophore 7-amino-4-trifluoromethyl coumarin (AFC) from the substrate conjugate DEVD-AFC. Cells were plated (63105 NEO; 33105 SOD-H) in 100 mm dishes and treated with 100 mM antimycin for 0 – 48 h. Cells (13106) were collected at various time points after treatment, lysed on ice for 10 min, centrifuged (12,000 rpm for 3 min), and the supernatants were collected and stored at 270°C until further use. Cell lysates were incubated with DEVD-AFC 1603
TABLE 1. Doubling time (HR)a Cell line
FSa-NEO FSa-SOD-L FSa-SOD-H
Control
25 mM Antimycin
100 nM Rotenone
10 mM NaCN
19.1 6 2.4 17.4 6 1.7 18.1 6 1.7
41.8 6 9.7* 25.6 6 1.6*† 23.4 6 1.1*†
30.3 6 2.3* 15.5 6 0.9† 17.9 6 2.4†
23.0 6 2.8 22.6 6 2.5 22.5 6 1.9
a In vitro growth analysis of NEO and MnSOD FSa-II cells after treatment with antimycin, rotenone, or sodium cyanide. Cells were seeded in 60 mm dishes and counted daily using a hemacytometer. * P , 0.05 compared to corresponding control value; 1 P , 0.05 compared to NEO value.
in the presence of DTT for 1 h at 37°C. Detection of AFC was measured using a 400 nm excitation filter and a 530 nm emission filter in a Cytofluor 2300 fluorometer (Millipore, Bedford, Mass.). Caspase-3 activity is expressed as percent control. Data analysis Data were evaluated using analysis of variance for multiple comparisons of each dependent variable. A P value ,0.05 was considered to be statistically significant.
RESULTS Effect of electron transport chain inhibitors on growth kinetics and cell survival Growth curves were performed by seeding cells as described in Materials and Methods and then counting the cells daily. Table 1 shows the results of experiments performed in triplicate with each of the respiratory inhibitors. As previously reported, the doubling time between the MnSOD transfectants and the control (NEO) line was not significantly
different (31). However, upon treatment with 25 mM antimycin, the doubling time of the MnSOD transfectants increased ;1.4 fold whereas the doubling time of the NEO line increased by a factor of 2. Rotenone treatment (100 nM) had no effect on the growth of the MnSOD transfectants whereas the doubling time of the control cells increased ;1.5 fold. Sodium cyanide (NaCN) was equally toxic to all three lines. Concentrations of NaCN above 20 mM were lethal to all cell lines. Colony formation analysis (Fig. 1A, B) show increased protection against inhibition of complex I or III with MnSOD overexpression. With 25 nM antimycin or 10 nM rotenone, the surviving fraction for SOD-L and SOD-H were significantly greater compared with the nontransfected control (P,0.05). It should be noted that both antimycin and rotenone toxicities were dependent on cell density. Therefore, lower concentrations of both inhibitors were used for the survival assay in which only 300 (SOD-L, SOD-H) or 500 (NEO) cells were plated/60 mm dish as compared to the growth kinetics experiment.
Figure 1. Cell survival assay of NEO and MnSOD-transfectants after A) antimycin or B) rotenone treatment. Cells were plated onto 60 mm dishes, 500 cells/dish for the NEO control and 300 cells/dish for the MnSOD transfectants. Vales are the mean 6 se. *P , 0.05, significant difference from NEO; **P , 0.0001, significant difference from NEO. 1604
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Generation of ROS by antimycin To estimate the level of ROS production by antimycin in the FSa cell lines, we used 29,79-dichlorofluorescin diacetate (DCFH-DA), a membrane-permeable nonfluorescent probe that is sequestered in living cells after deacetylation by endogenous esterases. Reactive oxygen species oxidize the diesterified product to generate a fluorescent compound, 29,79dichlorofluorescein (DCF), which can be analyzed by flow cytometry (32, 33). Analysis of fluorescence of untreated cells served as a baseline for assessment of increased ROS levels as a result of antimycin treatment. Upon treatment with 100 mM antimycin, both the control (NEO) and SOD-H cell lines exhibited a time-dependent increase in DCF fluorescence (Fig. 4). Mitochondrial bcl-xL is increased upon treatment with antimycin; cellular bax remains unchanged Figure 2. Ultrastructural analysis of MnSOD-transfected murine FSa cell lines treated with 100 mM antimycin. a) 36 h treatment of NEO cells-majority of cells were unremarkable. b) NEO, 48 h treatment: multiple areas of condensed heterochromatin is observed both in viable (arrow, cell on left) and dead (arrows, nuclei of injured cell on right) cells. Mitochondria show focal swelling with loss of cristae. c, d) SOD-L cells at 36 and 48 h, respectively; cell morphology similar to NEO cells at 36 h. e, f ) SOD-H cells at 36 and 48 h, respectively; cell morphology similar to NEO cells at 36 h. a, 38400; b, 34300; c, 33500; d, 38400; e, 33500; f, 33500.
Electron microscopy and DNA fragmentation analysis To determine whether cells were dying by apoptosis, we examined cellular morphology by electron microscopy after antimycin treatment. In addition, we extracted cellular DNA and used electrophoresis to analyze for fragmentation patterns characteristic of apoptotic cell death. Figure 2, an electron micrograph of FSa cells after exposure to 100 mM antimycin, shows morphological changes characteristic of apoptosis, such as multiple areas of heterochromatin condensation, are not present in the control cells (NEO) at 36 h after treatment (Fig. 2a), but appear by 48 h (Fig. 2b). In addition, at 48 h the mitochondria of the NEO cells were swollen with loss of cristae. No evidence of apoptosis was seen in the SOD-L (Fig. 2c, d) or SOD-H (Fig. 2e, f ) cell lines at either 36 or 48 h after treatment. In Fig. 3, neither NEO or MnSOD transfectants showed evidence of DNA fragmentation with 25 mM antimycin treatment after 48 h (lanes 2– 4). Increasing the concentration of antimycin to 100 mM resulted in DNA fragmentation in the NEO cell line at 48 h, suggesting a timeand dose-dependent effect on cell death (lane 5). MnSOD PROTECTS AGAINST ANTIMYCIN-MEDIATED CELL DEATH
We previously reported a decrease in mitochondrial bcl-xL content in the murine FSa cell line, which overexpressed MnSOD (31). Since bcl-xL is known to be an antiapoptotic protein and bax is a proapoptotic protein, we used Western analysis to examine the cellular content of these two proteins after treatment with antimycin. Within 24 h, antimycin treatment lead to an increase in mitochondrial bcl-xL protein, most noticeably in the NEO cell line (Fig. 5A). The increase in mitochondrial bcl-xL was apparent with both 25 (lane 2) and 100 mM (lane 3) antimycin in the NEO cells. Bax levels did not change in either cell line upon treatment with antimycin (Fig. 5B).
Figure 3. Agarose gel of DNA fragmentation. FSa cells were exposed to 25–100 mM antimycin for 0 – 48 h. Lanes: 1, 1 kb DNA marker; 2, NEO 25 mM antimycin; 3, SOD-L 25 mM antimycin; 4, SOD-H 25 mM antimycin; 5, NEO 100 mM antimycin; 6, SOD-L 100 mM antimycin; 7, SOD-H 100 mM antimycin; 8, positive control NEO 1 1 mM A23187. 1605
Figure 5. Examination of A) bcl-xL in mitochondrial fraction (50 mg/lane) and B) bax in total cell lysate (100 mg/lane) of antimycin-treated cells. Lane 1: NEO control; 2: NEO 25 mM antimycin, 24 h; 3: NEO 100 mM antimycin, 24 h; 4: SOD-H control; 5: SOD-H 25 mM antimycin, 24 h; 6: SOD-H 100 mM antimycin, 24 h.
mitochondria from cells treated with antimycin for 0 – 48 h. Figure 6 shows retention of cytochrome c within the mitochondria of both the NEO (lanes 1–3) and SOD-H (lanes 4 – 6) cell lines after treatment with antimycin for either 24 or 48 h. Caspase 3 (CPP-32) activity is increased in NEO cells after antimycin treatment
Figure 4. FACScan analysis of dichlorofluorescein (DCF) fluorescence by antimycin-treated A) NEO and B) SOD-H cells. Analyses were performed on a Becton Dickinson FACScan. Each histogram of fluorescent intensity vs. number of cells was obtained from 10,000 cells. The cells were incubated at 37°C in media containing 10 mM DCFH-DA. The following histograms are shown: I) Cells without antimycin 1 DCF; II) 100 mM antimycin 24 h, DCF; III) 100 mM antimycin 48 h, DCF.
p53 is not detectable in the FSa cell line Protein expression of p53 in nuclear extracts of antimycin-treated cells was below the level of detection using Western analysis (data not shown).
The role of CPP-32 in antimycin-induced apoptosis in FSa cells was measured using the ApoAlert assay kit by Clontech. Cells were treated with 100 mM antimycin and samples were collected at various time points from 0 to 48 h. Figure 7 shows a timedependent increase in CPP-32 activity. Within 48 h, CPP-32 activity had increased . 200% in the NEO cells. Only a slight increase (140%) was noted in the SOD-H clone, suggesting that MnSOD reduces antimycin-induced activation of CPP-32. Poly(ADP-ribose) polymerase (PARP) cleavage in antimycin-treated cells PARP cleavage has been identified as an early event in programmed cell death. PARP has also been shown to serve as a substrate for CPP-32. In the present study we used Western analysis to detect the parental enzyme (116 kDa) as well as its proteolytically cleaved fragment(s). We were able to detect degradation of the parental enzyme in nuclear extracts obtained from NEO cells treated with antimy-
Cytochrome c is retained by the mitochondria after antimycin treatment Cytochrome c is a mitochondrial protein released upon exposure to a variety of agents. Interaction of cytochrome c with various cytoplasmic proteins has been shown to activate caspases, resulting in apoptotic cell death (34). To determine whether antimycin leads to release of cytochrome c, we isolated the 1606
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Figure 6. Examination of cytochrome c in the mitochondrial fraction after treatment with 100 mM antimycin. Lane 1: NEO control; 2: NEO, 24 h; 3: NEO, 48 h; 4: SOD-H control; 5: SOD-H, 24 h; 6: SOD-H, 48 h.
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Figure 7. Caspase 3 activity after antimycin treatment over a time course of 0 – 48 h. Black bars (NEO); light bars (SODH).Values are the mean 6 se *P , 0.05 compared with the corresponding NEO value.
cin (Fig. 8A, lane 3). In addition, we were able to detect accumulation of a 64 kDa fragment as early as 24 h in both nuclear extracts (Fig. 8A, lanes 2 and 3) and total cell lysates (Fig. 8B, lanes 2 and 3) from NEO cells. The accumulation of the 64 kDa fragment paralleled the degradation of the parental enzyme. Furthermore, minor accumulation of the 64 kDa fragment was seen in the SOD-H cell line at 24 (Fig. 8B, lane 5) and 48 h (Fig. 8B, lane 6) after antimycin treatment, consistent with the slight increase in CPP-32 activity (Fig. 7).
neomycin-transfected cells (NEO) were used as the control. Overexpression of MnSOD significantly protected cells against rotenone- and antimycin-mediated cytotoxicity; however, no advantage in cellular resistance to cyanide-mediated cell death was observed in either the SOD-L or SOD-H cell lines (see Table 1 and Fig. 1). The reason for selectivity of protection by MnSOD upon inhibition of complexes I and III, but not IV, is currently unknown. Possible explanations may be due to the reaction rate of superoxide generation vs. ATP depletion upon treatment with cyanide. Since expression of MnSOD protects against rotenone and antimycin similarly, we examined the mode of cell death upon exposure to antimycin. Two distinct modes of cell death, apoptosis and necrosis, have been described in a variety of cell types exposed to respiratory chain inhibitors. Endonuclease activation with subsequent DNA fragmentation was observed in LLC-PK1 cells upon exposure to antimycin (36). Treatment of a series of human lymphoblastoid cell lines (BM 13674, BMX 13674, Daudi, BL29, K562), human melanoma cell lines (MEL 3, MEL28, MM96), and a mouse fibroblast cell line (L929) with antimycin lead to apoptotic cell death visualized by DNA fragmentation analysis and Hoechst 33258 staining (37). In the present study, apoptotic cell death was confirmed by DNA fragmentation and ultrastructural analysis using electrophoresis and electron microscopy (Figs. 2, 3). The control (NEO) cells exhibited DNA fragmentation and morphological changes characteristic of apoptosis at 48 h. Cells overexpressing MnSOD showed no evidence of apo-
DISCUSSION During cellular metabolism, mitochondria produce superoxide radicals either through electron leakage from the flavin component of complex I or through ubisemiquinone at complex III. Production of these oxyradicals can be exacerbated by inhibition at one or more sites along the electron transport chain (35). MnSOD is the primary antioxidant defense against superoxide radicals within the mitochondria. In the presence of MnSOD, superoxide radicals are rapidly converted to hydrogen peroxide and molecular oxygen within the mitochondrial matrix. The objective of the present work was to investigate the role of mitochondrial-derived ROS in programmed cell death. To accomplish this, we treated an MnSOD-transfected murine fibrosarcoma cell line (FSaII) with a series of respiratory chain inhibitors; MnSOD PROTECTS AGAINST ANTIMYCIN-MEDIATED CELL DEATH
Figure 8. Western analysis of PARP cleavage in A) nuclear extracts and B) total cell lysates of antimycin-treated cells. Lane 1: NEO control; 2: NEO, 24 h; 3: NEO, 48 h; 4: SOD-H control; 5: SOD-H, 24 h; 6: SOD-H, 48 h. 116 kDa band corresponds to parental PARP. Arrows indicate accumulation of 64 kDa proteolytic fragment. 1607
ptosis. This finding strongly suggests a role for the superoxide radical in the antimycin-mediated death cascade. Antimycin-mediated ROS production was confirmed using DCFH-DA, which is an indicator of intracellular oxidant production (38). Figure 4 shows a time-dependent increase in DCF fluorescence in both the NEO and SOD-H cell line; however, a consistently higher level of DCF fluorescence was noted in the NEO cell line at 48 h. We were not surprised to see an increase in DCF fluorescence in the MnSOD overexpressing cells after antimycin treatment because overexpression of MnSOD should not prevent the increase in superoxide radicals formed as a result of inhibition of respiration, and dismutation of superoxide radicals leads to the formation of hydrogen peroxide, which can contribute to DCF fluorescence (38). This study confirms that treatment with antimycin leads to an increase in ROS production and suggests that cells overexpressing MnSOD can compensate for the increase in superoxide radical production, thereby protecting the cells from ROS-mediated cell death. To identify a pathway that might be involved in the antimycin-mediated cell death, we examined the antiapoptotic protein bcl-xL and the proapoptotic protein bax after antimycin treatment. Bcl-xL, the predominant bcl-2 family member in mice (39), has been shown to protect cells against a variety of insults that cause apoptosis. Previously we reported an increase in the mitochondrial level of bcl-xL in the control (NEO) cells compared to the MnSOD overexpressing cells (31). However, in this study the control cells were not protected by their bcl-xLenriched mitochondria, suggesting a bcl-xL-independent cell death pathway. Furthermore, upon treatment with antimycin, an increase was consistently noted in the mitochondrial content of bcl-xL in the NEO cell line (Fig. 5A, lanes 2, 3). This finding is consistent with results by Wolvetang et al. (37), who reported apoptotic cell death in a series of human tumor lines upon treatment with antimycin independent of their bcl-2 levels. Bax is a cytosolic protein that redistributes to the mitochondria in cells undergoing apoptosis (40). Although the mechanism is still unclear, studies have shown that overexpression of bax can accelerate apoptosis after a death signal (41). Bax can form homodimers or heterodimerize with bcl-2 proteins, leading to speculation that it may function as an inhibitor of bcl-2. In addition, bax can regulate cell death independent of bcl-2, perhaps by forming ion channels or inducing the release of apoptogenic factors from the mitochondria (42). In the present study, we saw no change in bax levels upon exposure to antimycin in either cell line (Fig. 5B). Next, we tried to establish whether the antimycin1608
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mediated cell death pathway in FSa-II cells involved p53. Polyak et al. (43) have shown that p53 can induce apoptosis through a multistep process involving transcriptional induction of p53-induced genes, formation of ROS, and the oxidative degradation of mitochondrial components. However, the p53 levels in both the NEO and SOD-H cell line were below the limits of detection. This was not too surprising since the FSa-II cell line is highly aggressive. Miyashita et al. (44) identified p53 as a regulator of bcl-2 and bax gene expression. In a murine leukemia cell line (M1), a temperature-sensitive p53 induced a temperature-dependent decrease in bcl-2 while simultaneously stimulating an increase in bax. In addition, mice deficient in p53 showed elevated levels of bcl-2 and decreased levels of bax in several tissues. In Fig. 5 the levels of bcl-xL actually increased with antimycin treatment, whereas no change was observed in bax expression. Taken together, these results indicate that p53 may not play a major role in this cell death model. Cytochrome c is a mitochondrial protein that induces apoptosis when released into the cytosol in response to various stress inducers. The addition of cytochrome c to cytosolic fractions from growing cells initiates a cascade, including activation of caspases and induction of DNA fragmentation (45). A report by Chauhan et al. (4) showed that apoptotic cell death could occur in the absence of cytochrome c release from the mitochondria upon treatment with dexamethasone or anti-Fas monoclonal antibody in multiple myeloma cells. Therefore, we sought to determine whether the antimycin-mediated cell death was occurring in response to cytochrome c release from the mitochondria. Examination of cytochrome c was performed by Western analysis over a 48 h time course after antimycin treatment. We compared levels in the mitochondrial and S-100 fractions and found no accumulation of cytochrome c in the cytosol after treatment in either the NEO or SOD-H cell lines (data not shown). Figure 6 shows no loss of cytochrome c from the mitochondria on antimycin treatment over the course of 48 h. This suggests that the cell death pathway initiated in the FSa cells on antimycin treatment does not require cytochrome c release from the mitochondria. Studies of the nematode Caenorhabditis elegans have provided significant insight into the molecular control of apoptosis (46, 47). Mammalian counterparts to numerous C. elegans genes associated with programmed cell death have been identified. Caspase 3 (CPP-32), the ced-3 homologue, has been shown to be one of the major activated cysteine proteases present in cells undergoing apoptosis, suggesting an important role in the cell death process (48). Recent work by Woo et al. (49) suggests that the require-
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ment for CPP-32 activation in apoptotic cells is system and stimulus dependent. In the present study, CPP-32 activity increased in the NEO cell line as early as 24 h after antimycin treatment. Exposure for 48 h resulted in a greater than 200% increase in CPP-32 activity in the NEO cells, which was significantly higher than the SOD-H cells (P,0.05). These results show an increase in CPP-32 activity prior to any evidence of DNA fragmentation or morphological changes consistent with apoptosis. These results also suggest that the antimycin generated ROS function upstream of CPP-32 activation. Our results compliment studies by Higuchi et al. (50), who treated ML-1a cells with a mixture of xanthine and xanthine oxidase and showed that the generated ROS act upstream of the CPP-32 activity. Taken together, these results identify CPP-32 as a redox-responsive protein. Activation of the caspase enzymes leads to proteolytic cleavage of a number of cellular targets including proteins involved in RNA splicing, DNA repair, and scaffolding of the cytosol and nucleus (51). One DNA repair enzyme, PARP, is a substrate for proteolytic cleavage by several cellular proteases including CPP-32 (52). Cleavage of PARP results in decreased enzyme activity, which compromises the cell’s response to damage. Furthermore, ROS including peroxides and nitric oxide have been shown to inactivate PARP, contributing to cell death cascades (53, 54). Using Western analysis of both total cell lysates and nuclear extracts (Fig. 8A, B) from antimycin-treated cells, we were able to detect cleavage of parental PARP (116 kDa) to a 64 kDa fragment, primarily in the NEO cell line. In both the nuclear extracts and total cell lysates, the 64 kDa fragment can be seen by 24 h, with significant accumulation by 48 h. Only minor accumulation of the 64 kDa fragment was seen in the SOD-H cell line at 24 and 48 h (Fig. 8A, B). These results suggest that CPP-32 activation and PARP cleavage are closely associated events. In conclusion, this study establishes a role for mitochondrial-derived ROS, primarily superoxide radical and its products in programmed cell death. Inhibition of mitochondrial respiration leads to an increase in cellular ROS, which can cause cell death independent of bcl-2 or cytochrome c release, and identifies CPP-32 and the PARP-linked pathway as targets of mitochondrial-derived ROS-induced cell death. Furthermore, modulation of a cell’s mitochondrial antioxidant status may provide significant protection against toxic insults involving a compromise to mitochondrial respiration. The authors wish to thank Ketah Doty, Dr. Sonya Carlson, Jennifer Strange, Greg Bauman, Dr. Hsiu-Chuan Yen, and Dr. Hideyuki Majima for their technical contributions to this manuscript. This work was supported by NIH grants MnSOD PROTECTS AGAINST ANTIMYCIN-MEDIATED CELL DEATH
HL03544, CA49797, CA59835, and CA73599, and by Veterans Affairs Research Service.
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Received for publication February 23, 1999. Accepted for publication without revision April 17, 1999.
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