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Cip1
Experimental Cell Research 250, 75– 85 (1999) Article ID excr.1999.4504, available online at http://www.idealibrary.com on
MPP 1 Inhibits Proliferation of PC12 Cells by a p21 WAF1/Cip1-Dependent Pathway and Induces Cell Death in Cells Lacking p21 WAF1/Cip1 Frank Soldner,* Michael Weller,† Sibylle Haid,* Stefan Beinroth,* Scott W. Miller,‡ Ullrich Wu¨llner,* Robert E. Davis,‡ Johannes Dichgans,* Thomas Klockgether,* and Jo¨rg B. Schulz* ,1 *Laboratory of Experimental Neuropharmacology and †Laboratory of Molecular Neuro-Oncology, Department of Neurology, University of Tu¨bingen, Hoppe-Seyler-Str. 3, D-72076 Tu¨bingen, Germany; and ‡MitoKor Corporation, San Diego, California 92121
cal abnormalities that characterize the disease. The cause of neuronal loss in the substantia nigra is not known. However, recent advances have been made in defining morphological and biochemical events in the pathogenesis of the disease. Inhibition of oxidative phosphorylation, excitotoxicity, and generation of reactive oxygen species (ROS) are considered important mediators of neuronal cell death in Parkinson’s disease [1]. A defect of complex I of the mitochondrial electron transport chain has been identified in substantia nigra of PD patients and is thought to be specific for PD and selective for the substantia nigra in the central nervous system [2, 3]. Evidence implicating apoptosis in PD is controversial. Some studies found evidence for apoptosis based on morphologic criteria or in situ end labeling [4, 5], whereas others did not [6, 7]. In humans and nonhuman primates, the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces irreversible clinical, biochemical, and neuropathologic effects which closely mimic those observed in idiopathic PD [8]. This meperidine analog is metabolized to 1-methyl-4-phenylpyridinium (MPP 1) by the enzyme monoamine oxidase B. MPP 1 is subsequently selectively taken up by dopaminergic terminals and concentrated in neuronal mitochondria in the substantia nigra. MPP 1 binds to and inhibits complex I of the electron transport chain [9], thereby producing the same biochemical defect as detected in the substantia nigra of PD patients. MPP 1 can deplete ATP levels both in vitro and in vivo [10 –12]. It has been suggested that under certain conditions MPP 1 may induce apoptosis in PC12 cells [12] and apoptosis in cerebellar granule neurons by release of cytochrome c and activation of caspases [13]. Rat pheochromocytoma PC12 cells have been used extensively to study the mechanisms of MPP 1 toxicity [12, 14 –16]. Here, we used undifferentiated PC12 cells to study the molecular mechanisms governing cell growth arrest and death in MPP 1 toxicity and putative mechanisms of dopaminergic cell death in PD. We tried to clarify the role of MPP 1 -induced energy depletion by studying the effects of MPP 1 on anaerobic glycolysis, repletion of energy substrates,
The molecular and biochemical mode of cell death of dopaminergic neurons in Parkinson’s disease (PD) is uncertain. In an attempt at further clarification we studied the effects of 1-methyl-4-phenylpyridinium (MPP 1), the active metabolite of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), on dopaminergic PC12 cells. In humans and nonhuman primates MPTP/ MPP 1 causes a syndrome closely resembling PD. MPP 1 toxicity is thought to be mediated by the block of complex I of the mitochondrial electron transport chain. Treatment of undifferentiated PC12 cells with MPP 1 primarily inhibited proliferation of PC12 cells and secondarily led to cell death after the depletion of all energy substrates by glycolysis. This cell death showed no morphological characteristics of apoptosis and was not blocked by treatment with caspase inhibitors. The inhibition of cell growth was not dependent on an inhibition of complex I activity since MPP 1 also inhibited cell proliferation in SH-SY5Y cells lacking mitochondrial DNA and complex I activity (p 0 cells). As shown by flow cytometric analysis, MPP 1 induced a block in the G 0/G 1 to S phase transition that correlated with increased expression of the cyclin-dependent kinase inhibitor p21 WAF1/Cip1 and growth arrest. Since treatment with 1 mM MPP 1 caused apoptotic cell death in p21 WAF1/Cip1 -deficient (p21 2/2 ) but not in parental (p21 1/1) mouse embryo fibroblasts, our data suggest that in an early phase MPP 1-induced p21 WAF1/Cip1 expression leads to growth arrest and prevents apoptosis until energy depletion finally leads to a nonapoptotic cell death. © 1999 Academic Press Key Words: MPP 1; p21; PC12; cell cycle; Parkinson’s disease; mitochondria.
INTRODUCTION
Pathologically, the hallmark of Parkinson’s disease (PD) is loss of dopaminergic neurons in the substantia nigra, leading to the major clinical and pharmacologi1 To whom correspondence and reprint requests should be addressed. Fax: **49-7071-295260. E-mail: [email protected].
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0014-4827/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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and the effects on cells with a mitochondrial DNA depletion. Since these studies suggested that MPP 1 primarily inhibited cell proliferation, and only secondarily lead to cell death after depletion of energy substrates, we further investigated the effects of MPP 1 on cell cycle progression and the cyclindependent kinase inhibitor p21 WAF1/Cip1 as well as the effects of MPP 1 on cells lacking p21 WAF1/Cip1 (p21 2/2 ). METHODS Materials RPMI 1640 cell culture medium, Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s modified phosphate-buffered saline (PBS), fetal calf serum (FCS), donor horse serum, penicillin, streptomycin, and supplements were obtained from GIBCO Life Technologies (Heidelberg, Germany). MPP 1 was purchased from RBI (Cologne, Germany). Cell Culture PC12 cells were maintained at 37°C, 5% CO 2, in RPMI 1640 medium, supplemented with 5% heat-inactivated FCS, 10% heatinactivated horse serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were split every 5–7 days 1:10. Confluent cultures were washed with prewarmed PBS, detached with a trypsin/EDTA solution, centrifuged, and subcultured. If not otherwise stated, PC12 cells were seeded at a density of 50,000 or 200,000/ml on poly-Llysine-coated 24- or 96-well plates for cytotoxicity and proliferation experiments. MPP 1 and drugs were added to cultures 24 h later. A clonal stock of human SH-SY5Y neuroblastoma cells containing no mtDNA (p 0 cells, MitoKor Corp., San Diego, CA) was created by prolonged exposure of these cells to ethidium bromide [17]. SH-SY5Y parental cells were grown at 37°C, 5% CO 2, in medium consisting of DMEM, supplemented with 10% FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. For p 0 cells the growth medium was additionally supplemented with 100 mg/ml of pyruvate and 50 mg/ml of uridine because p 0 cells are auxotrophic for pyruvate and uridine [18]. p21 1/1 , p21 1/2 , and p21 2/2 primary mouse embryo fibroblasts (MEFs) [19] were grown to subconfluence in DME medium, 10% FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were plated at a density of 1.5 3 10 5 cells/ml and were treated with MPP 1 at 24 h. Cell Viability Trypan blue exclusion assay. Cells were removed from the culture dish by trituration in their culture medium, centrifuged, and resuspended, and 50 ml was added to 50 ml of trypan blue solution (0.4% in PBS). The numbers of trypan blue-excluding (live) cells and trypan blue-staining (dead) cells were counted using a counting chamber. Crystal violet assay. Cell culture medium was removed, and cells were exposed for 10 min to a solution of 0.5% crystal violet in 20% methanol. After incubation, the plates were rinsed with distilled water as previously described and air dried for 24 h. After drying, cells were lysed with 0.1 M sodium citrate buffer and scanned at 550 nm on a plate photometer (Dynex, Denkendorf, Germany). Background readings were performed by staining of collagen-precoated cell-free wells. LDH release assay. LDH activity was measured as optical density (OD) at 490 nm using a plate photometer and expressed according to the manufacturer’s (Boehringer, Mannheim, Germany) directions: specific LDH release (%) 5 [(experimental value-spontaneous release)/(maximum release-spontaneous release)] 3 100. The spontaneous release was defined as the amount of LDH released from
untreated PC12 cells and the maximum release obtained after exposure of PC12 cells to 1% Triton X-100 for 30 min at 37°C. Apoptosis Assays DNA fragmentation. DNA fragmentation was quantified by DNA fluorometry [20]. In brief, cells were harvested and lysed on ice, and fragmented DNA was separated from nucleus-attached DNA by high-speed centrifugation. After disruption of the pellets by brief sonication and RNase digestion, fragmented and pelleted DNA was measured by ethidium bromide. The percentage of fragmentation was calculated by dividing fragmented DNA by the total sum of fragmented and pelleted DNA. TUNEL staining. For TUNEL staining [21] cells were seeded at a density of 200,000 cells/ml on 35-mm dishes. After culturing and treatment at conditions as indicated, medium was removed, and cells were rinsed twice in PBS and fixed for 15 min in 4% paraformaldehyde and 1.5% glutaraldehyde. After a wash in PBS, endogenous peroxidase was inactivated by covering the cells with 0.1% H 2O 2 for 10 min. After two PBS washes, cells were preincubated for 10 min in sodium cacodylate (TdT) buffer containing 140 mM sodium cacodylate, 30 mM Tris–HCl, pH 7.2, and 1.25 mg/ml bovine serum albumin. Cells then were exposed for 20 min to the reaction mixture (50 U/ml terminal transferase, 10 mM biotin-dUTP, 25 mM cobalt chloride in TdT buffer) at 37°C. The reaction was stopped by incubating the dishes for 10 min with sodium citrate buffer. After blocking with 2% bovine serum albumin (BSA), cells were incubated for 30 min with streptavidin alkaline phosphatase conjugate and developed with 0.41 mM nitroblue tetrazolium chloride and 0.38 mM 5-bromo-4-chloro-3-indolyl phosphate in 200 mM Tris–HCl, pH 9.5, containing 10 mM MgCl 2. Western Blot Expression of p21 WAF1/Cip1 and p53 was detected by Western blot. PC12 cells were seeded on 100-mm dishes. Soluble protein was harvested from cells lysed for 15 min on ice in 50 mM Tris–HCl, pH 8, containing 120 mM NaCl, 5 mM EDTA, 0.5% NP-40, 2 mg/ml aprotinin, 100 mg/ml PMSF, and 10 mg/ml leupeptin, followed by high-speed centrifugation at 4°C. Twenty micrograms of protein per lane was separated by 12% SDS–PAGE. After electrophoresis and electroblotting to nitrocellulose membranes, the blots were blocked in 250 mM Tris–HCl, pH 8.0, 120 mM NaCl, 10% nonfat dry milk, 5 % BSA, 1% of serum corresponding to the secondary antibody, 0.5% Tween 20, and 0.1% azide for 60 min. The blots were incubated with the first antibody at 4°C overnight. The following antisera were used: monoclonal anti-p53 (Ab-3, Oncogene, Bad Soden, Germany), polyclonal anti-p21 WAF1/Cip1 (C19, Santa Cruz,), and monoclonal anti-cdk5 (J-3, Santa Cruz). After three washes in PBS containing 0.05% Tween 20, the membranes were incubated with secondary alkaline phosphatase-conjugated antibody for 1 h, washed three times in PBS, and stained with 0.2 mg/ml nitroblue tetrazolium chloride and 0.3 mg/ml 5-bromo-4-chloro-3-indolyl-phosphate in 0.1 M Tris–HCl, pH 9.5, containing 50 mM MgCl 2 and 100 mM NaCl. DNA Synthesis PC12 cells were seeded in 96-well microtiter plates at the densities indicated and treated with various concentrations of MPP 1. At 6 h before the end of the respective study [ 3H]thymidine (Amersham, Braunschweig, Germany) was added to a final concentration of 3.3 mCi/ml and cells were incubated at 37°C for 6 h. Then cells were harvested and incorporated radioactivity was determined with a liquid scintillation counter (Wallac, Turku, Finland). For each condition a ratio of incorporated [ 3H]thymidine to the number of live cells was calculated. Cell Cycle Analysis For cell cycle analysis [22] cells were seeded at a density of 200,000/ml in 35-mm dishes and treated with MPP 1 as described
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FIG. 2. Effects of MPP 1 on DNA synthesis. MPP 1 concentration dependently inhibits [ 3H]thymidine incorporation. PC12 cells were treated with 250 mM MPP 1 and 6 h before the end of the respective measurement [ 3H]thymidine was added.
equipped with a 15-mW, 488-nm air-cooled argon–ion laser. Emission fluorescence was measured with a 514-nm bandpass filter for FITC and a 600-nm wavelength filter for propidium iodide (PI). Data aquisition and data analysis were performed using the Cell Quest software (Becton–Dickinson). Measurement of Glucose, Lactate, and pH Concentrations of glucose and lactate were measured using the glucose oxidase assay and the lactate oxidase assay, respectively, following the manufacturer’s directions (Boehringer). FIG. 1. Effects of MPP 1 on growth and survival of PC12 cells. (A) Cells were treated with varying concentrations of MPP 1 and cell densities were measured by crystal violet staining (OD units) at time points indicated. (B) PC12 cells were treated with 250 mM MPP 1 and the trypan blue assay was used to detect viable (trypan blue-negative) and dead (trypan blue-positive) cells compared with controls (C). (C) Treatment with 100 mM MPP 1 for 48 or 96 h did not cause cell death but inhibited proliferation. (D) Treatment with varying concentrations of MPP 1 for 48 h did not lead to LDH release from PC12 cells. above. At the end of the experiment cultures were incubated with 5-bromodeoxyuridine (BrdU) 30 min at a final concentration of 10 mM. Cells were removed from the culture dish by trituration in their culture medium and pelleted by centrifugation. Ice-cold 70% ethanol was added to a final concentration of 1 3 10 6 cells/100 ml and cells were incubated for 20 min. Aliquots of 100 ml were transferred to test tubes, centrifuged, and washed with 1 ml wash buffer containing PBS and 0.5% BSA. Cells were resuspended in denaturating solution (2 M HCl, 0.5% Tween 20) and incubated for 20 min. After washing of cells 0.5 ml of 0.1 M sodium borate (Na 2B 4O 7) was added to neutralize any residual acid. After washing cells were incubated for 20 min at room temperature with a FITC-conjugated anti-BrdU monoclonal antibody (Boehringer, Mannheim, Germany). PC12 cells were washed, centrifuged, resuspended in 0.5 ml propidium iodide solution containing 10 mg/ml of propidium iodide and 100 mg/ml of RNase A dissolved in PBS, and incubated for 30 min. Cells were analyzed on a FACScan (Becton–Dickinson, Heidelberg, Germany),
Statistics Data are expressed as means 6 SD. Significance was assessed by two-tailed Student’s t test (comparison of two groups) or one-way ANOVA followed by Dunnett’s posthoc test (comparison of more than two groups). All experiments represent at least three independent replications performed in triplicate.
FIG. 3. Absence of DNA fragmentation after MPP 1 treatment. DNA fragmentation was measured by a fluorometric fragmentation assay after treatment with MPP 1 or VM26 at the time points indicated. *P , 0.001.
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RESULTS
MPP 1 Inhibits Proliferation of PC12 Cells and Abolishes DNA Synthesis Initially, we studied the effects of increasing concentrations of MPP 1 on undifferentiated PC12 cells seeded at a density of 200,000 cells/ml at 24, 48, 72, and 96 h. After exposure to 100, 250, and 1000 mM MPP 1 for 48 h or longer cell density was lower than that with vehicletreated controls, as measured by cresyl violet staining (Fig. 1A). To study whether the difference in cell density was caused by MPP 1-induced inhibition of cell proliferation, loss of viability, or both, we studied the effects of MPP 1 using trypan blue exclusion to distinguish viable and dead cells. Treatment with 250 mM MPP 1 inhibited cell proliferation and, in addition, led to cell death at 72 and 96 h (Fig. 1B). Treatment with 100 mM MPP 1 for up to 96 h inhibited cell proliferation but did not cause cell death (Fig. 1C). The lack of cell death up to 48 h after addition of 50 to 1000 mM MPP 1 was confirmed by the absence of LDH release into the culture medium (Fig. 1D). Since MPP 1 blocked cell proliferation, we studied whether MPP 1 affected DNA synthesis of PC12 cells. We studied [ 3H]thymidine incorporation as an index for DNA synthesis and S phase activity. MPP 1 concentration and time dependently blocked [ 3H]thymidine incorporation (Fig. 2). PC12 cells treated with 250 mM MPP 1 for 48 h essentially showed no incorporation of [ 3H]thymidine, corresponding to the inhibition of cell proliferation (Figs. 1A and 1B). MPP 1-Induced Cell Death Does Not Show Characteristics of Apoptosis It has been suggested that treatment with 10 –50 mM MPP 1 for 48 h may induce apoptosis and DNA fragmentation in PC12 cells [12]. We did not observe MPP 1-induced cell death at concentrations lower than 100 mM as assayed by light microscopy, trypan blue exclusion assay, and LDH release assay. To investigate whether MPP 1 may induce apoptosis in PC12 cells, we studied DNA fragmentation after treatment with 50 and 250 mM MPP 1 for 48, 72, or 96 h. MPP 1 did not lead to DNA fragmentation, as measured by a fluorometric fragmentation assay (Fig. 3). Since we were able to detect DNA fragmentation after treatment of PC12 cells with 100 mM VM26 (Fig. 3), the absence of DNA fragmentation after MPP 1 treatment cannot be explained by the lack of sensitivity of this assay. Treatment of PC12 cells with VM26 leads to a decreased
FIG. 5. Effects of caspase inhibitors on MPP 1 treatment of PC12 cells. Cells were treated with 250 mM MPP 1 and the effects of caspase inhibitors (DEVD-CHO, zVAD-fmk, YVAD-cmk) on cell viability were studied by staining with crystal violet (CV) at (A) 24, (B) 48, and (C) 72 h.
viability of 49 6 3% at 48 h and complete cell loss at 96 h. We also did not find any increase of TUNEL-positive cells after treatment with 50 or 250 mM MPP 1 for 48 h (data not shown) or 72 h (Fig. 4). Although cell death already occurs after 72 h of treatment with 250 mM MPP 1, we could not detect an increase of TUNELpositive cells compared with controls. Since most types of neuronal apoptosis involve activation of caspases we studied the ability of pseudosubstrate caspase inhibitors to block cell death induced by treatment with 250 mM MPP 1. Treatment with 10 mM DEVD-CHO, 100 mM zVAD-fmk, and 100 mM YVAD-cmk, which were read every 24 h, had no effect on cell proliferation of controls, on the inhibitory effects of MPP 1 on cell proliferation at 24, 48, or 72 h, or on MPP 1-induced cell death effects at 72 h (Fig. 5). The concentrations of
FIG. 4. Absence of TUNEL staining of MPP 1-treated compared with VM 26-treated PC12 cells. PC12 cells treated with (A) vehicle, (B) 50 mM MPP 1, or (C) 250 mM MPP 1 for 72 h were stained for TUNEL and compared with (D) PC12 cells treated with 100 mM VM 26. FIG. 12. Effects of p21 WAF1/Cip1 on MPP 1 induced TUNEL staining. TUNEL staining of p21 1/1 MEFs treated with (A) vehicle or (B) 1 mM MPP 1 and of p21 2/2 MEFs treated with (C) vehicle or (D) 1 mM MPP 1 for 72 h.
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drugs used for caspase inhibition have been proven to inhibit apoptosis of PC12 cells after serum or NGF withdrawal or oxidative stress [20, 23]. Again, we used VM26-induced apoptosis of PC12 cells as a positive control. Treatment with 100 mM zVAD-fmk blocked cell death induced by 100 mM VM26 after 24 h (viability 52 6 4% vs 93 6 3%, P , 0.01). Altogether, we did not find any evidence that MPP 1 induces apoptosis in undifferentiated PC12 cells. MPP 1-Induced Cell Death Depends on Energy Depletion and a Lack of Energy Substrates The toxic effects of MPP 1 are thought to depend on its ability to inhibit complex I of the mitochondrial electron transport chain [9]. Interestingly, treatment of PC12 cells with 200 mM or higher concentrations of MPP 1 for 48 h leads to a depletion of ATP of at least 50% [12]. To study the relationship of MPP 1-induced cell death and energy depletion, we investigated the effects of culture conditions on cell survival after MPP 1 treatment. When cells were seeded at a density of 50,000 instead of 200,000 cells/ml, cell death between 48 and 96 h after treatment with 250 mM MPP 1 was attenuated (Figs. 6A and 6B). Inhibition of oxidative phosphorylation should lead to a compensatory increase in glycolysis with a rise of lactate concentrations and a decrease of the pH. The better survival of cells seeded at lower densities is paralleled by a delayed increase of lactate and a delayed decrease of glucose concentrations (Figs. 6C and 6D). It can be calculated from the graphs that in cells treated with MPP 1, a consumption of 1 mmol glucose yielded an increase of lactate concentration of 2 mmol at either seeding density, suggesting a complete switch from oxidative phosphorylation to nonaerobic glycolysis. In vehicle treated controls only 1.2 to 1.4 mmol lactate is produced per consumption of 1 mmol glucose. When the medium was supplemented with Hepes and glucose in cultures seeded at a density of 200,000 PC12 cells/ml, cell survival after 72 h of MPP 1 treatment was significantly increased compared to normal culture conditions (Fig. 7A). Under low seeding condition of 50,000 cells/ml no cell death occured until day 8; however, cell proliferation was effectively reduced (Fig. 7B). These results suggest that cell death occurs only when energy substrates are depleted [33].
FIG. 6. Effects of MPP 1 on the temporal profile of cell density, glucose, and lactate. PC12 cells were seeded at densities of (A and C) 50,000 or (B and D) 200,000 cells/ml. The effects of treatment with 250 mM MPP 1 on (A and B) cell density (crystal violet, CV) and pH and the effects on (C and D) glucose consumption and lactate production were measured at time points indicated.
MPP 1-Induced Growth Arrest Is Independent of Mitochondrial Complex I Activity To study whether MPP 1-induced growth arrest is mediated by inhibition of complex I of the electron transport chain we used SH-SY5Y neuroblastoma cells and compared parental with cells containing no mitochondrial DNA (p 0 cells, MitoKor Corp., San Diego, CA). Similar to PC12 cells, treatment of undifferenti-
ated SH-SY5Y cells with 250 mM MPP 1 led to growth arrest without cell death (Fig. 8A). Despite the lack of any detectable complex I and IV activity (data not shown), MPP 1 lead to the same growth arrest in p 0 cells (Fig. 8B). Note that growth in p 0 cells in general is slower than in the parental cell line.
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ble 1), suggesting that MPP 1 induces blockage of the G 0/G 1 to S phase transition. MPP 1 Induces p21 by a p53-Independent Pathway p21 WAF1/Cip1 is the prototype inhibitor of cyclin-dependent kinases [24]. It has been suggested that the blockage of cell proliferation elicited by the addition of NGF to exponentially growing PC12 cells results, in part, from the negative regulator of cell cycle progression p21 WAF1/Cip1 [25, 26]. Treatment of PC12 cells with MPP 1 resulted in a marked increase of p21 expression, as detected by Western blot (Fig. 10). Theoretically, p21 WAF1/Cip1 induction may occur as a p53-dependent process activated by DNA damage or as p53 independent [27–29]. However, we did not detect any MPP 1induced expression of p53 in PC12 cells, although we were able to detect p53 expression in PC12 cells after treatment with 0.5 mM doxorubicin for 24 h (Fig. 10). This suggests that MPP 1 induces p21 WAF1/Cip1 expression independently of p53. p21 WAF1/Cip1 Expression Prevents Apoptosis Induced by MPP 1 To study the effects of p21 WAF1/Cip1 expression on MPP 1 treatment we used mouse embryo fibroblasts
FIG. 7. MPP 1-induced cell death depends on energy depletion. (A) Effects of Hepes and glucose supplementation on PC12 cells treated with 250 mM MPP 1. (B) At a low seeding density of 50,000 cells/ml, treatment with 250 mM MPP 1 inhibits cell proliferation (trypan blue-negative cells) but does not induce cell death (trypan blue-positive cells). *P , 0.01 compared with MPP 1-treated PC12 cells in normal RPMI medium not supplemented.
MPP 1 Induces Blockage of the G 0/G 1 to S Phase Transition Subsequently, flow cytometric analysis was carried out using PI to label DNA and BrdU incorporation as a specific marker for new DNA synthesis. Vehicletreated PC12 cells show strong BrdU incorporation (Fig. 9). BrdU incorporation of PC12 cells decreased after treatment with 1000 mM MPP 1 for 48 h (Fig. 9). Cell cycle analysis was performed by standard FACScan software automatically. The percentage of cells in the G 0/G 1 phase increased and the percentage of cells in the S phase decreased after exposure to MPP 1 (Ta-
FIG. 8. MPP 1-induced growth arrest is independent of mitochondrial complex I activity of SH-SY5Y cells. Effects of treatment with 250 mM MPP 1 on (A) SH-SY5Y parental neuroblastoma cells and (B) p 0 cells lacking mitochondrial DNA. In both, MPP 1 treatment leads to growth arrest of living cells (trypan blue-negative) but not to an increase of dead (trypan blue-positive) cells.
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to the effects on PC12 cells, MPP 1 did not lead to cell death of p21 1/1 MEFs but to growth arrest (Fig. 11B). However, in p21 2/2 MEFs MPP 1 did not lead to growth arrest (Fig. 11B) but caused accelerated cell death after treatment for 72 and 96 h (Fig. 11C). The similar kinetics of MPP 1-induced cell death of p21 1/2 cells to that of p21 2/2 cells (Fig. 11C) may be explained by the low expression of p21 WAF1/Cip1 protein in p21 1/2 cells (Fig. 11A) and is consistent with the incomplete growth arrest of p21 1/2 cells after MPP 1 treatment (Fig. 11B). Further, MPP 1 induced TUNEL staining in p21 2/2 (Fig. 12D) but not p21 1/1 (Fig. 12B) cells, suggesting that in the absence of p21 WAF1/Cip1 MPP 1 will lead to apoptosis, whereas in the presence of p21 WAF1/Cip1 MPP 1 will lead to a growth arrest in proliferating cells. DISCUSSION
In the present study, we investigated the effects of MPP 1 on undifferentiated dopaminergic PC12 cells. We found that MPP 1 did not primarily induce cell death but a cessation of cell proliferation that was due to a block in the G 0/G 1 to S phase transition. MPP 1 treatment resulted in the induction of p21 WAF1/Cip1 expression by a p53-independent pathway. Since p21 WAF1/Cip1 controls the G 0/G 1 to S phase transition, we hypothesized that the induction of p21 expression observed after MPP 1 treatment arrested cell cycle progression. The induced expression of p21 WAF1/Cip1 seems to be required for the survival of MPP 1-treated cells, since p21 WAF1/Cip1-deficient fibroblasts died after treatment with equal concentrations of MPP 1. In the presence of p21 WAF1/Cip1, cell death occured only secondarily when energy substrates were depleted due to enhanced anaerobic glycolysis following inhibition of oxidative phosphorylation. Several reports have suggested that MPP 1 may induce apoptosis in PC12 cells at concentrations ranging from 10 mM [12] to 1 mM MPP 1 [16]. In contrast, we did not observe any cell death after treatment with MPP 1 for 96 h at concentrations lower than 250 mM (Fig. 1). At concentrations between 250 mM and 1 mM of MPP 1, cell death was induced between 48 and 96 h. This cell death was not apoptotic, since we could not FIG. 9. MPP 1 induced a block in G 0/G 1 to S phase transition. Contour blots of bivariate DNA/BrdU distributions in PC12 cells untreated (top) or treated for 24 (middle) or 48 (bottom) h with 1000 mM MPP 1. Propidium iodide (PI) was used to label DNA and 5-bromodeoxiuridine (BrdU) incorporation as a specific marker for new DNA synthesis. Cells in the left cluster with the smallest amount of DNA are in G 0/G 1 phase, cells in the right cluster with double this amount are in either G 2 or M phase, while cells in S phase have intermediate amounts.
(MEFs) with different genetically regulated expression of p21 WAF1/Cip1 [19]. p21 1/1, p21 1/2, and p21 2/2 MEFs showed different expression of p21 WAF1/Cip1 protein, with no detectable protein of p21 2/2 cells (Fig. 11A). Similar
TABLE 1 Cell Cycle Analysis Based on Flow Cytometric Analysis MPP 1
G 0/G 1 phase S phase G 2 phase
Controls
24 h
48 h
64.9 6 0.4 24.9 6 0.8 6.8 6 0.4
79.4 6 2.8*** 5.4 6 1.4*** 10.2 6 1.6
83.1 6 1.4*** 4.5 6 0.4*** 9.5 6 1.0
*** P , 0.001 compared with controls (ANOVA followed by Dunnett’s posthoc test).
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treatment led to a sharp rise of lactate production in PC12 cells which died only after energy substrates were depleted. As suggested earlier [33] supplying more energy substrates led to a longer survival (Fig. 7). The cell cycle-related response of PC12 cells to treatment with MPP 1 is similar to their response to NGF treatment. In the presence of serum, NGF induces a block in the G1 phase of the cell cycle and upregulation of p21 WAF1/Cip1 protein levels [26, 36]. Removal of serum from differentiated PC12 cells results in loss of p21WAF1/Cip1. However, this loss of p21 WAF1/Cip1 expression has no effect
FIG. 10. MPP 1 induces expression of p21 WAF1/Cip1 but not of p53. Treatment with 250 mM MPP 1 time dependently induces expression of p21 (top) but not of p53 (bottom). In contrast, treatment with 0.5 mM doxorubicin induces expression of p53.
detect DNA fragmentation or TUNEL-positive staining. We also could not block cell death with peptide inhibitors of caspases (Fig. 5), which are thought to be involved in almost all paradigms of neuronal apoptosis. Compared to the toxicity of MPP 1 against dopaminergic neurons in mesencephalic cultures, only substantially higher concentrations of MPP 1 show effects in PC12 cells. MPP 1 is toxic to dopaminergic neurons of mesencephalic cultures at concentrations as low as 0.5 to 2.0 mM [30, 31], but shows effects in PC12 cells only at concentrations exceeding 100 mM. We report here that MPP 1 arrests cell cycle progression at concentrations of 100 mM and higher but does not affect cell survival for 96 h at concentrations lower than 250 mM. The requirement of high MPP 1 concentrations to induce death in PC12 cells supports earlier reports, in which 1 to 3 mM treatments for 24 h were necessary to induce cell death [32, 33]. Adrenal chromaffin cells express a high-affinity catecholamine uptake system and have been demonstrated to accumulate [ 3H]MPP 1 with relatively little toxicity [34]. It has been argued that adrenal chromaffin cells may be relatively resistant to MPP 1 due to a reserpine-sensitive sequestration of the drug within a distinct intracellular compartment [34, 35]. These and our data are in sharp contrast to a report by Hartley et al. [12], who provided evidence that MPP 1 induces apoptosis in PC12 cells at concentrations ranging from 10 to 100 mM as detected morphologically by electron microscopy and biochemically by DNA fragmentation and DNA laddering on agarose gel electrophoresis. At concentrations higher than 100 mM, the predominant mode of cell death was necrosis. After treatment with 250 mM MPP 1 for 48 h they observed a 60% LDH release relative to the total LDH activity. There may be substantial differences between different PC12 cell lines used. We show that inhibition of complex I of the electron transport chain can be compensated by glycolysis and is apparently not sufficient to kill PC12 cells. MPP 1
FIG. 11. Expression of p21 WAF1/Cip1 blocks apoptosis induced by MPP 1. (A) Expression of p21 WAF1/Cip1 in p21 1/1, p21 1/2, and p21 2/2 mouse embryo fibroblasts. (B) Effects of 1 mM MPP 1 on growth arrest depends on p21 WAF1/Cip1 expression in p21 1/1, p21 1/2, and p21 2/2 mouse embryo fibroblasts. ***P , 0.001 compared with untreated p21 1/1 cells. (C) Effects of p21 WAF1/Cip1 on the treatment of MEFs with 1 mM MPP 1.
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on differentiation or the nonproliferative state in the presence of NGF, suggesting that p21 WAF1/Cip1 serves in part to neutralize the mitogenic stimulus of serum [26]. In the absence of the mitogenic signal, cells no longer require this response and could use other means to stop or prevent cell cycle progression. Using mouse embryo fibroblasts with varying expression of p21 WAF1/Cip1 we show here that p21 WAF1/Cip1 expression after MPP 1 treatment is required to inhibit cell proliferation and to prevent apoptosis. Since MPP 1 leads to the same growth arrest in SH-SY5Y p 0 cells, in which no complex I activity is detectable, this block in cell proliferation is not due to inhibition of complex I. This paper provides evidence for a new mechanism of MPP 1 toxicity. We demonstrate that in PC12 cells MPP 1 does not primarily lead to cell death by inhibition of oxidative phosphorylation but interferes with cell cycle progression. Survival of cells may depend on their ability to induce p21 WAF1/Cip1 expression. Since MPTP/MPP 1 toxicity in humans and animals resembles PD in many aspects, including the selectivity for dopaminergic neurons, the major clinical syndrome, and complex I inhibition of the electron transport chain, these results may lead to new insights into the pathogenesis of PD. We thank Dr. Tyler Jacks for providing p21-deficient mouse embryo fibroblasts. This study was supported by grants from the Deutsche Forschungsgemeinschaft (Kl 782/3-1) and from the fortu¨ne-program of the University of Tu¨bingen (Project 214) to T.K. and J.B.S.
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Tipton, K. F., and Singer, T. P. (1993). Advances in our understanding of the mechanisms of the neurotoxicity of MPTP and related compounds. J. Neurochem. 61, 1191–1206.
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Chan, P., DeLanney, L. E., Irwin, I., Langston, J. W., and DiMonte, D. (1991). Rapid ATP loss caused by 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine in mouse brain. J. Neurochem. 57, 348 –351.
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Storey, E., Hyman, B. T., Jenkins, B., Brouillet, E., Miller, J. M., Rosen, B. R., and Beal, M. F. (1992). 1-Methyl-4-phenylpyridinium produces excitotoxic lesions in rat striatum as a result of impairment of oxidative metabolism. J. Neurochem. 58, 1975–1978.
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Hartley, A., Stone, J. M., Heron, C., Cooper, J. M., and Schapira, A. H. V. (1994). Complex I inhibitors induce dose-dependent apoptosis in PC12 cells. J. Neurochem. 63, 1987–1990.
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Du, Y., Dodel, R. C., Bales, K. R., Jemmerson, R., HamiltonByrd, E., and Paul, S. M. (1997). Involvement of caspase-3-like cysteine protease in 1-methyl-4-phenylpyridinium-mediated apoptosis of cultured cerebellar granule neurons. J. Neurochem. 69, 1382–1388.
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Desole, M. S., Sciola, L., Delogu, M. R., Sircana, S., Migheli, R., and Miele, E. (1996). Role of oxidative stress in the manganese and 1-methyl-4-(2’-ethylphenyl)-1,2,3,6-tetrahydropyridineinduced apoptosis in PC12 cells. Neurochem. Int. 31, 169 –176.
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Marongiu, M. E., Piccardi, M. P., Bernardi, F., Corsini, G. U., and Del Zompo, M. (1988). Evaluation of the toxicity of the dopaminergic neurotoxins MPTP and MPP 1 in PC12 pheochromocytoma cells: Binding and bilogical studies. Neurosci. Lett. 94, 349 –354.
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Itano, Y., Kitamura, Y., and Nomura, Y. (1994). 1-Methyl-4phenylpyridinium (MPP 1)-induced cell death in PC12 cells: inhibitory effects of several drugs. Neurochem. Int. 25, 419 – 424.
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Miller, S. W., Trimmer, P. A., Parker, W. D. Jr., and Davis, R. E. (1996). Creation and characterization of mitochondrial DNAdepleted cell lines with “neuronal-like” properties. J. Neurochem. 67, 1897–1907.
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King, M. P., and Attardi, G. (1989). Human cells lacking mtDNA: Repopulation with exogenous mitochondria by complementation. Science 246, 500 –503.
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Brugarolas, J., Chandrasekaran, C., Gordon, J. I., Beach, D., Jacks, T., and Hannon, G. J. (1995). Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature 377, 552– 557.
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Schulz, J. B., Bremen, D., Reed, J. C., Lommatzsch, J., Takayama, S., Wu¨llner, U., Lo¨schmann, P. A., Klockgether, T., and Weller, M. (1997). Cooperative interception of neuronal apoptosis by BCL-2 and BAG-1 expression: Prevention of caspase activation and reduced production of reactive oxygen species. J. Neurochem. 69, 2075–2086.
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Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. (1992). Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493–501.
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Dolbeare, F., Gratzner, H., Pallavicini, M. G., and Gray, J. W. (1983). Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Proc. Natl. Acad. Sci. USA 80, 5573–5577.
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Troy, C. M., Stefanis, L., Prochiantz, A., Greene, L. A., and Shelanski, M. L. (1996). The contrasting roles of ICE family proteases and interleukin-1b in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation. Proc. Natl. Acad. Sci. USA 93, 5635–5640.
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Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993). The p21 Cdk-interacting protein Cip1 is a
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Received November 19, 1998 Revised version received March 8, 1999
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MPP 1 Inhibits Proliferation of PC12 Cells by a p21 WAF1/Cip1-Dependent Pathway and Induces Cell Death in Cells Lacking p21 WAF1/Cip1 Frank Soldner,* Michael Weller,† Sibylle Haid,* Stefan Beinroth,* Scott W. Miller,‡ Ullrich Wu¨llner,* Robert E. Davis,‡ Johannes Dichgans,* Thomas Klockgether,* and Jo¨rg B. Schulz* ,1 *Laboratory of Experimental Neuropharmacology and †Laboratory of Molecular Neuro-Oncology, Department of Neurology, University of Tu¨bingen, Hoppe-Seyler-Str. 3, D-72076 Tu¨bingen, Germany; and ‡MitoKor Corporation, San Diego, California 92121
cal abnormalities that characterize the disease. The cause of neuronal loss in the substantia nigra is not known. However, recent advances have been made in defining morphological and biochemical events in the pathogenesis of the disease. Inhibition of oxidative phosphorylation, excitotoxicity, and generation of reactive oxygen species (ROS) are considered important mediators of neuronal cell death in Parkinson’s disease [1]. A defect of complex I of the mitochondrial electron transport chain has been identified in substantia nigra of PD patients and is thought to be specific for PD and selective for the substantia nigra in the central nervous system [2, 3]. Evidence implicating apoptosis in PD is controversial. Some studies found evidence for apoptosis based on morphologic criteria or in situ end labeling [4, 5], whereas others did not [6, 7]. In humans and nonhuman primates, the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces irreversible clinical, biochemical, and neuropathologic effects which closely mimic those observed in idiopathic PD [8]. This meperidine analog is metabolized to 1-methyl-4-phenylpyridinium (MPP 1) by the enzyme monoamine oxidase B. MPP 1 is subsequently selectively taken up by dopaminergic terminals and concentrated in neuronal mitochondria in the substantia nigra. MPP 1 binds to and inhibits complex I of the electron transport chain [9], thereby producing the same biochemical defect as detected in the substantia nigra of PD patients. MPP 1 can deplete ATP levels both in vitro and in vivo [10 –12]. It has been suggested that under certain conditions MPP 1 may induce apoptosis in PC12 cells [12] and apoptosis in cerebellar granule neurons by release of cytochrome c and activation of caspases [13]. Rat pheochromocytoma PC12 cells have been used extensively to study the mechanisms of MPP 1 toxicity [12, 14 –16]. Here, we used undifferentiated PC12 cells to study the molecular mechanisms governing cell growth arrest and death in MPP 1 toxicity and putative mechanisms of dopaminergic cell death in PD. We tried to clarify the role of MPP 1 -induced energy depletion by studying the effects of MPP 1 on anaerobic glycolysis, repletion of energy substrates,
The molecular and biochemical mode of cell death of dopaminergic neurons in Parkinson’s disease (PD) is uncertain. In an attempt at further clarification we studied the effects of 1-methyl-4-phenylpyridinium (MPP 1), the active metabolite of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), on dopaminergic PC12 cells. In humans and nonhuman primates MPTP/ MPP 1 causes a syndrome closely resembling PD. MPP 1 toxicity is thought to be mediated by the block of complex I of the mitochondrial electron transport chain. Treatment of undifferentiated PC12 cells with MPP 1 primarily inhibited proliferation of PC12 cells and secondarily led to cell death after the depletion of all energy substrates by glycolysis. This cell death showed no morphological characteristics of apoptosis and was not blocked by treatment with caspase inhibitors. The inhibition of cell growth was not dependent on an inhibition of complex I activity since MPP 1 also inhibited cell proliferation in SH-SY5Y cells lacking mitochondrial DNA and complex I activity (p 0 cells). As shown by flow cytometric analysis, MPP 1 induced a block in the G 0/G 1 to S phase transition that correlated with increased expression of the cyclin-dependent kinase inhibitor p21 WAF1/Cip1 and growth arrest. Since treatment with 1 mM MPP 1 caused apoptotic cell death in p21 WAF1/Cip1 -deficient (p21 2/2 ) but not in parental (p21 1/1) mouse embryo fibroblasts, our data suggest that in an early phase MPP 1-induced p21 WAF1/Cip1 expression leads to growth arrest and prevents apoptosis until energy depletion finally leads to a nonapoptotic cell death. © 1999 Academic Press Key Words: MPP 1; p21; PC12; cell cycle; Parkinson’s disease; mitochondria.
INTRODUCTION
Pathologically, the hallmark of Parkinson’s disease (PD) is loss of dopaminergic neurons in the substantia nigra, leading to the major clinical and pharmacologi1 To whom correspondence and reprint requests should be addressed. Fax: **49-7071-295260. E-mail: [email protected].
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and the effects on cells with a mitochondrial DNA depletion. Since these studies suggested that MPP 1 primarily inhibited cell proliferation, and only secondarily lead to cell death after depletion of energy substrates, we further investigated the effects of MPP 1 on cell cycle progression and the cyclindependent kinase inhibitor p21 WAF1/Cip1 as well as the effects of MPP 1 on cells lacking p21 WAF1/Cip1 (p21 2/2 ). METHODS Materials RPMI 1640 cell culture medium, Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s modified phosphate-buffered saline (PBS), fetal calf serum (FCS), donor horse serum, penicillin, streptomycin, and supplements were obtained from GIBCO Life Technologies (Heidelberg, Germany). MPP 1 was purchased from RBI (Cologne, Germany). Cell Culture PC12 cells were maintained at 37°C, 5% CO 2, in RPMI 1640 medium, supplemented with 5% heat-inactivated FCS, 10% heatinactivated horse serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were split every 5–7 days 1:10. Confluent cultures were washed with prewarmed PBS, detached with a trypsin/EDTA solution, centrifuged, and subcultured. If not otherwise stated, PC12 cells were seeded at a density of 50,000 or 200,000/ml on poly-Llysine-coated 24- or 96-well plates for cytotoxicity and proliferation experiments. MPP 1 and drugs were added to cultures 24 h later. A clonal stock of human SH-SY5Y neuroblastoma cells containing no mtDNA (p 0 cells, MitoKor Corp., San Diego, CA) was created by prolonged exposure of these cells to ethidium bromide [17]. SH-SY5Y parental cells were grown at 37°C, 5% CO 2, in medium consisting of DMEM, supplemented with 10% FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. For p 0 cells the growth medium was additionally supplemented with 100 mg/ml of pyruvate and 50 mg/ml of uridine because p 0 cells are auxotrophic for pyruvate and uridine [18]. p21 1/1 , p21 1/2 , and p21 2/2 primary mouse embryo fibroblasts (MEFs) [19] were grown to subconfluence in DME medium, 10% FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were plated at a density of 1.5 3 10 5 cells/ml and were treated with MPP 1 at 24 h. Cell Viability Trypan blue exclusion assay. Cells were removed from the culture dish by trituration in their culture medium, centrifuged, and resuspended, and 50 ml was added to 50 ml of trypan blue solution (0.4% in PBS). The numbers of trypan blue-excluding (live) cells and trypan blue-staining (dead) cells were counted using a counting chamber. Crystal violet assay. Cell culture medium was removed, and cells were exposed for 10 min to a solution of 0.5% crystal violet in 20% methanol. After incubation, the plates were rinsed with distilled water as previously described and air dried for 24 h. After drying, cells were lysed with 0.1 M sodium citrate buffer and scanned at 550 nm on a plate photometer (Dynex, Denkendorf, Germany). Background readings were performed by staining of collagen-precoated cell-free wells. LDH release assay. LDH activity was measured as optical density (OD) at 490 nm using a plate photometer and expressed according to the manufacturer’s (Boehringer, Mannheim, Germany) directions: specific LDH release (%) 5 [(experimental value-spontaneous release)/(maximum release-spontaneous release)] 3 100. The spontaneous release was defined as the amount of LDH released from
untreated PC12 cells and the maximum release obtained after exposure of PC12 cells to 1% Triton X-100 for 30 min at 37°C. Apoptosis Assays DNA fragmentation. DNA fragmentation was quantified by DNA fluorometry [20]. In brief, cells were harvested and lysed on ice, and fragmented DNA was separated from nucleus-attached DNA by high-speed centrifugation. After disruption of the pellets by brief sonication and RNase digestion, fragmented and pelleted DNA was measured by ethidium bromide. The percentage of fragmentation was calculated by dividing fragmented DNA by the total sum of fragmented and pelleted DNA. TUNEL staining. For TUNEL staining [21] cells were seeded at a density of 200,000 cells/ml on 35-mm dishes. After culturing and treatment at conditions as indicated, medium was removed, and cells were rinsed twice in PBS and fixed for 15 min in 4% paraformaldehyde and 1.5% glutaraldehyde. After a wash in PBS, endogenous peroxidase was inactivated by covering the cells with 0.1% H 2O 2 for 10 min. After two PBS washes, cells were preincubated for 10 min in sodium cacodylate (TdT) buffer containing 140 mM sodium cacodylate, 30 mM Tris–HCl, pH 7.2, and 1.25 mg/ml bovine serum albumin. Cells then were exposed for 20 min to the reaction mixture (50 U/ml terminal transferase, 10 mM biotin-dUTP, 25 mM cobalt chloride in TdT buffer) at 37°C. The reaction was stopped by incubating the dishes for 10 min with sodium citrate buffer. After blocking with 2% bovine serum albumin (BSA), cells were incubated for 30 min with streptavidin alkaline phosphatase conjugate and developed with 0.41 mM nitroblue tetrazolium chloride and 0.38 mM 5-bromo-4-chloro-3-indolyl phosphate in 200 mM Tris–HCl, pH 9.5, containing 10 mM MgCl 2. Western Blot Expression of p21 WAF1/Cip1 and p53 was detected by Western blot. PC12 cells were seeded on 100-mm dishes. Soluble protein was harvested from cells lysed for 15 min on ice in 50 mM Tris–HCl, pH 8, containing 120 mM NaCl, 5 mM EDTA, 0.5% NP-40, 2 mg/ml aprotinin, 100 mg/ml PMSF, and 10 mg/ml leupeptin, followed by high-speed centrifugation at 4°C. Twenty micrograms of protein per lane was separated by 12% SDS–PAGE. After electrophoresis and electroblotting to nitrocellulose membranes, the blots were blocked in 250 mM Tris–HCl, pH 8.0, 120 mM NaCl, 10% nonfat dry milk, 5 % BSA, 1% of serum corresponding to the secondary antibody, 0.5% Tween 20, and 0.1% azide for 60 min. The blots were incubated with the first antibody at 4°C overnight. The following antisera were used: monoclonal anti-p53 (Ab-3, Oncogene, Bad Soden, Germany), polyclonal anti-p21 WAF1/Cip1 (C19, Santa Cruz,), and monoclonal anti-cdk5 (J-3, Santa Cruz). After three washes in PBS containing 0.05% Tween 20, the membranes were incubated with secondary alkaline phosphatase-conjugated antibody for 1 h, washed three times in PBS, and stained with 0.2 mg/ml nitroblue tetrazolium chloride and 0.3 mg/ml 5-bromo-4-chloro-3-indolyl-phosphate in 0.1 M Tris–HCl, pH 9.5, containing 50 mM MgCl 2 and 100 mM NaCl. DNA Synthesis PC12 cells were seeded in 96-well microtiter plates at the densities indicated and treated with various concentrations of MPP 1. At 6 h before the end of the respective study [ 3H]thymidine (Amersham, Braunschweig, Germany) was added to a final concentration of 3.3 mCi/ml and cells were incubated at 37°C for 6 h. Then cells were harvested and incorporated radioactivity was determined with a liquid scintillation counter (Wallac, Turku, Finland). For each condition a ratio of incorporated [ 3H]thymidine to the number of live cells was calculated. Cell Cycle Analysis For cell cycle analysis [22] cells were seeded at a density of 200,000/ml in 35-mm dishes and treated with MPP 1 as described
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FIG. 2. Effects of MPP 1 on DNA synthesis. MPP 1 concentration dependently inhibits [ 3H]thymidine incorporation. PC12 cells were treated with 250 mM MPP 1 and 6 h before the end of the respective measurement [ 3H]thymidine was added.
equipped with a 15-mW, 488-nm air-cooled argon–ion laser. Emission fluorescence was measured with a 514-nm bandpass filter for FITC and a 600-nm wavelength filter for propidium iodide (PI). Data aquisition and data analysis were performed using the Cell Quest software (Becton–Dickinson). Measurement of Glucose, Lactate, and pH Concentrations of glucose and lactate were measured using the glucose oxidase assay and the lactate oxidase assay, respectively, following the manufacturer’s directions (Boehringer). FIG. 1. Effects of MPP 1 on growth and survival of PC12 cells. (A) Cells were treated with varying concentrations of MPP 1 and cell densities were measured by crystal violet staining (OD units) at time points indicated. (B) PC12 cells were treated with 250 mM MPP 1 and the trypan blue assay was used to detect viable (trypan blue-negative) and dead (trypan blue-positive) cells compared with controls (C). (C) Treatment with 100 mM MPP 1 for 48 or 96 h did not cause cell death but inhibited proliferation. (D) Treatment with varying concentrations of MPP 1 for 48 h did not lead to LDH release from PC12 cells. above. At the end of the experiment cultures were incubated with 5-bromodeoxyuridine (BrdU) 30 min at a final concentration of 10 mM. Cells were removed from the culture dish by trituration in their culture medium and pelleted by centrifugation. Ice-cold 70% ethanol was added to a final concentration of 1 3 10 6 cells/100 ml and cells were incubated for 20 min. Aliquots of 100 ml were transferred to test tubes, centrifuged, and washed with 1 ml wash buffer containing PBS and 0.5% BSA. Cells were resuspended in denaturating solution (2 M HCl, 0.5% Tween 20) and incubated for 20 min. After washing of cells 0.5 ml of 0.1 M sodium borate (Na 2B 4O 7) was added to neutralize any residual acid. After washing cells were incubated for 20 min at room temperature with a FITC-conjugated anti-BrdU monoclonal antibody (Boehringer, Mannheim, Germany). PC12 cells were washed, centrifuged, resuspended in 0.5 ml propidium iodide solution containing 10 mg/ml of propidium iodide and 100 mg/ml of RNase A dissolved in PBS, and incubated for 30 min. Cells were analyzed on a FACScan (Becton–Dickinson, Heidelberg, Germany),
Statistics Data are expressed as means 6 SD. Significance was assessed by two-tailed Student’s t test (comparison of two groups) or one-way ANOVA followed by Dunnett’s posthoc test (comparison of more than two groups). All experiments represent at least three independent replications performed in triplicate.
FIG. 3. Absence of DNA fragmentation after MPP 1 treatment. DNA fragmentation was measured by a fluorometric fragmentation assay after treatment with MPP 1 or VM26 at the time points indicated. *P , 0.001.
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RESULTS
MPP 1 Inhibits Proliferation of PC12 Cells and Abolishes DNA Synthesis Initially, we studied the effects of increasing concentrations of MPP 1 on undifferentiated PC12 cells seeded at a density of 200,000 cells/ml at 24, 48, 72, and 96 h. After exposure to 100, 250, and 1000 mM MPP 1 for 48 h or longer cell density was lower than that with vehicletreated controls, as measured by cresyl violet staining (Fig. 1A). To study whether the difference in cell density was caused by MPP 1-induced inhibition of cell proliferation, loss of viability, or both, we studied the effects of MPP 1 using trypan blue exclusion to distinguish viable and dead cells. Treatment with 250 mM MPP 1 inhibited cell proliferation and, in addition, led to cell death at 72 and 96 h (Fig. 1B). Treatment with 100 mM MPP 1 for up to 96 h inhibited cell proliferation but did not cause cell death (Fig. 1C). The lack of cell death up to 48 h after addition of 50 to 1000 mM MPP 1 was confirmed by the absence of LDH release into the culture medium (Fig. 1D). Since MPP 1 blocked cell proliferation, we studied whether MPP 1 affected DNA synthesis of PC12 cells. We studied [ 3H]thymidine incorporation as an index for DNA synthesis and S phase activity. MPP 1 concentration and time dependently blocked [ 3H]thymidine incorporation (Fig. 2). PC12 cells treated with 250 mM MPP 1 for 48 h essentially showed no incorporation of [ 3H]thymidine, corresponding to the inhibition of cell proliferation (Figs. 1A and 1B). MPP 1-Induced Cell Death Does Not Show Characteristics of Apoptosis It has been suggested that treatment with 10 –50 mM MPP 1 for 48 h may induce apoptosis and DNA fragmentation in PC12 cells [12]. We did not observe MPP 1-induced cell death at concentrations lower than 100 mM as assayed by light microscopy, trypan blue exclusion assay, and LDH release assay. To investigate whether MPP 1 may induce apoptosis in PC12 cells, we studied DNA fragmentation after treatment with 50 and 250 mM MPP 1 for 48, 72, or 96 h. MPP 1 did not lead to DNA fragmentation, as measured by a fluorometric fragmentation assay (Fig. 3). Since we were able to detect DNA fragmentation after treatment of PC12 cells with 100 mM VM26 (Fig. 3), the absence of DNA fragmentation after MPP 1 treatment cannot be explained by the lack of sensitivity of this assay. Treatment of PC12 cells with VM26 leads to a decreased
FIG. 5. Effects of caspase inhibitors on MPP 1 treatment of PC12 cells. Cells were treated with 250 mM MPP 1 and the effects of caspase inhibitors (DEVD-CHO, zVAD-fmk, YVAD-cmk) on cell viability were studied by staining with crystal violet (CV) at (A) 24, (B) 48, and (C) 72 h.
viability of 49 6 3% at 48 h and complete cell loss at 96 h. We also did not find any increase of TUNEL-positive cells after treatment with 50 or 250 mM MPP 1 for 48 h (data not shown) or 72 h (Fig. 4). Although cell death already occurs after 72 h of treatment with 250 mM MPP 1, we could not detect an increase of TUNELpositive cells compared with controls. Since most types of neuronal apoptosis involve activation of caspases we studied the ability of pseudosubstrate caspase inhibitors to block cell death induced by treatment with 250 mM MPP 1. Treatment with 10 mM DEVD-CHO, 100 mM zVAD-fmk, and 100 mM YVAD-cmk, which were read every 24 h, had no effect on cell proliferation of controls, on the inhibitory effects of MPP 1 on cell proliferation at 24, 48, or 72 h, or on MPP 1-induced cell death effects at 72 h (Fig. 5). The concentrations of
FIG. 4. Absence of TUNEL staining of MPP 1-treated compared with VM 26-treated PC12 cells. PC12 cells treated with (A) vehicle, (B) 50 mM MPP 1, or (C) 250 mM MPP 1 for 72 h were stained for TUNEL and compared with (D) PC12 cells treated with 100 mM VM 26. FIG. 12. Effects of p21 WAF1/Cip1 on MPP 1 induced TUNEL staining. TUNEL staining of p21 1/1 MEFs treated with (A) vehicle or (B) 1 mM MPP 1 and of p21 2/2 MEFs treated with (C) vehicle or (D) 1 mM MPP 1 for 72 h.
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drugs used for caspase inhibition have been proven to inhibit apoptosis of PC12 cells after serum or NGF withdrawal or oxidative stress [20, 23]. Again, we used VM26-induced apoptosis of PC12 cells as a positive control. Treatment with 100 mM zVAD-fmk blocked cell death induced by 100 mM VM26 after 24 h (viability 52 6 4% vs 93 6 3%, P , 0.01). Altogether, we did not find any evidence that MPP 1 induces apoptosis in undifferentiated PC12 cells. MPP 1-Induced Cell Death Depends on Energy Depletion and a Lack of Energy Substrates The toxic effects of MPP 1 are thought to depend on its ability to inhibit complex I of the mitochondrial electron transport chain [9]. Interestingly, treatment of PC12 cells with 200 mM or higher concentrations of MPP 1 for 48 h leads to a depletion of ATP of at least 50% [12]. To study the relationship of MPP 1-induced cell death and energy depletion, we investigated the effects of culture conditions on cell survival after MPP 1 treatment. When cells were seeded at a density of 50,000 instead of 200,000 cells/ml, cell death between 48 and 96 h after treatment with 250 mM MPP 1 was attenuated (Figs. 6A and 6B). Inhibition of oxidative phosphorylation should lead to a compensatory increase in glycolysis with a rise of lactate concentrations and a decrease of the pH. The better survival of cells seeded at lower densities is paralleled by a delayed increase of lactate and a delayed decrease of glucose concentrations (Figs. 6C and 6D). It can be calculated from the graphs that in cells treated with MPP 1, a consumption of 1 mmol glucose yielded an increase of lactate concentration of 2 mmol at either seeding density, suggesting a complete switch from oxidative phosphorylation to nonaerobic glycolysis. In vehicle treated controls only 1.2 to 1.4 mmol lactate is produced per consumption of 1 mmol glucose. When the medium was supplemented with Hepes and glucose in cultures seeded at a density of 200,000 PC12 cells/ml, cell survival after 72 h of MPP 1 treatment was significantly increased compared to normal culture conditions (Fig. 7A). Under low seeding condition of 50,000 cells/ml no cell death occured until day 8; however, cell proliferation was effectively reduced (Fig. 7B). These results suggest that cell death occurs only when energy substrates are depleted [33].
FIG. 6. Effects of MPP 1 on the temporal profile of cell density, glucose, and lactate. PC12 cells were seeded at densities of (A and C) 50,000 or (B and D) 200,000 cells/ml. The effects of treatment with 250 mM MPP 1 on (A and B) cell density (crystal violet, CV) and pH and the effects on (C and D) glucose consumption and lactate production were measured at time points indicated.
MPP 1-Induced Growth Arrest Is Independent of Mitochondrial Complex I Activity To study whether MPP 1-induced growth arrest is mediated by inhibition of complex I of the electron transport chain we used SH-SY5Y neuroblastoma cells and compared parental with cells containing no mitochondrial DNA (p 0 cells, MitoKor Corp., San Diego, CA). Similar to PC12 cells, treatment of undifferenti-
ated SH-SY5Y cells with 250 mM MPP 1 led to growth arrest without cell death (Fig. 8A). Despite the lack of any detectable complex I and IV activity (data not shown), MPP 1 lead to the same growth arrest in p 0 cells (Fig. 8B). Note that growth in p 0 cells in general is slower than in the parental cell line.
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ble 1), suggesting that MPP 1 induces blockage of the G 0/G 1 to S phase transition. MPP 1 Induces p21 by a p53-Independent Pathway p21 WAF1/Cip1 is the prototype inhibitor of cyclin-dependent kinases [24]. It has been suggested that the blockage of cell proliferation elicited by the addition of NGF to exponentially growing PC12 cells results, in part, from the negative regulator of cell cycle progression p21 WAF1/Cip1 [25, 26]. Treatment of PC12 cells with MPP 1 resulted in a marked increase of p21 expression, as detected by Western blot (Fig. 10). Theoretically, p21 WAF1/Cip1 induction may occur as a p53-dependent process activated by DNA damage or as p53 independent [27–29]. However, we did not detect any MPP 1induced expression of p53 in PC12 cells, although we were able to detect p53 expression in PC12 cells after treatment with 0.5 mM doxorubicin for 24 h (Fig. 10). This suggests that MPP 1 induces p21 WAF1/Cip1 expression independently of p53. p21 WAF1/Cip1 Expression Prevents Apoptosis Induced by MPP 1 To study the effects of p21 WAF1/Cip1 expression on MPP 1 treatment we used mouse embryo fibroblasts
FIG. 7. MPP 1-induced cell death depends on energy depletion. (A) Effects of Hepes and glucose supplementation on PC12 cells treated with 250 mM MPP 1. (B) At a low seeding density of 50,000 cells/ml, treatment with 250 mM MPP 1 inhibits cell proliferation (trypan blue-negative cells) but does not induce cell death (trypan blue-positive cells). *P , 0.01 compared with MPP 1-treated PC12 cells in normal RPMI medium not supplemented.
MPP 1 Induces Blockage of the G 0/G 1 to S Phase Transition Subsequently, flow cytometric analysis was carried out using PI to label DNA and BrdU incorporation as a specific marker for new DNA synthesis. Vehicletreated PC12 cells show strong BrdU incorporation (Fig. 9). BrdU incorporation of PC12 cells decreased after treatment with 1000 mM MPP 1 for 48 h (Fig. 9). Cell cycle analysis was performed by standard FACScan software automatically. The percentage of cells in the G 0/G 1 phase increased and the percentage of cells in the S phase decreased after exposure to MPP 1 (Ta-
FIG. 8. MPP 1-induced growth arrest is independent of mitochondrial complex I activity of SH-SY5Y cells. Effects of treatment with 250 mM MPP 1 on (A) SH-SY5Y parental neuroblastoma cells and (B) p 0 cells lacking mitochondrial DNA. In both, MPP 1 treatment leads to growth arrest of living cells (trypan blue-negative) but not to an increase of dead (trypan blue-positive) cells.
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to the effects on PC12 cells, MPP 1 did not lead to cell death of p21 1/1 MEFs but to growth arrest (Fig. 11B). However, in p21 2/2 MEFs MPP 1 did not lead to growth arrest (Fig. 11B) but caused accelerated cell death after treatment for 72 and 96 h (Fig. 11C). The similar kinetics of MPP 1-induced cell death of p21 1/2 cells to that of p21 2/2 cells (Fig. 11C) may be explained by the low expression of p21 WAF1/Cip1 protein in p21 1/2 cells (Fig. 11A) and is consistent with the incomplete growth arrest of p21 1/2 cells after MPP 1 treatment (Fig. 11B). Further, MPP 1 induced TUNEL staining in p21 2/2 (Fig. 12D) but not p21 1/1 (Fig. 12B) cells, suggesting that in the absence of p21 WAF1/Cip1 MPP 1 will lead to apoptosis, whereas in the presence of p21 WAF1/Cip1 MPP 1 will lead to a growth arrest in proliferating cells. DISCUSSION
In the present study, we investigated the effects of MPP 1 on undifferentiated dopaminergic PC12 cells. We found that MPP 1 did not primarily induce cell death but a cessation of cell proliferation that was due to a block in the G 0/G 1 to S phase transition. MPP 1 treatment resulted in the induction of p21 WAF1/Cip1 expression by a p53-independent pathway. Since p21 WAF1/Cip1 controls the G 0/G 1 to S phase transition, we hypothesized that the induction of p21 expression observed after MPP 1 treatment arrested cell cycle progression. The induced expression of p21 WAF1/Cip1 seems to be required for the survival of MPP 1-treated cells, since p21 WAF1/Cip1-deficient fibroblasts died after treatment with equal concentrations of MPP 1. In the presence of p21 WAF1/Cip1, cell death occured only secondarily when energy substrates were depleted due to enhanced anaerobic glycolysis following inhibition of oxidative phosphorylation. Several reports have suggested that MPP 1 may induce apoptosis in PC12 cells at concentrations ranging from 10 mM [12] to 1 mM MPP 1 [16]. In contrast, we did not observe any cell death after treatment with MPP 1 for 96 h at concentrations lower than 250 mM (Fig. 1). At concentrations between 250 mM and 1 mM of MPP 1, cell death was induced between 48 and 96 h. This cell death was not apoptotic, since we could not FIG. 9. MPP 1 induced a block in G 0/G 1 to S phase transition. Contour blots of bivariate DNA/BrdU distributions in PC12 cells untreated (top) or treated for 24 (middle) or 48 (bottom) h with 1000 mM MPP 1. Propidium iodide (PI) was used to label DNA and 5-bromodeoxiuridine (BrdU) incorporation as a specific marker for new DNA synthesis. Cells in the left cluster with the smallest amount of DNA are in G 0/G 1 phase, cells in the right cluster with double this amount are in either G 2 or M phase, while cells in S phase have intermediate amounts.
(MEFs) with different genetically regulated expression of p21 WAF1/Cip1 [19]. p21 1/1, p21 1/2, and p21 2/2 MEFs showed different expression of p21 WAF1/Cip1 protein, with no detectable protein of p21 2/2 cells (Fig. 11A). Similar
TABLE 1 Cell Cycle Analysis Based on Flow Cytometric Analysis MPP 1
G 0/G 1 phase S phase G 2 phase
Controls
24 h
48 h
64.9 6 0.4 24.9 6 0.8 6.8 6 0.4
79.4 6 2.8*** 5.4 6 1.4*** 10.2 6 1.6
83.1 6 1.4*** 4.5 6 0.4*** 9.5 6 1.0
*** P , 0.001 compared with controls (ANOVA followed by Dunnett’s posthoc test).
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treatment led to a sharp rise of lactate production in PC12 cells which died only after energy substrates were depleted. As suggested earlier [33] supplying more energy substrates led to a longer survival (Fig. 7). The cell cycle-related response of PC12 cells to treatment with MPP 1 is similar to their response to NGF treatment. In the presence of serum, NGF induces a block in the G1 phase of the cell cycle and upregulation of p21 WAF1/Cip1 protein levels [26, 36]. Removal of serum from differentiated PC12 cells results in loss of p21WAF1/Cip1. However, this loss of p21 WAF1/Cip1 expression has no effect
FIG. 10. MPP 1 induces expression of p21 WAF1/Cip1 but not of p53. Treatment with 250 mM MPP 1 time dependently induces expression of p21 (top) but not of p53 (bottom). In contrast, treatment with 0.5 mM doxorubicin induces expression of p53.
detect DNA fragmentation or TUNEL-positive staining. We also could not block cell death with peptide inhibitors of caspases (Fig. 5), which are thought to be involved in almost all paradigms of neuronal apoptosis. Compared to the toxicity of MPP 1 against dopaminergic neurons in mesencephalic cultures, only substantially higher concentrations of MPP 1 show effects in PC12 cells. MPP 1 is toxic to dopaminergic neurons of mesencephalic cultures at concentrations as low as 0.5 to 2.0 mM [30, 31], but shows effects in PC12 cells only at concentrations exceeding 100 mM. We report here that MPP 1 arrests cell cycle progression at concentrations of 100 mM and higher but does not affect cell survival for 96 h at concentrations lower than 250 mM. The requirement of high MPP 1 concentrations to induce death in PC12 cells supports earlier reports, in which 1 to 3 mM treatments for 24 h were necessary to induce cell death [32, 33]. Adrenal chromaffin cells express a high-affinity catecholamine uptake system and have been demonstrated to accumulate [ 3H]MPP 1 with relatively little toxicity [34]. It has been argued that adrenal chromaffin cells may be relatively resistant to MPP 1 due to a reserpine-sensitive sequestration of the drug within a distinct intracellular compartment [34, 35]. These and our data are in sharp contrast to a report by Hartley et al. [12], who provided evidence that MPP 1 induces apoptosis in PC12 cells at concentrations ranging from 10 to 100 mM as detected morphologically by electron microscopy and biochemically by DNA fragmentation and DNA laddering on agarose gel electrophoresis. At concentrations higher than 100 mM, the predominant mode of cell death was necrosis. After treatment with 250 mM MPP 1 for 48 h they observed a 60% LDH release relative to the total LDH activity. There may be substantial differences between different PC12 cell lines used. We show that inhibition of complex I of the electron transport chain can be compensated by glycolysis and is apparently not sufficient to kill PC12 cells. MPP 1
FIG. 11. Expression of p21 WAF1/Cip1 blocks apoptosis induced by MPP 1. (A) Expression of p21 WAF1/Cip1 in p21 1/1, p21 1/2, and p21 2/2 mouse embryo fibroblasts. (B) Effects of 1 mM MPP 1 on growth arrest depends on p21 WAF1/Cip1 expression in p21 1/1, p21 1/2, and p21 2/2 mouse embryo fibroblasts. ***P , 0.001 compared with untreated p21 1/1 cells. (C) Effects of p21 WAF1/Cip1 on the treatment of MEFs with 1 mM MPP 1.
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on differentiation or the nonproliferative state in the presence of NGF, suggesting that p21 WAF1/Cip1 serves in part to neutralize the mitogenic stimulus of serum [26]. In the absence of the mitogenic signal, cells no longer require this response and could use other means to stop or prevent cell cycle progression. Using mouse embryo fibroblasts with varying expression of p21 WAF1/Cip1 we show here that p21 WAF1/Cip1 expression after MPP 1 treatment is required to inhibit cell proliferation and to prevent apoptosis. Since MPP 1 leads to the same growth arrest in SH-SY5Y p 0 cells, in which no complex I activity is detectable, this block in cell proliferation is not due to inhibition of complex I. This paper provides evidence for a new mechanism of MPP 1 toxicity. We demonstrate that in PC12 cells MPP 1 does not primarily lead to cell death by inhibition of oxidative phosphorylation but interferes with cell cycle progression. Survival of cells may depend on their ability to induce p21 WAF1/Cip1 expression. Since MPTP/MPP 1 toxicity in humans and animals resembles PD in many aspects, including the selectivity for dopaminergic neurons, the major clinical syndrome, and complex I inhibition of the electron transport chain, these results may lead to new insights into the pathogenesis of PD. We thank Dr. Tyler Jacks for providing p21-deficient mouse embryo fibroblasts. This study was supported by grants from the Deutsche Forschungsgemeinschaft (Kl 782/3-1) and from the fortu¨ne-program of the University of Tu¨bingen (Project 214) to T.K. and J.B.S.
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