Experimental Cell Research 250, 75–85 (1999)Article ID excr.1999.4504, available online at http://www.idealibrary.com on
MPP Inhibits Proliferation of PC12 Cells by a p21 -DependentPathway and Induces Cell Death in Cells Lacking p21WAF1/Cip1
Frank Soldner,* Michael Weller,† Sibylle Haid,* Stefan Beinroth,* Scott W. Miller,‡ Ullrich Wullner,*Robert E. Davis,‡ Johannes Dichgans,* Thomas Klockgether,* and Jorg B. Schulz*,1
*Laboratory of Experimental Neuropharmacology and †Laboratory of Molecular Neuro-Oncology, Department of Neurology,University of Tubingen, Hoppe-Seyler-Str. 3, D-72076 Tubingen, Germany; and ‡MitoKor Corporation, San Diego, California 92121
cal abnormalities that characterize the disease. Theckdpptm[tosvPal
1ppioesctesnbtpgv
eiccpPea
The molecular and biochemical mode of cell death ofopaminergic neurons in Parkinson’s disease (PD) isncertain. In an attempt at further clarification wetudied the effects of 1-methyl-4-phenylpyridiniumMPP1), the active metabolite of 1-methyl-4-phenyl-,2,3,6-tetrahydropyridine (MPTP), on dopaminergicC12 cells. In humans and nonhuman primates MPTP/PP1 causes a syndrome closely resembling PD.PP1 toxicity is thought to be mediated by the block
f complex I of the mitochondrial electron transporthain. Treatment of undifferentiated PC12 cells withPP1 primarily inhibited proliferation of PC12 cells
nd secondarily led to cell death after the depletion ofll energy substrates by glycolysis. This cell deathhowed no morphological characteristics of apoptosisnd was not blocked by treatment with caspase inhib-tors. The inhibition of cell growth was not dependentn an inhibition of complex I activity since MPP1 alsonhibited cell proliferation in SH-SY5Y cells lacking
Pathologically, the hallmark of Parkinson’s diseasePD) is loss of dopaminergic neurons in the substantiaigra, leading to the major clinical and pharmacologi-
1 To whom correspondence and reprint requests should be ad-ressed. Fax: **49-7071-295260. E-mail: [email protected].
75
ause of neuronal loss in the substantia nigra is notnown. However, recent advances have been made inefining morphological and biochemical events in theathogenesis of the disease. Inhibition of oxidativehosphorylation, excitotoxicity, and generation of reac-ive oxygen species (ROS) are considered importantediators of neuronal cell death in Parkinson’s disease
1]. A defect of complex I of the mitochondrial electronransport chain has been identified in substantia nigraf PD patients and is thought to be specific for PD andelective for the substantia nigra in the central ner-ous system [2, 3]. Evidence implicating apoptosis inD is controversial. Some studies found evidence forpoptosis based on morphologic criteria or in situ endabeling [4, 5], whereas others did not [6, 7].
In humans and nonhuman primates, the neurotoxin-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)roduces irreversible clinical, biochemical, and neuro-athologic effects which closely mimic those observedn idiopathic PD [8]. This meperidine analog is metab-lized to 1-methyl-4-phenylpyridinium (MPP1) by thenzyme monoamine oxidase B. MPP1 is subsequentlyelectively taken up by dopaminergic terminals andoncentrated in neuronal mitochondria in the substan-ia nigra. MPP1 binds to and inhibits complex I of thelectron transport chain [9], thereby producing theame biochemical defect as detected in the substantiaigra of PD patients. MPP1 can deplete ATP levelsoth in vitro and in vivo [10–12]. It has been suggestedhat under certain conditions MPP1 may induce apo-tosis in PC12 cells [12] and apoptosis in cerebellarranule neurons by release of cytochrome c and acti-ation of caspases [13].Rat pheochromocytoma PC12 cells have been used
xtensively to study the mechanisms of MPP1 toxic-ty [12, 14 –16]. Here, we used undifferentiated PC12ells to study the molecular mechanisms governingell growth arrest and death in MPP1 toxicity andutative mechanisms of dopaminergic cell death inD. We tried to clarify the role of MPP1-inducednergy depletion by studying the effects of MPP1 onnaerobic glycolysis, repletion of energy substrates,
and the effects on cells with a mitochondrial DNAdposMde
M
dfadm
C
mitwscle
nppDmsb
(FpM
C
tr(tc
wmwcngc
sitrt
untreated PC12 cells and the maximum release obtained after expo-s
A
flfhsmwf
dmr1iP(pfbwbiamc
W
Ph8ahlei%Ttmc(TpP0p
D
ibBmhldc
C
2
76 SOLDNER ET AL.
epletion. Since these studies suggested that MPP1
rimarily inhibited cell proliferation, and only sec-ndarily lead to cell death after depletion of energyubstrates, we further investigated the effects ofPP1 on cell cycle progression and the cyclin-
ependent kinase inhibitor p21WAF1/Cip1 as well as theffects of MPP1 on cells lacking p21WAF1/Cip1 (p212/2).
METHODS
aterials
RPMI 1640 cell culture medium, Dulbecco’s modified Eagle’s me-ium (DMEM), Dulbecco’s modified phosphate-buffered saline (PBS),etal calf serum (FCS), donor horse serum, penicillin, streptomycin,nd supplements were obtained from GIBCO Life Technologies (Hei-elberg, Germany). MPP1 was purchased from RBI (Cologne, Ger-any).
ell Culture
PC12 cells were maintained at 37°C, 5% CO2, in RPMI 1640edium, supplemented with 5% heat-inactivated FCS, 10% heat-
nactivated horse serum, 100 U/ml penicillin, and 100 mg/ml strep-omycin. Cells were split every 5–7 days 1:10. Confluent culturesere washed with prewarmed PBS, detached with a trypsin/EDTA
olution, centrifuged, and subcultured. If not otherwise stated, PC12ells were seeded at a density of 50,000 or 200,000/ml on poly-L-ysine-coated 24- or 96-well plates for cytotoxicity and proliferationxperiments. MPP1 and drugs were added to cultures 24 h later.A clonal stock of human SH-SY5Y neuroblastoma cells containing
o mtDNA (p0 cells, MitoKor Corp., San Diego, CA) was created byrolonged exposure of these cells to ethidium bromide [17]. SH-SY5Yarental cells were grown at 37°C, 5% CO2, in medium consisting ofMEM, supplemented with 10% FCS, 100 U/ml penicillin, and 100g/ml streptomycin. For p0 cells the growth medium was additionallyupplemented with 100 mg/ml of pyruvate and 50 mg/ml of uridineecause p0 cells are auxotrophic for pyruvate and uridine [18].p211/1, p211/2, and p212/2 primary mouse embryo fibroblasts
MEFs) [19] were grown to subconfluence in DME medium, 10%CS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells werelated at a density of 1.5 3 105 cells/ml and were treated withPP1 at 24 h.
ell Viability
Trypan blue exclusion assay. Cells were removed from the cul-ure dish by trituration in their culture medium, centrifuged, andesuspended, and 50 ml was added to 50 ml of trypan blue solution0.4% in PBS). The numbers of trypan blue-excluding (live) cells andrypan blue-staining (dead) cells were counted using a countinghamber.Crystal violet assay. Cell culture medium was removed, and cellsere exposed for 10 min to a solution of 0.5% crystal violet in 20%ethanol. After incubation, the plates were rinsed with distilledater as previously described and air dried for 24 h. After drying,
ells were lysed with 0.1 M sodium citrate buffer and scanned at 550m on a plate photometer (Dynex, Denkendorf, Germany). Back-round readings were performed by staining of collagen-precoatedell-free wells.LDH release assay. LDH activity was measured as optical den-
ity (OD) at 490 nm using a plate photometer and expressed accord-ng to the manufacturer’s (Boehringer, Mannheim, Germany) direc-ions: specific LDH release (%) 5 [(experimental value-spontaneouselease)/(maximum release-spontaneous release)] 3 100. The spon-aneous release was defined as the amount of LDH released from
ure of PC12 cells to 1% Triton X-100 for 30 min at 37°C.
poptosis Assays
DNA fragmentation. DNA fragmentation was quantified by DNAuorometry [20]. In brief, cells were harvested and lysed on ice, andragmented DNA was separated from nucleus-attached DNA byigh-speed centrifugation. After disruption of the pellets by briefonication and RNase digestion, fragmented and pelleted DNA waseasured by ethidium bromide. The percentage of fragmentationas calculated by dividing fragmented DNA by the total sum of
ragmented and pelleted DNA.TUNEL staining. For TUNEL staining [21] cells were seeded at a
ensity of 200,000 cells/ml on 35-mm dishes. After culturing and treat-ent at conditions as indicated, medium was removed, and cells were
insed twice in PBS and fixed for 15 min in 4% paraformaldehyde and.5% glutaraldehyde. After a wash in PBS, endogenous peroxidase wasnactivated by covering the cells with 0.1% H2O2 for 10 min. After twoBS washes, cells were preincubated for 10 min in sodium cacodylate
TdT) buffer containing 140 mM sodium cacodylate, 30 mM Tris–HCl,H 7.2, and 1.25 mg/ml bovine serum albumin. Cells then were exposedor 20 min to the reaction mixture (50 U/ml terminal transferase, 10 mMiotin-dUTP, 25 mM cobalt chloride in TdT buffer) at 37°C. The reactionas stopped by incubating the dishes for 10 min with sodium citrateuffer. After blocking with 2% bovine serum albumin (BSA), cells werencubated for 30 min with streptavidin alkaline phosphatase conjugatend developed with 0.41 mM nitroblue tetrazolium chloride and 0.38M 5-bromo-4-chloro-3-indolyl phosphate in 200 mM Tris–HCl, pH 9.5,
ontaining 10 mM MgCl2.
estern Blot
Expression of p21WAF1/Cip1 and p53 was detected by Western blot.C12 cells were seeded on 100-mm dishes. Soluble protein wasarvested from cells lysed for 15 min on ice in 50 mM Tris–HCl, pH, containing 120 mM NaCl, 5 mM EDTA, 0.5% NP-40, 2 mg/mlprotinin, 100 mg/ml PMSF, and 10 mg/ml leupeptin, followed byigh-speed centrifugation at 4°C. Twenty micrograms of protein per
ane was separated by 12% SDS–PAGE. After electrophoresis andlectroblotting to nitrocellulose membranes, the blots were blockedn 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%ween 20, and 0.1% azide for 60 min. The blots were incubated withhe first antibody at 4°C overnight. The following antisera were used:onoclonal anti-p53 (Ab-3, Oncogene, Bad Soden, Germany), poly-
lonal anti-p21WAF1/Cip1 (C19, Santa Cruz,), and monoclonal anti-cdk5J-3, Santa Cruz). After three washes in PBS containing 0.05%ween 20, the membranes were incubated with secondary alkalinehosphatase-conjugated antibody for 1 h, washed three times inBS, and stained with 0.2 mg/ml nitroblue tetrazolium chloride and.3 mg/ml 5-bromo-4-chloro-3-indolyl-phosphate in 0.1 M Tris–HCl,H 9.5, containing 50 mM MgCl2 and 100 mM NaCl.
NA Synthesis
PC12 cells were seeded in 96-well microtiter plates at the densitiesndicated and treated with various concentrations of MPP1. At 6 hefore the end of the respective study [3H]thymidine (Amersham,raunschweig, Germany) was added to a final concentration of 3.3Ci/ml and cells were incubated at 37°C for 6 h. Then cells werearvested and incorporated radioactivity was determined with a
iquid scintillation counter (Wallac, Turku, Finland). For each con-ition a ratio of incorporated [3H]thymidine to the number of liveells was calculated.
ell Cycle Analysis
For cell cycle analysis [22] cells were seeded at a density of00,000/ml in 35-mm dishes and treated with MPP1 as described
a5mcwwtP(on2mwsRa
esFas
M
gf
S
tAtr
(caMbwdwr
dtm
Dac
77MPP1 INDUCES p21WAF1/Cip1 AND INHIBITS CELL PROLIFERATION
bove. At the end of the experiment cultures were incubated with-bromodeoxyuridine (BrdU) 30 min at a final concentration of 10M. Cells were removed from the culture dish by trituration in theirulture medium and pelleted by centrifugation. Ice-cold 70% ethanolas added to a final concentration of 1 3 106 cells/100 ml and cellsere incubated for 20 min. Aliquots of 100 ml were transferred to test
ubes, centrifuged, and washed with 1 ml wash buffer containingBS and 0.5% BSA. Cells were resuspended in denaturating solution
2 M HCl, 0.5% Tween 20) and incubated for 20 min. After washingf cells 0.5 ml of 0.1 M sodium borate (Na2B4O7) was added toeutralize any residual acid. After washing cells were incubated for0 min at room temperature with a FITC-conjugated anti-BrdUonoclonal antibody (Boehringer, Mannheim, Germany). PC12 cellsere washed, centrifuged, resuspended in 0.5 ml propidium iodide
olution containing 10 mg/ml of propidium iodide and 100 mg/ml ofNase A dissolved in PBS, and incubated for 30 min. Cells werenalyzed on a FACScan (Becton–Dickinson, Heidelberg, Germany),
FIG. 1. Effects of MPP1 on growth and survival of PC12 cells.A) Cells were treated with varying concentrations of MPP1 andell densities were measured by crystal violet staining (OD units)t time points indicated. (B) PC12 cells were treated with 250 mMPP1 and the trypan blue assay was used to detect viable (trypan
lue-negative) and dead (trypan blue-positive) cells comparedith controls (C). (C) Treatment with 100 mM MPP1 for 48 or 96 hid not cause cell death but inhibited proliferation. (D) Treatmentith varying concentrations of MPP1 for 48 h did not lead to LDH
elease from PC12 cells.
quipped with a 15-mW, 488-nm air-cooled argon–ion laser. Emis-ion fluorescence was measured with a 514-nm bandpass filter forITC and a 600-nm wavelength filter for propidium iodide (PI). Dataquisition and data analysis were performed using the Cell Questoftware (Becton–Dickinson).
easurement of Glucose, Lactate, and pH
Concentrations of glucose and lactate were measured using thelucose oxidase assay and the lactate oxidase assay, respectively,ollowing the manufacturer’s directions (Boehringer).
tatistics
Data are expressed as means 6 SD. Significance was assessed bywo-tailed Student’s t test (comparison of two groups) or one-wayNOVA followed by Dunnett’s posthoc test (comparison of more than
wo groups). All experiments represent at least three independenteplications performed in triplicate.
FIG. 2. Effects of MPP1 on DNA synthesis. MPP1 concentrationependently inhibits [3H]thymidine incorporation. PC12 cells werereated with 250 mM MPP1 and 6 h before the end of the respectiveeasurement [3H]thymidine was added.
FIG. 3. Absence of DNA fragmentation after MPP1 treatment.NA fragmentation was measured by a fluorometric fragmentationssay after treatment with MPP1 or VM26 at the time points indi-ated. *P , 0.001.
78 SOLDNER ET AL.
RESULTS
M
taAot(spegMt1bdwc
wWftiM[p
M
MmM1ewsalmtcfpm
v9
c(aMpostmmrcld
ccb4
5
M
79MPP1 INDUCES p21WAF1/Cip1 AND INHIBITS CELL PROLIFERATION
PP1 Inhibits Proliferation of PC12 Cells andAbolishes DNA Synthesis
Initially, we studied the effects of increasing concen-rations of MPP1 on undifferentiated PC12 cells seededt a density of 200,000 cells/ml at 24, 48, 72, and 96 h.fter exposure to 100, 250, and 1000 mM MPP1 for 48 hr longer cell density was lower than that with vehicle-reated controls, as measured by cresyl violet stainingFig. 1A). To study whether the difference in cell den-ity was caused by MPP1-induced inhibition of cellroliferation, loss of viability, or both, we studied theffects of MPP1 using trypan blue exclusion to distin-uish viable and dead cells. Treatment with 250 mMPP1 inhibited cell proliferation and, in addition, led
o cell death at 72 and 96 h (Fig. 1B). Treatment with00 mM MPP1 for up to 96 h inhibited cell proliferationut did not cause cell death (Fig. 1C). The lack of celleath up to 48 h after addition of 50 to 1000 mM MPP1
as confirmed by the absence of LDH release into theulture medium (Fig. 1D).
Since MPP1 blocked cell proliferation, we studiedhether MPP1 affected DNA synthesis of PC12 cells.e studied [3H]thymidine incorporation as an index
or DNA synthesis and S phase activity. MPP1 concen-ration and time dependently blocked [3H]thymidinencorporation (Fig. 2). PC12 cells treated with 250 mM
PP1 for 48 h essentially showed no incorporation of3H]thymidine, corresponding to the inhibition of cellroliferation (Figs. 1A and 1B).
PP1-Induced Cell Death Does Not ShowCharacteristics of Apoptosis
It has been suggested that treatment with 10–50 mMPP1 for 48 h may induce apoptosis and DNA frag-entation in PC12 cells [12]. We did not observePP1-induced cell death at concentrations lower than
00 mM as assayed by light microscopy, trypan bluexclusion assay, and LDH release assay. To investigatehether MPP1 may induce apoptosis in PC12 cells, we
tudied DNA fragmentation after treatment with 50nd 250 mM MPP1 for 48, 72, or 96 h. MPP1 did notead to DNA fragmentation, as measured by a fluoro-
etric fragmentation assay (Fig. 3). Since we were ableo detect DNA fragmentation after treatment of PC12ells with 100 mM VM26 (Fig. 3), the absence of DNAragmentation after MPP1 treatment cannot be ex-lained by the lack of sensitivity of this assay. Treat-ent of PC12 cells with VM26 leads to a decreased
FIG. 4. Absence of TUNEL staining of MPP1-treated compared w0 mM MPP1, or (C) 250 mM MPP1 for 72 h were stained for TUNEFIG. 12. Effects of p21WAF1/Cip1 on MPP1 induced TUNEL stainingPP1 and of p212/2 MEFs treated with (C) vehicle or (D) 1 mM MP
iability of 49 6 3% at 48 h and complete cell loss at6 h.We also did not find any increase of TUNEL-positive
ells after treatment with 50 or 250 mM MPP1 for 48 hdata not shown) or 72 h (Fig. 4). Although cell deathlready occurs after 72 h of treatment with 250 mMPP1, we could not detect an increase of TUNEL-
ositive cells compared with controls. Since most typesf neuronal apoptosis involve activation of caspases wetudied the ability of pseudosubstrate caspase inhibi-ors to block cell death induced by treatment with 250M MPP1. Treatment with 10 mM DEVD-CHO, 100M zVAD-fmk, and 100 mM YVAD-cmk, which wereead every 24 h, had no effect on cell proliferation ofontrols, on the inhibitory effects of MPP1 on cell pro-iferation at 24, 48, or 72 h, or on MPP1-induced celleath effects at 72 h (Fig. 5). The concentrations of
FIG. 5. Effects of caspase inhibitors on MPP1 treatment of PC12ells. Cells were treated with 250 mM MPP1 and the effects ofaspase inhibitors (DEVD-CHO, zVAD-fmk, YVAD-cmk) on cell via-ility were studied by staining with crystal violet (CV) at (A) 24, (B)8, and (C) 72 h.
VM 26-treated PC12 cells. PC12 cells treated with (A) vehicle, (B)nd compared with (D) PC12 cells treated with 100 mM VM 26.NEL staining of p211/1 MEFs treated with (A) vehicle or (B) 1 mM
for 72 h.
ithL a. TUP1
drugs used for caspase inhibition have been proven toiwVccinu
M
ieoM5cet54apcasicfclspcc
aPmcth7w
M
mtacC
aaascs
g52ad
80 SOLDNER ET AL.
nhibit apoptosis of PC12 cells after serum or NGFithdrawal or oxidative stress [20, 23]. Again, we usedM26-induced apoptosis of PC12 cells as a positive
ontrol. Treatment with 100 mM zVAD-fmk blockedell death induced by 100 mM VM26 after 24 h (viabil-ty 52 6 4% vs 93 6 3%, P , 0.01). Altogether, we didot find any evidence that MPP1 induces apoptosis inndifferentiated PC12 cells.
PP1-Induced Cell Death Depends on EnergyDepletion and a Lack of Energy Substrates
The toxic effects of MPP1 are thought to depend onts ability to inhibit complex I of the mitochondriallectron transport chain [9]. Interestingly, treatmentf PC12 cells with 200 mM or higher concentrations ofPP1 for 48 h leads to a depletion of ATP of at least
0% [12]. To study the relationship of MPP1-inducedell death and energy depletion, we investigated theffects of culture conditions on cell survival after MPP1
reatment. When cells were seeded at a density of0,000 instead of 200,000 cells/ml, cell death between8 and 96 h after treatment with 250 mM MPP1 wasttenuated (Figs. 6A and 6B). Inhibition of oxidativehosphorylation should lead to a compensatory in-rease in glycolysis with a rise of lactate concentrationsnd a decrease of the pH. The better survival of cellseeded at lower densities is paralleled by a delayedncrease of lactate and a delayed decrease of glucoseoncentrations (Figs. 6C and 6D). It can be calculatedrom the graphs that in cells treated with MPP1, aonsumption of 1 mmol glucose yielded an increase ofactate concentration of 2 mmol at either seeding den-ity, suggesting a complete switch from oxidative phos-horylation to nonaerobic glycolysis. In vehicle treatedontrols only 1.2 to 1.4 mmol lactate is produced peronsumption of 1 mmol glucose.
When the medium was supplemented with Hepesnd glucose in cultures seeded at a density of 200,000C12 cells/ml, cell survival after 72 h of MPP1 treat-ent was significantly increased compared to normal
ulture conditions (Fig. 7A). Under low seeding condi-ion of 50,000 cells/ml no cell death occured until day 8;owever, cell proliferation was effectively reduced (Fig.B). These results suggest that cell death occurs onlyhen energy substrates are depleted [33].
PP1-Induced Growth Arrest Is Independent ofMitochondrial Complex I Activity
To study whether MPP1-induced growth arrest isediated by inhibition of complex I of the electron
ransport chain we used SH-SY5Y neuroblastoma cellsnd compared parental with cells containing no mito-hondrial DNA (p0 cells, MitoKor Corp., San Diego,A). Similar to PC12 cells, treatment of undifferenti-
ted SH-SY5Y cells with 250 mM MPP1 led to growthrrest without cell death (Fig. 8A). Despite the lack ofny detectable complex I and IV activity (data nothown), MPP1 lead to the same growth arrest in p0
ells (Fig. 8B). Note that growth in p0 cells in general islower than in the parental cell line.
FIG. 6. Effects of MPP1 on the temporal profile of cell density,lucose, and lactate. PC12 cells were seeded at densities of (A and C)0,000 or (B and D) 200,000 cells/ml. The effects of treatment with50 mM MPP1 on (A and B) cell density (crystal violet, CV) and pHnd the effects on (C and D) glucose consumption and lactate pro-uction were measured at time points indicated.
M
ost(aCSti
ble 1), suggesting that MPP1 induces blockage of theG
M
datfprdppdiwtTs
p
M
d2pti
(tc(bc
81MPP1 INDUCES p21WAF1/Cip1 AND INHIBITS CELL PROLIFERATION
PP1 Induces Blockage of the G0/G1 to S PhaseTransition
Subsequently, flow cytometric analysis was carriedut using PI to label DNA and BrdU incorporation as apecific marker for new DNA synthesis. Vehicle-reated PC12 cells show strong BrdU incorporationFig. 9). BrdU incorporation of PC12 cells decreasedfter treatment with 1000 mM MPP1 for 48 h (Fig. 9).ell cycle analysis was performed by standard FAC-can software automatically. The percentage of cells inhe G0/G1 phase increased and the percentage of cellsn the S phase decreased after exposure to MPP1 (Ta-
FIG. 7. MPP1-induced cell death depends on energy depletion.A) Effects of Hepes and glucose supplementation on PC12 cellsreated with 250 mM MPP1. (B) At a low seeding density of 50,000ells/ml, treatment with 250 mM MPP1 inhibits cell proliferationtrypan blue-negative cells) but does not induce cell death (trypanlue-positive cells). *P , 0.01 compared with MPP1-treated PC12ells in normal RPMI medium not supplemented.
0/G1 to S phase transition.
PP1 Induces p21 by a p53-Independent Pathway
p21WAF1/Cip1 is the prototype inhibitor of cyclin-depen-ent kinases [24]. It has been suggested that the block-ge of cell proliferation elicited by the addition of NGFo exponentially growing PC12 cells results, in part,rom the negative regulator of cell cycle progression21WAF1/Cip1 [25, 26]. Treatment of PC12 cells with MPP1
esulted in a marked increase of p21 expression, asetected by Western blot (Fig. 10). Theoretically,21WAF1/Cip1 induction may occur as a p53-dependentrocess activated by DNA damage or as p53 indepen-ent [27–29]. However, we did not detect any MPP1-nduced expression of p53 in PC12 cells, although weere able to detect p53 expression in PC12 cells after
reatment with 0.5 mM doxorubicin for 24 h (Fig. 10).his suggests that MPP1 induces p21WAF1/Cip1 expres-ion independently of p53.
To study the effects of p21WAF1/Cip1 expression onPP1 treatment we used mouse embryo fibroblasts
FIG. 8. MPP1-induced growth arrest is independent of mitochon-rial complex I activity of SH-SY5Y cells. Effects of treatment with50 mM MPP1 on (A) SH-SY5Y parental neuroblastoma cells and (B)0 cells lacking mitochondrial DNA. In both, MPP1 treatment leadso growth arrest of living cells (trypan blue-negative) but not to anncrease of dead (trypan blue-positive) cells.
(osn
to the effects on PC12 cells, MPP1 did not lead to celldHatktl1aF(taw
MWdttpphsgtsmewap
dfdM(oT
GSG
n
CtMosie
82 SOLDNER ET AL.
MEFs) with different genetically regulated expressionf p21WAF1/Cip1 [19]. p211/1, p211/2, and p212/2 MEFshowed different expression of p21WAF1/Cip1 protein, witho detectable protein of p212/2 cells (Fig. 11A). Similar
FIG. 9. MPP1 induced a block in G0/G1 to S phase transition.ontour blots of bivariate DNA/BrdU distributions in PC12 cells un-
reated (top) or treated for 24 (middle) or 48 (bottom) h with 1000 mMPP1. Propidium iodide (PI) was used to label DNA and 5-bromode-
xiuridine (BrdU) incorporation as a specific marker for new DNAynthesis. Cells in the left cluster with the smallest amount of DNA aren G0/G1 phase, cells in the right cluster with double this amount are inither G2 or M phase, while cells in S phase have intermediate amounts.
eath of p211/1 MEFs but to growth arrest (Fig. 11B).owever, in p212/2 MEFs MPP1 did not lead to growthrrest (Fig. 11B) but caused accelerated cell death afterreatment for 72 and 96 h (Fig. 11C). The similarinetics of MPP1-induced cell death of p211/2 cells tohat of p212/2 cells (Fig. 11C) may be explained by theow expression of p21WAF1/Cip1 protein in p211/2 cells (Fig.1A) and is consistent with the incomplete growthrrest of p211/2 cells after MPP1 treatment (Fig. 11B).urther, MPP1 induced TUNEL staining in p212/2
Fig. 12D) but not p211/1 (Fig. 12B) cells, suggestinghat in the absence of p21WAF1/Cip1 MPP1 will lead topoptosis, whereas in the presence of p21WAF1/Cip1 MPP1
ill lead to a growth arrest in proliferating cells.
DISCUSSION
In the present study, we investigated the effects ofPP1 on undifferentiated dopaminergic PC12 cells.e found that MPP1 did not primarily induce cell
eath but a cessation of cell proliferation that was dueo a block in the G0/G1 to S phase transition. MPP1
reatment resulted in the induction of p21WAF1/Cip1 ex-ression by a p53-independent pathway. Since21WAF1/Cip1 controls the G0/G1 to S phase transition, weypothesized that the induction of p21 expression ob-erved after MPP1 treatment arrested cell cycle pro-ression. The induced expression of p21WAF1/Cip1 seemso be required for the survival of MPP1-treated cells,ince p21WAF1/Cip1-deficient fibroblasts died after treat-ent with equal concentrations of MPP1. In the pres-
nce of p21WAF1/Cip1, cell death occured only secondarilyhen energy substrates were depleted due to enhancednaerobic glycolysis following inhibition of oxidativehosphorylation.Several reports have suggested that MPP1 may in-
uce apoptosis in PC12 cells at concentrations rangingrom 10 mM [12] to 1 mM MPP1 [16]. In contrast, weid not observe any cell death after treatment withPP1 for 96 h at concentrations lower than 250 mM
Fig. 1). At concentrations between 250 mM and 1 mMf MPP1, cell death was induced between 48 and 96 h.his cell death was not apoptotic, since we could not
TABLE 1
Cell Cycle Analysis Based on Flow Cytometric Analysis
*** P , 0.001 compared with controls (ANOVA followed by Dun-ett’s posthoc test).
diii
nsio0ohccmiwieawtttmtttpbgmAoad
ti
treatment led to a sharp rise of lactate production inPwe
mtbofH
Tom
Mmamp1
83MPP1 INDUCES p21WAF1/Cip1 AND INHIBITS CELL PROLIFERATION
etect DNA fragmentation or TUNEL-positive stain-ng. We also could not block cell death with peptidenhibitors of caspases (Fig. 5), which are thought to benvolved in almost all paradigms of neuronal apoptosis.
Compared to the toxicity of MPP1 against dopami-ergic neurons in mesencephalic cultures, only sub-tantially higher concentrations of MPP1 show effectsn PC12 cells. MPP1 is toxic to dopaminergic neuronsf mesencephalic cultures at concentrations as low as.5 to 2.0 mM [30, 31], but shows effects in PC12 cellsnly at concentrations exceeding 100 mM. We reportere that MPP1 arrests cell cycle progression at con-entrations of 100 mM and higher but does not affectell survival for 96 h at concentrations lower than 250M. The requirement of high MPP1 concentrations to
nduce death in PC12 cells supports earlier reports, inhich 1 to 3 mM treatments for 24 h were necessary to
nduce cell death [32, 33]. Adrenal chromaffin cellsxpress a high-affinity catecholamine uptake systemnd have been demonstrated to accumulate [3H]MPP1
ith relatively little toxicity [34]. It has been arguedhat adrenal chromaffin cells may be relatively resis-ant to MPP1 due to a reserpine-sensitive sequestra-ion of the drug within a distinct intracellular compart-ent [34, 35]. These and our data are in sharp contrast
o a report by Hartley et al. [12], who provided evidencehat MPP1 induces apoptosis in PC12 cells at concen-rations ranging from 10 to 100 mM as detected mor-hologically by electron microscopy and biochemicallyy DNA fragmentation and DNA laddering on agaroseel electrophoresis. At concentrations higher than 100M, the predominant mode of cell death was necrosis.fter treatment with 250 mM MPP1 for 48 h theybserved a 60% LDH release relative to the total LDHctivity. There may be substantial differences betweenifferent PC12 cell lines used.We show that inhibition of complex I of the electron
ransport chain can be compensated by glycolysis ands apparently not sufficient to kill PC12 cells. MPP1
FIG. 10. MPP1 induces expression of p21WAF1/Cip1 but not of p53.reatment with 250 mM MPP1 time dependently induces expressionf p21 (top) but not of p53 (bottom). In contrast, treatment with 0.5M doxorubicin induces expression of p53.
C12 cells which died only after energy substratesere depleted. As suggested earlier [33] supplying morenergy substrates led to a longer survival (Fig. 7).The cell cycle-related response of PC12 cells to treat-ent with MPP1 is similar to their response to NGF
reatment. In the presence of serum, NGF induces alock in the G1 phase of the cell cycle and upregulationf p21WAF1/Cip1 protein levels [26, 36]. Removal of serumrom differentiated PC12 cells results in loss of p21WAF1/Cip1.owever, this loss of p21WAF1/Cip1 expression has no effect
FIG. 11. Expression of p21WAF1/Cip1 blocks apoptosis induced byPP1. (A) Expression of p21WAF1/Cip1 in p211/1, p211/2, and p212/2
ouse embryo fibroblasts. (B) Effects of 1 mM MPP1 on growthrrest depends on p21WAF1/Cip1 expression in p211/1, p211/2, and p212/2
ouse embryo fibroblasts. ***P , 0.001 compared with untreated211/1 cells. (C) Effects of p21WAF1/Cip1 on the treatment of MEFs withmM MPP1.
on differentiation or the nonproliferative state in theppIrsbwtpgIn
MMtctMbdacp
bDtJ
9. Tipton, K. F., and Singer, T. P. (1993). Advances in our under-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
84 SOLDNER ET AL.
resence of NGF, suggesting that p21WAF1/Cip1 serves inart to neutralize the mitogenic stimulus of serum [26].n the absence of the mitogenic signal, cells no longerequire this response and could use other means totop or prevent cell cycle progression. Using mouse em-ryo fibroblasts with varying expression of p21WAF1/Cip1
e show here that p21WAF1/Cip1 expression after MPP1
reatment is required to inhibit cell proliferation and torevent apoptosis. Since MPP1 leads to the samerowth arrest in SH-SY5Y p0 cells, in which no complexactivity is detectable, this block in cell proliferation isot due to inhibition of complex I.This paper provides evidence for a new mechanism ofPP1 toxicity. We demonstrate that in PC12 cellsPP1 does not primarily lead to cell death by inhibi-
ion of oxidative phosphorylation but interferes withell cycle progression. Survival of cells may depend onheir ability to induce p21WAF1/Cip1 expression. SincePTP/MPP1 toxicity in humans and animals resem-
les PD in many aspects, including the selectivity foropaminergic neurons, the major clinical syndrome,nd complex I inhibition of the electron transporthain, these results may lead to new insights into theathogenesis of PD.
We thank Dr. Tyler Jacks for providing p21-deficient mouse em-ryo fibroblasts. This study was supported by grants from theeutsche Forschungsgemeinschaft (Kl 782/3-1) and from the for-
une-program of the University of Tubingen (Project 214) to T.K. and.B.S.
REFERENCES
1. Beal, M. F. (1995). Aging, energy and oxidative stress in neu-rodegenerative diseases. Ann. Neurol. 38, 357–366.
2. Schapira, A. H. V. (1994). Evidence for mitochondrial dysfunc-tion in Parkinson’s disease: A critical appraisal. Mov. Disord. 9,125–138.
3. Schulz, J. B., and Beal, M. F. (1994). Mitochondrial dysfunctionin movement disorders. Curr. Opin. Neurol. 7, 333–339.
4. Mochizuki, H., Goto, K., Mori, H., and Mizuno, Y. (1996). His-tochemical detection of apoptosis in Parkinson’s disease.J. Neurol. Sci. 137, 120–123.
5. Anglade, P., Vyas, S., Javoy-Agid, F., Herrero, M. T., Michel,P. P., Marquez, J., Mouatt-Prigent, A., Ruberg, M., Hirsch,E. C., and Agid, Y. (1997). Apoptosis and autophagy in nigralneurons of patients with Parkinson’s disease. Histol. His-topathol. 12, 25–31.
6. Kosel, S., Egensperger, R., Voneitzen, U., Mehraein, P., andGraeber, M. B. (1997). On the question of apoptosis in theparkinsonian substantia nigra. Acta Neuropathol. 93, 105–108.
7. Wullner, U., Kornhuber, J., Weller , M., Schulz, J. B., Losch-mann, P.-A., Riederer, P., and Klockgether, T. (1998). Celldeath and apoptosis regulating proteins in Parkinson’s dis-ease—A cautionary note. Acta Neuropathol. 97, 408–412.
8. Bloem, B. R., Irwin, I., Buruma, O. J. S., Haan, J., Roos,R. A. C., Tetrud, J. W., and Langston, J. W. (1990). The MPTPmodel: Versatile contributions to the treatment. J. Neurol. Sci.97, 273–293.
standing of the mechanisms of the neurotoxicity of MPTP andrelated compounds. J. Neurochem. 61, 1191–1206.
0. Chan, P., DeLanney, L. E., Irwin, I., Langston, J. W., andDiMonte, D. (1991). Rapid ATP loss caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mouse brain. J. Neuro-chem. 57, 348–351.
1. Storey, E., Hyman, B. T., Jenkins, B., Brouillet, E., Miller,J. M., Rosen, B. R., and Beal, M. F. (1992). 1-Methyl-4-phe-nylpyridinium produces excitotoxic lesions in rat striatum as aresult of impairment of oxidative metabolism. J. Neurochem.58, 1975–1978.
2. Hartley, A., Stone, J. M., Heron, C., Cooper, J. M., and Scha-pira, A. H. V. (1994). Complex I inhibitors induce dose-depen-dent apoptosis in PC12 cells. J. Neurochem. 63, 1987–1990.
3. Du, Y., Dodel, R. C., Bales, K. R., Jemmerson, R., Hamilton-Byrd, E., and Paul, S. M. (1997). Involvement of caspase-3-likecysteine protease in 1-methyl-4-phenylpyridinium-mediatedapoptosis of cultured cerebellar granule neurons. J. Neuro-chem. 69, 1382–1388.
4. Desole, M. S., Sciola, L., Delogu, M. R., Sircana, S., Migheli, R.,and Miele, E. (1996). Role of oxidative stress in the manganeseand 1-methyl-4-(2’-ethylphenyl)-1,2,3,6-tetrahydropyridine-induced apoptosis in PC12 cells. Neurochem. Int. 31, 169–176.
5. Marongiu, M. E., Piccardi, M. P., Bernardi, F., Corsini, G. U.,and Del Zompo, M. (1988). Evaluation of the toxicity of thedopaminergic neurotoxins MPTP and MPP1 in PC12 pheochro-mocytoma cells: Binding and bilogical studies. Neurosci. Lett.94, 349–354.
6. Itano, Y., Kitamura, Y., and Nomura, Y. (1994). 1-Methyl-4-phenylpyridinium (MPP1)-induced cell death in PC12 cells:inhibitory effects of several drugs. Neurochem. Int. 25, 419–424.
7. Miller, S. W., Trimmer, P. A., Parker, W. D. Jr., and Davis, R. E.(1996). Creation and characterization of mitochondrial DNA-depleted cell lines with “neuronal-like” properties. J. Neuro-chem. 67, 1897–1907.
8. King, M. P., and Attardi, G. (1989). Human cells lackingmtDNA: Repopulation with exogenous mitochondria by comple-mentation. Science 246, 500–503.
9. Brugarolas, J., Chandrasekaran, C., Gordon, J. I., Beach, D.,Jacks, T., and Hannon, G. J. (1995). Radiation-induced cellcycle arrest compromised by p21 deficiency. Nature 377, 552–557.
0. Schulz, J. B., Bremen, D., Reed, J. C., Lommatzsch, J.,Takayama, S., Wullner, U., Loschmann, P. A., Klockgether, T.,and Weller, M. (1997). Cooperative interception of neuronalapoptosis by BCL-2 and BAG-1 expression: Prevention ofcaspase activation and reduced production of reactive oxygenspecies. J. Neurochem. 69, 2075–2086.
1. Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. (1992). Iden-tification of programmed cell death in situ via specific labelingof nuclear DNA fragmentation. J. Cell Biol. 119, 493–501.
2. Dolbeare, F., Gratzner, H., Pallavicini, M. G., and Gray, J. W.(1983). Flow cytometric measurement of total DNA content andincorporated bromodeoxyuridine. Proc. Natl. Acad. Sci. USA80, 5573–5577.
3. Troy, C. M., Stefanis, L., Prochiantz, A., Greene, L. A., andShelanski, M. L. (1996). The contrasting roles of ICE familyproteases and interleukin-1b in apoptosis induced by trophicfactor withdrawal and by copper/zinc superoxide dismutasedown-regulation. Proc. Natl. Acad. Sci. USA 93, 5635–5640.
4. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., andElledge, S. J. (1993). The p21 Cdk-interacting protein Cip1 is a
potent inhibitor of G1 cyclin-dependent kinases. Cell 75, 805–
2
2
2
2
2
3
31. Sanchez-Ramos, J., Barrett, J. N., Goldstein, M., Weiner, W. J.,
3
3
3
3
3
RR
85MPP1 INDUCES p21WAF1/Cip1 AND INHIBITS CELL PROLIFERATION
816.5. Yan, G.-Z., and Ziff, E. B. (1995). NGF regulates the PC12 cell
cycle machinery through specific inhibition of the cdk kinasesand induction of cyclin D1. J. Neurosci. 15, 6200–6212.
6. van Grunsven, L. A., Billon, N., Savatier, P., Thomas, A.,Urdiales, J. L., and Rudkin, B. B. (1996). Effect of nerve growthfactor on the expression of cell cycle regulatory proteins in PC12cells: Dissection of the neurotrophic response from the antimi-togenic response. Oncogene 12, 1347–1356.
7. Michieli, P., Chedid, M., Lin, D., Pierce, J. H., Mercer, W. E.,and Givol, D. (1994). Induction of WAF1/CIP1 by a p53-inde-pendent pathway. Cancer Res. 54, 3391–3395.
8. Macleod, K. F., Sherry, N., Hannon, G., Beach, D., Tokino, T.,Kinzler, K., Vogelstein, B., and Jacks, T. (1995). p53-dependentand independent expression of p21 during cell growth, differ-entiation, and DNA damage. Genes Dev. 9, 935–944.
9. Halevy, O., Novitch, B. G., Spicer, D. B., Skapek, S. X., Rhee, J.,Hannon, G. J., Beach, D., and Lassar, A. B. (1995). Correlationof terminal cell cycle arrest of skeletal muscle with induction ofp21 by MyoD. Science 267, 1018–1021.
0. Krieglstein, K., Suter-Crazzolara, C., Fischer, W. H., and Un-sicker, K. (1995). TGF-b superfamily members promote sur-vival of midbrain dopaminergic neurons and protect themagainst MPP1 toxicity. EMBO J. 14, 736–742.
eceived November 19, 1998
and Hefti, F. (1986). 1-Methyl-4-phenylpyridinium (MPP1) butnot 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) se-lectively destroys dopaminergic neurons in cultures of dissoci-ated rat mesencephalic neurons. Neurosci. Lett. 72, 215–220.
2. Greene, L. A., and Rein, G. (1977). Release, storage and uptakeof catecholamines by a clonal cell line of nerve growth factor(NGF) responsive pheo-chromocytoma cells. Brain Res. 129,247–263.
3. Denton, T., and Howard, B. D. (1987). A dopaminergic cell linevariant resistant to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. J. Neurochem. 49, 622–630.
4. Reinhard, J. F., Diliberto Jr., E. J., Viveros, O. H., and Daniels,A. J. (1987). Subcellular compartmentalization of 1-methyl-4-phenylpyridinium with catecholamines in adrenal medullarychromaffin vesicles may explain the lack of toxicity to adrenalchromaffin cells. Proc. Natl. Acad. Sci. USA 84, 8160–8164.
5. Liu, Y., Roghani, A., and Edwards, R. H. (1992). Gene transferof a reserpine-sensitive mechanism of resistance to N-methyl-4-phenylpyridinium. Proc. Natl. Acad. Sci. USA 89, 9074–9078.
6. Poluha, W., Schonhoff, C. M., Harrington, K. S., Lachyankar,M. B., Crosbie, N. E., Bulseco, D. A., and Ross, A. H. (1997). Anovel, nerve growth factor-activated pathway involving nitricoxide, p53, and p21WAF1 regulates neuronal differentiation ofPC12 cells. J. Biol. Chem. 272, 24002–24007.
