Caspase-3-Dependent Proteolytic Cleavage of Protein Kinase C? Is
Essential for Oxidative Stress-Mediated Dopaminergic Cell Death
after Exposure to Methylcyclopentadienyl Manganese Tricarbonyl
Vellareddy Anantharam, Masashi Kitazawa, Jarrad Wagner, Siddharth Kaul, and Anumantha G. Kanthasamy
Parkinson Disorders Research Program, Department of Biomedical Sciences, Iowa Sate University, Ames, Iowa 50011
In the present study, we characterized oxidative stress-dependent
cellular events in dopaminergic cells after exposure to an organic
form of manganese compound, methylcyclopentadienyl manga-
nese tricarbonyl (MMT). In pheochromocytoma cells, MMT expo-
sure resulted in rapid increase in generation of reactive oxygen
species (ROS) within 5–15 min, followed by release of mitochon-
drial cytochrome C into cytoplasm and subsequent activation of
caspase-3 (15- to 25-fold), but not caspase-8, in a time- and
dose-dependent manner. Interestingly, we also found that MMT
exposure induces a time- and dose-dependent proteolytic cleav-
age of native protein kinase C? (PKC?, 72–74 kDa) to yield 41 kDa
catalytically active and 38 kDa regulatory fragments. Pretreatment
with caspase inhibitors (Z-DEVD-FMK or Z-VAD-FMK) blocked
MMT-induced proteolytic cleavage of PKC?, indicating that cleav-
age is mediated by caspase-3. Furthermore, inhibition of PKC?
activity with a specific inhibitor, rottlerin, significantly inhibited
caspase-3 activation in a dose-dependent manner along with a
reduction in PKC? cleavage products, indicating a possible posi-
tive feedback activation of caspase-3 activity by PKC?. The pres-
ence of such a positive feedback loop was also confirmed by
delivering the catalytically active PKC? fragment. Attenuation of
ROS generation, caspase-3 activation, and PKC? activity before
MMT treatment almost completely suppressed DNA fragmenta-
PKC?K376R(dominant-negative mutant) prevented MMT-induced
apoptosis in immortalized mesencephalic dopaminergic cells. For
the first time, these data demonstrate that caspase-3-dependent
proteolytic activation of PKC? plays a key role in oxidative stress-
mediated apoptosis in dopaminergic cells after exposure to an
environmental neurotoxic agent.
Key words: apoptosis; oxidative stress; Parkinson’s disease;
environmental factors; manganese; dopaminergic degeneration
Parkinson’s disease (PD) is an idiopathic neurodegenerative dis-
order characterized by profound loss of dopaminergic neurons in
the nigrostriatal tract. Although debated, most studies have con-
cluded that aging, environmental neurotoxicant exposures, and
genetic alterations are potential risk factors in the development of
PD (Oertel and Kupsch, 1993; Langsten and Hill, 1998; Aschner,
2000; Simon et al., 2000). Recently, a study conducted on
thousands of twins concluded that genetic factors do not play a role
in the pathogenesis of geriatric onset of PD, which further supports
the view that environmental factors are dominant risk factors
in the etiology of PD (Tanner et al., 1999). Results of several
epidemiological studies conducted in rural areas have also sug-
gested that certain pesticides and other environmental factors,
including transition metals such as manganese, have a positive
association with increased incidences of PD (Seidler et al., 1996;
Liou et al., 1997; Gorell et al., 1999). Occupational exposure to
manganese during mining was shown to cause a Parkinson’s-like
syndrome known as Manganism (Mena et al., 1967; Barbeau, 1984;
Donaldson, 1987; Gorell et al., 1999). Furthermore, exposure to
manganese-containing compounds such as manganese ethylene-
bis-dithiocarbamate (a fungicide) and Bazooka (a cocaine-based
drug) among farm workers and abusers, respectively, has been
shown to result in adverse neurological defects (Roels et al., 1987;
Ferraz et al., 1988; Wang et al., 1989; Thiruchelvam et al., 2000).
Methylcyclopentadienyl manganese tricarbonyl (MMT) has
been used in Canada as an anti-knock gasoline agent and has
been recently legalized for use in the United States as a replace-
ment for tetraethyl lead [(CH3CH2)4Pb] in gasoline (Lynam et al.,
1999; Zayed et al., 1999). Because MMT is a manganese-
containing compound, its use has raised great a concern regarding
increased exposure to the public and its possible adverse health
effects (Frumkin and Solomon, 1997; Davis, 1998; Lynam et al.,
1999; Zayed et al., 1999). Exposure to MMT produces a pro-
longed and more pronounced accumulation of manganese in rat
brain as compared with manganese derived from an inorganic
source, for example, MnCl2(Zheng et al., 2000). Administration
of MMT produces seizures in mice (Fishman et al., 1987) and
also results in depletion of dopamine in the mouse striatum
(Gianutsos and Murray, 1982). Furthermore, MMT administra-
tion has been shown to be an effective inhibitor of complex I in
mitochondrial electron transport chain (Autissier et al., 1977), an
action similar to the pyridinium metabolite of 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP), a Parkinsonian toxin. Re-
cently, we demonstrated that MMT exposure induces reactive
oxygen species (ROS) generation, dopamine depletion, and cell
death in dopamine-producing rat pheochromocytoma (PC12)
Received Oct. 26, 2001; revised Dec. 7, 2001; accepted Dec. 12, 2001.
This work was supported by the National Institute of Environmental Health
Sciences Grant RO1-ES10586. We acknowledge Dr. Palur Gunasekar (Operational
Toxicology, Air Force Research Laboratories, Dayton, OH) for his initial assistance
in some experiments. We thank Dr. Michael L. Kirby, Dr. Arthi Kanthasamy, and
Mr. Siddharth Ranade in the preparation of this manuscript and Dr. Donghui Cheng
for help with flow cytometry.
Correspondence should be addressed to Dr. A. G. Kanthasamy, Parkinson
Disorders Research Laboratory, Department of Biomedical Sciences, 2062 Vet-
erinary Medicine Building, Iowa Sate University, Ames, IA 50011. E-mail:
J. Wagner’s present address: Department of Chemistry, California State Univer-
sity, Fresno, CA.
Copyright © 2002 Society for Neuroscience 0270-6474/02/221738-14$15.00/0
The Journal of Neuroscience, March 1, 2002, 22(5):1738–1751
cells, which can be protected by pretreatment with antioxidants
(Wagner et al., 2000). To further understand the cellular mech-
anism of MMT-mediated apoptosis, we investigated whether
oxidative stress induced by MMT can activate a series of cellular
factors associated with apoptotic pathways, which could subse-
quently lead to programmed cell death in dopaminergic cells.
Herein, we report that MMT exposure activates a novel apoptotic
pathway in dopaminergic cells through caspase-3-dependent pro-
teolytic cleavage of PKC?.
MATERIALS AND METHODS
Reagents. MMT was obtained from Sigma-Aldrich (St. Louis, MO);
rottlerin was purchased from Calbiochem (San Diego, CA); acetyl-Asp-
Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), acetyl-Iso-Glu-Thr-Asp-7-
amino-4-methylcoumarin (Ac-IETD-AMC), acetyl-Leu-Glu-His-Asp-7-
amino-4-methylcoumarin (Ac-LEHD-AMC), and Z-Asp-Glu-Val-Asp-
fluoromethyl ketone (Z-DEVD-FMK) were obtained from Alexis
Biochemicals (San Diego, CA); Z-Val-Ala-Asp-fluoromethyl ketone (Z-
VAD-FMK) was obtained from Enzyme Systems (Livermore, CA).
was obtained from Bachem (King of Prussia, PA); fluorescein isothiocya-
nate conjugated to VAD-FMK (FITC-VAD-FMK) was purchased from
Promega (Madison, WI); antibodies to PKC?, PKC?, PKC?I, and PKC?II
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), cyto-
chrome C (mouse monoclonal) from PharMingen (San Diego, CA), green
fluorescent protein (GFP) (mouse monoclonal) from Clontech (Palo Alto,
CA), and ?-actin (mouse monoclonal) from Sigma (St. Louis, MO). ECL
chemiluminescence kit was purchased from Amersham Pharmacia Biotech
(Piscataway, NJ). PC12 cells were purchased from American Type Culture
Collection (ATCC) (Rockville, MD), and immortalized rat mesencephalic
dopaminergic neuronal cell line (1RB3AN27) was a kind gift of Dr. Kedar
N. Prasad (University of Colorado Health Sciences Center, Denver, CO).
Hydroethidine and Hoechst 33342 were purchased from Molecular Probes
(Eugene, OR). Cell Death Detection ELISA Plus assay kit was purchased
from Roche Molecular Biochemicals (Indianapolis, IN). PKC? catalytic
fragment, acridine orange, histone H1, ?-glycerophosphate, superoxide
dismutase (SOD), ATP, Protein-A-Sepharose, phosphatidylserine, and dio-
leoylglycerol were purchased from Sigma. Mn(III)tetrakis(4-Benzoic acid-
)porphyrin chloride (MnTBAP) was purchased from Oxis Health Products
(Portland, OR). [?-32P]ATP was purchased from NEN (Boston, MA).
Bradford protein assay kit was purchased from Bio-Rad (Hercules, CA).
Lipofectamine Plus reagent, Roswell Park Memorial Institute (RPMI)-
1640 medium, horse serum, fetal bovine serum, L-glutamine, penicillin,
streptomycin, and PCEP4 plasmid were purchased from Invitrogen (Gaith-
ersburg, MD). BioPORTER, protein delivery reagent was purchased from
Gene Therapy Systems (San Diego, CA), and plasmids PKC?K376-GFP
fusion protein and pEGFP-N1 were kind gifts of Dr. Stuart Yuspa (Na-
tional Cancer Institute, Bethesda, MD).
Cell culture. PC12 (ATCC CRL1721) cells were grown in RPMI
medium supplemented with 10% horse serum, 5% fetal bovine serum,
1% L-glutamine, penicillin (100 U/ml), and streptomycin (100 U/ml) and
maintained at 37°C in a humidified atmosphere of 5% CO2. Immortal-
ized rat mesencephalic cells (1RB3AN27) were grown in RPMI medium
supplemented with 10% fetal bovine serum, 1% L-glutamine, penicillin
(100 U/ml), and streptomycin (100 U/ml), maintained at 37°C in a
humidified atmosphere of 5% CO2(Prasad et al., 1998).
Stable transfection. Plasmid pPKC?K376R-GFP encodes protein kinase
C?-GFP fusion protein, the number K376R refers to the mutation of
lysine residue at position 376 to arginine in the catalytic site of PKC?
rendering it inactive (Li et al., 1999). Plasmid pEGFP-NI encodes the
green fluorescent protein alone and used as vector control. pEGFP-N1
and pPKC?K376R were transfected into 1RB3AN27cells using Lipo-
fectamine Plus reagent according to the procedure recommended by the
manufacturer. In brief, 8 ?g of DNA, 24 ?l of lipid, and 24 ?l of Plus
reagent were used to transfect 1RB3AN27cells in 100 mm tissue culture
dishes at 50% confluency in 4 ml of culture medium without serum. Fresh
medium containing serum was added 3 hr later. For stable cell lines, the
1RB3AN27cells were selected in 400 ?g/ml hygromycin, 48 hr after
cotransfection with PCEP4 plasmid, which confers hygromycin resis-
tance. Colonies were isolated with trypsin and glass cloning cylinders,
and they were then replated and grown to confluence in T75 flasks.
Subsequently, the stable cell lines were maintained in 200 ?g/ml
Treatment paradigm. After 2–4 d in culture, PC12 cells and 1RB3AN27
were harvested and resuspended in serum-free growth medium at a cell
density of 1–3 ? 106/ml. Cell suspensions were treated with varying
concentrations of MMT (30–500 ?M) over a period of 0.5–5 hr at 37°C.
In inhibitor studies SOD (ROS inhibitor, 100 U/ml), MnTBAP (ROS
inhibitor, 10 ?M), rottlerin (PKC? inhibitor, 5–20 ?M), Ac-DEVD-CHO
(caspase-3-specific inhibitor, 100–300 ?M), Z-DEVD-FMK (caspase-3-
specific inhibitor, 10–50 ?M), or Z-VAD-FMK (a broad spectrum
caspase inhibitor, 30–100 ?M) were added 30–90 min before the addition
of MMT. The reaction samples were removed at 0.25, 0.5, 1, 2, 3, and 5
hr, then spun at 200 ? g, and after 5 min, the cell pellets were used for
assessing cytochrome C release, caspase-3, caspase-8, and caspase-9
enzymatic activities, extent of PKC? cleavage, and DNA fragmentation.
Dimethylsulfoxide (DMSO) (0.5–1%) was used as a vehicle in control
Lactate dehydrogenase assay. Lactate dehydrogenase (LDH) activity in
the cell-free extracellular supernatant was quantified as an index of cell
death (Vassault, 1983). We modified the original method to a 96-well
format (Kitazawa et al., 2001). Briefly, PC12 cells were plated in 96-well
plate, and after treatment 10 ?l of the extracellular supernatant was
added to 200 ?l of 0.08 M Tris buffer, pH 7.2, containing 0.2 M NaCl, 0.2
mM NADH, and 1.6 mM sodium pyruvate. LDH activity was measured
continuously by monitoring the decrease in the rate of absorbance at 339
nm using a microplate reader (Molecular Devices, Sunnyvale, CA), and
the temperature was maintained at 37°C during reading. Changes in
absorbance per minute (?A/?T) were used to calculate LDH activity
(U/I), using the following equation: U/I ? (?A/?T) ? 9682 ? 0.66, where
9682 was a coefficient factor, and 0.66 was a correction factor at 37°C.
Detection of reactive oxygen species and lipid peroxidation by flow cytom-
etry. Flow cytometry analysis was performed on a Becton Dickinson (San
Francisco, CA) FACScan flow cytometer. Hydroethidine, a sodium
borohydride-reduced derivative of ethidium bromide, is used to detect
ROS produced specifically inside the cell (Narayanan et al., 1997). When
hydroethidine is loaded in the cells, it binds to cellular macromolecules.
and increases red fluorescence (620 nm). A 15 mW air-cooled argon–ion
laser was used as an excitation source for hydroethidine at 488 nm, and
the optical filter was 585/42 nm bandpass. Cells were detected and
distinguished from the background by forward-angle light scattering
and orthogonal light scattering characteristics. All the flow cytometric
data were analyzed by Cellquest data analysis software to determine the
significant increase or decrease of fluorescence intensity.
PC12 cells and engineered 1RB3AN27cells expressing kinase inactive
PKC? protein were resuspended with HBSS with 2 mM calcium at a
density of 0.5 ? 106cells/ml. Cells were then incubated with 10 ?M
hydroethidine for 15 min at 37°C in the dark to allow dye loading into the
cells. After incubation with dye, excess dye was removed, and the cells
were resuspended with HBSS. After addition of MMT (30–500 ?M) ROS
generation was measured at 0, 5, 15, 30, and 45 min after the exposure.
In inhibitor studies, cells were incubated with SOD (100 U/ml) and
MnTBAP (10 ?M) 10–30 min before MMT exposure.
Quantification of cytochrome C release. Cytochrome C release was
quantified using a recently developed ELISA kit developed by MBL
(Watertown, MA). This is a fast, highly sensitive and reliable assay for
the detection of early changes in cytochrome C levels. Briefly, after 2–4
d in culture, PC12 cells were harvested and resuspended in serum-free
growth medium at a cell density of 5 ? 106/ml. Cell suspensions were
exposed to 200 and 500 ?M MMT for 15–30 min at 37°C. After treatment
the cells were spun at 200 ? g, and after 5 min, washed once with 1?
ice-cold PBS and resuspended in 1 ml of ice-cold homogenization buffer
(10 mM Tris HCl, pH 7.5, 0.3 M sucrose, 1 mM phenylmethylsulfonyl
fluoride, 25 ?g/ml aprotinin, and 10 ?g/ml leupeptin) and homogenized
on ice. Cells were then centrifuged for 10,000 ? g for 60 min at 4°C. The
resulting supernatants were collected as cytoplasmic fraction and used
for cytochrome C release measurements. The MBL ELISA kit measures
cytochrome C by one-step sandwich ELISA. The assay uses affinity-
purified two polyclonal antibodies against cytochrome C. The cytoplas-
cytochrome C polyclonal antibody in the 96-well microtiter for 60 min at
room temperature (RT). After washing with buffer (provided with the
kit), the peroxidase substrate is mixed with the chromogen and allowed
to incubate for an additional 15 min. An acid solution provided with the
kit is then added to each well to terminate the enzyme reaction and to
stabilize the developed color. The optical density of each well is then
measured at 450 nm using a microplate reader. The concentration of
?is generated, it converts hydroethidine to ethidium bromide
Anantharam et al. • MMT Cleaves Protein Kinase C?
J. Neurosci., March 1, 2002, 22(5):1738–1751 1739
cytochrome C is calibrated from a standard curve based on reference
Confocal analysis of in situ caspase activity. For this study, we used
CaspACE kit (Promega) to label PC12 cells. The kit uses FITC-VAD-
FMK, an FITC conjugate of the cell-permeable caspase inhibitor
Z-VAD-FMK, which binds to activated caspase and serves as an in situ
marker for apoptosis. The experiment was performed as per the manu-
facturer’s protocol with slight modifications. Briefly, PC12 cells were
grown on laminin (5 ?g/ml)-coated slides for 2–3 d in a 37°C, 5% CO2
incubator. Cells were then exposed to 200 ?M MMT for 1 hr in the dark.
After exposure, the cells were treated with 10 ?M FITC-VAD-FMK for
20 min at 37°C. Cells were then rinsed with 1? PBS and fixed in 10%
buffered formalin for 30 min at RT in the dark. After fixing, the cells
were washed three times with PBS to remove formalin and then mounted
with medium and coverslips, and observed under a Leica TCS-NT
confocal microscope (Leica Microsystems Inc., Exton, PA).
Enzymatic assay for caspases. Caspase-3, caspase-8, and caspase-9
activities were performed as previously described by Yoshimura et al.
(1998). Briefly, after treatment cells were spun and the cell pellets were
lysed with Tris buffer, pH 7.4 (50 mM Tris HCl, 1 mM EDTA, and 10 mM
EGTA) containing 10 ?M digitonin for 20 min at 37°C. Lysates were
centrifuged at 900 ? g for 3 min, and the resulting supernatants were
incubated with specific fluorogenic caspase substrates at 37°C for 1 hr.
Ac-DEVD-AMC (50 ?M), Ac-IETD-AMC (50 ?M), and Ac-LEHD-
AMC (50 ?M) were used as substrates for determining caspase-3-,
caspase-8-, and caspase-9-like protease activities, respectively. Levels of
cleaved (active) caspase substrate were monitored at excitation ? 380 nm
and emission ? 460 nm using a fluorescence plate reader (model: Fluo-
roskan-11; Titertek). Caspase activities were expressed as fluorescence
units per milligram of protein per hour. The protein concentrations were
determined using the Bio-Rad protein assay kit.
Isolation of cytoplasmic fractions. After incubation, the PC12 cells were
spun at 200 ? g for 5 min. Cell pellets were then washed once with
ice-cold Ca2?-free PBS saline and resuspended in 2 ml of homogeniza-
tion buffer (20 mM Tris-HCl, pH 8.0, 10 mM EGTA, 2 mM EDTA, 2 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 ?g/ml aprotinin,
and 10 ?g/ml leupeptin). The suspensions were sonicated for 10 sec, and
centrifuged at 100,000 ? g for 1 hr at 4°C. The supernatants were
collected as cytosolic fractions. Protein concentration of each sample was
determined, and the SDS-gel electrophoresis was performed as described
Western blotting. Cytoplasmic fractions containing equal amounts of
protein were loaded in each lane and separated on a 10–12% SDS-
polyacrylamide gel. Proteins were then transferred to nitrocellulose
membrane by electroblotting for 75–90 min at 100 V. Nonspecific binding
sites were blocked by treating the nitrocellulose membranes with 5%
nonfat dry milk powder for 2 hr before treatment with primary antibod-
ies. The nitrocellulose membranes containing the proteins were incu-
bated with primary antibodies for 1 hr at RT with antibody directed
against PKC? (1:2000 dilution), PKC? (1:2000 dilution), cytochrome C
(1:2000 dilution), or GFP (1:1000 dilution). The primary antibody treat-
ments were followed by treatment with secondary HRP-conjugated anti-
rabbit or anti-mouse IgG (1:2000 dilution) for 1 hr at RT. Secondary
antibody-bound proteins were detected using an ECL chemilumines-
cence kit (Amersham). To confirm equal protein loading, blots were
reprobed with a ?-actin antibody (1:5000 dilution). Gel photographs were
taken with a gel imaging system and quantification of bands was per-
formed using the imaging software from Scion Corp. (Frederick, MD).
Immunoprecipitation kinase assays. PKC? enzymatic activity was as-
sayed using an immunoprecipitation kinase assay as described by Rey-
land et al. (1999) and Vancurova et al. (2001). Briefly, after treatment
with MMT, PC12 cells were washed once with PBS and resuspended in
1 ml of PKC lysis buffer (25 mM HEPES, pH 7.5, 20 mM
?-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1% Triton X-100,
0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 10 mM NaF,
and 4 ?g/ml each aprotonin and leupeptin). In inhibition experiments,
cells were pretreated with 10 ?M rottlerin before the addition of 200 ?M
MMT. The cell lysates were allowed to sit on ice for 30 min and
centrifuged at 13,000 ? g for 5 min, and the supernatants were collected
as cytosolic fraction. Protein concentration was determined using a
Bradford assay. Cytosolic protein (0.25–0.5 mg) was immunoprecipitated
overnight at 4°C using 2 ?g of anti-PKC?, anti-PKC?, anti-PKC?I, or
anti-PKC?II antibodies. The immunoprecipitates were then incubated
with Protein-A Sepharose (Sigma) for 1 hr at 4°C. The protein A bound
antigen–antibody complexes were then washed three times with PKC
lysis buffer, three times with 2? kinase buffer (40 mM Tris, pH 7.4, 20 mM
MgCl2,20 ?M ATP, and 2.5 mM CaCl2), and resuspended in 20 ?l of 2?
kinase buffer. Reaction was started by adding 20 ?l of reaction buffer
containing 0.4 mg Histone H1, 50 ?g/ml phosphatidylserine, 4.1 ?M
dioleoylglycerol, and 5 ?Ci of [?-32P] ATP (3000 Ci/mM) to the immu-
noprecipitated samples and incubated for 10 min at 30°C. SDS gel-
loading buffer (2?) was added to terminate the reaction, the samples
were boiled for 5 min, and the products were separated on a 12.5%
SDS-PAGE gel. For in vitro inhibition of PKC? kinase activity, 5–20 ?M
rottlerin was added to 200 ?M MMT-treated immunoprecipitated sample
15 min before the addition of 2? reaction buffer containing 5 ?Ci of
[?-32P] ATP (3000 Ci/mmol). The H1 phosphorylated bands were de-
tected using a Personal Molecular Imager (FX model; Bio-Rad), and
quantification was done using Quantity One 4.2.0 software.
Intracellular delivery of PKC? catalytic fragment. Intracellular delivery
of PKC? fragment was performed using a recently developed lipid-
mediated delivery system (BioPORTER; Gene Therapy Systems, San
Diego, CA). This is a fast and reliable procedure that delivers proteins in
a functionally active form into the cytoplasm of cells (Zelphati et al.,
2001). The protein delivery system is composed of a new trifluoroacety-
lated lipopolyamine (TFA-DODAPL) and dioleoyl phosphatidylethano-
lamine. This cationic formulation has recently been used for delivery of
various bioactive molecules, including antibodies, enzymes (caspase-3,
caspase-8, ?-galactosidase, and granzyme B), cytochrome C, dextran
sulfates, phycobiliproteins, and albumins into the cytoplasm of numerous
adherent and suspension cells (Zelphati et al., 2001). Active PKC?
catalytic fragments were delivered into cells using the protein delivery
reagent by following the manufacturer’s protocol. PC12 cells (?1–2 ?
105cells/ml) were subcultured in 24-well tissue culture plate for 24 hr.
PKC? catalytic fragment (5 ng) was mixed with 3 ?l of protein delivery
reagent and 300 ?l of serum-free DMEM media and added to each well.
The cells were incubated at 37°C for 4 hr. Cells were then lysed, and
caspase-3 activity measured as described above. Heat-inactivated PKC?
catalytic fragment was used as negative control and inactivation was
performed by incubating the active PKC? fragment at 95°C for 15 min.
The delivery efficiency was ?70% in PC12 cells as determined using a
FITC-tagged antibody control (supplied with the assay kit). Also, the
protein delivery system produced no significant cytotoxic response as
measured by Trypan blue dye exclusion method.
In situ assessment of apoptosis. To assess nuclear morphology and DNA
damage, we stained the cells with fluorescent DNA-binding dyes acridine
orange and Hoechst 33342. Acridine orange, a useful probe for detecting
apoptotic cells, exhibits metachromatic fluorescence that is sensitive to
DNA conformation. Apoptotic cells stained with acridine orange show
reduced green and enhanced red fluorescence in comparison with nor-
mal cells (Pulliam et al., 1998). Briefly, PC12 cells were grown on laminin
(5 ?g/ml)-coated slides for 2–3 d in a 37°C, 5% CO2incubator. Cells
were washed twice with PBS and after 1 hr treatment with 200 ?M MMT,
the cells were incubated with 10 ?M acridine orange for 15 min at RT in
the dark. The cells were again washed with PBS, mounted with cover-
slips, and observed under a Nikon DiaPhot microscope, and pictures
were captured with a SPOT digital camera (Diagnostic Instruments,
Sterling Heights, MI).
Morphological changes associated with apoptosis were also assessed by
staining with Hoechst 33342. Cells stained with Hoechst 33342 dye
fluoresce bright blue after binding to DNA in the nucleus. The nucleus
of apoptotic cells exhibit strong blue staining and staining pattern is
heterogeneous and occurs in patches, indicative of chromatin condensa-
tion, whereas the nucleus of nonapoptotic cells exhibit more diffused,
weak and homogenous staining (Shimizu et al., 1996; Du et al., 1997).
Briefly, PC12 cells were plated on collagen (6 ?g/cm2)-coated cover
slides and treated with 200 ?M MMT. After 1 hr of exposure, the cells
were fixed with 10% buffered formaldehyde for 30 min at room temper-
ature and stained with Hoechst 33342 (10 ?g/ml) for 3 min in dark. The
cells were again washed three times with PBS, mounted with coverslips,
and observed under a Nikon DiaPhot microscope under UV illumina-
tion, and pictures were captured with a SPOT digital camera (Diagnostic
Quantification assay for DNA fragmentation. DNA fragmentation assay
was performed using a recently developed Cell Death Detection ELISA
Plus assay kit. This is a fast, highly sensitive and reliable assay for the
detection of early changes in apoptotic cell death and measures the
appearance and amount of histone-associated low molecular weight
DNA in the cytoplasm of cells. This assay has been recently used in
quantitation of apoptosis because of its reliability and high sensitivity
1740 J. Neurosci., March 1, 2002, 22(5):1738–1751Anantharam et al. • MMT Cleaves Protein Kinase C?
(Reyland et al., 1999). Briefly, PC12 and engineered 1RB3AN27cells
were exposed to 200–500 ?M MMT for 1–3 hr. In inhibitor studies, SOD
(100 U/ml), MnTBAP (10 ?M), rottlerin (10 ?M), Z-DEVD-FMK (50
?M), or Z-VAD-FMK (100 ?M) were treated for 30 min at 37°C before
the addition of MMT. After MMT treatment, cells were spun down at
200 ? g for 5 min and washed once with 1? PBS. Cells were then
incubated with a lysis buffer (supplied with the kit) at RT. After 30 min,
samples were centrifuged, and 20 ?l aliquots of the supernatant were
then dispensed into streptavidin-coated 96-well microtiter plates fol-
lowed by addition of 80 ?l of antibody cocktail and incubated for 2 hr at
RT with mild shaking. The antibody cocktail consisted of a mixture of
anti-histone biotin and anti-DNA-HRP directed against various histones
and antibodies to both single-stranded DNA and dsDNA, which are
major constituents of the nucleosomes. After incubation, unbound com-
ponents were removed by washing with the incubation buffer supplied
with the kit. Quantitative determination of the amount of nucleosomes
retained by anti-DNA-HRP in the immunocomplex was determined
spectrophotometrically with 2,2?-azino-di-(3-ethylbenzthiazoline sulfo-
nate (6)) diammonium salt (ABTS) as an HRP substrate (supplied with
the kit). Measurements were made at 405 nm against an ABTS solution
as a blank (reference wavelength ?490 nm) using a Molecular Devices
Spectramax Microplate Reader.
Data analysis. Data analysis was performed using Prism 3.0 software
(GraphPad Software, San Diego, CA). Data from caspase enzymatic
activities and DNA fragmentation assays were first analyzed using one-
way ANOVA. Neuman–Keuls or Dunnett’s post-tests were then per-
formed to compare control with MMT-treated groups, and differences
with p ? 0.05 were considered significant. For individual comparisons,
student t test or Welch’s corrected t test where appropriate was used.
PC12 cells were exposed to 200 and 500 ?M MMT for varying
amounts of time. The amount of LDH released into the extra-
cellular media was measured as an index of cytotoxicity (Vassault,
1983; Kanthasamy et al., 1995). Extracellular LDH activity
showed a dose- and time-dependent increase with MMT treat-
ment ranging from 2- to 20-fold over the control group (Fig. 1).
For example, exposure to 200 ?M MMT resulted in 3-, 10-, and
17-fold increase in LDH release over untreated cells at 1, 3, and
5 hr, respectively. To determine the percent cell death, total LDH
content of untreated cells (?2000 U/I) was normalized to 100%.
PC12 cells exposed to 200 ?M MMT for 3 hr produced ?50% cell
death. Similarly, exposure to 500 ?M MMT resulted in a signifi-
cant ( p ? 0.05) toxicity in MMT-treated groups as compared
with vehicle-treated cells at all time points. There were no sig-
nificant differences in LDH release between vehicle-treated and
untreated PC12 cells during the 5 hr exposure. These data suggest
that MMT induces a dose- and time-dependent cell death in
Generation of ROS after MMT treatment
Flow cytometric analysis using the ROS-sensitive fluorescence
probe hydroethidine revealed that MMT treatment induces ROS
generation. Figure 2A depicts a representative flow cytometric
histogram of 200 ?M MMT-treated PC12 cells exhibiting time-
dependent increases in red fluorescence. MMT treatment in-
creased ROS production in a dose- and time-dependent manner
(Fig. 2B). For example, a 15 min exposure to 30, 100, and 200 ?M
MMT resulted in a 10, 41, and 76% increase in ROS production,
respectively. Exposure to 200 ?M MMT resulted in 40, 76, and
80% increase in ROS production over vehicle treatment at 5, 15,
and 30 min, respectively. The time course study also revealed that
500 ?M MMT treatment induced a rapid and dramatic increase in
ROS generation (?200% of control) within 5 min and then the
response rapidly declined over time (data not shown). Pretreat-
ment with SOD (100 U/ml) or MnTBAP (SOD mimetic, 10 ?M)
significantly ( p ? 0.05) reduced MMT-induced ROS production,
indicating that MMT predominantly generates superoxide spe-
cies (Fig. 2C).
Accumulation of cytochrome C in the cytosol after
ROS production in the cells is known to activate many cellular
factors including cytochrome C, which subsequently triggers ap-
optotic cell death (Tan et al., 1998; Cassarino et al., 1999).
Release of cytochrome C from the mitochondria into the cyto-
plasm is an early event that occurs during programmed cell death
(Muller-Hocker, 1992; Crompton, 1999), and therefore we deter-
mined whether MMT induces release of cytochrome C in PC12
cells. Figure 3A shows a time-dependent increase of cytochrome
C in the cytoplasmic fractions of PC12 cells treated with MMT.
No detectable levels of cytochrome C were detected in the cytosol
of vehicle (DMSO)-treated cells up to 3 hr, whereas a profound
release of cytochrome C was observed as early as 1 hr in MMT-
treated cells. Nitrocellulose membranes were reprobed with
?-actin antibody, and the density of 43 kDa ?-actin bands was
identical in all lanes confirming equal protein loading. To further
accurately quantify how soon cytochrome C is released, we used
a highly sensitive cytochrome C ELISA sandwich assay. MMT
exposure resulted in a dose-dependent increase in cytosolic cy-
tochrome C as early as 15 min (Fig. 3B). A 15 min exposure to 200
and 500 ?M MMT resulted in an increase in cytosolic cytochrome
C by 40 and 200% over the vehicle-treated group, respectively,
and a 30 min exposure resulted in an increase in cytosolic cyto-
chrome C by 70 and 170%, respectively. The reason for the
decrease in amount of cytochrome C released at 30 min after 500
?M MMT exposure might be attributed to loss of cellular integ-
rity caused by the observed necrotic cell death (Fig. 1).
Activation of caspase-3 and caspase-9 but not
caspase-8 after MMT treatment
Because the release of cytochrome C is known to activate a group
of cysteine proteases, namely caspases (Cohen, 1997; Earnshaw et
al., 1999; Schultz and Andreasen, 1999; Jellinger, 2000), we ex-
amined whether caspase-8, caspase-9, and caspase-3 are activated
during MMT exposure. PC12 cells exposed to MMT showed a
200 and 500 ?M of MMT for 0.5–5 hr at 37°C. After the exposure, cell-free
extracellular supernatants were collected, and LDH activity was mea-
sured by spectrophotometer. Values represent mean ? SEM for three to
five separate experiments in triplicate. Significance was determined by
ANOVA followed by Dunnett’s post-test between the vehicle-treated
group and each treatment group (*p ? 0.05).
MMT exposure induces cell death. PC12 cells were exposed to
Anantharam et al. • MMT Cleaves Protein Kinase C?
J. Neurosci., March 1, 2002, 22(5):1738–1751 1741
significant increase in caspase-9 activity, however, no significant
increase in caspase-8 enzyme activity was observed (data not
shown). A 30 min exposure to 200 and 500 ?M MMT produced a
threefold and twofold increase in caspase-9 activity, respectively.
The lack of dose–response in caspase-9 enzymatic activity at 500
fluorescent intensity was measured at 0, 5, 15, and 30 min. Data represent
the mean ? SEM of two to five separate experiments in triplicate.
Asterisks (*p ? 0.5 and **p ? 0.01) indicate significant differences
compared with the time-matched vehicle-treated cells. C, Effect of SOD
and MnTBAP on ROS production. Cells were pretreated with ROS
inhibitors, SOD (100 U/ml) and MnTBAP (10 ?M), and then exposed to
100 or 200 ?M MMT for 15 min. The value of each treatment group is the
mean ? SEM from two to three separate experiments performed in
triplicate. Asterisks (*p ? 0.05) indicate significant differences compared
with MMT-treated cells.
suspended in HBSS supplemented with 2 mM Ca2?at a density of
0.5–0.75 ? 106cells/ml. A concentration of 10 ?M hydroethidine was
added to the cells and incubated for 15 min at 37°C in the dark. A,
Time-dependent change in hydroethidine fluorescent intensity in PC12
cells treated with MMT. A concentration of 200 ?M MMT was added, and
fluorescent intensity was measured at 0, 15, and 30 min by flow cytometry
as described in Materials and Methods. The data are a representative flow
cytometric histogram of MMT-treated PC12 cells exhibiting a time-
dependent increase in red fluorescence. B, Dose- and time-dependent
increase in ROS production. Various doses of MMT were added, and
MMT treatment generates ROS in PC12 cells. PC12 cells were
chrome C in MMT-treated PC12 cells. A, Western blot. B, Cytochrome C
ELISA assay. A, Subconfluent cultures of undifferentiated PC12 cells were
harvested at 1 and 3 hr after treatment with 200 or 500 ?M MMT. The
cytosolic fractions were obtained as described in Materials and Methods.
Cytosolic fractions were separated by 12% SDS-PAGE, transferred to a
nitrocellulose membrane, and cytochrome C (Cyt C) was detected using
polyclonal antibody raised against cytochrome C. For ?-actin measure-
ments, the membrane used for cytochrome C was reprobed with ?-actin
antibody to confirm equal protein loading in each lane. The immunoblots
were visualized using ECL detection agents from Amersham. B, Subcon-
fluent cultures of undifferentiated PC12 cells were harvested at 15 and 30
min after treatment with 200 or 500 ?M MMT. The cytosolic fractions
were obtained as described in Materials and Methods. The value of each
treatment group is the mean ? SEM from two separate experiments in
triplicate. Asterisks (*p ? 0.05) indicate significant differences compared
with vehicle-treated cells.
Dose- and time-dependent accumulation of cytosolic cyto-
1742 J. Neurosci., March 1, 2002, 22(5):1738–1751 Anantharam et al. • MMT Cleaves Protein Kinase C?
?M MMT concentration is probably caused by acute cytotoxic
effects of the toxic compound at higher doses.
MMT treatment in PC12 cells resulted in dramatic increase in
caspase-3 enzymatic activity (Fig. 4). After exposure to 200 ?M
MMT, caspase-3-specific activity was increased 12-, 17-, 7-, and
6-fold over the vehicle-treated groups at 0.5, 1, 2, and 3 hr after
treatment, respectively (Fig. 4A). Similarly, exposure to 500 ?M
MMT resulted in an increase in caspase-3 specific activity by 20-,
27-, 20-, and 6-fold over the vehicle-treated groups after 0.5, 1, 2,
and 3 hr exposure, respectively. The overall pattern of the time
course study of caspase-3 activity revealed a clear pattern of dra-
matic increase in enzyme activity peaking at 1 hr and then pro-
gressively decreasing over time, returning nearly to that of vehicle-
treated cells at 3 hr. To further confirm the activation of caspase-3,
in situ fluorometric analysis was performed using FITC-VAD-
FMK in live cells. In these experiments, we found majority of PC12
cells were labeled within 1 hr of exposure to 200 ?M MMT,
indicating a profound increase in caspase-3 activity in situ, whereas
no labeling was seen in vehicle-treated cells (Fig. 4B).
Proteolytic cleavage of protein kinase C? but not
PKC? by MMT
Recent studies have indicated PKC? to be one of the endogenous
substrates for caspase-3, which cleaves the kinase to yield a 41
kDa catalytically active and a 38 kDa regulatory PKC? fragments
in non-neuronal cell lines, salivary gland acinar cells, (Reyland et
al., 1999), rat fibroblasts (Dal Pra et al., 1999), and neutrophils
(Pongracz et al., 1999). Because MMT exposure resulted in a
profound activation of caspase-3, we decided to examine the
proteolytic cleavage of PKC? in MMT-treated PC12 cells. After
treatment of PC12 cells with MMT at 37°C, we observed over a 5
hr period a significant proportion of native PKC? (72–74 kDa)
protein was proteolytically cleaved to yield 38 kDa regulatory and
41 kDa catalytically active fragments when immunoblotted with
an antibody raised against PKC? (Fig. 5A). The time course study
revealed that almost all of the native PKC? (72–74 kDa) protein
was cleaved within 3 hr of incubation with MMT, evidenced by a
reduction in the intensity of the native 72–74 kDa band and a
concomitant increase in the catalytically active 41 kDa cleaved
fragment. PC12 cells exposed to increasing concentrations of
MMT (200 and 500 ?M) showed a dose-dependent cleavage of
PKC?. However, no cleavage of PKC? was observed in vehicle-
treated cells during the entire 5 hr experimental time period at
the doses tested. Nitrocellulose membranes were reprobed with
?-actin antibody, and the density of 43 kDa ?-actin band was
identical in all lanes confirming equal protein loading.
MMT-induced proteolytic cleavage of PKC was also isoform-
specific, because exposure of PC12 cells to 200 or 500 ?M MMT
enzymatic activity. B, In situ caspase-3 activity. A, Subconfluent cultures
of undifferentiated PC12 cells were harvested at 30 min, 1, 2, and 3 hr
after MMT treatment. Caspase-3 activity was assayed using specific
fluorogenic substrate, Ac-DEVD-AMC (50 ?M), as described in Materi-
als and Methods. The data represent mean ? SEM of nine individual
measurements from three separate experiments. Asterisks (**p ? 0.01;
*p ? 0.05) indicate significant differences compared with temporally
matched vehicle (DMSO)-treated cells. B, PC12 cells were grown on
laminin-coated slides for 2–3 d and then exposed to 0.5% DMS0 (vehicle)
and 200 ?M MMT for 1 hr in the dark. After exposure, cells were treated
with 10 ?M FITC-VAD-FMK (Promega caspACE, in situ marker for
caspase-3 activity) and processed as described in Materials and Methods.
Confocal images were obtained using a Leica TCS-NT microscope.
MMT treatment increases caspase-3 activity. A, Caspase-3
treated PC12 cells. A, PKC?; B, PKC?. Subconfluent undifferentiated
PC12 cells were harvested at 1, 3, and 5 hr after treatment of 200 or 500
?M MMT. Cytosolic fractions were obtained as described in Materials
and Methods, and were separated by 10% SDS-PAGE, transferred to
nitrocellulose membrane, and PKC? and PKC? were detected using
antibodies directed against their catalytic subunits. To confirm equal
protein loading in each lane, the membranes were reprobed with ?-actin
antibody. The immunoblots were visualized using ECL detection agents
from Amersham. C, PKC? catalytic subunit; R, PKC? regulatory subunit.
Proteolytic cleavage of PKC? but not of PKC? in MMT-
Anantharam et al. • MMT Cleaves Protein Kinase C?
J. Neurosci., March 1, 2002, 22(5):1738–1751 1743
for up to 5 hr failed to induce proteolytic cleavage of PKC? (Fig.
5B). Membranes were reprobed with ?-actin antibody, and the
density of 43 kDa ?-actin band was identical in all lanes confirm-
ing equal protein loading. Additionally, MMT exposure did not
result in the translocation of either PKC? or ? from the cytoplasm
to the membrane for their activation (data not shown).
MMT-induced proteolytic cleavage of PKC?
To further confirm that PKC? cleavage is mediated by caspase-3,
we used caspase-3-specific inhibitors Ac-DEVD-CHO (Fig. 6A),
Z-DEVD-FMK (Fig. 6B) or a broad-spectrum caspase inhibitor,
Z-VAD-FMK (Fig. 6A), to block the cleavage. Pretreatment of
PC12 cells for 30 min with any of the three inhibitors used here
before 3 hr exposure of cells to 200 ?M MMT prevented the
appearance of the 41 kDa catalytically active PKC? fragment, and
effects of all three inhibitors were dose-dependent. Furthermore,
Z-DEVD-FMK and Z-VAD-FMK appeared to be more potent
in blocking PKC? cleavage than Ac-DEVD-CHO. Membranes
were reprobed with ?-actin antibody (Fig. 6A,B), and the density
of 43 kDa ?-actin bands was identical in all lanes confirming equal
Rottlerin blocks MMT-induced caspase-3 enzymatic
activity: possible feedback activation of caspase-3
As reported above, we observed a dramatic increase in caspase-3
(6- to 27-fold) (Fig. 4A) enzymatic activity in MMT-treated PC12
cells at 1 hr after treatment. These results prompted us to deter-
mine the cause for the dramatic increase in MMT-induced
caspase-3 activity, and so we investigated whether PKC? is capa-
ble of activating caspase-3 by a positive feedback mechanism.
To address this hypothesis, we tested the ability of PKC? spe-
cific inhibitor rottlerin to modulate caspase-3 activity in MMT-
treated PC12 cells by pre- and post-treatment. Pretreatment
with rottlerin 30 min before the addition of 200 ?M MMT
suppressed caspase-3 activity in a dose-dependent manner (Fig.
7A). Rottlerin at 5, 10, and 20 ?M suppressed MMT-induced
caspase-3 activity by 38, 64, and 70%, respectively, whereas the
basal caspase-3 activity was not altered by treatment with rot-
tlerin alone. Post-treatment with rottlerin 30 min after the addi-
tion of 200 ?M MMT also suppressed caspase-3 activity in a
dose-dependent manner (Fig. 7B). Rottlerin at 5 and 20 ?M
suppressed MMT-induced caspase-3 activity by 60 and 98%,
respectively, whereas the basal caspase-3 activity was unaltered by
post-treatment with rottlerin alone. The extent of MMT-induced
caspase-3 inhibition by 5 ?M rottlerin was not statistically signif-
icant ( p ? 0.05) between pre- and post-treatments, whereas
inhibition with 20 ?M rottlerin post-treatment was very significant
( p ? 0.01) as compared with pre-treatment, reducing the
caspase-3 activity to almost the basal level. Overall, these results
indicate that there may be a positive feedback activation of
caspase-3 by PKC?, and this activation can be blocked by
Rottlerin blocks caspase-3 mediated proteolytic
cleavage of PKC?
Because the pretreatment study with rottlerin blocked caspase-3
enzymatic activity, we further tested whether rottlerin pretreat-
ment attenuates caspase-3-dependent proteolytic cleavage of
PKC?. Pretreatment with rottlerin before the addition of 200 ?M
MMT prevented the accumulation of PKC? cleavage product in a
dose-dependent manner (Fig. 8). Rottlerin at 20 ?M markedly
reduced the appearance of PKC? cleavage product in PC12 cells
exposed to 200 ?M MMT, indicating that the activation of PKC?
is essential to its cleavage by caspase-3. Membranes were re-
probed with ?-actin antibody (Fig. 8), and the density of 43 kDa
?-actin band was identical in all lanes, confirming equal protein
Rottlerin inhibits MMT-induced increases in PKC?
kinase activity in PC12 cells
To determine whether the MMT induced caspase-3- and PKC?-
dependent accumulation of PKC? cleaved product is attributed to
an increase in PKC? enzyme activity, we performed kinase assays
in immunoprecipitated samples from cytosolic fractions using
PKC? specific polyclonal antibody and by examining the ability
of PKC? to phosphorylate histone H1. The enzymatic activity of
PKC? increased after 1 hr exposure to MMT in dose-dependent
manner (Fig. 9A). Densitometric analysis of phosphorylated his-
tone H1 bands revealed a three-fold and five-fold increase in
protein kinase activity in cells exposed to 200 and 500 ?M MMT
for 1 hr, respectively, and was coincident with generation of PKC?
cleavage. We attribute this increased kinase activity to the per-
sistently active PKC? catalytic fragment, because activation of
MMT-treated PC12 cells. A, Effect of Ac-DEVD-CHO and Z-VAD-
FMK on PKC? cleavage. B, Effect of Z-DEVD-FMK on PKC? cleavage.
Subconfluent undifferentiated PC12 cells were treated with 200 ?M MMT,
with or without the inclusion of caspase inhibitors Ac-DEVD-CHO,
Z-VAD-FMK, or Z-DEVD-FMK. Inhibitors were added 30 min before
the addition of MMT. Cells were harvested 3 hr after the addition of
MMT. The cytosolic fractions were obtained as described in Materials
and Methods, and were analyzed by 10% SDS-PAGE and Western blot.
To confirm equal protein loading in each lane, the membranes were
reprobed with ?-actin antibody. C, PKC? catalytic subunit; R, PKC?
Caspase-3 mediates the proteolytic cleavage of PKC? in
1744 J. Neurosci., March 1, 2002, 22(5):1738–1751 Anantharam et al. • MMT Cleaves Protein Kinase C?
intact PKC? by translocation to the membrane does not occur
during MMT treatment (data not shown). There was no increase
in kinase activity of PKC?, PKC?I, and PKC?II in immunopre-
cipitated samples of treated cells (data not shown) suggesting that
the MMT-induced increase in kinase activity is isoform specific
for PKC?. Pretreatment with 10 ?M rottlerin resulted in 80%
reduction in the kinase activity (Fig. 9B), suggesting that the
activation of PKC? is essential for MMT-induced increases in
kinase activity, and this may be facilitated via the positive-
feedback activation of caspase-3 by the catalytically active PKC?
Rottlerin directly inhibits PKC? kinase activity in vitro
Rottlerin was originally reported to inhibit PKC? kinase activity
by competing for the ATP-binding site (Gschwendt et al., 1994).
This inhibitor has been used to implicate PKC? in a variety of
cellular events, including apoptosis (Chen et al., 1999; Reyland et
al., 1999; Dempsey et al., 2000; Way et al., 2000; Basu et al., 2001;
Vancurova et al., 2001). To further confirm the inhibitory potency
of rottlerin on PKC? activity, we tested various concentrations of
rottlerin on PKC? enzyme activity using an in vitro kinase assay.
PKC? was immunoprecipitated from MMT-treated cytosolic frac-
tions using PKC? specific polyclonal antibody and incubated with
rottlerin in vitro for 15 min before the addition of histone H1 and
[32P]ATP. For the in vitro reaction, we used same rottlerin con-
centrations that blocked MMT-stimulated PKC? kinase activity
in intact PC12 cells. Rottlerin at 5, 10, and 20 ?M inhibited PKC?
activity in vitro by 63, 77, and 84%, respectively (Fig. 9C), and is
consistent with rottlerin inhibition of MMT-induced PKC? activ-
ity in intact PC12 cells (Fig. 9B). Our data are also in agreement
with previously published values for direct inhibition of PKC? by
rottlerin in vitro kinase assays (Gschwendt et al., 1994; Way et al.,
2000; Vancurova et al., 2001).
Activation of caspase-3 after intracellular delivery of
PKC? catalytic fragment
To further confirm the existence of a positive feedback loop
between caspase-3 and proteolytic cleavage PKC?, we investi-
gated the effect of intracellular delivery of PKC? catalytic frag-
ment on caspase-3 activity in PC12 cells. We used a recently
developed lipid-mediated delivery system to introduce the cata-
lytically active PKC? fragment into the cytoplasm of PC12 cells
(Zelphati et al., 2001). The cells were treated with the delivery
reagent with or without the PKC? catalytic fragment for 4 hr at
37°C. As shown in Table 1, PC12 cells delivered with PKC?
catalytic fragment showed increases in caspase-3 activity to 341%
of reagent control. Neither the intracellular delivery of heat-
inactivated PKC? catalytic fragment nor the delivery reagent
alone produced any increases in caspase-3 enzymatic activity.
These results strongly suggest that catalytic fragment of PKC? is
capable of mediating caspase-3 activation, further supporting our
hypothesis that proteolytic cleavage of PKC? can augment
caspase-3 activity by a positive feedback loop during MMT
PC12 cells. A, Pre-treatment; B, post-treatment. Subconfluent undifferen-
tiated PC12 cells were treated with 200 ?M MMT with or without the
inclusion of rottlerin (Rot; 5–20 ?M) for 1 hr. Rottlerin was added 30 min
before or 30 min after the addition of MMT. Caspase-3 activity was
assayed using Ac-DEVD-AMC (50 ?M) as substrate, as described in
Materials and Methods. The data represent an average of four to nine
individual measurements from two or three separate experiments ?
SEM. Asterisk (*p ? 0.05) indicates significant difference compared with
cells exposed to 200 ?M MMT.
Suppression of caspase-3 activity by rottlerin in MMT-treated
MMT-treated PC12 cells. Subconfluent undifferentiated PC12 cells were
treated with 200 ?M MMT with or without the inclusion of rottlerin (5–20
?M). Rottlerin was added 90 min before the addition of MMT. Cytosolic
fractions were obtained as described in Materials and Methods and were
separated by 10% SDS-PAGE, transferred to nitrocellulose membrane,
and PKC? was detected using an antibody directed against its catalytic
subunit. The immunoblots were visualized using ECL detection agents
from Amersham. To confirm equal protein loading in each lane, the
membranes were reprobed with ?-actin antibody. C, PKC? catalytic
subunit; R, PKC? regulatory subunit.
Rottlerin pretreatment blocks proteolytic cleavage of PKC? in
Anantharam et al. • MMT Cleaves Protein Kinase C?
J. Neurosci., March 1, 2002, 22(5):1738–1751 1745
In situ fluorometric detection of apoptosis
To understand the functional consequence of the activation of
many apoptotic factors, we tested whether MMT induces DNA
fragmentation. Chromosomal breakdown of DNA into 200 bp
nucleosomal fragments and DNA condensation are hallmarks of
cells undergoing apoptosis. We used in situ fluorometric analysis
to identify apoptotic cells using acridine orange and Hoechst
33342 to detect nuclear condensation and DNA damage after
MMT treatment. In these experiments, we found the majority of
PC12 cells exposed to 200 ?M MMT for 1 hr showed enhanced
red fluorescence and reduced green fluorescence, suggesting that
acridine orange dye is bound to single-stranded or highly con-
densed DNA (Fig. 10B), whereas little or no enhanced red
fluorescence was seen in vehicle-treated cells (Fig. 10A). Simi-
larly, the nucleus of PC12 cells exposed to 200 ?M MMT for 1 hr
and subsequently stained with Hoechst 33342 dye showed nuclear
condensation as the dye bound to the highly condensed DNA
(Fig. 10D). The Hoechst 33342 staining of vehicle-treated cells
showed a weak and diffused staining, indicating that there is no
nuclear condensation in these cells (Fig. 10C).
Oxidative stress, caspase-3, and PKC? mediate
MMT-induced DNA fragmentation
To further confirm the results obtained by in situ fluorometric
detection of live apoptosis and to assess the involvement of
caspases and PKC? in mediating apoptosis, a quantitative DNA
fragmentation assay was performed. PC12 cells treated with 200
?M MMT showed DNA fragmentation within 1 hr of exposure
(Fig. 11). MMT treatment resulted in more than a twofold in-
crease over the levels of basal (vehicle-treated) DNA fragmenta-
tion. Pretreatment with an ROS inhibitor, SOD (100 U/ml),
almost completely blocked MMT-induced DNA fragmentation
(Fig. 11A), indicating that SOD is capable of blocking MMT-
induced apoptosis. To further confirm the anti-apoptotic effect of
SOD in MMT treatment, a cell-permeable SOD mimetic, MnT-
BAP, was used. Pretreatment with 10 ?M MnTBAP also almost
completely attenuated MMT-induced apoptosis (Fig. 11A). SOD,
but not MnTBAP, when treated alone also significantly attenu-
ated the basal apoptosis in vehicle-treated PC12 cells.
Pretreatment for 30 min with PKC? inhibitor rottlerin (10 ?M)
completely prevented 200 ?M MMT-induced DNA fragmentation
(Fig. 11B). Similarly, pretreatment with caspase inhibitors
Z-DEVD-FMK (50 ?M) or Z-VAD-FMK (100 ?M) almost com-
pletely blocked MMT-induced DNA fragmentation (Fig. 11B).
Rottlerin, Z-DEVD-FMK, and Z-VAD-FMK when treated
alone did not significantly attenuate the basal apoptosis in
vitro. A, Dose-dependent increase in PKC? activity. B, Rottlerin sup-
presses MMT-induced increase in PKC? kinase activity in intact cells. C,
Rottlerin inhibits PKC? kinase activity in vitro. Subconfluent undifferen-
tiated PC12 cells were treated with 200 ?M MMT for 1 hr at 37°C with or
without the inclusion of rottlerin (Rot; 5–20 ?M). Rottlerin was added 30
min before the addition of MMT. For in vitro inhibition of PKC? activity,
rottlerin (5–20 ?M) was added to the immunoprecipitated samples from
MMT-treated cells and incubated for 30 min before the addition of
substrate (histone H1) and [?-32P]ATP. The immunoprecipitation kinase
assay was performed as described in Materials and Methods. The bands
were quantified by a PhosphoImager after scanning the dried gel and
expressed as a percentage of control (untreated cells) (A), percentage of
MMT treatment (B), or percentage of PKC? kinase activity (C). The
data represent an average of three individual measurements from two
separate experiments ? SEM. Asterisks (*p ? 0.05) indicate significant
differences compared with control, MMT-treated cells, or PKC? kinase
Rottlerin inhibits PKC? kinase activity in intact cells and in in
Table 1. Activation of caspase-3 after intracellular delivery of PKC?
catalytic fragment in PC12 cells
protein/hr)% Reagent control
Catalytic active PKC?
25,293 ? 5726100
86,296 ? 34,116 341 ? 135*
29,814 ? 6851118 ? 27
*Asterisk indicates significant difference (p ? 0.05) compared with reagent control.
The data are given as the mean ? SEM from two separate experiments performed
1746 J. Neurosci., March 1, 2002, 22(5):1738–1751 Anantharam et al. • MMT Cleaves Protein Kinase C?
vehicle-treated PC12 cells, suggesting that caspase-3 and PKC?
are participants specifically in MMT-stimulated, and not basal
programmed cell death.
MMT treatment does not induce apoptosis in
mesencephalic cells overexpressing mutant
Pretreatment with a PKC?-specific inhibitor rottlerin significantly
reduced MMT-induced DNA fragmentation, supporting the idea
that the catalytic activity of PKC? enzyme is vital for induction of
apoptosis. If the kinase activity of PKC? is essential for apoptosis,
then overexpression of a kinase inactive PKC? mutant protein
should suppress MMT-induced DNA fragmentation, which oc-
curs downstream of caspase-3 dependent PKC? activation. Alter-
natively, overexpression of a kinase inactive PKC? mutant pro-
tein may not interfere with ROS production, an event that occurs
before caspase-3 dependent PKC? activation. To explore these
possibilities, we engineered a rat-immortalized mesencephalic
(1RB3AN27) cell line to express a dominant-negative PKC? mu-
tant by stably transfecting with plasmids pPKC?K376R-GFP (in
which a lysine at 376 position is mutated to arginine) and
pEGFP-N1 (Fig. 12A). The plasmid pPKC?K376R-GFP codes for
a catalytically inactive PKC? mutant fused to GFP and
pEGFP-N1 plasmid encodes the green fluorescent protein alone,
which was used as a vector control. Figure 12B shows stable GFP
expression in cell lines transfected with kinase inactive mutant
PKC?K376R-GFP and GFP alone. Antibody directed against GFP
detected ?100 and 27 kDa bands in cell lines expressing kinase
inactive mutant PKC?K376R-GFP and GFP alone, respectively.
Similarly, antibody directed against PKC? detected ?100 and 72
kDa bands in cell line expressing PKC?K376R-GFP fusion,
whereas only a 72 kDa band was detected in cells expressing GFP
alone. The 100, 72, and 27 kDa bands obtained in Western blots
correspond to the expression of intact mutant PKC?K376R-GFP
treated cells; B, D, 200 ?M MMT-treated cells. PC12 cells were grown on
laminin-coated slides for 2–3 d and then exposed to 200 ?M MMT for 1
hr. A, B, For acridine orange staining, cells were treated with acridine
orange (10 ?M) for 15 min in the dark at RT after exposure to MMT.
Arrows indicate enhanced red fluorescence and reduced green fluores-
cence in MMT-treated cells, which are undergoing apoptosis, whereas
little or no enhanced red fluorescence was seen in vehicle-treated cells. C,
D, For Hoechst 33342 staining, cells were stained with Hoechst 33342 (10
?g/ml) for 3 min in dark after exposure to MMT. Arrows indicate
apoptotic cells containing condensed chromatin.
MMT treatment increases apoptosis in situ. A, C, Vehicle-
ROS inhibitors, SOD and MnTBAP. B, Caspase-3 inhibitors, Z-VAD-
FMK and Z-DEVD-FMK and PKC? inhibitor, rottlerin. Subconfluent
cultures of undifferentiated PC12 cells were treated with MMT (200 ?M)
with or without the inclusion of the following inhibitors: ROS inhibitors
SOD (100 U/ml) or MnTBAP (10 ?M); caspase inhibitors Z-VAD-FMK
(100 ?M) or Z-DEVD-FMK (50 ?M); and PKC? inhibitor rottlerin (10
?M). Inhibitors were added 30 min before addition of MMT. Cells were
harvested 1 hr after MMT treatment. Apoptosis was assayed using ELISA
assay as described in Materials and Methods. The data are expressed as
percentage of apoptosis observed in vehicle-treated cells. The data rep-
resent the mean ? SEM of six individual measurements from three
separate experiments. Asterisks (*p ? 0.01) indicate significant differ-
ences when compared with cells exposed to 200 ?M MMT.
Suppression of MMT-induced apoptosis in PC12 cells. A,
Anantharam et al. • MMT Cleaves Protein Kinase C?
J. Neurosci., March 1, 2002, 22(5):1738–1751 1747
fusion protein, native PKC? and GFP protein, respectively. In
apoptotic measurements, MMT-induced DNA fragmentation was
completely abolished in cells stably expressing kinase inactive
PKC? protein but not in GFP-alone (vector) transfected
1RB3AN27cells (Fig. 12C). However, MMT-induced ROS pro-
duction was not significantly different between the kinase inactive
PKC?-GFP and GFP-alone expressing cell lines. A 15 min expo-
sure of 1RB3AN27cells stably expressing kinase inactive PKC?-
GFP and GFP alone to 200 ?M MMT resulted in a 157 ? 20%
and 148 ? 11% increase in ROS production, respectively. These
results suggest that the kinase activity of PKC? is essential for
MMT-induced DNA fragmentation. These data also indicate that
the suppression of apoptosis in PKC? dominant-negative cells
was not caused by a change in the amount of ROS generated in
the PKC?-GFP overexpressing cells versus GFP-vector cells.
We recently reported that exposure of PC12 cells to MMT
induces dopamine depletion and cytotoxic cell death in a dose-
and time-dependent manner (Wagner et al., 2000). The present
study extends these observations by demonstrating that MMT
induces apoptosis in dopamine-producing cells through ROS
production and activation of a series of specific cell death signal-
ing events, including release of cytochrome C into the cytosol,
activation of caspase-9 and caspase-3, proteolytic cleavage of
PKC?, and nuclear DNA breakdown. To our knowledge, this is
the first report demonstrating that caspase-3-dependent proteo-
lytic cleavage of PKC? mediates oxidative stress-induced apopto-
tic cell death in dopaminergic cells after exposure to an environ-
In this study, MMT treatment elevated intracellular ROS levels
over 45 min in a time- and dose-dependent manner. ROS gener-
ation was observed as early as 5 min after MMT exposure,
indicating that ROS generation precedes the cytotoxic response.
ROS has been shown to induce cytochrome C release from
mitochondria in both neuronal and non-neuronal systems by
activation of mitochondrial transition pore opening, which results
in swelling and rupturing of mitochondrial membrane (Liu et al.,
1996; Petit et al., 1996; Blackstone and Green, 1999; Hollens-
worth et al., 2000; Lee and Wei, 2000). We observed an accumu-
lation of cytosolic cytochrome C in PC12 cells within 15 min after
MMT treatment, suggesting that ROS may be an initial signal for
events in MMT-induced apoptosis. 1, Increased ROS production can be
blocked by pretreatment with antioxidants, superoxide dismutase and
MnTBAP; 2, cytochrome C is released into the cytosol from the mito-
chondria; 3, cytosolic cytochrome C activates caspase-9; 4, caspase-9
activates caspase-3; 5, caspase-3 mediates proteolytic cleavage of PKC?,
which can be blocked by pretreatment with the caspase inhibitors Ac-
DEVD-CHO, Z-VAD-FMK, and Z-DEVD-FMK; 6, pretreatment with
rottlerin, a PKC? inhibitor, reduces caspase-3 activity indicating a possi-
ble feedback activation; 7, both caspase-3 and PKC? inhibitors block
MMT-induced DNA fragmentation; and 8, dopaminergic cells stably
overexpressing catalytically inactive PKC? [dominant-negative mutant
(DNM) PKC?K376RGFP] completely blocked MMT-induced DNA frag-
mentation. In conclusion, our data suggest that caspase-3-dependent
proteolytic activation of PKC? plays a key role in MMT-induced dopa-
minergic cell death.
A model describing the sequence of cell death signaling
MMT-induced apoptosis in immortalized dopaminergic neuronal cell
line (1RB3AN27). A, Plasmid description, pEGFP-NI construct codes for
the green fluorescent protein (GFP) mRNA transcribed under the 5?
human cytomegalovirus (CMV) immediate early promoter, and the
mRNA is stabilized with the 3? SV40 mRNA polyadenylation signal ( pA)
and was used as vector control. PKC?K376R-GFP construct codes for the
kinase inactive PKC?-GFP fusion transcript. B, Stable expression of GFP
and PKC?K376R-GFP fusion protein in 1RB3AN27cells. The cells were
viewed under a fluorescence microscope, and images were obtained with
a SPOT digital camera. C, Subconfluent cultures of undifferentiated
1RB3AN27cells stably expressing vector or PKC?K376R-GFP fusion pro-
tein were treated with MMT (200 and 500 ?M) for 3 hr. Apoptosis was
assayed using ELISA assay as described in Materials and Methods. The
data are expressed as percentage of apoptosis observed in vehicle-treated
cells. The data represent a mean ? SEM of four to six individual
measurements from two separate experiments. Asterisks (*p ? 0.01)
indicate significant differences when compared with MMT-treated cells.
Overexpression of catalytically inactive PKC? protein blocks
1748 J. Neurosci., March 1, 2002, 22(5):1738–1751Anantharam et al. • MMT Cleaves Protein Kinase C?
the release of cytochrome C. Our data are also consistent with the
actions of other dopaminergic toxins, 1-methyl-4-phenylpyri-
dinium (MPP?) (Leist et al., 1998; Cassarino et al., 1999) and
6-hydroxydopamine (6-OHDA) (Dodel et al., 1999) in their abil-
ity to induce ROS-mediated cytochrome C release. Cytochrome
C, once released into the cytoplasm, forms a complex with apo-
ptotic protease activating factor, and together they activate a
series of caspases.
Activation of caspases by cytosolic cytochrome C is an early
and essential step in the apoptotic-signaling pathway (Earnshaw
et al., 1999; Jellinger, 2000). Several lines of evidence indicate
that caspase-3 plays a major role in the regulation and execution
phase of both in vitro and in vivo models of apoptosis (Cohen,
1997; Schultz and Andreasen, 1999). In this study, we demon-
strate that MMT exposure to PC12 cells results in a dramatic
activation of caspase-3, indicating that caspase-3 may play a key
role in MMT-induced dopaminergic degeneration. Our data are
further supported by a recent study in which caspase-3 activation
was observed in neuronal cultures after MPP?and 6-OHDA
treatment (Dodel et al., 1999). The importance of caspase-3
activation as an indicator of apoptosis is further underscored by
a recent study from Hartmann et al. (2000), who demonstrated
caspase-3 to be a vulnerability factor and a critical effector of
apoptotic death in dopaminergic neurons in both MPTP mouse
model and in human patients with Parkinson’s disease.
Biochemical consequences of caspase-3 activation are proteo-
lytic cleavage of cellular targets associated with apoptosis. Poly
(ADP-ribose) polymerase, a DNA cleaving enzyme, has been
established as one of the important apoptotic substrates of
caspase-3 (Earnshaw et al., 1999; Schultz and Andreasen, 1999).
In the present study, we demonstrated that PKC? is an emerging
putative endogenous substrate for caspase-3 and show that MMT
exposure induces PKC? cleavage and increases PKC? activity in
a dose- and time-dependent manner in dopaminergic cells. Ad-
ditionally, MMT does not induce cleavage of PKC?, suggesting
that the cleavage is isoform specific. Proteolytic cleavage of PKC?
in MMT-treated PC12 cells is blocked by specific caspase inhib-
itors, indicating that the cleavage is mediated by caspase-3. Pro-
teolytic cleavage of PKC? by caspase-3 results in persistent acti-
vation of PKC? in cytosol, which might initiate a myriad of vital
signaling cascades. In a previous study, proteolytic cleavage of
PKC? was observed in KCl-deprived cerebellar granule cell ap-
optosis, however, this study did not characterize the caspase-3
dependency of PKC? cleavage (Villalba, 1998). Recent studies
have additionally implicated the persistently active catalytic frag-
ment of PKC? in apoptotic cell death in non-neuronal systems
(Earnshaw et al., 1999; Schultz and Andreasen, 1999).
We further determined a possible interaction between PKC?
and caspase-3 activation in MMT-treated PC12 cells using the
PKC?-specific inhibitor rottlerin. Pre- and post-rottlerin treat-
ment effectively blocked MMT-induced caspase-3 activation in
PC12 cells in a dose-dependent manner, suggesting a positive
feedback modulatory role of PKC? on caspase-3 activity. Al-
though rottlerin suppressed MMT-induced caspase-3 activity in
both pre-and post-treatments (Fig. 7), the inhibition was more
pronounced in post-treatment at higher concentrations. The rea-
son for the pronounced inhibition is not completely clear at the
present time, and we attribute that this might be caused by action
of rottlerin on other cellular targets including other kinases
(Davies et al., 2000; Way et al., 2000). However, there was no
significant difference in caspase activity between pre- and post-
treatments of rottlerin at lower dose 5 ?M, the concentration at
which a pronounced inhibition of PKC? activity in vitro (Fig. 9C)
was observed. Furthermore, delivery of the catalytically active
PKC? fragment alone into PC12 cells increased the caspase-3
activity, confirming the presence of such a positive feedback
mechanism. It appears that maximal caspase-3 activity requires
the kinase activity of cleaved PKC? fragment and is made possi-
ble by the existence of a positive feedback activation loop. A
positive feedback loop between PKC? and caspase-3 activation
has recently been shown to exist in an etoposide-induced salivary
cell apoptosis model (Reyland et al., 1999). Thus, the existence of
such a positive feedback loop discovered independently by two
research groups in two different apoptotic models, MMT-induced
PC12 apoptosis (this study) and in etoposide-induced salivary cell
apoptosis, suggests that this may be an important regulatory
mechanism, allowing for the amplification of apoptotic signaling
processes. Further studies are needed to understand the cellular
mechanisms of caspase-3 regulation by PKC? and their role in
DNA fragmentation and condensation resulting from intranu-
cleosomal cleavage have long been considered biochemical hall-
marks of apoptosis and are terminal events in the apoptotic
process (Cohen, 1997). In situ fluorometric experiments using
two different fluorescent dyes revealed that MMT exposure of
PC12 cells induces chromatin condensation in the nucleus. We
also took advantage of a recently developed ELISA method that
provides a better quantitative measurement of DNA fragmenta-
tion. MMT exposure to PC12 cells induced DNA fragmentation,
which could be suppressed under conditions where caspase or
PKC? activities were inhibited. Suppression of MMT-induced
DNA fragmentation by either caspase inhibitors or rottlerin in the
present study indicates that both caspase-3 and PKC? activities
are essential for MMT-induced DNA fragmentation. Further-
more, caspase-3-mediated promotion of DNA fragmentation may
be amplified via feedback activation of caspase-3 by the catalyti-
cally active PKC? fragment. To additionally confirm the role of
PKC? in MMT-induced apoptosis, we conducted dominant-
negative experiments by stably expressing catalytically inactive
PKC? protein (PKC?K376R) in immortalized rat mesencephalic
neurons. MMT treatment produced a significant increase in DNA
fragmentation in vector control cells, whereas MMT failed to
induce DNA fragmentation in catalytically inactive PKC? over-
expressing cells, thus confirming the key functional role of PKC?
in MMT-induced apoptotic cell death.
Although the events downstream of PKC? and those that lead
to apoptosis remain unclear, recent studies from many research
groups have shown that catalytically active PKC? fragment can
regulate the activity of a host of cell signaling molecules such
as scrambalase, a membrane phosphatidylserine translocator
(Frasch et al., 2000), DNA protein kinase, a DNA repair enzyme
(Bharti et al., 1998), heat-shock proteins-25/27 (Maizels et al.,
1998), histone H2B (Ajiro, 2000), and lamin kinase (Cross et al.,
2000). In addition, PKC? has been shown to phosphorylate other
signaling molecules such as MAP kinases (Chen et al., 1999),
Jak2, a tyrosine kinase (Kovanen et al., 2000), and Stat3, signal
transducers and activators of transcription (Jain et al., 1999).
Most recently, it has been demonstrated that PKC? activates
redox-sensitive transcription factor, NF-?B, and thereby pro-
motes apoptosis in neutrophils (Vancurova et al., 2001). Further-
more, PKC? has been shown to translocate to cytosol and a
variety of cellular organelles to initiate apoptosis (Sawai et al.,
1997; Chen et al., 1999; Dal Pra et al., 1999; Li et al., 1999;
Dempsey et al., 2000; Majumder et al., 2000). Hence, constitu-
Anantharam et al. • MMT Cleaves Protein Kinase C?
J. Neurosci., March 1, 2002, 22(5):1738–1751 1749
tively active PKC? fragment can promote loss of cellular regula-
tory function in many of its substrates, resulting in rapid apopto-
sis. Currently, our laboratory is focusing on identifying critical
cellular targets of PKC? that might contribute to apoptotic cell
death in dopaminergic cells.
In conclusion, we demonstrate for the first time that an envi-
ronmental neurotoxicant, MMT, induces dopaminergic degener-
ation by a novel oxidative stress-mediated apoptotic mechanism
in which caspase-3-dependent proteolytic cleavage of PKC? plays
a critical role (Fig. 13). Our data also demonstrate a positive
feedback amplification loop between PKC? and capsase-3, which
has a regulatory role in the promotion of apoptosis. Further
research into identifying molecules that participate in this loop
might provide very exciting information regarding cell signaling
and neuronal apoptosis. Finally, this study emphasizes the im-
portance of characterizing oxidative stress-induced cell signaling
molecules after neurotoxicant exposure to better understand the
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