From the William T. Gossett Neurology Laboratories of
Henry Ford Hospital, Detroit, Michigan 48202, ¶ Cellular and
Clinical Neurobiology, Department of Psychiatry and Behavioral
Neurosciences, Wayne State University, Detroit, Michigan
48201, and
John D. Dingle Veterans Administration Medical
Center, Detroit, Michigan 48201
Received for publication, July 24, 2000, and in revised form, December 18, 2000
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ABSTRACT |
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(6R)-Tetrahydro-L-biopterin
(BH4) is the rate-limiting cofactor in the production of
catecholamine and indoleamine neurotransmitters and is also essential
for the synthesis of nitric oxide by nitric-oxide synthase. We have
previously reported that BH4 administration induces PC12
cell proliferation and that nerve growth factor- or epidermal
growth factor-induced PC12 cell proliferation requires the elevation of
intracellular BH4 levels. We show here that BH4 accelerates apoptosis in undifferentiated PC12 cells deprived of serum
and in differentiated neuron-like PC12 cells after nerve growth factor
withdrawal. Increased production of catecholamines or nitric oxide
cannot account for the enhancement of apoptosis by BH4.
Furthermore, increased calcium influx by exogenous BH4 administration is not involved in the BH4 proapoptotic
effect. Our data also argue against the possibility that increased
oxidative stress, due to BH4 autoxidation, is responsible
for the observed BH4 effects. Instead, they are consistent
with the hypothesis that BH4 induces apoptosis by
increasing cell cycle progression. Elevation of intracellular
BH4 during serum withdrawal increased c-Myc (and especially
Myc S) expression earlier than serum withdrawal alone. Furthermore,
N-acetylcysteine and the cyclin-dependent kinase inhibitor olomoucine ameliorated the BH4
proapoptotic effect. These data suggest that BH4 affects
c-Myc expression and cell cycle-dependent events, possibly
accounting for its effects on promoting cell cycle progression or apoptosis.
Apoptotic cell death is important for normal nervous system
development, where neurons that make proper connections and receive sufficient trophic support survive, whereas neurons that are deprived of trophic support die via apoptosis (for a review, see Ref. 1). The
requirement for neurotrophic support is thought to continue in mature
neurons (for a review, see Ref. 2). During normal aging and in
Parkinson's disease (PD),1
nigrostriatal dopamine neurons preferentially degenerate. The etiology
of this selective cell loss remains unknown. Local availability of
trophic factors is able to protect cells from degeneration in animal
models of PD (3-6), suggesting that lack of trophic support could
contribute to the observed neurodegeneration in normal aging and in PD.
Furthermore, morphological characteristics of apoptotic cell death have
been reported in brain sections from PD patients (7, 8), but the
involvement of apoptosis in PD is still controversial.
PC12 cells have been extensively used as a model of catecholaminergic
neurons in culture as well as for the study of neuronal apoptotic
death. Naive (undifferentiated) PC12 cells grown in the presence of
serum undergo apoptosis upon serum withdrawal (9-11). Furthermore,
PC12 cells that become neuronally differentiated and postmitotic
following prolonged incubation with NGF undergo apoptosis upon NGF and
serum withdrawal (9, 10). Undifferentiated PC12 cell death upon serum
withdrawal has been linked to an inappropriate progression through the
cell cycle, while apoptotic death of "neuronal" PC12 cells
following NGF withdrawal was linked to illegitimate cell cycle reentry
(12-15). This interpretation favors the hypothesis that during normal
cell growth trophic factors are required for proper cell cycle
progression, and in their absence cells die as they try to progress
through the cell cycle.
We have previously reported that
(6R)-tetrahydro-L-biopterin (BH4)
enhances PC12 cell growth by inducing cell cycle progression rather
than cell survival (16, 17). BH4 is known for its role as
an essential and rate-limiting cofactor in the synthesis of catecholamine and indoleamine neurotransmitters (18, 19) and nitric
oxide (20). Nevertheless, the induction of PC12 cell proliferation by
BH4 was not mediated by catecholamine or nitric oxide
synthesis (16, 21). In addition, the induction of PC12 cell
proliferation after a 24-h exposure to epidermal growth factor or nerve
growth factor (NGF) required the elevation of intracellular BH4 and activation of its initial biosynthetic enzyme, GTP
cyclohydrolase (17). Since BH4 enhanced PC12 cell cycle
progression and mediated the proliferative effect of NGF, we tested its
effects on apoptotic death of naive and neuronally differentiated PC12
cells and the effects on NGF-mediated PC12 cell survival.
Cell Culture
Rat PC12 cells were maintained in tissue culture flasks as
previously described (16). Logarithmically growing cells were harvested
by mechanical dislodging, and after centrifugation the pellet was
resuspended in Dulbecco's modified Eagle's medium (DMEM). Cell
viability was determined by trypan blue (0.01%) exclusion, and cells
were resuspended to the desired final density in the appropriate
medium and replated. 24 h later, test conditions were added
to the medium.
Models of PC12 Apoptotic Cell Death
Model A: Undifferentiated, Dividing PC12 Cells--
Apoptosis
was induced by serum withdrawal. 24 h following plating of
nonsynchronized, logarithmically growing PC12 cells in 75-cm2 tissue culture flasks (7 × 106
cells/flask), cells were washed twice with serum-free DMEM and incubated for 24 h in serum-free medium containing 1% bovine
serum albumin. Cells were also treated for 24 h with test
conditions in serum-free medium.
Model B: Differentiated, Neuron-like PC12 Cells--
PC12 cells
were plated in 75-cm2 tissue culture flasks (1 × 106 cells/flask) and incubated for 8 days in reduced serum
medium (DMEM ( Cell Counting
Cells were plated in 75-cm2 flasks (7 × 106 cells/flask), equilibrated for 24 h, incubated in
test conditions (10.36 × 106 cells/flask) for 24 h, and dislodged with trypsin. Cells were then pelleted and resuspended
in Dulbecco's phosphate-buffered saline without calcium and magnesium;
cell number was determined by direct counting using a hemacytometer. In
some cases, cell number was estimated by the use of the CellTiter 96 Aqueous cell proliferation assay by Promega. Control experiments
verified that color formation was dependent on cell number and was not
affected by intracellular BH4 or other treatments. In
contrast, intracellular BH4 levels affected color formation
when the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based CellTiter 96-cell proliferation assay (Promega) was used.
Results with the colorimetric assay were verified by direct cell counting.
DNA Fragmentation
PC12 cells were trypsinized and counted using a hemacytometer as
described earlier. Soluble, cytoplasmic DNA was extracted routinely
from 4 × 106 cells or in certain cases (to better
resolve DNA laddering) from 8 × 106 cells, run on
agarose gels, and visualized as previously described (22). Control
experiments excluded the possibility that genomic DNA fragmentation
occurred during cell counting. Similarly, trypsinization did not
adversely affect laddering, since identical results were obtained when
cytoplasmic DNA was directly extracted from plated cells and controlled
later for total protein levels.
Thymidine Incorporation
PC12 cells were cultured in 60-mm dishes (2 × 106 cells/dish), and 24 h later, cells were treated
with test conditions for an additional 24 h. Tritiated methyl
thymidine incorporation was measured as previously described (23).
Lactate Dehydrogenase Assay
PC12 cells were plated in 96-well plates at 15 × 103 cells/well. After 24 h, cells were washed twice
with serum-free DMEM and test conditions were added. Following a
24-h incubation, the activity of lactate dehydrogenase (LDH) was
measured in the medium as a measure of total dead, burst cells using a
kit from Promega (Cytotox 96). The results represent extracellular LDH
activity and were expressed as a percentage of the total LDH activity
in the well.
Quantitation of Soluble DNA
In some cases, cytotoxicity was also measured by the total
amount of double-stranded DNA released in the culture medium during incubation with test conditions. PicoGreen (Molecular Probes, Inc.,
Eugene, OR) was used for double-stranded DNA quantitation as previously
described (17).
In Vitro Hydrogen Peroxide Assay
Hydrogen peroxide generation was monitored both in
vitro and in PC12 cell medium in the presence or absence of
BH4 by dichlorofluorescin oxidation and subsequent
spectrophotometric analysis according to Royall and Ischiropoulos
(24).
Western Blot Analysis of c-Myc Expression
PC12 cells were cultured in test conditions for appropriate
incubation times and harvested at indicated times (1.5-24 h) by trypsinization. After cell counting, lysates were obtained by sonication at 4 °C in 20 mM Tris (pH 7.5), 50 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate,
0.5% SDS, 1 mM EDTA, and 0.5% aprotinin. Proteins (50 µl equivalent to 625,000 cells) were electrophoretically separated on
a SDS-polyacrylamide gel (7.5% separating, 4% stacking) and
electroblotted onto a nitrocellulose membrane (BA 85; Schleicher & Schuell). Immunodetection was performed by blocking in TBST (20 mM Tris (pH 7.5), 150 mM NaCl, 0.2% Tween 20)
containing 3% nonfat dry milk, incubating with affinity-purified
anti-c-Myc rabbit polyclonal antibody raised against the full-length
murine c-Myc (provided by Dr. S. R. Hann) and
peroxidase-conjugated goat anti-rabbit IgG (Jackson
Immunoresearch), followed by ECL detection (Amersham Pharmacia
Biotech). Control experiments established that trypsinization did not
adversely affect c-Myc protein levels or cleavage, since identical
results were obtained when cell lysates were controlled for total
protein content (without trypsinization) rather than cell number.
BH4 and DNA Fragmentation during Serum
Withdrawal--
We initially tested the effect of BH4 on
serum withdrawal-induced PC12 apoptotic cell death. To selectively
enhance intracellular BH4 levels and minimize degradation
of BH4 in the culture medium, we routinely treated PC12
cells with sepiapterin. Sepiapterin enters the cells readily and is
converted to BH4 intracellularly through the sequential
actions of sepiapterin reductase and dihydrofolate reductase (salvage
BH4 biosynthetic pathway) (16, 25). Incubation of PC12
cells for 24 h in the absence of serum induces DNA fragmentation (Fig. 1) as reported previously (9-11).
Elevation of intracellular BH4 levels with sepiapterin
enhances DNA fragmentation (soluble DNA was extracted from an equal
number of cells in all cases), resulting in characteristic DNA
laddering, one of the hallmarks of apoptotic cell death (Fig.
1A). A range of sepiapterin concentrations (0, 25, 50, and
100 µM) were then tested for their effect on DNA fragmentation, and, as depicted in Fig. 1B, increasing
concentrations of sepiapterin resulted in proportional increases in DNA
fragmentation.
We next tested whether inhibition of BH4 biosynthesis could
block fragmentation following serum withdrawal.
Diaminohydroxypyrimidine (DAHP), an inhibitor of GTP cyclohydrolase
(26, 27), the first enzyme in de novo BH4
biosynthetic pathway (25, 28, 29), did not alter the serum withdrawal
effect on DNA fragmentation (Fig. 1C). Similar results were
obtained with N-acetylserotonin (NAS) (28), an inhibitor of
sepiapterin reductase, which is the last enzyme in de novo
BH4 biosynthesis (25, 28, 29) (data not shown). To explain
why inhibitors of BH4 synthesis did not alter DNA
fragmentation following serum withdrawal, we considered the possibility
that inhibition of endogenous BH4 prevents the generation
of nitric oxide (NO) by nitric-oxide synthase, which can protect PC12
cells from apoptosis (30-32). Indeed, we observed an increase in DNA
fragmentation by inhibiting NO synthesis (discussed in detail below).
In preliminary studies, the minimum dose of sodium nitroprusside (an NO
donor) able to protect against apoptotic DNA fragmentation following
serum withdrawal was reduced 10-fold from 100 to 10 µM in
the presence of 1 mM DAHP. These data suggest that
inhibition of endogenous BH4 affects PC12 cell survival by at least two pathways; it inhibits the proapoptotic
BH4 effects but at the same time blocks the antiapoptotic
effects of NO. Thus, it is possible that the lack of an effect on DNA
fragmentation with BH4 biosynthetic inhibitors is due to
the concomitant inhibition of NO synthesis.
We then tested whether BH4 enhances serum
withdrawal-induced cell death via a caspase-dependent
pathway. Previous studies have established a role for cysteine
proteases (caspases) in the apoptotic death of PC12 cells (32-35).
Fig. 1D shows that the sepiapterin-induced enhancement of
DNA fragmentation is completely blocked by zVAD-fmk, a caspase
inhibitor, which is thought to inhibit apoptotic cell death induced by
either withdrawal of trophic support or oxidative stress in PC12 cells
(36). Trophic factors also reverse the BH4 effect, since
NGF (50 ng/ml) treatment eliminated DNA fragmentation induced by serum
withdrawal in the presence or absence of sepiapterin (Fig. 1,
C and D).
Effect of Intracellular BH4 on PC12 Cell Number and
Extracellular LDH Activity during Serum Withdrawal--
Incubation of
PC12 cells for 24 h in serum-free medium caused a reduction of
cell number to ~50% of control (Fig.
2A). Sepiapterin did not
significantly affect cell counts compared with serum-deprived medium
alone, whereas NGF treatment partially reversed the effect of serum
withdrawal. Both NAS and DAHP, inhibitors of de
novo BH4 biosynthesis (26-28), also increased PC12
cell survival after 24 h of serum withdrawal in an apparent
contradiction with prior DNA fragmentation results (Fig.
1C). Finally, N-
Since enhancement of DNA fragmentation is likely to be an irreversible
effect and has been shown to be a relatively late event in the
progression of apoptotic PC12 cell death (10), we expected that
elevation of intracellular BH4 would also increase cell
death. To further explore this possibility, we tested the activity of extracellular LDH as a measure of total cell death during the 24-h
incubation period. Extracellular LDH activity was expressed as
percentage of total activity (both in the medium and in living cells),
which represents the death of cells that have released LDH following
loss of membrane integrity. In control experiments, the presence of
serum (but not NGF or other treatments) altered LDH activity, and for
this reason the effects of test compounds on extracellular LDH
accumulation were controlled against the NGF protective effect in the
absence of serum. At 24 h, extracellular LDH activity was
increased to 28% following serum withdrawal as compared with 17% of
control (50 ng/ml NGF in serum-free medium; Fig. 2B). LDH
activity was further increased to 56% by sepiapterin treatment,
indicating that increased intracellular BH4 indeed enhances
PC12 cell death. Incubation with NAS protected PC12 cells from the
serum withdrawal effects on cell death, while in combination with NGF,
NAS further potentiated the NGF protective effect. These results were
verified by measuring total DNA in the medium as another indication of
cytotoxicity (data not shown) and are in agreement with the previous
results on DNA fragmentation.
The fact that sepiapterin significantly increased cell death (LDH
results) without decreasing the number of intact cells when compared
with serum deprivation alone (Fig. 2A) indicates that total
cell number (intact and burst) increased upon BH4
treatment, suggesting that BH4 can induce cell
proliferation in the absence of serum. To test whether BH4
increases the number of dying PC12 cells following serum withdrawal or
accelerates the death of already committed or sensitive cells, we
tested LDH accumulation following 48 h in test conditions (Fig.
2B). Sepiapterin treatment of serum-deprived PC12 cells for
48 h did not alter extracellular LDH activity when compared with
serum deprivation alone, suggesting that BH4 accelerates the death of cells that are already predisposed to death under conditions of serum withdrawal. NAS still protected cells from death
and potentiated the NGF trophic effect at 48 h.
Incubation of PC12 cells for 3 days in serum-free medium results in the
selection of growth-arrested, apoptosis-resistant PC12 cells (40).
Subsequent treatment of these cells with sepiapterin for an additional
24 h caused a dose-dependent increase in cell number
evidenced by the use of a colorimetric cell proliferation/cytotoxicity assay (Fig. 2C). This result strongly suggests that
BH4 can induce cell proliferation in the absence of serum.
Effect of Altered Intracellular BH4 on NGF
Withdrawal-induced DNA Fragmentation--
PC12 cells that have
acquired a neuron-like phenotype and are postmitotic following a
prolonged treatment with NGF undergo apoptotic cell death following NGF
withdrawal (9, 10). As shown in Fig. 3,
DNA fragmentation is enhanced 24 h after NGF withdrawal, and this
effect is markedly enhanced by elevation of intracellular
BH4 with sepiapterin. In addition, inhibition of the
intracellular conversion of sepiapterin to BH4 by NAS
blocked the sepiapterin effect, while DAHP, which inhibits only
de novo BH4 synthesis and does not block
conversion of sepiapterin to BH4, had no effect on
sepiapterin's proapoptotic effect. DAHP and, to a lesser extent, NAS
protected neuronal PC12 cells from DNA fragmentation following NGF
withdrawal and further potentiated the NGF protective effect. The
effect of sepiapterin on DNA fragmentation was blocked by continued NGF
treatment. Fig. 3 also shows the effects of actinomycin D (which
inhibits DNA transcription) on neuron-like PC12 cell DNA fragmentation.
Actinomycin D protected neuron-like PC12 cells both from NGF
withdrawal-induced and sepiapterin-potentiated DNA fragmentation,
indicating that active DNA transcription is required for the
sepiapterin effect on neuron-like PC12 cell death. Similar experiments
under serum withdrawal-induced apoptosis failed to show a protective
effect and rather resulted in enhanced cytotoxicity both for control
(as reported previously) (41) and for sepiapterin treated
cells2 in the presence of
actinomycin D.
Total cell counts following NGF withdrawal-induced apoptotic death
were also tested. After 24 h of NGF withdrawal, the PC12 cell
number dropped to 60% of control (NGF-treated cells), whereas the
addition of sepiapterin, in the absence of NGF, only produced a
decrease to 80% of control (not shown). Despite the increased cell
number in sepiapterin-treated cells when compared with cells subjected
to NGF withdrawal alone, extracellular soluble DNA accumulation during
the 24-h incubation was significantly elevated in sepiapterin-treated cells (data not shown). These data are also consistent with an increase
in cellular proliferation following sepiapterin treatment (16),
accompanied by an acceleration of apoptotic cell death in sensitive
cells. Finally, both cell counts and total DNA levels verified that NAS
and DAHP fully protect neuron-like PC12 cells from NGF
withdrawal-induced apoptotic death.
Effect of Nitric Oxide, Catecholamine, or Hydrogen Peroxide
Metabolism and Effect of Calcium Influx on Serum
Withdrawal-induced DNA Fragmentation--
Since toxic effects
resulting from NO production and catecholamine accumulation have been
reported (42-45) and since BH4 enhances their synthesis
(46, 47), we examined whether elevation of intracellular
BH4 enhances apoptotic death of PC12 cells through these
mechanisms. Treatment of serum-deprived PC12 cells with 5 mM N
To test whether BH4 induces apoptotic cell death by
increasing intracellular catecholamine levels, we treated PC12 cells
with 20 µM
Using a spectrophotometric assay to quantify hydrogen peroxide (24), we
have verified that BH4 autoxidation in medium can lead to
hydrogen peroxide formation (48, 49). Hydrogen peroxide formation is as
high as 30% of the initial BH4 concentration within 30 min
of incubation in cell-free medium or PBS and was blocked by 1500 units/ml of catalase or 1 mM dithiothreitol (DTT). However, DTT treatment does not inhibit the sepiapterin-induced enhancement of
apoptotic death (Fig. 5A),
although it prevents BH4 degradation to hydrogen peroxide
in vitro. Treatment of PC12 cells for 24 h with
catalase (1500 units/ml) or superoxide dismutase (1000 units/ml) also
fails to block the sepiapterin-induced enhancement in PC12 cell DNA
fragmentation following serum deprivation (Fig. 5B). In
contrast, catalase inhibits BH4 degradation in
vitro and hydrogen peroxide-induced cytotoxicity (Fig.
5C) and DNA
fragmentation3 in PC12 cells.
Superoxide dismutase and catalase also inhibit the cytotoxic effect of
BH4 in serum-maintained PC12 cells treated in the absence
of DTT (data not shown). As reported previously, in the presence of DTT
(it has no effect by itself), oxidative degradation is minimized, and
BH4 induces cell growth like sepiapterin (16), while
hydrogen peroxide induces cell death under the same conditions. These
results suggest that hydrogen peroxide production cannot account for
the enhancement of apoptotic death by elevating intracellular
BH4 levels during withdrawal of trophic support.
Since calcium influx can induce apoptotic PC12 cell death (10, 50, 51)
and previous reports suggested that BH4 induces calcium
influx (52, 53), we also tested the possibility that calcium influx is
responsible for the enhancement of apoptotic PC12 cell death
following sepiapterin treatment. Treatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid or with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid/acetoxymethylester (54) to chelate extracellular or
intracellular calcium, respectively, failed to show a reversal of the
sepiapterin-induced enhancement of DNA fragmentation and cell death
(data not shown).
Effect of BH4 on c-Myc Expression under Conditions of
Serum Deprivation--
We then tested the effect of BH4 on
c-Myc expression under conditions of serum withdrawal, since c-Myc
expression has been linked to apoptosis in several model systems
(55-57). Fig. 6 shows that incubation of
serum-starved PC12 cells in the presence of sepiapterin (100 µM) significantly induced the expression c-Myc S proteins
at early time points when compared with control (minus serum). At later
time points (9-24 h) c-Myc S expression was inhibited by sepiapterin
treatment. Similar results were evident for c-Myc 1 (68 kDa) and c-Myc
2 (65 kDa) expression under longer exposures (data not shown).
Nonetheless, sepiapterin treatment preferentially induced the
expression of c-Myc S proteins (predicted sizes between 45 and 50 kDa),
which are amino-terminally truncated versions of full-length c-Myc
arising through the use of downstream AUG codons (58). Thus,
alterations in c-Myc expression may be responsible for the enhancement
of PC12 cell apoptosis by elevated intracellular BH4 during
withdrawal of trophic support.
Effect of N-Acetylcysteine or Inhibition of
Cyclin-dependent Kinases on BH4-induced DNA
Fragmentation--
Previous reports have established the ability of
N-acetylcysteine (NAC) to prevent apoptotic death of PC12
cells (59-61). This protective effect is not mediated by the ability
of NAC to affect glutathione metabolism (60) or to inhibit cell cycle
progression (61) but rather by a mechanism that involves the activation of the MAP kinase pathway (61). We found that NAC at moderate concentrations (5 mM) is able to fully inhibit the
sepiapterin-induced DNA fragmentation under conditions of serum
withdrawal (Fig. 7A).
We also tested the effect of the cyclin-dependent kinase
inhibitor, olomoucine, on the proapoptotic effect of sepiapterin. Olomoucine is reportedly capable of blocking apoptotic cell death of
differentiated, neuron-like PC12 cells following NGF withdrawal (14,
36, 63), while it increases cell death of serum-deprived PC12 cells.
Indeed, in repeated experiments, olomoucine moderately increased DNA
fragmentation in serum-deprived PC12 cells (Fig. 7B).
Interestingly, while the addition of sepiapterin alone potently enhanced DNA fragmentation, upon the addition of 200 µM
olomoucine, the sepiapterin-induced DNA fragmentation was reduced to
levels observed with the addition of olomoucine alone (Fig.
7B). This result suggests that olomoucine blocks
sepiapterin-induced apoptosis even under conditions of serum withdrawal.
We have previously reported that enhanced proliferation of PC12
cells by epidermal growth factor or NGF requires an increase in
intracellular BH4 levels, which are raised ~3-fold (17). To achieve the same proliferative effect by exogenous administration, intracellular BH4 levels need to be increased 12-fold (16). Doses of sepiapterin up to 100 µM raise intracellular
BH4 levels in a dose-dependent manner up to
~12-fold, and we have seen enhancement of apoptosis throughout the
range of sepiapterin concentrations up to 100 µM. Thus,
the concentrations of sepiapterin used previously and in the current
study are likely to generate BH4 levels that are
physiologically relevant. The relevance of endogenous, intracellular BH4 to apoptotic cell death is also supported by our
observations that BH4 biosynthetic inhibitors prevent
apoptosis in neuronally differentiated PC12 cells and potentiate the
antiapoptotic effects of NO and NGF.
Elevation of intracellular BH4 was shown to increase PC12
cell number in a manner consistent with enhancing cell cycle
progression rather than increasing cell survival (16). We now show that elevated intracellular BH4 enhances apoptotic death of
undifferentiated PC12 cells in a dose-dependent manner
following serum withdrawal and of neuron-like PC12 cells in a
transcription-dependent manner following NGF withdrawal. We
also show that BH4 biosynthetic inhibitors protect
differentiated neuron-like PC12 cells from apoptotic death. We found no
evidence implicating catecholamine or NO metabolism (which are known to
be induced by elevated BH4) in mediating the BH4 effect, since inhibitors of their synthesis did not
alter the apoptotic profile.
Generation of superoxide radicals or hydrogen peroxide during
BH4 autoxidation and induction of calcium influx are
also not likely to be involved in the BH4 effect. Our
results are consistent with the hypothesis that BH4
enhances the rate of death in cells that are already responsive to
apoptotic death following withdrawal of trophic support. Cells that
escape apoptotic death under these conditions respond to
BH4 with increased cell growth (Fig. 2C). Increased cell proliferation is also the likely explanation for increased cell counts in sepiapterin-treated neuron-like PC12 cells
after NGF withdrawal (when compared with NGF withdrawal alone), despite
increased cytotoxicity and DNA fragmentation. Indeed, treatment of
neuron-like PC12 cells with BH4 in the absence of NGF and
serum results in increased cell number after 5 days in culture (64).
One possibility, in view of our data, is that increased cell number is
the result of increased proliferation of apoptosis-resistant PC12 cells
in the presence of BH4.
Elevation of intracellular BH4 in PC12 cells with
sepiapterin increased c-Myc expression (especially c-Myc S) at early
time points compared with serum deprivation alone. Interestingly, c-Myc S proteins are often found constitutively expressed in tumors or
transiently expressed during rapid cell growth (58). It is possible
that the early increase in c-Myc proteins is not relevant to the
enhancement of apoptotic cell death by BH4, but prior data indicate that both c-Myc and c-Myc S can induce apoptosis in several cell types via the illegitimate progression of the cell cycle (65, 66).
In PC12 cells, exogenous c-Myc expression can block NGF-induced growth
arrest and differentiation (67, 68). It is thus possible that the
effects of BH4 both on PC12 cell proliferation and
apoptosis are mediated by c-Myc (or c-Myc S).
The BH4-induced enhancement of cell death was completely
abrogated by the cysteine protease inhibitor zVAD-fmk, indicating that
caspases are involved in the BH4 pro-apoptotic effect.
Interestingly, NAC also completely blocked the BH4 effect
(Fig. 7A). Previous studies have demonstrated that the
anti-apoptotic effects of NAC in PC12 cells are not due to its
antioxidant capacity (60) but are instead dependent on new
transcription and the activation of the Ras-ERK signaling pathway (61).
Given our data with inhibitors of oxidative stress, we feel that NAC
prevents BH4-induced DNA fragmentation by a mechanism that
does not involve its antioxidant potential. In support of this,
administration of reduced glutathione was unable to block the
BH4 effect following serum withdrawal (not shown). Although
not tested, it is likely that the BH4 effect is blocked by
the activation of the Ras-ERK signaling pathway. Finally, olomoucine,
an inhibitor of cyclin-dependent kinases, also reversed the
BH4 effect on DNA fragmentation. By itself, olomoucine is
able to block NGF withdrawal but not serum deprivation-induced cell
death (14).
The above results also allow us to postulate that BH4
affects one of three major pathways involved in PC12 cell apoptotic death. Previous studies have demonstrated that PC12 cell death by
superoxide dismutase 1 depletion (leading to oxidative stress) can be
reversed by zVAD-fmk but not by olomoucine (36). Furthermore, apoptosis
induced by DNA-damaging agents is reversed by olomoucine but not by
zVAD-fmk or NAC (69, 70). The only pathway of apoptotic cell death that
is blocked by either zVAD-fmk, olomoucine, or NAC is that of trophic
factor deprivation (36, 60). Previous reports suggested that
BH4 can induce cell proliferation (16, 71) and that growth
factor-induced proliferation correlates with endogenous BH4
levels in PC12 cells (17). In view of these data, we postulate that
BH4 affects a growth factor-dependent signaling
event, which leads to increased proliferation in the presence of
sufficient trophic support or enhances cell death in its absence. Fig.
8 summarizes our current understanding of the effects of BH4 on the proliferation and survival of
PC12 cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
); without fetal bovine serum and containing only 1%
heat-inactivated horse serum) containing 50 ng/ml 2.5 S NGF. After 8 days, apoptotic death was induced by NGF withdrawal. Cells were washed
twice with DMEM (
) (without NGF) and incubated for 24 h in DMEM
(
) medium without NGF containing a 1:2000 dilution of anti-NGF
antibody (Sigma catalog no. N5142). At this concentration, the antibody completely blocked neurite outgrowth following a 3-day NGF treatment. The effect of various treatments on apoptotic death was tested by
incubating cells for 24 h in the presence of test compounds in
DMEM (
) containing anti-NGF antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of intracellular BH4
levels on PC12 DNA fragmentation upon serum withdrawal.
A, 24 h after plating PC12 cells were washed free of
serum and cultured in DMEM with serum (serum) or without
serum (( ) serum) or in serum-free medium containing 100 µM sepiapterin ((
)/SEP). 24 h later,
soluble DNA was extracted and resolved on a 1.2% agarose gel. Each
lane was loaded with DNA extracted from 8 × 106 cells. B, serum withdrawal-induced DNA
fragmentation was blocked by 50 ng/ml NGF, while increasing
concentrations of sepiapterin (25, 50, and 100 µM)
induced a dose-dependent increase in serum
withdrawal-induced DNA fragmentation. Soluble DNA was extracted from
4 × 106 cells per condition and resolved on a 1.2%
agarose gel. C, after washing with serum-free medium, cells
were incubated for 24 h in either serum-containing medium
(control), serum-free medium alone, or serum-free medium supplemented
with either 50 ng/ml NGF, 1 mM DAHP, 100 µM
sepiapterin, NGF plus DAHP, or NGF plus sepiapterin. Soluble DNA was
extracted from 4 × 106 cells/condition and resolved
on a 1.2% agarose gel. D, cells were serum-starved as
before and incubated in the presence of sepiapterin alone, zVAD-fmk (50 µM) alone, or a combination of sepiapterin and zVAD-fmk
for 24 h. Soluble DNA was extracted from an equal number of cells
per condition and resolved on a 1.2% agarose gel. Comparative results
for all figures were obtained in at least three independent
experiments. bp, base pair.
-nitroarginine, an inhibitor
of nitric-oxide synthase (37), did not affect death by serum withdrawal
at a concentration that totally blocks NO production and NGF-induced
neurite formation (38). These results suggest that inhibition of
BH4 biosynthesis can protect from or delay apoptotic death
but fail to demonstrate an elevation in cell death following
sepiapterin treatment.
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Fig. 2.
Effect of BH4 on cell number and
extracellular LDH activity. A, cells were plated in
75-cm2 flasks at 7 × 106 cells/flask.
24 h later, cells were washed free of serum and then placed in
fresh serum-containing medium (DMEM) or in serum-free DMEM in the
absence or presence of additional test compounds. Test compounds
included NGF, sepiapterin, DAHP, NAS, and
N- -nitro-arginine (NNA). After a 24-h
incubation, total cells were counted using a hemacytometer as described
under "Experimental Procedures." Values are expressed as percentage
of control and represent the mean ± S.E. of 3-6 independent
determinations performed in duplicate. *, p < 0.05;
**, p < 0.01; ***, p < 0.001;
analysis of variance as compared with DMEM. B, lactate
dehydrogenase activity in the culture medium was measured using the Cytotox 96 kit from Promega as suggested by the manufacturer.
Since the addition of serum significantly increased the assay
background, results with test conditions were compared with the
protective effect of NGF on serum withdrawal-induced cytotoxicity.
Cells were incubated for 24 or 48 h in serum-free DMEM alone or
containing either NGF, sepiapterin, NAS, or a combination of NGF and
NAS. Extracellular LDH activity is expressed as a percentage of total
cellular LDH activity. Values represent the mean ± S.E. of 2-4
independent determinations performed in triplicate. *,
p < 0.05; **, p < 0.01; ***,
p < 0.001; Student's t test as compared
with results with NGF at 24 h. +, p < 0.05; ++, p < 0.01; Student's t test as
compared with results with NGF at 48 h. C, PC12
cells were plated in 96-well plates and incubated for 72 h in
serum-free medium. Cells were then treated for an additional 24 h
with sepiapterin and/or NAS in serum-free DMEM. Absorbance at 490 nm
was measured after incubation of cells in CellTiter 96 Aqueous reagent
as described under "Experimental Procedures." Color formation
linearly correlated with PC12 cell number under these conditions.
Values represent the mean ± S.E. of three independent
determinations performed in triplicate. *, p < 0.05;
**, p < 0.01; Student's t test as compared
with results without serum treatment. +, p < 0.05; Student's t test as compared with results without
serum in the presence of sepiapterin 100 µM.
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Fig. 3.
Effect of BH4 on NGF
withdrawal-induced DNA fragmentation. PC12 cells were plated in
75-cm2 flasks and incubated for 8 days in DMEM containing
1% horse serum and 50 ng/ml NGF. Apoptotic cell death was induced in
differentiated PC12 cells by washing cells free of NGF and incubating
for another 24 h in the presence of an anti-NGF antibody as
described in detail under "Experimental Procedures." Similar
results were obtained after extensive washing in the absence of the
anti-NGF antibody. Conditions containing NGF were treated with NGF in
the absence of the NGF antibody. , absence of NGF and presence of the
anti-NGF antibody. Sepiapterin was used to elevate intracellular
BH4 levels, while NAS and DAHP were used to selectively
inhibit different steps in either salvage or de novo
BH4 biosynthesis. Actinomycin D (AD) was used to
block DNA transcription. Similar results were obtained in three
independent experiments.
-nitro-L-arginine methyl
ester (NAME), which totally blocks NO production in PC12
cells (38), failed to reduce and instead potentiated control and
sepiapterin-induced DNA fragmentation (Fig.
4A).
N-
-Nitro-L-arginine, another nitric-oxide
synthase inhibitor, also failed to alter serum withdrawal-induced cell death (Fig. 2A), indicating that the effect of
BH4 on the potentiation of apoptotic PC12 cell death is not
mediated by increased NO metabolism.
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Fig. 4.
Involvement of nitric oxide or catecholamine
synthesis in BH4-mediated induction of PC12 apoptotic cell
death. 24 h after plating, PC12 cells were treated with test
compounds for 24 h in the absence of serum (( )
serum). A, the effect of
N
-nitro-L-arginine methyl ester
(NAME) on BH4-mediated induction of DNA
fragmentation was tested.
N
-Nitro-L-arginine methyl ester
was used at 5 mM to inhibit nitric oxide synthesis.
B, The effect of catecholamine synthesis on the
BH4-mediated induction of DNA fragmentation was tested.
Catecholamine synthesis was blocked by incubating PC12 cells with 20 µM
-methyl-L-p-tyrosine
(
-MPT). Controls for both experiments included
cells grown in serum-containing medium and cells where apoptosis was
induced by serum withdrawal. Both experiments were repeated twice with
similar results.
-methyl-para-L-tyrosine, which blocks
catecholamine production (16) by inhibiting tyrosine hydroxylase, the
BH4-requiring and rate-limiting enzyme in catecholamine
synthesis (18).
-Methyl-para-L-tyrosine (
-MPT) failed to reduce DNA fragmentation
following serum withdrawal in the absence or presence of sepiapterin,
suggesting that this BH4 effect is independent from the
production of PC12 cell catecholamines (Fig. 4B).
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Fig. 5.
Effect of superoxide dismutase or catalase
treatment on BH4-induced DNA fragmentation following serum
withdrawal or hydrogen peroxide cytotoxicity. PC12 cells were
allowed to attach for 24 h after plating prior to the addition of
test compounds. A, sepiapterin and/or DTT were added in
serum-free medium, and 24 h later soluble DNA was extracted and
resolved in an agarose gel. B, sepiapterin, catalase, or
superoxide dismutase was added alone or in combination in serum-free
medium, and soluble DNA was isolated 24 h later from an equal
number of cells and resolved in an agarose gel. Comparative results
were obtained in two more experiments. C, effect of catalase
on hydrogen peroxide-mediated cytotoxicity. PC12 cells were plated in
96-well plates and were allowed to attach for 24 h in
serum-containing medium. A range of hydrogen peroxide concentrations
(100-800 µM) in serum-containing medium with or without
catalase were added to appropriate wells, and cells were incubated for
an additional 24 h. Absorbance at 490 nm was measured after
incubation of cells in CellTiter 96 Aqueous reagent as described under
"Experimental Procedures." Color formation linearly correlated with
PC12 cell number. Values represent the mean ± S.E. of two or
three independent determinations performed in sixplicate. *,
p < 0.05; **, p < 0.01 compared with
DMEM (+ serum) treatment alone; one-way analysis of variance and
post hoc analysis using the Newman-Keuls test .
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Fig. 6.
Temporal effects of sepiapterin treatment on
c-Myc protein levels. Cells were cultured in test conditions
(serum-containing DMEM, serum-free DMEM, and serum-free DMEM containing
100 µM sepiapterin) for 1.5, 3, 4.5, 6, or 9 h.
After trypsinization, cells were counted and lysed. Western blot
analysis of electrophoretically separated proteins from equal number of
cells for each condition was performed as described under
"Experimental Procedures." ECL exposure shown was for 5 min.
Increased expression of Myc S proteins is evident following incubation
with sepiapterin for 1.5, 3, and 4.5 h. A similar pattern was
evident for Myc-1 and Myc-2 expression at longer (15 and 30 min) ECL
exposure times (not shown). Comparative results were obtained in two
experiments.
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Fig. 7.
Effect of N-acetylcysteine
or cyclin-dependent kinase inhibition by olomoucine on
BH4-induced DNA fragmentation. A, cells
were grown as previously described and treated for 24 h with 5 mM NAC alone or together with sepiapterin in the absence of
serum. B, cells were grown as previously described and
treated for 24 h with 200 µM olomoucine alone or
together with sepiapterin in the absence of serum. Both NAC and
olomoucine inhibited sepiapterin-induced DNA fragmentation. Comparative
results on DNA fragmentation were obtained in three independent
experiments. bp, base pair.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Schematic diagram depicting our current
understanding of the effects of BH4 on PC12 cell growth or
death. In the presence of sufficient trophic support,
BH4 promotes PC12 cell growth by a mechanism that involves
increased cell cycle progression. BH4 biosynthetic
inhibitors inhibited growth factor-induced proliferation, suggesting
that BH4 inhibition affects a growth factor-mediated
signaling event. In the absence of trophic factors, BH4
accelerates DNA fragmentation and PC12 cell death. This BH4
effect can be blocked by NAC, olomoucine, and zVAD-fmk, agents that
were previously shown to inhibit PC12 apoptotic death due to loss of
trophic support. Together with data showing that BH4
affects the expression of Myc-related proteins and indications that
BH4 may promote cell cycle progression even under
conditions of serum withdrawal, our results suggest that the effects of
BH4 on PC12 cell growth or death, are mediated by the same
mechanism.
An interesting possibility not addressed by our data is whether treatment of serum-deprived cells with BH4 enhances apoptotic cell death by activating the same pathway that induces cell death following NGF withdrawal. It has been reported, for example, that prolonged NGF treatment "primes" PC12 cells for apoptotic death, most likely due to the time-dependent accumulation of caspase-3 (72). Consequently, the rate of cell death following NGF withdrawal is increased according to the length of NGF pretreatment. To avoid problems with early effects of NGF treatment on cell growth and gene expression, we tested the effect of BH4 after 8 days of NGF pretreatment, which results in a postmitotic, neuronally differentiated cellular phenotype. An interesting focus for future investigation would be to characterize the effects of BH4 treatment on caspases, Bcl-related genes, mitogen-activated protein kinases, and Ras N-nitrosylation (which are known to play important roles in growth factor-mediated survival). By comparing these BH4 effects with the basal responses of our cells to serum or NGF deprivation, we hope to better address the mechanism by which BH4 affects cell death.
The loss of nigrostriatal dopaminergic neurons in Parkinson's disease is reflected by a decrease of BH4 levels in CSF of Parkinsonian patients (62). These neurons contain as much as 100-fold higher BH4 levels than surrounding nondopaminergic cells within the substantia nigra (39), but it is still not known whether BH4 plays an active role in the neurodegenerative process. More recently, morphological characteristics of apoptotic death were reported in dopamine neurons within the substantia nigra pars compacta of Parkinson's disease patients (7, 8). Loss of trophic support can induce apoptosis and is hypothesized to play a role in several neurological diseases, including Parkinson's and Alzheimer's disease. While BH4 is essential in dopamine neurons for neurotransmitter synthesis under normal conditions, it is possible that with insufficient trophic support, endogenous BH4 levels become detrimental to dopamine cell viability. There is no evidence supporting a decrease in BH4 levels within surviving dopaminergic neurons. Instead, a direct correlation exists between cell loss and the respective reduction in BH4 levels during aging or Parkinson's disease (62), or in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. If the local environment or striatal target cells cease providing an appropriate level of trophic support, dopaminergic neurons may become preferentially susceptible to apoptotic death promoted by their increased BH4 metabolism. In support of this hypothesis, we have preliminary evidence in rats that removal of striatal target cells by excitotoxic lesions causes delayed apoptotic death of dopamine neurons in the substantia nigra.4
Whether a shift in the balance of BH4 from being supportive
of neuronal function to becoming destructive for neuron viability occurs in neurodegenerative disorders warrants further investigation. The validity of this hypothesis is relevant to patient management, since BH4 has been used as a therapeutic agent for the
treatment of neurodegenerative disorders where monoaminergic deficits
are evident, including Parkinson's and Alzheimer's disease. Our data may also be relevant to the development of novel experimental approaches including the engineering of cells expressing high levels of
BH4 and catecholamines for transplantation therapy in Parkinson's disease.
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ACKNOWLEDGEMENTS |
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We thank Marisa C. Louie, Bruce Imerman, Jeniffer Blitz, and Gissela F. Claassen for technical help. Dr. Steven R. Hann generously provided the c-Myc antiserum.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Cell Biology, Vanderbilt Univ, 1161 21st Ave. S., MCN #C-2310, Nashville, TN 37232-2175.
** Supported by National Institutes of Health Grant NS39132 and a Merit Award from the Veterans Administration.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M006570200
2 P. Z. Anastasiadis, H. Jiang, L. Bezin, D. M. Kuhn, and R. A. Levine, unpublished results.
3 P. Z. Anastasiadis, H. Jiang, L. Bezin, D. M. Kuhn, and R. A. Levine, unpublished data.
4 P. Z. Anastasiadis, H. Jiang, L. Bezin, D. M. Kuhn, and R. A. Levine, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PD, Parkinson's disease; BH4, tetrahydrobiopterin; NGF, nerve growth factor; NAS, N-acetylserotonin; DAHP, diaminohydroxypyrimidine; kb, kilobase pair; LDH, lactate dehydrogenase; DTT, dithiothreitol; NAC, N-acetylcysteine; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone.
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