(Received for publication, November 10, 1995; and in revised form, January 26, 1996)
From the
Previous studies have demonstrated that multiple agents that promote survival of PC12 cells and sympathetic neurons deprived of trophic support also block cell cycle progression. Presently, we address whether inhibition of cell cycle-related cyclin-dependent kinases (CDKs) prevents neuronal cell death. We show that two distinct CDK inhibitors, flavopiridol and olomoucine, suppress the death of neuronal PC12 cells and sympathetic neurons. In addition, we demonstrate that inhibitor concentrations required to promote survival correlate with their ability to inhibit proliferation. Promotion of survival, however, does not correlate with inhibition of extracellular signal-regulated kinase or c-Jun kinase activities or with interference with the activation of c-Jun kinase that accompanies serum/nerve growth factor deprivation. In contrast to their actions on nerve growth factor-differentiated PC12 cells, the CDK inhibitors do not prevent the death of proliferation-competent PC12 cells and, in fact, promote their cell death. These findings support the hypothesis that post-mitotic neuronal cells die after removal of trophic support due to an attempt to re-enter the cell cycle in an uncoordinated and inappropriate manner. We speculate that cycling PC12 cells are not saved by these agents due to a signaling conflict between an inherent oncogenic signal and the inhibition of CDK activity.
Neuronal apoptosis is an important aspect of nervous system development and a component of neuronal injury and disease. The most generally accepted model of the developmental regulation of neuronal death states that limiting quantities of target-derived neurotrophic support control the optimum number of neuron-target interactions(1) . Neurotrophins also play a role in ameliorating the effects of oxidative stress and many forms of neuronal injury(2, 3) .
In an effort to define the
mechanisms of neurotrophin action in neuronal survival, two model
systems, the PC12 pheochromocytoma cell line and cultured primary
sympathetic neurons, have been exploited. The PC12 cell line was
initially derived from a rat adrenal medullary
pheochromocytoma(4) . When grown in serum-containing medium,
PC12 cells divide and resemble precursors of adrenal chromaffin cells
and sympathetic neurons. Upon addition of NGF, ()these
``naive'' cells gradually attain the phenotypic properties of
sympathetic neurons. Both naive and neuronally differentiated PC12
cells undergo apoptosis upon removal of trophic support (i.e. serum or serum/NGF)(5, 6) . The response of PC12
cells to withdrawal of trophic support is quite analogous to that of
sympathetic neurons. In vivo(7, 8, 9) and in vitro(10, 11) evidence demonstrates that sympathetic
neurons require NGF for survival. Studies in this laboratory (6, 12) and by others(13, 14) have
shown that both PC12 cells and sympathetic neurons undergo apoptotic
death upon NGF deprivation.
Although the mechanisms by which neurotrophins suppress apoptosis are not fully understood, it has been hypothesized that neurotrophins prevent apoptotic death by acting to coordinate cell cycle progression and/or prevent inappropriate cell cycle re-entry(12, 15, 16, 17) . Accordingly, this hypothesis predicts that cells that attempt to enter or traverse the cell cycle without a set of proper mitogenic signals will undergo apoptosis. In support of this model, numerous observations of apoptosis in the presence of conflicting cell cycle signals have been reported in non-neuronal systems(18, 19, 20, 21) . We have applied the cell cycle/apoptosis hypothesis to interpret the characteristics of apoptotic death in PC12 cells, sympathetic neurons, and other cells of neuronal origin(15, 22) . In this view, withdrawal of serum from naive proliferating PC12 cells leads to an uncoordinated and disastrous attempt to continue to cycle, whereas in post-mitotic differentiated PC12 cells and sympathetic neurons, withdrawal of NGF results in an inappropriate attempt to re-enter the cell cycle and consequent death.
Findings from this and other
laboratories have provided some evidence for this interpretation and
for the cell cycle/apoptosis model in neuronal cells. While apoptotic
death of sympathetic neurons and post-mitotic PC12 cells is delayed by
protein synthesis inhibitors(11, 14, 23) ,
such inhibitors do not block cell death of naive PC12 cells (24) . One interpretation of this discrepancy is that the
proteins needed for apoptosis are regulators of the general cell cycle
mechanism. Since naive PC12 cells continually synthesize cell cycle
proteins, they may utilize a pre-existing pool of cell cycle regulators
to enter the cell cycle even in the absence of new protein synthesis.
Without appropriate coordinating mitogenic signals such as provided by
growth factors, apoptosis would result. In contrast, post-mitotic cells
would require de novo synthesis of cell cycle proteins prior
to inappropriate cell cycle re-entry. In accordance with this view,
Freeman et al.(17) showed that NGF removal from
sympathetic neurons results in an induction of the cell cycle
regulatory protein cyclin D1 along with transcription factors c-Fos and
c-Jun(25) , typically induced prior to cell division.
Furthermore, the activation of another cell cycle protein, Cdc2, has
been reported in differentiated PC12 cells as a consequence of NGF
withdrawal(26) . It has also been reported that expression of
SV40 T antigen in Purkinje cells results in apoptotic death (27) concurrent with DNA synthesis. ()
Initial
attempts to test the cell cycle/apoptosis model by blocking cell cycle
progression have produced additional support for this hypothesis.
Induction of dominant-negative Ras expression in both naive and
post-mitotic PC12 cells inhibits cell cycle progression and death
induced by withdrawal of trophic support(15) . A similar
correlation between survival and blockade of the cell cycle has been
shown in PC12 cells and sympathetic neurons treated with N-acetylcysteine (28) . In addition, we have recently
reported that the G/S blockers mimosine, ciclopirox, and
deferoxamine are effective in preventing cell death of both
post-mitotic and dividing PC12 cells as well as of sympathetic
neurons(29) . In these cases, the mechanisms by which the cell
cycle is inhibited are unknown.
The cyclin-dependent kinase (CDK)
family, which among others includes Cdk2-4/6 and Cdc2 (Cdk1), is
an important group of cell cycle regulatory molecules whose inhibition
represents a more defined means to block cell cycle progression or
re-entry. Cdc2 is a well characterized M phase regulator and may also
serve to mediate progression through the S phase(30) . Cdk2 and
Cdk3 activities are required for progression through the
G/S phases of the cycle(30, 31) . In the
present studies, we investigated whether inhibitors of the CDK family
of kinases would prevent apoptotic death induced by trophic factor
withdrawal from PC12 cells and sympathetic neurons. We report that the
CDK inhibitors flavopiridol and olomoucine are effective in blocking
the death of trophic factor-deprived, post-mitotic PC12 cells and
sympathetic neurons, but not of dividing PC12 cells. These observations
thus support and further refine the cell cycle/apoptosis model.
We first examined whether these inhibitors
inhibit DNA synthesis by naive PC12 cells. As shown in Fig. 1A and 2A, flavopiridol and olomoucine inhibited
[H]thymidine incorporation with IC
values of
0.3 and
100 µM, respectively.
Figure 1:
Flavopiridol inhibits
[H]thymidine incorporation by dividing PC12 cells
and promotes survival of neuronally differentiated PC12 cells in
serum-free medium following withdrawal of NGF. The neuronal PC12 cell
phenotype was obtained by treatment with NGF in serum-free medium for 8
days. A, relationship between the drug dose required for
promotion of day 2 survival of NGF-deprived neuronally differentiated
PC12 cells and inhibition of thymidine incorporation by dividing
NGF-untreated (naive) PC12 cells. Naive PC12 cells were pretreated with
the indicated concentrations of flavopiridol for 16 h in serum- or
insulin-containing RPMI 1640 medium prior to measurement of thymidine
incorporation (determined as described under ``Experimental
Procedures''). Cell survival data are normalized so that survival
without flavopiridol (51%) is defined as zero and 100% survival is
defined as the number of cells initially present. B, effect of
flavopiridol (3 µM) on the time course of survival of
neuronally differentiated PC12 cells following withdrawal of NGF. Each
data point is the mean ± S.E. of three
samples.
We next determined the ability of flavopiridol and olomoucine to block death induced by NGF withdrawal from PC12 cells that had been neuronally differentiated by pre-exposure to NGF in serum-free medium. Maximal protection was observed at a concentration of 1 µM for flavopiridol and 200 µM for olomoucine. Flavopiridol was more effective in long-term protection of NGF-differentiated PC12 cells than olomoucine, with good maintenance of survival even 5 days after NGF withdrawal (Fig. 1B and 2B). Both drugs showed progressive toxicity even in the presence of NGF, which appears to limit their long-term efficacy. As shown in Fig. 1and Fig. 2, both drugs significantly delayed death, and this correlated well with inhibition of thymidine incorporation.
Figure 2:
Olomoucine inhibits
[H]thymidine incorporation by dividing PC12 cells
and promotes survival of neuronally differentiated PC12 cells in
serum-free medium following withdrawal of NGF. A, relationship
between the drug dose required for promotion of day 2 survival of
NGF-deprived neuronally differentiated PC12 cells and inhibition of
thymidine incorporation by naive PC12 cells. Naive PC12 cells were
pretreated with the indicated concentrations of olomoucine for 16 h in
serum- or insulin-containing medium prior to measurement of thymidine
incorporation. Cell survival data are normalized so that survival
without olomoucine (43%) is defined as zero and 100% survival is
defined as the number of cells initially present. B, effect of
olomoucine (200 µM) on the time course of survival of
neuronally differentiated PC12 cells following withdrawal of NGF. Each
data point is the mean ± S.E. of three
samples.
As a control for nonspecific effects of olomoucine,
the analog isoolomoucine was also tested for its ability to inhibit
cell cycle progression and neuronal death. This derivative is identical
to olomoucine with the exception of the location of a substituent
methyl group on the imidazole ring of the purine backbone. As reported
by the manufacturer(51) , this change severely reduces
inhibition of Cdk1 activity (IC > 500 µM isoolomoucine versus IC
= 7
µM olomoucine). As shown in Fig. 3, isoolomoucine
was far less effective than olomoucine in inhibiting both thymidine
incorporation and cell death.
Figure 3:
Isoolomoucine is less effective than
olomoucine in inhibiting [H]thymidine
incorporation and maintaining survival of neuronally differentiated
PC12 cells following withdrawal of NGF. A, comparison of
inhibition of thymidine incorporation by olomoucine and isoolomoucine.
Naive PC12 cells were pretreated with the indicated concentrations of
olomoucine/isoolomoucine for 16 h in serum- or insulin-containing
medium prior to measurement of thymidine incorporation. B,
time course of survival of neuronally differentiated PC12 cells
following withdrawal of NGF. Each data point is the mean ± S.E.
of three samples.
Fig. 4shows the morphology of
neuronally differentiated PC12 cells treated with the CDK inhibitors in
serum-free medium with and without NGF. The cells rescued by the drugs
showed the typical phase-bright morphology of living cells, but did not
regenerate neurites. In the presence of NGF, the drugs appeared to
partially suppress neurite regeneration. Potential reasons for this
effect on regeneration include inhibition of Cdk5, a kinase linked to
neurite formation(38, 39) , and/or of ERK1 kinase,
activation of which also appears to be required for neurite
outgrowth(40) . Olomoucine is reported to inhibit GST-ERK1
activity in vitro (IC = 30
µM).
Figure 4:
Phase-contrast micrographs of neuronally
differentiated PC12 cells. Neuronally differentiated PC12 cells were
maintained in serum-free medium for 2 days and treated with no
additives (A), 100 ng/ml NGF (B), 1 µM flavopiridol (C), 1 µM flavopiridol +
NGF (D), 200 µM olomoucine (E), and 200
µM olomoucine + NGF (F). Magnification
375.
To test whether ERK inhibition by olomoucine might contribute to its actions on survival (by either promoting or blocking death), we treated naive and neuronally differentiated PC12 cells with PD98059 (20-100 µM), an inhibitor of the mitogen-activated protein kinase/ERK kinase that mediates NGF-promoted activation of ERKs(41) . We then examined the behavior of the cells when passaged into serum-free medium with or without NGF. Although the drug effectively suppressed NGF-stimulated neurite regeneration (as anticipated from its inhibition of ERK activation), it neither blocked nor mimicked the capacity of NGF to promote survival of naive or neuronally differentiated PC12 cells (data not shown). These findings suggest that inhibition of ERK activity does not account either for the survival-promoting actions of olomoucine in the absence of NGF or for its toxicity in the presence of NGF. They also indicate that ERK activation is not required for NGF-promoted survival.
Figure 5: Effects of flavopiridol and olomoucine on JNK activity. JNK activity was determined using GST-c-Jun protein as substrate as described under ``Experimental Procedures.'' A, neuronally differentiated PC12 cultures (first through fifth lanes) or naive PC12 cells (sixth through eleventh lanes) were deprived of serum or NGF, respectively, for the indicated times. The densitometric values at each time point are normalized such that the zero time point is defined as 1. B, neuronally differentiated PC12 cultures were deprived of NGF in the presence or absence of flavopiridol (fl) or olomoucine (olo) for the indicated times. The densitometric values at each time point are normalized such that the zero time point is defined as 1. C, constant amounts of activated JNK activity were assayed in vitro in the presence or absence of the indicated concentrations of flavopiridol or olomoucine. The densitometric values at each concentration of inhibitor are normalized such that the JNK activity at time 0 in the absence of drug is defined as 1.
We determined whether flavopiridol and
olomoucine might promote survival of neuronal PC12 cells by preventing
the induction of JNK activity through the JNK-specific
mitogen-activated protein kinase cascade (see (43) for
review). As shown in Fig. 5B, treatment of neuronal
PC12 cultures with either flavopiridol (1 µM) or
olomoucine (200 µM) did not decrease the level of JNK
activation that occurred in vivo when the cells were deprived
of NGF. We next determined whether the CDK inhibitors might directly
affect JNK activity in vitro. At concentrations similar to
that at which it promotes survival in culture, olomoucine inhibited
direct phosphorylation of GST-c-Jun by activated JNK in vitro.
As shown in Fig. 5C, olomoucine inhibited GST-c-Jun
phosphorylation with an IC of
100 µM. In
contrast, a 12 µM concentration of flavopiridol (12-fold
higher than that required for survival of neuronal cultures) had no
effect on the in vitro phosphorylation of GST-c-Jun, while 50
µM flavopiridol significantly inhibited phosphorylation (Fig. 5C). These observations indicate that it is
unlikely that the CDK inhibitors used here promote survival by
preventing activation of JNK and that at least flavopiridol does not
work by inhibiting JNK activity.
Figure 6: Flavopiridol and olomoucine promote survival of rat sympathetic neurons following withdrawal of NGF, whereas isoolomoucine does not. Primary cultures of neonatal rat superior cervical ganglion neurons were grown in the presence of NGF for 3 days prior to withdrawal. Concurrent with withdrawal of NGF, the cultures were treated with the appropriate agents. Each data point is the mean ± S.E. of three samples and is expressed relative to the number of neurons present in each well at the time of NGF withdrawal. A, effects of flavopiridol (1 µM) on the time course of survival of sympathetic neurons after withdrawal of NGF; B, effects of various doses of flavopiridol on day 2 survival of NGF-deprived sympathetic neurons; C, effects of olomoucine and isoolomoucine (200 µM) on the time course of survival of sympathetic neurons after withdrawal of NGF; D, effects of various doses of olomoucine on day 2 survival of NGF-deprived sympathetic neurons.
Figure 7:
Phase-contrast micrographs of rat
sympathetic neurons. Rat sympathetic neurons were maintained in
NGF-free medium for various times and treated with the following: no
additives, day 2 (A); NGF, day 2 (B); 1 µM flavopiridol, day 2 (C); 1 µM flavopiridol,
day 6 (D); 200 µM olomoucine, day 2 (E);
and 200 µM olomoucine, day 6 (F). Magnification
375.
Fig. 7illustrates the morphology of NGF-deprived neurons cultured with flavopiridol and olomoucine. We have previously noted that a number of agents that support the survival of such neurons do not maintain neurites and that these degenerate within several days(6, 28) . However, as shown in Fig. 7, healthy processes are clearly visible in the drug-treated cultures 6 days after NGF depletion. In contrast, as with most other agents that rescue sympathetic neurons after NGF withdrawal, somatic hypertrophy was not maintained by the drugs (Fig. 7).
Because inhibition of protein synthesis promotes survival of
neuronally differentiated PC12 cells and sympathetic
neurons(11, 14, 23) , we examined the effect
of flavopiridol (Fig. 8A) and olomoucine (Fig. 8B) on leucine incorporation. As shown in Fig. 8A, 1 µM flavopiridol inhibited
leucine incorporation by 25% in cultures of either neuronally
differentiated PC12 cells or sympathetic neurons. Olomoucine (200
µM) inhibited leucine incorporation in sympathetic neuron
cultures by
30%, but had no effect on protein synthesis in PC12
cell cultures (Fig. 8B). Martin et al.(44) reported that at least 80% inhibition of protein synthesis
is required to protect sympathetic neurons from NGF withdrawal.
Accordingly, it is therefore unlikely that the mechanism by which
flavopiridol and olomoucine rescue post-mitotic neurons and PC12 cells
is by inhibition of protein synthesis.
Figure 8: Effects of flavopiridol (A) and olomoucine (B) on protein synthesis by neuronally differentiated PC12 cells and rat primary sympathetic neurons. Data are expressed relative to untreated cultures. Each data point is the mean ± S.E. of three samples.
Figure 9: Flavopiridol and olomoucine do not promote survival of naive PC12 cells following withdrawal of serum and also cause death in presence of serum. A, replicate naive PC12 cell cultures were preincubated with or without the indicated inhibitor for 16 h prior to withdrawal of serum and then treated for additional days as indicated; B, replicate naive PC12 cell cultures were grown as indicated in the presence of serum-containing RPMI 1640 medium without drug pretreatment and assessed at various times for numbers of live cells. Each data point is the mean ± S.E. of three samples.
We have hypothesized that trophic factors such as NGF prevent
the death of proliferating neuroblasts by guiding them through the cell
cycle, as is the case with naive PC12 cells, and inhibit the death of
post-mitotic neurons by suppressing their inappropriate re-entry into
the cell cycle. Testing this model has involved examining whether
agents that are known to prevent cell cycle progression promote
neuronal survival. In previous studies, we showed that multiple agents
that induce cell cycle arrest, including G blockers(29) , N-acetylcysteine(28) ,
and chlorphenylthio-cAMP(33) , as well as induction of
dominant-negative Ras (15) all suppress the death of neuronal
cells caused by withdrawal of trophic support. Although these findings
support the cell cycle/apoptosis model, the mechanisms by which such
agents block the cell cycle remain largely unclear, and in each case,
there was the possibility that alternative actions might be responsible
for preventing death. To further and more directly evaluate the effects
of cell cycle inhibition on death of neurons, we employed two known
inhibitors of the cell cycle-related CDK family of kinases.
As
predicted from the cell cycle/apoptosis model and consistent with
previous findings with agents that induce cell cycle arrest, both
flavopiridol and olomoucine suppressed the death of post-mitotic PC12
cells and sympathetic neurons caused by NGF deprivation. Significantly,
the dose relationship for inhibition of thymidine incorporation by
flavopiridol and olomoucine correlates very closely with their
abilities to block cell death. In accordance with these observations,
several lines of evidence support the potential role of cyclins and
CDKs in the process of neuronal death. Brooks et al.(26) reported that elevated Cdc2 activity is observed
concurrent with NGF deprivation and death of neuronally differentiated
PC12 cells. In addition, NGF treatment of PC12 cells leads to a
reduction of both Cdk2 and Cdc2 activities (45) and increases
the levels of p21, a G phase CDK inhibitor(46) .
Outside the nervous system, Cdc2 activity has been shown to be required
for lymphocyte granule protease-mediated cell death(21) .
Furthermore, cyclin A induction and an increase in cyclin A/Cdc2
activity occur in association with apoptosis induced pharmacologically
or by Myc overexpression(47, 48) .
Surprisingly,
the CDK inhibitors were not effective in preventing the death of
proliferation-competent naive PC12 cells after withdrawal of trophic
support. This is in sharp contrast to our earlier observations with
other cell cycle-blocking agents. The G/S blockers
mimosine, deferoxamine, and ciclopirox as well as N-acetylcysteine are equally effective in protecting naive
PC12 cells, post-mitotic neuronally differentiated PC12 cells, and
sympathetic neurons from loss of trophic
support(28, 29) . This indicates that the manner in
which the cell cycle is blocked may be critical in determining whether
an agent promotes neuronal survival. For instance, we showed that
G
blockers, and not S, G
, or M phase blockers,
promote survival of neuronal cells (29) . This, however, does
not imply that cells die in a cell cycle stage-specific manner, as
Lindenboim et al.(49) have shown that apoptosis of
PC12 cells occurs at each phase of the cell cycle.
How might
flavopiridol and olomoucine act to save post-mitotic cells and not
proliferation-competent cells? It is possible that nonspecific toxicity
may negate any potential attenuation of cell death in naive PC12
cultures. The increased degree of CDK inhibitor-induced toxicity when
compared with neuronal PC12 cells, however, suggests additional causes
for this difference. One attractive explanation is that inhibition of
CDKs in cycling PC12 cells forces a conflict between this aspect of
cell cycle arrest and one or more endogenous proliferative signals.
Naive PC12 cells, by origin, are transformed and therefore must possess
oncogenic signals that have yet to be defined. The cell cycle/apoptosis
hypothesis would predict that the resulting conflict would lead to
apoptotic death. In support of this view, flavopiridol promotes the
death not only of naive PC12 cells, but also of other transformed cell
lines even in the presence of trophic support. ()After
long-term exposure to NGF, PC12 cells attain a nondividing phenotype
lacking the oncogenic cue present in proliferating cultures and thereby
eliminate the signaling conflict present in naive cultures.
Although
the presently demonstrated effects of flavopiridol and olomoucine on
survival are consistent with their ability to prevent cell cycle
progression and are therefore consistent with the cell cycle/apoptosis
theory, alternative actions could account for these observations. The
most likely of these is the inhibition of kinases other than CDKs. For
instance, as discussed above, olomoucine inhibits ERKs only somewhat
less potently than CDKs. However, our data presented here, as well as
those of Virdee and Tolkovsky(50) , indicate that ERKs are not
required for evocation or prevention of neuronal cell death. Another
possibility is that these agents inhibit either the activation or
activity of JNK family members. This possibility, however, does not
appear to account for the differences we observed here between
post-mitotic and naive PC12 cells. In addition, we show that
flavopiridol and olomoucine do not prevent the intracellular activation
of JNK. While olomoucine does suppress JNK activity in vitro at concentrations similar to those at which it blocks death,
flavopiridol proved to be a relatively poor inhibitor of JNK (IC > 12 µM). Thus, it seems unlikely that inhibition
of either JNK activation or activity accounts for the protective
effects of flavopiridol or olomoucine.
In summary, we have found that two distinct CDK inhibitors prevent the death of post-mitotic neuronal cells. Proliferating PC12 cells, however, were not saved by these agents. These results are consistent with the model that conflicts between concurrent proliferative and nonproliferative signals may be one important factor in apoptosis due to trophic withdrawal. These findings may have important implications for developing therapeutic strategies for the treatment of neuronal injury and degeneration.