By
From The Amgen Institute, the Ontario Cancer Institute, and the Department of Medical Biophysics and the Department of Immunology, University of Toronto, Toronto, Ontario, Canada M5G 2C1
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Abstract |
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Aberrant activation of cell cycle molecules has been postulated to play a role in apoptosis ("catastrophic cell cycle"). Here we show that in noncycling developing thymocytes, the cyclin- dependent kinase Cdk2 is activated in response to all specific and nonspecific apoptotic stimuli tested, including peptide-specific thymocyte apoptosis. Cdk2 was found to function upstream of the tumor suppressor p53, transactivation of the death promoter Bax, alterations of mitochondrial permeability, Bcl-2, caspase activation, and caspase-dependent proteolytic cleavage of the retinoblastoma protein. Inhibition of Cdk2 completely protected thymocytes from apoptosis, mitochondrial changes, and caspase activation. These data provide the first evidence that Cdk2 activity is crucial for the induction of thymocyte apoptosis.
Key words: cyclin-dependent kinase 2; apoptosis; cell cycle; thymocyte ![]() |
Introduction |
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Apoptosis or programmed cell death (PCD) is required for all multicellular organisms to maintain the homeostasis of their organ systems. Failure to invoke effective programmed cell death can result in developmental abnormalities, cancer, or autoimmune diseases, whereas increased apoptosis produces degenerative diseases of the brain or immunodeficiencies (1). Apoptosis can be triggered by numerous different stimuli, all of which converge at the common checkpoint of mitochondrially regulated death induction (1) and caspase activation (5).
Apoptosis and mitosis have many features in common,
including cytoskeletal changes, nuclear envelope breakdown, and chromatin condensation, and it has been speculated that apoptosis may result from a form of aberrant cell
cycling called "catastrophic mitosis" (10). This idea is
supported by the fact that various gene products that have
marked effects on cell cycle control, such as p53, retinoblastoma protein (Rb),1 Cdc25, Max, c-Myc, or E2F-1,
also regulate susceptibility to apoptosis (13). Thus, it has
been shown that overexpression of the tumor suppressor
p53 can induce either growth arrest or apoptosis, depending on the cell type (21). Conversely, loss of p53 function
in mice produces resistance to apoptotic stimuli such as
-irradiation (22, 23). Furthermore, deficiency of another
cell cycle regulator, E2F-1, in mice resulted in an enlarged thymus in these mutant mice, implying a possible role of
E2F-1 as a proapoptotic molecule (19, 20). Inversely, the
loss of the tumor suppressor and cell cycle regulator Rb in
mice leads to increased cell death, further confirming the
close interaction between apoptotic pathways and cell cycle
pathways (24).
Members of the cyclin-dependent kinase (Cdk) family of serine/threonine kinases are known to be key regulators of eukaryotic cell cycle progression (25). Different Cdk catalytic subunits and their activating cyclin subunits operate as control checkpoints during cell cycle progression. Cdk2 is crucial for the progression from the G1 to the S phase of the cell cycle. Inhibition of Cdk2 activity in vitro has been shown to protect cultured sympathetic neurons and heart muscle cells from apoptosis (26, 27). However, Cdk2 inhibition can also lead to cell death in tumor cell lines (28). Whether Cdk2 and the aberrant activation of the cell cycle machinery have an apoptotic function necessary for normal development has yet to be addressed.
CD4+CD8+ thymocytes are noncycling cells which are
sensitive to many apoptotic stimuli in vitro and in vivo, including glucocorticoids, ionizing irradiation, heat shock,
and CD95 (9). Physiologically, CD4+CD8+ thymocytes
undergo negative selection and clonal deletion required for
the induction and maintenance of immunological tolerance
(29). We report in developing thymocytes that Cdk2 is activated in response to all specific and nonspecific apoptotic
stimuli tested, including -irradiation and peptide-specific
negative thymocyte selection. Inhibition of Cdk2 completely prevented all aspects of thymocyte apoptosis and
blocks peptide-specific thymocyte death.
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Materials and Methods |
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Mice.
p53 gene-deficient, Bcl-2 transgenic (Tg), DO.11.10 OVA TCR-Apoptosis Induction and Inhibitors.
Freshly isolated thymocytes from BALB/c mice were cultured in RPMI 1640 medium (10% FCS, 10Detection of Apoptosis.
Total cell numbers of viable and apoptotic cells were determined by trypan blue exclusion. Relative percentages of viable and apoptotic CD4+CD8+ thymocytes were determined by triple staining with anti-CD4-PE, anti-CD8-FITC, and the vital chromogenic dye, 7AAD (34). The results were expressed as the percentage of viable thymocytes remaining after 22 h, calculated as follows: (number of viable CD4+CD8+ thymocytes after stimulation)/(number of viable CD4+CD8+ thymocytes cultured under the same conditions in the absence of stimulation) × 100. For the detection of cycling and apoptotic cells, thymocytes were stained with propidium iodide (PI). After different periods of stimulation, thymocytes were harvested, washed once in PBS (0.5% glucose), and fixed in cold 70% ethanol overnight. Fixed cells were pelleted to remove ethanol and stained with PI (final concentration 50 µmol/ml) for 30 min at room temperature. Apoptosis-mediated membrane changes were determined via staining with Annexin V (R&D Systems). PI and Annexin V staining of thymocytes was determined by cytofluorometry using a FACSCaliburTM (Becton Dickinson).Kinase Assays.
After different periods of stimulation, thymocytes were harvested and lysed, and proteins were immunoprecipitated using Abs against Cdk2 (amino acids [aa] 283-298), Cdk4 (aa 282-303), and Cdc2 (aa 278-297) (all from Santa Cruz Biotechnology). Cdk2 and Cdc2 kinase activities in immunoprecipitates were assayed using [Immunoprecipitations and Western Blotting.
Thymocytes were lysed in 1% NP-40 lysis buffer. Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with Abs reactive to Cdc2, Cdk2, Cdk4, Cdk7, Pctaire-2, Cdc25A, cyclins A, E, D1, D2, B, and D3, E2F-1, p27Kip1, caspase 2, and p53 (clone 240) (all from Santa Cruz Biotechnology), p21 (Calbiochem), Bcl-XL (Transduction Laboratories), Bcl-2 (PharMingen), caspase 3 (gift of Dr. R. Sekaly, McGill University, Montreal, Quebec, Canada), and Rb (clone G3-245 reactive to an aa 300-380 epitope of Rb, PharMingen; and clone C-15 reactive against aa 914-928, Santa Cruz Biotechnology). The anti-caspase 8-specific Abs were developed in our Institute and were a kind gift of Dr. R. Hakem (Amgen Institute). Immunoprecipitations were performed using protein A-Sepharose. Optimal Ab concentrations and conditions for immunoprecipitations were determined in pilot studies.m Disruption.
In Vitro Negative Thymocyte Selection.
Thymocytes were purified from P14 Tg mice, which express anFetal Thymic Organ Culture.
DO.11.10 males were mated with estrous BALB/c females. On day 16 of gestation, pregnant females were killed and embryonic thymi were harvested and placed in culture. Thymi were microdissected and placed on the surface of 0.8-mm filters (Nucleopore) resting on Gelfoam gelatin sponges (Upjohn) in RPMI 1640 medium supplemented with 10% FCS. Each sponge was placed into a 3.5-cm plastic dish in 2 ml of medium. Cultures were incubated at 37°C. Chicken (c)OVA protein was added at 1 mg/ml on day 1 of culture, and thymi were analyzed 20 and 40 h later. Cell numbers were determined by counting in the presence of trypan blue. ![]() |
Results |
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To determine
whether the cell cycle machinery has a role in apoptosis of
noncycling G1 CD4+CD8+ thymocytes, we analyzed the
activity of several Cdks in these cells. Freshly isolated thymocytes were treated with different death stimuli such as
dexamethasone, heat shock, -irradiation, and CD95 for
different time points, and Cdk activities of stimulated as
well as control thymocytes were assessed in in vitro kinase assays. Surprisingly, increased activity of the cyclin-dependent kinase Cdk2 was detected within 30 min of dexamethasone activation and peaked at 5 h (Fig. 1 A). Cdk2 activity was rapidly increased in response to all apoptotic
stimuli tested, including dexamethasone, anti-Fas (CD95)
cross-linking, heat shock, or
-irradiation (Fig. 1 B). No
changes in the kinase activities of Cdk4 or Cdc2 were observed after induction of apoptosis (Fig. 1, A and C). Cdc2 activation was also not observed using an anti-Cdc2 phosphorylation epitope-specific Ab indicative of Cdc2 activation. Cdc2 and Cdk4 activities were readily detectable in
cycling T lymphoma cells (not shown). The expression
levels of molecules involved in the cell cycle, such as Cdk2,
Cdk4, Cdc2, Cdk7, Pctaire-2, Cdc25A, cyclins A, B, D1,
D2, D3, and E, p21, p27Kip1, and E2F-1, did not change 5 h
after stimuli (not shown). Cdk2 was found to bind to cyclin A and E thymocytes after treatment with dexamethasone and
-irradiation. Immunoprecipitations of both cyclin A and cyclin E showed that after
-irradiation or dexamethasone, histone H1 was phosphorylated, suggesting that both cyclin A and cyclin E have a role in Cdk2 activation (Fig. 1 E). These results show that the induction of
thymocyte apoptosis by dexamethasone,
-irradiation, heat
shock, or CD95 cross-linking leads to the activation of the
cell cycle regulator Cdk2.
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To test
whether Cdk2 activity was required for the induction of
thymocyte apoptosis, the effects of two specific inhibitors of Cdk2, olomoucine and roscovitine (39, 40), were examined. These inhibitors are purine analogues that selectively
inhibit the activity of Cdk2 and Cdc2 by specific binding
to the ATP-binding pocket. Both molecules completely
inhibited dexamethasone-induced Cdk2 activation in thymocytes (Fig. 1 D), and blocked thymocyte apoptosis after
stimulation with dexamethasone, heat shock, -irradiation, PMA, or the DNA damaging agent, etoposide (Fig. 2 A).
PI staining confirmed that thymocytes were in the G1
phase of the cell cycle and that induction of apoptosis did
not correlate with cell cycle progression (Fig. 2 D). A
"point of no return" was reached between 15 and 30 min
after treatment with the apoptotic stimulus such that a
Cdk2 blocker added after this time was unable to prevent apoptosis (Fig. 2 B). Addition of other cell cycle blockers
such as TGF-
1 or rapamycin, used at the optimal concentrations, had no effect on the kinetics or extent of thymocyte death (Fig. 2 C). Interestingly, although CD95
cross-linking led to strong Cdk2 activation (Fig. 1 B),
Cdk2 blockers did not inhibit CD95-mediated thymocyte
death (Fig. 2 A). In fact, CD95-mediated apoptosis was consistently enhanced in the presence of the Cdk2 blockers. These results imply that CD95 uses a pathway other
than the one used by the other inducers, i.e., a receptor/
caspase 8 pathway instead of a nucleus/mitochondrial/
caspase 9 pathway (41). It should be noted that in our
screen, CD95 activation is the only thymocyte death stimulus so far that cannot be blocked by Cdk2 inhibition.
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The process of clonal deletion and selection-triggered thymocyte death is a fundamental mechanism required for the maintenance of lymphocyte homeostasis and immunotolerance. CD4+CD8+ thymocytes expressing TCRs which recognize self-antigens with high affinity/ avidity are clonally deleted via apoptosis, leading to the removal of T cells that express TCRs with potentially harmful self-reactivity (thymic tolerance [29]). To test whether Cdk2 is a physiological regulator of thymocyte apoptosis, we induced apoptosis of CD4+CD8+ immature thymocytes by anti-CD3 cross-linking (42). Fig. 2 A shows that Cdk2 inhibitors were able to block anti-CD3-mediated apoptosis of immature thymocytes.
To further investigate the role of Cdk2 in clonal deletion, we used an in vitro negative selection system using
thymocytes from P14 Tg mice. P14 Tg mice express a rearranged TCR /
chain reactive to the p33 peptide of
LCMV. P14 Tg CD4+CD8+ thymocytes undergo apoptosis after culture with APCs pulsed with different concentrations of the deleting p33 peptide. Thymocytes from P14
Tg mice underwent apoptosis in a p33 peptide dose-
dependent fashion which was inhibited by the addition of
Cdk2 blockers (Fig. 3 A). Importantly, induction of peptide-specific apoptosis of P14 Tg thymocytes triggered
Cdk2 kinase activity (Fig. 3 B). Inhibition of Cdk2 did not
interfere with TCR-mediated proximal signaling events or
with TCR internalization, which is a functional measure of
antigen receptor-mediated activation (not shown). Moreover, inhibition of Cdk2 by olomoucine blocked OVA-mediated negative selection of CD4+CD8+ OVA-specific TCR Tg thymocytes in fetal thymic organ cultures (FTOCs; Fig. 3 C). Cdk2 blockers did not interfere with
positive thymocyte selection in reaggregation culture assays
(not shown), indicating that Cdk2 has a specific role in
peptide-specific thymocyte apoptosis.
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Where does Cdk2 function in
the hierarchy of apoptosis? Disruption of m due to the
opening of mitochondrial pores has been invariably associated with apoptosis and is an early common denominator of cell death (43). Alterations in mitochondria lead to the release through the outer mitochondrial membrane of molecules such as cytochrome c and the apoptosis-inducing
factor (AIF), and the activation of the caspase cascade (7,
35, 36, 44).
To assess whether Cdk2 acts upstream or downstream of
mitochondrial events, we examined changes in m using
cytometry and the fluorochromic dye, DiOC6(3). Thymocytes were stimulated either with dexamethasone or
anti-CD95, and the mitochondria changes of
m were
assessed at different time points. The first changes in thymocyte
m were observed 2 h after dexamethasone treatment, and
m was significantly disrupted after 5 h (Fig. 4
A). Addition of Cdk2 inhibitors blocked dexamethasone-induced losses of
m (Fig. 4 A). CD95-mediated
m
disruption and apoptosis still occurred in the presence of
Cdk2 inhibitors (Figs. 2 A and 4 A), implying that Cdk2
inhibition per se does not interfere with opening of mitochondrial pores. Since
m is regulated by Bcl-2 family members (43, 49), we also tested Cdk2 activation in Bcl-2 Tg thymocytes (31, 50). Although overexpression of Bcl-2
protected thymocytes from dexamethasone- and irradiation-induced cell death and disruption of
m (31, 50),
Cdk2 was still activated in Bcl-2 Tg thymocytes in response to these apoptotic stimuli (not shown).
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Caspase activation is a crucial event in apoptosis, and
caspases can function upstream or downstream of mitochondrial m disruption (8, 9). To determine where
Cdk2 acts during apoptosis with regard to the caspase activation cascade, processing of different caspases was assessed
in thymocytes after treatment with different apoptotic
stimuli in the presence or absence of Cdk2 inhibitors. Within 2 h after dexamethasone and
-irradiation, caspase
3 (Cpp32) and caspase 8 activation was observed in thymocytes whereas caspase 2 (nedd2) processing was first observed
3 h after death induction. Caspase activation peaked at 5 h
after induction of cell death (Fig. 4 B, and data not shown).
However, activation of caspase 3 (Fig. 4 B), caspase 2 (Fig.
4 C), or caspase 8 (not shown) did not occur after blocking
of Cdk2 kinase activity. These results show that Cdk2 acts
upstream of Bcl-2,
m, and caspases.
During apoptosis, various cell cycle regulatory molecules such as p21 and Rb are proteolytically
cleaved by caspases. In particular, proteolytic processing of
the G1 to S cell cycle gatekeeper Rb (Rb) has been previously reported in TNF- and CD95-treated tumor cell
lines (51, 52). Rb and Cdk2 were found to coimmunoprecipitate in developing thymocytes (not shown). Induction
of thymocyte apoptosis in response to dexamethasone, irradiation, heat shock, or anti-CD95 correlated with the appearance of a second smaller Rb protein (
Rb; Fig. 4 D).
Although it has been shown that Rb is cleaved by caspase 3 (53) and in thymocytes
Rb was found to be a proteolytic cleavage product of Rb mediated by caspases,
Rb was still
observed in caspase 3 gene-deficient mice (not shown).
The earliest detectable Rb cleavage (
Rb) occurred 5 h
after dexamethasone treatment (Fig. 4 D). Cdk2 inhibitors
or transgenic overexpression of Bcl-2 in thymocytes prevented cleavage of Rb in response to dexamethasone (Fig.
4 D, and data not shown). These results demonstrate that Cdk2 acts upstream of mitochondrial pore opening, Bcl-2,
caspase activation, and proteolytic cleavage of the cell cycle
regulator Rb. The functional consequences of Rb cleavage
are not known. Since
Rb can only be observed downstream of the caspase effector phase and still binds to Cdk2
(not shown), the generation of
Rb might function as a
regulatory feedback loop that could influence Cdk2 and/or
E2F-1 activity.
How is Cdk2 activity mechanistically linked
to apoptotic mitochondrial events? Various members of the
Bcl-2 family of mitochondrial gatekeepers are phosphorylated on serine/threonine residues (9, 49). Although Bcl-2
and Bcl-XL contain consensus sites for Cdk2 activity, we
could not detect Cdk2-mediated phosphorylation of either
Bcl-2 or Bcl-XL in in vitro kinase assays (not shown). The
tumor suppressor p53 is a substrate for Cdk2 in the DNA
repair response (54), and thymocytes mutated in p53 are resistant to -irradiation-induced apoptosis but still susceptible to dexamethasone and antigen receptor-mediated cell
death (22, 23). The effect of the p53 mutation has been
mapped upstream of apoptotic mitochondrial events (55).
Therefore, we tested whether p53 is a target for Cdk2
activity during thymocyte apoptosis after -irradiation. In
vitro kinase assays using immunoprecipitated Cdk2 from
-irradiated and dexamethasone-treated thymocytes showed
that Cdk2 can phosphorylate p53 (Fig. 5 A). Moreover,
p53 was found to associate with Cdk2 in thymocytes (Fig. 5 B). To test the effect of Cdk2 activity on p53 expression,
we analyzed the levels of p53 protein in
-irradiated thymocytes in the presence or absence of Cdk2 inhibitors. Although p53 protein accumulated to significant levels after
treatment of cells with
-irradiation alone, little p53 accumulation was observed when cells were treated with
-irradiation in the presence of Cdk2 blockers (Fig. 5 C). Irradiation-induced p53 protein accumulation was caused by
enhanced p53 protein stability but not by p53 gene transactivation (not shown).
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To further corroborate the regulation of p53 by Cdk2,
we examined the expression of the p53-inducible death
promoter Bax (56) by Northern blotting. Induction of
thymocyte apoptosis by -irradiation led to an increase in
Bax transcripts, and Bax transactivation was found to depend on Cdk2 activity (Fig. 5 D). In dying thymocytes we
found only induction of the p53-regulated death promoter
Bax but not transactivation of the p53-regulated gene p21
(not shown). Cdk2 kinase activity was normally induced in
-irradiated p53
/
thymocytes (not shown), indicating
that p53 is downstream of Cdk2 in the thymocyte death
signaling cascade. Since thymocytes from p53
/
mice are
not resistant to dexamethasone or antigen receptor-mediated apoptosis, other molecules must exist that link Cdk2
activation to cell death.
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Discussion |
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The identification of Cdk2 as a master regulator of cell
death provides the first evidence for a shared signaling
pathway that integrates multiple death signaling pathways
into a common death effector cascade in developing thymocytes. The hierarchy of Cdk2 action suggests that Cdk2
is the earliest known common signaling element required
for thymocyte apoptosis in response to environmental and
developmental cues such as negative selection. This hypothesis is based on the following findings: (a) all nonspecific (-irradiation, heat shock, dexamethasone) and specific (peptide-mediated thymocyte cell death) apoptotic
stimuli tested induce rapid activity of the cyclin-dependent
kinase Cdk2 in noncycling thymocytes; (b) Cdk2 acts upstream of the opening of mitochondrial pores, Bcl-2 family
proteins, caspase activation, p53, and proteolytic processing
of Rb; (c) inhibition of Cdk2 completely protects thymocytes from
-irradiation, heat shock, dexamethasone,
PMA, anti-CD3, and peptide-mediated cell death; (d) Cdk2
and the tumor suppressor, p53, constitutively associate in
thymocytes and activated Cdk2 isolated from apoptotic
thymocytes can phosphorylate p53; (e) Cdk2 regulates p53
protein accumulation and transactivation of the p53-inducible death promoter, Bax, after
-irradiation. These data
provide the first link between the cell cycle machinery and apoptosis in normal development and differentiation and
indicate that Cdk2 is a crucial kinase that mediates cell
death in thymocyte maturation and thymocyte selection.
Our results indicate that after -irradiation, Cdk2 phosphorylates and stabilizes p53, which then transactivates the
death promoter Bax. Interestingly, in dying thymocytes we
found only induction of the p53-regulated death promoter
Bax but not transactivation of the p53-regulated gene p21
(59), suggesting that only certain gene loci are accessible for
p53 transactivation or that other cofactors act in concert
with p53 to modulate gene expression in a tissue- and lineage-specific manner. Although p53 protein levels were increased in thymocytes after dexamethasone and
-irradiation, it has been shown in p53 gene-deficient mice that
p53 protects thymocytes only from DNA damage, and not
from dexamethasone or antigen receptor-mediated cell
death (22, 23), implying that other downstream molecules
exist that link Cdk2 to apoptosis. Preliminary evidence
from our laboratory implies that the glucocorticoid receptor which is required for dexamethasone-mediated cell
death can be phosphorylated by activated Cdk2 and coimmunoprecipitates with Cdk2 in dying thymocytes. Besides
association with the ligand, phosphorylation of the glucocorticoid receptor is required for its translocation from the
cytoplasm into the nucleus (60). In addition to the glucocorticoid receptor, other orphan steroid receptors such as
Nur77 might be molecular targets for Cdk2 kinase activity.
It has been shown that mitochondria are early checkpoints that integrate multiple death signaling pathways into
a common Ced4/caspase-regulated effector mechanism.
Opening of mitochondrial pores, mitochondrial swelling,
disruption of m, and release of proapoptotic molecules, including cytochrome c and apoptosis-inducing factor (AIF),
from the mitochondrial intermembrane spaces into the cytoplasm have all been implicated as fundamental mechanisms
that initiate and propel the effector phase of apoptosis.
Posttranslational modification and the balance between death
suppressors, such as Bcl-2 and Bcl-XL, and death promoters, including Bax and Bad, are crucial mechanisms of
mitochondrial integrity and the apoptotic effector phase
(9, 49). Our results show that Cdk2 acts upstream of
m
disruption, Bax and Bcl-2, and caspase activation in developing thymocytes. Moreover, whereas loss of the mitochondrial transmembrane potential can only be observed 2 h
after addition of death stimuli, Cdk2 kinase activity is induced very rapidly and a "point of no return" was reached
between 15 and 30 min after treatment with the apoptotic stimulus, such that a Cdk2 blocker added after this time
was unable to prevent apoptosis. Thus, our results indicate
that Cdk2 is the earliest known common denominator that
can integrate many independent apoptotic signals into one
common effector pathway.
Although CD95 (Fas) stimulation induced Cdk2 activity
in thymocytes, inhibition of Cdk2 did not block CD95-mediated apoptosis. In fact, Cdk2 inhibition enhanced the
susceptibility to CD95 killing. So far, CD95-mediated apoptosis is the only death signal in thymocytes that does not
rely on Cdk2 activation. Although apoptosis in response to
-irradiation, heat shock, dexamethasone, or peptide-specific negative thymocyte selection requires active transcription of death genes, apoptosis after CD95 killing can occur
in the presence of RNA or protein synthesis inhibitors (9).
Moreover, enucleated cells can undergo apoptosis after
CD95 activation, suggesting that all components necessary
for CD95-mediated apoptosis are present in cells and that
CD95 activation can directly trigger the apoptotic machinery. It should be noted that both Cdk2 inhibitors roscovitine and olomoucine do not prevent transcription or
translation in noncycling neurons (63) and that inhibition
of Cdk2 did not regulate transactivation of the FasL in thymocytes (not shown).
The specific Cdk2 blockers olomoucine and roscovitine
are purine analogues that inhibit Cdk2 kinase activity by
specifically binding to the Cdk2 ATP-binding site (39, 40).
At concentrations at which olomoucine and roscovitine
blocked apoptosis in in vivo thymocyte cultures, these inhibitors had no effects on in vitro kinase activity of PKC-
,
Cdk4, Cdk6, Abl, cAMP- or GMP-dependent protein kinases (PKA, PKG), mitogen-activated protein kinase (MAPK; extracellular signal regulatory kinase [ERK]1, ERK2), Src
family kinases, glycogen synthase kinase 3 (GSK3), casein
kinase, receptor tyrosine kinases, myosin light chain kinase,
p38/HOG, or stress-activated protein kinase (SAPK)/c-Jun
NH2-terminal kinases (JNKs) (39; and data not shown).
Through their unique selectivity for Cdk2, roscovitine and
olomoucine provide a unique opportunity to study the role of Cdk2 in thymocyte apoptosis. However, we cannot exclude that roscovitine and olomoucine inhibit a yet unidentified kinase, and our results need to be confirmed using
genetic model systems. Thus, genetic systems for inducible
and thymocyte-specific inactivation/activation of Cdk2
need to be developed in the future. However, our results showing that all death stimuli lead to Cdk2 kinase activity
in thymocytes and that two different Cdk2 kinase inhibitors, but not other inhibitors that block G1 to S progression, inhibit thymocyte apoptosis strongly suggest that
Cdk2 is a key kinase involved in thymocyte apoptosis.
Cdk2 is crucial for the progression from the G1 to the S
phase of the cell cycle. Inhibition of Cdk2 activity in vitro
has been shown to protect cultured sympathetic neurons
and heart muscle cells from apoptosis (26, 27). Our results
in noncycling CD4+CD8+ thymocytes provide the first
evidence that Cdk2 has a crucial role in the induction of
cell death during normal development. However, it has
been shown that Cdk2 inhibition can also lead to cell death
in tumor cell lines, and Cdks are frequently deregulated in
tumors (28). Similarly, we found that in contrast to developing, noncycling thymocytes, inhibition of Cdk2 in four
different thymic lymphoma cell lines led to rapid apoptosis
and sensitized T cell tumors to anti-CD3, dexamethasone,
or -irradiation-mediated cell death (not shown). These
results suggest that Cdk2 has functions in the apoptotic
processes that regulate normal development which are
distinct from those in tumorigenesis and transformation.
Thus, specific inhibition of Cdk2 could be exploited to
sensitize tumor cells to apoptosis by anticancer drugs,
whereas molecular inhibition of Cdk2 might protect normal, noncycling cells from the adverse effects of the same drugs.
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Footnotes |
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Address correspondence to Josef M. Penninger, The Amgen Institute, 620 University Ave., Suite 706, Toronto, Ontario, Canada M5G 2C1. Phone: 416-204-2241; Fax: 416-204-2278; E-mail: jpenning{at}amgen.com
Received for publication 12 November 1998 and in revised form 23 December 1998.
A. Hakem and J.M. Penninger are supported by a grant from the Medical Research Council of Canada.We thank L. Meijer and R. Sekaly for reagents; R. Hakem, K. Bachmaier, Y.Y. Kong, H. Nishina, A. Oliveira-dos-Santos, L. Zhang, V. Stambulic, L. Harrington, J. Krassopolous, D. Bentley, J. Woodgett, P. Ohashi, T.W. Mak, and G. Kroemer for helpful discussions; and M. Saunders for scientific editing.
Abbreviations used in this paper
aa, amino acid(s);
Cdk, cyclin-dependent
kinase;
m, mitochondrial transmembrane potential;
FTOC, fetal thymic organ culture;
GST, glutathione S-transferase;
LCMV, lympholytic
choriomeningitis virus;
PI, propidium iodide;
PK, protein kinase;
Rb, retinoblastoma protein;
Tg, transgenic.
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References |
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1. | White, E.. 1993. Death-defying acts: a meeting review on apoptosis. Genes Dev. 7: 2277-2284 [Medline]. |
2. | Russell, J.H., C.L. White, D.Y. Loh, and R.-P. Meleedy. 1991. Receptor-stimulated death pathway is opened by antigen in mature T cells. Proc. Natl. Acad. Sci. USA. 88: 2151-2155 [Abstract]. |
3. | Green, D.R., and D.W. Scott. 1994. Activation-induced apoptosis in lymphocytes. Curr. Opin. Immunol. 6: 476-487 [Medline]. |
4. | King, L.B., and J.D. Ashwell. 1993. Signaling for death of lymphoid cells. Curr. Opin. Immunol. 5: 368-373 [Medline]. |
5. | Martin, J.S., and R.D. Green. 1995. Protease activation during apoptosis: death by thousand cuts? Cell. 82: 349-352 [Medline]. |
6. | Miller, D.K.. 1997. The role of the Caspase family of cysteine proteases in apoptosis. Semin. Immunol. 9: 35-49 [Medline]. |
7. | Cohen, G.M.. 1997. Caspases: the executioners of apoptosis. Biochem. J. 326: 1-16 [Medline]. |
8. | Salvesen, S.G., and M.V. Dixit. 1997. Caspases: intracellular signaling by proteolysis. Cell. 91: 443-446 [Medline]. |
9. | Penninger, M.J., and G. Kroemer. 1998. Molecular and cellular mechanisms of T lymphocyte apoptosis. Adv. Immunol. 68: 51-144 [Medline]. |
10. | Raff, M.C.. 1992. Social controls on cell survival and cell death. Nature. 356: 397-400 [Medline]. |
11. | Evan, G.I., L. Brown, M. Whyte, and E. Harrington. 1995. Apoptosis and the cell cycle. Curr. Opin. Cell Biol. 7: 825-834 [Medline]. |
12. | Shi, L., W.K. Nishioka, J. Thang, E.M. Bradbury, D.W. Litchfield, and A.H. Greenberg. 1994. Premature p34cdc2 activation required for apoptosis. Science. 263: 1143-1145 [Medline]. |
13. | Evan, G.I., A.H. Wyllie, C.S. Gilbert, T.D. Littlewood, H. Land, M. Brooks, C.M. Waters, L.Z. Penn, and D.C. Hancock. 1992. Induction of apoptosis in fibroblasts by c-myc protein. Cell. 69: 119-128 [Medline]. |
14. | Mazel, S., D. Burtrum, and H.T. Petrie. 1996. Regulation of cell division cycle progression by Bcl-2 expression: a potential mechanism for inhibition of programmed cell death. J. Exp. Med. 183: 2219-2226 [Abstract]. |
15. |
Linette, G.P.,
Y. Li,
K. Roth, and
S.J. Korsmeyer.
1996.
Cross talk between cell death and cell cycle progression:
BCL-2 regulates NFAT-mediated activation.
Proc. Natl.
Acad. Sci. USA.
93:
9545-9552
|
16. | Vairo, G., K.M. Innes, and J.M. Adams. 1996. Bcl-2 has a cell cycle inhibitory function separable from its enhancement of cell survival. Oncogene. 13: 1511-1519 [Medline]. |
17. | Bates, S., and K.H. Vousden. 1996. p53 in signaling checkpoint arrest or apoptosis. Curr. Opin. Genet. Dev. 6: 12-18 [Medline]. |
18. | Brady, H.J., G. Gil-Gomez, J. Kirberg, and A.J. Berns. 1996. Bax alpha perturbs T cell development and affects cell cycle entry of T cells. EMBO (Eur. Mol. Biol. Organ.) J. 15: 6991-7001 [Abstract]. |
19. | Field, S.J., F.Y. Tsai, F. Kuo, A.M. Zubiaga, W.G. Kaelin Jr., D.M. Livingston, S.H. Orkin, and M.E. Greenberg. 1996. E2F-1 functions in mice to promote apoptosis and suppress proliferation. Cell. 85: 549-561 [Medline]. |
20. | Yamasaki, L., T. Jacks, R. Bronson, E. Goillot, E. Harlow, and N.J. Dyson. 1996. Tumor induction and tissue atrophy in mice lacking E2F-1. Cell. 85: 537-548 [Medline]. |
21. |
Lotem, J., and
L. Sachs.
1997.
Cytokine suppression of protease activation in wild-type p53-dependent and p53-independent apoptosis.
Proc. Natl. Acad. Sci. USA.
94:
9349-9353
|
22. | Clarke, A.R., C.A. Purdie, D.J. Harrison, R.G. Morris, C.C. Bird, M.L. Hooper, and A.H. Wyllie. 1993. Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature. 362: 849-852 [Medline]. |
23. | Lowe, S.W., E.M. Schmitt, S.W. Smith, B.A. Osborne, and T. Jacks. 1993. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature. 362: 847-849 [Medline]. |
24. | Lee, E.Y., C.-Y. Chang, N. Hu, Y.-C. Wang, C.-C. Lai, K. Herrup, W.-H. Lee, and A. Bradley. 1992. Mice deficient for Rb are nonviable and show defect in neurogenesis and haematopoiesis. Nature. 359: 288-294 [Medline]. |
25. | Morgan, O.D.. 1997. Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu. Rev. Cell Dev. Biol. 13: 261-291 . [Medline] |
26. | Wang, J., and K. Walsh. 1996. Resistance to apoptosis conferred by Cdk inhibitors during myocyte apoptosis. Science. 273: 359-361 [Abstract]. |
27. |
Park, D.S.,
B. Levine,
G. Ferrari, and
L.A. Greene.
1997.
Cyclin dependent kinase and dominant negative cyclin dependent kinase 4 and 6 promote survival of NGF-deprived
sympathetic neurons.
J. Neurosci.
17:
8975-8983
|
28. | Meikrantz, W., S. Gisselbrecht, S.W. Tam, and R. Schlegel. 1994. Activation of cyclin A-dependent protein kinases during apoptosis. Proc. Natl. Acad. Sci. USA. 91: 3754-3758 [Abstract]. |
29. | Von Boehmer, H.. 1996. CD4/CD8 lineage commitment: back to instruction? J. Exp. Med. 183: 713-715 [Medline]. |
30. | Murphy, K.M., A.B. Heimberger, and D.Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+CD8+ TCRlo thymocytes in vivo. Science. 250: 1720-1723 [Medline]. |
31. | Strasser, A., A.W. Harris, and S. Cory. 1991. Bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship. Cell. 67: 889-899 [Medline]. |
32. | Donehower, L.A., M. Harvey, B.L. Slagle, M.J. McArthur, C.A. Montgomery Jr., J.S. Butel, and A. Bradley. 1992. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature. 356: 215-221 [Medline]. |
33. | Pircher, H., T.W. Mak, R. Lang, W. Ballhausen, E. Rueedi, H. Hengartner, R.M. Zinkernagel, and K. Buerki. 1989. T cell tolerance to Mlsa encoded antigens in T cell receptor V beta 8.1 chain transgenic mice. EMBO (Eur. Mol. Biol. Organ.) J. 8: 719-727 [Abstract]. |
34. | Nishina, H., K.D. Fischer, L. Radvanyi, A. Shahinian, R. Hakem, E.A. Rubie, A. Bernstein, T.W. Mak, J.R. Woodgett, and J.M. Penninger. 1997. Stress-signalling kinase Sek1 protects thymocytes from apoptosis mediated by CD95 and CD3. Nature. 385: 350-353 [Medline]. |
35. | Zamzami, N., P. Marchetti, M. Castedo, T. Hirsch, S.A. Susin, B. Masse, and G. Kroemer. 1996. Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis. FEBS Lett. 384: 53-57 [Medline]. |
36. | Susin, S.A., N. Zamzami, M. Castedo, T. Hirsch, P. Marchetti, A. Macho, E. Daugas, M. Geuskens, and G. Kroemer. 1996. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J. Exp. Med. 184: 1331-1341 [Abstract]. |
37. | Pircher, H., K. Brduscha, U. Steinhoff, M. Kasai, T. Mizuochi, R.M. Zinkernagel, H. Hengartner, B. Kyewski, and K.P. Muller. 1993. Tolerance induction by clonal deletion of CD4+8+ thymocytes in vitro does not require dedicated antigen-presenting cells. Eur. J. Immunol. 23: 669-674 [Medline]. |
38. | Sebzda, E., V.A. Wallace, J. Mayer, R.S. Yeung, T.W. Mak, and P.S. Ohashi. 1994. Positive and negative thymocyte selection induced by different concentrations of a single peptide. Science. 263: 1615-1618 [Medline]. |
39. | Meijer, L., and S.H. Kim. 1997. Chemical inhibitors of cyclin-dependent kinases. Methods Enzymol. 283: 113-128 [Medline]. |
40. |
Meikrantz, W., and
R. Schlegel.
1996.
Suppression of apoptosis by dominant negative mutants of cyclin-dependent protein kinases.
J. Biol. Chem.
271:
10205-10209
|
41. | Rouquet, N., K. Carlier, P. Briand, J. Wiels, and V. Joulin. 1996. Multiple pathways of Fas-induced apoptosis in primary culture of hepatocytes. Biochem. Biophys. Res. Commun. 229: 27-35 [Medline]. |
42. | Smith, C.A., G.T. Williams, R. Kingston, E.J. Jenkinson, and J.J. Owen. 1989. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature. 337: 181-184 [Medline]. |
43. | Kroemer, G., N. Zamzami, and S.A. Susin. 1997. Mitochondrial control of apoptosis. Immunol. Today. 18: 44-51 [Medline]. |
44. | Vander Heiden, M.G., N.S. Chandel, E.K. Williamson, P.T. Schumacker, and C.B. Thompson. 1997. Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell. 91: 627-637 [Medline]. |
45. |
Yang, J.,
X. Liu,
K. Bhalla,
C.N. Kim,
A.M. Ibrado,
J. Cai,
T.I. Peng,
D.P. Jones, and
X. Wang.
1997.
Prevention of
apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.
Science.
275:
1129-1132
|
46. |
Kluck, R.M.,
W.-E. Bossy,
D.R. Green, and
D.D. Newmeyer.
1997.
The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis.
Science.
275:
1132-1136
|
47. |
Liu, X.,
C.N. Kim,
J. Pohl, and
X. Wang.
1996.
Purification
and characterization of an interleukin-1beta-converting enzyme family protease that activates cysteine protease P32
(CPP32).
J. Biol. Chem.
271:
13371-13376
|
48. | Liu, X., C.N. Kim, J. Yang, R. Jemmerson, and X. Wang. 1996. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 86: 147-157 [Medline]. |
49. | Reed, J.C.. 1997. Double identity for protein of the Bcl-2 family. Nature. 387: 773-776 [Medline]. |
50. | Korsmeyer, S.J.. 1995. Regulators of cell death. Trends Genet. 11: 101-105 [Medline]. |
51. | Dou, Q.P., B. An, K. Antoku, and D.E. Johnson. 1997. Fas stimulation induces RB dephosphorylation and proteolysis that is blocked by inhibitors of the ICE protease family. J. Cell. Biochem. 64: 586-594 [Medline]. |
52. |
Tan, X.,
S.J. Martin,
D.R. Green, and
J.Y.J. Wang.
1997.
Degradation of retinoblastoma protein in tumor necrosis factor- and CD95-induced cell death.
J. Biol. Chem.
272:
9613-9616
|
53. | Chen, W.D., G.A. Otterson, S. Lipkowitz, S.N. Khleif, A.B. Coxon, and F.J. Kaye. 1997. Apoptosis is associated with cleavage of a 5 kDa fragment from RB which mimics dephosphorylation and modulates E2F binding. Oncogene. 14: 1243-1248 [Medline]. |
54. | Wang, Y., and C. Prives. 1995. Increased and altered DNA binding of human p53 by S and G2/M but not G1 cyclin- dependent kinases. Nature. 376: 88-91 [Medline]. |
55. | Marchetti, P., M. Castedo, S.A. Susin, N. Zamzami, T. Hirsch, A. Macho, A. Haeffner, F. Hirsch, M. Geuskens, and G. Kroemer. 1996. Mitochondrial permeability transition is a central coordinating event of apoptosis. J. Exp. Med. 184: 1155-1160 [Abstract]. |
56. | Boehme, S.A., and M.J. Lenardo. 1996. TCR-mediated death of mature T lymphocytes occurs in the absence of p53. J. Immunol. 156: 4075-4078 [Abstract]. |
57. |
McCurrach, M.E.,
T.M. Connor,
C.M. Knudson,
S.J. Korsmeyer, and
S.W. Lowe.
1997.
Bax-deficiency promotes drug
resistance and oncogenic transformation by attenuating p53-dependent apoptosis.
Proc. Natl. Acad. Sci. USA.
94:
2345-2349
|
58. | Miyashita, T., S. Krajewski, M. Krajewska, H.G. Wang, H.K. Lin, D.A. Liebermann, B. Hoffman, and J.C. Reed. 1994. Tumor suppressor p53 is a regulator of Bcl-2 and bax gene expression in vitro and in vivo. Oncogene. 9: 1799-1805 [Medline]. |
59. | Attardi, L.D., S.W. Lowe, J. Brugarolas, and T. Jacks. 1996. Transcriptional activation by p53, but not induction of the p21 gene, is essential for oncogene-mediated apoptosis. EMBO (Eur. Mol. Biol. Organ.) J. 15: 3693-3701 [Medline]. |
60. | Krstic, D.M., I. Rogatsky, R.K. Yamamoto, and J.M. Garabedian. 1997. Mitogen-activated and cyclin-dependent protein kinases selectively and differentially modulate transcriptional enhancement by the glucocorticoid receptor. Mol. Cell. Biol. 17: 3947-3954 [Abstract]. |
61. | De Franco, D.B., M. Qi, K.C. Borror, M.J. Garabedian, and D.L. Brautigan. 1991. Protein phosphatase types 1 and/or 2A regulate nucleocytoplasmic shuttling of glucocorticoid receptors. Mol. Endocrinol. 5: 1215-1228 [Abstract]. |
62. |
Hoecks, W., and
B. Groner.
1990.
Hormone-dependent
phosphorylation of the glucocorticoid receptor occur mainly
in the amino-terminal transactivation domain.
J. Biol. Chem.
265:
5403-5408
|
63. | Krucher, N.A., L. Meijer, and M.H. Roberts. 1997. The cyclin dependent kinase (cdk) inhibitors, olomoucine and roscovitine, alter the expression of a molluscan circadian pacemaker. Cell. Mol. Neurobiol. 17: 495-507 [Medline]. |