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Mol Cell Biol, May 1998, p. 2912-2922, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Commitment and Effector Phases of the Physiological
Cell Death Pathway Elucidated with Respect to Bcl-2, Caspase, and
Cyclin-Dependent Kinase Activities
Kevin J.
Harvey,
James F.
Blomquist, and
David S.
Ucker*
Department of Microbiology and Immunology,
University of Illinois College of Medicine, Chicago, Illinois 60612
Received 20 November 1997/Accepted 3 February 1998
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ABSTRACT |
Physiological cell deaths occur ubiquitously throughout biology and
have common attributes, including apoptotic morphology with
mitosis-like chromatin condensation and prelytic genome digestion. The
fundamental question is whether a common mechanism of dying underlies
these common hallmarks of death. Here we describe evidence of such a
conserved mechanism in different cells induced by distinct stimuli to
undergo physiological cell death. Our genetic and quantitative biochemical analyses of T- and B-cell deaths reveal a conserved pattern
of requisite components. We have dissected the role of cysteine
proteases (caspases) in cell death to reflect two obligate classes of
cytoplasmic activities functioning in an amplifying cascade, with
upstream interleukin-1
-converting enzyme-like proteases activating
downstream caspase 3-like caspases. Bcl-2 spares cells from death by
punctuating this cascade, preventing the activation of downstream
caspases while leaving upstream activity undisturbed. This observation
permits an operational definition of the stages of the cell death
process. Upstream steps, which are necessary but not themselves lethal,
are modulators of the death process. Downstream steps are effectors of,
and not dissociable from, actual death; the irreversible commitment to
cell death reflects the initiation of this downstream phase. In
addition to caspase 3-like proteases, the effector phase of death
involves the activation in the nucleus of cell cycle kinases of the
cyclin-dependent kinase (Cdk) family. Nuclear recruitment and
activation of Cdk components is dependent on the caspase cascade,
suggesting that catastrophic Cdk activity may be the actual effector of
cell death. The conservation of the cell death mechanism is not
reflected in the molecular identity of its individual components,
however. For example, we have detected different cyclin-Cdk pairs in
different instances of cell death. The ordered course of events that we
have observed in distinct cases reflects essential thematic elements of
a conserved sequence of modulatory and effector activities
comprising a common pathway of physiological cell death.
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INTRODUCTION |
Although interest in the process of
physiological cell death has grown enormously in recent years, the
mechanism of death has remained enigmatic. While the induction of
physiological death in diverse cell types is effected by a wide variety
of stimuli, a common morphology, described as apoptosis, ensues in all
cases. The commonality of morphology has led to the belief that
disparate inducers trigger distinct signaling events which ultimately
converge in a common biochemical pathway of death. This hypothesis
suggests a division of the biochemical process into upstream events
that are specific for individual inducers and downstream steps,
comprising the common pathway, which bring about the actual demise of
the cell.
Since most cell deaths in the nematode Caenorhabditis
elegans are induced in a lineage-determined program, the simple
pathway of death elucidated in that species (17) is likely
to be revealing of downstream steps. Cell death in C. elegans is dependent on the activation of Ced3, a cysteine
protease (77, 79), and is inhibited by Ced9 (27).
In mammalian cells, a group of Ced3 homologs, termed caspases
(1), appears to play a role in virtually all of the
physiological cell deaths studied to date. These enzymes cleave on the
carboxyl-terminal side of aspartate residues within distinct
recognition motifs. Each caspase is synthesized as a proenzyme and
activated by cleavage at internal sites, potentially by the same or
another caspase class (66, 77). This leads to the notion
that caspases function in an ordered cascade, with members of one
family activating members of the next. Data consistent with this
pattern have been obtained from studies in vitro (41, 60,
65).
Of the large family of mammalian caspases, caspase 3 is closely
homologous to Ced3 and appears to be involved widely in cell deaths
(50, 65). Nonetheless, specific caspases seem not to be
associated uniquely with distinct cases of death, and gene-targeting experiments reveal that the absence of a single caspase has extremely limited consequences for cell death responsiveness (38, 39).
Similarly, a family of ced9-related death response
modulatory genes exists in mammals; the most closely related homolog,
bcl-2, is functionally interchangeable with ced9
in the worm (28, 73). These gene products do not function in
all mammalian cell deaths (61, 72). Moreover, while the
products of some bcl-2 gene family members have
death-sparing activity (6, 7), others exert the opposite
effect (52, 78).
Several cellular proteins, among them poly(ADP-ribose) polymerase
(PARP), nuclear lamins, fodrin, and DNA-dependent protein kinase
(10, 16, 34), are targets for cleavage by various caspases.
In cells spared from death, for example by Bcl-2, these proteolytic
events do not occur (9, 13, 18). Still, the cleavage of none
of these proteins has been shown to be essential for the cell death
response (42, 54, 74). The specific consequences of caspase
activation which are lethal are unknown.
It may be that the consequence of protease activity is the specific
activation of distinct death effectors. We have proposed that essential
genes involved in cell division may be critically involved in cell
death as well and that the difficulty in identifying distal effector
steps genetically reflects the indispensable function of those gene
products in cell life (67). Data from several groups have
shown that cell cycle catastrophes, the precocious expression of
mitosis-like cyclin-dependent histone kinases (Cdks), are
associated with a variety of physiological cell deaths and that the
inhibition of death by Bcl-2 is associated with alterations in the
expression and localization of these Cdk proteins (22, 23, 29, 36,
40, 46, 47, 58, 59, 70).
We have taken advantage of the death-sparing activities of Bcl-2 and
two viral caspase inhibitors, CrmA and p35 (64, 77), to
dissect the mechanism of cell death in two separate cellular paradigms.
These studies allow us to draw a generalized skeletal pathway of the
death-associated biochemical activities discussed above and demonstrate
the requisite involvement of these different classes of activities in a
conserved and ordered pathway by which cells die physiologically.
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MATERIALS AND METHODS |
Cell culture.
Freshly cloned cells were grown in RPMI 1640 medium (Whittaker Bioproducts, Walkersville, Md.) supplemented with
glutamine (2 mM), 2-mercaptoethanol (50 µM), and heat-inactivated
fetal bovine serum (10% [vol/vol]; Tissue Culture Biologicals,
Tulare, Calif.). Cell death was assessed routinely as trypan blue dye uptake.
Transfections.
Transfectant clones were selected in 1.5 mg
of G418 sulfate (Gibco BRL, Grand Island, N.Y.) per ml added 24 h
after electroporation (250 V and 960 µF) of 107 cells
with 20 µg of linearized pSFFV-Neo/Bcl-2 (30) or
constructs of CrmA, CrmAmut, or p35 in pcDNA3 vector
(64). For each construct, at least 10 independent
transfectant clones were isolated; in each case, the cell death
responses of these clones were similar. Expression of transfected Bcl-2
and CrmA proteins was confirmed by Western blot analysis (data not
shown) with antibodies specific for Bcl-2 (Santa Cruz Biotechnology;
Santa Cruz, Calif.) and CrmA (Pharmingen, San Diego, Calif.). The cell
death responses of vector-only controls were unaltered from those of
untransfected parental cells.
Cell cycle analysis.
Cell cycle analysis was performed by
staining with propidium iodide (37). Briefly,
106 cells were washed twice with phosphate-buffered saline
(PBS) and fixed in 1 ml of 50% ethanol for 30 min on ice. The cells were pelleted and treated in the dark with 1 mg of RNase A per ml and
50 µg of propidium iodide per ml in 400 µl of PBS for 30 min at
room temperature. Stained cells were analyzed cytofluorimetrically on
an EPICS 753 or Elite ESP sorter (Coulter, Hialeah, Fla.). Cell cycle
distributions were quantified with Multicycle AV software (Phoenix Flow
Systems, Inc., San Diego, Calif.). The fraction of cells with
subdiploid DNA content was consistent with that found by other measures
of cell death.
Extract preparation.
Crude cytoplasmic and nuclear extracts
were prepared from cells after two washes in PBS and one in buffer HKEB
(100 mM HEPES, 10 mM MgCl2, 5 mM EGTA, 100 µM
phenylmethylsulfonyl fluoride [PMSF], 1 mM dithiothreitol, 50 µM
-S-ATP). The cells then were incubated (10 min at 25°C) in buffer
HKEB with 50 µg of digitonin per ml (35). Following
centrifugation (20 min at 13,000 × g), the resulting cytoplasmic supernatants were frozen at 
70°C. Nuclear supernatants were prepared by dispersing the pellets in the same buffer with three
1-s 50-W pulses of a Vibra-cell sonicator (Sonics and Materials, Danbury, Conn.) and centrifuging the suspension (10 min at 13,000 × g). Protein concentrations were determined by the
Bradford (Bio-Rad, Richmond, Calif.) or bicinchoninic acid
(55) microtiter plate assay. Preferential extraction of
dying cells can lead to an augmentation of apparent specific enzyme
activity; we have avoided this artifact by monitoring the extent of
extraction as indicated by trypan blue dye inclusion during digitonin
treatment. Typically, complete permeabilization was achieved with 300 µl of HKEB-digitonin buffer per 5 × 106 cells. The
efficacy of cellular fractionation was monitored as well. The activity
of the cytoplasmic enzyme L-lactate dehydrogenase (Sigma
Chemical Co., St. Louis, Mo.) was assayed in cellular fractions. We
generally recovered 80% of lactate dehydrogenase activity
(approximately 250 pmol of -NADH/min/105 cells) in
cytoplasmic fractions and the remainder (approximately 60 pmol of
-NADH/min/105 cells) in nuclear fractions. The total
protein content of cytoplasmic extracts was typically threefold greater
than that of nuclear extracts from both dying and viable populations.
Caspase assay and reagents.
Interleukin-1-converting
enzyme (ICE)-like and caspase 3-like activities were assayed in 50-µl
reaction mixtures with fluorogenic reporter substrate peptides specific
for ICE (acetyl-Tyr-Val-Ala-Asp-4-methylcoumaryl-7-amide [YVAD-MCA;
Peninsula Laboratories, Belmont, Calif.]) (66) and the
mammalian Ced3 homolog caspase 3 (acetyl-Asp-Glu-Val-Asp-4-methylcoumaryl-7-amide [DEVD-MCA; Peptides
International, Louisville, Ky.]). The substrate peptide (200 µM) was
incubated at 37°C with cytoplasmic extract (5 µg of extract for the
DEVD-MCA reaction and 20 µg of extract for the YVAD-MCA assay) in 100 mM HEPES-10% sucrose-10 mM dithiothreitol-0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Fluorescence was measured after 120 min (excitation wavelength, 360 nm;
emission wavelength, 460 nm) with a Cytofluor 2350 fluorescence plate
reader (Millipore, Marlborough, Mass.). A standard curve was prepared
with 7-amino-4-methyl-coumarin (AMC; Peninsula Laboratories). Stock
solutions of peptide reagents, including aldehyde-derivatized inhibitors (Peptides International), were prepared in anhydrous dimethyl sulfoxide.
Histone kinase assay.
Histone (Sigma; type VS) was modified
by addition of 2-iminobiotin as described previously (31).
Nuclear extracts (5 µg diluted to 10 µl in 80 mM sodium
-glycerophosphate, 10 mM MgCl2, 5 mM EGTA [pH 7.5]
[EB]) were mixed with 10 µl of assay mix consisting of 40 mM HEPES
(pH 7.3), 10 mM EGTA (pH 8.0), 20 mM MgCl2, 400 µl of
[-32P]ATP (5 µCi/mmol), and 150 µg of
2-iminobiotinyl histone per ml. After incubation (30 min at 25°C),
the reactions were stopped by the addition of 1 ml of 50 mM
NaCO3 (pH 10.5)-0.5 M NaCl-5 mM EDTA-50 µl of
avidin-agarose, and the mixtures were rotated for 60 min at 4°C.
Protein A-agarose (Sigma) was used as a negative control. Agarose
pellets were washed three times with 1 ml of the same buffer and once
with 1 ml of PBS and were eluted twice with 100 µl of 100 mM sodium
acetate (pH 4.0)-0.5 M NaCl. The eluates were pooled, and
32P-incorporation was determined by scintillation analysis.
In addition, Cdk activity was assayed with a synthetic substrate
peptide derived from the site of H1 phosphorylation mediated by Cdk1
(Cdc2). In these assays, 440 µM histone H1 peptide (Upstate Biotechnology, Inc., Lake Placid, N.Y.) was substituted for
2-iminobiotinyl histone. The reactions were stopped after 10 min by the
addition of 5 µl of 5% H3PO4, and the
mixtures were spotted on duplicate 1.5- by 1.5-cm squares of P-81
paper, washed three times for 15 min in 1%
H3PO4, dried, and subjected to scintillation
analysis. Purified p34cdk1 complex (from
starfish oocytes [Promega, Madison, Wis.]) was used as a positive
control.
Western immunoblot analysis.
Nuclear and cytoplasmic extract
proteins (100 µg/well) were run on 10% polyacrylamide gels and
transferred to Immobilon P (Millipore, Bedford, Mass.). The blots were
probed with rabbit antisera raised against either a C-terminal peptide
of cyclin A or a peptide corresponding to the conserved PSTAIRE domain
of Cdk1 and Cdk2 (Santa Cruz Biotechnology). The bands were visualized by the luminol reaction (Renaissance; DuPont NEN, Boston, Mass.).
Cdk analysis by p9cksHs2 adsorption and
by immunodepletion.
p9cksHs2 agarose (20 µl; Promega) was incubated at 4°C for 60 min with 40 µg of
nuclear extract in 100 µl of EB supplemented with 10 mg of bovine
serum albumin per ml. The p9cksHs2 agarose was
pelleted, washed three times with the same buffer, and analyzed for
histone activity with biotinylated H1 substrate. Supernatants also were
analyzed for activity. For immunodepletions, 100 µg of nuclear
extract in 200 µl of buffer HKEB was incubated overnight at 4°C
with 1 µg of specific antibody (Santa Cruz Biotechnology). Immune
complexes were cleared by incubation for 2 h with 20 µl of
protein A-agarose (Sigma); the kinase activity remaining in the
supernatant was assayed. Immunodepletion of Cdk components was
confirmed by Western blot analysis of both immune complex pellets and
supernatants.
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RESULTS |
In distinct cases, Bcl-2 sparing of cell death is independent of
the associated G1 arrest.
In an effort to test the
hypothesis that diverse cases of cell death have a common effector
pathway, we sought to identify general features by correlating results
from two independent cases of physiological cell death. One is the
classical model of glucocorticoid-mediated death of T cells,
represented here by the DO11.10 T-cell hybridoma. The second, in cells
of the B-lymphocyte lineage, reflects a connection between cell death
and enforced cell cycle progression. For this model, we used DE, a
pre-B-cell line transformed by a temperature-sensitive v-Abl
oncoprotein (12).
DE cells proliferate at the permissive temperature (34°C), but at the
restrictive temperature (39.5°C) they spontaneously
arrest in the
G
1 phase of the cell cycle and undergo physiological
cell
death (Table
1; Fig.
1A) (
12). Arrest in a
postmitotic
(especially G
1) compartment of the cell cycle
(
2,
26) is
a common feature of physiological cell death
(
67). The glucocorticoid-mediated
death response of the T
hybridoma DO11.10 also involves a G
1 arrest
(Table
1).
Figure
2A reveals the kinetics of death
in DO11.10
cells treated with the glucocorticoid dexamethasone.
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FIG. 1.
In B cells, physiological cell death and associated
caspase 3-like protease and Cdk activities are inhibited by Bcl-2, but
upstream ICE-like protease activity is not. The kinetics of induction
of death (A), cytoplasmic YVAD-specific ICE-like (B) and DEVD-specific
caspase 3-like (C) activities, and nuclear H1-specific Cdk activity (D)
upon shift to the nonpermissive temperature were monitored in DE cells
and DE/Bcl-2 cells. Cysteine protease activities were not detectable in
nuclear extracts, and only basal levels of H1 kinase activity were
present in cytoplasmic extracts of dying cells. The data presented are
derived from one experiment in which a single set of extracts was used
for all activity determinations. These data are representative of three
separate experiments. In other experiments, elevated DEVD-specific
caspase activity was not detectable at time points before 3 h of
temperature shift.
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FIG. 2.
Glucocorticoid-mediated cell death of T cells and
associated caspase 3-like protease and Cdk activities are inhibited by
Bcl-2, but upstream ICE-like protease activity is not. The kinetics of
induction of death (A), cytoplasmic YVAD-specific ICE-like (B) and
DEVD-specific caspase 3-like (C) activities, and nuclear H1-specific
Cdk activity (D) were monitored after treatment with 10 6
M dexamethasone in DO11.10 cells and in representative clones of
DO11.10 transfected with bcl-2 and crmA. The
behaviors of DO11.10 cells transfected with p35 and with CrmA were
identical; data for the DO11.10/p35 transfectants is omitted for
clarity of presentation. Note that histone kinase activity in panel D
was measured with histone H1 substrate peptide (see Materials and
Methods). Comparable kinetics of appearance of kinase activity,
although with lower apparent specific activity, were observed with
biotinylated H1 substrate (see Fig. 6 for a comparison). The data
presented are derived from one experiment in which a single set of
extracts was used for all activity determinations. These data are
representative of three separate experiments.
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Bcl-2 expression does not relieve the G
1 arrest associated
with the induction of death in DE cells, but it significantly inhibits
the actual cell death response upon temperature shift (Table
1;
Fig.
1A). Bcl-2 also inhibits the death of DO11.10 cells (Fig.
2A) but has
no effect on the associated G
1 arrest (Table
1).
These data
map critical effector steps of cell death sensitive
to Bcl-2 inhibition
downstream of cell cycle arrest.
ICE-like protease activity is necessary for cell death but is not
itself lethal.
We characterized quantitatively the appearance of
caspase activities in the cell death responses of these lymphocyte cell lines. Among caspases, those most closely related to ICE generally exhibit activity directed to the tetrapeptide sequence YVAD, modified from ICE cleavage site 2 in the interleukin-1 precursor (43, 63, 66). We used a fluorogenic substrate derived from this tetrapeptide, YVAD-MCA, to assay ICE-like activity.
A transient twofold induction of ICE-like activity in DE cells occurs
within 2 h of the death-inducing shift to the restrictive
temperature and precedes death by several hours (Fig.
1A and B).
Permeabilization with digitonin (see Materials and Methods) revealed
that virtually all of the death-associated ICE-like activity is
soluble
and cytoplasmic (data not shown). Strikingly, although
it spares DE
cells from death upon temperature shift, Bcl-2 exerts
no effect on the
appearance (Fig.
1A and B) or localization (data
not shown) of ICE-like
protease activity.
We obtained similar results when examining DO11.10 cells treated with
the glucocorticoid dexamethasone (Fig.
2B). A modest
twofold induced
peak of ICE-like activity appears early in the
DO11.10 death response,
roughly following the period of required
macromolecular synthesis
(
5,
11,
69). The YVAD-specific
activity in DO11.10 cells,
like that in DE cells, represents an
authentic caspase by several
criteria. It is inactivated in vitro
by the cysteine-reactive reagent
N-ethylmaleimide (NEM) and is
relatively insensitive to
inhibition by PMSF, a preferential inhibitor
of serine proteases (Fig.
3A). Moreover, two viral gene products
which have been shown to inhibit ICE-like proteases, the poxvirus
serpin CrmA and the baculovirus p35 protein (
76) (see
below),
inhibit this YVAD-specific activity in vivo (Fig.
2B) and in
vitro
(data not shown). Consistent with a previous characterization
of
ICE-like protease inhibition profiles (
43), the
aldehyde-derivatized
YVAD tetrapeptide (YVAD-CHO), as well as the
aldehyde-derivatized
DEVD tetrapeptide (DEVD-CHO), inhibits the
death-associated ICE-like
activities we detected (Fig.
3A) (see below).
The biphasic nature
of the YVAD-CHO inhibition curve suggests that this
activity may
comprise two (or more) individual caspases; for brevity,
we refer
to YVAD-specific caspase activity below as class I activity.
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FIG. 3.
Characterization in vitro of cell death-associated
cysteine protease activities. YVAD-specific ICE-like (A) and
DEVD-specific caspase 3-like (B) activities were assayed in the
presence of the protease inhibitors NEM and PMSF and the specific
aldehyde-derivatized tetrapetide inhibitors YVAD-CHO and DEVD-CHO.
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In addition to inhibiting class I activity, expression of CrmA and p35
inhibit glucocorticoid-mediated death in transfected
DO11.10 cells
(Fig.
2A) (also see reference
56). As with Bcl-2,
inhibition of death is not associated with relief from G
1
arrest
(Table
1). A mutant of the poxvirus
crmA gene that
lacks protease-inhibiting
activity (
65) fails to spare
transfected cells from cell death
(data not shown). These data
implicate class I caspase activity
as being critically involved in the
cell death response. In contrast
to CrmA and p35, the death-sparing
action of Bcl-2 has no effect
on the appearance of the class I activity
in DO11.10 cells (Fig.
2B). These results suggest that Bcl-2 acts to
interfere with cell
death at a step(s) distinct from cell cycle arrest
and CrmA-inhibitable
class I protease activation. Most simply, this is
consistent with
the biochemical action of Bcl-2 occurring downstream of
class
I activity.
Induction of caspase 3-like activity occurs downstream of the
action of Bcl-2.
We next asked where caspase 3-like activity,
representing the other major caspase family, mapped in the cell death
process. The protease activity of many members of this family of
caspases is directed to the peptide sequence DEVD, representing the
sequence of the caspase 3 cleavage site of PARP. We found that caspase 3-like protease activity, detected quantitatively with the fluorogenic peptide substrate, DEVD-MCA, also was activated in the cytoplasm of
lymphocytes induced to die. Again for ease, we refer to this DEVD-specific caspase activity as class III activity.
In contrast to the early peak of class I activity following a
death-inducing temperature shift, cytoplasmic class III activity
increases eightfold over a more prolonged course and is sustained
at
that higher level in dying DE cells (Fig.
1B and C). Still,
class III
activity appears early in the cell death process, well
before the
terminal manifestations of death such as chromatin
condensation, genome
digestion, and loss of plasma membrane integrity
(Fig.
1A). The pattern
we observe, in which the rise of class
III activity follows the class I
protease peak in vivo, is consistent
with previous work in vitro
demonstrating that caspase 3-like
proteases can be activated by
ICE-like proteases (
41,
60,
65). In cells spared from death
by expression of Bcl-2, class
III activity is not induced (Fig.
1C).
This is in striking contrast
to the ability of Bcl-2 to dissociate
class I activity and cell
death (Fig.
1B).
A similar pattern is seen in the death response of DO11.10 T cells
(Fig.
2B and C). A sustained 20-fold induction of class
III activity
follows the earlier induction of class I activity
triggered by
glucocorticoid treatment. By the criteria outlined
above for class I
activity, the class III activity detected is
the product of one or more
authentic caspases (Fig.
3B). Specifically,
class III activity in vitro
is inactivated by NEM but not by PMSF,
and it is preferentially
inhibited by the aldehyde-derivatized
DEVD tetrapeptide inhibitor
(DEVD-CHO) and not by the aldehyde-derivatized
YVAD-tetrapeptide
inhibitor (YVAD-CHO).
As in the case of DE cells induced to die, the death-sparing expression
of Bcl-2 in DO11.10 cells precludes the induction
of class III protease
activity (Fig.
2C). That class I activity
but not class III activity is
induced in the presence of death-sparing
Bcl-2 reveals that the
upstream activity itself is not lethal
and implies that downstream
class III activity must be necessary
for cell death.
Class III activity is necessary for death and is dependent on
upstream class I activity.
Class I and class III activities are
absent in cells spared from death by virtue of expression of the
transfected baculovirus protease inhibitor p35 (data not shown), which
interferes directly with class I and class III proteases
(76). Both classes of activities also are absent in
transfectants spared from death by the expression of CrmA (Fig. 2B and
C). Because CrmA is a specific inhibitor of class I proteases and a
very poor inhibitor of class III proteases (50), these data
are consistent with the notion, founded on in vitro studies (13,
20, 41, 60), that the appearance of class III protease activity
is dependent on upstream activation of class I proteases.
To explore further the dependence of class III activity on upstream
class I activity and the necessity of class III activity,
distinct from
class I activity, for cell death, we used the specific
tetrapeptide
inhibitors in vivo. Although these pharmacologic
agents are only
partially effective at inhibiting death, the involvement
of class I and
class III proteases in several cases of physiological
cell death has
been inferred from the limited death-sparing effects
afforded by in
vivo treatment (
15,
33,
49). Simultaneous
treatment of
DO11.10 cells with the DEVD-CHO inhibitor and death-inducing
glucocorticoid hormone inhibited class III activity and spared
cells
from glucocorticoid-mediated death (Fig.
4). Surprisingly,
the YVAD-CHO inhibitor
was much less effective in blocking the
appearance of class III
activity and cell death (Fig.
4) when
added at low (
1 µM)
concentrations, where its unique specificity
for class I proteases is
manifest (Fig.
3). YVAD-CHO inhibition
at high concentrations (
10
µM) may be due to cross-reactive inhibition
of the DEVD-specific
activity (
43).
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FIG. 4.
The appearance of class III caspase activity and cell
death is dependent on upstream class I activity. The ability of
aldehyde-derivatized tetrapeptide protease inhibitors to block in vivo
DEVD-specific class III caspase activity (A) and cell death (B) induced
by glucocorticoid treatment was monitored in DO11.10 cells. The
derivatized DEVD tetrapeptide itself (left) or the derivatized YVAD
tetrapeptide specific for class I proteases (right) was added at the
indicated concentrations simultaneously with or 1 h before the
addition of 106 M dexamethasone. DEVD-specific activity
was assayed 12 h after glucocorticoid addition. Cell death was
quantified by measurement of trypan blue inclusion 24 h after
hormone addition.
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Our kinetic data prompted us to explore whether the early induction of
class I activity precludes its effective inhibition
by added YVAD-CHO,
while the consequent and more delayed appearance
of class III activity
allows the more effective action of its
cognate inhibitor. Indeed, a
1-h pretreatment of cells with YVAD-CHO
before the application of the
death-inducing stimulus significantly
enhanced the effectiveness of the
class I inhibitor at low concentrations
to avert class III activity and
death (Fig.
4). Pretreatment had
little effect on the efficacy of the
DEVD-CHO inhibitor (Fig.
4). A similar pattern of inhibition was seen
with DE cells (data
not shown). We interpret these results to suggest
that pharmacologic
inhibition of the protease cascade and actual death
demand that
sufficient YVAD-CHO inhibitor be present in the cell at the
time
of class I protease activation to prevent subsequent class III
protease activation and its lethal consequences.
Together, these data indicate that both class I and class III protease
activities are necessary for cell death. However, class
I protease
activity is not sufficient for cell death and is not
itself lethal. The
upstream, transient class I activity appears
to function to activate
the downstream class III proteases; Bcl-2
acts to interfere with the
cell death response by punctuating
this caspase cascade. The appearance
of class III protease activity
downstream of Bcl-2 and operationally
not dissociable from actual
death in these cases is consistent with a
role within the effector
phase of the cell death process.
Induction of death-associated nuclear cyclin-dependent kinase
activity is dependent on the cytoplasmic caspase cascade.
Having
characterized class I caspase activity as an activator of class III
caspases and class III activity as a downstream effector, we sought to
determine where Cdks might act within the cell death process. Cdk
activity was quantified by a novel assay which employs biotinylated
histone H1 as the substrate, allowing for rapid and quantitative
retrieval of the 32P-phosphorylated product with
avidin-agarose. Since many different Cdks phosphorylate histone H1,
this assay allows the most inclusive assessment of Cdk activities while
effectively eliminating the experimental background of phosphorylation
of endogenous proteins. Because significant quantities of endogenous H1
are liberated with chromatin condensation and genome digestion in dying
cells (3), solubilized H1 substrate may mask
death-associated Cdk activity.
Histone kinase activity appeared transiently in the nuclei of DE cells,
with kinetics of induction similar to the cytoplasmic
class III
protease activity (Fig.
1D). The appearance of nuclear
histone kinase
also was associated with the induction of cell
death in DO11.10 cells
and was indistinguishable kinetically from
that of class III activity
(Fig.
2D). The presence of this three-
to fourfold-elevated level of H1
kinase activity in cells arrested
in the G
1 compartment is
closely linked to ultimate death. Proliferating
cells traversing
G
1 exhibit only a basal level of nuclear activity,
and the
induction of kinase activity does not ensue when cells
refractile to
death by virtue of Bcl-2 expression are subjected
to treatments that
induce death in susceptible cells (Fig.
1D
and
2D). Furthermore,
inhibitors of the caspase cascade, including
CrmA (Fig.
2D) and p35
(data not shown), also prevent the appearance
of nuclear kinase. These
data suggest that death-associated H1
kinase activity maps to the
effector phase of the cell death process.
Immunoprecipitation and immunoblot analyses reveal the death-associated
nuclear H1 kinase activities to represent authentic
cyclin-dependent
kinases. The H1 kinase activities in both DE
B cells and DO11.10 T
cells are composed of 60-kDa cyclin A protein
(Fig.
5A) complexed with ca. 34-kDa
polypeptides reactive with
antibodies specific for the conserved
PSTAIRE motif of the Cdk
family (Fig.
5B). In these lymphocyte cell
lines, all H1 kinase
activity appears to be dependent on cyclin A
exclusively. No other
cyclin species are detectable in nuclear extracts
containing death-associated
H1 kinase activity (data not shown), and
virtually all death kinase
activity is specifically and quantitatively
depleted by antibody
to cyclin A (Fig.
6A).
View larger version (51K):
[in this window]
[in a new window]
|
FIG. 5.
Immunoblot analysis of cell death-associated nuclear
cyclin A-dependent kinases. Cyclin A (A) and Cdk (B) components were
identified by immunoblot analysis. Cytoplasmic and nuclear extracts
were prepared from DE and DE/Bcl-2 cells (upper panels) incubated at
the permissive temperature (34°C; ) and at the restrictive
temperature (39.5°C; +) for 4 h and from DO11.10 cells and
representative clones of DO11.10 transfected with bcl-2
(DO/Bcl-2) and crmA (DO/CrmA) (lower panels) that were
untreated () or after treatment with 106 M
dexamethasone for 6 h (+). Analysis of a nuclear extract from
DO11.10 cells treated for 12 h with 2 µg of aphidicolin per ml
(Aph.) is included for comparison in panel A. The densitometric
quantitation of nuclear cyclin A expression is given in Table 2.
|
|
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 6.
Cell death-associated histone H1 kinase activities
represent cyclin A-dependent Cdks. (A) Death kinase activity was
analyzed by immunodepletion analysis. Nuclear extracts from untreated
DO11.10 cells or cells treated for 12 h with 106 M
dexamethasone were analyzed for histone kinase activity following
depletion with antibody specific for the indicated cyclin ( cyc) or
Cdk (Cdk 2) or with irrelevant anti-Myc (Myc) antibody. (B)
Nuclear extracts from DE cells incubated at the restrictive temperature
for 6 h and from DO11.10 cells treated with dexamethasone (Dex.)
as above, as well as viable control (Ctrl.) cells, were treated with
p9cksHs2 agarose to adsorb nuclear Cdk activity.
Adsorbed and unadsorbed Cdk activities from 1 µg of nuclear extract
were quantified. Note that for the experiments in panel A, H1 substrate
peptide was used, while the activity in panel B was assayed with
biotinylated H1. Comparison of DO11.10 activity in the two panels
demonstrates the difference in apparent specific activity obtained with
the different substrates.
|
|
By comparison, the identities of the multiple Cdk components are more
ambiguous. Although antigenically similar, subtle structural
differences among the death-associated Cdks exist and are manifest
as
differences in substrate specificities and accessory molecule
interactions. A peptide sequence that is phosphorylated by the
mitotic
kinase complex of cyclin B and Cdk1 serves as a substrate
for the death
kinase from DO11.10 cells but not that from DE cells
(Fig.
6). The
death kinase of DE cells is not adsorbed by
p9
cksHs2, the mammalian homolog of
p13
suc1, which has been shown to interact
specifically with Cdk1, Cdk2,
and Cdk3 (
48). On the other
hand, about half of the DO11.10
kinase activity is adsorbed by
p9
cksHs2 (Fig.
6B). Antibody to Cdk2 similarly
immunodepletes about half
of the total H1 kinase activity (data not
shown) and all of the
peptide-specific activity (Fig.
6A). These
results reveal that
different Cdks contribute to death kinases in
different cells
and that death kinase activity may be composed of more
than one
Cdk in the same cell.
Death-associated Cdk activity results from the recruitment of
cyclin molecules to the nucleus.
The appearance of cyclin
A-dependent death kinase in the nucleus correlates with elevated
nuclear cyclin A levels. In cells spared
from death by virtue of Bcl-2 or viral protease inhibitor expression,
nuclear cyclin A levels (Fig. 5A; Table 1), like cyclin A-dependent
kinase activity (Fig. 1D and 2D), do not accumulate. The approximately
twofold increase evident in dying DO11.10 cells and the more subtle
change in DE cells reveal the specificity of this death-associated
nuclear cyclin A recruitment (Fig. 5A; Table 2). Analysis of whole-cell
and cytoplasmic cyclin A levels (Fig. 5A) suggests that these
death-specific changes are not a consequence of the relatively small
changes in gross levels of total cellular cyclin A.
DE cells exhibit a substantial basal level of nuclear cyclin A, which
appears to change little with the induction of cell
death. This basal
level of nuclear cyclin A expression reflects
the steady-state fraction
(35%) of S-phase cells (or cells at
the G
1/S transition)
within the DE cell culture (Table
1); cyclin
A expression is maximal
within that compartment of the cell cycle
(
53). When the
entire population resides within the S phase,
due to arrest induced by
aphidicolin treatment, cyclin A levels
are elevated proportionately
(threefold greater by densitometric
analysis [Table
2]). Bcl-2
expression causes DE cells to transit
the cell cycle more slowly (also
see references
8 and
44),
and as
a consequence, the fraction of cells in the S phase (80%
of control
cells [Table
1]) and the levels of nuclear cyclin
A (90% of control
[Table
2]) both are diminished. The presence
of cyclin A in the
nucleus of viable cells therefore reflects
the extent of productive
cell cycle transit.
Under conditions of death induction, the presence of cyclin A in the
nucleus instead appears to reflect the extent to which
cells execute
the death process. In dying DE cells, nuclear cyclin
A levels appear
similar to those seen in proliferating cells (Fig.
5A; Table
2);
however, when DE cells spared from death by Bcl-2
are arrested in
G
1 upon temperature shift, nuclear cyclin A expression
is
lost (Fig.
5A; Table
2). Thus, the termination of cycle transit
with
stable G
1 arrest uncovers the apparently rapid turnover to
which nuclear cyclin A is subject. The sustained level of
death-associated
nuclear cyclin A represents the active recruitment of
cyclin A
from the cytoplasm above turnover. Death-specific nuclear Cdk
activity appears to depend on this active import process.
It is worth noting that the death-specific nuclear cyclin is present in
cells before the execution of the death process as
a function of normal
cell cycle transit and growth arrest. Indeed,
total cellular levels of
cyclin A protein (Fig.
5A), as well as
cyclin A mRNA (
11),
change little (less than 20%) during the
cell death response. These
data, together with those for class
III activity, suggest that
posttranslational activation of resident
products of the caspase 3 and
Cdk families is a common theme in
the effector phase of cell death.
| |
DISCUSSION |
While late events, such as the digestion of genomic DNA and the
loss of plasma membrane integrity, unambiguously mark a cell as dead,
the process by which a cell reaches that state physiologically has not
been well characterized. Consequently, it has not been possible to
define precisely the steps of dying or to identify the point at which a
cell is irreversibly committed to death. Our studies have sought to
address these issues by examining quantitatively the activities of
families of gene products with respect to both their requisite function
and their order of action during distinct physiological cell death
responses.
The data reported here define and map a conserved process comprising
the physiological cell death pathway. Death effector activities,
including cytoplasmic class III (DEVD-specific caspase 3-like) caspases
and nuclear Cdks, act in an ordered sequence between upstream
modulatory steps, including the activation of class I (YVAD-specific
ICE-like) caspase activity, and downstream posteffector events.
An amplifying cytoplasmic caspase cascade, punctuated by Bcl-2, is
necessary for cell death.
Studies in vitro have led to the notion
that a cascade of caspases is involved in the physiological cell death
process. The pattern of appearance of caspase activities observed in
cells induced to die via the CD95 death-signaling receptor
(20) also supports this view; however, the functional
demonstration of such a cascade in vivo has been lacking.
On the basis of several criteria, including the use of death-inhibitory
gene products, such as the viral serpin CrmA, as well
as pharmacologic
protease inhibitors, we found that both class
I and class III caspases
are required for these cell death responses.
The most striking
conclusion from our characterization of caspase
activities in these
cells is that the early transient class I
activity is necessary but not
sufficient for cell death. Because
class I activity is induced normally
in cells spared from death
by Bcl-2 expression, the presence of
activated class I protease
cannot itself be lethal. Its function as the
activator of downstream
class III activity in cell death identifies the
class I activity
as serving a requisite regulatory function. The
amplified activation
of caspase 3-like activity in response to a
death-inducing stimulus
is the obligate function of the caspase
cascade; our results extend
more general observations that terminal
proteolytic events attributed
to class III proteases ensue only in
dying cells and not in cells
spared from death by Bcl-2 family members
(
7,
9,
13,
18).
The ability of Bcl-2 and CrmA to spare cells from
glucocorticoid-mediated death rebuts proposed models which invoke
alternate
pathways of death either sensitive to Bcl-2 inhibition or
dependent
on ICE-like caspase activation (
32,
62). Even in
cases where
pharmacologic inhibitors are inactive (
45,
75),
we have found
that the viral inhibitors are effective at sparing cells
from
death and reveal the same pattern of activities (
5).
The caspase
activities we detect appear to reflect the products of
multiple
genes; we have yet to identify the specific caspases involved.
The aldehyde-derivatized tetrapeptide inhibition profiles for
ICE-like
activity both in T cells (Fig.
3A) and in B cells (data
not shown) are
consistent at least with ICE itself and caspase
4 (
43).
An operational definition of commitment leads to the discrimination
of modulatory and effector phases of the cell death process.
Steps
of the cell death process up to and including class I activity are not
lethal and cannot themselves be effectors of death. The commitment of a
cell to die must succeed class I activation. We define precommitment
events as modulatory steps within the death response. On the other
hand, we have not been able to dissociate class III activity from cell
death. This implies that the irreversible commitment to die, at least
operationally, is equivalent (or closely linked) to class III
activation. More precisely, at our current level of discrimination, we
can define the step inhibitable by Bcl-2 to represent the point of cell
death commitment.
Commitment signifies the point of initiation of the essential lethal
steps of cell death. We define this stage as the effector
phase of cell
death. Our previous work identified genome digestion
as a common and
dispensable consequence of physiological cell
death (
51,
71). We can describe the effector phase as lying
between such
downstream consequences and the upstream point of
commitment. By this
mapping analysis, class III caspase activity
functions within the
effector phase of cell death.
The lethal effector phase of cell death involves nuclear Cdk
activity.
Our results with nuclear Cdks map Cdk activity as
another component of the effector phase of physiological cell death.
The appearance of effector-phase nuclear Cdk activity is kinetically indistinguishable from the appearance of cytoplasmic class III activity
in our experiments. However, the CrmA and p35 transfectants reveal that
the caspase cascade is requisite for Cdk induction, suggesting that Cdk
activity acts downstream of the caspases within a sequential and linear
pathway.
That Cdks play an essential role in the cell death process has been
inferred from genetic studies with transiently expressed
antisense and
dominant negative Cdk mutants (
22,
47) and a
somatic cell
mutant conditionally defective in Cdk1 (
59). However,
those
studies did not allow the function of Cdk activities identified
to be
mapped with respect to the effector phase of the cell death
pathway.
Cdks certainly can play a role in the modulation of a
death response;
this seems to be the case, for example, with
myc-transfected
cells subjected to serum deprivation (
24). Independent of
such
a particular function in signaling, our findings demonstrate that
Cdk components play a role generally within the effector phase
of cell
death. We imagine that the link between cell cycle arrest
and cell
death serves to ensure the presence in dying cells of
specific Cdk
components, especially cyclins. It is notable that
our transfectant
studies do not exclude cycle arrest as a requisite
step in the cell
death pathway but do reveal that cell cycle arrest
occurs by a
signaling process that is independent of caspases
and not inhibitable
by Bcl-2.
The manner in which resident Cdk components are recruited and activated
within the nucleus during the effector phase of cell
death remains to
be understood. We find that constitutive levels
of PSTAIRE-reactive
nuclear Cdks are unaltered as a function of
the cell death response, in
contrast to other reports (
46).
On the other hand, a
significant augmentation in nuclear cyclin
A levels precedes the
appearance of Cdk activity and death. That
cyclin A recruitment to the
nucleus does not occur in cells spared
from death by Bcl-2 or caspase
inhibitors suggests the possibility
that cyclin recruitment occurs via
a caspase-dependent process.
The caspase-specific proteolytic
activation of other kinases,
such as protein kinase C-
(
25) and the Cdk-related PITSLRE
kinase (
4,
40),
during cell death also presents an attractive
precedent for an
alternative mode of kinase activation.
Physiological cell death occurs by a thematically conserved and
ordered pathway.
The data reported here support a model for the
cell death process in which diverse stimuli act afferently to modulate
a common effector pathway of death. That components of the effector
phase, caspase 3-like caspases (13, 19, 21, 57) and Cdks
(this work), appear to be resident in cells independent of new gene expression (11, 68) suggests that any requirement for
macromolecular synthesis in cell death must be limited to the
modulatory phase of death signaling.
While previous studies have suggested that specific caspases can
function in an ordered manner (
13,
20,
60), the variety
of
interactions among individual caspases, especially differences
in the
abilities of caspase inhibitors to interfere with particular
cell death
responses (
14,
62), has obscured the generality
of this
pattern. Our data reveal that in different cases of cell
death, a
variety of biochemical activities, including but not
limited to
caspases, function in a conserved and requisite order.
Differences in
caspases as well as effector Cdks in disparate
cases of cell death
inform our view of the cell death pathway
(
68) as conserved
in theme but not in the molecular identity
of individual components.
| |
ACKNOWLEDGMENTS |
We are grateful to Naomi Rosenberg for DE and DE/Bcl-2 cells and
to Stan Korsmeyer and Vishva Dixit for bcl-2 and viral
inhibitor clones, respectively. We thank our colleagues Raj Belani,
Sandra Chang, Bill Hendrickson, Phil Matsumura, and William Walden for constructive comments.
This work was supported by grants to D.S.U. from the National
Institutes of Health and as a Scholar of the Leukemia Society of
America.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, University of Illinois College of
Medicine, Rm. E803 (M/C 790), 835 South Wolcott, Chicago, IL 60612. Phone: (312) 413 1102. Fax: (312) 996 6415. E-mail:
DUCK{at}UIC.EDU.
| |
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Mol Cell Biol, May 1998, p. 2912-2922, Vol. 18, No. 5
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
