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J Biol Chem, Vol. 273, Issue 43, 28378-28383, October 23, 1998
From the Department of Pediatric Oncology, Dana-Farber Cancer
Institute and Children's Hospital, Harvard Medical School, Boston,
Massachusetts 02115
The mechanism by which p53 modulates apoptosis in
cancer therapy is incompletely understood. Here, cell-free extracts
from irradiated tumor cells are described in which endogenous p53
protein is shown to participate in caspase activation. This apoptotic activity is also oncogene-dependent, but independent of
transcription in general or the presence of Bax or cytochrome
c. A general use for this system is as a cell-free screen
for apoptosis modulators. In this way, profound effects of protein
kinase A were identified and corroborated in vivo by the
protection conferred by cAMP against diverse triggers of
p53-dependent apoptosis. This system provides direct
biochemical evidence that p53 protein can transduce apoptotic signals
through protein-protein interactions and reveals a modulator kinase
pathway capable of regulating p53-dependent caspase
activation.
p53 plays a major role in modulating the apoptotic response in
tumor cells following signals such as DNA damage, growth factor deprivation, and hypoxia (1-3). Its regulation of apoptosis in the
context of oncogene overexpression suggests that apoptosis constitutes
an important component of p53's tumor suppressor activity. In
transgenic mice inactivation of p53, either by germ-line mutation or by
expression of the p53-binding domain of SV40 large T antigen leads to
attenuation of apoptosis and rapid tumor progression (4). p53-mediated
apoptosis has also been implicated as an important mechanism by
which many antitumor treatments kill cancer cells (5, 6). Thus, studies
of p53-mediated apoptosis may have important implications for cancer
treatment.
The mechanism by which p53 modulates apoptosis is incompletely
understood. Cell-based studies have provided evidence that p53 can
induce apoptosis through either transcription-dependent or
transcription-independent mechanisms (1-3). In the
transcription-dependent pathway(s), the pro-apoptotic
proteins Bax, the insulin-like growth factor-binding protein-3, the
death receptor KILLER/DR5, the zinc finger protein PAG608, and proteins
involved in generation or response to oxidative stress are potentially
important transcriptional targets of p53 (7-12). Little is currently
known about the mechanistic basis for p53-mediated apoptosis that is
independent of transcription, which may be part of the
transcription-independent activities of p53 involved in tumor
suppression (13-15).
A cell-free p53-regulated apoptosis system would be useful for
biochemically dissecting this medically important pathway. The
possibility that a cell-free system may be able to recapitulate p53-dependent apoptosis is suggested by evidence that p53
can induce apoptosis independent of its transcription function
(16-19). In these studies, p53-dependent apoptosis was not
blocked by inhibition of transcription or translation, and p53
mutations have been defined whose behavior uncouples transcriptional
activity from apoptotic activity within cells. In different cellular
contexts, apoptosis and transcriptional activities of p53 appeared to
be more tightly coupled (20, 21).
The measurement of cell-free apoptosis requires a biochemical surrogate
for active apoptotic death, which has been facilitated by the discovery
that apoptosis is executed by the activation of caspases. Caspases
mediate apoptosis by cleaving a number of intracellular proteins
("death substrates") (22, 23). One of the first identified caspase
substrates is poly(ADP-ribose) polymerase
(PARP),1 a target of
caspase-3 (CPP32, apopain, YAMA) (24, 25). Because activation of this
caspase is a common step in apoptosis, PARP cleavage by caspase-3 is
now widely used as a biochemical marker for apoptosis, including in
studies of cell-free systems (26-28).
Here we report that cell-free extracts from Cell Culture, DNA Fragmentation Analysis, and Extract
Preparation--
E1A/Ras-transformed mouse embryo fibroblasts (MEFs)
were grown in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum and 10% newborn calf serum. Primary rat embryo
fibroblasts (REFs) and Myc/Ras-transformed REFs were maintained in
minimal essential medium supplemented with 10% fetal bovine serum and 10% newborn calf serum. Exponentially growing cells at 60-80% confluence were either untreated or treated with 10 Gy of ionizing radiation using a Gammacell 40 equipped with a 137Cs
source. At various times after irradiation, the cells were harvested,
collected by centrifugation, and washed once with ice-cold phosphate-buffered saline. For DNA fragmentation analysis, cells were
harvested at 16 h after irradiation, and apoptotic DNA was isolated and analyzed as described (29). To make cell-free extracts, the cell pellet was resuspended at 108 cells per 0.25 ml of
ice-cold buffer A (20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 3 mM
dithiothreitol, 4 mM Pefabloc, 5 µg/ml pepstatin A, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). After incubating on ice
for 15 min, the cells were disrupted by douncing 15 times in a Wheaton
douncer with a tight pestle. The lysates were directly centrifuged at
100,000 × g for 1 h in a Sorvall RC M120
ultracentrifuge. The resulting supernatants (protein concentration ~8
mg/ml) were collected and stored at PARP Cleavage Assay--
For in vitro induction of
PARP-cleaving activity, 8-10 µl of extracts were incubated at
32 °C for various times, after which 80 ng of recombinant PARP (a
truncated form comprising the first 338 N-terminal amino acids and
including the CPP32 cleavage site) was added for 5 min at 37 °C. For
assays of in vivo activated PARP-cleaving caspases, 10 µl
of extract was incubated with 80 ng of recombinant PARP for 15 min at
37 °C. At the end of the reaction, 4× SDS sample buffer was added.
The samples were boiled for 4 min, subjected to 12% SDS-polyacrylamide
gel electrophoresis, and transferred to a nitrocellulose membrane. The
membrane was then probed with a monoclonal antibody (C2.10) against
PARP (G. Poirier, Université Laval, Canada) and visualized by ECL
(Amersham Pharmacia Biotech). Recombinant SV40 large T antigen, either
as lysates (200-400 ng of total proteins) from recombinant T antigen baculovirus-infected Sf9 cells (generously provided by J. DeCaprio, Dana-Farber Cancer Institute) or as purified, bacterially
produced T antigen (0.5-1 µg, Chimerx), was added to extracts at
various time points during the 32 °C incubation period. Lysates from
wild-type baculovirus-infected Sf9 cells or bovine serum albumin
were used as controls. 200-400 ng of purified, polyclonal IgG against
full-length p53 or the control IgG against the tyrosine kinase Fyn
(both from Santa Cruz) was used in the indicated PARP cleavage assays.
For cytochrome c experiments, 200 ng of cytochrome
c (Sigma) was added to extracts at the beginning of the
32 °C incubation period. Nucleotides ATP, ATP Immunodepletion--
5 µg of monoclonal antibodies against
mouse p53 (equal mixture of Ab-1, -3, -4, and -5; Oncogene Science),
Bax (Trevigen), cytochrome c (Pharmingen), or microphthalmia
(as a control) were incubated with 200 µl of extracts for 2 h on
ice. The immune complexes were then cleared by two sequential rounds of
incubation with a precoated and washed protein A/G-agarose bead pellet
(from 30 µl of 50% suspension, Life Technologies, Inc.) for 2 h
in a rotator at 4 °C. The beads were subsequently pelleted by
centrifugation for 2 min in a microcentrifuge at 4 °C, and the
resulting supernatants were collected and used for in vitro
PARP cleavage assays.
Flow Cytometry Analysis--
Myc/Ras-transformed REFs were
plated at 4 × 105 per 60-mm plate. 8 h after
plating, 10 µM forskolin in Me2SO or the same
volume of Me2SO was added, and the cells were either
untreated or immediately treated with 5 Gy of ionizing radiation. In
serum starvation experiments, E1A/Ras-transformed p53 wild-type MEFs
were plated at 4 × 105 per 60-mm plate. 8 h
after plating, the cells were washed with serum-free Dulbecco's
modified Eagle's medium and maintained in Dulbecco's modified
Eagle's medium containing either 20% or 2% serum (equal parts fetal
bovine and newborn calf sera) with or without forskolin (10 µM). At 72 h after irradiation or 48 h after serum deprivation, adherent and floating cells were pooled, collected by centrifugation, and washed once with ice-cold phosphate-buffered saline. Staining with fluorescein isothiocyanate-annexin V or propidium
iodide were performed according to the manufacturer's instructions
(Trevigen).
Latent Caspase Activation in Extracts of Irradiated, Transformed
Fibroblasts--
Oncogene-dependent Regulation of Caspase Activation
by p53 Protein in a Cell-free System*
,
ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
-irradiated, transformed
p53 wild-type mouse or rat embryo fibroblasts induce caspase activation
after a latent phase. This caspase activation requires functional p53:
immunodepletion or inactivation of p53 either by recombinant SV40 large
T antigen or anti-p53 IgG blocks caspase activation early, but not
late, in cell-free incubations. Caspase activation is not blocked by
immunodepletion of Bax, a transcriptional target of p53 (8), or
cytochrome c, a co-activator of caspase-3 (27). Moreover an
ATP analog, AMP-PNP, blocks p53-dependent caspase
activation via cAMP/protein kinase A (PKA)-mediated repression, an
effect which was corroborated in vivo by evidence that cAMP rescues cells from diverse triggers of p53-dependent
apoptosis. These results demonstrate that p53 can transduce apoptotic
signals through a Bax/cytochrome c-independent pathway.
EXPERIMENTAL PROCEDURES
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Abstract
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80 °C.
S, AMP-PNP, and cAMP
were purchased from Boehringer Mannheim, and 1 mM final
concentration was used in the indicated experiments. Catalytic subunits
of PKA and a specific peptide inhibitor of PKA (PKI 6-22) were
purchased from New England Biolabs and Calbiochem, respectively. 1 unit
of PKA or 200 nM PKI 6-22 was used in the indicated
experiments.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
-Irradiation triggers apoptosis in
oncogene-transformed MEFs and REFs, as indicated by the characteristic
fragmentation of genomic DNA (Fig.
1a, lanes 3-6)
(30). Although E1A/Ras-transformed p53 wild-type fibroblasts undergo
radiation-induced apoptosis, genetically related E1A/Ras-transformed
p53
/ MEFs do not (lanes 1 and 2)
(17, 30). Irradiated primary fibroblasts also fail to undergo apoptosis
(lanes 7 and 8), instead undergoing cell cycle
arrest (data not shown) (30). Consistent with the role of caspases in
apoptosis, by 4 h after irradiation, extracts from transformed
p53+/+ MEFs, but not from transformed p53/
MEFs, contain active caspases that readily cleave the death substrate PARP in vitro (exogenous truncated PARP, Fig.
1b).
View larger version (25K):
[in a new window]
Fig. 1.
Panel a, induction of DNA fragmentation
by -irradiation of transformed p53+/+ MEFs
and REFs. MEFs were transformed by E1A and Ras (30), and REFs were
transformed by Myc and Ras or were used as represented primary
embryonic fibroblasts. DNA was isolated from the indicated cells at
16 h after 10-Gy irradiation and subjected to 1.2% agarose gel
electrophoresis. The DNA was visualized by ethidium bromide staining.
Panel b, activation of PARP-cleaving caspase by
-irradiation of transformed p53+/+ MEFs.
Extracts were prepared from untreated (U) or irradiated
cells at the indicated time points (hours) after 10-Gy
irradiation. Recombinant truncated PARP was added for 15 min at
37 °C. PARP cleavage was monitored by immunoblot with the C2.10
monoclonal antibody against PARP. Molecular weight markers and the
positions of PARP and its cleaved product are indicated.
/
MEFs, whether or not they had been irradiated, or from unirradiated transformed p53+/+ MEFs as long as 24 h after
incubating at 32 °C (Fig. 2b), suggesting that the
caspase activity was specific for in vivo irradiation rather
than in vitro incubation per se. Caspase
induction with similar kinetics was also observed in extracts from
irradiated Myc/Ras-transformed REFs (Fig. 2c). Consistent
with the notion that irradiation leads to p53-driven cell cycle arrest
(rather than apoptosis) in primary cells, extracts from untransformed REFs showed no caspase activity even after 24 h of incubation (Fig. 2d). This was not due to the absence of caspase
precursors in the extracts because PARP-cleaving activity could be
stimulated by addition of cytochrome c (Fig. 2d,
lane 7), a co-activator of caspase-3 (27). The PARP-cleaving
activity correlated with the appearance of the p20 subunit of activated
caspase-3/CPP32 (Fig. 2e).
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Functional p53 Protein Is Required for Cell-free Caspase Activation-- To determine whether activation of caspases in the cell-free extracts remains p53 dependent, we first performed immunodepletion experiments using monoclonal antibodies against p53. As shown in Fig. 3a, immunodepletion of p53 from extracts before the 32 °C incubation period completely abrogated the caspase activation (lane 3). Identical p53 immunodepletions at later time points (following 4 or 8 h at 32 °C) failed to block the caspase activation (lanes 4 and 5). No effect on the caspase activation was observed with control immunodepletions (lane 2). These results suggest that p53 function is required at an early stage upstream of caspase activation in the cell-free extracts. Addition of recombinant p53 derived from p53-baculovirus-infected Sf9 cells to extracts depleted of endogenous p53 has so far failed to activate caspases in this system (data not shown), which could reflect lack of proper post-translational modifications or other critical associations.
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p53 and Cytochrome c Function in Distinguishable Apoptotic Pathways in the Cell-free Extracts-- Failure of the extracts to induce PARP cleavage upon immunodepletion or inactivation of p53 might in principle have been caused by direct disruption or depletion of PARP-cleaving caspases by these manipulations, although this is unlikely because p53 depletion or T antigen addition at later time points did not inhibit caspase activity (Fig. 3, a and b). Further evidence of the specificity of the p53 inactivations came from the observation that exogenous cytochrome c restores caspase activation when added to extracts depleted of p53 (Fig. 4a, lanes 4 and 5) or in the presence of T antigen (lanes 1-3). Thus, these treatments are unlikely to directly inactivate caspases but are more likely to block an upstream activity, consistent with an early requirement for p53.
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/ mouse thymocytes (33), although it could play a
role in setting cellular apoptosis thresholds because measurable
effects have been detected in Bax-deficient fibroblasts (34). The
finding that cytochrome c depletion did not block the
ability of the extracts to activate caspases suggests that cytochrome
c is not linearly downstream of p53 in this apoptosis
pathway. In fact, subcellular fractionation in the presence of sucrose
shows that cytochrome c is not cytosolic 1 h after
irradiation, but only emerges during extract preparation (data not
shown), probably as a result of disrupting mitochondrial integrity by
hypotonic lysis. Therefore, extracts prepared from both
p53/ and p53+/+ cells with or without
irradiation contained similar levels of released cytochrome
c (data not shown), which was not capable of inducing
caspases, although high levels of exogenous cytochrome c can
activate caspases in these extracts.
Protein Kinase A Blocks p53-dependent Apoptosis in
Vitro and in Vivo--
The activation of caspases in this cell-free
system is unlikely to require hydrolysis of ATP, whose addition even
partially inhibited induction of PARP-cleaving activity (Fig.
5a, lane 2). The
nonhydrolyzable ATP analog, ATPS, had minimal effect on caspase activation (Fig. 5a, lane 3). However, another
nonhydrolyzable ATP analog, AMP-PNP, completely abrogated the
activation of caspases (Fig. 5a, lane 4). This
inhibitory effect was observed when AMP-PNP was added at the beginning
but not later in the 32 °C incubation period (Fig. 5a,
lanes 5-7), suggesting that AMP-PNP inhibited an activity
upstream of caspases.
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S, can be converted
to cAMP (35), which in turn can activate PKA. Three lines of evidence
suggested essential roles for cAMP and PKA in the AMP-PNP-mediated
inhibition of caspase activation. First, a purified specific peptide
inhibitor of PKA (PKI 6-22) strongly relieved the inhibitory effect of
AMP-PNP (Fig. 5b, lanes 1-3). Second, when added
to the cell-free extracts at the beginning of the 32 °C incubation
period, cAMP inhibited the caspase activation as potently as AMP-PNP
(Fig. 5b, lane 4-6). Finally, purified PKA catalytic subunit completely abolished the induction of caspase activity in the presence of ATP, but not its nonhydrolyzable analog ATPS (Fig. 5b, lanes 7-10), and this
inhibition was reversed by the specific PKA peptide inhibitor. Thus
cAMP/PKA appear to inhibit the p53-dependent apoptosis
reflected in these extracts.
Finally, the relevance of these in vitro cAMP effects was
examined in cell-based apoptosis studies. The cAMP-inducing agent forskolin was examined for its effects on p53-dependent
apoptosis in vivo. As shown in Fig. 5c, forskolin
significantly protected radiation-induced apoptosis in transformed
fibroblasts. In addition, the apoptotic response to serum starvation
was significantly blunted by forskolin (Fig. 5c). Of note,
the activation of PKA by forskolin is short-lived (because of
homeostatic down-regulation, see Ref. 36) and may therefore
underestimate the magnitude of the protection because of this
experimental system. Nonetheless, these results demonstrate that
the cell-free apoptosis assay described here predicts effects
that are in agreement with in vivo behavior, and that the
cAMP/PKA pathway can negatively regulate p53-dependent apoptosis.
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DISCUSSION |
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This report demonstrates p53-mediated apoptosis in a cell-free system. Significantly, the p53-dependent activation of caspases in this system does not require the presence of Bax or cytochrome c. These findings suggest that a distinct set of factors relay the p53-dependent apoptotic signals to caspases. A recent study has shown that Bax may act upstream of cytochrome c; overexpression of Bax induces the release of cytochrome c, which in turn activates caspase-3 and results in cell death (37). Cytochrome c may well be an essential component in Bax-dependent apoptosis. According to the death cycle model (38, 39), it is also possible that within cells cytochrome c could play a role in caspase activation through a Bax-independent pathway in which p53-mediated activation of caspases promotes the release of cytochrome c, which in turn amplifies the apoptotic cascade. This hypothesis is also consistent with the report that there is synergy between transcription-dependent and -independent functions of p53 in apoptosis (40).
One reproducible feature of the system described here is the relatively long delay (8-12 h) before in vitro induction of caspase activity. There are several important potential reasons for these kinetics. First, caspase activation requires approximately 4 h even within intact cells, thereby representing a relatively slow cascade (or multistep process) in vivo. Second, this assay specifically employs 32 °C incubation periods which, although kinetically suboptimal, diminishes nonspecific degradation of exogenous PARP. Third, components within the extracts are significantly diluted relative to intracellular conditions. Finally, the in vitro latent period before caspase activation was found to shorten with increasing in vivo incubation after irradiation, eventually passing the p53-dependent stage (data not shown). The same is true for other stimuli of apoptosis such as growth factor deprivation, which in the extreme gives "spontaneous" caspase activation upon extract preparation. Thus for this system the viability of starting cell populations is crucial for the ability to discriminate the slower in vitro p53-dependent activity.
The tumor cells used here have been transformed by two oncogenes, Ras plus either E1A or Myc. Therefore, the precise contribution of each oncogene to the apoptotic pathway is uncertain at this time. Stable overexpression of Myc alone in REFs has been shown to produce immortalization without transformation (as measured by solid tumor formation) (41), but the apoptotic propensity of these cells has not been reported. In contrast, p53+/+ cells transiently and forcibly overexpressing E1A readily undergo apoptosis, even to a degree that limits generation of stable lines (42). It would be of interest to examine any such immortalized cells for the apoptotic activity reported here as they could provide clues to the mechanistic connection between oncogene activity and p53-dependent apoptosis.
In this tumor cell system, PKA blocks p53-dependent caspase
activation in cell-free extracts and protects transformed embryo fibroblasts from apoptosis triggered by -irradiation or by serum deprivation. Activation of PKA has been reported to either inhibit or
promote apoptosis depending on experimental systems (43). The ability
of PKA to inhibit the caspase activation in vitro suggests
that it functions by phosphorylation of a protein(s) present in the
extracts, which is consistent with a report that cAMP prevents
apoptosis by translation- and transcription-independent mechanisms
(44). p53 itself has been shown to serve as a PKA substrate in
vitro (45), but it is unclear whether this correlates with its
apoptotic activity. Identification of the PKA target(s) may reveal
important components of this pathway. Finally, recognition that
elevated cAMP may repress p53-dependent apoptosis carries clinical implications for patients with p53 wild-type malignancies undergoing curative antineoplastic therapy, because numerous common agents (for example, caffeine) regulate cAMP metabolism.
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ACKNOWLEDGEMENTS |
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We thank S. Lowe, T. Jacks, and D. Housman (Massachusetts Institute of Technology) for the E1A/Ras-transformed MEF cell lines, J. DeCaprio (Dana-Farber Cancer Institute) for the baculoviral-recombinant SV40 T antigen and control baculoviral lysates, and J. Collier (Harvard Medical School) for the PARP overexpression plasmid.
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FOOTNOTES |
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* This work was supported in part by grants from the National Institutes of Health (to D. E. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Novartis Fellow.
§ Recipients of Howard Hughes Medical Institute predoctoral fellowships and Sandoz Fellows.
¶ Recipient of a scholarship from the Deutsche Forschungs- gemeinschaft.
Nirenberg Fellow at Dana-Farber Cancer Institute and a Fellow
of the Pew Foundation and the James S. McDonnell Foundation. To whom
correspondence should be addressed. Tel.: 617-632-4916; Fax:
617-632-2085; E-mail: david_fisher{at}dfci.harvard.edu.
The abbreviations used are:
PARP, poly(ADP-ribose) polymerase; MEF, mouse embryo fibroblast; REF, rat embryo fibroblast; PKA, protein kinase A; AMP-PNP, adenosine
5'-(
,-imino)triphosphate or adenosine
5'-(,-iminotriphosphate); ATPS, adenosine 5'-O-
(thiotriphosphate).
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REFERENCES |
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References
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