Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 9349–9353, August 1997
Medical Sciences
Cytokine suppression of protease activation in wild-type
p53-dependent and p53-independent apoptosis
JOSEPH L OTEM
AND
LEO SACHS*
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
Contributed by Leo Sachs, June 23, 1997
ICE-like proteases are involved in wild-type p53-induced
apoptosis (11). We have now shown that overexpression of
wild-type p53 activates intracellular proteases, resulting in
cleavage of protein substrates of ICE-like proteases, and that
suppressors of this apoptosis also suppress protease activation.
We also determined whether such proteases are activated in
p53 expressing and nonexpressing cells under conditions in
which some protease inhibitors did not suppress apoptosis.
ABSTRACT
M1 myeloid leukemic cells overexpressing
wild-type p53 undergo apoptosis. This apoptosis can be suppressed by some cytokines, protease inhibitors, and antioxidants. We now show that induction of apoptosis by overexpressing wild-type p53 is associated with activation of interleukin-1b-converting enzyme (ICE)-like proteases, resulting
in cleavage of poly(ADP- ribose) polymerase and the proenzyme of the ICE-like protease Nedd-2. Activation of these
proteases and apoptosis were suppressed by the cytokine
interleukin 6 or by a combination of the cytokine interferon
g and the antioxidant butylated hydroxyanisole, and activation of poly(ADP-ribose) polymerase and apoptosis were
suppressed by some protease inhibitors. In a clone of M1 cells
that did not express p53, vincristine or doxorubicin induced
protease activation and apoptosis that were not suppressed by
protease inhibitors, but were suppressed by interleukin 6. In
another myeloid leukemia (7-M12) doxorubicin also induced
protease activation and apoptosis that were not suppressed by
protease inhibitors, but were suppressed by granulocyte–
macrophage colony-stimulating factor. The results indicate
that (i) overexpression of wild-type p53 by itself or treatment
with cytotoxic compounds in wild-type p53-expressing or
p53-nonexpressing myeloid leukemic cells is associated with
activation of ICE-like proteases; (ii) cytokines exert apoptosis-suppressing functions upstream of protease activation;
(iii) the cytotoxic compounds induce additional pathways in
apoptosis; and (iv) cytokines can also suppress these other
components of the apoptotic machinery.
MATERIALS AND METHODS
Cells and Cell Culture. The cells used were mouse M1
myeloid leukemic cells, which do not express p53, transfected
with plasmids containing the neomycin-resistance gene (M1neo) or both the neomycin-resistance gene and a temperaturesensitive mutant p53 gene (M1-t-p53) (8), and cells from
another mouse myeloid leukemia, 7-M12 (15), which accumulate p53 protein after g-irradiation and treatment with other
cytotoxic agents (10). In the M1-t-p53 cells the temperaturesensitive p53 gene codes for a protein [Val-135]p53, which
behaves like a tumor-suppressing wild-type p53 at 32°C and
like a mutant p53 at 37°C (16). The cells were cultured in
DMEM (GIBCO) with 10% heat-inactivated (56°C, 30 min)
horse serum (GIBCO) in a 10% CO2y90% air atmosphere at
37°C or 32°C.
Compounds. The compounds used to induce apoptosis were
doxorubicin (Sigma) and vincristine (Teva Pharmaceutical,
Jerusalem). The compounds used to suppress apoptosis were the
antioxidant butylated hydroxyanisole (BHA; Sigma); the protease inhibitors N-tosyl-L-phenylalanine chloromethyl ketone
(TPCK; Sigma) and benzyloxycarbonyl-Phe-Ala fluoromethyl
ketone (Z-FAfmk) and benzyloxycarbonyl-Ala-Ala-Asp chloromethyl ketone (Z-AADcmk; Enzyme Systems Products, Dublin, CA); and the recombinant mouse cytokines IL-6 (obtained
from J. Van Snick, Ludwig Institute for Cancer Research, Brussels), granulocyte–macrophage colony-stimulating factor (GMCSF, Immunex), and IFN-g (Genzyme).
Determination of Protease Activation. Activation of ICElike proteases during apoptosis is associated with intracellular
cleavage of specific protein substrates, including 116-kDa
poly(ADP-ribose) polymerase (PARP) to 85-kDa and 30-kDa
fragments (4–7). The 51-kDa proenzyme of the ICE-like
protease Nedd-2 (neural precursor cell-expressed, developmentally down-regulated gene 2; ref. 17) is also cleaved during
induction of apoptosis by certain treatments (18), reflecting
activation of Nedd-2 proteolytic activity (6). The intracellular
cleavage of these two protein substrates was determined by
The Ced-3 gene that is required for programmed cell death
during development in the nematode Caenorhabditis elegans
(1) codes for a cysteine aspartase (2, 3) homologous to the
mammalian cysteine protease interleukin-1b-converting enzyme (ICE) and other ICE-like cysteine proteases, now collectively called caspases (reviewed in refs. 4–7). Induction of
apoptosis can be associated with activation of different members of the ICE family (4–7). Some ICE-like proteases activate
other members of this family, creating a cascade of protease
activations during apoptosis (4–7). We have previously shown
that overexpression of the tumor suppressor wild-type p53 is
sufficient to induce apoptotic cell death even without prior
DNA damage by other agents (8–11). Wild-type p53 is also
required for apoptosis induced in normal thymocytes (12–14)
and bone marrow myeloid precursor cells (12) by DNAdamaging agents, but not by some other compounds (12–14).
We also showed that wild-type p53-mediated apoptosis, without prior DNA damage, can be effectively suppressed by the
cytokine interleukin 6 (IL-6) (8–11) and to a lesser extent by
interferon g (IFN-g) (9), by certain antioxidants (10), and by
some protease inhibitors (11). The suppression of apoptosis by
cleavage-site-directed protease inhibitors (11) indicated that
Abbreviations: BHA, butylated hydroxyanisole; caspase, cysteine aspartase; GM-CSF, granulocyte–macrophage colony-stimulating factor; ICE, interleukin-1b-converting enzyme; IFN-g, interferon g; IL-6,
interleukin 6; Nedd-2, neural precursor cell-expressed, developmentally down-regulated gene 2; PARP, poly(ADP-ribose) polymerase;
TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; Z-AADcmk,
benzyloxycarbonyl-Ala-Ala-Asp chloromethyl ketone; Z-FAfmk, benzyloxycarbonyl-Phe-Ala fluoromethyl ketone.
*To whom reprint requests should be addressed. e-mail:
lgsachs@weizmann.weizmann.ac.il.
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
© 1997 by The National Academy of Sciences 0027-8424y97y949349-5$2.00y0
PNAS is available online at http:yywww.pnas.org.
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SDSypolyacrylamide gel electrophoresis of 100 mg of whole
cell extract proteins prepared from cells at different times after
initiation of apoptosis-inducing treatments as described (10),
followed by Western blotting using a 1:200 dilution of antibody
to the amino-terminal portion of mouse PARP (A20) or the
p12 carboxy-terminal portion of mouse Nedd-2 (C20) (Santa
Cruz Biotechnology). After 5 washes in TBS blocking buffer
(50 mM TriszHCl, pH 7.5y150 mM NaCl) containing 0.2%
Tween 20 and 5% low-fat milk, the blots were further incubated with a 1:2000 dilution of horseradish peroxidaseconjugated anti-IgG secondary antibody (Santa Cruz Biotechnology), washed five times in TBS containing 0.2% Tween 20
and developed with the enhanced chemiluminescence detection kit (ECL; Amersham). Bands corresponding to the appropriate proteins or protein fragments were visualized after
exposing the blots to Fuji RX medical x-ray film. After
determination of PARP cleavage, the same blots were stripped
and then reprobed with anti-Nedd-2 antibody.
RESULTS
Cleavage of PARP and Pro-Nedd-2 in Apoptosis Induced by
Overexpression of Wild-Type p53. M1-t-p53 cells overexpress
the Val-135 temperature-sensitive mutant p53 protein when
cultured at 37°C (8). After transfer of cells to 32°C, the
conformation of the mutant protein changes so that it behaves
like wild-type p53 (16), and this is sufficient to induce apoptotic cell death (8–11). While expression of a p53-inducible
gene such as p21yWAF1 is induced and a p53-suppressible
gene, c-myc, is suppressed within 1–2 hr after M1-t-p53 cell
transfer to 32°C (9, 19), apoptotic cells start to be recognizable
after about 10 hr (8, 10). Analysis of PARP and pro-Nedd-2 in
cells cultured at 32°C has shown that the 116-kDa and 51-kDa
proteins were cleaved to produce fragments of 30 kDa (aminoterminal) and 12 kDa (carboxyl-terminal), respectively. These
fragments were barely detectable in cells cultured at 37°C, and
after cell transfer to 32°C showed a readily detectable increase
at 10 hr (Fig. 1). This indicates that induction of apoptosis by
overexpression of wild-type p53, even without DNA damage by
another agent, is sufficient to activate ICE-like proteases.
Suppression of Protease Activation by Different Suppressors of Wild-Type p53-Induced Apoptosis. Addition of 10
ngyml IL-6, which suppresses p53-mediated apoptosis in M1t-p53 cells cultured at 32°C (8–11), suppressed production of
FIG. 1. Cleavage of PARP and pro-Nedd-2 during apoptosis
induced by wild-type p53. M1-t-p53 cells cultured at 37°C (0 hr at 32°C)
were transferred to 32°C, and cleavage of PARP and pro-Nedd-2 was
determined by Western blotting. The 116-kDa PARP and 51-kDa
pro-Nedd-2 and their respective cleavage products 30-kDa PARP and
12-kDa Nedd-2 are indicated. The same procedure to detect the
cleaved products was used in all the following figures.
Proc. Natl. Acad. Sci. USA 94 (1997)
the 30-kDa PARP and 12-kDa Nedd-2 fragments after culture
at 32°C (Fig. 2). The cytokine IFN-g, the antioxidant BHA,
and the protease inhibitor Z-AADcmk also suppressed wildtype p53-induced apoptosis in M1-t-p53 cells, but they were
less effective than IL-6 in suppressing apoptosis (9–11). IFN-g,
BHA, and Z-AADcmk also showed a lower protective effect
on PARP cleavage (Fig. 2). The results with two other
apoptosis-suppressing protease inhibitors, TPCK and ZFAfmk (11), were similar to those obtained with Z-AADcmk.
Combined treatment with IFN-g and BHA, or IFN-g, BHA,
and Z-AADcmk, which were almost as effective as IL-6 in
protection against wild-type p53-induced apoptosis (10, 11),
also effectively suppressed production of the 30-kDa PARP
and the 12-kDa Nedd-2 fragments (Fig. 2). Although IFN-g,
BHA, or the protease inhibitors by themselves suppressed
cleavage of PARP, they did not suppress production of the
12-kDa Nedd-2 fragment (Fig. 2).
Activation of Proteases by Cytotoxic Compounds in Other
p53-Expressing and in p53-Nonexpressing Myeloid Leukemic
Cells. Myeloid leukemic 7-M12 cells do not express detectable
p53 protein, but addition of doxorubicin induces accumulation
of endogenous p53 protein, and this is associated with induction of apoptosis starting at 3 hr (10). Unlike M1-t-p53 cells
induced to undergo apoptosis by overexpression of wild-type
p53 at 32°C, doxorubicin-induced apoptosis in 7-M12 cells was
not suppressed by different protease inhibitors (11). Apoptosis
induced in 7-M12 cells by 1.5 mgyml doxorubicin was also
associated with cleavage of PARP and pro-Nedd-2 (Fig. 3).
The cytokine GM-CSF, which suppressed induction of apoptosis by doxorubicin in 7-M12 cells (10, 11), also suppressed
cleavage of PARP and pro-Nedd-2 (Fig. 3). The protease
inhibitor Z-FAfmk, which did not suppress this induction of
apoptosis by doxorubicin (11), showed no suppression of
cleavage of PARP or pro-Nedd-2 (Fig. 3), and similar results
were obtained with TPCK. These data indicate that although
induction of apoptosis by doxorubicin in the p53-expressing
7-M12 cell was not suppressed by some protease inhibitors
(11), induction of apoptosis by this DNA-damaging agent in
FIG. 2. The suppressibility of wild-type p53-induced cleavage of
PARP and pro-Nedd-2 by cytokines, Z-AADcmk, BHA, or combined
treatments. M1-t-p53 cells were cultured with no additions (None) for
16 hr at 37°C (2) or 32°C (1) with 10 ngyml IL-6, 1 ngyml IFN-g, 0.1
mM BHA, 10 mM Z-AADcmk, or combined treatments. Results with
1 mM TPCK or 50 mM Z-FAfmk were similar to those with ZAADcmk.
Medical Sciences: Lotem and Sachs
Proc. Natl. Acad. Sci. USA 94 (1997)
9351
suppressed by IL-6 (Fig. 4), which protected the cells from
vincristine-induced apoptosis (11, 20), but there was no suppression of cleavage of PARP and pro-Nedd-2 by TPCK (Fig.
4) or by Z-FAfmk, which did not protect the cells from this
apoptosis (11). Similar results were obtained in M1-neo cells
treated with 1.2 mgyml doxorubicin (Fig. 5). PARP- and
pro-Nedd-2-cleaving proteases are thus activated during apoptosis mediated by wild-type p53-dependent and p53independent pathways, apoptosis-suppressing cytokines such
as IL-6 or GM-CSF, and a combination of IFN-g with the
antioxidant BHA suppress protease activation, but addition of
various protease inhibitors by themselves was not always
sufficient to suppress apoptosis. This indicates that the apoptosis-suppressing cytokines can suppress other components of
the apoptotic machinery in addition to suppressing activation
of ICE-like proteases.
DISCUSSION
FIG. 3. Cleavage of PARP and pro-Nedd-2 induced in 7-M12
myeloid leukemic cells by doxorubicin and its suppression by GM-CSF
but not by Z-FAfmk. 7-M12 cells were cultured for 3 or 4 hr, without
(2) or with (1) 1.5 mgyml doxorubicin with no further additions, or
with 10 ngyml GM-CSF or 50 mM Z-FAfmk. Results with 1 mM TPCK
were similar to those with Z-FAfmk.
these cells was associated with activation of PARP- and
pro-Nedd-2-cleaving proteases.
We also determined whether PARP- and pro-Nedd-2cleaving proteases are activated in M1-neo myeloid leukemic
cells, which do not express p53 before or after treatment with
cytotoxic agents (11). As in doxorubicin-treated 7-M12 cells,
apoptosis induced in M1-neo cells by 60 ngyml vincristine was
associated with cleavage of PARP and pro-Nedd-2 (Fig. 4).
Activation of proteases in M1-neo cells by vincristine was
FIG. 4. Cleavage of PARP and pro-Nedd-2 induced in M1-neo cells
by vincristine and its suppression by IL-6 but not by TPCK. M1-neo
cells were cultured for 4 or 16 hr without (2) or with (1) 60 ngyml
vincristine with no further additions (None) or with 10 ngyml IL-6 or
1 mM TPCK. Results with 50 mM Z-FAfmk were similar to those with
TPCK.
The present results have shown that overexpression of wildtype p53 in M1 myeloid leukemic cells without prior DNA
damage by another agent is sufficient for activation of intracellular proteases, resulting in cleavage of PARP and proNedd-2 proteins. ICE-like proteases are activated from their
proenzymes by cleavage after aspartyl residues at sites similar
to ICE-like substrates, generating fragments of 10–20 kDa
(p10-p20), which then assemble into proteolytically active
heterodimeric or tetrameric enzymes (4–7). Activation of
Nedd-2 proteolytic activity follows assembly of a p12 carboxylterminal subunit with another p19 subunit (6). It is, therefore,
suggested that the 12-kDa carboxyl-terminal Nedd-2 fragment
cleaved from the 51-kDa pro-Nedd-2 during wild-type p53mediated apoptosis in the present experiments reflects acti-
FIG. 5. Cleavage of PARP and pro-Nedd-2 induced in M1-neo cells
by doxorubicin and its suppression by IL-6 but not by TPCK. M1-neo
cells were cultured for 16 hr without (2) or with (1) 1.2 mgyml
doxorubicin with no further additions (None) or with 10 ngyml IL-6
or 1 mM TPCK. Results with 50 mM Z-FAfmk were similar to those
with TPCK.
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Proc. Natl. Acad. Sci. USA 94 (1997)
Medical Sciences: Lotem and Sachs
vation of the Nedd-2 protease. This activation of Nedd-2 may
follow its physical interaction with the ubiquitously expressed
death-domain-containing adaptor molecule R A IDDy
CRADD (21, 22), similar to the interaction between the
ICE-like protease MACHyFLICEycaspase 8 with the adaptor
molecule FADDyMORT1 in the FAS and tumor necrosis
factor pathways to apoptosis (23, 24).
Cleavage of PARP was described as being carried out by the
ICE-like protease prICE (25), also known as CPP32, apopain,
yama, or caspase 3 (4–7). CPP322/2 mice showed abnormalities in the apoptotic pathway during neuronal development,
indicating the involvement of this ICE-like protease in this
apoptotic process (26). Further experiments indicated that
PARP can also be cleaved by other ICE-like proteases, including ICH-1yNedd-2 (caspase 2), ICE-rel II (caspase 4),
Mch 3 (caspase 7), and ICE-LAP6 (caspase 9) (4–7). PARP
was also shown to be cleaved during induction of apoptosis by
different stimuli even in thymocytes from CPP322/2 mice (26).
It is thus possible that wild-type p53-induced cleavage of
PARP in M1-t-p53 cells was mediated, at least in part, by
activated Nedd-2, although other proteases of the ICE family
may also be involved in this PARP cleavage.
Various cytokines can suppress apoptosis (reviewed in refs.
27–29). We have now shown that suppression of wild-type
p53-induced apoptosis by IL-6 results in suppression of cleavage of PARP and pro-Nedd-2. IL-6 can thus act upstream of
activation of ICE-like proteases in the apoptotic pathway (Fig.
6). Our results have also shown that different cytotoxic agents
can activate ICE-like proteases during apoptosis in p53expressing and p53-nonexpressing cells and that this protease
activation and apoptosis are suppressed by the cytokines
GM-CSF or IL-6. Different cytokines can thus suppress activation of cytotoxic compound-induced p53-dependent and
p53-independent pathways of apoptosis by acting at a point
upstream of activation of ICE-like proteases, as in apoptosis
induced by overexpression of wild-type p53 (Fig. 6).
We have previously shown that induction of apoptosis by
overexpression of wild-type p53 as well as by certain DNA-
damaging agents involves an oxidative pathway that can be
suppressed by different antioxidants, including BHA (10). We
have now found that BHA also suppresses PARP cleavage
induced by overexpression of wild-type p53, but not as effectively as the cytokines IL-6 or GM-CSF. This indicates that
BHA also acts upstream of protease activation (Fig. 6). This
agrees with other results showing that antioxidants can suppress activation of PARP cleavage (30). However, unlike
suppression of pro-Nedd-2 cleavage by these cytokines, we did
not observe suppression of cleavage of pro-Nedd-2 by BHA or
protease inhibitors. It is therefore possible that activation of
different proteases during apoptosis can be selectively regulated by different apoptosis-suppressing compounds acting
through different pathways. This may also explain the lower
apoptosis-suppressing effect of BHA compared with IL-6 or
GM-CSF, and the cooperative activity of BHA, IFN-g, and
different protease inhibitors in suppression of protease activation and apoptosis.
Unlike apoptosis induced by overexpression of wild-type
p53, induction of apoptosis by doxorubicin or vincristine,
which was also associated with protease activation, was not
suppressed by the protease inhibitors Z-VADfmk, Z-FAfmk,
Z-AADcmk, or TPCK (11). These results indicate that apoptosis induced by these cytotoxic compounds is associated with
activation of additional components of apoptosis that are not
suppressed by the protease inhibitors used in our experiments.
But cytokines can suppress these other components of the
apoptotic machinery. The ability to suppress different components of apoptosis is supported by the ability of the proteaseinhibiting viral proteins CrmA and p35 to suppress apoptosis
induced by some but not other apoptosis-inducing agents (31)
and by the observation that overexpression of Bcl-2 and
Bcl-XL, which act upstream of protease activation (32–34),
does not always prevent apoptosis (33, 34).
We thank Nurit Dorevitch and Rachel Kama for skillful technical
assistance. This work was supported by the National Foundation for
Cancer Research (Bethesda), by the Esther Mazor Family (Washington, DC), and by the Ebner Family Biomedical Research Foundation
at the Weizmann Institute of Science in memory of Alfred and Dolfi
Ebner.
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FIG. 6. Regulation of ICE-like protease activation and apoptosis
induced by overexpression of wild-type p53, doxorubicin, or vincristine. Apoptosis induced by overexpression of wild-type p53 (W.T.p53,
Upper) and by doxorubicin or vincristine (Lower). Cooperation between different apoptosis-suppressing compounds, such as IFN-g and
an antioxidant, is shown in Upper. Arrow indicates apoptotic pathway;
', suppression of pathway; and 3, pathway not suppressed. p, The
protease inhibitors tested were Z-AADcmk, Z-FAfmk, and TPCK.
These protease inhibitors suppressed activation of ICE-like proteases
and apoptosis induced by wild-type p53, but showed no suppression of
this protease activation or apoptosis induced by doxorubicin or
vincristine.
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