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(Received for publication, December 19, 1996, and in revised form, March 21, 1997)
From the Dana-Farber Cancer Institute and the Department of
Pathology, Harvard Medical School, Boston, Massachusetts 02115
ABSTRACT Proteolysis by the ubiquitin/proteasome pathway
controls the intracellular levels of a number of proteins that regulate
cell proliferation and cell cycle progression. To determine whether this pathway of protein turnover was also linked to apoptosis, we
treated Rat-1 and PC12 cells with specific proteasome inhibitors. The
peptide aldehydes PSI and MG115, which specifically inhibit the
chymotrypsin-like activity of the proteasome, induced apoptosis of both
cell types. In contrast, apoptosis was not induced by inhibitors of
lysosomal proteases or by an alcohol analog of PSI. The tumor
suppressor p53 rapidly accumulated in cells treated with proteasome
inhibitors, as did the p53-inducible gene products p21 and Mdm-2. In
addition, apoptosis induced by proteasome inhibitors was inhibited by
expression of dominant-negative p53, whereas overexpression of
wild-type p53 was sufficient to induce apoptosis of Rat-1 cells in
transient transfection assays. Although other molecules may also be
involved, these results suggest that stabilization and accumulation of
p53 plays a key role in apoptosis induced by proteasome inhibitors.
INTRODUCTION Many types of mammalian cells undergo apoptosis during normal
development or in response to a variety of stimuli, including DNA
damage, growth factor deprivation, and abnormal expression of oncogenes
or tumor suppressor genes (1-3). Apoptosis induced by these various
agents appears to be mediated by a common set of downstream elements
that act as regulators and effectors of apoptotic cell death. In many
cases, apoptosis requires the p53 tumor suppressor protein (4).
Overexpression of p53 is not only induced by several cell death stimuli
but is itself sufficient to induce apoptosis in gene transfer assays
(5). Apoptosis is also regulated by members of the Bcl-2 family (2, 6), which act upstream of a family of cysteine proteases known as the
interleukin-1 The ubiquitin/proteasome pathway is a major pathway of proteolysis in
eukaryotic cells and may contribute to controlling the intracellular
levels of a variety of short-lived proteins (8-10). The proteasome is
a large multicatalytic complex that catalyzes the degradation of
ubiquitinated cellular proteins. Substrates of the ubiquitin/proteasome
pathway include a number of cell regulatory molecules, such as cyclins,
the Myc oncogene protein, and p53, and the regulated degradation of
these molecules has been linked to the control of cell proliferation
and cell cycle progression (8-10).
By controlling the intracellular levels of such proteins, the activity
of the ubiquitin/proteasome pathway might also be linked to apoptosis.
In the present study, we have tested this possibility and demonstrate
that inhibition of the chymotrypsin-related activity of the proteasome
induces apoptosis of proliferating Rat-1 cells and of PC12 cells. This
induction of apoptosis is associated with increased intracellular
levels of p53 and is blocked by dominant negative p53 mutants,
suggesting that it results from inhibition of p53 degradation.
MATERIALS AND METHODS Rat-1 cells were cultured in Dulbecco's
modified Eagle's medium supplemented with 10% calf serum. PC12 cells
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum and 5% horse serum. Cells overexpressing
Bcl-2 and CrmA were kindly provided by J. Yuan (Massachusetts General
Hospital). Cells overexpressing dominant-negative p53 were obtained by
transfection with plasmids expressing the p53 mutant Val143
N-Benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal
(PSI) and carbobenzoxyl-leucinyl-leucinyl-norvalinal-II (MG115) were
purchased from the Peptide Institute Inc. and diluted in
Me2SO. The alcohol analog of PSI,
N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinol, was kindly supplied by S. Wilk (Mount Sinai School of Medicine, New
York, NY). Calpain inhibitor II was purchased from Boehringer Mannheim.
The peptidyl aldehydes were added to cultures, whereas control plates
were treated with the diluent. Treated cells were collected by
centrifugation, and cytoplasmic DNA was isolated, electrophoresed in
1.8% agarose gels, and visualized by ethidium bromide staining (12).
Cytological characterization of apoptotic cells was performed by
staining with propidium iodide and TUNEL assay (Boehringer
Mannheim).
Cell lysates (20 µg) were
electrophoresed in SDS-polyacrylamide gels and transferred to
nitrocellulose membranes. Primary monoclonal antibodies against p53,
p21, and Mdm2 were purchased from Santa Cruz Biotechnology Inc. The
secondary antibody was horseradish peroxidase-conjugated anti-mouse IgG
and proteins were detected using the ECL system (Amersham Corp.).
Rat-1 cells growing on
coverslips were transfected with 5 µg of human wild-type or
Val143 RESULTS The
proteasome contains three distinct proteolytic activities: a
chymotrypsin-related activity, a trypsin-related activity, and a
peptidylglutamyl-peptide hydrolyzing activity (13). The chymotrypsin-related activity is specifically inhibited by the peptide
aldehydes PSI and MG115 (14), so we initially tested the possibility
that these inhibitors would induce apoptosis. Actively proliferating
Rat-1 fibroblasts and PC12 pheochromocytoma cells were treated with PSI
and MG115 at concentrations selected based on the IC50
described for inhibition of proteasome degradation of I
Both Rat-1 and PC12 cells underwent apoptosis following treatment with
PSI at concentrations of 15 or 25 µM (Fig.
1A). Apoptosis was similarly induced by treatment of Rat-1
cells with 30 µM MG115 (Fig. 1B, lane
4). These data were confirmed by the TUNEL assay, which provides
an in situ assay for DNA fragmentation. In a typical experiment, 10-15% of cells were TUNEL positive following 5 h of
treatment with PSI compared with 1-2% of untreated control cells
(data not shown).
In contrast to the activity of PSI and MG115, apoptosis was not induced
by treatment with NH4Cl, which nonspecifically inhibits lysosomal proteolysis, or with a specific inhibitor of calpain II (Fig.
1B, lanes 5 and 6). Thus, inhibition
of lysosomal proteases did not lead to apoptosis. As a further
specificity control, cells were treated with an alcohol analog of PSI,
which only weakly inhibits proteasome function (14). The alcohol analog
failed to induce apoptosis of Rat-1 cells (Fig. 1, B,
lane 3, and C, lane 2), indicating
that apoptosis induced by PSI and MG115 resulted from specific
inhibition of the proteasome.
Apoptosis induced by a variety of agents can be inhibited by
overexpression of Bcl-2 or by inhibition of ICE family proteases. We
therefore tested the effects of Bcl-2 and CrmA, a cowpox virus-encoded ICE inhibitor, on apoptosis induced by proteasome inhibitors. Transfected Rat-1 cells overexpressing either Bcl-2 or CrmA were resistant to treatment with PSI and MG115 (Fig. 1C),
indicating that apoptosis induced by these proteasome inhibitors was
regulated by Bcl-2 and mediated by ICE family proteases.
Induction of apoptosis by proteasome inhibitors contrasted with recent
results, indicating that inhibition of the proteasome prevents
apoptosis of thymocytes and sympathetic neurons (24, 25). It appeared
possible that this difference was due to the fact that the cells used
in the present study were actively proliferating, in contrast to
terminally differentiated thymocytes and neurons. We therefore tested
the effects of proteasome inhibitors on nonproliferating Rat-1 cells,
which had been rendered quiescent by serum deprivation, and on PC12
cells, which had been induced to differentiate by treatment with nerve
growth factor. Treatment with MG115 failed to induce apoptosis of
quiescent Rat-1 cells but was still able to induce apoptosis of
nonproliferating differentiated PC12 cells (Fig. 1D). Thus,
cell proliferation was not the sole determinant of cellular sensitivity
to proteasome inhibitors.
p53
is required for apoptosis in a number of models and stabilization of
p53 leads cells to undergo apoptosis (4, 5). Degradation of p53 occurs
through the ubiquitin/proteasome pathway (19, 20). If this involved the
chymotrypsin-related function of the proteasome, PSI and MG115 might
induce apoptosis by inhibiting p53 degradation.
We therefore tested the levels of p53 in Rat-1 and PC12 cells following
treatment with proteasome inhibitors (Fig. 2). In both
cell types, PSI and MG115 lead to the accumulation of p53, which could
be detected as early as 2 h after initiation of treatment (Fig.
2A, lane 2). In contrast, treatment with the
lysosomal protease inhibitor calpain II did not result in p53
accumulation (Fig. 2A, lanes 6 and 7),
nor did treatment with the alcohol analog of PSI (Fig. 2B,
lanes 2, 3, 7, and 8). Most
of the p53 accumulated following treatment with the proteasome
inhibitors migrated at its normal electrophoretic mobility,
corresponding to 53 kDa, and therefore appeared to be nonubiquitinated.
However, higher molecular mass bands that reacted with anti-p53
monoclonal antibody were also observed after treatment with proteasome
inhibitors (for example, Fig. 2A, lanes 2-4,
8, and 9). These bands might correspond to
accumulation of ubiquinated p53 complexes.
p53 is a transcriptional activator that stimulates expression of
several genes, including the cyclin-dependent kinase
inhibitor p21 and the p53 negative regulator Mdm2 (21). To determine
whether the p53 accumulated in response to treatment with proteasome
inhibitors was functional, we assayed expression of these genes in
cells treated with MG115. Treatment with MG115-induced expression of both p21 (Fig. 3A) and Mdm-2 (Fig.
3B). Inhibition of the chymotrypsin-related function of the
proteasome thus led not only to stabilization of p53 but also to
transactivation of at least two p53 target genes.
To determine whether p53 was functionally
required for apoptosis induced by proteasome inhibitors, we transfected
Rat-1 and PC12 cells with a plasmid expressing a dominant-negative p53
mutant (Val143
To determine whether accumulation of p53 was sufficient to
induce apoptosis, Rat-1 cells were transiently transfected with wild-type or mutant human p53 expression plasmids. Cells expressing human p53 were identified by immunofluorescence and nuclear morphology was assessed by staining with propidium iodide. Transfected cells expressing wild-type p53, but not the Val143
DISCUSSION The results of the present study demonstrate that treatment of
proliferating Rat-1 cells and of PC12 cells with inhibitors of the
chymotrypsin-like function of the proteasome induces apoptosis. A
number of previous investigations have demonstrated that the peptidyl
aldehydes employed in this study are specific inhibitors of the
proteasome chymotrypsin-related activity (14-18). In contrast, neither
inhibitors of lysosomal proteases nor a peptide alcohol analog that is
ineffective as a proteasome inhibitor was able to induce apoptosis. It
thus appears that inhibition of this function of the proteasome
specifically induces apoptosis, linking the ubiquitin/proteasome
pathway of protein degradation to regulation of apoptotic cell
death.
Induction of apoptosis by proteasome inhibitors implies that cell death
is induced as a result of increased intracellular concentrations of a
regulatory molecule(s) normally degraded by the ubiquitin/proteasome
pathway. A prime candidate for such a regulator of apoptosis appeared
to be p53, which is known to be ubiquitinated and degraded by the
proteasome (19, 20). Not only is p53 induced by a variety of apoptotic
stimuli, but overexpression of p53 has been demonstrate to induce
apoptosis in a variety of cell types (4, 5). Consistent with this
hypothesis, we found that treatment with proteasome inhibitors resulted
in stabilization and rapid accumulation of p53 in both Rat-1 and PC12
cells. Moreover, the accumulated p53 was shown to be biologically
active, based on transactivation of the p53 target genes encoding p21
and Mdm2. Finally, we have shown that apoptosis induced by proteasome
inhibitors is blocked by expression of dominant-negative p53, and that
overexpression of wild-type p53 is sufficient to induce apoptosis of
Rat-1 cells in transient transfection assays. Although other molecules
may also be involved in cell death, these data suggest that modulation of p53 turnover is a key event in apoptosis induced by proteasome inhibitors.
Our finding that apoptosis is induced by inhibitors of the
chymotrypsin-like activity of the proteasome is consistent with a
previous report showing that lactacystin, which inhibits all three
proteasome activities (22), induces apoptosis of U937 myeloid leukemia
cells (23). On the other hand, it has recently been reported that
inhibition of the proteasome by either lactacystin or peptide aldehydes
prevents apoptosis of thymocytes induced by radiation, glucocorticoids,
or phorbol esters and of sympathetic neurons induced by nerve growth
factor deprivation (24, 25). In these systems, proteasome activity
appears to be required for induction of apoptosis upstream of the ICE
family proteases. It appeared possible that this apparent discrepancy
was due to the fact that U937 cells and the cells used in the present
study were actively proliferating, in contrast to the nonproliferating
thymocytes and sympathetic neurons in which proteasome activity was
required for induction of apoptosis by other agents. Indeed, Grimm
et al. (24) noted that prolonged exposure to proteasome
inhibitors actually increased cell death in thymocytes that were not
treated with other inducers of apoptosis. Consistent with this
possibility, we found that nonproliferating Rat-1 cells were no longer
susceptible to apoptosis induced by proteasome inhibitors. However,
inhibition of the proteasome still induced apoptosis of
nonproliferating PC12 cells that had been induced to differentiate by
treatment with nerve growth factor. These results indicate that cell
proliferation is one but not the only determinant of cellular response
to inhibition of the proteasome. It thus appears that the proteasome
can function to promote either cell survival or cell death, depending
on both proliferative state and other factors that may be cell
type-specific.
FOOTNOTES REFERENCES
Volume 272, Number 20,
Issue of May 16, 1997
pp. 12893-12896
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
,
converting enzyme (ICE)1
protease family (3, 7). Members of the ICE family, which are activated
by proteolytic cleavage of proenzymes, appear to act as common
executioners of apoptosis induced by a variety of cell death
stimuli.
Ala from a cytomegalovirus promoter (11).
Ala mutant p53 plasmids (11) using the
liptofectamine method. 40 h after transfection, cells were fixed
with 2% paraformaldehyde for 10 min and permeabilized with 0.5%
Triton X-100 for 5 min. Blocking and incubation with antibodies were
carried out in phosphate-buffered saline containing 1% bovine serum
albumin and 5% goat serum. Anti-p53 DO-1 primary antibody (Santa Cruz
Biotechnology) and fluorescein isothiocyanate-conjugated secondary
antibody (Sigma) were applied for 1 h in 1:1000 and 1:500
dilutions, respectively. Cells were simultaneously stained with
propidium iodide and then examined with a fluorescence microscope.
B-
and
processing of major histocompatability complex class I molecules
(15-18). After 4 h of treatment, apoptosis was assayed by DNA
fragmentation (Fig. 1).
Fig. 1.
Induction of apoptosis by proteasome
inhibitors. Cells were treated for 4 h either with the
diluent Me2SO or with the indicated concentrations of
inhibitors. Apoptosis was assayed by oligonucleosomal fragmentation of
DNA. A, proliferating Rat-1 fibroblasts (R) or
PC12 pheochromocytoma cells were treated with Me2SO or the
proteasome inhibitor PSI. B, proliferating Rat-1 fibroblasts
were treated with PSI (lanes 1 and 2), the
alcohol analog of PSI (Pep. Mod., lane 3), the
proteasome inhibitor MG115 (lane 4), the lysosomal inhibitor
NH4Cl (lane 5), calpain II inhibitor (lane
6), or Me2SO (lane 7). C, Rat-1
cells were treated with PSI (lane 1), the alcohol analog of
PSI (lane 2), or Me2SO (lane 7).
Transfected Rat-1 cells expressing CrmA (lanes 3 and
4) or Bcl2 (lanes 5 and 6) were
treated with PSI and MG115. D, Rat-1 cells were
growth-arrested by incubation in medium containing 0.5% calf serum for
36 h and treated with Me2SO (lane 1) or
MG115 (lane 2). Parallel cultures of proliferating Rat-1
cells were treated with MG115 (lane 3) or Me2SO
(lane 4). Proliferating PC12 cells were treated with MG115
(lane 5). Nonproliferating differentiated PC12 cells were
obtained by treatment with nerve growth factor (100 ng/ml) in medium
containing 0.5% horse serum for 8 days and treated with MG115
(lane 6) or Me2SO (lane 7).
DMSO, Me2SO.
[View Larger Version of this Image (27K GIF file)]
Fig. 2.
Accumulation of p53 in cells treated with
proteasome inhibitors. A, proliferating Rat-1 cells were
treated with Me2SO, PSI, calpain II (CII)
inhibitor, or MG115 for the indicated times. Cell lysates were
immunoblotted with anti-p53 monoclonal antibody. B,
proliferating Rat-1 and PC12 cells were treated with Me2SO (DMSO), PSI, MG115, or the alcohol analog of PSI (Pep.
Mod.) for 4 h.
[View Larger Version of this Image (54K GIF file)]
Fig. 3.
Induction of p21 and Mdm-2 in cells treated
with proteasome inhibitors. Proliferating Rat-1 cells were treated
with the proteasome inhibitor MG115 for the indicated times. Cell
lysates were analyzed by immunoblotting with antibodies against p21
(A) and Mdm-2 (B). Positions of p21 and Mdm-2 are
indicated by arrows. DMSO,
Me2SO.
[View Larger Version of this Image (42K GIF file)]
Ala), which is defective in DNA binding
and transactivation (11). Transfected cell lines were screened by
immunoblotting to identify cells that expressed high levels of the
mutant p53 (data not shown). These cells were then tested for apoptosis
following treatment with MG115. Both PC12 and Rat-1 subclones
expressing the mutant p53 (143.32 and 143.3 cells, respectively) were
resistant to induction of apoptosis (Fig. 4), indicating
that p53 is required for apoptosis induced by inhibition of proteasome
function.
Fig. 4.
Cells expressing dominant negative p53 are
resistant to apoptosis induced by proteasome inhibitors.
Proliferating wild-type PC12 and Rat-1 cells or PC12 and Rat-1 cells
overexpressing dominant negative p53 (designated 143.32 and 143.3, respectively) were treated with MG115. Apoptosis was assayed by DNA
fragmentation. DMSO, Me2SO; WT, wild
type.
[View Larger Version of this Image (91K GIF file)]
Ala
mutant, displayed the fragmented nuclei typical of apoptotic cells
(Fig. 5). Overexpression of wild-type p53 is thus
sufficient to induce apoptosis of proliferating Rat-1 cells, consistent
with the involvement of p53 accumulation in apoptosis induced by
proteasome inhibitors.
Fig. 5.
Induction of apoptosis by wild-type p53.
Proliferating Rat-1 cells were transfected with human wild-type or
Val143 Ala mutant p53 expression plasmids. 40 h
after transfection, cells were stained with anti-p53 antibody
(left panels) and with propidium iodide (right
panels). Nuclei of p53-expressing cells are indicated by
arrows.
[View Larger Version of this Image (102K GIF file)]
Recipient of a Brazilian Council of Research research fellowship.
Present address: Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janiero, Brazil.
§
To whom correspondence should be addressed: Dana-Farber Cancer
Institute, 44 Binney Street, Boston, MA 02115.
1
The abbreviation used is: ICE, interleukin-1
converting enzyme.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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N. Allaman-Pillet, J. Storling, A. Oberson, R. Roduit, S. Negri, C. Sauser, P. Nicod, J. S. Beckmann, D. F. Schorderet, T. Mandrup-Poulsen, et al. Calcium- and Proteasome-dependent Degradation of the JNK Scaffold Protein Islet-brain 1 J. Biol. Chem., December 5, 2003; 278(49): 48720 - 48726. [Abstract] [Full Text] [PDF]
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S. A. Williams and D. J. McConkey The Proteasome Inhibitor Bortezomib Stabilizes a Novel Active Form of p53 in Human LNCaP-Pro5 Prostate Cancer Cells Cancer Res., November 1, 2003; 63(21): 7338 - 7344. [Abstract] [Full Text] [PDF]
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L. Chen, L. Smith, Z. Wang, and J. B. Smith Preservation of Caspase-3 Subunits from Degradation Contributes to Apoptosis Evoked by Lactacystin: Any Single Lysine or Lysine Pair of the Small Subunit Is Sufficient for Ubiquitination Mol. Pharmacol., August 1, 2003; 64(2): 334 - 345. [Abstract] [Full Text] [PDF]
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Y.-H. Ling, L. Liebes, J.-D. Jiang, J. F. Holland, P. J. Elliott, J. Adams, F. M. Muggia, and R. Perez-Soler Mechanisms of Proteasome Inhibitor PS-341-induced G2-M-Phase Arrest and Apoptosis in Human Non-Small Cell Lung Cancer Cell Lines Clin. Cancer Res., March 1, 2003; 9(3): 1145 - 1154. [Abstract] [Full Text] [PDF]
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ABSTRACT