Clemens1 1 Translational Control Group, Department of Basic Medical Sciences, St George’s Hospital Medical School, Cranmer Terrace, London;2Biochemistry Group, School of Biological Scien
Trang 1p53-induced inhibition of protein synthesis is independent
of apoptosis
Constantina Constantinou1, Martin Bushell1*, Ian W Jeffrey1, Vivienne Tilleray1, Matthew West1,
Victoria Frost2†, Jack Hensold3and Michael J Clemens1
1
Translational Control Group, Department of Basic Medical Sciences, St George’s Hospital Medical School, Cranmer Terrace, London;2Biochemistry Group, School of Biological Sciences, University of Sussex, Falmer, Brighton, UK;3Department of Hematology and Oncology, Case Western Reserve University and the Veterans Administration, Cleveland, Ohio, USA
Activation of a temperature-sensitive form of p53 in murine
erythroleukaemia cells results in a rapid impairment of
protein synthesis that precedes inhibition of cell proliferation
and loss of cell viability by several hours The inhibition of
translation is associated with specific cleavages of
polypep-tide chain initiation factors eIF4GI and eIF4B, a
pheno-menon previously observed in cells induced to undergo
apoptosis in response to other stimuli Although caspase
activity is enhanced in the cells in which p53 is activated, both
the effects on translation and the cleavages of the initiation
factors are completely resistant to inhibition of caspase
activity Moreover, exposure of the cells to a combination of the caspase inhibitor z-VAD.FMK and the survival factor erythropoietin prevents p53-induced cell death but does not reverse the inhibition of protein synthesis We conclude that the p53-regulated cleavages of eIF4GI and eIF4B, as well as the overall inhibition of protein synthesis, are caspase-inde-pendent events that can be dissociated from the induction of apoptosis per se
Keywords: caspases; erythroleukaemia; p53; protein synthe-sis; temperature-sensitive mutants
The tumour suppressor protein p53 is a key regulator of
both cell cycle progression and cell death by apoptosis [1–5]
Inactivating mutations of p53 have been found with high
frequency in a broad spectrum of tumours and the
inactivation of p53 is central to the transforming function
of several viral oncoproteins [6–8] Primarily, p53 functions
as a transcription factor controlling expression of genes that
affect cell proliferation, induce DNA repair or regulate cell
survival [9–12] Expression of p53 in p53-negative cell lines
induces a cell cycle block and in many cases results in cell death by apoptosis [1,13] p53 has also been demonstrated
to control the activity of RNA polymerases I and III, suggesting that p53 regulates the synthesis of ribosomes and tRNAs [14] Furthermore, the tumour suppressor protein has been found in association with ribosomes [15,16] and has been shown to have an effect on the translation of specific mRNAs, such as those encoding cdk4, fibroblast growth factor (FGF) 2 and p53 itself [14,17–21]
Recently, we reported that p53 down-regulates overall translation at the level of polypeptide chain initiation [22] In those studies we utilized a murine erythroleukaemia (MEL) cell line expressing a temperature-sensitive p53 mutant (Val135) [23] and showed that activation of p53 by placing the cells at 32C caused a rapid decrease in the overall rate
of protein synthesis However it has not been established whether this translational inhibition is an early part of the programme of induced cell death or whether it is associated with the block to cell cycle progression mediated by the activation of p53 There are strong precedents for the former as several studies have shown that the induction of apoptosis by other agents is accompanied by a substantial down-regulation of translation and the caspase-mediated cleavage of certain polypeptide chain initiation factors [24–29]
In the work described here we have employed the same MEL cell system to address some of these issues Careful comparisons of the kinetics of translational down-regula-tion vs the inhibidown-regula-tion of cell cycle progression and induction of apoptosis show that the effect of p53 activation on protein synthesis is an early event that precedes both overt inhibition of cell proliferation and the loss of cell viability We show that although caspase
Correspondence to M J Clemens, Department of Basic Medical
Sciences, St George’s Hospital Medical School, Cranmer Terrace,
London SW17 0RE, UK.
Fax: + 44 (0)20 87252992, Tel.: + 44 (0)20 8725 5762,
E-mail: M.Clemens@sghms.ac.uk
Abbreviations: 4E-BP1, eIF4E binding protein 1; Ac-DEVD-AMC,
acetyl-Asp-Glu-Val-Asp7-amino-4-methylcoumarin;
Ac-IETD-AMC, acetyl-Ile-Glu-Thr-Asp7-amino-4-methylcoumarin;
Ac-LEHD-AMC,
acetyl-Leu-Glu-His-Asp7-amino-4-methylcou-marin; eIF, eukaryotic initiation factor; Epo, erythropoietin; MEL,
murine erythroleukaemia; mTOR, mammalian target of rapamycin;
PARP, poly(ADP-ribose) polymerase; RFU, relative fluorescence
units; TNF-a, tumour necrosis factor a; TRAIL, tumour necrosis
factor-related apoptosis-inducing ligand; z-VAD.FMK,
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone.
*Present address: Department of Biochemistry, University of
Leicester, University Road, Leicester, LE1 7RH, UK.
Present address: School of Biological Sciences, University of
Manchester, 2.205 Stopford Building, Oxford Road,
Manchester M13 9PT, UK.
(Received 11 March 2003, revised 21 May 2003,
accepted 27 May 2003)
Trang 2activities increase within a few hours of activating p53, and
specific proteolytic cleavages of some polypeptide chain
initiation factors are observed, the factor cleavages do
not depend on caspase activity Clonogenicity assays have
established that the cells do not become irreversibly
committed to apoptosis until several hours after the initial
inhibition of translation Moreover, conditions that block
apoptosis do not prevent the p53-induced translational
down-regulation Our results are consistent with a
mech-anism whereby the p53-mediated inhibition of protein
synthesis in MEL cells is at least partially mediated by
initiation factor cleavages However caspase activity is not
required for these cleavages and the down-regulation of
translation can be dissociated from the p53-induced
apoptotic programme
Materials and methods
Cell culture conditions
The Val135 and Pro190 MEL cell lines were obtained from
S Benchimol [30] and were grown in stationary suspension
culture in DMEM medium supplemented with glutamine
(300 mgÆL)1) and 10% (v/v) fetal bovine serum in a 5%
CO2atmosphere at 38C Under these conditions the cells
had a doubling time of approximately 12 h Cultures were
maintained at densities between 2 and 8· 105 cells per
milliliter Continued expression of p53 was assured by
weekly selection of the cells in G418 (200 lgÆmL)1) For
activation of p53 in the Val135 cells the cultures were
transferred to 32C for the times indicated The control
Pro190 cells were treated similarly Where indicated, the
cells were treated with erythropoietin (Epo) and caspase
inhibitors at the concentrations described in Table 2 and
legends to Figs 4–6
Analysis of cell proliferation, the cell cycle and clonal
growth potential
Cells were counted in triplicate using a haemocytometer and
cell viability was determined by trypan blue exclusion For
cell cycle analysis cells were examined by flow cytometry as
described in [31] Cells that had been grown at 38C or
incubated at 32C for various periods of time (107cells per
sample) were centrifuged at 1000 g for 5 min and washed
three times in 5 mL NaCl/Pi The pellets were resuspended
in approximately 500 lL of NaCl/Pi, 5 mL of cold ethanol
were added and the cells were fixed at 4C overnight
The fixed cells were washed in NaCl/Pi and stained with
propidium iodide (500 lgÆmL)1) After treatment with
boiled RNAse A the cells were analyzed on a FACS flow
cytometer (Beckton Dickinson)
To determine the ability of the cells to proliferate clonally
after exposure to the p53 permissive temperature, Pro190
and Val135 cells were diluted 1· 105 such that the final
concentration was three cells per milliliter Aliquots of
100 lL were added to the wells of 96-well microtiter plates
(giving an average of one cell for every three wells) The
plates were incubated for various times up to 72 h at 32C
and then returned to 38C The wells were observed
microscopically after a total of 10 days and were scored for
the number of clones that had proliferated
Measurement of protein synthesis rates Overall rates of protein synthesis were measured by pulse-labeling intact cells for up to 1 h with 10–15 lCiÆmL)1of [35S]methionine (in the presence of the normal level of methionine in the cell culture medium) The cells were centrifuged briefly at 1000 g, washed once in cold NaCl/Pi, dissolved in 0.3 M NaOH and precipitated with 10% trichloroacetic acid in the presence of 0.5 mg bovine serum albumin carrier protein Precipitates were harvested on GF/
C filters under suction and washed with 5% trichloroacetic acid and industrial methylated spirit The acid-insoluble radioactivity was determined by scintillation counting
Preparation of cell extracts and analysis
by immunoblotting Cytoplasmic extracts were prepared for immunoblotting by washing the cells in NaCl/Piand lyzing them in a buffer containing a cocktail of protease and protein phosphatase inhibitors [24] The extracts were analyzed by SDS gel electrophoresis using equal amounts of protein in each lane
of the gel (3–10 lg protein per sample) After transfer of the proteins to poly(vinylidene difluoride) membranes the blots were blocked and incubated with the appropriate primary antibodies against polypeptide chain initiation factors eIF4B and eIF4GI The blots were developed with alkaline phosphatase-linked secondary antibodies using nitroblue tetrazolium as the substrate [24], or with horseradish peroxidase-linked secondary antibodies followed by enhanced chemiluminescence As a positive control for the effects of z.VAD-FMK, the same initiation factors were also examined in extracts from Jurkat cells treated with
an agonistic anti-Fas (CD95) antibody, as described previously [32,33]
Measurements of apoptosis The progress of apoptosis in the MEL cells was assessed
by measuring the activities of caspases-3, -8 and -9 in cell extracts At appropriate times after incubation at 32C, in the absence or presence of z.VAD-FMK, aliquots of 107 cells were washed with NaCl/Pi, resuspended in 1 mL cell lysis buffer (10 mM Hepes, pH 7.3, 2 mM EDTA, 0.1% NP-40, 5 mM dithiothreitol, 1 mMphenylmethanesulfonyl fluoride, 10 lgÆmL)1 pepstatin A, 20 lgÆmL)1 leupeptin,
10 lgÆmL)1aprotinin) and incubated on ice for 10–15 min After centrifugation of the extracts at 10 000 g for 1 min at
4C the supernatants were frozen at )80 C Caspase activities using fluorogenic substrates were determined in a Packard Fusion microplate reader Twenty microliters of each cell extract was incubated with 200 lL of reaction buffer [100 mMHepes, pH 7.3, 20% (v/v) glycerol, 0.5 mM EDTA, 5 mM dithiothreitol] and 2 lL of substrate for caspases-3, -8 or -9 (Ac-DEVD-AMC, Ac-IETD-AMC or Ac-LEHD-AMC, respectively) (Biosource International), each at 5 mM Reactions were incubated at 37C for 1 h and the product was quantified by fluorescence using an excitation wavelength of 380 nm and an emission wave-length of 460 nm Protein concentrations were determined and caspase activities expressed in relative fluorescence units (RFU) per microgram of protein Apoptosis was
Trang 3also assessed by the cleavage of the caspase substrates
poly(ADP-ribose) polymerase (PARP) and p27KIP1, using
immunoblotting procedures as described elsewhere
[24,34,35]
Results
Inhibition of protein synthesis and cell proliferation
following activation of p53
Activation of the temperature-sensitive Val135 p53 mutant
in MEL cells (or of the equivalent Val138 mutant in human
cells) can be achieved by reducing the incubation
tempera-ture from 38 to 32C and results in inhibition of cell
proliferation and subsequent induction of apoptosis [36–39]
Figure 1A shows the kinetics of cell growth at the two
temperatures of the Val135 cells, containing the
tempera-ture-sensitive p53, in comparison with that of Pro190 cells
which express a mutant form of p53 that is inactive at either
temperature Both cell lines grew at approximately equal
rates at 38C, with a doubling time of about 12 h At 32 C
the growth rates were slower but again approximately the
same for the first 24 h, during which both cell lines
completed at least one traverse of the cell cycle After this
time, whereas the Pro190 cells continued to proliferate, the
Val135 cells showed no further increase in number and
indeed exhibited a decline over the ensuing 24–48 h Cell
cycle analysis of the Val135 cells (Fig 1B) indicates that
after about 24 h at 32C there was a substantial decrease in
the fraction of cells in G2/M relative to G1, consistent with a
cell cycle block in the G1 phase At this time very few cells
showed a sub-G1 DNA content, in contrast to the situation
at later times (Fig 1B), suggesting that overt apoptosis does
not begin until after 24 h Pro190 cells showed neither any
significant shift in cell cycle distribution nor any evidence of
apoptosis, even after 72 h at 32C (data not shown)
Consistent with the cell cycle analysis, the viability of the
Val135 cells remained high up to 20 h at 32C but declined
substantially thereafter, as judged by trypan blue exclusion
assays (Fig 2A)
In contrast to the delayed effects of p53 activation on cell
proliferation and viability, the shift to the lower temperature
resulted in an early inhibition of the overall rate of protein
synthesis in the Val135 cells, relative to that in the Pro190
cells (Fig 2A) Thus comparison of the kinetics of inhibition
with the rate of decline in cell viability and the appearance of
apoptotic cells shows that the p53-mediated decrease in
translational activity preceded cell death by several hours
We also investigated whether the translational inhibition,
although preceding overt apoptosis, may nevertheless act
as a signal to commit cells to death To test this, we
measured the ability of Val135 cells to recover when
replaced at 38C after various lengths of exposure to the
p53-permissive temperature Figure 2B shows that the
majority of cells retained the ability to survive and recover
after incubation at 32C for up to 16 h (as judged by their
potential for subsequent clonal growth at 38C) This was
in spite of the fact that the overall rate of protein synthesis
progressively declined by up to 50% over this time period
However, after 20 h or more at 32C the ability of the
cells to recover declined sharply, coinciding with the onset
of cell death indicated by the failure to exclude trypan blue
Taken together with the data in Fig 1 these results suggest that the effect of p53 on protein synthesis cannot merely be
a consequence of either the cessation of cell proliferation or the loss of cell viability as it precedes both these events in the temperature-sensitive MEL cells incubated at the permissive temperature Moreover, translational inhibition per se for
up to 16 h is not sufficient to induce cell death Neverthe-less, as we have not yet identified a means of preventing the down-regulation of protein synthesis, we cannot exclude
a requirement for longer periods of inhibition for the p53-mediated induction of subsequent apoptosis
Fig 1 Inhibition of cell proliferation and changes in cell cycle distri-bution following activation of p53 (A) Exponentially growing Val135 and Pro190 MEL cells were diluted to 1.3 · 10 5 cellsÆmL)1and incu-bated at 38 C or 32 C for the times indicated Total cell numbers were determined in quadruplicate in a haemocytometer The values shown are means ± SEM (B) Exponentially growing Val135 MEL cells were maintained at 38 C or transferred to 32 C for 24 h or 48 h The cells were fixed with ethanol and then stained with propidium iodide as described in Materials and methods The distribution of the cells in the cell cycle was determined by FACS analysis of DNA content The peaks corresponding to cells with a sub-G1, G1 or G2/M DNA content are indicated.
Trang 4Initiation factor cleavages following activation of p53
Previously several changes to the protein synthetic
machin-ery have been observed to occur during the early stages of
apoptosis in a range of cell types These include the specific,
caspase-dependent cleavage of polypeptide chain initiation
factors such as eIF4GI, eIF4B and 4E-BP1 [24–29] We
have therefore examined extracts from Val135 and Pro190
MEL cells for the integrity of eIF4GI and eIF4B following
the shift to 32C Figure 3 shows that, although the effect was variable from one experiment to another, both eIF4GI and eIF4B underwent partial cleavage within 6–8 h following the activation of p53 in the Val135 cells, giving rise to discrete fragments These changes were not seen in the Pro190 cells after the shift to 32C The fragments that were generated correspond in size to the eIF4GI cleavage products N-FAG + M-FAG and M-FAG alone [27] (Fig 3A,B) and to the eIF4B cleavage product DeIF4B [28,29] (Fig 3C) These products have previously been observed in both human and mouse cells induced to undergo apoptosis in response to treatment with cyclo-heximide, anti-Fas (CD95) antibody, TNF-a, TRAI L, staurosporine or etoposide [24–29,39] Partial cleavage was also seen in the case of the eIF4E binding protein 4E-BP1 in the Val135 cells (data not shown)
Fig 2 Activation of p53 results in rapid impairment of protein synthesis
that precedes the loss of cell viability and irreversible commitment to cell
death (A) Exponentially growing Val135 and Pro190 cells were
counted, transferred from 38 C to 32 C and incubated for the times
shown Rates of protein synthesis per 105cells were then measured by
pulse-labeling the cells with [ 35 S]methionine (10 lCiÆmL)1) for 15 min
(two incubations, each in triplicate), as described in Materials and
methods Methionine incorporation in the Val135 cells is shown as a
percentage of that in the Pro190 cells at the same temperature (s) The
cell viabilities were determined by trypan blue exclusion and are
plotted on the same time scale (d,m) (B) Val135 and Pro190 cells were
extensively diluted and placed in multiwell plates such that an average
of only one cell was present in every three wells of the plates After
incubation for various times at 32 C, the cells were shifted back to
38 C and allowed to proliferate The wells were observed
micro-scopically 10 days later and scored for the numbers of colonies formed.
The data are the means ± the ranges of duplicate determinations.
Dark-shaded bars, Pro190 cells; light-shaded bars, Val135 cells.
Fig 3 Activation of p53 causes cleavage of initiation factors eIF4GI and eIF4B (A) Characterization of cleavage products of eIF4GI: Pro190 and Val135 cells were incubated at 32 C for 15 h and extracts prepared and analyzed by immunoblotting for initiation factor
eIF4-GI The positions of migration of the intact factor ( 200 kDa), an intermediate cleavage product comprising the N-terminal and middle fragments of eIF4GI (N-FAG plus M-FAG) ( 150 kDa) and the middle fragment of eIF4GI (M-FAG) ( 76 kDa) [27] are indicated (B) Time-course of cleavage of eIF4GI following activation of p53 at
32 C Pro190 and Val135 cells were incubated at 32 C for the times indicated and extracts were analyzed for the disappearance of intact eIF4GI and the appearance of M-FAG as in (A) (C) Time-course of cleavage of eIF4B following activation of p53 at 32 C Pro190 and Val135 cells were incubated at 32 C for the times indicated and extracts were analyzed for the presence of eIF4B (80 kDa) and its cleavage product DeIF4B (60 kDa) as described previously [28,29] No cleavage of eIF4B was observed when either cell line was maintained at
38 C (see Fig 5C).
Trang 5Effects of p53 on protein synthesis and initiation factor
cleavages are independent of caspases
To determine whether caspases are activated under the same
conditions that result in the initiation factor cleavages we
have assayed caspases-3, -8 and -9 in extracts from the two
cell lines As shown in Table 1, within 6 h at 32C the
activities of all three caspases increased significantly in the
Val135 cells but not in the Pro190 cells, reaching a
maximum at about 20 h In addition, we determined the
extent of cleavage of two well characterized caspase
substrates, PARP and the cyclin-dependent protein kinase
inhibitor p27KIP1(Fig 4A,B) Consistent with the
activa-tion of caspase-3, processing of PARP to give rise to the
characteristic p89 cleavage product was observed (Fig 4A)
A proportion of p27KIP1 was also cleaved to produce a
discrete fragment, Dp27 (Fig 4B) Both the p53-induced
increase in caspase-3 activity and the cleavages of PARP
and p27KIP1were inhibited by the broad specificity caspase inhibitor z-VAD.FMK (Table 2, Fig 4A,B)
The above results show that, although cell viability does not decline until about 20 h (Fig 2), p53 activation does cause increased caspase activity within 6 h We therefore investigated whether caspase activity is responsible for the early changes in protein synthesis and the initiation factor cleavages that occur following the activation of p53 Figure 5(A,B) shows that the p53-induced inhibition of protein synthesis was completely resistant to treatment of the cells with z-VAD.FMK, both at early and late times after the temperature shift Moreover, neither the cleavage
of eIF4GI nor that of eIF4B was prevented by the caspase inhibitor, even when these cleavages involved only a relatively small fraction of the respective proteins (Fig 5C) This was the case even though the z-VAD.FMK was able to block completely the activity of caspase-3 (Table 2), as well
as that of caspases-8 and -9 (data not shown), and prevented the cleavages of PARP and p27KIP1 induced by p53 activation (Fig 4A,B) Moreover, the same z-VAD.FMK preparation inhibited the extensive cleavages of eIF4GI and eIF4B that occur in another apoptotic system, viz Jurkat cells treated with an agonistic anti-Fas antibody [32,33] (Fig 5C) In some experiments where more extensive cleavage of eIF4GI occurred z-VAD-FMK had a very slight protective effect but a substantial level of M-FAG was still generated in the presence of the caspase inhibitor
Table 1 Activation of p53 rapidly enhances caspase activity Pro190 and Val135 cells were incubated at 32 C for the times indicated Cell extracts were prepared and the activities of caspases-3, -8 and -9 were assayed as described in Materials and methods The data are expressed as RFU per lg
of protein and show the means ± the ranges of duplicate determinations.
Cell line
Caspase activity (RFUÆlg)1protein)
Hours at 32 C
Fig 4 Activation of p53 in MEL cells causes caspase-dependent
cleavages of PARP and p27KIP1 (A) Pro190 and Val135 cells were
incubated at 32 C in the presence and absence of z-VAD.FMK for
15 h Extracts were prepared and immunoblotted for (A) the apoptotic
cleavage product of PARP (89 kDa) and (B) p27 KIP1 and its caspase
cleavage product Dp27 as described previously [33–35].
Table 2 p53-induced caspase activity is sensitive to inhibition by z.VAD-FMK Val135 cells were incubated at 38 C or at 32 C for 6 h
in the presence and absence of the caspase inhibitor z.VAD-FMK (10 l M and 50 l M ) Extracts were prepared and assayed for caspase-3 activity by cleavage of the substrate Ac-DEVD.AMC as described in Materials and methods The data are expressed as RFU per micro-gram of protein and are the means ± the ranges of duplicate deter-minations.
Condition
Caspase-3 activity (RFUÆlg protein)1)
32 C plus z.VAD-FMK (10 l M ) 50.7 ± 1.6
38 C plus z.VAD-FMK (50 l M ) 17.5 ± 1.6
Trang 6(Fig 5D) These data therefore suggest that p53 regulates
protein synthesis by mechanism(s) that do not require
caspase activity and are consistent with the conclusion that
the translational inhibition occurs independently of the
induction of apoptosis
It is likely that other proteases are involved in causing the
initiation factor cleavages and these may be responsible for
regulating translation following p53 activation [40,41]
Incubation of the Val135 cells with a range of protease
inhibitors, viz the chymotrypsin inhibitor TPCK, the
calpain inhibitors N-acetyl-Leu-Leu-Nle-CHO (ALLN),
calpain inhibitor IV (z-LLY.FMK) and calpeptin
(z-Leu-Nle-CHO), and the cathepsin B inhibitor z-FA.FMK,
prevented neither the p53-induced cleavage of eIF4GI nor
the inhibition of protein synthesis at 32C (data not
shown) Further investigations utilizing a wider range of
protease inhibitors will therefore be necessary to identify the
enzyme(s) involved
Although the inhibition of protein synthesis by p53
activation can be dissociated temporally from the progress
of apoptosis, we wanted to determine whether the
preven-tion of cell death had an effect on the down-regulapreven-tion of
translation To address this question we took advantage
of the observation that treatment of cells either with
z-VAD.FMK or with cytokines that function as survival
factors inhibits p53-induced apoptosis [13,30,42–46] In our
hands, although z-VAD.FMK and the erythroid cell-specific survival factor erythropoietin (Epo) each had a marked antiapoptotic effect, both were required together to prevent completely the loss of viability of Val135 cells at
32C (Fig 6A) In spite of this dramatic protective effect, however, neither z-VAD.FMK nor Epo, alone or in combination, showed any ability to rescue protein synthesis from p53-induced inhibition (Fig 6B) This again suggests that the down-regulation of translation by p53 does not require the activity of caspases or other apoptotic mediators
It also indicates that the inhibition does not involve other pathways that are inactivated in the presence of Epo
Discussion
The tumour suppressor protein p53 is activated in cells by
a number of stresses, including UV irradiation, chemically induced DNA damage and hypoxia [47–50] Activation of p53 results in a variety of cellular responses, notably inhibition of cell cycle progression and stimulation of DNA repair [51] If p53 activity is sustained it can also lead to cell death by apoptosis [52] Many of these effects require nuclear translocation of p53 and subsequent transcriptional activation of a large number of target genes [10,53] However there is also evidence for direct cytoplasmic effects
of activated p53, including association of the protein with
Fig 5 p53-induced translational inhibition and initiation factor cleavages do not require caspase activity (A and B) Protein synthesis measurements Pro190 and Val135 cells were incubated at 32 C for (A) 4 h or 6 h or (B) 15 h in the presence and absence of z-VAD.FMK (50 l M ) and protein synthesis was determined as described in Materials and methods (C) Pro190 and Val135 cells were incubated at 32 C for 15 h in the presence and absence of z-VAD.FMK and cell extracts were immunoblotted for eIF4GI or eIF4B and their cleavage products As a positive control for the efficacy of the z-VAD.FMK, Jurkat cells were incubated for 2 h with or without an agonistic anti-Fas antibody [32], in the presence or absence of the same preparation of the inhibitor, and extracts were blotted for the same initiation factors (D) Val135 cells were incubated at 38 C or 32 C for
6 h in the presence or absence of z-VAD.FMK as indicated Cell extracts were immunoblotted for eIF4GI and its cleavage products.
Trang 7mitochondria and ribosomes [15,16,54], and several studies
have shown that pro-apoptotic effects of p53 do not
necessarily require transcriptional transactivation activity of
the protein [55–57]
Well documented reports have revealed a role for p53 in
the control of translation of individual mRNA species such
as those encoding cdk4, FGF-2 and even p53 itself [14,17–
21] However the mechanisms responsible have not been
elucidated Our data show that p53 can also control the rate
of global protein synthesis Although the inhibition of
translation precedes the impairment of cell cycle progression
such an effect may ultimately contribute to the growth
inhibitory effects of the activated tumour suppressor
protein Detailed analysis of the kinetics of the inhibition
of translation has shown that this effect begins within 2–4 h
of activating wild-type p53 [22] (manuscript in preparation)
Thus it is unlikely that the regulation of protein synthesis is
simply a consequence of either the impairment of cell cycle
progression or the induction of apoptosis, both of which are
associated with inhibition of translation in other systems
[58–60] However the question of whether common
signal-ing pathways are involved in the control of translation and
in the effects of p53 on the cell cycle and/or apoptosis will require the use of further mutants of p53 defective in inducing one or other of the latter effects It is possible that the effects of p53 on protein synthesis are a result of new gene transcription events, although the early response time would tend to mitigate against this Unfortunately we have been unable to use transcription inhibitors to investigate this possibility directly because such agents alone affect p53 function [61]
We have reported elsewhere that the extent of phos-phorylation of the inhibitor of polypeptide chain initiation factor eIF4E, 4E-BP1, is reduced following activation of p53 in the Val135 cells and that this results in sequestra-tion of eIF4E away from the eIF4F initiasequestra-tion complex [22] No changes in the phosphorylation state of other key protein synthesis initiation factors such as eIF2a or eIF4E could be observed The possibility that the dephosphory-lation of 4E-BP1 results in the inhibition of transdephosphory-lation of specific mRNAs, including those known to be regulated at the translational level by p53, remains to be tested The changes in 4E-BP1 function, in combination with the partial cleavages of eIF4GI and eIF4B reported here, may be sufficient to bring about the overall inhibition of protein synthesis by p53 However the present data do not address this issue directly In many experiments a significant proportion of both eIF4GI and eIF4B remained intact following p53 activation; nevertheless it
is possible that the cleavage products that accumulate could exert an inhibitory (dominant negative) effect on the activity of the remaining full-length protein A further consequence of the cleavage of eIF4GI could be the stimulation of cap-independent translation Fragments that are generated in apoptotic cells from both eIF4GI itself [62] and the related protein DAP5 [63,64] have been shown to enhance the utilization for translation of mRNAs with internal ribosome entry sites
At first sight the specific cleavages of initiation factors eIF4GI and eIF4B, both of which are known substrates for proteolysis in apoptosing cells, would seem to be in accord with the established pro-apoptotic effects of p53 These factors have been shown previously to be cleaved in cells undergoing apoptosis in response to treatment with anti-Fas antibody [28,32,33], cycloheximide [24,28], staurosporine or tumour necrosis factor a [26] However, as shown in Figs 2 and 3, the initial down-regulation of translation, as well as the cleavages of eIF4GI and eIF4B, occurs at a time when there is little loss of cell viability and during a period when the p53-induced inhibition of cell growth is reversible [65] This indicates that neither the translational inhibition nor the initiation factor modifications are simply consequences
of apoptosis Moreover these events are clearly not sufficient
to commit the cells to death, although of course later changes that affect translation may be Consistent with these conclusions, progression into apoptosis is not required for translational inhibition by p53 as essentially complete protection of the Val135 cells against death at 32C by the combination of z.VAD-FMK and Epo did not rescue protein synthesis (Fig 6)
Both eIF4GI and eIF4B can be cleaved by caspase-3
in vivo and in vitro [27,29] As caspase-3 is activated in the Val135 cells following the temperature shift, and the cleavage products of the two initiation factors appear to be
Fig 6 The p53-induced inhibition of protein synthesis can be dissociated
fromapoptosis (A) Pro190 and Val135 cells were incubated at 38 C or
32 C for 48 h in the absence or presence of Epo (10 unitsÆmL)1) and/
or z.VAD-FMK (10 l M ) as indicated At the end of this period cell
viability was determined by trypan blue exclusion (B) Cells were
incubated as in (A) After 24 h, protein synthesis was monitored by the
incorporation of [ 35 S]methionine (10 lCiÆmL)1) into acid-insoluble
material during the last 1 h of incubation The data are expressed as a
percentage of the incorporation in Pro190 cells incubated at 38 C in
the absence of Epo and z.VAD-FMK.
Trang 8very similar, if not identical, to those seen as a result of
caspase-dependent degradation in other systems, we were
surprised to find that the appearance of M-FAG and
DeIF4B was not inhibited by the broad specificity caspase
inhibitor z.VAD-FMK This was not due to a failure of the
latter to act on MEL cells as the compound inhibited the
activation of caspase-3 and completely blocked the cleavage
of the caspase substrates PARP and p27KIP1in Val135 cells
shifted to 32C Moreover the same z.VAD-FMK
prepar-ation was effective in inhibiting the cleavage of eIF4GI and
eIF4B in Jurkat cells treated with anti-Fas Along with an
inability to prevent the factor cleavages in the Val135 cells
z.VAD-FMK was also unable to prevent the overall
inhibition of protein synthesis
Although we cannot rule out the possibility that eIF4GI
and eIF4B are cleaved by caspase(s) that are at least
partially active even in the presence of z.VAD-FMK [66] it
is possible that other proteases that are activated directly or
indirectly by p53 are responsible [40,41] Such alternative
pathways may also operate in other systems Whereas p53 is
required for radiation-induced neuronal cell death, caspase
activity is not required for this process [67] Several studies
have established the phenomenon of caspase-independent
cell death Moreover noncaspase proteases are involved in
some forms of apoptosis mediated by p53 and other
pathways, and specific protein cleavages occur in some cases
[68–72] Morley and Pain [73] reported that eIF4GI and
eIF4GII can be cleaved by a z.VAD-FMK-resistant
mech-anism in cells undergoing apoptosis in response to treatment
with the immunosuppressant drugs FTY720 and
cyclo-sporin A If noncaspase mediated proteolytic events are
responsible for the cleavage of eIF4GI and eIF4B the
enzyme(s) involved must presumably act on sites that are
identical or very close to those targeted by the caspases
[27,28] These sites may lie in relatively accessible or
unstructured regions of the proteins In spite of using a
wide range of protease inhibitors we have not yet identified
the protease(s) responsible for the initiation factor cleavages
following p53 activation
The effects of p53 on the translational machinery are
very similar to those seen following treatment of cells with
DNA damaging agents such as etoposide, mitomycin-C or
cisplatin [32,39,74] Common features include the
caspase-independent nature of the inhibition of overall translation,
the lack of effect on eIF2a phosphorylation and, in contrast,
the marked dephosphorylation of 4E-BP1 [22] These
observations suggest that the effects of DNA-damaging
agents on translation could be mediated, at least in part, by
p53 The p53-regulated effects we observe are also similar to
those seen following inhibition of proteasome activity [75]
Proteasome inhibition not only induces p53-dependent
apoptosis [76–78] but also causes dephosphorylation of
4E-BP1 and the cleavage of initiation factors, effects which
are partially caspase-independent (S Morley, personal
communication) Cyclosporin A, which can induce eIF4G
cleavage [73], also inhibits proteasome activity [79,80]
Moreover, inhibition of proteasome-mediated proteolysis
induces p53 expression and caspase-independent apoptosis
[78,81]
Another potential mechanism of action of p53 may
involve signaling by ceramide as a second messenger
Ceramide causes caspase-independent apoptosis and also
induces p53 in at least one system [82,83] Irradiation-induced DNA damage activates ceramide production [84], and p53 is required for the induction of ceramide by some cell stresses [85] Whether the tumour suppressor protein
is required for ceramide-induced growth inhibition and apoptosis remains controversial however, [85–87] If p53 functions upstream of ceramide then the latter may indeed contribute to the down-regulation of translation observed in this study However ceramide has been reported to activate the eIF2a-specific protein kinase PKR and thereby inhibit translation [88], whereas p53 activation has no effect on eIF2a phosphorylation [22] Few other studies of the effects
of ceramide on protein synthesis or initiation factor modifications have been reported and the possibility of regulation by this second messenger in cells expressing active p53 remains to be evaluated
In summary, we have reported the novel observation that activation of p53 results in the caspase-independent down-regulation of translation, together with the cleavages of at least two polypeptide chain initiation factors that are critical for protein synthesis Moreover, we have shown that these events are not simply the consequences of p53-induced apoptosis and indeed occur independently of this process Further details of the mechanisms involved await future study
Acknowledgements This research was supported by grants to M J Clemens from the Wellcome Trust (056778), the Leukaemia Research Fund and Glaxo-Wellcome and by grants to J Hensold from the Office of Research and Development, Medical Research Service, Department of Veterans’ Affairs and the NIH (DK43414) J Hensold was also funded during a period of sabbatical leave by an award from Burroughs-Wellcome.
C Constantinou is supported by a PhD studentship from the Cancer Prevention Research Trust, with additional funding from the AG Leventis Foundation and an Overseas Research Scholarship from Universities UK M Bushell is supported by a Fellowship from The Wellcome Trust (063233).
References
1 Johnson, P., Gray, D., Mowat, M & Benchimol, S (1991) Expression of wild-type p53 is not compatible with continued growth of p53-negative tumor cells Mol Cell Biol 11, 1–11.
2 Burns, T.F & El-Deiry, W.S (1999) The p53 pathway and apoptosis J Cell Physiol 181, 231–239.
3 Sionov, R.V & Haupt, Y (1999) The cellular response to p53: the decision between life and death Oncogene 18, 6145–6157.
4 Appella, E (2001) Modulation of p53 function in cellular regu-lation Eur J Biochem 268, 2763–2763.
5 Vousden, K.H (2000) p53: death star Cell 103, 691–694.
6 Farrell, P.J., Allan, G.J., Shanahan, F., Vousden, K.H & Crook,
T (1991) p53 Is frequently mutated in Burkitt’s lymphoma cell lines EMBO J 10, 2879–2887.
7 Bates, S & Vousden, K.H (1999) Mechanisms of p53-mediated apoptosis Cell Mol Life Sci 55, 28–37.
8 Wang, X.W (1999) Role of p53 and apoptosis in carcinogenesis Anticancer Res 19, 4759–4771.
9 Owen-Schaub, L.B., Zhang, W., Cusack, J.C., Angelo, L.S., Santee, S.M., Fujiwara, T., Roth, J.A., Deisseroth, A.B., Zhang, W.W., Kruzel, E & Radinsky, R (1995) Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression Mol Cell Biol 15, 3032–3040.
Trang 910 Miyashita, T & Reed, J.C (1995) Tumor suppressor p53 is a
direct transcriptional activator of the human bax gene Cell 80,
293–299.
11 Kato, M.V., Sato, H., Anzai, H., Nagayoshi, M & Ikawa, Y.
(1997) Up-regulation of cell cycle-associated genes by
p53 in apoptosis of an erythroleukemic cell line Leukemia 11,
389–392.
12 Tokino, T & Nakamura, Y (2000) The role of p53-target genes in
human cancer Crit Rev Oncol Hematol 33, 1–6.
13 Lin, Y.P & Benchimol, S (1995) Cytokines inhibit p53-mediated
apoptosis but not p53-mediated G 1 arrest Mol Cell Biol 15,
6045–6054.
14 Ewen, M.E & Miller, S.J (1996) p53 and translational control.
Biochim Biophys Acta Rev Cancer 1242, 181–184.
15 Fontoura, B.M.A., Atienza, C.A., Sorokina, E.A., Morimoto, T.
& Carroll, R.B (1997) Cytoplasmic p53 polypeptide is associated
with ribosomes Mol Cell Biol 17, 3146–3154.
16 Marechal, V., Elenbaas, B., Piette, J., Nicolas, J.C & Levine, A.J.
(1994) The ribosomal L5 protein is associated with mdm-2 and
mdm-2-p53 complexes Mol Cell Biol 14, 7414–7420.
17 Ewen, M.E., Oliver, C.J., Sluss, H.K., Miller, S.J & Peeper, D.S.
(1995) p53-dependent repression of CDK4 translation in
TGF-b-induced G 1 cell-cycle arrest Genes Dev 9, 204–217.
18 Miller, S.J., Suthiphongchai, T., Zambetti, G.P & Ewen, M.E.
(2000) p53 binds selectively to the 5¢ untranslated region of cdk4,
an RNA element necessary and sufficient for transforming growth
factor b- and p53-mediated translational inhibition of Cdk4 Mol.
Cell Biol 20, 8420–8431.
19 Galy, B., Cre´ancier, L., Zanibellato, C., Prats, A.C & Prats, H.
(2001) Tumour suppressor p53 inhibits human fibroblast growth
factor 2 expression by a post-transcriptional mechanism
Onco-gene 20, 1669–1677.
20 Galy, B., Cre´ancier, L., Prado-Lourenco, L., Prats, A.-C & Prats,
H (2001) p53 directs conformational change and translation
initiation blockade of human fibroblast growth factor 2 mRNA.
Oncogene 20, 4613–4620.
21 Mosner, J., Mummenbrauer, T., Bauer, C., Sczakiel, G., Grosse,
F & Deppert, W (1995) Negative feedback regulation of
wild-type p53 biosynthesis EMBO J 14, 4442–4449.
22 Horton, L.E., Bushell, M., Barth-Baus, D., Tilleray, V.J.,
Clemens, M.J & Hensold, J (2002) p53 activation results in rapid
dephosphorylation of the eIF4E-binding protein 4E-BP1,
inhibi-tion of ribosomal protein S6 kinase and inhibiinhibi-tion of translainhibi-tion
initiation Oncogene 21, 5325–5334.
23 Michalovitz, D., Halevy, O & Oren, M (1990) Conditional
inhibition of transformation and of cell proliferation by a
tem-perature-sensitive mutant of p53 Cell 62, 671–680.
24 Clemens, M.J., Bushell, M & Morley, S.J (1998) Degradation of
eukaryotic polypeptide chain initiation factor (eIF) 4G in response
to induction of apoptosis in human lymphoma cell lines Oncogene
17, 2921–2931.
25 Marissen, W.E & Lloyd, R.E (1998) Eukaryotic translation
initiation factor 4G is targeted for proteolytic cleavage by caspase
3 during inhibition of translation in apoptotic cells Mol Cell Biol.
18, 7565–7574.
26 Bushell, M., McKendrick, L., Janicke, R.U., Clemens, M.J &
Morley, S.J (1999) Caspase-3 is necessary and sufficient for
clea-vage of protein synthesis eukaryotic initiation factor 4G during
apoptosis FEBS Lett 451, 332–336.
27 Bushell, M., Poncet, D., Marissen, W.E., Flotow, H., Lloyd, R.E.,
Clemens, M.J & Morley, S.J (2000) Cleavage of polypeptide
chain initiation factor eIF4GI during apoptosis: characterisation
of an internal fragment generated by caspase-3-mediated cleavage.
Cell Death Differ 7, 628–636.
28 Bushell, M., Wood, W., Clemens, M.J & Morley, S.J (2000)
Changes in integrity and association of eukaryotic protein
synthesis initiation factors during apoptosis Eur J Biochem 267, 1083–1091.
29 Bushell, M., Wood, W., Carpenter, G., Pain, V.M., Morley, S.J & Clemens, M.J (2001) Disruption of the interaction of mammalian protein synthesis eukaryotic initiation factor 4B with the poly (A)-binding protein by caspase- and viral protease-mediated cleavages.
J Biol Chem 276, 23922–23928.
30 Johnson, P., Chung, S & Benchimol, S (1993) Growth suppres-sion of Friend virus-transformed erythroleukemia cells by p53 protein is accompanied by hemoglobin production and is sensitive
to erythropoietin Mol Cell Biol 13, 1456–1463.
31 Spector, D.L., Goldman, R.D & Leinwand, L.A (1998) Cells:
a Laboratory Manual Cold Spring Harbor Laboratory Press, New York.
32 Morley, S.J., McKendrick, L & Bushell, M (1998) Cleavage of translation initiation factor 4G (eIF4G) during anti-Fas IgM-induced apoptosis does not require signalling through the p38 mitogen-activated protein (MAP) kinase FEBS Lett 438, 41–48.
33 Morley, S.J., Jeffrey, I., Bushell, M., Pain, V.M & Clemens, M.J (2000) Differential requirements for caspase-8 activity in the mechanism of phosphorylation of elF2a, cleavage of eIF4GI and signaling events associated with the inhibition of protein synthesis
in apoptotic Jurkat T cells FEBS Lett 477, 229–236.
34 Frost, V & Sinclair, A.J (2000) p27 KIP1 is down-regulated by two different mechanisms in human lymphoid cells undergoing apop-tosis Oncogene 19, 3115–3120.
35 Frost, V., Al-Mehairi, S & Sinclair, A.J (2001) Exploitation of a non-apoptotic caspase to regulate the abundance of the cdkI p27 KIP1 in transformed lymphoid cells Oncogene 20, 2737–2748.
36 Martinez, J., Georgoff, I & Levine, A.J (1991) Cellular localiza-tion and cell cycle regulalocaliza-tion by a temperature-sensitive p53 pro-tein Genes Dev 5, 151–159.
37 Yamato, K., Yamamoto, M., Hirano, Y & Tsuchida, N (1995) A human temperature-sensitive p53 mutant p53Val-138: modulation
of the cell cycle, viability and expression of p53-responsive genes Oncogene 11, 1–6.
38 Kato, M.V (1999) The mechanisms of death of an erythroleuke-mic cell line by p53: involvement of the erythroleuke-microtubule and mito-chondria Leuk Lymphoma 33, 181–186.
39 Jeffrey, I.W., Bushell, M., Tilleray, V.J., Morley, S & Clemens, M.J (2002) Inhibition of protein synthesis in apoptosis: Differ-ential requirements by the tumour necrosis factor a family and a DNA damaging agent for caspases and the double-stranded RNA-dependent protein kinase Cancer Res 62, 2272–2280.
40 Lotem, J & Sachs, L (1996) Differential suppression by protease inhibitors and cytokines of apoptosis induced by wild-type p53 and cytotoxic agents Proc Natl Acad Sci USA 93, 12507–12512.
41 Yuan, X.M., Li, W., Dalen, H., Lotem, J., Kama, R., Sachs, L & Brunk, U.T (2002) Lysosomal destabilization in p53-induced apoptosis Proc Natl Acad Sci USA 99, 6286–6291.
42 Abrahamson, J.L., Lee, J.M & Bernstein, A (1995) Regulation of p53-mediated apoptosis and cell cycle arrest by Steel factor Mol Cell Biol 15, 6953–6960.
43 Li, P.F., Dietz, R & von Harsdorf, R (1999) p53 regulates mitochondrial membrane potential through reactive oxygen spe-cies and induces cytochrome c-independent apoptosis blocked by Bcl-2 EMBO J 18, 6027–6036.
44 Gao, C.F., Ren, S., Zhang, L., Nakajima, T., Ichinose, S., Hara, T., Koike, K & Tsuchida, N (2001) Caspase-dependent cytosolic release of cytochrome c and membrane translocation of Bax in p53-induced apoptosis Exp Cell Res 265, 145–151.
45 Yamaguchi, A., Tamatani, M., Matsuzaki, H., Namikawa, K., Kiyama, H., Vitek, M.P., Mitsuda, N & Tohyama, M (2001) Akt activation protects hippocampal neurons from apoptosis by inhibiting transcriptional activity of p53 J Biol Chem 276, 5256–5264.
Trang 1046 Di Bacco, A.M.A & Cotter, T.G (2002) p53 expression in K562
cells is associated with caspase-mediated cleavage of c-ABL and
BCR-ABL protein kinases Br J Haematol 117, 588–597.
47 Lutzker, S.G., Mathew, R & Taller, D.R (2001) A p53 dose–
response relationship for sensitivity to DNA damage in isogenic
teratocarcinoma cells Oncogene 20, 2982–2986.
48 Lakin, N.D & Jackson, S.P (1999) Regulation of p53 in response
to DNA damage Oncogene 18, 7644–7655.
49 Oren, M (1999) Regulation of the p53 tumor suppressor protein.
J Biol Chem 274, 36031–36034.
50 Xie, S., Wu, H., Wang, Q., Cogswell, J.P., Husain, I , Conn, C.W.,
Stambrook, P., Jhanwar-Uniyal, M & Dai, W (2001) Plk3 links
DNA damage to cell cycle arrest and apoptosis at least in part via
the p53 pathway J Biol Chem 276, 43305–43312.
51 Ryan, K.M., Phillips, A.C & Vousden, K.H (2001) Regulation
and function of the p53 tumor suppressor protein Curr Opin Cell
Biol 13, 332–337.
52 Lowe, S.W., Schmitt, E.M., Smith, S.W., Osborne, B.A & Jacks,
T (1993) p53 is required for radiation-induced apoptosis in mouse
thymocytes Nature 362, 847–849.
53 Rodriguez-Lopez, A.M., Xenaki, D., Eden, T.O., Hickman, J.A.
& Chresta, C.M (2001) MDM2-mediated nuclear exclusion of
p53 attenuates etoposide-induced apoptosis in neuroblastoma
cells Mol Pharmacol 59, 135–143.
54 Marchenko, N.D., Zaika, A & Moll, U.M (2000) Death
signal-induced localization of p53 protein to mitochondria – a potential
role in apoptotic signaling J Biol Chem 275, 16202–16212.
55 Caelles, C., Helmberg, A & Karin, M (1994) p53-dependent
apoptosis in the absence of transcriptional activation of p53-target
genes Nature 370, 220–223.
56 Gao, C & Tsuchida, N (1999) Activation of caspases in
p53-induced transactivation-independent apoptosis Jpn J Cancer
Res 90, 180–187.
57 Ding, H.F., Lin, Y.L., McGill, G., Juo, P., Zhu, H., Blenis, J.,
Yuan, J.Y & Fisher, D.E (2000) Essential role for caspase-8 in
transcription-independent apoptosis triggered by p53 J Biol.
Chem 275, 38905–38911.
58 Pyronnet, S., Dostie, J & Sonenberg, N (2001) Suppression of
cap-dependent translation in mitosis Genes Dev 15, 2083–2093.
59 Pyronnet, S & Sonenberg, N (2001) Cell-cycle-dependent
trans-lational control Curr Opin Genet Dev 11, 13–18.
60 Clemens, M.J., Bushell, M., Jeffrey, I.W., Pain, V.M & Morley,
S.J (2000) Translation initiation factor modifications and the
regulation of protein synthesis in apoptotic cells Cell Death Differ.
7, 603–615.
61 David-Pfeuty, T., Nouvian-Dooghe, Y., Sirri, V., Roussel, P &
Hernandez-Verdun, D (2001) Common and reversible
regulation of wild-type p53 function and of ribosomal
bio-genesis by protein kinases in human cells Oncogene 20, 5951–
5963.
62 Nevins, T.A., Harder, Z.M., Korneluk, R.G & Holcik, M.
(2003) Distinct regulation of internal ribosome entry site-mediated
translation following cellular stress is mediated by apoptotic
fragments of eIF4G translation initiation factor family members
eIF4GI and p97/DAP5/NAT1 J Biol Chem 278, 3572–3579.
63 Henis-Korenblit, S., Strumpf, N.L., Goldstaub, D & Kimchi, A.
(2000) A novel form of DAP5 protein accumulates in apoptotic
cells as a result of caspase cleavage and internal ribosome entry
site-mediated translation Mol Cell Biol 20, 496–506.
64 Henis-Korenblit, S., Shani, G., Sines, T., Marash, L., Shohat, G.
& Kimchi, A (2002) The caspase-cleaved DAP5 protein supports
internal ribosome entry site-mediated translation of death
pro-teins Proc Natl Acad Sci USA 99, 5400–5405.
65 Geske, F.J., Lieberman, R., Strange, R & Gerschenson, L.E.
(2001) Early stages of p53-induced apoptosis are reversible Cell
Death Differ 8, 182–191.
66 MacFarlane, M., Cohen, G.M & Dickens, M (2000) JNK (c-Jun N-terminal kinase) and p38 activation in receptor-mediated and chemically-induced apoptosis of T-cells: differential requirements for caspase activation Biochem J 348, 93–101.
67 Johnson, M.D., Xiang, H., London, S., Kinoshita, Y., Knudson, M., Mayberg, M., Korsmeyer, S.J & Morrison, R.S (1998) Evi-dence for involvement of Bax and p53, but not caspases, in radiation-induced cell death of cultured postnatal hippocampal neurons J Neurosci Res 54, 721–733.
68 Denecker, G., Vercammen, D., Declercq, W & Vandenabeele, P (2001) Apoptotic and necrotic cell death induced by death domain receptors Cell Mol Life Sci 58, 356–370.
69 Sperandio, S., De Belle, I & Bredesen, D.E (2000) An alternative, nonapoptotic form of programmed cell death Proc Natl Acad Sci USA 97, 14376–14381.
70 Belmokhtar, C.A., Hillion, J & Se´gal-Bendirdjian, E (2001) Staurosporine induces apoptosis through both caspase-dependent and caspase-incaspase-dependent mechanisms Oncogene 20, 3354–3362.
71 Foghsgaard, L., Wissing, D., Mauch, D., Lademann, U., Bast-holm, L., Boes, M., Elling, F., Leist, M & Ja¨a¨ttela¨, M (2001) Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor J Cell Biol 153, 999–1009.
72 Jin, S., Kalkum, M., Overholtzer, M., Stoffel, A., Chait, B.T & Levine, A.J (2003) CIAP1 and the serine protease HTRA2 are involved in a novel p53-dependent apoptosis pathway in mam-mals Genes Dev 17, 359–367.
73 Morley, S.J & Pain, V.M (2001) Proteasome inhibitors and immunosuppressive drugs promote the cleavage of eIF4GI and eIF4GII by caspase-8-independent mechanisms in Jurkat T cell lines FEBS Lett 503, 206–212.
74 Tee, A.R & Proud, C.G (2000) DNA-damaging agents cause inactivation of translational regulators linked to mTOR signalling Oncogene 19, 3021–3031.
75 Wojcik, C (1999) Proteasomes in apoptosis: villains or guardians? Cell Mol Life Sci 56, 908–917.
76 Chen, F., Chang, D., Goh, M., Klibanov, S.A & Ljungman, M (2000) Role of p53 in cell cycle regulation and apoptosis following exposure to proteasome inhibitors Cell Growth Differ 11, 239–246.
77 MacLaren, A.P., Chapman, R.S., Wyllie, A.H & Watson, C.J (2001) p53-dependent apoptosis induced by proteasome inhibition
in mammary epithelial cells Cell Death Differ 8, 210–218.
78 Kurland, J.F & Meyn, R.E (2001) Protease inhibitors restore radiation-induced apoptosis to Bcl-2-expressing lymphoma cells Int J Cancer 96, 327–333.
79 Meyer, S., Kohler, N.G & Joly, A (1997) Cyclosporine A is an uncompetitive inhibitor of proteasome activity and prevents NF-kappaB activation FEBS Lett 413, 354–358.
80 Marienfeld, R., Neumann, M., Chuvpilo, S., Escher, C., Kneitz, B., Avots, A., Schimpl, A & Serfling, E (1997) Cyclosporin A interferes with the inducible degradation of NF-kappa B inhibitors, but not with the processing of p105/NF-kappa B1 in T cells Eur J Immunol 27, 1601–1609.
81 Monney, L., Otter, I., Olivier, R., Ozer, H.L., Haas, A.L., Omura,
S & Borner, C (1998) Defects in the ubiquitin pathway induce caspase-independent apoptosis blocked by Bcl-2 J Biol Chem.
273, 6121–6131.
82 Belaud-Rotureau, M.-A., Lacombe, F., Durrieu, F., Vial, J.-P., Lacoste, L., Bernard, P & Belloc, F (1999) Ceramide-induced apoptosis occurs independently of caspases and is decreased by leupeptin Cell Death Differ 6, 788–795.
83 Lopez-Marure, R., Ventura, J.L., Sanchez, L., Montano, L.F & Zentella, A (2000) Ceramide mimics tumour necrosis factor-alpha
in the induction of cell cycle arrest in endothelial cells Induction of