We have demonstrated previously that TRAIL has an inhibitory effect on protein synthesis [Jeffrey IW, Bushell M, Tilleray VJ, Morley S & Clemens MJ 2002 Cancer Res 62, 2272–2280] and we
Trang 1protein synthesis by tumour necrosis factor-related
apoptosis-inducing ligand
Ian W Jeffrey, Androulla Elia, Ste´phanie Bornes*, Vivienne J Tilleray, Karthiga Gengatharan and Michael J Clemens
Translational Control Group, Centre for Molecular and Metabolic Signalling, Division of Basic Medical Sciences, St George’s, University of London, UK
Members of the tumour necrosis factor-a (TNFa)
fam-ily are well known as inhibitors of cell growth and
inducers of apoptosis in a wide variety of systems [1]
We have previously shown that both TNFa and
tumour necrosis factor-related apoptosis-inducing
ligand (TRAIL) cause rapid downregulation of global
protein synthesis in MCF-7 breast cancer cells [2] In
addition, studies with embryonic fibroblasts deficient
in the interferon (IFN)-inducible, double-stranded
RNA-dependent protein kinase (PKR) demonstrated
that expression of this protein is essential for the
TNFa-induced inhibition of translation [2] Consistent
with these observations, the a subunit of polypeptide chain eukaryotic initiation factor eIF2, which is a sub-strate for PKR, becomes more highly phosphorylated
in cells exposed to TRAIL or TNFa It is well estab-lished that the phosphorylation of eIF2a by PKR results in inhibition of polypeptide chain initiation [3] There are, however, additional events that impinge
on the translational machinery in TNFa-treated or TRAIL-treated cells In particular, we have observed increased association of the inhibitory protein eukary-otic initiation factor 4E-binding protein (4E-BP1) with the mRNA cap-binding factor eIF4E in cells
Keywords
caspases; interferon-a; polypeptide chain
initiation; protein synthesis; TRAIL
Correspondence
M J Clemens, Division of Basic Medical
Sciences, St George’s, University of
London, Cranmer Terrace, London SW17
0RE, UK
Fax: +44 20 8725 2992
Tel: +44 20 8725 5762
E-mail: M.Clemens@sgul.ac.uk
*Present address
De´partement d’Oncoge´ne´tique, Centre
Biome´dicale de Recherche et Valorisation,
Clermont-Ferrand, France.
(Received 15 March 2006, revised 19 May
2006, accepted 12 June 2006)
doi:10.1111/j.1742-4658.2006.05374.x
Tumour cells are often sensitized by interferons to the effects of tumour necrosis factor-a-related apoptosis-inducing ligand (TRAIL) We have demonstrated previously that TRAIL has an inhibitory effect on protein synthesis [Jeffrey IW, Bushell M, Tilleray VJ, Morley S & Clemens MJ (2002) Cancer Res 62, 2272–2280] and we have therefore examined the consequences of prior interferon-a treatment for the sensitivity of transla-tion to inhibitransla-tion by TRAIL Interferon treatment alone has only a minor effect on protein synthesis but it sensitizes both MCF-7 cells and HeLa cells to the downregulation of translation by TRAIL The inhibi-tion of translainhibi-tion is characterized by increased phosphorylainhibi-tion of the a subunit of eukaryotic initiation factor eIF2 and dephosphorylation of the eIF4E-binding protein 4E-BP1 Both of these effects, as well as the decrease in overall protein synthesis, require caspase-8 activity, although they precede overt apoptosis by several hours Interferon-a enhances the level and⁄ or the extent of activation of caspase-8 by TRAIL, thus provi-ding a likely explanation for the sensitization of cells to the inhibition of translation
Abbreviations
4E-BP, eukaryotic initiation factor 4E binding protein; BID, Bcl-2-interacting death protein; eIF, eukaryotic initiation factor; FADD, Fas-associated death domain; IFN, interferon; PARP, poly(ADP-ribose) polymerase; PKR, RNA-dependent protein kinase; TNFa, tumour necrosis factor-a; TRAIL, tumour necrosis factor-a-related apoptosis-inducing ligand; zIETD.FMK, zIle-Glu-Thr-Asp-fluoromethyl ketone.
Trang 2treated with TNFa [2] Competition between 4E-BP1
and eIF4G for binding to eIF4E regulates the extent
of formation of the eIF4F initiation complex and
hence the rate of 5¢-cap-dependent protein synthesis
[4–6]
Exposure to IFNs often alters the sensitivity of cells
to agents such as TRAIL, although this varies with cell
type (reviewed in [7]) In some cases, IFNs can be
proapoptotic in their own right [8–12], but more
usu-ally these cytokines are cytostatic rather than cytotoxic
when applied as single agents [13,14] However,
numer-ous reports indicate that prior treatment with IFNs
(either type I or type II) can sensitize cells to the
effects of members of the TNFa family [15–21]
(reviewed in [7,22]) In this study, we have investigated
whether IFNa also has effects on the sensitivity of cells
to TRAIL-induced downregulation of protein
synthe-sis Our data indicate that IFNa treatment sensitizes
both MCF-7 and HeLa cells to the translational
inhib-itory effect of TRAIL This inhibition of translation
precedes by several hours the appearance of overtly
apoptotic or nonviable cells
Binding of TRAIL to its active receptors,
TRAIL-R1 (DR4) and TRAIL-R2 (DR5), results in the
recruitment of procaspase-8 to the death-inducing
sig-nalling complex at the cell membrane, a process
mediated by the Fas-associated death domain
(FADD) protein [23] Procaspase-8 then undergoes
proteolytic processing that converts it from p53 and
p55 forms to p41 and p43 intermediates [24], and the
latter give rise to the large and small subunits of
act-ive caspase-8 [25,26] Caspase-8 in turn is responsible
for initiating a cascade of activation of effector
casp-ases that ultimately leads to the multiple changes in
cells characteristic of TRAIL-induced apoptosis [27]
The process of activation of caspase-8, and the
down-stream consequences that arise from it, are blocked
by the caspase-8-specific peptide inhibitor
zIle-Glu-Thr-Asp-fluoromethyl ketone (zIETD.FMK) [17] We
show here that the effects of TRAIL on overall
pro-tein synthesis and the phosphorylation of eIF2a
require the activity of caspase-8 Moreover, TRAIL
also causes extensive dephosphorylation of 4E-BP1,
and this too is a caspase-8-dependent phenomenon
Consistent with its effects on the regulation of protein
synthesis, IFNa enhances the extent of activation of
caspase-8 by TRAIL in MCF-7 and HeLa cells Our
data therefore suggest that the degree to which this
apical caspase is activated determines not only the
extent of apoptosis but also the ability of TRAIL to
regulate the initiation of translation at the level of
eIF2a phosphorylation and 4E-BP1
dephosphoryla-tion
Results
Effects of IFNa treatment on the sensitivity of cells to inhibition of protein synthesis by TRAIL
We have previously shown that protein synthesis is rapidly downregulated following exposure of cells to TRAIL and other inducers of apoptosis [2,28,29] In most cases, such inhibition precedes the loss of cell viability and is not simply a consequence of cell death However, the influence of IFNs on the regulation of translation by TRAIL has not previously been investi-gated We therefore examined the effect of increasing concentrations of TRAIL on the incorporation of [35S]methionine into total protein in cells that had or had not been pretreated with IFNa The data shown
in Fig 1A indicate that the combination of the two cytokines had a marked effect on overall protein syn-thesis in MCF-7 cells This was manifested as a sensiti-zation by IFNa pretreatment to the effect of TRAIL
In MCF-7 cells not previously exposed to IFNa,
25 ngÆmL)1 TRAIL was required to inhibit protein synthesis by 50% within 5 h, whereas when the cells had been pretreated with IFNa, only 10 ngÆmL)1 TRAIL was required to produce the same extent of inhibition at this time-point (inset to Fig 1A) This sensitization was largely due to a permissive effect of IFNa, since the latter had only a relatively small effect
on protein synthesis in the absence of TRAIL We observed a similar sensitizing effect of IFN in HeLa cells (Fig 1B), although in this case the cells were
two-to three-fold more sensitive than MCF-7 cells two-to TRAIL As shown previously [2], the downregulation
of translation by TRAIL was not a secondary conse-quence of the loss of cell viability, since, during the times examined, viability remained close to 100% as judged by trypan blue exclusion (I W Jeffrey, unpub-lished results) Moreover, very few cells became overtly apoptotic at these early times after initiation of TRAIL treatment (see below)
Role of caspase-8 in the regulation of protein synthesis by TRAIL
We have examined whether caspase-8, which is activa-ted following the binding of TRAIL to its receptors and the formation of the death-inducing signalling complex [30], is required for the inhibition of transla-tion The data in Fig 2A show that in MCF-7 cells the caspase-8-specific inhibitor zIETD.FMK largely prevented the inhibitory effect of TRAIL on protein synthesis This was the case whether or not the cells had been pretreated with IFNa (I W Jeffrey, unpublished
Trang 30 10 25 50 75 100 200 500
0
1
2
3
4
5
TRAIL (ng/ml)
3- )
A
0 25 50 75 100
TRAIL (ng/ml) (log scale)
0
1
2
3
4
TRAIL (ng/ml )
0.1 1 10 100 0
25 50 75 100
TRAIL (ng/ml) (log scale)
3-B
Fig 1 Effects of IFNa on the sensitivity of MCF-7 and HeLa cells
to inhibition of protein synthesis by TRAIL MCF-7 cells (A) and
HeLa cells (B) were cultured for 72 and 24 h, respectively, in the
absence (light-shaded bars) or presence (dark-shaded bars) of
human IFNa 2b (1000 UÆmL)1) and then further treated with the
indi-cated concentrations of TRAIL for the last 5 h (A) or 3 h (B) Protein
synthesis was measured by the incorporation of [ 35 S]methionine
into acid-insoluble material for the last 40 min The data are the
means ± SEM of three determinations Insets: percentage
inhibi-tion of protein synthesis as a funcinhibi-tion of TRAIL concentrainhibi-tion in
cells without IFN (squares) or with prior IFN treatment (triangles).
The arrows indicate the concentrations of TRAIL producing 50%
inhibition of protein synthesis.
0 25 50 75 100
A
TRAIL Control TRAIL + z-IETD.FMK
p18
full length (p53/55) p41/43
B
TRAIL Control z-IETD.FMK
full length t-BID
TRAIL + z-IETD.FMK
C
BID
(Pro)caspase-8
αα-tubulin
Fig 2 Effects of zIETD.FMK on TRAIL-induced inhibition of protein synthesis and caspase-8 activity in MCF-7 cells (A) MCF-7 cells were incubated with or without TRAIL (167 ngÆmL)1) for 5 h as indicated Where shown, zIETD.FMK was present at 10 l M Protein synthesis was then measured as described in Fig 1 The data are expressed as percentage of the value obtained with untreated con-trol cells and are the means ± SEM of three determinations (B) Total cytoplasmic extracts were prepared and subjected to SDS gel electrophoresis, and this was followed by immunoblotting for procaspase-8 and processed forms of the enzyme The positions of the full-length (p53 ⁄ p55) forms of the protein and the p41 ⁄ p43 and p18 cleavage products are indicated The samples were also immu-noblotted for a-tubulin as a loading control (C) A similar experiment was performed as in (B) and extracts were immunoblotted for BID The positions of the full-length protein and the cleavage product t-BID are indicated.
Trang 4results) Although peptide inhibitors containing the
IETD sequence preferentially inhibit caspase-8 [31], it
was possible that zIETD.FMK might directly affect
other caspases as well However, a specific requirement
for caspase-8 for the effect on protein synthesis is
indi-cated by the fact that caspase-8-deficient Jurkat cells
[32] are also largely resistant to the inhibition of
methionine incorporation by TRAIL, in contrast to
wild-type Jurkat cells (Table 1) In MCF-7 cells,
zIETD.FMK impaired the TRAIL-induced cleavage of
the p53 and p55 forms of procaspase-8 to the p41 and
p43 intermediates and the p18 subunit by only about
50% (Fig 2B) However, the effect of zIETD.FMK
was sufficient to restore protein synthesis to about
80% of the control rate Moreover, the
caspase-8-mediated cleavage of the Bcl-2 family member BID
[33,34] was completely inhibited by zIETD.FMK
under the same conditions (Fig 2C)
TRAIL treatment strongly enhanced the
phosphory-lation of the a subunit of polypeptide chain initiation
factor eIF2 in MCF-7 cells, in the presence or absence
of prior IFN treatment (Fig 3A,B) Neither TRAIL
nor IFNa had any effect on the level of total eIF2a
TRAIL treatment also decreased the extent of
phos-phorylation of 4E-BP1, as revealed by a shift in the
migration of the latter protein on SDS gels from the
b and c forms to the hypophosphorylated a form
(Fig 3C,D) and by the loss of immunoreactivity with
a phosphospecific antibody directed at residue Ser65
(Fig 3D, right panel) In view of the effect of
zIE-TD.FMK on the inhibition of protein synthesis by
TRAIL (Fig 2A), the requirement for caspase-8 for
these events was determined Both the increase in
phosphorylation of eIF2a and the decrease in
phos-phorylation of 4E-BP1 caused by TRAIL were
com-pletely blocked by treatment of MCF-7 cells with
zIETD.FMK (Fig 3B,C) Similar results were
obtained with HeLa cells The caspase-8 inhibitor had
no effect on the total levels of these factors (A Elia, unpublished results) Moreover, treatment of caspase-8-deficient Jurkat cells with TRAIL failed to cause any dephosphorylation of 4E-BP1 (Fig 3D) or any change
in the phosphorylation of eIF2a (A Elia, unpublished results), in contrast to the effects of TRAIL on wild-type Jurkat cells
Since caspase-8 activity is required for the regulation
of translation by TRAIL, it was also of interest to determine whether IFN affected the level or extent of activation of caspase-8 in MCF-7 and HeLa cells
Table 1 Requirement for caspase-8 for inhibition of protein
synthe-sis by TRAIL Wild-type and caspase-8-deficient Jurkat cells
were incubated for 3 h in the absence or presence of TRAIL
(400 ngÆmL)1) Protein synthesis was measured by the
incorpor-ation of [35S]methionine into acid-insoluble material for the last
60 min The data are the means ± SEM of four to six
determina-tions.
Cell line
[ 35 S]methionine incorporation (counts per min per 10 5 cells) (· 10)3)
Inhibition by TRAIL (%)
Wild type 4.44 ± 0.08 1.11 ± 0.05 75.0
Caspase-8 deficient 3.89 ± 0.11 3.43 ± 0.11 11.8
eIF2 αα((P)
A TRAIL - + - +
eIF2 αα(P)
TRAIL - + - + Z.IETD.FMK - - + +
B
α
βγ
C
Total 4E-BP1
TRAIL - + + Z.IETD.FMK - - +
TRAIL - + - + TRAIL - + - + D
Total 4E-BP1
4E-BP1
Wild-type C8-deficient Wild-type C8-deficient
Fig 3 Caspase-8 requirement for TRAIL-induced changes in the state of phosphorylation of eIF2a and 4E-BP1 (A) MCF-7 cells were grown for 72 h in the absence or presence of human IFNa 2b (1000 UÆmL)1) and further treated with or without TRAIL (167 ngÆmL)1) for the last 5 h as indicated Total cytoplasmic extracts were prepared and analysed by SDS gel electrophoresis followed by immunoblotting for phosphorylated eIF2a (Ser51) and total eIF2a as indicated (B,C) MCF-7 cells were incubated for 5 h
in the absence or presence of TRAIL (167 ngÆmL)1) and zIETD.FMK (10 l M ) as indicated Extracts were prepared as in (A) and analysed
by immunoblotting for (B) phosphorylated eIF2a (Ser51) and (C) 4E-BP1 The hypophosphorylated (a) and the b and c forms of 4E-BP1 are indicated in (C) (D) Wild-type and caspase-8-deficient Jurkat cells were incubated for 3 h in the absence (-)or presence (+) of TRAIL (150 ngÆmL)1) Extracts were prepared and analysed by immunoblotting for total 4E-BP1 (left panel) and phosphorylated 4E-BP1 (Ser65) (right panel).
Trang 5Examination of the levels of procaspase-8 in MCF-7
cells by immunoblotting and quantitative densitometry
(Fig 4A,C) showed that IFNa treatment resulted in a c
40% increase in the immunoreactive signals, but without
any activation of the enzyme (as indicated by the lack of
processing to the p41⁄ p43 or p18 products) TRAIL
treatment led to processing of the basal and elevated
amounts of procaspase-8 in both control and
IFN-trea-ted cells and there was an approximately two-fold
increase in the amount of the p18 large subunit of active
caspase-8 in cells treated with IFN and TRAIL,
com-pared to the amount in cells treated with TRAIL alone
(Fig 4A,C) Enhancement of the level of p18 was also
observed after IFN and TRAIL treatment of HeLa cells,
although densitometry of the immunoblots showed that
in this case there was no measurable increase in the level
of the proenzyme in cells treated with IFNa in the
absence of TRAIL (Fig 4B,C) In contrast to these
effects on caspase-8, there were no IFN-induced or
TRAIL-induced changes in the levels of other proteins
involved in TRAIL signalling (i.e FADD and the
TRAIL receptors DR4 and DR5), or in levels of the
caspase-8 antagonist cellular FLICE-like inhibitory
protein (I W Jeffrey, unpublished results)
To investigate whether IFNa could enhance the activity of caspase-8 in cells subsequently treated with TRAIL, we examined the extent of cleavage of the caspase-8 substrate Bcl-2-interacting death protein (BID) to form truncated BID (t-BID) [33,34] We also monitored the cleavage of the 116 kDa caspase sub-strate poly(ADP-ribose) polymerase (PARP) to pro-duce its characteristic 89-kDa cleavage product The results in Fig 5 show that TRAIL alone induced partial cleavage of BID and PARP within 5 h IFNa alone had no effect on BID or PARP cleavage, but enhanced the effect of TRAIL such that very little of the full-length form of either protein remained in the IFN-treated cells after 5 h of exposure to TRAIL Thus, the activity of caspase-8, and most likely that of downstream effector caspases also, is enhanced in cells treated with the combination of IFNa and TRAIL, relative to TRAIL alone
In view of the cleavage of caspase substrates such as BID and PARP, the effect of IFNa and TRAIL on the DNA content of MCF-7 cells was also assessed, using fluorescence-activated cell sorting Figure 6 shows that, in spite of the activation of caspase-8 and the cleavage of BID and PARP, TRAIL alone had
full length (p53/55) p41/43
p18
A Control TRAIL IFNααα IFNαα
+ TRAIL
Caspase-8
B
full length (p53/55) p41/43
p18
Control TRAIL IFNα IFNα
+TRAIL
αα-tubulin
0 25 50 75
100
p53/p55 0
25 50 75
100
MCF-7 cells
HeLa cells
Control + TRAIL + IFNαα
(141%)
(88%) (103%)
(90%)
(143%) C
Fig 4 Effects of IFNa and TRAIL on levels and activation of caspase-8 in MCF-7 and HeLa cells MCF-7 cells (A) and HeLa cells (B) were incubated for 72 h and 24 h, respectively, in the absence or presence of IFNa (1000 unitsÆmL)1), and then treated with or without TRAIL as indicated (MCF-7 cells, 5 h at 167 ngÆmL)1; HeLa cells, 3 h at 10 ngÆmL)1) Total cytoplasmic extracts were prepared and analysed by SDS gel electrophoresis followed by immunoblotting for caspase-8 and a-tubulin In (A) the samples were analysed in duplicate The positions of the full-length (p53 ⁄ p55) forms of caspase-8 and the p41 ⁄ p43 and p18 cleavage products are indicated (C) The intensities of the caspase-8 bands were determined by quantitative densitometry The values in brackets above the histograms show the relative intensities of the appropriate bands in the IFN-treated cells, as a percentage of the values seen in the absence of IFNa treatment.
Trang 6very little effect on the appearance of cells with a
sub-G1 DNA content Approximately 1 and 6% of the
total cell population showed a decreased DNA content
after 4 and 16 h, respectively In cells pretreated with
IFNa, the corresponding figures were approximately 2
and 16% at 4 and 16 h after exposure to TRAIL,
respectively A recent report has analysed the basis for
the relative insensitivity of MCF-7 cells to these and
other apoptotic effects of TRAIL, and it has been
sug-gested that this is due to the absence of caspase-3 [35]
However, in MCF-7 cells, the cleavage of DNA during
apoptosis may also be effected through the activity of
caspase-6 and⁄ or caspase-7 [36,37] Our data therefore
indicate that, although the substantial inhibition of
protein synthesis caused by TRAIL within 4–5 h
requires caspase-8 activity, it precedes the loss of
cellu-lar DNA and is not a consequence of overt apoptosis
However, the sensitizing effect of IFNa for
caspase-mediated substrate cleavages in cells exposed to
TRAIL is reflected in the increased number of cells
with a sub-G1 content of DNA appearing at later
times, confirming the reports that IFNa can sensitize
cells to TRAIL-induced apoptosis [15–21]
Discussion
Effects of TRAIL on protein synthesis
Previous studies have shown that a rapid decrease in
the rate of overall protein synthesis occurs in cells
exposed to various proapoptotic stimuli, including
treatment with members of the TNFa family [2,28]
Using MCF-7 and HeLa cells, we have now shown
that the TRAIL-induced inhibition of translation is a
caspase-8-dependent event that is modified by IFNa
treatment The effect of IFN is to sensitize MCF-7
and HeLa cells to the effects of TRAIL, and the enhanced downregulation of translation seen in the presence of IFN correlates with increased caspase activity Although the inhibition of protein synthesis requires caspase activity, it precedes the appearance of
an overtly apoptotic phenotype and the loss of cell viability (Fig 6) In contrast to the effects of TRAIL, IFN treatment alone has relatively little effect on translation; it also does not significantly activate caspase-8 (Fig 4) or result in any cleavage of BID or PARP (Fig 5)
In TRAIL-treated cells, both the increased phos-phorylation of eIF2a and the modulation of 4E-BP1 activity are blocked by the broad-specificity caspase inhibitor zVal-Ala-Asp-fluoromethyl ketone [2] We have now extended those findings to demonstrate a specific requirement for caspase-8 activity for these changes The caspase-8 inhibitor zIETD.FMK was able to prevent completely both the phosphorylation
of eIF2a and the dephosphorylation of 4E-BP1 in cells exposed to TRAIL (Fig 3B,C) Moreover, in Jurkat cells, deficiency for caspase-8 [32] rendered the cells resistant to the effects of TRAIL on initiation factor phosphorylation (Fig 3D) and overall protein synthe-sis (Table 1) Caspase-8 is intimately involved in the function of the TRAIL-activated death-inducing sig-nalling complex [27,30], and so it is not surprising that its activity is required However, it is of interest that caspase-8 plays a specific role in the regulation of translation, particularly as the inhibition of polypep-tide chain initiation by TRAIL precedes apoptosis by several hours The requirement for caspase-8 activity
in MCF-7 cells, as revealed by the inhibitor studies, is confirmed by the inability of caspase-8-deficient cells
to show extensive inhibition of translation in response
to TRAIL treatment (Table 1)
The IFN-induced sensitization of MCF-7 and HeLa cells to TRAIL is consistent with the enhancement by IFN of the level of TRAIL-induced active caspase-8 (Fig 4) Our data suggest that, at least in MCF-7 cells, IFN pretreatment induces cells to express a higher level of procaspase-8 We have not determined the molecular basis for this, but others have shown that the promoter for procaspase-8 contains an IFN-stimu-lated response element and responds to both IFNa and IFNc with transcriptional upregulation [38–40] In Huh7 hepatoma cells, IFNa treatment results in enhancement of the expression of procaspase-8 at both the RNA and protein levels, and this sensitizes the cells to the proapoptotic effects of TRAIL Interest-ingly, our data suggest that only relatively small changes in caspase-8 activity appear to be sufficient to alter substantially the cellular sensitivity to TRAIL
Control TRAIL IFNα IFNα
+ TRAIL
α−
α−tubulin
BID
full length protein
cleavage product (t-BID) full length protein
Fig 5 TRAIL-induced caspase activity is enhanced by IFNa
pre-treatment MCF-7 cells were grown for 72 h in the absence or
presence of human IFNa 2b (1000 UÆmL)1) and further incubated
with or without TRAIL (167 ngÆmL)1) for the last 5 h as indicated.
Total cytoplasmic extracts were prepared and subjected to SDS gel
electrophoresis, followed by immunoblotting for BID, PARP and
a-tubulin The positions of the full-length proteins and their caspase
cleavage products are indicated.
Trang 7zIETD.FMK caused only a partial reduction in
TRAIL-induced cleavage of procaspase-8 (Fig 2B),
and IFN treatment caused at best only a two-fold
increase in the level of the catalytically active form of
caspase-8 in cells subsequently exposed to TRAIL
(Fig 4) Nevertheless, zIETD.FMK was able to
decrease the inhibition of protein synthesis by TRAIL
by 80% (Fig 2A) and, conversely, IFNa enhanced the
sensitivity of protein synthesis to TRAIL in MCF-7
cells and HeLa cells by 2.5-fold and 10-fold,
respect-ively (Fig 1) These results are consistent with the
con-cept that the activity of caspase-8 is rate-limiting for
the biological effects of TRAIL [40] and that relatively
small changes in caspase-8 activity can be amplified by
downstream events, including the activation of effector caspases
Mechanisms of inhibition of protein synthesis
We have shown that TRAIL treatment causes both phosphorylation of eIF2a and dephosphorylation of 4E-BP1 The latter change results in increased associ-ation of 4E-BP1 with eIF4E (S Bornes, unpublished results) The question therefore arises as to which mechanism is responsible for the inhibition of overall protein synthesis Since Kim et al [41] have previously reported that MCF-7 cells are relatively insensitive to the effects of eIF2a phosphorylation, it is likely that
Control
DNA content
Sub-G1 0.3%
DNA content
Sub-G1 0.4%
DNA content
+TRAIL (4h)
Sub-G1 0.8%
DNA content
+TRAIL (4h)
Sub-G1 1.6%
DNA content
+TRAIL (16h)
Sub-G1 6.1%
DNA content
+TRAIL (16h)
Sub-G1 16.0%
Fig 6 Effects of IFNa and TRAIL on cellular DNA content MCF-7 cells were incubated for 24 h in the absence or presence of human IFNa2b (1000 UÆmL)1) and then further treated with or without TRAIL (100 ngÆmL)1) for the last 4 h or 16 h as indicated The cells were fixed, stained with propidium iodide and analysed for DNA content by fluorescence-activated cell sorting The percentage of cells with a sub-G1 DNA content is indicated in each panel.
Trang 8the regulation of 4E-BP1 activity is the more
import-ant change for the downregulation of translation
following TRAIL treatment Nevertheless, TRAIL
treatment enhances the level of the transcription factor
ATF4 (I W Jeffrey, unpublished results), the
expres-sion of which is known to be upregulated at the
trans-lational level in response to increased phosphorylation
of eIF2a [42] This suggests that increased eIF2a
phos-phorylation does have a role to play in the cellular
response to TRAIL TRAIL may be able to activate
the IFN-inducible protein kinase PKR, which targets
eIF2a as a substrate and is required for inhibition of
protein synthesis by TNFa [2] However, it is possible
that other eIF2a kinases are also stimulated by
TRAIL
Relationship of translational inhibition to
apoptosis
A striking synergistic effect on the induction of
apoptosis is often observed when cells are treated
with members of the IFN and TNF families together
(reviewed in [7,22]), and the enhanced inhibition of
protein synthesis by TRAIL observed in IFN-treated
MCF-7 and HeLa cells is clearly related to this
However, this inhibition is an early effect of TRAIL
treatment and, at least in MCF-7 cells, precedes
apoptosis by several hours Compared to HeLa cells,
MCF-7 cells are in fact relatively insensitive to the
apoptosis-inducing effect of TRAIL This may be
because they lack caspase-3 activity [35]
Interest-ingly, although caspase-3 is clearly not essential for
the inhibition of protein synthesis, MCF-7 cells are
also much less sensitive than HeLa cells to this effect
of TRAIL (Fig 1)
As indicated above, our data are consistent with a
role for caspase-8 regulation in mediating the effect of
IFNa on the sensitivity of protein synthesis to
inhibi-tion by TRAIL In other systems, increased apoptosis
seen in response to IFNa plus TRAIL is characterized
by elevated caspase-8 and caspase-9 activity, with
enhanced degradation of BID and translocation of
Bax to mitochondria [15], and we have also observed
similar phenomena As well as the induction of
ca-spase-8 by IFNs [40,43–45], there are several other
potential mechanisms that could also operate to bring
about such synergism, including IFN-induced
enhance-ment of the expression of TRAIL receptors [15] IFN
treatment might also inhibit the activity of
antiapop-totic mechanisms that counteract the death-inducing
effects of TNF family members [19] However, we have
not observed any consistent IFN-induced changes in
the levels of TRAIL receptor proteins or the large or
small forms of the caspase-8 antagonist protein c-FLIP (I W Jeffrey, unpublished results)
Exactly how the levels of phosphorylation of eIF2a
or 4E-BP1 are regulated by caspase activity remains to
be determined In the case of eIF2a, there is a preced-ent for caspase-induced cleavage and activation of PKR [46] This enzyme is present in both MCF-7 and HeLa cells, and its level is enhanced by IFN treatment (I W Jeffrey, unpublished results) As a basis for the dephosphorylation of 4E-BP1, there may be caspase-mediated inhibition of one or more protein kinases and⁄ or activation of protein phosphatase(s) such as PP2A that act on 4E-BP1 [47] A substantial body of evidence suggests involvement of protein phosphatases
in mediating the effects of apoptotic stimuli [48–50], but further work will be needed to determine whether regulation of these enzymes by TRAIL (via caspase-8)
is responsible for the changes in 4E-BP1 phosphoryla-tion identified here
Experimental procedures Materials
Materials for tissue culture were obtained from Sigma (Poole, UK) Monoclonal antibody against PARP (C2-10) was obtained from Trevigen (Gaithersburg, MD, USA) Antibodies against 4E-BP1 and a-tubulin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Sigma, respectively Monoclonal antibodies to eIF2a and phosphorylated eIF2a (Ser51) were as previously des-cribed [2,51] Antibodies to phosphorylated 4E-BP1 (Ser65), caspase-8 and BID were obtained from Cell Signalling Technology (Beverley, MA, USA) PVDF paper (Hybond P) was obtained from GE Healthcare (Chalfont St Giles, UK) TRAIL was obtained from PeproTech EC (London, UK) and IFNa2b (Intron A) was obtained from
inhibitor zIETD.FMK was obtained from Calbiochem (Nottingham, UK) All other chemicals were from Sigma
Cell culture and cytokine treatments
The human breast cancer cell line MCF-7 was kindly provi-ded by R Ja¨nicke (University of Dusseldorf, Germany) These cells, as well as HeLa cells, were cultured under the conditions previously described [2] Both cell lines were treated with IFNa2b (1000 International reference unitsÆmL)1) for the times shown in the legends to Figs 1,3,4,5 and 6 No significant differences in the effects of IFN were noted between 24 h and 72 h of treatment Wild-type and caspase-8-deficient Jurkat cells were grown as previously described [28] For all cell lines TRAIL was added at the concentrations stated for the last 4–5 h of the incubations
Trang 9Cell growth and viability measurements
The cells were harvested by trypsinization, resuspended and
counted in a haemocytometer Cell viability was determined
by trypan blue exclusion Cells were fixed with ethanol,
stained with propidium iodide, treated with ribonuclease A
and analysed for DNA content and the appearance of a
sub-G1 fraction by fluorescence-activated cell sorting, as
described previously [29]
Determination of protein synthesis rates
Overall rates of protein synthesis in intact cells were
meas-ured by the incorporation of [35S]methionine (10 lCiÆmL)1)
into trichloroacetic acid-insoluble material for 40 min
Radioactivity was determined as previously described [2]
Protein content was determined and rates of protein
synthe-sis are expressed as counts per min incorporated per lg
protein
Immunoblotting of cell extracts
Cells were harvested, washed in NaCl⁄ Piand lysed as
des-cribed previously [2] Samples containing equal amounts of
protein were subjected to electrophoresis on SDS
polyacryl-amide gels and the proteins transferred to PVDF
mem-branes using a semidry blotting apparatus (Bio-Rad, Hemel
Hempstead, UK) Blots were blocked, incubated with the
appropriate primary antibodies and developed using
horse-radish peroxidase-linked secondary antibodies Enhanced
chemiluminescence was performed using Lumiglo reagent
(Cell Signaling Technology) according to the
manufac-turer’s instructions Quantitative densitometry of
appropri-ate bands was performed using Scion image software
(Scion Corporation, Frederick, MD)
Acknowledgements
We are grateful to Bill Newman for assistance with the
fluorescence-activated cell sorter analysis This work
was supported by grants from the Association for
International Cancer Research, the Leukaemia
Research Fund and the Cancer Prevention Research
Trust SB was funded by a fellowship from the
Fonda-tion pour la Recherche Me´dicale
References
1 Schulze-Osthoff K, Ferrari D, Los M, Wesselborg S &
Peter ME (1998) Apoptosis signaling by death receptors
Eur J Biochem 254, 439–459
2 Jeffrey IW, Bushell M, Tilleray VJ, Morley S &
Clemens MJ (2002) Inhibition of protein synthesis
in apoptosis: differential requirements by the tumor
necrosis factor a family and a DNA-damaging agent for caspases and the double-stranded RNA-dependent protein kinase Cancer Res 62, 2272–2280
3 Clemens MJ, Bushell M, Jeffrey IW, Pain VM & Morley SJ (2000) Translation initiation factor modifica-tions and the regulation of protein synthesis in apopto-tic cells Cell Death Differ 7, 603–615
4 Gingras AC, Raught B & Sonenberg N (1999) eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation Annu Rev Biochem 68, 913–963
5 Clemens MJ (2004) Targets and mechanisms for the regulation of translation in malignant transformation Oncogene 23, 3180–3188
6 Avdulov S, Li S, Michalek V, Burrichter D, Peterson
M, Perlman DM, Manivel JC, Sonenberg N, Yee D, Bitterman PB et al (2004) Activation of translation complex eIF4F is essential for the genesis and mainten-ance of the malignant phenotype in human mammary epithelial cells Cancer Cell 5, 553–563
7 Clemens MJ (2003) Interferons and apoptosis J Inter-feron Cytokine Res 23, 277–292
8 Nanbo A, Inoue K, Adachi-Takasawa K & Takada K (2002) Epstein–Barr virus RNA confers resistance to interferon-a-induced apoptosis in Burkitt’s lymphoma EMBO J 21, 954–965
9 Chawla-Sarkar M, Lindner DJ, Liu YF, Williams BR, Sen GC, Silverman RH & Borden EC (2003) Apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis Apoptosis 8, 237–249
10 Abadie A & Wietzerbin J (2003) Involvement of TNF-related apoptosis-inducing ligand (TRAIL) induction in interferon gamma-mediated apoptosis in Ewing tumor cells Ann NY Acad Sci 1010, 117–120
11 Takada E, Shimo K, Hata K, Abiake M, Mukai Y, Moriyama M, Heasley L & Mizuguchi J (2005) Inter-feron-beta-induced activation of c-Jun NH2-terminal kinase mediates apoptosis through up-regulation of CD95 in CH31 B lymphoma cells Exp Cell Res 304, 518–530
12 Panaretakis T, Pokrovskaja K, Shoshan MC & Grande´r
D (2003) Interferon-a-induced apoptosis in U266 cells is associated with activation of the proapoptotic Bcl-2 family members Bak and Bax Oncogene 22, 4543–4556
13 Tiefenbrun N, Melamed D, Levy N, Resnitzky D, Hoffmann I, Reed SI & Kimchi A (1996) Alpha interferon suppresses the cyclin D3 and cdc25A genes, leading to a reversible G0-like arrest Mol Cell Biol
16, 3934–3944
14 Thomas NS, Pizzey AR, Tiwari S, Williams CD & Yang J (1998) p130, 107, and pRb are differentially regulated in proliferating cells and during cell cycle arrest
by alpha-interferon J Biol Chem 273, 23659–23667
Trang 1015 Shigeno M, Nako K, Ichikawa T, Suzuki K, Kawakami
A, Abiru S, Miyazoe S, Nakagawa Y, Ishikawa H,
Hamasaki K et al (2003) Interferon-a sensitizes human
hepatoma cells to TRAIL-induced apoptosis through
DR5 upregulation and NF-kappaB inactivation
Oncogene 22, 1653–1662
16 Langaas V, Shahzidi S, Johnsen JI, Smedsrod B &
Sveinbjornsson B (2001) Interferon-gamma modulates
TRAIL-mediated apoptosis in human colon carcinoma
cells Anticancer Res 21, 3733–3738
17 Fulda S & Debatin KM (2002) IFNgamma sensitizes
for apoptosis by upregulating caspase-8 expression
through the Stat1 pathway Oncogene 21, 2295–2308
18 Suk K, Kim YH, Chang I, Kim JY, Choi YH, Lee KY
& Lee MS (2001) IFNa sensitizes ME-180 human
cervi-cal cancer cells to TNFa-induced apoptosis by
inhibit-ing cytoprotective NF-kappa-B activation FEBS Lett
495, 66–70
19 Leaman DW, Chawla-Sarkar M, Vyas K, Reheman M,
Tamai K, Toji S & Borden EC (2002) Identification of
X-linked inhibitor of apoptosis-associated factor-1 as an
interferon-stimulated gene that augments TRAIL
Apo2L-induced apoptosis J Biol Chem 277, 28504–
28511
20 Chawla-Sarkar M, Leaman DW, Jacobs BS & Borden
EC (2002) IFN-b pretreatment sensitizes human
melan-oma cells to TRAIL⁄ Apo2 ligand-induced apoptosis
J Immunol 169, 847–855
21 Ruiz-Ruiz C & Lo´pez-Rivas A (2002)
Mitochondria-dependent and -inMitochondria-dependent mechanisms in tumour
necrosis factor-related apoptosis-inducing ligand
(TRAIL)-induced apoptosis are both regulated by
interferon-gamma in human breast tumour cells
Biochem J 365, 825–832
22 Ruiz d A, Lopez-Rivas A & Ruiz-Ruiz C (2004)
Inter-feron-gamma and TRAIL in human breast tumor cells
Vitam Horm 67, 291–318
23 Kuang AA, Diehl GE, Zhang JK & Winoto A (2000)
FADD is required for DR4-and DR5-mediated
apopto-sis) lack of trail-induced apoptosis in FADD-deficient
mouse embryonic fibroblasts J Biol Chem 275, 25065–
25068
24 Scaffidi C, Medema JP, Krammer PH & Peter ME
(1997) FLICE is predominantly expressed as two
func-tionally active isoforms, caspase-8⁄ a and caspase-8 ⁄ b
J Biol Chem 272, 26953–26958
25 Sohn D, Schulze-Osthoff K & Janicke RU (2005)
Cas-pase-8 can be activated by interchain proteolysis
with-out receptor-triggered dimerization during drug-induced
apoptosis J Biol Chem 280, 5267–5273
26 Shin S, Lee Y, Kim W, Ko H, Choi H & Kim K (2005)
Caspase-2 primes cancer cells for TRAIL-mediated
apoptosis by processing procaspase-8 EMBO J 24,
3532–3542
27 Ho PK & Hawkins CJ (2005) Mammalian initiator apoptotic caspases FEBS J 272, 5436–5453
28 Morley SJ, Jeffrey I, Bushell M, Pain VM & Clemens
MJ (2000) Differential requirements for caspase-8 activ-ity 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
29 Constantinou C, Bushell M, Jeffrey IW, Tilleray V, West M, Frost V, Hensold J & Clemens MJ (2003) p53-induced inhibition of protein synthesis is independent of apoptosis Eur J Biochem 270, 3122–3132
30 Bratton SB, MacFarlane M, Cain K & Cohen GM (2000) Protein complexes activate distinct caspase cas-cades in death receptor and stress-induced apoptosis Exp Cell Res 256, 27–33
31 Garcia-Calvo M, Peterson EP, Leiting B, Ruel R, Nicholson DW & Thornberry NA (1998) Inhibition of human caspases by peptide-based and macromolecular inhibitors J Biol Chem 273, 32608–32613
32 Juo P, Kuo CJ, Yuan JY & Blenis J (1998) Essential requirement for caspase-8⁄ FLICE in the initiation of the Fas-induced apoptotic cascade Curr Biol 8, 1001– 1008
33 Werner AB, De Vries E, Tait SWG, Bontjer J & Borst J (2002) TRAIL receptor and CD95 signal to mitochon-dria via FADD, caspase-8⁄ 10, Bid, and Bax but differ-entially regulate events downstream from truncated Bid
J Biol Chem 277, 40760–40767
34 Yamada H, Tada-Oikawa S, Uchida A & Kawanishi S (1999) TRAIL causes cleavage of bid by caspase-8 and loss of mitochondrial membrane potential resulting in apoptosis in BJAB cells Biochem Biophys Res Commun
265, 130–133
35 Engels IH, Totzke G, Fischer U, Schulze-Osthoff K & Janicke RU (2005) Caspase-10 sensitizes breast carci-noma cells to TRAIL-induced but not tumor necrosis factor-induced apoptosis in a caspase-3-dependent manner Mol Cell Biol 25, 2808–2818
36 Semenov DV, Aronov PA, Kuligina EV, Potapenko
MO & Richter VA (2004) Oligonucleosome DNA frag-mentation of caspase 3 deficient MCF-7 cells in palmi-tate-induced apoptosis Nucleosides Nucleotides Nucleic Acids 23, 831–836
37 Mooney LM, Al Sakkaf KA, Brown BL & Dobson
PR (2002) Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells Br J Cancer 87, 909–917
38 Ruiz-Ruiz C, De Almodo´var CR, Rodrı´guez A, Ortiz-Ferro´n G, Redondo JM & Lo´pez-Rivas A (2004) The up-regulation of human caspase-8 by interferon-gamma
in breast tumor cells requires the induction and action
of the transcription factor interferon regulatory factor-1
J Biol Chem 279, 19712–19720