Tài liệu Báo cáo khoa học: Interferon-a induces sensitization of cells to inhibition of protein synthesis by tumour necrosis factor-related apoptosis-inducing ligand ppt

Interferon-a induces sensitization of cells to inhibition of
protein 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:
*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 inhibition 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 translation is characterized by increased phosphorylation 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.
3698 FEBS Journal 273 (2006) 3698–3708 ª 2006 The Authors Journal compilation ª 2006 FEBS
treated 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
[
35
S]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 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
I. W. Jeffrey et al. Control of protein synthesis by IFNa and TRAIL
FEBS Journal 273 (2006) 3698–3708 ª 2006 The Authors Journal compilation ª 2006 FEBS 3699
0 10 25 50 75 100 200 500
0
1
2
3
4
5
TRAIL (ng/ml)
[
53
noitaroprocni teM]S
/nim rep stnuoc( μ
μ
01 x nietorp g
3
-
)
A
10 100
0
25

50
75
100
fo noiti
b
i
hnI
%
sisehtnys nietorp
TRAIL (ng/ml) (log scale)
0 0.1 0.5 1.0 5.0 10 25
0
1
2
3
4
TRAIL (n
g
/ml)
0.1 1 10 100
0
25
50
75
100
TRAIL (ng/ml) (log scale)
f
o
n
oitib

i
hnI%
s
i
s
e
ht
n
ys
nie
to
r
p
[
5
3
noitar
oproc
ni
teM]S
/nim
rep stnuoc( μ
μ
01 x )nietorp g
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 function of TRAIL concentration 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
TRAIL - + +
Z-IETD.FMK - - +
sisehtnyS nietorP
)lortnoc fo %(
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
α
α
-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.
Control of protein synthesis by IFNa and TRAIL I. W. Jeffrey et al.
3700 FEBS Journal 273 (2006) 3698–3708 ª 2006 The Authors Journal compilation ª 2006 FEBS
results). 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 [
35
S]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 (%)
–TRAIL +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
Total eIF2α
α
eIF2α
α
(
(
P)
A

TRAIL - + - +
IFNα
α
- - + +
eIF2α
α
(P)
TRAIL - + - +
Z.IETD.FMK - - + +
B
α
β
γ
C
Total
4E-BP1
TRAIL - + +
Z.IETD.FMK - - +
TRAIL - + - + TRAIL - + - +
D
Total
4E-BP1
4E-BP1
(P)Ser
65
Wild-type C8-deficient Wild-type C8-deficient
Jurkat Jurkat Jurkat Jurkat
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).
I. W. Jeffrey et al. Control of protein synthesis by IFNa and TRAIL
FEBS Journal 273 (2006) 3698–3708 ª 2006 The Authors Journal compilation ª 2006 FEBS 3701
Examination 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 IFNαα
α
IFNα
α
+ TRAIL
Caspase-8
B
full length
(p53/55)
p41/43
p18

Control TRAIL IFNα IFNα
+TRAIL
α
α
-tubulin
0
25
50
75
100
ytis
n
et
n
i d
n
aB
)s
ti
n
u
yr
art
i
br
a
(
p53/p55
0
25

50
75
100
yt
i
s
ne
t
ni

dnaB
)st
i
n
u
yr
a
rtibra
(
p41/p43 p18
p53/p55 p41/p43 p18
MCF-7 cells
HeLa cells
Control
+ TRAIL
+ IFNα
α
+ IFNα
α
+ TRAIL

(141%)
(137%)
(227%)
(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.
Control of protein synthesis by IFNa and TRAIL I. W. Jeffrey et al.
3702 FEBS Journal 273 (2006) 3698–3708 ª 2006 The Authors Journal compilation ª 2006 FEBS
very 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.
PARP
cleavage product
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.
I. W. Jeffrey et al. Control of protein synthesis by IFNa and TRAIL
FEBS Journal 273 (2006) 3698–3708 ª 2006 The Authors Journal compilation ª 2006 FEBS 3703
zIETD.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
0 200 400 0 200 400 600
0 200 400 600
0 200 400 6000 200 400 600
0 200 400 600
0
180 360 540
720
0 180 360 540 7200 180 360 540 720
0 180 360 540 7200 180 360 540 720
0 180 360 540 720
Sub-G1
0.3%
DNA content
tnuoclleC
+ IFN

α
Sub-G1
0.4%
DNA content
+TRAIL (4h)
Sub-G1
0.8%
DNA content
tnuoclleC
+ IFNα
+TRAIL (4h)
Sub-G1
1.6%
DNA content
Cell count Cell count Cell count
+TRAIL (16h)
Sub-G1
6.1%
DNA content
tnuoclleC
+ IFNα
+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
IFNa
2b
(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.
Control of protein synthesis by IFNa and TRAIL I. W. Jeffrey et al.
3704 FEBS Journal 273 (2006) 3698–3708 ª 2006 The Authors Journal compilation ª 2006 FEBS
the 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 IFNa
2b
(Intron A) was obtained from Schering-
Plough (Welwyn Garden City, UK). The caspase-8
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 IFNa
2b
(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.
I. W. Jeffrey et al. Control of protein synthesis by IFNa and TRAIL
FEBS Journal 273 (2006) 3698–3708 ª 2006 The Authors Journal compilation ª 2006 FEBS 3705
Cell 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 [
35
S]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 ⁄ P
i
and 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.
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