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-

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

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

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

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

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

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

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

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

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

(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

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

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

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

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

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,

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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