Interferon-a induces sensitization of cells to inhibition of protein synthesis by tumour necrosis factor-related apoptosis-inducing ligand ´ Ian W Jeffrey, Androulla Elia, Stephanie 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 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 ´ ´ ´ Departement d’Oncogenetique, Centre ´ Biomedicale de Recherche et Valorisation, Clermont-Ferrand, France (Received 15 March 2006, revised 19 May 2006, accepted 12 June 2006) 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 translation 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 inhibition 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 providing a likely explanation for the sensitization of cells to the inhibition of translation doi:10.1111/j.1742-4658.2006.05374.x Members of the tumour necrosis factor-a (TNFa) family 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 substrate for PKR, becomes more highly phosphorylated in cells exposed to TRAIL or TNFa It is well established 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 eukaryotic initiation factor 4E-binding protein (4E-BP1) with the mRNA cap-binding factor eIF4E in cells Abbreviations 4E-BP, eukaryotic initiation factor 4E binding protein; BID, Bcl-2-interacting death protein; eIF, eukaryotic initiation factor; FADD, Fasassociated 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 I W Jeffrey et al 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 usually these cytokines are cytostatic rather than cytotoxic when applied as single agents [13,14] However, numerous 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 synthesis Our data indicate that IFNa treatment sensitizes both MCF-7 and HeLa cells to the translational inhibitory 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, TRAILR1 (DR4) and TRAIL-R2 (DR5), results in the recruitment of procaspase-8 to the death-inducing signalling 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 active caspase-8 [25,26] Caspase-8 in turn is responsible for initiating a cascade of activation of effector caspases that ultimately leads to the multiple changes in cells characteristic of TRAIL-induced apoptosis [27] The process of activation of caspase-8, and the downstream consequences that arise from it, are blocked by the caspase-8-specific peptide inhibitor zIle-GluThr-Asp-fluoromethyl ketone (zIETD.FMK) [17] We show here that the effects of TRAIL on overall protein 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 dephosphorylation Control of protein synthesis by IFNa and TRAIL 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 investigated 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 synthesis in MCF-7 cells This was manifested as a sensitization by IFNa pretreatment to the effect of TRAIL In MCF-7 cells not previously exposed to IFNa, 25 ngỈmL)1 TRAIL was required to inhibit protein synthesis by 50% within h, whereas when the cells had been pretreated with IFNa, only 10 ngỈmL)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 twoto three-fold more sensitive than MCF-7 cells to TRAIL As shown previously [2], the downregulation of translation by TRAIL was not a secondary consequence 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, unpublished 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 activated following the binding of TRAIL to its receptors and the formation of the death-inducing signalling complex [30], is required for the inhibition of translation 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 FEBS Journal 273 (2006) 3698–3708 ª 2006 The Authors Journal compilation ª 2006 FEBS 3699 Control of protein synthesis by IFNa and TRAIL A I W Jeffrey et al A 100 %Inhibition of protein synthesis 75 100 50 10 Pr o t e i n Sy n t h e s i s (% of c ont rol ) [35S]Met incorporation (counts per min/μg protein x 10-3) 25 100 TRAIL (ng/ml) (log scale) 75 50 25 TRAIL Z-IETD.FMK B 0 10 25 50 75 100 200 - + + - - + TRAIL 500 Control TRAIL + z-IETD.FMK TRAIL (ng/ml) full length (p53/55) 100 %Inhibition of protein synthesis [35S]Met incorporation (counts per min/μg protein) x 10-3 B 75 p41/43 50 (Pro)caspase-8 25 0.1 10 100 TRAIL (ng/ml) (log scale) p18 α-tubulin C TRAIL Control TRAIL + z-IETD.FMK z-IETD.FMK full length 0 0.1 0.5 1.0 5.0 10 25 BID t-BID TRAIL (ng/ml) Fig 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 IFNa2b (1000 mL)1) and then further treated with the indicated concentrations of TRAIL for the last h (A) or h (B) Protein synthesis was measured by the incorporation of [35S]methionine into acid-insoluble material for the last 40 The data are the means ± SEM of three determinations Insets: percentage inhibition 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 3700 Fig 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 ngỈmL)1) for h as indicated Where shown, zIETD.FMK was present at 10 lM Protein synthesis was then measured as described in Fig The data are expressed as percentage of the value obtained with untreated control 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 immunoblotted 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 FEBS Journal 273 (2006) 3698–3708 ª 2006 The Authors Journal compilation ª 2006 FEBS I W Jeffrey et al Control of protein synthesis by IFNa and TRAIL Table Requirement for caspase-8 for inhibition of protein synthesis by TRAIL Wild-type and caspase-8-deficient Jurkat cells were incubated for h in the absence or presence of TRAIL (400 ngỈmL)1) Protein synthesis was measured by the incorporation of [35S]methionine into acid-insoluble material for the last 60 The data are the means ± SEM of four to six determinations [35S]methionine incorporation (counts per per 105 cells) (· 10)3) A - TRAIL IFNα α + - Total eIF2α α - TRAIL Z.IETD.FMK –TRAIL +TRAIL Inhibition by TRAIL (%) Wild type Caspase-8 deficient 4.44 ± 0.08 3.89 ± 0.11 1.11 ± 0.05 3.43 ± 0.11 + + eIF2α(P) α B Cell line + + - + + + 75.0 11.8 eIF2α(P) α C - TRAIL Z.IETD.FMK 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 indicated 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-8mediated 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 phosphorylation 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 phosphorylation 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 zIETD.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 phosphorylation of 4E-BP1 caused by TRAIL were completely 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 + - + + γ β α Total 4E-BP1 D TRAIL - + - + Total 4E-BP1 TRAIL - + - + 4E-BP1 (P)Ser65 Wild-type C8-deficient Jurkat Jurkat Wild-type C8-deficient Jurkat Jurkat Fig 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 IFNa2b (1000 mL)1) and further treated with or without TRAIL (167 ngỈmL)1) for the last 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 h in the absence or presence of TRAIL (167 ngỈmL)1) and zIETD.FMK (10 lM) as indicated Extracts were prepared as in (A) and analysed by immunoblotting for (B) phosphorylated eIF2a (Ser51) and (C) 4EBP1 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 h in the absence (-)or presence (+) of TRAIL (150 ngỈmL)1) Extracts were prepared and analysed by immunoblotting for total 4E-BP1 (left panel) and phosphorylated 4E-BP1 (Ser65) (right panel) no effect on the total levels of these factors (A Elia, unpublished results) Moreover, treatment of caspase8-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 wildtype 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 FEBS Journal 273 (2006) 3698–3708 ª 2006 The Authors Journal compilation ª 2006 FEBS 3701 Control of protein synthesis by IFNa and TRAIL Control TRAIL IFNα α IFNα α + TRAIL C Control + TRAIL + IFNα α + IFNα + TRAIL α full length (p53/55) p41/43 Caspase-8 100 p18 α-tubulin Band intensity (arbitrary units) A I W Jeffrey et al 75 MCF-7 cells (141%) 50 25 (227%) (137%) B Control TRAIL IFNα IFNα +TRAIL p53/p55 full length (p53/55) p18 Band intensity (arbitrary units) p41/43 100 p41/p43 p18 HeLa cells (143%) 75 50 (88%) (90%) (103%) 25 p53/p55 p41/p43 p18 Fig 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 unitsỈmL)1), and then treated with or without TRAIL as indicated (MCF-7 cells, h at 167 ngỈmL)1; HeLa cells, h at 10 ngỈmL)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 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-treated 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, compared 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) 3702 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 substrate poly(ADP-ribose) polymerase (PARP) to produce its characteristic 89-kDa cleavage product The results in Fig show that TRAIL alone induced partial cleavage of BID and PARP within 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 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 shows that, in spite of the activation of caspase-8 and the cleavage of BID and PARP, TRAIL alone had FEBS Journal 273 (2006) 3698–3708 ª 2006 The Authors Journal compilation ª 2006 FEBS I W Jeffrey et al Control Control of protein synthesis by IFNa and TRAIL TRAIL IFNα IFNα + TRAIL full length protein BID PARP cleavage product (t-BID) full length protein cleavage product α−tubulin α− Fig TRAIL-induced caspase activity is enhanced by IFNa pretreatment MCF-7 cells were grown for 72 h in the absence or presence of human IFNa2b (1000 mL)1) and further incubated with or without TRAIL (167 ngỈmL)1) for the last 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 very little effect on the appearance of cells with a subG1 DNA content Approximately and 6% of the total cell population showed a decreased DNA content after and 16 h, respectively In cells pretreated with IFNa, the corresponding figures were approximately and 16% at 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 suggested 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 cellular DNA and is not a consequence of overt apoptosis However, the sensitizing effect of IFNa for caspasemediated 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 phosphorylation 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 synthesis (Table 1) Caspase-8 is intimately involved in the function of the TRAIL-activated death-inducing signalling 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 polypeptide 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-stimulated 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 Interestingly, our data suggest that only relatively small changes in caspase-8 activity appear to be sufficient to alter substantially the cellular sensitivity to TRAIL FEBS Journal 273 (2006) 3698–3708 ª 2006 The Authors Journal compilation ª 2006 FEBS 3703 I W Jeffrey et al C ell count Sub-G1 0.3% + IFNα 180 360 540 720 180 360 540 720 Control Sub-G1 0.4% 0 Cell count Control of protein synthesis by IFNa and TRAIL 200 400 200 600 C ell count Sub-G1 0.8% + IFNα +TRAIL (4h) 180 360 540 720 180 360 540 720 +TRAIL (4h) Sub-G1 1.6% 0 200 400 600 200 DNA content 600 Ce ll c ount Sub-G1 6.1% + IFNα +TRAIL (16h) 180 360 540 720 +TRAIL (16h) 180 360 540 720 400 DNA content Sub-G1 16.0% 0 Cell count 400 DNA content Cell count DNA content 200 400 600 DNA content 200 400 600 DNA content Fig 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 mL)1) and then further treated with or without TRAIL (100 ngỈmL)1) for the last 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 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, respectively (Fig 1) These results are consistent with the concept 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 3704 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 association 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 FEBS Journal 273 (2006) 3698–3708 ª 2006 The Authors Journal compilation ª 2006 FEBS I W Jeffrey et al the regulation of 4E-BP1 activity is the more important 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 expression of which is known to be upregulated at the translational level in response to increased phosphorylation of eIF2a [42] This suggests that increased eIF2a phosphorylation 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] Interestingly, 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 inhibition 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 caspase-8 by IFNs [40,43–45], there are several other potential mechanisms that could also operate to bring about such synergism, including IFN-induced enhancement of the expression of TRAIL receptors [15] IFN treatment might also inhibit the activity of antiapoptotic 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 Control of protein synthesis by IFNa and TRAIL 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 precedent 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 caspasemediated 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 phosphorylation 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 described [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 ScheringPlough (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 provided by R Janicke (University of Dusseldorf, Germany) ă 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 unitsỈmL)1) for the times shown in the legends to Figs 1,3,4,5 and 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 FEBS Journal 273 (2006) 3698–3708 ª 2006 The Authors Journal compilation ª 2006 FEBS 3705 Control of protein synthesis by IFNa and TRAIL I W Jeffrey et al 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 measured by the incorporation of [35S]methionine (10 lCiỈmL)1) into trichloroacetic acid-insoluble material for 40 Radioactivity was determined as previously described [2] Protein content was determined and rates of protein synthesis are expressed as counts per incorporated per lg protein Immunoblotting of cell extracts Cells were harvested, washed in NaCl ⁄ Pi and lysed as described previously [2] Samples containing equal amounts of protein were subjected to electrophoresis on SDS polyacrylamide gels and the proteins transferred to PVDF membranes using a semidry blotting apparatus (Bio-Rad, Hemel Hempstead, UK) Blots were blocked, incubated with the appropriate primary 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concentrations of TRAIL producing 50% inhibition of protein synthesis 3700 Fig Effects of zIETD.FMK on TRAIL-induced inhibition of protein synthesis and caspase-8 activity in MCF-7 cells (A) MCF-7 cells