Cancer Research Prevention Award Frontiers in Basic Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jeffrey, I. W.
Right arrow Articles by Clemens, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jeffrey, I. W.
Right arrow Articles by Clemens, M. J.
[Cancer Research 62, 2272-2280, April 15, 2002]
© 2002 American Association for Cancer Research

Regular Articles

Inhibition of Protein Synthesis in Apoptosis

Differential Requirements by the Tumor Necrosis Factor {alpha} Family and a DNA-damaging Agent for Caspases and the Double-stranded RNA-dependent Protein Kinase1

Ian W. Jeffrey, Martin Bushell2, Vivienne J. Tilleray, Simon Morley and Michael J. Clemens3

Department of Biochemistry and Immunology, Cellular and Molecular Sciences Group, St George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE [I. W. J., M. B., V. J. T., M. J. C.], and Biochemistry Group, School of Biological Sciences, University of Sussex, Brighton BN1 9QG, United Kingdom [S. M.], United Kingdom


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of mammalian cells to agents that induce apoptosis results in a rapid and substantial inhibition of protein synthesis. In MCF-7 breast cancer cells, tumor necrosis factor {alpha} (TNF{alpha}) and TNF-related apoptosis-inducing ligand inhibit overall translation by a mechanism that requires caspase (but not necessarily caspase-3) activity. This inhibition is associated with the increased phosphorylation of eukaryotic initiation factor (eIF2) {alpha}, increased association of eIF4E with the inhibitory eIF4E-binding protein (4E-BP1), and specific cleavages of eIF4B and eIF2{alpha}. All of these changes require caspase activity. The cleavage of eIF4GI, which specifically needs caspase-3 activity, is dispensable for the inhibition of translation in MCF-7 cells. Similar experiments with embryonic fibroblasts from control mice and animals defective for expression of the double-stranded RNA-regulated protein kinase (PKR) reveal requirements for both caspase activity and PKR for inhibition of protein synthesis in response to TNF{alpha}. In contrast, treatment of cells with the DNA-damaging agent etoposide inhibits protein synthesis equally well in the presence of a pan-specific caspase inhibitor and in the presence or absence of PKR. Surprisingly, the ability of etoposide to cause increased association of eIF4E with 4E-BP1 does require PKR activity. However, our data suggest that neither increased phosphorylation of eIF2{alpha} nor increased [eIF4E.4E-BP1] complex formation is essential for the inhibition of overall translation by the DNA-damaging agent.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein synthesis in mammalian cells is subject to rapid regulation after exposure of cells to a wide variety of growth-promoting, growth-inhibitory, and stress-inducing conditions. Aberrant control of translation can result in cell transformation, and changes in the expression of key initiation factors or the signaling pathways that regulate them are often observed in tumors (reviewed in Refs. 1 and 2 ). The initiation of protein synthesis is controlled at a number of levels in cells that are induced to undergo apoptosis after serum deprivation (3) or after treatment with TNF{alpha}4 (4) , agonistic Fas antibodies (5) , DNA-damaging agents (6 , 7) , or staurosporine (4 , 7) . Changes in the protein synthetic machinery associated with early stages of apoptosis (reviewed in Ref. 8 ) include increased phosphorylation of initiation factor eIF2{alpha} (5) , decreased phosphorylation of 4E-BP1 and increased association of the latter with eIF4E (6 , 7 , 9) , caspase-mediated cleavages of certain initiation factors (eIF4GI, eIF4GII, eIF4B, the j subunit of eIF3, and the {alpha} subunit of eIF2; Refs. 3 and 9, 10, 11, 12 ), and specific cleavage of the 28S rRNA component of the larger ribosomal subunit (13) . However, the signal transduction pathways by which these events are activated and the relative contributions of each of these changes to the overall inhibition of translation remain to be established.

Members of the TNF family, including TNF{alpha}, TRAIL, and Fas ligand, inhibit growth and induce programmed cell death in a wide variety of target cells. Association of these ligands with their specific cell surface receptors (14) initiates a sequence of intracellular events that results in the recruitment of adapter proteins, such as TNF receptor-associated death domain protein and FADD, to form a DISC (15) . TNF receptor-associated death domain protein and FADD in turn recruit procaspase-8, the proteolytic activation of which leads to the activation of effector caspases (14 , 16) and is essential for subsequent apoptosis (17 , 18) .

In contrast to cytokine-mediated apoptosis, ionizing radiation and DNA-damaging agents, such as etoposide, exert their effects through the activation of downstream factors, such as the DNA-dependent protein kinase; the protein kinases c-Abl, ATM, and ATR; and the tumor suppressor protein p53 (19 , 20) . DNA damage-dependent or p53-induced apoptosis may also involve TNF family receptors and/or components of the DISC complex (21, 22, 23) , as well as changes in mitochondrial function, and the patterns of caspase activation and protein cleavages are very similar to those seen after exposure of cells to members of the TNF family (21) .

The requirement for caspase activation in the process of cell death varies with different stimuli, and caspase-independent mechanisms can also cause loss of viability (24, 25, 26) . Consistent with this, the inhibition of protein synthesis resulting from exposure of cells to inducers of apoptosis is prevented by caspase inhibitors in some cases but not others (8) .

Recent studies have identified some of the signaling pathways by which protein synthesis may be regulated by cellular stresses and conditions that induce cell death. The cleavage of initiation factor eIF4GI shows a specific requirement for caspase-3 (4) . In addition, important roles have been suggested for the protein kinase PKR, which phosphorylates polypeptide chain initiation factor eIF2{alpha} (27 , 28) , for the rapamycin-sensitive protein kinase mTOR, which is involved in the phosphorylation of the 4E-BPs (29) , and for stress-activated protein kinases, which activate the phosphorylation of eIF4E itself (29) . Previous work has shown that cells that are deficient in PKR expression or that contain a dominant-negative form of PKR are more resistant than control cells to the proapoptotic effects of TNF{alpha} (30, 31, 32) . However, the regulation of protein synthesis itself has not been investigated previously in these systems. Changes in the activity of mTOR rather than PKR have been suggested to be important for the inhibition of translation in response to DNA-damaging agents (6) . Thus, although we know a great deal both about the mechanisms of induction of apoptosis and the changes in the translational machinery associated with the early stages of programmed cell death, little is understood concerning how the two series of events are linked mechanistically. With this in mind, we have begun to dissect the requirements for the various events associated with translational down-regulation in cells induced to undergo apoptosis by a variety of treatments. We have used genetically well-characterized MCF-7 breast cancer cell lines (33) and MEFs (34) to determine the roles of caspases and PKR in the response of protein synthesis to treatment of cells with TNF{alpha}, TRAIL, and etoposide.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
Antiserum against the caspase cleavage product of PARP was from Promega (Southampton, United Kingdom). Monoclonal antibody to eIF2{alpha} and rabbit antisera to eIF4G (raised against the COOH-terminal fragment of eIF4GI expressed in bacteria), eIF4E (raised against a COOH-terminal peptide), and phosphorylated eIF2{alpha} were as described previously (3 , 5) . Immobilon polyvinylidene difluoride was from Millipore, and human TNF{alpha} and TRAIL were from PeproTech EC Ltd. Etoposide and z-VAD.FMK were from Calbiochem, m7GTP-Sepharose was from Pharmacia-LKB, and [35S]methionine was from New England Nuclear.

Tissue Culture and Treatment with Inducers of Apoptosis.
MCF-7 cells (caspase-3 deficient) and MCF-7.3.28 cells (stably transfected to express caspase-3; Ref. 33 ) were kindly provided by Dr. R. Jänicke (University of Singapore) and cultured as described previously (4 , 33 , 35) . MEFs from PKR knockout (PKR-/-) and control (PKR+/+) mice were a gift from Professor C. Weissmann and were maintained in DMEM with 10% FCS and 0.1 mM 2-mercaptoethanol (34) . The cell lines were treated with TNF{alpha}, TRAIL, or etoposide at concentrations of 5 ng/ml, 0.5 µg/ml, and 100 µg/ml, respectively. Cell viability was determined by trypan blue exclusion.

Measurement of Protein Synthesis.
Overall protein synthesis in intact cells was measured by the incorporation of [35S]methionine into trichloroacetic acid-insoluble material. After pulse labeling with 10 µCi/ml of the radioactive amino acid (in methionine-free DMEM with 10% dialyzed FCS), cells were washed in cold PBS and dissolved in 0.3 M NaOH. The protein content was determined, and total proteins were then precipitated, washed, and analyzed by scintillation counting. Rates of protein synthesis were calculated as counts per min of radioactivity incorporated per µg of total protein.

Preparation of Cell Extracts and Analysis by Immunoblotting.
Approximately 2–3 x 106 cells/64-cm2 dish were harvested. The medium containing any free cells was retained, and monolayers were scraped into this with 20 ml of PBS. The cells were washed in PBS twice by centrifugation in the cold and resuspended in 200 µl of buffer A [50 mM 3-(N-morpholino)propanesulphonic acid (pH 7.4), 50 mM NaCl, 2 mM EDTA, 50 mM ß-glycerophosphate, 1 mM microcystin, 2 mM benzamidine, 2 mM Na vanadate, 5 mM p-nitrophenylphosphate, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM GTP, 50 mM Na fluoride, and 7 mM 2-mercaptoethanol]. Cells were lysed by adding NP40 and Triton X-100 to final concentrations of 1.2 and 2.4% (volume for volume), respectively, and centrifuged at 10,000 x g for 10 min at 4°C. For the isolation of eIF4E, cell extracts of equal protein concentration were subjected to m7GTP-Sepharose chromatography as described previously (3 , 9) .

Samples containing equal amounts of protein were subjected to electrophoresis on SDS polyacrylamide gels, and the proteins were analyzed by immunoblotting using alkaline phosphatase-linked secondary antibodies with nitroblue tetrazolium as substrate (3 , 9) . Blots of total and phosphorylated eIF2{alpha} were analyzed by scanning densitometry using an AlphaImager (Alpha Innotech Corp.), and the relative extent of phosphorylation of the factor was calculated from the ratio between the values obtained for each pair of samples.

Analysis of Caspase-8 Activation.
The extent of activation of caspase-8 in MEFs was analyzed by immunoblotting for the active (p20) fragment of this enzyme and by assay of the cleavage of the caspase-8 substrate acetyl-isoleucyl-glutamyl-threonyl-aspartyl-p-nitroanilide by cell extracts (36) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TNF{alpha}- and TRAIL-induced Down-Regulation of Protein Synthesis and Concurrent Initiation Factor Modifications Require Caspase Activity.
MCF-7 cells are sensitive to a number of apoptotic inducers despite being deficient in caspase-3 activity (33 , 35 , 37) . As shown in Fig. 1ACitation , treatment of MCF-7 cells with TNF{alpha} results in a progressive inhibition of protein synthesis, culminating in an 80–90% decrease after 24 h. No greater down-regulation is observed in MCF-7.3.28 cells stably transfected with the caspase-3 gene (33) , which express pro-caspase-3 to high levels (data not shown). Treatment of the two cell lines with TRAIL has a much more rapid effect, such that by 4 h, protein synthesis is inhibited by 70–75% (Fig. 1B)Citation . Again, caspase-3 is not essential for the effect, but the presence of caspase-3 in the MCF-7.3.28 cells does accelerate the response to TRAIL. Nonetheless, it is clear from these results that the inhibition of protein synthesis by TNF{alpha} or TRAIL is not dependent on caspase-3 activity.



View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Caspase-dependent inhibition of translation by TNF{alpha} or TRAIL in MCF-7 breast cancer cells. Exponentially growing MCF-7 cell lines (MCF-7, caspase-3 deficient and MCF-7.3.28, expressing stably transfected caspase-3) were incubated in the absence or presence of TNF{alpha} (A, 5 ng/ml) or TRAIL (B, 0.5 µg/ml) for the times indicated. Cells were also incubated in the presence or absence of z-VAD.FMK (10 µM) and TNF{alpha} for 16 h (C) or z-VAD.FMK and TRAIL for 8 h (D). Protein synthesis was measured by the incorporation of [35S]methionine into acid-insoluble material for the last 60 (A and C), 30 (B), or 12 min (D) of the incubation, as described in "Materials and Methods." Rates of protein synthesis were calculated as the radioactivity incorporated per µg total protein. The data are expressed as a percentage of the untreated control values and are the means +/-SE of six determinations.

 
To determine whether other caspases may be involved, cells were exposed to TNF{alpha} or TRAIL in the presence of the broad spectrum caspase inhibitor z-VAD.FMK. Fig. 1, C and DCitation show that z-VAD.FMK largely prevents the down-regulation of protein synthesis that is otherwise observed after 16-h treatment with TNF{alpha} or 8-h treatment with TRAIL. A similar effect was also observed with another caspase inhibitor, N-tert-butoxycarbonyl-Asp-fluoromethylketone (data not shown). Other proteases may also be inhibited by z-VAD.FMK, and cathepsin-B, in particular, is a target for this compound (38) . However, benzyloxycarbonyl-Phe-Ala-fluoromethylketone, which inhibits cathepsin-B but not caspases, was unable to rescue cells from the inhibitory effect of TRAIL (data not shown). Collectively, these data suggest that the inhibition of general protein synthesis brought about by proapoptotic members of the TNF family requires active caspases but not caspase-3 specifically. The early down-regulation of protein synthesis by TNF{alpha} or TRAIL is not a consequence of a possible loss of cell viability associated with apoptosis, because more than half of the cells treated with TNF{alpha} and >90% of the cells treated with TRAIL are viable (as judged by trypan blue exclusion) at 16 and 4 h, respectively (Table 1)Citation , a time when protein synthesis had been inhibited by >=75%.


View this table:
[in this window]
[in a new window]

  		Table 1 Effects of TNF{alpha} and TRAIL on viability of cell lines

Exponentially growing cells were incubated in the absence or presence of TNF{alpha} (5 ng/ml; A) or TRAIL (0.5 µg/ml; B) for the times indicated, and cell viabilities were determined by trypan blue exclusion. The results of several different experiments are summarized.

 
Fig. 2ACitation shows that there is partial cleavage of eIF4B in MCF-7 cells treated with TNF{alpha} for 16 h, whereas eIF4G remains intact, as reported previously (4) . The well-characterized caspase substrate PARP is also cleaved to give rise to an Mr 89,000 fragment (Fig. 2A)Citation . The eIF4B and PARP cleavages are blocked when z-VAD.FMK is included in the culture medium. Because MCF-7 cells are deficient in caspase-3, we can conclude that neither PARP cleavage nor eIF4B cleavage is dependent on this enzyme. In extracts from TNF{alpha}-treated MCF-7.3.28 cells, the eIF4GI cleavage product M-FAG was clearly visible (Fig. 2A)Citation , confirming our earlier observations (4) .



View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Caspase-dependent initiation factor modifications after TNF{alpha} treatment of MCF-7 cells. In A, exponentially growing MCF-7 and MCF-7.3.28 (C3+) cells (see Fig. 1Citation ) were incubated in the absence or presence of TNF{alpha} for 16 h, without or with z-VAD.FMK as indicated. Total cytoplasmic extracts were prepared as described in "Materials and Methods" and analyzed by immunoblotting for eIF4G, the Mr 89,000 caspase cleavage product of PARP, eIF4B, total eIF2{alpha}, and phosphorylated eIF2{alpha} as indicated. Bottom two panels, eIF4E and associated proteins were isolated by m7GTP-Sepharose chromatography, and the recovered eIF4E and bound 4E-BP1 were visualized by immunoblotting. In B, the levels of total eIF2{alpha} and phosphorylated eIF2{alpha} were measured by scanning densitometry, and the relative extent of phosphorylation of the initiation factor (in arbitrary units) was calculated from the ratio between the two values for each condition.

 
We have shown previously that, in Jurkat cells, stimulation of the Fas receptor results in increased phosphorylaton of the {alpha} subunit of eIF2 (5) . This blocks the ability of the factor to participate in successive rounds of initiation complex formation, leading to a decrease in the rate of global protein synthesis (39) . A similar phenomenon occurs in response to TNF{alpha} treatment, the level of phosphorylated eIF2{alpha} being increased by 50–80% in both MCF-7 and MCF-7.3.28 cells (Fig. 2, A and B)Citation . Interestingly, this increase is abolished when z-VAD.FMK is present. In apoptotic cells, a small fraction of eIF2{alpha} is cleaved by one or more caspases (5 , 9 , 10 , 12) , and we have also observed this, particularly for phosphorylated eIF2{alpha}, after TNF{alpha} treatment (Fig. 2A)Citation . Thus, both the phosphorylation and cleavage of eIF2{alpha} occur by mechanisms that require caspase (but not specifically caspase-3) activity under these conditions.

Protein synthesis may also be inhibited as a result of dephosphorylation of 4E-BP1, causing sequestration of eIF4E away from eIF4G and impairment of cap-dependent initiation of translation (29) . As shown in Fig. 2ACitation (bottom panels), the level of eIF4E isolated from TNF{alpha}-treated cells remains unchanged, but the binding of 4E-BP1 to eIF4E is increased dramatically in both MCF-7 cell types. Again, this change is prevented by z-VAD.FMK.

Essentially similar results are seen when MCF-7 cells are treated with TRAIL (Fig. 3)Citation . Phosphorylation of eIF2{alpha} is enhanced by >=50% within 4 h in both MCF-7 and MCF-7.3.28 cells (Fig. 3, A and B)Citation , and eIF4B is cleaved in both cases as well (Fig. 3C)Citation . However, the cleavage of eIF4GI, as well as that of the j subunit of eIF3 (p35), requires the specific expression of caspase-3 (Fig. 3, B and C)Citation . The association of 4E-BP1 with eIF4E also increases in a z-VAD.FMK-sensitive manner when MCF-7 or MCF-7.3.28 cells are treated with TRAIL (Fig. 3D)Citation . All of the above observations thus support the notion of links between modifications of translation initiation factors and caspase activation in MCF-7 breast cancer cells undergoing apoptosis in response to members of the TNF family.



View larger version (64K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Caspase-dependent initiation factor modifications after TRAIL treatment of MCF-7 cells. In A, exponentially growing MCF-7 and MCF-7.3.28 (C3+) cells (see Fig. 1Citation ) were incubated in the absence or presence of TRAIL (0.5 µg/ml) for 2 or 4 h as indicated. Total cytoplasmic extracts were prepared as described in "Materials and Methods" and analyzed by immunoblotting for the Mr 89,000 caspase cleavage product of PARP, total eIF2{alpha}, and phosphorylated eIF2{alpha}. In B, cells were incubated with TRAIL for the times indicated, and extracts were analyzed similarly for cleavage of PARP and eIF4G and for levels of total and phosphorylated eIF2{alpha}. In C, cells were incubated with or without TRAIL or TRAIL and z-VAD.FMK for 8 h as indicated. Cytoplasmic extracts were prepared and analyzed by immunoblotting for PARP, eIF4B, and eIF3j (p35) as indicated. In D, cells were incubated with TRAIL for the times shown, and eIF4E and associated proteins were isolated from extracts by m7GTP-Sepharose chromatography. The recovered eIF4E and bound 4E-BP1 were visualized by immunoblotting.

 
TNF{alpha}-induced Inhibition of Protein Synthesis in Murine MEFs Requires Both Caspase and PKR Activity.
The protein kinase PKR plays an important role not only in the regulation of translation but also in the control of apoptosis (reviewed in Refs. 28 and 40 ). However, the requirement for PKR in the regulation of protein synthesis by members of the TNF family has not been investigated previously. Fig. 4ACitation shows that TNF{alpha} inhibits protein synthesis by ~50% in control (PKR+/+) MEFs but has no effect on cells that are deficient in the protein kinase. The TNF{alpha} effect in control MEFs was again sensitive to the caspase inhibitor z-VAD.FMK, suggesting roles for both PKR and caspase activity in the translational response to the cytokine. There is no significant loss of cell viability in the MEFs under these conditions of TNF{alpha} treatment (Table 1)Citation .



View larger version (24K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Caspase and PKR requirements for TNF{alpha}-induced inhibition of protein synthesis and activation of caspase-8 in murine embryo fibroblasts. In A, MEFs from PKR knockout (PKR-/-) mice and control (PKR+/+) mice were incubated in the absence or presence of TNF{alpha} (5 ng/ml) or TNF{alpha} and z-VAD.FMK (10 µM), as indicated, for 21 h. Protein synthesis was then measured as described in Fig. 1ACitation . The data are expressed as a percentage of untreated control values and are the means +/-SE of six determinations. In B, MEFs from control (PKR+/+) mice and PKR knockout (PKR-/-) mice were incubated in the absence or presence of TNF{alpha} (5 ng/ml) for the times indicated. Cell extracts were analyzed for the presence of the active, cleaved p20 form of caspase-8 by immunoblotting. As controls, similar extracts were prepared from wild-type and caspase-8-deficient Jurkat cells, treated where indicated with an agonistic anti-Fas antibody as described previously (6) . In C, extracts were prepared from PKR+/+ MEFs (shaded bars) and PKR-/- MEFs (open bars), treated with TNF{alpha} for the times indicated. Caspase-8 activity was assayed using acetyl-isoleucyl-glutamyl-threonyl-aspartyl-p-nitroanilide as the substrate, as described in "Materials and Methods."

 
To determine whether the loss of sensitivity of PKR-/- cells to TNF{alpha}-induced inhibition of translation is accompanied by a lack of response of these cells to other TNF-induced signaling events, we assayed the state of phosphorylation of the stress-activated protein kinases c-Jun-NH2-terminal kinase and p38, enzymes that are known to be activated by TNF{alpha} in fibroblasts (14) . As has been reported by others (41) , activation of c-Jun-NH2-terminal kinase and p38 by TNF{alpha} treatment was defective in PKR-/- cells (data not shown). We also investigated whether the apical caspase, caspase-8, was activated in these cells. This enzyme associates with the DISC complex and is activated rapidly by autocatalytic cleavage in cells treated with members of the TNF{alpha} family (15) . Fig. 4BCitation shows that PKR+/+ cells respond to TNF{alpha} with appearance of the active, cleaved form of caspase-8 (p20) within 15 min, although the effect is transient. A substantial but transient increase in caspase-8 activity can also be detected in extracts of these cells over a similar period of time (Fig. 4C)Citation . In contrast, neither caspase-8 cleavage nor caspase-8 activity could be detected in TNF{alpha}-treated PKR-/- cells. These data suggest that PKR is required for many of the cellular responses to activation of the TNF{alpha} receptor.

The DNA-damaging Agent Etoposide Inhibits Protein Synthesis by a PKR- and Caspase-independent Mechanism.
Agents and conditions that cause DNA damage are well known as inducers of apoptosis (42) , and, although there are conflicting reports as to whether TNF family receptors are involved (23 , 43) , the apoptotic response activates downstream pathways similar to those stimulated by TNF{alpha} and TRAIL (42) . Protein synthesis is inhibited rapidly in response to exposure of cells to the DNA topoisomerase II inhibitor etoposide and other DNA-damaging agents (5 , 6) . Similarly, etoposide is a potent inhibitor of protein synthesis in MCF-7 cells, irrespective of whether caspase-3 is expressed in the cells (Fig. 5)Citation . Again, this is not because of any extensive loss of cell viability (data not shown). In contrast to the situation with TNF{alpha} or TRAIL, z-VAD.FMK does not interfere with translational inhibition by etoposide (Fig. 5B)Citation . Moreover, immunoblot analysis of MCF-7 cells treated with etoposide showed that apoptosis, as assayed by PARP cleavage, was induced at 20 h but not at 4 h (data not shown), suggesting that the translation rate is depressed significantly before caspase activation in this system. Etoposide treatment inhibits protein synthesis in MEFs by ~30% at 4 h and ~80% at 20 h (Fig. 5C)Citation . This effect does not require PKR activity because PKR-/- cells are inhibited equally. Overall, these results suggest that the effects of etoposide on translation are independent of both caspase and PKR activity.



View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Lack of requirement for caspases or PKR for etoposide-induced inhibition of protein synthesis. In A, MCF-7 cell lines (MCF-7, caspase-3 deficient and MCF-7.3.28, expressing stably transfected caspase-3) were incubated in the absence or presence of etoposide (100 µg/ml) for 4 or 20 h. Protein synthesis was measured as described for Fig. 1DCitation . The data are expressed as a percentage of untreated control values and are the means +/-SE of six determinations. In B, MCF-7 cells were incubated with etoposide in the absence or presence of z-VAD.FMK (10 µM) for the times indicated, and protein synthesis was measured as in A. In C, MEFs from PKR knockout (PKR-/-) mice and control (PKR+/+) mice were incubated in the absence or presence of etoposide (100 µg/ml) for 4 or 20 h. Protein synthesis was measured as described for Fig. 1DCitation . The data are expressed as a percentage of untreated control values and are the means +/-SE of six determinations.

 
Etoposide-induced Association of eIF4E with 4E-BP1 Requires PKR.
In further contrast to the effects of TNF{alpha} and TRAIL, etoposide does not cause any consistent increase in the extent of phosphorylation of eIF2{alpha} in MCF-7 cells (Fig. 6, A and C)Citation . However, MEFs do respond to etoposide with an increase in eIF2{alpha} phosphorylation, and this requires the presence of PKR (Fig. 6, B and D)Citation . These findings suggest that the ability of DNA-damaging agents to activate PKR may be cell-type specific. Regardless of this, etoposide is clearly capable of acting by other mechanisms also because there is a lack of requirement for PKR for the overall inhibition of protein synthesis.



View larger version (51K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Effects of etoposide treatment on phosphorylation of eIF2{alpha} and association of eIF4E with 4E-BP1 in MCF-7 cells and MEFs. In A, MCF-7 and MCF-7.3.28 (C3+) cells (see Fig. 1Citation ) were incubated with etoposide (100 µg/ml) for the times shown. Total cytoplasmic extracts were prepared as described in "Materials and Methods" and analyzed by immunoblotting for total eIF2{alpha} and phosphorylated eIF2{alpha}. In addition, eIF4E and associated proteins were isolated by m7GTP-Sepharose chromatography, and the recovered eIF4E, eIF4G, and 4E-BP1 were visualized by immunoblotting. In B, MEFs from PKR knockout (PKR-/-) and control (PKR+/+) mice were incubated with etoposide for the indicated times, and extracts were analyzed as in A. In C, the relative extent of phosphorylation of eIF2{alpha} in the MCF-7 cells was determined by densitometry as described in Fig. 2Citation . In D, the relative extent of phosphorylation of eIF2{alpha} in the MEFs was determined by densitometry as described in Fig. 2Citation .

 
Etoposide also causes a dramatic increase in the association of eIF4E with 4E-BP1 after 20 h in both MCF-7 and MCF-7.3.28 cells, with no change in the levels of extractable eIF4E, and this is accompanied by a decrease in the association of eIF4G with eIF4E (Fig. 6A)Citation . However, [eIF4E.4E-BP1] complex formation is only increased marginally at 4 h, in agreement with data reported for Swiss 3T3 cells (6) . A similar late increase in binding of 4E-BP1 to eIF4E occurs in etoposide-treated PKR+/+ MEFs (Fig. 6B)Citation . Strikingly, however, 20 h of etoposide treatment completely fails to stimulate 4E-BP1 binding to eIF4E in PKR-/- MEFs. This finding suggests a novel requirement for PKR activity in the regulation of eIF4E function.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Members of the TNF family are well-known inducers of cellular growth inhibition and apoptosis in many systems. However, in comparison with several other cytokines and hormones, their effects on overall protein synthesis have received relatively little attention. TNF{alpha} inhibits translation in skeletal muscle (44) , and stimulation of the Fas receptor down-regulates translation in Jurkat cells (5) , but little is known of the mechanisms by which these effects are mediated.

Previously, we reported that eIF4GI cleavage in response to TNF{alpha} and other proapoptotic agents in MCF-7 cells requires caspase-3 (4) . However, apoptosis itself can occur in MCF-7 cells in the absence of caspase-3 (33 , 37) , and other caspases have been implicated in TNF{alpha} effects on cells (22) . Here we have shown that protein synthesis is inhibited in both caspase-3-positive and -negative cells in response to TNF{alpha} or TRAIL. Thus, mechanisms other than eIF4GI cleavage must be sufficient for the inhibition. Nevertheless, translational down-regulation is likely to be a caspase-dependent process in this system because it is prevented by z-VAD.FMK. It is possible that the cleavages of eIF4B (Refs. 9 and 45 ; and perhaps eIF4GII; Refs. 9 and 46 ), which are mediated by other caspases, and/or the caspase-dependent phosphorylation of eIF2{alpha} and increased association of 4E-BP1 with eIF4E may be involved. There could also be a role for caspase-dependent 28S rRNA cleavage, which occurs in response to apoptotic stimuli (13) . In 293 cells, cleavage of eIF4G by an inducible poliovirus 2A protease is sufficient to cause apoptosis (47) .

Numerous studies have implicated PKR in both the inhibition of protein synthesis and the induction of stress responses and apoptosis (28 , 31 , 32 , 41 , 48 , 49) . One report (50) indicates that PKR can be activated by ceramide, a lipid second messenger generated during cellular stress and associated with apoptotic responses (51 , 52) . PKR is also involved in the activation of transcription factors of the NF{kappa}B family, although the kinase is not essential for the activation of NF{kappa}B by either TNF{alpha} or double-stranded RNA (32 , 53, 54, 55, 56, 57) . Both eIF2{alpha} phosphorylation and NF{kappa}B activation are necessary for PKR-induced apoptosis, and translational inhibition alone is not sufficient (32 , 48) . Taken in conjunction with these earlier observations, our present findings with PKR-/- MEFs imply that TNF{alpha}-induced activation of NF{kappa}B alone is not sufficient to inhibit translation.

In the case of MCF-7 cells, PKR is present at relatively high levels and is active (58) . Our observations of substantial phosphorylation of eIF2{alpha} in the exponentially growing cells are in agreement with this. MCF-7 cells seem able to tolerate significant phosphorylation of eIF2{alpha}, perhaps because they contain high eIF2B activity (58) , and this may be a property that contributes to their malignant phenotype. Although it is not clear whether the enhanced phosphorylation of eIF2{alpha} seen after TNF{alpha} or TRAIL treatment (Figs. 2Citation and 3Citation and Ref. 32 ) is sufficient to affect overall protein synthetic rates in MCF-7 cells, both the TNF{alpha}-stimulated phosphorylation of eIF2{alpha} and the inhibition of protein synthesis show a common requirement for caspase activity.

It is possible that the effects of TNF{alpha} on apoptosis in the MCF-7 cell system involve the tumor suppressor protein p53 (59) . Cell lines (including MCF-7 cell variants) that contain mutant or inhibited p53 show resistance to TNF{alpha}-induced cell death, and this can be reversed by the expression of wild-type p53 (22 , 59) . TNF{alpha} is also able to induce p53 expression (60 , 61) . We have found recently that activation of a temperature-sensitive form of p53 leads to a rapid inhibition of overall translation. However there are some differences between the actions of TNF{alpha} or TRAIL and the effects of p53 activation.5

Taken together, our data are compatible with a model in which PKR is essential for the expression or activation of caspase-8 and/or other components of a signaling pathway that leads from TNF family receptors to the translational machinery (Fig. 7)Citation . This conclusion is consistent with work showing that the receptor-associated adapter protein FADD is required for the apoptotic response to PKR activation and is down-regulated in cells expressing dominant-negative PKR (62) . Our observation that caspase-8 is not activated by TNF{alpha} treatment in PKR-/- cells places the kinase (or a PKR-dependent event) upstream of FADD and caspase-8. Interestingly, PKR can activate caspase-8 by a FADD-mediated mechanism downstream of TNF{alpha} receptors (63) and can itself be activated after cleavage by effector caspases (64) . This suggests the existence of a positive feedback loop in the regulation of apoptosis by the kinase. Our data on the effects of etoposide, on the other hand, suggest that DNA damage regulates PKR more distally, at a point beyond that requiring any caspase activity (Fig. 7)Citation .



View larger version (41K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. Proposed signal transduction pathways for the regulation of protein synthesis in response to TNF{alpha}/TRAIL and DNA damage. The data in this study, together with the work of others (62 , 63) , are compatible with a model in which PKR or a PKR-dependent signaling component lies upstream of FADD and the initiator caspase (caspase-8) on the pathway leading from TNF family receptors to the translational machinery. In contrast, DNA damage resulting from etoposide treatment can inhibit protein synthesis more distally, at steps beyond those requiring either PKR or caspase activity. However, the increased association of 4E-BP1 with eIF4E after etoposide treatment does show a requirement for PKR, suggesting that the protein kinase may be involved additionally in translational regulation at this level after the DNA damage response.

 
One of the most striking changes in the protein synthetic machinery in cells treated with either TNF family members or etoposide is the increased association of 4E-BP1 with eIF4E. This is a common response to conditions that cause cell growth inhibition or induce stress responses (29) , and it is not associated exclusively with apoptosis. Nevertheless, the z-VAD.FMK sensitivity of [eIF4E.4E-BP1] complex formation in cells treated with TNF{alpha} or TRAIL indicates a caspase dependency that links the effect with apoptosis. In contrast, Tee and Proud (6) found that z-VAD.FMK was unable to block the effect of etoposide on 4E-BP1 binding to eIF4E. This is consistent with our observation of a lack of effect of the caspase inhibitor on the inhibition of protein synthesis by this DNA-damaging agent. The requirement for PKR for the association of 4E-BP1 with eIF4E after etoposide treatment (Fig. 6)Citation indicates that the protein kinase is involved in the regulation of 4E-BP1 phosphorylation downstream of the DNA damage pathway (Fig. 7)Citation . This may reflect the activation of protein phosphatase PP2A as a result of the phosphorylation of its regulatory subunit B56{alpha} by PKR (65) . Thus, although DNA damage can inhibit the activity of the 4E-BP1 protein kinase mTOR (66) , PKR-mediated activation of PP2A may also bring about the dephosphorylation of 4E-BP1 (67) .

Finally, the differential requirements for PKR for increased eIF2{alpha} phosphorylation and 4E-BP1 association with eIF4E, versus the inhibition of methionine incorporation after etoposide treatment, suggest that inhibition of the availability of eIF2 and eIF4E cannot alone account for the effect of the DNA-damaging agent on overall translation. Therefore, it is likely that etoposide has additional mechanisms of action on the protein synthetic machinery, the nature of which remain to be determined.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the Wellcome Trust (040800, 058915, 057494, 045619, and 056778), the Leukaemia Research Fund, the Cancer Prevention Research Trust, and Glaxo-Wellcome. S. J. M. is a Senior Research Fellow of the Wellcome Trust. Back

2 Present address: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305. Back

3 To whom requests for reprints should be addressed, at Department of Biochemistry and Immunology, Cellular and Molecular Sciences Group, St George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, United Kingdom. Phone: 44-20-8725-5762; Fax: 44-20-8725-2992; E-mail: M.Clemens{at}sghms.ac.uk Back

4 The abbreviations used are: TNF, tumor necrosis factor; 4E-BP1, eukaryotic initiation factor 4E-binding protein; DISC, death-inducing signaling complex; eIF, eukaryotic initiation factor; FADD, Fas-associated death domain protein; MEF, murine embryonic fibroblast; PARP, poly(ADP-ribose) polymerase; PKR, double-stranded RNA-regulated protein kinase; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; z-VAD.FMK, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; mTOR, mammalian target of rapamycin; NF{kappa}B, nuclear factor {kappa}B. Back

5 J. Hensold, V. Tilleray, M. Bushell, and M. J. Clemens, unpublished data. Back

Received 6/18/01. Accepted 2/ 5/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Clemens M. J., Bommer U. A. Translational control: the cancer connection. Int. J. Biochem. Cell Biol., 31: 1-23, 1999.[Medline]
  2. Hershey J. W. B., Miyamoto S. Translational control and cancer Sonenberg N. Hershey J. W. B. Mathews M. B. eds. . Translational Control of Gene Expression, : 637-654, Cold Spring Harbor Laboratory Cold Spring Harbor, NY 2000.
  3. Clemens M. J., Bushell M., Morley S. J. Degradation of eukaryotic polypeptide chain initiation factor (eIF) 4G in response to induction of apoptosis in human lymphoma cell lines. Oncogene, 17: 2921-2931, 1998.[Medline]
  4. Bushell M., McKendrick L., Jänicke R. U., Clemens M. J., Morley S. J. Caspase-3 is necessary and sufficient for cleavage of protein synthesis eukaryotic initiation factor 4G during apoptosis. FEBS Lett., 451: 332-336, 1999.[Medline]
  5. Morley S. J., Jeffrey I., Bushell M., Pain V. M., Clemens M. J. Differential requirements for caspase-8 activity in the mechanism of phosphorylation of elF2{alpha}, cleavage of eIF4GI and signaling events associated with the inhibition of protein synthesis in apoptotic Jurkat T cells. FEBS Lett., 477: 229-236, 2000.[Medline]
  6. Tee A. R., Proud C. G. DNA-damaging agents cause inactivation of translational regulators linked to mTOR signaling. Oncogene, 19: 3021-3031, 2000.[Medline]
  7. Tee A. R., Proud C. G. Staurosporine inhibits phosphorylation of translational regulators linked to mTOR. Cell Death Differ., 8: 841-849, 2001.[Medline]
  8. Clemens M. J., Bushell M., Jeffrey I. W., Pain V. M., Morley S. J. Translation initiation factor modifications and the regulation of protein synthesis in apoptotic cells. Cell Death Differ., 7: 603-615, 2000.[Medline]
  9. Bushell M., Wood W., Clemens M. J., Morley S. J. Changes in integrity and association of eukaryotic protein synthesis initiation factors during apoptosis. Eur. J. Biochem., 267: 1083-1091, 2000.[Medline]
  10. Satoh S., Hijikata M., Handa H., Shimotohno K. Caspase-mediated cleavage of eukaryotic translation initiation factor subunit 2{alpha}. Biochem. J., 342: 65-70, 1999.
  11. Bushell M., Poncet D., Marissen W. E., Flotow H., Lloyd R. E., Clemens M. J., Morley S. J. Cleavage of polypeptide chain initiation factor eIF4GI during apoptosis: Characterization of an internal fragment generated by caspase-3-mediated cleavage. Cell Death Differ., 7: 628-636, 2000.[Medline]
  12. Marissen W. E., Guo Y. W., Thomas A. A. M., Matts R. L., Lloyd R. E. Identification of caspase 3-mediated cleavage and functional alteration of eukaryotic initiation factor 2{alpha} in apoptosis. J. Biol. Chem., 275: 9314-9323, 2000.[Abstract/Free Full Text]
  13. Houge G., Robaye B., Eikhom T. S., Golstein J., Mellgren G., Gjertsen B. T., Lanotte M., Doskeland S. O. Fine mapping of 28S rRNA sites specifically cleaved in cells undergoing apoptosis. Mol. Cell. Biol., 15: 2051-2062, 1995.[Abstract]
  14. Baud V., Karin M. Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol., 11: 372-377, 2001.[Medline]
  15. Kuang A. A., Diehl G. E., Zhang J. K., Winoto A. FADD is required for DR4-and DR5-mediated apoptosis–lack of trail-induced apoptosis in FADD-deficient mouse embryonic fibroblasts. J. Biol. Chem., 275: 25065-25068, 2000.[Abstract/Free Full Text]
  16. Budihardjo I., Oliver H., Lutter M., Luo X., Wang X. D. Biochemical pathways of caspase activation during apoptosis. Annu. Rev. Cell Dev. Biol., 15: 269-290, 1999.[Medline]
  17. Bodmer J-L., Holler N., Reynard S., Vinciguerra P., Schneider P., Juo P., Blenis J., Tschopp J. TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nature Cell Biol., 2: 241-243, 2000.[Medline]
  18. Seol D. W., Li J. R., Seol M. H., Park S. Y., Talanian R. V., Billiar T. R. Signaling events triggered by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL): caspase-8 is required for TRAIL-induced apoptosis. Cancer Res., 61: 1138-1143, 2001.[Abstract/Free Full Text]
  19. Durocher D., Jackson S. P. DNA-PK, ATM, and A. T. R. as sensors of DNA damage: variations on a theme?. Curr. Opin. Cell Biol., 13: 225-231, 2001.[Medline]
  20. Shiloh Y. ATM and ATR: networking cellular responses to DNA damage. Curr. Opin. Genet. Dev., 11: 71-77, 2001.[Medline]
  21. Boesen-de Cock J. G. R., Tepper A. D., De Vries E., Van Blitterswijk W. J., Borst J. Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and {gamma}-radiation downstream from caspase-8 activation. J. Biol. Chem., 274: 14255-14261, 1999.[Abstract/Free Full Text]
  22. Rokhlin O. W., Gudkov A. V., Kwek S., Glover R. A., Gewies A. S., Cohen M. B. p53 is involved in tumor necrosis factor-{alpha}-induced apoptosis in the human prostatic carcinoma cell line LNCaP. Oncogene, 19: 1959-1968, 2000.[Medline]
  23. Gibson S. B., Oyer R., Spalding A. C., Anderson S. M., Johnson G. L. Increased expression of death receptors 4 and 5 synergizes the apoptosis response to combined treatment with etoposide and TRAIL. Mol. Cell. Biol., 20: 205-212, 2000.[Abstract/Free Full Text]
  24. Daugas E., Susin S. A., Zamzami N., Ferri K. F., Irinopoulou T., Larochette N., Prévost M. C., Leber B., Andrews D., Penninger J., Kroemer G. Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. FASEB J., 14: 729-739, 2000.[Abstract/Free Full Text]
  25. Susin S. A., Daugas E., Ravagnan L., Samejima K., Zamzami N., Loeffler M., Costantini P., Ferri K. F., Irinopoulou T., Prévost M. C., Brothers G., Mak T. W., Penninger J., Earnshaw W. C., Kroemer G. Two distinct pathways leading to nuclear apoptosis. J. Exp. Med., 192: 571-579, 2000.[Abstract/Free Full Text]
  26. Denecker G., Vercammen D., Declercq W., Vandenabeele P. Apoptotic and necrotic cell death induced by death domain receptors. Cell. Mol. Life Sci., 58: 356-370, 2001.[Medline]
  27. Clemens M. J., Elia A. The double-stranded RNA-dependent protein kinase PKR: structure and function. J. Interferon Cytokine Res., 17: 503-524, 1997.[Medline]
  28. Williams B. R. G. PKR, a sentinel kinase for cellular stress. Oncogene, 18: 6112-6120, 1999.[Medline]
  29. Gingras A. C., Raught B., Sonenberg N. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu. Rev. Biochem., 68: 913-963, 1999.[Medline]
  30. Der S. D., Yang Y. L., Weissmann C., Williams B. R. G. A double-stranded RNA-activated protein kinase-dependent pathway mediating stress-induced apoptosis. Proc. Natl. Acad. Sci. USA, 94: 3279-3283, 1997.[Abstract/Free Full Text]
  31. Yeung M. C., Liu J., Lau A. S. An essential role for the interferon-inducible, double-stranded RNA-activated protein kinase PKR in the tumor necrosis factor-induced apoptosis in U937 cells. Proc. Natl. Acad. Sci. USA, 93: 12451-12455, 1996.[Abstract/Free Full Text]
  32. Srivastava S. P., Kumar K. U., Kaufman R. J. Phosphorylation of eukaryotic translation initiation factor 2 mediates apoptosis in response to activation of the double-stranded RNA-dependent protein kinase. J. Biol. Chem., 273: 2416-2423, 1998.[Abstract/Free Full Text]
  33. Janicke R. U., Sprengart M. L., Wati M. R., Porter A. G. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J. Biol. Chem., 273: 9357-9360, 1998.[Abstract/Free Full Text]
  34. Kumar A., Yang Y-L., Flati V., Der S., Kadereit S., Deb A., Haque J., Reis L., Weissmann C., Williams B. R. G. Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF-1 and NF-kB. EMBO J., 16: 406-416, 1997.[Medline]
  35. Janicke R. U., Ng P., Sprengart M. L., Porter A. G. Caspase-3 is required for {alpha}-fodrin cleavage but dispensable for cleavage of other death substrates in apoptosis. J. Biol. Chem., 273: 15540-15545, 1998.[Abstract/Free Full Text]
  36. Stennicke H. R., Salvesen G. S. Caspase assays. Methods Enzymol., 322: 91-100, 2000.[Medline]
  37. Liang Y., Yan C. H., Schor N. F. Apoptosis in the absence of caspase 3. Oncogene, 20: 6570-6578, 2001.[Medline]
  38. Schotte P., Declercq W., Van Huffel S., Vandenabeele P., Beyaert R. Non-specific effects of methyl ketone peptide inhibitors of caspases. FEBS Lett., 442: 117-121, 1999.[Medline]
  39. Kimball S. R. Eukaryotic initiation factor eIF2. Int. J. Biochem. Cell Biol., 31: 25-29, 1999.[Medline]
  40. Gil J., Esteban M. Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis, 5: 107-114, 2000.[Medline]
  41. Goh K. C., DeVeer M. J., Williams B. R. G. The protein kinase PKR is required for p38 MAPK activation and the innate immune response to bacterial endotoxin. EMBO J., 19: 4292-4297, 2000.[Medline]
  42. Norbury C. J., Hickson I. D. Cellular responses to DNA damage. Annu. Rev. Pharmacol. Toxicol., 41: 367-401, 2001.[Medline]
  43. Ferraro C., Quemeneur L., Fournel S., Prigent A. F., Revillard J. P., Bonnefoy-Berard N. The topoisomerase inhibitors camptothecin and etoposide induce a CD95-independent apoptosis of activated peripheral lymphocytes. Cell Death Differ., 7: 197-206, 2000.[Medline]
  44. Cooney R., Kimball S. R., Eckman R., Maish G., Shumate M., Vary T. C. TNF-binding protein ameliorates inhibition of skeletal muscle protein synthesis during sepsis. Am. J. Physiol., 276: E611-E619, 1999.[Abstract/Free Full Text]
  45. Bushell M., Wood W., Carpenter G., Pain V. M., Morley S. J., Clemens M. J. Disruption of the interaction of mammalian protein synthesis eukaryotic initiation factor 4B with the poly(A)-binding protein by caspase- and viral protease-mediated cleavages. J. Biol. Chem., 276: 23922-23928, 2001.[Abstract/Free Full Text]
  46. Marissen W. E., Gradi A., Sonenberg N., Lloyd R. E. Cleavage of eukaryotic translation initiation factor 4GII correlates with translation inhibition during apoptosis. Cell Death Differ., 7: 1234-1243, 2000.[Medline]
  47. Goldstaub D., Gradi A., Bercovitch Z., Grosmann Z., Nophar Y., Luria S., Sonenberg N., Kahana C. Poliovirus 2A protease induces apoptotic cell death. Mol. Cell. Biol., 20: 1271-1277, 2000.[Abstract/Free Full Text]
  48. Gil J., Alcamí J., Esteban M. Induction of apoptosis by double-stranded-RNA-dependent protein kinase (PKR) involves the {alpha} subunit of eukaryotic translation initiation factor 2 and NF-{kappa}B. Mol. Cell. Biol., 19: 4653-4663, 1999.[Abstract/Free Full Text]
  49. Williams B. R. G. Signal integration via PKR. Science’s STKE, : 1-10, 2001.
  50. Ruvolo P. P., Gao F., Blalock W. L., Deng X., May W. S. Ceramide regulates protein synthesis by a novel mechanism involving the cellular PKR activator RAX. J. Biol. Chem., 276: 11754-11758, 2001.[Abstract/Free Full Text]
  51. Sortino M. A., Condorelli F., Vancheri C., Canonico P. L. Tumor necrosis factor-{alpha} induces apoptosis in immortalized hypothalamic neurons: involvement of ceramide-generating pathways. Endocrinology, 140: 4841-4849, 1999.[Abstract/Free Full Text]
  52. Sawada N., Nakashima S., Banno Y., Yamakawa H., Hayashi K., Takenaka K., Nishimura Y., Sakai N., Nozawa Y. Ordering of ceramide formation, caspase activation, and Bax/Bcl-2 expression during etoposide-induced apoptosis in C6 glioma cells. Cell Death Differ., 7: 761-772, 2000.[Medline]
  53. Gil J., Alcamí J., Esteban M. Activation of NF-{kappa}B by the dsRNA-dependent protein kinase. PKR involves the I{kappa}B kinase complex. Oncogene, 19: 1369-1378, 2000.[Medline]
  54. Bonnet M. C., Weil R., Dam E., Hovanessian A. G., Meurs E. F. PKR stimulates NF-{kappa}B irrespective of its kinase function by interacting with the I{kappa}B kinase complex. Mol. Cell. Biol., 20: 4532-4542, 2000.[Abstract/Free Full Text]
  55. Iordanov M. S., Wong J., Bell J. C., Magun B. E. Activation of NF-{kappa}B by double-stranded RNA (dsRNA) in the absence of protein kinase R and RNase L demonstrates the existence of two separate dsRNA-triggered antiviral programs. Mol. Cell. Biol., 21: 61-72, 2001.[Abstract/Free Full Text]
  56. Maran A., Maitra R. K., Kumar A., Dong B., Xiao W., Li G., Williams B. R. G., Torrence P. F., Silverman R. H. Blockage of NF-{kappa}B signaling by selective ablation of an mRNA target by 2–5A antisense chimeras. Science (Wash. DC), 265: 789-792, 1994.[Abstract/Free Full Text]
  57. Zamanian-Daryoush M., Mogensen T. H., DiDonato J. A., Williams B. R. G. NF-{kappa}B activation by double-stranded-RNA-activated protein kinase (PKR) is mediated through NF-{kappa}B-inducing kinase and I{kappa}B kinase. Mol. Cell. Biol., 20: 1278-1290, 2000.[Abstract/Free Full Text]
  58. Kim S. H., Forman A. P., Mathews M. B., Gunnery S. Human breast cancer cells contain elevated levels and activity of the protein kinase, PKR. Oncogene, 19: 3086-3094, 2000.[Medline]
  59. Cai Z., Capoulade C., Moyret-Lalle C., Amor-Gueret M., Feunteun J., Larsen A. K., Bressac-de Paillerets B., Chouaib S Resistance of MCF-7 human breast carcinoma cells to TNF-induced cell death is associated with loss of p53 function. Oncogene, 15: 2817-2826, 1997.[Medline]
  60. Jeoung D., Tang B., Sonenberg M. Effects of tumor necrosis factor-{alpha} on antimitogenicity and cell cycle-related proteins in MCF-7 cells. J. Biol. Chem., 270: 18367-18373, 1995.[Abstract/Free Full Text]
  61. Yin D., Tamaki N., Kokunai T. Wild-type p53-dependent etoposide-induced apoptosis mediated by caspase-3 activation in human glioma cells. J. Neurosurg., 93: 289-297, 2000.[Medline]
  62. Balachandran S., Kim C. N., Yeh W. C., Mak T. W., Bhalla K., Barber G. N. Activation of the dsRNA-dependent protein kinase. PKR induces apoptosis through FADD-mediated death signaling. EMBO J., 17: 6888-6902, 1998.[Medline]
  63. Gil J., Esteban M. The interferon-induced protein kinase (PKR), triggers apoptosis through FADD-mediated activation of caspase 8 in a manner independent of Fas and TNF-{alpha} receptors. Oncogene, 19: 3665-3674, 2000.[Medline]
  64. Saelens X., Kalai M., Vandenabeele P. Translation inhibition in apoptosis: caspase-dependent PKR activation and eIF2-{alpha} phosphorylation. J. Biol. Chem., 276: 41620-41628, 2001.[Abstract/Free Full Text]
  65. Xu Z., Williams B. R. G. The B56{alpha} regulatory subunit of protein phosphatase 2A is a target for regulation by double-stranded RNA-dependent protein kinase PKR. Mol. Cell. Biol., 20: 5285-5299, 2000.[Abstract/Free Full Text]
  66. Kumar V., Sabatini D., Pandey P., Gingras A. C., Majumder P. K., Kumar M., Yuan Z. M., Carmichael G., Weichselbaum R., Sonenberg N., Kufe D., Kharbanda S. Regulation of the rapamycin and FKBP-target 1/mammalian target of rapamycin and cap-dependent initiation of translation by the c-Abl protein-tyrosine kinase. J. Biol. Chem., 275: 10779-10787, 2000.[Abstract/Free Full Text]
  67. Nanahoshi M., Nishiuma T., Tsujishita Y., Hara K., Inui S., Sakaguchi N., Yonezawa K. Regulation of protein phosphatase 2A catalytic activity by {alpha}4 protein and its yeast homolog Tap42. Biochem. Biophys. Res. Commun., 251: 520-526, 1998.[Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
H. L. Eley and M. J. Tisdale
Skeletal Muscle Atrophy, a Link between Depression of Protein Synthesis and Increase in Degradation
J. Biol. Chem., March 9, 2007; 282(10): 7087 - 7097.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Y. Wang, S.-A. Xue, G. Hallden, J. Francis, M. Yuan, B. E. Griffin, and N. R. Lemoine
Virus-Associated RNA I-Deleted Adenovirus, a Potential Oncolytic Agent Targeting EBV-Associated Tumors
Cancer Res., February 15, 2005; 65(4): 1523 - 1531.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X. Zhang, J. Li, D. P. Sejas, K. R. Rathbun, G. C. Bagby, and Q. Pang
The Fanconi Anemia Proteins Functionally Interact with the Protein Kinase Regulated by RNA (PKR)
J. Biol. Chem., October 15, 2004; 279(42): 43910 - 43919.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
H.-L. Yang, J.-X. Pan, L. Sun, and S.-C. J. Yeung
p21 Waf-1 (Cip-1) Enhances Apoptosis Induced by Manumycin and Paclitaxel in Anaplastic Thyroid Cancer Cells
J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 763 - 772.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jeffrey, I. W.
Right arrow Articles by Clemens, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jeffrey, I. W.
Right arrow Articles by Clemens, M. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online