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Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in Jurkat T lymphoma cells. One of the characteristics of the phase preceding overt apoptosis is the marked downregulation of protein synthesis. We have investigated factors that can influence this response and have explored some of the signaling pathways involved. We show that interferon-α (IFNα) pretreatment desensitizes Jurkat cells to TRAIL-induced inhibition of protein synthesis, such that the concentration of TRAIL required for 50% inhibition is increased by 10-fold. The inhibition of translation is characterized by dephosphorylation of the eIF4E-binding protein 4E-BP1 and IFNα desensitizes Jurkat cells to this effect. IFNα also inhibits TRAIL-mediated dephosphorylation of the growth-promoting protein kinase B (Akt). Since Jurkat cells are defective for phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and therefore have constitutive phosphoinositide 3-kinase (PI3K) activity, we investigated the consequences for protein synthesis of inhibiting PI3K using LY294002. Inhibition of PI3K partially inhibits translation, but also enhances the effect of a suboptimal concentration of TRAIL. However, LY294002 does not block the ability of IFNα to protect protein synthesis from TRAIL-induced inhibition. Data are presented suggesting that IFNα impairs the process of activation of caspase-8 within the TRAIL death-inducing signaling complex. [ABSTRACT FROM AUTHOR]

Background Information Tumour cells can be induced to undergo apoptosis after treatment with the tumour necrosis factor α-related death-inducing ligand (TRAIL). Although human pancreatic cancer cells show varying degrees of response they can be sensitised to the pro-apoptotic effects of TRAIL in the presence of celastrol, a natural compound extracted from the plant Tripterygium wilfordii Hook F. One important aspect of the cellular response to TRAIL is the control of protein synthesis, a key regulator of which is the eukaryotic initiation factor 4E-binding protein, 4E-BP1. Results We examined the effects of celastrol and TRAIL in several pancreatic cancer cell lines. In cells that are normally resistant to TRAIL, synergistic effects of TRAIL plus celastrol on commitment to apoptosis and inhibition of protein synthesis were observed. These were associated with a strong up-regulation and dephosphorylation of 4E-BP1. The enhancement of 4E-BP1 expression, which correlated with a threefold increase in the level of the 4E-BP1 transcript, was blocked by inhibitors of reactive oxygen species and the JNK protein kinase. When the expression of 4E-BP1 was reduced by an inducible micro-RNA, TRAIL-mediated apoptosis was inhibited. Conclusion These results suggest that 4E-BP1 plays a critical role in the mechanism by which TRAIL and celastrol together cause apoptotic cell death in human pancreatic tumour cells. [ABSTRACT FROM AUTHOR]

The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely. [ABSTRACT FROM AUTHOR]

Tumour cells are often sensitized by interferons to the effects of tumour necrosis factor-α-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-α 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 α 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-α 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. [ABSTRACT FROM AUTHOR]

Abstract: Activation of an over-expressed mutant form of the tumour suppressor protein p53 has been shown to inhibit protein synthesis. To determine whether this effect is due only to high level expression or the mutant nature of the protein, we have used a doxycycline-inducible lung carcinoma cell line capable of expressing wild-type p53. We now show that levels of wild-type p53 similar to those expressed endogenously also inhibit protein synthesis. The mechanism involves dephosphorylation and accumulation of the translational inhibitor 4E-BP1, and increased association of 4E-BP1 with initiation factor eIF4E. The inhibition of translation is not a consequence of p53-mediated apoptosis. [Copyright &y& Elsevier]

Activation of a temperature-sensitive form of mouse p53 in murine erythroleukaemia cells rapidly inhibits protein synthesis and causes early dephosphorylation and cleavage of protein synthesis initiation factor eIF4GI and the eIF4E-binding protein 4E-BP1. Dephosphorylated 4E-BP1 and the cleaved products of 4E-BP1 and eIF4GI associate with eIF4E under these conditions, concomitant with decreased interaction of full-length eIF4GI with eIF4E. These changes may play an important role in preventing formation of the eIF4F complex and thus the initiation of protein synthesis. As observed previously for eIF4GI, the cleavage of 4E-BP1 is insensitive to the general caspase inhibitor z-VAD.FMK, consistent with a caspase-independent mechanism of factor modification and regulation of protein synthesis. Comparison of the p53-induced patterns of eIF4GI and 4E-BP1 dephosphorylation and cleavage with those caused by the mTOR inhibitor rapamycin indicates that p53 activation and rapamycin have distinct but additive effects. Moreover, p53 activation inhibits rapamycin-insensitive protein kinase activity against 4E-BP1. P53 and rapamycin have additive effects on the inhibition of overall protein synthesis. These data suggest that the inhibition of protein synthesis by p53 is largely independent of the regulation of rapamycin-sensitive mTOR in the system under investigation.Oncogene (2005) 24, 4839–4850. doi:10.1038/sj.onc.1208648; published online 9 May 2005 [ABSTRACT FROM AUTHOR]

Abstract: Protein synthesis is regulated at the translational level by a variety of mechanisms in virus-infected cells. Viruses often induce the shut-off of host translation in order to favour the expression of their own genetic information, but cells possess a number of strategies for counteracting such effects of infection. Important regulatory mechanisms include the phosphorylation of the α subunit of polypeptide chain initiation factor eIF2, RNA degradation mediated by the 2′5′-oligoadenylate/RNase L system, control of availability of the cap-binding protein eIF4E by its interaction with the 4E-binding proteins and specific proteolytic cleavage of several key initiation factors. Most of these mechanisms are also utilised in uninfected cells in response to a variety of physiological stresses and during the early stages of apoptosis. Thus, mechanisms of translational control during virus infection can provide models for the cellular stress responses observed in a wide range of other circumstances. [Copyright &y& Elsevier]

Activation of a temperature-sensitive form of p53 in murine erythroleukaemia cells results in a rapid impairment of protein synthesis that precedes inhibition of cell proliferation and loss of cell viability by several hours. The inhibition of translation is associated with specific cleavages of polypeptide chain initiation factors eIF4GI and eIF4B, a phenomenon previously observed in cells induced to undergo apoptosis in response to other stimuli. Although caspase activity is enhanced in the cells in which p53 is activated, both the effects on translation and the cleavages of the initiation factors are completely resistant to inhibition of caspase activity. Moreover, exposure of the cells to a combination of the caspase inhibitor z-VAD.FMK and the survival factor erythropoietin prevents p53-induced cell death but does not reverse the inhibition of protein synthesis. We conclude that the p53-regulated cleavages of eIF4GI and eIF4B, as well as the overall inhibition of protein synthesis, are caspase-independent events that can be dissociated from the induction of apoptosis per se . [ABSTRACT FROM AUTHOR]

Examines the eukaryotic protein synthesis initiation factors during apoptosis. Inhibition of the rate of overall protein synthesis in a variety of eukaryotic cell types; Occurrence of cleavages for the binding proteins; Result of the induction of apoptosis in biphasic change.

Examines the interaction between the La antigen and double-stranded RNA. Regulation of protein synthesis in eukaryotic cells; Role of the protein in termination and initiation of transcription; Binding of single-stranded RNA.

1. Addition of haem to cell-free extracts from Ehrlich ascites tumour cells stimulates protein synthesis only in extracts from cells previously incubated in nutritionally complete conditions. Extracts from amino-acid-deprived cells do not respond to haem. The stimulation of protein synthesis in fed cell extracts is due to increased initiation on endogenous mRNA mediated by an increase in the levels of 40-S-subunit. Met-tRNA initiation complexes. Extracts from starved cells exhibit a defect in 40-S initiation complex formation which cannot be overcome by haem. 2. Experiments to test for the presence of an inhibitor of initiation in Ehrlich cell extracts by monitoring effects on translation in haem-supplemented reticulocyte lysates have revealed that extracts from both fed and starved cells contain one or more inhibitory activities which shut off protein synthesis, dissagregate polysomes and reduce the level of 40-S initiation complexes in the lysate. Extracts from starved cells are more inhibitory for protein synthesis than those from fed cells. 3. Initiation factor eIF-2 is phosphorylated by an endogenous Ehrlich cell protein kinase in vitro, but this occurs to the same extent in extracts from fed and starved cells. 4. We propose a possible model for the role of eIF-2 in the control of protein synthesis by amino acid supply in Ehrlich cells. [ABSTRACT FROM AUTHOR]

The oligonucleotide 5′-triphosphoadenylyl(2′-5′)adenylyl(2′-5′)adenosine (pppA2′p5′A2′p5′A) is synthesised by reticulocyte lysates and extracts from interferon-treated cells in the presence of double-stranded RNA. It inhibits the translation of both viral and non-viral mRNAs and of poly-(rU,rC) in the micrococcal-nuclease-treated reticulocyte lysate system. The translation of poly(rU) is insensitive in this assay, however. Inhibition develops in vitro after a lag period, the length of which depends on the nature of the mRNA present and its concentration. The degradation of radioactive Mengo virus RNA or L cell cytoplasmic RNA is accelerated in reticulocyte lysates incubated with the oligonucleotide. This is due to activation of one or more endonucleases which degrade(s) RNA to large oligonucleotide fragments. Our results suggest that the endonuclease is responsible for most (if not all) of the inhibitory effects of the oligonucleotide on protein synthesis in the reticuiocyte lysate. In the presence of pppA2′p5fA2′pS′A, protein synthesis can be reactivated after it has ceased by addition of fresh mRNA. In contrast, initiation factor eIF-2 is unable to prevent inhibition by the oligonucleotide under conditions where it protects the system from the effects of the haem-controlled repressor and partially protects against inhibition by double-stranded RNA. These findings are discussed in relation to the regulation of protein synthesis by double-stranded RNA and by interferon. [ABSTRACT FROM AUTHOR]

The rate of initiation of protein synthesis in rabbit reticulocyte lysates is regulated by a translational inhibitor protein which is activated in the absence of added haemin. The effects of this inhibitor on amino acid incorporation are overcome by the protein synthesis initiation factor IF-MP which binds Met-tRNAf in a ternary complex with GTP and which can transfer this complex to small ribosomal subunits. Addition of this factor to haemin-deficient lysates prevents loss ofpolysomes and regenerates polysomes from 80-S single ribosomes, thus confirming an effect at the level of polypeptide initiation. The ability of the initiation factor to overcome the effects of various concentrations of the translational inhibitor suggests that the inhibitor inactivates the factor catalytically rather than stoichiometrically. In a system in vitro consisting of salt-washed 40-S ribosomal subunits, initiator Met-tRNAf and GTP, the initiation factor IF-MP transfers Met-tRNAf to the subunits in the absence of any other factor or mRNA. Equilibrium buoyant density gradient analysis in CsCl shows that formaldehyde- fixed subunits carrying Met-tRNAf bound under these conditions have a buoyant density approximately 0.02 g/cm³ lower than the bulk of salt-washed subunits, suggesting that approximately I00000 daltons of additional protein are associated with these subunits. This is in marked contrast to the amounts of protein bound to subunits incubated with Met-tRNAf and GTP in the presence of a crude ribosomal salt-wash fraction. The translational inhibitor has no effect on formation of the ternary complex IF-MP - Met-tRNAf o GTP but does impair the factor-catalysed transfer of Met-tRNAf to washed subunits. The possible mechanisms of action of the inhibitor on polypeptide chain initiation are reviewed in the light of these results. [ABSTRACT FROM AUTHOR]

The inhibition by double-stranded RNA (dsRNA) of the translation of encephalomyocurditis virion RNA (EMC RNA) in extracts from interferon-treated mouse L-cells has been investigated. In particular, we have found that the addition of small amounts of dialysed cell sap from interferon-treated cells to cell-free systems from control ceils renders them more sensitive to inhibition by dsRNA. This has provided an assay for the component(s) involved in the inhibition. The translation of both viral and non-viral messenger RNAs is inhibited by dsRNA in the presence of interferon cell sap. The concentration of dsRNA required for inhibition, however, varies with the suit concentration used. The lower sensitivity of the control cell-free system to dsRNA does not reflect a higher level of dsRNA-specific nuclease, nor is the inhibition by interferon cell sap and dsRNA reversed by the addition of tRNA. There is an enhanced breakdown of the EMC RNA message in the inhibited systems. It is not yet clear, however, whether this reflects the activity of a dsRNA-activated single-strand-RNA-specific nuclease, or merely the degradation of unused message m the dsRNA-inhibited system. Interestingly, the abnormal distribution of polypeptide products synthesised in response to EMC RNA in systems inhibited by interferon cell sap and dsRNA is apparently identical to that obtained on translation of this RNA in cell-free systems from interferon-treated, vacciniavirus-infected L-cells. [ABSTRACT FROM AUTHOR]

A role for the Epstein-Barr virus small RNA species EBER-1 in the regulation of protein synthesis has been investigated in the reticulocyte-lysate cell-free translation system. Recombinant EBER-1 was synthesized by in vitro transcription of a plasmid containing the viral gene and purified by CF11-cellulose chromatography and ribonuclease III treatment. When added to the reticulocyte lysate at 10–20 μg/ml or more, EBER-1 prevents the inhibition of protein synthesis caused by low concentrations of synthetic double-stranded RNA, poly(I) · poly(C). This effect is eliminated by treatment of the recombinant EBER-1 with ribonuclease T1. Disruption of the secondary structure of EBER-1 by substitution of inosine for guanosine in the in-vitro-synthesized RNA impairs the ability of EBER-1 to prevent the poly(I) · poly(C)-mediated inhibition of protein synthesis. These results suggest that high concentrations of EBER-1 regulate protein synthesis by blocking the activation of the double-stranded RNA-dependent eukaryotic initiation factor 2α (eIF-2α) protein kinase DAI (p68), and that this property is dependent on the secondary structure of the small RNA molecule. [ABSTRACT FROM AUTHOR]

Exposure of the temperature-sensitive leucyl-tRNA synthetase mutant of Chinese hamster ovary cells, tsH1, to the non-permissive temperature of 39.5°C results in a rapid inhibition of polypeptide chain initiation. This inhibition is caused by a reduced ability of the eukaryotic initiation factor eIF-2 to participate in the formation of eIF-2 · GTP · Met-tRNAf ternary complexes and thus in the formation of 43S ribosomal pre-initiation complexes. Associated with this decreased eIF-2 activity is an increased phosphorylation of the eIF-2α subunit. It has previously been shown in other systems that phosphorylation of eIF-2α slows the rate of recycling of eIF-2 · GDP to eIF-2 · GTP catalysed by the guanine nucleotide exchange factor eIF-2B. We show here that phosphorylation of eIF-2α by the reticulocyte haem-controlled repressor also inhibits eIF-2B activity in cell-free extracts derived from tsH1 cells. Thus the observed increased phosphorylation of eIF-2α at the non-permissive temperature in this system is consistent with impaired recycling of eIF-2 in vivo. Using a single-step temperature revertant of tsH1 cells, TR-3 (which has normal leucyl-tRNA synthetase activity at 39.5°C), we demonstrate here that all inhibition of eIF-2 function reverts together with the synthetase mutation. This establishes the close link between synthetase function and eIF-2 activity. In contrast, recharging tRNALeu in vivo in tsH1 cells at 39.5 °C by treatment with a low concentration of cycloheximide failed to reverse the inhibition of eIF-2 function. This indicates that tRNA charging per se is not involved in the regulatory mechanism. Our data indicate a novel role for aminoacyl-tRNA synthetases in the regulation of eIF-2 function mediated through phosphorylation of the α subunit of this factor. However, in spite of the fact that cell-free extracts from Chinese hamster ovary cells contain protein kinase and phosphatase activities active against either exogenous or endogenous eIF-2α, we have been unable to show any activation of kinase or inactivation of phosphatase following incubation of the cells at 39.5°C. [ABSTRACT FROM AUTHOR]

1. A cell-free protein-synthesising system has been prepared from the liver of adult male Xenopus laevis which shows several adaptive changes following induction of egg-yolk protein synthesis by administration of oestradiol-17β in vivo, 2. The product of the cell-free system from oestrogen-treated animals is characterised by elevated incorporation of radioactive serine relative to phenylalanine. This is taken as an indication of the synthesis of phosvitin, which has a high serine content, in vitro. 3. Increased protein-synthetic activity of Xenopus liver ribosomes following oestrogen treatment can be demonstrated and is maintained after the particles are washed in 0.5 M KCl. This effect of the hormone is also apparent when the ribosomes are first stripped of endogenous mRNA by preincubation and then added to a fresh cell-free system containing poly (U). It is concluded that oestrogen treatment produces an intrinsic activation of synthetic ability of ribosomes in the liver. 4. Increased ability of ribosomes to translate poly(U) does not result from changes in their ability to bind the polynucleotide, either before or after preincubation. 5. Cell sap from liver of oestrogen-treated animals shows increased ability to support amino acid incorporation by ribosomes from control or treated animals in vitro. This is not due to enhanced initiation of peptide synthesis or to increased availability of endogenous aminoacyl- tRNA in vitro. 6. The effects of oestradiol on Xenopus liver are considered in relation to the translational control of protein synthesis by hormones and other agents in a variety of systems. [ABSTRACT FROM AUTHOR]

3′-Deoxyadenosine, also known as cordycepin, is a known polyadenylation inhibitor with a large spectrum of biological activities, including anti-proliferative, pro-apoptotic and anti-inflammatory effects. In this study we confirm that cordycepin reduces the length of poly(A) tails, with some mRNAs being much more sensitive than others. The low doses of cordycepin that cause poly(A) changes also reduce the proliferation of NIH3T3 fibroblasts. At higher doses of the drug we observed inhibition of cell attachment and a reduction of focal adhesions. Furthermore, we observed a strong inhibition of total protein synthesis that correlates with an inhibition of mammalian target of rapamycin (mTOR) signaling, as observed by reductions in Akt kinase and 4E-binding protein (4EBP) phosphorylation. In 4EBP knock-out cells, the effect of cordycepin on translation is strongly reduced, confirming the role of this modification. In addition, the AMP-activated kinase (AMPK) was shown to be activated. Inhibition of AMPK prevented translation repression by cordycepin and abolished 4EBP1 dephosphorylation, indicating that the effect of cordycepin on mTOR signaling and protein synthesis is mediated by AMPK activation. We conclude that many of the reported biological effects of cordycepin are likely to be due to its effects on mTOR and AMPK signaling. [ABSTRACT FROM AUTHOR]

Recent studies have suggested a role for the Epstein–Barr virus-encoded RNA EBER-1 in malignant transformation. EBER-1 inhibits the activity of the protein kinase PKR, an inhibitor of protein synthesis with tumour suppressor properties. In human 293 cells and murine embryonic fibroblasts, transient expression of EBER-1 promoted total protein synthesis and enhanced the expression of cotransfected reporter genes. However reporter gene expression was stimulated equally well in cells from control and PKR knockout mice. NIH 3T3 cells stably expressing EBER-1 exhibited a greatly increased frequency of colony formation in soft agar, and protein synthesis in these cells was relatively resistant to inhibition by the calcium ionophore A23187. Nevertheless clones containing a high concentration of EBER-1 were not invariably tumourigenic. We conclude that EBER-1 can enhance protein synthesis by a PKR-independent mechanism and that, although this RNA may contribute to the oncogenic potential of Epstein–Barr virus, its expression is not always sufficient for malignant transformation. [Copyright &y& Elsevier]