Interferon-alpha induces sensitization of cells to inhibition of protein synthesis by tumour necrosis factor-related apoptosis-inducing ligand.

Tumour cells are often sensitized by interferons to the effects of tumour necrosis factor-alpha-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 Res62, 2272-2280] and we have therefore examined the consequences of prior interferon-alpha 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 alpha 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-alpha 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.

Regulation of protein synthesis by inducible wild-type p53 in human lung carcinoma cells.

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.

p53-induced inhibition of protein synthesis is independent of apoptosis.

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.

p53 activation results in rapid dephosphorylation of the eIF4E-binding protein 4E-BP1, inhibition of ribosomal protein S6 kinase and inhibition of translation initiation.

p53 is an important regulator of cell cycle progression and apoptosis, and inactivation of p53 is associated with tumorigenesis. Although p53 exerts many of its effects through regulation of transcription, this protein is also found in association with ribosomes and several mRNAs have been identified that are translationally controlled in a p53-dependent manner. We have utilized murine erythroleukemic cells that express a temperature-sensitive p53 protein to determine whether p53 also functions at the level of translation. The data presented here demonstrate that p53 causes a rapid decrease in translation initiation. Analysis of several potential mechanisms for regulating protein synthesis shows that p53 has selective effects on the phosphorylation of the eIF4E-binding protein, 4E-BP1, and the activity of the p70 ribosomal protein S6 kinase. These data provide evidence that modulation of translational activity constitutes a further mechanism by which the growth inhibitory effects of p53 may be mediated.

In vivo effects of the Epstein-Barr virus small RNA EBER-1 on protein synthesis and cell growth regulation.

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.

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 kinase.

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 (TNFalpha) 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 eIF2alpha. 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 TNFalpha. 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 eIF2alpha nor increased [eIF4E.4E-BP1] complex formation is essential for the inhibition of overall translation by the DNA-damaging agent.