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Chronic exposure to trichothecenes is known to disturb insulin-like growth factor 1 and signaling of insulin and leptin hormones and causes considerable growth retardation in animals. However, limited information was available on mechanisms underlying trichothecene-induced growth retardation. In this study, we employed an integrated transcriptomics, proteomics, and RNA interference (RNAi) approach to study the molecular mechanisms underlying trichothecene cytotoxicity in rat pituitary adenoma GH3 cells. Our results showed that trichothecenes suppressed the synthesis of growth hormone 1 (Gh1) and inhibited the eukaryotic transcription and translation initiation by suppressing aminoacyl-tRNA synthetases transcription, inducing eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2) and reducing eukaryotic translation initiation factor 5 a. The sulfhydryl oxidases , protein disulfide isomerase,and heat shock protein 90 (were greatly reduced, which resulted in adverse regulation of protein processing and folding. Differential genes and proteins associated with a decline in energy metabolism and cell cycle arrest were also found in our study. However, use of RNAi to interfere with hemopoietic cell kinase (Hck) and EIF2AK2 transcriptions or use of chemical inhibitors of MAPK, p38, Ras, and JNK partially reversed the reduction of Gh1 levels induced by trichothecenes. It indicated that the activation of MAPKs, Hck, and EIF2AK2 were important for trichothecene-induced growth hormone suppression. Considering the potential hazards of exposure to trichothecenes, our findings could help to improve our understanding regarding human and animal health implications., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The translation factor eIF5 is an important partner of eIF2, directly modulating its function in several critical steps. First, eIF5 binds eIF2/GTP/Met-tRNA(i)(Met) ternary complex (TC), promoting its recruitment to 40S ribosomal subunits. Secondly, its GTPase activating function promotes eIF2 dissociation for ribosomal subunit joining. Finally, eIF5 GDP dissociation inhibition (GDI) activity can antagonize eIF2 reactivation by competing with the eIF2 guanine exchange factor (GEF), eIF2B. The C-terminal domain (CTD) of eIF5, a W2-type HEAT domain, mediates its interaction with eIF2. Here, we characterize a related human protein containing MA3- and W2-type HEAT domains, previously termed BZW2 and renamed here as eIF5-mimic protein 1 (5MP1). Human 5MP1 interacts with eIF2 and eIF3 and inhibits general and gene-specific translation in mammalian systems. We further test whether 5MP1 is a mimic or competitor of the GEF catalytic subunit eIF2Bε or eIF5, using yeast as a model. Our results suggest that 5MP1 interacts with yeast eIF2 and promotes TC formation, but inhibits TC binding to the ribosome. Moreover, 5MP1 is not a GEF but a weak GDI for yeast eIF2. We propose that 5MP1 is a partial mimic and competitor of eIF5, interfering with the key steps by which eIF5 regulates eIF2 function.

RNA interference and the microRNA (miRNA) pathway can induce sequence-specific mRNA degradation and/or translational repression. The human genome encodes hundreds of miRNAs that can post-transcriptionally repress thousands of genes. Using reporter constructs, we observed that degradation of mRNAs bearing sites imperfectly complementary to the endogenous let-7 miRNA is considerably stronger in human HEK293 than HeLa cells. The degradation did not result from the Ago2-mediated endonucleolytic cleavage but it was Dicer- and Ago2-dependent. We used this feature of HEK293 to address the size of a pool of transcripts regulated by RNA silencing in a single cell type. We generated HEK293 cell lines depleted of Dicer or individual Ago proteins. The cell lines were used for microarray analyses to obtain a comprehensive picture of RNA silencing. The 3'-untranslated region sequences of a few hundred transcripts that were commonly up-regulated upon Ago2 and Dicer knock-downs showed a significant enrichment of putative miRNA-binding sites. The up-regulation upon Ago2 and Dicer knock-downs was moderate and we found no evidence, at the mRNA level, for activation of silenced genes. Taken together, our data suggest that, independent of the effect on translation, miRNAs affect levels of a few hundred mRNAs in HEK293 cells.

Interferon-alpha (IFNalpha) can induce apoptosis, a process regulated by a complex network of cell factors. Among these, eukaryotic initiation factor-5A (eIF-5A) is peculiar because its activity is modulated by the post-translational formation of the amino acid hypusine. Here we report the effects of IFNalpha and epidermal growth factor (EGF) on apoptosis and eIF-5A activity in human epidermoid oropharyngeal KB and lung H1355 cancer cells. We found that 48-h exposure to 1000 and 2000 IU/ml IFNalpha induced about 50% growth inhibition and apoptosis in H1355 and KB cells, respectively, and the addition of EGF completely antagonized this effect. When IFNalpha induced apoptosis, a hyperactivation of MEK-1 and ERK signalling and a decrease of the hypusine-containing form and, thus, of eIF-5A activity were recorded. The latter effect was again antagonized by the addition of EGF to IFNalpha-pretreated cells, probably through the activation of the EGF-->ERK-dependent pathway, since the addition of the specific MEK-1 inhibitor PD098059 abrogated the recovery of intracellular hypusine content induced by EGF in IFNalpha-pretreated cancer cells. Subsequently, we evaluated if the hypusine synthesis inhibitor (and eIF-5A inactivator) N1-guanyl-1,7-diaminoheptane (GC7) synergized with IFNalpha in the induction of cell growth inhibition and apoptosis. The analysis of the isobologram of IFNalpha and GC7 demonstrated a strong synergism between the two drugs in inducing cell growth inhibition. We also found that GC7 and IFNalpha had a synergistic effect on apoptosis. These data suggest that the apoptosis induced by IFNalpha could be regulated by eIF-5A that, therefore, could represent a useful target for the potentiation of IFNalpha antitumor activity.