Defining the protein complexome of translation termination factor eRF1: Identification of four novel eRF1-containing complexes that range from 20S to 57S in size.

The eukaryotic eRF1 translation termination factor plays an important role in recognizing stop codons and initiating the end to translation. However, which exact complexes contain eRF1 and at what abundance is not clear. We have used analytical ultracentrifugation with fluorescent detection system to identify the protein complexome of eRF1 in the yeast Saccharomyces cerevisiae. In addition to eRF1 presence in translating polysomes, we found that eRF1 associated with five other macromolecular complexes: 77S, 57S, 39S, 28S, and 20S in size. Generally equal abundances of each of these complexes were found. The 77S complex primarily contained the free 80S ribosome consistent with in vitro studies and did not appear to contain significant levels of the monosomal translating complex that co-migrates with the free 80S ribosome. The 57S and 39S complexes represented, respectively, free 60S and 40S ribosomal subunits bound to eRF1, associations not previously reported. The novel 28S and 20S complexes (containing minimal masses of 830 KDa and 500 KDa, respectively) lacked significant RNA components and appeared to be oligomeric, as eRF1 has a mass of 49 KDa. The majority of polysomal complexes containing eRF1 were both substantially deadenylated and lacking in closed-loop factors eIF4E and eIF4G. The thirteen percent of such translating polysomes that contained poly(A) tails had equivalent levels of eIF4E and eIF4G, suggesting these complexes were in a closed-loop structure. The identification of eRF1 in these unique and previously unrecognized complexes suggests a variety of new roles for eRF1 in the regulation of cellular processes.

Significance of MNK1 in prognostic prediction and chemotherapy development of epithelial ovarian cancer

Background. Ovarian cancer is the most lethal gynecologic malignancy worldwide with surgery as the only curative treatment. Long-term overall survival (OS) of ovarian cancer is far from satisfactory, even though significant improvement has been made in post-operative chemotherapy. One of the most important death cause is the chemoresistance due to consecutive chemotherapy. Therefore, understanding the molecular mechanisms involved in ovarian cancer development and identification of novel therapeutic targets are urgently required. Methods. Immunohistochemical (IHC) staining was used to explore the expression pattern of mitogen-activated protein kinase (MAPK)-interacting kinase 1 (MNK1) in tumor tissues from 138 epithelial ovarian cancer (EOC) patients. Clinicopathological data were subjected to Kaplan-Meier survival and Cox multivariate analyses to evaluate the prognostic value of MNK1 in EOC. Overexpression and silencing procedures were performed on OVCAR-5 cells to investigate the mechanisms of MNK1 in regulating EOC development. The anti-tumor effects of CGP57380, a specific MNK inhibitor, were examined by cell viability assay. Results. Higher MNK1 expression showed significant relationship with advanced FIGO stage and positive lymph node metastasis of EOC. Univariate and multivariate analyses revealed that MNK1 was an independent prognostic factor for OS of EOC patients. In vitro study demonstrated that MNK1 can promote cell proliferation through regulating the phosphorylation level of eukaryotic initiation factor 4E. In addition, inhibition of MNK1 by CGP57380 significantly down-regulated the OVCAR-5 cell viability. Conclusion. High MNK1 expression in EOC tissues indicates poor clinical outcomes, and MNK1 can act as a potential target for novel chemotherapy development towards EOC (AU)

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Distinct recruitment of human eIF4E isoforms to processing bodies and stress granules.

BACKGROUND: Eukaryotic translation initiation factor 4E (eIF4E) plays a pivotal role in the control of cap-dependent translation initiation, modulates the fate of specific mRNAs, occurs in processing bodies (PBs) and is required for formation of stress granules (SGs). In this study, we focused on the subcellular localization of a representative compendium of eIF4E protein isoforms, particularly on the less studied members of the human eIF4E protein family, eIF4E2 and eIF4E3. RESULTS: We showed that unlike eIF4E1, its less studied isoform eIF4E3_A, encoded by human chromosome 3, localized to stress granules but not PBs upon both heat shock and arsenite stress. Furthermore, we found that eIF4E3_A interacts with human translation initiation factors eIF4G1, eIF4G3 and PABP1 in vivo and sediments into the same fractions as canonical eIF4E1 during polysome analysis in sucrose gradients. Contrary to this finding, the truncated human eIF4E3 isoform, eIF4E3_B, showed no localization to SGs and no binding to eIF4G. We also highlighted that eIF4E2 may exhibit distinct functions under different stresses as it readily localizes to P-bodies during arsenite and heat stresses, whereas it is redirected to stress granules only upon heat shock. We extended our study to a number of protein variants, arising from alternative mRNA splicing, of each of the three eIF4E isoforms. Our results surprisingly uncovered differences in the ability of eIF4E1_1 and eIF4E1_3 to form stress granules in response to cellular stresses. CONCLUSION: Our comparison of all three human eIF4E isoforms and their protein variants enriches the intriguing spectrum of roles attributed to the eukaryotic initiation translation factors of the 4E family, which exhibit a distinctive localization within different RNA granules under different stresses. The localization of eIF4E3_A to stress granules, but not to processing bodies, along with its binding to eIF4G and PABP1 suggests a role of human eIF4E3_A in translation initiation rather than its involvement in a translational repression and mRNA decay and turnover. The localization of eIF4E2 to stress granules under heat shock but not arsenite stress indicates its distinct function in cellular response to these stresses and points to the variable protein content of SGs as a consequence of different stress insults.

Multiple myeloma and bone marrow mesenchymal stem cells' crosstalk: Effect on translation initiation.

Multiple myeloma (MM) malignant plasma cells reside in the bone marrow (BM) and convert it into a specialized pre-neoplastic niche that promotes the proliferation and survival of the cancer cells. BM resident mesenchymal stem cells (BM-MSCs) are altered in MM and in vitro studies indicate their transformation by MM proximity is within hours. The response time frame suggested that protein translation may be implicated. Thus, we assembled a co-culture model of MM cell lines with MSCs from normal donors (ND) and MM patients to test our hypothesis. The cell lines (U266, ARP-1) and BM-MSCs (ND, MM) were harvested separately after 72 h of co-culture and assayed for proliferation, death, levels of major translation initiation factors (eIF4E, eIF4GI), their targets, and regulators. Significant changes were observed: BM-MSCs (ND and MM) co-cultured with MM cell lines displayed elevated proliferation and death as well as increased expression/activity of eIF4E/eIF4GI; MM cell lines co-cultured with MM-MSCs also displayed higher proliferation and death rates coupled with augmented translation initiation factors; in contrast, MM cell lines co-cultured with ND-MSCs did not display elevated proliferation only death and had no changes in eIF4GI levels/activity. eIF4E expression was increased in one of the cell lines. Our study demonstrates that there is direct dialogue between the MM and BM-MSCs populations that includes translation initiation manipulation and critically affects cell fate. Future research should be aimed at identifying therapeutic targets that may be used to minimize the collateral damage to the cancer microenvironment and limit its recruitment into the malignant process. © 2015 Wiley Periodicals, Inc.

Immunohistochemical characterization of the mTOR pathway in stage-I non-small-cell lung carcinoma.

BACKGROUND: Dysregulation of mammalian target of rapamycin (mTOR) pathway has been linked with malignant tumorigenesis. This study explored the expression profiles of proteins involved in the mTOR pathway and their relationships with clinicopathologic characteristics in stage-I non-small-cell lung carcinoma (NSCLC). METHODS: The protein expression profiles of PTEN, p-Akt, p-mTOR, p-S6, and eIF4E were examined using immunohistochemical staining and tissue microarray method in 408 patients with stage-I NSCLC (250 adenocarcinomas [ADC] and 158 squamous cell carcinomas). RESULTS: Retained PTEN expression (P<0.001), p-mTOR expression (P<0.001), and p-S6 expression (P=0.007) were associated with ADC histology. Expression of PTEN (P=0.001), p-Akt (P=0.005), p-mTOR (P=0.007), p-S6 (P<0.001) were correlated with lower pathologic T stage. PTEN loss was correlated with male gender and smoking history and p-mTOR expression was inversely correlated with these factors (P<0.001). Subgroup analysis of ADCs indicated that male gender, high pT stage, lymphovascular invasion, and PTEN loss were poor prognostic factors. Multivariate analysis revealed that the PTEN(-)/p-Akt(+)/p-mTOR(+) combination more effectively determined the prognosis of ADC (hazard ratio=2.2, P=0.004) than PTEN alone. CONCLUSIONS: Activation of the mTOR pathway in early-stage ADCs suggests a significant role for the mTOR axis in early carcinogenesis. The combination of PTEN(-)/p-Akt(+)/p-mTOR(+) expression was correlated with poor overall survival in patients with stage-I lung ADC.

Translation initiation complex eIF4F is a therapeutic target for dual mTOR kinase inhibitors in non-Hodgkin lymphoma.

Deregulated mRNA translation has been implicated in disease development and in part is controlled by a eukaryotic initiation complex eIF4F (composed of eIF4E, eIF4G and eIF4A). We demonstrate here that the cap bound fraction from lymphoma cells was enriched with eIF4G and eIF4E indicating that lymphoma cells exist in an activated translational state. Moreover, 77% (110/142) of diffuse large B cell lymphoma tumors expressed eIF4E and this was associated with an inferior event free survival. Over-expression of wild-type eIF4E (eIF4E(WT)) but not cap-mutant eIF4E (eIF4E(cap mutant)) increased the activation of the eIF4F complex. Treatment with the active-site dual mTOR inhibitor CC214-1 reduced the level of the eIF4F complex by decreasing the cap bound fraction of eIF4G and increasing the levels of 4E-BP1. CC214-1 inhibited both the cap dependent and global protein translation. CC214-1 inhibited c-Myc, and cyclin D3 translation by decreasing polysomal fractions from lymphoma cells. Inhibition of eIF4E with shRNA further decreased the CC214-1 induced inhibition of the eIF4F complex, c-Myc, cyclin D3 translation, and colony formation. These studies demonstrate that the eIF4F complex is deregulated in aggressive lymphoma and that dual mTOR therapy has therapeutic potential in these patients.

Maternal low-protein diet affects myostatin signaling and protein synthesis in skeletal muscle of offspring piglets at weaning stage.

PURPOSE: We tested the hypothesis that maternal low-protein (LP) diet during gestation and lactation can program myostatin (MSTN) signaling and protein synthesis in skeletal muscle of offspring at weaning stage (35 days). METHODS: Fourteen Meishan sows were fed either LP or standard-protein diets throughout gestation and lactation, male offspring piglets were killed at weaning stage and longissimus dorsi (LD) muscles were taken. The cross-sectional areas (CSA) of LD muscles were measured by hematoxylin and eosin staining. The levels of free amino acids in plasma were measured by amino acid auto-analyzer. Proteins and mRNA were determined by Western blot and RT-qPCR, respectively. RESULTS: Body weight, LD muscle weight and the myofiber CSA were significantly decreased (P < 0.05) in LP piglets; meanwhile, the concentration of branched-chain amino acids was also significantly decreased (P < 0.001). MSTN protein content tended to be higher (P = 0.098) in LP piglets, while the expression of MSTN receptors, activin type II receptor-beta and transforming growth factor type-beta type I receptor kinase, was significantly up-regulated (P < 0.05). Furthermore, p38 mitogen-activated protein kinase, the downstream signaling factor of MSTN, was also enhanced significantly (P < 0.05). In addition, key factors of translation initiation, phosphorylated eukaryotic initiation factor 4E and the 70 kDa ribosomal protein S6 kinase, were significantly decreased (P < 0.05) in LP piglets. CONCLUSIONS: Our results suggest that maternal LP diet during gestation and lactation affects MSTN signaling and protein synthesis in skeletal muscle of offspring at weaning stage.

Eukaryotic translation initiation factor 4E (eIF4E) expression is associated with breast cancer tumor phenotype and predicts survival after anthracycline chemotherapy treatment.

Abnormal translation of mRNAs frequently occurring during carcinogenesis is among the mechanisms that can affect the expression of proteins involved in tumor development and progression. Eukaryotic initiation factor eIF4E is a key regulator of translation of many cancer-related transcripts and its expression is altered in various cancers and has been associated with worse survival. We determined the eIF4E protein levels using immunohistochemistry (IHC) in 1,233 breast tumors on tissue microarrays. We analyzed the effects of the IHC expression level on tumor characteristics and patient survival, also with stratification by adjuvant chemotherapy treatment. In 1,085 successfully stained tumors, high level of eIF4E protein expression was associated with features of aggressive tumor phenotype, namely grade, estrogen and progesterone receptor negativity, HER2 receptor positivity, and high expression of p53 and Ki67, and with triple negative subtype (p < 0.001). High eIF4E expression was associated with worse breast cancer-specific survival with a hazard ratio (HR) of 1.99 (95 % CI 1.32-3.00, p = 0.0008) and was in a multivariate analysis an independent prognostic factor. High eIF4E expression was associated with worse outcome also after detection of distant metastasis (HR = 1.88, 95 % CI 1.20-2.94, p = 0.0060). In the subgroup analysis the survival effect was strongest among patients treated with anthracycline chemotherapy (HR = 3.34, 95 % CI 1.72-6.48, p = 0.0002), whereas no such effect was seen among patients who had not received anthracycline with significant difference in heterogeneity between the two groups (p = 0.0358). High expression of eIF4E is associated with adverse tumor characteristics and predicts poor breast cancer-specific survival. This effect is emphasized in patients treated with anthracycline chemotherapy. eIF4E as a treatment predictive factor warrants further studies.

Tracking a refined eIF4E-binding motif reveals Angel1 as a new partner of eIF4E.

The initiation factor 4E (eIF4E) is implicated in most of the crucial steps of the mRNA life cycle and is recognized as a pivotal protein in gene regulation. Many of these roles are mediated by its interaction with specific proteins generally known as eIF4E-interacting partners (4E-IPs), such as eIF4G and 4E-BP. To screen for new 4E-IPs, we developed a novel approach based on structural, in silico and biochemical analyses. We identified the protein Angel1, a member of the CCR4 deadenylase family. Immunoprecipitation experiments provided evidence that Angel1 is able to interact in vitro and in vivo with eIF4E. Point mutation variants of Angel1 demonstrated that the interaction of Angel1 with eIF4E is mediated through a consensus eIF4E-binding motif. Immunofluorescence and cell fractionation experiments showed that Angel1 is confined to the endoplasmic reticulum and Golgi apparatus, where it partially co-localizes with eIF4E and eIF4G, but not with 4E-BP. Furthermore, manipulating Angel1 levels in living cells had no effect on global translation rates, suggesting that the protein has a more specific function. Taken together, our results illustrate that we developed a powerful method for identifying new eIF4E partners and open new perspectives for understanding eIF4E-specific regulation.

The involvement of eukaryotic translation initiation factor 4E in extravillous trophoblast cell function.

UNLABELLED: Extravillous trophoblast cells (EVT) are major players in placental implantation. They differentiate in the villous cell column, invade to the uterus and remodel the uterine spiral arteries. Trophoblast and tumor cells have similar invasion mechanisms, share similar biochemical mediators (e.g. c-myc, MMP9) and growth-factors (e.g. VEGF). The mRNA of these proteins has extremely structured 5-UTR and their translation is highly dependent on eukaryotic-translation-initiation-factor-4E (eIF4E). Cancer cells have elevated eIF4E and are more vulnerable to its silencing than normal cells. We speculated that like cancer, trophoblast function is highly eIF4E dependent. OBJECTIVE: Analyze eIF4E involvement in EVT differentiation and function. STUDY DESIGN: EIF4E levels were assessed in first-trimester human placentae and in placental explants before and after EVT differentiation. The effect of eIF4E knockdown (siRNA, ribavirin) on the phenotype of placental explant and EVT cell lines (HTR-8/SVNEO) was evaluated. Tested parameters included eIF4E and its target levels, migration, invasion, cell death, cell cycle and cell count. RESULTS: High eIF4E levels were found in cytotrophoblast and especially EVT cells during their differentiation in the villi, compared to other placental cell types. EIF4E silencing increased cell death and cell cycle arrest in placental explants and HTR-8/SVNEO cells. Although it induced EVT outgrowth in the placental explants, it reduced HTR-8/SVNEO motility, reflecting the importance of using ex vivo models that include an intact placental microenvironment in its original architecture. CONCLUSIONS: Our results suggest that eIF4E prevents final EVT differentiation and supports placental cell proliferation and survival. A balance between cell proliferation and differentiation is crucial for placental development and implantation.

Mapping protein surface accessibility via an electron transfer dissociation selectively cleavable hydrazone probe.

A protein's surface influences its role in protein-protein interactions and protein-ligand binding. Mass spectrometry can be used to give low resolution structural information about protein surfaces and conformations when used in combination with derivatization methods that target surface accessible amino acid residues. However, pinpointing the resulting modified peptides upon enzymatic digestion of the surface-modified protein is challenging because of the complexity of the peptide mixture and low abundance of modified peptides. Here a novel hydrazone reagent (NN) is presented that allows facile identification of all modified surface residues through a preferential cleavage upon activation by electron transfer dissociation coupled with a collision activation scan to pinpoint the modified residue in the peptide sequence. Using this approach, the correlation between percent reactivity and surface accessibility is demonstrated for two biologically active proteins, wheat eIF4E and PARP-1 Domain C.

Predictive value of eIF4E reduction after neoadjuvant therapy in breast cancer.

INTRODUCTION: Initiation factor 4E (eIF4E) overexpression has prognostic significance in breast cancer. Up-regulation of downstream gene products associated with high eIF4E overexpression has been linked to chemoresistance. We hypothesize patients whose tumors had eIF4E reduction after neoadjuvant chemotherapy will have lower cancer recurrence compared with those who did not. METHODS: Seventeen locally advanced breast cancer patients were accrued, and tumor specimens were obtained before and after neoadjuvant therapy. eIF4E was quantified by Western blots. Primary end-point was cancer recurrence. RESULTS: Low eIF4E was defined as < or =7.5-fold elevation and high eIF4E was >7.5-fold elevation. Of 17 patients, six tumors remained low after neoadjuvant therapy, six dropped from high to low eIF4E, and five remained high. With a median follow-up of 29 mo, four of five patients with tumors that remained high have recurred while only three of 12 patients in the low eIF4E post-therapy group have recurred (P=0.05, chi(2)). Survival analysis using the Kaplan-Meier method showed a higher rate of cancer recurrence in patients with post-treatment high eIF4E overexpression (P=0.026, log rank test). CONCLUSIONS: Breast cancer patients whose tumors had low eIF4E overexpression after neoadjuvant chemotherapy had lower cancer recurrence compared with those who did not.

Phosphorylated eukaryotic translation initiation factor 4 (eIF4E) is elevated in human cancer tissues.

Eukaryotic translation initiation factor 4E (eIF4E) is a rate-limiting factor for cap-dependent protein synthesis and is regulated by PI3 kinase/mTOR and mitogen-activated protein kinase (MAPK)/Mnk signaling pathways. Recent studies have shown that Mnk-mediated eIF4E phosphorylation is absolutely required for eIF4E's oncogenic function. Overexpression of eIF4E has been reported in many types of cancers; however, the expression of phosphorylated eIF4E (p-eIF4E) in human cancer tissues, particularly solid tumor tissues, has not been reported. The current study focused on evaluating p-eIF4E expression patterns in the tumor tissues obtained from patients with a variety of malignancies. Using three different tissue microarrays consisting of a total of 380 cases of human cancers and 146 cases of adjacent normal tissues, we detected p-eIF4E positive staining in 63.4% (241/380) of cancers, but only in 30.1% (44/146) of adjacent normal tissues. Thus, p-eIF4E expression is significantly higher in cancers than in adjacent normal tissues (p < 0.001). In general, there was no major difference in p-eIF4E staining between cancers with and without lymph node metastasis. In certain types of maligancies such as lung, gastric and colorectal cancers, p-eIF4E staining was significantly higher in the early stage (T1) than in the late stage (T3) disease (p < 0.05). Collectively, these findings suggest that p-eIF4E may play a critical role in cancer development, particularly early stages of tumorigenesis and support p-eIF4E as a good cancer therapeutic target.

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs.

Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.

Dietary arginine supplementation increases mTOR signaling activity in skeletal muscle of neonatal pigs.

Dietary arginine supplementation increases growth of neonatal pigs, but the underlying mechanisms are unknown. This study was conducted to test the hypothesis that the arginine treatment activates translation initiation factors and protein synthesis in skeletal muscle. Piglets were fed milk-based diets supplemented with 0 or 0.6% L-arginine between 7 and 14 d of age. Following a 7-d period of arginine supplementation, at 1 h after the last meal, jugular venous blood samples were obtained for metabolite analysis, whereas longissimus muscle and liver were collected to determine the abundance and phosphorylation state of the mammalian target of the rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1), eIF4E, and eIF4G. Fractional rates of protein synthesis were measured in muscle and liver using the [(3)H]phenylalanine flooding-dose technique. Arginine supplementation increased (P < 0.05) daily gain, the plasma insulin concentration, and protein synthesis in skeletal muscle but not in liver. The arginine treatment enhanced the formation of the active eIF4E x eIF4G complex but reduced the amount of the inactive 4E-BP1 x eIF4E complex in muscle. These changes were associated with elevated levels of phosphorylated mTOR and 4E-BP1 in muscle of arginine-supplemented piglets (P < 0.05). Neither the total amounts nor phosphorylation levels of the translation initiation factors in the liver differed between control and arginine-supplemented piglets. Collectively, these results suggest that dietary arginine supplementation increases mTOR signaling activity in skeletal muscle, but not in liver, of milk-fed neonatal pigs. The findings provide a molecular mechanism for explaining the previous observation that increased circulating arginine stimulated muscle protein synthesis and promoted weight gain in neonatal pigs.

Chromium(III) triggers the DNA-damaged checkpoint of the cell cycle and induces a functional increase of 4E-BP.

Using sea urchin early embryos as a pertinent model, chromium(III) provoked cell cycle arrest and induced apoptosis. The molecular machinery of translation initiation was investigated. Chromium provoked a time- and dose-dependent increase in the level of 4E-BP protein, the natural regulator of the cap-dependent initiation factor 4E (eIF4E). The 4E-BP increase was the result of 4E-BP stabilization and appeared functional for physiological eIF4E binding, removal of eIF4E from the initiation factor eIF4G, and almost full inhibition of cap-dependent translation in vivo. The protein 4E-BP may be involved in the biological pathway of apoptosis associated with the activation of the DNA-damaged checkpoint of the cell cycle.

Initiation factors for translation of proteins in the rectus abdominis muscle from patients on overnight standard parenteral nutrition before surgery.

Previous studies have provided conflicting conclusions concerning the efficacy of improving protein balance in patients by standard intravenous nutrition [TPN (total parenteral nutrition)], which is either explained by suboptimal nutritional regimens or insensitive clinical methods. The aim of the present study was therefore to evaluate the effects on the initiation of translation of skeletal muscle proteins by standard overnight TPN. A total of 12 patients who underwent standard surgery were included. TPN was provided as an all-in-one treatment by constant infusion [0.16 gN.kg(-1) of body weight.day(-1) (30 kcal.kg(-1) of body weight.day(-1))]. Saline-infused patients served as controls. Rectus abdominis muscle biopsies were taken at the time of the operation. The phosphorylation state of the proteins for initiation of translation was quantified. Plasma glucose, and serum insulin, glycerol, triacylglycerols (triglycerides) and NEFAs (non-esterified fatty acids; 'free fatty acids') were not significantly altered during TPN infusion, whereas total plasma amino acids increased, as shown by increases in methionine, phenylalanine, threonine, alanine, arginine, aspartic acid, glycine and histidine (P<0.05). Overnight TPN increased the formation of active eIF4G-eIF4E (where eIF is eukaryotic-initiation factor) complexes (P<0.05), whereas the inhibitory complex 4E-BP1 (eIF4E-binding protein)-eIF4E was moderately decreased (P<0.06). TPN increased the amount of the most phosphorylated form of 4E-BP1 (P<0.05), and increased the amount (P<0.04) and phosphorylation (P<0.01) of p70(S6K) (70 kDa ribosomal protein S6 kinase). In conclusion, an overnight pre-operative constant infusion of standard TPN altered initiation factor complexes, indicating activation of the initiation of protein translation in rectus abdominis muscle in the presence of increased plasma amino acid levels, but without a concomitant increase in energy substrates and insulin. In contrast with our results from previous studies, the methodology used in the present study appears to be more sensitive in reflecting directional changes in human muscle protein synthesis compared with traditional methods, particularly based on measurements of amino acid flux.

High eIF4E, VEGF, and microvessel density in stage I to III breast cancer.

OBJECTIVE: In a prospective trial, to determine if eIF4E overexpression in breast cancer specimens is correlated with VEGF elevation, increased tumor microvessel density (MVD) counts, and a worse clinical outcome irrespective of nodal status. SUMMARY AND BACKGROUND DATA: In vitro, the overexpression of eukaryotic initiation factor 4E (eIF4E) up-regulates the translation of mRNAs with long 5'-untranslated regions (5'-UTRs). One such gene product is the vascular endothelial growth factor (VEGF). METHODS: A total of 114 stage I to III breast cancer patients were prospectively accrued and followed with a standardized clinical surveillance protocol. Cancer specimens were quantified for eIF4E, VEGF, and MVD. Outcome endpoints were cancer recurrence and cancer-related death. RESULTS: eIF4E overexpression was found in all cancer specimens (mean +/- SD, 12.5 +/- 7.6-fold). Increasing eIF4E overexpression correlated with increasing VEGF elevation (r = 0.24, P = 0.01, Spearman's coefficient), and increasing MVD counts (r = 0.35, P < 0.0002). Patients whose tumor had high eIF4E overexpression had shorter disease-free survival (P = 0.004, log-rank test) and higher cancer-related deaths (P = 0.002) than patients whose tumors had low eIF4E overexpression. Patients with high eIF4E had a hazard ratio for cancer recurrence and cancer-related death of 1.8 and 2.1 times that of patients with low eIF4E (respectively, P = 0.009 and P = 0.002, Cox proportional hazard model). CONCLUSIONS: In breast cancer patients, increasing eIF4E overexpression in the cancer specimens correlates with higher VEGF levels and MVD counts. Patients whose tumors had high eIF4E overexpression had a worse clinical outcome, independent of nodal status. Thus, eIF4E overexpression in breast cancer appears to predict increased tumor vascularity and perhaps cancer dissemination by hematogenous means.

eIF4E as a marker for cervical neoplasia.

Eukaryotic translation initiation factor 4E (eIF4E) is upregulated in cancers of the breast and head and neck. The authors have shown that eIF4E is increased in cervical neoplasia and that eIF4E upregulates human papillomavirus (HPV) oncoprotein E7. The aim of this study was to quantitate eIF4E in tissues representing a wide range of cervical pathology. The potential correlation between dysplastic grade or tumor stage with eIF4E upregulation and/or HPV genotype was analyzed for 10 normal, 27 cancer, and 37 dysplasia cases. A progressive increase in eIF4E staining intensity was found with increasing cervical pathology (P < 0.0001). No difference was seen in eIF4E stain intensity by either tumor type--squamous cell cancer (n = 18), adenocarcinoma (n = 4), or other types of cancer (n = 5) (P = 0.97)--or by tumor grade--II (n = 5) versus III (n = 7). Likewise, neither an HPV typing result of HPV 16 (n = 10) versus non-HPV 16 (n = 4) nor single HPV infection (n = 11) versus dual HPV infection (n = 3) significantly altered the eIF4E stain results (P = 0.86 and 0.97, respectively). These results indicate that eIF4E stain intensity may be useful as a marker for cervical neoplasia.

Identification in the ancient protist Giardia lamblia of two eukaryotic translation initiation factor 4E homologues with distinctive functions.

Eukaryotic translation initiation factor 4E (eIF4E) binds to the m(7)GTP of capped mRNAs and is an essential component of the translational machinery that recruits the 40S small ribosomal subunit. We describe here the identification and characterization of two eIF4E homologues in an ancient protist, Giardia lamblia. Using m(7)GTP-Sepharose affinity column chromatography, a specific binding protein was isolated and identified as Giardia eIF4E2. The other homologue, Giardia eIF4E1, bound only to the m(2,2,7)GpppN structure. Although neither homologue can rescue the function of yeast eIF4E, a knockdown of eIF4E2 mRNA in Giardia by a virus-based antisense ribozyme decreased translation, which was shown to use m(7)GpppN-capped mRNA as a template. Thus, eIF4E2 is likely the cap-binding protein in a translation initiation complex. The same knockdown approach indicated that eIF4E1 is not required for translation in Giardia. Immunofluorescence assays showed wide distribution of both homologues in the cytoplasm. But eIF4E1 was also found concentrated and colocalized with the m(2,2,7)GpppN cap, 16S-like rRNA, and fibrillarin in the nucleolus-like structure in the nucleus. eIF4E1 depletion from Giardia did not affect mRNA splicing, but the protein was bound to Giardia small nuclear RNAs D and H known to have an m(2,2,7)GpppN cap, thus suggesting a novel function not yet observed among other eIF4Es in eukaryotes.