Bevacizumab attenuates major signaling cascades and eIF4E translation initiation factor in multiple myeloma cells

Multiple myeloma (MM), a malignancy of plasma cells, remains fatal despite introduction of novel therapies, partially due to humoral factors, including vascular endothelial growth factor (VEGF), in their microenvironment. The aim of this study was to explore the efficacy of anti-VEGF treatment with bevacizumab directly on MM cells. Particular attention was directed to the affect of VEGF inhibition on protein translation initiation. Experiments were conducted on MM cells (lines, bone marrow (BM) samples) cultured on plastic. Inhibition of VEGF was achieved with the clinically employed anti-VEGF antibody, bevacizumab, as a platform and its consequences on viability, proliferation, and survival was assessed. VEGF downstream signals of established importance to MM cell biology were assayed as well, with particular emphasis on translation initiation factor eIF4E. We showed that blocking VEGF is deleterious to the MM cells and causes cytostasis. This was evidenced in MM cell lines, as well as in primary BM samples (BM MM). A common bevacizumab-induced attenuation of critical signaling effectors was determined: VEGFR1, mTOR, c-Myc, Akt, STAT3, (cell lines) and eIF4E translation initiation factor (lines and BM). ERK1/2 displayed a variegated response to bevacizumab (lines). Utilizing a constitutively Akt-expressing MM model, we showed that the effect of bevacizumab on viability and eIF4E status is Akt-dependent. Of note, the effect of bevacizumab was achieved with high concentrations (2 mg/ml), but was shown to be specific. These findings demonstrate that bevacizumab has a direct influence on major pathways critically activated in MM that is independent from its established effect on angiogenesis. The cytostatic effect of VEGF inhibition on MM cells underscores its potential in combined therapy, and our findings, regarding its influence on translation initiation, suggest that drugs that unbalance cellular proteostasis may be particularly effective.     

Functional characterization of the translation initiation factor eIF4E of Echinococcus granulosus

Parasitology Research - The eukaryotic initiation factor 4E (eIF4E) specifically recognizes the 5′ mRNA cap, a rate-limiting step in the translation initiation process. Although the...     

ISG15 modification of the eIF4E cognate 4EHP enhances cap structure-binding activity of 4EHP

The expression of the ubiquitin-like molecule ISG15 and protein modification by ISG15 (ISGylation) are strongly activated by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. 4EHP is an mRNA 5' cap structure-binding protein and acts as a translation suppressor by competing with eIF4E for binding to the cap structure. Here, we report that 4EHP is modified by ISG15 and ISGylated 4EHP has a much higher cap structure-binding activity. These data suggest that ISGylation of 4EHP may play an important role in cap structure-dependent translation control in immune responses.     

Neutralizing innate host defenses to control viral translation in HSV-1 infected cells

Lytic replication of many viruses activates an innate host response designed to prevent the completion of the viral lifecycle, thus impeding the spread of the infection. One branch of the host's complex reaction functions to incapacitate the cellular translational machinery on which the synthesis of viral polypeptides completely depends. This is achieved through the activation of specific protein kinases that phosphorylate eIF2 on its alpha subunit and inactivate this critical translation initiation factor. However, as continued synthesis of viral proteins is required to assemble the viral progeny necessary to transmit the infection to neighboring cells, viruses have developed a variety of strategies to counter this cellular response. Genetic and biochemical studies with herpes simplex virus type 1 (HSV-1) have revealed that the virus produces at least two discrete products at different times during its replicative program that act to prevent the accumulation of phosphorylated eIF2alpha. The gamma(1)34.5 gene product is expressed first, encoding a regulatory subunit that binds the cellular protein phosphatase 1alpha and regenerates pools of active eIF2 by removing the inhibitory phosphate from the alpha subunit. The second function, encoded by the product of the Us11 gene, specifies a double-stranded RNA-binding protein that prevents activation of PKR, a cellular eIF2alpha kinase. Together, both proteins cooperate to overcome the antiviral response of the host and properly regulate translation in HSV-1-infected cells.     

Dissecting eIF4E action in tumorigenesis

Genetically engineered mouse models are powerful tools for studying cancer genes and validating targets for cancer therapy. We previously used a mouse lymphoma model to demonstrate that the translation initiation factor eIF4E is a potent oncogene in vivo. Using the same model, we now show that the oncogenic activity of eIF4E correlates with its ability to activate translation and become phosphorylated on Ser 209. Furthermore, constitutively activated MNK1, an eIF4E Ser 209 kinase, promotes tumorigenesis in a manner similar to eIF4E, and a dominant-negative MNK mutant inhibits the in vivo proliferation of tumor cells driven by mutations that deregulate translation. Phosphorylated eIF4E promotes tumorigenesis primarily by suppressing apoptosis and, accordingly, the anti-apoptotic protein Mcl-1 is one target of both phospho-eIF4E and MNK1 that contributes to tumor formation. Our results provide insight into how eIF4E contributes to tumorigenesis and pinpoint a level of translational control that may be suitable for therapeutic intervention.     

Oxidant-induced hypertrophy of A549 cells is accompanied by alterations in eukaryotic translation initiation factor 4E and 4E-binding protein-1

Control of protein synthesis resides at the level of eukaryotic translation initiation (eIF) complex formation. Complex formation is regulated by the mRNA cap-binding protein, eIF4E, whose activity is influenced by phosphorylation and binding to 4E-binding protein 1 (4E-BP1). To provide a link between alterations in protein synthesis and the pathogenesis of oxidant-mediated lung disease, we investigated the effect of hydrogen peroxide (H2O2) on actively growing A549 cells. Cells were exposed to 200 or 400 microM H2O2 for 4 h and then assessed for changes in proliferation, protein synthesis, and eIF4E and 4E-BP1 status over 72 h. We found that both concentrations of H2O2 inhibited [3H]thymidine incorporation and cell division while inducing a G2/M-predominant growth arrest within 24 h. In addition, H2O2 increased cell size, [3H]leucine incorporation/cell, and total cell protein. Although time had little effect on eIF4E and 4E-BP1 expression and phosphorylation state of control cells, H2O2 induced a 2- to 3-fold increase in eIF4E and 4E-BP1 expression, a 5-fold increase in eIF4E phosphorylation, and a shift in the distribution of 4E-BP1 phosphorylation favoring lesser phosphorylated forms. These findings suggest that oxidant-mediated alterations in protein synthesis and cell morphology occur in concert with changes in factors known to regulate translation kinetics.     

The eukaryotic translation initiation factor eIF4E in the nucleus: taking the road less traveled

The eukaryotic translation initiation factor eIF4E is a potent oncogene. Although eIF4E has traditional roles in translation initiation in the cytoplasm, it is also found in the nucleus, suggesting that it has activities beyond its role in protein synthesis. The road less traveled has been taken to study these nuclear activities and to understand their contribution to the oncogenic potential of eIF4E. The molecular features and biological pathways underpinning eIF4E's nuclear mRNA export are described. New classes of eIF4E regulators have been identified and their relevance to cancer shown. The studies presented here reveal the molecular, biophysical, and structural bases for eIF4E regulation. Finally, recent clinical work targeting eIF4E in acute myeloid leukemia patients with ribavirin is discussed. In summary, these findings provide a novel paradigm for eIF4E function and the molecular basis for targeting it in leukemia patients.     

Effect of recombinant hybrid human interferon on replication and morphogenesis of HSV-1 in monkey cells

Human recombinant alpha interferon, A/D, significantly reduced the replication and cell fusion induced by herpes simplex virus type 1 in monkey cells. Thin-section electron microscopy of interferon-treated monkey cells showed distinct assembly of nucleocapsids within the nucleus. Analysis of virus-specific proteins by the immunoblot technique confirmed that A/D interferon had no significant effect on the expression of major nucleocapsid proteins, although the expression of glycoproteins B and D was reduced in interferon-treated cells. The possibility of an interferon-induced block at a late stage in virus morphogenesis is discussed.     

Functional impairment of eIF4A and eIF4G factors correlates with inhibition of influenza virus mRNA translation

Influenza virus mRNAs contain a 5′-cap structure followed by short cell-derived heterogeneous oligonucleotides and they are polyadenylated. However, selective translation of viral mRNAs occurs upon infection. Thus, we have studied whether differential requirements for the eIF4F components on viral and cellular translation could mediate this selectivity. We have previously reported that influenza virus infection proceeds efficiently upon functional impairment of the cap-binding factor eIF4E. Now, the requirements for the eIF4A helicase and the eIF4G scaffolding factor have been examined. The two proteins are essential for viral translation both in in vivo and in vitro analysis. Consequently, viral mRNAs do not contain cis-acting signals that could mediate eIF4A and eIF4G independence and trans-acting viral proteins do not replace their function. Thus, eIF4A and eIF4G proteins are not responsible for the selective translation of viral mRNAs and the translational shut-off of cellular protein synthesis observed in influenza virus infected cells.     

Phosphorylation of translation initiation factor eIF2alpha in the brain during pilocarpine-induced status epilepticus in mice

In this work, we show extensive phosphorylation of the alpha subunit of translation initiation factor 2 (eIF2alpha) occurring in the brain of mice subjected to 30 min of status epilepticus induced by pilocarpine. eIF2alpha(P) immunoreactivity was detected in the hippocampal pyramidal layer CA1 and CA3, cortex layer V, thalamus and amygdala. After 2 h of recovery, there was a marked decrease in total brain eIF2alpha(P), with the cortex layer V showing the most pronounced loss of anti-eIF2alpha(P) labeling, whereas the CA1 subregion had a significant increase in eIF2alpha(P). These results indicate that inhibition of protein synthesis in experimental models of epilepsy might be due to low levels of eIF2-GTP caused by the phosphorylation of eIF2alpha, and suggest that translational control may contribute to cell fate in the affected areas.     

Acute adrenal infection by HSV-1: role of apoptosis in viral replication

Summary.  Replication of herpes simplex virus type 1 (HSV-1) in the adrenal gland of mice was observed 12 h after intravenous inoculation, peaked at 48 h (7 × 10⁷ PFU/tissue), and was maintained until death. Virus spread to the bilateral intermediolateral column of the thoracic spinal cord. Infected cells appeared in the fascicular zone of the adrenal cortex 12 h after infection, and cell death was evident in lesions found in the adrenal cortex. Lesions involved the medulla 48 h after inoculation. In cortical lesions, cell nuclei were fragmented or shrunken with little damage to the cytoplasm. DNA fragmentation appeared 12 h after inoculation and increased mainly in cortical lesions, which were characterized by apoptosis induced by HSV-1 infection. In the adrenal medulla, cells were fused and formed multinucleated giant cells but rarely displayed cell death. Macrophages, which serve as a frontal barrier to viral infection in the adrenal gland, especially the cortex, were fewer in number than those found in the liver or spleen. It is likely that HSV-1 easily infects the adrenal gland, resulting in suppression of local immunity, and that adrenal cell apoptosis serves as a primitive type of immunity to limit viral replication. Received March 6, 2000 Accepted August 23, 2000     

Influenza virus polymerase confers independence of the cellular cap-binding factor eIF4E for viral mRNA translation

The influenza virus mRNAs are structurally similar to cellular mRNAs nevertheless; the virus promotes selective translation of viral mRNAs despite the inhibition of host cell protein synthesis. The infection proceeds normally upon functional impairment of eIF4E cap-binding protein, but requires functional eIF4A helicase and eIF4G factor. Here, we have studied whether the presence of cis elements in viral mRNAs or the action of viral proteins is responsible for this eIF4E-independence. The eIF4E protein is required for viral mRNA translation in vitro, indicating that cis-acting RNA sequences are not involved in this process. We also show that PB2 viral polymerase subunit interacts with the eIF4G protein. In addition, a chimeric mRNA containing viral UTR sequences transcribed by the viral polymerase out of the infection is successfully translated independently of an impaired eIF4E factor. These data support that the viral polymerase is responsible for the eIF4E independence of influenza virus mRNA translation.     

Dendritic eIF4E-binding Protein 1 (eIF4E-BP1) mRNA Is Upregulated by Neuronal Activation

Long-term synaptic plasticity requires addition of new proteins at the synaptic site. The local protein synthesis at subsynaptic sites confers advantageous mechanisms that would regulate the protein composition in local domains on a moment-by-moment basis. However, our information on the identities of ''dendritic'' mRNAs is very limited. In this study we investigated the expression of the protein and mRNA for eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) in cultured rat hippocampal neurons. Immunocytochemistry (ICC) showed that 4EBP1 protein is highly localized to the nucleus. In dendrites most 4EBP1 punctae were not colocalized with those of eIF4E. In situ hybridization (ISH) and Fluorescence ISH (FISH) revealed that 4EBP1 mRNA was present in dendrites. The FISH signals formed clusters along dendrites that colocalized with ICC signals for Staufen, a marker for RNA granules. The neuronal activation by KCl (60 mM, 10 min) significantly increased the density of 4EBP1 FISH signals in the nucleus after 2 hr, and both in the nucleus and dendrites after 6 hr. Our results indicate that 4EBP1 and its mRNA are present in dendrites, and the mRNA is upregulated and transported to dendritic domains in RNA granules upon neuronal activation.     

Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E

The coordinated action of cell cycle progression and cell growth (an increase in cell size and cell mass) is critical for sustained cellular proliferation, yet the biochemical signals that control cell growth are poorly defined, particularly in mammalian systems. We find that cell growth and cell cycle progression are separable processes in mammalian cells and that growth to appropriate cell size requires mTOR- and PI3K-dependent signals. Expression of a rapamycin-resistant mutant of mTOR rescues the reduced cell size phenotype induced by rapamycin in a kinase-dependent manner, showing the evolutionarily conserved role of mTOR in control of cell growth. Expression of S6K1 mutants that possess partial rapamycin-resistant activity or overexpression of eIF4E individually and additively partially rescues the rapamycin-induced decrease in cell size. In the absence of rapamycin, overexpression of S6K1 or eIF4E increases cell size, and, when coexpressed, they cooperate to increase cell size further. Expression of a phosphorylation site-defective mutant of 4EBP1 that constitutively binds the eIF4E-Cap complex to inhibit translation initiation reduces cell size and blocks eIF4E effects on cell size. These data show that mTOR signals downstream to at least two independent targets, S6K1 and 4EBP1/eIF4E, that function in translational control to regulate mammalian cell size.     

eIF4E与c-myc在喉癌中的表达及相关性研究

目的：检测真核细胞翻译起始因子4E（eIF4E）与原癌基因c—myc蛋白在喉癌组织中的表达，探讨二者与喉癌的关系及其相互之间的内在关系。方法：采用Western blot法分析36例喉癌标本中肿瘤核心区、癌旁组织区（过渡区）及无肿瘤手术切缘（无癌区）eIF4E与c—myc基因蛋白的表达水平，并进行统计学分析。结果：eIF4E与c-myc在喉癌无癌区、过渡区、核心区的表达水平呈递增趋势；且二者之间密切相关。结论：过度表达的eIF4E与c—myc均可导致喉癌细胞恶性转化，且二者在喉癌组织中的表达具有相关性，可为喉癌的基因治疗提供一定的理论基础。     

Phosphorylation of the human papillomavirus type 1 E4 proteins in vivo and in vitro

Following incubation of HPV 1-induced warts in the presence of [32P] phosphate several of the E4-encoded proteins were found to be radiolabeled. Two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 17K E4 polypeptides had incorporated [32P]phosphate whereas those of 16K were unlabeled. Purified E4 gene products were separated by ion exchange chromatography into a large number of different species, which were of similar size but of different charge due to varying extents of phosphorylated peptides have been isolated and identified. Phosphoserine and phosphothreonine were identified in all 16/17K E4 fractions but not phosphotyrosine. Both HPV 1 E4 16K and 17K fractions were phosphorylated in vitro by cAMP-dependent protein kinase but not by myosin light chain kinase or by phosphorylase kinase. Incubation with cAMP PK gave incorporation of approx. 0.5 mole phosphate/mol of protein indicating that the cAMP-dependent protein kinase site(s) was partially phosphorylated in vivo. This view was supported by the fact that species which were more heavily phosphorylated in vivo incorporated less phosphate after cAMP-dependent protein kinase phosphorylation. HPV 1 E4 was also phosphorylated at serine and threonine residues by a crude cytoplasmic extract prepared from cultured human keratinocytes and cultured human retinoblasts. These results are discussed in the light of the known effects of phosphorylation on the interactions of other keratinocyte-specific proteins.     

The 65K DNA binding protein appears early in HSV-1 replication

Summary The infectivity of influenza A viruses like fowl plague virus (FPV) with a cleaved hemagglutinin (HA) is highly sensitive to treatment at pH 5, while strains like PR 8 or virus N with a noncleaved HA survive under this condition. After double infection of chick embryo cells with FPV and PR 8 or virus N, the yield of virus with the HA gene of FPV is greatly reduced. However, it can now survive treatment at pH 5, and the surviving FPV particles form plaques only in the presence of trypsin, indicating that they were coated by the HA of PR 8 or virus N, depending on the coinfecting virus. The results are discussed with respect to the build-up and maintenance of a large reservoir of nonpathogenic influenza A viruses with noncleavable HA in water fowl.