Phosphorylation Levels Of 4EBP1 Research Articles

Effects and cell signaling mechanism of glutamine on rat cardiomyocytes intervened with serum from burned rat

Effects and cell signaling mechanism of glutamine on rat cardiomyocytes intervened with serum from burned rat

MYC-binding lncRNA EPIC1 promotes AKT-mTORC1 signaling and rapamycin resistance in breast and ovarian cancer.

AKT-mTORC1 (mammalian target of rapamycin complex 1) signaling pathway plays a critical role in tumorigenesis and can be targeted by rapamycin. However, the underlying mechanism of how long noncoding RNA (lncRNAs) regulate the AKT-mTORC1 pathway remains unclear. EPIC1 (epigenetically-induced lncRNA 1) is a Myc-binding lncRNA, which has been previously demonstrated to be overexpressed in multiple cancer types. In a pathway analysis including 4962 cancer patients, we observed that lncRNA EPIC1 expression was positively correlated with the AKT-mTORC1 signaling pathway in more than 10 cancer types, including breast and ovarian cancers. RNA-seq analysis of breast and ovarian cancer cells demonstrated that EPIC1-knockdown led to the downregulation of genes in the AKT-mTORC1 signaling pathway. In MCF-7, OVCAR4, and A2780cis cell lines, EPIC1 knockdown and overexpression, respectively, inhibited and activated phosphorylated AKT and the downstream phosphorylation levels of 4EBP1 and S6K. Further knockdown of Myc abolished the EPIC1's regulation of AKT-mTORC1 signaling; suggested that the regulation of phosphorylation level of AKT, 4EBP1, and S6K by EPIC1 depended on the expression of Myc. Moreover, EPIC1 overexpressed MCF-7, A2780cis, and OVCAR4 cells treated with rapamycin showed a significant decreasing in rapamycin mediated inhibition of p-S6K and p-S6 comparing with the control group. In addition, Colony Formation assay and MTT assay indicated that EPIC1 overexpression led to rapamycin resistance in breast and ovarian cancer cell lines. Our results demonstrated the lncRNA EPIC1 expression activated the AKT-mTORC1 signaling pathway through Myc and led to rapamycin resistance in breast and ovarian cancer.

Chronic Binge Alcohol Exposure During Pregnancy Alters mTOR System in Rat Fetal Hippocampus.

Gestational alcohol exposure can contribute to fetal alcohol spectrum disorders (FASD), an array of cognitive, behavioral, and physical developmental impairments. Mammalian target of rapamycin (mTOR) plays a key role in regulating protein synthesis in response to neuronal activity, thereby modulating synaptic plasticity and long-term memory formation in the brain. Based on our previous quantitative mass spectrometry proteomic studies, we hypothesized that gestational chronic binge alcohol exposure alters mTOR signaling and downstream pathways in the fetal hippocampus. Pregnant Sprague-Dawley rats were assigned to either a pair-fed control (PF-Cont) or a binge alcohol (Alcohol) treatment group. Alcohol dams were acclimatized via a once-daily orogastric gavage of 4.5g/kg alcohol (peak BAC, 216mg/dl) from GD 5-10 and progressed to 6g/kg alcohol (peak BAC, 289mg/dl) from GD 11-21. Pair-fed dams similarly received isocaloric maltose dextrin. In the Alcohol group, following this exposure paradigm, fetal body weight and crown-rump length were decreased. The phosphorylation level of mTOR (P-mTOR) in the fetal hippocampus was decreased in the Alcohol group compared with controls. Alcohol exposure resulted in dysregulation of fetal hippocampal mTORC1 signaling, as evidenced by an increase in total 4E-BP1 expression. Phosphorylation levels of 4E-BP1 and p70 S6K were also increased following alcohol exposure. P-mTOR and P-4E-BP1 were exclusively detected in the dentate gyrus and oriens layer of the fetal hippocampus, respectively. DEPTOR and RICTOR expression levels in the fetal hippocampus were increased; however, RAPTOR was not altered by chronic binge alcohol exposure. We conclude that chronic binge alcohol exposure during pregnancy alters mTORC1 signaling pathway in the fetal hippocampus. We conjecture that this dysregulation of mTOR protein expression, its activity, and downstream proteins may play a critical role in FASD neurobiological phenotypes.

Molecular and functional characterization of Raptor in mTOR pathway from Litopenaeus vannamei

Raptor, a member of the target of rapamycin complex 1 (TORC1), participates in the formation of complex proteins related to the mechanistic target of rapamycin (mTOR) signalling. In this study, a 5,020 bp cDNA of Raptor with an open reading frame (ORF) of 3,804 bp encoding for 1,267 amino acids was cloned from Litopenaeus vannamei. The protein contains three conserved domains: Raptor N, HEAT and WD40 domains. The expression of Raptor gene was detected by qRT‐PCR in different tissues of L. vannamei, including hepatopancreas, intestinal, stomach, eyestalk, gill and muscle. The mRNA expression profiles of Raptor in muscle were also analysed under suppression or stimulation of mTOR signalling pathway. The level of Raptor mRNA significantly increased either at 0.5–6 hr after an injection of rapamycin (RAPA) or after 3 days starvation. Leucine or arginine alleviated the up‐regulation of Raptor gene expression caused by RAPA or starvation. The Raptor gene was successfully suppressed using RNA interference (RNAi) technology, and the gene expression and the protein phosphorylation level of 4EBP1 and S6K were significantly decreased. The results of the study suggested that the expression of Raptor was sensitive to the immunology status of L. vannamei and participated in nutritional metabolism.

Methylphenidate alters Akt-mTOR signaling in rat pheochromocytoma cells

The exponential increase in methylphenidate (MPH) prescriptions in recent years has worried researchers about its misuse among individuals who do not meet the full diagnostic criteria for attention-deficit/hyperactivity disorder (ADHD) such as young children and students in search of cognitive improvement or for recreational reasons. The action of MPH is based mainly on inhibition of dopamine transporter, but the complete cellular effects are still unknown. Based upon prior studies, we attempted to determine whether the treatment with MPH (1μM) influences protein kinase B-mammalian target of rapamycin complex 1 signaling pathways (Akt-mTOR), including translation repressor protein (4E-BP1) and mitogen activated protein kinase (S6K), in rat pheochromocytoma cells (PC12), a well characterized cellular model, in a long or short term. MPH effects on the Akt substrates [cAMP response element-binding protein (CREB), forkhead box protein O1 (FoxO1), and glycogen synthase kinase 3 beta (GSK-3β)] were also evaluated. Whereas short term MPH treatment decreased the pAkt/Akt, pmTOR/mTOR and pS6K/S6K ratios, as well as pFoxO1 immunocontent in PC12 cells, long term treatment increased pAkt/Akt, pmTOR/mTOR and pGSK-3β/GSK-3β ratio. Phosphorylation levels of 4E-BP1 were decreased at 15 and 30 min and increased at 1 and 6 h by MPH. pCREB/CREB ratio was decreased. This study shows that the Akt-mTOR pathway, as well as other important Akt substrates which have been described as important regulators of protein synthesis, as well as being implicated in cellular survival, synaptic plasticity and memory consolidation, was affected by MPH in PC12 cells, representing an important step in exploring the MPH effects.

AAV1.NT-3 gene therapy increases muscle fiber diameter through activation of mTOR pathway and metabolic remodeling in a CMT mouse model.

Neurotrophin 3 (NT-3) has well-recognized effects on peripheral nerve and Schwann cells, promoting axonal regeneration and associated myelination. In this study, we assessed the effects of AAV.NT-3 gene therapy on the oxidative state of the neurogenic muscle from the TremblerJ (Tr J ) mice at 16 weeks post-gene injection and found that the muscle fiber size increase was associated with a change in the oxidative state of muscle fibers towards normalization of the fiber type ratio seen in the wild type. NT-3-induced fiber size increase was most prominent for the fast twitch glycolytic fiber population. These changes in the Tr J muscle were accompanied by increased phosphorylation levels of 4E-BP1 and S6 proteins as evidence of mTORC1 activation. In parallel, the expression levels of the mitochondrial biogenesis regulator PGC1α, and the markers of glycolysis (HK1 and PK1) increased in the TrJ muscle. In vitro studies showed that recombinant NT-3 can directly induce Akt/mTOR pathway activation in the TrkC expressing myotubes but not in myoblasts. In addition, myogenin expression levels were increased in myotubes while p75 NTR expression was downregulated compared to myoblasts, indicating that NT-3 induced myoblast differentiation is associated with mTORC1 activation. These studies for the first time have shown that NT-3 increases muscle fiber diameter in the neurogenic muscle through direct activation of mTOR pathway and that the fiber size increase is more prominent for fast twitch glycolytic fibers.

Combination treatment of RAD001 and BEZ235 exhibits synergistic antitumor activity via down-regulation of p-4E-BP1/Mcl-1 in small cell lung cancer.

Small cell lung cancer (SCLC) is a highly malignant cancer with few targeted therapies. In the study, by mining the Cancer Cell Line Encyclopedia (CCLE) database, we found that PI3K/AKT/mTOR pathway was aberrant in 92% of SCLC cell lines. Moreover, we found that the phosphorylation level of 4E-BP1 was significantly correlated with SCLC sensitivity to RAD001 (mTOR inhibitor) and BEZ235 (PI3K/mTOR dual inhibitor). Combination of RAD001 and BEZ235 synergistically inhibited the growth of SCLC cells, which was accompanied by enhanced induction of cell cycle arrest and apoptosis. Such a combination dramatically inhibited the activation of AKT, and strongly reduced the phosphorylation of 4E-BP1 and its downstream target Mcl-1. Knock-down of Mcl-1 enhanced the growth inhibition of SCLC cells induced by RAD001 and BEZ235 co-treatment, whereas over-expression of Mcl-1 reduced the growth inhibitory effect. Furthermore, in vivo study demonstrated that the combination treatment suppressed tumor growth more effectively than RAD001 or BEZ235 treatment alone. In summary, our study suggests that combination of BEZ235 and RAD001 may be an effective regimen for SCLC treatment, and p-4E-BP1 may serve as a predictive biomarker for SCLC response to mTOR inhibitor.

Curcumin inhibits urothelial tumor development by suppressing IGF2 and IGF2-mediated PI3K/AKT/mTOR signaling pathway

We have previously reported that curcumin inhibits urothelial tumor development in a rat bladder carcinogenesis model. In this study, we report that curcumin inhibits urothelial tumor development by suppressing IGF2 and IGF2-mediated PI3K/AKT/mTOR signaling pathway. Curcumin inhibits IGF2 expression at the transcriptional level and decreases the phosphorylation levels of IGF1R and IRS-1 in bladder cancer cells and N-methyl-N-nitrosourea (MNU)-induced urothelial tumor tissue. Ectopic expression of IGF2 and IGF1R, but not IGF1, in bladder cancer cells restored this process, suggesting that IGF2 is a target of curcumin. Moreover, introduction of constitutively active AKT1 abolished the inhibitory effect of curcumin on cell proliferation, migration, and restored the phosphorylation levels of 4E-BP1 and S6K1, suggesting that curcumin functions via suppressing IGF2-mediated AKT/mTOR signaling pathway. In summary, our results reveal that suppressing IGF2 and IGF2-mediated PI3K/AKT/mTOR signaling pathway is one of the mechanisms of action of curcumin. Our findings suggest a new therapeutic strategy against human bladder cancer caused by aberrant activation of IGF2, which are useful for translational application of curcumin.

Effects of tiletamine on the adenosine monophosphate-activated protein kinase signaling pathway in the rat central nervous system

ObjectiveThe dissociative anesthetic tiletamine, which acts on the central nervous system (CNS), is widely used in veterinary medicine and animal experiments. Recent studies indicate that adenosine 5′-monophosphate activated protein kinase (AMPK) plays a key role in the analgesic action of tiletamine. In the present study, the effects of tiletamine on the AMPK signaling pathway in rats were investigated. MethodsSprague-Dawley rats were injected intraperitoneally with tiletamine and executed at 10, 20, 40 and 60min post injection. The cerebral cortex, hippocampus, thalamus, cerebellum and brainstem were immediately taken out to evaluate the mRNA and protein phosphorylation levels of liver kinase B1 (LKB1), AMPKα and eIF4E-binding protein 1 (4EBP1) using quantitative real-time polymerase chain reaction and western blot analysis. ResultsTiletamine increased AMPK mRNA expression in the rat brain (P<0.01). Increased mRNA expression of AMPK was accompanied by an increase in phosphorylation of LKB1, resulting in significant decreases in the phosphorylation levels of 4EBP1 in the corresponding brain regions (P<0.01). ConclusionIn summary, the findings indicate that tiletamine regulates the mRNA expression and protein phosphorylation levels of LKB1, AMPK and 4EBP1 in the CNS, suggesting that the analgesic effect of the anesthetic is mediated, at least in part, by the AMPK signaling pathway.

Phosphorylated 4E-BP1 is associated with tumor progression and adverse prognosis in colorectal cancer.

Phosphorylation of eukaryotic translation initiation factor 4E (eIF4E)- binding protein (4E-BP1) results in release of eIF4E, relieving translational repression and enhancing cancerigenic protein synthesis. This study aim to evaluate the level of phosphorylated 4E-BP1 (p-4E-BP1) in colorectal cancer (CRC) and to assess the cor-relation with clinicopathological factors and patient survival. The level of p-4E-BP1 was detected by immunohistochemistry and western bolt in patients with CRC. Then Cox regression model was used to evaluate the prognostic value of all covariates. Among 164 assessed patients, 95 (57.9%) patients showed high level of p-4E-BP1. We noted that the level of p-4E-BP1 was significantly associated with tumor differentiation, invasive depth, lymph node metastasis and TNM stage. Then we compared the mRNA and protein expressions of 4E-BP1 in tumor regions and paired adjacent normal colorectal mucosal tissues in CRC patients. mRNA and protein expressions of 4E-BP1 did not differ between colorectal cancer and corresponding normal tissues, while the phosphorylation level of 4E-BP1 was markedly increased in CRC. Survival analysis and Cox proportional hazards model revealed that p-4E-BP1 was an independent adverse prognostic factor for both overall survival (OS) (HR = 5.414, p = 0.029) and progression-free survival (PFS) (HR = 4.754, p = 0.042). Herein, our results indicate that high p-4E-BP1 level is associated with tumor progression and adverse prognosis. p-4E-BP1 might be a novel biomarker to predict the clinical outcome of patients with CRC.

Modulation of Doxorubicin Actions in Hepatocellular Carcinoma Cells by Insulin-Like Growth Factor-I

Hepatocellular carcinoma (HCC) is typically advanced at presentation and doxorubicin is the chemotherapeutic agent most frequently used in chemoembolization. Tumor microenvironment contains many cell types, including platelets, as well as cytokines and growth factors, potentially capable of modulating doxorubicin actions. Insulin-like growth factor I (IGF1) and its receptor (IGF1-R) signaling is important in cellular proliferation and prevention of apoptosis and the acquisition of chemotherapy resistance. IGF1 is present in platelets, which have been shown to alter doxorubicin actions in HCC cells. The effects on growth and motility after IGF1 pre-treatment in doxorubicin-treated HCC cells were thus examined, as well as the IGF1-R downstream pathways, PI3/Akt and MAPK kinases. We found that IGF1 antagonized the doxorubicin-mediated decrease in cell growth and motility, as well as the doxorubicin-mediated decrease in levels of the proliferation-associated proteins phospho-IGF1-R, phospho-ERK, phospho-p38 and phospho-STAT3. The simultaneous induction of PI3K/Akt pathway, mediated by IGF1 receptor, regulated an increase in levels of Bcl-2, BclxL and survivin through the Akt activation. Furthermore, PI3K/Akt signaling modulated several downstream targets, including phosphorylation levels of 4EB-P1, p70S6K and GSK-3β. Up-regulated IGF1 signaling antagonized the doxorubicin-mediated changes cell proliferation, motility and apoptosis, thus contributing to drug resistance. Therefore IGF1-R may be a promising target for HCC management.

Inhibition of placental mTORC1 signalling may explain decreased placental nutrient transport and contribute to restricted fetal growth in placental malaria

Inhibition of placental mTORC1 signalling may explain decreased placental nutrient transport and contribute to restricted fetal growth in placental malaria

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Phosphorylation of 4E-BP1 predicts sensitivity to everolimus in gastric cancer cells

We studied the effect of everolimus, an inhibitor of the mammalian target of rapamycin (mTOR) on human gastric cancer cell lines. Cell proliferation in 3 of 8 cell lines was effectively inhibited by everolimus. Basal phosphorylation level of 4E-BP1 (T37/46, T70) was significantly higher in everolimus-sensitive cells than in everolimus-resistant cells. In subcutaneous xenograft model, immunohistochemistry analysis revealed that everolimus-sensitive cells expressed high levels of phospho-4E-BP1 (T37/46). In conclusion, phosphorylation of 4E-BP1 may be a predictive biomarker of everolimus sensitivity in gastric cancer.

Increased expression of tuberin in human uterine leiomyoma

Female Eker rats harboring an insertional deletion in one copy of the tuberous sclerosis complex 2 (Tsc2) gene develop uterine leiomyoma, but the underlying mechanism of human uterine leiomyoma is not completely understood. To examine whether down-regulation of tuberin, a TSC2 gene product, is present in human uterine leiomyoma, we analyzed leiomyoma and matched myometrium tissues from 22 Chinese patients with Western blotting and real-time polymerase chain reaction analyses, and found that the expression of tuberin was significantly increased in leiomyoma tissues compared with matched myometrium tissues with inhibition of both the mammalian target of rapacmycin pathway and mitogen-activated protein kinase pathways.

Translation Regulatory Protein 4E-Binding Protein (BP)-1 Phosphorylation Identifies Diffuse Large B Cell Lymphoma Patients with Low IPI Scores Who Experience Short Survival After Chemotherapy.

Abstract 1925Poster Board I-948Eukaryotic initiation factor 4E binding protein 1 (4E-BP1) regulates the function of eukaryotic initiation factor 4E (eIF-4E) which mediates nucleo-cytoplasmic transport and cap-dependent translation of specific transcripts. eIF-4E expression itself is elevated in several carcinomas and hematopoietic malignancies. Hypophosphorylated 4E-BP1 binds eIF-4E and inhibits cap-dependent translation. Phosphorylated 4E-BP1 dissociates from eIF-4E, thereby allowing translation of several cap-dependent transcripts responsible for cell cycling, survival and angiogenesis. Hyper-phosphorylation of 4E-BP1 may thus be associated with oncogenesis and the malignant phenotype; its influence on the response to chemotherapy and prognosis of diffuse large B cell lymphoma (DLBCL) is unknown. We examined diagnostic tissues from 85 patients with DLBCL for 4E-BP1 expression and phosphorylation, and correlated these with clinical outcomes. Samples were stained for total and phosphorylated 4E-BPI using immunohistochemistry (IHC). Slides were scored for percent cells positive for the two markers (0% to 100%) and the intensity of expression (0 to 3+) by hematopathologists (A.R. and H.M.) blinded to the clinical data. An Expression Score was calculated from their product ([% cells positive × intensity]/10), thus ranging from 0-30. In parallel, expression of bcl-2 was determined by IHC on the same samples. Patients had a median age of 75 years (range, 25-91). The majority received CHOP-based chemotherapy with/without rituximab. Total 4E-BP1 was expressed uniformly (by 97% of the cases). However, there was marked heterogeneity in the level of phosphorylation of 4E-BP1 (none [Expression Score = 0] in 35%, uniformly high [Expression Score = 30] in 34%, and variable [Expression Score = 1-29] in the remainder [31%]). We therefore examined if the level of phosphorylation of 4E-BP1 varied with clinical features at diagnosis, and whether it correlated with response to chemotherapy and survival. The level of expression or phosphorylation of 4E-BP1 did not correlate with the clinical stage, IPI score or bcl-2 expression at diagnosis, and did not influence the likelihood of achieving complete remission with CHOP-based chemotherapy. However, in patients with low IPI scores (IPI 0-2), the level of phosphorylation of 4E-BP1 correlated significantly with overall survival (OS). In these patients, the OS was significantly different (P = 0.031) in patients with low levels of phosphorylation of 4E-BP1 (Expression Score = 0-15) compared to those with higher levels of 4E-BP1 phosphorylation (Expression Score = 16-30). The median survival was >146.4 months vs 20.6 months, and the 5-year survival was 73% vs 31%, in patients with low and high levels of 4E-BP1 phosphorylation, respectively. The difference in survival between the two groups was not a consequence of differences in age or IPI score at diagnosis or inclusion of rituximab in the treatment regimen. The level of phosphorylation of 4E-BP1 did not have any further impact on the survival of patients with high IPI scores (IPI 4-5). We conclude that while the large majority of DLBCL express the translation regulatory protein 4E-BP1, the level of phosphorylation (indicative of biological activity) of this protein varies widely. Importantly, the level of phosphorylation of 4E-BP1 further stratifies DLBCL patients who would be expected to have a favorable prognosis based on low IPI scores, and identifies a subset of these patients who do poorly following standard CHOP-based chemotherapy. Since hyper-phosphorylation of 4E-BP1 increases the activity of eIF-4E, this study provides the rationale for trials incorporating novel targeted agents that inhibit eIF-4E into the therapeutic regimen, specifically in DLBCL patients with low IPI scores and high levels of 4E-BP1 phosphorylation. Disclosures:No relevant conflicts of interest to declare.

Diagnostic and Therapeutic Implications of Expression and Phosphorylation of the Translation Regulatory Protein 4E-Binding Protein (BP)-1 in B-Cell Lymphomas and Reactive Lymphoid Tissues.

Abstract 3919Poster Board III-855Identifying pathogenic mechanisms that contribute to the development of lymphomas and influence clinical behavior is critical for developing targeted therapies, and selecting patients who may benefit from such drugs. An important level of control of gene expression occurs during initiation of cap-mediated mRNA translation by the eukaryotic initiation factor-4F (eIF-4F) trimolecular complex (eIF-4E, eIF-4G and eIF-4A), in which eIF-4E is rate limiting and oncogenic. eIF-4F hyperactivity plays a key role in human cancers by mediating expression of proteins critical for cell growth, transformation and tumorigenesis. eIF-4F activity is controlled by repressor eIF-4 binding proteins (BPs). 4E-BP1 activity is regulated by phosphorylation. Hypo/non-phosphorylated 4E-BP1 is active, binds eIF-4E and impedes eIF-4F formation, blocking translation and inducing apoptosis. Phosphorylation of 4E-BP1 (p4E-BP1) releases bound eIF-4E, which initiates cap-dependent translation. Because only limited information is available on the expression and phosphorylation of 4E-BP1 in lymphomas, and since agents (e.g., antisense oligonucleotides and small molecules) that target eIF-4E have been developed, we examined the frequency and level of expression of 4E-BP1 and its phosphorylation in various subtypes of mature B cell non-Hodgkin's lymphomas (BCL). Forty-six BCLs (12 follicular [FL], 13 diffuse large B-cell [DLBCL], 7 mantle cell, 5 extranodal marginal zone, and 9 small lymphocytic [SLL] lymphomas), 4 FL with incipient/partial lymph node involvement, and 11 reactive lymphoid tissues were examined using immunohistochemistry for total and phosphorylated 4E-BP1. Staining intensity was graded as from 0 to 3+. Western immunoblotting (WB) was performed on lysates of 5 mature BCLs (2 FL, 3 DLBCL) and 2 reactive lymph nodal tissues for eIF-4G (total), eIF-4E and 4E-BP1 (total and phosphorylated) expression. In reactive lymphoid tissues, there was regional and cellular specificity of expression of 4E-BP1, with either lack of, or minimal (0 to 1+) cytoplasmic expression in follicular center cells and paracortical T-cells, 2+ expression in follicular dendritic cells and paracortical zone Langerhan cells, and 3+ expression in mantle and marginal zones. p4E-BP1 expression was inverted, with 3+ cytoplasmic immunoreactivity in reactive follicular center cells and no expression in the mantle and marginal zone cells or T-cells, and 2+ or 3+ immunoreactivity in follicular dendritic cells and paracortical zone Langerhan cells. In BCLs, a consistently high level (2+ or 3+) of cytoplasmic 4E-BP1 expression was seen in neoplastic lymphocytes in 45/46 (98%) cases. In contrast, p4E-BP1 was moderately or strongly expressed in 19/46 (41%) cases of BCL, being negative in 17 (37%) cases, and only dimly expressed in the remaining 10 (22%) cases. Three of 4 cases with incipient/partial involvement by FL were easily distinguishable from reactive germinal centers by strong, diffuse staining with 4E-BP1 (and 1+ staining in the 4th case) in neoplastic follicles, distinct from negative/weak staining of adjacent reactive germinal centers. In SLL, slightly higher 4E-BP1 expression was noted in proliferation centers in comparison to surrounding small mature lymphocytes. WB confirmed that non-phosphorylated and p4E-BP1 were expressed in reactive nodes, FL and DLBCL. Other components of the eIF-4F complex including eIF-4G, total and p-eIF-4E and total 4E-BP1 were detectable in whole tissue lysates from BCL samples. We conclude that (a) while 4E-BP1 is almost uniformly expressed in various subtypes of BCL, its level of phosphorylation (indicative of activity) varies widely and has regional and cellular specificity, and (b) 4E-BP1 expression may identify minimal/early lymphomatous involvement in tissues. We speculate that 4E-BP1 phosphorylation may influence the biological behavior of BCLs, since in other investigations we found that the level of phosphorylation of 4E-BP1 correlates with survival after CHOP-based chemotherapy in DLBCL. Our findings support therapeutic trials targeting the eIF-4E pathway in many BCL subtypes, particularly in patients where immunostaining identifies high levels of 4E-BP1 phosphorylation. Disclosures:No relevant conflicts of interest to declare.

Matrine Inhibits the Activity of Translation Factor eIF4E through Dephosphorylation of 4E-BP1 in Gastric MKN45 Cells

Matrine, from Sophora flavescens, could remarkably inhibit tumor growth and induce apoptosis in various cancer cells in vitro. eIF4E and its inhibitor 4E-BP1 play key roles in regulating mRNA translation and cell proliferation. However, it remained elusive whether matrine inhibited cancer cells growth through attenuating the activity of 4E-BP1. In this study, we analyzed the effects of matrine on 4E-BP1 and eIF4E in gastric cancer MKN45 cells. Immunoblots showed that matrine inhibited the activity of eIF4E through dephosphorylation of 4E-BP1 in a dose- and time-dependent manner. We found that matrine inactivated Erk1/2, an upstream regulator of 4E-BP1 and eIF4E, and remarkably reduced the phosphorylation level of 4E-BP1 and eIF4E, whereas 4E-BP1 was little influenced by JNK, p38 or Akt/mTOR. Inactivation of PP2A obviously decreased the phosphorylation of 4E-BP1 in matrine-treated cells. These findings suggested that matrine inhibits the activity of eIF4E by dephosphorylating 4E-BP1, which partly counts for the growth inhibition in gastric MKN45 cells.

Lack of evidence for a role of TRB3/NIPK as an inhibitor of PKB-mediated insulin signalling in primary hepatocytes.

The protein TRB3 (tribbles 3), also called NIPK (neuronal cell death-inducible protein kinase), was recently identified as a protein-protein interaction partner and an inhibitor of PKB (protein kinase B). To explore the hypothesis that TRB3/NIPK might act as a negative regulator of insulin signalling in the liver, this protein was overexpressed by adenoviral transduction of primary cultures of rat hepatocytes, and various aspects of insulin action were investigated. The insulin-induced phosphorylation of Ser-473 and Thr-308 of PKB was found to be undiminished in transduced hepatocytes with a molar excess of TRB3/NIPK over PKB of more than 25-fold. Consistent with unimpaired insulin activation of PKB, the stimulation of Ser-21 and Ser-9 phosphorylation of glycogen synthase kinase 3-alpha and -beta, and the apparent phosphorylation level of 4E-BP1 (eukaryotic initiation factor 4-binding protein 1), were similar in transduced and control hepatocytes. The induction by insulin of the mRNAs encoding glucokinase and SREBF1 (sterol-regulatory-element-binding factor 1) were also normal in TRB3/NIPK hepatocytes. In contrast, the insulin-dependent induction of these two genes, as well as the activation of PKB, were shown to be suppressed in hepatocytes treated with the lipid ether compound PIA6 (phosphatidylinositol ether lipid analogue 6), a recently discovered specific inhibitor of PKB. Since TRB3/NIPK was reported to be increased in the liver of fasting mice, the effects of glucagon, glucocorticoids and insulin on the level of endogenous TRB3/NIPK mRNA in primary hepatocytes were investigated. No significant change in mRNA level occurred under any of the hormonal treatments. The present study does not support the hypothesis that the physiological role of TRB3/NIPK might be to put a brake on insulin signalling in hepatocytes.

Impaired translational response and increased protein kinase PKR expression in T cells from lupus patients.

Activation of peripheral blood T cells results in a rapid and substantial rise in translation rates and proliferation, but proliferation in response to mitogen stimulation is impaired in systemic lupus erythematosus (SLE). We have investigated translation rates and initiation factor activities in T cells from SLE patients in response to activating signals. Activation by PMA plus ionomycin strongly increased protein synthesis in control T cells but not in T cells from SLE patients. The rate of protein synthesis is known to be strongly dependent on the activity of two eukaryotic translation initiation factors, eIF4E and eIF2alpha. We show that following stimulation, eIF4E expression and phosphorylation increased equivalently in control and SLE T cells. Expression of eIF4E interacting proteins - eIF4G, an inducer, and 4E-BP1 and 4E-BP2, two specific repressors of eIF4E function - and the phosphorylation level of 4E-BP1, were all identical in control and SLE T cells. In contrast, the protein kinase PKR, which is responsible for the phosphorylation and consequent inhibition of eIF2alpha activity, was specifically overexpressed in activated SLE T cells, correlating with an increase in eIF2alpha phosphorylation. Therefore, high expression of PKR and subsequent eIF2alpha phosphorylation is likely responsible, at least in part, for impaired translational and proliferative responses to mitogens in T cells from SLE patients.