ABSTRACT
Here, we have identified the interaction site of the contraceptive drug gamendazole using computational modeling. The drug was previously described as a ligand for eukaryotic translation elongation factor 1-α 1 (eEF1A1) and found to be a potential target site for derivatives of 2-phenyl-3-hydroxy-4(1 H)-quinolinones (3-HQs), which exhibit anticancer activity. The interaction of this class of derivatives of 3-HQs with eEF1A1 inside cancer cells was confirmed via pull-down assay. We designed and synthesized a new family of 3-HQs and subsequently applied isothermal titration calorimetry to show that these compounds strongly bind to eEF1A1. Further, we found that some of these derivatives possess significant in vitro anticancer activity.
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Indazoles/metabolism , Peptide Elongation Factor 1/drug effects , Quinolones/chemical synthesis , Quinolones/pharmacology , Binding Sites/drug effects , Cell Line, Tumor , Computational Biology , Humans , Ligands , Models, Molecular , Molecular Conformation , Peptide Elongation Factor 1/biosynthesis , Structure-Activity RelationshipABSTRACT
BACKGROUND: Tick-borne Babesia bigemina is responsible for acute and potentially lethal hemolytic disease in cattle. The development of genetic manipulation tools necessary to the better understanding of parasite biology is currently limited by the lack of a complete parasite genome and experimental tools such as transfection. Effective promoters, required to regulate expression of transgenes, such as the elongation factor-1 alpha (ef-1α), have been identified in other apicomplexans such as Babesia bovis and Plasmodium falciparum. METHODS: The B. bigemina ef-1a locus was defined by searching a partial genome library of B. bigemina (Sanger Institute). Presence of an intron in the 5' untranslated region was determined by 5' Rapid Amplification of cDNA Ends (RACE) analysis. Promoter activity was determined by measurement of luciferase expression at several time points after electroporation, efficiency of transfections and normalization of data was determined by quantitative PCR and by the percentage of parasitized erythrocytes. RESULTS: The ef-1α locus contains two identical head to head ef-1α genes separated by a 1.425 kb intergenic (IG) region. Significant sequence divergence in the regions upstream of the inverted repeats on each side of the B. bigemina IG region suggest independent regulation mechanisms for controlling expression of each of the two ef-1α genes. Plasmid constructs containing the 5' and 3' halves of the IG regions controlling the expression of the luciferase gene containing a 3' region of a B. bigemina rap-1a gene, were generated for the testing of luciferase activity in transiently transfected parasites. Both halves of the ef-1α IG region tested showed the ability to promote high level production of luciferase. Moreover, both B. bigemina ef-1α promoters are also active in transiently transfected B. bovis and conversely, a B. bovis ef-1α promoter is active in transiently transfected B. bigemina. CONCLUSIONS: Collectively these data demonstrate the existence of two distinct promoters with homologous and heterologous promoter function in B. bigemina and B. bovis which is described for the first time in Babesia species. This study is of significance for development of interspecies stable transfection systems for B. bigemina and for B. bovis.
Subject(s)
Babesia/genetics , Peptide Elongation Factor 1/biosynthesis , Peptide Elongation Factor 1/genetics , Promoter Regions, Genetic , Artificial Gene Fusion , Gene Expression Profiling , Genes, Reporter , Luciferases/analysis , Luciferases/genetics , Real-Time Polymerase Chain Reaction , TransfectionABSTRACT
Synaptic dysfunction may represent an early and crucial pathophysiology in Alzheimer's disease (AD). Recent studies implicate a connection between synaptic plasticity deficits and compromised capacity of de novo protein synthesis in AD. The mRNA translational factor eukaryotic elongation factor 1A (eEF1A) is critically involved in several forms of long-lasting synaptic plasticity. By examining postmortem human brain samples, a transgenic mouse model, and application of synthetic human Aß42 on mouse hippocampal slices, we demonstrated that eEF1A protein levels were significantly decreased in AD, particularly in the hippocampus. In contrast, brain levels of eukaryotic elongation factor 2 were unaltered in AD. Further, upregulation of eEF1A expression by the adenylyl cyclase activator forskolin, which induces long-lasting synaptic plasticity, was blunted in hippocampal slices derived from Tg2576 AD model mice. Finally, Aß-induced hippocampal long-term potentiation defects were alleviated by upregulation of eEF1A signaling via brain-specific knockdown of the gene encoding tuberous sclerosis 2. In summary, our findings suggest a strong correlation between the dysregulation of eEF1A synthesis and AD-associated synaptic failure. These findings provide insights into the understanding of molecular mechanisms underlying AD etiology and may aid in identification of novel biomarkers and therapeutic targets.
Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Neuronal Plasticity/physiology , Peptide Elongation Factor 1/biosynthesis , Aged, 80 and over , Alzheimer Disease/genetics , Animals , Female , Gene Expression , Hippocampus/metabolism , Hippocampus/pathology , Humans , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Organ Culture Techniques , Peptide Elongation Factor 1/geneticsABSTRACT
Paclitaxel (Taxol), a potent drug of natural origin isolated from the bark of the Pacific yew, is widely used for treating ovarian, lung and breast cancers. Currently, there is little information regarding the specific mechanism underlying the anticancer activity of paclitaxel. In the present study, we found that 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR), a well-known activator of adenosine monophosphate (AMP)-activated protein kinase (AMPK), downregulated the protein and mRNA levels of elongation factor 1 α (EF1α) in breast cancer MCF7 cells. Paclitaxel increased the phosphorylation of AMPK and also downregulated the expression of EF1α in MCF7 cells. In addition, paclitaxel increased the expression, as well as the phosphorylation of forkhead box O3a (FOXO3a). Phosphorylation of FOXO3a was suppressed in the presence of compound C, a specific AMPK inhibitor, suggesting the involvement of AMPK in paclitaxel-induced FOXO3a phosphorylation. The induction and phosphorylation of FOXO3a by paclitaxel were not observed in EF1α and AMPK knockdown cells. Co-treatment with AICAR resulted in increased susceptibility of cancer cells to paclitaxel-induced suppression of their viability and further enhanced paclitaxel-induced FOXO3a phosphorylation. These results suggest that the antitumor effects of paclitaxel in breast cancer are mediated by activation of the AMPK/EF1α/FOXO3a signaling pathway.
Subject(s)
AMP-Activated Protein Kinases/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Forkhead Transcription Factors/genetics , Peptide Elongation Factor 1/genetics , AMP-Activated Protein Kinases/biosynthesis , Aminoimidazole Carboxamide/administration & dosage , Aminoimidazole Carboxamide/analogs & derivatives , Breast Neoplasms/pathology , Cell Survival/drug effects , Female , Forkhead Box Protein O3 , Forkhead Transcription Factors/biosynthesis , Gene Expression Regulation, Neoplastic/drug effects , Humans , MCF-7 Cells , Paclitaxel/administration & dosage , Peptide Elongation Factor 1/biosynthesis , Signal Transduction/drug effectsABSTRACT
The mammalian timeless (TIM) protein interacts with proteins of the endogenous clock and essentially contributes to the circadian rhythm. In addition, TIM is involved in maintenance of chromosome integrity, growth control and development. Thus, we hypothesized that TIM may exert a potential protumorigenic function in human hepatocarcinogenesis. TIM was overexpressed in a subset of human HCCs both at the mRNA and the protein level. siRNA-mediated knockdown of TIM reduced cell viability due to the induction of apoptosis and G2 arrest. The latter was mediated via CHEK2 phosphorylation. In addition, siRNA-treated cells showed a significantly reduced migratory capacity and reduced expression levels of various proteins. Mechanistically, TIM directly interacts with the eukaryotic elongation factor 1A2 (EEF1A2), which binds to actin filaments to promote tumor cell migration. siRNA-mediated knockdown of TIM reduced EEF1A2 protein levels thereby affecting ribosomal protein biosynthesis. Thus, overexpression of TIM exerts oncogenic function in human HCCs, which is mediated via CHEK2 and EEF1A2.
Subject(s)
Carcinogenesis/genetics , Carcinoma, Hepatocellular/genetics , Cell Cycle Proteins/genetics , Intracellular Signaling Peptides and Proteins/genetics , Liver Neoplasms/genetics , Adolescent , Adult , Aged , Carcinoma, Hepatocellular/pathology , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/biosynthesis , Checkpoint Kinase 2/biosynthesis , Female , Gene Expression Regulation, Neoplastic , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/biosynthesis , Liver Neoplasms/pathology , Male , Middle Aged , Peptide Elongation Factor 1/biosynthesis , RNA, Small InterferingABSTRACT
It is thought that Shine-Dalgarno-like sequences, which exhibit complementarity to the nucleotide sequences at the 3'-end of 18S rRNA, are not present in eukaryotic mRNAs. However, complementary sequences consisting of more than 5 nucleotides to the 3'-end of 18S rRNA, i.e., a CR sequence, are present at -17 to -32 upstream from the initiation codon AUG in 18 mRNAs involved in protein synthesis except eEF1A mRNA. Thus, effects of the CR sequence in mRNAs and polyamines on protein synthesis were examined using control and polyamine-reduced FM3A and NIH3T3 cells. Polyamines did not stimulate protein synthesis encoded by 18 mRNAs possessing a normal CR sequence. When the CR sequence was deleted, protein synthetic activities decreased to less than 70% of intact mRNAs. In eEF1A mRNA, the CR sequence was located at -33 to -39 upstream from the initiation codon AUG, and polyamines stimulated eEF1A synthesis about threefold. When the CR sequence was shifted to -22 to -28 upstream from the AUG, eEF1A synthesis increased in polyamine-reduced cells and the degree of polyamine stimulation decreased greatly. The results indicate that the CR sequence exists in many eukaryotic mRNAs, and the location of a CR sequence in mRNAs influences polyamine stimulation of protein synthesis.
Subject(s)
Codon, Initiator/metabolism , Peptide Elongation Factor 1/biosynthesis , Polyamines/pharmacology , Protein Biosynthesis/drug effects , RNA, Ribosomal, 18S/metabolism , Animals , Cell Line, Tumor , Codon, Initiator/genetics , Mice , Peptide Elongation Factor 1/genetics , Protein Biosynthesis/physiology , RNA, Ribosomal, 18S/geneticsABSTRACT
Silicone rubber (SR) is a common soft tissue filler material used in plastic surgery. However, it presents a poor surface for cellular adhesion and suffers from poor biocompatibility. In contrast, hydroxyapatite (HA), a prominent component of animal bone and teeth, can promote improved cell compatibility, but HA is an unsuitable filler material because of the brittleness in mechanism. In this study, using a simple and economical method, two sizes of HA was applied to coat on SR to counteract the poor biocompatibility of SR. Surface and mechanical properties of SR and HA/SRs confirmed that coating with HA changes the surface topology and material properties. Analysis of cell proliferation and adhesion as well as measurement of the expression levels of adhesion related molecules indicated that HA-coated SR significantly increased cell compatibility. Furthermore, mass spectrometry proved that the biocompatibility improvement may be related to elongation factor 1-beta (EF1ß)/γ-actin adjusted cytoskeletal rearrangement.
Subject(s)
Actins/metabolism , Cell Adhesion/physiology , Durapatite/chemistry , Peptide Elongation Factor 1/metabolism , Silicone Elastomers/chemistry , Actins/biosynthesis , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Proliferation/physiology , Cells, Cultured , Coated Materials, Biocompatible/chemistry , Cytoskeleton/metabolism , Fibroblasts/cytology , Humans , Materials Testing/methods , Microscopy, Electron, Scanning , Peptide Elongation Factor 1/biosynthesis , Surface PropertiesABSTRACT
The promoter is a major element in the expression cassette of gene therapy vectors. Optimal promoter selection can enhance target specificity and gene expression. Recently, we evaluated three different human elongation factor 1 alpha (EF1α) promoters. The three promoters were put into the same expression vector, pAC-luc, driving expression of the luciferase cDNA. The activity from one EF1α promoter (termed EF1α -3), obtained in a commercial vector, was markedly lower when tested in vitro (from 50 - 500 x) in four cell lines and in vivo in rat submandibular glands (~250 x). Sequence differences in the EF1α -3 promoter likely account for the activity differences seen. Investigators need to recognize that all promoters of the same name may not be equivalent in driving transgene expression.
Subject(s)
Gene Expression Regulation , Peptide Elongation Factor 1/genetics , Promoter Regions, Genetic , Animals , Cell Line , DNA, Complementary/genetics , Genetic Therapy , Genetic Vectors , Humans , Luciferases/biosynthesis , Peptide Elongation Factor 1/biosynthesis , RatsABSTRACT
The eukaryotic elongation factor 1 gamma (eEF1γ) is a multi-domain protein, which consist of a glutathione transferase (GST)-like N-terminus domain. In association with α, ß and δ subunits, eEF1γ forms part of the eukaryotic elongation factor complex, which is mainly involved in protein biosynthesis. The N-terminus GST domain of eEF1γ interacts with the ß subunit. eEF1γ subunit is over-expressed in human carcinoma. The role of human eEF1γ (heEF1γ) is poorly understood. A successful purification of recombinant heEF1γ is the first step towards determining unknown properties of the protein, including putative GST-like activities and the structure of the protein. This paper describes the over-expression, purification and characterisation of recombinant full-length, and the N- and C-terminus domains of heEF1γ. All three recombinant heEF1γ constructs over-expressed in the soluble Escherichia coli cell fraction and were purified to homogeneity. Secondary structure analysis indicates that the heEF1γ constructs have high α-helical structural character. The full-length and N-terminus domain are dimeric, while the C-terminus is monomeric. Both full-length and N-terminus domain interact with 8-anilino-1-naphthalene sulfonate (ANS) with KD=70.0 (±5.7) µM and with reduced glutathione (GSH). Glutathione sulfonate displaced ANS bound to hydrophobic binding sites in the recombinant N-terminus domain. Using the standard GSH-1-chloro-2,4-dinitrobenzene conjugation assay, the N-domain showed some enzyme activity (0.03µmolmin(-1) mg(-1) protein), while the full-length heEF1γ did not catalyse the GSH-CDNB conjugation. Consequently, we hypothesize the presence of a presumed GST-like active site structure in the heEF1γ, which comprises a glutathione binding site and a hydrophobic substrate binding site.
Subject(s)
Peptide Elongation Factor 1/isolation & purification , Peptide Elongation Factor 1/metabolism , Binding Sites , Dinitrochlorobenzene/metabolism , Escherichia coli/metabolism , Glutathione/metabolism , Glutathione Transferase/metabolism , Humans , Peptide Elongation Factor 1/biosynthesis , Protein Structure, Quaternary , Protein Structure, Secondary , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
BACKGROUND: Eukaryotic elongation factor 1 alpha-2 (eEF1A2) has been recently shown to be a putative oncogene of lung cancer. MATERIALS AND METHODS: We analyzed the expression and prognostic significance of eEF1A2 in 69 primary non-small cell lung cancer (NSCLC) cases. We also suppressed eEF1A2 expression using RNA interference and then analyzed cell proliferation, migration and invasion of five adenocarcinoma cell lines. RESULTS: eEF1A2 protein expression was positive in 84.1%. Negative immunostaining for eEF1A2 was shown to be an independent prognostic factor and significantly correlated with lymph node metastasis. There was no significant correlation between eEF1A2 protein and mRNA expression levels. Among the five examined cell lines, transfection of eEF1A2 siRNA inhibited cell migration in only one cell line while it did not change cell proliferation and invasion. CONCLUSION: Negative immunostaining of eEF1A2 predicted for poor prognosis of NSCLC. The mechanism of this result could not be elucidated by cell proliferation, migration and invasion assays.
Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Lung Neoplasms/metabolism , Peptide Elongation Factor 1/biosynthesis , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Growth Processes/physiology , Cell Line, Tumor , Cell Movement/physiology , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Neoplasm Invasiveness , Neoplasm Staging , Peptide Elongation Factor 1/genetics , Prognosis , RNA, Messenger/biosynthesis , RNA, Messenger/geneticsABSTRACT
BACKGROUND: Eukaryotic translation elongation factor 1A2 (eEF1A2) is a known proto-oncogene. We proposed that stimulation of the eEF1A2 expression in cancer tissues is caused by the loss of miRNA-mediated control. METHODS: Impact of miRNAs on eEF1A2 at the mRNA and protein levels was examined by qPCR and western blot, respectively. Dual-luciferase assay was applied to examine the influence of miRNAs on 3'-UTR of EEF1A2. To detect miRNA-binding sites, mutations into the 3'-UTR of EEF1A2 mRNA were introduced by the overlap extension PCR. RESULTS: miR-663 and miR-744 inhibited the expression of luciferase gene attached to the 3'-UTR of EEF1A2 up to 20% and 50%, respectively. In MCF7 cells, overexpression of miR-663 and miR-744 reduced the EEF1A2 mRNA level by 30% and 50%. Analogous effects were also observed at the eEF1A2 protein level. In resveratrol-treated MCF7 cells the upregulation of mir-663 and mir-744 was accompanied by downregulation of EEF1A2 mRNA. Both miRNAs were able to inhibit the proliferation of MCF7 cells. CONCLUSION: miR-663 and miR-744 mediate inhibition of the proto-oncogene eEF1A2 expression that results in retardation of the MCF7 cancer cells proliferation. Antitumour effect of resveratrol may include stimulation of the miR-663 and miR-744 expression.
Subject(s)
Breast Neoplasms/enzymology , Breast Neoplasms/therapy , MicroRNAs/administration & dosage , Peptide Elongation Factor 1/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Growth Processes/genetics , Cell Movement/genetics , Down-Regulation , Female , Humans , MCF-7 Cells , MicroRNAs/genetics , Peptide Elongation Factor 1/antagonists & inhibitors , Peptide Elongation Factor 1/biosynthesis , Proto-Oncogene Mas , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Resveratrol , Stilbenes/pharmacology , TransfectionABSTRACT
In order to better understand vitamin D3 in cattle metabolism, we quantified 1alpha-HYD and 24-HYD gene expression. In the kidneys of 35 male Nellore cattle, these were divided into a control group and two treatment groups (2 x 10(6) international units of vitamin D3 administered for 2 or 8 consecutive days pre-slaughter). Vitamin D3 supplementation resulted in a significant increase in 1alpha-HYD gene expression; however, significantly increased 24-HYD gene expression was only detected in cattle that had 8 days of supplementation. The finding of upregulation of 24-HYD due to vitamin D supplementation is in line with the expected rise in 24,25-di-hydroxy-vitamin D3 synthesis observed when plasma vitamin D3 concentrations are high, stimulating excretion by the organism. On the other hand, upregulation of 1alpha-HYD was unexpected, since vitamin D3 supplementation has been reported to impact these two genes in opposite directions. We conclude that vitamin D3 metabolism in these animals is more complex than previously reported.
Subject(s)
25-Hydroxyvitamin D3 1-alpha-Hydroxylase/biosynthesis , Cattle/metabolism , Cholecalciferol/pharmacology , Kidney/metabolism , Steroid Hydroxylases/biosynthesis , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/genetics , Animals , Calcium/blood , Dietary Supplements , Environmental Exposure , Gene Expression , Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)/biosynthesis , Male , Meat , Peptide Elongation Factor 1/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Ribosomal Proteins/biosynthesis , Steroid Hydroxylases/genetics , Sunlight , Vitamin D3 24-HydroxylaseABSTRACT
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total proteins contained in encystment-induced Colpoda cucullus showed that a 50-kDa protein (p50) disappeared, whereas the expression of a 49-kDa protein (p49) was enhanced in early phase of morphogenetic transformation into the resting cyst (i.e. 2-5 h after the onset of encystment induction). Puromycin or actinomycin D inhibited the alteration in the expression of p50 and p49 by the induction of encystment. These results suggest that the encystment-specific alteration in expression of these proteins is performed by a transcriptional regulation. Liquid chromatography tandem mass spectrometry analysis revealed that p50 is mitochondrial ATP synthase ß chains, and that p49 is elongation factor 1α.
Subject(s)
Ciliophora/growth & development , Gene Expression Regulation , Mitochondrial Proton-Translocating ATPases/biosynthesis , Peptide Elongation Factor 1/biosynthesis , Chromatography, Liquid , Ciliophora/enzymology , Ciliophora/genetics , Electrophoresis, Polyacrylamide Gel , Mitochondrial Proton-Translocating ATPases/chemistry , Molecular Weight , Peptide Elongation Factor 1/chemistry , Proteome/analysis , Protozoan Proteins/analysis , Tandem Mass SpectrometryABSTRACT
BACKGROUND: One of the fundamental questions in olfaction is whether olfactory receptor neurons (ORNs) behave as independent entities within the olfactory epithelium. On the basis that mature ORNs express multiple connexins, I postulated that gap junctional communication modulates olfactory responses in the periphery and that disruption of gap junctions in ORNs reduces olfactory sensitivity. The data collected from characterizing connexin 43 (Cx43) dominant negative transgenic mice OlfDNCX, and from calcium imaging of wild type mice (WT) support my hypothesis. RESULTS: I generated OlfDNCX mice that express a dominant negative Cx43 protein, Cx43/ß-gal, in mature ORNs to inactivate gap junctions and hemichannels composed of Cx43 or other structurally related connexins. Characterization of OlfDNCX revealed that Cx43/ß-gal was exclusively expressed in areas where mature ORNs resided. Real time quantitative PCR indicated that cellular machineries of OlfDNCX were normal in comparison to WT. Electroolfactogram recordings showed decreased olfactory responses to octaldehyde, heptaldehyde and acetyl acetate in OlfDNCX compared to WT. Octaldehyde-elicited glomerular activity in the olfactory bulb, measured according to odor-elicited c-fos mRNA upregulation in juxtaglomerular cells, was confined to smaller areas of the glomerular layer in OlfDNCX compared to WT. In WT mice, octaldehyde sensitive neurons exhibited reduced response magnitudes after application of gap junction uncoupling reagents and the effects were specific to subsets of neurons. CONCLUSIONS: My study has demonstrated that altered assembly of Cx43 or structurally related connexins in ORNs modulates olfactory responses and changes olfactory activation maps in the olfactory bulb. Furthermore, pharmacologically uncoupling of gap junctions reduces olfactory activity in subsets of ORNs. These data suggest that gap junctional communication or hemichannel activity plays a critical role in maintaining olfactory sensitivity and odor perception.
Subject(s)
Gap Junctions/physiology , Olfactory Receptor Neurons/physiology , Smell/physiology , Animals , Blotting, Western , Brain Mapping , Calcium/metabolism , Cell Membrane/metabolism , Connexin 43/genetics , Connexins/biosynthesis , Connexins/genetics , Electrophysiology , Gap Junctions/metabolism , Humans , Immunohistochemistry , In Situ Hybridization , Mice , Mice, Inbred C57BL , Mice, Transgenic , Odorants , Olfactory Mucosa/physiology , Olfactory Receptor Neurons/metabolism , Peptide Elongation Factor 1/biosynthesis , Peptide Elongation Factor 1/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , beta-Galactosidase/geneticsABSTRACT
The eukaryotic elongation factor 1A2 (eEF1A2) is known to retain oncogenic potential and is recognized as a novel target for cancer prevention and therapy. Resveratrol (trans-3,4',5-trihydroxystilbene), a phytoalexin present in grapes, has been reported to possess chemopreventive and chemotherapeutic activities. In the present study, we examined the growth-inhibitory effects of resveratrol in human ovarian cancer PA-1 cells, considering eEF1A2 as a potential molecular target. Pretreatment with resveratrol attenuated proliferation of serum-starved PA-1 cells stimulated with insulin or serum. Resveratrol also activated caspase-9, -7, and -3 and induced apoptosis in PA-1 cells in the presence of insulin or serum. Insulin or serum stimulation of PA-1 cells resulted in the marked induction of eEF1A2, which was suppressed by pretreatment with resveratrol. Moreover, resveratrol inhibited insulin- or serum-induced soft-agar colony formation in eEF1A2-transfected NIH3T3 cells. An antibody array directed to assess the phosphorylation of protein kinases revealed that treatment with insulin or serum induced the phosphorylation of Akt in PA-1 cells. Pharmacologic inhibition of Akt with LY294002 abrogated insulin- or serum-induced eEF1A2 expression and increased the caspase-3 activity. In another experiment, i.p. administration of resveratrol retarded the growth of PA-1 cell xenograft and the expression of eEF1A2 in athymic nude mice in association with decreased bromodeoxyuridine positivity, reduced expression of proliferating cell nuclear antigen, increased the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and caspase-3 staining, and diminished CD31 positivity. Taken together, eEF1A2 may be considered as a potential molecular target for the antiproliferative effects of resveratrol in PA-1 ovarian cancer cells.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Ovarian Neoplasms/drug therapy , Peptide Elongation Factor 1/biosynthesis , Stilbenes/pharmacology , Animals , Apoptosis/drug effects , Cell Growth Processes/drug effects , Down-Regulation/drug effects , Female , Humans , Insulin/pharmacology , Mice , Mice, Inbred BALB C , NIH 3T3 Cells , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Resveratrol , Xenograft Model Antitumor AssaysABSTRACT
The systematics of the green algal class Ulvophyceae have been difficult to resolve with ultrastructural and molecular phylogenetic analyses. Therefore, we investigated relationships among ulvophycean orders by determining the distribution of two discrete genetic characters previously identified only in the order Dasycladales. First, Acetabularia acetabulum uses the core translation GTPase Elongation Factor 1alpha (EF-1alpha) while most Chlorophyta instead possess the related GTPase Elongation Factor-Like (EFL). Second, the nuclear genomes of dasycladaleans A. acetabulum and Batophora oerstedii use a rare non-canonical genetic code in which the canonical termination codons TAA and TAG instead encode glutamine. Representatives of Ulvales and Ulotrichales were found to encode EFL, while Caulerpales, Dasycladales, Siphonocladales, and Ignatius tetrasporus were found to encode EF-1alpha, in congruence with the two major lineages previously proposed for the Ulvophyceae. The EF-1alpha of I. tetrasporus supports its relationship with Caulerpales/Dasycladales/Siphonocladales, in agreement with ultrastructural evidence, but contrary to certain small subunit rRNA analyses that place it with Ulvales/Ulotrichales. The same non-canonical genetic code previously described in A. acetabulum was observed in EF-1alpha sequences from Parvocaulis pusillus (Dasycladales), Chaetomorpha coliformis, and Cladophora cf. crinalis (Siphonocladales), whereas Caulerpales use the universal code. This supports a sister relationship between Siphonocladales and Dasycladales and further refines our understanding of ulvophycean phylogeny.
Subject(s)
Chlorophyta , Genetic Code , Peptide Elongation Factor 1/biosynthesis , Phylogeny , Chlorophyta/classification , Chlorophyta/genetics , Chlorophyta/metabolism , Evolution, Molecular , Gene Expression Regulation , Peptide Elongation Factor 1/genetics , RNA, Algal/analysis , RNA, Algal/biosynthesis , RNA, Algal/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis , Species SpecificityABSTRACT
The metastatic nature of breast cancer has been well recognized, yet the mechanisms through which breast cancer cells acquire their invasive properties have not been clearly elucidated. Our previous study indicates that BMP-6 restores E-cadherin-mediated EMT through repressing deltaEF1 in breast cancer. However, the mechanism by which BMP-6 regulates deltaEF1 expression remains unclear. In this study, we confirmed the significant role of BMP-6 in inhibiting MDA-MB-231 migration through decreasing deltaEF1 expression which subsequently relieves deltaEF1-mediated invasion. The inhibitory effect of BMP-6 through deltaEF1 regulation was supported by an inverse correlation of BMP-6/miR-192 and deltaEF1 expressions observed in both MDA-MB-231 and MCF-7 cells and clinical tumor specimens. Moreover, BMP-6 treatment or miR-192 transfection decreased the reporter activity of the deltaEF1 3'-UTR-luc, validating that deltaEF1 is a target of miR-192. Meanwhile, we also found that BMP-6 acted as a potent transcriptional repressor of the human deltaEF1 promoter. Mutation of the AP-1 binding site on this promoter abolished BMP-6-induced transrepression of deltaEF1. Depletion of BMP-6 expression by RNAi resulted in a significant increase in the promoter activity of deltaEF1. Our study has provided novel findings of a dual mechanism for BMP-6-regulated deltaEF1 expression in breast cancer cells, involving cross-talks between AP-1-mediated transcriptional repression and miRs-mediated translational inhibition.
Subject(s)
Bone Morphogenetic Protein 6/metabolism , Breast Neoplasms/genetics , Carcinoma, Ductal/genetics , Gene Expression Regulation, Neoplastic , Peptide Elongation Factor 1/biosynthesis , 3' Untranslated Regions , Bone Morphogenetic Protein 6/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cadherins/genetics , Cadherins/metabolism , Carcinoma, Ductal/metabolism , Carcinoma, Ductal/pathology , Cell Line, Tumor , Electrophoresis, Agar Gel , Female , Humans , MicroRNAs/biosynthesis , MicroRNAs/genetics , Peptide Elongation Factor 1/antagonists & inhibitors , Peptide Elongation Factor 1/genetics , Promoter Regions, Genetic , Repressor Proteins/genetics , Repressor Proteins/metabolism , Signal Transduction , Transcriptional Activation , Up-RegulationABSTRACT
When duplicated sister chromatids are not properly compacted in mitosis, chromosomes are mis-segregated, inducing genetically unstable tetraploidy known to facilitate aneuploid malignancies. Here, we show that tetraploid cells produced by impaired chromosomal condensation are eliminated by a novel type of cell death different from caspase-dependent apoptosis. The cell death was associated with downregulation of eukaryotic translation elongation factor-1 alpha 1 (eEF1A1/EF-1alpha) expression in conjunction with accumulation of its mRNA in processing bodies (P bodies). Importantly, expression of exogenous eEF1A1 was shown to inhibit the caspase-independent cell death, and a similar cell death was observed after inducing the expression of short hairpin RNA specific for eEF1A1. Furthermore, the number of spontaneously arising binucleated cells was indicated to increase several fold during 1- to 2-week cultivation after initiation of exogenous eEF1A expression. Taken together, the novel cell death machinery should help to eliminate abnormal tetraploid cells and inhibit tumorigenesis.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Chromosome Segregation , Down-Regulation , Peptide Elongation Factor 1/biosynthesis , Polyploidy , Aneuploidy , Animals , BALB 3T3 Cells , Base Sequence , CHO Cells , Cell Death , Chromatids/metabolism , Cricetinae , Cricetulus , Humans , Mice , Mitosis , Molecular Sequence Data , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , Time FactorsABSTRACT
In cultured hippocampal neurons and in adult brain, the splicing regulatory protein Sam68 is partially relocated to the somatodendritic domain and associates with dendritic polysomes. Transfer to the dendrites is activity-dependent. We have investigated the repertoire of neuronal mRNAs to which Sam68 binds in vivo. By using coimmunoprecipitation and microarray screening techniques, Sam68 was found to associate with a number of plasticity-related mRNA species, including Eef1a1, an activity-responsive mRNA coding for translation elongation factor eEF1A. In cortical neuronal cultures, translation of the Eef1a1 mRNA was strongly induced by neuronal depolarisation and correlated with enhanced association of Sam68 with polysomal mRNAs. The possible function of Sam68 in Eef1a1 mRNA utilization was studied by expressing a dominant-negative, cytoplasmic Sam68 mutant (GFP-Sam68DeltaC) in cultured hippocampal neurons. The level of eEF1A was lower in neurons expressing GFP-Sam68DeltaC than in control neurons, supporting the proposal that endogenous Sam68 may contribute to the translational efficiency of the Eef1a1 mRNA. These findings are discussed in the light of the complex, potentially crucial regulation of eEF1A biosynthesis during long-term synaptic change.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation/physiology , Neurons/metabolism , Peptide Elongation Factor 1/biosynthesis , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Animals , Cells, Cultured , Embryo, Mammalian , Hippocampus/cytology , Humans , Immunoprecipitation/methods , Protein Binding/physiology , Protein Biosynthesis , Rats , Receptors, N-Methyl-D-Aspartate/metabolism , Transfection/methodsABSTRACT
The potential role of PTI-1, in the natural story of prostate adenocarcinoma remains to be fully determined. PTI-1 expression was evaluated in human prostate cancer cell lines and in paraffin-embedded archive tissues. PTI-1 expression was found in Mycoplasma infected but not in non-infected cells. The lack of PTI-1 expression was also confirmed in fixed and paraffin-embedded human cancer prostate biopsies. The overall data indicate that, in prostate tumor cell lines, PTI-1 presence parallels Mycoplasma infection suggesting that PTI-1 might not necessarily play a major role in the onset of prostate tumorigenesis.