Emetine-treated Cells Research Articles

Efficacy and Mechanism of Action of Low Dose Emetine against Human Cytomegalovirus.

Infection with human cytomegalovirus (HCMV) is a threat for pregnant women and immunocompromised hosts. Although limited drugs are available, development of new agents against HCMV is desired. Through screening of the LOPAC library, we identified emetine as HCMV inhibitor. Additional studies confirmed its anti-HCMV activities in human foreskin fibroblasts: EC50−40±1.72 nM, CC50−8±0.56 μM, and selectivity index of 200. HCMV inhibition occurred after virus entry, but before DNA replication, and resulted in decreased expression of viral proteins. Synergistic virus inhibition was achieved when emetine was combined with ganciclovir. In a mouse CMV (MCMV) model, emetine was well-tolerated, displayed long half-life, preferential distribution to tissues over plasma, and effectively suppressed MCMV. Since the in vitro anti-HCMV activity of emetine decreased significantly in low-density cells, a mechanism involving cell cycle regulation was suspected. HCMV inhibition by emetine depended on ribosomal processing S14 (RPS14) binding to MDM2, leading to disruption of HCMV-induced MDM2-p53 and MDM2-IE2 interactions. Irrespective of cell density, emetine induced RPS14 translocation into the nucleus during infection. In infected high-density cells, MDM2 was available for interaction with RPS14, resulting in disruption of MDM2-p53 interaction. However, in low-density cells the pre-existing interaction of MDM2-p53 could not be disrupted, and RPS14 could not interact with MDM2. In high-density cells the interaction of MDM2-RPS14 resulted in ubiquitination and degradation of RPS14, which was not observed in low-density cells. In infected-only or in non-infected emetine-treated cells, RPS14 failed to translocate into the nucleus, hence could not interact with MDM2, and was not ubiquitinated. HCMV replicated similarly in RPS14 knockdown or control cells, but emetine did not inhibit virus replication in the former cell line. The interaction of MDM2-p53 was maintained in infected RPS14 knockdown cells despite emetine treatment, confirming a unique mechanism by which emetine exploits RPS14 to disrupt MDM2-p53 interaction. Summarized, emetine may represent a promising candidate for HCMV therapy alone or in combination with ganciclovir through a novel host-dependent mechanism.

Impairment of HuR-Mediated FOS mRNA Stabilization in Granulocytes From Myelodysplastic Syndrome Patients.

Abstract 2805Infection is a major cause of death in patients with myelodysplastic syndromes (MDS). Although qualitative and quantitative granulocyte defects have been described, the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. Recently, we found that FOS mRNA elevation following translation inhibition was significantly smaller in MDS granulocytes than in healthy granulocytes (Shikama et al, Br J Haematol 154, 525–527, 2011). FOS is an immediate early gene that is rapidly expressed in response to various stimuli and cellular stresses, and possesses an AU-rich element (ARE) in its mRNA 3’UTR, to which mRNA stability-regulating proteins, such as HuR, bind. The aim of this study was to clarify the cause of impaired FOS induction following translation inhibition in MDS.We first confirmed impaired FOS induction in MDS by comparing 17 patients with MDS (refractory anemia) with 17 age-matched healthy controls. Granulocyte fractions, enriched to >90% neutrophils, were cultured with 200 ng/mL of the translation inhibitor emetine at 37 °C for 30 min. FOS mRNA increased 6.2±1.1-fold in controls, vs. 3.5±0.8-fold in MDS (p<0.01). In contrast, when 5 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) or 100 ng/mL lipopolysaccharide (LPS) was added instead of emetine, there were no significant differences in the FOS mRNA elevation between controls and MDS (GM-CSF: 2.4±0.5-fold vs. 2.3±1.3-fold, LPS: 2.2±0.9-fold vs. 2.4±1.1-fold), suggesting that FOS mRNA induction by a translation inhibitor was specifically impaired in MDS granulocytes.To study the mechanisms of FOS mRNA elevation by emetine, we next examined the involvement of (1) MAPK signaling pathways, (2) FOS transcription and mRNA stabilization, and (3) ARE-binding protein HuR, using healthy granulocytes. Inhibition of MAPK p38, which completely blocked the FOS-upregulating effect of LPS, reduced FOS elevation by emetine to nearly 50%. Neither inhibition of JNK nor ERK altered the effects of emetine. Thus, emetine increases FOS mRNA via MAPK p38-dependent and MAPK-independent mechanisms. Emetine treatment for 30 min increased nascent FOS mRNA 3.5±0.8-fold, which was reduced to 1.1±0.8-fold by MAPK p38 inhibition (p<0.01). When transcription was inhibited, FOS mRNA decayed to 19.2±9.1% of the initial level in 45 min. In the presence of emetine, 78.1±23.5% of FOS mRNA remained at 45 min, and was not affected by p38 inhibition. These data indicate that FOS mRNA elevation by emetine results from both upregulation of transcription via MAPK p38 and p38-independent mRNA stabilization. RNA-protein complex immunoprecipitation showed that 2.5±1.2-fold more FOS mRNA 3’UTR coprecipitated with HuR in emetine-treated cells than in untreated cells (p<0.05). GAPDH mRNA 3’UTR without ARE was not detected regardless of emetine treatment. Although HL60 cells treated with HuR and control siRNA showed similar FOS mRNA decay rates in the absence of emetine, emetine treatment led to a significantly lower FOS mRNA at 45 min in HuR-reduced cells (48.0±12.8%) than in control cells (77.4±13.9%, p<0.05). Thus, emetine stabilizes FOS mRNA via upregulated interaction of HuR with FOS mRNA.Finally, we investigated which process was impaired in MDS, p38-mediated FOS transcription, or HuR-mediated mRNA stabilization. The increase of FOS transcription in MDS (2.0±0.3-fold) did not significantly differ from that in controls (2.4±0.3-fold). When FOS transcription was blocked by a MAPK p38 inhibitor, FOS mRNA increase by emetine was significantly smaller in MDS (2.9±0.7-fold) than in controls (4.4±1.5-fold, p<0.05), suggesting that the aberrant FOS induction in MDS was not due to impaired FOS transcription. FOS mRNA, which decayed similarly in MDS and control cells in the absence of emetine, remained 41.9±24.6% and 61.0±19.9% in MDS and control granulocytes, respectively, at 45 min in the presence of emetine (p<0.05).In conclusion, the aberrant FOS mRNA induction by emetine in MDS results from the impairment of HuR-mediated FOS mRNA stabilization. Since nearly 15% of the transcriptome in mammalian cells is targeted by HuR, these results may provide new insights into the functional defects in MDS. Disclosures:No relevant conflicts of interest to declare.

Antiviral Activity of Emetine Dihydrochloride Against Dengue Virus Infection

Dengue fever has become a global concern as dengue infections are prevalent in the tropics and the subtropics, and can lead to a severe life-threatening illness. Currently, there is a widespread interest to search for vaccine or antiviral therapy to combat DENV infection. In this study, we have identified emetine dihydrochloride with potent antiviral activity against DENV infection. Emetine was also shown to inhibit DENV replication consistently in all the dengue serotypes. Experiments were designed to define the stage of viral replication cycle at which emetine blocked DENV infection. Emetine did not target the entry process of DENV into cells as inhibitory effect of emetine on DENV infectivity remained when naked dengue viral RNA were transfected directly into the cells. Thus, we further investigated the inhibitory effect of emetine through timecourse studies and emetine was shown to strongly inhibit DENV infection at early stage of viral replication cycle by either affecting the synthesis viral RNA pathway or viral protein translation pathway.Quantitative RT-PCR assay indicated that emetine strongly reduced the production of positive-strand and negative-strand of DENV RNA. Ultrastructural analysis of emetine-treated cells further revealed that the formations of membranous replication complexes of DENV within cells were aborted in the presence of emetine. Together, these results suggest that emetine can inhibit DENV infection by impeding viral RNA synthesis therefore emetine could be further assessed and developed as a potential antiviral therapeutic agent against DENV infection.

Nuclei may anchor at specific locations during nuclear determination in Paramecium caudatum

Summary During the conjugation of Paramecium caudatum, the fates of postzygotic nuclear division products are closely associated with their anteroposterior locations at a critical time. Soon after the third division of the synkaryon, one of the four anterior nuclei becomes a micronucleus and the other three degenerate, while all the posterior ones differentiate into macronuclear anlagen. Recently, we reported that heat shock induced abnormal nuclear determination. To understand the mechanism of this phenomenon, the behaviour of spindles in postzygotic cells was studied using an anti-alpha tubulin antibody. In control cells without the heat shock, the spindle lengthened in a fairly straight axial line at first and then curved at the two poles of the cells, elongating continuously but no longer moving the nuclei. The eight nuclei kept their 4 to 4 anteroposterior localization, though the nuclei themselves were rotated by the attached spindles during the third telophase. In heat-treated or emetine-treated cells, spindles also elongated, but they U-turned or wound in the center region of the cells; as a result, all eight nuclei seemed to be passively moved by the spindles and lost anteroposterior localization. All of these results strongly suggest a nuclear anchoring mechanism that controls the anteroposterior nuclear localization and plays an essential role in normal nuclear determination.

Multiple sites of replication initiation in the human beta-globin gene locus.

The cell cycle-dependent, ordered assembly of protein prereplicative complexes suggests that eukaryotic replication origins determine when genomic replication initiates. By comparison, the factors that determine where replication initiates relative to the sites of prereplicative complex formation are not known. In the human globin gene locus previous work showed that replication initiates at a single site 5' to the ss-globin gene when protein synthesis is inhibited by emetine. The present study has examined the pattern of initiation around the genetically defined ss-globin replicator in logarithmically growing HeLa cells, using two PCR-based nascent strand assays. In contrast to the pattern of initiation detected in emetine-treated cells, analysis of the short nascent strands at five positions spanning a 40 kb globin gene region shows that replication initiates at more than one site in non-drug-treated cells. Quantitation of nascent DNA chains confirmed that replication begins at several locations in this domain, including one near the initiation region (IR) identified in emetine-treated cells. However, the abundance of short nascent strands at another initiation site approximately 20 kb upstream is approximately 4-fold as great as that at the IR. The latter site abuts an early S phase replicating fragment previously defined at low resolution in logarithmically dividing cells.

Role of Newly Synthesized MHC Class II Molecules in Antigen-Specific Antigen Presentation by B cells

Using a B lymphoma, A20-HL, bearing IgM receptors for TNP, we have shown that presentation of an Ag taken up through the receptors is highly sensitive, whereas that of an Ag taken up nonspecifically is resistant to inhibition of protein synthesis or protein transport from the endoplasmic reticulum. To analyze the difference, we have examined the effect of protein synthesis inhibition on A20-HL cells in terms of internalization and fragmentation of a specific Ag, TNP-OVA, distribution of MHC class II molecules. Inhibition of protein synthesis in A20-HL cells with emetine, an irreversible protein synthesis inhibitor, did not decrease the surface expression of anti-TNP receptors, or the kinetics of internalization of 125I-TNP-OVA. To detect fragmentation of TNP-OVA, A20-HL cells were incubated at 37°C in the presence of 125I-TNP-OVA, the cell lysate was analyzed in SDS-PAGE. The number of detectable fragments increased with the incubation period, inhibition of protein synthesis did not change the electrophoretic pattern. Expression of MHC class II molecules on the surface of A20-HL cells was not affected by inhibition of protein synthesis. However, intracellular MHC class II molecules markedly decreased in amount in the emetine-treated cells. Thus, presentation of an Ag taken up through Ag receptors seems to be dependent on intracellular MHC class II molecules, whereas that of an Ag taken up nonspecifically

M-phase-specific protein kinase from mitotic sea urchin eggs: Cyclic activation depends on protein synthesis and phosphorylation but does not require DNA or RNA synthesis

Histone H 1 kinase (H 1K) undergoes a transient activation at each early M phase of both meiotic and mitotic cell cycles. The mechanisms underlying the transient activation of this protein kinase were investigated in mitotic sea urchin eggs. Translocation of active H 1K from particulate to soluble fraction does not seem to be responsible for this activation. H 1K activation cannot be accounted for by the transient disappearance of a putative H 1K inhibitor present in soluble fractions of homogenates. Aphidicolin, an inhibitor of DNA synthesis, and actinomycin D, an inhibitor of RNA synthesis, do not impede the transient appearance of H 1K activity. H 1K activation therefore does not require DNA or RNA synthesis. Fertilization triggers a rise in intracellular pH responsible for the increase of protein synthesis. H 1K activation is highly dependent on the intracellular pH. Ammonia triggers an increase of intracellular pH and stimulates protein synthesis and H 1K activation. Acetate lowers the intracellular pH, decreases protein synthesis, and blocks H 1K activation. Protein synthesis is an absolute requirement for H 1K activation as demonstrated by their identical sensitivities to emetine concentration and to time of emetine addition. About 60 min after fertilization, H 1K activation and cleavage become independent of protein synthesis. The concentration of p34, a homolog of the yeast cdc2 gene product which has been recently shown to be a subunit of H 1K, does not vary during the cell cycle and remains constant in emetine-treated cells. H 1K activation thus requires the synthesis of either a p34 postranslational modifying enzyme or another subunit. Finally, phosphatase inhibitors and ATP slow down in the in vitro inactivation rate of H 1K. These results suggest that a subunit or an activator of H 1K is stored as an mRNA in the egg before mitosis and that full activation of H 1K requires a phosphorylation.

Ribosomal aggregates in amebae exposed to the protein synthesis inhibitor emetine

Amebae were studied with the electron microscope after up to 5 days exposure to emetine, which inhibits protein synthesis in eukaryotic cells. Clusters of particles, identified as aggregates of ribosomes, appeared in large numbers in the cytoplasm of emetine-treated cells. The clusters contained as many as 16 ribosomes, which appeared to be arranged in a helical pattern. Control cells and normal amebae from stock cultures contained a coarsely granular cytoplasmic matrix with scattered small clumps of ribosomes, but they lacked large ribosomal aggregates comparable to those found in emetine-treated cells. The results are discussed in relation to previous biochemical studies on the effects of emetine on ribosomes and protein synthesis.

Synthesis of trachoma agent proteins in emetine-treated cells.

Trachoma agent develops in emetine-treated cells in the absence of host cell protein synthesis. The time course of protein synthesis throughout the trachoma developmental cycle and the effect of antibiotics on this process were investigated.