Addition Of Cycloheximide Research Articles

Functional and Structural Analyses of CYP1B1 Variants Linked to Congenital and Adult-Onset Glaucoma to Investigate the Molecular Basis of These Diseases.

Glaucoma, the leading cause of irreversible blindness, appears in various forms. Mutations in CYP1B1 result in primary congenital glaucoma (PCG) by an autosomal recessive mode of inheritance while it acts as a modifier locus for primary open angle glaucoma (POAG). We investigated the molecular basis of the variable phenotypes resulting from the defects in CYP1B1 by using subclones of 23 CYP1B1 mutants reported in glaucoma patients, in a cell based system by measuring the dual activity of the enzyme to metabolize both retinol and 17β-estradiol. Most variants linked to POAG showed low steroid metabolism while null or very high retinol metabolism was observed in variants identified in PCG. We examined the translational turnover rates of mutant proteins after the addition of cycloheximide and observed that the levels of enzyme activity mostly corroborated the translational turnover rate. We performed extensive normal mode analysis and molecular-dynamics-simulations-based structural analyses and observed significant variation of fluctuation in certain segmental parts of the mutant proteins, especially at the B-C and F-G loops, which were previously shown to affect the dynamic behavior and ligand entry/exit properties of the cytochrome P450 family of proteins. Our molecular study corroborates the structural analysis, and suggests that the pathologic state of the carrier of CYP1B1 mutations is determined by the allelic state of the gene. To our knowledge, this is the first attempt to dissect biological activities of CYP1B1 for correlation with congenital and adult onset glaucomas.

Biodegradation of zearalenone by Saccharomyces cerevisiae: Possible involvement of ZEN responsive proteins of the yeast

The mycotoxin zearalenone, also known as F-2 mycotoxin or RAL is a potent estrogenic metabolite produced by some Gibberella and Fusarium species. It is a common contaminant of cereal crops, livestock and poultry products. However, detoxification of zearalenone (ZEN) remains a challenge. Recently, biological approach for ZEN detoxification is being explored. In this study, we investigated the biodegradation of ZEN by using Saccharomyces cerevisiae and the possible mechanisms involved. The findings revealed that, after 48h of incubation of S. cerevisiae in combination with ZEN, the ZEN was completely degraded by S. cerevisiae. On the contrary, heat-killed cells and cell-free culture filtrates of S. cerevisiae could not degrade ZEN. Furthermore, addition of cycloheximide to S. cerevisiae combined with ZEN at time 0h prevented ZEN degradation, while addition of cycloheximide at 12h significantly slowed down degradation. The results also indicated cellular proteomics of S. cerevisiae. Several differential proteins were identified, most of which were related to basic metabolism. Biological significanceThe findings revealed that, after 48h of incubating ZEN together with S. cerevisiae, ZEN was completely degraded by S. cerevisiae. The mechanisms involved in the degradation of ZEN by S. cerevisiae may be the production of associated intracellular and extracellular enzymes, which have the ability to degrade ZEN. In addition, there were some functional proteins produced by S. cerevisiae, indicating that the basic metabolism of S. cerevisiae was improved when ZEN was added. This novel discovery by the authors, will greatly contribute to the field of biodegradation of mycotoxin by antagonists. The authors also believed this innovation will open the grounds for further research and improvement of S. cerevisiae in the field of biodegradation.

Cycloheximide Chase Analysis of Protein Degradation in <em>Saccharomyces cerevisiae</em>

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae.

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Hairy and Groucho mediate the action of juvenile hormone receptor Methoprene-tolerant in gene repression

The arthropod-specific juvenile hormone (JH) controls numerous essential functions. Its involvement in gene activation is known to be mediated by the transcription factor Methoprene-tolerant (Met), which turns on JH-controlled genes by directly binding to E-box-like motifs in their regulatory regions. However, it remains unclear how JH represses genes. We used the Aedes aegypti female mosquito, in which JH is necessary for reproductive maturation, to show that a repressor, Hairy, is required for the gene-repressive action of JH and Met. The RNA interference (RNAi) screen for Met and Hairy in the Aedes female fat body revealed a large cohort of Met- and Hairy-corepressed genes. Analysis of selected genes from this cohort demonstrated that they are repressed by JH, but RNAi of either Met or Hairy renders JH ineffective in repressing these genes in an in vitro fat-body culture assay. Moreover, this JH action was prevented by the addition of the translational inhibitor cycloheximide (CHX) to the culture, indicating the existence of an indirect regulatory hierarchy. The lack of Hairy protein in the CHX-treated tissue was verified using immunoblot analysis, and the upstream regions of Met/Hairy-corepressed genes were shown to contain common binding motifs that interact with Hairy. Groucho (gro) RNAi silencing phenocopied the effect of Hairy RNAi knockdown, indicating that it is involved in the JH/Met/Hairy hierarchy. Finally, the requirement of Hairy and Gro for gene repression was confirmed in a cell transfection assay. Thus, our study has established that Hairy and its cofactor Gro mediate the repressive function of JH and Met.

Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast.

Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX) to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics.

TNF Apoptosis Protection Fraction (TAPF) prevents apoptosis induced by TNF, but not by Fas or TRAIL, via NF-κB-induced increase in cFLIP

Tumor Necrosis Factor α (TNFα) induces both the apoptotic pathway and anti-apoptotic factors. Incubation of human dermal fibroblasts with TAPF (TNF Apoptosis Protection Fraction) protects them from apoptosis induced by the subsequent addition of TNF and cycloheximide (CHX). TAPF does not protect against apoptosis induced by CHX in combination with either TRAIL (TNF related apoptosis inducing ligand) or an agonistic Fas antibody, or against apoptosis induced by the chemotherapeutic agent doxorubicin. Incubation with TAPF does not affect the quantity of TNF that binds to the cell. TAPF prevents TNF-induced cleavage of caspases 8, 9, 3 and 7 and the apoptotic substrate PARP (poly-ADP ribose polymerase), but has no effect when these molecules are induced by an agonistic Fas antibody. TAPF induces rapid phosphorylation of the NF-κB/p65 (nuclear factor-κB) transcription factor at serine 536 which is indicative of its activation. TAPF increases the expression of cFLIP (cellular FLICE-inhibitory protein) which is a potent inhibitor of apoptosis that acts by preventing the cleavage of caspase 8. This increase in cFLIP is coincident with protection from TNF-induced apoptosis. Decreasing cFLIP levels using shRNA (short hairpin RNA) decreases protection by TAPF. TAPF also induced the anti-apoptotic A20 protein. These data indicate that TAPF protects human dermal fibroblasts from TNF-induced apoptosis by induction of cFLIP and subsequent inhibition of caspase 8 cleavage.

Mechanical Force–induced TGFB1 Increases Expression of SOST/POSTN by hPDL Cells

The aim of this study was to investigate the response of human periodontal ligament (hPDL) fibroblasts to an intermittent compressive force and its effect on the expression of SOST, POSTN, and TGFB1. A computerized cell compressive force loading apparatus was introduced, and hPDL cells were subjected to intermittent compressive force. The changes in messenger RNA (mRNA) and protein expression were monitored by real-time polymerase chain reaction and Western blot analysis, respectively. An increased expression of SOST, POSTN, and TGFB1 was observed in a time-dependent fashion. Addition of cycloheximide, a transforming growth factor (TGF)–β inhibitor (SB431542), or a neutralizing antibody against TGF-β1 attenuated the force-induced expression of SOST and POSTN as well as sclerostin and periostin, indicating a role of TGF-β1 in the pressure-induced expression of these proteins. Enzyme-linked immunosorbent assay analysis revealed an increased level of TGF-β1 in the cell extracts but not in the medium, suggesting that intermittent compressive force promoted the accumulation of TGF-β1 in the cells or their surrounding matrix. In conclusion, an intermittent compressive force regulates SOST/POSTN expression by hPDL cells via the TGF-β1 signaling pathway. Since these proteins play important roles in the homeostasis of the periodontal tissue, our results indicate the importance of masticatory forces in this process.

Identification of two juvenile hormone inducible transcription factors from the silkworm, Bombyx mori

Juvenile hormone (JH) regulates many physiological processes in insects. However, the signal cascades in which JH is active have not yet been fully elucidated, particularly in comparison to another major hormone ecdysteroid. Here we identified two JH inducible transcription factors as candidate components of JH signaling pathways in the silkworm, Bombyx mori. DNA microarray analysis showed that expression of two transcription factor genes, E75 and Enhancer of split mβ (E(spl)mβ), was induced by juvenile hormone I (JH I) in NIAS-Bm-aff3 cells. Real time RT-PCR analysis confirmed that expression of four E75 isoforms (E75A, E75B, E75C and E75D) and E(spl)mβ was 3–8 times greater after JH I addition. Addition of the protein synthesis inhibitor cycloheximide did not suppress JH-induced expression of the genes, indicating that they were directly induced by JH. JH-induced expression of E75 and E(spl)mβ was also observed in four other B. mori cell lines and in larval hemocytes of final instar larvae. Notably, E75A expression was induced very strongly in larval hemocytes by topical application of the JH analog fenoxycarb; the level of induced expression was comparable to that produced by feeding larvae with 20-hydroxyecdysone. These results suggest that E75 and E(spl)mβ are general and direct target genes of JH and that the transcription factors encoded by these genes play important roles in JH signaling.

Stress-induced expression of NICOTINE2-locus genes and their homologs encoding Ethylene Response Factor transcription factors in tobacco

Plants have evolved diverse defense metabolites as adaptations to biotic and abiotic stresses. The defense alkaloid nicotine is produced in Nicotiana tabacum (tobacco) and its biosynthesis is elicited by jasmonates in the roots. At least seven jasmonate-responsive genes that encode transcription factors of the Ethylene Response Factor (ERF) family are clustered at the nicotine-regulatory locus NICOTINE2 (NIC2) in the tobacco genome. A subset of the NIC2-locus ERFs and their homologs, including ERF189 and ERF199, have been shown to be most effective in controlling nicotine biosynthetic pathway genes. Herein reported is that the ERF genes of this group, other than ERF189 and ERF199, were strongly induced by NaCl in tobacco hairy roots, although salt stress had no effect on expression of nicotine biosynthesis genes. Abscisic acid and osmotic stress also increased expression of a subset of these NaCl-inducible ERF genes. Promoter expression analysis in transgenic tobacco hairy roots confirmed that while methyl jasmonate (MJ) activated the promoters of ERF29, ERF210 and ERF199, salt stress up-regulated the promoters of only ERF29 and ERF210, but not ERF199. The protein biosynthesis inhibitor cycloheximide induced expression of the ERFs, and simultaneous addition of MJ and cycloheximide showed synergistic effects. These results indicate that, after several gene duplication events, the NIC2-locus ERFs and possibly their homologs appear to have diverged in their responses to jasmonates and various environmental inputs, including salt stress, and may have evolved to regulate distinct metabolic processes and cellular responses.

Activation of c-jun N-terminal kinase upon influenza A virus (IAV) infection is independent of pathogen-related receptors but dependent on amino acid sequence variations of IAV NS1.

A hallmark cell response to influenza A virus (IAV) infections is the phosphorylation and activation of c-jun N-terminal kinase (JNK). However, so far it is not fully clear which molecules are involved in the activation of JNK upon IAV infection. Here, we report that the transfection of influenza viral-RNA induces JNK in a retinoic acid-inducible gene I (RIG-I)-dependent manner. However, neither RIG-I-like receptors nor MyD88-dependent Toll-like receptors were found to be involved in the activation of JNK upon IAV infection. Viral JNK activation may be blocked by addition of cycloheximide and heat shock protein inhibitors during infection, suggesting that the expression of an IAV-encoded protein is responsible for JNK activation. Indeed, the overexpression of nonstructural protein 1 (NS1) of certain IAV subtypes activated JNK, whereas those of some other subtypes failed to activate JNK. Site-directed mutagenesis experiments using NS1 of the IAV H7N7, H5N1, and H3N2 subtypes identified the amino acid residue phenylalanine (F) at position 103 to be decisive for JNK activation. Cleavage- and polyadenylation-specific factor 30 (CPSF30), whose binding to NS1 is stabilized by the amino acids F103 and M106, is not involved in JNK activation. Conclusively, subtype-specific sequence variations in the IAV NS1 protein result in subtype-specific differences in JNK signaling upon IAV infection. Influenza A virus (IAV) infection leads to the activation or modulation of multiple signaling pathways. Here, we demonstrate for the first time that the c-jun N-terminal kinase (JNK), a long-known stress-activated mitogen-activated protein (MAP) kinase, is activated by RIG-I when cells are treated with IAV RNA. However, at the same time, nonstructural protein 1 (NS1) of IAV has an intrinsic JNK-activating property that is dependent on IAV subtype-specific amino acid variations around position 103. Our findings identify two different and independent pathways that result in the activation of JNK in the course of an IAV infection.

Isolation, Purification, and Characterization of Antimicrobial Substances from Endophytic Actinomycetes

Antimicrobial active substances produced by endophytic actinomycetes were isolated and purified. Plant samples were obtained from four different medicinal plants namely Curcuma domestica, Phaleria macrocarpa, Isotoma longiflora, and Symplocos cocchinensis. Isolation of actinomycetes was conducted using HV agar with the addition of cycloheximide, nystatin, nalidixic acid, and rifamycin. A total of 21 actinomycete isolates were obtained and tested for antimicrobial activity against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853, and Bacillus subtilis ATCC 66923. Among the isolates, isolate KY01 was the most active to Gram-positive and Gram-negative bacteria. Morphological observation and identification using 16S rRNA gene sequence showed that the isolate KY01 was similar to Streptomyces antibioticus. An active compound from the isolate KY01 was produced using yeast peptone medium. The active compound was purified using silica-gel-column chromatography and preparative high performance liquid chromatography (HPLC). A single peak of the active compound was detected with HPLC and LCMS, which showed a retention time of 26.6 min and molecular weight (MW) 906.4474 g/mol, respectively.

Induction and carbon catabolite repression of isoamylase production in Rhizopus oryzae PR7

The growth and the extracellular isoamylase production by Rhizopus oryzae PR7 MTCC 9642 were studied in a stationary culture at 28°C, with maximum isoamylase production obtained after 72 hours. Glycogen was found to be the best inducer for isoamylase synthesis, followed by maltose and dextrin. The enzyme was found to be repressed by glucose and this repression was not overcome by the addition of cGMP. The abrupt reduction in enzyme synthesis after the addition of exogenous glucose in a glycogen-induced culture medium confirmed the repressive action of glucose. An almost similar rate of repression was found to be exerted by α- and β-cyclodextrins. The inhibition of enzyme production after the addition of cycloheximide, a translation blocker, indicated the existence of de novo synthesis of the enzyme.

N2O emission from cropland field soil through fungal denitrification after surface applications of organic fertilizer

Agricultural fields are one of the largest anthropogenic sources of atmospheric nitrous oxide (N2O). Fungi have been suggested to contribute substantially to N2O emission in terrestrial environments; however, the extent of the fungal contribution and the phylogenetic and physiologic nature of related fungal communities in agricultural fields are largely unknown. This study focused on the large N2O emission from cropland soil that occurs after granular organic fertilizers were applied to the surface. The granular organic fertilizers applied were evidently covered by fungal mycelia. An experiment using a soil microcosm was established to imitate the field observations. N2O emissions following surface organic fertilization were suppressed by 84 and 20% after the addition of cycloheximide (a fungal inhibitor) and streptomycin (a bacterial inhibitor), respectively, suggesting that fungi provide the main contribution to the observed N2O emission. The population density and community composition of fungi in the surface-fertilized and non-fertilized soils in the field were determined using colony counting, denaturing gradient gel electrophoresis and subsequent phylogenetic analyses. Thirty-four fungal strains were isolated from the soils, and their N2O producing activities were analyzed. Fungal population density in the surface-fertilized soil (2.6 × 106 CFU/g) was much higher than that in the non-fertilized soil (1.0 × 105 CFU/g). In addition, the fungal community compositions of the soils differed. Actinomucor elegans, Bionectria ochroleuca, Fusarium avenaceum, Fusarium equiseti, Fusarium oxysporum, Fusarium solani and Nectria sp. dominated the surface-fertilized soil, and their activity in producing N2O was confirmed. These results suggested that N2O emission after the surface application of granular organic fertilizers in the cropland field mainly resulted from fungal denitrification.

Dissecting the Heat Stress Response in Chlamydomonas by Pharmaceutical and RNAi Approaches Reveals Conserved and Novel Aspects

To study how conserved fundamental concepts of the heat stress response (HSR) are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotolerance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenuation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cell's entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP70B-antisense strains.

The significant contribution of fungi to soil N2O production across diverse ecosystems

Sporadic observations from pure culture study and direct soil measurement have indicated that fungi can substantially contribute to soil N2O production. Yet, it is still uncertain whether this fungal significance is a more general ecological phenomenon. In this study, relative contributions of fungi and bacteria to soil N2O production were examined in five ecosystems, including conventional farming (CON), integrated crop and livestock system (ICL), organic farming (ORG), plantation forestry (PF), and abandoned agriculture field subjected to natural succession (SUCC). Soil N2O production was measured at 90% water-filled pore space from antibiotic-free controls and soils amended with streptomycin, cycloheximide, or both. Streptomycin and cycloheximide additions significantly reduced soil N2O fluxes from the five systems, ranging from 31% to 54% and 40% to 51%, respectively. Fungi contributed more to soil N2O fluxes than bacteria in PF, whereas fungi and bacteria made comparable contributions in other four systems. Furthermore, soil pH was correlated positively with the percentage of bacterial contribution to soil N2O flux, but negatively with the percentage of fungal contribution to soil N2O flux as well as the ratio of fungal-to-bacterial contributions. Our results showed that fungi could potentially contribute to soil N2O production in diverse agroecosystems and their contribution might be more pronounced in the acidic plantation forestry.

Spatial Reorganization of Saccharomyces cerevisiae Enolase To Alter Carbon Metabolism under Hypoxia

Hypoxia has critical effects on the physiology of organisms. In the yeast Saccharomyces cerevisiae, glycolytic enzymes, including enolase (Eno2p), formed cellular foci under hypoxia. Here, we investigated the regulation and biological functions of these foci. Focus formation by Eno2p was inhibited temperature independently by the addition of cycloheximide or rapamycin or by the single substitution of alanine for the Val22 residue. Using mitochondrial inhibitors and an antioxidant, mitochondrial reactive oxygen species (ROS) production was shown to participate in focus formation. Focus formation was also inhibited temperature dependently by an SNF1 knockout mutation. Interestingly, the foci were observed in the cell even after reoxygenation. The metabolic turnover analysis revealed that [U-(13)C]glucose conversion to pyruvate and oxaloacetate was accelerated in focus-forming cells. These results suggest that under hypoxia, S. cerevisiae cells sense mitochondrial ROS and, by the involvement of SNF1/AMPK, spatially reorganize metabolic enzymes in the cytosol via de novo protein synthesis, which subsequently increases carbon metabolism. The mechanism may be important for yeast cells under hypoxia, to quickly provide both energy and substrates for the biosynthesis of lipids and proteins independently of the tricarboxylic acid (TCA) cycle and also to fit changing environments.

Abstract 2940: Statin induced apoptosis in human melanoma cells is prevented by inhibition of caspase 2 and translational activity.

Abstract Background: Statins may trigger apoptosis in tumour cells in vitro and, particularly, melanoma cells are susceptible to statin induced apoptosis. Lipophilic statins (e.g. Atorvastatin, Simvastatin) induce strong caspase 3 activation after 48h of treatment, whereas hydrophilic statins (e.g. Pravastatin, Rosuvastatin) do not trigger caspase activation. Recently, we demonstrated the presence of an autocrine amplification loop which increases apoptosis by a caspase 8 dependent pathway after treatment with lipophilic statins (Minichsdorfer and Hohenegger, Br J Ph 2009). It is known that activation of caspase 2 may prime melanoma cells for death receptor induced apoptosis, however, although an association between caspase 2 activation and RhoB upregulation was shown, the significance of this interaction is still not clear. Methods: We investigated the activity of caspase 2, 3 and 8 in A375 and 518A2 human metastatic melanoma cells after statin treatment in the presence and absence of a caspase 2 inhibitor (Z-VDVAD-FMK) or cycloheximide by cleavage of specific fluorescent caspase substrates. Upregulation of RhoB after statin treatment as well as the influence of the co-treatment with the isoprenoids farnesylpyrophosphate (FPP) or geranylgeranylpyrophosphate (GGPP) was studied by Western blot. Results: Exposure of these cells to simvastatin led to increased caspase 2 activation which is abrogated by co-application of the caspase 2 inhibitor Z-VDVAD-FMK. Moreover, treatment with Z-VDVAD-FMK also prevented caspase 8 and caspase 3 activation. We could show that production of an autocrine factor needs an intact translation machinery. Inhibition of translation by co-administration of cycloheximide prevented apoptotic morphological changes as well as caspase 3 and caspase 8 activation in A375 and 518A2 cells. Since caspase 2 may interact with RhoB, we also investigated the effect of statin treatment on RhoB expression and observed an upregulation of RhoB which was inhibited by the addition of cycloheximide in both cell lines. However, isoprenoids were not able to reverse RhoB upregulation or PARP cleavage in 518A2 cells. Conclusion: Overall, our data provide evidence for an important role of caspase 2 in statin induced apoptosis of 2 human metastatic melanoma cell lines. These findings may help to understand the function of caspase 2 in the induction of apoptosis. Moreover, we observed upregulation of RhoB following statin treatment in these cell lines. However, additional experiments are necessary to fully understand the role of RhoB in statin induced apoptosis. Citation Format: Christoph Minichsdorfer, Christine Wasinger, Evelyn Sieczkowski, Atil Bihter, Gerwin Heller, Sabine Zöchbauer-Müller, Martin Hohenegger. Statin induced apoptosis in human melanoma cells is prevented by inhibition of caspase 2 and translational activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2940. doi:10.1158/1538-7445.AM2013-2940

Measles virus nucleocapsid protein, a key contributor to Paget's disease, increases IL-6 expression via down-regulation of FoxO3/Sirt1 signaling

Measles virus plays an important role as an environmental factor in the pathogenesis of Paget's disease (PD). Previous studies have shown that IL-6 is increased in the bone marrow of Paget's patients and that measles virus nucleocapsid protein (MVNP) induces IL-6 secretion by pagetic osteoclasts. Further, IL-6 plays a critical role in the development of pagetic osteoclasts and bone lesions induced by PD, but the mechanisms regulating IL-6 production by MVNP remain unclear. Our current studies revealed that MVNP expression in osteoclast precursors down-regulated Sirt1 mRNA and protein, a negative regulator of NF-κB activity, which is a key factor for IL-6 expression. MVNP expression in NIH3T3 cells also elevated Il-6 transcription and impaired the expression of Sirt1 mRNA both under basal conditions and upon activation of the Sirt1 upstream regulator FoxO3 by LY294002 (a PI3K/AKT inhibitor). Luciferase activity assays showed that constitutively active FoxO3 abolished the repressive effect of MVNP on reporters driven by either FoxO3 response elements or the Sirt1 promoter. Further, protein stability assays revealed that FoxO3 was degraded more rapidly in MVNP-expressing cells than in control cells following the addition of cycloheximide. Similarly, co-transfection of MVNP and FoxO3 into HEK293 cells demonstrated that MVNP decreased the protein levels of over-expressed FoxO3 in a dose-dependent manner. Treatment with the proteasome inhibitor, MG132, blocked the MVNP-triggered decrease of FoxO3, and the treatment with the serine/threonine phosphatase inhibitor, calyculin A, revealed that MVNP increased phosphorylation of FoxO3. Further, over-expression of Sirt1 or treatment with the Sirt1 activator resveratrol blocked the increase in Il-6 transcription by MVNP. Finally, resveratrol reduced the numbers of TRAP positive multi-nuclear cells in bone marrow cultures from TRAP-MVNP transgenic mice to wild type levels. These results indicate that MVNP decreases FoxO3/Sirt1 signaling to enhance the levels of IL-6, which in part mediate MVNP's contribution to the development of Paget's disease.

The Pias-Like Coactivator ZMIZ1 Drives C-MYC Induction in Collaboration with NOTCH1 in T-Cell Acute Lymphoblastic Leukemia.

Abstract 2380Activating NOTCH1 mutations are found in 50–60% of human T-cell acute lymphoblastic leukemia (T-ALL) samples. In mouse models, these mutations generally fail to induce leukemia. Cooperating oncogenes must be recruited by NOTCH1 to fully induce leukemia. Murine insertional mutagenesis screens previously implicated ZMIZ1 as a possible NOTCH1 collaborator in leukemia (Uren et al., Cell, 2008; Dupuy et al., Nature, 2005; Berquam-Vrieze et al., Blood, 2011). ZMIZ1 is a transcriptional co-activator of the Protein Inhibitor of Activated STAT (PIAS)-like family. It shares a zinc finger domain, the MIZ domain, with PIAS proteins. The MIZ domain mediates interactions with DNA-binding transcription factors and sumoylation. Previously, we showed that ZMIZ1 promotes T-ALL in collaboration with leukemia-associated NOTCH1 alleles in mouse models. ZMIZ1 and activated NOTCH1 were co-expressed in a subset of human patients. Genetic ZMIZ1 inhibition slowed leukemic cell growth and overcame resistance of some T-ALL cell lines to NOTCH inhibitors. ZMIZ1 may be a new clinically relevant oncogene. Here we sought to determine the downstream target genes of ZMIZ1 in leukemia. Validation of gene expression profiling data identified C-MYC and IL7RA as downstream targets of ZMIZ1. Targeting the C-MYC or IL-7 pathways using genetic and pharmacological inhibitors partly phenocopied the growth inhibitory effects we previously saw with ZMIZ1 inhibition. In order to determine whether these genes are direct or indirect targets of ZMIZ1, we generated an estrogen fusion protein, ZMIZ1-ER. ZMIZ1-ER induced C-MYC and IL7RA expression in the presence of tamoxifen, but failed to induce these genes with the addition of cycloheximide. These data suggest that C-MYC and IL-7RA are indirect targets. Like the PIAS proteins, ZMIZ1 appeared to have a broad effect on transcription to exert its functions. We next sought to elucidate the biochemical mechanism of ZMIZ1. Ectopic expression of ZMIZ1 or NOTCH1 had weak effects on endogenous c-Myc expression and failed to rescue a C-MYC-dependent T-ALL cell line after withdrawal of ectopic C-MYC. In contrast, ZMIZ1 in combination with NOTCH1 dramatically induced C-MYC expression by several fold and rescued the C-MYC dependent cell line. ZMIZ1 enhanced the ability of even weak NOTCH1 mutants to induce C-MYC, suggesting a mechanism by which ZMIZ1 may increase resistance to NOTCH inhibitors. ZMIZ1 did not influence C-MYC expression post-transcriptionally. It functioned primarily as a transcriptional activator. Although both C-MYC and IL7RA are both NOTCH1 target genes, ZMIZ1 did not directly interact with NOTCH1 or influence the expression of several other NOTCH1 target genes such Ptcra, Hes1, Dtx1, and Cd25. Thus, ZMIZ1 did not pan-activate NOTCH signaling. Based on bioinformatic analysis, we generated mutants that deleted individual domains of ZMIZ1. All mutants expressed at high levels by Western blot. Deletion of the transcriptional activation domain or the N-terminal domain (NTD) abolished the ability of ZMIZ1 to induce c-Myc and drive proliferation. Surprisingly, deletion of the PAT-like, Proline-rich, and MIZ domains or all three domains simultaneously had no effect on ZMIZ1 function. The 120-amino acid NTD has a predicted helical structure without significant sequence homology to any known domain. It is not found in ZMIZ2 or PIAS proteins. In summary, the mechanism of ZMIZ1 appears to be novel, indirect, transcriptional, and independent of canonical NOTCH and PIAS functions. Our study demonstrates the importance of characterizing genetic collaborations between parallel leukemic pathways that may be therapeutically targeted. They also raise new inquiries into potential NOTCH-ZMIZ1 collaboration in a variety of C-MYC-driven cancers. Disclosures:No relevant conflicts of interest to declare.