scholarly journals Proteomic Analysis of Trichoderma atroviride Reveals Independent Roles for Transcription Factors BLR-1 and BLR-2 in Light and Darkness

2011 ◽  
Vol 11 (1) ◽  
pp. 30-41 ◽  
Author(s):  
Alejandro Sánchez-Arreguín ◽  
Ana Silvia Pérez-Martínez ◽  
Alfredo Herrera-Estrella
Keyword(s):  
Transcription Factors ◽  
Blue Light ◽  
Light Pulse ◽  
Mechanical Damage ◽  
Pathogenic Fungi ◽  
Mrna Levels ◽  
Trichoderma Atroviride ◽  
Proteomic Profiling ◽  
Content Type ◽  
Protein Levels

ABSTRACT The genus Trichoderma is one of the most widely used biological control agents of plant-pathogenic fungi. The main mechanism for survival and dispersal of Trichoderma is through the production of asexual spores (conidia). The transition from filamentous growth to conidiation can be triggered by light, nutrient deprivation, and mechanical damage of the mycelium. We conducted proteomic profiling analyses of Trichoderma atroviride after a blue light pulse. The use of two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) analysis allowed us to identify 72 proteins whose expression was affected by blue light. Functional category analysis showed that the various proteins are involved in metabolism, cell rescue, and protein synthesis. We determined the relationship between mRNA levels of selected genes 30 min after a light pulse and protein expression levels at different times after the pulse and found this correlation to be very weak. The correlation was highest when protein and mRNA levels were compared for the same time point. The transcription factors BLR-1 and BLR-2 are vital to the photoconidiation process; here we demonstrate that both BLR proteins are active in darkness and affect several elements at both the transcript and protein levels. Unexpectedly, in darkness, downregulation of proteins prevailed in the Δ blr-1 mutant, while upregulation of proteins predominated in the Δ blr-2 mutant. Our data demonstrate that the BLR proteins play roles individually and as a complex.

scholarly journals Restoration of Noradrenergic Function in Parkinson’s Disease Model Mice

ASN NEURO ◽  
2021 ◽  
Vol 13 ◽  
pp. 175909142110097
Author(s):  
Kui Cui ◽  
Fan Yang ◽  
Turan Tufan ◽  
Muhammad U. Raza ◽  
Yanqiang Zhan ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Transcription Factors ◽  
Disease Model ◽  
Neuronal Loss ◽  
Mrna Levels ◽  
Gene Therapies ◽  
Protein Levels ◽  
Dopaminergic Systems ◽  
And Function

Dysfunction of the central noradrenergic and dopaminergic systems is the primary neurobiological characteristic of Parkinson’s disease (PD). Importantly, neuronal loss in the locus coeruleus (LC) that occurs in early stages of PD may accelerate progressive loss of dopaminergic neurons. Therefore, restoring the activity and function of the deficient noradrenergic system may be an important therapeutic strategy for early PD. In the present study, the lentiviral constructions of transcription factors Phox2a/2b, Hand2 and Gata3, either alone or in combination, were microinjected into the LC region of the PD model VMAT2 Lo mice at 12 and 18 month age. Biochemical analysis showed that microinjection of lentiviral expression cassettes into the LC significantly increased mRNA levels of Phox2a, and Phox2b, which were accompanied by parallel increases of mRNA and proteins of dopamine β-hydroxylase (DBH) and tyrosine hydroxylase (TH) in the LC. Furthermore, there was considerable enhancement of DBH protein levels in the frontal cortex and hippocampus, as well as enhanced TH protein levels in the striatum and substantia nigra. Moreover, these manipulations profoundly increased norepinephrine and dopamine concentrations in the striatum, which was followed by a remarkable improvement of the spatial memory and locomotor behavior. These results reveal that over-expression of these transcription factors in the LC improves noradrenergic and dopaminergic activities and functions in this rodent model of PD. It provides the necessary groundwork for the development of gene therapies of PD, and expands our understanding of the link between the LC-norepinephrine and dopamine systems during the progression of PD.

scholarly journals Mammalian ECD Protein Is a Novel Negative Regulator of the PERK Arm of the Unfolded Protein Response

2017 ◽  
Vol 37 (18) ◽  
Author(s):  
Appolinaire A. Olou ◽  
Aniruddha Sarkar ◽  
Aditya Bele ◽  
C. B. Gurumurthy ◽  
Riyaz A. Mir ◽  
...  
Keyword(s):  
Er Stress ◽  
Translation Initiation Factor ◽  
Negative Regulator ◽  
Initiation Factor ◽  
Mrna Levels ◽  
Produce Cell ◽  
Stress Induction ◽  
Content Type ◽  
Protein Levels ◽  
The Unfolded Protein Response

ABSTRACT Mammalian Ecdysoneless (ECD) is a highly conserved ortholog of the Drosophila Ecd gene product whose mutations impair the synthesis of Ecdysone and produce cell-autonomous survival defects, but the mechanisms by which ECD functions are largely unknown. Here we present evidence that ECD regulates the endoplasmic reticulum (ER) stress response. ER stress induction led to a reduced ECD protein level, but this effect was not seen in PKR-like ER kinase knockout (PERK-KO) or phosphodeficient eukaryotic translation initiation factor 2α (eIF2α) mouse embryonic fibroblasts (MEFs); moreover, ECD mRNA levels were increased, suggesting impaired ECD translation as the mechanism for reduced protein levels. ECD colocalizes and coimmunoprecipitates with PERK and GRP78. ECD depletion increased the levels of both phospho-PERK (p-PERK) and p-eIF2α, and these effects were enhanced upon ER stress induction. Reciprocally, overexpression of ECD led to marked decreases in p-PERK, p-eIF2α, and ATF4 levels but robust increases in GRP78 protein levels. However, GRP78 mRNA levels were unchanged, suggesting a posttranscriptional event. Knockdown of GRP78 reversed the attenuating effect of ECD overexpression on PERK signaling. Significantly, overexpression of ECD provided a survival advantage to cells upon ER stress induction. Taken together, our data demonstrate that ECD promotes survival upon ER stress by increasing GRP78 protein levels to enhance the adaptive folding protein in the ER to attenuate PERK signaling.

Pax5 Activates Oncogenic Transcription Factors of the Ets Family by Repressing the mir-15a/16-1 microRNA Cluster.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 346-346
Author(s):  
Elaine Y. Chung ◽  
Diana Cozma ◽  
Duonan Yu ◽  
Michael Dews ◽  
Erik A. Wentzel ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cell ◽  
Western Blotting ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Down Regulation ◽  
Protein Levels ◽  
Ets Family ◽  
Post Transcriptional Regulation

Abstract We have recently demonstrated that Pax5 promotes B-lymphomagenesis by upregulating key components of B-cell receptor signaling [Cozma et al, J Clin Inv, 117 (8), 2007]. Gene regulation by Pax5 often involves complex formation with other oncogenic transcription factors of the Ets family, namely Myb and Ets1. We determined that expression of these proteins themselves depends on the presence of Pax5, as seen in human diffuse large B-cell lymphomas with Pax5 knockdown and murine lymphomas with epigenetic silencing of Pax5 [Yu et al, Blood, 101:1950–1955, 2003; Johnson et al, Nat Immunol, 5:853–861, 2004]. Upon reconstitution with the Pax5 gene, Myb and Ets1 levels increase sharply. This occurs with little increase in steady-state mRNA levels, suggesting post-transcriptional regulation, possibly by microRNAs. To test this hypothesis, we compared miRNA profiles of Pax5-deficieint and sufficient cells and discovered that several miRNAs are indeed repressed by Pax5. Among them is the miR-15a/16-1 cluster whose predicted targets include both Myb and Ets1. Consistent with this prediction, forced expression of miR-15a/16 brings down Myb and Ets1 protein levels. This is accompanied by impaired Pax5 function and overall suppression of B-lymphomagenesis. Thus, Ets family members (along with previously identified bcl-2) are key targets of the miR-15a/16 locus, a tumor suppressor in chronic lymphocytic leukemia. Interplay between Pax5, Myb/Ets1, and miR-15a/16-1. (A) Upregulation of Myb and Ets 1 in tumors over-expressing Pax5ER fusion, as compared to control GFP-only neoplasms. (B) Down-regulation of Myb and Ets1 in Pax5 tumors engineered to over-express the miR-15a/16-1 cluster. All panels depict Western blotting. Interplay between Pax5, Myb/Ets1, and miR-15a/16-1. (A) Upregulation of Myb and Ets 1 in tumors over-expressing Pax5ER fusion, as compared to control GFP-only neoplasms. (B) Down-regulation of Myb and Ets1 in Pax5 tumors engineered to over-express the miR-15a/16-1 cluster. All panels depict Western blotting.

scholarly journals Role of UBC9 in the Regulation of the Adipogenic Program in 3T3-L1 Adipocytes

Endocrinology ◽  
2010 ◽  
Vol 151 (11) ◽  
pp. 5255-5266 ◽  
Author(s):  
Angelo Cignarelli ◽  
Mariangela Melchiorre ◽  
Alessandro Peschechera ◽  
Antonella Conserva ◽  
Lucia Adelaide Renna ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Small Interfering Rna ◽  
Protein Modification ◽  
Covalent Attachment ◽  
Mrna Levels ◽  
Protein Levels ◽  
Mrna Induction ◽  
Induction Of Differentiation ◽  
Interfering Rna

The small ubiquitin-like modifier-conjugating enzyme UBC9, involved in protein modification through covalent attachment of small ubiquitin-like modifier and other less defined mechanisms, has emerged as a key regulator of cell proliferation and differentiation. To explore the role of UBC9 in adipocyte differentiation, the UBC9 protein levels were examined in differentiating 3T3-L1 cells. UBC9 mRNA and protein levels were increased 2.5-fold at d 2 and then gradually declined to basal levels at d 8 of differentiation. In addition, UBC9 was expressed predominantly in the nucleus of preadipocytes but shifted to cytoplasmic compartments after d 4, after induction of differentiation. UBC9 knockdown was then achieved in differentiating 3T3-L1 preadipocytes using a specific small interfering RNA. Oil-Red-O staining demonstrated accumulation of large triglyceride droplets in approximately 90% of control cells, whereas lipid droplets were smaller and evident in only 30% of cells treated with the UBC9-specific small interfering RNA. CCAAT/enhancer-binding protein (C/EBP)-δ, peroxisome proliferator-activated receptor-γ, and C/EBPα mRNA levels were increased severalfold 2–6 d after induction of differentiation in control cells, whereas the expression of these transcription factors was significantly lower in the presence of UBC9 gene silencing. Adenovirus-mediated overexpression of a catalytically inactive mutant UBC9 protein in 3T3-L1 cells resulted in no changes in expression of adipogenic transcription factors and conversion to mature adipocytes as compared with control. In conclusion, UBC9 appears to play an important role in adipogenesis. The temporal profile of UBC9 induction and its ability to affect C/EBPδ mRNA induction support a role for this protein during early adipogenesis.

scholarly journals Bronchial Epithelial Tet2 Maintains Epithelial Integrity during Acute Pseudomonas aeruginosa Pneumonia

2020 ◽  
Vol 89 (1) ◽  
pp. e00603-20
Author(s):  
Wanhai Qin ◽  
Xanthe Brands ◽  
Cornelis van't Veer ◽  
Alex F. de Vos ◽  
Brendon P. Scicluna ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Bronchial Epithelial Cell ◽  
Mrna Levels ◽  
Wild Type ◽  
Bronchial Epithelial ◽  
Epithelial Integrity ◽  
Content Type ◽  
Protein Levels

ABSTRACTRespiratory epithelial cells are important for pulmonary innate immune responses during Pseudomonas aeruginosa infection. Tet methylcytosine dioxygenase 2 (Tet2) has been implicated in the regulation of host defense by myeloid and lymphoid cells, but whether Tet2 also contributes to epithelial responses during pneumonia is unknown. The aim of this study was to investigate the role of bronchial epithelial Tet2 in acute pneumonia caused by P. aeruginosa. To this end, we crossed mice with Tet2 flanked by two Lox-P sites (Tet2fl/fl mice) with mice expressing Cre recombinase under the bronchial epithelial cell-specific Cc10 promoter (Cc10Cre mice) to generate bronchial epithelial cell-specific Tet2-deficient (Tet2fl/fl Cc10Cre) mice. Six hours after infection with P. aeruginosa,Tet2fl/fl Cc10Cre and wild-type mice had similar bacterial loads in bronchoalveolar lavage fluid (BALF). At this time point, Tet2fl/fl Cc10Cre mice displayed reduced mRNA levels of the chemokines Cxcl1, Cxcl2, and Ccl20 in bronchial brushes. However, Cxcl1, Cxcl2, and Ccl20 protein levels and leukocyte recruitment in BALF were not different between groups. Tet2fl/fl Cc10Cre mice had increased protein levels in BALF after infection, indicating a disturbed epithelial barrier function, which was corroborated by reduced mRNA expression of tight junction protein 1 and occludin in bronchial brushes. Differences detected between Tet2fl/fl Cc10Cre and wild-type mice were no longer present at 24 h after infection. These results suggest that bronchial epithelial Tet2 contributes to maintaining epithelial integrity by enhancing intracellular connections between epithelial cells during the early phase of P. aeruginosa pneumonia.

scholarly journals TcI Isolates of Trypanosoma cruzi Exploit the Antioxidant Network for Enhanced Intracellular Survival in Macrophages and Virulence in Mice

2016 ◽  
Vol 84 (6) ◽  
pp. 1842-1856 ◽  
Author(s):  
María Paola Zago ◽  
Yashoda M. Hosakote ◽  
Sue-jie Koo ◽  
Monisha Dhiman ◽  
María Dolores Piñeyro ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Biological Behavior ◽  
Data Sets ◽  
Proteomic Profiling ◽  
Two Dimensional Gel Electrophoresis ◽  
Content Type ◽  
Transmission Cycle ◽  
Protein Levels ◽  
Flight Mass Spectrometry ◽  
And Control

Trypanosoma cruzispecies is categorized into six discrete typing units (TcI to TcVI) of which TcI is most abundantly noted in the sylvatic transmission cycle and considered the major cause of human disease. In our study, the TcI strains Colombiana (COL), SylvioX10/4 (SYL), and a cultured clone (TCC) exhibited different biological behavior in a murine model, ranging from high parasitemia and symptomatic cardiomyopathy (SYL), mild parasitemia and high tissue tropism (COL), to no pathogenicity (TCC). Proteomic profiling of the insect (epimastigote) and infective (trypomastigote) forms by two-dimensional gel electrophoresis/matrix-assisted laser desorption ionization–time of flight mass spectrometry, followed by functional annotation of the differential proteome data sets (≥2-fold change,P< 0.05), showed that several proteins involved in (i) cytoskeletal assembly and remodeling, essential for flagellar wave frequency and amplitude and forward motility of the parasite, and (ii) the parasite-specific antioxidant network were enhanced in COL and SYL (versus TCC) trypomastigotes. Western blotting confirmed the enhanced protein levels of cytosolic and mitochondrial tryparedoxin peroxidases and their substrate (tryparedoxin) and iron superoxide dismutase in COL and SYL (versus TCC) trypomastigotes. Further, COL and SYL (but not TCC) were resistant to exogenous treatment with stable oxidants (H2O2and peroxynitrite [ONOO−]) and dampened the intracellular superoxide and nitric oxide response in macrophages, and thus these isolates escaped from macrophages. Our findings suggest that protein expression conducive to increase in motility and control of macrophage-derived free radicals provides survival and persistence benefits to TcI isolates ofT. cruzi.

scholarly journals A Comparative Quantitative Proteomic Study Identifies New Proteins Relevant for Sulfur Oxidation in the Purple Sulfur Bacterium Allochromatium vinosum

2014 ◽  
Vol 80 (7) ◽  
pp. 2279-2292 ◽  
Author(s):  
Thomas Weissgerber ◽  
Marc Sylvester ◽  
Lena Kröninger ◽  
Christiane Dahl
Keyword(s):  
Sulfur Metabolism ◽  
Sulfur Oxidation ◽  
Hypothetical Protein ◽  
Mrna Levels ◽  
Reduced Sulfur Compounds ◽  
Allochromatium Vinosum ◽  
Purple Sulfur Bacterium ◽  
Content Type ◽  
Protein Levels ◽  
New Genes

ABSTRACTIn the present study, we compared the proteome response ofAllochromatium vinosumwhen growing photoautotrophically in the presence of sulfide, thiosulfate, and elemental sulfur with the proteome response when the organism was growing photoheterotrophically on malate. Applying tandem mass tag analysis as well as two-dimensional (2D) PAGE, we detected 1,955 of the 3,302 predicted proteins by identification of at least two peptides (59.2%) and quantified 1,848 of the identified proteins. Altered relative protein amounts (≥1.5-fold) were observed for 385 proteins, corresponding to 20.8% of the quantifiedA. vinosumproteome. A significant number of the proteins exhibiting strongly enhanced relative protein levels in the presence of reduced sulfur compounds are well documented essential players during oxidative sulfur metabolism, e.g., the dissimilatory sulfite reductase DsrAB. Changes in protein levels generally matched those observed for the respective relative mRNA levels in a previous study and allowed identification of new genes/proteins participating in oxidative sulfur metabolism. One gene cluster (hyd; Alvin_2036-Alvin_2040) and one hypothetical protein (Alvin_2107) exhibiting strong responses on both the transcriptome and proteome levels were chosen for gene inactivation and phenotypic analyses of the respective mutant strains, which verified the importance of the so-called Isp hydrogenase supercomplex for efficient oxidation of sulfide and a crucial role of Alvin_2107 for the oxidation of sulfur stored in sulfur globules to sulfite. In addition, we analyzed the sulfur globule proteome and identified a new sulfur globule protein (SgpD; Alvin_2515).

scholarly journals Differential Penicillin-Binding Protein 5 (PBP5) Levels in the Enterococcus faecium Clades with Different Levels of Ampicillin Resistance

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Maria Camila Montealegre ◽  
Jung Hyeob Roh ◽  
Meredith Rae ◽  
Milya G. Davlieva ◽  
Kavindra V. Singh ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Binding Protein ◽  
Mrna Levels ◽  
Nucleotide Polymorphisms ◽  
Penicillin Binding Protein ◽  
Ampicillin Resistance ◽  
Content Type ◽  
Protein Levels ◽  
Clade B ◽  
Wide Range

ABSTRACT Ampicillin resistance in Enterococcus faecium is a serious concern worldwide, complicating the treatment of E. faecium infections. Penicillin-binding protein 5 (PBP5) is considered the main ampicillin resistance determinant in E. faecium. The three known E. faecium clades showed sequence variations in the pbp5 gene that are associated with their ampicillin resistance phenotype; however, these changes alone do not explain the array of resistance levels observed among E. faecium clinical strains. We aimed to determine if the levels of PBP5 are differentially regulated between the E. faecium clades, with the hypothesis that variations in PBP5 levels could help account for the spectrum of ampicillin MICs seen in E. faecium. We studied pbp5 mRNA levels and PBP5 protein levels as well as the genetic environment upstream of pbp5 in 16 E. faecium strains that belong to the different E. faecium clades and for which the ampicillin MICs covered a wide range. Our results found that pbp5 and PBP5 levels are increased in subclade A1 and A2 ampicillin-resistant strains compared to those in clade B and subclade A2 ampicillin-susceptible strains. Furthermore, we found evidence of major clade-associated rearrangements in the region upstream of pbp5, including large DNA fragment insertions, deletions, and single nucleotide polymorphisms, that may be associated with the differential regulation of PBP5 levels between the E. faecium clades. Overall, these findings highlight the contribution of the clade background to the regulation of PBP5 abundance and point to differences in the region upstream of pbp5 as likely contributors to the differential expression of ampicillin resistance.

scholarly journals MEF2 Cooperates With Forskolin/cAMP and GATA4 to Regulate Star Gene Expression in Mouse MA-10 Leydig Cells

Endocrinology ◽  
2015 ◽  
Vol 156 (7) ◽  
pp. 2693-2703 ◽  
Author(s):  
Caroline Daems ◽  
Mickaël Di-Luoffo ◽  
Élise Paradis ◽  
Jacques J. Tremblay
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Leydig Cells ◽  
Regulatory Protein ◽  
Mrna Levels ◽  
Small Interfering Rnas ◽  
Inner Mitochondrial Membrane ◽  
Protein Levels ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

In Leydig cells, steroidogenic acute regulatory protein (STAR) participates in cholesterol shuttling from the outer to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis. Steroid hormone biosynthesis and steroidogenic gene expression are regulated by LH, which activates various signaling pathways and transcription factors, including cAMP/Ca2+/CAMK (Ca2+/calmodulin-dependent kinase)–myocyte enhancer factor 2 (MEF2). The 4 MEF2 transcription factors are essential regulators of cell differentiation and organogenesis in numerous tissues. Recently, MEF2 was identified in Sertoli and Leydig cells of the testis. Here, we report that MEF2 regulates steroidogenesis in mouse MA-10 Leydig cells by acting on the Star gene. In MA-10 cells depleted of MEF2 using siRNAs (small interfering RNAs), STAR protein levels, Star mRNA levels, and promoter activity were significantly decreased. On its own, MEF2 did not activate the mouse Star promoter but was found to cooperate with forskolin/cAMP. By chromatin immunoprecipitation and DNA precipitation assays, we confirmed MEF2 binding to a consensus element located at −232 bp of the Star promoter. Mutation or deletion of the MEF2 element reduced but did not abrogate the MEF2/cAMP cooperation, indicating that MEF2 cooperates with other DNA-bound transcription factor(s). We identified GATA4 (GATA binding protein 4) as a partner for MEF2 in Leydig cells, because mutation of the GATA element abrogated the MEF2/cAMP cooperation on a reporter lacking a MEF2 element. MEF2 and GATA4 interact as revealed by coimmunoprecipitation, and MEF2 and GATA4 transcriptionally cooperate on the Star promoter. Altogether, our results define MEF2 as a novel regulator of steroidogenesis and Star transcription in Leydig cells and identify GATA4 as a key partner for MEF2-mediated action.

scholarly journals Mebendazole Exerts Potent Anti-Leukemic Effects By Downregulating Protein Levels of Hedgehog Transcription Factors GLI1 and GLI2

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5145-5145
Author(s):  
Fabian Freisleben ◽  
Hauke Stamm ◽  
Jana Muschhammer ◽  
Alexander Krispien ◽  
Vanessa Thaden ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Proteasomal Degradation ◽  
26S Proteasome ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Advisory Committees ◽  
Protein Levels

Abstract The relevance of the Hedgehog signaling pathway in the pathophysiology of acute myeloid leukemia (AML) has been demonstrated by us and others. Inhibition of the downstream Hedgehog transcription factors GLI1 and GLI2 results in strong anti-leukemic effects. Therefore, Hedgehog pathway inhibitors represent a promising therapeutic approach in AML. Mebendazole is an anthelmintic drug commonly used for the treatment of various parasitic worm infections. Recently, mebendazole has been shown to exhibit strong anti-tumor effects in different cancer entities including AML. In the work presented here, we investigated the effect of mebendazole on expression and activity of GLI transcription factors and its anti-leukemic activity. To determine the effect of mebendazole on GLI transcription factors, we treated the AML cell lines MV4-11, MOLM-13, THP-1 and OCI-AML3 with different concentrations of mebendazole and analyzed its impact on GLI1 and GLI2 protein- and mRNA levels. Furthermore, GLI reporter assays (Cignal GLI Reporter (luc) Kit, Qiagen) were performed to determine the effect of mebendazole on the GLI1 and -2 transcriptional activity. Mebendazole strongly inhibited GLI1 and GLI2 signaling activity in a dose-dependent manner. Exemplarily, treatment with 500 nM mebendazole reduced the GLI1 and -2 transcriptional activity in all cell lines tested by 54.8 % (± 9.6) after 24h and 73.2 % (± 11.6) after 48h. We could demonstrate by Western Blotting that GLI1 and -2 protein levels were clearly reduced 24h and 48h after mebendazole exposure, whereas GLI1 and -2 mRNA levels did not decrease. These data suggest that mebendazole may increase degradation of GLI proteins via the proteasome pathway. Therefore, we evaluated the influence of the 26s proteasome inhibitor bortezomib on GLI levels after mebendazole treatment. Inhibiting the 26s proteasome with 2 nM, 5 nM and 10 nM of bortezomib increased GLI signaling activity by 13.6 % (± 8.0), 84.6 % (± 39.2) and 137.1 % (± 37.9), respectively. Furthermore, 10 nM bortezomib abolished the effect of mebendazole on GLI protein levels. Taken together, mebendazole increased the proteasomal degradation of GLI1 and GLI2. These observations were extended to samples from AML patients. After mebendazole treatment for 24h or 48h all analyzed patients had reductions of GLI1 protein levels as confirmed by Western blotting (n=4), whereas GLI1 and GLI2 mRNA levels were not changed (n=7), indicating that proteasomal degradation was operational in primary blasts as well. Evaluating the anti-leukemic effects of mebendazole, we also investigated its combination with the small molecule GLI inhibitor GANT61. We treated the AML cell lines MV4-11, MOLM-13, THP-1 and OCI-AML3 with combinations of mebendazole and GANT61 and analyzed cell proliferation, apoptosis and colony formation. Mebendazole treatment alone already resulted in decreased proliferation and colony forming capacity as well as increased apoptosis rates in a dose-dependent manner. The combination of mebendazole with the GLI inhibitor GANT61 synergistically increased the anti-proliferative effects of mebendazole on all 4 AML cell lines tested. Additionally, GANT61 further increased the effect of mebendazole on colony formation significantly. Incubation with 100 nM, 200 nM and 500 nM mebendazole inhibited the proliferation of primary blasts from AML patients by 15.1 % (± 7.5), 31.6 % (± 16.8) and 66.0 % (± 17.4), respectively (n=8). Moreover, the combination with GANT61 significantly increased these anti-proliferative effects. This work indicates that mebendazole exerts profound anti-leukemic effects by decreasing GLI1 and GLI2 intracellular levels by promoting its proteasomal degradation. Combining mebendazole with GLI1 and GLI2 inhibitors such as GANT61 enhances this effect considerably. These observations may lead to the introduction of novel treatment strategies in AML. Disclosures Stamm: Amgen Research (Munich) GmbH / Amgen Inc.: Patents & Royalties; Astellas GmbH: Other: Travel Grant. Wellbrock:Amgen Research (Munich) GmbH: Patents & Royalties. Fiedler:GSO: Other: support for meeting attendance; Gilead: Other: support for meeting attendance; Amgen: Other: support for meetíng attendance; Pfizer: Research Funding; Amgen: Research Funding; Amgen: Patents & Royalties; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees, support for meeting attendance; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Other: support for meeting attendance; JAZZ Pharmaceuticals: Other: support for meeting attendance; Teva: Other: support for meeting attendance.

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