scholarly journals CSIG-15. INHIBITION OF THE bHLH TRANSCRIPTIONAL NETWORKS BY A MUTATED E47 PROTEIN LEADS TO A STRONG ANTI-GLIOMA ACTIVITY IN VITRO AND IN VIVO

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi47-vi47
Author(s):  
Marilin Koch ◽  
Stefan Czemmel ◽  
Felix Lennartz ◽  
Sarah Beyeler ◽  
Justyna Przystal ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Clonogenic Survival ◽  
Dominant Negative ◽  
Molecular Profile ◽  
Transcriptional Networks ◽  
Molecular Alterations ◽  
Helix Loop Helix

Abstract OBJECTIVE The transcription factor E47 heterodimerizes with helix-loop-helix (HLH) and basic helix-loop-helix transcription (bHLH) factors like ID-1 and Olig2 that are overexpressed in glioblastoma. A dominant-negative variant of the E47 (dnE47) lacking the nuclear translocation signal, leads to cytoplasmatic sequestration of HLH and bHLH transcription factors. Here, we investigated combinations of dnE47-mediated inhibition of the bHLH transcriptional network with temozolomide and irradiation and explored the underlying molecular mechanisms. METHODS Long-term and stem cell glioma lines were transduced with a Doxycycline-inducible dnE47 lentivirus. Functional characterizations included immunocytochemistry, immunoblots, cytotoxicity and clonogenicity assays in vitro and latency until the onset of symptoms in vivo. CAGE and RNASeq were conducted for analyzing the dnE47-induced molecular profile. RESULTS The induction of dnE47 led to cytoplasmatic sequestration of HLH/bHLH transcription, reduced proliferation, increased cytotoxicity and reduced clonogenic survival in vitro and a prolonged latency until the onset of neurological symptoms in vivo. CAGE and RNASeq data revealed alterations in several cancer-relevant pathways. CONCLUSIONS A dnE47-mediated inhibition of the bHLH transcription network induced actionable molecular alterations in glioma cells that could be exploited for the design of novel therapies.

scholarly journals Experimental glioma with high bHLH expression harbor increased replicative stress and are sensitive toward ATR inhibition

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Marilin Sophia Koch ◽  
Stefan Czemmel ◽  
Felix Lennartz ◽  
Sarah Beyeler ◽  
Srinath Rajaraman ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Clonogenic Survival ◽  
Dominant Negative ◽  
Molecular Subgroup ◽  
Replicative Stress ◽  
Helix Loop Helix ◽  
Molecular Events ◽  
Dominant Negative Variant

Abstract Background The overexpression of (basic)helix-loop-helix ((b)HLH) transcription factors (TFs) is frequent in malignant glioma. We investigated molecular effects upon disruption of the (b)HLH network by a dominant-negative variant of the E47 protein (dnE47). Our goal was to identify novel molecular subgroup-specific therapeutic strategies. Methods Glioma cell lines LN229, LNZ308, and GS-2/GS-9 were lentivirally transduced. Functional characterization included immunocytochemistry, immunoblots, cytotoxic, and clonogenic survival assays in vitro, and latency until neurological symptoms in vivo. Results of cap analysis gene expression and RNA-sequencing were further validated by immunoblot, flow cytometry, and functional assays in vitro. Results The induction of dnE47-RFP led to cytoplasmic sequestration of (b)HLH TFs and antiglioma activity in vitro and in vivo. Downstream molecular events, ie, alterations in transcription start site usage and in the transcriptome revealed enrichment of cancer-relevant pathways, particularly of the DNA damage response (DDR) pathway. Pharmacologic validation of this result using ataxia telangiectasia and Rad3 related (ATR) inhibition led to a significantly enhanced early and late apoptotic effect compared with temozolomide alone. Conclusions Gliomas overexpressing (b)HLH TFs are sensitive toward inhibition of the ATR kinase. The combination of ATR inhibition plus temozolomide or radiation therapy in this molecular subgroup are warranted.

Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1056-1067 ◽  
Author(s):  
Mira T. Kassouf ◽  
Hedia Chagraoui ◽  
Paresh Vyas ◽  
Catherine Porcher
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Experimental Systems ◽  
Dna Binding Activity ◽  
Independent Functions

Abstract Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding–independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

Role of the ligand in intracellular receptor function: receptor affinity determines activation in vitro of the latent dioxin receptor to a DNA-binding form

1991 ◽  
Vol 11 (1) ◽  
pp. 401-411
Author(s):  
S Cuthill ◽  
A Wilhelmsson ◽  
L Poellinger
Keyword(s):  
Dna Binding ◽  
Cellular Response ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Rank Order ◽  
Receptor Ligands ◽  
Free System ◽  
Dioxin Receptor

To reconstitute the molecular mechanisms underlying the cellular response to soluble receptor ligands, we have exploited a cell-free system that exhibits signal- (dioxin-)induced activation of the latent cytosolic dioxin receptor to an active DNA-binding species. The DNA-binding properties of the in vitro-activated form were qualitatively indistinguishable from those of in vivo-activated nuclear receptor extracted from dioxin-treated cells. In vitro activation of the receptor by dioxin was dose dependent and was mimicked by other dioxin receptor ligands in a manner that followed the rank order of their relative affinities for the receptor in vitro and their relative potencies to induce target gene transcription in vivo. Thus, in addition to triggering the initial release of inhibition of DNA binding and presumably allowing nuclear translocation, the ligand appears to play a crucial role in the direct control of the level of functional activity of a given ligand-receptor complex.

scholarly journals TIE2 Associates with Caveolae and Regulates Caveolin-1 To Promote Their Nuclear Translocation

2017 ◽  
Vol 37 (21) ◽  
Author(s):  
Mohammad B. Hossain ◽  
Rehnuma Shifat ◽  
Jingyi Li ◽  
Xuemei Luo ◽  
Kenneth R. Hess ◽  
...  
Keyword(s):  
Dna Repair ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Subcellular Fractionation ◽  
Tyrosine Kinase Receptor ◽  
Histone H4 ◽  
Small Interfering Rnas ◽  
Caveolin 1

ABSTRACT DNA repair pathways are aberrant in cancer, enabling tumor cells to survive standard therapies—chemotherapy and radiotherapy. Our group previously reported that, upon irradiation, the membrane-bound tyrosine kinase receptor TIE2 translocates into the nucleus and phosphorylates histone H4 at Tyr51, recruiting ABL1 to the DNA repair complexes that participate in the nonhomologous end-joining pathway. However, no specific molecular mechanisms of TIE2 endocytosis have been reported. Here, we show that irradiation or ligand-induced TIE2 trafficking is dependent on caveolin-1, the main component of caveolae. Subcellular fractionation and confocal microscopy demonstrated TIE2/caveolin-1 complexes in the nucleus, and using inhibitor or small interfering RNAs (siRNAs) against caveolin-1 or Tie2 inhibited their trafficking. TIE2 was found in caveolae and directly phosphorylated caveolin-1 at Tyr14 in vitro and in vivo. This modification regulated the generation of TIE2/caveolin-1 complexes and was essential for TIE2/caveolin-1 nuclear translocation. Our data further demonstrate that the combination of TIE2 and caveolin-1 inhibitors resulted in significant radiosensitization of malignant glioma cells, which will guide the development of combinatorial treatment with radiotherapy for patients with glioblastoma.

scholarly journals Molecular Mechanisms of Pollen-Pistil Interactions in Interspecific Crossing Barriers in the Tomato Family

2000 ◽  
Author(s):  
Yoram Eyal ◽  
Sheila McCormick
Keyword(s):  
Pollen Germination ◽  
Molecular Mechanisms ◽  
Functional Characterization ◽  
Dominant Negative ◽  
Peptide Sequence ◽  
Peptide Ligand ◽  
Hybrid Breakdown ◽  
Degenerate Primers

During the evolutionary process of speciation in plants, naturally occurring barriers to reproduction have developed that affect the transfer of genes within and between related species. These barriers can occur at several different levels beginning with pollination-barriers and ending with hybrid-breakdown. The interaction between pollen and pistils presents one of the major barriers to intra- and inter-specific crosses and is the focus of this research project. Our long-term goal in this research proposal was defined to resolve questions on recognition and communication during pollen-pistil interactions in the extended tomato family. In this context, this work was initiated and planned to study the potential involvement of tomato pollen-specific receptor-like kinases (RLK's) in the interaction between pollen and pistils. By special permission from BARD the objectives of this research were extended to include studies on pollen-pistil interactions and pollination barriers in horticultural crops with an emphasis on citrus. Functional characterization of 2 pollen-specific RLK's from tomato was carried out. The data shows that both encode functional kinases that were active as recombinant proteins. One of the kinases was shown to accumulate mainly after pollen germination and to be phosphorylated in-vitro in pollen membranes as well as in-vivo. The presence of style extract resulted in dephosphorylation of the RLK, although no species specificity was observed. This data implies a role for at least one RLK in pollination events following pollen germination. However, a transgenic plant analysis of the RLK's comprising overexpression, dominant-negative and anti-sense constructs failed to provide answers on their role in pollination. While genetic effects on some of the plants were observed in both the Israeli and American labs, no clear functional answers were obtained. An alternative approach to addressing function was pursued by screening for an artificial ligand for the receptor domain using a peptide phage display library. An enriched peptide sequence was obtained and will be used to design a peptide-ligand to be tested for its effect o pollen germination and tube growth. Self-incompatibility (SI) in citrus was studied on 3 varieties of pummelo. SI was observed using fluorescence microscopy in each of the 3 varieties and compatibility relations between varieties was determined. An initial screen for an S-RNase SI mechanism yielded only a cDNA homologous to the group of S-like RNases, suggesting that SI results from an as yet unknown mechanism. 2D gel electrophoresis was applied to compare pollen and style profiles of different compatibility groups. A "polymorphic" protein band from style extracts was observed, isolated and micro-sequenced. Degenerate primers designed based on the peptide sequence date will be used to isolate the relevant genes i order to study their potential involvement in SI. A study on SI in the apple cultivar Top red was initiated. SI was found, as previously shown, to be complete thus requiring a compatible pollinator variety. A new S-RNase allele was discovered fro Top red styles and was found to be highly homologous to pear S-RNases, suggesting that evolution of these genes pre-dated speciation into apples and pears but not to other Rosaceae species. The new allele provides molecular-genetic tools to determine potential pollinators for the variety Top red as well as a tool to break-down SI in this important variety.

Molecular mechanisms of platelet exocytosis: role of SNAP-23 and syntaxin 2 in dense core granule release

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 921-929 ◽  
Author(s):  
Dong Chen ◽  
Audrey M. Bernstein ◽  
Paula P. Lemons ◽  
Sidney W. Whiteheart
Keyword(s):  
Molecular Mechanisms ◽  
Cell Types ◽  
Dominant Negative ◽  
Dense Core ◽  
Protein Receptors ◽  
Dense Core Granules ◽  
Platelet Exocytosis ◽  
Granule Release

To characterize the molecular mechanisms of platelet secretion, we focused on the calcium-induced exocytosis of dense core granules. Platelets contain several known t-SNAREs (soluble N-ethylmaleimide sensitive factor [NSF] attachment protein receptors) such as syntaxins 2, 4, and 7 and SNAP-23 (synaptosomal associated protein 23). By using an in vitro exocytosis assay, we have been able to assign roles for some of these t-SNAREs in dense core granule release. This calcium-induced secretion relies on the SNARE proteins because it is stimulated by the addition of recombinant -SNAP and inhibited by a dominant negative -SNAP–L294A mutant or by anti–-SNAP and anti-NSF antibodies. SNAP-23 antibodies and an inhibitory C-terminal SNAP-23 peptide both blocked dense core granule release, demonstrating a role for SNAP-23. Unlike other cell types, platelets contain a significant pool of soluble SNAP-23, which does not partition into Triton X-114. Of the anti-syntaxin antibodies tested, only anti–syntaxin 2 antibody inhibited dense core granule release. Immunoprecipitation studies showed that the 2 t-SNAREs syntaxin 2 and SNAP-23 do form a complex in vivo. These data clearly show that SNAPs, NSF, and specific t-SNAREs are used for dense core granule release; these data provide a greater understanding of regulated exocytosis in platelets.

β2-AR Activation promotes cleavage and nuclear translocation of Her2 and metastatic potential of cancer cells

2020 ◽  
Author(s):  
Dan Liu ◽  
Xiyue Xu ◽  
Shuci Liu ◽  
Xuan Zhao ◽  
Anqun Tang ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Biological Response ◽  
Metastatic Potential ◽  
Adrenergic Signaling ◽  
Signaling Activation

Abstract Background The prolonged hypersecretion of catecholamine induced by chronic stress may correlate with various steps of malignant progression of cancer and β2-AR overexpressed in certain cancer cells may translate the signals from neuroendocrine system to malignant signals by interacting with oncoproteins such as Her2. Crosstalk of the cell signaling pathways mediated by β2-AR and Her2 may promote a stronger or more sustained biological response. However, the molecular mechanisms underlying cross-communication between β2-AR and Her2 mediated signaling pathways are not fully understood. Methods In this study, the effects of adrenergic signaling on Her2 cleavage were evaluated by various assays, such as western blot, immunofluorescence and immunohistochemistry. In order to reveal the mechanism about Her2 cleavage triggered by β2-AR activation, the molecular and pharmacological means were employed. By using in vitro and in vivo assay, the influences of the crosstalk between β2-AR and Her2 on the bio-behaviors of tumor cells were demonstrated. Results Our data demonstrate that catecholamine stimulation activates the expression and proteolytic activity of ADAM10 by modulating the expression of miR-199a-5p and SIRT1 and also confirm that catecholamine induction triggers the activities of γ-secretase, leading to shedding of Her2 ECD by ADAM10 and subsequent intramembranous cleavage of Her2 ICD by presenilin-dependent γ-secretase, nuclear translocation of Her2 ICD and enhanced transcription of tumor metastasis-associated gene COX-2 . Chronic stimulation of catecholamine strongly promotes the invasive activities of cancer cells in vitro and spontaneous tumor lung metastasis in mice. Furthermore, the nuclear localization of Her2 was significantly correlated with overexpression of β2-AR in human breast cancer tissues. Conclusion This study illustrates that adrenergic signaling activation triggers Her2 cleavage, resulting in enhanced invasive and metastasis activities of cancer cells. Our data also reveal that an unknown mechanism by which the regulated intramembrane proteolysis (RIP) initiated by β2-AR activation controls a novel Her2-mediated signaling transduction under physiological and pathological conditions.

Identification of Key Cellular Regulators That Interact with Id1 To Control Hematopoietic Stem Cell Behavior.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1319-1319
Author(s):  
Vladimir Jankovic ◽  
Alessia Ciarrocchi ◽  
Tony DeBlasio ◽  
Robert Benezra ◽  
Stephen D. Nimer
Keyword(s):  
Transcription Factors ◽  
Transcriptional Repression ◽  
Genetic Interaction ◽  
Dominant Negative ◽  
Double Knockout ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Helix Loop Helix

Abstract The ability of hematopoietic stem cells to tightly regulate the transition from relative quiescence and self-renewal to the transiently amplifying, differentiating progenitor fate is critical for HSC homeostasis as well as their regenerative capacity. We have recently described the diminished frequency and rapid exhaustion of HSC self-renewal capacity in the absence of the dominant negative helix-loop-helix molecule Id1. Furthermore, Id1 null HSCs have an increased rate of cycling, coupled with accelerated myeloid commitment both in vivo and in vitro. This is reflected in the elevated expression of myelo-erythroid transcription factors (c/EBPalpha and GATA1) within the Lin−c-kit+Sca-1+ population - “myeloid priming”. The major targets of Id1 mediated transcriptional repression are the ubiquitous E protein E2A as well as Ets transcription factors (Ets1 and Ets2). We hypothesized that the unrestrained activity of these and/or other targets of Id1 transcriptional repression leads to premature HSC commitment in Id1 null animals. Indeed, we show that HSC differentiation in culture can be delayed by transduction of E2A directed shRNA specifically in Id1 null, but not in wild-type Id1 expressing cells. This indicates an abnormal E2A activity in Id1 null HSCs that could be responsible for their increased differentiation status. To further define the transcriptional deregulation in Id1 null HSCs, we have used the Affymetrix microarray technology. We observed ~3 fold increased expression of the CDK inhibitor p21 in freshly isolated Id1 null HSCs and have confirmed this result by multiple independent qPCR measurements. The transcriptional induction of p21 by E2A as well as its repression by Id1 have been well established. Therefore, the observed p21 induction could be explained by the elevated level of E2A activity in HSCs in the absence of Id1 expression. To explore the functional significance of Id1 mediated p21 regulation in HSCs, we have generated p21/Id1 double knockout animals. Surprisingly, despite its reported function in restricting the cell cycle entry of normal HSCs, we show that in the context of Id1 loss, p21 expression is required for the accelerated HSC cycling, and unlike Id1 single null HSCs, p21/Id1 double knockout HSCs do not show accelerated myeloid differentiation in culture. Therefore, we propose that Id1 actively represses E2A activity in HSCs, as well as the induction of p21, which could be an important component of the HSC commitment program. Further studies will be presented defining the in vivo relevance of the Id1/p21 genetic interaction for HSC growth and differentiation.

High glucose promotes nascent nephron apoptosis via NF-κB and p53 pathways

2011 ◽  
Vol 300 (1) ◽  
pp. F147-F156 ◽  
Author(s):  
Yun-Wen Chen ◽  
Isabelle Chenier ◽  
Shiao-Ying Chang ◽  
Stella Tran ◽  
Julie R. Ingelfinger ◽  
...  
Keyword(s):  
High Glucose ◽  
Nuclear Translocation ◽  
Dominant Negative ◽  
In Vivo Studies ◽  
Ros Generation ◽  
Small Interfering Rnas ◽  
Kidney Size ◽  
P53 Signaling

A hyperglycemic environment in utero reduces kidney size and nephron number due to nascent nephron apoptosis. However, the underlying mechanisms are incompletely understood. The present study investigated whether the nascent nephron apoptosis promoted by high glucose is mediated via the transcription factor NF-κB and p53 signaling pathways. Neonatal mouse kidneys from the offspring of nondiabetic, diabetic, and insulin-treated diabetic dams were used for in vivo studies, and MK4 cells, an embryonic metanephric mesenchymal (MM) cell line, were used for in vitro studies. Neonatal kidneys of the offspring of diabetic mothers exhibited an increased number of apoptotic cells and reactive oxygen species (ROS) generation, enhanced NF-κB activation, and nuclear translocation of its subunits (p50 and p65 subunits) as well as phosphorylation (Ser 15) of p53 compared with kidneys of offspring of nondiabetic mothers. Insulin treatment of diabetic dams normalized these parameters in the offspring. In vitro, high-glucose (25 mM) induced ROS generation and significantly increased MK4 cell apoptosis and caspase-3 activity via activation of NF-κB pathway, with p53 phosphorylation and nuclear translocation compared with normal glucose (5 mM). These changes in a high-glucose milieu were prevented by transient transfection of small interfering RNAs for dominant negative IκBα or IKK or p53. Our data demonstrate that high glucose-induced nascent nephron apoptosis is mediated, at least in part, via ROS generation and the activation of NF-κB and p53 pathways.

scholarly journals Network models of primary melanoma microenvironments identify key melanoma regulators underlying prognosis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Won-Min Song ◽  
Praveen Agrawal ◽  
Richard Von Itter ◽  
Barbara Fontanals-Cirera ◽  
Minghui Wang ◽  
...  
Keyword(s):  
Large Scale ◽  
Molecular Mechanisms ◽  
Immune Cell ◽  
Primary Melanoma ◽  
Network Models ◽  
Transcriptional Networks ◽  
Melanoma Progression ◽  
Skin Malignancy

AbstractMelanoma is the most lethal skin malignancy, driven by genetic and epigenetic alterations in the complex tumour microenvironment. While large-scale molecular profiling of melanoma has identified molecular signatures associated with melanoma progression, comprehensive systems-level modeling remains elusive. This study builds up predictive gene network models of molecular alterations in primary melanoma by integrating large-scale bulk-based multi-omic and single-cell transcriptomic data. Incorporating clinical, epigenetic, and proteomic data into these networks reveals key subnetworks, cell types, and regulators underlying melanoma progression. Tumors with high immune infiltrates are found to be associated with good prognosis, presumably due to induced CD8+ T-cell cytotoxicity, via MYO1F-mediated M1-polarization of macrophages. Seventeen key drivers of the gene subnetworks associated with poor prognosis, including the transcription factor ZNF180, are tested for their pro-tumorigenic effects in vitro. The anti-tumor effect of silencing ZNF180 is further validated using in vivo xenografts. Experimentally validated targets of ZNF180 are enriched in the ZNF180 centered network and the known pathways such as melanoma cell maintenance and immune cell infiltration. The transcriptional networks and their critical regulators provide insights into the molecular mechanisms of melanomagenesis and pave the way for developing therapeutic strategies for melanoma.

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