scholarly journals Decrease of Nibrin expression in chronic hypoxia is associated with hypoxia-induced chemoresistance in medulloblastoma cells

2017 ◽  
Author(s):  
Sophie Cowman ◽  
Yuen Ngan Fan ◽  
Barry Pizer ◽  
Violaine Sée
Keyword(s):  
Dna Damage ◽  
Cell Line ◽  
Induced Resistance ◽  
Brain Tumours ◽  
Molecular Mechanisms ◽  
Chronic Hypoxia ◽  
Acute Hypoxia ◽  
Adult Brain ◽  
Cell Type ◽  
Hypoxic Conditions

AbstractSolid tumours are less oxygenated than normal tissues. This is called tumour hypoxia and leads to resistance to radiotherapy and chemotherapy. The molecular mechanisms underlying such resistance have been investigated in a range of tumour types, including the adult brain tumours glioblastoma, yet little is known for paediatric brain tumours. Medulloblastoma (MB) is the most common malignant brain tumour in children. Here we used a common MB cell line (D283-MED), to investigate the mechanisms of chemo and radio-resistance in MB, comparing to another MB cell line (MEB-Med8A) and to a widely used glioblastoma cell line (U87MG). In D283-MED and U87MG, chronic hypoxia (5 days), but not acute hypoxia (24 h) induced resistance to etoposide and X-ray irradiation. This acquired resistance upon chronic hypoxia was much less pronounced in MEB-Med8A cells. Using a transcriptomic approach in D283-MED cells, we found a large transcriptional remodelling upon long term hypoxia, in particular the expression of a number of genes involved in detection and repair of double strand breaks (DSB) was altered. The levels of Nibrin (NBN) and MRE11, members of the MRN complex (MRE11/Rad50/NBN) responsible for DSB recognition, were significantly down-regulated. This was associated with a reduction of Ataxia Telangiectasia Mutated (ATM) activation by etoposide, indicating a profound dampening of the DNA damage signalling in hypoxic conditions. As a consequence, p53 activation by etoposide was reduced, and cell survival enhanced. Whilst U87MG shared the same dampened p53 activity, upon chemotherapeutic drug treatment in chronic hypoxic conditions, these cells used a different mechanism, independent of the DNA damage pathway. Together our results demonstrate a new mechanism explaining hypoxia-induced resistance involving the alteration of the response to DSB, but also highlight the cell type to cell type diversity and the necessity to take into account the differing tumour genetic make-up when considering re-sensitisation therapeutic protocols.

scholarly journals Hypoxia Transcriptomic Modifications Induced by Proton Irradiation in U87 Glioblastoma Multiforme Cell Line

2021 ◽  
Vol 11 (4) ◽  
pp. 308
Author(s):  
Valentina Bravatà ◽  
Walter Tinganelli ◽  
Francesco P. Cammarata ◽  
Luigi Minafra ◽  
Marco Calvaruso ◽  
...  
Keyword(s):  
Glioblastoma Multiforme ◽  
Cell Line ◽  
Acute Hypoxia ◽  
Hypoxic Condition ◽  
Biological Processes ◽  
Microarray Gene Expression ◽  
Hypoxic Conditions ◽  
Gene Expression Analyses ◽  
Shed Light ◽  
New Strategies

In Glioblastoma Multiforme (GBM), hypoxia is associated with radioresistance and poor prognosis. Since standard GBM treatments are not always effective, new strategies are needed to overcome resistance to therapeutic treatments, including radiotherapy (RT). Our study aims to shed light on the biomarker network involved in a hypoxic (0.2% oxygen) GBM cell line that is radioresistant after proton therapy (PT). For cultivating cells in acute hypoxia, GSI’s hypoxic chambers were used. Cells were irradiated in the middle of a spread-out Bragg peak with increasing PT doses to verify the greater radioresistance in hypoxic conditions. Whole-genome cDNA microarray gene expression analyses were performed for samples treated with 2 and 10 Gy to highlight biological processes activated in GBM following PT in the hypoxic condition. We describe cell survival response and significant deregulated pathways responsible for the cell death/survival balance and gene signatures linked to the PT/hypoxia configurations assayed. Highlighting the molecular pathways involved in GBM resistance following hypoxia and ionizing radiation (IR), this work could suggest new molecular targets, allowing the development of targeted drugs to be suggested in association with PT.

scholarly journals A Method to Fluorescently Label the CRISPR/Cas9-gRNA RNP Complexes Enables Enrichment of Clinical-Grade Gene-Edited Primary Hematopoietic Stem Cells and iPSCs

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1108-1108
Author(s):  
Masoud Nasri ◽  
Perihan Mir ◽  
Benjamin Dannenmann ◽  
Diana Amend ◽  
Yun Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Hematopoietic Stem Cells ◽  
Cell Line ◽  
Gene Editing ◽  
Jurkat Cells ◽  
Cell Type ◽  
Hematopoietic Stem ◽  
Editing Efficiency ◽  
Human Ipscs

Abstract Although proven to be an excellent method for gene editing, CRISPR/Cas9-mediated technology still has some limitations for the applications in primary hematopoietic stem cells and progenitor cells (HSPCs) as well as in human induced pluripotent stem cells (hiPSCs). Delivery of Cas9 protein in a form of ribonucleoprotein (RNP) in a complex with guide RNA (gRNA) provides a DNA free methodology, but a big hinderance of this application is that it is not possible to sort and enrich gene edited cells for further applications. Here we report the establishment of a new protocol of fluorescent labeling of the Cas9/gRNA ribonucleoprotein complex (CRISPR/Cas9-gRNA RNP). We designed crRNA for exon 1 of GADD45b gene, annealed this crRNA with transactivating crRNA (tracrRNA) to form gRNA and covalently introduced one fluorchrome agent (CX-rhodamine or fluorescein) per approximately every 20 nucleotides. HEK293FT cells, Jurkat T-ALL cell line, bone marrow CD34+ HSPCs, and iPSCs were transfected with fluorescently-labeled GADD45b CRISPR/Cas9-gRNA RNP by means of cathionic polymer based transfection reagent for HEK293FT cells and Lonza 4D nucleofection for Jurkat T-ALL cell line, CD34+ HSPCs, and iPSCs. We detected CX-rhodamine- or fluorescein intracellular signals 12 hours after transfection that disappeared approximately 48 hours post transfection. Transfection efficiency varied between 40 % and 80 %, depending on the cell type. Labeling did not affect integrity of crRNA/tracRNA duplex formation, gene editing efficiency and off-target activities of CRISPR/Cas9-gRNA RNP, as assessed by Sanger sequencing and TIDE assay of transfected HEK293FT cells, Jurkat cells, CD34+ HSPCs and human iPSCs. Using fluorescein- or CX-rhodamine signal of labeled CRISPR/Cas9-gRNA RNP, we sorted and enriched gene-edited cells. Gene modification efficiency in sorted cells was between 40 and 70 %, based on the cell type. Of note, we detected much lower transfection and editing efficiency of the fused Cas9-EGFP protein assembled with GADD45b targeting gRNA, as compared to CRISPR/Cas9-gRNA RNP. Most probably, conjugation of EGFP tag is affecting functions of CRISPR/Cas9- gRNA RNP. GADD45b (Growth Arrest And DNA Damage Inducible Beta), also termed myeloid differentiation primary response 118 gene (MyD118), belongs to a family of evolutionarily conserved GADD45 proteins (GADD45a, GADD45b and GADD45g) that function as stress sensors regulating cell cycle, survival and apoptosis in response to stress stimulus as ultraviolet (UV)-induced DNA damage and genotoxic stress. We further performed functional studies of the effect of GADD45b knockout on cell growth and sensitivity to UV-induced DNA damage. Remarkably, we detected severe diminished viability of GADD45b-deficient HEK293FT, Jurkat cells, iPSCs and CD34+ HSPCs as compared to control transfected cells. We also found markedly elevated susceptibility of GADD45b-deficient Jurkat cells, CD34+ HSPCs and iPSCs to UV induced DNA damage, as documented by elevated levels of γH2AX (pSer139). Based on these observations, we conclude that GADD45b knockout using transfection of cells with labeled GADD45b-targeting CRISPR/Cas9-gRNA RNP led to increased susceptibility to DNA damage. Moreover, GADD45b deficient iPSCs retained pluripotency, but they failed to differentiate to mature neutrophils in embryoid body (EB)-based culture. Taken together, this is the first report describing transfection and sorting of primary hematopoietic cells and iPSCs using fluorescently-labeled CRISPR/Cas9-RNP, which is simple, safe and efficient method, and therefore may strongly expand the therapeutic avenues for gene-edited cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Studies of Acute Hypoxia and Reoxygenation on Oxygen Sensors, Respiratory Metabolism, Oxidative Stress, and Apoptosis in Hybrid Yellow Catfish "Huangyou-1"

2021 ◽  
Author(s):  
Xueying Pei ◽  
Hongyan Zhang ◽  
Xinyu Zhang ◽  
Xiang Zheng ◽  
Jie Li ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Molecular Mechanisms ◽  
Acute Hypoxia ◽  
Oxygen Sensors ◽  
Hypoxic Exposure ◽  
Regulation Mechanism ◽  
Respiratory Metabolism ◽  
Yellow Catfish ◽  
Hypoxic Conditions ◽  
Cyt C

Abstract The regulation mechanism of the hybrid yellow catfish “Huangyou-1” was assessed under conditions of hypoxia and reoxygenation by examination of oxygen sensors and by monitoring respiratory metabolism, oxidative stress, and apoptosis. The expressions of genes related to oxygen sensors (HIF-1α, HIF-2α, VHL, HIF-1β, PHD2, and FIH-1) were upregulated in the brain and liver during hypoxia, and recovered compared with control upon reoxygenation. The expressions of genes related to glycolysis (HK1, PGK1, PGAM2, PFK, and LDH) were increased during hypoxia and then recovered compared with control upon reoxygenation. The expressions of CS and SDH were lower than those of control during hypoxia and increased upon reoxygenation. Under hypoxic conditions, the expressions of genes related to oxidative stress (SOD1, SOD2, GSH-Px, and CAT) and the activity of antioxidant enzymes (SOD, CAT, and GSH-Px) and MDA were upregulated compared with control. The expressions of genes related to apoptosis (Apaf-1, Bax, Caspase 3, Caspase 9, and p53) were higher than those in control during hypoxic exposure, while the expressions of Bcl-2 and Cyt C were decreased. The findings of the transcriptional analyses will provide insights into the molecular mechanisms of hybrid yellow catfish "Huangyou-1" under conditions of hypoxia and reoxygenation.

INTERACTION OF OXYGEN-SENSING MECHANISMS IN CELLS

2019 ◽  
pp. 52-62 ◽  
Author(s):  
A.N. Vetosh
Keyword(s):  
Potassium Channels ◽  
Human Body ◽  
Plasma Membranes ◽  
Molecular Mechanisms ◽  
Chronic Hypoxia ◽  
Oxygen Sensing ◽  
Acute Hypoxia ◽  
Cell Responses ◽  
Pubmed Database ◽  
In Cells

Reactions of the human body to chronic, acute or interval hypoxic hypoxia are different and may be triggered by certain intracellular molecular mechanisms. The authors analyzed PubMed database using the keywords “intracellular oxygen sensing” to verify the assumption. In 1977–2019, almost 1000 papers were published on the issue including more than 50 reviews. For their analysis, the authors chose articles on molecular oxygen sensing Metazoan tissue cells, mainly animals. Cell responses to chronic hypoxia are determined by HIF-pool localized in the cytoplasm. Oxygen-sensing to acute hypoxia in cells is preconditioned by molecular mechanisms involving potassium channels of plasma cell membranes and associated juxtamembrane complexes. Molecular intracellular reactions to interval hypoxia are triggered by the prooxidant process activation in the mitochondria of cells. This review discusses the interactional characteristics of the three mechanisms of oxygen-sensing cells. Keywords: oxygen, HIF, potassium channels of plasma membranes, mitochondria, ROS. Реакции организма человека на хроническую, острую или интервальную гипоксическую гипоксию различны и, возможно, запускаются отдельными внутриклеточными молекулярными механизмами. Для проверки этого предположения был проведён анализ литературных данных базы PubMed по ключевым словам «intracellular oxygen sensing». За период 1977–2019 гг. по данному вопросу было опубликовано почти 1000 работ, среди которых более 50 обзоров. Для анализа выбирались публикации, касающиеся молекулярной чувствительности к кислороду клеток тахитрофных тканей Metazoa, по преимуществу животных. Реакции клеток на хроническую гипоксию определяются HIF-пулом, локализованным в их цитоплазме. Кислородная чувствительность клеток к острой гипоксии обусловлена молекулярными механизмами при участии калиевых каналов плазматических клеточных мембран и ассоциированных с ними околомембранных комплексов. Молекулярные внутриклеточные реакции на интервальную гипоксию запускаются путём активизации прооксидантных процессов в митохондриях клеток. В данном обзоре обсуждаются особенности взаимодействия этих трёх механизмов кислородной чувствительности клеток. Ключевые слова: кислород, HIF, калиевые каналы плазматических мембран, митохондрии, АФК.

scholarly journals Gene expression of the liver in response to chronic hypoxia

2010 ◽  
Vol 41 (3) ◽  
pp. 275-288 ◽  
Author(s):  
Monica M. Baze ◽  
Karen Schlauch ◽  
Jack P. Hayes
Keyword(s):  
Gene Expression ◽  
Chronic Hypoxia ◽  
Inbred Mice ◽  
Acute Hypoxia ◽  
Transcript Abundance ◽  
Differentially Expressed ◽  
Severe Hypoxia ◽  
Protein Amino Acid ◽  
Hypoxic Conditions ◽  
False Discovery

Hypoxia is an important ecological, evolutionary, and biomedical stressor. While physiological acclimatization of mammals to hypoxia is well studied, the variation in gene expression that underlies acclimatization is not well studied. We acclimatized inbred mice for 32 days to hypoxic conditions that simulated altitudes of 1400, 3000, and 4500 m. We used oligonucleotide microarrays to measure changes in steady-state abundance of mRNA in the livers of these mice. Mice exposed to more severe hypoxia (simulated altitude of 4500 m) were smaller in mass and had higher hematocrit than mice exposed to less severe hypoxia. ANOVA and false discovery rate tests indicated that 580 genes were significantly differentially expressed in response to chronic hypoxia. Few of these 580 genes have previously been reported to respond to hypoxia. In contrast, many of these 580 genes belonged to same functional groups typically respond to acute hypoxia. That is, both chronic and acute hypoxia elicit changes in transcript abundance for genes involved in angiogenesis, glycolysis, lipid metabolism, carbohydrate metabolism, and protein amino acid phosphorylation, but the particular genes affected by the two types of hypoxia were mostly different. Numerous genes affecting the immune system were differentially expressed in response to chronic hypoxia, which supports recently proposed hypotheses that link immune function and hypoxia. Furthermore, our results discovered novel elevated mRNA abundance of genes involved in hematopoiesis and oxygen transport not reported previously, but consistent with extreme hematocrits found in hypoxic mice.

scholarly journals Metabolism of asymmetric dimethylarginine in hypoxia: from bench to bedside

2020 ◽  
Vol 10 (1_suppl) ◽  
pp. 31-41 ◽  
Author(s):  
Juliane Hannemann ◽  
Julia Zummack ◽  
Jonas Hillig ◽  
Rainer Böger
Keyword(s):  
Nitric Oxide ◽  
Pulmonary Hypertension ◽  
Animal Models ◽  
High Altitude ◽  
Vascular Function ◽  
Molecular Mechanisms ◽  
Asymmetric Dimethylarginine ◽  
Acute Hypoxia ◽  
Hypoxic Conditions ◽  
Pulmonary Vasoconstriction

Acute hypoxia and chronic hypoxia induce pulmonary vasoconstriction. While hypoxic pulmonary vasoconstriction is a physiological response if parts of the lung are affected, global exposure to hypoxic conditions may lead to clinical conditions like high-altitude pulmonary hypertension. Nitric oxide is the major vasodilator released from the vascular endothelium. Nitric oxide-dependent vasodilation is impaired in hypoxic conditions. Inhibition of nitric oxide synthesis is the most rapid and easily reversible molecular mechanism to regulate nitric oxide-dependent vascular function in response to physiological and pathophysiological stimuli. Asymmetric dimethylarginine is an endogenous, competitive inhibitor of nitric oxide synthase and a risk marker for major cardiovascular events and mortality. Elevated asymmetric dimethylarginine has been observed in animal models of hypoxia as well as in human cohorts under chronic and chronic intermittent hypoxia at high altitude. In lowlanders, asymmetric dimethylarginine is high in patients with pulmonary hypertension. We have recently shown that high asymmetric dimethylarginine at sea level is a predictor for high-altitude pulmonary hypertension. Asymmetric dimethylarginine is a highly regulated molecule, both by its biosynthesis and metabolism. Methylation of L-arginine by protein arginine methyltransferases was shown to be increased in hypoxia. Furthermore, the metabolism of asymmetric dimethylarginine by dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2) is decreased in animal models of hypoxia. Whether these changes are caused by transcriptional or posttranslational modifications remains to be elucidated. Current data suggest a major role of asymmetric dimethylarginine in regulating pulmonary arterial nitric oxide production in hypoxia. Further studies are needed to decipher the molecular mechanisms regulating asymmetric dimethylarginine in hypoxia and to understand their clinical significance.

Glibenclamide does not reverse attenuated vasoreactivity to acute or chronic hypoxia

1995 ◽  
Vol 79 (4) ◽  
pp. 1173-1180 ◽  
Author(s):  
M. R. Eichinger ◽  
T. C. Resta ◽  
D. S. Balderrama ◽  
G. M. Herrera ◽  
L. A. Richardson ◽  
...  
Keyword(s):  
Chronic Hypoxia ◽  
Channel Blocker ◽  
Acute Hypoxia ◽  
Peripheral Resistance ◽  
Katp Channels ◽  
Hypoxic Exposure ◽  
Conscious Rat ◽  
Hypoxic Conditions ◽  
Before And After

Recent studies from our laboratory have shown that acute and chronic hypoxic exposures are associated with attenuated systemic vasoreactivity in conscious rats. The present studies examined the role of adenosine triphosphate-sensitive potassium channels (KATP channels) in modulating the pressor and vasoconstrictor responses to phenylephrine (PE) in conscious instrumented rats 1) during acute hypoxia or 2) after chronic hypoxic exposure. Mean arterial pressure, mean cardiac output, and total peripheral resistance were assessed before and after graded infusions of PE in both groups of rats under normoxic or hypoxic conditions. Additionally, the role of KATP channels in attenuating vasoreactivity was determined by administration of glibenclamide (KATP channel blocker) before PE infusions. Acute hypoxia (12% O2) was associated with reduced pressor and constrictor responses to PE in control animals. Furthermore, acute return to room air did not restore the pressor and constrictor responses in the chronically hypoxic rats. Glibenclamide infusion did not influence the pressor or vasoconstrictor responses to PE in either group of animals during normoxia or acute hypoxia. Therefore, our data suggest that opening of KATP channels is not involved in the attenuated vasoreactivity associated with acute and chronic hypoxia in the conscious rat.

scholarly journals Comprehensive functional genomic resource and integrative model for the human brain

Science ◽  
2018 ◽  
Vol 362 (6420) ◽  
pp. eaat8464 ◽  
Author(s):  
Daifeng Wang ◽  
Shuang Liu ◽  
Jonathan Warrell ◽  
Hyejung Won ◽  
Xu Shi ◽  
...  
Keyword(s):  
Psychiatric Disorders ◽  
Molecular Mechanisms ◽  
Genome Wide Association Study ◽  
Expression Profiles ◽  
Cell Types ◽  
Adult Brain ◽  
Population Variation ◽  
Integrative Model ◽  
Risk Scores ◽  
Cell Type

Despite progress in defining genetic risk for psychiatric disorders, their molecular mechanisms remain elusive. Addressing this, the PsychENCODE Consortium has generated a comprehensive online resource for the adult brain across 1866 individuals. The PsychENCODE resource contains ~79,000 brain-active enhancers, sets of Hi-C linkages, and topologically associating domains; single-cell expression profiles for many cell types; expression quantitative-trait loci (QTLs); and further QTLs associated with chromatin, splicing, and cell-type proportions. Integration shows that varying cell-type proportions largely account for the cross-population variation in expression (with >88% reconstruction accuracy). It also allows building of a gene regulatory network, linking genome-wide association study variants to genes (e.g., 321 for schizophrenia). We embed this network into an interpretable deep-learning model, which improves disease prediction by ~6-fold versus polygenic risk scores and identifies key genes and pathways in psychiatric disorders.

scholarly journals Aging, inflammation and DNA damage in the somatic testicular niche with idiopathic germ cell aplasia

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Massimo Alfano ◽  
Anna Sofia Tascini ◽  
Filippo Pederzoli ◽  
Irene Locatelli ◽  
Manuela Nebuloni ◽  
...  
Keyword(s):  
Dna Damage ◽  
Germ Cell ◽  
Leydig Cells ◽  
Molecular Mechanisms ◽  
Transcriptome Profiling ◽  
Cell Type ◽  
Infertile Men ◽  
Damage Response ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

AbstractMolecular mechanisms associated with human germ cell aplasia in infertile men remain undefined. Here we perform single-cell transcriptome profiling to highlight differentially expressed genes and pathways in each somatic cell type in testes of men with idiopathic germ cell aplasia. We identify immaturity of Leydig cells, chronic tissue inflammation, fibrosis, and senescence phenotype of the somatic cells, as well markers of chronic inflammation in the blood. We find that deregulated expression of parentally imprinted genes in myoid and immature Leydig cells, with relevant changes in the ratio of Lamin A/C transcripts and an active DNA damage response in Leydig and peritubular myoid cells are also indicative of senescence of the testicular niche. This study offers molecular insights into the pathogenesis of idiopathic germ cell aplasia.

scholarly journals DDRE-28. MECHANISTIC AND THERAPEUTIC LINKS BETWEEN PURINE BIOSYNTHESIS AND DNA DAMAGE IN GLIOBLASTOMA

2021 ◽  
Vol 3 (Supplement_1) ◽  
pp. i12-i12
Author(s):  
Andrew Scott ◽  
Weihua Zhou ◽  
Kari Wilder-Romans ◽  
Jiane Feng ◽  
Zhe Wu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Brain Tumors ◽  
Cell Line ◽  
De Novo ◽  
Radiation Treatment ◽  
Adult Brain ◽  
Purine Biosynthesis ◽  
Purine Synthesis ◽  
Normal Cortex

Abstract Glioblastoma (GBM) is the most common and aggressive adult brain cancer. Radiation therapy (RT) is a critical treatment modality, and development of RT resistance is the predominant cause of recurrence and mortality in GBM patients. Using cell line models as well as patient-derived xenografts and neurospheres in orthotopic brain tumor models, we have identified increased rates and dependence upon de novo purine biosynthesis as a hallmark of GBM RT resistance. More recently, we have discovered that radiation treatment acutely stimulates flux through de novo purine synthesis in cell line and neurosphere models of GBM. This RT-induced increase in de novo purine synthesis is dependent on signaling through the DNA damage response and thus appears to be an adaptive mechanism to supply purines to repair radiation-induced DNA damage. To determine whether this regulatory mechanism also exists in vivo, we have used advanced metabolomic and metabolic tracing techniques with 13C-labeled glucose and 15N-labeled glutamine in mice bearing RT-resistant GBM patient-derived orthotopic brain tumors. We found that that orthotopic GBM PDXs had elevated activity of de novo purine synthesis that increased further after RT, while normal cortex had little activity even after RT. These observations have therapeutic relevance, as targeting this metabolic pathway with the FDA-approved purine biosynthesis inhibitor mycophenolate mofetil (MMF) overcomes GBM radiation resistance in mouse models in vivo. The lack of de novo purine synthesis in normal cortex suggests that targeting this pathway may be tumor specific. Collectively our data suggest that de novo synthesis of purines mediates RT resistance in GBM and that treatment of brain tumors with MMF in combination with RT may be a promising therapeutic strategy in patients.

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