Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

AbstractBackgroundStaufen2 (STAU2) is an RNA binding protein involved in the posttranscriptional regulation of gene expression. In neurons, STAU2 is required to maintain the balance between differentiation and proliferation of neural stem cells through asymmetric cell division. However, the importance of controlling STAU2 expression for cell cycle progression is not clear in non-neuronal dividing cells. We recently showed that STAU2 transcription is inhibited in response to DNA-damage due to E2F1 displacement from theSTAU2gene promoter. We now study the regulation of STAU2 steady-state levels in unstressed cells and its consequence for cell proliferation.ResultsCRISPR/Cas9-mediated and RNAi-dependent STAU2 depletion in the non-transformed hTERT-RPE1 cells both facilitate cell proliferation suggesting that STAU2 expression influences pathway(s) linked to cell cycle controls. Such effects are not observed in the CRISPR STAU2-KO cancer HCT116 cells nor in the STAU2-RNAi-depleted HeLa cells. Interestingly, a physiological decrease in the steady-state level of STAU2 is controlled by caspases. This effect of peptidases is counterbalanced by the activity of the CHK1 pathway suggesting that STAU2 partial degradation/stabilization fines tune cell cycle progression in unstressed cells. A large-scale proteomic analysis using STAU2/biotinylase fusion protein identifies known STAU2 interactors involved in RNA translation, localization, splicing, or decay confirming the role of STAU2 in the posttranscriptional regulation of gene expression. In addition, several proteins found in the nucleolus, including proteins of the ribosome biogenesis pathway and of the DNA damage response, are found in close proximity to STAU2. Strikingly, many of these proteins are linked to the kinase CHK1 pathway, reinforcing the link between STAU2 functions and the CHK1 pathway. Indeed, inhibition of the CHK1 pathway for 4 h dissociates STAU2 from proteins involved in translation and RNA metabolism.ConclusionsThese results indicate that STAU2 is involved in pathway(s) that control(s) cell proliferation, likely via mechanisms of posttranscriptional regulation, ribonucleoprotein complex assembly, genome integrity and/or checkpoint controls. The mechanism by which STAU2 regulates cell growth likely involves caspases and the kinase CHK1 pathway.

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TCF19 Impacts a Network of Inflammatory and DNA Damage Response Genes in the Pancreatic β-Cell

Metabolites ◽

10.3390/metabo11080513 ◽

2021 ◽

Vol 11 (8) ◽

pp. 513

Author(s):

Grace H. Yang ◽

Danielle A. Fontaine ◽

Sukanya Lodh ◽

Joseph T. Blumer ◽

Avtar Roopra ◽

...

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Dna Damage Response ◽

Association Studies ◽

Cell Cycle Gene ◽

Genome Wide Association Studies ◽

Β Cell ◽

Nucleotide Incorporation ◽

Damage Response ◽

The Impact

Transcription factor 19 (TCF19) is a gene associated with type 1 diabetes (T1DM) and type 2 diabetes (T2DM) in genome-wide association studies. Prior studies have demonstrated that Tcf19 knockdown impairs β-cell proliferation and increases apoptosis. However, little is known about its role in diabetes pathogenesis or the effects of TCF19 gain-of-function. The aim of this study was to examine the impact of TCF19 overexpression in INS-1 β-cells and human islets on proliferation and gene expression. With TCF19 overexpression, there was an increase in nucleotide incorporation without any change in cell cycle gene expression, alluding to an alternate process of nucleotide incorporation. Analysis of RNA-seq of TCF19 overexpressing cells revealed increased expression of several DNA damage response (DDR) genes, as well as a tightly linked set of genes involved in viral responses, immune system processes, and inflammation. This connectivity between DNA damage and inflammatory gene expression has not been well studied in the β-cell and suggests a novel role for TCF19 in regulating these pathways. Future studies determining how TCF19 may modulate these pathways can provide potential targets for improving β-cell survival.

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Dissecting the Role of Thyrotropin in the DNA Damage Response in Human Thyrocytes after 131I, γ Radiation and H2O2

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgz185 ◽

2019 ◽

Vol 105 (3) ◽

pp. 839-853

Author(s):

Aglaia Kyrilli ◽

David Gacquer ◽

Vincent Detours ◽

Anne Lefort ◽

Frédéric Libert ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Dna Damage ◽

Dna Damage Response ◽

Iodide Uptake ◽

Transcriptomic Response ◽

Uptake Inhibition ◽

Γ Radiation ◽

Damage Response

Abstract Background The early molecular events in human thyrocytes after 131I exposure have not yet been unravelled. Therefore, we investigated the role of TSH in the 131I-induced DNA damage response and gene expression in primary cultured human thyrocytes. Methods Following exposure of thyrocytes, in the presence or absence of TSH, to 131I (β radiation), γ radiation (3 Gy), and hydrogen peroxide (H2O2), we assessed DNA damage, proliferation, and cell-cycle status. We conducted RNA sequencing to profile gene expression after each type of exposure and evaluated the influence of TSH on each transcriptomic response. Results Overall, the thyrocyte responses following exposure to β or γ radiation and to H2O2 were similar. However, TSH increased 131I-induced DNA damage, an effect partially diminished after iodide uptake inhibition. Specifically, TSH increased the number of DNA double-strand breaks in nonexposed thyrocytes and thus predisposed them to greater damage following 131I exposure. This effect most likely occurred via Gα q cascade and a rise in intracellular reactive oxygen species (ROS) levels. β and γ radiation prolonged thyroid cell-cycle arrest to a similar extent without sign of apoptosis. The gene expression profiles of thyrocytes exposed to β/γ radiation or H2O2 were overlapping. Modulations in genes involved in inflammatory response, apoptosis, and proliferation were observed. TSH increased the number and intensity of modulation of differentially expressed genes after 131I exposure. Conclusions TSH specifically increased 131I-induced DNA damage probably via a rise in ROS levels and produced a more prominent transcriptomic response after exposure to 131I.

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Ancient Sturgeons Possess Effective DNA Repair Mechanisms: Influence of Model Genotoxicants on Embryo Development of Sterlet, Acipenser ruthenus

International Journal of Molecular Sciences ◽

10.3390/ijms22010006 ◽

2020 ◽

Vol 22 (1) ◽

pp. 6

Author(s):

Ievgeniia Gazo ◽

Roman Franěk ◽

Radek Šindelka ◽

Ievgen Lebeda ◽

Sahana Shivaramu ◽

...

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Dna Repair ◽

Embryo Development ◽

Hatching Rate ◽

Embryo Survival ◽

Dna Repair Mechanisms ◽

Acipenser Ruthenus ◽

Repair Mechanisms ◽

Sterlet Acipenser Ruthenus

DNA damage caused by exogenous or endogenous factors is a common challenge for developing fish embryos. DNA damage repair (DDR) pathways help organisms minimize adverse effects of DNA alterations. In terms of DNA repair mechanisms, sturgeons represent a particularly interesting model due to their exceptional genome plasticity. Sterlet (Acipenser ruthenus) is a relatively small species of sturgeon. The goal of this study was to assess the sensitivity of sterlet embryos to model genotoxicants (camptothecin, etoposide, and benzo[a]pyrene), and to assess DDR responses. We assessed the effects of genotoxicants on embryo survival, hatching rate, DNA fragmentation, gene expression, and phosphorylation of H2AX and ATM kinase. Exposure of sterlet embryos to 1 µM benzo[a]pyrene induced low levels of DNA damage accompanied by ATM phosphorylation and xpc gene expression. Conversely, 20 µM etoposide exposure induced DNA damage without activation of known DDR pathways. Effects of 10 nM camptothecin on embryo development were stage-specific, with early stages, before gastrulation, being most sensitive. Overall, this study provides foundational information for future investigation of sterlet DDR pathways.

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Female ageing affects the DNA repair capacity of oocytes in IVF using a controlled model of sperm DNA damage in mice

Human Reproduction ◽

10.1093/humrep/dez308 ◽

2020 ◽

Vol 35 (3) ◽

pp. 529-544 ◽

Cited By ~ 6

Author(s):

F Horta ◽

S Catt ◽

P Ramachandran ◽

B Vollenhoven ◽

P Temple-Smith

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Dna Repair ◽

Age Groups ◽

Oocyte Quality ◽

Repair Capacity ◽

Sperm Dna Damage ◽

Dna Repair Capacity ◽

Sperm Dna ◽

Damage Response

Abstract STUDY QUESTION Does female ageing have a negative effect on the DNA repair capacity of oocytes fertilised by spermatozoa with controlled levels of DNA damage? SUMMARY ANSWER Compared to oocytes from younger females, oocytes from older females have a reduced capacity to repair damaged DNA introduced by spermatozoa. WHAT IS KNOWN ALREADY The reproductive lifespan in women declines with age predominantly due to poor oocyte quality. This leads to decreased reproductive outcomes for older women undergoing assisted reproductive technology (ART) treatments, compared to young women. Ageing and oocyte quality have been clearly associated with aneuploidy, but the range of factors that influence this change in oocyte quality with age remains unclear. The DNA repair activity prior to embryonic genomic activation is considered to be of maternal origin, with maternal transcripts and proteins controlling DNA integrity. With increasing maternal age, the number of mRNAs stored in oocytes decreases. This could result in diminished efficiency of DNA repair and/or negative effects on embryo development, especially in the presence of DNA damage. STUDY DESIGN, SIZE, DURATION Oocytes from two age groups of 30 super-ovulated female mice (young: 5–8 weeks old, n = 15; old: 42–45 weeks old, n = 15) were inseminated with sperm from five males with three different controlled DNA damage levels; control: ≤10%, 1 Gray (Gy): 11–30%, and 30 Gy: >30%. Inseminated oocytes (young: 125, old: 78) were assessed for the formation of zygotes (per oocyte) and blastocysts (per zygote). Five replicates of five germinal vesicles (GVs) and five MII oocytes from each age group were analysed for gene expression. The DNA damage response (DDR) was assessed in a minimum of three IVF replicates in control and 1 Gy zygotes and two-cell embryos using γH2AX labelling. PARTICIPANTS/MATERIALS, SETTING, METHODS Swim-up sperm samples from the cauda epididymidis of C57BL6 mice were divided into control (no irradiation) and 1- and 30-Gy groups. Treated spermatozoa were irradiated at 1 and 30 Gy, respectively, using a linear accelerator Varian 21iX. Following irradiation, samples were used for DNA damage assessment (Halomax) and for insemination. Presumed zygotes were cultured in a time-lapse incubator (MIRI, ESCO). Gene expression of 91 DNA repair genes was assessed using the Fluidigm Biomark HD system. The DNA damage response in zygotes (6–8 h post-fertilisation) and two-cell embryos (22–24 h post-fertilisation) was assessed by immunocytochemical analysis of γH2AX using confocal microscopy (Olympus FV1200) and 3D volumetric analysis using IMARIS software. MAIN RESULTS AND THE ROLE OF CHANCE The average sperm DNA damage for the three groups was statistically different (control: 6.1%, 1 Gy: 16.1%, 30 Gy: 53.1%, P < 0.0001), but there were no significant differences in fertilisation rates after IVF within or between the two age groups [(young; control: 86.79%, 1 Gy: 82.75%, 30 Gy: 76.74%) (old; control: 93.1%, 1 Gy: 70.37%, 30 Gy: 68.18%) Fisher’s exact]. However, blastocyst rates were significantly different (P < 0.0001) among the groups [(young; control: 86.95%, 1 Gy: 33.33%, 30 Gy: 0.0%) (old; control: 70.37%, 1 Gy: 0.0%, 30 Gy: 0.0%)]. Between the age groups, 1-Gy samples showed a significant decrease in the blastocyst rate in old females compared to young females (P = 0.0166). Gene expression analysis revealed a decrease in relative expression of 21 DNA repair genes in old GV oocytes compared to young GV oocytes (P < 0.05), and similarly, old MII oocytes showed 23 genes with reduced expression compared to young MII oocytes (P < 0.05). The number of genes with decreased expression in older GV and MII oocytes significantly affected pathways such as double strand break (GV: 5; MII: 6), nucleotide excision repair (GV: 8; MII: 5) and DNA damage response (GV: 4; MII: 8). There was a decreased DDR in zygotes and in two-cell embryos from old females compared to young regardless of sperm treatment (P < 0.05). The decrease in DNA repair gene expression of oocytes and decreased DDR in embryos derived from older females suggests that ageing results in a diminished DNA repair capacity. LARGE-SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION Ionising radiation was used only for experimental purposes, aiming at controlled levels of sperm DNA damage; however, it can also damage spermatozoa proteins. The female age groups selected in mice were intended to model effects in young and old women, but clinical studies are required to demonstrate a similar effect. WIDER IMPLICATIONS OF THE FINDINGS Fertilisation can occur with sperm populations with medium and high DNA damage, but subsequent embryo growth is affected to a greater extent with aging females, supporting the theory that oocyte DNA repair capacity decreases with age. Assessment of the oocyte DNA repair capacity may be a useful diagnostic tool for infertile couples. STUDY FUNDING/COMPETING INTEREST(S) Funded by the Education Program in Reproduction and Development, Department of Obstetrics and Gynaecology, Monash University. None of the authors has any conflict of interest to report.

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Gene Expression Profiling after Radiation-Induced DNA Damage Is Strongly Predictive of BRCA1 Mutation Carrier Status

Clinical Cancer Research ◽

10.1158/1078-0432.ccr-1067-3 ◽

2004 ◽

Vol 10 (3) ◽

pp. 958-963 ◽

Cited By ~ 27

Author(s):

Zsofia Kote-Jarai ◽

Richard D. Williams ◽

Nicola Cattini ◽

Maria Copeland ◽

Ian Giddings ◽

...

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Gene Expression Profiling ◽

Mutation Carrier ◽

Expression Profiling ◽

Brca1 Mutation ◽

Brca1 Mutation Carrier ◽

Carrier Status ◽

Radiation Induced

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In vitro assessment of DNA damage after short- and long-term exposure to benzo(a)pyrene using RAPD and the RTG-2 fish cell line

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis ◽

10.1016/j.mrfmmm.2004.06.010 ◽

2004 ◽

Vol 552 (1-2) ◽

pp. 141-151 ◽

Cited By ~ 48

Author(s):

Argelia Castaño ◽

Concepción Becerril

Keyword(s):

Dna Damage ◽

Cell Line ◽

Fish Cell Line ◽

Fish Cell ◽

Short And Long Term ◽

In Vitro Assessment

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Genotoxicity and Gene Expression in the Rat Lung Tissue following Instillation and Inhalation of Different Variants of Amorphous Silica Nanomaterials (aSiO2 NM)

Nanomaterials ◽

10.3390/nano11061502 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1502

Author(s):

Fátima Brandão ◽

Carla Costa ◽

Maria João Bessa ◽

Elise Dumortier ◽

Florence Debacq-Chainiaux ◽

...

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Amorphous Silica ◽

Oxidative Dna Damage ◽

Intratracheal Instillation ◽

Dna Lesions ◽

Lung Cells ◽

Rat Lung ◽

Inhalation Study

Several reports on amorphous silica nanomaterial (aSiO2 NM) toxicity have been questioning their safety. Herein, we investigated the in vivo pulmonary toxicity of four variants of aSiO2 NM: SiO2_15_Unmod, SiO2_15_Amino, SiO2_7 and SiO2_40. We focused on alterations in lung DNA and protein integrity, and gene expression following single intratracheal instillation in rats. Additionally, a short-term inhalation study (STIS) was carried out for SiO2_7, using TiO2_NM105 as a benchmark NM. In the instillation study, a significant but slight increase in oxidative DNA damage in rats exposed to the highest instilled dose (0.36 mg/rat) of SiO2_15_Amino was observed in the recovery (R) group. Exposure to SiO2_7 or SiO2_40 markedly increased oxidative DNA lesions in rat lung cells of the exposure (E) group at every tested dose. This damage seems to be repaired, since no changes compared to controls were observed in the R groups. In STIS, a significant increase in DNA strand breaks of the lung cells exposed to 0.5 mg/m3 of SiO2_7 or 50 mg/m3 of TiO2_NM105 was observed in both groups. The detected gene expression changes suggest that oxidative stress and/or inflammation pathways are likely implicated in the induction of (oxidative) DNA damage. Overall, all tested aSiO2 NM were not associated with marked in vivo toxicity following instillation or STIS. The genotoxicity findings for SiO2_7 from instillation and STIS are concordant; however, changes in STIS animals were more permanent/difficult to revert.

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Pseudorabies virus infection triggers NF-κB activation via the DNA damage response, but actively inhibits NFkB-dependent gene expression

Journal of Virology ◽

10.1128/jvi.01666-21 ◽

2021 ◽

Author(s):

Nicolás Romero ◽

Herman W. Favoreel

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Dna Damage Response ◽

Pseudorabies Virus ◽

Critical Role ◽

Genome Replication ◽

Viral Protein Synthesis ◽

Damage Response ◽

Signaling Axis ◽

Inside Out

The nuclear factor kappa B (NF-κB) pathway is known to integrate signaling associated with very diverse intra- and extracellular stressors including virus infections, and triggers a powerful (pro-inflammatory) response through the expression of NF-κB-regulated genes. Typically, the NF-κB pathway collects and transduces threatening signals at the cell surface or in the cytoplasm leading to nuclear import of activated NF-κB transcription factors. In the current work, we demonstrate that the swine alphaherpesvirus pseudorabies virus (PRV) induces a peculiar mode of NF-κB activation known as “inside-out” NF-κB activation. We show that PRV triggers the DNA damage response (DDR) and that this DDR response drives NF-κB activation since inhibition of the nuclear ataxia telangiectasia-mutated (ATM) kinase, a chief controller of DDR, abolished PRV-induced NF-κB activation. Initiation of the DDR-NF-κB signaling axis requires viral protein synthesis but occurs before active viral genome replication. In addition, the initiation of the DDR-NF-κB signaling axis is followed by a virus-induced complete shutoff of NF-κB-dependent gene expression that depends on viral DNA replication. In summary, the results presented in this study reveal that PRV infection triggers a non-canonical DDR-NF-κB activation signaling axis and that the virus actively inhibits the (potentially antiviral) consequences of this pathway, by inhibiting NF-κB-dependent gene expression. IMPORTANCE: The NF-κB signaling pathway plays a critical role in coordination of innate immune responses that are of vital importance in the control of infections. The current report generates new insights in the interaction of the alphaherpesvirus pseudorabies virus (PRV) with the NF-κB pathway, as they reveal that (i) PRV infection leads to NF-κB activation via a peculiar “inside-out’ nucleus-to-cytoplasm signal that is triggered via the DNA damage response (DDR), (ii) the DDR-NF-κB signaling axis requires expression of viral proteins but is initiated before active PRV replication, and (iii) late viral factor(s) allow PRV to actively and efficiently inhibit NF-κB-dependent (pro-inflammatory) gene expression. These data suggest that activation of the DDR-NF-κB during PRV infection is host-driven and that its potential antiviral consequences are actively inhibited by the virus.

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