Assessing the DNA Damaging Effectiveness of Ionizing Radiation Using Plasmid DNA
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ABSTRACT:
SUBMITTER: Maayah Y
PROVIDER: S-EPMC9604049 | biostudies-literature | 2022 Oct
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:DNA damage response (DDR) to double strand breaks is coordinated by 3 phosphatidylinositol 3-kinase-related kinase (PIKK) family members: the ataxia-telangiectasia mutated kinase (ATM), the ATM and Rad3-related (ATR) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). ATM and ATR are central players in activating cell cycle checkpoints and function as an active barrier against genome instability and tumorigenesis in replicating cells. Loss of ATM function is frequently reported in various types of tumors, thus placing more reliance on ATR for checkpoint arrest and cell survival following DNA damage. To investigate the role of ATR in the G2/M checkpoint regulation in response to ionizing radiation (IR), particularly when ATM is deficient, cell lines deficient of ATM, ATR, or both were generated using a doxycycline-inducible lentiviral system. Our data suggests that while depletion of ATR or ATM alone in wild-type human mammary epithelial cell cultures (HME-CCs) has little effect on radiosensitivity or IR-induced G2/M checkpoint arrest, depletion of ATR in ATM-deficient cells causes synthetic lethality following IR, which correlates with severe G2/M checkpoint attenuation. ATR depletion also inhibits IR-induced autophagy, regardless of the ATM status, and enhances IR-induced apoptosis particularly when ATM is deficient. Collectively, our results clearly demonstrate that ATR function is required for the IR-induced G2/M checkpoint activation and subsequent survival of cells with ATM deficiency. The synthetic lethal interaction between ATM and ATR in response to IR supports ATR as a therapeutic target for improved anti-cancer regimens, especially in tumors with a dysfunctional ATM pathway.
| S-EPMC4615027 | biostudies-literature
Project description:DNA repair is an essential cellular process required to maintain genomic stability. Every cell is subjected to thousands of DNA lesions daily under normal physiological conditions. Ionizing radiation (IR) is a major DNA damaging agent that can be produced by both natural and man-made sources. A common source of radiation exposure is through its use in medical diagnostics or treatments such as for cancer radiotherapy where relatively high doses are received by patients. To understand the detailed DNA repair gene transcription response to high dose IR, gene expression exon array studies have been performed and the response to radiation in two divergent cell types, lymphoblastoid cell lines and primary fibroblasts, has been examined. These exon arrays detect expression levels across the entire gene, and have the advantage of high sensitivity and the ability to identify alternative transcripts. We found a selection of DNA repair genes, including some not previously reported, that are modulated in response to radiation. Detailed dose and time course kinetics of DNA repair transcription was conducted and results have been validated utilizing PCR methods. Alternative transcription products in response to IR were identified in several DNA repair genes including RRM2B and XPC where alternative initiation sites were found. These investigations have advanced the knowledge about the transcriptional response of DNA repair.
| S-EPMC3532210 | biostudies-literature
Project description:Ionizing radiation (IR) is a common mode of cancer therapy, where DNA damage is the major reason of cell death. Here, we use an assay based on fluorescence imaging of single damaged DNA molecules isolated from radiated lymphocytes, to quantify IR induced DNA damage. The assay uses a cocktail of DNA-repair enzymes that recognizes and excises DNA lesions and then a polymerase and a ligase incorporate fluorescent nucleotides at the damage sites, resulting in a fluorescent "spot" at each site. The individual fluorescent spots can then be counted along single stretched DNA molecules and the global level of DNA damage can be quantified. Our results demonstrate that inclusion of the human apurinic/apyrimidinic endonuclease 1 (APE1) in the enzyme cocktail increases the sensitivity of the assay for detection of IR induced damage significantly. This optimized assay also allowed detection of a cooperative increase in DNA damage when IR was combined with mild hyperthermia, which is sometimes used as an adjuvant in IR therapy. Finally, we discuss how the method may be used to identify patients that are sensitive to IR and other types of DNA damaging agents.
| S-EPMC7350159 | biostudies-literature
Project description:PURPOSE:Ionizing radiation induced foci (IRIF) known also as DNA repair foci represent most sensitive endpoint for assessing DNA double strand breaks (DSB). IRIF are usually visualized and enumerated with the aid of fluorescence microscopy using antibodies to ?H2AX and 53BP1. This study analyzed effect of low dose ionizing radiation on residual IRIF in human lymphocytes to the aim of potential biodosimetry and possible extrapolation of high-dose ?H2AX/53BP1 effects to low doses and compared kinetics of DSB and IRIF. We also analyzed whether DNaseI, which is used for reducing of clumps, affects the IRIF level. MATERIALS AND METHODS:The cryopreserved human lymphocytes from umbilical cord blood (UCB) were thawed with/without DNaseI, ?-irradiated at doses of 0, 5, 10, and 50 cGy and ?H2AX/53BP1 foci were analyzed 30 min, 2 h, and 22 h post-irradiation using appropriate antibodies. We also analyzed kinetics of DSB using PFGE. RESULTS:No significant difference was observed between data obtained by ?H2AX foci evaluation in cells that were irradiated by low doses and data obtained by extrapolation from higher doses. Residual 53BP1 foci induced by low doses significantly outreached the data extrapolated from irradiation by higher doses. 53BP1 foci induced by low dose-radiation remain longer at DSB loci than foci induced by higher doses. There was no significant effect of DNaseI on DNA repair foci. CONCLUSIONS:Primary ?H2AX, 53BP1 foci and their co-localization represent valuable markers for biodosimetry of low doses, but their usefulness is limited by short time window. Residual ?H2AX and 53BP1 foci are more useful markers for biodosimetry in vitro. Effects of low doses can be extrapolated from high dose using ?H2AX residual foci while ?H2AX/53BP1 foci are valuable markers for evaluation of initial DSB induced by ionizing radiation. Residual IRIF induced by low doses persist longer time than those induced by higher doses.
| S-EPMC7016656 | biostudies-literature
Project description:Ectoine plays an important role in protecting biomolecules and entire cells against environmental stressors such as salinity, freezing, drying and high temperatures. Recent studies revealed that ectoine also provides effective protection for human skin cells from damage caused by UV-A radiation. These protective properties make ectoine a valuable compound and it is applied as an active ingredient in numerous pharmaceutical devices and cosmetics. Interestingly, the underlying mechanism resulting in protecting cells from radiation is not yet fully understood. Here we present a study on ectoine and its protective influence on DNA during electron irradiation. Applying gel electrophoresis and atomic force microscopy, we demonstrate for the first time that ectoine prevents DNA strand breaks caused by ionizing electron radiation. The results presented here point to future applications of ectoine for instance in cancer radiation therapy.
| S-EPMC5681641 | biostudies-literature
Project description:DNA repair is an essential cellular process required to maintain genomic stability. Every cell is subjected to thousands of DNA lesions daily under normal changes in transcription. Transcription is a primary process where protein amount and function can be regulated. One aspect of the transcriptional IR response that little is known about on a whole genome basis is alternative transcription. These investigations focus on the response to IR at the exon level in human cells but also at the whole gene level. Whole genome exon arrays were utilized to comprehensively characterize radiation-induced transcriptional expression products in two human cell types, namely EBV-transformed lymphoblast and primary fibroblast cell lines.
2012-10-26 | GSE41840 | GEO
Project description:Exposure to radiation causes DNA damage; hence, continuous surveillance and timely DNA repair are important for genome stability. Epigenetic modifications alter the chromatin architecture, thereby affecting the efficiency of DNA repair. However, how epigenetic modifiers coordinate with the DNA repair machinery to modulate cellular radiosensitivity is relatively unknown. Here, we report that loss of the demethylase ribosomal oxygenase 1 (RIOX1) restores cell proliferation and reduces cell death after exposure to ionizing radiation. Furthermore, RIOX1 depletion enhances homologous recombination (HR) repair but not nonhomologous end-joining (NHEJ) repair in irradiated bone marrow cells and oral mucosal epithelial cells. Mechanistic study demonstrates that RIOX1 removes monomethylation at K491 of cyclic GMP-AMP synthase (cGAS) to release cGAS from its interaction with the methyl-lysine reader protein SAGA complex-associated factor 29 (SGF29), which subsequently enables cGAS to interact with poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase 1 (PARP1) at DNA break sites, thereby blocking PARP1-mediated recruitment of Timeless. High expression of RIOX1 maintains cGAS K491me at a low level, which impedes HR repair and reduces cellular tolerance to ionizing radiation. This study highlights a novel RIOX1-dependent mechanism involved in the non-immune function of cGAS that is essential for the regulation of ionizing radiation-elicited HR repair.
| S-EPMC8873214 | biostudies-literature
Project description:DNA repair is an essential cellular process required to maintain genomic stability. Every cell is subjected to thousands of DNA lesions daily under normal changes in transcription. Transcription is a primary process where protein amount and function can be regulated. One aspect of the transcriptional IR response that little is known about on a whole genome basis is alternative transcription. These investigations focus on the response to IR at the exon level in human cells but also at the whole gene level. Whole genome exon arrays were utilized to comprehensively characterize radiation-induced transcriptional expression products in two human cell types, namely EBV-transformed lymphoblast and primary fibroblast cell lines. 12 human primary fibroblast cell lines and 12 primary lymphoblast cell line samples were used for two doses (0 and 10 Gy) and two time points (0 and 4hr). Additional doses ( 1 Gy, 2 Gy, 5 Gy, and 20 Gy) and time points ( 1hr, 2hr, 8hr, 24hr and 48hr) were assessed in four primary lymphoblast cell lines and four primary fibroblasts.
2012-10-26 | E-GEOD-41840 | biostudies-arrayexpress
Project description:We report experimental results on damage induced by ionizing radiation to DNA origami triangles which are commonly used prototypes for scaffolded DNA origami nanostructures. We demonstrate extreme stability of DNA origami upon irradiation, which is caused by (i) the multi-row design holding the shape of the origami even after severe damage to the scaffold DNA and (ii) the reduction of damage to the scaffold DNA due to the protective effect of the folded structure. With respect to damage induced by ionizing radiation, the protective effect of the structure is superior to that of a naturally paired DNA double helix. Present results allow estimating the stability of scaffolded DNA origami nanostructures in applications such as nanotechnology, pharmacy or in singulo molecular studies where they are exposed to ionizing radiation from natural and artificial sources. Additionally, possibilities are opened for scaffolded DNA use in the design of radiation-resistant and radio-sensitive materials.
| S-EPMC8247635 | biostudies-literature
Project description:Ionizing radiation is known to cause DNA damage, yet the mechanisms underlying potential transgenerational effects of exposure have been scarcely studied. Previously, we observed effects in offspring of zebrafish exposed to gamma radiation during gametogenesis. Here, we hypothesize that these effects are accompanied by changes of DNA methylation possibly inherited by subsequent generations. We assessed DNA methylation in F1 embryos (5.5 hours post fertilization) with whole genome bisulfite sequencing following parental exposure to 8.7 mGy/h for 27 days and found 5658 differentially methylated regions (DMRs). DMRs were predominantly located at known regulatory regions, such as gene promoters and enhancers. Pathway analysis indicated the involvement of DMRs related to similar pathways found with gene expression analysis, such as development, apoptosis and cancers, which could be linked to previous observed developmental defects and genomic instability in the offspring. Follow up of 19 F1 DMRs in F2 and F3 embryos revealed persistent effects up to the F3 generation at 5 regions. These results indicate that ionizing radiation related effects in offspring can be linked to DNA methylation changes that partly can persist over generations. Monitoring DNA methylation could serve as a biomarker to provide an indication of ancestral exposures to ionizing radiation.
| S-EPMC6193964 | biostudies-literature