Assessment of Normal Tissue Radiosensitivity by Evaluating DNA Damage and Repair Kinetics in Human Brain Organoids

DNA-double strand break (DSB), detected by immunostaining of key proteins orchestrating repair, like γH2AX and 53BP1, is well established as a surrogate for tissue radiosensitivity. We hypothesized that the generation of normal brain 3D organoids (“mini-brains”) from human induced pluripotent stem cells (hiPSC) combined with detection of DNA damage repair (DDR) may hold the promise towards developing personalized models for the determination of normal tissue radiosensitivity. In this study, cerebral organoids, an in vitro model that stands in its complexity between 2D cellular system and an organ, have been used. To quantify radiation-induced response, immunofluorescent staining with γH2AX and 53BP1 were applied at early (30 min, initial damage), and late time points (18 and 72 h, residual damage), following clinical standard 2 Gy irradiation. Based on our findings, assessment of DDR kinetics as a surrogate for radiosensitivity in hiPSC derived cerebral organoids is feasible. Further development of mini-brains recapitulating mature adult neuronal tissue and implementation of additional signaling and toxicity surrogates may pave the way towards development of next-generation personalized assessment of radiosensitivity in healthy neuronal tissue.

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DNA double-strand break repair: a theoretical framework and its application

Journal of The Royal Society Interface ◽

10.1098/rsif.2015.0679 ◽

2016 ◽

Vol 13 (114) ◽

pp. 20150679 ◽

Cited By ~ 9

Author(s):

Philip J. Murray ◽

Bart Cornelissen ◽

Katherine A. Vallis ◽

S. Jon Chapman

Keyword(s):

Dna Damage ◽

Auger Electron ◽

Strand Break ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Histone H2ax ◽

Dna Double Strand Break ◽

A Cell

DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γ H2AX. Many copies of γ H2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti- γ H2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo . Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, 111 In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti- γ H2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti- γ H2AX-TAT and γ H2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti- γ H2AX antibody is labelled with Auger electron-emitting 111 In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti- γ H2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti- γ H2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage accumulation in the presence of Auger electron-emitting 111 In that is supported qualitatively by the available experimental data.

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ChK1 Activation Induces Reactive Astrogliosis Through CIP2A/PP2A/STAT3 Pathway In Alzheimer's Disease

10.21203/rs.3.rs-877963/v1 ◽

2021 ◽

Author(s):

Ying Zhou ◽

Xiaoyuan Liu ◽

Shuqing Ma ◽

Dichen Yang ◽

Nan Zhang ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Dna Damage ◽

Cognitive Deficits ◽

Immunofluorescent Staining ◽

Experimental Conditions ◽

Stat3 Pathway ◽

Reactive Astrogliosis

Abstract Background: In Alzheimer’s disease (AD), activation of astrocyte participates in the development of neurodegenerative diseases through neuroinflammation and disturbs glia-neuron interaction. Cancerous Inhibitor of PP2A (CIP2A) is an endogenous PP2A inhibitor. CIP2A upregulation specifically in astrocytes causes reactive astrogliosis, synaptic degeneration and cognitive deficits. However, the underlying mechanism of CIP2A upregulation remains unclear. Methods: In 3xTg-AD mice, we determined ChK1 was activated and related to DNA damage upregulating CIP2A by WB. We transfected EGFP-ChK1 plasmid into HEK293-T cell to determine ChK1 induces CIP2A upregulation and PP2A inhibition. We incubated Aβ and infected GFAP-ChK1-LV into primary astrocytes to confirm the signaling pathway in astrocytes and astrogliosis in AD. GFAP-ChK1-AAV was injected into C57/BL6 mice to induce specific expression of target protein in astrocytes. ChK1 inhibitor (SB) was performed to reverse the ChK1 activity. Outcomes were assessed using molecular (immunofluorescent staining, Western Blot and Golgi staining) measures to estimate symptomatic pathology and behavioral (NORT, OLT, MWM and FCT) measures to assess cognitive function. For most experiments, subjects were randomly assigned to experimental groups, and data were collected under blinded experimental conditions.Results: We demonstrated that DNA damage related Checkpoint kinase 1 (ChK1) was activated in 3xTg-AD mice. ChK1-mediated CIP2A overexpression drove inhibition of PP2A and activated STAT3, then led to reactive astrogliosis and neurodegeneration in vitro. Infection of mouse brain with GFAP-ChK1-AAV induced AD-like cognitive deficits and exacerbated AD pathologies in vivo. In conclusion, we showed that ChK1 activation induced reactive astrogliosis, degeneration of neurons and deterioration of AD through CIP2A-PP2A-STAT3 pathway, and inhibiting ChK1 might be a potential therapeutic approach for AD treatment.Conclusions: These results suggest that ChK1 is upregulated in 3xTg-AD mice, ChK1-mediated CIP2A overexpression drives inhibition of PP2A and activates STAT3, then leads to reactive astrogliosis, neurodegeneration and AD-like cognitive deficits in vitro and in vivo.

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DNA polymerase X from Deinococcus radiodurans implicated in bacterial tolerance to DNA damage is characterized as a short patch base excision repair polymerase

Microbiology ◽

10.1099/mic.0.029223-0 ◽

2009 ◽

Vol 155 (9) ◽

pp. 3005-3014 ◽

Cited By ~ 19

Author(s):

Nivedita P. Khairnar ◽

Hari S. Misra

Keyword(s):

Dna Damage ◽

Dna Polymerase ◽

Base Excision Repair ◽

Deinococcus Radiodurans ◽

Excision Repair ◽

Strand Break ◽

Klenow Fragment ◽

E Coli ◽

Base Excision

The Deinococcus radiodurans R1 genome encodes an X-family DNA repair polymerase homologous to eukaryotic DNA polymerase β. The recombinant deinococcal polymerase X (PolX) purified from transgenic Escherichia coli showed deoxynucleotidyltransferase activity. Unlike the Klenow fragment of E. coli, this enzyme showed short patch DNA synthesis activity on heteropolymeric DNA substrate. The recombinant enzyme showed 5′-deoxyribose phosphate (5′-dRP) lyase activity and base excision repair function in vitro, with the help of externally supplied glycosylase and AP endonuclease functions. A polX disruption mutant of D. radiodurans expressing 5′-dRP lyase and a truncated polymerase domain was comparatively less sensitive to γ-radiation than a polX deletion mutant. Both mutants showed higher sensitivity to hydrogen peroxide. Excision repair mutants of E. coli expressing this polymerase showed functional complementation of UV sensitivity. These results suggest the involvement of deinococcal polymerase X in DNA-damage tolerance of D. radiodurans, possibly by contributing to DNA double-strand break repair and base excision repair.

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Supramolecular Complex Formation between Rad6 and Proteins of the p53 Pathway during DNA Damage-Induced Response

Molecular and Cellular Biology ◽

10.1128/mcb.23.7.2463-2475.2003 ◽

2003 ◽

Vol 23 (7) ◽

pp. 2463-2475 ◽

Cited By ~ 33

Author(s):

Alex Lyakhovich ◽

Malathy P. V. Shekhar

Keyword(s):

Dna Damage ◽

Complex Formation ◽

26S Proteasome ◽

Induced Response ◽

Postreplication Repair ◽

Protein Levels ◽

P53 Response ◽

Mcf10a Cells

ABSTRACT The HR6A and -B genes, homologues of the yeast Rad6 gene, encode ubiquitin-conjugating enzymes that are required for postreplication repair of DNA and damage-induced mutagenesis. Using surface plasmon resonance, we show here that HR6 protein (referred as Rad6) physically interacts with p53. Analysis of proteins coimmunoprecipitated with Rad6 antibody from metabolically labeled normal MCF10A human breast epithelial cells not only confirmed Rad6-p53 interactions in vivo but also demonstrated for the first time that exposure of MCF10A cells to cisplatin or adriamycin (ADR) induces recruitment of p14ARF into Rad6-p53 complexes. Further analysis of ADR-induced p53 response showed that stable Rad6-p53-p14ARF complex formation is associated with a parallel increase and decrease in monoubiquitinated and polyubiquitinated p53, respectively, and arrest in G2/M phase of the cell cycle. Interestingly, the ADR-induced suppression of p53 polyubiquitination correlated with a corresponding decline in intact Hdm2 protein levels. Treatment of MCF10A cells with MG132, a 26S proteasome inhibitor, effectively stabilized monoubiquitinated p53 and rescued ADR-induced downregulation of Hdm2. These data suggest that ADR-induced degradation of Hdm2 occurs via the ubiquitin-proteasome pathway. Rad6 is present in both the cytoplasmic and nuclear compartments of normal MCF10A cells, although in response to DNA damage it is predominantly found in the nucleus colocalizing with ubiquitinated p53, whereas Hdm2 is undetectable. Consistent with in vivo data, results from in vitro ubiquitination assays show that Rad6 mediates addition of one (mono-) to two (multimono-) ubiquitin molecules on p53 and that inclusion of Mdm2 is essential for its polyubiquitination. The data presented in the present study suggest that Rad6-p53-p14ARF complex formation and p53 ubiquitin modification are important damage-induced responses that perhaps determine the fidelity of DNA postreplication repair.

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The DNA damage response regulates the oocyte pool in mammals

10.1101/816488 ◽

2019 ◽

Author(s):

Ana Martínez-Marchal ◽

Maria Teresa Guillot ◽

Mònica Ferrer ◽

Anna Guixé ◽

Montserrat Garcia-Caldés ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Dna Damage Response ◽

Adult Life ◽

Strand Break ◽

Oocyte Number ◽

Genome Wide ◽

Germ Cell Cysts ◽

Damage Response

SummaryMammalian oogonia proliferate without completing cytokinesis producing germ cell cysts. Within these cysts, oocytes differentiate and enter meiosis, promote genome-wide double-strand break (DSBs) formation which repair by homologous recombination leads to synapsis of the homologous chromosomes. Errors in homologous recombination or synapsis trigger the activation of surveillance mechanisms, traditionally called ‘pachytene checkpoint’, to either repair them or send the cells to programmed death. Contrary to what is found in spermatocytes, most oocytes present a remarkable persistence of unrepaired DSBs at pachynema. Simultaneously, there is a massive oocyte death accompanying the oocyte cyst breakdown. This oocyte elimination is thought to be required to properly form the follicles, which constitute the pool of germ cells females will use during their adult life. Based on all the above mentioned, we hypothesized that the apparently inefficient meiotic recombination occurring in mouse oocytes may be required to eliminate most of the oocytes in order to regulate the oocyte number, promote cyst breakdown and follicle formation in mammalian females. To test this idea, we analyzed perinatal ovaries to evaluate the oocyte population, cyst breakdown and follicle formation in control and mutant mice for the effector kinase of the DNA damage response, CHK2. Our results confirm the involvement of CHK2 in the elimination of oocytes that accumulate unrepaired DSBs and show that CHK2 regulates the number of oocytes in fetal ovaries. We also show that CHK2 is required to eliminate oocytes as a result of LINE-1 activation, which was previously shown to be responsible for fetal oocyte loss. Nonetheless, the number of oocytes found in Chk2 mutant ovaries three days after birth was similar to that of control ovaries, suggesting the existence of CHK2-independent mechanisms capable of eliminating oocytes. In vitro inhibition of CHK1 rescued the oocyte number in Chk2 mutant ovaries suggesting that CHK1 regulates postnatal oocyte death. Moreover, both CHK1 and CHK2 functions are required to timely breakdown cyst and form follicles. Altogether, we propose the DNA damage response controls the number of oocytes present perinatally and is required to properly break down oocyte cysts and form follicles, highlighting the importance of the DNA damage response in setting the reserve of oocytes each female will use during their entire lifespan.

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Modeling Cancer Genomic Data in Yeast Reveals Selection Against ATM Function During Tumorigenesis

10.1101/771253 ◽

2019 ◽

Author(s):

Marcel Hohl ◽

Aditya Mojumdar ◽

Sarem Hailemariam ◽

Vitaly Kuryavyi ◽

Fiorella Ghisays ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Tumor Suppression ◽

Chromosome Instability ◽

Genomic Data ◽

Major Axis ◽

Strand Break ◽

Cancer Center ◽

Mre11 Complex

AbstractThe DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene-induced carcinogenesis. The complex is integral to all modes of double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S.cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was defective while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells.Author SummaryA complex network of functions is required for suppressing tumorigenesis. These include processes that regulate cell growth and differentiation, processes that repair damage to DNA and thereby prevent cancer promoting mutations and signaling pathways that lead to growth arrest and programmed cell death. The Mre11 complex influences both signaling and DNA repair. To understand its role in tumor suppression, we characterized mutations affecting members of the Mre11 complex that were uncovered through cancer genomic analyses. The data reveal that the signaling functions of the Mre11 complex are important for tumor suppression to a greater degree than its role in DNA repair.

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The DNA damage response is required for oocyte cyst breakdown and follicle formation in mice

PLoS Genetics ◽

10.1371/journal.pgen.1009067 ◽

2020 ◽

Vol 16 (11) ◽

pp. e1009067

Author(s):

Ana Martínez-Marchal ◽

Yan Huang ◽

Maria Teresa Guillot-Ferriols ◽

Mònica Ferrer-Roda ◽

Anna Guixé ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Dna Damage Response ◽

Strand Break ◽

Double Strand Break ◽

Mutant Mice ◽

Oocyte Number ◽

Prophase I ◽

Damage Response

Mammalian oogonia proliferate without completing cytokinesis, forming cysts. Within these, oocytes differentiate and initiate meiosis, promoting double-strand break (DSBs) formation, which are repaired by homologous recombination (HR) causing the pairing and synapsis of the homologs. Errors in these processes activate checkpoint mechanisms, leading to apoptosis. At the end of prophase I, in contrast with what is observed in spermatocytes, oocytes accumulate unrepaired DSBs. Simultaneously to the cyst breakdown, there is a massive oocyte death, which has been proposed to be necessary to enable the individualization of the oocytes to form follicles. Based upon all the above-mentioned information, we hypothesize that the apparently inefficient HR occurring in the oocytes may be a requirement to first eliminate most of the oocytes and enable cyst breakdown and follicle formation. To test this idea, we compared perinatal ovaries from control and mutant mice for the effector kinase of the DNA Damage Response (DDR), CHK2. We found that CHK2 is required to eliminate ~50% of the fetal oocyte population. Nevertheless, the number of oocytes and follicles found in Chk2-mutant ovaries three days after birth was equivalent to that of the controls. These data revealed the existence of another mechanism capable of eliminating oocytes. In vitro inhibition of CHK1 rescued the oocyte number in Chk2-/- mice, implying that CHK1 regulates postnatal oocyte death. Moreover, we found that CHK1 and CHK2 functions are required for the timely breakdown of the cyst and to form follicles. Thus, we uncovered a novel CHK1 function in regulating the oocyte population in mice. Based upon these data, we propose that the CHK1- and CHK2-dependent DDR controls the number of oocytes and is required to properly break down oocyte cysts and form follicles in mammals.

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Abstract TP265: AATF Protects Neuron Against Ischemia via Regulating PAR/AIF Pathway

Stroke ◽

10.1161/str.48.suppl_1.tp265 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Wei Jin ◽

Wei Xu ◽

Jing Chen ◽

Xiaoxiao Zhang ◽

Chuancheng Ren

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Western Blot ◽

Dna Fragmentation ◽

Caspase 3 ◽

Immunofluorescent Staining ◽

Dependent Manner ◽

Cellular Death ◽

Cell Death Pathway

Abstract: Apoptosis antagonizing transcription factor (AATF) exerts an effect against oxidative stress, DNA damage and cellular apoptosis. However, its role in neuronal ischemia or hypoxia damage has not been elucidated yet. Present study investigated the neuroprotective roles and mechanisms of AATF under ischemia and hypoxia in vivo and in vitro. Focal cerebral ischemia of rat was generated by distal middle cerebral artery occlusion (dMCAO) model, SH-SY5Y cells were used to generate oxygen glucose deprivation (OGD) model in vitro. Western blot and immunofluorescent staining were used to investigate the expression changes of AATF. CCK-8 and LDH were performed to evaluate cellular survival and cytotoxicity. Overexpression and interference lentivirus vectors were performed to regulate the expression of AATF in SH-SY5Y cells. DHE staining that measured by flow cytometry was performed to investigate cellular superoxide anion levels. 8-OHdG expression and AP sites measurement were used to evaluate DNA damage. DNA Ladder and TUNEL staining were employed to evaluate DNA fragmentation. MNNG and DPQ were respectively used to agitate or antagonist caspase-3 independent PCD (programmed cell death) pathway, STS and Z-VAD-fmk were respectively used to agitate or antagonist caspase-3 dependent PCD pathway. Western blot was performed to investigate the expression of Poly(ADP-ribose) polymers (PAR) and apoptosis inducing factor (AIF) in different cellular components, Co-IP (co-immunoprecipitation) was used to test the interaction of AIF, H2AX and CypA (Cyclophilin A). We found that AATF was increased in cortical neurons after brain ischemia (P<0.001). Besides, AATF was upregulated in OGD-treated SH-SY5Y cells in a time-dependent manner (P=0.007). Additionally, overexpressing AATF ameliorated OGD-induced cellular death (P < 0.001) and cytotoxicity (P = 0.001), and AATF interference exacerbated OGD-induced cellular death (P=0.033) and cytotoxicity (P=0.006). We also found that AATF overexpression suppressed cellular DNA fragmentation (P=0.003) but did not ameliorate oxidative stress and DNA damage. Moreover, we discovered that overexpressing AATF suppressed PAR/AIF signaling pathway via binding with AIF.

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Nuclear localization of Beclin 1 promotes radiation-induced DNA damage repair independent of autophagy

Scientific Reports ◽

10.1038/srep45385 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 13

Author(s):

Fei Xu ◽

Yixuan Fang ◽

Lili Yan ◽

Lan Xu ◽

Suping Zhang ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Repair ◽

Strand Break ◽

Beclin 1 ◽

Dsb Repair ◽

Dna Topoisomerase ◽

Damage Repair ◽

Dna Double Strand Break ◽

Radiation Induced

Abstract Beclin 1 is a well-established core mammalian autophagy protein that is embryonically indispensable and has been presumed to suppress oncogenesis via an autophagy-mediated mechanism. Here, we show that Beclin 1 is a prenatal primary cytoplasmic protein but rapidly relocated into the nucleus during postnatal development in mice. Surprisingly, deletion of beclin1 in in vitro human cells did not block an autophagy response, but attenuated the expression of several DNA double-strand break (DSB) repair proteins and formation of repair complexes, and reduced an ability to repair DNA in the cells exposed to ionizing radiation (IR). Overexpressing Beclin 1 improved the repair of IR-induced DSB, but did not restore an autophagy response in cells lacking autophagy gene Atg7, suggesting that Beclin 1 may regulate DSB repair independent of autophagy in the cells exposed to IR. Indeed, we found that Beclin 1 could directly interact with DNA topoisomerase IIβ and was recruited to the DSB sites by the interaction. These findings reveal a novel function of Beclin 1 in regulation of DNA damage repair independent of its role in autophagy particularly when the cells are under radiation insult.

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Analysis of glutathione and vitamin C effects

The Scientific World JOURNAL ◽

10.1100/tsw.2006.205 ◽

2006 ◽

Vol 6 ◽

pp. 1191-1201 ◽

Cited By ~ 1

Author(s):

M. Pitarque ◽

A. Creus ◽

R. Marcos

Keyword(s):

Dna Damage ◽

Vitamin C ◽

Comet Assay ◽

Human Lymphocytes ◽

Fixed Dose ◽

Induced Response ◽

Antioxidant Vitamin ◽

Individual Level ◽

The Individual

The alkaline single-cell gel electrophoresis (or Comet) assay was applied to evaluate the eventual DNA damage induced by the triphenolic metabolite of benzene, 1,2,4-benzenetriol (BT), in isolated human lymphocytes. Prior to BT treatment, ranging from 5 to 50 μM, a supplementation with glutathione (GSH, 350 μg/ml) was carried out to assess whether GSH may have a modulating effect on the Comet response. The effect of a fixed dose of BT was also evaluated in the presence of the exogenous antioxidant vitamin C (40 and 200 μM). Additionally, we investigated whether the polymorphism of glutathione S-transferase T1 (GSTT1) gene may affect the individual level of BT-induced DNA damagein vitro. For all donors included in the present study, BT produced a significant dose-response relationship. No clear effect of GSH preincubation was seen on the BT-induced response. On the contrary, a significant reduction of DNA damage was observed in the presence of vitamin C (at least at 200 μM). Although our data suggest some individual differences according to theGSTT1genotype in the outcome of the Comet assay, a large number of individuals should be studied in further investigations to obtain reliable conclusions.

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