Inter-individual variation in DNA repair capacity: A need for multi-pathway functional assays to promote translational DNA repair research

Nagel, Zachary D.; Chaim, Isaac A.; Samson, Leona D.

doi:10.1016/j.dnarep.2014.03.009

Cited by 76 publications

(59 citation statements)

References 231 publications

(207 reference statements)

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“…Because the DDR network is a major defense system against environmental exposures linked to diseases, as well as a major target of somatic mutational inactivation in human cancers, being able to quantify and understand this variation has major clinical and public health implications (54). Unfortunately, there are few examples of translating knowledge of the DDR into the clinic or public health arenas, due in part to a paucity of quantitative tools for monitoring DDR activity (54,55). This study shows the feasibility of configuring highly multiplex, MRM-based assays to support basic, clinical, and epidemiological studies.…”

Section: Discussionmentioning

confidence: 99%

Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Enables Multiplex, Quantitative Pharmacodynamic Studies of Phospho-Signaling

Whiteaker

Zhao

Yan

et al. 2015

Molecular & Cellular Proteomics

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In most cell signaling experiments, analytes are measured one Western blot lane at a time in a semiquantitative and often poorly specific manner, limiting our understanding of network biology and hindering the translation of novel therapeutics and diagnostics. We show the feasibility of using multiplex immuno-MRM for phospho-pharmacodynamic measurements, establishing the potential for rapid and precise quantification of cell signaling networks. A 69-plex immuno-MRM assay targeting the DNA damage response network was developed and characterized by response curves and determinations of intra-and interassay repeatability. The linear range was >3 orders of magnitude, the median limit of quantification was 2.0 fmol/mg, the median intra-assay variability was 10% CV, and the median interassay variability was 16% CV. The assay was applied in proof-of-concept studies to immortalized and primary human cells and surgically excised cancer tissues to quantify exposure-response relationships and the effects of a genomic variant (ATM kinase mutation) or pharmacologic ( Because there is limited correlation between mRNA and protein levels/activity (1), quantification of proteins and posttranslational modifications is critical to understanding cellular signaling and determining pharmacodynamic (PD) 1 responses. Phosphorylation is a key post-translational modification used in signaling networks to modulate protein/pathway activity, protein interactions, and protein localization in response to extracellular and intracellular stimuli. Many diseases exhibit dysfunctions in signaling networks, and thus major efforts to identify novel drug targets (e.g. kinase inhibitors) are based on signal transduction pathways (2).Currently the research community lacks high throughput, quantitative tools for studying phospho-signaling networks, hindering our basic understanding of network biology and hence the translation of novel therapeutics and companion diagnostics. In most experiments, one analyte is measured one Western blot lane at a time in a semiquantitative and often nonspecific manner. These drawbacks limit our ability to extend knowledge beyond individual phosphorylation events to a system-wide study of phosphorylation dynamics, which is critical because signal transduction pathways act as interconnected networks, and the effects of mutations in individual genes (as well as the effects of pharmacologic compounds) spread throughout the network (3). Although Western blotting and related traditional immuno-assay platforms (e.g. ELISA) have been pushed brilliantly to their limits and have formed the basis of many advances in biomedical research, they are inadequate to support the needs of the postgenomic world, in which we need innovative technologies for determining the effects of any experimental condition (e.g. agonist or antagonist exposures, genetic variations) on the major signal transduction networks of the human cell, using precise, standardized, moderate-to-high throughput methods that can be reproduced across laboratories.Newer tec...

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Section: Discussionmentioning

confidence: 99%

Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Enables Multiplex, Quantitative Pharmacodynamic Studies of Phospho-Signaling

Whiteaker

Zhao

Yan

et al. 2015

Molecular & Cellular Proteomics

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“…These include population studies of the potential effects of carriership of allelic variants of genes coding for proteins of repair and maintenance of genomic integrity on the risk for late-onset disease [162,168,169] as well as personalised studies of capacity to repair genotoxic damage and/or the capacity for self-renewal in individually collected cultured cells [170][171][172][173].…”

Section: Don't Throw Away Repair It -Capacity To Repair Genotoxic Damentioning

confidence: 99%

APOE4, oxidative stress and decreased repair capacity - a no-brainer. Faulty lipid metabolism and increased levels of oxidative damage may be risk factors in the pathogenesis of late-onset dementia

Nayyar

Chakalova²

2015

Biodiscovery

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Keywords: Late-onset disease, dementia, APOE, mitochondrial DNA, individual repair capacity.Abbreviations: AD -Alzheimer's disease, APOE -apolipoprotein E, ATP -adenosine triphosphate, BER -base excision repair, CAA -cerebral amyloid angiopathy, CNS -central nervous system, FTD -frontotemporal dementia, HD -Huntington's disease, IMT -intimamedia thickness, IRC -individual repair capacity, MCI -mild cognitive impairment, MND -motor neuron disease, NER -nucleotide excision repair, NMDA -N-methyl-D-aspartate, PD -Parkinson's disease, PDAPP -PDGF promoter expressing amyloid precursor protein, ROS -reactive oxygen species; SOD -superoxide dismutase, VCI -vascular cognitive decline.* Corresponding Authors: Ashima Nayyar, email: a.nayyar@abertay.ac.uk Conflict of Interests:No potential conflict of interest was disclosed by any of the authors. AbstractDementia is very common in the elderly and its incidence increases in an age-dependent fashion. Alzheimer's disease and vascular cognitive decline are the most common cases of dementia in the elderly. Amyloid burden and increased levels of oxidative damage have been implicated to play significant roles in the pathogenesis of late-onset dementia. In this paper we propose that there are three major genetic factors that may modulate the risk for dementia in later life: carriership of APOE variant alleles, carriership of mitochondrial DNA of haplogroups associated with ineffective oxygen utilisation (specifically, haplogroup H) and carriership of genetic polymorphisms conferring subtly deficient DNA repair. All three factors are quite common in the European populations. Each of these three factors may not have significant effect on the phenotype when taken separately, but when combined in the same genotype, the effects may be cumulative. Further studies are needed in order to elucidate the genotype-phenotype relationships and provide a reliable basis for assessment of the genetic risk for sporadic late-onset dementia. Lifestyle alterations and therapies targeted at decreasing the oxidative burden to aging cells and tissues may decrease the risk for neurological decline in later life.

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“…These assays include the single cell gel electrophoresis assay (COMET) [33] [34] [35], mass spectrometry based quantification of specific DNA lesions [36], host extract based molecular beacons [37], microarray analysis of transcripts [38] and fluorescence-based multiplex flow cytometric host cell reactivation assays (FM-HCR) that measure the repair of specific lesions [39] [40] [41]. In general, these assays provide valuable data on the integrity of DNA or parts of the DDR, and each assay has proved to be extremely useful for research studies on the cellular DDR and DNA repair capacity.…”

Section: Introductionmentioning

confidence: 99%

Activation of DNA Damage Signaling Components by Diagnostic Computed Tomography (CT) Scans Detected in Patient Samples Using an Electrochemiluminescence-Based Assay Platform

Hseih¹,

Begley²,

Endres³

et al. 2017

ABB

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Technologies that measure activation of components of the DNA damage response (DDR) have applications in exposure assessment and personalized medicine. The DDR and associated DNA repair pathways encompass hundreds of proteins, making detailed measurement of activation technically challenging and laborious. The purpose of our study was to develop proteinspecific assays for certain DDR components on a high-throughput electrochemiluminescence (ECL)-based platform. We developed five working assay pairs for ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), phosphorylated-ATM S1981, phosphorylated-CHK2 T68 and phosphorylatedtumor protein p53 (p53) S15. We validated the ECL results against traditional immunoblot and γ-H2AX foci measures in cell and cancer models. In an effort to test the ECL-based technology in a clinical setting, we utilized peripheral blood mononuclear cells (PBMCs) from patients undergoing computed tomography (CT) scans. CT scans represent both a valuable medical imaging diagnostic and a controlled environmental exposure to ionizing radiation for research studies, as they deliver ~2 to 31 millisieverts (mSv) and are known to activate DDR components. In this study, we show that ECL-based technology can measure the basal and damage-induced levels of DDR components in patient PBMC samples. Using a blinded study design and patient matched preand post CT scan samples, we show that ECL-derived data can consistently 229(94% of the time, 15/16 patients) identify PBMCs that have been exposed to low dose ionizing radiation associated with CT scans. Ultimately, the results of our pilot clinical study support the idea that ECL-based technology is applicable for use in clinical and population cohorts that study components of the DDR.

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Inter-individual variation in DNA repair capacity: A need for multi-pathway functional assays to promote translational DNA repair research

Cited by 76 publications

References 231 publications

Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Enables Multiplex, Quantitative Pharmacodynamic Studies of Phospho-Signaling

Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Enables Multiplex, Quantitative Pharmacodynamic Studies of Phospho-Signaling

APOE4, oxidative stress and decreased repair capacity - a no-brainer. Faulty lipid metabolism and increased levels of oxidative damage may be risk factors in the pathogenesis of late-onset dementia

Activation of DNA Damage Signaling Components by Diagnostic Computed Tomography (CT) Scans Detected in Patient Samples Using an Electrochemiluminescence-Based Assay Platform

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