Replication Stress Induces Micronuclei Comprising of Aggregated DNA Double-Strand Breaks

BACKGROUND: Micronuclei (MN) in mammalian cells serve as a reliable biomarker of genomic instability and genotoxic exposure. Elevation of MN is commonly observed in cells bearing intrinsic genomic instability and in normal cells exposed to genotoxic agents. DNA double-strand breaks are marked by phosphorylation of H2AX at serine 139 (γ-H2AX). One subclass of MN contains massive and uniform γ-H2AX signals. This study tested whether this subclass of MN can be induced by replication stress. PRINCIPAL FINDINGS: We observed that a large proportion of MN, from 20% to nearly 50%, showed uniform staining by antibodies against γ-H2AX, a marker of DNA double-strand breaks (DSBs). Such micronuclei were designated as MN-γ-H2AX (+). We showed that such MN can be induced by chemicals that are known to cause DNA replication stress and S phase arrest. Hydroxyurea, aphidicolin and thymidine could all significantly induce MN-γ-H2AX (+), which were formed during S phase and appeared to be derived from aggregation of DSBs. MN-γ-H2AX (-), MN that were devoid of uniform γ-H2AX signals, were induced to a lesser extent in terms of fold change. Paclitaxel, which inhibits the disassembly of microtubules, only induced MN-γ-H2AX (-). The frequency of MN-γ-H2AX (+), but not that of MN-γ-H2AX (-), was also significantly increased in cells that experience S phase prolongation due to depletion of cell cycle regulator CUL4B. Depletion of replication protein A1 (RPA1) by RNA interference resulted in an elevation of both MN-γ-H2AX (+) and MN-γ-H2AX (-). CONCLUSIONS/SIGNIFICANCE: A subclass of MN, MN-γ-H2AX (+), can be preferentially induced by replication stress. Classification of MN according to their γ-H2AX status may provide a more refined evaluation of intrinsic genomic instabilities and the various environmental genotoxicants

Analysis of Lymphocytic DNA Damage in Early Multiple Sclerosis by Automated Gamma-H2AX and 53BP1 Foci Detection: A Case Control Study

Background In response to DNA double-strand breaks, the histone protein H2AX becomes phosphorylated at its C-terminal serine 139 residue, referred to as γ-H2AX. Formation of γ-H2AX foci is associated with recruitment of p53-binding protein 1 (53BP1), a regulator of the cellular response to DNA double-strand breaks. γ-H2AX expression in peripheral blood mononuclear cells (PBMCs) was recently proposed as a diagnostic and disease activity marker for multiple sclerosis (MS). Objective To evaluate the significance of γ-H2AX and 53BP1 foci in PBMCs as diagnostic and disease activity markers in patients with clinically isolated syndrome (CIS) and early relapsing-remitting MS (RRMS) using automated γ-H2AX and 53BP1 foci detection. Methods Immunocytochemistry was performed on freshly isolated PBMCs of patients with CIS/early RRMS (n = 25) and healthy controls (n = 27) with γ-H2AX and 53BP1 specific antibodies. Nuclear γ-H2AX and 53BP1 foci were determined using a fully automated reading system, assessing the numbers of γ-H2AX and 53BP1 foci per total number of cells and the percentage of cells with foci. Patients underwent contrast enhanced 3 Tesla magnetic resonance imaging (MRI) and clinical examination including expanded disability status scale (EDSS) score. γ-H2AX and 53BP1 were also compared in previously frozen PBMCs of each 10 CIS/early RRMS patients with and without contrast enhancing lesions (CEL) and 10 healthy controls. Results The median (range) number of γ-H2AX (0.04 [0–0.5]) and 53BP1 (0.005 [0–0.2]) foci per cell in freshly isolated PBMCs across all study participants was low and similar to previously reported values of healthy individuals. For both, γ-H2AX and 53BP1, the cellular focus number as well as the percentage of positive cells did not differ between patients with CIS/RRMS and healthy controls. γ-H2AX and 53BP1 levels neither correlated with number nor volume of T2-weighted lesions on MRI, nor with the EDSS. Although γ-H2AX, but not 53BP1, levels were higher in previously frozen PBMCs of patients with than without CEL, γ-H2AX values of both groups overlapped and γ-H2AX did not correlate with the number or volume of CEL. Conclusion γ-H2AX and 53BP1 foci do not seem to be promising diagnostic or disease activity biomarkers in patients with early MS. Lymphocytic DNA double-strand breaks are unlikely to play a major role in the pathophysiology of MS

Heterochromatin is refractory to γ-H2AX modification in yeast and mammals

Double-strand break (DSB) damage in yeast and mammalian cells induces the rapid ATM (ataxia telangiectasia mutated)/ATR (ataxia telangiectasia and Rad3 related)-dependent phosphorylation of histone H2AX (γ-H2AX). In budding yeast, a single endonuclease-induced DSB triggers γ-H2AX modification of 50 kb on either side of the DSB. The extent of γ-H2AX spreading does not depend on the chromosomal sequences. DNA resection after DSB formation causes the slow, progressive loss of γ-H2AX from single-stranded DNA and, after several hours, the Mec1 (ATR)-dependent spreading of γ-H2AX to more distant regions. Heterochromatic sequences are only weakly modified upon insertion of a 3-kb silent HMR locus into a γ-H2AX–covered region. The presence of heterochromatin does not stop the phosphorylation of chromatin more distant from the DSB. In mouse embryo fibroblasts, γ-H2AX distribution shows that γ-H2AX foci increase in size as chromatin becomes more accessible. In yeast, we see a high level of constitutive γ-H2AX in telomere regions in the absence of any exogenous DNA damage, suggesting that yeast chromosome ends are transiently detected as DSBs

Prolonged expression of the γ-H2AX DNA repair biomarker correlates with excess acute and chronic toxicity from radiotherapy treatment

The normal tissue tolerance levels to fractionated radiotherapy have been appreciated by a century of careful clinical observations and radiobiological studies in animals. During clinical fractionated radiotherapy, these normal tissue tolerance levels are respected, and severe sequelae of radiotherapy are avoided in the majority of patients. Notwithstanding, a minority of patients experience unexpectedly severe normal tissue reactions. The ability to predict which patients might form this minority would be important. We have conducted a study to develop a rapid and reliable diagnostic test to predict excessive normal tissue toxicity (NTT) in radiotherapy patients. A flow cytometric immunocytochemical assay was used to measure DNA damage in peripheral blood lymphocytes (PBL) from cancer patients exposed to 2-Gy gamma radiation. DNA damage and repair was measured by induction of cellular γ-H2AX in unirradiated and exposed cells at specific time points following exposure. In 12 cancer patients that experienced severe atypical NTT following radiotherapy, there was a failure to repair DNA double-strand breaks (DSB) as measured by γ-H2AX induction and persistence. In ten cancer patients that experienced little or no NTT and in seven normal (noncancer controls), efficient repair of DNA DSB was observed in the γ-H2AX assay. We conclude that a flow cytometric assay based on γ-H2AX induction in PBL of radiotherapy patients may represent a robust, rapid and reliable biomarker to predict NTT during radiotherapy. Further research is required with a larger patient cohort to validate this important study

BRCA1 Forms a Functional Complex with γ-H2AX as a Late Response to Genotoxic Stress

Following genotoxic stress, the histone H2AX becomes phosphorylated at serine 139 by the ATM/ATR family of kinases. The tumor suppressor BRCA1, also phosphorylated by ATM/ATR kinases, is one of several proteins that colocalize with phospho-H2AX (γ-H2AX) at sites of active DNA repair. Both the precise mechanism and the purpose of BRCA1 recruitment to sites of DNA damage are unknown. Here we show that BRCA1 and γ-H2AX form an acid-stable biochemical complex on chromatin after DNA damage. Maximal association of BRCA1 with γ-H2AX correlates with reduced global γ-H2AX levels on chromatin late in the repair process. Since BRCA1 is known to have E3 ubiquitin ligase activity in vitro, we examined H2AX for evidence of ubiquitination. We found that H2AX is ubiquitinated at lysines 119 and 119 in vivo and that blockage of 26S proteasome function stabilizes γ-H2AX levels within cells. When BRCA1 levels were reduced, ubiquitination of H2AX was also reduced, and the cells retained higher levels of phosphorylated H2AX. These results indicate that BRCA1 is recruited into stable complexes with γ-H2AX and that the complex is involved in attenuation of the γ-H2AX repair signal after DNA damage

Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay

Phosphorylation of the histone protein H2AX to form γ-H2AX foci directly represents DNA double-strand break formation. Traditional γ-H2AX detection involves counting individual foci within individual nuclei. The novelty of this work is the application of a time-resolved fluorescence assay using dissociation-enhanced lanthanide fluorescence immunoassay for quantitative measurements of γ-H2AX. For comparison, standard fluorescence detection was employed and analyzed either by bulk fluorescent measurements or by direct foci counting using BioTek Spot Count algorithm and Gen 5 software. Etoposide induced DNA damage in A549 carcinoma cells was compared across all test platforms. Time resolved fluorescence detection of europium as a chelated complex enabled quantitative measurement of γ-H2AX foci with nanomolar resolution. Comparative bulk fluorescent signals achieved only micromolar sensitivity. Lanthanide based immunodetection of γ-H2AX offers superior detection and a user-friendly workflow. These approaches have the potential to improve screening of compounds that either enhance DNA damage or protect against its deleterious effects

Expression of γ-H2AX, 53BP1 and Micronuclei as Genome Damage Biomarker of Population in Keang and Salumati Village, Mamuju West Sulawesi Province

The residents living in high background radiation area have risk to be exposed by ionizing radiation that also potentially cause their DNA damage. The aim of this study was to determine the expression of γ-H2AX, 53BP1 foci and micronuclei in the residentswho live in high background radiation area of Salumati village, Mamuju, West Sulawesi, Indonesia. Twenty one blood samples which consist of 11 from the study area and 10 from control were assessed for their expression of γ-H2AX and 53BPI foci by using specific antibodies and observed under fluorescence microscope whereas micronuclei was detected after being cultured and giemsa stainedaccording to standard procedures. Results showed that both γ-H2AX and 53BP1foci from high background area was lower than that of control area (0.37±0.24vs 0.19±0.11 (p=0.03) for γ-H2AX and 0.61±0.30vs 0.31±0.12 for 53BP1 (p=0.01)). The mean of micronuclei frequency in exposed area was 0.02 (0,01-0,03) while in control area was 0.02 (0.003-0.02). There was statistical significant in corellation between both γ-H2AX, 53BP1 foci with micronuclei index in exposed area (p=0.02, p=0,04 respectively).In conclusion,there was a positive correlation between γ-H2AX and 53BP1 foci to micronuclei and this might be a clue of the occurrence of genome repairing mechanism caused by natural radiation at low dose chronical exposure in thestudied area

γ-H2AX+CD8+ T lymphocytes cannot respond to IFN-α, IL-2 or IL-6 in chronic hepatitis C virus infection

Background & AimsAge is the dominant prognostic factor influencing the natural history of hepatitis C virus (HCV) infection and treatment response. Accelerated lymphocyte telomere shortening in HCV infection correlates with adverse clinical outcomes. Critical telomere shortening generates double-stranded DNA breaks (DSB) inducing the DNA damage response, leading to replicative senescence. The phenotype and function of CD8+ T lymphocytes and the in vitro response to IFN-α in relation to the DNA damage response were investigated in patients with chronic HCV infection.MethodsCD8+ T lymphocytes with DSB were identified by expression of γ-H2AX (Ser-139) in 134 HCV-exposed subjects and 27 controls. Telomere length was determined by flow-FISH; cytokine expression by intracellular cytokine staining; in vitro responses to IFN-α, IL-2 or IL-6 by phospho-STAT1 (Y701) or phospho-STAT5 (Y694) expression.ResultsThe proportion of circulating CD8+γ-H2AX+ T lymphocytes rose with increasing fibrosis stage (p=0.0023). CD8+γ-H2AX+ T lymphocytes were enriched in liver compared to blood (p=0.03). CD8+γ-H2AX+ T lymphocytes demonstrated increased IFN-γ (p=0.02) and reduced IL-2 expression (p=0.02). CD8+γ-H2AX+ T lymphocytes failed to phosphorylate STAT1 in response to IFN-α compared to unfractionated CD8+ T lymphocytes (p <0.0001). More widespread failure of Jak/Stat signalling in CD8+γ-H2AX+ T lymphocytes was suggested by impaired phosphorylation of STAT1 with IL-6 (p=0.002) and STAT5 with IL-2 (p=0.0039) compared to unfractionated CD8+ T-lymphocytes.ConclusionsIn chronic HCV infection, CD8+γ-H2AX+ T lymphocytes are highly differentiated with shortened telomeres, are more frequent within the liver, are associated with severe fibrosis and fail to activate Jak/Stat pathways in response to IFN-α, IL-2 or IL-6, perhaps explaining treatment failure in those with severe fibrosis