DNA Repair Foci Research Articles

Divalent and multivalent cations control liquid-like assembly of poly(ADP-ribosyl)ated PARP1 into multimolecular associates in vitro

The formation of nuclear biomolecular condensates is often associated with local accumulation of proteins at a site of DNA damage. The key role in the formation of DNA repair foci belongs to PARP1, which is a sensor of DNA damage and catalyzes the synthesis of poly(ADP-ribose) attracting repair factors. We show here that biogenic cations such as Mg2+, Ca2+, Mn2+, spermidine3+, or spermine4+ can induce liquid-like assembly of poly(ADP-ribosyl)ated [PARylated] PARP1 into multimolecular associates (hereafter: self-assembly). The self-assembly of PARylated PARP1 affects the level of its automodification and hydrolysis of poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase (PARG). Furthermore, association of PARylated PARP1 with repair proteins strongly stimulates strand displacement DNA synthesis by DNA polymerase β (Pol β) but has no noticeable effect on DNA ligase III activity. Thus, liquid-like self-assembly of PARylated PARP1 may play a critical part in the regulation of i) its own activity, ii) PARG-dependent hydrolysis of poly(ADP-ribose), and iii) Pol β–mediated DNA synthesis. The latter can be considered an additional factor influencing the choice between long-patch and short-patch DNA synthesis during repair.

Early and Late Effects of Low-Dose X-ray Exposure in Human Fibroblasts: DNA Repair Foci, Proliferation, Autophagy, and Senescence.

The effects of low-dose radiation exposure remain a controversial topic in radiation biology. This study compares early (0.5, 4, 24, 48, and 72 h) and late (5, 10, and 15 cell passages) post-irradiation changes in γH2AX, 53BP1, pATM, and p-p53 (Ser-15) foci, proliferation, autophagy, and senescence in primary fibroblasts exposed to 100 and 2000 mGy X-ray radiation. The results show that exposure to 100 mGy significantly increased γH2AX, 53BP1, and pATM foci only at 0.5 and 4 h post irradiation. There were no changes in p-p53 (Ser-15) foci, proliferation, autophagy, or senescence up to 15 passages post irradiation at the low dose.

Super-resolution GSDIM microscopy unveils distinct nanoscale characteristics of DNA repair foci under diverse genotoxic stress

DNA double-strand breaks initiate the DNA damage response (DDR), leading to the accumulation of repair proteins at break sites and the formation of the-so-called foci. Various microscopy methods, such as wide-field, confocal, electron, and super-resolution microscopy, have been used to study these structures. However, the impact of different DNA-damaging agents on their (nano)structure remains unclear. Utilising GSDIM super-resolution microscopy, here we investigated the distribution of fluorescently tagged DDR proteins (53BP1, RNF168, MDC1) and γH2AX in U2OS cells treated with γ-irradiation, etoposide, cisplatin, or hydroxyurea. Our results revealed that both foci structure and their nanoscale ultrastructure, including foci size, nanocluster characteristics, fluorophore density and localisation, can be significantly altered by different inducing agents, even ones with similar mechanisms. Furthermore, distinct behaviours of DDR proteins were observed under the same treatment. These findings have implications for cancer treatment strategies involving these agents and provide insights into the nanoscale organisation of the DDR.

Guanine-containing ssDNA and RNA induce dimeric and tetrameric structural forms of SAMHD1.

The dNTPase activity of tetrameric SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) plays a critical role in cellular dNTP regulation. SAMHD1 also associates with stalled DNA replication forks, DNA repair foci, ssRNAand telomeres. The above functions require nucleic acid binding by SAMHD1, which may be modulated by its oligomeric state. Here we establish in cryo-EM and biochemical studies that the guanine-specific A1 activator site of each SAMHD1 monomer is used to target the enzyme to guanine nucleotides within single-stranded (ss) DNA and RNA. Remarkably, nucleic acid strands containing a single guanine base induce dimeric SAMHD1, while two or more guanines with ∼20 nucleotide spacing induce a tetrameric form. A cryo-EM structure of ssRNA-bound tetrameric SAMHD1 shows how ssRNA strands bridge two SAMHD1 dimers and stabilize the structure. This ssRNA-bound tetramer is inactive with respect to dNTPase and RNase activity.

Effects of low-dose ionizing radiation on genomic instability in interventional radiology workers

Interventional radiologists are chronically exposed to low-dose ionizing radiation (IR), which may represent a health risk. The aim of the present study was to evaluate genomic instability by analyzing chromosomal aberrations, micronuclei, and 53BP1 DNA repair foci in peripheral blood lymphocytes of radiologists. Based on the IAEA guidelines on biodosimetry using dicentrics, the average protracted whole-body dose in radiologists were estimated. Since preleukemic fusion genes (PFG) are the primary events leading to leukemia, we also studied their presence by RT-qPCR and FISH. No significant difference in 53BP1 foci and incidence of PFG (MLL-AF4, MLL-AF9, AML1-ETO, BCR-ABL p190) was found in cells of interventional radiologists in comparison to controls. However, our results showed an increased frequency of micronuclei and various types of chromosomal aberrations including dicentrics in interventional radiologists. The average protracted whole body estimated dose was defined at 452.63 mGy. We also found a significantly higher amplification of the MLL gene segment and increased RNA expression in cells of interventional radiologists in comparison to controls. In conclusion, our results showed that long-term low-dose IR induces genomic instability in interventional radiologists.

Targeting lysine-specific demethylase 1 (KDM1A/LSD1) impairs colorectal cancer tumorigenesis by affecting cancer cells stemness, motility, and differentiation

Among all cancers, colorectal cancer (CRC) is the 3rd most common and the 2nd leading cause of death worldwide. New therapeutic strategies are required to target cancer stem cells (CSCs), a subset of tumor cells highly resistant to present-day therapy and responsible for tumor relapse. CSCs display dynamic genetic and epigenetic alterations that allow quick adaptations to perturbations. Lysine-specific histone demethylase 1A (KDM1A also known as LSD1), a FAD-dependent H3K4me1/2 and H3K9me1/2 demethylase, was found to be upregulated in several tumors and associated with a poor prognosis due to its ability to maintain CSCs staminal features. Here, we explored the potential role of KDM1A targeting in CRC by characterizing the effect of KDM1A silencing in differentiated and CRC stem cells (CRC-SCs). In CRC samples, KDM1A overexpression was associated with a worse prognosis, confirming its role as an independent negative prognostic factor of CRC. Consistently, biological assays such as methylcellulose colony formation, invasion, and migration assays demonstrated a significantly decreased self-renewal potential, as well as migration and invasion potential upon KDM1A silencing. Our untargeted multi-omics approach (transcriptomic and proteomic) revealed the association of KDM1A silencing with CRC-SCs cytoskeletal and metabolism remodeling towards a differentiated phenotype, supporting the role of KDM1A in CRC cells stemness maintenance. Also, KDM1A silencing resulted in up-regulation of miR-506-3p, previously reported to play a tumor-suppressive role in CRC. Lastly, loss of KDM1A markedly reduced 53BP1 DNA repair foci, implying the involvement of KDM1A in the DNA damage response. Overall, our results indicate that KDM1A impacts CRC progression in several non-overlapping ways, and therefore it represents a promising epigenetic target to prevent tumor relapse.

Stability of Rad51 recombinase and persistence of Rad51 DNA repair foci depends on post-translational modifiers, ubiquitin and SUMO

The DNA double-strand breaks are particularly deleterious, especially when an error-free repair pathway is unavailable, enforcing the error-prone recombination pathways to repair the lesion. Cells can resume the cell cycle but at the expense of decreased viability due to genome rearrangements. One of the major players involved in recombinational repair of DNA damage is Rad51 recombinase, a protein responsible for presynaptic complex formation. We previously showed that an increased level of this protein promotes the usage of illegitimate recombination. Here we show that the level of Rad51 is regulated via the ubiquitin-dependent proteolytic pathway. The ubiquitination of Rad51 depends on multiple E3 enzymes, including SUMO-targeted ubiquitin ligases. We also demonstrate that Rad51 can be modified by both ubiquitin and SUMO. Moreover, its modification with ubiquitin may lead to opposite effects: degradation dependent on Rad6, Rad18, Slx8, Dia2, and the anaphase-promoting complex, or stabilization dependent on Rsp5. We also show that post-translational modifications with SUMO and ubiquitin affect Rad51's ability to form and disassemble DNA repair foci, respectively, influencing cell cycle progression and cell viability in genotoxic stress conditions. Our data suggest the existence of a complex E3 ligases network that regulates Rad51 recombinase's turnover, its molecular activity, and access to DNA, limiting it to the proportions optimal for the actual cell cycle stage and growth conditions, e.g., stress. Dysregulation of this network would result in a drop in cell viability due to uncontrolled genome rearrangement in the yeast cells. In mammals would promote the development of genetic diseases and cancer.

Compartmentalization of the DNA damage response: Mechanisms and functions

Cells have evolved an arsenal of molecular mechanisms to respond to continuous alterations in the primary structure of DNA. At the cellular level, DNA damage response proteins accumulate at sites of DNA damage and organize into nuclear foci. As recounted by Errol Friedberg, pioneering work on DNA repair in the 1930 s was stimulated by collaborations between physicists and geneticists. In recent years, the introduction of ideas from physics on self-organizing compartments has taken the field of cell biology by storm. Percolation and phase separation theories are increasingly used to model the self-assembly of compartments, called biomolecular condensates, that selectively concentrate molecules without a surrounding membrane. In this review, we discuss these concepts in the context of the DNA damage response. We discuss how studies of DNA repair foci as condensates can link molecular mechanisms with cell physiological functions, provide new insights into regulatory mechanisms, and open new perspectives for targeting DNA damage responses for therapeutic purposes.

Chemotherapy and cryopreservation affects DNA repair foci in lymphocytes of breast cancer patients

Purpose Although breast cancer (BC) patients benefit from radiotherapy (RT), some radiosensitive (RS) patients suffer from side effects caused by ionizing radiation in healthy tissues. It is thought that RS is underlaid by a deficiency in the repair of DNA double-strand breaks (DSB). DNA repair proteins such as p53-binding protein 1 (53BP1) and phosphorylated histone H2AX (γH2AX), form DNA repair foci at the DSB locations and thus serve as DSB biomarkers. Peripheral blood lymphocytes (PBL) are commonly believed to be an appropriate cell system for RS assessment using DNA repair foci. The amount of DSB may also be influenced by chemotherapy (CHT), which is often chosen as the first treatment modality before RT. As it is not always possible to analyze blood samples immediately after collection, there is a need for cryopreservation of PBL in liquid nitrogen. However, cryopreservation may potentially affect the number of DNA repair foci. In this work, we studied the effect of cryopreservation and CHT on the amount of DNA repair foci in PBL of BC patients undergoing radiotherapy. Materials and methods The effect of cryopreservation was studied by immunofluorescence analysis of 53BP1 and γH2AX proteins at different time intervals after in vitro irradiation. The effect of chemotherapy was analyzed by fluorescent labelling of 53BP1 and γH2AX proteins in PBL collected before, during, and after RT. Results Higher number of primary 53BP1/γH2AX foci was observed in frozen cells indicating that cryopreservation affects the formation of DNA repair foci in PBL of BC patients. In CHT-treated patients, a higher number of foci were found before RT, but no differences were observed during and after the RT. Conclusions Cryopreservation is the method of choice for analyzing DNA repair residual foci, but only similarly treated and preserved cells should be used for comparison of primary foci. CHT induces DNA repair foci in PBL of BC patients, but this effect disappears during radiotherapy.

Residual Foci of DNA Damage Response Proteins in Relation to Cellular Senescence and Autophagy in X-Ray Irradiated Fibroblasts.

DNA repair (DNA damage) foci observed 24 h and later after irradiation are called "residual" in the literature. They are believed to be the repair sites for complex, potentially lethal DNA double strand breaks. However, the features of their post-radiation dose-dependent quantitative changes and their role in the processes of cell death and senescence are still insufficiently studied. For the first time in one work, a simultaneous study of the association of changes in the number of residual foci of key DNA damage response (DDR) proteins (γH2AX, pATM, 53BP1, p-p53), the proportion of caspase-3 positive, LC-3 II autophagic and SA-β-gal senescent cells was carried out 24-72 h after fibroblast irradiation with X-rays at doses of 1-10 Gy. It was shown that with an increase in time after irradiation from 24 h to 72 h, the number of residual foci and the proportion of caspase-3 positive cells decrease, while the proportion of senescent cells, on the contrary, increases. The highest number of autophagic cells was noted 48 h after irradiation. In general, the results obtained provide important information for understanding the dynamics of the development of a dose-dependent cellular response in populations of irradiated fibroblasts.

Replication stress drives chromosomal instability in fibroblasts of childhood cancer survivors with second primary neoplasms

New development and optimization of oncologic strategies are steadily increasing the number of long-term cancer survivors being at risk of developing second primary neoplasms (SPNs) as a late consequence of genotoxic cancer therapies with the highest risk among former childhood cancer patients. Since risk factors and predictive biomarkers for therapy-associated SPN remain unknown, we examined the sensitivity to mild replication stress as a driver of genomic instability and carcinogenesis in fibroblasts from 23 long-term survivors of a pediatric first primary neoplasm (FPN), 22 patients with the same FPN and a subsequent SPN, and 22 controls with no neoplasm (NN) using the cytokinesis-block micronucleus (CBMN) assay. Mild replication stress was induced with the DNA-polymerase inhibitor aphidicolin (APH). Fibroblasts from patients with the DNA repair deficiency syndromes Bloom, Seckel, and Fanconi anemia served as positive controls and for validation of the CBMN assay supplemented by analysis of chromosomal aberrations, DNA repair foci (γH2AX/53BP1), and cell cycle regulation. APH treatment resulted in G2/M arrest and underestimation of cytogenetic damage beyond G2, which could be overcome by inhibition of Chk1. Basal micronuclei were significantly increased in DNA repair deficiency syndromes but comparable between NN, FPN, and SPN donors. After APH-induced replication stress, the average yield of micronuclei was significantly elevated in SPN donors compared to FPN (p = 0.013) as well as NN (p = 0.03) donors but substantially lower than for DNA repair deficiency syndromes. Our findings suggest that mild impairment of the response to replication stress induced by genotoxic impacts of DNA-damaging cancer therapies promotes genomic instability in a subset of long-term cancer survivors and may drive the development of an SPN. Our study provides a basis for detailed mechanistic studies as well as predictive bioassays for clinical surveillance, to identify cancer patients at high risk for SPNs at first diagnosis.

Repair Foci as Liquid Phase Separation: Evidence and Limitations.

In response to DNA double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less condensates or “foci”. The formation of these foci and their disassembly within the proper time window are essential for genome integrity. However, how these membrane-less sub-compartments are formed, maintained and disassembled remains unclear. Recently, several studies across different model organisms proposed that DNA repair foci form via liquid phase separation. In this review, we discuss the current research investigating the physical nature of repair foci. First, we present the different models of condensates proposed in the literature, highlighting the criteria to differentiate them. Second, we discuss evidence of liquid phase separation at DNA repair sites and the limitations of this model to fully describe structures formed in response to DNA damage. Finally, we discuss the origin and possible function of liquid phase separation for DNA repair processes.

Inhibition of DNA Repair by Inappropriate Activation of ATM, PARP, and DNA-PK with the Drug Agonist AsiDNA.

AsiDNA is a DNA repair inhibitor mimicking DNA double-strand breaks (DSB) that was designed to disorganize DSB repair pathways to sensitize tumors to DNA damaging therapies such as radiotherapy and chemotherapy. We used the property of AsiDNA of triggering artificial DNA damage signaling to examine the activation of DSB repair pathways and to study the main steps of inhibition of DNA repair foci after irradiation. We show that, upon AsiDNA cellular uptake, cytoplasmic ATM and PARP are rapidly activated (within one hour) even in the absence of irradiation. ATM activation by AsiDNA leads to its transient autophosphorylation and sequestration in the cytoplasm, preventing the formation of ATM nuclear foci on irradiation-induced damage. In contrast, the activation of PARP did not seem to alter its ability to form DNA repair foci, but prevented 53BP1 and XRCC4 recruitment at the damage sites. In the nucleus, AsiDNA is essentially associated with DNA-PK, which triggers its activation leading to phosphorylation of H2AX all over chromatin. This pan-nuclear phosphorylation of H2AX correlates with the massive inhibition, at damage sites induced by irradiation, of the recruitment of repair enzymes involved in DSB repair by homologous recombination and nonhomologous end joining. These results highlight the interest in a new generation of DNA repair inhibitors targeting DNA damage signaling.

MEDB-50. Assessment of cellular radiosensitivity and DNA repair in medulloblastoma cell lines and patient-derivded xenograft slice cultures

Medulloblastoma (WHO grade 4) is the most common malignant brain tumor of childhood. Despite the high importance of radiotherapy for disease control, the mechanisms underlying response and resistance to radiotherapy are incompletely understood. Therefore, we assessed the radiosensitivity and DNA repair capacity of medulloblastoma cell lines in-vitro and of patient-derived xenograft (PDX) models ex-vivo. Cell survival after irradiation of seven medulloblastoma cell lines displaying different subgroups was assessed via colony formation assay (DAOY, UW228, UW473, SJMM4, ONS-76, HDMB-03, D283). The ONS-76 and the mouse SJMM4 cell line were the most radioresistant strains (surviving fraction after 6 Gy (SF6): 0.33 and 0.31, respectively), followed by UW473, UW 228 and DAOY cells (SF6 0.16-0.21). The non-WNT/non-SHH-activated cell lines HDMB-03 and D283 cells demonstrated profoundly higher cellular radiosensitivity (SF6 <0.05). Analysis of residual (24h after irradiation) DNA double-strand breaks (DSB) as assessed by co-localized γH2AX/53BP1-foci demonstrated a significant correlation between DSB repair capacity and cellular survival. To use a more reliable pre-clinical model for medulloblastoma, we further examined DNA repair foci in ex-vivo irradiated slice cultures of PDX models MED-113 (SHH) and NCH2194 & HT028 (Gr. 3). Immunofluorescence analyses of frozen sections demonstrated non-hypoxic (pimonidazole-negative) and proliferating (EdU-positive) cells at the outer rim of the tumor slices. Two hours after irradiation all three PDX models showed a strong increase in 53BP1-foci, clearly indicating DNA damage induction. Most radiation-induced DSB were repaired after 24h. In a first radiosensitization approach, we treated the HT028 model with the PARP inhibitor olaparib (1µM ± 2Gy irradiation). Twenty-four hours after treatment the sample displayed a strong increase in the amount and size of 53BP1-foci, indicating compromised DNA repair. Further in-vitro and ex-vivo investigations with the aim to predict individual radiosensitivity and effective radiosensitization strategies are ongoing.

Bloom helicase mediates formation of large single\u2013stranded DNA loops during DNA end processing

Bloom syndrome (BS) is associated with a profoundly increased cancer risk and is caused by mutations in the Bloom helicase (BLM). BLM is involved in the nucleolytic processing of the ends of DNA double–strand breaks (DSBs), to yield long 3′ ssDNA tails that serve as the substrate for break repair by homologous recombination (HR). Here, we use single–molecule imaging to demonstrate that BLM mediates formation of large ssDNA loops during DNA end processing. A BLM mutant lacking the N–terminal domain (NTD) retains vigorous in vitro end processing activity but fails to generate ssDNA loops. This same mutant supports DSB end processing in cells, however, these cells do not form RAD51 DNA repair foci and the processed DSBs are channeled into synthesis–dependent strand annealing (SSA) instead of HR–mediated repair, consistent with a defect in RAD51 filament formation. Together, our results provide insights into BLM functions during homologous recombination.

Classifier Spot Count Optimization of Automated Fluorescent Slide Scanning System

Abstract Purpose: Ionizing radiation induced foci (IRIF) known also as DNA repair foci represent the 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 γH2AX and 53BP1. Although several approaches and software packages were developed for the quantification of IRIF, not one of them was commonly accepted and inter-laboratory variability in the outputs was reported. In this study, the sensitization of Metafer software to counting also small appearing IRIF was validated. Materials and Methods: Human lymphocytes were γ-irradiated at a dose of 2 Gy. The cells were fixed at 0.5, 1, 2, and 18 hours post-irradiation, permeabilized and IRIF were immunostained using appropriate antibodies. Cell images were acquired with the automatic Metafer system. Radiation-induced γH2AX and 53BP1 foci were enumerated using either manual counting (JCountPro program) or the Metafer software (after its classifier optimization has been done) and compared. The statistical analysis was performed using One-way ANOVA. Results: The enumeration of 53BP1, γH2AX foci manually by JCountPro did not statistically significantly differ from the automatic one performed with the optimized Metafer classifier. A detailed step-by-step protocol of this successful optimization is described in this study. Conclusions: We concluded that the Metafer software after the optimization was efficient in objectively enumerating IRIF, having a potential for usage in clinics and molecular epidemiology.

Poly(ADP-ribose) potentiates ZAP antiviral activity.

Zinc-finger antiviral protein (ZAP), also known as poly(ADP-ribose) polymerase 13 (PARP13), is an antiviral factor that selectively targets viral RNA for degradation. ZAP is active against both DNA and RNA viruses, including important human pathogens such as hepatitis B virus and type 1 human immunodeficiency virus (HIV-1). ZAP selectively binds CpG dinucleotides through its N-terminal RNA-binding domain, which consists of four zinc fingers. ZAP also contains a central region that consists of a fifth zinc finger and two WWE domains. Through structural and biochemical studies, we found that the fifth zinc finger and tandem WWEs of ZAP combine into a single integrated domain that binds to poly(ADP-ribose) (PAR), a cellular polynucleotide. PAR binding is mediated by the second WWE module of ZAP and likely involves specific recognition of an adenosine diphosphate-containing unit of PAR. Mutation of the PAR binding site in ZAP abrogates the interaction in vitro and diminishes ZAP activity against a CpG-rich HIV-1 reporter virus and murine leukemia virus. In cells, PAR facilitates formation of non-membranous sub-cellular compartments such as DNA repair foci, spindle poles and cytosolic RNA stress granules. Our results suggest that ZAP-mediated viral mRNA degradation is facilitated by PAR, and provides a biophysical rationale for the reported association of ZAP with RNA stress granules.

Toxicological effects of silver nanoparticles and cadmium chloride in macrophage cell line (RAW 264.7): An in vitro approach

BackgroundSilver nanoparticles (AgNP) are largely used in nanotechnological products, but the real risks for human and environment are still poorly understood if we consider the effects of mixtures of AgNP and environmental contaminants, such as non-essential metals. MethodsThe aim of the present study was to investigate the cytotoxicity and toxicological interaction of AgNP (1−4 nm, 0.36 and 3.6 μg mL−1) and cadmium (Cd, 1 and 10 μM) mixtures. The murine macrophage cell line RAW 264.7 was used as a model. ResultsEffects were observed after a few hours (4 h) on reactive oxygen species (ROS) and became more pronounced after 24 h-exposure. Cell death occurred by apoptosis, and loss of cell viability (24 h-exposure) was preceded by increases of ROS levels and DNA repair foci, but not of NO levels. Co-exposure potentiated some effects (decrease of cell viability and increase of ROS and NO levels), indicating toxicological interaction. ConclusionThese effects are important findings that must be better investigated, since the interaction of Cd with AgNP from nanoproducts may impair the function of macrophages and represent a health risk for humans.

The evolving complexity of DNA damage foci: RNA, condensates and chromatin in DNA double-strand break repair

Formation of biomolecular condensates is increasingly recognized as a mechanism employed by cells to deal with stress and to optimize enzymatic reactions. Recent studies have characterized several DNA repair foci as phase-separated condensates, behaving like liquid droplets. Concomitantly, the apparent importance of long non-coding RNAs and RNA-binding proteins for the repair of double-strand breaks has raised many questions about their exact contribution to the repair process. Here we discuss how RNA molecules can participate in condensate formation and how RNA-binding proteins can act as molecular scaffolds. We furthermore summarize our current knowledge about how properties of condensates can influence the choice of repair pathway (homologous recombination or non-homologous end joining) and identify the open questions in this field of emerging importance.

Imaging flow cytometry and fluorescence microscopy in assessing radiation response in lymphocytes from umbilical cord blood and cancer patients.

DNA double strand breaks (DSB) induced by ionizing radiation (IR) are usually measured using γH2AX/53BP1 DNA repair foci, that is considered to be the most sensitive assay for DSB analysis. While fluorescence microscopy (FM) is the gold standard for this analysis, imaging flow cytometry (IFC) may offer number of advantages such as lack of the fluorescence background, higher number of cells analyzed, and higher sensitivity in detection of DNA damage induced by IR at low doses. Along with appearance of γH2AX foci, the variable fraction of the cells exhibits homogeneously stained γH2AX signal resulting in so-called γH2AX pan-staining, which is believed to appear at early stages of apoptosis. Here, we investigated incidence of γH2AX pan-staining at different time points after irradiation with γ-rays using IFC and compared the obtained data with the data from FM. Appearance of γH2AX pan-staining during the apoptotic process was further analyzed by fluorescence-activated cell sorting (FACS) of cells at different stages of apoptosis and subsequent immunofluorescence analysis. Our results show that IFC was able to reveal dose dependence of pan-staining, while FM failed to detect all pan-staining cells. Moreover, we found that γH2AX pan-staining could be induced by therapeutic, but not low doses of γ-rays and correlate well with percentage of apoptotic cells was analyzed using flow cytometric Annexin-V/7-AAD assay. Further investigations showed that γH2AX pan-staining is formed in the early phases of apoptosis and remains until later stages of apoptotic process. Apoptotic DNA fragmentation as detected with comet assay using FM correlated with the percentage of live and late apoptotic/necrotic cells as analyzed by flow cytometry. Lastly, we successfully tested IFC for detection of γH2AX pan-staining and γH2AX/53BP1 DNA repair foci in lymphocyte of breast cancer patients after radiotherapy, which may be useful for assessing individual radiosensitivity in a clinically relevant cohort of patients.