Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+βD²). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a ¹³⁷Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.     

Where did they come from? The origin of endogenous γ-H2AX foci in tumor cells

Comment on: Telomere-dependent and telomere-independent origins of endogenous DNA damage in tumor cells. Asako J. Nakamura, Christophe E. Redon, William M. Bonner and Olga A. Sedelnikova. Aging 2009; 212-8.     

Dynamics of γH2AX formation and elimination in mammalian cells after X-irradiation

Phosphorylation of the replacement histone H2AX occurs in megabase chromatin domains around DNA double-strand breaks (DSBs), and this modification called γH2AX can be used as an effective marker for DSB repair and DNA damage response. In this study, we examined a bystander effect (BE) in locally irradiated embryonic human fibroblasts. Using fluorescence microscopy, we found that BE could be observed 1 h after X-ray irradiation (IR) and was completely eliminated 24 h after IR. Using immunohistochemistry and immunoblotting, we also studied kinetics of γH2AX formation and elimination in Syrian hamster and mouse tissues after whole body IR of animals. Analysis of hamster tissues at different times after IR at the dose 5 Gy showed that γH2AX-associated fluorescence in heart was decreased slowly with about a half level remaining 24 h after IR; at the same time, in brain, the level of γH2AX was about 3 times increased over the control level, and in liver, γH2AX level decreased to control values. We also report that in mouse heart the level of γH2AX measured by immunoblotting is lower than in brain, kidney and liver at different times after IR at the dose 3 Gy. Our observations indicate that there are significant variations in dynamics of γH2AX formation and elimination between non-proliferating mammalian tissues. These variations in γH2AX dynamics in indicated organs partially correlated with the expression level of the major kinase genes involved in H2AX phosphorylation (ATM and DNA-PK).     

Measurement of DNA damage in peripheral blood by the γ-H2AX assay as predictor of colorectal cancer risk

The detection of γ-H2AX focus is one of the most sensitive ways to monitor DNA double-strand breaks (DSBs). Although changes in γ-H2AX activity have been studied in tumor cells in colorectal cancer (CRC), changes in peripheral blood lymphocytes (PBLs) have not been examined previously. We hypothesize that higher levels of irradiation-induced γ-H2AX in PBLs may be associated with an elevated risk of colorectal cancer (CRC). In a case-control study, the baseline and ionizing radiation (IR)-induced γ-H2AX levels in PBLs from frequency-matched 320 untreated CRC patients and 320 controls were detected by a laser scanning cytometer-based immunocytochemical method. We used unconditional multivariable logistic regression to evaluate CRC risk by using the ratio of IR-induced γ-H2AX to the baseline levels with adjustment of age, sex and smoking status. We found CRC cases had significantly higher γ-H2AX ratio (1.5 vs. 1.41, P < 0.0001) compared with controls. When using the median γ-H2AX ratio of controls as a cutoff point, we found higher γ-H2AX ratio was significantly associated with an increased risk of CRC (OR = 6.72, 95% CI = 4.54–9.94). Quartile analyses also showed significant dose–response relationship between higher γ-H2AX ratio and increased risk of CRC (P for trend < 0.0001). Age, sex, BMI and smoking status also influenced the association of γ-H2AX ratio with CRC risk; however, no interactions with γ-H2AX ratio were observed. These results support the premise that DSBs in peripheral blood as measured by γ-H2AX level might represent an intermediate phenotype to assess the risk of CRC. Future prospective studies are necessary to confirm our findings in independent populations.     

Structural basis of γH2AX recognition by human PTIP BRCT5-BRCT6 domains in the DNA damage response pathway

Human Pax2 transactivation domain-interacting protein (hPTIP), containing six BRCT domains, is an essential protein required for the IR induced DDR process with an unclear role. Here we report that the tandem BRCT5–BRCT6 domain of hPTIP recognizes the γH2AX tail, and this interaction depends on the phosphorylation of H2AX Ser139 and binding with the carboxyl ending peptide to the aminoacyl ending peptide. The 2.15 Å crystal structure of hPTIP BRCT5/6–γH2AX complex and mutation analysis provide molecular evidence for direct interactions between PTIP and γH2AX. This interaction proffers a new clue to identify the role of PTIP in DDR pathways.

Structured summary of protein interactions

PTIP and gamma H2AXbind by fluorescence polarization spectroscopy (View Interaction: 1, 2, 3, 4, 5, 6).PTIP and gamma H2AXbind by X-ray crystallography (View interaction).     

Expression of γ-H2AX and patient prognosis in breast cancer cohort

H2AX phosphorylation is a novel marker of DNA double-stranded breaks. In the present study, we assessed the γ-H2AX expression, its association with other clinicopathologic characteristics, and the prognosis in a cohort of 97 patients with breast cancer. Ninety-seven specimens of tumor tissue and 77 adjacent normal tissues from patients with breast cancer were examined. All patients underwent modified radical mastectomy or local tumor resection without lymph node dissection. γ-H2AX expression was assessed by standard immunohistochemistry. Patients were followed after surgery for a mean duration of 70.1 ± 18.7 months (range, 6-93 months). The γ-H2AX staining was positive in 27 (27.8%) patients. The positive rates of H2AX were 26.0% and 2.6% in tumor tissue and adjacent normal tissues, respectively. γ-H2AX positive status was negatively associated with TNM staging, with 24 positive cases (32.4%) in TNM staging I-II, while no positive cases in TNM staging III-IV (P = 0.026). Sixteen patients (16.5%) died during the follow-up. No significant association between γ-H2AX expression and patient survival was detected. The unadjusted HR (hazard ratio) for γ-H2AX positive was 0.84 (95% CI: 0.27, 2.60). In TNM staging subgroup analysis, death only occurred in γ-H2AX negative patients. Our study is the first study to demonstrate that expression of γ-H2AX is associated with TNM staging. Due to the small sample and limited follow-up time, we did not observe a significant association between γ-H2AX and patient survival. γ-H2AX expression could be a potential biomarker for cancer diagnosis and prediction, and further studies are in need.     

Quantitation of γH2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX, is an early response to DNA double-strand breaks¹. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)². Overall, DSB induction results in the formation of discrete nuclear γH2AX foci which can be easily detected and quantitated by immunofluorescence microscopy². Given the unique specificity and sensitivity of this marker, analysis of γH2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of γH2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, γH2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer³. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of γH2AX foci ex vivo and in vivo^4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of γH2AX foci in mouse tissues.Download video file.^{(284M, mp4)}     

Activation of DNA damage response pathways in human mesenchymal stem cells exposed to cisplatin or γ-irradiation

DNA damaging agents are widely used in treatment of hematogical malignancies and solid tumors. While effects on hematopoietic stem cells have been characterized, less is known about the DNA damage response in human mesenchymal stem cells (hMSCs) in the bone marrow stroma, progenitors of osteoblasts, chondrocytes and adipocytes. To elucidate the response of undifferentiated hMSCs to γ-irradiation and cisplatin, key DNA damage responses have been characterised in hMSCs from normal adult donors. Cisplatin and γ-irradiation activated the DNA damage response in hMSCs, including induction of p53 and p21, and activation of PI3 kinase-related protein kinase (PIKK)-dependent phosphorylation of histone H2AX on serine 139, and replication protein A2 on serine4/serine8. Chemical inhibition of ATM or DNA-PK reduced DNA damage-induced phosphorylation of H2AX, indicating a role for both PIKKs in the response of hMSCs to DNA damage. Consistent with repair of DNA strand breaks, γ-H2AX staining decreased by 24 hours following gamma-irradiation. γ-irradiation arrested hMSCs in the G₁ phase of the cell cycle, while cisplatin induced S-phase arrest, mediated in part by the ATR/Chk1 checkpoint pathway. In hMSCs isolated from a chronic lymphocytic leukemia (CLL) patient, p53 and p21 were induced by cisplatin and γ-irradiation, while RPA2 was phosphorylated on serine4/8 in particular following cisplatin. Compared to peripheral blood lymphocytes or the leukemia cell line K562, both normal hMSCs and CLL-derived hMSCs were more resistant to cisplatin and γ-irradiation. These results provide insights into key pathways mediating the response of bone marrow-derived hMSCs to DNA damaging agents used in cancer treatment.     

Are endogenous clustered DNA damages induced in human cells?

Although clustered DNA damages are induced in cells by ionizing radiation and can be induced artifactually during DNA isolation, it was not known if they are formed in unirradiated cells by normal oxidative metabolism. Using high-sensitivity methods of quantitative gel electrophoresis, electronic imaging, and number average length analysis, we found that two radiosensitive human cell lines (TK6 and WI-L2-NS) accumulated Fpg-oxidized purine clusters and Nth-oxidized pyrimidine clusters but not Nfo-abasic clusters. However, four repair-proficient human lines (MOLT 4, HL-60, WTK1, and 28SC) did not contain significant levels (<5/Gbp) of any cluster type. Cluster levels were independent of p53 status. Measurement of glycosylase levels in 28SC, TK6, and WI-L2-NS cells suggested that depressed hOGG1 and hNth activities in TK6 and WI-L2-NS could be related to oxybase cluster accumulation. Thus, individuals with DNA repair enzyme deficiencies could accumulate potentially cytotoxic and mutagenic clustered DNA damages. The absence of Nfo-detected endogenous clusters in any cells examined suggests that abasic clusters could be a signature of cellular ionizing radiation exposure.     

RhoB Promotes γH2AX Dephosphorylation and DNA Double-Strand Break Repair

Unlike other Rho GTPases, RhoB is rapidly induced by DNA damage, and its expression level decreases during cancer progression. Because inefficient repair of DNA double-strand breaks (DSBs) can lead to cancer, we investigated whether camptothecin, an anticancer drug that produces DSBs, induces RhoB expression and examined its role in the camptothecin-induced DNA damage response. We show that in camptothecin-treated cells, DSBs induce RhoB expression by a mechanism that depends notably on Chk2 and its substrate HuR, which binds to RhoB mRNA and protects it against degradation. RhoB-deficient cells fail to dephosphorylate γH2AX following camptothecin removal and show reduced efficiency of DSB repair by homologous recombination. These cells also show decreased activity of protein phosphatase 2A (PP2A), a phosphatase for γH2AX and other DNA damage and repair proteins. Thus, we propose that DSBs activate a Chk2-HuR-RhoB pathway that promotes PP2A-mediated dephosphorylation of γH2AX and DSB repair. Finally, we show that RhoB-deficient cells accumulate endogenous γH2AX and chromosomal abnormalities, suggesting that RhoB loss increases DSB-mediated genomic instability and tumor progression.     

Clustered DNA damage induces pan-nuclear H2AX phosphorylation mediated by ATM and DNA–PK

DNA double-strand breaks (DSB) are considered as the most deleterious DNA lesions, and their repair is further complicated by increasing damage complexity. However, the molecular effects of clustered lesions are yet not fully understood. As the locally restricted phosphorylation of H2AX to form γH2AX is a key step in facilitating efficient DSB repair, we investigated this process after localized induction of clustered damage by ionizing radiation. We show that in addition to foci at damaged sites, H2AX is also phosphorylated in undamaged chromatin over the whole-cell nucleus in human and rodent cells, but this is not related to apoptosis. This pan-nuclear γH2AX is mediated by the kinases ataxia telangiectasia mutated and DNA-dependent protein kinase (DNA–PK) that also phosphorylate H2AX at DSBs. The pan-nuclear response is dependent on the amount of DNA damage and is transient even under conditions of impaired DSB repair. Using fluorescence recovery after photobleaching (FRAP), we found that MDC1, but not 53BP1, binds to the nuclear-wide γH2AX. Consequently, the accumulation of MDC1 at DSBs is reduced. Altogether, we show that a transient dose-dependent activation of the kinases occurring on complex DNA lesions leads to their nuclear-wide distribution and H2AX phosphorylation, yet without eliciting a full pan-nuclear DNA damage response.     

Quantification of γH2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX¹. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)². Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB^2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete γH2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy². The loss of γH2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary^4-8. The disappearence of γH2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C^5,6. Further, removal of γH2AX by redistribution involving histone exchange with H2A.Z has been implicated^7,8. Importantly, the quantitative analysis of γH2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of γH2AX foci in γ-irradiated adherent human keratinocytes⁹.Download video file.^{(225M, mp4)}     

Deregulation of BRCA1 Leads to Impaired Spatiotemporal Dynamics of γ-H2AX and DNA Damage Responses in Huntington’s Disease

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder of mid-life onset characterized by involuntary movements and progressive cognitive decline caused by a CAG repeat expansion in exon 1 of the Huntingtin (Htt) gene. Neuronal DNA damage is one of the major features of neurodegeneration in HD, but it is not known how it arises or relates to the triplet repeat expansion mutation in the Htt gene. Herein, we found that imbalanced levels of non-phosphorylated and phosphorylated BRCA1 contribute to the DNA damage response in HD. Notably, nuclear foci of γ-H2AX, the molecular component that recruits various DNA damage repair factors to damage sites including BRCA1, were deregulated when DNA was damaged in HD cell lines. BRCA1 specifically interacted with γ-H2AX via the BRCT domain, and this association was reduced in HD. BRCA1 overexpression restored γ-H2AX level in the nucleus of HD cells, while BRCA1 knockdown reduced the spatiotemporal propagation of γ-H2AX foci to the nucleoplasm. The deregulation of BRCA1 correlated with an abnormal nuclear distribution of γ-H2AX in striatal neurons of HD transgenic (R6/2) mice and BRCA1(+/-) mice. Our data indicate that BRCA1 is required for the efficient focal recruitment of γ-H2AX to the sites of neuronal DNA damage. Taken together, our results show that BRCA1 directly modulates the spatiotemporal dynamics of γ-H2AX upon genotoxic stress and serves as a molecular maker for neuronal DNA damage response in HD.     

Multispectral imaging flow cytometry reveals distinct frequencies of γ-H2AX foci induction in DNA double strand break repair defective human cell lines

The measurement of γ-H2AX foci induction in cells provides a sensitive and reliable method for the quantitation of DNA damage responses in a variety of cell types. Accurate and rapid methods to conduct such observations are desirable. In this study, we have employed the novel technique of multispectral imaging flow cytometry to compare the induction and repair of γ-H2AX foci in three human cell types with different capacities for the repair of DNA double strand breaks (DSB). A repair normal fibroblast cell line MRC5-SV1, a DSB repair defective ataxia telangiectasia (AT5BIVA) cell line, and a DNA-PKcs deficient cell line XP14BRneo17 were exposed to 2 Gy gamma radiation from a (60)Cobalt source. Thirty minutes following exposure, we observed a dramatic induction of foci in the nuclei of these cells. After 24 hrs, there was a predictable reduction on the number of foci in the MRC5-SV1 cells, consistent with the repair of DNA DSB. In the AT5BIVA cells, persistence of the foci over a 24-hr period was due to the failure in the repair of DNA DSB. However, in the DNA-PKcs defective cells (XP14BRneo17), we observed an intermediate retention of foci in the nuclei indicative of partial repair of DNA DSB. In summary, the application of imaging flow cytometry has permitted an evaluation of foci in a large number of cells (20,000) for each cell line at each time point. This provides a novel method to determine differences in repair kinetics between different cell types. We propose that imaging flow cytometry provides an alternative platform for accurate automated high through-put analysis of foci induction in a variety of cell types.     

Coilin levels modulate cell cycle progression and γH2AX levels in etoposide treated U2OS cells

Coilin is considered the Cajal body (CB) marker protein. In this report, we investigated the role of coilin in the DNA damage response and found that coilin reduction correlated with significantly increased levels of soluble γH2AX in etoposide treated U2OS cells. Additionally, coilin levels influenced the proliferation rate and cell cycle distribution of cells exposed to etoposide. Moreover, coilin overexpression inhibited nucleolar localization of endogenous coilin in etoposide treated U2OS cells. Collectively, these data provide additional evidence for coilin and CBs in the DNA damage response.     

Protective effect of an antithyroid compound against γ-radiation-induced damage in human colon cancer cells

We have previously reported the radioprotective effect of propylthiouracil (PTU) on thyroid cells. The aim of the present study was to analyze whether tumor cells and normal cells demonstrate the same response to PTU. Human colon carcinoma cells were irradiated with γ-irradiation with or without PTU. We evaluated the clonogenic survival, intracellular reactive oxygen species levels, catalase, superoxide dismutase and glutathione peroxidase activities, and apoptosis by nuclear cell morphology and caspase-3 activity assays. Cyclic AMP (cAMP) levels were measured by radioimmunoassay. PTU treatment increased surviving cell fraction at 2 Gy (SF2) from 56.9 ± 3.6 in controls to 75.0 ± 3.5 (p < 0.05) and diminished radiation-induced apoptosis. In addition, we observed that the level of antioxidant enzymes’ activity was increased in cells treated with PTU. Moreover, pretreatment with PTU increased intracellular levels of cAMP. Forskolin (p < 0.01) and dibutyryl cAMP (p < 0.05) mimicked the effect of PTU on SF2. Co-treatment with H89, an inhibitor of protein kinase A, abolished the radioprotective effect of PTU. PTU reduces the toxicity of ionizing radiation by increasing cAMP levels and also possibly through a reduction in apoptosis levels and in radiation-induced oxidative stress damage. We therefore conclude that PTU protects both normal and cancer cells during exposure to radiation in conditions mimicking the radiotherapy.     

γH2AX foci formation in the absence of DNA damage: Mitotic H2AX phosphorylation is mediated by the DNA-PKcs/CHK2 pathway

Phosphorylated H2AX is considered to be a biomarker for DNA double-strand breaks (DSB), but recent evidence suggests that γH2AX does not always indicate the presence of DSB. Here we demonstrate the bimodal dynamic of H2AX phosphorylation induced by ionizing radiation, with the second peak appearing when G2/M arrest is induced. An increased level of γH2AX occurred in mitotic cells, and this increase was attenuated by DNA-PKcs inactivation or Chk2 depletion, but not by ATM inhibition. The phosphorylation-mimic CHK2-T68D abrogated the attenuation of mitotic γH2AX induced by DNA-PKcs inactivation. Thus, the DNA-PKcs/CHK2 pathway mediates the mitotic phosphorylation of H2AX in the absence of DNA damage.     

Global DNA methylation profile at LINE-1 repeats and promoter methylation of genes involved in DNA damage response and repair pathways in human peripheral blood mononuclear cells in response to γ-radiation

Molecular and Cellular Biochemistry - DNA methylation is an epigenetic mechanism, which plays an important role in gene regulation. The present study evaluated DNA methylation profile of LINE1...     

Adapting the γ-H2AX assay for automated processing in human lymphocytes. 1. Technological aspects

The immunofluorescence-based detection of γ-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this well-established biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated high-throughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure γ-H2AX yields from fingerstick-derived samples of blood. The RABIT workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparin-coated PVC capillaries to the immunolabeling of γ-H2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purpose-built robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of γ-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in γ-H2AX fluorescence in peripheral blood samples irradiated ex vivo with γ rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify γ-H2AX total fluorescence in irradiated peripheral lymphocytes.