Gamma histone 2AX (γ-H2AX)as a predictive tool in radiation oncology

Abstract

Ionizing radiation cause DNA damage to cells, leading them to cell death via DNA double-strand breaks (DSBs) formation. DSBs formation is followed immediately by histone H2AX phosphorylation (γ-H2AX) and multitude repair factors accumulation. Here we present the methods and the bio-sampling for γ-H2AX detection, γ-H2AX formation in normal cells and animal tissues, in cancer cell lines/tissues and in clinical trials after radiation treatment, alone or in combination with other factors. The purpose of this review is to highlight the use of γ-H2AX, as a marker to assess DNA damage and repair.     

γ-H2AX and other histone post-translational modifications in the clinic

Chromatin is a dynamic complex of DNA and proteins that regulates the flow of information from genome to end product. The efficient recognition and faithful repair of DNA damage, particularly double-strand damage, is essential for genomic stability and cellular homeostasis. Imperfect repair of DNA double-strand breaks (DSBs) can lead to oncogenesis. The efficient repair of DSBs relies in part on the rapid formation of foci of phosphorylated histone H2AX (γ-H2AX) at each break site, and the subsequent recruitment of repair factors. These foci can be visualized with appropriate antibodies, enabling low levels of DSB damage to be measured in samples obtained from patients. Such measurements are proving useful to optimize treatments involving ionizing radiation, to assay in vivo the efficiency of various drugs to induce DNA damage, and to help diagnose patients with a variety of syndromes involving elevated levels of γ-H2AX. We will survey the state of the art of utilizing γ-H2AX in clinical settings. We will also discuss possibilities with other histone post-translational modifications. The ability to measure in vivo the responses of individual patients to particular drugs and/or radiation may help optimize treatments and improve patient care. This article is part of a Special Issue entitled: Chromatin in time and space.     

Calibration of the γ-H2AX DNA Double Strand Break Focus Assay for Internal Radiation Exposure of Blood Lymphocytes

DNA double strand break (DSB) formation induced by ionizing radiation exposure is indicated by the DSB biomarkers γ-H2AX and 53BP1. Knowledge about DSB foci formation in-vitro after internal irradiation of whole blood samples with radionuclides in solution will help us to gain detailed insights about dose-response relationships in patients after molecular radiotherapy (MRT). Therefore, we studied the induction of radiation-induced co-localizing γ-H2AX and 53BP1 foci as surrogate markers for DSBs in-vitro, and correlated the obtained foci per cell values with the in-vitro absorbed doses to the blood for the two most frequently used radionuclides in MRT (I-131 and Lu-177). This approach led to an in-vitro calibration curve. Overall, 55 blood samples of three healthy volunteers were analyzed. For each experiment several vials containing a mixture of whole blood and radioactive solutions with different concentrations of isotonic NaCl-diluted radionuclides with known activities were prepared. Leukocytes were recovered by density centrifugation after incubation and constant blending for 1 h at 37°C. After ethanol fixation they were subjected to two-color immunofluorescence staining and the average frequencies of the co-localizing γ-H2AX and 53BP1 foci/nucleus were determined using a fluorescence microscope equipped with a red/green double band pass filter. The exact activity was determined in parallel in each blood sample by calibrated germanium detector measurements. The absorbed dose rates to the blood per nuclear disintegrations occurring in 1 ml of blood were calculated for both isotopes by a Monte Carlo simulation. The measured blood doses in our samples ranged from 6 to 95 mGy. A linear relationship was found between the number of DSB-marking foci/nucleus and the absorbed dose to the blood for both radionuclides studied. There were only minor nuclide-specific intra- and inter-subject deviations.     

Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation

The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G₀ and G₁ phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage.     

Ratio of γ-H2AX level in lymphocytes to that in granulocytes detected using flow cytometry as a potential biodosimeter for radiation exposure

This study aims to assess utilisation of the ratio of γ-H2AX in lymphocytes to that in granulocytes (RL/G of γ-H2AX) in blood as a rapid method for population triage and dose estimation during large-scale radiation emergencies. Blood samples from healthy volunteers exposed to 0–10 Gy of ⁶⁰Co irradiation were collected. The samples were cultured for 0–24 h and then analysed using flow cytometry to measure the levels of γ-H2AX in lymphocytes and granulocytes. The basal RL/G levels of γ-H2AX in healthy human blood, the response of RL/G of γ-H2AX to ionising radiation and its relationship with doses, time intervals after exposure and individual differences were also analysed. The level of γ-H2AX in lymphocytes increased in a dose-dependent manner after irradiation, whereas the level in granulocytes was not affected. A linear dose–effect relationship with low inter-experimental and inter-individual variations was observed. The RL/G of γ-H2AX may be used as a biomarker for population triage and dose estimation during large-scale radiation emergencies if blood samples can be collected within 24 h.     

Use of the γ-H2AX Assay to Investigate DNA Repair Dynamics Following Multiple Radiation Exposures

Radiation therapy is one of the most common and effective strategies used to treat cancer. The irradiation is usually performed with a fractionated scheme, where the dose required to kill tumour cells is given in several sessions, spaced by specific time intervals, to allow healthy tissue recovery. In this work, we examined the DNA repair dynamics of cells exposed to radiation delivered in fractions, by assessing the response of histone-2AX (H2AX) phosphorylation (γ-H2AX), a marker of DNA double strand breaks. γ-H2AX foci induction and disappearance were monitored following split dose irradiation experiments in which time interval between exposure and dose were varied. Experimental data have been coupled to an analytical theoretical model, in order to quantify key parameters involved in the foci induction process. Induction of γ-H2AX foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. This was compared to chromatin relaxation and cell survival. The time needed for full recovery of γ-H2AX foci induction was quantified (12 hours) and the 1:1 relationship between radiation induced DNA double strand breaks and foci numbers was critically assessed in the multiple irradiation scenarios.     

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.     

γH2AX foci as a measure of DNA damage: a computational approach to automatic analysis

The γH2AX focus assay represents a fast and sensitive approach for the detection of one of the critical types of DNA damage - double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised γH2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable way due to limitations of computational approaches. We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens.     

Investigating the Use of Support Vector Machine Classification on Structural Brain Images of Preterm–Born Teenagers as a Biological Marker

Preterm birth has been shown to induce an altered developmental trajectory of brain structure and function. With the aid support vector machine (SVM) classification methods we aimed to investigate whether MRI data, collected in adolescence, could be used to predict whether an individual had been born preterm or at term. To this end we collected T1–weighted anatomical MRI data from 143 individuals (69 controls, mean age 14.6y). The inclusion criteria for those born preterm were birth weight ≤ 1500g and gestational age < 37w. A linear SVM was trained on the grey matter segment of MR images in two different ways. First, all the individuals were used for training and classification was performed by the leave–one–out method, yielding 93% correct classification (sensitivity = 0.905, specificity = 0.942). Separately, a random half of the available data were used for training twice and each time the other, unseen, half of the data was classified, resulting 86% and 91% accurate classifications. Both gestational age (R = –0.24, p<0.04) and birth weight (R = –0.51, p < 0.001) correlated with the distance to decision boundary within the group of individuals born preterm. Statistically significant correlations were also found between IQ (R = –0.30, p < 0.001) and the distance to decision boundary. Those born small for gestational age did not form a separate subgroup in these analyses. The high rate of correct classification by the SVM motivates further investigation. The long–term goal is to automatically and non–invasively predict the outcome of preterm–born individuals on an individual basis using as early a scan as possible.     

γ-H2AX in Cancer Cells: A Potential Biomarker for Cancer Diagnostics,Prediction and Recurrence

Current advances in cancer biology have identified major pathways involved in tumorigenesis. The association of DNA damage with premalignant stages of tumor progression, genome instability and further oncogenic transformation opens the possibility of using common DNA damage markers for early cancer detection, prediction, prognosis, therapeutics and possibly for cancer prevention. Perhaps the most sensitive DNA damage marker is γ-H2AX formation in the chromatin flanking the free DNA double-stranded ends in double-strand breaks (DSBs) and eroded telomeres, both present during oncogenic transformation. Our group and others found elevated endogenous levels of γ-H2AX in various human cancer cell lines, premalignant lesions and solid tumors. These data suggest that increased DNA damage is a general characteristic of cancer development. γ-H2AX-based assay can be applied to human biopsies, aspirates and, possibly, to mononuclear cells of the peripheral blood. We propose that detection of γ-H2AX could benefit for the early cancer screening and to ascertain the efficiency of clinical treatment involving chemo- and radiotherapeutic protocols.     

γ-H2AX Kinetic Profile in Mouse Lymphocytes Exposed to the Internal Emitters Cesium-137 and Strontium-90

In the event of a dirty bomb scenario or an industrial nuclear accident, a significant dose of volatile radionuclides such as ¹³⁷Cs and ⁹⁰Sr may be dispersed into the atmosphere as a component of fallout and inhaled or ingested by hundreds and thousands of people. To study the effects of prolonged exposure to ingested radionuclides, we have performed long-term (30 day) internal-emitter mouse irradiations using soluble-injected ¹³⁷CsCl and ⁹⁰SrCl₂ radioisotopes. The effect of ionizing radiation on the induction and repair of DNA double strand breaks (DSBs) in peripheral mouse lymphocytes in vivo was determined using the γ-H2AX biodosimetry marker. Using a serial sacrifice experimental design, whole-body radiation absorbed doses for ¹³⁷Cs (0 to 10 Gy) and ⁹⁰Sr (0 to 49 Gy) were delivered over 30 days following exposure to each radionuclide. The committed absorbed doses of the two internal emitters as a function of time post exposure were calculated based on their retention parameters and their derived dose coefficients for each specific sacrifice time. In order to measure the kinetic profile for γ-H2AX, peripheral blood samples were drawn at 5 specific timed dose points over the 30-day study period and the total γ-H2AX nuclear fluorescence per lymphocyte was determined using image analysis software. A key finding was that a significant γ-H2AX signal was observed in vivo several weeks after a single radionuclide exposure. A mechanistically-motivated model was used to analyze the temporal kinetics of γ-H2AX fluorescence. Exposure to either radionuclide showed two peaks of γ-H2AX: one within the first week, which may represent the death of mature, differentiated lymphocytes, and the second at approximately three weeks, which may represent the production of new lymphocytes from damaged progenitor cells. The complexity of the observed responses to internal irradiation is likely caused by the interplay between continual production and repair of DNA damage, cell cycle effects and apoptosis.     

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.     

Mitochondria with Morphology Characteristic for Alzheimer’s Disease Patients Are Found in the Brain of OXYS Rats

Growing evidence suggests that mitochondrial dysfunction is closely linked to the pathogenesis of sporadic Alzheimer’s disease (AD). One of the key contributors to various aspects of AD pathogenesis, along with metabolic dysfunction, is mitochondrial dynamics, involving balance between fusion and fission, which regulates mitochondrial number and morphology in response to changes in cellular energy demand. Recently, Zhang et al. ((2016) Sci. Rep., 6, 18725) described a previously unknown mitochondrial phenotype manifesting as elongated chain-linked mitochondria termed “mitochondria-on-a-string” (MOAS) in brain tissue from AD patients and mouse models of AD. The authors associated this phenotype with fission arrest, but implications of MOAS formation in AD pathogenesis remain to be understood. Here we analyze the presence and number of MOAS in the brain of OXYS rats simulating key signs of sporadic AD. Using electron microscopy, we found MOAS in OXYS prefrontal cortex neuropil in all stages of AD-like pathology, including mani-festation (5-month-old rats) and progression (12–18-month-old rats). The most pronounced elevation of MOAS content (8–fold) in OXYS rats compared to Wistar controls was found at the preclinical stage (20 days) on the background of decreased numbers of non-MOAS elongated mitochondria. From the age of 20 days through 18 months, the percentage of MOAS-containing neuronal processes increased from 1.7 to 8.3% in Wistar and from 13.9 to 16% in OXYS rats. Our results support the importance of the disruption of mitochondrial dynamics in AD pathogenesis and corroborate the existence of a causal link between impaired mitochondrial dynamics and formation of the distinctive phenotype of “mitochondria-on-a-sting”.     

Progressive Induction of Type 2 Diabetes: Effects of a Reality–Like Fructose Enriched Diet in Young Wistar Rats

Purpose

The aim of this study was to characterize short and medium-lasting effects of fructose supplementation on young Wistar rats. The diet was similar to actual human consumption.

Methods

Three week old male rats were randomly divided into 2 groups: control (C; n = 16), fructose fed (FF; n = 16) with a fructose enriched drink for 6 or 12 weeks. Bodyweight, fasting glycemia and systolic blood pressure were monitored. Glucose tolerance was evaluated using an oral glucose tolerance test. Insulinemia was measured concomitantly and enable us to calculate insulin resistance markers (HOMA-IR, Insulin Sensitivity Index for glycemia: ISI-gly). Blood chemistry analyses were performed.

Results

After six weeks of fructose supplementation, rats were not overweight but presented increased fasting glycemia, reduced glucose tolerance, and lower insulin sensitivity compared to control group. Systolic blood pressure and heart weight were also increased without any change in renal function (theoretical creatinine clearance). After twelve weeks of fructose supplementation, FF rats had increased bodyweight and presented insulin resistance (higher HOMA-IR, lower ISI-gly). Rats also presented higher heart volume and lower ASAT/ALAT ratio (presumed liver lesion). Surprisingly, the Total Cholesterol/Triglycerides ratio was increased only after six weeks of fructose supplementation, predicting a higher LDL presence and thus a higher risk of developing cardiovascular disease. This risk was no longer present after twelve weeks of a fructose enriched diet.

Conclusion

On young Wistar rats, six weeks of fructose supplementation is sufficient to induce signs of metabolic syndrome. After twelve weeks of fructose enriched diet, rats are insulin resistant. This model enabled us to study longitudinally the early development of type 2 diabetes.     

Not all the same after all? Superdiversity as a lens for the study of past migrations

Due to its claim of contemporary exceptionalism, the notion of superdiversity raises suspicion among historians. However, historians would do well to not dismiss the entire superdiversity debate as more hype that does not concern them. As a multidimensional perspective on diversity, encouraging researchers to examine the interplay of many different factors that condition people's lives and to move beyond an ethno-focal perspective, superdiversity could be of interest to historians as well. This article shows how the notion can help historians debunk some of the homogenizing categories that tend to characterize the representation of past immigrant populations. The paper uses a superdiversity lens to examine migration to the city of Ghent from 1960 to 1980. It is an open invitation to historians to accept the challenges that superdiversity poses and to provide a proper historicization of the concept, thus furthering its theoretical development.     

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.     

Cell Therapy as a Way to Correct Impaired Neurogenesis in the Adult Brain in a Model of Alzheimer’s Disease

Journal of Evolutionary Biochemistry and Physiology - This work is based on the hypothesis of an impairment in neurogenesis during aging, which may be one of the causes of neurodegenerative...