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)}     

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.     

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.     

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.     

Structural Insights into Estrogen Receptor α Methylation by Histone Methyltransferase SMYD2, a Cellular Event Implicated in Estrogen Signaling Regulation

Estrogen receptor (ER) signaling plays a pivotal role in many developmental processes and has been implicated in numerous diseases including cancers. We recently showed that direct ERα methylation by the multi-specificity histone lysine methyltransferase SMYD2 regulates estrogen signaling through repressing ERα-dependent transactivation. However, the mechanism controlling the specificity of the SMYD2–ERα interaction and the structural basis of SMYD2 substrate binding diversity are unknown. Here we present the crystal structure of SMYD2 in complex with a target lysine (Lys266)-containing ERα peptide. The structure reveals that ERα binds SMYD2 in a U-shaped conformation with the binding specificity determined mainly by residues C-terminal to the target lysine. The structure also reveals numerous intrapeptide contacts that ensure shape complementarity between the substrate and the active site of the enzyme, thereby likely serving as an additional structural determinant of substrate specificity. In addition, comparison of the SMYD2–ERα and SMYD2–p53 structures provides the first structural insight into the diverse nature of SMYD2 substrate recognition and suggests that the broad specificity of SMYD2 is achieved by multiple molecular mechanisms such as distinct peptide binding modes and the intrinsic dynamics of peptide ligands. Strikingly, a novel potentially SMYD2-specific polyethylene glycol binding site is identified in the CTD domain, implicating possible functions in extended substrate binding or protein–protein interactions. Our study thus provides the structural basis for the SMYD2-mediated ERα methylation, and the resulting knowledge of SMYD2 substrate specificity and target binding diversity could have important implications in selective drug design against a wide range of ERα-related diseases.     

Displacement of Ca2+ by Na+ from the Plasmalemma of Root Cells : A Primary Response to Salt Stress?

A microfluorometric assay using chlorotetracycline (CTC) as a probe for membrane-associated Ca²⁺ in intact cotton (Gossypium hirsutum L. cv Acala SJ-2) root hairs indicated displacement of Ca²⁺ by Na⁺ from membrane sites with increasing levels of NaCl (0 to 250 millimolar). K⁺(⁸⁶Rb) efflux increased dramatically at high salinity. An increase in external Ca²⁺ concentration (10 millimolar) mitigated both responses. Other cations and mannitol, which did not affect Ca²⁺-CTC chelation properties, were found to have no effect on Ca²⁺-CTC fluorescence, indicating a Na⁺-specific effect. Reduction of Ca²⁺-CTC fluorescence by ethyleneglycol-bis-(β-aminoethyl ether) N,N′-tetraacetic acid, which does not cross membranes, provided an indication that reduction by Na⁺ of Ca²⁺-CTC fluorescence may be occurring primarily at the plasmalemma. The findings support prior proposals that Ca²⁺ protects membranes from adverse effects of Na⁺ thereby maintaining membrane integrity and minimizing leakage of cytosolic K⁺.     

Oxidative Stress,Redox Homeostasis and Cellular Stress Response in Ménière’s Disease: Role of Vitagenes

Ménière’s disease (MD) is characterized by the triad of fluctuating hearing loss, episodic vertigo and tinnitus, and by endolymphatic hydrops found on post-mortem examination. Increasing evidence suggests that oxidative stress is involved in the development of endolymphatic hydrops and that cellular damage and apoptotic cell death might contribute to the sensorineural hearing loss found in later stages of MD. While excess reactive oxygen species (ROS) are toxic, regulated ROS, however, play an important role in cellular signaling. The ability of a cell to counteract stressful conditions, known as cellular stress response, requires the activation of pro-survival pathways and the production of molecules with anti-oxidant, anti-apoptotic or pro-apoptotic activities. Among the cellular pathways conferring protection against oxidative stress, a key role is played by vitagenes, which include heat shock proteins (Hsps) as well as the thioredoxin/thioredoxin reductase system. In this study we tested the hypothesis that in MD patients measurable increases in markers of cellular stress response and oxidative stress in peripheral blood are present. This study also explores the hypothesis that changes in the redox status of glutathione, the major endogenous antioxidant, associated with abnormal expression and activity of carbonic anhydrase can contribute to increase oxidative stress and to disruption of systemic redox homeostasis which can be associated to possible alterations on vulnerable neurons such as spiral ganglion neurons and consequent cellular degeneration. We therefore evaluated systemic oxidative stress and cellular stress response in patients suffering from Meniere’s disease (MD) and in age-matched healthy subjects. Systemic oxidative stress was estimated by measuring protein oxidation, such as protein carbonyls (PC) and 4-hydroxynonenal (HNE) in lymphocytes of MD patients, as well as ultraweak luminescence (UCL) as end-stable products of lipid oxidation in MD plasma and lymphocytes, as compared to age-matched controls, whereas heat shock proteins Hsp70 and thioredoxin (Trx) expression were measured in lymphocytes to evaluate the systemic cellular stress response. Increased levels of PC (P < 0.01) and HNE (P < 0.05) have been found in lymphocytes from MD patients with respect to control group. This was paralleled by a significant induction of Hsp70, and a decreased expression of Trx (P < 0.01), whereas a significant decrease in both plasma and lymphocyte ratio reduced glutathione GSH) vs. oxidized glutathione (GSSG) (P < 0.05) were also observed. In conclusion, patients affected by MD are under condition of systemic oxidative stress and the induction of vitagenes Hsp70 is a maintained response in counteracting the intracellular pro-oxidant status generated by decreased content of GSH as well as expression of Trx. The search for novel and more potent inducers of vitagenes will facilitate the development of pharmacological strategies to increase the intrinsic capacity of vulnerable ganglion cells to maximize antidegenerative mechanisms, such as stress response and thus cytoprotection.     

Phosphatases Involved in Modulation of Gap Junctional Intercellular Communication and Dephosphorylation of Connexin43 in Hamster Fibroblasts: 2B or Not 2B?

12-O-Tetradecanoylphorbol-13-acetate (TPA) caused strong suppression of gap junctional intercellular communication, altered phosphorylation status of the gap junction protein, connexin43, and disappearance of immunorecognizible connexin43-containing gap junction plaques in V79 fibroblasts. When TPA was removed, all parameters normalized during a 3- to 4-h period. The normalizations were independent of protein synthesis, suggesting the possible involvement of phosphatases. None of the phosphatase inhibitors okadaic acid, calyculin A, cyclosporin A, or FK506 affected intercellular communication or connexin43 phosphorylation status on their own. In sequential exposures to TPA and phosphatase inhibitors, only the protein-phosphatase 2B (PP2B) inhibitors cyclosporin A and FK506 delayed the recovery of the studied parameters. Rapamycin binds to the same set of proteins as does FK506, but without inhibiting PP2B. Rapamycin did not affect the recovery of intercellular communication, but it delayed the normalization of connexin43 band pattern and immunorecognition of gap junction plaques. Dephosphorylation of immunoprecipitated connexin43 was studied using PP1, 2A, 2B, and 2C. PP2A was the most efficient (by 100-fold on a molar basis). Connexin43 immunoprecipitated from TPA-exposed cells was a poor substrate for PP1, 2B, and 2C. Thus, PP2B appeared to play a role in normalization of intercellular communication, but not necessarily in direct dephosphorylation of connexin43. Peptidyl-prolyl isomerase activity of cyclosporin/FK506/rapamycin-binding proteins may promote the dephosphorylation of connexin43 in cells.     

Cytokine Diversity in the Th1-Dominated Human Anti-Influenza Response Caused by Variable Cytokine Expression by Th1 Cells,and a Minor Population of Uncommitted IL-2+IFNγ- Thpp Cells

Within overall Th1-like human memory T cell responses, individual T cells may express only some of the characteristic Th1 cytokines when reactivated. In the Th1-oriented memory response to influenza, we have tested the contributions of two potential mechanisms for this diversity: variable expression of cytokines by a uniform population during activation, or different stable subsets that consistently expressed subsets of the Th1 cytokine pattern. To test for short-term variability, in vitro-stimulated influenza-specific human memory CD4+ T cells were sorted according to IL-2 and IFNγ expression, cultured briefly in vitro, and cytokine patterns measured after restimulation. Cells that were initially IFNγ+ and either IL-2+ or IL-2- converged rapidly, containing similar proportions of IL-2-IFNγ+ and IL-2+IFNγ+ cells after culture and restimulation. Both phenotypes expressed Tbet, and similar patterns of mRNA. Thus variability of IL-2 expression in IFNγ+ cells appeared to be regulated more by short-term variability than by stable differentiated subsets. In contrast, heterogeneous expression of IFNγ in IL-2+ influenza-specific T cells appeared to be due partly to stable T cell subsets. After sorting, culture and restimulation, influenza-specific IL-2+IFNγ- and IL-2+IFNγ+ cells maintained significantly biased ratios of IFNγ+ and IFNγ- cells. IL-2+IFNγ- cells included both Tbet^lo and Tbet^hi cells, and showed more mRNA expression differences with either of the IFNγ+ populations. To test whether IL-2+IFNγ-Tbet^lo cells were Thpp cells (primed but uncommitted memory cells, predominant in responses to protein vaccines), influenza-specific IL-2+IFNγ- and IL-2+IFNγ+ T cells were sorted and cultured in Th1- or Th2-generating conditions. Both cell types yielded IFNγ-secreting cells in Th1 conditions, but only IL-2+IFNγ- cells were able to differentiate into IL-4-producing cells. Thus expression of IL-2 in the anti-influenza response may be regulated mainly by short term variability, whereas different T cell subsets, Th1 and Thpp, may contribute to variability in IFNγ expression.     

USP11 Is a Negative Regulator to γH2AX Ubiquitylation by RNF8/RNF168

Ubiquitin modification at double strand breaks (DSB) sites is an essential regulator of signaling and repair. γH2AX extends from DSB sites and provides a platform for subsequent recruitment and amplification of DNA repair proteins and signaling factors. Here, we found that RNF8/RNF168 ubiquitylates γH2AX. We identified that USP11 is a unique deubiquitylation enzyme for γH2AX. USP11 deubiquitylates γH2AX both in vivo and in vitro but not the canonical (ub)-K119-H2A and (ub)-K120-H2B in vitro, and USP11 ablation enhances the levels of γH2AX ubiquitylation. We also found that USP11 interacts with γH2AX both in vivo and in vitro. We found that 53BP1 and ubiquitin-conjugated proteins are misregulated to be retained longer and stronger at DSB sites after knockdown of USP11. We further found that cells are hypersensitive to γ-irradiation after ablation of USP11. Together, our findings elucidate deeply and extensively the mechanism of RNF8/RNF168 and USP11 to maintain the proper status of ubiquitylation γH2AX to repair DSB.     

Two Strategies for Response to 14°C Cold-Water Immersion: Is there a Difference in the Response of Motor,Cognitive, Immune and Stress Markers?

Here, we address the question of why some people have a greater chance of surviving and/or better resistance to cold-related-injuries in prolonged exposure to acute cold environments than do others, despite similar physical characteristics. The main aim of this study was to compare physiological and psychological reactions between people who exhibited fast cooling (FC; n = 20) or slow cooling (SC; n = 20) responses to cold water immersion. Individuals in whom the T_re decreased to a set point of 35.5°C before the end of the 170-min cooling time were indicated as the FC group; individuals in whom the T_re did not decrease to the set point of 35.5°C before the end of the 170-min cooling time were classified as the SC group. Cold stress was induced using intermittent immersion in bath water at 14°C. Motor (spinal and supraspinal reflexes, voluntary and electrically induced skeletal muscle contraction force) and cognitive (executive function, short term memory, short term spatial recognition) performance, immune variables (neutrophils, leucocytes, lymphocytes, monocytes, IL-6, TNF-α), markers of hypothalamic–pituitary–adrenal axis activity (cortisol, corticosterone) and autonomic nervous system activity (epinephrine, norepinephrine) were monitored. The data obtained in this study suggest that the response of the FC group to cooling vs the SC group response was more likely an insulative–hypothermic response and that the SC vs the FC group displayed a metabolic–insulative response. The observations that an exposure time to 14°C cold water—which was nearly twice as short (96-min vs 170-min) with a greater rectal temperature decrease (35.5°C vs 36.2°C) in the FC group compared with the SC group—induces similar responses of motor, cognitive, and blood stress markers were novel. The most important finding is that subjects with a lower cold-strain-index (SC group) showed stimulation of some markers of innate immunity and suppression of markers of specific immunity.     

The α2L111R,N122D Isoform of the Na,K-ATPase Expressed in HeLa Cells Does Not Undergo an Adipocyte-like Increase in Activity in Response to Insulin

In the rat adipocyte, insulin increases potassium uptake by a preferential activation of the α2 isoform of the Na,K-ATPase. The question under consideration here is whether expression of the α2 isoform is sufficient to replicate its differential activation by insulin. Accordingly, we compared the effect of insulin on the activity of the ouabain resistant rat α1 and α2RD (α2L111R,N122D) isoforms in HeLa cells. In HeLa cells, in contrast to the rat adipocyte, insulin produces an increase of equal magnitude in the rate of⁸⁶Rb⁺/K⁺uptake by the ouabain resistant rat α1 and rat α2RD subunits. We conclude that the mechanism of insulin activation of the α2RD isoform in HeLa cells differs from that of the wild type α2 isoform in the rat adipocyte.     

γH2AX foci on apparently intact mitotic chromosomes: Not signatures of misrejoining events but signals of unresolved DNA damage

The presence of γH2AX foci on apparently intact mitotic chromosomes is controversial because they challenge the assumed relationship between γH2AX foci and DNA double-strand breaks (DSBs). In this work, we show that after irradiation during interphase, a variety of γH2AX foci are scored in mitotic cells. Surprisingly, approximately 80% of the γH2AX foci spread over apparently undamaged chromatin at Terminal or Interstitial positions and they can display variable sizes, thus being classified as Small, Medium and Big foci. Chromosome and chromatid breaks that reach mitosis are spotted with Big (60%) and Medium (30%) Terminal γH2AX foci, but very rarely are they signaled with Small γH2AX foci. To evaluate if Interstitial γH2AX foci might be signatures of misrejoining, an mFISH analysis was performed on the same slides. The results show that Interstitial γH2AX foci lying on apparently intact chromatin do not mark sites of misrejoining, and that misrejoined events were never signaled by a γH2AX foci during mitosis. Finally, when analyzing the presence of other DNA-damage response (DDR) factors we found that all γH2AX foci—regardless their coincidence with a visible break—always colocalized with MRE11, but not with 53BP1. This pattern suggests that these γH2AX foci may be hallmarks of both microscopically visible and invisible DNA damage, in which an active, although incomplete or halted DDR is taking place.     

Protein Arginine Methyltransferase 7 Regulates Cellular Response to DNA Damage by Methylating Promoter Histones H2A and H4 of the Polymerase δ Catalytic Subunit Gene, POLD1

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage.     

γH2AX foci analysis for monitoring DNA double-strand break repair: Strengths,limitations and optimization

DNA double-strand breaks (DSBs) represent an important radiation-induced lesion and impaired DSB repair provides the best available correlation with radiosensitivity. Physical techniques for monitoring DSB repair require high, non-physiological doses and cannot reliably detect subtle defects. One outcome from extensive research into the DNA damage response is the observation that H2AX, a variant form of the histone H2A, undergoes extensive phosphorylation at the DSB, creating γH2AX foci that can be visualised by immunofluorescence. There is a close correlation between γH2AX foci and DSB numbers and between the rate of foci loss and DSB repair, providing a sensitive assay to monitor DSB repair in individual cells using physiological doses. However, γH2AX formation can occur at single-stranded DNA regions which arise during replication or repair and thus does not solely correlate with DSB formation. Here, we present and discuss evidence that following exposure to ionising radiation, γH2AX foci analysis can provide a sensitive monitor of DSB formation and repair and describe techniques to optimise the analysis. We discuss the limitations and benefits of the technique, enabling the procedure to be optimally exploited but not misused.