Upon DNA double-strand break (DSB) induction in mammals, the histone H2A variant, H2AX, becomes rapidly phosphorylated at serine 139. This modified form, termed γ-H2AX, is easily identified with ...antibodies and serves as a sensitive indicator of DNA DSB formation. This review focuses on the potential clinical applications of γ-H2AX detection in cancer and in response to other cellular stresses. In addition, the role of H2AX in homeostasis and disease will be discussed. Recent work indicates that γ-H2AX detection may become a powerful tool for monitoring genotoxic events associated with cancer development and tumor progression.
Summary
Accumulation of DNA damage may play an essential role in both cellular senescence and organismal aging. The ability of cells to sense and repair DNA damage declines with age. However, the ...underlying molecular mechanism for this age‐dependent decline is still elusive. To understand quantitative and qualitative changes in the DNA damage response during human aging, DNA damage‐induced foci of phosphorylated histone H2AX (γ‐H2AX), which occurs specifically at sites of DNA double‐strand breaks (DSBs) and eroded telomeres, were examined in human young and senescing fibroblasts, and in lymphocytes of peripheral blood. Here, we show that the incidence of endogenous γ‐H2AX foci increases with age. Fibroblasts taken from patients with Werner syndrome, a disorder associated with premature aging, genomic instability and increased incidence of cancer, exhibited considerably higher incidence of γ‐H2AX foci than those taken from normal donors of comparable age. Further increases in γ‐H2AX focal incidence occurred in culture as both normal and Werner syndrome fibroblasts progressed toward senescence. The rates of recruitment of DSB repair proteins to γ‐H2AX foci correlated inversely with age for both normal and Werner syndrome donors, perhaps due in part to the slower growth of γ‐H2AX foci in older donors. Because genomic stability may depend on the efficient processing of DSBs, and hence the rapid formation of γ‐H2AX foci and the rapid accumulation of DSB repair proteins on these foci at sites of nascent DSBs, our findings suggest that decreasing efficiency in these processes may contribute to genome instability associated with normal and pathological aging.
A detailed analysis of TUBALL single‐walled carbon nanotubes (SWCNTs) after two‐step purification involving acid purification treatment with concentrated HCl, HNO3, H2SO4 and solution of HNO3:H2SO4 ...followed by magnetic processing is presented. The purity degree from iron higher than 99% is achieved, and the yield obtained by the above process is about 75 wt.%. Such high purity of the resultant sample is achieved by removing of the external iron nanoparticles by acid treatment and following extraction of nanotubes containing encapsulated nanoparticles under applied magnetic field. A comprehensive characterization of SWCNTs by Raman, optical absorption, and X‐ray photoelectron spectroscopies reveals purification‐induced changes in the integrity, composition, and electronic properties of SWCNT.
Contamination of single‐walled carbon nanotubes (SWCNTs) with residual catalyst particles restricts their practical applications. Here the effective post‐synthesis method for purification of SWCNTs from iron‐contained nanoparticles by an acid treatment followed by magnetic processing has been demonstrated. This route produces SWCNTs with high purity (≈99%) and yield (≈76%). Depending on acid used, defectiveness and length of nanotubes and entanglement of their bundles can be changed.
Ionizing radiation (IR) exposure is inevitable in our modern society and can lead to a variety of deleterious effects including cancer and birth defects. A reliable, reproducible and sensitive ...assessment of exposure to IR and the individual response to that exposure would provide much needed information for the optimal treatment of each donor examined. We have developed a diagnostic test for IR exposure based on detection of the phosphorylated form of variant histone H2AX (γ-H2AX), which occurs specifically at sites of DNA double-strand breaks (DSBs). The cell responds to a nascent DSB through the phosphorylation of thousands of H2AX molecules flanking the damaged site. This highly amplified response can be visualized as a γ-H2AX focus in the chromatin that can be detected
in situ with the appropriate antibody. Here we assess the usability of γ-H2AX focus formation as a possible biodosimeter for human exposure to IR using peripheral blood lymphocytes irradiated
ex vivo and three-dimensional artificial models of human skin biopsies. In both systems, the tissues were exposed to 0.2–5
Gy, doses of IR that might be realistically encountered in various scenarios such as cancer radiotherapies or accidental exposure to radiation. Since the γ-H2AX response is maximal 30
min after exposure and declines over a period of hours as the cells repair the damage, we examined the time limitations of the useful detectability of γ-H2AX foci. We report that a linear response proportional to the initial radiation dose was obtained 48 and 24
h after exposure in blood samples and skin cells respectively. Thus, detection of γ-H2AX formation to monitor DNA damage in minimally invasive blood and skin tests could be useful tools to determine radiation dose exposure and analyze its effects on humans.
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.
Cellular senescence is a state reached by normal mammalian cells after a finite number of cell divisions and is characterized by morphological and physiological changes including terminal cell-cycle ...arrest. The limits on cell division imposed by senescence may play an important role in both organismal aging and in preventing tumorigenesis. Cellular senescence and organismal aging are both accompanied by increased DNA damage, seen as the formation of gamma-H2AX foci (gamma-foci), which may be found on uncapped telomeres or at non-telomeric sites of DNA damage. However, the relative importance of telomere- and non-telomere-associated DNA damage to inducing senescence has never been demonstrated. Here we present a new approach to determine accurately the chromosomal location of gamma-foci and quantify the number of telomeric versus non-telomeric gamma-foci associated with senescence in both human and mouse cells. This approach enables researchers to obtain accurate values and to avoid various possible misestimates inherent in earlier methods.
Using combined immunofluorescence and telomere fluorescence in situ hybridization on metaphase chromosomes, we show that human cellular senescence is not solely determined by telomeric DNA damage. In addition, mouse cellular senescence is not solely determined by non-telomeric DNA damage. By comparing cells from different generations of telomerase-null mice with human cells, we show that cells from late generation telomerase-null mice, which have substantially short telomeres, contain mostly telomeric gamma-foci. Most notably, we report that, as human and mouse cells approach senescence, all cells exhibit similar numbers of total gamma-foci per cell, irrespective of chromosomal locations.
Our results suggest that the chromosome location of senescence-related gamma-foci is determined by the telomere length rather than species differences per se. In addition, our data indicate that both telomeric and non-telomeric DNA damage responses play equivalent roles in signaling the initiation of cellular senescence and organismal aging. These data have important implications in the study of mechanisms to induce or delay cellular senescence in different species.
Filling of single-walled carbon nanotubes (SWCNTs) and extraction of the encapsulated species from their cavities are perspective treatments for tuning the functional properties of SWCNT-based ...materials. Here, we have investigated sulfur-modified SWCNTs synthesized by the ampoule method. The morphology and chemical states of carbon and sulfur were analyzed by transmission electron microscopy, Raman scattering, thermogravimetric analysis, X-ray photoelectron and near-edge X-ray absorption fine structure spectroscopies. Successful encapsulation of sulfur inside SWCNTs cavities was demonstrated. The peculiarities of interactions of SWCNTs with encapsulated and external sulfur species were analyzed in details. In particular, the donor-acceptor interaction between encapsulated sulfur and host SWCNT is experimentally demonstrated. The sulfur-filled SWCNTs were continuously irradiated
with polychromatic photon beam of high intensity. Comparison of X-ray spectra of the samples before and after the treatment revealed sulfur transport from the interior to the surface of SWCNTs bundles, in particular extraction of sulfur from the SWCNT cavity. These results show that the moderate heating of filled nanotubes could be used to de-encapsulate the guest species tuning the local composition, and hence, the functional properties of SWCNT-based materials.
When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated ...within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (gamma-H2AX) demonstrates that gamma-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of (125)I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of (125)IdU and processed immunocytochemically to determine the number of gamma-H2AX foci. The numbers of (125)IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of (125)I decays and the number of foci per cell, consistent with the assumptions that each (125)I decay yields a DNA DSB and each DNA DSB yields a visible gamma-H2AX focus. Based on these findings, we conclude that gamma-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.
That irradiated cells affect their unirradiated 'bystander' neighbors is evidenced by reports of increased clonogenic mortality, genomic instability, and expression of DNA-repair genes in the ...bystander cell populations. The mechanisms underlying the bystander effect are obscure, but genomic instability suggests DNA double-strand breaks (DSBs) may be involved. Formation of DSBs induces the phosphorylation of the tumor suppressor protein, histone H2AX and this phosphorylated form, named gamma-H2AX, forms foci at DSB sites. Here we report that irradiation of target cells induces gamma-H2AX focus formation in bystander cell populations. The effect is manifested by increases in the fraction of cells in a population that contains multiple gamma-H2AX foci. After 18 h coculture with cells irradiated with 20 alpha-particles, the fraction of bystander cells with multiple foci increased 3.7-fold. Similar changes occurred in bystander populations mixed and grown with cells irradiated with gamma-rays, and in cultures containing media conditioned on gamma-irradiated cells. DNA DSB repair proteins accumulated at gamma-H2AX foci, indicating that they are sites of DNA DSB repair. Lindane, which blocks gap-junctions, prevented the bystander effect in mixing but not in media transfer protocols, while c-PTIO and aminoguanidine, which lower nitric oxide levels, prevented the bystander effect in both protocols. Thus, multiple mechanisms may be involved in transmitting bystander effects. These studies show that H2AX phosphorylation is an early step in the bystander effect and that the DNA DSBs underlying gamma-H2AX focus formation may be responsible for its downstream manifestations.
When cells are exposed to ionizing radiation (IR), unexposed cells that share media with damaged cells exhibit similar effects to irradiated cells including increased levels of DNA double-strand ...breaks (DSBs). Hypothesizing that this effect, known as the radiation-induced bystander effect, may be a specific instance of communication between damaged and undamaged cells regardless of damage source, we demonstrated that exposure of target cells to non-IR induces bystander damage in non-targeted cells as measured by γ-H2AX and 53BP1 focal formation. Initially, bystander damage was found primarily in S-phase cells, but at later times, non-S-phase cells were also affected. In addition, media from undamaged malignant and senescent cells also was found to induce DSBs in primary cultures. Media conditioned on cells targeted with either ionizing or non-IR as well as on undamaged malignant and senescent cells contained elevated levels of several cytokines. One of these, transforming growth factor beta (TGF-β), and nitric oxide (NO) were found to elevate numbers of γ-H2AX/53BP1 foci in normal cell cultures similar to levels found in bystander cells, and this elevation was abrogated by NO synthase inhibitors, TGF-β blocking antibody and antioxidants. These findings support the hypothesis that damage in bystander cells results from their exposure to cytokines or reactive compounds released from stressed cells, regardless of damage source. These results have implications for oncogenesis in that they indicate that damaged normal cells or undamaged tumor cells may induce genomic instability, leading to an increased risk of oncogenic transformation in other cells with which they share media or contact directly.
PDF
