Silver ions enhance UVB‐induced phosphorylation of histone H2AX

Silver (Ag) is used in a wide range of industries including healthcare, food, cosmetics, and environmental industries due to its antibacterial properties. The rapidly expanding use of Ag has raised issues concerning its toxicity in humans. However, studies investigating the effects of Ag on humans are very limited, and the combined effects of Ag and other environmental factors have not yet been determined. Ultraviolet (UV) radiation in sunlight is the most prominent and ubiquitous physical stressor in our natural environment. In this study, we investigated the genotoxic potential of combined exposure to Ag⁺ (AgNO₃) and UVB in the human keratinocyte cell line, HaCaT, by measuring the generation of phosphorylated histone H2AX, which is currently attracting attention as a biomarker for the detection of genotoxic insults. We found that the generation of γ‐H2AX was synergistically enhanced when cells were coexposed to Ag⁺ and UVB. Furthermore, we showed that the enhanced generation of γ‐H2AX could be attributed to the increased formation of UVB‐induced cyclobutane pyrimidine dimers and (6–4) photoproducts. These lesions, if not repaired properly, are the major causal factor for skin carcinogenesis. Our results provide an important insight into influence of Ag on the genotoxic potency of sunlight. Environ. Mol. Mutagen. 55:556–565, 2014. © 2014 Wiley Periodicals, Inc.     

Water soluble fraction of solar-simulated light-exposed crude oil generates phosphorylation of histone H2AX in human skin cells under UVA exposure

Crude oil contains compounds, which have toxic and cancer-causing properties to humans. The oil spilled in environments is usually exposed to sunlight; however, the toxicity of sunlight-exposed oil is poorly understood. In this study, we found that the water soluble fraction (WSF) of crude oil irradiated with solar-simulated light (SSL) generated phosphorylation of histone H2AX (gamma-H2AX) in human skin cells under UVA irradiation, which was due to the formation of DNA double strand breaks (DSBs). Crude oil was exposed to SSL for approximately 7 days. The WSF obtained from unexposed crude oil showed no toxicity, whereas the WSF obtained from crude oil pre-exposed to SSL induced acute cell death on exposure to UVA irradiation (induction of phototoxicity), which was more remarkable in human skin fibroblasts than human skin keratinocytes. gamma-H2AX was detected in both cell lines immediately after treatment with the WSF plus UVA. Interestingly, gamma-H2AX was detectable even at low SSL- and UVA-doses, which induced no cytotoxicity. The WSF of crude oil irradiated with SSL, generated DSBs under UVA irradiation, which were detected by biased sinusoidal field gel electrophoresis. This was confirmed using xrs-5 cells isolated from CHO-K1 cells, which are deficient in a repair enzyme for DSBs; the WSF plus UVA induced a more dramatic decrease in survival in xrs-5 cells than CHO-K1 cells. These findings demonstrate that exposure of crude oil to sunlight makes the WSF phototoxic, generating DSBs accompanying the appearance of gamma-H2AX in human skin cells.     

Conserved gammaherpesvirus kinase and histone variant H2AX facilitate gammaherpesvirus latency in vivo

Many herpesvirus-encoded protein kinases facilitate viral lytic replication. Importantly, the role of viral kinases in herpesvirus latency is less clear. Mouse gammaherpesvirus-68 (MHV68)-encoded protein kinase orf36 facilitates lytic replication in part through activation of the host DNA damage response (DDR). Here we show that MHV68 latency was attenuated in the absence of orf36 expression. Unexpectedly, our study uncovered enzymatic activity-independent role of orf36 in the establishment of MHV68 latency following intraperitoneal route of infection. H2AX, an important DDR protein, facilitates MHV68 lytic replication and may be directly phosphorylated by orf36 during lytic infection. In this study, H2AX deficiency, whether systemic or limited to infected cells, attenuated the establishment of MHV68 latency in vivo. Thus, our work reveals viral kinase-dependent regulation of gammaherpesvirus latency and illuminates a novel link between H2AX, a component of a tumor suppressor DDR network, and in vivo latency of a cancer-associated gammaherpesvirus.     

Follow-up evaluation of radiation-induced DNA damage in CSF disseminated high-grade glioma using phospho-histone H2AX antibody

Cytological examination of cerebrospinal fluid (CSF) is used not only for the diagnosis of spinal disease, but also to assess the postoperative effect of treatment. We experienced a case of high-grade glioma in disseminated CSF, and retrospectively examined the clinical, pathological and cytological features. We further investigated radiation-induced DNA damage in glioma cells using phospho-Histone H2AX antibody. A five-year-old boy received a clinical diagnosis of optic nerve glioma, and was followed-up for three months after chemotherapy. Magnetic resonance imaging was repeated, revealing abnormalities in other brain areas. The pathological diagnosis was anaplastic astrocytoma. CSF dissemination was detected, and increases in the number and mitosis of tumor cells were observed in CSF cytology. After radiotherapy the tumor cells in CSF decreased markedly. On cytomorphologic and immunocytochemical evaluation post-irradiation, tumor cells showed vacuolation of both the nucleus and cytoplasm, degeneration of nuclear chromatin, and alteration of the phospho-Histone H2AX expression, compared with tumor cells before the irradiation. CSF cytology is an effective means of evaluating DNA damage in tumor cells after irradiation, and may be useful in assessing the therapeutic response.     

Histone H3 phosphorylation of mammalian chromosomes

Inferences about the role and location of phosphorylated histone H3 are derived primarily from biochemical studies. A few direct observations at chromosome level have shown that phosphorylation begins at the site of heterochromatin and spreads throughout the chromosome. However, a comparative study of chromosomes of mouse (L929 cells), Chinese hamster (CHO 9 cells) and the Indian muntjac (male cells) reveals some distinguishable details among mammalian species. Whereas the L929 cells exhibit the typical pattern of phosphorylation at the region of centromeric heterochromatin associated with the active centromere, the heterochromatin blocks associated with the inactive centromeres also show label of about equivalent intensity. Throughout the cell cycle, heterochromatin exhibits sharper (denser) and better defined label than does euchromatin which expresses somewhat diffuse label. The centromere constriction on biarmed chromosomes, originating in Robertsonian translocations, appears phosphorylated in some, if not all chromosomes. A similar situation was found for the CHO 9 cells indicating that phosphorylation does include the region in which H3 is supposedly replaced by CENP-A. An interesting feature of the CHO cell line was the dense label at and near the telomeres; this feature was not observed in either the mouse or the Indian muntjac. The centromere regions of the Indian muntjac chromosomes showed three sites of label in the multicentric X chromosome and two each on chromosome pair number 1 and Y₂; the sites coinciding with the reaction sites of antikinetochore antibodies. Also, the X and Y₁ chromosomes of Indian muntjac show intense phosphorylation at the sites of secondary constrictions.The chromosomes of all three species were phosphorylated throughout the cell cycle. As the chromosomes started to decondense during anaphase, heavy phosphorylation was observed in the form of discontinuous beaded structures indicating partial despiralization of the chromosome. Interestingly, when cells had completed karyokinesis and resolved into two independent nuclei, the phosphorylation was observed at the midbody. At this stage, the cytoplasm appeared to be again phosphorylated.     

组蛋白H2AX在高氧致肺损伤新生大鼠肺组织的表达

目的 研究组蛋白H2AX在高氧致肺损伤新生大鼠肺组织中的表达,并探讨其在高氧肺损伤中的作用.方法 将80例新生大鼠随机分为实验组(高氧组)和对照组(空气组),建立高氧致新生大鼠肺损伤的模型,在实验开始后1、2、3、5、7天取肺组织标本,运用免疫荧光技术和Western-blot方法检测高氧肺损伤过程中H2AX的表达规律...     

Immunogold labeling of chromosomes for scanning electron microscopy: A closer look at phosphorylated histone H3 in mitotic metaphase chromosomes of Hordeum vulgare

High-resolution detection of phosphorylated histone H3 at serine 10 in mitotic barley chromosomes for scanning electron microscopy was shown using a novel application of indirect immunogold labeling with Nanogold. This method permits localization and quantification of signals in a three-dimensional context. Because the chromosome structure is well preserved, characterization of binding sites (chromomeres, parallel matrix fibers, solenoids), currently in the realm of nanometer decades, is possible. Quantification and three-dimensional localization of labels is possible with stereoscopic analysis. Limitations of the method pertain to the challenges in preservation of chromosome ultrastructure, accessibility of immunoreactants into the fixed chromatin and unspecific labeling. The differences between silver and gold enhancement and the current status of labeling efficiency are addressed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sumoylation precedes accumulation of phosphorylated H2AX on sex chromosomes during their meiotic inactivation

During meiosis in male mammals, X and Y chromosomes undergo the process of meiotic sex chromosome inactivation (MSCI). A crucial role in MSCI has recently been reported for BRCA1, ATR kinase, and phosphorylated histone H2AX, but the exact mechanism remains to be determined. Small ubiquitin-like modifier (SUMO) proteins have recently been shown to localize to the sex body in mouse meiotic spermatocytes, but the role they play during MSCI is unknown. In this study, in order to better understand the molecular events of MSCI, we followed dynamic changes in γH2AX and SUMO localization patterns during MSCI. Using confocal laser scanning microscopy (CLSM) as an analytical tool for visualizing numerous spermatocytes from the same development stage and for consecutively following the meiotic progression, we were able to demonstrate a very early appearance of SUMO-1, which preceded γH2AX accumulation on the sex chromosomes during their meiotic inactivation. In contrast to SUMO-1, SUMO-2/3 was undetectable in zygotene spermatocytes, suggesting a possible specific role for SUMO-1 in the initiation of MSCI.     

Increased level of DNA damage in some organs of obese Zucker rats by γ‐H2AX analysis

In a recent study, we showed that lymphocytes of obese Italian children/adolescents displayed levels of double strand breaks (DSB), assayed as serine 139‐phosphorylated histone H2AX (γ‐H2AX), about eightfold higher than normal weight controls, and that 30% of this damage‐generated micronuclei. These findings suggested that obese children could be at increased risk of obesity‐mediated cancer later in life. We therefore aimed to assess the level of γ‐H2AX in a genetic animal model of obesity (Zucker rat) to identify a genotoxic/carcinogenic risk in some organs. The DSB marker was studied in 3‐ to 4‐week‐old rats and in 9‐ to 13‐week‐old rats. Paraffin‐embedded sections of heart, thyroid, liver, pancreas, lung, kidney, esophagus, and gut from the fa?/fa? (obese) and the fa+/fa? (lean) control animals were processed for immunohistochemistry detection of γ‐H2AX. Pancreas (0.0624 ± 0.0195), lung (0.1197 ± 0.0217), esophagus (0.1230 ± 0.0351), kidney (0.1546 ± 0.0149), and gut (0.1724 ± 0.0352) of 9‐ to 13‐week‐old obese rats showed a higher proportion of γ‐H2AX‐positive nuclei, than their lean counterparts (0.0092 ± 0.0033, 0.0416 ± 0.0185, 0.0368 ± 0.0088, 0.0686 ± 0.0318, and 0.0703 ± 0.0239, respectively). No difference was seen in the 3‐ to 4‐week‐old age group with regard to obesity, indicating that the DNA damage increased with older age of the rats. We hypothesize that the organs of the obese animals showing high levels of DSB could represent target tissues for the development of obesity‐related cancers. Environ. Mol. Mutagen. 58:477–484, 2017. © 2017 Wiley Periodicals, Inc.     

Analysis of protein dynamics at active, stalled, and collapsed replication forks

Successful DNA replication and packaging of newly synthesized DNA into chromatin are essential to maintain genome integrity. Defects in the DNA template challenge genetic and epigenetic inheritance. Unfortunately, tracking DNA damage responses (DDRs), histone deposition, and chromatin maturation at replication forks is difficult in mammalian cells. Here we describe a technology called iPOND (isolation of proteins on nascent DNA) to analyze proteins at active and damaged replication forks at high resolution. Using this methodology, we define the timing of histone deposition and chromatin maturation. Class 1 histone deacetylases are enriched at replisomes and remove predeposition marks on histone H4. Chromatin maturation continues even when decoupled from replisome movement. Furthermore, fork stalling causes changes in the recruitment and phosphorylation of proteins at the damaged fork. Checkpoint kinases catalyze H2AX phosphorylation, which spreads from the stalled fork to include a large chromatin domain even prior to fork collapse and double-strand break formation. Finally, we demonstrate a switch in the DDR at persistently stalled forks that includes MRE11-dependent RAD51 assembly. These data reveal a dynamic recruitment of proteins and post-translational modifications at damaged forks and surrounding chromatin. Furthermore, our studies establish iPOND as a useful methodology to study DNA replication and chromatin maturation.     

Catalysis-dependent stabilization of Bre1 fine-tunes histone H2B ubiquitylation to regulate gene transcription

Monoubiquitylation of histone H2B on Lys123 (H2BK123ub1) plays a multifaceted role in diverse DNA-templated processes, yet the mechanistic details by which this modification is regulated are not fully elucidated. Here we show in yeast that H2BK123ub1 is regulated in part through the protein stability of the E3 ubiquitin ligase Bre1. We found that Bre1 stability is controlled by the Rtf1 subunit of the polymerase-associated factor (PAF) complex and through the ability of Bre1 to catalyze H2BK123ub1. Using a domain in Rtf1 that stabilizes Bre1, we show that inappropriate Bre1 levels lead to defects in gene regulation. Collectively, these data uncover a novel quality control mechanism used by the cell to maintain proper Bre1 and H2BK123ub1 levels, thereby ensuring proper control of gene expression.     

Alterations in the distribution of histone H3 phosphorylation in mitotic plant chromosomes in response to cold treatment and the protein phosphatase inhibitor cantharidin

The function of the phosphorylation of histone H3 at Ser 10 in plant cell division is uncertain. The timing correlates with chromosome condensation, and studies in plant meiosis suggest that it is involved in sister chromatid cohesion. In mitosis, plant chromosomes are highly phosphorylated in the pericentromeric region only. In order to modulate H3 phosphorylation, root meristems of different plant species were treated with the protein phosphatase inhibitor cantharidin or with ice-water. Immunostaining using an antibody specific to phosphorylated H3 at Ser 10 revealed a high level of H3 phosphorylation along the whole mitotic chromosome after cantharidin treatment, which resembles the distribution seen exclusively in first meiotic division. In chromosomes that were isolated from meristems treated with ice-water, the heterochromatic regions and nucleolar organizer regions, in addition to the pericentromeric region, were highly phosphorylated at H3. Cantharidin and ice-water also affected spindle assembly and chromosome length, but these effects did not seem to be directly linked to changes in H3 phosphorylation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Kinetics of gamma-H2AX focus formation upon treatment of cells with UV light and alkylating agents

Histone H2AX is rapidly phosphorylated in response to DNA double-strand breaks (DSBs) induced by ionizing radiation (IR). Here we show that DNA damage induced by alkylating agents [methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)] and ultraviolet light (UV-C) leads to a dose and time dependent accumulation of phosphorylated H2AX (gamma-H2AX). Time course experiments revealed that the number of gamma-H2AX foci reached peak levels 8 hr after MMS or MNNG treatment and declined to almost control values within 24 hr after exposure. Upon UV-C treatment, a biphasic response was observed with a maximum 12 hr after treatment. In 43-3B cells deficient in nucleotide excision repair (NER) the number of gamma-H2AX foci increased steadily. gamma-H2AX foci were preferentially formed in BrdU labeled cells. In proliferation compromised cells, the gamma-H2AX level was significantly reduced, indicating that most of the gamma-H2AX foci induced by UV-C and alkylating agent treatments were replication dependent. The data are in line with the view that DNA damage induced by UV-C light and simple alkylating agents, leads to the formation of DSBs during DNA replication giving rise to H2AX phosphorylation. In replicating NER defective cells, DSBs accumulate due to nonrepaired primary DNA lesions that produce a high level of DSBs during replication. The data support that gamma-H2AX foci are a useful marker of DSBs that are induced by S-phase dependent genotoxins during replication.