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Polycystic ovary syndrome (PCOS) appears to have a genetic predisposition and a fetal origin. We compared the expression levels of 25 PCOS candidate genes from adult control and PCOS human ovaries (n = 16) using microarrays. Only one gene was potentially statistically different. Using qRT-PCR, expression of PCOS candidate genes was examined in bovine fetal ovaries from early stages when they first developed stroma through to completion of development (n = 27; 60–270 days of gestation). The levels of ERBB3 mRNA negatively correlated with gestational age but positively with HMGA2, FBN3, TOX3, GATA4, and DENND1A.X1,2,3,4, previously identified as correlated with each other and expressed early. PLGRKT and ZBTB16, and less so IRF1, were also correlated with AMH, FSHR, AR, INSR, and TGFB1I1, previously identified as correlated with each other and expressed late. ARL14EP, FDFT1, NEIL2, and MAPRE1 were expressed across gestation and not correlated with gestational age as shown previously for THADA, ERBB4, RAD50, C8H9orf3, YAP1, RAB5B, SUOX, and KRR1. LHCGR, because of its unusual bimodal expression pattern, had some unusual correlations with other genes. In human ovaries (n = 15

When first formed, the ovary only has an established epithelium at its base or hilum. Later, an epithelium is established around the rest of the ovary. To examine this further, we conducted scanning electron microscopy of the surface of bovine fetal ovaries and immunohistochemistry of ovarian cross-sections. From the earliest time point, the cells on the surface of the base or hilum of the ovary were cuboidal. On the remainder of the ovary, the surface was more irregular. By mid-development, the surface was covered completely with either a stratified or simple epithelium of cuboidal cells. Clefts were observed in the surface and appeared to form due to the expansion of stroma surrounding each open ovigerous cord, elevating the areas surrounding each cord, while leaving the opening of the cord to form the base of each cleft. The continued expansion of the surrounding stroma below the surface appeared not only to close the ovigerous cords from the surface but to compress the clefts into the shape of a groove. Later, most of the ovarian surface was covered with a simple cuboidal epithelium. The changes to the ovarian surface during fetal development coincide with the remodeling of the stroma and cords below.

Phosphorylation of histone H2AX is a major contributor to efficient DNA repair. We recently reported neurobehavioral deficits in mice lacking H2AX. Here we establish that this neural failure stems from impairment of mitochondrial function and repression of the mitochondrial biogenesis gene PGC-1α. H2AX loss leads to reduced levels of the major subunits of the mitochondrial respiratory complexes in mouse embryonic fibroblasts and in the striatum, a brain region particularly vulnerable to mitochondrial damage. These defects are substantiated by disruption of the mitochondrial shape in H2AX mutant cells. Ectopic expression of PGC-1α restores mitochondrial oxidative phosphorylation complexes and mitigates cell death. H2AX knockout mice display increased neuronal death in the brain when challenged with 3-nitropronionic acid, which targets mitochondria. This study establishes a role for H2AX in mitochondrial homeostasis associated with neuroprotection.

Meiosis is a defining characteristic of eukaryotes, believed to have evolved only once, over one billion years ago. While the general progression of meiotic events is conserved across multiple diverse organisms, the specific pathways and proteins involved can be highly divergent, even within species from the same genus. Here we investigate the rapid evolution of Matrimony (Mtrm), a female meiosis-specific regulator of Polo kinase (Polo) in Drosophila. Mtrm physically interacts with Polo and is required to restrict the activity of Polo during meiosis. Despite Mtrm’s critical role in meiosis, sequence conservation within the genus Drosophila is poor. To explore the functional significance of this rapid divergence, we expressed Mtrm proteins from 12 different Drosophila species in the Drosophila melanogaster female germline. Distantly related Mtrm homologs are able to both physically interact with D. melanogaster Polo and rescue the meiotic defects seen in mtrm mutants. However, these distant homologs are not properly degraded after the completion of meiosis. Rather, they continue to inhibit Polo function in the early embryo, resulting in dominant maternal-effect lethality. We show that the ability of Mtrm to be properly degraded, and thus release Polo, is partially due to residues or motifs found within Mtrm’s least-conserved regions. We hypothesize that, while Mtrm regions critical for its meiotic function are under strong purifying selection, changes that occurred in its unconserved regions may have been advantageous, potentially by affecting the timing or duration of meiosis and/or the early embryonic divisions.

ATM drives DNA repair by phosphorylating the histone variant H2AX. While ATM mutations elicit prominent neurobehavioral phenotypes, neural roles for H2AX have been elusive. We report impaired motor learning and balance in H2AX-deficient mice. Mitigation of reactive oxygen species (ROS) with N-acetylcysteine (NAC) reverses the behavioral deficits. Mouse embryonic fibroblasts deficient for H2AX exhibit increased ROS production and failure to activate the antioxidant response pathway controlled by the transcription factor NRF2. The NRF2 targets GCLC and NQO1 are depleted in the striatum of H2AX knockouts, one of the regions most vulnerable to ROS-mediated damage. These findings establish a role for ROS in the behavioral deficits of H2AX knockout mice and reveal a physiologic function of H2AX in mediating influences of oxidative stress on NRF2-transcriptional targets and behavior., H2AX is a histone variant with an essential function in DNA double-strand break repair and genome stability. Here, Weyemi and colleagues show that loss of neuronal H2AX leads to locomotor dysfunction and alteration in oxidative stress response.

Studies suggest that exposure to potential endocrine disrupting chemicals (pEDCs) may contribute to adverse health outcomes, but pEDC exposures among firefighters have not been fully characterized. Previously, we demonstrated the military-style silicone dog tag as a personal passive sampling device for assessing polycyclic aromatic hydrocarbon exposures among structural firefighters. This follow-up analysis examined the pEDC exposures based on department call volume, duty shift, and questionnaire variables. Structural firefighters (n = 56) were from one high and one low fire call volume department (Kansas City, MO metropolitan area) and wore separate dog tags while on- and off-duty (ndogtags = 110). The targeted 1530 analyte semi-quantitative screening method was conducted using gas chromatography mass spectrometry (npEDCs = 433). A total of 47 pEDCs were detected, and several less-frequently-detected pEDCs (75%), off-duty pEDC concentrations were strongly correlated (r = 0.31–0.82, p < 0.05), suggesting co-applications of phthalates and fragrances in consumer products. Questionnaire variables of “regular use of conventional cleaning products” and “fireplace in the home” were associated with select elevated pEDC concentrations by duty shift (paired t-test). This suggested researchers should include detailed questions about consumer product use and home environment when examining personal pEDC exposures.

H2A.J is a poorly studied mammalian-specific variant of histone H2A. We used immunohistochemistry to study its localization in various human and mouse tissues. H2A.J showed cell-type specific expression with a striking enrichment in luminal epithelial cells of multiple glands including those of breast, prostate, pancreas, thyroid, stomach, and salivary glands. H2A.J was also highly expressed in many carcinoma cell lines and in particular, those derived from luminal breast and prostate cancer. H2A.J thus appears to be a novel marker for luminal epithelial cancers. Knocking-out the H2AFJ gene in T47D luminal breast cancer cells reduced the expression of several estrogen-responsive genes which may explain its putative tumorigenic role in luminal-B breast cancer.

The Fukushima Daiichi Nuclear Power Plant (FNPP) accident, the largest nuclear incident since the 1986 Chernobyl disaster, occurred when the plant was hit by a tsunami triggered by the Great East Japan Earthquake on March 11, 2011. The subsequent uncontrolled release of radioactive substances resulted in massive evacuations in a 20-km zone. To better understand the biological consequences of the FNPP accident, we have been measuring DNA damage levels in cattle in the evacuation zone. DNA damage was evaluated by assessing the levels of DNA double-strand breaks in peripheral blood lymphocytes by immunocyto-fluorescence-based quantification of γ-H2AX foci. A greater than two-fold increase in the fraction of damaged lymphocytes was observed in all animal cohorts within the evacuation zone, and the levels of DNA damage decreased slightly over the 700-day sample collection period. While the extent of damage appeared to be independent of the distance from the accident site and the estimated radiation dose from radiocesium, we observed age-dependent accumulation of DNA damage. Thus, this study, which was the first to evaluate the biological impact of the FNPP accident utilizing the γ-H2AX assays, indicated the causal relation between high levels of DNA damage in animals living in the evacuation zone and the FNPP accident.

Polo-like kinases (PLKs) have numerous roles in both mitosis and meiosis, including functions related to chromosome segregation, cohesin removal, and kinetochore orientation [1-7]. PLKs require specific regulation during meiosis to control those processes. Genetic studies demonstrate that the Drosophila PLK Polo kinase (Polo) is inhibited by the female meiosis-specific protein Matrimony (Mtrm) in a stoichiometric manner [8]. Drosophila Polo localizes strongly to kinetochores and to central spindle microtubules during prometaphase and metaphase I of female meiosis [9, 10]. Mtrm protein levels increase dramatically after nuclear envelope breakdown [11]. We show that Mtrm is enriched along the meiotic spindle and that loss of mtrm results in mislocalization of the catalytically active form of Polo. The mtrm gene is haploinsufficient, and heterozygosity for mtrm (mtrm/+) results in high levels of achiasmate chromosome missegregation [8, 12]. In mtrm/+ heterozygotes, there is a low level of sister centromere separation, as well as precocious loss of cohesion along the arms of achiasmate chromosomes. However, mtrm-null females are sterile [13], and sister chromatid cohesion is abolished on all chromosomes, leading to a failure to properly congress or orient chromosomes in metaphase I. These data demonstrate a requirement for the inhibition of Polo, perhaps by sequestering Polo to the microtubules during Drosophila melanogaster female meiosis and suggest that catalytically active Polo is a distinct subset of the total Polo population within the oocyte that requires its own regulation.

Polycystic ovary syndrome (PCOS) affects around 10% of young women, with adverse consequences on fertility and cardiometabolic outcomes. PCOS appears to result from a genetic predisposition interacting with developmental events during fetal or perinatal life. We hypothesised that PCOS candidate genes might be expressed in the fetal ovary when the stroma develops; mechanistically linking the genetics, fetal origins and adult ovarian phenotype of PCOS. In bovine fetal ovaries (n = 37) of 18 PCOS candidate genes only SUMO1P1 was not expressed. Three patterns of expression were observed: early gestation (FBN3, GATA4, HMGA2, TOX3, DENND1A, LHCGR and FSHB), late gestation (INSR, FSHR, and LHCGR) and throughout gestation (THADA, ERBB4, RAD50, C8H9orf3, YAP1, RAB5B, SUOX and KRR1). A splice variant of FSHB exon 3 was also detected early in the bovine ovaries, but exon 2 was not detected. Three other genes, likely to be related to the PCOS aetiology (AMH, AR and TGFB1I1), were also expressed late in gestation. Significantly within each of the three gene groups, the mRNA levels of many genes were highly correlated with each other, despite, in some instances, being expressed in different cell types. TGFβ is a well-known stimulator of stromal cell replication and collagen synthesis and TGFβ treatment of cultured fetal ovarian stromal cells inhibited the expression of INSR, AR, C8H9orf3 and RAD50 and stimulated the expression of TGFB1I1. In human ovaries (n = 15, < 150 days gestation) many of the same genes as in bovine (FBN3, GATA4, HMGA2, FSHR, DENND1A and LHCGR but not TOX3 or FSHB) were expressed and correlated with each other. With so many relationships between PCOS candidate genes during development of the fetal ovary, including TGFβ and androgen signalling, we suggest that future studies should determine if perturbations of these genes in the fetal ovary can lead to PCOS in later life.

During ovarian development stroma from the mesonephros penetrates and expands into the ovarian primordium and thus appears to be involved, at least physically, in the formation of ovigerous cords, follicles and surface epithelium. Cortical stromal development during gestation in bovine fetal ovaries (n=27) was characterised by immunohistochemistry and by mRNA analyses. Stroma was identified by immunostaining of stromal matrix collagen type I and proliferating cells were identified by Ki67 expression. The cortical and medullar volume expanded across gestation, with the rate of cortical expansion slowing over time. During gestation, the proportion of stroma in the cortex and total volume in the cortex significantly increased (P0.05). The expression levels of 12 genes out of 18 examined, including osteoglycin (OGN) and lumican (LUM), were significantly increased later in development (P

Ataxia telangiectasia (A-T), a rare autosomal recessive disorder characterized by progressive cerebellar degeneration and a greatly increased incidence of cancer among other symptoms, is caused by a defective or missing ataxia telangiectasia mutated (ATM) gene. The ATM protein has roles in DNA repair and in the regulation of reactive oxygen species (ROS). Here, we provide, to our knowledge, the first evidence that NADPH oxidase 4 (NOX4) is involved in manifesting A-T disease. We showed that NOX4 expression levels are higher in A-T cells, and that ATM inhibition leads to increased NOX4 expression in normal cells. A-T cells exhibit elevated levels of oxidative DNA damage, DNA double-strand breaks and replicative senescence, all of which are partially abrogated by down-regulation of NOX4 with siRNA. Sections of degenerating cerebelli from A-T patients revealed elevated NOX4 levels. ATM-null mice exhibit A-T disease but they die from cancer before the neurological symptoms are manifested. Injecting Atm-null mice with fulvene-5, a specific inhibitor of NOX4 and NADPH oxidase 2 (NOX2), decreased their elevated cancer incidence to that of the controls. We conclude that, in A-T disease in humans and mice, NOX4 may be critical mediator and targeting it will open up new avenues for therapeutic intervention in neurodegeneration.

Studies of selenium (Se) status indicate that Se is necessary for fertility but how precisely is not known. We aimed to show that Se was important in bovine female reproductive function. The elemental distribution in the bovine ovary (n = 45 sections) was identified by X-ray fluorescence (XRF) imaging. Se was consistently localized to the granulosa cell layer of large (>10 mm) healthy follicles. Inductively Coupled Plasma – Mass Spectrometry revealed tenfold higher Se in the bovine follicle wall compared to corpora lutea. Gene expression analysis of selenoprotein genes GPX1, GPX3, VIMP and SELM in bovine granulosa cells revealed that only GPX1 was significantly up-regulated in large healthy follicles compared to the small healthy or atretic follicles (P < 0.05). Western immunoblotting identified GPX1 protein in bovine granulosa cells of large healthy follicles, but not of small healthy follicles. To assess if GPX1 was important in human follicles, cumulus cells from women undergoing IVF/ICSI with single embryo transfer were collected. Oocytes and embryos were cultured and transferred independently in 30 patients undergoing elective single embryo transfer. Gene expression of GPX1 was significantly higher in human cumulus cells from cumulus–oocyte complexes yielding a pregnancy (P < 0.05). We present the first XRF imaging of mammalian ovaries showing that Se is consistently localized to the granulosa cells of large healthy follicles. We conclude that Se and selenoproteins are elevated in large healthy follicles and may play a critical role as an antioxidant during late follicular development.

The senescence of mammalian cells is characterized by a proliferative arrest in response to stress and the expression of an inflammatory phenotype. Here we show that histone H2A.J, a poorly studied H2A variant found only in mammals, accumulates in human fibroblasts in senescence with persistent DNA damage. H2A.J also accumulates in mice with aging in a tissue-specific manner and in human skin. Knock-down of H2A.J inhibits the expression of inflammatory genes that contribute to the senescent-associated secretory phenotype (SASP), and over expression of H2A.J increases the expression of some of these genes in proliferating cells. H2A.J accumulation may thus promote the signalling of senescent cells to the immune system, and it may contribute to chronic inflammation and the development of aging-associated diseases., Senescence of mammalian cells is characterized by proliferative arrest and expression of an inflammatory phenotype. Here the authors show the H2A variant H2A.J, found only in mammals, accumulates following persistent DNA damage or natural aging.

Recently we found that mice bearing subcutaneous non-metastatic tumors exhibited elevated levels of two types of complex DNA damage, i.e., double-strand breaks and oxidatively-induced clustered DNA lesions in various tissues throughout the body, both adjacent to and distant from the tumor site. This DNA damage was dependent on CCL2, a cytokine involved in the recruitment and activation of macrophages, suggesting that this systemic DNA damage was mediated via tumor-induced chronic inflammatory responses involving cytokines, activation of macrophages, and consequent free radical production. If free radicals are involved, then a diet containing an antioxidant may decrease the distant DNA damage. Here we repeated our standard protocol in cohorts of two syngeneic tumor-bearing C57BL/6NCr mice that were on a Tempol-supplemented diet. We show that double-strand break and oxidatively-induced clustered DNA lesion levels were considerably decreased, about two- to three fold, in the majority of tissues studied from the tumor-bearing mice fed the antioxidant Tempol compared to the control tumor-bearing mice. Similar results were also observed in nude mice suggesting that the Tempol effects are independent of functioning adaptive immunity. This is the first in vivo study demonstrating the effect of a dietary antioxidant on abscopal DNA damage in tissues distant from a localized source of genotoxic stress. These findings may be important for understanding the mechanisms of genomic instability and carcinogenesis caused by chronic stress-induced systemic DNA damage and for developing preventative strategies.

There is a paucity of large animal models to study both the extent and the health risk of ionizing radiation exposure in humans. One promising candidate for such a model is the minipig. Here, we evaluate the minipig for its potential in γ-H2AX-based biodosimetry after exposure to ionizing radiation using both Cs137 and Co60 sources. γ-H2AX foci were enumerated in blood lymphocytes and normal fibroblasts of human and porcine origin after ex vivo g-ray irradiation. DNA double-strand break repair kinetics in minipig blood lymphocytes and fibroblasts, based on the γ-H2AX assay, were similar to those observed in their human counterparts. To substantiate the similarity observed between the human and minipig we show that minipig fibroblast radiosensitivity was similar to that observed with human fibroblasts. Finally, a strong γ-H2AX induction was observed in blood lymphocytes following minipig total body irradiation. Significant responses were detected 3 days after 1.8 Gy and 1 week after 3.8 and 5 Gy with residual γ-H2AX foci proportional to the initial radiation doses. These findings show that the Gottingen minipig provides a useful in vivo model for validation of γ-H2AX biodosimetry for dose assessment in humans.

Phosphorylated H2AX (γ-H2AX) is a sensitive marker for DNA double-strand breaks (DSBs), but the variability of H2AX expression in different cell and tissue types makes it difficult to interpret the meaning of the γ-H2AX level. Furthermore, the assays commonly used for γ-H2AX detection utilize laborious and low-throughput microscopy-based methods. We describe here an ELISA assay that measures both phosphorylated H2AX and total H2AX absolute amounts to determine the percentage of γ-H2AX, providing a normalized value representative of the amount of DNA damage. We demonstrate the utility of the assay to measure DSBs introduced by either ionizing radiation or DNA-damaging agents in cultured cells and in xenograft models. Furthermore, utilizing the NCI-60 cancer cell line panel, we show a correlation between the basal fraction of γ-H2AX and cellular mutation levels. This additional application highlights the ability of the assay to measure γ-H2AX levels in many extracts at once, making it possible to correlate findings with other cellular characteristics. Overall, the γ-H2AX ELISA represents a novel approach to quantifying DNA damage, which may lead to a better understanding of mutagenic pathways in cancer and provide a useful biomarker for monitoring the effectiveness of DNA-damaging anticancer agents.

Allelic variation at the Cu–Zn superoxide dismutase (SOD1) locus has been shown to be associated with resistance of the snail, Biomphalaria glabrata, to infection by the trematode parasite, Schistosoma mansoni. SOD1 catalyses the production of hydrogen peroxide, a known cytotoxic component of the oxidative burst used in defence against pathogens. In our laboratory population of B. glabrata, the most resistant allele at SOD1 is over-expressed relative to the other two alleles. Because hydrogen peroxide also causes oxidative stress on host tissues, we hypothesised that over-expression of SOD1 might be compensated by epistatic interactions with other loci involved in oxidation–reduction (redox) pathways. Catalase, peroxiredoxins and glutathione peroxidases all degrade hydrogen peroxide. We tested whether alleles at each of these loci were in linkage disequilibrium with SOD1 in our population, as might be expected given strong epistatic selection. We found that SOD1, catalase (CAT) and a peroxiredoxin locus (PRX4) are in strong linkage disequilibrium in our population. We also found that these loci are tightly linked, within 1–2 cM of each other, which explains the high linkage disequilibrium. This result raises the possibility that there is a linked cluster of redox genes, and perhaps other defence-relevant genes, in the B. glabrata genome. Whether epistatic interactions for fitness actually exist among these loci still needs to be tested. However the close physical linkage among SOD1, PRX4 and CAT, and subsequent high disequilibrium, makes such interactions a plausible hypothesis.

Upon DNA damage or other stressors, the tumor suppressor p53 is activated, leading to transient expression of the cyclin-dependent kinase inhibitor (CKI) p21. This either triggers momentary G1 cell cycle arrest or leads to a chronic state of senescence or apoptosis, a form of genome guardianship. In the clinic, the presence of p21 has been considered an indicator of wildtype p53 activity. However, recent evidence suggests that p21 also acts as an oncogenic factor in a p53-deficient environment. Here, we discuss the controversial aspects of the two-faced involvement of p21 in cancer and speculate on how this new information may increase our understanding of its role in cancer pathogenesis. Prevailing notions indicate that p21 might also act as antiapoptotic agent, which may have relevant implications for future therapeutic strategies.

Fibrillins 1–3 are stromal extracellular matrix proteins that play important roles in regulating TGFβ activity, which stimulates fibroblasts to proliferate and synthesize collagen. In the developing ovary, the action of stroma is initially necessary for the formation of ovigerous cords and subsequently for the formation of follicles and the surface epithelium of the ovary.FBN3is highly expressed only in early ovarian development and then it declines. In contrast,FBN1and2are upregulated in later ovarian development. We examined the expression ofFBN1–3in bovine and human fetal ovaries. We used cell dispersion and monolayer culture, cell passaging and tissue culture. Cells were treated with growth factors, hormones or inhibitors to assess the regulation of expression ofFBN1–3. When bovine fetal ovarian tissue was cultured,FBN3expression declined significantly. Treatment with TGFβ-1 increasedFBN1andFBN2expression in bovine fibroblasts, but did not affectFBN3expression. Additionally, in cultures of human fetal ovarian fibroblasts (9–17weeks gestational age), the expression ofFBN1andFBN2increased with passage, whereasFBN3dramatically decreased. Treatment with activin A and a TGFβ family signaling inhibitor, SB431542, differentially regulated the expression of a range of modulators of TGFβ signaling and of other growth factors in cultured human fetal ovarian fibroblasts suggesting that TGFβ signaling is differentially involved in the regulation of ovarian fibroblasts. Additionally, since the changes inFBN1–3expression that occurin vitroare those that occur with increasing gestational agein vivo, we suggest that the fetal ovarian fibroblasts maturein vitro.

The epithelial–mesenchymal transition (EMT), considered essential for metastatic cancer, has been a focus of much research, but important questions remain. Here, we show that silencing or removing H2A.X, a histone H2A variant involved in cellular DNA repair and robust growth, induces mesenchymal-like characteristics including activation of EMT transcription factors, Slug and ZEB1, in HCT116 human colon cancer cells. Ectopic H2A.X re-expression partially reverses these changes, as does silencing Slug and ZEB1. In an experimental metastasis model, the HCT116 parental and H2A.X-null cells exhibit a similar metastatic behaviour, but the cells with re-expressed H2A.X are substantially more metastatic. We surmise that H2A.X re-expression leads to partial EMT reversal and increases robustness in the HCT116 cells, permitting them to both form tumours and to metastasize. In a human adenocarcinoma panel, H2A.X levels correlate inversely with Slug and ZEB1 levels. Together, these results point to H2A.X as a regulator of EMT., The histone H2A variants are involved in DNA repair, gene regulation and cancer development. In this study, the authors unravel an additional role for H2A.X in the regulation of mesenchymal-like traits and activation of the EMT transcription factors, Slug and ZEB1, in colon cancer cells.

Direct cellular DNA damage may lead to genome destabilization in unexposed, bystander, cells sharing the same milieu with directly damaged cells by means of the bystander effect. One proposed mechanism involves double strand break (DSB) formation in S phase cells at sites of single strand lesions in the DNA of replication complexes, which has a more open structure compared with neighboring DNA. The DNA in transcription complexes also has a more open structure, and hence may be susceptible to bystander DSB formation from single strand lesions. To examine whether transcription predisposes non-replicating cells to bystander effect-induced DNA DSBs, we examined two types of primary cells that exhibit high levels of transcription in the absence of replication, rat neurons and human lymphocytes. We found that non-replicating bystander cells with high transcription rates exhibited substantial levels of DNA DSBs, as monitored by γ-H2AX foci formation. Additionally, as reported in proliferating cells, TGF-β and NO were found to mimic bystander effects in cell populations lacking DNA synthesis. These results indicate that cell vulnerability to bystander DSB damage may result from transcription as well as replication. The findings offer insights into which tissues may be vulnerable to bystander genomic destabilization in vivo.

Meiotic recombination, which is necessary to ensure that homologous chromosomes segregate properly, begins with the induction of meiotic DNA double-strand breaks (DSBs) and ends with the repair of a subset of those breaks into crossovers. Here we investigate the roles of two paralogous genes, CG12200 and CG31053, which we have named Narya and Nenya, respectively, due to their relationship with a structurally similar protein named Vilya. We find that narya recently evolved from nenya by a gene duplication event, and we show that these two RING finger domain-containing proteins are functionally redundant with respect to a critical role in DSB formation. Narya colocalizes with Vilya foci, which are known to define recombination nodules, or sites of crossover formation. A separation-of-function allele of narya retains the capacity for DSB formation but cannot mature those DSBs into crossovers. We further provide data on the physical interaction of Narya, Nenya and Vilya, as assayed by the yeast two-hybrid system. Together these data support the view that all three RING finger domain-containing proteins function in the formation of meiotic DNA DSBs and in the process of crossing over., Author summary Errors in chromosome segregation during meiosis are the leading cause of miscarriages and can result in genetic abnormalities like Down syndrome or Turner syndrome. For chromosomes to segregate faithfully, they must recombine with their homolog during the early steps of meiosis. An essential component of the process of meiotic recombination is creating the lesions (double-strand breaks, DSBs) that are required to form a crossover with the homologous chromosome. Crossovers are required to ensure chromosomes segregate properly at the first meiotic division. In this study we have identified two genes, narya and nenya, that are essential in DSB formation. We found that narya arose from a duplication of nenya, and these two genes are functionally redundant. In addition to its role in DSB formation, narya also plays a role in processing DSBs into crossovers. Strengthening our knowledge about the mechanism by which Narya both creates DSBs and processes them into crossovers will lead to a better understanding of the process of meiotic chromosome segregation not only in flies but many other organisms, as these genes have homologs in yeast, worms, plants, mice and humans.

Purpose: Phosphorylated histone H2AX (γH2AX) serves as a biomarker for formation of DNA double-strand break repair complexes. A quantitative pharmacodynamic immunofluorescence assay for γH2AX was developed, validated, and tested in human tumor xenograft models with the use of clinically relevant procedures. Experimental Design: The γH2AX immunofluorescence assay uses a novel data quantitation and image processing algorithm to determine the extent of nuclear-specific γH2AX staining in tumor needle biopsies and hair follicles collected from mice bearing topotecan-responsive A375 xenografts. After method validation with the topoisomerase I (Top1) inhibitor topotecan, the assay was used to compare pharmacodynamic properties of three structurally related indenoisoquinoline Top1 inhibitors. Results: γH2AX response to topotecan was quantified over a 60-fold dose range (0.016-1.0 times the murine single-dose maximum tolerated dose), and significant pharmacodynamic response was measured at the mouse equivalent of the 1.5 mg/m2 clinical dose as well as the lowest dose tested. Responses were within a time window amenable for biopsy collection in clinical trials. These studies enabled characterization of dose and time responses for three indenoisoquinolines, resulting in selection of two for clinical evaluation. γH2AX response to Top1 inhibitors in hair follicles was also observable above a minimal dose threshold. Conclusions: Our γH2AX assay is sufficiently accurate and sensitive to quantify γH2AX in tumor samples and will be used in correlative studies of two indenoisoquinolines in a phase I clinical trial at the National Cancer Institute. Data suggest that hair follicles may potentially serve as a surrogate tissue to evaluate tumor γH2AX response to Top1 inhibitors. Clin Cancer Res; 16(22); 5447–57. ©2010 AACR.

Tumor cells are often deficient in DNA damage response (DDR) pathways, and anticancer therapies are commonly based on genotoxic treatments using radiation and/or drugs that damage DNA directly or interfere with DNA metabolism, leading to the formation of DNA double-strand breaks (DSB), and ultimately to cell death. Because DSBs induce the phosphorylation of histone H2AX (γH2AX) in the chromatin flanking the break site, an antibody directed against γH2AX can be employed to measure DNA damage levels before and after patient treatment. Poly(ADP-ribose) polymerases (PARP1 and PARP2) are also activated by DNA damage, and PARP inhibitors show promising activity in cancers with defective homologous recombination (HR) pathways for DSB repair. Ongoing clinical trials are testing combinations of PARP inhibitors with DNA damaging agents. Poly(ADP-ribosylation), abbreviated as PAR, can be measured in clinical samples and used to determine the efficiency of PARP inhibitors. This review summarizes the roles of γH2AX and PAR in the DDR, and their use as biomarkers to monitor drug response and guide clinical trials, especially phase 0 clinical trials. We also discuss the choices of relevant samples for γH2AX and PAR analyses. Clin Cancer Res; 16(18); 4532–42. ©2010 AACR.

The maintenance of genome stability requires efficient DNA double-stranded break (DSB) repair mediated by the phosphorylation of multiple histone H2AX molecules near the break sites. The phosphorylated H2AX (gammaH2AX) molecules form foci covering many megabases of chromatin. The formation of gamma-H2AX foci is critical for efficient DNA damage response (DDR) and for the maintenance of genome stability, however, the mechanisms of protein organization in foci is largely unknown. To investigate the nature of gammaH2AX foci formation, we analyzed the distribution of gammaH2AX and other DDR proteins at DSB sites using a variety of techniques to visualize, expand and partially disrupt chromatin. We report here that gammaH2AX foci change composition during the cell cycle, with proteins 53BP1, NBS1 and MRE11 dissociating from foci in G(2) and mitosis to return at the beginning of the following G(1). In contrast, MDC1 remained colocalized with gamma-H2AX during mitosis. In addition, while gammaH2AX was found to span large domains flanking DSB sites, 53BP1 and NBS1 were more localized and MDC1 colocalized in doublets in foci. H2AX and MDC1 were found to be involved in chromatin relaxation after DSB formation. Our data demonstrates that the DSB repair focus is a heterogeneous and dynamic structure containing internal complexity.

Despite the mouse being an important laboratory species, little is known about changes in its extracellular matrix (ECM) during follicle and corpora lutea formation and regression. Follicle development was induced in mice (29 days of age/experimental day 0) by injections of pregnant mare's serum gonadotrophin on days 0 and 1 and ovulation was induced by injection of human chorionic gonadotrophin on day 2. Ovaries were collected for immunohistochemistry (n=10 per group) on days 0, 2 and 5. Another group was mated and ovaries were examined on day 11 (n=7). Collagen type IV alpha1 and alpha2, laminin alpha1, beta1 and gamma1 chains, nidogens 1 and 2 and perlecan were present in the follicular basal lamina of all developmental stages. Collagen type XVIII was only found in basal lamina of primordial, primary and some preantral follicles, whereas laminin alpha2 was only detected in some preantral and antral follicles. The focimatrix, a specialised matrix of the membrana granulosa, contained collagen type IV alpha1 and alpha2, laminin alpha1, beta1 and gamma1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. In the corpora lutea, staining was restricted to capillary sub-endothelial basal laminas containing collagen type IV alpha1 and alpha2, laminin alpha1, beta1 and gamma1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. Laminins alpha4 and alpha5 were not immunolocalised to any structure in the mouse ovary. The ECM composition of the mouse ovary has similarities to, but also major differences from, other species with respect to nidogens 1 and 2 and perlecan.

Upon DNA double-strand break (DSB) induction in mammals, the histone H2A variant, H2AX, becomes rapidly phosphorylated at serine 139. This modified form, termed gamma-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 gamma-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 gamma-H2AX detection may become a powerful tool for monitoring genotoxic events associated with cancer development and tumor progression.

Background New strategies to combat the global scourge of schistosomiasis may be revealed by increased understanding of the mechanisms by which the obligate snail host can resist the schistosome parasite. However, few molecular markers linked to resistance have been identified and characterized in snails. Methodology/Principal Findings Here we test six independent genetic loci for their influence on resistance to Schistosoma mansoni strain PR1 in the 13-16-R1 strain of the snail Biomphalaria glabrata. We first identify a genomic region, RADres, showing the highest differentiation between susceptible and resistant inbred lines among 1611 informative restriction-site associated DNA (RAD) markers, and show that it significantly influences resistance in an independent set of 439 outbred snails. The additive effect of each RADres resistance allele is 2-fold, similar to that of the previously identified resistance gene sod1. The data fit a model in which both loci contribute independently and additively to resistance, such that the odds of infection in homozygotes for the resistance alleles at both loci (13% infected) is 16-fold lower than the odds of infection in snails without any resistance alleles (70% infected). Genome-wide linkage disequilibrium is high, with both sod1 and RADres residing on haplotype blocks >2Mb, and with other markers in each block also showing significant effects on resistance; thus the causal genes within these blocks remain to be demonstrated. Other candidate loci had no effect on resistance, including the Guadeloupe Resistance Complex and three genes (aif, infPhox, and prx1) with immunological roles and expression patterns tied to resistance, which must therefore be trans-regulated. Conclusions/Significance The loci RADres and sod1 both have strong effects on resistance to S. mansoni. Future approaches to control schistosomiasis may benefit from further efforts to characterize and harness this natural genetic variation., Author Summary Aquatic snails transmit schistosome blood flukes, causing a parasitic disease second only to malaria in its global health impact. The mechanisms by which some snails naturally resist infection are poorly understood, but if characterized could enable protocols to interfere with transmission of the disease. Here we identify a region of the snail genome that correlates with resistance to infection, and we examine its effect on immunity jointly with a previously described resistance gene. Variation at other candidate genes has no effect on parasite resistance in this laboratory population. The two resistance regions could serve as targets to block parasite transmission via snails.

X-ray fluorescence (XRF) was used to image 40 histological cross-sections of bovine ovaries (n=19), focusing on structures including: antral follicles at different stages of growth or atresia, corpora lutea at three stages of development (II–IV), and capillaries, arterioles, and other blood vessels. This method identified three key trace elements [iron (Fe), zinc (Zn), and selenium (Se)] within the ovarian tissue which appeared to be localized to specific structures. Owing to minimal preprocessing of the ovaries, important high-resolution information regarding the spatial distribution of these elements was obtained with elemental trends and colocalizations of Fe and Zn apparent, as well as the infrequent appearance of Se surrounding the antrum of large follicles, as previously reported. The ability to use synchrotron radiation to measure trace element distributions in bovine ovaries at such high resolution and over such large areas could have a significant impact on understanding the mechanisms of ovarian development. This research is intended to form a baseline study of healthy ovaries which can later be extended to disease states, thereby improving our current understanding of infertility and endocrine diseases involving the ovary.

Thymus, an important component of hematopoietic tissue, is a well-documented “target” of radiation carcinogenesis. Both acute and fractionated irradiation result in a high risk of leukemia and thymic lymphoma. However, the exact mechanisms underlying radiation-induced predisposition to leukemia and lymphoma are still unknown, and the contributions of genetic and epigenetic mechanisms in particular have yet to be defined. Global DNA hypomethylation is a well-known characteristic of cancer cells. Recent studies have also shown that tumor cells undergo prominent changes in histone methylation, particularly a substantial loss of trimethylation of histone H4-Lys20 and demethylation of genomic DNA. These losses are considered a universal marker of malignant transformation. In the present study, we investigated the effect of low-dose radiation exposure on the accumulation of DNA lesions and alterations of DNA methylation and histone H4-Lys20 trimethylation in the thymus tissue using an in vivo murine model. For the first time, we show that fractionated whole-body application of 0.5 Gy X-ray leads to decrease in histone H4-Lys20 trimethylation in the thymus. The loss of histone H4-Lys20 trimethylation was accompanied by a significant decrease in global DNA methylation as well as the accumulation of DNA damage as monitored by persistence of histone γH2AX foci in the thymus tissue of mice exposed to fractionated irradiation. Altered DNA methylation was associated with reduced expression of maintenance (DNMT1) and, to a lesser extent, de novo DNA methyltransferase DNMT3a in exposed animals. Expression of another de novo DNA methyltransferase DNMT3b was decreased only in males. Irradiation also resulted in ∼20% reduction in the levels of methyl-binding proteins MeCP2 and MBD2. Our results show the involvement of epigenetic alterations in radiation-induced responses in vivo. These changes may play a role in genome destabilization that ultimately leads to cancer.

Histone H2AX is rapidly phosphorylated in the chromatin micro-environment surrounding a DNA double-strand break (DSB). Although H2AX deficiency is not detrimental to life, H2AX is required for the accumulation of numerous essential proteins into irradiation induced foci (IRIF). However, the relationship between IRIF formation, H2AX phosphorylation (gamma-H2AX) and the detection of DNA damage is unclear. Here, we show that the migration of repair and signalling proteins to DSBs is not abrogated in H2AX(-/-) cells, or in H2AX-deficient cells that have been reconstituted with H2AX mutants that eliminate phosphorylation. Despite their initial recruitment to DSBs, numerous factors, including Nbs1, 53BP1 and Brca1, subsequently fail to form IRIF. We propose that gamma-H2AX does not constitute the primary signal required for the redistribution of repair complexes to damaged chromatin, but may function to concentrate proteins in the vicinity of DNA lesions. The differential requirements for factor recruitment to DSBs and sequestration into IRIF may explain why essential regulatory pathways controlling the ability of cells to respond to DNA damage are not abolished in the absence of H2AX.

Cells maintain genomic stability by the coordination of DNA-damage repair and cell-cycle checkpoint control. In replicating cells, DNA damage usually activates intra-S-phase checkpoint controls, which are characterized by delayed S-phase progression and increased Rad53 phosphorylation. We show that in budding yeast, the intra-S-phase checkpoint controls, although functional, are not activated by the topoisomerase I inhibitor camptothecin (CPT). In a CPT-hypersensitive mutant strain that lacks the histone 2A (H2A) phosphatidylinositol-3-OH kinase (PI(3) K) motif at Ser 129 (h2a-s129a), the hypersensitivity was found to result from a failure to process full-length chromosomal DNA molecules during ongoing replication. H2A Ser 129 is not epistatic to the RAD24 and RAD9 checkpoint genes, suggesting a non-checkpoint role for the H2A PI(3) K site. These results suggest that H2A Ser 129 is an essential component for the efficient repair of DNA double-stranded breaks (DSBs) during replication in yeast, particularly of those DSBs that do not induce the intra-S-phase checkpoint.

During meiotic prophase in male mammals, the X and Y chromosomes condense to form a macrochromatin body, termed the sex, or XY, body, within which X- and Y-linked genes are transcriptionally repressed. The molecular basis and biological function of both sex body formation and meiotic sex chromosome inactivation (MSCI) are unknown. A phosphorylated form of H2AX, a histone H2A variant implicated in DNA repair, accumulates in the sex body in a manner independent of meiotic recombination-associated double-strand breaks. Here we show that the X and Y chromosomes of histone H2AX-deficient spermatocytes fail to condense to form a sex body, do not initiate MSCI, and exhibit severe defects in meiotic pairing. Moreover, other sex body proteins, including macroH2A1.2 and XMR, do not preferentially localize with the sex chromosomes in the absence of H2AX. Thus, H2AX is required for the chromatin remodeling and associated silencing in male meiosis.

Translating research advances to natural systems using experimental laboratory studies is often difficult because of the variability between the natural environment and experimental conditions. Because environmental conditions have a large effect on an organism's physiology, responses to stressors like nutrient limitation, temperature, oxygen deprivation, predation, and parasite/pathogen infection are likely to be context dependent. Therefore, it is essential to examine the impact the study environment has on the experimental outcome. Here, we explored the effect of light exposure on susceptibility to parasite infection. The Biomphalaria glabrata / Schistosoma mansoni study system is a well-established model for studying schistosomiasis. It has been general practice to maintain the vector, B. glabrata, in dark conditions after exposure to miracidia of the human pathogen S. mansoni. We evaluated susceptibility of B. glabrata to S. mansoni under 3 different light conditions during the prepatent period, light (125 lx) on a 12-12 cycle, dim light (3 lx) on a 12-12 cycle, and no light (24 hr at 0 lx). We hypothesized that stress due to photoperiod disruption (24 hr of darkness) would result in compromised immune function and lead to higher susceptibility to infection. Prevalence of infected snails differed significantly between the light conditions, and higher susceptibility was observed in the full light and complete dark conditions compared with the low light conditions. The dim conditions are representative of current methods for evaluating susceptibility in this system. Our results indicate that light exposure during the prepatent period can affect infection outcomes, and environmental conditions must therefore be considered when assessing fitness and immune response due to interactions between host genotype and environment.

Reactive oxygen species (ROS) are essential for survival but also pose serious risks to that survival. A particularly striking example was the demonstration in 2003 by the Campisi group that primary mouse fibroblasts have an indefinite proliferative lifespan in 3% oxygen, the amount found in the capillaries feeding the tissues, but greatly shortened ones under normal in vitro culturing conditions, i. e., 20% oxygen [1]. Now, the same group has generated some insights into how oxidative stress contributes to cellular senescence and aging phenotypes in mouse skin [2]. ROS may be produced in a regulated manner during cellular metabolism but they can also arise in an unregulated manner by metabolic dysfunctions and by exogenous stresses. ROS production is typically localized in cellular compartments by NAD(P)H oxidases, lipoxygenases, cyclooxygenases among others as well as by the mitochondrial electron-transport chain [3]. At physiological levels, ROS play beneficial or even essential roles as regulatory mediators in signalling or defence processes, including the promotion of endothelium-dependent vasorelaxation, apoptosis, angiogenesis, erythropoietin production and destruction of bacteria and other foreign substances by macrophages [4]. However, compromised homeostatic pathways lead to elevated ROS levels that may result in the damage of cellular components (i.e., proteins, lipids, DNA). A growing body of evidence implicates oxidative stress in both aging and a wide spectrum of human diseases including diabetes, atherosclerosis, hypertension, cancer, cardiovascular diseases and neurodegenerative diseases among others [5]. The counterpoint of deleterious and useful ROS roles may have provided evolutionary pressure to develop robust pathways for intracellular redox homeostasis including antioxidants such as glutathione peroxidases, peroxiredoxins, catalases and SODs. The first line of defence against ROS appears to be one of the superoxide dismutases, SOD2, (aka manganese superoxide dismutase (MnSOD)). SOD2 is a mitochondrial matrix protein that converts the superoxide anion (O2−) to hydrogen peroxide (H2O2), which is in turn, converted to molecular oxygen and water by catalase and peroxiredoxins. In this Aging paper, the Campisi group showed that normal aged mice exhibit impaired mitochondrial complex II activity and increased frequencies of senescent cells. They observed similar phenotypes in SOD2 knockout (KO) mice at very young ages and also reported that these mice exhibit significant epidermal thinning which is an age-associated phenotype in humans as well as mice. The epidermal thinning appeared to be due to decreased cellular proliferation in the senescent skin tissue rather than increased apoptosis. In addition, senescence in SOD2 KO mouse skin correlated with increased levels of two senescence markers, SA-βgal activity and the p16INK4a tumor suppressor, as well as amplified keratinocyte terminal differentiation (Figure ​(Figure1).1). Moreover, these features are accompanied by the presence of nuclear DNA damage in the epidermis, a characteristic linked to senescence in both mouse and human cells in culture [6]. Figure 1 Scheme illustrating the putative role of SOD2 in skin aging Similar to the observations made in mice, other studies in yeast and Drosophila also show a predominant role for SOD2 in increasing the lifespan [7-9]. However, the evidence of a direct link between aging and elevated ROS levels is not undisputed. For example, mice heterozygous for SOD2 exhibited accrued DNA damage and subsequent predisposition to cancer, but without any impact on the lifespan [10]. In mice with a mutated mitochondrial DNA polymerase, PolG, ROS production was not significantly increased but the mitochondria accumulated mutations which, in turn, resulted in dysfunctions leading to several aging phenotypes and early death [11, 12]. Other studies failed to find a direct link between SOD2 and aging. A study with mice showed that SOD2 overexpression, while resulting in decreased lipid peroxidation, did not alter either lifespan or age-related pathology [13]. In the nematode, depletion of SOD2 increased oxidative stress but lifespan could still be dramatically prolonged [14]. Also, in the human colorectal cancer cell line HCT116, SOD2 overexpression unexpectedly induced growth arrest and increased senescence [15]. These discrepancies may be explained by the differential responses between species and cell types to a rise in ROS levels. The shorter lifespan observed in SOD2 KO mice compared to wild-type and heterozygous mice may be reminiscent of previous studies showing that rates of mitochondrial O2− and H2O2 generation were inversely correlated to maximum life span potential with shorter-lived species producing relatively higher amounts of ROS [16]. Because SOD2 plays a role in aging in mice, it would be interesting to look if a correlation exists between age-related pathologies and SOD2 alterations in human. For example, mitochondrial dysfunction was shown to contribute to the pathogenesis of aging-related neurodegenerative diseases [17]. Moreover, presently unresolved issues include the exact nature of oxidant species involved in the establishment of senescence, and the molecular pathways which link mitochondrial oxidative stress to the appearance of genomic DNA lesions. Further research will clarify how these lesions occur in the nucleus, their relationship to aging and which capabilities of cellular redox homeostasis are most important in minimizing them.

Higher order chromatin structure presents a barrier to the recognition and repair of DNA damage. Double-strand breaks (DSBs) induce histone H2AX phosphorylation, which is associated with the recruitment of repair factors to damaged DNA. To help clarify the physiological role of H2AX, we targeted H2AX in mice. Although H2AX is not essential for irradiation-induced cell-cycle checkpoints, H2AX−/−mice were radiation sensitive, growth retarded, and immune deficient, and mutant males were infertile. These pleiotropic phenotypes were associated with chromosomal instability, repair defects, and impaired recruitment of Nbs1, 53bp1, and Brca1, but not Rad51, to irradiation-induced foci. Thus, H2AX is critical for facilitating the assembly of specific DNA-repair complexes on damaged DNA.

Class switch recombination (CSR) is a region-specific DNA recombination reaction that replaces one immunoglobulin heavy-chain constant region (Ch) gene with another. This enables a single variable (V) region gene to be used in conjunction with different downstream Ch genes, each having a unique biological activity. The molecular mechanisms that mediate CSR have not been defined, but activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is required for this reaction. Here we report that the Nijmegen breakage syndrome protein (Nbs1) and phosphorylated H2A histone family member X (gamma-H2AX, also known as gamma-H2afx), which facilitate DNA double-strand break (DSB) repair, form nuclear foci at the Ch region in the G1 phase of the cell cycle in cells undergoing CSR, and that switching is impaired in H2AX-/- mice. Localization of Nbs1 and gamma-H2AX to the Igh locus during CSR is dependent on AID. In addition, AID is required for induction of switch region (S mu)-specific DNA lesions that precede CSR. These results place AID function upstream of the DNA modifications that initiate CSR.

In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent double-strand breaks (DSBs), a process that precedes homologous synapsis. Here we use an antibody specific for a phosphorylated histone (gamma-H2AX, which marks the sites of DSBs) to investigate the timing, distribution and Spo11-dependence of meiotic DSBs in the mouse. We show that, as in yeast, recombination in the mouse is initiated by Spo11-dependent DSBs that form during leptotene. Loss of gamma-H2AX staining (which in irradiated somatic cells is temporally linked with DSB repair) is temporally and spatially correlated with synapsis, even when this synapsis is 'non-homologous'.

The recent years have witnessed a rapid accumulation of experimental data showing that ionizing radiation elicits a plethora of biological effects in unirradiated cells receiving bystander signals from hit cells. This so-called radiation-induced bystander effect (RIBE) manifests in various ways including changes in gene expression, genetic and epigenetic alterations, as well as increases in cell transformation and cell death. Our group and others found that DNA double-stranded breaks (DSBs), directly measured by the gamma-H2AX focus formation assay, accumulate in bystander cells in a number of experimental systems such as human cultured cells, human three-dimensional tissue models and in mice. In addition, we recently found that various other sources of cell stress, including media from cancerous cells resulted in a DNA damage response (DDR) in normal human cells that is reminiscent of RIBE. These results suggest that the RIBE may be part of a more general stress response, however, the molecular mechanism underpinning the formation of DNA DSBs in bystander cells is still unclear. This extra view points to some possibilities that might explain why DDR in human cells can be observed under bystander conditions.

Nine catfish fillets, three catfish nuggets, two feed samples, and one pond sediment were analyzed for PCDD, PCDF, and PCB. Farm-raised catfish from Mississippi, Alabama, and Arkansas contained significant levels of 2,3,7,8-substituted PCDD and PCDF. In addition, a large number of non-2,3,7,8-substituted congeners were present in all samples. The catfish fillets and catfish nuggets also contained high concentrations of dioxin-like PCB, as well as a number of non-dioxin-like PCB. The TEQ based on PCDD and PCDF ranged from 9.5 to 43.0 pg/g lipid and the TEQ based on PCB ranged from 0.45 to 4.9 pg/g lipid for all catfish samples. The dioxin-like PCB contributed 4-16% to the total TEQ (PCDD/PCDF/PCB) for the catfish samples. The major source for the PCDD, PCDF, and PCB appears to be from feed and not from pond sediment. Immunoreactive CYP1A protein was elevated 2.5 fold in the pond-raised catfish compared to the aquarium-raised one. The results of this study suggest that the PCDD/PCDF are more important than the PCB in the CYP1A induction.

When mammalian cell cultures or mice are exposed to ionizing radiation in survivable or lethal amounts, novel mass components are found in the histone H2A region of two-dimensional gels. Collectively referred to as gamma, these components are formed in vivo by several procedures that introduce double-stranded breaks into DNA. gamma-Components, which appeared to be the only major novel components detected by mass or 32PO4 incorporation on acetic acid-urea-Triton X-100-acetic acid-urea-cetyltrimethylammonium bromide or SDS-acetic acid-urea-cetyltrimethylammonium bromide gels after exposure of cells to ionizing radiation, are shown to be histone H2AX species that have been phosphorylated specifically at serine 139. gamma-H2AX appears rapidly after exposure of cell cultures to ionizing radiation; half-maximal amounts are reached by 1 min and maximal amounts by 10 min. At the maximum, approximately 1% of the H2AX becomes gamma-phosphorylated per gray of ionizing radiation, a finding that indicates that 35 DNA double-stranded breaks, the number introduced by each gray into the 6 x 10(9) base pairs of a mammalian G1 genome, leads to the gamma-phosphorylation of H2AX distributed over 1% of the chromatin. Thus, about 0.03% of the chromatin appears to be involved per DNA double-stranded break. This value, which corresponds to about 2 x 10(6) base pairs of DNA per double-stranded break, indicates that large amounts of chromatin are involved with each DNA double-stranded break. Thus, gamma-H2AX formation is a rapid and sensitive cellular response to the presence of DNA double-stranded breaks, a response that may provide insight into higher order chromatin structures.

Drosophila melanogaster Polo kinase physically interacts with, and is repressed by, the Matrimony (Mtrm) protein during oogenesis. Females heterozygous for a deletion of the mtrm gene display defects in chromosome segregation at meiosis I. However, a complete absence of Mtrm results in both meiotic catastrophe and female sterility. We show that three phosphorylated residues in an N-terminal region in Mtrm are required for Mtrm::Polo binding. However, this binding is noncanonical; it does not require either a complete S-pS/pT-P motif in Mtrm or key residues in the Polo-box domain of Polo that allow Polo to bind phosphorylated substrates. By using fluorescence cross-correlation spectroscopy to characterize the Mtrm::Polo interaction in vivo, we show that a sterile α-motif (SAM) domain located at the C terminus of Mtrm increases the stability of Mtrm::Polo binding. Although Mtrm’s C-terminal SAM domain is not required to rescue the chromosome segregation defects observed in mtrm/+ females, it is essential to prevent both meiotic catastrophe and the female sterility observed in mtrm/mtrm females. We propose that Polo’s interaction with the cluster of phosphorylated residues alone is sufficient to rescue the meiosis I defect. However, the strengthening of Mtrm::Polo binding mediated by the SAM domain is necessary to prevent meiotic catastrophe and ensure female fertility. Characterization of the Mtrm::Polo interaction, as well as that of other Polo regulators, may assist in the design of a new class of Polo inhibitors to be used as targeted anticancer therapeutic agents.

Humans and animals undergo ageing, and although their primary cells undergo cellular senescence in culture, the relationship between these two processes is unclear. Here we show that gamma-H2AX foci (gamma-foci), which reveal DNA double-strand breaks (DSBs), accumulate in senescing human cell cultures and in ageing mice. They colocalize with DSB repair factors, but not significantly with telomeres. These cryptogenic gamma-foci remain after repair of radiation-induced gamma-foci, suggesting that they may represent DNA lesions with unrepairable DSBs. Thus, we conclude that accumulation of unrepairable DSBs may have a causal role in mammalian ageing.

ICA69 (islet cell autoantigen 69 kDa) is a protein implicated in type 1 diabetes mellitus in both the non-obese diabetic (NOD) mouse model and humans. ICA69 is encoded by the Ica1 gene on mouse chromosome 6 A1-A2. We previously reported reduced ICA69 expression in the thymus of NOD mice compared with thymus of several non-diabetic mouse strains. We propose that reduced thymic ICA69 expression could result from variations in transcriptional regulation of the gene and that polymorphisms within the Ica1 core promoter may partially determine this transcriptional variability. We characterized the functional promoter of Ica1 in NOD mice and compared it with the corresponding portions of Ica1 in non-diabetic C57BL/6 mice. Luciferase reporter constructs demonstrated that the NOD Ica1 promoter region exhibited markedly reduced luciferase expression in transiently transfected medullary thymus epithelial (mTEC(+)) and B-cell (M12)-derived cell lines. However, in a non-diabetic strain, C57BL/6, the Ica1 promoter region was transcriptionally active when transiently transfected into the same cell lines. We concomitantly identified five single nucleotide polymorphisms within the NOD Ica1 promoter. One of these single nucleotide polymorphisms increases the binding affinity for the transcription factor AIRE (autoimmune regulator), which is highly expressed in thymic epithelial cells, where it is known to play a key role regulating self-antigen expression. We conclude that polymorphisms within the NOD Ica1 core promoter may determine AIRE-mediated down-regulation of ICA69 expression in medullary thymic epithelial cells, thus providing a novel mechanistic explanation for the loss of immunologic tolerance to this self-antigen in autoimmunity.

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.

The importance of bystander effects is becoming more appreciated, as studies show they may affect the course of cancer and other chronic diseases. The term “bystander effects” refers to changes in naïve cells sharing the same milieu with cells that have been damaged. Bystander cells may be in contact with, or distant from, damaged cells. In addition, it has been shown in culture that not only physically damaged cells, but also cells that have become abnormal (i.e., cancerous or senescent) may induce bystander effects. Recently, we have shown a similar effect in animals. Mice harboring subcutaneous tumors exhibited elevated levels of DNA damage in distant organs. In contrast to cell culture, immune cells seemed to be involved in tumor-induced bystander effects in animals because CCL2-null tumor-bearing mice did not exhibit increased distant DNA damage. Here, we discuss some of the implications of these observations. Cancer Res; 71(10); 3437–41. ©2011 AACR.

The past year has seen considerable developments in the use of the DNA double-strand breaks (DSBs) to evaluate genome alterations in cells undergoing a variety of genotoxic stresses in vitro and in vivo. When the γ -H2AX foci which mark the DSBs are stained, individual breaks are detectible, making the assay suitable for situations requiring great sensitivity. While the methods for the detection of γ -H2AX foci are still evolving, particularly for in vivo detection, the basic assay has proven to be useful in several diverse areas of research. We will highlight recent developments of the assay in four areas: radiation biodosimetry, the evaluation or validation of new cancer drugs in clinical studies, chronic inflammation, and environmental genotoxicity.

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.

Measurement of DNA double-strand break (DSB) levels in cells is useful in many research areas, including those related to DNA damage and repair, tumorigenesis, anti-cancer drug development, apoptosis, radiobiology, environmental effects, and aging, as well as in the clinic. DSBs can be detected in the nuclei of cultured cells and tissues with an antibody to H2AX phosphorylated on serine residue 139 (γ-H2AX). DSB levels can be obtained either by measuring overall γ-H2AX protein levels in a cell population or by counting γ-H2AX foci in individual nuclei. Total levels can be obtained in extracts of cell populations by immunoblot analysis, and in cell populations by flow cytometry. Furthermore, with flow cytometry, the cell cycle distribution of a population can be obtained in addition to DSB levels, which is an advantage when studying anti-cancer drugs targeting replicating tumor cells. These described methods are used in genotoxicity assays of compounds of interest or in analyzing DSB repair after exposure to drugs or radiation. Immunocyto/immunohistochemical analysis can detect γ-H2AX foci in individual cells and is very sensitive (a single DSB can be visualized), permitting the use of extremely small samples. Measurements of γ-H2AX focal numbers can reveal subtle changes found in the radiation-induced tissue bystander response, low dose radiation exposure, and in cells with mutations in genomic stability maintenance pathways. In addition, marking DNA DSBs in a nucleus with γ-H2AX is a powerful tool to identify novel DNA repair proteins by their abilities to co-localize with γ-H2AX foci at the DSB site. This chapter presents techniques for γ-H2AX detection in a variety of human and mouse samples.