Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P16104 (H2AX)
 3,930 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the repair of clustered lesions within the DNA/chromatin, the focus formation and persistence of foci of the phosphorylated histone protein H2AX and the repair protein MRE11 were studied in normal cells and in cells lacking DNA-PKcs (M059J) or ATM (GM2052D) after irradiation with high-LET nitrogen ions or low-LET photons. There was a rapid formation of MRE11 and gamma-H2AX foci, and 0.5 h after high-LET irradiation, the number of foci in normal cells correlated well with the number of particle hits per cell nucleus. After 8 h of repair, there were significantly more gamma-H2AX foci than MRE11 foci remaining in the normal cells, independent of radiation quality. The difficulty in repairing clustered breaks was detected as slower rejoining of DSBs (measured by DNA fragmentation analysis), as quantification of the amount of gamma-H2AX over time, and as a larger fraction of repair foci remaining after 24 h in cells irradiated with high- LET ions. These data indicate that clustered lesions are repaired by a pathway involving the same proteins that repair sparsely distributed breaks. Further, for both low- and high- LET radiation, no reduction of the initial number of gamma-H2AX and MRE11 foci was detected in M059J cells up to 21 h after irradiation, which was in accordance with a complete absence of DSB rejoining in these cells. In the GM2052D cells there was also a higher level of foci remaining after 21 h; however, this was not accompanied by unrejoined DSBs, indicating that these foci not only represent DSBs but also may be a sign of persistent problems even when breaks are rejoined.
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PMID:Focus formation of DNA repair proteins in normal and repair-deficient cells irradiated with high-LET ions. 1516 72

Recently, cytolethal distending toxin V (CDT-V), a new member of the CDT family, was identified in Shiga toxin-producing Escherichia coli (STEC) O157 and particular non-O157 serotypes. Here we investigated the biological effects of CDT-V from STEC O157:H(-) (strain 493/89) on human endothelial cells, which are believed to be major pathogenetic targets in severe STEC-mediated diseases. CDT-V caused dose-dependent G(2)/M cell cycle arrest leading to distension, inhibition of proliferation, and death in primary human umbilical vein endothelial cells (HUVEC) and two endothelial cell lines, EA.hy 926 cells (HUVEC derived) and human brain microvascular endothelial cells (HBMEC). The cell cycle effects of CDT-V were cell type specific. In HUVEC and EA.hy 926 cells, CDT-V caused a slowly developing but persistent G(2)/M block which resulted in delayed nonapoptotic cell death. In contrast, in HBMEC, CDT-V induced a rapidly evolving but transient G(2)/M block which was followed by progressive, mostly apoptotic cell death. In both HBMEC and EA.hy 926 cells, G(2)/M arrest was preceded by the early accumulation of a phosphorylated inactive form of cdc2 kinase. Significant G(2)/M arrest and inhibition of proliferation in both HUVEC and each of the endothelial cell lines were induced by 2 to 15 min of exposure to CDT-V, indicating that the effects of the toxin are irreversible. CDT-V-treated HBMEC and EA.hy 926 cells displayed fragmented nuclei and expressed phosphorylated histone protein H2AX, indicative of DNA damage followed by a DNA repair response. Our data demonstrate that CDT-V causes irreversible damage to human endothelial cells and thus may contribute to the pathogenesis of STEC-mediated diseases.
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PMID:Cytolethal distending toxin from Shiga toxin-producing Escherichia coli O157 causes irreversible G2/M arrest, inhibition of proliferation, and death of human endothelial cells. 1561 95

We investigated the spatial distribution of the induction of the phosphorylated form of the histone protein H2AX (gamma-H2AX), known to be activated by DSBs. Following irradiation of human fibroblast cells with 600 MeV/nucleon silicon and 600 MeV/nucleon iron ions we observed the formation of gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in an x/y plane. Polyethylene shielding was used to achieve a Bragg curve distribution with beam geometry parallel to the monolayer of cells. We present data that highlights the formation of immunofluorescent gamma-H2AX tracks showing the ion trajectories across the Bragg peak of irradiated human fibroblast cells. Qualitative analyses of these distributions indicated potentially increased clustering of DNA damage before the Bragg peak, enhanced gamma-H2AX distribution at the peak, and provided visual evidence of high-linear energy transfer particle traversal of cells beyond the Bragg peak in agreement with one-dimensional transport approximations. Spatial assessment of gamma-H2AX fluorescence may provide direct insights into DNA damage across the Bragg curve for high charge and energy ions including the biological consequences of shielding and possible contributors to bystander effects.
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PMID:High LET-induced H2AX phosphorylation around the Bragg curve. 1593

We studied the spatial and temporal distributions of foci of the phosphorylated form of the histone protein H2AX (gamma-H2AX), which is known to be activated by double-strand breaks after irradiation of human fibroblast cells with high-energy silicon (54 keV/microm) and iron (176 keV/microm) ions. Here we present data obtained with the ion path parallel to a monolayer of human fibroblast cells that leads to gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in an x/y plane, thus enabling the analysis of the fluorescence distributions along the ion trajectories. Qualitative analyses of these distributions provide insights into DNA damage processing kinetics for high charge and energy (HZE) ions, including evidence of increased clustering of DNA damage and slower processing with increasing LET.
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PMID:Immunofluorescence detection of clustered gamma-H2AX foci induced by HZE-particle radiation. 1618 60

The histone protein family member X (H2AFX) is important in maintaining chromatin structure and genetic stability. Genetic variants in H2AFX may alter protein functions and thus cancer risk. In this case-control study, we genotyped four common single nucleotide polymorphisms (i.e., -1654A > G [rs643788], -1420G > A [rs8551], and -1187T > C [rs7759] in the H2AFX promoter region and 1057C > T [rs7350] in the 3' untranslated region (UTR)) in 467 patients with sporadic breast cancer and 488 cancer-free controls. All female subjects were non-Hispanic whites aged <or=55 years. We found that significantly increased risk of breast cancer was associated with variant genotypes in the H2AFX promoter: adjusted odds ratio [OR] = 1.80, 95% confidence interval [CI] = 1.38-2.34 for -1654AG/GG; OR = 1.40, 95% CI = 1.07-1.83 for -1420GA/AA; and OR = 1.65, 95% CI = 1.26-2.16 for -1187TC/CC. Furthermore, the number of variant alleles in the promoter haplotypes was associated with increased risks of breast cancer in a dose-response manner (OR = 6.08, 95% CI = 3.25-11.38; OR = 6.83, 95% CI = 3.83-12.18; and OR = 23.61, 95% CI = 3.95-140.99 for one, two, and three variant alleles, respectively) (P (trend) \ < 0.0001). Age at onset of breast cancer significantly decreased as the number of variant alleles increased (P (trend) = 0.024). However, these effects were not observed in the 3'UTR 1057C > T polymorphism. Therefore, we believe that H2AFX promoter polymorphisms may contribute to the etiology of sporadic breast cancer in young non-Hispanic white women. Larger association studies and related functional studies are warranted to confirm these findings.
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PMID:Genetic variants in the H2AFX promoter region are associated with risk of sporadic breast cancer in non-Hispanic white women aged <or=55 years. 1785 62

Phosphorylation of histone protein H2AX on serine 139 (gamma-H2AX) occurs at sites flanking DNA double-stranded breaks (DSBs) and can provide a measure of the number of DSBs within a cell. We describe a flow cytometry-based method optimized to measure gamma-H2AX in nonfixed mononuclear blood cells as well as in cultured cells, which is more sensitive and involves less steps compared with protocols involving fixed cells. This method can be used to monitor induction of gamma-H2AX in mononuclear cells from cancer patients undergoing radiotherapy and for detection of gamma-H2AX throughout the cell cycle in cultured cells. The method is based on the fact that H2AX like other histone proteins are retained in the nucleus when cells are lysed at physiological salt concentrations. Cells are therefore added without fixation to a solution containing detergent to lyse the cells along with a fluorescein isothiocyanate-labeled monoclonal gamma-H2AX antibody, DNA staining dye and blocking agents. The stained nuclei can be analyzed by flow cytometry to monitor the level of gamma-H2AX to determine the level of DSBs and DNA content and to determine the cell cycle stage. The omission of fixation simplifies staining and enhances the sensitivity. This protocol can be completed within 4-6 h.
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PMID:An optimized method for measurement of gamma-H2AX in blood mononuclear and cultured cells. 1860 Feb 24

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse, but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed <10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However, the low level of DNA-PK(cs) found in wild-type cells appeared sufficient to allow rejoining of dsb after doses <20Gy even in G1 phase cells. Inhibition of DNA-PK(cs) with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PK(cs) at low doses. In contrast to wild-type cells, mES cells lacking H2AX, a histone protein involved in the DNA damage response, were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining, H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs), an increase in dsb rejoining rate, and a decrease in Ku70/80. Unlike mouse ES, human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells, they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM, and they add to the growing list of differences in the way rodent and human cells deal with DNA damage.
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PMID:Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks. 1860 49

Chronic myelogenous leukemia (CML) is a hematological malignancy that begins as indolent chronic phase (CP) but inevitably progresses to fatal blast crisis (BC). p210BCR/ABL, a chimeric protein with enhanced kinase activity, initiates CML CP, and additional genetic alterations account for progression to BC, but the precise mechanisms underlying disease evolution are not fully understood. In the present study, we investigated the possible contribution of dysfunction of Bcl11b, a zinc-finger protein required for thymocyte differentiation, and of H2AX, a histone protein involved in DNA repair, to the transition from CML CP to BC. For this purpose, we crossed CML CP-exhibiting p210BCR/ABL transgenic (BA(tg/-)) mice with Bcl11b heterozygous (Bcl11b(+/-)) mice and H2AX heterozygous (H2AX(+/-)) mice. Interestingly, p210BCR/ABL transgenic, Bcl11b heterozygous (BA(tg/-)Bcl11b(+/-)) mice and p210BCR/ABL transgenic, H2AX heterozygous (BA(tg/-)H2AX(+/-)) mice frequently developed CML BC with T-cell phenotype and died in a short period. In addition, whereas p210BCR/ABL was expressed in all of the leukemic tissues, the expression of Bcl11b and H2AX was undetectable in several tumors, which was attributed to the loss of the residual normal allele or the lack of mRNA expression. These results indicate that Bcl11b and H2AX function as tumor suppressor and that haploinsufficiency and acquired loss of these gene products cooperate with p210BCR/ABL to develop CML BC.
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PMID:Haploinsufficiency and acquired loss of Bcl11b and H2AX induces blast crisis of chronic myelogenous leukemia in a transgenic mouse model. 1943 95

The gammaH2AX focus assay, based on phosphorylation of the variant histone protein H2AX, was evaluated as a genotoxicity test in immortalised wild-type mouse embryonic fibroblasts (MEFs) treated for 4h with a panel of reference compounds routinely used in genotoxicity testing. The topoisomerase II poison etoposide (0.006-60 microg/ml), the alkylating agent methyl methanesulfonate (1.3-65 microg/ml) and the direct DNA-damaging agent bleomycin (0.1-10 microg/ml) all produced a positive concentration-response relationship. The non-genotoxic compounds ampicillin (0.035-3500 microg/ml) and sodium chloride (0.058-580 microg/ml) showed no such response with increased concentrations. The H2AX phosphorylation results were compared with the outcome of two standard in vitro genotoxicity tests, namely the micronucleus and comet assays. Compounds that produced measurable DNA damage in the focus assay generated micronuclei at comparable concentrations. In this study, the focus assay identified genotoxic agents with the same specificity as the comet assay. These results were substantiated when H2AX phosphorylation was analysed using flow cytometry in the murine cell line L5178Y, growing in suspension. The data were in concordance with the manual scoring focus assay. To further this investigation, the gammaH2AX flow cytometry was compared to the in vitro micronucleus flow cytometry and mouse lymphoma assay using the same cell population after MMS treatment. The median gammaH2AX value increased significantly above the control at all four MMS concentrations tested. The percentage of micronucleus events in the in vitro micronucleus flow test and the mutation frequency in the mouse lymphoma assay were also significantly increased at each MMS concentration. The current data indicate that H2AX phosphorylation could be used as a biomarker of genotoxicity, which could predict the outcome of in vitro mammalian cell genotoxicity assays.
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PMID:H2AX phosphorylation as a genotoxicity endpoint. 1962 53

The phosphorylated form of the histone protein H2AX (gammaH2AX) plays a central role in sensing and repairing DNA damage and is a sensitive marker for DNA double-strand breaks (DSB). Although a wide range of genotoxic agents that do not initiate DSB induce gammaH2AX, the range of chemicals that cause H2AX phosphorylation is not clear. We designed a novel, whole cell enzyme-linked immunosorbent assay (cell-ELISA) that can accurately quantify gammaH2AX levels and identify chemical compounds that induce gammaH2AX formation; our novel assay is more convenient than microscopic examination of gammaH2AX foci or flow cytometry. We measured gammaH2AX levels in CHL, CHO and V79 cells exposed to DNA-damaging, non-genotoxic and aneugenic chemicals using the cell-ELISA assay. The cell-ELISA results for the DNA-damaging compounds (methyl methanesulfonate, N-ethyl-N'-nitro-N-nitrosoguanidine, mitomycin C, cisplatin, irinotecan, etoposide, methotrexate and 5-fluorouracil) assayed showed that there was a concentration-dependent increase in gammaH2AX, which was 1.5-fold greater than the negative control; the only exception was a negative response of CHO cells to 5-fluorouracil. None of the 10 non-genotoxic compounds assayed showed similar increases in gammaH2AX and all exhibited concentration-dependent growth inhibition of the cells. The highest levels of gammaH2AX found from treatment with aneugens (vincristine, colcemid, paclitaxel, griseofulvin, 17-allylaminogeldanamycin and CH3310395), which are compounds that cause spindle dysfunction and have no genotoxic activity in the Ames test, were 1.5-fold lower than the negative control. In contrast, mitomycin C and etoposide, which both have aneugenic and DNA-damaging activities, induced a positive response. None of the aneugens caused an increase in gammaH2AX at concentrations that induce micronuclei. The chemical classes that show positive results in the cell-ELISA are different from those that are positive in the Ames or in vitro micronucleus test. By using the cell-ELISA for the level of gammaH2AX, we were able to distinguish DNA-damaging agents from non-genotoxic compounds or aneugens.
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PMID:Whole cell-ELISA to measure the gammaH2AX response of six aneugens and eight DNA-damaging chemicals. 2058 Aug 54


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