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)

The ATR kinase phosphorylates both p53 and Chk1 in response to extreme hypoxia (oxygen concentrations of less than 0.02%). In contrast to ATR, loss of ATM does not affect the phosphorylation of these or other targets in response to hypoxia. However, hypoxia within tumors is often transient and is inevitably followed by reoxygenation. We hypothesized that ATR activity is induced under hypoxic conditions because of growth arrest and ATM activity increases in response to the oxidative stress of reoxygenation. Using the comet assay to detect DNA damage, we find that reoxygenation induced significant amounts of DNA damage. Two ATR/ATM targets, p53 serine 15 and histone H2AX, were both phosphorylated in response to hypoxia in an ATR-dependent manner. These phosphorylations were then maintained in response to reoxygenation-induced DNA damage in an ATM-dependent manner. The reoxygenation-induced p53 serine 15 phosphorylation was inhibited by the addition of N-acetyl-l-cysteine (NAC), indicating that free radical-induced DNA damage was mediated by reactive oxygen species. Taken together these data implicate both ATR and ATM as critical roles in the response of hypoxia and reperfusion in solid tumors.
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PMID:ATR/ATM targets are phosphorylated by ATR in response to hypoxia and ATM in response to reoxygenation. 1251 69

We have developed stable cell lines expressing green fluorescent protein fusion proteins containing polyglutamine repeats of various lengths under tetracycline control. The expression of the expanded (43Q) repeat protein resulted in aggregate formation in a time-dependent fashion. The accumulation of aggregates did not induce apoptosis, although the survival of these cells was critically dependent on the presence of serum and growth factors. However, the expression of 43Q expanded protein strongly activated the ataxia telangiectasia mutated kinase/ATM and Rad3-related kinase (ATM/ATR)-dependent DNA damage response, as shown by selective phosphorylation of ATM substrates. This activation was dependent on 43 CAG protein expression, reversible and sensitive to caffeine and reducing agents. Similarly, we found phosphorylated ATM substrates in fibroblasts from Huntington's disease or SCA-2 patients. Oxidative stress induced accumulation of ATM/ATR phosphorylated protein in HD and SCA-2 patients, but not in normal controls. Furthermore, a significant phosphorylation of H2AX was shown by fibroblasts from patients. We conclude that polyglutamine induces ATM/ATR-dependent DNA damage response through accumulation of reactive oxygen species. ATM activation can be used to monitor the disease in vivo.
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PMID:DNA damage induced by polyglutamine-expanded proteins. 1291 85

Severe levels of hypoxia (oxygen concentrations of less that 0.02%) have been shown to induce a rapid S-phase arrest. The mechanism behind hypoxia-induced S-phase arrest is unclear, we show here that it was not mediated by a shortage of nucleosides and was not dependent on p53, p21 or Hif 1alpha status. The drugs aphidicolin and hydroxyurea both induce rapid replication arrest and have been used throughout the literature to study the ATR-mediated response to stalled replication. We have shown previously that hypoxia induces ATR-dependent phosphorylation of p53, Chk1 and histone H2AX. Using comet-assays to detect DNA-damage we found that both aphidicolin and hydroxyurea induced significant levels of DNA-damage while hypoxia did not. Here we show that like aphidicolin and hydroxyurea, hypoxia induces phosphorylation of Nbs1 at serine 343 and Rad17 serine 645. Hypoxia-dependent phosphorylation of Nbs1 and Rad17 was ATM-independent and therefore likely to be a result of the ATR kinase activity. In contrast, p53 was phosphorylated differentially in response to the three treatments considered here. p53 was phosphorylated at serine 15 in response to all three treatments but was only phosphorylated at serine 20 in response to the drug treatments. We propose that treatment with either aphidicolin or hydroxyurea leads to not only replication arrest but also DNA-damage and therefore both ATM and ATR-mediated signaling. In contrast replication arrest induced by severe hypoxia is sensed exclusively through ATR, with ATM only having a role to play after re-oxygenation.
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PMID:Comparison of hypoxia-induced replication arrest with hydroxyurea and aphidicolin-induced arrest. 1464 37

Previously, we showed that sulforaphane (SFN), a naturally occurring cancer chemopreventive agent, effectively inhibits proliferation of PC-3 human prostate cancer cells by causing caspase-9- and caspase-8-mediated apoptosis. Here, we demonstrate that SFN treatment causes an irreversible arrest in the G(2)/M phase of the cell cycle. Cell cycle arrest induced by SFN was associated with a significant decrease in protein levels of cyclin B1, cell division cycle (Cdc) 25B, and Cdc25C, leading to accumulation of Tyr-15-phosphorylated (inactive) cyclin-dependent kinase 1. The SFN-induced decline in Cdc25C protein level was blocked in the presence of proteasome inhibitor lactacystin, but lactacystin did not confer protection against cell cycle arrest. Interestingly, SFN treatment also resulted in a rapid and sustained phosphorylation of Cdc25C at Ser-216, leading to its translocation from the nucleus to the cytoplasm because of increased binding with 14-3-3beta. Increased Ser-216 phosphorylation of Cdc25C upon treatment with SFN was the result of activation of checkpoint kinase 2 (Chk2), which was associated with Ser-1981 phosphorylation of ataxia telangiectasia-mutated, generation of reactive oxygen species, and Ser-139 phosphorylation of histone H2A.X, a sensitive marker for the presence of DNA double-strand breaks. Transient transfection of PC-3 cells with Chk2-specific small interfering RNA duplexes significantly attenuated SFN-induced G(2)/M arrest. HCT116 human colon cancer-derived Chk2(-/-) cells were significantly more resistant to G(2)/M arrest by SFN compared with the wild type HCT116 cells. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in irreversible G(2)/M arrest by SFN. Activation of Chk2 in response to DNA damage is well documented, but the present study is the first published report to link Chk2 activation to cell cycle arrest by an isothiocyanate.
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PMID:Sulforaphane-induced G2/M phase cell cycle arrest involves checkpoint kinase 2-mediated phosphorylation of cell division cycle 25C. 1507 69

The requirement for the serine/threonine protein kinase ATM in coordinating the cellular response to DNA damage induced by ionizing radiation has been studied extensively. Many of the anti-tumor chemotherapeutics in clinical use today cause DNA double strand breaks; however, few have been evaluated for their ability to modulate ATM-mediated pathways. We have investigated the requirement for ATM in the cellular response to doxorubicin, a topoisomerase II-stabilizing drug. Using several ATM-proficient and ATM-deficient cell lines, we have observed ATM-dependent nuclear accumulation of p53 and ATM-dependent phosphorylation of p53 on seven serine residues. This was accompanied by an increased binding of p53 to its cognate binding site, suggesting transcriptional competency of p53 to activate its downstream effectors. Treatment of cells with doxorubicin led to the phosphorylation of histone H2AX on serine 139 with dependence on ATM for the initial response. Doxorubicin treatment also stimulated ATM autophosphorylation on serine 1981 and the ATM-dependent phosphorylation of numerous effectors in the ATM-signaling pathway, including Nbs1 (Ser(343)), SMC1 (Ser(957)), Chk1 (Ser(317) and Ser(345)), and Chk2 (Ser(33/35) and Thr(68)). Although generally classified as a topoisomerase II-stabilizing drug that induces DNA double strand breaks, doxorubicin can intercalate DNA and generate reactive oxygen species. Pretreatment of cells with the superoxide scavenger ascorbic acid had no effect on the doxorubicin-induced phosphorylation and accumulation of p53. In contrast, preincubation of cells with the hydroxyl radical scavenger, N-acetylcysteine, significantly attenuated the doxorubicin-mediated phosphorylation and accumulation of p53, p53-DNA binding, and the phosphorylation of H2AX, Nbs1, SMC1, Chk1, and Chk2, suggesting that hydroxyl radicals contribute to the doxorubicin-induced activation of ATM-dependent pathways.
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PMID:Doxorubicin activates ATM-dependent phosphorylation of multiple downstream targets in part through the generation of reactive oxygen species. 1548 21

Although radiation-induced bystander effects have been demonstrated in a number of cell types, the studies have largely been performed using high linear energy transfer (LET) radiation, such as alpha-particles. The literature is contradictory on whether fibroblasts show bystander responses, especially after low LET radiation such as X- or gamma-rays and whether the same signal transmission pathways are involved. Herein, a novel transwell insert culture dish method is used to show that X-irradiation induces medium-mediated bystander effects in AGO1522 normal human fibroblasts. The frequency of micronuclei formation in unirradiated bystander cells increases from a background of about 6.5% to about 9-13% at all doses from 0.1 to 10 Gy to the irradiated cells. Induction of p21Waf1 protein and foci of gamma-H2AX in bystander cells is also independent of dose to the irradiated cells above 0.1 Gy. In addition, levels of reactive oxygen species (ROS) were increased persistently in directly irradiated cells up to 60 h after irradiation and in bystander cells for 30 h. Adding Cu-Zn superoxide dismutase (SOD) and catalase to the medium decreases the formation of micronuclei and induction of p21Waf1 and gamma-H2AX foci in bystander cells, suggesting oxidative metabolism plays a role in the signaling pathways in bystander cells. The results of clonogenic assay of bystander cells showed that survival of bystander cells decreases from 0 to 0.5 Gy, and then is independent of the dose to irradiated cells from 0.5 to 10 Gy. Unlike the response with p21Waf1 expression, gamma-H2AX foci and micronuclei, adding SOD and catalase has no effect on the survival of bystander cells. The data suggest that irradiated cells release toxic factors other than ROS into the medium.
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PMID:Medium-mediated intercellular communication is involved in bystander responses of X-ray-irradiated normal human fibroblasts. 1568 9

Farnesyltransferase inhibitors (FTIs) possess antitumor activity. Based on recent findings, we hypothesized that FTIs induce reactive oxygen species (ROS) that damage DNA, leading to DNA damage responses. To test this hypothesis, we investigated the effects of FTIs on the generation of ROS, DNA double-strand breaks (DSB), DNA damage responses, and RhoB, and the effects of quenching ROS on these FTI effects. We evaluated four FTIs in human cancer cell lines of different tissue origins. We found that FTIs induced ROS and DSBs. Suppressing expression of the beta-subunit of farnesyltransferase with siRNA did not induce ROS, but slightly attenuated the ROS induced by FTIs. N-acetyl-L-cysteine (NAC), but not caspase inhibitors, blocked FTI-induced DSBs, suggesting that the DSBs were caused by ROS and did not result from apoptosis. The DSBs led to DNA damage responses. H2AX became phosphorylated and formed nuclear foci. The DNA-damage-sensing molecules involved were probably ataxia-telangiectasia mutated protein (ATM) and DNA-dependent protein kinase (DNA-PK) but not ATM- and Rad3-related protein (ATR). Key components of the homologous recombination and nonhomologous end joining repair pathways (DNA-PK, BRCA1, and NBS1) underwent phosphorylation and formed nuclear foci. RhoB, a mediator of the antineoplastic effect of FTIs and a protein inducible by DNA damage, was increased by FTIs. This increase was blocked by NAC. We concluded that FTIs induced oxidative DNA damage by inducing ROS and initiated DNA damage responses, including RhoB induction, and there was a complex relationship among FTIs, farnesyltransferase, ROS, and RhoB. Our data also imply that inhibitors of DNA repair may accentuate the clinical efficacy of FTIs.
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PMID:Farnesyltransferase inhibitors induce DNA damage via reactive oxygen species in human cancer cells. 1586 62

Bystander effects induced by low dose of ionizing radiation have been shown to widely exist in many cell types and may have a significant impact on radiation risk assessment. Though many studies have been reported on this phenomenological observation, the mechanisms underlying this process are not clear, especially on the questions of how soon after irradiation the bystander effects can be initiated and how far this bystander signal can be propagated once it is started. DNA double-strand breaks (DSBs) induced by ionizing radiation or carcinogenic chemicals can be visualized in situ using gamma-H2AX immunofluorescent staining. Our previous studies have shown that in situ visualization of DSBs could be used to assess irradiation-induced extranuclear/extracellular (bystander) effect at an early stage after irradiation. In the present studies, we used this method to investigate the time and spatial effects of damage signals on unirradiated bystander cells. The results showed that increased DSBs in irradiated and unirradiated bystander areas could be visualized 2 min after radiation and reached its maximum 30 min after radiation. The average levels of DSB formation at 30 min post-1cGy irradiation in the irradiated and unirradiated bystander areas were 3-fold and 2-fold higher than those of the sham-irradiated control cells, respectively. Afterwards, the formation of DSBs declined with incubation time and remained steady for at least 6 h at a level that was statistically higher than their controls. The results also showed that the bystander signal derived from irradiated cells could be transferred to anywhere in the dish and the percentage of DSBs in the unirradiated bystander cells was not dependent on the dose delivered. Moreover, the fraction of DSB positive cells in unirradiated bystander areas showed a time-dependent increase based on its distance to the irradiated area at very early stage post-irradiation. Both lindane and DMSO significantly suppressed the yield of DSBs in the cells of unirradiated bystander areas, which suggest that gap junctional intercellular communication and reactive oxygen species played important roles in the induction of the bystander effects, both in irradiated and unirradiated bystander areas.
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PMID:The time and spatial effects of bystander response in mammalian cells induced by low dose radiation. 1615 Aug 94

Phosphorylation of histone H2AX (termed gamma-H2AX) was recently identified as an early event after induction of DNA double strand breaks (DSBs). We have previously shown that co-exposure to benzo[a]pyrene (BaP), a wide-spread environmental carcinogen, and ultraviolet A (UVA), a major component of solar UV radiation, induced DSBs in mammalian cells. In the present study, we examined whether co-exposure to BaP and UVA generates gamma-H2AX in CHO-K1 cells. Single treatment with BaP (10(-9)-10(-7)M) or UVA ( approximately 2.4 J/cm(2)) did not result in gamma-H2AX, however, co-exposure drastically induced foci of gamma-H2AX in a dose-dependent manner. gamma-H2AX could be detected even at very low concentration of BaP (10(-9)M) plus UVA (0.6J/cm(2)), which did not change cell survival rates. NaN(3) effectively inhibited the formation of gamma-H2AX induced by co-exposure, indicating the contribution of singlet oxygen. This is the first evidence that co-exposure to BaP and UVA induced DSBs, involving gamma-H2AX.
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PMID:Co-exposure to benzo[a]pyrene and UVA induces phosphorylation of histone H2AX. 1625 11

Interferons are cytokines with potent antiviral and antiproliferative activities. We report that although a transient exposure to beta-interferon induces a reversible cell cycle arrest, a sustained treatment triggers a p53-dependent senescence program. Beta-interferon switched on p53 in two steps. First, it induced the acetylation of p53 at lysine 320 and its dephosphorylation at serine 392 but not p53 activity. Later on, it triggered a DNA signaling pathway, the phosphorylation of p53 at serine 15 and its transcriptional activity. In agreement, beta-interferon-treated cells accumulated gamma-H2AX foci and phosphorylated forms of ATM and CHK2. The DNA damage signaling pathway was activated by an increase in reactive oxygen species (ROS) induced by interferon and was inhibited by the antioxidant N-acetyl cysteine. More important, RNA interference against ATM inhibited p53 phosphorylation at serine 15, p53 activity and senescence in response to beta-interferon. Beta-interferon-induced senescence was more efficient in cells expressing either, p53, or constitutive allele of ERK2 or RasV12. Hence, beta-interferon-induced senescence targets preferentially cells with premalignant changes.
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PMID:DNA damage signaling and p53-dependent senescence after prolonged beta-interferon stimulation. 1643 15


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