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)

Paraquat (PQ; 1,1'-dimethyl-4,4'-bipyridinium dichloride) is widely used as a universal herbicide. Although systemic treatment with PQ gives rise to the highest level of the herbicide in the cerebral cortex, our knowledge of its effects in this brain region is very limited. We took advantage of rat cortical cell cultures to analyze how PQ affects cortical neurons. Lactate dehydrogenase (LDH) assay and propidium iodide (PI) staining showed that PQ was cytotoxic to cortical neurons with an IC50 on the third day after treatment of approximately 10 microM. PQ-treated cells had shrunken soma with condensed nuclei and disintegrated dendrites, typical signs of apoptosis. Immunocytochemistry of 8-day in vitro (DIV) cells one day after PQ treatment with anti-phospho-H2AX antibody showed that the average number of punctae per nucleus had increased several-fold, indicating substantial DNA fragmentation. Furthermore, double-staining of 7.5 DIV cultures (50 microM PQ) with PI and an antibody against annexin V (AN), an impermeable plasma protein which specifically binds to phosphatidylserine (PS), showed that the percentages of AN(+)/PI(-) cells had also increased several-fold, pointing to considerable movement of PS from the inner to the outer leaflet of the plasma membrane. Taken together, our data indicate that PQ induces apoptosis in cortical cell cultures.
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PMID:Paraquat induces apoptosis of cultured rat cortical cells. 1505 35

DNA damage that leads to formation of DNA double-strand breaks (DSBs) induces phosphorylation of histone H2AX on Ser-139 at sites flanking the breakage. Immunocytochemical detection of phosphorylated H2AX (denoted as gammaH2AX) thus provides a marker of DSBs. The method presented in this chapter describes the detection of gammaH2AX for revealing the presence of DSBs, combined with differential staining of cellular DNA for revealing the cell cycle phase. The detection of gammaH2AX is based on indirect immunofluorescence using secondary antibody tagged with fluorescein isothiocyanate (FITC) while DNA is counterstained with propidium iodide (PI). Intensity of cellular green (FITC) and red (PI) fluorescence is measured by flow cytometry and bivariate analysis of the data is used to correlate the presence of DSBs with the cell cycle phase.
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PMID:Cytometric assessment of histone H2AX phosphorylation: a reporter of DNA damage. 1667 75

Histone deacetylases (HDAC) have been identified as therapeutic targets due to their regulatory function in DNA structure and organization. LBH589 is a novel inhibitor of class I and II HDACs. We studied the effect of LBH589 and ionizing radiation (IR) on DNA repair in two human non-small cell lung cancer (NSCLC) cell lines (H23 and H460). gamma-H2AX foci present at DNA double-strand breaks (DSBs) were detected in the nuclei following 3 Gy irradiation for up to 6 hours. LBH589 administered before irradiation increased the duration of gamma-H2AX foci beyond 24 hours. Furthermore, radiation alone induced translocation of HDAC4 to the nucleus. In contrast, treatment with LBH589 followed by irradiation resulted in HDAC4 confinement to the cytoplasm, indicating that HDAC inhibition affects the nuclear localization of HDAC4. The findings that LBH589 confines HDAC4 to the cytoplasm and increases the duration of gamma-H2AX foci in irradiated cell lines suggest that HDAC4 participates in DNA damage signaling following IR. Annexin-propidium iodide flow cytometry assays, cell morphology studies, and cleaved caspase-3 Western blot analysis revealed a synergistic effect of LBH589 with IR in inducing apoptosis. Clonogenic survival showed a greater than additive effect when LBH589 was administered before irradiation compared with irradiation alone. In vivo tumor volume studies showed a growth delay of 20 days with combined treatment compared with 4 and 2 days for radiation or LBH589 alone. This study identifies HDAC4 as a biomarker of LBH589 activity and recognizes the ability of LBH589 to sensitize human NSCLC to radiation-induced DNA DSBs.
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PMID:Histone deacetylase (HDAC) inhibitor LBH589 increases duration of gamma-H2AX foci and confines HDAC4 to the cytoplasm in irradiated non-small cell lung cancer. 1714 76

Stage III nonsmall cell lung cancer is primarily treated with combined chemotherapy and radiation therapy. Relapses for progression of disease within irradiated sites remains a primary pattern of failure. To evaluate the interaction between histone deacetylase inhibitors and irradiation in nonsmall cell lung cancer, we studied NVP-LAQ824 in mouse models of human lung cancer. Colony formation assays were performed to determine whether LAQ824 sensitized nonsmall cell lung cancer to the cytotoxic effects of ionizing radiation. LAQ824 reduced clonogenic survival of the H23 and H460 cell lines five-fold compared with controls and four-fold compared with either agent alone (P<0.001). Western blot analysis of caspase cleavage, microscopic analysis of nuclei and Annexin-fluorescein isothiocyanate/propidium iodide flow cytometry assays showed that LAQ824 enhanced radiation-induced apoptosis and attenuated mitosis (P<0.001). Immunostaining for gamma-H2AX nuclear foci was performed to determine the effect of LAQ824 on radiation-induced DNA double-strand breaks. Combined modality treatment delayed the resolution of gamma-H2AX foci with over 30% of cells staining positive 6 h after treatment versus approximately 5 and 3% in cells treated with LAQ824 or radiation alone (P<0.001). Additionally, an in-vivo xenograft model was utilized to study the effects of fractioned irradiation and LAQ824 on tumor growth. Fractioned irradiation and LAQ824 delayed tumor growth by 19 days versus 7 and 4 days for treatment with LAQ824 and radiation alone. This study shows the effectiveness of histone deacetylase inhibitors to enhance the cytotoxic effects of radiation by attenuating DNA repair and inducing apoptosis in human nonsmall cell lung cancer.
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PMID:Histone deacetylase inhibitor NVP-LAQ824 sensitizes human nonsmall cell lung cancer to the cytotoxic effects of ionizing radiation. 1758 1

A number of methods have been developed to examine the morphologic, biochemical, and molecular changes that happen during the DNA damage response that may ultimately lead to death of cells through various mechanisms that include apoptosis. When cells are exposed to ionizing radiation or chemical DNA-damaging agents, double-stranded DNA breaks (DSB) are generated that rapidly result in the phosphorylation of histone variant H2AX. Because phosphorylation of H2AX at Ser 139 correlates well with each DSB, phospho-H2AX is a sensitive marker to used to examine the DNA damage and its repair. Apoptotic cells are characterized on the basis of their reduced DNA content and morphologic changes, including nuclear condensation, which can be detected by flow cytometry (sub-G1 DNA content), trypan blue, or Hoechst staining. The appearance of phosphatidylserine on the plasma membrane with annexin V-fluorochrome conjugates indicates the changes in plasma membrane composition and function. By combining it with propidium iodide staining, this method can also be used to distinguish early versus late apoptotic or necrotic events. The activation of caspases is another well-known biochemical marker of apoptosis. Finally, the Bcl-2 family of proteins and the mitochondria that play a critical role in DNA damage-induced apoptosis can be examined by translocation of Bax and cytochrome c in and out of mitochondria. In this chapter, we discuss the most commonly used techniques used in our laboratory for determining the DNA damage response leading to apoptosis.
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PMID:DNA damage response and apoptosis. 1860 18

This unit describes immunocytochemical detection of phosphorylated histone H2AX for revealing the presence of DNA double-strand breaks. Double-strand breaks indicate DNA damage induced by ionizing radiation or by treatment with antitumor drugs such as DNA topoisomerase inhibitors. However, double-strand breaks can also be intrinsic, occurring in healthy, nontreated cells for a variety of reasons, and are generated in the course of DNA fragmentation in apoptotic cells. The unit presents strategies to distinguish radiation- or drug-induced breaks from those intrinsically formed in untreated cells or associated with apoptosis. The protocol describes the immunocytochemical detection of histone H2AX phosphorylated on Ser-139 combined with measurement of DNA content to identify cells that have DNA double-strand breaks and to concurrently assess their cell cycle phase. The detection is based on indirect immunofluorescence using a FITC-labeled secondary antibody, and DNA is counterstained with propidium iodide (PI). Cellular RNA, which may be stained by PI, is removed with RNase A.
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PMID:Detection of histone H2AX phosphorylation on Ser-139 as an indicator of DNA damage (DNA double-strand breaks). 1877 Aug 4

We tested several classes of antioxidant manganese compounds for radioprotective effects using human lymphoblastoid cells: six porphyrins, three salens, and two cyclic polyamines. Radioprotection was evaluated by seven assays: XTT, annexin V and propidium iodide flow cytometry analysis, gamma-H2AX immunofluorescence, the neutral comet assay, dichlorofluorescein and dihydroethidium staining, resazurin, and colony survival assay. Two compounds were most effective in protecting wild-type and A-T cells against radiation-induced damage: MnMx-2-PyP-Calbio (a mixture of differently N-methylated MnT-2-PyP+ from Calbiochem) and MnTnHex-2-PyP. MnTnHex-2-PyP protected WT cells against radiation-induced apoptosis by 58% (p = 0.04), using XTT, and A-T cells by 39% (p = 0.01), using annexin V and propidium iodide staining. MnTnHex-2-PyP protected WT cells against DNA damage by 57% (p = 0.005), using gamma-H2AX immunofluorescence, and by 30% (p < 0.01), using neutral comet assay. MnTnHex-2-PyP is more lipophilic than MnMx-2-PyP-Calbio and is also >10-fold more SOD-active; consequently it is >50-fold more potent as a radioprotectant, as supported by six of the tests employed in this study. Thus, lipophilicity and antioxidant potency correlated with the magnitude of the beneficial radioprotectant effects observed. Our results identify a new class of porphyrinic radioprotectants for the general and radiosensitive populations and may also provide a new option for treating A-T patients.
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PMID:Radioprotective effects of manganese-containing superoxide dismutase mimics on ataxia-telangiectasia cells. 1938 72

DNA double-strand breaks (DSBs) are considered as one of the primary causes of cancer but their induction by hydrogen peroxide (H(2)O(2)) is still controversial. In this work, we studied whether the high levels of H(2)O(2) produced in the thyroid to oxidize iodide could induce DNA modifications. Scores of DNA damage, in terms of strand breaks, were obtained by comet assay (alkaline condition for single-strand breaks (SSBs) and neutral condition for DSBs). We demonstrated that in a rat thyroid cell line (PCCl3), non-lethal concentrations of H(2)O(2) (0.1-0.5 mmol/l) as well as irradiation (1-10 Gy) provoked a large number of SSBs ( approximately 2-3 times control DNA damage values) but also high levels of DSBs (1.2-2.3 times control DNA damage values). We confirmed the generation of DSBs in this cell line and also in human thyroid in primary culture and in pig thyroid slices by measuring phosphorylation of histone H2AX. L-Buthionine-sulfoximine, an agent that depletes cells of glutathione, decreased the threshold to observe H(2)O(2)-induced DNA damage. Moreover, we showed that DNA breaks induced by H(2)O(2) were more slowly repaired than those induced by irradiation. In conclusion, H(2)O(2) causes SSBs and DSBs in thyroid cells. DSBs are produced in amounts comparable with those observed after irradiation but with a slower repair. These data support the hypothesis that the generation of H(2)O(2) in thyroid could also play a role in mutagenesis particularly in the case of antioxidant defense deficiency.
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PMID:Hydrogen peroxide induces DNA single- and double-strand breaks in thyroid cells and is therefore a potential mutagen for this organ. 1950 65

The aim of this study was to investigate and quantify two biomarkers for radiation exposure (dicentrics and gamma-H2AX foci) in human lymphocytes after CT scans in the presence of an iodinated contrast agent. Blood samples from a healthy donor were exposed to CT scans in the absence or presence of iotrolan 300 at iodine concentrations of 5 or 50 mg ml(-1) blood. The samples were exposed to 0.025, 0.05, 0.1 and 1 Gy in a tissue equivalent body phantom. Chromosome aberration scoring and automated microscopic analysis of gamma-H2AX foci were performed in parts of the same samples. The theoretical physical dose enhancement factor (DEF) was calculated on the basis of the mass energy-absorption coefficients of iodine and blood and the photon energy spectrum of the CT tube. No significant differences in the yields of dicentrics and gamma-H2AX foci were observed in the absence or presence of 5 mg iodine ml(-1) blood up to 0.1 Gy, whereas at 1 Gy the yields were elevated for both biomarkers. At an iodine concentration of 50 mg ml(-1) serving as a positive control, a biological DEF of 9.5 +/- 1.4 and 2.3 +/- 0.5 was determined for dicentrics and gamma-H2AX foci, respectively. A physical DEF of 1.56 and 6.30 was calculated for 5 and 50 mg iodine ml(-1), respectively. Thus, it can be concluded that in the diagnostic dose range (radiation and contrast dose), no relevant biological dose-enhancing effect could be detected, whereas a clear biological dose-enhancing effect could be found for a contrast dose well outside the diagnostic CT range for the complete radiation dose range with both methods.
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PMID:The influence of x-ray contrast agents in computed tomography on the induction of dicentrics and gamma-H2AX foci in lymphocytes of human blood samples. 1977 23

Rhabdomyosarcoma, consisting of alveolar (aRMS) and embryonal (eRMS) subtypes, is the most common type of sarcoma in children. Currently, there are no targeted drug therapies available for rhabdomyosarcoma. In searching for new molecular therapeutic targets, we carried out genome-wide small interfering RNA (siRNA) library screens targeting human phosphatases (n = 206) and kinases (n = 691) initially against an aRMS cell line, RH30. Sixteen phosphatases and 50 kinases were identified based on growth inhibition after 72 hours. Inhibiting polo-like kinase 1 (PLK1) had the most remarkable impact on growth inhibition (approximately 80%) and apoptosis on all three rhabdomyosarcoma cell lines tested, namely, RH30, CW9019 (aRMS), and RD (eRMS), whereas there was no effect on normal muscle cells. The loss of PLK1 expression and subsequent growth inhibition correlated with decreased p-CDC25C and Cyclin B1. Increased expression of WEE 1 was also noted. The induction of apoptosis after PLK1 silencing was confirmed by increased p-H2AX, propidium iodide uptake, and chromatin condensation, as well as caspase-3 and poly(ADP-ribose) polymerase cleavage. Pediatric Ewing's sarcoma (TC-32), neuroblastoma (IMR32 and KCNR), and glioblastoma (SF188) models were also highly sensitive to PLK1 inhibition. Finally, based on cDNA microarray analyses, PLK1 mRNA was overexpressed (>1.5 fold) in 10 of 10 rhabdomyosarcoma cell lines and in 47% and 51% of primary aRMS (17 of 36 samples) and eRMS (21 of 41 samples) tumors, respectively, compared with normal muscles. Similarly, pediatric Ewing's sarcoma, neuroblastoma, and osteosarcoma tumors expressed high PLK1. We conclude that PLK1 could be a promising therapeutic target for the treatment of a wide range of pediatric solid tumors including rhabdomyosarcoma.
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PMID:Small interfering RNA library screen of human kinases and phosphatases identifies polo-like kinase 1 as a promising new target for the treatment of pediatric rhabdomyosarcomas. 1988 53


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