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)

Ions of high atomic number and energy (HZE particles) pose a significant cancer risk to astronauts on prolonged space missions. On Earth, similar ions are being used for targeted cancer therapy. The properties of these particles can be drastically altered during passage through spacecraft shielding, therapy beam modulators, or the human body. Here, we have used pertinent responses to DNA double-strand breaks (DSBs) to understand the consequences of energy loss versus nuclear fragmentation of Fe ions during passage through shielding or tissue-equivalent materials. Phosphorylation of histone H2AX and recruitment of 53BP1 were used to generate 3D reconstructions of DNA damage in human cells and to follow its repair. Human cells are unable to repair a significant portion of DNA damage induced by Fe ions. DNA-PK and ATM are required, to different extents, for the partial repair of Fe-induced DNA damage. Aluminum shielding has little effect on DNA damage or its repair, confirming that the hulls of the Space Shuttle and the International Space Station afford scant protection against these particles. Lead shielding, on the other hand, exacerbates the effects of Fe ions due to energy loss during particle traversal. In sharp contrast, polyethylene (PE), a favored hydrogenous shield, results in DNA damage that is more amenable to repair presumably due to Fe-ion fragmentation. Human cells are indeed able to efficiently repair DSBs induced by chlorine ions and protons that represent fragmentation products of Fe. Interestingly, activation of the tumor suppressor p53 in Fe-irradiated cells is uniquely biphasic and culminates in the induction of high levels of p21 (Waf1/Cip1), p16 (INK4a) and senescence-associated beta-galactosidase activity. Surprisingly, these events occur even in the absence of ATM kinase implying that ATR may be a major responder to the complex DNA damage inflicted by Fe ions. Significantly, fragmentation of the Fe beam through PE attenuates these responses and this, in turn, results in better long-term survival in a colony-forming assay. Our results help us to understand the biological consequences of ion fragmentation through materials, whether in space or in the clinic, and provide us with a biological basis for the use of hydrogenous materials like PE as effective space shields.
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PMID:Modulation of the DNA-damage response to HZE particles by shielding. 1867 98

As demonstrated recently, ionizing radiation (IR) can mediate phosphorylation of DNA-PKcs in human tumor cells through stimulation of the PI3K/Akt pathway. It is also known that DNA-PKcs directly interacts the X-ray repair cross-complementing group 1 protein (XRCC1) involved in base excision repair (BER). Therefore, in the present study we investigated the role of PI3K/Akt activity and DNA-PKcs on XRCC1 expression/stabilization. In contrast to the DNA-PKcs-deficient glioblastoma cell line MO59J, the DNA-PKcs-proficient counterpart MO59K as well as human lung adenocarcinoma A549 cells presented a high basal level of XRCC1 expression. Radiation doses of 3-12Gy did not stimulate a further enhanced expression of XRCC1 in DNA-PKcs-proficient cells (MO59K and A549) within 180min post-irradiation. However, a marked induction of XRCC1 expression was apparent in DNA-PKcs-deficient MO59J cells. Targeting of DNA-PKcs as well as PI3K/Akt pathway by specific kinase inhibitors and/or siRNA reduced basal XRCC1 expression in un-irradiated DNA-PKcs-proficient cells to the level observed in DNA-PKcs-deficient cells. Reduction of basal expression of XRCC1 by XRCC1-siRNA, AKT-siRNA as well as DNA-PKcs inhibitor facilitated IR-induced XRCC1 expression. XRCC1 expression induced by irradiation, however, was independent of PI3K/Akt signaling, but dependent of MAPK-ERK1/2. By immuno-precipitation experiments and confocal microscopy a complex formation of XRCC1 and DNA-PKcs was shown. Applying gamma-H2AX foci analysis it was shown that basal expression of XRCC1 is important for the repair of IR-induced DNA-double strand breaks (DNA-DSBs). These data indicate that IR-induced XRCC1 expression is dependent on the expression level of DNA-PKcs and basal activity status of PI3K/Akt signaling. Likewise, potential of IR-induced XRCC1 expression depends on its basal expression level.
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PMID:PI3K-Akt signaling regulates basal, but MAP-kinase signaling regulates radiation-induced XRCC1 expression in human tumor cells in vitro. 1867 86

Histone H2AX rapidly undergoes phosphorylation at Ser139 (gamma-H2AX) in response to DNA double-strand breaks. Although ATM kinase and DNA-PK phosphorylate Ser139 of H2AX in culture cells, the regulatory mechanism of gamma-H2AX level remains unclear in vivo. Here, we detected the phosphorylation of H2AX and the elimination of gamma-H2AX in the mouse skin after X-irradiation. Furthermore, following X-irradiation, the level of gamma-H2AX also increased in mice lacking either ATM or DNA-PK. Although the elimination after X-irradiation was detected in the skin of these mutant mice, the elimination in DNA-PK-deficient mice was slower than that in C3H and ATM knockout mice, suggesting that a fraction of gamma-H2AX in the skin is eliminated in a DNA-PK-dependent manner. Although the DNA-PK-dependent elimination of gamma-H2AX was also detected in the liver, kidney, and spleen, the DNA-PK-dependent phosphorylation of H2AX was detected in the spleen only. These results suggest that the regulatory mechanism of gamma-H2AX level is tissue-specific.
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PMID:Tissue-specific DNA-PK-dependent H2AX phosphorylation and gamma-H2AX elimination after X-irradiation in vivo. 1875 45

DNA double-strand breaks (DSBs) are extremely dangerous lesions with severe consequences for cell survival and the maintenance of genomic stability. In higher eukaryotic cells, DSBs in chromatin promptly initiate the phosphorylation of the histone H2A variant, H2AX, at Serine 139 to generate gamma-H2AX. This phosphorylation event requires the activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs, ATM, and ATR, and serves as a landing pad for the accumulation and retention of the central components of the signaling cascade initiated by DNA damage. Regions in chromatin with gamma-H2AX are conveniently detected by immunofluorescence microscopy and serve as beacons of DSBs. This has allowed the development of an assay that has proved particularly useful in the molecular analysis of the processing of DSBs. Here, we first review the role of gamma-H2AX in DNA damage response in the context of chromatin and discuss subsequently the use of this modification as a surrogate marker for mechanistic studies of DSB induction and processing. We conclude with a critical analysis of the strengths and weaknesses of the approach and present some interesting applications of the resulting methodology.
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PMID:Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. 1877 27

O(6)-methylguanine (O(6)MeG) is a highly critical DNA adduct induced by methylating carcinogens and anticancer drugs such as temozolomide, streptozotocine, procarbazine and dacarbazine. Induction of cell death by O(6)MeG lesions requires mismatch repair (MMR) and cell proliferation and is thought to be dependent on the formation of DNA double-strand breaks (DSBs) or, according to an alternative hypothesis, direct signaling by the MMR complex. Given a role for DSBs in this process, either homologous recombination (HR) or non-homologous end joining (NHEJ) or both might protect against O(6)MeG. Here, we compared the response of cells mutated in HR and NHEJ proteins to temozolomide and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The data show that cells defective in HR (Xrcc2 and Brca2 mutants) are extremely sensitive to cell death by apoptosis and chromosomal aberration formation and less sensitive to sister-chromatid exchange (SCE) induction than the corresponding wild-type. Cells defective in NHEJ were not (Ku80 mutant), or only slightly more sensitive (DNA-PK(cs) mutant) to cell death and showed similar aberration and SCE frequencies than the corresponding wild-type. Transfection of O(6)-methylguanine-DNA methyltransferase (MGMT) in all of the mutants almost completely abrogated the genotoxic effects in both HR and NHEJ defective cells, indicating the mutant-specific hypersensitivity was due to O(6)MeG lesions. MNNG provoked H2AX phosphorylation 24-48h after methylation both in wild-type and HR mutants, which was not found in MGMT transfected cells. The gammaH2AX foci formed in response to O(6)MeG declined later in wild-type but not in HR-defective cells. The data support a model where DSBs are formed in response to O(6)MeG in the post-treatment cell cycle, which are repaired by HR, but not NHEJ, in a process that leads to SCEs. Therefore, HR can be considered as a mechanism that causes tolerance of O(6)MeG adducts. The data implicate that down-regulation or inhibition of HR might be a powerful strategy in improving cancer therapy with methylating agents.
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PMID:Brca2/Xrcc2 dependent HR, but not NHEJ, is required for protection against O(6)-methylguanine triggered apoptosis, DSBs and chromosomal aberrations by a process leading to SCEs. 1884 May 49

Little information is available on the ability of terminally differentiated cells to efficiently repair DNA double strand breaks (DSBs), and one might reasonably speculate that efficient DNA repair of these threatening DNA lesions, is needed in cells of long life span with no or limited regeneration from precursor. Few tissues are available besides neurons that allow the study of DNA DSBs repair activity in very long-lived cells. Adipocytes represent a suitable model since it is generally admitted that there is a very slow turnover of adipocytes in adult. Using both Pulse Field Gel Electrophoresis (PFGE) and the disappearance of the phosphorylated form of the histone variant H2AX, we demonstrated that the ability to repair DSBs is increased during adipocyte differentiation using the murine pre-adipocyte cell line, 3T3F442A. In mammalian cells, DSBs are mainly repaired by the non-homologous end-joining pathway (NHEJ) that relies on the DNA dependent protein kinase (DNA-PK) activity. During the first 24 h following the commitment into adipogenesis, we show an increase in the expression and activity of the catalytic sub-unit of the DNA-PK complex, DNA-PKcs. The increased in DNA DSBs repair activity observed in adipocytes was due to the increase in DNA-PK activity as shown by the use of DNA-PK inhibitor or sub-clones of 3T3F442A deficient in DNA-PKcs using long term RNA interference. Interestingly, the up-regulation of DNA-PK does not regulate the differentiation program itself. Finally, similar positive regulation of DNA-PKcs expression and activity was observed during differentiation of primary culture of pre-adipocytes isolated from human sub-cutaneous adipose tissue. Our results show that DNA DSBs repair activity is up regulated during the early commitment into adipogenesis due to an up-regulation of DNA-PK expression and activity. In opposition to the general view that DNA DSBs repair is decreased during differentiation, our results demonstrate that an up-regulation of this process might be observed in post-mitotic long-lived cells.
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PMID:Positive regulation of DNA double strand break repair activity during differentiation of long life span cells: the example of adipogenesis. 1884 13

Cytotoxicity and DNA double strand breaks (DSBs) were studied in HeLa cells treated with sulforaphane (SFN), a well-known chemo-preventive agent. Cell survival was impaired by SFN in a concentration and treatment time-dependent manner. Both constant field gel electrophoresis (CFGE) and gamma-H2AX assay unambiguously indicated formation of DSBs by SFN, reflecting the cell survival data. These DSBs were predominantly processed by homologous recombination repair (HRR), judging from the SFN concentration-dependent manner of Rad51 foci formation. On the other hand, the phosphorylation of DNA-PKcs, a key non-homologous end joining (NHEJ) protein, was not observed by SFN treatment, suggesting that NHEJ may not be involved in DSBs induced by this chemical. G2/M arrest by SFN, a typical response for cells exposed to ionizing radiation was also observed. Our new data indicate the clear induction of DSBs by SFN and a useful anti-tumor aspect of SFN through the induction of DNA DSBs.
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PMID:Sulforaphane induces DNA double strand breaks predominantly repaired by homologous recombination pathway in human cancer cells. 1885 74

TRAIL is an endogenous death receptor ligand also used therapeutically because of its selective proapoptotic activity in cancer cells. In the present study, we examined chromatin alterations induced by TRAIL and show that TRAIL induces a rapid activation of DNA damage response (DDR) pathways with histone H2AX, Chk2, ATM, and DNA-PK phosphorylations. Within 1 h of TRAIL exposure, immunofluorescence confocal microscopy revealed gamma-H2AX peripheral nuclear staining (gamma-H2AX ring) colocalizing with phosphorylated/activated Chk2, ATM, and DNA-PK inside heterochromatin regions. The marginal distribution of DDR proteins in early apoptotic cells is remarkably different from the focal staining seen after DNA damage. TRAIL-induced DDR was suppressed upon caspase inhibition or Bax inactivation, demonstrating that the DDR activated by TRAIL is downstream from the mitochondrial death pathway. H2AX phosphorylation was dependent on DNA-PK, while Chk2 phosphorylation was dependent on both ATM and DNA-PK. Downregulation of Chk2 decreased TRAIL-induced cell detachment; delayed the activation of caspases 2, 3, 8, and 9; and reduced TRAIL-induced cell killing. Together, our findings suggest that nuclear activation of Chk2 by TRAIL acts as a positive feedback loop involving the mitochondrion-dependent activation of caspases, independently of p53.
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PMID:Death receptor-induced activation of the Chk2- and histone H2AX-associated DNA damage response pathways. 1895

Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax.Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased DNA-PK activity, and specific inhibition of DNA-PK prevented Tax-induced activation of Chk2 kinase activity. Expression of Tax induced foci formation and phosphorylation of H2AX. However, Tax-induced constitutive signaling of the DNA-PK pathway impaired cellular response to new damage, as reflected in suppression of ionizing radiation-induced DNA-PK phosphorylation and gammaH2AX stabilization. Tax co-localized with phospho-DNA-PK into nuclear speckles and a nuclear excluded Tax mutant sequestered endogenous phospho-DNA-PK into the cytoplasm, suggesting that Tax interaction with DNA-PK is an initiating event. We also describe a novel interaction between DNA-PK and Chk2 that requires Tax. We propose that Tax binds to and stabilizes a protein complex with DNA-PK and Chk2, resulting in a saturation of DNA-PK-mediated damage repair response.
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PMID:HTLV-1 Tax oncoprotein subverts the cellular DNA damage response via binding to DNA-dependent protein kinase. 1895 25

To identify the repair dynamics involved in high linear energy transfer (LET) radiation-induced DNA damage, phospho-H2AX (gammaH2AX) foci formation was analyzed after cellular exposure to iron ions (Fe-ions, 500 MeV u(-1), 200 KeV microm(-1)). The foci located at DNA damage sites were visualized using immunocytochemical methods. Since H2AX is phosphorylated at sites of radiation-induced double strand breaks (DSB), gammaH2AX foci were used to detect or illuminate tracks formed by DSB after exposure to various doses of ionizing radiation. Additional DSB-recognition proteins such as ATM phospho-serine 1981, DNA-PKcs phospho-threonine 2609, NBS1 phospho-serine 343 and CHK2 phospho-threonine 68 all co-localized with gammaH2AX at high LET radiation induced DSB. In addition, Fe-ion induced foci remained for longer times than X-radiation induced foci. These findings suggest that Fe-ion induced damage is repaired more slowly than X-radiation induced damage, possibly because Fe-ion induced damage or lesions are more complex or extensive. Antibodies for all these phosphorylated DNA DSB recognition proteins appear to be very effective for the detection and localization of DSB.
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PMID:DNA damage recognition proteins localize along heavy ion induced tracks in the cell nucleus. 1898 40


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