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)

Here we document the role of MDC1 (mediator of DNA damage checkpoint 1) in the detection and repair of human and mouse telomeres rendered dysfunctional through inhibition of TRF2. Consistent with its role in promoting DNA damage foci, MDC1 knockdown affected the formation of telomere dysfunction-induced foci (TIFs), diminishing the accumulation of phosphorylated ATM, 53BP1, Nbs1, and to a lesser extent, gamma-H2AX. In addition to this effect on TIFs, the rate of nonhomologous end-joining (NHEJ) of dysfunctional telomeres was significantly decreased when MDC1 itself or its recruitment to chromatin was inhibited. MDC1 appeared to promote a step in the NHEJ pathway after the removal of the 3' telomeric overhang. The acceleration of NHEJ was unlikely to be due to increased presence of 53BP1 and Mre11 in TIFs, since knockdown of neither factor affected telomere fusions. Furthermore, relevant cell cycle effectors (Chk2, p53, and p21) of the ATM kinase pathway were unaffected and there was no change in the rate of cell cycle progression. We propose that the binding of MDC1 to gamma-H2AX directly affects NHEJ in a manner that is independent of the ATM-dependent cell cycle arrest pathway.
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PMID:MDC1 accelerates nonhomologous end-joining of dysfunctional telomeres. 1715 42

Chk1 and Chk2 kinases are critically involved in modulating DNA damage checkpoints. In particular, Chk1, a key activator of the S-phase DNA damage response, may be involved in resistance to genotoxic therapies that target DNA synthesis. We studied the in vitro and in vivo effects of EXEL-9844 (XL844), a potent, orally available, and specific inhibitor of Chk1 and Chk2, in combination with gemcitabine. In clonogenic assays using multiple cell lines in vitro, EXEL-9844 had only minor effects as a single agent but substantially enhanced gemcitabine-induced cell killing. Correspondingly, in PANC-1 cells, EXEL-9844 increased gemcitabine-induced H2AX phosphorylation, blocked Cdc25A phosphorylation, and induced premature mitotic entry. In a PANC-1 xenograft model, EXEL-9844 significantly enhanced gemcitabine antitumor activity but had limited effect as a single agent. Together, these data show that cell cycle checkpoint inhibitors may have significant clinical utility in potentiating the activity of gemcitabine.
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PMID:Pharmacological abrogation of S-phase checkpoint enhances the anti-tumor activity of gemcitabine in vivo. 1724 19

Thermionic emission (TE)-type X-ray generators have been exclusively used in medicine, but there are many difficulties in making these X-ray sources compact. A field emission (FE)-type X-ray generator using carbon nanotubes is a newly-developed compact system that can be as small as several cm in length. Considering the compactness of the equipment, the FE-type X-ray generator may become a useful tool for endoscopic, intracavitary or intraoperative radiotherapy in the future. The aim of this study was to investigate the biological effects of X-rays generated by the FE-type X-ray source in comparison with those of conventional TE-type X-rays. Mouse thymic lymphoma 3SB cells were irradiated by an FE-type X-ray generator developed by our group and a conventional TE-type X-ray source under identical conditions. DNA damage after radiation was detected by foci formation of phospho-H2AX (gamma-H2AX). Effect on the cell cycle was analyzed by flow cytometry. Activation of the DNA damage checkpoint was analyzed by immunoblotting. Induction of apoptosis was studied using the TUNEL assay. In terms of induction of DNA damage (DNA double-strand breaks), activation of cell cycle checkpoints (p53 stabilization, p21 induction, Chk1 and Chk2 phosphorylations), and induction of apoptotic cell death, FE-type X-rays were as effective as TE-type X-rays, and FE-type X-rays appeared to be applicable to radiation therapy.
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PMID:Biological effects of field emission-type X-rays generated by nanotechnology. 1731 70

Here we investigated the cytotoxicity of JS-K, a prodrug designed to release nitric oxide (NO(*)) following reaction with glutathione S-transferases, in multiple myeloma (MM). JS-K showed significant cytotoxicity in both conventional therapy-sensitive and -resistant MM cell lines, as well as patient-derived MM cells. JS-K induced apoptosis in MM cells, which was associated with PARP, caspase-8, and caspase-9 cleavage; increased Fas/CD95 expression; Mcl-1 cleavage; and Bcl-2 phosphorylation, as well as cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (EndoG) release. Moreover, JS-K overcame the survival advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells. Mechanistic studies revealed that JS-K-induced cytotoxicity was mediated via NO(*) in MM cells. Furthermore, JS-K induced DNA double-strand breaks (DSBs) and activated DNA damage responses, as evidenced by neutral comet assay, as well as H2AX, Chk2 and p53 phosphorylation. JS-K also activated c-Jun NH(2)-terminal kinase (JNK) in MM cells; conversely, inhibition of JNK markedly decreased JS-K-induced cytotoxicity. Importantly, bortezomib significantly enhanced JS-K-induced cytotoxicity. Finally, JS-K is well tolerated, inhibits tumor growth, and prolongs survival in a human MM xenograft mouse model. Taken together, these data provide the preclinical rationale for the clinical evaluation of JS-K to improve patient outcome in MM.
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PMID:JS-K, a GST-activated nitric oxide generator, induces DNA double-strand breaks, activates DNA damage response pathways, and induces apoptosis in vitro and in vivo in human multiple myeloma cells. 1738 1

In addition to conferring an indefinite replicative life span, telomerase renders p16(-) human mammary epithelial cells (HMEC) resistant to growth arrest by TGFbeta or by loss of EGF or insulin signaling. In contrast to earlier reports, we recently found that growth factor signaling was not directly affected by telomerase expression. Rather, short dysfunctional or near-dysfunctional telomeres in proliferating telomerase(-) HMEC sensitized the cells to p53-dependent signals for growth arrest. We showed that during serial passage and before any signs of replicative senescence, HMEC lacking telomerase experience enhanced p53 stability and DNA damage signaling, as determined by increased phosphorylation on p53-Ser15 and Chk2-Thr68, and formation of 53BP1/phosphorylated histone H2AX foci at chromosome ends. This heightened activity of the p53 pathway enhanced the efficiency with which cells arrested growth in response to TGFbeta or to EGF or insulin withdrawal, and was abolished by ectopic expression of hTERT, the catalytic subunit of telomerase. Telomerase elongated short telomeres, thereby reducing the basal level of activated p53 and raising cellular tolerance for other p53-dependent signals, including those emanating from non-genotoxic sources. These findings explain a number of observed effects of telomerase expression on cell growth and survival without postulating additional functions for telomerase.
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PMID:Soothing the watchman: telomerase reduces the p53-dependent cellular stress response. 1753 47

DNA damage response (DDR) is emerging as a physiological anti-cancer barrier in early stages of cancer development, as shown for several types of solid cancers derived from somatic cells. Here we discuss our recently published and unpublished results on the exceptional paucity of such constitutive activation of the DDR machinery in human testicular germ cell tumours (TGCTs), including their common pre-invasive stage of carcinoma in situ (CIS). Our conclusions are supported by immunohistochemical analyses of multiple markers of activated DNA damage signalling, such as the phosphorylated ATM and Chk2 checkpoint kinases and phosphorylated histone H2AX. We propose that the unique lack of DDR activation in TGCTs reflects the biology of their cell of origin, the gonocyte. Furthermore, we propose that the lack of DDR activation avoids the pressure to select for mutations in DDR genes such as p53 or ATM, and the resulting intact DDR machinery may have implications for the exceptional curability of TGCTs by DNA damaging therapies.
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PMID:DNA damage response in human testes and testicular germ cell tumours: biology and implications for therapy. 1757 48

In this study, we investigated the cytotoxicity of 5-azacytidine, a DNA methyltransferase inhibitor, against multiple myeloma (MM) cells, and characterized DNA damage-related mechanisms of cell death. 5-Azacytidine showed significant cytotoxicity against both conventional therapy-sensitive and therapy-resistant MM cell lines, as well as multidrug-resistant patient-derived MM cells, with IC(50) of approximately 0.8-3 micromol/L. Conversely, 5-azacytidine was not cytotoxic to peripheral blood mononuclear cells or patient-derived bone marrow stromal cells (BMSC) at these doses. Importantly, 5-azacytidine overcame the survival and growth advantages conferred by exogenous interleukin-6 (IL-6), insulin-like growth factor-I (IGF-I), or by adherence of MM cells to BMSCs. 5-Azacytidine treatment induced DNA double-strand break (DSB) responses, as evidenced by H2AX, Chk2, and p53 phosphorylations, and apoptosis of MM cells. 5-Azacytidine-induced apoptosis was both caspase dependent and independent, with caspase 8 and caspase 9 cleavage; Mcl-1 cleavage; Bax, Puma, and Noxa up-regulation; as well as release of AIF and EndoG from the mitochondria. Finally, we show that 5-azacytidine-induced DNA DSB responses were mediated predominantly by ATR, and that doxorubicin, as well as bortezomib, synergistically enhanced 5-azacytidine-induced MM cell death. Taken together, these data provide the preclinical rationale for the clinical evaluation of 5-azacytidine, alone and in combination with doxorubicin and bortezomib, to improve patient outcome in MM.
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PMID:5-Azacytidine, a DNA methyltransferase inhibitor, induces ATR-mediated DNA double-strand break responses, apoptosis, and synergistic cytotoxicity with doxorubicin and bortezomib against multiple myeloma cells. 1757 3

The Bloom syndrome helicase (BLM) is critical for genomic stability. A defect in BLM activity results in the cancer-predisposing Bloom syndrome (BS). Here, we report that BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX (gamma-H2AX), Chk2 (p(T68)Chk2), and ATM (p(S1981)ATM) colocalizing in nuclear foci. Interestingly, the mitotic fraction of gamma-H2AX foci did not seem to be higher in BLM-deficient cells, indicating that these lesions form transiently during interphase. Pulse labeling with iododeoxyuridine and immunofluorescence microscopy showed the colocalization of gamma-H2AX, ATM, and Chk2 together with replication foci. Those foci costained for Rad51, indicating homologous recombination at these replication sites. We therefore analyzed replication in BS cells using a single molecule approach on combed DNA fibers. In addition to a higher frequency of replication fork barriers, BS cells displayed a reduced average fork velocity and global reduction of interorigin distances indicative of an elevated frequency of origin firing. Because BS is one of the most penetrant cancer-predisposing hereditary diseases, it is likely that the lack of BLM engages the cells in a situation similar to precancerous tissues with replication stress. To our knowledge, this is the first report of high ATM-Chk2 kinase activation and its linkage to replication defects in a BS model.
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PMID:Endogenous gamma-H2AX-ATM-Chk2 checkpoint activation in Bloom's syndrome helicase deficient cells is related to DNA replication arrested forks. 1763 26

In Xenopus laevis embryos, the midblastula transition (MBT) at the 12th cell division marks initiation of critical developmental events, including zygotic transcription and the abrupt inclusion of gap phases into the cell cycle. Interestingly, although an ionizing radiation-induced checkpoint response is absent in pre-MBT embryos, introduction of a threshold amount of undamaged plasmid or sperm DNA allows a DNA damage checkpoint response to be activated. We show here that undamaged threshold DNA directly participates in checkpoint signaling, as judged by several dynamic changes, including H2AX phosphorylation, ATM phosphorylation and loading onto chromatin, and Chk1/Chk2 phosphorylation and release from nuclear DNA. These responses on physically separate threshold DNA require gamma-H2AX and are triggered by an ATM-dependent soluble signal initiated by damaged DNA. The signal persists in egg extracts even after damaged DNA is removed from the system, indicating that the absence of damaged DNA is not sufficient to end the checkpoint response. The results identify a novel mechanism by which undamaged DNA enhances checkpoint signaling and provide an example of how the transition to cell cycle checkpoint activation during development is accomplished by maternally programmed increases in the DNA-to-cytoplasm ratio.
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PMID:Undamaged DNA transmits and enhances DNA damage checkpoint signals in early embryos. 1766 86

Whether Chk2 contributes to DNA damage-induced arrest in G2 has been controversial. To investigate this issue further, we generated Chk2-deficient DT40 B-lymphoma cells by gene targeting and compared their cell cycle response to ionizing radiation (IR) with wild-type (WT) and isogenic Chk1-deficient counterparts. After moderate doses of IR (4 Gy), we find that Chk2-/- cells which are in G1 or S phase at the time of irradiation arrest efficiently in G2. In contrast, Chk2-/- cells which are in G2 when DNA damage is incurred exhibit an impaired mitotic delay compared to WT, with the result that cells enter mitosis with damaged DNA as judged by the presence of numerous gamma-H2AX foci on condensed chromosomes. Impaired G2 delay as the result of Chk2 deficiency can be detected at very low doses of radiation (0.1 Gy), and may allow division with spontaneous DNA damage, since a higher proportion of mitotic Chk2-/- cells bear spontaneous gamma-H2AX foci and damaged chromosomes during unperturbed growth compared to WT. The contribution of Chk2 to G2/M delay is epistatic to that of Chk1, since Chk1-/- cells exhibit no measurable mitotic delay at any radiation dose tested. We suggest that this function of Chk2 could contribute to tumour suppression, since cell division with low levels of spontaneous damage is likely to promote genetic instability and thus carcinogenesis.
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PMID:Chk2 is required for optimal mitotic delay in response to irradiation-induced DNA damage incurred in G2 phase. 1768 83


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