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 tumor suppressor p53 binding protein 1 (53BP1) binds to the DNA-binding domain of p53 and enhances p53-mediated transcriptional activation. 53BP1 contains two breast cancer susceptibility gene 1 COOH terminus (BRCT) motifs, which are present in several proteins involved in DNA repair and/or DNA damage-signaling pathways. Thus, we investigated the potential role of 53BP1 in DNA damage-signaling pathways. Here, we report that 53BP1 becomes hyperphosphorylated and forms discrete nuclear foci in response to DNA damage. These foci colocalize at all time points with phosphorylated H2AX (gamma-H2AX), which has been previously demonstrated to localize at sites of DNA strand breaks. 53BP1 foci formation is not restricted to gamma-radiation but is also detected in response to UV radiation as well as hydroxyurea, camptothecin, etoposide, and methylmethanesulfonate treatment. Several observations suggest that 53BP1 is regulated by ataxia telangiectasia mutated (ATM) after DNA damage. First, ATM-deficient cells show no 53BP1 hyperphosphorylation and reduced 53BP1 foci formation in response to gamma-radiation compared with cells expressing wild-type ATM. Second, wortmannin treatment strongly inhibits gamma-radiation-induced hyperphosphorylation and foci formation of 53BP1. Third, 53BP1 is readily phosphorylated by ATM in vitro. Taken together, these results suggest that 53BP1 is an ATM substrate that is involved early in the DNA damage-signaling pathways in mammalian cells.
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PMID:Tumor suppressor p53 binding protein 1 (53BP1) is involved in DNA damage-signaling pathways. 1133 10

NFBD1/KIAA0170 is a nuclear factor with an N-terminal FHA (forkhead-associated) domain and a tandem repeat of BRCT (breast cancer susceptibility gene-1 C terminus) domains, both of which are present in a number of proteins involved in DNA repair and/or DNA damage signaling pathways. We have investigated the association of NFBD1 with DNA damage responses. We found that the NFBD1 transcript is abundant in the testis relative to other tissues. NFBD1 is a chromatin-associated protein and is modified in G(2)/M phase or after DNA damage. NFBD1 phosphorylation in response to ionizing radiation (IR) was ATM-dependent. NFBD1 exhibited diffuse nuclear staining in the majority of untreated cells analyzed by indirect immunofluorescence and formed discrete nuclear foci after exposure to IR, UV radiation, and hydroxyurea treatment. IR induced NFBD1 foci within 1 min. The foci colocalized with gamma-H2AX foci, which have been previously shown to localize at sites of DNA double-strand breaks. IR-induced NFBD1 foci also colocalized with 53BP1 and MRE11/RAD50 foci. Taken together, these results suggest that NFBD1 is a mediator of DNA damage-dependent signaling.
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PMID:NFBD1/KIAA0170 is a chromatin-associated protein involved in DNA damage signaling pathways. 1249 69

Signaling pathways in response to DNA double strand breaks involve molecular cascades consisting of sensors, transducers, and effector proteins that activate cell cycle checkpoints and recruit repair machinery proteins. NFBD1 (a nuclear factor with BRCT domains protein 1) contains FHA (forkhead-associated), BRCT (breast cancer susceptibility gene 1 carboxyl terminus) domains, and internal repeats and is an early participant in nuclear foci in response to IR. To elucidate its role in the response pathways, small interfering RNA (siRNA) directed against NFDB1 in human cells demonstrated that its absence is associated with increased radio-sensitivity and delayed G(2)/M transition, but not G(1) to S. NFBD1 associates with nuclear foci within minutes following IR, a property similar to histone H2AX, 53BP1, and Chk2, which are all early participants in the DNA damage signaling cascade. Temporal studies show that H2AX is required for the foci positive for NFBD1, but NFBD1 is not needed for 53BP1- and H2AX-positive foci. NFBD1, together with 53BP1, plays a partially redundant role in regulating phosphorylation of the downstream effector protein, Chk2, since abrogation of both diminishes phosphorylated Chk2 in IR-induced foci. These results place NFBD1 parallel to 53BP1 in regulating Chk2 and downstream of H2AX in the recruitment of repair and signaling proteins to sites of DNA damage.
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PMID:NFBD1, like 53BP1, is an early and redundant transducer mediating Chk2 phosphorylation in response to DNA damage. 1255 34

NFBD1/MDC1 (mediator of DNA damage checkpoint 1) is a nuclear factor with an amino-terminal FHA (forkhead-associated) domain and a tandem repeat of BRCT (breast cancer susceptibility gene-1 carboxyl terminus) domains. We have previously shown that NFBD1 is an early participant in DNA damage signaling pathways and that ionizing radiation-induced nuclear foci (IRIF) of NFBD1 colocalize with several DNA checkpoint signaling and repair factors. We report here that NFBD1 physically associates with ATM, p53, components of the MRE11-RAD50-NBS1 (MRN) complex, and gamma-H2AX. An overexpressed FHA domain-containing fragment of NFBD1 binds to endogenous NFBD1 and components of the MRN complex, but not to gamma-H2AX. This fragment interferes with IRIF formation by endogenous NFBD1, MRE11, or NBS1. A BRCT domain-containing fragment of NFBD1 binds to gamma-H2AX and 53BP1, but not to components of the MRN complex, and abolishes IRIF formation by NFBD1, MRE11, NBS1, 53BP1, CHK2 phospho-T68, gamma-H2AX, and possible ATM/ATR substrates recognized by anti-phospho-SQ/TQ antibody. These results suggest that NFBD1 is an ATM/ATR-dependent organizer that recruits DNA checkpoint signaling and repair proteins to the sites of DNA damage.
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PMID:NFBD1/MDC1 regulates ionizing radiation-induced focus formation by DNA checkpoint signaling and repair factors. 1451 63

Because treatment regimens for breast cancer commonly include gemcitabine, we evaluated two promising combinations in preclinical studies: gemcitabine (Gemzar; Eli Lilly and Company, Indianapolis, IN) with either ionizing radiation or docetaxel (Taxotere; Aventis Pharmaceuticals, Inc, Parsippany, NJ). In breast cancer cell lines that expressed either wild-type p53 (MCF-7) or mutant p53 (MCF-7/Adr), sensitivity to the cytotoxic effects of gemcitabine during a 24-hour incubation was similar (IC(50) values 80 and 60 nmol/L in MCF-7 and MCF-7/Adr, respectively). Both cell lines were well radiosensitized by gemcitabine at the corresponding IC(50), with radiation enhancement ratios of 1.6 to 1.7. Although the MCF-7 cells accumulated nearly twice as much gemcitabine triphosphate compared with the MCF-7/Adr cells, a similar reduction in 2'-deoxyadenosine 5'-triphosphate pools was observed. While the number of dying cells, as measured by sub-G1 DNA content or S-phase cells unable to replicate DNA, differed between the wild-type p53 or mutant p53-expressing cell lines, neither parameter correlated with radiosensitization. Docetaxel was a more potent cytotoxic agent than gemcitabine in MCF-7 cells (IC(50) = 1 nmol/L). Strong synergistic cytotoxicity was observed in cells treated with gemcitabine (24 hours) followed by docetaxel (24 hours) or the reverse sequence. However, simultaneous addition of the two drugs was antagonistic. To determine whether synergy with radiation or docetaxel was mediated by increased DNA damage, DNA double-strand breaks (double-strand breaks) were measured by immunostaining for phosphorylated H2AX. Ionizing radiation produced more double-strand breaks than gemcitabine alone, while no significant double-strand breaks formed with docetaxel alone. The addition of docetaxel or ionizing radiation to gemcitabine-treated cells did not increase H2AX foci formation. These results show that the combination of gemcitabine with ionizing radiation or docetaxel produces strong, schedule-dependent synergy in breast cancer cells that is not mediated through increasing DNA double-strand breaks.
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PMID:Promising combination therapies with gemcitabine. 1519 26

The phytochemical indole-3-carbinol (I3C), from cruciferous vegetables such as broccoli, has been shown to elicit a potent anti-proliferative response in human breast cancer cell lines. Treatment of the immortalized human mammary epithelial cell line MCF10A with I3C induced a G1 cell cycle arrest, elevated p53 tumor suppressor protein levels and stimulated expression of downstream transcriptional target, p21. I3C treatment also elevated p53 levels in several breast cancer cell lines that express mutant p53. I3C did not arrest MCF10A cells stably transfected with dominant-negative p53, establishing a functional requirement for p53. Cell fractionation and immunolocalization studies revealed a large fraction of stabilized p53 protein in the nucleus of I3C-treated MCF10A cells. With I3C treatment, phosphatidyl-inositol-3-kinase family member ataxia telangiectasia-mutated (ATM) was phosphorylated, as were its substrates p53, CHK2 and BRCA1. Phosphorylation of p53 at the N-terminus has previously been shown to disrupt the interaction between p53 and its ubiquitin ligase, MDM2, and therefore stabilizing p53. Coimmunoprecipitation analysis revealed that I3C reduced by 4-fold the level of MDM2 protein that associated with p53. The p53-MDM2 interaction and absence of p21 production were restored in cells treated with I3C and the ATM inhibitor wortmannin. Significantly, I3C does not increase the number of 53BP1 foci or H2AX phosphorylation, indicating that ATM is activated independent of DNA double-strand breaks. Taken together, our results demonstrate that I3C activates ATM signaling through a novel pathway to stimulate p53 phosphorylation and disruption of the p53-MDM2 interaction, which releases p53 to induce the p21 CDK inhibitor and a G1 cell cycle arrest.
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PMID:Indole-3-carbinol activates the ATM signaling pathway independent of DNA damage to stabilize p53 and induce G1 arrest of human mammary epithelial cells. 1615 27

on-homologous end joining (NHEJ) and homologous recombination (HR) are pathways that repair DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, the repair of these breaks is influenced by histone acetylation. Therefore, we tested mammalian cells deleted for NHEJ (Ku80 or DNA Ligase IV) or altered for HR (breast cancer associated gene, Brca2, or Bloom's syndrome, Blm) for sensitivity to trichostatin A (TSA), a histone deacetylase inhibitor that is being investigated as an anti-cancer therapeutic. We show that cells mutated for Ku80 (ku80-/-) or DNA Ligase IV (lig 4-/-), but not cells mutated for Brca2 (brca2lex1/lex2) or Blm (blm(tm3Brd/tm4Brd)), are hypersensitive to TSA in a dose-dependent manner. TSA-induced toxicity stimulates apoptosis and cell cycle checkpoint responses independent of p53, but does not increase phosphorylated histone H2AX (-H2AX) as compared with a clastogenic agent, camptothecin, indicating that the quantity of DSBs is not the primary cause of TSA-induced cell death. In addition, we show that potential anti-cancer drugs (LY-294002 and vanillin) that inhibit the family of phosphatidylinositol 3 kinases that include the NHEJ protein, DNA-PKCS act in synergy with TSA to reduce the viability of HeLa cells in tissue culture presenting the possibility of using the two drugs in combination to treat cancer.
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PMID:Non-homologous end joining, but not homologous recombination, enables survival for cells exposed to a histone deacetylase inhibitor. 1617 81

DNA-PK and ATM are members of the phosphatidylinositol 3'-kinase like kinase (PIKK) family of serine/threonine protein kinases and have critical roles in the cellular response to DNA double-strand breaks. Genetic loss of either activity leads to pronounced sensitivity to ionizing radiation (IR). Hence, these enzymes are potential targets to confer enhanced radiosensitivity on tumour cells. We show that novel inhibitors of either DNA-PK or ATM sensitize breast carcinoma cells to IR. Radiosensitization was accompanied by an apparent DNA repair deficit as measured by the persistence of IR-induced foci of phosphorylated histone H2AX (gammaH2AX foci). These specific inhibitors also allowed us to probe the biochemistry and kinetics of histone H2AX phosphorylation following gamma-irradiation in breast cancer cells with the aim of validating H2AX as a biomarker for DNA-PK or ATM inhibition in vivo. ATM inhibition reduced the initial average intensity of gammaH2AX foci while inhibition of DNA-PK had only a small effect on the initial phosphorylation of H2AX. However, simultaneous treatment with both compounds dramatically reduced gammaH2AX focus intensity, consistent with the reported role of ATM and DNA-PK in IR induced phosphorylation of H2AX.
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PMID:Sensitization of breast carcinoma cells to ionizing radiation by small molecule inhibitors of DNA-dependent protein kinase and ataxia telangiectsia mutated. 1629 33

The BRCA2 protein is involved in the maintenance of genomic stability through its key role in homologous recombination repair of DNA double strand breaks. Biallelic inactivation of BRCA2 leads to a defect in DNA repair and is associated with a chromosomal instability phenotype. Recent studies on familial breast cancer clusters revealed chromosomal rearrangements and higher rates of sister chromatid exchanges also in heterozygous BRCA2 mutation carriers. In the present study, lymphoblastoid cell lines of heterozygous BRCA2 mutation carriers and of wildtype relatives were compared with regard to BRCA2 mRNA and protein expression and capacity to repair DNA damage induced by gamma-irradiation and mitomycin C. BRCA2+/- cells showed lower amounts of the full-length BRCA2 protein compared to BRCA2+/+ cells. The kinetics of gamma-H2AX protein level revealed distinct defects in DNA double strand break repair in the BRCA2+/- cells. These results are indicative of a haploinsufficiency phenotype in BRCA2+/- cells, suggesting that reduced amounts of functional BRCA2 protein in BRCA2+/- carriers are insufficient for an efficient repair of DNA double strand breaks, a condition that could contribute to the impairment of genomic stability.
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PMID:Lower level of BRCA2 protein in heterozygous mutation carriers is correlated with an increase in DNA double strand breaks and an impaired DSB repair. 1644 46

The link between defects in BRCA1 and breast cancer development may be best understood by deciphering the role of associated proteins. BRCA1 associated C-terminal helicase (BACH1) interacts directly with the BRCA1 C-terminal BRCT repeats, which are important for BRCA1 DNA repair and are mutated in the majority of BRCA1 familial cancers. Thus, BACH1 is a likely candidate for mediating BRCA1 DNA repair and tumor suppression functions. Although previous evidence using overexpression of a dominant negative BACH1 has suggested that BACH1 is involved in BRCA1-DNA repair function, our results using BACH1 deficient cells provide direct evidence for involvement of BACH1 in DNA repair as well as for localizing BRCA1. Following DNA damage BACH1 is modified by phosphorylation, displays a BRCA1-like nuclear foci pattern and colocalizes with gamma-H2AX. Given that the BACH1/BRCA1 complex is unaltered by DNA damage and the intensity of BRCA1 foci is diminished in BACH1 deficient cells, BACH1 may serve to not only facilitate DNA repair, but also maintain BRCA1 in DNA damage foci.
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PMID:BACH1 is a DNA repair protein supporting BRCA1 damage response. 1646 73


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