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)

Chromosome segregation errors are a significant cause of aneuploidy among human neonates and often result from errors in female meiosis that occur during fetal life. For the latter reason, little is known about chromosome dynamics during female prophase I. Here, we analyzed chromosome reorganization, and centromere and telomere dynamics in meiosis in the human female by immunofluorescent staining of the SYCP3 and SYCP1 synaptonemal complex proteins and the course of recombinational DNA repair by IF of phospho-histone H2A.X (gamma-H2AX), RPA and MLH1 recombination proteins. We found that SYCP3, but not SYCP1, aggregates appear in the preleptotene nucleus and some persist up to pachytene. Telomere clustering (bouquet stage) in oocytes lasted from late-leptotene to early pachytene-significantly longer than in the male. Leptotene and zygotene oocytes and spermatocytes showed strong gamma-H2AX labeling, while gamma-H2AX patches, which colocalized with RPA, were present on SYCP1-tagged pachytene SCs. This was rarely seen in the male and may suggest that synapsis installs faster with respect to progression of recombinational double-strand break repair or that the latter is slower in the female. It is speculated that the presence of gamma-H2AX into pachytene highlights female-specific peculiarities of recombination, chromosome behavior and checkpoint control that may contribute to female susceptibility for aneuploidy.
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PMID:Female-specific features of recombinational double-stranded DNA repair in relation to synapsis and telomere dynamics in human oocytes. 1523 94

Human replication protein A (RPA p34), a crucial component of diverse DNA excision repair pathways, is implicated in DNA double-strand break (DSB) repair. To evaluate its role in DSB repair, the intranuclear dynamics of RPA was investigated after DNA damage and replication blockage in human cells. Using two different agents [ionizing radiation (IR) and hydroxyurea (HU)] to generate DSBs, we found that RPA relocated into distinct nuclear foci and colocalized with a well-known DSB binding factor, gamma-H2AX, at the sites of DNA damage in a time-dependent manner. Colocalization of RPA and gamma-H2AX foci peaked at 2 h after IR treatment and subsequently declined with increasing postrecovery times. The time course of RPA and gamma-H2AX foci association correlated well with the DSB repair activity detected by a neutral comet assay. A phosphatidylinositol-3 (PI-3) kinase inhibitor, wortmannin, completely abolished both RPA and gamma-H2AX foci formation triggered by IR. Additionally, radiosensitive ataxia telangiectasia (AT) cells harboring mutations in ATM gene product were found to be deficient in RPA and gamma-H2AX colocalization after IR. Transfection of AT cells with ATM cDNA fully restored the association of RPA foci with gamma-H2AX illustrating the requirement of ATM gene product for this process. The exact coincidence of RPA and gamma-H2AX in response to HU specifically in S-phase cells supports their role in DNA replication checkpoint control. Depletion of RPA by small interfering RNA (SiRNA) substantially elevated the frequencies of IR-induced micronuclei (MN) and apoptosis in human cells suggestive of a role for RPA in DSB repair. We propose that RPA in association with gamma-H2AX contributes to both DNA damage checkpoint control and repair in response to strand breaks and stalled replication forks in human cells.
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PMID:Replication protein A and gamma-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks. 1547 97

Human checkpoint kinase 1 (Chk1) is an essential kinase required to preserve genome stability. Here, we show that Chk1 inhibition by two distinct drugs, UCN-01 and CEP-3891, or by Chk1 small interfering RNA (siRNA) leads to phosphorylation of ATR targets. Chk1-inhibition triggered rapid, pan-nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in cancer treatment.
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PMID:Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage. 1583 61

The integrity of genomic DNA during the cell division cycle in eukaryotic cells is maintained by regulated chromosomal DNA replication and repair of damaged DNA. We have used fractionation and reconstitution experiments to purify essential factors for the initiation of human chromosomal DNA replication in late G1 phase template nuclei from human cells. Here, we report the identification of soluble PCNA as an essential initiation factor in this system. Recombinant histidine-tagged human PCNA can substitute for purified endogenous human PCNA to initiate human chromosomal DNA replication. It is recruited specifically to discrete DNA replication foci formed during initiation in vitro. The template nuclei also contain DNA breaks as result of the synchronisation procedure. A separate population of chromatin-bound PCNA is already present in these template nuclei at discrete DNA damage foci, co-localising with gamma-H2AX, RPA and Rad51. This DNA damage-associated PCNA population is marked by mono-ubiquitination, suggesting that it is involved in DNA repair. Importantly, the population of damage focus-associated PCNA is neither involved in, nor required for, the initiation of chromosomal DNA replication in the same nuclei.
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PMID:Distinct populations of human PCNA are required for initiation of chromosomal DNA replication and concurrent DNA repair. 1622 49

Spy1 is the originally identified member of the Speedy/Ringo family of vertebrate cell cycle regulators, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases. Here we report a role for Spy1 in apoptosis and checkpoint activation in response to UV irradiation. Using an inducible system allowing for regulated expression of Spy1, we show that Spy1 expression prevents activation of caspase-3 and suppresses apoptosis in response to UV irradiation. Spy1 expression also allows for UV irradiation-resistant DNA synthesis and permits cells to progress into mitosis, as demonstrated by phosphorylation on histone H3, indicating that Spy1 expression can inhibit the S-phase/replication and G2/M checkpoints. We demonstrate that Spy1 expression inhibits phosphorylation of Chk1, RPA, and histone H2A.X, which may directly contribute to the decrease in apoptosis and checkpoint bypass. Furthermore, mutation of the conserved Speedy/Ringo box, known to mediate interaction with CDK2, abrogates the ability of Spy1 to inhibit apoptosis and the phosphorylation of Chk1 and RPA. The data presented indicate that Spy1 expression allows cells to evade checkpoints and apoptosis and suggest that Spy1 regulation of CDK2 is important for the response to DNA damage.
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PMID:Spy1 expression prevents normal cellular responses to DNA damage: inhibition of apoptosis and checkpoint activation. 1695 7

Atypical retinoids, or retinoid-related molecules (RRMs), represent a class of proapoptotic agents with a promising potential in the treatment of neoplastic diseases. In the present work, the synthesis and structure-activity relationship of a series of 3'-adamantan-1-yl-biphenyl-4-yl-acrylic acids substituted in ring A were studied. The synthesized compounds were evaluated for their antiproliferative activity in a human promyelocitic leukemia cell line (NB4), and in an ovarian carcinoma cell system including IGROV-1, carrying a functional wild-type p53, and a cisplatin-resistant subline, IGROV-1/Pt-1. The presence of at least one oxygenated substituent in positions 4' or 5' appears determinant for the antiproliferative activity. With two substituents of this kind the activity increases, particularly in the case of alkylenedioxy compounds. The activation of DNA damage response as indicated by phosphorylation of H2AX histone, RPA-2 protein, and p53 at serine 15 by the most apoptotic compounds provides additional support to the hypothesis that the genotoxic stress is a critical event mediating apoptosis induction by compounds of this group.
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PMID:Synthesis and structure-activity relationships of new antiproliferative and proapoptotic retinoid-related biphenyl-4-yl-acrylic acids. 1751 4

The activation of the ataxia telangiectasia mutated (ATM) and ATM/Rad3-related (ATR) kinases triggers a diverse cellular response including the initiation of DNA damage-induced cell cycle checkpoints. Mediator of DNA Damage Checkpoint protein, MDC1, and H2AX are chromatin remodeling factors required for the recruitment of DNA repair proteins to the DNA damage sites. We identified a novel mediator protein, Cep164 (KIAA1052), that interacts with both ATR and ATM. Cep164 is phosphorylated upon replication stress, ultraviolet radiation (UV), and ionizing radiation (IR). Ser186 of Cep164 is phosphorylated by ATR/ATM in vitro and in vivo. The phosphorylation of Ser186 is not affected by RPA knockdown but is severely hampered by MDC1 knockdown. siRNA-mediated silencing of Cep164 significantly reduces DNA damage-induced phosphorylation of RPA, H2AX, MDC1, CHK2, and CHK1, but not NBS1. Analyses of Cep164 knockdown cells demonstrate a critical role of Cep164 in G2/M checkpoint and nuclear divisions. These findings reveal that Cep164 is a key player in the DNA damage-activated signaling cascade.
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PMID:Cep164 is a mediator protein required for the maintenance of genomic stability through modulation of MDC1, RPA, and CHK1. 1828 22

ST1968, a novel hydrophilic camptothecin analogue of the 7-oxyiminomethyl series, is characterised by the formation of stable DNA-topoisomerase I cleavable complex and by a promising profile of antitumour activity. The present study was designed to extend preclinical evaluation of the novel camptothecin in human squamous cell carcinoma (SCC) models. ST1968 exhibited an impressive activity with a high cure rate in SCC models. ST1968 produced 100% of complete response without evidence of regrowth in tumours characterised by susceptibility to drug-induced apoptosis (FaDu, A431 and A2780). In contrast to irinotecan, ST1968 still showed an excellent, persisting activity in models less susceptible to apoptosis induction (KB, Caski and SiHa), in which drug treatment elicited a persistent DNA damage response, as documented by phosphorylation of p53, RPA-2 and histone H2AX, resulting in delayed apoptosis and senescence. This behaviour was associated with a marked cellular/tumour drug accumulation. In conclusion, ST1968 exhibited an outstanding antitumour activity superior to that of irinotecan against SCC. A high intracellular accumulation, resulting in fast apoptosis or DNA damage persistence, appeared to be a critical determinant of SCC sensitivity to ST1968.
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PMID:Intracellular accumulation and DNA damage persistence as determinants of human squamous cell carcinoma hypersensitivity to the novel camptothecin ST1968. 1844 21

E1B-55K-associated protein 5 (E1B-AP5) is a cellular, heterogeneous nuclear ribonucleoprotein that is targeted by adenovirus (Ad) E1B-55K during infection. The function of E1B-AP5 during infection, however, remains largely unknown. Given the role of E1B-55K targets in the DNA damage response, we examined whether E1B-AP5 function was integral to these pathways. Here, we show a novel role for E1B-AP5 as a key regulator of ATR signaling pathways activated during Ad infection. E1B-AP5 is recruited to viral replication centers during infection, where it colocalizes with ATR-interacting protein (ATRIP) and the ATR substrate replication protein A 32 (RPA32). Indeed, E1B-AP5 associates with ATRIP and RPA complex component RPA70 in both uninfected and Ad-infected cells. Additionally, glutathione S-transferase pull-downs show that E1B-AP5 associates with RPA components RPA70 and RPA32 directly in vitro. E1B-AP5 is required for the ATR-dependent phosphorylation of RPA32 during infection and contributes to the Ad-induced phosphorylation of Smc1 and H2AX. In this regard, it is interesting that Ad5 and Ad12 differentially promote the phosphorylation of RPA32, Rad9, and Smc1 during infection such that Ad12 promotes a significant phosphorylation of RPA32 and Rad9, whereas Ad5 only weakly promotes RPA32 phosphorylation and does not induce Rad9 phosphorylation. These data suggest that Ad5 and Ad12 have evolved different strategies to regulate DNA damage signaling pathways during infection in order to promote viral replication. Taken together, our results define a role for E1B-AP5 in ATR signaling pathways activated during infection. This might have broader implications for the regulation of ATR activity during cellular DNA replication or in response to DNA damage.
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PMID:A role for E1B-AP5 in ATR signaling pathways during adenovirus infection. 1848 Apr 32

Irradiation of cell nuclei with charged particles leads to the spatially defined production of DNA damage along the particle trajectories, thus facilitating studies on the dynamics of radiation-induced protein foci associated with lesion processing. Here we used visual inspection and computational analysis of the track morphology after immunodetection to describe the patterns of formation of gamma-H2AX foci and the repair-related proteins 53BP1 and RPA. We addressed the influence of lesion density on gamma-H2AX formation and the mobility of damaged chromatin sites by using low-angle irradiation of cell monolayers with low-energy carbon or uranium ions. We show the discrete formation of gamma-H2AX foci and the recruitment of repair-related proteins along ion trajectories over an LET range from 200 to 14300 keV/microm in human fibroblasts and in HeLa cells. The marked DSBs exhibited a limited mobility that was independent of the LET. The moderate extent of mobility in human fibroblasts pointed to a relatively stable positioning of the damaged chromatin domains during repair, in contrast to HeLa cells, which showed significant changes in the streak patterns in a fraction of cells, suggesting greater mobility in the local processing of DSBs. Our data indicate that the presence of single or multiple DSBs is not associated with an altered potential for movement of damaged chromatin. We infer that the repair of high-LET radiation-induced DSBs in mammalian cells is not coupled to an increased motional activity of lesions enhancing the probability of translocations.
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PMID:Positional stability of damaged chromatin domains along radiation tracks in mammalian cells. 1939 41


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