UNIPROT:P04637 - FACTA Search

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Query: UNIPROT:P04637 (p53)
77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate double strand break (DSB) repair and signaling in human glioma cells, we stably transfected human U87 (ATM(+), p53(+)) glioma cells with a plasmid having a single I-SceI site within an inactive green fluorescent protein (GFP) expression cassette, allowing for the detection of homologous recombination repair (HRR) by GFP expression. HRR and nonhomologous end joining (NHEJ) were also determined by PCR. DSB repair was first detected at 12 h postinfection with an adenovirus expressing I-SceI with repair reaching plateau levels between 24 and 48 h. Within this time frame, NHEJ predominated over HRR in the range of 3-50-fold. To assess the involvement of ATM in DSB repair, we first examined whether ATM was associated with the DSB. Chromatin immunoprecipitation showed that ATM was present at the site of the DSB as early as 18 h postinfection. In cells treated with caffeine, an inhibitor of ATM, HRR was reduced, whereas NHEJ was not. In support of this finding, GFP flow cytometry demonstrated that caffeine reduced HRR by 90% under conditions when ATM kinase activity was inhibited. Dominant-negative ATM expressed from adenovirus inhibited HRR by 45%, also having little to no effect on NHEJ. Furthermore, HRR was inhibited by caffeine in serum-starved cells arrested in G(0)/G(1), suggesting that ATM is also important for HRR outside of the S and G(2) cell cycle phases. Altogether, these results demonstrate that HRR contributes substantially to DSB repair in human glioma cells, and, importantly, ATM plays a critical role in regulating HRR but not NHEJ throughout the cell cycle.
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PMID:Double strand break repair by homologous recombination is regulated by cell cycle-independent signaling via ATM in human glioma cells. 1474 54

Caffeine has been widely described as a chemo/radiosensitizing agent, presumably by inhibiting DNA repair, and affecting preferentially cells with an altered p53 status. We evaluated the effects of caffeine using isogenic and isophenotypic K1 cells derived from a papillary thyroid carcinoma and displaying either a wild type or a mutated p53 status. Apoptosis and clonogenic survival were examined after exposure of the cells to cisplatin or UVc irradiation. We find that at the most currently used concentration, 2mM, caffeine hinders cisplatin or UVc induced apoptosis in K1 cells. In addition, at this already barely achievable concentration in vivo, caffeine does not decrease their clonogenic survival. Hence in our cellular model, caffeine does not behave as a chemo- or a radiosensitizer. Although surprising, these results (1) are in agreement with the delayed G2/M block caused by caffeine that we previously observed in normal human fibroblasts and K1 cells and (2) allow us to elucidate some discrepancies concerning this molecule throughout the literature such as increase or decrease of apoptosis and clonogenic survival, activation or deactivation of molecules involved in DNA damage repair and proliferation inhibition but accelerated G2/M traverse.
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PMID:Conflicting effects of caffeine on apoptosis and clonogenic survival of human K1 thyroid carcinoma cell lines with different p53 status after exposure to cisplatin or UVc irradiation. 1475 Dec 46

Genistein, a soy isoflavone, has a wide range of biological actions that suggest it may be of use in cancer prevention. We have recently reported that it arrests hepatoma cells at G2/M phase and inhibits Cdc2 kinase activity. In the present study, we examined the signaling pathway by which genistein modulates Cdc2 kinase activity in HepG2 cells and leads to G2/M arrest, and found that it caused an increase in both Cdc2 phosphorylation and expression of the Cdc2-active kinase, Wee1. Genistein also enhanced the expression of the cell cycle inhibitor, p21waf1/cip1, which interacts with Cdc2. Furthermore, phosphorylation/inactivation of Cdc25C phosphatase, which dephosphorylates/activates Cdc2, was increased. Genistein enhanced the activity of the checkpoint kinase, Chk2, which phosphorylates/inactivates Cdc25C, induced accumulation of p53, and activated the ataxia-telangiectasia-mutated (ATM) gene. Caffeine, an ATM kinase inhibitor, inhibited these effects of genistein on Chk2, p53, and p21waf1/cip1. These findings suggest that the effect of genistein on G2/M arrest in HepG2 cells is partly due to ATM-dependent Chk2 activation, an increase in Cdc2 phosphorylation/inactivation as a result of induction of Wee1 expression, and a decrease in Cdc2 activity as a result of induction of p21waf1/cip1 expression.
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PMID:Genistein arrests hepatoma cells at G2/M phase: involvement of ATM activation and upregulation of p21waf1/cip1 and Wee1. 1475 71

Camptothecin and Adriamycin are clinically important inhibitors for topoisomerase (Topo) I and Topo II, respectively. The ataxia-telangiectasia mutated (ATM) product is essential for ionizing radiation-induced DNA damage responses, but the role of ATM in Topo poisons-induced checkpoints remains unresolved. We found that distinct mechanisms are involved in the activation of different cell cycle checkpoints at different concentrations of Adriamycin and camptothecin. Adriamycin promotes the G(1) checkpoint through activation of the p53-p21(CIP1/WAF1) pathway and decrease of pRb phosphorylation. Phosphorylation of p53(Ser20) after Adriamycin treatment is ATM dependent, but is not required for the full activation of p53. The G(1) checkpoint is dependent on ATM at low doses but not at high doses of Adriamycin. In contrast, the Adriamycin-induced G(2) checkpoint is independent on ATM but sensitive to caffeine. Adriamycin inhibits histone H3(Ser10) phosphorylation through inhibitory phosphorylation of CDC2 at low doses and down-regulation of cyclin B1 at high doses. The camptothecin-induced intra-S checkpoint is partially dependent on ATM, and is associated with inhibitory phosphorylation of cyclin-dependent kinase 2 and reduction of BrdUrd incorporation after mid-S phase. Finally, apoptosis associated with high doses of Adriamycin or camptothecin is not influenced by the absence of ATM. These data indicate that the involvement of ATM following treatment with Topo poisons differs extensively with dosage and for different cell cycle checkpoints.
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PMID:Topoisomerase poisons differentially activate DNA damage checkpoints through ataxia-telangiectasia mutated-dependent and -independent mechanisms. 1514 Oct 20

Although the link between transcription and DNA repair is well established, defects in the core transcriptional complex itself have not been shown to elicit a DNA damage response. Here we show that a cell line with a temperature-sensitive defect in TBP-associated factor 1 (TAF1), a component of the TFIID general transcription complex, exhibits hallmarks of an ATR-mediated DNA damage response. Upon inactivation of TAF1, ATR rapidly localized to subnuclear foci and contributed to the phosphorylation of several downstream targets, including p53 and Chk1, resulting in cell cycle arrest. The increase in p53 expression and the G(1) phase arrest could be blocked by caffeine, an inhibitor of ATR. In addition, dominant negative forms of ATR but not ATM were able to override the arrest in G(1). These results suggest that a defect in TAF1 can elicit a DNA damage response.
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PMID:Activation of a DNA damage checkpoint response in a TAF1-defective cell line. 1516 97

Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in controlling the cellular response to ionizing radiation and other DNA-damaging agents. ATM is a 3056 amino acid polypeptide that is present in low abundance in the nucleus of human cells. Here, we describe the purification and characterization of ATM from the nuclear fraction of HeLa cells. Microgram quantities of highly stable, kinase-active ATM were prepared. Purified ATM was phosphorylated on serine 1981 and was active towards a variety of known ATM substrates, including p53 and the Bloom Syndrome helicase, BLM. The protein kinase activity of ATM was selectively inhibited by wortmannin, caffeine and LY294002 and was stimulated by charged biological polymers, including single-stranded M13 DNA (ssDNA), sheared double-stranded calf thymus DNA, heparin sulfate and poly ADP-ribose (PAR), raising the possibility that charged structures may contribute to regulation of ATM activity. However, chemical inhibition of the formation of poly ADP-ribose in cells had no effect on the activation of ATM-dependent pathways by ionizing radiation. Using gel filtration chromatography, we also show that purified ATM, as well as ATM in crude nuclear extracts from unirradiated and irradiated cells elutes with an estimated native molecular weight of approximately 600 kDa. Moreover, dephosphorylation of serine 1981 did not affect the apparent molecular weight of ATM in irradiated extracts. Our results suggest that phosphorylation of serine 1981 alone may not directly regulate the subunit composition of ATM.
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PMID:Biochemical characterization of the ataxia-telangiectasia mutated (ATM) protein from human cells. 1517 84

Shaved male or female p53(-/-) C57BL/6J mice and their wild-type littermates were irradiated once with UVB (60 mJ/cm(2)). The UVB-induced increase in apoptotic sunburn cells in p53(-/-) mice at 6-10 h after exposure to UVB was only 10-30% of that observed after treatment of p53(+/+) mice with UVB. Topical applications of caffeine immediately after UVB irradiation in female p53(+/+) or p53(-/-) mice enhanced the UVB-induced increase in apoptotic sunburn cells 6 h later by 127% and 563%, respectively. In another study, shaved female Bax(-/-) C57BL/6J mice and their wild-type littermates were irradiated once with UVB (60 mJ/cm(2)). The UVB-induced increase in apoptotic sunburn cells in Bax(-/-) mice at 6 h after exposure to UVB was only 14% of that observed after treatment of Bax(+/+) mice with UVB. Topical application of caffeine immediately after irradiation of Bax(+/+) or Bax(-/-) mice with UVB enhanced the UVB-induced increases in apoptotic sunburn cells at 6 h by 214% and 467%, respectively, and topical application of caffeine immediately after irradiation of Bax(+/+) or Bax(-/-) mice with UVB enhanced the UVB-induced increase in caspase 3 (active form) positive cells at 6 h by 253% and 750%, respectively. The results indicate that UVB-induced increases in apoptosis in the epidermis of wild-type mice are predominantly (but not entirely) by p53- and Bax-dependent pathways and that topical application of caffeine can enhance UVB-induced increases in apoptosis by p53- and Bax-independent pathways.
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PMID:Stimulatory effect of topical application of caffeine on UVB-induced apoptosis in the epidermis of p53 and Bax knockout mice. 1525 77

Polo-like kinase 1 (Plk1) is an important regulator of several events during mitosis. Recent reports show that Plk1 is involved in both G2 and mitotic DNA damage checkpoints. Ataxia telangiectasia mutated kinase (ATM) is an important enzyme involved in G2 phase cell cycle arrest following interphase DNA damage, and inhibition of Plk1 by DNA damage during G2 occurs in an ATM-/ATM-Rad3-related kinase (ATR)-dependent fashion. However, it is unclear how Plk1 is regulated in response to M phase DNA damage. We found that treatment of mitotic cells with DNA damaging agents inhibits Plk1 activity primarily through dephosphorylation of Plk1, which occurred in both p53 wild-type and mutant cells. Inhibition of Plk1 is not prevented by caffeine pretreatment that inhibits ATM activity and also occurs in ATM mutant cell lines. Furthermore, ATM mutant cell lines, unlike wild-type cells, fail to arrest after mitotic DNA damaging treatments. The phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, reduces Plk1 dephosphorylation following mitotic DNA damaging treatments, suggesting that the PI3K pathway may be involved in regulating Plk1 activity. Earlier studies showed that inhibition of Plk1 by G2 DNA damage occurs in an ATM-dependent fashion. Our results extend the previous studies by showing that ATM is not required for dephosphorylation and inhibition of Plk1 activity following mitotic DNA damage, and also suggest that Plk1 is not a principal regulator or mediator of the mitotic DNA damage response.
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PMID:Polo-like kinase 1 inactivation following mitotic DNA damaging treatments is independent of ataxia telangiectasia mutated kinase. 1528 Apr 49

Ionizing radiation and mitotic inhibitors are used for the treatment of lymphoma. We have studied cell cycle arrest and apoptosis of three human B-lymphocyte cell lines after X irradiation and/or nocodazole treatment. Radiation (4 and 6 Gy) caused arrest in the G(2) phase of the cell cycle as well as in G(1) in Reh cells with an intact TP53 response. Reh cells, but not U698 and Daudi cells with defects in the TP53 pathway, died by apoptosis after exposure to 4 or 6 Gy radiation (>15% apoptotic Reh cells and <5% apoptotic U698/Daudi cells 24 h postirradiation). Lower doses of radiation (0.5 and 1 Gy) caused a transient delay in the G(2) phase of the cell cycle for the three cell lines but did not induce apoptosis (<5% apoptotic cells at 24 h postirradiation). Cells of all three cell lines died by apoptosis after exposure to 1 microg/ml nocodazole, a mitotic blocker that acts by inhibiting the polymerization of tubulin (>25% apoptotic cells after 24 h). When X irradiation with 4 or 6 Gy was performed at the time of addition of nocodazole to U698 and Daudi cells, X rays protected against the apoptosis-inducing effects of the microtubule inhibitor (<5% and 15% apoptotic cells, respectively, 24 h incubation). U698 and Daudi cells apparently have some error(s) in the signaling pathway inducing apoptosis after irradiation, and our results suggest that the arrest in G(2) prevents the cells from entering mitosis and from apoptosis in the presence of microtubule inhibitors. This arrest was overcome by caffeine, which caused U698 cells to enter mitosis (after irradiation) and become apoptotic in the presence of nocodazole (26% apoptotic cells, 24 h incubation). These results may have implications for the design of clinical multimodality protocols involving ionizing radiation for the treatment of cancer.
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PMID:The response of malignant B lymphocytes to ionizing radiation: cell cycle arrest, apoptosis and protection against the cytotoxic effects of the mitotic inhibitor nocodazole. 1544 42

We have shown previously that ionizing radiation (IR) induces a persistent G(2)-M arrest but not cell death in MCF-7 breast carcinoma cells that harbor functional p53 but lack caspase-3. In the present study, we investigated the mechanisms of apoptosis resistance and the roles of p53, caspase-3, and cell cycle arrest in IR-induced apoptosis. The methylxanthine caffeine and the staurosporine analog UCN-01, which can inhibit ATM and Chk kinases, efficiently abrogated the IR-induced G(2)-M arrest and induced mitochondrial activation as judged by the loss of the mitochondrial membrane potential and the release of cytochrome c and Smac/Diablo. However, despite these proapoptotic alterations, cell death and activation of the initiator caspase-9 were not induced in MCF-7 cells but were interestingly only observed after reexpression of caspase-3. Sensitization to IR-induced apoptosis by caffeine or UCN-01 was abrogated neither by cycloheximide nor by pifithrin-alpha, an inhibitor of the transcriptional activity of p53. Furthermore, suppression of p53 by RNA interference could not prevent caffeine- and IR-induced mitochondrial alterations and apoptosis but resulted in an even more pronounced G(2)-M arrest. Collectively, our results clearly show that the resistance of MCF-7 cells to IR-induced apoptosis is caused by two independent events; one of them is a caffeine- or UCN-01-inhibitable event that does not depend on p53 or a release of the G(2)-M arrest. The second event is the loss of caspase-3 that surprisingly seems essential for a fully functional caspase-9 pathway, even despite the previous release of mitochondrial proapoptotic proteins.
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PMID:Apoptosis resistance of MCF-7 breast carcinoma cells to ionizing radiation is independent of p53 and cell cycle control but caused by the lack of caspase-3 and a caffeine-inhibitable event. 1546 1

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