Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0004135 (ATM)
13,001 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA-dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortmannin on the catalytic subunit of PI 3-kinase, p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes.
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PMID:Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. 865 48

The glucose transporter isoform, GLUT4, has been expressed in Chinese hamster clones and its subcellular trafficking has been determined following labelling at the cell surface with the impermeant bis-mannose photolabel, 2-N-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos -4-yloxy)-2-propylamine (ATM-BMPA). ATM-BMPA-tagged GLUT4 leaves the cell surface rapidly and equilibrates to give an internal/surface distribution ratio of approx. 3.5 after 60 min. GLUT4 in which the N-terminal phenylalanine-5 and glutamine-6 are mutated to alanine-N-(FQ-AA) and in which the C-terminal leucine-489 and -490 are mutated to alanine C-(LL-AA) have low internal/surface ratios of 0.64 and 1.24 respectively. If all cell-surface transporters are able to recycle, as would be the case for a two-pool recycling model with a single intracellular pool, then analysis suggests that the wild-type GLUT4 distribution ratio is dependent on endocytosis and exocytosis rate constants of 0.074 and 0.023 min(-1). These values are similar, but not identical, to those found for GLUT4 trafficking in adipocytes. The distribution of the N-(FQ-AA) transporter appears to be due to a decrease in endocytosis with reduced intracellular retention, while the distribution of the C-(LL_AA) transporter appears to be mainly due to poor intracellular retention. These results are also considered in terms of a consecutive intracellular pool model in which GLUT4 targeting domains alter the distribution between recycling endosomes and a slowly recycling compartment. In this case the more rapid apparent exocytosis of the mutated GLUT4 is due to their failure to reach a slowly recycling compartment with a consequent return to the plasma membrane by default. It is suggested that overexpression of transporters increases the proportion that are recycled in this way. Wortmannin is shown to decrease glucose transport activity and cell-surface photolabelled transporters in a manner consistent with an inhibition of transporter recycling. Studies on the rate of loss of transport activity and ATB-BMPA-tagged transporter in wortmannin-treated cells confirm that the N-(FQ-AA) mutant is endocytosed more slowly than the wild-type GLUT4. Taken together, these results suggest that the mutation at either the N- or the C-terminal domain can reduce movement to a slowly recycling intracellular compartment but that neither domain alone is entirely sufficient to produce wild-type GLUT4 trafficking behaviour.
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PMID:Subcellular trafficking kinetics of GLU4 mutated at the N- and C-terminal. 867 Jan 1

ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have been shown to have sequences homologous to the catalytic domains of mammalian phosphatidylinositol 3-kinase (PI3-kinase). In order to determine the contribution of ATM and DNA-PKcs to the increased sensitivity of cells to DNA-damaging agents observed in the presence of PI3-kinase inhibitors, we examined the effects of a PI3-kinase inhibitor, wortmannin, on cellular sensitivity to bleomycin (BLM), mitomycin C (MMC), X-irradiation and ultraviolet (UV)-irradiation using 2 human tumor cell lines (T98G and A172), a human fibroblast cell line (LM217), an ataxia telangiectasia (AT) cell line (AT3BISV), a scid murine cell line (SCF) and a control murine cell line (CBF). Wortmannin sensitized all of the cells, including AT3BISV and SCF, to BLM and X-irradiation, but not to MMC or UV-irradiation. Hypersensitivity to BLM and X-irradiation and normal sensitivity to MMC and UV-irradiation are characteristic phenotypes of both AT and scid mice. DNA-dependent protein kinase (DNA-PK) activity was suppressed by wortmannin to 45-65% of the control values in all of the cells except SCF, in which DNA-PK activity was not detected. Wortmannin also induced radioresistant DNA synthesis, which is a cellular phenotype of AT, in T98G and SCF cells, but did not change the DNA synthesis rates after X-irradiation in AT3BISV. Our data suggest that wortmannin decreases the activities of both the ATM protein and DNA-PK, indicating that it might be of use as a sensitizing agent for radiotherapy and chemotherapy.
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PMID:A phosphatidylinositol 3-kinase inhibitor wortmannin induces radioresistant DNA synthesis and sensitizes cells to bleomycin and ionizing radiation. 980 36

Members of the phosphatidylinositol (PI) 3-kinase gene family, including the ataxia telangiectasia gene and the DNA-dependent protein kinase (DNA-PK), are involved in regulating cellular radiosensitivity. We have investigated two structurally unrelated PI 3-kinase inhibitors, wortmannin and LY294002, to determine whether they inhibit DNA-PK and increase cellular radiosensitivity. The PI 3-kinase inhibitors wortmannin and LY294002 were effective radiosensitizers of human tumor cells, with sensitizer enhancement ratios (at 10% survival) of 2.8 and 1.9, respectively, in SW480 cells. Wortmannin and LY294002 inhibited the kinase activity of purified DNA-PK and inactivated cellular DNA-PK kinase activity. Inhibition of cellular DNA-PK activity occurred at the same concentrations of wortmannin that caused radiosensitization, and this correlation was found in a range of tumor cell lines. However, cells deficient in either DNA-PK (scid cells) or the ataxia telangiectasia protein were also partly sensitized to radiation by wortmannin, indicating the involvement of more than one protein kinase in the mechanism of action of wortmannin. Wortmannin also affected the G2-M checkpoint. SW480 cells had a reversible G2-M delay of 20 h following irradiation. However, wortmannin-treated SW480 cells had a prolonged G2-M delay; more than 75% of cells were arrested in G2 at 50 h postirradiation. This suggests the accumulation of significant unrepaired DNA damage following inhibition of PI 3-kinase family members. Therefore, PI 3-kinase inhibitors may represent a new class of radiosensitizers that inhibit the repair of DNA damage.
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PMID:Radiosensitization of human tumor cells by the phosphatidylinositol3-kinase inhibitors wortmannin and LY294002 correlates with inhibition of DNA-dependent protein kinase and prolonged G2-M delay. 981 94

Wortmannin has been shown to be an efficient radiosensitizer. Since wortmannin is able to inhibit DNA-dependent protein kinase (DNA-PK) and double-strand break (DSB) rejoining, it is believed that its mechanism of radiation sensitization is through the inhibition of DNA-PK-mediated repair of DSBs. However, since wortmannin is not a specific inhibitor, the possibility that other kinases are inhibited and thereby may contribute to radiosensitization cannot be ruled out. Here we present data confirming the radiosensitizing effect of wortmannin on cells of different cell lines. In the same range of wortmannin concentrations, survival after exposure to ionizing radiation correlated well with DSB rejoining and the induction of micronuclei, suggesting that the inhibition of the processing of DSBs is involved in the sensitizing effect. Pretreatment with wortmannin enhanced the radiosensitivity of ataxia telangiectasia (AT) cells, thereby precluding the participation of ATM protein in the radiation sensitization by wortmannin. At the same time, irradiated DNA-PK-deficient cells were not significantly affected by pretreatment with wortmannin. These observations support a likely mechanism; that is, wortmannin sensitizes cells to radiation through inhibition of the DNA-PK-mediated rejoining of DSBs.
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PMID:Wortmannin sensitizes mammalian cells to radiation by inhibiting the DNA-dependent protein kinase-mediated rejoining of double-strand breaks. 995

Wortmannin is a potent inhibitor of phosphatidylinositol (PI) 3-kinase and PI 3-kinase-related proteins (e.g. ATM), but it does not inhibit the activity of purified calmodulin-dependent protein kinase II (CaMKII). In the present study, we compared the effects of wortmannin and the CaMKII inhibitor KN62 on the response of normal human dermal fibroblast cultures to gamma radiation. We demonstrate that wortmannin confers a phenotype on normal fibroblasts remarkably similar to that characteristic of cells homozygous for the ATM mutation. Thus wortmannin-treated normal fibroblasts exhibit increased sensitivity to radiation-induced cell killing, lack of temporary block in transition from G1 to S phase following irradiation (i.e. impaired G1/S checkpoint), and radioresistant DNA synthesis (i.e. impaired S phase checkpoint). Wortmannin-treated cultures display a diminished capacity for radiation-induced up-regulation of p53 protein and expression of p21WAF1, a p53-regulated gene involved in cell cycle arrest at the G1/S border; the treated cultures also exhibit decreased capacity for enhancement of CaMKII activity post-irradiation, known to be necessary for triggering the S phase checkpoint. We further demonstrate that KN62 confers a radioresistant DNA synthesis phenotype on normal fibroblasts and moderately potentiates their sensitivity to killing by gamma rays, without modulating G1/S checkpoint, p53 up-regulation and p21WAF1 expression following radiation exposure. We conclude that CaMKII is involved in the radiation responsive signalling pathway mediating S phase checkpoint but not in the p53-dependent pathway controlling G1/S checkpoint, and that a wortmannin-sensitive kinase functions upstream in both pathways.
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PMID:Effects of the protein kinase inhibitors wortmannin and KN62 on cellular radiosensitivity and radiation-activated S phase and G1/S checkpoints in normal human fibroblasts. 1057 51

Expression of the cyclin kinase inhibitor, p21, is regulated both transcriptionally and posttranscriptionally by the ubiquitin-proteasome degradation pathway. Recently, we reported that DNA damage is required for efficient p21 expression by demonstrating that enhanced p21 mRNA expression induced by DNA damage results in increased p21 protein, but enhanced p21 mRNA without DNA damage does not. In addition, we demonstrated that DNA damage suppressed the ubiquitination of p21. In this study, we analyze the link between p21 stabilization and DNA damage. Enhanced p21 protein expression in ML-1 cells resulting from 15 Gy gamma-irradiation was diminished by Wortmannin or LY294002 pretreatment of cells. However, the levels of p21 mRNA were not affected by inhibitor pretreatment. Wortmannin or LY294002 pretreatment reduces p53 expression after gamma-irradiation to a lesser degree than that of p21. In addition, we examined the involvement of DNA-PK, whose activity is inhibited by Wortmannin or LY294002, in p21 stabilization using the SCID fibroblast cell line and a DNA-PK targeting ML-1 cell line. Accumulation of p21 protein by gamma-irradiation was similar to that of DNA-PK intact cells and was reduced by Wortmannin or LY294002 pretreatment. Involvement of another DNA damage detecting enzyme, the ATM gene product, whose activity is also inhibited by Wortmannin or LY294002, was evaluated. ATM deficient cells induced p21 after gamma-irradiation, gamma-irradiation-induced p21 protein was diminished by pretreatment of cells with Wortmannin or LY294002. We conclude that the p21 stabilization mechanism functions after gamma-irradiation, was sensitive to Wortmannin or LY294002, and required neither DNA-PK nor ATM gene product for activity.
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PMID:Phosphatidylinositol 3-kinase inhibitors, Wortmannin or LY294002, inhibited accumulation of p21 protein after gamma-irradiation by stabilization of the protein. 1077 Oct 89

Genistein, a natural isoflavone found in soybeans, exerts a number of biological actions suggesting that it may have a role in cancer prevention. We have previously shown that it potently inhibits OCM-1 melanoma cell proliferation by inducing a G(2) cell cycle arrest. Here we show that genistein exerts this effect by impairing the Cdc25C-dependent Tyr-15 dephosphorylation of Cdk1, as the overexpression of this phosphatase allows the cells to escape G(2) arrest and enter an abnormal chromatin condensation stage. Caffeine totally overrides the genistein-induced G(2) arrest, whereas the block caused by etoposide is not bypassed and that caused by adriamycin is only partially abolished. We also report that genistein activates the checkpoint kinase Chk2 as efficiently as the two genotoxic agents and that caffeine may counteract the activation of Chk2 by genistein but not by etoposide. In contrast, caffeine abolishes the accumulation of p53 caused by all the compounds. Wortmannin does not suppress the Chk2 activation in any situation, suggesting that the ataxia telangiectasia-mutated kinase is not involved in this regulation. Finally, unlike etoposide and adriamycin, genistein induces only a weak response in terms of DNA damage in OCM-1 cells. Taken together, these results suggest that the G(2) checkpoints activated by genistein and the two genotoxic agents involve different pathways.
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PMID:Distinct Chk2 activation pathways are triggered by genistein and DNA-damaging agents in human melanoma cells. 1080 72

DNA damage response pathways coordinate the cellular response to DNA damage. To investigate the roles of tumor suppressor genes in these pathways, human lymphoblastoid cells (wild-type, p53-/-, ATM-/-) were treated for 1 h with 0-3 microg/ml of the radiomimetic compound bleomycin (BLM), and cells treated in G(2) were analyzed for chromatid aberrations. BLM-induced aberration frequencies were significantly increased, to the greatest extent in the ATM-/- cells and, to a lesser extent, in the p53-/- cells compared to wild-type cells. These observations are consistent with p53 and ATM acting in a damage response pathway activated by DNA strand breaks. The consequences of disrupting this pathway were further investigated by studies using wortmannin, a PI-3 kinase and DNA repair inhibitor. Wortmannin significantly increased the BLM-induced aberration frequencies in all but the ATM-/- cells, elevating the sensitivity of p53-/- cells to ATM-/- levels and that of wild-type cells to intermediate levels. These differential sensitivities suggest that the ATM phenotype is the result of dual cellular defects, one involving p53 and the other a wortmannin-sensitive component. Similar studies in Brca1+/- and Brca2+/- human lymphoblasts showed no increased sensitization to BLM in the absence of inhibitor, and differential sensitization by wortmannin. To determine if there was any substrate specificity for p53- and ATM-mediated DNA damage responses, chromatid aberrations were assessed in wild-type, p53-/-, and ATM-/- cells exposed to 0-0.4 microg/ml neocarzinostatin (NCS) for 1 h. In contrast to results with BLM, the p53-/- cells exhibited a low sensitivity to NCS-induced aberrations, similar to wild-type, while ATM-/- cells remained highly sensitive. This suggests that the response to BLM- and NCS-induced lesions involves different mechanisms.
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PMID:Increased sensitivity to chromatid aberration induction by bleomycin and neocarzinostatin results from alterations in a DNA damage response pathway. 1100 7

Here we found that wortmannin sensitized Chinese hamster V79 cells to hyperthermic treatment at 44.0 degrees C as determined either by colony formation assay or by dye exclusion assay. Wortmannin enhanced heat-induced cell death accompanying cleavage of poly (ADP-ribose) polymerases (PARP). Additionally, the induction of heat shock protein HSP70 was suppressed and delayed in wortmannin-treated cells. Heat sensitizing effect of wortmannin was obvious at more than 5 or 10 microM of final concentrations, while radiosensitization was apparent at 5 microM. Requirement for high concentration of wortmannin, i.e., order of microM, suggests a possible role of certain protein kinases, such as DNA-PK and/or ATM among PI3-kinase family. The sensitization was minimal when wortmannin was added at the end of heat treatment. This was similar to the case of X-ray. Since heat-induced cell death and PARP cleavage preceded HSP70 induction phenomenon, the sensitization to the hyperthermic treatment was considered mainly caused by enhanced apoptotic cell death rather than secondary to suppression or delay by wortmannin of HSP70 induction. Further, in the present system radiosensitization by wortmannin was also at least partly mediated through enhancement of apoptotic cell death.
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PMID:Sensitization by wortmannin of heat- or X-ray induced cell death in cultured Chinese hamster V79 cells. 1103 77


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