UMLS:C0017638 - FACTA Search

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Query: UMLS:C0017638 (glioma)
30,880 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Double-strand breaks (DSBs) can be efficiently removed from the DNA of higher eukaryotes by nonhomologous end-joining (NHEJ). Genetic studies implicate the DNA-dependent protein kinase (DNA-PK) in NHEJ, but the exact function of this protein complex in the rejoining reaction remains to be elucidated. We compared rejoining of DNA DSBs in a human glioma cell line, M059-J, lacking the catalytic subunit of DNA-PK (DNA-PKcs), and their isogenic but DNA-PK-proficient counterpart, M059-K. In both cell lines, rejoining of DNA DSBs was biphasic, with a fast and a slow component operating with a half-life of approximately 22 min and 12 h, respectively. Deficiency in DNA-PK activity did not alter the half-times of either of these components of rejoining but increased from 17 to 72% the proportion of DNA DSB rejoining with slow kinetics. DNA DSB rejoining was nearly complete in both cell lines, and there was only a small increase in the number of unrejoined breaks in M059-J as compared with M059-K cells after 30 h of incubation. Wortmannin radiosensitized to killing M059-K cells and strongly inhibited DNA DSB rejoining. Wortmannin did not affect the radiosensitivity to killing and produced only a modest inhibition in DNA DSB rejoining in M059-J cells, suggesting that, for these end points, DNA-PK is the principal target of the drug. These observations demonstrate that DNA-PK deficiency profoundly decreases the proportion of DNA DSB rejoining with fast kinetics but has only a small effect on the fraction remaining unrejoined. We propose that in higher eukaryotes, an evolutionarily conserved, independently active, but inherently slow NHEJ pathway is stimulated 30-fold by DNA-PKcs to rapidly remove DNA DSBs from the genome. The stimulation is expected to be of local nature and the presence of DNA-PKcs in the vicinity of the DNA DSB determines whether rejoining will follow fast or slow kinetics. Structural and regulatory functions of DNA-PKcs may mediate this impressive acceleration of DNA DSB rejoining, and regions of chromatin within a certain range from this large protein may benefit from these activities. We propose the term DNA-PK surveillance domains to describe these regions.
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PMID:DNA-dependent protein kinase stimulates an independently active, nonhomologous, end-joining apparatus. 1072 83

Anderson, C. W., Dunn, J. J., Freimuth, P. I., Galloway, A. M. and Allalunis-Turner, M. J. Frameshift Mutation in PRKDC, the Gene for DNA-PKcs, in the DNA Repair-Defective, Human, Glioma-Derived Cell Line M059J. Radiat. Res. 156, 2-9 (2001). The glioma-derived cell line M059J is hypersensitive to ionizing radiation, lacks DNA-PK activity, and fails to express protein for the catalytic subunit, DNA-PKcs, while a sister cell line, M059K, derived from the same tumor, has normal DNA-PK activity. Both cell lines are near pentaploid and have multiple copies of chromosome 8, the chromosome on which the DNA-PKcs gene, PRKDC, is located. Sequence analysis of PCR-amplified exons revealed the loss in M059J cells of a single "A" nucleotide in exon 32, corresponding to the first nucleotide of codon 1351 (ACC, Thr) of PRKDC. Loss of the "A" nucleotide would terminate the DNA-PKcs reading frame early in exon 33. DNA from M059K cells had only the wild-type sequence. An analysis of sequences surrounding PRKDC exon 32 from 87 unrelated individuals revealed no polymorphic nucleotides except for a triplet repeat near the 3' end of this exon; no individual had a frameshift mutation in exon 32. No other sequence differences in PRKDC between M059J and M059K cells were observed in approximately 15,000 bp of genomic sequence including the sequences of exons 5 through 38 and surrounding intron sequence, suggesting a possible reduction to homozygosity at this locus prior to acquisition of the mutation leading to the M059J cell line.
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PMID:Frameshift mutation in PRKDC, the gene for DNA-PKcs, in the DNA repair-defective, human, glioma-derived cell line M059J. 1141 67

Gene amplification is known to occur frequently in human glioma. Recently we reported cloning of a novel gene termed glioma-amplified sequence 16 (GAS16) by microdissection-mediated cDNA capture. In this article, we demonstrate that GAS16 results from an alternative splicing process of the Ku70 binding protein 3 (KUB3) that is essential for DNA double-strand break repair. The alternative splice product was found in glioblastoma and in normal fetal brain. We determined the amplification frequency of KUB3 in glioma with different grading. We analyzed a total of 102 glioma primary tumors and found KUB3 to be amplified in 12/82 (14%) glioblastomas, 4/13 anaplastic astrocytomas (30%), and 2/4 astrocytomas, but in none of three pilocytic astrocytomas. Northern blot analysis of glioblastoma shows a strong correlation between KUB3 amplification and overexpression. Amplification of KUB3 appears to be independent of other genetic changes frequently associated with the development of gliomas, including EGFR amplification, LOH of TP53, and LOH of chromosome 10. The KUB3 amplification and overexpression may interfere with the function of KUB3 in the DNA-PK complex involved in the maintenance of genome stability and reduction of mutation frequency.
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PMID:KUB3 amplification and overexpression in human gliomas. 1157 79

Ku is a heterodimer of M(r) 70,000 and M(r) 86,000 subunits. It binds with strong affinity to DNA ends and is indispensable for nonhomologous DNA end joining (NHEJ) and V(D)J recombination. In this study, we investigated whether down-regulation of the Ku86 gene, by 2'-O-methoxyethyl/uniform phosphorothioate chimeric antisense oligonucleotides (ASOs), increases the sensitivity of the DNA-protein kinase catalytic subunit (PKcs)-proficient human glioma cell line (M059K), and its isogenic DNA-PKcs-deficient counterpart (M059J), to ionizing radiation and anticancer drugs. Transfection of these cell lines with 200 nM Ku86 antisense ASOs was associated with a specific decrease in Ku86 mRNA levels (IC(50) <25 nM; n = 3) and a concomitant rapid decrease (<10% of control) in Ku86 protein expression. Moreover, transfection of M059K cells with Ku86 antisense ASOs markedly increased cell death after treatment with ionizing radiation, bleomycin, and etoposide. However, no sensitization to the DNA cross-linking agents chlorambucil and cisplatin was observed after Ku86 antisense transfection. As expected, transfection of M059J cells with Ku86 antisense ASOs did not result in any sensitization to ionizing radiation, bleomycin, or DNA cross-linking agents, but there was a 2-fold increase in sensitivity to etoposide. Thus, our results indicate that antisense ASOs targeted against Ku86 may increase the efficacy of radiotherapy and DNA-damaging agents in tumor treatment. Furthermore, Ku86 antisense ASOs may be used to create a temporal knockout in different human cell lines to further investigate the biological roles of Ku86.
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PMID:An antisense oligonucleotide targeted to human Ku86 messenger RNA sensitizes M059K malignant glioma cells to ionizing radiation, bleomycin, and etoposide but not DNA cross-linking agents. 1238 53

The human glioma cell line M059J is deficient in DNA-dependent protein kinase (DNA-PK) due to a frame-shift mutation in PRKDC, the gene for its catalytic subunit, while cell line M059K, isolated from the same malignant tumor, has normal DNA-PK activity. DNA-PK is required for double-strand DNA break repair, and its absence is responsible for increased radiosensitivity of M059J. We show that transcripts of several melanoma antigen subfamily A (MAGE-A) genes, the expression of which is restricted to tumor and germ-line cells,are present in M059K, but that their expression is strongly downregulated in M059J. Normal levels of MAGE-A expression are restored in the PRKDC-complemented cell line M059J/Fus1, suggesting that the presence of DNA-PK is required for MAGE-A gene transcription. We also show that the MAGE-A1 promoter is methylated in M059J, while the promoter is demethylated in M059K and M059J/Fus1. Other genes, including all three major histocompatibility class I (HLA) genes, BENE, and an unnamed gene related to CNIL(CORNICHON-like), display an opposite expression profile (i.e., they are upregulated in the DNA-PK-deficient cell line, but show low levels of expression in both M059K and in the PRKDC-complemented cell line). For these genes, differential expression does not correlate with DNA methylation in upstream promoter sequences. Our results suggest that the presence of DNA-PK can exert effects on gene expression by various mechanisms and pathways, thus affecting overall cell physiology even in the absence of DNA damage.
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PMID:Differential gene expression in human glioma cells: correlation with presence or absence of DNA-dependent protein kinase. 1269 24

DNA damage induces accumulation and activation of p53 via various posttranslational modifications. Among them, several lines of evidence indicated the phosphorylation of Ser46 as an important mediator of DNA damage-induced apoptosis but the responsible kinase remains to be clarified, especially in the case of ionizing radiation (IR). Here we showed that DNA-dependent protein kinase (DNA-PK) could phosphorylate Ser46 of p53 in addition to reported phosphorylation sites Ser15 and Ser37. However, IR-induced phosphorylation of Ser46 was seen even in M059J, a human glioma cell line lacking DNA-PKcs, and it was, at most, only slightly less than in control M059K. On the other hand, a related kinase ataxia-telangiectasia mutated (ATM), which was shown to be essential for IR-induced phosphorylation of Ser46, could poorly phosphorylate Ser46 by itself. These results collectively suggested two pathways for IR-induced phosphorylation of Ser46, i.e., direct phosphorylation by DNA-PK and indirect phosphorylation via ATM.
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PMID:Potentiality of DNA-dependent protein kinase to phosphorylate Ser46 of human p53. 1538 Oct 73

Previous reports have suggested a connection between reduced levels of the catalytic subunit of DNA-dependent protein kinases (DNA-PKcs), a component of the nonhomologous DNA double-strand breaks end-joining system, and a reduction in ATM. We studied this possible connection in other DNA-PKcs-deficient cell types, and following knockdown of DNA-PKcs with small interfering RNA, Chinese hamster ovary V3 cells, lacking DNA-PKcs, had reduced levels of ATM and hSMG-1, but both were restored after transfection with PRKDC. Atm levels were also reduced in murine scid cells. Reduction of ATM in a human glioma cell line lacking DNA-PKcs was accompanied by defective signaling through downstream substrates, post-irradiation. A large reduction of DNA-PKcs was achieved in normal human fibroblasts after transfection with two DNA-PKcs small interfering RNA sequences. This was accompanied by a reduction in ATM. These data were confirmed using immunocytochemical detection of the proteins. Within hours after transfection, a decline in PRKDC mRNA was seen, followed by a more gradual decline in DNA-PKcs protein beginning 1 day after transfection. No change in ATM mRNA was observed for 2 days post-transfection. Only after the DNA-PKcs reduction occurred was a reduction in ATM mRNA observed, beginning 2 days post-transfection. The amount of ATM began to decline, starting about 3 days post-treatment, then it declined to levels comparable to DNA-PKcs. Both proteins returned to normal levels at later times. These data illustrate a potentially important cross-regulation between the nonhomologous end-joining system for rejoining of DNA double-strand breaks and the ATM-dependent damage response network of pathways, both of which operate to maintain the integrity of the genome.
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PMID:Deficiency in the catalytic subunit of DNA-dependent protein kinase causes down-regulation of ATM. 1575 61

DNA-dependent protein kinase (DNA-PK) plays a major role in the repair of DNA double-strand breaks induced by ionizing radiation (IR). Lack of DNA-PK causes defective DNA double-strand break repair and radiosensitization. In general, the cell death induced by IR is considered to be apoptotic. On the other hand, nonapoptotic cell death, autophagy, has recently attracted attention as a novel response of cancer cells to chemotherapy and IR. Autophagy is a protein degradation system characterized by a prominent formation of double-membrane vesicles in the cytoplasm. Little is known, however, regarding the relationship between DNA-PK and IR-induced autophagy. In the present study, we used human malignant glioma M059J and M059K cells to investigate the role of DNA-PK in IR-induced apoptotic and autophagic cell death. Low-dose IR induced massive autophagic cell death in M059J cells that lack the catalytic subunit of DNA-PK (DNA-PKcs). Most M059K cells, the counterpart of M059J cells in which DNA-PKcs are expressed at normal levels, survived, and proliferated although a small portion of the cells underwent apoptosis. Low-dose IR inhibited the phosphorylation of p70(S6K), a molecule downstream of the mammalian target of rapamycin associated with autophagy in M059J cells but not in M059K cells. The treatment of M059K cells with antisense oligonucleotides against DNA-PKcs caused radiation-induced autophagy and radiosensitized the cells. Furthermore, antisense oligonucleotides against DNA-PKcs radiosensitized other malignant glioma cell lines with DNA-PK activity, U373-MG and T98G, by inducing autophagy. The specific inhibition of DNA-PKcs may be promising as a new therapy to radiosensitize malignant glioma cells by inducing autophagy.
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PMID:Inhibition of the DNA-dependent protein kinase catalytic subunit radiosensitizes malignant glioma cells by inducing autophagy. 1589 29

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumours (malignant gliomas). The mechanism of TMZ-induced glioma cell death is unknown. Here, we show that malignant glioma cells undergo apoptosis following treatment with the methylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and TMZ. Cell death determined by colony formation and apoptosis following methylation is greatly stimulated by p53. Transfection experiments with O(6)-methylguanine-DNA methyltransferase (MGMT) and depletion of MGMT by O(6)-benzylguanine showed that, in gliomas, the apoptotic signal originates from O(6)-methylguanine (O(6)MeG) and that repair of O(6)MeG by MGMT prevents apoptosis. We further demonstrate that O(6)MeG-triggered apoptosis requires Fas/CD95/Apo-1 receptor activation in p53 non-mutated glioma cells, whereas in p53 mutated gliomas the same DNA lesion triggers the mitochondrial apoptotic pathway. This occurs less effectively via Bcl-2 degradation and caspase-9, -2, -7 and -3 activation. O(6)MeG-triggered apoptosis in gliomas is a late response (occurring >120 h after treatment) that requires extensive cell proliferation. Stimulation of cell cycle progression by the Pasteurella multocida toxin promoted apoptosis whereas serum starvation attenuated it. O(6)MeG-induced apoptosis in glioma cells was preceded by the formation of DNA double-strand breaks (DSBs), as measured by gammaH2AX formation. Glioma cells mutated in DNA-PK(cs), which is involved in non-homologous end-joining, were more sensitive to TMZ-induced apoptosis, supporting the involvement of DSBs as a downstream apoptosis triggering lesion. Overall, the data demonstrate that cell death induced by TMZ in gliomas is due to apoptosis and that determinants of sensitivity of gliomas to TMZ are MGMT, p53, proliferation rate and DSB repair.
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PMID:Apoptosis in malignant glioma cells triggered by the temozolomide-induced DNA lesion O6-methylguanine. 1681 6

Radiotherapy is an important treatment for patients suffering from high-grade malignant gliomas. Non-targeted (bystander) effects may influence these cells' response to radiation and the investigation of these effects may therefore provide new insights into mechanisms of radiosensitivity and responses to radiotherapy as well as define new targets for therapeutic approaches. Normal primary human astrocytes (NHA) and T98G glioma cells were irradiated with helium ions using the Gray Cancer Institute microbeam facility targeting individual cells. Irradiated NHA and T98G glioma cells generated signals that induced gammaH2AX foci in neighbouring non-targeted bystander cells up to 48 h after irradiation. gammaH2AX bystander foci were also observed in co-cultures targeting either NHA or T98G cells and in medium transfer experiments. Dimethyl sulphoxide, Filipin and anti-transforming growth factor (TGF)-beta 1 could suppress gammaH2AX foci in bystander cells, confirming that reactive oxygen species (ROS) and membrane-mediated signals are involved in the bystander signalling pathways. Also, TGF-beta 1 induced gammaH2AX in an ROS-dependent manner similar to bystander foci. ROS and membrane signalling-dependent differences in bystander foci induction between T98G glioma cells and normal human astrocytes have been observed. Inhibition of ataxia telangiectasia mutated (ATM) protein and DNA-PK could not suppress the induction of bystander gammaH2AX foci whereas the mutation of ATM- and rad3-related (ATR) abrogated bystander foci induction. Furthermore, ATR-dependent bystander foci induction was restricted to S-phase cells. These observations may provide additional therapeutic targets for the exploitation of the bystander effect.
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PMID:ATR-dependent radiation-induced gamma H2AX foci in bystander primary human astrocytes and glioma cells. 1690 3

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