Differential stability of the DNA-activated protein kinase catalytic subunit mRNA in human glioma cells

Am, Galloway; Ca, Spencer; Cw, Anderson; Mj, Allalunis-Turner

doi:10.1038/sj.onc.1202433

Cited by 17 publications

(8 citation statements)

References 34 publications

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“…Human cells lacking ATR are not available. MO59J cells lack detectable levels of DNA-PK (Galloway et al, 1999), but have many 53BP1 foci even in the absence of exposure to IR, which has precluded us from obtaining reliable data regarding the kinetics of 53BP1 focus formation after irradiation (Fig. 7).…”

Section: Discussionmentioning

confidence: 99%

P53 Binding Protein 1 (53bp1) Is an Early Participant in the Cellular Response to DNA Double-Strand Breaks

Schultz

Chehab

Malikzay

et al. 2000

The Journal of Cell Biology

823

627

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p53 binding protein 1 (53BP1), a protein proposed to function as a transcriptional coactivator of the p53 tumor suppressor, has BRCT domains with high homology to the Saccharomyces cerevisiae Rad9p DNA damage checkpoint protein. To examine whether 53BP1 has a role in the cellular response to DNA damage, we probed its intracellular localization by immunofluorescence. In untreated primary cells and U2OS osteosarcoma cells, 53BP1 exhibited diffuse nuclear staining; whereas, within 5–15 min after exposure to ionizing radiation (IR), 53BP1 localized at discreet nuclear foci. We propose that these foci represent sites of processing of DNA double-strand breaks (DSBs), because they were induced by IR and chemicals that cause DSBs, but not by ultraviolet light; their peak number approximated the number of DSBs induced by IR and decreased over time with kinetics that parallel the rate of DNA repair; and they colocalized with IR-induced Mre11/NBS and γ-H2AX foci, which have been previously shown to localize at sites of DSBs. Formation of 53BP1 foci after irradiation was not dependent on ataxia-telangiectasia mutated (ATM), Nijmegen breakage syndrome (NBS1), or wild-type p53. Thus, the fast kinetics of 53BP1 focus formation after irradiation and the lack of dependency on ATM and NBS1 suggest that 53BP1 functions early in the cellular response to DNA DSBs.

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Section: Discussionmentioning

confidence: 99%

P53 Binding Protein 1 (53bp1) Is an Early Participant in the Cellular Response to DNA Double-Strand Breaks

Schultz

Chehab

Malikzay

et al. 2000

The Journal of Cell Biology

823

627

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“…In contrast, both DNA-PK cs and Ku protein were absent from neutrophils and were not synthesized in resting PBLs (Ajmani et al, 1995). Galloway et al (1998) measured a half-life of about 3 days for DNA-PK cs mRNA in the human glioblastoma cell line M059K. A consequence of the relatively long protein and mRNA half-lives and relatively low mRNA abundance is that reductions in the half-lives of either to more typical values (*1 day) would result in comparatively large changes in protein levels.…”

Section: Discussionmentioning

confidence: 99%

DNA-PK, the DNA-activated protein kinase, is differentially expressed in normal and malignant human tissues

et al. 1999

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DNA-PK is a nuclear, serine/threonine protein kinase required for repairing DNA double-strand breaks and for V(D)J recombination. To determine the distribution of DNA-PK in human tissues, we assayed paraffin-embedded sections of normal and cancerous tissues for DNA-PKcs and Ku80 by immunohistochemistry. We also assayed for Brca2, a human tumor suppressor gene that is implicated in the repair of DNA strand-breaks. Brca2 was strongly expressed in epithelial cells of the breast, endometrium, and thymus, in tingible body macrophages of follicular germinal centers of lymphoid tissue, and in reticuloendothelial cells in the spleen. DNA-PKcs and Ku80 expression was usually parallel, but both were expressed in a highly cell- and tissue-specific manner. The highest levels were observed in spermatogenic cells (but not in spermatozoa), and in neurons and glial cells of the central and autonomic nervous system. Neither protein was consistently expressed in liver nor in resting mammary epithelium, but lactating breast epithelium was strongly positive for DNA-PKcs and Ku80. In contrast to established human cell cultures, expression between cells in the same tissue was highly selective in the epidermis, exocrine pancreas, renal glomeruli, the red pulp of the spleen, and within cellular compartments of tonsils, lymph nodes, and thymus. Most cancerous tissues were consistently positive for DNA-PKcs and Ku80, except invasive carcinoma of the breast. DNA-PKcs, Ku80, and Ku70 mRNAs were expressed in all normal tissues with relatively little variation in levels. Our results suggest that the apparent absence of DNA-PKcs and Ku80 from some cells or tissues is a consequence of post-transcriptional mechanisms that regulate protein levels.

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“…M059J cells (kindly provided by Dr. J. AllalunisTurner) were derived from a human malignant glioma, as previously described, and found to be deficient in DNA-PKcs due to a frameshift mutation in the gene encoding for the protein Galloway et al, 1999;Anderson et al, 2001). They were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% L-glutamine, at 378C in a humidified incubator, in an atmosphere of 5% CO 2 and 95% air.…”

Section: Methodsmentioning

confidence: 99%

“…For further insights regarding the potential role and the interplay between these kinases in H2AX phosphorylation we studied M059J cells. These cells are radiosensitive to killing by IR and have no detectable DNA-PK activity Galloway et al, 1999) as a result of a mutation in the gene encoding DNA-PKcs (Anderson et al, 2001). The results obtained are shown in Figure 2A.…”

Section: H2ax Phosphorylation In Cells Deficient In Dna-pkmentioning

confidence: 99%

Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA‐PK in human cells exposed to ionizing radiation and treated with kinase inhibitors

Wang

et al. 2004

Journal Cellular Physiology

179

109

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In eukaryotic cells, DNA double strand breaks (DSBs) cause the prompt phosphorylation of serine 139 at the carboxy terminus of histone H2AX to generate gamma-H2AX, detectable by Western blotting or immunofluorescence. The consensus sequence at the phosphorylation site implicates the phosphatidylinositol 3-like family of protein kinases in H2AX phosphorylation. It remains open whether ATM (ataxia telangiectasia mutated) is the major H2AX kinase, or whether other members of the family, such as DNA-PK (DNA dependent protein kinase) or ATR (ATM and Rad3 related), contribute in a functionally complementary manner. To address this question, we measured global H2AX phosphorylation in cell lysates and foci formation in individual cells of either wild type or mutant (ATM or DNA-PK) genetic background. Normal global phosphorylation kinetics is observed after irradiation in cells defective either in ATM or DNA-PK alone, suggesting a complementary contribution to H2AX phosphorylation. This is further supported by the observation that initial H2AX phosphorylation is delayed when both kinases are inhibited by wortmannin, as well as when ATM is inhibited by caffeine in DNA-PK deficient cells. However, robust residual global phosphorylation is detectable under all conditions of genetic or chemical inhibition suggesting the function of additional kinases, such as ATR. Treatment with wortmannin, caffeine, or UCN-01 produces a strong DNA-PK dependent late global hyperphosphorylation of H2AX, uncoupled from DNA DSB rejoining and compatible with an inhibition of late steps in DNA DSB processing. Evaluation of gamma-H2AX foci formation confirms the major conclusions made on the basis of global H2AX phosphorylation, but also points to differences particularly several hours after exposure to IR. The results in aggregate implicate DNA-PK, ATM and possibly other kinases in H2AX phosphorylation. The functional significance and the mechanisms of coordination in space and time of these multiple inputs require further investigation.

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Differential stability of the DNA-activated protein kinase catalytic subunit mRNA in human glioma cells

Cited by 17 publications

References 34 publications

P53 Binding Protein 1 (53bp1) Is an Early Participant in the Cellular Response to DNA Double-Strand Breaks

P53 Binding Protein 1 (53bp1) Is an Early Participant in the Cellular Response to DNA Double-Strand Breaks

DNA-PK, the DNA-activated protein kinase, is differentially expressed in normal and malignant human tissues

Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA‐PK in human cells exposed to ionizing radiation and treated with kinase inhibitors

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