title: Glioblastoma cells deficient in DNA-dependent protein kinase are resistant to cell death.
abstract: DNA-dependent protein kinase (DNA-PK), a nuclear serine/threonine kinase, is responsible for the DNA double-strand break repair. Cells lacking or with dysfunctional DNA-PK are often associated with mis-repair, chromosome aberrations, and complex exchanges, all of which are known to contribute to the development of human cancers including glioblastoma. Two human glioblastoma cell lines were used in the experiment, M059J cells lacking the catalytic subunit of DNA-PK, and their isogenic but DNA-PK proficient counterpart, M059K. We found that M059K cells were much more sensitive to staurosporine (STS) treatment than M059J cells, as demonstrated by MTT assay, TUNEL detection, and annexin-V and propidium iodide (PI) staining. A possible mechanism responsible for the different sensitivity in these two cell lines was explored by the examination of Bcl-2, Bax, Bak, and Fas. The cell death stimulus increased anti-apoptotic Bcl-2 and decreased pro-apoptotic Bcl-2 members (Bak and Bax) and Fas in glioblastoma cells deficient in DNA-PK. Activation of DNA-PK is known to promote cell death of human tumor cells via modulation of p53, which can down-regulate the anti-apoptotic Bcl-2 member proteins, induce pro-apoptotic Bcl-2 family members and promote a Bax-Bak interaction. Our experiment also demonstrated that the mode of glioblastoma cell death induced by STS consisted of both apoptosis and necrosis and the percentage of cell death in both modes was similar in glioblastoma cell lines either lacking DNA-PK or containing intact DNA-PK. Taken together, our findings suggest that DNA-PK has a positive role in the regulation of apoptosis in human glioblastomas. The aberrant expression of Bcl-2 family members and Fas was, at least in part, responsible for decreased sensitivity of DNA-PK deficient glioblastoma cells to cell death stimuli.
authors.affiliation: Department of Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong. gchen@cuhk.edu.hk
authors.forename: George G
authors.initials: GG
authors.lastname: Chen
authors.forename: Fanny L F
authors.initials: FL
authors.lastname: Sin
authors.forename: Billy C S
authors.initials: BC
authors.lastname: Leung
authors.forename: Ho K
authors.initials: HK
authors.lastname: Ng
authors.forename: Wai S
authors.initials: WS
authors.lastname: Poon
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001932:Brain Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D020287:In Situ Nick-End Labeling; D007202:Indicators and Reagents; D008565:Membrane Proteins / Q000378:metabolism; D009687:Nuclear Proteins; D011419:Propidium; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D019014:fas Receptor / Q000378:metabolism
chemical_list: D017304:Annexin A5; C494772:BAK1 protein, human; C494826:BAX protein, human; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D008565:Membrane Proteins; D009687:Nuclear Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D011419:Propidium; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine