title: Role of Artemis in DSB repair and guarding chromosomal stability following exposure to ionizing radiation at different stages of cell cycle.
abstract: We analyzed the phenotype of cells derived from SCID patients with different mutations in the Artemis gene. Using clonogenic survival assay an increased sensitivity was found to X-rays (2-3-fold) and bleomycin (2-fold), as well as to etoposide, camptothecin and methylmethane sulphonate (up to 1.5-fold). In contrast, we did not find increased sensitivity to cross-linking agents mitomycin C and cis-platinum. The kinetics of DSB repair assessed by pulsed-field gel electrophoresis and gammaH2AX foci formation after ionizing irradiation, indicate that 15-20% of DSB are not repaired in Artemis-deficient cells. In order to get a better understanding of the repair defect in Artemis-deficient cells, we studied chromosomal damage at different stages of the cell cycle. In contrast to AT cells, Artemis-deficient cells appear to have a normal G(1)/S-block that resulted in a similar frequency of dicentrics and translocations, however, frequency of acentrics fragments was found to be 2-4-fold higher compared to normal fibroblasts. Irradiation in G(2) resulted in a higher frequency of chromatid-type aberrations (1.5-3-fold) than in normal cells, indicating that a fraction of DSB requires Artemis for proper repair. Our data are consistent with a function of Artemis protein in processing of a subset of complex DSB, without G(1) cell cycle checkpoint defects. This type of DSB can be induced in high proportion and persist through S-phase and in part might be responsible for the formation of chromatid-type exchanges in G(1)-irradiated Artemis-deficient cells. Among different human radiosensitive fibroblasts studied for endogenous (in untreated samples) as well as X-ray-induced DNA damage, the ranking order on the basis of higher incidence of spontaneously occurring chromosomal alterations and induced ones was: ligase 4> or =AT>Artemis. This observation implicates that in human fibroblasts following exposure to ionizing radiation a lower risk might be created when cells are devoid of endogenous damage.
authors.affiliation: Department of Toxicogenetics, Leiden University Medical Centre, Einthovenweg 20, 2300RC Leiden, The Netherlands. F.Darroudi@LUMC.NL
authors.forename: Firouz
authors.initials: F
authors.lastname: Darroudi
authors.forename: Wouter
authors.initials: W
authors.lastname: Wiegant
authors.forename: Matty
authors.initials: M
authors.lastname: Meijers
authors.forename: Anna A
authors.initials: AA
authors.lastname: Friedl
authors.forename: Mirjam
authors.initials: M
authors.lastname: van der Burg
authors.forename: Janna
authors.initials: J
authors.lastname: Fomina
authors.forename: Jacques J M
authors.initials: JJ
authors.lastname: van Dongen
authors.forename: Dik C
authors.initials: DC
authors.lastname: van Gent
authors.forename: Małgorzata Z
authors.initials: MZ
authors.lastname: Zdzienicka
mesh_terms: D002453:Cell Cycle / Q000235:genetics / Q000528:radiation effects; D002460:Cell Line; D002478:Cells, Cultured; D043171:Chromosomal Instability / Q000187:drug effects / Q000528:radiation effects; D002869:Chromosome Aberrations; D003114:Colony-Forming Units Assay; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins; D004720:Endonucleases; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009153:Mutagens / Q000633:toxicity; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D010641:Phenotype; D011836:Radiation Tolerance; D016511:Severe Combined Immunodeficiency / Q000235:genetics / Q000378:metabolism / Q000473:pathology
chemical_list: D004268:DNA-Binding Proteins; D009153:Mutagens; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C426344:DCLRE1C protein, human; D004720:Endonucleases