Role of Artemis in DSB repair and guarding chromosomal stability following exposure to ionizing radiation at different stages of cell cycle.
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.
Darroudi
Firouz
F
Department of Toxicogenetics, Leiden University Medical Centre, Einthovenweg 20, 2300RC Leiden, The Netherlands. F.Darroudi@LUMC.NL
Wiegant
Wouter
W
Meijers
Matty
M
Friedl
Anna A
AA
van der Burg
Mirjam
M
Fomina
Janna
J
van Dongen
Jacques J M
JJ
van Gent
Dik C
DC
Zdzienicka
Małgorzata Z
MZ
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
D004268:DNA-Binding Proteins; D009153:Mutagens; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C426344:DCLRE1C protein, human; D004720:Endonucleases