title: Use of the cytokinesis-block micronucleus assay to measure radiation-induced chromosome damage in lymphoblastoid cell lines.
abstract: To establish the optimal experimental conditions for the use of the micronuclei (MN) test to determine the level of chromosomal damage induced by ionising radiation (IR) exposure in lymphoblastoid cell lines, a time-course study was performed comparing a normal and an ataxia telangiectasia (AT) cell line, the latter being characterised by an extreme radiation sensitivity. Several parameters were analysed: the use of cytochalasin-B (Cyt-B) to quantify MN, the optimum fixation time to measure radiation-induced MN, the most appropriate treatment dose of IR to distinguish between the normal and the radiosensitive cells and the cell-cycle distribution after irradiation. The results obtained showed that the spontaneous as well as the radiation-induced levels of MN were significantly higher in the AT cell compared to the normal cells (P < 0.001 and P = 0.005, respectively). In both cell types the number of radiation-induced MN were lower in the cultures without Cyt-B than those with Cyt-B (P < 0.001), with the AT cells being distinguished in terms of IR-induced MN from the normal cells only with the addition of Cyt-B. The level of MN formation was independent of the dose of Cyt-B used (3 or 6 microg/ml). The optimum time for radiation-induced MN measured was found to be between 48 and 72 h post-irradiation, with 2 and 4 Gy exposures inducing similar levels of MN. However, as the higher dose caused a greater delay in the cell-cycle, treatment with 2 Gy with MN measurement at 48 h in the presence of 3 microg/ml Cyt-B were chosen as the optimum experimental conditions. This choice was validated using two additional normal and AT cell lines. In conclusion, our results show that the use of Cyt-B increases the sensitivity of the MN test for comparing differences in radiosensitivity between lymphoblastoid cell lines.
authors.affiliation: DNA Repair Group, International Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon Cedex 08, France.
authors.forename: Sara
authors.initials: S
authors.lastname: Gutiérrez-Enríquez
authors.forename: Janet
authors.initials: J
authors.lastname: Hall
mesh_terms: D001260:Ataxia Telangiectasia / Q000235:genetics; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002869:Chromosome Aberrations; D002877:Chromosomes, Human / Q000235:genetics / Q000528:radiation effects; D003571:Cytochalasin B / Q000494:pharmacology; D004249:DNA Damage; D004268:DNA-Binding Proteins; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000528:radiation effects; D015162:Micronucleus Tests / Q000379:methods*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011836:Radiation Tolerance / Q000235:genetics; D025521:Tumor Suppressor Proteins
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D003571:Cytochalasin B; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases