Use of the cytokinesis-block micronucleus assay to measure radiation-induced chromosome damage in lymphoblastoid cell lines.
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.
Gutiérrez-Enríquez
Sara
S
DNA Repair Group, International Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon Cedex 08, France.
Hall
Janet
J
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
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