Measurement of mutant frequency in T-cell receptor (TCR) gene by flow cytometry after X-irradiation on EL-4 mice lymphoma cells.
It is well known that somatic mutations are induced by ionizing irradiation. We have previously reported the measurement of mutant frequency (MF) on the T-cell receptor (TCR) gene in mouse T-lymphocytes after irradiation by flow cytometry. In this study, we developed an in vitro system using murine EL-4 lymphoma cells and observed frequency of cells defective in TCR gene expression after exposure to ionizing irradiation. EL-4 cells were stained with fluorescein-labeled anti-CD4 and phycoerythrin-labeled anti-CD3 antibodies. They were analyzed with a flow cytometer to detect mutant EL-4 cells lacking surface expression of TCR/CD3 complexes which showed CD3-, CD4+ due to a somatic mutation at the TCR genes. Mutant cells could be observed at 2 days after 3 Gy irradiation. MF of EL-4 cells was 6.7x10(-4) for 0 Gy and the value increased to the maximum level of 39x10(-4) between 4 and 8 days after 3 Gy irradiation and these data were found to be best fitted by a linear-quadratic dose-response model. After the peak value the TCR MF gradually decreased with a half-life of approximately 3.2 days. We also examined the hprt mutant frequencies at seven days after irradiation and the cytokinesis-blocked micronucleus frequency at 20 hrs after irradiation. The frequencies of hprt mutation and micronuclei were found to be best fitted by a linear-quadratic dose-response model and a linear dose-response model, respectively. The method to detect mutation on TCR gene is quick and easy in comparison with other methods and is considered useful for the mutagenicity test.
Kunugita
Naoki
N
Department of Health Information Science, School of Health Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan. kunugita@med.uoeh-u.ac.jp
Mei
Nan
N
Goncharova
Tatjana
T
Norimura
Toshiyuki
T
D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry; D015870:Gene Expression / Q000528:radiation effects*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D051379:Mice; D015162:Micronucleus Tests; D009154:Mutation; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D013601:T-Lymphocytes / Q000378:metabolism / Q000528:radiation effects*; D014965:X-Rays / Q000009:adverse effects
D011948:Receptors, Antigen, T-Cell; D007041:Hypoxanthine Phosphoribosyltransferase