Measurement of Mutant Frequency in T-Cell Receptor (Tcr) Gene by Flow Cytometry After X-Irradiation on El-4 Mice Lymphoma Cells

Abstract

-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 cytomery. 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 6.7 × 10 −4 for 0 Gy and the value increased to the maximum level of 39 × 10 −4 between 4 and 8 days after 3 Gy irradiation and these data were found to be best fitted by a linear-quadratic doseresponse 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 cytokinesisblocked 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.

Increased frequencies of micronucleated reticulocytes and T-cell receptor mutation in Aldh2 knockout mice exposed to acetaldehyde

“…We had previously developed a T-cell receptor (TCR) somatic cell mutant assay (Kunugita et al, 1996(Kunugita et al, , 2007.…”