Stimulated rapid expression in vitro for early detection of in vivo T-cell receptor mutations induced by radiation exposure

Ishioka, Nahoko; Umeki, Shigeko; Hirai, Yuko; Akiyama, Mitoshi; Kodama, Takashi; K, Ohama; Kyoizumi, Seishi

doi:10.1016/s1383-5718(97)00025-9

Cited by 19 publications

(9 citation statements)

References 33 publications

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“…3). These results are consistent with that for cultured human T-lymphocytes Ishioka et al, 1997), and support the observations of the linear-quadratic dose responses obtained from mouse in vivo Umeki et al, 1997), indicating that the in vitro mouse cell line model for TCR mutation studies is to facilitate the in vivo mouse screening of mutagenic substances.…”

Section: Discussionsupporting

confidence: 88%

“…The in vivo TCR mutant assays using T-lymphocytes from human (Kyoizumi et al, 1990;Sawada et al, 1998aSawada et al, , 1998b) and mice Umeki et al, 1997) have been established, and the assays have successfully been used to detect radiationinduced mutations in the patients receiving radiotherapy (Iwamoto et al, 1994;Ishioka et al, 1997) and in the population exposed to radiation during the 1986 Chernobyl accident (Saenko et al, 1998(Saenko et al, , 2000. For the in vitro TCR mutant assay, using cultured human Tlymphocytes, we previously reported that X-raysinduced TCR MF at D 37 (giving 37% survival) was 31.7 × 10 −4 and the relationship appears linear between the mutation induction and the doses .…”

Section: Discussionmentioning

confidence: 99%

“…Ionizing radiation increases the frequency of TCR mutants in T cells in a dose dependent fashion. This relationship provides a highly sensitive means for measuring genotoxic effect of radiation, and it has been used in both human (Iwamoto et al, 1994;Ishioka et al, 1997) and mouse Umeki et al, 1997) studies. Although the sensitivity of the assays of three biological endpoints including TCR assay, hprt assay, and micronuclei assay in EL-4 (C-4) cells may be different, the relationships appear to be linear between TCR and hprt MFs, and between TCR mutant cells and micronuclei cells (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The thorotrast patients who have been continuously irradiated with alpha-particles emitted from the internal deposition of thorium dioxide have a significantly higher frequency of mutants at the lymphocyte TCR gene (Umeki et al, 1991;. Although there was no significant doserelated effect observed in atomic bomb survivors, cancer patients who had received radiotherapy (Kyoizumi et al, 1992;Iwamoto et al, 1994;Ishioka et al, 1997) or chemotherapy (Hirota et al, 1994;Sawada et al, 1998a) showed a dose-related increase of TCR mutant frequency (MF). Thus, this assay has been applied to human lymphocytes from people who were directly or indirectly exposed to radiation during and after the Chernobyl accident in Ukraine (Akleyev et al, 1995;Saenko et al, 1998Saenko et al, , 2000Zamulaeva et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2007

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-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.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2007

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“…4). Previously, it had been shown that the cell growth rate declined appreciably in similar T-cell cultures 10-14 days after the beginning of culture [Ishioka et al, 1997]. This decline may have been simply due to growth arrest mediated by attenuated T-cell receptor and/or growth factor signals.…”

Section: Discussionmentioning

confidence: 95%

Short‐term culture and γH2AX flow cytometry determine differences in individual radiosensitivity in human peripheral T lymphocytes

Hamasaki

Imai

Nakachi

et al. 2006

Environ and Mol Mutagen

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Histone H2AX, a subfamily of histone H2A, is phosphorylated and forms proteinaceous repair foci at the sites of DNA double-strand breaks in response to genotoxic insults, such as ionizing radiation. This process is believed to play a key role in the repair of DNA damage. In this study, we established a flow cytometry (FCM) system for measuring radiation-induced phosphorylated histone H2AX (gammaH2AX) in cultured human T lymphocytes to evaluate individual radiation sensitivity in vitro. Irradiation of short-term ( approximately 7 days) cultured T lymphocytes exhibited significant interindividual, but not interexperimental, differences in the cellular content of gammaH2AX 6 hr after 4 Gy of X-irradiation in three independent experiments using peripheral blood lymphocytes from six healthy donors. However, these differences were not as marked in uncultured lymphocytes, or lymphocytes that were cultured for a prolonged period ( approximately 13 days). The variation of gammaH2AX focus formation in lymphocytes of individuals was reproducible, with differences reaching about 1.5-fold following 7 days of culture. Therefore, the FCM-based gammaH2AX measurement appeared to reflect both the temporal course and the amount of DNA damage within the irradiated lymphocytes. Further, we confirmed that the differences in residual lymphocyte subsets were not involved in individual radiosensitivity. These results suggest that the FCM-based gammaH2AX assay using cultured T lymphocytes might be useful for the rapid and reliable assessment of individual radiation sensitivity involved in DNA damage repair.

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T-Cell Receptor Mutation Assay for Monitoring Human Genotoxic Exposure

Kyoizumi

2014

Genotoxicity and DNA Repair

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Mutant T-cells defective in T-cell receptor (TCR) α or β gene expression among CD4+ T-cells can be detected as CD3 − CD4 + cells by two-color fl ow cytometry using anti-CD3 and anti-CD4 monoclonal antibodies labeled with different fl uorescent dyes. This is because incomplete TCR αβ/CD3 complexes are not transported to the surface membrane. TCR mutations are spontaneously generated at a frequency of about 2 × 10 −4 in peripheral CD4 + T-cells, and the mutant frequencies are dose-dependently increased by exposure to genotoxic substances such as ionizing radiation and chemicals. The TCR mutation assay was developed in 1990, and this method has since been applied to monitoring human genotoxic exposure. It has been proved that the assay has advantage in detection of chronic exposure in large-scale studies. Here, I will briefl y review past studies reporting the applications, and describe methods for both preparation of the target cells and detection of the mutant cells.

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Stimulated rapid expression in vitro for early detection of in vivo T-cell receptor mutations induced by radiation exposure

Cited by 19 publications

References 33 publications

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

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

Short‐term culture and γH2AX flow cytometry determine differences in individual radiosensitivity in human peripheral T lymphocytes

T-Cell Receptor Mutation Assay for Monitoring Human Genotoxic Exposure

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