Nature of nontargeted radiation effects observed during fractionated irradiation-induced thymic lymphomagenesis in mice

摘要

All animals were handled according to our institutional guidelines. Female 5-week-old C57BL/6J mice were irradiated four times with 1.8 Gy 137Cs γ-rays (RCS-50, Tokyo Shibaura, Japan) at 1-week intervals, which resulted in the development of thymic lymphomas in almost 100% of the mice [1]. Mice were maintained in a specific pathogen-free facility for 1 year to monitor thymic lymphomagenesis. To examine the thymocyte characteristics, thymocytes were isolated 0 (2 h) to 13 weeks after the final irradiation by mildly rubbing the thymus with frosted slide glass within 30 min after the thymuses were isolated and placed in Dulbecco's modified Eagle's medium (D-MEM) supplemented with 10% heat-inactivated foetal bovine serum at room temperature. The thymocytes were suspended in the medium at 4°C until use in the experiments. Immediately after isolation, the thymocytes (2.5 × 106 cells) were incubated in 2.5 ml of 5 μM freshly prepared 5-(and-6)-chloromethyl-2′, 7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA; Molecular Probes, Eugene, OR, USA) in phosphate-buffered saline (PBS) for 30 min at 37°C. After washing once with PBS, the cells were suspended in 2.5 ml PBS containing 5 μg/ml propidium iodide. Fluorescence intensity was analysed in 5 × 104 viable cells by FACSCalibur flow cytometry (BD Biosciences, Mountain View, CA, USA). Because ROS values fluctuated somewhat between experiments, values relative to those in five nonirradiated mice examined simultaneously are shown. Immediately after isolation, the thymocytes (5 × 106 cells) were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton-X 100 in PBS for 20 min, and then blocked with 10% goat serum for 1 h at room temperature to eliminate nonspecific antibody binding. The thymocytes were treated with 250-fold diluted anti-γ-H2AX mouse monoclonal antibody (Millipore, Billerica, MA, USA) in A-buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 0.1% Tween20 and 5% non-fat milk) for 2 h at 37°C, and then with 80-fold diluted tetramethylrhodamine B isothiocyanate-conjugated secondary antibody (Dako Cytomation, Glostrup, Denmark) in A-buffer for 1 h at 37°C. The thymocytes were washed with PBS three times, and then once with 0.5% Tween20 in PBS. The number of large γ-H2AX foci, not nascent small foci, was counted in 100 cells per sample under fluorescence microscopy (AX70, Olympus, Tokyo, Japan). The human colon cancer cell line HCT116 bearing a defective mismatch repair gene, hMLH1 [32], was obtained from ATCC and stocked at –85°C immediately after receipt. XRCC4?/? cells were isolated from HCT116 cells by gene targeting of XRCC4 loci [33]. Confirmation of the targeted loci by Southern blotting and western blotting, and the sensitivity to DNA damaging agents based on a cell survival assay have been described previously [33]. Mouse mutant cell lines, Ogg1?/? (OG7L line) cells with deficient repair of oxidized base lesions and Mth1?/? (T5 line) cells with deficient hydrolysis of oxidized nucleotides, were established from embryos of the respective mutant mice by long periods of in vitro culture, as described previously [34, 35]. Mutyh?/? mouse embryo fibroblasts lacking adenine/2-hydroxyadenine DNA glycosylase were isolated from embryos (13.5 days postcoital) obtained by mating Mutyh+/? mice (n = 12, C57BL/6J background), and maintained for 40 passages in D-MEM supplemented with 10% heat inactivated foetal bovine serum. Cells that spontaneously immortalized were designated as the Mutyh?/? (Y12L) line. The mutated loci in established cell lines were confirmed by genomic polymerase chain reaction (PCR) and western blotting, as described previously [34–36], and oxidative damage repair deficiency of the OG7L and Y12L lines was verified using a nicking assay, as described previously [34, 37]. Mth1 deficiency was verified using an 8-oxo-dGTPase assay, as described previously [35]. Once cell lines were established, the cells were stocked and used for experiments within 1 month after re-culture. To analyse chromosomal instability in vivo and in vitro, thymocytes (5 × 106 cells) were incubated with 100 ng/ml Colcemid in D-MEM for 3 h or cultured with 5 ml D-MEM for 48 h with 10 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO, USA), 250 ng/ml ionomycin (Calbiochem, San Diego, CA, USA), and 50 μM 2-mercaptoethanol at 37°C in a humidified 5% CO₂ atmosphere, and treated with Colcemid for the last 1 h of the culture [38]. Chromosomes were prepared according to the air-drying method. Forty metaphase cells were observed for aneuploidy induction in each sample. Chromosomal aberrations were scored on coded slides in 200 cells per sample according to the ISCN (1995) criteria [39]. Tra