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. 2017 May 1;8:449. doi: 10.3389/fimmu.2017.00449

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Copyright © 2017 Speckmann, Sahoo, Rizzi, Hirabayashi, Karow, Serwas, Hoemberg, Damatova, Schindler, Vannier, Boulton, Pannicke, Göhring, Thomay, Verdu-Amoros, Hauch, Woessmann, Escherich, Laack, Rindle, Seidl, Rensing-Ehl, Lausch, Jandrasits, Strahm, Schwarz, Ehl, Niemeyer, Boztug and Wlodarski.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Spontaneous chromosomal breakage and crosslink induced genomic instability. (A–D) Chromosomal aberrations in P1 fibroblasts exposed to 50 ng/ml mitomycin C (MMC), as indicated by arrows: (A) a multiradial and a triradial figure; (B) two atypical reunion figures as well as chromatid and chromosome breaks; (C,D) radial figures with chromatid breaks. (E) Spontaneous and MMC-induced G2 phase accumulation of RTEL1-deficient fibroblasts. Fibroblasts from healthy control (Ctrl) of Fanconi anemia patient (FA) run as controls. ^aDAPI stained cells from 48 h cultures w/o or w/10 ng/ml MMC; ^bone-tailed Student’s t-test, P1 or P3 compared with normal controls. (F) G2 phase accumulation of RTEL1-deficient fibroblasts of P1, DAPI staining. Compared to an untreated healthy control (G1 phase 72.5%, S phase 15.9%, G2 phase 11.6%), fibroblasts from P1 spontaneously show an elevated G2 phase proportion (G1 phase 72.8%, S phase 11.1%, G2 phase 16.1%; left panels). Exposure to 10 ng/ml MMC increases G2 phase in P1 disproportionately (G1 phase 57.4%, S phase 9.5%, G2 phase 33.1%, arrow) compared to a control (G1 phase 69.6%, S phase 7.5%, G2 phase 22.9%). By contrast, 1.5 Gy irradiation reveals comparable G2 phase fractions (P1: 16.0%; control: 20.2%; right panels; DAPI staining). (G) Chromosomal break distributions of RTEL1-deficient fibroblasts of P1. Fibroblasts were left untreated or exposed to MMC for the last 24 h. Breakage rates amounted to 0.28 (0 MMC), 0.52 (10 ng/ml MMC), 2.71 (50 ng/ml MMC) or 4.72 (100 ng/ml MMC), respectively. These were elevated compared to normal control fibroblasts with breakage rates of 0.05 (0 MMC; normal mean, 0.02), 0.12 (50 ng/ml MMC; normal mean, 0.18), or 0.55 (100 ng/ml MMC; normal mean, 0.36); the normal mean rate for 10 ng/ml is 0.06. Breakage rates of fibroblast P1 were more evenly distributed among all break classes and did not show the tendency toward metaphases with 10 or more breaks of FA fibroblasts at 100 ng/ml MMC.