Effect of irradiation on DNA synthesis, NBN gene expression and chromosomal stability in cells with NBN mutations

• Main Authors: Jerzy Nowak, Bogna Świątek-Kościelna, Ewelina M. Kałużna, Jolanta Rembowska, Agnieszka Dzikiewicz-Krawczyk, Mariola Zawada, Danuta Januszkiewicz-Lewandowska
• Format: Article
• Language: English
• Published: Termedia Publishing House 2017-01-01
• Series: Archives of Medical Science
• Subjects: DNA damage; genetic instability; <I>NBN</I>; nibrin; radiosensitivity
• Online Access: https://www.termedia.pl/Effect-of-irradiation-on-DNA-synthesis-NBN-gene-expression-and-chromosomal-stability-in-cells-with-NBN-mutations,19,29240,1,1.html

Introduction : The NBN gene product is part of the MRE11/RAD50/ NBN complex, which plays an essential role in genomic stability. In the study we try to answer the question what is the effect of irradiation on DNA synthesis, NBN gene expression and chromosomal stability in cells with homozygous c.657-661del, and heterozygous c.657-661del, p.I171V and p.R215W NBN gene mutations. Material and methods : Immortalized B-lymphocytes with NBN gene mutations were X-ray irradiated at doses of 1, 2, 5 and 8 Gy/min. Radioresistant DNA synthesis rate and the percentage of cells in phase S was analyzed by 3H thymidine and BrdU incorporation assays. NBN gene expression was quantified by real-time PCR with TaqMan fluorescent probe. Results : Increasing the irradiation dose resulted in gradual decrease of 3H thymidine incorporation in all cells, but significantly only in homo- and heterozygous c.657-661del cells (p-values < 0.0001). After irradiation the relative expression of NBN was significantly higher in homozygous c.657-661del and heterozygous p.R215W cells as compared to heterozygous c.657-661del, p.I171V and control cells (p < 0.01). All cells with NBN gene mutations showed significantly higher total number of chromosomal aberrations per metaphase as compared to control cells, with the highest number of aberrations in homozygous c.657-661del cells (p < 0.001). Conclusions : The results obtained indicate that homozygous c.657-661del mutation affects cell sensitivity to irradiation. Moreover, homozygous variant is associated with disturbance in the activation of cell cycle checkpoints and with defects in DNA repair. In turn, heterozygous c.657-661del, p.R215W and p.I171V mutations do not substantially alter the radiosensitivity.