title: Effect of irradiation on DNA synthesis,NBNgene expression and chromosomal stability in cells withNBNmutations.
abstract: BACKGROUND
  TheNBNgene 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,NBNgene expression and chromosomal stability in cells with homozygous c.657-661del, and heterozygous c.657-661del, p.I171V and p.R215WNBNgene mutations.
  
  
  METHODS
  Immortalized B-lymphocytes withNBNgene 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 by3H thymidine and BrdU incorporation assays.NBNgene expression was quantified by real-time PCR with TaqMan fluorescent probe.
  
  
  RESULTS
  Increasing the irradiation dose resulted in gradual decrease of3H 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 ofNBNwas 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 withNBNgene 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.
authors.affiliation: Department of Molecular Pathology, Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Jerzy
authors.initials: J
authors.lastname: Nowak
authors.affiliation: Department of Molecular Pathology, Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Bogna
authors.initials: B
authors.lastname: Świątek-Kościelna
authors.affiliation: Department of Molecular Pathology, Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Ewelina M
authors.initials: EM
authors.lastname: Kałużna
authors.affiliation: Department of Molecular Pathology, Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Rembowska
authors.affiliation: Department of Molecular Pathology, Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Dzikiewicz-Krawczyk
authors.affiliation: Department of Molecular Pathology, Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Mariola
authors.initials: M
authors.lastname: Zawada
authors.affiliation: Department of Molecular Pathology, Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland; Department of Paediatric Oncology, Haematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Poznan, Poland.
authors.forename: Danuta
authors.initials: D
authors.lastname: Januszkiewicz-Lewandowska
keywords: DNA damage; NBN; genetic instability; nibrin; radiosensitivity