title: Functional deficiency of NBN, the Nijmegen breakage syndrome protein, in a p.R215W mutant breast cancer cell line.
abstract: BACKGROUND
  Mutations in NBN, the gene for Nijmegen Breakage Syndrome (NBS), are thought to predispose women to developing breast cancer, but a breast cancer cell line containing mutations in NBN has not yet been described. The p.R215W missense mutation occurs at sub-polymorphic frequencies in several populations. We aimed to investigate its functional impact in breast cancer cells from a carrier of this NBN mutation.
  
  
  METHODS
  Breast cancer cell lines were screened by immunoblotting for NBN protein levels, and the NBN coding region was sequenced for mutation analysis. Radiosensitivity assays and functional studies were performed through immunocytochemistry and immunoblotting, and flow cytometry was employed to assess cell cycle progression. Impedance measurements were used to study the consequences of PARP1 inhibition. Statistical comparisons between cell lines were performed using t-tests.
  
  
  RESULTS
  HCC1395 breast cancer cells exhibited reduced NBN protein levels. Direct sequencing identified the NBN p.R215W mutation in the hemizygous state, in addition to a truncation in BRCA1. Mutations in both genes were already present in the heterozygous state in the patient's germline. HCC1395 cells were highly radiosensitive, susceptible to apoptosis and were deficient in the formation of NBN foci. There was also evidence for some impairment in the formation of γH2AX, MDC1, and 53BP1 foci after irradiation; these foci appeared smaller and irregular compared with repair foci in wild-type cells, although ATM signalling was largely unaffected. In line with their deficiency in NBN and BRCA1, HCC1395 cells were particularly sensitive to PARP1 inhibition.
  
  
  CONCLUSIONS
  Our results indicate that the p.R215W mutation in the HCC1395 breast cancer cell line impairs NBN function, making this cell line a potentially useful cellular model for studying defective NBN protein within a mutant BRCA1 background.
authors.forename: Bianca
authors.initials: B
authors.lastname: Schröder-Heurich
authors.forename: Natalia
authors.initials: N
authors.lastname: Bogdanova
authors.forename: Britta
authors.initials: B
authors.lastname: Wieland
authors.forename: Xiaoxi
authors.initials: X
authors.lastname: Xie
authors.forename: Monika
authors.initials: M
authors.lastname: Noskowicz
authors.forename: Tjoung-Won
authors.initials: TW
authors.lastname: Park-Simon
authors.forename: Peter
authors.initials: P
authors.lastname: Hillemanns
authors.forename: Hans
authors.initials: H
authors.lastname: Christiansen
authors.affiliation: Clinics of Obstetrics and Gynaecology, Hannover Medical School, Carl-Neuberg Straße 1, D-30625 Hannover, Germany. Doerk.Thilo@mh-hannover.de.
authors.forename: Thilo
authors.initials: T
authors.lastname: Dörk
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis; D004260:DNA Repair; D005260:Female; D006657:Histones / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D011836:Radiation Tolerance / Q000235:genetics; D014410:Tumor Stem Cell Assay
chemical_list: D018797:Cell Cycle Proteins; D006657:Histones; C112156:NBN protein, human; D009687:Nuclear Proteins