title: The radiosensitizing effect of Ku70/80 knockdown in MCF10A cells irradiated with X-rays and p(66)+Be(40) neutrons.
abstract: BACKGROUND
  A better understanding of the underlying mechanisms of DNA repair after low- and high-LET radiations represents a research priority aimed at improving the outcome of clinical radiotherapy. To date however, our knowledge regarding the importance of DNA DSB repair proteins and mechanisms in the response of human cells to high-LET radiation, is far from being complete.
  
  
  METHODS
  We investigated the radiosensitizing effect after interfering with the DNA repair capacity in a human mammary epithelial cell line (MCF10A) by lentiviral-mediated RNA interference (RNAi) of the Ku70 protein, a key-element of the nonhomologous end-joining (NHEJ) pathway. Following irradiation of control and Ku-deficient cell lines with either 6 MV X-rays or p(66)+Be(40) neutrons, cellular radiosensitivity testing was performed using a crystal violet cell proliferation assay. Chromosomal radiosensitivity was evaluated using the micronucleus (MN) assay.
  
  
  RESULTS
  RNAi of Ku70 caused downregulation of both the Ku70 and the Ku80 proteins. This downregulation sensitized cells to both X-rays and neutrons. Comparable dose modifying factors (DMFs) for X-rays and neutrons of 1.62 and 1.52 respectively were obtained with the cell proliferation assay, which points to the similar involvement of the Ku heterodimer in the cellular response to both types of radiation beams. After using the MN assay to evaluate chromosomal radiosensitivity, the obtained DMFs for X-ray doses of 2 and 4 Gy were 2.95 and 2.66 respectively. After neutron irradiation, the DMFs for doses of 1 and 2 Gy were 3.36 and 2.82 respectively. The fact that DMFs are in the same range for X-rays and neutrons confirms a similar importance of the NHEJ pathway and the Ku heterodimer for repairing DNA damage induced by both X-rays and p(66)+Be(40) neutrons.
  
  
  CONCLUSIONS
  Interfering with the NHEJ pathway enhanced the radiosensitivity of human MCF10A cells to low-LET X-rays and high-LET neutrons, pointing to the importance of the Ku heterodimer for repairing damage induced by both types of radiation. Further research using other high-LET radiation sources is however needed to unravel the involvement of DNA double strand break repair pathways and proteins in the cellular response of human cells to high-LET radiation.
authors.affiliation: Department of Structural and Functional Biology, Laboratory of Toxicology and Pharmacology, Università degli Studi dell' Insubria, via A, Da Guissano 10, 21052 Busto Arsizio, Italy.
authors.forename: Veerle
authors.initials: V
authors.lastname: Vandersickel
authors.forename: Monica
authors.initials: M
authors.lastname: Mancini
authors.forename: Jacobus
authors.initials: J
authors.lastname: Slabbert
authors.forename: Emanuela
authors.initials: E
authors.lastname: Marras
authors.forename: Hubert
authors.initials: H
authors.lastname: Thierens
authors.forename: Gianpaolo
authors.initials: G
authors.lastname: Perletti
authors.forename: Anne
authors.initials: A
authors.lastname: Vral
mesh_terms: D034961:Antigens, Nuclear / Q000235:genetics*; D001940:Breast / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects*; D049109:Cell Proliferation / Q000528:radiation effects; D002478:Cells, Cultured; D004249:DNA Damage / Q000528:radiation effects; D004260:DNA Repair / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D006801:Humans; D000072200:Ku Autoantigen; D015162:Micronucleus Tests; D008875:Middle Aged; D009502:Neutrons; D034741:RNA, Small Interfering / Q000494:pharmacology; D011836:Radiation Tolerance / Q000235:genetics*; D014965:X-Rays
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen