title: High LET radiation amplifies centrosome overduplication through a pathway of γ-tubulin monoubiquitination.
abstract: OBJECTIVE
  Radiation induces centrosome overduplication, leading to mitotic catastrophe and tumorigenesis. Because mitotic catastrophe is one of the major tumor cell killing factors in high linear energy transfer (LET) radiation therapy and long-term survivors from such treatment have a potential risk of secondary tumors, we investigated LET dependence of radiation-induced centrosome overduplication and the underlying mechanism.
  
  
  METHODS
  Carbon and iron ion beams (13-200 keV/μm) and γ-rays (0.5 keV/μm) were used as radiation sources. To count centrosomes after IR exposure, human U2OS and mouse NIH3T3 cells were immunostained with antibodies of γ-tubulin and centrin 2. Similarly, Nbs1-, Brca1-, Ku70-, and DNA-PKcs-deficient mouse cells and their counterpart wild-type cells were used for measurement of centrosome overduplication.
  
  
  RESULTS
  The number of excess centrosome-containing cells at interphase and the resulting multipolar spindle at mitosis were amplified with increased LET, reaching a maximum level of 100 keV/μm, followed by sharp decrease in frequency. Interestingly, Ku70 and DNA-PKcs deficiencies marginally affected the induction of centrosome overduplication, whereas the cell killings were significantly enhanced. This was in contrast to observation that high LET radiation significantly enhanced frequencies of centrosome overduplication in Nbs1- and Brca1-deficient cells. Because NBS1/BRCA1 is implicated in monoubiquitination of γ-tubulin, we subsequently tested whether it is affected by high LET radiation. As a result, monoubiquitination of γ-tubulin was abolished in 48 to 72 hours after exposure to high LET radiation, although γ-ray exposure slightly decreased it 48 hours postirradiation and was restored to a normal level at 72 hours.
  
  
  CONCLUSIONS
  High LET radiation significantly reduces NBS1/BRCA1-mediated monoubiquitination of γ-tubulin and amplifies centrosome overduplication with a peak at 100 keV/μm. In contrast, Ku70 and DNA-PKcs deficiencies mitigate centrosome overduplication, although deficiencies of both NBS1/BRCA1 and Ku70/DNA-PKcs markedly enhance cell killing.
authors: Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Kyoto, Japan.
authors: Mikio
authors: M
authors: Shimada
authors: Ryoichi
authors: R
authors: Hirayama
authors: Kenshi
authors: K
authors: Komatsu
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear; D019313:BRCA1 Protein / Q000172:deficiency / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000172:deficiency; D002244:Carbon; D018797:Cell Cycle Proteins / Q000172:deficiency / Q000502:physiology; D045744:Cell Line, Tumor; D018385:Centrosome / Q000502:physiology / Q000528:radiation effects*; D051747:DNA-Activated Protein Kinase / Q000172:deficiency; D004268:DNA-Binding Proteins / Q000172:deficiency; D063193:Heavy Ion Radiotherapy / Q000379:methods; D006801:Humans; D007501:Iron; D000072200:Ku Autoantigen; D018499:Linear Energy Transfer / Q000502:physiology*; D051379:Mice; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000172:deficiency / Q000502:physiology; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism*
chemical_list: D034961:Antigens, Nuclear; D019313:BRCA1 Protein; C472752:CETN2 protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C112156:NBN protein, human; D009687:Nuclear Proteins; D014404:Tubulin; D002244:Carbon; D007501:Iron; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen