High LET Radiation Amplifies Centrosome Overduplication Through a Pathway of gamma-Tubulin Monoubiquitination.

PURPOSE: 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.\nMETHODS AND MATERIALS: Carbon and iron ion beams (13-200 keV/um) and gamma-rays (0.5 keV/um) were used as radiation sources. To count centrosomes after IR exposure, human U2OS and mouse NIH3T3 cells were immunostained with antibodies of gamma-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.\nRESULTS: 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/um, 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 gamma-tubulin, we subsequently tested whether it is affected by high LET radiation. As a result, monoubiquitination of gamma-tubulin was abolished in 48 to 72 hours after exposure to high LET radiation, although gamma-ray exposure slightly decreased it 48 hours postirradiation and was restored to a normal level at 72 hours.\nCONCLUSIONS: High LET radiation significantly reduces NBS1/BRCA1-mediated monoubiquitination of gamma-tubulin and amplifies centrosome overduplication with a peak at 100 keV/um. In contrast, Ku70 and DNA-PKcs deficiencies mitigate centrosome overduplication, although deficiencies of both NBS1/BRCA1 and Ku70/DNA-PKcs markedly enhance cell killing