Abstract BACKGOUND. Radiotherapy induces tumor cell killing mainly by DNA damage and is widely used as one of the main therapeutic modalities for prostate cancer. However, a significant proportion of prostate cancer patients experiences tumor recurrence after radiotherapy and eventually dies of the disease. We previously observed that ionizing radiation (IR) initially damaged prostate cancer stem cells (pCSCs) but levels of CSCs were increased in the recurrent tumors of prostate cancer cell lines and human prostate cancer tissues. METHODS. To define the radioresistance mechanism of pCSCs, we examined expressions of DNA-repair proteins [Ku70, Ku80 and Ataxia telangiectasia and Rad3-related protein kinase (ATR)], cell cycle distribution, and levels of intracellular reactive oxygen species (ROS) in prostaspheres, which is one of pCSC models, and compared to those of total population of a prostate cancer cell line LNCaP. Expressions of DNA-repair proteins were also examined in human prostate cancer tissues harvested before and after radiotherapy. RESTULTS. In non-irradiated cells, the expressions of Ku70 and Ku80 were higher in prostaspheres compared to those of total population. ATR expression was inconspicuous in both cells. After IR with 10 Gy, the expressions of Ku 70, Ku 80, and ATR proteins were increased in both cells with higher levels in prostaspheres than those of total population. Although there was a tendency that prostaspheres belonged to more in G1 phase and less in S phase of cell cycle, it was not statistically significant. Apoptotic cell population and expressions of total and cleaved forms of caspase 3 were not different between prostaspheres and total population. Prostaspheres contained one group of cells that had low levels of ROS. In recurrent human prostate cancer tissue after radiotherapy, expressions of Ku70 and Ku80 were tended to increase. CONCLUSIONS. These results demonstrate that pCSCs contain more DNA-repair proteins and less amount of RCO compared to total population. They suggest that pCSCs have better DNA-repair mechanism and less vulnerability to oxidative damage compared to differentiated cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3459. doi:1538-7445.AM2012-3459