selective serotonin reuptake inhibitor In this study we test

In this study, we tested the hypothesis that high RAD51 selective serotonin reuptake inhibitor and RAD51 foci activation is specifically associated with GSCs and that small-molecule inhibitors are effective GSC radiosensitizers.
Results
Discussion Identifying targets in the DDR pathway as a means to sensitize cancers to DNA-damaging cytotoxic treatments including radiotherapy has become of increasing interest with the availability of new inhibitors and the demonstration of their effectiveness in specific contexts. A notable example is the use of PARP inhibitors in BRCA-deficient tumors, based on the synthetic lethality paradigm (Audeh et al., 2010; Helleday, 2011; Tutt et al., 2010). In glioma, attempts to improve the outcome by adding agents to increase radiation sensitivity have been the subject of much research, but none of these approaches have been successful. The stem cell model has added a new perspective to these investigations, since GSCs are believed to be the relevant target population. However, specific repair targets have not been linked with this cell population. In this study, we demonstrated that RAD51 and the HR pathway represent a specific DNA repair target in GSC. We first demonstrated that GSCs express high levels of RAD51 and that this is associated with robust foci formation after irradiation. This is surprising since in most circumstances HR is responsible for a small fraction of DSB repair. However, the complex regulation of the balance between repair pathways is still not fully understood and hyper-recombination with significant utilization of HR documented in tumor cell lines may be associated with loss of normal TP53 function, which can increase HR by promoting BRCA1 binding at DSBs (Dong et al., 2015). We further show that RAD51 expression is associated with markers of stemness both in vitro and in tumor material, and that a forced differentiation paradigm using BMP4/FBS reduces this expression within 72 hr. It has recently become clear that this manipulation is not equivalent to terminal differentiation and that associated changes in DNA methylation, chromatin structure, and transcription occur with variable and often delayed kinetics. Interestingly a failure to repress SOX2-driven transcription programs is suggested to be an important influence on the ability of these cells to re-enter the cell cycle (Caren et al., 2015). The transcriptional programs that underlie these changes, including those that may explain the close correlation between SOX2 and RAD51 expression are the subject of ongoing research. Interestingly, recent data suggest that SOX2 and RAD51 may be regulated by overlapping transcription factors, including FOXM1 (Lee et al., 2015; Zhang et al., 2012). The data presented here also demonstrate that inhibiting RAD51 is an effective means of sensitizing GSCs, as would be predicted from evidence that RAD51 is very active in contributing to DNA repair in these cells. This is consistent with previous data specifically examining the role of HR in GSCs (Lim et al., 2012, 2014) but may also explain the important contribution of cell-cycle checkpoint upregulation to resistance in these cells since homology-directed repair has a long half-time (Jeggo et al., 2011; Qin et al., 2014). When we examined our GSCs following treatment with a RAD51 inhibitor and radiation, we found that, contrary to our expectations, a RAD51-expressing population reappeared after treatment and that exposure to XR seemed to enhance this. Fascinatingly, however, combination treatment was effective in removing the SOX2-expressing population and rendered surviving cells non-clonogenic. The exact mechanisms that explain sensitivity to XR combined with RAD51 inhibition in these cells and the association between RAD51 expression and surviving, non-clonogenic cells after XR requires further investigation. Nevertheless, our study provides a strong rationale for targeting RAD51-mediated repair to specifically radiosensitize GSCs.