Most malignancy therapies involve a component of treatment which inflicts DNA

Most malignancy therapies involve a component of treatment which inflicts DNA damage in tumor cells such as double-strand breaks (DSBs) which are considered the most serious threat to genomic integrity. 20 0 Rabbit Polyclonal to ALK. compounds for molecules which selectively modulate NHEJ and HR repair in tumor cells. We identified a collection of novel hits which potently inhibit DSB repair and we have validated their functional activity in comprehensive panel of orthogonal secondary assays. A selection of these inhibitors were found to radiosensitize cancer cell lines assays with purified proteins. However there are numerous steps in key DSB repair pathways which have not yet been targeted. These findings suggest the need for additional efforts and also option drug screening strategies to identify new drugs which can inhibit DSB repair. Here we report on the results of a high-throughput cell-based screen for novel inhibitors of NHEJ and HR repair using a forward chemical genetics approach. The HR pathway utilizes homologous DNA sequences as a template for repair while NHEJ processes and re-ligates the ends of the breaks (10). NHEJ repair is considered more error prone than HR and occurs more frequently in cells. NHEJ is the predominant pathway in the G0/G1-phases of the cell cycle while HR increases during S-phase when a sister chromatid becomes available as a template for repair. As cells enter the G2/M-phase of the cell cycle NHEJ becomes more active and likely predominates over HR Benzoylpaeoniflorin repair (11). Emerging evidence indicates that many sub-pathways exist within both the NHEJ and HR pathways of repair. In particular NHEJ repair mainly is comprised of canonical NHEJ (cNHEJ) and non-canonical NHEJ repair. The latter process has been given many names including back-up NHEJ (bNHEJ) alternative NHEJ (aNHEJ) and microhomology-mediated NHEJ (MMEJ; (12)). This lack of consensus in part can be attributed to the fact that specific DSB repair proteins that mediate non-canonical NHEJ repair remain elusive. The cNHEJ pathway is usually well-defined and results in minimal processing of the DSB ends (13) while the latter process typically results in deletions with local sequence microhomology (14-17). cNHEJ proteins include Ku70/80 DNA-PK catalytic subunit (DNA-PKcs) X-ray repair cross-complementing protein 4 (XRCC4) and Ligase IV (13). As noted above the non-canonical pathway(s) are poorly defined but appear to require MRE11 (18) and PARP-1 (19). Ligase III and X-ray repair cross-complementing protein 1 (XRCC1) are also implicated in these processes (20 21 although more recent studies have questioned the requirement of these proteins (22-24). Examples of key HR proteins include breast malignancy 1 (BRCA1) BRCA2 and Rad51 (10). CtIP is usually a key HR factor Benzoylpaeoniflorin involved in the initial end-resection step of this process (25) but it also appears to play a role in NHEJ repair particularly in pathways distinct from cNHEJ (26). Collectively the non-canonical NHEJ repair processes share a common theme of higher rates of insertions deletions and microhomology usage. As Benzoylpaeoniflorin such we have termed this pathway mutagenic NHEJ (mNHEJ) repair previously in order to distinguish cNHEJ repair versus bNHEJ aNHEJ MMEJ which often are used interchangeably but sometimes distinctly (27). However MMEJ repair specifically may represent a subset of mNHEJ in which flanking sequence microhomology is commonly (if not exclusively) utilized. Another DSB repair pathway has been described single strand annealing (SSA) which is usually distinct from NHEJ repair and likely represents a sub-pathway of HR repair. SSA repair anneals adjacent sequence repeats flanking a DSB resulting in a deletion Benzoylpaeoniflorin between the Benzoylpaeoniflorin repeats (28). Numerous assays to measure DSB repair in cells have been described previously and they typically use IR or endonucleases to induce DNA cleavage events at chromosomal loci or in plasmid substrates. DSB repair proteins form discrete foci at DNA damage sites after treatment with IR which can be visualized by immunofluorescence microscopy. These foci patterns can be Benzoylpaeoniflorin used as markers for DSB repair in cultured cells (29 30 DSB repair can also be assayed using the neutral comet assay which relies on the altered mobility of cleaved DNA (31). Engleward and.