Small Molecule Screening for Novel Double-strand Break Repair Inhibitors using High-throughput Flow Cytometry

2011 ◽  
Vol 81 (2) ◽  
pp. S54
Author(s):  
R.S. Bindra ◽  
F. Glickman ◽  
S.N. Powell
Keyword(s):  
Flow Cytometry ◽  
Small Molecule ◽  
High Throughput ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Small Molecule Screening ◽  
Break Repair

scholarly journals The Mechanism of Radiosensitization by YM155, a Novel Small Molecule Inhibitor of Survivin Expression, is Associated with DNA Damage Repair

2015 ◽  
Vol 37 (3) ◽  
pp. 1219-1230 ◽  
Author(s):  
Songliu Hu ◽  
Songbin Fu ◽  
Xiangying Xu ◽  
Lin Chen ◽  
Jianyu Xu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Small Molecule ◽  
Cellular Localization ◽  
Survivin Expression ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Dna Double Strand Break ◽  
Molecule Inhibitor ◽  
Break Repair

Background/Aims: Survivin, a member of the inhibitor of apoptosis protein family, is an attractive target for cancer therapy. We investigated the effects of YM155, a small molecule inhibitor of survivin expression, on the radiosensitivity of human non-small cell lung cancer (NSCLC) cell lines and elucidated a relationship between the cellular localization of survivin and DNA double-strand break repair. Methods: The cellular distribution of survivin was determined by Western blotting of subcellular fractions and by immunofluorescent staining in A549 NSCLC cells. Radiation-induced DNA damage was evaluated based on histone H2AX phosphorylation and foci formation. The relationship between the cellular localization of survivin and DNA double-strand break repair was analyzed by Western blotting and co-immunoprecipitations. Results: YM155 down-regulated survivin expression in NSCLC cells in a concentration- and time-dependent manner. An in vitro clonogenic survival assay revealed that YM155 increased the sensitivity of NSCLC cells to radiation. After irradiation, we observed a rapid accumulation of survivin in the nucleus. An immunofluorescent analysis of histone γ-H2AX demonstrated that the inhibition of survivin expression by YM155 resulted in impaired DNA double-strand break repair. Co-immunoprecipitation assays using nuclear extracts revealed an interaction between survivin, Ku70, γ-H2AX, and DNA-PKcs. Furthermore, S2056 autophosphorylation of DNA-PKcs was reduced in survivin-depleted cells. Conclusions: These results suggested that YM155 sensitized NSCLC cells to radiation, at least in part by inhibiting DNA repair and enhancing apoptosis via the down-regulation of survivin expression. YM155 pretreatment inhibited DNA-PKcs autophosphorylation at S2056. Nuclear survivin was involved in DNA double-strand break repair via interactions with members of the DNA double-strand break repair machinery.

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