FIGURE 8.

WIP1 inhibits repair of DNA DSBs. A, recombination substrates pLB4 and pTNeo99-7 in stable cell lines, LB4 and TNeo99-7. Each substrate contains a functional hygromycin gene (hyg), used to select for stably transfected cells, and a tk-neo fusion gene that is disrupted by a 22-bp oligonucleotide containing the recognition site for endonuclease I-SceI. Substrate pLB4 contains a 2.5-kb HindIII fragment containing a complete HSV-1 tk gene that serves as a donor for the homologous recombination with the disrupted tk-neo gene. B, WIP1, but not phosphatase-dead WIP1 inhibits DSB repair. I-SceI and wild-type/mutant WIP1 or control expression vector DNA were transfected into cells, and the frequency of recovery of G418^R clones was measured. C, WIP1 has dose-dependent inhibitory effects on DSB repair. As indicated, varying amounts of WIP1 vector DNA or control vector DNA were introduced into cells, and the frequency of recovery of G418^R clones was measured. D, suppression of WIP1 activity by WIP1 shRNA and the WIP1 inhibitors arsenic trioxide (ATO) and CCT007093 enhances DSB repair. LB4 and TNeo99-7 cells were transduced with a lentiviral WIP1 shRNA vector or were incubated in 10 μm ATO or 25 μm CCT007093 prior to I-SceI introduction. Formation of G418^R clones was then measured. E, the effects of WIP1 on DSB repair are not solely dependent on ATM. LB4 and TNeo99-7 cells were transfected with control or WIP1 expression vector DNA, treated with neocarzinostatin (200 ng/ml) and with or without KU55933 (10 μm). Proteins were detected by their specific antibodies in immunoblotting (left panel). The effects of WIP1 on DSB repair were also examined in LB4 and TNeo99-7 cells in the presence or absence of KU55933 (right panel).