WRN protects against cytotoxicity of topotecan but not etoposide

Markus Christmann and colleagues recently reported their findings on whether DNA helicases, like WRN, are involved in the repair of topoisomerase inhibitor-induced DNA damage.

“[24] M. Poot, K.A. Gollahon and P.S. Rabinovitch, Werner syndrome lymphoblastoid cells are sensitive to camptothecin-induced apoptosis in S-phase, Hum. Genet. 104 (1999), pp. 10–14. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (97)Although the interplay between topo I and WRN has been reported and some early publications are available on WRN and resistance against topo II poisons, there is no systematic analysis of the role of WRN in the sensitivity against topo I and topo II poisons in human cancer cells.”

“… we utilized human U2-OS osteosarcoma cells stably transfected with siRNA specific for the wrn gene. Using this isogenic cell system, we studied for the first time comparatively the role of WRN in cellular resistance to topo I and topo II inhibitors and its involvement in the repair or processing of topo I inhibitor-induced DNA damage.”

Using three different methods (WST assay, colony forming assay, and quantification of apoptosis), they showed that the WRN helicase protects against the cytotoxic effects of the topo I inhibitor topotecan (TPT) but not the topo II inhibitor etoposide (ETO).

Fig. 4. Formation and repair of SSBs and DSBs following TPT treatment. (A) To analyze the TPT-induced formation and repair of DNA strand breaks, wrn-wt and wrn-kd cells were exposed to 1 μg/ml TPT (left panel) or etoposide (right panel). At the indicated time points, the formation of DNA single-strand breaks was detected via alkaline SCGE. OTM, olive tail moment. Data of the mean ± S.D. of three independent experiments are shown. (B) Determination of DSBs by neutral SCGE in wrn-wt and wrn-kd cells at various times after exposure to 1 μg/ml TPT. Data of at least three independent experiments are shown. (C) To determine the amount of H2AX phosphorylation, wrn-wt and wrn-kd cells were exposed to 1 μg/ml TPT. At different times after exposure, cells were harvested, protein extracts were prepared and 25 μg were subjected to Western blot analysis. The membrane was incubated with γH2AX specific antibodies. For loading control, ERK2 was detected. To quantify the amount H2AX phosphorylation, the intensity of the strongest band was set to 100%. (D) To determine the formation of γH2AX foci, wrn-wt and wrn-kd cells were exposed to 1 μg/ml TPT. After the indicated time points, cells were fixed, and the γH2AX foci were visualized using γH2AX specific antibodies using fluorescence microscopy. For each time point, foci in 40 cells were counted and the results of three independent experiments ± S.D. is shown (left panel). A representative experiment is provided in the right panel.