WRN protects against topo I but not topo II inhibitors by preventing DNA break formation

Abstract

The Werner syndrome helicase/3'-exonuclease (WRN) is a major component of the DNA repair and replication machinery. To analyze whether WRN is involved in the repair of topoisomerase-induced DNA damage we utilized U2-OS cells, in which WRN is stably down-regulated (wrn-kd), and the corresponding wild-type cells (wrn-wt). We show that cells not expressing WRN are hypersensitive to the toxic effect of the topoisomerase I inhibitor topotecan, but not to the topoisomerase II inhibitor etoposide. This was shown by mass survival assays, colony formation and induction of apoptosis. Upon topotecan treatment WRN deficient cells showed enhanced DNA replication inhibition and S-phase arrest, whereas after treatment with etoposide they showed the same cell cycle response as the wild-type. A considerable difference between WRN and wild-type cells was observed for DNA single- and double-strand break formation in response to topotecan. Topotecan induced DNA single-strand breaks 6h after treatment. In both wrn-wt and wrn-kd cells these breaks were repaired at similar kinetics. However, in wrn-kd but not wrn-wt cells they were converted into DNA double-strand breaks (DSBs) at high frequency, as shown by neutral comet assay and phosphorylation of H2AX. Our data provide evidence that WRN is involved in the repair of topoisomerase I, but not topoisomerase II-induced DNA damage, most likely via preventing the conversion of DNA single-strand breaks into DSBs during the resolution of stalled replication forks at topo I-DNA complexes. We suggest that the WRN status of tumor cells impacts anticancer therapy with topoisomerase I, but not topoisomerase II inhibitors.

Fig. 1

Characterization of wrn-kd cells and sensitivity against topo inhibitors. (A) To analyze the WRN status, wrn-wt and wrn-kd cells were exposed to 1 μg/ml TPT. At different times after exposure, cells were harvested and total RNA was isolated. 1 μg was subjected to cDNA synthesis, followed by RT-PCR with specific primers (c, non-exposed control). As internal control, gapdh was amplified. (B and C) To elucidate the role of WRN helicase in the sensitivity against TPT and ETO, wrn-wt and wrn-kd cells were exposed to different doses of TPT. Cellular viability was determined 72 h later by the metabolic WST-1 assay (B) and reproductive cell death was measured 7 days later by colony forming assay (C).

Fig. 2

Induction of apoptosis by TPT and ETO. (A) To analyze the induction of apoptosis wrn-wt and wrn-kd cells were exposed to different concentrations of TPT. 72 h later, cells were harvested and analyzed by FACS. Apoptosis was determined as sub-G1 fraction. (B) To analyze the time dependency of apoptosis, wrn-wt and wrn-kd cells were exposed to 1 μg/ml TPT or ETO. After the indicated time points, cells were harvested and analyzed by FACS. Apoptosis was determined as sub-G1 fraction.

Fig. 3

Cell cycle blockage induced by TPT and ETO. To analyze cell cycle progression, wrn-wt and wrn-kd cells were exposed to 1 μg/ml TPT or ETO. Upon the indicated times, cells were harvested and the distribution in G1, S and G2-phase was analyzed by FACS. (A) Percentage of cells in G2 after the addition of TPT or ETO to the culture medium. (B) Percentage of cells in S-phase after treatment with TPT or ETO. The data are the mean of at least three independent experiments. (C) DNA replication in wrn-wt and wrn-kd cells. Exponentially growing cells were treated with 1 μg/ml TPT (left panel) or ETO (right panel) and replication was analyzed by BrdU incorporation at different times after addition of the drugs to the medium. The data are the mean of at least three independent experiments.

Fig. 4

Formation and repair of SSBs and DSBs upon TPT. (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). After 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.

Fig. 5

Degradation of topoisomerase I upon TPT treatment. Wrn-wt and wrn-kd cells were exposed to 1 μg/ml TPT for the indicated times. (A) After exposure, the drug treated cells were either directly lysed to detect the formation of the cleavable complex by band depletion or (B) carefully rinsed with culture medium and incubated for another 30 min to reverse the cleavable complex. (C) Cells were exposed to 1 μg/ml TPT for the indicated times in the absence (left blot) or presence of 5 μM MG132. After exposure, the drug-treated cells were carefully rinsed with culture medium and incubated for another 30 min to reverse the cleavable complex. The amount of topo I was detected by Western blot analysis using anti-topo I pAb followed by ECL detection. For loading control, the filter was re-probed with ERK2.