for 48 h were analyzed for c-H2AX-containing foci that co-localized with TRF1 (Figure 9A and B). There was a background level of c- H2AX foci in untreated MRC5-V1/YFP-Topo III cells of 1466 foci/ nucleus; only 8% (62%) of these foci co-localized with TRF1. In the presence of telomestatin, we observed an increased number of c- H2AX foci to 6864 foci/nucleus and almost half co-localized with TRF1 (4664%), suggesting that telomestatin triggers a DNA damage response that mostly takes place at telomeres. However, the analysis of treated cells foci that contain both YFP-Topo III and c-H2AX indicated that most of the DNA damage response was not associated with Topo III foci (Figure 9C). We then evaluated the amount of G- overhang telomeric DNA signal in MRC5-V1/YFP-Topo III cells after treatment with telomestatin using solution hybridization experiments (Figure 10). A significant reduction of the hybridization was observed in telomestatin-treated cells (40% of the signal observed in untreated cells). This is in agreement with other reports where telomestatin alters telomeric G-overhang in telomerase-positive cell lines[41,44,46]. Together, these results indicate that telomestatin induces a massive DNA damage response at telomeres. Discussion Telomestatin binds specifically to G-quadruplex structures. In cells with telomerase activity, this binding results in telomere uncapping and leads to the G-overhang degradation and the release of telomeric proteins (POT1 and/or TRF2). Telomere uncapping is associated with a DNA damage response on a subset of telomere ends (for a recent review, see [15]). Little was known about the effect of telomestatin or other G-quadruplex ligands in telomerase-negative ALT cell lines, except that cell proliferation is inhibited [8,16,17,52]. The results discussed here indicate that telomestatin induces G-overhang degradation in MRC5-V1/YFP- Topo III cells, an effect also observed in another ALT cell line, WI38-VA13 cells (result not shown). Our data suggest that alteration of the conformation or length of the telomeric G- Telomestatin Uncaps Telomeres overhang is a relevant marker for telomestatin activity in both telomerase positive and ALT cells. Interestingly, telomestatin provoked a massive DNA damage response at telomeres in ALT cells, a result that contrasts with previous finding for this compound in telomerase-positive cell lines where only a small fraction of DNA damage foci corresponded to TIFs [49,53]. Either telomestatin-induced DNA damage mostly takes place in extra-telomeric regions where potential G- quadruplex forming sequences have been identified [4] or the G-quadruplexes stabilized by telomestatin may be tightly regulated in telomeric regions during telomere replication. Specific G- quadruplex resolvases may repair these G-quadruplex structures to avoid replication blocks and DNA damage accumulation. For example, the FANCJ helicase suppresses the DNA damage response induced by telomestatin in telomerase-positive cells [53]. The massive DNA damage response at telomeres in ALT cells after telomestatin treatment is accompanied by depletion of the Topo III/BLM/TRF2 complex and by telomere uncapping. These are the same effects observed after Topo III depletion by siRNA in ALT cells [39]. Telomestatin treatment also disrupted APBs, as evidenced by the segregation of Topo III, PML, and other shelterin components into separate foci (Figure 11). A close linkage between the formation of APBs, the presence of proteins from the shelterin complex and telomere maintenance by recombination has been suggested, although their structural organization is poorly understood [54,55]. These specialized bodies contain recombination proteins that are thought to support the processes that maintain telomere length in the absence of telomerase activity. Recent evidence indicates that APBs provide a platform (called telomere clusters) where post-replicative telomere recombination intermediates are resolved [56]. These structures may maintain a spatio-temporal organization of telomeres needed for the completion of these reactions. Our data are consistent with a redistribution of unprotected telomere ends due to the absence of the telomere bouquet after telomestatin treatment. Figure 10. Effect of telomestatin on G-overhang signal. A, DNA was extracted from untreated MRC5-V1/YFP-Topo III cells or cells treated for 48 h with telomestatin at indicated concentrations. The G-overhang signal was evaluated by non-denaturing solution hybridization with a telomeric probe (AATCCC)4. The gel was also stained with ethidium bromide. B, Quantification of the experiment presented in A. The G-overhang hybridization signal was normalized relative to the ethidium bromide signal. Results are expressed as the percentage of G-overhang signal in control untreated cells, which was defined as 100%. doi:10.1371/journal.pone.0006919.g010 PLoS ONE | www.plosone.org 11 September 2009 | Volume 4 | Issue 9 | e6919