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The protection and maintenance of the genome in human cells is critical. The cell uses signaling pathways that can down regulate the cell cycle when DNA-damage occurs, giving an opportunity for efficient repair before division.1 However, this raises the question of how cells manage to distinguish the telomeres from DNA double-strand breaks. A six-protein complex named "telosome" or "shelterin" (Figure 1) binds to the double-stranded telomeric DNA and single-stranded telomeric G-overhang at the chromosome ends.2,3 The complex shelters telomeres from the DNA-damage response machinery and protects chromosomes from shortening, nonhomologous end-joining, and homology-directed repair. Moreover, the shelterin component POT1 (protection of telomeres 1)4 has been shown to modulate the activity of telomerase,5 the enzyme capable of conferring infinite proliferative capacity on cells by extension of the G-overhang.6 It has been shown that the telomeric G-overhang, when folded into G-quadruplexes, is resistant to extension by telomerase,7 and that synthetic small molecules that stabilize these structures can decrease the enzyme efficiency.8-10 Gomez et al. showed that the potent G-quadruplex binding natural product telomestatin induces apoptosis of cancer cells via a mechanism proposed to involve the uncapping of POT1 from telomeres.11 Herein, we describe a novel synthetic small molecule (compound 1, Figure 1), which exhibits unprecedented G-quadruplex stabilization leading to an alteration of shelterin at the telomeres of human cancer cells.Compound 1 was designed following intensive research on the biology of G-quadruplex nucleic acids.12 The design rationale comprises certain structural features shared by known quadruplex binding small molecules, with particular emphasis on an electron rich aromatic surface, the potential for a flat conformation, and an ability to participate in hydrogen bonding.13 The small molecule is readily accessible in six synthetic steps that are easily scalable and amenable to molecular diversity (see Supporting Information).We first evaluated the potential for 1 to stabilize the telomeric G-quadruplex by FRETmelting experiments.14 Compound 1 stabilized the human telomeric G-quadruplex with a maximum ΔT m of 35 K in 60 mM K + and 44 K in 100 mM Na + at 0.18 and 0.34 μM compound, respectively. In contrast, the ligand-induced double-stranded DNA stabilization was negligible with a ΔT m of 0.5 K in 60 mM K + at 1 μM compound. It is noteworthy that the G-quadruplex melting profile was almost unaffected by the presence of 25 mol equiv of © 2008 American Chemical Society riou@mnhn.fr; sb10031@cam.ac.uk. Supporting Information Available: Experimental details for the synthesis of 1, FRET-melting, direct telomerase extension assay, in vitro POT1 uncapping assay, POT1 and γH2AX in cellulo experiments, growth inhibition assay. This material is available free of charge via the Internet at http://pubs.acs.org. The data recorded for 1 represent the highest induced shifts in melting temperature for the telomeri...

Here, we show that autophagy is activated in the intestinal epithelium in murine and human colorectal cancer and that the conditional inactivation of Atg7 in intestinal epithelial cells inhibits the formation of pre-cancerous lesions in Apc(+/-) mice by enhancing anti-tumour responses. The antibody-mediated depletion of CD8(+) T cells showed that these cells are essential for the anti-tumoral responses mediated by the inhibition of autophagy. We show that Atg7 deficiency leads to intestinal dysbiosis and that the microbiota is required for anticancer responses. In addition, Atg7 deficiency resulted in a stress response accompanied by metabolic defects, AMPK activation and p53-mediated cell-cycle arrest in tumour cells but not in normal tissue. This study reveals that the inhibition of autophagy within the epithelium may prevent the development and progression of colorectal cancer in genetically predisposed patients.

Telomestatin is a potent G-quadruplex ligand that interacts with the 3 telomeric overhang, leading to its degradation, and induces a delayed senescence and apoptosis of cancer cells. POT1 and TRF2 were recently identified as specific telomere-binding proteins involved in telomere capping and t-loop maintenance and whose interaction with telomeres is modulated by telomestatin. We show here that the treatment of HT1080 human tumor cells by telomestatin induces a rapid decrease of the telomeric G-overhang and of the doublestranded telomeric repeats. Telomestatin treatment also provokes a strong decrease of POT1 and TRF2 from their telomere sites, suggesting that the ligand triggers the uncapping of the telomere ends. The effect of the ligand is associated with an increase of the ␥-H2AX foci, one part of them colocalizing at telomeres, thus indicating the occurrence of a DNA damage response at the telomere, but also the presence of additional DNA targets for telomestatin. Interestingly, the expression of GFP-POT1 in HT1080 cells increases both telomere and G-overhang length. As compared with HT1080 cells, HT1080GFP-POT1 cells presented a resistance to telomestatin treatment characterized by a protection to the telomestatin-induced growth inhibition and the G-overhang shortening. This protection is related to the initial G-overhang length rather than to its degradation rate and is overcome by increased telomestatin concentration. Altogether these results suggest that telomestatin induced a telomere dysfunction in which G-overhang length and POT1 level are important factors but also suggest the presence of additional DNA sites of action for the ligand.

Ligands that stabilize the telomeric G-rich singlestranded DNA overhang into G-quadruplex can be considered as potential antitumor agents that block telomere replication. Ligand 12459, a potent G-quadruplex ligand that belongs to the triazine series, has been previously shown to induce both telomere shortening and apoptosis in the human A549 cell line as a function of its concentration and time exposure. We show here that A549 clones obtained after mutagenesis and selected for resistance to the short term effect of ligand 12459 frequently displayed hTERT transcript overexpression (2-6-fold). Overexpression of hTERT was also characterized in two resistant clones (JFD10 and JFD18) as an increase in telomerase activity, leading to an increase in telomere length. An increased frequency of anaphase bridges was also detected in JFD10 and JFD18, suggesting an alteration of telomere capping functions. Transfection of either hTERT or DN-hTERT cDNAs into A549 cells did not confer resistance or hypersensitivity to the short term effect of ligand 12459, indicating that telomerase expression is not the main determinant of the antiproliferative effect of ligand 12459. In contrast, transfection of DN-hTERT cDNA into resistant JFD18 cells restored sensitivity to apoptotic concentrations of ligand 12459, suggesting that telomerase does participate in the resistance to this G-quadruplex ligand. This work provides evidence that telomerase activity is not the main target for the 12459 G-quadruplex ligand but that hTERT functions contribute to the resistance phenotype to this class of agents.Telomeres are essential to maintain the stability of chromosome ends and are synthesized by a specialized enzyme called telomerase. Telomerase is overexpressed in a large number of tumors and is involved in cell immortalization and tumorigenesis, whereas it is not described as being expressed in most somatic cells (1). A recent work showed that telomerase was efficiently expressed in S phase from normal cycling cells and played an important function to delay the onset of replicative senescence by maintaining the 3Ј telomeric overhang integrity independently from overall telomere length regulation (2). Differential expression of telomerase between normal and cancer cells was the initial rationale for the evaluation of telomerase inhibitors as potential anticancer agents (3), and a highly specific catalytic telomerase inhibitor, BIBR1532, was described as impairing cancer cell proliferation without acute toxicity in a mouse xenograft model (4). Since telomerase is expressed in normal proliferating human cells, the useful therapeutic index of these inhibitors has to be carefully determined in future studies.Folding of the telomeric G-rich single-stranded overhang into a quadruplex DNA has been found to inhibit telomerase activity. Stabilization of G-quadruplexes can then be considered an original strategy to achieve antitumor activity (5-8).The intracellular existence of G-quadruplexes was recently demonstrated in the telomeres from ciliates (9). G...

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Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG) with a 3Ј single-stranded extension (the G-overhang). The stabilization of G-quadruplexes in the human telomeric sequence by small-molecule ligands inhibits the activity of telomerase and results in telomere uncapping, leading to senescence or apoptosis of tumor cells. Therefore, the search for new and selective G-quadruplex ligands is of considerable interest because a selective ligand might provide a telomere-targeted therapeutic approach to treatment of cancer. We have screened a bank of derivatives from natural and synthetic origin using a temperature fluorescence assay and have identified two related compounds that induce G-quadruplex stabilization: malouetine and steroid FG. These steroid derivatives have nonplanar and nonaromatic structures, different from currently known G-quadruplex ligands. Malouetine is a natural product isolated from the leaves of Malouetia bequaaertiana E. Woodson and is known for its curarizing and DNAbinding properties. Steroid FG, a funtumine derivative substituted with a guanylhydrazone moiety, interacted selectively with the telomeric G-quadruplex in vitro. This derivative induced senescence and telomere shortening of HT1080 tumor cells at submicromolar concentrations, corresponding to the phenotypic inactivation of telomerase activity. In addition, steroid FG induced a rapid degradation of the telomeric G-overhang and the formation of anaphase bridges, characteristics of telomere uncapping. Finally, the expression of protection of telomere 1 (POT1) induced resistance to the growth effect of steroid FG. These results indicate that these steroid ligands represent a new class of telomere-targeted agents with potential as antitumor drugs.

The intestinal epithelium acts as a barrier between the organism and its microenvironment, including the gut microbiota. It is the most rapidly regenerating tissue in the human body thanks to a pool of intestinal stem cells (ISCs) expressing Lgr5. The intestinal epithelium has to cope with continuous stress linked to its digestive and barrier functions. Epithelial repair is crucial to maintain its integrity, and Lgr5-positive intestinal stem cell (Lgr5+ISC) resilience following cytotoxic stresses is central to this repair stage. We show here that autophagy, a pathway allowing the lysosomal degradation of intracellular components, plays a crucial role in the maintenance and genetic integrity of Lgr5+ISC under physiological and stress conditions. Using conditional mice models lacking the autophagy gene Atg7 specifically in all intestinal epithelial cells or in Lgr5+ISC, we show that loss of Atg7 induces the p53-mediated apoptosis of Lgr5+ISC. Mechanistically, this is due to increasing oxidative stress, alterations to interactions with the microbiota, and defective DNA repair. Following irradiation, we show that Lgr5+ISC repair DNA damage more efficiently than their progenitors and that this protection is Atg7 dependent. Accordingly, we found that the stimulation of autophagy on fasting protects Lgr5+ISC against DNA damage and cell death mediated by oxaliplatin and doxorubicin treatments. Finally, p53 deletion prevents the death of Atg7-deficient Lgr5+ISC but promotes genetic instability and tumor formation. Altogether, our findings provide insights into the mechanisms underlying maintenance and integrity of ISC and highlight the key functions of Atg7 and p53.