Preferential binding of a G-quadruplex ligand to human chromosome ends

Abstract

The G-overhangs of telomeres are thought to adopt particular conformations, such as T-loops or G-quadruplexes. It has been suggested that G-quadruplex structures could be stabilized by specific ligands in a new approach to cancer treatment consisting in inhibition of telomerase, an enzyme involved in telomere maintenance and cell immortality. Although the formation of G-quadruplexes was demonstrated in vitro many years ago, it has not been definitively demonstrated in living human cells. We therefore investigated the chromosomal binding of a tritiated G-quadruplex ligand, 3H-360A (2,6-N,N′-methyl-quinolinio-3-yl)-pyridine dicarboxamide [methyl-3H]. We verified the in vitro selectivity of 3H-360A for G-quadruplex structures by equilibrium dialysis. We then showed by binding experiments with human genomic DNA that 3H-360A has a very potent selectivity toward G-quadruplex structures of the telomeric 3′-overhang. Finally, we performed autoradiography of metaphase spreads from cells cultured with 3H-360A. We found that 3H-360A was preferentially bound to chromosome terminal regions of both human normal (peripheral blood lymphocytes) and tumor cells (T98G and CEM1301). In conclusion, our results provide evidence that a specific G-quadruplex ligand interacts with the terminal ends of human chromosomes. They support the hypothesis that G-quadruplex ligands induce and/or stabilize G-quadruplex structures at telomeres of human cells.

G-Quadruplexes Induce Apoptosis in Tumor Cells

“…For example, GQ-ODNs may cause telomere deprotection by titrating a telomere binding protein and thus activate ATM-dependent apoptosis (19,27). As telomeric G-tails can readily adopt G-quadruplex conformation in vitro (2, 3) and G-quadruplexes have also been detected at telomeres in vivo (15)(16)(17)(18), it is tempting to speculate that G-quadruplex-induced cell death may occur during telomere deprotection and GQ-ODNs may mimic such a signal and hijack this death pathway. Our studies have shown that GQ-ODN-induced apoptosis is p53 independent.…”

“…Telomeric G-tails can readily adopt the thermodynamically stable G-quadruplex conformation in vitro (2, 3). G-quadruplexes have also been detected at telomeres in vivo (15)(16)(17)(18). Deprotection of telomeres has been shown to induce ataxia telangiectasia mutated (ATM)-dependent apoptosis (19).…”

Elevated telomere-telomere recombination in WRN-deficient, telomere dysfunctional cells promotes escape from senescence and engagement of the ALT pathway

“…This replication block may involve in vivo formation of G-quadruplexes, intramolecular structures containing G-quartets of four hydrogen-bonded guanines, on G-rich telomeric sequences (Schaffitzel et al 2001;C.C. Chang et al 2004;Granotier et al 2005). WRN preferentially localizes to telomeres during the S-phase (Opresko et al 2004;Crabbe et al 2004) and can efficiently unwind Gquadruplexes in a helicase-dependent manner (Mohaghegh et al 2001).…”

Inhibition of translation in living eukaryotic cells by an RNA G-quadruplex motif

“…Strong evidence for the existence of G-rich quadruplex structures in vivo was observed by staining of telomeres with quadruplex-specific antibodies (Schaffitzel et al 2001). Furthermore, selective binding of proteins and small molecules to G-rich mRNAs and telomeric DNA (Darnell et al 2001;Ramos et al 2003;Granotier et al 2005;Paeschke et al 2005) was reported and the formation of DNA GQ structures during transcription was observed (Duquette et al 2004). Moreover, small molecules that bind to and thereby stabilize DNA GQs have been shown to suppress gene expression of certain proto-oncogenes, hence providing a potential new therapeutic approach (Rangan et al 2001;Siddiqui-Jain et al 2002).…”