Mechanism of Action of G-Quadruplex–Forming Oligonucleotide Homologous to the Telomere Overhang in Melanoma

T-oligo, a guanine-rich oligonucleotide (GRO) homologous to the 3'-telomeric overhang of telomeres, elicits potent DNA-damage responses in melanoma cells; however, its mechanism of action is largely unknown. GROs can form G-quadruplexes (G4) which are stabilized by the hydrogen-bonding of guanine residues. In this study, we confirmed the G4-forming capabilities of T-oligo using non-denaturing PAGE, NMR and immunofluorescence. Using an anti-G-quadruplex antibody (BG4) we showed that T-oligo can form G4 in the nuclei of melanoma cells. Further, using DNase I in a nuclease degradation assay, G4-T-oligo was found more stable than single-stranded (SS)-T-oligo. G4-T-oligo had decreased anti-proliferative effects compared to SS-T-oligo. However, G4-T-oligo has similar cellular uptake as SS-T-oligo as demonstrated by FACS analysis. Inhibition of JNK, which causes DNA damage-induced apoptosis, partially reversed the anti-proliferative activity of T-oligo. T-oligo also inhibited mRNA expression of hTERT, a catalytic subunit of telomerase which was reversed by JNK inhibition. Furthermore, two shelterin complex proteins TRF2/POT1 were found to be upregulated and bound by T-oligo suggesting that T-oligo may mediate dissociation of these proteins from the telomere overhang. These studies demonstrate that T-oligo can form a G-quadruplex and the anti-tumor effects of T-oligo may be mediated through POT1/TRF2 as well as via hTERT-inhibition through JNK-activation.