Investigating the Effect of Mono- and Dimeric 360A G-Quadruplex Ligands on Telomere Stability by Single Telomere Length Analysis (STELA)

Abstract

Telomeres are nucleoprotein structures that cap and protect the natural ends of chromosomes. Telomeric DNA G-rich strands can form G-quadruplex (or G4) structures. Ligands that bind to and stabilize G4 structures can lead to telomere dysfunctions by displacing shelterin proteins and/or by interfering with the replication of telomeres. We previously reported that two pyridine dicarboxamide G4 ligands, 360A and its dimeric analogue (360A)_2A, were able to displace in vitro hRPA (a single-stranded DNA-binding protein of the replication machinery) from telomeric DNA by stabilizing the G4 structures. In this paper, we perform for the first time single telomere length analysis (STELA) to investigate the effect of G4 ligands on telomere length and stability. We used the unique ability of STELA to reveal the full spectrum of telomere lengths at a chromosome terminus in cancer cells treated with 360A and (360A)_2A. Upon treatment with these ligands, we readily detected an increase of ultrashort telomeres, whose lengths are significantly shorter than the mean telomere length, and that could not have been detected by other methods.

Keywords: G-quadruplex ligands; G-quadruplex structures; STELA; telomere.

Figure 1

Chemical structure of 360A and (360A)_2A and the effect of these ligands on the proliferation of A549 cells: (a) Chemical structure of G4 ligands 360A and (360A)_2A; (b) Cell growth curves plotting mean PDs and days in culture of non-treated A549 (NT), A549 treated with 0.1% DMSO (DMSO), with 5 μM of 360A (M), or with 5 μM of (360A)_2A (D) (n = 3 independent experiments; error bars, SD).

Figure 2

Treatment with 360A and (360A)_2A had no effect on mean telomere length: (a) Schematic representation of the principle of STELA [5]; (b) STELA at 17p telomere in non-treated A549 (NT), A549 treated with 0.1% DMSO (DMSO), 5 μM 360A (M), or 5 μM (360A)_2A (D) after 4, 8, and 11 days of treatment (D4, D8, D11) (representative STELA from experiment (1)). Each lane represents a PCR and each band a telomere. The mean and SD of the telomere length are shown below. Arrows show examples of telomere deletion events (TDEs) (i.e., telomeres shorter than 2.2 kb (red dashed line)).

Figure 3

360A and (360A)_2A induced telomere deletion events in A549 cells: (a) Representative experiment (from experiment (2)) of the scaled up STELA at 17p telomere in the last PD points of A549 treated with 5 μM of 360A (M) or 5 μM of (360A)_2A (D). Each lane represents a PCR and each band a telomere; (b) Histograms showing the proportion of TDEs in 2 independent experiments in the last PD points (i.e., at day 11 for both 360A and (360A)_2A in experiment 1; and at day 11 and 8 for 360A and (360A)_2A, respectively, in experiment 2) of A549 treated with 5 μM of 360A (M) or 5 μM of (360A)_2A (D). The Chi-square test was used to determine the p values.