Resistance to the Short Term Antiproliferative Activity of the G-quadruplex Ligand 12459 Is Associated with Telomerase Overexpression and Telomere Capping Alteration

Jean-François Riou,Frédérique Mégnin-Chanet,Dennis Gomez,Nassera Aouali,Chantal Trentesaux,Arturo Londoño-Vallejo,Hamid Morjani,Kazuo Shin-Ya,Patrick Mailliet,Thibault Lemarteleur,Jean-Louis Mergny,Laurent Lacroix,Céline Douarre

doi:10.1074/jbc.m308440200

Abstract

Ligands that stabilize the telomeric G-rich single-stranded 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.

Highlights

Ligands that stabilize the telomeric G-rich singlestranded DNA overhang into G-quadruplex can be considered as potential antitumor agents that block telomere replication
The level of hTERT transcripts was investigated in the resistant clones by RT-PCR analysis. hTERT presents a complex splicing pattern that includes an active ϩ␣,ϩ␤ transcript and several inactive species including one major Ϫ␤ transcript and two minor Ϫ␣ and Ϫ␣,Ϫ␤ transcripts that were barely detectable by RT-PCR (Fig. 3, a and b) [23, 27]
Three independent RT-PCR quantitative experiments indicated that hTERT expression was increased by 2-fold or 6-fold in JFD10 and JFD18, respectively, whereas it remained unchanged in JFD9 and JFD11 (Fig. 3c)

Summary

Introduction

Ligands that stabilize the telomeric G-rich singlestranded DNA overhang into G-quadruplex can be considered as potential antitumor agents that block telomere replication. 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 characterized in two resistant clones (JFD10 and JFD18) as an increase in telomerase activity, leading to an increase in telomere length.

Results

Conclusion