Please cite this article in press as: F. Berardinelli, et al., The G-quadruplex-stabilising agent RHPS4 induces telomeric dysfunction and enhances radiosensitivity in glioblastoma cells, DNA Repair (2014), http://dx.doi.org/10.1016/j.dnarep.2014.10.009 ARTICLE IN PRESS G Model DNAREP-2017; No. of Pages 12 DNA Repair xxx (2014) xxx–xxx Contents lists available at ScienceDirect DNA Repair j ourna l ho me pa ge: www.elsevier.com/locate/dnarepair The G-quadruplex-stabilising agent RHPS4 induces telomeric dysfunction and enhances radiosensitivity in glioblastoma cells F. Berardinelli a,d,∗,1 , S. Siteni a,b,1 , C. Tanzarella a , M.F. Stevens c , A. Sgura a,d , A. Antoccia a,d a Department of Science, Università “Roma Tre”, Rome, Italy b Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy c Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom d INFN Roma Tre, Rome, Italy a r t i c l e i n f o Article history: Received 10 June 2014 Received in revised form 21 October 2014 Accepted 24 October 2014 Available online xxx Keywords: G-quadruplex ligands RHPS4 Telomeres Astrocytoma cells Glioblastoma cells Ionising radiations a b s t r a c t G-quadruplex (G4) interacting agents are a class of ligands that can bind to and stabilise secondary structures located in genomic G-rich regions such as telomeres. Stabilisation of G4 leads to telomere architecture disruption with a consequent detrimental effect on cell proliferation, which makes these agents good candidates for chemotherapeutic purposes. RHPS4 is one of the most effective and well- studied G4 ligands with a very high specificity for telomeric G4. In this work, we tested the in vitro efficacy of RHPS4 in astrocytoma cell lines, and we evaluated whether RHPS4 can act as a radiosensitising agent by destabilising telomeres. In the first part of the study, the response to RHPS4 was investigated in four human astrocytoma cell lines (U251MG, U87MG, T67 and T70) and in two normal primary fibroblast strains (AG01522 and MRC5). Cell growth reduction, histone H2AX phosphorylation and telomere-induced dysfunctional foci (TIF) for- mation were markedly higher in astrocytoma cells than in normal fibroblasts, despite the absence of telomere shortening. In the second part of the study, the combined effect of submicromolar concentra- tions of RHPS4 and X-rays was assessed in the U251MG glioblastoma radioresistant cell line. Long-term growth curves, cell cycle analysis and cell survival experiments, clearly showed the synergistic effect of the combined treatment. Interestingly the effect was greater in cells bearing a higher number of dysfunc- tional telomeres. DNA double-strand breaks rejoining after irradiation revealed delayed repair kinetics in cells pre-treated with the drug and a synergistic increase in chromosome-type exchanges and telomeric fusions. These findings provide the first evidence that exposure to RHPS4 radiosensitizes astrocytoma cells, suggesting the potential for future therapeutic applications. © 2014 Elsevier B.V. All rights reserved. Abbreviations: G4, G quadruplex; TIF, telomere induced dysfunctional foci; GBM, glioblastoma multiforme; ALT, alternative lengthening of telomeres; hTERT, human telomerase catalytic subunit; PCC, premature chromosome condensation; cPDL, cumulative population doubling level; SER, sensitisation enhancement ratio; TRF1, telomere repeat binding factor 1; TRF2, telomere repeat binding factor 2; POT1, protection of telomeres 1; DSB, double strand break; CSC, cancer stem cells. ∗ Corresponding author at: Department of Science, University of Rome “Roma Tre”, Viale G. Marconi 446, 00146 Rome, Italy. Tel.: +39 06 57336337; fax: +39 06 57336321. E-mail address: francesco.berardinelli@uniroma3.it (F. Berardinelli). 1 These authors contributed equally to the work. 1. Introduction Telomeres are protein–DNA complexes that are located at the physical ends of linear eukaryotic chromosomes, and they con- fer protection against the action of exonucleases and ligases [1,2]. For this reason, telomeres are known to play a major role in the maintenance of genomic stability by preventing inappropriate chromosome end-to-end fusion [3]. Telomere attrition or telomeric dysfunction resulting from the loss of function of shelterin com- plex proteins [4] lead to cell cycle arrest, senescence, and apoptosis [5]. This makes telomeres and mechanisms involved in the main- tenance of their length promising targets for the development of selective molecules for cancer therapy [6–10]. In particular, in the past decade, considerable attention has been focused on telomerase, because the activation of this enzyme http://dx.doi.org/10.1016/j.dnarep.2014.10.009 1568-7864/© 2014 Elsevier B.V. All rights reserved.