Abstract
Most human tumors maintain telomere lengths by telomerase, whereas a portion of them (10–15%) uses a mechanism named alternative lengthening of telomeres (ALT). The telomeric G-quadruplex (G4) ligand RHPS4 is known for its potent antiproliferative effect, as shown in telomerase-positive cancer models. Moreover, RHPS4 is also able to reduce cell proliferation in ALT cells, although the influence of G4 stabilization on the ALT mechanism has so far been poorly investigated. Here we show that sensitivity to RHPS4 is comparable in ALT-positive (U2OS; SAOS-2) and telomerase-positive (HOS) osteosarcoma cell lines, unlinking the telomere maintenance mechanism and RHPS4 responsiveness. To investigate the impact of G4 stabilization on ALT, the cardinal ALT hallmarks were analyzed. A significant induction of telomeric doublets, telomeric clusterized DNA damage, ALT-associated Promyelocytic Leukaemia-bodies (APBs), telomere sister chromatid exchanges (T-SCE) and c-circles was found exclusively in RHPS4-treated ALT cells. We surmise that RHPS4 affects ALT mechanisms through the induction of replicative stress that in turn is converted in DNA damage at telomeres, fueling recombination. In conclusion, our work indicates that RHPS4-induced telomeric DNA damage promotes overactivation of telomeric recombination in ALT cells, opening new questions on the therapeutic employment of G4 ligands in the treatment of ALT positive tumors.
Highlights
Telomeres are nucleoprotein structures that protect the ends of linear chromosomes [1,2].In humans, telomeres are composed of 10–15 kb of tandem arrays of TTAGGG repeats [3,4] bound by specialized proteins [5] that cooperatively conceal chromosome ends, impeding their processing as DNA double-strand breaks (DSBs) and contributing to genome stability
The frequency of telomeric doublets, telomeric damage, alternative lengthening of telomeres (ALT)-associated PML bodies (APBs), telomeric sister chromatid exchanges (T-SCEs) and c-circles was significantly increased upon RHPS4 treatment exclusively in ALT cells
As shown by IC50 values, calculated from the dose-response curves obtained at 120 h after treatment (Figure 1b), sensitivity to the G4 ligand was comparable in ALT and telomerase positive cell lines (IC50 values: 1.6, 1.4, 1.2 μM for SAOS-2, U2OS, and HOS, respectively)
Summary
Telomeres are nucleoprotein structures that protect the ends of linear chromosomes [1,2]. Telomeres are composed of 10–15 kb of tandem arrays of TTAGGG repeats [3,4] bound by specialized proteins [5] that cooperatively conceal chromosome ends, impeding their processing as DNA double-strand breaks (DSBs) and contributing to genome stability. In cells lacking a telomere maintenance mechanism (TMM), telomeric DNA encounters progressive erosion at each cell duplication, eventually leading to replicative senescence. This represents a tumor-suppressive mechanism and, as a consequence, continuous dividing cells (e.g., cancer cells) need to avoid senescence by the maintenance of stable telomere lengths. Most human cancers maintain their telomeres by reactivating the reverse transcriptase telomerase [6].
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