Telomere damage induced by the G-quadruplex ligand RHPS4 has an antitumor effect
Erica Salvati, … , Gabriella Zupi, Annamaria Biroccio
Erica Salvati, … , Gabriella Zupi, Annamaria Biroccio
Published October 11, 2007
Citation Information: J Clin Invest. 2007;117(11):3236-3247. https://doi.org/10.1172/JCI32461.
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Research Article

Telomere damage induced by the G-quadruplex ligand RHPS4 has an antitumor effect

Abstract

Functional telomeres are required for the replicability of cancer cells. The G-rich strand of telomeric DNA can fold into a 4-stranded structure known as the G-quadruplex (G4), whose stabilization alters telomere function limiting cancer cell growth. Therefore, the G4 ligand RHPS4 may possess antitumor activity. Here, we show that RHPS4 triggers a rapid and potent DNA damage response at telomeres in human transformed fibroblasts and melanoma cells, characterized by the formation of several telomeric foci containing phosphorylated DNA damage response factors γ-H2AX, RAD17, and 53BP1. This was dependent on DNA repair enzyme ATR, correlated with delocalization of the protective telomeric DNA–binding protein POT1, and was antagonized by overexpression of POT1 or TRF2. In mice, RHPS4 exerted its antitumor effect on xenografts of human tumor cells of different histotype by telomere injury and tumor cell apoptosis. Tumor inhibition was accompanied by a strong DNA damage response, and tumors overexpressing POT1 or TRF2 were resistant to RHPS4 treatment. These data provide evidence that RHPS4 is a telomere damage inducer and that telomere disruption selectively triggered in malignant cells results in a high therapeutic index in mice. They also define a functional link between telomere damage and antitumor activity and reveal the key role of telomere-protective factors TRF2 and POT1 in response to this anti-telomere strategy.

Authors

Erica Salvati, Carlo Leonetti, Angela Rizzo, Marco Scarsella, Marcella Mottolese, Rossella Galati, Isabella Sperduti, Malcolm F.G. Stevens, Maurizio D’Incalci, Maria Blasco, Giovanna Chiorino, Serge Bauwens, Béatrice Horard, Eric Gilson, Antonella Stoppacciaro, Gabriella Zupi, Annamaria Biroccio

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Figure 2

RHPS4 triggers DNA damage response at telomeres.

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RHPS4 triggers DNA damage response at telomeres. (A) BJ-EHLT fibroblasts...
(A) BJ-EHLT fibroblasts expressing TRF2ΔBΔC or treated with either bleomycin or RHPS4 for 8 hours were fixed and processed for immunofluorescence using antibodies against γ-H2AX (green) and TRF1 (red). Representative confocal images are shown. (B) Higher-magnification view of A, bottom right. Shown are the nucleus and γ-H2AX foci, magnified ×2 and ×4, respectively, relative to the panel in A. (C) Formaldehyde-cross-linked chromatin fragments were immunoprecipitated with antibodies against TRF1, γ-H2AX, H3 (positive control of ChIP assay), or β-actin (negative control). To verify that an equivalent amount of chromatin was used in the immunoprecipitates, serial dilutions of sample the total chromatin (Input) were included in the blot. Specific (telomeric) and nonspecific (Alu) probes were used. (D) Untreated and RHPS4-treated BJ-EHLT cells stained with TRF1 (green) and 53BP1 or p-Rad17 (red). Boxes in the merged images of the RHPS4-treated samples indicate the location of the enlarged views. Original magnification, ×63; ×126 (enlarged panels). (E) TIF index, defined as foci of DNA damage response factors that coincide with TRF1, was calculated as the percentage of TIF-positive cells in the BJ-EHLT fibroblasts expressing TRF2ΔBΔC or treated with either bleomycin or RHPS4. Cells with 4 or more γ-H2AX/TRF1 (gray bars) or 53BP1/TRF1 (black bars) foci were scored as TIF positive. The mean of 3 independent experiments is reported. Error bars indicate SD.