Abstract 2865: Gemcitabine causes telomere shortening by stabilizing TRF2

Abstract

Gemcitabine is an effective anti-cancer agent against sold tumors. The pharmacological mechanism of gemcitabine is known as incorporation into DNA and thereby inhibition of DNA synthesis. Here, we report that gemcitabine treatment not only induced growth arrest and apoptosis in HeLa cells, but also caused telomere shortening without affecting telomerase activity. We performed cycloheximide chase assay to confirm that the half-life of TRF2, a telomere regulator, was prolonged in a dose-pendent manner under the treatment of gemcitabine. In addition, we did in vivo ubiquitination assay and found gemcitabine treatment increased TRF2 protein level by reducing the amount of ubiquitinated TRF2. And, the results of subcellular fractionation and immunostaining showed that the accumulated TRF2 indeed translocates into nucleus. Since TRF2 is a telomeric DNA binding protein, we then made an attempt to determine whether the accumulated TRF2 binds on telomeric DNA. We did chromatin immunoprecipitation and the result revealed that the accumulated TRF2 not only translocates into nucleus but also binds to telomeric DNA. Previous study has shown that XPF-ERCC1, a structure-specific endonuclease and negative mediator of telomere length, interacts with TRF2, also required for TRF2-promoting telomere shortening. To confirm this interaction also exists in HeLa cells under the treatment of gemcitabine, we carried out immunoprecipitation with anti-TRF2 and anti-XPF antibodies and a de-telomeric DNA pull-down assay. Consistently, the results of both assay indicated that increased nuclear TRF2 which was induced by gemcitabine treatment is capable of recruiting more XPF/ERCC1 complex onto telomeric DNA. Besides, the binding studies by experimental manipulation of TRF2 confirmed that the binding ability of XPF-ERCC1 to telomere closely relates to TRF2 and resulted in telomere shortening. Together, these results show that gemcitabine enhances the nuclear accumulation of TRF2 and increased the amount of DNA-bound TRF2, therefore recruiting more XPF/ERCC1 to telomeric DNA and strengthening the nuclease activity of XPF, and resulting in telomere shortening. Since telomere loss is closely related to occurrence of apoptosis, gemcitabine may exert its anti-cancer effect by inducing TRF2 and telomere shortening. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2865. doi:1538-7445.AM2012-2865