Abstract 4862: Histone deacetylase inhibition induces DNA damage, slows down replication forks and activates dormant origins

Abstract

Histone acetylation is often altered in cancers and histone deacetylases inhibitors (HDACi) are actively pursued as anti-cancer agents. SAHA (suberoylanilide hydroxamic acid) is the first HDACi approved for clinical use. To monitor the effects of SAHA on DNA replication and genomic integrity in cancer cells, we detected broken DNA with COMET assays and analyzed phosphorylation of histone H2AX (gH2AX). Nuclear replication factories in single cells were visualized after pulse labeling with two thymidine analogs and confocal immunofluorescence microscopy. Nascent strand reverse transcriptase polymerase chain reaction (RT-PCR) in the human g-globin locus was used to assess the effects of SAHA on replication fork origin firing. In addition, cellular replicons were analyzed by genome-wide molecular combing. Our experiments prove that pharmacological concentrations of SAHA induce replication damage, as shown by single-cell and single-DNA molecule analyses. Molecular combing demonstrated a slow-down of replicons and activation of dormant replication origins in response to SAHA treatment. Similar results were obtained with siRNA-mediated HDAC3 depletion indicating the selectivity of the effect of SAHA. Activation of dormant origins at the g-globin locus control region was confirmed by nascent strand RT-PCR. Our findings demonstrate specific replication alterations and DNA damage in response to SAHA and emphasize the importance of chromatin acetylation for DNA replication in human cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4862.