Protein acetylation is a reversible process regulated by histone deacetylases (HDAC) that is often altered in human cancers. Suberoylanilide hydroxamic acid (SAHA) is the first HDAC inhibitor to be approved for clinical use as an anticancer agent. Given that histone acetylation is a key determinant of chromatin structure, we investigated how SAHA may affect DNA replication and integrity to gain deeper insights into the basis for its anticancer activity. Nuclear replication factories were visualized with confocal immunofluorescence microscopy and single-replicon analyses were conducted by genome-wide molecular combing after pulse labeling with two thymidine analogues. We found that pharmacologic concentrations of SAHA induce replicationmediated DNA damage with activation of histone γH2AX. Single DNA molecule analyses indicated slowdown in replication speed along with activation of dormant replication origins in response to SAHA. Similar results were obtained using siRNA-mediated depletion of HDAC3 expression, implicating this HDAC member as a likely target in the SAHA response. Activation of dormant origins was confirmed by molecular analyses of the β-globin locus control region. Our findings demonstrate that SAHA produces profound alterations in DNA replication that cause DNA damage, establishing a critical link between robust chromatin acetylation and DNA replication in human cancer cells. Cancer Res; 70(11); 4470-80. ©2010 AACR.
DNA topoisomerase I (Top1) is essential for removing DNA supercoiling generated in transcribing and replicating chromatin (11,67). Top1 relaxes positively and negatively supercoiled DNA by introducing reversible DNA single-strand breaks associated with covalent Top1-DNA complexes. Camptothecin, a natural alkaloid, selectively targets the Top1-DNA complex by stabilizing the covalent Top1-DNA cleavage intermediate (33,47,65). Camptothecin and its derivatives, irinotecan and topotecan, are potent anticancer drugs currently being used successfully in the treatment of colon and ovarian cancer (4,46,64). The cytotoxic action of camptothecin is manifested when a replication fork encounters the drug-stabilized cleavage complex (31, 34). At these sites, extension of the replicating strand up to the end of the Top1-mediated break in the template strand generates a replication double-strand break ("replication runoff") as demonstrated by ligation-mediated PCR (62) and the induction of ␥-H2AX (23) (http://discover.nci.nih.gov/ pommier/pommier.htm). Camptothecin is, therefore, a wellcharacterized pharmacological tool for studying the molecular mechanisms involved in cellular responses to replicative stress (23,48,59,62). Top1 cleavage complexes and, therefore, replication double-strand breaks can form in response to common DNA lesions including abasic sites, mismatches, oxidative base lesions, base adducts, and strand breaks (49, 51).Histone H2AX phosphorylated on serine 139, termed ␥-H2AX, is one of the earliest known markers of camptothecin-induced replication-associated damage (23). More generally, ␥-H2AX is a marker of DNA double-strand breaks (45, 54). ␥-H2AX has been proposed to anchor the broken chromosome ends together and recruit DNA repair elements (5,20,23,45,53). We have shown previously that ␥-H2AX is critical for the recruitment of the Mre11-Rad50-Nbs1 (MRN) complex in camptothecin-treated cells and that H2AX deficiency renders cells hypersensitive to camptothecin (23,53). Using aphidicolin, we also showed that blocking replicative polymerases abrogates ␥-H2AX formation (23), indicating that ␥-H2AX forms in response to replication-associated doublestrand breaks induced by camptothecin.The causative gene of the cancer-predisposing genetic disease Bloom's syndrome, BLM, is a member of the RecQ family of DNA helicases (28). BLM is considered a caretaker of the genome (28, 39) and a key component in DNA damage response signaling (22,52). Evolutionarily conserved and essential for the maintenance of genomic stability, BLM promotes branch migration of Holliday junctions in vitro in an ATPdriven fashion (36,38,40,66,70). BLM functions in association with topoisomerase III␣ (Top3␣) (68), a type I class of topoisomerases (11,37,67,69). The BLM-Top3␣ complex can resolve recombination intermediates and prevent the collapse of replication forks and consequent DNA double-strand
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.
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