Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells. SIRT1 is an enzyme that catalyzes the deacetylation of acetyl-lysine residues by a mechanism in which NADϩ is cleaved and a unique product, O-acetyl ADP-ribose, is generated (4,19,21). In addition, the reaction results in the release of nicotinamide, which acts as an end product inhibitor (3). SIRT1 is distinct from the class I and II histone deacetylase (HDAC) enzymes that remove acetyl groups without hydrolysis of NAD ϩ (13). Indeed, SIRT1 catalytic activity is not affected by the class I and II HDAC inhibitor trichostatin A (TSA) (19).SIRT1 plays a role in a wide variety of processes including stress resistance, metabolism, differentiation, and aging (4). Overexpression of SIRT1 orthologs in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster leads to an increased life span of these organisms (23,43,49), and this has excited speculation that SIRT1 might also regulate life span in mammals (5). SIRT1 binds to and regulates the activity of several transcription factors, including FOXO1, FOXO3a, and FOXO4 (8,16,37,38,51), HES-1 and HEY-2 (48), MyoD (15), CTIP2 (46), PPAR␥ (40), NF-B (55), and PGC1␣ (42). SIRT1 has also been shown to interact with and deacetylate the p53 tumor suppressor protein (25,30,53). p53 is a key transcriptional regulator of genes involved in cell cycle progression, apoptosis, and DNA repair. Indeed, many human tumors have inactivated p53 protein (54). p53 becomes acetylated after DNA damage, and the acetylated form has been reported to have increased transcriptional activity, promote coactivator recruitment, and enhance site-specific DNA binding (2). Acetylation of p53 is also believed to increase p53 stability by preventing ubiquitination of key lysine residues and subsequent proteasom...