Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation but Does Not Alter Cell Survival following DNA Damage

Elixir Pharmaceuticals, Inc., One Kendall Square, Cambridge, MA 02139, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 02/2006; 26(1):28-38. DOI: 10.1128/MCB.26.1.28-38.2006
Source: PubMed


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.

Download full-text


Available from: Jonathan M Solomon, Aug 10, 2015
5 Reads
  • Source
    • "EX-527 is cell-permeable and selectively inhibits SIRT1 by occupying the NAD + binding site (Gertz et al. 2013). EX-527 has a reported IC 50 of 98 nM in vivo (Napper et al. 2005) and 38 nM in vitro (Solomon et al. 2006), however, it has much lower potency against other sirtuin deacetylase family members and does not inhibit histone deacetylase (HDAC) up to 100 μM (Napper et al. 2005). EX-527 is therefore ideal for inhibiting SIRT1 without influencing other sirtuins which may influence metabolism. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Silent information regulators (SIRT)s have been shown to deacetylate a range of metabolic enzymes, including those in glycolysis and the Krebs cycle, and thus alter their activity. SIRTs require NAD+ for their activity; this linking cellular energy status to enzyme activity. Here, to examine the impact of SIRT1 modulation on oxidative metabolism, we tested the effect of ligands which are either SIRT activating compounds (STACs; resveratrol and SRT1720) or inhibitors (STICs; EX527) on the metabolism of 13C-enriched substrates by Guinea pig brain cortical tissue slices using 13C and 1H NMR spectroscopy. Resveratrol increased lactate labelling but decreased incorporation of 13C into Krebs cycle intermediates consistent with effects on AMP-activated protein kinase (AMPK) and inhibition of the F0/F1ATPase. Testing resveratrol directly applied to astrocytes using a Seahorse analyzer found increased glycolytic shift and increased mitochondrial proton leak due to interactions of resveratrol with the mitochondrial electron transport chain. SRT1720, by contrast, stimulated incorporation of 13C into Krebs cycle intermediates and reduced incorporation into lactate, while the inhibitor EX527 paradoxically also increased Krebs cycle 13C incorporation. In summary, the various SIRT1 modulators show distinct, acute effects on oxidative metabolism. The strong effects of resveratrol on the mitochondrial respiratory chain and on glycolysis suggest caution should be used in attempts to increase bioavailability of this compound in the central nervous system.
    Journal of Neuroscience Research 02/2015; 93:1147-1156. DOI:10.1002/jnr.23570 · 2.59 Impact Factor
  • Source
    • "However, we found that other SIRT1 inhibitors such as EX527 and sirtinol asserted a small cytotoxic effect on MB cells and that once again a high concentration dose of EX527 [100 µM] was required to cause 50% cell viability after 48 hours treatment in D283-MED cells (Fig. S1). These findings are similar to the one described in [52], where the authors found that EX527 decreased SIRT1 deacetylase activity in epithelial cells but with no effect on cell viability. This result and the need of high concentrations of nicotinamide to induce cell death challenge the idea of using SIRT1 inhibitor alone as a therapeutic agent for brain tumour regression. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Chemotherapeutic drug resistance and relapse remains a major challenge for paediatric (medulloblastoma) and adult (glioblastoma) brain tumour treatment. Medulloblastoma tumours and cell lines with mutations in the p53 signalling pathway have been shown to be specifically insensitive to DNA damaging agents. The aim of this study was to investigate the potential of triggering cell death in p53 mutated medulloblastoma cells by a direct activation of pro-death signalling downstream of p53 activation. Since non-coding microRNAs (miRNAs) have the ability to fine tune the expression of a variety of target genes, orchestrating multiple downstream effects, we hypothesised that triggering the expression of a p53 target miRNA could induce cell death in chemo-resistant cells. Treatment with etoposide, increased miR-34a levels in a p53-dependent fashion and the level of miR-34a transcription was correlated with the cell sensitivity to etoposide. miR-34a activity was validated by measuring the expression levels of one of its well described target: the NADH dependent sirtuin1 (SIRT1). Whilst drugs directly targeting SIRT1, were potent to trigger cell death at high concentrations only, introduction of synthetic miR-34a mimics was able to induce cell death in p53 mutated medulloblastoma and glioblastoma cell lines. Our results show that the need of a functional p53 signaling pathway can be bypassed by direct activation of miR-34a in brain tumour cells.
    PLoS ONE 09/2014; 9(9):e108514. DOI:10.1371/journal.pone.0108514 · 3.23 Impact Factor
  • Source
    • "Rabbit polyclonal anti-TRF2 antibody was purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). ASSESSMENT OF SIRT1 ACTIVITY SIRT1 deacetylase activity was measured in nuclear extracts from MSCs using a histone deacetylase assay kit (AK-555), which was purchased from BIOMOL (Plymouth Meeting, PA, USA) (Solomon et al., 2006 "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mesenchymal stem cells (MSCs) senescence is an age-related process that impairs the capacity for tissue repair and compromises the clinical use of autologous MSCs for tissue regeneration. Here, we describe the effects of SIRT1, a NAD(+)-dependent deacetylase, on age-related MSCs senescence. Knockdown of SIRT1 in young MSCs induced cellular senescence and inhibited cell proliferation whereas overexpression of SIRT1 in aged MSCs reversed the senescence phenotype and stimulated cell proliferation. These results suggest that SIRT1 plays a key role in modulating age-induced MSCs senescence. Aging-related proteins, P16 and P21 may be downstream effectors of the SIRT1-mediated anti-aging effects. SIRT1 protected MSCs from age-related DNA damage, induced telomerase reverse transcriptase (TERT) expression and enhanced telomerase activity but did not affect telomere length. SIRT1 positively regulated the expression of tripeptidyl peptidase 1 (TPP1), a component of the shelterin pathway that protects chromosome ends from DNA damage. Together, the results demonstrate that SIRT1 quenches age-related MSCs senescence by mechanisms that include enhanced TPP1 expression, increased telomerase activity and reduced DNA damage.
    Frontiers in Aging Neuroscience 06/2014; 6:103. DOI:10.3389/fnagi.2014.00103 · 4.00 Impact Factor
Show more