Article

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

ABSTRACT

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

Full-text

Available from: Jonathan M Solomon, Aug 10, 2015
  • Source
    • "Some cultures were treated with the AT1 receptor antagonist ZD 7155 (1 µM; Sigma) for 30 minutes before treatment with AII to confirm the involvement of AT1 receptors. Cultures in the second series of experiments were exposed to the SIRT1 activator resveratrol (50 µM; Cayman chemical) or the SIRT1specific inhibitor Ex 527 (Tocris; 1µM) [69, 70] for 30 min before treatment with resveratrol. The cells were then washed and processed for WB or RT-PCR. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Local angiotensin II (AII) and sirtuin 1 (SIRT1) play a major role in the modulation of neuroinflammation, oxidative stress and aging-related dopaminergic vulnerability to damage. However, it is not known whether the modulation is related to reciprocal regulation between SIRT1 and AII. In the present study, a single intraventricular injection of AII increased nigral SIRT1 levels in young adult rats. Although AII activity is known to be increased in aged rats, levels of SIRT1 were significantly lower than in young controls. Treatment with the SIRT1-activating compound resveratrol increased nigral SIRT1 levels in aged rats. Levels of SIRT1 were significantly higher in aged wild type mice than in AII type-1 receptor (AT1) deficient mice. In cell culture studies, treatment with AII also induced a transitory increase in levels of SIRT1 in the MES 23.5 dopaminergic neuron and the N9 microglial cell lines. In aged rats, treatment with resveratrol induced a significant decrease in the expression of AT1 receptors and markers of NADPH-oxidase activation (p47phox). In aged transgenic mice over-expressing SIRT1, levels of AT1 and p47 phox were lower than in aged wild type controls. In vitro, the inhibitory effects of resveratrol on AII/AT1/NADPH-oxidase activity were confirmed in primary mesencephalic cultures, the N9 microglial cell line, and the dopaminergic neuron cell line MES 23.5, and they were blocked by the SIRT1 specific inhibitor EX527. The present findings show that SIRT1 and the axis AII/AT1/NADPH-oxidase regulate each other. This is impaired in aged animals and may be mitigated with sirtuin-activating compounds.
    Full-text · Article · Sep 2015 · Oncotarget
  • 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.
    Full-text · Article · Feb 2015 · Journal of Neuroscience Research
  • 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.
    Full-text · Article · Sep 2014 · PLoS ONE
Show more