MOLECULAR AND CELLULAR BIOLOGY, Jan. 2006, p. 28–38
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 26, No. 1
Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation
but Does Not Alter Cell Survival following DNA Damage
Jonathan M. Solomon, Rao Pasupuleti, Lei Xu, Thomas McDonagh, Rory Curtis,
Peter S. DiStefano, and L. Julie Huber*
Elixir Pharmaceuticals, Inc., One Kendall Square, Cambridge, Massachusetts 02139
Received 16 May 2005/Returned for modification 5 July 2005/Accepted 3 October 2005
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
SIRT1 is an enzyme that catalyzes the deacetylation of
acetyl-lysine residues by a mechanism in which NAD?is
cleaved and a unique product, O-acetyl ADP-ribose, is gener-
ated (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 hydroly-
sis of NAD?(13). Indeed, SIRT1 catalytic activity is not af-
fected by the class I and II HDAC inhibitor trichostatin A
SIRT1 plays a role in a wide variety of processes including
stress resistance, metabolism, differentiation, and aging (4).
Overexpression of SIRT1 orthologs in Saccharomyces cerevi-
siae, 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 pro-
gression, apoptosis, and DNA repair. Indeed, many human
tumors have inactivated p53 protein (54). p53 becomes acety-
lated after DNA damage, and the acetylated form has been
reported to have increased transcriptional activity, promote
coactivator recruitment, and enhance site-specific DNA bind-
ing (2). Acetylation of p53 is also believed to increase p53
stability by preventing ubiquitination of key lysine residues and
subsequent proteasomal degradation (27).
Compounds that activate p53 function have been success-
fully used to treat tumors in vivo (9, 20, 52). Therefore, small-
molecule inhibitors of SIRT1 might function as anticancer
agents if SIRT1 acts as an inhibitor of p53. This hypothesis is
supported by studies in cell lines in which overexpression of a
catalytically impaired variant of SIRT1 decreased survival and
increased p53 target gene expression after DNA damage (53).
Overexpression of wild-type SIRT1 had the opposite effect of
increasing cell survival after DNA damage (30). Moreover,
thymocytes derived from mice lacking Sir2? (mouse SIRT1)
exhibit increased sensitivity to ?-radiation although, interest-
ingly, embryonic stem cells and fibroblasts from these mice
showed no altered resistance to DNA damage-induced stress
In this study, the role of SIRT1 on p53 acetylation and
function is examined using a novel, potent, and specific small-
molecule inhibitor of SIRT1 catalytic activity, designated EX-
527. In cells subjected to DNA damage, inhibition of SIRT1
with EX-527 results in increased p53 acetylation but surpris-
ingly does not result in detectable effects on p53-controled
gene expression, cell survival, or cell proliferation. In contrast,
inhibition of class I/II HDAC-mediated p53 deacetylation with
TSA increases p53 acetylation after DNA damage, with re-
duced cell survival and growth. Although p53 acetylation is
synergistically increased by inhibition of both HDAC and
SIRT1, concomitant treatment with EX-527 and TSA does not
further affect cell proliferation or survival after DNA damage.
MATERIALS AND METHODS
Materials and cell culture. Etoposide was obtained from Calbiochem. TSA,
nicotinamide, adriamycin, hydroxyurea, hydrogen peroxide, and other reagent
quality chemicals were from Sigma. Tissue culture plastics were obtained from
* Corresponding author. Mailing address: Elixir Pharmaceuticals,
Inc., One Kendall Square, Cambridge, MA 02139. Phone: (617) 995-
7007. Fax: (617) 995-7050. E-mail: firstname.lastname@example.org.
Corning except where noted. NCI-H460, MCF-7, IMR-90, 293T, and U-2 OS
cells were obtained from the American Type Tissue Collection. NCI-H460 cells
were cultured in RPMI 1640 medium with 10 mM HEPES, 1 mM sodium
pyruvate, and 10% fetal bovine serum (FBS). MCF-7, IMR-90, and 293T cells
were grown in Dulbecco’s modified Eagle’s medium supplemented with 10%
FBS. U-2 OS cells were cultured in McCoy’s 5a medium with 1.5 mM L-glu-
tamine and 10% FBS. Primary human mammary epithelial cells (HMEC, derived
from normal adult mammary glands) and complete serum-free medium were
obtained from Cell Applications, Inc. HMEC were passaged no more than three
times before use in experiments.
Deacetylation assays and inhibitory compounds. Deacetylation was measured
using the Fluor de Lys kit (AK-555; Biomol) using a fluorogenic peptide encom-
passing residues 379 to 382 of p53, acetylated on lysine 382 (KI-177; Biomol).
The acetylated lysine residue was coupled to an aminomethylcoumarin moiety.
The peptide was deacetylated by SIRT1, followed by the addition of a proteolytic
developer that released the fluorescent aminomethylcoumarin. Briefly, enzyme
preparations were incubated with 170 ?M NAD?and 100 ?M p53 fluorogenic
peptide for 45 min at 37°C followed by incubation in developer for 15 min at
37°C. Fluorescence was measured by excitation at 360 nm and emission at 460
nm and enzymatic activity was expressed in relative fluorescence units.
Human SIRT5 (N-terminal glutathione S-transferase [GST] tag) was ex-
pressed in Escherichia coli and purified by affinity chromatography with gluta-
thione-Sepharose (G.E. Healthcare). Deacetylation of p53 lysine 382 was mea-
sured using the Fluor de Lys kit (as described above). SIRT5 activity was also
measured by the deacetylation of cytochrome c, prepared by chemical acetylation
with [3H]acetate. First, 1 mg cytochrome c (Sigma) was acetylated for 2 h at room
temperature in 140 ?l of 100 mM morpholineethanesulfonic acid (MES), pH 5.0,
with 250 ?Ci [3H]acetate (ICN) and 2.2 mg/ml 1-ethyl-3-[3-dimethylaminopro-
pyl]carbodiimide hydrochloride (Pierce). Unincorporated [3H]acetate was re-
moved on a G-50 microspin column (G.E. Healthcare). The labeled protein was
precipitated with 20% trichloroacetic acid for 20 min on ice, washed three times
with ice-cold acetone, air dried, and resuspended at 1 mg/ml in distilled water.
The deacetylation reaction was performed as described previously (45) using 10
EX-527 (see Fig. 1A) and additional SIRT1-inhibitory compounds (EX-519,
EX-586, EX-589, EX-622, and EX-635) were identified in a high-throughput
screen using the Fluor de Lys assay (J. Hixon, T. McDonagh, R. Curtis, P. S.
DiStefano, A. Napper, T. Hesterkamp, R. Thomas, K. Keavey, and J. Pons, Soc.
Biomol. Screen. 10th Annu. Meet., poster P10045, 2004). EX-527 was found to
be 200- to 500-fold more selective for SIRT1 than SIRT2 and SIRT3. EX-527 is
a racemic mixture, and its optical isomers were separated by chiral high-perfor-
mance liquid chromatography and designated EX-242 and EX-243. When tested
in the deacetylation assay, EX-243 was active in SIRT1 inhibition, whereas
EX-242 was inactive. The details of the synthesis and separation of EX-527 are
described elsewhere (37a). Compounds were stored at ?20°C in dimethyl sul-
foxide and were retested in the deacetylation assay prior to use in cell-based
Inhibition of GST-SIRT1 deacetylase activity. 293T cells were transiently
transfected with GST-tagged human SIRT1 in the pDEST27 Gateway vector
(Invitrogen) using FuGENE-6 (Roche). After 48 h, the cells were lysed with 50
mM Tris, pH 8.0, 120 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40, sup-
plemented with Complete Mini protease inhibitor cocktail tablets (Roche). GST-
SIRT1 was purified from lysates using glutathione-Sepharose beads (G.E.
Healthcare) and washed extensively in the above buffer. The deacetylation assay
was performed with approximately 30 ng of GST-SIRT1 in the presence of
EX-527 (48 pM to 100 ?M).
Yeast Sir2 silencing assay. The effect of compounds on Sir2-dependent silenc-
ing was assessed by measuring the growth of a yeast strain harboring a telomere-
proximal URA3 gene in the presence or absence of 5-fluoroorotic acid (5-FOA)
(17). Strain SL8c was constructed by mating PSY316AUTa to W303ARUT?
(35). Cells were grown overnight in SD medium (2% glucose, 0.67% yeast
nitrogen base, 0.39% Casamino Acids, 0.005% adenine, 0.005% tryptophan).
The culture was split 1:20 into fresh yeast extract-peptone-dextrose medium
containing 2% glucose and grown for 5 h. Cells were plated in 96-well plates
(Costar) in 150 ?l of SD containing 0.005% uracil either with or without 0.1%
5-FOA, containing 5 to 100 ?M EX-242 or EX-243. Plates were cultured at 30°C
for 18 to 24 h, and growth of resuspended cells was measured by light scattering
at 600 nm in a spectrophotometer.
Determination of acetylated p53 levels. NCI-H460 cells, MCF-7 cells, U-2 OS
cells, or HMEC (1 to 2 million) were plated in six-well tissue culture plates or
10-cm dishes. After 1 day (NCI-H460, MCF-7, and U-2 OS cells) or 2 days
(HMEC), p53 acetylation was induced for 6 h with one of the following DNA
damaging agents: etoposide (20 ?M), hydrogen peroxide (400 ?M), hydroxyurea
(1 mM), or adriamycin (0.2 ?g/ml for NCI-H460, MCF-7, and U-2 OS cells; 0.8
?g/ml for HMEC). To determine the effects of deacetylase inhibitors on p53
acetylation levels, cells were treated with 6.25 to 400 nM TSA and/or 1 ?M
EX-527. In some experiments, other SIRT1 inhibitors, EX-635, EX-622, EX-519,
EX-586, and EX-589, or the optical isomers, EX-242 and EX-243, were used at
1 ?M instead of EX-527. After washing with phosphate-buffered saline, cells
were lysed in 50 mM Tris, pH 7.8, 137 mM NaCl, 10 mM NaF, 1 mM EDTA, 1%
Triton X-100, 0.2% Sarkosyl, 1 mM dithiothreitol, and 10% glycerol containing
Complete Mini protease inhibitor cocktail tablets and supplemented with 10 ?M
TSA and 5 mM nicotinamide to prevent deacetylation after cell lysis.
Immunoprecipitation of total p53 was performed with agarose-conjugated p53
monoclonal antibody (Ab-6; Oncogene Research Products) for 2 h at 4°C, fol-
lowed by incubation with protein A-Sepharose (Pharmacia) for 1 h at 4°C. Beads
were washed extensively with lysis buffer. Cell lysates or immunoprecipitates
were resolved on 10% Criterion Tris-HCl gels (Bio-Rad). p53 acetylation was
detected with anti-acetylated-Lys382 p53 antibody (Cell Signaling Technology),
and total p53 levels were determined with antibodies Ab-6 or Ab-7 (Oncogene
Research Products). Bound antibodies were visualized with horseradish peroxi-
dase-conjugated secondary antibody and SuperSignal West Femto maximum
sensitivity substrate (Pierce). Luminescence was quantified by a Bioimaging
SIRT1 immunoblotting. SIRT1 in cell lysates was visualized by immunoblot-
ting using antibody 07-131 (Upstate Cell Signaling Solutions). Gels, secondary
antibodies, and horseradish peroxidase substrate were used as described above.
Cell viability and proliferation assays. NCI-H460 cells, MCF-7 cells, U-2 OS
cells, or HMEC were plated at 2,000 cells per well in opaque-walled 96-well
plates (Corning) for the viability assay and 800 cells per well in 96-well Cy-
tostar-T scintillating microplates (Amersham) for the proliferation assay. Cells
were incubated for 1 day (NCI-H460) or 2 days (MCF-7, U-2 OS, and HMEC)
prior to exposure to DNA-damaging agents and deacetylase inhibitors. All ex-
periments were performed in triplicate.
For viability assays, cells were treated with the indicated compounds for 48 h.
Cell viability was then determined using the Cell Titer-Glo luminescent assay
(Promega), which measures total ATP levels as an index of cell number. Lumi-
nescence was measured on a Luminoskan Ascent (ThermoLabSystems).
For the proliferation assay, 0.5 ?Ci/ml of [14C]thymidine was added to the
medium immediately after the genotoxins and deacetylase inhibitors. Plates were
counted at 48 h (HMEC) or 72 h (NCI-H460, MCF-7, and U-2 OS cells) in a
Microbeta liquid scintillation counter (Perkin-Elmer). Thymidine incorporated
by the cells was detected by proximity to the scintillant in the base of the
Cytostar-T tissue culture plate. Values were graphed as means ? standard
Effects on p53-controlled gene expression. NCI-H460 cells, MCF-7 cells, U-2
OS cells, or HMEC (0.5 to 2 million) were plated in 10-cm tissue culture plates.
After 1 to 2 days, cells were treated with various concentrations of etoposide (0
to 100 ?M) in the presence or absence of 1 ?M EX-527 for 6 h. RNA was
isolated from cells using the RNAqueous 4-PCR kit (Ambion). cDNA was
generated using the First-Strand cDNA synthesis kit (Roche). Quantitative PCR
was performed to determine mRNA levels using Lightcycler (Roche) and the
following primers: p21 forward, GCGACTGTGATGCGCTAATGG; p21 re-
verse, GCGTTTGGAGTGGTAGAAATCTG; BAX forward, GCGAGTGTC
TCAAGCGC; BAX reverse, GCACCAGTTTGCTGGCA; hypoxanthine phos-
phoribosyltransferase forward, TAGCCCTCTGTGTGCT; hypoxanthine phospho-
ribosyltransferase reverse, CTTCGTGGGGTCCTTT.
To detect p21 protein, NCI-H460 cells were treated with adriamycin (0.2
?g/ml) or etoposide (20 ?M) in the presence or absence of 1 ?M EX-527 for 6 h
and then lysed in 50 mM Tris buffer, pH 8.0, 120 mM NaCl, 1 mM EDTA, and
0.5% Nonidet P-40, supplemented with Complete Mini protease inhibitor cock-
tail tablets. The samples were resolved on Criterion 10% Tris-HCl gels, and blots
were probed with anti-p21 antibody F-5 (Santa Cruz Biotechnology) at a 1:1,000
dilution. Tubulin was visualized with antibody B-5-1-2 (Sigma) at a 1:5,000
dilution as a protein loading control. Secondary antibodies and horseradish
peroxidase substrate were used as described above.
SIRT1 overexpression. Constructs that express either SIRT1 or green fluores-
cent protein (GFP) were made using the pLP-LNCX vector (Clontech). The
constructs were packaged into virus by cotransfecting them using FuGENE 6 into
293T cells (500,000 cells per 60-mm dish) with the pVPack-VSV-G and
pVPack-GP vectors (Stratagene). After 24 h, virus was collected from the su-
pernatant and filtered through a 0.45-?m filter, and Polybrene (Sigma) was
added to a final concentration of 8 ?g/ml. IMR-90 and MCF-7 cells cultured in
six-well plates were infected with virus-containing medium for 24 h, after which
the medium was replaced with fresh medium containing G418. Following selec-
tion, cells were plated in 96-well plates at 5,000 cells per well and treated with 200
VOL. 26, 2006 INHIBITION OF SIRT1 DOES NOT ALTER CELL SURVIVAL29
to 1,600 ?M hydrogen peroxide for 24 h, and the cell viability was determined
using the Cell Titer-Glo kit (Promega) as described above.
Characterization of a potent inhibitor of SIRT1. EX-527
(Fig. 1A) is an inhibitor of SIRT1 enzymatic activity (50%
inhibitory concentration [IC50], 98 nM), identified in a high-
throughput screen using bacterially expressed human SIRT1
(37a). To confirm the activity of EX-527 against SIRT1 pro-
duced in mammalian cells, the potency of EX-527 was deter-
mined using the in vitro Fluor de Lys deacetylation assay. The
activity of GST-tagged human SIRT1, purified from 293T cells,
was measured in the presence of EX-527 (48 pM to 100 ?M).
As shown in Fig. 1B, EX-527 inhibited SIRT1 in a concentra-
tion-dependent manner with an IC50of 38 nM, in agreement
with the activity on bacterially expressed SIRT1.
EX-527 has much lower potency against SIRT2 (IC50, 19.6
?M) or SIRT3 (IC50, 48.7 ?M) but does not inhibit class I/II
HDAC activity at concentrations up to 100 ?M (37a). We and
others (39) have found that SIRT5 possesses low, albeit signif-
icant, deacetylase activity, and we sought to determine if this
can be inhibited by EX-527. SIRT5 had no effect on deacety-
lation of p53 lysine 382 peptide in the Fluor de Lys assay (data
not shown). Therefore, the release of [3H]acetate from chem-
ically acetylated cytochrome c was measured. SIRT5-mediated
deacetylation of cytochrome c was not inhibited by EX-527 at
concentrations up to 50 ?M (data not shown). No deacetylase
activity was detected for SIRT4, SIRT6, and SIRT7. Thus,
EX-527 is selective for SIRT1 compared to other SIRT family
members and class I/II HDAC enzymes.
To confirm that EX-527 acts on NAD-dependent deacety-
lase enzymes, the compound was tested in the well-character-
ized Sir2-dependent silencing assay in yeast. SIRT1 is the clos-
est human ortholog of the yeast silent information regulator-2
protein, Sir2 (12). Sir2 regulates transcriptional silencing at the
DNA near telomeres, the silent mating type locus, and in the
region encoding rRNA by deacetylating lysines on histone tails
(36). Inhibition of yeast Sir2 activity by the pure optical iso-
mers of EX-527 was determined (Fig. 1C). The optical isomers
are EX-243, which inhibits SIRT1 catalytic activity, and EX-
242, which does not. The inactive isomer serves as a control for
cellular effects of the compound that are unrelated to the
inhibition of catalytic activity. Sir2 activity was assayed by an-
alyzing the silencing of a URA3 gene inserted near a telomere
(17). 5-FOA in the medium blocks the growth of yeast only
when the URA3 gene is expressed. Sir2 activity silences the
URA3 gene near the telomere, and the cells grow normally in
the presence of 5-FOA. However, if Sir2-dependent silencing
is inhibited, the URA3 gene is expressed and growth in the
presence of 5-FOA is reduced. As expected, in the absence of
5-FOA, neither enantiomer had any growth inhibitory effect,
indicating that EX-243 and EX-242 are not toxic at the con-
centrations tested (Fig. 1C). In the presence of 5-FOA, the
active enantiomer EX-243 inhibited growth, whereas EX-242
had no effect, indicating that Sir2 silencing of URA3 is reduced
(Fig. 1C). EX-527 gave results similar to those of EX-243 (data
not shown). These results demonstrate that EX-527 can inhibit
NAD-dependent deacetylation in a cellular environment.
Inhibition of SIRT1 enhances p53 acetylation in response to
DNA damaging agents. Previous studies have shown that the
acetylation of lysine 382 of p53 is modulated by SIRT1 (25, 30,
53). To determine if inhibition of SIRT1 resulted in an in-
crease in p53 acetylation, NCI-H460 cells were treated with
FIG. 1. EX-527 is a potent inhibitor of SIRT1 that also inhibits
Sir2-dependent silencing in yeast. (A) Structure of EX-527. (B) EX-
527 inhibits deacetylase activity of purified human SIRT1. Deacetylase
activity of GST-SIRT1 in the presence of increasing concentrations of
EX-527 was measured by the Fluor de Lys assay. Samples were assayed
in triplicate and analyzed using Prism (Graphpad Software, Inc.).
(C) The active optical isomer of EX-527, EX-243, and not the inactive
optical isomer, EX-242, inhibits Sir2-dependent silencing in yeast. A
yeast strain constructed with a URA3 gene inserted near a telomere
was grown in the presence of compounds with or without 5-FOA.
Growth was measured by light scattering at 600 nm.
30 SOLOMON ET AL.MOL. CELL. BIOL.
EX-527. NCI-H460 cells were chosen because they have pre-
viously been shown to express abundant SIRT1 protein, as well
as wild-type p53, and they have an intact p53 response to DNA
damage (24, 30). EX-527 (1 ?M) had no detectable effect on
the acetylation of lysine 382 in the absence of genotoxic insult
(data not shown). In contrast, in cells treated with the DNA
damaging agent etoposide, EX-527 produced a concentra-
tion-dependent increase in the amount of acetylated p53,
with a maximal effect at 1 ?M (Fig. 2A). Nicotinamide (5
mM, a concentration commonly used to inhibit SIRT1) was
less effective at increasing p53 acetylation than 1 ?M EX-
527 (Fig. 2A).
To eliminate the possibility that EX-527 might influence p53
acetylation via nonspecific effects on p53, several compounds
from the same chemical series as EX-527 were tested. These
compounds varied in their ability to inhibit SIRT1 in the in
vitro deacetylation assay (IC50, 124 nM to ?80 ?M). Signifi-
FIG. 2. Inhibition of SIRT1 catalytic activity increases p53 acety-
lation after DNA damage. (A) Effect of EX-527 on p53 acetylation
after DNA damage. NCI-H460 cells were treated with etoposide in
combination with either nicotinamide (Nicot.), EX-527, or dimethyl
sulfoxide vehicle (DMSO) for 6 h. Blots were probed with an anti-
acetylated p53 Lys 382 antibody (upper panel) or p53 antibody (Ab-7)
that recognizes all forms of human p53 (lower panel). (B) Effect of
EX-527 and a series of closely related compounds on p53 acetylation
after DNA damage. Cells were treated with 1 ?M concentrations of
the indicated compounds for 6 h, and samples were prepared as de-
scribed for panel A, except that cells were treated additionally with 25
nM TSA to inhibit class I/II HDAC activity. (C) Effect of enantiomers
of EX-527 on p53 acetylation after DNA damage. Cells were treated
with EX-242 or EX-243 for 6 h. p53 was immunoprecipitated and
immunoblotted as described for panel A. ?, present; ?, absent.
FIG. 3. Inhibition of SIRT1 catalytic activity enhances p53 lysine
382 acetylation after a variety of DNA-damaging agents and is not cell
type specific. (A) NCI-H460 cells were treated with adriamycin, hy-
droxyurea, or hydrogen peroxide for 6 h. p53 was immunoprecipitated
and immunoblotted with an anti-acetylated p53 Lys 382 antibody or
p53 antibody (Ab-7). (B) U-2 OS and MCF-7 cells were treated with
etoposide in the presence or absence of 1 ?M EX-527 for 6 h. Immu-
noprecipitation and immunoblotting was performed as described for
panel A. (C) HMEC were treated with etoposide or adriamycin in the
presence or absence of 1 ?M EX-527 for 6 h. Immunoprecipitation
and immunoblotting of p53 was performed as described for panel A.
Total SIRT1 levels were measured by immunoblotting using antibody
07-131 (Upstate Cell Signaling Solutions). ?, present; ?, absent.
VOL. 26, 2006 INHIBITION OF SIRT1 DOES NOT ALTER CELL SURVIVAL31
cantly, inhibition of SIRT1 catalytic activity correlated well
with the ability to increase p53 acetylation in etoposide-treated
cells when the compounds were tested at 1 ?M in the presence
of 25 nM TSA (Fig. 2B). For additional validation, the optical
isomers of EX-527 were tested, with the inactive isomer EX-
242 serving as a control for SIRT1-independent cellular effects.
One micromolar EX-243, the active isomer, enhanced the
acetylation of p53 in the presence of etoposide, whereas 1 ?M
EX-242 had no effect (Fig. 2C). These results confirm that
EX-527 regulates p53 acetylation directly by inhibition of
SIRT1 catalytic activity.
Acetylation of p53 is known to occur in response to a wide
variety of DNA damaging agents (1). To determine whether
the catalytic activity of SIRT1 plays a general role in control-
ling DNA damage-induced acetylation, NCI-H460 cells were
treated with 1 ?M EX-527 in combination with a variety of
genotoxic agents. EX-527 increased acetylation of lysine 382 in
response to adriamycin, hydroxyurea, and hydrogen peroxide
(Fig. 3A) as well as etoposide (Fig. 2). We also investigated
whether inhibition of SIRT1 with EX-527 can regulate acetyl-
p53 in other cell lines as well as primary human cells. In
addition to NCI-H460 cells, EX-527 inhibited p53 deacetyla-
tion in U-2 OS and MCF-7 cells treated with etoposide
(Fig. 3B). Both of these cell lines express SIRT1 as assessed by
immunoblotting (data not shown).
HMEC were treated with etoposide or adriamycin in the
presence or absence of EX-527 (Fig. 3C). These cells express
SIRT1, as shown by immunoblotting, and inhibition with EX-
527 increased acetylation of p53 lysine 382 in response to both
etoposide and adriamycin. These results show that SIRT1 reg-
ulates p53 acetylation in several cell types, including primary
cells, under a broad range of conditions.
Effects of inhibition of SIRT1 on p53-controlled gene ex-
pression. As p53 carries out many of its effects by functioning
as a transcription factor, for instance, increasing the expression
of p21 and BAX (26), we assessed the role of SIRT1-mediated
FIG. 4. p53-controlled gene expression is not altered by inhibition of SIRT1 in several p53-positive cell lines. (A) NCI-H460, MCF-7, U-2 OS,
and HMEC were treated with various concentrations of etoposide for 6 h in the presence of 1 ?M EX-527 or dimethyl sulfoxide vehicle (DMSO).
Levels of p21 mRNA were measured using quantitative PCR and normalized to the expression levels of hypoxanthine phosphoribosyltransferase
(HPRT). (B) NCI-H460 cells were treated with etoposide or adriamycin in the presence or absence of EX-527 for 6 h. Cell lysates were prepared
for immunoblotting and were probed with antibodies directed against p21 and tubulin. ?, present; ?, absent.
32SOLOMON ET AL.MOL. CELL. BIOL.