Molecular Biology of the Cell
Vol. 19, 1210–1219, March 2008
SIRT1 Acts as a Nutrient-sensitive Growth Suppressor and
Its Loss Is Associated with Increased AMPK and
Swami R. Narala,* Richard C. Allsopp,†Trystan B. Wells,* Guanglei Zhang,*
Prerna Prasad,†Matthew J. Coussens,†Derrick J. Rossi,‡Irving L. Weissman,‡
and Homayoun Vaziri*
*Ontario Cancer Institute, Department of Medical Biophysics, University of Toronto, Toronto, ON, M5G-2M9,
Canada;‡Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford,
CA 94305; and†Institute for Biogenesis Research, University of Hawaii, Honolulu, HI 96813
Submitted September 25, 2007; Revised December 1, 2007; Accepted December 27, 2007
Monitoring Editor: Wendy Bickmore
SIRT1, the mammalian homolog of SIR2 in Saccharomyces cerevisiae, is an NAD-dependent deacetylase implicated in
regulation of lifespan. By designing effective short hairpin RNAs and a silent shRNA-resistant mutant SIRT1 in a
genetically defined system, we show that efficient inhibition of SIRT1 in telomerase-immortalized human cells enhanced
cell growth under normal and nutrient limiting conditions. Hematopoietic stem cells obtained from SIRT1-deficient mice
also showed increased growth capacity and decreased dependency on growth factors. Consistent with this, SIRT1
inhibition was associated with increased telomerase activity in human cells. We also observed a significant increase in
AMPK levels up on SIRT1 inhibition under glucose limiting conditions. Although SIRT1 suppression cooperated with
hTERT to promote cell growth, either overexpression or suppression of SIRT1 alone had no effect on life span of human
diploid fibroblasts. Our findings challenge certain models and connect nutrient sensing enzymes to the immortalization
process. Furthermore, they show that in certain cell lineages, SIRT1 can act as a growth suppressor gene.
Loss of silencing of mating type loci in Saccharomyces cerevi-
siae (Hopper and Hall, 1975; Haber and George, 1979) led to
discovery of a gene named MAR1 (mating-type regulator 1;
Klar et al., 1979) also known as SIR2 (silent information
regulator 2; Rine et al., 1979). Increased dosage of SIR2
extends replicative lifespan in certain strains of S. cerevisiae
(Kaeberlein et al., 1999) and increases longevity of Caenorhab-
ditis elegans (Tissenbaum and Guarente, 2001). Studies also
have shown that CR (calorie restriction) may mediate lifes-
pan extension through SIR2 (Lin et al., 2000) or HST2 (Lam-
ming et al., 2005). However, newer studies have challenged
these notions and shown that CR-dependent replicative
lifespan extension occurs in a SIR2/HST2-independent man-
ner (Kaeberlein et al., 2004; 2006).
In the case of chronological lifespan in yeast, SIR2 me-
diates the opposite effect (limiting lifespan). In one study,
deletion of SIR2 promoted chronological lifespan exten-
sion under CR (Fabrizio et al., 2005). Early studies on SIR2
of S. cerevisiae suggested that SIR2 has an ADP-ribosylat-
ing activity in vitro (Moazed, 2001). This subsequently led
to uncovering an activity capable of deacetylating syn-
thetic acetylated histone substrates in vitro (Imai et al.,
2000), generating O-acetyl ADP-ribose (Tanner et al., 2000;
Tanny and Moazed, 2001). The in vivo deacetylation tar-
gets of mammalian SIR2 homolog (SIRT1) are nuclear
factors such as p53 (Luo et al., 2001; Vaziri et al., 2001,
Langley et al., 2002, Michishita et al., 2005), FOXO (Brunet
et al., 2004; Nemoto et al., 2004), Ku (Cohen et al., 2004a),
acetylated histones (Vaquero et al., 2004), and nuclear
factor (NF)-?B (Yeung et al., 2004).
More recently novel activators of SIRT1 such as HIC1 and
AROS have also been identified that activate SIRT1 and
promote deacetylation of its targets such as p53 (Chen et al.,
2005; Kim et al., 2007). SIRT1 is also suggested to act as a
nutrient sensor in response to caloric restriction (Cohen et al.,
2004b; Nemoto et al., 2004). In S. cerevisiae, Sir proteins have
been shown to have critical roles in response to DNA dam-
age and are mobilized from telomeres to sites of DNA strand
breaks (McAinsh et al., 1999; Mills et al., 1999) and are
involved in maintenance of telomeric silencing (Moretti et
al., 1994). Synthesis of de novo telomere repeats is achieved
by telomerase an enzyme originally detected as an RNP
(ribosenucleotide protein) complex in Tetrahymena (Greider
and Blackburn, 1985) and subsequently in human cells
(Morin, 1989). The mammalian telomerase is composed of a
reverse transcriptase catalytic subunit (hTERT; Harrington
et al., 1997; Meyerson et al., 1997; Nakamura et al., 1997) and
an RNA template (hTR; Feng et al., 1995). Inactivation of
telomerase in Mus musculus has revealed roles in cell sur-
vival and maintenance of genomic integrity via telomere
maintenance (Blasco et al., 1997; Lee et al., 1998). Telomere
maintenance and regulation in mammals is achieved by
collaborative effects of telomerase and telomere-binding
proteins (van Steensel and de Lange, 1997). Protective effects
of telomeres on chromosome ends may be achieved via
function of specialized protein complexes including TRF1/
This article was published online ahead of print in MBC in Press
on January 9, 2008.
Address correspondence to: Homayoun Vaziri (firstname.lastname@example.org).
1210 © 2008 by The American Society for Cell Biology
TRF2/TIN2 (Smogorzewska and de Lange, 2004) and other
single-strand G-rich telomere-binding proteins such as Pot1
that regulate accessibility of telomeres to telomerase (Bau-
mann and Cech, 2001; Colgin et al., 2003). Human diploid
fibroblasts have a finite lifespan and undergo senescence
upon completion of a fixed number of cell doublings (Hay-
flick and Moorhead, 1961). At least a part of this molecular
clock is thought to operate through telomere erosion or
dysfunction with each division in normal cells that ulti-
mately triggers initiation of cellular senescence (Harley et al.,
1990). Consistent with this model telomerase is reactivated
in immortal human cells (Counter et al., 1992; Kim et al.,
Further direct findings indicate that reconstitution of te-
lomerase activity in vivo in primary mortal human fibro-
blasts causes bypass of senescence and leads to cell immor-
tality (Bodnar et al., 1998; Vaziri and Benchimol, 1998).
Consistent with this model, human germ cells maintain their
telomeres (Allsopp et al., 1992), and human embryonic and
adult hematopoietic stem cells express telomerase (Chiu et
al., 1996). This telomerase activity in hematopoietic stem
cells is not sufficient to prevent telomere shortening and may
confer a finite self-renewing capacity (Vaziri et al., 1994).
Telomerase has since been widely used as a marker for
identification of human pluripotent stem cells (Shamblott et
Here we investigate the role of SIRT1 in regulation of
replicative life span and cell growth in primary, telomerase-
immortalized human cells and murine hematopoietic stem
cells under normal and nutrient-limiting conditions. We
designed effective short hairpin RNA (shRNA) constructs
that are able to reduce SIRT1 protein expression signifi-
cantly. By suppressing endogenous SIRT1 in human cells we
show that SIRT1 can negatively regulate cell growth, and
this is associated with an increase in telomerase activity
levels. Extension of these findings to an animal model indi-
cates that hematopoietic stem cells from mice lacking SIRT1
show a greater proliferative capacity under conditions of
stress. We propose that SIRT1 is a nutrient-sensitive growth
suppressor in certain cell types. Therefore our findings have
implications for growth of normal and immortal cells.
MATERIALS AND METHODS
Cell Culture and Cell Lines
All cell strains were grown either in DMEM ? 10% fetal bovine serum (FBS)
in 60–100-mm Petri dishes (Greiner, Frickenhausen, Germany). A rapid plas-
mid-based system (Vaziri et al., 2001) was used to generate all retroviruses
(Imgenex, San Diego, CA). In brief, pSRP (pSUPER-Retro-Puro), pSRP-
shSIRT1(HS6), pSRP-shSIRT1(HS11) and pSRP-shControl, pBabe-Ires-Neo,
pBabe-Ires-Neo-SIRT1-R, and pBabe-puro-wtSIRT1 vectors and packaging
plasmid (Imgenex) were transfected into 293T cells using Fugene 6, and
supernatants were used to infect the target cells carrying mCAT1. Cells were
typically infected with pSRP-based viruses at multiplicity of infection (MOI)
of ?20, two times sequentially and subsequently selected in 1 ?g/ml puro-
mycin or 200–400 ?g of G418. Wild-type or SIRT1-R viruses were put in at
Cell Culture in Absence of Nutrients
Initially, cells were grown in growth medium (H21 medium, Invitrogen,
Carlsbad, CA; cat no 12800) with 10% FBS (Invitrogen) under low density in
60-mm dishes. Twenty-four hours later, the exponentially dividing cells were
washed once with phosphate-buffered saline (PBS; ?Ca and ?Mg). Media on
the cells was subsequently changed with 4 ml of ?-MEM without glucose and
serum (89-5118EF, Invitrogen, with base media to which aspargine, arginine,
methionine, isoleucine, l-valine, and ascorbic acid with antibiotics were added;
OCI, Toronto, Ontario, Canada, Media Department). Duplicate dishes were
used to estimate the total number of cells in the plates. For each cell line the
cells were trypsinized, neutralized by addition of ?-MEM without glucose
with 2% FBS, and counted on a hemocytometer using trypan blue exclusion.
The average live cell count was then calculated. Cell counts were performed
every 24 h after the addition of the ?-MEM without glucose. Cells were
collected at different time points (0, 4, 8, 12, and 15 h) after addition of ?-MEM
without glucose and subjected to lysis as described below under immuno-
TRAP (telomere repeat amplification protocol) assays were performed as
previously described (Kim and Wu, 1997). Typically, within 2–10 population
doublings (PDs) after selection, CHAPS lysates were prepared from cells, and
aliquots were frozen. For rescue experiments cells from ?PD 93 were used to
prepare lysates. On thawing, the lysates were subjected to protein quantifi-
cation using the quick-start Bradford assay system (Bio-Rad, Hercules, CA).
Twenty-six–cycle PCR-TRAPs were performed in linear range of the assay
using 50–300 ng of total protein lysate per reaction. TRAP products were
resolved on 15% polyacrylamide large gels and exposed to phosphorimager
Design of SIRT1 shRNA Expression Vectors,
shRNA-resistant Silent Mutant
More than 12 shRNAs were designed to find the most effective set. The most
effective we developed was HS6 (Qiagen, Chatsworth, CA). The second
sequence (HS11) was based on a published sequence (Ota et al., 2006).
The SIRT1 shRNA sequences (bold) used as insert in pSRP (pSuper-Retro-
Puro, OligoEngine, Seattle, WA) vector were as follows: HS6: GATC-
TCAAGAGATGAGGAGGTCAACTTCATCTTTTTA. The control shRNA
sequence was as follows: GATCCCCTTCTCCGAACGTGTCACGTTTCAA-
A PCR-based strategy was used to introduce six silent mutations in the
SIRT1 region targeted by the HS6 shRNA (for sequence, see Supplementary
Figure 1A). The resulting mutant named SIRT1-R was subcloned in the
PBabe-INeo vector. This vector PBIN-SIRT1-R and the backbone (PBIN)
were subsequently used to infect puromycin-resistant target cells express-
ing pSRPshControl and pSRPshSIRT1(HS6) for a genetic rescue experi-
Cells were harvested by trypsinization (0.05%) and neutralized with either
DMEM ? 10% FBS (for nutrient experiments, MEM without glucose ? 2%
FBS). Cells were spun and washed in PBS?/?twice, and the pellets were
lysed in 0.5% NP40, 150 mM NaCl, and 50 mM Tris in presence of 1?
complete miniprotease inhibitor mix (Roche, Indianapolis, IN; 10? stock, 1
tablet in 10 ml water), for 30 min with occasional vortexing. Cell lysates were
centrifuged at 12,000 rpm for 20 min at 4°C. Protein content of lysates was
measured by Bio-Rad Quick Start protein assay (500–0201). Protein, 10–50
?g, was resolved on NuPAGE (Novex, Encinitas, CA) 4–12% Bis-Tris gradient
gels, transferred to PVDF membranes (Bio-Rad) and blocked in 5% skim milk.
The membrane was incubated in: 1:5000 dilution for anti-SIRT1 (Vaziri et al.,
2001), 1:500 for 2 h for hTERT (Santa Cruz Biotechnology, Santa Cruz, CA;
H-231: SC-7212), 1:20,000 for ?-actin (Abcam, Cambridge, MA) 10–20 min,
1:2000 dilution of Phospho-AMPK-? (Thr172)(40H9) and total AMPK-?
(23A3) (Cell Signalling, Beverly, MA; kit 9957) for 2 h. For AMPK experiments
membranes were first immunoblotted with total anti-AMPK-? antibody, and
the levels were measured. To prevent residual carry over, the membrane was
subsequently stripped and after testing for clearance was subjected to the
phospho-AMPK-? antibody for detection of active form.
The membrane was washed twice in 0.05% TBST buffer for 20 min. Perox-
idase conjugated AffinPure goat anti-rabbit horseradish peroxidase IgG
(H?L) secondary antibody or anti-mouse (Jackson ImmunoResearch, West
Grove, PA) were used at a concentration of 1:30,000 for 45 min in 1% milk was
used. After washing, the membrane was then incubated with Super signal
west, dura, or femto maximum substrate (Pierce, Rockford, IL) for 2 min and
exposed to film for up to 30 min.
Cells (107) were cross-linked in plates by addition of 1% formaldehyde for
10 min, followed by the addition of glycine to a final concentration of 0.125
M to stop the cross-linking reaction. Hela-pSRP-controlshRNA and Hela-
pSRPshSIRT1 cells (n ? 107) were used per immunoprecipitation reaction
mixture. Cells were washed twice in PBS and lysed in 1 ml of cell lysis
buffer (5 mM PIPES, pH 8.0, 85 mM KCl, 0.5% NP-40, 1? protease inhibitors)
on ice for 10 min. The nuclei were pelleted at 5000 rpm and lysed in nuclei
lysis buffer (50 mM Tris, pH 8.1, 10 mM EDTA, and 1% SDS, including
protease inhibitors on ice for 10 min. The chromatin was sonicated eight
times, 15 s each on ice. The samples were precleared by incubating with 20 ?l
of blocked protein G agarose beads (Roche) containing 1.5 ?g of sea urchin
sonicated sperm DNA for 15 min. The protein-chromatin complexes were
incubated with no antibody, 3 ?l of antiacetylated histone H4 antibody
(06-866; Upstate Biotechnology, Lake Placid, NY), anti-SIR2 antibody (2 ?l), or
rabbit serum (2 ?l) at 4°C overnight. Each reaction mixture was then incu-
Suppression of Cell Growth by SIRT1
Vol. 19, March 2008 1211
bated with 20 ?l of protein G beads for 30 min at room temperature. The
protein G agarose beads were pelleted, and the supernatant from the no-
antibody sample was used as total input chromatin (input). The protein G
agarose pellets were washed twice in dialysis buffer (2 mM EDTA, 50 mM
Tris, pH 8.0) and four times in immunoprecipitation (IP) wash buffer (100 mM
Tris, pH 9.0, 500 mM LiCl, 1% NP-40, 1% deoxycholic acid). The protein-
chromatin complexes were eluted from the protein G agarose beads twice in
IP elution buffer (50 mM NaHCO3, 1% SDS), followed by reverse cross-linking
in 0.3 M NaCl along with 1 ?g of RNase-A at 67°C for 5 h. The reactions were
precipitated with 2.5 volumes of ethanol at ?20°C overnight. The reaction
mixtures were then centrifuged at 13,200 rpm for 20 min, and the pellets were
air-dried and resuspended in 100 ?l of Tris-EDTA–proteinase K buffer (final
reaction concentrations, 10 mM Tris, pH 7.5, 5 mM EDTA, 0.25% SDS, and
proteinase K (1 U) and incubated at 45°C for 2 h. Subsequently, the samples
were purified by phenol-chloroform extraction. NaCl (final concentration of
0.14 M), and 2.5 volumes of ethanol were then added, and the samples were
allowed to precipitate overnight at ?20°C. The samples were centrifuged at
13,200 rpm for 20 min, and the pellets were air dried and resuspended in 50
?l of water. Two microliters of the purified DNA was used for each PCR. In
addition the input DNA was diluted 1:20, and the same volume was used in
the PCR reaction. The PCR was performed with the following primers:
Forward : 5?-acgtggcggagggactg, and Reverse: 5?-gccagggcttcccacgt.
PCR conditions were as follows: 94°C for 3 min, followed by 32 cycles at;
94°C for 0.45 min; 65°C for 0.30 min; and 72°C for 0.30 min. The ChIP
(chromatin immunoprecipitation) PCR products were analyzed on a 2%
agarose gel and analyzed using the Bio-Rad imaging system.
Population Doubling Assays
Primary BJ cells infected with pSRPshControl and pSRPshSIRT1(HS6) were
grown in DMEM ? 10% FBS and were subjected to a standard replicative
lifespan assay. Late passage BJ fibroblasts strain ?7 PDs away from senes-
cence was infected with pM-hTERT-IRES-EGFP vector (MSCV-based vector,
Weinberg lab). Immediately after green fluorescent protein (GFP) was ex-
pressed these BJT cells were either infected with pSRP, pSRP-shControl, or
pSRP-shSIRT1(HS6) viruses. After selection in 1 ?g of puromycin for 4 d, the
resistant cells were split and grown for a standard population-doubling
For RT-PCR analysis, Trizol reagent was used to purify total RNA from cells.
First-strand cDNA synthesis was performed as described by manufacturer
(Amersham Biosciences, Piscataway, NJ). The sequence of primers used is
described elsewhere (Nakamura et al., 1997). The resulting cDNA were quan-
tified on a Turner fluorometer, and equal DNA amounts were used for the
PCR amplification. PCR amplification was performed using 25 cycles in
presence of a32P-labeled forward hTERT primer. Products were resolved on
15% polyacrylamide gels and exposed to phosphoimager screens, and bands
were quantified using Image Quant (Molecular Dynamics, Sunnyvale, CA/
Amersham). hTERT signals were normalized to the GAPDH signal. Quanti-
tative-PCR on an ABI 7900HT sequence detection system (Applied Biosys-
tems, Foster City, CA) with SYBR Green chemistry (Qiagen). The cDNA
preparation was similar to that of RT-PCR use; however, the template was
used at a final concentration of 500 ng/reaction in a 20 ?l total reaction
volume. Each sample had been run through the Q-PCR (quantitative PCR)
analysis in triplicate on freshly synthesized cDNA, using a no-template
negative control for each sample set of cDNA and primers. Each 20-?l
reaction contained 10 ?l of SYBR Green master mix, 2 ?l of template cDNA or
water, 1 ?l of forward and reverse primer mix at 0.6 ?M each/reaction, and
7 ?l of nuclease-free water.
Hematopoietic Stem Cell Analysis and Culture
The Sirt1 knockout strain (from Dr. Fred Alt, Harvard Medical School) was
back-crossed five times onto a C57BL6 background before performing this study.
In all experiments, young mice (3–9 wk old) were used. Mice were fed with a
standard diet and maintained in a temperature- and light-controlled room (228C,
14L:10D; light starting at 0700 h), in accordance with the guidelines of the
Laboratory Animal Services at the University of Hawaii and the Committee on
Care and Use of Laboratory Animals of the Institute of Laboratory Resources
National Research Council (DHEW publication 80-23, revised in 1985). The
protocol for animal handling and treatment procedures was reviewed and ap-
proved by the Animal Care and Use Committee at the University of Hawaii.
Hematopoietic stem cells (HSCs) were analyzed using flow cytometry as previ-
ously described (Allsopp et al., 2002; Rossi et al., 2005; Yilmaz et al., 2006). Briefly,
whole bone marrow (WBM) was flushed from the tibia and femur bones, and
cells were stained with antibodies to c-Kit, Sca-1, plus a lineage cocktail, as well
as either antibodies to Flk2 and CD34, or CD150 (SLAM). All analysis and cell
sorting was performed on a FACS Aria (Becton-Dickinson). For HSC culture,
complete media consisted of X-Vivo 15 media (BioWhittaker, Walkersville, MD)
plus 5 ? 10?5M 2-mercaptoethanol, Steel factor (10 ng/ml), IL-3 (30 ng/ml), IL-6
(10 ng/ml), IL-11 (10 ng/ml), Tpo (10 ng/ml), and Flt3 ligand (10 ng/ml). Cells
were cultured in standard tissue culture incubators at 5% CO2.
Inhibition of SIRT1 and Telomerase Activity
Low hTERT-expressing primary human BJT diploid fibro-
blasts, Hela cells, and Lovo cells were infected with pSRP-
shSIRT1(HS6), pSRP-ShSIRT1(HS11), and pSRP-shControl
(a control shRNA). Cells were selected in puromycin and
kept under selection for the remainder of experiments.
SIRT1 expression was effectively reduced to varying degrees
in all cell types by shSIRT1 (Figure 1, A–D). The HS6 shRNA
was more effective than HS11 (Figure 1D). In all cell lines in
which SIRT1 shRNA was stably expressed, we observed an
increase in telomerase activity as measured by TRAP (Figure
1, E–G). As an additional control we ran the TRAP reaction
in the presence of an internal control for which similar
results was observed (Figure 1H). Furthermore, in order to
rule out the possibility of off-target effects, we took two
strategies. First, we used a second shRNA (HS11) for SIRT1
suppression (Figure 1, D and I), and most importantly we
designed a shRNA-resistant SIRT1 gene, (SIRT1-R) in which
we introduced six silent mutations in the region targeted by
HS6. Expression of this shRNA-resistant mutant (Supple-
mentary Figure 1B) of SIRT1 in BJT cells blocked the ability
of shSIRT1(HS6) to induce telomerase activity (Figure 1J). It
is noteworthy to mention that we found that higher than
physiological quantities of wild-type SIRT1 or a SIRT1H363Y
mutant can lead to enhancement or suppression of telomerase
activity, respectively (Supplementary Figure 1C). Hence by
using two independent shRNAs and a genetic rescue experi-
ment we show that SIRT1 suppression is associated with an
increase in telomerase activity and is not due to off-target
SIRT1 Suppression and hTERT
To determine the mechanism through which SIRT1 sup-
presses hTERT activity, we performed RT-PCR and im-
munoblotting in shSIRT1-expressing cells. When SIRT1
was suppressed in Hela cells, an ?3-fold increase in the
level of hTERT protein was observed (Figure 2A), and this
was accompanied by a 0.3-fold increase in the levels of
hTERT mRNA both by in-gel RT-PCR (Figure 2B) and an
insignificant but reproducible increase of 0.3–0.5-fold by
real-time quantitative PCR (Figure 2, C and E). We con-
clude that SIRT1 controls endogenous and exogenous
hTERT expression possibly at the level of RNA stability
and/or through changes in chromatin structure at the
hTERT promoter. To test this model, we performed ChIP
experiments 240 nucleotides upstream of ATG in the
hTERT promoter (Figure 2F). We used a pan-acetyl anti-
body against acetylated histone H4 and found that Hela
cells in which SIRT1 was suppressed contained more total
acetylated H4 on hTERT promoter than control cells ex-
pressing endogenous SIRT1. Furthermore, a small amount
of SIRT1 was associated with hTERT promoter (Figure 2F)
in control cells but not in SIRT1 knockdown cells. These
results indicate that there is a transcriptional component
(albeit small) to the observed effect. When we performed
effective knockdown of SIRT1 in BJ-hTERT cells, we found
that compared with controls, the BJ-hTERT-pSRP-SIRT1
cells showed a slower migrating band (Figure 2D). Al-
though this suggests a posttranslational component by
acetylation in stabilization of hTERT, further experimen-
tation is required to show that the effect is directly
through posttranslational modification of hTERT.
S. R. Narala et al.
Molecular Biology of the Cell1212
Control- no lysate
Control- no lysate
Control- no lysate
Control- no lysate
1 2 3 4 5 6 7 8 9 10 11
Control- no lysate
were infected with a control shRNA expressing retrovirus pSRP-shControl or SIRT1 knockdown virus pSRP-shSIRT1(HS6). Cell lysates
were subjected to immunoblotting using an anti-SIRT1 antibody or a ?-actin antibody. (B) Suppression of SIRT1 in Lovo cells. The same
retroviral vectors as in A and in analysis were used. (C) Suppression of SIRT1 in Hela cells. The same retroviral constructs as in A were
Effect of SIRT1 suppression on telomerase activity in human cells. (A) Suppression of SIRT1 in BJ-hTERT cells (BJT). BJT cells
Suppression of Cell Growth by SIRT1
Vol. 19, March 20081213
SIRT1 Inhibition Cooperates with hTERT to Promote Cell
Growth under Normal and Low Nutrient Conditions
Having shown that SIRT1 suppression is associated with
increased telomerase activity, we wanted to determine if
SIRT1 and telomerase functionally cooperate in a replicative
lifespan assay in human cells. The shRNA-mediated repres-
sion of SIRT1 in primary BJ fibroblasts did not affect repli-
cative lifespan in a long-term assay (Figure 3A). Further-
more, no significant effect on replicative lifespan was
observed when SIRT1 was overexpressed (Figure 3B). Next,
we asked if SIRT1 repression affected the growth of ectopic
hTERT-expressing BJ cells. We first introduced hTERT in the
same primary BJ cells, and then subjected these telomerase-
positive cells to infection with the shSIRT1(HS6), control
shRNA virus, or the backbone virus (pSRP). We observed that
the population doubling time of cells expressing shSIRT1 was
significantly reduced compared with cells infected with pSRP
or pSRP-shControl (Figure 3, C and D) and that the endoge-
nous SIRT1 was effectively repressed in the shSIRT1-express-
ing cells (Supplementary Figure 1D.). Hence the ability of
SIRT1 to control the growth of BJ cells is observed only in the
presence of hTERT expression. Ectopic expression of SIRT1-R
hTERT and shSIRT1(HS6) (Figure 3D). Hence the results of this
genetic rescue experiment indicate that the effect of the SIRT1
shRNA in enhancement of cell growth is specific to SIRT1 and
is independent of any off-target effects of the shRNA used.
Effect of Glucose Withdrawal on SIRT1-depleted BJT Cells
When cells are exposed to glucose withdrawal they are known
to undergo cell cycle arrest followed by death. When we ex-
posed BJT-pSRP-shControl and BJT-pSRP-shSIRT1(HS6) cells
expressing telomerase to nutrient withdrawal by exposing
them to media containing no glucose, we found that BJT cells
expressing ectopic telomerase with no SIRT1 expression could
survive and divide much longer in the initial phases of glucose
withdrawal (Figure 3E). Although both control and knock-
down cells died at approximately the same time (5th day;
Figure 3E). In the control BJT cells, the levels of SIRT1 were
gradually increased after glucose depletion and activated
AMPK levels gradually increased with time. However, in
SIRT1 suppressed BJT cells subjected to glucose withdrawal
there was a significant increase early on in total AMPK levels
and phosphorylated AMPK protein levels at 4–8 h (AMPK-?
Thr 172 phosphorylation).
Increased Proliferative Capacity of Hematopoietic Stem
Cells in Animals Lacking SIRT1
To extend our findings to a more physiological system, we
assayed the effect of SIRT1 deficiency on the establishment
of the primitive HSC compartment, by quantitating the total
number of HSCs and multipotent progenitors in BM from
young (3–9 wk) Sirt1 knockout (Sirt11?/?) and control mice
by flow cytometry using rigorous cell surface criteria for
isolating HSCs and progenitor cells (Rossi et al., 2005) (Sup-
plementary Figure 1). These analyses showed that establish-
ment of neither the HSCs nor multiprogenitor subsets were
significantly impacted in the absence of Sirt1 (Figure 4A).
Similar results were observed when alternative markers for
isolating HSCs were used (Kiel et al., 2005; not shown).
These results suggest that Sirt1 does not play an important
role in establishing HSC homeostasis in young adult mice
housed in a stress-free environment.
To assess the capacity of young Sirt1-deficient HSCs to
proliferate in response to mitogenic stimuli, we purified
HSCs from Sirt1?/?and control mice by fluorescence-acti-
vated cell sorting, cultured the cells in cytokine-rich media,
and then quantitated the total number of progeny cells
generated after 7 d (Figure 4B). Strikingly, these experiments
revealed that the Sirt1?/?HSCs exhibited a three- to fivefold
(?20,000 cells) increased proliferative capacity compared
with Sirt1?/? HSC controls (Figure 4B). To address the
capacity of Sirt1-deficient HSCs to proliferate under condi-
tions of nutrient deprivation, we clone sorted HSCs from
Sirt1?/?or Sirt1?/?mice into individual wells of Terasaki
plates containing cytokine-deprived media and monitored
the number of wells in which cell proliferation could be
detected (i.e., wells containing two or more cells). As shown
in Figure 4, a significantly greater number of Sirt1?/?HSCs
were capable of proliferating in media in the presence of
single cytokines with either IL-3 (Figure 4C) or SCF (Figure
4D) compared with control HSCs, indicating that Sirt1-defi-
cient HSCs have a greater capacity than controls to prolifer-
ate under these restrictive conditions.
To determine whether Sirt1 expression per se is affected
by cell proliferation, we purified HSCs, as described above,
and either immediately isolated RNA from resting HSCs
(HSC-R), or cultured HSCs in complete media for 4 d before
RNA isolation from actively cytokine stimulated HSCs
(HSC-S). As shown in Figure 4E, real-time RT-PCR analysis
of Sirt1 mRNA levels relative to Hprt reveals a small (about
twofold) but significant increase in Sirt1 levels in cytokine
stimulated HSCs. Although this result suggests that Sirt1
expression may be cell cycle dependent in HSCs, it is im-
portant to note that cytokine-stimulated HSCs undergo ex-
tensive differentiation in vitro. Thus it remains to be deter-
mined to what extent the regulation of Sirt1 levels has
physiologically relevant affects on the proliferation of HSCs
Here we present results showing that SIRT1, the NAD-
utilizing deacetylase enzyme is a negative regulator of
growth under normal and restrictive conditions in certain
cell lineages. Consistent with this notion, efficient inhibition
of SIRT1 deacetylase was associated with an increase in
telomerase activity that is required for survival and long-
term cell growth. Our data indicate that the effect of SIRT1
on telomerase activity is mediated through the catalytic
Figure 1 (cont).
control shRNA retrovirus and two shSIRT1 (HS6) and shSIRT1
(HS11) constructs.(E) Parental BJ cells expressing ectopic pM
(MSCV)-hTERT-IEGFP were infected with pSRP-shControl RNA or
pSRP-shSIRT1 (HS6) virus. Within 6 PDs after selection in puromy-
cin, cells were lysed in CHAPS lysis buffer, and equal protein
quantities (50 and 300 ng) were subjected to TRAP analysis to
determine telomerase activity. Control RNase and heat treatments
all contained 300 ng of protein lysate. (F) Lovo cells were infected
twice with the viruses and were subjected to TRAP analysis. Protein
amounts of 50, 200, and 600 ng were used in the TRAP reaction. (G)
Same as F except Hela cells were used. (H) Same as in G except
internal controls were included using HeLa cell extracts (10, 50, and
200 ng). (I) A second shRNA (HS11) was used to suppress SIRT1 in
Hela cells and TRAP analysis was performed as in H. (J) Same cells
as in A (BJ-hTERT-IEGFP with and without shRNA against SIRT1)
were infected with pBabe-neo (PBN) control vector or with an
SIRT1-R) generating four additional lines. Two protein concentra-
tions (50 and 200 ng) from four cell line were subjected (total of 16)
to the TRAP analysis. The first six lanes are experimental controls.
Lanes 7 and 8 are results of rescue experiments. The last three lanes
are negative controls for the TRAP reaction.
used. (D) Suppression of SIRT1 in Hela cells using
S. R. Narala et al.
Molecular Biology of the Cell1214
subunit of telomerase, hTERT. On SIRT1 inhibition there is a
small increase in hTERT mRNA level and a significant in-
crease in levels of hTERT protein. This increase in mRNA
correlated with lack of SIRT1 at proximal regions of hTERT
promoter and an increase in total H4 acetylation at the
hTERT promoter. Cell lines expressing either endogenous
hTERT under its native promoter or primary human dip-
loid fibroblasts expressing ectopic hTERT showed in-
creased levels of hTERT protein and activity upon SIRT1
suppression. We find that the suppression of SIRT1 and its
effects on telomerase are independent of how hTERT is
expressed (i.e., under native or ectopic viral promoters).
Interestingly, we also observed an hTERT doublet in BJT
cells in which SIRT1 was expressed suggesting a post-
translational role for SIRT1 in regulation of hTERT protein
stability. However, further experimentation is required to
investigate if this is caused by increased hTERT acetyla-
The increased telomerase activity and cell growth pheno-
type observed could be rescued by a silent mutant SIRT1-R
protein that is resistant to repressive effect of shRNA di-
rected to SIRT1, showing that the effect observed was spe-
cific. Our results point toward a functional interaction be-
tween SIRT1 and hTERT; however, the basis for this genetic
interaction is unknown, and it is possible that the effect of
SIRT1 on hTERT is not direct and is mediated via other
proteins. Furthermore, given the diverse range of SIRT1
targets the effects observed on hTERT maybe one factor
that contributes to the observed cellular phenotype.
multicellular protostomes such as C. elegans. We reasoned that
if the lifespan-inducing functions of the mammalian SIR2 ho-
molog SIRT1 is conserved, this should reflect itself in either
survival or replicative lifespan of vertebrate cells with long life
spans, such as somatic cells of Homo sapiens.
When we overexpressed wild-type SIRT1 in mortal normal
human diploid BJ fibroblasts, we observed no significant effect
on replicative lifespan, consistent with published data (Mich-
ishita et al., 2005). We also performed the reverse experiments
by suppressing SIRT1 to near detection limits in primary BJ
fibroblasts, and we still did not observe any effects on replica-
tive life span. Because it has been shown before by us and
others that ectopic expression of hTERT and reconstitution of
its activity causes life span extension in human cells, we rea-
soned that inhibition of SIRT1 may have an effect on telomer-
ase-induced extension of lifespan. If primary BJ cells were first
infected with an hTERT-expressing virus and sequentially
were subjected to SIRT1 inhibition, there was an increased
efficiency in cell growth reflected by a decrease in the popula-
tion doubling time. This effect could be mediated through
telomeres or other indirect effects on cell survival. It is however
clear from our data that SIRT1 suppression promotes cell
growth in the presence of ectopic telomerase activity. Our
findings in human cells are consistent with that of others who
have shown that murine fibroblasts deficient for Sirt1(Sir2?)
have a higher frequency of immortalization (Chua et al., 2005).
In contrast, others have shown that in different cell types such
BJT pSRP shControl
- No Ab
- Input - Input
suppression on hTERT protein in Hela cells. Western
blot analysis was performed on cell lysates, and they
were subjected to immunoblotting with anti-SIRT1,
anti-hTERT, and anti-?-actin antibodies. (B) Effect of
SIRT1 suppression on hTERT mRNA in Hela cells.
Total RNA was isolated from Hela-pSRP-shControl
and Hela-pSRPshSIRT1 cells and hTERT mRNA was
quantified by quantitative radioactive in-gel PCR as
described (Nakamura et al., 1997). The ratio of hTERT/
GAPDH is shown. (C) Quantification of hTERT
mRNA in Hela and Hela-pSRPshSIRT1 cells by real-
time Q-PCR. The values shown are normalized to an
internal GAPDH control. (D) Regulation of hTERT
protein in BJT cells. BJT-pSRP-shControl and BJT-
pSRPshSIRT1 cell lysates were resolved on 4–12%
gradient gels, and immunoblotting was performed us-
ing an anti-hTERT rabbit antibody. Primary BJ cells in
the first lane were used as negative control. (E) Effect
of SIRT1 suppression on hTERT mRNA in BJT cells.
Same as in C except that BJT and BJT-pSRPshSIRT1
cells were used. (F) ChIP of hTERT promoter using the
antibodies shown. Hela control and SIRT1(HS6)
knockdown cells were used in each ChIP reaction as
shown. Antibodies used were against total acetylated
H4 and SIRT1. Controls were rabbit serum (RS) and no
antibody reactions. For details consult Materials and
Regulation of hTERT. (A) Effect of SIRT1
Suppression of Cell Growth by SIRT1
Vol. 19, March 2008 1215
0 20 406080 100120140
Population Doublings (PDs)
0 20 4060 80100 120
Population Doulbings (PDs)
pSRP cont shRNA
0 4 8 12 15
0 4 8 12 15
0 1020 30405060 7080
Population Doulbings (PDs)
pM-hTERT-IEGFP + pSRP ShSIRT1(HS6)
0 2040 6080 100 120
Population Doulbings (PDs)
1 2 3 4 5 6
BJT+p SRP-ControlBJT+pSRP-shSIRT1 (HS6)
1 2 3 4 5 6
lifespan of primary BJ fibroblasts. Late-passage BJ cells (?7 PD before senescence) were infected with either pSRP-shControl or pSRPshSIRT1
(HS6) and pSRP control shRNA vector viruses and cells were subjected to selection in puromycin. (B) Overexpression of wild-type SIRT1 in
primary BJ fibroblasts. Same as in A, except for the overexpression constructs (pBabe-Puro-wtSIRT1) and pBabe-Puro control vector were
used. (C) Effects of SIRT1 knockdown on growth of BJT (hTERT-IRES-EGFP) fibroblasts. Late-passage BJ fibroblasts were infected with an
hTERT containing virus, and the resulting BJT cells were subsequently infected by pSRP-shControl or BJ-pSRPshSIRT1 (HS6) viruses and
subjected to a standard replicative lifespan assay. (D) Rescue of the biological effect of shRNA by an shRNA-resistant mutant. BJT cells were
infected with pSRP-shControl or BJ-pSRPshSIRT1(HS6) viruses and subsequently infected with the rescue construct PBN-SIRT1-R or PBN
control alone. Cells were kept under puromycin and neomycin selection throughout the experiments. BJT cells and their rescue counterparts
generated were subjected to a long-term replicative assay as before to asses the ability of SIRT-R to rescue the phenotype of BJT cells
expressing the SIRT1 shRNA. (E) BJT cells expressing control of SIRT1 shRNA were subjected to glucose withdrawal on day 0 and cell
viability was measured for a week. Experiments were performed in duplicate dishes. Error bars, SEM. (F) Same strains as in E were subjected
to glucose withdrawal and at the shown time point (hours after withdrawal), and cell lysates were prepared and subjected to immunoblotting
with an anti-SIRT1, anti-phosphor-AMPK-? (Thr-172), total AMPK-? and ?-actin antibodies.
Cooperative effects of SIRT1 knockdown and hTERT expression on cell growth and survival. (A) SIRT1 suppression effects on
S. R. Narala et al.
Molecular Biology of the Cell1216
as endothelial cells SIRT1 suppression has the opposite effect:
its loss induces cell cycle arrest (Ota et al., 2007). Given the
range of substrates currently identified for SIRT1 and their
increasing number, it is possible that the contradicting growth-
promoting and growth-suppressing properties observed are
cell type or species specific.
Extension of our in vitro results to hematopoiesis under
adverse conditions caused by lack of growth factors is con-
sistent with the notion that SIRT1 is a growth suppressor.
Although we observed no appreciable difference in HSCs or
progenitor frequencies in young Sirt1?/?mice, the in vitro
proliferative capacity of Sirt1-deficient HSCs were signifi-
cantly elevated in both complete media and under cytokine-
deprived conditions containing a single growth factor. These
results were consistent with that of immortalized human BJT
cells lacking SIRT1 expression that showed higher prolifer-
ation under normal or glucose-deprived conditions. We
found that consistent with the role of activated AMPK in
response to low glucose (Salt et al., 1998), cells lacking SIRT1
showed an earlier peak in both total levels and activated
phospho-AMPK-? protein upon glucose deprivation. Ac-
tivation of AMPK hence may allow survival in response to
an energy shortage. Although this finding suggests that
SIRT1 may regulate AMPK, others have found that induc-
tion of AMPK by the SIRT1 activator resveratrol is SIRT1
independent (Dasgupta and Milbrandt, 2007). Although a
useful marker of energy status and survival, AMPK in-
duction observed here maybe due to a complex and indi-
rect effect of SIRT1 on cell survival under ATP-limiting
ciency on HSCs and progenitor frequency and
proliferation. (A) Analysis of the affect of Sirt1
deficiency on HSCs and progenitor numbers.
Bone marrow was harvested, red blood cells
were lysed, and the remaining white cells
were stained with fluorophor-conjugated an-
tibodies to markers for HSCs and progenitors
(see Materials and Methods for details). Multi-
potent progenitors and short-term and long-
term HSCs are defined as the c-Kit?Sca-
1?LinnegFlk2?CD34?fraction, the c-Kit?Sca-
WBM, respectively. The average total cell
number for each type of cell, normalized to
body weight, is shown (for each bar, n ? 3).
Error bars, SD. (B) Quantitative analysis of cell
numbers after 1 wk of growth in complete
media. BM was stained as described in Mate-
rials and Methods, and 100 HSCs were sorted
directly into individual wells of a 48-well plate
containing complete media. Bars represent av-
erage counts from five wells. HSCs are defined
as the c-Kit?Sca-1?SLAM(CD150)?LinnegFlk2neg
fraction of WBM. (C) Left, graph represents
read out over time of Terasaki plates contain-
ing single HSCs per well. The HSCs were
sorted into Terasaki plates containing serum
free X-Vivo media plus IL-3, and plates were
monitored daily for the number of wells con-
taining proliferating cells (i.e., more than one
cell). Right, graph represents average value of
frequency of proliferating HSCs from four
Terasaki plates. HSCs are defined as the
tion of WBM. For all experiments, mice were
3–9 wk old. p values represent results from
Student’s t test. (D) Same experiments as in C
were performed except that cells were sorted
into media containing Steel factor. (E) Analy-
sis of Sirt1 mRNA levels in resting and prolif-
erating HSCs. HSCs (n ? 1000) were purified
from young adult mice (n ? 3), and RNA was
either extracted immediately for analysis of
resting HSCs (HSC-R) or cells were stimulated
to proliferate in media (X-Vivo15 serum-free
media [Stem Cell] plus 20 ng/ml Steel factor,
10 ng/ml IL-6, 30 ng/?l IL-3, 2 mM l-Glu, and
50 ?M mercaptoethanol) for 5 d for analysis of
proliferating HSCs (HSC-S). Both T-cells (CD-
3?B220negMac1neg) and B-cells (B220?CD-3negMac1neg) were purified from the bone marrow as a reference. RNA was extracted using
Trizol, cDNA was synthesized using Superscript III (Invitrogen), and real-time PCR was performed using primers specific for Hprt
(reference) and Sirt1 yielding single amplicons of 100 and 150 bp, respectively. Forty cycles of PCR was performed in triplicate for all samples
using an iCycler real-time PCR machine (Bio-Rad). For each cell type, the average level of Sirt1 is shown, relative to Hprt.
Analysis of the effect of Sirt1 defi-
Suppression of Cell Growth by SIRT1
Vol. 19, March 20081217
It is possible that under nutrient-restrictive conditions,
SIRT1 acts as a growth suppressor to limit division in high-
capacity progenitor cells. This limitation may be a physio-
logical response to save on usage of macromolecules re-
quired for survival of pre-existing stem cells. Hence, SIRT1
can modulate the division and survival capacity of stem cells
in response to nutrient availability. Our results have signif-
icant implications for survival of adult stem cells under
stress and would be of interest to examine whether SIRT1
has similar effects in other types of stem cells. They also
indicate that specific chemical inhibitors of SIRT1 may en-
hance survival or pluripotency in adult or embryonic human
or murine stem cells.
Evidence suggests that calorie restriction is associated
with decreased age-associated tumor incidence (Weindruch,
1992). Furthermore, the beneficial biological effects of calorie
restriction in increasing lifespan have been well docu-
mented. Therefore, it is possible that in human cells, calorie
restriction can increase SIRT1 activity, which in turn can
suppress immortalizing genes such as telomerase. There-
fore increased SIRT1 activity would then suppress tumor
incidence and therefore only indirectly leads to extension
of lifespan. Hence the effects of induction of molecules
such as SIRT1 on longevity of complex multicellular ver-
tebrates may be mediated indirectly via stimulating its
tumor suppressor functions and hence reduce death due
to cancer. We predict that overexpression of SIRT1 in mice
would primarily result in suppression of certain types of
tumors. Based on our results and models, SIRT1 overex-
pression may have no functional effect on the network of
human genes promoting somatic cell chronological/rep-
licative survival, leading directly to increased longevity.
Current lack of a unifying evolutionary conservation in
longevity functions of SIR2 however should not detract
from its fundamental roles in cellular survival and growth
from yeast to mammals.
Our findings underscore the importance of nutrient-de-
pendent pathways and propose that SIRT1 is a nutrient-
sensitive growth suppressor that may act as an important
barrier to retard the growth of certain nutrient-sensitive
immortal tumor cells.
We thank Dr. Samuel Benchimol and Dr. Norman Iscove for comments on the
manuscript. The SIRT1 knockout mice used in this study were a kind gift from
Dr. Fred Alt. S.N., G.Z., and T.W. performed all experiments in Figures 1, 2,
and 3 and Supplementary Figure 1. R.A., M.C., P.P., and D.R. performed
experiments in Figure 4 and Supplementary Figure 2. This work was sup-
ported by an operating grant from the Canadian Institutes of Health Research
and Canada Research Chair program (H.V.) and National Institutes of Health
Grant P20 RR16467-05 (R.A.). Infrastructure support was provided by Cana-
dian Foundation for Innovation to H.V.
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