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HEMATOPOIESIS AND STEM CELLS

Salidroside stimulates DNA repair enzyme Parp-1 activity in mouse HSC

maintenance
Xue Li,1,2 Jared Sipple,1 Qishen Pang,1,3 and Wei Du1
1Division
of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 2College of Life Sciences, South China
Normal University, Guangzhou, China; and 3Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH

Salidroside is a phenylpropanoid glyco-
side isolated from the medicinal plant
Rhodiola rosea, which has potent antioxi-
dant properties. Here we show that
salidroside prevented the loss of hemato-
poietic stem cells (HSCs) in mice under
oxidative stress. Quiescent HSCs were
recruited into cell cycling on in vivo chal-
lenge with oxidative stress, which was
blocked by salidroside. Surprisingly,
salidroside does not prevent the produc-
tion of reactive oxygen species but re-
duces hydrogen peroxide–induced DNA-
strand breaks in bone marrow cells
enriched for HSCs. We tested whether
salidroside enhances oxidative DNA dam-
age repair in mice deficient for 5 DNA
repair pathways known to be involved in
oxidative DNA damage repair; we found
that salidroside activated poly(ADP-
ribose)polymerase-1 (PARP-1), a compo-
nent of the base excision repair pathway,
in mouse bone marrow HSCs as well as
primary fibroblasts and human lympho-
blasts. PARP-1 activation by salidroside
protects quiescent HSCs from oxidative
stress–induced cycling in native animals
and self-renewal defect in transplanted
recipients, which was abrogated by ge-
netic ablation or pharmacologic inhibi-
tion of PARP-1. Together, these findings
suggest that activation of PARP-1 by
salidroside could affect the homeostasis
and function of HSCs and contribute to
the antioxidant effects of salidroside.
(Blood. 2012;119(18):4162-4173)

Introduction
Hematopoietic stem cells (HSCs) are a rare population of
pluripotent cells that can self-renew and differentiate into
various types of cells of the blood lineage.1 Under steady
physiologic conditions, the most primitive HSCs are in a
quiescent state and reside in the bone marrow (BM) niche where
they preserve the capacity to self-renew and to continue to
produce all types of blood cells throughout a prolonged life span
without depleting the regenerative cell pool.2,3 In response to
stress or stimulation, the HSCs can move out of the BM niche,
entering cell cycle and undergoing division. In addition, the
cycling HSCs may return to the BM niche and regain their
quiescent state.4 Disruption of HSC quiescence prematurely
exhausts the stem cell pool and causes hematologic failure under
various stresses, such as oxidative stress, cell cycling, and
aging.5,6
Oxidative stress, defined as an imbalance between the produc-
tion of reactive oxygen species (ROS) and antioxidant defense, is
most evident in states of aging and diseases such as BM failure and
cancer.7,8 Even in a healthy state, HSCs are exposed to various
ROS, which are routinely generated during metabolic or inflamma-
tory process.9 ROS induce a variety of responses in HSCs,
including cellular proliferation and apoptosis.10,11 ROS can also
cause DNA damage and drive HSCs into cell division, which is
essential for DNA repair processes.12,13 Similar to stem cells from
other tissues, HSCs have developed several mechanisms to prevent
the damage induced by oxidative stress. Antioxidant enzymes,
including superoxide dismutases, catalase, glutathione peroxi-
dases, and peroxiredoxins, can directly eliminate ROS.10 Other
cellular enzymes can function to repair DNA damage induced by
ROS in hematopoietic tissues.8,14 There is strong evidence that
HSCs are activated and thus functionally exhausted by oxidative
stress. Mice with mutations in the ATM or FOXO genes, as well as
various DNA repair genes, exhibit premature exhaustion of HSCs
because of accumulation of ROS or DNA damage, indicating that
cellular balance between ROS and antioxidant defense, as well as
DNA repair, is crucial for the maintenance of HSC self-renewal and
hematopoietic function.15,16
Salidroside is a phenylpropanoid glycoside isolated from the
medicinal plant Rhodiola rosea that grows in high altitude or
cold regions of the world and was used as a folk medicine in
France, Germany, and many European countries to fight fatigue
in the 19th century.17 In present days, extract of R rosea has been
used to enhance both the physical and mental performance.18,19
Salidroside (2-[4-hydroxyphenyl]ethyl ␤-D-glucopyranoside) has
been reported to have anti-aging, anti-cancer, anti-inflammatory,
and antioxidative functions.20-23 A recent study shows that
salidroside promotes erythropoiesis and up-regulates the level
of antioxidative enzymes glutathione peroxidase-1 and thiore-
doxin-1 to counteract oxidative stress.24 In this study, we show
that salidroside prevented quiescent HSCs and progenitor cells
from being recruited into cell cycling on in vivo challenge with
oxidative stress in mice. Using several mouse models deficient
for DNA repair pathways known to be involved in oxidative
DNA damage repair (ODDR), we demonstrate that salidroside
protects quiescent hematopoietic stem progenitor cells (HSPCs)
from oxidative stress–induced cycling through stimulation activ-
ity of poly(ADP-ribose)polymerase-1 (PARP-1), a component
of the base excision repair pathway.

Submitted October 20, 2011; accepted March 6, 2012. Prepublished online as Blood The publication costs of this article were defrayed in part by page charge
First Edition paper, March 16, 2012; DOI 10.1182/blood-2011-10-387332. payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement. © 2012 by The American Society of Hematology

4162 BLOOD, 3 MAY 2012 䡠 VOLUME 119, NUMBER 18

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BLOOD, 3 MAY 2012 䡠 VOLUME 119, NUMBER 18
Methods
Mice and treatments
Parp1⫺/⫺ or Xpc⫺/⫺ mice were generated by backcrossing 129S-
Parp1tm1zqw/J or B6;129-Xpctm1Ecf/J (The Jackson laboratory), respectively,
with wild-type (WT) C57BL/6 mice. Fancd2⫺/⫺ mice (provided by
Dr Markus Grompe, Oregon Health & Science University) or DNA-
PKcs3A/3A mice (a gift from Benjamin P. C. Chen, University of Texas
Southwestern Medical Center at Dallas)25 were generated by interbreeding
heterozygous Fancd2⫾ or DNA-PKcs⫹/3A mice, respectively. Brca2⫺/⫺
mice were generated by Cre-mediated deletion of floxed alleles by crossing
Brca2tm1Brn (NCI)26 with a Cre-ERT2 strain.27
For H2O2 treatment, mice were first screened with increasing doses of
H2O2 (Sigma-Aldrich; 0, 0.05, 0.15, 0.25, 0.35, and 0.50 ␮mol/g body
weight), and that the optimal dose (0.25 ␮mol/g body weight) was chosen
for further experiments. For antioxidant treatment, mice were injected with
salidroside (PhytoLab; 75␮g/g body weight), manganese (III) tetrakis
(4-benzoic acid) porphyrin (MnTBAP, Sigma-Aldrich; 10 ␮g/g body
weight), N-acetyl-L-cysteine (NAC; Sigma-Aldrich; 50 ␮g/g body weight)
or saline vehicle, intraperitoneally followed by H2O2 (0.25 ␮mol/g body
weight). For NU1025 treatment, mice were injected with NU1025 (25 mg/kg
body weight; Sigma-Aldrich) followed by H2O2 treatment. All experimental
procedures conducted in this study were approved by the Institutional Animal
Care and Use Committee of Cincinnati Children’s Hospital Medical Center.
Parp1 activity
Parp1 activity was detected by flow cytometry as previously described.28
Briefly, cells were centrifuged and resuspended in 100% ethanol and left at
⫺20°C for at least 20 minutes. Cells were then resuspended in ⬃10 mL
buffer A (10mM Tris-HCl pH 7.8, 1mM EDTA [ethylenediaminetetraacetic
acid], 4mM MgCl2, and 30mM 2-mercaptoethanol). Then cells were
centrifuged again and resuspended in buffer A again and transferred to a
V-shaped 96-well plate on ice for at least 5 minutes. Then 20 ␮L of 3X
reaction buffer (with or with NAD⫹) plus 13 ␮L of 15mM NaCl
incorporating were added to the reaction mix followed by 37°C incubation
for 10 minutes. Then second fixation was done by adding 60 ␮L of 4%
formaldehyde/phosphate-buffered saline (PBS) for 20 minutes at room
temperature. PBS was then added to quench the reaction. Cells were then
centrifuged and resuspended in 100 ␮L primary antibody diluted in
fluorescence-activated cell sorter (FACS) buffer and incubated at 37°C for 1
hour or overnight at 4°C. Then the cells were washed and resuspended in
100 ␮L of diluted secondary antibody (Alexa 488–conjugated goat
anti–mouse; Invitrogen) followed by 37°C incubation for 30 minutes. Cells
were washed and resuspended for flow cytometry analysis.
For flow cytometry and cell cycle analysis, BrdU incorporation,
determination of ROS production, comet assay, and BM transplantation, see
supplemental Methods (available on the Blood Web site; see the Supplemen-
tal Materials link at the top of the online article).
Results
Salidroside prevents oxidative stress–induced HSC loss in
mice
In an attempt to search for new chemopreventive and antioxidant
agents that are effective and less toxic in hematopoietic improve-
ment for patients with BM failure syndromes, such as Fanconi
anemia (FA), in which oxidative stress is identified as a physiologic
mediator of HSC loss,7 we investigated the antioxidant effect of
salidroside on HSC maintenance. Salidroside (2-[4-hydroxyphenyl-
]ethyl ␤-D-glucopyranoside; supplemental Figure 1A), a phenylpro-
panoid glycoside found in the medicinal plant R rosea, has a wide
range of biologic activities, such as antiaging, anticancer, anti-
inflammatory, and antioxidative functions.20-23 To test the effect of
SALIDROSIDE STIMULATES PARP-1 ACTIVITY 4163
salidroside on oxidative stress in HSC in mice, we first determined
the optimal dose of hydrogen peroxide (H2O2) and found that
0.25 ␮mol/g body weight was the most effective dose that
effectively induced DNA damage without causing death (supple-
mental Figure 1B). In addition, this range of H2O2 doses gave an
inverse correlation between DNA damage and HSC repopulation
(supplemental Figure 1C). We then used this dose of H2O2 to treat
the mice and found that salidroside effectively antagonized H2O2-
induced effect on WT HSCs in vivo (Figure 1). Although both
salidroside and H2O2 treatments did not change absolute number of
total BM cells (supplemental Figure 3), H2O2-treated mice had a
much higher frequency of Lin-c-kit⫹Sca-1⫹ (LSK) cells in the BM
than untreated mice, and salidroside partially limited this expansion
(Figure 1A-B). Further analysis of the LSK compartment indicated
that H2O2-treated mice had much lower LSK CD34⫺Flt3⫺ cells, a
population enriched for long-term (LT)–HSCs,29,30 than untreated
control mice (Figure 1C-D). Significantly, salidroside almost
completely abrogated H2O2-induced loss of LT-HSCs. We also
determined the effect of salidroside on the BM HSC compartment
using the CD150 and CD48-based immunophenotyping.31,32 Simi-
larly, we observed that salidroside effectively rescued LT-HSCs
(CD150⫹CD48⫺ LSK) in H2O2-treated mice (Figure 1E-F). More-
over, salidroside protected H2O2-induced HSC loss in mice defi-
cient for the FA gene, Fanca (Fanca⫺/⫺) or Fancc (Fancc⫺/⫺;
supplemental Figure 2). Together, these data suggest that salidro-
side plays a selective role in the maintenance of LT-HSCs.
Salidroside enhances the ability of stressed HSCs to
repopulate mouse hematopoietic system
We next assessed whether salidroside improved the repopulating
ability of oxidative stressed HSCs. We transplanted LSK cells from
H2O2-treated WT C57BL/6 mice (CD45.2⫹) pretreated with or
without salidroside, along with BM cells (depleted of c-Kit⫹ cells
to provide short-term hematopoiesis after irradiation) obtained
from congenic WT Boy J mice (CD45.1⫹), into lethally irradiated
congenic recipients (CD45.1⫹). We observed a significant increase
in both repopulated hematopoiesis in the peripheral blood (Figure
2A) and HSC-enriched LSK cells in the BM of recipients that
received LSK cells from salidroside-treated mice (Figure 2B) at
4 months after transplantation. We also performed BM transplanta-
tion using total BM from stressed WT mice, which shows similar
results (supplemental Figure 4). Further, we performed competitive
reconstitution using limiting dilutions of LSK cells from stressed
mice with defined ratios of WT LSK competitor cells, and analyzed
hematopoietic reconstitution in recipient mice by donor cells at
8 and 16 weeks after transplantation. The results show that
donor-repopulated hematopoiesis was greater in the recipients that
received all doses of LSK cells from salidroside-treated mice than
in those that received LSK cells from untreated mice (Figure 2C).
Furthermore, the recipients transplanted with cells from salidroside-
treated mice showed greater abundance of donor-derived LSK cells
in the BM (Figure 2D). Analysis of the frequency of CD45.2⫹
lymphoid and myeloid as well as erythroid lineage cells in the
peripheral blood showed that both groups of donor LSK cells had a
similar multilineage-reconstitution capacity (supplemental Figure
5), indicating that salidroside does not affect cell fate or differentia-
tion potential of HSCs. To investigate the long-term-repopulation
abilities of salidroside-conditioned HSCs, we performed serial
transplantation experiments by transplanting primary recipients of
salidroside-treated or untreated mouse BM at 16 weeks after
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4164 LI et al
transplantation into lethally irradiated secondary recipients. Six-
teen weeks after transplantation, we analyzed hematopoiesis de-
rived from donor (CD45.2⫹) cells. The results show that donor-
derived hematopoiesis from primary recipients of salidroside-
treated BM was significantly greater in the secondary recipients
than that from primary recipients of untreated BM (Figure 2E).
Notably, this was correlated with an increased number of LSK cells
in the bone marrow (Figure 2F). Together, these results demon-
strate that salidroside enhances the ability of stressed HSCs to
repopulate the hematopoietic system.
Salidroside prevents oxidative stress–induced HSC cycling in vivo
To identify the mechanism of salidroside in HSC maintenance, we
asked whether salidroside prevented oxidative stress–induced
BLOOD, 3 MAY 2012 䡠 VOLUME 119, NUMBER 18
Figure 1. Salidroside prevents oxidative stress–
induced HSC loss. (A) salidroside partially limits H2O2-
induced LSK expansion. BM cells from WT C57BL/6
mice pretreated with or without salidroside (75␮g/g body
weight) followed by H2O2 (0.25 ␮mol/g body weight)
injection were harvested for LSK (Lin-Sca1⫹c-kit⫹) cell
frequency assessment using flow cytometry. (B) Quantifi-
cation of LSK frequency in mice described in panel A.
(C) SD prevents H2O2-induced LSK CD34-Flt3- cell loss.
BM cells described in panel A were subjected to clow
cytometry analysis for LSK CD34⫺Flt3⫺ frequency.
(D). Quantification of LSK CD34⫺Flt3⫺ frequency in mice
described in panel A. (E) SD prevents H2O2-induced LSK
CD150⫹CD48⫺ cell loss. BM cells described in panel A
were subjected to Flow Cytometry analysis for LSK
CD150⫹CD48⫺ frequency. (F) Quantification of
CD150⫹CD48⫺ frequency in mice described in panel A.
Results are means ⫾ standard deviation (SD) of
3 independent experiments (n ⫽ 9 per group).
apoptosis of HSCs in stressed animals. We observed increased
apoptosis of LSK cells in H2O2-treated WT C57BL/6 mice
compared with untreated mice, and salidroside partially reduced
the stress-induced apoptosis (Figure 3A). However, H2O2 treatment
did not induce significant apoptosis in the more primitive CD34⫺
LSK cell compartment29 and salidroside had no effect on the
apoptosis in the cell population (Figure 3B). This finding suggests
that the loss of HSCs in stressed mice may be because of the
recruitment of quiescent HSCs into cell cycle on challenge with
oxidative stress. To test whether salidroside played a role in
preventing quiescent HSCs from entering cell cycling or prolifera-
tion, we first analyzed the cell cycle profile of CD34⫺ LSK cells by
staining RNA and DNA with pyronin Y and Hoechst 33342,
respectively. Quiescent cell populations can be identified by
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BLOOD, 3 MAY 2012 䡠 VOLUME 119, NUMBER 18 SALIDROSIDE STIMULATES PARP-1 ACTIVITY 4165

Figure 2. Salidroside enhances HSC repopulation.

(A) Salidroside enhances the repopulating ability in recipi-
ent mice. LSK (Lin-Sca1⫹c-kit⫹) cells from WT C57BL/6
mice (CD45.2⫹) pretreated with or without salidroside
(75␮g/g body weight) followed by H2O2 (0.25 ␮mol/g
body weight) treatment were isolated by cell sorting using
FlowAria II. One thousand sorted LSK cells plus 1 million
c-kit–depleted competitors (CD45.1⫹) were injected to
lethally irradiated recipients. Donor chimerism was exam-
ined at 4 months after transplantation using peripheral
blood from recipients. Representative images (left) and
quantifications (right) were shown. Results are means
⫾ SD of 3 independent experiments (n ⫽ 15 per group).
(B) Salidroside increases HSC-enriched LSK cells in
recipient mice. One thousand sorted LSK cells from WT
C57BL/6 mice (CD45.2⫹) pretreated with or without
salidroside followed by H2O2 treatment were injected
along with 1 million c-kit–depleted competitors (CD45.1⫹)
to lethally irradiated recipients. BM cells from recipient
mice were harvested and subjected to flow cytometry
analysis for LSK frequency. (C) Salidroside enhances
competitive reconstitution of stressed HSCs. Various
numbers of donor (CD45.2⫹) LSK cells (50, 100, or
1000) from stressed WT C57BL/6 mice were mixed
with 1000 competitor LSK (CD45.1⫹) cells and the
mixtures were transplanted intravenously into lethally
irradiated congenic (CD45.1 ⫹) recipients. Donor-
derived hematopoietic reconstitution was analyzed by
flow cytometry 8 and 16 weeks after transplantation
using peripheral blood from recipients. (D) Salidroside
increases HSC-enriched LSK in recipient mice. BM
cells from recipient mice described in panel C were
subjected to flow cytometry analysis for LSK fre-
quency. (E-F) Salidroside enhances LT-HSC repopula-
tion. BM cells from primary recipient mice described in
panel B were used for secondary transplantation by
injecting 5 ⫻ 106 BM cells to lethally irradiated recipi-
ents. Donor-derived chimerism (E) and LSK (F) were
analyzed by flow cytometry 16 weeks after BMT.
Results are means ⫾ SD of 2 independent experi-
ments (n ⫽ 10 per group).

negative to low pyronin Y staining; whereas actively cycling cells
are positive for pyronin Y.33 There was a marked decrease in the
number of quiescent (pyronin Y-negative; G0) and an increase in
the number of cycling (pyronin Y-positive; G1⫹S) CD34⫺ LSK
cells in H2O2-treated mice compared with the unstressed animals
(Figure 3C). Salidroside treatment significantly decreased cycling,
and at the same time, increased quiescent (G0) CD34⫺ LSK cells in
the stressed mice. We also performed in vivo BrdU labeling in mice
to determine the proliferative status of HSCs in the BM. In line
with the cell-cycle data, the percentage of CD34⫺ LSK cells that
incorporated BrdU was elevated in H2O2-treated mice compared
with the unstressed animals, which was largely abrogated by
salidroside (Figure 3D). These data suggest that salidroside pre-
vents quiescent HSCs from oxidative stress–induced cycling.
Salidroside reduces H2O2-induced DNA-strand breaks in
HSPCs
Because salidroside has antioxidative activity,20,21,24 we tested
whether salidroside could function as a free radical scavenger in
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4166 LI et al BLOOD, 3 MAY 2012 䡠 VOLUME 119, NUMBER 18

Figure 3. Salidroside prevents HSC cycling in vivo. (A) Salidroside partially reduces H2O2-induced LSK cell apoptosis. BM cells were harvested from WT C57BL/6 mice
pretreated with or without salidroside (75␮g/g body weight) followed by H2O2 (0.25 ␮mol/g body weight), and gated for LSK population. Apoptotic LSK cells were determined by
annexin-V and 7AAD staining. (B) Salidroside and H2O2 have no effect on apoptosis of CD34-LSK cells. CD34 negative LSK cells were gated for apoptosis analysis.

BM HSCs of mice stressed with H2O2, which is a potent producer
of reactive oxygen species (ROS). We measured ROS by staining
BM CD34⫺ LSK cells from H2O2-injected mice with CM-H2
DCFDA, a cell-permeable fluorescent dye that reacts to a broad
spectrum of ROS. To accurately evaluate the effect of salidroside
on ROS, we used 2 small antioxidant molecules, NAC, and
manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP).
NAC stimulates the formation of the endogenous reducing agent
glutathione, which cells use to scavenge H2O2.10 MnTBAP has
catalytic activities similar to the ROS-scavenging enzymes super-
oxide dismutase (SOD) and catalase.34 We pretreated the mice with
salidroside, NAC, or MnTBAP, and then concomitantly with H2O2.
Compared with NAC and MnTBAP, both eliminated most of the
ROS generated in the BM LSK cells of H2O2-injected mice,
salidroside did not prevent the production of ROS in these cells
(Figure 4A). This finding suggests that salidroside antagonizes
oxidative stress through a distinct mechanism that does not involve
ROS-scavenging.
To further elucidate the action of mechanism by salidroside, we
determined whether salidroside played a role in preventing oxida-
tive DNA damage in HSPCs of the H2O2-treated mice. We used the
comet assay35 using a Fpg-FLARE (fragment length analysis using
repair enzymes) assay kit, which measures specifically oxidative
DNA damage including single and double-strand DNA breaks.36
There was significant accumulation of DNA damage in BM Lin-
cells freshly isolated from H2O2-treated mice, which was largely
eliminated by salidroside, as well as by NAC or MnTBAP (Figure
4B). Consistent with this, H2O2 induced expression of ␥-H2AX, a
robust indicator of DNA strand breaks,37 and all 3 antioxidants
suppressed ␥-H2AX expression in BM cells from H2O2-treated
mice (supplemental Figure 6). A similar increase in 8-oxo-
deoxyguanosin (8-oxodG), an established marker of oxidative
DNA damage,9 was also demonstrated in BM cells from H2O2-
injected mice (Figure 4C). Cotreatment of H2O2-injected mice with
salidroside, or the ROS scavenger NAC or MnTBAP reduced the
accumulation of 8-oxodG (Figure 4C).
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BLOOD, 3 MAY 2012 䡠 VOLUME 119, NUMBER 18 SALIDROSIDE STIMULATES PARP-1 ACTIVITY 4167

Figure 3. (continued) (C-D) Salidroside prevents HSC cycling in vivo. Cells described in panel B were subjected to Hochest and pyronin Y (C) or BrdU (D) staining.
Representative images (top) and quantifications (bottom) were shown. Results are means ⫾ SD of 3 independent experiments (n ⫽ 9 per group).

To distinguish the antioxidant property of salidroside from that
of NAC or MnTBAP in the context of oxidative DNA damage
repair, we pretreated fresh-isolated low-density BM cells with
salidroside, NAC, or MnTBAP for 2 hours, followed by H2O2 for
additional 2 hours, and then measured the remaining amounts of
8-oxodG and DNA strand breaks for a period of 12 hours after
H2O2 treatment. There was significant difference between the effect
of salidroside and that of NAC or MnTBAP in terms of the kinetics
of 8-oxodG removal. Specifically, the level of oxidative DNA
damage was not decreased until 4 hours after salidroside treatment
with almost no 8-oxodG remaining at 12 hours (Figure 4D).
However, NAC and MnTBAP reduced the level of 8-oxodG at each
time point after H2O2 incubation. This result indicates that the ROS
scavengers NAC and MnTBAP have no effect on the repair of
8-oxodG, which is consistent with their function in reducing ROS
formation and preventing DNA damage. We observed similar
repair kinetics of DNA strand breaks in a comet assay, in which
DNA strand breaks remained high in salidroside-treated cells
during 8 hours after H2O2 incubation (supplemental Figure 7).
Together, these results suggest that salidroside may use different
mechanism from that of the ROS scavenger NAC or MnTBAP in
repair of oxidative DNA damage.
Salidroside stimulates PARP-1 activity
To identify the mechanism by which salidroside antagonists the effect of
oxidative stress on HSC maintenance, we next tested whether salidro-
side enhanced ODDR in mice deficient for 5 different DNA repair
pathways known to be involved in ODDR, including homologous
recombination (HR), nonhomologous end-joining (NHEJ), base exci-
sion repair (BER), nucleotide excision repair (NER), and FA path-
ways.7,38-40 We observed reduction of H2O2-induced DNA strand breaks
by salidroside in BM Lin- cells from mice deficient for genes function-
ing in HR (Brca2⫺/⫺),41 NHEJ (DNA-PKcs3A/3A),42 NER (Xpc⫺/⫺),43 and
FA (Fancd2⫺/⫺)44 pathways (Figure 5A). However, salidroside failed to
reduce H2O2-induced DNA strand breaks in HSPCs from mice deleted
for the gene encoding poly(ADP-ribose)polymerase-1 (PARP-1), a
component of the BER pathway (Figure 5A). Analysis of H2O2-induced
8-oxodG in these gene-knockout BM cells showed similar results, with
salidroside accelerating clearance of the oxidative DNA adducts in
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4168 LI et al
Brca2⫺/⫺, DNA-PKc3A/3A, Fancd2⫺/⫺, and Xpc⫺/⫺ cells but having no
effect in Parp-1⫺/⫺ cells (Figure 5B). These results suggest that
salidroside may specifically target PARP-1 in response to oxidative
DNA damage. To test this notion, we used a flow-cytometric assay to
determine the effect of salidroside on the enzyme activity of PARP-1 in
BM LSK cells from WT or knockout mice deficient for each of the
5 repair pathways. We found that salidroside stimulated PARP-1 activity
in cells from all except Parp-1 knockout mice treated with or without
H2O2 (Figure 5C). We next determined whether salidroside stimulated
PARP-1 activity in other cell types. We isolate mouse embryonic
fibroblasts (MEFs) from WT mice and cotreated the cells with salidro-
side and H2O2. We observed similar stimulation of PARP-1 activity by
BLOOD, 3 MAY 2012 䡠 VOLUME 119, NUMBER 18
Figure 4. Salidroside reduces DNA strand breaks but
not ROS. (A) Salidroside does not reduce H2O2-induced
ROS. WT C57BL/6 mice were pretreated with one dose
of salidroside (75␮g/g body weight), NAC (50␮g/g body
weight), or MnTBAP (10␮g/g body weight), followed by
H2O2 (0.25 ␮mol/g body weight). BM cells were then
harvested and labeled with CM-H2DCFDA for flow cytom-
etry analysis of ROS in the LSK-gated cells. Representa-
tive images (left) and quantifications (right) were shown.
(B) Salidroside reduces DNA strand breaks. BM cells
from mice described in panel A were isolated by magnetic
beads depletion, and then subjected to analysis for DNA
strand breaks by the comet assay. The mean tail moment
of untreated vehicle sample is expressed as 100%.
Larger tail moment represents higher levels of DNA
damage. For each treatment, 50 cells were scored from
random sampling. (C) Salidroside reduces 8-oxodG.
Protein extractions were prepared using low density BM
cells from mice described in panel A. Whole cell lysates
were then subjected to SDS-PAGE and immunoblotted
with Western Blot antibodies for 8-oxodG and ␤-actin.
(D) Repair kinetics. Low-density BM cells were pre-
treated with salidroside (250␮M), NAC (500␮M), or
MnTBAP (250␮M) for 2 hours, followed by H2O2 for
additional 2 hours, and then released for indicated time
intervals. Whole cell lysates were prepared and sub-
jected to SDS-PAGE and immunoblotted with antibodies
for 8-oxodG and ␤-actin.
salidroside in these MEFs (supplemental Figure 8A). We also found that
salidroside enhanced PARP-1 activity in human lymphoblasts (supple-
mental Figure 8B). To substantiate these observations, we performed
immunoprecipitation to determine the stimulatory effect of salidroside
on PARP1 activity in vivo. We cotreated fresh-isolated BM cells with
H2O2 and salidroside, and subjected the cell lysates to immunoprecipita-
tion with PAR antibodies followed by Western blot with PARP1
antibodies. Consistent with the results obtained from the flow-
cytometric assay, we observed enhanced PARP1 poly-ADP-ribosylation
by salidroside in both stressed and unstressed cells (Figure 5D).
Together, these results indicate that salidroside reduces oxidative DNA
damage through stimulation of PARP-1 activity.
 From www.bloodjournal.org by guest on September 29, 2018. For personal use only.

BLOOD, 3 MAY 2012 䡠 VOLUME 119, NUMBER 18 SALIDROSIDE STIMULATES PARP-1 ACTIVITY 4169

Figure 5. Salidroside stimulates PARP-1 activity. (A) Salidroside fails to reduce H2O2-induced DNA strand breaks in Parp-1⫺/⫺ cells. Lin⫺ cells were isolated from Brca2⫺/⫺,
DNA-PKcs3A/3A, Fancd2⫺/⫺, Parp-1⫺/⫺, or Xpc⫺/⫺ mice. Cells were treated with or without salidroside, NAC, or MnTBAP in the presence of H2O2, and subjected to Comet
Assay. The mean tail moment of H2O2-treated WT sample is expressed as 100%. For each sample, 50 cells were scored from random sampling. (B) Salidroside fails to reduce
H2O2-induced 8-oxodG in Parp-1⫺/⫺ cells. Protein lysates were prepared from low-density BM cells treated as described in panel A. Whole cell lysates were subjected to
SDS-PAGE and immunoblotted with antibodies against 8-oxodG and ␤-actin. (C) Salidroside stimulates Parp-1 activity. Low-density BM cells isolated from mice described in
panel A were treated with or without H2O2 and salidroside, and BM cells were isolated and subjected to flow cytometry analysis for Parp-1 activity in the LSK population using
antibody against PARP-1. (D) Salidroside activates Parp-1 in vivo. Low-density BM cells were isolated from WT mice, and treated with or without SD and H2O2 for 2 hours.
Whole cell lysates were subjected to immunoprecipitation using PAR antibody. Precipitated samples were resolved by SDS-PAGE and blotted with antibody specific for
PARP-1.

Inhibition of PARP-1 abrogates the effect of salidroside on HSC
maintenance
We next sought to test whether PARP-1 activation by salidroside
protected quiescent HSCs from oxidative stress–induced cycling.
We took 2 approaches: genetic ablation of the Parp-1 gene and
pharmacologic inhibition of PARP-1 enzymatic activity in oxida-
tive stressed mice to further elucidate the specific action of
salidroside in PARP-1 stimulation. Compared with WT mice in
which salidroside increased quiescent HSC pool significantly,
salidroside had no discernible effect on oxidative stress–induced
HSC cycling in Parp-1⫺/⫺ mice (Figure 6A). To substantiate this
finding, we made use of a specific small molecule inhibitor of
PARP-1, 8-hydroxy-2-methylquinazolinone (NU1025).45 Consis-
tent with PARP-1 knockout, NU1025 treatment abolished salidroside-
mediated increase in quiescent HSC frequency in stressed mice
(Figure 6B). As expected, both salidroside and NU1025 had no
 From www.bloodjournal.org by guest on September 29, 2018. For personal use only.

4170 LI et al BLOOD, 3 MAY 2012 䡠 VOLUME 119, NUMBER 18

Figure 6. Inhibition of PARP-1 abrogates the effect of salidroside on HSC maintenance. (A) Deletion of Parp-1 abolishes salidroside-mediated increase in quiescent HSC
frequency in stressed mice. Parp1⫺/⫺ mice as well as their WT littermates were pretreated with or without salidroside (75 ␮g/g body weight) followed by H2O2 (0.25 ␮mol/g body
weight). BM cells were subjected to flow cytometry analysis for quiescent HSC (G0 phase). Results are means ⫾ SD of 3 independent experiments (n ⫽ 9 per group).
(B) NU1025 treatment abolishes salidroside-mediated increase in quiescent HSC frequency in stressed mice. H2O2-treated (0.25 ␮mol/g body weight) WT mice were treated
with or without salidroside (SD; 75␮g/g body weight) and NU1025 (25 mg/kg body weight). BM cells were subjected to flow cytometry analysis for quiescent HSC. Results are
means ⫾ SD of 3 independent experiments (n ⫽ 9 per group). (C) The maintenance of HSC quiescence by salidroside requires Parp-1. 1000 LSK cells from WT or Parp1⫺/⫺
mice were injected to lethally irradiated WT recipients. Recipient mice were then subjected to salidroside and H2O2 treatments. Cycling donor-derived (CD45.2⫹) cells were
assessed by Flow Cytometry analysis by pyronin Y staining. Representative images (top) and quantifications (bottom) were shown. Results are means ⫾ SD of 3 independent
experiments (n ⫽ 9 per group). (D) The enhancing effect of salidroside on the long-term repopulation abilities of stressed HSCs requires Parp1. Cells from primary recipients
described in panel C were used for second transplantation by injecting 10 million whole BM cells to lethally irradiated WT recipients. Donor-derived cells were determined by
flow cytometry analysis. Representative images (top) and quantifications (bottom) were shown. Results are means ⫾ SD of 3 independent experiments (n ⫽ 9 per group).

effect on oxidative stress–induced HSC cycling in Parp-1⫺/⫺ mice To provide functional evidence that HSC maintenance by
(supplemental Figure 9). Collectively, these results provided con- salidroside under oxidative stress requires PARP-1, we assessed the
vincing evidence to show that protection of quiescent HSCs by ability of salidroside to preserve the self-renewal and hematopoi-
salidroside from oxidative stress–induced cycling requires PARP-1. etic reconstitution capacity of HSCs in the context of PARP-1
 From www.bloodjournal.org by guest on September 29, 2018. For personal use only.
BLOOD, 3 MAY 2012 䡠 VOLUME 119, NUMBER 18
function. We transplanted BM LSK cells from WT or Parp-1⫺/⫺
mice into lethally irradiated WT recipient mice, which were treated
with salidroside and H2O2 at 4 months after transplantation. On
injection with H2O2, we observed that cell cycle progression
induced by oxidative stress was not prevented by salidroside in
donor-derived HSCs deficient for Parp-1 (Figure 6C), which
suggests that the action of salidroside in maintaining quiescent
HSCs requires PARP-1. To determine whether the effect of
salidroside on the long-term repopulation abilities of HSCs also
required PARP-1, we did serial transplantation experiments by
transplanting BM cells from the salidroside and H2O2-treated
primary recipients of Parp-1⫹/⫹ or Parp-1⫺/⫺ BM cells into
lethally irradiated WT secondary recipients. Four months after
transplantation, we obtained peripheral blood from the secondary
recipient mice and analyzed hematopoietic reconstitution derived
from donor (CD45.2⫹) cells. Our analysis indicated that salidroside
failed to enhance donor-derived hematopoiesis in recipients trans-
planted with Parp-1⫺/⫺ HSCs (Figure 6D). These findings suggest
that the effect of salidroside on the maintenance of quiescent HSCs
under oxidative stress requires PARP-1.
Discussion
Oxidative stress has been linked to aging and cancer as well as
other major human health problems. A promising strategy for
preventing oxidative damage to the cell is to use readily available
natural compounds isolated from vegetables, fruits, and herbs.
Many of the phytochemicals have already been identified to have
chemopreventive potential, capable of intervening in tumorigen-
esis. We investigated the potential of the natural antioxidant
salidroside as a new chemopreventive and antioxidant agent that
has therapeutic value for patients with BM failure or leukemia.
Salidroside has been reported to have antiaging, anticancer, anti-
inflammatory, and antioxidative functions.20-23 In this study, we
demonstrate that salidroside promotes the maintenance of mouse
HSCs under oxidative stress. There are several findings that
highlight the significance of our study: first, salidroside prevents
quiescent HSCs from oxidative stress–induced activation; second,
salidroside does not function as a ROS scavenger but enhances
oxidative DNA damage repair through a mechanism involving
stimulation of repair enzyme PARP-1 activity; and third, PARP-1
activation by salidroside protects quiescent HSCs from oxidative
stress–induced cycling and repopulating defect. Thus, salidroside
plays a significant role in inhibiting oxidative DNA damage; and
thereby regulates the homeostasis of HSCs.
Quiescence has been postulated to prevent HSC exhaustion.3 In
the BM, HSCs are kept in a low proliferative, relatively quiescent
state within the BM microenvironment termed niches. Whereas
numerous molecular factors that contribute to quiescence exist in
the HSCs and the BM niche, HSCs are inevitably exposed to stress,
such as accumulation of ROS and DNA damage, which can drive
HSCs into uncontrolled cell-cycle entry and excessive prolifera-
tion. Indeed, we found that on challenge with oxidative stress,
quiescent HSCs were recruited into cell cycle, evidenced by a
marked decrease in the number of quiescent and an increase in the
number of cycling CD34⫺ LSK cells in H2O2-treated mice
compared with the unstressed animals. We confirmed this finding
by showing elevated BrdU-positive CD34⫺ LSK cells in H2O2-
treated mice. Functionally, we demonstrate that oxidative stress
impaired long-term repopulation abilities of HSCs. These results
are consistent with the recent reports describing functional exhaus-
tion of HSCs, because of uncontrolled accumulation of ROS within
SALIDROSIDE STIMULATES PARP-1 ACTIVITY 4171
the HSC population, in mice deficient in several oxidative damage
response and repair pathways.15,46,47 It is in this context that we
identified salidroside as a potent small molecule that prevents
exhaustion of HSCs from oxidative stress–induced uncontrolled
cell-cycle entry and excessive proliferation.
Another interesting finding of our study is that unlike other
antioxidants, such as NAC, MnTBAP, and quercetin that function
as free radical scavengers, salidroside does not prevent the
production of ROS but acts to eliminate oxidative DNA damage in
BM cells enriched for HSCs, as demonstrated by a robust reduction
of DNA-stranded breaks and expression of ␥-H2AX and 8-oxodG.
More importantly, we present evidence of distinct kinetics of
8-oxodG clearance by salidroside compared with those by the ROS
scavenger NAC or MnTBAP. That is, the effect of salidroside on
repair kinetics consistently lags those of NAC and MnTBAP. We
propose that salidroside acts directly in the process of oxidative
DNA damage repair. Oxidative DNA damage by increased ROS
accumulation in HSCs has been documented in several recent
studies using age-related murine HSCs15,16 and more recently
human HSCs.13 Although these studies suggest a correlation
between oxidative DNA damage and impaired HSC maintenance,
little is known about the mechanism by which oxidative DNA
damage influences the function of HSCs in disease states, such as
BM failure and leukemia, which are commonly accompanied by
defective DNA repair. Our results suggest that oxidative DNA
damage drives HSCs into cell cycling, which may be a prerequisite
for DNA repair to proceed. Inefficient repair of oxidative DNA
damage in these disease conditions may prolong cell cycling,
which leads to HSC exhaustion. In addition, patients with BM
failure and leukemia often have an extremely low number of HSCs,
which critically limits the success of stem cell therapy. Therefore,
one of the key issues in these disease states is to identify conditions
to increase the number of HSCs under stressful conditions, either
in vivo or during ex vivo growth cultures. We show here that
salidroside promotes HSC self-renewal and preservation under
oxidative stress and thus may be valuable for HSC expansion.
Although further studies are required to reveal the underlying
molecular mechanisms, it is intriguing that salidroside promotes
HSC maintenance by reducing oxidative DNA damage through
stimulation of PARP-1 activity. Studies conducted using mutant
mice have underscored the importance of DNA repair pathways in
maintaining the functionality of HSCs. A number of studies
demonstrate hematopoietic defects in mice deficient for HR, NHEJ,
NER, and telomere maintenance pathways.15,16 In the context of
oxidative DNA damage response/repair in HSC maintenance,
recent studies in mice deficient for the ATM kinase or the Foxo
transcription factors showed impaired HSC function as a result of
increased accumulation of ROS in HSCs.47-50 Cumulatively, these
studies suggest that genomic DNA integrity is a limiting factor in
the maintenance of functional HSCs. Indeed we observed defective
long-term hematopoietic repopulation by oxidative stressed HSCs
from mice lacking the BER enzyme Parp-1. Furthermore, our
results indicate that salidroside reduces oxidative DNA damage
through stimulation of PARP-1 activity, and that inhibition of
PARP-1 abrogates the beneficial effect of salidroside on HSC
maintenance. These data suggest that stimulation of PARP-1
activity by salidroside could account for the accelerated repair of
the oxidative DNA damage and enhanced repopulating capacity of
the stressed HSCs.
Another interesting finding of this study is the observation that
salidroside was able to maintain the oxidative stressed HSCs from
WT but not Parp-1⫺/⫺ mice in quiescent state in transplanted mice.
Cell-cycle analysis shows that on challenge with oxidative stress,
 From www.bloodjournal.org by guest on September 29, 2018. For personal use only.

4172 LI et al BLOOD, 3 MAY 2012 䡠 VOLUME 119, NUMBER 18

donor-derived HSCs exited quiescence and underwent cycling.
However, these proliferating HSCs were able to regain quiescence
in recipient mice conditioned with salidroside. This phenomenon
was probably because of Parp-1 stimulation by salidroside, because
donor-derived HSCs deficient for Parp-1 failed to regain quiescent
in salidroside-conditioned recipient mice. These results might
suggest that cell-cycle status of HSCs could reversibly change from
oxidative stress–induced activation back to quiescence after repair
of the oxidative DNA damage. This finding raises important
questions as to whether activation is required for efficient repair of
oxidative DNA damage in the stressed HSCs and whether elimina-
tion of the DNA damage is sufficient for the activated HSCs to
return to quiescent state. Although further evidence needs to be
provided, it is tempting to speculate that reversibility between
quiescence and activation may be a physiologic function of
activated HSCs at the interface between damage repair and
reestablishment of homeostasis. In this context, it is noteworthy
that recent studies in both mice and human show that HSCs can
reversibly switch between dormancy and self-renewal at the
interface between homeostasis and repair.12,13
Acknowledgments
The authors thank Dr Markus Grompe (Oregon Health & Science
University) for Fancd2⫾ mice, Benjamin Chen (University of
Texas Southwestern Medical Center at Dallas) for DNA-PKcs⫹/3A
mice, Dr Liang Li for technical assistance, and the Comprehensive
Mouse and Cancer Core of the Cincinnati Children’s Research
Foundation (Cincinnati Children’s Hospital Medical Center) for
BM transplantation service.
This work was supported by a Visiting Scholarship from South
China Normal University (X.L.) and partially by National Institutes
of Health (NIH) grants R01 HL076712 and R01 CA157537. Q.P. is
supported by a Leukemia & Lymphoma Scholar award. W.D. is
supported by an NIH T32 training grant.
Authorship
Contribution: X.L. designed research and performed research and
analyzed data; J.S. performed research; Q.P. designed research and
analyzed data; and W.D. designed research, analyzed data, and
wrote the paper.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Wei Du, Division of Experimental Hematol-
ogy and Cancer Biology, Cincinnati Children’s Hospital Medical
Center, 3333 Burnet Ave, Cincinnati, OH 45229; e-mail:
wei.du@cchmc.org.

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2012 119: 4162-4173

doi:10.1182/blood-2011-10-387332 originally published
online March 16, 2012

Salidroside stimulates DNA repair enzyme Parp-1 activity in mouse HSC

maintenance
Xue Li, Jared Sipple, Qishen Pang and Wei Du

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