5-AED enhances survival of irradiated mice in a G-CSF-dependent
manner, stimulates innate immune cell function, reduces
radiation-induced DNA damage and induces genes that modulate
cell cycle progression and apoptosis
Marcy B. GRACE1, Vijay K. SINGH1,2, Juong G. RHEE4, William E. JACKSON, III1,
Tzu-Cheg KAO3and Mark H. WHITNALL1,*
1Radiation Countermeasures Program, Armed Forces Radiobiology Research Institute, Uniformed Services University of
the Health Sciences, 8901 Wisconsin Ave, Bethesda, MD 20889-5603, USA
2Department of Radiation Biology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health
Sciences, 8901 Wisconsin Ave, Bethesda, MD 20889-5603, USA
3Division of Epidemiology and Biostatistics, Department of Preventive Medicine and Biometrics, F. Edward Hébert School
of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
4Department of Radiation Oncology, University of Maryland School of Medicine, 655 West Baltimore St., Baltimore, MD
*Corresponding author. Radiation Countermeasures Program, Armed Forces Radiobiology Research Institute, Uniformed
Services University of the Health Sciences, 8901 Wisconsin Ave., Bethesda, MD 20889-5603. Phone: 1-301-295-9262;
Fax: 1-301-295-6503; E-mail: firstname.lastname@example.org
(Received 12 April 2012; revised 25 June 2012; accepted 26 June 2012)
The steroid androst-5-ene-3ß,17ß-diol (5-androstenediol, 5-AED) elevates circulating granulocytes and
platelets in animals and humans, and enhances survival during the acute radiation syndrome (ARS) in
mice and non-human primates. 5-AED promotes survival of irradiated human hematopoietic progeni-
tors in vitro through induction of Nuclear Factor-κB (NFκB)-dependent Granulocyte Colony-
Stimulating Factor (G-CSF) expression, and causes elevations of circulating G-CSF and interleukin-6
(IL-6). However, the in vivo cellular and molecular effects of 5-AED are not well understood. The aim of
this study was to investigate the mechanisms of action of 5-AED administered subcutaneously (s.c.) to
mice 24 h before total body γ- or X-irradiation (TBI). We used neutralizing antibodies, flow cytometric
functional assays of circulating innate immune cells, analysis of expression of genes related to cell cycle
progression, DNA repair and apoptosis, and assessment of DNA strand breaks with halo-comet assays.
Neutralization experiments indicated endogenous G-CSF but not IL-6 was involved in survival enhance-
ment by 5-AED. In keeping with known effects of G-CSF on the innate immune system, s.c. 5-AED
stimulated phagocytosis in circulating granulocytes and oxidative burst in monocytes. 5-AED induced
expression of both bax and bcl-2 in irradiated animals. Cdkn1a and ddb1, but not gadd45a expression,
were upregulated by 5-AED in irradiated mice. S.c. 5-AED administration caused decreased DNA
strand breaks in splenocytes from irradiated mice. Our results suggest 5-AED survival enhancement is
G-CSF-dependent, and that it stimulates innate immune cell function and reduces radiation-induced
DNA damage via induction of genes that modulate cell cycle progression and apoptosis.
Because of the increasing threat posed by nuclear weapons
, there is a pressing need for both (pre-irradiation) radio-
protectants and (post-irradiation) therapeutics, as recognized
by civilian and military government agencies [2–4]. 5-AED
is being developed as a radioprotectant and therapeutic for
ARS. The steroid induces resistance to a variety of infec-
tions in animals [5–10], enhances survival in mice and
rhesus macaques exposed to whole-body γ-irradiation [11,
Journal of Radiation Research, 2012, 53, 840–853
Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Therapeutic Radiology and Oncology
12], and induces hematopoiesis and hematopoietic growth
factor expression [13, 14]. Its administration causes
increases in circulating granulocytes, monocytes, NK cells
and platelets in irradiated animals [14–16]. In humans,
5-AED induces elevations in circulating granulocytes and
platelets, and exhibits minimal side effects . 5-AED
also displays beneficial effects after burn injury, trauma and
sepsis [18–20]. A radiation countermeasure having a
dose reduction factor (DRF) of 1.25 like 5-AED 
would increase the radiation dose at which 50% of
exposed personnel died by 25%. A mass casualty scenario
could involve exposure of hundreds of thousands of
people , and radiation dose-mortality curves are steep
. Hence a countermeasure with a DRF of 1.2–1.3
could reduce casualties by very large numbers. For
example, in canines, supportive care (DRF 1.3) resulted in
mortality falling from above 95% to below 5% at a radi-
ation dose of 2.9 Gy .
Effects of 5-AED on hematopoiesis have been demon-
strated by increases in granulocyte-macrophage colony-
forming cells (GM-CFC) in mouse bone marrow after s.c.
injections . This has been correlated with increases in
neutrophil progenitors in marrow as shown by histology
. We hypothesized that these effects were due to induc-
tion of hematopoietic growth factors and went on to show
that s.c. administration of 5-AED caused increased serum
concentrations of G-CSF in both unirradiated and irradiated
mice . The induction of G-CSF supported the hypothesis
that 5-AED’s survival-enhancing effects are due at least in
part to stimulation of hematopoiesis, resulting in increased
numbers of innate immune system cells in circulation. PCR
analysis of hematopoietic tissues showed  that 5-AED
administration was associated with elevation of mRNAs for
Granulocyte-Monocyte-CSF, Interleukin (IL)-2, IL-3, IL-6
and IL-10 in spleen, and GM-CSF and IL-2 in bone marrow.
The same study confirmed the 5-AED-stimulated increase in
circulating G-CSF, and also showed enhancement by 5-AED
of serum macrophage inflammatory protein-1γ (MIP-1 γ).
A recent study found 5-AED strongly synergized with
thrombopoietin at the level of immature hematopoietic pro-
genitor cells in irradiated mice, whereas pegylated G-CSF
amplified expansion of surviving progenitors .
A pharmacokinetic analysis of 5-AED in mice demon-
strated that plasma 5-AED peaked 2 h after s.c. injection,
remaining significantly above control after 4 days, but not
8 days. The time course of plasma 5-AED after buccal de-
livery (60 mg/kg) was similar, but levels were significantly
lower compared to s.c. delivery. Plasma 5-AED 24 h after
administration was not significantly different between s.c.
and buccal delivery. However, whereas 5-AED enhanced
survival when injected s.c. 24 h before irradiation, buccal
5-AED did not affect survival. Those results suggested
that the survival-enhancing effects of 5-AED are dependent
on events triggered during the first few hours after
administration. We postulated these survival-enhancing
early events involved induction of hematopoietic cytokines
[15, 24]. However, this speculation was based on correla-
tions, rather than a direct test of the hypothesis by blocking
hematopoietic cytokines. In addition, the immediate target
cells of 5-AED were not known.
To obtain more direct evidence regarding the mechan-
isms of 5-AED action, we tested the effects of 5-AED on
irradiated human hematopoietic progenitor (CD34+) cells
. We found that 5-AED protected CD34+ cells from radi-
ation damage, as shown by enhanced cell survival, clonogeni-
city, proliferation and differentiation. The data demonstrated
that hematopoietic progenitor cells are direct targets of the
steroid. Furthermore, 5-AED stabilized NFκB in irradiated
cells and induced NFκB gene expression and activation .
NFκB is a transcription factor that induces genes that inhibit
apoptosis, activate immune system cells and promote survival
in irradiated cells [27–29]. An NFκB-binding region of the
G-CSF promoter is required for induction of G-CSF by tumor
necrosis factor-α and IL-1β in human embryonic lung (HEL)
fibroblasts . 5-AED stimulated release of G-CSF from
human CD34+ cells, and this effect was blocked by the
NFκB inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal
(MG132) . Moreover, the survival-enhancing effects of
5-AED in vitro were dependent on both NFκB and G-CSF, as
shown by siRNA inhibition of NFκB and neutralizing anti-
body blocking of G-CSF.
Given the results of the in vitro study , and the previ-
ous demonstration of 5-AED-induced G-CSF expression in
vivo [13, 24], we wondered whether the survival enhance-
ment by 5-AED in irradiated mice was dependent on
G-CSF. Since our study of CD34+ cells only addressed one
cell type, and effects of drugs in in vitro studies are often
different from what is observed in whole animals, this
required a direct test in vivo. We compared the effects of
blocking G-CSF to blocking IL-6, since IL-6 is induced
by 5-AED [24, 26], but was not necessary for the
survival-enhancing effects of 5-AED in vitro . Since
the beneficial effects of 5-AED in vitro were dependent on
NFκB and G-CSF, we also tested whether known effects of
G-CSF and NFκB could be observed in irradiated animals
after 5-AED administration. These effects include activation
of innate immune system cells [28, 29, 31–38], modulation
of radiation-responsive genes related to cell cycle progres-
sion, DNA repair, apoptosis  and protection against
DNA damage .
MATERIALS AND METHODS
All studies were carried out in accordance with the princi-
ples and procedures of the National Research Council
Guide for the Care and Use of Laboratory Animals .
All research was approved by the Institutional Animal Care
Radiation countermeasure 5-AED 841
and Use Committee of the Armed Forces Radiobiology
Research Institute (AFRRI). For antibody blocking studies,
male CD2F1 mice were used (Harlan, Indianapolis, IN).
For phagocytosis, oxidative burst and PCR studies, animals
were male C3H/HeN mice (National Cancer Institute,
Frederick, MD). For the comet assays, mice were male
C3H/FeJ (Jackson Laboratory, Bar Harbor, Maine). Mouse
strains were chosen according to the evolving standard
practices of the laboratories performing the experiments. A
body of work has shown that responses to 5-AED in
various strains of mice, including B6D2F1/J [15, 16, 40],
C3H [13, 24, 40] and CD2F1 , are quite similar.
5-AED is used routinely at AFRRI as a positive control in
30 day survival assays in CD2F1 mice when screening
novel radiation countermeasure candidates (our unpublished
data). The C3H/HeN and C3H/FeJ strains are closely
Mice were male, 6–8 weeks of age, 22–35 g body
weight, and were held in quarantine for two weeks. Up to 8
mice were housed in sanitized 46 cm× 24 cm ×15 cm poly-
carbonate boxes with filter covers (MicroIsolator; Lab
Products, Inc., Maywood, NJ) on autoclaved hardwood
chip bedding in a facility accredited by the Association for
the Assessment and Accreditation of Laboratory Animal
Care International and were given feed and acidified (pH
2.5 −3.0) water freely. The animal holding room was pro-
vided with conditioned fresh air that was changed at least 10
times per h. Air was kept at approximately 21°C and 50% (±
20%) relative humidity. The animal room was maintained on
a 12-h light/dark full-spectrum lighting cycle. For each ex-
periment, mice for all groups were selected from the same
lot of animals (i.e. a cohort of mice delivered as a single
item from the vendor, with the same birth date).
NH), or vehicle (polyethylene glycol MW400, PEG-400,
Sigma, St. Louis, MO) was injected s.c. in a volume of 0.1
ml, 24 h before irradiation. Neutralizing antibodies to
G-CSF and IL-6 were used to determine the role of G-CSF
and IL-6 on the survival of irradiated mice. CD2F1 mice
received antibodies to either G-CSF or IL-6 (0.2 ml, 600
µg/mouse) or the appropriate isotype control antibody (0.2
ml, 600 µg/mouse) i.p. 16 h before irradiation. Monoclonal
anti-mouse G-CSF and IL-6 antibodies, and rat IgG1
isotype control antibodies, were purchased from R&D
Systems (R&D systems, Inc., Minneapolis, MN). Antibody
was diluted to 3000 µg/ml in Dulbecco’s phosphate-
buffered saline (D-PBS) and administered in a volume of
0.2 ml. Neutralizing antibody experiments were repeated
twice, n =16–20 mice/group in each experiment. Group
sizes for other experiments are shown in the figure legends.
For comet assays, mice were exposed to X-rays (250 kVp,
15 mA, 2 Gy/min) at the University of Maryland Medical
Center in Baltimore, receiving midline doses of 1, 3 or 5
Gy. For all other experiments, mice were placed in venti-
lated Plexiglas containers and exposed bilaterally to
γ-radiation from the AFRRI60Co source at 0.6 Gy/min. The
radiation type (γ-rays or X-rays) was chosen based on the
facilities of the laboratories performing the experiments.
These γ-rays and X-rays were both low Linear Energy
Transfer (LET) photons with similar energy profiles.
Irradiations took place between 9 am and noon. Exposure
time was adjusted so that animals received the midline
tissue-absorbed doses described. Radiation dosimetry was
based on the alanine/EPR (electron paramagnetic resonance)
system [42, 43], currently accepted as one of the most accur-
ate methods and used for intercomparison between national
metrology institutions. The calibration curves used in dose
measurements at AFRRI (spectrometer e-Scan, Burker
Biospin, Inc., Madison, WI, USA) are based on standard
alanine calibration sets purchased from the United States
National Instituteof Standards
Gaithersburg, MD, USA. The accuracy of the constructed
calibration curve was additionally verified by intercompari-
son with the National Physical Laboratory of the United
Kingdom. The dose rate was measured using small
tissue-equivalent alanine pellets in the cores of mouse phan-
toms positioned in the compartments of the mouse rack.
Optically stimulated luminescence (InLight system from
Landauer, Inc., Glenwood, IL, USA), traditional ionization
chambers, and new generation Gafchromic film colorimetry
were used as supporting and quality-control methods.
Phagocytosis and oxidative burst assays
For assays of phagocytosis, the reagents and instructions
from Phagotest® kits(Orpegen
Germany) were used. Heparanized blood was incubated
with FITC-labeled opsonized E. coli at 37°C. Negative
control samples were incubated on ice. FITC fluorescence
of surface-bound bacteria was quenched at the end of the
incubation. Bacteria were discriminated from blood cells
with a DNA label and appropriate gating during acquisition
and analysis. Data are presented as percentage of granulo-
cytes or monocytes having performed phagocytosis (i.e.
positive for FITC labeling).
For assays of oxidative burst, the reagents and instruc-
tions from BurstTest® kits (Orpegen Pharma, Heidelberg,
Germany) were used. The experimental design was similar
to that for phagocytosis, except that heparanized blood was
incubated with opsonized unlabeled bacteria, and then incu-
bated with dihydrorhodamine 123, which is converted to
rhodamine 123 and trapped in cells in the presence of react-
ive oxidants. Negative control samples were not incubated
M.B. Grace et al.
with bacteria. Samples were then analyzed in the flow cyt-
ometer. Data are presented as percentage of granulocytes or
monocytes having produced detectable reactive oxygen
species (i.e. positive for rhodamine 123 labeling).
In both assays, granulocytes and monocytes were distin-
guished on the basis of forward light scatter (size) and side
scatter (granularity). These experiments were repeated twice.
RNA isolation procedures
Mice were anesthetized to unconsciousness with Isoflurane
(Abbott Laboratories, Chicago, IL) before blood was collected
from the abdominal vena cava with a 23-gauge needle.
Anesthetized mice were humanely euthanized by cervical dis-
location after blood collection. Spleens were harvested after
euthanization. Whole spleen from each mouse was homoge-
nized in 1.0 ml RNA STAT-60 (Tel-Test Inc., Friendswood,
TX) in a polytron homogenizer (Brinkmann Instruments, Inc.,
Westbury, NY). Homogenized splenic suspension was imme-
diately frozen and kept at –70°C until further use. Total RNA
of each sample was extracted according to the RNA STAT-60
manufacturer’s protocol. Total RNAwas isolated by homogen-
ization in TRIzol reagent (Life Technologies, Gaithersburg,
MD) followed by QIAGEN® RNeasy® 96 Universal Tissue
Kits (Qiagen, Chatsworth, CA). The concentration and quality
of total RNAwere determined with the Agilent 2100 bioanaly-
zer with nanochips (Agilent Technologies, Palo Alto CA),
spectrophotometrically (260 nm/280nm ratio), and agarose gel
Total RNA was converted to complementary DNA (cDNA)
by a reverse transcription step with M-MLV reverse tran-
scriptase, OligodT primer, RNase inhibitor and dNTPs. For
PCR amplification, a maximum of 20 ng of cDNA was
used per 50 µL PCR reaction. PCR amplifications were per-
formed using the iCycler iQ Sequence Detection System
(Bio-Rad Laboratories, Hercules CA) on 96-well microtiter
plates with optical caps. Reactions were performed in a
total volume of 50 µL containing 20 ng cDNA. Real-time
RT-PCR assays were carried out using 1× buffer (10× PCR
buffer is 200 mM Tris-HCl, pH 8.4, 500 mM KCl). SYBR
Green primer pair validation experiments were carried out
in 50 µl using Taq (0.025 U/μl reaction volume), 5 µl of a
1/10000 SYBR Green dilution, 300 nM of each PCR
primer, 1× buffer, dNTPs (200 µM), and 3.5 mM MgCl2.
PCR was performed to determine levels of mRNA coding
for gadd45a, ddb1, ddb2, cdkn1a, bax and bcl-2, as well as
18S rRNA subunit which was used as an internal control.
All cDNA samples were amplified for 40–50 cycles using
denaturation at 94°C for 30 s, annealing and extension at
60°C for 60 s. The ratio of fluorescence intensity of target-
specific product to that of the internal control product
represents relative levels of target mRNA expression. The
performance of primers and probe sets included in our multi-
plex qRT-PCRs was evaluated using serial dilutions (one-
fifth or one-tenth dilutions) of murine cDNA spanning three
orders of magnitude. All components were maintained under
universal conditions. PCR amplifications were always per-
formed in duplicate or triplicate wells, using universal tem-
perature cycles: 10 min at 94°C, followed by 35–45
two-temperature cycles (15 s at 94°C and 1 min at 60°C).
Primers and probes
Amplicon lengths were set between 70 and 110 bp. To
avoid co-amplification of contaminating genomic DNA,
primers were (where possible) designed on different exons or
intron–exon boundaries. All primers and hydrolysis probe
sequences were designed with Primer Express software. The
(Gaithersburg MD). Selected fluorophores were FAM
(6-carbonylfluorescein) and TexasRed™. Appropriate Black
Hole dark quenchers were matched for each fluorophore.
Gene expression was measured in five mice per group.
by Loftstrand Laboratories
Mice were injected s.c. with 5-AED at different doses (10,
30 and 100 mg/kg), and exposed to X-rays (1, 3 or 5 Gy at
2 Gy/min) 24 h later. Within 2 min after irradiation, spleens
were excised and minced to separate splenocytes. These
cells were washed once and suspended in ice-cold PBS
(5×105cells/ml). For the halo-comet assay, a filter paper
was punched with 5 mm diameter holes, placed on a clean
glass slide, and moistened with PBS. The cell suspension
in PBS was mixed with the same volume of a pre-warmed
2% agarose solution (60°C), and a drop of the mixture (41°
C) was applied to the hole of a filter paper. In order to
remove lipids and proteins, the gel in a filter paper was
exposed to an NN (0.03 M NaOH and 1 M NaC1) lysis so-
lution for 60 min at 4°C, followed by washing with an NE
(0.03 M NaOH and 2 mM EDTA) solution for 30 min at 4°
C. This treated gel was transferred to a submarine gel ap-
paratus (E-C Apparatus Corp.), and DNA separation was
achieved by applying 16.5 DC volts for 6 min (3 volts/cm,
18 mA) to the NE solution. After electrophoresis, the gel
was washed with water for 15 min, and then stained with
2.5 µg/ml propidium iodide (PI) for 10 min, in a dark box.
The PI intercalated to the DNA strands was excited at 545
nm (BP-545), and the red fluorescence emission at 580 nm
(Olympus BH-2) with the aid of a 590 nm barrier filter.
The red fluorescence light (DNA image) was enhanced
with the Dark-Invader night vision system (Meyers & Co.
Inc., Redmond, WA), and was either digitized with a frame
grabber or recorded on a videotape for later analysis. To
quantify DNA damage, individual comet images were
Radiation countermeasure 5-AED 843
analyzed with Accuware (Automatic Visual Inspection,
Santa Clara, CA). The length of the halo-comet image cor-
responded to the extent of DNA damage . The analysis
was repeated for 300 nuclei for each group.
All results were expressed as means± SE. For antibody
neutralization experiments, survival data were analyzed
using Fishers Exact Test adjusted for multiple comparisons
using Bonferroni correction. Phagocytosis and oxidative
burst data were analyzed by ANOVA. PCR data were ana-
lyzed in terms of relative gene expression. The cycle
number at which the fluorescent signal of a given reaction
well crossed the threshold value was denoted as the thresh-
old cycle (CT). Target data from each well were normalized
to the internal standard, 18S ribosomal subunit, using the
formula ΔCT=target CT– internal standard CT.Radiation
effects on gene expression levels were analyzed further by
comparison to a reference standard (i.e. vehicle calibrator;
nonirradiated sham-treated samples). Microsoft Excel-fitted
lines were obtained using standard regression analysis pro-
grams within Excel XP (Microsoft Corporation, Redmond,
WA) and Sigma Plot 5.0 (SPSS Inc., Chicago, IL). Standard
errors of CTwere determined for each value. Analysis of
variance (ANOVA) was used to detect significant differences
among groups at each time point. If there was any significant
difference among the groups, the Tukey-Kramer method was
used for pair-wise comparisons. For comet assays, least
square means were adjusted for multiple comparisons using
Tukey-Kramer. Differences were considered significant if the
two-sided P value was <0.05. A software package, PC SAS,
was used for statistical analyses.
Effects of neutralization of G-CSF or IL-6
on survival of 5-AED-treated, irradiated mice
Mice were exposed to TBI at a dose (9.25 Gy, 0.6 Gy/min)
that resulted in 30-day survival of 44% in controls (drug
vehicle and isotype antibody injections, Fig. 1). Survival of
vehicle-treated irradiated mice after pre-treatment with neu-
tralizing antibody (anti-G-CSF, 0% or anti-IL-6, 25%) was
not significantly different from survival after vehicle and
isotype control antibody. When irradiated mice were pre-
treated with 5-AED and isotype control antibody, all
animals survived. Isotype control antibody injections had
no effect on survival of unirradiated mice receiving vehicle
or 5-AED injections (not shown). However, as shown in
Fig. 1, when irradiated mice were injected with neutralizing
antibody to G-CSF, the survival-enhancing effect of 5-AED
was blocked (31% survival, compared to 100% with
isotype control antibody). Survival in these mice was not
significantly different from mice receiving vehicle plus
isotype control (44%) or vehicle plus anti-G-CSF (0%). On
the other hand, pre-treatment of irradiated mice with neu-
tralizing antibody to IL-6 had no effect on the ability of
5-AED to enhance survival: all mice in this group survived
(Fig. 1). The results indicate endogenous G-CSF but not
IL-6 was involved in survival enhancement by 5-AED.
Phagocytosis and oxidative burst in innate immune
cells in irradiated mice
Phagocytosis of opsonized FITC-labeled bacteria was mea-
sured using flow cytometry of granulocytes and monocytes
in blood taken from sublethally irradiated or sham-
irradiated mice that had been injected s.c. with 5-AED or
vehicle 24 h before irradiation (3 Gy) or sham-irradiation
(Fig. 2). The radiation dose was relatively low in this experi-
ment to allow sufficient numbers of circulating cells to study.
A decrease in phagocytic activity was observed in both gran-
ulocytes and monocytes 1 day after irradiation (Fig. 2).
ANOVA revealed a significant effect of 5-AED on phagocyt-
ic activity in granulocytes 1 day after irradiation and 4 days
after sham-irradiation (Fig. 2). However, no effect of 5-AED
on phagocytosis was observed in monocytes (Fig. 2).
To measure oxidative burst, we analyzed baseline activity
in granulocytes and monocytes (i.e. in the absence of incu-
bation with bacteria), and the effect of incubation with
opsonized unlabeled bacteria (Fig. 3). In contrast to phago-
cytosis, which was stimulated by 5-AED in granulocytes
but not monocytes (Fig. 2), oxidative activity was stimulated
by 5-AED in monocytes but not granulocytes (Fig. 3). In
monocytes, both baseline and bacteria-stimulated oxidative
activity were higher in 5-AED-treated mice 1 and 4 days
on survival of irradiated mice. 5-AED or vehicle was injected s.c.
24 h before total body γ-irradiation (9.25 Gy). Antibodies were
injected i.p. 16 h before irradiation. *P< 0.05 compared to
vehicle, for both 5-AED+isotype control antibody and 5-AED+
anti-IL-6 antibody. Results from one of two experiments showing
similar results; n=16–20 mice/group in each experiment.
Effects of neutralizing anti-G-CSF or anti-IL-6 antibody
M.B. Grace et al.
after sham-irradiation or irradiation (Fig. 3). In summary, s.c.
5-AED stimulated phagocytosis in circulating granulocytes
and oxidative burst in monocytes.
Expression of genes related to cell cycle
progression, DNA repair and apoptosis
5-AED was administered s.c. (30 mg/kg) 24 h prior to irradi-
ation at a high sublethal dose (7.5 Gy) and spleens were col-
lected 4 h after 5-AED injection, or 4 or 24 h after irradiation.
The results presented in Fig. 4 show modulation of messages
of apoptotic genes by 5-AED in irradiated and sham-
irradiated mice: 5-AED induced pro-apoptotic bax expression
4 h after sham-irradiation and irradiation. Decreased expres-
sion of anti-apoptotic bcl-2 was observed 4 h after 5-AED in-
jection, as compared to vehicle-treated mice (Fig. 4, not
significant). However, 5-AED pre-treatment 24 h before ir-
radiation enhanced bcl-2 expression when compared to down-
regulated vehicle-treated controls 4 h post-irradiation (Fig. 4).
The results show increased levels of message for both bax
and bcl-2 gene expression in spleen tissue after 5-AED injec-
tion. No significant change in the bax/bcl-2 ratio was observed.
5-AED administration resulted in no change in transcrip-
tion levels of the regulator of cell cycle progression and
DNA repair, cdkn1a, in spleens of sham-irradiated mice
(Fig. 5). However, the steroid induced a rise in cdkn1a
mRNA 4 h post-irradiation (Fig. 5).
Ddb1, which is not up-regulated in response to radiation
in human or mouse models, is up-regulated in response to
5-AED treatment. DDB1 and DDB2 form a heterodimer
involved in DNA repair. 5-AED induced significant increases
in ddb1 expression in two groups as shown in Fig. 6: 4 h
post-irradiation, and 24 h after sham irradiation. Treatment
with 5-AED had no significant effect on the radiation-
responsive DNA repair genes gadd45a and ddb2 in spleno-
cytes (data not shown). The results of the gene regulation
study in spleen tissue show 5-AED-induced modulation of
genes that that regulate apoptosis, with no clear indication of
a shift in the balance of pro-apoptotic vs anti-apoptotic
genes. However, there was a significant induction of some
but not all genes involved in regulation of cell proliferation
and DNA repair (cdkn1a and ddb1).
Effects of radiation on whole blood gene expression
Changes in relative gene expression in whole blood are
shown in Table 1. mRNA for cdkn1a measured in whole
blood 24 h post-irradiation displayed an increase (fold
change) of 6.6 ±0.4. Expression of bcl-2 decreased 0.68 ±
0.1 fold, and bax increased 1.9 ±0.6 fold. Expression of
the DNA repair gene gadd45a increased 25.0 ±15 fold. As
was found for splenocytes, ddb2 transcription values in
whole blood remained unchanged.
(160 mg/kg) 24 h before sham-irradiation (0 Gy) or total body γ-irradiation (3 Gy) caused increases in
phagocytotic activity in granulocytes (P<0.05) as measured by flow cytometry. Blood was incubated with
FITC-labeled opsonized E. coli at 37°C. Negative control samples were incubated on ice. n=18–20 mice/group.
Effects of 5-AED on phagocytosis in circulating granulocytes and monocytes. Injections of 5-AED
Radiation countermeasure 5-AED845
Effects of 5-AED pre-treatment on DNA damage
in splenocytes of irradiated mice
Following treatment with X-rays (1–5 Gy) alone, the halo-
comet image length increased with increasing doses of
X-rays (Fig. 7), suggesting dose-dependent DNA damage.
When mice were treated with 5-AED 24 h before irradi-
ation, the image length was significantly shorter than what
was obtained with vehicle pre-treatment, indicating a pro-
tective effect of 5-AED. All tested doses of 5-AED (10, 30
and 100 mg/kg) produced the same protective effect
(Fig. 7). The data indicate 5-AED injected 24 h before ir-
radiation reduced radiation-induced DNA damage measured
immediately after irradiation.
Roles of cytokines in survival-enhancing effects
Previous reports showed administration of 5-AED induces
G-CSF expression in mice and in cultured human
hematopoietic progenitor cells [13, 26], and that the
survival-enhancing effects of 5-AED on hematopoietic pro-
genitor cells in vitro were dependent on G-CSF. Since
G-CSF promotes survival in irradiated mice , canines
 and NHP , and is recommended for use in human
radiation casualties , we tested whether the survival-
enhancing effects of 5-AED in vivo were dependent on
G-CSF using neutralizing antibodies. Our results are con-
sistent with studies documenting the essential role of en-
dogenous G-CSF in the mechanism of action of some other
radiation countermeasures [48, 49]. We also tested the role
of IL-6, since administration of 5-AED induces IL-6
expression in mice and in cultured human hematopoietic
progenitor cells [24, 26], and anti-IL-6 antibody reduces
the radioprotective effects of IL-1 and TNF in mice .
However, in our previous in vitro study, the survival-
enhancing effects of 5-AED on hematopoietic progenitor
cells in vitro were not dependent on IL-6 . Consistent
with the in vitro results, blocking IL-6 did not inhibit the
radioprotective effects of 5-AED in vivo.
AED (160 mg/kg) caused increases in oxidative activity in monocytes when injected 24 h before
sham-irradiation (0 Gy) or total body γ-irradiation (3 Gy) (P<0.001) as measured by flow cytometry.
Radiation induced an increase in monocyte baseline oxidative activity (in the absence of bacterial
stimulation) (P<0.05). The experimental design was similar to that for phagocytosis (Fig. 2), except that
blood was incubated with opsonized unlabeled bacteria, and then incubated with dihydrorhodamine 123,
which is converted to rhodamine 123 and trapped in cells in the presence of reactive oxidants. Negative
control samples were not incubated with bacteria. n= 18–20 mice/group.
Effects of s.c. 5-AED on oxidative burst in circulating granulocytes and monocytes. Injections of
M.B. Grace et al.
Effects of 5-AED on innate immune cell function
G-CSF is induced by the pro-survival transcription factor
NFκB , and the 5-AED-induced G-CSF we observed in
dependent . Increased levels of G-CSF and/or NFκB
after 5-AED administration to mice could enhance survival
by reducing apoptosis in hematopoietic progenitor cells
. However, G-CSF also induces activation of cells of
the innate immune system, as shown by gene microarray
analysis . Functional assays show G-CSF upregulates
phagocytosis in neutrophils, with conflicting results on
upregulation of oxidative burst [32–37]. Inhibition of
and irradiated mice. Six- to eight-week old C3H/HeN mice received s.c. injections of 5-AED (30 mg/kg)
24 h before 7.5 Gy γ-irradiation. Spleens were collected 4, 28 or 48 h after steroid administration. V =
vehicle, A =5-AED, Means± SEM, *P <0.05, 5-AED-treated vs vehicle-treated. n=10 mice for all
groups except 4 h post-vehicle, which contained 5 mice.
Quantitation of bax, bcl2 and bax/bcl2 ratio mRNA by QRT-PCR in spleens of 5-AED-treated
Radiation countermeasure 5-AED 847
NFκB decreased neutrophil phagocytosis and microbicidal
capacity . A large variety of NFκB target genes is acti-
vated in stimulated monocytes/macrophages, including
G-CSF and inducible nitric oxide synthase, which contri-
butes to the oxidative burst . Hence it has been postu-
lated that NFκB plays a central role in coordinately
controlling gene expression during monocyte/macrophage
activation . Since 5-AED induces both G-CSF and
NFκB in human hematopoietic progenitor cells, we
hypothesized that the radioprotective efficacy of 5-AED
in vivo may be due partially to activation of innate immune
cells by G-CSF and/or NFκB. Our finding that 5-AED ad-
ministration to mice increased phagocytotic activity of cir-
culating granulocytes and oxidative activity in circulating
monocytes supports this hypothesis. The data support our
hypothesis  that survival enhancement by 5-AED in
irradiated animals could be due, at least in part, to increased
effectiveness by innate immune cells at removing harmful
mice. Six- to eight-week-old C3H/HeN mice received s.c. injections of 5-AED (30 mg/kg) 24 h before 7.5
Gy γ-irradiation. Samples were collected 4, 28 or 48 h after steroid administration. V =vehicle, A =
5-AED, Means±SEM, *P <0.05, 5-AED-treated vs vehicle-treated. n= 10 mice for all groups except 4 h
post-vehicle, which contained 5 mice.
Quantitation of cdkn1a mRNA by QRT-PCR in splenocytes of 5-AED-treated and irradiated
eight-week-old C3H/HeN mice received s.c. injections of 5-AED (30 mg/kg) 24 h before 7.5 Gy γ-irradiation.
Samples were collected 4, 28 or 48 h after steroid administration. V =vehicle, A= 5-AED, Means±SEM *P<
0.05, 5-AED-treated vs vehicle-treated. n=10 mice for all groups except 4 h post-vehicle, which contained 5 mice.
Quantitation of ddb1 RNA by QRT-PCR in splenocytes of 5-AED-treated and irradiated mice. Six- to
M.B. Grace et al.
microorganisms, supplementing the effect of increased
numbers of circulating innate immune cells.
The striking dichotomy between granulocytes and mono-
cytes in terms of which function (phagocytosis vs oxidative
burst) was stimulated by 5-AED is intriguing. Further ex-
perimentation will be necessary to determine how 5-AED
affects the different intracellular signaling pathways that
regulate these two functions . Granulocytes (mainly
neutrophils) play an important role in the inflammatory re-
sponse . Since inflammation is thought to be a major
component of radiation injury , the lack of stimulation of
oxidative burst in granulocytes by 5-AED may serve to
prevent the steroid from aggravating this component of radi-
ation injury. In fact, 5-AED has been shown to inhibit inflam-
mation [19, 55, 56]. Moreover, nonoxidative killing is an
important function of both neutrophils and monocytes .
Monocytes also participate in inflammation, but they are
far fewer in number, so the 5-AED-induced increase in
monocyte oxidative burst may not be a major factor in ag-
responses in brain monocytes (microglia) . However,
stimulation of the monocyte oxidative burst by 5-AED
would facilitate killing of microorganisms by these cells.
The 5-AED-induced increase in oxidative activity in mono-
cytes with no modulation of phagocytotic ability may indi-
cate a stimulation of extracellular killing by these cells
. The increase in oxidative activity in monocytes of
5-AED-treated mice may be due to induced differentiation
of these cells, since G-CSF is known to cause differenti-
ation of monocytes .
Effects of 5-AED on gene expression in splenocytes
The balance between apoptosis and survival/proliferation
after irradiation is largely controlled by the Ataxia
Telangiectasia Mutated (ATM) protein, which activates pro-
survival factor NF-κB and pro-apoptotic P53 . The
balance between pro-survival and pro-apoptotic pathways
regulated by these signals determines whether cells will
survive, enter into a state of cell cycle arrest, or undergo
apoptosis. P53 is a transcription factor that induces a G1 or
G2 arrest via activation of cdkn1a (p21), creating extra
time for DNA repair mechanisms and impeding apoptosis
[61–64]. If DNA fails to repair, P53 initiates apoptosis by
activation of members of the pro-apoptotic members of the
bax/bcl-2 family [65–67]. We found increased levels of
message for both bax and bcl-2 gene expression after
5-AED injection, which did not provide a clear signal as to
whether 5-AED was promoting or inhibiting apoptosis.
This may be due to the heterogeneous population of cells
in the spleen, with different cell types responding different-
ly to radiation and 5-AED.
Expression of mRNA for the cell cycle control gene
cdkn1a is used as an indicator of growth arrest, which facil-
itates DNA repair . The clear increase in expression of
cdkn1a after 5-AED administration indicates that the overall
effect of the steroid on splenocytes was to promote cell
cycle arrest in G1 and G2, which would be consistent with
protection against apoptosis. However, the relationship
between cdkn1a and apoptosis is complex. Inhibition of
apoptosis is one outcome of overexpression of cdkn1a, but
senescence would be another since radiation-triggered
accelerated senescence is dependent on CDKN1A function
32Dc13 murine myeloblasts induced to differentiate into
to bepro-apoptotic in
splenocytes 2 min after X-irradiation. 5-AED was injected s.c. 24
h before whole-body irradiation at 10 mg/kg, 30 mg/kg and 100
mg/kg. Halo-comet lengths for each of the three doses were
significantly lower than for vehicle controls (P<0.05). There were
no statistical differences between results for the different 5-AED
doses, except at 5 Gy: halo-comet lengths for 30 mg/kg were
significantly shorter than for 10 mg/kg. n= 6 mice/group.
Quantification of halo-comet assay results in mouse
mice 24 h post-irradiation (7.5 Gy)
Average gene expression changes in blood of five
kinase inhibitor 1A (p21)
NM_009741 BCL2(−) 0.68± 0.1
NM_007527 BAX; BCL2-associated X
BC011141 GADD45A; growth arrest and
AY027937 DDB2 0.0±0.0
Radiation countermeasure 5-AED849
granulocytes by removal of IL-3 and addition of G-CSF
. The same group found that CDKN1A inhibited gran-
ulocytic differentiation in 32Dc13 cells . It remains to
be seen whether similar effects occur in vivo, in the
absence of IL-3 deprivation. Some of the protective effect
of 5-AED against DNA damage may be mediated by
CDKN1A’s promotion of chromosomal stability .
We observed significant up-regulation of ddb-1 gene ex-
pression 4 h post-irradiation in the 5-AED treatment group,
but no change was observed for the radiation-responsive
DNA repair genes ddb-2 and gadd45a [73, 74]. This is
worthy of note because gadd45a mRNA is a global indica-
tor of cellular stress. To our knowledge, this is the first
time a compound has been reported to alter ddb-1 gene ex-
pression. Depletion of DDB1 protein is known to result in
increased DSB in widely dispersed regions throughout the
genome as evidenced by γH2AX foci formation and activa-
tion of ATM and ATR damage response pathways .
Current evidence suggests that DDB1 is involved in
general cellular responses to DNA damage. DDB1 protein
is a component of the Cullin4A ubiquitin ligase and is
involved in DNA repair, cell cycle regulation, DNA replica-
tion, and maintenance of genome integrity . DDB1/
DDB2 complexesare known
UV-damaged DNA lesions, and initiate the nucleotide exci-
sion repair process [76, 77]. Although the mechanism is
not yet known, it appears that DDB assists in nucleotide ex-
cision repair in chromatin. Using mouse models, Cang
et al.  showed that ddb1 is required for embryogenesis,
and the conditional inactivation of ddb1 results in the apop-
tosis of proliferating cells owing to the accumulation of cell
cycle regulators and genomic instability. Cang et al. 
also reported that tissue-specific deletion of ddb1 in mouse
epidermis led to dramatic accumulation of CDKN1A, arrest
of cell cycle at G2/M, and selective apoptosis in mouse
skin. Upregulation of ddb1 by 5-AED but not by radiation
exposure suggests an important effect of 5-AED is to main-
tain genome integrity after radiation exposure. This hypoth-
esis is supported by the direct observation of reduced DNA
damage in splenocytes of 5-AED-treated mice in the comet
Effects of 5-AED on gene expression in whole
5-AED-induced changes in whole blood were similar to
splenocytes for cdkn1a, bax and ddb2 after irradiation,
were in the opposite direction for bcl2 post-irradiation, and
gadd45a was induced strongly in whole blood but not in
splenocytes. The results indicate an overall effect on circu-
lating cells of promoting DNA repair and preserving
genome integrity (cdkn1a, ddb2 and gadd45a). However,
whereas the regulation of genes in the spleen indicated both
pro-apoptotic and anti-apoptotic influences of 5-AED, the
effect on whole blood was clearly pro-apoptotic. This effect
of 5-AED may be related to removal of defective cells from
circulation. The pro-apoptotic effect of 5-AED on whole
blood does not interfere with its stimulation of increases in
numbers of circulating innate immune cells [15, 16]. It also
does not seem to be related to lymphocytes, since 5-AED
administration is not associated with changes in numbers of
lymphocytes [15, 16].
DNA damage elicits a multifaceted response that includes
cell cycle arrest, transcriptional activation of DNA repair
genes, and apoptosis. We demonstrate that in mouse spleen,
DNA damage leads to induction of specific alterations in
gene expression patterns. The expression of genes involved
in cell cycle control, repair of DSBs, and apoptosis were
modulated after treatment with 5-AED. These results are
consistent with our comet assay results showing decreased
DNA damage shortly after irradiation of 5-AED-pretreated
mice.The present findings
survival-enhancing effects of 5-AED are dependent on en-
dogenous G-CSF and are associated with functional activa-
tion of innate immune cells. Further studies are required to
elucidate the exact mode of radioprotection at the molecular
level, including a better understanding of the relative rates
of proliferation and apoptosis in conjunction with DNA
damage signaling, persistence of DNA damage versus tran-
sient DNA damage, and underlying molecular mechanisms
involved in the radiation response.
Radiobiology Research Institute (RAB4AO to M.B.G.,
RAB2AX to V.K.S. and RAB2AD to M.H.W.) and the
National Institute of Allergy and Infectious Diseases (Y1–
AI–3809–01 to M.H.W.)
work was supportedbythe ArmedForces
The authors acknowledge the excellent technical support of
Christopher McLeland, Vaishali Parekh and Cynthia Inal;
the staffs of the AFRRI Veterinary Sciences and Radiation
Sources Departments; Cheng-Min Chang for preparation of
high quality RNA samples; and Patrice Bolte for graphic
services. The views expressed here are those of the authors;
no endorsement by the US Department of Defense or any
US Government agency has been given and should not be
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