BIOLOGY CONTRIBUTION

DIFFERENTIAL EFFECTS OF X-RAYS AND HIGH-ENERGY

56
Fe IONS ON HUMAN
MESENCHYMAL STEM CELLS
KYLE KURPINSKI, PH.D.,* DEOK-JIN JANG, PH.D.,
y
SANCHITA BHATTACHARYA, M.S.,
y
BJORN RYDBERG, PH.D.,
y
JULIA CHU, B.S.,* JOANNA SO, B.S.,* ANDY WYROBEK, PH.D.,
y
SONG LI, PH.D.,*
AND DAOJING WANG, PH.D.
y
*Department of Bioengineering, University of CaliforniaBerkeley, Berkeley, CA; and
y
Life Sciences Division, Lawrence Berkeley
National Laboratory, Berkeley, CA
Purpose: Stem cells hold great potential for regenerative medicine, but they have also been implicated in cancer
and aging. How different kinds of ionizing radiation affect stem cell biology remains unexplored. This study
was designed to compare the biological effects of X-rays and of highlinear energy transfer (LET)
56
Fe ions on hu-
man mesenchymal stem cells (hMSC).
Methods and Materials: A multi-functional comparison was carried out to investigate the differential effects of
X-rays and
56
Fe ions on hMSC. The end points included modulation of key markers such as p53, cell cycle
progression, osteogenic differentiation, and pathway and networks through transcriptomic proling and bioinfor-
matics analysis.
Results: X-rays and
56
Fe ions differentially inhibited the cell cycle progression of hMSC in a p53-dependent man-
ner without impairing their in vitro osteogenic differentiation process. Pathway and network analyses revealed
that cytoskeleton and receptor signaling were uniquely enriched for low-dose (0.1 Gy) X-rays. In contrast,
DNA/RNA metabolism and cell cycle regulation were enriched for high-dose (1 Gy) X-rays and
56
Fe ions, with
more signicant effects from
56
Fe ions. Specically, DNA replication, DNA strand elongation, and DNA bind-
ing/transferase activity were perturbed more severely by 1 Gy
56
Fe ions than by 1 Gy X-rays, consistent with
the signicant G2/M arrest for the former while not for the latter.
Conclusions:
56
Fe ions exert more signicant effects on hMSC than X-rays. Since hMSC are the progenitors of
osteoblasts in vivo, this study provides new mechanistic understandings of the relative health risks associated
with low- and high-dose X-rays and high-LET space radiation. 2009 Elsevier Inc.
Radioresponse, High-LET, Cell cycle, Osteogenic differentiation, Transcriptomics.
INTRODUCTION
Stem cells hold great potential for regenerative medicine be-
cause they can self-renew and differentiate along tissue-spe-
cic lineages. Mesenchymal stem cells (MSCs), the
multipotent stromal cells from bone marrow, play important
roles in the maintenance and repair of various tissues, includ-
ing bone, cartilage, and muscle (1). The differentiation of
MSCs into osteoblasts is controlled by the transcription factor
Runx2 (2). Stem cells also respond to genotoxic stresses, for
example, ionizing radiation (IR), which may lead to the induc-
tion of mutations and chromosome aberrations, transient cell-
cycle arrest, apoptosis, mitotic catastrophe, cellular senes-
cence, or malignant transformation. Abnormalities in stem
cell biology have been implicated in cancer and aging, but
the mechanisms are poorly understood (3). For example,
MSCs have been shown as a target for neoplastic transforma-
tion following manipulations (4, 5). However, major chal-
lenges still remain to dene the normal molecular signaling
and tumorigenic pathways in stem cells.
Because of their high tissue-penetrating property, IR such
as X-rays/gamma rays is used in CT scans, cancer
Reprint requests to: Daojing Wang, Ph.D., Life Sciences Divi-
sion, Lawrence Berkeley National Laboratory, 1 Cyclotron Road,
MS 977-225A, Berkeley, CA 94720. Tel: (510) 486-6592; Fax:
(510) 495-2535; E-mail: djwang@lbl.gov
Conict of interest: none.
AcknowledgmentsThe authors thank Dr. M. Bissell for advice and
Drs. B. Sutherland and P. Guida for help with the logistics of Na-
tional Aeronautics and Space Administration (NASA) Space Radi-
ation Laboratory (NSRL) experiments. This work was supported by
the Low Dose Radiation Research Program jointly funded by the
U.S. Department of Energy (DOE) and NASA (to D.W.). A.W.
acknowledges the funding support from the DOE Low Dose Radi-
ation Research Program. S.L. acknowledges the funding support
from the National Institutes of Health (HL079419). This work
was performed under the auspices of the U.S. DOE, at the University
of California/Lawrence Berkeley National Laboratory under
contract No. DE-AC02-05CH11231.
Supplementary material for this article can be found at
www.redjournal.org.
Received May 12, 2008, and in revised form Aug 29, 2008.
Accepted for publication Sept 3, 2008.
869
Int. J. Radiation Oncology Biol. Phys., Vol. 73, No. 3, pp. 869877, 2009
Copyright 2009 Elsevier Inc.
Printed in the USA. All rights reserved
0360-3016/09/$see front matter
doi:10.1016/j.ijrobp.2008.10.002
radiotherapy, and nuclear medicine. However, IR is also
a well-known DNA-damaging agent, clastogen, and carcino-
gen (6, 7). During deep space missions, space crews encounter
an elevated radiation environment that includes high-energy
charged (HZE) particles, such as
56
Fe ions from the galactic
cosmic rays (8). Such densely ionizing radiation induces more
severe clustering of damage on DNA compared to X-rays,
and gives rise to complex DNA double-strand breaks (DSBs)
that are more prone to be misrepaired and cause permanent
genomic changes (9). Another complicatingfactor duringspace
travel is the possibility of synergistic relationships with other
risk factors, such as microgravity. The possibility that HZE
radiation affects the differentiation process of human mesen-
chymal stem cells (hMSC) into osteoblasts, important for
bone regeneration and loss in space because of microgravity,
has not been investigated.
Advances in systems biology have opened up new possi-
bilities for elucidating the molecular mechanisms underlying
cellular responses to IR. Global gene expression changes in
response to IR have been reported for Saccharomyces cerevi-
siae (10), murine brain (11), normal human skin broblasts
(12), and human lung broblasts (13). These studies sug-
gested that IR regulates gene expression at the transcriptional
level in a dose-dependent manner and depending on radiation
quality, for example, X-rays vs. HZE particles. On the other
hand, proteomic and transcriptomic studies of hMSC have
provided important insights into their proliferation and differ-
entiation (14, 15). However, no direct correlations between
phenotypic radioresponses and pathway regulations have
been reported for hMSC, particularly in the context of osteo-
genic differentiation.
Here we describe the rst comprehensive study of the
radioresponses of hMSC to X-rays and high energy
56
Fe
ions. We show that neither X-rays nor
56
Fe ions impaired
the in vitro osteogenic differentiation process of hMSC.
We further show that they differentially regulated the cell
cycle progression of hMSC in a dose-dependent and p53-
dependent manner. Pathway and network analyses of
transcriptomic proles revealed the underlying mechanisms.
MATERIALS AND METHODS
Cell culture
Human mesenchymal stem cells were obtained from Lonza
Group (Walkersville, MD). The log number was 4F1560, which
was derived from the bone marrow of a healthy 23-year-old woman.
We characterized expanded hMSC (up to passage 10) again by their
surface markers and differentiation potential as we described previ-
ously (14, 15). For cell proliferation without differentiation, hMSC
(typical passage number, ve to eight) were cultured in MSCgrowth
medium, supplemented with prescreened fetal bovine serum, L-glu-
tamine, and a penicillin/streptomycin mix (MSCGM, Lonza). For
osteogenic differentiation after irradiation, hMSC were expanded to
70% conuency and then supplemented with osteogenic differ-
entiation medium containing dexamethasone, b-glycerophosphate,
and ascorbate (Lonza) approximately 1.5 h before exposure. After
irradiation, cells were maintained in osteogenic differentiation
medium for 1, 3, 6, or 7 days and characterized. For growth
and proliferation after irradiation, hMSC were expanded to
35% conuency before exposure. After irradiation, cells were
maintained in MSCGM for 3, 5, or 24 h and characterized.
Irradiation with X-rays and
56
Fe ions
X-ray irradiation was performed at the Lawrence Berkeley
National Laboratory (LBNL) with 320 kVp X-rays (Pantak Inc.,
Branford, CT) with 0.5 mm copper ltration. Acalibrated ion cham-
ber with temperature and pressure correction (RadCal Corporation,
Monrovia, CA) was used for dosimetry.
56
Fe ion irradiation was per-
formed at the National Aeronautics and Space Administration
(NASA) Space Radiation Laboratory (NSRL) radiation facility at
Brookhaven National Laboratory (BNL) with 1 GeV/amu
56
Fe
ions, LET = 150 keV/mm. Dosimetry was carried out by NSRL per-
sonnel, as previously described (16). Triplicates of individual hMSC
cultures were exposed to 0 Gy (sham control), 0.1 Gy (low dose),
and 1 Gy (high dose) for X-rays and
56
Fe ions, respectively. For
both X-rays and
56
Fe ions, the dose rate used was 0.1 Gy/min and
1 Gy/min for 0.1 Gy and 1 Gy, respectively.
To estimate the number of
56
Fe traversals per cell nucleus, the
average nuclear area perpendicular to the beam was measured using
live Hoechst 33342 staining and the number of traversals per cell
was calculated as N = 6.24 dose (Gy) area (mm
2
) / LET
(keV/mm) (17). The nuclear diameter of hMSC grown on plastic
substrates was found under our growth conditions to be 20.2
3.2 mm (SD), corresponding to a nuclear area of 330 110 mm
2
(SD). The average number of traversals per cell is then calculated
to be 1.36 per nucleus for the dose of 0.1 Gy and 13.6 per nucleus
for the dose of 1 Gy. Applying Poisson statistics, 75% of the cell nu-
clei were hit by at least one
56
Fe particle for a dose of 0.1 Gy, with
25% of the cell nuclei exposed only to a low dose of delta rays. At
1 Gy, 100% of the cells were traversed by multiple
56
Fe particles.
Microscopy, alkaline phosphatase activity assay, and
calcium staining
Live-cell phase-contrast images were collected using a Nikon
TMS microscope and a Hamamatsu Orca100 cooled digital CCD
camera, usingC-ImagingSystemsoftware (CompixInc., Irvine, CA).
Osteogenic differentiation was assessed using a colorimetric ALP
activity assay and Alizarin Red S staining. Cells were lysed with
0.5% Triton X-100. SIGMAFAST p-nitrophenyl phosphate solution
(N1891-5SET, Sigma, St. Louis, MO) was added to the supernatant.
Absorbance at 405 nm was taken at several intervals from 0 to 60
min immediately after adding the substrate. Alkaline phosphatase
(ALP) activity was calculated as the rate of increasing absorbance
over time (slope value), and was normalized to the overall protein
concentration as determined by DC Protein Assay (Bio-Rad, Hercu-
les, CA). For calcium staining, cells after differentiation for 3 weeks
were xed with 70% ethanol and stained with 1% Alizarin Red
S solution (Sigma) at room temperature.
Flow cytometry and immunoblotting analysis
To determine the effects of IR on hMSC cell cycle progression,
a standard FACS cell cycle analysis protocol was used (15). Immu-
noblotting analysis of total cell lysates was performed as described
(14). The mouse monoclonal antibody against total actin (including
all isoforms) was from Chemicon (Temecula, CA). The phospho-
specic rabbit polyclonal anti-pSer15-p53 and mouse monoclonal
anti-pErk1/2 antibodies were from Cell Signaling (Danvers, MA).
870 I. J. Radiation Oncology d Biology d Physics Volume 73, Number 3, 2009
Microarray analysis and data processing
Global mRNA expression was proled by Affymetrix (Santa
Clara, CA) microarrays at the LBNL HTArray Genomics Core Fa-
cility. Samples were prepared, labeled, and hybridized according to
standard protocols (15). X-raytreated samples were hybridized to
U133AAofAv2 chips containing 22,944 probe-sets per chip, and
56
Fe ions-treated samples were hybridized to HT_HG-U133A chips
containing 22,277 probe-sets per chip. U133AAofAv2 was the ear-
lier version of HT_HG-U133A and was used because of chip-type
availability at the time. Signal intensities were obtained for all
probe-sets and were organized and sorted using GeneTrafc v.3.2
microarray analysis software (Iobion, La Jolla, CA).
Validation of microarray results by qRT-PCR. To verify gene
changes obtained from microarray analysis, we used quantitative re-
verse transcriptionpolymerase chain reaction (qRT-PCR) as previ-
ously described (15). Primers for cyclin A2 (CCNA2), cyclin B1
(CCNB1), cyclin E2 (CCNE2), cell division cycle 2 (CDC2), G-2
and S-phaseexpressed 1 (GTSE1), helicase, lymphoid-specic
(HELLS), antigen identied by monoclonal antibody Ki-67
(MKI67), RAD51 homolog (RAD51), topoisomerase II alpha
(TOP2A), and 18S ribosomal RNA (18S) were designed, using
the ABI Prism Primer Express software v.2.0 (Applied Biosystems,
Foster City, CA). A full list of primer sequences is shown in
Table E1. Standard curves for each gene were generated from
untreated hMSC. All gene levels for a given sample were normal-
ized to 18S levels from the same sample. Data were analyzed using
ABI Prism 7000 SDS software.
Statistical analysis
Data were rst analyzed with analysis of variance (ANOVA), fol-
lowed by a Holms t-test to nd the specic difference between
irradiation doses (p < 0.05, n = 3). To account for baseline variations
between experiments, values from each individual treated sample
(0.1 or 1 Gy) were rst normalized to values from the corresponding
sham control sample (0 Gy). After normalization, ratio values (radi-
ation/control) were pooled among all experiments and analyzed for
statistical signicance using a log-transformed one-sample t test (p <
0.05, n = 3). For qRT-PCR, differences were compared between
radiation (0.1 or 1 Gy) and control (0 Gy). Data are presented as
mean SD.
Bioinformatics analysis
To evaluate molecular interactions among regulated genes identi-
ed by microarray experiments, we performed network and pathway
analysis using Ingenuity program (Ingenuity Systems, Redwood
City, CA). The dataset contains the signicantly up- or downregu-
lated (at least twofold) genes. To select the most signicant
networks and pathways from our gene sets, we used the HG-
U133A_2 (www.affymetrix.com) as the reference sets to generate
sets of over-represented pathways that are known to be affected
by X-rays or
56
Fe ions at different doses. A p value for each network
and canonical pathway was then calculated. The score of network
was displayed as the negative log of the p value (-log
10
(p value)).
RESULTS
X-rays and
56
Fe ions inhibited proliferation without
impairing in vitro osteogenic differentiation process of
hMSC
Figure 1a demonstrates clear morphologic changes in
hMSC over a 7-day period when stimulated with osteogenic
differentiation media. These changes became noticeable
starting on Day 3 with an increasingly three-dimensional,
cobblestone-like morphology potentially indicative of osteo-
genic differentiation. However, no obvious morphologic dif-
ferences were detected in irradiated cultures when comparing
between radiation doses or types at any given time point.
Quantitative ALP activity data in Fig. 1b supported this qual-
itative observation. The hMSC showed minimal basal ALP
activity after 1 day (data not shown), moderate ALP activity
after 3 days, and a marked increase in ALP activity after ap-
proximately 1 week. Statistical analysis revealed no signi-
cant differences between radiation doses for any given time
point. The ALP is the early marker for osteogenic differenti-
ation; therefore, our results suggested that even after high
doses of radiation, the in vitro osteogenic differentiation
process of hMSC was not impaired. This was conrmed by
Alizarin Red S staining of hMSC in osteogenic media for
3 weeks, showing that calcium phosphate deposition (an os-
teogenic maturation marker) remained intact even after high
doses of IR (data not shown).
Despite the lack of morphologic changes and differences
in ALP activity after radiation exposure, the phase contrast
Fig. 1. Alkaline phosphatase (ALP) activity of human mesenchy-
mal stem cells (hMSC) in osteogenic media after exposure to
X-rays or
56
Fe ions. (a) Phase contrast images of hMSC after irradi-
ation from Day 0 to Day 7. (b) Bar graph representation of ALP ac-
tivity after irradiation. Activity is normalized to total protein
concentration. No statistically signicant differences were found
between doses (*p < 0.05 using Holms t test, n = 3).
X-ray and
56
Fe ions effects on mesenchymal stem cells d K. KURPINSKI et al. 871
images in Fig. 1a did reveal lower conuency of hMSC with
1 Gy X-rays and
56
Fe ions after several days in culture (most
noticeable at Days 3 and 7). This suggested that high doses of
IR inhibited the proliferation of hMSC, whereas the remain-
ing cells differentiated normally.
X-rays and
56
Fe ions induced differential effects on cell
cycle progression of hMSC
Figure 2a displays representative FACS dot plots for distri-
butions of G1/G0, S, and G2/M phases in hMSC, analyzed at
24 h after exposure to
56
Fe ions. Figures 2b and 2c showcom-
piled dot plot data for X-rays and
56
Fe ions in a dose-depen-
dent manner. Low-dose IR (0.1 Gy) did not cause any
detectable change in hMSC cell cycle distribution, whereas
1 Gy exposure induced signicant cell cycle modulation.
Both X-rays and
56
Fe ions at 1 Gy caused a decrease in the
population of S phase cells, suggesting a strong hindrance
to entering S-phase. 1 Gy
56
Fe ions also caused a signicant
increase in G2/M phase cells, whereas X-rays did not. In ad-
dition, 1 Gy
56
Fe ions decreased the fraction of cells in G1/G0
phase, whereas 1 Gy X-rays showed the opposite trend.
These ndings suggested that 1 Gy
56
Fe ions hindered the
cell cycle progression by impairing both the entry into
S phase and mitosis (G2/M), whereas 1 Gy X-rays mainly
inhibited the entry into S phase. The impaired mitotic function
(G2/M arrest) may account for the reduced number of cells in
G1/G0 after exposure to 1 Gy
56
Fe ions. Conversely, the
increased number of cells in G1/G0 after exposure to 1 Gy
X-rays may be caused by a reduced ability of these cells to
enter S phase (G1/G0 arrest), consistent with the decreased
number of cells in S phase.
We further characterized the kinetics of cell cycle pro-
gression of hMSC after exposure to IR. As shown in
Fig. E1, there was an apparent G2/M arrest starting at 5 h
for 1 Gy X-rays but not for 1 Gy
56
Fe ions. More detailed
kinetics for X-rays is shown in Fig. E2. G2/M arrest started
5 h for both 1 Gy and 3 Gy X-rays. For 1 Gy X-rays at 24
h, a portion of cells recovered from G2/M arrest and moved
into G1, whereas for 3 Gy X-rays, cells were in the persis-
tent G2/M arrest even after 24 h. Although 3 Gy X-rays
may give comparable levels of cell killing to 1 Gy
56
Fe
ions, their effects on cell cycle arrest were apparently
different. The 3 Gy X-rays induced G2/M arrest at early
PI intensity
b
0
0.2
0.6
0.4
0.8
1.0
*
*
X-rays
F
r
a
c
t
i
o
n

o
f

c
e
l
l
s
F
r
a
c
t
i
o
n

o
f

c
e
l
l
s
0.1 Gy
1 Gy
0 Gy
G1/G0 G2/M
c
G1/G0
0
0.2
0.6
0.4
0.8
1.0
S G2/M
0.1 Gy
1 Gy
0 Gy
*
*
*
56
Fe ions
S
a
0 Gy 0.1 Gy 1 Gy
S
(1.7%)
G1/
G0
(69.6%)
G2/M
(28.6%)
S
(13.6%)
G1/
G0
(77.9%)
G2/M
(8.5%)
S
G1/
G0
(73.3%)
G2/M
(9.4%)
F
I
T
C

(
B
r
d
U
)

i
n
t
e
n
s
i
t
y
10
1
10
2
10
0
10
1
10
2
10
0
10
1
10
2
10
0
10
1
10
2
10
0
10
1
10
2
10
0
10
1
10
2
10
0
(17.3%)
Fig. 2. Cell cycle distribution analysis of human mesenchymal stemcells (hMSC) exposed to ionizing irradiation. (a) Rep-
resentative dot plots of hMSC 24 h after exposure to
56
Fe ions. (b) Bar graph representation of cell cycle distribution 24 h
after exposure to X-rays. (c) Bar graph representation of cell cycle distribution 24 h after exposure to
56
Fe ions. *p < 0.05
using Holms t test (n = 3).
872 I. J. Radiation Oncology d Biology d Physics Volume 73, Number 3, 2009
time points (e.g., 5 h) but 1 Gy
56
Fe ions did not. Further-
more, the G2/M arrest at 24 h increased only 1.3-fold for
3 Gy X-rays (Fig. E2) while increasing more than 2.0-
fold for 1 Gy
56
Fe ions (Fig. 2c) when normalized with their
respective 0 Gy controls.
Taken together, our results suggested that although both
X-rays and
56
Fe ions seriously impaired the entry into
S phase,
56
Fe ions were a more potent inhibitor of mitosis
than were X-rays.
X-rays and
56
Fe ions induced differential p53 but not Erk1/
2 activation in hMSC
Figure 3 shows that p53 was activated as early as 1 min at
high dose but not at low dose, and
56
Fe ions induced higher
p53 activation than X-rays irrespective of growth media. Our
results conrmed that hMSC used in this study have normal
p53 functions. In contrast, Erk1/2 was not activated even at
high dose for either X-rays or
56
Fe ions. The apparent lack
of Erk1/2 activation was consistent with no apparent effects
of IR on in vitro osteogenic differentiation of hMSC (Fig. 1
and Discussion).
X-rays and
56
Fe ions induced differential transcriptome
responses in hMSC
The numbers of signicantly regulated genes are summa-
rized in Table 1. A total of 81 genes showed common
changes between X-rays and
56
Fe ions at 1 Gy. Complete de-
tails for each gene are listed in Table E2 (5 h) and Table E3
(24 h). For the 5-h gene set, fewer than 10 genes were up- or
downregulated. One of the major changes was the downregu-
lation of cyclin E2 (CCNE2), consistent with the cell cycle
arrest at this early time point. For the 24-h gene set, visual
inspection suggested that many of the common genes were
involved in cell cycle, DNA damage response and repair,
and other DNA interactions (e.g., MDM2, p21, and
Rad51). Some genes showed differential responses when
comparing between X-rays and
56
Fe ions (e.g., PIP5K1A,
DDX17, and AP1S1). The master osteogenic transcription
factor, Runx2, was not signicantly modulated by either X-
rays (0.951.14 fold) or
56
Fe ions (1.01.25 fold), based on
the averages of its duplicate probe sets. These results sug-
gested that X-rays and
56
Fe ions shared common damage re-
sponses, as well as having some distinct differential effects
on transcriptional regulation of hMSC.
qRT-PCR conrmed differential transcriptional regulation
of select genes in hMSC
We veried nine genes involved in cell cycle and DNAme-
tabolism by qRT-PCR (Fig. 4) The results were consistent
with the microarray ratios shown in Table E3, and conrmed
that IR induced downregulation of the cell cyclerelated
genes CCNA2, CCNB1, CCNE2, CDC2, GTSE1, and
MKI67 as well as HELLS and TOP2A, which are involved
in maintaining and modifying the coiled superstructure of
the DNA. Use of RT-PCR analysis also conrmed the down-
regulation of RAD51 which is involved in several facets of
DNA repair and cell cycle control (18).
The higher dose of irradiation had larger overall effects on
gene expression for all genes shown in Fig. 4, suggesting that
both X-rays and
56
Fe ions acted in a dose-dependent manner
for these genes. Several genes such as CCNB1, CCNE2, and
HELLS, were signicantly downregulated by 0.1 Gy of
56
Fe
ions but showed minimum response to the same dose of
X-rays. Furthermore, at a dose of 1 Gy, many genes were
signicantly downregulated with both radiation types, but
the overall changes were not of the same magnitude. For
example, CCNE2 and HELLS showed a greater average
decrease for
56
Fe ions than X-rays. Also, RAD51 was
signicantly downregulated with 1 Gy
56
Fe ions, but the cor-
responding decrease with X-rays was small and not statisti-
cally signicant.
Bioinformatics analysis shed lights on the underlying
mechanism for differential regulation of hMSC by X-rays
and
56
Fe ions
Figure 5 shows the comparisons of the top ranked path-
ways that were enriched in hMSC in response to IR. The sig-
nicance of over-representation of the top ranked pathways
Fig. 3. Immunoblotting analysis of human mesenchymal stem cells
(hMSC) exposed to ionizing radiation. Cells were irradiated with
4 Gy X-rays and lysed at different time points (a) or at 30 min after
exposure to different doses of X-rays (b). To compare the effects of
X-rays and
56
Fe ions, cells grown in basal media (c) or osteogenic
media (d) were exposed to IR and lysed at 3 to 5 h or 24 h after ion-
izing radiation (IR). Western blots were probed with anti-pSer15-
p53 and anti-pErk1/2 antibodies. Actin was used as the loading
control.
Table 1. Summary of microarray results: Number of genes
upregulated ($2.00 fold) or downregulated (#0.50 fold) in
human mesenchymal stem cells 24 h after exposure to
ionizing radiation
X-rays
56
Fe ions Common between
X-rays and
56
Fe ions
at 1 Gy 0.1 Gy 1 Gy 0.1 Gy 1 Gy
Upregulated 6 29 6 14 3
Downregulated 138 100 23 160 78
Total 144 129 29 174 81
X-ray and
56
Fe ions effects on mesenchymal stem cells d K. KURPINSKI et al. 873
was expressed as -log
10
(p value), and the pathways with
-log
10
(p value) above the threshold (p = 0.05; pink line)
were considered signicant. Several pathways, including
cytoskeleton signaling, IGF-1 signaling, integrin signaling,
estrogen receptor signaling, and insulin receptor signaling,
were enriched for low-dose (0.1 Gy) X-rays, and were not af-
fected signicantly under the other three radiation conditions.
In contrast, DNA/RNA metabolism and cell cycle regulation
were enriched for both high-dose (1 Gy) X-rays and
56
Fe
ions, with more signicant effects for
56
Fe ions. This is con-
sistent with a previous study showing that X-rays at low dose
preferentially modulate cytoskeleton genes (12).
The merged top-two networks shows similarities and dif-
ferences between 1 Gy X-rays (Fig. 6a, p < 10
63
10
32
)
and 1 Gy
56
Fe ions (Fig. 6b, p < 10
69
10
57
). Specically,
the
56
Fe ions network included genes involved in the follow-
ing: (1) chromosomal DNA replication and DNA repair:
DNA-directed polymerases (e.g., POLE2); (2) DNA replica-
tion and cell proliferation: DNA replication licensing factors
(e.g., MCM2); (3) DNA elongation: replication factors (e.g.,
RFC5); and (4) cell cycle control: cyclins (e.g., cyclin A) and
cyclin kinases/inhibitors (e.g., CDKN1A). The X-rays net-
work contained only a subset of the functions found in the
56
Fe ions network, specically genes associated with cell cy-
cle control, DNA replication, and cell proliferation. Both net-
works contained the DNA replication licensing factors MCM
2, 5, and 10. The MCM proteins are critical for DNA synthe-
sis and replication (19). Of particular interest is MCM2,
which is required for the entry into S phase and for cell divi-
sion. Reduced MCM2 expression was recently shown to re-
sult in severe stem/progenitor cell deciency and cancer (20).
We showed that MCM2 was downregulated by both 1 Gy
X-rays and
56
Fe ions, consistent with the S-phase reduction
in both cases, as shown in Fig. 2. The persistent G2/M arrest
for
56
Fe ions but not for X-rays may be caused by the more
severe downregulation of the four gene classes related to
DNA replication and cell proliferation, as shaded in Fig. 6.
Most of the genes in merged networks were downregu-
lated in hMSC after IR. One of the few upregulated genes
was CDKN1A, cyclin-dependent kinase inhibitor 1, or
CCNA2
*
*
* *
*
*
*
0.8
0.6
0.4
0.2
1.0
1.2
m
R
N
A

r
a
t
i
o
(
i
r
r
a
d
i
a
t
i
o
n

/

c
o
n
t
r
o
l
)
0
CCNB1 CCNE2 CDC2 GTSE1 HELLS MKI67 RAD51 TOP2A
*
*
*
*
*
*
*
*
*
*
*
* *
*
*
*
*
1 Gy
56
Fe ions
0.1 Gy
56
Fe ions
1 Gy X-rays
0.1 Gy X-rays
1.4
Fig. 4. Verication of select microarray genes using quantitative reverse transcriptionpolymerase chain reaction (qRT-
PCR). Bar graph shows relative changes in gene expression 24 h after irradiation when compared to respective 0 Gy control
(dotted line at 1.0 represents no change). *p < 0.05 using log-transformed one-sample t test (n = 3), for comparison between
irradiated samples and 0 Gy control only.
Fig. 5. Comparative pathway enrichments for human mesenchymal stem cells (hMSC) exposed to low and high doses of
X-rays and
56
Fe ions. Signicance is expressed as -log10 (p value). The threshold of p = 0.05 are shown as the pink line.
The bars that are above the line indicate signicant enrichment for the corresponding radiation exposure group.
874 I. J. Radiation Oncology d Biology d Physics Volume 73, Number 3, 2009
p21
cip1/waf1
(21). P21 prevents phosphorylation of critical cy-
clin-dependent kinase substrates and blocking cell cycle pro-
gression. We showed that p21 was upregulated by 2.1-fold by
1 Gy
56
Fe ions compared with 1.8-fold by 1 Gy X-rays, rel-
ative to sham control (0 Gy) (Table E3). These microarray
ndings were consistent with the data showing phenotypic
cell cycle arrest (Fig. 2), and p53 activation through Ser15
phosphorylation (Fig. 3).
DISCUSSION
Ionizing radiation induces a variety of lesions to DNA, in-
cluding single-strand breaks, double-strand breaks, and base
damages, depending on radiation quality and dose.
56
Fe ions
induce denser clustering of DNA lesions than X-rays, in-
creasing the likelihood of failed repairs. In addition, DSBs
in close proximity can misrejoin to give rise to chromosome
aberrations and permanent genetic damage (22). Our results
indicated that these biophysical events were associated with
perturbed DNA synthesis, DNA replication, and cell cycle
progression. Consistent with this view, drastic G2/M arrest
has been reported for other cell types, for example, Chinese
hamster cells after irradiation with other heavy-ion beams
(
13
C,
20
Ne,
40
Ar) (23). We showed that the majority of
hMSC exit G2/M arrest at 24 h after 1 Gy X-rays, whereas
a large portion is still in the persistent G2/M arrest from
1 Gy
56
Fe ions, causing more severe damage. We reason
that this may be caused by more efcient DNA damage re-
sponse and repair of hMSC after X-ray exposure. It has
been observed clinically that hMSC obtained from patients
after bone marrow transplantation originate from host tissue,
suggesting hMSC are inherently radioresistant in vivo (24,
25). Indeed, the radioresistance to g-rays of human bone mar-
rowderived, clonally expanded hMSC has been reported
(26). On the other hand, we observed minimum effects of
0.1 Gy
56
Fe ions on hMSC. This was consistent with the
fact that the cell nuclei of hMSC are large so that most cells
(75%) are hit at the dose of 0.1 Gy. For other smaller cells, the
majority of cells will not be hit but those that are hit get
a much higher dose than 0.1 Gy. For example, if the cells
are smaller and therefore only 25% of the cells are hit, the
hit cells will receive 0.4 Gy and the rest close to 0 Gy, for
an average of 0.1 Gy. In our case, the hit cells will receive
just a bit more than 0.1 Gy (0.133 Gy), which is still
a low dose.
Our study linked changes in gene expression at single pro-
tein and genome-wide levels with changes in cellular re-
sponses after IR. Phosphorylation of p53 at Ser15 is a key
indicator of DSBresponse and repair, and results in increased
activity and stability of p53 (27). The major p53 responses in-
cluded induction of growth arrest in the G1 and/or G2 phases
of the cell cycle through p21 and the induction of apoptosis
(28, 29). We showed that phosphorylation of p53 occurred
within minutes in response to IR. Our transcriptomic studies
showed that hMSC coordinated the transcriptional networks
of DNA replication, DNA strand elongation, and DNA bind-
ing/transferase activity for G2/Mand G1/G0 arrests 24 h after
a high dose of IR. Therefore, it is tempting to speculate that
the initial cellular response (minutes to hours) to IR is at
the posttranslational level (e.g., phosphorylation of p53),
whereas subsequent effects (hours to days) are mostly at
the transcriptional and translational levels after activation
Fig. 6. Comparison of interaction networks for genes regulated in human mesenchymal stem cells (hMSC) exposed to
1-Gy X-rays (a) and 1 Gy
56
Fe ions (b). Upregulated genes are coded in pink, whereas downregulated genes are coded
in green. Genes in shaded regions are four different function groups associated with cell cycle control, DNA strand
elongation, DNA binding/transferase activity, and replication fork formation.
X-ray and
56
Fe ions effects on mesenchymal stem cells d K. KURPINSKI et al. 875
of p53 and other transcription factors. Using a phosphopro-
teomics strategy to identify ATM/ATR substrates 1 h after
IR, it was demonstrated that interconnecting networks coor-
dinate during DNA damage responses (30). The work chal-
lenged the old paradigm of distinct DNA repair pathways
responding individually to different lesions after IR and val-
idated the merit of a systems biology approach.
Our results have implications for space exploration. We
found that IR, even 1 Gy
56
Fe ion irradiation, did not alter
the in vitro osteogenic differentiation process of hMSC.
Runx2, the master osteogenic transcription factor, is regu-
lated by pErk1/2 (2, 31). Lack of changes in osteogenic dif-
ferentiation was consistent with our observation that Runx2
expression and Erk1/2 activation did not change in hMSC af-
ter exposure to either X-rays or
56
Fe ions. Our results raise the
possibility of osteosarcoma formation after high doses of
56
Fe ion irradiation. Osteosarcoma develops during physio-
logical growth froman expanding cell population, suggesting
that the transformed cell is a bone-forming progenitor (32).
MSCs are physiological osteogenic progenitors, and osteo-
sarcoma may originate from MSCs. In vivo, hMSC differen-
tiate into osteoblasts which eventually become post-mitotic
bone cells. Because irradiated hMSC do not lose their in vitro
osteogenic differentiation potentials, we speculate that repo-
pulation of osteoblast/bone cells by these damaged MSCs
in vivo might trigger osteosarcoma. In fact, osteosarcoma
induction by high-LET radiation has been documented
(3336). We realized that the hMSC used in this study
were a subset of multipotent bone morrow stromal cells
and might contain heterogeneous populations of stem cells
and lineage-committed cells. Furthermore, IR may have
effects on in vivo bone formation because both proliferation
and osteogenic differentiation of MSC in vivo contribute to
the process. For example, both direct and indirect effects of
high-LET radiations on bone loss in rodents have been
reported (37, 38). Future studies of radioresponses of MSCs
in vivo using murine models and also along other lineages
(e.g., to adipocytes and chondrocytes) may provide more
insights into the origin of bone loss during space travel, as
well as cancer risks fromexposure to galactic cosmic rays (39).
CONCLUSION
In conclusion, we showed both phenotypically and mech-
anistically the differential effects of X-rays and high-energy
56
Fe ions on hMSC. Our results will assist the Department
of Energy with risk estimations for low-dose radiation,
and the National Aeronautics and Space Admininstration
with HZE risk estimations and countermeasures for space
exploration.
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