Rescue effects in radiobiology: unirradiated bystander cells assist irradiated cells through intercellular signal feedback.

Abstract

Mammalian cells respond to ionization radiation by sending out extracellular signals to affect non-irradiated neighboring cells, which is referred to as radiation induced bystander effect. In the present paper, we described a phenomenon entitled the "rescue effects", where the bystander cells rescued the irradiated cells through intercellular signal feedback. The effect was observed in both human primary fibroblast (NHLF) and cancer cells (HeLa) using two-cell co-culture systems. After co-culturing irradiated cells with unirradiated bystander cells for 24h, the numbers of 53BP1 foci, corresponding to the number of DNA double-strand breaks in the irradiated cells were less than those in the irradiated cells that were not co-cultured with the bystander cells (0.78±0.04foci/cell vs. 0.90±0.04foci/cell) at a statistically significant level. Similarly, both micronucleus formation and extent of apoptosis in the irradiated cells were different at statistically significant levels if they were co-cultured with the bystander cells. Furthermore, it was found that unirradiated normal cells would also reduce the micronucleus formation in irradiated cancer cells. These results suggested that the rescue effects could participate in repairing the radiation-induced DNA damages through a media-mediated signaling feedback, thereby mitigating the cytotoxicity and genotoxicity of ionizing radiation.

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Mutation Research 706 (2011) 59–64
Contents lists available at ScienceDirect
Mutation Research/Fundamental and Molecular
Mechanisms of Mutagenesis
journal homepage: www.elsevier.com/locate/molmut
Community address: www.elsevier.com/locate/mutres
Rescue effects in radiobiology: Unirradiated bystander cells assist irradiated cells
through intercellular signal feedback
S. Chena,b, Y. Zhaob, W. Hana, S.K. Chiud, L. Zhuc, L. Wub, K.N. Yua,∗
aDepartment of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong, Hong Kong
bKey Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031, People’s Republic of China
cOffice of Admission and Careers Advisory Service, Shenzhen University, Shenzhen 518060, People’s Republic of China
dDepartment of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong, Hong Kong
a r t i c l ei n f o
Article history:
Received 10 June 2010
Received in revised form 6 October 2010
Accepted 29 October 2010
Available online 10 November 2010
Keywords:
Radiation
Bystander effect
Radioresistance
53BP1
Signaling transduction
a b s t r a c t
Mammalian cells respond to ionization radiation by sending out extracellular signals to affect non-
irradiated neighboring cells, which is referred to as radiation induced bystander effect. In the present
paper, we described a phenomenon entitled the “rescue effects”, where the bystander cells rescued the
irradiated cells through intercellular signal feedback. The effect was observed in both human primary
fibroblast (NHLF) and cancer cells (HeLa) using two-cell co-culture systems. After co-culturing irradiated
cells with unirradiated bystander cells for 24h, the numbers of 53BP1 foci, corresponding to the num-
ber of DNA double-strand breaks in the irradiated cells were less than those in the irradiated cells that
were not co-cultured with the bystander cells (0.78±0.04foci/cell vs. 0.90±0.04foci/cell) at a statisti-
cally significant level. Similarly, both micronucleus formation and extent of apoptosis in the irradiated
cells were different at statistically significant levels if they were co-cultured with the bystander cells.
Furthermore, it was found that unirradiated normal cells would also reduce the micronucleus formation
in irradiated cancer cells. These results suggested that the rescue effects could participate in repairing
the radiation-induced DNA damages through a media-mediated signaling feedback, thereby mitigating
the cytotoxicity and genotoxicity of ionizing radiation.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
In radiation biology, radiation-induced bystander effect (RIBE)
refers to the phenomenon that irradiated cells send out signals to
unirradiated neighboring cells, and induce responses in these unir-
radiatedcellsasiftheyareirradiated[1].RIBEhasarousedimmense
research interests in the field and it has been reviewed in Refs.
[2–4]. Studies on the mechanisms on RIBE have suggested that gap
junction intercellular communication (GJIC) or soluble molecules
released by the irradiated cells are related to RIBE. Secreted solu-
ble molecules, including nitric oxide (NO), reactive oxygen species
(ROS),orTGF-?1,havebeenfoundtoplayimportantrolesinmedia-
mediated bystander effects [5–7]. It has also been pointed out that
bystander signaling after irradiation is likely an evolutionarily con-
served mechanism aimed at alerting nearby cells or organisms,
enablingapopulationresponsetobemountedeffectively[8].Gold-
berg and Lehnert made an assumption that bystander cells could
releasetheirownsignalingfactorsandaffectthedirectlyirradiated
cells [9]. However, there have been few researches on whether the
∗Corresponding author.
E-mail address: peter.yu@cityu.edu.hk (K.N. Yu).
irradiated cells themselves are deriving benefits from sending out
signals and thereby affecting and affected by the bystander cells, or
whether the bystander cells are in fact participating in some “res-
cue effects” which affect the fate of the irradiated cells. The present
work is devoted to studying such rescue effects.
Radiation insults result in the activation of intracellular sig-
naling event to regulate their responses and to determine the
fate of cells, i.e., apoptosis, survival or carcinogenesis. Signaling
transduction is essential for almost all aspects of eukaryotic cell
functions [10]. Understanding how intercellular and intracellular
signals are produced, activated and transmitted in an effective and
faithful manner inside a cell or between neighboring cells is vital
to the understanding of such important biological processes as cell
proliferation, differentiation, motility, survival, and tumorigenesis.
Focuses on intercellular signaling transduction under pathologi-
cal status or environment insults will be useful for treatment and
prevention of diseases, which might involve inflammation, hypo-
genesis, and carcinogenesis, and others.
Interestingly, there is evidence that signaling transduction
will not cease after the irradiated cells release the intercellular
molecule(s) to the bystander cells. The molecule(s) will continue
to stimulate intracellular signal pathway(s) and cause a series of
bystander response in bystander cells. It is most intriguing if the
0027-5107/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrfmmm.2010.10.011

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S. Chen et al. / Mutation Research 706 (2011) 59–64
bystander cells also release intercellular molecules of their own. In
fact, activation of MAPK pathway [11] and NF-?B related pathway
[12], and release of NO/ROS [7,13] in bystander cells have pro-
vided supporting evidence. It was therefore natural to ask what the
purposes and target recipients were of the intercellular molecules
released by bystander cells, on which we explored in the present
work. We studied whether the bystander cells were participating
in the “rescue effect”, e.g., to assist the irradiated cells in mitigat-
ing the radiation-induced damages. Sokolov et al. mentioned that
media from cancerous cells resulted in a DNA damage response
in normal human cells that is reminiscent of RIBE [14]. However,
if bystander rescue effects occurred between bystander normal
cells and irradiated cancer cells, these will have even more far-
reaching consequences for radiation therapy. For these studies, cell
co-culture systems were employed, and the studied biological end
points were 53BP1 induction, micronucleus (MN) induction, apop-
tosis and colony formation.
2. Materials and methods
2.1. Cell culture and conditioned media preparation
Normal human lung fibroblasts (NHLF) and human cervical cancer cells (HeLa)
were routinely cultured in ?-MEM containing 10% fetal bovine serum (FBS)
supplementedwith2mMl-glutamine,100units/mlpenicillin,and100?g/mlstrep-
tomycin. The cultures were maintained at 37◦C in a humidified 5% CO2incubator.
The medium was changed every 2 days, and the cells were passaged at 90% conflu-
ence.
A total of 4×105NHLF cells were inoculated into 100mm tissue culture dishes.
Twodayslater,whenthecellswereat50%confluence,themediawereaspiratedand
12ml of fresh growth media was added. After 24h, the media were collected and
stored at 4◦C as conditioned media for the co-culture system as outlined in Protocol
2.
2.2. Alpha-particle irradiation
The average energy and LET of ? particles derived from the241Am irradiation
source, as measured at the cell layer, were 3.5MeV and 128keV?m−1, respectively,
and the ? particles were delivered at a dose rate of 1.0cGys−1.
The co-culture systems for Protocols 1 and 2 were fabricated as described in
Refs. [15,16] with some modifications. According to Protocol 1, 3.5×104expo-
nentially growing cells were inoculated into a specially designed rectangular dish
(10mm×6mm, Fig. 1A). After 24h, the cells were irradiated with a 20cGy dose
of ?-particles. After incubation for another 24h, the cultures were fixed with 2%
paraformaldehyde solution for subsequent immunostaining experiments. The cells
inthedisheswithoutaluminumfoilswereallirradiatedwitha20cGydoseof?par-
ticles and were classified as irradiated cells (IRall). On the other hand, the cells in the
irradiated regions in dishes covered with aluminum foils were classified as “irradi-
atedcellspartneredwithbystandercells”(IRby),andshamirradiatedcellswereused
as controls. For Protocol 2, as shown in Fig. 1B, 8×104and 2.5×105exponentially
growing cells were inoculated in the outer and inner dishes, respectively. After 20h,
the cells in the inner dishes were irradiated with a predetermined dose. The inner
dishes were then slotted into the outer dishes, with the cells in the inner dishes fac-
ingthecellsintheouterdishes(Fig.1).Around2.2mloftheconditionedmediawere
added into the co-culture system to cover all the cells. After culturing for a desired
period of time, the cells in the inner dishes were trypsinized and prepared for the
subsequent endpoint experiments: micronucleus test, clonogenic survival test and
apoptosis assay. The cells in the inner dishes were IRallcells, and those in the outer
dishes were IRbycells, and sham irradiated cells were controls. All the dishes used in
Protocols 1 and 2 were made of stainless steel and each of them had a 3.5-mm-thick
replaceable Mylar bottom. The experiments were performed according to Protocol
2 unless otherwise stated.
2.3. Alpha particle traversals
Calculations for ?-particle irradiation were described in detail by Portess et al.
[17]. In the present work, poly-allyl diglycol carbonate (PADC) films (Page Mould-
ings, England) were used to record the positions of ?-particle traversals. After 20
or 40cGy ?-particle irradiation, the PADC films were etched for 2h in a 7N NaOH
solution at 70◦C. The cells for analyses were fixed using paraformaldehyde solu-
tion and stained with 5?g/ml Hoechst 33342 (Molecular Probes, OR, USA). The
images of ?-particle tracks revealed on the etched PADC films together with the
cell nuclei stained with Hoechst 33342 were captured under a microscope (Olym-
pus IX71 Inverted Research Microscope, Olympus, Japan). The average area of cell
nuclei was determined by Image-Pro Plus (Media Cybernetics, Bethesda, USA) as
142.5±6.9 and 224.9±10.1?m2for NHLF and HeLa cells, respectively. These fig-
ureswereusedtocalculatetheaveragenumber(n)of?-particletraversalsaccording
Fig.1. SchematicdiagramsforProtocols1and2.(A)InProtocol1,culturecellswere
grown on a piece of Mylar film attached to the bottom of a specially made 10mm
dish,andhalfofthebottomwascoveredwithanaluminumfoiltostopthe?-particle
irradiation. (B) In Protocol 2, the setup was made for coculturing the bystander cells
withtheirradiatedcells.ThebottomoftheouterdishwascoveredwithaMylarfilm
as in Protocol 1 but without being covered with an aluminum foil. The inner dish
with a smaller diameter which holds the irradiated cells can be put into the outer
dish with the two types of cells covered with a conditioned medium.
to
n =
DA
0.16L
where L is the LET (keV?m−1), A is the average area of the cell nuclei (?m2) and D
is the dose (Gy).
2.4. Immunostaining of 53BP1
53BP1 is a regulator protein of p53 and it has been considered as a biomarker of
DSBs,and?-H2AXhasbeenwidelyusedtoevaluatetheriskofenvironmentalinsults
[18,19]. Like NBS1 and BRCA1, 53BP1 is a member of the BRCT (BRCAI C-Terminal)
repeat family. BRCT domain is found in a large number of proteins involved in the
cellular responses to DNA damages. Staining of 53BP1 with immunocytochemistry
was used to monitor the radiation-induced DSBs in the irradiated cells.
The fixed cells were rinsed and permeabilised with TNBS solution (PBS sup-
plemented with 0.1% Triton X-100 and 1% FBS), followed by exposure to rabbit
polyclonal anti-53BP1 antibody (Novus Biologicals, Littleton, USA) for 1h. The cells
were then incubated with FITC-conjugated anti-rabbit secondary antibody (Zhong-
shan Goldenbridge Biotechnology Company, Beijing, China) for another 1h. After
washing 3 times with TNBS for 5min each, the cells were counterstained with
5?g/ml Hoechst 33342. Immunofluorescent images were captured by a TCS SP2
confocal laser scanning microscope (Leica, Wetzlar, Germany). At least 700 cells in
each sample were counted and statistical analyses were performed on the means of
the data obtained from three independent experiments.
2.5. Micronucleus (MN) test
The formation of MN was assayed using the cytokinesis block technique as
described by Fenech [20]. Briefly, cultures were dissociated by trypsinization, and
approximately 5×104cells were seeded in Ø35mm culture dishes. Cytochalasin B
(CB, Sigma, Steinheim, Germany) was added into the culture medium with the final
concentration of 2.5?g/ml at 4–6h post seeding. After incubation for 36h, the cells
were fixed with methanol/acetic acid [9:1 (v/v)], stained with 0.1% acridine orange
for 5min, and viewed under a fluorescence microscope. At least 1000 binucleated
cells were examined and the frequency of micronucleus formation was calculated
as the ratio of the number of binucleated cells with micronuclei to the total number
of binucleated cells. The experiment was repeated at least three times, with each
experiment including three samples.

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Fig. 2. Effect of bystander cells on the DSB formation or repair in the NHLF cells irradiated with 20cGy dose of ? particles. The cells were fixed after incubation for 30min
(A–C) or 24h (D–F) post-irradiation. The 53BP1 foci (colored red) were stained with FITC and the cell nuclei were stained with Hoechst 33342 (colored blue). (A and D) Sham
irradiated controls; (B and E) the irradiated cells with the bystander cells; (C and F) all irradiated cells. (G) The quantification of 53BP1 foci within a cell at two different time
points. The data were pooled from three independent experiments and the results were represented as mean±s.d.
2.6. Apoptosis assay
Theflippingofphosphatidylserinefromtheinsidetotheoutsideofplasmamem-
brane of apoptotic cells was determined by using labeled annexin V-fluorescein
isothiocyanate (FITC, BD Biosciences, San Jose, USA), a Ca2+-dependent phospho-
lipid binding protein with high affinity for phosphatidylserine [21]. The assay was
performed as outlined by the manufacturer and the cells were counterstained with
propidium iodide (PI) to distinguish apoptosis from necrosis. Flow cytometry was
employed to visualize the bound FITC and necrotic cells (which were stained with
bothPIandFITC)weregatedoutsothatanaccuratedeterminationofthepercentage
of apoptotic cells could be made.
2.7. Colony formation assays
The cultures were trypsinized, counted, and replated into tissue culture dishes
for colony formation (250cells/dish: Ø100mm culture dishes for NHLF cells;
Ø60mm culture dishes for HeLa cells). After incubation for 10d, the cells were fixed
with methanol/acetic acid [9:1 (v/v)], and stained with 1% crystal violet. The exper-
iment was repeated at least three times, each experiment including three samples.
2.8. Statistical analyses
The data were presented as means and standard derivations. The significance
levels were assessed using Student’s t-test. A p-value of 0.05 or less between groups
was considered statistically significant.
3. Results
3.1. Bystander cells decreased 53BP1 foci induction in irradiated
NHLF cells
To investigate the rescue effect on DSB formation or repair
in the cells irradiated by ? particles, the formation of foci
by the 53BP1 protein in NHLF cells was monitored according
to Protocol 1. As shown in Fig. 2, irradiation with ?-particle
induced a statistically significant increase in 53BP1-positive foci at
30min post-irradiation, not only in IRallcells (1.92±0.08foci/cell),
but also in IRbycells (1.79±0.20foci/cell), and there were no
statistically significant differences between these two types of
cells. At the 24h time point, the 53BP1 positive foci in either
IRallcells or IRbycells were decreased by a statistically signifi-
cant amount. It was most important that the number of 53BP1
foci in IRby cells was less than that in IRall cells at a sta-
tistically significant level (0.78±0.04foci/cell for IRbycells and
0.90±0.04foci/cell for IRallcells, p<0.02). These results indicated
that the bystander cells produced signals which might be involved
in DSB repair.

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Fig. 3. Effect of bystander cells on the micronuclei (MN) formation in NHLF cells irradiated by ? particles at two different doses. The cells were inoculated for the formation
of MN after the cells were co-cultured for 24h. The results showed that the bystander cells decreased the MN formation in irradiated cells at a statistically significant level
(p<0.05). The data were pooled from at least three independent experiments and the results were represented as mean±s.d.
3.2. Bystander cells decreased MN induction in irradiated NHLF
cells
To investigate the rescue effect on micronuclei (MN) formation
in the cells irradiated by ? particles, the NHLF cells were treated
according to Protocol 2. After irradiation with ? particles and fur-
ther incubation for 24h, the ratio of number of binucleated cells
with micronuclei to the number of binucleated cells increased.
Interestingly, if bystander cells were present, the MN induction
in the irradiated cells was reduced by a statistically significant
amount. As shown in Fig. 3, if all the cells were irradiated by 20 and
40cGy of ?-particles, the ratios of MN in IRallwere 4.8 and 10.2%,
respectively. However, the ratios in the bystander cells (IRby) were
reduced to 3.2 and 7.6%, respectively (Fig. 3, p<0.05). The results
indicated the presence of the rescue effect, i.e., the bystander cells
could help mitigate the micronuclei formation in the irradiated
cells.
3.3. Bystander cells decreased apoptosis of irradiated NHLF cells
To investigate the rescue effect on the cytotoxicity of radiation,
the extent of cell death in the irradiated cells was studied with
apoptosis assay and colony formation assay. As expected the num-
ber of apoptotic cells, which are annexin V-positive (FL1-H), was
increased by a statistically significant level at 72h post-irradiation
(see Fig. 4A–C). However, the presence of bystander cells signifi-
cantly decreased the radiation-induced apoptosis in the irradiated
cells (Fig. 4D, IRall=80%, IRby=73%, p<0.02). The colony formation
of the irradiated cells at 24h post-irradiation was also studied. The
surviving fraction (SF) is defined as [colonies counted/(colonies
seeded×plating efficiency)]. The SF of IRallwas lower than that
of IRbyin all repeated experiments (Fig. 4E). However, there were
no statistically significant differences between the IRalland the
IRbycells. These results indicated that the rescue effect might have
reduced the number of the radiation-induced apoptotic cells.
3.4. Bystander NHLF cells rescued irradiated HeLa cells
To study whether normal bystander cells would also exert the
rescue effect in irradiated cancer cells, HeLa cells were grown in
the inner dishes while NHLF cells were cultured in the outer dishes
according to Protocol 2. After irradiation of the HeLa cells, the inner
dishes were slotted into the outer dishes. The micronuclei for-
mation and the colony formation were examined after incubating
for 24h. As in Fig. 5A, the bystander cells decreased the ratio of
micronuclei in the irradiated HeLa cells at a statistically significant
level (p<0.05). The measurement of surviving fraction in the two
typesofcellsshowedasimilareffectinirradiatedNHLFcells,i.e.,the
surviving fraction of IRallwas lower than that of IRbyin all repeated
experiments (Fig. 5B). However, the difference was not statistically
significant. These results suggested that the rescue effect not only
couldoccurinnormalgrowingcells,butitcouldalsooccurbetween
irradiated cancer cells and unirradiated normal cells.
4. Discussion
RIBE has aroused immense interests in the field of cancer
research in the past decade because of its implications on carcino-
genesis. Irradiated cells release intercellular signaling molecules
which result in cell growth, cell proliferation, DNA damage, chro-
mosome aberration, transformation and carcinogenesis. In the
present study, we observed a phenomenon termed the “rescue
effect”, in which the unirradiated bystander cells send out “feed-
back signals” back to the irradiated cells.
Inordertostudythisphenomenon,thepopulationsoftheirradi-
atedcellsandtheunirradiatedbystandercellsshouldbeseparated.
We therefore came up with an experimental setup in which the
irradiated cells and the unirradiated bystander cells were cultured
in different parts of the irradiated systems (in Protocols 1 or 2). To
ensure that there are no bystander cells in the population of the
irradiated cells, the cells in the irradiated regions or dishes were
irradiated with 20 or 40cGy dose of ? particles, and thus all of
the cell nuclei were expected to be hit by at least one ? particle
(Table 1). Even when the random nature of radioactivity is taken
into account, or when the Poisson distribution was considered,
about 98% of the cell nuclei were hit by ? particles.
Under normal physiological conditions, cells not only absorb
nutrientsandreceivesignalingmoleculesfromothercells,butthey
also secrete intercellular molecules, such as metabolites, extracel-
lular proteins and cytokines. It is pertinent to study the locations
from which the signals causing the rescue effects are released, and
to find out whether these signals are products of normal living
cells or molecules released by the bystander cells. To eliminate the
effect of normal metabolites, and to focus on the signals released
by the bystander cells which had been stimulated by the signal-
ing molecules released by the irradiated cells, all the growth media

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Fig. 4. Effect of bystander cells on the cell death of NHLF cells irradiated by ? particles. The cells were stained with annexin V-FITC after co-cultured for 72h, and analyzed
by flow cytometry. FL1-H was the signal from annexin V-FITC labeled cells. (A) Sham-irradiated control; (B) irradiated cells with bystander cells; (C) irradiated cells without
bystandercells.(D)ThequantificationoftheapoptoticcellpopulationwithannexinVsignal.Theresultsshowedthatthebystandercellsdecreasedthepopulationofapoptotic
cells in the irradiated cells (p<0.05). (E) The surviving fraction of NHLF cells at 24h post-irradiation. The differences in the surviving fractions between IRalland IRbycells
were not statistically significant. Data were pooled from three independent experiments and the results were represented as mean±s.d.
usedinProtocol2post-irradiationwereconditionedmediaderived
from exponentially growing normal cells, as described in Section 2.
In this way, the intercellular signaling effects between the irradi-
ated cells and the bystander cells were clearly identified.
Based on the “traditional” theories of RIBE, the irradiated cells
can induce RIBE via GJIC or soluble molecules. In fact, the results
obtained by Mothersill et al. [22] showing that repair deficient
cells could be made to behave as though repair proficient using
mediumfromrepairproficientcellsalsosupportedtherescueeffect
reported in the present paper.
Recently, there is evidence that the bystander cells also can
affect their neighboring cells. Kadhim et al. [23] observed that
Fig. 5. Effect of bystander cells on the MN formation and the cell death of cancer cells (HeLa) irradiated by ? particles. (A) The frequency of the formation of micronuclei was
measured after the cells were irradiated with a 20cGy ?-particle. (B) The surviving fraction of the cells 24h after irradiation with a 20cGy ?-particle was also measured.
Data were normalized to sham irradiated cells in the MN test. The results showed that the bystander cells decreased the micronuclei (MN) formation in the irradiated cells
at a statistically significant level (p<0.05). Data were pooled from at least three independent experiments and the results were represented as mean±s.d.

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Table 1
Dosimetry for ?-particle radiation.
Dose (cGy) NHLF cellsHeLa cells
Average number of ?-particles
traversed a nucleus
Percent of cells with nuclei
traversed by ?-particles
Average number of ?-particles
traversed a nucleus
Percent of cells with nuclei
traversed by ?-particles
0.5
20
40
0.04
1.53
3.06
30.06
2.41
4.83
4
98
99
98
99
the unirradiated bystander cells could induce chromosome aber-
rations and adaptive response in other bystander cells via the
solublebystanderfactor(s).Inthisstudy,ourobjectivewastoshow
whetherthebystandercellswouldsendoutintercellularmolecules
which would have any effect (i.e., the rescue effect) on the irra-
diated cells. We found that the bystander cells can enhance the
repair of DNA damage and lessen the extent of apoptosis of the
irradiated cells by probably sending out intercellular signals. In
particular, the rescue effect reduced the radiation-induced MN
formation in the irradiated cells at statistically significant lev-
els (Figs. 3 and 4). Mackonis et al. [24] reported an interesting
“type III effect” which referred to an increase in the survival of
cells having received a high radiation when their nearby cells
received a low radiation dose. This might appear similar to the
rescue effect reported in the present paper. However, there is
one big difference between the two. For the “type III effect”,
all the cells, including the “nearby” cells, were irradiated. In
contrast, for the rescue effect, the bystander cells were not irra-
diated.
The bystander cells did not decrease the DSB formation at the
30min time point at a statistically significant level, as measured
by the numbers of the 53BP1 foci, but the frequency in the DSB
formation was decreased at the 24h time point at a statistically
significant level, possibly by facilitating DNA repair (Fig. 2). Similar
to the role of DNA repair proteins in radio-adaptation, activation
of the DNA repair pathway through intercellular signaling may be
responsible for these effects.
These findings led us to investigate whether the irradiated
cancer cells could involve the unirradiated normal cells in the
rescue effect. Accordingly, after co-culturing the irradiated HeLa
cells and unirradiated NHLF cells, the tests for micronuclei for-
mation as well as surviving fractions in HeLa cells were carried
out. The decrease in the formation of micronuclei in the irradi-
ated cells cocultured with the unirradiated cells suggested that
the NHLF cells without receiving any ?-particle irradiation were
involved in the rescue effect to assist the irradiated HeLa cells in
dealing with the radiation damages (Fig. 5). This has significant
implications on the effects of radiation therapy, as the irradiated
cancer cells would send out signals to coerce the bystander unir-
radiated normal cells to exercise rescue actions to assist them.
In other words, the intercellular communication network might
have a significant impact on the efficiency of radiation ther-
apy.
Conflict of interest statement
None.
Acknowledgement
This research was supported by the National Nature Science
Foundation of China under Grant No. Y05JM64506.
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