Bystander effect and adaptive
response in C3H 10T˝ cells
Mitchell et al / International
Journal of Radiation Biology v.80, n.7, July 2004 1jul04
S. A. Mitchell,1 G. Randers-Pehrson, D. J. Brenner and E. J. Hall
Center for Radiological Research, Columbia University, New York, New York
10032
1 Address for correspondence: Center for Radiological Research, Columbia
University, 630 West 168th Street, New York, NY 10032; e-mail: sm2104@columbia.edu.
Mitchell, S. A., Randers-Pehrson, G., Brenner, D. J. and Hall, E. J. The
Bystander Response in C3H 10T˝ Cells: The Influence of Cell-to-Cell Contact.
Radiat. Res. 161, 397–401 (2004).
Although radiation-induced heritable damage in mammalian cells was thought to
result from the direct interaction of radiation with DNA, it is now accepted
that biological effects may occur in cells that were not themselves traversed by
ionizing radiation but are close to those that were. However, little is known
about the mechanism underlying such a bystander effect, although cell-to-cell
communication is thought to be of importance. Previous work using the Columbia
microbeam demonstrated a significant bystander effect for clonogenic survival
and oncogenic transformation in C3H 10T˝ cells. The present study was
undertaken to assess the importance of the degree of cell-to-cell contact at the
time of irradiation on the magnitude of this bystander effect by varying the
cell density. When 10% of cells were exposed to a range of 2–12 
particles, a
significantly greater number of cells (P , 0.0001) were inactivated when cells
were irradiated at high density (.90% in contact with neighbors) than at low
density (<10% in contact). In addition, the oncogenic transformation frequency
was significantly higher in high-density cultures (P , 0.0004). These results
suggest that when a cell is hit by radiation, the transmission of the bystander
signal through cell-to-cell contact is an important mediator of the effect,
implicating the involvement of intracellular communication through gap
junctions.
INTRODUCTION
It has been a long-held tenet of radiation biology that cellular DNA damage
required direct interaction of radiation with DNA. This ‘‘targeted’’ DNA
damage occurred either by direct ionization or by production of hydroxyl
radicals in water molecules adjacent to DNA. However, over the past decade,
considerable evidence has emerged for the existence of a ‘‘non-targeted’’
phenomenon that has been termed the bystander effect (1). The bystander effect
is defined as the observation of a biological response in cells which have not
been directly traversed by ionizing radiation but which results from signals
initiating in cells in which energy has been deposited. This may be an important
phenomenon influencing the shape of the dose–response curve, particularly at
low doses, where there are many nontraversed cells, and several models are
available which assess the significance of such effects at low doses (2, 3).
Although the effect of such bystander responses on the low-dose cancer risk is
not fully understood (4), they are thought to represent a balance between
protective mechanisms such as apoptosis and differentiation (5) and potentially
harmful mechanisms, in which DNA damage and potential genomic instability are
mediated through bystander signals (3, 6).
To date, many studies, primarily using single cell in vitro systems but also
in vivo, have confirmed a bystander response for several end points including
clonogenic survival, oncogenic transformation, micronucleus induction and gene
expression (reviewed in ref. 7). These studies have employed three distinct
protocols: the conventional irradiation of cells with low fluences of
particles, the transfer of medium from irradiated onto unirradiated cells, and
the use of charged-particle microbeams, which have made it possible to define
precisely which cells are traversed by an exact number of
particles (reviewed
in ref. 8). However, the specific factor(s) produced by irradiated cells
responsible for the bystander response remains unknown, although several
candidate molecules have been suggested (9–11).
several candidate molecules have been suggested (9–11). It seems likely
that the bystander effect is mediated by two distinct mechanisms that depend on
the experimental protocol employed and that both may contribute to the final
response (12). The first involves the transmission of a secreted, soluble
extracellular factor from irradiated to unirradiated cells, often over some
considerable distance (13, 14). The second is direct communications between
adjacent cells through gap junctions (15, 16).
Previous studies using the Columbia microbeam have shown a significant
bystander effect for the end points of clonogenic survival and oncogenic
transformation in C3H 10T˝ cells (17, 18). The aim of the present study was to
assess whether the magnitude of this effect was dependent upon cell-to-cell
proximity at the time of irradiation. To achieve this, cells were plated at both
high and low density, targeted with a range of
particles aimed at the
centroid of the nucleus, and assessed for clonogenic survival and oncogenic
transformation.
MATERIALS AND METHODS
Microbeam Irradiation
The Columbia microbeam system and the irradiation procedures have been
described in detail previously (19). Briefly, cells were attached to the thin
bases (3.8 µm polypropylene) of 6.3-mm-diameter miniwells to give a final cell
density of either 200 or 2000 cells per well. Individual nuclei were then
identified and located with a computer-controlled optical image analysis system.
For each dish, a computer/microscope-based analysis system first automatically
locates and records the x,y coordinates of the nuclei of cells on the dish. Next
the dish is moved sequentially under computer control such that the first cell
nucleus is positioned over a highly collimated -particle
beam. The beam shutter
is opened until the required numbers of
particles are detected (with a
gas-filled ion counter mounted on the microscope lens) to have passed through
the nucleus. The shutter is then closed and the next cell is moved over the
beam. The overall spatial precision of the beam, including positioning and beam
spread, is about ±3.5 µm, compared with an average nuclear cross-sectional area
of the cells of approximately 200 µm2 and a cellular cross-sectional area of .500
µm2 (20). In the present study, 5.3 MeV
particles accelerated by a Van de
Graaff accelerator were used for the irradiations. The average stopping power of
the particles traversing the cells was 90
keV/µm. The search and irradiate
software can be instructed to expose any given proportion of the cells, selected
at random, to any desired number of
particles. To assess clonogenic survival,
either 10% or 100% of the cells were exposed to a range of
particles from 2–12
through the nucleus and for oncogenic transformation 10% of the cells were
exposed to 8 particles. Dishes used to assess plating efficiency were
sham-irradiated, i.e. handled in an identical fashion except that the beam
shutter was not opened. Irradiation times for each dish were approximately 6–10
min.
Cell Culture
Prior to irradiation, C3H 10T˝ mouse fibroblast cells between
passages 9 and 11 were grown in Eagle’s basal medium supplemented with 10% FBS
and penicillin/streptomycin. Eighteen hours before exposure, 200 or 2000
exponentially growing cells were plated into miniwells as described above. Prior
to irradiation, cells plated at the high density formed a con- fluent culture
with a high degree of cell-to-cell contact, in contrast to the low-density
cultures in which the cells were individuals, separated from their neighbors.
The attached cells were stained for 0.5 h with 50 nM of the vital nuclear dye
Hoechst 33342, enabling individual nuclei to be identified and located using the
optical image analysis system. Immediately prior to irradiation, cells were
washed with serum-free medium to avoid fluorescence from serum components and
irradiations were carried out in the presence of a thin film of serum-free
medium. After irradiation, cells were washed twice with PBS and trypsinized from
the irradiation container. For assessment of clonogenic survival, approximately
100 viable cells were plated into 100-mm culture dishes and incubated for 2
weeks, and the resulting colonies were stained with 2% crystal violet to
determine both the plating efficiencies and surviving fractions of the control
and irradiated cells. To assess oncogenic transformation, cells were replated at
a low density of about 300 viable cells per dish. The cells were incubated for 7
weeks with culture medium changed every 12 days before they were fixed and
stained with Giemsa to identify morphologically transformed type II and III
foci, as described elsewhere (21).
Data from a minimum of three independent experiments were pooled. All data
for clonogenic survival were presented as means with standard errors. Surviving
fractions measured at the doses tested were fitted with the linear-quadratic
equation. The statistical significance of differences between groups was tested
by Student’s t test and between survival curves using multiple regression
analysis.
FIG. 1. Clonogenic survival of C3H 10T˝ cells after nuclear traversals by
5.3 MeV particles. Data points represent the means
± SEM from at least three
repeat experiments. The triangles indicate survival when all cell nuclei on the
dish are exposed to a range of -particle
traversals, from 2 to 12 per nucleus.
The dotted line shows the percentage of cells that would be expected to survive
when 10% of the cells are irradiated and is calculated from the survival
observed when all cells are irradiated. The circles and squares show survival
when 10% of the cells are irradiated at high and low density, respectively. The
extent to which survival seen at the two densities falls below the dotted line
indicates the magnitude of the bystander effect.
RESULTS
The results of the experiments to compare clonogenic survival for
cells exposed at high and low density are shown in Fig. 1. The clonogenic
survival obtained when 100% of cells are hit by various numbers of
particles is indicated by the solid triangles and is in agreement with that seen
previously in C3H 10T˝ cells (18).
The dotted line shows the percentage of the cells that would be expected to
survive when 10% of the cells are irradiated in the absence of any bystander
effect. It is calculated by applying the 100% survival curve to the 10% of cells
that were actually irradiated. The results are compared with those obtained from
the irradiation of 10% of the cells at either high or low density, which are
shown by the solid circles and squares, respectively. The extent to which these
lines fall below the dotted line indicates the magnitude of the bystander
effect, although only the cell survival observed in high-density cultures is
significantly lower (P < 0.0001 compared to P = 0.11 at low density). At both
cell densities, the surviving fractions fall progressively as more
particles traverse the nucleus, but the amount of cell killing is significantly greater at
the high cell density compared with low-density cultures (P < 0.0001).
TABLE 1 Clonogenic Survival Rates, Number of Dishes Exposed, Numbers of Viable Cells
Exposed in Transformation Studies, Number of Transformed Clones Produced, and
Transformation Frequencies for Microbeam Irradiations
No. of cells Clonogenic surviving No.a of No. of Transformation
plated, no. of fraction (plating No. of dishes viable cells transformants frequency/104
particles efficiency)(±SEM) exposed exposed/104 produced surviving cells
200,0 (0.23±0.02) 71 0.9 1 1.1
2000,0 (0.17±0.03) 60 2.1 1 0.5
200,8 0.93±0.02 155 1.8 6 3.3
2000,8 0.81±0.01 73 1.5 14 9.6
a Estimated, accounting for plating efficiency and clonogenic survival.
Results from experiments conducted to evaluate the effect of cell density at
the time of irradiation on oncogenic transformation are shown in Table 1. In
these studies, a total of approximately 3.1 × 105 cells were individually
imaged, positioned and irradiated. At high density, a transformation frequency
of 9.6/104 viable cells was seen, which is similar to that found previously in
high-density cultures (17). Using previously published data (22), it is possible
to calculate that when 10% of the cells in a population are irradiated with 8
particles, the expected transformation frequency in the absence of a bystander
effect would be 2.1/104 viable cells. This is lower than that seen in the
present study at both cell densities, although again the difference is only
significant in the case of the high-density cultures (P < 0.0001 compared to
P = 0.28 at low density). A statistically significant threefold decrease in the
transformation frequency was observed in the low-density cultures relative to
high-density cultures (P < 0.0004).
DISCUSSION
It is now widely accepted that radiation-induced heritable effects in
mammalian cells are not solely the result of direct damage to DNA, and there is
now evidence for a number of non-targeted effects, including the bystander
response, which do not require a direct nuclear exposure (1).
Although reproducible bystander effects have now been demonstrated for a
range of biological end points, the mechanisms by which the biological insult is
transmitted from targeted to non-targeted cells have not been fully elucidated
(reviewed in ref. 8).
One causative agent may be the secretion from irradiated cells of a soluble
factor(s) into the medium that then elicits a biological response in adjacent,
unirradiated cells (13). Alternatively, when densely cultures are irradiated,
the signal may be transmitted through cell-to-cell communication between
adjacent cells (reviewed in ref. 7). When cells are in close contact, they can
communicate through gap junctions which are intercellular membrane channels that
permit the direct exchange of small molecules (< 1.2 kDa) between adjacent
cells (23)
It now seems apparent that for a given cell line, transmission of the
bystander signal by cell-to-cell contact elicits a more pronounced bystander
effect compared with the transfer of an extracellular factor through the medium,
suggesting that these are two separate phenomena. Evidence for this comes from
mutation studies in AL cells using two different protocols. Either 20% of
densely cultured cells (
70% of cells in contact) were irradiated with the
Columbia microbeam, or cells on one surface of a double Mylar dish were
irradiated while cells on the other side of the dish acted as bystanders. The
results suggest that the cytotoxic factor(s) released from the cells into the
culture medium using the latter protocol had a small, barely significant effect
on the mutagenic response of the bystander cells (24), whereas the microbeam-based
studies showed a threefold elevation of mutation incidence in bystander cells,
which was significantly reduced in the presence of the gap junction inhibitor
lindane (15).
A similar conclusion has been implied from experiments performed with V79
cells in two different laboratories. In one study carried out at Columbia
University, cells were irradiated with various numbers of
particles and a
considerable degree of cell killing was seen in non-hit cells, with survival
reduced to 60% at the highest dose of 16
particles per nucleus (25). This was
in contrast to data from the Gray Cancer Institute, UK, where only 5 to 10%
lethality was seen (B. Michael, personal communication, 2001), and it was
concluded that this discrepancy was a result of the cells being plated at a
lower density in the latter study. Here there was little contact between cells,
and therefore the bystander effect observed was assumed to be due to the release
of a soluble factor into the medium affecting nonirradiated cells.
However, there are several differences between the protocols used at Columbia
and the Gray Cancer Institute for microbeam experiments that, in addition to the
cell density at the time of irradiation, may have contributed to the observed
discrepancy in survival.
The present study is the first to examine the effect of cell density under
the same experimental conditions, allowing us to conclude that any observed
differences are solely a result of cell density at the time of irradiation. When
approximately 2000 cells were plated on a microbeam dish, the vast majority
(>90%) of the cells were in direct contact with neighbors through membranes
and intercellular gap junctions when they were irradiated 18 h later. In
contrast, when 200 cells were plated using the same protocol, very little
contact between cells (< 10%) was seen, with the majority of cells appearing
as isolated entities, separated by many tens of micrometers from their
neighbors.
The results of the present study confirm those seen previously in C3H 10T˝
cells when irradiation of 10% of the cells ( 80% confluent at time of
irradiation) on a dish with an exact number of
particles (>4
particles/nucleus) resulted in a surviving fraction of less than 90%, indicative
of a substantial bystander effect (17).
In the present study, at both high and low cell density, the surviving
fraction fell progressively as the number of
particles traversing 10% of the
cells increased. This suggests that as more damage is inflicted on the cells,
there is an increase in transmission of the bystander signal either through
increased gap junction communication (high density only) or increased secretion
of a cytotoxic factor(s) into the culture medium. However, the amount of cell
killing, and by implication the magnitude of the bystander effect, was
significantly greater in the high-density cultures, with an approximately
2.5-fold increase in the amount of cell killing at the highest dose tested. A
significant increase in the transformation frequency was also observed at the
high density. These data indicate that the magnitude of the bystander effect is
dependent on cell density in C3H 10T˝ cells, implicating the involvement of gap
junction-mediated intercellular communication in transmitting the bystander
effect. Several studies have now shown that inhibition of this gap junction
activity in cells irradiated in close contact results in decreased levels of the
bystander effects for a variety of biological end points (15, 16, 26). An
alternative, but unlikely, explanation is that the observed effect is due to
some factor released into the medium, which, because of a very short half-life,
can migrate only small distances from the irradiated cell. This is unlikely
because it has been estimated that for the irradiation protocol used in the
present study, any bystander signal induced could travel over a large distance
through the medium during irradiation (approximately 600–700 µm) (27).
The results obtained for low-density cultures did deviate from those expected
in the absence of a bystander effect, suggesting that such an effect may still
be operative. Considering oncogenic transformation, in the absence of a
bystander effect, a transformation frequency of 2.1/104 viable cells is expected
that is less (although not significantly) than the observed frequency of 3.3
(Table 1). A similar result was seen for clonogenic survival with a
non-significant increase in cell killing (Fig. 1). However, any bystander effect
evident in the low-density cultures is likely to result from interaction of a
secreted cytotoxic factor with unirradiated cells rather than from direct
communication due to the very low frequency of cell-to-cell contact. This has
been con- firmed in a previous study of low-density cells in which a random
distribution of damaged cells throughout the population was seen, suggestive of
an extracellular factor (14).
In conclusion, the present study confirms that when cells are exposed to low
doses of particles, the degree of
cell-to-cell contact at the time of
irradiation is important in transmission of the bystander signal. When cells are
in close contact, gap junctions play a major role, whereas if the degree of
contact is low, the bystander effect is mediated by the release of factors into
the surrounding environment.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Fu-ru Zhan and Mr. Steve Marino for their
help with the microbeam irradiations and Dr. Charles Geard for his help in the
preparation of this manuscript. The Columbia University Radiological Research
Accelerator Facility (RARAF) is an NIH-Supported Resource Center. This work was
supported by grants RR-11623, CA-49062 and CA-37967 from the U.S. National
Institutes of Health.
Received: July 22, 2003; accepted: November 6, 2003
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