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Proc Natl Acad Sci U S A. 2001 January 16; 98(2): 473–478. Published online 2001 January 9. | PMCID: PMC14611 |
Copyright © 2001, The National Academy of Sciences Biophysics Direct evidence for the participation of gap
junction-mediated intercellular communication in the transmission
of damage signals from α-particle irradiated to nonirradiated
cells Edouard I. Azzam,* Sonia M. de Toledo,* and John B. Little† Department of Cancer Cell Biology, Laboratory of Radiobiology, Harvard School of Public Health, Boston, MA 02115 Received August 30, 2000. It has generally been considered that important biological effects
of ionizing radiation arise as a direct consequence of DNA damage
occurring in irradiated cells. We have examined this hypothesis by
exposing cells to very low fluences of α-particles, similar to those
emitted by radon gas, such that as few as 1% of the cells in a
population are traversed by a particle and thus receive any radiation
exposure. By using the endpoints of changes in gene expression and
induction of DNA damage, we show that nonirradiated “bystander”
cells participate in the overall response of confluent
density-inhibited populations of cultured fibroblast and epithelial
cells. By in situ immunofluorescence techniques and the
use of cells genetically compromised in their ability to perform gap
junction intercellular communication, we present direct evidence for
the involvement of connexin43-mediated intercellular communication in
the transmission of damage signals to nonirradiated cells. Induction of
the stress-inducible p21Waf1 protein in aggregates of
neighboring cells far exceeding the fraction of cells whose nucleus has
been traversed occurred in gap junction-competent cells only. These
changes in p21Waf1 expression correlated with both the
induction of DNA damage (as measured by micronucleus formation) as well
as increased Ser-15 phosphorylation of p53. It has long been thought that
the important biological effects of radiation in a cell population are
a direct consequence of DNA damage occurring in the irradiated cells:
unrepaired or misrepaired DNA damage in these cells is responsible for
the genetic effects of radiation. Presumably, no effect would be
expected in cells in the population that received no direct radiation
exposure. Recently, however, evidence has been presented indicating
that genetic changes such as increased levels of sister chromatid
exchanges ( 1, 2), mutations ( 3, 4), micronuclei ( 5), and DNA
damage-inducible proteins ( 6, 7) occur in a greater-than-expected
number of cells in cultures exposed to very low fluences of
α-particles, fluences in which only a small fraction of the cells are
actually traversed by a particle track and thus directly exposed to
radiation. Finally, it has been shown that when some cells were labeled
with tritiated thymidine in a three-dimensional multicellular cluster
model, a cytotoxic effect was transmitted to adjoining nonlabeled cells
present in the same cluster ( 8). Overall, these studies indicate that radiation traversal through the
nucleus of a cell is not a necessary prerequisite to producing genetic
damage or a biological response; cells in a population that are in the
vicinity of directly hit cells can also respond to the radiation
exposure. These nonirradiated cells that express genetic damage or
changes in the expression of stress-induced genes have been termed
“bystander cells.” The present investigation was designed to
determine the mechanisms by which damage signals may be transmitted
from irradiated to nonirradiated bystander cells. We previously presented preliminary evidence for the involvement of gap
junction-mediated intercellular communication (GJIC) in the molecular
events leading to the modulation of gene expression in bystander cells
( 7). In these studies, confluent density-inhibited cultures of normal
human fibroblasts were exposed to low fluences of α-particles in the
presence or absence of lindane, a chemical inhibitor of GJIC. Changes
in gene expression were measured by Western blotting. The participation
of bystander cells in the overall cellular response to the radiation
stress was inferred from the observations that, the effect was
significantly greater than expected based on the fraction of directly
irradiated cells in the population, and that it was reduced in the
presence of lindane. In the present study, we further explored the involvement of GJIC in
the response of bystander cells in confluent cultures exposed to
fluences of α-particles where a very small fraction of cells' nuclei
were traversed by a particle track. By using the endpoints of p53 and
p21Waf1 expression and induction of DNA damage,
we present direct evidence for the involvement of connexin43-mediated
intercellular communication, by in situ immunofluorescence
techniques and by the use of cells genetically compromised in their
ability to perform GJIC. Cell Culture. Human cells. AG1522 normal human-diploid skin fibroblasts were obtained from the
Coriell Cell Repositories, Camden, NJ. HLF1 normal human-diploid lung
fibroblasts were obtained from the American Type Culture Collection.
Cells destined for α-particle irradiation were grown in 36-mm
stainless steel dishes with 1.5-μm-thick replaceable mylar bottoms
( 9) at a seeding density of about 1.2 × 10 5 cells per
dish. The mylar surface was precoated with fibronectin to facilitate
cell attachment. The cells were subsequently fed on days 5, 7, and 9
with Eagle's MEM supplemented with 15% (vol/vol) heat-inactivated
FCS, penicillin 50 units/ml, and streptomycin 50 μg/ml.
Experiments were started 48 h after the last feeding. At that
time, 95–98% of the cells were in G 0/G 1 as
determined by labeling with 3H thymidine and/or flow
cytometry. To eliminate complications in the interpretations of results
due to changes in cellular sensitivity to radiation at different phases
of the cell cycle, the cells were synchronized in
G 0/G 1 by confluent density-inhibition of
growth. Importantly, this protocol maximizes interaction among the
cells. Cells in passages 10–11 maintained in a 37°C humidified
incubator (atmosphere = 5% CO 2 in air) were used in
the experiments. Control cells were sham-irradiated and handled in
parallel with the test cells. Mouse embryo fibroblasts (MEFs). Wild-type and connexin43 −/− primary MEFs were
established in our laboratory with day 9 embryos from a pregnant female
mouse heterozygous for gap junction membrane-channel protein α-1
(commonly known as connexin43) that was mated to a male of the same
genotype. The mice were a generous gift from Caterina Sellito, Harvard
Medical School (Boston), who originally purchased them from The Jackson
Laboratory. Genetic typing of the established cells was carried out
according to a 3-primer protocol from The Jackson Laboratory. The
phenotype was further confirmed by transfer of Lucifer yellow dye
between cells in a confluent culture by the scrape-loading technique
( 10). Cells in passages 6–9 were used for the experiments. The cells
were grown in modified Eagle's medium with Earle's balanced salt
solution supplemented with 50% more vitamins and essential amino acids
(except glutamine), 100% more nonessential amino acids, and 1 mM
sodium pyruvate (D medium, GIBCO). The medium was supplemented with
15% (vol/vol) heat-inactivated FCS. For the experiments, cells were
seeded at a density of about 0.5 × 10 6
cells per 36-mm stainless steel dish with replaceable mylar bottom. The
cells were fed twice at 48-h intervals after seeding, and experiments
were started 48 h after the last feeding. At this stage, the cells
were confluent and were sensitive to contact inhibition of growth.
Furthermore, control cells from either strain did not loose contact
inhibition or express morphologically transformed foci ( 11) in
monolayer cultures (maintained in the confluent state for 7 weeks and
fed every 5 days with fresh medium). Rat liver epithelial cells. WB-F344 and WM-aB1 cell lines (both GJIC-competent and GJIC-deficient)
were a generous gift from James Trosko (Michigan State University, East
Lansing). The WM-aB1 cells were derived from a mutant clone of WB-F344
cells ( 12). The cells were cultured according to the protocol described
for MEFs and exposed to α-particles in the confluent
density-inhibited state. Both cell strains were very sensitive to
contact inhibition of growth in a manner similar to the human
fibroblasts and MEFs described above, and hence complications in the
interpretation of microdosimetric measurements for α-type experiments
are eliminated. Irradiation. For α-particle irradiation, cells were exposed to a
238Pu collimated source at a dose rate of 9.9
cGy/min, as described ( 9). The source was located inside of a
helium-filled Plexiglas box. Irradiation was carried out from below,
through the mylar base, with α-particles at an average energy of 3.65
million electronvolts at the cell layer. The source was fitted with a
photographic shutter to allow accurate delivery of the specific
radiation dose. Microscopic examination of pits etched in CR-39 plastic
after a 1-min exposure showed no source hot spots or cold spots down to
the 2,500 μm 2 level. The fraction of cells
whose nucleus was actually traversed by an α-particle was derived by
using Poisson statistics and estimates involving cell geometry,
α-particle fluence, and energy loss. Western Analysis. After irradiation, confluent density-inhibited cultures were held at
37°C in 5% CO 2 atmosphere for various time
intervals before harvesting for analysis. The cells were pelleted,
rinsed in PBS, repelleted, and lysed in chilled RIPA buffer (50 mM
Tris ![[center dot]](/corehtml/pmc/pmcents/middot.gif) Cl, pH 7.5/150 mM NaCl/1% Nonidet P-40/0.5% sodium
deoxycholate/0.1% SDS). The RIPA buffer was supplemented with the
following protease and phosphatase inhibitors: PMSF (1 mM), aprotinin
(1 μg/ml), pepstatin (1 μg/ml), leupeptin (1 μg/ml), sodium
fluoride (50 mM), and sodium orthovanadate (1 mM).
Anti-p21 Waf1 (Ab-1) and anti-p53 (Ab-6)
against-human proteins, anti-p53 (Ab-7) and
anti-p21 Waf1 (Ab-6) against-mouse and -rat
proteins, and anti-α-tubulin (Ab-1, used to verify equal loading of
the samples) were obtained from Oncogene Science. An anti-mouse Ig-G
secondary antibody conjugated with horseradish peroxidase was used to
detect the various proteins by chemiluminescence. Immunofluorescence. Confluent cell cultures grown on mylar surfaces were rinsed in PBS
supplemented with 1 mM MgCl 2 and 0.1 mM
CaCl 2 (PBS +), and were
fixed in 3% (vol/vol) paraformaldehyde in
PBS +. After a 5-min rinse in 50 mM
NH 4Cl and 2 PBS + rinses,
the cells were permeabilized in ice-cold Triton-X buffer (50 mM
NaCl/3 mM MgCl 2/200 mM sucrose/10 mM Hepes,
pH 7.4/0.5% Triton X-100 in water). The cell monolayers were
subsequently blocked in 1% BSA and reacted to the respective
antibody-against-human or -rodent p21 Waf1
according to the protocol of Eckner et al. ( 13). After
incubation with an FITC-conjugated goat anti-mouse IgG secondary
antibody (Sigma), the monolayers were washed at least five times with
PBS +. Antifade (Oncor) was used in mounting the
samples. Microscopy of coded samples was carried out by using a Leica
TCSNT scanning confocal microscope equipped with an argon laser
(excitation at 488 nm). Experiments were repeated at least three times. GJIC. The scrape-loading and dye-transfer techniques of El-Fouly et
al. ( 10) were used to measure GJIC. Briefly, confluent
density-inhibited cells in 60-mm polystyrene dishes were rinsed with
PBS +, and 0.05% Lucifer yellow in
PBS + was added to the cells. The cell monolayer
was scraped with a scalpel blade and kept in the dark for approximately
3–5 min. After incubation, the dye solution was decanted, and the
monolayer was rinsed three times with PBS + and
viewed with a Zeiss II UV-microscope. Micronucleus Assay. The frequency of micronucleus formation was measured by using the
cytokinesis-block technique developed by Fenech and Morley ( 14).
Briefly, after experimental treatments, confluent cultures were
dissociated by trypsinization. Approximately 3 ×
10 4 cells were plated in 2 ml of growth medium in
chamber flaskettes (Nunc) in the presence of 2 μg/ml cytochalasin B
and were incubated at 37°C. After 72 h, the cells were rinsed
twice with PBS + (pH 7.4) and fixed in cold
methanol. After air drying, the cells were stained with Hoechst 33342
solution (1 μg/ml) and viewed under a fluorescent microscope. At
least 500 cells were examined, and micronuclei in binucleate cells only
were considered for analysis. In Situ Detection of Bystander p21Waf1
Induction in Confluent Density-Inhibited Populations of Human-Diploid
Fibroblasts Exposed to Very Low Mean Doses of α-Particles: The Effect
of GJIC Inhibitors. We have shown ( 7) that induction of the p53 signaling pathway can be
detected by Western analyses in confluent human-diploid fibroblast
cultures exposed to mean doses of α-particles as low as 0.3 cGy. The
protein analyses data in Fig.
A extend our previous studies
and show that p53 and p21 Waf1 are also
significantly induced in AG1522 skin and HLF-1 lung fibroblasts exposed
to low mean doses. An increase of 2.0- to 2.5-fold in
p21 Waf1 levels measured by densitometry occurred
in AG1522 cells exposed to a mean dose as low as 0.16 cGy. These
results confirm our previous findings with other human fibroblast
strains ( 7) and show that induction of these proteins can be detected
by Western analyses at yet lower mean doses. At a dose of 0.16 cGy to
the overall culture, about 1% of the cells' nuclei would be traversed
by an α-particle. The results of an in situ
immunofluorescence study for p21 Waf1 expression
carried out in parallel in AG1522 cultures exposed to 0.3 cGy is shown
in Fig. B. As can be seen, staining occurs in aggregates of
cells. At this dose, about 2% of the nuclei would be irradiated. The
magnitude of the response at low-mean doses (shown in Fig.
A), coupled with the immunofluorescence studies
that showed that whenever p21 Waf1 was induced it
occurred in aggregates of neighboring cells (Fig. B),
strongly support the view that bystander cells participate in the
overall response of the exposed confluent cultures.
We have shown by Western blot analyses that cellular exposure to
lindane (γ-isomer of hexa-chloro-cyclo-hexane), an inhibitor of gap
junctions that mediates intercellular communication ( 15), prevented
detectable induction of p21 Waf1 in confluent
cultures exposed to low fluences of α-particles ( 7). As lindane may
have effects other than inhibition of connexin43-mediated intercellular
communication, we tested the generality of the response by using other
gap junction inhibitors. The data in Fig.
A indicate that, similarly to
lindane, the inhibitors DDT
[1,1′bis(pchlorophenyl)-2,2,2-trichloroethane] and dieldrin also
reduced p21 Waf1 induction by low fluence exposure
(1 cGy), albeit to different extents. Importantly, the in
situ immunofluorescence data in Fig. B show
clearly that exposure of confluent cultures to lindane beginning 2
h before irradiation (mean dose of 0.3 cGy) and continuing until
paraformaldehyde fixation 3 h later, resulted in the inhibition of
the aggregate pattern of p21 Waf1 induction (Fig.
B Right) that typically occurs in control
irradiated cultures (Fig. B Center). In
irradiated cultures treated with lindane, p21 Waf1
was induced primarily in single cells. These data further implicate
GJIC in the bystander p21 Waf1 response observed
after exposure to fluences where a very small fraction of cell nuclei
in the exposed culture is traversed by an irradiating α-particle. At
higher mean doses, where most of the cell nuclei in the exposed culture
is traversed by a particle track, lindane did not have a clear
attenuating effect on the p21 Waf1 response. The
data in Fig. C indicate that when confluent AG1522 cultures
are exposed to a mean dose of 10 cGy, in which a particle would
traverse the nucleus in about 50–60% of the cells,
p21 Waf1 was induced in the majority of cells
whether lindane was present or absent. Its induction in a greater
proportion of cells in the exposed control culture could be because of
a bystander effect that is inhibited in the presence of lindane.
The dye-transfer data in Fig. provide
evidence that AG1522 cells are GJIC-competent. Intact cells are
impermeable to Lucifer yellow; however, the dye permeates into cells
that are damaged, and owing to its low molecular mass (457.2 Da), the
dye subsequently migrates readily into neighboring cells through gap
junctions. Fig. A shows extensive transfer of Lucifer
yellow in control cultures where a row of cells was scraped with a
scalpel blade. The elongated morphology of AG1522 fibroblasts that
express connexin43, and the use of confluent density-inhibited
cultures, maximize interaction among cells and result in optimum dye
transfer. The lack of dye transfer beyond the damaged cells at the edge
of the scrape (when the cultures were incubated in the presence of 40
μM lindane) confirms the ability of lindane to inhibit
GJIC in human fibroblasts (Fig. B).
Differential Activation of p21Waf1 Expression in Low
Fluence α-Particle-Exposed Isogenic Cell Strains That Differ in
Their Ability to Perform Gap Junction Intercellular Communication. Inhibitors such as lindane may not, however, be specific in their
action. To examine the involvement of intercellular communication in
bystander effects after low fluence exposure in a direct manner,
confluent cultures of two related rat epithelial cell lines that differ
in their ability to perform gap junction communication were exposed to
α-particle radiation. The dye transfer experiments in Fig.
A indicate that WB-F344 cells
are GJIC competent; Lucifer yellow readily migrates to adjacent cells.
However, their ability to perform GJIC is sensitive to inhibition by
lindane (Fig. B). The WM-aB1 cells were derived from
WB-F344 cells and are deficient in GJIC, as indicated by their
inability to transfer Lucifer yellow to adjacent cells after scraping
of the monolayer (Fig. C). The Western blot analyses data
in Fig. show an increase in
p21Waf1 levels in confluent WB-F344 cultures
exposed to mean doses as low as 0.3 cGy. In WM-aB1 cultures, an
increase in p21Waf1 levels is significant only at
mean doses of 5 cGy or higher. Therefore, the magnitude of the response
in the GJIC-competent cells and the lack of
p21Waf1 up-regulation in WM-aB1 cultures that
have been exposed to low mean doses strongly support the involvement of
GJIC in the bystander gene-expression response. This is further
confirmed by the in situ immunofluorescence data in Fig.
Upper that shows the
induction of p21Waf1 in confluent cultures
exposed to a mean dose of 0.3 cGy. Whereas small clusters of responding
cells were observed in WB-F344 cells, only single isolated WM-aB1 cells
invariably exhibited up-regulation of p21Waf1
after exposure to 1.0 cGy (Fig. Lower). These rat cells
are much smaller than the AG1522 human fibroblasts, and at these mean
doses to the monolayer, 1% or less of the cells would have their
nuclei traversed by an α-particle.
The WM-aB1 cells were transformed by the mutagenesis of the WB-F344
parental-cell line ( 15). To exclude effects because of mutagenesis
other than loss of GJIC, we tested the induction of the
p53/p21 Waf1-signaling pathway after low fluence
α-particle irradiation of low passage MEFs from wild-type and
isogenic connexin43 knockout. The data in Fig.
are similar to those for WB-F344 and
WM-aB1 cells (Fig. ); they indicate a lack of detectable increase in
p21 Waf1 expression in
connexin43 −/− cells exposed to mean doses less
than 10 cGy. In contrast, p21 Waf1 was induced in
wild-type cells after exposure to mean doses as low as 0.6 cGy.
Collectively, these data strongly support the involvement of GJIC in
the bystander gene-expression response observed in confluent
density-inhibited cell cultures exposed to low fluences of
α-particles.
DNA Damage Is Induced at Levels Higher Than Expected in
Low-Fluence-Exposed Human-Diploid Fibroblast Cultures: Inhibition by
Lindane. To investigate whether the bystander induction of the stress-inducible
p21Waf1 protein (Figs. and ) is associated
with higher-than-expected levels of DNA damage after cellular exposure
to low fluences of α-particles, we measured the frequency of
micronucleus formation in confluent cultures of AG1522 fibroblasts held
in confluence for 3 h after the exposure. Compared with the
control (nonexposed cells), the data in Fig.
indicate a 3-fold increase in the
induction of micronuclei after exposure to mean doses in the range of
1–3 cGy, and only a 4-fold increase after exposure to 10 cGy. At a
mean dose of 10 cGy, 10-fold more cells in the population experience a
nuclear traversal by an α-particle than those at a mean dose of 1
cGy. Therefore, the magnitude of the response at low fluences suggests
that nontraversed bystander cells were also subject to DNA damage. To
investigate the involvement of GJIC, lindane was added to the cultures
2 h before exposure and remained for 3 h thereafter. A highly
significant reduction in the frequency of micronucleus formation was
observed in cultures exposed to 1 or 2 cGy. At 10 cGy, lindane did not
reduce the frequency of micronucleus formation in confluent cultures
exposed to this same mean dose (Fig. ). These data thus suggest that
DNA damage may be the signal for the bystander induction of
p21Waf1 in low fluence-exposed confluent-cell
cultures. However, both effects may also be independent consequences of
signals communicated from irradiated to bystander cells.
To further ascertain the involvement of DNA damage in the bystander
induction of p21 Waf1, we measured the levels of
phosphorylation of Ser-15 in p53. Phosphorylation of p53 in Ser-15 has
been shown to be dependent on the ataxia telangiectasia mutated gene in
cells exposed to DNA damaging agents. However, its role in the
activation of p53 is not clear (previously reviewed in ref. 16). The
data in Fig. indicate an increase in
p53 phosphorylation after exposure of AG1522 cells to mean doses as low
as 1 cGy. This increase at 1 cGy was attenuated in the presence of 40
μM lindane. Therefore, these data further support the occurrence of
DNA damage in bystander cells and implicate GJIC in its induction.
Although much evidence for the existence of bystander responses to
ionizing radiation has accumulated recently ( 1– 8, 17, 18), the
mechanisms underlying the observed effects remain obscure. The gene
expression data obtained in this study with confluent cultures of human
and rodent cells exposed to very low fluences of α-particles indicate
that the effects of these particles are not confined to cells whose DNA
is directly damaged by a traversing particle, and they provide direct
evidence for the involvement of GJIC in mediating the bystander
response. The observation that the induction of
p21 waf1 expression detected in situ
occurs in aggregates of neighboring gap junction-competent cells far
exceeding the expected number that would receive direct DNA damage
(Figs. B and A) indicates clearly that
nontraversed bystander cells participate in the overall response of the
exposed cultures, and suggests that this response could be mediated by
GJIC. The involvement of GJIC was confirmed by the lack of detectable
p21 waf1 induction by Western analyses in protein
lysates from cells that are genetically or chemically
gap-junction-compromised (Figs. A, B,
and ), and, importantly, by studies with in situ
immunostaining. In situ immunofluorescence analysis of
p21 Waf1 expression in cells lacking GJIC (after
exposure to a low mean dose of 0.3 cGy, by which 2% or less of nuclei
are traversed by an α-particle) revealed that it was induced only in
single cells that were well secluded from each other (Figs.
B and B), as opposed to its induction in
aggregates of cells (as did occur in competent cell cultures; Figs.
B and A). These data thus suggest that
cultures of cells exposed to low fluences of α-particles respond as a
whole to the radiation stress with similar signaling pathways induced
in nontraversed and traversed cells, and directly implicate GJIC as a
mechanism mediating these effects. These results imply that the
modeling of dose–response relationships based on the number of cells
hit, as has been commonly used in risk analysis, may not be a valid
approach. Gap junctions are dynamic structures that have been shown to be
critical for diverse functions such as early developmental events,
oncogenic transformation, cell growth, propagation of excitation in
muscle cells, and central nervous system neurons ( 19). Their role in
radiation-induced biological effects was postulated earlier ( 20), and
recent studies with chemical inhibitors of GJIC ( 4, 7, 8, 21) or
connexin43-transfected cells ( 22) have explored their involvement in
the induction and propagation of various radiation-induced effects such
as modulation of gene expression, cell survival, and mutation
induction. Through in situ techniques and the use of
knockout cells, this report provides direct evidence for the
participation of connexin43 gap junctions in the response to radiation
stress and their role in mediating radiation-induced bystander effects. Whereas the inhibition of the bystander response in cells that are
genetically compromised to perform connexin43-mediated GJIC confirms
the involvement of this specific mechanism in the observed effects, our
data do not preclude that the effects of gap junction inhibitors may
vary in different cell types. For example, it was recently shown ( 23)
that lindane differentially modulates calcium levels in peripheral
blood lymphocytes and phagocytes. Also, our findings do not exclude
mechanisms other than GJIC from having a role in the bystander response
in confluent cultures exposed to low fluences of α-particles ( 24).
With the endpoints of gene expression and micronucleus formation, our
work in progress is consistent with that of Narayanan et al.
( 25) and indicates that reactive oxygen species, as well as
membrane-originating pathways, participate in the bystander response.
We are investigating whether these various mechanisms share common
upstream signaling events, and whether there is crossover among them.
The results of such studies may be informative as to the signaling
events leading to activation of the connexin proteins. Importantly, the
nature of the communicated molecule or molecules and the in
situ identification of cells that have been traversed remain to be
investigated. The up-regulation of p21 Waf1 in bystander cells
was associated with a greater frequency of micronucleus induction than
expected at mean doses of 1–2 cGy (Fig. ). Such an increased
frequency is consistent with the findings of Prise et al.
( 5). Its reduction in the presence of lindane (Fig. ) suggests that
DNA damage also occurs in bystander cells, and that GJIC may be
involved in signaling the process. The induction of micronuclei and the
cyclin-dependent kinase inhibitor p21 Waf1 in
bystander cells is also consistent with the recently reported
G 1 arrest occurring in a greater-than-expected
number of cells in human fibroblasts exposed to a mean dose of 1 cGy of
α-particles ( 26). We studied micronucleus formation as a surrogate
measure of DNA damage. Micronuclei arise predominantly from nonrejoined
DNA double-strand breaks ( 27), which have been strongly implicated in
the process of cancer development in humans ( 28). If DNA damage were to
occur in bystander cells in vivo, and these cells survived
such damage, these results would impact significantly on the assessment
of cancer risk initiated by low fluence exposures to α-radiation. On
the other hand, dead cells stimulate compensatory hyperplasia in a
tissue in vivo, which is a potentially tumor-promoting
condition. The DNA-damage-dependent phosphorylation of Ser-15 in p53 (Fig. )
suggests that DNA damage in bystander cells precedes activation of the
signaling pathways leading to this specific event. It is not yet known
whether the transmitted signal or signals from irradiated cells to
bystander cells cause in these cells DNA damage that leads to the
alterations observed in gene expression (Figs. and ). Alternatively,
the transmitted signal or signals may directly activate
signal-transduction pathways leading to DNA damage and cell death in
the bystander cells. This study and others ( 1– 8, 17) indicate that the level of damage in
bystander cells measured several hours after the exposure of confluent
cultures to α-particles exceeds that arising from normal metabolism.
These findings are different from data obtained with sparsely ionizing
(low Linear Energy Transfer) radiation ( 29), whereby a γ-ray dose as
little as 0.1 cGy has been reported to induce a protective mechanism
against damage from endogenous metabolism or from a
subsequent radiation exposure. Whereas progress has occurred in
radiobiological research tools, much remains to be learned about
mechanisms underlying low and high Linear Energy Transfer radiations.
An understanding of the mechanisms underlying various biological
effects induced in vitro in cell populations exposed to low
fluences of α-particles are directly relevant to the assessment of
health risks to the public from exposure to radon. It is currently
estimated that 10–14% of lung cancer deaths in the U.S.A. are linked
to α-particles from radon gas in the environment ( 30). Moreover,
these studies are pertinent to the hot-particle problem, and the
biological effects of incorporated radionuclides used clinically in the
diagnosis and treatment of various medical conditions. These studies
are also pertinent to our understanding of the observations made during
experimental gene therapy, whereby cells transduced with a
drug-converting enzyme are cytotoxic to bystander cells ( 31), or
where bystander cells protect transduced cells exposed to the
pro-drug ( 32). We are grateful to Dr. James Trosko for providing the WB-F344 and
WM-aB1 cells and to Caterina Sellito for the connexin43-heterozygous
mice. We thank Tamara Gooding for dedicated technical support; Drs.
Hatsumi Nagasawa, William Dahlberg, Helene Hill, Roger Howell, and
Anupam Bishayee for helpful discussions; and Jean Lai for performing
confocal microscopy. This work was supported by Research Grant
FG02–98ER62685 from the United States Department of Energy and Center
Grant ES-00002 from the National Institutes of Health. | | GJIC | gap junction-mediated intercellular communication | | | MEF | mouse embryo fibroblast | | | PBS+ | PBS supplemented with 1
mM MgCl2 and 0.1 mM CaCl2 |
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