| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Carcinogenesis |
Particles1
Cell and Molecular Biology Group, Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
particles like those emitted by
radon/radon progeny stimulates their proliferation in
vitro, and this response also occurs when unirradiated cells
are treated with supernatants from -irradiated cells. We
attribute the promitogenic response to superoxide dismutase- and
catalase-inhibitable particle-induced increases in the
concentrations of transforming growth factor ß1 (TGF-ß1) in cell
supernatants. TGF-ß1 at concentrations commensurate with those in the
supernatants capably induces increases in intracellular reactive oxygen
species (ROS) in unirradiated cells. Furthermore, the addition of
supernatants from -irradiated cells to unirradiated cells decreases
cellular levels of TP53 and CDKN1A and increases CDC2 and proliferating
cell nuclear antigen in the latter. Like the increased intracellular
ROS bystander effect, this "decreased TP53/CDKN1A response" can be
mimicked in otherwise untreated cells by the addition of low
concentrations of TGF-ß1. Our results indicate that
particle-associated increases in cell growth correlate with
intracellular increases in ROS along with decreases in TP53 and CDKN1A,
and that these cellular responses are mechanistically coupled. As well,
the proliferating cell nuclear antigen and CDC2 increases that occur
along with the decreased TP53/CDKN1A bystander effect also would
expectedly favor enhanced cell growth. Such processes may account for
cell hyperplastic responses in the conducting airways of the lower
respiratory track that occur after inhalation exposure to radon/radon
progeny, as well as, perhaps, other ROS-associated environmental
stresses. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
3
particles like those emitted by 222radon and
radon progeny and several other radionuclides, e.g.,
241americium and
238,239plutonium, can cause DNA damage, mutation,
genomic instability, and oncogenesis (1, 2, 3, 4, 5, 6)
. Mounting
evidence, however, indicates that some biological effects can occur in
cells the nuclei of which do not experience nuclear or even whole-cell
encounters with the particles. Hickman et al.
(7)
, for example, reported that particles induced
accumulations of the tumor suppressor TP53 (p53) protein in rat lung
epithelial cells in a higher percentage of the exposed population than
that calculated to receive a direct nuclear traversal. Such increases
in TP53 in cells the nuclei of which were unirradiated provided early
evidence consistent with an extranuclear mechanism for the induction of
DNA damage in the form of strand breaks (8)
. The
apparently large target size that is indicated by investigations of
particle-induced genomic instability (9, 10, 11)
also suggests
a relationship with extranuclear and perhaps extracellular DNA
damage-inducing processes as well.
We and others (12, 13, 14, 15)
have found that particles in
the circa 1 cGy region can induce DNA damage, as indexed by increases
in SCEs, in the absence of direct nuclear or even whole cell hits. This
effect has been associated recently with the generation of ROS in
culture medium and the induction of intracellular ROS, particularly
superoxide anions and hydrogen peroxide (16)
. Both the
increased SCE and ROS responses can occur in unirradiated, normal human
cells via the actions of SOD-inhibitable transmissible factor(s)
present in -irradiated medium and in the supernatants of
-irradiated cells (14, 15, 16, 17)
. These and other
investigations (17)
implicate an important role(s) for ROS
in mediating at least some effects of particles, and they
demonstrate well that biological responses to particles can be
induced indirectly in unirradiated cells, i.e., as
"bystander" effects, to the same extent as found with directly
-irradiated cells (14, 15, 16)
.
Most recently, Azzam et al. (18)
extended upon
the findings of Hickman et al. (7)
by
demonstrating that exposure of human fibroblasts to low doses of
particles causes unexpectedly high increases in TP53 protein and
downstream CDKIN1A (p21Waf-1) protein in cell
populations in which only a low percentage of the cells experienced a
nuclear traversal. Moreover, these investigators showed that the
excessive occurrence of increases in TP53 and CDKN1A may involve direct
cell-cell communications in that disruption of intercellular gap
junctions eliminated the effect.
In the present study, we set out to examine the possibility that the
bystander responses reported by Hickman et al.
(7)
and Azzam et al. (18)
may be
mediated, at least in part, by fluid-phase, soluble factors generated
in response to -irradiation. We unexpectedly observed that the
transfer of supernatants from -irradiated cells to unirradiated
cells consistently caused decreases in basal levels of TP53 and
CDKN1A in the latter populations, not increases. We also found that
these decreases were accompanied by increases in PCNA and CDC2. In
follow-up experiments, we identified TGF-ß1 as a mediator of both the
particle-associated "increased intracellular ROS bystander
response" we have described elsewhere (16)
and the newly
discovered "decreased TP53/CDKN1A bystander effect." Furthermore,
we report that these bystander responses correlate with enhancements in
cell proliferation in a manner that suggests they collectively explain
the phenomenon of cell hyperplasia that occurs in response to the
inhalation of radon/radon progeny (19)
and, perhaps, cell
hyperplastic responses to other ROS-associated environmental stresses
as well (20, 21, 22, 23)
. Conceivably, such increases in cell
proliferation may play a critical role(s) in radon-induced
carcinogenesis.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Particles-MEM (Life Technologies, Inc., Grand Island, NY)
supplemented with 10% fetal bovine serum (Hyclone Laboratories, Inc.,
Logan, UT). All cell cultures were incubated at 37°C in humidified
5% CO2/95% air. Cells were harvested from the
flasks by trypsinization and seeded in 1.5-µm-thick Mylar-bottomed,
30-mm-diameter culture dishes (24)
at an initial
density of 1 x 104 to 2 x 105 cells/dish for 1–3 days prior to
exposure to particles or other treatments in experiments that
involved exponentially growing cells. Higher initial seeding densities
or longer growth periods were used in other experiments, depending on
experimental requirements. Fresh medium was added to the cultures prior
to all irradiation, unless indicated otherwise. Cells were exposed to
particles at doses ranging from 1.0 to 19 cGy at room temperature
using a collimated 238Pu particle exposure
system (25)
. The average energy of the particles at
the cell-Mylar interface is 3.5 MeV delivered at a dose rate of 3.65
cGy · s-1. Control HFL1 cells were sham
irradiated. In all experiments, precautions were taken to ensure that
the pH of the media did not change. Supernatants were transferred
quickly and efficiently, and no obvious changes in the media were
observed.
Intracellular Hydrogen Peroxide
Previous studies showed that the intracellular ROS response to
particles includes both superoxide anions and hydrogen peroxide
(16)
. In this study, we flow cytometrically assessed for
putative hydrogen peroxide to comparatively index the intracellular
generation of ROS (16)
. Untreated cells were
simultaneously incubated for 30 min with supernatants from sham- or
-irradiated cells and/or other reagents along with DCFH-DA (1
µM). The cells were then harvested, centrifuged, and
resuspended in the medium in which they were initially treated
(16)
. After cleavage of the acetate moieties by cell
esterases, the oxidative potentials of hydrogen peroxide, along with
peroxidases, are able to oxidize the trapped diacetate to form
fluorogenically active DCF (26)
. Flow cytometric data were
collected on a Becton Dickinson FACSCalibur flow cytometer (Becton
Dickinson, San Francisco, CA) using standard computer, optics, and
electronics. A 15-mW, air-cooled argon-ion laser provided excitation at
488 nm, and a 530/30 nm band pass optical filter was used for measuring
DCF fluorescence. Cells were distinguished from background events by
their forward angle and orthogonal light scattering characteristics,
and the cellular events were evaluated after applying a bitmap (gate).
Fluorescence signals collected in this gate were analyzed by the
Cellquest data analysis software to determine fluorescence differences
between control and test samples.
TGF-ß Analyses
TGF-ß1 concentrations were measured in cell supernatants by ELISA
(Genzyme, Cambridge, MA). The assay used does not show cross-reactivity
with TGF-ß2 or TGF-ß3, nor does it show cross-reactivity for other
cytokines, including platelet-derived growth factor, epidermal growth
factor, fibroblast growth factor, tumor necrosis factor-, and tumor
necrosis factor-ß. This ELISA kit requires activation of TGF-ß by
acidification prior to its measurement. To measure biologically active
TGF-ß1 samples, standards and controls must be acidified using HCL
for 1 h. It is unclear at this time whether the changes observed
in TGF-ß are attributable to the release of latent TGF-ß from the
cell surface or enhanced production and release of TGF-ß from the
irradiated cells. In some experiments, neutralizing antibodies to
TGF-ß1 (R&D Systems, Minneapolis, MN) were added to cell culture
media to selectively inhibit its activity. In other experiments, human
recombinant TGF-ß1 (R&D Systems) was added to cell cultures to mimic
responses observed with supernatants from -irradiated cells.
Cell Proliferation
Cells were seeded at 1 x 104 per
Mylar dish for 24 h prior to sham- or (1 cGy)-irradiation or
other treatments, as indicated. In other experiments, confluent HFL1
cells were irradiated with 0 or 1 cGy of particles, and immediately
or 1 h thereafter, we transferred their supernatants onto other
unirradiated, exponentially growing HFL1 cells. Cells were harvested
1–3 days thereafter for cell counts.
SDS-PAGE and Immunoblotting
Cells were harvested by trypsinization and washed twice with PBS. Cells
were counted and lysed in NP40 lysis buffer and incubated on ice for 30
min. Samples were centrifuged at 12,500 x g
for 20 min at 4°C, and the supernatants were collected. For direct
immunodetection, equal amounts of protein (measured by Bio-Rad Protein
assay) were loaded and resolved on 10% SDS Ready Gels (Bio-Rad) in a
mini-gel apparatus (Bio-Rad, Hercules, CA). Resolved proteins were
transferred to a nitrocellulose membrane (Amersham, Arlington Heights,
IL) using a wet mini-transfer unit (Bio-Rad). Membranes were stained
with 0.1% Ponceau S to verify loading and transfer efficiency.
Membranes were blocked by overnight incubation at 4°C in blocking
buffer (5% dry nonfat milk in 10 mM Tris, 150
mM NaCl, pH 7.2). Membranes were then incubated
with relevant antibodies in blocking buffer for 1 h at 25°C and
washed extensively with Tris-buffered saline (TBST; 0.05% Tween 20, pH
8.0). Blots were incubated with peroxidase-linked anti-rabbit/mouse
immunoglobulin (Amersham) in TBST for 1 h at 25°C and then
washed extensively with TBST. Blots were placed in ECL enhanced
chemiluminescence (ECL) reagents (Amersham) for 1 min, followed by
exposure to autoradiographic film (ECL film; Amersham) for the required
time, which was developed in an automated film processor (Kodak).
Antibodies used were as follows: DO-1 p53 antibody (1:1000 dilution;
Santa Cruz Biotechnology, Inc., Santa Cruz, CA),
p21Waf-1 mouse monoclonal (1:1000; Santa Cruz
Biotechnology), CDC2 (p34cdc2; 3:1000; Santa Cruz
Biotechnology), PCNA (2.5:1000; Santa Cruz Biotechnology), and
antimouse immunoglobulin horseradish peroxidase secondary antibody
(Amersham).
Data Presentations
Unless indicated otherwise, data shown from each series of experiments
are representative of results obtained from three and often more
replicate experiments.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Particles and the Decreased TP53/CDKN1A Bystander
Response particles, the cells were harvested 4 h later, and their TP53,
CDKN1A, and CDC2 contents were assessed by Western analysis.
Sham-irradiated cells served as controls. Similar to the study reported
by Azzam et al. (18)
, exposure of the cells to
only 1 cGy of particles resulted in markedly elevated levels in
TP53 and CDKN1A and a decrease in CDC2 (Fig. 1A)
. On the basis of our previous morphometric analyses
(13)
, 7% of the cells in this study actually received
one or more nuclear hits by the particles.
|
particles that could contribute to the greater than
expected TP53 and CDKN1A elevations. The increased intracellular ROS
bystander response we reported previously (16)
, for
example, conceivably could cause DNA damage, including DNA strand
breaks (27, 28, 29)
, and thereby induce increased
accumulations of TP53 and downstream CDKN1A via transcriptional
activation by TP53 (30
, 31)
. We irradiated confluent HFL1
cells with 1 cGy of particles; immediately and 1 h thereafter,
we transferred their supernatants onto other unirradiated confluent
HFL1 cultures. Four h later, the cells were harvested, and their TP53
and CDKN1A levels were analyzed by Western analysis. Control cell
populations were treated with supernatants from sham-irradiated HFL1
cells. In repeated experiments, the supernatants from -irradiated
cells consistently caused decreases in the basal levels of TP53 and
CDKN1A in the unirradiated cells (Fig. 1B)
. More pronounced
decreases in the cell cycle regulating proteins were obtained with
supernatants that were harvested from cells 1 h after exposure
than immediately after exposure (Fig. 1B)
. Furthermore, the
decrease in TP53 observed at 4 h was persistent at least up to
24 h after addition of the supernatants (Fig. 1C)
.
Hence, a soluble, transmissible factor(s) in the supernatants of
-irradiated cells induces a decreased TP53/CDKN1A bystander
response, and the decreases in TP53 are relatively persistent.
Moreover, such a transmissible factor(s) evidently progressively
increases shortly after exposure to particles. In further analyses,
we found that mdm-2 protein levels, which can be up-regulated by
TP53-mediated transcriptional activation (32)
, were also
decreased during the decreased TP53/CDKN1A bystander response (data not
shown). Similar experiments were performed using irradiated cell-free
media transferred 1 h after irradiation onto fresh unirradiated
cells. No changes in TP53/CDKN1A were observed with media alone,
excluding the possibility of any involvement of media constituents in
the observed effects.
Cell Growth in Response to Low-Dose Particles and Supernatants
from -Irradiated Cells
Although it is clear that exposure to high concentrations of ROS can
induce DNA damage, cell cycle arrests, senescence, and cell death
(33, 34, 35)
, numerous reports indicate that a relatively
low-level exposure to ROS can stimulate cell growth (33
, 36, 37, 38, 39)
by pathways that are not well understood. The reported
promitogenic effects of low levels of ROS suggested to us that low-dose
exposure to particles may induce a ROS-associated enhancement in
cell proliferation. To test this possibility, cultures of exponentially
growing HFL1 cells were irradiated with 1 cGy particles, and cell
counts were obtained over a 3-day period. On the first day after
irradiation, cell counts were reduced relative to those obtained with
sham-irradiated cultures in a manner that is consistent with
particle-induced cell cycle arrests (40)
. By day 3,
however, cell numbers in the irradiated cultures were significantly
higher than control values (Fig. 2A)
.
|
particle-associated bystander effect in
unirradiated HFL1 cells. In these experiments, confluent HFL1 cells
were exposed to 0 or 1 cGy particles and further incubated for
1 h at 37°C. Supernatants were then harvested from the cells and
transferred onto nonconfluent, exponentially growing HFL1 cells. Over a
3-day period thereafter, cells were harvested and counted. As
illustrated in Fig. 2B
, cell numbers from cultures that
received the supernatants from the irradiated cells were 135 and
150% higher that control cell numbers on assay days 2 and 3,
respectively. Thus, the supernatants from -irradiated cells, which
have been shown previously to induce intracellular ROS
(16)
and that coincidentally reduce TP53 and CDKN1A
protein levels, also increase cell growth.
PCNA and CDC2 in HFL1 Cells Treated with Supernatants from
-Irradiated Cells
PCNA is an essential DNA replication protein that is increased in
abundance in proliferating cells (41, 42, 43, 44)
. In that low
levels of TP53 activate PCNA gene expression
(45)
, we investigated the possibility that the levels of
PCNA in HFL1 cells treated with the TP53-decreasing supernatants from
-irradiated cells may become up-regulated. In these same
experiments, we also assessed how cellular levels of CDC2, a protein
kinase that complexes with mitotic cyclins and is required for mitotic
entry (46, 47, 48)
, might also be altered in a manner that
would favor an enhanced state of proliferation. As shown in Fig. 3A
, a 2-fold increase in PCNA levels was observed in HFL1
cells as early as 6 h and up to 24 h after treatment with the
supernatants from -irradiated cells relative to those of cells
treated with sham-irradiated supernatants. Increases in CDC2 protein
levels were also observed at these times in HFL1 that had been
incubated with the supernatants from -irradiated cells (Fig. 3B)
.
|
Particle-induced Increases in Extracellular TGF-ß1-irradiated fibroblasts
induce excessive SCE and intracellular ROS bystander responses when
transferred to unirradiated cells (16)
. That the
particle-induced factor(s) survives freeze-thawing and is heat labile
(14
, 15)
suggested to us that it may be a cytokine(s).
Because other investigators have reported that a low concentration of
TGF-ß can induce the production of ROS in human fibroblasts
(14
, 15
, 49, 50, 51, 52)
and because low concentrations of TGF-ß
can suppress both p53 and p21 mRNA levels and CDKN1A protein levels in
human lung fibroblasts (53)
, we especially focused on how
this particular cytokine might contribute to the bystander responses
described herein. In the next experiments, confluent HFL1 cells were
irradiated with 0.4–19 cGy doses of particles, their supernatants
were harvested over a 24-h period thereafter, and TGF-ß1
concentrations in the supernatants were measured. Exposure of cells to
the particles resulted in early dose-independent, step-like
function increases in TGF-ß1 (to a level of 1 ng/ml) at the
earliest postexposure time point (30 min; Fig. 4A
). The early increase in TGF-ß1 evidently was attributable
to a posttranscriptional and posttranslational mechanism, inasmuch as
the elevated TGF-ß1 concentrations measured as of 30 min after
exposure remained relatively stable for at least 8 h after their
initial increase. More dose-dependent, yet still modest, increases in
TGF-ß1 were observed in the supernatants over the remaining 8–24-h
period after irradiation; we have yet to further investigate this
latter component of the TGF-ß1 response to particles. Even so, it
should be noted that the levels of TGF-ß1 ultimately measured in the
supernatants rose from a background starting concentration of 0.5
ng/ml to no more than 2 ng/ml. Although such increases in TGF-ß1
concentrations do not appear to be dramatic elevations over control
levels, the addition of 1 ng/ml human recombinant TGF-ß1 to HFL1
cells in culture medium that already contains nearly half of this
concentration can induce an intracellular ROS response (to be
described). Hence, even relatively small but abrupt increases in
TGF-ß1 can have functional consequences.
|
-irradiated cells.
Conceivably, the TGF-ß1 increases could be related to oxidative
processes associated with the extracellular generation of relatively
short-lived ROS we have found in -irradiated medium
(16)
. Of relevance to this possibility, Barcellos-Hoff and
Dix (54)
have reported that latent TGF-ß can be
activated by ionizing radiation and other oxidizing conditions, Ehrhart
et al. (55)
have observed increases in TGF-ß
in vivo with radiation doses as low as 10 cGy, and other
investigators (56
, 57)
have found that the activation of
TGF-ß occurs concomitantly with the generation of ROS. On the basis
of these findings, experiments were performed using the antioxidant
enzymes SOD and catalase to determine how fluid phase
O2
2 and/or
H2O2 generated by
particles (16)
may contribute to the early increases in
TGF-ß1. As illustrated in Fig. 4B
, increases in TGF-ß1
in response to particles can be inhibited totally by adding SOD to
cells prior to exposure to the particles. As well, the addition of
catalase to the cell cultures before they were irradiated also
eliminated increases in TGF-ß1 (Fig. 4B)
. That the removal
of either O2 or
H2O2 prevents the increased
extracellular availability of TGF-ß1 in response to -irradiation
may be explained by a requirement for both species, with, perhaps, the
ultimate mediator of the TGF-ß1 release being the formation of the
highly reactive hydroxyl radical (·OH) via the Haber-Weiss reaction:
O2 + H2O2
transition metal
O2 + ·OH + OH-.
TGF-ß1 as a Mediator of the Increased Intracellular ROS and
Decreased TP53/CDKN1A Bystander Responses
A low concentration of TGF-ß, i.e., 1 ng/ml, can induce
the intracellular production of ROS in fibroblasts (51
, 52)
, as noted previously. In subsequent experiments, we assessed
the possibility that TGF-ß1 at concentrations commensurate with those
in the supernatants of -irradiated cells can induce an intracellular
ROS response in our cells. Clearly, the addition of recombinant
TGF-ß1 to HFL1 at a low concentration causes an increase in the
intracellular production of ROS (Fig. 4C)
, whereas much
higher concentration TGF-ß1 diminishes ROS production. TGF-ß1 may
also play a role in regulating basal levels of ROS production in HFL1
cells in that the addition of anti-TGF-ß neutralizing antibody to the
cells markedly decreases their ability to convert DCFH to DCF.
Regardless, the above findings indicate that the prompt increases in
TGF-ß1 in the supernatants of -irradiated cells can account for
the induction of the increased intracellular ROS bystander response.
To further confirm the involvement of TGF-ß1 in the -irradiated
bystander response, neutralizing antibodies to TGF-ß1 were added to
the supernatants from either sham or -irradiated cells, and the
supernatants were then transferred onto fresh, unirradiated cells.
Control experiments were done in parallel wherein cells were either
sham or -irradiated, followed by the transfer of their supernatants
to fresh cells. Treatment of supernatants from -irradiated cells
with neutralizing TGF-ß antibodies prior to addition to fresh cells
completely inhibited the ROS bystander response, whereas an increase in
the DCF fluorescence was observed in the cells treated with the
supernatants from -irradiated cells without TGF-ß antibody (Fig. 4D)
.
That TGF-ß1 may also be involved in mediating the decreased
TP53/CDKN1A bystander response is suggested by the recent report that
low concentrations of this cytokine can cause decreases in p53 and p21
mRNA levels and CDKN1A cellular protein levels (53)
. To
evaluate this possibility, HLF1 cells were either sham-irradiated or
irradiated with 1 cGy particles, or we incubated HFL1 with varying
concentrations of human recombinant TGF-ß1, i.e., 1 ng/ml
(40 pM), 5 ng/ml (200 pM),
10 ng/ml (400 pM), or 50 ng/ml (2
nM) for 4 h. The cells were then harvested
and processed for analysis of their TP53 contents by Western blotting.
Cell cultures that received no TGF-ß1 additions served as controls.
Similar to the supernatants from -irradiated cells, low
concentrations of TGF-ß1, i.e., 0.5, 1.0, 5, and 10 ng/ml,
caused decreases in the cellular levels of TP53 (Fig. 5A)
. Higher concentrations of TGF-ß1, on the other hand,
caused increases in TP53 protein levels (Fig. 5A)
.
|
-irradiated cells underlie the
abilities of the supernatants to decrease TP53 protein levels in HFL1,
cells were irradiated with 1 cGy particles, and their supernatants
were harvested after 1 h. The supernatants were then incubated
with 120 µg/ml of neutralizing TGF-ß1 antibodies 30 min prior to
being added to fresh cells. As shown in Fig. 5B
, TGF-ß1
neutralization prevented decreases in cellular TP53 and CDKN1A protein
levels that otherwise occur in HFL1 cells treated with the supernatants
from -irradiated cells. In addition to demonstrating how TGF-ß1
can affect TP53 protein levels, the above collective evidence suggests
a link between the increased intracellular ROS bystander response and
the decreased TP53 bystander responses. As well, the above results
suggest a possible inverse relationship between cellular oxidant levels
and TP53 protein levels. That is, a low concentration TGF-ß1-mediated
increases in intracellular ROS levels are associated with decreases in
TP53 like those observed in HFL1 cells treated with supernatants from
-irradiated cells, whereas decreases in intracellular ROS levels
mediated by higher concentrations of TGF-ß1 are associated with
increases in TP53 protein levels.
As shown by others (18
, 58
, 59)
, exposure of cells to
-rays and particles down-regulates CDC2 levels in a
TP53/CDKN1A-dependent manner. We speculated that CDC2 decreases that
occur when TP53 protein levels increase in response to ionizing
radiation may not occur in cells that are treated with low
concentrations of TGF-ß1, but instead, such treatment may actually
cause increases in CDC2. To examine this possibility, which would be
expected to favor cell growth, we measured CDC2 protein levels in the
cells that were treated with 1–50 ng of human recombinant TGF-ß1 for
4 h. As shown in Fig. 5C
, treating HFL1 with a low
concentration of TGF-ß1 indeed resulted in increases in basal CDC2
protein levels. High concentrations of TGF-ß1, i.e., 50
ng/ml, on the other hand, caused decreases in CDC2 levels (Fig. 5C)
, as might be expected under conditions when TP53 protein
is increased.
Relationships among ROS, TGF-ß1, and the Decreased TP53/CDKN1A
Bystander Response
Given the correlation between the ROS and the decreased TP53/CDKN1A
bystander responses, we next examined how extracellular and
intracellular ROS may be involved in TGF-ß1-mediated decreases in
TP53 protein. In the first experiments, HFL1 were incubated with SOD
(100 units/ml) or catalase (10 units/ml) 30 min prior to the addition
of recombinant TGF-ß1. Catalase inhibited the TGF-ß1-mediated TP53
decrease in HFL1 cells, as did SOD (Fig. 6A)
. We also pretreated HFL1 populations with the flavoprotein
inhibitor DPI (5 µM; Refs. 16
and
60
). The cells were then treated with recombinant TGF-ß1
(1 ng and 5 ng/ml) and harvested 4 h later for analysis of their
TP53 contents. DPI, which inhibits the particle-induced
intracellular ROS response (16)
, as well as the production
of H2O2 by surface membrane
NADH oxidase (51)
, completely inhibited the
TGF-ß1-mediated TP53 decrease (Fig. 6A)
. Furthermore, the
TGF-ß1 induced increase in CDC2 was inhibitable by pretreating the
cells with SOD, as shown in Fig. 6B
, implicating the
involvement of ROS not only in the TGF-ß1-induced decrease in p53 but
also in the TGF-ß1-stimulated increase in CDC2. The above collective
results suggest that extracellular
H2O2 and intracellular
O2 may both play roles in the decreased TP53 protein bystander
response. Apparently extracellular O2 contributes not only to
the early TGF-ß1 increases in the supernatants of -irradiated
cells but also to the decreased TP53 bystander response. Hence, both
increases in extracellular and intracellular ROS apparently play roles
in mediating the decreased TP53 protein bystander response.
|
particle-induced
bystander enhanced growth effect as our previous results indicate, then
treatment of cells with low concentrations of TGF-ß1 should have a
promitogenic effect and thereby mimic the effects of supernatants from
-irradiated cells. In the final set of experiments, cells were
treated with varying concentrations of TGF-ß1, i.e., 1, 5,
10, and 50 ng/ml. Cells were harvested on days 1, 2, and 3, and cell
numbers were counted. As expected, treatment of cells with 1 and 5
ng/ml of TGF-ß1 resulted in increase in cell numbers as early as day
1, with a significant increase becoming unequivocally evident on day 2
and thereafter (Fig. 7)
. However, only a moderate increase in cell numbers was observed at the
10 ng/ml concentration, and major decreases in cell numbers were found
with cells treated with 50 ng/ml of TGF-ß1 (Fig. 7)
. These results
indicate that low concentrations of TGF-ß1 are promitogenic, whereas
high concentrations inhibit cell growth, which correlates with the
inversely related TP53 and ROS dose response observed with TGF-ß1,
i.e., low levels of TGF-ß1 decrease TP53 protein levels
and increase ROS levels, whereas high concentrations of TGF-ß1 induce
elevations in TP53 protein and a decreased ROS response.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
particles can result in enhancements in cell
growth, and that this promitogenic effect can be transmitted to
unirradiated cells by incubating them with supernatants harvested from
the irradiated cells. Our earlier findings and the results from the
present study, along with observations made by other investigators,
suggest that these proliferative responses can be explained in the
context of ROS playing a central role in mediating a cascade of events
that ultimately lead to enhancements in cell growth.
Our results indicate that the pathway to increases in cell growth
begins with the generation of extracellular ROS attributable to the
interactions of particles with extracellular constituents, as we
have described elsewhere (68)
. The ROS cause a prompt
increase in availability of extracellular TGF-ß1, a process that we
have shown to be SOD and catalase inhibitable in a manner that implies
hydroxyl radicals as the primary oxidant species for its initiation.
The increases in TGF-ß1, which others have shown can occur in
response to even low-dose ionizing radiation and other oxidizing
conditions (54, 55, 56, 57)
, tentatively arise from the rapid
secretion of TGF-ß1 and/or the release of TGF-ß1 from betaglycan
receptors and/or possibly the extracellular matrix
(69, 70, 71)
. Regardless, the essential step function-like
increases in TGF-ß1 activates cell membrane-associated NADPH oxidase
that is present in human fibroblasts (72
, 73)
, which, in
turn, increases the intracellular production of superoxide anions and
hydrogen peroxide. TGF-ß1 may additionally activate cell surface
membrane-associated NADH oxidase and the release of extracellular and
cell-permeating H2O2 as a
later and perhaps sustaining event (51
, 74)
; as reported
previously (16)
, the intracellular ROS increases in
response to -irradiation have been noted to persist for at least
24 h after exposure. That TGF-ß1 increases in the supernatants
of irradiated cells can indeed mediate the activation of the metabolic
production of ROS is well illustrated by the fact that the increased
intracellular ROS bystander response can be mimicked by the addition of
recombinant TGF-ß1 to unirradiated cells, and the ability of the
cytokine to produce the response can be totally inhibited by
anti-TGF-ß1-neutralizing antibodies.
Existing evidence suggests that the production of intracellular
ROS by the above mechanisms is a key step in mediating the promitogenic
effects of low-dose particles. Unlike with high level exposure to
ROS, relatively low-level exposure to ROS or transient exposure to ROS
can stimulate cell growth (36
, 38
, 39
, 75)
, and the
addition of antioxidants such as SOD, glutathione, catalase,
inter alia, or decreases in endogenous cellular levels of
oxidants all have antiproliferative effects (38
, 39
, 76)
.
Moreover, some investigators have reported synergistic effects of
oxidants and mitogens on cell growth (33)
. Although the
mechanism(s) involved in ROS-stimulated cell proliferation are likely
multifarious, it is evident that a cells redox status can play
fundamental roles in cell growth-associated signal transduction
pathways (77
, 78)
, in the regulation of the expression of
genes whose products contribute to cell growth (74
, 79
, 80)
, and in posttranslational modifications of cell
cycle-regulating proteins (81
, 82)
.
Increases in ROS alone, however, may be only one component of the
promitogenic response to particles, given the finding that
supernatants from irradiated cells cause significant reductions in the
levels of TP53 and CDKN1A proteins in unirradiated cells, with the
latter reduction in CDKN1A perhaps being a result of initial decreases
in its transcriptional activator, TP53, a possibility that is currently
being investigated. Regardless, both of these proteins are well
recognized as negative regulators of cell cycle progression (31
, 83)
, perhaps to some extent even when expressed under normal
basal conditions. At least in vitro, a variety of cell
types, including fibroblasts, that are null for TP53 or CDKN1A show
higher proliferation rates than their wild-type counterparts
(e.g., Refs. 73
, 84,
and 85
). As
well, our findings that increases in PCNA and CDC2 protein levels
during the increased ROS and decreased TP53/CDKN1A responses underscore
how numerous regulators of cell proliferation may be contributing to
the overall promitogenic response to particles.
Although we have yet to elucidate the ultimate mechanism(s) that
underlie the decreased TP53 bystander response, our results indicate
that it is likely linked to the TGF-ß1-induced intracellular ROS
response in that it can be eliminated, like the intracellular ROS and
promitogenic bystander responses, by the addition of anti-TGF-ß1
neutralizing antibodies to the supernatants of -irradiated cells and
by inhibition of the intracellular ROS response. We currently
hypothesize that the diminished TP53 protein levels that occur as a
bystander effect result from ROS-associated alterations in one or more
processes that down-regulate TP53 levels in cells, which potentially
could include the transcription, translation, and/or degradation, with,
perhaps, transcription being the most affected, given the recent report
that low concentrations of TGF-ß can suppress both p53 and p21 mRNA
levels and CDKN1A protein levels in human lung fibroblasts
(53)
.
A question that arises as to how our findings that a low dose of
particles causes a promitogenic effect in directly irradiated and
unirradiated cells can be reconciled with the investigation reported by
Azzam et al. (18)
, in which low-dose
particles can induce increases in TP53 and CDKN1A in a manner that
expectedly would disfavor cell growth. Both sets of data may best be
accommodated by the existence of two different processes. In the study
by Azzam et al. (18)
, evidence was obtained
that is consistent with the transmission of TP53/CDKN1A up-regulating
signals from directly irradiated cells to nearby cells via gap
junctional communications. In our studies, we also observed greater
than expected increases in TP53 and CDKN1A in cells that were
irradiated with a low dose of particles, and this was associated with
evidence of decreases in cell growth as of 24 h after irradiation.
Thereafter, however, we observed growth rates in the irradiated
populations that, over time, exceeded control rates. Conceivably, the
bystander effects observed in our study may serve to reduce the effect
of the cell-cell communicated increased TP53/CDKN1A response, or
perhaps, the enhanced growth we observed occurred in cells that did not
experience the increased TP53/CDKN1A bystander effect.
Finally, ample evidence shows that the DNA-damaging effects of
excessive ROS can activate cell cycle checkpoints, induce a
senescence-like state, or cause cells to undergo apoptosis and even
necrosis in vitro (34
, 38
, 86)
. As we have
reported earlier, at least higher doses of particles,
i.e., 19 and 57 cGy, than those used in the present study
cause both G1 and G2 cell
cycle arrests in HFL1 cells (40)
. On the basis of these
findings, one might expect that exposure to ROS stresses, if anything,
would curtail cell proliferation in vivo as well.
Paradoxically, however, cells along the conducting airways often
undergo enhanced proliferation, i.e., hyperplasia, in
vivo in a background of ROS-associated stimuli, including
inhalation exposure to radon/radon progeny (19, 20, 21, 22, 23)
. In
this regard, our results provide a mechanistic explanation for such
hyperproliferative responses. Furthermore, excessive cell
proliferation, especially in a concurrent background of DNA-damaging
oxidative stress, expectedly could contribute to carcinogenic processes
(87, 88, 89, 90, 91)
. How our overall results may extend to other
important phenotypes, such as bronchial epithelial cells, remains to be
determined.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported in part by the Los Alamos National
Flow Cytometry Resource (NIH Grant p41-RR01315), NIH Grant CA82598, and
a grant from the Office of Biological and Energy Research, United
States Department of Energy. The work was conducted under the auspices
of the United States Department of Energy. 
2 To whom requests for reprints should be
addressed, at Bioscience Division, MS M888, Los Alamos National
Laboratory, Los Alamos, NM 87545. Phone: (505) 667-2753; Fax: (505)
665-3024; E-mail: lehnert{at}telomere.lanl.gov
3 The abbreviations used are: , high linear
energy transfer ; SCE, sister chromatid exchange; ROS, reactive
oxygen species; SOD, superoxide dismutase; PCNA, proliferating cell
nuclear antigen; TGF, transforming growth factor; DCFH-DA,
2',7'-dichlorofluorescin diacetate; DCF, 2',7'-dichlorofluorescein;
DPI, diphenyleneiodonium.
Received 7/ 6/99. Accepted 12/22/99.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
particles in mammalian cells [see comments]. Proc. Natl. Acad. Sci. USA, 94: 3765-3770, 1997.-particles. Carcinogenesis (Lond.)., 17: 1671-1676, 1996. particles through mammalian cell nuclei. Proc. Natl. Acad. Sci. USA, 96: 19-22, 1999.-Particle-induced p53 protein expression in a rat lung epithelial cell strain. Cancer Res., 54: 5797-5800, 1994.-particle irradiation [see comments]. Nature (Lond.), 355: 738-740, 1992.[Medline]
-Particle-induced chromosomal instability in human bone marrow cells. Lancet, 344: 987-988, 1994.[Medline]
-Particle aftereffects. Nature (Lond.), 355: 674-675, 1992.[Medline]
-particles [see comments]. Cancer Res., 52: 6394-6396, 1992.-Particle-induced sister chromatid exchange in normal human lung fibroblasts: evidence for an extranuclear target. Radiat. Res., 145: 260-267, 1996.[Medline]
particles such as those emitted by radon and radon progeny. Environ. Health Perspect., 105(Suppl.5): 1095-1101, 1997.
particles can cause sister chromatid exchanges in normal human cells. Cancer Res., 57: 2164-2171, 1997. particles initiate biological production of superoxide anions and hydrogen peroxide in human cells. Cancer Res., 57: 3963-3971, 1997. particles. Radiat Res., 150: 497-504, 1998.[Medline]
particles. III. Cell inactivation by -particle traversals of the cell nucleus. Radiat. Res., 128: 204-209, 1991.[Medline]
particles. I. Exposure system and dosimetry. Radiat. Res., 123: 304-310, 1990.[Medline]
particles or rays. Radiat. Res., 146: 414-424, 1996.[Medline]
auxiliary protein. Nature (Lond.), 326: 517-520, 1987.[Medline]
-irradiation. FASEB J., 11: 991-1002, 1997.[Abstract]
, and RAD51 expression in normal human diploid fibroblasts is dependent on p53/p21Waf1. Cell Growth Differ., 9: 887-896, 1998.[Abstract]
irradiation is not required to induce a bystander effect in normal human keratinocytes: evidence for release during irradiation of a signal controlling survival into the medium. Radiat. Res., 149: 256-262, 1998.[Medline]
-particle irradiation. Proc. Natl. Acad. Sci. USA, 95: 5730-5733, 1998. particles induce the production of interleukin-8 by human cells. Radiat. Res., 152: 57-63, 1999.[Medline]
This article has been cited by other articles:
|
|
 |
|
  B. Risek, P. Bilski, A. B. Rice, and W. T. Schrader Androgen Receptor-Mediated Apoptosis Is Regulated by Photoactivatable Androgen Receptor Ligands Mol. Endocrinol., September 1, 2008; 22(9): 2099 - 2115. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhou, V. N. Ivanov, Y.-C. Lien, M. Davidson, and T. K. Hei Mitochondrial Function and Nuclear Factor-{kappa}B-Mediated Signaling in Radiation-Induced Bystander Effects Cancer Res., April 1, 2008; 68(7): 2233 - 2240. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Sgouros, S. J. Knox, M. C. Joiner, W. F. Morgan, and A. I. Kassis MIRD Continuing Education: Bystander and Low Dose-Rate Effects: Are These Relevant to Radionuclide Therapy? J. Nucl. Med., October 1, 2007; 48(10): 1683 - 1691. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W Han, L Wu, B Hu, L Zhang, S Chen, L Bao, Y Zhao, A Xu, and Z Yu The early and initiation processes of radiation-induced bystander effects involved in the induction of DNA double strand breaks in non-irradiated cultures Br. J. Radiol., September 1, 2007; 80(Special_Issue_1): S7 - S12. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M Natarajan, C F Gibbons, S Mohan, S Moore, and M A Kadhim Oxidative stress signalling: a potential mediator of tumour necrosis factor {alpha}-induced genomic instability in primary vascular endothelial cells Br. J. Radiol., September 1, 2007; 80(Special_Issue_1): S13 - S22. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Tartier, S. Gilchrist, S. Burdak-Rothkamm, M. Folkard, and K. M. Prise Cytoplasmic Irradiation Induces Mitochondrial-Dependent 53BP1 Protein Relocalization in Irradiated and Bystander Cells Cancer Res., June 15, 2007; 67(12): 5872 - 5879. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Y. Tyurina, L. V. Basova, N. V. Konduru, V. A. Tyurin, A. I. Potapovich, P. Cai, H. Bayir, D. Stoyanovsky, B. R. Pitt, A. A. Shvedova, et al. Nitrosative Stress Inhibits the Aminophospholipid Translocase Resulting in Phosphatidylserine Externalization and Macrophage Engulfment: IMPLICATIONS FOR THE RESOLUTION OF INFLAMMATION J. Biol. Chem., March 16, 2007; 282(11): 8498 - 8509. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Huang, P. M. Kim, J. A. Nickoloff, and W. F. Morgan Targeted and Nontargeted Effects of Low-Dose Ionizing Radiation on Delayed Genomic Instability in Human Cells Cancer Res., February 1, 2007; 67(3): 1099 - 1104. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. J.Kim, K. Chandrasekaran, and W. F.Morgan Mitochondrial dysfunction, persistently elevated levels of reactive oxygen species and radiation-induced genomic instability: a review Mutagenesis, November 1, 2006; 21(6): 361 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Boyd, S. C. Ross, J. Dorrens, N. E. Fullerton, K. W. Tan, M. R. Zalutsky, and R. J. Mairs Radiation-Induced Biologic Bystander Effect Elicited In Vitro by Targeted Radiopharmaceuticals Labeled with {alpha}-, {beta}-, and Auger Electron-Emitting Radionuclides J. Nucl. Med., June 1, 2006; 47(6): 1007 - 1015. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Prise New advances in radiation biology Occup. Med., May 1, 2006; 56(3): 156 - 161. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Hu, L. Wu, W. Han, L. Zhang, S. Chen, A. Xu, T. K. Hei, and Z. Yu The time and spatial effects of bystander response in mammalian cells induced by low dose radiation Carcinogenesis, February 1, 2006; 27(2): 245 - 251. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhou, V. N. Ivanov, J. Gillespie, C. R. Geard, S. A. Amundson, D. J. Brenner, Z. Yu, H. B. Lieberman, and T. K. Hei Mechanism of radiation-induced bystander effect: Role of the cyclooxygenase-2 signaling pathway PNAS, October 11, 2005; 102(41): 14641 - 14646. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Shao, M. Folkard, B. D. Michael, and K. M. Prise Targeted cytoplasmic irradiation induces bystander responses PNAS, September 14, 2004; 101(37): 13495 - 13500. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. I Azzam and J. B Little The radiation-induced bystander effect: evidence and significance Human and Experimental Toxicology, February 1, 2004; 23(2): 61 - 65. [Abstract] [PDF]
|
|
|
|
|
|
E. G Wright Commentary on radiation-induced bystander effects Human and Experimental Toxicology, February 1, 2004; 23(2): 91 - 94. [Abstract] [PDF]
|
|
|
|
|
|
C. A Waldren Classical radiation biology dogma, bystander effects and paradigm shifts Human and Experimental Toxicology, February 1, 2004; 23(2): 95 - 100. [Abstract] [PDF]
|
|
|
|
|
|
C. Shao, V. Stewart, M. Folkard, B. D. Michael, and K. M. Prise Nitric Oxide-Mediated Signaling in the Bystander Response of Individually Targeted Glioma Cells Cancer Res., December 1, 2003; 63(23): 8437 - 8442. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Nagar, L. E. Smith, and W. F. Morgan Mechanisms of cell death associated with death-inducing factors from genomically unstable cell lines Mutagenesis, November 1, 2003; 18(6): 549 - 560. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. SHAO, Y. FURUSAWA, Y. KOBAYASHI, T. FUNAYAMA, and S. WADA Bystander effect induced by counted high-LET particles in confluent human fibroblasts: a mechanistic study FASEB J, August 1, 2003; 17(11): 1422 - 1427. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Limoli, E. Giedzinski, W. F. Morgan, S. G. Swarts, G. D. D. Jones, and W. Hyun Persistent Oxidative Stress in Chromosomally Unstable Cells Cancer Res., June 15, 2003; 63(12): 3107 - 3111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Nagar, L. E. Smith, and W. F. Morgan Characterization of a Novel Epigenetic Effect of Ionizing Radiation: The Death-Inducing Effect Cancer Res., January 15, 2003; 63(2): 324 - 328. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Deshpande, P. K. Narayanan, and B. E. Lehnert Silica-Induced Generation of Extracellular Factor(s) Increases Reactive Oxygen Species in Human Bronchial Epithelial Cells Toxicol. Sci., June 1, 2002; 67(2): 275 - 283. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B E Lehnert and R Iyer Exposure to low-level chemicals and ionizing radiation: reactive oxygen species and cellular pathways Human and Experimental Toxicology, February 1, 2002; 21(2): 65 - 69. [Abstract] [PDF]
|
|
|
|
|
|
E. Bortoletto, M. Mognato, P. Ferraro, S. Canova, R. Cherubini, L. Celotti, and A. Russo Chromosome instability induced in the cell progeny of human T lymphocytes irradiated in G0 with {gamma}-rays Mutagenesis, November 1, 2001; 16(6): 529 - 537. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Mothersill, D. Rea, E. G. Wright, S. A. Lorimore, D. Murphy, C. B. Seymour, and K. O'Malley Individual variation in the production of a `bystander signal' following irradiation of primary cultures of normal human urothelium Carcinogenesis, September 1, 2001; 22(9): 1465 - 1471. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
