Download Autocrine Effects of Fibroblast Growth Factor in Repair of Radiation

(CANCER RESEARCH 51, 2552-2558, May 15, 1991]
Autocrine Effects of Fibroblast Growth Factor in Repair of Radiation Damage in
Endothelial Cells'
Adriana Haimovitz-Friedman,
Israel Vlodavsky, Alpana Chaudhuri, Larry Witte, and Zvi Fuks2
Departments of Radiation Oncology; Memorial Sloan-Kettering Cancer Center, New York, New York (A. H-F., Z. F.); the Department of Oncology, Hadassah-Hebrew
University Hospital, Jerusalem, Israel (I. V.); and the Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, New York (A. C.,
L. W.)
ABSTRACT
The study demonstrates that basic fibroblast growth factor (bFGF)
serves as an inducer of radiation damage repair in bovine aortic endothelial cells (BAEC). Radiation dose-survival curves were generated with
plateau-phase BAEC using culture dishes precoated with HR9-bFGF/
extracellular matrix (ECM) for the postradiation colony formation assay.
This natural basement membrane-like ECM is enriched with ECMbound bFGF. Under these conditions the cells exhibited increased repair
of radiation damage as compared to cells plated on top of the bFGF-free
isotype of this extracellular matrix (the HR9/ECM). While the slopes
of the curves did not differ significantly (Do 107 ±6.8 cGy on the HR9/
ECM, compared to 112 ±1.3 cGy on the HR9-bFGF/ECM),
there was
a nearly complete elimination of the threshold shoulder in the curves
generated on the bFGF-free HR9/ECM (Dq 29 ±19 cGy, compared to
174 ±22 cGy on the HR9-bFGF/ECM;
P < 0.05). Delayed plating
experiments, in which the cells were irradiated under bFGF-free condi
tions (while adherent as contact-inhibited monolayers to the HR9/ECM
in bFGF-free medium) and maintained after irradiation in the same
culture for various periods of time, showed that the cells performed repair
of potentially lethal damage (PLDR) and restored clonogenic ability,
with a 24 h to immediate postradiation recovery ratio of 3.27. This
expression of PLDR was inhibited by neutralizing monoclonal antibodies
against bFGF, indicating that the irradiated cells secreted bFGF into
their conditioned medium. Northern blot hybridization showed a 5.6-fold
increase of the 3.7-kilobase species and a 4.7-fold increase of the 7.0kilobase species of the bFGF-specific mRNA within 6 h after delivery of
a single dose of 400 cGy. The data suggest that radiation induces a
complete cycle of an autoregulated damage-repair pathway in BAEC,
initiated by radiation-induced damage to cellular DNA and followed by
stimulation of bFGF synthesis and its secretion into the medium. The
newly synthesized bFGF stimulates the PLDR pathway, acting via an
extracellular autocrine loop (inhibitable by specific anti-bFGF antibod
ies), leading to recovery of cells from radiation lesions and restoration of
their clonogenic capacity.
INTRODUCTION
The current hypothesis concerning the nature of the lethal
lesions of ionizing irradiation in eukaryotic cells suggests that
the loss of the clonogenic capacity and cell death result from
damage to the structure and function of genomic DNA (1, 2).
Although mammalian cells are proficient in their capacity to
repair radiation damage to DNA (3), not all radiation lesions
undergo repair (2). Some lesions are irreparable and are consid
ered lethal (4, 5), while others are potentially repairable (6-9),
but their fate depends upon competing processes of repair and
lethal fixation (10-12). Thus, the proportional loss of the
clonogenic capacity at a given radiation dose results from the
production of nonrepairable lesions and from potentially re
pairable lesions that undergo lethal fixation (10, 12). The net
Received 11/30/90; accepted 3/7/91.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by NIH Grant CA-52462.
; To whom request for reprints should be addressed, at the Department of
Radiation Oncology, Memorial Sloan-Kettering Cancer Center. 1275 York Ave.,
New York, NY 10021.
effect of increasing doses of radiation on the clonogenic capacity
is classically described by dose-survival curves (13), which typ
ically consist of an initial curved component at the low dose
range (the threshold shoulder) and of a subsequent exponential
component. Several biophysical models simulate the cell sur
vival data, and parameters derived from the mathematical func
tions that describe these models are used to define the sensitivity
of mammalian cells to radiation and their potential for repair
of radiation damage (10, 14, 15). Parameters that describe the
exponential components are commonly used to define the in
herent radiosensitivity of a given cell, while those parameters
that describe the curvilinear threshold shoulder reflect the ca
pacity to repair radiation lesions and to restore clonogenicity.
While there are methods to quantitatively evaluate the repair
capacity of radiation-induced damage, there is little information
concerning the enzymatic pathways involved in repair of radia
tion lesions in eukaryotic cells and the genetic and epigenetic
mechanisms that control and regulate this cellular function
(16). Potentially lethal radiation damage repair3 has been re
ported to be an inducible process (17-20) and sensitive to
changes in the microenvironmental conditions of the culture.
Conditions leading to reduction of the metabolic rate of cells
or in their proliferative activity increase PLDR, while hypertonic media or chemical inhibitors of DNA synthesis promote
potentially lethal damage fixation and decreased repair (9, 11).
In recent experiments4 we showed that, when BAEC were
seeded after irradiation on top of their autologous natural
basement membrane-like ECM (the BAEC/ECM), they exhib
ited an improved capacity for repair of radiation damage com
pared to cells plated on other natural or artificial substrata.
Because the BAEC/ECM contains endogenous heparan sulfatebound bFGF (21), these experiments raised the possibility that
bFGF may be involved in regulation of radiation damage repair
in this cell type.
To evaluate this possibility, in the present study, we generated
radiation dose-survival curves for BAEC, using culture dishes
coated with two isotypes of ECM produced by variants of the
PF-HR9 mouse endodermal carcinoma cell lines for the postradiation colony formation assays (22). We recently showed (23)
that the wild-type PF-HR9 cells produce an FGF-free ECM
(the HR9/ECM), while the HR9-bFGF cell line, which was
cloned from PF-HR9 cells transfected with the bovine bFGF
gene (23), produces an identical ECM except for the inclusion
of bFGF in a bound form to heparan sulfate proteoglycans (the
HR9-bFGF/ECM). The ECM-bound bFGF has been shown to
confer normal biological activities in terms of mitogenic stim' The abbreviations used are: PLDR, potentially lethal damage repair; BAEC,
bovine aortic endothelial cells; ECM, extracellular matrix; BAEC/ECM, ECM
produced by BAEC; HR9/ECM, ECM produced by PF-HR9 cells; HR9-bFGF/
ECM, ECM produced by HR9-bFGF cells; FGF, fibroblast growth factor; bFGF,
basic fibroblast growth factor; DMEM, Dulbecco's modified Eagle's medium;
PBS, phosphate-buffered saline; STV. 0.05% trypsin and 0.02% EDTA in PBS;
BCS, bovine calf serum.
4 Z. Fuks. I. Vlodavsky. M. Andreeff. and A. Haimovitz-Friedman. Effects of
extracellular matrix on the response of endothelial cells to radiation in vitro,
submitted for publication.
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FGF INDUCTION OF PLDR
ulation of endothelial cells and of neuronal differentiation of
PC 12 cells (23). Utilizing these matrix systems, we demonstrate
in the present study that bFGF is capable of serving as an
inducer of PLDR in plateau-phase BAEC. Furthermore, our
data also show that the induction of PLDR by bFGF in this
cell type represents a component of an autoregulated damagerepair cycle. This cycle is initiated by radiation injury and
involves the synthesis and secretion of bFGF by the irradiated
cells, and the newly synthesized bFGF activates the PLDR
pathway via an extracellular autocrine loop.
cytofluorograph was interfaced with a Hewlet-Packard microcomputer
and the data were analyzed using the "Cell Fit" software package
(Becton-Dickinson, Mountainview, CA) and presented via a Tektronix
terminal.
Radiation Dose-Survival Curves. Survival after irradiation was de
fined as the ability of the cells to maintain their clonogenic ability and
to form colonies. Twenty-four h prior to irradiation the culture medium
was changed to 10% BCS-DMEM. Immediately prior to irradiation,
the cells were dissociated with STV, washed twice in 10% BCS-DMEM,
and resuspended in the same medium at a concentration of IO5 cells/
ml. Irradiation was carried out in a gamma cell 40 chamber containing
two sources of '"Cs (Atomic Energy of Canada) at a dose rate of 100
cGy/min. Single doses ranging from 200 to 600 cGy were delivered.
Immediately after irradiation the cells were seeded for colony formation
assays in 35-mm dishes (300-3000 cells/dish) coated with either the
Cell Cultures. Cloned populations of BAEC were established from
HR9/ECM or the HR9-bFGF/ECM. At designated times as described
the intima of bovine aorta as previously described (24). Stock cultures
in each experiment, a single dose of partially purified bovine brain
were grown in 35-mm culture dishes in DMEM supplemented with
bFGF (to a final concentration of 200 ng/ml) was added to the media
glucose (1 g/liter), 10% heat-inactivated BCS, penicillin (50 units/ml),
and streptomycin (50 Mg/ml). Partially purified bovine brain bFGF [200 of the HR9/ECM cultures, because in the absence of bFGF, BAEC
failed to form colonies on the HR9/ECM (23). The cells seeded on top
ng/ml; prepared as previously described (25)] or purified recombinant
of the HR9-bFGF/ECM did not require exogenous FGF supplements
bFGF (10 ng/ml; kindly provided by Dr. J. Abraham, California
Biotechnology, Inc.) was added every other day during the phase of to produce colonies, since this ECM was shown to contain biologically
active ECM-bound bFGF (23). To prove that the effects observed were
exponential growth. After 8-10 days in culture, the cells reached
conferred by bFGF and not by a contaminant present in the partially
confluence and exhibited features of contact-inhibited monolayers.
purified bFGF preparation, recombinant bovine bFGF (to a final con
These plateau-phase cells were either used for experiments or further
subcultured (up toa maximum of 10 subcultures) at a split ratio of 1:10 centration of 10 ng/ml) was used in some experiments in parallel to
200 ng/ml partially purified bFGF, and the results were similar. After
to expand the cell population for other experiments. For subculturing,
7-9 days, the cultures were fixed in 3.7% formaldehyde and stained
the monolayers were dissociated with 0.05% trypsin and 0.02% EDTA
in PBS (STV) (2-3 min at 22°Cor 4°C),washed twice in 10% BCS- with 1% crystal violet in ethanol. Colonies consisting of 50 cells or
more were scored, and 3-6 replicate dishes containing 50-150 colonies/
DMEM, and resuspended in DMEM with supplements as described
dish
were counted for each radiation dose tested. Survival curves were
above. These mild conditions of trypsinization were sufficient to detach
generated by a computer-assisted program (27). This program analyzes
the cells but did not injure, stimulate, or affect cell functions in a
the cellular survival after irradiation from the raw data of colony counts
detectable way. Cultures of the PF-HR9 mouse endoderma! carcinoma
of each culture dish included in the experiment. Using these data, the
cell line and its bFGF-HR9 variant were cultured as previously de
scribed (23) in DMEM supplemented with glucose (4.5 g/liter), 10% program calculates the surviving fractions for each dose point with
associated weights that are based on a Poisson distribution for the
fetal calf serum, and antibiotic supplements as described above. After
number of counts and the variation of colony counts for each data
reaching confluence, the cells were subcultured weekly at a split ratio
point. Using the calculated survival fractions and the associated
of 1:10 (after dissociation with STV). All cell cultures were maintained
weights, the program then fits the data according to the survival model
at 37°Cin 10% CO2 humidified incubators.
tested [the single-hit multitarget model (14) or the linear quadratic
Preparation of Dishes Coated with ECM. HR9/ECM and HR9model (15)1 D>'iteratively weighted least squares regression analysis of
bFGF/ECM were produced as previously described (24). PF-HR9 cells
all
data points, estimating the covariance of the survival curve param
or HR9-DFGF cells were seeded (105/35-mm dish) into culture dishes
eters and the corresponding confidence limits. The computer then plots
precoated with fibronectin (50 jig/dish) to enforce a firm adhesion of
the best fit survival curve and assigns an SE bar to each data point,
the secreted ECM onto the plastic substratum. The medium consisted
thus permitting statistical comparisons of corresponding points on
of DMEM supplemented with glucose (4.5 g/liter), 10% fetal calf different curves.
serum, and antibiotic supplements as described above. Ascorbic acid
Northern Blot Hybridization for Quantitative Evaluations of bFGF
(50 jig/ml) was added on days 2 and 4. After 6-7 days in culture, the
mRNA. Plateau-phase BAEC were irradiated while adherent as monocell monolayers were denuded by treatment with a solution containing
layers to their original culture dishes. For each experimental point, IO8
0.5% Triton X-100 and 20 mivi NH4OH in PBS (3 min at 22°C),
cells were used. For extraction of cellular DNA, the monolayers were
followed by 4 washes with PBS. While cellular debris and cytoskeletons
dissociated by scraping and the cellular RNA was extracted using the
of the lysed cells were effectively removed, the underlying ECM re
guanidine isothiocyanate-cesium chloride method (28). The total RNA
mained intact and adhered to the entire area of the tissue culture dish.
(20 Mg/ml) was sorted out according to size by formaldehyde-1%
The ECMs could be stored at 4°Cfor several months before being used
agarose gel electrophoresis (29), transferred to nitrocellulose mem
branes, and hybridized by standard techniques (30) to the 32P nickin experiments.
Cell Cycle Analysis. The cell cycle distribution of BAEC was deter
translated PJII-I probe containing a 1.4-kilobase fragment of bovine
mined by flow cytometry after acridine orange staining of DNA and
bFGF cDNA (31). Each sample was also hybridized with a human yRNA as previously described (26). Prior to staining the monolayers
actin probe to normalize for the amounts of mRNA involved in each
experiment. The blots were then autoradiographed for 16 h at —85°C
were dissociated with STV, washed twice with 10% BCS-DMEM, and
resuspended in PBS at a concentration of 2-4 x IO5cells/0.2 ml. The
with intensifying screens. Quantitative analysis was performed by film
MATERIALS AND METHODS
cells were then mixed with 0.4 ml of a solution containing 0.05 N HC1, densitometry measurements.
0.15 M NaCl, and 0.1% (v/v) Triton X-100. After 30 s, 1.2 ml of a
Statistical Methods. The statistical analysis of the dose-survival
solution containing 1 mM EDTA, 0.15 M NaCl, and 6 mg/ml of curves, parameters, and exponents was performed as previously de
chromatographically purified acridine orange in 0.2 M Na2HPO4-0.1 M scribed (27). Mean values were compared by Student's t test. All P
values were two sided.
citric acid buffer (pH 6.0) was added. The cells were then passed through
the focus of an excitation argon-ion laser beam (Lexel model 75), and
the red fluorescence (fluorescence >600) and green fluorescence (fluo
RESULTS
rescence, 530) emissions for each cell were measured by separate
Effects of bFGF on Parameters of the Dose-Survival Curves.
photomultipilers in a computer-interfaced research cytofluorograph
The radiation dose-survival experiments were performed with
(modified model FC201; Ortho Instruments, Westwood MA). The
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FGF INDUCTION
plateau-phase BAEC 7-9 days after reaching confluency. Cell
cycle analysis by flow cytometry showed that 91 ±1.13% (mean
±SE of 6 experiments) of the cells were in G0-G| phase, 3.66%
±0.71% in S phase, and 5.33 ±0.66% in G2-M phase. Fig. 1
shows the dose-survival curves generated with these cells. Im
mediately after irradiation the cells were seeded on either the
HR9/ECM or the HR9-bFGF/ECM for colony formation as
says. A single dose of the partially purified bovine brain bFGF
was added to the media of the HR9/ECM cultures 72 h after
irradiation (to a final concentration of 200 ng/ml, equivalent
to approximately 10 ng/ml of purified recombinant bFGF),
since control unirradiated BAEC failed to form colonies on the
HR9/ECM in the absence of bFGF (23). The intent of the
relatively long delay in bFGF addition was to permit enough
time for either repair or lethal fixation of radiation-induced
lesion, while the 72-h period was not considered deleterious,
because the cloning efficiencies of control unirradiated BAEC
remained essentially unchanged regardless of whether the bFGF
was added immediately or up to 96 h after seeding (Fig. 3, top
curve). The cells cultured on top of the HR9-bFGF/ECM did
not regularly receive exogenous bFGF supplements, because
this ECM was shown to contain biologically active ECM-bound
bFGF (23). However, in several control experiments partially
purified bovine bFGF (200 ng/ml) or recombinant bFGF was
added to the culture media of the cells plated on the HR9bFGF/ECM, and the resultant survival curves did not differ
from those observed without addition of exogenous bFGF (data
not shown). As shown in Fig. 1, the slopes of the dose-survival
curves did not differ significantly (the Do was 107 ±6.8 cGy
on the HR9/ECM, compared to 112 ±1.3 cGy on the HR9bFGF/ECM). On the other hand, the threshold shoulder was
nearly completely eliminated in the cells plated on the HR9/
ECM. The Dq on the HR9/ECM was 29 ±19 cGy, compared
to 174 ±22 cGy for the cells plated on the HR9-bFGF/ECM
(P < 0.05). Similarly, there was an increase in the n value from
1.3 ±0.7 observed on the HR9/ECM to 3.8 ±1.1 on the HR9bFGF/ECM (P < 0.05). When the data were analyzed accord
ing to the linear quadratic model (15), the «exponent was
0.304 ±0.16 Gy-' for the HR9-bFGF/ECM and 0.665 ±0.15
Gy~' for the HR9/ECM-plated cells, while the corresponding
ßexponents were 0.069 ± 0.03 and 0.03 ± 0.02 Gy"2,
respectively.
To evaluate whether the timing of bFGF presentation to the
irradiated cells accounted for the differences observed in Fig.
1, exogenous bFGF (200 ng/ml) was added to the HR9/ECM
cultures immediately after irradiation, instead of 72 h later.
Fig. 2 shows that such an application of bFGF resulted in
restoration of the shoulder region. The Dq for the cells plated
on the HR9-bFGF/ECM in this experiment was 133 ±45 cGy.
When plated on the HR9/ECM and bFGF (200 ng/ml) was
added immediately after irradiation, the Dq was 162 ±47 cGy,
while when bFGF was added 72 h after irradiation the Dq was
35 ±5.9 cGy. The «value from the linear quadratic model
changed from 0.17 ±0.048 Gy"1 when bFGF was added at 0'
time to 1.93 ±0.36 Gy~' for bFGF addition at 72 h, and the
corresponding change in the ßexponent was from 0.1388 ±
0.008 to 0.0114 ±0.06 Gy~2. With increasing delays in bFGF
addition, there was a progressive elimination of the threshold
shoulder. Fig. 3 shows that, when plateau-phase BAEC were
irradiated with a single dose of 200 cGy and plated for colony
formation on HR9/ECM with bFGF (200 ng/ml) added im
mediately after irradiation, the surviving fraction was 0.156.
When addition of bFGF was delayed, the surviving fractions
OF PLDR
I
I
M
co 10-2
10
-3
Radiation Dose (Gy)
Fig. 1. Radiation dose-survival curves of plateau-phase BAEC seeded for colony
formation assays on top of either the HR9-Ã’FGF/ECM (O) or on top of the
HR9/ECM (A). The cells seeded on top of the HR9/ECM received a single dose
of partially purified bovine brain bFGF (200 ng/ml) 72 h after irradiation, while
the cells plated on top of the HR9-bFGF/ECM did not receive exogenous bFGF
supplements. The best fit curves were calculated by the least squares regression
analysis of all data points, using the single-hit multitarget model (14, 27). The
data shown were pooled from 6 different experiments. Point, mean; bar, ±SE.
10
O
00
10
-2
co
-3
10
10
Radiation Dose (Gy)
Fig. 2. Effect of delay in exposure to FGF on the radiation dose-survival curves
of BAEC. Plateau-phase BAEC were irradiated and seeded for colony formation
assays on top of the HR9/ECM. Partially purified bFGF (200 ng/ml) was added
to the cultures at 0' time (A) or at 72 h after irradiation (O). For comparison, a
dose-survival curve of the cells plated on top of the bFGF-HR9/ECM (D) is also
shown. Dose survival was computed as described in Fig. 1. Point, mean of 5
dishes; bar, ±SE.
decreased progressively, with the maximal loss observed within
the first 24 h after irradiation. The capacity of the cells to
exhibit recovery from radiation damage at 200 cGy was de
pendent not only on the timing of bFGF addition after irradia
tion but also on the medium concentration of bFGF. When
increasing doses of the partially purified brain bFGF (10-600
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FGF INDUCTION OF PLDR
Delay of bFGF Addition
(hours)
Fig. 3. Effect of delay in addition to bFGF to BAEC plated after irradiation
on the HR9/ECM. Plateau-phase cells were irradiated with a single fraction 200
cGy and plated on the HR9/ECM. bFGF (200 ng/ml) was added at different
time points after irradiation (*). Top line, cloning efficiencies of control shamirradiated cells plated on the HR9/ECM and bFGF (200 ng/ml) added at different
time points after seeding (O). Point, mean; bar, ±SE.
bation to permit firm adhesion of the cells as a monolayer to
the HR9/ECM, cell cycle analysis revealed that >90% of the
cells showed RNA/DNA profiles characteristic of G0-G,
(mostly Go) cells. The cells were then washed and resuspended
in the same bFGF-free medium, and while still adherent as a
monolayer on the HR9/ECM, the cells were irradiated with a
single dose of 400 cGy on ice. Immediately after irradiation,
cells of sample dishes were dissociated and tested for colony
formation on either HR9/ECM (with a single dose of 200 ng/
ml bFGF given 72 h later) or on HR9-bFGF/ECM. The rest
of the irradiated cells were maintained at 37°Cwithout medium
change and samples were scored for colony formation at 2-h
intervals on either the HR9/ECM or the bFGF-HR9/ECM, as
described above. Fig. 5 shows that during the first 2 h after
irradiation there was a 2.19-fold difference in the clonal ability
of the cells tested on the HR9/ECM and the HR9-bFGF/ECM.
However, as the delay before being tested for the clonogenic
ability was extended, this difference gradually decreased, reach
ing a 1.07-fold difference 8 h after irradiation. These data
indicated that the cells were undergoing PLDR. The recovery
ratio, defined as the ratio of the surviving fraction for 24 h
delayed plating onto the HR9/ECM (with a single dose of 200
ng/ml bFGF given 72 h later) to immediate postradiation
plating, was 3.27. To evaluate whether this expression of PLDR
was associated with bFGF, the cells were irradiated and main
tained postirradiation in the presence of neutralizing monoclo
nal antibodies against bFGF. Fig. 6 shows that PLDR was
nearly completely inhibited by the addition of 12 /¿g/mlof Fl09 anti-bFGF mouse monoclonal antibody (kindly provided by
ImClone Systems, Inc., New York, NY). This concentration of
the F1-09 antibody was previously shown to neutralize the
mitogenic effects of bFGF on BAEC (32).
Northern Blot Hybridization Experiments. Since exogenous
bFGF was not added to the culture media during the postradiation maintenance of the cell on HR9/ECM, the PLDR experi
ments suggested that the irradiated cells themselves secreted
0.25-
0.0
200
400
FGF (ng/ml)
Fig. 4. Effect of bFGF concentration on the capacity of irradiated BAEC to
form colonies. Plateau-phase cells were irradiated with a single fraction 200 cGy
and plated on the HR9/ECM. bFGF was added at different concentrations
immediately after irradiation. The surviving fraction is presented as a function of
bFGF concentration.
ng/ml) were added immediately after irradiation, a dose-re
sponse effect was observed, reaching a nearly maximal effect
with 200 ng/ml of the partially purified bFGF (Fig. 4).
Involvement of bFGF in PLDR. To further characterize the
bFGF-dependent radiation damage repair observed in Fig. 1,
the capacity of the cells to carry out PLDR was tested by
delayed plating experiments (8, 9). Prior to irradiation, the cells
were transferred onto dishes coated with the bFGF-free HR9/
ECM at a density of 1.2 x 10" cells/35-mm dish (a density
typical for confluent contact-inhibited BAEC). The medium
consisted of DMEM supplemented with glucose (1 g/liter),
antibiotics as detailed in "Materials and Methods," and 10%
heat-inactivated bovine calf serum (which did not have a mitogenie effect on BAEC), but no FGF. After an overnight incu
4
8
12
16
20
24
Delay Before Plating (hours)
Fig. 5. Delayed plating (PLDR) experiments in plateau-phase BAEC main
tained after irradiation under bFGF-free conditions. Monolayers of plateau-phase
BAEC were irradiated with a single dose of 400 cGy while adherent as contactinhibited monolayers (1.2 x 10* cells/dish) to the HR9/ECM in bFGF-free
medium. Immediately after irradiation, and at the specified time intervals after
irradiation, cells of sample dishes were assayed for colony formation by plating
2000 cells on either the HR9/ECM (A) (with a single dose of 200 ng/ml partially
purified FGF given 72 h later) or on the HR9-bFGF/ECM (O). The surviving
fraction is shown as a function of the duration of delay in plating. Similar results
were observed in 3 independent experiments. Point, mean of 6-8 dishes; bar, ±
SE.
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FGF INDUCTION OF PLDR
thereafter to preirradiation levels (data not shown). Increased
levels of mRNA of bFGF (2.2-fold) and secretion of bFGF into
the conditioned media were observed even after a radiation dose
of 125 cGY, a dose which was not accompanied by detectable
acute postirradiation cell lysis (<2% at 24 and 48 h by dye
exclusion tests) and which retained the clonogenic capacity in
>75% of the irradiated BAEC.
0.20-
I
O
e
0.15-
0.10-
DISCUSSION
Our data demonstrate that bFGF serves as a potent inducer
of potentially lethal radiation damage repair in endothelial cells.
In fact, bFGF is the first well-defined physiological agent char
acterized as an inducer of PLDR in any type of cells. Our data
also suggest that bFGF activates the PLDR pathway via inter
0.00
action of extracellular bFGF with receptors located on the
24
plasma membrane of the cell, since neutralizing monoclonal
Delay Before Plating (hours)
antibodies against bFGF inhibited PLDR (Fig. 5). This exper
Fig. 6. Inhibition of PLDR in irradiated plateau-phase BAEC by monoclonal
antibodies against bFGF. PLDR experiments were performed with plateau-phase
iment suggested that the newly synthesized intracellular bFGF
BAEC as described in Fig. 5. White columns, delayed plating for colony formation
was
apparently incapable of stimulating PLDR via an internal
assays on the HR9-bFGF/ECM; hatched columns, delayed plating for colony
loop
and that bFGF secretion and stimulation via an extracel
formation on the HR9/ECM (with a single dose of 200 ng/ml partially purified
bovine brain bFGF given 72 h later); shaded columns, delayed plating conditions
lular autocrine loop were necessary to initiate the PLDR path
similar to those shown by the hatched bars except that 12 ¿ig/mlof the Fl-09
way. Another basic requirement for the induction of PLDR in
anti-bFGF mouse monoclonal antibodies were present in the medium, removed
our system was its critical dependence on the timing of bFGF
only when the cells were plated for the colony formation assays. Similar data
were obtained in 3 independent experiments. Column, mean of 3-5 dishes: bar,
presentation to irradiated cells, restricting it to the early pos±SE.
tradiation period. A delay resulted in an irreversible lethal
fixation of potentially repairable lesions, which progressively
developed
over the first 24 h after irradiation (Figs. 2 and 3).
A B
In contrast, a delay in bFGF application of up to 96 h after
irradiation did not affect its ability to induce a mitogenic
r 7.0response and replication of surviving cells (Fig. 3). Whether the
b-FGF
mitogenic response and the PLDR pathway share the same
bFGF receptor-binding specificities and the same signal trans-3.7
duction pathways in endothelial cells is unknown, but it is
currently being investigated.
The fact that PLDR is an inducible process was previously
demonstrated in several studies, which showed that conditioned
media from irradiated cells conferred PLDR in other irradiated
cells (17-20). These data suggested that after irradiation cells
Actin
2.2
secrete into the culture media substances that serve as autocrine
or paracrine inducers of PLDR. One study demonstrated that
conditioned media from irradiated cells accumulated an as yet
Fig. 7. Northern blot analysis of mRNA extracted from irradiated BAEC
unidentified temperature-labile factor which induced PLDR
hybridized with the PJII-1 bFGF cDNA probe. Plateau-phase BAEC (10' cells/
and
which was both DNase and RNase resistant (20). Our data
blot) were scored for the levels of bFGF mRNA 6 h after irradiation with a single
dose of 400 cGy. Hybridization was performed with the PJII-I probe containing
are consistent with this model, because we have demonstrated
a 1.4-kilobase fragment of bovine FGF cDNA (32). The blots were also probed
that radiation induced in BAEC a complete cycle of an autorewith a human -. .u-iin probe to normalize for the amounts of mRNA located in
gulated damage-repair pathway. This cycle was initiated by
each lane. Species of bFGF transcriptional mRNA are shown for control unirradiated BAEC (lane A) and for BAEC irradiated with a single dose of 400 cGy
DNA damage and was followed by an increase in bFGF-specific
(lane B).
mRNA, bFGF synthesis, and its secretion into the conditioned
medium. The newly synthesized bFGF induced the PLDR
bFGF into the medium. This presumption is consistent with pathway by the bFGF acting via an extracellular autocrine loop,
inhibitable by specific anti-bFGF antibodies. The cycle was
our previous publication (33), which showed that BAEC surviv
completed with recovery of damaged cells from radiation le
ing the acute lethal effects of radiation synthesize and secrete
biologically active bFGF into their conditioned media (33). To sions and with restoration of their clonogenic capacity. Evi
dence for the existence of this autoregulated damage-repair
further evaluate this possibility. Northern blot hybridization
experiments were performed using the PJII-I probe containing
cycle was derived not only from the experiments which tested
a 1.4-kilobase fragment of bovine bFGF cDNA (31). Fig. 7 the capacity of the cells to repair potentially lethal damage
shows that, following a single dose of 400 cGy, there was a 5.6- (Figs. 5 and 6) but perhaps also indirectly from the radiation
dose-survival curves. The minimal threshold shoulder observed
fold increase in the 3.7-kilobase species and a 4.7-fold increase
in the 7.0-kilobase species of bFGF mRNA. In contrast, the in the survival curves of the HR9/ECM-plated BAEC shown
same dose of radiation had no effect on the levels of human y- in Fig. 1 (Dq 29 ±19 cGy) probably represents an autocrineactin mRNA. Increased bFGF mRNA levels were detected as stimulated repair effect of bFGF. The low level of repair activity
early as 2 h after irradiation, peaked at 6-8 h, and decreased
in this experiment can be attributed to the small numbers of
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FGF INDUCTION OF PLDR
cells used in the colony formation assays (300-3000 cells/35mm dish, compared to 1.2 X 106/35-mm dish used in the PLDR
experiments), resulting in low culture media concentrations of
bFGF. The demonstration that PLDR is associated with bFGF
secreted into the culture medium by the irradiated cells them
selves is consistent with our previous data (33), which demon
strated the accumulation of increasing amounts of biologically
active bFGF in conditioned media of endothelial cells after
irradiation.
Whereas there is significant information concerning several
components of our proposed autoregulated damage-repair cycle
in irradiated endothelial cells, others remain essentially un
known. In particular, there is little information regarding the
molecular basis of potentially lethal damage lesions and the
signal transduction and biochemical pathways involved in their
repair. PLDR in mammalian cells has been shown to proceed
in two distinct phases, with an early fast phase that occurs
within the first few minutes after irradiation and a slower phase
which proceeds during 4-6 h after treatment, although residual
repair has been detected up to 24 h after irradiation (11). Kinetic
studies of rejoining of radiation-induced single- and doublestranded DNA breaks in mammalian cells have also demon
strated bipasic patterns (34, 35), with a tv, of 2-20 min for the
early phase and a tVtof 1-3 h for the late phase (36-39). It has
been suggested that the initial fast repair entails rejoining of
breaks by enzymes which are constitutively expressed in mam
malian cells, such as DNA ligases, exonucleases, and DNA
polymerases (35, 40, 41). The late phase, which proceeds at a
significantly slower rate, is characteristically blocked by inhib
itors of protein, RNA, and DNA synthesis (11), suggesting that
it may require the induction of specific genes and gene products
to repair more complex types of DNA lesions (41). Our data
are consistent with this hypothesis, demonstrating that at the
least the induction of bFGF synthesis may be required to initiate
the slow phase of PLDR in our system. Whether the induction
of specific enzymes is also necessary is as yet unknown, but the
well-defined and highly controlled BAEC/HR9 system may be
useful in elucidating the biochemical mechanisms of this repair
and the nature of potentially lethal damage lesions. The use of
neutralizing monoclonal antibodies against bFGF and chemical
agents capable of dissociating bFGF from its functional cell
surface receptors [e.g., suramin (42), protamine (43)] provides
opportunities to turn on and off the stimuli that lead to PLDR
in our system and may be useful in the study of individual
components of its pathway and in kinetic studies.
The radiation-induced activation of bFGF synthesis observed
in our study apparently represents a widespread biological
radiation-related phenomenon which is not specific to the
BAEC, nor is it specific to the bFGF gene. We have found that,
in addition to bFGF, irradiation of BAEC resulted in an in
crease of platelet-derived growth factor-specific mRNA.5
Boothman et al. (41) recently showed that ionizing irradiation
activated in confluence-arrested human malignant melanoma
cells a pleiotropic protein synthesis response detected by twodimensional polyacrylamide gel electrophoresis of whole cell
extracts. This inducible expression of protein synthesis seemed
to be specific to ionizing radiation, since it was not induced by
other stresses such as heat shock, hypoxia, or alkylation treat
ment with melphalan (41). Ionizing irradiation was also re
ported to induce increased tumor necrosis factor-a-specific
mRNA transcripts and enhanced production of tumor necrosis
5 L. Witte, A. Haimovitz-Friedman,
data.
A. Chaudhuri, and Z. Fuks, unpublished
factor-«protein in several human tumor cell lines (44), transcriptional and posttranscriptional expressions of the c-jun and
c-fos protooncogenes in HL-60 cells (45), transcription of the
ß-PKCgene in Syrian hamster embryo fibroblasts (46), tran
scription of the chloramphenicol acetyltransferase gene transfected into the genome of NIH-3T3 cells (47), and transcriptional expressions of the interleukin 1 gene in Syrian hamster
embryo fibroblasts (48). The pleiotropic gene expressions ob
served in mammalian cells after exposure to X-irradiation are
believed to represent a stress response to the damage induced
by radiation to cellular DNA (41, 45, 47-49). Although the full
details of this stress response are unknown, recent studies have
suggested that it is different in several of its expressions from
the stress responses induced by heat shock or by chemical injury
to DNA in mammalian cells (41, 45, 47-50). In analogy to the
SOS response to DNA damage in prokaryotic cells (51), a
hypothesis has been suggested that relates PLDR after Xirradiation of mammalian cells to the postradiation stress re
sponse (41). Our data provide the first direct confirmation for
this hypothesis. Furthermore, since many types of cells do not
express receptors to bFGF, we suggest that the capacity to
stimulate PLDR is not specific to bFGF. Other cell types may
respond to other inducers of PLDR, the synthesis of which may
also be activated as a stress response to radiation-induced DNA
damage, and the stimulation of PLDR by such agents may also
be conferred via autocrine/paracrine mechanisms. By analogy
to the patterns of the growth factor-mediated mitogenic path
ways, it is possible to envision that different cell systems re
spond to different autocrine/paracrine-regulated
"PLDR-stimulating factors," each functioning through a specific cell surface
receptor and through its corresponding signal transduction
pathway, but all leading to a common final pathway that
terminates with repair of radiation-induced lesions.
The demonstration that bFGF is an inducer of radiation
damage repair in endothelial cells may also have practical
clinical implications. Recent histopathological and ultrastruc
tural studies showed that damage to the endothelium of the
microvasculature is a predominant lesion leading to radiation
injury in slowly proliferating normal tissues (e.g., skin, heart,
lung, liver, and the central nervous system) (52,53). In contrast,
the endothelium of large vessels and of the endocardium are
significantly more resistant to the effects of irradiation in vivo
(52, 53). The variations in the sensitivities of different sections
of the vascular system to irradiation correlate with our recent
observations of the distribution of bFGF in the basement mem
branes of various types of blood vessels (32). Our immunohistochemical studies showed that bFGF is ubiquitously detected
in the basement membranes of all sizes of blood vessels (32),
but while homogeneous and intensive immunoreactivities were
observed in large and intermediate size blood vessels, hetero
geneity was found in capillaries. The most intense capillary
immunoreactivities were observed in the basement membranes
of branching points of mid-size capillary vessels, while staining
became progressively less intense in nonbranching sections of
capillaries and seemed to be absent in some nonbranching
regions (32). The latter regions of the the capillary endothelium
apparently exhibit the most severe changes of radiation damage
(52, 53). Whether the cells of these radiosensitive sections of
the capillary intima are capable of responding to irradiation
with synthesis and secretion of bFGF is unknown, but the idea
of using i.v. applications of bFGF as a mode of radioprotection
against radiation damage to the microvasculature is an objective
that warrants further consideration.
2557
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FGF INDUCTION OF PLDR
ACKNOWLEDGMENTS
We wish to acknowledge with thanks Maureen McLoughlin and
Roger S. Persaud for their excellent technical assistance.
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Autocrine Effects of Fibroblast Growth Factor in Repair of
Radiation Damage in Endothelial Cells
Adriana Haimovitz-Friedman, Israel Vlodavsky, Alpana Chaudhuri, et al.
Cancer Res 1991;51:2552-2558.
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