From the Section for Radiobiology and Molecular
Environmental Research, Röntgenweg 11, 72076 Tübingen, Germany, the ¶ Queensland Cancer Fund
Research Laboratory, The Queensland Institute of Medical Research,
P. O. Royal Brisbane Hospital, Brisbane,
Queensland 4029, Australia, the
Gifu University School of
Medicine, 40 Tsukasa-Machi, Gifu 500-8076, Japan, and the
** Department of Surgery, University of Queensland, P. O. Royal
Brisbane Hospital, Brisbane, Queensland 4029, Australia
Received for publication, July 13, 2000, and in revised form, October 20, 2000
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ABSTRACT |
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Epidermal growth factor (EGF) has been reported
to either sensitize or protect cells against ionizing radiation. We
report here that EGF increases radiosensitivity in both human
fibroblasts and lymphoblasts and down-regulates both ATM (mutated in
ataxia-telangiectasia (A-T)) and the catalytic subunit of
DNA-dependent protein kinase (DNA-PKcs). No further
radiosensitization was observed in A-T cells after pretreatment with
EGF. The down-regulation of ATM occurs at the transcriptional level.
Concomitant with the down-regulation of ATM, the DNA binding activity
of the transcription factor Sp1 decreased. A causal relationship was
established between these observations by demonstrating that
up-regulation of Sp1 DNA binding activity by granulocyte/macrophage
colony-stimulating factor rapidly reversed the EGF-induced decrease in
ATM protein and restored radiosensitivity to normal levels. Failure to
radiosensitize EGF-treated cells to the same extent as observed for A-T
cells can be explained by induction of ATM protein and kinase activity
with time post-irradiation. Although ionizing radiation damage to DNA
rapidly activates ATM kinase and cell cycle checkpoints, we have
provided evidence for the first time that alteration in the amount of
ATM protein occurs in response to both EGF and radiation exposure.
Taken together these data support complex control of ATM function that
has important repercussions for targeting ATM to improve
radiotherapeutic benefit.
Functional loss of the
ATM1 protein, mutated in the
human genetic disorder ataxia-telangiectasia (A-T) (1), leads to a
pleiotropic phenotype including neurological abnormalities,
immunodeficiency, and a predisposition to develop a number of
malignancies, primarily leukemias and lymphomas (2). The characteristic
most widely studied in A-T is hypersensitivity to ionizing radiation
(3-5) that appears to be due to an inability of ATM to recognize and facilitate the repair of a subcategory of double strand breaks or a
form of damage that is converted into a double strand break in DNA (6,
7). It is also likely that recognition of a specific form of DNA damage
represents the trigger for ATM to activate a number of cell cycle
checkpoints (8, 9). A-T cells are defective in the phosphorylation of
p53 on serine 15 and serine 20 and dephosphorylation at serine 376 after exposure of cells to ionizing radiation (10-12). Evidence for a
direct involvement of ATM with p53 was provided by the demonstration
that these two molecules interact directly (13) and ATM phosphorylates
p53 on serine 15 in response to DNA damage (14, 15). Pre-existing ATM
protein is rapidly activated by ionizing radiation and radiomimetic agents by an undescribed mechanism (13-15). Exposure of cells to ionizing radiation fails to change either the subcellular distribution or the total amount of cellular ATM protein (16-18). Furthermore, ATM
protein levels are relatively constant throughout the cell cycle in
human fibroblasts (17). These data point to a post-translational mechanism of activation for ATM. However, there is evidence for alteration in the amount of ATM under certain conditions. Although ATM
is not detectable by immunohistochemical staining in quiescent myoepithelial cells lining normal breast ducts, significant expression is observed in the proliferative myoepithelium of sclerosing adenosis (19). In addition the amount of ATM protein changes dramatically in
quiescent lymphocytes (PBMCs) in response to mitogenic agents (20). In
the latter study a 6-10-fold increase in ATM in PBMCs was observed in
response to PHA, reaching a maximum by 3-4 days. As the amount of ATM
protein increased, so too did its protein kinase activity. Clearly this
is a slow response compared with the rapid activation of ATM
post-irradiation. Alteration in the amount of the ATM protein could
occur by transcriptional control at the promoter. The housekeeping gene
NPAT/E14/CAND3 lies ~0.55 kb from the 5' end of the
ATM gene (21-23). These genes share a bidirectional promoter that
contains CCAAT boxes and 4 consensus sites for the Sp1 transcription
factor (21, 22). Further delineation of the ATM promoter is required to
assist in understanding the transcriptional regulation of ATM.
ATM is located predominantly in the nucleus of proliferating cells,
which is in keeping with its role in DNA damage recognition and cell
cycle control, but ATM has also been detected in cytoplasmic vesicles
(16, 17, 24). Since ATM has been implicated in more general
intracellular signaling and since it responds to mitogenic agents, we
studied its possible regulation by epidermal growth factor (EGF) that
alters cellular response to radiation. EGF enhances the
radiosensitivity of some cell types (25-28) and increases the
radioresistance of other cells (29). We show here that EGF enhanced the
radiosensitivity of both fibroblasts and lymphoblasts, and this was
associated with a decrease in ATM. Reduction in ATM protein was
accompanied by a decrease in the amount of Sp1 DNA binding activity. We
also demonstrate that ATM protein is rapidly restored to constitutive
levels by both granulocyte/macrophage colony-stimulating factor
(GM-CSF) and ionizing radiation, and this appears to be achieved by
increasing Sp1 DNA binding activity.
Materials--
Normal human skin fibroblasts HSF7 and NFF were
from healthy donors expressing ATM protein; A-T fibroblasts GM03395
were obtained from Coriell Cell Repositories. Lymphoblastoid cells were
established by Epstein-Barr virus transformation from healthy
individuals, C3ABR, C28ABR, C35ABR, C2ABR, and C31ABR. All cells were
grown at 37 °C in a humidified atmosphere of 5% CO2 and
95% air in RPMI 1640 medium supplemented with 10% fetal calf serum.
Irradiation of cells was performed at room temperature using a
137Cs source delivering gamma rays at a dose rate of 2.8 Gy/min. EGF (R & D Systems) was added at a concentration of 50 ng/ml to the culture medium of log-phase cultures or serum-reduced fibroblasts for various times as indicated. The antibodies used are as follows: p53
monoclonal antibody (polyclonal antibody 1801) and polyclonal Sp1
antibody (Sp1pEp2) were from Santa Cruz Biotechnology; monoclonal Cell Survival--
Logarithmically growing lymphoblastoid cells
were incubated with or without EGF (50 ng/ml) for 16 h prior to
exposure to ionizing radiation (2.5 Gy/min, 137Cs). Cell
viability was determined at daily intervals between 1 and 4 days by
adding 0.1 ml of 0.4% trypan blue to a 0.5-ml cell suspension as
described previously, and viable cells were counted (30).
Immunoblotting--
EGF treatment was carried out on log-phase
cells for different times as indicated. After washing in PBS, 5 × 106 cells were resuspended in 40 µl of nuclear extraction
buffer (25 mM Hepes, pH 8, 0.25 M sucrose, 1 mM EGTA, 5 mM MgSO4, 50 mM NaF, 1 mM DTT, 1 mM PMSF) and
disrupted by at least 4 freeze-thaw cycles. Insoluble material was
removed by centrifugation at 13,000 × g for 20 min.
Protein concentration was determined using a Bio-Rad DC protein assay
kit according to the manufacturer's recommendations, and 50-100 µg
was used per sample. Protein samples were separated on 5 or 10%
denaturing gels and blotted on nitrocellulose membranes. After blocking
in 4% milk powder, 0.1% Tween 20, PBS for at least 1 h, the blot
was incubated for 1 h at room temperature or overnight at 4 °C,
with the relevant primary antibody. The blot was washed three times in
0.1% Tween 20/PBS and incubated with secondary peroxidase-conjugated
antibody for 1 h. Following several washing steps, the blot was
developed using a chemiluminescence kit (DuPont).
mRNA Determination--
Changes in ATM mRNA in
lymphoblastoid cells were determined by quantitative PCR as described
(31). In short, known amounts of a 402-bp DNA fragment corresponding to
an ATM mutation lacking exon 38 (5319 G to A) was used as a competitor
during amplification of the corresponding region in ATM cDNA from
normal cells, which gives a 544-bp fragment (nucleotides 5122-5665). A
series of 2-fold dilutions were employed to find the equivalence point
and thus a measure of the amount of ATM mRNA. Expression of
mRNA was also determined by Northern blotting. Briefly, total RNA
was isolated from log-phase cells with an RNA isolation kit (Qiaquick,
Qiagen) according to the manufacturer's instructions, and 20 µg of
RNA/lane was separated on a 1% agarose gel. Blotting of RNA on nylon
membrane (Hybond-N, Amersham Pharmacia Biotech) was performed overnight by capillary force followed by hybridization with an ATM cDNA probe.
Gel Retardation Assay (Electrophoretic Mobility Shift
Assay)--
The DNA binding activity of transcription factor Sp1 was
determined by modification of a method described previously (32). Briefly, 107 cells were washed in ice-cold PBS, resuspended
in 400 µl of buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.2 mM PMSF), and incubated for 15 min
on ice. After addition of 25 µl of 10% Nonidet P-40, the cells were
subsequently vortexed for 10 s and the nuclei pelleted by
centrifugation (1 min, 13,000 × g at 4 °C). The
nuclear pellet was resuspended in 40 µl of buffer C (20 mM Hepes, pH 7.9, 400 mM KCl, 1 mM
EDTA, 1 mM EGTA, 10% glycerol, 1 mM DTT, 0.2 mM PMSF) and rotated for 30-60 min at 4 °C to elute
nuclear proteins. After additional centrifugation for 15 min at 4 °C
the supernatant containing the nuclear proteins was aliquoted, frozen
in liquid nitrogen, and stored at Luciferase Assays--
The ATM promoter was amplified
from human genomic DNA using primers (forward,
5-TCCCCCGGGGGAGATCAAAACCACAGCAGG, and reverse, 5-CCCAAGCTTGGGCGTTCTCTCGCCTCCTCCCGTG). The 615-bp amplification product was cloned into the pGL3-basic luciferase reporter vector (Promega) using a SmaI/HindIII digest. This
construct (pGL3-ATM) was cotransfected with the pRL-CMV vector
(Promega) at a ratio of 2:1 into lymphoblastoid cells by
electroporation (280 V, 960 microfarads, 1 pulse). Thirty six hours
after electroporation the cells were incubated with 50 ng/ml EGF for a
further 16 h before cell extracts were prepared. Luciferase
activity was measured using the Dual Luciferase Assay (Promega, E1910).
Briefly, 100 µl of firefly luciferase substrate (LARII) and 20 µl
of cell extract were mixed, and the reaction was immediately measured
for 10 s. Then 100 µl of Renilla luciferase substrate
including an inhibitor for firefly luciferase (Stop & Glow) was added,
and light emission was detected for another 10-s interval. The ratio of
both measurements (pGL/pRL) results in the relative luciferase
activity, avoiding variabilities in transfection. The absolute values
of luminescence measured (relative light unit) was generally
in the range of 5·104-5·106. The untreated
control was set to 1.
ATM Kinase Assay--
ATM kinase activity after EGF treatment
was determined using the method described by Canman et al.
(15). For whole cell lysate isolation, cells were lysed on ice in TGN
buffer (50 mM Tris, pH 7.5, 50 mM
EGF Sensitizes Cells to Radiation--
Engagement of the EGF
receptor by EGF and other ligands initiates signal transduction
pathways giving rise to mitogenesis and can also alter the
radiosensitivity status of cells (25-29). We determined whether EGF
might increase the sensitivity of human fibroblasts and lymphoblasts by
incubating cells in EGF for 16 h prior to exposure to radiation
(1-4 Gy). The results in Fig. 1A demonstrate that
EGF-treated lymphoblastoid cells are more sensitive to radiation, and
this is observed over the radiation dose range 1-4 Gy (Fig.
1B). The effect of EGF treatment was significant for both
dose and time course experiments for C3ABR; p = 0.0023 ( EGF Treatment Reduces ATM Protein Expression--
Since
radiosensitivity and abnormalities in mitogenesis and other signaling
pathways are characteristic of the A-T phenotype, we initially
determined whether the radiosensitizing effect of EGF might be due to
alteration in the amount of ATM protein in normal control cells.
Immunoblotting with anti-ATM antibodies showed that the amount of ATM
protein was reduced significantly with time after incubation with EGF
in normal fibroblasts (Fig. 2A). No ATM was detected in
negative control AT3ABR cells, as expected (16). The same pattern of
reduction was observed for the catalytic subunit of
DNA-dependent protein kinase, DNA-PKcs, after EGF
treatment, but Ku, the DNA damage recognition component of
DNA-dependent protein kinase, remained largely unchanged
(Fig. 2A). It is evident that the amount of DNA-PKcs is
normal in the A-T cell line, AT3ABR, when compared with C3ABR. EGF
treatment of C3ABR lymphoblastoid cells revealed a similar pattern of
loss of expression of ATM, down to 30% by 9 h post-treatment
(Fig. 2B). A similar pattern of decrease was also observed
for DNA-PKcs (Fig. 1B). To test the universality of these
observations we employed several control lymphoblastoid cell lines. In
all four control lines investigated, ATM protein decreased
significantly after EGF treatment (Fig. 2C). It is evident
from the positive control (100 µg of extract from the lymphoblastoid
cell line, C3ABR) that the amount of ATM protein in HSF7 fibroblasts
(at equal loading) is considerably lower than that in lymphoblastoid
cells (Fig. 2A).
Effect of EGF on mRNA Expression--
Since EGF caused a
marked decrease in the amount of ATM protein, it was possible that this
was due to transcriptional down-regulation. To monitor changes to ATM
mRNA in lymphoblastoid cells, we employed quantitative reverse
transcriptase-PCR as described previously (31). A DNA fragment (402 bp)
corresponding to an ATM mutation (G5319A), which lacks exon 38, was used as a competitor for ATM cDNA amplification (nucleotides
5122-5665, 544 bp in size) in a series of 2-fold dilutions from C3ABR
cells. After 8 h of treatment with EGF, an equivalent point was
reached at 0.625 attomol/µl of competitor compared with 1.25 attomol/µl for untreated cells representing an ~2-fold
reduction in ATM mRNA in response to EGF (Fig.
3A). After 16 h of
treatment with EGF the equivalent point was reached at ~0.4
attomol representing a 3-fold reduction in ATM mRNA.
Northern blot analysis revealed that ATM mRNA was reduced to 60%
after incubation of normal fibroblasts (HSF7) with EGF for 8 h and
reached a plateau at ~30% of the untreated value at 12-24 h
post-treatment, which parallels the changes shown by reverse transcriptase-PCR in lymphoblastoid cells (Fig. 3B).
Reduced Sp1 Binding Activity after EGF Treatment--
A similar
genomic organization at the ATM locus exists for the human and mouse
genes where ATM and nuclear protein at the A-T locus (NPAT) are
arranged ~0.5 kb apart in a head-to-head configuration (Fig.
5A) (21, 22). These two genes are transcribed from a central
bidirectional promoter that contains several binding sites for the
transcriptional factor, Sp1 (33). Since ATM mRNA and protein were
reduced after EGF treatment, we predicted that this might be due to
interference with or reduced Sp1 DNA binding activity. Use of gel-shift
analysis with an oligonucleotide-binding consensus sequence for Sp1
revealed the presence of a single, well defined, retarded band (Fig.
4A). The amount of Sp1 binding in extracts from C3ABR cells decreased with time after EGF treatment (Fig. 4A). A decrease of ~50% by 12 h
post-treatment, leveling off at later times, paralleled the decrease in
the amount of ATM mRNA and protein seen previously. To establish
the specificity of this binding, we added anti-Sp1 antibody to the
incubation mixture and observed a supershift of the retarded band (Fig.
4A, lower panel). A control antibody against p53 did not
alter migration of the band. In addition excess cold Sp1 binding
consensus oligonucleotide successfully competed for binding, whereas
cold AP-1 binding oligonucleotide failed to do so (results not shown).
Under these conditions the amount of Sp1 protein did not change (Fig.
4B). When Sp1 binding was determined in four additional cell
lines a similar decrease was observed in all cases (Fig.
4C).
Effect of EGF on ATM Promoter--
To confirm the effect of EGF on
the transcriptional regulation of ATM, we cloned the common promoter
region between ATM and NPAT into a luciferase reporter construct (Fig.
5) and carried out transient
transfections in control lymphoblastoid cells. Exposure of transfected
C3ABR cells to EGF for 16 h led to a significant down-regulation
of luciferase activity in cells transfected with the ATM promoter
construct in agreement with loss of Sp1 binding activity (Fig. 5).
Causal Relationship between Sp1 and ATM Down-regulation--
The
parallel decrease of ATM and Sp1 binding activity together with the
down-regulation of ATM promoter activity after EGF treatment suggested
that the decrease in Sp1 binding led to reduced ATM. To test this we
employed GM-CSF that has been shown to increase markedly the DNA
binding activity of Sp1 (34). As observed above, treatment of cells
with EGF for 16 h down-regulated the amount of Sp1 binding
activity (Fig. 6A, 3rd lane)
and subsequent addition of GM-CSF to these cells caused a rapid (within
1 h) restoration of Sp1 binding activity (Fig. 6A, 5th
lane). Incubation of untreated cells with GM-CSF also showed
evidence of increased Sp1 binding activity (Fig. 6A, 4th
lane). EGF caused a down-regulation of ATM protein (Fig. 6B,
2nd lane), and compatible with the increase in Sp1 binding
activity there was also a rapid return of ATM to normal levels when
cells were subsequently treated with GM-CSF (Fig. 6B, 4th
lane). Under these conditions Sp1 protein levels did not change
(Fig. 6B). To test whether the reduction in Sp1 binding
activity and ATM down-regulation were responsible for the EGF-induced
radiosensitivity, cells were exposed to radiation after pretreatment
with EGF followed by GM-CSF incubation for 1 h. A normal pattern
of radiosensitivity was observed compatible with restored levels of ATM
protein under these conditions (Fig. 6C).
Radiation Induces ATM Protein--
We investigated whether
radiation might also attenuate the response, since it has been shown
that Sp1 activity is up-regulated by radiation (32, 35), which in turn
should increase the amount of ATM protein. When C3ABR cells were
exposed to radiation (5 Gy) there was no change in the amount of Sp1
binding activity, but when these cells were pretreated with EGF
initially for 16 h, Sp1 binding activity decreased and subsequent
exposure to radiation restored the amount of Sp1 binding activity in
3 h (Fig. 7A). Under
these conditions incubation of cells with EGF followed by radiation
exposure led to an increase in the amount of ATM protein to untreated
levels by 3 h post-irradiation (Fig. 7B). ATM kinase activity increased by 30 min post-irradiation, and the extent of this
increase was somewhat lower in cells pretreated with EGF (Fig.
7B). By 3 h post-irradiation ATM kinase induction was
the same in cells either untreated or pretreated with EGF, at which time ATM protein levels were the same (Fig. 7B). These data
were further supported by radiation-induced stabilization of p53 and serine 15 phosphorylation of p53 with or without prior incubation with
EGF (Fig. 7B). The latter provides an in vivo
measure of ATM kinase activation.
Activation of ATM kinase by ionizing radiation and other
DNA-damaging agents is critical for modulating its activity in several DNA damage signaling pathways (36, 37). We have demonstrated here that
ATM can also be altered at the transcriptional level by continuous
incubation with EGF over a 16-h period. Under these conditions
EGF-treated cells were more sensitive to subsequent ionizing radiation
exposure than untreated, irradiated cells. Evidence exists for both
radiosensitizing and radioprotective effects of EGF, which is dependent
upon both the cell type and position in the cell cycle (25-29,
38-40). In human squamous carcinoma cells, radiosensitization by EGF
appears to be in part due to increasing the magnitude of
radiation-induced G2/M arrest (39). In CaSki cells the
enhancement of radiosensitivity was evident from a reduction in the
shoulder region of the survival curves (27). The degree of
radiosensitization in these cells by EGF was of the same order as that
observed in the present study. None of the previous studies have
provided a mechanistic explanation for radiosensitization, but the
down-regulation of ATM in this study provides a potential explanation.
Failure to observe a sensitizing effect of radiation in A-T cells
provides further support for an association between reduction of ATM
and radiosensitivity.
The catalytic component (DNA-PKcs) of a second protein involved in DNA
damage recognition, DNA-dependent protein kinase (42), was
also shown to be down-regulated by EGF, and this would also be expected
to increase the radiosensitivity of cells. Down-regulation of both ATM
and DNA-PKcs suggests that they may have common regulatory elements
responsive to EGF. Indeed this appears to be the case since both genes
are organized in head-to-head configuration with other genes, and their
putative promoters contain CCAAT boxes and Sp1 consensus sequences.
Approximately 0.5 kb separates the 5' end of the ATM transcript from
the 5' end of another NPAT/E14/CAND3 gene, indicating
that they share a bidirectional promoter (21, 22, 34). NPAT plays a
role in facilitating the entry of cells into S phase (43), and ATM
activates the p53 pathway to delay the passage of cells from
G1 to S phase after ionizing radiation exposure (44, 45).
DNA-PKcs and Cdc21 (coding for a protein involved in the initiation of
DNA replication (46)) also share a bidirectional promoter that contains
several CCAAT boxes and Sp1 consensus sites similar to the ATM/NPAT
promoter (34, 47). Clearly Sp1 is a candidate for common regulation of
these four genes, and its down-regulation or inactivation might account
for the effect of EGF in reducing the amounts of ATM and DNA-PKcs. Evidence to support this was provided in a recent report which demonstrated that nitric oxide causes a 4-5-fold increase in
expression of DNA-PKcs and protects cells from the toxic effects of a
number of agents including ionizing radiation (48). The increased
expression of DNA-PKcs was shown to be due to increased binding of Sp1
to the DNA-PKcs promoter. The mechanism by which nitric oxide enhanced Sp1 binding was not elucidated. We have shown here that a decrease in
Sp1 DNA binding activity paralleled the EGF-induced decrease in ATM
protein, and when the ATM promoter was linked to a reporter gene,
transcriptional activation was reduced by EGF. We have established here
that GM-CSF which enhances the amount of Sp1 DNA-binding activity
rapidly reverses the EGF-induced down-regulation of ATM. Thus there
appears to be a causal relationship between the reduction in Sp1
DNA binding activity in EGF-treated cells and the decrease in ATM
protein. Evidence for a role for EGF in reducing the amount of Sp1
protein in a rat pituitary cell line (GH4) has been
reported (49). This reduction was accompanied by a 50% reduction in
Sp1 binding to a GC-rich element in the gastrin promoter, a 50-60% decrease in Sp1-mediated transactivation of reporter genes and a
decrease in cell proliferation. The results obtained here with several
normal human fibroblasts and lymphoblastoid cells differ from the data
with GH4 cells, in that Sp1 protein remains unchanged as
the DNA binding activity decreases with time after EGF incubation, and
in this case also there was no decrease in cell proliferation (results
not shown). In our case, it is possible that modification of Sp1 might
account for decreased DNA binding activity (35). Several protein
kinases alter the DNA binding activity of Sp1 (50), and thus alteration
in specific sites of phosphorylation of Sp1 could account for the
changes in DNA binding activity reported here.
The human glioblastoma cell line MO59J is characterized by lack of
expression of DNA-PKcs and low levels of ATM protein and is extremely
sensitive to ionizing radiation (47). Thus it was somewhat surprising
in the present study that EGF did not increase markedly the
radiosensitivity of cells in which both ATM and DNA-PKcs were
down-regulated. This can be explained by the ability of ionizing radiation to both transcriptionally increase the amount of ATM protein
and activate its kinase activity. Compared with activation of
pre-existing ATM protein which reaches optimal levels by 20 min
post-irradiation (14, 15), EGF-pretreated cells respond more slowly to
radiation showing some kinase activity by 30 min but only reaching
maximum levels by 3 h post-irradiation. This delay in restoring
ATM protein to constitutive levels and in activating ATM kinase appears
to account for the reduction in survival in EGF-pretreated cells,
post-irradiation, since up-regulation of ATM by GM-CSF restored
survival to normal levels. Failure of A-T cells to show any additional
radiosensitization also supports this. Although it is likely that EGF
operates via Sp1 in reducing the amount of ATM protein, it is apparent
that radiation counteracts this. We have shown here that the amount of
Sp1 DNA binding activity is rapidly restored when EGF pretreated cells
are exposed to radiation, accounting for the increase in ATM. Previous
results have revealed that Sp1 DNA binding activity is increased
rapidly in human melanoma cells exposed to ionizing radiation (32,
35).
Clarke et al. (19) were the first to report alterations of
ATM protein in a specific tissue when they failed to detect ATM in
normal breast myoepithelial cells, but a significant level of
expression was present in the proliferative myoepithelium of sclerosing
adenosis. Immunoblotting extracts from freshly isolated PBMCs
established that ATM was either absent or present at low levels, but
increased ~10-fold in response to mitogenic stimuli over 3-4 days
(20). To add to this we have demonstrated here that ATM protein can be
down-regulated by EGF to radiosensitize cells, but radiation restores
ATM to normal levels and activates its kinase activity, and this is
mediated at least in part by the Sp1 transcription factor that has
several consensus binding sites in the ATM bidirectional promoter.
These observations provide greater insight into the regulation of ATM
that involves not only activation of pre-existing protein but also
alterations in the amount of ATM protein in response to different
stimuli. Determination of ATM expression levels in different
tissues/cell types may provide further insight into the response of
different tissues to radiation and the factors involved in
transcriptional control of the ATM/NPAT promoter. Finally, the results
described here raise a fundamental issue in relation to
radiosensitization by attenuating ATM protein. Reduction of ATM, in
this case by EGF, is countered by radiation exposure that rapidly
restores ATM to constitutive levels and would be expected to lessen any
therapeutic benefit of radiation. A combination of attenuating ATM,
together with inhibitors of its activity, may be a more effective solution.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin (A4700) was from Sigma; polyclonal anti-Ku and DNA-PKcs antibodies were obtained from Susan Lees-Miller, University of Calgary,
Canada; anti-phosphorylated p53 serine 15 was obtained from New England
Biolabs; and monoclonal ATM (CT1) and polyclonal ATM raised in sheep
(ATM5BA) antibodies were generated in this laboratory.
70 °C until used in binding
reactions. Approximately 35 fmol of 32P-radiolabeled
double-strand Sp1 consensus sequence (5'-ATTCGATCGGGGCGGGGCGAGC-3'; Promega) (labeled using T4 polynucleotide kinase) was incubated with 10 µg of nuclear extract in the presence of 1 µg of herring sperm DNA
and 20 µg of BSA in binding buffer (10 mM Hepes, pH 7.9, 50 mM KCl, 0.5 mM EDTA, 10% glycerol, 1%
Nonidet P-40, 5 mM DTT, 0.2 mM PMSF) in a
20-µl reaction. Binding was for 25 min at room temperature before
DNA-protein complexes were separated on a native 5% acrylamide gel.
Exposure time to x-ray film was usually 30-45 min.
-glycerophosphate, 150 mM NaCl, 10% glycerol, 1% Tween
20, 1 mM NaF, 1 mM
Na3VO4, 1 mM PMSF, 2 µg/ml
pepstatin, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM
DTT). After centrifugation at 13,000 × g for 1 min, 2 mg of extract was precleared with mouse immunoglobulin G and protein
A/G-Sepharose beads. ATM was immunoprecipitated with anti-ATM antibody
(ATM5BA) and kinase activity determined as described.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), p = 0.0053 (
) for dose and p = 0.0065 (
) and p = 0.07 (
) for time course. On
the other hand treatment of A-T cells with EGF failed to increase the
radiosensitivity of these cells (Fig. 1, A and
B). A similar sensitization was observed in EGF-treated control fibroblasts, but again no further increase in sensitivity was
revealed in EGF-treated A-T fibroblasts (Fig. 1C). We
observed that EGF receptor was activated under these conditions
(results not shown).
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Fig. 1.
EGF sensitizes human cells to ionizing
radiation. A, effect of increasing radiation dose on
survival of control (C3ABR) and A-T (AT1ABR)
lymphoblastoid cells untreated or pretreated with EGF (50 ng/ml) for
16 h prior to irradiation. Viability was determined by trypan blue
exclusion at 72 h post-irradiation. ( ) and
(+) refer to EGF addition. B, effect of
-radiation (4 Gy) on survival of C3ABR and AT1ABR cells with time
after irradiation. Cells were untreated and pretreated with EGF as in
A above. C, effect of EGF (50 ng/ml) for 16 h on radiation killing in control (NFF) and A-T (GM
3395) fibroblast cells. In this case survival was determined by colony
formation. In each case three independent experiments were carried out,
and values were fitted by nonlinear repression and calculated curve
parameters (
,
) subjected to Student's t test.
View larger version (47K):
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Fig. 2.
Effect of EGF incubation on the amount of ATM
protein in control cells. A, ATM protein levels in
fibroblasts (HSF7) treated with EGF (50 ng/ml). 200 µg of HSF7
protein was loaded in each lane and immunoblotted with anti-ATM
antibody (ATM-4BA) (16). Positive (C3ABR lymphoblastoid cell extracts,
100 µg) and negative (AT3ABR lymphoblastoid cell extracts, 100 µg)
controls were also loaded. DNA-PKcs, the catalytic subunit of
DNA-dependent protein kinase, was determined using the
antibody DPKI (kindly provided by Susan Lees-Miller, University of
Calgary). The same cell lines were employed as controls for DNA-PKcs.
Immunoblotting was also carried out for Ku70/86 using a mouse
polyclonal antibody that detects both forms, and this acted effectively
as a loading control. B, effect of EGF (50 ng/ml) incubation
on ATM protein in (C3ABR) lymphoblastoid cells. Immunoblotting was
carried out with anti-ATM antibody (CT-1) using 40 µg of extracts in
each lane. DNA-PKcs was determined using immunoblotting with DPKI, and
actin was used as a loading control. C, EGF reduces ATM
protein in different lymphoblastoid cell lines. The cells were
incubated with EGF (50 ng/ml) for 20 h and ATM levels determined
by immunoblotting in C3ABR, C28ABR, C35ABR, and C31ABR. Protein loading
in each lane was determined by Ponceau S staining (not shown).
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Fig. 3.
Determination of ATM mRNA transcript in
lymphoblastoid cells after incubation with EGF. A,
quantitative reverse transcriptase-PCR was carried out to determine ATM
mRNA in C3ABR lymphoblastoid cells as described previously (31). In
these experiments a DNA fragment (402 bp) corresponding to an A-T
mutant (G5319A) lacking exon 38 was employed as a competitor for
ATM cDNA amplification from C3ABR cells (nucleotides 5122-5665,
544 bp) in a series of 2-fold dilutions. The amount of competitor DNA
added to each sample was as follows: lane 1, 0.078 attomol/µl; lane 2, 0.156; lane 3,
0.313; lane 4, 0.625; lane 5, 1.25, lane
6, 2.5; lane 7, 5.0; and lane 8, 10 attomol/µl. Size markers appear on the left-hand
side of the figure. Cells were incubated for 8 and 16 h with
EGF (50 ng/ml) as indicated. These experiments were repeated three
times. B, Northern blot analysis of ATM mRNA form normal
human skin fibroblasts (HSF7) after treatment with EGF (50 ng/ml) for
various times up to 24 h. Each lane contained 20 µg of total
RNA, and glyceraldehyde-3-phosphate dehydrogenase was employed as a
loading control. The ATM/glyceraldehyde-3-phosphate dehydrogenase ratio
was determined by densitometry and represents the relative amounts of
ATM mRNA present with time after EGF treatment.
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Fig. 4.
Effect of EGF on nuclear protein binding to
an Sp1-binding consensus sequence in lymphoblastoid cells.
A, nuclear extracts (20 µg), prepared at different times
after EGF addition from C3ABR, were incubated with
32P-labeled double-stranded oligonucleotide (22-mer)
containing a single Sp1-binding site. Free and retarded fragments were
separated on native 5% acrylamide gels. 1st lane is free
fragment only. An Sp1 supershift assay was employed to establish the
specificity of binding. Incubation of nuclear extract and labeled
fragment was carried out followed by addition of 1 µg of anti-Sp1
antibody (Upstate Biotechnology) for 30 min on ice prior to gel
electrophoresis. Anti-p53 antibody (Oncogene Science) was used as a control.
Quantitation of binding activity was determined by densitometry.
B, Sp1 levels were determined by immunoblotting 20 µg of
nuclear extract from each sample in A. Actin was used as a
loading control. C, several different lymphoblastoid cells
(C3ABR, C28ABR, C35ABR, and C31ABR) were treated with EGF for 16 h
prior to preparation of extracts for binding to the Sp1 consensus
sequence as described above.
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Fig. 5.
Effect of EGF on luciferase activity in cells
transfected with the ATM promoter reporter construct. Common
bidirectional promoter for ATM and NPAT. Luciferase-reporter construct
for the ATM promoter (pGL3ATM). C3ABR were simultaneously transfected
with pGL3-ATM and pRL-CMV (containing the CMV promoter as an internal
control) at a ratio of 2:1. 36 h after transfection the cells were
incubated with 50 ng/ml EGF for 16 h. The activity of the ATM
promoter was calculated as ratio of firefly luciferase (pGL3-ATM) to
Renilla luciferase (pRL-CMV). The data represent four
independent experiments with three samples each. p value was
calculated using Student's t test.
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Fig. 6.
Restoration of Sp1 DNA binding activity,
amount of ATM, and radiosensitivity GM-CSF after EGF treatment of
lymphoblastoid cells. Cells were treated with EGF (50 ng/ml) for
16 h prior to addition of GM-CSF (10 ng/ml) for 2 h.
A, Sp1 DNA binding activity was determined by
electrophoretic mobility shift assay. B, changes in amount
of ATM protein detected with anti-ATM antibody. Sp1 protein levels were
measured with Sp1 antibody, and actin was used as a loading control.
C, radiosensitivity was determined as described in the
legend to Fig. 1. GM-CSF (10 ng/ml) was added to cells for 1 h
after 16 h of EGF treatment.
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Fig. 7.
Radiation induces Sp1 binding activity and
ATM protein/kinase in EGF-pretreated cells. A, effect
of radiation on Sp1 DNA binding activity. Cells were either treated
with EGF (50 ng/ml, 16 h), radiation (6 Gy, 3 h) alone, or
with EGF for 16 h followed by radiation (6 Gy) and subsequent
incubation for 3 h. Binding to an Sp1 consensus sequence was as
described in the legend to Fig. 4. B, effect of radiation on
ATM protein and ATM kinase. Untreated or EGF (50 ng/ml, 16 h)-treated cells were subsequently exposed to radiation (6 Gy) and
incubated for either 30 min or 3 h prior to preparation of
extracts. ATM kinase activity was determined using p531-40
as a substrate (14), and ATM protein was also determined in
immunoprecipitates. p53 protein and serine 15 phosphorylated p53 were
determined in parallel samples by immunoblotting with specific
antibodies. Actin was used as a loading control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We Thank to Aine Farrell for technical support and Ann Knight and Kylee Wallace for typing the manuscript.
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FOOTNOTES |
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* This work was supported in part by the Australian National Health and Medical Research Council, the Deutsche Forschungsgemeinschaft Grant 488/1-1, and the A-T Children's foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed: the Queensland
Cancer Fund Research Laboratory, Queensland Institute of Medical Research, P. O. Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia. Tel.: 61-7-33620341; Fax: 61-7-33620106; E-mail: martinL@qimr.edu.au.
Published, JBC Papers in Press, November 15, 2000, DOI 10.1074/jbc.M006190200
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ABBREVIATIONS |
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The abbreviations used are: ATM, protein mutated in ataxia-telangiectasia; EGF, epidermal growth factor, A-T, ataxia-telangiectasia; DNA-PKcs, catalytic substrate of DNA-dependent protein kinase; GM-CSF, granulocyte/macrophage colony-stimulating factor; IR, ionizing radiation; PBMCs, peripheral blood mononuclear cells; Gy, gray; PBS, phosphate-buffered saline; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; kb, kilobase pair; bp, base pair; NPAT, nuclear protein at the A-T locus; CMV, cytomegalovirus.
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