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INTRODUCTION |
A1-5 and B4 are two independently isolated cell lines that were
derived in the same laboratory from primary rat embryo
fibroblasts transformed with activated Ras (T24) and
mutant p53val-135 (1). Both A1-5 and B4 cells show the
typical phenotypes of the temperature-sensitive p53val-135
(1), but as reported here, only A1-5 cells show extreme
radioresistance to killing accompanied by a strong G2
checkpoint response.
G2 checkpoint activation plays an important role in
promoting cell survival following DNA damage (2). The mechanism that regulates G2 arrest after DNA damage is conserved among
species from yeast to human. The DNA damage checkpoint activated in
G2 is believed to be mediated, at least in part, by an
inhibition of the Cdc25C phosphatase that activates the Cdc2 kinase by
removing inhibitory phosphates, thus allowing entry into mitosis (3, 4). Cdc25C could be phosphorylated in vitro at serine 216 by either Chk1 or Chk2 (5-7). This phosphorylation creates a binding site
for the small acidic proteins 14-3-3 (3) that cause the transport of
Cdc25C to the cytoplasm and prevent Cdc2 activation. The Chk1 and
Chk2 (Chk2 is the homologue of Rad53 in Saccharomyces cerevisiae and Cds1 in Schizosaccharomyces pombe)
kinases, two important checkpoint regulators (3, 5, 6, 8-13), were initially cloned in yeast, but homologues were subsequently identified in mammalian cells. Following DNA damage, both Chk1 and Chk2 are activated in human cells; however, it is not clear which pathway, Chk1/Cdc25C/Cdc2 or Chk2/Cdc25C/Cdc2, has a dominant role in
G2 arrest. In this study we examine whether Chk1 or Chk2
plays the major role in the strong G2 arrest and the
radioresistance to killing of A1-5 cells.
UCN-01 and caffeine are two efficient inhibitors of G2
checkpoint activation (14-20) that act by targeting different
proteins. UCN-01, a protein kinase inhibitor, potentiates the
cytotoxicity of a variety of anticancer agents, including cisplatin,
camptothecin, and ionizing radiation (18-21). Therefore, it is
currently undergoing testing in clinical trials for the treatment of
human cancer. It is believed that UCN-01 sensitizes cells to DNA damage
by abrogating the G2 checkpoint. Although UCN-01 inhibits
multiple protein kinases (22), the way UCN-01 abrogates the
G2 checkpoint is mainly by inhibiting Chk1 and affecting
the Chk1-Cdc25C but not the Chk2-Cdc25C regulatory pathway (23, 24).
Caffeine, which sensitizes cells to ionizing radiation and other
genotoxic agents by abrogating DNA damage checkpoints, has been shown
to be an effective inhibitor of
ATM1 and ATR (25). ATM and
ATR, both members of the phosphatidylinositol 3-kinase family, are
upstream activators of Chk1 and Chk2 after DNA damage (8, 12, 13, 26).
Recently it has been reported that in mammalian cells, caffeine
abolishes the G2 checkpoint by inhibiting the ATM-Chk2
pathway (27). To determine the effects of Chk1 and Chk2 on
G2 checkpoint response and radioresistance to the killing
of A1-5 cells, we used these drugs (caffeine and UCN-01) and specific
Chk1 or Chk2 antisense oligonucleotides in our experiments. Our results
point to the Chk1 pathway as the major player for the extreme
radioresistance and the strong G2 checkpoint
response in A1-5 cells.
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EXPERIMENTAL PROCEDURES |
Cell Lines, Chemicals, and Irradiation--
The two cell lines,
A1-5 and B4 (obtained from Dr. A. Levine) (1), were grown in
Dulbecco's modified Eagle's medium supplemented with 10%
iron-supplemented calf serum (Sigma). All incubations were at 37 °C
in an atmosphere of 5% CO2 and 95% air. Caffeine (Sigma)
was dissolved in water, and UCN-01 (NSC 638850, obtained from the Drug
and Chemistry Branch, NCI, National Institutes of Health) was dissolved
in dimethyl sulfoxide. Drugs were added to the cells 30 min before
irradiation. Radiation exposures were carried out using an x-ray
machine (Pantak) operating at 310 kV and 10 mA with a 2-mm aluminum
filtration. The effective photon energy was about 90 keV.
Colony-forming Assay--
Cellular sensitivity to radiation was
determined by the loss of colony-forming ability. In brief, 2 × 105 cells were plated per 60-mm dish with 3 ml of
medium. Thirty h later, cultures were supplemented with drugs
(caffeine or UCN-01) for 30 min and then irradiated and returned to
37 °C for 18 h. Cells were collected and plated at 20-200
colonies per 100-mm dish. Two replicates were prepared for each datum
point and incubated for 1 week in the absence of drugs to allow
colonies to develop. Colonies were stained with crystal violet (100%
methanol solution) before counting.
Flow Cytometry--
2 × 105 cells were plated
in 60-mm dishes with 3 ml of growth medium. Thirty h later,
cultures were supplemented with either caffeine or UCN-01 for 30 min
and then exposed to 6 Gy and returned to 37 °C. At different times
thereafter, cells were trypsinized and fixed in 70% ethanol. Cells
were stained in the solution (62 µg/ml RNase A, 40 µg/ml propidium
iodide, and 0.1% Triton X-100 in phosphate-buffered saline buffer) at
room temperature for 1 h. The distribution of cells in the cell
cycle was measured in a flow cytometer (Coulter Epics Elite).
Induction and Repair of DNA Double Strand Breaks (DSB)--
As
described earlier (28), cells were labeled with 0.01 µCi/ml
[14C]thymidine plus 2.5 µM cold thymidine.
For measuring the repair of DNA DSB, cells were irradiated and then
returned to the incubator at 37 °C. After the cells were collected,
they were mixed with an equal volume of 1% agarose (InCert agarose)
(FMC) to prepare 3 × 5-mm cylindrical blocks containing ~1 × 105 cells. Blocks were placed in lysis buffer (10 mM Tris, pH 8.0, 50 mM NaCl, 0.5 M
EDTA, 2% N-lauryl sarcosyl, and 0.1 mg/ml proteinase E) for
16-18 h. Then the blocks were washed in a buffer containing 10 mM Tris, pH 8.0, and 0.1 M EDTA and treated for
1 h with 0.1 mg/ml RNase A in the same buffer. A similar protocol
was also employed to determine the induction of DNA DSB except that in this case cells were embedded in agarose blocks prior to irradiation and lysed immediately after irradiation. Asymmetric field inversion gel
electrophoresis was carried out in 0.5% SeaKem agarose (FMC) in
0.5× TBE (45 mM Tris, pH 8.2, 45 mM boric
acid, and 1 mM EDTA) at 10 °C for 40 h. Gels were
dried and DNA DSB were quantitated as the fraction of activity released
from the well into the lane by means of a PhosphorImager (Molecular
Dynamics). The rejoining of DNA DSB measured by this assay evaluates
the capacity of the cells to carry out nonhomologous end-joining repair.
Western Blotting--
Cells were treated with caffeine or UCN-01
for 30 min and then exposed to 6 Gy of x-rays. Cells were incubated for
various times at 37 °C as noted and then collected to prepare whole
cell lysates. For this purpose, cell pellets were suspended in lysis buffer (20 mM HEPES, pH 8.0, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 0.5% Nonidet
P-40, 20 mM
-glycerophosphate, 0.2 mM
phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol)
and subjected to three cycles of freeze-thaw. After centrifugation at
12,000 rpm for 5 min, the supernatant was collected and the protein
concentration was measured. Western blot analysis was performed using
enhanced chemiluminescence (ECL) according to the manufacturer's
instructions (Amersham Pharmacia Biotech). Antibodies against
ATR, Chk1, Chk2, Cdc2, and p53 (pAb240, which recognizes both wild type
and mutant p53 under denaturing conditions) were purchased from Santa
Cruz Biotechnology, Inc. The Ha-Ras antibody was purchased from
Oncogene Research Products Corp. The ATM antibody (2C1) was purchased
from GeneTex Inc. The phospho-Cdc2 (Tyr-15) antibody was
purchased from Cell Signaling Technology. The
glyceraldehyde-3-phosphate dehydrogenase antibody was purchased from
Chemicon International. The expression levels of these proteins were
quantitated in a PhosphorImager (Molecular Dynamics).
Chk1 and Chk2 Kinase Assays--
Whole cell lysate (200 µg)
was mixed with 1 µg of Chk1 or Chk2 antibody in the presence of 10 µl of a 50% (v/v) protein A-Sepharose slurry (Life Technologies,
Inc.) in 200 µl of Buffer A (0.5% Nonidet P-40, 1 mM
Na3VO4, 5 mM NaF, and 0.2 mM phenylmethylsulfonyl fluoride in phosphate-buffered
saline buffer) and gently rotated for 2 h at 4 °C. Immune
complexes were washed twice with Buffer A and then washed twice with
Buffer B (20 mM HEPES, pH 8.0, 50 mM KCl, 10 mM MgCl2, 0.5 mM dithiothreitol,
and 10 µM ATP). The kinase immunoprecipitate was
incubated at 30 °C for 30 min with 2 µg of GST-Cdc25C (kindly
provided by Dr. Bin-Bing Zhou, SmithKline Beecham, Philadelphia) (27)
in 20 µl of Buffer B and 5 µCi of [
-32P]ATP.
Samples were analyzed by 10% SDS-polyacrylamide gel electrophoresis, and the kinase activities were determined by the incorporation of
32P into the Cdc25C protein using the PhosphorImager.
Treatment of Cells with Oligonucleotides--
The antisense
oligonucleotides of Chk1 (5'-GGCACTGCCATGACTCCA-3') and Chk2
(5'-TGACTCTTCATATCCGAC-3') are designed to specifically target
at the sequence of the start codon region of Chk1 or Chk2 mRNA. The oligonucleotides used in this study are phosphorothioate oligodeoxynucleotides synthesized by Genemed Synthesis, Inc. The oligonucleotides were delivered to cells by
OligofectAMINETM (Life Technologies, Inc.) according to the
manufacturer's instructions. Briefly, 1.5 µM antisense
oligonucleotides were added to serum-free minimum Eagle's
medium containing 20 µl/ml OligofectAMINETM
reagent. This preparation (1 ml) was added to 30% confluent A1-5 cells cultured in a 60-mm plate. After 4.5 h, additional
Dulbecco's modified Eagle's medium (0.5 ml) with 30%
iron-supplemented calf serum was added to the culture. After 14 h,
the cells were irradiated and returned to 37 °C. The cells were
collected 10-14 h later for the colony-forming assay, the flow
cytometry assay (as described above), and Western blot by directly
lysing the cell pellet in 1× SDS-polyacrylamide gel loading buffer.
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RESULTS |
The Radioresistance of A1-5 Cells Can Be Diminished by Caffeine or
UCN-01--
A1-5 and B4 cells have a similar genetic background (1).
Indeed, in the absence of DNA damage, no differences in their phenotypes are apparent. However, after exposure to x-rays, A1-5 cells
are extremely radioresistant to killing as compared with B4 cells (Fig.
1). The difference in radiosensitivity is
greater than that observed in other transformed rat embryo fibroblast cell lines (29-31).

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Fig. 1.
Effects of caffeine or UCN-01 on the survival
of A1-5 and B4 cells. 105 A1-5 (filled
symbols) or B4 (open symbols) cells were grown for 2 days in 60-mm dishes and treated for 30 min with 2 mM
caffeine (triangles) or with 100 nM UCN-01
(squares) before exposure to the indicated doses of
radiation. Circles depict untreated cells. The surviving
fraction was determined using the clonogenic assay as described under
"Experimental Procedures." Data shown are the averages from four
independent experiments.
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The radioresistance of A1-5 could be diminished by either 2 mM caffeine or 100 nM UCN-01. After caffeine or
UCN-01 treatment, the survival of irradiated A1-5 cells decreased to
levels similar to that of B4 cells. The observation that inhibitors of
checkpoint activation sensitize A1-5 cells to radiation-induced
killing suggests that a strong checkpoint response underlies the
radioresistance of these cells.
A Strong G2 Delay in A1-5 Cells Can Be Abolished by
Caffeine or UCN-01--
The above results prompted experiments
investigating the relationship between checkpoint response and cell
radiosensitivity to killing in A1-5 cells. As shown in Fig.
2A, there is a large difference in the percentage of G2/M cells between
irradiated A1-5 and B4 cells. B4 cells show a modest accumulation in
G2 after exposure to 6 Gy, but cells overcame the arrest
and divided after ~12 h. However, after exposure to the same
dose, A1-5 cells experience a high accumulation in the G2
phase and an unusually long G2 delay. The delay is not
completely overcome during the 24-h follow-up period. This response is
the strongest we ever observed in a repair-proficient cell line. When
the cells were treated with either caffeine or UCN-01, the enhanced
G2 delay response in irradiated A1-5 cells is abolished
(Fig. 2B). The percentage of A1-5 cells in the
G2/M phase is similar to that of B4 cells, and the cells
overcome the arrest and divided after ~12 h.

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Fig. 2.
Effects of caffeine or UCN-01 on the
G2 delay of A1-5 and B4 cells. A, as
described under "Experimental Procedures," at various times after a
6-Gy radiation, cells were trypsinized, fixed with 70% ethanol, and
stained with propidium iodide. The distribution of cells through the
cell cycle was measured by flow cytometry, and the fraction of cells in
G1, S, and G2/M was determined. The fraction of
cells in the G2/M phase is plotted as a function of time
after irradiation (A1-5, filled circle; B4, open
circle). B, A1-5 (filled symbols) and B4
(open symbols) were treated with 2 mM caffeine
(circles) or 100 nM UCN-01
(triangles) for 30 min before and continuously after
irradiation until samples were collected for analysis.
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The Radioresistance of A1-5 Cells Is Not Attributable to Reduced
Induction or Increased Rejoining of DNA DSB--
DNA DSB are thought
to be severe lesions that if unrepaired or misrepaired will lead to
cell death (32-34). Therefore, we investigated whether the altered
induction or repair of DNA DSB underlies the radioresistance of A1-5
cells. There is no difference in the induction of DNA DSB between A1-5
and B4 cells (Fig. 3A). In
addition, the rates of rejoining of DNA DSB are similar in the two cell
lines, suggesting a similar capacity for nonhomologous end-joining
repair (Fig. 3B).

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Fig. 3.
Induction and rejoining of DNA DSB in A1-5
and B4 cells. A, dose-response curves for the induction
of DNA DSB. Cells were trypsinized, embedded in agarose blocks, and
exposed to various doses of x-rays while kept on ice. The amount of DNA
DSB was measured by asymmetric field inversion gel electrophoresis and
is expressed as the fraction of activity released (FAR). The
results shown are obtained by quantitating gels from three experiments.
The mean (A1-5, filled circles; B4, open
circles) and S.E. are plotted as a function of time. B,
rejoining of DNA DSB as a function of time. Cells were exposed to 30-Gy
x-rays and returned to 37 °C. At various times thereafter, cells
were trypsinized and prepared for asymmetric field inversion gel
electrophoresis. The value of fraction of activity released measured in
nonirradiated cells has been subtracted from all data points (A1-5,
filled circles; B4, open circles) (28).
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Ha-Ras or p53 Does Not Directly Mediate the Increased
Radioresistance and the Strong G2 Checkpoint Response of
A1-5 Cells--
We inquired whether the differences in the
radiosensitivity between A1-5 and B4 cells derive from differences in
the levels of expression of either the human Ha-Ras or the mouse mutant
p53val-135. Therefore, we measured the kinetics of
expression of these proteins after irradiation. There is no difference
in the levels of Ha-Ras or p53 expression between A1-5 and B4 cells.
In addition, the radiosensitivity and G2 response of A1-5
cells is not affected by a shift in temperature from 37 °C to
39 °C or to 32 °C (data not shown). Furthermore, neither caffeine
nor UCN-01 affects the expression of these proteins (data not shown).
Finally, several transformed cell lines generated in our laboratories
by transfection with the same genes have a similar radiosensitivity to
that of B4 cells (data not shown). These results in aggregate suggest that Ha-Ras and p53 expression are not directly related to the extreme
radioresistance with the strong G2 checkpoint response of
A1-5 cells. Because Chk1 and Chk2 are considered important regulators
of the G2 checkpoint, we investigated next whether they are
involved in the strong G2 checkpoint response in A1-5 cells.
High Expression of Chk1 Correlates with High Phosphorylation of
Cdc2 in A1-5 Cells--
Fig.
4A shows that the constitutive
levels of Chk1 were higher in A1-5 than in B4 cells. The difference is
much larger after a 6-Gy radiation. G2 arrest after DNA
damage is achieved by maintaining inhibitory phosphorylations on Cdc2
through the inactivation of Cdc25C. To further determine whether the
higher expression of Chk1 is related to the Cdc25C/Cdc2 pathway, we
compared Cdc2 expression as well as its phosphorylation status in A1-5
and B4 cells. Although there is no difference in Cdc2 expression
between A1-5 and B4 cells, and there is no change in Cdc2 expression
after irradiation (data not shown), there is a difference in the levels
of Cdc2 phosphorylation between A1-5 and B4 cells (Fig.
4B). The level of phosphorylated Cdc2 is higher in
nonirradiated A1-5 cells (0 h) than in B4 cells, which is consistent
with the higher Chk1 expression level and the percentage of cells in
G2 (Fig. 2B and Fig. 4, A and
B, at 0 h for controls). Also similar to the kinetics of Chk1 expression, the levels of phosphorylated Cdc2 increased in
irradiated A1-5 and B4 cells with a stronger effect observed in A1-5
cells (Fig. 4B). Caffeine or UCN-01 clearly reduces Chk1 expression in A1-5 cells starting at 6 h after irradiation (Fig. 4A) and correlates with the reduction of Cdc2
phosphorylation, which leads to the similar levels in A1-5 and B4
cells (Fig. 4, A and B). Similar results are also
obtained when the kinase activity of Chk1 is evaluated, but
quantitation is hampered by the parallel changes in the levels of the
protein (data not shown). Thus, a relationship is indicated between
Chk1 activation and G2 arrest in A1-5 cells.

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Fig. 4.
Effects of caffeine or UCN-01 on Chk1
expression and Cdc2 phosphorylation in A1-5 and B4 cells.
A, as described under "Experimental Procedures," cells
were treated with caffeine or UCN-01 for 30 min and irradiated
(R, 6 gy) as indicated. Cells were collected at different
times for extract preparation. The monoclonal Chk1 antibody was used
for Western blot analysis. B, all the procedures are similar
to those described in panel A. The only difference is that
instead of the Chk1 antibody, the phospho-Cdc2 (Tyr-15) antibody
was used for the Western blot analysis.
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Chk2 Expression and Kinase Activity Are Similar in A1-5 and B4
Cells--
Because Chk2 could also affect the phosphorylations of Cdc2
through the inactivation of Cdc25C, we then examined whether Chk2 also
contributes to the phosphorylation of Cdc2 in irradiated A1-5 cells.
Fig. 5A shows that there is no
apparent difference in the levels of Chk2 expression between A1-5 and
B4 cells, although Chk2 expression increased somewhat in both cell
lines after irradiation. In addition, neither caffeine nor UCN-01
affects Chk2 expression in A1-5 or B4 cells (Fig. 5A). To
determine whether this observation also holds at the level of kinase
activity, we measured the ability of Chk2 to phosphorylate Cdc25C
in vitro. The results obtained are consistent with those
obtained at the level of protein expression and suggest that there is
no difference in Chk2 activity between irradiated A1-5 and B4 cells
(Fig. 5B). In addition, neither caffeine nor UCN-01
apparently affects the Chk2 kinase activity (Fig. 5B). These
data suggest that Chk2 is not directly related to the phosphorylation of Cdc2 in irradiated A1-5 cells.

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Fig. 5.
Effects of caffeine or UCN-01 on the
expression and kinase activity of Chk2 in A1-5 and B4 cells.
A, as described under "Experimental Procedures," after
drug and radiation treatment (R, 6 gy), cells were collected
at different times for extract preparation. The polyclonal Chk2
antibody was used for Western blot analysis. B, the Chk2
kinase activities were measured as described under "Experimental
Procedures." The substrate is Cdc25C.
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To confirm that the higher expression of Chk1 is the major reason for
the extreme radioresistance with the stronger G2 arrest response in A1-5 cells, we then examined the effects of Chk1 or Chk2
antisense oligonucleotides on the survival and G2 arrest of
irradiated A1-5 cells.
Chk1 but Not Chk2 Antisense Oligonucleotides Can Reduce the
G2 Delay and Radiosensitize A1-5 Cells to
Killing--
Without irradiation, the Chk1 and Chk2 antisense
oligonucleotides show a different toxicity to A1-5 cells. Compared
with the nontreated cells, the cells treated with the Chk1 antisense
show a remarkable reduction of their number and reach about one-third of the control at 24 h after the treatment (Fig.
6A). On the other hand, Chk2
shows little effect on the cell number (Fig. 6A). We checked
whether the antisense oligonucleotides could specifically inhibit Chk1
or Chk2 expression. The Western blot data are shown in Fig.
6B; the Chk1 or Chk2 antisense oligonucleotide specifically reduces the protein expression. Next, as we expected, the anti-Chk1 oligonucleotide radiosensitizes the survival of A1-5 cells down to the
levels similar to that observed after UCN-01 treatment (Figs. 1 and
6C). Also, anti-Chk1 oligonucleotide reduces the strong
G2 delay response in A1-5 cells (Fig. 6D). The
percentage of the Chk1 antisense-treated A1-5 cells in the
G2/M phase is much less than that of non-antisense-treated
A1-5 cells, and cells overcome the arrest and divided ~14 h
after radiation (Fig. 6D). At the same time, the Chk2
antisense oligonucleotide has little effect on A1-5 cell survival and
G2 arrest (Fig. 6, B and C). These
observations provide the direct evidence that the Chk1 but not the Chk2
pathway plays the major role in the special phenotypes of A1-5
cells.

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Fig. 6.
Effects of Chk1 or Chk2 antisense
oligonucleotide on A1-5 cells. A, the effects of the
oligonucleotides on the number of A1-5 cells. After the Chk1
antisense (black bars) or the Chk2 antisense (gray
bars) treatment, the cells were counted at different times and
plotted as the percentages of nontreated control. Data shown are the
averages from three independent experiments. B, Western blot
data. As described under "Experimental Procedures," 20 h after
antisense oligonucleotide treatment, cells were collected and directly
lysed in 1× protein gel loading buffer. The glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) antibody was used as the internal
standard. C, survival data. Twelve h after antisense
oligonucleotide treatment, the cells were irradiated (6 Gy) and
incubated for another 12 h. Then the cells were collected for the
clonogenic assay as described under "Experimental Procedures." Data
shown are the averages from three independent experiments.
D, G2 delay data. Twelve h after antisense
oligonucleotide treatment, the cells were irradiated (6 Gy) and
incubated for another 14 h. The histograms represent
the distribution of cells through the cell cycle measured by flow
cytometry.
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ATM and ATR are considered upstream modifiers of Chk1. To determine
whether the activation of these upstream modifiers causes the
activation of the Chk1/Cdc2 pathway, we examined their expression in
A1-5 cells. Although the expression of ATM and ATR increases after
irradiation, there is no difference in the expression of these proteins
between A1-5 and B4 cells (data not shown). This result suggests that
the higher expression of Chk1 in A1-5 cells is not directly related to
the expression of ATM or ATR.
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DISCUSSION |
The activation of DNA damage checkpoints arrests the normal
progression through the cycle and facilitates repair, which in turn
increases the survival probability. Although the underlying mechanism
of checkpoint response remains unclear in its details, the essential
aspects have been elucidated. A1-5 cells with their extreme
radioresistance and strong G2 checkpoint response provide a
useful model for studying the relationship between radiosensitivity to
killing and checkpoint activation.
The results presented here indicate that the radioresistance to the
killing of A1-5 cells can be abolished by drugs preventing the
activation of the G2 checkpoint and suggest that
G2 checkpoint activation promotes cell survival.
Phosphorylated Cdc2 is in an inactive state but can be activated by
Cdc25C-mediated dephosphorylation. Therefore, the inactivation of
Cdc25C could lead to an arrest of cells in G2 (7). Chk1 or
Chk2 could phosphorylate Cdc25C after DNA damage and induce such
inactivation. It is important to establish whether both of them are
involved in the strong G2 arrest in A1-5 cells. The
use of this information will allow the mechanistic characterization of
the A1-5 phenotypes to DNA damage and also further our understanding
of the G2 checkpoint. Our results suggest that A1-5 cells
with higher levels of Cdc2 phosphorylation are accompanied by a
stronger arrest in G2. Drugs that abolish this arrest, such
as caffeine and UCN-01, also reduce the levels of Cdc2 phosphorylation.
Thus, as expected, the strong G2 arrest in irradiated A1-5
cells is associated with an increase in Cdc2 phosphorylation.
Interesting also is the observation that the increased phosphorylation
of Cdc2 correlates with the higher expression of Chk1 in A1-5 cells
and that caffeine or UCN-01 not only reduces the levels of Cdc2
phosphorylation but also affects the expression levels of Chk1. It has
been reported that Chk1 is phosphorylated and activated by upstream
modifiers following DNA damage (5). Our results don't show any
phosphorylation of Chk1 in irradiated A1-5 cells, which might be
attributable to the fact that the regular one-dimensional gel is not
sensitive enough to observe the phosphorylation of Chk1 (26). On the
other hand, the expression levels of Chk2 are not correlated with the
changes of Cdc2 phosphorylation. UCN-01 at 100 nM could
inhibit Chk1 but has no effect on Chk2 (23, 24). In our experiments,
this concentration of UCN-01 abolishes G2 arrest with
radioresistance in A1-5 cells, which correlates with the reduction of
Cdc2 phosphorylation. These results suggest that Chk1 but not Chk2 is
responsible for maintaining Cdc2 phosphorylation in irradiated A1-5 cells.
Caffeine is an inhibitor of checkpoint activation known to sensitize
cells to radiation (14, 35-39). However, the mechanism by which
caffeine abolishes the G2 checkpoint and causes cell radiosensitization to killing remained unknown until recently. The
discovery that caffeine inhibits the kinase activities of ATM and ATR
(25) suggested a mechanism for caffeine radiosensitization. Following
DNA damage, the activation of Chk2 requires ATM (8, 12, 13), whereas
the activation of Chk1 requires ATR (26). Caffeine down-regulates both
Chk1 expression and Cdc2 phosphorylation but has only a small effect on
Chk2. Also the Chk1 but not the Chk2 antisense oligonucleotide reduces
the G2 delay and sensitizes A1-5 cells to killing by
irradiation. These results suggest by mainly affecting the Chk1
pathway, caffeine abolishes the strong G2 arrest in
irradiated A1-5 cells. Our results differ from those of an earlier
study (27), which suggested that caffeine abolishes the G2
checkpoint by inhibiting the ATM/Chk2 pathway. The contrasting conclusions may be attributable to the different cell lines used and
the different time frame in which Chk2 activity was measured. However,
our results do not exclude the possibility that the Chk2 pathway plays
a role in maintaining the strong G2 arrest (40) in A1-5
cells. In fact, although Chk1 expression in A1-5 cells is
decreased to the level similar to that in B4 cells at 24 h after
irradiation (Fig. 4A), at the same time, the percentage of
G2 phase in A1-5 cells is still higher than that in B4
cells (Fig. 2A), which might depend upon Chk2 activation.
Because there is no difference in the expression of ATR between A1-5
and B4 cells (data not shown), the higher Chk1 expression may not be
directly related to ATR, the upstream regulator of Chk1. It remains
unclear why A1-5 and B4 cells, despite their similar genetic
background, display such a different DNA damage response. One
possibility is that during the process of transformation, Chk1 is
modified because it is directly or indirectly targeted by the specific
recombination in A1-5 cells. This hypothesis is under investigation in
our laboratory now.
As mentioned above, it is thought that checkpoint activation is
exploited by the cell to perform DNA repair and thus to reduce cell
killing. Although DNA DSB are repaired most efficiently through homologous recombination (HR) in yeast and through nonhomologous end-joining in mammalian cells, some experiments show that both repair
pathways are conserved from yeast to humans (41). The results presented
here indicate that A1-5 cells show a stronger G2
checkpoint response but no alterations in either the induction or the
nonhomologous end-joining repair of DNA DSB. It is not clear yet how
the G2 arrest promotes DNA repair and facilitates cell
survival. It was suggested that ATM links to HR activation (42, 43),
thus providing evidence to support the hypothesis that checkpoint
activation facilitates HR repair in irradiated cells. A new report
shows that caffeine cannot radiosensitize one HR-defective cell line
but can still radiosensitize ataxia telangiectasia cells (44). These
results suggest that an ATM-independent target of caffeine (which might
be ATR) is also involved in HR repair. The recently discovered
connections between checkpoint pathways and DNA repair and their
physiological effects on the cell prompted us to reevaluate the role of
checkpoint proteins within the context of the overall response to DNA
damage (45).
Here we show that caffeine and UCN-01 have similar effects on A1-5
cells, which suggests that both drugs target the same pathway. A
possible candidate is the ATR/Chk1 pathway. The ATR/Chk1 pathway is
essential for cell survival. Checkpoint activation and HR repair may be
two parallel functions controlled by this pathway, and the ATM/Chk2
pathway may play a supporting role for the ATR/Chk1 functions in
addition to the other independent roles it may have. To accommodate our
results with A1-5 cells, a model was developed; this model is shown in
Fig. 7. In this model, ATR/Chk1 is the major pathway responding to DNA damage and arresting cells in G2. This pathway maintains genomic integrity not only by
arresting cells in G2 but also by activating HR repair. On
the other hand, the ATM/Chk2 pathway plays a supporting role for the
ATR/Chk1 pathway. We hypothesize that these two pathways cooperate to
ensure the prompt and efficient repair of DNA damage and to maintain genomic integrity. The level or the type of this cooperation may be
cell-type-dependent, and it may differ for the different
types of DNA damage.

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Fig. 7.
A model for the DNA damage response pathway
in mammalian cells. The ATR/Chk1 pathway has a predominant role of
G2 arrest, whereas ATM/Chk2 plays a supporting role.
The ATR/Chk1 pathway can be interrupted either by caffeine
(inhibiting ATR and ATM) or by UCN-01 (inhibiting Chk1).
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