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Oncogene (2000) 19, 1386 ± 1391
2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00
www.nature.com/onc
Ionizing radiation activates the ATM kinase throughout the cell cycle
Tej K Pandita*,1, Howard B Lieberman1, Dae-Sik Lim2, Sonu Dhar1, Wei Zheng1, Yoichi Taya3
and Michael B Kastan2
1
Center for Radiological Research, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA;
Department of Hematology-Oncology, St. Jude Children's Research Hospital, Memphis, TN 38205-2794, USA; 3National Cancer
Center Research Institute, Tsukiji 5-Chome, Chuo-ku, Tokyo 104, Japan
2
The ATM protein kinase is a critical intermediate in a
number of cellular responses to ionizing irradiation (IR)
and possibly other stresses. ATM dysfunction results in
abnormal checkpoint responses in multiple phases of the
cell cycle, including G1, S and G2. Though downstream
targets of the ATM kinase are still being elucidated, it
has been demonstrated that ATM acts upstream of p53
in a signal transduction pathway initiated by IR and can
phosphorylate p53 at serine 15. The cell cycle stagespeci®city of ATM activation and p53Ser15 phosphorylation was investigated in normal lymphoblastoid cell
line (GM536). Ionizing radiation was found to enhance
the kinase activity of ATM in all phases of the cell cycle.
This enhanced activity was apparent immediately after
treatment of cells with IR, but was not accompanied by a
change in the abundance of the ATM protein. Since IR
activates the ATM kinase in all phases of the cell cycle,
DNA replication-dependent strand breaks are not
required for this activation. Further, since p53 protein
is not directly required for IR-induced S and G2-phase
checkpoints, the ATM kinase likely has di€erent
functional targets in di€erent phases of the cell cycle.
These observations indicate that the ATM kinase is
necessary primarily for the immediate response to DNA
damage incurred in all phases of the cell cycle. Oncogene
(2000) 19, 1386 ± 1391.
Keywords: ATM; p53; kinase activity; cell cycle
Introduction
Ataxia telangiectasia (A-T) is a rare, pleiotropic,
autosomal human recessive disorder characterized by
progressive neurological degeneration, growth retardation, premature aging, oculocutaneous telangiectasias,
speci®c immunode®ciencies, high sensitivity to ionizing
radiation, gonadal atrophy, genomic instability, defective telomere metabolism, and cancer predisposition
(Bridges and Harnden, 1982; Harnden 1994; Morgan
and Kastan 1997a; Shiloh 1995; Pandita et al., 1999;
Smilenov et al., 1999). Cells derived from A-T
individuals exhibit a variety of abnormalities in culture,
such as a higher requirement for serum factors,
hypersensitivity to ionizing radiation and cytoskeletal
defects (Morgan and Kastan 1997a; Shiloh 1995). The
gene that is mutated in A-T has been designated ATM
(A-T, mutated) and its product shares the PI-3 kinase
*Correspondence: TK Pandita
Received 24 September 1999; revised 29 December 1999; accepted
13 January 2000
signature of a growing family of proteins involved in
the control of cell cycle progression, processing of
DNA damage and maintenance of genomic stability
(Hawley and Friend, 1996; Keith and Schreiber, 1995;
Morrow et al., 1995; Savitsky et al., 1995a,b). ATM
appears to be required for initiation of multiple DNA
damage-dependent signal transduction cascades that
activate cell-cycle checkpoints (Morgan and Kastan,
1997a; Shiloh, 1995). One of the best characterized
mammalian cell cycle checkpoints involves accumulation/stabilization of p53 protein and subsequent G1
arrest or apoptosis (Morgan and Kastan, 1997a;
Kastan et al., 1992). Cells derived from A-T individuals
or Atm null mice are very poor at this induction of p53
following ionizing irradiation, though induction following UV irradiation appears relatively normal (Kastan
et al., 1992; Khanna and Lavin, 1993; Canman et al.,
1994; Xu and Baltimore, 1996). For this reason, it has
been suggested that ATM is involved in speci®c
signaling pathways induced by ionizing radiation
exposure (Kastan et al., 1992; Khanna and Lavin,
1993; Canman et al., 1994; Xu and Baltimore, 1996).
Recently, it has been reported that the phosphorylation of p53 on serine-15 is impaired in A-T cells after IR
(Siliciano et al., 1997) and that the ATM kinase is
capable of phosphorylating this site in p53 (Banin et al.,
1998; Canman et al., 1998; Khanna et al., 1998).
Though these results suggest the importance of ATM in
the phosphorylation of p53, it is not known whether
this activation of ATM by IR is cell cycle dependent.
For example, it is conceivable that ATM activation by
IR could be dependent on speci®c types of DNA lesions
introduced during a particular phase of the cycle, such
as DNA replication-dependent events during S phase.
However, since ATM signals to p53, and p53 is
involved in a G1 checkpoint after IR, this activation
is likely to occur at least during the G1 phase of the
cycle. Ataxia telangiectasia cells also exhibit speci®c
defects in S phase and G2 checkpoints which are intact
in SV-40 transformed cells from non-A-T patients and
in tumor cell lines with mutant p53 (Morgan and
Kastan, 1997b; Morgan et al., 1997). Thus, there are
p53-independent pathways in which ATM participates
following IR. These observations raised the question of
whether ATM activation after IR occurs in all phases of
the cell cycle. Therefore, cells were enriched at di€erent
phases of the cell cycle and tested for the IR-induced
activation of the ATM kinase and phosphorylation of
p53 at serine-15. We observed an enhancement of ATM
activity after IR in all phases of the cell cycle,
implicating a role for ATM activation and potentially
for p53 phosphorylation in multiple radiation-induced
transient delays in cell cycle progression.
ATM kinase activity during the cell cycle
TK Pandita et al
Results and discussion
Cells de®cient in ATM function have higher initial
chromosomal damage and greater amounts of residual
chromosomal damage in G1 as well as in G2 after
treatment with ionizing radiation, and are sensitive to
ionizing radiation induced cell killing in all phases of the
cell cycle (Pandita and Hittelman, 1992a,b; Morgan et
al., 1997). ATM might therefore play a critical role in all
phases of the cell cycle after ionizing radiation treatment.
We set out to determine the cell survival, ATM kinase
activity and, simultaneously, the serine-15-phosphorylation state of p53 in cell populations enriched in the G1, S
or G2/M-phases of the cell cycle. Asynchronous
exponentially growing populations of GM536 lymphoblastoid cells were fractionated by centrifugal elutriation
into populations enriched for di€erent phases of the cell
cycle (Pandita and Hittelman, 1992a). The quality of cell
cycle enrichment was monitored by ¯ow cytometry for
DNA content (Figure 1), and independent determinations of cell cycle stage were assessed by premature
chromosome condensation (Pandita and Hittelman,
1992a). The G1 phase enriched populations contained
greater than 98% of their cells in that phase. The S phase
and G2/M enriched populations were about 88 and 7080% pure, respectively.
To determine whether centrifugal elutriation in¯uences the physiology/reproductive capability of the
cells, we compared cells that did or did not undergo
this procedure for viability and survival after ionizing
radiation exposure. We elutriated the cells and pooled
fractions together. The ¯ow analysis of the pooled
fraction showed similar frequencies of G1, S and G2/M
phase cells as was found in the exponentially growing
asynchronous population of cells before fractionation.
GM536 cells, elutriated or not elutriated, were treated
with di€erent doses of gamma-rays. Cell viability as
determined by the trypan blue exclusion test showed no
di€erence in elutriated versus mock-treated cells (data
not shown).
Cell survival after ionizing radiation treatment was
determined by two independent assays, i.e., growth
curve analysis and limiting dilution analysis (Pandita
and Hittelman, 1992a). GM717 (ataxia telangiectasia)
lymphoblastoid cells were used as a control. No
di€erence in cell survival after irradiation was detected
between the cells that were elutriated versus not
elutriated (Figure 2). In addition, the normal cells
(GM536) were much less sensitive to IR than the
GM717 (ataxia telangiectasia, A-T) cells, (Figure 2),
with survival characteristics for all cell cycle dependent
results given in Table 1. The enhanced sensitivity of G1
phase cells to ionizing radiation (Figure 2) relative to
cells in other phases of the cell cycle are consistent with
previous studies (Pandita and Hittelman, 1992a).
Cells at di€erent phases of the cell cycle show
di€erences in radiosensitivity and those de®cient in
ATM function show abnormal checkpoint responses in
G1, S and G2. Recent studies revealed enhanced ATM
kinase activity in response to DNA damage (Banin et
al., 1998; Canman et al., 1998; Khanna et al., 1998).
We were interested to determine whether ATM protein
levels and/or kinase activity were enhanced in all
phases of the cell cycle following IR treatment. Cell
cycle phase enrichment was accomplished by centrifugal elutriation. First, we determined that centrifugal
elutriation had no in¯uence on the induction of ATM
protein or on its kinase activity. These features of the
ATM protein were compared in cells that were
elutriated and pooled versus those not subjected to
the enrichment procedure. Both cell preparations
(elutriated and nonelutriated) were treated with 10
Gy of gamma rays and incubated for di€erent times
1387
Figure 2 Gamma-ray survival through the cell cycle. Doseresponse curves are shown for GM536 cells treated with ionizing
radiation while growing exponentially and asynchronously (*),
or in G1 (&), S (D) or the G2/M (*) phases of the cell cycle. The
ataxia telangiectasia cell line GM717 was used as a positive
control to indicate radiosensitivity in an exponentially growing
asynchronous population (^)
Table 1 Survival characteristics after ionizing radiation
Cell phase
GM717
GM536
GM536
GM536
GM536
(A-T) (asynchronous population)
(normal) (asynchronous population)
(normal) (G1 phase)
(normal) (S phase)
(normal) (G2/M phase)
D0 (Gy)
n
0.37
0.93
0.69
1.04
0.89
1.00
1.37
1.25
1.42
1.25
Figure 1 Synchronization quality measurements. DNA content distribution measured by ¯ow cytometry in GM536 cell
populations enriched for di€erent cell cycle phase fractions separated by centrifugal elutriation. (a) The DNA content distribution of
an unsynchronized population, other panels represent the DNA content distribution of: (b) G1-phase enriched cell populations; (c)
represent the DNA content distribution of S phase enriched cell populations and (d) G2/M- phase enriched cell populations
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ATM kinase activity during the cell cycle
TK Pandita et al
1388
prior to cell lysis for determination of ATM protein
levels and kinase activity. Elutriation did not in¯uence
ATM protein levels or its kinase activity before or after
ionizing radiation treatment (Figure 3a). We then
analysed the G1, S and G2/M phase-enriched cell
populations separately after treatment with 10 Gy of
gamma-rays. ATM protein levels and kinase activity
were analysed at di€erent times post-irradiation. ATM
(c)
Figure 3 Kinase activity of ATM immunoprecipitates from cells treated with ionizing radiation. Cells were either mock-irradiated
or exposed 10 Gy of gamma rays (dose rate 1.1 Gy per min) and lysed at 0 or 60 min after irradiation. ATM was
immunoprecipitated and assayed with wild type GSTp531-101 protein as a substrate. Amounts of ATM present in each reaction were
determined by immunoblotting with anti-ATM (Ab-3) (upper most panel, (a) and (b)). The amount of radiolabel incorporated into
ATM during the kinase reaction was visualized with a PhosphorImager (middle panel, (a) and (b)). The amount of [g-32P]phosphate
incorporated into GSTp531-101 during each reaction (lower panel, (a) and (b) was quantitated with a PhosphorImager and
normalized to that obtained with immunoprecipitates from unirradiated cells. (a) Represents ATM protein, radiolabeled ATM
protein and 32P-GST-p531-101 levels in exponentially growing cells after treatment with 10 Gy at di€erent periods of post irradiation.
Note that ATM kinase activity was not seen in cells derived from ataxia telangiectasia individual (GM717). (b) Represents ATM
protein, labeled ATM protein and 32P-GST-p531-101 levels in G1, S and G2/M phase enriched cells after treatment with 10 Gy at
di€erent periods of post irradiation. (c) A histogram showing enhancement of ATM kinase activity in cells after irradiation with 10
Gy of gamma-rays and normalized to unirradiated control cells. Note that the enhancement of ATM kinase activity was seen in all
phases of cell cycle
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ATM kinase activity during the cell cycle
TK Pandita et al
protein levels were identical in all phases of the cell
cycle, and did not change after irradiation (Figure 3).
However, when ATM kinase activity was assessed,
enhanced activity after IR was found immediately in all
phases (G1, S or G2/M) (Figure 3b,c), and this
enhanced activity remained constant for 1 h postirradiation. These results suggest that ATM has a
critical role in sensing ionizing radiation induced DNA
damage in each of these phases of the cell cycle.
A known target of ionizing radiation DNA damage
induced phosphorylation is the p53 protein. Phosphorylation of p53 at serine-15 in response to ionizing
radiation correlates with both the accumulation of total
p53 protein as well as its transactivation of downstream
genes. The cell cycle phase dependence of p53 phosphorylation is not known. Activation of p53 results in a G1
cell cycle arrest or apoptosis that contributes to
suppression of malignant transformation and the
maintenance of genomic integrity (Hartwell and Kastan,
1994). The p53 tumor suppressor protein is a transcription factor that is activated in response to treatment with
a variety of DNA-damaging agents, including ionizing
radiation (Kastan et al., 1992). Since G1 cells are more
sensitive to killing by ionizing radiation, while the
enhancement of ATM kinase activity levels postirradiation is similar among G1, S and G2/M phase
cells, p53 accumulation and phosphorylation at serine-15
may be di€erentially altered in G1 cells.
We investigated p53 accumulation and phosphorylation in vivo following ionizing radiation treatment of
cells in di€erent phases of the cell cycle. As for the
other endpoints tested, we found that the enrichment
protocol had no e€ect on the radiation-induced
accumulation of p53. Cells were elutriated and all the
fractions pooled, treated with 5 Gy of ionizing
radiation and p53 levels determined at di€erent time
periods. No di€erence in the accumulation of p53 was
observed between cells elutriated versus those not
elutriated (Figure 4a).
To assess p53-serine-15 phosphorylation after treatment with ionizing radiation, p53 was ®rst immunoprecipitated using a speci®c monoclonal antibody (Figure
4b). The amount of p53 protein loaded was adjusted to
similar amounts per lane of the gel (Figure 4b). As a
control for the serine-15 antibody, some cells were
treated with the proteosome inhibitor acetyl-Leu-Leunorleucinal (ALLN), resulting in the stabilization of p53
protein levels by inhibiting its degradation (Maki et al.,
1996). Under these conditions, Western blot analysis of
lysates prepared from ALLN-treated or irradiated cells
demonstrated equivalent amounts of p53 protein
(Figure 4a). Western blot analysis demonstrated that
p53 was phosphorylated on serine-15 in response to
ionizing radiation. p53 serine-15 phosphorylation was
not observed in ALLN-treated cells even after irradiation. No di€erence was observed in the levels of p53
serine-15 phosphorylation after treatment with ionizing
radiation between cells elutriated and pooled versus
those not elutriated (Figure 4b).
To determine whether the p53 response to ionizing
radiation is cell cycle phase dependent, we examined
p53 protein levels in unirradiated or irradiated G1, S
and G2/M cells and found no cell cycle phase
di€erences (data not shown). Similarly, when p53
1389
Figure 4 p53 protein levels and p53 serine-15 phosphorylation in response to irradiation. (a) Western blot analysis of p53 after
exposure of elutriated and pooled cells to 5 Gy of gamma rays (0, 15, 60 min post-irradiation). Some cells were treated with ALLN.
Time after irradiation and gamma-ray dose are indicated. The amount of p53 accumulated after 1 h post-irradiation is almost
identical to that found in cells treated by 20 mM of ALLN for 1 h. Note that elutriation did not in¯uence the time-dependent
increase in p53 levels. (b) Post-translational modi®cation of p53 on serine-15 in response to ionizing radiation. p53 protein was
immunoprecipitated, subjected to SDS ± PAGE (7.5% gel), transferred to nitrocellulose, and immunoblotted with monoclonal
antibodies against phosphoserine-15 p53 (upper panel). Blots were stripped and probed with antibodies against p53 to con®rm that
equal amounts of this protein were loaded onto the gel (lower panel). Note that the serine-15 phosphorylation of p53 appears soon
after irradiation and p53 immunoprecipitated from ALLN treated cells did not show any enhanced serine-15 phosphorylation. (c)
Cells enriched in di€erent phases of the cell cycle were irradiated with 5 Gy and examined for serine-15 phosphorylation of p53.
Cells in all phases (G1, S and G2/M) show an immediate increase in the phosphorylation of the serine-15 residue of p53. C in all
panels represents unirradiated control
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ATM kinase activity during the cell cycle
TK Pandita et al
1390
serine-15 phosphorylation was compared among G1, S
and G2/M phase cells after gamma irradiation, no
signi®cant di€erences were detected (Figure 4c). p53 is
involved in a G1 checkpoint and phosphorylation of
p53 can be linked with the G1 checkpoint. It is not
clear whether the phosphorylation of p53 at serine-15
has any role in S and G2/M phase cells after treatment
with DNA damaging agents. Cells de®cient in p53
function have normal S phase as well as G2 phase
checkpoints after ionizing radiation treatment in
contrast to the altered G1 checkpoint, while A-T cells
exhibit speci®c defects in G1 as well as S phase and G2
phase checkpoints. These studies suggest that the ATM
kinase may have other functional targets in S phase
and G2 phase of the cell cycle, and that activation of
the ATM kinase by DNA damaging agents is
important for signaling in all phases of the cell cycle.
Materials and methods
Cell culture
Lymphoblastoid cell lines GM536 and GM717 were obtained
from the NIGMS Human Genetic Mutant Cell Repository
(Coriell Institute for Medical Research, Camden, NJ, USA).
Both cell lines were maintained according to procedures
described earlier (Pandita and Hittelman, 1992a). Cell
viability was monitored by the trypan blue exclusion test.
Cell population densities were determined by hemacytometer
and electronic counting (Coulter Counter Inc. Hialeah, FL,
USA).
Immunoprecipitation of ATM and kinase assay
Immunoprecitation of ATM and kinase assay were done with
a modi®ed procedure described earlier (Canman et al., 1998).
Brie¯y, cells were broken open in lysis bu€er (50 mM Tris
pH 7.5, 150 mM NaCl, 1% Tween 20, 0.2% NP-40, 1 mM
NaF, 1 mM NaVO4, 50 mM glycerophosphate, 10% glycerol,
1 mM phenylmethylsulfonyl ¯uoride, 2 mg/ml pepstatin A,
5 mg/ml leupeptin, 10 mg/ml aprotinin, and 1 mM dithiothreitol (DTT). Afterwards, lysates were pre-cleaned for 1 h at
48C with 10 mg rabbit puri®ed IgG and 40 ml protein A/G
beads. Endogenous ATM proteins were immunoprecipitated
with Anti-ATM antibodies (Ab-3 from Calbiochem) and
protein A/G Sepharose. Immunoprecipitants were washed
three times with lysis bu€er, once with lysis bu€er and 0.5 M
LiCl, and three times with kinase bu€er (50 mM HEPES
pH 7.5, 150 mM NaCl, 1% Tween 20, 0.2% NP-40, 1 mM
NaF, 1 mM NaVO4, 1 mM DTT, 10 mM MnCl2). Immunoprecipitant beads were mixed with kinase bu€er containing
10 mC [g-32P]ATP and 1 mg GST-p531 ± 101, incubated for
20 min at 308C. The kinase reactions were stopped with
SDS ± PAGE loading dye and reaction mixtures were
separated by SDS ± PAGE. Transferred ATM and substrates
were visualized and quantitated on a PhosphorImager. Then
ATM proteins were detected using anti-ATM antibodies.
Immunoprecipitation of p53 and immunoblot analysis
Cell irradiation
For immunoblot analysis of the p53 protein, cells were
treated with ice cold bu€er containing 50 mM Tris-HCl
pH 7.5, 150 mM NaCl, 1% NP40, 1 mM DTT, 1 mM EDTA,
50 mM glycerophosphate, 10% glycerol, 1 mM phenylmethylsulfonyl ¯uoride, 2 mg/ml pepstatin A, 5 mg/ml leupeptin,
10 mg/ml aprotinin, and 1 mM dithiothreitol (DTT) at 48C
for 30 min. p53 was immunoprecipitated with 50 ml of premade p53-agarose (OP43A, ONCOGENE) for 2 ± 3 h at 48C.
The resulting immune complexes were washed three times
with lysis bu€er, followed by three washes with TBS
containing protease inhibitors. 60 ml of 26 Laemmli sample
bu€er was added to immune complex and then boiled for
5 min. A similar amount of immunoprecipitated proteins
were fractionated by SDS/10% PAGE and electrophoretically
transferred to nitrocellulose. Nonspeci®c binding was blocked
by incubating the membrane in PBS solution containing
0.05% Tween-20 and 1% nonfat dried milk. For immunodetection of serine-15-phosphorylated p53 by anti-p53-ser-15
antibodies, the ECL procedure was used as described
(Siliciano et al., 1997; Canman et al., 1998).
Cells were irradiated with a 137Cs source at a dose rate of 1.1
Gy per min. The proteosome inhibitor ALLN (Sigma) was
added to cells at a ®nal concentration of 20 mM. Cells that
received both irradiation and ALLN were ®rst irradiated and
then immediately given ALLN. Cells were harvested at
indicated times after irradiation or ALLN treatment.
Abbreviations
IR, ionizing radiation; ATM, ataxia telangiectasia mutant.
Cell phase enrichment
To obtain fractions of lymphoblastoid cells enriched at
di€erent stages of the cell cycle, exponentially growing
populations were fractionated by centrifugal elutriation using
modi®cations of the procedure described previously (Pandita
and Hittelman, 1992a). After separation, the cell fractions
were placed on ice to prevent cell cycle progression. Cell
viability was not a€ected by elutriation, as monitored by
trypan blue exclusion and thymidine uptake. The cell cycle
distribution of the fractionated samples were determined by
the procedures described previously (Pandita and Hittelman,
1992a).
Cell survival measurements
Cells were irradiated in complete medium at a dose rate of
1.1 Gy per min. All experiments were performed at least
twice, and the mean values of the replicates are shown in the
®gures. Two independent methods (a regrowth curve assay
and a microtiter well assay), as described previously, were
used to determine cell survival after radiation exposure
(Pandita and Hittelman, 1992a). The survival values obtained
after irradiation, as determined by the two independent
assays, were found to be close to one another, with a
standard deviation of 7 ± 13%. The reported survival results
represent the averaged values of the two assays. The Do
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values were determined using the exponential portion of the
dose-response curves.
Acknowledgments
This work was supported by grants from NIH (NS34746,
GM52493, CA71387, CA75061, ES05777), DOE (DEFG07-96ER62309), and Columbia Cancer Center Breast
Cancer Research Program. MB Kastan was the Steven
Birbaum Scholar of the Leukemia Society of America and
is supported by the American Lebanese Syrian Associated
Charities (ALSAC) of St. Judes Children's Research
Hospital. HB Lieberman was supported by a NIH
Research Career Development Award CA68446. Thanks
are due to Lubomir B Smilenov, AS Balajee and Satin
Sawant for their technical help.
ATM kinase activity during the cell cycle
TK Pandita et al
1391
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