MOLECULAR AND CELLULAR BIOLOGY, Sept. 2002, p. 6521–6532
0270-7306/02/$04.00?0 DOI: 10.1128/MCB.22.18.6521–6532.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 22, No. 18
Chk2 Is a Tumor Suppressor That Regulates Apoptosis in both an
Ataxia Telangiectasia Mutated (ATM)-Dependent and an
Atsushi Hirao,1,2Alison Cheung,1Gordon Duncan,1Pierre-Marie Girard,3Andrew J. Elia,1
Andrew Wakeham,1Hitoshi Okada,1Talin Sarkissian,1Jorge A. Wong,1Takashi Sakai,1
Elisa de Stanchina,4Robert G. Bristow,5Toshio Suda,2Scott W. Lowe,4
Penny A. Jeggo,3Stephen J. Elledge,6and Tak W. Mak1*
Departments of Medical Biophysics and Immunology, Ontario Cancer Institute, University of Toronto, Toronto, Ontario M5G 2C1,1
and Department of Radiation Oncology and Department of Medical Biophysics, Ontario Cancer Institute, University of Toronto,
Toronto, Ontario M5G 2M9,5Canada; Genome Damage and Stability Centre, University of Sussex, Falmer, Sussex BN1 9RR,
United Kingdom3; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 117244; Verna and Marrs
McLean Department of Biochemistry and Molecular Biology and Department of Molecular and
Human Genetics, Howard Hughes Medical Institute, Baylor College of Medicine, Houston,
Texas 770306; and The Sakaguchi Laboratory of Developmental Biology, School of
Medicine, Keio University, Tokyo 160-8582, Japan2
Received 5 March 2002/Accepted 18 June 2002
In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle
arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation.
Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated
(ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a
linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-
targeted Chk2-deficient mice. Unlike ATM?/?and p53?/?mice, Chk2?/?mice do not spontaneously develop
tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from
Chk2?/?mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair
follicles, show significant defects in IR-induced apoptosis or impaired G1/S arrest. Quantitative comparison of
the G1/S checkpoint, apoptosis, and expression of p53 proteins in Chk2?/?versus ATM?/?thymocytes
suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced
apoptosis was restored in Chk2?/?thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2
gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3?related (ATR) were
mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that
distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.
DNA damage activates cellular responses that promote
DNA repair, arrest the cell cycle, and in some cases, induce
apoptosis (56). Cell cycle arrest allows time for the repair of
damaged DNA while apoptosis eliminates cells harboring ab-
normal DNA. It is widely believed that these DNA damage
responses are required for the maintenance of genomic stabil-
ity and prevention of tumor development (20).
The ataxia telangiectasia (A-T) mutated (ATM) gene, which
is homologous to the yeast checkpoint gene Tel1, plays a crit-
ical role in sensing DNA double strand breaks (DSBs) in mam-
malian DNA. ATM is a kinase involved in activating the ap-
propriate damage response pathway, leading to either cell
cycle arrest or apoptosis, and is therefore a key checkpoint
molecule in regulating cell cycle responses to DNA damage
(37, 45). Indeed, the majority of phosphorylation events in-
duced by ionizing radiation (IR) are carried out by ATM. Both
A-T patients and ATM-deficient mice show defective cell cycle
arrest, hypersensitivity to DNA DSBs, and tumor predisposi-
tion (4, 21, 52, 53). When cells are damaged by IR, ATM
phosphorylates and activates the protein kinase Chk2 (1, 35,
36, 55). Chk2 is a homologue of the Rad53 gene in budding
yeast and of the Cds1 gene in fission yeast. Once phosphory-
lated, activated Chk2 phosphorylates multiple Cdc25 mole-
cules which are thought to inhibit the activation of cyclin-
dependent kinases (7, 10, 34). However, in response to damage
induced by UV-irradiation or hydroxyurea, Chk2 is phosphor-
ylated in an ATM-independent manner, possibly by A-T and
rad3?related (ATR) (35, 46). Notably, ATM, ATR, and Chk2
are each able to phosphorylate the tumor suppressor gene p53
(2, 9, 11, 26, 42, 49).
p53 is the most frequently mutated cancer-associated gene
identified to date (29). In response to DNA damage, p53 un-
dergoes phosphorylation and conformational changes which
result in increased levels and activity of the protein (23). In-
creased p53 activity enhances the rate of transcription of nu-
merous target genes (such as p21, Mdm2, GADD45, and Bax)
that mediate the plethora of p53-dependent functions (19, 54).
These functions include the promotion of apoptosis and the
induction of G1cell cycle arrest. The p53 protein can be mod-
* Corresponding author. Mailing address: Ontario Cancer Institute,
University of Toronto, 620 University Ave., Suite 706, Toronto, On-
tario M5G 2C1, Canada. Phone: (416) 204-2236. Fax: (416) 204-5300.
ified by many different protein kinases and acetylases, resulting
in modulation of p53 function (39). In particular, the phos-
phorylation or dephosphorylation of various serine residues
can have a significant impact on p53 stability. Recent studies
with phospho-specific antibodies have established that serines
(Ser) 6, 9, 15, 20, 33, 37, and 46 of p53 are sites of de novo
phosphorylation in cells following DNA damage and that phos-
phorylation of different sites has different effects (2, 9, 12, 25,
38, 43, 44, 47). For example, it has been proposed that the
phosphorylation of the N-terminal Ser 15, 33, and 37 residues
permits subsequent modification of the distant C-terminal ly-
sine residues of p53 through enhanced recruitment of the co-
activator protein p300/CBP/PCAF (28, 41). In contrast, phos-
phorylation of Ser20 is required for stability of p53 in response
to DNA damage (11). Ser20 comprises part of the site used by
Mdm2 to bind p53 and target it for ubiquitination, and phos-
phorylation of Ser20 interferes with Mdm2 binding.
Previous studies have demonstrated that, in response to IR,
Ser15 on p53 is phosphorylated by ATM (2, 9), whereas Ser20
is phosphorylated by Chk2 (11, 26, 42). There is abundant
evidence that ATM controls p53 stabilization either directly or
indirectly via Chk2, and it is also now clear that p53-mediated
G1arrest is suppressed in ATM?/?thymocytes. However, it is
more controversial whether ATM is involved in p53-mediated
apoptosis of damaged cells. While some laboratories have
shown that ATM?/?thymocytes are resistant to IR-induced
apoptosis (51, 53), others have found that these cells exhibit
normal p53-mediated cell death (3, 21, 24). It appears that the
pathways governing p53-dependent cell cycle arrest and apo-
ptosis may be distinct and that ATM plays a major role in
regulating only the former.
Previous work on Chk2-deficient thymocytes reconstituted
from Chk2?/?embryonic stem (ES) cells in Rag1?/?mice
showed that Chk2 contributes to p53 stabilization following
exposure to IR (26). We have now created mutant mice in
which the Chk2 gene was disrupted through homologous re-
combination. We report on the phenotype of Chk2?/?mice
and compare it to that of ATM?/?mice. Using various
Chk2?/?cell types, we show that Chk2 is involved in both
ATM-dependent and ATM-independent regulation of IR-in-
duced p53-mediated apoptosis.
MATERIALS AND METHODS
Generation of Chk2?/?mice. To generate Chk2?/?mice, fragments of the
murine Chk2 gene were isolated from a mouse 129 genomic library by using the
mouse Chk2 cDNA as a probe. The targeting construct was designed to delete
the exons encoding the conserved kinase sequence as described previously (26).
ES clones heterozygous for the targeted mutation were isolated by drug selection
and confirmed by Southern blotting. Two independent heterozygous ES clones
were injected into 3.5-day C57BL/6 blastocysts which were subsequently trans-
ferred into pseudopregnant foster mothers. Chimeric mice were crossed into
C57BL/6 mice to produce heterozygous Chk2?/?mice which were subsequently
intercrossed to generate Chk2?/?animals. p53?/?mice were purchased from
Taconic. ATM?/?mice were the kind gift of Peter J. McKinnon (St. Jude
Children’s Research Hospital).
Apoptosis assays. To analyze apoptosis in the thymus in vivo, 6- to 8-week-old
mice were irradiated with 10 Gy of irradiation from a137Cs source (Gammacell
40) at a dose of 1.0 Gy/min. For apoptosis in the central nervous system (CNS)
or skin, mice at day 5 after birth (P5) and P7, respectively, were irradiated with
5 Gy of irradiation. Mice were anesthetized by cooling, and their hearts were
perfused with ice-cold saline followed by fixation in ice-cold 4% paraformalde-
hyde. The brains were removed and fixed for a further 24 to 48 h in 4%
paraformaldehyde at 4°C. The thymus and skin from the dorsal area were fixed
in 10% buffered formalin overnight at 4°C. Tissue samples were embedded in
wax by following standard procedures and sectioned at a thickness of 5 to 7 ?m.
Apoptosis in thymus, skin, and brain sections was analyzed by the terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay
(Boehringer-Mannheim). Sectioned material was prepared for the TUNEL assay
by following standard procedures (33). Briefly, sections were deparaffinized and
rehydrated through a graded series of ethanol solutions to phosphate-buffered
saline. Sections were then subjected to the TUNEL assay. To quantify the
apoptosis of thymocytes in vitro, thymocytes were isolated from 5- to 8-week-old
mice and irradiated with 1 to 4 Gy of irradiation. Apoptotic cells were detected
by flow cytometric analysis with Annexin V and propidium iodide (PI) staining
Cell cycle analysis. For analysis of G1arrest in the skin, P7 mice were irradi-
ated with 5 Gy of irradiation, and the mice were injected intraperitoneally with
bromodeoxyuridine (BrdU) (1 mg) 1 h before sacrifice (48). Skin sections were
treated with 3% hydrogen peroxidase for 15 min to quench endogenous perox-
idase. BrdU was detected by using a BrdU staining kit (Oncogene Research
Products) according to the manufacturer’s protocol. For analysis of the G1/S
checkpoint in the thymus, 5- to 8-week-old mice were irradiated with 10 Gy of
irradiation and injected with 2 mg of BrdU at 2 h postirradiation (3). At 1 h
postinjection, the mice were sacrificed and the thymi were dissected. Thymocytes
were fixed by using the BrdU flow kit (BD PharMingen). The fixed cells were
treated with 300 ?g of DNase/ml for 1 h at 37°C and then incubated with
anti-BrdU-fluorescein isothiocyanate for 30 min at room temperature. After
washing, the cells were counterstained with PI and subjected to flow cytometry.
To detect mitotic cells, skin sections were incubated with anti-phosphohistone
H3 antibody (Ab) (Upstate Biotechnology). After incubation with secondary Ab
followed by Vectastain ABC reagents, the slides were exposed to DAB substrate
(Vector Laboratories). Total cell numbers and the number of BrdU- or phos-
phohistone H3-positive cells were counted in several regions of the epidermis of
G1arrest was examined in cultured mouse embryonic fibroblasts (MEFs)
established as described previously (18). MEFs were synchronized at G0by
incubation for 4 days in Dulbecco’s minimal essential medium containing 0.1%
serum. The G0-synchronized cells were trypsinized and resuspended in growth
medium containing 65 ?M BrdU. The cells were irradiated with 0 to 20 Gy of
irradiation, replated, and cultured for 24 h prior to fixation in 70% ethanol. Cells
were then stained with anti-BrdU Ab (BD PharMingen) as described previously.
To evaluate the S-phase checkpoint, the inhibition of DNA synthesis was mea-
sured by using a previously described procedure (27). Briefly, exponentially
growing MEFs were irradiated with 10 Gy of irradiation and cultured for 0 to 3 h.
[3H]thymidine (2.5 ?Ci/ml) was added 30 min prior to sampling. Cells were lysed
in 0.5 ml of 2% sodium dodecyl sulfate (SDS) in 0.2 M NaOH, and 100 ?l of
lysate was adsorbed onto Whatman 17 CHR paper. The filters were washed with
5% trichloroacetic acid, rinsed in 95% ethanol, and air dried, and the incorpo-
rated radioactivity was measured in a liquid scintillation counter.
Northern blotting. Thymocytes were subjected to 5 Gy of ?-irradiation, and
total RNA was isolated by using Trizol according to the manufacturer’s protocol
(GIBCO BRL). Briefly, thymocytes (2 ? 107) were lysed with 1 ml of Trizol and
mixed with chloroform. Total RNA was precipitated with isopropyl alcohol, and
5 ?g/sample was electrophoresed on a 1% agarose gel followed by transfer to a
membrane (GeneScreen plus; NEN Life Science Products). Blots were hybrid-
ized with mouse p21, Bax, and ?-actin cDNA probes, and the intensity of each
band was quantified by densitometry (Storm860; Molecular Dynamics). Statisti-
cal analyses were carried out by using Student’s t test.
Western blotting. Tissues were lysed in lysis buffer (10 mM Tris-HCl [pH 7.5],
10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 5 mM Na2P2O7, 1 mM Na2VO4,
and 50 mM NaF plus protease inhibitor cocktail [Boehringer Mannheim]). Ly-
sates were boiled with SDS sample buffer at 100°C for 5 min and placed on ice.
Protein extracts were fractionated by SDS–10% polyacrylamide gel electro-
phoresis and transferred to a polyvinylidene difluoride membrane (Sigma). The
blot was incubated with 4% milk overnight followed by incubation with anti-p53
Ab (CM5; Novocastra Laboratories), anti-mouse Chk2 Ab (26), or anti-?-actin
Ab (Sigma). Signals were visualized by incubation with horseradish peroxidase-
conjugated secondary Ab (Amersham) followed by enhanced chemilumines-
Flow cytometric analysis. The preparation of samples for flow cytometry was
performed as described previously (26). Cells were analyzed on a Becton Dick-
DMBA-induced skin tumor formation. The backs of 6- to 8-week-old mice
were shaved and painted with 10 ?g of 7,12-dimethylbenzanthracene (DMBA)
(Sigma) in 0.2 ml of acetone once a week for 25 weeks. Scoring for tumors was
done once a week.
6522 HIRAO ET AL.MOL. CELL. BIOL.
Reintroduction of Chk2 genes. A BamHI site was introduced into the 5? end
of the coding region of the mouse Chk2 cDNA by PCR. The full-length mouse
Chk2 cDNA was cloned into pCAGGS (a gift of Junichi Miyazaki, Osaka Uni-
versity) with a blunt end. To create the mutated Chk2 allele, PCR-based mu-
tagenesis was used to change the seven Ser-Gln/Thr-Gln (SQ/TQ) sites (Ser 22,
24, 29, 36, 42, and 59 and Thr77) in Chk2 to Ala-Gln (AQ) followed by sub-
cloning into pCAGGS. To obtain thymocytes expressing wild-type or mutant
Chk2, somatic chimeras were generated by Rag1?/?blastocyst complementation
as previously described (26). pCAGGS-wild-type Chk2, pCAGGS-mutated
Chk2,or the empty vector was electrophoretically transfected along with pBS-
pgk-Hygro into Chk2?/?ES cells. At 48 h posttransfection, hygromycin (200
?g/?l) was added to the cultures. After 10 days, hygromycin-resistant clones were
isolated and expanded. Clones with the highest expression of Chk2 were selected
by Western blotting with anti-Chk2 Ab. Selected ES clones were injected into
3.5-day Rag1?/?blastocysts as described above to generate animals whose thy-
mocytes had recovered Chk2 expression.
Generation of Chk2?/?mice. The Chk2 gene was disrupted
by replacing a region of the genomic sequence containing ex-
ons 8 to 11 with a neomycin resistance cassette (Fig. 1A).
Following transfection, selection of targeted clones, and blas-
tocyst microinjection, chimeric mice were produced that suc-
cessfully transmitted the Chk2 mutation to the germ line. F1
heterozygote mice were intercrossed to generate animals ho-
mozygous for the mutant Chk2 allele. Chk2?/?mice were
viable and born at the expected Mendelian ratio (?/?:?/?:
?/? ? 0.20:0.53:0.26). The expression of Chk2 protein was
assessed in Chk2?/?and Chk2?/?mice. The Chk2 protein is
ubiquitously expressed in wild-type mouse tissues with its high-
est levels in the thymus, spleen, and colon (Fig. 1B). The
protein was not detected in Chk2?/?tissues, confirming the
Chk2 is not essential for somatic growth, fertility, or immu-
nological development. Since Chk2 activation in response to
DNA DSBs is dependent on ATM (34), we anticipated that
Chk2?/?mice might display overlapping phenotypes with
those of ATM?/?mice. We therefore investigated several of
the most obvious phenotypes of ATM?/?mice in Chk2?/?
ATM?/?mice are smaller in size and weigh less than their
wild-type or heterozygous littermates (4, 21, 52). However,
Chk2?/?mice were not smaller than control mice at birth, at
weaning, or through adulthood (male, ?/? versus ?/? versus
?/?: 19.5 ? 3.4 g versus 23.1 ? 2.1 g versus 22.0 ? 2.6 g,
respectively; female, ?/? versus ?/? versus ?/?: 18.2 ? 3.2 g
versus 17.8 ? 1.3 g versus 17.9 ? 3.1 g, respectively) (42 days
of age). In addition, while cultured MEFs from ATM?/?mice
showed extremely poor growth consistent with the animals’
growth retardation, cultured Chk2?/?fibroblasts did not show
an obvious growth deficit (data not shown).
ATM?/?mice are infertile due to a defect in germ cell
development. However, Chk2?/?male and female mice are
fertile, and their gonads are histologically normal (data not
Several immunological abnormalities have been reported in
ATM?/?mice, including a defect in T-lymphocyte maturation.
However, lymphoid tissues from Chk2?/?mice were similar in
gross size to those of their Chk2?/?and wild-type littermates,
and no histological abnormalities were observed in the thymus,
spleen, or lymph nodes. The numbers of thymocytes and spleen
cells isolated from Chk2?/?mice were comparable to those of
the controls at 8 to 10 weeks of age. Analysis of cell surface
markers of isolated Chk2?/?lymphoid cells showed that pop-
ulations of cells bearing CD3, CD4, CD8, Thy1, or T-cell
receptor ?? were not affected by the loss of Chk2 in either the
thymus or the spleen. The development of immature T cells, as
evaluated by CD25 and CD44 expression, was also normal.
Analysis of the expression of B220, immunoglobulin D (IgD),
IgM, and CD43 in spleen and bone marrow cells revealed no
abnormalities in B-cell development. The proliferation of pe-
ripheral lymph node T cells stimulated with anti-CD3 Ab,
anti-CD28 Ab, and interleukin-2 was normal, as was that of
splenic B cells stimulated with anti-CD40 Ab, anti-IgM Ab,
interleukin-4, or lipopolysaccharide. Immunoglobulin levels in
Chk2?/?mice were normal.
Apoptosis and cell cycle arrest induced by IR are defective
in Chk2?/?mice. In our previous work, we showed that CD4?
CD8?thymocytes isolated from Chk2?/?Rag1?/?chimeric
animals were resistant to IR-induced apoptosis because of a
lack of p53 stabilization. In this study, we evaluated the role of
Chk2 in regulating IR-induced cell death in vivo. Wild-type
and Chk2?/?mice were subjected to whole-body ?-irradiation,
and their thymic tissues were evaluated for apoptosis by using
the TUNEL assay. Thymi of unirradiated wild-type and
Chk2?/?mice showed very few apoptotic cells (Fig. 2A). As
expected, substantial apoptosis was observed in the thymi of
irradiated wild-type mice. However, the thymi of irradiated
Chk2?/?mice showed significantly less apoptosis. We next
examined the effect of the Chk2 mutation on hair follicular
FIG. 1. Targeted disruption of the Chk2 gene in mice. (A) Target-
ing strategy. The genomic configuration of the germ line Chk2 locus is
shown at the top. The targeting vector is shown in the center; exons 8
to 11 were replaced with a neomycin cassette (Neo). The mutated
Chk2 locus is shown at the bottom. (B) Western blot showing the
expression of Chk2 and ?-actin proteins in various tissues of wild-type
(Wild) and Chk2?/?mice.
VOL. 22, 2002REGULATION OF p53-MEDIATED APOPTOSIS BY Chk26523
FIG. 2. Induction of apoptosis in response to ?-irradiation. (A) Mice (8 weeks old) were mock irradiated (a and b) or irradiated with 10 Gy
of irradiation (c and d), and apoptosis in thymi was evaluated at 10 h post-IR. (B) Mice (P7) were mock irradiated (a and b) or irradiated with
5 Gy of irradiation (c and d), and apoptosis in skin was evaluated at 6 h post-IR by in situ TUNEL staining. E and F indicate epidermis and hair
follicle, respectively. (C) Mice (P5) were mock irradiated (a and b) or irradiated with 10 Gy of irradiation (c and d), and apoptosis in the
hippocampal dentate gyrus was evaluated at 24 h post-IR by in situ TUNEL staining. Arrows indicate apoptotic TUNEL-positive cells.
matrical cells since these cells have been shown to be suscep-
tible to IR-induced apoptosis (4, 48). Four hours after 5 Gy of
irradiation, a large number of cells in the wild-type matrix were
undergoing apoptosis, as evaluated by an in situ TUNEL assay
(Fig. 2B). In contrast, there was an almost complete absence of
IR-induced apoptosis in the follicular matrices of Chk2?/?
mice. Finally, we examined IR-induced apoptosis in the devel-
oping CNS. In wild-type perinatal mice (P5), significant apo-
ptosis was observed in many regions of the CNS 24 h after 5 Gy
of ?-irradiation, consistent with previous reports. Strikingly,
very little apoptosis was observed in the CNS, including the
cerebellum and dentate gyrus, of irradiated Chk2?/?mice
(Fig. 2C and data not shown).
Since IR induces cell cycle arrest at several distinct cell cycle
transitions, we studied the effect of the absence of Chk2 on the
G1/S, S, and G2/M checkpoints. First, we examined the G1/S
checkpoint in epidermal cells in vivo. P7 mice were irradiated
with 5 Gy of irradiation followed by BrdU injection 1 h prior to
sacrifice. The frequency of BrdU-positive cells in Chk2?/?
epidermis was comparable to that in wild-type tissue (wild type
versus Chk2?/?: 108 ? 13 per 1,000 cells versus 110 ? 15 per
1,000 cells). Although epidermal cells are not susceptible to
IR-induced apoptosis, there was a clear reduction in the BrdU-
positive population in the wild type at 24 h postirradiation (21
? 5 per 1,000 epidermal cells), likely due to the operation of
the G1/S checkpoint (Fig. 3A). In contrast, there was no re-
duction in the BrdU-positive population at 24 h in either
Chk2?/?epidermis (112 ? 10 per 1,000 cells) or in p53?/?
epidermis (132 ? 7 per 1,000 cells). G1arrest was also evalu-
ated in cultured wild-type, p53?/?, and Chk2?/?MEFs. Se-
rum-starved cells were irradiated and stimulated to enter the
cell cycle by the addition of serum. BrdU was added with the
serum to allow the detection of S-phase entrance. In response
to increasing doses of IR, p53?/?MEFs failed to arrest in G1,
as expected (Fig. 3B) (18). Wild-type MEFs arrested normally
in G1as evidenced by a dose-dependent reduction in the num-
ber of BrdU-positive cells. Interestingly, Chk2?/?MEFs were
significantly defective in their ability to arrest in G1at low IR
doses but behaved like wild-type cells at higher doses.
To examine the G2/M checkpoint, we used an anti-phospho-
histone H3 Ab which specifically recognizes mitotic cells to
evaluate the frequency of mitotic cells in the epidermis follow-
ing IR. In the absence of IR, the frequency of mitotic cells was
comparable in wild-type and Chk2?/?mice (wild type versus
Chk2?/?: 18 ? 4 per 1,000 cells versus 20 ? 4 per 1,000 cells)
(Fig. 3C). Almost no mitotic cells were observed in wild-type or
Chk2?/?mice 6 h after exposure to 5 Gy of irradiation, indi-
cating that the induction of G2arrest is intact in Chk2?/?mice.
However, mitosis had resumed in Chk2?/?cells by 24 h
post-IR (21 ? 4 per 1,000 cells), whereas wild-type cells still
showed suppression of mitosis (3 ? 2 per 1,000 cells) at this
time. These data indicate that the loss of Chk2 curtails the
duration of the G2/M checkpoint.
An intra-S-phase checkpoint occurs in MEFs in response to
IR, and this checkpoint requires ATM function (4). We eval-
uated the effect of the Chk2 mutation on this checkpoint by
examining the inhibition of DNA synthesis in MEFs. Both
wild-type and Chk2?/?primary MEFs showed equivalent lev-
els of DNA synthesis inhibition following IR, whereas
ATM?/?cells showed a characteristic profile of radiation-
VOL. 22, 2002 REGULATION OF p53-MEDIATED APOPTOSIS BY Chk26525
resistant DNA synthesis (Fig. 3D). These findings show that
the intra-S-phase checkpoint is ATM dependent but Chk2
Incidence of DMBA-induced skin tumors is enhanced in
Chk2-deficient mice. Since the loss of Chk2 leads to sup-
pressed apoptosis and cell cycle arrest in response to IR, one
would expect to find a higher incidence of tumor formation in
Chk2?/?mice, perhaps comparable to that in p53?/?mice.
However, by age 1 year, Chk2?/?mice had not developed
tumors of any kind. We speculate that tumors caused by the
loss of Chk2 are either too rare to be detected or require a
prolonged period to develop. We therefore challenged
FIG. 3. Cell cycle checkpoints induced by ?-irradiation of wild-type (wild) and Chk2?/?mice. (A) G1/S checkpoint in the epidermis. Mice (P7)
were irradiated with 5 Gy of irradiation, and BrdU was injected 1 h prior to sacrifice. BrdU-positive cells were taken as those in S phase. Each
value represents the mean number ? standard deviation (SD) of BrdU-positive cells from 3 mice per group. (B) G1/S checkpoint in MEFs. MEFs
were synchronized in G0by serum starvation and irradiated with 0 to 20 Gy of irradiation as indicated. The irradiated cells were incubated with
BrdU, and S-phase cells were detected by flow cytometric analysis of anti-BrdU Ab binding. Each value represents the mean percentage ? SD of
BrdU-positive cells present after IR in 5 cultures per group relative to the unirradiated controls. (C) G2/M checkpoint in epidermis. Mice (P7) were
irradiated with 5 Gy of irradiation, and mitotic cells in the epidermis were detected with anti-phosphohistone H3 Ab. Each value represents the
mean number ? SD of mitotic cells from 3 mice per group. (D) S-phase checkpoint in primary MEFs. Exponentially growing primary MEFs were
irradiated with 10 Gy of irradiation and sampled at the post-IR times indicated. [3H]thymidine was added for 30 min prior to sampling, and
[3H]thymidine incorporation was determined in the cell lysates. The results are expressed as percentages of DNA synthesis relative to label
incorporated in unirradiated cells. Each point represents the mean ? SD of 3 to 5 samples per group.
6526 HIRAO ET AL.MOL. CELL. BIOL.
Chk2?/?mice with the chemical carcinogen DMBA, an agent
that damages DNA and efficiently induces skin tumors (15, 40).
More Chk2?/?mice than wild-type animals developed skin
tumors in response to DMBA treatment (wild type versus
Chk2?/?: 6 of 14 versus 12 of 14 at 25 weeks) (Fig. 4A). The
onset of tumors in Chk2?/?mice occurred earlier than in
wild-type animals, and the total number of tumors was in-
creased in Chk2?/?mice compared to wild-type mice (Fig.
4B). However, the size of individual tumors and the frequency
of malignant tumors (carcinomas) were comparable between
wild-type and Chk2?/?mice. We conclude that Chk2 has a
suppressive effect on tumor development induced by at least
some types of DNA damage.
Chk2 selectively regulates apoptosis in an ATM-indepen-
dent manner. Although Chk2 acts downstream of ATM in
yeast and mammals, the loss of Chk2 does not result in many
of the phenotypes observed in ATM?/?mice. In fact, the only
shared phenotype is defective p53 function in response to IR.
This finding prompted us to explore the possibility that Chk2
and ATM might have different regulatory effects on p53 func-
tion. Whereas Chk2?/?cells have a clear defect in IR-induced
apoptosis, this phenotype is variable in ATM?/?cells. We
therefore carefully compared the effect of the loss of Chk2 or
ATM on the regulation of p53 activation in thymocytes. p53-
mediated cell cycle arrest and apoptosis have been well char-
acterized in thymocytes, and it is possible to precisely quantify
these phenotypes in this cell type. When wild-type mice were
subjected to 10 Gy of ?-irradiation, the number of thymic
BrdU-positive S-phase cells was reduced to 35% ? 4% of that
in the nonirradiated controls (Fig. 5A). The G1/S-phase check-
point was defective in ATM?/?and p53?/?mice (ATM?/?,
132% ? 10%; p53?/?, 128% ? 11%), consistent with previous
reports (3). In contrast to irradiated ATM?/?mice, irradiated
Chk2?/?mice showed only a partial defect that resulted in
milder G1arrest (65% ? 5%). However, when thymocytes
were isolated from Chk2?/?mice and subjected to IR in vitro,
apoptosis was dramatically impaired (Fig. 5B). ATM?/?thy-
mocytes were more resistant than wild-type thymocytes to IR-
induced apoptosis but considerably more sensitive than either
Chk2?/?or p53?/?thymocytes. It should be noted that the
IR-induced apoptosis and inhibition of the G1/S transition
observed in this study are p53 dependent because they are
completely inhibited by the loss of p53 function. Dying p53?/?
thymocytes can be detected after 48 h of IR treatment, and
irradiated Chk2?/?p53?/?thymocytes behave in the same
manner as irradiated p53?/?cells (data not shown). These
results indicate that Chk2 acts in the pathway leading to p53-
dependent apoptosis rather than in the general apoptosis pro-
Our findings led us to hypothesize that Chk2 selectively
regulates p53 activity leading to apoptosis. To address this
question, we used Northern blotting to evaluate the transacti-
vation of mRNA expression for known p53 target genes. Al-
though the expression of many molecules is induced by p53
activation, p21 and Bax are the most prominent p53-responsive
genes in mouse primary thymocytes (8). Loss of p21 in thymo-
cytes leads to a clear defect of the G1checkpoint induced by IR
(18). Although Bax?/?thymocytes are not resistant to IR-
induced apoptosis, Bax?/?Bak?/?thymocytes fail to die in
response to IR (32). Since Bak?/?thymocytes also show nor-
mal responses to IR, the induction of Bax must be critical for
IR-induced apoptosis. In wild-type thymocytes subjected to 5
Gy of IR, Bax mRNA was increased by (6.05 ? 1.44)-fold over
the baseline at 3 h and by (5.68 ? 1.64)-fold at 6 h (Fig. 5C, left
panel). Strikingly, irradiated Chk2?/?thymocytes showed de-
fective induction of Bax ([1.9 ? 0.45]-fold at 3 h and [1.80 ?
0.40]-fold at 6 h). These results represent statistically signifi-
cant decreases compared to the wild type at 3 h (P ? 0.01) and
6 h (P ? 0.01). In contrast, irradiated ATM?/?thymocytes
were slower than the wild-type to induce Bax mRNA synthesis
at 3 h (P ? 0.05) but had caught up by 6 h. p21 mRNA
induction was significantly suppressed compared to that of the
wild type (P ? 0.05) in both ATM?/?and Chk2?/?irradiated
thymocytes at 3 h (Fig. 5C, right panel), but there were no
significant differences in the level of suppression between these
two genotypes. Consistent with a previous report (3), neither
p21 nor Bax was induced in irradiated p53?/?thymocytes.
These results suggest that thymic apoptosis induced by IR
depends on p53 function and is controlled mainly by Chk2
rather than ATM. p53 was stabilized partially by IR in both
ATM?/?and Chk2?/?thymocytes (Fig. 5D). Taken together,
the data in Fig. 5 demonstrate that Chk2, rather than ATM,
controls p53-mediated apoptosis, and that p53-mediated apo-
ptosis does not correlate with stabilization of the p53 protein.
We next determined whether Chk2 phosphorylation is re-
FIG. 4. Skin tumor formation in mice treated with DMBA. The
back skin of 6- to 8-week-old mice was shaved and painted with 10 ?g
of DMBA once a week for 25 weeks. Tumors in skin were scored once
a week. (A) Kaplan-Meier plot of tumor incidence in wild-type (n ?
14) and Chk2?/?(n ? 14) mice. (B) Total numbers of tumors in mice
for which data are shown in panel A.
VOL. 22, 2002 REGULATION OF p53-MEDIATED APOPTOSIS BY Chk26527
quired for the activation of p53 leading to apoptosis. Mouse
Chk2 has seven N-terminal SQ/TQ sites in the N-terminal
region of the protein. In response to IR in vivo, ATM phos-
phorylates several of these SQ/TQ sites in Chk2, including
Thr68 (1, 35, 36). Phosphorylation of Chk2 following IR is
abolished by mutation of these SQ/TQ sites, and most of the
endogenous Chk2 is not phosphorylated in ATM?/?cells (Fig.
5D); nevertheless, p53-mediated apoptosis can occur under
these circumstances. To determine whether the phosphoryla-
tion of Chk2 SQ/TQ sites is required for Chk2-mediated reg-
ulation of p53-mediated apoptosis, we reintroduced into
Chk2?/?thymocytes a mutant form of Chk2 in which all N-
terminal SQ/TQ sites were replaced with AQ and analyzed
apoptosis. In a previous study (35), the mutated SQ/TQ kinase
had the same level of kinase activity as the wild-type enzyme
when transfected into unirradiated Chk2?/?MEFs. To rein-
troduce the mutated Chk2 gene into Chk2?/?thymocytes, we
generated somatic chimeras by Rag1?/?blastocyst comple-
mentation. An expression vector carrying wild-type or mutant
Chk2 was transfected into Chk2?/?ES cells, and clones with
FIG. 5. IR-induced p53 activation and p53 stabilization in Chk2?/?and ATM?/?thymocytes. (A) G1/S checkpoint induced by ?-irradiation.
The indicated strains of mice were irradiated with 10 Gy of irradiation and injected 1 h later with BrdU. At 2 h post-IR, thymocytes were isolated,
stained with anti-BrdU Ab, and subjected to flow cytometry. Each value represents the mean percentage ? standard deviation (SD) of
BrdU-positive (S phase) cells present after IR in 4 samples per group relative to the unirradiated controls. (B) Apoptosis induced by ?-irradiation.
Isolated thymocytes were treated with the indicated doses of ?-irradiation, and apoptotic cells were evaluated by flow cytometry after Annexin V
and PI staining. Each value represents the mean percentage ? SD of viable cells (Annexin V negative and PI negative) for 4 samples per group.
(C) Induction of p53 downstream molecules. Total RNA was isolated from thymocytes before and after treatment with 5 Gy of irradiation. The
expression of mRNA for p21, Bax, and ?-actin was evaluated by Northern blotting. The amount of p21 or Bax mRNA was quantified by using a
PhosphorImager analyzer and normalized to ?-actin expression. Each value represents the mean increase (n-fold) ? SD of the expression of Bax
or p21 mRNA after IR in 3 to 5 samples per group relative to the unirradiated controls. (D) p53 protein stabilization and Chk2 phosphorylation
in thymocytes after irradiation. Isolated thymocytes were irradiated with 5 Gy of irradiation and lysed at the indicated times. p53 and Chk2 proteins
were detected by Western blotting with anti-p53 or anti-Chk2 Ab. Wild, wild type.
6528HIRAO ET AL.MOL. CELL. BIOL.
high expression of Chk2 protein were selected and used for
blastocyst complementation. The size and cellularity of thymi
in Chk2?/?mice bearing an empty vector, Chk2?/?mice bear-
ing wild-type Chk2, and Chk2?/?mice bearing the mutated
Chk2 were all comparable to those of control wild-type mice
(data not shown). Moreover, the expression level of the exog-
enous Chk2 protein was comparable to that of the endogenous
Chk2 protein. Exogenous wild-type Chk2 was phosphorylated
in response to IR, whereas the mutated Chk2 protein failed to
show a mobility shift (Fig. 6A). Thus, the defective p53 stabi-
lization in Chk2?/?thymocytes was restored by the introduc-
tion of exogenous wild-type Chk2 but not by mutant Chk2.
Reintroduction of wild-type Chk2 also clearly restored the
defective apoptosis observed in Chk2?/?thymocytes (Fig. 6B),
whereas thymocytes receiving mutant Chk2 behaved similarly
to control Chk2?/?thymocytes. These data suggest that phos-
phorylation of the SQ/TQ sites in Chk2 is required for this
protein to induce ATM-independent p53 activation leading to
We have shown in this study that there are differences be-
tween Chk2?/?and ATM?/?cells in the IR-induced activation
of checkpoint and apoptotic responses, despite the fact that
ATM regulates IR-induced Chk2 activity. ATM and Chk2 both
modify p53 and activate it; however, our results show that at
least some p53 phosphorylation is Chk2 dependent and ATM
independent. Moreover, Chk2-dependent and ATM-depen-
dent p53 phosphorylation events may differentially affect
downstream p53-dependent transactivation targets. Specifi-
cally, we have demonstrated that Chk2 predominantly regu-
lates IR-induced apoptosis rather than the G1/S checkpoint in
thymocytes, whereas ATM is predominantly involved in the
regulation of the G1/S checkpoint.
Contradictory results have been reported regarding the ef-
fect of ATM on p53-mediated apoptosis (3, 21, 24, 53). The
discrepancies among these reports are not caused by strain
differences among experimental mice but could be due to dif-
ferences in methods used to detect apoptosis or to irradiation
conditions (in vivo versus ex vivo). The in situ TUNEL assay
may not be sensitive enough to detect differences in apoptosis
in a thymus taken from an ATM?/?mouse treated with whole-
body irradiation, whereas in vitro irradiation of isolated
ATM?/?thymocytes reveals an apoptotic defect. In contrast,
Chk2?/?thymocytes examined using either method show ob-
vious resistance to IR-induced apoptosis. In our study,
ATM?/?thymocytes were more resistant than the wild type to
IR but significantly less resistant than Chk2?/?thymocytes.
Consistent with the results of the apoptosis assay, the induction
of Bax mRNA was more profoundly impaired in Chk2?/?
thymocytes than in ATM?/?cells. This tight correlation be-
tween the induction of apoptosis and the transactivation of p53
downstream molecules suggests that the inhibition of apoptosis
induced by the loss of Chk2 is caused by the suppression of p53
activation itself and not by effects on molecules further down-
stream in the apoptosis pathway.
We and others have proposed a model of IR-induced p53
activation in which Chk2 phosphorylates Ser20 of p53, leading
to stabilization of p53 protein in an ATM-dependent manner
(11, 26). Certainly, p53 stabilization was suppressed in both
Chk2?/?and ATM?/?mice. However, the loss of ATM or
Chk2 had different effects on p53 activation leading to apopto-
sis, indicating that the regulation of IR-induced p53 activation
is not as simple as proposed above. Both ATM and Chk2 are
required for p53 stabilization, but Chk2 must have another
effect on p53 function in addition to stabilization that promotes
apoptosis in response to IR (Fig. 7). Loss of Chk2 does lead to
a defect of the G1/S checkpoint, but this could be caused by
defective p53 protein stabilization. A similar explanation could
hold for the partial effect of the ATM mutation on apoptosis.
The mechanism underlying the regulation of p53 activation
remains unknown, but a key candidate is phosphorylation since
p53 is phosphorylated on many sites in response to DNA
damage. Regulation of p53 activation by Chk2 could involve
the phosphorylation of additional sites on p53 or of other
molecules that affect p53 activation. For example, the phos-
phorylation of Ser46 on p53 is specifically required for the
induction of p53AIP1, a gene inducing apoptotic cell death.
Mutation of Ser46 abolishes the ability of p53 to induce apo-
FIG. 6. Effect of reintroduced mutant Chk2 on IR-induced apo-
ptosis. Chk2?/?ES cells transfected with either empty vector (vector),
vector containing wild-type Chk2 (wild), or vector containing the Chk2
SQ/TQ mutant gene (SQ/TQ mutant) were injected into blastocysts
from Rag1?/?mice to generate chimeric animals expressing the cor-
responding proteins in thymocytes. (A) Expression of reintroduced
Chk2 protein and stabilization of p53 following 5 Gy of ?-irradiation
for the indicated times. (B) Thymocytes isolated from the chimeric
mice for which data are shown in panel A were irradiated at the
indicated doses. Apoptosis was analyzed by Annexin V-PI staining at
24 h post-IR. Each value represents the mean percentage ? standard
deviation of viable cells in 3 cultures per group.
VOL. 22, 2002 REGULATION OF p53-MEDIATED APOPTOSIS BY Chk26529
ptosis and selectively blocks transcription of p53AIP (but not
other p53 target genes) (38). These data suggest that posttrans-
lational modification of the p53 protein may determine its
affinity for specific p53 binding sequences present in different
target genes. Unfortunately, we do not know if Chk2 affects
phosphorylation of Ser46 and p53AIP1 because Ser46 on hu-
man p53 is not conserved in mouse p53. It remains possible
that other types of p53 modifications such as acetylation could
lead to apoptosis via Chk2.
Mutation of the SQ/TQ sites on Chk2 abolished p53 activa-
tion leading to apoptosis, demonstrating that the phosphory-
lation of the SQ/TQ sites in the N-terminal region of Chk2 is
essential for this process. Most of the phosphorylation of Chk2
induced by IR is abolished if ATM is absent. However, our
data suggest that there is some ATM-independent phosphor-
ylation at SQ/TQ sites of Chk2 in response to IR. It is possible
that the very low level of ATM-independent phosphorylation
of Chk2 that occurs is insufficient to alter the mobility of the
protein. The most likely agent of ATM-independent Chk2
phosphorylation is ATR, although other members of the phos-
phatidylinositol 3-kinase family are also possibilities. ATR is a
phosphatidylinositol 3-kinase-related kinase which contains a
protein kinase domain similar in sequence to a region of
Schizosaccharomyces pombe rad3 (6, 13). Matsuoka et al. have
reported that ATR phosphorylates Thr26, Ser50, and Thr68 in
the SQ/TQ cluster domain of human Chk2 in vitro (35). Al-
though ATR is believed to act primarily in response to a DNA
replication block or UV-irradiation, it is also involved in re-
sponses to IR. Cells lacking ATR die within several days of
exposure to IR; however, prior to their deaths, a profound
defect in the IR-induced G2/M checkpoint can be demon-
strated (16). Other studies have shown that overexpression of
a kinase-dead mutation of ATR causes increased sensitivity to
IR and a defect in the G2/M arrest and S-phase checkpoints
(14). Furthermore, ATR mutation also abolishes DNA dam-
age-induced phosphorylation of Ser15 on p53 (49). These data
suggest a potential functional overlap between ATM and ATR
with respect to IR responses, consistent with our hypothesis
that ATM and ATR cooperate in regulating p53 activity (Fig.
7). We theorize that ATR selectively regulates p53 activation
leading to apoptosis via Chk2, while ATM governs cell cycle
arrest in a Chk2-independent manner.
In addition to phosphorylating p53, Chk2 is known to phos-
phorylate Cdc25. It has been reported that ATM and Chk2 are
required for the S-phase checkpoint induced by IR and that
this induction depends on Chk2-dependent phosphorylation of
Cdc25A (22). In our study, we confirm that ATM is required
for the S-phase checkpoint in primary MEFs but we also dem-
onstrate that Chk2 is dispensable for this checkpoint in this cell
type. In experiments with immortalized MEFs, we have ob-
served a slightly slower onset and shorter duration of the S-
phase checkpoint in the absence of Chk2 (unpublished data).
This small difference may reflect differences in cell cycle pa-
rameters between primary and transformed cells. On balance,
however, we believe that Chk2 is not essential for S-phase
arrest in normal cells. It is possible that Chk1 can substitute for
Chk2 in the phosphorylation of Cdc25 and that Chk2 thus has
a redundant function in the intra-S-phase checkpoint. Previous
reports concluding that Chk2 was required for the intra-S-
phase checkpoint utilized overexpression constructs containing
mutated Chk2 genes identified in sporadic colon cancer and as
a germ line mutation in Li-Fraumeni syndrome (LFS). The
data showed that these mutations had a dominant-negative
impact in wild-type cells. Constructs containing such mutations
could therefore have an inhibitory effect on a downstream
component of the S-phase checkpoint (such as Cdc25), thereby
preventing the operation of any compensatory Chk1-depen-
dent process. Alternatively, differences in Chk2 dependency in
different tissues or between mice and humans may underlie the
discrepancy between the previous reports and our data.
Failures in the transcriptional response to damage, cell cycle
arrest and apoptosis induced by IR should lead to a higher
incidence of tumor development. Unexpectedly, however,
Chk2?/?mice do not have obvious tumors, unlike ATM?/?
mice, which die within 4 months of birth with thymic lympho-
mas. Interestingly, the thymic lymphomas in ATM?/?mice are
critically dependent on V(D)J recombination, whereas thymic
lymphomas in p53?/?mice arise independent of V(D)J recom-
bination (30). These observations indicate that at least two
different mechanisms of lymphoma development are at work in
these mutant animals, such that ATM-mediated p53 activation
may not be required for the development of lymphomas. ATM
phosphorylates many target substrates in addition to Chk2 and
p53, including Nbs1 and Brca1 (17, 31). These molecules,
which act downstream of ATM, may contribute in an unknown
way to the prevention of spontaneous lymphoma development.
Mutations of Chk2 are found more frequently in patients
with variant LFS, which has a moderate phenotype, than in
patients with classical LFS (5). In contrast, mutations of p53
have been reported in 70% of classical LFS cases and 20% of
variant LFS patients (50). Although the tumor-suppressive ef-
FIG. 7. Model of the regulation of p53 activation by Chk2 in re-
sponse to IR. Chk2-mediated stabilization of p53 induced by IR and
leading to apoptosis is controlled independently of ATM, possibly by
ATR. ATM appears to stabilize p53, leading to cell cycle arrest without
6530 HIRAO ET AL.MOL. CELL. BIOL.
fect of Chk2 appears to be milder than that of p53, loss of Chk2
clearly increased the incidence of DMBA-induced skin tumors
in mice. DBMA treatment resembles UV-irradiation in that
both cause DNA adducts that are repaired by the nucleotide
excision repair system. This similarity in DNA lesions suggests
that the DBMA-induced skin tumors appearing in Chk2?/?
mice could be the result of a failure in the tumor-suppressive
effect by ATR.
In conclusion, the results of this study have shown that p53
activation leading to cell cycle arrest is regulated differently
from that leading to apoptosis. Chk2 is required for p53-me-
diated apoptosis of thymocytes and must undergo phosphory-
lation in order to function, but this phosphorylation is not
carried out by ATM. We propose a new model for the IR
pathway that emphasizes the independent effects of Chk2 and
We thank Malte Peters, Atsushi Togawa, and Katsuya Tsuchihara
for helpful discussions, Peter J. McKinnon for providing ATM?/?
mice, Junichi Miyazaki for providing pCAGGS, Mary Saunders for
scientific editing, Denis Bouchard for technical expertise, and Irene Ng
for excellent administrative support.
T.W.M. was supported by grants from the Canadian Institutes of
Health Research (CIHR) and the National Cancer Institute of Canada
(NCI). S.J.E. was supported by a DAMD grant. P.A.J. was supported
by the industry-funded UKCCCR Radiation Research Programme,
the Leukemia Research Fund, and European Union grant Figh CT
1. Ahn, J. Y., J. K. Schwarz, H. Piwnica-Worms, and C. E. Canman. 2000.
Threonine 68 phosphorylation by ataxia telangiectasia mutated is required
for efficient activation of Chk2 in response to ionizing radiation. Cancer Res.
2. Banin, S., L. Moyal, S. Shieh, Y. Taya, C. W. Anderson, L. Chessa, N. I.
Smorodinsky, C. Prives, Y. Reiss, Y. Shiloh, and Y. Ziv. 1998. Enhanced
phosphorylation of p53 by ATM in response to DNA damage. Science
3. Barlow, C., K. D. Brown, C. X. Deng, D. A. Tagle, and A. Wynshaw-Boris.
1997. Atm selectively regulates distinct p53-dependent cell-cycle checkpoint
and apoptotic pathways. Nat. Genet. 17:453–456.
4. Barlow, C., S. Hirotsune, R. Paylor, M. Liyanage, M. Eckhaus, F. Collins, Y.
Shiloh, J. N. Crawley, T. Ried, D. Tagle, and A. Wynshaw-Boris. 1996.
Atm-deficient mice: a paradigm of ataxia telangiectasia. Cell 86:159–171.
5. Bell, D. W., J. M. Varley, T. E. Szydlo, D. H. Kang, D. C. Wahrer, K. E.
Shannon, M. Lubratovich, S. J. Verselis, K. J. Isselbacher, J. F. Fraumeni,
J. M. Birch, F. P. Li, J. E. Garber, and D. A. Haber. 1999. Heterozygous
germ line hCHK2 mutations in Li-Fraumeni syndrome. Science 286:2528–
6. Bentley, N. J., D. A. Holtzman, G. Flaggs, K. S. Keegan, A. DeMaggio, J. C.
Ford, M. Hoekstra, and A. M. Carr. 1996. The Schizosaccharomyces pombe
rad3 checkpoint gene. EMBO J. 15:6641–6651.
7. Blasina, A., B. D. Price, G. A. Turenne, and C. H. McGowan. 1999. Caffeine
inhibits the checkpoint kinase ATM. Curr. Biol. 9:1135–1138.
8. Bouvard, V., T. Zaitchouk, M. Vacher, A. Duthu, M. Canivet, C. Choisy-
Rossi, M. Nieruchalski, and E. May. 2000. Tissue and cell-specific expression
of the p53-target genes: bax, fas, mdm2 and waf1/p21, before and following
ionising irradiation in mice. Oncogene 19:649–660.
9. Canman, C. E., D. S. Lim, K. A. Cimprich, Y. Taya, K. Tamai, K. Sakaguchi,
E. Appella, M. B. Kastan, and J. D. Siliciano. 1998. Activation of the ATM
kinase by ionizing radiation and phosphorylation of p53. Science 281:1677–
10. Chaturvedi, P., W. K. Eng, Y. Zhu, M. R. Mattern, R. Mishra, M. R. Hurle,
X. Zhang, R. S. Annan, Q. Lu, L. F. Faucette, G. F. Scott, X. Li, S. A. Carr,
R. K. Johnson, J. D. Winkler, and B. B. Zhou. 1999. Mammalian Chk2 is a
downstream effector of the ATM-dependent DNA damage checkpoint path-
way. Oncogene 18:4047–4054.
11. Chehab, N. H., A. Malikzay, M. Appel, and T. D. Halazonetis. 2000. Chk2/
hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53.
Genes Dev. 14:278–288.
12. Chehab, N. H., A. Malikzay, E. S. Stavridi, and T. D. Halazonetis. 1999.
Phosphorylation of Ser-20 mediates stabilization of human p53 in response
to DNA damage. Proc. Natl. Acad. Sci. USA 96:13777–13782.
13. Cimprich, K. A., T. B. Shin, C. T. Keith, and S. L. Schreiber. 1996. cDNA
cloning and gene mapping of a candidate human cell cycle checkpoint pro-
tein. Proc. Natl. Acad. Sci. USA 93:2850–2855.
14. Cliby, W. A., C. J. Roberts, K. A. Cimprich, C. M. Stringer, J. R. Lamb, S. L.
Schreiber, and S. H. Friend. 1998. Overexpression of a kinase-inactive ATR
protein causes sensitivity to DNA-damaging agents and defects in cell cycle
checkpoints. EMBO J. 17:159–169.
15. Corominas, M., J. Leon, H. Kamino, M. Cruz-Alvarez, S. C. Novick, and A.
Pellicer. 1991. Oncogene involvement in tumor regression: H-ras activation
in the rabbit keratoacanthoma model. Oncogene 6:645–651.
16. Cortez, D., S. Guntuku, J. Qin, and S. J. Elledge. 2001. ATR and ATRIP:
partners in checkpoint signaling. Science 294:1713–1716.
17. Cortez, D., Y. Wang, J. Qin, and S. J. Elledge. 1999. Requirement of ATM-
dependent phosphorylation of brca1 in the DNA damage response to dou-
ble-strand breaks. Science 286:1162–1166.
18. Deng, C., P. Zhang, J. W. Harper, S. J. Elledge, and P. Leder. 1995. Mice
lacking p21CIP1/WAF1 undergo normal development, but are defective in
G1 checkpoint control. Cell 82:675–684.
19. el-Deiry, W. S. 1998. Regulation of p53 downstream genes. Semin. Cancer
20. Elledge, S. J. 1996. Cell cycle checkpoints: preventing an identity crisis.
21. Elson, A., Y. Wang, C. J. Daugherty, C. C. Morton, F. Zhou, J. Campos-
Torres, and P. Leder. 1996. Pleiotropic defects in ataxia-telangiectasia pro-
tein-deficient mice. Proc. Natl. Acad. Sci. USA 93:13084–13089.
22. Falck, J., N. Mailand, R. G. Syljuasen, J. Bartek, and J. Lukas. 2001. The
ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA
synthesis. Nature 410:842–847.
23. Giaccia, A. J., and M. B. Kastan. 1998. The complexity of p53 modulation:
emerging patterns from divergent signals. Genes Dev. 12:2973–2983.
24. Herzog, K. H., M. J. Chong, M. Kapsetaki, J. I. Morgan, and P. J. McKin-
non. 1998. Requirement for Atm in ionizing radiation-induced cell death in
the developing central nervous system. Science 280:1089–1091.
25. Higashimoto, Y., S. Saito, X. H. Tong, A. Hong, K. Sakaguchi, E. Appella,
and C. W. Anderson. 2000. Human p53 is phosphorylated on serines 6 and
9 in response to DNA damage-inducing agents. J. Biol. Chem. 275:23199–
26. Hirao, A., Y. Y. Kong, S. Matsuoka, A. Wakeham, J. Ruland, H. Yoshida, D.
Liu, S. J. Elledge, and T. W. Mak. 2000. DNA damage-induced activation of
p53 by the checkpoint kinase Chk2. Science 287:1824–1827.
27. Jaspers, N. G., R. A. Gatti, C. Baan, P. C. Linssen, and D. Bootsma. 1988.
Genetic complementation analysis of ataxia telangiectasia and Nijmegen
breakage syndrome: a survey of 50 patients. Cytogenet. Cell Genet. 49:259–
28. Lambert, P. F., F. Kashanchi, M. F. Radonovich, R. Shiekhattar, and J. N.
Brady. 1998. Phosphorylation of p53 serine 15 increases interaction with
CBP. J. Biol. Chem. 273:33048–33053.
29. Levine, A. J. 1997. p53, the cellular gatekeeper for growth and division. Cell
30. Liao, M. J., and T. Van Dyke. 1999. Critical role for Atm in suppressing
V(D)J recombination-driven thymic lymphoma. Genes Dev. 13:1246–1250.
31. Lim, D. S., S. T. Kim, B. Xu, R. S. Maser, J. Lin, J. H. Petrini, and M. B.
Kastan. 2000. ATM phosphorylates p95/nbs1 in an S-phase checkpoint path-
way. Nature 404:613–617.
32. Lindsten, T., A. J. Ross, A. King, W. X. Zong, J. C. Rathmell, H. A. Shiels,
E. Ulrich, K. G. Waymire, P. Mahar, K. Frauwirth, Y. Chen, M. Wei, V. M.
Eng, D. M. Adelman, M. C. Simon, A. Ma, J. A. Golden, G. Evan, S. J.
Korsmeyer, G. R. MacGregor, and C. B. Thompson. 2000. The combined
functions of proapoptotic Bcl-2 family members bak and bax are essential for
normal development of multiple tissues. Mol. Cell 6:1389–1399.
33. Lomaga, M. A., J. T. Henderson, A. J. Elia, J. Robertson, R. S. Noyce, W. C.
Yeh, and T. W. Mak. 2000. Tumor necrosis factor receptor-associated factor
6 (TRAF6) deficiency results in exencephaly and is required for apoptosis
within the developing CNS. J. Neurosci. 20:7384–7393.
34. Matsuoka, S., M. Huang, and S. J. Elledge. 1998. Linkage of ATM to cell
cycle regulation by the Chk2 protein kinase. Science 282:1893–1897.
35. Matsuoka, S., G. Rotman, A. Ogawa, Y. Shiloh, K. Tamai, and S. J. Elledge.
2000. Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in
vitro. Proc. Natl. Acad. Sci. USA 97:10389–10394.
36. Melchionna, R., X. B. Chen, A. Blasina, and C. H. McGowan. 2000. Thre-
onine 68 is required for radiation-induced phosphorylation and activation of
Cds1. Nat. Cell Biol. 2:762–765.
37. Meyn, M. S. 1995. Ataxia-telangiectasia and cellular responses to DNA
damage. Cancer Res. 55:5991–6001.
38. Oda, K., H. Arakawa, T. Tanaka, K. Matsuda, C. Tanikawa, T. Mori, H.
Nishimori, K. Tamai, T. Tokino, Y. Nakamura, and Y. Taya. 2000. p53AIP1,
a potential mediator of p53-dependent apoptosis, and its regulation by Ser-
46-phosphorylated p53. Cell 102:849–862.
39. Prives, C. 1998. Signaling to p53: breaking the MDM2-p53 circuit. Cell
40. Quintanilla, M., K. Brown, M. Ramsden, and A. Balmain. 1986. Carcinogen-
VOL. 22, 2002REGULATION OF p53-MEDIATED APOPTOSIS BY Chk26531
specific mutation and amplification of Ha-ras during mouse skin carcinogen- Download full-text
esis. Nature 322:78–80.
41. Sakaguchi, K., J. E. Herrera, S. Saito, T. Miki, M. Bustin, A. Vassilev, C. W.
Anderson, and E. Appella. 1998. DNA damage activates p53 through a
phosphorylation-acetylation cascade. Genes Dev. 12:2831–2841.
42. Shieh, S. Y., J. Ahn, K. Tamai, Y. Taya, and C. Prives. 2000. The human
homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at
multiple DNA damage-inducible sites. Genes Dev. 14:289–300.
43. Shieh, S. Y., M. Ikeda, Y. Taya, and C. Prives. 1997. DNA damage-induced
phosphorylation of p53 alleviates inhibition by MDM2. Cell 91:325–334.
44. Shieh, S. Y., Y. Taya, and C. Prives. 1999. DNA damage-inducible phos-
phorylation of p53 at N-terminal sites including a novel site, Ser20, requires
tetramerization. EMBO J. 18:1815–1823.
45. Shiloh, Y. 1995. Ataxia-telangiectasia: closer to unraveling the mystery. Eur.
J. Hum. Genet. 3:116–138.
46. Shiloh, Y. 2001. ATM and ATR: networking cellular responses to DNA
damage. Curr. Opin. Genet. Dev. 11:71–77.
47. Siliciano, J. D., C. E. Canman, Y. Taya, K. Sakaguchi, E. Appella, and M. B.
Kastan. 1997. DNA damage induces phosphorylation of the amino terminus
of p53. Genes Dev. 11:3471–3481.
48. Song, S., and P. F. Lambert. 1999. Different responses of epidermal and hair
follicular cells to radiation correlate with distinct patterns of p53 and p21
induction. Am. J. Pathol. 155:1121–1127.
49. Tibbetts, R. S., K. M. Brumbaugh, J. M. Williams, J. N. Sarkaria, W. A.
Cliby, S. Y. Shieh, Y. Taya, C. Prives, and R. T. Abraham. 1999. A role for
ATR in the DNA damage-induced phosphorylation of p53. Genes Dev.
50. Varley, J. M., G. McGown, M. Thorncroft, M. F. Santibanez-Koref, A. M.
Kelsey, K. J. Tricker, D. G. Evans, and J. M. Birch. 1997. Germ-line muta-
tions of TP53 in Li-Fraumeni families: an extended study of 39 families.
Cancer Res. 57:3245–3252.
51. Westphal, C. H., S. Rowan, C. Schmaltz, A. Elson, D. E. Fisher, and P.
Leder. 1997. atm and p53 cooperate in apoptosis and suppression of tumor-
igenesis, but not in resistance to acute radiation toxicity. Nat. Genet. 16:397–
52. Xu, Y., T. Ashley, E. E. Brainerd, R. T. Bronson, M. S. Meyn, and D.
Baltimore. 1996. Targeted disruption of ATM leads to growth retardation,
chromosomal fragmentation during meiosis, immune defects, and thymic
lymphoma. Genes Dev. 10:2411–2422.
53. Xu, Y., and D. Baltimore. 1996. Dual roles of ATM in the cellular response
to radiation and in cell growth control. Genes Dev. 10:2401–2410.
54. Zhao, R., K. Gish, M. Murphy, Y. Yin, D. Notterman, W. H. Hoffman, E.
Tom, D. H. Mack, and A. J. Levine. 2000. Analysis of p53-regulated gene
expression patterns using oligonucleotide arrays. Genes Dev. 14:981–993.
55. Zhou, B. B., P. Chaturvedi, K. Spring, S. P. Scott, R. A. Johanson, R.
Mishra, M. R. Mattern, J. D. Winkler, and K. K. Khanna. 2000. Caffeine
abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting
ataxia-telangiectasia-mutated kinase activity. J. Biol. Chem. 275:10342–
56. Zhou, B. B., and S. J. Elledge. 2000. The DNA damage response: putting
checkpoints in perspective. Nature 408:433–439.
6532HIRAO ET AL.MOL. CELL. BIOL.