RESEARCH COMMUNICATION
ATR signaling can drive cells
into senescence in the absence
of DNA breaks
Luis I. Toledo, Matilde Murga,
Paula Gutierrez-Martinez, Rebeca Soria,
and Oscar Fernandez-Capetillo1
Genomic Instability Group, Spanish National Cancer
Research Center (CNIO), Madrid 28029, Spain
The ATR kinase is a key transducer of “replicative
stress,” the type of genomic damage that has been pos-
tulated to be induced by oncogenes. Here we describe a
cellular system in which we can unleash ATR activity at
will, in the absence of any actual damage or additional
signaling pathways triggered by DNA breaks. We dem-
onstrate that activating ATR is sufficient to promote cell
cycle arrest and, if persistent, triggers p53-dependent but
Ink4a/ARF-independent senescence. Moreover, we show
that an ectopic activation of ATR leads to a G1/S arrest
in ATM−/− cells, providing the first evidence of func-
tional complementation of ATM deficiency by ATR.
Our system provides a novel platform for the study of the
specific functions of ATR signaling and adds evidence for
the tumor-suppressive potential of the DNA damage re-
sponse.
Supplemental material is available at http://www.genesdev.org.
Received August 10, 2007; revised version accepted
November 30, 2007.
Three members of the PIKK family of protein kinases
(ATM, ATR, and DNA-PKcs) have been shown to be ac-
tivated in response to DNA double-strand breaks (DSBs)
(Abraham 2004). Among this group, the role of DNA-
PKcs seems to be limited to the repair of DSBs through
nonhomologous end-joining, which it stimulates
through the phosphorylation of targets at the break site
(Weterings and van Gent 2004). ATM and ATR also
stimulate DNA repair (Kuhne et al. 2004; Wang et al.
2004), but are additionally involved in regulating the cell
cycle in response to DNA damage. Moreover, in the case
of ATM and ATR, the transmission of the signal is fur-
ther amplified by the subsequent activation of Chk1 and
Chk2 kinases, which increases the spectrum and/or
amount of phoshorylation events triggered by DSBs
(Matsuoka et al. 1998; Guo et al. 2000; Liu et al. 2000).
This rapid phosphorylation-based transduction cascade
is generally known as the DNA damage response (DDR).
The function of the DDR is to delay cell cycle progres-
sion until damage is repaired, or to promote apoptosis
depending on the cell type and amount of damage. If
damage persists, the arrest can become permanent, lead-
ing the cells to an irreversibly quiescent state known as
senescence. At the organism level, cellular senescence
has been recently shown to constitute a strong anti-tu-
moral barrier, which is activated early on during the ini-
tial steps of malignant transformation (Braig et al. 2005;
Chen et al. 2005; Collado et al. 2005; Michaloglou et al.
2005). At the molecular level, all instances in which se-
nescence has been observed in vivo are associated with
an activation of the DDR, including oncogene activation
(Bartkova et al. 2006; Di Micco et al. 2006), telomere
attrition (Satyanarayana et al. 2003), or treatments with
DSB-inducing agents (te Poele et al. 2002). Thus, regard-
less of the context, the presence of an activated DDR
seems to be a common initiating signal that drives se-
nescence.
Oncogene-induced senescence has been recently pro-
posed to be due to generation of replicative damage (Bart-
kova et al. 2006; Di Micco et al. 2006), which should
impinge mainly on ATR rather than ATM. However,
performing clean genetic experiments on ATR has been
hampered by its essential role during replication, leading
to rapid cellular lethality in knockout mice or human
tumoral lines (Brown and Baltimore 2000; Cortez et al.
2001). The essential role of the pathway is further un-
derscored by the fact that no ATR- or Chk1-deficient
cells have been ever encountered in tumors. In this con-
text, our aim was to design a system in which we could
specifically activate ATR in the absence of DNA dam-
age, so that we could interrogate the contribution of this
pathway in the absence of additional signaling cascades
that are triggered by genomic lesions.
Results and Discussion
Based on the recent finding that a unique domain of
TopBP1 is able to stimulate the kinase activity of ATR
(Kumagai et al. 2006), we devised a retroviral system in
which the intracellular localization of this ATR-stimu-
lating fragment (AD*: amino acids 978–1286 of the hu-
man protein) could be controlled by 4-hydroxytamoxifen
(OHT) (Fig. 1A). To minimize the contribution of unex-
pected mutations found in cell lines, we first started our
analyses by infecting early-passage mouse embryonic fi-
broblasts (MEF). As expected, the chimeric protein
(TopBP1ER from now on) was kept cytoplasmic until the
addition of OHT, when it translocated to the nucleus
(Fig. 1B). In order to verify whether the nuclear translo-
cation of TopBP1ER was sufficient to promote the phos-
phorylation of targets of ATR, we first analyzed the dis-
tribution of the phosporylated isoform of histone H2AX
(
H2AX), a well-characterized target of the DDR kinases
(Burma et al. 2001; Ward and Chen 2001). Notably,
whereas small amounts of 
H2AX can be detected in
proliferating MEF, treatment of TopBP1ER-infected cells
with OHT led to a massive pan-nuclear phosphorylation
of H2AX (Fig. 1C, Supplemental Fig. S1). Moreover, the
level of 
H2AX observed per cell correlated with that of
GFP, indicating that the amount of DDR signaling was
proportional to that of TopBP1ER (data not shown).
Equivalent observations were made with other targets of
the DDR kinases such as SMC1 (Fig. 1D). Neither unin-
fected MEF treated with OHT nor TopBP1ER-infected
MEF that had not been treated with tamoxifen presented
[Keywords: ATR; DNA damage response; senescence; ARF; p53]
1Corresponding author.
E-MAIL ofernandez@cnio.es; FAX 34-91-7328033.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.452308.
GENES & DEVELOPMENT 22:297–302 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org 297
 Cold Spring Harbor Laboratory Press on February 9, 2024 - Published by genesdev.cshlp.orgDownloaded from 
the same staining pattern (Fig. 1C,E). Western blot analy-
sis led to equivalent observations (Fig. 1F). The phos-
phorylation of DDR targets was noticeable as early as 30
min after OHT addition, reaching a maximum at 24 h
post-treatment (data not shown). Of note, the phosphor-
ylation of direct ATR targets such as H2AX or Rad17
(Bao et al. 2001; Ward and Chen 2001) was more notice-
able than that of ATM targets like SMC1 (Kim et al.
2002; Yazdi et al. 2002). Along these lines, TopBP1ER-
induced H2AX phosphorylation was not affected by
ATM absence but was abrogated in ATR-depleted cells
(Supplemental Fig. S2), confirming the strict specificity
of the system for ATR. In summary, controlling the nu-
cleo/cytoplasmic localization of an activating domain of
TopBP1 provides a molecular switch by which the ki-
nase activity of ATR can be activated at will.
The fact that the fragment of TopBP1 used in this
study was able to stimulate ATR activity in vitro (Kuma-
gai et al. 2006) made it unlikely that our findings were
related to the generation of DNA damage. In order to
prove that this was the case, the following experiments
were performed. First, along with the fact that 
H2AX
signal is distributed throughout the nucleus, the analysis
of proteins that localize into foci at sites of DSBs such as
53BP1 or MDC1 did not show any focus formation after
the treatment with OHT (Fig. 2A; data not
shown). Second, and providing direct evidence
of the absence of DSBs, both neutral and alka-
line comet assays failed to detect any signs of
DNA breakage induced by TopBP1ER translo-
cation (Fig. 2B; Supplemental Fig. S3). Of note,
the amount of signaling induced by this sys-
tem was equivalent to that induced by 5 Gy of
ionizing radiation (IR) (Fig. 2C), which with
the available estimates should lead to >180
DSBs (Rothkamm and Lobrich 2003), and
should thus be readily detectable by any of the
used methodologies. Third, the activation of
ATR was not associated with any detectable
increase in the amount of RPA-coated ssDNA
regions (Fig. 2D; Supplemental Fig. S3). Hence,
the activation of ATR following the nuclear
translocation of TopBP1ER occurs in the ab-
sence of DNA damage.
To evaluate whether the activation of ATR
affected cell cycle progression, the growth of
TopBP1ER-infected MEF was evaluated in
the presence or absence of OHT. Whereas
the treatment with tamoxifen did not alter
the proliferation of wild-type MEF, it dramati-
cally reduced the growth of TopBP1ER-infected
cells (Fig. 3A). To determine whether this
arrest recapitulates a bona fide DDR check-
point, we analyzed the cell cycle distribu-
tion of TopBP1ER-infected cells. Addition of
OHT led to a rapid arrest at both G1 and G2,
which is comparable with that elicited by ion-
izing radiation (Fig. 3B,C). Importantly, an
equivalent G1/S arrest could be observed in
ATM-deficient MEF that had been infected
with TopBP1ER, once again consistent with
the fact that TopBP1 specifically activates
ATR rather than ATM (Fig. 3C). Notably,
these data provide the first experimental evi-
dence of functional complementation of ATM
deficiency by ATR. Nevertheless, p53-defi-
cient MEF could not be arrested by TopBP1ER transloca-
tion, which is in agreement with the critical role of p53
in the activation of the G1/S checkpoint (Fig. 3C; Kastan
et al. 1991). The analysis of cellular proliferation reca-
pitulated the ATM-independent and p53-dependent ef-
fect of TopBP1ER translocation on the growth of primary
cells (Fig. 3D). In summary, an ectopic activation of ATR
leads to a growth arrest that shows the same genetic
dependencies as the checkpoints induced by DNA dam-
age.
Recent data have suggested that oncogene-induced se-
nescence is linked to an activation of the DDR due to the
generation of “replicative stress” (Bartkova et al. 2006;
Di Micco et al. 2006). In particular, oncogene activation
would constitute a persistent source of replicative stress
that, among other things, activates ATR. We thus evalu-
ated whether a continuous activation of ATR, in the ab-
sence of additional pathways that are activated by onco-
genes, has the capability to induce senescence. Whereas
no induction of senescence could be detected in the first
48 h, both the size of the cells and nuclei (Supplemental
Fig. S4) as well as the percentage of senescence-associ-
ated -galactosidase-positive cells (Fig. 3E,F) dramati-
cally increased after 72 h of continuous OHT treatment.
In contrast to replicative senescence, which has been
Figure 1. Description of an inducible ATR system. (A) Scheme of the retroviral
construct used in this study. (B) OHT treatment translocates TopBP1ER to the
nucleus. The localization of TopBP1ER was visualized with an ER-recognizing an-
tibody (red). (C) Pan-nuclear H2AX phosphorylation in response to OHT treatment.
GFP identifies infected cells. Note the lack of response in uninfected cells (white
arrows). (D) Coincident phosphorylation of H2AX and SMC1 in response to OHT.
(E) HT microscopy-mediated quantification of the amount of 
H2AX per individual
nucleus in infected or uninfected MEF upon OHT treatment. (F) Western blot analy-
sis of SMC1, H2AX, and Rad17 phosphorylation in TopBP1ER-infected or uninfected
MEF. Conditions: control (C), hydroxyurea: 2 mM, 3 h (HU), and (IR) 10 Gy. OHT
was used at 500 nM throughout the study and for 4 h in this figure.
Toledo et al.
298 GENES & DEVELOPMENT
 Cold Spring Harbor Laboratory Press on February 9, 2024 - Published by genesdev.cshlp.orgDownloaded from 
associated with the accumulation of nonrepairable DSBs
(Sedelnikova et al. 2004), no 53BP1 foci could be ob-
served in TopBP1ER-infected MEF (Supplemental Fig.
S4). Thus, a persistent activation of ATR, even if no dam-
age is present, leads to senescence in primary cells.
The observation that the DDR is specifically activated
in early precancerous lesions in humans led to the pro-
posal that the DDR could constitute a strong initial bar-
rier against malignant transformation (Bartkova et al.
2005; Gorgoulis et al. 2005). However, in addition to ac-
tivating the DDR, oncogenic stress leads to a robust ac-
tivation of the ink4a/arf locus, which is also capable of
inducing senescence (Serrano et al. 1997). Importantly,
recent reports have challenged the contribution of the
DDR in the tumor suppression mediated by p53, which
would be entirely mediated by ARF (Christophorou et al.
2006; Efeyan et al. 2006). However, it is conceivable that
the type and persistence of damage used in these studies
might not reflect that induced by oncogenes in vivo. To
directly evaluate whether the DDR has the potential to
induce senescence independently of ARF, the behavior of
Ink4a−/−/ARF−/− MEF infected with our TopBP1ER sys-
tem was evaluated. In contrast to p53-deficient MEF,
Ink4a/ARF-deficient cells arrested and entered senescent
in response to continuous ATR activation (Fig. 3F;
Supplemental Fig. S5). Thus, our data indicate that p53-
dependent but ARF-independent senescence can be
achieved solely by activation of ATR, thereby reinforc-
ing the view of the DDR as a potential tumor-suppres-
sive barrier.
To avoid the heterogeneity in the strength of ATR
activation obtained by infection and to test whether
the system is able to induce senescence in immortalized
lines that are more refractory to senescence than MEF,
stable transfectants of TopBP1ER were obtained from
NIH3T3 cells. The experiments performed on the
isolated clone (3T3T-ER, from now on) recapitulated all
of our previous findings. At the molecular
level, the treatment with tamoxifen led to a
robust phosphorylation of targets of the DDR
kinases such as H2AX, Chk1, SMC1, and
Rad17 (Fig. 4A). Of note, TopBP1ER transloca-
tion did not induce the phosphorylation of
Chk2, which again is consistent with the
strict specificity of the system for ATR. Fur-
thermore, the depletion of factors that are spe-
cifically required for ATR activity, such as
ATRIP (Cortez et al. 2001), severely dimin-
ished the response to tamoxifen (Supplemen-
tal Fig. S6). At the cellular level, the addition
of tamoxifen was associated with a rapid acti-
vation of cell cycle checkpoints. Importantly,
depletion of Chk1 or treatment with the
Chk1-specific inhibitor UCN-01 impaired the
OHT-induced G2/M arrest, as proof that this
arrest was dependent on Chk1 activity (Fig.
4B; Supplemental Fig. S7). Whereas additional
signaling pathways emanating from DSBs
might be important in modulating the DDR,
our analysis provides formal proof that activa-
tion of ATR is sufficient to trigger a Chk1-
dependent cell cycle arrest. As a consequence
of the arrest, the addition of OHT led to a
sharp inhibition of cell proliferation and the
subsequent appearance of senescence (Fig.
4C,D).
We finally tested whether our system has the potential
to promote senescence in cell lines established from hu-
man cancer samples. To this extent, TopBP1ER-express-
ing clones of MCF7 cells were obtained. Once more, the
findings made in MCF7T-ER cells were equivalent to our
previous observations including the phosphorylation of
DDR targets, growth arrest, and, importantly, senes-
cence (Fig. 4E–G). Interestingly, ectopic ATR activation
did not lead to any detectable ubiquitinylation of
FANCD2, a previously reported ATR-dependent event
(Andreassen et al. 2004), which might suggest that DDR-
independent ubiquitin ligase activities are activated by
DNA damage (Supplemental Fig. S8). Finally, and in
agreement with our previous findings, the cell cycle pro-
file of senescent cells showed that cells had arrested at
both G1/S and G2/M boundaries (data not shown). In-
deed, high-throughput (HT) microscopy-mediated analy-
sis of 
H2AX and cyclin A nuclear levels confirmed that
OHT-induced H2AX phosphorylation was also detect-
able in G1 cells (Supplemental Fig. S9). Whereas we can-
not discard that additional layers of regulation of ATR
activity might exist in vivo, our data demonstrate that
promoting the interaction between TopBP1 and ATR is
sufficient to trigger ATR activity throughout the cell
cycle.
Besides p53, and in human cells in particular, the ret-
inoblastoma (Rb) tumor suppressor is a critical mediator
of senescence (Shay et al. 1991; Lowe and Sherr 2003). In
this context, whereas the phosphorylation of DDR tar-
gets occurred very rapidly, no dephosphorylation of Rb
was noticeable until at least 48 h of treatment. This de-
lay might reflect the amount of DDR signaling necessary
to achieve full inactivation of CDK activity and there-
fore the transition from cell cycle arrest to cell cycle exit.
Consistent with this, the induction of senescence started
to accumulate only after 2 or 3 d of continuous exposure
to OHT (Supplemental Fig. S10; data not shown), reach-
Figure 2. TopBP1ER translocation generates no damage. (A) Distribution of

-H2AX and 53BP1 localization in OHT-treated cells. (Insets represent a larger
image of a single nucleus). IR-induced 53BP1 foci are shown as control. (B) Neutral
and alkaline comet assay analysis in TopBP1ER-infected MEF exposed to OHT
(8 h) or to IR (10 Gy). At least 200 cells were counted for the analysis. (C) Compari-
son of H2AX phosphorylation levels between those obtained by OHT treatment or
exposure to increasing doses of IR. (D) HT-microscopy-mediated quantification of
chromatin-bound RPA-positive cells in TopBP1ER-infected MEF upon exposure to
HU (2 mM, 3 h) or OHT. Unless otherwise indicated, OHT was used for 4 h in all
panels.
Senescence by ATR activation
GENES & DEVELOPMENT 299
 Cold Spring Harbor Laboratory Press on February 9, 2024 - Published by genesdev.cshlp.orgDownloaded from 
ing its maximum after 1 wk of treatment. Remarkably,
the induction of senescence was accompanied by the ap-
pearance of senescence-associated heterochromatin foci
(SAHF), which are hallmarks of Rb-induced senescence
in human cells (Fig. 4H; Supplemental Fig. S10; Narita et
al. 2003). Furthermore, MCF7 cells present a silenced
Ink4a/ARF locus (data not shown), which demonstrates
that not only senescence but also DDR-induced SAHF
arise independently of Ink4a/ARF.
In summary, whereas several signaling cascades are
initiated by oncogenic stress or DNA damage, our data
demonstrate that ATR activation is sufficient to pro-
mote cell cycle arrest and, if persistent, to activate the
secondary pathways necessary to make the transition
into senescence (see model in Supplemental Fig. S11).
Additionally, it is tempting to propose that this approach
harbors the potential to develop into a nongenotoxic
prosenescence therapy, which might be particularly use-
ful in the case of ARF-deficient tumors that keep an in-
tact p53 gene. In any case, our data provide experimental
support for the tumor-suppressive potential of the DDR,
which occurs through p53-dependent activities that are
unlinked from Ink4a/ARF.
Materials and methods
Cell lines
MEF were isolated from 12.5-d-post-coitum embryos. p53-,
Ink4a/ARF-, and ATM-deficient mice have been described
before (Donehower et al. 1992; Barlow et al. 1996; Serrano
et al. 1996). P0/P1 MEF were used for the infections. NIH/
3T3 and MCF7 cell lines were obtained from the American
Type Culture Collection.
DNA transduction and generation of clones
TopBP1ER was constructed using the pMX-PIE (kind gift of
Dr. Andre Nussenzweig) vector as a backbone. All cell lines
were infected by standard procedures, selected with 2 mg/
mL puromycin for 2 d, and subsequently sorted in a
FACSAria (BD Biosciences) for GFP expression. NIH/3T3
and MCF7 clones were generated by limiting dilution of
parental infected lines.
RNAi
Three predesigned siRNA targeting oligos targeting Chk1
(or control duplexes) were purchased (Ambion, Inc.). Cells
at 30% confluency were transfected with the oligos at a
concentration of 50 nM using DharmaFECT transfection
reagent (Dharmacon). Analyses were performed 3 d after
transfection. Results were confirmed by at least two inde-
pendent siRNA duplexes.
Immunofluorescence and immunoblotting
The following primary antibodies were used in this study:

H2AX, Chk2, and H3K93xMe (Upstate Biotechnology);
53BP1 (Novus Biologicals); Chk1-S345P and Rad17-S645P
(Cell Signaling); Chk1 (Novocastra); SMC1-957P and H2AX
(Abcam); Smc1 (Bethyl); HP1
 (Chemicon); RPA (Lab Vi-
sion Corporation); Rb (BD-Pharmingen); -actin (Sigma-Al-
drich); and FANCD2 and CyclinA (Santa Cruz Biotechnol-
ogy). For immunofluorescence, secondary antibodies conju-
gated with Alexa 488, Alexa 594, or Alexa 647 (Molecular
Probes) were used. Image acquisition was done at room
temperature in a Zeiss Imager Z1 fluorescence microscope
with Apotome technology, using oil as an immersion me-
dia, an ORCA 1394 camera (Hamamatsu), and a 40× HCX
PL APO-0.75 N.A. objective. The HT-microscopy-mediated
analysis of the DDR has been described before (Murga et al.
2007). Briefly, cells were grown on µCLEAR-bottom 96-
well dishes (Greiner Bio-One) and analyzed on a BD Path-
way 855 BioImager (Beckton Dickinson). Image analysis
was performed with the AttoVision software (Beckton
Dickinson). All the images for quantitative analyses were acquired under
nonsaturating exposure conditions. All Western analyses shown in this
study were performed on the LICOR platform (Biosciences), which al-
lows linearly quantitative Western blots with the use of Alexa 680- and
Alexa 800-conjugated secondary antibodies (Molecular Probes).
Cell cycle arrest
G1/S checkpoint analyses were performed using standard procedures.
Briefly, cells were given a 60-min pulse of BrdU (3 µg/mL) at the end of
each of the treatments. Fixed cells were stained with a monoclonal anti-
BrdU-FITC antibody (BD-Pharmingen) and propidium iodide and ana-
lyzed by flow cytometry. For G2/M checkpoint analyses, fixed cells were
stained with an H3S10P antibody (Upstate Biotechnology) and propidium
iodide and analyzed by flow cytometry.
Cell proliferation and senescence
For proliferation measurements, cells were seeded at equal confluence at
day 0 and treated with OHT for different days. On the last day, cells were
trypsinized and counted in a Neubauer chamber. Senescence was evalu-
ated by a SA--galactosidase staining kit (Cell Signaling). For quantifica-
tions, senescent and nonsenescent cells were manually differentiated
according to shape and SA--gal staining.
Comet assay
Alkaline and neutral comet assays were performed with the Comet As-
say Kit (Trevigen) following the manufacturer’s instructions.
Figure 3. Cellular responses to ATR activation in MEF. (A) Proliferation of
TopBP1ER-infected or uninfected primary MEF in response to OHT. (B) Flow cytom-
etry profiles showing DNA content (propidium iodide, X-axis) and replication (BrdU,
Y-axis) of TopBP1ER-infected MEF exposed to OHT (8 h). (C) A similar analysis to the
one shown in B was performed in wild-type, ATM−/−, and p53−/− MEF. (D) Prolifera-
tion of wild-type, ATM−/−, and p53−/− MEF exposed to OHT for 2, 4, or 6 d. Data are
represented as the fraction of cells relative to the number of cells counted in the
absence of OHT. (E) SA--galactosidase staining (blue) in TopBP1ER-infected MEF
maintained either with or without OHT for 4 d. Black bar, 10 µm. (F) Proliferation
and senescence rates of wild-type, Ink4a−/−/ARF−/−, and p53−/− MEF upon a week of
culture in the presence of OHT.
Toledo et al.
300 GENES & DEVELOPMENT
 Cold Spring Harbor Laboratory Press on February 9, 2024 - Published by genesdev.cshlp.orgDownloaded from 
Acknowledgments
We thank Drs. M. Serrano and A. Nussenzweig for critical comments on
the manuscript. M.M. is supported by a Ramón y Cajal contract from the
Spanish Ministry of Science and Education (RYC-2003-002731) and from
a grant from Fondo de Investigaciones Sanitarias (PI05945). Work in
O.F.’s laboratory is supported by grants from the Spanish Ministry of
Science (RYC-2003-002731 and BFU2005-09429), Swiss Bridge, and Epi-
genome Network of Excellence (EU-FP6).
References
Abraham, R.T. 2004. PI 3-kinase related kinases: ‘Big’ players in stress-
induced signaling pathways. DNA Repair (Amst.) 3: 883–887.
Andreassen, P.R., D’Andrea, A.D., and Taniguchi, T. 2004. ATR couples
FANCD2 monoubiquitination to the DNA-damage response. Genes
& Dev. 18: 1958–1963.
Bao, S., Tibbetts, R.S., Brumbaugh, K.M., Fang, Y., Richardson,
D.A., Ali, A., Chen, S.M., Abraham, R.T., and Wang, X.F.
2001. ATR/ATM-mediated phosphorylation of human
Rad17 is required for genotoxic stress responses. Nature
411: 969–974.
Barlow, C., Hirotsune, S., Paylor, R., Liyanage, M., Eckhaus, M.,
Collins, F., Shiloh, Y., Crawley, J.N., Ried, T., Tagle, D., et
al. 1996. Atm-deficient mice: A paradigm of ataxia telangi-
ectasia. Cell 86: 159–171.
Bartkova, J., Horejsi, Z., Koed, K., Kramer, A., Tort, F., Zieger,
K., Guldberg, P., Sehested, M., Nesland, J.M., Lukas, C., et
al. 2005. DNA damage response as a candidate anti-cancer
barrier in early human tumorigenesis. Nature 434: 864–870.
Bartkova, J., Rezaei, N., Liontos, M., Karakaidos, P., Kletsas, D.,
Issaeva, N., Vassiliou, L.V., Kolettas, E., Niforou, K.,
Zoumpourlis, V.C., et al. 2006. Oncogene-induced senes-
cence is part of the tumorigenesis barrier imposed by DNA
damage checkpoints. Nature 444: 633–637.
Braig, M., Lee, S., Loddenkemper, C., Rudolph, C., Peters, A.H.,
Schlegelberger, B., Stein, H., Dorken, B., Jenuwein, T., and
Schmitt, C.A. 2005. Oncogene-induced senescence as an
initial barrier in lymphoma development. Nature 436: 660–
665.
Brown, E.J. and Baltimore, D. 2000. ATR disruption leads to
chromosomal fragmentation and early embryonic lethality.
Genes & Dev. 14: 397–402.
Burma, S., Chen, B.P., Murphy, M., Kurimasa, A., and Chen,
D.J. 2001. ATM phosphorylates histone H2AX in response
to DNA double-strand breaks. J. Biol. Chem. 276: 42462–
42467.
Chen, Z., Trotman, L.C., Shaffer, D., Lin, H.K., Dotan, Z.A.,
Niki, M., Koutcher, J.A., Scher, H.I., Ludwig, T., Gerald, W.,
et al. 2005. Crucial role of p53-dependent cellular senes-
cence in suppression of Pten-deficient tumorigenesis. Na-
ture 436: 725–730.
Christophorou, M.A., Ringshausen, I., Finch, A.J., Swigart, L.B.,
and Evan, G.I. 2006. The pathological response to DNA
damage does not contribute to p53-mediated tumour sup-
pression. Nature 443: 214–217.
Collado, M., Gil, J., Efeyan, A., Guerra, C., Schuhmacher, A.J.,
Barradas, M., Benguria, A., Zaballos, A., Flores, J.M., Bar-
bacid, M., et al. 2005. Tumour biology: Senescence in pre-
malignant tumours. Nature 436: 642.
Cortez, D., Guntuku, S., Qin, J., and Elledge, S.J. 2001. ATR and
ATRIP: Partners in checkpoint signaling. Science 294:
1713–1716.
Di Micco, R., Fumagalli, M., Cicalese, A., Piccinin, S., Gaspa-
rini, P., Luise, C., Schurra, C., Garre, M., Nuciforo, P.G.,
Bensimon, A., et al. 2006. Oncogene-induced senescence is
a DNA damage response triggered by DNA hyper-replica-
tion. Nature 444: 638–642.
Donehower, L.A., Harvey, M., Slagle, B.L., McArthur, M.J.,
Montgomery Jr., C.A., Butel, J.S., and Bradley, A. 1992. Mice
deficient for p53 are developmentally normal but suscep-
tible to spontaneous tumours. Nature 356: 215–221.
Efeyan, A., Garcia-Cao, I., Herranz, D., Velasco-Miguel, S., and
Serrano, M. 2006. Tumour biology: Policing of oncogene
activity by p53. Nature 443: 159.
Gorgoulis, V.G., Vassiliou, L.V., Karakaidos, P., Zacharatos, P., Kotsinas,
A., Liloglou, T., Venere, M., Ditullio Jr., R.A., Kastrinakis, N.G.,
Levy, B., et al. 2005. Activation of the DNA damage checkpoint and
genomic instability in human precancerous lesions. Nature 434: 907–
913.
Guo, Z., Kumagai, A., Wang, S.X., and Dunphy, W.G. 2000. Requirement
for Atr in phosphorylation of Chk1 and cell cycle regulation in re-
sponse to DNA replication blocks and UV-damaged DNA in Xenopus
egg extracts. Genes & Dev. 14: 2745–2756.
Kastan, M.B., Onyekwere, O., Sidransky, D., Vogelstein, B., and Craig,
R.W. 1991. Participation of p53 protein in the cellular response to
DNA damage. Cancer Res. 51: 6304–6311.
Kim, S.T., Xu, B., and Kastan, M.B. 2002. Involvement of the cohesin
protein, Smc1, in Atm-dependent and independent responses to DNA
damage. Genes & Dev. 16: 560–570.
Figure 4. ATR-induced senescence in immortalized and cancer cells. (A) West-
ern blot analysis of SMC1, Chk1, Chk2, H2AX, and Rad17 phosphorylation in
3T3T-ER cells. (HU) Hydroxyurea: 2 mM, 2 h. (B) Analysis of the G2/M check-
point induced by OHT (8 h) or IR (10 Gy) in 3T3T-ER cells. The numbers represent
the percentage of mitotic cells that were identified by flow cytometry as being
positive for H3-S10P and having a G2 DNA content. UCN-01 (50 nM) was added
for the last 4 h before collecting the data. (IR) 10 Gy. (C) Proliferation of 3T3T-ER
cells (in comparison with the parental NIH3T3 line used in this study) in re-
sponse to OHT. (D) SA--galactosidase staining (blue) in 3T3T-ER cells after a 4-d
exposure to OHT. Control image represents the same cells kept in the absence
of OHT. Black bar, 10 µm. (E) Western blot analysis of Rb, Chk1, and H2AX
phosphorylation in MCF7T-ER cells. (HU) Hydroxyurea: 2 mM, 2 h; (IR) 10 Gy. (F)
Proliferation of MCF7T-ER cells (in comparison with the parental MCF7 line used
in this study) in response to OHT. (G) SA--galactosidase staining (blue) in
MCF7T-ER cells after a 4-d exposure to OHT. Control image represents the same
clone kept in the absence of OHT. Black bar, 10 µm. (H) Distribution of HP1
 in
MCF7T-ER cells after a 4-d exposure to OHT. White bar, 3 µm.
Senescence by ATR activation
GENES & DEVELOPMENT 301
 Cold Spring Harbor Laboratory Press on February 9, 2024 - Published by genesdev.cshlp.orgDownloaded from 
Kuhne, M., Riballo, E., Rief, N., Rothkamm, K., Jeggo, P.A., and Lobrich,
M. 2004. A double-strand break repair defect in ATM-deficient cells
contributes to radiosensitivity. Cancer Res. 64: 500–508.
Kumagai, A., Lee, J., Yoo, H.Y., and Dunphy, W.G. 2006. TopBP1 acti-
vates the ATR–ATRIP complex. Cell 124: 943–955.
Liu, Q., Guntuku, S., Cui, X.S., Matsuoka, S., Cortez, D., Tamai, K., Luo,
G., Carattini-Rivera, S., DeMayo, F., Bradley, A., et al. 2000. Chk1 is
an essential kinase that is regulated by Atr and required for the
G(2)/M DNA damage checkpoint. Genes & Dev. 14: 1448–1459.
Lowe, S.W. and Sherr, C.J. 2003. Tumor suppression by Ink4a-Arf:
Progress and puzzles. Curr. Opin. Genet. Dev. 13: 77–83.
Matsuoka, S., Huang, M., and Elledge, S.J. 1998. Linkage of ATM to cell
cycle regulation by the Chk2 protein kinase. Science 282: 1893–1897.
Michaloglou, C., Vredeveld, L.C., Soengas, M.S., Denoyelle, C., Kuilman,
T., van der Horst, C.M., Majoor, D.M., Shay, J.W., Mooi, W.J., and
Peeper, D.S. 2005. BRAFE600-associated senescence-like cell cycle
arrest of human naevi. Nature 436: 720–724.
Murga, M., Jaco, I., Fan, Y., Soria, R., Martinez-Pastor, B., Cuadrado, M.,
Yang, S.M., Blasco, M.A., Skoultchi, A.I., and Fernandez-Capetillo, O.
2007. Global chromatin compaction limits the strength of the DNA
damage response. J. Cell Biol. 178: 1101–1108.
Narita, M., Nunez, S., Heard, E., Lin, A.W., Hearn, S.A., Spector, D.L.,
Hannon, G.J., and Lowe, S.W. 2003. Rb-mediated heterochromatin
formation and silencing of E2F target genes during cellular senes-
cence. Cell 113: 703–716.
Rothkamm, K. and Lobrich, M. 2003. Evidence for a lack of DNA double-
strand break repair in human cells exposed to very low x-ray doses.
Proc. Natl. Acad. Sci. 100: 5057–5062.
Satyanarayana, A., Wiemann, S.U., Buer, J., Lauber, J., Dittmar, K.E.,
Wustefeld, T., Blasco, M.A., Manns, M.P., and Rudolph, K.L. 2003.
Telomere shortening impairs organ regeneration by inhibiting cell
cycle re-entry of a subpopulation of cells. EMBO J. 22: 4003–4013.
Sedelnikova, O.A., Horikawa, I., Zimonjic, D.B., Popescu, N.C., Bonner,
W.M., and Barrett, J.C. 2004. Senescing human cells and ageing mice
accumulate DNA lesions with unrepairable double-strand breaks.
Nat. Cell Biol. 6: 168–170.
Serrano, M., Lee, H., Chin, L., Cordon-Cardo, C., Beach, D., and DePinho,
R.A. 1996. Role of the INK4a locus in tumor suppression and cell
mortality. Cell 85: 27–37.
Serrano, M., Lin, A.W., McCurrach, M.E., Beach, D., and Lowe, S.W.
1997. Oncogenic ras provokes premature cell senescence associated
with accumulation of p53 and p16INK4a. Cell 88: 593–602.
Shay, J.W., Pereira-Smith, O.M., and Wright, W.E. 1991. A role for both
RB and p53 in the regulation of human cellular senescence. Exp. Cell
Res. 196: 33–39.
te Poele, R.H., Okorokov, A.L., Jardine, L., Cummings, J., and Joel, S.P.
2002. DNA damage is able to induce senescence in tumor cells in
vitro and in vivo. Cancer Res. 62: 1876–1883.
Wang, H., Powell, S.N., Iliakis, G., and Wang, Y. 2004. ATR affecting cell
radiosensitivity is dependent on homologous recombination repair
but independent of nonhomologous end joining. Cancer Res. 64:
7139–7143.
Ward, I.M. and Chen, J. 2001. Histone H2AX is phosphorylated in an
ATR-dependent manner in response to replicational stress. J. Biol.
Chem. 276: 47759–47762.
Weterings, E. and van Gent, D.C. 2004. The mechanism of non-homolo-
gous end-joining: A synopsis of synapsis. DNA Repair (Amst.) 3:
1425–1435.
Yazdi, P.T., Wang, Y., Zhao, S., Patel, N., Lee, E.Y., and Qin, J. 2002.
SMC1 is a downstream effector in the ATM/NBS1 branch of the
human S-phase checkpoint. Genes & Dev. 16: 571–582.
Toledo et al.
302 GENES & DEVELOPMENT
 Cold Spring Harbor Laboratory Press on February 9, 2024 - Published by genesdev.cshlp.orgDownloaded from 
 10.1101/gad.452308Access the most recent version at doi:
 22:2008, Genes Dev. 
  
Luis I. Toledo, Matilde Murga, Paula Gutierrez-Martinez, et al. 
  
breaks
ATR signaling can drive cells into senescence in the absence of DNA
  
Material
Supplemental
  
 http://genesdev.cshlp.org/content/suppl/2008/01/16/22.3.297.DC1
  
References
  
 http://genesdev.cshlp.org/content/22/3/297.full.html#ref-list-1
This article cites 39 articles, 17 of which can be accessed free at:
  
License
Service
Email Alerting
  
 click here.right corner of the article or 
Receive free email alerts when new articles cite this article - sign up in the box at the top
Copyright © 2008, Cold Spring Harbor Laboratory Press
 Cold Spring Harbor Laboratory Press on February 9, 2024 - Published by genesdev.cshlp.orgDownloaded from