An extra allele of Chk1 limits oncogene-induced replicative stress and promotes transformation

Article (PDF Available)inJournal of Experimental Medicine 209(3):455-61 · February 2012with35 Reads
DOI: 10.1084/jem.20112147 · Source: PubMed
Abstract
Replicative stress (RS) is a type of endogenous DNA damage that cells suffer every time they duplicate their genomes, and which is further boosted by oncogenes. In mammals, the RS response (RSR) is coordinated by ATR and Chk1 kinases. We sought to develop a mammalian organism that is selectively protected from RS. To this end, mice carrying an extra copy of the Chk1 gene were generated. In vitro, Chk1 transgenic cells are protected from RS-inducing agents. Moreover, an extra Chk1 allele prolongs the survival of ATR-Seckel mice, which suffer from high levels of RS, but not that of ATM-deficient mice, which accumulate DNA breaks. Surprisingly, increased Chk1 levels favor transformation, which we show is associated with a reduction in the levels of RS induced by oncogenes. Our study provides the first example where supra-physiological levels of a tumor suppressor can promote malignant transformation, which is a result of the protection from the RS found in cancer cells.
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Brief Definitive Report
DNA damage is a common source of cancer and
aging. Therefore, organisms have evolved a co-
ordinated DNA damage response which detects,
signals, and promotes the repair of the lesions that
compromise genomic integrity (Jackson and
Bartek, 2009). Even though much emphasis has
been placed on how organisms respond to DNA
double-strand breaks (DSBs), recent works are
revealing that replicative stress (RS) might also be
a very relevant source of endogenous DNA dam-
age for mammalian disease. For instance, one of
the recent models of cancer progression is based
on thending that oncogenes generate RS, plac-
ing RS studies at the forefront of cancer research
(Halazonetis et al., 2008). At the same time, we
and others have previously shown that RS can
promote aging in mammals (Ruzankina et al.,
2007, 2009; Murga et al., 2009).
Whereas the nature of RS is still poorly
dened, it essentially stands for the accumulation
of recombinogenic stretches of single-stranded
DNA (ssDNA), which can form at processed
DSB, but which most frequently derive from
stalled replication forks. In mammals, the RS
response (RSR) is coordinated by ATR and Chk1
kinases (Cimprich and Cortez, 2008; López-
Contreras and Fernandez-Capetillo, 2010).
The essential nature of ATR (Brown and
Baltimore, 2000; de Klein et al., 2000) and Chk1
(Liu et al., 2000; Takai et al., 2000) kinases has
signicantly limited genetic studies addressing
the role of the RSR in mammals. To overcome
this limitation, we took a complementary genetic
approach and decided to generate a mouse model
that would be selectively protected from RS.
Given that RSR kinases are responsible for
the activation of cell cycle checkpoints, we
reasoned that an uncontrolled expression of these
kinases could be deleterious. Hence, to develop
a gain-of-function model of the RSR, we
decided to generate a mouse model with just
one additional copy of the Chk1 gene. Similar
strategies were shown to be successful for other
loci where regulation of protein levels is im-
portant such as TP53 (García-Cao et al., 2002)
or INK4a/ARF (Matheu et al., 2004). The rea-
son for choosing Chk1 was that, in contrast to
ATR, which naturally exists in a complex with
its binding partner ATRIP (Cortez et al., 2001),
Chk1 works in an autonomous manner. More-
over, Chk1 has been shown to be haploin-
sucient (Lam et al., 2004), indicating that
CORRESPONDENCE
Oscar Fernandez-Capetillo:
ofernandez@cnio.es
Abbreviations used: APH,
aphidicolin; DSB, double-strand
break; HTM, high-throughput
microscopy; HU, hydroxyurea;
IR, ionizing radiation; MEF,
mouse embryonic broblast;
RS, replicative stress; RSR, RS
response; ssDNA, single-
stranded DNA.
Andres J. López-Contreras and Paula Gutierrez-Martinez
contributed equally to this paper.
An extra allele of Chk1 limits
oncogene-induced replicative stress
and promotes transformation
Andres J. López-Contreras, Paula Gutierrez-Martinez, Julia Specks,
Sara Rodrigo-Perez, and Oscar Fernandez-Capetillo
Genomic Instability Group, Spanish National Cancer Research Centre (CNIO), E-28029 Madrid, Spain
Replicative stress (RS) is a type of endogenous DNA damage that cells suffer every time they
duplicate their genomes, and which is further boosted by oncogenes. In mammals, the RS
response (RSR) is coordinated by ATR and Chk1 kinases. We sought to develop a mammalian
organism that is selectively protected from RS. To this end, mice carrying an extra copy of
the
Chk1
gene were generated. In vitro,
Chk1
transgenic cells are protected from RS-inducing
agents. Moreover, an extra
Chk1
allele prolongs the survival of ATR-Seckel mice, which suffer
from high levels of RS, but not that of
ATM
-decient mice, which accumulate DNA breaks.
Surprisingly, increased Chk1 levels favor transformation, which we show is associated with a
reduction in the levels of RS induced by oncogenes. Our study provides the rst example
where supra-physiological levels of a tumor suppressor can promote malignant transformation,
which is a result of the protection from the RS found in cancer cells.
© 2012 López-Contreras et al. This article is distributed under the terms of an
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456 Chk1 facilitates transformation by oncogenes | López-Contreras et al.
Protection from RS in Chk1
Tg
cells
To determine whether increased Chk1 levels provide additional
Chk1 function, we rst analyzed the response of Chk1
Tg
MEF to
Chk1 inhibition. Transgenic MEFs were resistant to the cyto-
toxic eects of the Chk1 inhibitor UCN-01 and showed lower
amounts of H2AX in response to the drug (unpublished data),
illustrating that the extra Chk1 present on Chk1
Tg
cells is capable
of enhancing Chk1 function beyond WT levels. We next ana-
lyzed the response of Chk1
Tg
MEF to RS-inducing agents other
than UCN-01. Colony survival assays revealed that Chk1 trans-
genic MEFs were more resistant than their WT controls to HU
or aphidicolin (APH; Fig. 2 A). Noteworthy, and in agreement
with the normal overall size of Chk1
Tg
mice, replication was not
aected by the extra Chk1 allele (WT: 24.6 ± 1.6% vs. Chk1
Tg
:
24.93 ± 1.5%; P = 0.757; Fig. 2 B). Therefore, the observed
survival dierences reected an intrinsic resistance of Chk1
Tg
MEF to RS. In agreement with these observations, HU- or
APH-treated Chk1
Tg
MEF presented lower amounts of H2AX
(Fig. 2 C). Besides a pan-nuclear H2AX staining, RS also leads
to an accumulation of ssDNA, which can be detected as foci for
the ssDNA-binding protein RPA. High-throughput microscopy
(HTM) analyses revealed that HU- or APH-treated Chk1
Tg
MEFs presented signicantly lower amounts of chromatin-
bound RPA than their WT littermates (Fig. 2 D). Altogether,
these results illustrate that an additional allele of Chk1 can pro-
vide a supra-physiological protection against RS.
Chk1
Tg
alleviates the symptoms of ATR-Seckel
but not those of ATM-decient mice
Given the eects observed on isolated cells, we next sought to
evaluate whether an enhanced RSR could have an impact
in vivo. One of the most well understood models of an RS-driven
disease is ATR-Seckel Syndrome. Patients of this syndrome
present reduced levels of the ATR kinase (O’Driscoll et al.,
2003). We recently generated a mouse model
endogenous Chk1 levels are limiting and that therefore an
increased gene dosage could have an eect. We thus decided
to develop a mouse strain with one additional copy of the
Chk1 gene as a mean to enhance the RSR.
RESULTS AND DISCUSSION
Generation of a mouse model with supra-physiological
Chk1 function
Similar approaches in the past relied on the trans-genesis
of bacterial articial chromosomes (BAC) containing the gene
of interest (García-Cao et al., 2002; Matheu et al., 2004). How-
ever, Chk1 is a small gene and available BACs contained addi-
tional genes. Thus, we subcloned a 33.5-kb region from the
mouse genome including Chk1 and sequences 5and 3 until
the next conserved gene was found in either direction (Fig. 1 A).
The construct was used for the microinjection of fertilized
mouse oocytes and Chk1 transgenic founder lines were estab-
lished. Southern blot analyses revealed that one of the lines
carried a single integration site, for which the intensity of the
transgenic band was about half of that caused by endogenous
Chk1 (Fig. 1 B). We thus selected this line as a mouse model
carrying one additional allele of Chk1 (Chk1
Tg
).
Transgenic mice were viable with no obvious phenotype
that would distinguish them from their littermates (Fig. 1,
C and D). Nevertheless, tissues from Chk1
Tg
animals showed
increased protein levels of Chk1 (Fig. 1 E). This dierence
increased in proliferating cells such as B-lymphocytes or mouse
embryonic broblasts (MEFs; Fig. 1, E and F). Exposure of
Chk1
Tg
MEF to hydroxyurea (HU) or ionizing radiation (IR)
led to increased levels of phosphorylation of Chk1 but not
other ATR targets such as Rad17 or RPA (Fig. 1, F and G).
Hence, an extra allele of Chk1 is compatible with mouse
development and leads to increased Chk1 levels, which are
susceptible to phosphorylation by ATR.
Figure 1. Generation of a
Chk1
Tg
strain. (A) Region
including Chk1 that was subcloned from the mouse
genome. (B) Southern blot with an internal Chk1 probe,
illustrating the presence of an integration site (8 kb) on
the Chk1
Tg
strain. The 5-kb band corresponds to the
endogenous Chk1. (C) Representative picture of
4-mo-old WT and Chk1
Tg
littermates. (D) Weight distribu-
tion of 1-mo-old WT and Chk1
Tg
mice. (E) Chk1 Western
blot in testis, spleen, and puried B cells after a 2-d
stimulation with lipopolysaccharide from WT and
Chk1
Tg
mice. Data are representative of four indepen-
dent analyses. (F) ATR, Chk1-P, Chk1, RPA-P, and
Rad17-P Western blot in WT and Chk1
Tg
littermate MEF,
either untreated (C) or upon treatment with 2 mM HU
for 3 h or 10 Gy IR for 1 h. Data are representative of
two independent analyses. (G) Chk1-P, RPA-P, and
Rad17-P Western blot in WT and Chk1
Tg
littermate MEF
upon treatment with 2 mM HU for 3 h in the presence
or absence of 1 µM of ATR inhibitor (ATRi; ETP-46464).
Data are representative of two independent analyses.
-Actin was used as a loading control in all Western
blots. In D, center lines indicate mean values.
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JEM Vol. 209, No. 3
457
Br ief Definitive Repor t
progeroid disease to which animals succumb
early in life. Therefore, and given that Chk1 is
phosphorylated and activated by ATR, we in-
vestigated the effect of the Chk1 transgene on
ATR-Seckel mice.
Similar to our observations in MEF, the pres-
ence of an extra allele of Chk1 reduced the number
of cells presenting a pan-nuclear
H2AX distribution on
ATR-Seckel embryos (Fig. 3 A). This eect was not a result
of dierences in embryonic proliferation rates, as illustrated by
Ki67 staining (Fig. 3 B). In addition, there were no signicant
dierences on the weights of Chk1
Tg
and Chk1
+/+
animals at
of ATR-Seckel Syndrome (Murga et al., 2009). ATR mutant
animals present an accumulation of RS, which is particularly
abundant during embryonic development. Once born, ATR-
Seckel (ATR
S/S
) mice develop a pleiotropic disease, which is
manifested by a characteristic cranial appearance and a segmental
Figure 2. Resistance to RS in
Chk1
Tg
MEF. (A) Number
of colonies per plate in WT and Chk1
Tg
MEF after treatment
with 0.5 mM HU or 1 µM APH for 24 h (normalized to the
number of colonies found in the untreated control plates).
The experiment was repeated twice with three independent
MEF pairs. (B) Representative ow cytometry proles of WT
and Chk1
Tg
MEF after a 1-h BrdU pulse. Numbers indicate
the percentage of BrdU-positive cells. Data are representa-
tive of ve independent analyses. (C and D) HTM-mediated
quantication of H2AX (C) and RPA (D) intensities in WT
and Chk1
Tg
MEF treated with 0.5 mM HU for 3 h and 5 µM
APH for 4 h. Data are representative of four independent
analyses. In A, error bars indicate SD; in C and D, center lines
indicate mean values. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 3. Alleviation of the ATR-Seckel syndrome by
Chk1
Tg
. (A and B) Representative images of H2AX (A) and Ki67 (B) on the placenta and face
from ATR
S/S
and ATR
S/S
/Chk1
Tg
littermate embryos. Red arrows indicate cells showing a pan-nuclear H2AX staining. Numbers indicate the mean percentage
and SD of positive cells in each case (n = 3). (C) Kaplan-Meyer curves of ATR
S/S
(n = 23) and ATR
S/S
/Chk1
Tg
(n = 36) mice. The p-value was calculated with the
Mantel-Cox log-rank test. (D) Computerized Tomography-mediated reconstruction of the heads from WT, Chk1
Tg
, ATR
S/S
, and ATR
S/S
/Chk1
Tg
mice. Yellow
arrows indicate features that show evident rescue such as the shape of the crania (a), the micrognathia (b), and the decient closure of the fontanelle (c).
Data are representative of four independent analyses. (E) ATR, Chk1-P, and Chk1 protein levels in WT, ATR
S/S
, and ATR
S/S
/Chk1
Tg
MEF treated with 0.5 mM HU
for 3 h or 10 mM methyl methanesulfonate (MMS) for 4 h. -Actin was used as a loading control. Data are representative of two independent analyses.
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458 Chk1 facilitates transformation by oncogenes | López-Contreras et al.
mice, which present a deficient re-
sponse to DSB and die from lymphomas
initiated by chromosomal translocations
(Barlow et al., 1996). In humans, ATM
deciency leads to another hereditary
syndrome known as Ataxia telangiectasia
(AT), which is also associated with a high
incidence of cancer (Savitsky et al., 1995).
In contrast to the eect on ATR-Seckel animals, the Chk1 trans-
gene did not extend the lifespan of ATM
/
mice (Fig. 4 C),
the cause of death being lymphoma in both ATM
/
and
Chk1
Tg
/ATM
/
animals. The Chk1 transgene also failed to res-
cue the IR-induced G2/M checkpoint defect that is ob-
served on ATM
/
cells (Fig. 4 D), which is in agreement
with IR-induced Chk1 phosphorylation being ATM dependent
(Cuadrado et al., 2006; Jazayeri et al., 2006). In summary,
whereas the Chk1
Tg
allele can alleviate the symptoms of the
ATR-Seckel Syndrome, it has no detectable impact on a mouse
model of AT.
An extra allele of Chk1 promotes transformation by limiting
oncogene-induced RS
Beside external reagents, RS can also occur by endoge-
nous sources that perturb replication. According to the
oncogene-induced DNA damage model of cancer progres-
sion (Halazonetis et al., 2008), oncogenes would generate
substantial amounts of RS which, by activating the DNA
damage response, would limit the expansion of the tumor in
its initial stages. In this model, ATR and Chk1 would suppress
transformation through the activation of p53. We therefore
evaluated the response of Chk1
Tg
MEF to oncogenic trans-
formation. To this end, MEFs were infected with a retrovi-
ral plasmid that expressed H-Ras
V12G
and E1A oncogenes
(Ras/E1A), a system which is widely used to evaluate trans-
formation in MEF. Moreover, H-Ras
V12G
overexpression pro-
motes Chk1 phosphorylation (Gilad et al., 2010). Surprisingly,
transformation with Ras/E1A was consistently and signi-
cantly more ecient on Chk1
Tg
MEF (Fig. 5, A and B) than
on WT littermates.
birth (WT: 1.24 ± 0.19 g vs. Chk1
Tg
: 1.26 ± 0.16 g;
P = 0.801), reinforcing that Chk1
Tg
does not aect fetal
proliferation. Importantly, this reduction on embryonic RS
correlated with a significant extension of the lifespan on
ATR
S/S
/Chk1
Tg
animals (Fig. 3 C). Around one fifth of
ATR
S/S
/Chk1
Tg
animals lived longer than any ATR
S/S
mouse, with some reaching ages beyond 100 wk. In agree-
ment with this, ATR
S/S
/Chk1
Tg
animals showed an alleviation
of the craniofacial abnormalities that are present on ATR-
Seckel mice. This eect was also present at dierent degrees,
with some of the double mutant mice showing a clear rescue
of all measured parameters (Fig. 3 D). Consistent with the in
vivo ndings, ATR
S/S
/Chk1
Tg
MEF showed a partial rescue of
the decient Chk1 phosphorylation that is observed on ATR
S/S
cells (Fig. 3 E). In summary, all of these results demonstrate
that the Chk1 transgene is able to alleviate the symptoms asso-
ciated to the ATR-Seckel syndrome in mice.
We next evaluated the eect of the Chk1
Tg
in the context
of DNA DSB. In contrast to HU or APH, the presence of the
Chk1 transgene did not signicantly aect the levels of H2AX
induced by IR (Fig. 4 A). However, Chk1
Tg
cells presented a
hyperactive IR-induced G2/M checkpoint (unpublished data),
which is consistent with the role of Chk1 in checkpoint regu-
lation. In spite of this, Chk1
Tg
MEFs were not radioresistant
when compared with their WT littermates (Fig. 4 B). This
limited contribution of the G2/M checkpoint on survival to
radiation has been previously noticed (Löbrich and Jeggo,
2007) and is consistent with the fact that ATR or Chk1 de-
ciencies are not particularly radiosensitive.
To study the impact of the Chk1 transgene on the response
to DSB in vivo, Chk1
Tg
mice were crossed with ATM-decient
Figure 4.
Chk1
Tg
does not rescue ATM
deciency. (A) HTM-mediated quantication of
H2AX in WT and Chk1
Tg
MEF exposed to 5 Gy of
IR for 1 h. Data are representative of three inde-
pendent experiments. (B) Number of colonies per
plate in WT and Chk1
Tg
MEF after exposure to 1
or 4 Gy of IR (normalized to the number of colo-
nies found in control plates). The experiment was
repeated twice with two independent MEF pairs.
(C) Kaplan-Meyer curves of ATM
/
(n = 39) and
ATM
/
/Chk1
Tg
(n = 26) mice. The p-value was
calculated with the Mantel-Cox log-rank test.
(D) Activation of the IR-induced G2/M check-
point in WT, ATM
/
, and ATM
/
/Chk1
Tg
cells.
Images are representative of two independent
experiments. In A, center lines indicate mean
values; in B, error bars indicate SD.
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JEM Vol. 209, No. 3
459
Br ief Definitive Repor t
knockdown (Fig. 5 G), a situation where Myc-induced RS is
exacerbated (Murga et al., 2011). Altogether, our data suggest
that the RS induced by oncogenes is a toxic by-product of the
transformation process, which is not the required to promote
transformation, and which can be limited by selectively poten-
tiating the RSR.
Here, we provide proof of concept to show that the mam-
malian RSR is susceptible of improvement. At the same time,
and whereas ATR might have >700 targets (Matsuoka et al.,
2007), this study identies Chk1 as a critical mediator of ATR
in vivo, providing the rst example of a genetic context which
modies the severity of the Seckel Syndrome. It is possible
that the eect of the Chk1
Tg
might be more marked in the
context of other syndromes initiated by replication problems,
but which keep intact ATR levels. For instance, recent studies
have identied mutations in the pre-replication complex as
causative of Meier-Gorlin Syndrome (Klingseisen and Jackson,
2011), which is also characterized by craniofacial abnormali-
ties. To what extent these syndromes are also modulated by
the RSR remains to be elucidated.
In what relates to cancer, our work provides a more complex
view of the role of Chk1 and RS during carcinogenesis. Recent
To determine how Chk1 could facilitate oncogenic trans-
formation, we analyzed the levels of RS present at various
times after infection. Interestingly, whereas Ras/E1A expres-
sion led to a detectable increase in H2AX phosphorylation in
WT MEF, this was signicantly reduced on Chk1
Tg
cells
(Fig. 5 C). These data suggest that, whereas Chk1 might be in-
volved in activating p53 in response to oncogenes, it also has a
previous function in suppressing oncogene-induced RS. In this
context, the enhanced Chk1 levels present on Chk1
Tg
MEF
would be favoring transformation by decreasing the amount of
RS generated by the oncogenes, which is intrinsically cyto-
toxic. In agreement with this, Ras/E1A immortalized Chk1
Tg
MEFs presented a lower percentage of dead cells than their
WT littermates (Fig. 5 D). Moreover, Ras/E1A transformed
Chk1
Tg
colonies were signicantly larger in size than those
obtained from WT MEF (WT: 1.41 ± 0.68 mm
2
vs. Chk1
Tg
:
2.72 ± 1.48 mm
2
; P = 0.0003), which would support the idea
that Chk1 facilitates the proliferation of cells carrying onco-
gene-induced RS. In addition to Ras/E1A, Chk1
Tg
MEFs
were also protected from the RS and apoptosis induced by
Myc (Fig. 5, E and F). Noteworthy, Chk1
Tg
-mediated sup-
pression of Myc-induced RS was accentuated upon p53
Figure 5. Enhanced oncogenic transformation in
Chk1
Tg
MEF. (A) Plates of WT and Chk1
Tg
MEF 2 wk after infection with a Ras/E1A-expressing
retrovirus. Colonies were stained with Methylene blue. Images are representative of six independent experiments. (B) Numbers of transformed colonies
per plate in WT and Chk1
Tg
MEF infected with Ras/E1A. Data derive from three independent experiments. (C) HTM-mediated quantication of H2AX in
WT and Chk1
Tg
MEF 24 and 48 h after infection with a Ras/E1A-expressing retrovirus. Data are representative of three independent experiments. (D) Percentage
of apoptotic cells present in cultures of WT and Chk1
Tg
MEF 24 and 48 h after infection with a Ras/E1A-expressing retrovirus. The quantication derives
from three independent experiments. (E) HTM-mediated quantication of H2AX in WT and Chk1
Tg
MEF infected with MycER, treated or untreated with
4-OHT for 8 or 24 h. Data are representative of three independent experiments. (F) MycER-induced apoptosis in WT and Chk1
Tg
MEF infected with MycER,
treated or untreated with 4-OHT for 72 h. Data derive from three independent experiments. (G) HTM-mediated analysis of H2AX in WT and Chk1
Tg
MEF infected with MycER and treated with 4-OHT for 24 h, which were previously infected with lentiviruses expressing a p53-specic shRNA (sh p53)
or a control shRNA (sh C). Data are representative of three independent experiments. In B, D, and F, error bars indicate SD; in C–G, center lines indicate
mean values. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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460 Chk1 facilitates transformation by oncogenes | López-Contreras et al.
10 d. For the work on oncogenes, pBabe-RAS
V12
/E1A-puro or pBabe-puro
(gift from M. Barbacid, Spanish National Cancer Research Centre, Madrid,
Spain) were retrovirally transduced according to standard procedures. Infected
cells were selected with 2 µg/ml puromycin for 2 d, and then 2,000 cells were
seeded in 10-cm diameter plates. After 7 d, colony formation was assessed by
Methylene blue staining. Apoptosis was quantied by ow-cytometry as the
fraction of cells with a DNA content lower than G1. Lentiviruses expressing
p
53-specic shRNAs and their controls (gift from J.M. Silva, Institute for
Cancer Genetics, Columbia University, New York, NY) were infected
according to standard procedures. For all experiments, cells were grown in
5% oxygen to minimize the exposure to reactive oxygen species, and low
(less than three) passage MEFs were used.
Immunoblotting. For total protein extracts, cells were washed once with
PBS, collected by directly adding NuPAGE LDS Sample buer, and
incubated for 5 min at 95°C. For soluble protein extracts, cells were lysed in
RIPA buer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate,
150 mM NaCl, and 1 mM EDTA) containing protease and phosphatase
inhibitors (Sigma-Aldrich). Samples were resolved by SDS-PAGE and
analyzed by standard Western blotting techniques. Antibodies against p53,
RPA, Rad17-S645P, and Chk1-S345P (Cell Signaling Technology), H2AX
(Millipore), ATR (Serotec), -actin (Sigma-Aldrich), RPA-S4P/S8P (Bethyl
Laboratories), and Chk1 (Novocastra) were used. Protein blot analyses were
performed on the LICOR platform (eBioscience).
HTM analyses. MEFs were grown on CLEAR bottom 96-well plates
(Greiner Bio-One) and H2AX and RPA immunouorescence were performed
using standard procedures. In the case of RPA, a detergent extraction step was
done previous to xation which eliminates the nucleosoluble pool of RPA and
leaves the chromatin-bound fraction. Images were automatically acquired from
each well by an Opera High-Content Screening System (Perkin Elmer). A 40×
magnication lens was used and pictures were taken at nonsaturating conditions.
Images were segmented using the DAPI staining to generate masks matching cell
nuclei from which the mean H2AX and RPA signals were calculated.
Cell cycle analysis. Cells were resuspended in a PBS solution containing
1% (wt/vol) BSA, 10 µg/ml propidium iodide, and 0.5 mg/ml RNase A and
were analyzed by ow cytometry in a FACSCalibur machine (BD). To
monitor replication, cells were incubated with BrdU for 1 h at 37°C. After
xation with 4% (wt/vol) paraformaldehyde for 30 min, cells were processed
with a FITC BrdU Flow Cytometry kit as recommended by the manufac-
turer (BD). For G2/M checkpoint analysis, proliferating B-lymphocytes
were exposed to IR at the indicated doses, and the percentage of mitotic
cells was calculated 1 h after the treatment by a dual staining with propidium
iodide (DNA content) and the mitotic marker H3
S10P
(Millipore).
Whole body imaging. Whole-body imaging was performed on anesthetized
mice using the eXplore Vista PET-CT (GE Healthcare) and a 7-tesla Pharmascan
(Bruker). MMWKS software (GE Healthcare) was used for the quantications.
We thank Dr. M. Serrano and A. Nussenzweig for critical comments on the manuscript.
A.J. pez-Contreras is recipient of a Juan de la Cierva fellowship (JCI-
2009-05099) from the Spanish Ministry of Science. Work in the O. Fernandez-
Capetillo laboratory is supported by grants from the Spanish Ministry of
Science (CSD2007-00017 and SAF2011-23753), the Association for International
Cancer Research (12-0229), and the European Research Council (ERC-210520).
The authors declare no competing nancial interests.
Submitted: 10 October 2011
Accepted: 8 February 2012
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added at the indicated concentrations. For clonogenic survival analyses, 10
3
cells were plated per 10-cm plate and the number of colonies was counted after
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    • "While some siRNAs, under this condition, significantly increased cell death at 96 h post-transfection, we did not observe any significant difference between Polθ-overexpressing clones and the control cell line. Given that cancer cells are known to host disparate genetic rearrangements and rely on pathways involved in DNA checkpoints or repair (López-Contreras et al., 2012; Murga et al., 2011), we then decided to explore, by siRNA technology, the possibility of a functional relationship between up-regulated Polθ levels and the expression of specific genes within a tumorigenic phenotype. As a model, we chose the RKO colorectal cell line, which express high levels of endogenous Polθ, and knocked down Polθ Biology Open @BULLET Advance article prior to, 24 h later, a second knock-down round by the siRNA candidates previously selected as putative hits in fibroblasts (Fig. 2C). "
    [Show abstract] [Hide abstract] ABSTRACT: DNA polymerase theta (Polθ) is a specialized A-family DNA polymerase that functions in processes such as translesion synthesis (TLS), DNA double-strand break repair and DNA replication timing. Overexpression of POLQ, the gene encoding Polθ, is a prognostic marker for an adverse outcome in a wide range of human cancers. While increased Polθ dosage was recently suggested to promote survival of homologous recombination (HR)-deficient cancer cells, it remains unclear whether POLQ overexpression could be also beneficial to HR-proficient cancer cells.By performing a short interfering (si) RNA screen in which genes encoding druggable proteins were knocked down in Polθ-overexpressing cells as a means to uncover genetic vulnerabilities associated with POLQ overexpression, we could not identify genes that were essential for viability in Polθ-overexpressing cells in normal growth conditions. We also showed that, upon external DNA replication stress, Polθ expression promotes cell survival and limits genetic instability. Finally, we report that POLQ expression correlates with the expression of a set of HR genes in breast, lung and colorectal cancers. Collectively, our data suggest that Polθ upregulation, besides its importance for survival of HR deficient cancer cells, may be crucial also for HR-proficient cells to better tolerate DNA replication stress, as part of a global gene deregulation response, including HR genes.
    Article · Sep 2016
    • "Consistent with the fact that the expression of CHK1 and BRCA1 is not affected in human MCPH1 patient cell lines [42], the Mcph1 knockout affects neither the expression of Chk1 nor the phosphorylation of Chk1 upon DNA damage [23,49] . It is interesting to note that the ectopic expression of Chk1 generated by crossing Mcph1-del mice with Super-Chk1 mice [50] failed to correct the microcephaly phenotype in our Mcph1-del mice (Fig. 2), suggesting that MCPH1 may regulate the Cdc25-Cdk1-mediated mitotic entry through a pathway independent of Chk1. Finally, consistent with its role in DDR, Mcph1-del neuroprogenitors are hypersensitive to IR, which is associated with a massive apoptosis in the Mcph1-del neocortex and an increased embryonic lethality, likely due to compromised DNA repair and increased genome instability [49]. "
    [Show abstract] [Hide abstract] ABSTRACT: Microcephalin (MCPH1) is identified as being responsible for the neurodevelopmental disorder primary microcephaly type 1, which is characterized by a smaller-than-normal brain size and mental retardation. MCPH1 has originally been identified as an important regulator of telomere integrity and of cell cycle control. Genetic and cellular studies show that MCPH1 controls neurogenesis by coordinating the cell cycle and the centrosome cycle and thereby regulating the division mode of neuroprogenitors to prevent the exhaustion of the progenitor pool and thereby microcephaly. In addition to its role in neurogenesis, MCPH1 plays a role in gonad development. MCPH1 also functions as a tumor suppressor in several human cancers as well as in mouse models. Here, we review the role of MCPH1 in DNA damage response, cell cycle control, chromosome condensation and chromatin remodeling. We also summarize the studies on the biological functions of MCPH1 in brain size determination and in pathologies, including infertility and cancer.
    Full-text · Article · May 2016
    • "Loss of one copy of CHK1 causes spontaneous cell death even in the absence of external stress in mammalian cells which the authors interpreted as limiting endogenous Chk1 levels [28]. A recent study reported that expression of one extra-allele of Chk1 in transgenic mice protects against replication stress [56]. The viability of these cells was increased and was associated with a decrease of double strand breaks when transgenic cells were treated with hydroxyurea and aphidicolin. "
    [Show abstract] [Hide abstract] ABSTRACT: DNA replication in higher eukaryotes initiates at thousands of origins according to a spatio-temporal program. The ATR/Chk1 dependent replication checkpoint inhibits the activation of later firing origins. In the Xenopus in vitro system initiations are not sequence dependent and 2-5 origins are grouped in clusters that fire at different times despite a very short S phase. We have shown that the temporal program is stochastic at the level of single origins and replication clusters. It is unclear how the replication checkpoint inhibits late origins but permits origin activation in early clusters. Here, we analyze the role of Chk1 in the replication program in sperm nuclei replicating in Xenopus egg extracts by a combination of experimental and modelling approaches. After Chk1 inhibition or immunodepletion, we observed an increase of the replication extent and fork density in the presence or absence of external stress. However, overexpression of Chk1 in the absence of external replication stress inhibited DNA replication by decreasing fork densities due to lower Cdk2 kinase activity. Thus, Chk1 levels need to be tightly controlled in order to properly regulate the replication program even during normal S phase. DNA combing experiments showed that Chk1 inhibits origins outside, but not inside, already active clusters. Numerical simulations of initiation frequencies in the absence and presence of Chk1 activity are consistent with a global inhibition of origins by Chk1 at the level of clusters but need to be combined with a local repression of Chk1 action close to activated origins to fit our data.
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