The Mre11 Nuclease Is Critical for the Sensitivity of Cells
to Chk1 Inhibition
Ruth Thompson1,2, Ryan Montano1,2, Alan Eastman1,2*
1Department of Pharmacology and Toxicology, The Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America, 2Norris Cotton Cancer
Center, Lebanon, New Hampshire, United States of America
The Chk1 kinase is required for the arrest of cell cycle progression when DNA is damaged, and for stabilizing stalled
replication forks. As a consequence, many Chk1 inhibitors have been developed and tested for their potential to enhance
DNA damage-induced tumor cell killing. However, inhibition of Chk1 alone, without any additional exogenous agent, can be
cytotoxic. Understanding the underlying mechanisms of this sensitivity is critical for defining which patients might respond
best to therapy with Chk1 inhibitors. We have investigated the mechanism of sensitivity in U2OS osteosarcoma cells. Upon
incubation with the Chk1 inhibitor MK-8776, single-stranded DNA regions (ssDNA) and double-strand breaks (DSB) begin to
appear within 6 h. These DSB have been attributed to the structure-specific DNA endonuclease, Mus81. The Mre11/Rad50/
Nbs1 complex is known to be responsible for the resection of DSB to ssDNA. However, we show that inhibition of the Mre11
nuclease activity leads, not only to a decrease in the amount of ssDNA following Chk1 inhibition, but also inhibits the
formation of DSB, suggesting that DSB are a consequence of ssDNA formation. These findings were corroborated by the
discovery that Mre11-deficient ATLD1 cells are highly resistant to MK-8776 and form neither ssDNA nor DSB following
treatment. However, once complimented with exogenous Mre11, the cells accumulate both ssDNA and DSB when
incubated with MK-8776. Our findings suggest that Mre11 provides the link between aberrant activation of Cdc25A/Cdk2
and Mus81. The results highlight a novel role for Mre11 in the production of DSB and may help define which tumors are
more sensitive to MK-8776 alone or in combination with DNA damaging agents.
Citation: Thompson R, Montano R, Eastman A (2012) The Mre11 Nuclease Is Critical for the Sensitivity of Cells to Chk1 Inhibition. PLoS ONE 7(8): e44021.
Editor: Kerstin Borgmann, University Medical Center Hamburg-Eppendorf, Germany
Received January 12, 2012; Accepted July 31, 2012; Published August 24, 2012
Copyright: ? 2012 Thompson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH grant CA117874 (A. Eastman), and Cancer Center Support grant CA23108 to the Norris Cotton Cancer Center. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Alan.R.Eastman@Dartmouth.edu
High fidelity DNA replication is essential for the maintenance of
genomic stability and cell survival. Cells have therefore evolved
intricate checkpoint pathways to ensure the repair of any DNA
lesions prior to progression through the cell cycle. Checkpoint
kinase 1 (Chk1) is a vital mediator of the S and G2 checkpoints
and it is well characterized as being essential for cell survival in the
response to many DNA damaging agents [1–4]. However, more
recent studies have revealed a role for Chk1 in normal S phase
progression . Chk1 inhibition in unperturbed human cells can
result in the stabilization of Cdc25A and the activation of cyclin
dependent kinases (CDKs) . This increased CDK activity
causes increased replication origin firing, and DNA-damage
accumulates in S-phase most likely due to the aberrant upregula-
tion of replication initiation . Despite the increased origin firing
in Chk1-deficient cells, replication fork progression is dramatically
reduced [8,9] and consequently, it has been suggested that Chk1
promotes replication fork progression in normal S phase through
the control of replication origin firing .
Inhibition of Chk1 has been shown to induce regions of single-
stranded DNA (ssDNA), RPA binding to ssDNA and the
formation of double strand breaks (DSB) in normal S phase .
Replication fork collapse has been proposed as the reason behind
S phase-specific DNA damage, and the DNA endonuclease Mus81
has recently been demonstrated as the source of DSB following
Chk1 inhibition . However, the DNA substrate for Mus81
cleavage is unknown and this observation does not account for the
appearance of regions of ssDNA. The Mre11/Rad50/Nbs1
(MRN) complex functions as a DNA damage sensor and is
responsible for the recruitment of ATM to the sites of DSB .
The MRN complex also promotes the processing of DSB to
ssDNA . We therefore, hypothesized that the Mre11 nuclease
could play a role in the production of ssDNA following Chk1
Here we show that the Chk1 inhibitor MK-8776 (previously
known as SCH900776) induces phosphorylation of RPA and
H2AX in U2OS cells. The Mre11 inhibitor mirin suppresses both
these effects. Moreover, the Mre11-deficient cell line ATLD1 was
inherently resistant to Chk1 inhibition but could be sensitized
through ectopic expression of Mre11. These findings suggest a
novel role for Mre11 in the production of DNA DSB following
Chk1 inhibition results in rapid accumulation of ssDNA
and DSB in U2OS cells
Since discovering the checkpoint inhibitory activity of UCN-01
over 15 years ago , we have performed extensive experiments
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on the activation of Chk1 by DNA damaging agents and its
inhibition by UCN-01, and more recently by MK-8776 .
These observations led to the realization that some cell lines are
highly sensitive to the inhibition of Chk1 as a single agent.
To investigate the role of Chk1 in unperturbed cell cycle
progression we incubated U2OS cells with two concentrations of
MK-8776, selected based on our previous findings that 2 mM MK-
8776 enhances the cytotoxic effects of hydroxyurea in most cell
lines but 200 nM was sufficient in more sensitive cell lines such as
U2OS . Western blotting revealed that MK-8776 induced
phosphorylation of Chk1 at serine 345 at both concentrations as
early as 2 h after administration. It has been suggested that this
phosphorylation is due to the loss of Chk1-mediated feedback
inhibition of ATR . Phosphorylation of H2AX (indicative of
DSB formation; see below) and accumulation of RPA phosphor-
ylation at the S4/S8 site began to appear at 4 h and was
dramatically elevated by 16 h (Fig. 1A). The phosphorylation of
RPA is also observed as a band with retarded electrophoretic
mobility in blots of total RPA.
Analysis by confocal microscopy revealed dramatic cH2AX
pan-nuclear staining in response to just 6 h of Chk1 inhibition
(Fig. 2A, second row), a phenomenon which has been previously
documented following the inhibition or depletion of Chk1 .
Furthermore, the cells showing pan-nuclear cH2AX staining
(approximately 20%) were, for the most part, positive for RPA foci
(.10 foci/cell). The fact that ssDNA and DSB were occurring in
the same cells led us to hypothesize that one may be a precursor
for the other. There were a few cells positive for RPA but not
cH2AX suggesting that RPA foci may appear first.
Our previous work has shown that, compared to a panel of ten
cell lines, U2OS cells are very sensitive to Chk1 inhibition by MK-
8776 as a single agent . Here, U2OS cells were incubated with
MK-8776 for 24 or 48 h, then allowed to grow for an additional
5–6 days (Fig. 1B). Alternatively, cells were incubated continuously
for 7 days. The growth curves are fairly similar in all cases
demonstrating that the maximum growth suppression is elicited
within the first 24 h, and the cells are unable to recover thereafter.
It has been shown that the cH2AX induced by Chk1 inhibition
occurs exclusively in cells actively replicating their DNA . We
confirmed this was the case by pulse-labeling cells with EdU to
stain S phase cells and then incubating with MK-8776 for 6 hours.
Cells were then stained for cH2AX and EdU and analyzed by
confocal microscopy (data not shown). Collectively, these results
suggest that inhibition of Chk1 causes DNA damage in cells
actively replicating their DNA.
The Mre11 inhibitor mirin prevents MK-8776-induced
Chk1 inhibition induces DSB which have been attributed to the
Mus81 endonuclease . Whether the production of ssDNA
regions is a cause or consequence of DSB remains unknown. It is
well documented that the MRN complex is recruited to DSB and
is required for the processing of DSB to ssDNA to which RPA
binds [13,18–20]. To determine whether the ssDNA formed
following Chk1 inhibition is Mre11 dependent, we co-incubated
cells with MK-8776 and the Mre11 inhibitor mirin. Immunoflu-
orescence showed that the cH2AX pan-nuclear staining and the
RPA foci induced by 1 mM MK-8776 were completely inhibited
by co-treatment with 100 mM mirin (Fig. 2A). Western blotting
revealed that, while there was little change in the amount of
phospho-Chk1 at serine-345, the higher concentrations of mirin
reduced phospho-RPA, phospho-ATM and cH2AX induced by
MK-8776 (Fig. 2B). These concentrations are consistent with those
previously shown to inhibit the Mre11 nuclease in cells .
To determine whether the damage previously shown to be
induced by other Chk1 inhibitors such as UCN-01 is also inhibited
Figure 1. MK-8776 induces DNA damage and S phase arrest in U2OS cells. A. Cells were incubated with 200 nM or 2 mM MK-8776 for 0–
24 h then analyzed by western blotting for markers of DNA damage. B. Cells were incubated with MK-8776 for 24 h or 48 h then allowed to recover
in drug-free media, or incubated continuously for 7 days with MK-8776. Total DNA content per well was then assessed as a measure of cell growth.
Error bars (shown only in one direction for clarity) represent the standard error of 3 independent experiments.
Sensitivity of Cells to Chk1 Inhibition
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