We have shown that the DNA damage induced by the Chk1 inhibitor MK-8776 is dependent on the action of the Mre11 nuclease. In particular we have demonstrated that inhibition of Mre11 activity prevents formation of ssDNA and DSB induced by MK-8776 and UCN-01. These data and the fact that an Mre11-deficient cell line is resistant to Chk1 inhibition, suggests that Mre11 is involved in the production of DNA damage following treatment with Chk1 inhibitors.
Following treatment with MK-8776 there is rapid phosphorylation of Chk1 at the serine-345 site. This is consistent with the report that Chk1 induces feed-back dephosphorylation of ATR by protein-phosphatase 2A; when Chk1 is inhibited, ATR becomes active and phosphorylates Chk1 
. This Chk1 phosphorylation was also seen in the ATLD cells in response to MK-8776, indicating that the resistance to MK-8776 in this cell line is not attributable to a failure to inhibit Chk1.
Our study shows that Chk1 inhibition induces rapid phosphorylation of RPA and H2AX in U2OS cells, indicative of the appearance of regions of ssDNA and DSB respectively. We also found that MK-8776 treatment induces RPA foci and γH2AX pan-nuclear staining in about 25% of the cells. Pan-nuclear γH2AX staining, as opposed to foci has been previously documented in response to Chk1 inhibition and has been shown to be associated with DSB following Chk1 inhibition 
. It has been suggested that this widespread phosphorylation of H2AX is due to the inappropriate firing of replication origins, leading to fork collapse at numerous points distributed throughout the genome 
. The comet assay was performed to further confirm that DSB were responsible for the phosphorylation of H2AX. Flow cytometry revealed that the cells expressing the phosphorylated form of H2AX were in S phase of the cell cycle. This is consistent with previous reports that Chk1 is required in normal S phase to protect against DNA breakage 
In response to DSB induced by ionizing radiation (IR), the MRN complex acts as a DNA damage sensor and is responsible for recruiting ATM to the sites of DNA damage 
. The MRN complex promotes the initial processing of DSB to ssDNA by the action of the Mre11 nuclease in concert with its partner CtIP 
. We therefore hypothesized that Mre11 could be involved in the production of ssDNA following Chk1 inhibition as it has been shown to promote the processing of DSB to ssDNA following IR 
. Inhibition of Mre11 did indeed reduce the amount of ssDNA but importantly Mre11 inhibition also led to a decrease in γH2AX and a decrease in DSB. It is of note that while levels of phospho-RPA and γH2AX were dramatically decreased by the presence of mirin, phospho-Chk1 levels were not altered, indicating that the ability of MK-8776 to inhibit Chk1 was not affected.
The DNA endonuclease Mus81 is required for DSB following Chk1 inhibition 
. Mus81 is a substrate-specific endonuclease which cleaves structures mimicking Holliday junctions 
but has been shown to cleave model replication forks and other “nicked” substrates 75-fold more efficiently than stationary Holliday junctions 
. Our data demonstrate that ssDNA occurs upstream of DSB following Chk1 inhibition and we therefore hypothesize that Mre11 may act to provide the substrate for Mus81 cleavage.
Chk1 controls the cell cycle by targeting the protein phosphatase Cdc25A for degradation 
, thereby preventing Cdc25A from dephosphorylating and activating CDK2 
. Following Chk1 inhibition, Cdc25A accumulates leading to increased active CDK2. The means by which Mre11 is activated following Chk1 inhibition remains unclear. CtIP promotes DSB end resection and has been shown to interact with the MRN complex 
. CtIP is phosphorylated by CDK2 
and DSB resection by MRN/CtIP has recently been shown to be CDK2 dependent in Xenopus eggs 
. CDK2 has recently been reported to bind directly to Mre11 and this interaction is required for CtIP phosphorylation 
. Following initial resection by MRN/CtIP, efficient long range strand resection is taken over by the DNA nucleases Exo1 and Dna2. The activation of these two nucleases is also mediated by phosphorylation by CDK2 
. The ssDNA produced by these nucleases is then coated with RPA which recruits ATR and subsequently activates Chk1 
. These findings taken together with the fact that Chk1-induced DNA damage is prevented by the addition of the CDK inhibitor roscovitine, and by depletion of Cdc25A 
led us to propose a pathway for the induction of DNA damage following Chk1 inhibition (). In this pathway, the aberrant activation of CDK2 following Chk1 inhibition leads to activation of Mre11.
There are still many unknown steps in this pathway; in particular why CDK2 does not activate Mre11 in normal S phase. One possibility is that Mre11 activity is suppressed in normal S phase by other factors; for example the Ku and RPA proteins have both been shown to regulate the nuclease activity of the MRN complex 
. The ability of BRCA2 to stabilize Rad51 filaments has also been shown to protect stalled replication forks from degradation by Mre11 
. As Chk1 is required for the interaction of BRCA2 and Rad51 
, it is possible that in the absence of Chk1 activity, BRCA2 is unable to protect stalled forks from Mre11 degradation. Another possible explanation is that, as Chk1 restrains CDK activity in normal DNA replication 
, the high levels of CDK2 which result from the inhibition of Chk1 in normal S phase could result in over-activation of Mre11. Further work is required to elucidate the finer points of this pathway but it will be of importance to determine whether Mre11 function could act as a determinant of cellular sensitivity to Chk1 inhibitors.
In our recent analysis, we observed a large range of sensitivity to MK-8776 following a 24 h incubation 
. Interestingly some cell lines resembled the ATLD1 cells in that they continued to grow when incubated with MK-8776. However, these cells are not deficient in Mre11 
, suggesting there must be alternate mechanisms by which cells can avoid growth inhibition when Chk1 is inhibited. We anticipate that the other steps in the pathway described in can also vary between cell lines, and thereby avoid activation of Mre11. It is also worth recognizing that many previous studies have used U2OS cells as if they are representative of the majority of cell lines, yet our results suggest that in comparison to many other cell lines, U2OS are very sensitive to short-term pharmacologic inhibition of Chk1 
. If normal cells are relatively resistant to MK-8776 as has been suggested 
, our hope is that some tumors will be highly responsive and thereby provide a therapeutic window for successful administration of Chk1 inhibitors to patients.