Checkpoint Control and Cancer

RH Medema1,3 and L Macu˚rek2

1Department of Medical Oncology, University Medical Center, Utrecht, The Netherlands and 2Department of Genome Integrity, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic

DNA-damaging therapies represent the most frequently efficient DNA repair has been recently reviewed used non-surgical anticancer strategies in the treatment of extensively elsewhere (Bekker-Jensen and Mailand, human tumors. These therapies can kill tumor cells, but at 2010; Ciccia and Elledge, 2010; Polo and Jackson, the same time they can be particularly damaging and 2011). This review will focus on the mechanisms that mutagenic to healthy tissues. The efficacy of DNA- activate checkpoints after genotoxic stress and silencing damaging treatments can be improved if tumor cell death of checkpoints during the recovery process that can is selectively enhanced, and the recent application of poly- occur after successful repair of the damage. (ADP-ribose) polymerase inhibitors in BRCA1/2-deficient tumors is a successful example of this. DNA damage is known to trigger cell-cycle arrest through activation of Activation of DNA-damage checkpoints DNA-damage checkpoints. This arrest can be reversed once the damage has been repaired, but irreparable In general, activation of DNA-damage checkpoints is damage can promote apoptosis or senescence. Alterna- enabled by recognition of DNA damage by sensors, tively, cells can reenter the cell cycle before repair has followed by an ordered activation of upstream and been completed, giving rise to mutations. In this review we effector kinases, the latter of which can directly target discuss the mechanisms involved in the activation and the major cell-cycle control machinery (Figure 1a). inactivation of DNA-damage checkpoints, and how the DNA damage can induce a cell-cycle arrest in the G1, transition from arrest and cell-cycle re-entry is controlled. intra-S or G2 phase of the cell cycle. Depending on the In addition, we discuss recent attempts to target the phase of the cell cycle when damage occurs, and the checkpoint in anticancer strategies. mode of DNA damage, cells can activate distinct Oncogene (2012) 31, 2601–2613; doi:10.1038/onc.2011.451; pathways in response to genotoxic stress. published online 3 October 2011 The primary response to DNA damage is accom- plished by upstream kinases of the phosphatidylinositol- Keywords: DNA damage; checkpoint; cancer; check- 3-OH kinase-related family, which includes ATM point recovery (ataxia teleangiectasia mutated kinase), ATR (ATM- and Rad3-related kinase) and DNA-PK (DNA-dependent protein kinase). Whereas ATM and DNA-PK are activated by double-strand breaks (DSBs), ATR re- Introduction sponds to a broader spectrum of DNA-damaging lesions that generate single-stranded DNA (including stalled To maintain genome integrity, cells need to adequately replication forks caused by bulky base adducts or UV respond to various modes of genotoxic stress. This is photoproducts). This simplistic view, however, is in fact achieved by activation of evolutionarily conserved more complex because DSBs are processed during the DNA-damage response (DDR) pathways that abrogate S/G2 phases of the cell cycle by the endonuclease CtIP cell-cycle progression when the genome is damaged and and generate single-stranded DNA lesions leading to a stimulate DNA repair. Depending on the extent of secondary activation of the ATR (Jazayeri et al., 2006; DNA damage, cells either manage to repair all lesions Sartori et al., 2007). All phosphatidylinositol-3-OH and re-enter the cell cycle (checkpoint recovery), or they kinases responding to DNA damage show similar are eliminated by programmed cell death (apoptosis). substrate specificities, and recent phosphoproteomic Alternatively, cells can remain permanently arrested screens reveal a substantial overlap of their substrates after a DNA-damaging insult (senescence). Activation (Matsuoka et al., 2007; Bennetzen et al., 2010; Bensimon of the components of DDR pathway that allows et al., 2010). The best example is probably the histone variant H2AX, which is rapidly phosphorylated at Correspondence: Dr L Macu˚rek, Department of Genome Integrity, Ser-139 (producing gH2AX) by ATM/ATR or DNA-PK Institute of Molecular Genetics, Academy of Sciences of the Czech in the chromatin flanking the damage site. On the other Republic, Videnska 1083, Prague 4 14200, Czech Republic. hand, these kinases also have some exclusive substrates, E-mail: [email protected] which are not shared by the other members. Thus ATM 3Current address: Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands phosphorylates checkpoint kinase-2 (Chk2) at Thr-68, Received 10 July 2011; revised 16 August 2011; accepted 29 August 2011; whereas ATR phosphorylates Chk1 at two residues, published online 3 October 2011 Ser-317 and Ser-345 (Bartek and Lukas, 2003). In general, Checkpoint control and cancer RH Medema and L Macu˚rek 2602 IR or chemotherapy UV or replication stress (Falck et al., 2005). Active ATM generates gH2AX at the surrounding chromatin, which is recognized by DSB ssDNA CtIP (S or G2 phase) RPA phosphopeptide-binding BRCT domains that are present MRN 9-1-1 H2AX ATRIP in many proteins involved in the DDR, and serves as a MDC1 TopBP1 docking site for a large adaptor protein, MDC1 (Stucki RHINO et al., 2005). Recruitment of multiple additional compo- ATM ATR p38 nents of the checkpoint then follows in an orderly Claspin Chk2 Mdm2 manner, including RNF8, RNF168, HERC2, 53BP1 and MK2 Chk1 BRCA1 (reviewed elsewhere Bekker-Jensen and Mai- p53 land, 2010). In general, recruitment of these proteins to Nek11 the chromatin is essential for efficient DNA repair and Bax GADD45 p21 Cdc25A/B/C Wee1 may also have some role in further amplification of the Noxa checkpoint signaling (that is, by recruiting more ATM) (Lee et al., 2010; Shibata et al., 2010). ATM is rapidly cyclin autophosphorylated at Ser-1981, which was originally APOPTOSIS Cdk proposed to induce the dissociation of an inactive CHECKPOINT MAINTENANCE homodimer into active monomers (Bakkenist and & SURVIVAL CHECKPOINT ARREST Kastan, 2003). However, mice lacking the corresponding ATM autophosphorylation sites are still able to fully Aurora-A activate the checkpoint after exposure to ionizing H2AX hBora radiation (IR), strongly suggesting that ATM autopho- ATM sphorylation is not an essential step for its activation Plk1 (but a useful marker of ATM activity) (Pellegrini et al., Mdm2 Wip1 MdmX 2006). Instead, DNA damage-induced acetylation of ATR ATM at Lys-3016 by the acetyltransferase Tip60 was Claspin recently proposed to activate the ATM (Sun et al., 2005, 2007). In this model, Tip60 is recruited to DSBs by a GTSE1 p53 Chk1 direct interaction with the Mre11–Rad50–Nbs1 complex and its enzymatic activity toward ATM is increased after binding of its chromodomain to trimethylated histone p21 Cdc25A/B/C Wee1 H3 (H3K9me3) (Sun et al., 2009; Chailleux et al., 2010). By contrast, single-stranded DNA lesions (such as the cyclin B stalled replication forks or resected DSBs) are rapidly Cdk1 coated by replication protein-A complexes, which further recruit and activate ATR in a stable complex CHECKPOINT RECOVERY with its cofactor ATRIP (Zou and Elledge, 2003). In parallel, replication protein-A binds to Rad17 and Figure 1 (a) Checkpoint activation. DNA damage is recognized by various sensor and adaptor proteins, which leads to activation of recruits a ring-shaped trimeric complex, Rad9–Hus1– ATM and ATR kinases and allows establishing of a DNA-damage Rad1 (9-1-1), to the site of damage. This 9-1-1 complex checkpoint. The major components activated during this response is then phosphorylated by ATR and can bind to the are Chk1 and p53 (green). Chk1 regulates cdc25A/B/C and Wee1, BRCT domains of an adaptor protein, TopBP1, which which leads to inhibitory phosphorylation of Cdk (yellow dots), further boosts the activity of ATR (Cimprich and prevents its activation (red) and causes cell-cycle arrest. p53 is stabilized by multiple posttranslational modifications (dots), and by Cortez, 2008). RHINO, a recently identified 9-1-1- and increasing the transcription of a Cdk inhibitor, p21, and repressing TopBP1-interacting protein, is required for efficient the transcription of cyclin-B, it contributes to inhibition of cyclin– activation of ATR (Cotta-Ramusino et al., 2011). Active Cdk. In addition, cells activate the Chk2 and p38/MK2 pathways ATR has several hundreds of substrates, including that are involved in apoptosis and checkpoint maintenance, Chk1, which is essential for induction of the checkpoint respectively. (b) Recovery from the G2 checkpoint. After DNA repair, multiple proteins are de-phosphorylated by Wip1 phospha- response. Importantly, Chk1 needs to form a complex tase, which leads to silencing of the checkpoint signaling and with a mediator protein, claspin, to be efficiently inactivation of p53. In parallel, Plk1 kinase targets claspin and activated by ATR (Chini and Chen, 2003; Kumagai Wee1 for degradation, and activates Cdc25A/B/C, which allows et al., 2004). Upon phosphorylation, Chk1 is released activation of cyclin-B–Cdk1 and checkpoint recovery. from the chromatin and can freely diffuse through the nucleus to phosphorylate multiple substrates to establish phosphorylation of Chk2 and Chk1 leads to their the checkpoint response (see below) (Smits et al., 2006). activation and further transmission of the checkpoint signal (see below). Activation of the upstream checkpoint kinases requires the recognition of the DNA lesion. In case of Checkpoint kinases—effectors of the checkpoint DSBs, this is achieved by the Mre11–Rad50–Nbs1 complex that directly binds to the exposed ends of Proliferating cells repeatedly pass through the G1 the DNA, recruits ATM and initiates its activation (growth phase), S (replication of DNA) and G2 phases

Oncogene Checkpoint control and cancer RH Medema and L Macu˚rek 2603 of the cell cycle, followed by nuclear division (mitosis) from HCT116-Chk2À/À human colon cancer cells and cellular division (cytokinesis). Transitions between indicate that, in contrast to yeast, Chk2 is functionally the different phases of the cell cycle are facilitated by redundant in checkpoint activation in higher eukaryotes evolutionarily conserved cyclin-dependent kinases (Jack et al., 2002; Jin et al., 2008). Instead, Chk2 seems (Cdks), which act in complex with various cyclins to adopt a unique role in the regulation of the apoptotic À/À (mainly Cdk2/cyclin-E in G1/S and Cdk1/cyclin-B in pathways induced by DNA damage, as Chk2 mice are G2/M transition). Activity of a Cdk is negatively more resistant to IR compared with the control animals regulated by phosphorylation of Thr-14 and Tyr15 by (Hirao et al., 2002; Takai et al., 2002). IR stimulates Wee1 and Myt1 kinases. Conversely, Cdks are activated ATM to activate Chk2 by phosphorylation at Thr-68, by de-phosphorylation of these residues by the Cdc25 which in turn leads to stabilization of the tumor family of phosphatases. The cell-cycle arrest induced in suppressor p53 and promotes the transcription of response to DNA damage is at least in part established several pro-apoptotic genes such as Bax, Puma and through inhibition of Cdc25 activity. Human cells Noxa (Ahn et al., 2000; Hirao et al., 2000, 2002; Takai express three distinct isoforms of Cdc25, Cdc25A, B et al., 2002). Phosphorylation of p53 at Ser-20 was and C, all of which can dephosphorylate and activate originally proposed to be responsible for the Chk2- Cdks. After DNA damage, Cdc25A is rapidly degraded, dependent activation of p53; however, this modification which leads to increased inhibitory phosphorylation at was retained in Chk2À/À cells as well as in Chk2-depleted Tyr-15 of Cdk2 or Cdk1, and causes a G1 or G2 cells, suggesting that additional mechanisms may exist checkpoint arrest, respectively (Mailand et al., 2000). by which Chk2 modulates apoptotic responses (Ahn Destruction of Cdc25A is executed by bTrCP–SCF- et al., 2003; Jallepalli et al., 2003). Indeed, Chk2 has also dependent ubiquitination and proteasomal degradation, been reported to regulate apoptosis after treatment with and is induced by Cdc25A phosphorylation at Ser-76 etoposide in an p53-independent manner through mainly by Chk1 (Jin et al., 2003, 2008). Chk1 also phosphorylation of E2F1 (Stevens et al., 2003). Chk2 autophosphorylates at Ser-296, which generates a motif seems to be specifically involved in regulating apoptotic recognized by 14-3-3g that mediates the interaction of responses to agents causing DSBs as UV-induced Chk1 with Cdc25A (Kasahara et al., 2010). In parallel, apoptosis is not affected in Chk2À/À cells (Hirao et al., Chk1 also activates Nek11 (Never in mitosis gene-A- 2000). This is in good agreement with Chk2 being related kinase-11), which further phosphorylates specifically activated by ATM but not ATR. Strikingly, Cdc25A at Ser-82 and enables its recognition by the functional redundancy of Chk2 for checkpoint the bTrCP–SCF (SKP/Cullin/F-box protein) complex activation is lost when cells lose p53 (Jiang et al., (Melixetian et al., 2009). The ATM–Chk2 pathway 2009). In contrast to p53-proficient cells, depletion of was originally also suggested to be involved in the Chk2 in p53-deficient cells interferes with the IR-induced degradation of Cdc25A (Falck et al., 2001); however checkpoint arrest (Jiang et al., 2009). Reasons for the finding that IR-induced degradation of Cdc25A in rewiring of checkpoint-activating pathways in p53- HCT116-Chk2À/À cells occurs with the same kinetics as deficient cells are currently unclear; however, an inter- in wt-HCT116 cells indicates that the contribution of esting possibility is that, by losing p53, higher eukaryotic Chk2 to degradation of Cdc25A is either functionally cells simply regain similar molecular pathways that act in redundant or occurs in a cell type-specific manner (Jin et al., yeast and that were suppressed during evolution. 2008). In addition, Cdc25B and Cdc25C are phosphory- Another mechanism implicated in G1 checkpoint lated by Chk1/2 at Ser-323 and Ser-216, respectively, and induction is rapid degradation of cyclin-D1 after are functionally inactivated by binding to 14-3-3 proteins exposure to IR (Agami and Bernards, 2000). Degrada- (Peng et al., 1997; Sanchez et al., 1997; Forrest and tion of cyclin-D1 is believed to release the Cdk inhibitor Gabrielli, 2001). Nonetheless, mice deficient in Cdc25B p21 from Cdk2/cyclin-D1 complexes, which in turn and Cdc25C are still able to fully activate the DNA- allows p21 to inhibit Cdk2/cyclin-E and prevent the damage checkpoints, indicating that mere regulation of G1/S transition. Degradation of cyclin-D1 was originally Cdc25A is sufficient to establish the checkpoint (Fergu- reported to depend on a destruction motif within cyclin- son et al., 2005). This observation is in line with the D1 and ubiquitination by the APC E3 ligase complex current view of functional redundancy in the molecular (Agami and Bernards, 2000). However, recent evidence machinery that controls the G2/M transition (Lindqvist suggests that after DNA damage, cyclin-D1 is ubiqui- et al., 2009b). In addition to its effect on Cdc25A, Chk1 tinated by the SCF E3 ligase complex and this depends can also phosphorylate and activate Wee1 kinase, which both on phosphorylation of cyclin-D1 at Thr-286 by can directly increase the inhibitory modification of Cdk2 glycogen synthase kinase-3b and a direct phosphoryla- and Cdk1 (O’Connell et al., 1997). tion of the F-box protein FBXO31 by ATM (Santra Importantly, the role of Chk1 in checkpoint control is et al., 2009). Thus, it is possible that the precise evolutionarily conserved from yeast to humans and mechanism by which cells degrade cyclin-D1 after human Chk1 operates by the same molecular mechan- DNA damage is cell type-dependent. Moreover, it also isms as its yeast homolog (Sanchez et al., 1997). Chk2 appears that DNA damage specifically induces the (homologous to Rad53 and Cds1 in yeast) is structurally degradation of cyclin-D1 (but not cyclins D2 and D3) distinct from Chk1, but both kinases share similar indicating that the role of this pathway in the DDR in substrate specificity and may therefore have overlapping G1 might depend on the expression profile of D-type substrates. Data from Chk2-knockout mice and also cyclins in various tissues.

Oncogene Checkpoint control and cancer RH Medema and L Macu˚rek 2604 Finally, p38 and its downstream effector MAPKAP transcriptional coactivators (or co-repressors); and kinase-2 (MK2) have recently been implicated in the binding of p53 to the promoters of its multiple target DDR in G2 after exposure to UV (Bulavin et al., 2001; genes (for a recent review on p53 posttranslational Manke et al., 2005). The contribution of p38 to the modifications see Dai and Gu (2010)). One of these, the DDR was attributed to the phosphorylation of cdc25B cyclin-dependent kinase inhibitor p21, binds to and and cdc25C by MK2 (Manke et al., 2005). Moreover, inhibits the cyclin-E/Cdk2 and cyclin-A/Cdk2 com- rapid and transient activation of the p38/MK2 pathway plexes, and has a major role in the control of G1 arrest. was also reported after IR, or treatment with doxor- Similarly, p21 also contributes to maintenance of the G2 ubicin or alkylating agents such as methyl methanesul- checkpoint (Bunz et al., 1998). In addition, p53 can also fonate (MMS) (Mikhailov et al., 2004; Raman et al., control the G2 checkpoint independently of p21 through 2007; Lafarga et al., 2009; Phong et al., 2010). transcriptional repression of mitotic inducers, including Activation of the p38/MK2 branch of the checkpoint cyclin-B, cdc25B and polo-like kinase-1 (Plk1) (Imbriano has been reported to involve ATM/ATR-dependent et al., 2005; McKenzie et al., 2010; Dalvai et al., 2011). activation of Thousand and one amino acid protein Interestingly, recent data indicate that the dynamics of kinases (TAO) that belong to the MAPKKK family. p53’s response to IR does not occur in a linear manner TAO-dependent phosphorylation of MEK3 and MEK6 but instead appears in multiple repeating pulses with fixed than induces the activity of p38 (Raman et al., 2007). In amplitude and duration (Lahav et al., 2004; Batchelor addition, the p38 pathway might be involved in the et al., 2008). These oscillations had long remained masked initiation of the G1 checkpoint by a rapid stabilization owing to averaging across a population of cells, but their of p21 mRNA caused through phosphorylation of the observation became possible with recent advances in mRNA-binding protein HuR by p38 at Thr-118 single-cell imaging techniques (Batchelor et al., 2009). The (Lafarga et al., 2009). Despite all this, the role of the oscillatory behavior of p53 is induced by activation of p53 p38 pathway in checkpoint activation has recently been by ATM at the time DNA damage is present, which challenged when p38 pathway was suggested to have an subsequently results in the transcriptional activation of essential role in survival after DNA damage through Mdm2 and Wip1 (see below), both negative regulators of induction of multiple survival genes (Phong et al., 2010). p53, which establish negative feedback loops (Batchelor In addition, p38/MK2 seems to be involved in check- et al., 2009) (Figure 2). In response to DSBs, cells stabilize point maintenance (see below). Thus, the precise role of p53, generating the first pulse, which is then followed by the ATM/ATR–p38–MK2 pathway in DDRs remains pulses of a similar intensity repeating in 4- to 7-h intervals incompletely understood and it is possible that it largely until the DNA is fully repaired. Strikingly, spontaneous depends on the mode and amplitude of DNA damage. pulses of p53 expression of comparable intensity can also occur in normal cycling cells; however, posttranslational modifications keep p53 inactive in the absence of sustained damage (Loewer et al.,2010).Bycontrast,UV Checkpoint maintenance irradiation causes an induction of p53, but does not induce oscillations as seen after IR. Instead, UV induces An appropriate checkpoint response to genotoxic stress a single p53 pulse the amplitude of which increases with needs to be fast enough to prevent transition to the next the dose of UV (Batchelor et al., 2011). The role of IR- phase in the cell cycle with damaged DNA, but durable induced p53 pulses in determining cell fate still needs to enough to allow time for efficient DNA repair. Recent be addressed fully; however, it opens up an exciting work has made it clear that induction and maintenance possibility that, by recurring phases of strong check- of the checkpoints are governed by distinct molecular point output with weak checkpoint output, cells can mechanisms. As discussed above, the fast checkpoint sense the actual strength of the DNA damage and can induction largely depends on the transmission of the prevent inappropriate initiation of apoptotic cell death signal through phosphorylation of multiple substrates (Batchelor et al., 2009). by ATM/ATR and Chk1/2 kinases, affecting their Importantly, the DDR in G1 is primarily dependent activity, protein stability or both. Conversely, pathways on p53. As a consequence, cancer cells that have lost p53 that depend on changes in the transcription of target fail to establish a G1 arrest in response to DNA damage, genes act considerably slower and are mainly involved in but arrest in G2 instead, a response that remains checkpoint maintenance. The best-studied example of relatively intact also in cancer cells (Kuntz and checkpoint maintenance is the contribution of the tumor O’Connell, 2009). This implies that, apart from p53, suppressor p53 and its transcriptional target p21. p53 is additional mechanisms must exist to prevent p53- extensively modified posttranslationally, especially in deficient cells from entry into mitosis with damaged the N-terminal transactivation domain (including phos- DNA. Survival of p53-deficient cells after DNA damage phorylation of Ser-15 and Ser-20 by ATM/ATR/DNA-PK was recently reported to depend on the p38/MK2 and Chk1/2, respectively) and in the C-terminal regu- pathway (Reinhardt et al., 2007). Whereas the Chk1 latory domain (including acetylation of Lys-382 and pathway was found to be important to establish the G2 Lys-320 by CBP/p300 and PCAF, respectively), which checkpoint, the p38/MK2 pathway was shown to be together reduce the affinity of p53 for its negative critical for long-term maintenance of the G2 checkpoint regulator Mdm2. This allows stabilization and tetra- (Reinhardt et al., 2010). Strikingly, Chk1 and MK2 merization of p53; promotes association of p53 with kinases share the same optimal phosphorylation motif

Oncogene Checkpoint control and cancer RH Medema and L Macu˚rek 2605 cytosolic sequestration of cdc25B/C (Reinhardt et al., IR 2010). This ﬁnding is in line with the observation that the pro-survival activity of GADD45a in hematopoietic cells exposed to UV depends on the p38 pathway (Gupta et al., 2006). However, GADD45a was also reported to ATM directly associate with and inhibit cyclin-B/Cdk1 (Wang et al., 1999; Zhan et al., 1999), and thus it is possible that GADD45a prevents premature mitotic entry through multiple independent mechanisms. In addition, the p38 pathway was also reported to regulate checkpoint maintenance through stabilization of p27Kip, which p53 can further suppress any residual Cdk activity in case the DNA damage persists (Cuadrado et al., 2009; Liontos et al., 2010). As checkpoints need to be maintained until the DNA damage is fully repaired, DNA-repair pathways are Wip1 Mdm2 likely to be functionally connected with checkpoint signaling. Indeed, short interfering RNA screens for DNA repair factors involved in checkpoint regulation revealed that two homologous recombination-repair proteins, BRCA2 and PALB2, are required for G2 checkpoint maintenance (Cotta-Ramusino et al., 2011; Menzel et al., 2011). It is possible that by using DNA- repair factors, cells coordinate checkpoint signaling with ongoing DNA repair; however, the exact mechanism by p53 levels which BRCA2 and PALB2 communicate with the checkpoint still remains to be explored.

time Silencing of checkpoints by phosphatases and checkpoint IR repair recovery Figure 2 Dynamics of the p53 response induced by DNA damage. In unstressed cells, basal levels of p53 are kept low by Mdm2- After successful DNA repair, cells re-enter the cell cycle dependent degradation. In response to IR-induced damage, ATM in a process called checkpoint recovery. Owing to the is activated and phosphorylates p53 and Mdm2 both resulting in inactivation and degradation of a multitude of cell- stabilization of p53. Active p53 stimulates the transcription of Mdm2 and Wip1 mRNA, which leads to a delayed increased cycle-regulatory proteins, which occurs in response to expression of these proteins. In turn, Wip1 de-phosphorylates p53, DNA damage, cells that are arrested in response to ATM and Mdm2, generating negative feedback loops that lead to DNA damage are biochemically different from unda- destabilization of p53. This response results in regular oscillations maged cells in the same phase of the cell cycle. This has of p53 levels until the DNA damage is repaired. Spontaneous pulses of p53 transcription can be generated by transient damage the consequence that the cell-cycle machinery operates that occurs during the cell-cycle phase associated with intrinsic differently during recovery as compared with control DNA damage. The green lines indicate activation, red lines of the cell cycle in unperturbed cells. Currently, the inactivation. best understood mechanisms that promote recovery are mechanisms responsible for checkpoint recovery from the G2 checkpoint (Figure 1b). Whereas there (Arg-X-X-pSer/Thr) and thus may have overlapping is a considerable degree of functional redundancy substrates (Manke et al., 2005). However, this does not in mechanisms regulating unperturbed G2/M progres- appear to be the entire story, as both kinases show a sion, certain pathways become essential when cells distinct subcellular distribution after treatment with recover from DNA damage (Lindqvist et al., 2009b). doxorubicin. Whereas Chk1 remains mainly nuclear In particular, this is true for Plk1 (and its yeast homolog after DNA damage, activated MK2 rapidly redistributes cdc5) depletion or inhibition of which does not affect to the cytoplasm, where it phosphorylates distinct normal mitotic entry but completely blocks checkpoint substrates (Reinhardt et al., 2010). By phosphorylating recovery (Toczyski et al., 1997; van Vugt et al., 2004; an RNA-binding protein hnRNPA0 and a ribonuclease Le´na´rt et al., 2007). Plk1 contains a conserved threonine PARN, activated MK2 induces the accumulation of residue (Thr-210) within the T-loop, which has to be growth arrest and DNA-damage-inducible protein-a phosphorylated to fully activate Plk1 (Jang et al., 2002). (GADD45a) mRNA, leading to increased protein In late G2, Aurora-A kinase, together with its cofactor, levels of GADD45a (Reinhardt et al., 2010). In turn, hBora, phosphorylates Plk1 at Thr-210, resulting in GADD45a was suggested to act in a positive feedback activation Plk1 (Macurek et al., 2008; Seki et al., 2008). loop and to induce p38/MK2-dependent phosphoryla- In response to DNA damage, activity of Plk1 is kept low tion of cdc25B/C, binding of 14-3-3 protein and through inhibition of Thr-210 phosphorylation (Smits

Oncogene Checkpoint control and cancer RH Medema and L Macu˚rek 2606 et al., 2000; Tsvetkov and Stern, 2005). Plk1 activation More is known about the contribution of phospha- can promote mitotic entry in an unperturbed cell cycle, tases to checkpoint silencing and recovery. Recent data but following a DNA-damaging insult, cells come to from several groups indicate that a PP2Cd phosphatase completely rely on Plk1 to re-enter the mitotic cycle (van (PPM1D, hereafter referred to as Wip1) has a central Vugt et al., 2004). To promote recovery, Plk1 targets role in termination of the checkpoint signaling and in claspin and Wee1 for bTrCP–SCF-dependent degrada- checkpoint recovery (Lu et al., 2008; Le Guezennec and tion (Watanabe et al., 2004; Mailand et al., 2006; Bulavin, 2010). Several lines of evidence suggest that the Mamely et al., 2006; Peschiaroli et al., 2006), promotes function of Wip1 is tightly linked with regulation of the nuclear translocation of Cdc25B/C (Toyoshima-Mor- DDR. First, depletion of Wip1 by RNA interference imoto et al., 2002; Lobjois et al., 2009) and directly results in prolonged checkpoint activation whereas inhibits Chk2 (van Vugt et al., 2010). As a result, Plk1 overexpression of Wip1 causes efficient checkpoint effectively prevents further activation of both Chk1/2 override, indicating that Wip1 has the potential to and contributes to activation of cyclin-B/cdk1 com- regulate checkpoint signaling (Lu et al., 2005; Lindqvist plexes, stimulating checkpoint silencing and recovery at et al., 2009a). Second, the substrate specificity of the multiple levels (van Vugt et al., 2004). In addition, Plk1 Wip1 seems to correspond with the motifs phosphory- phosphorylates G2- and S-phase-expressed protein-1, lated during the DDR, namely pSQ/pTQ motifs that are which acts as a negative regulator of p53 and thus Plk1 selectively phosphorylated by ATM/ATR and the activity contributes to suppression of p53 during pTxpY motif in p38 that is also phosphorylated in checkpoint recovery (Liu et al., 2010). response to DNA damage (Yamaguchi et al., 2007). Plk1 activity is essential but not sufficient for By recognizing such motifs, Wip1 could efficiently target checkpoint recovery, indicating that additional control all of the substrates of ATM/ATR, and this is has thus mechanisms exist. Among them, phosphatases, which far been confirmed for ATM–pSer-1981, Chk1–pSer-317, counteract the multitude of protein phosphorylations Chk2–pThr-68, p53–pSer-15, Mdm2–pSer-395 and implemented by the checkpoint machinery, are likely gH2AX (Takekawa et al., 2000; Fujimoto et al., 2005; candidates to be involved in silencing the checkpoint Lu et al., 2005, 2007; Shreeram et al., 2006a; Cha et al., and promoting checkpoint recovery (Freeman and 2010; Macurek et al., 2010; Moon et al., 2010). Third, Monteiro, 2010). Data analysis from large phosphopro- PPM1D is a target gene of p53 and expression of Wip1 teomic screens indicates that phosphatases may have a is increased after genotoxic stress (Fiscella et al., 1997). more active role in the regulation of DDRs than Like this, cells can cycle with low levels of Wip1 originally anticipated (Bensimon et al., 2010). So far (limiting the risk of an undesired de-phosphorylation of the only well-characterized phosphatase that has been any phosphoprotein involved in normal cell progres- described to actively participate to establish the cell- sion), whereas DNA damage releases a negative feed- cycle arrest is PP2A in complex with its regulatory back loop in which expression of Wip1 will limit the subunit, B56g. Thus, B56g is stabilized in an ATM- amplitude of the checkpoint to prevent an irreversible dependent manner and activates p53 through de- arrest. Fourth, premature translation of Wip1 mRNA is phosphorylation of pThr-55 (Li et al., 2007; Shouse blocked by miR-16, which is also induced by genotoxic et al., 2011). On the other hand, the major cellular stress (Zhang et al., 2010). This mechanism assures that phosphatase PP2A has also been shown to limit basal early after DNA damage Wip1 levels are kept low to phosphorylation of ATM–pSer-1981, Chk2–pThr-68, allow efficient checkpoint induction and DNA repair. Chk1–pSer-317 and gH2AX (Goodarzi et al., 2004; Fifth, Wip1 is a nuclear protein and is enriched at the Chowdhury et al., 2005; Leung-Pineda et al., 2006; chromatin, where it can get in close contact with the Carlessi et al., 2010; Freeman et al., 2010). By keeping a proteins of the DDR pathway (Macurek et al., 2010). relatively high basal activity of PP2A, cells can therefore Finally, although Wip1 is not present in yeast, it appears prevent inadequate activation of the checkpoint under that low eukaryotes use the same family of PP2C normal conditions. DNA damage (perhaps when phosphatases (namely Ptc2 and Ptc3) to regulate exceeding a certain threshold) rapidly disrupts the checkpoint recovery, indicating partial conservation in interaction of PP2A with ATM, Chk1 and Chk2, evolution (Leroy et al., 2003). allowing full activation of the checkpoint (Goodarzi Among many substrates of Wip1, p53 seems to have a et al., 2004; Carlessi et al., 2010; Freeman et al., 2010). special role in checkpoint recovery. We have noted that Similarly, PP1 phosphatase in complex with its chroma- even complete inhibition of Chk1/2, ATM/ATR and tin-targeting subunit, Repo/Man, was recently demon- p38 is not sufficient to promote recovery in Wip1- strated to inhibit the activity of ATM under unstressed depleted cells (Lindqvist et al., 2009a). On the other conditions, thus determining the threshold for activation hand, cells lacking both Wip1 and p53 recovered of the DNA-damage checkpoint (Peng et al., 2010). normally, indicating that p53 is the crucial target of It is possible that after successful DNA repair, the re- Wip1 to control recovery. This also implies that de- established interaction of PP2A and/or PP1 with various phosphorylation of all other Wip1 substrates is less components of the DDR pathway helps to revert protein relevant for efficient recovery. What is more, we could phosphorylation back to the original level, contributing show that Wip1 is required throughout the checkpoint to the silencing of the checkpoint. However, it remains response, not just at the stage that the checkpoint is unclear to what extent cells actively regulate these definitively terminated (Lindqvist et al., 2009a). This phosphatases during the DDR. means that the activity of Wip1 is required throughout

Oncogene Checkpoint control and cancer RH Medema and L Macu˚rek 2607 the DNA-damage checkpoint to prevent excessive p53 therapies, such as radiotherapy and a large proportion activation and is essential to retain checkpoint recovery of chemotherapeutic therapies. This treatment targets competence (Lindqvist et al., 2009a). Interestingly, Wip1 genetically instable cancer cells (that are prone to cell can regulate p53 by multiple mechanisms. Apart from death caused by genotoxic stress) but also hits normal direct de-phosphorylation of pSer15 on p53, Wip1 has tissues, especially those with high proliferative rates been shown to activate the ubiquitin E3 ligase mdm2, (such as epithelia in the gastrointestinal tract, hair which targets p53 for proteasomal degradation, and follicles and bone marrow). Undesired targeting of activate MdmX, which directly inhibits the transcrip- healthy tissues by DNA-damaging agents represents a tional activity of p53 (Lu et al., 2005, 2007; Zhang et al., major problem in the clinics and limits efficiency in 2009). Which one of these mechanisms is the most curing cancer. physiologically relevant is currently unclear; however, A large subset of cancer types is incapable to establish counteracting p53’s function seems to be the major role the G1 checkpoint, most often because of mutation or for Wip1 to prevent an irreversible cell-cycle arrest. In loss of the p53 tumor suppressor. Survival of these cells addition, the two negative feedback loops in which after a DNA-damaging treatment depends on activation Wip1 inactivates p53 and ATM are required to establish of the G2 checkpoint by the ATR/Chk1, p38/MK2 and the dynamic behavior of the p53 response that follows ATM/Chk2 pathways (Wang et al., 1996; Zhao et al., after DSBs (see above). 2002; Reinhardt et al., 2007; Jiang et al., 2009). As a Importantly, Wip1 is overexpressed (usually because of consequence, cells deficient of p53 are more vulnerable amplification of the 17q23 locus) in multiple human to inactivation of the G2 checkpoint. This finding led to cancers, including medulloblastomas, neuroblastomas, a novel strategy of chemosensitization, which combines breast, ovarian and gastric carcinomas (Bulavin et al., treatment with a DNA-damaging agent with checkpoint 2002; Li et al., 2002; Saito-Ohara et al.,2003;Castellino inhibitor(s), thus preventing cell-cycle arrest and pro- et al., 2008; Tan et al., 2011). Moreover, overexpression moting cell death through mitotic catastrophe (Zhou of Wip1 is linked to poor prognosis in lung adenocarci- and Bartek, 2004) (Figure 3). Previous studies showed noma (Satoh et al., 2011). High expression levels of Wip1 that disruption of the Chk1 pathway, either by short are usually found in tumors that retain wild-type p53, interfering RNA or by treatment with the Chk1 whereas overexpression of Wip1 is rare in tumors inhibitor UCN-01, abrogates G2 checkpoint activation carrying mutations or lacking p53 (Bulavin et al., 2002). induced by various DNA-damaging agents (including Strikingly, Wip1-knockout mice are viable and are IR, 5-fluorouracil, cisplatin and campotecin), and leads resistant to oncogene-induced as well as spontaneous to premature mitotic entry and cell death (Zhao et al., cancer development (Bulavin et al., 2004; Harrison et al., 2002; Xiao et al., 2003). Subsequently, UCN-01 was 2004; Belova et al., 2005; Nannenga et al., 2006; Shreeram used in phase-I trials in combination with cisplatin et al., 2006b), suggesting that inhibition of Wip1 activity or irinotecan (Lara et al., 2005; Fracasso et al., 2010; may potentially be beneficial in cancer therapy. During prolonged checkpoint activation, cells need to Activated maintain expression of critical cell-cycle regulators above Treatment pathway Outcome a minimal level to remain competent for eventual checkpoint recovery after successful repair. This has been ATM/Chk2/p53 shown to be the most crucial function of Wip1 during a Healthy cells IR Checkpoint arrest ATR/Chk1 checkpoint response in G2. When cells depleted of Wip1 are treated with DNA-damaging agents, expression of cyclin-B (as well as a number of other cell-cycle- p53 mutated ATM/Chk2/p53 IR Checkpoint arrest regulatory proteins) decreases below the minimal level cancer cells required for recovery (Lindqvist et al., 2009a). The ATR/Chk1 excessive reduction in cyclin-B expression is due to disproportionate activation of p53, which occurs in cells ATM/Chk2/p53 lackingWip1(Lindqvistet al., 2009a). When expression IR/Chk1i Mitotic catastrophe of cyclin-B reduces below a critical threshold, cells lose ATR/Chk1 the competence to recover. This demonstrates the IR/Wip1i importance of continued expression of the G cluster of p53wt ATM/Chk2/p53 2 or Apoptosis cancer cells cell-cycle-regulated genes during a DNA-damage-induced IR/Nutlin ATR/Chk1 arrest in G2. Indeed, it was demonstrated recently that minimal basal Cdk activity and transactivation by the Figure 3 Checkpoint regulators and cancer therapy. Healthy cells with intact checkpoint components are able to fully activate the transcriptional factor FoxM1 is needed to sustain cyclin- checkpoint after genotoxic stress. Tumor cells lacking functional B expression at a level that can facilitate recovery once the p53 show a weak G1 checkpoint but are still able to arrest in G2 checkpoint is silenced (Alvarez-Fernandez et al.,2010). thanks to the ATR/Chk1 pathway. Exposure of these cells to DNA damage together with inhibition of Chk1 prevents checkpoint Targeting checkpoint components in cancer therapy activation and leads to cell death by mitotic catastrophe. Tumor cells with wild-type p53 activate the checkpoint when exposed to DNA damage; however, stimulation of p53 by treatment with Induction of cell death by excessive DNA damage nutlin-3 or by inhibition of Wip1 phosphatase may lead to represents the general principle of various cancer extensive p53 response and apoptotic cell death.

Oncogene Checkpoint control and cancer RH Medema and L Macu˚rek 2608 Ma et al., 2011). However, low specificity of UCN-01 the establishment of reliable predictive biomarkers (such for Chk1, as well as unfavorable pharmacokinetic as the status of the p53 and ATM pathways) and profiles, prevented further use of UCN-01 in the clinic. arguably will be limited to situations where tumor tissue A new generation of ATP-competitive inhibitors of samples are available (Jiang et al., 2009). The observa- Chk1 shows higher selectivity toward Chk1/2 than tion that survival of p53-deficient cells exposed to UCN-01 (Ma et al., 2011). For example, AZD7762 is genotoxic stress critically depends on the p38/MK2 comparably efficient toward Chk1 and Chk2 (IC50 ¼ 5– pathway (Reinhardt et al., 2007) opens an intriguing 10 nM), whereas the PF477736 and SCH900776 com- possibility that patients receiving chemotherapy could pounds selectively inhibit Chk1 (IC50 ¼ 0.49 and 3 nM, potentially benefit from adjuvant therapy with p38 respectively). Preclinical testing showed that these novel inhibitors; however this novel concept awaits experi- Chk1 inhibitors efficiently abrogate the G2 checkpoint mental testing. and sensitize p53-deficient cells to various DNA-dama- Importantly, chemosensitization seems to work effi- ging agents (Ma et al., 2011), and it will be interesting to ciently only in cells with a deficient p53 pathway (Zhou see how these inhibitors will perform in ongoing clinical and Bartek, 2004; Ma et al., 2011). Thus, although this trials. An important aspect that needs to be considered strategy could be useful in cancers with mutated p53, when using Chk1 inhibitors in the clinics is the role of there is a need to develop other strategies suitable for the Chk1 outside the checkpoint control. Essential cancers that retain wild-type p53. Nutlin-3a was the first functions of Chk1 at the organismal level are well- identified small-molecule antagonist of MDM2 that documented by the early embryonic lethality of Chk1- specifically blocks interaction between MDM2 and p53, knockout mice as well as by severe defects in the leading to stabilization of p53 and inducing apoptosis or mammary epithelia in the conditional Chk1 haploinsuf- cellular senescence (Vassilev et al., 2004). Strikingly, ficiency mouse model (Liu et al., 2000; Takai et al., 2000; Nutlin-3a has a strong antitumor activity in tumor cells Lam et al., 2004). Chk1 appears to be essential for that retain wild-type p53, and promising preclinical maintenance of genome integrity by monitoring replica- results for Nutlin-3a were reported with various tumor tion forks during normal S-phase progression (Takai types (Secchiero et al., 2011; Shen and Maki, 2011). et al., 2000; Syljuasen et al., 2005; Maya-Mendoza et al., Similarly to Nutlin-3a, MI-219 blocks the MDM2–p53 2007). Thus inhibition of Chk1 together with treatments interaction, but it shows higher affinity to MDM2 and that cause stalling of replication forks may lead to excellent pharmacokinetic properties (Shangary et al., irreversible replication fork collapse (Maya-Mendoza 2008). Another potential pharmacological target in p53- et al., 2007). In addition, recent reports suggest that positive tumors is Wip1, which acts in silencing of the Chk1 may also contribute to the regulation of multiple G2 checkpoint primarily through blocking the activity of aspects of unperturbed mitotic progression and cell p53 (Lindqvist et al., 2009a). Depletion of Wip1 by division (Kramer et al., 2004; Zachos et al., 2007; antisense oligonucleotides or by RNA interference Wilsker et al., 2008; Peddibhotla et al., 2009). In this decreased viability in cell lines derived from neuroblas- respect it might be beneficial to pharmacologically target toma, glioma, breast adenocarcinoma and ovarian clear downstream effectors of Chk1 specifically acting in the cell carcinoma, indicating that a subset of cancer checkpoint pathway. A promising example is the Wee1 patients suffering from p53-positive tumors could kinase, which acts downstream from Chk1 and regulates benefit from blocking the activity of Wip1 (Saito-Ohara the G2/M transition. MK1775, a small-molecule inhi- et al., 2003; Rayter et al., 2008; Tan et al., 2009; Wang bitor of Wee1, has recently been developed and showed et al., 2011). Indeed, PPM1D-knockout mice are viable chemosensitization of p53-deficient tumor cells and and are protected from tumor development, indicating xenografts to gemcitabine, cisplatin and 5-fluorouracil, that loss of Wip1 activity is well-tolerated and may basically mimicking the effects of Chk1 inhibition (Hirai block tumor growth (Bulavin et al., 2004; Harrison et al., 2009, 2010; Rajeshkumar et al., 2011). Similarly, et al., 2004). However, development of specific Wip1 the PD0166285 compound, which also inhibits Wee1, inhibitors represents a major challenge owing to the showed radiosensitization of various cancer cell lines relatively shallow groove in the active site and the (Wang et al., 2001). As discussed above, efficient high homology with other PP2C family phosphatases checkpoint activation in cells that lack p53 requires (Harrison et al., 2004). Cyclic phosphopeptides that also activity of Chk2, and it has been suggested that mimic the binding of a substrate to the active site of inhibition of the ATM/Chk2 pathway might sensitize Wip1 were shown to inhibit the activity of Wip1 in vitro; p53-deficient tumors to DNA-damaging treatments or however their efficiency in inhibiting Wip1 in cells to poly-(ADP-ribose) polymerase inhibitors (Jiang et al., still requires further testing (Yamaguchi et al., 2006). 2009; Anderson et al., 2011). The use of Chk2 inhibitors Chemical library screening yielded a compound that might also bring additional benefits to surrounding inhibited Wip1 activity in vitro and also increased the healthy tissues expressing intact p53, as this could phosphorylation of p38, JNK and extracellular signal- prevent an apoptotic response when Chk2 is inhibited regulated kinase in transformed mouse embryonic fibro- (Zhou and Bartek, 2004). By contrast, using Chk2 blasts (Belova et al., 2005). However, as this compound inhibitors on tumors, which retain wild-type p53, would affected the phosphorylation status of all mitogen- dramatically increase their resistance to irradiation, activated protein kinases, concerns remain about its leading to unfavorable clinical outcomes. Thus transla- specificity toward Wip1 and further modification may be tion of this treatment strategy into the clinic will require necessary to generate a more selective inhibitor of Wip1

Oncogene Checkpoint control and cancer RH Medema and L Macu˚rek 2609 (Belova et al., 2005). Another cell-permeable, small- components of the overall DDR can be lost in cancer molecule inhibitor of Wip1 (CCT007093, IC50 ¼ 8.4 mM) cells, rendering them more dependent on the remaining has recently been reported to decrease the viability of intact pathways to promote repair and arrest the cell the MCF7, KPL1 and MCF3B tumor cell lines (Rayter cycle. This has already led to the development of et al., 2008). Interestingly, this cytotoxic effect of Wip1 tailored therapies that exploit the cancer-specific DDR inhibition seems to be specific for tumors that over- defect(s). These promising approaches call for a more express Wip1, whereas cells with normal Wip1 levels advanced molecular understanding of the DDR as tolerate the loss of Wip1 activity (Rayter et al., 2008). well as the development of small-molecule inhibitors The cell death induced by CCT007093 was dependent on targeting various components of the DNA-damage p38, which is a well-described substrate of Wip1, and checkpoint, which can open additional avenues for mimicked the effect of Wip1 RNA interference, indicat- personalized cancer treatment. ing that the observed phenotype indeed results from specific inhibition of Wip1. It will be interesting to see how this promising Wip1 inhibitor will perform on a broader spectrum of tumors and in xenograft tumor Conflict of interest models. The authors declare no conflict of interest.

Concluding remarks Acknowledgements Over the last decades, we have gained a lot of insight into the mechanisms that act to coordinate the cellular RHM was funded by the Netherlands Genomics Initiative of response to DNA damage. It has become clear that the Netherlands Organization for Scientiﬁc Research and by various pathways can act in parallel to promote DNA the Dutch Cancer Society (Grant UU2009-4478). LM was repair and several others act together to inhibit cell-cycle supported by the Grant Agency of the Czech Republic (P301/ progression. We have also learned that different 10/1525 and P305/10/P420).

References

Agami R, Bernards R. (2000). Distinct initiation and maintenance Belova G, Demidov ON, Fornace AJ, Bulavin DV. (2005). Chemical mechanisms cooperate to induce G1 cell cycle arrest in response to inhibition of Wip1 phosphatase contributes to suppression of DNA damage. Cell 102: 55–66. tumorigenesis. Cancer Biol Ther 4: 1154–1158. Ahn J, Schwarz J, Piwnica-Worms H, Canman C. (2000). Threonine Bennetzen MV, Larsen DH, Bunkenborg J, Bartek J, Lukas J, 68 phosphorylation by ataxia telangiectasia mutated is required for Andersen JS. (2010). Site-specific phosphorylation dynamics of the efficient activation of Chk2 in response to ionizing radiation. Cancer nuclear proteome during the DNA damage tesponse. Mol Cell Res 60: 5934–5936. Proteomics 9: 1314–1323. Ahn J, Urist M, Prives C. (2003). Questioning the role of checkpoint Bensimon A, Schmidt A, Ziv Y, Elkon R, Wang S-Y, Chen DJ et al. kinase 2 in the p53 DNA damage response. J Biol Chem 278: (2010). ATM-dependent and -independent dynamics of the nuclear 20480–20489. phosphoproteome after DNA damage. Sci Signal 3: rs3. Alvarez-Fernandez M, Halim VA, Krenning L, Aprelia M, Bulavin D, Higashimoto Y, Popoff I, Gaarde W, Basrur V, Potapova Mohammed S, Heck AJ et al. (2010). Recovery from a DNA- O. (2001). Initiation of a G2/M checkpoint after ultraviolet damage-induced G2 arrest requires Cdk-dependent activation of radiation requires p38 kinase. Nature 411: 102–107. FoxM1. EMBO Rep 11: 452–458. Bulavin DV, Demidov ON, Saito Si, Kauraniemi P, Phillips C, Anderson VE, Walton MI, Eve PD, Boxall KJ, Antoni L, Caldwell JJ Amundson SA et al. (2002). Amplification of PPM1D in et al. (2011). CCT241533 is a potent and selective inhibitor of CHK2 human tumors abrogates p53 tumor-suppressor activity. Nat Genet that potentiates the cytotoxicity of PARP inhibitors. Cancer Res 71: 31: 210–215. 463–472. Bulavin DV, Phillips C, Nannenga B, Timofeev O, Donehower LA, Bakkenist C, Kastan M. (2003). DNA damage activates ATM through Anderson CW et al. (2004). Inactivation of the Wip1 phosphatase intermolecular autophosphorylation and dimer dissociation. Nature inhibits mammary tumorigenesis through p38 MAPK-mediated 421: 499–506. activation of the p16Ink4a–p19Arf pathway. Nat Genet 36: 343–350. Bartek J, Lukas J. (2003). Chk1 and Chk2 kinases in checkpoint Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP control and cancer. Cancer Cell 3: 421–429. et al. (1998). Requirement for p53 and p21 to sustain G2 arrest after Batchelor E, Loewer A, Lahav G. (2009). The ups and downs of p53: DNA damage. Science 282: 1497–1501. understanding protein dynamics in single cells. Nat Rev Cancer 9: Carlessi L, Buscemi G, Fontanella E, Delia D. (2010). A protein 371–377. phosphatase feedback mechanism regulates the basal phosphoryla- Batchelor E, Loewer A, Mock C, Lahav G. (2011). Stimulus- tion of Chk2 kinase in the absence of DNA damage. Biochim dependent dynamics of p53 in single cells. Mol Syst Biol 7: 488. Biophys Acta 1803: 1213–1223. Batchelor E, Mock CS, Bhan I, Loewer A, Lahav G. (2008). Recurrent Castellino R, De Bortoli M, Lu X, Moon S-H, Nguyen T-A, Shepard initiation: a mechanism for triggering p53 pulses in response to M et al. (2008). Medulloblastomas overexpress the p53-inactivating DNA damage. Mol Cell 30: 277–289. oncogene WIP1/PPM1D. J Neurooncol 86: 245–256. Bekker-Jensen S, Mailand N. (2010). Assembly and function of DNA Cha H, Lowe JM, Li H, Lee J-S, Belova GI, Bulavin DV et al. (2010). double-strand break repair foci in mammalian cells. DNA Repair 9: Wip1 directly dephosphorylates g-H2AX and attenuates the DNA 1219–1228. damage response. Cancer Res 70: 4112–4122.

Oncogene Checkpoint control and cancer RH Medema and L Macu˚rek 2610 Chailleux C, Tyteca S, Papin C, Boudsocq F, Puget N, Courilleau C Hirai H, Iwasawa Y, Okada M, Arai T, Nishibata T, Kobayashi M et al. (2010). Physical interaction between the histone acetyl et al. (2009). Small-molecule inhibition of Wee1 kinase by MK-1775 transferase Tip60 and the DNA double-strand breaks sensor selectively sensitizes p53-deficient tumor cells to DNA-damaging MRN complex. Biochem J 426: 365–371. agents. Mol Cancer Ther 8: 2992–3000. Chini C, Chen J. (2003). Human claspin is required for replication Hirao A, Cheung A, Duncan G, Girard P, Elia A, Wakeham A. checkpoint control. J Biol Chem 278: 30057–30062. (2002). Chk2 is a tumor suppressor that regulates apoptosis in both Chowdhury D, Keogh M, Ishii H, Peterson C, Buratowski S, an ataxia telangiectasia mutated (ATM)-dependent and an ATM- Lieberman J. (2005). Gamma-H2AX dephosphorylation by protein independent manner. Mol Cell Biol 22: 6521–6532. phosphatase 2A facilitates DNA double-strand break repair. Mol Hirao A, Kong Y-Y, Matsuoka S, Wakeham A, Ruland J, Yoshida H Cell 20: 801–809. et al. (2000). DNA damage-induced activation of p53 by the Ciccia A, Elledge SJ. (2010). The DNA damage response: making it checkpoint kinase Chk2. Science 287: 1824–1827. safe to play with knives. Mol Cell 40: 179–204. Imbriano C, Gurtner A, Cocchiarella F, Di Agostino S, Basile V, Cimprich K, Cortez D. (2008). ATR: an essential regulator of genome Gostissa M et al. (2005). Direct p53 transcriptional repression: integrity. Nat Rev Mol Cell Biol 9: 616–627. in vivo analysis of CCAAT-containing G2/M promoters. Mol Cell Cotta-Ramusino C, McDonald ER, Hurov K, Sowa ME, Harper JW, Biol 25: 3737–3751. Elledge SJ. (2011). A DNA damage response screen identifies Jack MT, Woo RA, Hirao A, Cheung A, Mak TW, Lee PWK. (2002). RHINO, a 9-1-1 and TopBP1 interacting protein required for ATR Chk2 is dispensable for p53-mediated G1 arrest but is required for a signaling. Science 332: 1313–1317. latent p53-mediated apoptotic response. Proc Natl Acad Sci USA Cuadrado M, Gutierrez-Martinez P, Swat A, Nebreda AR, Fernan- 99: 9825–9829. dez-Capetillo O. (2009). p27Kip1 stabilization is essential for the Jallepalli PV, Lengauer C, Vogelstein B, Bunz F. (2003). The Chk2 maintenance of cell cycle arrest in response to DNA damage. Cancer tumor suppressor is not required for p53 responses in human cancer Res 69: 8726–8732. cells. J Biol Chem 278: 20475–20479. Dai C, Gu W. (2010). p53 post-translational modification: deregulated Jang Y-J, Ma S, Terada Y, Erikson RL. (2002). Phosphorylation of in tumorigenesis. Trends Mol Med 16: 528–536. threonine 210 and the role of serine 137 in the regulation of Dalvai M, Mondesert O, Bourdon JC, Ducommun B, Dozier C. mammalian Polo-like kinase. J Biol Chem 277: 44115–44120. (2011). Cdc25B is negatively regulated by p53 through Sp1 and NF- Jazayeri A, Falck J, Lukas C, Bartek J, Smith GCM, Lukas J et al. Y transcription factors. Oncogene 30: 2282–2288. (2006). ATM- and cell cycle-dependent regulation of ATR in Falck J, Coates J, Jackson SP. (2005). Conserved modes of recruitment response to DNA double-strand breaks. Nat Cell Biol 8: 37–45. of ATM, ATR and DNA-PKcs to sites of DNA damage. Nature Jiang H, Reinhardt HC, Bartkova J, Tommiska J, Blomqvist C, 434: 605–611. Nevanlinna H et al. (2009). The combined status of ATM and p53 Falck J, Mailand N, Syljuasen RG, Bartek J, Lukas J. (2001). The link tumor development with therapeutic response. Genes Dev 23: ATM–Chk2–Cdc25A checkpoint pathway guards against radio- 1895–1909. resistant DNA synthesis. Nature 410: 842–847. Jin J, Ang XL, Ye X, Livingstone M, Harper JW. (2008). Differential Ferguson AM, White LS, Donovan PJ, Piwnica-Worms H. (2005). roles for checkpoint kinases in DNA damage-dependent Normal cell cycle and checkpoint responses in mice and cells degradation of the Cdc25A protein phosphatase. J Biol Chem 283: lacking Cdc25B and Cdc25C protein phosphatases. Mol Cell Biol 19322–19328. 25: 2853–2860. Jin J, Shirogane T, Xu L, Nalepa G, Qin J, Elledge S. (2003). Fiscella M, Zhang H, Fan S, Sakaguchi K, Shen S, Mercer W. (1997). SCFbeta–TRCP links Chk1 signaling to degradation of the Cdc25A Wip1, a novel human protein phosphatase that is induced in protein phosphatase. Genes Dev 17: 3062–3074. response to ionizing radiation in a p53-dependent manner. Proc Kasahara K, Goto H, Enomoto M, Tomono Y, Kiyono T, Inagaki M. Natl Acad Sci USA 94: 6048–6053. (2010). 14-3-3[gamma] mediates Cdc25A proteolysis to Forrest A, Gabrielli B. (2001). Cdc25B activity is regulated by 14-3-3. block premature mitotic entry after DNA damage. EMBO J 29: Oncogene 20: 4393–4401. 2802–2812. Fracasso P, Williams K, Chen R, Picus J, Ma C, Ellis M et al. (2010). Kramer A, Mailand N, Lukas C, Syljuasen R, Wilkinson C, Nigg E. A phase 1 study of UCN-01 in combination with irinotecan in (2004). Centrosome-associated Chk1 prevents premature activation patients with resistant solid tumor malignancies. Cancer Chemother of cyclin-B–Cdk1 kinase. Nat Cell Biol 6: 884–891. Pharmacol 67: 1225–1237. Kumagai A, Kim S, Dunphy W. (2004). Claspin and the activated Freeman A, Dapic V, Monteiro A. (2010). Negative regulation of form of ATR–ATRIP collaborate in the activation of Chk1. J Biol CHK2 activity by protein phosphatase 2A is modulated by DNA Chem 279: 49599–49608. damage. Cell Cycle 9: 736–747. Kuntz K, O’Connell MJ. (2009). The G2 DNA damage checkpoint: Freeman A, Monteiro A. (2010). Phosphatases in the cellular response could this ancient regulator be the Achilles heel of cancer? Cancer to DNA damage. Cell Commun Signal 8: 27. Biol Ther 8: 1433–1439. Fujimoto H, Onishi N, Kato N, Takekawa M, Xu XZ, Kosugi A et al. Lafarga V, Cuadrado A, Lopez de Silanes I, Bengoechea R, (2005). Regulation of the antioncogenic Chk2 kinase by the Fernandez-Capetillo O, Nebreda AR. (2009). p38 mitogen- oncogenic Wip1 phosphatase. Cell Death Differ 13: 1170–1180. activated protein kinase- and HuR-dependent stabilization of Goodarzi A, Jonnalagadda J, Douglas P, Young D, Ye R, p21Cip1 mRNA mediates the G1/S checkpoint. Mol Cell Biol 29: Moorhead G. (2004). Autophosphorylation of ataxia-telangiectasia 4341–4351. mutated is regulated by protein phosphatase 2A. EMBO J 23: Lahav G, Rosenfeld N, Sigal A, Geva-Zatorsky N, Levine AJ, Elowitz 4451–4461. MB et al. (2004). Dynamics of the p53–Mdm2 feedback loop in Gupta M, Gupta SK, Hoffman B, Liebermann DA. (2006). Gadd45a individual cells. Nat Genet 36: 147–150. and Gadd45b protect hematopoietic cells from UV-induced Lam MH, Liu Q, Elledge SJ, Rosen JM. (2004). Chk1 is haploinsuffi- apoptosis via distinct signaling pathways, including p38 activation cient for multiple functions critical to tumor suppression. Cancer and JNK inhibition. J Biol Chem 281: 17552–17558. Cell 6: 45–59. Harrison M, Li J, Degenhardt Y, Hoey T, Powers S. (2004). Wip1- Lara PN, Mack PC, Synold T, Frankel P, Longmate J, Gumerlock PH deficient mice are resistant to common cancer genes. Trends Mol et al. (2005). The cyclin-dependent kinase inhibitor UCN-01 plus Med 10: 359–361. c–isplatin in advanced solid tumors: a California Cancer Con- Hirai H, Arai T, Okada M, Nishibata T, Kobayashi M, Sakai N et al. sortium Phase I Pharmacokinetic and Molecular Correlative Trial. (2010). MK-1775, a small molecule Wee1 inhibitor, enhances anti- Clin Cancer Res 11: 4444–4450. tumor efficacy of various DNA-damaging agents, including Le Guezennec X, Bulavin DV. (2010). WIP1 phosphatase at the 5-fluorouracil. Cancer Biol Ther 9: 514–522. crossroads of cancer and aging. Trends Biochem Sci 35: 109–114.

Oncogene Checkpoint control and cancer RH Medema and L Macu˚rek 2611 Lee J-H, Goodarzi AA, Jeggo PA, Paull TT. (2010). 53BP1 promotes Mamely I, van Vugt MATM, Smits VAJ, Semple JI, Lemmens B, ATM activity through direct interactions with the MRN complex. Perrakis A et al. (2006). Polo-like kinase-1 controls proteasome- EMBO J 29: 574–585. dependent degradation of claspin during checkpoint recovery. Curr Leńa´rt P, Petronczki M, Steegmaier M, Di Fiore B, Lipp JJ, Hoffmann Biol 16: 1950–1955. M et al. (2007). The small-molecule inhibitor BI 2536 reveals Manke IA, Nguyen A, Lim D, Stewart MQ, Elia AEH, Yaffe MB. novel insights into mitotic roles of Polo-like kinase 1. Curr Biol 17: (2005). MAPKAP kinase-2 is a cell cycle checkpoint kinase that 304–315. regulates the G2/M transition and S phase progression in response Leroy C, Lee S, Vaze M, Ochsenbien F, Guerois R, Haber J. (2003). to UV irradiation. Mol Cell 17: 37–48. PP2C phosphatases Ptc2 and Ptc3 are required for DNA checkpoint Matsuoka S, Ballif BA, Smogorzewska A, McDonald III ER, Hurov inactivation after a double-strand break. Mol Cell 11: 827–835. KE, Luo J et al. (2007). ATM and ATR substrate analysis reveals Leung-Pineda V, Ryan C, Piwnica-Worms H. (2006). Phosphorylation extensive protein networks responsive to DNA damage. Science of Chk1 by ATR is antagonized by a Chk1-regulated protein 316: 1160–1166. phosphatase 2A circuit. Mol Cell Biol 26: 7529–7538. Maya-Mendoza A, Petermann E, Gillespie DAF, Caldecott KW, Li H-H, Cai X, Shouse GP, Piluso LG, Liu X. (2007). A specific PP2A Jackson DA. (2007). Chk1 regulates the density of active replication regulatory subunit, B56[gamma], mediates DNA damage-induced origins during the vertebrate S phase. EMBO J 26: 2719–2731. dephosphorylation of p53 at Thr55. EMBO J 26: 402–411. McKenzie L, King S, Marcar L, Nicol S, Dias S, Schumm K et al. Li J, Yang Y, Peng Y, Austin RJ, van Eyndhoven WG, Nguyen KCQ (2010). p53-dependent repression of polo-like kinase-1 (PLK1). Cell et al. (2002). Oncogenic properties of PPM1D located Cycle 9: 4200–4212. within a breast cancer amplification epicenter at 17q23. Nat Genet Melixetian M, Klein DK, Sorensen CS, Helin K. (2009). NEK11 31: 133–134. regulates CDC25A degradation and the IR-induced G2/M check- Lindqvist A, de B, Macurek L, Bras A, Mensinga A, Bruinsma W. point. Nat Cell Biol 11: 1247–1253. (2009a). Wip1 confers G2 checkpoint recovery competence by Menzel T, Nahse-Kumpf V, Kousholt AN, Klein DK, Lund-Andersen counteracting p53-dependent transcriptional repression. EMBO J C, Lees M et al. (2011). A genetic screen identifies BRCA2 and 28: 3196–3206. PALB2 as key regulators of G2 checkpoint maintenance. EMBO Lindqvist A, Rodrı´guez-Bravo V, Medema RH. (2009b). The decision Rep 12: 705–712. to enter mitosis: feedback and redundancy in the mitotic entry Mikhailov A, Shinohara M, Rieder CL. (2004). Topoisomerase II and network. J Cell Biol 185: 193–202. histone deacetylase inhibitors delay the G2/M transition by Liontos M, Velimezi G, Pateras IS, Angelopoulou R, Papavassiliou triggering the p38 MAPK checkpoint pathway. J Cell Biol 166: AG, Bartek J et al. (2010). The roles of p27Kip1 and DNA damage 517–526. signalling in the chemotherapy-induced delayed cell cycle check- Moon S, Lin L, Zhang X, Nguyen T, Darlington Y, Waldman A. point. J Cell Mol Med 14: 2264–2267. (2010). Wildtype p53-induced phosphatase 1 dephosphorylates Liu Q, Guntuku S, Cui X, Matsuoka S, Cortez D, Tamai K. (2000). histone variant {gamma}-H2AX and suppresses DNA double Chk1 is an essential kinase that is regulated by Atr and required for strand break repair. J Biol Chem 285: 12935–12947. the G(2)/M DNA damage checkpoint. Genes Dev 14: 1448–1459. Nannenga B, Lu X, Dumble M, Van Maanen M, Nguyen T-A, Liu XS, Li H, Song B, Liu X. (2010). Polo-like kinase 1 phosphoryla- Sutton R et al. (2006). Augmented cancer resistance and DNA tion of G2 and S-phase-expressed 1 protein is essential for damage response phenotypes in PPM1D null mice. Mol Carcinog p53 inactivation during G2 checkpoint recovery. EMBO Rep 11: 45: 594–604. 626–632. O’Connell M, Raleigh J, Verkade H, Nurse P. (1997). Chk1 is a wee1 Lobjois V, Jullien D, Bouche´J-P, Ducommun B. (2009). The polo-like kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 kinase 1 regulates CDC25B-dependent mitosis entry. Biochim phosphorylation. EMBO J 16: 545–554. Biophys Acta 1793: 462–468. Peddibhotla S, Lam MH, Gonzalez-Rimbau M, Rosen JM. (2009). Loewer A, Batchelor E, Gaglia G, Lahav G. (2010). Basal dynamics of The DNA-damage effector checkpoint kinase 1 is essential for p53 reveal transcriptionally attenuated pulses in cycling cells. Cell chromosome segregation and cytokinesis. Proc Natl Acad Sci USA 142: 89–100. 106: 5159–5164. Lu X, Ma O, Nguyen T-A, Jones SN, Oren M, Donehower LA. (2007). Pellegrini M, Celeste A, Difilippantonio S, Guo R, Wang W, The Wip1 phosphatase acts as a gatekeeper in the p53–Mdm2 Feigenbaum L. (2006). Autophosphorylation at serine 1987 is autoregulatory loop. Cancer Cell 12: 342–354. dispensable for murine Atm activation in vivo. Nature 443: Lu X, Nannenga B, Donehower L. (2005). PPM1D dephosphorylates 222–225. Chk1 and p53 and abrogates cell cycle checkpoints. Genes Dev 19: Peng A, Lewellyn AL, Schiemann WP, Maller JL. (2010). Repo-Man 1162–1174. controls a protein phosphatase 1-dependent rhreshold for DNA Lu X, Nguyen T, Moon S, Darlington Y, Sommer M, Donehower L. damage checkpoint activation. Curr Biol 20: 387–396. (2008). The type 2C phosphatase Wip1: an oncogenic regulator of Peng C-Y, Graves PR, Thoma RS, Wu Z, Shaw AS, Piwnica-Worms tumor suppressor and DNA damage response pathways. Cancer H. (1997). Mitotic and G2 checkpoint control: regulation of 14-3-3 Metastasis Rev 27: 123–135. protein binding by phosphorylation of Cdc25C on serine-216. Ma CX, Janetka JW, Piwnica-Worms H. (2011). Death by releasing Science 277: 1501–1505. the breaks: CHK1 inhibitors as cancer therapeutics. Trends Mol Peschiaroli A, Dorrello NV, Guardavaccaro D, Venere M, Halazonetis Med 17: 88–96. T, Sherman NE et al. (2006). SCF[beta]TrCP-mediated degradation Macurek L, Lindqvist A, Lim D, Lampson MA, Klompmaker R, of claspin regulates recovery from the DNA replication checkpoint Freire R et al. (2008). Polo-like kinase-1 is activated by aurora A to response. Mol Cell 23: 319–329. promote checkpoint recovery. Nature 455: 119–123. Phong MS, Van Horn RD, Li S, Tucker-Kellogg G, Surana U, Ye XS. Macurek L, Lindqvist A, Voets O, Kool J, Vos H, Medema R. (2010). (2010). p38 mitogen-activated protein kinase promotes cell Wip1 phosphatase is associated with chromatin and dephosphor- survival in response to DNA damage but is not required for the ylates gammaH2AX to promote checkpoint inhibition. Oncogene G2 DNA damage checkpoint in human cancer cells. Mol Cell Biol 29: 2281–2291. 30: 3816–3826. Mailand N, Bekker-Jensen S, Bartek J, Lukas J. (2006). Destruction of Polo SE, Jackson SP. (2011). Dynamics of DNA damage response claspin by SCF[beta]TrCP restrains Chk1 activation and facilitates proteins at DNA breaks: a focus on protein modifications. Genes recovery from genotoxic stress. Mol Cell 23: 307–318. Dev 25: 409–433. Mailand N, Falck J, Lukas C, Syljua˚sen RG, Welcker M, Bartek J Rajeshkumar NV, De Oliveira E, Ottenhof N, Watters J, Brooks D, et al. (2000). Rapid destruction of human Cdc25A in response to Demuth T et al. (2011). MK-1775, a potent Wee1 inhibitor, DNA damage. Science 288: 1425–1429. synergizes with gemcitabine to achieve tumor regressions, selectively

Oncogene Checkpoint control and cancer RH Medema and L Macu˚rek 2612 in p53-deficient pancreatic cancer xenografts. Clin Cancer Res 17: Sun Y, Jiang X, Chen S, Fernandes N, Price BD. (2005). A role for the 2799–2806. Tip60 histone acetyltransferase in the acetylation and activation of Raman M, Earnest S, Zhang K, Zhao Y, Cobb MH. (2007). TAO ATM. Proc Natl Acad Sci USA 102: 13182–13187. kinases mediate activation of p38 in response to DNA damage. Sun Y, Jiang X, Xu Y, Ayrapetov MK, Moreau LA, Whetstine JR EMBO J 26: 2005–2014. et al. (2009). Histone H3 methylation links DNA damage detection Rayter S, Elliott R, Travers J, Rowlands MG, Richardson TB, Boxall to activation of the tumour suppressor Tip60. Nat Cell Biol 11: K et al. (2008). A chemical inhibitor of PPM1D that selectively kills 1376–1382. cells overexpressing PPM1D. Oncogene 27: 1036–1044. Sun Y, Xu Y, Roy K, Price BD. (2007). DNA Damage-Induced Reinhardt HC, Aslanian AS, Lees JA, Yaffe MB. (2007). p53-deficient Acetylation of Lysine 3016 of ATM Activates ATM Kinase cells rely on ATM- and ATR-mediated checkpoint signaling Activity. Mol Cell Biol 27: 8502–8509. through the p38MAPK/MK2 pathway for survival after DNA Syljuasen RG, Sorensen CS, Hansen LT, Fugger K, Lundin C, damage. Cancer Cell 11: 175–189. Johansson F et al. (2005). Inhibition of human Chk1 causes Reinhardt HC, Hasskamp P, Schmedding I, Morandell S, van Vugt increased initiation of DNA replication, phosphorylation of ATR MATM, Wang X et al. (2010). DNA damage activates a spatially targets, and DNA breakage. Mol Cell Biol 25: 3553–3562. distinct late cytoplasmic cell-cycle checkpoint network controlled by Takai H, Naka K, Okada Y, Watanabe M, Harada N, Saito S. (2002). MK2-mediated RNA stabilization. Mol Cell 40: 34–49. Chk2-deficient mice exhibit radioresistance and defective p53- Saito-Ohara F, Imoto I, Inoue J, Hosoi H, Nakagawara A, Sugimoto mediated transcription. EMBO J 21: 5195–5205. T et al. (2003). PPM1D is a potential target for 17q gain in Takai H, Tominaga K, Motoyama N, Minamishima Y, Nagahama H, neuroblastoma. Cancer Res 63: 1876–1883. Tsukiyama T. (2000). Aberrant cell cycle checkpoint function Sanchez Y, Wong C, Thoma RS, Richman R, Wu Z, Piwnica-Worms and early embryonic death in Chk1(À/À) mice. Genes Dev 14: H et al. (1997). Conservation of the Chk1 checkpoint pathway in 1439–1447. mammals: linkage of DNA damage to Cdk regulation through Takekawa M, Adachi M, Nakahata A, Nakayama I, Itoh F, Tsukuda Cdc25. Science 277: 1497–1501. H et al. (2000). p53-inducible Wip1 phosphatase mediates a negative Santra MK, Wajapeyee N, Green MR. (2009). F-box protein FBXO31 feedback regulation of p38 MAPK–p53 signaling in response to UV mediates cyclin D1 degradation to induce G1 arrest after DNA radiation. EMBO J 19: 6517–6526. damage. Nature 459: 722–725. Tan DSP, Iravani M, McCluggage WG, Lambros MBK, Milanezi F, Sartori AA, Lukas C, Coates J, Mistrik M, Fu S, Bartek J et al. (2007). Mackay A et al. (2011). Genomic analysis reveals the molecular Human CtIP promotes DNA end resection. Nature 450: 509–514. heterogeneity of ovarian clear cell carcinomas. Clin Cancer Res 17: Satoh N, Maniwa Y, Bermudez VP, Nishimura K, Nishio W, 1521–1534. Yoshimura M et al. (2011). Oncogenic phosphatase Wip1 is a Tan DSP, Lambros MBK, Rayter S, Natrajan R, Vatcheva R, Gao Q novel prognostic marker for lung adenocarcinoma patient survival. et al. (2009). PPM1D is a potential therapeutic target in ovarian Cancer Sci 102: 1101–1106. clear cell carcinomas. Clin Cancer Res 15: 2269–2280. Secchiero P, Bosco R, Celeghini C, Zauli G. (2011). Recent advances Toczyski DP, Galgoczy DJ, Hartwell LH. (1997). CDC5 and CKII in the therapeutic perspectives of nutlin-3. Curr Pharm Des 17: control adaptation to the yeast DNA damage checkpoint. Cell 90: 569–577. 1097–1106. Seki A, Coppinger JA, Jang C-Y, Yates JR, Fang G. (2008). Bora and Toyoshima-Morimoto F, Taniguchi E, Nishida E. (2002). Plk1 the kinase aurora A cooperatively activate the kinase Plk1 and promotes nuclear translocation of human Cdc25C during prophase. control mitotic entry. Science 320: 1655–1658. EMBO Rep 3: 341–348. Shangary S, Qin D, McEachern D, Liu M, Miller RS, Qiu S et al. Tsvetkov L, Stern D. (2005). Phosphorylation of Plk1 at S137 (2008). Temporal activation of p53 by a specific MDM2 inhibitor is and T210 is inhibited in response to DNA damage. Cell Cycle 4: selectively toxic to tumors and leads to complete tumor growth 166–171. inhibition. Proc Natl Acad Sci USA 105: 3933–3938. van Vugt MATM, Bra´s A, Medema RH. (2004). Polo-like kinase-1 Shen H, Maki C. (2011). Pharmacologic activation of p53 by small- controls recovery from a G2 DNA damage-induced arrest in molecule MDM2 antagonists. Curr Pharm Des 17: 560–568. mammalian cells. Mol Cell 15: 799–811. Shibata A, Barton O, Noon AT, Dahm K, Deckbar D, Goodarzi AA van Vugt MATM, Gardino AK, Linding R, Ostheimer GJ, et al. (2010). Role of ATM and the damage response mediator Reinhardt HC, Ong S-E et al. (2010). A mitotic phosphorylation proteins 53BP1 and MDC1 in the maintenance of G2/M checkpoint feedback network connects Cdk1, Plk1, 53BP1, and Chk2 to arrest. Mol Cell Biol 30: 3371–3383. inactivate the G2/M DNA damage checkpoint. PLoS Biol 8: Shouse GP, Nobumori Y, Panowicz MJ, Liu X. (2011). ATM- e1000287. mediated phosphorylation activates the tumor-suppressive function Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z of B56[gamma]-PP2A. Oncogene 30: 3755–3765. et al. (2004). In vivo activation of the p53 pathway by small-molecule Shreeram S, Demidov O, Hee W, Yamaguchi H, Onishi N, Kek C. antagonists of MDM2. Science 303: 844–848. (2006a). Wip1 phosphatase modulates ATM-dependent signaling Wang P, Rao J, Yang H, Zhao H, Yang L. (2011). PPM1D silencing pathways. Mol Cell 23: 757–764. by lentiviral-mediated RNA interference inhibits proliferation and Shreeram S, Hee WK, Demidov ON, Kek C, Yamaguchi H, Fornace invasion of human glioma cells. J Huazhong Univ Sci Technolog Jr AJ. et al. (2006b). Regulation of ATM/p53-dependent suppres- Med Sci 31: 94–99. sion of myc-induced lymphomas by Wip1 phosphatase. J Exp Med Wang Q, Fan S, Eastman A, Worland PJ, Sausville EA, O0Connor 203: 2793–2799. PM. (1996). UCN-01: a potent abrogator of G2 checkpoint Smits VAJ, Klompmaker R, Arnaud L, Rijksen G, Nigg EA, Medema function in cancer cells with disrupted p53. J Natl Cancer Inst 88: RH. (2000). Polo-like kinase-1 is a target of the DNA damage 956–965. checkpoint. Nat Cell Biol 2: 672–676. Wang XW, Zhan Q, Coursen JD, Khan MA, Kontny HU, Yu L et al. Smits VAJ, Reaper PM, Jackson SP. (2006). Rapid PIKK-dependent (1999). GADD45 induction of a G2/M cell cycle checkpoint. Proc release of Chk1 from chromatin promotes the DNA-damage Natl Acad Sci USA 96: 3706–3711. checkpoint response. Curr Biol 16: 150–159. Wang Y, Li J, Booher RN, Kraker A, Lawrence T, Leopold WR et al. Stevens C, Smith L, La Thangue N. (2003). Chk2 activates E2F-1 in (2001). Radiosensitization of p53 mutant cells by PD0166285, a response to DNA damage. Nat Cell Biol 5: 401–409. novel G2 checkpoint abrogator. Cancer Res 61: 8211–8217. Stucki M, Clapperton J, Mohammad D, Yaffe M, Smerdon S, Jackson Watanabe N, Arai H, Nishihara Y, Taniguchi M, Watanabe N, S. (2005). MDC1 directly binds phosphorylated histone H2AX to Hunter T et al. (2004). M-phase kinases induce phospho-dependent regulate cellular responses to DNA double-strand breaks. Cell 123: ubiquitination of somatic Wee1 by SCFIˆ2–TrCP. Proc Natl Acad Sci 1213–1226. USA 101: 4419–4424.

Oncogene Checkpoint control and cancer RH Medema and L Macu˚rek 2613 Wilsker D, Petermann E, Helleday T, Bunz F. (2008). Essential Zhan Q, Antinore MJ, Wang XW, Carrier F, Smith ML, Harris CC function of Chk1 can be uncoupled from DNA damage et al. (1999). Association with Cdc2 and inhibition of Cdc2/cyclin checkpoint and replication control. Proc Natl Acad Sci USA 105: B1 kinase activity by the p53-regulated protein Gadd45. Oncogene 20752–20757. 18: 2892–2900. Xiao Z, Chen Z, Gunasekera AH, Sowin TJ, Rosenberg SH, Fesik S Zhang X, Lin L, Guo H, Yang J, Jones SN, Jochemsen A et al. (2009). et al. (2003). Chk1 mediates S and G2 arrests through Cdc25A Phosphorylation and degradation of MdmX is inhibited by degradation in response to DNA-damaging agents. J Biol Chem Wip1 phosphatase in the DNA damage response. Cancer Res 69: 278: 21767–21773. 7960–7968. Yamaguchi H, Durell SR, Chatterjee DK, Anderson CW, Appella E. Zhang X, Wan G, Mlotshwa S, Vance V, Berger FG, Chen H et al. (2007). The Wip1 phosphatase PPM1D dephosphorylates SQ/TQ (2010). Oncogenic Wip1 phosphatase is inhibited by miR-16 in the motifs in checkpoint substrates phosphorylated by PI3K-like DNA damage signaling pathway. Cancer Res 70: 7176–7186. kinases. Biochemistry 46: 12594–12603. Zhao H, Watkins JL, Piwnica-Worms H. (2002). Disruption of the Yamaguchi H, Durell SR, Feng H, Bai Y, Anderson CW, Appella E. checkpoint kinase 1/cell division cycle 25A pathway abrogates (2006). Development of a substrate-based cyclic phosphopeptide ionizing radiation-induced S and G2 checkpoints. Proc Natl Acad inhibitor of protein phosphatase 2Cd, Wip1. Biochemistry 45: Sci USA 99: 14795–14800. 13193–13202. Zhou B-BS, Bartek J. (2004). Targeting the checkpoint kinases: Zachos G, Black EJ, Walker M, Scott MT, Vagnarelli P, Earnshaw chemosensitization versus chemoprotection. Nat Rev Cancer 4: 216–225. WC et al. (2007). Chk1 is required for spindle checkpoint function. Zou L, Elledge SJ. (2003). Sensing DNA damage through ATRIP Dev Cell 12: 247–260. recognition of RPA–ssDNA complexes. Science 300: 1542–1548.

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