Inhibition of Chk1-dependent G2 DNA damage checkpoint ... - Nature

Oncogene (2001) 20, 7453 ± 7463 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

ORIGINAL PAPERS

Inhibition of Chk1-dependent G2 DNA damage checkpoint radiosensitizes p53 mutant human cells Kalli Koniaras1,3, Andrew R Cuddihy1,3, Helen Christopoulos1, Annette Hogg1 and Matthew J O'Connell*,1,2 1

Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Locked Bag 1, A'Beckett St, Melbourne, VIC 8006 Australia; 2Department of Genetics, University of Melbourne, Parkville VIC 3052, Australia

Cell cycle checkpoints are surveillance mechanisms that monitor and coordinate the order and ®delity of cell cycle events. When defects in the division program of a cell are detected, checkpoints prevent the pursuant cell cycle transition through regulation of the relevant cyclincdk complex(es). Checkpoints that respond to DNA damage have been described for the G1, S and G2 phases of the cell cycle. The p53 tumour suppressor is a key regulator of G1/S checkpoints, and can promote cell cycle delay or apoptosis in response to DNA damage. The importance of these events to cellular physiology is highlighted by the fact that tumours, in which p53 is frequently mutated, have widespread defects in the G1/S DNA damage checkpoints and a heightened level of genomic instability. G2/M DNA damage checkpoints have been de®ned by yeast genetics, though the genes in this response are conserved in mammals. We show here using biochemical and physiological assays that p53 is dispensable for a DNA damage checkpoint activated in the G2 phase of the cell cycle. Moreover, upregulation of p53 through serine 20 phosphorylation, does not occur in G2. Conversely, we show that the Chk1 protein kinase is essential for the human G2 DNA damage checkpoint. Importantly, inhibition of Chk1 in p53 de®cient cells greatly sensitizes them to radiation, validating the hypothesis of targeting Chk1 in rational drug design and development for anti-cancer therapies. Oncogene (2001) 20, 7453 ± 7463. Keywords: checkpoint; Chk1; p53; Cds1; DNA damage Introduction Cells replicate themselves with remarkable ®delity. Signal transduction pathways that control the major events in the cell cycle are well understood, and centre on the activation of cyclin-dependent kinases (Cdks). *Correspondence: MJ O'Connell, Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, St Andrew's Place, East Melbourne, VIC 3002, Australia; E-mail: [email protected] 3 These authors contributed equally to the work Received 25 June 2001; revised 15 August 2001; accepted 30 August 2001

Whilst Cyclin-Cdk complexes accumulate in interphase, they are kept in an inactive state by either inhibitory phosphorylations or by binding to Cdk inhibitor proteins (CKIs) (Dunphy, 1994; Hunter and Pines, 1994; Sherr and Roberts, 1999). Checkpoints are in place to ensure both the interdependency and ®delity of S phase and mitosis. When defects in the division program of a cell are detected, checkpoints are evoked to inhibit or to prevent the activation of Cdks until DNA replication or mitosis are completed, or when perturbed, recti®ed to ensure stable genomic inheritance (Elledge, 1996; O'Connell et al., 2000). In contrast to the ®delity of normal cell division, cancer cells exhibit a high degree of genomic instability, widely thought to promote a heightened rate of mutation, allowing cancer cells to evolve (Lengauer et al., 1998). The loss of checkpoint controls operative in the G1 and S phases are essentially universally seen in human tumours. A prime example is the high rate of inactivation of the p53 tumour suppressor. DNA damage-activated p53 can promote cell cycle arrest, in order to allow time for DNA repair, through transactivation of the CKI p21cip1 gene (ElDeiry et al., 1993), which inhibits the Cdk2/CyclinE and Cdk2/CyclinA complexes ultimately required for entry into and passage through S phase. In other cases, either when DNA repair cannot be completed successfully or if cells are not programmed to respond to stresses by viable growth arrest, p53 may destine the cells for apoptosis (White, 1996). Inactivation of p53 thus promotes the evolution of cancer in two ways: genomic instability and the absence of apoptotic programs that would normally be triggered in response to genetic abnormalities. From the point of view of clinical management of many tumour types, the absence of p53 function thwarts the ecacy of genotoxic anti-cancer therapies. The archetypal cdk, Cdc2, together with A and Btype cyclins, controls the transition from G2 into mitosis (Dunphy, 1994). Unlike the G1/S Cdk2 complexes, these are not inhibited by CKIs, but rather are under the control of inhibitory phosphorylations on T14 and Y15 (Poon et al., 1996). Phosphorylation on Y15 is a highly conserved event, with identical regulation spanning back to ®ssion yeast (Nurse, 1990). Studies of G2/M DNA damage checkpoints in this

Human G2 DNA damage checkpoint K Koniaras et al

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yeast have identi®ed that Y15 phosphorylation of Cdc2 is the mechanism of checkpoint-mediated arrest (O'Connell et al., 1997; Rhind et al., 1997). In human cells, expression of a non-phosphorylatable mutant of Cdc2 (Y15F) greatly abrogates the ecacy of the checkpoint (Blasina et al., 1997; Jin et al., 1996). The upstream checkpoint proteins are also highly conserved between yeast and humans. In yeast these proteins send a signal that results in the activation of a protein kinase, Chk1, which in turn regulates Y15 phosphorylation through the activation of the Wee1 kinase and inactivation of the Cdc25 phosphatase that regulate Y15 phosphorylation (O'Connell et al., 2000; Raleigh and O'Connell, 2000). Studies with relatively nonspeci®c protein kinase inhibitors in cell lines (Graves et al., 2000), combined with in vitro biochemical experiments (Sanchez et al., 1997) have suggested that human Chk1 behaves in an analogous way to yeast chk1, however, loss-of-function experiments have yet to be reported. Chk17/7 mice display early embryonic lethality (Liu et al., 2000), which may be due to a prevalence of G2/M checkpoints over the relative absence of G1/S checkpoints early in development as suggested by studies in Xenopus (Masui and Wang, 1998) and murine embryonic stem cells (SchmidtKastner et al., 1998). The PI3-K related protein kinases, Ataxia Telangiectasia Mutated ATM, and a related protein, ATR, are thought to activate Chk1 by phosphorylation (Guo et al., 2000; Liu et al., 2000; O'Connell et al., 2000; Zhao and Piwinica-Worms, 2001). While ATR7/7 mice are also embryonic lethal (Brown and Baltimore, 2000), expression of dominant negative ATR in somatic cells is not lethal, although it does abrogate G2/M DNA damage checkpoints (Cliby et al., 1998). This suggests that the essential nature of ATR and Chk1 may be con®ned to the early embryonic cell cycles. Does p53 play a role in G2/M checkpoints? The answer to this question is surprisingly unclear. Many studies have shown that p53 mutant cells are capable of G2 arrest in response to DNA damage (Kastan et al., 1991; Taylor and Stark, 2001). However, such studies have looked at cells in which DNA damage is in¯icted either when growing asynchronously or synchronized in S phase. Moreover, many of these studies have used high doses of DNA damage from which the cells cannot recover. Under these conditions, studies with human cell lines in which p53 has been deleted by homologous recombination suggest that p53 plays a role in maintaining a sustained G2 arrest (Bunz et al., 1999). However, under these regimes, it may well be that inhibition of CyclinA/Cdk2 complexes in late S phase to early G2 are halting cell cycle progression. Hence, the relationship between p53 and the checkpoint that signals through the ATM/R-Chk1 pathway to maintain Y15 phosphorylation on Cdc2 is unclear. There are few reports of mutations in regulators and checkpoint genes involved in G2/M in tumours. With the abundance of G1/S abnormalities in tumours, G2/ M checkpoint genes may become essential in somatic tumour cells as seen in murine embyrogenesis. Should

this be the case, one would predict that abrogation of G2/M checkpoints in tumour cells lacking G1/S checkpoint control should greatly sensitize these cells to genotoxic stress. We show here that p53 is dispensable for a DNA damage mediated checkpoint arrest when the checkpoint is evoked in G2 cells. Conversely, interference with the ATM/R-Chk1 pathway by expression of a dominant negative Chk1 mutant signi®cantly abrogates the ability of cells to undergo G2/M arrest. The combined eect of interfering with Chk1 signalling in the absence of p53 is a profound radiosensitization that indicates the utility in targeting Chk1 during genotoxic stress to kill p53 mutant tumour cells.

Results p53 is dispensable for DNA damage-induced cell cycle arrest in G2 Instability of the genome is seen in the vast majority, if not all, of human tumours. Several mechanisms have been shown to contribute to this, including failure of DNA repair mechanisms and perturbations to cell cycle checkpoints, both in interphase and mitosis. In the G1 phase of the cell cycle, the tumour suppressor protein p53 is a key checkpoint protein that is upregulated in response to DNA damage, and signals cell cycle arrest through transactivation of cell cycle inhibitory genes such as p21. The transition from S phase into G2 and through to mitosis in human cells sees a `passing of the baton' of various cyclin/cdk complexes. Early on in the transition, cdk2/cyclinA complexes predominate, and act upstream of the cdc2/cyclinA and B complexes that ultimately are required for mitotic entry. Cdk2 complexes are inhibited by p21, and ternary complexes consisting of p21, cdk2 and cyclinA are found following DNA damage. Conversely, Cdc2 containing complexes are neither inhibited by p21, nor does Cdc2 interact with p21, either during G2 or a checkpoint arrest (Poon et al., 1996). As p21 is a major transcriptional target of p53, the phase of the cell cycle at which a checkpoint is activated may be important in discerning the relevant target cyclin-cdk complex. Based on this, we reasoned that some of the discrepancy in the conclusions regarding the role of p53 in G2 arrest could be due to the point in the cell cycle at which the checkpoint is activated. We therefore assessed whether p53 mutant cell lines that had been synchronized in G2 prior to irradiation still retained G2 checkpoint function. A signi®cant enrichment of G2 cells in all experiments was assayed by FACS DNA pro®les, the appearance of inactive, Y15 phosphorylated, cyclinB1/Cdc2 complexes and the absence of Cdc2 activity. Initially, we looked at DNA damage-induced checkpoint control in G2 synchronized HeLa cells, which lack p53 function due to the presence of E6 and E7 oncoproteins from the human papilloma virus. These cells have been shown by many


groups to be capable of G2 arrest when irradiated during S phase or when growing asynchronously. In G2 synchronized HeLa cells, pilot experiments using a range of doses showed that 6 Gy delayed entry into mitosis for 6 ± 8 h, and cells maintained *30% viability in clonogenic assays (data not shown). Figure 1 shows an analysis of G2 cell cycle proteins from cells prepared by this protocol. Under these conditions, inactive, Y15 phosphorylated Cdc2 accumulated to high levels. However, this accumulation can be accounted for by concomitant accumulation of cyclins A and B1, which would provide a larger substrate pool for the Y15 kinases Wee1 and Myt1, which only phosphorylate Cdc2 when bound to a cyclin (Booher et al., 1997; Fattaey and Booher, 1997; Liu et al., 1997; Parker et al., 1991). This was notable because when cells are irradiated at higher doses, from which fewer cells will recover, the transcription of cyclin B1 is repressed and the proteins levels are unable to accumulate or be maintained (Muschel et al., 1993). Altered sub-cellular localization of cell cycle proteins has been proposed to be a contributing factor to checkpoint control in yeast and human cells, though follow-up experiments have questioned the initial conclusions (Lopez-Girona et al., 2001). Wee1 is a nuclear protein, and its localization has been proposed to protect the nucleus from precocious mitotic events (Heald et al., 1993). However, one study found that Wee1 co-immunoprecipitated with the cytoplasmic and p53-regulated 14-3-3s protein in cells treated with Adriamycin (Chan et al., 1999). We investigated the eects of ionizing radiation on the localization of Wee1 by indirect immuno¯uorescence, and found its nuclear location to be unaected by irradiation (Figure 2) and

Figure 1 Accumulation of G2 cell cycle proteins in checkpoint arrested cells. Protein extracts were prepared from HeLa cells that were growing asynchronously (A), synchronized in S phase (S) or G2 (G2). The G2 cells were either mock irradiated (U) or irradiated with 6 Gy ionizing radiation, and protein preparation made 8 h later, whence the mock irradiated cells had cycled through mitosis and the irradiated cells were still arrested in G2. The levels of the indicated proteins were then assayed by Western blotting

so it is still in position to protect the nucleus from Cdk activity. Whilst these experiments show biochemically and physiologically that G2 DNA damage checkpoint arrest occurs in G2 synchronized cells in the absence of p53 function, the lack of isogenic wildtype p53 controls makes it dicult to assess the overall validity of these experiments. Recently, cell lines based on an HCT116 colonic carcinoma background have been generated in which p53 and some target genes such as p21 and 14-3-3s have been deleted by homologous recombination (Bunz et al., 1999; Chan et al., 1999). We again performed pilot experiments to de®ne appropriate doses at which HCT116 cells arrest and recover. We found a dose of 8 Gy delayed mitotic entry by *4 h, though greater doses lead to signi®cant death and failure to recover over several days, as has previously been reported when these cells were irradiated with 12 Gy. The checkpoint kinetics of wildtype and p537/7 in response to irradiation were indistinguishable: both cell lines remained in G2 for an additional *4 h following irradiation (Figure 3), then passed through morphologically normal mitoses (data not shown) and exited into G1 (Figure 3). We next examined the levels of cyclins A and B1, together with the Y15 phosphorylation status of Cdc2. In the presence or absence of p53, Y15 phosphorylated Cdc2 increased over the duration of the arrest. CyclinB1 continued to accumulate in the checkpointarrested cells, which may account for the increase in Cdc2 Y15 phosphorylation, though cyclin A levels did not change to a signi®cant extent. The more modest changes in protein levels compared to HeLa cells most probably re¯ected the shorter duration of the arrest. We also found a concomitant delay to Cdc2 kinase activation (Figure 3c). These observations unequivocally show that, under conditions where the checkpoint is activated in G2

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Figure 2 Wee1 is nuclear, and remains nuclear following irradiation. HCT116 cells were grown on coverslips, mock irradiated (7IR) or irradiated with 8 Gy ionizing radiation (+IR), and Wee1 localized by indirect immuno¯uorescence. Note with the exception of the mitotic cell in the mock-irradiated cells (arrowed), Wee1 is a nuclear protein that is not relocalized by DNA damage Oncogene


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Figure 3 p53 is dispensable for the G2 DNA damage checkpoint in human cells. (a) Parental HCT116 cells (p53+/+, shaded histograms), and a derivate harbouring targeted alleles of p53 (p537/7, open histograms) were synchronized by a double-thymidine block and release protocol. DNA FACS pro®les of asynchronously growing cells (Asyn) and G2 synchronized cells (Oh) were irradiated with 8 Gy of ionizing radiation (+IR) or mock irradiated (7IR). Cells were then cultured for the indicated times, and DNA pro®les assayed. Both populations of mock irradiated cells progressed through mitosis into G1 after *4 h in culture. Both populations of irradiated cells were delayed in G2 until *6 h in culture, (i.e. an *2 h checkpoint delay) and thereafter entered mitosis. (b) Proteins were extracted from the cells in a, and the levels of the indicated proteins were assayed by Western blotting. The radiation induced checkpoint in both cell lines correlated with an accumulation of inactive, Y15 phosphorylated Cdc2, accumulation of cyclin B1, and maintenance of cyclin A. The small dierences in FACS pro®les of mock-irradiated cells represent subtle dierences between individual synchronizations. (c) Cdc2 kinase activity was assayed at the indicated timepoints following irradiation in an independent timecourse (FACS pro®les shown) that had slightly dierent kinetics to those in a. Both lines show a radiation induced delay to Cdc2 kinase activation Oncogene


cells, p53 is entirely dispensable for checkpoint delay, leading to the possibility that inhibiting the mechanisms that control G2 checkpoints may aid in killing p53 mutant cells. S20 phosphorylation and up-regulation of p53 does not occur in G2 cells A key event in the p53-dependent signalling of a G1 DNA damage checkpoint is the accumulation of p53 protein levels in response to DNA damage (Oren, 1999). Interestingly, during the course of our experiments we observed that, while asynchronously growing cells (predominately G1 and S) showed a radiationdependent increase in p53 protein levels, such an increase was not observed in G2 synchronized cells (Figure 4). Amongst the multitude of post-translational modi®cations that aect the activation and stabilization of p53, two N-terminal phosphorylation events are under the control of known checkpoint kinases. Serine 15 (S15) is phosphorylated by the ATM and ATR kinases (Canman et al., 1998; Khanna et al., 1998; Tibbetts et al., 1999), and although initial data implicated this event in the stabilization of p53, it has subsequently been shown that phosphorylation of S20 by Cds1 (also known as Chk2) is the critical event to inhibit the interaction of p53 with the E3 ubiquitin ligase Mdm2 (Chehab et al., 2000). We reasoned that the inability of p53 to accumulate in response to DNA damage in G2 synchronized cells might result from the absence of one or more N-terminal phosphorylation events. Using phospho-speci®c antibodies in extracts from independently synchronized cells, we observed that S15

Figure 4 Radiation-induced stabilization of p53 does not occur in G2 cells. Asynchronously growing (A) or G2 synchronized (G2) HCT116 cells were irradiated with 8 Gy of ionizing radiation at t=0 h, and p53 levels assayed by Western blotting at the indicated timepoints

phosphorylation was not cell cycle speci®c and increased in both asynchronous and G2 cells in response to irradiation. This is consistent with the widespread roles for ATM and ATR in DNA damage responses. Conversely, while an increase in S20 phosphorylation was observed in asynchronously growing cells following irradiation, no such eect was seen in G2 synchronized cells (Figure 5). The lack of upregulation of p53 in G2 is consistent with the observations that p53 is, minimally, dispensable for this arrest.

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Abrogation of G2 checkpoint arrest by dominant-negative chk1 Pathways involving p53 are essentially universally mutated in human cancer. The independence of p53mediated G1 checkpoints inhibiting Cdk2 through p21, and G2 checkpoints resulting from inhibition of Cdc2 by Y15 phosphorylation, makes the G2 checkpoint an attractive target for drug development to treat p53 mutant tumours. Data in ®ssion yeast indicate that the only function for the Chk1 protein kinase is in signalling a DNA damage-mediated checkpoint arrest, whereas other proteins that act upstream from Chk1 are implicated in other cellular processes including telomere maintenance and DNA replication (O'Connell

Figure 5 Radiation-induced phosphorylation of p53 on S20 does not occur in G2 cells. Asynchronously growing (A), or G2 synchronized (G2) HCT116 cells were irradiated with 8 Gy of ionizing radiation and t=0 h, and protein samples were prepared from one aliquot of each immediately. Two hours later, irradiated asynchronous cells (A+IR), G2 mock irradiated cells (G2) and G2 irradiated cells (G2+IR) were harvested and protein extracts made. The levels of total p53, and S15 or S20 phosphorylated p53 were assayed by Western blotting. Note that S15 phosphorylation does not vary between irradiated samples (when corrected for total p53 levels), though only residual S20 phosphorylation, probably catalyzed by Cds1/Chk2, is seen in the G2+IR sample, possibly due to the residual G1/S peak shown in the FACS pro®les below, and correlates with the small degree of p53 stabilization (n.b. Figure 4) Oncogene


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et al., 2000). This unique function of Chk1, together with the amenity of serine-threonine protein kinases to pharmacological inhibition makes Chk1 a key molecule for such drug development programs. Is human Chk1 required for G2 checkpoints in a similar manner to its homologue in ®ssion yeast? The broad-spectrum kinase inhibitor UCN-01 (7-hydroxystuarosporine) has been shown to inhibit Chk1 in vitro (Graves et al., 2000), and whilst it also abrogates checkpoint responses of cells in culture, it is dicult to attribute all its eects simply to Chk1 inhibition. Another related inhibitor of Chk1, SB-218078, has been developed (Jackson et al., 2000), though similar arguments regarding in vivo speci®city apply, and knock-out mice have not been informative due to embryonic lethality (Liu et al., 2000). We therefore chose to more speci®cally address the requirement for Chk1 in human G2 checkpoint signalling using overexpression of a dominant negative mutant (D148E), which we had shown to phenocopy deletion of chk1 in ®ssion yeast (K Koniaras and M O'Connell, unpublished). A similar approach has been used to show G2 checkpoint function for ATR. Stable HeLa-based cell lines were selected that overexpressed dominant-negative Chk1, and two were retained for further analysis (Figure 6). Although both lines grew adequately in culture, a higher proportion of spontaneous apoptosis was observed (data not shown). However, attempts to synchronize these cells with a double-thymidine block and release protocol greatly increased cell death, possibly due to an S-M coordination defect as seen in Xenopus (Kumagai et al., 1998). We therefore, by necessity, resorted to nocodazole trapping experiments in asynchronously growing cells to assess the checkpoint status of these clones. In the presence of the topoisomerase II poison Etoposide, which causes G2 arrest in HeLa cells, both clones expressing dominant negative Chk1 continued to enter mitosis (Figure 6b). In similar experiments using ionizing radiation (Figure 6c), over the ®rst 6 h following irradiation, again a signi®cantly higher proportion of cells expressing dominant negative Chk1 continued to enter mitosis compared to control parental cells. These timepoints correspond to the cells that were in G2 at the point of irradiation, and was concomitant with a lesser degree of inhibition of Cdc2 kinase activity (Figure 6d). In latter timepoints, the dierence was less signi®cant, perhaps due to Chk1independent mechanisms of delay in G1/S, or to completion of DNA repair. These data are consistent with Chk1 being required for the G2 DNA damage checkpoint in human cells, as was suggested by the inhibitor studies, albeit without the complications caused by the lack of in vivo inhibitor speci®city. On the basis of these data, we then tested the eect of dominant negative chk1 expression on clonogenic survival following irradiation. Both clones were profoundly radiosensitive compared to the HeLa parental cells (Figure 6e), and colonies that did grow generally contained less cells. Thus, the checkpoint defects caused by inhibition of Chk1 manifest as a

signi®cantly radiosensitizing event. These data con®rm the model that Chk1 may be a prime anti-cancer drug target in cells that lack p53 function, perhaps in combination with radiation or genotoxic chemotherapy. Discussion The last decade has seen an enormous increase in our understanding of the molecular mechanisms that regulate cell growth and division. In more recent years there has been a concerted attempt to exploit this knowledge to design more eective anti-cancer treatments. The next generation of anti-cancer treatments will speci®cally target individually characterized cancer cells by exploiting a molecular `Achilles heel'. Such a strategy would ideally leave normal somatic cells relatively unscathed, which would in turn reduce the severity of side eects encountered using adjuvant chemotherapy and/or radiotherapy. Inactivation of the p53 tumour suppressor, leading to the loss of G1/S checkpoints, is a key ingredient for neoplastic transformation in the majority of human cancers. Unfortunately, another key ingredient in the ability of cancer cells to proliferate even in the face of genomic instability is the continued presence of a G2/ M checkpoint. This prevents cancer cells from accumulating the types of destabilizing chromosomal aberrations that would eventually prevent cells from replicating successfully (Levine, 1997). Hence, an attractive strategy for anti-cancer therapy would both exploit the absence of a G1/S checkpoint and abrogate the remaining G2/M checkpoint to kill cancer cells. Indeed, several reports have identi®ed novel compounds that are capable of inhibiting the ability of cells lacking functional p53 to undergo G2/M arrest (Curman et al., 2001; Jackson et al., 2000; Roberge et al., 1998). Such studies have demonstrated that a much lower dose of radiation or a chemotherapeutic agent is required to eectively kill the cells. In contrast, normal cells with functional p53 are not aected as they retain the ability to undergo a transient G1 arrest in response to the treatments. Often, however, the pathways aected by the compounds are not known, and may not necessarily target any one molecule in particular. This study has taken a twofold approach to addressing the molecular mechanisms behind a DNA damage-induced G2/M arrest in order to more accurately determine which molecule(s) involved would present a logical target for rational drug design aimed at inhibiting G2/M phase arrest. First, we wanted to investigate more closely the molecular events that occur in cells that lack functional p53, but are known to retain the ability to undergo G2/M arrest in response to DNA damage. Speci®cally, unlike previous studies, we were interested in what would happen to such cells if they were synchronized to the G2 phase of the cell cycle when subject to DNA damage. This protocol was employed so that the regulation of a predominant Cdk,


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Figure 6 Abrogation of the G2 DNA damage checkpoint by expression of dominant negative Chk1. HeLa cells were stably transfected with plasmids expressing a dominant negative, HA-tagged Chk1 cDNA. Two clones (Clone 1 and Clone 2) were selected for further analysis. (a) Western blot analysis using anti-Chk1 antibodies (upper panel) showed overexpression of Chk1, and with 12CA5 (lower panel) showed expression of the dominant negative allele. Reactivity to a non-speci®c band (*) is included as a loading control. (b) Cells were incubated in the presence of 2 mM Etoposide for 3 h, and then nocodazole was added to trap cells entering mitosis. After a further 24 h, mitotic cells were assayed by FACS using MPM-2 antibodies, and the per cent MPM-2 positive normalized against cultures without Etoposide. Data is mean+s.d. (c) The indicated lines were irradiated with 6 Gy ionizing radiation at time = 0, or mock irradiated, and then cultured in the presence of nocodazole. Cells were then assayed by FACS for the percentage of MPM-2 positive cells. Data represents the percentage of MPM-2 positive cells in the mock-irradiated population, divided by that for the irradiated cultures. Data points are mean+s.d. (d) The indicated lines were irradiated with 6 Gy ionizing radiation at time = 0, or mock irradiated, and then cultured in the presence of nocodazole. Cdc2 kinase activity was assayed, and is displayed as a percentage of that in mock irradiated cultures. (e) Clonogenic survival assays for the indicated strains were performed as described in Materials and methods. Data points are mean+s.d.

Cdc2, could be studied without the confusing eects of upstream regulation mediated through Cdk2. Initially, we used HeLa cells, which not only lack p53 due to enforced degradation due to the presence of the HPVE6 oncoprotein, but are known to retain the ability to undergo G2/M arrest. An increase in Y15-phosphorylated Cdc2, one of the de®nitive markers indicating G2/M arrest, along with an increase in cyclins A and B1, was observed in G2 irradiated cells. This indicates

that the upstream signalling pathways leading to phosphorylation of Cdc2 were intact in these cells. Even though p53 is continually degraded in HeLa cells by HPV-E6, there is still a small amount of the protein that is expressed. We could not discount the possibility that this small amount of p53 could be sucient to aect the signalling pathways culminating in Y15 phosphorylation of Cdc2. Also, the eect of this small amount of p53 leading to the induction of Oncogene


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G2 arrest independently of Cdc2 phosphorylation had to be taken into account. We thus sought another model system that would provide a clearer picture of whether p53 is involved in some aspect of G2 arrest. To this end we used a HCT116 human colon carcinoma cell line in which both p53 alleles had sequentially been knocked out by homologous recombination (Bunz et al., 1999). In addition, the wildtype parental HCT116 cell line provided an isogenic control for these experiments. Both cell lines, when irradiated in G2, arrested for the same period of time, showed similar patterns of Cdc2 Y15 phosphorylation, concomitant with a similar increase in cyclin B1 expression. Therefore, under these conditions, the presence or absence of p53 does not appear to either enhance or hinder G2 arrest, nor the molecular events associated with it. Furthermore, we observed that, in wildtype HCT116 cells, p53 levels did not increase in response to DNA damage whilst in G2; this appears to be attributed to the inability of p53 to undergo phosphorylation at S20 in G2, which promotes dissociation of the p53-Mdm2 complex and allows for the stabilization of p53 (Chehab et al., 2000; Hirao et al., 2000; Shieh et al., 2000). The lack of signalling to p53 in G2, together with its dispensable nature for the G2 checkpoint arrest, suggests p53 is not a major player in human G2 DNA damage checkpoints. Previous reports have suggested that one of the mechanisms by which p53 might play a role in G2 arrest is through its ability to down-regulate genes such as cyclin B1 (Muschel et al., 1993). These reports have relied on enforced overexpression of p53 or high-level irradiation, each leading to a sustained arrest of up to several days. Under such conditions of sustained arrest, studies with the HCT116-based cell lines implicate p53 in its maintenance (Bunz et al., 1999). While not discounting the possibility that such an event may occur in a physiological system, we have shown here that under conditions where cells arrest transiently and recover to pass through mitosis, the p53-independent arrest was accompanied by cyclins A and B1 accumulation. The increased pool of inactive mitotic cyclin/cdk complexes, otherwise known as pre-MPF, which accumulate during this G2 delay results in the accumulation of Y15 phosphorylated Cdc2. Coupling these observations with the checkpoint-abrogating eects of expressing non-phosphorylatable mutants of Cdc2 (Blasina et al., 1997; Jin et al., 1996) highlights the importance of the checkpoint to ensure inactivation of accumulating pre-MPF. Many studies have focused on inactivation of Cdc25C by Chk1 phosphorylation as a mechanism of checkpoint arrest. However, the absence of the relevant site in murine Cdc25C (Ogg et al., 1994), and the lack of phenotype of Cdc25C nullizygous mice (Chen et al., 2001) suggest that this is insucient to mediate a G2 arrest. In ®ssion and budding yeasts, Wee1 protein levels are upregulated due to inhibition of proteolysis following DNA damage checkpoint activation (Leu and Roeder, 1999; Raleigh and O'Connell, 2000). In our experiments, however, Wee1 levels were upregulated by the

synchronization protocol, which is based on evoking the DNA replication checkpoint. Studies in Xenopus have shown the replication checkpoint leads to Wee1 accumulation through inhibition of Cdc34-dependent ubiquitination (Michael and Newport, 1998); it is probable that similar mechanisms may occur in human cells. Cells arrested in G2 with Etoposide also showed high levels of Wee1 despite not having been through the synchronization protocol (data not shown). The nuclear pools of Wee1, which we have shown to remain nuclear following irradiation, may be another important eector, as seen in ®ssion yeast. We have attempted to test this with constitutive and tetregulated expression of kinase dead Wee1 mutants, but have been unable to gain evidence of dominant negative eect over endogenous Wee1 (K Koniaras and M O'Connell, unpublished). Recent studies with tyrosine kinase inhibitors show that Wee1 activity is required for G2 arrest following DNA damage (Alan J Kraker, personal communication; Kraker et al., 2000), and so maintaining or increasing pools of nuclear Wee1 may be an important aspect of human G2 checkpoints. Cdc25B, which is also phosphorylated by Chk1 in vivo (Sanchez et al., 1997), may also be an in vitro target, and although extensive screens in yeast have not found other G2 checkpoint eectors, other yet to be identi®ed human proteins may contribute to the p53-independent G2 arrest in response to DNA damage. Targeting core cell cycle regulators such as Wee1 or Cdc25 to modulate a G2 checkpoint arrest, however, would be likely to have profound side eects in a clinical setting. Using a dominant negative Chk1 mutant, however, we have shown here both checkpoint abrogation, and most importantly, clonogenic radiosensitization of HeLa cells, without dramatic eects on cell proliferation in the absence of extrinsic DNA damage. These observations validate Chk1 as a target for drug development, particularly when p53 function is absent or diminished by viral oncoproteins. We propose that human DNA damage checkpoints, both of which are essential in maintaining genomic stability, consist of at least two independent and nonoverlapping signalling pathways (Figure 7). In G1 and S phase, p53 plays a crucial role to ensure inhibition of cyclin/Cdk2 complexes, which may extend into early G2 phase. At least one important eector of p53 function is p21. In later G2, where Cdc2 is the predominant Cdk, Chk1 dependent signalling to ensure maintenance of the G2 state through Y15 phosphorylation of Cdc2 is the key event. Although the adjacent threonine residue, T14, is also phosphorylated in inactive cyclin A, B/Cdc2 complexes, the localization of the kinase responsible, Myt1, to the cytoplasm (Booher et al., 1997; Fattaey and Booher, 1997; Liu et al., 1997) makes it an unlikely eector of DNA damage signalling, though there is no data to disprove this. The almost universal inactivation of G1/S checkpoints in human tumours is contrasted by the apparently universal retention of active G2 checkpoints. These observations, together with the observed eects


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Figure 7 Model for major DNA damage checkpoint pathways in human cells. In G1 and S phase, p53, through p21 transactivation, is the major eector of cell cycle arrest. p53 is upregulated by phosphorylation on S15 and S20, the latter catalyzed by Cds1 and prevents binding to Mdm2. The upregulated p21 prevents cell cycle progression by binding to and inhibiting CyclinE/ Cdk2 (E/K2) and CyclinA/Cdk2 (A/K2) complexes. The dashed line represents that the inhibitory eects over CyclinA/Cdk2 may continue to manifest after S phase completion into early G2. In G2 cells, Cds1-mediated upregulation of p53 does not occur, and p53 is dispensable for cell cycle arrest. In this phase of the cell cycle, ATM and ATR activate Chk1, probably through phosphorylation of S345. Chk1 can then down regulate Cdc25 family members or ensure they remain inactive, and may upregulate Wee1, or alternatively Wee1 activity is maintained throughout the arrest. The net eect of this maintenance of G2 is inhibition of accumulating CyclinA/Cdc2 (A/C2) and CyclinB/Cdc2 (B/C2) complexes through inhibitory phosphorylation on Y15. Inactivation of both pathways has a synergistic eect, greatly radiosensitizing the cells

reported here of interfering with Chk1 function, strongly implicate the necessity of the G2 checkpoint for the variability of p53-dependent checkpoint de®cient tumours. We eagerly await the development of speci®c Chk1 inhibitors, which we would predict to have enormous ecacy in clinical oncology.

Materials and methods DNA manipulations Human Chk1 cDNA was tagged with its 5' end with six histidines and one haemagglutinin tag by PCR with Deep Vent polymerase (NEB) with the following oligonucleotide: 5'-TACTG CAGACGC GTACCAT GGCACAT CACCATCATCA CCACTATCCCTA TGACGTCCCGGACTATGCAGTGCCCTTTGTGGAAGAGCTG-3'. A 3' primer of 5'CCTGCCATGAGTTGATGGAAGAATCTCTGA-3' was used for these reactions, and the resulting PCR products were cloned into pLitmus28 (NEB) and the ®delity of PCR con®rmed by sequencing. Aspartic acid 148 was mutated to glutamic acid with the following primer 5'-CTCAAAATCTCAGAATTCGGCTTGGCAACA-3', using previously described methods (Kunkel et al., 1987), and the ®delity of the reaction again con®rmed by sequencing. This mutation phenocopies the deletion of chk1 in ®ssion yeast (K Koniaras and M O'Connell, unpublished). The remaining portion of Chk1 was added to this construct, and the inserts were then subcloned into pIRES-Puro (Hobbs et al., 1998).

Cell culture HeLa cells, and stably transfected derivatives, were propagated in DMEM (Sigma) supplemented with 10% FCS (CSL) and 100 units/ml of penicillin/streptomycin in 5% CO2. HCT116 and HCT116-p537/7 cells (a kind gift of Dr B Vogelstein) were propagated in McCoys medium (Sigma) supplemented with 10% FCS (CSL) and 100 units/ml of penicillin/streptomycin in 5% CO2. For synchronizations, a double-thymidine block and release protocol was employed. Brie¯y, sub-con¯uent cells were incubated in the presence of 2 mM thymidine (Sigma) for 17 h, allowing the majority of cells to accumulate in early S phase. Cells were then washed with PBS and incubated in medium without thymidine for 9 h to allow release into the cell cycle. A second S phase block was imposed by a further 14-h incubation in the presence of 2 mM thymidine. These S phase synchronized cells were again washed with PBS, incubated with fresh medium, allowed to progress into G2 (approximately 5 h following release, as assessed by FACS, appearance of cyclinB1/Cdc2 complexes, the absence of mitotic ®gures and the absence of Cdc2 kinase activity, analysis prior to irradiation). Stably transfected HeLa cell lines were obtained by electroporating cells with pIRESPuro based plasmids described above followed by sequential selection in 0.4, 1, 2, 5 and 10 mg/ml Puromycin (Sigma) as described (Hobbs et al., 1998). Irradiation and survival assays For irradiation timecourse experiments, subcon¯uent synchronized or asynchronously growing cells were irradiated in medium with a 137Cs source at a dose rate of 0.75 Gy/minute. Oncogene


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For nocodazole trapping experiments, following irradiation cells were incubated in medium containing 150 ng/ml nocodazole. For survival assays, cells were irradiated, harvested and replated at varying densities depending on dose. Colonies were allowed to form over 4 weeks, whence surviving colonies were ®xed and stained in neutral formalin/ 0.01% Crystal violet. Colonies were counted from 6 ± 12 dishes, and the data normalized to unirradiated cells, which corrects for varying plating eciencies between lines. FACS analysis For analysis of DNA content, cells were harvested with trypsin, washed in PBS and ®xed with 70% ethanol. Fixed cells were rehydrated with PBS, and stained with Propidium Iodide (PI) as described. To assay the fraction of cells in mitosis, cells were ®xed in 0.5% formaldehyde/PBS for 15 min, post-®xed in 70% ethanol and stained with the MPM-2 monoclonal antibody (1 : 2000, a kind gift of Dr P Rao, University of Texas, USA), and with FITC-conjugated anti-mouse IgG secondary antibodies (Sigma) diluted 1 : 100. Cells were counter stained with PI. FACS analysis was carried out with a Becton Dickinson FACSCalibur, and data analysed with CellQuest software. Estimates of mitotic fractions in MPM-2 stained cells were veri®ed by direct counting on an epi¯uorescence microscope. Protein extractions, kinase assays and Western blotting For protein analysis cells were harvested with trypsin, washed in PBS and snap frozen in liquid nitrogen. Extracts were prepared in 50 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40. Electrophoresis and Western blotting was performed as previously described (O'Connell et al., 1997). Antigens were detected with horseradish peroxidase coupled secondary antibodies and ECL detection reagents according

to the manufacturer's speci®cations (Amersham). The following primary antibodies were obtained from the following providers and used at the indicated concentrations: Cdc2 (Santa Cruz no SC-954) 1 : 1000, Cdc2-Y15 (Cell Signalling Technologies no 9111) 1 : 100, CyclinA (Santa Cruz no SC751) 1 : 500, CyclinB1 (Santa Cruz no SC-752) 1 : 100, Wee1 (Santa Cruz no SC-325) 1 : 100, Chk1 (Santa Cruz no SC7234), 12CA5 (a gift of Dr R Pearson, PMCI) 1 : 500, p53 (Ab-6, Calbiochem no OP43) 1 : 2000, p53-S15 (Cell Signalling Technologies no 9284) 1 : 100, p53-S20 (Cell Signalling Technologies no 9287) 1 : 100. Cdc2 activity was assayed from Cdc2 (SC-954) immunoprecipitates as described (O'Connell et al., 1994). Indirect immunofluorescence Cells were grown on poly-L-lysine coated cover slips, irradiated with 6 Gy or mock irradiated, and ®xed in 4% formaldehyde/PBS 1 h following irradiation. Cells were then processed for indirect immuno¯uorescence using anti-Wee1 antibodies at 1 : 100, counter-stained with 10 mg/ml DAPI and mounted in 50% glycerol, 1 mg/ml p-Phenylenediamine. Cells were viewed on a Zeiss Axiophot microscope, and images captured with a Spot2 camera.

Acknowledgements We thank Drs B Vogelstein and P Rao for the prevision of reagents, Alan J Kraker for communication of results prior to their publication, and Doris Germain for critical reading of the manuscript. This work was supported by grants from the NHMRC. A Cuddihy is a Fellow, and M O'Connell a Scholar, of the Leukemia and Lymphoma Society.

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