WO2009047567A1

WO2009047567A1 - Assay for identifying agents which inhibit the atr and/or dna-pk pathways

Info

Publication number: WO2009047567A1
Application number: PCT/GB2008/050930
Authority: WO
Inventors: Carl Smythe
Original assignee: University Of Sheffield
Priority date: 2007-10-10
Filing date: 2008-10-10
Publication date: 2009-04-16

Abstract

The present invention relates to method and assay useful for identifying an active agent useful for treating neoplasia. The method involves determining the ability of a text agent to inhibit the ATR pathway, determining the ability of the text agent to inhibit the DNA-PK pathway; and selecting an agent which is able to inhibit one of the ATR-PK pathways, but which does not significantly inhibit the other.

Description

ASSAY FOR IDENTIFYING AGENTS WHICH INHIBIT THE ATR AND/OR DNA-PK PATHWAYS

The present invention relates to an assay which is suitable for identifying agents which may be useful in the treatment of neoplasia, especially cancer. In particular the present invention relates to an assay for the identification of agents which are able to selectively interfere with regulation of histone production.

In simple terms, neoplasias develop when cells loose their ability to regulate proliferation. Neoplasia is a general term encompassing conditions which involve pathological proliferation of cells in a tissue or organ, and includes cancers as well as other non-malignant pathologies.

The development of new treatments for cancer is one the most significant issues for the medical and pharmaceutical fields. Development of such new treatments relies on the identification and exploitation of novel pathways and targets with which to selectively target cancer cells with active agents, while minimising the effect of these agents on healthy cells. Identifying targets and pathways with which to combat cancer (and other neoplasias) is significantly more difficult than when treating infections by heterologous organisms, e.g. bacteria or viruses; cancerous cells may be almost identical to normal healthy cells, except for the loss of one or more regulatory systems. Thus, identifying treatments which can selectively target cancer cells without also targeting normal cells is a significant problem for the development of therapies.

It is therefore desirable to identify ways in which neoplastic cells differ from healthy cells, and to understand the effect that these changes have upon the cell. Based on such an understanding it is possible to formulate novel approaches to tackling neoplasia.

Checkpoints are signaling pathways which maintain appropriate temporal order during the complex set of events which comprise the cell division cycle. For example, checkpoints ensure that chromosome duplication precedes cell division, and that cell division must occur before the next round of DNA replication. The present invention is founded upon research which focuses on a checkpoint which is activated when cells are unable to progress with chromosomal duplication.

Chromosomal duplication involves the coordinated regulation of two very different processes. The primary process involves the replication of DNA double strands within every chromosome. Additionally, however, eukaryotes must successfully assemble newly synthesised DNA into a higher-order structure termed chromatin, which involves the organised assembly of nucleosomes, comprising DNA and histone proteins.

DNA replication and histone protein synthesis are essential S-phase events. These two processes must be finely balanced; disturbances can result in mis-regulation of gene expression, cell cycle arrest and chromosome instability (Han et al., 1987; Meeks-Wagner and Hartwell, 1986; Wyrick et al., 1999), any one of which may result in developmental failure.

Eukaryotic DNA replication is initiated from many origins that fire in a precise temporal sequence in S-phase. The overall rate of DNA synthesis is governed by the number of active origins together with the intrinsic catalytic rate of the replication machinery operating at a replication fork; this in turn creates specific demands for timely delivery of additional components, such as histones, required for chromatin assembly. DNA and histone synthesis are coupled. Inhibition of DNA synthesis causes rapid destabilisation of histone mRNA, resulting in a swift shutdown of histone synthesis (DeUsIe et al., 1983; Heintz et al., 1983), raising the possibility that molecular components that control histone mRNA levels may be regulated by checkpoint machinery. Histone mRNA stability is dependent on an RNA hairpin in the 3' untranslated region (Graves et al., 1987) bound by the histone mRNA binding protein SLBP/HBP (Martin et al., 1997; Wang et al., 1996; Zhao et al., 2004) which is necessary for histone mRNA expression.

Checkpoints control the timing and order of cell cycle events following genomic insult (Zhou and Elledge, 2000). Several checkpoints may be activated in S-phase. Failure in DNA replication blocks entry into mitosis (Rao and Johnson, 1970). Similarly replicational stress or DNA damage incurred early in S-phase prevents any further initiation from later-firing replication origins (Painter and Young, 1980) and stabilizes arrested replisome components arising from previously-fired origins (Dimitrova and Gilbert, 2000; Feijoo et al., 2001 ). In yeast, this origin firing checkpoint is mediated by Med and Rad53 (Santocanale and Diffley, 1998; Shirahige et al., 1998), the homologues of human checkpoint kinases ATR/ATM and Chk2 respectively.

Activation of the p53-dependent G(1 ) checkpoint in response to ionizing radiation in human cells results in inhibition of histone gene transcription, indicating that, like yeast, human histone gene expression is subject to checkpoint control (Su et al., 2004). Checkpoint proteins have also been implicated in the coordination of histone and DNA synthesis. In yeast, Rad53 is required for degradation of excess histone protein that is not packaged into chromatin (Gunjan and Verreault, 2003). Rad53 may also play a role in histone metabolism and chromatin assembly after DNAdamage (Emili et al., 2001 ).

A novel cellular checkpoint has been identified which coordinates the synthesis of DNA with the production of histone protein. Coordination is achieved by regulating the levels of histone messenger RNA (mRNA) in a cell which, in turn, controls the average level of histone protein. It has been found that this coordination is controlled by two inter-dependent signaling pathways. Cancer cells frequently mutate components of one or other of these pathways in the process of neoplasia.

It follows from the above that cancers which lack one or other of the pathways coordinating DNA replication with histone production would be peculiarly vulnerable to a particular cocktail of chemotherapeutic agents which would have the property of inhibiting DNA replication (as many chemotherapeutic drugs do) and blocking one or other, but not both, of the pathways involved in controlling histone protein production. Noncancerous cells which retain molecular components of both signaling pathways would retain a mechanism for ensuring viability in the presence of such drugs, while treatment of tumour cells which only have one pathway would result in an uncoupling of histone production from DNA synthesis. This condition is notoriously toxic for cells and is the likely reason that cells have developed this robust failsafe double pathway for co-ordinating DNA and histone synthesis.

According to a first aspect of the present invention, there is provided a method for identifying an active agent useful for treating neoplasia, the method comprising: - determining the ability of a test agent to inhibit the ATR pathway; - determining the ability of the test agent to inhibit the DNA-PK pathway; and

- selecting an agent which is able to inhibit one of the ATR and DNA-PK pathways, but which does not significantly inhibit the other.

As mentioned above, healthy cells typically have functioning ATR and DNA-PK pathways and, as such, when one pathway is inhibited for some reason, the other pathway effectively compensates. However, where a cell has lost one or other of the pathways, inhibition of the other pathway will lead to cell death due to accumulation of toxic levels of histones within cells in which DNA replication has been disrupted. This represents a novel approach to targeting neoplastic cells.

By the term "ATR pathway", it is intended to mean the entire pathway within which ATR exhibits the effect of providing a checkpoint to halt replication. It is well known that in most biological signalling pathways there are many active members, which may have varying roles in converting an input signal into a desired output. Evidence suggests the ATR pathway has a number of members, and thus it is envisaged that, although ATR is a key component in the pathway, there will likely be other proteins or other moieties with key roles in providing the checkpoint. The ATR pathway could be inhibited by agents which target any such member of the ATR pathway. The full complement of the ATR pathway is yet to be elucidated, though this will undoubtedly be achieved in due course.

The term "DNA-PK pathway", in an analogous manner, is intended to mean the entire pathway within which DNA-PK exhibits the effect of providing a checkpoint to halt replication. Some likely targets for a test agent, in addition to ATR and DNA-PK themselves, would be components of the major DNA replication and repair pathways that they are believed to regulate. In the case of ATR, any components of the DNA replication machinery, such as PCNA, RPA, and DNA polymerases, and any component of the homologous recombination pathway (such as Mus81 nuclease, Rad51 recombinase and the RecQ helicase). In the case of DNA-PK, they might be any additional components ( DNA-PK plays a major direct role in NHEJ) of the Nonhomologous end joining (NHEJ) pathway, such as the XRCC4, ligase IV complex.

Where a member of the ATR pathway also has a role in the DNA-PK pathway, or vice versa, then it would generally be unsuitable as a therapeutic target, as agents which inhibit such a member would inhibit both pathways.

Proteins homogenous to ATR and DNA-PK, and other members of their associated pathways, exist in organisms other than humans. In such organisms they may be known by other names. Such homologous proteins and pathways are intended to be within the scope of the present invention. However, inhibition of the human pathway is of primary concern.

An agent is able to inhibit one or other of the pathways when the activity of the pathway is substantially reduced. Inhibition of a pathway may be considered to occur wherein there is a 50% or greater reduction in activity of the pathway, preferably a 75% or greater reduction of activity, especially a 90% or greater reduction in activity. Preferably the step of determining the ability of a test agent to inhibit the ATR or DNA-PK pathways comprises determining the effect of the agent on an indicator of histone levels or histone production within a cell. Generally inhibition will be demonstrated by a reduction in histone levels or histone production.

A particularly suitable indicator of histone protein levels and/or histone production within a cell is the level of histone mRNA. Histone mRNA levels are closely liked to histone protein levels and modulation of these levels is a key control mechanism by which cells control replication. Histone H2A, H2B and H3 mRNA is a particularly suitable mRNA for analysis. Histone mRNA levels can determined by a number of conventional techniques, which include, but are not limited to, northern blotting, micro-array technology, or quantitative reverse transcriptase PCR (RT-PCT).

The test agent may be essentially any substance which would be suitable for delivery to a patient as a component of a pharmaceutical preparation. Examples of suitable agents include small molecules, peptides, proteins, and poly- or oligo-nucleotides. Delivery technologies are constantly developing and, as such, agents which are currently difficult or impossible to deliver to a patient will likely become deliverable in future.

In a preferred embodiment of the present invention there is provided a method for identifying an active agent useful for treating neoplasia, the method comprising: a) providing a first cell-line in which the activity of one of the ATR pathway or the DNA-PK pathway is abrogated, the other pathway being substantially functional; b) providing a second cell-line in which the pathway which is abrogated in the first cell-line is substantially functional; c) administering a test agent to said cell lines; d) determining the ability of the test agent to inhibit the ATR and DNA-PK pathways; and e) selecting an agent which selectively inhibits one or other, but not both of the ATR and DNA-PK pathways.

In the second cell line both the ATR and DNA-PK pathways may suitably be functional. Alternatively, the pathway which is functional in the first cell line may be abrogated. The significant issue is that the method allows the identification of agents which inhibit one pathway, but which do not substantially inhibit the other pathway.

It is generally preferred that the second cell line has substantially functional ATR and DNA-PK pathways. Such a cell line may suitably be a "wild-type" cell line.

Abrogation of the ATR or the DNA-PK pathways can achieved by inhibiting or knocking-out the expression or function of one or more members of the pathways. Members of the respective pathways are discussed above, and would provide possible targets for abrogating the relevant pathway. Means to achieve such abrogation are well known in the art, and the person skilled in the art would be able to achieve such abrogation of known pathway members using conventional techniques. These techniques could be applied to members of the pathway which are currently known or yet to be elucidated. Such techniques may be transient or permanent and include, but are not limited to, gene knock out, RNA interference (RNAi), anti-sense RNA (asRNA), and expression of a dominant negative mutant protein. It is envisaged that the ATR or the DNA-PK pathways may be most conveniently abrogated using techniques which target the ATR or DNA-PK proteins themselves, or their respective genes or mRNAs.

Abrogation of the ATR or the DNA-PK pathways may suitably involve total ablation of the function of the pathway. However, this is not essential and it may be sufficient to substantially abrogate the pathway, such that the activity of the pathway is reduced to 40% or less of normal activity for the duration of the assay, preferably 20% or less, especially 10% or less.

It is generally preferred that the cell-lines used in the present method are animal cell lines, preferably mammalian cell lines, especially human cell- lines. For use in the method the cell line would typically be maintained in suitable culture medicine and under suitable culture conditions as appropriate.

A particularly suitable cell-line in which the ATR pathway has been abrogated is a cell line which over-expresses a kinase-dead form of ATR, for example the U20S/kd-ATR cell-line.

A particularly suitable cell-line in which the DNA-PK pathway has been abrogated is the M059J cell line.

In a preferred embodiment of the present invention, a single cell-line can be used in which the ATR and/or the DNA-PK pathways can be selectively abrogated. Thus in the method described said first and second cell-lines may actually be in principle the same cell-line, but manipulated such that in the first cell line one or other of the ATR and DNA-PK pathways is abrogated, and in the other it is active. This can be conveniently achieved using RNAi technology, though other techniques will be apparent to the person skilled in the art. Conveniently the single cell line may be a HeLa cell-line, though other cell lines may be suitable.

Suitably the method is adopted for high throughput systems.

According to a further aspect the present invention provides an assay, the assay comprising: a) a first cell-line in which the activity of one of the ATR pathway or the DNA-PK pathway is abrogated, the other pathway being substantially functional; b) a second cell-line in which the pathway which is abrogated in the first cell line is substantially functional; and c) means to assess the ability of the test agent to inhibit the ATR and DNA-PK pathways.

Details of suitable cell lines and means by which inhibition of the ATR and DNA-PK pathways can be assessed are described above.

Suitably the assay is a high throughput assay.

According to a further aspect the present invention provides an active agent identified using the method or assay as described above. Such active agents would provide highly attractive lead compounds for further investigation in a drug development program.

Furthermore the invention provides a pharmaceutical composition comprising such an active agent in combination with a pharmaceutically acceptable excipient. The invention will now be described, by way of example only, with reference to the accompanying drawings, which show the following:

Figure 1 - Caffeine abrogates the replication checkpoint and inhibits histone mRNA decay induced by repiicationai stress in HeLa ceiis.

(A) Caffeine abrogates the replication checkpoint. (Top) Asynchronous HeLa cells were pulsed with CIdU for 20 min, and then incubated with drugs absent (mock), in the presence of APH alone or in the presence of both APH and caffeine. At different times (typically 6-16 h) after the CIdU pulse, cells were washed free of drugs and pulsed with IdU for 20 min. CIdU or IdU incorporation was visualised by immunofluorescence confocal microscopy. The schematic shows the initial CIdU pulse (green) incorporated into an early replication pattern and, at times thereafter, IdU pulses (red) either co-localised (yellow) with the CIdU pulse in the presence of APH or being incorporated into progressively later replication patterns in mock- treated cells and in cells where the replication checkpoint has been abrogated by the addition of caffeine. (Middle) Graph summarises typical data in the experiment depicted above and shows the fraction of those cells with an early pattern of replication at the first (CIdU) pulse proceeding into the indicated pattern (early, early/mid, mid or late) visualised by the second (IdU) pulse following each treatment. (Bottom) Nocodazole-arrested HeLa cells were released into drug-free medium for 14 h (to ensure cells were in S-phase) Then cells were either mock-treated (Control), or treated with 5 mM caffeine alone (+caffeine) for 1 h, prior to being treated for a further 2 h either with (+ HU, + caffeine + HU) or without (control, + caffeine) HU to induce repiicationai stress. Cells lysates were then immunoblotted for Chk1. (B) Caffeine inhibits histone mRNA decay. Asynchronous HeLa cells were treated with indicated caffeine concentration or mock-treated. After 1 h, 2 mM HU or water was added and incubation continued for 30 min, followed by isolation of RNA. RNA was analysed by Northern blotting using probes detecting histone H2B mRNA, H3 mRNA, or

GAPDH mRNA. Histone mRNA levels were standardised with respect to GAPDH mRNA levels, and the graph shows histone mRNA levels in HU-treated cells expressed as % of level in untreated cells.

Figure 2 - Inhibition of ATR signalling does not interfere with replicational stress-induced histone mRNA decay.

(A) Time-course of HU-induced decay of histone mRNA in cells expressing kd-ATR.

Expression of wt-ATR or kd-ATR was either induced (+Dox) or not (- Dox) by treatment of U2OS/ATR and U2OS/kd-ATR cells with doxycycline. Then, cells were treated with 2 mM HU and RNA was isolated at the indicated times after HU addition. Histone RNA was analysed as in Fig. 1. Changes in histone H2A, H2B and H3 mRNA levels were compared to the level at 0 min. (B) Inactivation of ATR signaling using cells expressing kd-ATR suppresses phosphorylation and activation of Chk1 in response to replicational stress.

Expression of kd-ATR was either induced (+kd-ATR) or not (-kd-ATR) by treatment of U2OS/kd-ATR cells with doxycycline or buffer. Cells were then treated with 50 μg/ml APH for 16 h to induce replicational stress. Subsequently, cell lysates were subjected to Chk1 immunoprecipitation kinase assay (graph) or immunoblotted using Chk1 antibodies (anti-phospho-Ser345; upper panel; total Chk1 antibody; lower panel). (C) Ablation of ATR by RNAi does not interfere with HU-induced histone mRNA decay. Asynchronous HeLa cells were transfected with siRNA targeting ATR, control siRNA 1 (targeting luciferase), control siRNA 2 (targeting a non-relevant gene), or mock-transfected. 32 h post-transfection, cells were treated +/- 2 mM HU for 0, 20 or 60 min, or left untreated for 60 min and then lysed for RNA analysis (right hand panel). Histone H3 mRNA levels were analysed as in Fig.1. Graph shows changes in H3 mRNA levels compared to the level at 0 min. In parallel, cell lysates were immunoblotted for ATR (to confirm knockdown) and nucleolin (loading; left hand panel).

Figure 3 - Replicational stress-induced histone mRNA decay occurs efficiently in cells lacking both ATM and ATR.

(A) ATM is not required for histone mRNA decay induced by replicational stress.

AT22IJE-T/pEBS (AT/pEBS) cells lacking ATM and AT22IJE-T/pEBS- YZ5 (AT/pEBS-YZ5) cells complemented with ATM were treated with 2 mM HU for the times indicated, and RNA was isolated and analysed by Northern blotting, as in Fig. 1. Histone H3 mRNA levels were normalised to GAPDH mRNA levels and graphed compared to the level at O min (100 %).

(B) Histone mRNA decay occurs normally in AT cells treated with ATR siRNA. AT221 JE-T/pEBS cells were transfected with siRNA targeting ATR or a control. 48 h post-transfection, 2 mM HU was added and cells were incubated with HU for the indicated times prior to RNA isolation and analysis. Graph shows changes in histone H3 mRNA levels compared to the level at 0 min.

Figure 4 - Wortmannin induces a delay in replicational stress-induced histone mRNA decay. (A) Concentration-dependence of delay in histone mRNA decay induced by wortmannin. Asynchronous HeLa cells were treated with the indicated concentration of wortmannin or mock (DMSO). After 1 h, cells were treated +/- 2 mM HU and RNA was isolated after a further 30 min for analysis by Northern blotting. Bar graph shows histone mRNA levels in HU-treated cells compared to levels in corresponding non-HU-treated cells.

(B) Kinetic analysis of delay on histone mRNA decay induced by wortmannin. Asynchronous HeLa cells were treated with 100 μM wortmannin or mock (DMSO). After 1 h, cells were treated +1-2 mM

HU. RNA was isolated at indicated times following HU addition and from untreated cells after 50 min. Graph shows the changes in histone mRNA levels compared to the level at 0 min.

Figure 5 - Inhibition ofATR/ATM signalling by caffeine results in a DNA-PK mediated activation of Chk2.

HeLa, MO59J (DNA-PK(-)) or MO59K (DNA-PK(+)) cells were synchronised in metaphase by treatment with nocodazole for 14 h. Mitotic cells were removed by shake-off, plated in fresh medium containing APH for 24 h (0 h) . Then, caffeine or buffer was added and cells incubated for a further 5h. Cells lysates were subjected to Chk2 immunoprecipitation kinase assay (upper graphs) or immunoblotted with Chk2 antibody (lower panels). Chk2 band-shifting (pChk2) is indicative of phosphorylation (Feijoo et al., 2001 ).

Figure 6 - DNA-PK is a regulator of histone mRNA stability.

(A) Asynchronous HeLa cells were treated with 5 mM caffeine LY294002 together, or alone, 200 μM LY294002 alone, 5mM caffeine and 200 μM mock-treated (DMSO). After 1 h, cells were treated +/- 2 mM HU and RNA was isolated after further 30 min for analysis as in Fig. 1. Graph shows histone mRNA levels in HUtreated cells, with 100 % defined as histone mRNA levels in non-HU-treated cells.

(B) Asynchronous HeLa cells were treated with 5 mM caffeine alone, 200 μM LY294002 alone, or 5 mM caffeine and 200 μM LY294002 together, or mock-treated (DMSO). After 1 h, cells were treated +/- 2 mM HU and RNA was isolated at indicated times after HU addition. RNA was also isolated from non-HU-treated cells incubated for 60 min and all samples were analysed by Northern blotting as in Figure 1. Graphs show changes in histone mRNA levels compared to the level at 0 min.

(C) M059J (DNA-PK(-)) and M059K (DNA-PK(+)) cells were treated with caffeine alone, 20 μM LY294002 alone, 5 mM caffeine and 200 μM LY294002 together, or mock-treated (DMSO). After 1 h, cells were treated +/- 2 mM HU and RNA was isolated after a further 1 h incubation and analysed as in Fig. 1. Graph shows histone mRNA levels in HU-treated cells compared to histone mRNA levels in non-HU treated cells.

Figure 7 - Inhibition of Chk1 function results in increased phosphorylation of Chk2. Chk1 and Chk2 are not functionally limiting for the control of replicational stress-induced histone mRNA decay.

(A) Asynchronous HeLa cells were transfected with siRNA targeting Chk1 , control siRNA 1 (targeting luciferase), control siRNA 2 (targeting a non-relevant gene), or were mock-transfected. After 32 h, cells were treated +/- 2 mM HU for 0, 20 and 60 min, or left untreated for 60 min and then lysed for RNA analysis (right hand panel). Histone H3 mRNA levels were analysed as in Fig. 1. Graph shows changes in H3 mRNA levels compared to the level at 0 min. Cell lysates were immunoblotted for Chk1 to confirm knockdown and actin (loading control).

(B) Asynchronous HeLa cells were incubated +/- Chk1 -selective inhibitor UCN-01 (300 nM). 1 h later, cells were incubated +/- 2 mM HU for indicated times and lysed for RNA analysis as before. Graph shows changes in histone mRNA levels compared to the level at 0 min.

(C) Left-hand panel: HeLa cells were synchronized in metaphase by treatment with nocodazole for 14 h and then released. 14 h post- release, when cells were in S-phase (determined by flow cytometry, not shown), cells were treated +/- 300 nM UCN-01. After 1 h, cells were treated +/- 2 mM HU and incubations were continued for a further 2 h. Cell lysates were immunoblotted with phospho-specific Chk2 antibody (upper panels) and total anti-Chk2 antibody (lower panels). Middle and Right-hand panel: asynchronous HeLa cells were treated for 1 h +/- 25 μM DBH and then incubated +/- 2 mM HU for indicated times and lysed for RNA analysis by Northern blotting as before. Graph shows the changes in histone mRNA levels compared to the level at 0 min.

Figure 8 - Model for the coordination of DNA replication and histone production in mammalian cells

Exposure of cells to replicational stress induces histone mRNA decay via a currently poorly understood pathway (upper right hand arrow). Replicational stress results in stabilisation of slowed or stalled replication forks via a pathway involving ATR and Chk1 (upper left- hand arrow). Replication restart from stalled replication forks occurs predominantly via a pathway involving homologous recombination. At some frequency, replication forks either encounter complex DNA damage or fail to be stabilised by the ATR/Chk1 pathway, which results in replication fork collapse, generating DNA double-strand breaks. These may be repaired either via ATM dependent homologous recombination-induced replication restart or via nonhomologous end-joining (NHEJ), mediated by DNA-PK. Interference with ATR signalling (such as occurs in the presence of caffeine) will result in higher levels of fork collapse, generating an increased level of substrate for DNA-PK-mediated NHEJ. The coordinated regulation of histone mRNA decay by both ATR/ATM and DNA-PK during replicational stress ensures that, irrespective of the extent to which each pathway operates in any given circumstance, supply of histones will remain closely coupled to the demand required for the assembly of newly synthesised chromosomes. Upon relief from replicational stress, histone mRNA stability is restored to normal levels, presumably by a mechanism linked to the restart of replication forks.

Figure 9 - Effect of Caffeine and Ly 294002 on DNA-PK and ATR Depleted HeLa Cells

(A) Graph showing how knock down of either DNA-PK or ATR affected the efficiency of replication stress-induced mRNA decay. (B) Graph showing the effect of adding either caffeine at low concentration (5 mM) or LY294002 to cells which lack a functional DNA-PK. As expected, in such cells, caffeine but not LY294002, was capable of stabilising histone mRNA levels over the time course of the assay. (C) Graph showing the effects of both compounds on cells lacking

ATR. In this case, LY294002 stabilised histone mRNA levels over the time course of the assay, while caffeine (5 mM) had little or no effect (Fig. 9C).

The Role of ATR and DNA-PK in Controlling Cellular Histone Levels ATM and ATR are checkpoint kinases that belong to the phosphatidylinositol 3-kinase-like kinase (PIKK) family and become activated in response to various forms of DNA damage. ATM activation is mainly triggered by the formation of DNA double-strand breaks, whereas ATR is activated by aberrant DNA structures induced by UV light or DNA synthesis inhibitors (Abraham, 2001 ). Another PIKK member, DNA- activated protein kinase (DNA-PK) is required primarily for DNA doublestrand break repair by non-homologous end joining (NHEJ) and telomere maintenance. It is also required for down-regulation of histone H2B gene transcription in response to ionizing radiation (Schild-Poulter et al., 2003), reflecting a potential role in a DNA damage response.

Chk1 and Chk2 are kinases activated by ATM/ATR, with partially overlapping functions. Targets common to both include Cdc25A and p53. Known Chk1 functions include the prevention of premature mitosis (Zachos et al., 2005), activation of the homologous recombination repair machinery (Sorensen et al., 2005), and in metazoans, activation of the origin firing and replisome integrity checkpoint (Feijoo et al., 2001 ; Zachos et al., 2005), operating downstream of ATR/ATM (Dimitrova and Gilbert, 2000).

We have investigated whether checkpoint components involved in monitoring DNA replication and effecting the origin firing checkpoint, also control the stability of histone mRNA. Using a combination of checkpoint- deficient cell lines, checkpoint kinase inhibitors and RNAi, we show for the first time a novel role for DNA-PK in an intra S-phase checkpoint pathway regulating histone mRNA decay. We find that DNA replication and histone mRNA stability are linked by two parallel and interdependent pathways, one of which is sensitive to caffeine and the other to LY294002. ATR appears to be the mediator of the caffeine-sensitive pathway and our results argue strongly that DNA-PK is the LY294002-sensitive mediator. We show that DNA-PK is activated during replicational stress, by virtue of its ability to phosphorylate Chk2, and that, together with a caffeine- sensitive protein, it plays a critical role in the regulation of histone mRNA stability. Chk1 is a molecular target of ATR-mediated checkpoint signalling previously implicated in abrogation of the S-phase replication checkpoint controlling replisome stability. Checkpoint regulation of histone mRNA decay occurring via either ATR or DNA-PK does not require either Chk1 or Chk2 activity. Our data are consistent with the notion that cellular machinery controlling histone mRNA stability is a direct target of ATR and DNA-PK or that an unidentified downstream effector of ATR and DNA-PK controls this process.

Results

Caffeine abrogates the replication origin firing checkpoint and inhibits histone mRNA decay induced by replicational stress in HeLa cells.

Small molecule inhibitors of the ATR/Chk1 pathway, such as caffeine, abrogate an S-phase checkpoint controlling replication origin firing in CHO cells (Dimitrova and Gilbert, 2000; Feijoo et al., 2001 ), which are particularly amenable to protocols involving multiple cell cycle arrests. To begin to investigate a potential linkage between known checkpoint signalling pathways with a role in origin firing and the regulation of histone mRNA decay in response to replicational stress, we investigated whether caffeine could abolish this checkpoint response in human cells amenable to the analysis of histone mRNA decay. Eukaryotic DNA replication takes place at discrete sites that may be visualized by pulse-labelling cells with halogenated derivatives of deoxyuridine (O'Keefe et al., 1992) and stained with labelled antibodies specific to each dU derivative (Dimitrova and Gilbert, 2000). The spatial pattern of replication sites reveals their temporal position within S-phase allowing the dynamics of groups of co-ordinately replicated chromosomal domains to be investigated.

To determine if abrogation of ATR function in HeLa cells arrested in S- phase would allow the initiation of replication at later replicating sites, asynchronously growing cells were briefly pulse-labelled with CIdU and then either left untreated (mock) or treated with the DNA polymerase inhibitor aphidicolin (APH) for 6 -16 h in the absence or presence of 5 mM caffeine. Thereafter, cells were washed free of inhibitors, briefly pulse- labelled with IdU, then fixed and stained with anti-CldU (green) or anti-ldU (red) antibodies (Fig. 1A).

Cells in early S-phase at the time of the CIdU pulse (identified by the presence of many sites of CIdU incorporation distributed throughout euchromatin, Fig. 1A, top left-hand panel) initiated replication at mid-S replicating sites (identified by peripheral and peri-nucleolar staining in the IdU pulse) at 6 h, and finally at late-replicating heterochromatin within 12 h (Fig. 1A, mock: top panels and bar graph). In APHtreated cells without added caffeine, IdU labelling largely co-localised with CIdU at all times investigated, indicating that, as expected, there was no progression through S-phase or initiation within later replicating domains under these conditions (Fig. 1A, APH, middle panels and bar graph). In contrast, in cells treated with caffeine during exposure to APH, IdU was incorporated into progressively later-replicating domains and was accompanied by a lack of DNA synthesis from previously initiated sites (Fig. 1A, APH + caffeine, lower panels). Quantitation of data in Figure 1A indicates that caffeine abrogates the origin firing replication checkpoint in HeLa cells, albeit with slightly reduced kinetics of progression through S-phase in comparison to untreated cells or compared with the same experiment in CHO cells (Dimitrova and Gilbert, 2000). Although the reason for this is unknown, it may be related to cell-type differences in rates of recovery from replication arrest (Jackson, 1995). To confirm that at the caffeine concentration used, ATR was indeed inhibited, HeLa cells were arrested in metaphase, released into fresh medium and after 14 h incubation (to ensure cells were in S-phase) treated with 5 mM caffeine in the presence or absence of the replication inhibitor hydroxyurea (HU). Cell lysates were immunoblotted for the presence of phosphorylated Chk1 , a direct target of ATR (Feijoo et al., 2001 ). Under these conditions, cellular Chk1 from HU treated cells migrated with reduced mobility characteristic of phosphorylation. As expected, inclusion of 5 mM caffeine, in addition to HU, eliminated the phosphorylated form of Chk1 , indicating that ATR was inhibited in cells exposed to this caffeine concentration.

As well as inducing replicational stress, treatment with either HU or APH results in histone mRNA decay (Baumbach et al., 1987; Levine et al.,

1987). To test if DNA replication checkpoint components also regulate the stability of histone mRNA, we investigated the effect of caffeine on replicational stress-induced decay of histone mRNA. Asynchronous HeLa cells were pre-treated with a range of caffeine concentrations, prior to inducing replicational stress by HU addition. Subsequently, total RNA was isolated and analysed by Northern blotting using probes that detect either histone H2B or H3 mRNA. As expected, HU addition alone initiated efficient destabilisation of histone H2B and H3 mRNA which after 30 min were reduced to ~ 40 % of control levels in untreated cells (Fig. 1 B, mock, upper panels and bar graph). The presence of caffeine affected histone mRNA decay induced by replicational stress. At a concentration where it inhibits ATR (Hall-Jackson et al., 1999) (and the related PIKK ATM (Blasina et al., 1999)), and abrogates the origin firing replication checkpoint (Fig. 1A and (Dimitrova and Gilbert, 2000)), caffeine partially inhibited histone mRNA decay induced by replicational stress, with -70 % of histone mRNA remaining after 30 min exposure to HU. Inhibition of replicational stress-induced histone mRNA decay by caffeine was dose-dependent and, in the presence of 20 mM caffeine, 85-95 % of histone mRNA remained after 30 min of HU treatment. These results indicate that destabilisation of histone mRNA induced by replicational stress is sensitive to the PIKK inhibitor caffeine and suggest that a PIKK might be involved in the control of histone mRNA stability.

Inhibition of ATR signalling does not interfere with replicational stress induced histone mRNA decay.

ATR has been implicated in a variety of pathways that respond to replication stress (reviewed in (Abraham, 2001)). To investigate a specific role for this caffeine sensitive kinase in regulating histone mRNA decay, we tested cells (U2OS/kd-ATR) that conditionally over-express a kinase- dead (kd) form of ATR (D2475A) which acts in a dominant-negative manner to block normal ATR function (Nghiem et al., 2001 ). Control and doxycycline-treated cells overexpressing either kd-ATR or wild-type (wt) ATR were treated with HU for various times, total RNA was isolated, and histone H2A, H2B and H3 mRNA levels were determined by Northern blotting (Fig. 2A).

In the absence of over-expressed protein, the pattern of histone mRNA decay was the same for all histone variants tested and, as expected, HU addition resulted in rapid (< 60 min) destabilisation of histone mRNA (Fig. 2A, upper panels and line graphs). However, over-expression of kd-ATR (or wt-ATR) did not significantly affect either the rate or extent of decay of any of the histone mRNA variants tested. Over-expression of kd-ATR in this cell line has been shown previously to interfere with ATR function. To confirm that kd-ATR over-expression did indeed interfere with ATR signalling, we determined the activity and phosphorylation state of Chk1 in response to replicational stress. Chk1 phosphorylation and activation was completely suppressed in the presence of over-expressed kd-ATR but not by wt-ATR (Fig. 2B).

In an alternative approach we used siRNA to deplete ATR in HeLa cells which reduced ATR levels to -12 % of that observed in control cells (Fig. 2C, left-hand panels). The rate and extent of histone H3 mRNA decay in ATR-depleted cells exposed to replicational stress was almost identical to that in control or mock-treated cells (Fig. 2C, right-hand panels and graph). Taken together, the results in Figure 2 indicate that ATR is not functionally limiting for the rate or extent of histone mRNA decay induced by replicational stress.

Replicational stress-induced histone mRNA decay occurs efficiently in cells lacking both ATM and ATR.

ATM is also a caffeine-sensitive component of a DNA damage checkpoint (Blasina et al., 1999). To establish if ATM was necessary for replicational stress-induced histone mRNA decay, we compared histone mRNA decay induced by replicational stress in ATM-null cells (AT221 JE-T) transfected either with empty vector or vector encoding wt-ATM (Fig. 3A). Histone H3 mRNA decay in these cells occurred with similar kinetics to decay in HeLa and U2OS cells. Re-introduction of ATM had no effect on either the rate or extent of replicational stress-induced histone H3 mRNA decay, indicating that ATM, like ATR, is not functionally limiting for the rate or extent of histone mRNA decay induced by replicational stress. To test if ATM and ATR are functionally redundant, we depleted ATR in ATM-null cells using siRNA, as in Figure 2. Western blotting analysis confirmed that, as above, the level of ATR protein was efficiently depleted using this approach (data not shown). The rate and extent of replicational stress-induced histone mRNA decay was indistinguishable in ATRdepleted ATM-null cells from that in control siRNA-treated cells (Fig. 3B), indicating that ATM and ATR together are not sufficient to account for the observed decay in histone mRNA.

The general PIKK family Inhibitor wortmannln affects the efficiency of replication stress-induced histone mRNA decay

Wortmannin brings about the inhibition of PIKK family members by covalent inactivation. To investigate if PIKKs play any role in histone mRNA decay, we tested wortmannin over a range of concentrations and determined its effect on histone H2B and H3 mRNA decay. Whereas pre- treatment with 3 IM or 30 IM wortmannin had little or no effect, 100 IM wortmannin significantly inhibited histone mRNA decay occurring during the first 30 min following HU addition (Fig. 4A). To determine the kinetics of replicational stress-induced histone mRNA decay +/- wortmannin, histone mRNA H3 and H2B mRNA levels were measured 25 and 50 min after HU addition (Fig. 4B). Normal histone mRNA decay was observed in mock-treated cells. Interestingly, histone mRNA decay was inhibited by 100 IM wortmannin for up to 25 min after HU addition. Subsequently decay resumed at a rate approximating that observed in mock-treated cells exposed to HU. These data suggest PIKK family members do play a role in controlling histone mRNA decay and that they modulate the kinetics of this process by delaying the onset of replication stress-induced histone mRNA decay.

DNA-PK is a component of the checkpoint mechanism sensing replicational stress and controlling histone mRNA stability

Exposing cells to replicational stress results in activation of Chk1 and Chk2 (Feijoo et al., 2001 ). Activation of Chk1 is believed to be dependent on its phosphorylation by ATR (Fig. 2B and (Abraham, 2001 )), while the activation of Chk2 occurs independently of both ATR (data not shown) and ATM (Feijoo et al., 2001). To investigate more fully the effects of PIKK family inhibitors, we tested the effects of caffeine (Fig. 5A) on replicational stress-induced activation of Chk2 in HeLa cells. Interestingly, while caffeine abolishes replicational stress-induced activation of Chk1 ((Feijoo et al., 2001 ), data not shown), the presence of caffeine in cells experiencing replicational stress resulted in a progressive increase in Chk2 phosphorylation and activation (Fig. 5), which was independent of ATM (data not shown). To investigate whether another PIKK could be responsible for the activation of Chk2 in response to replicational stress, we examined the Chk2 response to caffeine in the DNA-PK deficient cell line M059J, alongside its DNA-PK wild-type counterpart, M059K under conditions of replicational stress. Exposure of M059K cells to replicational stress resulted in the previously observed activation of Chk2 in response to caffeine (Fig. 5). In contrast, in DNA-PK deficient cells, M059J, Chk2 was not activated under identical conditions. Together, these data indicate that Chk2 activation in response to replicational stress is dependent on DNA-PK and suggest that activation of this pathway may be a consequence of a failure of ATR/ATM signalling. Concentrations of caffeine which inhibit ATR and ATM signalling in vivo are ineffective against DNA-PK (Sarkaria et al., 1999), while LY294002 has been reported to be a selective inhibitor of DNA-PK (Stiff et al., 2004). To investigate a role for DNA-PK in the regulation of replicational stress- induced histone mRNA decay, we 200determined the effect of LY294002 on this process. Treatment of cells with μM LY294002, a concentration which blocks DNA-PK function (Stiff et al., 2004), inhibited histone H2B and H3 mRNA decay during the first 30 min following HU addition. As before, addition of 5 mM caffeine also partially restored histone mRNA stability. Interestingly, the effects of caffeine and LY294002 combined were additive (Fig. 6A). Cells exposed to 5 mM caffeine and 200 μM LY294002 together retained close to maximum levels of histone mRNA following a 30 min treatment with HU (Fig. 6A). Kinetic analysis showed that, as for wortmannin, either caffeine or LY294002 alone significantly delayed the onset of histone H2B and H3 mRNA decay induced by replicational stress (Fig. 6B, upper panels and line graphs). Treatment with caffeine and LY294002 together completely abolished the rapid initiation of histone mRNA decay normally observed on imposition of replicational stress. Decay resumed after -30 min in the presence of caffeine alone, LY294002 alone, or caffeine and LY294002 combined (Fig. 6B). However, even after extended periods of replicational stress, the effects of caffeine and LY294002 combined remained additive with mRNA levels remaining at -70 % of control values after 1 h, indicating that these compounds are affecting at least two distinct pathways regulating histone mRNA decay. Given the target specificity of caffeine and LY294002 on PIKK family members, these data strongly implicate DNA-PK in the control of replicational stressinduced histone mRNA decay (Fig. 6B).

To establish if histone mRNA decay in response to replicational stress is indeed regulated by a DNA-PK dependent pathway, we tested whether the additive effect of caffeine and LY294002 on inhibiting replication stress- induced histone mRNA decay was dependent on the presence of DNA- PK. We reasoned that if the effect of LY294002 is to potentiate the ability of caffeine to both delay the onset and inhibit the progressive destabilisation of histone mRNA via the inhibition of DNAPK, it follows that, in cells lacking a functional DNA-PK signalling pathway, LY294002 would fail to inhibit histone mRNA decay in caffeine-treated cells exposed to replicational stress. Therefore, M059J and M059K cells were pre- treated with 5 mM caffeine alone, 200 μM LY294002 alone, 5 mM caffeine and 200 μM LY294002 together, or mock-treated. All cells were treated +/- 2 mM HU, incubated for 1 h (to maximise to relative contribution of DNA- PK compared to a caffeine-sensitive checkpoint component) and RNA was isolated and analysed by Northern blotting (Fig. 6C). As before, HU induced significant mRNA decay, with 20 % of histone H2B or H3 mRNA remaining after 1 h of HU treatment. At this time, the effect of caffeine on the extent of mRNA decay was minimal, as would be expected from the data in Fig. 6B. Consistent with a role for DNA-PK in this pathway, LY294002 significantly increased the levels of histone mRNA remaining in M059K cells, compared with M059J cells, after 1 h exposure to HU. Importantly, we found that, in M059K cells, but not M059J cells, LY294002 dramatically potentiated the effect of caffeine in inhibiting histone mRNA decay in response to replicational stress. Combined treatment of M059K cells with LY294002 and caffeine severely reduced the extent of mRNA decay, with -70 % of mRNA remaining after 1 h exposure to HU. In comparison, M059J cells showed significantly less sensitivity to combined LY294002 and caffeine treatment (Fig. 6C). Taken together, the data in Figure 6 indicate that DNA-PK plays an important role in regulating histone mRNA stability. Our data support roles in the regulation of histone mRNA stability for both a caffeine-sensitive pathway, presumably mediated by ATR (Fig. 1 and (Kaygun and Marzluff, 2005)), and an LY294002-dependent pathway involving DNA-PK (Fig. 6). Chk1 is a well-characterised mediator of ATR signalling during replicational stress and we show here that Chk2 is a target for DNA-PK dependent signalling (Fig. 5). Interfering with downstream effectors of ATM/ATR signalling abrogates the replication checkpoint controlling origin firing and replisome stability in both CHO cells (Feijoo et al., 2001) and HeLa cells (J. B. and CS. , unpubl.). We therefore wished to address if abrogation of Chk1 and/or Chk2 function interferes with histone mRNA decay during replicational stress.

Depletion of Chk1 to very low levels using siRNA ((Rodriguez and Meuth, 2006), Fig. 7A, left-hand panels) had no effect on replicational stress- induced histone mRNA decay (Fig. 7A, right hand panels and bar graphs). In an alternative approach we used the Chk1 inhibitor, UCN-01 , which interferes with the replication checkpoint ((Feijoo et al., 2001); J. B. and CS. , unpublished). Again, kinetics of replicational stress induced histone mRNA decay were unaffected by Chk1 inhibition (Fig. 7B). These data show that Chk1 is not necessary for histone mRNA decay, but do not exclude the possibility that it is involved in mediating a component of the caffeine-sensitive element of histone mRNA stability control. As shown here (Fig. 2, 5 and 6), it is conceivable that when this pathway is compromised, up-regulation of a DNA-PK dependent pathway acts to control histone mRNA decay, potentially, though not necessarily, through Chk2. Indeed, Chk1 inhibition with UCN-01 , like caffeine (Fig. 5), results in increased phosphorylation of Chk2 in response to replicational stress (Fig. 7C, left-hand panel). To address this issue directly we utilised the small molecule inhibitor debromohymenialdisine (DBH), shown previously to inhibit both Chk1 and Chk2 in vivo (Curman et al., 2001 ). The kinetics of HU-induced histone mRNA decay were unaffected by the presence of DBH at a concentration known to inhibit both Chk1 and Chk2 function (Fig. 7C), indicating that neither Chk1 nor Chk2 activity is functionally limiting for this process.

Discussion

The coordination of chromatin assembly with DNA replication, which is essential for genomic stability, requires close-coupling between the firing of replication origins, which determines the overall rate of DNA synthesis in S-phase, and the mechanics of histone production and deposition. Such coordination is likely to operate at multiple levels. However, a key mechanism in regulating the delivery of histone protein to newly synthesised DNA occurs via control of histone mRNA transcription and degradation (Marzluff and Duronio, 2002; Schϋmperli, 1986).

Exposure of cells to replicational stress induces histone mRNA decay via a currently poorly understood pathway. The occurrence of replicational stress arising from the presence of DNA damage results in a variety of checkpoint responses, which include suppression of new DNA synthesis via inhibition of replication origin firing, stabilisation of slowed or arrested replication forks as well as activation of DNA repair pathways to enable subsequent replication restart at stalled replication forks (Abraham, 2001 ; Lundin et al., 2002). Homologous recombination repair (HRR) and non- homologous end joining (NHEJ) pathways both operate on lesions associated with arrested or collapsed replication forks to effect replication restart (Arnaudeau et al., 2001 ; Lundin et al., 2002); see Fig. 8). In eukaryotes, HRR requires the activation of ATR/ATM and downstream effector Chk1 signalling component (Sorensen et al., 2005), while NHEJ requires the activity of the related PIKK, DNA-PK. NHEJ is required for replication restart at forks where a DNA double-strand break has been generated, but appears to be inessential for repair associated with slowed or arrested replication forks (Lundin et al., 2002). Failure of, or interference with, ATR signaling (which occurs in the presence of caffeine) will result in higher levels of fork collapse (Dimitrova and Gilbert, 2000), resulting in activation of DNA-PK and consequent NHEJ.

Our data support a model in which coordinate regulation of replication- dependent histone mRNA levels with DNA replication requires DNA-PK in addition to a caffeine-sensitive pathway (presumably mediated by ATR or ATM) (Fig. 8). The relative contribution from each signalling arm would be based on the nature of the DNA lesion generating replicational stress and thus the relevant replication restart pathway.

Consistent with this, over-expression of kd-ATR or significant ablation of ATR function by siRNA had little or no effect on the efficiency of histone mRNA decay (Fig. 2). This was presumably because, in such circumstances, the elevated levels of resultant fork collapse result in increased NHEJ activity and thus activation of a DNA-PK-mediated mechanism. In contrast caffeine, at concentrations believed to selectively inhibit ATR/ATM, which blocked Chk1 phosphorylation and abrogated the origin firing checkpoint (Fig. 1A), did partially affect histone mRNA decay in response to replicational stress. Maximal inhibition, however, required significantly higher caffeine concentrations, well in excess of those required to interfere with predicted ATR/ATM-mediated checkpoint responses (Hall-Jackson et al., 1999; Sarkaria et al., 1999). This observation is consistent with the fact that all PIKK family members share a related kinase domain and those tested exhibit some sensitivity to inhibition by this xanthine alkaloid (Sarkaria et al., 1999). Importantly, we found that low doses of caffeine, or UCN-01 , which affect replisome stability via inhibition of ATR/Chk1 signalling resulting in replication fork collapse (Dimitrova and Gilbert, 2000; Feijoo et al., 2001 ), gave rise to elevated Chk2 phosphorylation that was dependent on the presence of active DNA-PK. Such data support the hypothesis that interference with ATR/ATM-dependent pathways results in elevated signalling through DNA-PK.

Expression of a dominant-negative form of ATR at levels sufficient to efficiently block the phosphorylation and activation of a known substrate (Chk1) had no effect on replicational stress-induced mRNA decay. While this might be expected, it does contrast with results obtained with these cells elsewhere (Kaygun and Marzluff, 2005), in which over-expression of ATR did delay histone mRNA decay. Although reasons for this discrepancy are unknown, one possibility concerns differences in the extent of kd-ATR over-expression. Elledge and colleagues have suggested, given the relatedness between PIKKs, that over-expression of kinase-dead alleles of specific PIKKs might result in interference with other PIKK-dependent pathways (Zhou and Elledge, 2000). Here, over- expression of kd-ATR was optimized to block phosphorylation of Chk1. It is conceivable that robust over-expression (Kaygun and Marzluff, 2005) may result in interference not only with ATR-mediated events but functions associated with other PIKKs also.

These data together with other published observations (Kaygun and Marzluff, 2005) strongly implicate PIKKs in an S-phase checkpoint controlling the efficiency of histone mRNA decay in response to replicational stress. Screening experiments using a broad range of small molecule inhibitors indicate that complete uncoupling of histone mRNA decay from replicational stress may be achieved, and preliminary data indicate that active compounds do not directly target known checkpoint components (B. M. and CS. , unpublished observations). As suggested previously (Brown and Baltimore, 2003), cells lacking known checkpoint signalling function nonetheless may develop residual checkpoint responses for critical cellular pathways that may well involve further key signalling molecules in addition to ATR and DNA-PK. The linkage between replication and histone decay may be such a critical pathway.

UPF1 , an RNA helicase involved in degradation of nonsense messenger RNAs and in mRNA stability control (Kim et al., 2005), has recently been implicated in the regulation of histone mRNA decay (Kaygun and Marzluff, 2005). UPF1 contains multiple S/TQ motifs, substrate recognition motifs common to both ATR/ATM and DNA-PK (Hall-Jackson et al., 1999) as well as hSMG1 , a PIKK directly implicated in nonsense-mediated decay (NMD). Conceivably, direct phosphorylation of UPF1 by other PIKK family members such as ATR and DNA-PK may play a role in coordinating DNA replication and histone mRNA levels. Consistent with this notion, and in contrast to multiple other checkpoint signalling pathways, blocking Chk1 and Chk2 signalling had no effect on histone mRNA decay. Unlike their upstream activators (DNA-PK, ATR, ATM), whose activation is dependent on their physical association with the originating DNA lesion, Chk1 and Chk2 are believed to relay checkpoint signals to cellular targets removed from the site of damage. UPF1 is a predominantly cytoplasmic protein, associated with poly-ribosomes when hyperphosphorylated by hSMG1 (Yamashita et al., 2001 ), and histone mRNA decay is believed to be a cytoplasmic event. Hyper-phosphorylated UPF1 has been found associated with chromatin, and this association was seen to increase in response to DNA damage (Azzalin and Lingner, 2006). Whatever the molecular details underpinning the coordination of replication with histone mRNA levels, it is likely that sensing components, perhaps UPF1 , or another unknown factor, are required to transduce signals emanating from DNA lesions in the nucleus to decay machinery residing in the cytoplasm. Future work will focus on establishing how this may be achieved.

Materials and methods

Cell lines and culture conditions - HeLa (Feijoo et al., 2001 ), AT fibroblasts (Ziv et al., 1997), and U2OS cells (Nghiem et al., 2001 ) were grown as described. Induction of ATR expression in U2OS cells was by incubation with 1.5 ιg/ml doxycycline for 3 days. DNA-PK deficient (M059J) and proficient cells (M059K) were grown in DMEM or in F- 12/DMEM medium supplemented with 10 % (v/v) FCS, 100 units/ml penicillin, 100 ιg/ml streptomycin. Unless stated otherwise, experiments were performed with asynchronous cells. Synchronized cells were arrested in metaphase by adding 40 ng/ml nocodazole (Calbiochem) for 12 h-14 h. Mitotic cells were harvested by shake-off, collected by centrifugation, washed three times with PBS, and released into fresh medium.

Drug treatment - Caffeine (Sigma) was used, unless stated otherwise, at a final concentration of 5 mM, hydroxyurea (HU) (Sigma) at 2 mM, APH

(Sigma) at 50 μg/ml, debromohymenialdisine (DBH) (gift from D. Home,

University of Oregon) at 25 μM and UCN-01 , (NCI) at 300 nM.

Wortmannin and LY294002 (Calbiochem) were used at indicated concentrations. Hydroxyurea and APH were used in different experiments to induce replicational stress. No differences were observed in the timing or magnitude of checkpoint responses in response to either agent.

RNA analysis - Total RNA was prepared using Trizol (Invitrogen) and analysed by Northern blotting as described (Zhao et al., 2004). Human GAPDH cDNA for probing Northern blots spans nucleotides 2041-3239 of the open reading and was PCR amplified from genomic DNA. RNAs were visualized by autoradiography or using a Fujifilm FLA3000 Phosphorimager with Aida 2.0 software (Raytest GmbH).

Cell lysates, immunoblotting and kinase assays - Cell lysates for Chk1 activity and immunoblotting were as described (Feijoo et al., 2001 ). Chk1 and Chk2 antibodies were generated as described (Feijoo et al., 2001 ) and used at 1 μg/ml; rabbit polyclonal anti-phospho-Chk1 (Ser345) and anti-phospho-Chk2 (Thr68) (Cell Signalling), rabbit anti-ATR (Oncogene) used at 1 :2000. Monoclonal anti-actin (Sigma) and anti-nucleolin (Santa Cruz) were used at 1 :1000. HRP-conjugated antirabbit (Sigma) and anti- mouse antibodies (Sigma) were used at 1 :5000. Chk1 and Chk2 kinase activity were assayed as described(Feijoo et al., 2001 ).

RNA interference - RNA interference was carried out using ATR siRNA (target sequence AACCTCCGTGATGTTGCTTGA - Seq ID No 1), Chk1 siRNA (target sequence AAGAAGCAGTCGCAGTGAAGA - Seq ID No 2), control siRNA 1 targeting luciferase and control siRNA 2 targeting an unrelated gene, TAPP1 (target sequence GGTCAAGCCAGGGAACTTC - Seq ID No 3) from Dharmacon Research Inc. Cells were transfected with siRNA using Oligofectamine (Invitrogen), as described by the manufacturer. 48 h post-transfection (unless otherwise stated) cells were treated with HU for indicated times, and RNA or protein samples were prepared.

Assay to monitor S-phase progression - Asynchronous HeLa cells were pulsed with chlorodeoxyuridine (CIdU) (30 μM) for 20 min, washed with PBS and subsequently incubated for increasing lengths of time (typically 6, 12 or 16 h) either in the absence of drugs (mock), in the presence of APH alone (50 μg/ml), or in the presence of both APH and caffeine (5 mM). Cells were then washed free of drugs with PBS and pulsed with iodo-deoxyuridine (IdU) (30 μM) for 20 min. Differential staining of DNA sites substituted with halogenated derivatives of dU was performed essentially as described (Feijoo et al., 2001 ).

An Assay Suitable for the Assessment of the Ability of Test Substances to Abrogate the ATR and/or DNA-PK Pathways

Below is a discussion of an assay according to the present invention.

1. Culture of U2OS cell line (GK41 ; (Nghiem et al., 2001 )) in the absence and presence of doxycyclin. In the presence of the latter compound, these cells are induced to express a catalytically inactive form of ATR which acts in a dominant negative fashion to interfere with the function of the endogenous wild-type ATR protein, rendering the cells nonfunctional for ATR while retaining normal DNA-PK function. In the absence of doxycyclin, the cell line retains both ATR and DNA-PK function and thus act a relevant negative control for the specificity of any test agent on ATR/DNA-PK regulated pathways.

2. Culture of MO59J cells which lack functional DNA-PK, but retain a wild- type ATR. MO59K cells are the control parental cell line which retain both wild-type ATR and DNA-PK, and may be used as a relevant negative control for the specificity of any test agent having an effect on this cell type (Allalunis-Turner et al., 1995).

3. Cells are exposed for 24h to replication inhibitor (such as 2 mM hydroxurea) in the presence and absence of test agent.

4. Treated cells are harvested and total RNA extracted using standard methods (Zhao et al., 2004). 5. Total RNA is then subjected to electrophoresis and levels of histone mRNA determined using a radio-labelled probe which specifically and quantitatively detects a replication dependent form of histone mRNA (Zhao et al., 2004). 6. Levels of histone mRNA may be quantified using detection and quantification system (Zhao et al., 2004).

7. Under normal circumstances, the imposition of replicational stress in wild-type cells (i.e. U2OS cells not exposed to doxycyclin, or MO59 K cells) which retain functional ATR AND DNA-PK, results in rapid and specific decrease in the cellular levels of histone mRNA (baseline value).

8. In cells lacking functional ATR (such as U2OS cells in the presence of doxycyclin), DNA-PK is solely responsible for the rapid down-regulation of histone mRNA levels following the imposition of replicational stress and the ability of any test agent to interfere with DNA-PK function may be assessed by the effect of the test agent on HU-induced mRNA decay in such a cell line. Conversely, cells lacking DNA-PK (MO59J) rely on ATR to induce histone mRNA degradation in response to replicational stress and the ability of any test agent to interfere with ATR function may be assessed by the effect of the test agent on HU- induced mRNA decay in such a cell line.

9. It follows that in the above assay set, U2OS cells lacking functional ATR should only show replicational stress-induced decrease in histone mRNA levels when DNA -PK is functional and thus test agents which block this protein function when applied to this system will inhibit histone mRNA decay. Similarly MO59J cells which lack DNA-PK should only show replicational stress-induced decrease in histone mRNA levels when ATR is functional and thus test agents which block this protein function will inhibit histone mRNA decay in this system. 10. Test agents which have no selectivity between ATR and DNA-PK in assays in vivo (and are consequently of less interest in the search for pathway-selective inhibitors) will block histone mRNA decay in both experimental systems. 11.Test agents which interfere non-specifically (either positively or negatively) with aspects of histone mRNA metabolism would be expected to do so in the indicated control cell lines in addition to the specific lines outlined above.

An Alternative Assay Suitable for the Assessment of the Ability of Test Substances to Abrogate the ATR and/or DNA-PK Pathways

To provide an improved assay for use as a potential screen for compounds which are truly selective in vivo for one or other checkpoint pathways, it would be beneficial to have the assay in a single cell line, which normally contains functional ATR and DNA-Pk pathways, but which can be experimentally manipulated to eliminate one or other. The availability of so-called small interfering RNA (siRNA) technology for the specific inactivation of individual gene pathways makes this approach feasible and rapid.

We therefore set out to use siRNA technology to knock down the expression of either ATR or DNA-PK in HeLa cells and subsequently investigate the effects of known inhibitors of these pathways to interfere with replication-stress induced histone mRNA decay.

DNA-PK, but not ATR, is selectively inhibited by the compound LY294002. Caffeine (at low millimolar concentrations) has been shown to inhibit ATR (and ATM) but not DNA-PK. However, the selectivity of caffeine is relatively poor and DNA-PK can be inhibited by caffeine at higher concentrations. It follows that in cells lacking functional DNA-PK, the induction of replication stress-induced mRNA decay will be largely dependent on ATR, and would be expected to be sensitive to caffeine but not LY294002.

Conversely, in cells lacking functional ATR, the induction of replication stress induced mRNA decay should be dependent on DNA-PK, and in this circumstance, histone mRNA decay would be expected to be sensitive to a DNAPK selective inhibitor such as LY294002, but not low concentrations of caffeine.

However as DNA-PK is known to be sensitive to inhibition by high concentrations of caffeine, then such concentrations might be expected to inhibit histone mRNA decay in cells lacking ATR (and indeed in cells which have a functional ATR).

Results and Discussion

HeLa cells were treated with siRNA which target either ATR or DNA-PK or an irrelevant control siRNA and the knock-down of each protein was verified by SDS-PAGE and immunoblotting (not shown). Cells were then treated with the replication inhibitor camptothecin for indicated lengths of time and histone mRNA levels were measured by Northern blotting. As expected, knock down of either DNA-PK or ATR affected the efficiency of replication stress-induced mRNA decay (Fig 9A).

We then determined the effects of adding either caffeine at low concentration (5 mM) or LY294002 to cells which lack a functional DNA- PK. As expected, in such cells, caffeine but not LY294002, was capable of stabilising histone mRNA levels over the time course of the assay (Fig. 9B). We next investigated the effects of both compounds on cells lacking ATR. In this case, LY294002 stabilised histone mRNA levels over the time course of the assay, while caffeine (5 mM) had little or no effect (Fig. 9C). Importantly when such cells were treated with higher (20 mM) concentrations of caffeine, histone mRNA largely remained stable for the length of the assay, consistent with the notion that, at these higher concentrations, caffeine inhibits both ATR and DNA-PK dependent signalling pathways (Fig. 9C).

Our results show that high concentrations of caffeine inhibit histone mRNA decay even in cells which lack ATR, consistent with its known ability to inhibit DNA-PK at these concentrations. In contrast LY294002 is effective in cells lacking ATR, but has no effect in cells where ATR is the major pathway regulating histone mRNA decay suggesting that LY294002, at least at the concentrations used, and unlike caffeine at high concentration, is a selective inhibitor.

These data support the notion that the analysis of histone mRNA decay in cells which have been depleted for either ATR or DNA-PK permits the analysis of specificity in vivo of potential new ("druggable") inhibitors of either pathway.

References

Abraham, RT. (2001 ) Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev, 15, 2177-2196.

Allalunis-Turner, M.J., L. G. Lintott, G. M. Barron, R.S. Day, 3rd, and S. P. Lees-Miller. 1995. Lack of correlation between DNA-dependent protein kinase activity and tumor cell radiosensitivity. Cancer Res. 55:5200-2. Arnaudeau, C, Lundin, C. and Helleday, T. (2001 ) DNA double-strand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells. J MoI Biol, 307, 1235-1245. Azzalin, CM. and Lingner, J. (2006) The Human RNA Surveillance Factor

UPF1 Is Required for S Phase Progression and Genome Stability.

Curr Biol, 16, 433-439. Baumbach, L. L., Stein, G.S. and Stein, J. L. (1987) Regulation of human histone gene expression: transcriptional and posttranscriptional control in the coupling of histone messenger RNA stability with DNA replication. Biochemistry, 26, 6178-6187. Blasina, A., Price, B. D., Turenne, G.A. and McGowan, CH. (1999)

Caffeine inhibits the checkpoint kinase ATM. Curr Biol, 9, 1135-

1138. Brown, E.J. and Baltimore, D. (2003) Essential and dispensable roles of

ATR in cell cycle arrest and genome maintenance. Genes Dev, 17,

615-628. Curman, D., Cinel, B., Williams, D. E., Rundle, N., Block, W.D., Goodarzi,

A.A., Hutchins, J. R., Clarke, P. R., Zhou, B. B., Lees-Miller, S. P., Andersen, R.J. and Roberge, M. (2001 ) Inhibition of the G2 DNA damage checkpoint and of protein kinases Chk1 and Chk2 by the marine sponge alkaloid debromohymenialdisine. J Biol Chem, 276,

17914-17919.

DeLJsIe, A.J., Graves, R.A., Marzluff, W.F. and Johnson, L. F. (1983) Regulation of histone mRNA production and stability in serum- stimulated mouse 3T6 fibroblasts. MoI Cell Biol, 3, 1920-1929. Dimitrova, D. S. and Gilbert, D. M. (2000) Temporally coordinated assembly and disassembly of replication factories in the absence of DNA synthesis. Nat Cell Biol, 2, 686-694. Emili, A., Schieltz, D. M., Yates, J. R., Ill and Hartwell, L. H. (2001 ) Dynamic interaction of DNA damage checkpoint protein Rad53 with chromatin assembly factor Asfl . MoI Cell, 7, 13-20.

Feijoo, C, Hall-Jackson, C, Wu, R., Jenkins, D., Leitch, J., Gilbert, D. M. and Smythe, C. (2001) Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing. J Cell Biol, 154, 913-923.

Graves, R.A., Pandey, N. B., Chodchoy, N. and Marzluff, W.F. (1987)

Translation is required for regulation of histone mRNA degradation. Ce//, 48, 615-626.

Gunjan, A. and Verreault, A. (2003) A Rad53 kinase-dependent surveillance mechanism that regulates histone protein levels in S. cerevisiae. Cell, 115, 537-549.

Hall-Jackson, C.A., Cross, D.A., Morrice, N. and Smythe, C. (1999) ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK. Oncogene, 18, 6707-6713.

Han, M., Chang, M., Kim, U.J. and Grunstein, M. (1987) Histone H2B repression causes cell-cycle-specific arrest in yeast: effects on chromosomal segregation, replication, and transcription. Cell, 48, 589-597.

Heintz, N., Sive, H. L. and Roeder, R.G. (1983) Regulation of human histone gene expression: kinetics of accumulation and changes in the rate of synthesis and in the half-lives of individual histone mRNAs during the HeLa cell cycle. MoI Cell Biol, 3, 539-550. Jackson, D.A. (1995) S-phase progression in synchronized human cells. Exp Cell Res, 220, 62-70.

Kaygun, H. and Marzluff, W.F. (2005) Regulated degradation of replication-dependent histone mRNAs requires both ATR and Upfl . Nat Struct MoI Biol, 12, 794-800. Kim, Y.K., Furic, L., Desgroseillers, L. and Maquat, L. E. (2005)

Mammalian Staufeni recruits Upf1 to specific mRNA 3'UTRs so as to elicit mRNA decay. Cell, 120, 195-208.

Levine, B.J., Chodchoy, N., Marzluff, W.F. and Skoultchi, A.I. (1987) Coupling of replication type histone mRNA levels to DNA synthesis requires stem-loop sequences at the 3' end of the mRNA. Proc Natl Acad Sci USA, 84, 6189-6193.

Lundin, C, Erixon, K., Arnaudeau, C, Schultz, N., Jenssen, D., Meuth, M. and Helleday, T. (2002) Different roles for nonhomologous end joining and homologous recombination following replication arrest in mammalian cells. MoI Cell Biol, 22, 5869-5878.

Martin, F., Schaller, A., Eglite, S., Schϋmperli, D. and Mϋller, B. (1997) The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein. EMBO J, 16, 769-778.

Marzluff, W.F. and Duronio, R.J. (2002) Histone mRNA expression: multiple levels of cell cycle regulation and important developmental consequences. Curr Opin Cell Biol, 14, 692-699.

Meeks-Wagner, D. and Hartwell, L. H. (1986) Normal stoichiometry of histone dimer sets is necessary for high fidelity of mitotic chromosome transmission. Cell, 44, 43-52.

Nghiem, P., Park, P. K., Kim, Y., Vaziri, C. and Schreiber, S. L. (2001 ) ATR inhibition selectively sensitizes G1 checkpoint-deficient cells to lethal premature chromatin condensation. Proc Natl Acad Sci USA, 98, 9092-9097.

O'Keefe, R.T., Henderson, S.C. and Spector, D. L. (1992) Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha- satellite DNA sequences. J Cell Biol, 116, 1095-1110. Painter, R. B. and Young, B. R. (1980) Radiosensitivity in ataxia- telangiectasia: a new explanation. Proc Natl Acad Sci USA, 77, 7315-7317.

Rao, P.N. and Johnson, RT. (1970) Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis. Nature, 225, 159-164.

Rodriguez, R. and Meuth, M. (2006) Chk1 and p21 cooperate to prevent apoptosis during DNA replication fork stress. MoI Biol Cell, 17, 402- 412.

Santocanale, C. and Diffley, J. F. (1998) A Med- and Rad53-dependent checkpoint controls late-firing origins of DNA replication. Nature,

395, 615-618.

Sarkaria, J. N., Busby, E.C., Tibbetts, R.S., Roos, P., Taya, Y., Karnitz, L. M. and Abraham, RT. (1999) Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res, 59, 4375-4382.

Schild-Poulter, C, Shih, A., Yarymowich, N.C. and Hache, R.J. G. (2003) Down-Regulation of Histone H2B by DNA-Dependent Protein Kinase in Response to DNA Damage through Modulation of Octamer Transcription Factor 1. Cancer Research, 63, 7197-7205. Schumperli, D. (1986) Cell-cycle regulation of histone gene expression. Cell, 45, 471-472.

Shirahige, K., Hori, Y., Shiraishi, K., Yamashita, M., Takahashi, K., Obuse, C, Tsurimoto, T. and Yoshikawa, H. (1998) Regulation of DNA- replication origins during cell-cycle progression. Nature, 395, 618- 621.

Sorensen, CS. , Hansen, LT., Dziegielewski, J., Syljuasen, R. G., Lundin, C, Bartek, J. and Helleday, T. (2005) The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair. Nat Cell Biol, 7, 195-201. Stiff, T., O'Driscoll, M., Rief, N., Iwabuchi, K., Lobrich, M. and Jeggo, P.A. (2004) ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation. Cancer Res, 64, 2390- 2396. Su, C, Gao, G., Schneider, S., HeIt, C, Weiss, C, O'Reilly, M.A., Bohmann, D. and

Zhao, J. (2004) DNA damage induces downregulation of histone gene expression through the G(1 ) checkpoint pathway. EMBO J, 23, 1133-1143. Wang, Z. F., Whitfield, M. L., Ingledue, T.C., III, Dominski, Z. and Marzluff, W. F. (1996) The protein that binds the 3' end of histone mRNA: A novel RNAbinding protein required for histone pre-mRNA processing. Genes Dev, 10, 3028-3040.

Wyrick, J.J., Holstege, F. C, Jennings, E.G., Causton, H. C, Shore, D., Grunstein, M., Lander, E. S. and Young, R.A. (1999) Chromosomal landscape of nucleosomedependent gene expression and silencing in yeast. Nature, 402, 418-421.

Yamashita, A., Ohnishi, T., Kashima, I., Taya, Y. and Ohno, S. (2001 )

Human SMG-1 , a novel phosphatidylinositol 3-kinase-related protein kinase, associates with components of the mRNA surveillance complex and is involved in the regulation of nonsense- mediated mRNA decay. Genes Dev, 15, 2215-2228.

Zachos, G., Rainey, M. D. and Gillespie, D.A. (2005) Chk1 -dependent s-m checkpoint delay in vertebrate cells is linked to maintenance of viable replication structures. MoI Cell Biol, 25, 563-574.

Zhao, X., McKillop-Smith, S. and Mϋller, B. (2004) The human histone gene expression regulator HBP/SLBP is required for histone and DNA synthesis, cell cycle progression and cell proliferation in mitotic cells. J Cell Science, 117, 6043-6051. Zhou, B. B. and Elledge, S.J. (2000) The DNA damage response: putting checkpoints in perspective. Nature, 408, 433-439.

Ziv, Y., Bar-Shira, A., Pecker, I., Russell, P., Jorgensen, T.J., Tsarfati, I. and Shiloh, Y. (1997) Recombinant ATM protein complements the cellular A-T phenotype. Oncogene, 15, 159-167.

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Claims

1. A method for identifying an active agent useful for treating neoplasia, the method comprising: - determining the ability of a test agent to inhibit the ATR pathway;

- determining the ability of the test agent to inhibit the DNA-PK pathway; and

2. The method of claim 1 wherein the step of determining the ability of a test agent to inhibit the ATR or DNA-PK pathways comprises determining the effect of the agent on an indicator of histone levels or histone production within a cell.

3. The method of claim 2 wherein the step of determining the ability of a test agent to inhibit the ATR or DNA-PK pathways comprises determining the effect of the agent on the level of histone mRNA.

4. The method of claim 3 wherein the levels of at least one of histone H2A, H2B or H3 mRNA is determined.

5. The method of any preceding claim wherein the test agent is selected from the group consisting of small molecules, peptides, proteins, and poly- or oligo-nucleotides.

6. The method of any preceding claim comprising the steps of: a) providing a first cell-line in which the activity of one of the ATR pathway or the DNA-PK pathway is abrogated, the other pathway being substantially functional; b) providing a second cell-line in which the pathway which is abrogated in the first cell line is substantially functional; c) administering a test agent to said cell lines; d) determining the ability of the test agent to inhibit the ATR and DNA-PK pathways; and e) selecting an agent which selectively inhibits one or other, but not both of the ATR and DNA-PK pathways.

7. The method of claim 6 wherein in the second cell line both the ATR and DNA-PK pathways are functional.

8. The method of claim 6 wherein in the second cell line the pathway which is functional in the first cell line is abrogated.

9. The method of any one of claims 5 to 8 wherein abrogation of the

ATR or the DNA-PK pathways is achieved by inhibiting or knocking- out the expression or function of one or more members of the pathways.

10. The method of claim 9 wherein inhibiting or knocking-out the expression or function of one or more members of the pathways is achieved by gene knock-out, RNA interference, anti-sense RNA, or expression of a dominant negative mutant protein.

11. The method of any one of claims 5 to 10 wherein the cell-lines used in the method are human cell-lines.

12. The method of any one of claims 5 to 11 wherein the cell-line in which the ATR pathway has been abrogated is a cell line which over- expresses a kinase-dead form of ADR

13. The method of claim 12 wherein the cell line is the U20S/kd-ATR cell-line.

14. The method of any one of claims 5 to 13 wherein the cell-line in which the DNA-PK pathway has been abrogated is the M059J cell line.

15. The method of any one of claims 5 to 12 wherein a single cell-line is used in which the ATR and/or the DNA-PK pathways can be selectively abrogated, thus providing the first and second cell-lines.

16. An assay comprising: a) a first cell-line in which the activity of one of the ATR pathway or the DNA-PK pathway is abrogated, the other pathway being substantially functional; b) a second cell-line in which the pathway which is abrogated in the first cell line is substantially functional; and c) means to asses the ability of the test agent to inhibit the ATR and DNA-PK pathways.

17. An active agent identified using the method or assay as described above.

18. A pharmaceutical composition comprising an active agent according to claim 17 in combination with a pharmaceutically acceptable excipient.