Background to the Invention E2F is central to the coordination of cell cycle progression at the G1 to S phase transition (reference 1; numbers in parentheses herein refer to the references numbered at the end of the example). By integrating the control of retinoblastoma (pRb) tumour suppressor protein phosphorylation by cyclin- dependent kinases (Cdks) with the transcriptional activity of E2F target-genes, E2F orchestrates gene activity within the cell cycle. The pathway of control involved in regulating E2F becomes aberrant in most, if not all, human tumour cells, either through mutation and inactivation of the pRb gene or through altering Cdk-inhibitor genes, resulting in increased activity of Cdks and the subsequent inactivation of pRb activity (2). These events release the inhibitory effect of pRb on E2F activity and thereby deregulate E2F in favour of cell cycle progression.

Under physiological conditions, E2F exists as a heterodimer with members of the DP protein family (3). Currently, six E2F and two DP family members are known, and considerable evidence suggests that different family members have distinct, perhaps overlapping, physiological roles. For example, the analysis of knock-out mice carrying inactivated E2F genes indicates that diverse phenotypic differences reflect the loss of specific E2F genes (3). Thus, E2F-1-/-mice exhibit defects in apoptosis and an increased incidence of tumours (4,5), whereas mice lacking other E2F genes have quite distinct phenotypes (3). Moreover, <BR> <BR> although the concerted action of E2F-1, -2 and-3 promotes cell cycle progression (6,7) E2F proteins can augment apoptosis under

certain conditions (8-11). The induction of apoptosis by E2F-1 may involve the activation of target-genes, such as arf (12), and subsequent signalling of apoptosis through the p53 pathway (13). Alternatively, the transcriptional activation of p53 family members, such as the P73 gene, and the Apafl gene may contribute to E2F-mediated apoptosis (14-17). Although apoptosis is less easily observed with other members of the family, both E2F-4 and E2F-5 induce apoptosis with an appropriate DP partner (18).

Although regulation of E2F and its association with pRb is under cell cycle control, with active E2F appearing as cells progress through G1 towards S phase, there is growing evidence that E2F is induced in the absence of cell cycle progression by diverse forms of cellular stress, including DNA damage (19). In addition, the related phosphatidylinositol-3-OH-kinase-like kinases (PIKK) ATM and ATR may be involved (20).

The DNA damage signalling pathway is a highly conserved response to genotoxic stress (21). In mammalian cells, the integrity of the pathway has a protective role in overcoming the effects of agents that induce cellular transformation, where it functions in checkpoint control to cause cell cycle arrest or apoptosis (22). Although ATM/ATR PIKKs are generally regarded as sensors of DNA damage, the checkpoint kinases Chkl and Chk2 probably function as effectors of the response through phosphorylation of key substrates, such as p53 (22-24), the cell-cycle-regulating phosphatases Cdc25A (25) and Cdc25C (26), and Mdm2 (24), BRCA1 (27) and PML (28).

Disclosure of the Invention.

In this study, the present inventors have explored the role of the DNA damage signalling pathway, specifically Chk2, in the regulation of E2F-1 activity and have discovered that Chk2 functions as a key effector of E2F-1 induction during DNA

damage. The regulation of E2F-1 activity through the DNA damage signalling pathway, and specifically by Chk2, indicates a role for E2F-1 in checkpoint control. Such a role for E2F-1 may contribute to the tumour suppressor activity of E2F-1.

Accordingly, the present invention provides an assay for the activity of Chk2 which assay comprises: providing Chk2 and an E2F-1 polypeptide comprising residue Ser 364 under conditions in which Chk2 is able to phosphorylate said polypeptide; and determining whether said Ser 364 residue has been phosphorylated.

Phosphorylation of the Ser 364 residue is indicative of the activity of Chk2.

Our results also provide indication that there are also therapeutic applications of the discovery described here.

Specifically, pharmacological approaches that compromise Chk2- mediated activation of E2F-1 may interfere with the limited checkpoint activity that remains intact during DNA damage in tumour cells. By blocking Chk2 activity and inactivating checkpoint responses, tumour cells may remain in the proliferative cycle, and thus become more sensitive to chemo- therapeutic drugs, such as etoposide, that function through DNA damage. This approach may increase the therapeutic window between tumour and normal cells and enhance the efficacy of conventional anti-cancer drugs.

Thus in another aspect, the invention provides an assay for an inhibitor of E2F-1 phosphorylation by Chk2, which assay comprises:

providing Chk2 and an E2F-1 polypeptide comprising residue Ser 364 under conditions in which Chk2 is able to phosphorylate said polypeptide; providing a putative modulator compound; and determining whether said putative modulator compound affects the phosphorylation of said peptide.

In a further aspect, the invention provides peptides derived from the E2F-1 phosphorylation site as agents which potentiate the effects of genotoxic agents.

These and further aspects of the invention are described herein.

Description of the Drawings.

Figure 1 E2F-1 is stabilised after DNA damage and requires Chk2 kinase. a, b, Extracts from MCF7 cells were exposed to 10 uM etoposide (a) or 50 J m-2 UV (b) and immunoblotted for endogenous E2F-1 (top), p53 (middle) or PCNA (bottom). c, MCF7 cells were transfected Flag-tagged dominant-negative Chk2 or vector alone, as indicated. Cells were then exposed to etoposide (10 pM) or UV (50 J m-2) and cell extracts were prepared 6 h later. Levels of endogenous E2F-1 (top), p53 (middle), PCNA (bottom) and Flag-tagged Chk2 were analysed by immunoblotting.

In all cases, equal amounts of total protein were loaded in each lane.

Figure 2 Chk2 phosphorylates E2F-1 on Ser 364 in vitro. a, A schematic representation of E2F-1. The location of the Chk2 phosphorylation site is indicated between the marked box and activation/pocket protein (PP)-binding domain. b, Comparison of the Chk2 phosphorylation sites in Cdc25A, Cdc25C, PML, BRCA1 and p53 with E2F-1. Residues underlined fit the consensus phosphorylation motif. c, Recombinant Chk2 protein (0.2 ug) was incubated with or without 32P-ATP in the presence or absence of

1 ug recombinant E2F-lwt or E2F-1S364A. Recombinant Cdc25C was used as a positive control for phosphorylation by Chk2. Reaction products were resolved on denaturing gels and visualised by autoradiography. d, Recombinant cyclinA/Cdk2 (0.2 ug) was incubated with or without 32P-ATP in the presence or absence of 1 ug recombinant E2F-lwt or E2F-1S364A. Recombinant pRb was used as positive control for cyclin A/Cdk2 activity. Reaction products were resolved as described above.

Figure 3 E2F-1 is phosphorylated on Ser 364 in vivo. a, Sequence of the phospho-peptide derived from E2F-1 used to prepare the phospho-specific antiserum. b, Recombinant Chk2 (0. 2 ug) was incubated with recombinant E2F-1 (1 ug) in the presence or absence of ATP (50 uM). The reaction products were immunoblotted with either the non-specific anti-E2H-1 antibody KH95 (lanes 1 and 2) or the anti-P-Ser 364 antibody (lanes 3 and 4). E2F-1 is indicated, together with the increased specificity of anti-P-Ser 364 for Chk2-phosphorylated E2F-1. c, MCF7 cells were left untreated or exposed to etoposide (10 uM). Cell extracts were prepared 2 h later. Phosphorylation of endogenous E2F-1 on Ser 364 was monitored by immunoblotting with the anti-P-Ser 364 antibody (lanes 2 and 4). The amounts of extracts used have similar levels of total E2F-1, as shown by immunoblotting with the KH95 antibody (lanes 1 and 3). d, MCF7 cells were treated as described in c and probed with either affinity purified anti-P- Ser 364 antibody (top left) or affinity depleted antiserum containing antibodies against the non-phosphorylated epitope (top right). Underneath each blot, the level of E2F-1 determined by immunoblotting with antibody KH95 is shown. The E2F-1 polypeptide is shown. e, MCF7 cells were treated as in c. For immunoprecipitation of E2F-1, cell extract was incubated with the KH95 antibody bound to protein A beads. Phosphorylation of endogenous E2F-1 on Ser 364 was monitored by immunoblotting with the anti-P-Ser 364 antibody (top). The level of

immunoprecipitated E2F-1 (middle) and input E2F-1 protein (bottom) is also shown.

Figure 4 Dependence on Ser 364 for E2F-1 stabilisation in response to DNA damage. a, MCF7 cells were transfected with HA- E2F-lwt or HA-E2F-1S364A, along with ß-gal to monitor transfection efficiency, and then treated with etoposide (10 uM) for the indicated times. Extracts were then immunoblotted with an anti-HA antibody. b, U20S cells were transfected as in a and treated with or without etoposide (10 uM) for 4 h, as indicated.

Cycloheximide (50 ug) was added and extracts were then immunoblotted with an anti-HA antibody at the indicated times.

The level of PCNA is shown below each treatment as a loading control. To analyse the half-life of E2F-1, the exposure time of E2F-lwt in the presence of etoposide was reduced such that each immunoblot was of similar intensity. c, U20S cells were transfected with E2F-lwt or E2F-1S364A (100,250, 500 or 1, 000 ng) together with DP-1 (1 ug), cyclinE-luc (1 ug) and the internal control CMV-p-gal (500 ng). After 48 h, cells were harvested and the indicated data derived from triplicate readings. The levels of the exogenous E2F-1 and S364A protein levels determined by immunoblotting with an anti-HA antibody are shown.

Figure 5 Role of Chk2 in the stabilisation of E2F-1 after DNA damage. a, HCT-15 cells were left untreated, or exposed to etoposide (10 uM) or UV irradiation (50 J m-2) for 3 or 6 h.

Levels of endogenous E2F-1 (top), p53 (middle) or PCNA (bottom) were analysed by immunoblotting. Equal levels of total protein were loaded in each lane. b, U20S cells were transfected with vector alone (lanes 1 and 2) or Flag-tagged Chk2 (lanes 3 and 4) and exposed to 10 uM etoposide for 6 h. Extracts were prepared and immunoblotted for endogenous E2F-1 (top), Flagtagged Chk2 (middle) and PCNA (bottom), as described. c, HCT15 cells were transfected with vector alone (lanes 1-3) or Flag-tagged Chk2

(lanes 4-6) and exposed to 10 uM etoposide for 3 or 6 h.

Extracts were prepared and immunoblotted « for endogenous E2F-1, Flag-tagged Chk2, P-Thr 68-Chk2 and PCNA, as indicated.

Figure 6 Apoptosis and cell cycle arrest through E2F-1 requires Chk2. a, U20S cells were cotransfected with E2F-1, DP-1 and dominant-negative Chk2, as indicated. After transfection, cells were incubated for 48 h and then treated with etoposide (20 uM, 12 h) before harvesting and analysis by flow cytometry. The graph shows the percentage change in cell cycle profile with respect to cells transfected with vector alone and not treated with etoposide, where the flow cytometry profile was sub-G1, 11. 6% ; G1, 46% ; S, 6. 7% ; G2/M, 32%. b, U20S cells were transfected with E2F-lwt or E2F-1S364A and DP-1, together with dominant-negative Chk2, as indicated, and treated as described above. The graph shows the percentage of cells in the apoptotic (sub-G1 DNA content) population with respect to transfection with vector alone and incubation in the absence of etoposide (sub-Gl, 14. 1%). c, SOAS2 (p53-/-) cells were transfected with E2F-lwt or E2F-1S364A and DP-1, together with dominant-negative Chk2, as indicated, and treated with etoposide for 6 h. The graph shows the percentage of cells in the apoptotic (sub-G1 DNA content) population with respect to transfection with vector alone and incubation in the absence of etoposide (sub-G1, 16. 7%). d, Cells were treated with etoposide (10 uM) for 0, 8 or 16 h. Extracts were prepared and immunoblotted for endogenous Apafl (top), E2F-1 (middle) and PCNA (bottom). e, A summary of the pRb/E2F pathway (left) and p53 response to DNA damage (right), together with ATM/ATR and Chk2 kinases involved in DNA damage signalling to p53. Targets that may result in E2F- dependent apoptosis, such as ARF, p73 and Apaf-1, are indicated.

The arrow connecting Chk2 directly with E2F-1 is derived from the conclusions in this study and emphasises the interplay between the DNA damage signalling pathway and the pRb/E2F

pathway. The connection between ATM/ATR and E2F-1 is based on previous reports (50).

Detailed Description of the Invention.

The wild type Chk2 amino acid and nucleic acid sequences are disclosed in Matsuoka et al (47), Chaturvedi et al, (48), and Blasina et al (49). Chk2 nucleotide and amino acid sequences from many different sources are also available on Genbank, including mouse chk2 protein (Genbank AAC83694), mouse chk2 nucleotide (AF086905), human chk2 protein (AAD48504), human chk2 nucleotide (AF086904), C. elegans chk2 protein (AAD55890), C. elegans chk2 nucleotide (AB049441), rat chk2 protein (AAD55890), rat chk2 nucleotide (AF134054), zebrafish chk2 protein (BAB15803), zebrafish chk2 nucleotide (AF265346).

Constructs suitable for use in the present invention may be based upon those described by Chehab et al (22).

In performing assays of the present invention, it may not be necessary to use the entire full-length Chk2 protein provided the Chk2 protein retains the ability to phosphorylate the E2F-1 peptide.

The Chk2 may be provided in the form of a tagged protein, in order to assist the tracking of the protein in assay methods of the invention. Suitable tags include those conventional in the art such as a polyhistidine tag, a GST tag, an HA tag and the like.

An E2F-1 protein may be any human or other mammalian or eukaryotic E2F-1. The human E2F-1 may be found by reference to Helin et al, 1992, Cell, 70,337-350, and homologous polypeptides are found in other organisms, the sequences of many of which are available on databases such as Genbank. The E2F-1 polypeptide for use in assays of the invention will comprise at the least the Ser-364 residue together with sufficient

surrounding sequence of E2F-1 to act as a target for Chk2.

Generally this will comprise a fragment of at least 10, such as at least 50, preferably at least 100 amino acids which include the Ser-364 residue. In one embodiment, the full length E2F-1 protein will be used.

In performing assay methods Chk2 and E2F-1 polypeptide comprising residue Ser 364 may be contacted under conditions in which Chk2 is able to phosphorylate E2F-1 polypeptide.

Suitable conditions for performing assay methods can be readily determined by those of skill in the art, and with reference to the accompanying examples.

In order to determine whether Chk2 has phosphorylated the Ser- 364 of E2F-1, a radioactively labelled phosphate group may be used, e. g. in the form of 32P-ATP. This will provide a direct signal on the E2F-1 which may be determined by counting incorporated radiolabel or other means, such as immuno- precipitating the E2F-1, separating the E2F-1 on a gel and subjecting the gel to autoradiography to determine the signal from the E2F-1.

Another aspect of the invention provides an antibody which distinguishes between phosphorylated Ser-364 and un- phosphorylated Ser-364. This may be produced, for example, using the methods described in the accompanying examples. Such antibodies may bind preferentially E2F-1 polypeptide which is phosphorylated at Ser 364 relative to E2F-1 polypeptide which is un-phosphorylated at Ser 364. Such antibodies, which may be polyclonal, monoclonal or binding fragments of complete antibody molecules (e. g. single chain Fv fragments) may also be used in determining the extent to which the residue has been phosphorylated. Kits comprising such antibodies form another aspect of the invention.

Thus the invention provides a kit comprising a first antibody capable of binding the Ser-364 in unphosphorylated form of E2F-1 and a second antibody capable of binding phosphorylated Ser-364 of E2F-1. The first antibody may bind preferentially to the unphosphorylated form of Ser 364 of E2F-1 polypeptide and the second antibody may bind preferentially to the phosphorylated form of Ser 364 of E2F-1 polypeptide. The antibodies may be packed in the kit together with accompanying instructions for their use.

Antibodies of the present invention may be used to determine whether Ser 364 of E2F-1 in samples obtained from patients is in a phosphorylated or unphosphorylated form. Such prognosis may be useful to determine appropriate treatments for patients.

The assay of the present invention may be performed in the presence of a putative modulator compound, in order to examine whether or not the modulator compound alters the ability of Chk2 to phosphorylate the E2F-1 peptide. There is considerable commercial interest in the drug discovery industry in developing novel targets for possible therapeutic intervention of processes in the cell cycle. Thus performing the assay of the invention provides a commercially valuable resource to the drug discovery industry, regardless of the outcome of the assay (i. e. whether or not the phosphorylation of E2F-1 is affected). This is particularly the case for the present invention as kinase inhibitors are a class of compounds of general interest, and thus it will be of use to perform the assay of the invention using kinase inhibitors developed against other targets, in order to determine to what extent if any the inhibitors inhibit the phosphorylation of E2F-1 by Chk2. In the case of kinase inhibitors developed against other targets in the cell, where it may be desirable that they do not show activity against the part of the cell cycle under investigation in the present invention.

Thus in another aspect of the invention the assay may be performed with a kinase inhibitor for a target other than Chk2 in order to assess the selectivity of the inhibitor for such a target.

Assays of the invention may include a negative control, particularly a modified E2F-1 peptide in which Ser-364 has been modified to another residue which is not phosphorylated by Chk2, for example an aliphatic amino acid such as alanine.

The present invention also relates to the identification of polypeptides which direct or modulate the phosphorylation of Ser 364 of E2F-1. The identification of such polypeptides may be of considerable interest to the drug discovery industry in identifying novel targets and may be useful, for example, therapeutically augmenting E2F-1 induced apoptosis. Thus in a further aspect, the invention provides an assay for the identification of an agent which phosphorylates or modulates the phosphorylation of E2F-1 polypeptide, which assay comprises: contacting a test compound and an E2F-1 polypeptide comprising residue Ser 364; and determining the phosphorylation of Ser 364 of the E2F-1 polypeptide.

The compound and the E2F-1 polypeptide may be contacted under conditions in which Chk2 is able to phosphorylate said E2F-1 polypeptide. Preferably the assay is performed in vitro. An increase or decrease in the phosphorylation of Ser 364 may be indicative that the compound directs or modulates E2F-1 phosphorylation. Preferably the compound phosphorylates and increases phosphorylation of E2F-1.

The above assays of the invention rely on the activity of Chk2 or a test compound in phosphorylating E2F-1. However our finding also suggest that there is a direct physical interaction between Chk2 and E2F-1 when these polypeptides are contacted,

and in an alternative assay format, the direct interaction may be examined, either in the absence or presence of phosphorylation. An assay for a modulator of the interaction of E2F-1 with Chk2 may comprise: providing Chk2 and an E2F-1 polypeptide comprising residue Ser 364; providing a putative modulator compound; and determining whether said putative modulator compound affects ability of Chk2 and the E2F-1 polypeptide to form a complex.

For example, the interaction between Chk2 and the E2F-1 polypeptide may be studied by labelling one with a detectable label and bringing it into contact with the other which has been immobilised on a solid support. Suitable detectable labels include 35S-methionine which may be incorporated into recombinantly produced Chk2 and/or the E2F-1 polypeptide. The recombinantly produced Chk2 and/or the E2F-1 polypeptide may also be expressed as a fusion protein containing an epitope which can be labelled with an antibody.

The protein which is immobilized on a solid support may be immobilized using an antibody against that protein bound to a solid support or via other technologies which are known per se.

A preferred in vitro interaction may utilise a fusion protein including glutathione-S-transferase (GST). This may be immobilized on glutathione agarose beads. In an in vitro assay format of the type described above the putative modulator compound can be assayed by determining its ability to modulate the amount of labelled Chk2 or the E2F-1 polypeptide which binds to the immobilized GST-E2F-1 polypeptide or GST-Chk2, as the case may be. This may be determined by fractionating the glutathione-agarose beads by SDS-polyacrylamide gel electrophoresis. Alternatively, the beads may be rinsed to remove unbound protein and the amount of protein which has bound can be determined by counting the amount of'label present in, for example, a suitable scintillation counter.

Alternatively an antibody attached to a solid support and directed against one of Chk2 or the E2F-1 polypeptide may be

used in place of GST to attach the molecule to the solid support. Antibodies against Chk2 and the E2F-1 polypeptide may be obtained in a variety of ways known as such in the art, and as discussed herein.

In an alternative mode, one of Chk2 and the E2F-1 polypeptide may be labelled with a fluorescent donor moiety and the other labelled with an acceptor which is capable of reducing the emission from the donor. This allows an assay according to the invention to be conducted by fluorescence resonance energy transfer (FRET). In this mode, the fluorescence signal of the donor will be altered when Chk2 and the E2F-1 polypeptide interact. The presence to a candidate modulator compound which modulates the interaction will increase the amount of unaltered fluorescence signal of the donor.

FRET is a technique known per se in the art and thus the precise donor and acceptor molecules and the means by which they are linked to Chk2 and the E2F-1 polypeptide may be accomplished by reference to the literature.

Suitable fluorescent donor moieties are those capable of transferring fluorogenic energy to another fluorogenic molecule or part of a compound and include, but are not limited to, coumarins and related dyes such as fluoresceins, rhodols and rhodamines, resorufins, cyanine dyes, bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazines such as luminol and isoluminol derivatives, aminophthalimides, aminonaphthalimides, aminobenzofurans, aminoquinolines, dicyanohydroquinones, and europium and terbium complexes and related compounds.

Suitable acceptors include, but are not limited to, coumarins and related fluorophores, xanthenes such as fluoresceins, rhodols and rhodamines, resorufins, cyanines, difluoroboradiazaindacenes, and phthalocyanines.

A preferred donor is fluorescein and preferred acceptors include rhodamine and carbocyanine. The isothiocyanate derivatives of these fluorescein and rhodamine, available from Aldrich Chemical

Company Ltd, Gillingham, Dorset, UK, may be used to label Chk2 and the E2F-1 polypeptide. For attachment of carbocyanine, see for example Guo et al, J. Biol. Chem. , 270; 27562-8,1995.

Assays of the invention may also be performed in vivo. Such an assay may be performed in any suitable host cell, e. g a bacterial, yeast, insect or mammalian host cell. Yeast and mammalian host cells are particularly suitable.

To perform such an assay in vivo, constructs capable of expressing Chk2 and the E2F-1 polypeptide and a reporter gene construct may be introduced into the cells. This may be accomplished by any suitable technique, for example calcium phosphate precipitation or electroporation. The three constructs may be expressed transiently or as stable episomes, or integrated into the genome of the host cell.

In vivo assays may also take the form of two-hybrid assays wherein Chk2 and the E2F-1 polypeptide are expressed as fusion proteins, one being a fusion protein comprising a DNA binding domain (DBD), such as the yeast GAL4 binding domain, and the other being a fusion protein comprising an activation domain, such as that from GAL4 or VP16. In such a case the host cell (which again may be bacterial, yeast, insect or mammalian, particularly yeast or mammalian) will carry a reporter gene construct with a promoter comprising a DNA binding elements compatible with the DBD. The reporter gene may be a reporter gene as disclosed above. The promoters for the genes may be those discussed above.

Chk2 and the E2F-1 polypeptide and the reporter gene, may be introduced into the cell and expressed transiently or stably.

The assays described herein may be performed in the presence of additional protein components involved in the interaction of Chk2 and E2F-1, particularly a Cdc25 protein, such as Cdc25A or Cdc25C, or a DP protein such as DP-1 which forms a heterodimer with E2F-1.

In an additional aspect, the invention provides a peptide derived from the E2F-1 Chk2 phosphorylation site which is capable of acting as an agent which potentiates the effect of a genotoxic agent. Such peptides may comprise the Chk2 phosphorylation site of E2F-1 polypeptide. Such peptides are based around the Ser-364 residue and will compete with E2F-1 for Chk2 binding.

Such peptides may, for example, be based upon the sequence: P-L-L-S-R-M-G-X-L-R-A-P Where X is any amino acid. In one embodiment, X is serine or threonine, preferably serine. In another embodiment, X is an straight or branch-chained aliphatic amino acid such as alanine, valine, leucine or isoleucine, or is glycine.

Peptides may also be based on the sequence : R-M-G-X-L-R-A-P-V-D-E-D-R.

Where X is as defined above.

The peptides may comprise from 10 to 40, such as from amino acids, including the sequences above together with additional sequence which may be derived from wild-type E2F-1 sequence or may alternatively be a sequence intended to facilitate uptake of the above sequence into a cell. Example of such sequences include the HSV-1 VP22 protein, the HIV Tat protein (for example residues 1-72,37-72 or 48-60) or the sequence derived from the Drosophila melanogaster antennapedia protein. All of these are well known in the art as such.

The identification of the phosphorylation site on E2F-1 also provides the potential for the rational design of agents based on the phosphorylation site. Thus the structure of the phosphorylated E2F-1 may be determined in order to define a pharmacophore e. g. by modelling the tertiary structure of the

phosphorylated residue. This may be followed by devising chemical analogues which mimic the structure.

Such chemical analogues may be peptidomimetics of the incorporating the structure of the phosphorylated serine residue.

Thus, the results described here establish E2F-1 as a physiological target for Chk2 kinase in response to DNA damage signalling induced by etoposide treatment and argue that Chk2- mediated phosphorylation augments cell cycle arrest and apoptosis triggered by E2F-1. The fact that phosphorylation of E2F-1 is induced by DNA damage defines E2F-1 as a probable effector protein in the cellular response to DNA damage. In this respect, although it is well-established that E2F-1 can elicit proliferative effects and stimulate cell cycle progression, a variety of reports have defined a role for E2F-1 in cell cycle arrest and apoptosis (3). For example, E2F-1 can function as an oncogene in vitro and in vivo (34-37), and cells lacking E2F-1 have cell cycle defects (11). In contrast, negative growth control by E2F-1 is suggested from reports describing its apoptotic activity (8-10), which may in part be responsible for the phenotype of E2F-1-/-mice, which exhibit increased tumour incidence with reduced levels of apoptosis in the immune system (4,5). The pathway described here for Chk2 in regulating apoptosis and the abundance of E2F-1 may be involved in eliciting the tumour-suppressor-like activity previously ascribed to E2F-1.

Established substrates for Chk2 include p53, Cdc25A, BRCA1 and PML (24,25, 27,28). Although phosphorylation of Cdc25A facilitates cell cycle delay (25), the p53 response has been ascribed to both cell cycle arrest and apoptosis (38). Our results argue that Chk2 induces E2F-1, which, in turn provides a

signal for cell cycle arrest and apoptosis. In this respect, the damage induction of E2F-1 resembles the p53 response.

Together with other studies (20), we have established that both Chk2 and ATM/ATR kinase families signal to E2F-1, and in turn suggest that E2F-1 is a critical component in executing the physiological effects of the DNA damage response (Fig. 8e). It is known that a variety of E2F target-genes contribute to apoptosis. For example, E2F-1 activates transcription from the ink4a/arf locus, where ARF signals apoptosis through p53 (12).

The p53-related gene P73 is a direct target for E2F-1 and increased levels of p73 can induce cell cycle arrest and apoptosis (14-16). The gene for Apafl, which is mechanistically involved in apoptosis, is also a direct target for E2F-1 (17).

Therefore, we suggest that genes such as those mentioned above are induced through damage-responsive signalling to E2F-1, which, in turn and perhaps in concert with the p53 response, results in cell cycle arrest or apoptosis. The results suggest that E2F-1 phosphorylated at Ser 364 can reside in discrete nuclear structures. Although the significance of these structures remains to be determined, it is possible that their presence is causally related to the activity of phosphorylated E2F-1.

In this respect, p53 can accumulate in discrete nuclear bodies and colocalize with other nuclear proteins, including PML39, which may be related to p53 function (39). The P53 gene is frequently inactivated in tumour cells (40). Additionally, the Chk2 gene can be mutated in tumour cells (31), and studies on the familial inheritance of mutant genes in Li Fraumeni syndrome suggests that the Chk2 gene is inactivated in patients that do not harbour mutations in P53 (31). However, some tumour cells, such as the HCT15 cell line, concomitantly lack p53 and Chk2 (25).

Despite the inactivation of p53, checkpoint response to DNA damage remains intact in many tumour cells, allowing tumour cells to undergo transient cell cycle arrest (41). In contrast to the P53 gene, and despite the apparent tumour suppressor activity, E2F-1 rarely, if ever, becomes inactivated in tumour cells (1). This situation may reflect the positive function of E2F-1 in regulating cell cycle progression (7,37). Nevertheless, the presence of E2F-lwt and its activation by Chk2 kinase in DNA damage signalling could be in part the basis of the checkpoint response to DNA damage that remains intact in tumour cells.

Thus, the requirement of tumour cells to deregulate the pRb/E2F pathway to release active E2F and promote growth may be counterbalanced by the negative role of E2F-1 in proliferation control through activation by the DNA damage signalling pathway.

Tumour cells harbouring inactive Chk2 would be expected to carry a damage response that becomes more overtly disabled than those harbouring inactive p53, and perhaps retain E2F-1 activity in a state more favourable for growth.

Example The following example illustrates the invention.

E2F-1 is regulated by the DNA damage signalling pathway.

To investigate the relationship between regulation of endogenous E2F-1 and cellular stress, we treated human MCF7 cells, which express wild-type p53, with DNA-damaging agents and monitored the levels of E2F-1 and p53. Although some functional redundancy exists, etoposide and ultraviolet (UV) irradiation are thought to activate different arms of the DNA damage signalling pathway (21). Both treatments induced expression of E2F-1 with kinetics that closely followed the induction of p53 (Fig. la, b). Chk2 has previously been shown to regulate p53 (22,24). Given that E2F-1 and p53 were induced with similar kinetics, we reasoned that E2F-1 activity may be subject to regulation by Chk2. Thus,

we studied the effect of a dominant-negative derivative of Chk2 (22) on the induction of endogenous E2F-1 by DNA damage. Because of the established role of Chk2 in the p53 response (22-24,29), we anticipated that the dominant-negative variant would hinder induction of p53. In etoposide-treated MCF7 cells, p53 induction was inhibited, whereas there was negligible effect on UV-treated cells (Fig. lc). Most interestingly, however, the Chk2 dominant- negative mutant blocked induction of E2F-1 in response to etoposide (Fig. lc), suggesting that Chk2 is also involved in signalling to E2F-1.

Chk2 kinase phosphorylates E2F-1.

Chk2 is a Ser/Thr-specific kinase for which a small number of physiological substrates have been identified (24,25, 27,28). A consensus sequence motif for phosphorylation, derived from Chkl and Chk2 phosphorylation sites in different substrates, has been identified (Fig. 2b). A site that closely resembles this consensus phosphorylation site is found in the carboxy-terminal region of E2F-1, centred on Ser 364 (Fig. 2a, b), although the E2F-1 site was more similar to sites in Cdc25 and PML than sites in p53 and BRCA1 (Fig. 2a, b).

We assessed whether E2F-1 functions as an in vitro substrate for Chk2. Therefore, we prepared a mutant E2F-1 derivative in which Ser 364 was altered to an alanine (A) residue. We found that wild-type E2F-1 was phosphorylated by Chk2 and that the efficiency of phosphorylation was similar to the level observed for Cdc25C (Fig. 2c). However, the E2F-1S364A mutant was not phosphorylated (Fig. 2c), suggesting that Ser 364 is phosphorylated by Chk2.

As a control, we assessed the specificity of the S364A mutant by determining whether E2F-1S364A could be phosphorylated by the cyclinA/Cdk2 kinase, which phosphorylates E2F-1 (1). We found

that E2F-lwt and E2F-1S364A were phosphorylated with approximately equivalent efficiency by cyclinA/Cdk2 (Fig. 2d).

E2F-1 is phosphorylated at Ser 364 under physiological conditions.

To establish whether Ser 364 is phosphorylated by Chk2 in vivo, we prepared an anti-phospho-specific peptide antiserum directed against the phosphorylated Ser 364 residue (referred to as P-Ser 364 ; Fig. 3a). After peptide affinity purification (see Methods), the reactivity of the antiserum was much more specific for the phosphorylated Ser 364 peptide compared with the nonphosphorylated Ser 364.

We assessed the reactivity of anti-P-Ser 364 on in-vitro- phosphorylated E2F-1. Anti-P-Ser 364 reacted strongly with Chk2- phosphorylated E2F-1 when compared with unphosphorylated E2F-1, in contrast to a general E2F-1 antibody which exhibited similar reactivity to both un-phosphorylated and phosphorylated E2F-1 (Fig. 3b). In addition, we found that the anti-P-Ser 364 antibody had greatly reduced activity on the S364A mutant. These results suggest that the Ser 364 site is phosphorylated by Chk2 and that anti-P-Ser 364 recognises the phosphorylated site in E2F-1.

Next, we used the anti-P-Ser 364 antibody to study the regulation of endogenous E2F-1 under DNA damage conditions to ascertain whether Ser 364 is subject to phosphorylation-mediated control in vivo. We treated MCF7 cells with etoposide for 2 h and monitored E2F-1 with the anti-P-Ser 364 antibody and the non-phospho-specific anti-E2F-1 antibody. Under these experimental conditions, there was little change in the steady- state level of E2F-1 (Fig. 3c). In non-stressed MCF7 cells, anti-P-Ser 364 exhibited little reactivity, whereas in etoposide-treated MCF7 cells, the antiserum clearly reacted with E2F-1 (Fig. 3c). Furthermore, when antibody that had been

affinity purified using the P-Ser 364 peptide was used on the same cell extracts, E2F-1 was detected only in etoposide treated cells (Fig. 3d, top). In contrast, the P-Ser 364 peptide affinity depleted antiserum, which should include antibodies against the non-phosphorylated Ser 364 residue, recognised an equivalent level of E2F-1 in treated and untreated cells (Fig.

3d, top).

Finally, immunoprecipitated E2F-1 from etoposide-treated cells was recognised by anti-P-Ser 364, in contrast to E2F-1 from non- stressed cells (Fig. 3e). Thus, Ser 364-E2F-1 undergoes DNA damage responsive phosphorylation in vivo.

Chk2 regulates E2F-1 through increased half-life.

We compared the level of exogenous wild-type E2F-1 to E2F-1S364A in MCF7 cells. After etoposide treatment for 6 h, the level of accumulated E2F-1S364A was approximately four times less than wild-type E2F-1 (Fig. 4a). One possible explanation for our results on the regulation of E2F-1 by Chk2 is that Chk2 controls the abundance of E2F-1 through an extended half-life. Although the half-life of E2F-lwt and E2F-1S364A was similar in the absence of etoposide, the mutant was significantly less stable in the presence of etoposide (Fig. 4b). On the basis of several experiments in the presence of etoposide, we determined the half-lives of the S364A mutant (60-120 min) and the wild-type protein (120-180 min). Thus, phosphorylation of Chk2 augments E2F-1 levels through an extended half-life.

Functional consequences of phosphorylation on E2F-1 activity.

Next, we assessed the functional importance of the Ser 364 site for E2F-1 transcriptional activity by comparing the properties of E2F-1 wt with E2F-1S364A on E2F-responsive promoters. We used several promoters, including the cyclin E promoter, which is directly responsive to E2F-1 (30). The activity of E2F-1S364A was significantly lower than that of E2F-lwt, even though the

protein levels were similar (Fig. 4c). We conclude that Ser 364 is a functionally important site that contributes to the stability of E2F-1 and the ability of E2F-1 to activate transcription.

Regulation of E2F-1 in human tumour cells.

We explored the regulation of E2F-1 in HCT15 colon carcinoma cells, which express mutant forms of Chk2 and p53 (31, 32).

Strikingly, E2F-1 levels were not induced by etoposide (Fig.

5a). This was in marked contrast to the clear induction of E2F-1 by UV irradiation in the same cells, which exhibited similar kinetics in HCT15 and MCF7 cells (Figs. 5a and lb). Together with the earlier observations on the effects of dominant- negative Chk2, these results provide an indication of a physiological role for Chk2 in regulating the response of E2F-1 to etoposide. This hypothesis was further supported by studies in which Chk2wt was introduced into either U20S (carrying wild- type Chk2) or HCT15 cells and the level of endogenous E2F-1 was measured after etoposide treatment. In U20S cells, introduction of Chk2wt allowed a greater level of E2F-1 to be induced by etoposide (Fig. 5b). In HCT15 cells, introducing wild-type Chk2 re-instated the ability of E2F-1 to respond to etoposide (Fig.

5c). Under these conditions, recognition of exogenous Chk2 with an antibody specific for the phosphorylated Thr 68 residue, which is required for Chk2 to become catalytically active (33), occurred in etoposide-treated cells and correlated with the induction of E2F-1 (Fig. 5c).

Location of E2F-1-Ser 364-P.

We used the affinity purified anti-PSer 364 antibodies to study the location of E2F-1 protein phosphorylated on Ser 364. In normal cells (MCF7 cells), immunostaining with the affinity purified antibody produced a low level of diffuse but evenly distributed nuclear staining. The immunostaining pattern

produced by anti-P-Ser 364 was specific, as nuclear staining was competed out only by inclusion of the phosphorylated Ser 364 peptide, and not by the un-phosphorylated E2F-1 peptide. This indicates that a low level of E2F-1 phosphorylated on Ser 364 exists in non-stressed cells. In etoposide-treated MCF7 cells, although the overall level of nuclear stain exhibited a moderate increase, the increase occurred in conjunction with the appearance of discrete nuclear structures with intense staining.

Again, this staining pattern was specific for E2F-1 phosphorylated on Ser 364. Peptide competition was performed with the affinity purified anti-phospho Ser 364 antibody either in the presence of 2 ug phosphorylated peptide or 2 ug non- phosphorylated peptide. Discrete nuclear structures stained by anti-P-Ser 364 were seen only after etoposide treatment. These results imply the existence of specific nuclear sub-structures in which the phosphorylated species of E2F-1 can reside. Chk2 is required for E2F-dependent apoptosis. E2F-1 can induce apoptosis in U20S cells (18). We assessed whether dominant-negative Chk2 interferes with E2F-1 apoptotic activity using flow cytometry to examine cell cycle profiles. Introduction of E2F-1 into etoposide treated cells increased the level of sub-Gl (apoptosing) cells, although it lowered the proportion of cells in G2/M phase. Increasing the amount of exogenous E2F-1 further enhanced the level of apoptosis and reduced the size of the G2/M population (Fig. 6a). In contrast, introduction of Chk2S364A reduced the level of apoptotic cells, although it restored the S and G2/M population (Fig. 6a). Further evidence that Chk2 regulates E2F-1 activity came from comparing the effects of E2F- 1wt and E2F-1S364A in U20S cells, where apoptosis induced by E2F-lwt was considerably greater than that observed with E2F- 1S364A (Fig. 6b).

Moreover, although co-expression of dominant-negative Chk2 with E2F-1 reduced the level of apoptosis, this was not apparent with apoptosis induced by E2F-1S364A (Fig. 6b). To rule out a

contribution from p53 to apoptotic activity, we investigated apoptosis in SAOS2 cells (p53-/-), in which E2F-lwt possesses greater apoptotic activity than E2F-1S364A. Furthermore, although co-expression of dominant-negative Chk2 reduced the apoptotic activity of E2F-lwt, it failed to significantly affect the activity of E2F-1S364A (Fig. 6c). Thus, Chk2-mediated regulation of E2F-1 augments apoptosis.

Finally, in SAOS2 cells stimulated to enter apoptosis by treatment with etoposide, the induction of endogenous E2F-1 correlated well with the induction of proteins encoded by E2F- responsive genes involved in apoptosis, such as Apafl. These results support a role, independent of p53, for E2F-1 in etoposide-induced apoptosis.

Methods Recombinant plasmids and proteins The following plasmids have been previously described : pCMV- égal42, pG4-DP1 (43), pCMVCD20 (44) and pCyclinE-luciferase30. pCMVHA-E2F-lwt was used as a template to construct pCMVHA-E2F- 1S364A using the Quickchange in vitro mutagenesis system (Stratagene, CA). The primers used were: Fwd 5'-TTGTCCCGGATGGGCGCCCTGCGGGCTCCC ; Rev 5'-AACAGGGCCTACCCGCGGGACGCCCGAGGG.

For expression in Escherichia coli, pCMVHA-E2Flwt and pCMVHAE2FlS364A were digested with BamHI and SacI and the resulting insert was cloned into pGEXKG at the same sites. 6- His-tagged Chk2wt, Flag-CMV Chk2wt and Flag-CMV-Chk2 A347 were gifts from T. Halazonetis (22). Baculovirus vectors for His-cycA and His-Cdk2 were as described (44). Glutathione S-transferase (GST) -tagged E2F-1 protein was purified from crude E. coli lysates with glutathione beads (Amersham Pharmacia Biotech (Amersham, UK). 6-Histagged Chk2 protein was purified from crude

E. coli lysates with nickel-NTA agarose beads (Qiagen). Sf9 insect cells were co-infected with the cycA and Cdk2 recombinant baculoviruses and proteins were purified from whole-cell extracts 48 h after infection with nickel-NTA Agarose beads.

Purified Cdc25C protein was purchased from Biodiagnostics.

Cell culture and transfection MCF7, U20S and SAOS2 cells were grown in DMEM (Invitrogen) supplemented with 10% FCS. HCT15 cells were grown in RPMI-40 (Invitrogen, Carlsbad, CA) supplemented with 10% FCS. Transient transfections were carried out using calcium phosphate precipitation as described (45) or Lipofectamine (Invitrogen).

Sf9 cells were grown in TC100 (Invitrogen) supplemented with 5% FCS.

Immunoblotting Cell lysates were resolved by denaturing gel electrophoresis before electrotransfer to Protran nitrocellulose membrane. Equal protein loading was confirmed with Ponceau S staining. The E2F-1 antibody (KH-95), PCNA antibody (PC 10) and p53 antibody (DO-1) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

The Apafl antibody (NT) was purchased from Upstate Biotechnology. The Flag M2 antibody was purchased from Sigma (St Louis, MO). The HA-11 antibody was purchased from Cambridge Bioscience. Chk2 phosphorylated at Thr 68 was purchased from Cell Signalling Technology. The anti-P-Ser 364 antibody was generated by Eurogentec (Belgium). Briefly, two peptides were generated on the basis of sequence around Ser 364, one of which was chemically phosphorylated at Ser 364. Peptides were conjugated to keyhole limpet haemocyanin (KLH) carrier protein and the phosphorylated peptide was used for immunisation.

Antibodies were affinity purified over a column containing the immobilised phosphorylated peptide. Next, the bound material was passed over a second column containing immobilised non-

phosphorylated peptide. Phospho-Ser 364-specific antibodies were eluted in the flow-through of the second peptide column.

Immunostaining For immunofluorescence staining, MCF7 cells were seeded on 13-mm borosilicate glass coverslips at a density of 2 x 104 cells.

After 24 h, cells were treated with 10 uM etoposide, where indicated. After 16 h of etoposide treatment, cells were washed with twice with PBS, fixed with 4% paraformaldehyde, permeabilized with 0. 1% Triton X-100 and blocked with PBS containing 10% foetal calf serum (FCS). The affinity purified anti-P-Ser 364 polyclonal antibody was used to detect endogenous phosphorylated E2F-1. For peptide competition, antibody and peptide were pre-incubated for 10 min at room temperature.

Secondary anti-rabbit Alexa 594 antibody was from Molecular Probes (Eugene, OR). Cells were counterstained with 4', 6'- diamidino-2-phenylindole (DAPI). Images were captured using an Olympus BX60 system microscope with Hamamatsu C4742-95 digital camera. Images were analysed using Improvision Openlab digital imaging software.

In vitro kinase assays Purified recombinant wild-type or mutant E2F-1 proteins (1 pg), and Cdc25C (lug) were incubated with purified His-tagged Chk2 protein (0.2 ug) for 30 min in kinase buffer A (50 mM Hepes at pH 7.4, 10 mM magnesium chloride, 10 mM manganese chloride and <BR> <BR> 10 mM dithiothreitol (DTT) ) in the presence or absence of 200 nM labelled ATP and 200 nM 32P-ATP. Purified recombinant wild- type or mutant E2F-1 proteins (lpg), and pRb (1 ug) were incubated with purified cyclinA-Cdk2 (0.2 ug) from baculovirus for 30 min in kinase buffer (20 mM Hepes at pH 7.6, 10 mM magnesium chloride, 1 mM EGTA, 2 mM manganese chloride, 10% glycerol and 1 mM DTT) s in the presence or absence of 200 nM unlabelled ATP and 200 nM 32P-ATP. Reactions were stopped by the addition of 3 x SDS loading buffer and the protein mixtures were

resolved by denaturing gel electrophoresis. Gels were subjected to autoradiography to monitor 32P incorporation or immunoblotted with antibody anti-P-Ser 364 to monitor phosphorylation of E2F-1 on Ser 364. Equal E2F-1 levels were confirmed by immunoblotting with antibody KH-95.

E2F-1, p53 and Apafl protein levels MCF7, SAOS2 and HCT15 cells were exposed to 50 J m-2 UV irradiation or 10 uM etoposide. Whole cell extracts were prepared 1-16 h after exposure to UV or etoposide by lysis in extraction buffer (50 mM Tris at pH 7.4, 120 mM sodium chloride, 5 mM EDTA, 0. 5% NP-40, 1 mM PMSF, 5 mM sodium fluoride, 1 mM sodium vanadate and 1 x Protease Inhibitor Cocktail. DNA damage- induced stabilisation of endogenous E2F-1 and p53 was monitored by immunoblotting with KH-95 and anti-p53 antibodies, respectively, and Apafl with the NT monoclonal antibody. Flag- tagged Chk2 transfection efficiency was monitored by immunoblotting with Flag M2 antibody. Quantification was performed with a BioRad imaging densitometer (GS-670) using Molecular Analyst software.

Effects of dominant-negative Chk2 MCF7 cells were transfected with 5 ug of plasmid expressing Flag-tagged mutant Chk2 or 5 ug of the expression plasmid without insert. The transfected cells were exposed to 50 J m-2 UV irradiation or 10 uM etoposide 36 h after transfection.

Whole-cell extracts were prepared 6 h after exposure to UV or etoposide by lysis in extraction buffer. The levels of endogenous E2F-1 and p53 were monitored by immunoblotting with KH-95 and anti-p53 antibodies, respectively. Flag-tagged mutant Chk2 transfection efficiency was monitored by immunoblotting with the Flag-M2 antibody.

E2F-1-Ser 364 phosphorylation in intact cells MCF7 cells were left untreated or exposed to 10 pM etoposide.

Whole-cell extracts were prepared 2 h after exposure to etoposide by lysis in extraction buffer. Phosphorylation of endogenous E2F-1 on Ser 364 was assayed by immunoblotting with anti-P-Ser 364 antibody. The amounts of extracts used had similar E2F-1 levels, as shown by immunoblotting with antibody KH-95.

Flow cytometry 1 x 106 U20S cells seeded in a 10-cm dish were transfected with the indicated amounts of expression vectors together with 8 pg of pCMV-CD20. After 48 h, cells were exposed to 20 uM etoposide.

Cells were harvested 12 h after exposure to etoposide and analysed by flow cytometry (46). The data shown were derived from at least three independent experiments. For SAOS2 cells, 8 x 105 cells were transfected as described above and exposed to 20 pM etoposide 65 h after transfection. Cells were then harvested 6 h later.

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