WO2003091385A2 - Assay methods relating to the action of geminin in the licensing of dna replication complexes in transformed cells - Google Patents

Abstract

The invention is based on the finding that the presence of the protein geminin in the G1 phase in transformed cells leads to a reduction in the number of licensed replication complexes but does not prevent the DNA of the attempting to replicate by entering S phase, resulting in a proportion of the cells undergoing apoptosis. This is in contrast to untransformed cells, where the lack of sufficient replication complexes will prevent the entry of the cells into S phase. The differential effect of geminin provides a basis for a cell based assay for drug discovery, comprising: providing a candidate compound; providing a sample of transformed and a sample of untransformed cells; and determining whether said compound is capable of reducing the number of licensed replication origins in said cells present in the G1 phase.

Description

ASSAY METHODS RELATING TO THE ACTION OF GEMININ IN THE LICENSING OF DNA REPLICATION COMPLEXES IN TRANSFORMED CELLS

Field of the Invention.

The present invention relates to cell-based assay systems for compounds which affect licensing of DNA replication in a manner differential between untransformed, particularly primary and transformed cells.

Background to the Invention.

Cells ensure the precise duplication of chromosomal DNA in each cell cycle by dividing the replication process into two distinct stages. In the first stage, which occurs in late mitosis and early Gl, replication origins are "licensed" -for replication by being loaded with complexes of 6 miriichromosome maintenance proteins, Mcm2-7 (Blow and Laskey, 1988; Diffley, 2001; Lei and Tye, 2001; Blow and Hodgson, 2002) . These Mcm2-7 proteins form an important part of the pre-replicative complex (pre-RC) bound at replication origins in Gl of the cell cycle (Diffley et al., 1994; Labib et al., 2001).

In yeast, a 'pre-replicative complex' (pre-RC) forms a footprint over replication origins during Gl (Diffley et al, 1994) which may well correspond to the Mcm2-7 loading that represents the licensed state, although it is possible that other factors in addition to those required for origin licensing are involved.

Following activation of CDK {cyclin-dependent kinase) and Cdc7 protein kinases at the onset of S phase, replication forks are initiated only at these licensed replication origins. As initiation occurs at each origin, the licence is removed. In order to prevent replicated origins from becoming re- licensed and hence re-replicated, the ability to load new Mcm2-7 complexes onto origins is prevented during late Gl, S, G2 and early M. This inhibition is brought about by the combined activity of CDKs and an inhibitory protein called geminin (Diffley, 2001; Lei and Tye, 2001; Blow and Hodgson, 2002) . Mcm2-7 play an essential part in DNA replication, possibly providing the DNA helicase activity necessary to unwind the DNA ahead of the replication fork (Labib and

Diffley, 2001) . Following displacement from origins, Mcm2-7 appear to move along with the replication fork (Aparicio et al, 1997), consistent with their providing essential helicase activity for the progression of the replication fork (Labib et al, 2000) .

At least three other proteins are required for origins to load Mcm2-7 and become licensed (Blow and Hodgson, 2002; Diffley, 2001; Lei and Tye, 2001) . The Origin Recognition Complex (ORC) first binds to each replication origin, and then recruits two other proteins, Cdcδ and Cdtl. These act together to load Mcm2-7 complexes and functionally license the origin. The licensing of Xenopus sperm nuclei has recently been reconstituted with purified proteins (Gillespie et al., 2001). A crucial feature is that although ORC, Cdcβ and Cdtl are all essential for Mcm2-7 loading, none of them are subsequently required to maintain the binding of Mcm2-7 to origins (Donovan et al., 1997; Hua and Newport, 1998; Rowles et al., 1999; Maiorano et al., 2000). An important consequence is that re- licensing of replicated DNA can be prevented by inhibition or . removal of ORC, Cdcβ or Cdtl once S phase has started, without displacing functional Mcm2-7 at licensed origins. Evidence from a range of different organisms and cell types suggest that when cells withdraw from the cell cycle, either reversibly into GO or irreversibly, they lose Mcm2-7 proteins and become functionally unlicensed (Tsuruga et al., 1997; Musahl et al . , 1998; Stoeber et al . , 2001; Sun et al., 2000; Blow and Hodgson, 2002; Tan et al., 2001). A similar reduction in Cdcβ protein is seen in GO and permanently arrested cells (Stoeber et al., 1998). We have recently proposed that the presence or absence of licensed origins provides a functionally important distinction between cells in Gl and GO (Blow and Hodgson, 2002). Cancer cells, with a high .proportion of proliferating cells (high growth fraction) , stain strongly for Mcm2-7 (Williams et al., 1998; Stoeber et al., 1999; Stoeber et al., 2001; Tan et al., 2001).

Summary of the Invention.

In replicating cells, geminin is not usually present in late mitosis or Gl phase. Our experiments have investigated the action of geminin when it is present in cell-free replication systems and in transformed and untransformed primary cell lines.

Thus the presence of geminin in late mitosis or Gl phase of replicating cells inhibits the formation of licensed replication origins. This means that insufficient licensed origins are present for the whole genome of the cell to be fully replicated.

We have found that the effect of this in transformed cell lines is that cells still enter S phase and attempt, unsuccessfully, to replicate their DNA. This process leads to a proportion of the cells undergoing apoptosis. In contrast, it appears that surprisingly, in primary cell lines the effect of an insufficient number of licensed origins is to prevent the cell entering S phase. Thus such cells are effectively frozen in the cell cycle and should be able to continue licensing their origins once the presence of geminin has been removed. This property of geminin has not previously been described and is unexpected in view of the action of geminin in transformed cells.

Thus geminin, or compounds mimicking its activity, should have the ability to specifically kill cancer cells. The replication licensing system is regulated in .such a way as to prevent re- replication of DNA in a single cell cycle (Blow and Laskey, 1988; Blow and Hodgson, 2002; Diffley, 2001; Lei and Tye, 2001) . It is activated in late mitosis and is turned off again in late Gl to ensure that replicated origins do not become re- licensed during S phase. The inactivation of licensing in S, G2 and M phases appears to be due to the high CDK levels present at these cell cycle stages. Therefore once cells have entered S phase with an insufficient number of licensed origins to complete replication, they will never be able to license the inactive origins.

Thus, while not wishing to be bound by any one particular theory, we believe that once cells such as U20S and Saos2 had arrested in S phase following geminin overexpression, subsequent removal of the geminin would not save them from death. In contrast, cells arresting in Gl with an insufficient number of licensed origins remain in a cell cycle state where they should, still be able to license new origins . Thus our results are consistent with the theory that after primary cells had arrested in Gl by geminin over-expression, subsequent removal of the geminin would allow them to finish licensing their origins before progressing into a normal S phase.

This differential effect of geminin is an attractive target for the development of cancer therapeutics. Compounds which are able to prevent licensing of replication origins in the same manner as geminin will be useful therapeutics because normal cells in a patient which are undergoing replication (e.g. cells of the immune system, keratinocytes, etc) will be unaffected after removal of geminin.

Thus in one aspect, the invention provides assay methods for screening compounds which are able to inhibit complete licensing of replication complexes such that untransformed cells are substantially prevented from entering S phase whereas transformed cells are able to do so.

Thus in a first aspect, the invention provides a method comprising: providing a candidate compound; providing a sample of transformed and a sample of untransformed cells; and determining whether said compound is capable of reducing the number of licensed replication origins in said cells present in the Gl phase.

The determination may be made directly on cellular DNA, for example by analysing the amount of one or more Mem 2-7 proteins bound to the DNA or indirectly by determining whether there is a reduction in the number of licensed origins such that transformed cells enter S phase whilst substantially preventing untransformed cells from entering S phase. Candidate compounds can be identified in a cell-free assay system we have previously developed for investigating the licensing reaction (Gillespie et al, 2001) . This system is useful in the identification of candidate compounds for screening in the first aspect of the invention. Thus the invention also provides a method comprising: providing a cell-free DNA template containing at least one eukaryotic origin of replication; providing a mixture of proteins capable of forming a licensing complex comprising ORC, Cdcβ, Cdtl and Mcm2-7, in the presence of ATP, providing a candidate compound whose ability to inhibit formation of a licensed origin is to be determined; determining whether the presence of said compound inhibits the formation of a licensing complex; and selecting a compound capable of so inhibiting the formation of the licensing complex as a candidate compound for the first aspect of the invention described above.

These and other aspects of the invention are described further herein below.

Description of the Drawings.

Figure 1. Inhibition of cell proliferation by geminin and rescue by Cdtl. a, U20S cells were transfected with empty pcDNA3 vector, pcDNA3 containing geminin or pcDNA3 containing a mutant form of geminin. Cells were grown under selection for 21 days and then stained with giemsa. b, U20S cells transfected with empty pcDNA3 or pcDNA3 expressing geminin or pcDNA3 expressing geminin plus pcDNA3 expressing Cdtl at a ratio of 1:1, 1:2 or 1:4. Cells were grown under selection for 21 days and then stained with giemsa. Figure 2. Expression of geminin by adenovirus vector, a, U20S cells were treated by mock infection or infection with Ad5GFP or Ad5GFPgeminin. Geminin expression was assessed by immunoblotting 48h and 72h later. Blots were also probed with actin antibodies as a loading control, b, U20S and Saos2 cells were infection with either Ad5GFP or Ad5GFP-geminin . After 72 hr, total cell extracts were prepared (T) and fractionated into a soluble supernatants (S) or chromatin-containing pellets (P) , and then immunoblotted for Mcm2.

Figure 3. Analysis of Ad5GFP-geminin infected U20S . U20S cells were infected with either Ad5GFP or Ad5GFP-geminin or were mock-infected, a, At different times, cell number was measured and expressed as the percentage change over mock-infected controls at time 0 h. Numbers represent the mean of three independent experiments, b, 96 h after infection cellular DNA was stained with propidium iodide and analysed by flow cytometry .

Figure 4. PCNA and BrdU staining of Ad5GFP-geminin infected U20S. U20S cells were infected with either Ad5GFP or Ad5GFP- geminin and were incubated for 96 h. a, Cells were fixed and stained for PCNA using a fluorescein labelled secondary antibody. DNA was stained with DAPI. b, Cells were pulsed for 30 min with BrdU and counterstained using a Texas Red labelled secondary antibody. DNA was stained with DAPI.

Figure 5. Cell cycle status of Ad5GFP-geminin infected U20S . U20S cells were infected with either Ad5GFP or Ad5GFP-geminin or were mock-infected, a, At 72 or 96 h (72 h for mock- infected cells) cell extracts were prepared and stained with antibodies against cyclin A, cyclin E, p53, Cipl/Wafl or actin. b, An in situ TUNEL assay was performed 9βh post infection. Total DNA was stained with DAPI. Figure 6. Cell cycle analysis in U20S cells infected with increasing MOl of geminin expressing adenovirus. U20S cells were infected with either Ad5GFP or Ad5GFP-geminin or were mock-infected, using the indicated MOl. 96 h after infection cellular DNA was stained with propidium iodide and analysed by flow cytometry.

Figure 7. Analysis of Ad5GFP-geminin infected Saos2. Saos2 cells were infected with either Ad5GFP or Ad5GFP-geminin or were mock-infected, a, At different times, cell number was measured and expressed as the percentage change over mock- infected controls at time 0 h. Numbers represent the mean of three independent experiments, b, 9β h after infection cellular DNA was stained with propidium iodide and analysed by flow cytometry. c, At 96 h, infected cells were pulsed for 30 min with BrdU and counterstained using a Texas Red labelled secondary antibody. DNA was stained with DAPI. d, At 72 or 96 h cell extracts were prepared and stained with antibodies against cyclin A or actin.

Figure 8. Analysis of Ad5GFP-geminin infected primary fibroblasts. IMR90 cells were infected with either Ad5GFP or Ad5GFP-geminin or were mock-infected, a, At different times, cell number was measured and expressed as the percentage change over mock-infected controls at time 0 h. Numbers represent the mean of three independent experiments, b, 72 h after infection cellular DNA was stained with propidium iodide and analysed by flow cytometry. C, At 72 h cells were fixed and PCNA was detected by indirect immunofluorescence with an anti-PCNA antibody and fluorescein-labelled secondary antibody. DNA was stained with DAPI. d, At 72 h cell extracts were prepared and immunoblotted with antibodies against cyclin E, cyclin A, p53, Cipl/Wafl or actin. Detailed Description of the Invention.

Candidate Compound

A candidate compound may be any compound under development for pharmaceutical use. Compounds which may be used may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants or microorganisms which contain several characterised or uncharacterised components may also be used. Generally such compounds will be organic molecules which are typically from about 100 to 2000, more preferably from about 100 to 1000 Da in molecular weight. Such compounds include peptides and derivatives thereof. Peptides may include peptide fragments of geminin, for example fragment of from 5 to 40 amino acids. In principle, any compound under development in the field of pharmacy can be used in the present invention in order to facilitate its development or to allow further rational drug design to improve its properties.

Transformed Cells

The transformed cells may be any animal, preferably vertebrate and more preferably mammalian, particularly human, cell which is capable of uncontrolled proliferation in culture. Many cell lines fulfilling this requirement are available in the art. These include cell lines derived from human or animal tumours. Such tumour cell lines include cells from bone, lung, colon, stomach, liver, pancreatic, kidney, breast, cervical, and testicular cancer cell lines. Examples of tumour cell lines available in the art include Saos2, U20S and HeLa cell lines. Untransformed Cells

Untransformed cells may likewise be any animal, preferably vertebrate and more preferably mammalian, particularly human, cell. The cells are desirably primary cells, e.g. fibroblasts obtained from human or animal donors, or cultures of cells such as IMR-90 cells which are widely used in the art.

Performing the Method

In conducting the method of the first aspect of the invention described above, the cells may be provided in culture dishes in accordance with conventional procedures in the art. The cells may be, for example, in wells of a 96-well microtitre plate or in larger wells or in single culture dishes. Typically between 10,000 and 1,000,000 cells may be plated, though the numbers may be varied according to the configuration of the method, the nature of the cells and routine experimental preferences of those of skill in the art.

The cells will be plated under normal laboratory conditions in culture media suitable to support the growth and replication of the cells in the absence of a candidate compound. The cells may be synchronous or unsynchronised. If the latter, the cells may be synchronised prior to plating, or synchronised once plated for example by using a thymidine block and mitotic arrest by nocodazole in accordance with procedures well known as such in the art. The cells will then be released from arrest and allowed to enter the cell cycle prior to or simultaneously with addition of the candidate compound.

Unsynchronised or pre-synchronised cells can be plated out into wells containing candidate compounds, or alternatively the cells may be plated in the well and the candidate compound may be added to the cells. The amount of compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about lpM to lmM or more concentrations of the candidate compound may be used, for example from 0.01 nM to 100 μM, e.g. 0.1 to 50 μM, such as about 10 μM. Greater concentrations may be used when a peptide is the test substance. Even a molecule with weak activity may be a useful lead compound for further investigation and development.

The candidate compound is added to the cells and incubated for a test period, typically between 4 and 96 hours. A series of time points may be used in conducting the method. For example, where multiple wells of cells have been plated, samples may be incubated at 12, 24, 48 and 96 hours.

The cells taken at each time point will then be analysed to determine whether the DNA is licensed and/or to what extent it is licensed.

To determine whether, or to what extent, the DNA is licensed, a number of possible methods are available. These methods are in themselves based on procedures and reagents known as such in the art and are further exemplified in the accompanying examples, though it will be understood by those of skill in the art that the precise reaction conditions illustrated below may be varied within the bounds of routine experimental practice.

In summary, the methods may comprise any of:

i) Isolating DNA-bound proteins, for example by breaking open the cells and recovering chromatin, then analysing the proteins for the presence of any one or more of the proteins Mem 2-7. Immunoblotting is one suitable way to perform this analysis .

ii) Immunostaining directly the cells to reveal one or more of DNA-bound Mem 2-7 using antibodies in the presence of a non-ionic detergent (e.g. NP-40 or Triton X-100) that washes away any Mem 2-7 not bound to chromatin.

iii) Isolating chromatin or nuclei and incubating in a eukaryotic cell-free system that is capable of initiating DNA replication only on licensed DNA templates (such as Xenopus egg extract supplemented with geminin) and assessing the initiation DNA replication by standard means such as measuring the incorporation of a labelled nucleotide (32PdATP or BrdUTP or biotin-dUTP) into DNA, or the loading of a replication protein (such as Cdc45, RPA or PCNA) onto the DNA.

iv) Measuring DNA synthesis (e.g. incorporation of 32P-dATP or BrdUTP or biotin-dUTP) . A compound which inhibits licensing will block synthesis of DNA in normal cells but, as illustrated herein, allow some synthesis in transformed cells. The DNA content of cells may be measured by flow cytometry methods (e.g. FACs analysis). This has the advantage of revealing whether cells are undergoing apoptosis, indicated by cells with a sub-GO content of DNA. The extent to which the transformed and untransformed cells undergo cell death can therefore be examined, particularly in the embodiment of the invention mentioned above where the cells are sampled at different time points. Thus at the first or one of the early time points the extent to which the cells are licensed may be determined. Following this, the fate of the cells may be examined, to determine whether, where a compound reduces licensing, the transformed^' cells proceed to die at a greater rate than untransformed cells. v) Measuring markers of replication initiation, e.g. PCNA, DNA polymerase or RPA loading onto DNA.

The assay may be performed in the presence of controls in which geminin is used in samples of transformed and/or untransformed cells, or both, in order to compare the effects of the candidate compound with that of geminin. The geminin may be supplied in a manner similar to that described in the accompanying examples, e.g. in the form of an expression vector such as an adenoviral expression vector.

Embodiments in which the extent to which Mem 2-7 proteins are present are preferred, as these indicate a direct effect of the compound on the formation of the replication complex.

It is not necessary to make a precise quantitation of the number or amount of replication licensing complexes formed, as a qualitative comparison between the transformed and untransformed cells may be made.

Usually, the method will be conducted such that the transformed and untransformed cells are grown and tested in parallel, though the assays may be performed sequentially. For example, it may be possible to determine first whether a compound is active in inhibiting licensing in transformed cells, and then further testing compounds with such activity in primary cells.

The method may be performed using automated or semi-automated procedures, for example by utilising 96- or 384- well plate readers, e.g. the Biotrak Visible Plate Reader (Amersham Biosciences, UK) or similar products. Cell-free Assay System

The present finding relating to licensing of DNA replication are also based upon our previous development of a cell-free assay system. The use of this system for determining the effect of a candidate compound on licensing of DNA replication as a selection method for candidate compounds thus forms a further aspect of the invention.

The assay may be performed using a naked DNA (such as plasmid DNA) or chromatin or nuclear templates which are not licensed. Plasmid DNA may be in circular or linear form containing DNA of eukaryotic origin, preferably of mammalian origin.

Unlicensed nuclei and chromatin may be prepared from cells (such as Xenopus sperm) in the GO or G2 phases of the cell cycle, or from cell-free extracts not competent to support replication licensing.

The DNA templates will then be incubated in cell-free extracts (such as Xenopus egg extracts) or in mixtures of proteins capable of licensing the chromatin, plus or minus the candidate inhibitor. After suitable incubation (typically 0.5 hours) , the degree of chromatin licensing will be assessed by any of the techniques described above.

The proteins added to the cell free extracts will include Mcm2-7, Cdcβ, ORC and Cdt-1. These proteins may be isolated from cells or produced recombinantly. MCM protein used in the system of the invention may be mammalian such as human MCM protein or mouse MCM protein, amphibian such as Xenopus MCM protein or other eukaryotic such as Saccharomyces cerevisiae

MCM protein.

Any one of MCM2, MCM3 , MCM4 , MCM5, MCM6 or MCM7 can be used either in isolation or in combination. Human MCM2 sequence is disclosed in Todorov et al . , 1994, J. Cell Sci . , 107, 253- 265, GenBank Ace. No. X67334. Human MCM3 sequence is disclosed in Thommes et al . , 1992, Nucl . Acid Res . , 20, 1069- 1074, GenBank Ace. No. P25205. Human MCM4 sequence is disclosed in Ishimi et al . , 1996, J. Biol . Chem . , 271, 24115- 24122, GenBank Ace. No. X74794. Human MCM5 sequence is disclosed in Hu et al . , 1993, Nucleic Acids Res . , 21, 5289- 5293, GenBank Ace. No. X74795. Human MCM6 sequence is disclosed in Holthoff et al . , 1996, Genomics, 37, 131-134, GenBank Ace. No. X67334. Human MCM7 sequence is disclosed in Hu et al . , - 1993, Nucleic Acids Res . , 21, 5289-5293. Alternative names of MCM family members together with references to the sequences are catalogued in Chong et al . , (1996) Trends Bioche . Sci. 21 102-106.

In particular examples of the system of the present invention, Xenopus Cdcβ (XCdcβ) was used. However, Cdc6 which may be used in the system of the present invention is not limited to Xenopus Cdcβ. Cdcβ proteins from any appropriate species e.g. mammalian such as human Cdcβ, amphibian such as Xenopus Cdcβ or other eukaryotic such as Saccharomyces cerevisiae Cdcβ.

Similarly, we have used Xenopus Cdt-1 obtained by recombinant expression, though other sources of this protein and means for its isolation or production are available in the art.

ORC protein may be isolated as described in the accompanying example or produced by recombinant means:

Geminin is described in McGarry and Kirschner, 1998, and is also Genbank reference NM_015895.

Reference may be made to Gillespie et al (2001) for guidance ^'as to the appropriate concentrations of the various components of the assay method, though the precise amounts and concentration will be dependent upon the precise experimental conditions and may be determined on a case by case basis by those of skill in the art. The amounts and concentrations will be selected to allow assembly of licensing complexes at the origins of replication of the DNA provided in the method, such that in the absence of a compound which inhibits licensing, the DNA will be capable of undergoing at least one cycle of replication.

Compounds obtained by the Methods .

A compound found to have the ability to affect licensing of DNA replication has therapeutic and other potential in a number of contexts. For therapeutic treatment such a compound may be used in combination with another active substance, e.g. for anti-tumour therapy with an anti-tumour compound or therapy, such as radiotherapy or chemotherapy. A compound having the ability to inhibit licensing of untransformed cells such that such cells do not attempt to exit Gl phase will allow anti-tumour compounds to be administered to a patient more effectively, e.g. at higher doses or for longer periods.

Thus following identification of a compound which affects licensing of DNA replication, the compound may be investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals, e.g. for any of the purposes discussed elsewhere herein.

In a further aspect, the present invention provides the use of a compound identified or obtained using the above methods of the invention in methods of designing or screening for mimetics of the substance.

Accordingly, the present invention provides a method of designing mimetics of a compound able to modulate licensing of DNA replication identified or obtained using the above- described methods, said method comprising:

(i) analysing the substance to determine groups responsible for the modulating activity to define a pharmacophore; and,

(ii) modelling the pharmacophore to design and/or screen candidate mimetics having the modulating activity.

The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a target property.

There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, -the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its "pharmacophore".

Once the pharmacophore has been found, its structure is modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.

A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.

The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.

Mimetics of this type together with their use in therapy form a further aspect of the invention.

Generally, such a compound or mimetic thereof according to the present invention is provided in an isolated and/or purified form, i.e. substantially pure. This may include being in a composition where it represents at least about 90% active ingredient, more preferably at least about 95%, more preferably at least about 98%. Such a composition may, however, include inert carrier materials or other pharmaceutically and physiologically acceptable excipients. A composition according to the present invention may include in addition to a compound as discussed above, one or more other molecules of therapeutic use, such as an anti-tumour agent. The present invention thus extends in various aspects not only to a substance identified as a modulator of licensing of DNA replication, in accordance with what is disclosed herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a substance, a method comprising administration of such a composition to a patient, e.g. for a use in a method of treatment of the human or animal body by therapy which affects DNA replication in cells, e.g. tumour cells. Other purposes of a method of treatment employing a substance in accordance with the present invention are discussed elsewhere herein.

Whether it is a polypeptide, antibody, peptide, nucleic acid molecule, small molecule, mimetic or other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors .

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

The invention is illustrated by the following example.

EXAMPLE.

In this example, we determine how human cells respond to inhibition of replication licensing - in particular whether they respond by entering a GO-like state, or whether they plough into S phase regardless. To specifically inhibit replication licensing we used geminin, a specific inhibitor of Cdtl (McGarry and Kirschner, 1998; Tada et al . , 2001; Wohlschlegel et al. , 2000). Over-expression of geminin in Drosophila leads to inhibition of DNA synthesis, increased numbers of metaphase cells and increased apoptosis (Quinn et al. , 2001) . This combination of features should be consistent with cells entering mitosis with unreplicated DNA and entering apoptosis as a consequence of being unable to correctly segregate the unreplicated chromosomes. Here, we show that different mammalian cells respond in different ways to geminin over-expression. U20S cells arrest in early S phase with down- regulated cyclin A and undergo apoptosis. Soas2 cells in contrast continue to synthesise DNA and appear to try to continue through the cell cycle in the presence of geminin. Most dramatically, primary cells arrest with unreplicated DNA and low levels of cyclin E, as though capable of sensing that they have insufficient licensed origins to complete S phase. The differential response of cells to geminin over-expression suggests that the replication licensing system is a promising new target for anti-cancer drugs.

Materials and methods

Cell lines and culture conditions . The human osteosarcoma cell lines U20S, Saos2, lung carcinoma H1299, breast carcinoma BT 549 and primary fibroblast IMR-90 were obtained from the American type culture collection. These cells were cultured in Dulbecco modified Eagle medium supplemented with 10% heat inactivated fetal calf serum and 0.5% gentamycin at 37 °C In 5% C02.

Adenoviral vectors and infection Xenopus laevis geminin H lacking the destruction box sequence (RRTLKVIQP) was cloned into the pAdTrack-CMV vector. Replication defective recombinant adenoviruses expressing either GFP as control (Ad5GFP) or GFP and geminin (Ad5GFP- gemlnln) were generated as previously reported (He et al., 1998) . For infections, lxlO⁶ cells were plated in 10 cm tissue culture plates. The following day the media was removed and cells were infected by adding the adenoviral vectors in 1 ml of DMEM at the Multiplicity of Infection (MOl) of 100 plaque forming units/ cell. Mock infection was performed by treatment of cells with vehicle (media) only. One hour after incubation at 37 °C, 9 ml of fresh medium was added. Cells were harvested at specific time points for analysis.

Western blot analysis

Cells were treated by mock infection or infection with Ad5GFP or Ad5GFP-geminin at the MOl of 100. Cells were harvested at selected time points and lysed in lysis buffer (50mM Tris-HCl, 150mM NaCl, 1%NP40, 0.5% sodium deoxycholate, 01%SDS, ImM EDTA, ImM PMSF, ImM Na3V04, ImM NaF and lug/ml each of aprotinin, pepstatin and leupeptin) for 15 minutes on ice. Cell lysates were centrifuged and protein concentration was determined by BIO-RAD protein assay kit (Bio-Rad laboratories GmbH, DE) . Equal amounts of cellular protein were electrophoresed in SDS-polyacryla ide gels and transferred to a Hybond-PVDF membrane (Amersham Corp., Arlington Heights IL) . The membrane was first blocked with 5% non-fat dried milk and then incubated with the following primary antibodies: mouse anti-human BM28, rabbit anti-human cyclin A (transduction Laboratories, Lexington, KY) mouse anti-human cyclin E

(PharMingen, San Diego, CA) rabbit anti-human p21 (C-19) , rabbit anti-human PARP (H250) (Santa Cruz Biotechnoloy, Santa Cruz, CA) mouse anti-human actin (Oncogene Research Products , Boston MA) and then with peroxidase-linked anti-mouse Immunoglobulin or anti-rabbit immunoglobulin antibody (Sigma Corp.). Enhanced chemiluminescence reagents were used to detect the signals according to the manufacturer's Instruction (Pierce, Rockford, IL) . For BM28 immunoblotting, cells were treated prior to lysis as described (Todorov et al . , 1995).

Cell proliferation assay

Cell proliferation was assessed at 24, 48, 72 and 96 h after infection by measuring the conversion of tetrazolium salt, MTT to formazan according to manufacturer's instruction (Promega Corp., Madison, WI) . Briefly cells were plated into 96 well plates and infected with the adenoviral vectors 24 h later. At each time point 15 μl of dye solution was added to each well and cultured at 37 °C for 4h. The reaction was stopped by the addition of 100 μl of stop solution for 1 h. The absorbance was read at 570nm using a 96 well plate reader (Dynex technologies) . The results were expressed as the percentage of the absorbance of control (uninfected) cells. Cell cycle analysis Both adherent and non-adherent cells were harvested, washed once with PBS and fixed in ice-cold 70% ethanol with vigorous mixing. Cells were pelleted washed once with PBS and resuspended in 1 ml of PBS containing 25 μg/ml propidium iodide and 0.1 mg/ml RNase for 30min in the dark at 30-37C. Flow cytometric analysis was performed on a FACScan Flow cytometer (Beckton Dickinson, San Jose, CA) . The data from 10,000 cells were collected for analysis. The sub-diploid population was calculated as an estimate of the apoptotic cell population.

Immuno fluorescence analysis 96 h following adenoviral infection indirect immuno- fluorescence of chromatin bound PCNA was done as described (Miura and Sasaki, 1999). For 5-bromo-2 ' -deoxyuridine (BrdU) staining, infected cells growing on a 2-well chamber slide (Nunc, Naperville, II) were pulsed with 10 μm BrdU for 30 min at 37c followed by formaldehyde fixation, permeabilisation with 1% NP40 and DNA, acid denatured. Cells were then stained with anti-BrdU antibody (Becton Dickinson) followed by Texas- red labelled donkey anti-mouse secondary antibody. TdT - mediated dUTP-x nick end labelling (TUNEL) assay to detect apoptotic cells was done using the in situ cell death detection kit, fluorescein (Roche diagnostics, East Sussex) according to the manufacturers protocol. Methanol fixation quenched GFP expression and did not interfere with fluorescein ^■ labelled secondary antibodies.

Trans fections and colony formation assay Transfections were done using the effectene reagent (Qiagen) according to manufacturer's protocol. For colony formation assays, 24 h post transfection with either empty pcDNA3, pcDNA3 expressing the destruction box deleted geminin or a mutant form of geminin lacking the C-terminus 126 amino acid residues (cl26) or XCdtl, cells were selected with lmg/ml G418 (Life technologies, GIBCO) over 3 weeks. Surviving cells were then stained with Giemsa.

Results

To establish whether geminin could block proliferation of human tissue culture cells, we transfected U20S and Saos2 cells with an expression vector containing a nondegradable form of geminin (McGarry and Kirschner, 1998), and investigated the ability of transfected cells to form colonies after a 3 week selection. As controls, cells were transfected with either empty vector or with vector containing a truncated version of geminin (gemininCl26) (McGarry and Kirschner, 1998) incapable of inhibiting DNA replication. Geminin expression significantly abolished the colony forming ability of both cell lines compared to controls (Fig. la) . To demonstrate that this effect of geminin was specific to inhibition of Cdtl , geminin was co-transfected with a Cdtl expressing plasmid at increasing concentrations. Cdtl efficiently rescued the inhibitory effect of geminin at a ratio of 1:4 (Fig.l b) . In order to demonstrate that geminin inhibits DNA replication we microinjected geminin protein into HeLa cells synchronized in metaphase, and assessed BrdU incorporation 12 hr after metaphase release. This caused a profound reduction in the number of BrdU-positive cells, consistent with geminin inhibiting the DNA replication by inhibition of Cdtl.

In order to examine the effect of geminin overexpression in a variety of different cell lines, we constructed an adenoviral delivery vector containing the geminin-DEL gene a mutant form of geminin resistant to cell cycle degradation (Ad5GFP- geminin) . As a control, the Ad5GFP vector lacking geminin was used. To estimate the efficiency of infection, cell lines U20S, Saos2, H1299 and BT 549 were infected with Ad5GFP using a multiplicity-of infection (MOl) of 100. Greater than 95% infection efficiency, as determined by GFP expression, was detected in these cell lines. No significant cytotoxic effect was noted at this viral concentration. Infection of U20S cells with Ad5GFP~geminin resulted in substantial overexpression of geminin at 48 and 72 h (Fig. 2a) . Similar levels were seen in all other cell lines. In contrast, the baseline expression of geminin was undetectable in mock-infected and Ad5GFP infected control groups .

Previous work has shown that geminin inhibits DNA replication by preventing the loading of the Mcm2-7 proteins onto chromatin (McGarry and Kirschner, 1998; Tada et al . , 2001; Wohlschlegel et al., 2000). We therefore infected U20S and Saos2 cells with either Ad5GFP or Ad5GFP-geminin and monitored the presence of Mcm2 on chromatin. Fig 2b shows that, as expected, Ad5GFP-geminin infection causes a strong reduction of chromatin-bound Mcm2.

Effect of geminin on U20S cells We next used the adenoviral expression system to characterise in detail the effect of geminin overexpression in U20S cells, an osteosarcoma-derived cell line that expresses both Rb and p53. Ad5GFP-geminin caused a cessation of cell proliferation and a reduction in cell number 72-96 hr after infection (Fig 3a) . Flow cytometry showed that the geminin expressing cells had a range of DNA contents between Gl and G2 levels, consistent with cells being unable to complete S phase (Fig 3b) . In addition, the geminin expressing cells .showed a marked sub-Gl population, consistent with geminin expression inducing apoptosis. In contrast, Ad5GFP infected cells demonstrated no significant abnormalities in cell cycle profile. To confirm that the geminin-expressing cells were arresting in S phase, cells were infected with Ad5GFP and Ad5GFP-geminin, and 96 hr later were either immunostained with anti-PCNA antibodies, or pulsed with BrdU and immunostained with anti-BrdU antibodies (Fig 4b) . The majority of the geminin-expressing cells stained strongly for PCNA, as expected if they were arrested in S phase. In contrast, the geminin expressing cells were incorporating only low levels of BrdU into DNA.

Cell lysates were immunoblotted for cyclin A and E (Fig 5a) . This showed that the geminin-expressing cells contained high levels of cyclin E, consistent with an S phase arrest. In contrast, cyclin A levels were low. Although cyclin A is normally expressed during S phase, its levels are frequently down-regulated in response to genotoxic stress (Knudsen et al., 1998; Guo et al . , 2000; Knudsen et al., 2000; Sever- Chroneos et al . , 2001), so this may indicate that the geminin- expressing cells had activated a cell stress checkpoint as a consequence of being unable to complete S phase. Consistent with this interpretation, the geminin-expressing cells contained high levels of p53 and Cipl/Wafl (Fig 5a) .

The flow cytometry profile (Fig 3b) showed a significant number of cells with a subGl DNA content indicative of cells undergoing apoptosis. We took several approaches to verify that geminin induced cell death was the result of apoptosis. First, cell morphology demonstrated typical changes characteristic of apoptotic cell death, including cell shrinkage, cytoplasmic blebbing, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies. These changes were not seen in mock infected or AdSCMV-GFP infected cells. Apoptosis is associated with inter-nucleosomal degradation of genomic DNA, which can be assessed by in si tu TUNEL staining. At 96 h after infection, Ad5GFP-geminin infected cells demonstrated abundant TUNEL staining compared to controls (Fig. 5b) . In addition, we detected the typical apoptotic cleavage product of poly ADP ribose polymerase in the geminin-expressing cells.

To investigate the relationship between S phase arrest and apoptosis, we varied the MOl of the Ad5GFP-geminin from 1 to 100, and then analysed the cellular DNA content by flow cytometry. The results (Fig. 6) showed that even at an MOl of 1, there was an increase in the number of early S phase cells as well as the appearance of apoptotic cells. As the MOl was increased, the proportion of cells in early S phase also increased, coupled with an increase in apoptotic cells. Taken together, these results suggest that as a consequence of forced geminin expression, U20S cells enter, but cannot complete, S phase. The inability to complete S phase triggers checkpoint pathways that lead to the down-regulation of cyclin A, the induction of Cipl/Wafl and the induction of apoptosis.

Effect of geminin overexpression in other cell lines We next investigated whether the response of U20S cells to geminin over expression is typical of other cell lines, or whether significant differences exist. H1299 cells, a lung cancer-derived cell line, responded to Ad5GFP-geminin infection in a similar way to U20S. H1299 cells arrested with a Gl/S DNA content and high Cipl/Wafl levels, with a significant proportion of apoptotic cells being present. Since H1299 are p53 null, this shows that p53 is not necessary for the induction of apoptosis following geminin overexpression. However, when Ad5GFP-geminin infections were repeated with Saos2 cells, somewhat different results were obtained. U20S and Saos2 are both osteosarcomaderived cell lines, but unlike U20S, Saos2 contains neither p53 or Rb. Ad5GFP-geminin infection caused a slower decline in Saos2 cell numbers (Fig 7a) than was seen in U20S cells (Fig 3a) , despite a similar reduction in chromatin bound Mcm2 (Fig 2b) . Flow cytometry of Ad5GFP-geminin infected Saos2 cells showed a slight increase in cells in late S and ^• G2 (Fig 7b) instead of an accumulation of early S phase cells as was seen with U20S. A lack of clear S phase arrest was demonstrated by BrdU staining, which revealed high levels of DNA synthesis occurring in a significant number of cells (Fig 7c) . Consistent with this, the geminin-expressing Saos2 cells had low but detectable levels of cyclin A (Fig 7d) . However a significant proportion of the Ad5GFP-geminin infected Saos2 cells had a sub-Gl DNA content (Fig 7b) . TUNEL staining confirmed that these were apoptotic cells. Similar results were obtained with BT549 breast cancer-derived cells that are also p53 and Rb null. This suggests that Saos2 and BT549 cells have lost the ability to down-regulate cyclin A and undergo the early S phase arrest seen in U20S and H1299 cells.

Given the differences in the response to geminin shown by U20S and- Saos2 cells, we next examined how primary fibroblasts responded to ge inin-overexpression. Unlike the established cell lines, Ad5GFP-geminin caused the primary fibroblasts to stop proliferating but did not significantly reduce total cell number. Flow cytometry showed that geminin expression in primary cells led to an accumulation of cells with a Gl DNA content, with few cells in S phase, and no sign of apoptosis (Fig 8a) . The Ad5GFP-geminin infected cells contained low levels of both cyclin E and A, consistent with them arresting prior to entry into S phase (Fig 8b) . Further, virtually no immunostaining with anti-PCNA antibodies was seen in the geminin-expressing primary cells showing that they had not initiated DNA synthesis (Fig 8c) . However, the geminin- expressing cells showed a modest induction of Cipl/Wafl , which may be one cause of the cell cycle arrest.

Staining for Mcm2 protein . Asynchronously growing U20S cells were infected at 100 MOl with adenovirus expressing geminin or GFP, or were mock infected. 72 hr post-infection cells were treated with buffer containing 0.5% Triton X-100 then fixed and stained for chromatin-bound Mcm2 using an Mcm2 antibody, BM28. Cells were also stained with DAPI to show total DNA.

It was observed that the presence of geminin in the cells completely obliterated any signal from the BM28 antibody, showing that chromatin-bound Mcm2 was absent from the cells. Thus compounds which mimic this effect of geminin in transformed cells will be valuable in the provision of anti- cancer therapies.

Summary of Example

We have used geminin to inhibit replication licensing in a variety of different cell types. This has revealed that different cell types respond to geminin over-expression in at least 3 different ways. Analysis of these results suggest that compounds mimicking the action of geminin could make highly specific anti-cancer agents.

Requirement for Cdtl and Mcm2-1 in human cell prolifera tion

Work from yeast and Xenopus has shown that Mem 2-7 play an essential role in DNA replication, possibly by providing the helicase activity that opens up single-stranded DNA ahead of the replication fork (Labib and Diffley, 2001) . The loading of Mcm2-7 onto origin DNA, which results in the origin being "licensed" for replication, also depends on ORC, Cdc6 and Cdtl proteins (Blow and Hodgson, 2002; Diffley, 2001; Lei and Tye, 2001) . We have used geminin, a specific inhibitor of Cdtl (McGarry and Kirschner, 1998; Tada et al . , 2001; Wohlschlegel et al., 2000), to inhibit Mcm2-7 loading and origin licensing in human cells . Since geminin is normally unstable during late mitosis and early Gl (McGarry and Kirschner, 1998) , we used a non-degradable mutant of geminin to stably inhibit Cdtl throughout the cell cycle. Introduction of non-degradable geminin into human cells, by transfection, microinjection or adenovirus infection efficiently blocked cell proliferation. The inhibition was reversed by overexpression of Cdtl and was not seen with a deletion mutant incapable of inhibiting Cdtl, suggesting that inhibition was mediated by inhibition of origin licensing.

Precisely how inhibition of origin licensing blocks cell proliferation is much more complex. The response of Saos2 and BT549 cells raises a number of important questions. Although infection with Ad5GFP-geminin decreased the quantity of chromatin-bound Mcm2-7 at least 5-fold, Saos2 cells could still synthesize DNA at approximately normal rates as judged by BrdU incorporation. Despite this, geminin still induced apoptosis in the Saos2 cells. A likely explanation for this effect concerns the number of Mcm2-7 complexes present at each replication origin. Previous work in yeast (Lei et al., 1996), humans (Burkhart et al., 1995) and Xenopus (Mahbubani et al . , 1997) has estimated that more than 10 copies of the Mcm2-7 complex can be loaded onto each replication origin. Experiments in Xenopus showed that when the levels of Mcm2-7 were lowered to about 1 copy per origin, overall replication rates were still maximal; only when Mcm2- 7 was present at less than these levels was the bulk DNA replication rate lowered (Mahbubani et al., 1997). Studies in yeast, however, have shown that defects in the Mcm2-7 proteins affect different replication origins to different degrees (Maine et al., 1984), and halving the dosage of Mcm2 can dramatically decrease the replication from specific origins (Lei et al., 1996) . Therefore in response to lowered Mcm2-7, bulk DNA synthesis may be occurring at almost normal rates whilst certain chromosomal origins are inactivated.

Eukaryotes appear to use many more replication origins than would appear necessary to replicate their entire genomes. A likely explanation for the large number of origins is that any individual replication fork may irreversibly stall, perhaps as a consequence of encountering DNA damage. So long as only one fork stalls between two adjacent origins, all the intervening DNA can be replicated by the other replication fork; however, if both of the forks irreversibly stall, the intervening DNA can never be replicated. The larger the distance between adjacent origins, the more frequently this type of double- stall will occur. Since overexpression of geminin is expected to reduce the number of licensed replication origins, it will increase the probability that such double-stalls occur, and is likely to lead to the sort of cell cycle failure seen in Saos2 cells. It -is likely that these stalled forks could themselves trigger an apoptotic response. Alternatively, apoptosis could be triggered as a consequence of the cells trying to undergo mitosis in the presence of unreplicated DNA, as appears to occur when geminin is over-expressed in Drosophila (Quinn et al., 2001) . Checkpoint-like responses to geminin overexpression Unlike Saos2 and H1299 cells, most U20S and H1299 cells infected with Ad5GFPgeminin arrested with most of their DNA unreplicated. Although PCNA was present in foci, only low levels of BrdU incorporation were seen. This would be consistent with replication forks having stalled in the process of replication, but not having disassembled. In yeast, DNA damage or replication fork inhibition in early S phase suppresses the ability of late firing replication origins to initiate. The suppression of late origin firing is dependent on the checkpoint kinases Mecl and Rad53 (Santocanale and Diffley, 1998; Shirahige et al., 1998). Previous work has shown that in mammalian cells, Rb is required for- both Gl/S and intra-S arrest in response to genotoxic stress (Chew et al., 1998; Harrington et^'al., 1998; Knudsen et al., 1998;

Knudsen et al., 2000; Saudan et al . , 2000). Activation of this Rb-dependent checkpoint is associated with a down-regulation of cyclin A (Knudsen et al., 1998; Guo et al . , 2000; Knudsen et al., 2000; Sever-Chroneos et at., 2001). The response of U20S and H1299 cells to geminin expression suggests that this intra-S checkpoint has been activated. Both cell lines arrest in S phase with high cyclin E level but low cyclin A. U20S and Saos2 are both osteosarcoma-derived cell lines, but Saos2 lacks Rb. Similarly, H1299 and BT549 are both cancer-derived cell lines, but BT549 lacks Rb. The response of these cells to geminin over-expression is therefore consistent with the idea that an early S-phase arrest is dependent on Rb-dependent inhibition of late origin firing. In contrast, p53 is unnecessary because whilst U20S contains wild-type p53, H1299 are p53 null.

The response of primary fibroblasts to geminin overexpression is even more striking. Although geminin blocked cell proliferation and reduced the levels of Mcm2 levels on chromatin, there was no sign of these cells entering S phase. Flow cytometry showed a sharp Gl peak whilst no PCNA was associated with the chromatin. Most strikingly, both cyclin E and cyclin A were extremely low in the geminin expressing cells. These results suggest that primary cells expressing geminin arrest before entry into S phase. p53 and Cipl/Wafl levels were both moderately elevated, and could potentially mediate the Gl arrest. In contrast to the established cell lines, however, there is no simple explanation for the trigger that brings about the arrest. It is unlikely that it is triggered by DNA damage, since there is no reason to think that geminin will damage DNA if replication forks are not initiated. Another possibility is that geminin itself directly blocks entry into S phase. However, this also seems unlikely because geminin is normally expressed in unperturbed S phases (McGarry and Kirschner, 1998; Wohlschlegel et al., 2000; Nishitani et al . , 2001). The most likely explanation at present is that primary cells can respond directly to the absence of licensed replication origins by blocking entry into S phase. This makes teleological sense since it would cause arrest before there is the danger of DNA becoming damaged as a consequence of trying to replicate DNA in the absence of sufficient licensed origins. When cells withdraw into GO, their replication origins become unlicensed, and we have suggested that this can provide a functional definition of the GO state (Blow and Hodgson, 2002) . The ability to judge whether origins have become re-licensed may be important for successfully re-entering the cell cycle from the GO state. Ad5GFP-geminin infected primary cells may therefore be behaving like cells early in the process of GO exit. References

Aparicio OM, Weinstein DM, Bell SP: Components and dynamics of DNA replication complexes in S-cerevisiae: Redistribution of MCM proteins and Cdc45p during S phase. Cell 1997, 91:59-69

Blow, J.J., and B. Hodgson. 2002. Replication Licensing - defining the proliferative state? Trends Cell Biol . 12:72-78.

Blow, J.J., and R.A. Laskey. 1988. A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature . 332:546-548.

Burkhart, R., D. Schulte, D. Hu, C. Musahl, F. Gohring, and R. Knippers. 1995. Interactions of human nuclear proteins PI Mcm3 and PI Cdc46. Eur. J. Biochem . 228:431-438. Chew, Y.P., M. E/lis, S. Wilkie, and S. Mittnacht. 1998. pRB phosphorylation mutants reveal role of pRB in regulating S phase completion by a mechanism independent of E2F. Oncogene . 17:2177-2186.

Diffley, J.F., J.H. Cocker, S.J. Dowell, and A. Rowley. 1994. Two steps in the assembly of complexes at yeast replication origins in vivo. Cell . 78 : 303-316.

Diffley, J.F.X. 2001. Building the perfect switch. Current Biol . 11 :R367-370.

Donovan, S., J. Harwood, L.S. Drury, and J.F. Diffley. 1997. Cdcβp-dependent loading of Mem proteins onto pre- replicative chromatin in budding yeast. Proc . Natl . Acad. Sci . U. S . A. 94 : 5611 -561 6.

Gillespie, P.J., A. Li, and J.J. Blow. 2001. Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins. BioMed Central Biochem . 2 : 15.

Guo, N., D.V. Faller, and C. Vaziri. 2000. A novel DNA damage checkpoint involving posttranscriptional regulation of cyclin A expression. J. Biol . Chem . 275: 1715-1722. Harrington, E.A., J.L. Bruce, E. Harlow, and N. Dyson. 1998. pRB plays an essential role in cell cycle arrest induced by DNA damage. Proc . Na tl . Acad. Sci . U. S . A . 95:11945- 11950. He, T.C., S. Zhou, L. T. da Costa, J. Vu, K.W. Kinzler, and B. Vogelstein. 1998. A simplified system for generating recombinant adenoviruses . Proc . Na tl . Acad. Sci . U. S . A. 95:2509-2514.

Hua, X.H., and J. Newport. 1998. Identification of a preinitiation step in DNA ^'replication that is independent of origin recognition complex and cdcδ, but dependent on cdk2. J. Cell Biol . 140:271-281.

Knudsen, E.S., C. Buckmaster, T.T. Chen, J.R. Feramisco, and J.V. Wang. 1998. Inhibition of DNA synthesis by RB: effects on Gl/S transition and S-phase progression. Genes Dev. 12:2278-2292.

Knudsen, K.E., D. Booth, S. Naderi, Z. Sever-Chroneos, A.F. Fribourg, I.C. Hunton, J.R. Feramisco, J.V. Wang, and E.S. Knudsen. 2000. RB-dependent S-phase response to DNA damage. Mol . Cell . Biol . 20:7751-7763.

Labib, K., and J.F. Diffley. 2001. Is the MCM2-7 complex the eukaryotic DNA replication fork helicase? Curr. Opin . Genet . Dev. 11 : 64-70.

Labib K, Tercero JA, Diffley JFX: Uninterrupted MCM2-7 function required for DNA replication fork progression. Science 2000, 288:1643-1647.

Labib, K., S.E. Kearsey, and J.F. Diffley. 2001. MCM2-7 proteins are essential components of prereplicative complexes that accumulate cooperatively in the nucleus during Gl-phase and are required to establish, but not maintain, the S phase checkpoint. Mol . , Biol . Cell . 12:3658-3667.

Lei, M., V. Kawasaki, and B.K. Tye. 1996. Physical interactions among Mem proteins and effects of M cm dosage on DNA replication in Saccharomyces cerevisiae. Mol . Cell . Biol . 16:5081-5090. Lei, M., and B.K. Tye. 2001. Initiating DNA synthesis: from recruiting to activating the MCM complex. J. Cell Sci . 114:1447-1454.

Mahbubani, H.M., J.P. Chong, S. Chevalier, P. Thommes, and J.J. Blow. 1997. Cell cycle regulation of the replication licensing system: involvement of a Cdk-dependent inhibitor. J. Cell Biol . 136:125-135.

Maine, G.T., P. Sinha, and B.K. Tye. 1984. Mutants of S. cerevisiae defective in the maintenance of minichromosomes . Genetics . 106:365-385.

Maiorano, D., J. Moreau, and M. Mechali. 2000. XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis. Na ture . 404:622-625.

McGarry, T.J., and M.W. Kirschner. 1998. Geminin, an inhibitor of DNA replication, is degraded during mitosis. Cell . 93 : 1043-1053.

Miura, M. , and T. Sasaki. 1999. Detection of chromatin-bound

PCNA in cultured cells following exposure to DNA-damaging agents. Methods Mol . Biol . 113:577-582. Musahl, C, H.P. Holthoff, R. Lesch, and R. Knippers . 1998. Stability of the replicative Mcm3 protein in proliferating and differentiating human cells. Exp . Cell Res . 241:260-264.

Nishitani, H., S. Taraviras, Z. Lygerou, and T. Nishimoto. 2001. The human licensing factor for DNA replication Cdtl accumulates in Gl and is destabilized after initiation of S phase. J. Biol . Chem . 276:44905-44911.

Quinn, L.M., A. Herr, T.J. McGarry, and H. Richardson. 2001. The Drosophila Geminin homolog: roles for Geminin in limiting DNA replication, in anaphase and in neurogenesis . Genes Dev. 15: 2741-2754.

Rowles, A., S. Tada, and J.J. Blow. 1999. Changes in association of the Xenopus origin recognition complex with chromatin on licensing of replication origins. J. Cell Sci . 112:2011-2018. Santocanale C, and J.F.X. Diffley. 1998. A Mecl- and Rad53- dependent checkpoint controls late-firing origins of DNA replication. Na ture . 395:615-618.

Saudan, P., J. Vlach, and P. Beard. 2000. Inhibition of S- phase progression by adenoassociated virus Rep78 protein is mediated by hypophosphorylated pRb. EMB0 J. 19:4351- 4361.

Sever-Chroneos, Z., S.P. Angus, A.F. Fribourg, H. Wan, I. Todorov, K.E. Knudsen, and E.S. Knudsen. 2001. Retinoblastoma tumor suppressor protein signals through inhibition of cyclin-dependent kinase 2 activity to disrupt PCNA function in S phase. Mol . Cell . Biol . 21 .^•4032-4045.

Shirahige, K. , Y. Hori, K. Shiraishi, M. Yamashita, K. Takahashi, C. Obuse, T. Tsurimoto, and H. Yoshikawa.

1998. Regulation of DNA-replication origins during cell- cycle progression. Na ture . 395:618-621.

Stoeber, K. , Halsall, A. Freeman, R. Swinn, A. Doble, L.

Morris, N. Coleman, N. Bullock, R.A. Laskey, CN. Hales, and G.H. Williams. 1999. Immunoassay for urothelial cancers that detects DNA replication protein Mcm5 in urine. Lancet . 354: 15241525.

Stoeber, K., A.D. Mills, Y. Kubota, T. Krude, P. Romanowski,

K. Marheineke, R.A. Laskey, and G.H. Williams. 1998. Cdc6 protein causes premature entry into S phase in a mammalian cell-free system. EMBO J. 11 : 1219-1229 .

Stoeber, K., T.D. Tisty, L. Happerfield, G.A. Thomas, S.

Romanov, L. Bobrow, E.D. Williams, and G.H. Williams. 2001. DNA replication licensing and human cell proliferation. J. Cell Sci . 114:2027-2041.

Sun, W., M. Holat K. Pedley, S. Tada, J.J. Blow, T. Todorov, S.E. Kearsey, and R.F. Brooks. 2000. The replication capacity of intact mammalian nuclei in Xenopus egg extracts declines with quiescence, but the residual DNA synthesis is independent of Xenopus MCM proteins. J. Cell Sci . 113:683-695. Tada, S., A. Lit D. Maiorano, M. Mechali, and J.J. Blow. 2001. Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdtl by geminin. Na ture .Cell Biol .2 : 107-113. Tan, D.-F., J.A. Hubermant A. Hyland, G.M. Loewen, J.S.J. Brooks, A.F. Beck, I T. Todorov, and G. Bepler. 2001. MCM2 - a promising marker for premalignant lesions of the lung: a cohort study. Biol Med Central Cancer. 1 :6.

Todorov, I.T., A. Attaran, and S.E. Kearsey. 1995. BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication. J. Cell Biol . 129:1433- 1445.

Tsuruga, H., N. Yabuta, K. Hashizume, M. Ikeda, Y. Endo, and H. Nojima. 1997. Expression, nuclear localization and interactions of human MCM/P1 proteins. Biochem . Biophys . Res . Commun . 236:118-125.

Williams, G.H., P. Romanowski, L. Morris, M. Madine, A.D.

Mills, K. Stoeber, J. Marr, R.A. Laskey, and N. Coleman. 1998. Improved cervical smear assessment using antibodies against proteins that regulate DNA replication. Proc . Natl . Acad. Sci . U. S . A. 95:14932-14937.

Wohlschlegel, J.A., B.T. Dwyer, S.K. Dhar, C. Cvetic, J.C. Walter, and A. Dutta. 2000. Inhibition of eukaryotic replication by geminin binding to Cdtl. Science . 290:23092312.

Claims

Claims

1. A method comprising: providing a candidate compound; providing a sample of transformed and a sample of untransformed cells; and determining whether said compound is capable of reducing the number of licensed replication origins in said cells present in the Gl phase.

2. The method of claim 1 wherein said determining is performed by analysing the amount of one or more of Mem 2-7 proteins bound to the DNA in said cells.

3. The method of claim 1 wherein the determining is done by examining the ability of the transformed and untransformed cells to enter S phase.

4. The method of claim 1 which further comprises determining the extent to which cell death occurs in a sample of the transformed and untransformed cells.

5. The method of any one of the preceding claims wherein a control sample of transformed or untransformed cells to which geminin is added is provided.

6. The method of any one of claims 1 to 5 wherein the candidate compound is identified in a cell-free assay method comprising: providing a cell-free DNA template containing at least one eukaryotic origin of replication; providing a mixture of proteins capable of forming a licensing complex comprising ORC, Cdc6, Cdtl and Mcm2-7, in the presence of ATP, providing a candidate compound whose ability to inhibit formation of a licensed origin is to be determined; and determining whether the presence of said compound inhibits the formation of a licensing complex.

7. The method of any one of the preceding claims wherein said candidate compound is a peptide fragment of geminin.