CA2224940A1 - Interaction of p53 with transcription factor dp-1 - Google Patents

Abstract

Complexes of p53 with the transcription factors DP-1 (and other members of the DP family) or E2F-1 (or other E2F proteins, up to and including E2F-5), are disclosed. These novel complexes can be used to assay for potential chemotherapeutic agents that are p53 agonists or antagonists, particularly agonists or antagonists which modulate progression through the cell cycle by disruption of the normal binding of p53 to DP-1 and/or E2F-1.

Description

INTERACTION OF pS3 WITH TRANSCRIPTION FACTOR DP-1 This invention relates to complexes of the protein p53, which is a transc}iption modulator, with DP
proteins (e.g. DP-1) or E2F proteins (e.g. E2F-1). In particular, the invention relates to use of these complexes in assays for potential therapeutic agents.

The cellular transcription factor DRTFI/E2F and the ~umour suppressor protein pS3 play important roles in controlling early cell cycle events. DRTF1/E2F is believed to co-ordinate and integrate the transcription of cell cycle regulating genes, for example those involved in DNA synthesis. with the activity of regulatory proteins, such as the retinoblastoma tumour suppressor gene product (pRb), which modulate its transcriptionai activity. In contrast, p53 is thought to monitor the integrity of 10 chromosomal DNA and when appropriate interfere with cell cvcle progression, for example, in response to DNA damage. Generic DRTFI/E2F DNA bindin_ activity and ~ldns~ Lional activation arise when a member of two distinct families of proteins interact as DP/E2F heterodimers, such as DP-I and E2F-1. In many cell-types DP-I is a widespread component of DRTF1/E2F DNA binding activity which when expressed at high levels oncogenically transforms embryonic fibroblasts.

15 The transition from Gl into S phase is an important regulatory point in cell cycle progression since that is when a number of genes need to be transcriptionally acIivated in order that cells may continue through the cell cycle. Many of these genes contain within their control sequences binding sites for the cellular transcription factor DRTF1/E2F which is widely believed to play an important role in regulating transcription during early cell cycle progression (La Thangue, 1994).
20 The potential role of DRTF1/E~F in cell cycle control is underscored by the properties of the proteins which are known to influence its transcriptional activity. For example, a group of proteins which negatively regulate the cell cycle, including the retinoblastoma lumour suppressor protein (pRb) and its relatives plO7 and pl30 (collectively known as pocket proteins) bind to and inactivate the transcriptional activity of DRTF1/E2F (Bandara and La Thangue. 1991; Chellappan et a/., 1991;
25 Schwarz et a/., 1993: Cobrinik et a/., 1993). These interactions can be de-regulated in tumour cells, for example, through the action of viral oncoplotehls, such as adenovirus Ela (Nevins, 1992), and furthermore are known to be temporally influenced by cell cycle progression (Shirodkar et a/., 1992;
Cobrinik et a/.. 1993). Another group of molecules, the cyclins and their catalytic regulatory subunits, which regulate cell cycle transitions, interact with DRTF1/E2F (Bandara et a/., 1991;
30 Mudryj et a/., 1991; Devoto et a/., 1992; Lees et a/., 1992). Cyclins A, E and D together with an aL~ u~liate catalytic subunit are believed to infln~n- e the activity of pocket proteins (Hinds et al., 1992; Sherr et a/., 1993), and direct phosphorylation by a cyclin A-cdk2 kinase reduces the DNA

binding activity of DRTF1/E2F (Dynlacht et al., 1994; Krek el al., 1994). Overall, the nature of the targel genes together with the physiological properties of the afferent ~ign~11ing proteins suggest that the activity of DRTF1/E2F plays a pivotal role in regulating and co-Oldillalillg early cell cycle progression.

5 It is now known that generic DRTFI/E2F DNA binding activity defined in m~mm~ n cell extracts results from an array of heterodimers made up from two distinct families of proteins, E2F and DP
(E2F/DP heterodimers constituting physiological DRTF1/E2F). To date five members of the E2F
family have been defined, from E2F-1 to E2F-5 which, in the context of an E2F/DP heterodimer, dictate the interacting pocket prorein (Helin et al., 1992; Ivey-Hoyle et al., 1993; Kaelin et al., 1992;
10 Shan et al., 1992; Lees et al.. 1993; Beijersbergen et al., 1994; Ginsberg et al., 1994; Sardet et al., 1995; Buck et al., 1995; Hijmans et al., 1995). The proteins E2F~ and E2F-5 are the subject of International Patent Application Nos. PCT/GB95/00868 and PCT/GB95/00869, respectively. Three members of the DP family are known to exist namely DP-I, DP-2 and DP-3 (Girling et al., 1993 and 1994; Ormondroyd et al., 1995~. From these, DP-1 is the most widespread member of DRTF1/E2F
15 yet defined (Bandara et al., 1993 and 1994) being for example, in all the various forms of DRTFl/E2F which occur during the cell cycle in 3T3 cells (Bandara et al., 1994).
Both the E2F and DP proteins are endowed with growth-promoting activity since in a variety of assays they have been shown to possess proto-oncogenic activity (Singh et al., 1994; Ginsberg et al., 1994;
Johnson et al., 1994, Xu et al.. 1995; Jooss et al., 1995). For example, over-expression of DP-1 or 20 DP-2 together with activated Ha-ras causes transformation of rat embryo fibroblasts which, hlLelc~lillgly, is d~JdlcllL in the absence of a co-transfected E2F family member (Jooss et al., 1995).

Martin et al., 1995, report that the proto-oncogene MDM2 is able to form complexes with the E2F
family members E2Fl and DP-I. This interaction thus stimnl~tP~ S-phase progression of the cell cycle. The MDM2 gene product is believed to downmodulate p53 function and the downmodulation 25 of this tumour suprssor protein is believed to play a part in a number of tumour types where over-expression of MDM2 is found. Xiao et al., 1995 show an interaction between MDM2 and pRB.

In this application it is demonstrated that DP-l exists in at least two distinct forms which differ in DNA binding properties. Further, we have found that p53 interacts with DP-l. Functionally, p53 regulates lldlls.,lil,Lion driven by the DP- 1/E2F-1 heterodimer by repression. Thus DP-1 is a c~
30 cellular target in two distinct pdLllwdy~ of growth control mP~ tPd through the activities of the pRb and p53 tumour ~u~lessor proteins. Moreover, the integration of p53 and MDM2 with DP-1 defines a potential pd~ dy through which p53 and MDM2 can inflllPn-~e cell cycle progression.

W O 97/02354 PCT/GB96/OlS60 Thus the functional con~equen~s of the interaction of p53 with a DP-1/E2F heterodimer is inactivation of transcription driven by the E2F binding site whereas, in contrast, the MDM2 protein activates DP-l/E2F-dependent transcription. Thus DP-l appears to be a comml n cellular target in two distinct pdLllwdys of growth control, and these pathways, regulated bv the pRb and p53 tumour 5 :,u~ ssor proteins, now appear to be functionally integrated.

The invention is thus based on ~he surprising finding that p53 is able to interac~ with DPl, and thereby modulate the transactivation activity of the DP/E2F complexes.

Thus the invention in a first aspect provides a complex (preferably in a suhst~nti~lly isolated form) which comprises p53 and a DP or E2F protein.

10 The term "DP protein" is intended to encompass not only DP-l but additionally DP-2 and DP-3 and related proteins of similar activity. By the term "E2F protein" it is intenried to encompass the five known members of the E2F family, E2F-I to E2F-5 inclusive, as well as related proteins of similar activity. However DP-1 protein of m:~mm~ n preferably human or murine origin, is preferred.

These both additionally include mutants. allelic variants, fusions (with another protein) or species 15 homologues of the naturally occurring proteins. They additionally include proteins that are at least 70% homologous to the naturally occurring protein, where that homologous protein is able to form a complex with p53.

Thus the expression "DP protein" comprises:
(a) a DP protein. such as DP-I, DP-2 or DP-3;
(b) a mutant. allelic variant or species homologue of (a);
(c) a protein at least 70% homologous to (a) or (b);
(d) a fragment of any of (a) to (c) capable of forming a complex with p 53 or MDM2;
(e) a fragment of any of (a) to (d) of at least 18 amino acids long; or (f~ a fusion protein Cul~ illg a protein or defined in any of (a) to (e) fused to another (e.g. heterologous) protein.

The expression "E2F protein" is to be construed likewise although of course in (a) above one would instead insert the E2F proteins, E2F-1 to E2F-5, inclusive. ~mm~ n preferably human or murine, E2F-l is preferred.

Mutants will possess one or more mutations which are additions, deletions, or 5~hsrin-tions of amino acids residues. Preferably these mutations will not affect, or not ~ul.~ lly affect, the structure and/or function and or properties of the protein. Mutants will generally still possess the ability to be able to complex with p53. as the context requires. Mutants can either be naturally occurring (that is 5 to say, purified or isolated from a natural source) or synthetic (for example, by perforrning a site-directed mutagenesis on the encoding DNA). It will be a~al~ that the proteins used in the complexes of the invention can either be naturally occurring or reco-lll,h~dll~.
The term "p53" likewise includes fragments. mutants. allelic variants and species homologues etc. in the same manner as described and defined for the DP and E2F proteins. Particular pS3 mutants 10 include those p53 mutants which are found in tumours, for example substitutions at Rl75, G245, R248,R249, R273,R28~ and also mutants in the region lO0-lS0.

For simplicity, p53 proteins land their mutants, allelic variants and species homologues etc) will be referred to as tldnscli,vtional modulators. Proteins that fall within the terms "DP protein" and "E2F
protein" are referred to as transcription factors. Thus, in complexes of the invention, the 15 transcription mo~ tQr is (e.g. re~ersibly) bound to the transcription factor. Proteins unable to bind, or to complex with, one of the transcription modulator or transcription factor will not be inr~ d in complexes of the invention.

We have determined that DP- l exists in two forms defined using immunochemical reagents. The two forms may bè different by virtue of the degree of phosphorylation. p53 binds pl~l~lenLially to the 20 form and the resulting complex appears to contribute to the growth inactivating effects of DP-l.

It is possible to determine whether a transcription modulator or transcription factor will form a complex with the other by providing the two proteins and determining whether or not a complex has formed, for example by determining the molecular weight of the complex by methods such as SDS-PAGE or more preferably by using a tag or label to detect a fusion protein forming part of the 25 complex.

A complex of the invention will be in snbsr~nti~lly isolated form if it is in a form in which it is free of other polypeptides with which it may be associated in its natural em~i.o~ llL (eg the body). It will be understood that the complex may be mixed with carriers or diluents which will not interfere with the inten~lPd purpose of the complex and yet still be regarded as ~b~;."li~lly isolated.

CA 02224940 l997-l2-l7 The complex of the invention may also be in a s~-bst:~n~i~lly purified form, in which case it will generally comprise the complex in a ~ a.dLion in which more than 90%, eg. 95%, 98% or 99% of the proteins in the plcl)a.dlion is a con~timted by a complex of the invention.

An allelic variant will be a variant which will occur naturally in the same animal and which will 5 function in a ~ u~ lly similar manner to the proteins described herein.

Similarly, a species homologue will be the equivalent protein which occurs naturally in another species, and which performs the equivalent function in that species to the protein described herein.
Within any one species, a homologue may exist as several allelic variants, and these will all be considered homologues.

10 Proteins at least 70% homologous to the naturally occurring protein are also contemplated for use in the complexes of the invention, as are proteins at least 80 or 90% and more preferably at least 95%
homologous. This will generally be over a region of at least 20, preferably at least 30, for instance at least 40, 60 or 100 or more contiguous amino acids. Methods of measuring pro~ein homology are well known in the art and it will be understood by those of skill in the art that in the present context.
15 Homology is usuallv calculated on the basis of amino acid idemity (sometimes referred to as "hard homology ") .

Generally, mutants. fragments, allelic variants or species homologues thereof capable of forming a complex will be at least lO, preferably at least 15, for example at least 20, 25, 30, 40, 50 or 60 amino acids in length.

20 A complex of the invention may be labelled with a revealing or detectable label. The (revealing) label may be any suitable label which allows the complex to be detected. Suitable labels include radioisotopes, e.g. '~51, enzymes, antibodies and linkers such as biotin. Labelled complexes of the invention may be used in diagnostic procedures such as immunoassays.

A complex (or labelled complex) according to the invention may also be fixed to a solid phase, for 25 example the wall of an immunoassay dish.

A second aspect of the invention relates to a polynucleotide (suitably in a substantially isolated form) which ~ol~ es:
(a) a sequence encoding a transcription modulator and a transcription factor as defined in ~he first aspect;

W 097/02354 PCT/~,5/~1560 (b) a secll~Pn~nP complementary to (a);
(c) a seqnPnre at least 80% (e.g. 90%) homologous to a sequence in (a) or (b).

The polynucleotide may also comprise RNA. It may also be a polynucleotide which includes within it synthetic or modified nucleotides. A number of clirrelcllL types of m~.-1ific~ti~n to oligonucleotides 5 are known in the art. These include methylphosphonate and pho.~holoLhionate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of polynucleotides of the invention used in methods of therapy.

10 A polynucleotide of the invention will be in substantially isolated form if it is in a form in which it is free of other polynucleotides with which it may be associated in its natural envhull.llcllL (usually the body). It will be understood that the polynucleotide may be mixed with carriers or diluents which will not hllclrclc with the intended purpose of the polynucleotide and it may still be regarded as suhst~nti~lly isolated.

15 A polynucleotide according to the invention may be labelled, e.g. with a revealing or detectable label, by conventional means using radioactive or non-radioactive labels. or may be cloned into a vector.

Polynucleotides, such as a DNA polynucleotides according to the invention, may be produced recombinantly, synthetically. or by any means available to those of skill in the art. It may be also cloned by reference to the techniques disclosed herein.

20 The invention includes a double stranded polynucleotide comprising a polynucleotide according to the invention and its complement.

A third aspect of the invention relates to an (eg. expression) vector suitable for the replication and expression of the polynucleotide, in particular a DNA or RNA polynucleotide, according to the second aspect of the invention. The vector may be, for example, a plasmid, virus or phage vector provided 25 with an origin of replication, optionally a promoter for the expression of the polynucleotide and optionally a regulator of the promoter. The vector may contain one or more selectable marker genes, for example an ampicillin rPcict~n~~e gene in the case of a bacterial plasmid or a neomycin l~ re gene for a m~mm~ n vector. The vector may be used in vitro, for example for the production of RNA or used to Lldl~.rc-;. or ~ldl~.rolll- a host cell. The vector may also be adapted to be used in ~.~ivo, 30 for example in a method of gene therapy.

~7-Vectors of the third aspect are preferably lecollllJilldllI replicable vectors. The vector may thus be used to replicate the DNA. Preferably, the DNA in the vector is operably linked to a control seqnl~nre which is capable of providing for the expression of the coding seqnenre by a host cell. The terrn "operably linked" refers to a juxtaposition wherein the components described are in a relationship ~- 5 permitting them to function in their int~n-l~d manner. A control sequence "operably linked" to a coding seqnPnre is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences. Such vectors may be transformed or transfected into a suitable host cell to provide for expression of component proteins in complexes of the invention.

The vector of the invention may comprise separate promoters operably linked to the sequences 10 encoding the transcription modulalor and the transcription factor or the vector may comprise a single coding sequence comprising both the modulator and factor. In this event, the factor and mt-~lnlAtor sequences may be in either order. The sequenr~c are optionally linked by a further seq -~nre such as a linker sequence d--ci~nPd to allow the two encoded polypeptide seqn~nrec to fold in a manner found in nature or it may be a linker sequence which can be selectively cleaved allowing the two encoded 15 polypeptide sequences to be separated. An example of the former type of linker includes an IgG hinge region whereas cleavable sequences of the latter type are well known per se in the art.

A fourth aspect of the invention thus relates to host cells transformed or transfected with the vectors of the third aspect, or able to express DNA sequenre(s) encoding both the transcription modulator and transcription factor, as defined for the first aspect. This may allow for the replication and expression 20 of a polynucleotide according to the invention. The cells will be chosen to be compatible with the vector and may for example be bacrerial, yeast, insect or mAmm~ n. Preferred hosts include 3T3 and SAOS-2 cells (the latter cells lack functional retinoblastoma (pRb) protein and pS3).

The host cells of the fourth aspect express. and preferably secrete. both a transcription modulator and transcription factor, those terms already having been defined in the first aspect. Preferably the 25 transcription modulator and factor will combine to form a complex of the first aspect, such interaction occurring either inside the cell or outside in the medium surrounding the host cells (if any).

Such expression can be achieved either by using a single vector, such as of the third aspect, which encodes both a transcription modulator and a lldns~ Lion factor. Alternatively, a host cell can be L,dl~rolllled or transfected with two or more vectors. A first vector will encode the transcription 30 m~ tor, while a second vector will encode the transcription factor. Both vectors will then be co-~IA~rt~-ted into a host cell to produce the cells of the fourth aspect.

A polynucleotide according to the invention may also be inserted into the vectors described above in an ~nticence orient~rir~n in order to provide for the production of ~nticf~nce RNA. Antisense RNA or other ~nticen.ce polynucleotides may also be produced by synthetic means. Such ~ C~ e polynucleotides may be used in a method of controlling the levels of the transcription modulator, a 5 DP protein and/or E2F protein in a cell. Such a method may include introducing iMo the cell the ~nricence polynucleotide in an amount effective to inhibit or reduce the level of translation of the E2F
mRNA into protein. The cell may be a cell which is proliferating in an uncontrolled manner such as a tumour cell.

Thus, in a fifth aspect the invention provides a process for preparing a complex according to the 10 invention which comprises cultivating a host cell of the fourth aspect, preferably lldn~ru~ ed or transfected with an (expression) vector of the third aspect, under conditions providing for expression of coding sequence(s) encoding the component proteins of the complex, allowing the transcription modulator to bind to the transcription factor. and recovering the complex.

A sixth aspect of the invention relates to complexes of the first aspect, a polynucleotide of the second 15 aspect, vectors of the third aspect and host cells of the fourth aspect for use in medicine. In other words, all such substances (from the first to fourth aspects inclusive) find use in a method for treatment of the human or animal body by therapy.

The uses contemplated are the inhibition of cell growth, or the interference with transcription. End uses here would therefore include the treatment of proliferative diseases, such as cancer.

20 The transcription modulator need not be the naturally occurring p53 protein, as has been explained for the first aspect. Indeed. using the screening methods of the invention (which are ~iiccllcsed later) substances other than the naturally occurring proteins, which have similar properties, can be identified.
These too may then be used in the same or similar manner to the p53 protein themselves.

Thus, according to a seventh aspect of the present invention there is provided a pharrn~renti~l or 25 veterinary composition comprising a complex of the first aspect, a polynucleotide of the second aspect, a vector of the third aspect, or a host cell of the fourth aspect, together with a pharrn~rentir ~lly, or ~ Lelil-dlily, acceptable carrier or diluent, respectively.

ph~rm~t~ellti~11y or ~ lh.dlily acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal, topical (inl~lu~ling buccal and sublingual), vaginal or pal~ eldl 30 (inrlu~ling 5nbcllt~npouc~ i"ll~"~ c~ r, intravenous, intr~Prrn~1, intr~thPc~l and epidural) g a-l.,-i"i~.~,dLion. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which co~ es one or more accessory ingredients. In general the formulations are prepared by uniformly and inrim~3t~1y bringing 5 into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

For example, formulations suitable for parenteral ~mini~tration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants. buffers, bacteriostatis and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous 10 sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the polypeptide to blood components or one or more organs.

Complexes according to the invention or transcriptional modulators may be used for the L~~~l",~
regulation or diagnosis of conditions. including proliferative diseases, in a m~mm~l in~ ing man.
15 Such conditions include those associated with abnormal (eg at an unusually high or low level) and/or aberrant (eg due to a mutation in the gene sequence) expression of one or more transcription factors such as the DP or E F proteins or related family members Treatment or regulation of conditions with the above-mentioned complexes or peptides will usually involve ~lminictl~ring to a recipient in need of such treatment an effective amount of that complex or peptide as appropriate.

20 Vectors carrying a polynucleotide according to the invention or a nucleic acid encoding a complex according to the invention may be used in a method of gene therapy. Such gene ~herapy may aim to repress or inhibit cell growth and so treat uncontrolled proliferation of cells, for example a tumour cell. Alternatively the complexes may be used to encourage or activate cell growth. Methods of gene therapy may also include delivering to a cell in a patient in need of treatment an effective amount of 25 a vector capable of expressing in the cell either an antisense polynucleotide of the invention in order to inhibit or reduce the translation of E2F or DP mRNA into the corresponding protein which may hl~e,r~,~ with the binding of an E2F protein to a DP protein or a related family member.

Such a vector is suitably a viral vector. The viral vector may be any suitable vector available in the art for targetin~ tumour cells. For example, Huber et al (Proc. Natl. Acac. Sci. USA (1991) 88, 30 8039) report the use of amphotrophic retroviruses for the transformation of hepatoma, breast, colon or skin cells. Culver et al (Science (1992) 256; 1550-1552) also describe the use of retroviral vectors in virus-directed enzyme prodrug therapy, as do Ram et al (Cancer Research (1993) 53; 83-88).

:

F.n~leh"rdt et al (Nature Genetics (1993) _; 27-34 describe the use of adenovirus based vectors in the delivery of the cystic fibrosis tran~mt-nnhrane con~ ct:~nre product (CFTR) into cells.

According to an eighth aspect of the invention there is provided:
(a) a complex of the first aspect;
(b) a polynucleotide of the second aspect;
(c) a vector of the third aspect; or (d) a host cell of the fourth aspect of the invention;
in the m"nnf~etllre of a medic:lment for treating uncontrolled proliferation of cells, for example in the treatment of cancer. vi}al disease. heart disease, self proliferation disorders as well as auto-immune disorders such as psoriasis.

In another aspect, the invention provides a novel assay for identifying putative chemotherapeutic agents for the treatment of proliferative or viral disease which comprises bringing into contact a DP
protein, an E2F protein and a putative chemotherapeutic agent, and measuring the degree of inhibition of formation of the DP/E2F protein complex caused by the agent. It may not be necessary to use the complete DP and/or E F protein in the assay, as long as sufficient of each protein is provided such that under the conditions of the assay in the absence of agent, they form a heterodimer.

The cloning and sequencing of DP-I (and E2F 1,2 and 3) are known in the art and methods for the recombinant expression of these proteins will be known to those in the art.

In a niMh aspect of the invention there is provided a screening method or assay for identifying potential or putative chemotherapeutic agents, the method or assay COlllpl i~7hlg providing a Lldl~ Lion modulator and a po~ential or putative chemotherapeutic agent, and measuring the extent to which the agent is able to bind to the transcription modulator, or whether the agent forms a complex, of the invention with the modulator.

The ninth aspect also extends to a similar method where the transcription modulator is replaced by a transcription factor. The transcription modulators and factors are as defined for the first aspect of the invention.

Thus, in the tenth aspect the invention provides a screening method or assay for identifying potential or putative chemotherapeutic agents, the method or assay comprlsing:-(A) providing the following components:
30 (i) a DP protein and/or an E2F protein;

(ii) a p53 polypeptide; and (iii) a potential or putative rhpmcth~ ulic agent;
and bringing them into contact under conditions in which the Co.ll~u~ lL~ (i) and (ii) in the absence of (iii) form a complex; and (B) measuring the extent to which component (iii) is able to disrupt or interfere with the complex, inhibit or encourage the binding of components (i) or (ii), or affect the activity of the complex.

In the assays, any one or more of the components may be labelled, eg with a radioactive or colorimetric label, to allow measurement of the result of the assay. Potential chemotherapeutic agents include transcription modulators and factors as described in the first aspect of the invention.

Mutants. homologues and fragments of the DP and E2F proteins have been defined earlier in a corresponding manner to the mutants. homologues and fragments of the MDM2 and p53 Lldns~ Lion modulator proteins, all of which find use in the assays of the invention.

Such an assay can be performed without the need for a DP protein. However, if both a DP and E2F
protein are present the complex of (i) and (ii) may be measured, for example, by its ability to bind an E2F DNA binding site in vilro. Alternatively, the assay may be an in vivo assay in which the ability of the complex to activate a promoter colll~ ing an E2F binding site linked to a reporter gene is measured. The in vivo assay may be performed in yeast. insect. amphibian or m~mm~ n cells.

It will thus be seen that the assays of the invention extend to both to two component assays (in the ninth aspect) as well as three component assays (as in the tenth aspect).

In either a two component or three component system. but preferably the former. one or both of the components may comprise a fusion protein. That fusion protein. as mentioned earlier, is encompassed by the terms p53, DP and E2F proteins.

For example, a first protein of interest may be fused to a protein coulyli~illg a foreign DNA binding domain, such as a GAL-4 domain. That protein on its own would be inactive, and although it would bind DNA, it would not activate transcription. A second protein of interest (for example a DP
protein, if the first protein was a transcription modulator) may then be fused to be a protein t COIII~ hlg a foreign activation domain, such as a GAL~ activator. In the absence of the chemothe.a~GuLic agent, the first and second proteins of interest (which would usually be a transcription mn~lnl~tl-r and a L,dnsc.il.Lion factor) will form a complex. When respective fusion proteins are present, such as a foreign DNA binding domain and foreign activation domain, they will CA 02224940 l997-l2-l7 W O 97/02354 PCT/~_.SI~l560 -12 -cooperate to cause activation. The affect of the chemotherapeutic agent on this process can then be monitored. The agent will usually be added to medium surrounding cells, while inside the cell the two fusion proteins are expressed.

Thus the proteins that find use in the complex of the invention may themselves be fusion proteins.
They be fused with another (different) protein which can act as a label or tag for detection of that fusion protein. Suitable tags include GST and a series of hictirlin~c residues (e.g. six), the latter being able to bind nickel.

Such fusion proteins may be used to purify the protein, or allow it to be used in assays. The fusion protein or tag may have an affinitv for another protein or substance (which may be present on a 10 column) or an antibody. For example, if the protein is fused to GST, this may aid its purific~ti- n using glutathione sepharose ~" beads. Alternatively an antibody specific for the tag on the fusion protein may be employed.

~ n~ fe therapeutic agen~s which may be measured by the assay include not only the transcription modulators and transcription factors mentioned in the first aspect. but in particular fragments of 10~5 or more amino acids of:
(a) a p53 protein;
(b) an allelic variant or species homologue thereof; or (c) a protein at least 70% homologous to (a).

The assays of the invention can be performed using standard techniques. the components (i) and (ii) being taken from known. publicly available sources. One preferred method, however, in values providing the proteins in recombinant form. expressed by a host cell (e.g. of the fourth aspect). l:~ost cells can be transfected or transt'ormed in the assay performed in vitro.

The invention contemplates a number of assays. Broadly, these can be classified as follows.

1. PGlrollllhlg an assay to identify a substance that affects the interaction between the transcription modulator and the transcription factor. Such substances may interfere with or alter the tr~nC~rtivation IG~lGssillg properties of p53. Such substances may be more effective than the natural molecules for themselves. For example, they may be able to repress transcriptional activity more GrrG~;LivGly than p53. On the other hand, they may have lower levels of LldlLsclilJlion IG~lGs~hlg activity than p53. Such 5nhct:~nrps may therefore act as either agonists or antagonists of p53, as 30 d~ U~UlidlG. The sllbst~nrrc may hlLGIrelc with the interaction or binding between the transcription modulator and transcription factor, or alternatively c~ LiLi~ely inhibit this interaction, or the formation of a complex between the transcription modulator and factor. by mimirkin~ either of the transcriptional modulator or factor proteins.

Such an assay may be particularly useful in identifying compounds which are agonists of p53 5 or other substances which promote the presence in the cell of the form of DP-l recognised by p53 which is likely to be growth inhibiting. For example such compounds may bind to DP-1 in a manner which mimics p53. Alternatively the compound may promote the binding of p53 to DP-I. Such assays may also be used with pS3 mutants such as those described above to assay for compounds which restore the ability of such p53 mutants to bind to DP- l where such p53 mutants have lost the 10 ability to do so in tumour cells.

2. Con~lucting an assay to find an inhibitor of an E'F protein trans-activation (that is to say, inhibition of activation of transcription). This inhibitor may therefore inhibit binding of an E2F
protein to DNA (usually at the E2F binding site). Potentially suitable inhibitors are proteins, and may have a similar or same effecl as p53. Thus suitable inhibitory molecules may comprise fragments, 15 mutants, allelic variants, or species homologues of p53 in the same manner as defined for complexes of the first aspect.

3. Assaying for inhibitors of (hetero)dimerisation which mimic the binding of p53 to DP-1. The binding of DP-I to an E2F protein promotes cell proliferation and thus pS3 competes with E2F for binding to DP-l. Such inhibitors may prevent dimerisation of an E F protein (eg. E2F-l) with a DP
20 protein, such as DP-l. The assay can measure the degree of inhibition of binding by deL~ll--il-illg the ability of pS3 and/or E2F to compete with each other and the potential inhibitor. Of course the inhibitor can be a fra_ment. mutant. allelic variant or species homologue of a DP or E2F protein as defined for the complexes of the first aspect.

The invention thus contemplates the identification of substances for the treatment or prophylaxis of 25 diseases that are based on the uncontrolled proliferation of cells, or where uncontrolled proliferation is an important or essential pathological aspect of the disease. This includes cancer, viral disease, self proliferation itself as well as auto immune diseases such psoriasis. Compounds contemplated here would include p53 agonists.

One may also wish to temporarily inhibit the growth of dividing cells, for exarnple haematopoietic 30 stem cells and/or bone marrow cells. In these aspects one is generally seeking to prevent, inhibit or hlLt;lrei~ with the activity of an E2F protein.

4 PCT/GB96/01~60 In cont}ast some diseases and conditions can be treated by h~ c~illg E2F ~ ;on, for example by promoting or inti-lrin~ overexpression. That could be achieved through the use of p53 antagonists.
This preferably results in apoptosis, somelillles known as programmed cell death. Ove~lc~lJle~ion of the E2F protein can result in death of the cell (Qin et Rl ,1994) and therefore this aspect can also be 5 used in the treatment of cancer. Thus E2F and pS3 appear to mnd~ te the activity of each other and disruption of the interaction by antagonists may promote the observed apoptotic effects of E2F.

The invention will now be described by way of example with respect to the acculll~llyhlg Examples.

Immunorh~",i~
10 Monoclonal antibodies 421 and SMP 14 have been previously described (Harlow et al., 1981; Picksley et al., 1994). Anti-DP-l(A) is a rabbit polyclonal anti-peptide serum raised against a peptide representing an N-terminal region in DP-I, and has been previously described (Girling et al., 1993;
Bandara et al., 1994). Anti-DP-l(D) rabbit polyclonal anti-peptide serum (Bandara et al., 1994) and monoclonal antibody 32.3 were raised against a peptide representing a C-terminal region (residues 385 15 to 400) in DP-l. Irnmunoblotting with anti-DP-l(A), anti-DP-I (D) or 32.3 was performed as previously described. Either the homologous peptide. or a control unrelated peptide, peptide 1, was added to assess specificity.

For immunoprecipitation, cells were harvested in LSL buffer (50mM Tris-HC1 pH 8.0, 150mM NaCI, 0.1 % NP40, 2~g/ml aprotinin. 0.5rnM PMSF), to which 32.3 was added in the presence of peptide 20 and inrub~tf~cl for lhr on ice. Immune complexes were collected with protein A-Sepharose and washed extensively (at least 3 times) in LSL buffer. released in SDS sample buffer, electrophoresed and immunoblotted with anti-DP-I (A). The procedure for sequential imtnunoprecipitation and immunoblotting has been previously described (Bandara et al., 1993).

For the anti-DP-1 immuno-affinity chromatography, 2ml anti-DP-l(A) Ab was pl~:ci~iL~t~d with 25 ammonium sulphate (45%) and ~,dla..~lively dialysed in lOmM sodium phosphate (pH7.5). The resulting imtnunoglobulin was coupled to 3ml CNBr-activated Sepharose (as recommPn~lPd by the m~mlf~rnlrer) and inrllb~t--d with 3ml F9 EC extract for 24h at 4~C. The column was washed with NEP buffer (Girling et al., 1993) containing 0.5 % NP40, pre-elution buffer and bound proteins eluted using O.lM glycine (pH2.5). Samples were collected and neutralized. The peak fractions were 30 ~ dLed with trichloroacetic acid and washed with acetone before being solubilized in SDS sample buffer and immlln~blotted with either 32.3 or SMP14.

CA 02224940 l997-l2-l7 ~ Lar.laLiu~l:
Gel retardation with F9 EC cell extracts using an E2F binding site taken from the adenovirus E2a promoter was performed as described previously (Girling et al., 1993). Monoclonal antibody 32.3 was added to the binding reaction. together with competing peptide, and inrllh~ted for 10 min at 30~C.
To assess the DRTF1/E2F DNA binding activity immunop-cc;i~!iL~led by 32.3. ii~ u~ Lions were performed as described above in the presence of either the homologous or ullleldLed peptide.
Competing homologous peptide in LSL was added after washing the irnmullul,le~ in LSL buffer, the supernatant being subsequently assayed for DRTF1/E2F DNA binding acIivity.

The effect of pS3 on the E2F-site binding activity of the E2F-I/DP-1 heterodimer was assayed using 10 in vitro transcribed and translaled DP-l (pG4DP-1; 4) and E2F-1 (pSP72;27). In vitro transcription and translation was carried out in a TNT T7/SP6 coupled reticulocyte Iysate system (promega). pH6-mmp53 wt encodes a His-tagged complete mouse p53 protein (kindly supplied by Gunnar Weidt and Wolfgang Deppert). His-tagged murine p53 was purified from a 500ml pellet of IPTG-induced bacterial culture. The bacterial pellet was resuspended in lOmls denaturing buffer (lOOmM sodium 15 phosphate, lOmM tris base, 6.0M gll~niflin~ hydrochloride, 30mM imidazole) pH 8.0, and gently stirred for two hours at room temperature. MgCI2 was added to a final concentration of 5mM and cellular debris were cleared by repeated centrifugation at 4~C. 400~1 of nickel agarose (solid) QIAGEN) was added to the supernatant and rotated for one hour at room temperature. The resin was washed stepwise with two 50ml volumes each of; denaturing buffer pH8.0, denaturing buffer pH 6.4 20 and renaturing buffer (25rnM sodium phosphate pH 7.0, 300rnM NaCI, lOrnM ,~-mercaptoethanol) containing lM then O.lM and lastly no gu~ni~iin~ hydrochloride. Protein was eluted off the resin by sequential washes with imidazole buffer (150 mM imidazole, lOOmM NaCI, 50mM Tris-HCI pH 8.0) and analysed by SDS-PAGE. An equal amount of heat-denatured or non-denatured His-tagged p53 was added to the binding reaction.

25 Fractionation:
Heparin-Sepharose and E2F binding site-affinity chromatography of F9 EC cell extracts was performed as previously described (Girling et al., 1993). Fractions were assayed for DRTF1/E2F
DNA binding activity and immunoblotted as described.
-Rin-lin~ assay for pS3:
pH6-mmp53 wt encodes a His-tagged complete mouse p53 protein (kindly supplied by Gunnar Weidt and Wolfgang Deppert). pH6-mmp53, GST-pRb (Bandara et al., 1991) and GST alone were induced and purified by cu~ llLional procedures. For the in vitro binding assay, 15~1 of fusion protein bound to the ~lu~liaLe agarose (glutathione-agarose or nickel-agarose) was in~llh~ted with F9 EC whole W 097/02354 PCT/~,.'~1560 cell extract with constant rotation for 2hrs at 4~C. The suspension was ceMrifuged and repeatedly washed with LSL buffer. resuspended in SDS loading buffer and immunoblotted with anti-DP-l(A).

Binding assay for DP-1:
GST-DP-l encodes a complete DP-l protein fused to GST in pGEX-3X and was induced and purified by conventional procedures. E2F-I, E2F-4 and wild-type p53 were ~ ibed and translated in the presence 35S methionine in a TNT coupled Iysate (Promega) as leco~ d by the m~n-lf~rtllrer.
For the in vitro binding assay in Fig. 5~ fusion protein was added to the translate in PBSA containing ImM EDTA, lmM DTT. 0.5 % Tween 20 and inr~lb:lred for 30 min at 30~C. Glutathione beads were snhsequPntly added and incubated for a further 30 min. Beads were collected and washed four times 10 in the same buffer before being solubilized in SDS ample buffer. In order to map the region in p53 to which DP-l binds a panel of p53 mutants were made. GST-p53 1-73, 1-143 and 1-235 were made by PCR using human p53 as template 9php53CI; 50). PCR products were cloned in frame into a pGEX vector (Pharmacia). Fusion proteins were induced and purified by conventional procedures.
For in vitro binding reactions approximately 10,ug of GST or GST-p53 fusion protein bound to 15 glutathion-agarose beads was added to 15~1 of in vitro translated DP-l in Iysis buffer (50mM Tris pH
8.0, 150mM NaCI, containing 10mg/ml Iysozyme, 0.5mM PMSF, 50~g/ml leupeptin, 50~g/ml protease inhibitor, 50~g/ml aprotinin and 40mM DTT). After incubation for 2.5h at 4~C the beads were collected and washed 4 times in Iysis buffer. Proteins were released in SDS sample buffer, electrophoresed and immunoblotted with anti-DP-l(A) or anti-DP-I (D).

20 Binding assay for MDl\~2:
pGST-MDM2 encodes a complete MDM2 protein fused to GST in pGEX-3X and was induced and purified by conventional procedures. DP-I and wild-type p53 were transcribed and translated in the presence 35S methionine in a TNT coupled Iysate (Promega) as recomm--n-ird by the m~mlf~rtllrer.
For the in vitro binding assay. fusion protein was added to the translate in PBSA containing lmM
25 EDTA, lrnM DTT, 0.5% Tween 20 and inr-lhat--d for 30 min at 30~C. Glutathione beads were 5~lhseqllrntly added and inrnh~t~d for a further 30 min. Beads were collected and washed four times in the same buffer before being solubilized in SDS sample buffer.

T~ L 1-d~r~-liOn:
The ~c~ollc. construct p3xWT-GL. p3xMT-GL, pCMV-,Bgal, pCMV-E2F-1 and pCMV-DP-1 have 30 all been described previously (Girling et al., 1994; Jooss et al., 1995). pC53-SN3 encodes wild-type p53 driven by the CMV .onh~nrerjpromoter region (Baker et al., 1990). pJ4~2-MDM2 encodes the complete MDM2 protein (Oliner et al., 1992). The total amount of DNA in each l~ xr~ on was made up with empty vector which, in the case of the p53 titration was with pCMV-neoBam, and for CA 02224940 l997-l2-l7 the MDM2 titration waswith pJ4S2. Cells were transfected by the conventional calcium phosphate procedure. Luciferase and ,~-galactosidase assays were peformed as described previously (Girling et al., 1994). Each treatment was performed in duplicate.

Res~lts 5 Distinct forms of DP-l.
Two distinct DP-l polypeptides of 55kD can be resolved during cell cycle progression in 3T3 cells, referred to as p55L (lower) and p55U (upper), pS5L appearing towards the end of G1 as cells begin to enter S phase (Bandara et al., 1994). ln order to characterise the two forms of pSS in greater detail an anti-peptide was prepared monoclonal antibody, 32.3. which recognises pSSL. By imrnuno-blotting 10 extracts prepared from a synchronous cultures of F9 EC cells a polyclonal antiserum, anti-DP-l (A), revealed both forms of p55 in contrast to monoclonal antibody 32.3 which defined p55L. Further evidence that 32.3 recognises p55L was provided by immunoprecipitating with 32.3 in the presence and absence of the homologous peptide, and subsequently probing the immunoprecipitate with polyclonal anti-DP-l(A). The immunoprecipitated polypeptide co-migrated with p55L.

15 The effect of 32.3 on DRTFI/E2F DNA binding activity was examined. In extracts prepared from F9 embryonal carcinoma (EC) cells (obtained from the European Culture Collection) the addition of 32.3 to the binding reaction caused an almost complete shift of DRTFI/E2F compared to the reaction performed in the presence of a homologous peptide; similar results were observed in extracts prepared from a wide variety of other types of cells. Furthermore. 3'.3 immunoprecipitated DRTFl/E2F DNA
20 binding activity frorn F9 EC cell extracts in which pS5L was the predominant form of DP-I present in the immunoprecipilate. These data suggesl that p55L is a major component of DRTF1/E2F DNA
binding activity.

In order to substantiate this idea the chromatographic properties of p55U and p55L were studied during the fractionation of F9 EC cell extracts, and the presence of each polypeptide with the presence 25 of DRTF1/E2F DNA binding activity within each fraction was correlated. Extracts prepared from F9 EC cells were fractionated over heparin-Sepharose and subsequently assayed for DNA binding activity by gel retardation. Both p55U and p55L were present in the F9 EC cell extract, although an analysis performed on fractions after passage over heparin-Sepharose infli(~ oci that p55L, rather than p55U, correlated with DRTF1/E2F DNA binding activity. Thus, p55U was predominant in fractions 30 which lacked DNA binding activity. In contrast, DRTF1/E2F DNA binding activity both before and after further purification by E2F binding site-affinity chromatography (Girling et al., 1993), correlated with p55L. More rounds of E2F binding site-affinity chromatography did not alter the correlation.

CA 02224940 l997-l2-l7 W O 97/02354 PCT/~i'r~1560 When fractions containing pS5L and pS5U (with and without DRTF1/E2P DNA binding activity) were added together the original composition of p55U and L in the unfractionated cell extract was l~co~ l These data, combined with the results derived from the studies performed with monoclonal antibody 32.3, suggest that pS5L is present in DRTFI/E2F DNA binding activity and, further, that p55U is likely to be a form of DP-1 which either cannot bind or binds a less efflciently to the E2F site.

DP-l ~ccori~ff~c with p~3:
A number of polypeptides with distinct molecular weights were detected when dirr~clll polyclonal anti-DP-l peptide antisera were used to sequentially immuno-pl~ JiL~te DP-1 from 35S-methionine 10 radiolabelled extracts prepared from F9 EC cells . Thus, anti-DP- I (A) specifically immunoprecipitated a group of polypeptides with a range of molecular weights. When the anti-DP-l(A) immunoprc:cil,itaL~
was released and subsequently re-inrnunoprecipitated with anti-DP-l(D), which possesses similar specificity to 32.3 for p55L, a subset of the anti-DP-l(A) associated polypeptides were apparent.
These data suggest therefore that a number of cellular polypeptides associate with p55U.

15 In order to characterise these DP-I-associated polypeptides in greater detail it was ~cc.o~ced whether antisera directed against previously identified polypeptides with similar molecular weights recognised them. A range of antisera were studied, two of which were found to be of particular interest. Thus, when an anti-p53 monoclonal antibody, 421, was used in an immun~ e~ ,iL~Lion and subsequently immunoblotted with anti-DP-l(A), p55 was revealed. However, p55U rather than p55L ~ r~-~llLially 20 co-immunoprecipitated with p53. To subst,.nri~te this result, further evidence for an interaction between pS3 and DP-l was obtained from an in virro binding assay in which a p53 fusion protein was incubated in an F9 EC cell extract In these conditions, pS3 specifically associated with DP-l in the F9 extract. As expected. an pRb fusion protein known to bind to DRTF1/E2F in F9 EC cell extracts (Bandara et al., 1991) interacted with DP-1 in the same assay conditions. Overall, these results 25 suggest that p53 associates with DP-l in physiological conditions and, further, the preferred form of DP-l that interacts with p53 is p55U.

Similar experiments were performed with a monoclonal antibody recognising MDM2, encoded by the MDM2 oncogene, which is widely believed to be a transcriptional antagonist of p53 trans activation (Oliner et al., 199~) These e~ liln.,.lLs (data not shown) confirmed the interaction which has also 30 been reported by Martin et al., 1995, and Xiao et al., 1995.

The eff;ei~nry of interaction of DP-1 and MDM2 was compared with the binding of p53 and MDM2.
An assay was used in which the ability of MDM2, expressed as a GST fusion protein, to bind to in CA 02224940 l997-l2-l7 vitro translated DP-l and p53 was ~c.~.oc~eA It was found that p53 and MDM2 were able to associate in the conditions of this assay and likewise, DP-1 could interact with MDM2, similar results being obtained with the other members of the DP family, DP-2 and DP-3. The efficiency of interaction between DP-1 and MDM2 appeared to be somewhat lower than the pS3/MDM2 interaction although 5 both interactions were specific since neither DP-1 or p53 could bind to a control GST protein. Similar e~ on the specific binding of in vitro translated DP-1 to a p53 fusion protein were obtained.
Overall, these in vitro binding data support the results from the immuno~lcci~ lion studies on the specific interaction of DP-1 with MDM2 and p53 in m~mm~ n cells.

Mutational analysis of DP-l.
10 In order to determine if DP-I and p53 can interact in vitro and compare the efficiency of interaction with the binding of DP-I to E2F family members, we used an assay in which the ability of DP-l, expressed as a GST fusion protein. to bind to in vitro translated p53 and E2F proteins was ~Csf~cse~l As expected. DP-l could associate with either E2F-1 or E2F4 in the conditions of the assay used.
Likewise, DP-1 could interact with pS3. similar results being obtained with the other members of the 15 DP family, DP-2 and DP-3. The interaction between DP-1 and p53 was as efficient as the interaction between DP-1 and E2F-1 or E2F4~ and specific since p53 failed to bind to a control GST protein.
These in vitro binding data support the conclusion from the immunoprecipitation studies on the specific interaction of DP-1 with p53 in m~mm~ n cells.

Using a similar assay, we determined the region in DP- I required to bind to p53. A panel of mutant 20 proteins derived from DP-1 represencing N- and C-terminal truncations, together with a DP-1 protein altered at residues 172 and 173, were studied. In vitro translated wild-type DP-l bound to p53 about 20% of the input DP-l being retained by the wild-type p53 fusion protein. This binding efficiency was not significantly affected by removing up to 171 amino acid residues from the N-terminal region of DP-1, up to 79 amino acid residues from the C-terminal region, or by mnt~ting residues 172 and 25 173. However, further N-terminal deletion from residue 171 to 205. or C-terminal deletion from residue 331 to 238, signific~ntly reduced the binding activity of DP-l with pS3. From these data, the minimal region in DP-l capable of efficiently binding to p53 occurs within residue 171 to 331. This region of DP-1 contains several domains which are conserved between other members of the DP
family, notably DCB l and DCB2 (Girling et al 1994, Ormondroyd et al 1995), together with the DEF
30 box, a critical region involved in heterodillleli~Lion between DP-1 and E2F family members.
Importantly, in the conditions of this assay in vitro translated E2F-1 failed to interact with -53 since the amount of E2F-1 retained by pS3 was at back~loulld level. In the context of DRTFl/E2F, the DP protein is the principal component which is capable of interacting with pS3.

An i~ lly distinct form of DP~ o ~ . with p53.
To define the region in p53 required for the association with DP-l, a similar in vitro binding assay was developed in which the ability of p53 to interact with in vitro translated DP-l was monitored.
The data presented above in-lic~ed that p53 c~ c~ Les with p55U from m~mm~ n cell extracts 5 and, further, that p55U is a forrn of DP-I recognised by anti-DP-1 (A) but not anti-DP-1 (D). In a similar fashion, two immuno~h~nnic~11y distinct forrns of DP-I could be defined after in vitro trancl~tion using these same two antisera. Specifically, in the absence of translated exogenous DP-l, anti-DP-1 (A), but not anti-DP-I- (D), recognised the endogenous DP-1 protein. After translation, both antisera recognised the itt ~~itro translated DP-l protein, the exogenous polypeptide being resolved 10 with marginally faster mobility.

Evidence that at least two irnrnunochPmic~lly distinct forms of DP-I were present after in vitro translation was obtained upon studying the interaction with p53. When p53 was added to the in vitro translate, the DP-l form recognised by anti-DP-l(A), but not anti-DP-l(D), was retained by p53 although DP-l immunoreactive with both antisera was present in the input translate; the GST portion 15 failed to interact with DP-l. These data indicate that two distinct forms of DP-l are present after in vitro translation and further thal pS3 preferentially interacts with the form defined by anti-DP-l(A).
~ Importantly, this result reflects the data derived by immunoprecipitation from m~mm~ n cell extracts where p53 co-immunoprecipitated with pS5U. a DP-l protein recognised by anti-DP-l(A) but not anti-DP-l(D). The specificity of p53 for DP-I in the in vitro binding assay therefore possesses some similarity with the interaction in m~mm~ n cells, and supports the conclusion that p53 interacts with an immunochemcially distinct form of DP-l.

An N-te~ninal region in pS3 is req~uired for binding to DP-I.
We used the interaction of p53 and DP-I to define the domain in p53 required for the association.
As much as 250 amino acid residues could be deleted from the C-~e.",;".lc of p53 without any 2~ d~L~ L~LI effect on the interaction with DP-l. A further deletion from residue 143 to 73 abolished the interaction, thus defining a region in p53 required to bind DP-I. Since the N-terrninal region of p53 contains the MDM2 binding domain (Picksley et al, 1994) a domain in p53 necessary for the interaction with DP-l can therefore be distinguished from the MDM2 binding domain.

p53 and MDM2 mf~r~ tP E2F site--lPpPnrlPnt Ir ~ lion.
Since DP-l is a frequent colll~ of DRTFl/E2F (Bandara et al., 1993 and 1994), the functional cnnceql-en~e of an inte}action of either p53 with DP-1 was ~cs--cced by studying the effects on E2F
site-dependent Lldllsc,li~ion driven by DP-1 and E2F-1 a cihl~tion in which it is known that both proteins co-operate in lldlls~ ional activation as a DNA binding heterodimer (Bandara et al., 1993.

W O 97/02354 PCT/~b~.~1560 In these assay conditions DP-l alone possesses inci~nific~nt tld~ ional activity (Bandara et al., 1993). When a wild-type pS3 expression vector was co-transfected into 3T3 cells the level of trans-activation mPrli~t~d by either E2F-1 alone, or DP-l together with E2F-l, was c~ "o"lised in a pS3 concentration-dependent fashion. This inactivating effect of wild-type p53 was also a~.~a~c;"L in human SAOS-2 cells which contain a mutant p53 allele. The activity of a co~ alal)le promoter COII:,llu-;~
driven by mutant E2F binding sites, p3xM T-GL, was not significantly affected by pS3.

p53 and E2F-I compete for DP-l.
It was of interest to determine if the interaction of p53 with DP-I was mutually exclusive with the interaction of DP-l and E2F-l. To address this question we assessed whether E2F-l, expressed as 10 a GST fusion protein, would compete with pS3 for DP- l in the in litro binding assay, conditions in which in vitro translated DP-I binds to p53. As the amount of GST-E2F-l was increased there was a concomitant reduction in the level of DP-l bound to p53, an effect not apparent in the control GST
treatment. These data indicate that pS3 and E2F-l compete for binding to DP-l, and is consistent with the earlier data inr1ic~ring that p53 interacts with the dimerization domain of DP-I. If p53 and E2F-l 15 compete for DP-1, reduced DNA binding activity due to the DP-l/E2F-l heterodimer may be ~ a,~"~
in the presence of pS3. To test this possibility a band shift assay which measured the DNA binding activity of the DP-1/E2F-I heterodimer was supplemented with p53. After in vitro translation DP-l or E2F-l alone have little DNA binding activity although, when assessed together, co-operate. As the level of p53 was titrated into the reaction, reduced DP-l/E2F-l DNA binding activity was 20 apparent. In contrast, inactivated pS3 had little effect. In conclusion, pS3 and E2F-l compete for DP-l and, as a result. p53 can reduce the level of DNA binding activity of the DP-l/E2F-l heterodimer.

DISCUSSION

Distinct forms of DP-I.
25 It is known that DP-l is the most widespread DNA binding component of physiological DRTFl/E2F
DNA binding activity (Bandara et al., 1993, 1994) where it exists in a heterodimer with an E2F
family member (Bandara et al., 1993; Helin et al., 1993; Krek et al., 1993). Previous studies have shown that the polypeptide encoded by DP-l, p55, is subject to cell cycle control since it is regulated during cell cycle progression, the level of pS5L il-~-~i--g as cells progress through the cycle (Bandara 30 et al., 1994). Using antisera which efficiently recognise pS5L it has been possible to show that this form of DP-I is the predominant DNA binding component in DRTFl/E2F, a physiological ~ihl~tior' which is ~ aL~ in a wide variety of cell-types. In CoMraSt, the data suggested that pS5U is unlikely to be a frequent c~ ulle~l of DRTF1/E2F since antisera which preferentially recognise p55L react efficiently with physiological DRTFl/E2F, and the abundance of p55U inversely correlates with DRTF1/E2F DNA binding activity. Although no direct evidence that p55U and L are post-translational derivatives of each other is presented here, such an idea is a clear possibility given 5 previous results on the effect of phosphatase on DP-1 (Bandara et al., 1994).

pS3 ~o~ with DP-l.
The region in p53 required for the interaction with DP-1 exists within the N-terrninal 143 arnino acid residues. The first 73 residues. which contains the MDM2 binding domain are not sufflcient for the interaction. Although previous studies have suggested that MDM2 can interact with DRTFl/E2F, our 10 results imply that this interaction is unlikely to be responsible for the association of DP-1 with p53.
Interestingly, the region between residue 73 and 143 which is necessary for pS3 to bind DP-1 contains residues frequently altered in human tumour cells carrying mutant 53 alleles (Harris, 1993).

A variety of cellular polypeptides co-precipitated with DP-1. Two of these were i~ ntified as p53 and MDM2, proteins cnown to influence cell cycle progression. Thus, a piop~lly of the pS3 protein is 15 the negative regulation of the cell cycle in response to, for example, DNA damage and its gene is frequently mutated in human tumoLIr cells (Levine et al., 1991). A currently popular model for a merh~nicm through which p53 m~ teS its growth-regulating properties is that it functions as a transcriptional activator of genes involved in arresting cell cycle progression (El-Deiry e~ al., 1993).
In contrast, MDM2 is encoded by the MDM2 oncogene, MDM2 being amplified in certain human 20 tumour cells (Oliner et al., 199-'). It is believed in part to mediate its growth promoting activity by hlt~lrclillg with the transcription activating properties of p53 (Oliner et al., 1993).

Although it was found that p53 and MDM2 associate with DP-I, it has not been possible to detect either protein in physiological DRTFI/E2F DNA binding complexes. It is possible, given the data presented here, that these proteins target a population of DP-I which is not frequently present in the 25 DP-1/E2F DNA binding heterodimer, an idea which is consistent with the result that pS3 co-ple~ at~d with p55U, a forrn of DP-1 which is not signific~ntly present in DRTF1/E2F DNA
binding activity. Further experiments are required to clarify the signific~n~e of these observations although a possible physiological explanation may be that these interactions allow p53 and MDM2 to inflllrnre the levels of functional DP-1/E2F DNA binding heterodimers and hence transcriptionally 30 active DRTFI/E2F, and thus regulate the cl~wll~L-ealll transcriptional activity of target genes.

Co-expression of p53 specifically inactivated transcription driven by the DP-1/E2F-I heterodimer.
Given the earlier conclusion a potential model to explain these results would be that p53 holds DP-1 in a state which prevents it interacting with an E2F family member to form a DP-1/E2F heterodimer.
Indeed, the region is DP-l required to interact with pS3 is necessary to form a DP-l/E2F-1 heterodimer and thus binding of p53 to DP-l could be mutually exclusive with ~he interaction of DP-1 with E2F family members. Evidence for such a possibility was obtained by drlllull~ dLhlg that pS3 5 and E2F-1 can compete for DP-l and, consequently, reduce the level of DP-1/E2F-1 DNA binding activity. These data are compatible with a model in which 53 targets an immunochrmic~lly distinct form of DP-l, regulating the formation of DP-1/E2F heterodimers and hence the level of DRTF1/E2F
DNA binding activity.

The region in p53 necessary for the interaction with DP-l incudes residues frequently altered in 10 naturally-occurring mutant alleles. Based on the results reported here, a potential biological rationale for these mutations could be envisaged to be that they prevent p53 from interacting with DP-1 and thus relieve the negative regulation imposed by p53 on the formation of functional DRTFl/E2F and hence cell cycle progression.

A ~ .dy for p53-m~ te~l growth arrest.
15 Although p53 is believed to possess the properties of a transcription factor and the trans activation of target genes. such as gadd45 and WAFI (Kastan et al.. 1992; El-Deiry et al., 1993), thought to be important in p53-mr~ ted growth arrest, the interaction of p53 with DP-I provides another potential pathway through which p53 theoretically can infl~ nre cell cycle progression. Thus, since many of the genes regulated by DRTFl/E2F encode proteins required for cell cycle progression, their 20 transcriptional down-regulation would be expected to impede cell cycle progression. Conversely, an increase in the activity of DRTFI/E'F may be important in mrrii~ting the physiological effects of the products of the MDM2 oncogene.

Indeed, a variety of previous studies already have suggested that the pathways regulated by DRTFl/E2F and p53 are integrated. For example, ovel~ ession of E2F-l in cells induces apoptosis 25 in a p53-dependent fashion (Wu and Levine, 1994), and increased levels of apoptosis in the lens fibre cells of Rb-'- mice is overcome in embryos which are doubly-null in Rb and pS3. Similar conclusions have been made from studies in which the oncoproteins of tumour viruses, which can inactivate pRb - or p53, are sequentially targetted to defined physiological sites (Howes et al., 1994; Morgenbesser et al., 1994; Pan and Griep, 1994). Overall, such studies are compatible with a model in which p53 30 monitors in some way the status of the DRTF1/E2F pathway. It is possible that the association between DP-l and pS3 is involved this process.

Finally, the interaction of DP-1 and p53 may help explain the ~ ." through which DP-1 exerts proto-oncogenic activity, a property shared by other ~ lllb~l~ of the DP family, and one lll~irt~ in the absence of a co-transfected E2F family member (Jooss et al., 1995). Possibly an increased level of DP-l sequesters p53, titrating out its activity, and thus over-riding the growth-regulating effects 5 of p53. In this respect, DP-I may act in an analogous fashion to certain viral onco~lote~ s, such as the adenovirus Elb and papilloma virus E6 proteins, since their ability to inactivate pS3 correlates with oncogenic ac~ivity (Moran. 1993).

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Claims (16)

1. An assay for identifying a potential or putative chemotherapeutic agent, the assay comprising:
(i) bringing into contact DP protein, a p53 protein and said agent under conditions in which the DP and p53 protein in the absence of said agent are capable of forming a complex; and (ii) measuring the extent to which said agent is able to disrupt or interfere with the complex, inhibit or encourage the binding of said DP and p53 proteins to each other, or affect the activity of said complex.

2. An assay according to claim 1 wherein said DP protein is DP- 1.

3. An assay for identifying a potential or putative chemotherapeutic agent, the assay comprising:
(i) bringing into contact an E2F monomer protein, a p53 protein and said agent under conditions in which the E2F and p53 protein in the absence of said agent are capable of forming a complex; and (ii) measuring the extent to which said agent is able to disrupt or interfere with the complex, inhibit or encourage the binding of said E2F and p53 protein to each other, or affect the activity of said complex.

4. An assay according to claim 1, 2 or 3 wherein said p53 protein is brought into contact with a heterodimer of a Dp and E2F protein.

5. An assay according to claim 4 wherein the complex is measured its ability to bind to an E2F DNA binding site in vitro.

6. An assay according to claim 4 wherein the complex is measured by its ability to activate in vivo a promoter comprising an E2F DNA binding site linked to a reporter gene.

7. An assay according to claim 6 wherein the assay is performed in a yeast, insect or mammalian cell.

8. An assay according to any one of claims 1 to 7 wherein said agent is a fragment of 10 or more amino acids of 1 DP- or E2F monomer- protein, or a mutant, allelic variant or species homologue thereof.

9. A polynucleotide vector comprising a first coding sequence encoding a DP protein and a second coding sequence encoding a p53 protein.

10. A polynucleotide vector comprising a first coding sequence encoding E2F
monomer protein and a second coding sequence encoding a p53 protein.

11. A vector according to claim 9 or 10 wherein said first and second coding sequences are each operably linked to a promoter.

12. A vector according to claim 9 or 10 wherein said first and second coding sequences are linked by a linker sequence encoding a polypeptide linker which joins the proteins encoded by the first and second coding sequences.

13. A host cell which comprises a vector according to any one of claims 9 to 12

14. A host cell which comprises a first recombinant vector comprising a coding sequence encoding a DP protein and a second recombinant vector comprising a coding sequence encoding a p53 protein.

15. A host cell which comprises a first recombinant vector comprising a coding sequence encoding an E2F monomer protein and a second recombinant vector comprising a coding sequence encoding a p53 protein.

16. All isolated complex comprising one or both of a DP and/or E2F monomer protein in association with a p53 protein.