WO1999029334A1 - CtIP, A NOVEL PROTEIN THAT INTERACTS WITH CtBP AND USES THEREFOR - Google Patents

Abstract

A novel human protein, CtIP, that interacts with the human cellular protein CtBP, and the nucleotide sequence encoding CtIP are provided. CtIP is useful in diagnostic methods for determining malignancy of cells and in methods for inhibiting neoplasia in patients. Use of CtIP in a method for identifying agents that can inhibit neoplasia of cells is also disclosed.

Description

CtIP, A Novel Protein that Interacts with CtBP and Uses Therefor

Reference to Government Support

This invention was made with U.S. Government support under Grant No. CA-33616 from the National Cancer Institute. The U.S. Government has certain πghts in the invention.

Reference to Related Applications

This application claims priority to, and incorporates herein by reference, U.S. Provisional Application Serial No. 60/069362 filed December 12, 1997

Background of the Invention ( 1 ) Field of the Invention

This invention generally relates to the field of cancer and, more particularly, to a method and compositions for diagnosing the malignancy of a tumor as well as to compositions and methods for suppressing tumor growth.

(2) Descπption of Related Art The Ela region of human adenovirus types 2 and 5 encodes two major proteins of 289 amino acids and 243 ammo acids (289R and 243R) that differ only by an internal 46 ammo acid region unique to the 289R protein. These El A proteins immortalize pπmary animal cells and cooperate with other cellular and viral oncogenes in oncogemc transformation. These activities are dictated by the binding of several cellular proteins with the El A proteins. For example, the N-termmal half of El A proteins, which is encoded by exon 1 of the El a gene, interacts with cellular growth-regulatory proteins such as the retmoblastoina gene product (pRb) and related proteins (pl07 and pl30), as well as p300, a CREB binding protem-related transcription factor implicated in transcπptional repression of certain genes (Moran, supra; Eckner et al., Genes Dev 8:867-884, 1993; Arany et al., Nature 374:81-84, 1995; Lundblad et al., Nature 374:85-88, 1995). The interaction between E1A proteins and the cellular proteins pRb, pi 07 and pi 30 cause these cellular proteins to release the E2F transcription factor, thus activating gene expression. For review, see Dyson and Harlow, Cancer Surveys 12: 161-195,

1992, Nevms, J. R., Science 258. 424-429, 1992; Moran, E , Curr Op Gen Dev 3, 63-70,

1993, Mymryk, J S., et al., Int. J One 4, 2131-2141, 1994 Interaction of E1A proteins with p300 releases C-CAF, a cellular acetyl transferase, from the p300/C-CAF complex resulting m activation of transcription by chromatm remodeling (Yang et al., Nature 382:319-324, 1996). Thus, the transforming activities encoded by exon 1 of the adeno virus El a region appear to be linked to interactions with cellular proteins and the resulting regulation of transcription.

Although the functions of exon 2-encoded domains of El A proteins have been studied less intensively, these domains have been implicated m certain positive and negative transcπptional regulatory activities (Linder et al., Oncogene 7:439-443, 1992; Bondesson et Ά\., EMBO J 11:3347-3354, 1992; Mymryk, J.S. et al., J Virol 67 6922-2928, 1993). Exon 2 is required for immortalization (Subramanian, T., et al., Oncogene 4:415-520, 1989; Qumlan, M.P. et al., J Virol 66:2020-2040, 1992) and induction of Ad2/5-specιfic cytotoxic lymphocytes (Urbanelli et al, Virol 173.607-614, 1989). In addition, exon 2 influences the extent of oncogemc transformation. Deletions within the C-termmal 67 ammo acids of the El A 243R protein enhance El A/T24 ras cooperative transformation (Subramanian, supra, Douglas, J.L., et al., Oncogene 6:2093-2103, 1991), and tumoπgenesis of transformed cells m syngeneic and athymic rodent models (Subramanian, supra). Importantly, exon 2 also plays a role in tumor metastasis. Expression of wt El A efficiently suppresses the metastatic potential of tumor cells (Pozzatti, R. et al., Science 232:223-227, 1986; Pozzatti, R., et al., Mol Cell Bwl 8:2984-2988, 1988, Steeg et al., Cancer Res 48:6550-6554, 1988). In contrast, cells expressing El A proteins lacking the C-termmal 67 ammo acids are highly metastatic (Linder et al , supra; Subramanian, supra). Thus, the E1A protein region encoded by exon 2 appears to negatively modulate in vitro transformation, tumoπgenesis and metastasis. These activities have been localized within a 14 ammo acid region (residues 225 to 238) near the C-termmus of the 243R protein (Boyd, J. M., et al., EMBO J 12:469-478, 1993). These transformation restraining activities of the C-termmal region of ElA correlate with the interaction of a 48 kD cellular phosphoprotem termed CtBP (ElA C-termmal Binding Protein) (Boyd et al., supra) CtBP binds to ElA proteins via a 5 ammo acid motif, PLDLS, which corresponds to residues 233-237 of the 243R protein. ElA mutants having amino acid substitutions withm this motif do not form complexes with CtBP (Schaeper, U., et al., Proc Natl Acad Sci 92:10667- 10671, 1995) and do not have oncogenesis-restraming activities (Schaeper et al., J Bwl Chem. 273:8549-8552, 1998).

Although interaction of adenovirus ElA proteins with the cellular protein CtBP appears to lead to suppression of tumoπgenesis and tumor metastasis of cells transformed with ElA and activated T24 Ras oncogene, over expression of CtBP by itself does not appear to exert a significant effect on tumoπgenesis and tumor metastasis. One explanation for the inability to detect the effect of CtBP activity could be the fact that expression levels of endogenous CtBP are relatively high CtBP was found to be abundantly expressed in a vaπety of human and mouse tissues as well as tissue culture cell lines (Boyd et al., supra). If CtBP functions m a complex with other cellular proteins, overexpression of recombmant CtBP may not cause an effect if cellular cofactors are rate limiting. Thus, to understand the role of CtBP in modulating oncogenesis, it would be useful to identify and characterize cellular proteins that interact with CtBP.

Summary of the Invention

Briefly, therefore, the present invention is directed to the identification and isolation of substantially puπfied proteins that bind to the cellular protein CtBP. Accordingly, the inventor herein has succeeded in discoveπng a novel human CtBP-bmdmg protein, which is designated CtIP for CtBP-Interactmg Protein. The inventor herein has also discovered that CtIP contains the same five ammo acid motif (PLDLS) found in adenovirus ElA proteins to which CtBP binds and that deletion of this binding motif in CtIP abolishes its binding to CtBP.

The invention thus provides isolated and puπfied CtIP polypeptides. A preferred CtIP polypeptide identified herein comprises the human CtIP ammo acid sequence shown in Fig. 3 (SEQ ID NO:2).

The present invention also provides isolated polynucleotides encoding a CtIP polypeptide. Preferred polynucleotides identified herein encode the ammo acid sequence shown in Fig. 3. A particularly preferred polynucleotide compπses SEQ ID NO:3. A recombinant cell compπsmg a polynucleotide encoding for expression a CtIP polypeptide is also withm the scope of this invention. The recombinant cell can be used in a method for producing CtIP.

In another embodiment, the invention provides isolated polynucleotides compπsmg a human nucleotide sequence complementary to a nucleotide sequence encoding a CtIP polypeptide or CtIP fragment. A preferred complementary sequence is SEQ ID NO:4 (Fig. 2B, bottom strand). The invention also provides isolated polynucleotides that specifically hybridize to polynucleotides consisting of SEQ ID NO:l, SEQ ID NO:3 or SEQ ID NO:4 These complementary and hybridizing polynucleotides can be used m methods for detecting the CtIP gene and transcription products thereof, as well as m isolating CtlP-encodmg polynucleotides from other mammalian and nonmammahan species.

In yet another embodiment, the present invention provides a composition compπsmg a CtIP polypeptide or fragment and a carrier that facilitates delivery of the CtIP polypeptide or fragment into a target cell. The present invention also provides polyclonal and monoclonal antibodies that specifically react with CtIP or CtIP fragments and methods for purifying CtIP or detecting its expression using such antibodies.

A method for determining malignancy of a cell in a patient is also provided. The method compπses detecting CtIP expression m the cell, wherein an amount of CtIP expression that is lower than the amount m normal cell indicates the cell is malignant. In one embodiment, the method comprises detecting a CtIP polypeptide with an antibody that specifically reacts with CtIP or a fragment thereof. In other embodiments, the method comprises detecting CtIP mRNA with a polynucleotide probe or by amplifying a target sequence in CtIP mRNA. In another embodiment, the invention provides a method for inhibiting neoplasia of target cells in a patient which compπses treating the patient with an effective amount of a CtIP polypeptide or fragment. The patient may be treated by admmisteπng the CtIP polypeptide or biologically active fragment to the patient or by admmisteπng to the patient a polynucleotide encoding the CtIP polypeptide or fragment, through which CtIP or fragment is expressed in the target cells. The invention also provides a method for identifying agents that inhibit neoplasia of cells which involves determining whether a candidate agent disrupts binding of CtIP and CtBP.

Among the several advantages found to be achieved by the present invention, therefore, may be noted the provision of a new protein, CtIP, which is useful in a method for inhibiting neoplasia of cells; the provision of polynucleotides encoding CtIP; the provision of methods for obtaining CtIP by recombinant techniques; the provision of methods for determining the malignancy of a target cell in a patient; the provision of methods for identifying agents that inhibit neoplasia, and the provision of methods that can detect alterations in the CtIP gene. Brief Description of the Drawings

Figure 1 illustrates expression of CtIP m human cancer cell lines showing a northern blot of RNA prepared from human cancer cell lines (Clontech) analyzed using [³²P]-labeled probes derived from the cDNA clone pGAD#15 or Actm cDNA

Figure 2A illustrates the nucleotide sequence of a CtIP cDNA (SEQ ID NO:l). Figure 2B illustrates the nucleotide sequence encoding CtIP and its complementary sequence (SEQ ID NOS:3 and 4, respectively). Figure 3 illustrates the predicted am o acid sequence of CtIP.

Figure 4 illustrates that CtBP binds to immobilized GST-CtIP protein Figure 5 illustrates a sequence alignment of CtIP with ElA proteins from different adenoviruses, with the boxed residues designating the PLDLS sequence motif that is conserved among ElA proteins and essential for CtBP interaction Figure 6 illustrates the effect of a PLDLS mutation on CtBP interaction, showing m Fig

6A the substitution of residues 490 to 494 of CtIP (PLDLS) with non conserved residues

(LASQC) in a CtIP mutant (CtlPΔ) and m Fig. 6B an autoradiograph of binding products detected in an in vitro binding assay and analyzed by SDS/polyacrylamide gel (10%) elecfrophoresis. Figure 7 illustrates coimmunoprecipitation of CtBP and CtIP from BSC40 cells co- expressmg T7-epιtope tagged CtBP and CtIP or CtlPΔ in BSC40 cells showing an autoradiograph of proteins precipitated with T7 mAb (Novagen) or CtIP antiserum (anti-CtIP) and analyzed by SDS/polyacrylamide gel (8%) electrophoresis.

Figure 8 illustrates that ElA and CtIP compete for CtBP interaction showing an autoradiograph of bound proteins analyzed by SDS/polyacrylamide gel (10%) electrophoresis.

Detailed Description of the Preferred Embodiments

The present invention is based upon the identification, isolation and sequencing of cDNA clones that encode a novel human protein that binds the cellular protein CtBP, which the inventor has named CtIP. As descπbed in more detail below, CtIP was discovered by yeast- two hybrid screening of a cDNA library for GAL4 fusion proteins that bind to CtBP which identified a partial cDNA encoding a human cellular protein that binds to CtBP. The 5 'end of the CtIP cDNA sequence was obtained by rapid amplification of the 5 'ends of the human mRNA. The composite cDNA sequence consists of 3247 bp (Fig. 2) and contains a nucleotide sequence (Fig. 2A) which encodes the CtIP protein of 897 amino acids (Fig. 3)

As shown in the examples below, CtIP binds with CtBP and this binding is disrupted by adenovirus ElA proteins binding to CtBP. As the CtBP-bindmg region of El A has been implicated in transcπptional regulatory activity encoded by exon 1 (Sollerbrant, et al., Nucl Acids Res , 24:2578—2584, 1996), it is believed that CtIP has a transcπptional regulatory activity that plays a role in the observed oncogenesis-restrammg activity of the C-termmal region of ElA proteins. A data bank search based on sequence properties (PROPSEARCH, Hobohm et al., J. Mol BioL. 251:390-399, 1995) has revealed that CtIP shares similaπties with certain mammalian proteins involved in DNA repair. Thus, it is believed that CtIP may suppress neoplasia or oncogenesis through DNA repair and/or transcπptional regulatory activities.

In one embodiment, the invention provides CtIP polypeptides and fragments thereof. As used herein, the term "CtIP" means both CtIP polypeptides and CtIP fragments. Reference to a CtIP polypeptide herein is intended to be construed to include polypeptides of any origin which are substantially homologous to and which are biologically equivalent to the human CtIP characterized and descπbed herein. Such substantially homologous polypeptides may be native to any tissue or species and, similarly, biological activity can be characteπzed in any of a number of biological assay systems. The term "biologically equivalent" is intended to mean that a CtIP polypeptide of the present invention is capable of demonstrating some or all of the tumor suppressing or CtBP- binding properties m a similar fashion, although not necessaπly to the same degree, as the recombmantly produced human CtIP identified herein. By "substantially homologous" it is meant that the degree of sequence identity between human CtIP and a CtIP ortholog from another mammalian species is at least about 75% sequence identity and between human and non-mammalian orthologs is at least about 65% identity.

Sequence identity or percent identity is intended to mean the percentage of same residues between two sequences aligned using the Clustal method (Higgins et al, Cabios 5:189-191, 1992) of multiple sequence alignment in the Lasergene biocomputmg software (DNASTAR, INC, Madison, WI). In this method, multiple alignments are carried out in a progressive manner, in which larger and larger alignment groups are assembled using similarity scores calculated from a series of pairwise alignments. Optimal sequence alignments are obtained by finding the maximum alignment score, which is the average of all scores between the separate residues in the alignment, determined from a residue weight table representing the probability of a given amino acid change occurπng in two related proteins over a given evolutionary interval Penalties for opening and lengthening gaps in the alignment contribute to the score The default parameters used with this program are as follows: gap penalty for multiple alignment = 10; gap length penalty for multiple alignment = 10, k-tuple value in pairwise alignment = 1; gap penalty in pairwise alignment = 3; window value in pairwise alignment ^•= 5; diagonals saved in pairwise alignment = 5. The residue weight table used for the alignment program is PAM250 (Dayhoff et al., in Atlas of Protein Sequence and Structure, Dayhoff, Ed., NBRF, Washington, Vol. 5, suppl. 3, p. 345, 1978).

To determine percent sequence identity between two sequences, the number of identical ammo acids m the aligned sequences is divided by the total number of amino acids in the reference sequence. As used herein, the reference sequence is human CtIP when determining its percent identity with an ortholog from another species or with an engineered CtIP polypeptide. Percent conservation is calculated by adding the number of identical residues to the number of positions at which the two residues represent a conservative substitution (defined as having a log odds value of greater than or equal to 0.3 in the PAM250 residue weight table) and dividing by the total number of ammo acids in the reference sequence Preferred conservative ammo acid changes are: R-K; E-D, Y-F, L-M; V-I, Q-H.

Also included withm the meaning of substantially homologous is any CtIP polypeptide isolated by virtue of cross-reactivity with antibodies specific to human CtIP or whose encoding nucleotide sequences, including genomic DNA, mRNA or cDNA, may be isolated through hybridization with the complementary sequences shown m Fig. 2B or fragments thereof. It will also be appreciated by one skilled m the art that naturally occurπng allehc variants of the human CtIP sequence disclosed herein may exist and such allehc vaπants are also intended to be included in the present invention.

Conservatively substituted CtIP proteins retaining the biological activity of naturally occuπing CtIP are also withm the scope of the present invention. Conservative ammo acid substitutions refer to the lnterchangeabihty of residues having similar side chains. Conservatively substituted ammo acids can be grouped according to the chemical properties of their side chains. For example, one grouping of ammo acids includes those amino acids have neutral and hydrophobic side chains (A, V, L, I, P, W, F, and M); another grouping is those ammo acids having neutral and polar side chains (G, S, T, Y, C, N, and Q); another grouping is those amino acids having basic side chains (K, R, and H); another grouping is those ammo acids having acidic side chains (D and E); another grouping is those ammo acids having aliphatic side chains (G, A, V, L, and I); another grouping is those ammo acids having ahphatic-hydroxyl side chains (S and T); another grouping is those ammo acids having amine-contammg side chains (N, Q, K, R, and H); another grouping is those ammo acids having aromatic side chains (F, Y, and W); and another grouping is those ammo acids having sulfur-contaming side chains (C and M). Preferred conservative ammo acid substitutions groups are: R-K; E-D, Y-F, L-M; V-I, and Q-H

As used herein, a CtIP polypeptide can also include modifications of the human CtIP ammo acid sequence identified herein, including sequences m which one or more ammo acids have been inserted, deleted or replaced with a different ammo acid or a modified or unusual ammo acid, as well as modifications such as glycosylation or phosphorylation of one or more ammo acids so long as the polypeptide containing the modified sequence retains the biological activity of CtIP. Inserted or deleted ammo acιd(s) can be added to or removed from the N-termmus, C-termmus or withm the naturally-occumng amino acid sequence. By retaining the biological activity, it is meant that the modified polypeptide can suppress neoplasia when expressed in a cell although not necessarily to the same degree as that of naturally occurπng human CtIP. As used herein "neoplasia" means the conversion of normal cells into benign or malignant tumor cells and thus includes tumoπgenesis, oncogenesis and related terms, and is also intended to include metastasis.

Fragments of CtIP are also encompassed by the present invention. Such fragments may be of any length but preferably retain the biological activity of CtIP or are antigenic. The minimum length of such biologically active or antigenic fragments can readily be determined by those skilled in the art using known techniques. Antigenic fragments are capable of eliciting CtlP-specific antibodies when administered to a host animal and includes those smaller fragments that require conjugation to a carrier molecule to be lmmunogenic. Typically, antigenic fragments will be at least 5 or 6 ammo acids in length and may be any length up to the length of human CtIP. Preferably, an antigenic fragment comprises 10 to 12 ammo acids of SEQ ID NO: 2 and more preferably, an antigenic fragment will compπse at least 15 to 20 ammo acids, or more, or SEQ ID NO:2.

It is also believed that particular discrete fragments of CtIP comprising the CtBP-bindmg motif PLDLS, or analogues thereof, can serve to inhibit CtBP from binding to CtIP, thereby making more unbound CtIP available to suppress neoplasia of cells. Such CtBP-bmdmg inhibitor fragments are also included withm the scope of the invention. One preferred binding inhibitor fragment is shown m Fig. 5 and consists of SEQ ID NO: 11. Another preferred fragment consists of the pentapeptide PLDLS (SEQ ID NO: 12).

The present invention also includes non-peptidal substances such as peptide mimetics which possess the bmdmg-mhibitmg activity of CtIP fragments The techniques for development of peptide mimetics are well known in the art. (See for example, Navia and Peattie, Trends Pharm Sci 14 189-195, 1993; Olson et al, J Med Chem 36-3039-3049 which are incorporated by reference) Typically this involves identification and characteπzation of the interaction between a protein target and its peptide hgand using X-ray crystallography and nuclear magnetic resonance technology For example, it is believed that at least one target protein for CtIP peptides is CtBP. Using information on a normal peptide-protem complex along with computeπzed molecular modeling, a pharmacophore hypothesis is developed and analogue compounds are made and tested in an assay system.

A prefeπed CtIP polypeptide according to the present invention is prepared by recombinant DNA technology although it is believed that CtIP can be isolated in purified form from human cells. By "pure form" or "puπfied form" or "substantially puπfied form" it is meant that a CtIP composition is substantially free of other proteins which are not CtIP

Preferably, a substantially puπfied CtIP composition comprises at least about 50 percent CtIP on a molar basis compared to total proteins or other macromolecular species present More preferably, a substantially punfied CtIP composition will comprise at least about 80 to about 90 mole percent of the total protein or other macromolecular species present and still more preferably, at least about 95 mole percent or greater.

Recombinant CtIP may be made by expressing the DNA sequences encoding CtIP in a suitable transformed host cell Using methods well known m the art, the DNA encoding CtIP may be linked to an expression vector, transfected into a host cell and conditions established that are suitable for expression of CtIP by the transfected cell. Any suitable expression vector may be employed to produce recombinant CtIP such as, for example, the mammalian expression vector pCB6 (Brewer, Meth Cell Biol 43.233-245, 1994) or the E coh pET expression vectors, specifically, pET-30a (Studier et al., Methods Enzymol 185:60-89, 1990) Other suitable expression vectors for expression in mammalian and bacterial cells are known in the art as are expression vectors for use in yeast or insect cells Baculovirus expression systems can also be employed.

A number of cell types may be suitable as host cells for expression of recombinant CtIP. Mammalian host cells include, but are not limited to, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo 205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of pπmary tissue, pπmary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK and Jurkat cells. Yeast strains that may act as suitable host cells include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, and any other yeast strain capable of expressing heterologous proteins. Host bacterial strains include Escherichia coh, Bacillus subtihs, Salmonella typhimurium and any other bacterial strain capable of expressing heterologous proteins If the polypeptide is made in yeast or bacteria, it may be necessary to modify the polypeptide, for example, by phosphorylation or glycosylation of the appropriate sites using known chemical or enzymatic methods, to obtain a biologically active polypeptide.

A polypeptide according to the invention may also be expressed m transgenic animals, e.g., cows, goats, pigs, or sheep whose somatic or germ cells contain a nucleotide sequence encoding human CtIP or vaπant thereof.

The expressed CtIP polypeptide can be purified using known purification procedures, such as gel filtration and ion exchange chromatography. Purification may also include affinity chromatography using an agent that will specifically bind the CtIP polypeptide, such as a polyclonal or monoclonal antibody raised against CtIP or fragment thereof. Other affinity resms typically used in protein purification may also be used such as concanavahn A- agarose, heparm-toyopearl^® or Cibacrom blue 3GA Sepharose^®. Purification of CtIP can also include one or more steps involving hydrophobic interaction chromatography using such resms as phenyl ether, butyl ether, or propyl ether. It is also contemplated that a CtIP polypeptide may be expressed as a fusion protein to facilitate purification. Such fusion proteins, for example, include a CtIP amino acid sequence fused to a histidine tag such as when expressed m the pET bacterial expression system as well as the CtIP ammo acid sequence fused to the ammo acid sequence of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxm (TRX). Similarly, the polypeptide of the invention can be tagged with a heterologous epitope and subsequently purified by lmmunoaffmity chromatography using an antibody that specifically binds such epitope. Kits for expression and purification of such fusion proteins and tagged proteins are commercially available.

CtIP and fragments thereof may also be produced by chemical synthesis using methods known to those skilled m the art.

The present invention also encompasses isolated polynucleotides comprising nucleotide sequences that encode any of the CtIP polypeptides descπbed herein. As used herein, a polynucleotide includes DNA and/or RNA and thus the nucleotide sequences recited m the Sequence Listing as DNA sequences also include the identical RNA sequences with uracil substituted for thymine residues. Prefeπed nucleotide sequences included m the invention are those encoding the human CtIP ammo acid sequence shown in Fig. 3. Particularly preferred polynucleotides comprise SEQ ID NO:3. It is understood by the skilled artisan that degenerate nucleotide sequences can encode the CtIP ammo acid sequences described herein and these are also intended to be included withm the present invention. Such degenerate nucleotide sequences include modifications of naturally-occumng sequences m which at least one codon is substituted with a coπespondmg redundant codon preferred by a given host cell, such as E coh or insect cells, so as to improve expression of recombinant CtIP therein. Polynucleotides withm the scope of this invention do not include isolated chromosomes. The present invention also encompasses vectors comprising an expression regulatory element operably linked to any of the CtlP-encodmg nucleotide sequences included withm the scope of the invention. This invention also includes host cells, of any variety, that have been transformed with such vectors.

In yet another embodiment, a polynucleotide which specifically hybridizes to a human CtlP-encodmg polynucleotide or to its complement is provided. Specific hybridization is defined herein as the formation of hybπds between a polynucleotide, including ohgonucleotides, and a specific reference polynucleotide (e.g., a polynucleotide comprising a nucleotide sequence encoding human CtIP ) wherein the polynucleotide preferentially hybridizes to the specific reference polynucleotide over other non CtIP polynucleotides. Specifically hybπdizmg ohgonucleotides are typically at least 15 nucleotides m length and are preferably at least 17 to at least 20 nucleotides long Other preferred lengths include at least 22 to at least 25 nucleotides. Specific hybπdization is preferably done under high stπngency conditions which, as well understood by those skilled in the art, can readily be determined by adjusting several factors during hybridization and during the washing procedure, including temperature, ionic strength, length of hybridization or washing times, and concentration of formamide (see for example, Sambrook et al , 1989, supra)

The present invention also includes nucleic acid sequences which encode for CtIP polypeptides that have CtBP binding activity and that preferentially bind anti -human CtIP antibodies over other antibodies that do not bind to human CtIP.

Methods are also provided herein for producing recombinant CtIP polypeptides The method involves culturmg a cell which contains an expression vector comprising a nucleotide sequence encoding a CtIP polypeptide and isolating the expressed CtIP polypeptide. The present invention also includes therapeutic or pharmaceutical compositions comprising a CtIP polypeptide in an effective amount for suppressing neoplasia of target cells in a patient and a method comprising administering a therapeutically effective amount of the CtIP polypeptide to a cell ex vivo or in vivo. The compositions and methods are useful for treating a vaπety of diseases including but not limited to hyperplasia, neoplasia, lymphoprohferative diseases, autoimmune disorders, transplant rejection, and the like.

In certain circumstances, it may be desirable to modulate or decrease the amount of CtIP expressed. Thus, m another aspect of the present invention, CtIP anti-sense ohgonucleotides can be made and a method utilized for diminishing the level of expression of CtIP, respectively, by a cell comprising admmisteπng one or more CtIP anti-sense ohgonucleotides By CtIP anti-sense ohgonucleotides reference is made to ohgonucleotides that have a nucleotide sequence that interacts through base paiπng with a specific complementary nucleic acid sequence involved in the expression of CtIP such that the expression of CtIP is reduced. Preferably, the specific nucleic acid sequence involved in the expression of CtIP is a genomic DNA molecule or mRNA molecule that contains sequences of the CtIP gene. Thus, the invention contemplates CtIP anti-sense ohgonucleotides that can base pair to flankmg regions of the CtIP gene, untranslated regions of CtIP mRNA or the coding sequence for CtIP. The term complementary to a nucleotide sequence m the context of CtIP antisense ohgonucleotides and methods therefor means sufficiently complementary to such a sequence as to allow hybridization to that sequence in a cell, l e., under physiological conditions. The CtIP antisense-ohgonucleotides preferably comprise a sequence containing from about 8 to about 100 nucleotides and more preferably the CtIP antisense ohgonucleotides compπse from about 15 to about 30 nucleotides The CtIP antisense ohgonucleotides can also contain a variety of modifications that confer resistance to nucleolytic degradation such as, for example, modified mternucleoside linkages (Uhlmann and Peyman, Chemical Reviews 90:543-548, 1990; Schneider and Banner, Tetrahedron Lett 57:335, 1990), modified nucleic acid bases and/or sugars and the like.

The therapeutic or pharmaceutical compositions of the present invention can be administered by any suitable route known m the art including for example intravenous, subcutaneous, intramuscular, transdermal, mtrathecal or mtracerebral. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulation. For treating tissues in the central nervous system, administration can be by injection or infusion into the cerebrospmal fluid (CSF) When it is intended that CtIP be administered to cells m the central nervous system, administration can be with one or more agents capable of promoting penetration of CtIP across the blood-bram barrier.

CtIP can also be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. For example, CtIP can be coupled to any substance known in the art to promote penetration or transport across the blood-bram barrier such as an antibody to the transferrm receptor, and administered by intravenous injection. (See for example, Fπden et al., Science 259:373-377, 1993). Furthermore, CtIP can be stably linked to a polymer such as polyethylene glycol to obtain desirable properties of solubility, stability, half-life and other pharmaceutically advantageous properties. (See for example Davis et al Enzyme Eng 4. 169-73, 1978; Burnham, Am JHosp Pharm 57:210-218, 1994). Preferably, CtIP is administered with a carrier such as hposomes or polymers containing a targeting moiety to facilitate delivery of CtIP to targeted cells. Examples of targeting moieties include but are not limited to antibodies, hgands or receptors to specific cell surface molecules. The CtIP polypeptide or fragment can also be modified to include a specific transit peptide that facilitates dehveπng CtIP into the cytoplasm of cells Examples of such transit peptides include but are not limited to the TAT protein from HIV-1 (Frankel et al., Cell 55: 1189-1193, 1988; Fawell et al, Proc. Natl. Acad. Sci USA 91:664-668, 1994; Ezhevsky Proc Natl Acad. Sci. USA 94: 10699-10704, 1997), the third helix of the Antennapedia homeodomam (Derossi et al., J. Biol Chem 271:18188-18193, 1996), and penetratms, which are 16 mer peptides derived from the Antennapedia homeodomam (Derossi et al. Trends Cell Biol 8-84-87, 1998). Alternatively, CtIP can be delivered directly into target cells by micromjection.

For nonparenteral administration, the compositions can also include absorption enhancers which increase the pore size of the mucosal membrane. Such absorption enhancers include sodium deoxycholate, sodium glycocholate, dimethyl-β-cyclodextrm, lauroyl-1- lysophosphatidylcholme and other substances having structural similaπties to the phosphohpid domains of the mucosal membrane.

The compositions are usually employed in the form of pharmaceutical preparations Such preparations are made a manner well known in the pharmaceutical art. One preferred preparation utilizes a vehicle of physiological saline solution, but it is contemplated that other pharmaceutically acceptable caπiers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, steπle water or the like may also be used. It may also be desirable that a suitable buffer be present in the composition. Such solutions can, if desired, be lyophihzed and stored m a steπle ampoule ready for reconstitution by the addition of steπle water for ready injection. The pπmary solvent can be aqueous or alternatively non- aqueous. CtIP can also be incorporated into a solid or semi-solid biologically compatible matrix which can be implanted into tissues requiring treatment

The earner can also contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolaπty, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation. Similarly, the earner may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-bram barπer. Such excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion into the cerebrospmal fluid by continuous or peπodic infusion. Dose administration can be repeated depending upon the pharmacokmetic parameters of the dosage formulation and the route of administration used.

It is also contemplated that certain formulations containing CtIP are to be administered orally. Such formulations are preferably encapsulated and formulated with suitable earners in solid dosage forms. Some examples of suitable earners, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, algmates, calcium silicate, microcrystallme cellulose, polyvmylpyrrohdone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like The formulations can additionally include lubneating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoπng agents. The compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art. The formulations can also contain substances that dimmish proteolytic degradation and promote absorption such as, for example, surface active agents The specific dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied. The dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropπate dosage for treatment is routinely made by those of ordinary skill in the art. Exact dosages are determined in conjunction with standard dose-response studies It will be understood that the amount of the composition actually administered will be determined by a practitioner, m the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the seventy of the patient's symptoms, and the chosen route of administration. The invention also provides a method for determining malignancy of at least one cell in a patient which compπses detecting CtIP expression in the cell. Typically, the method involves detecting CtIP expression in a sample obtained from a patient tissue known or suspected to be cancerous. The term "determining malignancy" as used herein m the context of a patient with neoplastic disease is intended to include the estimation of prognosis in terms of probable outcome of the disease and prospect for recovery, the momtoπng of the disease status or the recurrence of the disease, or the determining of a prefeπed therapeutic regimen for the patient. For example, as discussed above, it is believed that Adenovirus ElA proteins compete with CtIP for interaction with CtBP and the resulting increase m the amount of CtIP that is free of CtBP plays a role m the oncogenesis-restraming activity of ElA proteins. Thus, the amount of CtIP that is unbound to CtBP m a cancer cell relative to the amount in normal cells would be expected to be indicate whether a cell is malignant and/or the degree of malignancy. For example, a cancer which contains no detectable or only low amounts of CtIP as compared to normal cells would be expected to be more malignant and thus the prognosis poorer than for cancers m which the amount of CtIP detected is closer to or equal to the amount present in normal cells. Thus the term "detecting CtIP expression" as used herein in the context of determining malignancy of cells means detecting and/or quantifying CtIP and/or mRNA encoding CtIP, although quantifying does not require actual measurement of amounts of the protein or mRNA and may include qualitative compansons.

In one embodiment of the method, CtIP expression in a cell is detected by contacting proteins from the cell with an antibody which specifically reacts with CtIP or a CtIP fragment and detecting binding of the antibody to CtIP. Any method known in the art for detecting specific proteins can be used. Such methods include, but are not limited to lmmunodiffusion, lmmunoelectrophoresis, immunochemical methods, bmder-hgand assays, immunohistochemical techniques, agglutination and complement assays, (for example see Basic and Clinical Immunology, Sites and Ten, eds, Appleton & Lange, Norwalk, Conn, pp 217-262, 1991).

Preferred methods for detecting CtIP are bmder-hgand immunoassay methods using an antibody to human CtIP or fragment thereof. Numerous competitive and non-competitive protein binding immunoassays are well known in the art. Antibodies employed in such assays may be unlabeled, for example as used in agglutination tests, or labeled for use m a wide variety of assay methods. Labels that can be used include radionuchdes, enzymes, fluorescers, chemilummescers, enzyme substrates or co-factors, enzyme inhibitors, particles, dyes and the like for use in radioimmunoassay (RIA), enzyme immunoassays, e.g., enzyme- linked immunosorbent assay (ELISA), fluorescent immunoassays and the like. Polyclonal or monoclonal antibodies to CtIP or to an epitope thereof can be made for use in immunoassays by any of a number of methods known m the art. By epitope reference is made to an antigenic determinant of a polypeptide. An epitope could compπse 3 ammo acids in a spatial conformation which is unique to the epitope. Methods of determining the spatial conformation of ammo acids are known m the art, and include, for example, x-ray crystallography and 2 dimensional nuclear magnetic resonance. Generally an epitope comprises at least 6 contiguous ammo acids of a polypeptide.

In another embodiment, malignancy of a cell is determined by detecting CtIP mRNA the cell. CtIP mRNA may be detected by hybndizmg a polynucleotide probe to mRNA of the cell or to cDNA prepared from this mRNA. High stπngency conditions can be used in order to prevent false positives, that is hybridization to non-CtIP nucleotide sequences. When using sequences that are not perfectly complementary to a CtlP-encodmg polynucleotide or a fragment thereof, less stringent conditions could be used, however, this would be a less preferred approach because of the likelihood of false positives. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, length of time and concentration of formamide. These factors are outlined in, for example, Sambrook et al. (Sambrook, et al, 1989, supra).

In order to increase the sensitivity of detection of CtIP mRNA in a cell, a target CtIP sequence in cDNA prepared from mRNA of the cell can be amplified using any technique known in the art. Such techniques include reverse transcription/polymerization chain reaction (RT/PCR), hgase chain reaction methods, including gap LCR (G-LCR) and other vanations, or self- sustained sequence replication (3SR) and its various modifications In addition, the CtIP mRNA can be detected directly by asymmetπc gap LCR (AG-LCR). See, e.g., Leckie et al , "Infectious Disease Testing by Ligase Cham Reaction" in Molecular Biology and Biotechnology, R. A. Myers, ed, pp. 463-466, VCH Publishers, 1995.

In some instances it is desirable to determine whether the CtIP gene is intact in the patient's genome or in a particular tissue withm the patient. For example, a number of cancers are caused by or are made more malignant by the mactivation of a tumor suppressor gene such as p53, NF1, MCC, and the retmoblastoma (RB) gene. By an intact CtIP gene it is meant that there are no alterations in the gene such as point mutations, deletions, insertions, chromosomal breakage, chromosomal reaπangements and the like wherein such alteration might alter production of CtIP or alter its biological activity, stability or the like to cause cancer or make the patient more susceptible to neoplastic diseases Conversely, by a non- mtact CtIP gene it is meant that such alterations are present. Thus, one embodiment of the present invention a method is provided for detecting and characterizing any alterations in the CtIP gene. The method compnses providing a polynucleotide that specifically hybridizes to a CtIP cDNA, genomic DNA or a fragment thereof. Typically, patient genomic DNA is isolated from a cell sample from the patient and digested with one or more restriction endonucleases such as, for example, TaqI and Alul. Using the Southern blot protocol, which is well known in the art, this assay determines whether a patient or a particular tissue in a patient has an intact CtIP gene or an abnormality m the CtIP gene Hybridization to the CtIP gene would involve denaturing the chromosomal DNA to obtain a single-stranded DNA; contacting the single-stranded DNA with a gene probe associated with the CtIP gene sequence; and identifying the hybridized DNA-probe to detect chromosomal DNA containing at least a portion of the human CtIP gene. The term "probe" as used herein refers to a structure comprised of a polynucleotide which forms a hybrid structure with a target sequence, due to complementaπty of probe sequence with a sequence in the target region. The probes need not contain the exact complement of the target sequence, but must be sufficiently complementary to selectively hybridize with the strand being detected. By selective hybridization or specific hybπdization it is meant that a polynucleotide preferentially hybridizes to a target polynucleotide. Ohgomers suitable for use as probes may contain a minimum of about 8-12 contiguous nucleotides which are complementary to the targeted sequence and preferably a minimum of about 15 or 17 nucleotides although polynucleotide probes of about 20 to 25 nucleotides and up to about 100 nucleotides or even greater are withm the scope of this invention.

The CtIP gene probes of the present invention can be DNA or RNA ohgonucleotides and can be made by any method known in the art such as, for example, excision, transcription or chemical synthesis Probes may be labeled with any detectable label known m the art such as, for example, radioactive or fluorescent labels or enzymatic marker. Labeling of the probe can be accomplished by any method known in the art such as by PCR, random pnmmg, end labeling, nick translation or the like. One skilled in the art will also recognize that other methods not employing a labeled probe can be used to determine the hybridization. Examples of methods that can be used for detecting hybridization include Southern blotting, fluorescence in situ hybπdization, and smgle-strand conformation polymorphism with PCR amplification.

Hybridization is typically earned out at 25-45°C, more preferably at 32-40°C and more preferably at 37-38°C. The time required for hybπdization is from about 0.25 to about 96 hours, more preferably from about one to about 72 hours, and most preferably from about 4 to about 24 hours. CtIP gene abnormalities can also be detected by using the PCR method or any other known DNA amplification method which uses ohgonucleotides to identify a target sequence withm a longer sequence to be amplified. The term "ohgonucleotide" as used herein refers to a short strand of DNA or RNA typically ranging m length from about 8 to about 30 bases and are preferably bout 15 nucleotides to about 25 nucleotides The pnmers are selected to be substantially complementary to the strand of DNA being amplified Therefore, the primers need not reflect the exact sequence of the template, but must be sufficiently complementary to selectively hybridize or specifically hybπdize with the strand being amplified. By selective hybridization or specific hybndization it is meant that a polynucleotide preferentially hybridizes to a target polynucleotide. After amplification, the amplification product may be sequenced and the sequence analyzed by comparison with the CtIP nucleotide sequences disclosed herein to identify alterations which might change activity or expression levels or the like.

The invention also provides methods for inhibiting neoplasia of target cells in a patient comprising treating the patient with CtIP. In one embodiment, the treating step compπses administering to the patient a polynucleotide comprising a nucleotide sequence encoding CtIP operably linked to a promoter that produces expression of CtIP m the target cells The polynucleotide can comprise an expression plasmid, a retrovirus vector, an adenovirus vector, an adenovirus associated vector (AAV) or other vector used m the art to deliver genes into cells. Alternatively, the polynucleotide can be administered to the cell by micromjection. It is also contemplated that CtlP-encodmg polynucleotide can be administered by comfection with a replication-defective adenovirus expressing CtIP and another replication competent adenovirus that complements the replication defective virus to increase the expression of CtIP m the infected cells

Preferably, the polynucleotide is selectively delivered to target cells withm the patient so as not to affect other tissues. Targeted delivery of the polynucleotide can be done for example by using delivery vehicles such as polycations, hposomes or viral vectors containing targeting moieties that recognizes and binds a specific marker on the target cell. Such methods are known m the art, see, e.g., U.S. Patent No. 5,635,383 Another targeted delivery approach uses viral vectors that can only replicate in specific cell types which is accomplished by placing the viral genes necessary for replication under the transcπptional control of a response element for a transcπption factor that is only active in the target cell. See, e.g , U S Patent No. 5,698,443

In another embodiment, the patient is treated with CtIP by administering a CtIP polypeptide or CtIP fragment to the patient. Preferably, the CtIP polypeptide or CtIP fragment is administered with a carrier that facilitates its delivery into the cell, such as hposomes. The hposomes may have targeting moieties exposed on the surface such as antibodies, hgands or receptors to specific cell surface molecules to limit delivery of CtIP to targeted cells. Liposome drug delivery is known in the art (see, e.g, Amselem et al, Chem Phys Lipid (54:219-237, 1993) Alternatively, one or more of the polypeptides of the complex can be modified to include a specific transit peptide that is capable of delivering CtIP into the cytoplasm of a cell or CtIP can be delivered directly into a cell by micromjection.

Also included withm the invention is a method for identifying agents that inhibit neoplasia of cells. The method compπses determining whether a candidate agent disrupts binding of CtIP and CtBP Any binding assay known in the art may be used Typically, binding assays involve one of two formats: an immobilized CtIP polypeptide can be used to bind labeled CtBP, or conversely, immobilized CtBP can be used to bind labeled CtIP polypeptides. In each case, the labeled polypeptide is contacted with the immobilized polypeptide under conditions that permit specific binding of the polypeptide to form a CtIP:CtBP complex in the absence of the candidate agent Particular conditions may be selected by the skilled artisan according to conventional methods The method can be used for high-throughput screening of agent banks such as compound hbraπes, peptide hbraπes and the like Alternatively, the binding assay is performed in vivo in a cell, such as a yeast two-hybrid system in which a reporter gene is expressed when a complex between CtIP and CtBP is formed. Such assays are descnbed m U.S. Patent No. 5,834,209. An agent that inhibits binding would reduce the amount of reporter gene expression as compared to the amount of reporter gene expression m the absence of the agent

Prefeπed embodiments of the invention are described in the following examples Other embodiments withm the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples.

Brief Description of the Materials and Methods Used in the Examples Yeast two hybrid screening.

Screenings were earned out using the yeast stain Y153 (MATa, gal4, gal80, leu2, ura3, trpl, hιs3, ade2, URA3::GALl-lacZ, Lys2::GAL-HIS3) (Durfee et al. Genes Dev 7:555-569, 1993) Yeast cells were grown in YPD medium (1% yeast extract, 2% bacto- peptone, 2% sucrose), or selective minimal medium, SD medium lacking specified ammo acids (2% sucrose, 0.67% nitrogen base plus ammo acid dropout solution; Rose et al, 1990)

Yeast two hybrid screens were earned out as described previously (Durfee et al, supra, Chien et al, Proc Natl Acad. Sci USA 88:9578-9582, 1991). A plasmid (pGb-CtBP) expressing a fusion protein consisting of the DNA binding domain of GAL4 (ammo acids 1- 147) and the entire CtBP protein was used as the bait. A GAL4 activation domain tagged

HeLa cell cDNA library (Clontech) was used as the prey. The yeast cells (strain Y153) were cotransformed with the bait plasmid and the cDNA library (prey) by the Li/Ac method (Rose et al, 1990). The transformants were selected for growth m SD medium that lacked histidme and leucine and also supplemented with 25 mM 3AT. After incubation at 30°C for 3 to 5 days, yeast colonies were transfeπed onto nitrocellulose filters and screened for β- galactosidase activity by X-gal blue/white filter lift assay (Breeden and Nasmyth, 1985). Positive colonies were subcultured in selective media and plasmid DNA was recovered (Hoffmann and Winston, 1987). cDNA plasmids were identified by their ability to complement the leuB mutation of E. coli HB101 (Chien et al, 1991). HB101 cells transformed by electroporation were grown on M9 plates supplemented with 40 μg/ml prolme, 1 mM thiamme and 100 μg/ml ampicilhn. cDNA plasmids recovered by this method were retested individually by yeast two hybrid studies for interaction with CtBP and various heterologous protein baits (Boyd et al, 1994; Kamine et al, 1996). cDNA clones that interacted only with the CtBP bait were chosen for further studies.

5' RACE reactions

5 ' sequences of CtIP cDNA were isolated by rapid amplification of cDNA ends (RACE) reaction using a commercially available kit (Gibco/BRL) First strand cDNA synthesis was earned out with the CtIP specific primer AS2 using poly(A) RNA from Raji cells (provided by Eric Uhlmann) as template. PCR reactions were performed with CtIP specific primers Cip-RACEl or Cιp-RACE-2 and anchor primers provided by the manufacturer (Gibco/BRL). PCR conditions were modified to optimize results: 35 cycles, 1 mm at 94°C, 30 s at 63 °C, 2 mm at 72° C For sequence analysis, the final PCR products were cloned into Bluescnpt KS+ (Stratagene) between Spel and EcoRl (taking advantage of the internal EcoKS. site present in the cDNA of CtIP).

DNA sequence analysis

Both strands of various cDNA clones were sequenced by dideoxy chain termination method using Sequenase version 2.0 (United States Biochemical Corporation) or the frnol sequencing system (Promega).

Northern blot analysis

Human multiple tissue northern (MTN) blots or human cancer cell line MTN blots (Clontech), containing approximately 2 μg of poly(A) RNA per lane, were probed with ³²P- labeled cDNA probes (CtBP, CtIP, or human Actin) under high stringency conditions. Mouse (MTN) blots were probed under low stringency conditions following the instructions of the manufacturer (Clontech). ³²P-dCTP labeled probes were prepared using a random pπmer extension labeling kit (DuPont/NEN). cDNA fragments isolated from pGAD#9 (BamHl/Bgtϊl fragment) served as template for labeling reactions. Expression and purification of GST-fusion proteins

Fresh overnight cultures of E. coh B121 cells (Promega) transformed pGEX-5X3 or pGST-CtBP or pGST-CtIP or GST-Cter (Boyd et al, supra) or GST-Cter (dll 135). Transformed cells were diluted 1/10 m LB medium plus 0.2 mM IPTG and 100 μg/ml ampicilhn and grown at 25° C for 12-16 hours Bacteπal cultures were collected by centπfugation at 4° C and the pellets were resuspended in 1/100 of original volume NETN buffer (50 mM Tπs, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP40) containing 0.5% milk. Bacterial cultures expressing GST-CtBP (GST-30) were resuspended m 1/20 of culture volume and stored at -70 C prior to purification (to reduce proteolysis of GST-CtBP). Cells expressing GST-CtIP were resuspended m 1/20 volume NETN plus 1% Tπton X100. All cells were lysed by somcation (2X 1 mm pulses) and precleared by centπfugation at high speed (15 mm at 10000 rpm). Glutathione agarose beads (Sigma) were prewashed m NETN buffer and incubated with bacterial lysate for 15 mm at 4° C. Beads were then washed 3X with NETN buffer and stored at 4° C. An aliquot of the beads was resuspended in 2X SDS sample buffer (0.125 M Tris/HCl, pH 6.8, 4% SDS, 20% glycerol, 2% β-mercaptoethanol, 10 μg/ml Bromophenol Blue), boiled for two mm, and the supernatant analyzed by SDS-PAGE. Gels were stained with coomassie blue solution (0.1% coomassie brilliant blue, 40% methanol) to estimate protein concentrations. 1 ml of protem-conjugated beads was prepared from 500 ml to 2000 ml of bacteπal culture depending on protein expression and recovery. Protein concentrations ranged from 20 mg/ml beads for GST to approximately 0.2 mg/ml beads for GST-CtIP. For binding expeπments, protein concentrations of GST-beads were normalized by mixing with glutathione agarose beads.

Preparation of polyclonal antisera Polyclonal antisera were raised against GST-CtIP as descnbed by Harlow and Lane

(1988). Affinity puπfied GST-fusion proteins were eluted by boiling m 2 X SDS- electrophoresis sample buffer and separated on SDS-polyacrylamide (8%) gels. Gels were rmsed in H₂0 and stained with coomassie blue (0.2% in H₂0). Protein bands were excised and mixed with Freund's adjuvant pπor to injection. New Zealand rabbits were injected subcutaneously with 50 to 100 μg protein every three to four weeks. Serum was collected 10- 14 days after injection and stored at -20° C. The IgG fraction was purified from GST-CtBP antiserum by protein A column chromatography (Pierce). In vitro protein binding and affinity chromatography

Proteins expressed by in vitro transcription/translation (using TNT transcription/translation expression system, Promega) were labeled by incorporation of [³⁵S]- methionme. The translation mixtures (50 μl) were claπfied and diluted m 1 ml of respective binding buffer (ElA lysis buffer. 250 mM NaCl, 0.1% NP40, 50 mM Hepes, pH 7.0; buffer A. 150 mM NaCl, 0.1% NaCl, 0.1% NP40, 50 mM Tπs, pH 7 5 containing protease inhibitors aprotmin (20 μg/ml) and leupeptin (200 μg/ml) and premcubated with GST beads for 1 to 2 hours at 4° C on rotor. For binding experiments using labeled cell extracts, lysates were premcubated with GST and glutathione agarose beads for 12 to 16 hours. The precleared lysates were divided equally among the immobilized GST or GST-fusion proteins (5 μg protein, 15 μl beads per binding reaction) After incubation at 4° C for 1 to 2 hours, beads were washed six times with binding buffer, resuspended in 2X electrophoresis sample buffer and boiled for 2 mm. Beads were pelleted and the supernatant was analyzed by SDS- Polyacrylamide gel electrophoresis. Gels were soaked in 1 M sodium sahcylate as a fluorographic enhancer, dπed and analyzed by autoradiography.

Competition binding experiments

Soluble ElA peptides representing the C-termmal 67 ammo acids of ElA 243R or dll 135 were prepared from immobilized GST-Cter and GST-dll 135 by cleavage with factor X_a (New England Biolabs). Beads containing 1 mg of protein (200 to 400 μl) were resuspended in 400 μl factor X_a buffer (20 mM Tπs pH 8.0, 100 mM NaCl, 2 mM CaCl₂) containing 8 units of factor X_a (New England Biolabs) and incubated for 6 to 12 hours at room temperature. Beads and supernatants were analyzed for cleavage products by SDS- PAGE and visualized by coomassie blue staining to estimate peptide concentrations. The supernatant (containing cleaved ElA peptides) was mixed with in vitro labeled CtBP and added to GST-Cter and GST-CtIP immobilized on glutathione beads Binding expeπments were then carried out as descnbed above.

Coimmunoprecipitation For in vivo coimmunoprecipitation analyses, recombinant proteins (CtIP and CtBP) were coexpressed in BSC40 cells using the vaccinia vιrus/T7 RNA polymerase system (Ausubel, et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1992) BSC40 cells (5X 10⁶ cells/T75 cm² flask) were infected with recombinant vaccinia virus vTF7-3 (which expresses T7 RNA polymerase; Fuerst et al, Proc. Natl Acad Sci USA 83:8122-8126, 1986) m serum free medium (DMEM) at 10 PFU per cell and then transfected with 10 μg of appropriate expression plasmids using LipofectAMINE (Gibco/BRL). Five hours later cell culture medium was supplemented with 10% fetal calf serum. At 18 to 20 hours post infection, cells were labeled with ³⁵S-methιonme/cysteme mixture (500 μCι/T75 cm² flask). After labeling, all procedures were carried out on ice or at 4° C Cells were rmsed twice with PBS and lysed by addition of ElA lysis buffer (1 ml/T75 cm² flask) supplemented with protease inhibitors aprotmm (20 μg/ml) and leupeptin (200 μg/ml). Cells were incubated for 30 mm in lysis buffer to solubihzed proteins. Lysates were claπfied by centnfugation in an eppendorf centrifuge at high speed for 15 mm and premcubated with protein A sepharose beads (Sigma) for at least one hour The precleared lysate was then divided and mixed with respective monoclonal antibodies (MAb) or polyclonal antiserum and incubated for at least one hour Protein A sepharose beads (Sigma) were then added and the incubation continued for one to two hours Beads were pelleted and washed at least seven times with ElA lysis buffer Samples were analyzed by gel electrophoresis and fluorography as descπbed previously (Boyd et al, supra)

Example 1

This example illustrates isolation of the cDNA clone

The cDNA clones that code for proteins which bind with CtBP were cloned by two hybrid screening m yeast The yeast reporter strain Y153 (Durfee et al, supra) was transformed with the bait plasmid (pGB-CtBP), expressing the entire coding sequence of CtBP fused m frame with the Gal4 DNA binding domain together with a Gal4-actιvatιon domain tagged HeLa cell cDNA library (Clontech) To identify putative CtBP interacting cDNA clones the yeast transformants were grown in selective media and screened for activation of the LacZ reporter gene by X gal filter lift assays Six cDNA clones were isolated by this method. They were further tested for interaction with heterologous baits in yeast Three clones, pGad#8, -#9 and #15 interacted specifically with CtBP, but not with the vector pGBT9 or three heterologous baits including ElA exon 2 (data not shown), suggesting that the proteins encoded by these cDNA clones interact specifically with CtBP. Sequence analysis revealed that clone #8 encoded the C-termmus of a known protein, the 70kD subunit of KU (Reeves and Sthoeger, 1989), while pGAD#9 and pGAD#15 represent partial cDNA clones of a novel protein, the CtBP interacting protein, CtIP (CtBP-Interactmg Protein).

Example 2 This examples illustrates the isolation of full length cDNA of CtIP. The cDNAs of the two other CtBP interacting cDNA clones, pGAD#9 and pGAD#15, were identical except for 153 base pair 5' coding sequences unique to clone #9, suggesting that they represent partial cDNA clones of the same protein, refeπed to as CtBP Interacting protein, CtIP. This was confirmed by northern blot analysis using multiple tissue blots (Clonetech). A ³²P-labeled probe specific for the cDNA of clone #15 hybridized to a single mRNA species present in preparations from several tumor cell lines (Figure 1). However, the transcript size of 3.7 kb was about 800 bp larger than the cDNA represented by clone #9. The predicted reading frame of clone #9 and clone #15, established in relation to the coding sequence of the GAL4 DNA binding domain contained several stop codons 3 ' to the coding region, suggesting that both cDNA clones encoded the carboxy-termmus of CtIP. To isolate additional 5 ' coding sequences of CtIP, 5 ' RACE reactions were performed using a commercially available kit (Clontech, Inc.) according to the manufacturer's protocol. 5 ' cDNA sequences were isolated that encoded an additional 31 amino acids in frame with the cDNA of clone #9 with a putative ATG start codon at position 300. The complete cDNA sequence is presented m Figure 2 A. The reading frame upstream of this ATG contains several stop codons, indicating that the ATG initiates the open reading frame. Thus, this sequence contains the complete 5 ' coding sequence of CtIP. The coding sequence of CtIP (Fig. 2B) codes for a protein of 897 ammo acids (Figure 3), while clone #9 and clone #15 encode the C-termmal 864 and 835 residues of CtIP respectively.

Example 3 This example illustrates in vitro binding of CtBP and CtIP.

To confirm that CtIP could directly bind with CtBP, in vitro binding experiments were earned out. The cDNA of pGAD-#15 encoding residues 70 to 897 of CtIP was cloned into the expression vector pGEX-5X3 (Pharmacia) in frame with the GST gene Bacteπally expressed and affinity puπfied GST-CtIP as well as GST control were immobilized on glutathione agarose beads and tested for interaction with in vitro translated ³⁵S -labeled CtBP. Bound proteins were eluted in SDS sample buffer and analyzed by SDS-PAGE and fluorography (Figure 4). CtBP interacted specifically with GST-CtIP but not with GST, confirming that it could directly associate with CtIP in vitro.

Example 4 This example illustrates the effect of PLDLS mutation on CtIP interaction, with CtBP Comparison of CtIP coding sequences with DNA sequences m the data base using BLAST analysis (Altschul et al, 1990) did not reveal significant sequence homologies to known proteins. However, upon close examination it was discovered that CtIP shares a five ammo acid motif, PLDLS, with ElA (Figure 5). These five residues are well conserved among ElA proteins of vanous adenovirus serotypes. By mutational analysis of Ad2 ElA, it had been previously shown that this region is required for efficient interaction with CtBP. To determine if these five residues are also essential for interaction of CtIP with CtBP, a CtIP mutant, CtlPΔ, was constructed in which PLDLS was substituted to LASQC (Figure 6A). CtIP was expressed as GST-fusion protein and tested for interaction with in vitro translated CtBP (Figure 6B). Binding of CtBP to CtlPΔ was significantly reduced compared to the interaction with wild type GST-CtIP, suggesting that the PLDLS sequence constitutes the CtBP binding motif of CtIP as well as ElA.

Example 5 This example illustrates the in vivo interaction of CtIP and CtBP.

To venfy that CtIP could interact with CtBP in vivo a coimmunoprecipitation analysis was earned out. A T7 epitope tagged CtBP and full length CtIP (CtIP wt or mutant CtlPΔ) were coexpressed m BSC40 cells using the recombinant vaccinia vιrus/T7 expression system. After metabolic labeling with ³⁵S-methιomne/cysteme, cells were lysed m ElA lysis buffer (Harlow et al, 1986). The precleared lysate was divided and subjected to immunoprecipitation with either T7 mAb (Novagen) or CtIP antiserum (raised against GST- CtIP). As seen m Figure 8 the majonty of CtIP protein migrated with an apparent molecular weight of 125kD on SDS gels, which is slightly larger than the predicted molecular weight of lOOkD. In addition, smaller CtIP products were also detected. Most likely they represent degradation products of the 125kD CtIP protein. Importantly, CtIP but not CtlPΔ, which lacks the CtBP binding motif, coprecipitated with CtBP. CtBP migrated with a molecular weight (48kD) close to that of some of the smaller CtIP products, but could be detected coprecipitatmg with CtBP antibody, but not with CtIP antibody (marked as dot in Figure 7). These data confirm that CtBP interacts with full length CtIP and that this interaction can occur in vivo.

Example 6 This example illustrates that ElA competes with CtIP for CtBP interaction. Since CtIP contains the same CtBP binding motif as ElA, an expeπment was performed to determine if ElA would compete with CtIP for CtBP interaction. It had been shown m earlier expeπments that the GST-El A fusion protein, GST-Cter, containing only the C- termmal 67 ammo acids of ElA is capable of binding CtBP (Boyd et al, supra). Soluble ElA peptides were generated by proteolytic cleavage of immobilized GST-Cter fusion protein and utilized in competition binding expeπments. CtBP was expressed by in vitro transcription/translation and then analyzed for binding to immobilized GST-CtIP or GST-Cter in the presence or absence of ElA peptide competitors. As demonstrated in Figure 9, CtBP interacts well with GST-CtIP or the El A fusion protein, GST-Cter However, these interactions were significantly reduced m the presence of 200 fold molar concentration of wt ElA peptide (Cter), but not ElA dll35 which lacks the C6BP binding region This result demonstrates that CtIP and ElA, which carry identical CtBP binding motifs, can compete for CtBP interaction

Example 7

This example illustrates the construction of AD-CtIP, an adenovirus vector for expressing CtIP.

The starting vector for the virus construction is pAd5LendCMV. This plasmid contains the Xho I C fragment (nucleotide number 1 to 5788 of adenovirus 5 (Ad5) with a deletion of the ElA gene and most of the ElB coding regions [Sac II (354) to Bgl II (3328)]. As a result of this deletion, this construct will not make any El polypeptides. The deleted region is substituted with the CMV immediate early (IE) promoter and a multiple cloning site containing Hind III, Kpn I and BamH I sites (other sites are not available because their multiple occuπence m the vector). The transcπpt from the CMV promoter will use the ElB polyadenylation site. Normally this vector is used for making recombinant adenoviruses that express proteins under the control of CMV promoter. For making AD-CtIP, an Apa I blunt- Kpn I fragment of pcDNA3-CtIP (CtIP cDNA) is cloned into BamHI bhmt-Hind III digested pAd5Lend CMV and 5μg of the resultant plasmid is transfected into human 293 cells (60mm dish) along with 5μg of pBHGE3 (Bett et.al , 1994) by the calcium phosphate method. The transfected cells are overlaid with growth medium containing 0 8% noble agar after 5 hours of transfection. After 7 days, the visible plaques are picked up, and screened for the expression of CtIP by western blot using rabbit polyclonal antibody raised against CtIP. After confirming, the correct virus (AD-CtIP) is amplified and titrated in 293 cells by plaque assay method. (Bett, A.J, Haddara, W, Prevec, L. and Graham, F.L. (1994). An efficient and flexible system for construction of adenovirus vectors with insertions or deletions m early regions 1 and 3, Proc. Natl. Acad. Sci. USA 91:8802-8806).

In view of the above, it will be seen that the several advantages of the invention are achieved and other advantageous results attained. As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not m a limiting sense.

All references cited in this specification, including patents and patent applications, are hereby incorporated by reference. The discussion of references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art. Applicants reserve the πght to challenge the accuracy and pertinency of the cited references.

Claims

What is Claimed is:

1. An isolated and purified CtIP polypeptide or a fragment thereof.

2. The isolated and punfied CtIP polypeptide of claim 1 which comprises SEQ ID NO:2 or a conservatively substituted vaπant thereof.

3. A composition compnsmg the isolated and purified CtIP polypeptide or fragment of claim land a caπier that facilitates delivery of the CtIP polypeptide or fragment into a target cell.

4 An isolated and purified polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a CtIP polypeptide;

(b) a nucleotide sequence encoding a fragment of CtIP;

(c) a nucleotide sequence consisting of at least 15 nucleotides of SEQ ID NO: l or SEQ ID NO:3; and

(d) a nucleotide sequence complementary to the nucleotide sequence of (a), (b) or (c). .

5. The isolated and puπfied polynucleotide of claim 4 wherein the CtIP polypeptide consists of SEQ ID NO:2 or a conservatively substituted vanant thereof.

6. The isolated and puπfied polynucleotide of claim 5 wherein the nucleotide sequence is SEQ ID NO:3 or SEQ ID NO:4.

7. An isolated and purified polynucleotide which specifically hybndizes to a polynucleotide consisting of SEQ ID NO:3 or SEQ ID NO:4.

8. A vector comprising expression regulatory elements operably linked to a nucleotide sequence encoding a CtIP polypeptide or a CtIP fragment.

9. A host cell transformed with the vector of claim 10.

10. An isolated and purified antibody which specifically reacts with the CtIP polypeptide or CtIP fragment of claim 1.

1 l.A method for determining malignancy of a cell in a patient which compπses detecting CtIP expression in the cell, wherein an amount of CtIP expression that is below the amount for normal cells indicates the cell is malignant.

12. The method of claim 11, wherein the detecting step comprises contacting proteins from the cell with an antibody which specifically reacts with CtIP or a CtIP fragment and detecting binding of the antibody to CtIP.

13. The method of claim 11, wherein the detecting step compπses: (a) contacting mRNA of the cell with a polynucleotide that specifically hybndizes to a polynucleotide consisting of SEQ ID NO:3; and (b) detecting the existence of a hybndization complex between the polynucleotide and CtIP mRNA.

14. The method of claim 12, wherein the detecting step comprises: (a) producing a cDNA from CtIP mRNA using the reverse transcnption method; (b) contacting the cDNA with at least two ohgonucleotides that specifically hybndize to the cDNA to define a region of the cDNA to be amplified;

(c) amplifying the cDNA region; and

(d) detecting the amplified cDNA region.

15. A method for inhibiting neoplasia of target cells in a patient comprising treating the patient with a CtIP polypeptide or a CtIP fragment, wherein said CtIP fragment has neoplasia suppressing activity.

16. The method of claim 15, wherein the treating step comprises admmisteπng to the patient a polynucleotide encoding the CtIP polypeptide or CtIP fragment and wherein the polynucleotide is internalized in the target cells and the CtIP polypeptide or CtIP fragment is expressed.

17. The method of claim 16, wherein the polynucleotide compnses a recombinant adenovirus vector.

18. The method of claim 17, wherein the treating step comprises admimstenng the CtIP polypeptide or CtIP fragment to the patient.

19. The method of claim 18, wherein the CtIP polypeptide or CtIP fragment is administered with a caπier that facilitates delivery of the CtIP polypeptide or CtIP fragment into the target cells.

20. The method of claim 19 wherein the CtIP fragment compnses PLDLS. 2 l.A method for identifying an agent that inhibits neoplasia of cells, which compnses determining whether the agent disrupts binding of CtIP and CtBP