WO2003073105A2

WO2003073105A2 - Use of plag1 and plagl2 in cancer diagnosis and drug screening

Info

Publication number: WO2003073105A2
Application number: PCT/EP2003/002150
Authority: WO
Inventors: Willem Jan Marie Van De Ven; Marianne Léontine VOZ; Karen Hensen
Original assignee: Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw
Priority date: 2002-02-28
Filing date: 2003-02-28
Publication date: 2003-09-04
Also published as: WO2003073105A9; WO2003073105A3; AU2003215622A1

Abstract

The present invention relates to the use of the PLAG1 consensus binding motif to screen for molecules that inhibit binding of PLAG1 and PLAGL2 to the latter consensus binding motif. The present invention further relates to a method to diagnose neoplastic transformation via measuring PLAG1 and PLAGL2 expression.

Description

Use of PLAG1 and PLAGL2 in cancer diagnosis and drug screening

Field of the invention

The present invention relates to the use of the PLAG1 consensus binding motif to screen for molecules that interfere with PLAG1 and PLAGL2-mediated neoplastic transformation. The present invention further relates to a method to diagnose neoplastic transformation via measuring PLAG1 and PLAGL2 expression.

Background of the invention

Pleomorphic adenoma of the salivary gland is a benign epithelial tumor, which usually arises as a result of activation of the PLAGl gene on chromosome 8q12 (1). In most cases, this activation result from recurrent chromosomal translocations that lead to promoter substitution between PLAG1, a gene mainly expressed in fetal tissue, and more broadly expressed genes. The three translocation partners characterized so far are the β-catenin gene (1), the leukemia inhibitory factor receptor gene (2) and the elongation factor Sll gene (3). Breakpoints invariably occur in the 5' noncoding part of the PLAG1 gene, leading to an exchange of regulatory control elements while preserving the PLAG1 coding sequence. The replacement of the PLAGl promoter, which is inactive in adult salivary glands, by a strong promoter derived from the translocation partner, leads to ectopic expression of PLAGl in the tumor cells. This abnormal PLAG1 expression presumably results in a deregulation of PLAG1 target genes, causing salivary gland tumorigenesis.

Recently, it has been demonstrated that PLAG1 is a genuine transcription factor that binds a bipartite element containing a Core sequence, GRGGC, and a G-cluster, RGGK, separated by 7 random nucleotides (4). Potential PLAG1 binding sites were found in promoter 3 of the human insulin-like growth factor II (IGF-II) gene. Moreover, IGF-II transcripts deriving from the P3 promoter are highly expressed in salivary gland adenomas overexpressing PLAGl while they are not detectable in adenomas without abnormal PLAGl expression or in normal salivary gland tissue (4). Two novel cDNAs encoding C₂H₂ zinc finger proteins, PLAGL1 and PLAGL2, which show high homology to PLAG1 , have been identified, constituting by this way a novel subfamily of zinc finger proteins (5). PLAGL1 has also been isolated independently and referred to as LOT1 and ZAC1 (6, 7). The homology between these 3 PLAG proteins resides mainly in their amino-terminal zinc finger domain (73 % and 79 % identity for PLAGL1 and PLAGL2 respectively) while the C-terminal region is much more divergent. Although PLAGL1 show high homology to PLAG1 in the DNA binding domain, the DNA binding specificity's of these 2 proteins seem to differ slightly. Indeed the consensus binding site for PLAGL1 has been identified as the sequence GGGGGGCCCC (8). This sequence contains the extended PLAG1 core GGRGGCC but does not include the G-Cluster 7 nucleotides downstream. A second difference between these two related factors is that PLAG1 , when consistently overexpressed in pleomorphic adenomas, is thought to act as a proto-oncogene while PLAG 1 seems to act as a tumor suppressor gene. Indeed, expression of PLAGL1 prevented proliferation of tumor cells as measured by colony formation, growth rate, and cloning in soft agar, and precluded tumor formation in nude mice (7, 8). Moreover it encodes a protein that regulates apoptosis and cell cycle arrest (7), maps to a chromosomal region frequently lost in cancer (6) and decrease or loss of expression has been observed in breast tumors (9). PIAS1 , the first member of the Protein Inhibitor of Activated STAT (PIAS) family, was identified in a yeast two-hybrid screening using a fragment of STAT-1 (Signal Transducers and Activators of Transcription 1) as bait. When expressed in mammalian cells, PIAS1 and PIAS3 inhibited STAT-1 and STAT-3 mediated gene activation, respectively. STATs are transcription factors that mediate cytokine and growth factor signals culminating in various biological responses. Since these initial findings, PIAS proteins have been linked to other biological events such as the transcriptional modulation of hormone-dependent nuclear receptors and SUMOylation of diverse proteins (Liao et al., Proc Natl Acad Sci USA 2000, 97(10): 5267-72, which is herein incorporated by reference). In the present invention, it has been determined that PLAG1 is a genuine proto-oncogene, by evaluating the in vitro transforming capacity of PLAG1 via analyzing the growth profile of NIH3T3 cells overexpressing PLAGl, their ability to form foci, to grow in soft agar and to form tumors in nude mice. These analyses were extended to PLAGL2, the third member of the PLAG family. Surprisingly, it was found that both PLAG1 and PLAGL2 are able to transform NIH3T3 cells and therefore can be considered proto-oncogenes. Furthermore by investigating the DNA binding specificity's of the PLAG proteins and comparing their mode of DNA recognition, it was found that PLAG1 and PLAGL2 have indistinguishable binding capacities which are different from that of PLAGL1. Their similar binding capacities are reflected in their ability to induce common target genes; it was shown here that IGF-II is a common target of PLAG1 and PLAGL2. Moreover, the transformation of NIH3T3 cells by these factors is accompanied by a drastic upregulation of IGF-II expression, indicating that PLAGl and PLAGL2 stimulate cell proliferation by activating the IGF-II mitogenic pathway. A yeast two hybrid screening with PLAGL2 revealed that members of a family of PIAS bind to it. We have confirmed these interactions in GST (Glutatione-S-Transferase)-pull down assays and proved that PLAG1 can also crosslink members of the PIAS proteins. Hence, the present invention relates to the use of the PLAG1 consensus binding motif as described in ref. (4) to screen for molecules that suppress PLAG1 and/or PLAGL2-mediated neoplastic transformation via binding, or interaction with, said consensus binding motif, PLAG1 and/or PLAGL2 or PIAS proteins. The present invention further relates to a method to differentially diagnose neoplastic transformation.

Brief description of figures Figure 1: PLAGL1 binds a different consensus sequence to PLAG1 and PLAGL2.

EMSAs were performed with recombinant PLAG proteins produced in vitro in reticulocyte lysates. PLAG proteins were incubated with the probes WT2 (lanes 1 and 8), mCLU2 (lanes 2 and 9), mCO2 (lanes 3 and 10), mCLUmCO2 (lanes 4 and 11), WT2m1 (lanes 5 and 12), Zac (lanes 6 and 13) and Zac mut (lanes 7 and 14) as described in Material and Methods. Equal efficiencies of protein expression were obtained for the different constructs as demonstrated by SDS-PAGE of proteins labeled with ³⁵S methionine (data not shown). The percentage of binding of the PLAG proteins to the different probes were compared to the binding of the wild- type PLAG to the probe WT2 and are the means of at least two experiments. The asterix indicates a band due to binding to the WT2 probe of a protein present in the reticulocyte lysate.

Figure 2: NIH3T3 cells infected with recombinant retrovirus containing human PLAG1 or PLAGL2 express high levels of functional protein.

A) Total RNA from NIH3T3 cells infected with control virus (lane 1 and 3), human Flag- PLAG1 retrovirus (lane 2) and human Flag-P 4G 2 retrovirus (lane 4) were hybridized with a ³²P-labeled human PLAG1 cDNA (lanes 1 and 2) or with a ³²P-labeled human PLAGL2 cDNA (lanes 3 and 4). Expression of β-actin monitored the integrity and yield of RNA.

B) Western blot analysis of whole cell lysates from NIH3T3 cells infected with Flag- PLAG1 retrovirus (lane 1), control retrovirus (lane 2) and F\ag-PLAGL2 retrovirus (lane 3) using an α-FLAG mouse monoclonal antibody C) PLAG1 and PLAGL2 can stimulate transcription through the binding to the consensus sequence WT2. Mock, PLAG1 and PLAGL2 expressing NIH3T3 cells were transfected with 400 ng of the (mCOmCLU2)₃ TK-luc reporter construct (solid bars) and 400 ng of the (WT2)₃TK-luc reporter construct (hatched bars), respectively. 200ng of RSV-βgal were cotransfected as internal control. The results correspond to the mean of the corrected luciferase value +/- S.E. of at least 2 independent transfections performed in triplicate.

Figure 3: The Mitogenic stimulation of NIH3T3 cells overexpressing PLAG1 and PLAGL2.

Mock, PLAG1 and PLAGL2 expressing NIH3T3 cells were grown in DMEM medium supplemented with either 5% (A) or 1% (B) fetal calf serum and counted daily. After 7 days, PLAG1 and PLAGL2 expressing cells reached a maximum cell density. The data correspond to the mean of three independent experiments. Figure 4: Overexpressed PLAG1 and PLAGL2 proteins are able to transform NIH3T3 cells.

(A) The mock, PLAG1 and PLAGL2 expressing NIH3T3 cells were used for focus forming assay in medium containing 1% FCS or 10% FCS and for soft agar assay as described in materials and methods. The photographs (100X magnification) were taken on day 17 (focus assay) and day 25 (agar).

(B) Table of the transforming properties of the NIH3T3 transfectants:

The number of foci per 300 000 puromycin-resistant cells. Average of 3 dishes from 2 independent infection experiments from cells grown in 1% FCS (1). Colonies have been counted 24 days after seeding and results are expressed as the ratio [(number of colonies formed/number of plated cells) X 100] (2). The number of tumors per site of inoculation. (3).

Figure 5: IGF-II is upregulated in NIH3T3 cells overexpressingP AGf and PLAGL2.

Northern Blot analysis of total RNA extracted from NIH3T3 cells infected with empty virus (a), human F\ag-PLAG1 (b) and human Flag-P AGL2 (d) grown in 10% FCS. The membrane was hybridized with a ³²P-labeled mouse IGF-II probe specific for exon 6, a ³²P-labeled PLAGl probe or a ³²P-labeled PLAGL2 probe. Expression of β-actin transcript monitored the integrity and yield of RNA.

Figure 6: IGF-II is a direct target of PLAG1 and PLAGL2.

Northern blot analysis of total RNA isolated from clones, deriving from the human epithelial kidney 293 cell line, containing a zinc-inducible expression vector (11) either for PLAGl (clones P1-8 and P1-32), PLAGL2 (clones PL2-2 and PL2-24) or the lacZ gene (clones B-1 , B21 and B-4) grown without (-) or with (+) 100μM ZnCI₂. Blots were hybridized sequentially with ³²P-labeled human PLAG1 cDNA, ³²P-labeled human PLAGL2 cDNA, ³²P-labeled lacZ , ³²P-labeled human IGF-II probe and ³²P-labeled human actin probe.

Figure 7: Comparison of the zinc finger motifs in the three PLAG proteins.

A) Alignment of the seven zinc fingers motifs in PLAG1 , PLAGL1 and PLAGL2. Only the amino acids different in PLAGL1 and PLAGL2 are indicated. The key residues (highlighted in the figure) are defined on the basis that each finger module folds to form a compact ββα structure with the α-helix fitting in the major grove, (reviewed in (17)) Residues -1 , +2, +3 and +6 (numbering with respect to the start of the α-helix) typically make key base contacts that are responsible for defining sequence specificity B) Comparison of the DNA binding consensus sequence of PLAG1 and PLAGL1. The consensus reported here for PLAG1 is not the minimal consensus GRGGC(N)7RGGK as described previously (4) but has been extended on both sides of the Core by the more frequent bases found in the CASTing experiments.

Figure 8: PIAS interacts with both PLAG1 and PLAGL2 Using GST-pull down assays, PIAS1 (³⁵S-PIAS1) interacts with both PLAG1 and PLAGL2.

Figure 9: PIAS modulates PLAG1 and PLAGL2 transactivation

PLAG1 and PLAGL2 stimulate luciferase expression from a reporter construct containing several copies of PLAG1/PLAGL2 DNA binding sites (WT2)₃. We investigated whether PIASs influence this ability by cotransfecting cell lines of different origins with the various PIAS and PLAG constructs. The findings show that cotransfection in the fibroblast cell line 293 with especially PIAS-beta and PLAG1 result in a more than synergistic enhancement. Enhancement was also detected when PIAS1 was cotransfected with either of the PLAGs.

Detailed description of invention

The present invention relates to the usage of the PLAG1 consensus binding motif to screen for molecules which suppress PLAG1 and/or PLAGL2-mediated neoplastic transformation comprising:

-exposing the PLAG1 consensus binding motif to at least one molecule whose ability to suppress neoplastic transformation is sought to be determined,

-determining binding or hybridising of said molecule(s) to the PLAG1 consensus binding motif, or to PLAG1 and/or PLAGL2 proteins, or to Protein Inhibitor of Activated

STAT (PIAS) proteins, and

-monitoring said neoplastic transformation when administering said molecules as a medicament.

The PLAG1 consensus binding motif as described in ref. (4) and more specifically to the consensus binding motif comprising a core sequence consisting of the nucleotides GGGGGCCC, a G-cluster consisting of the nucleotides GGGG, and seven random nucleotides between said core sequence and said G-cluster. The invention thus provides methods for identifying compounds or molecules which bind to said consensus binding motif or to PLAG1 or PLAGL2 or to Protein Inhibitor of Activated STAT (PIAS) proteins and which prevent or suppress neoplastic transformation. With "suppression" it is understood that said suppression of transformation can occur for at least 20%, 30%, 30%, 50%, 60%, 70%, 80%, 90% or even 100% compared to non-suppressed neoplastic transformation.

The latter methods are also referred to as 'drug screening assays' or 'bioassays' and typically include the step of screening a candidate/test compound or agent for the ability to interact with said consensus binding motif or to PLAG1 or PLAGL2 or to nucleic acids encoding PLAG1 or PLAGL2 or to Protein Inhibitor of Activated STAT (PIAS) proteins. Candidate compounds or agents which have this ability, can be used as drugs to combat or prevent neoplastic transformation. Candidate/test compounds such as small molecules, e.g. small organic molecules, and other drug candidates can be obtained, for example, from combinatorial and natural product libraries.

Typically, the assays are cell-free assays which include the steps of combining said consensus binding motif or PLAG1 or PLAGL2 or nucleic acids encoding PLAG1 or PLAGL2 or PIAS proteins and a candidate/test compound, e.g., under conditions which allow for interaction of (e.g. binding of) the candidate/test compound with said consensus binding motif or PLAG1 or PLAGL2 or nucleic acids encoding PLAG1 or PLAGL2 or PIAS proteins to form a complex, and detecting the formation of a complex, in which the ability of the candidate compound to interact with said consensus binding motif or PLAG1 or PLAGL2 or nucleic acids encoding PLAG1 or PLAGL2 or PIAS proteins is indicated by the presence of the candidate compound in the complex. Formation of complexes between said consensus binding motif or PLAG1 or PLAGL2 or PIAS proteins and the candidate compound can be quantitated, for example, using standard (immuno)assays. The said consensus binding motif or PLAG1 or PLAGL2 or nucleic acids encoding PLAG1 or PLAGL2 or PIAS proteins employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located extracellularly or even intracellularly.

To perform the above described drug screening assays, it is feasible to immobilize said consensus binding motif or PLAG1 or PLAGL2 or nucleic acids encoding PLAG1 or PLAGL2 or PIAS proteins or its (their) target molecule(s) to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Interaction (e.g., binding of) of said consensus binding motif or PLAG1 or PLAGL2 or nucleic acids encoding PLAG1 or PLAGL2 or PIAS proteins to a target molecule, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, PLAG1 or PLAGL2 -His tagged can be adsorbed onto Ni-NTA microtitre plates, or PLAG1 or PLAGL2 -ProtA fusions adsorbed to IgG, which are then combined with the cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the plates are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of PLAG1 or PLAGL2 -binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. Other techniques for immobilizing protein on matrices can also be used in the drug screening assays of the invention. For example, PLAG1 or PLAGL2 can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated PLAG1 or PLAGL2 can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, III.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Another technique for drug screening which provides for high throughput screening of compounds having suitable binding affinity to said consensus binding motif or PLAG1 or PLAGL2 is described in detail in "Determination of Amino Acid Sequence Antigenicity" by Geysen HN, WO 84/03564, published on 13/09/84. In summary, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The protein test compounds are reacted with fragments of said consensus binding motif or PLAG1 or PLAGL2 or nucleic acids encoding PLAG1 or PLAGL2 or PIAS proteins and washed. Bound said consensus binding motif or PLAG1 or PLAGL2 or nucleic acids encoding PLAG1 or PLAGL2 is then detected by methods well known in the art. Purified PLAG1 or PLAGL2 can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support. This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding PLAG1 or PLAGL2 specifically compete with a test compound for binding PLAG1 or PLAGL2. In this manner, the antibodies can be used to detect the presence of any protein, which shares one or more antigenic determinants with PLAG1 or PLAGL2.

According to the invention molecules that comprise a region specifically binding to PLAG1 or PLAGL2 or said consensus binding motif or nucleic acids encoding PLAG1 or PLAGL2 or PIAS proteins which can be used for the manufacture of a medicament to treat neoplastic transformation can be chosen from the list comprising an antibody or any fragment thereof binding to PLAG1 or PLAGL2, a (synthetic) peptide, a protein, a small molecule specifically binding to PLAG1 or PLAGL2 or said consensus binding motif or nucleic acids encoding PLAG1 or PLAGL2, anti-sense nucleic acids hybridising with said consensus binding motif or nucleic acids encoding PLAG1 or PLAGL2, a ribozyme against nucleic acids encoding PLAG1 or PLAGL2, and short interference RNA molecules as disclosed in WO 0244321 which is herein incorporated by reference.

The term 'antibody' or 'antibodies' relates to an antibody characterized as being specifically directed against PLAG1 or PLAGL2 or PIAS proteins or any functional derivative thereof, with said antibodies being preferably monoclonal antibodies; or an antigen-binding fragment thereof, of the F(ab')₂, F(ab) or single chain Fv type, or any type of recombinant antibody derived thereof. These antibodies of the invention, including specific polyclonal antisera prepared against PLAG1 or PLAGL2 or PIAS proteins or any functional derivative thereof, have no cross-reactivity to others proteins. The monoclonal antibodies of the invention can for instance be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly of a mouse or rat immunized against PLAG1 or PLAGL2 or PIAS proteins or any functional derivative thereof, and of cells of a myeloma cell line, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing PLAG1 or PLAGL2 or PIAS proteins or any functional derivative thereof which have been initially used for the immunization of the animals. The monoclonal antibodies according to this embodiment of the invention may be humanized versions of the mouse monoclonal antibodies made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains. Alternatively the monoclonal antibodies according to this embodiment of the invention may be human monoclonal antibodies. Such human monoclonal antibodies are prepared, for instance, by means of human peripheral blood lymphocytes (PBL) repopulation of severe combined immune deficiency (SCID) mice as described in PCT/EP 99/03605 or by using transgenic non-human animals capable of producing human antibodies as described in US patent 5,545,806. Also fragments derived from these monoclonal antibodies such as Fab, F(ab)'₂ and ssFv ("single chain variable fragment"), providing they have retained the original binding properties, form part of the present invention. Such fragments are commonly generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases. It is well known to the person skilled in the art that monoclonal antibodies, or fragments thereof, can be modified for various uses. The antibodies involved in the invention can be labeled by an appropriate label of the enzymatic, fluorescent, or radioactive type. In a specific embodiment the antibodies against PLAG1 or PLAGL2 or PIAS proteins can be derived from animals of the camelid family. In said family immunoglobulins devoid of light polypeptide chains are found. Heavy chain variable domain sequences derived from camelids are designated as VHH's. "Camelids" comprise old world camelids (Camelus bactrianus and Camelus dromaderius) and new world camelids (for example Lama paccos, Lama glama and Lama vicugna). EP0656946 describes the isolation and uses of camelid immunoglobulins and is incorporated herein by reference.

Small molecules, e.g. small organic molecules, and other drug candidates can be obtained, for example, from combinatorial and natural product libraries. Also within the scope of the invention are oligoribonucleotide sequences, that include anti- sense nucleic acids that bind to said consensus binding motif or nucleic acids encoding PLAG1 or PLAGL2. Anti-sense nucleic acids of the invention may be prepared by any method known in the art for the synthesis of nucleic acids. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize anti-sense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines. The terms 'medicament to treat' or 'administering said molecules as a medicament' relate to a composition comprising molecules as described above and a pharmaceutically acceptable carrier or excipient (both terms can be used interchangeably) to 'treat' neoplastic transformation. Suitable carriers or excipients known to the skilled man are saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives. Other suitable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers. The 'medicament' may be administered by any suitable method within the knowledge of the skilled man. The preferred route of administration is parenterally. In parental administration, the medicament of this invention will be formulated in a unit dosage injectable form such as a solution, suspension or emulsion, in association with the pharmaceutically acceptable excipients as defined above. However, the dosage and mode of administration will depend on the individual. Generally, the medicament is administered so that the protein, polypeptide, peptide of the present invention is given at a dose between 1 μg/kg and 10 mg/kg, more preferably between 10 μg/kg and 5 mg/kg, most preferably between 0.1 and 2 mg/kg. Preferably, it is given as a bolus dose. Continuous infusion may also be used and includes continuous subcutaneous delivery via an osmotic minipump. If so, the medicament may be infused at a dose between 5 and 20 μg/kg/minute, more preferably between 7 and 15 μg/kg/minute.

In another embodiment antibodies or functional fragments thereof can be used for the manufacture of a medicament for the treatment of the above-mentioned disorders. As a non- limiting example there are the antibodies described in US 5,843,633. In a specific embodiment said antibodies are humanized (Rader et al., 2000, J. Biol. Chem. 275, 13668) and more specifically human antibodies are used to manufacture a medicament to treat neoplastic transformation. In yet another specific embodiment antibodies derived from camelids are used to manufacture a medicament to treat neoplastic transformation. Another aspect of administration for treatment is the use of gene therapy to deliver the above- mentioned anti-sense nucleic acids. Gene therapy means the treatment by the delivery of therapeutic nucleic acids to patient's cells. This is extensively reviewed in Lever and Goodfellow 1995; Br. Med Bull.,51 , 1-242; Culver 1995; Ledley, F.D. 1995. Hum. Gene Ther. 6, 1129. To achieve gene therapy there must be a method of delivering genes to the patient's cells and additional methods to ensure the effective production of any therapeutic genes. There are two general approaches to achieve gene delivery; these are non-viral delivery and virus-mediated gene delivery.

The present invention further relates to a method to diagnose neoplastic transformation comprising measuring PLAG1 and PLAGL2 expression.

Hence, the present invention provides a diagnostic method for determining if a subject bears increased PLAG1 and PLAGL2 expression comprising the steps of (1) providing a biological sample of said subject, and (2) detecting in said sample increased PLAG1 and PLAGL2 expression. As will be appreciated by one of ordinary skill in the art, the choice of diagnostic methods of the present invention will be influenced by the nature of the available biological samples to be tested and the nature of the information required. When the diagnostic assay is to be based upon nucleic acids from a sample, either mRNA or cDNA may be used. With either mRNA or cDNA, standard methods well known in the art may be used to detect the presence of a particular sequence either in situ or in vitro (see, e.g. Sambrook et al., eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). A significant advantage of the use of either DNA or mRNA is the ability to amplify the amount of genetic material using the polymerase chain reaction (PCR). Other nucleotide sequence amplification techniques may be used, such as ligation-mediated PCR, anchored PCR and enzymatic amplification as will be understood by those skilled in the art. PLAG1 and PLAGL2 protein levels can be measured by any method well known in the art. Some examples of the latter method can be found further in the 'Examples' section.

Examples

1. PLAGL1 binds a different consensus sequence than PLAG1 and PLAGL2 In order to compare the DNA binding specificity's of the PLAG proteins, we performed EMSA analysis on double strand probes containing either the PLAG1 consensus binding site (WT2 probe), the PLAGL1 consensus binding site (zac probe) (8) and or on five mutated probes (see Figure 1). The proteins used for this study were full length PLAG proteins translated in vitro in reticulocyte lysates. As shown in a previous study (4), we found that PLAG1 binds strongly to the PLAG1 consensus binding motif (Figure 1A, lane 1). Mutations in the G-cluster reduce drastically the binding (lane 2) while destruction of the Core abolishes it (lane 3) as do mutations in both (lane 4). The distance between the G-cluster and the Core was also shown to be important, as PLAG1 only binds weakly to a probe containing the G-cluster separated by 2 bp instead of 7 bp from the Core (lane 5). In contrast, the binding of PLAG1 to the zac probe (lane 6) is much less efficient than to the WT2 probe. A similar binding profile is observed with PLAGL2 (Figure 1C, lanes 1 to 7) suggesting that PLAGL2 has binding capacities comparable to PLAGl In contrast, PLAGL1 show different binding specificity's (Figure 1B) as it binds more efficiently to the zac probe than to the WT2 probe (compare lane 6 to lane 1). Moreover, mutations in the cluster of WT2 only slightly affect PLAGL1 binding (lane 2), suggesting that a G-cluster is not critical for the binding of PLAGL1. This hypothesis is corroborated by the fact that the zac probe does not contain any G-cluster. Mutations in the core motif of the PLAG1 consensus, on the other hand, completely prevent the binding of PLAGL1 (lane 3) as well as mutations in the core of the zac probe. Comparable results were also obtained using chimeric proteins containing the complete zinc finger domain of PLAG proteins fused in-frame to Glutathione-S transferase (data not shown). All these results indicate that both motifs, the core and the G-cluster are critical for PLAG1 and PLAGL2 binding while only the core motif is recognized by PLAGL1.

2. Fingers 3 of PLAG1 and PLAGL2, but not of PLAGL1 are critical for the DNA binding capacities

Recently, we have shown that finger 3 of PLAG1 interacts with the G-cluster while the core is recognized by fingers 6 and 7 (4). As the G-cluster is critical for the binding of PLAG1 and

PLAGL2 but not for PLAGL1 , we were interested in determining whether finger 3 could play different roles in these three proteins. To answer this question, we produced mutant PLAG proteins where finger 3 was destroyed by replacing the first histidine of the C₂H₂ motif with an alanine. This mutation hinders the co-ordination of zinc and has been shown to prevent the formation of a functional zinc finger (16). The destruction of finger 3 in PLAG1 and in PLAGL2 drastically reduces the affinity of these proteins for the WT2 probe (compare lanes 8 with lanes 1 in Figure 1A and 1C). The specificity is also completely modified since the F3mut protein binds equally well to WT2, mCLU2 and WT2m1 (lanes 9,10 and 12 in Figure 1A and 1C). Moreover, the F3mut binds preferentially to the zac probe compared to the WT2 probe (compare lanes 13 and 8 in Figure 1A and 1C). These results indicate that, as for PLAG1 , finger 3 of PLAGL2 is required for interaction with the G-cluster. On the contrary, destruction of finger 3 of PLAGL1 does not affect the binding specificity of this protein and only slightly decreases its affinity (compare lanes 8 to 14 in figure 1 B with lanes 1 to 7). This indicates that in contrast to PLAG1 and PLAGL2, finger 3 of PLAGL1 is not directly involved in DNA recognition.

3. NIH3T3 cells infected with retrovirus containing human PLAG1 and PLAGL2 cDNA's express high levels of functional proteins.

To generate NIH3T3 cell lines overexpressing PLAG1 and PLAGL2, we infected the cells with the pMSCV retroviral vector encoding FLAG-tagged human PLAGl or PLAGL2. Empty pMSCV vector was used as a negative control. Positive transformants were obtained by puromycin selection and checked for gene expression by Northern blot and Western blot analyses. As shown on figure 2A, the exogenous PLAGl and PLAGL2 messages expressed from the viral 5' Long Terminal Repeat (5'LTR) could be detected as a 4 kb transcript in infected cells (lanes 2 and 4, respectively), but not in mock infected control cells (lanes 1 and 3). These are clearly overexpressed compared to the endogenous transcripts. The endogenous PLAGl transcript is visible as a 7.5 kb band (lanes 1 and 2) while the endogenous PLAGL2 transcript, which is also expected to be 7.5 kb, was undetectable (lanes 3 and 4), suggesting extremely low expression, if any, of endogenous PLAGL2. Western blot analysis was performed to detect the presence of PLAG1 and PLAGL2 recombinant proteins in infected NIH3T3 cells (Figure 2B). A 56 kDa and a 50 kDa protein corresponding to PLAG1 (lane 1) and PLAGL2 (lane 3) respectively were present in infected cells but absent in mock infected control cells (lane 2). These proteins were shown to be functional since PLAG1 and PLAGL2 overexpressing cells can stimulate the luciferase activity of a reporter construct containing three copies of the consensus binding site (WT2)₃-TK luc (27- and 10-fold respectively) (Figure 2C). This stimulation is completely abolished when the core and cluster are mutated (mCOmCLU). In contrary, mock infected cells only slightly induced the (WT2)₃-Tk luc reporter construct (~ 2,5 fold).This induction is probably due to endogenous PLAG1 present in the NIH3T3 cells.

All these data indicate that infected NIH3T3 cells produce high level of functional PLAG1 and PLAGL2 proteins. Moreover it is the first demonstration that PLAGL2 is also a genuine transcription factor.

4. Mitogenic stimulation of NIH3T3 cells overexpressing PLAG1 and PLAGL2. Transformed cells, unlike normal cells, are able to proliferate in culture medium containing low levels of serum, as they have become independent of growth factors. In order to prove that PLAG1 and PLAGL2 are transforming factors, we investigated the growth of NIH3T3 cells overexpressing PLAG1 and PLAGL2 in low serum culture conditions. Figure 3A shows the growth curve of a population of cells grown in 5% fetal calf serum (FCS). The proliferation rate of cells overexpressing PLAGl and PLAGL2 was significantly higher than mock infected NIH3T3 cells, (about 3-fold for PLAG1 and 2.5 fold for PLAGL2 expressing cells). The effect was even more striking when cells were cultured in 1% fetal calf serum. PLAG1 and PLAGL2 expressing cells proliferate, whereas mock infected NIH3T3 cells are unable to grow (Figure 3B). The PLAGl overexpressing cells consistently show higher growth rates than the PLAGL2 expressing cells. These data suggest that NIH3T3 cells overexpressing PLAGl and PLAGL2 behave like transformed cells and loose dependence on growth factors present in fetal calf serum.

5. Overexpressed PLAG1 and PLAGL2 proteins are able to transform NIH3T3 cells. To evaluate the transforming activity of PLAG1 and PLAGL2 when overexpressed, infected cells were grown as a monolayer to confluence in medium containing either 1% or 10% serum and the formation of foci was analyzed (Figure 4A). Contact inhibition of growth was lost leading the cells to pile up and form foci. Cells in the foci presented various morphologies, ranging from an elongated shape at the foci border, to a rounded and refractile appearance in the center. PLAG1 expressing cells seemed to have a higher efficiency in forming foci than PLAGL2 expressing cells (Figure 4B); Foci appeared sooner and became much bigger. Foci were more rapidly observed in cells grown in medium containing 1% serum. In contrast, mock infected NIH3T3 cells in both FCS concentrations exhibited contact inhibition and formed no foci. To test if PLAGl and PLAGL2 mediated effects on growth and transformation are dependent upon their DNA binding activity, we made retroviral constructs of PLAG1 and PLAGL2 containing a mutation in finger 7 in order to destroy their binding to DNA (4). NIH3T3 cells were infected with these constructs and focus-forming assay was performed. NIH3T3 cells were infected with these retroviruses and focus-forming assay was performed. Transformation of the NIH3T3 cells was almost completely abolished supporting the hypothesis that the transformation is caused by binding and subsequently activation of the target genes of PLAG1 and PLAGL2 (data not shown).

The cell lines overexpressing PLAGl and PLAGL2 were also tested for anchorage- independent growth, another tumorigenic feature. For this, cells were seeded in a soft agar suspension and colonies counted 3 weeks after growth. Colonies first appeared microscopically 10 days after seeding. Colonies of PLAG1 expressing cells remained small compared to colonies formed in PLAGL2 overexpressing cells where cells were able to form colonies visible by the naked eye within 4 weeks of incubation (Figure 4A). PLAGL2 expressing cells showed a colony-forming efficiency of 44% while for PLAG1 expressing cells, 26% of seeded cells give rise to colonies (Figure 4B). As expected, control cells failed to form such colonies. These results are summarized in Figure 4B. 6. PLAG1 and PLAGL2 expressing NIH3T3 cells are tumo αenic in nude mice.

To determine whether the PLAGl and PLAGL2 expressing NIH3T3 cells are tumorigenic in nude mice, cells were injected subcutaneously into athymic nu/nu NMRI mice and examined every week for tumor development. PLAG1 overexpressing cells induced rapidly growing tumors at the site of inoculation within 3 weeks, while tumor formation originating from cells overexpressing PLAGL2 was apparent a few days later. The mock infected cells did not form any tumors during this time period (Figure 4B). The mice were sacrificed after 5 weeks and histological analysis identified tumors induced by the PLAG1 expressing cell line as fibrosarcomas (data not shown). Metastatic spread to other organs was not detected macroscopically during the time period of observation, but formation of microscopic metastasis cannot be ruled out. To verify whether PLAG1 and PLAGL2 were still expressed in the tumor, northern blot analysis was performed. High levels of retroviral PLAGl and PLAGL2 transcripts could be detected indicating that the tumors produced in the nude mice originated from the injected cells (data not shown).

7. IGF-II is upregulated in cells overexpressing PLAG1 and PLAGL2.

As shown in this report, PLAGl and PLAGL2 have common transcriptional properties as they activate transcription via binding to the same DNA consensus sequence. These observations indicate that PLAG1 and PLAGL2 could be transcription factors that regulate common genes. As IGF-II has been identified as a putative target gene of PLAG1, we analyzed IGF-II expression in PLAGl and PLAGL2 overexpressing NIH3T3 cells. As shown in figure 5, a significant up-regulation of the major IGF-II transcript was observed in cells overexpressing PLAG1 and PLAGL2 (lane b and c). In addition, two minor transcripts of 2.0 and 1.2 KB, not present in the control cells, were also detected in cells overexpressing PLAG1 and PLAGL2. Moreover PLAGL2 induces IGF-II expression in the same range as PLAGL To exclude the possibility that IGF-II upregulation in transformed NIH3T3 cells could be a secondary effect occurring in the process of the transformation, we determined whether IGF-II constitutes a transcriptional target for PLAG1 and PLAGL2. For this purpose, we generated PLAG inducible cell lines where IGF-II expression could be analyzed shortly after induction of PLAG1 and PLAGL2 expression. We isolated independent clones, deriving from the human epithelial kidney 293 cell line, containing a zinc-inducible expression vector (11) encoding either PLAG1 (clones P1-8 and P1-32), PLAGL2 (clones PL2-2 and PL2-24) or the lacZ gene (clones B-1 and B-4). When clones were grown in the absence of zinc ions, no exogenous PLAG1 or PLAGL2 could be detected while only low level of β-galactosidase (β-gal) were detected. Upon induction with 100 μM of ZnCI₂, PLAG1 and PLAGL2 transcripts are efficiently synthesized. This expression results in a drastic upregulation of IGF-II expression (Figure 6). This stimulation is specific as it is not observed either with the βgal expressing clones used as control or with the parental cell line. Furthermore, IGF-II upregulation by PLAGL2 goes not via the induction of endogenous PLAG1 and vice versa as no stimulation of endogenous PLAG1 and PLAGL2 transcript could be detected after zinc induction (data not shown). Moreover, the detected 6 kb IGF-II transcript corresponds to the one deriving from the P3 promoter, which is known to contain PLAG1 binding sites (4) as hybridization with a probe specific for the P3 transcript (exon 5 probe) detects the same band (data not shown). All these results demonstrate a strong correlation between PLAGl, PLAGL2 and IGF-II expression, suggesting that IGF-II is a target gene not only for PLAG1 but also for PLAGL2.

Materials and Methods Plasmid Construction. pCDNA3-FLAG-PLAG1 (=pKH26) was constructed by ligating in frame the Mscl/Xhol fragment isolated from the pCDNA3-PLAG1 expression construct (4) into pCDNA3.1FLAG (a kind gift of Stefan Pype of the laboratory of Cell Growth, Differentiation and Development K.U.Leuven, VIB). This enables expression of a chimeric protein containing the FLAG epitope fused to amino acids 2 to 500 of PLAGL The complete PLAGL2 ORF except the first ATG was amplificated using pfu DNA polymerase (Stratagene) with the sense primer P3N2 (5'CCCGAATTCTGACCACA l l l l l CACCAGCG3') and the antisense primer P3C496 (5TTTCCATCAAGCATTCCAGTAGCTCGAG3'). The PCR product was cloned in frame into the pCDNA3.1 FLAG vector (=pKH32). pMSCV-FLAG-PLAG1 and pMSCV-FLAG- PLAGL2 were constructed by cloning a blunt Nhel/Xbal fragment of pKH26 or pKH32 into the Hpal site of the pMSCVpuro retroviral vector respectively (kindly provided by Jan Cools, CME, KULeuven and described by (10). All constructs were sequenced to confirm the fidelity of the PCR and the site-specific mutagenesis. The PLAG1 expression construct pCDNA3-PLAG1 has been described elsewhere (4). Expression plasmids pCDNA3-PLAGL1 and pCDNA3-PLAGL2 were constructed by inserting into EcoRI-Xhol digested pCDNA3 (Invitrogen) the complete ORFs of PLAGL1 or PLAGL2 including their own Kozak consensus translation start site. These fragments were generated by PCR using Pfu polymerase (Stratagene) with the 5' primer P2N-3 (5'- CCCGAATTCGCAAAGCCCATGGCCACGTTC-3') and the 3' primer P2C463 (5'-GGGCTCGAGTTATCTGAATGCATGATGGAAATGAG-3') for PLAGL1 and with the 5' primer P3N-3 (5'-CCCGAATTCAGCCTTGCCATGACCACATTT-3') and the 3' primer P3C496 (5'-

GGGCTCGAGCTACTGGAATGCTTGATGGAAA-3') for PLAGL2. The three mutant PLAG cDNA's (PLAG1-F3mut, PLAGL1-F3mut and PLAGL2-F3mut) were produced by altering the first codon for the histidine in the C₂H₂ motif of the corresponding zinc finger to that for alanine. This was performed using the QuickChange Site-directed Mutagenesis kit (Stratagene) according to the instructions of the supplier. The pSAR-MT- FLAG-PLAG1 and pSAR-MT- FLAG-PLAGL2 constructs were generated by inserting the Nhel/Xbal blunted fragments of pKH26 and pKH32 respectively into the blunted BamHI site of the pSAR-MT vector (11). (WT2)₃-TK-luc and (mCLUmCO2)₃-TK-luc have been obtained by inserting 3 copies of the corresponding ds oligonucleotides WT2 and mCOmCLU2 (4)into pTK81 luc (12).

Cell lines: The human fetal kidney epithelial cell line 293 (ATCC, CRL 1573) was cultured according to the suppliers' protocols. Inducible PLAG1 , PLAGL2 and β-galactosidase expressing cell lines were obtained by transfecting 293 cell line with 2 μg of pSAR-MT-FLAG- PLAG1 , pSAR-MT-FLAG-PLAGL2 or pSAR-MT-β-gal (11) respectively together with 400 ng of the neomycin resistance vector pCDNA3 using FuGENE™ 6 Transfection Reagent (Boehringer Mannheim) according to the manufacturer's protocol. After 10 days selection with G418 (Life technologies) at a concentration of 700 μg /ml, individual clones were isolated and expanded. Individual colonies were screened for zinc inducible expression of PLAG1, PLAGL2 or β-galactosidase by Western blot analysis. Cells were induced with 100 μM ZnCI₂ for 16 hours.

The pMSCV retroviral constructs were cotransfected with the pIK 6.1 Ecopac vector (kindly provided by Jan Cools, CME, KULeuven), coding for the gag, pol and env viral proteins, in 293T cells using Superfect (qiagen) according to the manufacturer's protocol. 48 hours after transfection, 1 ml of the supernatant containing replication incompetent-retroviruses was used to infect NIH3T3 cells (ATCC CRL1658). Cells expressing the gene of interest were obtained after 2 days culturing under puromycin selection.

Western blot analysis. Cells were harvested in PBS/EDTA. The cell pellets were lysed in SDS-PAGE sample buffer (60mM TRIS-HCL pH 6.8, 12% glycerol, 4% SDS), and sonicated. Samples containing equal protein amounts were heated at 95°C for 5 min and were electrophored in a 10% polyacrylamide gel and blotted onto nitrocellulose membranes. FLAG- tagged proteins were detected with a mouse anti-FLAG monoclonal antibody (SIGMA) (0.8 μg/ml) followed by a peroxidase-labeled rabbit α-mouse polyclonal antibody (PROSAN, DAKO) (0.4 μg/ml). Blots were revealed using the Renaissance detection kit (NEN Life Science products) according to the supplier's instructions.

Transfections and luciferase assay. The puromycin-resistant cells, obtained after infection with recombinant retroviruses or empty pMSCV vectors, were plated in 6-well plates. The next day, they were transiently cotransfected in triplicate with 400ng of (WT2)₃TKIuc or (mCLUmCO2)₃TKIuc reporter plasmids together with the Rouse sarcoma virus β-galactosidase DNA as internal control using 3 μl of FuGENE 6 Transfection Reagent (Boehringer Mannheim) according to the manufacturer's protocol. Cells were harvested 40 h after the transfection, and luciferase activity measured using a Monolight 2010 luminometer (Analytical Luminescence Laboratory).

Preparation of RNA and Northern blot analysis. Total RNA was extracted using the guanidine thiocyanate method (13). Northern blot analysis was performed according to standard procedures (Sambrook et al 1989). For filter hybridization's, probes were radiolabeled with α-³²P-dCTP using the megaprime DNA labeling system (Amersham). A 1.5 kb cDNA probe containing the complete PLAGl or PLAGL2 ORF was used for the detection of the respective transcripts. A probe containing exon 6 of the mouse IGF-II, which is common to the different transcripts generated by the three different promoters, was generated by PCR using sense primer mIGFII-up (5'CAGATACCCCGTGGGCAAGTTCTTCCAATA3') and antisense primer mIGFII-low (5TGAAGGGGGGGGGGCGCCGAATTATTTGA3'). The human IGF-II exon 9 probe, common to the 4 different transcripts P1 , P2, P3 and P4, was generated by PCR and contained nucleotides 7970 to 8774 of the published gene sequence (14) (GenBank/EMBL, accession number X03562). A human IGF-II exon 5 probe specific for the P3 transcript was a kind gift of Dr. P.E. Holthuizen. The lacZ probe was generated by isolation of a 2.3 kb Clal-EcoRI fragment from SDKIacZpA (15) (kindly given by Dr. J. Rossant).

EMSA: The full-length PLAG proteins as well as the mutants were expressed by in vitro transcription and translation using the TNT Kit (Promega). Quality of translation was monitored by SDS PAGE analysis of [³⁵S] Met-labeled proteins. The EMSAs were performed as described previously (4)and 3 μl of translation reaction products were used per lane.

Focus formation assay. Puromycin-resistant cells, obtained after infection with recombinant retroviruses, were plated at a density of 3X10⁵ cells in a 60mm tissue culture dish and grown in DMEM/F12 medium supplemented with 10% or 1% fetal calf serum (FCS) and puromycin. Medium was changed every two or three days and foci of densely growing cells appeared after 2-3 weeks. Some of the foci were cloned and grown to mass culture for RNA and protein analysis.

Cell proliferation assay. Puromycin-resistant cells, obtained after infection with recombinant retroviruses, were plated at a density of 1X10⁵ cells in a 60mm tissue culture dish and cultured in of 5% or 1% FCS. Cells were counted every day for 10 days using a coulter counter. Soft agar assay. 1X10⁵ of Puromycin-resistant cells, obtained after infection with recombinant retroviruses, were resuspended in 2ml 0.3%agar/DMEM/15%FCS and plated on a base of 0.6%agar/DMEM/15%FCS in 6-well plates. After 5 days the cells were fed with a fresh agar overlay in DMEM containing 15%FCS. Colonies were counted three weeks after plating.

Tumorigenicity in nude mice. Tumorigenicity was evaluated by injecting the puromycin- resistant cells, obtained after infection with recombinant retroviruses carrying pMSCV-FLAG- PLAG1, pMSCV-FLAG-PLAGL2 or empty pMSCV vectors (5X10⁶ cells in 0.1ml PBS) subcutaneously into both flanks of athymic nude mice. The mice were examined for tumor development once a week for 2 months. All tumors were clearly macroscopically visible within 3-4 weeks after inoculation. Mice were sacrificed after 5 weeks and the tumors were excised for RNA extraction or histological analysis.

References

1. Kas, K., Voz, M. L, Roijer, E., Astrom, A. K., Meyen, E., Stenman, G., and Van de Ven, W. J. Promoter swapping between the genes for a novel zinc finger protein and beta- catenin in pleiomorphic adenomas with t(3;8)(p21 ;q12) translocations, Nat Genet. 15:

170-4, 1997.

2. Voz, M. L, Astrom, A. K., Kas, K., Mark, J., Stenman, G., and Van de Ven, W. J. The recurrent translocation t(5;8)(p13;q12) in pleomorphic adenomas results in upregulation of PLAG1 gene expression under control of the LIFR promoter, Oncogene. 16: 1409- 16, 1998.

3. Astrom, A. K., Voz, M. L, Kas, K., Roijer, E., Wedell, B., Mandahl, N., Van de Ven, W., Mark, J., and Stenman, G. Conserved mechanism of PLAG1 activation in salivary gland tumors with and without chromosome 8q12 abnormalities: identification of SI I as a new fusion partner gene [In Process Citation], Cancer Res. 59: 918-23, 1999. 4. Voz, M. L, Agten, N. S., Van de Ven, W. J., and Kas, K. PLAG1 , the main translocation target in pleomorphic adenoma of the salivary glands, is a positive regulator of IGF-II, Cancer Res. 60: 106-13, 2000.

5. Kas, K., Voz, M. L., Hensen, K., Meyen, E., and Van de Ven, W. J. M. Transcriptional activation capacity of the novel PLAG family of zinc finger proteins [In Process Citation], J Biol Chem. 273: 23026-32, 1998.

6. Abdollahi, A., Roberts, D., Godwin, A. K., Schultz, D. C, Sonoda, G., Testa, J. R., and Hamilton, T. C. Identification of a zinc-finger gene at 6q25: a chromosomal region implicated in development of many solid tumors, Oncogene. 14: 1973-9, 1997.

7. Spengler, D., Villalba, M., Hoffmann, A., Pantaloni, O, Houssami, S., Bockaert, J., and Journot, L. Regulation of apoptosis and cell cycle arrest by Zac1 , a novel zinc finger protein expressed in the pituitary gland and the brain, Embo J. 16: 2814-25, 1997.

8. Varrault, A., Ciani, E., Apiou, F., Bilanges, B., Hoffmann, A., Pantaloni, C, Bockaert, J., Spengler, D., and Journot, L. hZAC encodes a zinc finger protein with antiproliferative properties and maps to a chromosomal region frequently lost in cancer, Proc Natl Acad Sci U S A. 95: 8835-40, 1998.

9. Bilanges, B., Varrault, A., Basyuk, E., Rodriguez, O, Mazumdar, A., Pantaloni, C, Bockaert, J., Theillet, C, Spengler, D., and Journot, L. Loss of expression of the candidate tumor suppressor gene ZAC in breast cancer cell lines and primary tumors, Oncogene. 18: 3979-88, 1999. 10. Hawley, R. G., Lieu, F. H., Fong, A. Z., and Hawley, T. S. Versatile retroviral vectors for potential use in gene therapy, Gene Ther. 1: 136-8, 1994. 11. Morin, P. J., Vogelstein, B., and Kinzler, K. W. Apoptosis and APC in colorectal tumorigenesis, Proc Natl Acad Sci U S A. 93: 7950-4, 1996.

12. Nordeen, S. K. Luciferase reporter gene vectors for analysis of promoters and enhancers, Biotechniques. 6: 454-8, 1988. 13. Chomczynski, P. and Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal Biochem. 162: 156-9., 1987. 14. Dull, T. J., Gray, A., Hayflick, J. S., and Ullrich, A. Insulin-like growth factor II precursor gene organization in relation to insulin gene family, Nature. 310: 777-81 , 1984. 15. Sanchez, M. P., Silos-Santiago, I., Frisen, J., He, B., Lira, S. A., and Barbacid, M. Renal agenesis and the absence of enteric neurons in mice lacking GDNF, Nature. 382: 70-3, 1996.

16. Ikeda, K. and Kawakami, K. DNA binding through distinct domains of zinc-finger- homeodomain protein AREB6 has different effects on gene transcription, Eur J Biochem. 233: 73-82, 1995.

17. Choo, Y. and Klug, A. Physical basis of a protein-DNA recognition code, Curr Opin Struct Biol. 7: 117-25, 1997.

Claims

1. Use of the PLAG1 consensus binding motif to screen for molecules which suppress PLAG1 and/or PLAGL2-mediated neoplastic transformation comprising: -exposing the PLAG1 consensus binding motif to at least one molecule whose ability to suppress neoplastic transformation is sought to be determined, -determining binding or hybridising of said molecule(s) to the PLAG1 consensus binding motif, or to PLAG1 and/or PLAGL2 proteins, or to Protein Inhibitor of Activated STAT (PIAS) proteins, and -monitoring said neoplastic transformation when administering said molecules as a medicament.

2. Use according to claim 1 , wherein said PLAG1 consensus binding motif comprises 1) a core sequence consisting of the nucleotides GGGGGCCC, 2) a G-cluster consisting of the nucleotides GGGG, and 3) seven random nucleotides between said core sequence and said G-cluster.

3. Use according to claims 1 and 2, wherein said PIAS proteins are chosen from the group consisting of: PIAS1 , PIAS3, PIASβ and PIASγ.

4. Use according to claims 1 to 3, wherein said molecule which suppresses PLAG and/or PLAGL2-mediated transformation is an antibody or a fragment thereof, a peptide, a protein, a small molecule, an anti-sense nucleic acid, a ribozyme or a short interference RNA molecule.

5. Use of PLAG1 and PLAGL2, or PLAGL2 for the preparation of a diagnostic kit to diagnose neoplastic transformation.