WO2003018037A2

WO2003018037A2 - Regulation method of cell growth

Info

Publication number: WO2003018037A2
Application number: PCT/IB2002/003656
Authority: WO
Inventors: Stephen Cohen; Aurelio Teleman
Original assignee: European Molecular Biology Laboratory
Priority date: 2001-08-09
Filing date: 2002-08-08
Publication date: 2003-03-06
Also published as: AU2002330693A1; WO2003018037A3

Abstract

The invention relates to methods of regulating the rate of growth of cells, tissues and organisms by regulating the rate of cell mass accumulation and the rate of cell cycle progression in a co-ordinated manner. In particular, the invention relates to methods of regulating the rate of growth of cells, tissues and organisms by modulating the level and the activity of specific proteins. The invention also relates to the use of these proteins, for example, in the treatment of diseases and conditions associated with an aberrant rate of cell growth.

Description

REGULATION METHOD

All publications, patents and patent applications cited herein are incorporated in full by reference.

One of the fundamental processes in the biology of multicellular organisms is the control of the rate of growth of individual cells, tissues and whole organisms. The rate of growth of a cell, tissue or organism is determined both by the rate of cell cycle progression and by the rate of cell mass accumulation of individual cells. The rate of cell cycle progression, or cell proliferation, is the rate at which cells divide. The rate of cell mass accumulation, which is sometimes referred to as the rate of cell growth, is the rate at which individual cells grow in size. In order to achieve normal tissue growth, cells must both grow and divide in a co-ordinated manner. The rate of cell cycle progression and the rate of cell mass accumulation are therefore very tightly controlled and co-ordinated during normal development. This control and co-ordination ensures, for example, that the organs are properly proportioned in the adult organism. When the control and/or co-ordination of either of these two processes is faulty, pathological conditions may arise. For example, a mutation in the FGFR3 gene that results in an inhibition of cell division is the most common form of inherited human dwarfism. The rate of growth of individual cells and tissues is tightly controlled and co-ordinated not only during development but also in adult, fully-grown organisms. Loss of control and coordination of the rate of cell cycle progression and/or the rate of cell mass accumulation in an organism can also lead to pathological conditions. For example, an aberrantly high rate of cell proliferation which is uncontrolled can cause cancer. This loss of control of the rate of cell cycle progression often occurs in combination with a change in cell fate and can cause metastases. There is thus an evident medical need for a greater understanding of the complex mechanisms that control the rate of growth of individual cells, tissues and organisms with the aim of developing drugs that can restore normal control when such control is lost.

Additionally, in agriculture, there are great benefits that can be derived from the ability to control the rate of growth of animals and plants and hence the overall size of such animals and plants. For example, expression levels of components of growth control pathways have previously been manipulated to yield larger organisms, such as larger and faster growing cows (Conlon and Raf, 1999) or larger tomatoes (Frary et al, 2000).

Despite these considerable medical and commercial implications for the understanding of growth control, little is known about how the rate of cell cycle progression and the rate of cell mass accumulation are co-ordinately regulated.

In the past few years, a number of proteins involved in regulating the rate of cell mass accumulation have been identified. For example, particular, all components of the insulin- receptor/PI3K pathway, including the Insulin receptor, the p60 adaptor protein (known as chico or insulin receptor substrate IRS4), PI3K, PTEN and Akt/PKB have been shown to influence the final size of a developing tissue (reviewed in Stacker and Hafen 2000). Overexpression of the components of this pathway speeds up the rate of cell mass accumulation without affecting the rate of cell cycle progression and cell division, resulting in cells that are no larger than normal. Many components of this pathway, notably PTEN, are also oncogenes (Li et al, 1997). The oncogenes Ras, Myc, Tscl and Tsc2 have also been shown to be potent drivers of tissue growth. These proteins also increase the rate of cell mass accumulation, resulting in larger than normal cells and it is thought that they exert their effects via the PI3K pathway (Johnston, Prober et al. 1999; Prober and Edgar 2000; Gao and Pan 2001). A number of proteins involved in regulating the rate of cell cycle progression have also been identified. For example, overexpression of E2F or cdc25/string results in an increase in the rate of cell cycle progression. However, because overexpression of these proteins does not affect the rate of cell mass accumulation, the result is cells that are smaller than normal because although the cells are dividing faster, there is no increase in size prior to division. Conversely, mutations in cdc2 decrease the rate of cell cycle progression, resulting in cells that are larger than normal because although cells are dividing more slowly, the rate of mass accumulation is unchanged. The majority of the proteins that have been identified to date are involved either in the regulation of the rate of cell cycle progression or in the regulation of the rate of mass accumulation. However, in order to achieve normal tissue growth, cells must both grow (increase in mass) and divide in a co-ordinated way. To date, only one protein complex that can regulate both the rate of cell cycle progression and the rate of mass accumulation has been identified, the cyclinD/cdk4 complex (Datar, Jacobs et al. 2000; Meyer, Jacobs et al. 2000).

In view of the medical and agricultural applications, there is thus a need for methods of regulating the rate of growth of cells, tissues and organisms by regulating both the rate of accumulation of cell mass and the rate of cell cycle progression in a co-ordinated fashion.

Summary of the invention

According to the invention, there is provided a method of regulating the rate of growth of a cell comprising modulating in said cell the activity or the level of a Melted protein or a functional equivalent thereof. The inventors have established that proteins termed Melted proteins are involved in the control of tissue growth. Overexpression of a gene encoding a Melted protein causes tissues to grow larger than normal by making cells grow and divide in a co-ordinated manner. In contrast to the effects of the oncogenes Ras, Myc, Tscl, 2 and the components of insulin signalling pathway which only increase the rate of mass accumulation and hence induce the formation of abnormally large cells, cells which result from overexpression of Melted are normal in size. Thus, Melted overexpression appears to increase tissue growth by way of a co-ordinated increase in both the rate of cell mass accumulation and the rate of cell cycle progression.

By "modulating the rate of growth of a cell" is meant modulating both the rate of mass accumulation of a cell and the rate of cell cycle progression of the cell in a co-ordinated manner. By the "rate of mass accumulation" is meant the rate at which the cell grows in size. By the "rate of cell cycle progression" is meant the rate at which the cell progresses through the cell cycle, which can also be described as the rate of cell proliferation or the rate of cell division. "Modulating the rate of growth of a cell" includes either up-regulating the rate of growth of a cell or down-regulating the rate of growth of a cell. Up-regulating the rate of growth

mace arrπmπlatinn arid the rate of cell cycle progression in a co-ordinated manner. Conversely, down-regulating the rate of growth of a cell involves down-regulating both the rate of mass accumulation and the rate of cell cycle progression in a co-ordinated manner.

The methods of the invention for modulating the rate of growth of cells have a wide variety of applications. In particular, methods of up-regulating the rate of growth of a cell have numerous applications in the field of stem cell research. Recently, there has been considerable interest in the possibility that the differentiation of stem cells could be controlled in vitro to enable specific types of cells and tissues to be produced for transplantation puiposes (see Odorico et al, 2001 for review). However, understanding the processes involved in multilineage differentiation of stem cells is hampered by the inability to produce and maintain homogeneous stem cell cultures. The methods of the invention may enable researchers to modulate the rate of growth of stem cells so that homogeneous stem cell cultures can be produced and maintained. This would enable researchers to gain a better understanding of the processes that are involved in the differentiation of stem cells with the eventual aim that the cultures would be used as the starting point for the production of a variety of cell and tissue types.

The invention is not limited to methods of regulating the rate of growth of single cells. The cells of the invention may form part of a tissue or even part of an organism. The invention therefore includes methods of modulating the rate of growth of a tissue and/or the rate of growth of an organism by modulating the level or activity of a Melted protein or a functional equivalent thereof. Methods of regulating the rate of growth of tissues and organisms clearly have many applications in the medical and agricultural fields. In particular, methods of modulating the rate of growth of cells in organisms that result in the production of bigger or smaller organisms, particularly bigger organisms, will be useful in the agricultural field.

The rate of cell growth may be modulated in any organism, animal or plant. Examples of plants in which it may be desirable to increase the rate of cell growth include crop plants, including vegetable plants and fruit trees, and also flowering plants.

Preferably, the rate of cell growth is modulated in an animal. Examples of animals in which the rate of cell growth may be modulated include mammals, insects and fish.

Examples of mammals in which it may be desirable to modulate the rate of cell growth include, in particular, domesticated animals such as sheep, cows, pigs, goats, deer, buffalo, crocodiles, ostriches and rodents. The rate of cell growth may also be modulated in other organisms such as fish and insects. The methods of the invention for modulating the rate of growth of cells, as outlined above, will also be useful in research into growth regulation in general. These methods will enable researchers to determine the role of the Melted protein and other proteins that function in the same signalling pathways as the Melted protein not only in normal development but also in diseases and conditions that are associated with abnormal cell mass accumulation, abnormal cell cycle progression or both. For example, it is possible that increased Melted protein activity or increased levels of Melted protein may be involved in the development of tissue malignancy. By "Melted protein" is meant any protein exhibiting an amino acid sequence as presented in Figure 5, and functional equivalents of this sequence, such as homologues and active fragments of this amino acid sequence. The amino acid sequence in Figure 5 is the amino acid sequence of the human Melted protein.

The term "functional equivalent" includes reference to homologues of the Melted protein. By "homologue" is meant a protein exhibiting a high degree of similarity or identity to the amino acid sequence of the Melted proteins of the invention. By "similarity" is meant that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. By "identity" is meant that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. Preferably, homologues possess greater than 30% identity with the sequence in Figure 5. More preferably, homologues show greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% sequence identity with the sequence of the wild type human Melted protein, as aligned using, for example, BLAST version 2.1.3 using the default parameters specified by the NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; gap open penalty=l 1 and gap extension penalty=l], provided that these homologues retain the ability to act as regulators of the rate of growth of a cell. Homologues may include proteins in which one or more of the amino acid residues are substituted with another amino acid residue. Any such substituted amino acid residue may or may not be a naturally occurring amino acid. Tools such as PROSITE (httpJ/expasy.hcuge.ch/sprot/prosite.html), PRINTS http://iupab.leeds.ac.uk/bmb5dp/prints.html), Profiles (http://ulrec3.unil.ch/software/ PFSCAN_form.html), Pfam (http://www.sanger.ac.uk/software/pfam), Identify (http://dna.stanford.edu/identify/) and Blocks (http://www.blocks.fhcrc.org) databases may also be used to identify homologues of Melted, as well as hidden Markov models (HMMs; preferably profile HMMs).

In particular, the term "homologue" includes Melted proteins from species other than the human. Preferably, such homologues not only possess at least 30% identity with the Melted protein from humans but also contain regions of homology which are common to Melted proteins. Preferably, said homologues may contain a pleckstrin homology domain. Homologues from other species may also contain additional regions of homology outside the pleckstrin homology domain, such as the MH1 and MH2 domains, as illustrated in Figure 8.

Functional equivalents of the Melted proteins of the invention also include active fragments of the naturally-occurring Melted protein sequence in Figure 5 and homologues thereof. The term "active fragment" include any fragment of a Melted protein or a Melted homologue that retains the ability to modulate the rate of growth of a cell. Functional equivalents of the Melted proteins of the invention also include natural biological variants of the naturally-occurring Melted protein sequence presented in Figure 5 and homologues thereof (e.g. allelic variants or geographical variants within the species from which the Melted proteins are derived), provided that such variants retain the ability to act as regulators of the rate of growth of a cell. Active fragments of such biological variants are also included in the invention.

Variants of the Melted proteins of the invention also include mutants containing amino acid substitutions, insertions or deletions from the wild type sequence of the Melted protein or its homologues. Such mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code. Typical such substitutions are among Ala, Nal, Leu and lie; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr. Particularly preferred are variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or added in any combination. Especially preferred are silent substitutions, additions and deletions, which do not alter the properties and activities of the protein. Also especially preferred in this regard are conservative substitutions. Active fragments of such mutants are also included in the invention.

Variants with altered function from that of the wild type sequence of the Melted proteins of the invention, are also included in the term "functional equivalent". Such variants may be designed through the systematic or directed mutation of specific residues in the protein sequence. Alterations in function that may be desired will include an altered ability to modulate the rate of growth of a cell compared to naturally-occurring Melted proteins. For example, variants may demonstrate an improved ability to up-regulate the rate of growth of a cell as compared to naturally-occurring Melted proteins. The term "variants" also includes dominant negative mutants of naturally-occurring Melted proteins that down-regulate the rate of growth of a cell. In addition, variants having improved stability in the cell compared to the naturally-occurring Melted proteins may be desired. Active fragments of these variants are also included in the invention.

As the skilled reader will appreciate, numerous methods are available for the random or systematic mutation of proteins, including in particular, site-directed mutagenesis. Furthermore, fragments may be generated using well known techniques of genetic engineering, through truncation and deletion of encoding gene sequences.

The term "functional equivalent" also refers to molecules that are structurally similar to the Melted proteins of the present invention or that contain similar or identical tertiary structure, and that possess the ability to act as regulators of the rate of growth of a cell. Such functional equivalents may be derived from the natural Melted proteins or they may be prepared synthetically or using techniques of genetic engineering. In particular, synthetic molecules such as peptide mimetics that are designed to mimic the tertiary structure or active site of the naturally-occurring Melted proteins of the invention are considered to be functional equivalents. In addition, molecules that are structurally similar to the variants of the Melted proteins of the invention, as described above, are included in the invention. Molecules that are structurally similar to dominant negative mutants of the Melted proteins of the invention are also considered to be functional equivalents.

The function of these functionally equivalent variants of the Melted protein may be tested in a suitable assay system that detects the effect of the variant on the regulation of cell growth.

Suitable assay systems may be cell culture-based, or may utilise genetically-modified organisms that have been manipulated to express a candidate functionally-equivalent variant. Such techniques include those exemplified herein for the full length Melted protein.

Preferably, the method of the invention for regulating the rate of growth of a particular cell type may comprise modulating the level or the activity of a Melted protein that is endogenous to that particular cell type. By "endogenous Melted protein" is meant that the genome in the cell naturally comprises a gene encoding the Melted protein of the type that is used in the method. For example, the cell may be a murine cell, in which case the modulated Melted protein may be a murine Melted protein. However, the invention also includes methods of regulating the rate of cell growth by modulating the level or activity of a Melted protein that is not endogenous to the cell whose rate of growth is being modulated, using, for example, a Melted protein from another species. For example, a nucleic acid molecule encoding a Drosophila Melted protein may be introduced into a mammalian cell.

According to one aspect of the invention, there is provided a method of up-regulating the rate of growth of a cell by increasing the activity or the level of a Melted protein or a functional equivalent thereof which acts as an up-regulator.

In one embodiment of this aspect, the method may involve supplying the cell with a Melted protein or a functional equivalent thereof which acts as an up-regulator. This may be done by any of several means which will be clear to the person skilled in the art. For example, the level of the Melted protein or functional equivalent thereof may be increased simply by introducing the protein directly into the cell. This may be done, for example, by scrape loading, microinjection, or by using liposomes or other carriers, such as the third helix of homeodomain. Alternatively, the level of the Melted protein or the functional equivalent may be increased by introducing a nucleic acid molecule encoding the Melted protein or functional equivalent into the cell and expressing the protein from the nucleic acid.

The level of the Melted protein or functional equivalent may also be increased by increasing the level of expression of the endogenous Melted protein or functional equivalent in the cell. The level of expression of this endogenous Melted protein or functional equivalent may, for example, be increased by supplying the cell with factors that promote transcription or translation of the endogenous gene encoding the Melted protein or functional equivalent. Such factors may, for example, be transcription factors that bind to the enhancer elements upstream of the genes coding for these proteins. Such transcription factors may be endogenous proteins that normally activate transcription in the cell. Alternatively, they may be artificial transcription factors, designed to bind to promoter and enhancer regions upstream of the genes encoding the Melted protein or functional equivalent to stimulate transcription. In some cases, for example, it may be desirable to introduce an enhancer element upstream of a gene encoding the Melted protein or functional equivalent. A transcription factor that binds to the newly introduced enhancer region can then be introduced into the cell to promote transcription of the gene. For example, in a Drosophila cell, the insertion of an EP element (a P element containing a UAS enhancer) upstream of the Melted gene followed by introduction of a GAL4 driver, results in increased transcription of the endogenous Melted gene.

Mechanisms by which translation may be controlled are also well known in the art and according to the invention, it may be possible to adapt the natural mechanisms in the cell to increase the translation of the desired nucleic acid molecule, either directly or by increasing the stability of the RNA. For example, it may be possible to introduce an iron response element into the 5' region of an mRNA, similar to that found in ferritin mRNA, to increase translation.

The level of the Melted protein or functional equivalent thereof may also be increased by increasing the stability of the protein itself. Variants with improved stability compared to the wild-type Melted proteins are included within the term "functional equivalents" as indicated above. The stability of the Melted protein may also be increased by using factors that bind to the protein and stabilise it.

According to a further aspect of the invention, there is provided a method of up-regulating the rate of growth of a cell by decreasing the level or activity of a functional equivalent of a Melted protein that acts as a down-regulator, such as a dominant negative mutant of a naturally-occurring Melted protein. The level of such a functional equivalent may be decreased by any of the methods outlined above.

According to a further aspect of the invention, there is provided a method of down- regulating the rate of growth of a cell by decreasing the level of the Melted protein, or by increasing the level or the expression of a form of the Melted protein that acts as a down- regulator. Such as, for example, a dominant negative mutant of a naturally-occurring Melted protein. The levels of such proteins may be altered using any of the methods that are outlined above.

According to one embodiment of this aspect, decreasing the level of a Melted protein in a cell may comprise decreasing the expression of an endogenous Melted protein. This may involve supplying the cell with a factor that decreases the level of transcription or translation of the endogenous gene encoding the Melted protein. The level of expression of Melted protein may also be decreased by introducing an antisense nucleic acid molecule or ribozyme into the cell.

In one example, an antisense nucleic acid molecule may inhibit the transcription or translation of a gene encoding the Melted protein by hybridising to the appropriate encoding mRNA or genomic DNA. An antisense nucleic acid molecule, such as described above, may be delivered to the cell directly. This may be done employing the methods described below for introducing nucleic acids directly into cells. Alternatively, an expression vector, such as a plasmid may be delivered to the cell, such that when the nucleic acid in the plasmid is transcribed in the cell, it produces an mRNA molecule which is complementary to a unique portion of the cellular mRNA which encodes the Melted protein.

In an alternative example, the level of translation of the Melted protein may be decreased by using ribozymes that are specific to the encoding mRNA sequence. Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al., Curr. Opin. Struct. Biol (1996) 6(4), 527-33.) Synthetic ribozymes can be designed specifically to cleave mRNAs that encode the Melted protein at selected positions, thereby preventing translation of the mRNAs into functional Melted polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribozymes may be synthesised with non-natural backbones to provide protection from ribonuclease degradation, for example, 2'-O-methyl RNA, and may contain modified bases.

The level of the Melted protein or functional equivalent may also be decreased by supplying the cell with factors that decrease the level of the protein directly. For example, enzymes which degrade the Melted protein or factors that decrease the stability of the Melted protein and make it more susceptible to degradation may be introduced into the cell. The methods of the invention may also involve modulating the rate of growth of a cell by modulating the activity of a Melted protein or functional equivalent thereof. As indicated above, the invention provides a method of up-regulating the rate of growth of a cell by increasing the activity of a Melted protein or a functional equivalent thereof that acts as a up-regulator. Conversely, there is provided a method of down-regulating the rate of growth of a cell by increasing the activity of a functional equivalent of a Melted protein that acts as a down-regulator. In addition, the invention provides a method of upregulating that rate of growth of a cell by decreasing the activity of a Melted protein or a functional equivalent thereof that acts as an up-regulator. Conversely, there is provided a method of downregulating the rate of growth of a cell by increasing the activity of a functional equivalent of a Melted protein that acts as a down-regulator.

The activity of a Melted protein or a functional equivalent thereof may be modulated in these methods using a variety of means. Although the Applicant does not wish to be limited by this theory, it is thought that the activity of the Melted protein may be related to its subcellular localisation. Therefore, to one aspect of the invention, there is provided a method regulating the rate of growth of a cell by modulating the activity of a Melted protein or a functional equivalent by altering its subcellular localisation. For example, the subcellular localisation of a Melted protein or functional equivalent thereof may be altered by means of a signal sequence, such as, a nuclear localisation signal that can be incorporated into a Melted protein that is introduced into a cell. In particular, it is postulated that the activity of the Melted protein or functional equivalent thereof may be modulated by localisation of the protein to a cellular membrane, although the Applicant does not wish to be limited by this theory. The invention therefore provides a method of regulating the rate of growth of a cell by modulating the activity of a Melted protein or a functional equivalent by altering the localisation of the protein towards membranes within the cell. Preferably, the activity of the Melted protein or a functional equivalent may be increased by localisation of the protein to a membrane.

Alternatively, the activity of the Melted protein or functional equivalent thereof may be increased or decreased through covalent modification. Any of the methods of modulating the rate of growth of a cell described above may be inducible or repressible within the cell. In particular, in the case of methods which involve introducing a nucleic acid molecule encoding a Melted protein or a functional equivalent into the cell, it may be useful to insert control elements into the promoter region of the encoding gene such that the expression of the protein in the cell be controlled. For example, a hormone binding domain might be inserted in the promoter region of the gene such that the expression of the nucleic acid encoding the Melted protein could be increased or decreased in the cell by the addition or hormones that bind to those domains. In particular, it is preferred to use hormones which will have negligible side-effects in the cell. Control of gene expression in this way may also be tissue-specific. Alternatively, the activity of a Melted protein or a functional equivalent thereof may be controlled by introducing the protein into the cell in a proprotein form such that a further factor must be added to the cell for the active protein to be generated.

Introduction of nucleic acid molecules into cells according to the aspects of the invention that are described above may be achieved by a variety of means. Suitable transformation or transfection techniques are well described in the literature (see, for example, Sambrook et al, Molecular cloning: a laboratory manual New York: Cold Spring Harbour Laboratory Press, 2000; Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience, New York, 1991; Spector, Goldman & Leinwald, Spector et al Cells, a laboratory manual; Cold Spring Harbour Laboratory Press, 1998). In eukaryotic cells, expression systems may either be transient (e.g. episomal) or permanent (such as by chromosomal integration) according to the needs of the system. The nucleic acid molecules of the invention may be introduced into cells using either viral or non- viral means. The nucleic acid molecule may be in the form of a conventional non- viral vector, such as a plasmid vector. Methods for introduction of such vectors into animal cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotide(s) in liposomes or direct microinjection of the DNA into nuclei. Plasmid vectors can be introduced into plant cells by direct DNA transformation, for example using electroporation, or by pathogen-mediated transfection. (See, e.g., Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, NY; pp. 191-196.) Other methods may be employed to introduce the nucleic acid molecules of the invention into the cells. For example, the DNA may be in the form of polycationic condensed DNA linked or unlinked to killed adenovirus (US Serial No. 08/366,787, filed December 30, 1994). The nucleic acid molecules may be in the form of ligand-linked DNA (Wu (1989) J Biol Chem 264:16985-16987). Alternatively, the nucleic acid molecules may be introduced into cells using a hand-held gene transfer particle gun, as described in US Patent 5,149,655 or ionizing radiation as described in US5,206,152 and in WO92/11033. Additional approaches are described in Philip (1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994) Proc Natl Acad Sci 91:1581-1585.

Particle-mediated nucleic acid transfer may also be employed, for example see US Serial No. 60/023,867. The sequence can be inserted into a conventional vector that contains conventional control sequences for high level expression, and then incubated with synthetic nucleic acid transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu & Wu (1987) J. Biol. Chem. 262:4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol 40:253-263, galactose as described in Plank (1992) Bioconjugate Chem 3:533-539, lactose or transferrin. Naked DNA may also be employed. Exemplary naked DNA introduction methods are described in WO90/11092 and US 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.

Exemplary liposome and polycationic nucleic acid delivery vehicles are those described in US 5,422,120 and 4,762,915; in WO 95/13796; WO94/23697; and WO91/14445; in EP- 0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Francisco; Szoka (1980) Biochem Biophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem 176:420.

Alternatively, the nucleic acid of the invention may be introduced into the cell using a viral vector. Preferably, the viral vector may be a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vector. The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, or togavirus viral vector. See generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) Nature Genetics 6:148-153.

Retroviral vectors and their use are well known in the ait. Portions of the retroviral vector may be derived from different retro viruses. For example, retro vector LTRs may be derived from a Murine Sarcoma Virus, a tRΝA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Vims.

These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see US patent 5,591,624). Retro virus vectors can be constructed for site-specific integration into host cell DΝA by incorporation of a chimeric integrase enzyme into the retroviral particle (see WO96/37626). It is preferable that the recombinant viral vector is a replication defective recombinant vims.

Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see WO95/30763 and WO92/05266), and can be used to create producer cell lines (also termed vector cell lines or "VCLs") for the production of recombinant vector particles.

Retroviruses which may be used for the constmction of retroviral vectors include Avian Leukosis Vims, Bovine Leukemia, Vims, Murine Leukemia Vims, Mink-Cell Focus-Inducing Vims, Murine Sarcoma Vims, Reticuloendotheliosis Vims and Rous Sarcoma Vims. Particularly preferred Murine Leukemia Vimses include 4070 A and 1504A (Hartley and Rowe (1976) J Virol 19:19-25), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey Sarcoma Vims and Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Vims (ATCC No. VR-190). Such retrovimses may be obtained from depositories or collections such as the American Type Culture Collection ("ATCC") in Rockville, Maryland or isolated from known sources using commonly available techniques.

Human adenoviral vectors are also known in the art and employable in this invention. See, for example, Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and WO93/07283, WO93/06223, and WO93/07282. Alternatively, administration of DNA linked to killed adenovirus as described in Curiel (1992) Hum. Gene Ther. 3:147-154 may be employed. The nucleic acid molecules may also be delivered using adenovims associated vims (AAV) vectors. Examples of such vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, WO93/09239. Additional AAV vectors are described in US 5,354,678, US 5,173,414, US 5,139,941, and US 5,252,479.

The nucleic acid molecules may also be introduced in the cell using herpes vectors. Leading and preferred examples are herpes simplex vims vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in US 5,288,641 and EP0176170 (Roizman). Additional exemplary herpes simplex vims vectors include HFEM/ICP6-LacZ disclosed in WO95/04139 (Wistar), pHSVlac described in Geller (1988) Science 241:1667-1669 and in WO90/09441 & WO92/07945, HSV Us3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with ATCC as accession numbers ATCC VR-977 and ATCC VR-260.

Alpha vims vectors can also be employed in this invention. Preferred alpha vims vectors are Sindbis vi ses vectors. Togavimses, Semliki Forest vims, Middleberg vims, Ross River vims, Venezuelan equine encephalitis vims, and those described in US patents 5,091,309, 5,217,879, and WO92/10578.

Other viral vectors which are suitable for introducing the nucleic acid molecules encoding the Melted protein, functional equivalent thereof or protein in the same signalling pathway as Melted into a cell will be known to those of skill in the art. The methods outlined above are merely examples of how the rate of growth of a cell might be regulated by modulating the activity or the level of a Melted protein or functional equivalent thereof. It will be understood that the invention includes any method of regulating the rate of growth of a cell using a modulator of a Melted protein or functional equivalent thereof. By the term "modulator" is meant any compound which modulates the activity or the level of a Melted protein or its functional equivalents. Modulator compounds may be molecules such as proteins which are naturally found in cells or they may be synthetic compounds.

The Melted protein is believed to form part of a signalling complex which is a functional node for a number of pathways that are involved in cell growth. In particular, it is believed that Melted forms part of a signalling complex which is a functional node at which cell signalling pathways controlling the rate of cell mass accumulation and the rate of cell cycle progression meet. It is possible that this signalling complex is one point at which these two pathways convene and/or branch.

Accordingly, the term modulator includes proteins which function upstream of this signalling complex and which act to modulate the level or the activity of the Melted protein, either directly or indirectly via other proteins found upstream of Melted or via other proteins in the signalling complex. The term modulator also includes proteins that function in the same signalling complex as Melted which may modulate the rate of growth of a cell by modulating the level or activity of the Melted protein itself. The term modulator also includes compounds that modulate the level or the activity of proteins found in the same signalling complex as a Melted protein or its functional equivalents, or that modulate the level or activity of proteins upstream of the signalling complex.

Modulator . compounds may be divided into activators and inhibitors compounds. Activators act to increase the level or activity of the Melted protein, and will therefore increase the rate of growth of a cell. Of course, activators of dominant negative forms of the Melted protein will decrease the rate of growth of a cell. Inhibitors act to decrease the level or activity of a Melted protein, and will therefore decrease the rate of growth of a cell of course, inhibitors of dominant negative forms of the Melted protein will act to increase the rate of growth of a cell.

According to a further aspect of the invention, there is provided a method of screening for a modulator compound, as described above, that regulates the rate of growth of a cell by modulating the level or activity of a Melted protein or a functional equivalent thereof . Preferably, said method comprises supplying a cell with a candidate compound and assessing the effect of said compound on the level or the activity of a Melted protein or functional equivalent. The screening methods of the invention may be conducted in a single cell or in a cell that is part of a tissue or of an organism.

The candidate compound may be selected as a result of a screening method for proteins that interact directly with the Melted protein or functional equivalent. Such interactions can be detected, for example, using methods such as the yeast 2-hybrid screen.

The effect of candidate modulator compounds on the rate of cell growth, and whether this effect occurs through modulation of the level or activity of a Melted protein or functional equivalent thereof can be assessed in a variety of techniques. For example, the effect of a candidate compound on the rate of growth of a cell may be assessed simply by counting the number and size of cells following incubation with the candidate compound, and comparing this to the number and size of control cells which have not been incubated with the candidate compound. If the candidate compound acts to upregulate the rate of growth of a cell, both the rate of cell mass accumulation and the rate of cell cycle progression will be upregulated in a co-ordinated manner, resulting in a larger number of cells of normal size. In the case of a screening method conducted in a tissue or organism, it may be possible to compare the size of the organism before and after supplying it with the candidate modulator compound. It may be necessary to check that the size of the cells in the organism supplied with the candidate compound is normal. Alternatively, it may be possible to compare the size of a tissue or organism treated with a candidate compound to "control" tissues or organisms that have not been contacted with the candidate modulator compound and check that the size of the cells in the tissue or organism supplied with the candidate compound is normal.

However, it is also necessary to show that any effect of the candidate compound on the rate of growth of a cell is the result of the candidate compound modulating the level or the activity of a Melted protein or a functional equivalent thereof. Alterations in the levels of the Melted protein may be assessed using any one of a number of techniques, including assessment of the level of the protein and/or assessment of the level of the mRNA encoding the Melted protein. The level of Melted protein in a cell can be measured by a number of methods, as will be clear to the skilled reader. Such a method may be by separating a cell extract on a matrix, such as a gel, and then adding an antibody that binds to the Melted protein. Following removal of unbound antibody, the level of the Melted protein can be determined by assessing the level of the antibody bound to it. This may be done by using a second labelled antibody that binds to the first antibody. Alternatively, the first antibody may be labelled directly, for example with a fluorescent label, to enable direct detection. This method may be used to compare the level of Melted protein in a cell before and after incubation with a candidate modulator compound. This method can be adapted to measure the levels of functional equivalents of Melted proteins. The level of mRNA encoding the Melted protein or functional equivalent can be assessed, for example, by traditional blotting techniques described in Sambrook et al [supra]. mRNA can be purified and separated using gel electrophoresis. The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose. The solid support is exposed to a labelled probe and then washed to remove any unhybridized probe. Next, the duplexes containing the labelled probe are detected. Typically, the probe is labelled with a radioactive moiety. Alternatively, the polymerase chain reaction (PCR) may be used to amplify the amount of mRNA encoding the Melted protein. One assay is described in: Mullis et al. [Meth. Enzymol (1987) 155: 335-350]; also US patents 4,683,195 & 4,683,202. After a threshold amount of mRNA is generated by PCR, the level can be detected by Southern blots, as described above. An increase or decrease in the level of the Melted protein or functional equivalent thereof in cells contacted with the candidate compound can be compared to the level in cells which have not been contacted with a candidate compound.

An alteration in the activity of a Melted protein or a functional equivalent thereof can also be assessed in a number of ways. For example, if the activity of one of these proteins is modulated by covalent modification, such as phosphorylation, it may be possible to assess the activity of the protein by detecting the presence of the activated or inhibited form, such as a phosphorylated or dephosphorylated form present when the cell has been treated with a candidate compound.

As noted above, it is proposed herein that the activity of the Melted protein may be regulated by changing its subcellular localisation. According to a further aspect of the invention, there is therefore provide a method of screening for a modulator of a Melted protein that alters the subcellular localisation of said Melted protein. In particular, there is provided a method of screening for an activator of a Melted protein that localises said Melted protein to a membrane in the cell. Preferably, said method comprises adding a candidate modulator compound to a cell and detecting a change in the subcellular location of a Melted protein in said cell.

In order to detect a change in the subcellular location of a Melted protein, it is necessary to visualise the subcellular location of these proteins prior to and subsequent to the addition of a candidate modulator compound. For example, the subcellular location of a Melted protein or a functional equivalent thereof can be visualised using antibodies that bind to the Melted protein or a functional equivalent thereof. An alternative method of visualizing the subcellular location of a Melted protein or a functional equivalent thereof may comprise expressing a fusion protein comprising a Melted protein or a functional equivalent thereof fused to a marker domain in a cell and detecting the presence of the marker domain. Preferably, the marker domain in the fusion protein comprises a green fluorescent protein (GFP) or a fluorescent derivative thereof such as YFP or CFP (see Prasher et al, (1995), Trends in Genetics, 11(8), 320-329). Constructs comprising GFP are preferred since it is inherently fluorescent and easily detectable in the cell, for example by fluorescence microscopy, confocal microscopy, spectroscopy (quantitative) or fluorescence activated cell sorting (FACS). Furthermore, it is small, non-toxic and easy to generate in the cell.

According to a further embodiment of this aspect of the invention, there is provided a method of screening for modulators that alter the activity of a Melted protein or a functional equivalent thereof by screening for modulators of Melted-dependent overgrowth in vivo. One example of a suitable method is the EP screening system originally described by Rorth et al, 1998.

Suitable modulator compounds for testing in the methods of the invention may be any one of a number of different compounds. Where the screening method is being conducted in an organism, particularly suitable compounds are small molecules that are suitable for oral delivery, including natural or modified substrates, hormones, small organic molecules, such as small natural or synthetic organic molecules of up to 2000Da, preferably 800Da or less, and inorganic molecules. Other suitable compounds might include polypeptides, such as enzymes, receptors, antibodies, and stmctural or functional mimetics of these polypeptides, including peptides and peptidomimetics, although these compounds would not be suitable for oral delivery.

As will be apparent to the skilled reader, candidate polypeptide and peptide compounds for testing in the method of the invention may either be natural compounds, isolated from natural sources, or may be synthetic or recombinant. The invention further provides a modulator compound identified or identifiable by any one of the screening methods described above. Modulator compounds may alter the level of a Melted protein or a functional equivalent thereof by modulating expression at the level of transcription or translation. Alternatively, such compounds may act to modulate the level of the proteins directly. For example, inhibitors may include enzymes which digest the Melted protein or functional equivalent thereof , reducing the level of Melted protein or functional equivalent thereof. Modulator compounds may act to alter the activity of the Melted protein or functional equivalent in a number of ways.

Modulator compounds may act by binding directly to the Melted protein or functional equivalent thereof and such binding may either increase or decrease the activity of the protein. For example, an inhibitor may comprise an antibody which binds to a Melted protein or functional equivalent thereof, resulting in a decrease in the activity of the Melted protein or functional equivalent thereof.

Modulator compounds may also act to alter the activity of a Melted protein or functional equivalent thereof through covalent modification. For example, a modulator compound may be an enzyme which can alter the phosphorylation state of the Melted protein or functional equivalent thereof.

Alternatively, modulator compounds may alter the activity of a Melted protein or functional equivalent thereof by altering the subcellular location of said Melted protein or a functional equivalent thereof. The modulator compounds of the invention may alter the activity of the Melted protein or functional equivalent thereof directly or they may act indirectly, for example, by modulating the level or activity of a protein in the same signalling complex as the Melted protein or functional equivalent thereof or of a protein upstream of said signalling complex. According to a further aspect of the invention, there is provided a method of screening for an effector of a Melted protein or a functional equivalent thereof. As described above, the Melted protein is thought to act as a functional node at which multiple pathways that are involved in regulating the rate of growth of a cell come together. The Melted protein may act as one point at which the cell signalling pathways that are involved in regulating the rate of cell mass accumulation and the rate of cell cycle progression convene and branch. By an "effector" is meant any compound that functions downstream of the Melted protein or functional equivalents, via which the Melted protein exerts its effect. An effector protein may modulate both the rate of cell cycle progression and the rate of cell mass accumulation and hence regulate the rate of growth of a cell. However, effectors which only modulate the rate of cell mass accumulation or which only regulate the rate of cell cycle progression are also included in the invention. Effectors may be divided into inhibitors and activators of the pathways in which they are involved. Increasing the level or the activity of an effector compound that is an activator will increase the rate of cell cycle progression or the rate of mass accumulation or both. Increasing the level or the activity of an effector compound that is an inhibitor will decrease the rate of cell cycle progression or the rate of cell mass accumulation or both, according to the pathways in which the effector compound is involved. In addition, the level and the activity of effector compounds may be either increased or decreased by increasing the level or the activity of a Melted protein or functional equivalent thereof.

One method of screening for effectors of Melted action may be to identify genes and/or proteins whose levels or activity are altered by changes in the levels or activity of the

Melted protein. Examples of suitable methods include methods for monitoring the differential expression of genes, including techniques such as indexing differential display reverse transcriptase polymerase chain reaction (DDRT-PCR; Mahadeva et al. (1998) J.

Mol.Biol. 284, 1391-1398; International patent application WO94/01582), subtractive mRNA hybridisation (see "Advanced Molecular Biology", R. M. Twyman (1998) Bios

Scientific Publishers, Oxford, p336; "Nucleic Acid Hybridization", M. L. M. Anderson

(1999) Bios Scientific Publishers, Oxford, ppl99-202; Sagerstrom et al. (1997) Annu. Rev.

Biochem. 66: 751-783), the use of nucleic acid arrays or microarrays (see Nature Genetics,

(1999), vol 21 suppl; 1-61), and the serial analysis of gene expression (SAGE; Velculescu et al, Science (1995) 270; 484-487).

Preferably, candidate compounds are identified as a result of a yeast two-hybrid screen for compounds that interact with a Melted protein or a functional equivalent thereof. Preferably, a suitable method of screening for the activity of candidate effector molecules comprises supplying a cell with a candidate effector compound and assessing whether there is any change in the rate of cell mass accumulation or the rate of cell cycle progression.

The invention also includes effector molecules identifiable or identified using any of the screening methods described above. Particular examples of effectors included in the invention are Tscl and 14-3-3ε. Tscl appears to an effector of Melted that is involved in regulating the rate of cell mass accumulation. Although the Applicant does not wish to be bound by this theory, it appears that Melted antagonises Tscl to modulate the rate of cell mass accumulation. 14-3-3ε appears to be an effector of Melted that is involved in regulating the rate of cell cycle progression. It appears that Melted acts via 14-3-3ε which may interact with CDC25 to control the rate of cell cycle progression.

The ability to screen for and identify effectors of the Melted protein will be useful to researchers because it will enable the development of methods for targeting effectors so that they can differentially regulate the rate of cell mass accumulation and the rate of cell cycle progression. The ability to regulate these pathways differentially is likely to present numerous medical applications. In addition, the identification of effectors will enable the development of methods for re-establishing control of the rate of growth of a cell where such control is lost. For example, in view of the fact that Melted appears to antagonise Tscl, defects due to a reduced level or activity of Melted could be compensated to some extent by reduction of Tscl expression or activity. Conversely, defects due to an increased level or activity of Melted could be compensated for in part by increasing the level or the activity of Tscl.

The invention also provides a growth regulating protein comprising a Melted protein, a functional equivalent thereof, or a modulator or an effector of a Melted protein or functional equivalent. Where the growth regulating protein is a Melted protein, a functional equivalent thereof or a modulator, it will modulate both the rate of cell mass accumulation and the rate of cell cycle progression. Where the growth regulating is protein an effector, it may modulate either the rate of cell mass accumulation or the rate of cell cycle progression or both.

The Melted protein of the invention is preferably derived from a vertebrate, a metazoan or from an insect. Where the Melted protein is from an insect, it is preferably from Drosophila. Preferably, if from Drosophila, the Melted protein may comprise the sequence of Figure 6 (complete dMelt sequence) functional equivalent thereof. Where the Melted protein is from a vertebrate, it may preferably be isolated from a mammal, such as a human. If from a human, the Melted protein may comprise the sequence of Figure 5 (complete hMelt sequence) or a functional equivalent thereof. Where the Melted protein is isolated from a metazoan, it is preferably isolated from a nematode, such as Caenorhabditis elegans. If from C. elegans, Melted protein may comprise the sequence of Figure 7 (complete nMelt sequence) or a functional equivalent thereof.

The growth regulating protein may be prepared in recombinant form by expression in a host cell. Suitable expression methods are well known to those of skill in the art and many are described in detail by Sambrook J. et al "Molecular cloning: a laboratory manual" New York: Cold Spring Harbour Laboratory Press, 2000) and Fernandez J.M. & Hoeffler J.P. (Gene expression systems. Using nature for the art of expression ed. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto, 1998.) The growth regulating protein can also be prepared using conventional techniques of protein chemistry, for example, by chemical synthesis.

According to a further embodiment, the invention provides antibodies which bind to a growth regulating protein of the invention, as described above. Antisera and monoclonal antibodies can be made by standard protocols using a growth regulating protein as an immunogen (see, for example, Antibodies: A Laboratory Manual ed. By Harlow and Lane, Cold Spring Harbor Press, 1988). As used herein, the term "antibody" includes fragments of antibodies that also bind specifically to a growth regulating protein. The term "antibody" further includes chimeric and humanised antibody molecules having specificity for growth regulating proteins. Antibodies which bind growth regulating proteins are useful in a variety of methods for elucidating the function of these proteins in regulating the rate of cell growth and in methods of screening for modulators, as outlined above. In some cases, it will be desirable to attach a label group to the antibody in order to facilitate detection. Preferably, the label is an enzyme, a radiolabel or a fluorescent tag.

According to a further aspect of the invention, there is provided a nucleic acid molecule encoding a growth regulating protein as described above. Examples of nucleic acid molecules according to this aspect of the invention are presented in Figure 9. The invention includes nucleic acid molecules that are homologous to these nucleic acid molecules, including those that are least 70% identical, preferably, at least 80% identical more preferably at least 90%, more preferably at least 95%, even more preferably at least 98% or 99% identical over their entire length to the same, and nucleic acid molecules that are substantially complementary to such nucleic acid molecules. Percentage identity, as referred to herein, is as determined using BLAST version 2.1.3 using the default parameters specified by the NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/). Nucleic acid molecules encoding a growth regulating protein include single- or double-stranded DNA, cDNA and RNA, as well as synthetic nucleic acid species. Preferably, the nucleic acid molecule is a DNA or cDNA molecule.

Preferably, the nucleic acid molecule is in the form of an expression vector. In addition to a nucleic acid sequence encoding the growth regulating protein, such an expression vector may incorporate regulatory sequences such as enhancers, promoters, ribosome binding sites and termination signals in the 5' and 3' untranslated regions of genes, that are required to ensure that the coding sequence is properly transcribed and translated, or to regulate the expression of the protein relative to the growth of the cell in which it is expressed. The regulatory sequences included in the vector will depend on the type of host cell in which it is expressed. Also, control sequences may be included that encode signal peptides or leader sequences. These leader or control sequences may be removed by the host during post-transiational processing. Vectors according to the invention include plasmids and vimses (including both bacteriophage and eukaryotic vimses), as well as other linear or circular DNA carriers, such as those employing transposable elements or homologous recombination technology. Many such vectors and expression systems are known and documented in the art (see, for example, Fernandez J.M. & Hoeffler J.P. in Gene expression systems. Using nature for the art of expression ed. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto, 1998). Suitable viral vectors include baculovirus-, adenovims- and. vaccinia vims- based vectors.

A variety of techniques may be used to introduce the vectors into the cell in which expression is desired. Some of these methods are outlined in some detail above. The invention further provides an antisense nucleic acid molecule that hybridises to a nucleic acid encoding a growth regulating protein, as described above. Such molecules may be useful as diagnostic probes or as antisense nucleic acid molecules, active to alter the level of encoding nucleic acid. Preferably, an antisense nucleic acid molecule according to this aspect of the invention hybridises under high stringency hybridisation conditions to the nucleic acid molecules encoding the growth regulating protein. High stringency hybridisation conditions are defined herein as overnight incubation at 42° C in a solution comprising 50% formamide, 5XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5xDenhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0. IX SSC at approximately 65 ° C.

Such antisense nucleic acid molecules may be modified oligonucleotides which are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and are therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der Krol et al. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668.

The growth regulating proteins, along with nucleic acid molecules, antisense nucleic acid molecules and antibodies will have many medical applications.

Accordingly, the invention further provides a growth regulating protein, a nucleic acid molecule encoding said growth regulating protein, an antisense nucleic acid molecule which hybridises to the nucleic acid molecule encoding said growth regulating protein or an antibody that binds to said growth regulating protein , as described above, for use as a pharmaceutical. In particular, the invention provides a growth regulating protein, a nucleic acid molecule encoding said growth regulating protein, an antisense nucleic acid molecule which hybridises to the nucleic acid molecule encoding said growth regulating protein, or an antibody that binds to said growth regulating protein, as described above, for use as a regulator of the rate of cell mass accumulation or the rate of cell cycle progression or both.

A further aspect of the invention includes a pharmaceutical composition comprising a growth regulating protein, a nucleic acid molecule encoding said growth regulating protein, an antisense nucleic acid molecule which hybridises to the nucleic acid molecule encoding said growth regulating protein or an antibody that binds to said growth regulating protein, according to any one of the embodiments of the invention recited above, in conjunction with a pharmaceutically-acceptable carrier molecule.

Carrier molecules may be genes, polypeptides, antibodies, liposomes, polysaccharides, polylactic acids, polyglycolic acids and inactive vims particles or indeed any other agent provided that the carrier does not itself induce toxicity effects or cause the production of antibodies that are harmful to the individual receiving the pharmaceutical composition. Carriers may also include pharmaceutically acceptable salts such as mineral acid salts (for example, hydrochlorides, hydrobromides, phosphates, sulphates) or the salts of organic acids (for example, acetates, propionates, malonates, benzoates). Pharmaceutically acceptable carriers may additionally contain liquids such as water, saline, glycerol, ethanol or auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like. Carriers may enable the pharmaceutical compositions to be formulated into tablets, pills, dragees, capsules, liquids, gels, symps, slurries, suspensions to aid intake by the patient. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). The amount of the active compound in the composition should also be in a therapeutically- effective amount. The phrase "therapeutically effective amount" used herein refers to the amount of agent needed to treat or ameliorate a targeted disease or condition. An effective initial method to determine a "therapeutically effective amount" may be by carrying out assays in the transgenic organism model, although more accurate tests must be carried out on the target organism if initial tests are successful. The transgenic organism model may also yield relevant information such as the preferred routes of administration that will lead to maximum effectiveness. The exact therapeutically-effective dosage will generally be dependent on the patient's status at the time of administration. Factors that may be taken into consideration when determining dosage include the severity of the disease state in the patient, the general health of the patient, the age, weight, gender, diet, time and frequency of administration, drug combinations, reaction sensitivities and the patient's tolerance or response to the therapy. The precise amount can be determined by routine experimentation but may ultimately lie with the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg (mass of drag compared to mass of patient) to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.

Uptake of a pharmaceutical composition by a patient may be initiated by a variety of methods including, but not limited to enteral, intra-arterial, intrathecal, intramedullary, intramuscular, intranasal, intraperitoneal, intravaginal, intravenous, intraventricular, oral, rectal (for example, in the form of suppositories), subcutaneous, sublingual, transcutaneous applications (for example, see WO98/20734) or transdermal means. Gene guns or hyposprays may also be used to administer pharmaceutical compositions. Typically, however, the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Direct delivery of the compositions can generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.

The invention also includes the use of a growth regulating protein as described above, a nucleic acid molecule encoding said growth regulating protein, an antisense nucleic acid molecule which hybridises to the nucleic acid molecule encoding said growth regulating protein, an antibody that binds to said growth regulating protein, or a composition, as described above, in the manufacture of a medicament for treating a disease or condition associated with aberrant cell growth.

The disease or condition may be associated with either an aberrantly low rate of cell mass accumulation or an aberrantly low rate of all cycle progression or both. Alternatively, the disease or condition may be associated with an abnormally high rate of cell mass accumulation or an abnormally high rate of cell cycle progression or both. The disease or condition may be associated with an abnormally high rate of cell mass accumulation and an abnormally low rate of cell cycle progression or vice versa. The skilled reader will be able to determine what suitable medicaments should comprise as active agent, according to the nature of the disease or condition. For example, if the disease involves an aberrantly low rate of cell cycle progression and an aberrantly low rate of cell mass accumulation, the medicament may comprise a Melted protein or a functional equivalent thereof that acts as an up-regulator, or a modulator that increases the level or the activity of a Melted protein. If the disease or condition is associated with an abnormally high level of cell cycle progression and/or cell mass accumulation, the medicament may comprise a dominant negative form of Melted, an effector that decreases the level of cell mass accumulation or an antisense molecule that inhibits the expression of an effector that increases the level of cell mass accumulation. Diseases and conditions which are associated with an abnormal rate of cell mass accumulation or cell cycle progression or both include dwarfism, spondyloepimetaphyseal dysplasia type II, acromicric dysplasia, Russell-Silver syndrome, benign growths such as harmatomas and cancer.

According to a still further aspect of the invention, there is provided a method of treating a disorder or disease in which an aberrant rate of cell growth is implicated in a patient, comprising administering to the patient a growth regulating protein, a nucleic acid molecule encoding said growth regulating protein, an antisense nucleic acid molecule which hybridises to the nucleic acid molecule encoding said growth regulating protein, an antibody that binds to said growth regulating protein, or a composition as described above in a therapeutically-effective amount. Preferred patients are mammals, more preferably humans and domesticated animals. According to a further aspect of the invention, there is provided a method of diagnosing a disease or condition associated with an aberrant rate of growth of a cell by determining the level or the activity of a Melted protein, a functional equivalent thereof, a modulator or an effector, as described above, in a cell sample from a patent. A variety of diseases may be associated with an aberrant level or activity of Melted. The level or the activity of a Melted protein or a functional equivalent thereof in a sample from a patient can be determined using any of the methods outlined above. In some cases, diseases or conditions may be associated with a mutation in the DNA coding for the Melted protein. Such mutations may be detected by sequencing chromosomal DNA from a patient.

According to a still further aspect of the invention, there is provided a transgenic organism comprising a Melted protein or a functional equivalent thereof which acts as an up- regulator, as described above. Preferably, the transgenic organism comprises a Melted protein or functional equivalent thereof that acts as an up-regulator, which is overexpressed relative to the level of expression under normal physiological conditions. As indicated previously, overexpression of Melted protein or a functional equivalent thereof which acts as an up-regulator leads to an increase in the rate of growth of cells and a consequent increase in the size of the transgenic organism.

The invention also provides a transgenic organism comprising a functional equivalent of a Melted protein that acts as a down-regulator. Preferably, the functional equivalent that acts as a down-regulator may be a dominant negative mutant of a naturally-occurring Melted protein. Preferably, the transgenic organism comprises a dominant negative mutant of a naturally-occurring Melted protein that is overexpressed relative to the level of expression of a normal Melted protein. Overexpression of a dominant negative mutant of a Melted protein leads to a decrease in the rate of growth of cells and a consequent decrease in the size of the transgenic organism. The transgenic organism may be any non-human transgenic system. It may be a plant system or an animal system. Examples or plant systems include crop plants, flowering plants, fruit trees and vegetables. Preferably, the transgenic system is an animal system. Examples of animal systems include: mammals, such as mice, rats, rabbits, chickens, and pigs; amphibians such as frogs; fish, such as Zebrafish, Medakafish; insects such as Drosophila; and basic organisms such as the nematode Caenorhabditis elegans. Where the transgenic organism is being used for research work in the laboratory, organisms which have a rapid lifecycle and thus short generation time, such as Drosophila and fish, have a particular utility. Preferably, the organism is a mouse, Zebrafish, Medakafish or insect, such as Drosophila.

The term "transgenic organism" includes any organism into which a nucleic acid encoding a Melted protein or functional equivalent thereof has been introduced and has integrated into the genome, resulting in overexpression of the Melted protein or functional equivalent thereof in comparison to a non-transgenic organism. Transgenic organisms can easily be generated according to methods known in the art. Local changes can be incorporated by modification of somatic cells, whilst germ line therapy may be used to incorporate heritable modifications. Examples of suitable techniques include using retroviral vector infection (see Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, 2nd Ed.), microinjection of DNA into a fertilized egg, transfection of and homologous recombination in embryonic stem cells and nuclear transfer (see Anderson and Seidel, (1998) Science 280: 1400; Wilmut, (1998), Scientific American 279(6): 58). The exact technique that is used will depend on the particular organism that is chosen.

Preferably, the nucleic acid incorporated into the genome in the transgenic organism encodes a Melted protein or functional equivalent thereof which is endogenous to that animal. In the case of a functional equivalent that acts as a down-regulator, this means that the functional equivalent should be derived from a Melted protein endogenous to that organism, for example by mutation. However, the Melted protein or functional equivalent that is encoded by the nucleic acid and integrated into the genome of the transgenic organism may be from a different organism providing that overexpression of the Melted protein or functional equivalent thereof results in a modulation in the rate of growth of cells. The term "transgenic organism" also encompasses animals in which a nucleic acid has been introduced in the genome which promotes the overexpression of the endogenous gene encoding Melted, or a functional equivalent thereof that acts as an up-regulator in situ. For example, in Drosophila, insertion of a P-element upstream of the endogenous Melted gene can be used to generate a strain which is then crossed with a Gal driver line resulting in a transgenic insect in which the endogenous Melted gene is overexpressed.

The invention also provides a method of screening for compounds that modulate the rate of cell growth using the transgenic organisms of the invention as defined above. The method comprises administering a candidate modulator compound, as described previously, to a transgenic organism overexpressmg a Melted protein or a functional equivalent thereof and assessing the effect of said candidate compound on the rate of growth of cells in the transgenic organism compared to the rate of growth of the cells of a transgenic organism which have not been treated with the candidate compound.

Various aspects and embodiments of the present invention will now be described in more detail by way of example. It will be appreciated that modification of detail may be made without departing from the scope of the invention.

Brief description of the Figures: Figure 1: Melt overexpression causes tissue overgrowth

Figure 2: dMelt overexpression does not perturb cell size or cell cycle parameters.

Figure 3: Location of dMelt in Drosophila

Figure 4: dMelt interacts with Tscl

Figures 5: Amino acid sequence of human Melted protein (hMelt) Figure 6: Amino acid sequence of Drosophila Melted protein (dMelt)

Figure 7: Amino acid sequence of Nematode Melted protein (nMelt)

Figure 8: Multiple sequence alignment of the sequences of dMelt, hMelt and nMelt. Additional regions of similarity outside the PH domain are shown. The MH1 domain is underlined. The MH2 domain is in bold. The PH domain is in italic. Figure 9 : a) The ORF of a human Melted protein (hMelt) . b) The ORF of a Drosophila Melted protein (dMelt). Figure 10: Subcellular localisation of dMelt, Tscl and Tsc2. Tscl is predominantly cytoplasmic in S2 cells transfected with Tscl expression vector DNA (a-c). When DNA for expression of dMelt is additionally transfected into S2 cells, both Tscl and dMelt are localized cortically (d-g). Tsc2 is also predominantly cytoplasmic in S2 cells transfected with Tsc2 expression vector DNA (h-k), however no alteration in its cytoplasmic localization is detectable when DNA for dMelt expression is also cotransfected (l-o). S2 cells transfected with both Tscl and Tsc2 (p-s) have cytoplasmic Tcsl and Tsc2. When dMelt DNA is additionally transfected in (t-x) all three proteins are found to localize cortically. Both the transfected Tscl and Tsc2 were epitope tagged (myc-Tscl, His-V5- Tsc2), and localization of the transfected proteins was detected using antibodies directed against the epitope tags.

Examples Drosophila Melted overexpression causes tissue overgrowth

We performed an overexpression screen in D. melanogaster looking for genes which, when overexpressed, cause tissue to grow larger than normal. We generated thousands of fly lines, each containing an EP element (a P transposon containing a UAS enhancer) inserted randomly in the genome as described by (Rorth, Szabo et al 1998). When crossed to a tissue specific GAL4 driver line, this caused overexpression of the gene located downstream of the EP element. Using the engrailed-GAL4 driver which resulted in the overexpression of genes in the posterior half of the wing, and searching for overgrowth of the posterior relative to the anterior half of the wing, we isolated EP line 31685 whose overgrowth phenotype was significant and is shown in Figure la. EP31685 was located roughly 50bp upstream from the start of a transcription unit defined by an EST (HL03627) and by a gene predicted by the Drosophila genome project called Melted (CG8624) with a predicted mRNA length of 2979bp. Sequencing of HL03627 revealed that its first 1.2kb agreed roughly with the sequence of CG8624. The remaining lOObp of HL03627 were determined by BLAST search to be identical to sequence from the Farinelli gene at 66B, suggesting that a cloning artefact occurred during creation of the Genome Project's cDNA library. (See Figure 1 of PI(5)P binding protein application.) To obtain the remaining 1.8kb of melt sequence, RT-PCR was done from a larval cDNA library. Our resulting cDNA for Drosophila Melt (dMelt) differed from CG8624. Compared to CG8624, our cDNA contains an extra 6bp in the ORF and is missing 18bp from the ORF. There are 24 nucleotide differences between our dMelt protein and CG8624 so 8 amino acids differ. This results in a 4 aa difference in length between our dMelt protein compared to CG8624. We generated transgenic flies containing the entire dMelt open reading frame cloned into the pUAST P-element vector for germ-line transformation of Drosophila. Overexpression of dMelt in the posterior of the wing produced a similar overgrowth phenotype to that caused by the original EP31685 element, formally demonstrating that dMelt is the gene responsible for the tissue overgrowth in the EP31685 Drosophila strain. By measuring the area of the posterior half of wings of wild-type flies and of flies overexpressing either EP31685 or UAS-dMelt with en-GAL4 we calculated that the overgrowth is statistically significant (Figure lc).

Overexpression of dMelt causes tissue overgrowth not only of the posterior of the wing, but also of the anterior of the wing (Figure Id) and, when expressed ubiquitously with Tubulin-GAL4, of the entire fly (Figure lb). Measurement of fly weights revealed a roughly 10% increase in body mass of dMelt overexpressing flies relative to flies of the parental genotypes (Figure le). dMelt overexpressing cells have normal cell cycles and normal cellsize Tissue size reflects both cell size and cell number. Tissue overgrowth can result from cells in the tissue being larger, or from an increase in cell number, or from both. Ras, Myc and the insulin signalling pathway cause tissue growth by increasing cell size with little or no effect on cell number. In order to understand the effect of dMelt overexpression we generated, in two parallel experiments, clones of cells in the Drosophila wing imaginal disc that overexpressed GFP (as a clone marker for use in the fluorescence activated cell sorter FACS) or GFP and dMelt. Imaginal discs were dissociated to produce a single cell suspension, stained for DNA with Hoechst 33342, and analyzed by FACS. Data from the cell sorter revealed the percentage of cells in each of the stages of the cell cycle for both GFP+ and GFP- cells, as well as a measurement of cell sizes (Figure 2a-d). Cells expressing GFP alone were roughly the same size as control cells not expressing GFP (Figure 2b) and the cell cycle profiles for both populations were roughly the same (Figure 2a). Similarly, cells expressing GFP and dMelt also showed no change in average cell size or in the cell cycle profile compared to control cells (Fig 2c-d). This result was corroborated by analyzing the wings of adult animals expressing dMelt and comparing them to wings from control animals. Each cell in the wing secretes a hair. Thus the number of cells per unit area in the wing can be counted. Although the tissue area was increased, the density of cells in wings of animals overexpressing Melt was not found to be significantly different from control wings, suggesting that dMelt overexpressing cells are not larger than normal cells. Thus dMelt causes tissue overgrowth by generating more cells of normal size. Among the currently-know tissue growth genes only CyclinD-cdk4 coexpression causes a comparable effect. Ras, Myc and the insulin signalling pathway cause abnormal cell cycle control and abnormal increases in cell size. dMelt activity is required for growth and survival dMelt mRNA is present in all cells of the embryo and in all imaginal discs, the precursors of the adult tissues of the fly (Figure 3b-i), suggesting that it normally has a role in growth control of such tissues. A P-element insertional mutant that dismpts Melted function had been described previously, although at that time its function in tissue growth was not recognized (melt^S144114, Salzberg et al 1997; Prokopenko et al 2000). Most animals mutant for melt^8144114 die during embryonic development. A small number survive to larval stages. These animals display a range of growth-related defects. They grow slowly, have less fat and lack imaginal discs (the progenitors of the adult). Larval life is extended beyond the stage when siblings would enter pupation. When mutant larvae pupate they fail to develop adult cuticle. These symptoms resemble the defects associated with nutrient deprivation and insufficient insulin pathway activity (Weinkove et al 1999; Britton, Banka Cohen and Edgar et al in preparation).

We also made use of the melt^S144114 mutant to generate clones of cells in the wing disc lacking dMelt activity. dMelt mutant clones fail to grow and mutant cells die by apoptosis within a few days, indicating that dMelt activity is autonomously required for cell growth and survival, in addition to being required for growth of the animal as a whole. dMelt acts in part via Tscl and 14-3-3ε

In order to understand molecularly how dMelt causes tissue overgrowth we performed a yeast 2-hybrid screen using the first 565 amino acids of dMelt as a bait and a tested a drosophila cDNA library. We screened 2 million clones and found that 14-3-3ε and Tuberous Sclerosis Complex Gene 1 (Tscl) interact specifically with dMelt.

To test whether we could reproduce the interaction between dMelt and Tscl in a different system, we transfected S2 cells with vectors for expression of full-length dMelt and an epitope tagged version of Tscl and were able to co-immunoprecipitate dMelt with antibodies to the epitope tag on Tscl. This indicates that the amino-terminal domain of dMelt can bind to Tscl protein in Drosophila cells and in yeast. Due to high sequence similarity in the amino terminal domain (Figure 9) , it can be expected that human Melted protein will bind to human Tscl.

Tscl is a tumour suppressor gene that has been shown to interact with Tsc2 in Drosophila and humans (Gao and Pan 2001; Potter et al. 2001; Tapon et al. 2001; and references therein). When expressed together TSC1 and TSC2 suppress cell growth. Humans or mice heterozygous mutant for either one develop harmatomas. Homozygous mutant mice die. In Drosophila cells homozygous mutant for either gene exhibit cell overgrowth and perturbations in cell cycle profile. Tscl and Tsc2 proteins bind together to form a complex. Since dMelt has a particular subcellular localization, and since it binds Tscl, we investigated whether dMelt can influence the subcellular localization of Tscl and Tsc2. S2 cells transfected with DNA for expression of epitope-tagged Tscl have cytoplasmic Tscl staining (Figure 10 a-c). Since S2 cells have very little dMelt protein (not shown), this can be presumed to be the localization of Tscl in the absence of dMelt. In S2 cells expressing both Tscl and dMelt, Tscl is predominantly cortical (Figure 10 d-g). These data are in agreement with the 2-hybrid data and the co-IP data, indicating that Tscl binds to dMelt. S2 cells transfected with an expression vector for Tsc2 only have cytoplasmic Tsc2 staining (Figure 10 h-k). The subcellular localization of Tsc2 does not seem to be affected by cotransfection of dMelt (Figure 10 l-o). Both Tscl and Tsc2 subcellular localization is cytoplasmic in S2 cells expressing both Tscl and Tsc2 (Figure 10 p-s). However, both Tscl and Tsc2 become localized cortically when S2 cells are additionally transfected with a dMelt expression vector (Figure 10 t-x). Thus, in this system, dMelt can change the subcellular localization of both Tscl (directly) and of Tsc2 (indirectly via Tscl).

14-3-3ε has been shown previously to interact with Raf , Raf with Ras , and Ras is a well- known oncogene and driver of cell growth (Avmch et al, 2001). Thus, it is probable that part of the effect of dMelt on tissue growth is mediated by Ras signalling.

Signalling through both Tscl/Tsc2 and Ras have been shown to drive cells through the Gl/S checkpoint, causing tissue overgrowth to be accompanied with an accumulation of cells in the G2 phase of the cell cycle. As we have noted above, when dMelt is overexpressed there is little if no accumulation of cells in G2. Rather, the entire cell cycle is accelerated. This suggests dMelt must also be driving cells through the G2/M transition. In this context, it is of note that 14-3-3ε has been shown to bind to CDC25 in a yeast 2- hybrid assay. Since CDC25 is the key regulator of the G2/M checkpoint, it is possible that dMelt signals via CDC25 to accelerate the G2/M transition and coordinately increase cell growth and progression through the cell cycle.

References Avmch J, et al (2001) Recent Prog Horm Res. 56: 127-55

Conlon and Raf (1999) Cell, 96: 235-244

Datar, S. A., H. W. Jacobs, et al. (2000). "The drosophila cyclin D-cdk4 complex promotes cellular growth [In Process Citation]." Embo J 19(17): 4543-54.

Frary et al, 2000, Science, 289: 85 Gao, X. and D. Pan (2001). "TSCl and TSC2 tumor suppressors antagonize insulin signalling in cell growth." Genes Dev 15(11): 1383-92.

Johnston, L. A., D. A. Prober, et al (1999). "Drosophila myc regulates cellular growth during development." Cell 98(6): 779-90.

Li et al, (1997) "PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast and protate cancer" Science 275: 1943-1947.

Meyer, C. A., H. W. Jacobs, et al (2000). "Drosophila cdk4 is required for normal growth and is dispensable for cell cycle progression [In Process Citation]." Embo J 19(17): 4533- 42.

Odorico, J.S. et al (2001), Stem Cells 19: 193-204 Potter, C. J., H. Huang, et al. (2001). "Drosophila Tscl functions with Tsc2 to antagonize insulin signalling in regulating cell growth, cell proliferation, and organ size." Cell 105(3): 357-68.

Prober, D. A. and B. A. Edgar (2000). "Rasl promotes cellular growth in the Drosophila wing." Cell 100(4): 435-46. Rorth, P., K. Szabo, et al. (1998). "Systematic Gain-of-Function Genetics in Drosophila." Development 125: 1049-1057.

Salzberg, A., S. N. Prokopenko, et al (1997). "P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: mutations affecting embryonic PNS development." Genetics 147(4): 1723-41. Stocker, H. and E. Hafen (2000). "Genetic control of cell size." Curr Opin Genet Dev 10(5): 529-35.

Tapon, N., N. Ito, et al. (2001). "The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation." Cell 105(3): 345-55.

Claims

CLAIMS:

1. A method of regulating the rate of growth of a cell comprising modulating in said cell the activity or the level of a Melted protein or a functional equivalent thereof.

2. A method according to claim 1 wherein the rate of growth of said cell is up-regulated.

3. A method according to claim 1 wherein the rate of growth of said cell is down- regulated.

4. A method according to claim 2, comprising increasing the level or the activity of a Melted protein or a functional equivalent thereof that acts as an up-regulator.

5. A method according to claim 2 comprising decreasing the level or the activity of a functional equivalent of a Melted protein that acts as a down-regulator.

6. A method according to claim 3 comprising decreasing the level or the activity of a Melted protein or a functional equivalent thereof that acts as an up-regulator.

7. A method according to claim 3 comprising increasing the level or the activity of a functional equivalent of a Melted protein that acts as a down-regulator.

8. A method of screening for a modulator compound that regulates the rate of growth of a cell by modulating the level or activity in the cell of a Melted protein or a functional equivalent thereof, said method comprising contacting a cell expressing a Melted protein or functional equivalent with a candidate modulator compound and testing the effect of said compound on the level or activity of the Melted protein or the functional equivalent.

9. A method of screening for an effector of a Melted protein or a functional equivalent thereof, said method comprising screening for a gene and/or protein whose level or activity is altered by a change in the level or activity of a Melted protein or functional equivalent.

10. A method according to any one of claims 1-9 wherein said cell is part of a tissue.

11. A method according to any one of claims 1-10 wherein said cell is part of an organism.

12. A modulator compound identifiable by the method of claim 8.

13. A modulator compound according to claim 12 that is part of the same signalling complex as the Melted protein or functional equivalent thereof.

14. A modulator according to claim 12 which is an antibody.

15. An effector compound identifiable by the method of claim 9.

16. An effector compound according to claim 15 that modulates the rate of cell cycle progression.

17. An effector according to claim 15 that modulates the rate of cell mass accumulation.

18. An effector according to claim 15 that modulates the rate of cell cycle progression and the rate of cell mass accumulation.

19. A growth regulating protein comprising the amino acid sequence of a Melted protein, a functional equivalent thereof, a modulator compound according to claim 12 or an effector compound according to claim 15.

20. A nucleic acid molecule encoding a growth regulating protein according to claim 19.

21. A vector comprising a nucleic acid molecule according to claim 20.

22. An antisense nucleic acid molecule that hybridises under stringent conditions to a nucleic acid molecule according to claim 20.

23. An antibody that binds to a growth regulating protein according to claim 19.

24. A growth regulating protein according to claim 19, a nucleic acid molecule according to claim 20, an antisense nucleic acid molecule according to claim 22 or an antibody according to claim 23 for use as a pharmaceutical.

25. A growth regulating protein, a nucleic acid molecule, an antisense nucleic acid molecule, an antibody or a modulator compound as recited in claim 24, for use as a regulator of the rate of cell mass accumulation, the rate of cell cycle progression or both.

26. A pharmaceutical composition comprising a growth regulating protein, a nucleic acid molecule, an antisense nucleic acid molecule or an antibody as recited in claim 24, in conjunction with a pharmaceutically acceptable carrier.

27. Use of a growth regulating protein, a nucleic acid molecule, an antisense nucleic acid molecule or an antibody as recited in claim 24 or a composition according to claim 26 in the manufacture of a medicament for treating a disease associated with aberrant cell growth.

28. Use according to claim 27 wherein said disease is dwarfism, spondyloepimetaphyseal dysplasia type II, acromicric dysplasia, Russell-Silver syndrome, benign growths such as harmatomas or cancer.

29. A transgenic organism comprising a Melted protein or a functional equivalent thereof.

30. A transgenic organism according to claim 29 which is a mammal, insect or fish.

31. A method of screening for a modulator compound that modulates the rate of growth of a cell using a transgenic animal according to claim 29 or claim 30.