WO2002081514A2

WO2002081514A2 - A novel f-box protein

Info

Publication number: WO2002081514A2
Application number: PCT/GB2002/001632
Authority: WO
Inventors: Alison Banham; Jacqueline Cordell; Margaret Jones; David Mason; Karen Pulford
Original assignee: Isis Innovation Limited
Priority date: 2001-04-05
Filing date: 2002-04-05
Publication date: 2002-10-17
Also published as: WO2002081514A3

Abstract

There is disclosed a novel F-Box protein and variants thereof that are useful in the diagnosis and treatment of cancers, including solid tumours or haematoporetic malignancies. Nucleic acid molecules encoding said F-Box proteins are also disclosed in addition to antisense molecules and antibodies capable of binding to the novel F-Box proteins.

Description

A NOVEL F-BOX PROTEIN

The present invention is concerned with a novel protein, and in particular with a protein that contains an F-box, a motif that mediates protein- protein interactions.

The F-box, so called because this domain was found in cyclin F (Bai, et al., 1996), is a protein motif of approximately 50 amino acids that functions as a site of protein-protein interactions. This motif is commonly found in the amino-terminal half of the protein and is often coupled with other C-terminal repeats such as WD repeats and leucine-rich repeats. This is an expanding family of eukaryotic proteins many of which still have unknown functions. There are 11 F-box proteins in Saccharomyces cerevisiae, 326 predicted in Caenorhabdi tis elegans, 22 in Drosophila , and at least 38 in humans (Kipreos & Pagano, 2000) .

F-box proteins were first identified as components of the SCF ubiquitin ligase complex (Feldman, et al., 1997; Skowyra, et al., 1997) which function in phosphorylation dependent ubiquitination of proteins (reviewed in (Elledge & Harper, 1998; Patton, et al., 1998) . The F-box is functionally defined as a motif that can interact with Skpl (Bai, et al., 1996; Winston, et al., 1999a). The functions of the majority of F-box proteins are still unknown but the family has already been assigned roles in controlling SCF substrate selection and are likely to be key regulators in cell signalling, transcription and the cell cycle (Kipreos & Pagano, 2000) . The range of SCF targets includes cell cycle regulators e.g. Gi-phase cyclins, cyclin dependent kinase inhibitors, DNA replication factors and transcription factors that promote cell cycle progression, as well as non-cell- cycle functions, such as cytoskeletal regulators, cell surface receptors, transcription-factor inhibitors, and non-cell-cycle transcription factors (Kipreos & Pagano, 2000) .

A role for an F-box protein outside of SCF substrate selection has recently been demonstrated for the yeast F-box protein Rcylp. Rcylp is required for recycling of the v-SNARE Snclp and this study implicated a complex of Rcylp and Skplp but not other SCF components in the recycling of internalised proteins (Galan et al. , 2001) .

The F-box protein Skp2 controls the entry into S-phase and it has been shown to be required for the ubiquitination and consequent degradation of the cell cycle inhibitor p27^Kιpl both in vivo (Carrano, et al., 1999; Sutterluty, et al., 1999) and in vi tro (Carrano, et al., 1999; Tsvetkov, et al., 1999).

p27^Kιpl was first identified as an inhibitor in cells arrested by transforming growth factor-β (TGF-b) and is regulated by both growth inhibitory cytokines and contact inhibition (Hengst, et al., 1994; Koff, et al., 1993; Polyak, et al., 1994a; Polyak, et al . , 1994b; Slingerland, et al., 1994). In most normal tissues, during both foetal development and in adults, there is a negative correlation between p27^Kιp expression and proliferation (Fredersdorf, et al.,

1997; Yatabe, et al . , 1998). While the p27^κipl protein is strongly expressed in non-proliferating cells its levels decrease when cells are stimulated by growth factors (Kato, et al., 1994; Nourse, et al., 1994). Recent studies suggest that p27^Kιpl functions in cellular differentiation and development (Hengst & Reed, 1996), apoptosis induction (Wang, et al., 1997) and chemotherapy resistance (St Croix, et al., 1996). p27^Kιpl knockout mice manifest altered differentiation programs (Casaccia-Bonnefil, et al., 1997), and p27^Kιp expression increases during differentiation in many cell types both in tissue culture and in vivo (Durand, et al., 1997; Koyama, et al., 1996). There is some disagreement over the association between p27^Kιpl and proliferation in human cancer. The presence of elevated levels of p27^Kιp protein expression in subsets of tumours with a high proliferative index has been explained by suggesting that the abnormally expressed protein lacks the functional capacity to inhibit cell cycle progression (Fredersdorf, et al., 1997; Quintanilla-Martinez, et al., 1998; Sgambata, et al., 1997; Yatabe, et al., 1998).

Evidence that p27^Kιpl may be involved in human tumour progression comes largely from studies that have directly measured the expression of p27^Kιp protein in clinical tumour samples using immunohistochemical assays. Absent or low levels of p27^Kιp expression in a subset of cancers of colon (Loda, et al., 1997; Tenjo, et al., 2000) , breast (Catzavelos, et al., 1997; Porter, et al., 1997; Tan, et al., 1997), lung (Catzavelos, et al., 1999; Esposito, et al., 1997), Barrett's esophagus (Singh, et al., 1998), melanoma (Flørenes, et al., 1998), stomach (Mori, et al., 1997), hypopharyngeal cancer (Mineta, et al., 1999), oral squamous cell carcinoma (Ito, et al., 1999), gastric (Mori, et al., 1997) and prostate (Cote, et al., 1998; Tsihlias, et al., 1998; Yang, et al., 1998) cancers are associated with cases that have a poor prognosis. Loss of p27^Kιp protein could contribute to resistance to growth inhibitory factors (Hunter & Pines, 1994; Kato, et al., 1994; Koff, et al., 1993; Polyak, et al., 1994b; Sandhu, et al., 1997; Slingerland, et al., 1994), deregulation of cell proliferation, and oncogenic change (Hunter & Pines, 1994; Sherr, 1996). Thus it is a common feature that low levels of p27^Kιpl protein correlate with a poor prognosis in cancers (reviewed in (Clurman & Porter, 1998)), and p27^Kιpl is becoming an important clinical marker for tumour progression. As down regulation of p27^Kιpl has been reported to increase sensitivity to anti cancer agents (Katayose, et al., 1997; Zhu, et al., 1996), it has been suggested that patients with weak p27^Kιp expression in their tumours may be the best candidates for adjuvant chemoradiation therapy, considering their poor prognosis (Shamma, et al., 2000) .

The only direct evidence that p27^Klpl is a tumour suppressor comes from the studies of p27^Kιpl-null mice. p27^Kιpl-deficient mice display a generalised increase in body size, pituitary tumours and multiple organ hyperplasia (Kiyokawa, et al . , 1996). Unlike classical tumour suppressors, in these mice, only a reduction in p27^Kιpl levels are necessary to predispose tissues to secondary tumours (Teixeira, et al., 2000).

Although it is a putative tumour suppressor gene, mutations or deletions in the p27^Klpl gene occur only rarely in human cancers (Kawamata, et al., 1995; Morosetti, et al., 1995; Pietenpol, et al., 1995) while another CKI, pl6INK4a, is rivalling p53 for being the most frequently altered gene in human cancer. Regulation of p27^Kιpl is complex and reports on transcriptional (Kolluri, et al., 1999), translational (Hengst & Reed, 1996; Millard, et al., 1997), proteolytic (Catzavelos, et al., 1997; Loda, et al., 1997; Nguyen, et al., 1999; Pagano, et al., 1995), and mis-localisation (Orend, et al., 1998; Soucek, et al., 1998) mechanisms are reported. However, it has been suggested that p27^Kιpl may be a useful clinical tool even before the mechanisms of p27^Kιp inactivation are completely understood and the routine use of p27^κipl as a prognostic indicator for cancer has been recommended (Moller, et al. , 1999) .

Skp2-deficient cells have a phenotype that demonstrate this proteins importance in positively regulating cell proliferation, including high levels of p27^Kιpl and free cyclin E, polyploidy and centrosome overduplication (Nakayama, et al., 2000). Skp2 overexpression has been observed in transformed cells (Zhang, et al . , 1995) and a recent study has implicated this protein in oncogenesis (Latres, et al., 2001). An immunohistochemical study of the expression of Skp2 in low-grade lymphomas and high- grade diffuse large cell lymphomas found a significant direct correlation between Skp2 expression and high- grade malignancy and that both parameters inversely correlated with p27 levels (Latres, et al., 2001). Transgenic mice carrying a CDC4-Skp2 construct to target expression of this protein in the T-lymphoid lineage were generated and studies using these mice showed that co-expression of N-Ras was needed to induce neoplasia (Latres, et al., 2001). This study claims to have provided evidence for an F-box protein in oncogenesis and establishes SKP2 as a protooncogene causally involved in the pathogenesis of lymphomas. Increased levels of Skp2 have recently also been associated with reduced p27 in a subset of oral epithelial dysplasias and carcinomas (Gstaiger et al., 2001) . Moreover this study demonstrated that Skp2 has oncogenic potential and cooperates with H-RasG12V to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumour formation in nude mice. A further study has demonstrated that high expression of the Skp2 protein correlated with poor prognosis in oral squamous cell carcinomas (Kado et al., 2001).

A study that mapped five human F-box genes to several chromosomal loci frequently altered in tumors, screened 42 human tumor cell lines and 48 human tumor samples by Southern hybridization and FISH for alterations in these genes. While no gross alterations of the genes encoding beta-Trcp/Fbwla, Fbw2, Fbx4 and Fbx5 were found, heterozygous deletion of the FBXL3A gene was found in four of 13 small cell carcinoma cell lines (Chiaur, et al., 2000).

An F-box protein, FBWla (FWD1, β-Trcp) , mediates ubiquitin-dependent proteolysis of β-catenin (Kitagawa, et al., 1999) and IκBα (Winston, et al . ,

1999b) . The accumulation of β-catenin as a result of mutations in the adenomatous polyposis coli (APC) tumour suppressor protein is believed to initiate colorectal neoplasia (Ilyas & Tomlinson, 1997). As FBWla is involved in β-catenin instability then loss of FBWla function could lead to cancer. β-Trcp also plays a central role in coupling β-catenin phophorylation-degradation and, in Xenopus, dorsal axis formation (Liu, et al., 1999). β-Trcp is the vertebrate homologue of the Drosphila Slimb protein. The Slimb protein has been shown to be required to restrict centrosome duplication during the cell cycle (Wojcik, et al., 2000). Defects in centrosome duplication are thought to contribute to genomic instability (Lengauer, et al., 1998) and are a hallmark of certain transformed cells and human cancer (Fukasawa, et al., 1996; Salisbury, et al., 1999).

Recently a murine F-box protein mHOS (a homologue of HOS/beta TrCP2) was shown to be over expressed in chemically inducted mouse skin tumours. This overexpression coincided with accelerated degradation of Ikappaβ in vivo and is implicated in the constitutive activation of NF-kappa β (Bhatia et al . , 2002) .

Elevated levels of cyclin E have been associated with a variety of malignancies and constitutive expression of cyclin E leads to genomic instability. Recently, an F-box protein hcdcH/Ago has been identified that regulates the level of cyclin E and mutations within gene have been found in human cancer cell lines that express high levels of cyclin E (Moberg et al., 2001, and Strohmaier, et al., 2001.

The von Hippel-Lindau (VHL) disease is a hereditory cancer syndrome caused by inactivation of the VHL tumour suppressor protein. The VHL protein has a function analagous to an F-box protein and through it's beta-domain binds the hypoxia-inducible factor

(HIF) targeting this for ubiquitination (Ohh, et al., 2000) .

Recent studies on F-box proteins from the fission yeast have identified some interesting roles for these proteins. PoflO overexpression causes loss of viability that can be rescued by overexpression of Skpl. The lethality of overexpression is thought to result from the sequestration of Skpl from other F-box proteins by the stable F-box protein PoflO (Ikebe et al., 2002). In the absence of the F-box protein Pof3 yeast cells exhibit a number of phenotypes reminiscent of genome integrity defects highlighting a role for Pof3 in genome integrity via maintaining chromatin structures (Katayama et al., 2002). These studies indicate that although this is a relatively new field of scientific research there is already sufficient evidence available to demonstrate that F-box proteins have a role in human cancer.

The present inventors have now, advantageously, identified, cloned and sequenced a novel gene encoding a protein, which contains an F-box motif and which is expressed widely in normal cells, but which may provide a useful prognostic indicator of clinical outcome in certain cancers. This novel protein was previously designated JC9 but has since been renamed FBX031 (F-Box only protein 31) by the Hugo Gene Nomenclature Committee. Any reference to FBX031 includes any of the amino acid sequences defined herein according to the invention including the variants of FBX031 identified below.

Therefore, according to a first aspect of the invention, there is provided an isolated nucleic acid molecule encoding an isolated protein having an amino acid sequence as set forth in Figure 5b or Figure 6b or an amino acid sequence which differs from those sequences only in conservative amino acid changes . The protein encoded by the nucleic acid molecule according to the invention as aforementioned has been designated as FBX031.

As outlined in the example provided, the F-box protein motif is located at position 68-117, as shown in Figure 7.

Furthermore, a long form and a short form of the FBX031 protein may exist, although it is likely that the long form is the actual protein expressed in vitro since the first methionine codon in the long form has a considerably better Kozak consensus sequence than the internal methionine potentially giving the short form of the protein. Thus, in a preferred embodiment, the nucleic acid molecule according to the invention preferably encodes the amino acid sequence of Figure 6b lacking the first 5 amino acids with translation starting from the first methionine codon.

As set out more fully in the examples provided, sequencing clones deposited in Genbank initially as EST clones identified a significant number of variant FBX031 proteins. Some of the variants were found to be expressed in human tumours which clearly suggests a role for these proteins in such conditions. The invention therefore also advantageously comprises the nucleic acid molecules encoding those FBX031 variant proteins having the amino acid sequences identified or set forth in Figures 24 to 33.

The nucleic acid molecule is preferably DNA and more preferably cDNA, which is preferably of mammalian and even more preferably of human origin. Even more preferably the nucleic acid molecule comprises the sequence of nucletides set forth in Figure 5a or 6a, and more preferably the sequence of Figure 6a.

A further aspect of the invention comprises nucleic acid molecules capable of hybridising to the nucleic acid molecules of the invention, under conditions of high stringency.

Stringency of hybridisation as used herein refers to conditions under which polynucleic acids are stable. The stability of hybrids is reflected in the melting temperature (Tm) of the hybrids. Tm can be approximated by the formula:

81.5°C+16.6(log₁₀[Na⁺]+0.41 (%G&C) -600/1 wherein 1 is the length of the hybrids in nucleotides. Tm decreases approximately by 1-1.5°C with every 1% decrease in sequence homology.

The term "stringency" refers to the hybridisation conditions wherein a single-stranded nucleic acid joins with a complementary strand when the purine or pyrimidine bases therein pair with their corresponding base by hydrogen bonding. High stringency conditions favour homologous base pairing whereas low stringency conditions favour non-homologous base pairing.

"Low stringency" conditions comprise, for example, a temperature of about 37°C or less, a formamide concentration of less than about 50%, and a moderate to low salt (SSC) concentration; or, alternatively, a temperature of about 50°C or less, and a moderate to high salt (SSPE) concentration, for example IM NaCl.

"High stringency" conditions comprise, for example, a temperature of about 42°C or less, a formamide concentration of less than about 20%, and a low salt (SSC) concentration; or, alternatively, a temperature of about 65°C, or less, and a low salt (SSPE) concentration. For example, high stringency conditions comprise hybridization in 0.5 M NaHP0₄, 7% sodium dodecyl sulfate (SDS) , 1 mM EDTA at 65°C (Ausubel, F.M. et al. Current Protocols in Molecular Biology, Vol. I, 1989; Green Inc. New York, at 2.10.3) .

"SSC" comprises a hybridization and wash solution. A stock 20X SSC solution contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0. "SSPE" comprises a hybridization and wash solution. A IX SSPE solution contains 180 mM NaCl, 9mM Na₂HP0₄ and 1 mM EDTA, pH 7.4.

There are other conditions, reagents and so forth which can be used, which result in stringent hybridisation and the skilled practitioner is familiar with such conditions.

The nucleic acid capable of hybridising to nucleic acid molecules according to the invention will generally be at least 70%, preferably at least 80, 85 or 90% and more preferably at least 95% and even more preferably at least 97% homologous to the nucleotide sequences according to the invention.

An antisense molecule capable of hybridising to the nucleic acid according to the invention may be used as a probe or as a medicament or may be included in a pharmaceutical composition with a pharmaceutically acceptable carrier, diluent or excipient therefor.

The term "homologous" describes the relationship between different nucleic acid molecules or amino acid sequences wherein said sequences or molecules are related by partial identity or similarity at one or more blocks or regions within said molecules or sequences. Homology may be determined by means of computer programs known in the art .

Substantial homology preferably carries with it that the nucleotide and amino acid sequences of the protein of the invention comprise a nucleotide and amino acid sequence fragment, respectively, corresponding and displaying a certain degree of sequence identity to the amino acid and nucleic acid sequences identified in the figures. Preferably they share an identity of at least 30%, preferably 40%, more preferably 50%, still more preferably 60%, most preferably 70%, and particularly an identity of at least 80%, preferably more than 90% and still more preferably more than 95% is desired with respect to the nucleotide or amino acid sequences depicted in Figures 5 or 6 or Figures 24 to 33. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using, for example, the Blast program described in Altschul, S.T., et al., (1990) Basic Local Alignment Search Tool, J. Mol. Biol., 215, 403- 410. In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Further programs that can be used in order to determine homology/identity are described below and in the examples. The sequences that are homologous to the sequences described above are, for example, variations of said sequences which represent modifications having the same biological function, in particular encoding proteins with the same or substantially the same specificity, e.g. binding specificity. They may be naturally occurring variations, such as sequences from other mammals, or mutations. These mutations may occur naturally or may be obtained by mutagenesis techniques. The allelic variations may be naturally occurring allelic variants as well as synthetically produced or genetically engineered variants. In a preferred embodiment the sequences are derived from human .

The nucleic acid molecules according to the invention may, advantageously, be included in a suitable expression vector to express the proteins encoded therefrom in a suitable host. Incorporation of cloned DNA into a suitable expression vector for subsequent transformation of said cell and subsequent selection of the transformed cells is well known to those skilled in the art as provided in Sambrook et al.

(1989), Molecular cloning: A Laboratory Manual, Cold Spring Harbour Laboratory.

An expression vector, according to the invention, includes a vector having a nucleic acid according to the invention operably linked to regulatory sequences, such as promoter regions, that are capable of effecting expression of said DNA fragments. A vector can include a large number of nucleic acids which can have a desired sequence inserted therein by, for example, using an appropriate restriction enzyme and ligating the sequence in the vector, for transport between cells of different genetic composition. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. Such vectors may be transformed into a suitable host cell to provide for expression of a protein according to the invention. The vectors are usually capable of replicating within a host environment and they also comprise one of a number of restriction sites for endonucleases which permits them to be cut in a selective manner at a particular location for insertion of a new nucleic acid molecule or sequence therein. Thus, in a further aspect, the invention provides a process for preparing polypeptides according to the invention, which comprises cultivating a host cell, transformed or transfected with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and recovering the expressed protein. In this regard, the nucleic acid molecule may encode a mature protein or a protein having a prosequence, including encoding a leader sequence on the preprotein which is cleaved by the host cell to form a mature protein.

The vectors may be, for example, plasmid, virus or phagemid vectors provided with an origin of replication, and optionally a promoter for the expression of said nucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable markers, such as, for example, an antibiotic resistance.

Regulatory elements required for expression include promoter sequences to bind RNA polymerase and to direct an appropriate level of transcription initiation and also translation initiation sequences for ribosome binding. For example, a bacterial expression vector may include a promoter such as the lac promoter and for translation initiation the Shine- Dalgarno sequence and the start codon AUG. Similarly, a eukaryotic expression vector may include a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. However, the precise regulatory elements required for expression of a gene of interest may vary between different cell types but generally include 5' non-transcribing and non-translating regions which are required for initiation of translation and transcription. Such vectors may be obtained commercially or be assembled from the sequences described by methods well known in the art.

Transcription of DNA encoding the polypeptides of the present invention by higher eukaryotes is optimised by including an enhancer sequence in the vector. Enhancers are cis-acting elements of DNA that act on a promoter to increase the level of transcription. Vectors will also generally include origins of replication in addition to the selectable markers.

Nucleic acid molecules according to the invention may be inserted into the vectors described in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense nucleic acids, including antisense peptide nucleic acid (PNA) , may be produced by synthetic means.

In accordance with the present invention, a defined nucleic acid includes not only the identical nucleic acid but also any minor base variations including in particular, substitutions in cases which result in a synonymous codon (a different codon specifying the same amino acid residue) due to the degenerate code in conservative amino acid substitutions. The term "nucleic acid sequence" also includes the complementary sequence to any single stranded sequence given regarding base variations.

As used herein with respect to nucleic acids

"isolated" means any of a) amplified in vi tro by, for example, polymerase chain reaction (PCR) , b) recombinantly produced by cloning, c) purified by, for example, gel separation, or d) synthesised, such as by chemical synthesis.

The present invention also advantageously provides oligonucleotides comprising at least 10 consecutive nucleotides of a nucleic acid according to the invention and preferably from 10 to 40 consecutive nucleotides of a nucleic acid according to the invention. As would be appreciated by one of skill in the art, it is also possible to use as primers those untranslated regions (UTR's) of the gene encoding the polypeptide of the invention. For example, 3' and 5' UTR's can be used to identify homologues of the polypeptide of the invention. The oligonucleotides of the invention may, advantageously be used as probes or primers to initiate replication, or the like. Oligonucleotides having a defined sequence may be produced according to techniques well known in the art, such as by recombinant or synthetic means. They may also be used in diagnostic kits or the like for detecting the presence of a nucleic acid according to the invention. These tests generally comprise contacting the probe with the sample under hybridising conditions and detecting for the presence of any duplex or triplex formation between the probe and any nucleic acid in the sample.

According to the present invention these probes may be anchored to a solid support. Preferably, they are present on an array so that multiple probes can simultaneously hybridize to a single biological sample. The probes can be spotted onto the array or synthesised in si tu on the array. (See Lockhart et al., Nature Biotechnology, vol. 14, December 1996 "Expression monitoring by hybridisation to high density oligonucleotide arrays".

The nucleic acid sequences according to the invention may be produced using recombinant or synthetic techniques, such as for example using PCR which generally involves making a pair of primers, which may be from approximately 10 to 50 nucleotides to a region of the gene which is desired to be cloned, bringing the primers into contact with cDNA, or genomic DNA from a human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified region or fragment and recovering the amplified DNA. Generally, such techniques are well known in the art, such as described in Sambrook et al. (Molecular Cloning: a Laboratory Manual, 1989) .

The nucleic acids or oligonucleotides according to the invention may carry a revealing label. Suitable labels include radioisotopes such as ³²P or ³⁵S, enzyme labels or other protein labels such as biotin or fluorescent markers. Such labels may be added to the nucleic acids or oligonucleotides of the invention and may be detected using known techniques per se .

Advantageously, human allelic variants or polymorphisms of the nucleic acid according to the invention may be identified by, for example, probing cDNA or genomic libraries from a range of individuals, for example, from different populations. Furthermore, nucleic acids and probes according to the invention may be used to sequence genomic DNA from patients using techniques well known in the art, such as the Sanger Dideoxy chain termination method, which may, advantageously, ascertain any predisposition of a patient to disorders associated with variants of the FBX031 polypeptide of the invention.

In the very least, the nucleotide sequences can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to "subtract- out" known sequences in the process of discovering novel nucleotide sequences, for selecting and making oligomers for attachment to a "gene chip" or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response. The nucleotide sequences identified herein according to the invention can be used in numerous ways as a reagent. The following description should be considered exemplary and utilizes known techniques. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. The FBX031 sequence has been found to map to chromosome 16. Thus, nucleotide sequences encoding FBX031 can be used in linkage analysis as a marker for chromosome 16. The sequence has been mapped to a particular region 16 pl3.3 of the chromosome using mapped sequence tags (STS markers) and localisation of the genomic sequence. Other mapping techniques that may be used include in si tu hybridization to chromosomal spreads, flow-sorted chromosomal preparations, or artificial chromosome constructions such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial Pi constructions or single chromosome cDNA libraries as reviewed in Price (Blood Rev. 7 (1993) , 127-134) and Trask (Trends Genet. 7 (1991), 149-154). The technique of fluorescent in si tu hybridization of chromosome spreads has been described, among other places, in Verma, (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York NY. Fluorescent in si tu hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data. Examples of genetic map data can be found in the art. Correlation between the location of the gene encoding a FBX031 polypeptide on a physical chromosomal map and a specific feature, e.g., a disease related to the dysfunction of the gene may help to delimit the region of DNA associated with this feature. The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals. Furthermore, the means and methods described herein can be used for marker-assisted animal breeding.

In si tu hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. For example a sequence tagged site based map of the human genome was recently published by the Whitehead-MIT Center for Genomic Research (Hudson, Science 270 (1995), 1945-1954) and is also available on the internet. Often the placement of a gene on the chromosome of another species may reveal associated markers even if the number or arm of a particular chromosome is not known. New sequences can be assigned to chromosomal arms, or parts thereof, by physical mapping. This provides valuable information to investigators searching for interacting genes using positional cloning or other gene discovery techniques. Once such gene has been crudely localized by genetic linkage to a particular genomic region, any sequences mapping to that area may represent associated or regulatory genes for further investigation. The nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier or affected individuals.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in any of Figures 5a or 6a of Figures 24 to 33. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene of interest corresponding to the above sequences will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping the nucleotide sequences to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the nucleotide sequences can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow- sorted chromosomes, and preselection by hybridization to construct chromosome specific cDNA libraries.

Precise chromosomal location of the nucleotide sequences can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses nucleotide sequences as short as 300 to 600 bases; however, nucleotide sequences 1,000-4,000 bp are preferred. For a review of this technique, see Verma et al., "Human Chromosomes: a Manual of Basic Techniques," Pergamon Press, New York (1988) .

For chromosome mapping, the nucleotide sequences can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes) .

Once a nucleotide sequence has been mapped to a precise chromosomal location, the physical position of the nucleotide sequence can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. (Disease mapping data are found, for example, in McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library) ) . Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes .

Thus, once coinheritance is established, differences in the nucleotide sequences of the invention and the corresponding gene between affected and unaffected individuals can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide encoded and the corresponding gene from several normal individuals is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.

According to a further aspect, the invention comprises an isolated FBX031 protein encoded by the nucleic acid molecules of the invention. Preferably, the protein comprises the sequence of amino acids set forth in Figures 5b or 6b, and more preferably 6b, which constitutes the long form of the FBX031 protein. Alternatively, the protein comprises any of the amino acid sequences in Figures 24 to 33 depicting variants of the FBX031 protein.

According to a further aspect, the invention comprises an isolated FBX031 polypeptide comprising an amino acid sequence exhibiting at least 70% sequence homology to the amino acid sequence illustrated in Figure 5b, or 6b or the amino acid sequences of Figures 24 to 33 or a functional equivalent or derivative thereof. Preferably, the invention comprises an isolated polypeptide exhibiting at least 75% preferably 80, more preferably 85, even more preferably 90, 95 or 97% sequence homology to the sequence illustrated in Figure 5b or 6b and preferably the amino acid of 6b or the amino acid sequences illustrated in Figures 24 to 33. Functional homologues or equivalents of the polypeptide of the invention can be prepared according to methods known in the art, and which comprise, amongst others, altering the polypeptide sequence as set out in Molecular Cloning, A Laboratory Manual, Sambrook et al . Conservative amino acid substitutions can be performed by altering the nucleic acid encoding the polypeptide, using, for example, PCR or site directed mutagenesis or by chemical synthesis of the nucleic acid molecule. Computer algorithms can also be utilised which predict the amino acid sequences that may be altered or substituted to prepare said functional equivalents.

A polypeptide according to the invention includes all possible amino acid variants encoded by its corresponding nucleic acid molecule, including a polypeptide encoded by said molecule and having conservative amino acid changes. Proteins or polypeptides according to the invention further include variants of such sequences, including naturally occurring allelic variants which are substantially homologous to said proteins or polypeptides. In this context, substantial homology is regarded as a sequence which has at least 60%, 70%, preferably 80 or 90%, more preferably 95% and even more preferably 97% amino acid homology with the proteins or polypeptides encoded by the nucleic acid molecules according to the invention. The protein according to the invention may be recombinant, synthetic or naturally occurring, but is preferably recombinant .

As used herein with respect to polypeptides, "isolated" means separated from its native environment and present in sufficient quantity to permit its identification or use. Isolated, when referring to a protein or polypeptide, means, for example: (i) selectively produced by expression cloning or (ii) purified as by chromatography or electrophoresis .

Isolated proteins or polypeptides may, but need not be, substantially pure. The term "substantially pure" means that the proteins or polypeptides are essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use.

As set out in the example in more detail, FBX031 mRNA expression is frequently found to be reduced in non- haematopoietic tumour cells when compared to adjacent normal tissues, suggesting FBX031 as a putative tumour suppressor. However, in some instances a higher level of FBX031 mRNA is to be found in some cases of rectal tumours and FBX031 protein in some lymphomas, leukaemias and lung tumours. Accordingly, altering expression of FBX031 may be advantageous in treating malignancies of these types. Therefore, the present invention is further directed to inhibiting expression or activity of the polypeptides of the invention in vivo by, for example, inhibiting transcription by, for example, the use of antisense technology. However, as would be appreciated by the skilled practitioner any other suitable method may be utilised. Other methods of inhibiting protein expression may utilise antibodies or binding polypeptides or other small molecules which, for example, bind or block the binding region of the polypeptides of the invention. As used herein, the term "antisense nucleotide" or "antisense" describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridises under physiological conditions to DNA encoding FBX031 polypeptide or to an mRNA transcript of the gene and, thereby, inhibits the transcription of that gene and/or translation of mRNA. Antisense technology can be used to control gene expression through triple- helix formation of antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion or the mature protein sequence, which encodes for the protein of the present invention, is used to design an antisense RNA oligonucleotide of from 10 to 40 base pairs in length. The antisense RNA oligonucleotide hybridises to the mRNA in vivo and blocks translation of an mRNA molecule into the protein (antisense - Okano, J. Neurochem., 56:560 (1991);

Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple- helix - see Lee et al. Nucl. Acids Res., 6:3073

(1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991), thereby preventing transcription and the production of the FBX031 polypeptide.

The term "modified oligonucleotide" as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide. Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, peptides, and carboxymethyl esters.

The term "modified oligonucleotide" also encompasses oligonucleotides with a covalently modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus, modified oligonucleotides may include a 2 ' -O-alkylated ribose group. In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose. Modified oligonucleotides also can include base analogs such as C-5 propyne modified bases (Wagner et al., Na ture Biotechnology 14:840-844, 1996) . The present invention, thus, contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding FBX031 polypeptide together with pharmaceutically acceptable carriers.

The hybrid and modified forms include, for example, when certain amino acids have been subjected to some modification or replacement, such as for example, by point mutation and yet which results in a polypeptide or protein which possesses the same function as the FBX031 polypeptides of the invention.

The antisense oligonucleotide described above can be delivered to cells by procedures in the art such that the anti-sense RNA and DNA may be expressed in vivo to inhibit production of the protein in the manner described above.

A further aspect of the invention provides a host cell or organism, transformed or transfected with an expression vector according to the invention. The cell or organism may be transformed or transfected using techniques that are well known in the art, such as, electroporation or by using liposomes. The host cell or organism may advantageously be used in a method of producing FBX031 polypeptide, which comprises recovering any expressed polypeptide from the host or organism transformed or transfected with the expression vector.

According to a further aspect of the invention there is also provided a transgenic cell, tissue or organism comprising a transgene capable of expressing a polypeptide according to the invention. The term "transgene capable of expressing" as used herein encompasses any suitable nucleic acid sequence which leads to expression of a polypeptide (s) having the same function and/or activity as FBX031. The transgene, may include, for example, genomic nucleic acid isolated from human cells or synthetic nucleic acid, including DNA integrated into the genome or in an extrachromosomal state. Preferably, the transgene comprises the nucleic acid sequence encoding the polypeptide according to the invention as described herein, or a functional fragment of said nucleic acid. A functional fragment of said nucleic acid should be taken to mean a fragment of the gene comprising said nucleic acid coding for the polypeptides according to the invention or a functional equivalent, derivative or a non-functional derivative such as a dominant negative mutant of said polypeptides.

Transgenic non-human organisms are being utilised as model systems for studying both normal and disease cell processes. In general, to create such transgenic animals an exogenous gene with or without a mutation is transferred to the animal host system and the phenotype resulting from the transferred gene is observed. Other genetic manipulations can be undertaken in the vector or host system to improve the gene expression leading to the observed phenotype

(phenotypic expression) . The gene may be transferred on a vector under the control of different inducible or constitutive promoters, may be overexpressed or the endogenous homologous gene may be rendered unexpressible, and the like (WO 92/11358) . The vector may be introduced by transfection or other suitable techniques such as electroporation, for example, in embryonic stem cells. The cells that have the exogenous DNA incorporated into their genome, for example, by homologous recombination, may subsequently be injected into blastocytes for generation of the transgenic animals with the desired phenotype. Successfully transformed cells containing the vector may be identified by well known techniques such as lysing the cells and examining the DNA, by, for example, Southern blotting or using the polymerase chain reaction.

Knock-out organisms may be generated to further investigate the role of the polypeptide of the invention in vivo. By "knock-out" it is meant an animal which has its endogenous gene knocked out or inactivated. Typically, homologous recombination is used to insert a selectable gene into an essential exon of the gene of interest. Furthermore, the gene of interest can be knocked out in favour of a homologous exogenous gene to investigate the role of the exogenous gene (Robbins, J., GENE TARGETING. The Precise Manipulation of the Mammalian Genome Circ. Res. 1993, J.W.; 73; 3-9). Transgenic animals, such as mice or Drosophila or the like, may therefore be used to over or under express the FBX031 protein according to the invention to further investigate its role in vivo .

The polypeptide expressed by said transgenic cell, tissue or organism or a functional equivalent thereof also forms part of the present invention. Recombinant proteins or polypeptides may be recovered and purified from host cell cultures by methods known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose, chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography.

The polypeptide of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial yeast, higher plant, insect and mammalian cells in culture) . Depending upon the host employed in a recombinant production procedure, the expressed polypeptide may lack the initiating methionine residue as a result of post-translational cleavage. Proteins or polypeptides which have been modified in this way are also included within the scope of the invention. In a still further aspect the invention provides a binding polypeptide which is capable of binding to the polypeptide of the invention or an epitope thereof. In one embodiment, the binding polypeptide comprises an antibody, for example, or a polypeptide exhibiting regions of homology with the polypeptide of the invention and capable of binding thereto. Such an antibody may be polyclonal, for example, and may be raised according to standard techniques well known to those skilled in the art by using the polypeptide of the invention or a fragment or single epitope thereof as the challenging antigen. Alternatively, the antibody may be monoclonal in nature and may be produced according to the techniques described by Kohler & Milstein (Nature (1975) 256, 495-497).

When the binding protein or polypeptide is an antibody the present invention includes not only complete antibody molecules but fragments thereof. Antibody fragments which contain the idiotype of the molecule can be generated by known techniques, for example, such fragments include but are not limited to the F(ab')₂ fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragments and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent. Chimeric, humanized and fully humanized monoclonal antibodies can now be made by recombinant engineering. By addition of the human constant chain to F(ab')₂ fragments it is possible to create a humanized monoclonal antibody which is useful in immunotherapy applications where patients making antibodies against the mouse Ig would otherwise be at a disadvantage. Breedveld F.C. Therapeutic Monoclonal Antibodies. Lancet 2000 Feb 26; 335, P735-40. Furthermore, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epiptope (see, in general, Clark, W.R. (1986) The

Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford) . The pFc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')₂ fragment, retains both of the antigen binding sites of an intact antibody.

Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope- binding ability in isolation.

Polypeptides that bind to the polypeptide of the invention may be identified by Phage Display. In this technique a phage library is prepared, displaying inserts from between about 4 to 80 amino acid residues using techniques which are well known in the art. It is then possible to select those Phage bearing inserts that bind to the polypeptide of the invention. DNA sequence analysis is then performed to identify the nucleic acid sequences encoding the expressed polypeptides .

Antibody fragments of predetermined binding specificity may also be constructed using Phage Display technology, which obviates the need for hybridoma technology and immunization. These antibody fragments are created from repertoires of antibody V genes which are harvested from populations of lymphocytes, or assembled in vi tro, and cloned for display of associated heavy and light chain variable domains on the surface of filamentous bacteriophage. The process mimics immune selection and antibodies with many different binding specificities have been isolated from the same Phage repertoire. (Winter et al . , Annu. Rev. Immunol. 1994; 12 : 433-55). Such antibodies are also embraced within the scope of the binding polypeptides of the present invention.

Other types of binding polypeptides that may be utilised in accordance with the invention are termed immunoadhesins . Immunoadhesins are a class of fusion proteins, which combine the target-binding region of a receptor, an adhesion molecule, a ligand or an enzyme, with the Fc portion or an immunoglobulin. Production of immunoadhesins is described in Byrn et al (1990) Nature 344, pp 667-670.

In a preferred embodiment an antibody according to the invention is that which is produced by the hybridoma deposited under Accession No. 01012314 of the European Collection of Cell Cultures.

The nucleic acid molecules or the polypeptides of the invention may also be included in a pharmaceutical composition together with any suitable pharmaceutically acceptable carrier diluent or excipient therefor. The nucleic acid molecule or polypeptides may be encapsulated and/or combined with suitable carriers in solid dosage forms for oral administration which would be well known to those of skill in the art or alternatively with suitable carriers for administration in an aerosol spray.

In the pharmaceutical composition of the invention, preferred compositions include pharmaceutically acceptable carriers including, for example, non-toxic salts, sterile water or the like. A suitable buffer may also be present allowing the compositions to be lyophilized and stored in sterile conditions prior to reconstitution by the addition of sterile water for subsequent administration. The carrier can also contain other pharmaceutically acceptable excipients for modifying other conditions such as pH, osmolarity, viscosity, sterility, lipophilicity, somobility or the like. Pharmaceutical compositions which permit sustained or delayed release following administration may also be used.

Furthermore, as would be appreciated by the skilled practitioner, the specific dosage regime may be calculated according to the body surface area of the patient or the volume of body space to be occupied, dependent on the particular route of administration to be used. The amount of the composition actually administered will, however, be determined by a medical practitioner based on the circumstances pertaining to the disorder to be treated, such as the severity of the symptoms, the age, weight and response of the individual .

The invention also contemplates gene therapy. This involves introduction in vi tro of a functional copy of a gene into a cell(s) of a subject which contains a defective copy of the gene and returning the genetically engineered cell(s) to the subject. The functional copy of the gene is under operable control of regulatory elements which permit expression of the gene in the genetically engineered cells. Numerous transfection and transduction techniques as well as appropriate expression vectors for carrying out such procedures are well known in the art. In vivo gene therapy using plasmids or viral vectors, such as adenovirus, vaccina virus and the like, is also contemplated according to the invention. Thus, incorporation of a nucleic acid molecule encoding FBX031 using gene therapy should permit replacement of any defective FBX031 protein and which should resume its normal function.

A further aspect of the present invention also provides a method of identifying a polypeptide of the invention in a sample, which method comprises contacting said sample with a binding polypeptide as described herein and monitoring for any specific binding of any polypeptides to said binding polypeptide. A kit for identifying the presence of such polypeptides in a sample is also provided comprising a binding polypeptide as described above and means for contacting said binding polypeptide with said sample.

In a further aspect the invention provides an in vi tro method of detecting expression of a polypeptide of the invention which method comprises contacting a sample of tissue, cells or cell lysates from a subject with a binding protein as previously described and detecting any binding of said binding polypeptide to a protein in the sample.

Preferably the method of the invention is performed on cells or tissues removed from a human subject. However, it is also within the scope of the invention to perform the method on cells or tissues removed from non-human mammals such as mouse or monkey by using an antibody which is cross-reactive against a homologous protein expressed in the non-human mammalian species.

In certain circumstances it may be particularly beneficial to alter the activity or function of FBX031 protein, particularly, for example, where the FBX031 protein is in some way defective and therefore unable to perform its putative role as a tumour suppressor.

Thus, the invention also comprises a method of modulating activity/function of FBX031 polypeptide, which method comprises inhibiting or enhancing expression or activity in a cell of a FBX031 polypeptide according to the invention. Enhancing expression of FBX031 may alleviate or remove any susceptibility of an individual to certain cancers as set out in the examples provided. Numerous methods and techniques are available in the art for inhibiting expression or function of the polypeptide of the invention which would be known to the skilled practitioner. For example, increased expression of FBX031 may be achieved by transformation of a suitable expression vector incorporating the nucleic acid sequence of FBX031, whereas inhibiting its function or expression may be accomplished using antisense techniques described herein or by using a blocking or binding protein. Furthermore, as would be well known to the skilled practitioner, other small molecules, such as binding peptides or polypeptides or other compounds may be synthesised or produced which can inhibit function or activity of the polypeptides of the invention.

FBX031 mDNA has been found by the inventors to be widely expressed in normal human tissue, both adult and foetal, but expression at different levels occurred when compared to tumour tissue taken from the same individuals (Figure 9b) , and generally less FBX031 mRNA is found in tumours when compared to normal tissue. These results, in addition to the existing knowledge regarding other F-box proteins, suggest a role as a tumour suppressor gene whose mRNA expression is reduced in solid tumours. Therefore, these tumours which reflect a reduction in FBX031 expression may be treated by increasing expression or activity of FBX031 or its variants or by administering the protein itself.

Therefore in a further aspect, the invention comprises a method of treating a disease or condition in an individual which is associated with under expression or activity of a polypeptide according to the invention which method comprises administering to said individual a polypeptide as described herein or an expression vector according to the invention.

A further aspect comprises a method of diagnosing the medical significance of a polypeptide according to the invention in a disease condition, which comprises monitoring expression or activity levels of said polypeptide and comparing said levels to those which are found in a non-disease state. As set out in the example provided, differential expression of the FBX031 protein or its variants may be of diagnostic relevance in a number of disease types, such as ALCL, providing a prognostic indication of the clinical outcome in such tumours.

In an even further aspect, the invention comprises a method for diagnosing or monitoring the progression of a disorder that is characterised by expression of a FBX031 polypeptide of the invention comprising contacting a biological sample isolated from a subject with an agent specific for the polypeptide to detect the presence of the polypeptide in the biological sample.

In view of the identification of overexpression of the FBX031 mRNA or protein or the variants thereof in some malignancies, identification of agonistic or antagonistic compounds may be useful for treating patients with such malignancies.

Therefore, also provided is a method of identifying compounds capable of modulating activity of a FBX031 polypeptide or the variants thereof comprising administering said compound to a transgenic cell, tissue or organism according to the invention, and monitoring the effect of said compound on said transgenic cell, tissue or organism compared to a cell tissue or organism that has not been contacted with said compound. An even further aspect of the invention comprises a method of producing a compound that modulates the activity or function of a polypeptide according to the invention, comprising i) synthesising the compound obtained or identified in the invention, or a physiologically acceptable analogue or derivative thereof, in an amount sufficient to provide said modulators in a therapeutically effective amount to a patient, and/or ii) combining the compound obtained or identified according to the invention or an analogue or derivative thereof, with a pharmaceutically acceptable carrier.

The compounds isolated by the above methods also form part of the invention and may be used in treating the human or animal body or in the manufacture of a medicament for treating cancer, such as prostate, pancreas, colon, stomach or lung cancer,

The compounds identified may also, as would be appreciated by those of skill in the art, serve as lead compounds for the development of analogue compounds. The analogues should have a stabilized electronic configuration and molecular conformation that allows key functional groups to be presented to the polypeptides of the invention in substantially the same way as the lead compound. In particular, the analogue compounds have spatial electronic properties which are comparable to the binding region, but can be smaller molecules than the lead compound, frequently having a molecular weight below about 2 kD and preferably below about 1 kD. Identification of analogue compounds can be performed through use of techniques such as self-consistent field (SCF) analysis, configuration interaction (CI) analysis, and normal mode dynamics analysis. Computer programs for implementing these techniques are available; e.g., Rein, Computer-Assisted Modelling of Receptor-Ligand Interactions (Alan Liss, New York, 1989) . Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York, USA. Furthermore, said derivatives and analogues can be tested for their effects according to methods known in the art; see also supra. Furthermore, peptidomimetics and/or computer aided design of appropriate derivatives and analogues can be used.

According to a further aspect of the invention there is provided a method of diagnosing the medical significance of a condition or a cancer in a patient which method comprises detecting for abnormal mRNA transcripts or abnormal levels of mRNA expression, nucleotide sequences or gene copy numbers encoding a FBX031 polypeptide or variant thereof according to the invention.

A further aspect of the invention comprises a method of screening for predisposition to cancer in an individual which comprises screening for an inherited genetic mutation in a nucleic acid sequence from said individual encoding a FBX031 polypeptide or its variants and a method of detecting or diagnosing cancer in an individual which is associated with reduced levels of expression of a FBX031 polypeptide which method comprises testing in a cell of said individual for increased levels of methylation of a regulatory region of a nucleic acid sequence from said individual encoding said protein. Reduced expression may also be caused by alternative splicing of the FBX031 mRNA. Therefore, a method of detecting or diagnosing cancer may also include testing for the expression of FBX031 splice variants.

According to a further aspect of the invention there is provided a method of treating cancer associated with reduced levels of expression of a FBX031 protein which method comprises administering to an individual in need thereof a therapeutic amount of a methylation inhibitor.

An even further aspect of the invention comprises a method of detecting or diagnosing cancer in an individual which is associated with increased levels of expression of a FBX031 protein which method comprises testing in a cell of said individual for decreased levels of methylation of a regulatory region of a nucleic acid molecule encoding a protein.

According to a further aspect of the invention there is also provided a method of treating a disease or condition in a patient associated with over or under expression of a FBX031 polypeptide, which method comprises administering to an individual in need thereof a therapeutic amount of an antisense molecule or an antibody or a blocking peptide or a nucleic acid molecule encoding said FBX031.

In an even further aspect of the invention, there is provided a method of identifying minimal residual disease in a cancer patient which method comprises detecting for the presence of neoplastic cells in an individual by identifying a change of expression level pattern, sub-cellular localisation or function of a FBX031 protein in a patient.

A method of identifying minimal residual disease in a cancer patient which method comprises detecting for the presence of neoplastic cells in an individual by identifying abnormal levels of mRNA expression or nucleotide sequences or gene copy number encoding a protein according to any of claims 10 to 12.

The invention may be more clearly understood from the following example with reference to the accompanying Figures wherein:

Figure la is an illustration of insoluble inclusion bodies prepared from plasmid pAB372 and which were used to immunise rabbits for polyclonal antibody production.

Figure lb is an illustration of the results of a

Western Blot of the proteins from Figure 1A. The reactivity of the rabbit polyclonal antibodies produced was confirmed by Western blotting of the bacterially expressed recombinant protein.

Figure 2 is an illustration of the results obtained from peroxidase immunostaining of routinely fixed tissue sections using the FBX031 rabbit polyclonal antibodies and a goat anti-rabbit HRP conjugated secondary antibody. The ALK180 monoclonal antibody staining of these cases is also illustrated to demonstrate the similar staining pattern.

Figure 3a is an illustration of the results obtained from Western blotting of tonsil cell lysate with the polyclonal antibodies generated in Figure lb, and which identified a reactive protein with a molecular weight of approximately 66 kDa that was specifically detected by the immune serum.

Figure 3b is an illustration of the results obtained from Western Blotting using a monoclonal antibody to FBX031, (JC9 antibody) on recombinant FBX031 protein expressed from plasmid pAB372. These confirm that the JC9 antibody recognised the FBX031 recombinant protein.

Figure 3c is an illustration of results obtained from Western blotting using antibody JC9 carried out on cytoplasmic (T-C) and nuclear (T-N) protein extracts prepared from tonsil and lymphoma cell lines.

Figure 4a is an illustration of the sequence of the fragment of the FBX031 cDNA in plasmid pAB172.

Figure 4b is an illustration of the corresponding protein encoded by the sequence from Figure

4a.

Figure 5a is an illustration of the nucleotide sequence of the short form of FBX031.

Figure 5b is an illustration of the protein sequence encoded by the cDNA of Figure 5a.

Figure 6a is an illustration of the nucleotide sequence of the long form of FBX031.

Figure 6b is an illustration of the protein encoded by the nucleotide sequence of Figure 6a.

Figure 7 is an illustration of the sequence of the F-

Box domain at position 68-117 of the long form of FBX031 identified in the Pfam database.

Figure 8 is an illustration of the protein sequence of the long form of FBX031 indicating the position of predicted domains.

Figure 9 is an illustration of the results obtained following hybridisation of a 5' 2.1 kb £coRI fragment from pABl72 labelled with ³²P Clontech's human (a) multiple tissue expression (MTE) array and (b) matched tumour/normal (MTN) expression array.

Figure 10 is a legend of the results in Figure 9. Figure 11 is an illustration of the results obtained from cryostat tissue sections immunostained with monoclonal antibody JC9 for expression of FBX031.

Figure 12 is an illustration of APAAP immunostaining of paraffin embedded tissues with monoclonal antibody JC9 demonstrating expression of the FBX031 protein in various human tissues A - N.

Figure 13 is an illustration of the results obtained from renal tumours stained with the JC9 antibody.

Figure 14 is an illustration of the results obtained from JC9 immunostaining of six cases of small cell lung cancer indicating that all cases expressed FBX031 protein to some degree.

Figure 15 is an illustration of the results obtained from JC9 immunostaining of pancreatic tumours and breast tumours.

Figure 16 is an illustration of the results obtained from immunostaining of cases ALCL (either T or null cell type) , ALK positive (A-E) and ALK negative (F-J), with JC9 antibody.

Figure 17 is an illustration of the results obtained from JC9 immunostaining of CLL, DLBCC, and follicular lymphomas (FL) .

Figure 18 is an illustration of the results obtained from JC9 immunostaining Hodgkins disease and MALT lymphomas. Figure 19 is an illustration of the results obtained from double immunofluorescent labelling of haematopoietic neoplasms.

Figure 20 is a cDNA sequence of FLJ22477 and its corresponding amino acid sequence.

Figure 21 illustrates cDNA sequences of putative genes upstream of FBX031 (EST BG724256 and EST AW014292 and its corresponding amino acid sequence .

Figure 22 is an illustration of the sequence of EST clone BE269540/IMAGE clone 3542631 containing full length FBX031 cDNA with an N

Terminal deletion.

Figure 23 is an illustration of the nucleotide and predicted protein sequences of FBX031 alternate form 1 (FBX031-1) .

Figure 24 is an illustration of the nucleotide and predicted protein sequences of partial and full length FBX031 alternate form 2 (FBX031- 2) .

Figure 25 is an illustration of the nucleotide and predicted protein sequence of partial and full length FBX031 alternate form 3 and 4 (FBX031-3 and 4) .

Figure 26 is an illustration of the nucleotide and amino sequences of partial and full length FBX031 alternate form 4.

Figure 27 is an illustration of the nucleotide sequence of FBX031 alternate form 5 (FBX031- 5) aligned to genomic FBX031 cDNA.

Figure 28 is an illustration of the nucleotide sequence of FBX031 alternate form 6 (FBX031- 6) .

Figure 29 is an illustration of the nucleotide and predicted protein sequence of FBX031 alternate form 7 (FBX031-7).

Figure 30 is an illustration of the nucleotide and predicted protein sequences of partial FBX031 alternate form 8 (FBX031-8).

Figure 31 is an illustration of the nucleotide and predicted protein sequences of partial and full length FBX031 alternate form 9 (FBX031- 9) .

Figure 32 is an illustration of the nucleotide and predicted protein sequences of partial and full length FBX031 alternate form 10 (FBXO31-10) .

Figure 33 shows that the FBX031 protein can bind Skp-

1. Panel A illustrates a COS cell transfected with a cDNA expressing the FBX031 tagged with the Xpress epitope tag. Panel B illustrates that the Skp-1 protein is co-immunoprecipitated with the FBX031 protein.

Figure 34 is a schematic illustration of the variant FBX031 mRNAs.

Figure 35 is a schematic illustration of the variant FBX031 proteins. Figure 36 is an illustration of the results obtained following expression of the FBX031-1 variant protein in COS cells. COS cells, transfected with either the Xpress tagged FBX031 long cDNA (A & B) or the Xpress tagged FBX031-1 variant cDNA C & D) , were immunostained with the anti-Xpress antibody. Lower levels of recombinant protein were observed in COS cells transfected with the FBX031-1 cDNA construct C & D) .

Figure 37 is an alignment of the FBX031 long cDNA to the FBX031 genomic sequence identified in the clone, accession no. NT_ 019609.4.

Figure 38 illustrates the sequence of the EST clone BE903394/IMAGE clone 3958783 containing a full length FBX031 cDNA.

Figure 39 illustrates the cDNA sequence in EST clone

BI5613051.

Figure 40 illustrates the (a) cDNA sequence in EST clone BI520057 and (b) the predicted protein sequence.

Figure 41 is a sequence of the protein encoded by the cDNA in the clone in AL117444.

Figure 42 is a nucleotide sequence of a full length cDNA sequence from EST clone BG180443/IMAGE clone 4432569 and (b) an amino acid sequence of the protein encoded by the nucleotide sequence of BG180443/IMAGE clone 443259.

Figure 43 shows the 5' amino acid sequence encoded by the cDNA in clone AL528102. Figure 44 is an illustration of the results obtained after incubating various cell lines with FBX031 monoclonal antibody JC9.

Figure 45 is an illustration of the results obtained following peroxidase immunostaining of NSCLC with the FBX031 antibody, JC9.

Panel A shows both nuclear and cytoplasmic staining of lung epithelium adjacent to a

NSCLC tumour. Panel B shows dysplasia of the lung epithelium with cells also having both nuclear and cytoplasmic staining. Panels C and D show NSCLC tumour cells underlying the epithelium with both cell types having similar levels of nuclear and cytoplasmic staining. Panel E shows weak cytoplasmic staining of both the tumour and the epithelium, this is not the result of a technical artifact as strong staining of plasma cells is observed (bottom left) . Panel F demonstrates increased cytoplasmic staining of the tumour cells compared to the adjacent epithelium. Panel G is a largely negative tumour with positive staining of plasma cells (top) . Panels H and I show weak cytoplasmic staining of the tumour cells and strong staining of plasma cells used as an internal control. Panels J and K show moderate cytoplasmic staining of the tumour cells while L shows additional nuclear labelling of the tumour cells. Panels M and N show strong cytoplasmic staining of the tumour cells comparable to that of the adjacent plasma cells. In panel 0 nuclear staining was also observed in the tumour cells . Figure 46 is an illustration of the results obtained following immunoprecipitation experiments using COS cells transfected with either the Xpress tagged full length FBX031 cDNA, the

Xpress tagged lacZ cDNA or an empty vector control which were lysed and immunoprecipitated with the anti-Xpress antibody Western Blotting with an anti-cdk2 antibody was performed to demonstrate co- immunoprecipitation of this protein.

EXAMPLE

Aberrant expression of Anaplastic Lymphoma Kinase (ALK) protein, largely as a result of the 2:5 translocation, is the oncogenic event causing anaplastic large cell lymphoma (ALCL) (Morris, et al., 1994) .

Monoclonal antibody ALK180 was raised against a peptide sequence corresponding to amino acids 265-278 of the NPM-ALK protein and recognises the peptide in an ELISA. The reactivity of this antibody with the ALK protein was confirmed, however it recognised several additional molecular weight bands in Western blotting experiments (150kDa, 90kDa and 80kDa) . Production of a specific anti-ALK monoclonal antibody, ALKl, confirmed that the ALK180 antibody recognised additional proteins and had a much wider immunostaining pattern on human tissues.

Identification of ALKl80 antigens using bacterial expression cloning

An oligo dT primed cDNA library from testis in lambda ZAP Express was purchased from Stratagene. 10 000 plaques per 15cm plate were grown on E. coli strain XL1 Blue MRF' (Stratagene) and protein expression was induced by overlaying IPTG (lOmM) soaked nitrocellulose filters overnight. Filters were removed next day (after approximately 16 hours) and rinsed in PBST (PBS + 0.05% Tween 20) for 5 minutes. Filters were washed in fresh PBST for 30 minutes before blocking in PBST + 5% Marvel at room temperature for 30 minutes. ALK180 tissue culture supernatant was then added at a 1/100 dilution at room temperature for 30 minutes. Filters were rinsed and then washed for 10 minutes with PBST. Filters were incubated for 30 minutes with a 1/750 dilution of goat anti-mouse peroxidase conjugated secondary antibody (DAKO) in PBST, before four 15 minute washes in PBST. Antigen- antibody complexes were visualised using diaminobenzidine with metal ion enhancement. A second screen was performed to isolate individual positive clones .

We originally isolated a single positive clone from the ALK180 library screening. The cDNA encoding the ALK180 antigen in plasmid pBK-CMV was excised in vivo from the lambda ZAP Express vector (Stratagene) using the manufacturer's instructions to yield the plasmid pAB172.

Subsequent expression cloning experiments with the ALK180 antibody isolated an additional cDNA cloned in plasmid pAB376 whose protein product was also recognised by the ALK180 antibody. Database searches using BLAST at NCBI identified this cDNA as that encoding human nuclear domain protein 52 (NDP52) . The NDP52 mRNA is reported to be widely expressed in human tissues and the NDP52 protein was originally reported to colocalise with the nuclear domain proteins SplOO and PML (Korioth, et al., 1995). Subsequently this has been shown to be due to cross reactivity of the original antibody with SplOO and the expression pattern of the NDP52 protein has been shown to be both nuclear and cytoplasmic (Sternsdorf, et al., 1997) without the presence of nuclear dots. We did however identify some nuclear dots in endothelial cells in tonsil using the NDP52 polyclonal antibodies (a kind gift from Dr Hans Will) . The mRNA from a bovine homolog of the NDP52 gene has also been shown to be overexpressed in a bovine T lymphoma cell line (Onodera, et al., 1998) indicating that differential expression of this molecule may be linked to cancer.

Reactivity of the ALKl80 antibody with the recombinant protein expressed from plasmid pAB172.

E. coli strain XLOLR (Stratagene) containing either plasmid pAB172 or the empty vector pBK-CMV was grown overnight in LB medium containing kanamycin at 37°C.

Next day cultures were diluted 1/10 into fresh medium and were allowed to grow for a further 1.5 hours. Protein expression was induced by the addition of ImM IPTG. After 3 hours at 37°C 1ml samples were removed and the cell pellets were resuspended in lOOμl of SDS- PAGE sample buffer. Samples were boiled for 2 minutes and run on a 12% SDS-PAGE gel alongside high molecular weight rainbow markers (Amersham) . Proteins were transferred electrophoretically using a Semi-Phor (Hoeffer) apparatus to PVDF membrane (Millipore) . The filter was then incubated with undiluted ALK180 hybridoma culture supernatant followed by secondary goat anti-mouse peroxidase conjugate as above. Antibody-antigen complexes were detected using an ECL kit (Amersham) following manufacturer's instructions. The ALK180 antibody specifically recognised the protein expressed by plasmid pAB172 in Western blotting while the ALKl antibody (which is specific for ALK) did not (Butler, 1997).

Reactivity of the mammalian expressed protein with ALKl80

To confirm that the ALK180 antibody was able to recognise the protein expressed by pAB172 in immunostaining techniques the plasmid was transfected into COS cells.

Plasmids pAB172 and pBK-CMV were transfected into the COS-1 monkey fibroblast cell line using the DEAE dextran method (Seed & Aruffo, 1987). Cells were used for transfection at 75% confluence and 5μg of plasmid DNA was used for each 25cm2 flask. After 3 days in culture the cells were recovered by EDTA treatment and cytocentrifuge preparations were made for immunocytochemical staining (Erber, et al., 1984). Cytospins were incubated with the ALK180 tissue culture supernatant for 30 mins then washed in PBS for 5 mins. Secondary antibody goat anti-mouse HRP (DAKO) was added at a 1/50 dilution for 30 minutes. After a 5 min wash in PBS, DAB substrate was added for 10 minutes. Slides were counterstained in haematoxylin and then mounted in Aquamount (BDH) .

This experiment confirmed the reactivity of the ALK180 antibody with this protein when expressed in mammalian cells (Butler, 1997) .

DNA sequencing was performed using M13 Universal and Reverse primers, a Cy5 Autoread sequencing kit and an ALF DNA sequencer (Pharmacia) . The cDNA in pAB172 was identified as a gene of unknown function for which only partial cDNAs were available (e.g. yc92h06.sl, EST56854, ye72cl0.sl, yg27f07.sl, yf90h01. si) (Butler, 1997) .

Expression of recombinant protein from the pABl72 cDNA and production of polyclonal antibodies

The 2.1 kb EcoRl fragment from plasmid pAB172 containing the entire coding region from this cDNA clone was cloned into the EcoRl site of pGEX-3X for bacterial protein expression as a GST fusion protein (plasmid pAB372) . Insoluble inclusion bodies were prepared (Figure 1A) and were used to immunise rabbits for polyclonal antibody production by Harlan Seralabs. The reactivity of these antibodies was confirmed by Western blotting of the recombinant protein (Figure IB) .

Peroxidase immunostaining of routinely fixed tissue sections was carried out with this reagent as described previously, using a goat anti-rabbit HRP conjugated secondary antibody. The polyclonal antibodies specifically immunostained the cytoplasm of neoplastic cells in both anaplastic large cell lymphoma (ALCL) and Hodgkin' s disease-mixed cellularity (HD/MC) . These results are similar to those obtained with the ALK180 monoclonal antibody (Figure 2) . However, in the case of ALCL the ALK180 monoclonal antibody also immunostained the ALK kinase and therefore the immunostaining due to the additional protein detected by the polyclonal antibody would be impossible to separate from the other reactivity of this non-specific reagent. These data provided the first experimental evidence linking the protein partially encoded by the pAB172 cDNA with human malignancy. Despite the positive immunostaining of haematopoietic malignancies with the ALK180 antibody this reactivity was not obviously due to this protein as the ALK180 antibody could also detect both ALK and NDP52 proteins.

These reagents demonstrated that the novel partial cDNA that the inventors had isolated did indeed encode a protein expressed in haematopoietic malignancies. Western blotting of the polyclonal antibody on tonsil cell lysate (Figure 3A) identified a reactive protein with a molecular weight of approximately 66 kDa (Imm) which was not detected with the pre-immune serum

(Pre) . These results also confirmed that the inventors did not have a full length cDNA clone as the protein product from the pAB172 cDNA was considerably smaller than 66 kDa.

Production of monoclonal antibody JC9

Specific monoclonal antibodies were produced from mice immunised with the recombinant protein produced from plasmid pAB172. One antibody, JC9, was further characterised as a potential diagnostic reagent.

In Western blotting the JC9 antibody detected the recombinant protein expressed from plasmid pAB172 (Figure 3B) . Cytoplasmic (T-C) and nuclear (T-N) protein extracts were prepared from tonsil. Two fractionation methods were used. 1) Pierce' s NE-PER Nuclear and Cytoplasmic extraction kit was used according to the manufacturer's instructions. 2) Tonsil cells were washed twice in PBS and counted. 1 xlO⁷ cells were lysed in lysis buffer (lOmM Tri HC1 pH 7.4, 1% (w/v) NP40, 150mM NaCl, ImM EDTA + protease inhibitors) for 30 min on ice. The nuclei were then pelleted (δOOOrpm for 3 mins in microfuge) . The supernatant (cytoplasmic extract) was transferred to a clean tube and stored at —70°C. The nuclei were then washed twice with lysis buffer and then repelleted as before. The pelleted nuclei were then resuspended in lOOμl of lysis buffer and 425mM NaCl and left on ice for 20 minutes with occasional agitation. The nuclear extract was then microfuged at 14,000 rpm for 20 minutes and then the supernatant (nuclear extract) was stored at —70°C. Western blotting with antibody JC9 showed that this reagent detected a cytoplasmic protein of molecular weight approximately 66 kDa. This is the same as the molecular weight obtained with the polyclonal antibodies confirming that these reagents recognise the same protein. Western blotting of extracts from DLBCL cell lines detected high levels of protein expression in the Mieu cell line while only small amounts were detected in the HLY-1 line. Small amounts of protein were also detected in the Sudhl-1 (DHL1) cell line an anaplastic large cell lymphoma line that harbours the 2; 5 translocation. Additional higher and lower molecular weight bands were seen in the HLY-1 and DHLl cell lines. These may reflect modifications of the protein or expression of the variant forms in these cell lines.

Sequence analysis of the cDNA in pABl72

The complete sequencing of the pAB172 cDNA was carried out the data for which is illustrated in figure 4.

Database searching using BLAST at NCBI has indicated that there were four cDNAs submitted to the non- redundant database which encode this cDNA. They have the following accession numbers human sequences AL117444.1 & BC002985 and mouse sequences AB041586.1 & AK009859.1.

The protein sequence encoded by AL117444.1 is shown in Figure 41.

These cDNAs are grouped in UniGene Cluster Hs.7970 which describes the DKFZP434B027 protein. There are no scientific papers describing this molecule and all cDNAs are described as unpublished and of unknown function. The gene has been mapped to chromosome 16 and using the map feature indicates that this gene maps to band pl3.3. Searching the TIGR Human Gene Index identified a theroretical clone THC537675 that corresponds to the JC9 antigen. This sequence did not provide any additional protein coding sequence to that provided by the AL117444 cDNA. The gene encoding the JC9 antigen (DKF2P434B027) has subsequently been named FBX031 by the Hugo Gene Nomenclature Committee.

Search for EST sequences to extend the FBX031 coding sequence

The 5' sequence from the AL117444 cDNA clone was used to BLAST search the EST database. This approach enabled us to identify additional EST sequences which extended the FBX031 cDNA sequence. Blast searches with the 5' of these cDNAs were performed to confirm that all the available cDNA sequence had been obtained.

Several ESTs including those with the following accession numbers, BF311960, AL561192 and AL528102 contained upstream stop codons. The amino acid sequences translated from the cDNAs with accession numbers AL561192 and AL528102 were exactly the same suggesting that the coding sequence started with the sequence MYLPPHDPH.

The 5' sequence from AL528102 is shown in Figure 43.

This is consistent with the sequence of a mouse homologue of the DKFZP434B027 protein with the accession number AB041586 which claims to be a full length cDNA sequence and starts with the sequence MYLPPHDPH. The sequence of the short form of the FBX031 protein is illustrated in figure 5.

In contrast human cDNAs submitted under accession numbers BG180443 and BE903394 did not have any upstream stop codons and therefore contained additional protein coding sequence. Comparison of the proteins encoded by cDNAs submitted under accession numbers BG180443, BE903394, AL528102, BF311960 and AL561192 suggests that the indicated that the differences in predicted reading frames may be the result of small sequencing errors as these sequences are almost identical.

The extra protein coding sequence predicted from translation of the BE903394 cDNA seems to be the consensus sequence. BG180443 has no upstream stop codons but a single C at position 108-109 in the BE903394 cDNA where there are two CCs (two CCs are also present in AL528102, BF311960 & AL561192) changes the very N terminal protein coding sequence of this clone. The stops in cDNAs AL528102 and AL561192 which as mentioned above might suggest the existence of a short form of the FBX031 protein are more likely to be a result of problems sequencing nucleotides 427 and 428 of the BE903394 cDNA sequence. This sequence should read GAAGCTG but AL528102 has GAASTG and AL561192 has GAACTG respectively. This does not conclusively rule out the use of the internal methionine to generate a short form of the protein. The stops in BF311960 are the result of missing Ts which are present in the other four sequences. Significantly the first methionine codon in the long form of the FBX031 protein has a considerably better Kozak consensus sequence than the internal methionine potentially giving the short form of the protein (Kozak, 1987a; Kozak, 1987b) . The first methionine has purines at both positions —3 and +4 while the internal methionine which may start the short form of the protein has pyrimidine residues at both positions. In summary these data suggest that the long form of the FBX031 protein is more likely to be translated in vivo.

The 5' sequence encoded by BE903394 when added to the sequence from clone AL117444 gives the cDNA and protein sequence of the FBX031 long form illustrated in figure 6. The predicted molecular weight for this protein of 61 kDa is consistent with the results of our Western blotting experiments with the FBX031 antibody (66 kDa) indicating that this is likely to represent the full length coding sequence.

Sequence analysis of the long form of the FBX031 protein

Sequence analysis using the PSORT program found a single potential transmembrane domain, aa 164-180, with predicted membrane topology type lb with the C- terminus being inside. The PSORT manual reports that type lb membrane proteins are generally localised to the endoplasmic reticulum (ER) . This program predicted that the FBX031 protein would have a cytoplasmic localisation. Sequence analysis of the FBX031 protein using MotifFinder found no motifs in the prosite pattern, no motifs in the prosite profile and no motifs in ProDom.

Prints identified the presence of Rhodopsin —like GPCR superfamily signatures at positions, 48-69, 74-98, 79- 103, 145-166, 150-174, 158-179 and 162-183. G protein- coupled receptors (GPCR) are seven transmembrane proteins that have diverse roles in signal transduction (Ferguson, 2001). The presence of only one potential transmembrane domain in the FBX031 protein makes it unlikely that these findings are significant unless the discovery of additional coding sequence identifies the presence of additional transmembrane domains.

Pfam identified the presence of an F-box at position 68-117 (Figure 7) . The F-box is a protein motif generally found in the amino-terminal part of the protein comprising approximately 50 amino acids that functions as a site of protein-protein interaction (reviewed in (Kipreos & Pagano, 2000) ) . The alignment of this domain to that in the Pfam database is illustrated in figure 7. There are very few invariant positions in the F-box the least variable are position 8 ( 92% L/M), 9 (92% P) , 16 (86% I/V) , 20 (81% L/M) and 32 (92% S/C) (Kipreos & Pagano, 2000), all of these positions are conserved within the FBX031 F-box motif.

F-box proteins contain a wide range of secondary motifs including zinc fingers, cyclin domains, leucine zippers, ring fingers, tetratricopeptide (TPR) repeats, proline rich regions, helix-loop-helix domains and Src homology domains (SH2) (Cenciarelli, et al., 1999; Kipreos & Pagano, 2000). The C-terminus of the FBX031 protein contains a proline rich region (26% P) between aa 402-432 and has helix turn helix structures including this region.

The F-box proteins found to function in SCF complexes have so far been those with WD repeats (FBW) or leucine rich repeats, LRRs, (FBI) in their C-termini (Kipreos & Pagano, 2000) . However, all three classes of F-box proteins including the FBX family have been shown to associate with a ubiquitin ligase activity in vivo (Cenciarelli, et al., 1999) and to bind Skpl in vitro and in vivo (Winston, et al., 1999a).

Sequence analysis using patmatmotifs identified the position of an Asn-glycosylation site at aa 235-239. PKC phosphorylation sites at aa 135-137, 232-234, 290- 292, 458-460, 467-469, 520-522. CK2 phosphorylation sites at aa 38-41, 41-45, 71-74, 135-138, 262-265, 283-286, 299-302, 424-427, 459-462, 484-487. N- myristoylation sites were predicted at aa 5-10, 14-19, 54-59, 56+-61, 57-62, 121-126, 162-167, 308-313, 312- 317, 333-338, 389-394, 429-434, 476-481. A single C- terminal amidation site was predicted between aa 327- 330.

The present inventors have also identified a potential cyclin dependent kinase (cdk) phosphorylation site at aa 485-488 within the FBX031 protein. These consist of a serine/threonine-proline (S/T-P) phosphoacceptor site and a preference for a basic residue at position +3 (where S/T is position 0, (Zhang, et al., 1994) (Srinivasan, et al., 1995)). The F-box protein Skp2 has been shown to be phosphorylated on serine 76 by the cyclinA-cdk2 complex (Yam, et al . , 1999). Physical association may also play a role in establishing substrate specificity and cyclin-cdk2 complexes bind stably to a number of cell cycle regulatory proteins. The ZRXL motif (where Z and X are typically basic) has been identified as the cyclin-cdk2 binding motif in a number of these proteins including E2F1, pl07 and p21

(Adams, et al., 1996; Schulman, et al., 1998; Zhu, et al., 1995). In both p45/Skp2 and pRB the sequence KXL is used instead of RXL (Lisztwan, et al., 1998)

(Adams, et al., 1999). A potential RXL motif at aa 104-106 and a potential KXL motif at aa 142-144 have been identified within the FBX031 protein. These motifs in combination with the F-box motif raise the possibility that the FBX031 protein may have a role in regulating the ubiquitin-mediated degradation of cell cycle associated proteins.

The PESTfind (Rechsteiner & Rogers, 1996) analysis results identified a very high scoring potential PEST sequence between amino acids 28 and 48. These regions are rich in proline, glutamate, serine and threonine residues. They occur with high frequency in short lived proteins and are thought to target proteins for rapid degradation via the proteasome, although the exact mechanism by which this occurs is unclear (Rechsteiner & Rogers, 1996) .

An annotated figure showing the position of predicted domains in the long form of the FBX031 protein is provided (Figure 8) .

Expression of the FBX031 mRNA in human tissues and tumours

The 5' 2.1 kb EcoRl fragment from plasmid pAB172 was labelled with ³²P using the High Prime Labelling kit according to the manufacturer's instructions (Roche Diagnostics) . The labelled cDNA probe was then hybridised to Clontech' s human multiple tissue expression (MTE) array and to Clontech' s Matched tumor /normal (MTN) expression array according to the manufacturer's instructions. These arrays comprise normalised amounts of cDNAs (normalised using several house keeping genes) prepared from a range of tissues. Thus differential hybridisation of a cDNA probe represents differential gene expression.

The arrays were exposed to photographic film and the results are illustrated in figure 9 together with a legend illustrated in figure 10 (additional data can be obtained from the Clontech web site) . On both arrays there was strong hybridisation to E. coli DNA [A) 12D; B) 24G) ] and to genomic DNA [A) 12G, 12H; B) 24M] . The former is probably the result of hybridisation to an E . coli homologue and the latter occurs if the gene is highly abundant or belongs to a multi-gene family. However, all of the controls which should remain negative did so with this probe.

Hybridisation of the FBX031 cDNA to Clontech' s MTE array (Figure 9A) indicated that the FBX031 mRNA was widely expressed in normal human tissues, both adult and foetal. The FBX031 mRNA was particularly strongly expressed in the cerebellum and was expressed at relatively low levels in transformed cell lines.

Interestingly another F box protein, NFB42, has been found to be strongly expressed in neurons (Erhardt, et al., 1998).

Hybridisation of the FBX031 cDNA to Clontech' s MTN array (Figure 9B) indicated that there was a difference in the signals observed in the normal tissues (rows A, D, G, J, M) when compared to tumour tissue taken from the same patient (rows B, E, H, K, N) . Generally there is less FBX031 mRNA expression in the tumours when compared to the normal tissues with the exception of two rectal tumours where the reverse appears to be the case (N13, 14). These data suggest that FBX031 is a putative tumour suppressor gene whose mRNA expression is reduced in solid tumours. The observation that there is often relatively little FBX031 mRNA in the normal control tissues (while expression is observed in the corresponding tissue on the MTE array) raises the possibility that this loss of expression may be an early event occurring during neoplastic progression and that phenotypically normal tissues may already have altered FBX031 mRNA expression. One limitation of these arrays is that they do not enable closely related mRNAs to be distinguished. The splicing of the FBX031 mRNA is complex thus there may be additional FBX031 mRNAs present and that are not bound by the 3' FBX031 cDNA fragment used to probe the array.

Chromosomal aberrations at chromosome 16pl3

The FBX031 gene has been localised to chromosome 16pl3.3. The Mitelman Database of Chromosome Aberrations in Cancer (2001) . Mitelman F, Johansson B and Mertens F (Eds.), http: //cαap.nci . nih. ov/Chromosomes/Mitelman was searched for chromosome 16pl3 through NCBI. There are a large number of chromosome aberrations mapping to chromosome 16pl3 with changes in adenocarcinomas, leukaemias (CLL, ALL, AML) and diffuse large B-cell lymphomas being particularly evident.

The PubMed scientific publication database was searched through NCBI for references to chromosome 16pl3. LOH at 16pl3 has been detected in a number of tumour types including, papillary (Lininger, et al., 1998) and apocrine (Lininger, et al., 1999) breast cancers, hepatocellular carcinomas (Sakai, et al., 1992), pancreatic endocrine (Chung, et al., 1998) and acinar carcinomas (Taruscio, et al., 2000), primary ependymomas (Zheng, et al., 2000) and anaplastic thyroid carcinoma (Kadota, et al., 2000) suggesting that a tumour suppressor gene does map to this location. Rearrangements at 16pl3 have been detected in colorectal adenocarcinomas (Bardi, et al., 1995). A chromosome abnormality involving 16pl3 and 16q22 has also been reported to define a good prognosis subset of myeloid leukemia despite morphological variations (Campbell, et al., 1991).

The novel genetic disorder microhydranencephaly maps to chromosome 16pl3.3-12.1 (Kavaslar, et al., 2000). Phenotypic features were severe mental and motor retardation, as well as very small body size and very small occipital-frontal circumference (Kavaslar, et al., 2000). Skp2-/- mice which do not express the F- box protein Skp2 were markedly smaller and it has been proposed that a reduced rate of cell growth may be the cause of the small body size of these mice (Nakayama, et al., 2000). This combined with the high degree of FBX031 expression in the cerebellum (see below) makes FBX031 a candidate gene for this disorder. A possible link between manic depressive illness and a locus on 16pl3 has also been reported (Ewald, et al., 1995).

Tuberous sclerosis complex (TSC) is an autosomal dominant multi-system disorder with two known disease loci on chromosomes 9q34 (TSC1) and 16pl3.3 (TSC2) (Gilbert, et al., 1998) and the PKD1 gene mutations in which cause polycystic kidney disease also maps to this locus (reviewed in (Sessa, et al., 1997)).

Immunostaining with the FBX031 monoclonal antibody JC9

Tissues obtained from the Histopathology Department at the John Radcliffe Hospital were snap frozen in liquid nitrogen and stored at -70°C. Human cell lines were obtained from either the Sir William Dunn School of Pathology, Oxford or the American Type Culture Collection (ATCC, Rockville, MD) . Cells were cultured in RPMI 1640 medium containing 10% fetal calf serum (GIBCO Biocult Ltd) at 37°C in 5% C0₂. Cryostat tissue sections (5-8 μm) on glass multiwell slides were dried overnight at room temperature, fixed in acetone for 10 minutes at room temperature and then stored, wrapped in aluminium foil, at -20°C before use. Formalin fixed, paraffin wax embedded sections were cut at approximately 5 μm and collected on superfrost plus glass slides (Snowcoat X-tra, Surgipath, U.S.A.). Cytocentrifuge preparations of cell lines were prepared as previously described (Erber, et al., 1984) and then stored wrapped in aluminium foil, at -20°C before use.

Immunostaining of tissue sections

Peroxidase Immunostaining. Cryostat sections were allowed to reach room temperature before being unwrapped and incubated for 30 minutes with the JC9 antibody. Slides were washed for 5 minutes in PBS before incubation for 30 minutes with 1/100 dilution of goat anti-mouse HRP conjugate (DAKO) . After a 5 minute wash in PBS sections were incubated for 8 minutes with DAB+ substrate (DAKO) before being counterstained with haematoxylin and then mounted in Aquamount (BDH) . Paraffin embedded tissues were dewaxed before antigen retrieval by pressure cooking in the microwave for 3 minutes in 50mM Tris, 2mM EDTA, pH9. Tissues were then immunostained using Dako' s EnvisionPlus kit (mouse) according to the manufacturer' s instructions and mounted as described above .

APAAP staining. Tissue sections were dewaxed and subjected to the same antigen retrieval procedure as described above. Tissues were then immunostained with the JC9 antibody following the modified APAAP staining protocol described in (Falini, et al . , 1998) and mounted as described above.

Normal tissue staining using the JC9 monoclonal antibody

Cryostat sections were immunostained as described above. Results of these studies are illustrated in figure 11. The FBX031 protein is widely expressed in normal human tissues and we have observed a predominantly cytoplasmic localisation of this protein. There are however considerable variations in the subcellular staining patterns observed with this antibody. For example the nuclear staining of the distal tubules in kidney (H) , cells at the base of crypts in gut (I) and some tonsil epithelium (B) ; the nuclear dots seen in a number of tissues including spleen [C) shown at high power in inset] and thymus, and the perinuclear staining seen in other tissues including the thymus cortex (D) and granular layer of the cerebellum. The strong JC9 staining of duct-lining cells in the breast (F) is particularly striking. In cerebellum, which expressed high levels of FBX031 mRNA, we also see high levels of FBX031 protein expression. Particularly in the granular layer where it is largely perinuclear, in the cytoplasm of purkinjie cells and in the nuclei of neurones. The weak staining of heart may possibly be the result of using an old tissue section and this result will need to be confirmed. F-box proteins have been found to be distributed in both the cytoplasm and in the nucleus. For example, yeast F-box proteins Cdc4 and Met30 are nuclear (Rouillon, et al., 2000) while Grrl is nuclear and cytoplasmic (Blondel, et al., 2000). The human proteins Fbwla/b-Trcp and Fbx4 are cytoplasmic and nuclear while Fbw2 was detected mainly in the cytoplasm and Fbl3a mainly in the nucleus (Cenciarelli, et al., 1999). Fbl2 was localised primarily in the cytoplasm concentrated around the nucleus in punctate foci (Ilyin, et al., 1999). Compartmentalized degradation means that specific protein degradation can be regulated by the proteins subcellular localisation.

Routinely fixed paraffin embedded normal tissues were stained using the APAAP technique (Figure 12). The only significant difference observed between frozen and routinely fixed material is that the subcellular localisation of the FBX031 protein in paraffin embedded tissues shows less of the nuclear, perinuclear and nuclear dot staining seen in frozen tissue. This phenomenon is known to occur with other antibodies and may be caused by fixation artifacts and partly by the difficulty of seeing nuclear staining with APAAP. In general the same cell types in different tissues are immunostained in either frozen or routinely fixed tissue. In tonsil a subset of large germinal center B-cells particularly those next to the mantle zone are strongly cytoplasmically stained as are the basal layer of the epithelium. Plasma cells are particularly strongly stained by the FBX031 antibody. In frozen tonsil we have observed both predominantly nuclear or cytoplasmic labelling of basal epithelium while in routinely fixed material the labelling is cytoplasmic. The expression of the FBX031 protein in cells which are actively proliferating such as the germinal center and basal epithelium suggests that the level of FBX031 protein expression may be related to cell proliferation. In addition double immunofluorescent labelling of tonsil with the JC9 antibody and CD30 antibody have demonstrated that the large blast cells stained predominantly in the germinal centre (but also in the interfollicular areas) coexpress both these proteins. CD30 is an activation marker which is particularly strongly expressed by Hodgkins lymphomas and anaplastic large cell lymphomas. Thus, the expression of the FBX031 protein may be related to the cells activation status. The FBX031 protein is also strongly expressed in the majority of other tissues as illustrated (Figure 12) . We have observed strong

FBX031 expression in reproductive tissues including ovary, testis, prostate and placenta which is consistent with the mRNA expression in these tissues. The bronchiolar epithelium in lung is strongly stained (L) while two lung tumours shown as insets (M & N) express much lower amounts of the FBX031 protein.

Immunostaining of solid tumours with the JC9 antibody

Solid tumours were immunostained using DAKO' s Envision peroxidase system as described.

Figure 13 shows the results from renal tumours stained with the JC9 antibody. The top row, (i) , shows the corresponding normal tissue from the same patient while the lower row, (ii), shows the tumour tissue. In normal kidney the FBX031 protein is expressed more strongly in the tubules than in the glomeruli and the distal tubules are frequently more strongly labelled than the proximal. The two renal clear cell tumours (Aii, VHL mutant & Bii, VHL disease) show decreased expression of the FBX031 protein. A papillary RCC (Cii) and chromophobe tumour (Dii) both show high levels of cytoplasmic FBX031 expression. Another chromophobe tumour case shows loss of FBX031 expression (E) indicating that these tumours show variable FBX031 expression. These results are consistent with those obtained using the MTN array which indicated that the majority of the renal tumours on the array had decreased expression of the FBX031 mRNA. However, the adjacent normal tissue does appear to express normal levels of the FBX031 protein despite the apparent reduction in mRNA levels on the array. These discrepancies may reflect the complex splicing of the FBX031 mRNA as some variants may not be measured by the different techniques.

The JC9 immunostaining of six cases of small cell lung cancer (SCLC) indicated that all of these expressed some FBX031 protein (Figure 14). However the Envision staining system is more sensitive than APAAP staining with JC9 indicating that there was likely to be generally less expression than was observed in normal bronchiolar epithelium. The MTN array data indicated that there was also a reduction in FBX031 mRNA in lung tumours. The SCLC case illustrated in low power (Ci) shows that the FBX031 expression is stronger towards the top of the picture which corresponds to the outer edge of the tumour. This may reflect either better fixation of the outer tissue or that the center of the tumour mass may be hypoxic and that either of these two phenomenon may affect FBX031 expression. Tumour hypoxia is known to be an adverse prognostic indicator in cancer as it modulates both tumour progression and treatment (Dachs & Tozer, 2000; Semenza, 2000) . The case illustrated in D) shows that the apoptotic cells illustrated to the left of the tumour do not express the FBX031 protein. Normal pancreas strongly expresses the FBX031 protein particularly in the exocrine acini while less expression is seen in the endocrine islets and the ducts. The majority of pancreatic adenocarcinomas arise from the exocrine cells and both of the pancreatic tumours illustrated (Figure 15 Aii & Bii) show considerable loss of FBX031 expression compared to adjacent normal tissue (Ai & Bi) . The breast tumours illustrated (Figure 15 C-F) show expression of the FBX031 protein while the MTN array data suggested that both adjacent normal and tumours had only very low levels of FBX031 mRNA. Discrepancies between mRNA and protein levels can be caused by a number of factors particularly mRNA and protein turnover rates and translational control mechanisms. One study has suggested that a defect in the SCF complex may occur in 15-20% of breast cancers and that the resulting coordinated elevation of cyclins Dl and D3 overcomes the inhibition of cell cycle progression by p21 (Russell, et al., 1999).

Immunostaining of haematopoietic malignancies using the JC9 monoclonal antibody

Anaplastic large cell lymphoma (ALCL) accounts for 5- 10% of adult non-Hodgkin' s lymphomas and 30-40% of paediatric large cell lymphomas. These tumours exhibit a wide variety of different immunophenotypes, morphological and clinical subforms (Harris, et al., 1994; Kadin, 1994). Approximately 53-89% of ALCL are associated with the (2; 5) (p23;q35) translocation which results in the expression, by the neoplastic cells, of the tumour-associated fusion protein nucleophosmin- anaplastic lymphoma kinase (NPM-ALK) consisting of the N-terminus of nucleophosmin and the entire intracytoplasmic region (containing the kinase domain) of the receptor tyrosine kinase ALK (Morris, et al., 1994; Shiota, et al., 1994). The production and the widespread use of a monoclonal antibody specific for ALK by the inventors (Pulford, et al., 1997) has led to the identification of the tumour entity ALK- positive lymphoma or ^λAlkoma' (Benharroch, et al., 1998) . Retrospective studies have shown that ALK- positive lymphoma has an improved prognostic outlook when compared with ALK-negative ALCL with 71-80 % of patients with ALK+ lymphoma achieving complete remission compared to only 15-35% of patients with ALK-negative tumours (Falini, et al., 1999; Gascoyne, et al., 1999; Shiota, et al., 1995)

14 cases of ALCL either T or null cell type were immunostained with the JC9 antibody. ALK positive cases:- 5 showed weak staining (Figure 16, A, B, C) and 4 showed moderate staining (D, E) . In E) the small strongly stained cells are plasma cells while the large tumour cells are diffusely stained. ALK negative cases:- one showed no cytoplasmic staining in the tumour cells (while nuclear staining was observed in the surrounding lymphocytes, J) , one weak staining (F) one moderate staining (G) and two strong staining (H, I) . The two strongly stained ALK negative cases expressed considerably more of the FBX031 protein than the ALK positive cases. The antigen recognised by antibody JC9 therefore appears to differentiate between cases of ALK-positive and ALK-negative ALCL. The distribution of the FBX031 protein was also different, showing a localised granular/blobby staining pattern in the ALK negative cases and a more diffuse pattern in the ALK positive cases. The differential expression of the FBX031 protein may, therefore, be of clinical relevance in ALCL providing evidence of prognostic outcome in these tumours. In addition, FBX031 expression in ALK-positive lymphomas may be of use in identifying the subgroup of these patients who do not respond to conventional treatment. For example, those with strong JC9 staining.

B-cell chronic lymphocytic leukaemia (B-CLL) , characterised by the clonal expansion of CD5 B cells, is the most common adult leukaemia in the Western world. The disease course is heterogeneous and survival times range from several months to in excess of twenty years.

Currently clinical staging remains the strongest predictor of survival (Zweibel & Cheson, 1998). Genetic alterations involving chromosome bands 17pl3 (lacking p53) , llq22.3-llq23.1 and trisomy 12 have been correlated with rapid disease progression, shorter survival times and resistance to therapy while patients with deletions in band 13ql4 had a more favourable outcome (Dόhner, et al., 1995; Dohner, et al., 1999; Dόhner, et al., 1997; Matutes, et al., 1996) . Immunophenotyping for markers such as CD38 expression (Damle, et al., 2000; Hamblin, et al., 2000) and other potential prognostic markers whose expression have been related to poor prognosis are also being evaluated. There remain, however, no definitive prognostic markers in routine use at present .

The JC9 immunostaining of CLL cases indicated that some cases weakly expressed the FBX031 protein while others strongly expressed this protein (Figure 17, A & B) . CLL can transform into a DLBCL in Richter's syndrome and immunostaining of a Richter's DLBCL case showed strong expression of the FBX031 protein (C) . The differential FBX031 expression levels may help to define subtypes of CLL with different survival rates. Diffuse large B-cell lymphoma accounts for 30-40% of all adult non-Hodgkin' s lymphomas and is heterogeneous in terms of it ' s morphology and clinical features (Harris, et al., 1994) with approximately 50% of patients relapsing after treatment (Project., 1997). The genetic abnormalities underlying DLBCL remain poorly understood. An important related question is whether more than one subtype exists.

Initial studies linked the BCL-6 gene to DLBCL, with translocations involving the BCL-6 gene occurring in approximately 25-40% of cases (Dalla-Favera, et al., 1994; Offit, et al., 1995) while point mutations in the 5 ' non-coding region of this gene were identified in the majority of the remaining cases (Migliazza, et al., 1995). However, since these point mutations are also found in normal tonsillar germinal centre B cells (Pasqualucci, et al., 1998), the role of the BCL-6 gene in DLBCL remains uncertain. Thus, in contrast to a number of other lymphoma types (e.g. follicular or Burkitt' s lymphoma), no characteristic genetic alteration has been found in DLBCL.

There have been studies suggesting that DLBCL can be divided into subtypes. The Kiel classification scheme, using morphological criteria, proposed that some DLBCL arise from germinal centre B cells and others from extracellular B cells ("centroblastic" and "immunoblastic" lymphomas respectively (Lennert, et al., 1975). There is, however, controversy as to whether subtypes differ in term of their clinical behaviour (Baars, et al., 1999; Kwak, et al . , 1991; Stein & Dallenbach, 1992) . The absence of objective criteria for these categories, (for example, immunophenotypic details) combined with the lack of reproducibility in their diagnosis means that few centres attempt to subdivide DLBCL morphologically and no such distinction is described in the REAL classification (Harris, et al., 1994).

Recent genetic studies have provided evidence for the possibility of at least two sub-categories of DLBCL. The presence of the (14; 18) translocation in a minority of cases suggests that some DLBCL represent transformed/diffuse follicular lymphomas (possibly associated with the acquisition of MYC and p53 abnormalities), in contrast to the commoner "de novo" cases (in which the BCL-6 gene may be (Dalla-Favera, et al., 1994)). The lack of cytogenetic and/or genetic data in most centres, however, means that recognition of these subtypes and their clinical relevance remains difficult in routine practice. A recent study using microarray-based techniques to study DLBCL gene expression claims to have identified two subgroups of DLBCL, those with a germinal centre-like profile having a better prognosis than those with an activated B-like profile (Alizadeh, et al., 2000). However, this approach again is not yet practical in the routine clinical context.

The expression of the FBX031 protein was investigated in 22 cases of DLBCL. Cases with both weak (D) and strong (E & F) FBX031 expression were observed (Figure 17). The differential expression of the FBX031 protein may define clinically relevant subtypes of DLBCL, as the expression of this protein correlated with the immunophenotyping of these cases as of either germinal centre or post-germinal centre origin. Interestingly follicular lymphomas (FL) , which are also derived from germinal center B-cells, are more indolent than DLBCls and can in fact transform into DLBCL. We have immunostained cases of FL and* have observed weak to moderate FBX031 expression in these tumours (Figure 17, G & H) . Immunostaining DLBCL cell lines that were gene expression profiled as belonging to either germinal centre (SUDHL-6) or activated (OCI-LylO & OCI-Ly3) classes (Alizadeh et al., 2000) with the JC9 antibody showed that all three cell lines expressed the FBX031 protein at comparable levels. An independent study using gene-expression profiling and supervised machine learning to predict the outcome of DLBCL patients has recently been published (Shipp et al., 2002). This study used 6,817 genes represented on Affymetrix HU6800 oligonucleotide arrays. Ninety of the cell-of-origin signature genes studied by Alizadeh et al. were also represented in this study. While Shipp et al. confirmed the association of the expression of these genes with clinical outcome in the cases from the prior study they did not find a significant correlation of the germinal centre-like and activated-like DLBCL subtypes with survival in their study. The genes that Shipp et al. identified as implicated in DLBCL outcome included some regulate responses to B-cell-receptor signalling, critical serine/threonine phosphorylation pathways and apoptosis. They were able to use 13 genes in a supervised DLBCL outcome predictor, only three of which, NORl, PDE4B and PKC-b, were represented on the original version of the Lymphochip. All three of these genes regulate apoptotic responses to antigen- receptor engagement and, potentially, cytotoxic chemotherapy. Significantly the clinical relevance of the PKC-b protein was confirmed by immunohistochemistry. While both gene-expression profiling studies were able to identify clinically relevant subtypes of DLBCL further studies are required to resolve the differences in their findings. Classical Hodkin's disease comprises three main categories, nodular sclerosing (ns) , mixed cellularity (mc) and lymphocyte depletion. These three types share features which distinguish them from lymphocyte predominance (lp) Hodgkin' s disease (Harris, et al., 1994). The tumour comprises scattered bi-nucleate or multi-nucleate Reed-Sternberg cells and mono-nuclear Hodgkin' s cells. These cells are often difficult to visualise and markers such as CD30 are often used to locate these tumour cells. Both classical and lymphocyte predominance Hodgkin' s cases were immunostained with the FBX031 antibody (Figure 18 A- D) . The FBX031 antigen is more strongly expressed by the tumour cells than by surrounding lymphocytes and this enables the tumour cells to be identified (shown using arrows) . FBX031 may therefore be an additional marker that can be used to immunostain neoplastic Hodgkin' s lymphoma cells. The inset in A) shows that in this additional case of HD ns much of the FBX031 protein is localised around the outside of the cell, towards the membrane.

Marginal zone (MALT) lymphomas are thought to represent the neoplastic equivalent of the marginal zone cells found in spleen and lymph nodes. FBX031 immunostaining of MALT lymphomas (Figure 18, E & F) indicated that these expressed lower levels of the FBX031 protein than many of the large cell lymphoma cases. The presence of strongly stained plasma cells confirms that there were no technical problems with the immunostaining. However, because the APAAP technique is weaker than the Envision kit it may be that these lymphomas do express low levels of the FBX031 protein.

Preliminary staining studies have identified weak staining in 2/3 mantle cell lymphomas and 2/3 hairy cell leukaemias (one case of each possibly negative) . Two NHL cases weakly expressed the FBX031 protein. One Burkitt' s lymphoma weakly expressed the FBX031 protein. Myeloma, three cases were weakly positive while one was very strongly positive.

Interestingly expression of the F-box protein Skp2 has been shown to correlate directly with the grade of malignancy, being expressed in two thirds of DLBCls (Latres, et al., 2001). Overexpression of Skp2 and activated N-ras induced T-cell lymphomas with shorter latency, higher penetrance and decreased survival times in a mouse model (Latres, et al., 2001). Therefore the FBX031 overexpression in lymphomas may also have clinical significance.

Double immunofluorescent labelling of haematopoietic neoplasms

Double labelling techniques enable the labelling of tissues with two different antibodies which are distinguished using individual fluorochromes . This technique frequently provides additional data to that obtained using colourimetric immunostaining particularly information on subcellular localisation or protein co-localisation.

Paraffin embedded tissues were dewaxed and antigens were retrieved using Tris/EDTA as described previously for APAAP and immunoperoxidase labelling. Tissue sections were incubated with both primary antibodies for approximately one hour. After washing for 2 minutes in PBS sections were then incubated with mix containing a 1/25 dilution of FITC conjugated anti-IgM (which recognises JC9) and 1/50 dilution of Texas-Red conjugated anti-IgGl (CD30 or VS38c) or anti-IgG2a (L26) for approximately one hour protected from light) . After a 2 minute wash in PBS slides were mounted in DAKO's fluorescent mounting medium containing DAPI. Results are illustrated in figure 19.

Figure 19, panel A B and C illustrate a case of classical Hodgkin 's disease showing double labelling of tumour cells with CD30 in red and JC9 in green (A-arrow) . Note that CD30 is present on the cell membrane while FBX031 is present in the cytoplasm and sometimes in the nucleus of these cells. The Golgi apparatus is not labelled by JC9 (B-arrow) but is strongly labelled by CD30 (C-arrow) . Panel D and E illustrate a case of ALK negative ALCL double labelled with CD30 in red and JC9 in green (D) . The distribution of the antigens in the tumour cells is similar to that seen in A. Nuclear labelling can clearly be seen in the tumour cells in the high power view (E-arrows) . Panel F and G show a case of ALK positive ALCL double labelled with CD30 in red and JC9 in green. In the low power view (F) some normal cells show strong perinuclear labelling (arrows) . In the high power view the JC9 labelling is clearly much weaker than that seen in the ALK negative case shown in panel E, although the CD30 labelling is of comparable intensity. Panel H illustrates normal tonsil labelled with CD20 (B-cell marker) in red and JC9 in green. The germinal centre B cells are double labelled although the JC9 labelling is weak except on a few larger cells towards the edge of the germinal centre (arrows) where stronger labelling can be seen. Panel I shows normal plasma cells in tonsil labelled with VS38c (a plasma cell marker that recognises the rough ER p63 protein) in red and FBX031 in green. These are strongly double labelled (yellow) reflecting the high FBX031 protein levels detected in this cell type. The expression of CD30 and FBX031 has also been observed in normal tonsil cells.

Genomic DNA encoding FBX031

Blast searching the FBX031 sequence through NCBI for recent submissions to Genbank identified Accession AC010531, Homo sapiens chromosome 16 clone RP11-178L8 as a genomic clone containing FBX031 and the adjacent 5' and 3' sequences from putative upstream and downstream genes that fuse to FBX031 as alternative exons . Blast searching the human genome through NCBI aligns FBX031 and adjacent sequences to Homo sapiens chromosome 16 working draft sequence segments (accession NT_019609.3) . The coordinates for the FBX031 long alignments are illustrated below. Where there is overlap between coordinates this represents sequence homology at either end of the junction and this will be resolved by predicting the most likely intron-exon boundary sequences. Figure 37 illustrates the alignment of the FBX031 long cDNA sequence to the genomic sequence in the more current version accession NT_019609.4.

FBX031 AC101531 NT_019609.3

7-356 143366-143017 347200-346851

356-428 119976-119904 323934-323862

429-507 106859-106781 310817-310739

505-673 103306-103198 307325-307157

672-749 102553-102476 306512-306435 747-858 95866-95755 299825-299714

859-1013 95057-94903 299016-298862

1012-1413 93887-93486 297846-297445

1413-3586 91111-88938 295070-292895

Analysis of the genomic DNA upstream of the FBX031 long sequence Translation of the genomic DNA upstream of the FBX031 long cDNA sequence identified multiple upstream stop codons and no additional in frame methionine codons indicating that the FBX031 long cDNA sequence encoded the full length FBX031 long protein.

Prediction of a promoter region for the FBX031 long form

To identify potential promoter regions for the FBX031 gene the reversed and complemented sequence (FBX031 coding strand) comprising nt 15000-143017 (nt 1-6984 in results from these analyses) from the RP11-178L8 genomic clone (AC101531) was analysed using a variety of web based tools. This sequence comprised the first exon from the FBX031 long form and approximately 6kb of upstream genomic sequence.

The programs Promoterlnspector and PromoterScan on

Zeno both identified a promoter 5' to the start of the first exon (nt 6635) in an overlapping region between nucleotides 6393-6908 and 6276-6526 respectively. Computational prediction of eukaryotic polll promoters is still not an exact science and this may explain the difference in the coordinates assigned by each program.

Almost all housekeeping and up to 50% of all genes have CpG islands at the 5' end of the transcript.

These are stretches of DNA with a higher frequency of CpG di-nucleotides than is found in the whole genome. A recent publication from Celera Genomics reports that CpG islands are the most dominant signals in eukaryotic promoters (Hannenhalli and Levy, 2001) . CpG island methylation is correlated with the level of gene expression and a common mechanism for loss of mRNA expression of tumour suppresser genes in human cancer is through promoter methylation of their CpG islands. The program CPGPlot from the EMBL European Bioinformatics Institute detected the presence of two CpG islands in the 6984bp genomic fragment (FBX031 coding strand, nt 15000-143017 from AC101531) between nucleotides 5109-5340 and 5593-6928 with the larger CpG island extending almost to the end of the first exon in FBX031.

Analysis of the 3' 1884 nucleotides from the above sequence for transcription factor binding sites, using Matlnspector, identified a vast array of potential transcription factor binding sites. Multiple sites were found for a number of these including API, 2 & 4, CETS1P54, GC_01, HFH2 & 3, HNF3B, IK2, LM02, MYOD, MZF1, SP1, SRY, TCFII and USF (Figure 22) .

These data all support the existence of a promoter upstream of the FBX031 gene.

Sequencing of EST clone BE903394/IMAGE clone 3958783 containing a full length FBX031 cDNA. (Figure 38)

This potentially full length cDNA was provided by the IMAGE Consortium through the UK HGMP Resource Centre. The cDNA insert was fully sequenced. The original FBX031 cDNA sequence was predicted from overlapping cDNA clones therefore this sequence represents the first sequence from a single full length cDNA clone. The predicted polypeptide from this sequence is identical to the FBX031 long form of the protein. However there are two single base changes in the cDNA sequence outside the coding region. The first change occurs at nt 7 of the FBX031 long sequence where a C is inserted into the sequence. This matches the genomic sequence and that in IMAGE clone 3542631 and therefore is likely to be the correct sequence. The second is nt 1880 of the FBX031 sequence which is changed from an A to a G. This is not present in the sequence from IMAGE clone 3542631 and may be a nucleotide polymorphism or a sequencing error and this will be further investigated.

Sequencing of EST clone BE269540/IMAGE clone 3542631 containing a full length FBX031 cDNA with an N- terminal internal deletion. (Figure 22)

This potentially full length cDNA was also provided by the IMAGE Consortium through the UK HGMP Resource

Centre and the cDNA insert was fully sequenced (Figure 22) . This cDNA contained an N-terminal deletion corresponding to nucleotides 303-1176 of the FBX031 long cDNA sequence. This does not correspond to the intron-exon boundaries in the FBX031 long sequence with the boundaries of the deletion occurring internally in exon 1 and exon 8. This cDNA was cloned from a Burkitt 's lymphoma and this raises the possibility that this deletion is a cancer related event. In the cDNA sequence there are two single nucleotide mismatches to the original FBX031 long cDNA sequence nt 16 changing from T to C and nucleotide 1250 from C to G. Again these could both be polymorphisms or sequencing errors. This deletion truncates the FBX031 long protein. The first 95 amino acids are the same but then there are 44 amino acids which are not homologous to the FBX031 protein. This disrupts the F-box domain and is likely to inactivate the FBX031 protein.

Current sequences deposited in Genbank for the DKFZP434B027 protein and cDNA

Two additional sequences (presumably predicted from the genomic and/or EST sequences as there is no information on the mRNA cloning) for the DKFZP434B027 (FBX031) mRNA were submitted to Genbank by the NCBI annotation project on 16th July 2001. XM_051337 corresponds to nucleotides 460-3586 of the FBX031 long cDNA and aa 173-539 of the corresponding protein encoding a polypeptide product that lacks the F-box

(equivalent to FBX031 short form Fig 5 (b) ) . XM_051338 corresponds to nucleotides 7-296 and 1171-3586 of the FBX031 long cDNA (having an N-terminal deletion) and encodes aa 465-539 of the predicted protein product.

Partial sequencing of EST clones that potentially encode the full length FBX031 gene

The cDNA clones deposited in Genbank as EST sequences with the following accession numbers BG180443,

BE269540 and BE903394 were obtained as IMAGE clones 4432569, 3542631, and 3958783 from the human genome project as potentially full length cDNA clones. Partial sequencing both ends of these clones to confirm their identify led to the discovery that there were variants of the originally described FBX031 short and long sequences. Analysis of all the EST sequences assigned to FBX031 subsequently identified an astonishing number of different cDNA sequences for this gene.

Sequencing the EST clone BE269540 (from a Burkitt 's lymphoma) identified an N-terminal deletion within the FBX031 long cDNA. Sequencing an EST clone (Accession: BG180443, from a prostate adenocarcinoma library) encoding the N-terminus of FBX031 unexpectedly led to the discovery that the 3' end of this cDNA belonged to a different gene FLJ22477 (Unigene Hs.118944) (Figure 20) . The genomic DNA encoding FLJ22477 is adjacent to what was thought to be the FBX031 gene in Homo sapiens chromosome 16 clone RP11-178L8 (Accession: AC010531) and the mRNA sequence encoding this gene is predicted.

FLJ22477

Of the 21 EST sequences in Unigene Cluster Hs.118944 corresponding to FLJ22477, five sequences (4 cDNAs) represent the predicted full length cDNA (Accession: NM_024735), two of which are from human tonsillar B- cells. The remainder are not sequenced far enough into the cDNA to reach the more N-terminal fusion point (nt 326 of FLJ22477) with FBX031 thus may represent either the wild type FLJ22477 gene or the FBX031-FLJ22477 fusion gene. Interestingly many of these cDNAs are cloned from human tumours suggesting that the 3' end of the mRNA is widely expressed in human malignancies.

Blast searching the human genome with the FLJ22477 mRNA sequence identified the following coordinates for the exons in the NT_019609.3 genomic sequence from Homo sapiens chromosome 16 working draft sequence adjacent to FBX031.

FLJ22477 NT_109609.3 These correspond to the following approx.

coordinates in the AC010531 genomic clone

1-327 281002-280676 77043-76717 325-378 279411-279358 75452-75399 378-504 278683-278557 74724-74598 503-691 269492-269304 65533-65345 691-953 266661-266399 62702-62440

Promoter analysis of the genomic sequence between FBX031 and the end of the first exon in FLJ22477 (nt 76741- 88980 reversed and complemented to give the coding strand sequence nt 1-12240) using PromoterScan on Zeno and Promoterlnspector did not find any potential promoter sequences. However a small CpG island was detected between nt 11607-12031. The proximity of gene starts to CpG islands is well documented and CpG are the most dominant signals associated with promoters (Hannenhalli and Levy, 2001) suggesting that there is a potential promoter region for the FLJ22477 gene around the proposed transcriptional start site.

1. The sequence in EST accesssion AI349351 from fetal lung has an extended 3' sequence to the mRNA corresponding to nucleotides 268304-268046 of the genomic sequence in NM_019609.4. This directly follows on from the polyadenylation site identified in the FLJ22477 sequence which ends at nt 268305 of the NM_019609.4 genomic sequence.

2. The following EST sequences derived from mRNAs expressed in normal tissues BI460660 (testis), BI520057 (medulla) and BI52956 (brain) contain two additional exons within the FLJ22477 protein coding sequence. These will alter the amino acid coding sequence and potentially the function of the FLJ22477 protein and provide additional exons which may fuse to FBX031 (FBX031).

Genomic coordinates (NM_019609.4) for BI52956 for FLJ22477

282907-282581 282633-282586 281316-281263 281316-281263 280588-280462 280588-280462 276789-276641 275826-275790

271398-271210 271398-271210 268567-268305 268567-268754

Analysis of EST sequences for variant FBX031 mRNAs

FBX031 Alternate form 1 (FBX031-1) : fusion to FLJ22477 (Figure 23)

Alignment of EST sequences in Unigene Hs.7970 (FBX031) identified another cDNA clone (Accession: AL528101, from a neuroblastoma cell line) with homology to FBX031 (nt 1026-1413) and FLJ22477 (nt 326-891) spanning the fusion point between the two genes in addition to the EST BG180443 which also shows the same fusion. Nucleotide 1413 in FBX031 corresponds to the end of an exon and the fusion results in a loss of the last exon encoding the FBX031 long form which is then replaced by the FLJ22477 sequence causing a change in the C-terminal protein coding sequence. In this splice variant the majority of the FBX031 C-terminus

(including the proline rich region) is retained with a change in sequence possibly affecting the protein:protein interactions potentially mediated by this region of the protein. A change in the 3' UTR also occurs and this may affect the level of protein expression from this mRNA.

FBX031 Alternate form 2 (FBX031-2) : fusion to FLJ22477 (Figure 24)

Only one FLJ22477 EST sequence (cloned from prostate) is a fusion gene with FBX031 (Accession BF437610) . This 3' sequence corresponds to the 3' end of FLJ22477 from nt 647-poly(A) tail then runs into FBX031 from nt 923 towards the 5' end of FBX031. The partial sequence from FLJ22477 from poly (A) tail to nt 691 is claimed in patent WO0112659 (Sequence 493 Accession: AX086541) as the C-terminus of the hypothetical protein DKFZp434J1815 thus the extra nucleotides between 691- 647 that fuse to FBX031 are not covered by these claims. This replaces the FBX031 C-terminal protein sequence from aa 307 with an in-frame C-terminal protein sequence encoded by the FLJ22477 gene. Unexpectedly this protein sequence is not that predicted for the FLJ22477 gene and is translated in an alternate reading frame to give a completely novel C-terminal protein sequence. This replaces the proline rich region in FBX031, predicted to be potentially important for mediating protein:protein interactions with proteins targeted for degradation, with a different sequence.

FBX031 Alternate forms 3 (FBX031-3) and 4 (FBX031-4) : last intron sequence replaces last exon (Figures 25 and 26)

There are 5 EST sequences in Unigene Hs.7970 (FBX031) that match the FBX031 sequence before the last exon and then lose homology and all of these have a similar 3' fusion sequence (accessions: AI432386, AI087321, AI093960, AI962454, AW196322). These clones have been derived from a pooled germ cell tumour library and a pooled melanocyte, fetal heart and pregnant uterus library indicating that both normal and neoplastic tissues express this variant mRNA. In all these clones the additional new sequence corresponds to the FBX031 long sequence from the intron before the last exon (FBX031-3) . In addition EST AI432386 has a change in the FBX031 cDNA sequence from nt 1300 GGATGCC to GAATTGCC in good quality sequence therefore there is the possibility that this sequence may encode a different C-terminal protein sequence (FBX031-4) to that encoded by FBX031-3. Both of these mRNAs change the protein coding sequence and the 3' UTR. Thus these changes may affect both the level of protein expression and its function.

FBX031 Alternate form 5 (FBX031-5) : extended 3' UTR (Figure 27)

There are 7 EST sequences in Unigene Hs.7970 (FBX031) that match the 3' FBX031 cDNA sequence to the start of it's poly (A) tail at which point these cDNA sequences continue to run into the FBX031 genomic sequence extending the 3' untranslated region (3' UTR) of the FBX031 long mRNA (accessions: BG770753, AL553647, BG764159, BG768045, BG768052, BE245587, BF798082).

Figure shows the sequence of EST BG764159 aligned to the genomic DNA clone, the small differences between the two sequences are probably due to sequencing errors in either the genomic clone or the EST sequence (therefore claim both aligned versions of this sequence) . While this does not change the FBX031 protein sequence itself, the extended 3' untranslated sequence may change the level of expression of the FBX031 protein through altering characteristics of the mRNA such as it's stability, localization, translation efficiency or coding capacity. Four of these EST sequences have been cloned from a multi drug resistant melanoma cell line and another from a pre-B ALL (leukaemia) indicating that this extended untranslated 3' region may be more common in cancer tissues. FBX031 Alternate form 6 (FBX031-6) : truncated 3 'UTR (Figure 28)

Two EST sequences in Unigene Hs.7970 (FBX031) have a truncated 3' UTR with the poly (A) tail being added after nt 3474 of the FBX031 long cDNA sequence (accession: R42132, infant brain and AA009560, fetal heart). As described, for variant form number 5 above, this may modify the same mRNA characteristics and ultimately the level of FBX031 protein expression. See below for a summary of how 3 ' UTR sequences can regulate gene expression.

FBX031 Alternate form 7 (FBX031-7) : deletion within 3' UTR (Figure 29)

The EST sequence from Unigene Hs.7970 with accession R51182 (cloned from infant brain) has a deletion within the 3' UTR region (between nucletides 2549-2551 to 3420-3422 of the FBX031 long cDNA sequence) which does not alter the protein coding sequence but, as described below, may have regulatory functions affecting FBX031 expression.

FBX031 Alternate form 8 (FBX031-8) : deletion within the last exon (Figure 30)

The EST sequence AI283060 from Unigene Hs.7970 (cloned from placenta) matches the FBX031 long sequence from nt 3249 to the poly (A) tail with nt 3513 being an A in place of the G in the FBX031 long sequence. The remaining 46 nucleotides correspond to sequence from the intron preceding the last exon (nt 91211-91256 of the human chromosome 16 clone RP11-178L8, accession AC010531) of the FBX031 long cDNA. This will alter both the sequence of the predicted protein product and the 3 ' UTR .

FBX031 Alternate form 9 (FBX031-9) : additional exon within the FBX031 sequence (Figure 31)

The EST sequence BG896799 (this sequence has been recently withdrawn from Genbank other than as an EST sequence) from Unigene Hs.7970 (cloned from human osteoarthritic cartilage) matches the FBX031 long sequence from nt 356-525 with there being 220 5' nucleotides that do not match the FBX031 long cDNA sequence. The first 85 nucleotides are derived from the cloning vector while nucleotides 86-221 match the genomic sequence (nt 127116-126981) of the human chromosome 16 clone RP11-178L8, accession AC010531. This EST identifies an additional exon within the FBX031 genomic clone within the first intron sequence. This additional exon truncates the FBX031 long protein and produces upstream stops before encoding the FBX031 short protein.

FBX031 Alternate form 10 (FBXO31-10) : alternate first exon (Figure 32)

The EST sequence BG718342 (cloned from testis) from Unigene Hs.7970 (FBX031) matches the FBX031 long sequence from nt 356. However, the 5' 135 nucleotides do not match this sequence. In this EST the first exon from the FBX031 long cDNA is replaced with a novel upstream exon (nt 22-136 of BG718342) corresponding to nt 353296-353183 of the Homo sapiens chromosome 16 working draft sequence segment (accession NT- _019609.3). This results in a protein product with an N-terminal methionine codon flanked by upstream stop codons which has 17 unique amino acids before being identical to the FBX031 long protein from aa 114 of that sequence. This results in the removal of the F- box and RXL motif from the FBX031 long protein and this form may negatively regulate the ability of FBX031 to target proteins for proteolytic degradation by competitively binding these proteins without targeting them to the ubiquitination machinery.

In addition to providing an additional exon for FBX031 there are other EST sequences containing the unique sequence from FBXO31-10 (accessions BG724256,

AI347047, AI346955, AI281097, AI273357, AW014292, AW139282) indicating that this region of the genomic clone may encode a separate gene in its own right and/or that there may be additional exons that fuse to the FBX031 sequence. EST BG724256 nt 6-427 aligns to the Homo sapiens chromosome 16 working draft sequence segments (accession NT_019609.3) nt 355584-355529, 353296-353187, 352205-352135, 349652-349470. The 3' sequence in EST AW014292 nt 2-288 aligns to the Homo sapiens chromosome 16 working draft sequence segments (accession NT_019609.3) nt 355184-355042, 353361- 353216. This encodes a potential partial protein sequence GTRPTDCSHL LNAVLDHVKA QLQRLKISES GNQRECLSTL LY*» which has a C-terminal stop codon. This partial protein sequence has some homology to a C/EBP-induced protein (accession NP_110429) see below.

Query: 1 GTRPTDCSHLLNA VLDH VKAQ QRLKISESGNQRECLSTLL 41

G R + C+H +A DH +K Q QR K+S SG ++E S LL C/EBP: 132 GERASSCAHKRSAS GSTDHRKEISKLKQQLQRTK SRSGKEKERGSPLL 181

FBX031 Alternate form 11 (FBX031-11) : deletion within protein coding region causing truncation (Figure 22)

While sequencing a potentially full length FBX031 EST (accession BE269540) a deletion within the N-terminus was unexpectedly discovered within known exons and occurring next to a sequence GCACGA which is repeated within the FBX031 cDNA sequence at either end of the deleted region. The full length sequence of this cDNA has been described above.

Schematic diagrams illustrating the variant FBX031 mRNAs and proteins are illustrated in Figure 34 and Figure 35 respectively.

Biological importance of the variant forms of FBX031 protein and mRNA

1. Regulation of gene expression by 3 ' UTRs

A number of the variant forms of the FBX031 mRNA have altered 3 'UTR regions. These variants do not change the predicted amino acid coding sequence of the FBX031 protein but these regions can have an important role in regulating gene expression. Some of these mechanisms are summarised below to provide information on how these changes may affect the level of protein expression from the FBX031 mRNAs.

Control of translation efficiency. Both spatial or temporal regulation of translation of a specific mRNA is mediated by its 3' UTR (reviewed by Decker and Parker, 1995). The 3' UTR can contain specific negative regulatory elements that interact with transacting components to repress translation either by stimulating the loss of the poly (A) tail (these can stimulate translation) or they can repress translation independently of the poly (A) tail (reviewed by Decker and Parker, 1995). Sequences in the 3' UTR can also enhance translation by relieving repression or mediating cytoplasmic adenylation of poly (A) -deficient mRNAs (reviewed by Wickens, 1990; Richter, 1991) . Control of coding capacity. A conserved RNA secondary structure within the 3' UTR of mammalian selenoproteins can direct the incorporation of selenocysteine at UGA rather than this specifying transcriptional termination (Berry et al., 1991).

Control of mRNA stability. mRNA molecules are stabilised by posttranscriptional modifications including a 7-methylguanosine cap structure at the 5' end and a poly (A) tail at the 3' end. In addition ongoing translation is also important as many mRNAs are stabilized by inhibitors of translation elongation. However, premature and aberrant translation termination , as well as impaired translation initiation, can promote mRNA degradation. Many elements that control mRNA stability have been localised to the 3' UTR (reviewed by Decker and Parker, 1994) . It is well established that the degradation rate of mRNA is related to poly (A) tails and specific sequences in 3' untranslated regions which increase or decrease access of ribosome-bound nucleases to mRNAs. One of these sequences A(U)nA has an established role in destabilizing the mRNAs of oncogene, cytokine, and growth factor transcripts and is also required for rapid decay of mRNA following deadenylation and RNA degradation via a 5' to 3' exonuclease. Other UTRs can affect mRNA turnover by containing sequence-specific endonucleolytic cleavage sites with mRNA stability being regulated by the binding of protective factors within the 3' UTR at or near the cleavage site. A recent report studying the complex regulation of nitric oxide synthase has suggested that RNA-binding proteins may be important factors in regulating the stability of its mRNA (Bloch, 1999) . Control of mRNA localisation and the cytoskeleton. Some mRNAs are localised within particular regions of the cytoplasm and the 3' UTR has been shown to be necessary for its proper localisation. There is also some evidence that mRNAs have a functionally significant association with the cytoskeleton and that they can dissociate from the 'cytoskeleton' when not being translated (reviewed by Decker and Parker, 1995) .

2. Variant FBX031 proteins

In addition to changing the level of FBX031 protein expression several of the FBX031 variant mRNAs are predicted to encode proteins with altered functions. For example FBX031 variant proteins -1, -2, -3 and 4 have a different C-terminus and this may affect the affinity for or identity of the substrate proteins that are targeted for ubiquitin mediated degradation. This could either increase or decrease the expression of substrate proteins. Other proteins e.g. FBX031 short, -9 and 10 lack the F-box motif and may act to regulate the degradation of substrate proteins by blocking their binding to a FBX031 protein with an F- box that would target the substrates for degradation. Different proteins may also have different subcellular localisations (observed in immunostaining studies) and this would affect the identity of the substrate proteins that they could interact with.

Cell line staining using the JC9 monoclonal antibody

Cytospin preparations of cell lines were acetone fixed for 10 minutes and then stored at -20°C. After incubation with MoAb JC9, DAKO's Envision kit was used for immunostaining as described previously. A variety of different staining patterns were observed with the JC9 MoAb and these are illustrated in Figure 44. In the MOLT-4 T-cell line cytoplasmic staining was observed, with a higher concentration of the FBX031 protein being observed around the edges of unstained areas giving the appearance of bubbles/vesicles in the cytoplasm (arrow heads). In contrast dividing cells contained very little FBX031 protein suggesting that it's expression and function may be, at least in part, related to the eukaryotic cell cycle/ cell division process. For example if the FBX031 protein targets cell cycle/cell division proteins for degradation then its reduced expression may enable the increased expression of these proteins during cell division. A reduction in the FBX031 expression by dividing cells was also observed in the MIEU and SUDHL-6 diffuse large B-cell lymphoma cell lines. In the JURKAT T-cell line cytoplasmic expression of the FBX031 protein was observed while additional nuclear staining was predominantly concentrated within distinct regions or nuclear bodies (arrow heads) . This staining of nuclear bodies was also a feature of the FBX031 expression in the HLY-1 diffuse large B-cell line and the SUDHL-1 anaplastic large cell lymphoma line (T-cell) . Dividing JURKAT cells showed some reduced cytoplasmic staining and there appeared to be FBX031 protein associated with the condensed chromosomes with possibly more

FBX031 staining at the ends of each chromosome. This DNA associated staining in dividing cells was also observed in the SUDHL-1 and HLY-1 cell lines. The SUDHL-1 cell line showed comparatively weak cytoplasmic expression of the FBX031 protein but perinuclear staining was observed indicating that the FBX031 protein may be associated with the nuclear membrane. The HLY-1 cell line expressed significantly higher levels of the FBX031 protein than any of the other cell lines tested. In the MIEU cell line there was some evidence of cell membrane staining indicating that FBX031 may be associated with the cell surface in certain cell types. In the majority of cell types e.g. MOLT-4, MIEU, HLY-1, DEAU (arrow heads) and SUDHL-6 the golgi area appeared to be unstained. This heterogeneity in FBX031 subcellular localisation was consistent with the results obtained from immunostaining normal and neoplastic human tissue sections with the JC9 antibody. The subcellular localisation of the FBX031 protein is likely to play a significant role in bringing this protein into contact with it's potential substrates and may regulate the proteins which are targeted for degradation by this protein. It is also possible that the wide range of variant FBX031 proteins encoded by the multiple forms of this mRNA may be, at least in part, responsible for these different expression patterns.

The FBX031 protein binds to Skpl

The F-box is functionally defined as a motif that can interact with Skpl (Bai et al., 1996). One approach to test the ability of the FBX031 protein to interact with Skpl was to perform a co-immunoprecipitation experiment. Due to the inability of the JC9 monoclonal antibody to immunoprecipitate the FBX031 protein the FBX031 protein was tagged with the Xpress epitope for immunoprecipitation studies.

The FBX031 entire cDNA sequence in IMAGE clone: 3958783 (accession BE903394) was excised using £coRI and then subcloned into the EcoRl site of pcDNA4/HisMax version A (Invitrogen). This plasmid (FBX031-HisMax) was then transfected into COS cells using a standard DEAE dextran transfection protocol. To confirm that the transfected COS cells expressed a tagged protein cytospin preparations of the transfectants were immunostained with the anti-Xpress antibody (Invitrogen) using DAKO's Envision kit as described previously. Immunostained transfected cells were clearly visible (Figure 33, panel A, transfected cell labelled with an arrow) confirming the expression of the tagged FBX031 protein. The transfected cells were lysed and then the Xpress tagged FBX031 proteins were immunoprecipitated with the anti-Xpress antibody. After SDS-PAGE separation, proteins were transferred to Immobilon and Western blotted with either al/5000 dilution of the anti-Skpl antibody or the JC9 hybridoma supernatant. Panel B (lane Ab: Skpl) clearly shows that the 19 kDa Skpl protein was co- immunoprecipitated with FBX031 indicating a close interaction between the two proteins. Blotting with JC9 (Panel B, lane Ab: JC9) identified a weak band at approximately 70 kDa that was consistent with the expected m.wt of the tagged FBX031 protein. These data confirmed that the FBX031 long protein is able to interact with the Skpl protein and from this the authors conclude that the F-box motif is functional and that the FBX031 protein is able to interact with the ubiquitination machinery. Control experiments using COS cells transfected with a LacZ fusion protein or empty vector did not co-immunoprecipitate Skp-1.

Expression of the FBX031 Variant-1 Protein in COS Cells

To investigate the expression of the protein encoded by the FBX031-1 cDNA variant, a Notl/Sall fragment containing the entire cDNA from IMAGE clone : 4432569 (accession BG180443) was cloned into pcDNA4/HisMax (Notl/Xhol) in reading frame C. This vector adds in- frame polyhistidine and Xpress epitope tags to the N- terminus of the FBX031-1 recombinant protein. These can be used to detect the expression of this variant protein, even in cells containing other FBX031 proteins. This FBX031-1 expression plasmid, an empty vector control and a similar construct containing the FBX031 long cDNA were then transfected into COS cells using the Fugene reagent (Roche) according to the manufacturer's instructions. After 24 hours cells were harvested and cytospin preparations were made as described previously. The cytospins were then immunostained using a 1/150 dilution of the anti-

Xpress antibody (Roche) and DAKO's Envision peroxidase staining kit.

The results indicated in figure 36 show that the COS cells transfected with the FBX031-1 cDNA construct C and D) appear to express much lower amounts of tagged recombinant protein than COS cells transfected with the tagged FBX031 long cDNA construct (A and B) . No staining was observed for the vector control (data not shown) . This experiment was repeated and the same results were obtained.

There are several explanations for these data. For example, the FBX031-1 mRNA/protein may be less efficiently transcribed/translated or another possibility is that the mRNA or protein may be less stable. A time course experiment where the FBX031-1 transfected cells were harvested up to three days after transfection was performed (data not shown) . This did not yield higher expression of the FBX031-1 protein. Thus slower protein synthesis alone is not likely to be the cause of the low abundance of this protein. This finding may be clinically significant as both of the EST clones encoding the FBX031-1 variant are derived from cancer patients. The expression of the FBX031-1 mRNA may represent a cancer related event (probably through alternative splicing) which not only changes the C-terminal FBX031 protein sequence but also reduces the abundance of the FBX031 F-box protein.

Expression of FBX031 in lung tumours

Paraffin embedded tissue sections from 129 non small cell lung tumours (NSCLC) were immunostained with the undiluted JC9 hybridoma supernatant using the Dako Envision kit as previously described. Of these, 28 slides were subsequently discarded as non informative e.g. many had weak staining due to a technical problem with the microwave pressure cooker. The preliminary data from this series are summarized below.

Negative 3 cases

Cytoplasmic staining

Weak 28

Weak-Moderate 16

Moderate 35

Moderate-Strong 3

Strong 4

Cytoplasmic + nuclear staining

Weak 3

Weak-Moderate 2

Moderate 5

Moderate-Strong 2

Strong 0

The majority of JC9 immunostaining in the NSCLC was cytoplasmic with either weak or moderate staining, 79 cases. Only 3 cases were negative (expression in lymphocytes confirmed that this was not a technical artifact) and 9 were moderate to strongly, or strongly stained. These data are illustrated in figure 45. In this series 28 cases had adjacent 'normal' lung epithelium. Of these 18 cases showed increased expression in the tumour compared to the normal epithelium while 8 were unchanged and 2 appeared to have decreased expression in the tumour. Normal lung epithelium commonly showed both nuclear and cytoplasmic staining. In cases where nuclear staining was observed in the normal epithelium 13 of these appeared to have only cytoplasmic staining of the tumour cells. Nuclear staining was only detected in 12 tumours .

These data demonstrate that FBX031 is differentially expressed in NSCLC. More than half of the cases with adjacent lung epithelium appeared to have increased FBX031 expression in the tumour cells, and most tumours did not have nuclear FBX031 expression. Further studies are required to investigate whether these findings have clinical significance in this malignancy.

Investigation of cell cycle related proteins that may bind to the FBX031 protein.

To identify proteins that FBX031 may bind and target for ubiquitination, the Xpress tagged JC9 protein was immunoprecipitated from transfected COS cells using the anti-Xpress antibody as done previously to identify the interaction between the FBX031 and Skpl proteins. The immunoprecipitated proteins were resolved by SDS-PAGE and transferred to an immobilon membrane. A variety of commercial antibodies to proteins involved in the cell cycle were then used in Western blotting experiments on these immunoprecipitated proteins.

Antibodies used in Western blotting experiments

Cyclin D3 : Cocktail of anti-D3 mAb (cat, C28620) Transduction Labs diluted 1/2000 and anti-D3 mAb (cat, 554195) Becton Dickinson Co at 0.5μg/μl.

p57: Cocktail of Kip2 p57 (H-91) diluted 1/200 and Kip2 p57 (C-20) diluted 1/200 from Santa Cruz.

pl6: pl6 (50.1) Santa Cruz diluted 1/200

p27: Anti-Kipl/p27 mAb (cat, K25020) Transduction Labs diluted 1/2500

Cyclin E: Cyclin E rabbit polyclonal antibody (C-19) Santa Cruz diluted 1/200

Cdk2: Cdk2 (cat C18520) Transduction Labs diluted 1/2500

Cyclin A: Cyclin A (H-432) Santa Cruz diluted 1/200

The antibodies to p21 and pl6 did not identify a protein of the expected molecular weight when COS cell lysates were Western blotted and thus these were not further investigated. Antibodies to p27, cyclin D3, p57 and cyclin E, identified a protein of the expected molecular weight in COS cell lysates but these were not detected in the immunoprecipitates suggesting that they did not interact with the FBX031 protein (data not shown) .

An initial experiment with an antibody against the cdk2 protein kinase suggested that this protein might bind FBX031. As shown in figure 46, panel A, the cdk2 protein was detected by Western blotting in the COS cell lysate and in the immunoprecipitated proteins from COS cells transfected with FBX031 but not the empty vector. A repeat of this experiment using an X- press lacZ fusion protein as a more appropriate control, for non-specific protein binding, also looked promising but was inconclusive as only low level binding was detected (Figure 46, panel B) . This experiment was performed again and cdk2 was observed in all the immunoprecipitated lanes suggesting that the binding was not specific (Figure 46, panel C) . These data are still inconclusive and further experiments will be undertaken to determine whether the FBX031 protein binds cdk2.

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Claims

1. An isolated nucleic acid molecule encoding an isolated protein designated FBX031 having an amino acid sequence as set forth in Figure 5b or Figure 6b, or an amino acid sequence which differs from those sequences only in conservative amino acid changes.

2. An isolated nucleic acid molecule encoding any of the protein variants of FBX031 1-11 having the sequences set forth in Figures 23 to 33 or an amino acid sequence which differs from those sequences only in conservative amino acid changes.

3. An isolated nucleic acid molecule according to claim 1 or 2 which is a DNA molecule.

4. An isolated nucleic acid molecule according to any of claims 1 to 3 which is a cDNA molecule.

5. An isolated nucleic acid molecule according to any of claims 1, 3 or 4 comprising the nucleotide sequence set forth in Figure 5a or 6a.

6. An isolated nucleic acid molecule according to any of claims 2 to 4 comprising the nucleotide sequences set forth in Figures 23 to 33.

7. A nucleic acid molecule which is capable of hybridising to a nucleic acid molecule according to any of claims 1 to 6 under conditions of high stringency.

8. An oligonucleotide comprising a sequence of 10 or more consecutive nucleotides of any of the nucleotide sequences set forth in Figures 5a or 6a or Figures 23 to 33 .

9. An antisense nucleic acid molecule which is capable of hybridising to the sequence of nucleotides set forth in either of Figures 5a or 6a or Figures 23 to 33 under conditions of high stringency.

10. An expression vector comprising the nucleic acid molecule of any of claims 1 to 7.

11. A host cell transformed or transfected with the expression vector of claim 10.

12. An isolated protein encoded by a nucleic acid molecule according to any of claims 1 and 3 to 7.

13. An isolated protein according to claim 12 comprising an amino acid sequence as set forth in Figure 5b or 6b.

14. An isolated protein encoded by a nucleic acid molecule according to any of claims 2 to 4, 6 and 7.

15. An isolated protein according to claim 14 comprising any of the amino acid sequences set forth in Figures 23 to 33.

16. An isolated protein designated FBX031 comprising an amino acid sequence as set forth in

Figure 5b or 6b.

17. An isolated FBX031 variant protein comprising an amino acid sequence as set forth in any of Figures 23 to 33.

18. An antibody which is capable of binding to the protein claimed in any of claims 12 to 17.

19. An antibody according to claim 18 which is a monoclonal antibody obtainable from the hybridoma deposited with the European Collection of Cell Cultures under Accession No. 01012314.

20. An antibody according to claim 18 or 19 for use in a method of treating the human or animal body.

21. Use of an antibody according to claims 18 or 19 in the manufacture of a medicament for treating a disease or condition mediated or associated with expression of function of a protein according to any of claims 12 to 17.

22. A hybridoma deposited with the European Collection of Cell Cultures under Accession No. 01012314.

23. An in vi tro method of detecting expression of a protein according to any of claims 12 to 17 in a mammalian subject which method comprises contacting a sample of tissue, cells or cell lysates removed from said mammalian subject with an antibody which is capable of binding to said protein, or an equivalent thereof, and detecting specific binding of the antibody to the target protein or equivalent thereof in said tissue, cell or cell lysate.

24. A method according to claim 23, wherein the antibody is a monoclonal antibody obtainable from the hydridoma deposited with the European Collection of Cell Cultures under Accession No. 01012314.

25. A nucleic acid molecule according to any of claims 1 to 7, or a fragment thereof, for use in a method of treatment of the human or animal body.

26. Use of a nucleic acid molecule according to any of claims 1 to 7 in the manufacture of a medicament for treating cancer.

27. Use of a nucleic acid molecule according to claim 27, wherein said cancer comprises any of solid tumours or haematopoietic malignancies.

28. Use according to claim 27 wherein said solid tumours comprise any of renal, lung, pancreatic, breast, prostate or stomach cancer.

29. Use of a nucleic acid molecule according to any of claims 1 to 7 in the manufacture of a medicament for treating microhydranencephaly.

30. A protein according to any of claims 12 to 17, or a functional equivalent, derivative or fragment thereof for use in a method of treating the human or animal body.

31. Use of a protein according to any of claims 12 to 17 or a functional equivalent, derivative or fragment thereof, in the manufacture of a medicament for treating cancer.

32. Use of a protein according to claim 31, wherein said cancer comprises any of solid tumours or haematopoietic malignancies.

33. Use according to claim 32, wherein said solid tumours comprise any of renal tumours, lung cancer, pancreatic, breast, prostate or stomach cancer.

34. Use of a protein according to any of claims 12 to 17, or a functional equivalent, derivative or fragment thereof, in the manufacture of a medicament for treating microhydranencephaly.

35. A pharmaceutical composition comprising any of an isolated nucleic acid molecule according to any of claims 1 to 7, an isolated protein according to any of claims 12 to 17, or an antibody according to claims 18 or 19, together with a pharmaceutically acceptable carrier, diluent or excipient therefor.

36. A method of treating cancer in a patient which method comprises administering to said patient an effective amount of a nucleic acid molecule according to any of claims 1 to 7, or a protein according to any of claims 12 to 17.

37. A method according to claim 36 wherein said cancer comprises solid tumours or haematopoietic malignancies .

38. A method according to claim 37 wherein said solid tumours are derived from any of kidney, lung, pancreas, breast, prostate or stomach cancer.

39. A method of treating cancer in a patient which method comprises administering to said patient an effective amount of an antisense molecule according to claim 9 or an antibody according to claim 18 or 19.

40. A method according to claim 39, wherein said cancer comprises any of β cell chronic lymphocytic leukemia (B-CLL) ALK negative or ALK positive Anaplastic large cell lymphoma (ALCL) , diffuse large B-cell lymphoma (DLBCL) , NSCL tumours, rectal tumours or Hodgkins disease (HD) .

41. A method of treating microhydranencephaly in a patient which method comprises administering to said patient an effective amount of a nucleic acid molecule according to any of claims 1 to 7, or a protein according to any of claims 12 to 17.

42. A method of diagnosing the medical significance of premalignant lesions in a patient which method comprises detecting a change of expression pattern or function of a protein according to any of claims 12 to 17 in said protein, wherein a change of expression pattern or function of said protein is indicative of the likelihood of said patients lesions developing into a malignant tumour.

43. A method of diagnosing the medical significance of a condition or cancer in a patient which method comprises detecting varying expression patterns or function of a protein according to any of claims 12 to 17 in said patient.

44. A method of diagnosing the medical significance of a condition or cancer in a patient which method comprises detecting for abnormal mRNA transcripts or abnormal levels of mRNA expression, nucleotide sequences or gene copy numbers encoding a protein according to any of claims 12 to 17, where abnormal levels of said protein are indicative of a disease condition or cancer.

45. A method of screening for predisposition to cancer in an individual which comprises screening for an inherited genetic mutation in a nucleic acid sequence from said individual encoding a protein according to any of claims 12 to 17.

46. A method of detecting or diagnosing cancer in an individual which is associated with reduced levels of expression of a protein according to any of claims 12 to 17, which method comprises testing in a cell of said individual for increased levels of methylation of a regulatory region of a nucleic acid sequence from said individual encoding a protein according to any of claims 12 to 17.

47. A method of treating cancer associated with reduced levels of expression of a protein according to any of claims 12 to 17 which method comprises administering to an individual in need thereof a therapeutic amount of a methylation inhibitor.

48. A method of detecting or diagnosing cancer in an individual which is associated with increased levels of expression of a protein according to any of claims 12 to 17, which method comprises testing in a cell of said individual for decreased levels of methylation of a regulatory region of a nucleic acid molecule encoding a protein according to any of claims 12 to 17.

49. A method of treating a disease or condition in a patient associated with over expression or activity of a FBX031 protein according to any of claims 12 to 17, which method comprises administering to an individual in need thereof a therapeutic amount of an antisense molecule according to claims 7 or a nucleic acid according to claim 18 or 19 or a blocking peptide.

50. A method of treating a disease or condition in a patient associated with under expression or activity of a FBX031 protein according to any of claims 12 to 17, which method comprises administering to an individual in need thereof, a therapeutic amount of a nucleic acid molecule according to any of claims 1 to 7, or a protein according to any of claims 12 to 17.

51. A method of identifying minimal residual disease in a cancer patient which method comprises detecting for the presence of neoplastic cells in an individual by identifying a change of expression pattern or function of a protein according to any of claims 12 to 17 in a patient.

52. A method of identifying minimal residual disease in a cancer patient which method comprises detecting for the presence of neoplastic cells in an individual by identifying abnormal levels of mRNA expression or nucleotide sequences or gene copy number encoding a protein according to any of claims 12 to 17.

53. A method of establishing cases of both ALK- negative and ALK positive anaplastic large cell lymphoma (ALCL) , with poor prognosis which method comprises contacting an antibody according to claim 18 or 19 with an ALCL lymphoma tissue sample and monitoring the level of immunostaining, wherein increased immunostaining is indicative of increased protein expression and increased risk of short survival time.

: 285863: CDM: CAR: ONDOCS