WO2001098459A2 - Acides nucleiques codant hei-c, polypeptides hei-c, anticorps hei-c et leurs methodes d'utilisation - Google Patents

Acides nucleiques codant hei-c, polypeptides hei-c, anticorps hei-c et leurs methodes d'utilisation Download PDF

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WO2001098459A2
WO2001098459A2 PCT/US2001/019746 US0119746W WO0198459A2 WO 2001098459 A2 WO2001098459 A2 WO 2001098459A2 US 0119746 W US0119746 W US 0119746W WO 0198459 A2 WO0198459 A2 WO 0198459A2
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hei
nucleic acid
sequence
protein
polypeptide
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WO2001098459A3 (fr
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Margret B. Einarson
Joanne Estojak
Ilya Serebriiskii
Erica A. Golemis
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Fox Chase Cancer Center
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • This invention relates to the fields of molecular biology and cell-cell adhesion. Specifically, nucleic acids encoding HEI-C, HEI-C polypeptides, HEI-C- specific antibodies, and methods of use thereof are provided.
  • extracellular matrix a complex network of extracellular macromolecules referred to as the extracellular matrix.
  • cells in direct physical contact with one another are often linked at specialized regions of their plasma membranes referred to as cell junctions. Some of these junctions serve mainly to hold cells together, ,while others facilitate the passage of small molecules from one cell to another.
  • the cadherins comprise a family of molecules which play a central role in the formation and regulation of cell-cell contacts ' .
  • Cadherins are single pass tra smembrane glycoproteins which mediate Ca++ dependent cellular adhesion.
  • Cadherins regulate cell- cell adhesion via both homotypic and heterotypic interactions between cadherins expressed on the cell surface. These molecules become clustered at the cell surface and are linked to the actin cytoskeleton to form adherens junctions. The molecular composition of the adherens junction determines the strength of the junction.
  • Cadherin function is also related to various interactions with proteins on the cytoplasmic side of the plasma membrane.
  • Cadherin binds to either ⁇ - catenin or plakoglobin via its C-terminal domain. These molecules mediate the linkage to the cytoskeleton via complex formation with ⁇ -catenin and -actinin. Another catenin family member, pl20 ctn , binds to the juxta embrane domain of cadherin. Cadherin function is regulated by its interactions with these various cytoskeletal/plasma membrane elements. For example, growth factor treatment results in the tyrosine phosphorylation of ⁇ -catenin, plakoglobin, and pl20 ctn .
  • Regulated cell-cell adhesion is thus essential for control of fundamental biological processes including embryogenesis, formation and maintenance of tissue structure, cellular migration through tissues, and etastatic invasion and establishment of malignant cells.
  • Plasma membrane related changes include, without limitation, enhanced transport of metabolites and increased amounts of extracellular matrix proteolysis.
  • Malignant cells demonstrate diminished adherence to surfaces and therefore retain a rounded shape when cultured. Additionally, lowered requirements for growth factors are observed, actin fails to organize into large bundles and extracellular deposition of fibronectin is low.
  • each cell undergoes a series of events to partition DNA, organelles and membranes.
  • nuclear cell cycle events must be coordinated with the reprogramming of cell shape, cell polarity and cell fate to allow normal proliferation.
  • cells transit through the cell cycle most undergo a progressive shape change which is characterized by cell rounding and partial release from the extracellular matrix followed by reattachment following cytokinesis.
  • Control of cell polarity and attachment is a particularly acute issue for cells organized in tissues, as tissues must regulate the integration of new cells into specific cell layers in a heterogeneous environment made of multiple cell types that collaborate in complex functions.
  • cells in tissues regulate the integration of newly divided cells into the tissue layer by maintaining cell-cell junctions and by regulating the plane of cleavage
  • EBl is localized to the plus ends of microtubules at the cell cortex as well as to the spindle during mitosis.
  • zyxin is a known regulator of the actin cytoskeleton at focal adhesions.
  • Zyxin is also found at the mitotic spindle in association with a tumor suppressor protein H-warts.
  • EBl and zyxin are representatives of a class of regulatory molecules which transit between the cell periphery and the mitotic spindle to coordinate processes such as cell shape, spindle positioning, plane of cell division and regulation of cell adhesion during cell division.
  • an isolated nucleic acid molecule which includes a sequence encoding an adhesion protein of a size between about 40 and 20 kilodaltons.
  • the encoded protein referred to herein as HEI-C (Human Enhancer of invasion, Cluster) comprises an approximately 278 amino acid protein which contains three coiled coil domains.
  • an isolated nucleic acid molecule in a preferred embodiment, includes a nucleic acid sequence encoding a human cellular adhesion protein, HEI-C, that also functions as a microtubule associated protein (MAP) .
  • HEI-C human cellular adhesion protein
  • MAP microtubule associated protein
  • the HEI-C protein has an amino acid sequence the same as Sequence ID NO: 2.
  • An exemplary nucleic acid molecule of the invention comprises Sequence ID NO: 1.
  • an isolated nucleic acid molecule which has a sequence selected from the group consisting of: (1) Sequence ID NO: 1; (2) a sequence specifically hybridizing with preselected portions or all of the complementary strand of Sequence ID NO: 1; (3) a sequence encoding preselected portions of
  • Sequence ID NO: 1 (4) a sequence encoding part or all of a polypeptide having amino acid Sequence I ' D NO: 2. Such partial sequences are useful as probes to identify and isolate homologues of the HEI-C gene of the invention. Accordingly, isolated nucleic acid sequences encoding natural allelic variants of Sequence ID NO: 1 are also contemplated to be within the scope of the present invention. The term natural allelic variants will be defined hereinbelow.
  • HEI-C polypeptide may conveniently be obtained by introducing expression vectors into host cells in which the vector is functional, culturing the host cells so that the HEI-C polypeptide is produced and recovering the HEI-C polypeptide from the host cells or the surrounding medium. Vectors comprising nucleic acid according to the present invention and host cells comprising such vectors or nucleic acid form further aspects of the present invention.
  • an isolated human HEI-C protein which has a deduced molecular weight of between about 20 kDa and 40 kDa.
  • the protein comprises three coiled coil domains which may play a role in cytoskeletal interactions .
  • the protein is of human origin, and has an amino acid sequence the same as Sequence ID NO: 2.
  • the protein may be encoded by natural allelic variants of Sequence ID NO: 1. Inasmuch as certain amino acid variations may be present in a HEI-C protein encoded by a natural allelic variant, such proteins are also contemplated to be within the scope of the invention.
  • antibodies immunologically specific for the proteins described hereinabove are provided.
  • methods are provided for identifying alterations in HEI-C nucleic acids. Methods are also disclosed for identifying agents which disrupt HEI-C protein-protein interactions such as those observed at microtubules and the spindle body. Also provided are methods for disrupting HEI-C interactions at the plasma membrane.
  • nucleic acids, proteins/polypeptides, peptides and antibodies of the present invention may be used to advantage for identifying therapeutic agents having efficacy in the modulation of cell adhesion and the regulation of mitosis.
  • the molecules may also be useful in the diagnosis and/or treatment of malignancy.
  • the HEI-C molecules of the invention may also be used as research tools and will facilitate the elucidation of the mechanistic action of the novel genetic and protein interactions involved in the maintenance of cellular adhesion and morphology.
  • the present invention also provides nucleic acid molecules, polypeptides and/or antibodies as mentioned above for use in medical treatment.
  • the present invention provides use of a nucleic acid molecule, polypeptide and/or antibody in the preparation of a medicament for treating cancer or aberrant tissue formation.
  • kits for detecting mutations in the HEI-C gene associated with aberrant cellular morphology, or a susceptibility to cancer comprising one or more nucleic acid probes capable of binding and/or detecting a mutated HEI-C nucleic acid.
  • the kit may comprise one or more antibodies capable of specifically binding and/or detecting a mutated HEI-C nucleic acid or amino acid sequence or a pair of oligonucleotide primers having sequences corresponding to, or complementary to a portion of the nucleic acid sequence set out in Sequence I. D. No.
  • transgenic animals are provided which are useful for elucidating the role HEI-C plays in growth and development. Isolation of mouse genomic DNA encoding HEI-C also facilitates the production of HEI-C knock-out mice.
  • Figures IA and IB show the results of a pseudohyphal growth screening assay showing colonies of yeast transformed with human cDNA and isolated via two hybrid analysis.
  • Figure IB depicts the altered morphology of the indicated transformants.
  • Figure 2A depicts a Northern blot showing the localization of HEI-C mRNA in the indicated tissues.
  • Figures 3A-3F show confocal immunofluorescent micrographs revealing that HEI-C is localized at cell- cell contacts and co-localizes with E-cadherin.
  • MDCK cells were grown on collagen coated coverslips for 24 hours, fixed with paraformaldehyde and stained with anti-HEI-C, and anti-E-cadherin antibodies.
  • Confocal images shown in Figs. 3D, 3E and 3F represent Z series images which indicate that HEI-C was at regions of cell-cell contact, primarily on the basolateral surface.
  • Figures 4A-4F show confocal immunofluorescent micrographs revealing that HEI-C was localized at some cell-cell junctions in a monolayer. MDCK cells were grown on collagen coated coverslips for 7 days, fixed in paraformaldehyde and stained with anti-HEI-C and anti-E cadherin antibodies.
  • Figs. 4D, 4E and 4F are Z series images.
  • Figures 5A-5C show fluorescent micrographs revealing that HEI-C is localized to interior cell-cell contacts following wounding. MDCK monolayer was wounded with a pasteur pipette. Cells which migrated away from the wound edges are shown. HEI-C was found at interior cell-cell contacts, but not at lamellipodia. Staining was proximal to E-cadherin, but not generally co-incident.
  • Figures 6A-6D depict a series of fluorescent micrographs showing that HEI-C was localized to the spindle in mitotic cells.
  • Upper panel MCF7 cells were permeabilized with 0.02% Triton, fixed with paraformaldehyde and stained with anti-HEI-C and anti- ⁇ tubulin antibodies.
  • Lower panel MCF7 cells fixed in - 20°C methanol and stained with anti-HEI-C and anti- dynamitin antibodies. The data show the intracellular colocalization of these two molecules confirming data obtained from two hybrid analysis.
  • Figures 7A-7F depict a series of immunofluorescent micrographs showing localization of HEI-C to the midbody of dividing cells. HEI-C remained associated with the spindle until the end of mitosis.
  • Figure 8 shows an alignment of the nucleic acid sequence of SEQ ID NO: 1 and amino acid sequence of the HEI-C protein (SEQ ID NO: 2) encoded by SEQ ID NO: 1.
  • Figure 9 shows additional HEI-C upstream nucleotide sequences identified by 5' RACE.
  • Figures 9A and 9B show the nucleic acid sequence of HEI-C including an in-frame stop codon upstream of the ATG codon. Both the coding (SEQ ID NO: 7) and the non- coding (SEQ ID NO: 8) strands are shown.
  • Figure 10 depicts full length HEI-C and truncated HEI-C domain constructs and their activity in the yeast invasion assay.
  • the regions of HEI-C indicated in the diagram at right were subcloned into the yeast expression vector pJG4-4.
  • the regions of HEI-C represented are full length HEI-C (aa. 1-278), 79CC2/3 (aa. 79-278), 124CC2/3 (aa. 124-278), 177CC3 (aa. 177- 278) .
  • These expression plasmids were transformed into yeast and grown for 24 hours on galactose containing media to induce expression, and their appearance was recorded (prewash) .
  • the plates were then washed with running water and photographed (postwash) . Yeast remaining on the plate after washing have invaded the agar substrate.
  • Figure 11 is a Western blot revealing the association of HEI-C with microtubules.
  • Precleared HeLa cell extracts were allowed to associate with polymerized microtubules for 30 minutes at 37°C.
  • the reactions were centrifuged through a sucrose cushion and the pellet and supernatant fractions were harvested.
  • the fractions were subjected to SDS-PAGE electrophoresis on a 10% gel. Proteins were transferred to a PVDF membrane and probed with anti- HEI-C antibodies.
  • Figure 12 is a Western blot showing that HEI-C undergoes a cell cycle dependent post-translational modification.
  • Asynchronous U20S cells or cells treated with thymidine or nocodazole were lysed and the extracts subjected to SDS-PAGE electrophoresis on a 12% gel. Proteins were transferred to a PVDF membrane and probed with polyclonal HEI-C antibodies.
  • HEI-C human enhancer of invasion cluster
  • HEI-C encodes a 278 amino acid protein containing three coiled coil domains and a putative nuclear export sequence (NES) . Based on the pseudohyphal phenotype induced by HEI-C overexpression in yeast, HEI-C appears to be involved in cell morphology and/or cellular adhesion regulation in mammalian cells.
  • HEI-C endogenous HEI-C in MDCK and MCF-7 cells is localized to sites of cell- cell contact.
  • MDCK monolayers the pattern of HEI-C localization at cell-cell junctions is partially coincident with E-cadherin.
  • HEI- C staining is specifically increased at sites of cell- cell contact but is absent in the lamellipodia.
  • HEI-C In order to gain insight into the role of HEI-C in cell-cell contact, two-hybrid analysis of HEI-C was performed. A number of interacting proteins were identified including Ost and dynamitin. Ost is a protooncogene which encodes a Rho/Cdc42 guanine nucleotide exchange factor or GEF. Dynamitin is a component of the dynein-dynactin motor complex known to be involved in spindle formation at mitosis, membrane trafficking and organelle maintenance. The interaction of HEI-C with two regulators of cytoskeletal dynamics and function suggests that HEI-C plays a role in these pathways. The identification of these interacting partners and the dynamic subcellular localization of HEI-C in mammalian cells indicates that HEI-C is a regulator of the cytoskeleton at cell-cell junctions.
  • chemotherapeutic drugs commonly used in the treatment of cancer patients include, for example, taxanes, such as paclitaxel and docetaxel, and Vinca alkyloids, such as vinblastine and vincristine. These drugs are among the most active chemotherapeutic agents in the treatment of cancers, including those affecting the breast and lung, but only 30-50% of previously untreated patients respond to treatment with these agents (DeVita et al., 1997). As clinicians have few assays with which to predict the responsiveness of a patient to a therapeutic protocol, patients may be exposed to toxic drugs and suffer from the side effects of such treatment without experiencing any benefit.
  • MAP4 protein level correlates with sensitivity to paclitaxel, which stabilizes polymerized microtubules, and Vinca alkyloids, which promote microtubule depolymerization (Zhang et al., 1999).
  • increased expression of MAP4 facilitated increased microtubule polymerization, paclitaxel binding, and sensitivity to paclitaxel. Binding of Vinca alkyloids to microtubules and sensitivity to these agents, however, decreased upon increased expression of MAP4.
  • HEI-C may also play a role in cellular sensitivity to drugs which target microtubules.
  • endogenous HEI-C expression levels in cancer cells could be utilized as a predictor of beneficial response to different therapeutic agents, thereby providing attending physicians valuable information with regard to the efficacy of a therapeutic protocol.
  • isolated nucleic acid refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it originates.
  • the "isolated nucleic acid” may comprise a DNA or cDNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the DNA of a prokaryote or eukaryote.
  • RNA molecules of the invention the term “isolated nucleic acid” primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above.
  • the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues) , such that it exists in a “substantially pure” form (the term “substantially pure” is defined below) .
  • the term “isolated protein” or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in "substantially pure” form.
  • promoter region refers to the transcriptional regulatory regions of a gene, which may be found at the 5' or 3 ' side of the coding region, or within the coding region, or within introns .
  • vector refers to a small carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell where it will be replicated.
  • expression vector is a specialized vector that contains a gene with the necessary regulatory regions needed for expression in a host cell.
  • operably linked means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector.
  • transcription control elements e.g. promoters, enhancers, and termination elements
  • substantially pure refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like) .
  • the term "immunologically specific” refers to antibodies that bind to one or more epitopes of a protein of interest (e.g., HEI-C), but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
  • a protein of interest e.g., HEI-C
  • the term “specifically hybridizing” refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non- complementary sequence.
  • percent identical is used herein with reference to comparisons among nucleic acid or amino acid sequences .
  • Nucleic acid and amino acid sequences are often compared using computer programs that align sequences of nucleic or amino acids thus defining the differences between the two.
  • comparisons of nucleic acid sequences are performed using the GCG Wisconsin Package version 9.1, available from the Genetics Computer Group in Madison, Wisconsin.
  • the Blastn 2.0 program provided by the National Center for Biotechnology Information (at http://www.ncbi.nlm.nih.gov/blast/ ; Altschul et al., 1990, J Mol Biol 215:403-410) using a gapped alignment with default parameters, may be used to determine the level of identity and similarity between nucleic acid sequences and amino acid sequences .
  • the present invention also includes active portions, fragments, derivatives and functional mimetics of the HEI-C polypeptide or protein of the invention.
  • An "active portion" of HEI-C polypeptide means a peptide which is less than said full length HEI-C polypeptide, but which retains its essential biological activity, e.g., microtubule association and dynamitin and/or ost binding.
  • a "fragment" of the HEI-C polypeptide means a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous amino acids, typically at least about nine to thirteen contigous amino acids and, most preferably, at least about twenty to thirty or more contiguous amino acids. Fragments of the HEI-C polypeptide sequence, antigenic determinants or epitopes are useful for raising antibodies to a portion of the HEI-C amino acid sequence.
  • a “derivative" of the HEI-C polypeptide or a fragment thereof means a polypeptide modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion or substitution of one or more amino acids, without fundamentally altering the essential activity of the wildtype HEI-C polypeptide.
  • “Functional mimetic” means a substance which may not contain an active portion of the HEI-C amino acid sequence, and probably is not a peptide at all, but which retains the essential biological activity of natural HEI-C polypeptide.
  • Nucleic acid molecules encoding the HEI-C protein of the invention may be prepared by two general methods: (1) Synthesis from appropriate nucleotide triphosphates, or (2) Isolation from biological sources. Both methods utilize protocols well known in the art.
  • nucleotide sequence information such as the full length cDNA having
  • Sequence ID NO: 1 enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis.
  • Synthetic oligonucleotides may be prepared by the phosphora idite method employed in the Applied Radios.
  • Biosystems 38A DNA Synthesizer or similar devices may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC) .
  • HPLC high performance liquid chromatography
  • a 1.4 kb double-stranded molecule may be synthesized as several smaller segments of appropriate complementarity. Complementary segments thus produced may be annealed such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment.
  • Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct an entire 1.4 kb double-stranded molecule.
  • a synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.
  • Nucleic acid sequences encoding HEI-C may be isolated from appropriate biological sources using methods known in the art.
  • a cDNA clone is isolated from a cDNA expression library of human origin.
  • genomic clones encoding HEI-C may be isolated.
  • cDNA or genomic clones having homology with HEI-C may be isolated from other species, such as mouse, using oligonucleotide probes corresponding to predetermined sequences within the HEI-C gene.
  • nucleic acids having the appropriate level of sequence homology with the protein coding region of Sequence ID NO: 1 may be identified by using hybridization and washing conditions of appropriate stringency.
  • hybridizations may be performed, according to the method of Sambrook et al., (supra) using a hybridization solution comprising: 5X SSC, 5X Denhardt's reagent, 0.5-1.0% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% for amide.
  • Hybridization is carried out at 37-42°C for at least six hours.
  • filters are washed as follows: (1) 5 minutes at room temperature in 2 X SSC and 0.5-1% SDS; (2) 15 minutes at room temperature in 2 X SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37°C in 1 X SSC and 1% SDS;
  • T m 81 . 5°C + 16. 6Log [Na+] + 0 . 41 ⁇ % G+C) - 0 . 63 ( % formamide ) - 600 /#bp in duplex
  • [Na+] [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T m is 57 °C.
  • the T m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology.
  • targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.
  • Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention.
  • Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector.
  • clones are maintained in a plasmid cloning/expression vector, such as pBluescript (Stratagene, La Jolla, CA) , which is propagated in a suitable E. coli host cell.
  • a plasmid cloning/expression vector such as pBluescript (Stratagene, La Jolla, CA)
  • Genomic clones of the invention encoding the human or mouse HEI-C gene may be maintained in lambda phage FIX II (Stratagene) .
  • HEI-C-encoding nucleic acid molecules of the invention include cDNA, genomic DNA, RNA, and fragments thereof which may be single- or double- stranded.
  • this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule of the present invention, such as selected segments of the cDNA having Sequence ID NO: 1.
  • Such oligonucleotides are useful as probes for detecting or isolating HEI-C genes.
  • variants e.g., allelic variants
  • HEI-C sequences disclosed herein or the oligos targeted to specific locations on the respective genes or RNA transcripts.
  • naturally allelic variants is used herein to refer to various specific nucleotide sequences and variants thereof that would occur in a human population. Genetic polymorphisms giving rise to conservative or neutral amino acid substitutions in the encoded protein are examples of such variants.
  • substantially complementary refers to oligo sequences that may not be perfectly matched to a target sequence, but the mismatches do not materially affect the ability of the oligo to hybridize with its target sequence under the conditions described.
  • the coding sequence may be that shown in Sequence ID NO: 1, or it may be a mutant, variant, derivative or allele of this sequence.
  • the sequence may differ from that shown by a change which is one or more of addition, insertion, deletion and subsitution of one or more nucleotides of the sequence shown. Changes to a nucleotide sequence may result in an amino acid change at the protein level, or not, as determined by the genetic code.
  • nucleic acid according to the present invention may include a sequence different from the sequence shown in Sequence ID NO: 1 yet encode a polypeptide with the same amino acid sequence.
  • the encoded polypeptide may comprise an amino acid sequence which differs by one or more amino acid residues from the amino acid sequence shown in Sequence ID NO: 2.
  • Nucleic acid encoding a polypeptide which is an amino acid sequence mutant, variant, derivative or allele of the sequence shown in Sequence ID NO: 2 is further provided by the present invention.
  • Nucleic acid encoding such a polypeptide may show greater than 60% homology with the coding sequence shown in Sequence ID NO: 1, greater than about 70% homology, greater than about 80% homology, greater than about 90% homology or greater than about 95% homology.
  • antisense oligonucleotide sequences based on the HEI-C nucleic acid sequences described herein.
  • Antisense oligonucleotides may be designed to hybridize to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of polypeptides encoded by a given DNA sequence (e.g. either native HEI-C polypeptide or a mutant form thereof) , so that its expression is reduced or prevented altogether.
  • a given DNA sequence e.g. either native HEI-C polypeptide or a mutant form thereof
  • antisense techniques can be used to target control sequences of the HEI-C gene, e.g. in the 5' flanking sequence of the HEI-C coding sequence, whereby the antisense oligonucleotides can interfere with HEI-C control sequences.
  • the construction of antisense sequences and their use is described in Peyman and Ulman, Chemical Reviews, 90:543-584, (1990), Crooke, Ann. Rev. Pharmacol.
  • the present invention provides a method of obtaining nucleic acid of interest, the method including hybridization of a probe having part or all of the sequence shown in Sequence ID NO: 1 or a complementary sequence, to target nucleic acid. Hybridization is generally followed by identification of successful hybridization and isolation of nucleic acid which has hybridized to the probe, which may involve one or more steps of PCR.
  • Such oligonucleotide probes or primers, as well as the full-length sequence (and mutants, alleles, variants, and derivatives) are useful in screening a test sample containing nucleic acid for the presence of alleles, mutants or variants, especially those that confer susceptibility or predisposition to cancers, via hybridization with a target sequence from a sample obtained from the individual being tested.
  • the conditions of the hybridization can be controlled to minimize non-specific binding, and preferably stringent to moderately stringent hybridization conditions are used.
  • the skilled person is readily able to design such probes, label them and devise suitable conditions for hybridization reactions, assisted by textbooks such as Sambrook et al (1989) and Ausubel et al (1992) .
  • oligonucleotides according to the present invention that are fragments of the sequence shown in Sequence ID NO: 1, or any allele associated with cancer susceptibility or aberrant cellular adhesion, are at least about 10 nucleotides in length, more preferably at least 15 nucleotides in length, more preferably at least about 20 nucleotides in length. Such fragments themselves individually represent aspects of the present invention. Fragments and other oligonucleotides may be used as primers or probes as discussed but may also be generated (e.g. by PCR) in methods concerned with determining the presence in a test sample of a sequence indicative of cancer susceptibility. Methods involving use of nucleic acids in diagnostic and/or prognostic contexts, for instance in determining susceptibility to cancer or aberrant cell-cell contact regulation, and other methods concerned with determining the presence of such sequences are discussed below.
  • Nucleic acids according to the present invention may be used in methods of gene therapy, for instance in treatment of individuals with the aim of preventing or curing (wholly or partially) cancer as discussed below.
  • HEI-C protein demonstrates association with microtubules and localization to the spindle and some cell-cell contacts.
  • a full-length HEI-C protein of the present invention may be prepared in a variety of ways, according to known methods.
  • the protein may be purified from appropriate sources, e.g., transformed bacterial or animal cultured cells or tissues, by immunoaffinity purification. However, this is not a preferred method due to the low amount of protein likely to be present in a given cell type at any time.
  • the availability of nucleic acid molecules encoding HEI-C enables production of the protein using in vi tro expression methods known in the art.
  • a cDNA or gene may be cloned into an appropriate in vi tro transcription vector, such as pSP64 or pSP65 for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocyte lysates.
  • vi tro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wisconsin or BRL, Rockville, Maryland.
  • larger quantities of HEI-C may be produced by expression in a suitable prokaryotic or eukaryotic system.
  • a DNA molecule such as the cDNA having Sequence ID NO: 1
  • a plasmid vector adapted for expression in a bacterial cell, such as E. coli .
  • Such vectors comprise the regulatory elements necessary for expression of the DNA in the host cell (e.g. E. coli ) positioned in such a manner as to permit expression of the DNA in the host cell.
  • regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences.
  • the HEI-C produced by gene expression in a recombinant prokaryotic or eukaryotic system may be purified according to methods known in the art.
  • a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium.
  • an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein or nickel columns for isolation of recombinant proteins tagged with 6-8 histidine residues at their N-terminus or C- terminus.
  • Alternative tags may comprise the FLAG epitope, GST or the hemagglutinin epitope. Such methods are commonly used by skilled practitioners.
  • the HEI-C proteins of the invention prepared by the aforementioned methods, may be analyzed according to standard procedures. For example, such proteins may be subjected to amino acid sequence analysis, according to known methods.
  • a convenient way of producing a polypeptide according to the present invention is to express nucleic acid encoding it, by use of the nucleic acid in an expression system.
  • the use of expression systems has reached an advanced degree of sophistication today.
  • the present invention also encompasses a method of making a polypeptide (as disclosed) , the method including expression from nucleic acid encoding the polypeptide (generally nucleic acid according to the invention) .
  • This may conveniently be achieved by growing a host cell in culture, containing such a vector, under appropriate conditions which cause or allow production of the polypeptide.
  • Polypeptides may also be produced in in vitro systems, such as reticulocyte lysate.
  • Polypeptides which are amino acid sequence variants, alleles, derivatives or mutants are also provided by the present invention.
  • a polypeptide which is a variant, allele, derivative, or mutant may have an amino acid sequence that differs from that given in Sequence ID NO: 2 by one or more of addition, substitution, deletion and insertion of one or more amino acids.
  • Preferred such polypeptides have HEI-C function, that is to say have one or more of the following properties: microtubule association and dynamitin and/or Ost binding; immunological cross-reactivity with an antibody reactive with the polypeptide for which the sequence is given in Sequence ID NO: 2; sharing an epitope with the polypeptide for which the sequence is given in Sequence ID NO: 2 (as determined for example by immunological cross-reactivity between the two polypeptides.
  • a polypeptide which is an amino acid sequence variant, allele, derivative or mutant of the amino acid sequence shown in Sequence ID NO: 2 may comprise an amino acid sequence which shares greater than about 35% sequence identity with the sequence shown, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90% or greater than about 95%.
  • Particular amino acid sequence variants may differ from that shown in
  • Sequence ID NO: 2 by insertion, addition, substition or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20, 20-30, 30-40, 40-50, 50-100, 100-150, or more than 150 amino acids.
  • a polypeptide according to the present invention may be used in screening for molecules which affect or modulate its activity or function. Such molecules may be useful in a therapeutic (possibly including prophylactic) context.
  • the present invention also provides antibodies capable of immunospecifically binding to proteins of the invention.
  • Polyclonal antibodies directed toward HEI-C may be prepared according to standard methods.
  • monoclonal antibodies are prepared, which react immunospecifically with various epitopes of HEI-C.
  • Monoclonal antibodies may be prepared according to general methods of K ⁇ hler and Milstein, following standard protocols. Polyclonal or monoclonal antibodies that immunospecifically interact with
  • HEI-C can be utilized for identifying and purifying such proteins.
  • antibodies may be utilized for affinity separation of proteins with which they immunospecifically interact.
  • Antibodies may also be used to immunoprecipitate proteins from a sample containing a mixture of proteins and other biological molecules. Other uses of anti-HEI-C antibodies are described below.
  • Antibodies according to the present invention may be modified in a number of ways. Indeed the term “antibody” should be construed as covering any binding substance having a binding domain with the required specificity. Thus, the invention covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including synthetic molecules and molecules whose shape mimics that of an antibody enabling it to bind an antigen or epitope.
  • Exemplary antibody fragments capable of binding an antigen or other binding partner, are Fab fragment consisting of the VL, VH, Cl and CHI domains; the Fd fragment consisting of the VH and CHI domains; the Fv fragment consisting of the VL and NH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and F(ab')2 fragments, a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragments are also included.
  • Humanized antibodies in which CDRs from a non- human source are grafted onto human framework regions, typically with alteration of some of the framework amino acid residues, to provide antibodies which are less immunogenic than the parent non-human antibodies, are also included within the present invention.
  • the HEI-C molecules of the invention may be used to advantage in genetic screening assays to identify mutations in the protein associated with aberrant cellular morphology and the metastatic invasion of normal tissues by malignant cells. Such screening assays may also be used to identify agents which disrupt and/or augment HEI-C function.
  • HEI-C nucleic acids, proteins and antibodies thereto may be used as a research tool to identify other proteins that are intimately involved in spindle formation and cell-cell adhesion.
  • HEI-C-encoding nucleic acids may be used for a variety of purposes in accordance with the present invention.
  • HEI-C-encoding DNA, RNA, or fragments thereof may be used as probes to detect the presence of and/or expression of genes encoding HEI-C proteins.
  • Methods in which HEI-C-encoding nucleic acids may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR) .
  • the HEI-C-encoding nucleic acids of the invention may also be utilized as probes to identify related genes from other animal species.
  • hybridization stringencies may be adjusted to allow hybridization of nucleic acid probes with complementary sequences of varying degrees of homology.
  • HEI-C-encoding nucleic acids may be used to advantage to identify and characterize other genes of varying degrees of relation to HEI-C, thereby enabling further characterization of spindle formation and cell-cell adhesion. Additionally, they may be used to identify genes encoding proteins that interact with HEI-C (e.g., by the "interaction trap” technique), which should further accelerate identification of the components involved in spindle formation and cell-cell adhesion.
  • Nucleic acid molecules, or fragments thereof, encoding HEI-C may also be utilized to control the production of HEI-C, thereby regulating the amount of protein available to participate in cell-cell adhesion reactions. Alterations in the physiological amount of HEI-C protein may dramatically affect the activity of other protein factors involved in maintenance of cellular morphology and junction formation.
  • the availability of HEI-C encoding nucleic acids enables the production of strains of laboratory mice carrying part or all of the HEI-C gene or mutated sequences thereof. Such mice may provide an in vivo model for HEI-C function in growth and development.
  • the HEI-C sequence information provided herein enables the production of knockout mice in which the endogenous gene encoding HEI-C has been specifically inactivated.
  • Methods of introducing transgenes in laboratory mice are known to those of skill in the art. Three common methods include: 1. integration of retroviral vectors encoding the foreign gene of interest into an early embryo; 2. injection of DNA into the pronucleus of a newly fertilized egg; and
  • a transgenic mouse carrying the human HEI-C gene is generated by direct replacement of the mouse HEI-C gene with the human gene. These transgenic animals are useful for drug screening studies as animal models for human diseases and for eventual treatment of disorders or diseases associated with biological activities modulated by HEI-C.
  • a transgenic animal carrying a "knock out" of HEI-C is useful for assessing the role of HEI-C in maintaining fidelity of adherens junctions and spindle formation.
  • mice may be generated that cannot make HEI-C protein because of a targeted mutational disruption of the HEI-C gene.
  • the term "animal” is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages.
  • a "transgenic animal” is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus.
  • transgenic animal is not meant to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant DNA molecule. This molecule may be specifically targeted to a defined genetic locus, be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • the term "germ cell line transgenic animal” refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the genetic information to offspring. If such offspring, in fact, possess some or all of that alteration or genetic information, then they, too, are transgenic animals.
  • the alteration or genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.
  • the altered HEI-C gene generally should not fully encode the same HEI-C protein native to the host animal and its expression product should be altered to a minor or great degree, or absent altogether. However, it is conceivable that a more modestly modified HEI-C gene will fall within the compass of the present invention if it is a specific alteration.
  • the DNA used for altering a target gene may be obtained by a wide variety of techniques that include, but are not limited to, isolation from genomic sources, preparation of cDNAs from isolated mRNA templates, direct synthesis, or a combination thereof.
  • ES cells may be obtained from pre-implantation embryos cultured in vitro (Evans et al., (1981) Nature 292:154-156; Bradley et al., (1984) Nature 309:255- 258; Gossler et al., (1986) Proc. Natl. Acad. Sci. 83:9065-9069).
  • Transgenes can be efficiently introduced into ES cells by standard techniques such ( as DNA transfection or by retrovirus-mediated transduction.
  • the resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal.
  • the introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
  • One approach to the problem of determining the contributions of individual genes and their expression products is to use isolated HEI-C genes to selectively inactivate the wild-type gene in totipotent ES cells (such as those described above) and then generate transgenic mice.
  • the use of gene- targeted ES cells in the generation of gene-targeted transgenic mice was described, and is reviewed elsewhere (Frohman et al . , (1989) Cell 56:145-147; Bradley et al., (1992) Bio/Technology 10:534-539).
  • Nonhomologous plasmid-chromosome interactions are more frequent occurring at levels 10 5 -fold to 10 2 - fold greater than comparable homologous insertion.
  • various strategies have been developed to detect or select rare homologous recombinants.
  • One approach for detecting homologous alteration events uses the polymerase chain reaction (PCR) to screen pools of transformant cells for homologous insertion, followed by screening of individual clones.
  • PCR polymerase chain reaction
  • a positive genetic selection approach has been developed in which a marker gene is constructed which will only be active if homologous insertion occurs, allowing these recombinants to be selected directly.
  • PNS positive-negative selection
  • HSV-TK Herpes Simplex virus thymidine kinase
  • GANC gancyclovir
  • FIAU FIAU
  • a “targeted gene” or “knockout” is a DNA sequence introduced into the germline or a non-human animal by way of human intervention, including but not limited to, the methods described herein.
  • the targeted genes of the invention include DNA sequences which are designed to specifically alter cognate endogenous alleles.
  • transgenic mice of the invention Methods of use for the transgenic mice of the invention are also provided herein.
  • Therapeutic agents for the treatment or prevention of cancer metastasis may be screened in studies using HEI-C transgenic mice.
  • HEI-C knockout mice may be used to produce an array of monoclonal antibodies specific for HEI-C protein.
  • HEI-C-encoding nucleic acids are also used to advantage to produce large quantities of substantially pure HEI-C protein, or selected portions thereof.
  • Purified HEI-C may be used to produce polyclonal or monoclonal antibodies which also may serve as sensitive detection reagents for the presence and accumulation of HEI-C (or complexes containing HEI-C) in mammalian cells.
  • Recombinant techniques enable expression of fusion proteins containing part or all of the HEI-C protein.
  • the full length protein or fragments of the protein may be used to advantage to generate an array of monoclonal antibodies specific for various epitopes of the protein, thereby providing even greater sensitivity for detection of the protein in cells .
  • Polyclonal or monoclonal antibodies immunologically specific for HEI-C may be used in a variety of assays designed to detect and quantitate the protein. Such assays include, but are not limited to: (1) flow cytometric analysis; (2) immunochemical localization of HEI-C in tumor cells; and (3) immunoblot analysis (e.g., dot blot, Western blot) of extracts from various cells. Additionally, as described above, anti-HEI-C can be used for purification of HEI-C (e.g., affinity column purification, immunoprecipitation) .
  • HEI-C-encoding nucleic acids, HEI-C expressing vectors, HEI-C proteins and anti-HEI-C antibodies of the invention can be used to detect HEI-C gene expression and alter HEI-C protein accumulation for purposes of assessing the genetic and protein interactions involved in maintenance of cell-cell contacts and spindle formation.
  • Exemplary approaches for detecting HEI-C nucleic acid or polypeptides/proteins include: a) comparing the sequence of nucleic acid in the sample with the HEI-C nucleic acid sequence to determine whether the sample from the patient contains mutations; or b) determining the presence, in a sample from a patient, of the polypeptide encoded by the HEI-C gene and, if present, determining whether the polypeptide is full length, and/or is mutated, and/or is expressed at the normal level; or c) using DNA restriction mapping to compare the restriction pattern produced when a restriction enzyme cuts a sample of nucleic acid from the patient with the restriction pattern obtained from normal HEI-C gene or from known mutations thereof; or, d) using a specific binding member capable of binding to a HEI-C nucleic acid sequence (either normal sequence or known mutated sequence) , the specific binding member comprising nucleic acid hybridizable with the HEI-C sequence, or substances comprising an antibody domain
  • a “specific binding pair” comprises a specific binding member (sb ) and a binding partner (bp) which have a particular specificity for each other and which in normal conditions bind to each other in preference to other molecules.
  • specific binding pairs are antigens and antibodies, ligands and receptors and complementary nucleotide sequences. The skilled person is aware of many other examples and they do not need to be listed here.
  • the term "specific binding pair" is also applicable where either or both of the specific binding member and the binding partner comprise a part of a large molecule.
  • the specific binding pair are nucleic acid sequences, they will be of a length to hybridize to each other under conditions of the assay, preferably greater than 10 nucleotides long, more preferably greater than 15 or 20 nucleotides long.
  • the HEI-C nucleic acid in the sample will initially be amplified, e.g. using PCR, to increase the amount of the analyte as compared to other sequences present in the sample.
  • the identification of the HEI-C gene and its association with aberrant cellular morphology and tissue structure paves the way for aspects of the present invention to provide the use of materials and methods, such as are disclosed and discussed above, for establishing the presence or absence in a test sample of a variant form of the gene, in particular an allele or variant specifically associated with cancer. This may be useful for diagnosing a predisposition of an individual to cancer. It may be useful for diagnosing cancer of a patient with a form of cancer associated with the HEI-C gene.
  • the approach further facilitates treatment by identifying those patients most likely to benefit.
  • methods of screening drugs for cancer therapy to identify suitable drugs for restoring HEI-C product functions are provided. Loss of HEI-C function/expression may be associated with tissue invasion by metastatic cancer cells. Restoration of HEI-C function by gene transfer or by pharmacological means may prevent such metastatic events.
  • the HEI-C polypeptide or fragment employed in drug screening assays may either be free in solution, affixed to a solid support or within a cell.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • a known ligand such as Ost or dynamitin
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the HEI-C polypeptides and is described in detail in Geysen, PCT published application WO 84/03564, published on Sep. 13, 1984. Briefly stated, large numbers of different, small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with HEI-C polypeptide and washed. Bound HEI-C polypeptide is then detected by methods well known in the art . Purified HEI-C can be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies to the polypeptide can be used to capture antibodies to immobilize the HEI-C polypeptide on the solid phase.
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of specifically binding the HEI-C polypeptide compete with a test compound for binding to the HEI-C polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants of the HEI-C polypeptide.
  • a further technique for drug screening involves the use of host eukaryotic cell lines or cells (such as described above) which have a nonfunctional HEI-C gene. These host cell lines or cells are defective at the HEI-C polypeptide level. The host cell lines or cells are grown in the presence of drug compound. The rate of growth of the host cells and/or cellular morphology is assessed to determine if the compound is capable of regulating the growth of HEI-C defective cells.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo . See, e.g., Hodgson, (1991) Bio/Technology 9:19-21.
  • one first determines the three- dimensional structure of a protein of interest (e.g., HEI-C polypeptide) or, for example, of the HEI-C/microtubule containing complex, by x-ray crystallography, by nuclear magnetic resonance, by computer modeling or most typically, by a combination of approaches. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous proteins.
  • An example of rational drug design is the development of HIV protease inhibitors (Erickson et al., (1990) Science 249:527-533).
  • peptides may be analyzed by an alanine scan Wells, (1991) Meth. Enzy . 202:390-411. In this technique, an amino acid residue is replaced by Ala, and its effect on the peptide' s activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.
  • drugs which have, e.g., improved HEI-C polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of HEI-C polypeptide activity.
  • improved HEI-C polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of HEI-C polypeptide activity.
  • sufficient amounts of the HEI-C polypeptide may be made available to perform such analytical studies as x- ray crystallography.
  • the knowledge of the HEI-C protein sequence provided herein will guide those employing computer modeling techniques in place of, or in addition to x-ray crystallography.
  • HEI-C in target cell sensitivity to anti-microtubule drugs, including, but not limited to paclitaxel, docetaxel, vinblastine, and vincristine, can be assessed using a number of in vitro assays well known to skilled artisans.
  • Such assays include, but are not limited to, cellular assays in which the active uptake or exclusion of cellular dyes (i.e., MTT, trypan blue) indicates target cell viability, binding assays in which the drug of choice is tagged with a detectable moiety (i.e., fluorescein-conjugated paclitaxel or vinblastine) , which facilitates visualization of drug binding to microtubules in living or fixed cells, and in vi tro microtubule binding assays such as microtubule pulldown assays as described herein. As described above, such assays can be used to screen drugs which modulate HEI-C polypeptide activity.
  • cellular assays in which the active uptake or exclusion of cellular dyes (i.e., MTT, trypan blue) indicates target cell viability
  • binding assays in which the drug of choice is tagged with a detectable moiety (i.e., fluorescein-conjugated paclitaxel or vin
  • the HEI-C polypeptides/proteins, antibodies, peptides and nucleic acids of the invention can be formulated in pharmaceutical compositions.
  • These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other material well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes .
  • administration is preferably in a "prophylactically effective amount" or a
  • therapeutically effective amount (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • the nucleic acid encoding the authentic biologically active HEI-C polypeptide could be used in a method of gene therapy, to treat a patient who is unable to synthesize the active "normal” polypeptide or unable to synthesize it at the normal level, thereby providing the effect elicited by wild-type HEI-C and suppressing the activity of "abnormal" HEI-C lacking the ability to effectively form adherens junctions.
  • Vectors such as viral vectors have been used in the prior art to introduce genes into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transformation can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide.
  • the transfected nucleic acid may be permanently incorporated into the genome of each of the targeted tumor cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically.
  • viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, herpes viruses including HSV and EBV, and retroviruses .
  • papovaviruses such as SV40
  • vaccinia virus vaccinia virus
  • herpes viruses including HSV and EBV
  • retroviruses retroviruses .
  • Many gene therapy protocols in the prior art have employed disabled murine retroviruses .
  • Gene transfer techniques which selectively target the HEI-C nucleic acid to affected tissues are preferred. Examples of this include receptor- mediated gene transfer, in which the nucleic acid is linked to a protein ligand via polylysine, with the ligand being specific for a receptor present on the surface of the target cells. Other delivery methods are available for targeted delivery of nucleic acids to specific cells. Such compositions and methods are provided for example in U.S. Patent Nos. 6,248,363 and 5,879,713, the entire disclosures of which are incorporated by reference herein. The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way. EXAMPLE I
  • a HeLa cell cDNA library constructed in pJG4-4 (TRP+;cDNAs inducibly expressed under the control of the GAL1 promoter; gift of J.Gyuris) was transformed into the Saccharomyces cerevisiae strain CGX74 (MATa/a trpl/trpl; gift of J. Gyuris) a diploid strain capable of forming pseudohyphae.
  • Plasmids The cDNA corresponding to HEI22 was subcloned into pucll9 by PCR (nt 220-nt 837) 5'CCC GGA TCC CAA TTG GAC CTT CTC ATG GAG AGT GTG 3' (SEQ ID NO: 3); 5' GCG CTC GAG GGG AAA ATG TTT ATT TGG C 3' (SEQ ID NO: 4) generating a 5' Muni site and utilizing a 3' Xhol site. This cDNA insert was subcloned into pEG202 to construct pEG202-HEI22.
  • DSIEAELTRRVDMMEL (SEQ ID NO: 5) was conjugated to KLH and used as an immunogen to induce production of polyclonal rabbit antiserum.
  • the antibodies were affinity purified prior to use.
  • oligonucleotide probe [5' TTT TAG AAA GTC CAT GTT CTG ACG ACG 3' (SEQ ID NO: 6)] was 5' end labeled with ⁇ - 32 P and used to probe a multiple tissue northern blot (MTN, CLONTECH) containing poly
  • Anti-HEI-C antipeptide antibodies were affinity purified prior to use. These antibodies were used at a 1:100 dilution for Western analysis and immunofluorescence. Monoclonal anti-tubulin antibody (DMlA, Sigma) was used at a 1:2000 dilution for immunofluorescence. Anti-p50 dynamitin antibodies (Transduction Labs) were used at a 1:500 dilution for immunofluorescence .
  • MCF7, HeLa, and COS cells were grown in DMEM +10% FBS
  • MDCK cells were grown in DMEM low glucose +10% FBS. All media contained penicillin and streptomycin.
  • MDCK cells were grown on coverslips thinly coated with collagen (rat tail collagen type I, Becton Dickinson) . Collagen was diluted to 50 ⁇ g/ml using 0.02N acetic acid and added to coverslips to attain 5 ⁇ g/cm 2 . The coverslips were incubated for 1 hour at room temperature at which time the collagen solution was aspirated and the coverslips were washed extensively with PBS.
  • the blot was probed with affinity purified anti-HEI-C antibodies at a 1:100 dilution for 1 hour at room temperature in 1% nonfat milk in TBST. The blot was washed 3 times in excess TBST for 5 minutes each wash. Secondary horseradish peroxidase (HRP) conjugated anti-rabbit antibody (Amersham) was added at a dilution of 1:4000 in 1% nonfat milk for 1 hour at room temperature. The blot was washed as before and developed with chemoluminescence (NEN; Renaissance) .
  • HRP horseradish peroxidase conjugated anti-rabbit antibody
  • a two hybrid screen was performed using pEG202- HEI22 in EGY191.
  • a screen of approximately 288,000 transformants was performed with standard reagents as previously described. Positive clones were isolated and transformed into KC8 bacteria and inserts were sequenced.
  • Paraformaldehyde fixation Cells were washed with PBS, incubated in 4% paraformaldehyde in PBS pH 7.2 for 10 minutes. The cells were permeabilized in PBS + 0.2% Triton for 5 minutes.
  • MDCK cells were allowed to grow to confluence and form a monolayer on coverslips. The narrow end of a pasteur pipette was dragged across the surface of the coverslip. Cells were then incubated for varying amounts of time and fixed in 4% paraformaldehyde. All samples were processed simultaneously for immunofluorescence as described above.
  • a HeLa cell library was transformed into CGX74 (MATa/a trpl/trpl; gift of J. Gyuris) a diploid strain capable of forming pseudohyphae.
  • the library vector contains the TRP locus to allow for selection on -TRP plates, and the human cDNA inserts are inducibly expressed in the presence of galactose. Greater than 500,000 primary transformed colonies were screened by comparing colony invasiveness on -Trp glucose versus -Trp galactose by washing the plates vigorously with running water. As previously reported, dipoid yeast are not expected to invade agar on rich media and the vast majority of cells plated in this experiment were washed away.
  • Trp-GAL plates Twenty four patches were observed on the Trp-GAL plates which on visual inspection contained highly filamentous cells. Fourteen of the original twenty four positives repeated the invasive and filamentous phenotypes after restreaking. Plasmid DNA was prepared from these colonies and used to retransform naive CGX74 yeast which were assayed for enhanced filamentation and invasion on Trp-GAL and SLAGR (low nitrogen media with Galactose/Raffinose as the carbon source) . All plasmids recapitulated the original phenotype.
  • HEF1 a gene previously cloned in the Golemis lab in a yeast screen for human genes which enhance filamentation.
  • Three of the original clones represented regions of PRK2 ( Figure IA) .
  • the region of PRK2 isolated encompasses the C-terminal half of the protein including the kinase domain.
  • Four of the original clones represented clones of a novel gene, termed human enhancer of invasion- cluster (HEI-C; Figure IA) .
  • the sequence of HEI-C has a coiled coil region and a putative nuclear export sequence in its C- terminus .
  • a search of the Genbank does not reveal homology to any other protein of known function.
  • HEF1 causes pseudohyphal growth in diploids only
  • HEI-C and PRK2 caused filamentation and invasiveness in both haploids and diploids ( Figure IB) .
  • the ability of PRK2 to induce this phenotype was dependent on an intact kinase domain as a kinase dead version of the invasive clone reduced the pseudohyphal and invasive phenotype .
  • HEI-C Confocal analysis of MDCK cells plated on collagen coated coverslips for 24 hours was also performed. HEI-C was found localized to regions of cell-cell contact on the basolateral surface. The HEI-C staining pattern was proximal to E-cadherin, and in some regions colocalized with that of E- cadherin. The HEI-C staining pattern was, however, heterogeneous, as it was not observed at all cell- cell contacts. When MDCK cells plated on collagen coated coverslips were allowed to grow to a monolayer, the majority of cells had a very low diffuse cytoplas ic staining pattern for HEI-C. A subset of cells in the monolayer exhibited prominent HEI-C staining at cell-cell contacts, partially colocalizing with E-cadherin staining ( Figures 4A- F).
  • FIG. 5A-C show a group of cells located between the wound edges.
  • HEI-C was localized to the interior cell-cell contacts in the multi-cellular island.
  • HEI-C staining has not been observed at the lamellipodia of the wound edges or in the cells which have migrated from the wound.
  • HEI-C staining at the cell-cell contacts in multi- cellular islands was generally not coincident with
  • the mitotic spindle is a cytoskeletal apparatus required to properly segregate chromosomes during cell division.
  • the spindle consists primarily of microtubules, specialized motors, and accessory factors which provide the mechanical force needed to congregate and segregate the chromosomes, as well as providing the checkpoints necessary for proper chromosome segregation.
  • One of the motors known to participate in the function of the mitotic spindle is the dynein/dynactin motor complex (Gaglio et al, 1997; Quintyne et al . 1999) .
  • the dynein/dynactin motor complex consists of the motor protein dynein and the accessory factors in the dynactin complex which act as processivity factors for the dynein motor.
  • One component of the dynactin complex is dynamitin. Injection of anti-dynein antibodies into cultured cells disrupts the formation and maintenance of a preformed spindle (Gaglio et al. 1997). Overexpression of dynamitin in cultured cells leads to the misalignment of spindles (Echeverri et al . 1996). The role of dynein/dynactin at the spindle is not fully understood, however it is thought to counteract other motors at the spindle to allow for the creation of the appropriate mechanical force needed to facilitate proper spindle function.
  • HEI-C appears to function as a regulator of dynein/dynactin at the spindle and may also participate as a constituent of the motor complex in all of its cellular functions, including endocytosis and organelle maintenance.
  • the localization of HEI- C at cell-cell contacts in interphase cells, a site which has not been characterized for dynein/dynactin) suggests that it plays a specialized role through its interaction with dynamitin at the mitotic spindle.
  • HEF-1 a docking protein found at focal adhesions and known to be involved in the regulation of the dynamics of focal adhesions has been shown to be localized to the mitotic spindle.
  • Zyxin a proline-rich zinc finger protein is found at focal adhesion sites, and recently has been localized to the mitotic spindle in a complex with the h-warts/LATSl tumor suppressor protein (Hirota et al, 2000) .
  • chromosome segregation is crucial, because chromosome loss or aneuploidy is a hallmark of transformed cells. Elucidation of the molecular components which play a role in the convergence of cell adhesion and mitotic pathways facilitates the development of therapeutic agents which regulate this process.
  • HEI-C was immunolocalized to the spindle in mitotic cells, including HeLa, MDCK (data not shown) and MCF7 cells ( Figures 6A-D) .
  • MCF7 cells arrested with l ⁇ M nocadozole for 14 hours and released into fresh media lacking nocodazole, HEI-C associated with spindles upon spindle formation.
  • HEI-C remained on the spindle throughout the cell cycle, and was observed at the midzone during cytokinesis and at the midbody after cytokinesis ( Figures 7A-F) . Two Hybrid Analysis .
  • HEI-C colocalizes with dynamitin at the spindle.
  • Immunofluorescence analysis of HEI-C and p50 dynamitin immunofluorescence demonstrates that these proteins do not colocalize in resting cells but do co-localize at the mitotic spindle. This colocalization continues throughout the cell cycle and includes staining of the midbody ( Figures 7A- 7F) .
  • HEI-C is enriched in aster preparations from mitotic cells .
  • Extracts were prepared from HeLa cells which had been arrested in nocodazole, lysed, and taxol supplemented.
  • the asters formed were pelleted by centrifugation, and the pellet and supernatant were subjected to electrophoresis and western analysis. Approximately 30% of the HEI-C was in the microtubule containing pellet.
  • RACE 5' Rapid Amplification of cDNA Ends
  • an internal gene specific primer [EG984: 5' AGA TCA GAG GTC AAA TCA TTC ACT GCA GGG 3' (SEQ ID NO: 9)] was used to prime cDNA synthesis.
  • the products of this reaction were subcloned and sequenced.
  • Regions of HEI-C were subcloned into the yeast expression vector pJG4-4 by PCR. The fragments generated were flanked on the 5' end with a Muni site and on the 3' end by an Xhol site, and were ligated to EcoRI and Xhol digested pJG4-4.
  • the resulting yeast HEI-C expression constructs corresponded to the following amino acids: full length HEI-C: aa. 1-278; 79CC2/3: aa. 79-278; 124CC2/3: aa. 124-278; 177CC3: aa . 177-278. Results
  • RACE was performed on total RNA derived from HeLa cells utilizing a gene specific primer to the known HEI-C ATG. The fragments obtained were cloned and sequenced, and one clone was obtained which contained additional 5' sequence [ Figures 9A and B, coding strand (SEQ ID NO: 7) and non-coding strand (SEQ ID NO: 8)]. This sequence contained an in- frame STOP codon. This result confirmed that the HEI-C cDNA cloned as a result of the invasion screen was indeed the full length coding region of the gene.
  • the primary sequence motifs found in the HEI-C cDNA are the three coiled coil domains. In order to assess the contribution of these domains to the enhancement of invasion, each coiled coil was sequentially deleted from the cDNA.
  • the resulting constructs 79CC2/3 (amino acids 79-278); 124CC2/3 (amino acids 124-278) and 177CC3 (amino acids 177- 278) were compared to full length HEI-C and vector alone for their ability to confer invasiveness in the agar invasion assay.
  • the yeast strains overexpressing each construct were grown on galactose containing media for 24 hours to induce expression of the HEI-C protein and the appearance of the yeast was recorded.
  • Microtubule Pulldown Microtubules were polymerized from purified tubulin (Molecular Probes, Eugene OR) by incubation in G-PEM buffer (100 mM Pipes pH 6.8, 1 mM EGTA, 1 mM MgS0 4 , 1 mM GTP, and 30 % glycerol) at 37°C for 10 minutes and subsequent addition of paxlitaxel (SIGMA, St. Louis, MO) to a final concentration of G-PEM buffer (100 mM Pipes pH 6.8, 1 mM EGTA, 1 mM MgS0 4 , 1 mM GTP, and 30 % glycerol) at 37°C for 10 minutes and subsequent addition of paxlitaxel (SIGMA, St. Louis, MO) to a final concentration of G-PEM buffer (100 mM Pipes pH 6.8, 1 mM EGTA, 1 mM MgS0 4 , 1 mM GTP, and 30 % glycerol)
  • HeLa cells were lysed by dounce homogenization in PHEM buffer (100 mM Pipes pH 6.9, 25 mM Hepes pH 6.9, 1 mM EGTA, 2 mM MgS0 4 ) plus 0.1 mg/ml aprotinin, O.lmg/ml leupeptin.
  • the cell lysate was precleared by centrifugation at 52,000 x g for 1 hour at 4°C. The supernatant was incubated for 30 minutes at 37°C with 5 ⁇ g polymerized microtubules.
  • the reactions were layered on a 15 % sucrose solution in PB buffer (80 mM Pipes pH 6.9, 1 mM EGTA, 1 mM CaCl 2 ) and centrifuged at 30,000 x g for 30 minutes at room temperature. Pellets were resuspended in PHEM buffer and the respective supernatants stored at -20°C until use.
  • PB buffer 80 mM Pipes pH 6.9, 1 mM EGTA, 1 mM CaCl 2
  • HEI-C is a microtubule associated protein.
  • HEI-C may be able to associate with microtubules, the primary structural component of the spindle apparatus.
  • a microtubule pulldown assay was used to determine if HEI-C was competent to associate with microtubules.
  • HeLa cell extracts were incubated at 37°C with purified, polymerized microtubules. These reactions were then layered onto a sucrose cushion and centrifuged to separate the polymerized microtubules and associated proteins recovered in the pellet from the remainder of the lysate found in the supernatant.
  • HEI-C as a MAP is intriguing in view of the correlation between the regulation of MAP4 and sensitivity of cells to chemotherapeutic drugs (Zhang et al., 1999). Thus, alterations in HEI-C levels within a cell have the potential to alter sensitivity of such a cell to drugs which target microtubules. In view of its specific association with the mitotic spindle, HEI-C may have a particularly profound influence on the drug sensitivity of cells undergoing mitosis.
  • U20S cells were treated for 14 hours with 2 mM thymidine. After 14 hours, the cells were washed twice with excess drug- free media and incubated in drug-free media for 9 hours. Following this incubation, 2 mM thymidine was added to the media for an additional 15 hours. The cells were washed and lysed. Cells were blocked in mitosis following 15 hours of treatment with 1 ⁇ M nocodazole. Cells were harvested by mitotic shake off, washed and lysed.
  • HEI-C was posttranslationally modified in mitotic cells.
  • HEI-C was posttranslationally modified during different phases of the cell cycle.
  • U20S cells were arrested in S phase by double thymidine block or in mitosis by treatment with nocodazole. Lysates from S phase arrested cells were compared by Western analysis with lysates from asynchronous cells.
  • HEI-C isolated from double thymidine blocked cells co- migrated with the HEI-C species observed in asynchronously growing cells.
  • HEI-C isolated from cells arrested in mitosis migrated as a doublet indicating that HEI-C was specifically posttranslationally modified in mitotic cells.
  • HEI-C post-translational modification of HEI-C which occurred in response to the microtubule destabilizing drug nocodazole provides further support for a correlation between HEI-C activity and microtubule status.
  • treatment of cells with nocodazole also causes mitotic cell cycle arrest, in addition to destabilization of microtubules.
  • the mitotic cell cycle arrest rendered by nocodazole treatment is due to the engagement of a mitotic checkpoint.
  • HEI-C may be a target of at least one enzyme, a kinase for example, which regulates this mitotic checkpoint.
  • HEI-C could, therefore, be used as an in vi tro substrate to monitor the activity of such a mitotic enzyme.
  • the kinases involved in the mitotic checkpoint, and substrate targets thereof, are of particular interest as the genes encoding many of these kinases have been mutated in human cancer cells.
  • HEI-C encoding nucleic acids, polypeptides and antibodies immunologically specific thereto may be used to advantage in assays to identify therapeutic agents which influence cellular adhesion interactions and mitosis.

Abstract

La présente invention concerne des acides nucléiques codant HEI-C, des polypeptides HEI-C et des anticorps HEI-C immunologiquement spécifiques. Par ailleurs, cette invention concerne leurs méthodes d'utilisation.
PCT/US2001/019746 2000-06-20 2001-06-20 Acides nucleiques codant hei-c, polypeptides hei-c, anticorps hei-c et leurs methodes d'utilisation WO2001098459A2 (fr)

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DATABASE GENBANK [Online] 14 July 1998 KIMMERLY W. ET AL., XP001055018 Database accession no. AC005214 *

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