WO2001018019A1 - Proteine epitheliale perdue dans le neoplasme (eplin) - Google Patents

Proteine epitheliale perdue dans le neoplasme (eplin) Download PDF

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WO2001018019A1
WO2001018019A1 PCT/US2000/024689 US0024689W WO0118019A1 WO 2001018019 A1 WO2001018019 A1 WO 2001018019A1 US 0024689 W US0024689 W US 0024689W WO 0118019 A1 WO0118019 A1 WO 0118019A1
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eplin
cell
nucleic acid
seq
polypeptide
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PCT/US2000/024689
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English (en)
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David D. Chang
Raymond S. Maul
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The Regents Of The University Of California
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Priority to AU73610/00A priority Critical patent/AU7361000A/en
Publication of WO2001018019A1 publication Critical patent/WO2001018019A1/fr

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates generally to gene expression in normal and neoplastic cells, and specifically to a novel tumor suppressor gene, EPLIN (epithelial protein lost in neoplasm), and its gene products.
  • Cancer genes are broadly classified into “oncogenes” which, when activated, promote tumorigenesis, and “tumor suppressor genes” which, when nonfunctional, fail to suppress tumori genesis. While these classifications provide a useful method for conceptualizing tumorigenesis, it is also possible that a particular gene may play differing roles depending upon the particular allelic form of that gene, its regulatory elements, the genetic background and the tissue environment in which it is operating.
  • Oncogenes are somatic cell genes that are mutated from their wild-type alleles (the art refers to these wild-type alleles as protooncogenes) into forms which are able to induce tumorigenesis under certain conditions.
  • Tumor suppressor genes are genes that, in their wild-type alleles, express proteins that suppress abnormal cellular proliferation. When the gene coding for a tumor suppressor protein is mutated, deleted or transcriptionally nonfunctional, the resulting absence of wild- type tumor suppressor protein expression promotes abnormal cellular proliferation. A number of well-studied human tumors and tumor cell lines have been shown to have missing or nonfunctional tumor suppressor genes.
  • tumor suppression genes include, but are not limited to, the retinoblastoma susceptibility gene or RB gene, the p53 gene, the deleted in colon carcinoma (DCC) gene and the neurof ⁇ bromatosis type 1 (NF-1) tumor suppressor gene (Weinberg, R. A. Science, 1991, 254:1138). Loss of function or inactivation of tumor suppressor genes may play a central role in the initiation and/or progression of a significant number of human cancers.
  • the present invention shows that many cancers exhibit decreased EPLIN expression relative to their tissues of origin.
  • the limitation and failings of the prior art to provide meaningful markers which correlate with the presence of cell proliferative disorders, such as cancer, has created a need for markers which can be used diagnostically, prognostically, and therapeuticaUy over the course of such disorders.
  • the present invention fulfills such a need.
  • the present invention is based on the seminal discovery of a novel tumor suppressor gene, EPLIN (epithelial protein lost in neoplasm), the expression of which is altered in multiple common human tumor types.
  • EPLIN epidermal protein lost in neoplasm
  • the invention provides EPLIN polypeptides (SEQ ID NO:2 and SEQ ID NO:4) as well as polynucleotide sequences encoding the polypeptides (SEQ ID NO: 1 and SEQ ID NO:3) and antibodies which bind to the polypeptides set forth in SEQ ID NO:2 and SEQ ID NO:4.
  • an expression vector containing EPLIN nucleic acid is provided. Also included is a method for producing the EPLIN polypeptides and antibodies which bind to the EPLIN polypeptides.
  • the invention provides a method for identifying a compound which binds to EPLIN polypeptide that includes incubating components comprising the compound and EPLIN polypeptide under conditions sufficient to allow the components to interact and measuring the binding of the compound to EPLIN polypeptide.
  • the present invention provides a method of detecting a neoplastic cell in a sample by contacting a sample suspected of containing a neoplastic cell with a reagent that binds to an EPLIN-specific cell component and detecting binding of the reagent to the component.
  • the invention provides a method of detecting a cell proliferative disorder in a sample from a subject by contacting a first sample having, or suspected of having, a cell proliferative disorder with a reagent that binds to an EPLIN-specific cell component and detecting binding of the reagent to the component; contacting a second cell not having a cell proliferative disorder with a reagent that binds to an EPLIN-specific cell component and detecting binding of the reagent to the component; comparing the level of binding in the first sample with the level of binding in the second sample, wherein a decreased level of binding of the reagent to an EPLIN-specific cell component from the first sample is indicative of a cell proliferative disorder.
  • a kit useful for the detection of an EPLIN-specific cell component comprising carrier means containing one or more containers comprising a first container containing an
  • the present invention provides a method of ameliorating a cell proliferative disorder associated with EPLIN, comprising administering to a subject with the disorder, a therapeuticaUy effective amount of reagent which regulates EPLIN activity.
  • the invention provides a method of gene therapy comprising introducing into cells of a host subject, an expression vector comprising a nucleotide sequence encoding EPLIN, in operable linkage with a promoter.
  • Figure 1A shows a schematic diagram of two EPLIN cDNAs. The sequence of two isoforms diverge at the 5' end (indicated by the stripped and dotted boxes).
  • Figure IB shows the deduced amino acid sequence of EPLIN- ⁇ .
  • Figure 1C shows the alignment of the EPLIN LIM domain sequence with the LIM domain of the mutant SREBP-2, KIAA0750, plant transcription factor SF3, and muscle LIM protein.
  • Figure 2A shows that EPLIN is preferentially expressed in epithelial cells as determined by Northern analysis.
  • Figure 2B shows the expression of EPLIN in different human primary cells by an immunoblot analysis.
  • Figure 3 A shows, by Northern analysis, that the expression of EPLIN transcript is lost in epithelial cancer cells.
  • Figure 3B shows the expression of EPLIN proteins in different prostate cancer cell lines and xenograft tumors as determined by an immunoblot analysis.
  • Figure 3C shows the expression of EPLIN proteins in different breast cancer cell lines as determined by an immunoblot analysis.
  • Figure 3D shows the expression of EPLIN transcripts in different breast cancer cell lines as determined by a Northern analysis.
  • Figure 4A shows the relative amount of EPLIN isoforms in HOK18C (a HPV- immortalized human oral keratinocyte cell line) and BeWo (a human choriocarcinoma cell line) as determined by an immunoblot analysis.
  • Figure 4B shows the subcellular localization of EPLIN as determined by in situ immunofluorescence using anti-EPLIN antibodies and Texas Red-conjugated goat anti-rabbit IgG secondary antibody.
  • Figure 4C shows the staining of actin stress fibers with Oregon Green-phalloidin.
  • Figure 4D shows the subcellular localization of EPLIN as determined by in situ immunofluorescence using anti-EPLIN antibodies and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG secondary antibody.
  • Figure 4E shows the staining of actin stress fibers with Texas Red-phalloidin.
  • Figure 5 A shows U2-OS osteosarcoma cultured without expression of EPLIN- ⁇ .
  • Figure 5B shows U2-OS osteosarcoma cultured with expression of EPLIN- .
  • Figure 5C shows U2-OS osteosarcoma cultured without expression of EPLIN- ⁇ .
  • Figure 5D shows U2-OS osteosarcoma cultured with expression of EPLIN- ⁇ .
  • Figure 5E shows the levels of EPLIN expression in the U2-OS cells cultures minus (no induction) and plus (induction) doxycycline as determined by an immunoblot analysis.
  • Figure 5F shows the growth of U2-OS cells presented as the ratio of cell numbers with and without EPLIN induction.
  • the present invention provides the novel tumor suppressor gene EPLIN (epithelial protein lost in neoplasm) encoding novel cytoskeletal proteins preferentially expressed in human epithelial cells.
  • EPLIN epithelial protein lost in neoplasm
  • the invention further provides a polynucleotide (SEQ ID NO: 1) encoding the amino acid sequence for EPLIN- ⁇ and a polynucleotide (SEQ ID NO: 3) encoding the amino acid sequence for EPLIN- ⁇ .
  • EPLIN protein The EPLIN protein, fragments thereof, and derivatives and other variants of the sequence in SEQ ID NO:2 or SEQ ID NO:4 thereof are collectively referred to as "polypeptides or proteins of the invention” or “EPLIN polypeptides or proteins.”
  • Nucleic acid molecules encoding such polypeptides or proteins are collectively referred to as “nucleic acids of the invention” or “EPLIN nucleic acids.”
  • EPLIN molecules refer to EPLIN nucleic acids, polypeptides, and antibodies.
  • nucleic acid molecule includes DNA molecules (e.g. , a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA generated, e.g. , by the use of nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • isolated or purified nucleic acid molecule includes nucleic acid molecules that are separated from other nucleic acid molecules that are present in the natural source of the nucleic acid.
  • isolated includes nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated.
  • an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and/or 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of 5' and/or 3' nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • hybridizes under stringent conditions describes conditions for hybridization and washing.
  • Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used.
  • a preferred example of stringent hybridization conditions includes hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50°C.
  • SSC sodium chloride/sodium citrate
  • stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 55°C.
  • a further example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C.
  • stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C.
  • Particularly preferred stringency conditions are 0.5M Sodium Phosphate, 1% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1%) SDS at 65°C.
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NOs:l or 3, corresponds to a naturally occurring nucleic acid molecule.
  • a "naturally occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g. , encodes a natural protein).
  • gene and “recombinant gene” refer to nucleic acid molecules that include an open reading frame encoding an EPLIN protein, preferably a mammalian EPLIN protein, and further can include non-coding regulatory sequences and introns.
  • an “isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free” means preparation of EPLIN protein having less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-EPLIN protein (also referred to herein as a "contaminating protein”), or of chemical precursors or non-EPLIN chemicals.
  • the EPLIN protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i. e.
  • culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.
  • non-essential amino acid residue is a residue that can be altered from the wild- type sequence of EPLIN (e.g., the sequence of SEQ ID NO:2 or 4) without abolishing or more preferably, without substantially altering a biological activity of the EPLIN protein, whereas an "essential" amino acid residue results in such a change.
  • amino acid residues that are conserved among the polypeptides of the present invention are predicted to be particularly unamenable to alteration.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • a predicted nonessential amino acid residue in an EPLIN protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of an EPLIN coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for EPLIN biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 1 or 3, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
  • a "biologically active portion" of an EPLIN protein includes a fragment of an EPLIN protein that participates in an interaction between an EPLIN molecule and a non-EPLIN molecule.
  • Biologically active portions of an EPLIN protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the EPLIN protein, e.g., the amino acid sequence shown in SEQ ID NO:2, which include less amino acids than the full length EPLIN protein and exhibit at least one activity of an EPLIN protein, such as tumor suppressor activity.
  • biologically active portions comprise a domain or motif with at least one activity of the EPLIN protein.
  • a biologically active portion of an EPLIN protein can be a polypeptide that is, for example, 10, 25, 50, 100, 200, 300 or more amino acids in length.
  • Biologically active portions of an EPLIN protein can be used as targets for developing agents that modulate an EPLIN mediated activity. Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%), more preferably at least 50%), even more preferably at least 60%, and even more preferably at least 70%>, 80%, 90%, 100% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol, 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters is using a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J Mol. Biol, 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res., 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST See http://www.ncbi.nlm.nih.gov.
  • “Misexpression or aberrant expression,” as used herein, refers to a non-wild type pattern of gene expression at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over or underexpression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post- transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the
  • the present invention provides substantially pure EPLIN polypeptides consisting essentially of the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:4.
  • substantially pure refers to EPLIN polypeptide that is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • One skilled in the art can purify EPLIN using standard techniques for protein purification.
  • the substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel.
  • the purity of an EPLIN polypeptide can also be determined by amino-terminal amino acid sequence analysis.
  • the invention includes functional polypeptides of EPLIN- ⁇ and EPLIN- ⁇ , and functional fragments thereof.
  • functional polypeptide refers to a polypeptide which possesses a biological function or activity which is identified through a defined functional assay and which is associated with a particular biologic, morphologic, or phenotypic alteration in the cell.
  • Functional fragments of the EPLIN polypeptide includes fragments of EPLIN as long as the activity, e.g., tumor suppressor activity, of EPLIN remains. Smaller peptides containing the biological activity of EPLIN are included in the invention.
  • the biological function can vary from a polypeptide fragment as small as an epitope to which an antibody molecule can bind to a large polypeptide which is capable of participating in the characteristic induction or programming of phenotypic changes within a cell.
  • a "functional polynucleotide” denotes a polynucleotide that encodes a functional polypeptide as described herein.
  • EPLIN primary amino acid sequence may result in proteins that have substantially equivalent activity as compared to the EPLIN polypeptide described herein.
  • modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as the tumor suppressor activity of EPLIN is present.
  • deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its activity. This can lead to the development of a smaller active molecule that would have broader utility. For example, it is possible to remove amino or carboxy terminal amino acids that may not be required for EPLIN activity.
  • the invention also provides an isolated polynucleotide sequence consisting essentially of a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
  • isolated includes polynucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • Polynucleotide sequences of the invention include DNA, cDNA and RNA sequences that encode EPLIN. It is understood that all polynucleotides encoding all or a portion of EPLIN are also included herein, as long as they encode a polypeptide with EPLIN activity.
  • Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides.
  • EPLIN polynucleotide may be subjected to site-directed mutagenesis.
  • the polynucleotide sequence for EPLIN also includes antisense sequences.
  • the polynucleotides of the invention include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of EPLIN polypeptide encoded by the nucleotide sequence is functionally unchanged.
  • the invention also includes a polynucleotide consisting essentially of a polynucleotide sequence encoding a polypeptide having an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 and having at least one epitope for an antibody immunoreactive with EPLIN polypeptide.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:l or 3. Such differences can be due to degeneracy of the genetic code (and result in a nucleic acid that encodes the same EPLIN proteins as those encoded by the nucleotide sequence disclosed herein.
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence that differs by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues than that shown in SEQ ID NO:2 or 4. If alignment is needed for this comparison the sequences should be aligned for maximum homology. "Looped" out sequences from deletions, insertions, or mismatches, are considered differences.
  • Nucleic acids of the invention can be chosen for having codons, which are preferred or non-preferred, for a particular expression system.
  • the nucleic acid can be one in which at least one codon, at preferably at least 10%, or 20% of the codons has been altered such that the sequence is optimized for expression in E. coli, yeast, human, insect, or Chinese hamster ovary (CHO) cells.
  • Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non-naturally occurring.
  • Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms.
  • the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared with the encoded product).
  • the nucleic acid differs from that of S ⁇ Q ID NO: 1 or 3 as follows: by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 1%), 5%, 10%> or 20%) of the nucleotides in the subject nucleic acid. If necessary for this analysis, the sequences should be aligned for maximum homology. "Looped" out sequences from deletions, insertions, or mismatches, are considered differences. Orthologs, homologs, and allelic variants can be identified using methods known in the art.
  • variants comprise a nucleotide sequence encoding a polypeptide that is 50%, at least about 55%), typically at least about 10-15%, more typically at least about 80-85%, and most typically at least about 90-95%) or more identical to the amino acid sequence shown in S ⁇ Q ID NO:2 or a fragment of this sequence.
  • nucleic acid molecules can readily be identified as being able to hybridize under stringent conditions, to the nucleotide sequence shown in S ⁇ Q ID NO 1 or a fragment of the sequence.
  • Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of the ⁇ PLLN cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the ⁇ PLIN gene.
  • Preferred variants include those that are correlated with a tumor suppressor activity.
  • Allelic variants of ⁇ PLIN e.g. , human ⁇ PLIN, include both functional and nonfunctional proteins.
  • Functional allelic variants are naturally occurring amino acid sequence variants of the ⁇ PLLN protein within a population that maintain the ability to function as a tumor suppressor protein.
  • Functional allelic variants typically will contain only conservative substitution of one or more amino acids of S ⁇ Q ID NO:2 or 4, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein.
  • Non-functional allelic variants are naturally occurring amino acid sequence variants of the ⁇ PLIN, e.g., human EPLIN.
  • Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation of the amino acid sequence of SEQ ID NO:2, or a substitution, insertion, or deletion in critical residues or critical regions of the protein.
  • Polynucleotides encoding EPLIN include the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO:3, as well as nucleic acid sequences complementary to those sequences sequence.
  • a complementary sequence may include an antisense nucleotide.
  • the sequence is RNA, the deoxynucleotides A, G, C, and T of SEQ ID NO:l are replaced by ribonucleotides A, G, C, and U, respectively.
  • fragments of the above-described nucleic acid sequences that are at least 15 bases in length, which is sufficient to permit the fragment to selectively hybridize to DNA that encodes the polypeptide of SEQ ID NO:2 or SEQ ID NO:4 under physiological conditions.
  • an isolated nucleic acid molecule that is antisense to EPLIN.
  • An "antisense" nucleic acid can include a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g. , complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.
  • the antisense nucleic acid can be complementary to an entire EPLIN coding strand, or to only a portion thereof (e.g., the coding region of EPLIN corresponding to SEQ ID NO:3).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding EPLIN (e.g., the 5' and 3' untranslated regions).
  • An antisense nucleic acid can be designed such that it is complementary to the entire coding region of EPLIN mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of EPLIN mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of EPLIN mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence.
  • An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
  • an antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions with procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • the antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • antisense nucleic acid molecules of the invention are typically administered to a subject (e.g. , by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an EPLIN protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
  • vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong polymerase II or polymerase III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. , 15:6625- 6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res., 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett., 215:321-330).
  • an antisense nucleic acid of the invention is a ribozyme.
  • a ribozyme having specificity for an EPLIN-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of an EPLIN cDNA disclosed herein (i.e., SEQ ID NO:l or 3), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature, 334:585-591).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an EPLIN-encoding mRNA.
  • EPLIN mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W.
  • EPLIN gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the EPLIN (e.g., the EPLIN promoter and/or enhancers) to form triple helical structures that prevent transcription of the EPLIN gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the EPLIN e.g., the EPLIN promoter and/or enhancers
  • the EPLIN promoter and/or enhancers e.g., the EPLIN promoter and/or enhancers
  • Switchback molecules are synthesized in an alternating 5' -3', 3 '-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • DNA sequences of the invention can be obtained by several methods. For example, the DNA can be isolated using hybridization techniques that are well known in the art.
  • the EPLIN polynucleotide of the invention is derived from a mammalian organism, and most preferably from human. Screening procedures that rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available. Oligonucleotide probes, which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically. This requires that short, oligopeptide stretches of amino acid sequence must be known.
  • the DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. It is possible to perform a mixed addition reaction when the sequence is degenerate. This includes a heterogeneous mixture of denatured double-stranded DNA. For such screening, hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA. Hybridization is particularly useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present.
  • EPLIN DNA sequences encoding EPLIN can also be obtained by: (1) isolation of double-stranded DNA sequences from the genomic DNA; (2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest; and (3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double- stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA.
  • genomic DNA isolates are the least common. This is especially true when it is desirable to obtain the microbial expression of mammalian polypeptides due to the presence of introns.
  • DNA sequences are frequently the method of choice when the entire sequence of amino acid residues of the desired polypeptide product is known.
  • the direct synthesis of DNA sequences is not possible and the method of choice is the synthesis of cDNA sequences.
  • the standard procedures for isolating cDNA sequences of interest is the formation of plasmid- or phage-carrying cDNA libraries that are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned.
  • the production of labeled single or double- stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single-stranded form (Jay et al, Nucl Acid Res., ⁇ :2325, 1983).
  • a cDNA expression library, such as lambda gtl 1 can be screened indirectly for
  • EPLIN peptides having at least one epitope using antibodies specific for EPLIN.
  • Such antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of EPLIN cDNA.
  • DNA sequences encoding EPLIN can be expressed in vitro by DNA transfer into a suitable host cell.
  • "Host cells” are cells in which a vector can be propagated and its DNA expressed.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell” is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
  • the EPLIN polynucleotide sequences may be inserted into a recombmant expression vector.
  • recombinant expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the EPLIN genetic sequences.
  • Such expression vectors contain a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.
  • Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg et al, Gene, 56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) and baculovirus-derived vectors for expression in insect cells.
  • the DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g. , T7, metallothionein I, or polyhedrin promoters).
  • Polynucleotide sequences encoding EPLIN can be expressed in either prokaryotes or eukaryotes.
  • Hosts can include microbial, yeast, insect, and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art.
  • Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention.
  • an EPLIN chimeric or fusion protein includes an EPLIN polypeptide linked to a non-EPLIN polypeptide.
  • a "non-EPLIN polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the EPLIN protein, e.g., a protein that is different from the EPLIN protein and that is derived from the same or a different organism.
  • the EPLIN polypeptide of the fusion protein can correspond to all or a portion e.g. , a fragment described herein of an EPLIN amino acid sequence.
  • an EPLIN fusion protein includes at least one (e.g. two) biologically active portion of an EPLIN protein.
  • the non-EPLIN polypeptide can be fused to the N-terminus or C-terminus of an EPLIN polypeptide.
  • the fusion protein can include a moiety that has high affinity for a ligand.
  • the fusion protein can be a GST-EPLIN fusion protein in which the EPLIN sequences are fused to the C-terminus of the GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant EPLIN.
  • the fusion protein can be an EPLIN protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of EPLIN can be increased through use of a heterologous signal sequence.
  • Fusion proteins can include all or a part of a serum protein, e.g., an IgG constant region, or human serum albumin.
  • the EPLIN fusion proteins of the invention can be inco ⁇ orated into pharmaceutical compositions and administered to a subject in vivo.
  • the EPLIN fusion proteins can be used to affect the bioavailability of an EPLIN substrate.
  • EPLIN fusion proteins may be useful therapeuticaUy for the treatment of disorders caused by, for example: (i) aberrant modification or mutation of a gene encoding an EPLIN protein; (ii) mis-regulation of the EPLIN gene; and (iii) aberrant post-translational modification of an EPLIN protein.
  • EPLIN-fusion proteins of the invention can be used as immunogens to produce anti-EPLIN antibodies in a subject, to purify EPLIN ligands, and in screening assays to identify molecules that inhibit the interaction of EPLIN with an EPLIN substrate.
  • Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • An EPLIN-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the EPLIN protein.
  • the invention features a variant of an EPLIN polypeptide, e.g., a polypeptide that functions as an agonist (mimetic) or as an antagonist of EPLIN activities.
  • Variants of the EPLIN proteins can be generated by mutagenesis, e.g., discrete point mutations, the insertion or deletion of sequences or the truncation of an EPLIN protein.
  • An agonist of the EPLIN protein retains substantially the same, or a subset, of the biological activities of the naturally occurring form of an EPLIN protein.
  • An antagonist of an EPLIN protein can inhibit one or more of the activities of the naturally occurring form of the EPLIN protein by, for example, competitively modulating an EPLIN-mediated activity of an EPLIN protein.
  • treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the EPLIN protein.
  • Variants of an EPLIN protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of an EPLIN protein for agonist or antagonist activity.
  • Libraries of fragments e.g., N terminal, C terminal, or internal fragments, of an EPLIN protein coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of an EPLIN protein.
  • Variants in which a cysteine residue is added or deleted or in which a residue that is glycosylated is added or deleted are particularly preferred.
  • a library of expression vectors can be transfected into a cell line, e.g., a cell line, which ordinarily responds to EPLIN in a substrate-dependent manner.
  • the transfected cells are then contacted with EPLIN and the effect of the expression of the mutant on signaling by the EPLIN substrate can be detected, e.g., by measuring tumor suppressor activity in an appropriate assay.
  • Plasmid DNA can then be recovered from the cells that score for inhibition, or alternatively, potentiation of signaling by the EPLIN substrate, and the individual clones further characterized.
  • the invention features a method of making an EPLIN polypeptide, e.g. , a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring EPLIN polypeptide, e.g., a naturally occurring EPLIN polypeptide.
  • the method includes: altering the sequence of an EPLIN polypeptide, e.g. , by substitution or deletion of one or more residues of a non-conserved region, a domain, or residue disclosed herein, and testing the altered polypeptide for the desired activity.
  • the invention features a method of making a fragment or analog of an EPLIN polypeptide that retains at least one biological activity of a naturally occurring EPLIN polypeptide.
  • the method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of an EPLIN polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.
  • EPLIN nucleic acids, proteins, and derivatives of the present invention also have uses in screening assays to detect molecules that specifically bind to EPLIN nucleic acids, proteins, or derivatives and thus have potential use as agonists or antagonists of EPLIN, in particular, molecules that affect cell proliferation.
  • such assays are performed to screen for molecules with potential utility as anti-cancer drugs or lead compounds for drug development.
  • the invention provides assays to detect molecules that specifically bind to EPLIN nucleic acids, proteins, or derivatives.
  • recombinant cells expressing EPLIN nucleic acids can be used to recombinanfiy produce EPLIN proteins in these assays, to screen for molecules that bind to an EPLIN protein.
  • Molecules e.g., putative binding partners of EPLIN
  • Molecules are contacted with the EPLIN protein (or fragment thereof) under conditions conducive to binding, and then molecules that specifically bind to the EPLIN protein are identified. Similar methods can be used to screen for molecules that bind to EPLIN derivatives or nucleic acids. Methods that can be used to carry out the foregoing are commonly known in the art.
  • diversity libraries such as random or combinatorial peptide or nonpeptide libraries can be screened for molecules that specifically bind to EPLIN.
  • libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries.
  • agonists and antagonists of EPLIN can be identified using “biochip” technology.
  • Biochips or arrays of binding agents, such as oligonucleotides and peptides, have become an increasingly important tool in the biotechnology industry and related fields.
  • binding agent arrays in which a plurality of binding agents are deposited onto a solid support surface in the form of an array or pattern, find use in a variety of applications, including drug screening, nucleic acid sequencing, mutation analysis, and the like.
  • One important use of biochips is in the analysis of differential gene expression, where the expression of genes in different cells, normally a cell of interest and a control, is compared and any discrepancies in expression are identified. In such assays, the presence of discrepancies indicates a difference in the classes of genes expressed in the cells being compared.
  • arrays find use by serving as a substrate to which is bound polynucleotide "probe” fragments.
  • probe polynucleotide fragments.
  • the targets are then hybridized to the immobilized set of polynucleotide "probe” fragments. Differences between the resultant hybridization patterns are then detected and related to differences in gene expression in the two sources.
  • the present invention provides nucleic acid and amino acid sequences useful for screening for differential expression of EPLIN in a cell.
  • the invention includes antibodies immunoreactive with EPLIN polypeptide or functional fragments thereof.
  • Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided.
  • Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art (Kohler et al, Nature, 256:495, 1975).
  • the term antibody as used in this invention is meant to include intact molecules as well as fragments thereof, such as Fab and F(ab') 2 , which are capable of binding an epitopic determinant on EPLIN.
  • Monoclonal antibodies used in the method of the invention are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • the monoclonal antibodies in these immunoassays can be detectably labeled in various ways.
  • Examples of types of immunoassays which can utilize monoclonal antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay.
  • Detection of the antigens using the monoclonal antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.
  • EPLIN proteins, analogues, derivatives, and subsequences thereof, EPLIN nucleic acids (and sequences complementary thereto), anti-EPLIN antibodies have uses in diagnostics.
  • Such molecules can be used in assays, such as immunoassays, to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders affecting EPLIN expression, or monitor the treatment thereof.
  • immunoassay is carried out by a method comprising contacting a sample derived from a patient with an anti-EPLIN antibody under conditions such that immunospecific binding can occur, and detecting or measuring the amount of any immunospecific binding by the antibody.
  • such binding of antibody, in tissue sections can be used to detect aberrant EPLIN localization or aberrant (e.g., low or absent) levels of EPLIN.
  • antibody to EPLIN can be used to assay in a patient tissue or serum sample for the presence of EPLIN where an aberrant level of EPLIN is an indication of a diseased condition.
  • aberrant levels is meant increased or decreased levels relative to that present, or a standard level representing that present, in an analogous sample from a portion of the body or from a subject not having the disorder.
  • the invention provides a method of detecting a cell proliferative disorder in a sample from a subject by contacting a first sample having, or suspected of having, a cell proliferative disorder with a reagent that binds to an EPLIN-specific cell component and detecting binding of the reagent to the component; contacting a second cell not having a cell proliferative disorder with a reagent that binds to an EPLIN-specific cell component and detecting binding of the reagent to the component; comparing the level of binding in the first sample with the level of binding in the second sample, wherein a decreased level of binding of the reagent to an EPLIN-specific cell component from the first sample is indicative of a cell proliferative disorder.
  • cell proliferative disorder refers to a condition characterized by abnormal cell growth.
  • the condition can include both hypertrophic (the continual multiplication of cells resulting in an overgrowth of a cell population within a tissue) and hypotrophic (a lack or deficiency of cells within a tissue) cell growth or an excessive influx or migration of cells into an area of a body.
  • the cell populations are not necessarily transformed, tumorigenic or malignant cells, but also can include normal cells.
  • an "EPLIN-specific cell component” includes, but is not limited to, RNA and DNA encoding an EPLIN protein, the EPLIN protein and fragments thereof, and EPLIN variants including translocations in EPLIN nucleic acids, truncations in the EPLIN gene or protein, changes in nucleotide or amino acid sequence relative to wild-type EPLIN .
  • the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement- fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement- fixation assays, immunoradiometric
  • EPLIN genes and related nucleic acid sequences and subsequences, including complementary sequences can also be used in hybridization assays.
  • EPLIN nucleic acid sequences, or subsequences thereof comprising about at least 8 nucleotides can be used as hybridization probes.
  • Hybridization assays can be used to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with aberrant changes in EPLIN expression and/or activity as described.
  • such a hybridization assay is carried out by a method comprising contacting a sample containing nucleic acid with a nucleic acid probe capable of hybridizing to EPLIN DNA or RNA, under conditions such that hybridization can occur, and detecting or measuring any resulting hybridization.
  • diseases and disorders involving over-proliferation of cells can be diagnosed, or their suspected presence can be screened for, or a predisposition to develop such disorders can be detected, by detecting increased levels of EPLIN protein, EPLIN RNA, or EPLIN functional activity or by detecting mutations in EPLIN RNA, DNA or protein (e.g. , translocations in EPLIN nucleic acids, truncations in the EPLIN gene or protein, changes in nucleotide or amino acid sequence relative to wild-type EPLIN) that cause increased expression or activity of EPLIN.
  • levels of EPLIN protein can be detected by immunoassay
  • levels of EPLIN RNA can be detected by hybridization assays (e.g., Northern blots, dot blots)
  • translocations and point mutations in EPLIN nucleic acids can be detected by Southern blotting, RFLP analysis, PCR using primers that preferably generate a fragment spanning at least most of the EPLIN gene, sequencing of the EPLIN genomic DNA or cDNA obtained from the patient.
  • levels of EPLIN mRNA or protein in a patient sample are detected or measured, in which increased levels indicate that the subject has, or has a predisposition to developing, a malignancy or hyperproliferative disorder; in which the increased levels are relative to the levels present in an analogous sample from a portion of the body or from a subject not having the malignancy or hyperproliferative disorder, as the case may be.
  • diseases and disorders involving a deficiency in cell proliferation or in which cell proliferation is desirable for treatment are diagnosed, or their suspected presence can be screened for, or a predisposition to develop such disorders can be detected, by detecting decreased levels of EPLIN protein, EPLIN RNA, or EPLIN functional activity, or by detecting mutations in EPLIN RNA, DNA or protein (e.g., translocations in EPLIN nucleic acids, truncations in the gene or protein, changes in nucleotide or amino acid sequence relative to wild-type EPLIN) that cause decreased expression or activity of EPLIN.
  • levels of EPLIN protein, levels of EPLIN RNA, EPLIN binding activity, and the presence of translocations or point mutations can be determined as described.
  • levels of EPLIN mRNA or protein in a patient sample are detected or measured, in which decreased levels indicate that the subject has, or has a predisposition to developing, a malignancy or hyperproliferative disorder; in which the decreased levels are relative to the levels present in an analogous sample from a portion of the body or from a subject not having the malignancy or hyperproliferative disorder, as the case may be.
  • the detectably labeled monoclonal antibody is given in a dose which is diagnostically effective.
  • diagnostically effective means that the amount of detectably labeled monoclonal antibody is administered in sufficient quantity to enable detection of the site having the EPLIN antigen for which the monoclonal antibodies are specific.
  • concentration of detectably labeled monoclonal antibody which is administered should be sufficient such that the binding to those cells having EPLIN is detectable compared to the background. Further, it is desirable that the detectably labeled monoclonal antibody be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.
  • the dosage of detectably labeled monoclonal antibody for in vivo diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual.
  • the dosage of monoclonal antibody can vary from about 0.001 mg/m 2 to about 500 mg/m 2 , preferably 0.1 mg/m 2 to about 200 mg/m 2 , most preferably about 0.1 mg/m 2 to about 10 mg/m 2 .
  • Such dosages may vary, for example, depending on whether multiple injections are given, tumor burden, and other factors known to those of skill in the art.
  • the type of detection instrument available is a major factor in selecting a given radioisotope.
  • the radioisotope chosen must have a type of decay which is detectable for a given type of instrument.
  • Still another important factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the host is minimized.
  • a radioisotope used for in vivo imaging will lack a particle emission, but produce a large number of photons in the 140- 250 keV range, which may be readily detected by conventional gamma cameras.
  • radioisotopes may be bound to immunoglobulin, either directly or indirectly, by using an intermediate functional group.
  • Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions to immunoglobulins are the bifunctional chelating agents such as diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • metallic ions which can be bound to the monoclonal antibodies of the invention are In, Ru, Ga, 68 Ga, 72 As, 89 Zr, and 201 T1.
  • a monoclonal antibody useful in the method of the invention can also be labeled with a paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR).
  • MRI magnetic resonance imaging
  • ESR electron spin resonance
  • any conventional method for visualizing diagnostic imaging can be utilized.
  • gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI.
  • Elements which are particularly useful in such techniques include 157 Gd, 55 Mn, 162 Dy, 52 Cr, and 56 Fe.
  • kits may comprise a carrier means being compartmentalized to receive one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
  • container means such as vials, tubes, and the like
  • each of the container means comprising one of the separate elements to be used in the method.
  • one of the container means may comprise an EPLIN or EPLIN binding reagent, such as an antibody or nucleic acid, respectively.
  • the constituents may be present in liquid or lyophilized form, as desired.
  • the present invention also provides a kit useful for the detection of an EPLIN-specific cell component, the kit comprising carrier means containing one or more containers comprising a first container containing an EPLIN-specific binding reagent.
  • an "EPLIN-specific binding reagent” includes nucleic acids, such as probes, which hybridize to an EPLIN-specific cell component, such as DNA or RNA encoding the EPLIN protein.
  • An EPLIN-specific binding reagent also includes proteins, such as antibodies, which bind to an EPLIN protein or fragment or derivative thereof. It is understood that an EPLIN-specific binding reagent includes any molecule which binds to an EPLIN-specific cell component such that the component can be identified.
  • One of the container means may comprise a probe which is or can be detectably labeled.
  • Such probe may be an antibody or nucleotide specific for a target protein, or fragments thereof, or a target nucleic acid, or fragment thereof, respectively, wherein the target is indicative, or correlates with, the presence of EPLIN protein or EPLIN transcript.
  • oligonucleotide probes of the present invention can be included in a kit and used for examining the presence of EPLIN nucleic acid, as well as the quantitative (relative) degree of binding of the probe for determining the lack of binding (hybridizing) to the sequences, thus indicating the likelihood for an subject having a cell proliferation-associated pathology, such as, for example, cancer.
  • the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence.
  • nucleotide(s) for amplification of the target nucleic acid sequence.
  • the kit may contain reagents necessary to perform RT-PCR on a sample containing, or suspected of containing, a cell harboring a pathogenic lentivirus such as HIV.
  • Oligonucleotide primers based upon identification of the flanking regions contiguous with the target nucleotide sequence can be included in the kit such that the primers bind to an EPLIN transcript in the presence of, and under conditions that promote RT-PCR.
  • the level of EPLIN transcript in a sample can be quantitated by means known to those of skill in the art.
  • the method of the invention provides the basis for a kit useful for the detection, or lack thereof, of a target EPLIN nucleic acid sequence in a sample obtained from a subject having, or suspected of having, a neoplasia.
  • the absence, or under-production of, EPLIN transcript obtained from such a sample is indicative of the presence of a neoplasia.
  • the kit includes a carrier means being compartmentalized to receive therein one or more containers.
  • a first container contains a nucleic acid primers which hybridize to the target nucleic acid (e.g., EPLIN RNA) for the purpose of performing semi-quantitative RT-PCR.
  • the kit can provide a nucleic acid probe for detection of an EPLIN RNA transcript.
  • a first container contains a nucleic acid hybridization probe which hybridizes to the target nucleic acid.
  • Other target nucleic acid sequences of EPLIN can be determined by those of skill in the art.
  • the kit may include a second container containing a means for detecting hybridization of the probe with the target nucleic acid.
  • reporter means include a biotin- binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent, or radionuclide label. Other reporter means and labels are well known in the art.
  • the kit may also include an amplification polymerase and deoxyribo- nucleotide(s).
  • the kit may further include nucleic acid amplification buffer.
  • the reagent that modifies unmethylated cytosine is bisulfite.
  • the kit of the invention is intended to provide the reagents necessary to perform nucleic acid hybridization analysis as described herein.
  • tissue containing, or believed to contain, neoplastic cells are usually based on collection of tissues containing such cells.
  • tissue can include, for example, blood, lymph or other tissue.
  • the method of the invention is useful for detecting a neoplasia in any sample believed to contain such cells.
  • Sample acquisition can be accomplished by any means which allows for the isolation of a sample from a subject that results in a sufficient quantity of fluid being obtained for testing.
  • the kit may also include a container containing antibodies which bind to a target protein, or fragments thereof.
  • antibodies which bind to EPLIN, or fragments thereof can be included in a kit.
  • the kit may include a second container containing a means for detecting binding of the antibody with the target EPLIN protein, or fragment thereof.
  • reporter means include a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent, or radionuclide label.
  • Other reporter means and labels are well known in the art.
  • the invention provides a method for converting a neoplastic cell to a non-neoplastic state through the expression of wild-type levels of EPLIN.
  • EPLIN encoding gene expression vectors consisting of appropriate transcriptional/translational control signals and the desired EPLIN cDNA sequence downstream from the first in-frame AUG codon.
  • These methods may include in vitro DNA recombinant and synthetic techniques and in vivo genetic recombination.
  • Expression of a nucleic acid sequence encoding EPLIN may be regulated by a second nucleic acid sequence so that EPLIN is expressed in a host infected or transfected with the recombinant DNA molecule.
  • expression of EPLIN may be controlled by any promoter/enhancer element known in the art.
  • the promoter activation may be tissue specific or inducible by a metabolic product or administered substance.
  • Promoters/enhancers which may be used to control EPLIN gene expression include, but are not limited to, the native EPLIN promoter, the cytomegalovirus (CMV) promoter/enhancer (Karasuyama, H. et al, 1989, J. Exp. Med., 169:13), the human beta-actin promoter (Gunning, P. et al. , 1987, Proc. Natl. Acad. Sci. USA, 84:4831), the glucocorticoid- inducible promoter present in the mouse mammary tumor virus long terminal repeat (HHTV LTR) (Klessig, D. F. et al, 1984, Mol.
  • CMV cytomegalovirus
  • HHTV LTR mouse mammary tumor virus long terminal repeat
  • MULV LTR Moloney murine leukemia virus
  • MULV LTR Moloney murine leukemia virus
  • SV40 early region promoter Bernoist and Chambon, 1981, Nature, 290:304
  • RSV Rous sarcoma virus
  • HSV herpes simplex virus
  • HSV herpes simplex virus
  • Expression vectors compatible with mammalian host cells for use in genetic therapy of tumor or cancer cells include, but are not limited to: plasmids, retroviral vectors, adenovirus vectors, herpes viral vectors, and non-replicative avipox viruses, as disclosed, for example, by U.S. Pat. No. 5,174,993, incorporated herein by reference. Methods of administering viral vectors are well known. In general, the skilled artisan will appreciate that a retroviral vector, an adenovirus vector, a plasmid vector, or any other appropriate vector capable of expressing the EPLIN protein can be administered in vivo to a neoplastic cell by a wide variety of manipulations.
  • Neoplastic cells present in the epithelial linings of hollow organs may be treated by infusing the vector suspension into a hollow fluid filled organ, or by spraying or misting into a hollow air filled organ.
  • the tumor cell may be present in or among the epithelial tissue in the lining of pulmonary bronchial tree, the lining of the gastrointestinal tract, the lining of the female reproductive tract, genitourinary tract, bladder, the gall bladder and any other organ tissue accessible to contact with the vector.
  • the EPLIN encoding gene construct of the present invention may be placed by methods well known to the art into an expression vector such as a plasmid or viral expression vector.
  • a plasmid expression vector may be introduced into a tumor cell by calcium phosphate transfection, liposome (for example, LIPOFECTIN)-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation and any other method of introducing DNA into a cell.
  • a viral expression vector may be introduced into a target cell in an expressible form by infection or transduction.
  • a viral vector includes, but is not limited to: a retrovirus, an adenovirus, a herpes virus and an avipox virus.
  • EPLIN When EPLIN is expressed in any abnormally proliferating cell, the cell replication cycle is arrested, thereby resulting in senescence and cell death and ultimately, reduction in the mass of the abnormal tissue, i.e., the tumor or cancer.
  • a vector able to introduce the gene construct into a target cell and able to express EPLIN therein in cell proliferation-suppressing amounts can be administered by any effective method.
  • a physiologically appropriate solution containing an effective concentration of active vectors can be administered topically, intraocularly, parenterally, orally, intranasally, intravenously, intramuscularly, subcutaneously or by any other effective means.
  • the vector may be directly injected into a target cancer or tumor tissue by a needle in amounts effective to treat the tumor cells of the target tissue.
  • a cancer or tumor present in a body cavity such as in the eye, gastrointestinal tract, genitourinary tract (e.g., the urinary bladder), pulmonary and bronchial system and the like can receive a physiologically appropriate composition (e.g., a solution such as a saline or phosphate buffer, a suspension, or an emulsion, which is sterile except for the vector) containing an effective concentration of active vectors via direct injection with a needle or via a catheter or other delivery tube placed into the cancer or tumor afflicted hollow organ.
  • a physiologically appropriate composition e.g., a solution such as a saline or phosphate buffer, a suspension, or an emulsion, which is sterile except for the vector
  • Any effective imaging device such as X-ray, sonogram, or fiberoptic visualization system may be used to locate the target tissue and guide the needle or catheter tube.
  • a physiologically appropriate solution containing an effective concentration of active vectors can be administered systemically into the blood circulation to treat a cancer or tumor that cannot be directly reached or anatomically isolated.
  • target tumor or cancer cells can be treated by introducing
  • EPLIN protein into the cells by any known method.
  • liposomes are artificial membrane vesicles that are available to deliver drugs, proteins and plasmid vectors both in vitro or in vivo (Mannino, R. J. et al, 1988, Biotechniques, 6:682) into target cells (Newton, A.C. and Huestis, W.H., Biochemistry, 1988, 27:4655; Tanswell, A.K. et al, 1990, Biochmica et Biophysica Acta, 1044:269; and Ceccoll, J. et al. , Journal of Investigative Dermatology, 1989, 93:190).
  • EPLIN protein can be encapsulated at high efficiency with liposome vesicles and delivered into mammalian cells in vitro or in vivo.
  • Liposome-encapsulated EPLIN protein may be administered topically, intraocularly, parenterally, intranasally, intratracheally, intrabronchially, intramuscularly, subcutaneously or by any other effective means at a dose efficacious to treat the abnormally proliferating cells of the target tissue.
  • the liposomes may be administered in any physiologically appropriate composition containing an effective concentration of encapsulated EPLIN protein.
  • a tumor cell is transduced with a retrovirus vector, an adenovirus vector, a plasmid vector or any other appropriate vector capable of expressing the EPLIN protein in that tumor cell.
  • the cancer cell may be present in a blood or bone marrow sample collected from a leukemia patient.
  • a dose of EPLIN protein expressing retrovirus vector or adenovirus vector or plasmid vector or any other appropriate vector is administered to the sample of blood or bone marrow at a dose sufficient to transduce enough cells in the sample to produce a reduction in tumor cell numbers.
  • the cell proliferation of the treated cancer cells will be slowed or terminated followed by a process similar to normal cellular differentiation or cell senescence.
  • blood or bone marrow or other tissue is treated ex vivo using an effective dose of a liposome-encapsulated EPLIN protein. Thereafter the sample may be returned to the donor or infused into another recipient.
  • MEC Human mammary epithelial cells
  • normal human dermal fibroblasts were purchased from Clonetics.
  • Breast cancer cell lines HBL-100, BT-20, SK-Br-3 and T-47D cells were cultured in RPMI 1640 medium supplemented with 10%> fetal bovine serum (FBS) with T-47D cells receiving lx ITS supplement (Sigma).
  • FBS fetal bovine serum
  • MCF-7 and MDA-MB-231 cells were cultured in DMEM supplemented with 10% FBS.
  • BeWo cells were cultured in Ham's
  • F12K medium supplemented with 15%> FBS.
  • RNA STAT-60 Tel-Test
  • Filter membranes were probed with cDNA clone #21 (corresponding to amino acids 268-462 of EPLLN- ⁇ ) and hybridization signals were quantified on a phosphorimager (Molecular Dynamics). All probes were labeled with [ P]- ⁇ -dCTP using a random prime labeling kit (Stratagene). Multiple tissue mRNA blots was purchased from Clontech and used in hybridization following the manufacturer's protocol.
  • the cDNA insert from clone #21 was used as a probe to isolate full length EPLLN- ⁇ and - ⁇ cDNAs from a HeLa cell cDNA library. Two representative clones were fully sequenced to obtain approximately 3.6 kb of sequence. Antibodies and Protein Analysis
  • the carboxy terminal region of EPLIN (aa 680-759 of EPLIN- ⁇ ) was cloned into the pQE-30 vector (Qiagen) and expressed as a 6xHis-tagged fusion protein in E. coli strain XL-1 Blue.
  • the recombinant protein was purified on Ni-NTA agarose under native conditions following the manufacturer's recommendations and used as immunogen for polyclonal rabbit anti-EPLIN antibodies (Covance Research Products).
  • Cell lysates used in immunoblot analyses were prepared by boiling tissue culture cells or minced tissues in 0.2 % SDS in TE (25 mM Tris-HCl, pH 7.5, 1 mM EDTA). 20 ⁇ g of cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. EPLIN isoforms were detected with polyclonal anti-EPLIN antibodies (1 : 10,000). To control the amounts of protein lysates, the filter membrane was also probed with a monoclonal anti- ⁇ -tubulin antibody (Sigma) at 1 :2,000 dilution. Following incubation with a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch), the immunoblots were developed using enhanced chemiluminescence (NEN).
  • NNN enhanced chemiluminescence
  • HOK18C and BeWo cells cultured on fibronectin-coated glass coverslips for 18 h were fixed in 3.7%> formaldehyde (LADD Research) in PBS for 10 min and permeabilized in 0.2%) Triton X-100 in PBS for 5 min.
  • the slides were preincubated in a blocking buffer (0.1%) Tween-20 + 10%) goat serum in PBS) for 30 min before the addition of polyclonal anti-EPLIN antibodies (1 :200 dilution). All incubations were performed at room temperature.
  • HOK18C cells were labeled with Texas Red-conjugated goat anti-rabbit IgG secondary antibody (Jackson ImmunoResearch) and Oregon Green phalloidin (Molecular Probes) while BeWo cells were labeled with fluorescein isothiocyanate-conjugated goat anti- rabbit IgG secondary antibody (Jackson ImmunoResearch) and Texas Red phalloidin (Molecular Probes). Coverslips were mounted with ProLong Antifade (Molecular Probes) and viewed under a fluorescence microscope (Nikon). Pre-immune sera did not produce a staining pattern.
  • U2-OS cells were transfected with the plasmid pTet-On (Clontech) to create U2-OS Tet-On cells expressing tetracycline-inducible transactivator.
  • EPLLN- ⁇ and - ⁇ cDNAs were cloned into the pTRE vector (Clontech) that has been modified by the insertion of an amino terminal FLAG epitope and multiple cloning sites.
  • pTRE-FLAG-EPLIN- ⁇ or - ⁇ and pBABEpuro (Morgenstern and Land, Nucleic Acids Res., 18:587, 1990) plasmids were co- transfected into the U2-OS cells and the stable transfectants were selected with puromycin (1 mg/ml).
  • EPLIN in stable cell lines was induced by the addition of 0.5 mg/ml doxycycline (Sigma). Morphological changes were observed 48 h after the induction of EPLIN. Cell growth was determined by a tetrazolium dye colorimetric assay (Denizot and Lang, J Immunological Methods, 89:271, 1986) with the following modifications.
  • Figure 1 A is a schematic diagram of two EPLIN cDNAs.
  • the sequence of two isoforms diverges at the 5' end (indicated by the stripped and dotted boxes).
  • the EPLIN- ⁇ unique sequences allow the extension of the ORF by 160 aa at the amino terminus of EPLIN- ⁇ .
  • the positions of in frame stop codons upstream to the AUG start codons for two EPLIN isoforms and the termination codons are denoted.
  • Figure IB shows the deduced amino acid sequence of EPLLN- ⁇ .
  • the ORF of EPLLN- ⁇ starts at aa position 161 of EPLIN- ⁇ .
  • the aa sequences of two EPLIN isoforms are identical except for Arg344 of EPLIN- ⁇ which has been replaced by Prol84Glyl85 in EPLIN- ⁇ .
  • the 52 aa sequence of a LIM domain is underlined.
  • Figure 1C shows the alignment of the EPLIN LIM domain sequence with the LIM domain of the mutant SREBP-2, KIAA0750, plant transcription factor SF3, and muscle LIM protein.
  • the signature cysteine and histidine residues of LIM domain are indicated by bold lettering. Amino acid sequence identities (o) and similarities (underlined) are indicated.
  • Figure 2 A shows the distribution of EPLIN expression in different human adult tissues as determined by a Northern analysis. Filters containing mRNA from multiple human tissues (Clontech) were used for Northern blotting. The positions of ⁇ 8 kb and ⁇ 3.8 kb transcripts hybridized by the EPLIN probe are indicated (top). The same blot was re-probed with human b-actin cDNA (bottom).
  • Figure 2B shows the expression of EPLIN in different human primary cells were examined by an immunoblot analysis.
  • MEC mammary epithelial cells.
  • PrEC prostate epithelial cells.
  • NHOK normal human oral keratinocytes.
  • Ao. Endo. aortic endothelial cells.
  • Fibroblasts Dermal fibroblasts.
  • Myocardium human left ventricle.
  • the positions of EPLIN- ⁇ and - ⁇ are noted. The loading of equivalent amounts of cell lysates was confirmed by probing the filter membrane with anti-a tubulin antibody.
  • Figure 3 A shows the expression of EPLIN transcripts in HPV-immortalized oral keratinocyte cell lines (HOK18A-C and HOK16B), tumorigenic HPV-transformed oral keratinocyte cell line (HOK16B-BapT), and oropharyngeal cancer cells (Tu-177, HEp2, and SCC-9) was determined by a Northern analysis (top). The filter membrane was re-probed with human G3PDH cDNA (bottom). The expression of EPLIN, normalized against the G3PDH, in each cell line is indicated.
  • Figure 3B shows the expression of EPLIN proteins in different prostate cancer cell lines and xenograft tumors was determined by an immunoblot analysis. PrEC: prostate epithelial cells.
  • PC3 and DU145 PSA-negative prostate cancer cell lines.
  • LnCAP, LAPC3, LAPC4, and LAPC9 PSA-positive prostate cancer cells or xenograft tumors.
  • the positions of EPLIN- ⁇ and - ⁇ are noted.
  • the loading of equivalent amounts of cell lysates was confirmed by probing the filter membrane with anti-a tubulin antibody.
  • Figure 3C shows the expression of EPLIN proteins in different breast cancer cell lines was determined by an immunoblot analysis.
  • MEC mammary epithelial cells.
  • IMEC immortalized mammary epithelial cells.
  • HBL-100 is a non-tumorigenic breast cancer cells
  • BT-20, SK-Br-3, MCF-7, T-47D, and MDA-MB-231 are tumorigenic breast cancer cell lines.
  • the positions of EPLIN- ⁇ and - ⁇ are noted.
  • the loading of equivalent amounts of cell lysates was confirmed by probing the filter membrane with anti-a tubulin antibody.
  • Figure 3D shows the expression of EPLIN transcripts in different breast cancer cell lines was determined by a Northern analysis (top). The filter membrane was re-probed with human G3PDH cDNA (bottom). The expression of EPLIN, normalized against the G3PDH, in each cell line is indicated.
  • Figure 4A shows the relative amount of EPLIN isoforms in HOK18C (an HPV- immortalized human oral keratinocyte cell line) and BeWo (a human choriocarcinoma cell line) was determined by an immunoblot analysis.
  • EPLIN- ⁇ is expressed as the major isoform in HOK18C, while EPLIN- ⁇ is the major isoform in BeWo.
  • Figures B-E show the subcellular localization of EPLIN was determined by in situ immunofluorescence using anti- EPLIN antibodies (B and D).
  • Texas Red-conjugated goat anti- rabbit IgG secondary antibody (B) and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG secondary antibody (D) were used to detect EPLIN.
  • the stress fibers were stained with Oregon Green-phalloidin ⁇ and Texas Red-phalloidin (E).
  • FIGS A-D show the U2-OS osteosarcoma cells were engineered to express either EPLIN- ⁇ or - ⁇ isoform under the control of a tetracycline-inducible promoter (Tet-On).
  • Tet-On tetracycline-inducible promoter
  • the appearance of U2-OS cells cultured with (B and D) and without (A and C) the induction of EPLIN are shown.
  • the expression of EPLIN changed the morphology of the U2-OS cells from round polygonal cells to fusiform cells characterized by asymmetric cytoplasmic extensions.
  • Figure 5E shows the levels of EPLIN expression in the U2-OS cells cultures minus (no induction) and plus (induction) doxycycline were determined by an immunoblot analysis using anti-EPLIN antisera.
  • Lane 1 parental U2-OS (Tet-on) cells; lane 2, U2-OS (EPLIN- ⁇ ) cells before the induction; lane 3 and 4, U2-OS (EPLIN- ⁇ ) and U2-OS (EPLIN- ⁇ ) cells 48 h after the induction.
  • Figure 5F shows the growth of U2-OS cells is presented as the ratio of cell numbers with and without EPLIN induction. For each time point, cell growth in triplicates were determined by a tetrazolium dye inclusion method.
  • EXAMPLE 2 cDNA fragments containing a partial open reading frame (ORF) were identified by the presence of a LIM domain. Clone #21 was used as a probe to isolate several cDNA clones from a HeLa cell cDNA library. Sequence analysis of these cDNA clones allowed us to assemble an ORF of 600 aa (EPLIN- ⁇ ) and an isoform (EPLJN- ⁇ ) that extended an additional 160 aa at the amino terminus (Fig 1A and IB). The EPLIN- ⁇ mRNA also contained a deletion of 3 nucleotides within the coding region, introducing an Arg in place of ProGly at the corresponding position of EPLIN- ⁇ .
  • EPLIN is a single copy gene, suggesting that the two EPLIN isoforms are generated by an alternative pre-RNA processing event.
  • the predicted amino acid sequence of EPLIN was notable for a single centrally located LIM domain that is homologous to the partial ORF of a hamster gene of an unknown function.
  • the EPLIN LIM domain is distantly related to the LIM domains of plant transcription factors SF-3 and the muscle LIM protein (Fig 1C). Outside the LIM domain, EPLIN is unique in sequence, displaying no significant homology to known proteins or recognizable motifs.
  • anti-EPLIN antisera directed against the carboxy-terminal region common to both ⁇ and ⁇ isoforms was prepared.
  • MEC normal primary mammary
  • PrEC prostate
  • NHOK oral epithelial cells
  • anti-EPLIN antisera detected a major protein band of 90 kD and a second minor species of 110 kD in molecular weight (Fig 2B). These two species were assigned EPLIN- ⁇ and EPLIN- ⁇ , respectively.
  • a Northern analysis of immortalized or transformed oropharyngeal cell lines confirmed a consistent down-regulation of EPLIN transcripts to 10 to 60 % of the level seen in the NHOK (Fig 3A).
  • An immunoblot analysis demonstrated a reduction in EPLIN protein in these cell lines.
  • An immunoblot analysis using cell lysates prepared from 4 human prostate cancer cell lines showed significant changes of EPLIN expression (Fig 3B).
  • PC3 and DU145 In two PSA-negative prostate cancer cell lines, PC3 and DU145, EPLIN expression was detectable, but at significantly reduced levels compared to the level seen in the normal primary prostate epithelial cells (PrEC).
  • EPLIN- ⁇ In two PSA-positive prostate cancer cell lines, LnCap and LAPC4, the expression of EPLIN- ⁇ was not detectable, while EPLIN- ⁇ continued to be expressed at a level comparable to that in the PrEC.
  • Examination of human prostate tumors propagated in SCID mice also demonstrated the loss of EPLLN- ⁇ expression in LAPC3, LAPC4, and LAPC9 xenografts.
  • a survey of breast cancer cell lines revealed a similar change in EPLIN expression
  • EPLIN is a cytoskeletal protein that can alter cell morphology and suppress cell proliferation.
  • EPLIN- ⁇ is expressed as the predominant form in HOK18C, while EPLIN- ⁇ is the predominant form in BeWo (Fig 4A).
  • In situ immunofluorescence analysis demonstrated the localization of both EPLLN- ⁇ and - ⁇ to the cytoplasm in a fibrillar pattern at the periphery of the cell (Fig 4B and D).
  • each isoform was expressed in U2-OS osteosarcoma cells under the control of a tetracycline-inducible promoter.
  • U2-OS cells like most other cells, express EPLIN- ⁇ as the major isoform and a small amount of EPLIN- ⁇ isoform (Fig 5E).
  • Ectopic expression of either EPLIN isoform altered the morphology of the U2-OS cells from round polygonal cells with a cobblestone appearance to larger fusiform cells with spindle cell features and cytoplasmic extensions (Fig 5A-D).
  • the EPLIN overexpressing cells required a longer incubation time in trypsin for detachment, suggesting a change in the cell-matrix interaction.
  • the present invention provides a novel gene, EPLIN, that is down-regulated in human cancer cells.
  • EPLIN a novel gene
  • the expression of EPLIN varied considerably in different adult tissues, there was a general tendency of higher expression in tissues rich in epithelial cells.
  • This preferential expression of EPLIN in epithelial cells was substantiated by an immunoblot analysis demonstrating high levels of EPLIN expression in the normal epithelial cells (e.g., MEC, PrEC, NHOK). Low levels of EPLIN were also detected in the primary aortic endothelial cells and fibroblasts, but not in the myocardium.
  • LMO-2 (formerly called RBTN2/TTG2), which interacts with the basic-helix-loop-helix protein Tal/Scl, is aberrantly expressed in acute T-cell leukemia as a result of chromosomal rearrangement and can promote T-cell tumors (Rabbitts, Genes andDevel, 12:2651, 1998).
  • ril and DRAL are proteins of unknown function that are transcriptionally down-regulated in Ras-transformed cells and rhabdomyosarcoma cells, respectively (Kiess et al, Oncogene, 10:61, 1995; Genini et al, DNA and Cell Biol., 16:433, 1997).
  • LIM domain proteins are involved in cell lineage determination as DNA-binding transcription factors or accessory factors that associate with DNA-binding transcription factors to modulate gene transcription (Dawid et al , Trends in Genetics, J_4: 156, 1998). Other LIM domain proteins interact with cytoskeletal proteins or localize to the site of cell-matrix attachment. This class of LIM domain proteins includes zyxin (Beckerle, Bioessays, 19:949, 1997); paxillin, hic-5, and leupaxin (Brown et al, J. Cell Biol, 135:1109, 1996); LIM-kinase (Yang et al, J. Biol.
  • the amino acid sequence of the EPLIN LIM domain is closely related to the LIM domain of the plant transcription factor SF3 (48% aa identity; 73% aa similarity within the 52 aa LIM domain).
  • EPLIN expression is down-regulated in the majority of cancer cell lines examined in the present study, indicating that the loss of EPLIN expression is directly linked to cellular transformation.
  • the loss of EPLIN- ⁇ isoform was accompanied by an increase in EPLIN- ⁇ isoform in 2/6 breast cancer cell lines (e.g., BT-20 and SK-Br-3).
  • the combined levels of the two EPLIN isoforms were lower in BT-20 and SK-Br-3 cells.
  • the major difference between the two EPLIN isoforms is at the amino terminus where the b isoform contains an extension of 160 aa.
  • the sequence divergence may be an alternative pre-mRNA splicing event involving a single pre-mRNA that utilizes alternative exons.
  • EPLIN is transcribed from two distinct promoters to generate two pre- mRNA species both of which are spliced to the common 3' exons.
  • the relative increase in EPLIN- ⁇ in breast cancer cell lines BT-20 and Sk-Br-3, which have lost the expression of EPLLN- ⁇ , indicates that the expression of the two EPLIN isoforms can be regulated independently.

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Abstract

La présente invention concerne des séquences de polynucléotides et de polypeptides codant une nouvelle protéine de suppression de tumeurs, l'EPLIN. En outre, cette invention concerne une méthode de détection de troubles de prolifération cellulaire associé à l'EPLIN. L'EPLIN est un marqueur qui peut être utilisé de manière diagnostique, pronostique, et thérapeutique lors de troubles de prolifération cellulaire associés à l'EPLIN.
PCT/US2000/024689 1999-09-08 2000-09-08 Proteine epitheliale perdue dans le neoplasme (eplin) WO2001018019A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001092578A2 (fr) * 2000-05-26 2001-12-06 Board Of Trustees Of The University Of Illinois Reactifs et methodes destines a l'identification et a la modulation de l'expression de genes regules par les retinoides

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4708948A (en) * 1984-04-20 1987-11-24 The United States Of America As Represented By The Department Of Health And Human Services Substantially purified tumor growth inhibitory factor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4708948A (en) * 1984-04-20 1987-11-24 The United States Of America As Represented By The Department Of Health And Human Services Substantially purified tumor growth inhibitory factor

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN ET AL.: "Characterization of the human EPLIN (epithelial protein lost in neoplasm) gene reveals distinct promoters for the two EPLIN isoforms", GENE, vol. 248, 2 May 2000 (2000-05-02), pages 69 - 76, XP002933577 *
MAUL ET AL.: "EPLIN, epithelial protein lost in neoplasm", ONCOGENE, vol. 18, October 1999 (1999-10-01), pages 7838 - 7841, XP002933578 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001092578A2 (fr) * 2000-05-26 2001-12-06 Board Of Trustees Of The University Of Illinois Reactifs et methodes destines a l'identification et a la modulation de l'expression de genes regules par les retinoides
WO2001092578A3 (fr) * 2000-05-26 2003-08-07 Univ Illinois Reactifs et methodes destines a l'identification et a la modulation de l'expression de genes regules par les retinoides
US6767705B2 (en) 2000-05-26 2004-07-27 The Board Of Trustees Of The University Of Illinois Reagents and methods for identifying and modulating expression of genes regulated by retinoids

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