WO2005005663A1 - Identification de crash, un gene deregule dans des tumeurs gynecologiques - Google Patents

Identification de crash, un gene deregule dans des tumeurs gynecologiques Download PDF

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WO2005005663A1
WO2005005663A1 PCT/EP2004/007560 EP2004007560W WO2005005663A1 WO 2005005663 A1 WO2005005663 A1 WO 2005005663A1 EP 2004007560 W EP2004007560 W EP 2004007560W WO 2005005663 A1 WO2005005663 A1 WO 2005005663A1
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expression
marker gene
sample
nucleic acid
protein
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PCT/EP2004/007560
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Vesna Evtimova
Ulrich Weidle
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F. Hoffmann-La Roche Ag
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention discloses methods wherein a nucleic acid molecule (CRASH) with the nucleic acid sequence SEQ ID NO: 1 is used for the evaluation of the risk of tumor progression and/or metastasis, primarily in uterine, mammary, prostatic or ovarian carcinoma. Uses of the CRASH protein of SEQ ID NO:2 are also provided.
  • a kit for determining whether cells in a tumor sample has metastatic potential is provided.
  • Breast cancer is the most frequent tumor in women and has a mortality rate of 65% (Schwirzke, M., et al., Anticancer Res. 19 (1999) 1801-1814). It is commonly accepted that breast cancer is a genetically based disease with sporadic and hereditary manifestation. Risk factors are extent of exposure to estrogens (Pike, M.C., et al., Epidemiol. Rev. 15 (1993) 17-35) and exogenous factors such as lifestyle and physical enviromental factors such as ionizing radiation (McGregor, H., et al, J. Natl. Cancer Inst. 59 (1977) 799-811). Patients with stage I and II mammary carcinoma can be treated very successfully with surgery and radiation.
  • Treatment options are adjuvant therapy, the use of cytotoxic and antihormonal drugs after surgery and neoadjuvant therapy as well as treatment with cytotoxic drugs before surgery to shrink the tumor.
  • estrogen-receptors ER
  • progesterone-receptors PR
  • estrogen blocking drugs such as Tamoxifen, a non-steroidal estrogen antagonist.
  • the presence or absence of estrogen and progesterone-receptor protein in primary or metastatic tumor tissue is used nowadays to predict which patient may be expected to respond to additive or ablative endocrine therapy.
  • the presence of ER is correlated with expected responsivity of the cancer to the hormonal therapy.
  • the absence of ER is correlated with expected nonresponsiveness to the hormonal therapy.
  • Microarray analysis for breast cancer was e.g. performed by Ahr et al. (Lancet (2002) 359:131-2) or Worsham et al. (Cytometry (2002) 47(l):56-9).
  • the present invention provides a gene with an expression profile that correlates with the metastatic propensity of cancer cell lines and which is upregulated in gynecological cancers.
  • the present invention provides uses of the new marker gene CRASH, a human asparaginase-like protein which is composed of 308 aa and exhibits 32 % homology to human aspartylglucosaminadase at the amino acid level.
  • CRASH a human asparaginase-like protein which is composed of 308 aa and exhibits 32 % homology to human aspartylglucosaminadase at the amino acid level.
  • Database analysis revealed that the gene corresponding to CRASH is composed of seven exons and six introns.
  • Steady-state level of CRASH mRNA was found to be increased in five cell lines derived from metastatic lesions compared with two cell lines derived from primary mammary carcinoma and HMEC (human mammary epithelial cells).
  • CRASH The mRNA level of CRASH corresponds to the metastatic propensity of several isogenic human colon cancer and pancreatic carcinoma cell lines. CRASH corresponds to a recently identified sperm autoantigen (Bush, L.A., et al., Mol. Reprod. Dev. 62 (2002) 233-247). Investigation of several types of human cancers and their corresponding normal tissues revealed high levels of CRASH mRNA in uterine, mammary and ovarian tumors compared with the corresponding normal tissues. CRASH mRNA expression was analysed in breast cancer samples with disclosed clinico-pathological features and corresponding normal tissues. The levels of CRASH mRNA were significantly up-regulated in tumors compared with normal breast tissues and correlate with the ER " (ER: estrogen receptor) status of the tumors.
  • ER estrogen receptor
  • the invention discloses a method of determining whether or not a human cancer cell containing patient sample has potential for tumor progression, the method comprising comparing: a) the level of expression a marker gene with a nucleic acid sequence according to SEQ ID NO: 1 in the patient sample, and b) the normal level of expression the marker gene in a sample from a control subject not afflicted with cancer or the normal level of expression of the marker gene in a sample containing cells from a human mammary epithelial cell line,
  • a significant difference between the level of expression of the marker gene in the patient sample and the normal level of the marker gene in the sample from a control subject not afflicted with cancer or in the sample containing cells from a human mammary epithelial cell line is an indication that the patient sample has potential for tumor progression.
  • the invention discloses a method of determining the estrogen receptor status of a human cancer cell in a human cancer cell containing patient sample, the method comprising comparing: a) the level of expression a marker gene with a nucleic acid sequence according to SEQ ID NO: 1 in the patient sample, and b) the normal level of expression the marker gene in a sample from a control subject not afflicted with cancer or the normal level of expression of the marker gene in a sample containing cells from a human mammary epithelial cell line,
  • a significant difference between the level of expression of the marker gene in the patient sample and the normal level of the marker gene in the sample from a control subject not afflicted with cancer or in the sample containing cells from a human mammary epithelial cell line is an indication of the estrogen receptor status of the human cancer cell.
  • a method of selecting a composition for inhibiting the progression of cancer in a patient comprising:
  • kits for assessing whether a patient has a risk for progression of cancer comprising reagents for assessing expression of a marker gene, wherein the marker gene has a nucleic acid sequence according to SEQ ID NO: 1.
  • kits for assessing the estrogen receptor status of a human breast cancer cell comprising reagents for assessing expression of a marker gene, wherein the marker gene has a nucleic acid sequence according to SEQ ID NO: 1.
  • kits for assessing the suitability of each of a plurality of compounds for inhibiting progression of cancer in a patient comprising:
  • marker gene has a nucleic acid sequence according to SEQ ID NO: 1.
  • a method of deriving a candidate agent comprising:
  • the marker gene has a nucleic acid sequence according to SEQ ID NO: 1,
  • an at least 1.5 fold difference or a less than 0.75 fold difference between the level of expression of the marker gene according to the inventionin the sample contacted with the candidate agent and the level of expression of the same marker gene in the sample not contacted with the candidate agent is an indication of an effect of the candidate agent.
  • a candidate agent derived by the method according to the invention is provided and a pharmaceutical preparation comprising an agent according to the invention.
  • the agent according to the invention may be used for the preparation of a composition for the inhibition of progression of cancer.
  • the invention is further related to a method of producing a drug comprising the steps of the method of the invention.
  • step (i) synthesizing the candidate agent identified in step (d) or an analog or derivative thereof in an amount sufficient to provide said drug in a therapeutically effective amount to a subject;
  • step (ii) combining the drug candidate the candidate agent identified in step (d) or an analog or derivative thereof with a pharmaceutically acceptable carrier.
  • nucleic acid molecule with a nucleic acid sequence according to SEQ ID NO: 1 or fragments thereof are used for the determination of the potential of a human cancer cell for tumor progression or for the determination of the estrogen receptor status of a human breast cancer cell.
  • SEQ ID NO: 2 or fragments thereof, or antibodies specifically binding to the protein with an amino acid sequence according to SEQ ID NO: 2 are used for the determination of the potential of a human cancer cell for tumor progression or for the determination of the estrogen receptor status of a human breast cancer cell.
  • a "solid phase” may be controlled pore glass (CPG), polystyrene or silica gel as used for oligonucleotide synthesis.
  • CPG controlled pore glass
  • polystyrene polystyrene
  • silica gel as used for oligonucleotide synthesis.
  • array is meant an arrangement of addressable locations on a device (see e.g. US 5,143,854, US 6,022,963, US 6,156,501, WO 90/15070, WO 92/10092).
  • the locations can be arranged in two dimensional arrays, three dimensional arrays, or other matrix formats.
  • the number of locations can range from several to at least hundreds of thousands.
  • each location represents a totally independent reaction site.
  • Each location carries a nucleic acid as e.g. an "oligomeric compound", which can serve as a binding partner for a second nucleic acid, in particular a target nucleic acid. Methods for the manufacturing thereof are described in EP-A-0 476 014 and Hoheisel, J.
  • Microfabricated arrays of large numbers of oligonucleotide probes, called “DNA chips” offer great promise for a wide variety of applications (see e.g. US 6,156,501 and US 6,022,963).
  • hybridization shall mean hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleotides.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • “Complementary,” as used herein, also refers to sequence complementarity between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
  • oligonucleotide and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood that an oligonucleotide need not be 100% complementary to its target DNA sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e.
  • marker gene is meant to include a gene which is useful according to this invention for the evaluation of the potential of (metastasis or) progression of cancer, particularly breast cancer. It could be also termed cancer, preferably breast cancer, marker gene.
  • marker polynucleotide is meant to include nucleotide transcript (hnRNA or mRNA) encoded by a cancer, preferably breast cancer, marker gene according to the invention, or cDNA derived from the nucleotide transcript, or a segment of said transcript or cDNA.
  • marker protein is meant to include protein or polypeptide encoded by a cancer, preferably breast cancer, marker gene, preferably the marker gene according to the invention, or a polypeptide or protein fragment comprising said marker protein.
  • gene product is meant to include marker polynucleotide and marker protein encoded by the referenced gene.
  • polynucleotide is synonymous with “nucleic acid.”
  • the term facedecological shall be understood in its broadest sense, i.e. of or pertaining to gynecology that is the science which treats of the structure and diseases of women. Therefore, the term regarding the term regarding the structure and diseases of women. Therefore, the term regarding the term regarding the structure and diseases of women. Therefore, the term regarding the term regarding the structure and diseases of women. Therefore, the term regarding the term regarding the structure and diseases of women. Therefore, the term regarding the term regarding installinggynecological tumor” is related to tumors that only afflicts women, in particular ovarian cancer, uterine cancer or breast cancer.
  • breast cancer and “mammary cancer” are used interchangeably throughout the application.
  • progression of cancer shall include the event of recurrence or metastasis which is regarded to be a more specific event during progression of cancer.
  • the progression of cancer preferably breast cancer, is "inhibited” if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.
  • a polynucleotide "corresponds to" another (a first) polynucleotide if it is related to the first polynucleotide by any of the following relationships: the second polynucleotide comprises the first polynucleotide and the second polynucleotide encodes a gene product; the second polynucleotide is the complement of the first polynucleotide and, the second polynucleotide is 5' or 3' to the first polynucleotide in cDNA, RNA, genomic DNA, or fragment of any of these polynucleotides.
  • a second polynucleotide may be a fragment of a gene that includes the first and second; polynucleotides.
  • the first and second polynucleotides are related in that they are components of the gene coding for a gene product, such as a protein or antibody.
  • the second polynucleotide comprises or overlaps with the first polynucleotide to be encompassed within the definition of "corresponding to" as used herein.
  • the first polynucleotide may be a segment of a 3' untranslated region of the second polynucleotide.
  • the first and second polynucleotide may be fragments of a gene coding for a gene product.
  • the second polynucleotide may be an exon of the gene while the first polynucleotide maybe an intron of the gene.
  • probe refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example a marker gene of the invention. Probes can either be synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, proteins, antibodies, organic monomers, RNA, DNA, and cDNA.
  • breast-associated body fluid is a fluid which, when in the body of a patient, contacts or passes through breast cells or into which cells, nucleic acids or proteins shed from breast cells are capable of passing.
  • Exemplary breast-associated body fluids include blood fluids, lymph, and cystic fluid.
  • the "normal” level of expression of a marker gene is the level of expression of the marker gene in (breast) cells or (breast-associated) body fluids of a subject, e.g a human, not afflicted with cancer, preferably breast cancer,, or a suitable cell line as human mammary epiethial cells (HMEC).
  • HMEC human mammary epiethial cells
  • promoter/regulatory sequence means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence.
  • this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue-specific manner.
  • a “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell under most or all physiological conditions of the cell.
  • An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • tissue-specific promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only if the cell is a cell of thetissue type corresponding to the promoter.
  • a “transcribed polynucleotide” is a polynucleotide (e.g an RNA, a cDNA, or an analog of one of an RNA or cDNA) which is complementary to or homologous with all or a portion of a mature RNA made by transcription of a gene, such as the marker gene of the invention, and normal post-transcriptional processing (e.g. splicing), if any, of the transcript.
  • a polynucleotide e.g an RNA, a cDNA, or an analog of one of an RNA or cDNA
  • normal post-transcriptional processing e.g. splicing
  • “Complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of fortning specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil.
  • a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine.
  • a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
  • the first region comprises a first portion and the second region comprises a second portion, whereby when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the 5 nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • Homologous refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. By way of example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC- 3' share 50% homology.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.
  • a nucleic acid or protein is "fixed" to a substrate if it is covalently or non covalently associated with the substrate such that the substrate can be rinsed with a fluid (e.g standard saline citrate, pH 7.4) without a substantial fraction of the nucleic acid or protein dissociating from the substrate.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature.
  • Expression of a marker gene in a patient is "significantly" altered from the level of expression of the marker gene in a control subject if the level of expression of the marker gene in a sample from the patient differs from the level in a sample from the control subject by an amount greater than the standard error of the assay employed to assess expression, and preferably at least twice, and more preferably three, four, five or ten times that amount.
  • Expression of a marker gene in a patient is "significantly" higher than the level of expression of the marker gene in a control subject if the level of expression of the marker gene in a sample from the patient is greater than the level in a sample from the control subject by an amount greater than the standard error of the assay employed to assess expression, and preferably at least 1.4 and more preferably twice, three, four, five or ten times that amount.
  • expression of the marker gene in the patient can be considered "significantly" lower than the level of expression in a control subject if the level of expression in a sample from the patient is lower than the level in a sample from the control subject by an amount greater than the standard error of the assay employed to assess expression, and preferably at least twice, and more preferably three, four, five or ten times that amount.
  • a “kit” is any manufacture (e.g a package or container) comprising at least one reagent, e.g a probe, for specifically detecting a marker gene or peptide of the invention.
  • the manufacture is preferably promoted, distributed, or sold as a unit for performing the methods of the present invention.
  • a method of determining whether or not a human cancer cell containing patient sample has potential for tumor progression comprising comparing: a) the level of expression a marker gene with a nucleic acid sequence according to SEQ ID NO: 1 in the patient sample, and b) the normal level of expression the marker gene in a sample from a control subject not afflicted with cancer or the normal level of expression of the marker gene in a sample containing cells from a human mammary epithelial cell line,
  • a significant difference between the level of expression of the marker gene in the patient sample and the normal level of the marker gene in the sample from a control subject not afflicted with cancer or in the sample containing cells from a human mammary epithelial cell line is an indication that the patient sample has potential for tumor progression.
  • the invention discloses a method of determining the estrogen receptor status of a human cancer cell in a human cancer cell containing patient sample, the method comprising comparing:
  • a significant difference between the level of expression of the marker gene in the patient sample and the normal level of the marker gene in the sample from a control subject not afflicted with cancer or in the sample containing cells from a human mammary epithelial cell line is an indication of the estrogen receptor status of the human cancer cell.
  • the human cancer cell is preferably a human breast cancer cell.
  • the significant difference comprises an at least 1.4 fold difference between the level of expression of the marker gene in the patient sample and the normal level of expression of the marker gene in the sample from the control subject or in the sample containing cells from a human mammary epithelial cell line. More preferably, the significant difference comprises an at least two fold, preferably 4 fold, difference between the level of expression of the marker gene in the patient sample and the normal level of expression of the same marker gene in the sample from the control subject or in the sample containing cells from a human mammary epithelial cell line.
  • the cancer is a gynecological cancer, more preferably mammary, breast or uterine cancer, most preferred is breast cancer.
  • This preferred embodiment shall apply to all methods, uses, kits, agents or other products according to the invention.
  • the sample is a breast tissue sample, a breast-associated fluid or urine.
  • the level of expression of the marker gene in the sample is assessed by detecting the presence in the sample of a protein encoded by the marker gene or a polypeptide or protein fragment comprising said protein.
  • the presence of said protein, polypeptide or protein fragment is detected using a reagent which specifically binds with said protein, polypeptide or protein fragment. More preferably, the reagent is selected from the group consisting of an antibody, an antibody derivative, and an antibody fragment.
  • the level of expression the marker gene in the sample is assessed by detecting the presence in the sample of a transcribed polynucleotide encoded by the marker gene or a portion of said transcribed polynucleotide.
  • the transcribed polynucleotide is a cDNA, mRNA or hnRNA. More preferably, the step of detecting further comprises amplifying the transcribed polynucleotide.
  • the level of expression of the marker gene in the samples is assessed by detecting the presence in the samples of a transcribed polynucleotide which anneals with the marker gene or anneals with a portion of said transcribed polynucleotide, under stringent hybridization conditions.
  • a method of selecting a composition for inhibiting the progression of cancer in a patient comprising: a) providing a sample comprising cancer cells from the patient; b) separately exposing aliquots of the sample in the presence of a plurality of test compositions; c) comparing expression of the marker gene according to the invention in each of the aliquots; and d) selecting one of the test compositions which alters the level of expression of the marker gene with a nucleic acid sequence according to SEQ ID NO: 1 in the aliquot containing that test composition, relative to other test compositions.
  • kits for assessing whether a patient has a risk for progression of cancer or for the determination of the estrogen receptor status of a human breast cancer cell comprising reagents for assessing expression of a marker gene, wherein the marker gene has a nucleic acid sequence according to SEQ ID NO: 1.
  • a kit for assessing the suitability of each of a plurality of compounds for inhibiting progression of cancer in a patient is provided, the kit comprising a) the plurality of compounds; and b) a reagent for assessing expression of a marker gene, wherein the marker gene has a nucleic acid sequence according to SEQ ID NO: 1.
  • the kit comprises a plurality of reagents, each of; which is capable of binding specifically with a protein or nucleic acid encoded by a marker gene of the invention.
  • Suitable reagents for binding with a protein encoded by a marker gene of the invention include antibodies, antibody derivatives, antibody fragments, and the like.
  • Additional reagents for specifically binding with a protein encoded by a marker gene include any natural ligands of the protein and derivatives of such ligands.
  • Suitable reagents for binding with a nucleic acid encoded by a marker gene include complementary nucleic acids.
  • the nucleic acid reagents may include oligonucleotides (labeled or non-labeled) fixed to a substrate, labeled oligonucleotides not bound with a substrate, pairs of PCR primers, molecular beacon probes, and the like.
  • the kit of the invention may optionally comprise additional components useful for performing the methods of the invention.
  • the kit may comprise; fluids (e.g SSC buffer) suitable for binding an antibody with a protein with which it specifically binds or, for annealing complementary nucleic acids one or more sample compartments, instructional material which describes performance of a method of the invention, a sample of control cells, a sample of breast cancer cells, and the like.
  • fluids e.g SSC buffer
  • instructional material which describes performance of a method of the invention, a sample of control cells, a sample of breast cancer cells, and the like.
  • a significant difference comprises an at least 1.4 fold difference between the level of expression of the marker gene according to the invention in the patient sample and the normal level of expression of the same marker gene. More preferably, the significant difference comprises an at least 2 or 5 fold difference between the level of expression of the marker gene according to the invention in the patient sample and the normal level of expression of the same marker gene.
  • a nucleic acid molecule with a nucleic acid sequence according to SEQ ID NO: 1 or fragments thereof is used for the determination of the potential of a human cancer cell for tumor progression or for the determination of the estrogen receptor status of a human breast cancer cell.
  • the level of expression of a marker gene in a sample can be determined, for example, by detecting the level in the sample of: a protein encoded by the marker gene, or a polypeptide or a fragment comprising the protein (e.g using a reagent, such as an antibody, an antibody derivative, which binds specifically with the protein or a fragment thereof); a metabolite which is produced directly by catalysis or indirectly by; the protein encoded by the marker gene; and/or a polynucleotide (e.g. an mRNA, hnRNA, cDNA) produced by or derived from the expression of the marker gene or a fragment of the polynucleotide (e.
  • a polynucleotide e.g. an mRNA, hnRNA, cDNA
  • marker gene or combination of marker genes may be used in the compositions, kits, and methods of the present invention.
  • marker genes for which the difference between the level of expression of the marker gene in breast cancer cells or breast- associated body fluids and the normal level of expression of the same marker gene is known.
  • the difference can be as small as the limit of detection of the method for assessing expression of the marker gene, it is preferred that the difference be at least greater than the standard error of the assessment method, and preferably a difference of at least 1.4, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-,
  • a significantly altered, preferably increased, level of expression in the patient sample of the marker gene according to the invention relative to the marker gene expression levels in samples from subjects serving as a control, is an indication that the patient has a higher risk of progression of cancer, preferably breast cancer,.
  • Preferred in vivo techniques for detection of a protein encoded by marker gene of the invention include introducing into a subject an antibody that specifically binds the protein, or a polypeptide or protein fragment comprising the protein.
  • the antibody can be labeled with a radioactive molecule whose presence and location in a subject can be detected by standard imaging techniques.
  • Expression of a marker gene of the invention may be assessed by any of a wide variety of well known methods for detecting expression of a transcribed molecule or protein.
  • Non-limiting examples of such methods include immunological methods for detection of secreted, cell-surface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.
  • Such method may also include physical methods such as liquid and gas chromatography, mass spectroscopy, and nuclear magnetic resonance.
  • expression of the marker gene according to the invention is assessed using an antibody (e.g a radio-labeled, chromophore-labeled, fluorophore- labeled, or enzyme-labeled antibody), an antibody derivative (e.g.
  • an antibody conjugated with a substrate or with the protein or ligand of a protein- ligand pair e.g biotin-skeptavidin
  • an antibody fragment e.g a single-chain antibody, an isolated antibody hypervariable domain, etc.
  • the protein may have undergone none, all or a portion of its normal post-translational modification and/or proteolysis during the course of its secretion or release from preferably colorectal cells, cancerous or otherwise.
  • expression of the marker gene according to the invention is assessed by preparing mRNA/cDNA (i. e. a transcribed polynucleotide) from cells in a patient sample, and by hybridizing the mRNA/cDNA with a reference polynucleotide which comprises the marker gene sequence or its complement, or a fragment of said sequence or complement.
  • cDNA can, optionally, be amplified using any of a variety of polymerase chain reaction methods prior to hybridization with the reference polynucleotide.
  • Expression of the marker gene according to the invention can likewise be detected using quantitative PCR to assess the level of RNA transcripts encoded by the marker gene.
  • a mixture of transcribed polynucleotides obtained from the sample is contacted with a substrate having fixed thereto a polynucleotide complementary to or homologous with at least a portion (e.g at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or more nucleotide residues) of a RNA transcript encoded by the marker gene of the invention.
  • polynucleotides complementary to or homologous with a RNA transcript encoded by the marker gene of the invention are differentially detectable on the substrate (e.g detectable using radioactivity, different chromophores or fluorophores), are fixed to different selected positions, then the levels of expression of a plurality of marker genes can be assessed simultaneously using a single substrate (e.g a "gene chip" microarray of polynucleotides fixed at selected positions).
  • a method of assessing marker gene expression which involves hybridization of one nucleic acid with another, it is preferred that the hybridization be performed under stringent hybridization conditions.
  • compositions, kits, and methods of the invention rely on detection of a difference in expression levels of the marker gene of the invention, it is preferable that the level of expression of the marker gene is greater, preferably significantly greater, than the minimum detection limit of the method used.
  • the methods of the present invention may be practiced using the marker gene of the invention in combination with one or more known marker genes. It will be appreciated that the methods and kits of the present invention may also include known cancer marker genes including known marker genes. It will further be appreciated that the methods and kits may be used to identify cancers other than cancer, preferably breast cancer,.
  • the panel of marker genes if the marker gene according to the invention is combined with known marker genes, whose expression level is assessed, is selected such that a positive result is obtained in at least about 20%, and preferably at least about 40%, 60%, or 80%, and more preferably in substantially all patients afflicted with cancer, preferably breast cancer,.
  • the panel of marker genes if the marker gene according to the invention is combined with known marker genes is selected such that a positive predictive value (PPV) of greater than about 10% is obtained for the general population.
  • the marker gene of the invention may also be used in a panel of marker genes, for example. It is well known that certain types of genes, such as oncogenes, tumor suppressor genes, growth factor-like genes, protease- like genes, and protein kinase- like genes are often involved with development of cancers of various types.
  • Known oncogenes and tumor suppressor genes include, for example, abl, abr, akt2, ape, bcl2a, bcl2,C, bcl3, bcr, brcal, brca2, cbl, ccudl, cdc42, cdk4, crk- II, csprlfins, dbl.
  • Known growth factors include platelet-derived growth factor alpha, platelet derived growth factor beta (simian sarcoma viral lysis) oncogene homolog), thrombopoletin (myeloproliferative leukemia virus oncogene ligand, megakaryocyte growth and development factor), erythropoletin, B cell growth factor, macrophage stimulating factor 1 (hepatocyte growth factorlike protein), hepatocyte growth factor (hepapoietin A), insulin-like growth factor 1 (somatomedia C), hepatoma- derived growth factor, amphiregulin (schwannoma- derived growth factor), bone morphogenetic; proteins 1, 2, 3, 3 beta, and 4, bone morphogenetic protein 7 (osteogenic protein 1), bone morphogenetic protein 8
  • osteogenesis protein 2 connective tissue growth factor
  • connective tissue activation peptide 3 epidermal growth factor (EGF)
  • EGF epidermal growth factor
  • teratocarcinoma derived growth factor 1 endothelin, endothelin 2, endothelin 3
  • stromal cell-derived factor 1 vascular endothelial growth factor (VEGF), VEGF-B, VEGF-C
  • placental growth factor vascular endothelial growth factor-related protein
  • transforming growth 25 factor alpha transforming growth factor beta 1 and its precursors
  • transforming growth factor beta 2 and its precursors fibroblast growth factor 1 (acidic), fibroblast growth factor 2 (basic), fibroblast growth factor 5 and its precursors, fibroblast growth factor 6 and its precursors, fibroblast growth factor 7 (keratinocyte growth factor), fibroblast growth factor 8 (androgen-induced), fibroblast growth factor 9
  • proteases include interleukin- 1 beta convertase and its precursors, Mch6 and its precursors, Mch2 isoform alpha, Mch4, Cpp32 isoform alpha, Lice2 gamma cysteine protease, Ich-1 S. Ich- 1 L, Ich-2 and its precursors,
  • TY protease matrix 35 metalloproteinase 1 (interstitial collagenase), matrix metalloproteinase 2 (gelatinase A, 72kD gelatinase, 72kD type IV collagenase), matrix metalloproteinase 7 (matrilysin), matrix metalloproteinase 8 (neutrophil collagenase), matrix metalloproteinase 12 (macrophage elastase), matrix metalloproteinase 13 (collagenase 3), metallopeptidase 1, cysteine-rich metalloprotease (disintegrin) and its precursors, subtilisin-like protease Pc8 and its precursors, chymotrypsin, snake venom-like protease, cathepsin 1, cathepsin D (lysosomal aspartyl protease), skomelysin, aminopeptidase N. plasminogen, tissue 5 plasminogen activator,
  • the level of expression of each marker gene in a patient sample can be compared with the normal level of expression of each of the plurality of marker genes in non- cancerous samples of the same type, either in a single reaction mixture (i. e. using reagents, such as different fluorescent probes, for each marker gene or a mixture of similiarly labeled probes to access expression level of a plurality of marker genes whose probes are fixed to a single substrate at different positions) or in individual reaction mixtures corresponding to one or more of the marker genes.
  • a significantly enhanced level of expression of more than one of the plurality of marker genes in the sample, relative to the corresponding normal levels, is an indication that the patient is at risk that cancer, preferably breast cancer, progresses.
  • cancer preferably breast cancer
  • the expression level of a plurality of marker genes it is preferred that the expression level of 1, 2, 3, or 4, individual marker genes according to the invention is assessed or even more when the marker genes according to the invention are combined with known cancer, preferably breast cancer, marker genes.
  • the invention is related to uses of a nucleic acid molecule (CRASH) which has upregulated expression in metastatic tumor cells and which is induced during tumor progression and/or metastasis, especially in mammary carcinoma cells.
  • CRASH nucleic acid molecule
  • the nucleic acid CRASH has the sequence SEQ ID NO:l or it is a nucleic acid which, because of the degeneracy of the genetic code, differs from SEQ ID NO:l, but which encodes preferably the amino acid sequence encoded by the nucleic acid of SEQ ID NO:l.
  • the present invention provides a nucleic acid which is upregulated in metastatic tumor cells and which codes for a polypeptide which is induced during tumor progression or metastasis, the nucleic acid being selected from the group consisting of:
  • the marker gene according to the invention has a sequence according to SEQ ID NO: 1.
  • nucleic acid molecules that correspond to the marker gene according to the invention.
  • nucleic acid molecules comprise sequences of RNA transcripts encoded by the marker gene according to the invention or portions of such transcripts.
  • isolated nucleic acids of the invention also include nucleic acid molecules sufficient for use as hybridization probes to identify of RNA transcripts encoded by the marker gene according to the invention or portions of such transcripts, and fragments of such nucleic acid molecules, e.g., those suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g.
  • RNA molecules e.g., mRNA
  • the nucleic acid molecule can be single-stranded or double- stranded, but preferably is double- stranded DNA.
  • the invention also encompasses polynucleotides which differ from that of the polynucleotides described herein, but which produce the same phenotypic effect, such as an allelic variant. These altered, but phenotypically equivalent polynucleotides are referred to as "equivalent nucleic acids.”
  • This invention also encompasses polynucleotides characterized by changes in non-coding regions that do not alter the polypeptide produced therefrom when compared to the polynucleotide herein.
  • This invention further encompasses polynucleotides, which hybridize to the polynucleotides of the subject invention under conditions of moderate or high stringency. Alternatively, the polynucleotides are at least 85%, or at least 90%, or more preferably, greater or equal to 95% identical as determined by a sequence alignment program when run under default parameters.
  • an “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an “isolated” nucleic acid molecule comprises a protein-coding sequence and is free of sequences which naturally flank the coding sequence 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 4 kB, 3 B, 2 B, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which 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.
  • a nucleic acid molecule of the present invention e.g., a nucleotide transcript encoded by the marker gene according to the invention, can be isolated using standard molecular biology techniques.
  • a nucleic acid molecule of the present invention also encompasses the nucleic acid molecules, which can be isolated using standard hybridization and cloning techniques (e.g. as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • the marker gene of the invention can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to all or a portion of the marker gene according to the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises a nucleotide sequence of a
  • RNA transcript encoded by a the marker gene according to the invention or a complement of said sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex.
  • a nucleic acid molecule of the invention can comprise only a portion of the nucleotide sequence (RNA or cDNA) of a RNA transcript encoded by the marker gene according to the invention or a complement of said sequence.
  • Such nucleic acids can be used, for example, as a probe or primer.
  • the probe/primer typically is used as one or more substantially purified oligonucleotides.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about; 7, preferably about 15, more preferably about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutive nucleotides of the marker gene according to the invention.
  • Probes based on the sequence of the marker gene according to the invention can be used to detect transcripts or genomic sequences of the marker gene according to the invention.
  • the probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which mix-express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sampleof cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted.
  • the invention further encompasses nucleic acid molecules that differ, due to degeneracy of the genetic code, from the nucleotide sequence of nucleic acids encoding a protein which corresponds to the marker gene according to the invention, and thus encode the same protein.
  • DNA sequence polymorphisms that lead to changes in the amino acid sequence can exist within a population (e.g., the human population). Such genetic polymorphisms can exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes which occur alternatively at a; given genetic locus. In addition, it will be appreciated that DNA polymorphisms that affect RNA expression levels can also exist that may affect the overall expression level ofthat gene (e.g. by affecting regulation or degradation). As used herein, the phrase "allelic variant" refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide by the marker gene according to the invention.
  • Such natural allelic variations can typically result in 0.1-0.5% variance in the nucleotide sequence of a given gene.
  • Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.
  • an isolated nucleic acid molecule of the invention is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400 or more nucleotides in length and hybridizes under stringent conditions to a RNA transcript of the marker gene according to the invention or a portion of said transcript or a cDNA corresponding to said transcript or portion thereof.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 75% (80%, 85%, preferably 90%) identical to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in sections 6.3.1 -6.3.6 of Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989).
  • DNA each of which is at least about 100 bases in length and/or for annealing a single-stranded DNA and a single-stranded RNA each of which is at least about 100 bases in length means hybridization at 65°C in a hybridization buffer consisting of 250 mmol/1 sodium phosphate buffer pH 7.2, 7% (w/v) SDS, 1% (w/v) BSA, 1 mmol/1 EDTA and 0.1 mg/ml single-stranded salmon sperm DNA. A final wash is performed at 65°C in 125 mmol/1 sodium phosphate buffer pH 7.2, 1 mmol/1 EDTA and 1% (w/v) SDS.
  • sequence changes can be introduced by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein encoded thereby.
  • sequence changes can be made by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein encoded thereby.
  • a "non essential” amino acid residue is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are not conserved or only semi-conserved among homologs of various species may be non essential for activity and thus would be likely targets for alteration.
  • amino acid residues that are conserved among the homologs of various species e.g. murine and human
  • another aspect of the invention pertains to nucleic acid molecules encoding a polypeptide of the invention that contain changes in amino acid residues that are not essential for activity.
  • Such polypeptides differ in amino acid sequence from the; naturally- occurring proteins encoded by the marker gene according to the invention, yet retain biological activity.
  • such a protein has an amino acid sequence that is at least about 40% identical, 50%, 60%, 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of one of the proteins encoded by the marker gene according to the invention.
  • An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of nucleic acids of the invention, such that one or more amino acid residue substitutions, additions, or deletions are introduced into the encoded protein.
  • Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • 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 deemed 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
  • non-polar side chains e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, mefhionine, tryptophan
  • beta-branched side chains e.g. threonine, valine, isoleucine
  • aromatic side chains e.g. tyrosine, phenylalanine, tryptophan, histidine.
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
  • the present invention encompasses antisense nucleic acid molecules, i. e., molecules which are complementary to a sense nucleic acid of the invention, e.g. complementary to the coding strand of a double-skanded cDNA molecule corresponding to the marker gene according to the invention or complementary to an mRNA sequence corresponding to the marker gene according to the invention.
  • an antisense nucleic acid of the invention can hydrogen bond to (i. e. anneal with a sense nucleic acid of the invention.
  • the antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g. all or part of the protein coding region (or open reading frame).
  • An antisense nucleic acid molecule can also be antisense to all or part of a non- coding region of the coding strand of a nucleotide sequence encoding a polypeptide of the invention.
  • the non- coding regions (“5' and 3' untranslated regions") are the 5' and 3' sequences which flank the coding region and are not translated into amino acids.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g. an antisense oligonucleotide
  • an antisense nucleic acid e.g. an antisense oligonucleotide
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5- bromouracil, 5 chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5 (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5 carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2 methyladenine, 2-methylguanine, 3- methylcytosine, 5-methylcytosine, N6-adenine, 7 methylguanine, 5- methylaminomethyluracil, 5-methoxya
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been sub- cloned 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).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a polypeptide of the invention to thereby inhibit expression of the gene, e.g. by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to forth a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site or infusion of the antisense nucleic acid into a cancer cell, e.g. a mammary cell, associated body fluid.
  • 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 which 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 pol II or pol III promoter are preferred.
  • An antisense nucleic acid molecule of the invention can be an a-anomeric nucleic acid molecule.
  • An anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual a- units, the strands run parallel to each other (Gautier, C, et al., Nucleic Acids Res. 15 (1987)
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue, H., et al., Nucleic Acids Res. 15 (1987) 6131-6148) or a chimeric RNA-DNA analogue (Inoue, H., et al, FEBS Lett. 215 (1987) 327-330).
  • the invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes as described in Haseloff, J., and Gerlach, W.L., Nature 334 (1988) 585-591
  • a ribozyme having specificity for a nucleic acid molecule of the invention can be designed based upon the nucleotide sequence of a cDNA corresponding to the nucleic acid molecule.
  • a derivative of a Tetrahymena L-19 INS R ⁇ A can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved (see U.S. Patent No. 4,987,071; and U.S. Patent No. 5,116,742).
  • an mRNA encoding a polypeptide ofthe invention can beused to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see, e.g. Bartel, D.P., and Szostak, J.W., Science 261 (1993) 1411-1418).
  • the invention also encompasses nucleic acid molecules which form triple helical structures.
  • expression of a polypeptide of the invention can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g. the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide e.g. the promoter and/or enhancer
  • the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup, B., and Nielsen, P.E., Bioorg. Med. Chem. 4 (1996) 5-23).
  • peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural bases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-O'Keefe, H., et al., Proc. Natl. Acad. Sci. USA 93 (1996) 14670-14675.
  • PNAs can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or anti-gene agents for sequence-specific modulation of gene expression by, e.g. inducing transcription or translation arrest or inhibiting replication.
  • PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g. PNA directed PCR clamping, as artificial restriction enzymes when used in combination with other enzymes, e.g. SI nucleases (Hyrup, 1996, supra; or as probes or primers for DNA sequence and hybridization (Hyrup, 1996, supra, Perry O'Keefe et al, 1996, supra).
  • PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras can be generated which can combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, e.g. RNASE H and DNA polymerases, to interact with the DNA 3s portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the bases, and orientation
  • PNA monomers are then coupled in a step-wise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al., 1996, supra).
  • chimeric molecules can be synthesized with a 5' DNA segment and a
  • the oligonucleotide can include other appended groups such as peptides (e.g. for targeting host cell receptors in viva), or agents facilitating transport across the cell membrane (see, e.g. Letsinger, R.L., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 6553-6556; Lemaitre, M., et al., Proc. Natl. Acad. Sci. USA 84 (1987) 648-652; WO 88/09810) or the blood-brain barrier (see, e.g.,
  • oligonucleotides can be modified with hybridization- triggered cleavage agents (see, e.g., van der Krol, A.R., et al, Bio/Techniques 6 (1988) 958-976) or intercalating agents (see, e.g., Zon, G., Pharm. Res. 5 (1988) 539-549).
  • the oligonucleotide can be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the invention also includes molecular beacon nucleic acids having at least one region which is complementary to a nucleic acid of the invention, such that the molecular beacon is useful for quantitating the presence of the marker gene according to the invention in a sample.
  • a "molecular beacon" nucleic acid is a nucleic acid comprising a pair of complementary regions and having a fluorophore and a fluorescent quencher associated therewith. The fluorophore and quencher are associated with different portions of the nucleic acid in such an orientation that when the complementary regions are annealed with one another, fluorescence of the fluorophore is quenched by the quencher.
  • nucleic acids are the double-labelled digestible oligonucleotides used in the TaqMan ® format as principally set out in US 5,210,015, US 5,487,972 or
  • the invention further comprises a recombinant polypeptide or protein with an amino acid sequence according to SEQ ID NO: 2 which is encoded by the marker gene according to the invention, preferably by the DNA sequence shown in
  • the invention further comprises a method for producing a protein whose expression is correlated with tumor metastasis, by expressing an exogenous DNA in prokaryotic or eukaryotic host cells and isolation of the desired protein, wherein the protein is coded by the marker gene according to the invention, preferably with the nucleic acid sequence as shown in SEQ ID NO:l.
  • the protein can be isolated from the cells or the culture supernatant and purified by chromatographic means, preferably by ion exchange chromatography, affinity chromatography and/or reverse phase HPLC.
  • the isolated CRASH polypeptide can occur in natural allelic variations which differ from individual to individual. Such variations ofthe amino acids are usually amino acid substitutions. However, they may also be deletions, insertions or additions of amino acids to the total sequence.
  • the CRASH protein according to the invention - depending, both in respect of the extent and type, on the cell and cell type in which it is expressed- can be in glycosylated or non-glycosylated form. This will be described in more detail below.
  • Polypeptide with CRASH activity means also proteins with minor amino acid variations but with substantially the same CRASH activity. Substantially the same means that the activities are of the same biological properties and the polypeptides show (at least 90%, preferably more than 95%) homology, or preferably identity, in amino acid sequence. Homology can be examined by using the BLAST algorithm described by Altschul, S.F., et al., Nucleic Acids Res. 25 (1997) 3389-3402. This is also described in more detail below.
  • the polypeptide according to the invention can be produced by recombinant means in host cells, using an expression vector, or can be produced synthetically.
  • Non-glycosylated CRASH polypeptide is obtained when it is produced recombinantly in prokaryotes.
  • nucleic acid sequences provided by the invention it is possible to search for the CRASH gene or its variants in genomes of any desired cells (e.g. apart from human cells, also in cells of other mammals), to identify these and to isolate the desired gene coding for the MMX-1 protein.
  • Such processes and suitable hybridization conditions are known to a person skilled in the art and are described, for example, by Sambrook et al.,
  • CRASH can be purified after recombinant production by affinity chromatography using known protein purification techniques, including immunoprecipitation, gel filtration, ion exchange chromatography, chromatofocussing, isoelectric focussing, selective precipitation, electrophoresis, or the like. This will be described in more detail below.
  • One aspect of the invention pertains to isolated proteins encoded by the nucleic acid sequence of the invention, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise antibodies directedagainst a polypeptide encoded by the nucleic acid sequence of the invention.
  • the native polypeptide encoded by the nucleic acid sequence can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • a polypeptide encoded by the nucleic acid sequence ofthe invention are produced by recombinant DNA techniques.
  • a polypeptide encoded by the marker gene according to the invention can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof 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 of chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5 % (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").
  • the 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 %,10 %, or 5 % of the volume of the protein preparation.
  • culture medium represents less than about 20 %,10 %, or 5 % of the volume of the protein preparation.
  • the protein is produced by chemical synthesis, it is preferably substantially free of chemicalprecursors or other chemicals, i. e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations ofthe protein have less than about 30 %,20 %, 10 %, 5 % (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.
  • Biologically active portions of a polypeptide encoded by nucleic acid molecule of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence ofthe protein encoded by the marker gene according to the invention which include fewer amino acids than the full length protein, and exhibit at least one activity ofthe corresponding full-length protein.
  • biologically active portions comprise a domain or motif with at least one activity of the corresponding protein.
  • a biologically active portion of the protein of the invention can be a polypeptide which is, for example, 10, 25,50, 100 or more amino acids in length.
  • biologically active portions in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the invention.
  • Other useful proteins are substantially identical (e.g. at least about 40%, preferably 50%, 60%, 70%, 80%, 90%, 95%, or 99%) to one of these sequences and retain the functional activity of the protein of the corresponding naturally- occurring protein yet differ in amino acid sequence due to natural allelic variation or mutagenesis.
  • the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid 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.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin, S., and Altschul, S.F., Proc. Natl. Acad. Sci. USA 87 (1990)
  • Gapped BLAST can be utilized as described in Altschul, S.F., et al., Nucleic Acids Res. 25 (1997) 3389-3402.
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See http://www.ncbi.nlm.nih.gov.
  • Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS or 11 - 17.
  • ALIGN program version 2.0
  • a PAM120 weight residue table version 2.0
  • a gap length penalty of 12 can be used.
  • a gap penalty of 4 can be used.
  • Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson, W.R., and Lipman, D.J., Proc. Natl. Acad. Sci. USA 85 (1988) 2444-2448.
  • a PAM120 weight residue table can, for example, be used with a k-tuple value of 2.
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
  • the invention also provides chimeric or fusion proteins corresponding to a the marker gene according to the invention.
  • a "chimeric protein” or “fusion protein” comprises all or part (preferably a biologically active part) of the polypeptide encoded by the marker gene according to the invention operably linked to a heterologous polypeptide (i e., a polypeptide other than the polypeptide encoded by the nucleic acid molecule).
  • a heterologous polypeptide i e., a polypeptide other than the polypeptide encoded by the nucleic acid molecule.
  • the term "operably linked” is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused in-frame to each other.
  • the heterologous polypeptide can be fused to the amino-terminus or the carboxyl-terminus of the
  • One useful fusion protein is a GST fusion protein in which a polypeptide encoded by the marker gene according to the invention is fused to the carboxyl terminus of GST sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide ofthe invention.
  • the fusion protein contains a heterologous signal sequence at its amino terminus.
  • the native signal sequence of a polypeptide encoded by the marker gene according to the invention can be removed and replaced with a signal sequence from another protein.
  • the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Ausubel et al., ed., Current Protocols in Molecular
  • eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California).
  • useful prokaryotic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech;
  • the fusion protein is an immunoglobulin fusion protein in which all or part of a polypeptide encoded by the marker gene according to the invention is fused to sequences derived from a member of the immunoglobulin protein family.
  • the immunoglobulin fusion proteins of the invention can be inco ⁇ orated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand (soluble or membrane-bound) and a protein on the surface of a cell (receptor), to thereby suppress signal transduction in vivo.
  • the immunoglobulin fusion protein can be used to affect the bioavailability of a cognate ligand of a polypeptide of the invention. Inhibition of ligand/receptor interaction can be useful therapeutically, both for treating proliferative and differentiative disorders and for modulating (e.g promoting or inhibiting) cell survival.
  • the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies directed against a polypeptide of the invention in a subject, to purify ligands and in screening assays to identify molecules which inhibit the interaction of receptors with ligands.
  • Chimeric and fusion proteins ofthe invention can be produced by standard recombinant DNA techniques.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, e.g. Ausubel et al., supra).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g. a GST polypeptide).
  • a nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide ofthe invention.
  • a signal sequence can be used to facilitate secretion and isolation of the secreted protein or other proteins of interest.
  • Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events.
  • Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway.
  • the invention pertains to the described polypeptides having a signal sequence, as well as to polypeptides from which the signal sequence has been proteolytically cleaved (i. e., the cleavage products).
  • a nucleic acid sequence encoding a signal sequence can be operably linked in an expression vector to a protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate.
  • the signal sequence directs secretion ofthe protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved.
  • the protein can then be readily purified from the extracellular medium by art recognized methods.
  • the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.
  • the present invention also pertains to variants of the polypeptides encoded by nucleic acid molecule of the invention.
  • Such variants have an altered amino acid sequence which can function as either agonists (mimetics) or as antagonists.
  • Variants can be generated by mutagenesis, e. g. discrete point mutation or truncation.
  • An agonist can retain substantially the same, or a subset, of the biological activities of the naturally occurring protein.
  • An antagonist of a protein can inhibit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein can have fewer side effects in a subject relative to treatment with the naturally occurring form ofthe protein.
  • Variants of a protein ofthe invention which function as either agonists (mimetics) or as antagonists can be identified by screening combinatorial libraries of mutants, e.g. truncation mutants, of the protein of the invention for agonist or antagonist activity.
  • a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).
  • methods which can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g.
  • libraries of fragments of the coding sequence of a polypeptide encoded by the marker gene according to the invention can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes amino terminal and internal fragments of various sizes ofthe protein of interest.
  • An isolated polypeptide encoded by the marker gene according to the invention, or a fragment thereof, can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full- length polypeptide or protein can be used or, alternatively, the invention provides antigenic peptide fragments for use as immunogens.
  • the antigenic peptide of a protein of the invention comprises at least 8 (preferably 10, 15, 20, or 30 or more) amino acid residues ofthe amino acid sequence ofthe polypeptide ofthe invention, and encompasses an epitope ofthe protein such that an antibody raised against the peptide forms a specific immune complex with a protein encoded by the marker gene according to the invention.
  • Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g. hydrophilic regions. Hydrophobicity sequence analysis, hydrophilicity sequence analysis, or similar analyses can be used to identify hydrophilic j regions.
  • An immunogen typically is used to prepare antibodies by immunizing a suitable (i.e. immunocompetent) subject such as a rabbit, goat, mouse, or other mammal or vertebrate.
  • An appropriate immunogenic preparation can contain, for example, recombinantly-expressed or chemically-synthesized polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent.
  • antibody and “antibody substance”; as used interchangeably herein refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i. e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention, e.g., an epitope of a polypeptide of the invention.
  • a molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g. a biological sample, which naturally contains the polypeptide.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies.
  • the term "monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope.
  • Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide of the invention as an immunogen.
  • Preferred polyclonal antibody compositions are ones that have been selected for antibodies directed against a polypeptide or polypeptides of the invention.
  • Particularly preferred polyclonal antibody preparations are ones that contain only antibodies directed against a polypeptide or polypeptides of the invention.
  • Particularly preferred immunogen compositions are those that contain no other human proteins such as, for example, immunogen compositions made using a non-human host cell for recombinant expression of a polypeptide of the invention. In such a manner, the only human epitope or epitopes recognized by the resulting antibody compositions raised against this immunogen will be present as part of a polypeptide or polypeptides ofthe invention.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules can be harvested or isolated from the subject (e.g., from the blood or serum of the subject) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibodies specific for a protein or polypeptide of the invention can be selected or (e.g., partially purified) or purified by, e.g. affinity chromatography.
  • a recombinantly expressed and purified (or partially purified) protein of the invention is produced as described herein, and covalently or non-covalently coupled to a solid support such as, for example, a chromatography column.
  • the column can then be used to affinity purify antibodies specific for the proteins ofthe invention from a sample containing antibodies directed against a large number of different epitopes, thereby generating a substantially purified antibody composition, i. e., one that is substantially free of contaminating antibodies.
  • a substantially purified antibody composition is meant, in this context, that the antibody sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those of the desired protein or polypeptide of the invention, and preferably at most 20%, yet more preferably at most 10%, and most preferably at most 5% (by dry weight) of the sample is contaminating antibodies.
  • a purified antibody composition means that at least 99% of the antibodies in the composition are directed against the desired protein or polypeptide ofthe invention.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler, G., and Milstein, C, Nature 256 (1975) 495-497, the human B cell hybridoma technique (see Kozbor et al., Immunol. Today 4 (1983) 72), the EBV hybridoma technique (see Cole et al., In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985, pp. 77-96) or trioma techniques.
  • the technology for producing hybridomas is well known (see generally
  • Hybridoma cells producing amonoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.
  • a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g. an antibody phage display library) with the polypeptide of interest.
  • Kits for generating and screening phage display libraries are commercially available (e.g. the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; end the Stratagene
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region (see, e.g., U.S. Patent No. 4,816,567; and U.S. Patent No. 4,816,397, which are incorporated herein by reference in their entirety).
  • Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule (see, e.g., U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety).
  • CDRs complementarity determining regions
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in WO 87/02671; EP-A 0 184 187; EP-A 0 171 496; EP-A 0 173 494; WO 86/01533; U.S. Patent No. 4,816,567; EP-A 0 125 023; Better, M., et al., Science 240 (1988)
  • Antibodies of the invention may be used as therapeutic agents in treating cancers.
  • completely human antibodies ofthe invention are used for therapeutic treatment of human cancer patients, particularly those having breast cancer.
  • Such antibodies can be produced, for example, using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g. all or a portion of a polypeptide encoded by the marker gene according to the invention.
  • Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • An antibody directed against a polypeptide encoded by the marker gene according to the invention can be used to isolate the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation.
  • an antibody can be used to detect the polypeptide (e.g., in a cellular lysate or cell supernatant) in order to evaluate the level and pattern of expression of the marker gene according to the invention.
  • the antibodies can also be used diagnostically to monitor protein levels in tissues or body fluids (e.g. in an ovary- associated body fluid) as part of a clinical testing procedure, e.g. to, for example, determine the efficacy of a given treatment regimen.
  • Detection can be facilitated by coupling the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, 6-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein iso hiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I, P, S or H.
  • an antibody can be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B.
  • gramicidin D ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dThydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-fhioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine, thioepa chlorambucil,melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-fhioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine, thioepa chlor
  • daunorubicin (formerly daunomycin) and doxorubicin
  • antibiotics e.g. dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)
  • anti-mitotic agents e.g. vincristine and o vinblastine.
  • the drug moiety can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor,.alpha.-interferon,.beta.-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4, 676,980.
  • the invention provides substantially purified antibodies or fragments thereof, and non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting ofthe amino acid sequences ofthe present invention, an amino acid sequence encoded by the cDNA of the present invention, a fragment of at least 15 amino acid residues of an amino acid sequence of the present invention, an amino acid sequence which is at least 95% identical to the amino acid sequence of the present invention (wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting ofthe nucleic acid molecules ofthe present invention, or a complement thereof, under conditions of hybridization of
  • the substantially purified antibodies of the invention, or fragments thereof can be human, non-human, chimeric and/or; humanized antibodies.
  • the invention provides non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence of the present invention, an amino acid sequence encoded by the cDNA ofthe present invention, a fragment of at least 15 amino acid residues of the amino acid sequence of; the present invention, an amino acid sequence which is at least 95% identical to the amino acid sequence of the present invention (wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting ofthe marker gene according to the invention, or a complement thereof, under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C.
  • non-human antibodies can be goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies.
  • the non-human antibodies of the invention can be chimeric and/or humanized antibodies.
  • the non human antibodies of the invention can be polyclonal antibodies or monoclonal antibodies.
  • the invention provides monoclonal antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences ofthe present invention, an amino acid sequence encoded by the cDNA ofthe present invention, a fragment of at least 15 amino acid residues of an amino acid sequence of the present invention, an amino acid sequence which is at least 95% identical to an amino acid sequence ofthe present invention (wherein the percent identity is determined using the ALIGN program ofthe GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of the nucleic acid molecules of the present invention or a complement thereof, under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C.
  • the monoclonal antibodies can be human, humanized,
  • the substantially purif ed antibodies or fragments thereof may specifically bind to a signal peptide, a secreted sequence, an extracellular domain, a kansmembrane or a cytoplasmic domain or cytoplasmic membrane of a polypeptide ofthe invention.
  • the substantially purified antibodies or fragments thereof, the non-human antibodies or fragments thereof, and/or the monoclonal; antibodies or fragments thereof, of the invention specifically bind to a secreted sequence or an extracellular domain of the amino acid sequences of the present invention.
  • any ofthe antibodies ofthe invention can be conjugated to a therapeutic moiety; or to a detectable substance.
  • detectable substances that can be conjugated to the antibodies ofthe invention are an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, and a radioactive material.
  • the invention also provides a kit containing an antibody of the invention conjugated to a detectable substance, and instructions for use.
  • Still another aspect of the invention is a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition contains an antibody of the invention, a therapeutic moiety, and a pharmaceutically acceptable carrier.
  • Still another aspect of the invention is a method of making an antibody that specifically recognizes a polypeptide of the present invention, the method comprising immunizing a mammal with a polypeptide.
  • the polypeptide used as an immunogen comprises an amino acid sequence selected from the group consisting of the amino acid sequence of the present invention, an amino acid sequence encoded by the cDNA of the nucleic acid molecules of the present invention, a fragment of at least 15 amino acid residues of the amino acid sequence of the present invention, an amino acid sequence which is at least 95% identical to the amino acid sequence of the present invention (wherein the percent identity is determined using the ALIGN program of the GCG 3s software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 and an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting ofthe nucleic acid molecules ofthe present invention, or a complement thereof, under conditions of hybridization of 6X SSC at 45°C
  • a sample is collected from the mammal that contains an antibody that specifically recognizes the polypeptide.
  • the polypeptide is recombinantly produced using a non-human host cell.
  • the antibodies can be further purified from the sample using techniques well known to those of skill in the art.
  • the method can further comprise producing a monoclonal antibody- producing cell from the cells of the mammal.
  • antibodies are collected from the antibody-producing cell.
  • Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide encoded by the marker gene according to the invention (or a portion of such a polypeptide). Therefore, the invention further provides an expression vector comprising a nucleic acid being selected from the group consisting of:
  • nucleic acid sequence which, because ofthe degeneracy ofthe genetic code, is not a sequence of (a) or (b), but which codes for a polypeptide having exactly the same amino acid sequence as a polypeptide encoded by a sequence of (a) or (b); and (d) a nucleic acid sequence which is a fragment of any ofthe sequences of (a), (b) or (c).
  • the expression vector comprises a nucleic acid with a nucleic acid sequence according to SEQ ID NO: 1.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g.
  • non-episomal mammalian vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors namely expression vectors, are capable of directing the expression of genes to which they are operably linked.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors).
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e. g.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell.
  • the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to fhe nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression ofthe nucleotide sequence (e.g.
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Methods in Enzymology: Gene Expression Technology, Vol. 185, Academic Press, San Diego, CA, 1991. Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression ofthe nucleotide sequence only in certain host cells (e.g. tissue-specific regulatory sequences).
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as; described herein.
  • the recombinant expression vectors of the invention can be designed for expression of a polypeptide encoded by the marker gene according to the invention in prokaryotic (e.g. E. coli) or eukaryotic cells (end insect cells using baculovirus expression vectors, yeast cells or mammalian cells). Suitable host cells are discussed further in Goeddel, supra.
  • the recombinant expression vector can be transcribed; and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B., and Johnson, K.S., Gene 67 (1988) 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Phannacia, Piscataway, NJ) which fuse glutathione S- transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E.
  • coli expression vectors include pTrc (Amann, E., et al., Gene 69 (1988) 301-315) and pET 1 Id (Studier et al., In: Gene Expression Technology: Methods in Enzymology, Vol. 85, Academic Press, San Diego, CA, 1991, pp. 60-89).
  • Target gene expression from the pTrc vector relies on host RNApolymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET 1 Id vector relies on transcription from a T7 gulO-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gal). This viral polymerase is supplied by host strains BL21(DE3) or HMS 174(DE3) from a resident prophage harboring a T7 gal gene under the transcriptional control ofthe lacUVpromoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, In: Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Pres, San Diego, CA, 1990, pp. 119-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada, K., et al., Nucleic Acids Res. 20 (1992) 2111- 2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.
  • the expression vector is a yeast expression vector.
  • vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari,
  • the expression vector is a baculovirus expression vector.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith, G.E., et al., Mol. Cell Biol. 3 (1983) 2156- 2165) and the pVL series (Luckow, V.A., and Summers, M.D., Virology 170 (1989) 31-39).
  • the marker gene according to the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B., Nature 329 (1987) 840- 842) and pMT2NOPC (Kaufman, R.J., et al., EMBO J. 6 (1987) 187-193).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both; prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al., supra.
  • the recombinant mammalian expression vector is capable of directing expression ofthe nucleic acid preferentially in a particular cell type (e.g. tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitabletissue-specific promoters include the albumin promoter (liver-specific; Pinkert, C.A., et al., Genes Dev. 1 (1987) 268-277), lymphoid-specific promoters
  • pancreas-specific promoters Eslund, T., et al., Science 230 (1985) 912-916
  • mammary gland-specific promoters e.g. milk whey promoter, U.S. Patent No. 4,873,316 and EP-A 0 264 166
  • Developmentally regulated promoters are also encompassed, for example the murine box promoters (Kessel, M., and Gruss, P., Science 249 (1990) 374-379) and the alpha-fetoprotein promoter (Camper, S.A., and Tilghman, S.M., Genes Dev. 3 (1989) 537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner which allows for expression (by transcription ofthe DNA molecule) of an RNA molecule which is antisense to the mRNA encoding a polypeptide of the invention.
  • Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue- specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic (e.g. E. coli) or eukaryotic cell (e.g. insect cells, yeast or mammalian cells).
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art- recognized; techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran- mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.
  • SM selective marker
  • a gene that encodes a "selectable marker” (SM) gene is generally introduced into the host cells along with the gene of interest.
  • SM selective marker
  • Preferred SM genes include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g. cells that have incorporated the SM gene will survive, while the other cells die).
  • a host cell ofthe invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce a polypeptide encoded by the marker gene according to the invention.
  • the invention further provides methods for producing a polypeptide encoded by the marker gene according to the invention using the host cells ofthe invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide encoded by the marker gene according to the invention is produced.
  • the method further comprises isolating the polypeptide from the medium or the host cell.
  • the host cells of the invention can also be used to produce nonhuman transgenic animals.
  • a host cell ofthe invention is a fertilized oocyte or an embryonic stem cell into which a sequences encoding a polypeptide ofthe marker gene according to the invention have been introduced.
  • Such host cells can then be used to create non- human transgenic animals in which exogenous sequences encoding the marker gene according to the invention have been introduced into their genome or homologous recombinant animals in which endogenous gene(s) encoding a polypeptide corresponding to the marker gene according to the invention have been altered.
  • Such animals are useful for studying the function and/or activity of the polypeptide corresponding to the marker gene and for identifying and/or evaluating modulators of polypeptide activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the s animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal; develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • an "homologous recombinant animal” is a non human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g. an embryonic cell of the animal, prior to development ofthe animal.
  • a transgenic animal of the invention can be created by introducing a nucleic acid encoding a polypeptide encoded by the marker gene according to the invention into the male pronuclei of a fertilized oocyte, e.g. by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the polypeptide of the invention to particular cells.
  • a transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of mRNA encoding the transgene in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying the transgene can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of the marker gene according to the invention into which a deletion, addition or substitution has been introduced to thereby alter, e.g. functionally disrupt, the gene.
  • the vector is designed such that, upon homologous recombination, the endogenous gene is functionally disrupted (i. e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous protein).
  • the altered portion ofthe gene is flanked at its 5' and 3' ends by additional nucleic acid of the gene to allow for homologous recombination to occur between the exogenous gene carried by the vector and an endogenous gene in an embryonic stem cell.
  • the additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • the vector is introduced into an embryonic stem cell line (e.g.
  • cells in which the introduced gene has homologously recombined with the endogenous gene are selected (see, e.g. Li, E., et al., Cell 69 (1992) 915-926).
  • the selected cells are then injected into a blastocyst of an animal (e.g. a mouse) to form aggregation chimeras (see, e.g. Bradley, Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, Robertson, ed., IRL, Oxford, 1987, pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, A., Curr. Opin. Biotechnol. 2 (1991) 823-829 and in WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.
  • transgenic non-human animals can be produced which contain selected systems which allow for regulated expression ofthe transgene.
  • One example of such a system is the cre/loxP recombinase system of bacteriophage Pi.
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman, S., et al., Science 251 (1991) 1351-1355). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g. by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones ofthe non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I., et al., Nature 385 (1997) 810-813 and WO 97/07668 and WO 97/07669.
  • the present invention pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining the level of expression of the protein or the nucleic acid according to the invention, in order to determine whether an individual is at risk of metastatic cancer. Such assays can be used for prognostic or predictive purposes to thereby prophylactically treat an individual prior to the metastasis ofthe cancer.
  • Yet another aspect of the invention pertains to monitoring the influence of agents (e.g. drugs or other compounds administered either to inhibit the metastasis of breast cancer or to treat or prevent any other disorder) on the expression or activity of the marker gene according to the invention in clinical trials.
  • agents e.g. drugs or other compounds administered either to inhibit the metastasis of breast cancer or to treat or prevent any other disorder
  • the CRASH nucleic acid is present in a greater amount in a metastasized tumor sample than in a sample free from metastasis.
  • a test sample having potential for tumor progression or metastasis will have a greater amount ofthe CRASH nucleic acid ofthe present invention than does a cancer cell sample which is free from metastasis.
  • the test sample it is preferable that the test sample have an approximate amount of CRASH nucleic acid which is appreciably greater that the approximate amount in a non-metastasized sample.
  • a test sample having an upregulated CRASH gene may have approximately 15- to approximately 60- fold greater amount of CRASH gene than a non-metastasized sample or at least 3-fold greater amount than a housekeeping gene like glyceraldehyde-3-phosphate dehydrogenase (GPDH) or porpho- bilinogendeaminase (PBGD).
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • PBGD porpho- bilinogendeaminase
  • the present invention provides a process for detecting the presence or absence of at least one specific nucleic acid or mixture of nucleic acids, or distinguishing between two different sequences in said sample, wherein the sample is suspected of containing said sequence or sequences, which process comprises the following steps in order:
  • nucleic acid probe which is selected from the group consisting of: (i) a nucleic acid sequence of SEQ ID NO: 1 ; (ii) a nucleic acid sequence which is complementary to a nucleic acid sequence of (i); (iii) a nucleic acid sequence which hybridizes under stringent conditions with the sequence of (i); and (iv) a nucleic acid sequence which hybridizes under stringent conditions with the sequence of (ii); and
  • the present invention provides a process for determining whether or not a cancer cell-containing test sample has potential for tumor progression or metastasis or a method for assessing the estrogen receptor status of a human breast cancer cell in a human cancer cell containing patient sample, wherein the test sample and a cancer cell-containing sample which is free from metastasis are obtained from the same individual or different individuals of the same species, which process comprises the following steps:
  • nucleic acid probe which is selected from the group consisting of: (i) a nucleic acid sequence of SEQ ID NO:l; (ii) a nucleic acid sequence which is complementary to a nucleic acid sequence of (i); (iii) a nucleic acid sequence which hybridizes under stringent conditions with the sequence of (i); and (iv) a nucleic acid sequence which hybridizes under stringent conditions with the sequence of (ii); and (b) determining the approximate amount of hybridization of each respective sample with said probe, and (c) comparing the approximate amount of hybridization ofthe test sample to an approximate amount of hybridization of the sample which is free from metastasis, to identify whether or not the test sample contains a greater amount of the specific nucleic acid or mixture of nucleic acids than does the sample which is free from metastasis.
  • the marker gene according to the invention or the corresponding in the sample containing cancer cells is overexpressed at least 1.25 fold, preferably 2 fold, more preferably 4 fold 10 fold or higher than in the test sample containing cells which are free from metastasis.
  • nucleic acids provided by the invention it is possible to provide a test which can be used to detect nucleic acids with upregulated expression in metastatic human tumor cells.
  • a test can be carried out by means of nucleic acid diagnostics.
  • the sample to be examined is contacted with a probe that is selected from the group comprising
  • nucleic acid sequence shown in SEQ ID NO:l or a nucleic acid sequence which is complementary to said nucleic acid sequence, and b) nucleic acids which hybridize under stringent conditions with one of the nucleic acids from a), wherein
  • the nucleic acid probe is incubated with the nucleic acid of the sample and the hybridization is detected optionally by means of a further binding partner for the nucleic acid ofthe sample and/or the nucleic acid probe. Hybridization between the probe used and nucleic acids from the sample indicates the presence of the RNA of such proteins.
  • the invention also includes a method for the detection of the metastatic potential of carcinoma cells or for the determination of the estrogen receptor status of a human breast cancer cell, comprising
  • a nucleic acid probe which is selected from the group consisting of (i) the nucleic acid shown in SEQ ID NO: 1 or a nucleic acid which is complementary to said nucleic acid sequence, and (ii) nucleic acids which hybridize with the nucleic acids from (i) and b) detecting hybridization by
  • the invention comprises a process for determining whether or not a test sample originating from or containing human cells has a tumor progression potential or a method for assessing the estrogen receptor status of a human breast cancer cell in a test sample originating from or containing human cells, which process comprises the following steps:
  • nucleic acid probe which is selected from the group consisting of: (i) a nucleic acid with a sequence of SEQ ID NO: 1; (ii) a nucleic acid with a sequence which is complementary to the nucleic acid of (i); (iii) a nucleic acid with a sequence which hybridizes under stringent conditions with the nucleic acid of (i); and (iv) a nucleic acid with a sequence which hybridizes under stringent conditions with the nucleic acid of (ii); and
  • test sample contains an at least 3-fold amount of nucleic acid hybridizing with the first probe in comparison to the amount of nucleic acid hybridizing with the second probe.
  • the nucleic acid probe is incubated with the nucleic acid of the sample and the hybridization is detected optionally by means of a further binding partner for the nucleic acid of the sample and/or the nucleic acid probe.
  • the nucleic acids according to the invention are hence valuable prognostic markers in the diagnosis ofthe metastatic and progression potential of tumor cells of a patient.
  • An exemplary method for detecting the presence or absence of a polypeptide or nucleic acid encoded by the marker gene according to the invention in a biological sample involves obtaining a biological sample (e.g. a biopsy) from a test subject and contacting the biological sample with a compound or an agent capable of detecting the polypeptide or nucleic acid (e.g. mRNA, genomic DNA, or cDNA).
  • a biological sample e.g. a biopsy
  • a compound or an agent capable of detecting the polypeptide or nucleic acid e.g. mRNA, genomic DNA, or cDNA.
  • the detection methods ofthe invention can thus be used to detect mRNA, protein, cDNA, or genomic DNA, for example, in a biological sample in vitro as well as in viva.
  • in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of a polypeptide encoded by the marker gene according to the invention include enzyme linked immunosorbent
  • in vivo techniques for detection of a polypeptide encoded by a the marker gene according to the invention include introducing into a subject a labeled antibody directed against the polypeptide.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • a general principle of such diagnostic and prognostic assays involves preparing a sample or reaction mixture that may contain a protein or polynucleotide encoded by a the nucleic acid molecule, and a probe, under appropriate conditions and for a time sufficient to allow the protein or nucleotide and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture.
  • These assays can be conducted in a variety of ways.
  • one method to conduct such an assay would involve anchoring the protein or nucleotide on the one hand or probe on the other onto a solid phase supports also referred to as a substrate, and detecting complexes comprising the target nucleic acid molecule or protein and the probe anchored on the solid phase at the end of the reaction.
  • a sample from a subject which is to be assayed for presence and/ or concentration ofthe proteins or nucleotides encoded by the the nucleic acid molecule, can be anchored onto a carrier or solid phase support.
  • the reverse situation is possible, in which the probe can be anchored to a solid phase and a sample from a subject can be allowed to react as an unanchored component ofthe assay.
  • biotinylated assay components can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g. biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin- coated 96 well plates (Pierce Chemical).
  • biotin-NHS N-hydroxy-succinimide
  • the surfaces with immobilized assay components can be prepared in advance and stored.
  • suitable carriers or solid phase supports for such assays include any material capable of binding the class of molecule to which the protein according to the invention or nucleotide or probe belongs.
  • Well-known supports or carriers include, but are not limited to, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the non immobilized component is added to the solid phase upon which the second component is anchored.
  • uncomplexed components may be removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized upon the solid phase.
  • the detection of complexes comprising the protein of the invention or nucleotide sequence and the probe anchored to the solid phase can be accomplished in a number of methods outlined herein.
  • the probe when it is the unanchored assay component, can be labeled for the purpose of detection and readout of the assay, either directly or indirectly, with detectable labels discussed herein and which are well-known to one skilled in the art.
  • a fluorophore label on the first, 'donor' molecule is selected such that, upon excitation with incident light of appropriate wavelength, its emitted fluorescent energy will be absorbed by a fluorescent; label on a second 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy.
  • the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that of the 'donor'.
  • a PET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g. using a fluorimeter).
  • determination of the ability of a probe to recognize a protein or nucleotide encoded by a nucleic acid molecule can be accomplished without labeling either assay component (probe or nucleic acid molecule) by utilizing a technology such as real time Biomolecular Interaction Analysis (BIA) (see, e.g. Sjolander, S. and Urbaniczky, C, Anal. Chem. 63 (1991) 2338-2345 and Szabo, A., et al., Curr. Opin. Struct. Biol. 5 (1995) 699-705).
  • BIOA Biomolecular Interaction Analysis
  • BIOA or "surface plasmon resonance” is a technology for studying biospecific interactions in real time, without labeling any ofthe interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable which can be used as an indication of real-time reactions between biological molecules.
  • SPR surface plasmon resonance
  • analogous diagnostic and prognostic assays can be conducted with the protein of the invention or nucleotide and the probe as solutes in a liquid phase.
  • complexes comprising the protein ofthe invention or nucleotide and the probe are separated from uncomplexed components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation.
  • such complexes may be separated from uncomplexed assay components through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A.P., Trends Biochem Sci. 18 (1993 ) 284-287).
  • Standard chromatographic techniques may also be utilized to separate such complexes from uncomplexed components. For example, gel f ltration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complexes may be separated from the relatively smaller uncomplexed components.
  • the level of mRNA encoded by the marker gene according to the invention can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art.
  • biological sample is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated RNA.
  • RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from cancer cells (see, e.g. Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (U.S. Patent
  • the isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays.
  • One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected.
  • the nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA encoded by the marker gene according to the invention.
  • probes for use in the diagnostic assays ofthe invention are described herein. Hybridization of a mRNA with the probe indicates that the the nucleic acid molecule in question is expressed.
  • the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose.
  • the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array.
  • a skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the the marker gene according to the invention.
  • An alternative method for determining the level of mRNA encoded by the marker gene according to the invention in a sample involves the process of nucleic acid amplification, e.g., by rtPCR (the experimental embodiment set forth in U.S. Patent No. 4,683,202), ligase chain reaction (Barany, F., Proc. Natl. Acad. Sci. USA 88 (1991) 189-193), self sustained sequence replication (Guatelli, J.C., et al., Proc. Natl.
  • amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice- versa) and contain a short region in between.
  • amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
  • mRNA does not need to be isolated from the cancer cells prior to detection.
  • a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA encoded by the the nucleic acid molecule.
  • determinations may be based on the normalized expression level of the the nucleic acid molecule.
  • Expression levels are normalized by correcting the absolute expression level of the nucleic acid molecule by comparing its expression to the expression of a gene that is not the nucleic acid molecule, e.g. a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison ofthe expression level in one sample, e.g., a patient sample, to another sample, e.g. a non mammary cancer sample, or between samples from different sources. Alternatively, the expression level can be provided as a relative expression level.
  • the level of expression of the nucleic acid molecule is determined for 10 or more samples of normal versus cancer cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question.
  • the mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the the nucleic acid molecule.
  • the expression level of the marker gene according to the invention determined for the test sample is then divided by the mean expression value obtained for that the nucleic acid molecule. This provides a relative expression level.
  • the samples used in the baseline determination will be from mammary cancer or from non-mammary cancer cells of mammary tissue.
  • the choice of the cell source is dependent on the use ofthe relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether the nucleic acid molecule assayed is mammary specific (versus normal cells).
  • the mean expression value can be revised, providing improved relative expression values based on accumulated data. Expression data from mammary cells provides a means for grading the severity of the mammary cancer state.
  • a polypeptide encoded by the nucleic acid molecule is detected.
  • a preferred agent for detecting a polypeptide of the invention is an antibody capable of binding to a polypeptide encoded by the marker gene according to the invention, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g. Fab or F(ab') 2) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.
  • a detectable substance to the probe or antibody
  • indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • Proteins from mammary cells can be isolated using techniques that are well known to those of skill in the art. The protein isolation methods employed can, for example, be such as those described in Harlow and Lane, Antibodies: Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988).
  • a variety of formats can be employed to determine whether a sample contains a protein that binds to a given antibody.
  • formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis, immunohistochemistry and enzyme linked immunoabsorbant assay (ELISA).
  • a skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether cancer cells express a nucleic acid molecule of the present invention.
  • antibodies, or antibody fragments can be used in methods such as Western blots, immunohistochemistry or immunofluorescence techniques to detect the expressed proteins.
  • Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • protein isolated from cancer cells can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support such as nitrocellulose.
  • the support can then be washed with suitable buffers followed by treatment with the detectably labeled antibody.
  • the solid phase support can then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on the solid support can then be detected by conventional means.
  • Patent Nos. 4,996,143; 5,565,322; 5,849,489; and 6,162,603 are based on the fact that when a donor and a corresponding acceptor fluorescent label are positioned within a certain distance of each other, energy transfer takes place between the two fluorescent labels that can be visualized or otherwise detected and/or quantitated.
  • two probes, each containing a fluorescent label whereby the probes hybridize to the nucleic acid according to the invention being the target nucleic acid, can hybridize to an amplification product at particular positions determined by the complementarity of the probes to the target nucleic acid. Upon hybridization of the probes to the amplification product at the appropriate positions, a FRET signal is generated.
  • Fluorescent analysis can be carried out using, for example, a photon counting epifluorescent microscope system (containing the appropriate dichroic mirror and filters for monitoring fluorescent emission at the particular range), a photon counting photomultiplier system, or a fluorometer.
  • Excitation to initiate energy transfer can be carried out with an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiber optic light source, or other high intensity light source appropriately filtered for excitation in the desired range.
  • Hg high intensity mercury
  • corresponding refers to an acceptor fluorescent label having an excitation spectrum that overlaps the emission spectrum of the donor fluorescent label. Accordingly, efficient non-radiative energy transfer can be produced there between.
  • the preferred fluorescent label is fluorescein as the donor fluorescent label, whereby the acceptor fluorescent label is rhodamine, however, preferred is a cyanine dye, preferably Cy5 as described in US 6,174,670.
  • a method for detecting the presence or absence of the target nucleic acid, which is the marker gene according to the invention in a sample comprising the steps of:
  • a cycling step comprises an amplifying step and a hybridizing step
  • said amplifying step comprises contacting said sample with primers to produce an amplification product if target nucleic acid is present in said sample
  • said hybridizing step comprises contacting said sample with a pair of probes, wherein the members of said pair of probes hybridize to said amplification product within no more than five nucleotides of each other, wherein a first probe of said pair of probes is labeled with a donor fluorescent label and wherein a second probe of said pair of probes is labeled with a corresponding acceptor fluorescent label, and detecting the presence or absence of fluorescence resonance energy transfer between said donor fluorescent label of said first probe and said acceptor fluorescent label of said second probe, wherein the presence of FRET is indicative of the presence of the target nucleic acid in the sample, and wherein the absence of FRET is indicative ofthe absence ofthe target nucleic acid in the sample.
  • a method for detecting a target nucleic acid in a sample comprising the steps of amplifying the nucleic acid according to the inventionby polymerase chain reaction in the presence of two nucleic acid probes, , that hybridize to adjacent regions ofthe target nucleic acid, one of said probes being labeled with an acceptor fluorescent label and the other probe labeled with a donor fluorescent label of a fluorescence energy transfer pair such that upon hybridization ofthe two probes with the target nucleic acid, the donor and acceptor fluorescent labels are within 25 nucleotides of one another, said polymerase chain reaction comprising the steps of adding a thermostable polymerase, nucleotides and primers for the target nucleic acid to the sample and thermally cycling the sample between at least a denaturation temperature and an elongation temperature; exciting the biological sample with light at a wavelength absorbed by the donor fluorescent label and detecting fluorescent emission from the fluorescence energy transfer pair.
  • a method for the detection of a target nucleic acid in sample comprising the steps of amplifying the nucleic acid according to the invention by polymerase chain reaction in the presence of two nucleic acid probes, , that hybridize to adjacent regions of the nucleic acid, one of said probes being labeled with an acceptor fluorescent label and the other probe labeled with donor fluorescent label of a fluorescence energy transfer pair such that upon hybridization ofthe two probes with the target nucleic acid, the donor and acceptor fluorescent labels are within 25 nucleotides of one another, said polymerase chain reaction comprising the steps of adding a thermostable polymerase, nucleotides and primers for the target nucleic acid to the sample and thermally cycling the sample between at least a denaturation temperature and an elongation temperature; exciting the sample with light at a wavelength absorbed by the donor label and monitoring temperature dependent fluorescence from the fluorescence energy transfer pair.
  • a “homogeneous” assay system comprises reporter molecules or labels which generate a signal while the target sequence is amplified.
  • An example for a “homogeneous” assay system is the TaqMan® system that has been detailed in U.S. Patent Nos. 5,210,015; 5,804,375; and 5,487,972.
  • the method is based on a double-labelled probe and the 5'-3' exonuclease activity of Taq DNA polymerase.
  • the probe is complementary to the target sequence to be amplified by the PCR process and is located between the two PCR primers during each polymerisation cycle step.
  • the probe has two fluorescent labels attached to it.
  • One is a reporter dye, such as 6-carboxyfluorescein (FAM), which has its emission spectra quenched by energy transfer due to the spatial proximity of a second fluorescent dye, 6-carboxy-tetramethyl-rhodamine (TAMRA).
  • FAM 6-carboxyfluorescein
  • TAMRA 6-carboxy-tetramethyl-rhodamine
  • the Taq DNA polymerase in the process of elongating a primed DNA strand displaces and degrades the annealed probe, the latter due to the intrinsic 5' -3' exonuclease activity ofthe polymerase.
  • the mechanism also frees the reporter dye from the quenching activity of TAMRA.
  • the fluorescent activity increases with an increase in cleavage of the probe, which is proportional to the amount of PCR product formed. Accordingly, amplified target sequence is measured detecting the intensity of released fluorescence label.
  • a method for the detection of a target nucleic acid, which is the marker gene according to the invention, in a sample comprising the steps of
  • step (c) contacting the sample with the oligomeric compound under conditions for binding the oligomeric compound to the target nucleic acid, (d) determining the binding product or the degree of hybridization between the target nucleic acid and the oligomeric compound as a measure of the presence, absence or amount ofthe target nucleic acid.
  • the degree of hybridization is determined by the quantity of the first or second fluorescent label that is released from the oligonucleotide hybridized to the target nucleic acid by exonuclease hydrolysis by the template-dependent DNA polymerase.
  • the olignucleotide comprises two labels, preferably two fluorescent labels.
  • a method for the detection ofthe marker gene according to the invention, being the target nucleic acid in a sample comprising the steps of
  • step (a) contacting a sample comprising single-stranded nucleic acids with a first oligonucleotide containing a sequence complementary to a region of the target nucleic acid and a secong oligonucleotide containing a first and a second fluorescent label, and whereby said first oligonucleotide contains a sequence complementary to a second region of the same target nucleic acid sequence strand, but not including the nucleic acid sequence defined by the second oligonucleotide, to create a mixture of duplexes during hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the first and second oligonucleotide such that the 3' end of the first oligonucleotide is upstream ofthe 5' end ofthe oligomeric compound, (b) maintaining the mixture of step (a) having a 5' to 3' nuclease activity under conditions sufficient to permit the 5' to 3' nuclease activity ofthe
  • kits for detecting the presence of a polypeptide or nucleic acid encoded by the marker gene according to the invention in a biological sample e.g a mammary-associated body fluid.
  • a biological sample e.g a mammary-associated body fluid
  • kits for detecting the presence of a polypeptide or nucleic acid encoded by the marker gene according to the invention in a biological sample can be used to determine if a subject is suffering from or is at increased risk of developing metastatic mammary cancer or for the determination of the estrogen receptor status of a human breast cancer cell.
  • the kit can comprise a labeled compound or agent capable of detecting a polypeptide or an mRNA encoding a polypeptide encoded by the marker gene according to the invention in a biological sample and means for determining the amount ofthe polypeptide or mRNA in the sample (e.g. an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptid
  • the kit can comprise, for example: (1) a first antibody (e.g. attached to a solid support) which binds to a polypeptide corresponding to the marker gene according to the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable label.
  • a first antibody e.g. attached to a solid support
  • a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable label.
  • the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide encoded by the marker gene according to the invention or (2) a pair of primers useful for amplifying the marker gene according to the invention.
  • the kit can also comprise, e.g, a buffering agent, a preservative, or a protein stabilizing agent.
  • the kit can further comprise components necessary for detecting the detectable label (e.g. an enzyme or a substrate).
  • the kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample.
  • Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results ofthe assays performed using the kit.
  • the invention also includes an array comprising the nucleotide sequence of the marker gene of the present invention.
  • the array can be used to assay expression of one or more genes, including the marker gene according to the invention, in the array.
  • the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array. In this manner, 12000 and more genes can be simultaneously assayed for expression. This allows a profile to be developed showing a battery of genes specifically expressed in one or more tissues.
  • the invention allows the quantitation ofthe marker gene according to the invention.
  • tissue specificity but also the level of expression of a battery of genes in the tissue is ascertainable.
  • marker genes can be grouped on the basis of their tissue expression per se and level of expression in that tissue. This is useful, for example, in ascertaining the relationship of gene expression between or among tissues.
  • one tissue can be perturbed and the effect on marker gene expression in a second tissue can be determined.
  • the effect of one cell type on another cell type in response to a biological stimulus can be determined.
  • Such a determination is useful, for example, to know the effect of cell-cell interaction at the level of gene expression.
  • the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect.
  • undesirable biological effects can be determined at the molecular level.
  • the effects of an agent on expression of other than the target gene can be ascertained and counteracted.
  • the array can be used to monitor the time course of expression of one or more marker genes in the array. This can occur in various biological contexts, as disclosed herein, for example in development and differentiation of colon cancer, tumor progression, progression of other diseases, in vitro processes, such a cellular transformation and senescence, autonomic neural and neurological processes, such as, for example, pain and appetite, and cognitive functions, such as learning or memory.
  • the array is also useful for ascertaining the effect ofthe expression of a marker gene on the expression of other genes in the same cell or in different cells. This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.
  • the array is also useful for ascertaining differential expression patterns of one or more marker genes in normal and abnormal cells. This provides a battery of marker genes that could serve as a molecular target for diagnosis or therapeutic intervention.
  • array is meant an arrangement of addressable locations on a device (see e.g. US 5,143,854, US 6,022,963, US 6,156,501, WO 90/15070, WO 92/10092).
  • the locations can be arranged in two dimensional arrays, three dimensional arrays, or other matrix formats.
  • the number of locations can range from several to at least hundreds of thousands.
  • each location represents a totally independent reaction site.
  • Each location carries a nucleic acid as e.g. an "oligomeric compound", which can serve as a binding partner for a second nucleic acid, in particular a target nucleic acid. Methods for the manufacturing thereof are described in EP-A-0 476 014 and Hoheisel, J.
  • DNA chips Microfabricated arrays of large numbers of oligonucleotide probes, called “DNA chips” offer great promise for a wide variety of applications (see e.g. US 6,156,501 and US 6,022,963)
  • a method for determining the presence or the amount of a target nucleic acid, i.e. the marker gene according to the invention, in a sample comprising the steps of
  • step b) and c) of the method according to the invention whereby double stranded cDNA is synthesized with a primer comprising the bacterial T7-Promoter and labeled RNA is transcribed in the presence of ribonucleoside triphosphates whereby labels are attached to some ofthe nucleoside triphosphates.
  • the target nucleic acid in this case is preferably in the form of ribonucleic acid.
  • inhibitors for the expression of CRASH can be used to inhibit tumor progression/metastasis, preferably of malignant mammary carcinomas, in vivo, preferably by somatic gene therapy.
  • the invention further provides methods for identifying and isolation of antagonists of CRASH or inhibitors for the expression of CRASH (e.g. antisense nucleotides).
  • Such antagonists or inhibitors can be used to inhibit tumor progression or metastasis and cause massive apoptosis of tumor cells in vivo.
  • CRASH antagonists which have utility in the treatment of cancer, especially in inhibiting metastasis and related disorders. These methods include methods for modulating the expression ofthe polypeptides according to the invention, methods for identifying CRASH antagonists which can selectively bind to the proteins according to the invention, and methods of identifying CRASH antagonists which can modulate the activity of said polypeptides.
  • the methods further include methods for modulating, preferably inhibiting, the transcription of CRASH gene to mRNA, which preferably down-regulates the metastatic potential of a tumor cell. These methods can be conducted in vitro or in vivo and may make use of and establish cell lines and transgenic animal models of the invention.
  • a CRASH antagonist is defined as a substance or compound which decreases or inhibits the biological activity of CRASH, a polypeptide and/or inhibits the transcription or translation of CRASH gene.
  • screening procedures for CRASH antagonists involve contacting candidate substances with host cells in which invasiveness is mediated by expression of CRASH under conditions favorable for measuring CRASH activity.
  • CRASH activity may be measured in several ways. Typically, the activation is apparent by a change in cell physiology, such as increased mobility and invasiveness in vitro, or by a change in the differentiation state, or by a change in cell metabolism leading to an increase of proliferation.
  • CRASH gene and protein ofthe invention can be used to identify and design drugs which interfere with proliferation and dissemination of tumor cells. This will be described in more detail below.
  • a method of deriving a candidate agent comprising:
  • the marker gene has a nucleic acid sequence according to SEQ ID NO: 1,
  • an at least 1.5 fold difference or a less than 0.75 fold difference between the level of expression of the marker gene according to the inventionin the sample contacted with the candidate agent and the level of expression of the same marker gene in the sample not contacted with the candidate agent is an indication of an effect ofthe candidate agent.
  • the candidate agent is a candidate inhibitory agent.
  • the candidate agent is a candidate enhancing agent.
  • a candidate agent derived by the method according to the invention is provided or a pharmaceutical preparation comprising an agent according to the invention.
  • the agent according to the invention can be used for the preparation of a composition for the inhibition of progression of cancer.
  • a method of producing a drug comprising the steps ofthe method ofthe invention.
  • step (i) synthesizing the candidate agent identified in step (d) or an analog or derivative thereof in an amount sufficient to provide said drug in a therapeutically effective amount to a subject; and/or (ii) combining the drug candidate the candidate agent identified in step (d) or an analog or derivative thereof with a pharmaceutically acceptable carrier.
  • compositions suitable for administration typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • the invention includes methods for preparing pharmaceutical compositions for modulating the expression or activity of a polypeptide or nucleic acid encoded by the marker gene according to the invention.
  • Such methods comprise formulating a pharmaceutically; acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid encoded by the marker gene according to the invention.
  • Such compositions can further include additional active agents.
  • the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid encoded by the marker gene according to the invention and one or more additional active compounds.
  • the invention also provides methods (also referred to herein as "screening assays") for identifying modulators, i. e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, peptoids, small molecules or other drugs) which (a) bind to the nucleic acid molecule or its gene products, or (b) have a modulatory (e.g. stimulatory or inhibitory) effect on the activity ofthe nucleic acid molecule or, more specifically, (c) have a modulatory effect on the interactions of a protein encoded by the nucleic acid molecule with one or more of its natural substrates (e.g.
  • modulators i. e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, peptoids, small molecules or other drugs) which (a) bind to the nucleic acid molecule or its gene products, or (b) have a modulatory (e.g. stimulatory or inhibitory) effect on the activity of
  • Such assays typically comprise a reaction between the nucleic acid molecule or the protein of the invention and one or more assay components.
  • the other components may be either the test compound itself, or a combination of test compound and a natural binding partner ofthe protein according to the invention.
  • the test compounds of the present invention may be obtained from any available source, including systematic libraries of natural and/or synthetic compounds.
  • Test s compounds may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g. Zuckermann, R.N., et al., J. Med. Chem. 37 (1994) 2678-2685); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the One-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S., Anticancer Drug Des. 12 (1997) 145-167).
  • the invention provides assays for screening candidate or test; compounds which are substrates of the protein of the invention or biologically active portion thereof.
  • the invention provides assays for screening candidate or test compounds which bind to a protein according to the invention or biologically active portion thereof. Determining the ability of the test compound to directly bind to a protein of the invention can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to the protein can be determined by detecting the protein compound in a labeled complex.
  • compounds e.g. substrates ofthe protein ofthe invention
  • assay components can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate s substrate to product.
  • the invention provides assays for screening candidate or test compounds which modulate the activity of the protein or a biologically active portion thereof.
  • the protein can, in viva, interact with one or more molecules, such as but not limited to, peptides, proteins, hormones, cofactors and nucleic acids.
  • binding partners such cellular and extracellular molecules are referred to herein as “binding partners” or protein "substrate”.
  • One necessary embodiment of the invention in order to facilitate such screening is the use of the protein according to the invention to identify its natural in viva binding partners.
  • the protein according to the invention to identify its natural in viva binding partners.
  • There are many ways to accomplish this which are known to one skilled in the art.
  • One example is the use of the protein of the invention as "bait protein" in a two-hybrid assay or three-hybrid assay; (see, e.g. U.S. Patent No. 5,283,317; Zervos, A.S., et al., Cell 72 (1993) 223-232; Madura, K., et al., J. Biol. Chem.
  • binding partners proteins which bind to or interact with the protein (binding partners) and, therefore, are possibly involved in the natural function of the protein.
  • binding partners are also likely to be involved in the propagation of signals by the protein or downstream elements of a gene-mediated signaling pathway. Alternatively, such protein binding partners may also be found to be inhibitors of the protein.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that encodes a protein fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample” is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be readily detected and cell clones containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the protein ofthe invention.
  • a reporter gene e.g., LacZ
  • assays may be devised through the use of the invention for the purpose of identifying compounds which modulate (e.g. affect either positively or negatively) interactions between the protein of the inventon and its substrates and/or binding partners.
  • compounds can include, but are not limited to, molecules such as antibodies, peptides, hormones, oligonucleotides, nucleic acids, and analogs thereof.
  • Such compounds may also be obtained from any available source, including systematic libraries of natural and/or synthetic compounds.
  • the preferred assay components for use in this embodiment is the protein of the invention, the known binding partner and/ or substrate of same, and the test compound. Test compounds can be supplied from any source.
  • the basic principle of the assay systems used to identify compounds that interfere with the interaction between the protein and its binding partner involves preparing a reaction mixture containing the protein and its binding partner under conditions and for a time sufficient to allow the two products to interact and bind, thus forming a complex.
  • the reaction mixture is prepared in the presence and absence of the test compound.
  • the test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the protein and its binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the protein and its binding partner is then detected.
  • the assay for compounds that interfere with the interaction of the protein with its binding partner may be conducted in a heterogeneous or homogeneous format.
  • Heterogeneous assays involve anchoring either the protein of the invention or its binding partner onto a solid phase and detecting complexes anchored to the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the protein of the invention and the binding partners (e.g. by competition) can be identified by conducting the reaction in the presence of the test substance, i. e., by adding the test substance to the reaction mixture prior to or simultaneously with the protein and its interactive binding partner.
  • test compounds that disrupt preformed complexes e.g., compounds with higher binding constants that displace one of the components from the complex
  • test compounds that disrupt preformed complexes e.g., compounds with higher binding constants that displace one of the components from the complex
  • either the protein of the invention or its binding partner is anchored onto a solid surface or matrix, while the other corresponding non-anchored component may be labeled, either directly or indirectly.
  • microtitre plates are often utilized for this approach.
  • the anchored species can be immobilized by a number of methods, either non-covalent or covalent, that are typically well known to one who practices the art. Non-covalent attachment can often be accomplished simply by coating the solid surface with a solution of the protein or its binding partner and drying. Alternatively, an immobilized antibody specific for the assay component to be anchored can be used for this purpose. Such surfaces can often be prepared in advance and stored.
  • a fusion protein can be provided which adds a domain that allows one or both of the assay components to be anchored to a matrix.
  • glutafhione-S-transferase/protein of the invention fusion proteins or glutathione-S transferase/binding partner can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed protein or its binding partner, and the mixture incubated under conditions conducive to complex formation (e.g:, physiological conditions).
  • the beads or microtiter plate wells are washed to remove any unbound assay components, the immobilized complex assessed either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of protein binding or activity determined using standard techniques.
  • Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a protein ofthe invention or its binding partner can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated protein or target molecules can be prepared from biotin-NHS (N-hydroxy succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • biotin-NHS N-hydroxy succinimide
  • Pierce Chemicals Pierce Chemicals
  • Rockford, IL e.g., Pierce Chemicals, Rockford, IL
  • the protein-immobilized surfaces can be prepared in as advance and stored.
  • the corresponding partner of the immobilized assay component is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted assay components are removed (e.g. 1 by washing) and any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways.
  • the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes anchored on the surface; e.g. using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled).
  • test compounds which modulate (inhibit or enhance) complex formation or which disrupt preformed complexes can be detected.
  • a homogeneous assay may be used. This is typically a reaction, analogous to those mentioned above, which is conducted in a liquid phase in the presence or absence of the test compound. The formed complexes are then separated from unreacted components, and the amount of complex formed is determined. As mentioned for heterogeneous assay systems, the order of addition of reactants to the liquid phase can yield information about which test compounds modulate (inhibit or enhance) complex formation and which disrupt preformed complexes. In such a homogeneous assay, the reaction products may be separated from unreacted assay components by any of a number of standard techniques, including but not limited to differential centrifugation, chromatography, electrophoresis and immunoprecipitation.
  • complexes of molecules may be separated from uncomplexed molecules through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A.P., Trends Biochem. Sci. 18 (1993) 284-287).
  • Standard chromatographic techniques may also be utilized to separate; complexed molecules from uncomplexed ones.
  • gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex may be separated from the relatively smaller uncomplexed components.
  • the relatively different charge properties of the complex as compared to the uncomplexed molecules may be exploited to differentially separate the complex from the remaining individual reactants, for example through the use of ion- exchange chromatography resins.
  • ion- exchange chromatography resins Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard, N.H., J. Mol. Recognit. 11
  • Gel electrophoresis may also be employed to separate complexed molecules from unbound species (see, e.g., Ausubel et al. (eds.), In: Current Protocols in Molecular Biology, J. Wiley & Sons, New York, 1999). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example.
  • Immunoprecipitation is another common technique utilized for the isolation of a protein-protein complex from solution (see, e.g., Ausubel et al. (eds.), In: Current Protocols in Molecular Biology, J. Wiley 8c Sons, New York, 1999). In this technique, all proteins binding to an antibody specific to one of the binding molecules are precipitated from solution by conjugating the antibody to a polymer bead that may be readily collected by centrifugation.
  • the bound assay components are released from the beads (through a specific proteolysis event or other technique well known in the art which will not disturb the protein-protein interaction in the complex), and a second immunoprecipitation step is performed, this time utilizing antibodies specific for the correspondingly different interacting assay component. In this manner, only formed complexes should remain attached to the beads. Variations in no complex formation in both the presence and the absence of a test compound can be compared, thus offering information about the ability of the compound to modulate interactions between the protein ofthe invention and its binding partner.
  • a technique for direct detection of interactions between the protein of the invention and its natural binding partner and/or a test compound in a homogeneous or heterogeneous assay system without further sample manipulation.
  • the technique of fluorescence energy transfer may be utilized (see, e.g., U.S. Patent No. 5,631,169; U.S. Patent No. 4,868,103).
  • this technique involves the addition of a fluorophore label on a first 'donor' molecule (edge, test compound) such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, 'acceptor' molecule (e.g. test compound), which in turn is able to fluoresce due to the absorbed energy.
  • the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that ofthe 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g. using a fluorimeter).
  • test substance which either enhances or hinders participation of one of the species in the preformed complex will result in the generation of a signal variant to that of background.
  • test substances that modulate interactions between a protein of the invention and its binding partner can be identified in controlled assays.
  • modulators of nucleic acid molecule expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of of mRNA or protein encoded by a nucleic acid molecule is determined. The level of expression of mRNA or protein in the presence of the candidate compound is compared to the level of expression of mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid molecule expression based on this comparison. For example, when expression of nucleic acid molecule or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid molecule expression.
  • nucleic acid molecule mRNA or protein when expression of nucleic acid molecule mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid molecule expression.
  • the level of nucleic acid molecule expression in the cells can be determined by methods described herein for detecting nucleic acid molecule mRNA or protein.
  • the invention pertains to a combination of two or more of the assays described herein.
  • a modulating agent can be identified using a cell- based or a cell free assay, and the ability ofthe agent to modulate the activity of a protein ofthe invention can be further confirmed in viva, e.g., in a whole animal model for cellular transformation and/or tumorigenesis.
  • This invention further pertains to novel agents identified by the above- described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g. a gene or protein modulating agent, an antisense gene nucleic acid molecule, an protein specific antibody, or an protein binding partner according to the invention
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • Exemplary doses of a small molecule include milligram or microgram amounts per kilogram of subject or sample weight (e.g about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram).
  • Exemplary doses of a protein or polypeptide include gram, milligram or microgram amounts per kilogram of subject or sample weight (e.g about 1 microgram per kilogram to about 5 grams per kilogram, about 100 micrograms per kilogram to about 500 milligrams per kilogram, or about 1 milligram per kilogram to about 50 milligrams per kilogram).
  • appropriate doses of one of these agents depend upon the potency of the agent with respect to the expression or activity to be modulated. Such appropriate doses can be determined using the assays described herein.
  • a physician, veterinarian, or researcher can, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • a pharmaceutical composition ofthe invention is formulated to be compatible with its intended route of administration.
  • routes of administration include; parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g. inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine-tetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF; Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfacta,ts.
  • Prevention ofthe action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid,, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g. a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium, and then incorporating the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze- drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • compositions can be included as part ofthe composition.
  • the tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin, an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch, a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a suitable propellant e.g. a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g. with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g. with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics ofthe active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the preferred dosage is 0.1 mg/kg to 100 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of
  • lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration.
  • a method for lipidation of antibodies is described by Cruikshank, W.W., et al., J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14 (1997) 193-203.
  • the nucleic acid molecules corresponding to the marker gene according to the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Patent No. 5,328,470), or by stereotactic injection (see, e.g., Chen, S.H., et al., Proc. Natl. Acad. Sci. USA 91 (1994) 3054-3057).
  • the pharmaceutical preparation ofthe gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • Agents or modulators which have a stimulatory or inhibitory effect on expression of the marker gene of the invention can be administered to individuals to treat mammary cancer in the patient.
  • the pharmacogenomics i. e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
  • the pharmacogenomics of the individual permits the selection of effective agents (e.g. drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype.
  • Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the level of expression of the marker gene of the invention in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • Pharmacogenomics deals with clinically significant variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Linder, M.W., et al., Clin. Chem. 43 (1997) 254-266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body are referred to as “altered drug action.” Genetic conditions transmitted as single factors altering the way the body acts on drugs are referred to as "altered drug metabolism”. These pharmacogenetic conditions can occur either as rare defects or as polymorphisms.
  • G6PD glucose-6-phosphate dehydrogenase
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • drug metabolizing enzymes e.g. N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • the level of expression of the marker gene according to the invention in an individual can be determined to thereby select appropriate agent(s) for therapeutic treatment of the individual.
  • pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a modulator of expression of the marker gene of the invention.
  • agents e.g. drug compounds
  • the effectiveness of an agent to affect gene expression can be monitored in clinical trials of subjects receiving treatment for mammary cancer.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) comprising the steps of (i) obtaining a pre- administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of the marker gene according to the invention in the pre-administration sample; (iii) obtaining one or more post- administration samples from the subject; (iv) detecting the level of expression ofthe the nucleic acid molecule in the post-administration samples; (v) comparing the level of expression of the the nucleic acid molecule in the pre-administration sample with the level of expression of the the nucleic acid molecule in the post- administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small
  • increased administration of the agent can be desirable to increase expression of the the nucleic acid molecule to higher levels than detected, i.e., to increase the effectiveness ofthe agent.
  • decreased administration of the agent can be desirable to decrease expression of the marker gene according to the invention to lower levels than detected, i. e., to decrease the effectiveness ofthe agent.
  • the cells in a sample wherefrom the normal level of expression of the marker gene of the invention is determined are either cells from a control subject not afflicted with cancer or cells from a human mammary epithelial cell line.
  • the cells from a control subject should be obtained in the same manner as from the patient afflicted with cancer.
  • the cells from a human mammary epithelial cell line are preferably those that can be commercially obtained from Cambrex Bio Science Walkersville, Inc., Walkersville, Maryland, USA. Methods for obtaining these or other suitable cell human mammary cell lines are described in US4,423,145. Other suitable cell lines are described in US4,808,532.
  • SEQ ID NO:l Nucleic acid sequence including translation into the amino acid sequence of CRASH
  • SEQ ID NO:2 Amino acid sequence of CRASH
  • Figure 1 Northern blot analysis of mammary carcinoma cell lines for analysis of expression of mRNA corresponding to EST RC_AA476944.
  • the membrane was exposed to X-ray film at -80°C for 24h. Equal loading of the gel was assessed by hybridization to [ ⁇ - 32 P]dATP labeled GAPDH probe.
  • Figure 2 Sequence analysis of CRASH cDNA and genomic organisation of the CRASH gene.
  • A Nucleotide sequence of the CRASH cDNA and the predicted amino acid sequence. Stop codon is indicated by an asterisk and the polyadenylation signal is underlined.
  • B Exon-intron organization ofthe CRASH gene. Screening for CRASH in genomic databases showed that the gene is localized on chromosome llql3.1.
  • Figure 3 Amino acid alignment of human CRASH and rat asparaginase- like protein. Identical amino acids are highlighted by bars, amino acid exchanges are shown by one (non-conservative) or two dots (conservative).
  • Figure 4 Comparison of the amino acid sequences of human CRASH and human aspartylglucosaminidase. For optimal sequence alignment, several gaps had to be introduced as indicated by dotted lines. Identical amino acids are highlighted by bars, amino acid exchanges are displayed by one (non-conservative) or two dots (conservative).
  • Figure 5 Analysis of expression of CRASH mRNA in different human tissues and cell lines. Clontech MTE Blot was hybridized with an [ ⁇ - 32 P]dATP CRASH cDNA probe and exposed to X-ray film at -80°C for 24h. The coding is resolved below. E6 corresponds to 1 ⁇ g, F6 to 0.1 ⁇ g and G6 to 0.01 ⁇ g poly A + RNA from cell line KM22.
  • Figure 6 Relative expression of CRASH mRNA in metastasizing versus non-metastasizing human tumor cell lines. mRNA levels were determined by real-time PCR (LightCycler Technology) as described in the Materials and Methods section and normalized to 18S ribosomal RNA as an internal standard.
  • Figure 7 Expression of CRASH mRNA in tumor and corresponding normal tissues.
  • An [ ⁇ - 32 P]dATP labeled cDNA probe corresponding to CRASH was hybridized to the Clontech cancer profiling array and RNA levels were quantified as described in the Materials and Methods section. The blot was exposed to X-ray film at -80°C for 17 hours.
  • Figure 8 Inducibility of CRASH mRNA by hormones.
  • BT474 cells were exposed to ethanol, norgestrel, 5 ⁇ - dihydrotestosterone (DHT) and estradiol.
  • the RNA was isolated at different times after induction, all performed as described in the Materials and Methods section.
  • CRASH mRNA levels were determined by real-time PCR (LightCycler Technology). Induction of CRASH mRNA is referred to the basal level of CRASH mRNA in non-induced BT-474 cells.
  • Figure 9 Relative expression of CRASH mRNA in normal breast tissues and primary breast cancer samples. mRNA levels were determined by real-time PCR (LightCycler Technology) as described in the Materials and Methods section and normalized to 18S ribosomal RNA as an internal standard.
  • FIG. 10 Immunohistochemical analysis of CRASH expression in invasive breast carcinomas and normal breast tissues. Making use of a polyclonal antibody directed against CRASH, immunohistochemical analysis was performed as described in the Materials and Methods section. Panels A and B: normal breast tissues Panels C and D: invasive ductal breast carcinomas Magnification: 20 fold
  • Mammary carcinoma cell lines AR, WA, KM22, HG15, 1590, 2928 and KS were propagated as a monolayer in DMEM with 10% FCS.
  • Human mammary epithelial cells (HMEC) were obtained from BioWhittaker, Inc (Walkersville, Maryland, USA) and cultured in MEGM (Mammary Epithelial Cell Medium) as specified by the supplier.
  • RPMI 1640 supplemented with 10% fetal bovine serum, 2mM L-glutamine and no antibiotics.
  • the breast cancer cell line BT474 was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and was cultured in RPMI 1640 media supplemented with 10% fetal bovine serum, 2mM glutamine, 10 mg/1 bovine insulin.
  • Experimental cultures were grown in phenol red-free RPMI 1640 media containing 10% charcoal-stripped FCS (HyClone Laboratories, Logan, UT, USA).
  • Human normal breast total RNA was obtained from Clontech (Palo Alto, CA,
  • RNA (10 ⁇ g) was electrophoresed through a 1% denaturing formaldehyde gel and blotted to BrightStar-PlusTM positively-charged nylon membranes (Ambion Inc., Austin, Texas, USA) by capillary downward transfer and fixed by exposure to ultraviolet light. Membranes were prehybridized for 30 min at 68°C with ExpressHyb hybridization solution (Clontech, Palo Alto, CA, USA). To detect
  • a 183-bp probe was generated from the EST sequence AA476944 by using specific oligonucleotides (forward primer: 5-TCCGTCCTCACCCTGTC- 3; reverse primer: 5-TTGAGCACGGATGATACAGC-3).
  • the probe was labeled with ⁇ -[ 32 P]dATP using the StripEZTM DNA Kit (Ambion Inc., Austin, Texas, USA). Denatured probe was added to prewarmed hybridization solution to result in
  • MTETM Human Multiple Tissue Expression array
  • cancer profiling array MTETM and cancer profiling array
  • the human MTE Array (Clontech, Palo Alto, CA, USA) and the cancer profiling array membranes (Clontech) were hybridized with a specific CRASH cDNA probe according to the manufacturer's protocol.
  • the cDNA probe was generated with the CRASH specific oligonucleotide primers (CRASH forward primer, 5- TCCGTCCTCACCCTGTC-3; reverse primer, 5-TTGAGCACGGATGATACAGC- 3).
  • the filters were exposed to X-ray film for documentation.
  • the cancer profiling array was scanned on a StormTM 860 image reader (Molecular
  • Tissue samples from primary human breast carcinomas were frozen in liquid nitrogen immediately after surgery and stored in it until RNA extraction was carried out. They were homogenized in a dismembrator (Braun-Melsungen, Melsungen, Germany) for 2 min. Frozen powder obtained from tissue samples weighing 1 g or less was suspended in 2.5 ml Gl-solution (4 M GuaSCN, 5 mM sodium citrate, 10 mM EDTA, 0.5% sarkosyl, 100 ⁇ M ⁇ -mercaptoethanol) by shaking for several seconds. The suspension was transferred to a sterile tube. The homogenization cup was rinsed with another 0.5 ml Gl-solution and the combined lysates were either processed immediately or stored frozen at -20°C.
  • Gl-solution 4 M GuaSCN, 5 mM sodium citrate, 10 mM EDTA, 0.5% sarkosyl, 100 ⁇ M ⁇ -mercaptoethanol
  • the suspensions were incubated at 50°C for up to 15 min. This resulted in a complete disintegration of tissue and lysis of cells.
  • the lysates were mixed with 2.5 ml of a CsTFA solution (2.0 gm/1) (Pharmacia, Molecular Biology Division, Uppsala,
  • RNA quality and quantity were determined by UN spectrophotometry and denaturing formaldehyde gel electrophoresis.
  • l ⁇ g R ⁇ A was reverse transcribed into first strand cD ⁇ A following the protocol of the First Strand cD ⁇ A Synthesis Kit (Roche Diagnostics, Penzberg, Germany) using random hexanucleotide primers. The reaction mixture was incubated at 25°C for 10 min followed by incubation at 42°C for 60 min and inactivation of the reverse transcriptase by heating at 99°C for 5 min and cooling at 4°C for 5 min.
  • Real-time RT-PCR LightCycler-Technology
  • Quantitative PCR was performed with a Roche LightCycler system (Roche Diagnostics, Mannheim, Germany). PCR reactions were carried out using the DNA Master SYBR Green I Kit (Roche Diagnostics, Mannheim, Germany) reaction mix. The PCR reaction was performed in a volume of 20 ⁇ l containing 2 ⁇ l cDNA, 2 ⁇ l
  • An anchor priming site at the 3' end of the single-stranded cDNA was created using the SMART II A oligonucleotide (Clontech, Palo Alto, CA, USA). 2.5 ⁇ l of the reaction product was amplified by PCR using gene-specific primers, for the AK025969 and BC006267 overlapping sequence, CRASH-R4 5-CCCAACGCGGCCGACCATTTTATTAAC-3 and an adapter-specific primer UPM. The 3' end of the human CRASH transcript was cloned using the 3' RACE method (Clontech, Palo Alto, CA, USA).
  • RNA from the cell line KM22 was reverse-transcribed using an oligo-dT adapter primer and PowerScript Reverse Transcriptase (Clontech, Palo Alto, CA, USA).
  • a total of 2.5 ⁇ l of the reaction product was amplified by PCR using CRASH gene- specific primer CRASH-F5 5-GAGATTCTTCCCACCTCCCCATCCTCA-3 and an adapter-specific primer UPM.
  • the PCR products ofthe 5' RACE and 3' RACE were cloned into the pCR 4-TOPO vector (TA Cloning Kit, Invitrogen).
  • the ligation products were transformed into One Shot competent cells (Invitrogen, Carlsbad, CA, USA). Positive clones were identified by PCR.
  • the plasmids of the positive clones were purified by using QIAGEN Plasmid Mini Kit. DNA was sequenced by means of an automated sequencer (310-DNA Sequencer, Applied Biosystems, Germany) with universal primers.
  • BT474 cells were plated in growth medium into 6-well plates at 2xl0 5 cells/well.
  • the culture media were changed to depleted media containing the steroid hormones 17 ⁇ -estradiol, 5 ⁇ -dihydrotestosterone and norgestrel (Sigma Chemical Co, St Louis, MO, USA) at a final concentration of 10 nM.
  • Cells stimulated with 100% ethanol were included as controls.
  • Cells were harvested after 0, 24, 48, 72 and 96 hours for total RNA extraction.
  • Cell conditioning was achieved by microwaving in 0.01 M sodium citrate solution for 15 min, followed by cooling of the slides in the citrate solution at room temperature for 20 min.
  • the polyclonal antibody CRASH was dispensed at a 1:50 dilution onto the sections and allowed to incubate for 20 min at room temperature.
  • the slides were subsequently incubated with a biotinylated immunoglobulin secondary antibody followed by the addition of streptavidin- alkaline phosphatase (Ventana). After the development of colour with naphthol substrate, the slides were counterstained with hematoxylin. After dehydration in graded alcohol and xylene, coverslips were mounted with Permount and the slides were examined by conventional optical microscopy.
  • Cell lines AR and WA are derived from primary mammary carcinomas; KM22,
  • HG15 and 1590 originate from bone marrow micrometastasis, whereas 2928 and KS are derived from a lymph-node metastasis and malignant ascites, respectively (Evtimova, V., et al, Anticancer Res. 21 (2001) 3799-3806). Transcriptional profiling of the cell lines has been described previously (Evtimova et al., supra). A 2.5 kb mRNA corresponding to EST RC AA476944 is expressed in all mammary carcinoma cell lines derived from metastatic lesions, but not in the cell lines derived from primary mammary tumors as well as non-transformed human mammary gland epithelial cells (Fig. 1).
  • CRASH a full-length cDNA was cloned, denoted as CRASH.
  • Screening for CRASH in genomic databases revealed that the gene is localized on chromosome llql3.1. Data analysis indicated that the CRASH gene consists of 7 exons separated by 6 introns (Fig. 2B).
  • Nucleotide sequence analysis reveals a cDNA encoding a protein composed of 308 aa (Fig. 2A).
  • a homologous rat cDNA in GenBank AF329099
  • sequence alignment reveals 77% homology at the aa level between asparaginase-like protein and
  • CRASH CRASH.
  • Amino acid alignment between CRASH and human aspartylglucosaminidase reveals homology of 32% (Fig. 4).
  • a gap of 33 aa had to be introduced for optimal alignment.
  • the human MTE Array (Clontech, Palo Alto, CA, USA) was used to screen for the presence and relative abundance of CRASH mRNA in a broad spectrum of fetal and adult human tissues. This analysis indicated strongest expression of CRASH mRNA in brain, kidney and testes (Fig. 5). Also tissues of the gastrointestinal tract scored positively.
  • a comparative quantification of CRASH mRNA in several cellular systems with different metastatic potential Fig. 6 was carried out.
  • CRASH mRNA is not expressed in the non-metastasizing colon cancer cell line KM12C, whereas KM12SM and KM12L4A (7, 8), which metastasize to the liver, scored positive for CRASH mRNA.
  • Colon cancer cell line HCT116U55 was selected from cell line HCT116 (9) because of its increased metastatic capacity and in this system also increased mRNA levels of CRASH correlate with metastatic capacity.
  • a similar correlation for metastatic versus non-metastatic human pancreatic carcinoma cell lines SUIT 007 and SUIT 028 (10, 11) was observed. These results prompted to analyse CRASH mRNA levels in a larger number of different normal and tumor tissues.
  • a cancer profiling array (Clontech) was used, which contains normalized cDNA from 241 tumors and the corresponding normal tissues. The strongest signals were found in samples of uterine, mammary, prostatic and ovarian carcinomas (Fig. 7A). Quantification of the results showed a more than 1.5-fold increase of CRASH mRNA levels in about 70% of the uterine, 28% of the mammary, 25% of the prostatic and 21% of the ovarian carcinomas (Fig. 7B). Scoring the tumor samples in which CRASH mRNA is overexpressed, we noted the following average fold overexpression: 4.3-fold for uterine carcinoma, 2.5-fold for ovarian carcinoma, 2.5-fold for mammary carcinoma and 1.4- fold for prostatic carcinoma.
  • CRASH mRNA was upregulated by 5 -dihydrotestosterone (4- fold) and progesterone (norgestrel) (3.5-fold) at 24 hours after treatment in BT474 cells (Fig. 8).
  • Total RNA derived from 36 breast cancer tissue samples and three samples derived from normal breast tissues were analyzed for expression of CRASH mRNA. It was significantly overexpressed in 11 breast cancer samples (Fig. 9). Two of these were ER + and nine were scored as ER " , indicating that CRASH might be predominantly expressed in ER " mammary carcinomas. 15 of the tumors investigated were classified as ER + and 18 as ER " , the estrogen receptor status ofthe others is unknown. Representative immunohistochemistry staining for CRASH with of a polyclonal antibody directed against CRASH is shown in Fig. 10. Evaluation ofthe results
  • Affymetrix GeneChip R based transcriptional profiling resulted in the identification of an EST, whose corresponding mRNA is expressed in five mammary carcinoma cell lines derived from bone marrow micrometastases, ascites fluid and lymph nodes, but not in two cell lines derived from primary mammary carcinoma (Fig. 1).
  • CRASH mRNA in 14 out of 50 mammary carcinomas, in 3 out of 14 ovarian carcinomas and in 28 out of 42 uterine carcinomas (Fig. 7).
  • 11 out of 36 mammary carcinomas significantly overexpressed CRASH mRNA compared with three normal breast tissues (15 ER + , 18 ER " , estrogen receptor status of the others unknown).
  • the finding that CRASH mRNA was overexpressed in only two ER + breast carcinomas, in contrast to nine ER " breast carcinomas indicates preferential expression of CRASH mRNA in ER " mammary carcinomas (Fig. 9).
  • a correlation with respect to the lymph node status. Immunohistochemistry analysis (Fig. 10) confirmed that CRASH is predominantly expressed in ER " tumor tissues.
  • ALP asparaginase-like protein
  • rats Bosset, L.A., et al., Mol. Reprod. Dev. 62 (2002) 233-247.
  • the sequence of human CRASH corresponds to the sequence ofthe rat orthologue ALP (Bush et al., supra).
  • Anti-sperm antibodies have been observed in a rat model system as a response of the male reproductive tract to obstruction (Alexander, N.J., and Anderson, D.J., Fertil. Steril. 32 (1979) 253-260).
  • Autoantigenic sperm proteins have also been identified in men, e.g. glycoprotein FA-1 (Naz, R.K., et al., Biol.
  • Lam K.S., et al., Nature 354 (1991) 82-84 Lee, C.Y., et al., Am. J. Reprod. Immunol. 3 (1983) 183-187

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Abstract

L'invention concerne des procédés consistant à employer une molécule d'acides nucléiques (CRASH) présentant la séquence d'acides nucléiques SEQ ID NO:1, afin d'évaluer le risque de progression tumorale et/ou de métastases, notamment dans le cas de carcinome de l'utérus, du sein, de la prostate ou de l'ovaire. L'invention concerne également des utilisations de protéines CRASH présentant une séquence SEQ ID NO:2. L'invention concerne par ailleurs un ensemble destiné à déterminer si des cellules d'un échantillon tumoral présentent un potentiel métastatique.
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EP2787350A1 (fr) 2013-04-05 2014-10-08 Atlas Antibodies AB ASRGL1 dans le cancer de l'endomètre
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EP2787350A1 (fr) 2013-04-05 2014-10-08 Atlas Antibodies AB ASRGL1 dans le cancer de l'endomètre
WO2014161980A1 (fr) * 2013-04-05 2014-10-09 Atlas Antibodies Ab Asrgl1 dans le cancer endométrial
CN111936627A (zh) * 2018-01-18 2020-11-13 奥斯瓦道·克鲁兹基金会 天冬酰胺酶活性多肽、表达盒、表达载体、宿主细胞、组合物、方法、癌症预防或治疗用途
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