WO1997038085A2 - Genes amplifies dans des cellules cancereuses - Google Patents

Genes amplifies dans des cellules cancereuses Download PDF

Info

Publication number
WO1997038085A2
WO1997038085A2 PCT/US1997/005930 US9705930W WO9738085A2 WO 1997038085 A2 WO1997038085 A2 WO 1997038085A2 US 9705930 W US9705930 W US 9705930W WO 9738085 A2 WO9738085 A2 WO 9738085A2
Authority
WO
WIPO (PCT)
Prior art keywords
rna
seq
cancer
cells
gene
Prior art date
Application number
PCT/US1997/005930
Other languages
English (en)
Other versions
WO1997038085A3 (fr
Inventor
Helene Smith
Ling-Chun Chen
Original Assignee
California Pacific Medical Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US1996/009286 external-priority patent/WO1996039516A2/fr
Priority claimed from US08/678,280 external-priority patent/US5776683A/en
Application filed by California Pacific Medical Center filed Critical California Pacific Medical Center
Priority to EP97920293A priority Critical patent/EP0944651A2/fr
Priority to JP9536486A priority patent/JP2000509256A/ja
Priority to AU24521/97A priority patent/AU2452197A/en
Publication of WO1997038085A2 publication Critical patent/WO1997038085A2/fr
Publication of WO1997038085A3 publication Critical patent/WO1997038085A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/48—Ergoline derivatives, e.g. lysergic acid, ergotamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates generally to the field of human genetics. More specifically, it relates to the identification of novel genes associated with overabundance of RNA in human cancer such as breast cancer. It pertains especially to those genes and the products thereof which may be important in diagnosis and treatment.
  • Cancer is a heterogeneous disease. It manifests itself in a wide variety of tissue sites, with different degrees of de-differentiation, invasiveness, and aggressiveness. Some forms of cancer are responsive to traditional modes of therapy, but many are not. For most common cancers, there is a pressing need to improve the arsenal of therapies available to provide more precise and more effective treatment in a less invasive way.
  • breast cancer has an unsatisfactory morbidity and mortality, despite presently available forms of medical intervention.
  • Traditional clinical initiatives are focused on early diagnosis, followed by surgery and chemotherapy. Such interventions are of limited success, particularly in patients where the tumor has undergone metastasis.
  • the heterogeneous nature of cancer arises because different cancer cells achieve their growth and pathological properties by different phenotypic alterations. Alteration of gene expression is intimately related to the uncontrolled growth and de-differentiation that are hallmarks of cancer. Certain similar phenotypic alterations in turn may have a different genetic base in different tumors. Yet, the number of genes central to the malignant process must be a finite one.
  • new pharmaceuticals that are tailored to specific genetic alterations in an individual tumor may be more effective.
  • the first type is the decreased expression of recessive genes, known as tumor suppresser genes, that apparently act to prevent malignant growth.
  • the second type is the increased expression of dominant genes, such as oncogenes, that act to promote malignant growth, or to provide some other phenotype critical for malignancy.
  • alteration in the expression of either type of gene is a potential diagnostic indicator.
  • a treatment strategy might seek to reinstate the expression of suppresser genes, or reduce the expression of dominant genes.
  • the present invention is directed to identifying genes of either type, particularly those of the second type.
  • amplification The most frequently studied mechanism for gene overexpression in cancer cells is sometimes referred to as amplification.
  • This is a process whereby the gene is duplicated within the chromosomes of the ancestral cell into multiple copies.
  • the process involves unscheduled replications of the region of the chromosome comprising the gene, followed by recombination of the replicated segments back into the chromosome (Aiitalo et al.). As a result, 50 or more copies of the gene may be produced.
  • the duplicated region is sometimes referred to as an "amplicon".
  • the level of expression of the gene escalates in the transformed cell in the same proportion as the number of copies of the gene that are made (Aiitalo et al.).
  • oncogenes have been described, some of which are duplicated, for example, in a significant proportion of breast tumors.
  • a prototype is the ⁇ rbB2 gene (also known as HER-2/neu), which encodes a 185 kDa membrane growth factor receptor homologous to the epidermal growth factor receptor.
  • erbB2 is duplicated in 61 of 283 tumors (22%) tested in a recent survey (Adnane et al.).
  • Other oncogenes duplicated in breast cancer are the b ⁇ k gene, duplicated in 34 out of 286 (12%); the fig gene, duplicated in 37 out of 297 (12%), the myc gene, duplicated in 43 out of 275 (16%) (Adnane et al.).
  • chromosome abnormalities such as double minute (DM) chromosomes and homogeneously stained regions (HSRs)
  • DM double minute
  • HSRs homogeneously stained regions
  • DM double minute
  • Giemsa staining intermediate density Giemsa staining throughout their length, rather than with the normal pattern of alternating dark and light bands. They correspond to multiple gene repeats.
  • HSRs are particularly abundant in breast cancers, showing up in 60-65% of tumors surveyed (Dutrillaux et al., Zafrani et al.).
  • CGH comparative genomic hybridization
  • the images are computer-processed for the fluorescence ratio, revealing chromosomal regions that have undergone amplification or deletion in the cancer cells (Kallioniemi et al. 1992). This method was recently applied to 15 breast cancer cell lines (Kallioniemi et al. 1994). DNA sequence copy number increases were detected in all 23 chromosome pairs.
  • oncogenes have been identified by hybridizing with probes for other known growth-promoting genes, particularly known oncogenes in other species.
  • the ⁇ rbB2 gene was identified using a probe from a chemically induced rat neuroglioblastoma (Slamon et al.).
  • genes with novel sequences and functions will evade this type of search.
  • genes may be cloned from an area identified as containing a duplicated region by CGH method. Since CGH is able to indicate only the approximate chromosomal region of duplicated genes, an extensive amount of experimentation is required to walk through the entire region and identify the particular gene involved.
  • Genes may also be overexpressed in cancer without being duplicated. Methods that rely on identification from genetic abnormalities necessarily bypass such genes. Increased expression can come about through a higher level of transcription of the gene; for example, by up-regulation of the promoter or substitution with an alternative promoter. It can also occur if the transcription product is able to persist longer in the cell; for example, by increasing the resistance to cytoplasmic RNase or by reducing the level of such cytoplasmic enzymes.
  • Two examples are the epidermal growth factor receptor, overexpressed in 45% of breast cancer tumors (Klijn et al.), and the IGF-1 receptor, overexpressed in 50-93% of breast cancer tumors (Berns et al.). In almost all cases, the overexpression of each of these receptors is by a mechanism other than gene duplication.
  • RNA preparations are prepared from only a subpopulation of each RNA preparation, and expanded via the polymerase chain reaction using primers of particular specificity. Similar subpopulations are compared across several RNA preparations by gel autoradiography for expression differences. In order to survey the RNA preparations entirely, the assay is repeated with a comprehensive set of PCR primers. The screening strategy more effectively includes multiple positive and negative control samples (Sunday et al.). The method has recently been applied to breast cancer cell lines, and highlights a number of expression differences (Liang et al. 1992b; Chen et al., McKenzie et al., Watson et al. 1994 & 1996, Kocher et al.). By excising the corresponding region of the separating gel, it is possible to recover and sequence the cDNA.
  • differential display highlights high copy number mRNAs and shorter RNAs (Bertioli et al., Yeatman et al.) , and may therefore miss critical cancer-associated transcripts when used as a survey technique.
  • a number of adjustments are made to gene expression levels when a cell undergoes malignant transformation or cultured in vitro. Most of these adjustments are secondary, and not part of the transformation process. Thus, even when a novel sequence is obtained from the differential display, it is far from certain that the corresponding gene is at the root of the disease process.
  • An early step in developing gene-specific therapeutic approaches is the identification of genes that are more central to malignant transformation or the persistence of the malignant phenotype.
  • the method can be used for any type of cancer, providing a plurality of cell populations or cell lines of the type of cancer are available, in conjunction with a suitable control cell population.
  • the method is highly effective in identifying genes and gene products that are intimately related to malignant transformation or maintenance of the malignant properties of the cancer cells.
  • cDNA and cDNA fragments corresponding to the cancer-associated gene can be used inter alia to determine the nucleotide sequence of the gene and mRNA, the amino acid sequence of any encoded protein, or to retrieve from a cDNA or genomic library additional polynucleotides related to the gene or its transcripts. Since the genes are typically involved in the malignant process of the cell, the polynucleotides, polypeptides, and antibodies derived by using this method can in turn be used to design or screen important diagnostic reagents and therapeutic compounds.
  • Another objective of this invention to provide isolated polynucleotides, polypeptides, and antibodies derived from four novel genes which are associated with several different types of cancer, including breast cancer.
  • the genes are designated CH1-9a11-2, CH8-2a13-1, CH13-2a12-1 , and CH14-2a16-1. These designations refer to both strands of the cDNA and fragments thereof; and to the respective corresponding messenger RNA, including splice variants, allelic variants, and fragments of any of these forms.
  • These genes show RNA overabundance in a majority of cancer cell lines tested. A majority of the cells showing RNA overabundance also have duplication of the corresponding gene.
  • Another object of this invention is to provide materials and methods based on these polynucleotides, polypeptides, and antibodies for use in the diagnosis and treatment of cancer, particularly breast cancer.
  • one embodiment of this invention is an isolated polynucleotide comprising a linear sequence contained in a polynucleotide selected from the group consisting of CH1-9a11-2, CH8-2a13-1, CH13-2a12-1, and CH14-2a16-1.
  • the linear sequence is contained in a duplicated gene or overabundant RNA in cancerous cells.
  • the RNA may be overabundant due to gene duplication, increased RNA transcription or processing, increased RNA persistence, any combination thereof, or by any other mechanism, in a proportion of breast cancer cells.
  • the RNA is overabundant in at least about 20% of .a representative panel of breast cancer cell lines, such as the panels listed herein; more preferably, it is overabundant in at least about 40% of the panel; even more preferably, it is overabundant in at least 60% or more of the panel.
  • the RNA is overabundant in at least about 5% of spontaneously occurring breast cancer tumors; more preferably, it is overabundant in at least about 10% of such tumors; more preferably, it is overabundant in at least about 20% of such tumors; more preferably, it is overabundant in at least about 30% of such tumors; even more preferably, it is overabundant in at least about 50% of such tumors.
  • a sequence of at least 10 nucleotides is essentially identical between the isolated polynucleotide of the invention and a cDNAfrom CH1-9a11-2, CH8-2a13-1, CH13-2a12-1, and CH14- 2a16-1; more preferably, a sequence of at least about 15 nucleotides is essentially identical; more preferably, a sequence of at least about 20 nucleotides is essentially identical; more preferably, a sequence of at least about 30 nucleotides is essentially identical; more preferably, a sequence of at least about 40 nucleotides is essentially identical; even more preferably, a sequence of at least about 70 nucleotides is essentially identical; still more preferably, a sequence of about 100 nucleotides or more is essentially identical.
  • a further embodiment of this invention is an isolated polynucleotide comprising a linear sequence essentially identical to a sequence selected from the group consisting of SEQ. ID NO:15, SEQ. ID NO:18, SEQ. ID NO:21, SEQ. ID NO:23, SEQ. ID NO:26, SEQ. ID NO:29, SEQ. ID NO:31,. SEQ. ID NO:33, and SEQ. ID NO:35.
  • These embodiments include an isolated polynucleotide which is a DNA polynucleotide, an RNA polynucleotide, a polynucleotide probe, or a polynucleotide primer.
  • This invention also provides an isolated polypeptide comprising a sequence of amino acids essentially identical to the polypeptide encoded by or translated from a polynucleotide selected from the group consisting of CH1-9a11-2, CH8-2a13-1, CH13-2a12-1, and CH14-2a16-1.
  • a sequence of at least about 5 amino acids is essentially identical between the polypeptide of this invention and that encoded by the polynucleotide; more preferably, a sequence of at least about 10 amino acids is essentially identical; more preferably, a sequence of at least 15 amino acids is essentially identical; even more preferably, a sequence of at least 20 amino acids is essentially identical; still more preferably, a sequence of about 30 amino acids or more is essentially identical.
  • the polypeptide comprises a linear sequence of at least 15 amino acids essentially identical to a sequence encoded by said polynucleotide.
  • Another embodiment of this invention is a polypeptide comprising a linear sequence essentially identical to a sequence selected from the group consisting of SEQ. ID NO:17, SEQ. ID NO:20, SEQ. ID NO:25, SEQ. ID NO:28, SEQ. ID NO:30, SEQ. ID NO:32, SEQ. ID NO:34; and SEQ. ID NO:37.
  • a further embodiment of this invention is an antibody specific for a polypeptide embodied in this invention. This encompasses both monoclonal and isolated polyclonal antibodies.
  • a further embodiment of this invention is a method of using the polynucleotides of this invention for detecting or measuring gene duplication in cancerous cells, especially but not limited to breast cancer cells, comprising the steps of reacting DNA contained in a clinical sample with a reagent comprising the polynucleotide, said clinical sample having been obtained from an individual suspected of having cancerous cells; and comparing the amount of complexes formed between the reagent and the DNA in the clinical sample with the amount of complexes formed between the reagent and DNA in a control sample.
  • a further embodiment is a method of using the polynucleotides of this invention for detecting or measuring overabundance of RNA in cancerous cells, especially but not limited to breast cancer cells, comprising the steps of reacting RNA contained in a clinical sample with a reagent comprising the polynucleotide, said clinical sample having been obtained from an individual suspected of having cancerous cells; and comparing the amount of complexes formed between the reagent and the RNA in the clinical sample with the amount of complexes formed between the reagent and RNA in a control sample.
  • Another embodiment of this invention is a diagnostic kit for detecting or measuring gene duplication or RNA overabundance in cells contained in an individual as manifest in a clinical sample, comprising a reagent and a buffer in suitable packaging, wherein the reagent comprises a polynucleotide of this invention.
  • Another embodiment of this invention is a method of using a polypeptide of this invention for detecting or measuring specific antibodies in a clinical sample, comprising the steps of reacting antibodies contained in the clinical sample with a reagent comprising the polypeptide, said clinical sample having been obtained from an individual suspected of having cancerous cells, especially but not limited to breast cancer cells; and comparing the amount of complexes formed between the reagent and the antibodies in the clinical sample with the amount of complexes formed between the reagent and antibodies in a control sample.
  • Another embodiment of this invention is a method of using an antibody of this invention for detecting or measuring altered protein expression in a clinical sample, comprising the steps of reacting a polypeptide contained in the clinical sample with a reagent comprising the antibody, said clinical sample having been obtained from an individual suspected of having cancerous cells, especially but not limited to breast cancer cells; and comparing the amount of complexes formed between the reagent and the polypeptide in the clinical sample with the amount of complexes formed between the reagent and a polypeptide in a control sample.
  • kits for detecting or measuring a polypeptide or antibody present in a clinical sample comprising a reagent and a buffer in suitable packaging, wherein the reagent respectively comprises either an antibody or a polypeptide of this invention.
  • Yet another embodiment of this invention is a host cell transfected by a polynucleotide of this invention.
  • a further embodiment of this invention is a method for using a polynucleotide for screening a pharmaceutical candidate, comprising the steps of separating progeny of the transfected host cell into a first group and a second group; treating the first group of cells with the pharmaceutical candidate; not treating the second group of cells with the pharmaceutical candidate; and comparing the phenotype of the treated cells with that of the untreated cells.
  • This invention also embodies a pharmaceutical preparation for use in cancer therapy, comprising a polynucleotide or polypeptide embodied by this invention, said preparation being capable of reducing the pathology of cancerous cells, especially for but not limited to breast cancer cells.
  • Further embodiments of this invention are methods for treating an individual bearing cancerous cells, such as breast cancer cells, comprising administering any of the aforementioned pharmaceutical preparations.
  • Still another embodiment of this invention is a pharmaceutical preparation or active vaccine comprising a polypeptide embodied by this invention in an immunogenic form and a pharmaceutically compatible excipient.
  • a further embodiment is a method for treatment of cancer, especially but not limited to breast cancer, either prophylactically or after cancerous cells are present in an individual being treated, comprising administration of the aforementioned pharmaceutical preparation.
  • Another series of embodiments of this invention relate to methods for obtaining cDNA corresponding to a gene associated with cancer, comprising the steps of: a) supplying an RNA preparation from uncultured control cells; b) supplying RNA preparations from at least two different cancer cells; c) displaying cDNA corresponding to the RNA preparations of step a) and step b) such that different cDNA corresponding to different RNA in each preparation are displayed separately; d) selecting cDNA corresponding to RNA that is present in greater abundance in the cancer cells of step b) relative to the control cells of step a); e) supplying a digested DNA preparation from control cells; f) supplying digested DNA preparations from at least two different cancer cells; g) hybridizing the cDNA of step d) with the digested DNA preparations of step e) and step f); and h) further selecting cDNA from the cDNA of step d) corresponding to genes that are duplicated in the cancer cells of step f) relative to the
  • One or more enhancements may optionally be included in the methods of this invention, including the following: 1. Cancer cells are preferably used for step b) that share a duplicated gene in the same region of a chromosome. If desired, the practitioner may test cancer cells beforehand to detect the duplication or deletion of chromosome regions; or cancer cell lines may be used that have already been characterized in this respect.
  • a higher plurality of cancer cells are preferably used to provide DNA for step b), step f), or preferably both step b) and step f)-
  • the use of three cancer cells is preferred over two; the use of four cancer cells is more preferred, about five cancer cells is still more preferred, about eight cancer cells is even more preferred.
  • the cDNA of each cancer cell population is displayed or hybridized separately, in accordance with the method.
  • a higher plurality of control cells are preferably used to provide DNA for step a), step e), or preferably both step a) and step e).
  • the use of two control cell populations is preferred; the use of three or more is even more preferred. Both proliferating and non- proliferating populations are preferably used, if available.
  • control cells are preferably supplied fresh from a tissue source, and are not cultured or transformed into a cell line. This is increasingly important when the control cell populations used in step a) is only one or two in number. Freshly obtained cancer cells may also be used as an alternative to cancer cell lines, although this is less critical.
  • An additional screening step is preferably conducted in which the cDNA corresponding to the putative cancer-associated gene is additionally hybridized with a digested mitochondrial DNA preparation, to eliminate mitochondrial genes. This screening step may be conducted before, between, subsequent to, or simultaneously with the other screening steps of the method.
  • RNA is supplied from a plurality of cancer cells, and one or preferably more control cell populations; the RNA is contacted with cDNA corresponding to the putative cancer-associated gene under conditions that permit formation of a stable duplex, and cDNA is selected corresponding to RNA that is present in greater abundance in a proportion of the cancer cells relative to the control cells.
  • the plurality of cancer cells is a panel of at least five, preferably at least ten cells.
  • at least three, more preferably at least five of the cancer cells show greater abundance of RNA.
  • At least one and preferably more of the cancer cells shows a greater abundance of RNA compared with control cells, but does not show duplication of the corresponding gene in step h) of the method.
  • Other embodiments of the invention are methods for obtaining cDNA corresponding to a gene that is deleted or underexpressed in cancer, comprising the steps of a) supplying an RNA preparation from control cells; b) supplying RNA preparations from at least two different cancer cells that share a deleted gene in the same region of a chromosome; c) displaying cDNA corresponding to the RNA preparations of step a) and step b) such that different cDNA corresponding to different RNA in each preparation are displayed separately; and d) selecting cDNA corresponding to RNA that is present in lower abundance in the cancer cells of step b) relative to the control cells of step a).
  • Such methods typically comprise the following further steps: e) supplying a digested DNA preparation from control cells; f) supplying digested DNA preparations from at least two different cancer cells; g) hybridizing the cDNA of step d) with the digested DNA preparations of step e) and step f); and h) further selecting cDNA from the cDNA of step d) corresponding to a gene that is deleted in the cancer cells of step f) relative to the control cells of step e).
  • Such methods for identifying deleted or underexpressed genes may also comprise enhancements such as those described above.
  • Additional embodiments of this invention are methods for characterizing cancer genes, comprising obtaining cDNA corresponding to a cancer-associated gene according to a method of this invention, particularly those highlighted above, and then sequencing the cDNA.
  • the cDNA may be used to rescue additional polynucleotides corresponding to a cancer- associated gene from an mRNA preparation, or a cDNA or genomic DNA library.
  • Additional embodiments of this invention are methods for screening candidate drugs for cancer treatment, comprising obtaining cDNA corresponding to a gene that is duplicated, overexpressed, deleted, or underexpressed in cancer, and comparing the effect of the candidate drug on a cell genetically altered with the cDNA or fragment thereof with the effect on a cell not genetically altered.
  • Cancers of particular interest include lung cancer, glioblastoma, pancreatic cancer, colon cancer, prostate cancer, hepatoma, myeloma, and breast cancer.
  • Figure 1 is a half-tone reproduction of an autoradiogram of a differential display experiment, in which radiolabeled cDNA corresponding to a subset of total messenger RNA in different cells are compared. This is used to select cDNA corresponding to particular RNA that are overabundant in breast cancer.
  • Figure 2 is a half-tone reproduction of an autoradiogram of electrophoresed DNA digests from a panel of breast cancer cell lines probed with a CH8-2a13-1 insert (Panel A) or a loading control (Panel B).
  • Figure 3 is a half-tone reproduction of an autoradiogram of electrophoresed total RNA from a panel of breast cancer cell lines probed with a CH8-2a13-1 insert (Panel A) or a loading control (Panel B).
  • Figure 4 is a half-tone reproduction of an autoradiogram of electrophoresed DNA digests from a panel of breast cancer cell lines probed with a CH13-2a12-1 insert.
  • Figure 5 is a half-tone reproduction of an autoradiogram of electrophoresed total RNA from a panel of breast cancer cell lines probed with a CH13-2a12-1 insert.
  • Figure 6 is a map of cDNA fragments obtained for the breast cancer associated genes CH1-9a11-2, CH8-2a13-1, CH13-2a12-1 and CH14-2a16-1. Regions of the fragments used to deduce sequence data listed in the application are indicated by shading. Nucleotide positions are numbered from the left-most residue for which double-strand sequence data has been obtained, which is not necessarily the 5' terminus of the corresponding message.
  • Figure 7 is a listing of primers used for obtaining the cDNA sequence data for CH1-9a11-2.
  • Figure 8 is a listing of cDNA sequence obtained for CH1-9a11-2.
  • Figure 9 is a listing of the amino acid sequence corresponding to the longest open reading frame of the DNA sequence of CH1-9a11-2 shown in Figure 8. The single-letter amino acid code is used.
  • Stop codons are indicated by a dot (•).
  • the upper panel shows the complete amino acid translation; the lower panel shows the predicted gene product protein sequence.
  • a possible transmembrane region is indicated by underlining.
  • Figure 10 is a listing of primers used for obtaining the cDNA sequence data for CH8-2a13-1.
  • Figure 11 is a listing of cDNA sequence obtained for CH8-2a13-1.
  • Figure 12 is a listing of the amino acid sequence corresponding to the longest open reading frame of the DNA sequence of CH8-2a13-1 shown in Figure 11.
  • the upper panel shows the complete amino acid translation; the lower panel shows the predicted gene product protein sequence.
  • Figure 13 is a listing of the nucleotide sequence predicted for a full-length CH8-2a13-1 cDNA.
  • Figure 14 is a listing of the amino acid sequence corresponding to the longest open reading frame of the DNA sequence of CH8-2a13-1 shown in Figure 13.
  • Figure 15 is a listing of primers used for obtaining the cDNA sequence data for CH13-2a12-1.
  • Figure 16 is a listing of cDNA sequence obtained for CH13-2a12-1.
  • Figure 17 is a listing of the amino acid sequence corresponding to the longest open reading frame of the DNA sequence of CH13-2a12-1 shown in Figure 16.
  • the upper panel shows the complete amino acid translation; the lower panel shows the predicted gene product protein sequence.
  • Figure 18 is a listing of primers used for obtaining cDNA sequence data for CH13-2a12-1..
  • Figure 19 is a listing of the cDNA sequence data obtained by two-directional sequencing for CH14- 2a16-1.
  • Figure 20 is a listing of the amino acid sequence corresponding to the longest open reading frame of the DNA sequence of CH14-2a16-1 shown in Figure 19.
  • the upper panel shows the complete amino acid translation; the lower panel shows the predicted gene product protein sequence. Residues corresponding to three zinc finger motifs are underlined, indicating that the protein may have DNA or RNA binding activity.
  • Figure 21 is a listing of additional DNA sequence data towards the 5' end of CH14-2a16-1 obtained by one-directional sequencing of the fragment pCH14-1.3. First two panels show nucleotide and amino acid sequence from the 5' end of the fragment; the second two panels show nucleotide and amino acid sequence from the 3' end of the fragment. Regions of overlap with pCH 14-800 are underlined.
  • Figure 22 is a listing of the nucleotide sequences of initial fragments obtained corresponding to the four breast cancer associated genes, along with their amino acid translations.
  • Figure 23 is a listing of additional cDNA sequence obtained for CH1-9a11-2, comprising approximately 1934 base pairs 5' from the sequence of Figure 8.
  • Figure 24 is a listing of the amino acid sequence corresponding to the longest open reading frame of the DNA sequence of CH1-9a11-2 shown in Figure 23. The single-letter amino acid code is used. Stop codons are indicated by a dot (•).
  • Figure 25 is a listing of additional cDNA sequence obtained for CH14-2a16-1, comprising approximately 1934 base pairs 5' from the sequence of Figure 19.
  • Figure 26 is a listing of the amino acid sequence corresponding to the longest open reading frame of the DNA sequence of CH1-9a11-2 shown in Figure 25. The single-letter amino acid code is used. Stop codons are indicated by a dot (•). The upper panel shows the complete amino acid translation; the lower panel shows zthe predicted gene product protein sequence.
  • This invention relates to the discovery and characterization of four novel genes associated with breast cancer.
  • the cDNA of these genes, and their sequences as disclosed below, provide the basis of a series of reagents that can be used in diagnosis and therapy.
  • each of the four genes was found to be duplicated in 40-60% of the cells tested. Surprisingly, each of the four genes was duplicated in at least one cell line where studies using comparative genomic hybridization had not revealed any amplification of the corresponding chromosomal region.
  • RNA overabundance without gene duplication indicating that the malignant cells had used some mechanism other than gene duplication to promote the abundance of RNA corresponding to these genes.
  • All four of the breast cancer genes have open reading frames, and likely are transcribed at various levels in different cell types. Overabundance of the corresponding RNA in a cancerous cell is likely associated with overexpression of the protein gene product.
  • Such overexpression may be manifest as increased secretion of the protein from the cell into blood or the surrounding environment, an increased density of the protein at the cell surface, or an increased accumulation the protein within the cell, in comparison to the typical level in no ⁇ cancerous cells of the same tissue type.
  • RNA overabundance by several mechanisms, because they are more likely to be directly involved in the pathogenic process, and therefore suitable targets for pharmacological manipulation.
  • All four genes sequences are unrelated to other genes known to be overexpressed in breast cancer, including the ent>B2 gene (Adnane et al.), tissue factor (Chen et al ), mammaglobulin (Watson et al.), and DD96 (Kocher et al.).
  • the four mRNA sequences each comprise an open reading frame.
  • the CH1-9a11-2 gene is expressed at the mRNA level at relatively elevated levels in pancreas and testis.
  • the CH8-2a13-1 gene is expressed at relatively elevated levels in adult heart, spleen, thymus, small intestine, colon, and tissues of the reproductive system; and at higher levels in certain tissues of the fetus.
  • the CH13- 2a12-1 gene is expressed at relatively elevated leves in heart, skeletal muscle, and testis.
  • the CH14- 2a16-1 gene is expressed at relatively elevated levels in testis.
  • the level of expression of all four genes is especially high in a substantial proportion of breast cancer cell lines.
  • the CH1-9a11-2 gene encodes a protein with a putative transmembrane region, and may be expressed as a surface protein on cancer cells.
  • the CH13-2a12-1 gene is distantly related to a C. elegans gene implicated in cell cycle regulation, and may play a role in the regulation of cell proliferation.
  • the protein encoded by CH13-2a12-1 is distantly related to a vasopressin-activated calcium binding receptor, and may have Ca* + binding activity.
  • the CH14-2a16-1 comprises at least five domains of a zinc finger binding motif and is distantly related to a yeast RNA binding protein.
  • the CH14-2a16-1 gene product is suspected of having DNA or RNA binding activity, which may relate to a role in cancer pathogenesis.
  • the four genes described here are exemplars of genes that undergo altered expression in cancer, identifiable using the gene screening methods of the invention.
  • the method involves an analysis for both DNA duplication and altered RNA abundance relating to the same gene. Since abnormal gene regulation is central to the malignant process, the identification method may be brought to bear on any type of cancer.
  • the screening method is superior to any previously available approach in several respects.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotides a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • polynucleotide refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form, and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a “linear sequence” or a “sequence” is an order of nucleotides in a polynucleotide in a 5' to 3' direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polynucleotide.
  • a “partial sequence” is a linear sequence of part of a polynucleotide which is known to comprise additional residues in one or both directions.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding is sequence-specific, and typical!/ occurs by Watson-Crick base pairing.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Hybridization reactions can be performed under conditions of different "stringency". Relevant conditions include temperature, ionic strength, time of incubation, the presence of additional solutes in the reaction mixture such as formamide, and the washing procedure. Higher stringency conditions are those conditions, such as higher temperature and lower sodium ion concentration, which require higher minimum complementarity between hybridizing elements for a stable hybridization complex to form. Conditions that increase the stringency of a hybridization reaction are widely known and published in the art: see, for example, "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook, Fritsch & Maniatis, 1989).
  • a double-stranded polynucleotide can be “complementary” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.
  • Complementarity (the degree that one polynucleotide is complementary with another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules.
  • a linear sequence of nucleotides is "identical" to another linear sequence, if the order of nucleotides in each sequence is the same, and occurs without substitution, deletion, or material substitution. It is understood that purine and pyrimidine nitrogenous bases with similar structures can be functionally equivalent in terms of Watson-Crick base-pairing; and the inter-substitution of like nitrogenous bases, particularly uracil and thymine, or the modification of nitrogenous bases, such as by methylation, does not constitute a material substitution.
  • RNA and a DNA polynucleotide have identical sequences when the sequence for the RNA reflects the order of nitrogenous bases in the polyribonucleotides, the sequence for the DNA reflects the order of nitrogenous bases in the polydeoxyribonucleotides, and the two sequences satisfy the other requirements of this definition. Where one or both of the polynucleotides being compared is double-stranded, the sequences are identical if one strand of the first polynucleotide is identical with one strand of the second polynucleotide.
  • a linear sequence of nucleotides is "essentially identical" to another linear sequence, if both sequences are capable of hybridizing to form a duplex with the same complementary polynucleotide. Sequences that hybridize under conditions of greater stringency are more preferred. It is understood that hybridization reactions can accommodate insertions, deletions, and substitutions in the nucleotide sequence. Thus, linear sequences of nucleotides can be essentially identical even if some of the nucleotide residues do not precisely correspond or align.
  • essentially identical sequences of about 40 nucleotides in length will hybridize at about 300C in 10 x SSC (0.15 M NaCI, 15 mM citrate buffer); preferably, they will hybridize at about 400C in 6 x SSC; more preferably, they will hybridize at about 500C in 6 x SSC; even more preferably, they will hybridize at about 600C in 6 x SSC, or at about 400C in 0.5 x SSC, or at about 300C in 6 x SSC containing 50% formamide; still more preferably, they will hybridize at 400C or higher in 2 x SSC or lower in the presence of 50% or more formamide.
  • the rigor of the test is partly a function of the length of the polynucleotide; hence shorter polynucleotides with the same homology should be tested under lower stringency and longer polynucleotides should be tested under higher stringency, adjusting the conditions accordingly.
  • the relationship between hybridization stringency, degree of sequence identity, and polynucleotide length is known in the art and can be calculated by standard formulae; see, e.g., Meinkoth et al. Sequences that correspond or align more closely to the invention disclosed herein are comparably more preferred. Generally, essentially identical sequences are at least about 50% identical with each other, after alignment of the homologous regions.
  • the sequences are at least about 60% identical; more preferably, they are at least about 70% identical; more preferably, they are at least about 80% identical; more preferably, the sequences are at least about 90% identical; even more preferably, they are at least 95% identical; still more preferably, the sequences are 100% identical.
  • Percent identity is calculated as the percent of residues in the sequence being compared that are identical to those in the reference sequence, which is usually one of those listed or described in this application, unless stated otherwise. No penalty is imposed for introduction of gaps in the reference or comparison sequence for purposes of alignment, but the resulting fragments must be rationally derived — small gaps may not be introduced to trivially improve the identity score.
  • polynucleotide sequences are essentially identical, a sequence that preserves the functionality of the polynucleotide with which it is being compared is particularly preferred. Functionality may be established by different criteria, such as ability to hybridize with a target polynucleotide, and whether the polynucleotide encodes an identical or essentially identical polypeptides. Thus, nucleotide substitutions which cause a non-conservative substitution in the encoded polypeptide are preferred over nucleotide substitutions that create a stop codon; nucleotide substitutions that cause a conservative substitution in the encoded polypeptide are more preferred, and identical nucleotide sequences are even more preferred.
  • Insertions or deletions in the polynucleotide that result in insertions or deletions in the polypeptide are preferred over those that result in the down-stream coding region being rendered out of phase.
  • the relative importance of hybridization properties and the polypeptide encoded by a polynucleotide depends on the application of the invention.
  • a “reagent” polynucleotide, polypeptide, or antibody is a substance provided for a reaction, the substance having some known and desirable parameters for the reaction.
  • a reaction mixture may also contain a "target", such as a polynucleotide, antibody, or polypeptide that the reagent is capable of reacting with.
  • a target such as a polynucleotide, antibody, or polypeptide that the reagent is capable of reacting with.
  • the amount of the target in a sample is determined by adding a reagent, allowing the reagent and target to react, and measuring the amount of reaction product.
  • a “target” may also be a cell, collection of cells, tissue, or organ that is the object of an administered substance, such as a pharmaceutical compound.
  • cDNA or “complementary DNA” is a single- or double-stranded DNA polynucleotide in which one strand is complementary to a messenger RNA.
  • Full-length cDNA is cDNA comprised of a strand which is complementary to an entire messenger RNA molecule.
  • a "cDNA fragment” as used herein generally represents a sub-region of the full-length form, but the entire full-length cDNA may also be included. Unless explicitly specified, the term cDNA encompasses both the full-length fomn and the fragment form.
  • RNA corresponds to the gene from which it is transcribed.
  • cDNA corresponds to the RNA from which it has been produced, such as by a reverse transcription reaction, or by chemical synthesis of a DNA based upon knowledge of the RNA sequence.
  • cDNA also corresponds to the gene that encodes the RNA.
  • Polynucleotides may be said to correspond even when one of the pair is derived from only a portion of the other.
  • a "probe” when used in the context of polynucleotide manipulation refers to a polynucleotide which is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target.
  • a probe will comprise a label or a means by which a label can be attached, either before or subsequent to the hybridization reaction.
  • Suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and enzymes.
  • a “primer” is a short polynucleotide, generally with a free 3' -OH group, that binds to a target potentially present in a sample of interest by hybridizing with the target, and thereafter promoting polymerization of a polynucleotide complementary to the target.
  • a “polymerase chain reaction” (“PCR”) is a reaction in which replicate copies are made of a target polynucleotide using one or more primers, and a catalyst of polymerization, such as a reverse transcriptase or a DNA polymerase, and particularly a thermally stable polymerase enzyme. Methods for PCR are taught in U.S. Patent Nos.
  • An “operon” is a genetic region comprising a gene encoding a protein and functionally related
  • promoter regions include but are not limited to promoter regions, enhancer regions, repressor binding regions, transcription initiation sites, ribosome binding sites, translation initiation sites, protein encoding regions, introns and exons, and termination sites for transcription and translation.
  • a "promoter” is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region located downstream (in the 3' direction) from the promoter.
  • “Operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.
  • Gene duplication is a term used herein to describe the process whereby an increased number of copies of a particular gene or a fragment thereof is present in a particular cell or cell line. "Gene amplification” generally is synonymous with gene duplication.
  • RNA overexpression reflects the presence of more RNA (as a proportion of total RNA) from a particular gene in a cell being described, such as a cancerous cell, in relation to that of the cell it is being compared with, such as a non-cancerous cell.
  • the protein product of the gene may or may not be produced in normal or abnormal amounts.
  • RNA overabundance or “overabundance of RNA” describes RNA that is present in greater proportion of total RNA in the cell type being described, compared with the same RNA as a proportion of the total RNA in a control cell.
  • a number of mechanisms may contribute to RNA overabundance in a particular cell type: for example, gene duplication, increased level of transcription of the gene, increased persistence of the RNA within the cell after it is produced, or any combination of these.
  • “lower abundance” or “underabundance” describes RNA that is present in lower proportion in the cell being described compared with a control cell.
  • polypeptide polypeptide
  • peptide protein
  • polymers of amino acids of any length may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • a "linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an N-terminal to C-terminal direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
  • a “partial sequence” is a linear sequence of part of a polypeptide which is known to comprise additional residues in one or both directions.
  • a linear sequence of amino acids is "essentially identical" to another sequence if the two sequences have a substantial degree of sequence identity. It is understood that the functional proteins can accommodate insertions, deletions, and substitutions in the amino acid sequence. Thus, linear sequences of amino acids can be essentially identical even if some of the residues do not precisely correspond or align.
  • Sequences that correspond or align more closely to the invention disclosed herein are more preferred. It is also understood that some amino acid substitutions are more easily tolerated. For example, substitution of an amino acid with hydrophobic side chains, aromatic side chains, polar side chains, side chains with a positive or negative charge, or side chains comprising two or fewer carbon atoms, by another amino acid with a side chain of like properties can occur without disturbing the essential identity of the two sequences. Methods for determining homologous regions and scoring the degree of homology are well known in the art; see for example Altschul et al. and Henikoff et al. Well-tolerated sequence differences are referred to as "conservative substitutions".
  • amino acid sequences that are essentially identical are at least about 15% identical, and comprise at least about another 15% which are either identical or are conservative substitutions, after alignment of homologous regions. More preferably, essentially identical sequences comprise at least about 50% identical residues or conservative substitutions; more preferably, they comprise at least about 70% identical residues or conservative substitutions; more preferably, they comprise at least about 80% identical residues or conservative substitutions; more preferably, they comprise at least about 90% identical residues or conservative substitutions; more preferably, they comprise at least about 95% identical residues or conservative substitutions; even more preferably, they contain 100% identical residues.
  • polypeptide sequences are essentially identical, a sequence that preserves the functionality of the polypeptide with which it is being compared is particularly preferred. Functionality may be established by different parameters, such as enzymatic activity, the binding rate or affinity in a receptor-ligand interaction, the binding affinity with an antibody, and X-ray crystallographic structure.
  • an “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a polypeptide, through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
  • a target such as a polypeptide
  • the term encompasses not only intact antibodies, but also fragments thereof, mutants thereof, fusion proteins, humanized antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.
  • the term "antigen” refers to the target molecule that is specifically bound by an antibody through its antigen recognition site.
  • the antigen may, but need not be chemically related to the immunogen that stimulated production of the antibody.
  • the antigen may be polyvalent, or it may be a monovalent hapten.
  • antigens examples include polypeptides, polynucleotides, other antibody molecules, oligosaccharides, complex lipids, drugs, and chemicals.
  • An "immunogen” is an antigen capable of stimulating production of an antibody when injected into a suitable host, usually a mammal. Compounds may be rendered immunogenic by many techniques known in the art, including crosslinking or conjugating with a carrier to increase valency, mixing with a mitogen to increase the immune response, and combining with an adjuvant to enhance presentation.
  • An "active vaccine” is a pharmaceutical preparation for human or animal use, which is used with the intention of eliciting a specific immune response. The immune response may be either humoral or cellular, systemic or secretory.
  • the immune response may be desired for experimental purposes, for the treatment of a particular condition, for the elimination of a particular substance, or for prophylaxis against a particular condition or substance.
  • An "isolated" polynucleotide, polypeptide, protein, antibody, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially obtained from.
  • an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture.
  • a substance can also be provided in an isolated state by a process of artificial assembly, such as by chemical synthesis or recombinant expression.
  • a polynucleotide used in a reaction such as a probe used in a hybridization reaction, a primer used in a PCR, or a polynucleotide present in a pharmaceutical preparation, is referred to as “specific” or “selective” if it hybridizes or reacts with the intended target more frequently, more rapidly, or with greater duration than it does with alternative substances.
  • an antibody is referred to as “specific” or “selective” if it binds via at least one antigen recognition site to the intended target more frequently, more rapidly, or with greater duration than it does to alternative substances.
  • a polynucleotide or antibody is said to "selectively inhibit” or “selectively interfere with” a reaction if it inhibits or interferes with the reaction between particular substrates to a greater degree or for a greater duration than it does with the reaction between alternative substrates.
  • An antibody is capable of "specifically delivering” a substance if it conveys or retains that substance near a particular cell type more frequently or for a greater duration compared with other cell types.
  • the "effector component" of a pharmaceutical preparation is a component which modifies target cells by altering their function in a desirable way when administered to a subject bearing the cells.
  • Some advanced pharmaceutical preparations also have a "targeting component", such as an antibody, which helps deliver the effector component more efficaciously to the target site.
  • the effector component may have any one of a number of modes of action. For example, it may restore or enhance a normal function of a cell, it may eliminate or suppress an abnormal function of a cell, or it may alter a cell's phenotype. Altematively, it may kill or render dormant a cell with pathological features, such as a cancer cell. Examples of effector components are provided in a later section.
  • a “pharmaceutical candidate” or “drug candidate” is a compound believed to have therapeutic potential, that is to be tested for efficacy.
  • the “screening” of a pharmaceutical candidate refers to conducting an assay that is capable of evaluating the efficacy and/or specificity of the candidate.
  • efficacy refers to the ability of the candidate to effect the cell or organism it is administered to in a beneficial way: for example, the limitation of the pathology of cancerous cells.
  • a “cell line” or “cell culture” denotes higher eukaryotic cells grown or maintained in vitro. It is understood that the descendants of a cell may not be completely identical (either morphologically, genotypically, or phenotypically) to the parent cell. Cells described as "uncultured” are obtained directly from a living organism, and have been maintained for a limited amount of time away from the organism: not long enough or under conditions for the cells to undergo substantial replication.
  • Genetic alteration refers to a process wherein a genetic element is introduced into a cell other than by mitosis or meiosis.
  • the element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell.
  • Genetic alteration may be effected, for example, by transfecting a cell with a recombinant plasmid or other polynucleotide through any process known in the art, such as electroporation, calcium phosphate precipitation, or contacting with a polynucleotide-liposome complex, or by transduction or infection with a DNA or RNA virus or viral vector.
  • the alteration is preferably but not necessarily inheritable by progeny of the altered cell.
  • a “host cell” is a cell which has been genetically altered, or is capable of being genetically altered, by administration of an exogenous polynucleotide.
  • a frequent feature of cancer cells is the tendency to grow in a manner that is uncontrollable by the host, but the pathology associated with a particular cancer cell may take another form, as outlined infra.
  • Primary cancer cells that is, cells obtained from near the site of malignant transformation
  • the definition of a cancer cell includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • the "pathology" caused by a cancer cell within a host is anything that compromises the well-being or normal physiology of the host.
  • This may involve (but is not limited to) abnormal or uncontrollable growth of the cell, metastasis, release of cytokines or other secretory products at an inappropriate level, manifestation of a function inappropriate for its physiological milieu, interference with the normal function of neighboring cells, aggravation or suppression of an inflammatory or immunological response, or the harboring of undesirable chemical agents or invasive organisms.
  • Treatment of an individual or a cell is any type of intervention in an attempt to alter the natural course of the individual or cell.
  • treatment of an individual may be undertaken to decrease or limit the pathology caused by a cancer cell harbored in the individual.
  • Treatment includes (but is not limited to) administration of a composition, such as a pharmaceutical composition, and may be performed either prophylactically, or subsequent to the initiation of a pathologic event or contact with an etiologic agent.
  • Effective amounts used in treatment are those which are sufficient to produce the desired effect, and may be given in single or divided doses.
  • control cell is an alternative source of cells or an alternative cell line used in an experiment for comparison purposes. Where the purpose of the experiment is to establish a base line for gene copy number or expression level, it is generally preferable to use a control cell that is not a cancer cell.
  • cancer gene refers to any gene which is yielding transcription or translation products at a substantially altered level or in a substantially altered form in cancerous cells compared with non-cancerous cells, and which may play a role in supporting the malignancy of the cell. It may be a normally quiescent gene that becomes activated (such as a dominant proto-oncogene), it may be a gene that becomes expressed at an abnormally high level (such as a growth factor receptor), it may be a gene that becomes mutated to produce a variant phenotype, or it may be a gene that becomes expressed at an abnormally low level (such as a tumor suppresser gene). The present invention is directed towards the discovery of genes in all these categories.
  • a "clinical sample” encompasses a variety of sample types obtained from a subject and useful in an in vitro procedure, such as a diagnostic test.
  • the definition encompasses solid tissue samples obtained as a surgical removal, a pathology specimen, or a biopsy specimen, tissue cultures or cells derived therefrom and the progeny thereof, and sections or smears prepared
  • Non-limiting examples are samples obtained from breast tissue, lymph nodes, and tumors.
  • the definition also encompasses blood, spinal fluid, and other liquid sample of biologic origin, and may refer to either the cells or cell fragments suspended therein, or to the liquid medium and its solutes.
  • the term "relative amount" is used where a comparison is made between a test measurement and a control measurement.
  • the relative amount of a reagent forming a complex in a reaction is the amount reacting with a test specimen, compared with the amount reacting with a control specimen.
  • the control specimen may be run separately in the same assay, or it may be part of the same sample (for example, normal tissue surrounding a malignant area in a tissue section).
  • a “differential” result is generally obtained from an assay in which a comparison is made between the findings of two different assay samples, such as a cancerous cell line and a control cell line.
  • “differential expression” is observed when the level of expression of a particular gene is higher in one cell than another.
  • “Differential display” refers to a display of a component, particularly RNA, from different cells to determine if there is a difference in the level of the component amongst different cells. Differential display of RNA is conducted, for example, by selective production and display of cDNA corresponding thereto. A method for performing differential display is provided in a later section.
  • a polynucleotide derived from or corresponding to CH1-9a11-2, CH8-2a13-1, CH13-2a12-1, or CH14-2a16-1 is any of the following: the respective cDNA fragments, the corresponding messenger RNA, including splice variants and fragments thereof, both strands of the corresponding full-length cDNA and fragments thereof, and the co ⁇ esponding gene. Isolated allelic variants of any of these forms are included.
  • This invention embodies any polynucleotide corresponding to CH1-9a11- 2, CH8-2a13-1, CH13-2a12-1 , or CH14-2a16-1 in an isolated form.
  • displaying cDNA is any technique in which DNA copies of RNA (not restricted to mRNA) is rendered detectable in a quantitative or relatively quantitative fashion, in that DNA copies present in a relatively greater amount in a first sample compared with a second sample generates a relatively stronger or weaker signal compared with that of the second sample due to the difference in copy number.
  • a preferred method of display is the differential display technique, and enhancements thereupon described in this disclosure and elsewhere.
  • the term "digested" DNA encompasses DNA (particularly chromosomal DNA) that has been fragmented by any suitable chemical or enzymatic means into fragments conveniently separable by standard techniques, particularly gel electrophoresis. Digestion with a restriction endonuclease specific for a particular nucleotide sequence is preferred.
  • Hybridizing in this context refers to contacting a first polynucleotide with a second polynucleotide under conditions that permit the formation of a multi-stranded polynucleotide duplex whenever one strand of the first polynucleotide has a sequence of sufficient complementarity to a sequence on the second polynucleotide.
  • the duplex may be a long-lived one, such as when one DNA molecule is used as a Iabeled probe to detect another DNA molecule, that may optionally be bound to a nitrocellulose filter or present in a separating gel.
  • the duplex may also be a shorter- lived one, such as when one DNA molecule is used to prime an amplification reaction of the other DNA molecule, and the amplified product is subsequently detected.
  • the practitioner may alter the conditions of the reaction to alter the degree of complementarity required, as long as sequence specificity remains a determining factor in the reaction.
  • steps of a method of this invention may be performed in any order, or combined where desired and appropriate.
  • steps a) through h) that is described above, it is entirely appropriate to conduct steps a) to c) of the method either before or after steps e) to g) of the method, as long as the cDNA ultimately selected fulfills the criteria of both steps d) and step h).
  • screening against different digested DNA preparations even if outlined separately, may optionally be done at the same time. All permutations of this kind are within the scope of the invention.
  • the cancer gene screening methods of this invention may be brought to bear to discover novel genes associated with cancer. Exemplars of cancer-associated genes identified by this method are described below. The exemplars were identified using breast cancer cell lines and tissue, but the strategy can be applied to any cancer type of interest. A central feature of the cancer gene screening method of this invention is to look for both
  • the present invention provides a way of detecting genes that may be present in an amplicon from a functional basis. Because an early part of the method involves detecting RNA, the method avoids genes that may be duplicated in an amplicon but are quiescent (and therefore irrelevant) in the cancer cells. Furthermore, it recruits active genes from a duplicated region of the chromosome too small to be detectable by the techniques used to describe amplicons. Near the heart of this approach are several concepts.
  • RNA expression refers to expression at the RNA transcription level. Most typically, the RNA is in turn be translated into a protein with a particular enzymatic, binding, or regulatory activity which increases after malignant transformation. In a less common example, the RNA may encode or participate as a ribozyme, antisense polynucleotide, or other functional nucleic acid molecule during malignancy. In a third example, RNA expression may be incidental but symptomatic of an important event in transformation.
  • RNA overabundance without gene duplication such as by increasing the rate of transcription of the gene (e.g., by upregulation of the promoter region), by enhancing transcript promotion or transport, or by increasing mRNA survival.
  • the method entails screening at the RNA level, several cancer cell lines or tumors, and several normal cell lines or tissue samples at the same time.
  • RNA are selected that show a consistent elevation amongst the cancer cells as compared with normal cells. Additional strategies may be employed in combination with the RNA screening to improve the success rate of the method.
  • One such strategy is to use several cancer cell lines that are all known to have duplicated genes in the same region of a particular chromosome. Thus, the RNA that emerge from the screen are more likely to represent a deliberate overexpression event, and the overexpressed gene is likely to be within the duplicated region.
  • a supplemental strategy is to use freshly prepared tissue samples rather than cell lines as controls for base-line expression. This avoids selection of genes that may alter their expression level just as a result of tissue culturing.
  • Another supplemental strategy is to conduct an additional level of screening, following identification of shared, overexpressed RNA.
  • the selected RNA are used to screen DNA from suitable cancer cells and normal cells, to ensure that at least a proportion of the cells achieved the overexpression by way of gene duplication.
  • the strategy for detecting such genes comprises a number of innovations over those that have been used in
  • the first part of the method is based on a search for particular RNAs that are overabundant in cancer cells.
  • a first innovation of the method is to compare RNA abundance between control cells and several different cancer cells or cancer cell lines of the desired type.
  • the cDNA fragments that emerge in a greater amount in several different cancer lines, but not in control cells, are more likely to reflect genes that are important in disease progression, rather than those that have undergone secondary or coincidental activation. It is particularly preferred to use cancer cells that are known to share a common duplicated chromosomal region.
  • a second innovation of this method is to supply as control, not RNA from a cell line or culture, but from fresh tissue samples of non-malignant origin.
  • the tissue will provide the spectrum of expression that is typical to the normal cell phenotype, rather than individual differences that may become more prominent in culture. This establishes a more reliable baseline for normal expression levels.
  • the tissue will be devoid of the effects that in vitro culturing may have in altering or selecting particular phenotypes. For example, proto-oncogenes or growth factors may become up-regulated in culture. When cultured cells are used as the control for differential display, these up-regulated genes would be missed.
  • a third innovation of this method is to undertake a subselection for cDNA corresponding to genes that achieve their RNA overabundance in a substantial proportion of cancer cells by gene duplication.
  • appropriate cDNA corresponding to overabundant RNA identified in the foregoing steps are used to probe digests of cellular DNA from a panel of different cancer cells, and from normal genomic DNA. cDNA that shows evidence of higher copy numbers in a proportion of the panel are selected for further characterization.
  • An additional advantage of this step is that cDNA corresponding to mitochondrial genes can rapidly be screened away by including a mitochondrial DNA digest as an additional sample for testing the probe. This eliminates most of the false-positive cDNA, which otherwise make up a majority of the cDNA identified.
  • RNA is prepared from both cancerous and control cells by standard techniques.
  • Cancer-associated genes may affect cellular metabolism by any one of a number of mechanisms. For example, they may encode ribozymes, anti-sense polynucleotides, DNA-binding polynucleotides, altered ribosomal RNA, and the like.
  • the gene screening methods of this invention may employ a comparison of RNA abundance levels at the total RNA level, not strictly limited to mRNA.
  • the vast majority of cancer- associated genes are predicted to encode a protein gene whose up-regulation is closely linked to the metabolic process.
  • the four exemplary breast cancer genes described elsewhere in this application all comprise an open reading frame.
  • a focus on mRNA enriches the selectable pool for candidate cancer-associated genes.
  • Focus towards mRNA can be conducted at any step in the method. It is particularly convenient to use a display method that displays cDNA copied only from mRNA. In this case, whole RNA may be prepared and analyzed from cancer and control cell populations without separating out mRNA.
  • RNA source it is particularly advantageous to use a plurality of cancer cells known to contain a duplicated gene or chromosomal segment in the same region of the chromosome.
  • the duplicated segment need not be the same size in all the cells, nor is it necessary that the number of duplications be the same, so long as there is at least some part of the duplicated segment that is shared amongst all the cancer cells used in the screen.
  • a minimum of two, and preferably at least three cancer cells are used that are sufficiently characterized to identify a shared duplicated region, and can be used as a source of RNA for the screening test.
  • the control cell population will not comprise chromosomal duplications.
  • RNA transcribed from the duplicated region is expected to be overabundant compared with that of the control cell. Accordingly, a highly effective strategy is to identify overabundant RNA that is present in all (or at least several) of the cancer cell preparations, but none of the control preparations.
  • the RNA comparison will be strongly biased in favor of RNA overabundance transcribed from the shared duplicated region.
  • RNA abundance differences resulting from normal metabolic variations between cells and/or b) RNA abundance differences related to cancer cell malignancy, but occurring secondarily to malignant transformation.
  • This is important, because it considerably minimizes the chief deficiency in the use of RNA comparison methods, particularly differential display, for the screening of potential cancer genes: namely, the onerous number of false-positives that such techniques generate.
  • Shared duplicated regions in cancer cells may be identified by a relevant analytical technique, or by reference to such analysis already conducted and published.
  • One approach that has been highly effective in mapping approximate sub-chromosomal locations of duplicated segments is comparative genomic hybridization (CGH). This technique involves extracting, amplifying and labeling DNA from the subject cell; hybridizing to reference metaphase chromosomes treated to remove repetitive sequences; and observing the position of the hybridized DNA on the chromosomes (WO 93/18186; Gray et al.). The greater the signal intensity at a given position, the greater the copy number of the sequences in the subject cell. Thus, regions showing elevated staining correspond to genes duplicated in the cancer cells, while regions showing diminished staining correspond to genes deleted in the cancer cells.
  • CGH comparative genomic hybridization
  • chromosomal mapping approach is irrelevant, especially once knowledge of the duplicated region is known. If the location of the chromosome duplication is already established for a cell line to be used in RNA comparison during the course of the present invention, then it is unnecessary to conduct a mapping technique de novo. For example, established cancer cell lines exist for which mapping data is already available in the public domain.
  • a plurality of cancer cells is chosen for the screening panel based on such data, so that they share a duplicated chromosomal region.
  • the chromosomal location of a suspected duplication may be confirmed by hybridization analysis, if desired, using a probe specific for the location.
  • the cancer cells used for RNA comparison are also generally (but not necessarily) derived from the same type of cancer or the same tissue. Using cells derived from the same type of cancer increases the probability that the gene ultimately identified will be common in that type of cancer, and suitable as a type-specific diagnostic marker. Using cells derived from different types of cancer is in effect a search for cancer-related genes that are less tissue specific and more related to the malignant process in general. Both types of genes are of interest for both diagnostic and therapeutic purposes. In one illustration highlighted in Example 1, RNA was screened from the three breast cancer cell lines BT474, SKBR3, and MCF7, which have been determined by CGH or Southern analysis to share a duplicated genetic regions in chromosomes 1, 8, 14, 17, and 20.
  • RNA overabundant in all three cancer cell lines corresponded to cancer-associated genes located on chromosomes 1, 8, and 14 that are listed in Table 1.
  • the chromosome 13 gene (CH13-2a12-1) was overexpressed in 2 of the 3 cell lines; namely BT474 and SKBR3. Southern analysis subsequently established that the chromosome 13 gene was duplicated in the same two cell lines (Example 6, Table 5).
  • control cell RNA can be derived from in vitro cultures of non-malignant cells, or established cell lines derived from a non-malignant source
  • the transforming event may, in turn, be shared with that of certain cancer cells, at least at the level of RNA abundance
  • companson of the RNA levels in cancer cells with so-called control cell lines may lead the practitioner to miss genes that are related to malignancy
  • control cells may be maintained in culture for a brief period before the experiment, and even stimulated, however, multiple rounds of cell division are to be avoided if possible
  • Use of both stimulated and unstimulated cells as controls may help provide RNA patterns corresponding to the normal range of abundance within various metabolic events of the cell cycle In one illustration highlighted in Example 1,
  • RNA is preserved until use in the comparison expenment in such a way to minimize fragmentation
  • RNA display methods For displaying relative overabundance of RNA in the cancer cells, compared with the control cells, many standard techniques are suitable These would include any form of subtractive hybridization or comparative analysis Preferred are techniques in which more than two RNA sources are compared at the same time, such as various types of arbitrarily primed PCR fingerprinting techniques (Welsh et al , Yoshikawa et al ) Particularly preferred are differential mRNA display methods and variations thereof, in which the samples are run in neighbonng lanes in a separating gel These techniques are focused towards mRNA by using primers that are specific for the poly-A tail characteristic of mRNA (Liang et al , 1992a, U S Patent 5,262,311 )
  • RNA is first reverse transcribed by standard techniques Short primers are used for the selection, preferably chosen such that alternative primers used in a series of like assays can complete a comprehensive survey of the mRNA
  • primers can be used for the 3' region of the mRNAs which have an oligo-dT sequence, followed by two other nucleotides (TiNM, where ⁇ * 11, N e ⁇ A,C,G ⁇ , and M ⁇ ⁇ A,C,G,T ⁇ ).
  • TiNM oligo-dT sequence
  • a random or arbitrary primer of minimal length can then be used for replication towards what corresponds in the sequence to the 5' region of the mRNA.
  • the optimal length for the random primer is about 10 nucleotides.
  • the product of the PCR reaction is Iabeled with a radioisotope, such as 35 S.
  • the Iabeled cDNA is then separated by molecular weight, such as on a polyacrylamide sequencing gel.
  • a polyacrylamide sequencing gel If desired, variations on the differential display technique may be employed.
  • one-base oligo-dT primers may be used (Liang et al., 1993 & 1994), although this is generally less preferred because the display pattern is correspondingly more complex.
  • Selection of primers may be optimized mathematically depending on the number of RNA species in a tissue of interest (Bauer et al.). The method may be adapted for non-denaturing gels, and for use with automatic DNA sequencers (Bauer et al.).
  • Radioisotopes may be used for labeling the differential display.
  • Differential display may optionally be combined with a ribonuclease protection assay (Yeatman et al.).
  • PCR primers may optionally incorporate a restriction site to facilitate cloning (Linskens et al., Ayala et al.).
  • Taqr polymerase from multiple manufacturers can increase the amount of variation under otherwise identical conditions (Haag et al ).
  • Nested PCR primers may be used in differential display to decrease background created by oligo-dT primers (WO 95/33760).
  • Other variants of the differential display technique are known in the art and described inter alia in the references cited in this disclosure. The use of such modifications are within the scope of the present invention, but are not required, as evidenced by the examples described below.
  • RNAs are chosen which are present as a higher proportion of the RNA in cancerous cells, compared with control cells.
  • the cDNA corresponding to overabundant RNA will produce a band with greater proportional intensity amongst neighboring cDNA bands, compared with the proportional intensity in the control lanes.
  • Desired cDNAs can be recovered most directly by cutting the spot in the gel corresponding to the band, and recovering the DNAs therefrom. Recovered cDNA can be replicated again for further use by any technique or combination of techniques known in the art, including PCR and cloning into a suitable carrier.
  • An optional but highly beneficial additional screening step is aimed at identifying genes that are duplicated in a substantial proportion of cancers. This is conducted by using cDNA such as selected from differential display to probe digests of chromosomal DNA obtained from two or more cancerous cells, such as cancer cell lines. Chromosomal DNA from non-cancerous cells that essentially reflects the germ line in terms of gene copy number is used for the control. A preferred source of control DNA in experiments for human cancer genes is placental DNA, which is readily obtainable. The DNA samples are cleaved at sequence-specific sites along the chromosome, most usually with a suitable restriction enzyme into fragments of appropriate size.
  • the DNA can be blotted directly onto a suitable medium, or separated on an agarose gel before blotting.
  • the latter method is preferred, because it enables a comparison of the hybridizing chromosomal restriction fragment to determine whether the probe is binding to the same fragment in all samples.
  • the amount of probe binding to DNA digests from each of the cancer cells is compared with the amount binding to control DNA.
  • One method is to administer a second probe to the same blot, probing for a second chromosomal gene unlikely to be duplicated in the cancer cells. This method is preferred, because it standardizes not only for differences in the amount of DNA provided, but also for differences in the amount transferred during blotting. This can be accomplished by using alternative labels for the two probes, or by stripping the first probe with a suitable eluant before administering the second.
  • cDNA for mitochondrial genes it is preferable to include in a parallel analysis a mitochondrial DNA preparation digested with the same restriction enzyme. Any cDNA probe that hybridizes to the appropriate mitochondrial restriction fragments can be suspected of corresponding to a mitochondrial gene.
  • the random primer may bind at any location along the
  • the copied and replicated segment may be a fragment of the full-length RNA.
  • Longer cDNA corresponding to a greater portion of the sequence can be obtained, if desired, by several techniques known to practitioners of ordinary skill. These include using the cDNA fragment to isolate the corresponding RNA, or to isolate complementary DNA from a cDNA library of the same species.
  • the library is derived from the same tissue source, and more preferably from a cancer cell line of the same type.
  • a preferred library is derived from breast cancer cell line BT474, constructed in lambda GT10.
  • Sequences of the cDNA can be determined by standard techniques, or by submitting the sample to commercial sequencing services.
  • the chromosomal locations of the genes can be determined by any one of several methods known in the art, such as in situ hybridization using chromosomal smears, or panels of somatic cell hybrids of known chromosomal composition.
  • the cDNA obtained through the selection process outlined can then be tested against a larger panel of cancer cell lines and/or fresh tumor cells to determine what proportion of the cells have duplicated the gene. This can be accomplished by using the cDNA as a probe for chromosomal DNA digests, as described earlier. As illustrated in the Example section, a preferred method for conducting this determination is Southern analysis.
  • the cDNA can also be used to determine what proportion of the cells have RNA overabundance. This can be accomplished by standard techniques, such as slot blots or blots of agarose gels, using whole RNA or messenger RNA from each of the cells in the panel. The blots are then probed with the cDNA using standard techniques. It is preferable to provide an internal loading and blotting control for this analysis. A preferred method is to re-probe the same blot for transcripts of a gene likely to be present in about the same level in all cells of the same type, such as the gene for a cytoskeletal protein. Thus, a preferred second probe is the cDNA for beta-actin. Using a novel cDNA found by this selection procedure, it is anticipated that essentially all cancer cells showing gene duplication will also show RNA overabundance, but that some will show RNA overabundance without gene duplication.
  • RNA overabundance may be reversed appropriately to screen for genes that are deleted and/or associated with RNA underabundance.
  • the principles are essentially the same. Genes that are frequently down-regulated in cancer (such as tumor suppresser genes) may be down-regulated by different mechanisms in different cells, and a gene with this behavior is more likely to be central to malignant transformation or persistence of the malignant state.
  • RNA is prepared from a plurality of tumors or cancer cell lines and the abundance is compared with RNA preparation from control cells.
  • cancer cells that share a deleted gene in the same chromosomal region, in order to focus any differences at the RNA level towards particular alterations in cancer cells and away from normal variations or coincidental changes.
  • the CGH technique may be used to identify deletions in previously uncharacterized cancer cells.
  • cancer cells may be chosen on the basis of previous knowledge of deleted regions; there is no need to conduct methods such as CGH on previously characterized lines.
  • cDNA from the RNA of cancer cells is displayed (preferably by differential display) alongside cDNA copied from (preferably uncultured) control cells, and cDNA is selected that appears to be underrepresented in at least two (preferably more) of the cancer cells compared with the control cells.
  • cDNA thus selected may optionally be further screened against digested DNA preparations, to confirm that the RNA underabundance observed in the cancer cell populations is attributable in at least a proportion of the cells to an actual gene deletion.
  • the cDNA may be used for sequencing or rescuing additional polynucleotides, in this case not from the cancer cells but from cells containing or expressing the gene at normal levels.
  • Pharmaceuticals based on deleted genes or those associated with underexpressed RNA are typically oriented at restoring or upregulating the gene, or a functional equivalent of the encoded gene product.
  • RNA has been compared between breast cancer cells and control cells.
  • the amount of total cellular RNA was compared using a modified differential display method.
  • Primers were used for the 3' region of the mRNAs which have an oligo-dT sequence, followed by two other nucleotides as described in the previous section. Random or arbitrary primers of about 10 nucleotides were used for replication towards what corresponds in the sequence to the 5' region of the mRNA.
  • the Iabeled amplification product was then separated by molecular weight on a polyacrylamide sequencing gel.
  • mRNAs were chosen that were present in a higher proportion of the RNA in cancerous cells, compared with control cells, according to the proportional intensity amongst neighboring cDNA bands.
  • the cDNA was recovered directly from the gel and amplified to provide a probe for screening.
  • Candidate polynucleotides were screened by a number of criteria, including both Northern and Southern analysis to determine if the corresponding genes were duplicated or responsible for to RNA overabundance in breast cancer cells. Sequence data of the polynucleotides was obtained and compared with sequences in GenBank.
  • Novel polynucleotides with the desired expression patterns were used to probe for longer cDNA inserts in a ⁇ gt10 library constructed from the breast cancer cell line BT474, which were then sequenced. Further description of the actual experimental events that occurred during identification of the four exemplary genes, and sequence data for CH1-9a11-2, CH8-2a13-1, CH13-2a12-1, and CH14- 2a 16-1 are provided in the Example section.
  • Polynucleotides based on the cDNA of CH1-9a11-2, CH8-2a13-1, CH13-2a12-1, CH14- 2a 16-1 can be rescued from cloned plasmids and phage provided as part of this invention. They may also be obtained from breast cancer cell libraries or mRNA preparations, or from normal human tissues such as placenta, by judicious use of primers or probes based on the sequence data provided herein. Altematively, the sequence data provided herein can be used in chemical synthesis to produce a polynucleotide with an identical sequence, or that incorporates occasional variations.
  • Polypeptides encoded by the corresponding mRNA can be prepared by several different methods, all of which will be known to a practitioner of ordinary skill.
  • the appropriate strand of the full-length cDNA can be operably linked to a suitable promoter, and transfected into a suitable host cell. The host cell is then cultured under conditions that allow transcription and translation to occur, and the polypeptide is subsequently recovered.
  • Another convenient method is to determine the polynucleotide sequence of the cDNA, and predict the polypeptide sequence according to the genetic code.
  • a polypeptide can then be prepared directly, for example, by chemical synthesis, either identical to the predicted sequence, or incorporating occasional variations.
  • Antibodies against polypeptides of this invention may be prepared by any method known in the art.
  • the immunogen is injected into a suitable experimental animal: preferably a rodent for the preparation of monoclonal antibodies; preferably a larger animal such as a rabbit or sheep for preparation of polyclonal antibodies. It is preferable to provide a second or booster injection after about 4 weeks, and begin harvesting the antibody source no less than about 1 week later.
  • Sera harvested from the immunized animals provide a source of polyclonal antibodies.
  • Detailed procedures for purifying specific antibody activity from a source material are known within the art. Unwanted activity cross-reacting with other antigens, if present, can be removed, for example, by running the preparation over adsorbants made of those antigens attached to a solid phase, and collecting the unbound fraction.
  • the specific antibody activity can be further purified by such techniques as protein A chromatography, ammonium sulfate precipitation, ion exchange chromatography, high-performance liquid chromatography and immunoaffinity chromatography on a column of the immunizing polypeptide coupled to a solid support.
  • immune cells such as splenocytes can be recovered from the immunized animals and used to prepare a monoclonal antibody-producing cell line.
  • immune cells such as splenocytes can be recovered from the immunized animals and used to prepare a monoclonal antibody-producing cell line.
  • Harrow & Lane (1988) U.S. Patent Nos. 4,491,632 (J.R. Wands et al.), U.S. 4,472,500 (C. Milstein et al.), and U.S. 4,444,887 (M.K. Hoffman et al.)
  • an antibody-producing line can be produced inter alia by cell fusion, or by transfecting antibody-producing cells with Epstein Barr Virus, or transforming with oncogenic DNA.
  • the treated cells are cloned and cultured, and clones are selected that produce antibody of the desired specificity.
  • Specificity testing can be performed on culture supematants by a number of techniques, such as using the immunizing polypeptide as the detecting reagent in a standard immunoassay, or using cells expressing the polypeptide in immunohistochemistry.
  • a supply of monoclonal antibody from the selected clones can be purified from a large volume of tissue culture supernatant, or from the ascites fluid of suitably prepared host animals injected with the clone.
  • Effective variations of this method include those in which the immunization with the polypeptide is performed on isolated ceils.
  • Antibody fragments and other derivatives can be prepared by methods of standard protein chemistry, such as subjecting the antibody to cleavage with a proteolytic enzyme.
  • Genetically engineered variants of the antibody can be produced by obtaining a polynucleotide encoding the antibody, and applying the general methods of molecular biology to introduce mutations and translate the variant.
  • Novel cDNA sequences corresponding to genes associated with cancer are potentially useful as diagnostic aids.
  • polypeptides encoded by such genes, and antibodies specific for these polypeptides, are also potentially useful as diagnostic aids.
  • RNA in particular cells can help identify those cells as being cancerous, and thereby play a part in the initial diagnosis.
  • Increased levels of RNA corresponding to CH1-9a11-2, CH8-2a13-12, CH13-2a12-1, and CH14-2a16-1 are present in a substantial proportion of breast cancer cell lines and primary breast tumors.
  • preliminary Northern analysis using probes for CH8-2a13-12, CH13-2a12-1 , and CH14-2a16-1 indicates that these genes may be duplicated or be associated with RNA overabundance in certain cell lines derived from cancers other than breast cancer, including colon cancer, lung cancer, prostrate cancer, glioma, and ovarian cancer.
  • RNA can assist with clinical management and prognosis.
  • overabundance of RNA may be a useful predictor of disease survival, metastasis, susceptibility to various regimens of standard chemotherapy, the stage of the cancer, or its aggressiveness. See generally the article by Blast, U.S. Patent No. 4,968,603 (Slamon et al.) and PCT Application WO 94/00601 (Levine et al.). All of these determinations are important in helping the clinician choose between the available treatment options.
  • a particularly important diagnostic application contemplated in this invention is the identification of patients suitable for gene-specific therapy, as outlined in the following section.
  • treatment directed against a particular gene or gene product is appropriate in cancers where the gene is duplicated or there is RNA overabundance.
  • a diagnostic test specific for the same gene is important in selecting patients likely to benefit from the pharmaceutical.
  • diagnostic tests for each gene are important in selecting which pharmaceutical is likely to benefit a particular patient.
  • the polynucleotide, polypeptide, and antibodies embodied in this invention provide specific reagents that can be used in standard diagnostic procedures.
  • one of the compositions of this invention is provided as a reagent to detect a target in a clinical sample with which it reacts.
  • the polynucleotide of this invention can be used as a reagent to detect a DNA or RNA target, such as might be present in a cell with duplication or RNA overabundance of the corresponding gene.
  • the polypeptide can be used as a reagent to detect a target for which it has a specific binding site, such as an antibody molecule or (if the polypeptide is a receptor) the corresponding ligand.
  • the antibody can be used as a reagent to detect a target it specifically recognizes, such as the polypeptide used as an immunogen to raise it.
  • the target is supplied by obtaining a suitable tissue sample from an individual for whom the diagnostic parameter is to be measured.
  • Relevant test samples are those obtained from individuals suspected of containing cancerous cells, particulariy breast cancer cells. Many types of samples are suitable for this purpose, including those that are obtained near the suspected tumor site by biopsy or surgical dissection, in vitro cultures of cells derived therefrom, blood, and blood components.
  • the target may be partially purified from the sample or amplified before the assay is conducted.
  • the reaction is performed by contacting the reagent with the sample under conditions that will allow a complex to form between the reagent and the target.
  • the reaction may be performed in solution, or on a solid tissue sample, for example, using histology sections.
  • the formation of the complex is detected by a number of techniques known in the art.
  • the reagent may be supplied with a label and unreacted reagent may be removed from the complex; the amount of remaining label thereby indicating the amount of complex formed. Further details and alternatives for complex detection are provided in the descriptions that follow.
  • the assay result is compared with a similar assay conducted on a control sample. It is generally preferable to use a control sample which is from a non-cancerous source, and otherwise similar in composition to the clinical sample being tested. However, any control sample may be suitable provided the relative amount of target in the control is known or can be used for comparative purposes. Where the assay is being conducted on tissue sections, suitable control cells with normal histopathology may surround the cancerous cells being tested. It is often preferable to conduct the assay on the test sample and the control sample simultaneously. However, if the amount of complex formed is quantifiable and sufficiently consistent, it is acceptable to assay the test sample and control sample on different days or in different laboratories.
  • a polynucleotide embodied in this invention can be used as a reagent for determining gene duplication or RNA overabundance that may be present in a clinical sample.
  • the binding of the reagent polynucleotide to a target in a clinical sample generally relies in part on a hybridization reaction between a region of the polynucleotide reagent, and the DNA or RNA in a sample being tested.
  • the nucleic acid may be extracted from the sample, and may also be partially purified.
  • the preparation is preferably enriched for chromosomal DNA; to measure RNA overabundance, the preparation is preferably enriched for RNA.
  • the target polynucleotide can be optionally subjected to any combination of additional treatments, including digestion with restriction endonucleases, size separation, for example by electrophoresis in agarose or polyacrylamide, and affixed to a reaction matrix, such as a blotting material.
  • Hybridization is allowed to occur by mixing the reagent polynucleotide with a sample suspected of containing a target polynucleotide under appropriate reaction conditions. This may be followed by washing or separation to remove unreacted reagent. Generally, both the target polynucleotide and the reagent must be at least partly equilibrated into the single-stranded form in order for complementary sequences to hybridize efficiently. Thus, it may be useful (particularly in tests for DNA) to prepare the sample by standard denaturation techniques known in the art. The minimum complementarity between the reagent sequence and the target sequence for a complex to form depends on the conditions under which the complex-forming reaction is allowed to occur.
  • Such conditions include temperature, ionic strength, time of incubation, the presence of additional solutes in the reaction mixture such as formamide, and washing procedure.
  • Higher stringency conditions are those under which higher minimum complementarity is required for stable hybridization to occur. It is generally preferable in diagnostic applications to increase the specificity of the reaction, minimizing cross-reactivity of the reagent polynucleotide alternative undesired hybridization sites in the sample. Thus, it is preferable to conduct the reaction under conditions of high stringency: for example, in the presence of high temperature, low salt, formamide, a combination of these, or followed by a low-salt wash. In order to detect the complexes formed between the reagent and the target, the reagent is generally provided with a label.
  • radioisotopes such as 32 P and M P
  • chemiluminescent or fluorescent reagents such as fluorescein
  • enzymes such as alkaline phosphatase that are capable of producing a colored solute or precipitant.
  • the label may be intrinsic to the reagent, it may be attached by direct chemical linkage, or it may be connected through a series of intermediate reactive molecules, such as a biotin-avidin complex, or a series of inter-reactive polynucleotides.
  • the label may be added to the reagent before hybridization with the target polynucleotide, or afterwards.
  • RNA in affected cells This relies on the fact that overabundance of RNA in affected cells is often associated with increased production of the corresponding polypeptide.
  • genes up-regulated in cancer cells encode for cell surface receptors A for example, ert>B-2, c-myc and epidermal growth factor.
  • the RNA may encode a protein kept inside the cell, or it may encode a protein secreted by the cell into the surrounding milieu.
  • any such protein product can be detected in solid tissue samples and cultured cells by immunohistological techniques that will be obvious to a practitioner of ordinary skill.
  • the tissue is preserved by a combination of techniques which may include cooling, exchanging into different solvents, fixing with agents such as paraformaldehyde, or embedding in a commercially available medium such as paraffin or OCT.
  • a section of the sample is suitably prepared and overlaid with a primary antibody specific for the protein.
  • the primary antibody may be provided directly with a suitable label. More frequently, the primary antibody is detected using one of a number of developing reagents which are easily produced or available commercially. Typically, these developing reagents are anti-immunoglobulin or protein A, and they typically bear labels which include, but are not limited to: fluorescent markers such as fluorescein, enzymes such as peroxidase that are capable of precipitating a suitable chemical compound, electron dense markers such as colloidal gold, or radioisotopes such as 125 l. The section is then visualized using an appropriate microscopic technique, and the level of labeling is compared between the suspected cancer cell and a control cell, such as cells surrounding the tumor area or those taken from an alternative site.
  • fluorescent markers such as fluorescein
  • enzymes such as peroxidase that are capable of precipitating a suitable chemical compound
  • electron dense markers such as colloidal gold
  • radioisotopes such as 125 l.
  • the amount of protein corresponding to the cancer-associated gene may be detected in a standard quantitative immunoassay. If the protein is secreted or shed from the cell in any appreciable amount, it may be detectable in plasma or serum samples. Altematively, the target protein may be solubilized or extracted from a solid tissue sample. Before quantitating, the protein may optionally be affixed to a solid phase, such as by a blot technique or using a capture antibody.
  • the protein may be mixed with a pre-determined non-limiting amount of the reagent antibody specific for the protein.
  • the reagent antibody may contain a directly attached label, such as an enzyme or a radioisotope, or a second Iabeled reagent may be added, such as anti-immunoglobuiin or protein A.
  • a solid-phase assay unreacted reagents are removed by washing.
  • unreacted reagents are removed by some other separation technique, such as filtration or chromatography.
  • the amount of label captured in the complex is positively related to the amount of target protein present in the test sample.
  • a variation of this technique is a competitive assay, in which the target protein competes with a Iabeled analog for binding sites on the specific antibody.
  • the amount of label captured is negatively related to the amount of target protein present in a test sample. Results obtained using any such assay on a sample from a suspected cancer-bearing source are compared with those from a non-cancerous source.
  • a polypeptide embodied in this invention can also be used as a reagent in cancer diagnosis, or for determining gene duplication or RNA overabundance that may be present in a clinical sample.
  • Overabundance of RNA in affected cells may result in the corresponding polypeptide being produced by the cells in an abnormal amount.
  • overabundance of RNA may occur concurrently with expression of the polypeptide in an unusual form. This in turn may result in stimulation of the immune response of the host to produce its own antibody molecules that are specific for the polypeptide.
  • a number of human hybridomas have been raised from cancer patients that produce antibodies against their own tumor antigens.
  • an immunoassay is conducted. Suitable methods are generally the same as the immunoassays outlined in the preceding paragraphs, except that the polypeptide is provided as a reagent, and the antibody is the target in the clinical sample which is to be quantified.
  • human IgG antibody molecules present in a serum sample may be captured with solid-phase protein A, and then overlaid with the Iabeled polypeptide reagent. The amount of antibody would then be proportional to the label attached to the solid phase.
  • cells or tissue sections expressing the polypeptide may be overlaid first with the test sample containing the antibody, and then with a detecting reagent such as Iabeled anti-immunoglobulin.
  • the amount of antibody would then be proportional to the label attached to the cells.
  • the amount of antibody detected in the sample from a suspected cancerous source would be compared with the amount detected in a control sample.
  • diagnostic procedures may be performed by diagnostic laboratories, experimental laboratories, practitioners, or private individuals. This invention provides diagnostic kits which can be used in these settings.
  • the presence of cancer cells in the individual may be manifest in a clinical sample obtained from that individual as an alteration in the DNA, RNA, protein, or antibodies contained in the sample.
  • An alteration in one of these components resulting from the presence of cancer may take the form of an increase or decrease of the level of the component, or an alteration in the form of the component, compared with that in a sample from a healthy individual.
  • the clinical sample is optionally pre-treated for enrichment of the target being tested for.
  • the user then applies a reagent contained in the kit in order to detect the changed level or alteration in the diagnostic component.
  • Each kit necessarily comprises the reagent which renders the procedure specific: a reagent polynucleotide, used for detecting target DNA or RNA; a reagent antibody, used for detecting target protein; or a reagent polypeptide, used for detecting target antibody that may be present in a sample to be analyzed.
  • the reagent is supplied in a solid form or liquid buffer that is suitable for inventory storage, and later for exchange or addition into the reaction medium when the test is performed. Suitable packaging is provided.
  • the kit may optionally provide additional components that are useful in the procedure. These optional components include buffers, capture reagents, developing reagents, labels, reacting surfaces, means for detection, control samples, instructions, and interpretive information.
  • Embodied in this invention are modes of treating subjects bearing cancer cells that have overabundance of the particular RNA described.
  • the strategy used to obtain the cDNAs provided in this invention was deliberately focused on genes that achieve RNA overabundance by gene duplication in some cells, and by alternative mechanisms in other cells.
  • These alternative mechanisms may include, for example, translocation or enhancement of transcription enhancing elements near the coding region of the gene, deletion of repressor binding sites, or altered production of gene regulators.
  • Such mechanisms would result in more RNA being transcribed from the same gene.
  • the same amount of RNA may be transcribed, but may persist longer in the cell, resulting in greater abundance. This could occur, for example, by reduction in the level of ribozymes or protein enzymes that degrade RNA, or in the modification of the RNA to render it more resistant to such enzymes or spontaneous degradation.
  • RNA overabundance of these genes is central to the cancer process in the affected cells. Interfering with the specific gene or gene product would consequently modify the cancer process. It is an objective of this invention to provide pharmaceutical compositions that enable therapy of this kind.
  • the general screening strategy is to apply the candidate to a manifestation of a gene associated with cancer, and then determine whether the effect is beneficial and specific.
  • a composition that interferes with a polynucleotide or polypeptide corresponding any of the novel cancer-associated genes described herein has the potential to block the associated pathology when administered to a tumor of the appropriate phenotype. It is not necessary that the mechanism of interference be known; only that the interference be preferential for cancerous cells (or cells near the cancer site) but not other cells.
  • a preferred method of screening is to provide cells in which a polynucleotide related to a cancer gene has been transfected. See, for example, PCT application WO 93/08701.
  • a suitable vector such as a viral vector
  • conveying the vector into the cell such as by electroporation
  • selecting cells that have been transformed such as by using a reporter or drug sensitivity element.
  • a cell line which has a phenotype desirable in testing, and which can be maintained well in culture.
  • the cell line is transfected with a polynucleotide corresponding to one of the cancer-associated genes identified herein. Transfection is performed such that the polynucleotide is operably linked to a genetic controlling element that permits the correct strand of the polynucleotide to be transcribed within the cell.
  • Successful transfection can be determined by the increased abundance of the RNA compared with an untransfected cell. It is not necessary that the cell previously be devoid of the RNA, only that the transfection result in a substantial increase in the level observed.
  • RNA abundance in the cell is measured using the same polynucleotide, according to the hybridization assays outlined earlier.
  • Drug screening is performed by adding each candidate to a sample of transfected cells, and monitoring the effect.
  • the experiment includes a parallel sample which does not receive the candidate drug.
  • the treated and untreated cells are then compared by any suitable phenotypic criteria, including but not limited to microscopic analysis, viability testing, ability to replicate, histological examination, the level of a particular RNA or polypeptide associated with the cells, the level of enzymatic activity expressed by the cells or cell lysates, and the ability of the cells to interact with other cells or compounds. Differences between treated and untreated cells indicates effects attributable to the candidate.
  • the effect of the drug on the cell transfected with the polynucleotide is also compared with the effect on a control cell.
  • Suitable control cells include untransfected cells of similar ancestry, cells transfected with an alternative polynucleotide, or cells transfected with the same polynucleotide in an inoperative fashion.
  • the drug has a greater effect on operably transfected cells than on control cells.
  • Desirable effects of a candidate drug include an effect on any phenotype that was conferred by transfection of the cell line with the polynucleotide from the cancer-associated gene, or an effect that could limit a pathological feature of the gene in a cancerous cell. Examples of the first type would be a drug that limits the overabundance of RNA in the transfected cell, limits production of the encoded protein, or limits the functional effect of the protein.
  • the effect of the drug would be apparent when comparing results between treated and untreated cells.
  • An example of the second type would be a drug that makes use of the transfected gene or a gene product to specifically poison the cell.
  • the effect of the drug would be apparent when comparing results between operably transfected cells and control cells.
  • This invention also provides gene-specific pharmaceuticals in which each of the polynucleotides, polypeptides, and antibodies embodied herein as a specific active ingredient in pharmaceutical compositions.
  • Such compositions may decrease the pathology of cancer cells on their own, or render the cancer cells more susceptible to treatment by the non-specific agents, such as classical chemotherapy or radiation.
  • polynucleotides embodied in this invention can be effectively used in treatment. See, for example, Morgan et al., Culver et al., and U.S. Patent No. 5,399,346 (French et al.).
  • the general principle is to introduce the polynucleotide into a cancer cell in a patient, and allow it to interfere with the expression of the corresponding gene, such as by complexing with the gene itself or with the RNA transcribed from the gene. Entry into the cell is facilitated by suitable techniques known in the art as providing the polynucleotide in the form of a suitable vector, or encapsulation of the polynucleotide in a liposome.
  • the polynucleotide may be provided to the cancer site by an antigen-specific homing mechanism, or by direct injection.
  • a preferred mode of gene therapy is to provide the polynucleotide in such a way that it will replicate inside the cell, enhancing and prolonging the interference effect.
  • the polynucleotide is operably linked to a suitable promoter, such as the natural promoter of the corresponding gene, a heterologous promoter that is intrinsically active in cancer cells, or a heterologous promoter that can be induced by a suitable agent.
  • the construct is designed so that the polynucleotide sequence operably linked to the promoter is complementary to the sequence of the corresponding gene.
  • the transcript of the administered polynucleotide will be complementary to the transcript of the gene, and capable of hybridizing with it.
  • an antibody that blocks the ligand binding site or causes endocytosis of the receptor would decrease the ability of the receptor to provide its signal to the cell. It is unnecessary to have knowledge of the mechanism beforehand; the effectiveness of a particular antibody can be predicted empirically by testing with cultured cancer cells expressing the corresponding protein.
  • Monoclonal antibodies may be more effective in this form of cancer therapy if several different clones directed at different determinants of the same cancer-associate gene product are used in combination: see PCT application WO 94/00136 (Kasprzyk et al.). Such antibody treatment may directly decrease the pathology of the cancer cells, or render them more susceptible to non-specific cytotoxic agents such as platinum (Lippman).
  • the protein product of the cancer-associated gene is expected to appear in high frequency on cancer cells compared to unaffected cells, due to the overabundance of the corresponding RNA.
  • the protein therefore provides a marker for cancer cells that a specific antibody can bind to.
  • An effector component attached to the antibody therefore becomes concentrated near the cancer cells, improving the effect on those cells and decreasing the effect on non-cancer cells. This concentration would generally occur not only near the primary tumor, but also near cancer cells that have metastasized to other tissue sites.
  • the antibody is able to induce endocytosis, this will enhance entry of the effector into the cell interior.
  • an antibody specific for the protein of the cancer-associated gene is conjugated with a suitable effector component, preferably by a covalent or high-affinity bond.
  • suitable effector components in such compositions include radionuclides such as 13, l, toxic chemicals such as vincristine, and toxic peptides such as diphtheria toxin.
  • Other suitable effector components include peptides or polynucleotidescapable of altering the phenotype of the cell in a desirable fashion: for example, installing a tumor suppressergene, or rendering them susceptible to immune attack.
  • polypeptides embodied in this invention can be effectively used in treatment.
  • the growth of cancer cells is naturally limited in part due to immune surveillance. This refers to the recognition of cancer cells by immune recognition units, particularly antibodies and T cells, and the consequent triggering of immune effector functions that limit tumor progression. Stimulation of the immune system using a particular tumor-specific antigen enhances the effect towards the tumor expressing the antigen.
  • an active vaccine comprising a polypeptide encoded by the cDNA of this invention would be appropriately administered to subjects having overabundance of the corresponding RNA.
  • Peptides may be capable of eliciting an immune response on their own, or they may be rendered more immunogenic by chemical manipulation, such as cross-linking or attaching to a protein carrier like KLH.
  • the vaccine also comprises an adjuvant, such as alum, muramyl dipeptides, liposomes, or DETOXTM
  • the vaccine may optionally comprise auxiliary substances such as wetting agents, emulsifying agents, and organic or inorganic salts or acids, It also comprises a pharmaceutically acceptable excipient which is compatible with the active ingredient and appropriate for the route of administration
  • the desired dose for peptide vaccines is generally from 10 ⁇ g to 1 mg, with a broad effective latitude
  • the vaccine is preferably administered first as a priming dose, and then again as a boosting dose, usually at least four weeks later Further boosting doses may be given to enhance the effect
  • the dose and its timing are usually determined by the person responsible for the treatment
  • Certain embodiments of this invention may be practiced by polynucleotide synthesis according to the data provided herein, by rescuing an appropnate insert corresponding to the gene of interest from one of the deposits listed below, or by isolating a corresponding polynucleotide from a suitable tissue source
  • Various useful probes and primers for use in polynucleotide isolation are provided herein, or may be designed from the sequence data
  • Three deposits have been made on May 31 , 1996 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 under terms of the Budapest treaty. The deposits are outlined in Table 2:
  • BCGF1 Mixture of E. coli with recombinant plasmids of cDNA fragments of genes associated with breast cancer.
  • the 8 recombinant plasmids may be separated
  • BCGF2 Mixture of ⁇ gtl 0 recombinant phages with cDNA inserts of genes associated with breast cancer.
  • the 2 phages may be separated by growing in the E. coli
  • plaques can be 97595 distinguished by PCR using ⁇ gt10 reverse and forward primers.
  • the cDNA insert sizes range from about 0.5 to 5 kb. 97594 ⁇ BCBT474 is a source of additional cDNA inserts corresponding to CH1-9a11-2, CH8-2a13-1 , CH13-2a12-1 , or CH14-2a16-1 not present in BCGF-1 or BCGF-2.
  • Sequence databases contain sequences of polynucleotide and polypeptide fragments with varyous degrees of identity and overlap with certain embodiments of this invention.
  • accession numbers is provided for the interest of the reader; it is not intended to be comprehensive or a limitation on the invention.
  • the database disclosures do not typically indicate use in cancer diagnosis, drug development, or disease treatment.
  • GenBank accession numbers are listed in relation to CH1-9a11-2: dbEST N32686; N45113; N36176; N22982; AA278830; H88670; AA235936; AA236951; H26301; N28026; H88063; H88064; D61948; H88718; H26460; AA137920; AA145308; W12952; AA200687; N44164; T27279; dbSTS G22044; G04961.
  • GenBank accession numbers are listed in relation to CH8-2a13-1 : dbNR D83780 The following GenBank accession numbers are listed in relation to CH13-2a12-1 : dbNR
  • Example 1 Selecting cDNA for messenger RNA that is overabundant In breast cancer cells
  • RNA was isolated from each breast cancer cell line or control cell by centrifugation through a gradient of guanidine isothiocyanate/CsCI. The RNA was treated with RNase-free DNase
  • RNA preparations were stored at -70°C. Oligo-dT polynucleotides for priming at the 3 * end of messenger RNA with the sequence
  • T NM (where N e ⁇ A,C,G ⁇ and M e ⁇ A.C.G.T ⁇ ) were synthesized according to standard protocols.
  • RNA was reverse-transcribed using AMV reverse transcriptase (obtained from BRL) and an anchored oligo-dT primer in a volume of 20 ⁇ L, according to the manufacturer's directions.
  • the reaction was incubated at 370C for 60 min and stopped by incubating at 950C for 5 min.
  • the cDNA obtained was used immediately or stored frozen at -70°C.
  • Differential display was conducted according to the following procedure: 1 ⁇ L cDNA was replicated in a total volume of 10 ⁇ L PCR mixture containing the appropriate T ⁇ NM sequence, 0.5 TM of a decamer primer, 200 TM dNTP, 5 TCi [ 3S S]-dATP (Amersham), Taq polymerase buffer with 2.5 mM MgCI 2 and 0.3 unit Taq polymerase (Promega).
  • FIG. 1 provides an example of an autoradiogram from such an experiment
  • Lane 1 is from non-proliferating normal breast cells
  • lane 2 is from proliferating normal breast ceils
  • lanes 3 to 5 are from breast cancer cell lines BT474, SKBR3, and MCF7
  • the left and right side shows the pattern obtained from experiments using the same T ⁇ NM sequence (T ⁇ AC), but two different decamer primers
  • T ⁇ AC T ⁇ NM sequence
  • the arrows indicate the cDNA fragments that were more abundant in all three tumor lines compared with controls
  • RNA derived from uncultured normal mammary epithelial cells are obtained from surgical samples resected from healthy breast tissue, which are then coaxed apart by blunt dissection techniques and mild enzyme treatment Using organoids as the negative control, 33 cDNA fragments were isolated from 15 displays
  • Example 2 Sub-selecting cDNA that corresponds to genes that are duplicated in breast cancer cells
  • BT474, SKBR3 and ZR-75-30 were used to prepare Southern blots to screen the cloned cDNA fragments
  • the cloned cDNA fragments were Iabeled with [32P ⁇ -dCTP, and used individually to probe the blots A larger relative amount of binding of the probe to the lanes corresponding to the cancer cell DNA indicated that the corresponding gene had been duplicated in the cancer cells
  • the Iabeled cDNA probes were also used in Northern blots to verify that the corresponding RNA was overabundant in the appropriate cell lines
  • the fragments were used as probes to screen a cDNA library from breast cancer cell line BT474, constructed in lambda GT10.
  • the longer cDNA obtained from lambda GT10 were sequenced using lambda GT10 primers.
  • the chromosomal locations of the cDNAs were determined using panels of somatic cell hybrids.
  • Example 3 Using the cDNA to test panels of breast cancer cells
  • the four cDNA obtained according to the selection procedures described were used to probe a panel of breast cancer cell lines and primary tumors.
  • the standardized ratio calculated as described underestimates the gene copy number, although it is expected to rank in the same order
  • the standardized ratio obtained for the c-myc gene in the SKBR3 breast cancer cell was 5 0
  • SKBR3 has approximately 50 copies of the c-myc gene
  • RNA from breast cancer cell lines or primary breast cancer tumors were electrophoresed on 0 8% agarose in the presence of the denaturant formamide, and then transferred to a nylon membrane
  • the membrane was probed first with 32P-labeled cDNA corresponding to the putative breast cancer gene, then stripped and reprobed with 32P-labeled cDNA for the beta-actin gene to adjust for differences in sample loading
  • Ratios of densities between the candidate gene and the beta-actin gene were calculated RNA from three different cultured normal epithelial cells were included in the analysis as a control for the normal level of gene expression
  • the highest ratio obtained from the normal cell samples was set at 1 0, and the ratios in the vanous tumor cells were standardized accordingly
  • Example 4 Chromosome 1 gene CH1-9a11-2
  • Table 4 summarizes the results of the analysis for gene duplication and RNA overabundance
  • RNA overabundance is reported relative to the highest level observed for several cultures of normal epithelial cells. Two hybridizing species of RNA are calculated and reported separately.
  • the gene corresponding to the CH1-9a11-2 cDNA was duplicated in 9 out of 15 (60%) of the breast cancer cell lines tested, compared with placental DNA digests (P3 and P12).
  • the sequence of the 115 bases from the 5' end of the cDNA fragment (SEQ. ID NO:1) is shown in Figure 22. There was no substantial homology to any known gene in GenBank. One of the three possible reading frames was found to be open, with the predicted amino acid shown in Figure 22 (SEQ. ID NO:2).
  • the CH1-9a11-2 gene was further characterized by obtaining additional sequence information.
  • a ⁇ -GT10 cDNA library from the breast cancer cell line BT474 (Example 2) was screened using the initial cDNA insert, and a clone with a 2.5 kilobase insert was identified. The identified clone was subcloned into plasmid vector pCRII. T7 and Sp6 primers for regions flanking the cDNA inserts were used as initial sequencing primers:
  • T7 primer (SEQ. ID NO:42)
  • Sequencing continued by walking along the region of interest by standard techniques, using sequencing primers based on data already obtained. Primers used in sequencing are designated 1- 16 in Figure 7. A second clone (designated pCH1-1.1) overlapping on the 5' end was obtained using
  • the sequence of 3452 base pairs between the 5' end of pCH 1 -1.1 and the poly-A tail of CH 1 - 9a11-2 was determined by standard sequencing techniques.
  • the DNA sequence is shown in Figure 8 (SEQ. ID NO: 15).
  • the longest open reading frame is in frame 1 (bases 1-1875), and codes for 624 amino acids before the stop codon.
  • the corresponding amino acid sequence of this frame is shown in the upper panel of Figure 9 (SEQ. ID NO:16).
  • the partial sequence predicted for the translated protein is listed the low panel of Figure 9 (SEQ. ID NO: 17). Bases 1876 to the end of the sequence are believed to be a 3' untranslated region.
  • a hydrophobicity analysis identified a putative membrane insertion or membrane spanning region at about amino acids 382-400, indicated in Figure 9 by underlining.
  • Figure 23 is a listing of additional cDNA sequence obtained for CH1-9a11-2, comprising approximately 1934 base pairs 5' from the sequence of Figure 8.
  • the additional sequence data was obtained by rescuing and amplifying two further fragments of CH1-9a11-2 cDNA.
  • Nested primers were designed -100 base pairs downstream from the 5' end of the known sequence. The primers were used in a nested amplification assay using AP1 and AP2, using the CLONTECH MarathonTM cDNA Amplification Kit as described above.
  • the template for the first upstream fragment was reverse-transcribed polyadenylated RNA from breast cancer cell line 600PE , as described earlier. This fragment was sequenced, and another set of nested primers was designed.
  • the template for the next upstream fragment was a MarathonTM ready cDNA preparation from human testes, also supplied by CLONTECH.
  • the nucleotide sequence shown in Figure 23 comprises an open reading frame through to the 5' end.
  • Figure 24 shows the corresponding protein translation. Between about another 500-1000 bases are predicted to be present in the CH1-9a11-2 direction, with the protein encoding sequence beginning somewhere within this additional sequence. Sequencing of the encoding region is completed by obtaining additional CH1-9a11-2 fragments in this direction.
  • a CH1-9a11-2 cloned insert has been used to probe the level of relative expression in polyadenylated RNA from a panel of tissue sources.
  • the RNA was obtained already prepared for Northern blot analysis (CLONTECH Catalog # 7759-1, 7760-1 and 7756-1.) The manufacturer produced the blots from approximately 2 ⁇ g of poly-A RNA per lane, run on a denaturing formaldehyde 1-2% agarose gel, transferred to a nylon membrane, and fixed by UV irradiation.
  • the relative CH1-9a11-2 expression observed at the RNA level is shown in Table 5:
  • the outermost primer is used to synthesize a first cDNA strand complementary to the mRNA in the upstream direction.
  • Second strand synthesis is performed using reagents in a CLONTECH MarathonTM cDNA amplification kit according to manufacturer's directions.
  • the double-stranded DNA is then ligated at the 5' end of the coding sequence with the double-stranded adaptor fragment provided in the kit.
  • a first PCR amplification (about 30 cycles) is performed using the first adapter primer from the kit and the outermost RNA-specific primer, and a second amplification (about 30 cycles) is performed using the second adapter primer and the innermost RNA-specific primer.
  • a CLONTECH RACE-READY single-stranded cDNA from human placenta is PCR amplified using nested 5' anchor primers in combination with the outermost and innermost RNA- specific primers.
  • Amplified DNA obtained using either approach is analyzed by gel electrophoresis, and cloned into plasmid vector pCRII. Clones are screened, as necessary, using the 2.5 kilobase CH1-9a11-2 insert. Clones corresponding to full-length mRNA (4.5 kb or 5.5 kb; Table 1), or cDNA fragments overlapping at the 5' end are selected for sequencing.
  • additional polynucleotide segments may be present in the 5.5 kb form within the encoding region, or in the 5' or 3' untranslated region.
  • FIG. 1 shows the Southern blot analysis for the corresponding gene in various DNA digests.
  • Lane 1 (P12) is the control preparation of placental DNA; the rest show DNA obtained from human breast cancer cell lines.
  • Panel A shows the pattern obtained using the 32P-labeled CH8-2a13-1 cDNA probe.
  • Panel B shows the pattern obtained with the same blot using the 32P-labeled D2S6 probe as a loading control. The sizes of the restriction fragments are indicated on the right.
  • Figure 3 shows the Northern blot analysis for RNA overabundance. Lanes 1-3 show the level of expression in cultured normal epithelial cells. Lanes 4-19 show the level of expression in human breast cancer cell lines. Panel A shows the pattern obtained using the CH8-2a13-1 probe; panel B shows the pattern obtained with beta-actin cDNA, a loading control.
  • the gene corresponding to CH8-2a13-1 showed clear evidence of duplication in 12 out of 17 (71%) of the cells tested. RNA overabundance was observed in 14 out of 17 (82%). Thus, 11% of the cells had achieved RNA overabundance by a mechanism other than gene duplication. Since the known oncogene c-myc is located on Chromosome 8, the Southern analysis was also conducted using a probe for c-myc. At least 2 of the breast cancer cells showing duplication of the gene corresponding to CH8-2a13-1 gene did not show duplication of c-myc. This indicates that the gene corresponding to CH8-2a13-1 is not part of the myc amplicon.
  • the CH8-2a13-1 gene was further characterized by obtaining additional sequence information.
  • a ⁇ -GT10 cDNA library from the breast cancer cell line BT474 (Example 2) was screened using the initial cDNA insert, and clones with a 3.0 kb and a 4.0 kb insert were identified. The two identified clones were subcloned into plasmid vector pCRII. T7 and Sp6 primers for regions flanking the cDNA inserts were used as initial sequencing primers. Sequencing continued by walking along the region of interest by standard techniques, using sequencing primers based on data already obtained. The two inserts were found to overlap (Figure 6). Primers used are those designated 1-25 in Figure 10.
  • a third clone of about 600 bp (designated pCH8-600) overlapping on the 5' end ( Figure 6) was obtained using CLONTECH MarathonTM cDNA Amplification Kit. Briefly, two DNA primers CH8a and CH8b ( Figure 10) were synthesized. Polyadenylated RNA from breast cancer cell line BT474 was reverse transcribed using CH8b primer. After second strand synthesis, adaptor DNA provided in the kit was ligated to the double-stranded cDNA. The 5' end cDNA of CH8-2a13-1 was then amplified by PCR using primers CH8a and AP1 (provided in the kit).
  • the first PCR products were PCR reamplified using nested primers CH8a and AP2 (provided in the kit).
  • the PCR products were cloned into pCRII vector (Invitrogen) and screened with CH8-2a13-1 probe.
  • pCRII vector Invitrogen
  • bases 1-152 are believed to be a 5' untranslated region.
  • the longest open reading frame is in frame 3 from base 153 to 3911, and codes for 1252 amino acids before the stop codon.
  • the corresponding amino acid sequence of this frame is shown in the upper panel of Figure 12 (SEQ. ID NO:19).
  • the sequence predicted for the translated protein is shown in the lower panel of Figure 12(SEQ. ID NO:20).
  • KIAA0196 was one of 200 different cDNA cloned at random from an immature male human myeloblast cell line. KIAA0196 has no known biological function, and is described by Nomura et al. as being ubiquitously expressed.
  • a fourth clone of about 600 bp overlapping pCH8-600 at the 5' end has also been obtained.
  • a DNA primer was synthesized corresponding to about the first 20 nucleotides at the 5' of the predicted cDNA sequence, and used along with a primer based on the pCH8-600 sequence to reverse-transcribe RNA from breast cancer cell line BT474.
  • the product was cloned into pCRII vector (Invitrogen) and screened with a CH8-2a13-1 probe. The new clone is sequenced along both strands to obtain additional 5' untranslated sequence data for the cDNA.
  • the predicted compiled cDNA nucleotide sequence of CH8-2a13-1 cDNA is shown in Figure 13 (SEQ. ID NO:21).
  • the corresponding amino acid sequence of this frame is shown in Figure 14 (SEQ. ID NO:22).
  • a polynucleotide comprising the compiled sequence is assembled by joining the insert of this fourth clone to pCH8-4k within the shared region. Briefly, CH8-4k is cut with Xbal and ⁇ fofl. The fourth clone is cut with SamHI and Xbal. The ligated polynucleotide is then inserted into pCRII cut with BamHI and ⁇ fofl.
  • a CH8-2a13-1 cloned insert has been used to probe the level of relative expression in polyadenylated RNA from a panel of tissue sources obtained from CLONTECH, as in Example 4.
  • the relative CH8-2a13-12 expression observed at the mRNA level is shown in Table 7:
  • Relative levels of expression observed were as follows: Low levels of expression were observed in adult peripheral blood leukocytes (PBL), brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. Medium levels of expression were observed in adult heart, spleen, thymus, prostate, testis, ovary, small intestine, and colon. High levels of expression were observed in four fetal tissues tested: brain, lung, liver and kidney. The level of expression in breast cancer cell lines is relatively high (about ++++ on the scale), since the Northern analysis performed on these lines was conducted on total cellular RNA. It is likely that the CH8-2a13-1 gene is involved in a biological process that is typical to the tissue types showing medium to high levels of expression, which may relate to increased tissue growth or metabolism.
  • Figure 4 shows the Southern blot analysis for the corresponding gene in various DNA digests. Lanes 1 and 2 are control preparations of placental DNA; the rest show DNA obtained from human breast cancer cell lines. Panel A shows the pattern obtained using the CH13-2a12-1 cDNA probe; panel B shows the pattern using D2S6 probe as a loading control. The sizes of the restriction fragments are indicated on the right.
  • Figure 5 shows the Northern blot analysis for RNA overabundance of the CH13-2a12-1 gene.
  • Lanes 1-3 show the level of expression in cultured normal epithelial cells.
  • Lanes 4-19 show the level of expression in human breast cancer cell lines.
  • Panel A shows the pattern obtained using the CH13-2a12-1 probe;
  • panel B shows the pattern obtained with beta-actin cDNA, a loading control.
  • the apparent size of the mRNA varied depending upon conditions of electrophoresis. Full-length mRNA is believed to occur at sizes of about 3.2 and 3.5 kb.
  • Table 8 The results of the RNA abundance comparison are summarized in Table 8. The scoring method is the same as for Example 4.
  • the gene corresponding to CH13-2a12-1 was duplicated in 7 out of 16 (44%) of the cells tested.
  • Three of the positive cell lines (600PE, BT474, and MDA435) had been studied previously by comparative genomic hybridization, but had not shown amplified chromatin in the region where CH13- 2A12-1 has been mapped in these studies.
  • RNA overabundance was observed in 13 out of 16 (81%) of the cell lines tested. Thus, 37% of the cells had achieved RNA overabundance by a mechanism other than gene duplication.
  • Cells from primary breast tumors have also been analyzed them for duplication of the chromosome 13 gene. Ten of the 82 tumors analyzed (12%) were positive, confirming that duplication of this gene is not an artifact of in vitro culture.
  • the CH13-2a12-1 gene was further characterized by obtaining additional sequence information.
  • a ⁇ -GT10 cDNA library from the breast cancer cell line BT474 (Example 2) was screened using the initial cDNA insert, and clones with a 3.5 kilobase and a 1.6 kilobase insert were identified. The two identified clones were subcloned into plasmid vector pCRII. T7 and Sp6 primers for regions flanking the cDNA inserts were used as initial sequencing primers. Sequencing continued by walking along the region of interest by standard techniques, using sequencing primers based on data already obtained. The two inserts were found to overlap (Figure 6). Primers used during sequencing are shown in Figure 15.
  • nucleic acid sequence of 3339 base pairs between the 5' end and the poly-A tail of CH13-2a12-1 was determined.
  • the DNA sequence is shown in Figure 16 (SEQ. ID NO:23).
  • Bases 1-520 are believed to be a 5' untranslated region.
  • the longest open reading frame is in frame 2 from base 521 to 1838, and codes for 611 amino acids before the stop codon.
  • the corresponding amino acid sequence of this frame is shown in the upper panel of Figure 17 (SEQ. ID NO:24).
  • sequence predicted for the translated protein is shown in the lower panel of Figure 17 (SEQ. ID NO:25).
  • Bases 1838 to 3339 of the nucleotide sequence are believed to be a 3' untranslated region, which is present in the 3.5 kb insert.
  • the 3.5 kb insert appears to be a splice variant ( Figure 6), in which the 3' untranslated region consists of bases 1838-2797 in the sequence.
  • Controlling cell proliferation in this context means that an abnormally high or low level of gene expression at the RNA or protein level results in a higher or lower rate of cell proliferation, or vice versa, compared with cells with an otherwise similar phenotype.
  • CH13-2a12-1 a vasopressin-activated, calcium-mobilizing receptor from rabbit kidney medulla (Burnatowska-Hledin et al).
  • VACM-1 has a transmembrane sequence, whereas none has been detected in CH13-2a12-1. Nevertheless, it is possible that the CH13-2a12-1 protein product has a Ca ++ binding or Ca ++ mobilizing function.
  • a CH13-2a12-1 cloned insert has been used to probe the level of relative expression in polyadenylated RNA from a panel of tissue sources obtained from CLONTECH, as in Example 4.
  • the relative CH13-2a12-1 expression observed at the mRNA level is shown in Table 9:
  • the gene corresponding to CH14-2a16-1 was duplicated in 8 out of 15 (53%) of the cells tested.
  • the sequence of 114 bases from the 5' end of the cDNA fragment is shown in Figure 22 (SEQ ID NO:7). There was no substantial homology to any known gene in GenBank. One of the three possible reading frames was found to be open, with the predicted amino acid sequence shown in Figure 22 (SEQ ID NO:8).
  • the CH14-2a16-1 gene was further characterized by obtaining additional sequence information.
  • a ⁇ -GT10 cDNA library from the breast cancer cell line BT474 (Example 2) was screened using the initial cDNA insert, and two clones were identified: one with a 1.6 kb insert, and the other with a 2.5 kb insert.
  • the identified clones were subcloned into plasmid vector pCRII.
  • the 1.6 kb insert was sequenced by using T7 and Sp6 primers for regions flanking the cDNA inserts as initial sequencing primers. Sequencing continued by walking along the region of interest by standard techniques, using sequencing primers based on data already obtained. Primers used are those designated 1-11 in Figure 18.
  • the first PCR products were PCR reamplified using nested primers CH14a (or CH14d) and AP2 (provided in the kit).
  • the PCR products were cloned into pCRII vector (Invitrogen) and screened with CH14-2a16-1 probe.
  • NAB2 Saccharomyces cerevisiae with the designation NAB2.
  • NAB2 is one of the major proteins associated with nuclear polyadenylated RNA in yeast cells, as detected by UV light-induced cross-linking and immunofluorescence. NAB2 is strongly and specifically associated with nuclear poly(A)+ RNA in vivo.
  • a fourth clone (pCH14-1.3) has been obtained that overlaps the pCH 14-800 clone at the 5' end ( Figure 6).
  • the method of isolation was similar to that for pCH 14-800, using primers based on the pCH14-800 sequence.
  • Partial sequence data for pCH14-1.3 has been obtained by one- directional sequencing from the 5' and 3' ends of the pCH14-1.3 clone.
  • Figure 21 shows the nucleotide sequence of the sequence of the 5' end (SEQ. ID NO:29) and the amino acid translation of the likely open reading frame (SEQ. ID NO:30); the nucleotide sequence of the 3' end (SEQ. ID NO:31) and the likely open reading frame (SEQ.
  • the additional sequence data was obtained by rescuing and amplifying further fragments of CH14-2a16-1 cDNA.
  • Nested primers were designed -100 base pairs downstream from the 5' end of the known sequence. The primers were used in a nested amplification assay using AP1 and AP2, using the CLONTECH MarathonTM cDNA Amplification Kit as described above. The template was a MarathonTM ready cDNA preparation from human testes, also supplied by CLONTECH.
  • the nucleotide sequence shown in Figure 25 is closed at the the 5' end.
  • the lower panel of Figure 26 shows what is predicted to be the sequence of the gene product, beginning at the first methionine residue.
  • the nucleotide sequence shown contains a point difference at the position indicated by the underlining in Figure 25.
  • a base determined to be A from the previously obtained polynucleotide fragment was a G in the one used in this part of the experiment. This corresponds to a change from E (glutamic acid) to G (glycine) in the protein sequence, at the position underlined in Figure 26. This may represent a natural allelic variation.
  • a CH14-2a16-1 cloned insert has been used to probe the level of relative expression in polyadenylated RNA from a panel of tissue sources obtained from CLONTECH, as in Example 4.
  • the relative CH14-2a16-1 expression observed at the mRNA level is shown in Table 11 :
  • CH14-2a16-1 mRNA was particulariy high in testis.
  • the level of expression in breast cancer cell lines is also quite high, since the Northem analysis performed on these lines was conducted on total cellular RNA. It is likely that the CH14-2a16-1 gene is involved in a biological process that is typical to the tissue types showing medium to high levels of expression, which may relate to increased tissue growth or metabolism.
  • Zinc finger motifs Five motifs corresponding to a zinc finger protein have been found in the CH14-2a16-1 nucleotide sequence. Further zinc finger motifs may be present in CH14-2a16-1 in the upstream direction. Zinc finger motifs are present, for example, in RNA polymerases I, II, and ill from S. cerevisiae, and are related to the zinc knuckle family of RNA/ssDNA-binding proteins found in the HIV nucleocapsid protein. The actual sequence observed in each of the five zinc finger motifs of CH14-2a16-1 is:
  • cDNA fragments corresponding to additional cancer-associated genes are obtained by applying the techniques of Examples 1 & 2 with appropriate adaptations.
  • cancer cells are selected for use in differential display of RNA, based on whether they share a duplicated chromosomal region according to Table 12:
  • Control RNA is prepared from normal tissues to match that of the cancer cells in the experiment. Normal tissue is obtained from autopsy, biopsy, or surgical resection. Absence of neoplastic cells in the control tissue is confirmed, if necessary, by standard histological techniques. cDNA corresponding to RNA that is overabundant in cancer cells and duplicated in a proportion of the same cells is characterized further, as in Examples 3-7. Additional cDNA comprising an entire protein-product encoding region is rescued or selected according to standard molecular biology techniques as described elsewhere in this disclosure.
  • SEQ ID NO:11 CAATCGCCGT 10
  • SEQ ID NO: 12 TCGGCGATAG 10
  • SEQ ID NO: 13 CAGCACCCAC 10
  • SEQ ID NO: 14 AGCCAGCGAA 10

Abstract

L'invention concerne des nouveaux procédés pour détecter des gènes associés au cancer et produire des séquences d'ADNc correspondantes. Lesdits procédés consistent à produire des préparations d'ARN à partir de cellules de régulation et de plusieurs cellules cancéreuses différentes partageant un gène dupliqué ou modifié dans la même région d'un chromosome. Des copies d'ADNc amplifié sont visualisées puis sélectionnées en fonction des différences d'abondance d'ARN entre les préparations. Les étapes de criblages supplémentaires optionnelles consistent à rechercher dans des séries de cellules cancéreuses au moyen de l'ADNc la surabondance d'ARN avec ou sans duplication génique. Les gènes identifiés peuvent être utilisés à leur tour pour mettre au point des matériaux et des techniques de traitement et de diagnostic du cancer sous-jacent. On a identifié quatre nouveaux gènes associés au cancer. Dans environ 60 % des lignées de cellules du cancer du sein testées, l'ARN s'hybridant avec les ADN était sensiblement plus abondant que dans des cellules normales. La plupart des lignées cellulaires présentaient également une duplication du gène correspondant, ce qui semble avoir contribué au fait que le niveau d'ARN dans la cellule est accru. Néanmoins, pour chacun des quatre gènes, certaines cellules présentaient une surabondance sans duplication génique, ce qui semble indiquer que le produit génique est suffisamment important dans le processus cancéreux pour que les cellules utilisent plusieurs mécanismes différents pour amplifier leur expression.
PCT/US1997/005930 1996-04-10 1997-04-09 Genes amplifies dans des cellules cancereuses WO1997038085A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP97920293A EP0944651A2 (fr) 1996-04-10 1997-04-09 Genes amplifies dans des cellules cancereuses
JP9536486A JP2000509256A (ja) 1996-04-10 1997-04-09 ガン細胞において増幅される遺伝子
AU24521/97A AU2452197A (en) 1996-04-10 1997-04-09 Genes amplified in cancer cells

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US1516796P 1996-04-10 1996-04-10
USPCT/US96/09286 1996-06-05
PCT/US1996/009286 WO1996039516A2 (fr) 1995-06-05 1996-06-05 Cibles pour le diagnostic et le traitement du cancer du sein
US1920296P 1996-06-06 1996-06-06
US60/019,202 1996-07-11
US08/678,280 1996-07-11
US08/678,280 US5776683A (en) 1996-07-11 1996-07-11 Methods for identifying genes amplified in cancer cells
US60/015,167 1996-07-11

Publications (2)

Publication Number Publication Date
WO1997038085A2 true WO1997038085A2 (fr) 1997-10-16
WO1997038085A3 WO1997038085A3 (fr) 1998-02-19

Family

ID=27486464

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/005930 WO1997038085A2 (fr) 1996-04-10 1997-04-09 Genes amplifies dans des cellules cancereuses

Country Status (4)

Country Link
EP (1) EP0944651A2 (fr)
JP (1) JP2000509256A (fr)
AU (1) AU2452197A (fr)
WO (1) WO1997038085A2 (fr)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000036420A2 (fr) * 1998-12-11 2000-06-22 Ludwig Institute For Cancer Research Expression differentielle dans le cas du cancer primaire du sein
US7101984B2 (en) 1999-02-12 2006-09-05 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7119173B2 (en) 2001-12-06 2006-10-10 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7118887B2 (en) 1999-03-08 2006-10-10 Genentech, Inc. Nucleic acid overexpressed in esophageal tumor, normal stomach and melanoma
US7160985B2 (en) 1997-10-29 2007-01-09 Genentech, Inc. Pro180 polypeptide
US7208321B2 (en) 1998-06-02 2007-04-24 Genentech Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7244817B2 (en) 1998-09-10 2007-07-17 Genentech, Inc. Pro1357 polypeptide
US7244428B2 (en) 1998-09-10 2007-07-17 Genentech, Inc. PRO1357 antibodies
US7288384B2 (en) 1999-12-09 2007-10-30 Genentech, Inc. Antibodies to polypeptides encoded by a nucleic acid underexpressed in esophageal tumor
US7300758B2 (en) 1999-12-09 2007-11-27 Genetech, Inc. Nucleic acid underexpressed in esophageal tumor
US7319008B2 (en) 1998-06-02 2008-01-15 Genentech, Inc. Nucleic acid underexpressed in melanoma
US7351543B2 (en) 1998-06-02 2008-04-01 Genentech, Inc. Antibodies to a polypeptide encoded by a nucleic acid underexpressed in melanoma
US7399828B2 (en) 1998-09-24 2008-07-15 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7402661B2 (en) 1998-10-06 2008-07-22 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7423127B2 (en) 1999-12-09 2008-09-09 Genentech, Inc. Antibodies to a polypeptide encoded by a nucleic acid overexpressed in melanoma
US7507404B2 (en) 1999-03-08 2009-03-24 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7989167B2 (en) * 2006-11-13 2011-08-02 Val-Chum L.P. Method of prognosing and diagnosing hereditary spastic paraplegia, mutant nucleic acid molecules and polypeptides

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4968603A (en) * 1986-12-31 1990-11-06 The Regents Of The University Of California Determination of status in neoplastic disease
WO1991002062A2 (fr) * 1989-08-04 1991-02-21 Triton Biosciences, Inc. Domaine externe du c-erbb-2: gp75
WO1992021771A1 (fr) * 1991-06-03 1992-12-10 Triton Diagnostics, Inc. Ectodomaine de recepteur du facteur de croissance epidermique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4968603A (en) * 1986-12-31 1990-11-06 The Regents Of The University Of California Determination of status in neoplastic disease
WO1991002062A2 (fr) * 1989-08-04 1991-02-21 Triton Biosciences, Inc. Domaine externe du c-erbb-2: gp75
WO1992021771A1 (fr) * 1991-06-03 1992-12-10 Triton Diagnostics, Inc. Ectodomaine de recepteur du facteur de croissance epidermique

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
CANCER RES, SEP 1 1994, 54 (17) P4598-602, UNITED STATES, XP002019977 WATSON MA ET AL: "Isolation of differentially expressed sequence tags from human breast cancer." *
CANCER RES;52(4):1036-9 1992, XP002037748 BERNS EM ET AL: "SPORADIC AMPLIFICATION OF THE INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR GENE IN HUMAN BREAST TUMORS" *
CLINICAL CANCER RESEARCH, 1995, 1, 123-127, XP000609027 BIECHE I ET AL: "LOSS AND GAIN OF DISTINCT REGIONS OF CHROMOSOME 1Q IN PRIMARY BREAST-CANCER" *
DNA RES, FEB 29 1996, 3 (1) P17-24, JAPAN, XP002037754 NAGASE T ET AL: "Prediction of the coding sequences of unidentified human genes. V. The coding sequences of 40 new genes (KIAA0161-KIAA0200) deduced by analysis of cDNA clones from human cell line KG-1." *
EMBL database ion number D83780 16-03- Nomura n. Human mRNA for KIA XP002037749 *
EMBL database ion number H15342 Hillie t al. The Washu-Me T project 03-07-95 XP002037750 *
MOL MED, JAN 1995, 1 (2) P153-60, UNITED STATES, XP000607858 CHEN Z ET AL: "Differential expression of human tissue factor in normal mammary epithelial cells and in carcinomas." *
ONCOGENE, APR 1991, 6 (4) P659-63, ENGLAND, XP000607852 ADNANE J ET AL: "BEK and FLG, two receptors to members of the FGF family, are amplified in subsets of human breast cancers." *
SCIENCE, MAY 12 1989, 244 (4905) P707-12, UNITED STATES, XP002019974 SLAMON DJ ET AL: "Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer." *
SCIENCE, vol. 257, no. 5072, 14 August 1992, pages 967-971, XP000508268 LIANG P ET AL: "DIFFERENTIAL DISPLAY OF EUKARYOTIC MESSENGER RNA BY MEANS OF THE POLYMERASE CHAIN REACTION" *

Cited By (86)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7160985B2 (en) 1997-10-29 2007-01-09 Genentech, Inc. Pro180 polypeptide
US7351543B2 (en) 1998-06-02 2008-04-01 Genentech, Inc. Antibodies to a polypeptide encoded by a nucleic acid underexpressed in melanoma
US7319008B2 (en) 1998-06-02 2008-01-15 Genentech, Inc. Nucleic acid underexpressed in melanoma
US7208321B2 (en) 1998-06-02 2007-04-24 Genentech Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7244428B2 (en) 1998-09-10 2007-07-17 Genentech, Inc. PRO1357 antibodies
US7244817B2 (en) 1998-09-10 2007-07-17 Genentech, Inc. Pro1357 polypeptide
US7399828B2 (en) 1998-09-24 2008-07-15 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7402661B2 (en) 1998-10-06 2008-07-22 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7405269B2 (en) 1998-10-08 2008-07-29 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7196167B2 (en) 1998-10-20 2007-03-27 Genentech, Inc. Polypeptide underexpressed in lung tumor
US7189821B2 (en) 1998-10-20 2007-03-13 Genentech, Inc. Antibodies to a polypeptide encoded by a nucleic acid underexpressed in lung tumor
US7193046B2 (en) 1998-10-20 2007-03-20 Genentech, Inc. Polypeptide encoded by a nucleic acid overexpressed in esophageal and lung tumor, and underexpressesd in kidney tumor and melanoma
US7193062B2 (en) 1998-10-20 2007-03-20 Genentech, Inc. Antibodies to a polypeptide encoded by a nucleic acid over expressed in esoprageal and lung tumor, and under expressed in kidney tumor and melanoma
US7220841B2 (en) 1998-10-27 2007-05-22 Genentech, Inc. Antibody to PRO1557 polypeptide encoded by a nucleic acid highly expressed in esophageal and kidney tumors
US7196174B2 (en) 1998-10-27 2007-03-27 Genentech, Inc. PRO1557 polypeptide encoded by a nucleic acid highly expressed in esophageal and kidney tumors
WO2000036420A2 (fr) * 1998-12-11 2000-06-22 Ludwig Institute For Cancer Research Expression differentielle dans le cas du cancer primaire du sein
WO2000036420A3 (fr) * 1998-12-11 2000-10-19 Ludwig Inst Cancer Res Expression differentielle dans le cas du cancer primaire du sein
US7101984B2 (en) 1999-02-12 2006-09-05 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7230077B2 (en) 1999-03-08 2007-06-12 Genentech, Inc. Polypeptide encoded by a nucleic acid underexpressed in melanoma
US7253255B2 (en) 1999-03-08 2007-08-07 Genentech, Inc. Polypeptide encoded by a nucleic acid underexpressed in esophageal tumor and melanoma
US7193074B2 (en) 1999-03-08 2007-03-20 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7507404B2 (en) 1999-03-08 2009-03-24 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7468424B2 (en) 1999-03-08 2008-12-23 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7465785B2 (en) 1999-03-08 2008-12-16 Genentech, Inc. Polypeptide encoded by a nucleic acid over-expressed in melanoma
US7193063B2 (en) 1999-03-08 2007-03-20 Genentech, Inc. Antibodies to a polypeptide encoded by a nucleic acid overexpressed in esophageal tumor, normal stomach and melanoma
US7193059B2 (en) 1999-03-08 2007-03-20 Genentech Inc. Antibodies that recognize a polypeptide overexpressed in rectal tumors
US7435798B2 (en) 1999-03-08 2008-10-14 Genentech, Inc. Antibodies to a polypeptide encoded by a nucleic acid overexpressed in normal stomach, normal skin and kidney tumor
US7202337B2 (en) 1999-03-08 2007-04-10 Genentech, Inc. Polypeptide encoded by a nucleic acid overexpressed in esophageal tumor, normal stomach and melanoma
US7423130B2 (en) 1999-03-08 2008-09-09 Genentech, Inc. PRO1158 polypeptides
US7202336B2 (en) 1999-03-08 2007-04-10 Genentech, Inc. Polypeptide encoded by a nucleic acid overexpressed in kidney tumor and underexpressed in lung tumor
US7205389B2 (en) 1999-03-08 2007-04-17 Genentech Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7205391B2 (en) 1999-03-08 2007-04-17 Genentech, Inc. Antibodies to a polypeptide suppressed in stomach tumors
US7193061B2 (en) 1999-03-08 2007-03-20 Genentech, Inc. Antibodies to a polypeptide encoded by a nucleic acid underexpressed in esophageal tumor and melanoma
US7214777B2 (en) 1999-03-08 2007-05-08 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7217787B2 (en) 1999-03-08 2007-05-15 Genentech, Inc. Polypeptide encoded by a nucleic acid underexpressed in rectal tumor
US7220850B2 (en) 1999-03-08 2007-05-22 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7220830B2 (en) 1999-03-08 2007-05-22 Genentech, Inc. Polypeptide underexpressed in stomach tumors
US7193060B2 (en) 1999-03-08 2007-03-20 Genentech, Inc. Antibodies to a polypeptides encoded by a nucleic acid overexpressed in kidney tumor and underexpressed in lung tumor
US7223841B2 (en) 1999-03-08 2007-05-29 Genentech, Inc. PR0994 antibodies
US7223838B2 (en) 1999-03-08 2007-05-29 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7227000B2 (en) 1999-03-08 2007-06-05 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7230076B2 (en) 1999-03-08 2007-06-12 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7189803B2 (en) 1999-03-08 2007-03-13 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7230082B2 (en) 1999-03-08 2007-06-12 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7232889B2 (en) 1999-03-08 2007-06-19 Genentech, Inc. PRO300 antibodies
US7232882B2 (en) 1999-03-08 2007-06-19 Genentech, Inc. Polypeptide uder-expressed in melanoma
US7235630B2 (en) 1999-03-08 2007-06-26 Genentech, Inc. PRO994 polypeptides
US7238787B2 (en) 1999-03-08 2007-07-03 Genentech, Inc. Antibodies to a polypeptide encoded by a nucleic acid underexpressed in melanoma
US7423119B2 (en) 1999-03-08 2008-09-09 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7189564B2 (en) 1999-03-08 2007-03-13 Genentech, Inc. PRO1335 nucleic acids
US7189805B2 (en) 1999-03-08 2007-03-13 Genetech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7193047B2 (en) 1999-03-08 2007-03-20 Genentech, Inc. Polypeptide encoded by a nucleic acid underexpressed in esophageal tumor
US7253256B2 (en) 1999-03-08 2007-08-07 Genentech, Inc. Polypeptides encoded by a nucleic acid are expressed in esophageal and kidney tumor
US7256261B2 (en) 1999-03-08 2007-08-14 Genentech, Inc. Polypeptides encoded by a nucleic acid over expressed in normal stomach normal skin and kidney tumor
US7259238B2 (en) 1999-03-08 2007-08-21 Genentech, Inc. Polypeptide encoded by a nucleic acid under-expressed in stomach and lung tumor
US7276586B2 (en) 1999-03-08 2007-10-02 Genentech, Inc. Antibodies to a polypeptide encoded by a nucleic acid under-expressed in stomach and lung tumor
US7285624B2 (en) 1999-03-08 2007-10-23 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7417125B2 (en) 1999-03-08 2008-08-26 Genentech, Inc. Antibodies that bind a PRO1158 polypeptide
US7294690B2 (en) 1999-03-08 2007-11-13 Genentech, Inc. Polypeptides encoded by a nucleic acid differentially expressed in lung tumor
US7411037B2 (en) 1999-03-08 2008-08-12 Genentech, Inc. Polypeptides encoded by a nucleic acid underexpressed in melanoma
US7189532B2 (en) 1999-03-08 2007-03-13 Genentech, Inc. Nucleic acid underexpressed in stomach tumor and lung tumor
US7345145B2 (en) 1999-03-08 2008-03-18 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7189804B2 (en) 1999-03-08 2007-03-13 Genentech Inc. PRO1335 polypeptides
US7354997B2 (en) 1999-03-08 2008-04-08 Genentech, Inc. Polypeptide encoded by a nucleic acid underexpressed in stomach tumor
US7358339B2 (en) 1999-03-08 2008-04-15 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7371814B2 (en) 1999-03-08 2008-05-13 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7378491B2 (en) 1999-03-08 2008-05-27 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7390876B2 (en) 1999-03-08 2008-06-24 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7393931B2 (en) 1999-03-08 2008-07-01 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7189822B2 (en) 1999-03-08 2007-03-13 Genentech, Inc. Antibodies to a polypeptide encoded by a nucleic acid underexpressed in stomach and lung tumor
US7186801B2 (en) 1999-03-08 2007-03-06 Genentech, Inc. Polypeptide encoded by a nucleic acid underexpressed in stomach tumor and lung tumor
US7118887B2 (en) 1999-03-08 2006-10-10 Genentech, Inc. Nucleic acid overexpressed in esophageal tumor, normal stomach and melanoma
US7300758B2 (en) 1999-12-09 2007-11-27 Genetech, Inc. Nucleic acid underexpressed in esophageal tumor
US7288384B2 (en) 1999-12-09 2007-10-30 Genentech, Inc. Antibodies to polypeptides encoded by a nucleic acid underexpressed in esophageal tumor
US7423127B2 (en) 1999-12-09 2008-09-09 Genentech, Inc. Antibodies to a polypeptide encoded by a nucleic acid overexpressed in melanoma
US7405268B2 (en) 2000-08-24 2008-07-29 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7241872B2 (en) 2000-08-24 2007-07-10 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7442765B2 (en) 2000-08-24 2008-10-28 Genentech, Inc. Secreted transmembrane polypeptides and nucleic acids encoding the same
US7196166B2 (en) 2001-12-06 2007-03-27 Genentech Inc. Secreted polypeptide overexpressed in rectal tumor
US7202335B2 (en) 2001-12-06 2007-04-10 Genentech, Inc. PRO300 polypeptides
US7196173B2 (en) 2001-12-06 2007-03-27 Genentech, Inc. PRO1315 polypeptide encoded by a nuclear acid highly expressed in esophageal tumors
US7189815B2 (en) 2001-12-06 2007-03-13 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7119173B2 (en) 2001-12-06 2006-10-10 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7989167B2 (en) * 2006-11-13 2011-08-02 Val-Chum L.P. Method of prognosing and diagnosing hereditary spastic paraplegia, mutant nucleic acid molecules and polypeptides
US8518657B2 (en) 2006-11-13 2013-08-27 Universite De Montreal Method of prognosing and diagnosing hereditary spastic paraplegia, mutant nucleic acid molecules and polypeptides
US9523698B2 (en) 2006-11-13 2016-12-20 Val-Chum L.P. Method of prognosing and diagnosing hereditary spastic paraplegia, mutant nucleic acid molecules and polypeptides

Also Published As

Publication number Publication date
AU2452197A (en) 1997-10-29
EP0944651A2 (fr) 1999-09-29
JP2000509256A (ja) 2000-07-25
WO1997038085A3 (fr) 1998-02-19

Similar Documents

Publication Publication Date Title
US5776683A (en) Methods for identifying genes amplified in cancer cells
US5759776A (en) Targets for breast cancer diagnosis and treatment
WO1996039516A9 (fr) Cibles pour le diagnostic et le traitement du cancer du sein
WO1997038085A2 (fr) Genes amplifies dans des cellules cancereuses
JPH11502404A (ja) 細胞表面発現分子および形質転換関連遺伝子に特異的なｄｎａプローブおよび免疫学的試薬の開発
WO2001009317A1 (fr) Gene associe au cancer de l'estomac
EP0578909B1 (fr) Prohibitine humaine et DNA codant pour la même
EP3095791B1 (fr) Produits geniques exprimes de maniere differentielle dans des tumeurs et leur utilisation
US20040014192A1 (en) PR/SET-domain containing nucleic acids, polypeptides, antibodies and methods of use
Dragani et al. Major urinary protein as a negative tumor marker in mouse hepatocarcinogenesis
EP0831148B1 (fr) Occludine de molecule d'adhesion humaine
EP0974652A1 (fr) Gene associe a la metastase cancereuse
WO2000009655A2 (fr) Genes amplifies dans des cellules cancereuses
JP2004512809A (ja) Ｔ細胞受容体γ代替リーディングフレームタンパク質、（ＴＡＲＰ）およびその用途
EP0727486A2 (fr) Gène supprimant les tumeurs
WO1997028193A9 (fr) Compositions et procedes utiles dans la detection et/ou le traitement d'etats cancereux
WO1997028193A1 (fr) Compositions et procedes utiles dans la detection et/ou le traitement d'etats cancereux
US20040204574A1 (en) TSA305 gene
US7365158B2 (en) PR-domain containing nucleic acids, polypeptides, antibodies and methods
WO1999003883A1 (fr) Compositions et procedes faisant appel au gene de la sclerose tubereuse de bourneville-1 (tsc1) et produit genique
US5559023A (en) Tumor suppressor gene
US7112419B2 (en) Human hepatoma associated protein and the polynucleotide encoding said polypeptide
US20030049623A1 (en) PR/SET-domain containing nucleic acids, polypeptides, antibodies and methods of use
JP2554045B2 (ja) 哺乳類細胞系の複合薬剤耐性及び決定因子糖蛋白質ｄｎａの単離
DE69635526T2 (de) Biomarker und ziele für die diagnose, prognose und handhabung von prostatkrebs

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LK LR LS LT LU LV MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA

CFP Corrected version of a pamphlet front page
CR1 Correction of entry in section i

Free format text: PAT.BUL.44/97, UNDER INID(81)"DESIGNATED STATES",ADD"BA,CU,GH,LC,YU"

121 Ep: the epo has been informed by wipo that ep was designated in this application
CFP Corrected version of a pamphlet front page
CR1 Correction of entry in section i

Free format text: PAT.BUL.44/97, UNDER INID(30)"PRIORITY DATA",REPLACE"960409"BY"960410"AND"960710"BY"960711"

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 332691

Country of ref document: NZ

Ref document number: 1997920293

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1997920293

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: CA

WWW Wipo information: withdrawn in national office

Ref document number: 1997920293

Country of ref document: EP