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WO1998004697A1 - Proteine et gene suppresseur de tumeur cdo - Google Patents

Proteine et gene suppresseur de tumeur cdo Download PDF

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WO1998004697A1
WO1998004697A1 PCT/US1997/014418 US9714418W WO9804697A1 WO 1998004697 A1 WO1998004697 A1 WO 1998004697A1 US 9714418 W US9714418 W US 9714418W WO 9804697 A1 WO9804697 A1 WO 9804697A1
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cdo
nucleic acid
acid molecule
protein
purified
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WO1998004697A9 (fr
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Robert S. Krauss
Min Gao
Jong-Sun Kang
Jessica Feinleib
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Icahn School of Medicine at Mount Sinai
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Mount Sinai School of Medicine
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a newly discovered tumor suppressor gene, termed "cdo" (for Cam-related gene down-regulated by ⁇ .ncogenes) and, in particular, to cdo nucleic acids and proteins.
  • cdo tumor suppressor gene
  • the invention is also directed to methods and compositions for detecting cdo nucleic acids and proteins in vertebrate samples and to methods of treating malignancies and other disorders of cell proliferation.
  • Carcinogenesis involves multiple, independent somatic mutations in proto- oncogenes and tumor suppressor genes.
  • the number of proto-oncogenes isolated and characterized now totals more than 70 (Bishop, 1991, Cell 64:235-248).
  • only about 10 candidate tumor suppressor genes have been identified (Knudson, 1993, Proc. Natl. Acad. Sci. U.S.A. 2Q: 1091 -1092).
  • a tumor suppressor gene in essence, is a "recessive oncogene", a gene whose role in the development of neoplasms becomes apparent only after inactivating mutations have occurred in both alleles of the gene.
  • Tumor suppressor genes are generally divided into two classes.
  • Tumor suppressor genes of the Class II type are genes which are unaltered by mutation in tumor cells, but rather are transcriptionally down-regulated by mutations in Class I genes or proto- oncogenes.
  • maspin a putative protease inhibitor
  • tropomyosin I a putative protease inhibitor
  • ⁇ -actinin a putative proteinase inhibitor
  • vinculin a putative proteinase inhibitor
  • NO3 DAN Zaou, et al., 1994, Science 26_3 526-529; Prasad, et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 20:7039-7043; Gluck, et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:383- 387; Fernandez, et al., 1992, J. Cell.
  • CAMs cell adhesion molecules
  • DCC down-regulation of a gene designated "DCC” has been associated with colorectal cancers (Fearon et al., 1990, Science 242:49-56; Hedrick et al., 1994, Genes Develop. 8: 1 174-1183; Reale et al., 1994, Cancer Res. £4:4493- 4501 ; Narayanan et al., 1992, Oncogene 7:553-561; Tanaka et al., 1991, Nature 349:340-342: and Pierceall et al., 1994, J. Cell. Biol. 124:1017-1027).
  • DCC located on human chromosome 18, is a member of the IgSF, and has been found to be the target of somatic mutations in several colorectal cancers. Additionally,
  • DCC mRNA and protein expression have been observed to be decreased or absent in a majority of colorectal cancer specimens studied, relative to normal colonic mucosa. Subsequent studies indicated that DCC expression may also be involved in additional types of cancers, including malignancies of the breast and prostate. Furthermore, it was found that expression of DCC anti-sense RNA in Rat 1 fibroblasts induced transformation of these cells, as measured by growth in soft agar and tumorigenicity in nude mice. Unlike N-CAM, DCC is expressed at extremely low levels in adult tissues. This observation, as well as recent experimental data, suggest that DCC may function not simply to aggregate cells physically, but to act as a transducer of signals that regulate cell growth and, particularly, differentiation.
  • the present invention relates to a tumor suppressor gene, termed "cdo", and its encoded protein.
  • cdo tumor suppressor gene
  • the invention is based, at least in part, on the discovery of the cdo gene, which is related to, but distinct from, the DCC gene disclosed above, and on the discovery that expression of cdo is decreased in transformed cells. Accordingly, the present invention also provides for methods of using cdo nucleic acids and proteins in the diagnosis and treatment of malignant diseases as well as proliferative disorders. 4. Description of the Figures
  • FIGURE 1 Nucleic acid and amino acid sequences of rat cdo (complete cdo-encoding sequence designated as "form ⁇ ", SEQ ID NO:l). In an alternately spliced form (“form ⁇ ", SEQ ID NO:2) of rat cdo, nucleic acids 2694-3044 (indicated by brackets) are deleted.
  • FIGURE 2 Diagram showing the relative positions of rat cdo-encoding nucleic acid and human clones.
  • FIGURE 3 Comparison of human (SEQ ID NO:3) and rat (SEQ ID NO:l) nucleic acid sequences.
  • FIGURE 4A-B Northern blot analysis showing total RNA from parental rat 6 cells transformed by the H-ras, neu, v-src, v-raf, protein kinase C e, v-fos or c-myc oncogenes hybridized to (A) cdo probe or (B) GAPDH probe.
  • FIGURE 5 Northern blot analysis of RNA prepared from confluent, serum-starved rat 6 cells stimulated with serum, and (A) hybridized with cdo probe or (B) stained with ethidium bromide.
  • FIGURE 6 Expression of cdo mRNA in various tissues, as detected by PCR-based "exon connection” assays.
  • FIGURE 7 Autoradiogram of SDS-PAGE showing products of in vitro transcription/translation of rat ⁇ and ⁇ cdo-encoding cDNAs.
  • the reporter gene luciferase was used as a positive control.
  • FIGURE 8 Coomassie stained gel showing bacterial synthesis of fusion protein comprising glutathione S transferase and the intracellular domain of cdo.
  • FIGURE 9A-E (A) Western blot demonstrating that antisera-recognized cdo protein is present in 293 cells transfected with cdo-containing expression vector, but not with control vector. (B) Western blot demonstrating cdo protein expression in parental rat 6 cells and ras-transformed C1-T24 cells. (C) Western blot showing time course of cdo expression in serum-stimulated rat 6 cells. (D) Western blot showing expression of cdo protein in adherent cultures (lane P) and non-adherent cultures (lane M). (E) Northern blot showing cdo RNA expression in adherent cultures (lane P) and non-adherent cultures (lane M).
  • FIGURE 10A-B Depicts an autoradiogram showing the results of PCR- based exon-connection assay for cdo expression in transformed breast cells.
  • FIGURE 11 Nucleic acid and amino acid sequence of human cdo (SEQ ID NO: 1
  • the present invention relates to nucleic acid molecules encoding cdo, cdo proteins, peptide fragments and derivatives, and antibodies directed toward cdo.
  • the invention relates to pharmacological compositions and diagnostic and therapeutic uses of cdo nucleic acids and proteins.
  • the present invention relates to purified and isolated nucleic acid molecules encoding cdo. It is based, at least in part, on the cloning and characterization of rat and human cdo-encoding nucleic acids. It is also based on the discovery that alternative splicing gives rise to multiple forms of cdo-encoding nucleic acids.
  • the present invention provides for a purified and isolated nucleic acid encoding rat cdo.
  • the invention provides for a nucleic acid molecule having a sequence as set forth in FIGURE 1, which sets forth two alternatively spliced forms of rat cdo.
  • the present invention also provides for nucleic acid molecules which are at least 90 percent (and preferably at least 95 percent) homologous to forms ⁇ and ⁇ of the cdo-encoding sequence set forth in FIGURE 1 (SEQ ID No: 1 and SEQ ID NO: 2, respectively), wherein the percent homology is defined as the percentage of identical nucleic acids occurring in molecules which have been aligned in a manner which pairs residues, for comparison, with the greatest degree of similarity (e.g., MacVector. Version 4.1 , "Sequence Analysis Software for the Macintosh", International Biotechnologies, Inc., a subsidiary of Eastman Kodak Co., New Haven, Connecticut).
  • the present invention provides for a nucleic acid molecule, at least 30 and preferably at least 50 nucleotides in length, which hybridizes with a nucleic acid molecule having a sequence as set forth for form ⁇ or form ⁇ in FIGURE 1
  • the present invention also provides for purified and isolated nucleic acid molecules which encode a protein having an amino acid sequence as set forth for form a or ⁇ in FIGURE 1 and to (i) nucleic acid molecules at least 90 percent (and preferably at least 95 percent) homologous and (ii) nucleic acid molecules (at least 30 or at least 50 nucleotides in length) which hybridize under stringent conditions, thereto.
  • the present invention provides for human cdo- encoding nucleic acids, as comprised in human cDNA clones pHC12, pkSHA3-4, and pTA7.
  • a nucleic acid sequence of human cdo (FIGURE 11; SEQ ID NO:3), as obtained by sequencing the cloned DNA (for much, but not all, of the cloned DNA, both strands were sequenced), corresponding to rat cdo, is set forth in FIGURE 3.
  • the present invention also provides for nucleic acid molecules that are at least 90 percent (and preferably at least 95 percent) homologous to the human cdo-encoding nucleic acids as contained in the deposited clones or having the sequence set forth in FIGURE 11 (SEQ ID NO:3), or that are at least 30 or at least 50 nucleotides in length and hybridize under stringent conditions (as set forth above) thereto.
  • the present invention yet further provides for the cloning of a cdo cDNA or genomic sequence using nucleic acid sequences disclosed herein.
  • a cdo-encoding nucleic acid may be cloned by a combination of procedures, comprising the derivation of an oligonucleotide probe based on the sequence information provided herein, construction of a cDNA or genomic library, and selection, isolation and cloning of the cdo-encoding nucleic acid.
  • a preferred procedure utilizes the polymerase chain reaction (PCR; Saiki et al., 1985, Science 23Q: 1350-1354) to expand the number of cdo sequences for cloning.
  • cdo mRNA, cDNA or genomic DNA from a vertebrate species (as discussed in Section 6, below, a "Zoo" Southern blot showed the presence of cdo genes in all vertebrate species tested).
  • a synthetic DNA probe consisting of a 10-30 nucleotide segment of a cdo nucleic acid molecule, as set forth above, and use the probe to screen at high stringency a cDNA library from an appropriate cell line presumed to carry cdo- encoding sequences.
  • PCR primers based upon the disclosed nucleic acid sequences, and generate a cdo cDNA either from the same library, or directly from the mRNA of that cell line. Both of these procedures are standard in the art (Benton and Davis, 1977, Science 196:180-182: Maniatis et al., 1978, Cell 15_:687-701).
  • cdo-encoding nucleic acid may be readily produced by inserting the nucleic acid into an appropriate cloning vector and introducing that vector into a suitable host cell, such as a bacterial cell. 5.2. Expression of cdo
  • the cdo-encoding nucleic acid molecules set forth above may be expressed in a suitable host cell, for example, a bacterial, yeast, fungal, plant, insect, or vertebrate host cell.
  • a cdo-encoding nucleic acid molecule may be inserted into a suitable expression vector, including a plasmid, cosmid, phage, or virus vector.
  • the vector may further comprise control elements which aid in the transcription, translation, and/or processing of cdo, as well as one or more selection marker.
  • useful control elements include one or more of the following: a promoter/enhancer element, polyadenylation signal, transcriptional terminator, translational initiation site and terminator, ribosome binding site, nuclear localization signal, and secretory signal sequence.
  • the vector may then be introduced, using standard techniques, into a suitable host cell for expression.
  • a cdo nucleic acid molecule may be incorporated into a pMV12 retroviral vector, which contains a hygromycin resistance gene as a selection marker.
  • the pMV12/cdo vector may then be packaged to form retrovirus suitable for transduction and then may be transduced into an appropriate cell line.
  • a pMV12-cdo plasmid vector may be transiently transfected into the ecotropic packaging mutant helper cell line, BOSC, to generate replication-defective viral particles; then, transduction of pMV12/cdo retrovirus into ras-transformed rat 6 or 3T3 cells (for example, C1/T24 cells), is preferred.
  • Hygromycin-resistant transfectants may then be selected. Ectopic expression of cdo in the transfectants may be confirmed by immunoprecipitation and/or Western blot analyses of total cellular protein with antibodies specific for extracellular and intracellular regions of cdo.
  • a mammalian cell line having inducible expression of cdo may be prepared utilizing an expression system based on the tetracycline-resistance (tet) operon of E. coli (Gossen and Bujard, 1992, Proc. Natl. Acad. Sci. U.S.A. 89:5547-5551).
  • This system employs a tetracycline- controlled, hybrid trans-activator (tTA) that consists of the tet-repressor and the transcriptional trans-activating domain of herpes simplex virus protein 16 (VP16).
  • tTA tetracycline- controlled, hybrid trans-activator
  • VP16 herpes simplex virus protein 16
  • the tTA can bind to tet operator sequences placed in front of a minimal mammalian promoter and thereby repress transcription in the presence of tetra- cycline. Removal of tetracyciine from the culture medium causes rapid induction of cDNAs placed downstream of the tet operator sequences (Gossen and Bujard, 1992, Proc. Natl. Acad. Sci. U.S.A. 89:5547-5551; Schmid, 1995, Trends Cell Biol.
  • nucleic acid encoding cdo protein may be incorporated into an expression vector so as to produce a fusion protein.
  • cDNA encoding cdo protein may be fused, in frame, into the vector pGEX-5Xl , which enables the IPTG-inducible production of a cdo/glutathione S-transferase fusion protein.
  • Escherichia coli strain XL-1 Blue may then be transformed with this vector, and cultured in the presence of IPTG, to produce the fusion protein, which may then be captured on glutathione beads.
  • a cdo nucleic acid may be incorporated into the pGEX-KG vector (Guan and Dixon, 1991 , Anal. Biochem. 192:262-267).
  • This vector directs EPTG-inducible expression of glutathione S- transferase (GST) fusion proteins that contain an "improved" thrombin cleavage region between the GST and fusion partner proteins. It also provides a one-step purification procedure of cleaved recombinant protein. Lysates from IPTG-treated bacteria that harbor the PGEX-KG/cdo vector may be incubated with glutathione agarose beads, and the beads extensively washed.
  • the GST/cdo-bound beads may then be transferred to thrombin cleavage buffer and incubated with thrombin, followed by collection of the supernatant containing the cleaved, purified cdo portion of the fusion protein.
  • the homogeneity of the recombinant cdo protein may be confirmed by SDS-PAGE and used for generation of antibodies. If necessary, a final acrylamide gel purification step may be added.
  • the present invention relates to purified and isolated cdo proteins encoded by the nucleic acid molecules described in section 5.1, supra. Such proteins may be produced by techniques set forth in section 5.2.
  • the present invention provides for a purified and isolated rat cdo protein.
  • the invention provides for a protein having an amino acid sequence as set forth in FIGURE 1, which sets forth the amino acid sequences of the two alternatively spliced forms of rat cdo.
  • the present invention also relates to proteins having amino acid sequences which are functionally equivalent to the amino acids sequences set forth in
  • FIGURE 1 and 1 For example, one or more of the amino acid residues within these sequences may be substituted with another amino acid residue of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • cdo proteins that have been modified by glycosylation, proteolytic cleavage, or incorporation into a larger molecule.
  • the present invention provides for human cdo proteins, as encoded by (i) nucleic acids comprised in human cDNA clones pHC12, pkSHA3-4, and pTA7; (ii) nucleic acid sequence SEQ ID NO:3 (FIGURE 1 1); and (iii) nucleic acid molecules which are at least 90 percent homologous (preferably at least 95 percent homologous), or which hybridize under stringent conditions, to cdo-encoding nucleic acids comprised in human cDNA clones pHC12, pkSHA3-4, and pTA7, or to nucleic acid sequence SEQ ID NO:3 (FIGURE 11).
  • the present invention also provides for protein fragments of cdo, for example fragments comprising the intracellular portion, and for fusion proteins comprising this intracellular portion of cdo.
  • the intracellular portion of cdo may have a sequence (i) as set forth for form ⁇ of rat cdo in FIGURE 1 (SEQ ID NO:l), from amino acid encoded by nucleic acid residue 4455-5222 to the end of the protein coding sequence in FIGURE 1 ; (ii) as encoded by nucleic acid residues 2893-3675 in FIGURE 11 (SEQ ID NO:3); or (iii) at least 90 percent, and preferably at least 95 percent, homologous to the sequences of (i) or (ii).
  • SEQ ID NO:l sequence as set forth for form ⁇ of rat cdo in FIGURE 1
  • SEQ ID NO:3 amino acid encoded by nucleic acid residue 4455-5222 to the end of the protein coding sequence in FIGURE 1
  • (ii) as encode
  • a cdo protein as set forth above, or an immunogenic fragment thereof, may be used as an immunogen to generate anti-cdo antibodies.
  • the amino acid sequence of cdo may be analyzed in order to identify portions of the cdo molecule which may be associated with greater immunogenicity.
  • the amino acid sequence may be subjected to computer analysis to identify surface epitopes, according to the method of Hopp and Woods, 1981, Proc. Natl. Acad.
  • polyclonal antibodies directed toward cdo may be prepared by methods known in the art.
  • Various adjuvants may be used to increase the immunological response, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, and keyhole limpet hemocyanin.
  • the present invention further provides for nucleic acids encoding immunoglobulin molecules directed toward cdo, including nucleic acids encoding single chain antibodies as well as conventional antibody molecules.
  • Antibody molecules may be purified by known techniques, such as immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC, or combinations thereof.
  • the present invention also provides for antibody fragments directed toward cdo, including, but not limited to, F(ab') 2 and Fab fragments. 5.5. Screening for cdo Expression in Cells
  • a nucleic acid probe comprising a portion of a cdo-encoding nucleic acid, as described in Section 5.1, may be used as a probe to detect cdo mRNA in a cell sample prepared from a subject who is suspected to suffer from a malignancy or defect in cell proliferation.
  • a probe used in such methods hybridizes specifically and selectively to nucleic acid encoding cdo; more preferably, the probe hybridizes to a nucleic acid having a sequence as set forth in FIGURE 1 (SEQ ID NOS 1 or 2) or FIGURE 11 (SEQ ID NO:3) under stringent conditions, but does not hybridize, under stringent conditions, to non- cdo-encoding nucleic acid.
  • the probe may be a single-stranded DNA or RNA molecule which is complementary to a cdo-encoding nucleic acid molecule.
  • the probe may be produced by chemical synthetic or recombinant DNA methods, and may be labeled such that it is directly or, alternatively, indirectly, detectable.
  • the probe may preferably be at least 10 nucleotides long, and more preferably, may be 20-50 nucleotides long.
  • the assay may be carried out by standard methods such as in situ hybridization, or Northern analysis, using hybridization conditions appropriate for the degree of homology estimated between the probe and its target nucleic acid.
  • Such techniques may be practiced, in a manner known to the skilled artisan, to allow quantitative or semi-quantitative measurement of cdo levels.
  • amplification methods such as the PCR exon- connection as described in Fearon et al., 1990, Science 247: 49-56.
  • cdo expression may be measured by assessing the amount of cdo protein produced using, for example, standard Western blot, ELISA, or RIA assays.
  • immunohistochemistry may be performed, as described in Hedrick et al., 1994, Genes Develop. &:1174-1183.
  • a cell sample may be fixed on a glass microscope slide, depleted of endogenous peroxidase activity by incubation in 0.3% H 2 O 2 in methanol, and blocked with non-immune goat serum.
  • the sample may then be incubated with anti-cdo antibody, which is, for example but not by way of limitation, directed against the intracellular domain of cdo.
  • detection of this antibody may be with a secondary, bio- tinylated, goat anti-rabbit antibody, followed by Vectastain Elite (Vector
  • anti-cdo antibodies are desirably affinity purified by passage over Sepharose linked to cdo protein or a relevant fragment thereof. Staining with pre-immune rabbit serum in place of anti- cdo antibody may be used as a control.
  • detecting, in a cell sample, cdo mRNA or protein levels which are substantially decreased or absent, compared to a level of cdo mRNA or protein measured in a control cell sample indicates that the sample cells may be malignant or may exhibit a defect in proliferation.
  • the present invention provides for a method of diagnosing a disorder of cell proliferation in a subject, comprising measuring the amount of cdo expression in a test sample of cells collected from the subject, and comparing the level of cdo expression in the test sample to a control sample of cells from a normal subject, wherein a decreased amount of cdo expression in the test sample is indicative of a disorder associated with increased cell proliferation in the subject.
  • a decreased amount of cdo expression in the test sample is indicative of a disorder associated with increased cell proliferation in the subject.
  • decreased or absent cdo expression correlated positively with the transformed phenotype in breast cells.
  • An increased amount of cdo expression may conversely indicate a disorder associated with decreased cell proliferation in the subject.
  • the present invention provides for a method of diagnosing a malignant disorder in a subject, comprising measuring the amount of cdo expression in a test sample of cells collected from the subject, and comparing the level of cdo expression in the test sample to a control sample of cells from a normal subject, wherein a decreased amount of cdo expression in the test sample is indicative of a malignant disorder in the subject.
  • the detection, by such methods, of a cdo mRNA or protein of abnormal size may indicate the presence of a malignancy or proliferative disorder.
  • Cdo expression may have an inhibitory effect on cell proliferation and on the expression of the transformed phenotype. Accordingly, cdo protein or cdo- encoding nucleic acid, or a portion thereof, may be introduced into a cell in order to inhibit the proliferation or transformation of the cell, or to reverse the transformed phenotype in a malignant cell.
  • Such methods may be useful for use in cell culturing techniques, where it may be desirable to retard the proliferation of cells (for example, but not by way of limitation, in a feeder culture).
  • expression of cdo may be controlled (for example, by an inducible promoter)
  • cells may be synchronized.
  • Such methods may be applied (in vitro or in vivo) to a cell of a subject in need of such treatment.
  • a subject may be a human or non-human subject.
  • a subject suffering from a malignant disorder or a proliferative disorder may be considered to be in need of such treatment.
  • a suitable expression vector as set forth above, may be used.
  • cdo nucleic acid is used for gene therapy, any vector system known in the art of gene therapy may be used.
  • a cdo protein or a portion thereof, is to be introduced into a cell
  • the protein may be incorporated into a vesicle, or may be fused to a second protein or other molecule, to promote uptake. 6.
  • a cDNA library was constructed from a transformation-resistant mutant rat 6 embryo fibroblast cell line that fails to form colonies in soft agar when infected with v-H-ras expressing retrovirus.
  • the library was screened by differential hybridization with 32 P-labelled cDNA prepared from both mutant and control cell mRNA. From this screening procedure, an approximately 3.5 kb cDNA clone, designated cdo, was isolated.
  • the cDNA hybridized to an approximately 8.5 kb mRNA species and showed a mild (about 2 fold) up-regulation in mutant cells.
  • the 3.5 kb cDNA clone represented the 3' most portion of the full length cdo mRNA.
  • a random-primed library was prepared from confluent, serum-starved rat 6 cells, and cDNAs covering the full sequence were isolated in three rounds of walking.
  • the nucleic acid sequence of the full length cdo cDNA was then determined and is disclosed in FIGURE 1 (form ⁇ ; SEQ ID NO:l).
  • FIGURE 4A-B depicts a Northern blot analysis of cdo mRNA expression (FIGURE 4A) in parental rat 6 cells transformed by the H-ras, neu, v-src, v-raf, protein kinase C e, v-fos or c-myc oncogenes. Twenty micrograms of total cellular RNA from designated cell lines was fractionated on agarose/formaldehyde gels, blotted to a nylon membrane and hybridized with a 32 P-labelled rat cdo cDNA probe. The lane designations in FIGURE 4A refer to the oncogene expressed in the given rat 6 cell derivative.
  • the lower panel shows signal generated when the same filter was hybridized to a rat GAPDH probe, as a control for the integrity of the RNA in each lane.
  • Expression of cdo mRNA was significantly down-regulated by transformation.
  • protein kinase C ⁇ l which is a very weak transforming gene, had little effect on cdo expression.
  • down-regulation of cdo mRNA expression correlated with establishment of the transformed phenotype by several different oncogenes. This evidence suggests that cdo is a candidate Class II tumor suppressor gene in that it is down regulated in transformed cells.
  • FIGURE 5 A shows the time course of cdo expression in serum-stimulated rat 6 cells.
  • Confluent cultures of wild-type rat 6 cells were rendered quiescent by incubation in medium containing 0.1 % serum for 48 hours and then stimulated by refeeding with fresh-serum-containing medium.
  • RNA was isolated at various time points thereafter, and cdo expression was analyzed by Northern blotting techniques as described for FIGURE 4A-B. The numbers above the lanes indicate the time, in hours, after refeeding of the cultures.
  • An ethidium bromide stained gel, shown in FIGURE 5B serves as a loading control.
  • cdo mRNA A decrease in steady state cdo mRNA levels was apparent within 2 hours. Four hours after serum stimulation, cdo expression was nearly extinguished. Expression of cdo returned in 8-12 hours, and in 24-48 hours had been restored to the level of the original cultures prior to stimulation. Thus, steady state levels of cdo mRNA were transiently down- regulated as quiescent, G 0 -arrested cells reentered the cell cycle. Finally, it should be noted that cdo mRNA is ordinarily present in low amounts in rat 6 cells (the transformable parent cell line of the mutant cell line in which cdo was first discovered), representing no more than 0.003% of total cellular mRNA, even when cells are confluent and starved of serum.
  • RNA from brain, liver, kidney, heart, large and small intestines, spleen, thymus, lung, stomach, breast and skeletal muscle was analyzed in this manner.
  • a PCR product of the predicted size (1883 bp) was detected in most tissues, but no such product was found in liver, kidney and skeletal muscle.
  • This expression pattern is consistent with the related gene, DCC.
  • DCC the related gene
  • cdo is expressed at extremely low levels. It is possible, therefore, that the role of cdo, like DCC, is not merely to bind cells together physically, but as a transducer of signals involved in cell growth and differentiation.
  • a Southern "zoo" blot was prepared containing DNA from various species using the rat cdo cDNA as a probe. This test revealed that mouse and human DNA displayed strong cross- species hybridization to the rat cdo cDNA, while chicken, frog (Xenopus laevis. and zebra fish DNA displayed a clearly detectable, but weaker, signal. This data indicates that cdo has been conserved throughout vertebrate evolution.
  • Both and ⁇ forms of rat cdo-encoding nucleic acids were expressed in an in vitro transcription/translation reaction.
  • the two cDNAs encoding the ⁇ and ⁇ forms were subcloned into the Bluescript vector, pSK (Stratagene) and added to a Promega TNT kit reaction.
  • a product of the predicted molecular weight (approximately 136,000 and 124,000 for ⁇ and ⁇ forms, respectively) was produced, as shown in FIGURE 7, in the lanes designated cdo ⁇ and cdo ⁇ . Three higher molecular weight forms were also detected, which probably represent glycosylation products.
  • FIGURE 8 shows the synthesis in bacteria of a recombinant fusion protein made between glutathione S transferase and the intracellular portion of rat cdo (encoded by nucleic acid residues 4666-5267. Nucleic acid encoding the intracellular portion of cdo was incorporated, in frame, into the vector pGEX-5Xl (Pharmacia). This vector enables IPTG-inducible production of a glutathione S- transferase (GST) fusion protein. E. coli strain XL-1 Blue was transformed with the recombinant vector, and then a large culture was grown and induced with IPTG.
  • GST glutathione S- transferase
  • the cells were then harvested and the GST-cdo fusion protein was purified by capture on glutathione beads, followed by SDS-gel electrophoresis and electroelution from a slice of the gel. The homogeneity of the recombinant cdo protein was confirmed by SDS-PAGE (see FIGURE 8).
  • the resulting purified protein was used to produce polyclonal antisera in rats by standard techniques (by Zymed Labs, Inc.). The polyclonal antisera was then used to demonstrate that the antisera-recognized cdo protein exhibited expression patterns which paralleled cdo RNA expression, as discussed above (FIGURE 9A-E).
  • FIGURE 9 A shows that transfection of 293 cells with an expression vector pBabePuro containing rat cdo-encoding cDNA resulted in production of antisera- recognized cdo protein.
  • FIGURE 9B shows that antisera-recognized cdo protein is present in parental rat 6 cells but not in ras-transformed rat 6 cells (line C1-T24).
  • FIGURE 9C depicts a Western blot which demonstrates that serum stimulation of serum-starved rat 6 cells leads to transient down-regulation of antisera-recognized cdo protein.
  • FIGURES 9D-E demonstrate that cdo RNA (FIGURE 9E) and antisera-recognized cdo protein (FIGURE 9D) are associated with cell substratum adhesion; lane P in these figures represents adherent cultures; lane M represents non-adherent cultures grown in methylcellulose.
  • Human cloned cDNA corresponding to more than 98 percent of the rat open reading frame has been isolated.
  • the human cloned cDNA is contained in clones pHC12, pkSHA3-4, and pTA7, prepared from two lambda phage libraries, one derived from fetal lung (a gift from Stuart Aaronson) and the second derived from human fetal brain (Clonetech).
  • FIGURE 2 provides a map of clones pHC12, pkSHA3-4 and pTA7 relative to rat cdo.
  • FIGURE 3 provides a comparison between the obtained human sequence and rat cdo.
  • the sequence data indicates that splice variant forms of cdo, in addition to forms ⁇ and ⁇ , are likely to exist.
  • human nucleotides 3304-3318 have no counterpart in the rat sequence; this likely represents an alternatively spliced exon encoding five amino acids. 8B.
  • human and rat cdo have virtually identical domain structures in their extracellular region, and their intracellular region are highly related, but neither resemble other known proteins.
  • Human and rat cdo are ⁇ 81% identical and 96% similar at the amino acid level, and the relative positions of amino acids that define the Ig and FN III- like domain consensus are fully conserved.
  • FIGURE 10A-B depicts an autoradiogram showing the results of these experiments. Briefly, equal amounts of RNA from each cell type were reverse-transcribed and gave similar yields of first-strand synthesis. Equal amounts of cDNA were then amplified by PCR with cdo-specific primers. The products were fractionated on an agarose gel, blotted to a nylon filter and hybridized to a 32 P-labelled human cdo cDNA probe (FIGURE 10A).
  • FIGURE 10B A PCR reaction with primers specific for ⁇ -actin was also performed on each sample as a control, and the ethidium bromide-stained gel demonstrating production of the appropriately-sized product is shown in FIGURE 10B.
  • the lane designations represent the different normal and tumor cell lines.
  • RM refers to an epithelial cell culture derived from a reduction mammoplasty. As shown in FIGURE 10A, Four out of seven transformed cell lines showed a substantial decrease in CDO levels. 10. Deposit of Microorganisms
  • GAATGCCCAT CAACGCCTAT TTCGTGAAGT ACCGAAAGCT GGACGACGGC AGTGGTGCGG 3360
  • CTATTATCTT AATTTACAAA ATGGCCACCA CGAGTTCTTT GCACTACTTG CAGAGGTATA 6000 TAATAAATAC AAAAGTAAGG CCTTTAAACT GATAGTTTG 6039
  • TTCCCCGTGC AAATGGCGGC TCTCCCATCA CTGCCTTCAA GGTGGAATAT AAGCGGATGA 3420
  • TGGAAGGTTC AAAGCAGTGG CACACCATTG GTCACCTGCA GCCAGAGACC TCCTATGACA 3840 TTAAGATGCA GTGCTTTAAT GAAGGAGGAG AGAGCGAGTT CAGCAACGTG ATGATCTGCG 3900
  • TGGAGTTCGA ACATCCTCAC CATCTAGTGA ACGGTGGAGC AGTGTACACG GCTGTCCCTC 4440 AGATGGACCC ACTGGAATGC ATTAATTGTC GGAATTGCCG GAACAACAAT AGGTGTTTCA 4500
  • GTACATGCCG CCAGGACAAC ACAAGCGACA TCAATTCTGA TTCCACAGAA GACACAGCAG 4740 AGTTCAACAG AGGAGACAGC AGCGGTCATT CAGAAGCAGA GGACAAAGTT TTCAGTTGGA 4800
  • GTCCTCTTAT TTTATCACCT GTCTTGGAGG CTGCAGTGAG AAGACAGCGT GGTCTCCTCC 4860 TGGCCCCCCT CTAGACGGGC TGTCAGTGGT CCTTCAGCAA GCCCAAGAGA CCTGAGAGGA 4920
  • ATAGTTGTGA TCATATGTTT ATAATAGCCA ACCCAGCTGA CACACTTTTG AGTACCTTCC 5400 AGAAAAATAC TAATACTGAC TTATTTTCTC TCTGTGCCTG GGTACAAGTA GCGATCAATC 5460
  • GTAGTACTGC ATTGTTCTGC TCAACCTGTG ACCACTCGTA TCTCATGGCT GCATAACGGA 120 AAAACATTGG ATGGAAACCT GGAACATATT AAGATTCATC AGGGGACTCT GACAATTCTT 180
  • CATGGTACCA CACAGGCAGA AGCATCTCTC ATGGTTGTTC CTTTTGAAA CAAATACAAAA 1500 GCAGAGACAG TCACACTTCC TGATGCTGCT CAGAATGATG ACAGAAGTA AGAGAGATGGT 1560
  • GAGCCATCTA GTCTTTATGA AGTCTTGATG GTAGCAAGAA GCGCAGCAG GTGAAGGCCAA 1920
  • GCATCCTCTC CACCCGTGGG CATCCCTAAG TATCCCGTTG TTTCAGAGG CTGCAAACAAC 2040
  • TATTCCCACC TTCACCATAA GGTCCCCAAT GCAGTCAATG GAATTGTGA ATGGGAGCCTA 3120
  • CATCCTCATC ATCTAGTGAA TGGTGGTGGA ATGTACACGG CCGTGCCTC AGATTGACCCT 3240
  • AACTGTGTGA AGGACCTTAA TTCAAATCAG AGAAAATCAT TATTTATTT TTTTGTAGTAG 3840

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Abstract

L'invention concerne un gène suppresseur de tumeur de Classe II, baptisé cdo (Cam-related gene/down-regulated by oncogenes/gene apparent au gène d'adhésion cellulaire/rétrorégulé par des oncogènes), qui est rétrorégulé au niveau de l'ARNm dans des cellules transformées, et est exprimé à des niveaux extrêmement faibles dans des tissus d'adultes.
PCT/US1997/014418 1996-07-26 1997-07-11 Proteine et gene suppresseur de tumeur cdo Ceased WO1998004697A1 (fr)

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US68772796A 1996-07-26 1996-07-26
US08/687,727 1996-07-26

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001027277A3 (fr) * 1999-10-13 2002-04-18 Curagen Corp Proteines et polynucleotides codes par ces proteines
WO2011117328A1 (fr) * 2010-03-23 2011-09-29 Netris Pharma Méthodes et compositions pour induire une apoptose des cellules tumorales exprimant la shh

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030166132A1 (en) * 1998-08-26 2003-09-04 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
JP2005267152A (ja) 2004-03-18 2005-09-29 Seiko Instruments Inc 電子歩数計
US7625759B2 (en) 2005-12-19 2009-12-01 Genentech, Inc. Method for using BOC/CDO to modulate hedgehog signaling

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, 01 January 1995, Volume 55, HSIEH J.-T. et al., "Tumor Suppressive Role in Androgen-Regulated Epithelial Cell Adhesion Molecule (C-CAM) in Prostate Carcinoma Cell Revealed by Sense and Antisense Approaches", pages 190-197. *
ONCOGENE, 1995, Volume 10, KLINGELHUTZ A.J. et al., "The DCC Gene Suppresses the Malignant Phenotype of Transformed Human Epithelial Cells", pages 1581-1586. *
SCIENCE, 25 April 1997, Volume 276, ROUSH W., "Putative Cancer Gene Shows Up in Development Instead", pages 534-535. *
SCIENCE, 25 August 1995, Volume 269, MARSHALL E., "Gene Therapy's Growing Pains", pages 1050-1055. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001027277A3 (fr) * 1999-10-13 2002-04-18 Curagen Corp Proteines et polynucleotides codes par ces proteines
WO2011117328A1 (fr) * 2010-03-23 2011-09-29 Netris Pharma Méthodes et compositions pour induire une apoptose des cellules tumorales exprimant la shh
EP2407173A1 (fr) * 2010-07-13 2012-01-18 Netris Pharma Procédé et compositions pour induire l'apoptose de cellules tumorales exprimant le gène SHH

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