WO1995032214A1 - Nouveau gene suppresseur des tumeurs de la prostate et du colon situe sur le chromosome humain 8 - Google Patents

Nouveau gene suppresseur des tumeurs de la prostate et du colon situe sur le chromosome humain 8 Download PDF

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Publication number
WO1995032214A1
WO1995032214A1 PCT/US1995/006593 US9506593W WO9532214A1 WO 1995032214 A1 WO1995032214 A1 WO 1995032214A1 US 9506593 W US9506593 W US 9506593W WO 9532214 A1 WO9532214 A1 WO 9532214A1
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Prior art keywords
protein
vector
ptsg
dna
cell
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PCT/US1995/006593
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English (en)
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Robert Bookstein
William B. Isaacs
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Canji, Inc.
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Priority to EP95920649A priority Critical patent/EP0714401A4/fr
Priority to SK246-96A priority patent/SK24696A3/sk
Priority to AU26034/95A priority patent/AU686815B2/en
Publication of WO1995032214A1 publication Critical patent/WO1995032214A1/fr
Priority to NO961539A priority patent/NO961539L/no
Priority to FI961723A priority patent/FI961723A0/fi

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • This invention is in the field of tumor suppressor genes (anti-oncogenes) and relates in general to products and methods for practicing broad-spectrum tumor suppressor gene therapy of various human cancers.
  • the invention relates to methods for treating tumor cells by: (1) administering vectors comprising a nucleic acid sequence coding for the novel proteins referred to herein as prostate tumor suppressor gene products (PTSG products); or, (2) administering an effective amount of a protein coded for by the nucleic acid sequence.
  • PTSG products prostate tumor suppressor gene products
  • the invention also relates to diagnosis of certain cancers such as prostate and colon cancer using the cloned nucleic acids of this invention. Cancers and tumors are the second most prevalent cause of death in the United States, causing 547,000 deaths per year. One in three Americans will develop cancer, and one in five will die of cancer
  • cancer genes i.e., genes that have been implicated in the etiology of cancer, have been identified in connection with hereditary forms of cancer and in a large number of well-studied tumor cells. Study of cancer genes has helped provide some understanding of the process of tumorigenesis. While a great deal more remains to be learned about cancer genes, the known cancer genes serve as useful models for
  • oncogenes which, when activated, promote tumorigenesis
  • tumor suppressor genes which, when damaged, fail to suppress tumorigenesis. While these classifications provide a useful method for conceptualizing
  • a particular gene may play differing roles depending upon the particular allelic form of that gene, its regulatory elements, the genetic background and the tissue environment in which it is operating.
  • mutant versions of normal cellular growth regulatory genes or viral or other foreign genes in mammalian cells that cause inappropriate, untimely, or ectopic expression of other classes of vital growth-regulatory genes.
  • a simplistic view of the biologic basis for neoplasia is that there are two major classes of cancer genes. The first class consists of mutated or otherwise aberrant alleles of normal cellular genes that are involved in the control of cellular growth or
  • protooncogenes When mutated, they can encode new cellular functions that disrupt normal cellular growth and replication. The consequence of these changes is the production of dominantly expressed tumor phenotypes.
  • this model of dominantly expressed oncogenes a view that has predominated since the emergence of the concept of the genetic and mutational basis for neoplasia, it is imagined that the persistence of a single wild-type allele is not sufficient to prevent neoplastic changes in the developmental program or the growth properties of the cell.
  • the genetic events responsible for the activation of these oncogenes therefore might be envisioned as
  • single-hit events The activation of tumorigenic activities of the myc oncogene in Burkitt lymphoma, the expression of bcr-abl chimeric gene product in patients with chronic myelogenous leukemia, the activation of the H-ras and K-ras oncogenes in other tumors represent some of the evidence for the involvement of such transforming oncogenes in clinical human cancer.
  • An approach to genetic-based therapy for dominantly expressed neoplastic disease presumably would require specific shutdown or inactivation of expression of the responsible gene.
  • Tumor suppressor genes A more recently discovered family of cancer- related genes are the so-called tumor-suppressor genes, sometimes referred to as antioncogenes, growth- suppressor, or cancer-suppressor genes. Recent research suggests strongly that it is loss-of-function mutations in this class of genes that is likely to be involved in the development of a high percentage of human cancers; more than a dozen good candidate human tumor-suppressor genes have been identified in several human cancers.
  • the tumor suppressor genes involved in the pathogenesis of retinoblastoma (RB), breast, and other carcinomas (p53), Wilm's tumors (wt 1, 2) and colonic carcinoma (DCC) have been identified and cloned.
  • the retinoblastoma gene is the prototype tumor suppressor.
  • the RB gene encodes a nuclear protein which is phosphorylated on both serine and threonine residues in a cell cycle dependent manner (Lee et al., Nature, 329:642-645 (1987); Buchkovich et al., Cell,
  • the familial breast cancer gene, BRCA-1 has been mapped at chromosome 17 q21-22 by linkage analysis. It is not clear whether this gene will behave as a tumor suppressor or dominant oncogene.
  • the gene involved in human familial cancer syndrome such as Li- Fraumeni syndrome, p53, apparently acts as the classical tumor suppressor; similarly, the loss of RB gene is associated with hereditary retinoblastoma (Knudson, 1993, supra).
  • mammalian cells are thought to represent a complex set of interactions between these two kinds of genes.
  • carcinogenesis occurs through the selection of several genetic changes, each modifying the expression or function of genes controlling cell growth or
  • the suppressor locus may be relatively close to LPL or NEFL.
  • This invention is based on the discovery of a nucleic acid molecule encoding a novel prostate/colon tumor suppressor gene product (PTSG protein) having tumor suppression capability.
  • the nucleic acid molecule has been mapped to the p22 region of chromosome 8.
  • the newly disclosed full length cDNAs encode two novel 348 and 347 amino acid proteins.
  • This invention establishes for the first time that inactivation of PTSG or PTSG product is responsible for prostate adenocarcinoma, colon cancer and other related cancerous pathologies, as provided herein.
  • oligonucleotide fragments capable of hybridizing with the PTSG gene, and assays utilizing such fragments, are provided. These oligonucleotides can contain as few as 5 nucleotides, while those consisting of about 20 to about 30
  • oligonucleotides being preferred. These oligonucleotides may optionally be labelled with radioisotopes (such as tritium, 32 phosphorus and 35 sulfur), enzymes (e.g., alkaline phosphatase and horse radish peroxidase), fluorescent compounds (for example, fluorescein,
  • Ethidium, terbium chelate or chemiluminsecent compounds (such as the acridinium esters, isoluminol, and the like). These and other labels, such as the ones
  • oligonucleotides can also be used as primers in polymerase chain reaction techniques, as techniques are described in, for example, "PCR
  • loss of the wild-type PTSG is
  • the loss may be due to either deletional and/or point mutational events.
  • the PTSG alleles which are not deleted can be screened for point mutations, such as missense, and frameshift mutations. Both of these types of mutations would lead to non-functional PTSG products.
  • point mutational events may occur in regulatory regions, such as in the promoter of the PTSG leading to loss or diminution of expression of the PTSG mRNA. In order to detect the loss of the PTSG wild- type in a tissue, it is helpful to isolate the tissue free from surrounding normal tissues. Means for
  • the tissue may be isolated from paraffin or cryostat sections. Cancer cells may also be separated from normal cells by flow cytometry. These as well as other techniques for separating tumor from normal cells are well known in the art. If the tumor tissue is highly contaminated with normal cells, detection of mutations is more difficult.
  • Detection of point mutations may be
  • the polymerase chain reaction can be used to amplify PTSG sequences directly from a genomic DNA preparation from the tumor tissue. The DNA sequence of the amplified sequence can then be determined.
  • the polymerase chain reaction itself is well known inthe art. See e.g., Saiki et al., Science, 239:487 (1988); U.S. Patent 4,683,203; and U.S. Patent 4,683,195.
  • PTSG specific deletions of PTSG can also be detected.
  • restriction fragment length polymorphism (RFLP) probes for the PTSG or surrounding marker genes can be used to score loss of PTSG allele.
  • Other techniques for detecting deletions, as are known in the art can be used.
  • Loss of wild-type PTSG may also be detected on the basis of the loss of a wild-type expression product of the PTSG.
  • Such expression products include both the mRNA as well as the PTSG protein product itself.
  • POint mutations may be detected by sequencing the mRNA directly or via molecular cloning of cDNA made from the mRNA. The sequence of the cloned cDNA can be determined using DNA sequencing techniques which are well known in the art.
  • the cDNA can also be sequenced via the polymerase chain reaction (PCR) which will be discussed in more detail below.
  • PCR polymerase chain reaction
  • mismatch detection can be used to detect point mutations in the PTSG or its mRNA
  • the method involves the use of a labeled RNA probe which is complementary to the human wild-type PTSG.
  • the riboprobe and either mRNA or DNA isolated from the tumor tissue are annealed (hybridized) together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNA structure. If a mismatch is detected by RNase A, it cleaves at the site of the mismatch.
  • RNA product when the annealed RNA preparation is separated on an electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product will be seen which is smaller than the full-length duplex RNA for the riboprobe and the PTSG mRNA or DNA.
  • the riboprobe need not be the full length of the PTSG mRNA or gene but can be a segment of either. If the riboprobe comprises only a segment of the PTSG mRNA or gene it will be desirable to use a number of these probes to screen the whole mRNA sequence for mismatches.
  • D ⁇ A probes can be used to detect mismatches, through enzymatic or chemical
  • mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes. See, e.g., Cariello, Human Genetics, 42:726 (1988).
  • riboprobes or DNA probes the cellular mRNA or DNA which might contain a mutation can be amplified using PCR (see below) before hybridization.
  • DNA sequences of the PTSG from the tumor tissue which have been amplified by use of polymerase chain reaction may also be screened using allele-specific probes. These probes are nucleic acid oligomers, each of which contains a region of the PTSG sequence DNA sequence harboring a known mutation.
  • one oligomer may be about 30 nucleotides in length, corresponding to a portion of the PTSG DNA sequence. At the position coding for the 175th codon of the oligomer encodes an alanine, rather than the wild-type codon valine.
  • amplification products can be screened to identify the presence of a previously identified mutation in the PTSG.
  • Hybridization of allele-specific probes with amplified PTSG sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe indicates the presence of the same mutation in the tumor tissue as in the allele-specific probe.
  • the kit of the present invention is useful for determination of the nucleotide sequence of the PTSG using the polymerase chain reaction.
  • the kit comprises a set of pairs of single stranded DNA primers which can be annealed to sequences within or surrounding the PTSG in order to prime amplifying DNA synthesis of the PTSG itself.
  • the complete set allows synthesis of all of the nucleotides of the PTSG coding sequences.
  • the set of primers may or may not allow synthesis of both intron and exon sequences. However, it should allow synthesis of all exon sequences.
  • the present invention is also directed to the administration of wild-type PTSG tumor suppressor gene or protein to suppress, eradicate or reverse the neoplastic phenotype in established cancer cells having no
  • the wild-type PTSG gene can be used to suppress or reverse the neoplastic phenotype or properties of established human cancer cells lacking wild-type PTSG protein.
  • This suppression of the neoplastic phenotype in turn suppressed or eradicated the abnormal mass of such cancer cells, i.e. tumors, which in turn can reduce the burden of such tumors on the animal which in turn can increase the survival of the treated animals.
  • the neoplastic properties which are monitored and reversed included the morphology, growth, and most significantly, the tumorigenicity of cancer cells lacking the normal PTSG protein.
  • Neoplastic phenotype is understood to refer to the phenotypic changes in cellular
  • the invention provides PTSG encoding vectors and PTSG proteins for use in treatment of tumors or cancers, and methods of preparing PTSG proteins and vectors suitable for use in methods of treatment.
  • the invention also provides methods for assaying for
  • the invention also provides methods of treatment for mammals such as humans, as well as methods of treating abnormally proliferating cells, such as cancer, such as prostate tumors and colon cancer or other tumor cells or suppressing the neoplastic phenotype.
  • the invention contemplates treating abnormally proliferating cells, or mammals having a disease
  • the invention comprises a method of treating a disease characterized by abnormally proliferating cells, in a mammal, by administering an expression vector coding for PTSG to the mammal having a disease characterized by abnormal proliferating cells, inserting the expression vector into the abnormally proliferating cells, and expressing PTSG in the
  • the invention comprises a method of treating abnormally proliferating cells of a mammal by inserting a PTSG encoding expression vector into the abnormally proliferating cells and expressing PTSG product therein in amounts effective to suppress proliferation of those cells. The treatment is repeated as needed.
  • the invention provides a DNA molecule able to suppress growth of an abnormally proliferating cell.
  • a prostate/colon tumor suppressor protein is PTSG protein product having an amino acid sequence substantially according to SEQ ID NO. 1.
  • the DNA molecule has the DNA sequence of SEQ ID NO. 1, and is expressed by an expression vector.
  • the expression vector may be any host cell-compatible vector.
  • the vector is preferably selected from the group
  • the DNA molecule has the DNA sequence of SEQ. ID No. 2, and is expressed by an expression vector.
  • the expression vector may be any host cell-compatible vector.
  • the vector is preferably selected from the group consisting of a retroviral vector, an adenoviral vector and a herpesviral vector.
  • the DNA molecule has the DNA sequence of SEQ. ID No. 2, and is expressed by an expression vector.
  • the expression vector may be any host cell-compatible vector.
  • the vector is preferably selected from the group
  • the invention consisting of a retroviral vector, an adenoviral vector and a herpes viral vector.
  • the retroviral vector consisting of a retroviral vector, an adenoviral vector and a herpes viral vector.
  • inventions provide a PTSG protein product having an amino acid sequence substantially according to SEQ ID NO. 2 and biologically active fragments thereof.
  • the invention provides a PTSG protein having an amino acid sequence substantially according to Seq. ID No. 4 and biologically active fragments thereof.
  • the invention provides a method of producing a PTSG protein product by the steps of : inserting a compatible expression vector comprising a PTSG encoding gene into a host cell and causing the host cell to express PTSG protein.
  • the invention comprises a method of treating abnormally proliferating cells of a mammal ex vivo by the steps of: removing a tissue sample in need of treatment from a mammal, the tissue sample comprising abnormally
  • the tissue treated ex vivo is blood or bone marrow tissue.
  • the tissue treated ex vivo is blood or bone marrow tissue.
  • invention comprises a method of treating a disease characterized by abnormal cellular proliferation in a mammal by a process comprising the steps of administering PTSG protein to a mammal having a disease characterized by abnormally proliferating cells, such that the PTSG protein is inserted into the abnormally proliferating cells in amounts effective to suppress abnormal
  • the PTSG protein fragments or derivatives thereof is liposome encapsulated for insertion into cells to be treated. The treatment is repeated as necessary.
  • FIG 1 shows KSR2 (8p22) Southern analysis in human prostate cancer. Paired purified prostate cancer DNA (T) and noncancerous DNA (N) from the same patients. The 1.9-kilobase allele is lost in the tumor tissue of patient 4, the 3.3-kilobase allele is lost in the tumor tissues of patients 5 and 6. Patient 7 is not
  • Figure 2 shows the percentage of prostate cancers with loss at loci studied on chromosome 8.
  • Figure 3 shows homozygous deletion of MSR in human prostate cancer.
  • Primary tumor 23 has retained both alleles at D8S201, is uninformative at D8S163, has lost the 6.3-kilobase allele at MSR, and is uninformative at D8S39.
  • Metastatic tumor N2 has lost one allele at D8S201 and at D8S163, while demonstrating complete loss of sequences at MSR. Re-probing the same blot with the 15-65 probe for DCC (18q), a strong signal is obtained at 8 kilobases (kb), demonstrating the presence of high molecular weight DNA in the tumor lane.
  • Both D8S39 alleles are present in tumor N2 and the intensity of the lower allele is multiplied 3-fold.
  • Figure 4 shows deletion map in human prostate cancer. Only tumors demonstrating chromosome 8p loss are illustrated. Samples 1-27 are primary tumors. Figure legend: Nl through N5 are metastatic prostate cancers. O, retained alleles, loss of heterozygosity, X,
  • FIG. 1 Yeast artificial chromosome and radiation hybrid map of loci in chromosome band 8p22, a common region of allelic loss in multiple human cancers. Genomics 24:317-323.
  • Figure 6 shows homologous integration of the conversion vector, which results in amplification of a 1855 bp band.
  • Figure 7 shows the Southern blot of yeast DNA with radiolabeled hygro-gene probe that confirms the presence of the hygro R gene in the YAC arm.
  • Figure 8. Long-range restriction map of YACs encompassing markers on chromosome band 8p22.
  • DNA from YACs 946_c_9, 877_f_2, 932_e_9, and 766_a_12 embedded in agarose beads was digested with various rare-cutting restriction enzymes (A: Asc I, M: Mlu I; N: Not I; Nr: Nru I; Sf : Sfi I) and separated by PFGE as described in Methods. Southern blotting with selected cDNA (i talic) and genomic DNA (roman) probes was performed to identify restriction fragments containing each probe (brackets) . Probes found to be homozygously deleted in Tumor N2
  • Figure 9 Nucleotide sequence and selected restriction sites of the insert of plasmid pBS-N33C(7), derived by cloning into pBluescript the 1.3 kb EcoRI- EcoRI insert from lambda phage clone ⁇ N33C (SEQ ID NO. 5), which was obtained by screening a human placenta cDNA library with selected cDNA probe N33. Selected
  • FIG. 10 Annotated double stranded sequence of N33 cDNA deduced from sequencing phage clone N33C(7) and RT-PCR clones A4 and A5.
  • a 65-bp segment from nt 1186 to 1250 of N33C(7) and A4 sequence is absent from the A5 clone, so N33C(7) and A4 clones represent the longer Form 1 whereas A5 represents the shorter Form 2 mRNA.
  • the presumptive alternative splice results in the utilization of either of two translational stop sites as indicated.
  • the predicted translational start site is also shown preceded by an in-frame stop codon (*).
  • FIG. 11 ORF map for clone N33C(7) representing mRNA form 1.
  • the longest ORF is nt 158 - 1202.
  • the predicted 348 amino-acid polypeptide has MW 39674.13 daltons (SEQ ID NO. 3). The last 5 amino acids differ from the form 2 polypeptide.
  • Figure 14 Translation of longest ORF from mRNA form 2 (SEQ. ID NO 2).
  • the predicted 347 amino-acid polypeptide has MW 39556.18 daltons (SEQ ID NO. 4).
  • the last four amino acids diverge from the form 1
  • Figure 15 Alignment of N33 form 1 and 2 polypeptides with hypothetical 37.7 kD protein encoded by ORF ZK686.3 from C. elegans . Four gaps were introduced into N33 to optimize alignment. 42% of residues were identical between human and C. elegans (underlined). The protein encoded by ORF ZK686.3 has MW 37.7 kD.
  • Figure 16 Northern blot of mRNA from normal human tissues (Clontech) hybridized with selected cDNA probes J2, J28 and N33. N33 mRNA is about 1.5 kb in size and is expressed in most tissues including heart, placenta, lung, liver, pancreas, prostate, testis, ovary and colon. Expression in spleen, thymus, small intestine and peripheral lymphocytes was low.
  • FIG. 17 Northern blot of mRNA from human tumor cell lines hybridized with selected cDNA probes N33, P10, J2 and P16. Actin was used as a control for mRNA loading. N33 expression was not detected in 13 out of 14 colorectal carcinoma cell lines (SW480, SW837, SW1417, HT-29, SW403, LS174T, DLD-1, CACO-2, EB, SK-CO-1, RKO, HCT116 and COLO-302).
  • Figure 18. Northern blot of mRNA from tumor lines PPC-1, WI-38, H460, A549 (lanes 1 - 4), normal colonic mucosa (lane 5), and colon tumor lines SW837 and SW480 (lanes 6 and 7). N33 is expressed in mucosa dissected from colon.
  • Figure 19 RT-PCR assay for N33 expression in
  • RNA from nine prostate cancer specimens (lanes 1-9).
  • C PCR control.
  • N33 primers were N33GEX-f and -r. Primers for the p53, Rb, and G3PD genes were used as controls for RNA/cDNA quality.
  • N33, Rb and p53 primers span exon boundaries and do not specifically amplify genomic DNA. Markedly decreased N33 expression was seen in cases 3, 6 and 9. In tissues expressing N33, both the upper (form 1) and lower (form 2) mRNAs can be seen.
  • Figure 20 Predicted sequence of N33 form 1 polypeptide. The conserved C-terminal 16 amino acids (boxed) was coupled to KLH and used to generate a rabbit polyclonal antibody.
  • Figure 21 Predicted sequence of N33 form 1 polypeptide. The conserved C-terminal 16 amino acids (boxed) was coupled to KLH and used to generate a rabbit polyclonal antibody.
  • Figure 21 Predicted sequence of N33 form 1 polypeptid
  • N33 RT-PCR products from placenta mRNA (primers N33GEX-f and -r) were cloned into pGEX-2T (Pharmacia).
  • Clones A4 and A5 were isolated representing form 1 and form 2 mRNAs, respectively.
  • Protein expression was induced by IPTG and cell lysates were separated by PAGE and transferred to membrane.
  • the Western blot was incubated with affinity- purified polyclonal anti-N33 peptide antibody, and reactive bands were visualized by an alkaline-phosphatase conjugated secondary antibody and NBT/BCIP substrate.
  • a fusion protein band of ⁇ 57 kD was detected in induced cells containing clone A4 but not A5 or other clones.
  • PTSG refers to two proteins: one composed of 348 amino acids and a second of 347 amino acids, each having a molecular weight of approximately 40kD.
  • nucleic acid shall mean single and double stranded genomic DNA, cDNA, mRNA and cRNA.
  • isolated when used to describe the state of the nucleic acids, denotes the nucleic acids free of at least a portion of the molecules associated with or occurring with the nucleic acid in its native environment.
  • Also provided by this invention is a recombinant expression vector or a recombinant
  • replication vector comprising an isolated nucleic acid molecule corresponding to a tumor suppressor gene as well as host cells, e.g., bacterial cells, containing these vectors.
  • Retroviral vectors in this context are retroviruses from which all viral genes have been removed or altered so that no viral proteins are made in cells infected with the vector. Viral replication functions are provided by the use of retrovirus 'packaging' cells that produce all of the viral proteins but that do not produce infectious virus. Introduction of the retroviral vector DNA into packaging cells results in production of virions that carry vector RNA and can infect target cells, but no further virus spread occurs after
  • a delivery system for insertion of a nucleic acid is a replication-incompetent retroviral vector.
  • retroviral includes, but is not limited to, a vector or delivery vehicle having the ability to selectively target and introduce the nucleic acid into dividing cells.
  • replication-incompetent is defined as the inability to produce viral proteins, precluding spread of the vector in the infected host cell.
  • LNL6 Miller, A.D. et al.
  • retroviral vectors for gene therapy are the high efficiency of gene transfer into replicating cells, the precise integration of the transferred genes into cellular DNA, and the lack of further spread of the sequences after gene transduction (Miller, A.D., Nature, 357:455-460 (1992)).
  • retroviral vectors remains a concern, although for practical purposes this problem has been solved. So far, all FDA-approved retroviral vectors have been made by using PA317 amphotropic retrovirus packaging cells
  • Non-retroviral vectors have been considered for use in genetic therapy.
  • One such alternative is the adenovirus (Rosenfeld, M.A., et al., Cell. 68:143-155 (1992); Jaffe, H.A. et al., Proc.
  • adenovirus vectors Major advantages of adenovirus vectors are their potential to carry large segments of DNA (36 kb genome), a very high titre (10 11 ml -1 ), ability to infecting tissues in situ, especially in the lung. The most striking use of this vector so far is to deliver a human cystic fibrosis transmembrane conductance
  • CTR CFTR gene by intratracheal instillation to airway epithelium in cotton rats
  • CFTR herpes viruses
  • herpes viruses may also prove valuable for human gene therapy (Wolfe, J.H., et al., Nature Genetics 1:379-384 (1992)).
  • any other suitable viral vector may be used for genetic therapy with the present invention.
  • Plasmid DNA should be easy to certify for use in human gene therapy because, unlike retroviral vectors, it can be purified to homogeneity. In addition to liposome- mediated DNA transfer, several other physical DNA
  • the PTSG of the present invention may be placed by methods well know to the art into an expression vector such as a plasmid or viral expression vector.
  • a plasmid expression vector may be introduced into a tumor cell by calcium phosphate transfection, liposome (for example, LIPOFECTIN) -mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection,
  • a viral expression vector may be introduced into a target cell in an expressible form by infection or transduction.
  • a viral vector includes, but is not limited to: a retrovirus, an adenovirus, a herpes virus and an avipox virus.
  • PTSG When PTSG is expressed in any abnormally proliferating cell, the cell replication cycle is arrested, thereby resulting in senescence and cell death and ultimately, reduction in the mass of the abnormal tissue, i.e., the tumor or cancer.
  • a vector able to introduce the gene construct into a target cell and able to express H-NUC therein in cell proliferation- suppressing amounts can be administered by any effective method.
  • a physiologically appropriate solution containing an effective concentration of active vectors can be administered topically, intraocularly, parenterally, orally, intranasally, intravenously, intramuscularly, subcutaneously or by any other effective means.
  • the vector may be directly
  • injected into a target cancer or tumor tissue by a needle in amounts effective to treat the tumor cells of the target tissue.
  • a cancer or tumor present in a body cavity such as in the eyes, gastrointestinal tract, genitourinary tract (e.g., the urinary bladder),
  • pulmonary and bronchial system and the like can receive a physiologically appropriate composition (e.g., a solution such as a saline or phosphate buffer, a suspension, or an emulsion, which is sterile except for the vector) containing an effective concentration of active vectors via direct injection with a needle or via a catheter or other delivery tube placed into the cancer or tumor afflicted hollow organ.
  • a physiologically appropriate composition e.g., a solution such as a saline or phosphate buffer, a suspension, or an emulsion, which is sterile except for the vector
  • Any effective imaging device such as X-ray, sonogram, or fiberoptic visualization system may be used to locate the target tissue and guide the needle or catheter tube.
  • a physiologically appropriate solution containing an effective concentration of active vectors via direct injection with a needle or via a catheter or other delivery tube placed into the cancer or tumor afflicted hollow organ.
  • Any effective imaging device such as X-ray, sonogram, or fiber
  • concentration of active vectors can be administered systemically into the blood circulation to treat a cancer or tumor which cannot be directly reached or anatomically isolated.
  • target tumor or cancer cells can be treated by introducing PTSG protein into the cells by any known method.
  • liposomes are artificial membrane vesicles that are available to deliver drugs, proteins and plasmid vectors both in vitro or in vivo (Mannino, R.J., et al.,
  • PTSG protein can be encapsulated at high efficiency with liposome vesicles and delivered into mammalian cells in vitro or in vivo.
  • Liposome-encapsulated PTSG protein may be administered topically, intraocularly, parenterally, intranasally, intratracheally, intrabronchially, means at a dose efficacious to treat the abnormally proliferating cells of the target tissue.
  • the liposomes may be administered topically, intraocularly, parenterally, intranasally, intratracheally, intrabronchially, means at a dose efficacious to treat the abnormally proliferating cells of the target tissue.
  • the liposomes may be administered topically, intraocularly, parenterally, intranasally, intratracheally, intrabronchially, means at a dose efficacious to treat the abnormally proliferating cells of the target tissue.
  • the liposomes may be administered topically, intraocularly, parenterally, intranasally, intratracheally, intrabronchially, means at a dose efficacious to treat the abnormally proliferating cells of the target tissue.
  • composition containing an effective concentration of encapsulated PTSG protein composition containing an effective concentration of encapsulated PTSG protein.
  • "Host-vector system” refers to host cells which have been transfected with vectors constructed using recombinant DNA techniques. Insertion of the vector or DNA can be accomplished by microcell transfer, retrovirus-mediated gene transfer, transfection, cell fusion, etc.
  • the vectors and methods disclosed herein are suitable for use in host cells over a wide range of prokaryotic and eukaryotic organisms. Additionally, this invention provides a method of transforming a cell by contacting the cell with the vector or DNA of this invention, under suitable conditions.
  • recombinant DNA methods currently used by those skilled in the art include the polymerase chain reaction (PCR) which, combined with the synthesis of oligonucleotides, allows easy reproduction of DNA sequences.
  • PCR polymerase chain reaction
  • a DNA segment of up to approximately 6000 base pairs in length can be amplified exponentially starting from as little as a single gene copy by means of PCR.
  • a denatured DNA sample is incubated with two oligonucleotide primers that direct the DNA polymerase-dependent synthesis of new
  • thermostable DNA polymerase eliminates the necessity of adding new enzyme for each cycle, thus permitting fully automated DNA amplification. Twenty- five amplification cycles increase the amount of target sequence by approximately 10 6 -fold.
  • the PCR technology is the subject matter of United States Patent Nos.
  • the probes are useful to detect the predisposition to a cancer caused by the malfunction of this gene.
  • the isolated nucleic acid fragments also are useful to generate novel peptides. These peptides, in turn, are useful as immunogens for the generation of polyclonal and monoclonal antibodies useful in diagnostic methods outlined below. Methods of preparing and using the probes and immunogens are well known in the art, and are briefly described below. Also included within the scope of this
  • nucleic acid molecules that hybridize under stringent conditions to an isolated nucleic acid molecule encoding this tumor suppressor protein.
  • Such hybridizing nucleic acid molecules or probes can by prepared, for example, by random priming of this nucleic acid molecule.
  • For methodology for the preparation of such fragments see Sambrook et al. (Sambrook et al., "Molecular
  • Purified tumor suppressor polypeptide or protein also is provided by this invention. These polypeptides and/or proteins are useful to prepare antibodies, which in turn are useful for diagnosis. They can be produced by recombinantly expressing an isolated nucleic acid molecule of this invention using well known molecular biology techniques. "Purified”, when used to describe the state of the protein, polypeptide, or antibody, denotes such protein free of a portion of the other proteins and molecules normally associated with or occurring with the tumor suppressor polypeptide, protein or antibody in its native environment. As used herein the term “native” refers to the form of a protein, polypeptide, antibody or a fragment of thereof that is isolated from nature or that which is without an intentional amino acid
  • immunoglobulin refers to a protein that is produced in response to immunization with an antigen and specifically reacts with the antigen. This includes polyclonal as well as monoclonal antibodies. Human and mammalian, for example, mouse, rat, rabbit and goat, are intended to be included in this definition. The most predominant human antibody produced is of the IgG isotype, having two light and two heavy chains linked by disulfide bonds, which constitute about 80% of total serum antibodies.
  • Anti-tumor suppressor antibodies can be any anti-tumor suppressor antibodies.
  • Purified fusion protein is useful as an antigen in generating specific anti-PTSG antibody.
  • Rabbits can be repeatedly injected, preferably at 14 day intervals with 1-20 ⁇ g, preferably 10 ⁇ g, of purified fusion protein mixed with complete Freund's adjuvant (initial injection) and then given booster injections of the same amount of the fusion protein in incomplete Freund's adjuvant (repeated injections).
  • Complete Freund's adjuvant generally consists of an emulsion of the antigen, in this case the fusion protein, in saline and a mixture of an emulsifying agent, such as for example Arlacel A, in mineral oil with killed
  • Incomplete Freund's adjuvant is the same except that it does not have the mycobacteria.
  • two or more affinity columns can be prepared using a method generally described in Harlow and Lane (Harlow and Lane,
  • At least one column is coupled with glutathione S transferase (GST) protein and at least one column is loaded with the fusion protein. Both columns are appropriately precycled.
  • GST glutathione S transferase
  • Antibody is passed first through the fusion protein- Sepharose column and eluted with glycine buffer of pH 2.3. The eluate is neutralized and passed through the GST column several times to remove antibody specifically directed against GST.
  • the purified anti-prostate tumor suppressor protein is useful for immunoprecipitation or immunostaining, for localization of prostate tumor suppressor protein and will be equally useful for
  • the purified proteins also are within the scope of this invention. It can be labeled with a detectable marker such as radioisotypes, dyes, enzymes and biotin.
  • the fusion proteins also can be used to
  • this invention provides a monoclonal antibody directed to an epitope on the prostate tumor suppressor protein or polypeptide.
  • the monoclonal antibody is a mouse monoclonal antibody.
  • the monoclonal antibody is a human monoclonal antibody.
  • mice For the isolation of mouse monoclonal antibodies, eight week old mice can be injected
  • mice interperitoneally with about 50 micrograms of a purified prostate tumor suppressor polypeptide (prepared as described above) in complete Freund's adjuvant 1:1 volume. Mice are then boosted, at monthly intervals, with the polypeptide, mixed with incomplete Freund's adjuvant, and bled through the tail vein. On days 4, 3 and 2 prior to fusion, mice are boosted intravenously with 50 micrograms of the polypeptide in saline.
  • Splenocytes are fused with non-secreting myeloma cells according to procedures which have been described and are known to those of ordinary skill in the art to which this invention pertains. Some time later, approximately two weeks later, hybridoma supernatant are screened for binding activity against the prostate tumor polypeptide as described hereinafter. Positive clones are isolated and propagated.
  • this invention also provides the monoclonal antibody described hereinabove conjugated to a therapeutic agent.
  • suitable therapeutic agents include, but are not limited to, a therapeutic agent selected from the group
  • chemotherapeutic agents Also provided by this invention is the monoclonal antibody described hereinabove
  • detectable markers include, but are not limited to, enzymes,
  • radioisotopes such as 32 P, 35 S and 131 I.
  • compositions comprising the purified prostate tumor suppressor polypeptide or protein
  • compositions comprising the monoclonal antibody described hereinabove alone, or conjugated to any one of the following: a detectable marker, a therapeutic agent, or an imaging agent, as described hereinabove and a pharmaceutically acceptable carrier.
  • pharmaceutical compositions comprising the monoclonal antibody described hereinabove alone, or conjugated to any one of the following: a detectable marker, a
  • phosphate buffered saline solution water
  • emulsions such as an oil/water emulsion
  • wetting agents various types
  • antibody also encompasses fragments of antibodies.
  • the antibody fragments retain at least some ability to selectively bind with its antigen.
  • antibody fragments that have been recombinantly or chemically synthesized that retain the ability to bind the antigen of the corresponding native antibody.
  • the ability to bind with an antigen or hapten is determined by antigen- binding assays known in the art such as antibody capture assays (See, for example, Harlow and Lane, (Harlow and Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988)).
  • Antibody fragments retaining some binding affinity include, but are not limited to: Fab (the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion with the enzyme papain to yield an intact light chain and a portion of one heavy chain); Fab' (the fragment of an antibody molecule obtained by treating with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule); (Fab') 2 , the fragment of the antibody that is obtained by treating with the enzyme pepsin without subsequent reduction; F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds; Fv and single chain antibodies (SCA). Also within the scope of this invention are CDR grafted and chimeric antibodies retaining the ability to bind prostate tumor suppressor protein.
  • chimeric antibody refers to an antibody in which the variable regions of antibodies derived from one species are combined with the constant regions of antibodies derived from a different species. Chimeric antibodies are constructed by
  • CDR grafted antibody refers to an antibody having an amino acid sequence in which at least parts of one or more CDR sequences in the light and/or variable domain have been replaced by analogous parts of CDR sequences from an antibody having a different binding specificity for a given hapten or antigen.
  • the analogous CDR sequences are said to be “grafted” onto the substrate or recipient antibody (see European Patent Publication No. 0 239 400).
  • the "donor” antibody is the antibody providing the CDR sequence, and the antibody receiving the substituted sequences is the "substrate” antibody.
  • a method of detecting the presence or absence, in a sample, of a protein, the absence of which is associated with a neoplasm, is provided by this
  • the method will include cell staining with polyclonal or monoclonal antibodies raised against the protein.
  • this method comprises the steps of obtaining a suitable sample from a subject. Suitable samples include, but are not limited to: prostate tumor tissue, colon tumor tissue, lymph node tissue and bone marrow cells.
  • the method requires contacting the sample with an agent specifically unique to the tumor suppressor protein under conditions favoring the formation of a complex with the agent then detecting the presence of any complex formed.
  • the absence of complex indicating the absence of a protein, which is associated with a neoplastic state such as prostate adenocarcinoma.
  • this method is useful to diagnose prostate adenocarcinoma.
  • suitable labeling agents are
  • radioisotopes such as 32 P, 35 S and 131 I, but also includes, but is not limited to dyes and enzymes.
  • the agent can be an antibody raised against the protein or a unique subregion of the protein, the absence of which is associated with prostate cancer.
  • a method of detecting the presence or absence, in a sample, of a tumor suppressor gene or nucleic acid, the absence of which is associated with a neoplasm is provided by this invention. This method comprises the steps of obtaining a suitable sample from a subject.
  • Detection methods for the presence of nucleic acid in cells include hybridization of a nucleic acid probe with the nucleic acid of a cell. Such techniques are
  • Suitable samples include, but are not limited to: prostate tumor tissue, colon tumor tissue, lymph node tissue and bone marrow cells.
  • the method requires contacting the sample with an agent specifically unique to wild-type or normal tumor suppressor gene under conditions favoring the formation of a complex with the agent then detecting the presence of any complex formed.
  • the absence of complex indicating the absence of a wild- type gene, which is associated with a neoplastic state such as prostate adenocarcinoma.
  • suitable detectable labels include radioisotopes e.g., 32 P, 35 S and 131 I, and includes, but not limited to additional labeling agents, such as dyes and enzymes.
  • the agent can be a nucleic acid molecule corresponding to the tumor suppressor protein or a unique subregion thereof.
  • a kit for the detection, diagnosis or prognosis of prostate cancer is provided by this invention.
  • the kit includes the reagents useful to carry out the methods described above and instructions for their use in the methods.
  • a kit can be used for the direct genetic detection of pathological alterations in the prostate tumor suppressor gene, and can include oligonucleotides, primers for PCR analysis, reagents for SSCP, or
  • kits, reagents and methods also are useful for prognosis. For example, deletion may be indicative of a less favorable prognosis for recovery.
  • compositions containing, at least, any of the above- references nucleic acids, peptides, or antibodies.
  • These compositions also can contain carriers or diluents such as phosphate buffered saline, emulsions or various wetting agents.
  • Prostate cancer tissue was obtained from patients undergoing radical prostatectomy for clinically localized prostate cancer between August 1988 and
  • tissue Paired noncancerous tissue (seminal vesicle, prostate, or blood lymphocytes) was obtained from each patient. Seminal vesicle or prostate tissue serving as source material for noncancerous DNA was examined every 300 ⁇ m by PTSG section, and all tissue containing
  • Prostate specific antigen containing greater than 70% prostate cancer nuclei was isolated from surrounding tissue (containing benign prostate epithelia, stroma, lymphocytes, etc.) as much as possible using a cryostat sectioning technique described in Bos, J.L. et al., (1987). All prostate carcinomas studied were of the usual acinar type and were ⁇ 2 cm in diameter. DNA isolation and quantification were performed as described in Carter, B.S. et al., (1990) and Burton, K., (1968).
  • Samples were cleaved with restriction endonucleases (BRL and New England Biolabs) with the buffers recommended by the supplier, using 10 units of enzyme/ ⁇ g of DNA for Mspl digests and 7.5 units/ ⁇ g for Taql digests. Samples were electrophoresed in 0.8% agarose gels and transferred to Nytran nylon membranes (Schleicher & Schuell) in 0.4 M sodium hydroxide/0.6 M sodium chloride after depurination in 0.25 N HCl for 10 minutes.
  • restriction endonucleases BRL and New England Biolabs
  • DNA probes KSR2, NF 5.1, and MCT 128.2 were obtained from the American Type Culture Collection.
  • Probes CI8-1, MSR-32 (MSR-macrophage scavenger receptor), CI8-319, and CI8-277 are cosmid probes that have been described in Emi, M. et al., (1993). Probes were labeled using random hexamer priming and incorporation of [ ⁇ - 32 P]dCTP (Amersham) with the Klenow fragment of DNA polymerase I (Amersham).
  • Probes CI8-1, MSR-32, CI8-319, and CI8-277 were boiled with sheared human placental DNA (Sigma), (0.2 mg/ml), cooled briefly on ice, and hybridized at 65°C overnight.
  • Probes KSR2, NEFL, and MCT 128.2 were boiled with 0.5 ml of 2 mg/ml denatured sonicated salmon sperm DNA, briefly cooled on ice, and hybridized at 65°C overnight. After hybridization, membranes were washed in 0. IX standard saline-phosphate-EDTA 0.1% sodium dodecyl sulfate for 15 minutes and were subsequently exposed to Kodak XAR-5 film at -80°C in cassettes with amplifying screens.
  • Allelic loss was defined as the absence of one allele in prostate tumor DNA compared to the noncancerous paired control DNA. In some cases, when there was residual signal from contaminating normal tissue, densitometry was used for analysis. A sample was scored as having allelic loss if a 60% reduction was present in the diminished allele compared to its normalized retained counterpart.
  • Allelic multiplication using probe MCT 128.2 was defined as an increase in intensity of greater than 100% of one of two alleles present in tumor samples, or intensity differences of greater than 100% between tumor and normal alleles in homozygous cases when prior probing of the same blots demonstrated equal loading of DNA in tumor and normal lanes. 4. Microsatellite Analysis
  • LPL lipoprotein lipase
  • GZ 14 and Mfd 199 primer sets were as previously published in Tomfohrde, J. et al., (1992).
  • One of each pair of primers (LPL GZ 14 and Mfd 199R) was end-labeled with [ ⁇ - 32 P]ATP (ICN Biomedicals) using polynucleotide kinase (Boehringer-Manneheim) and 5X kinase buffer [0.25 M Tris, (pH 9.0), 50mM MgCl 2 , 50mM dithiothreitol, and 0.25 mg/ml bovine serum albumin].
  • One ⁇ l labeled primer was added to 1 ⁇ l unlabeled primer (10 ⁇ M), 0.5 ml deoxynucleotide triphosphate mix (equal volumes of dATP, dCTP, dGTP, and dTTP each at 10mM), 5.5 ⁇ l sterile deionized water, and 10X Taq DNA polymerase buffer (Perkin-Elmer), 10 ⁇ l genomic DNA were added (2.5 ng/ ⁇ l), and the mixture was heated to 94°C.
  • thermocycling was then performed with 30 cycles of denaturation at 94°C for 30 seconds, annealing at 62°C (LPL) or 58°C (Mfd 199) for 30 seconds, and extension at 72°C for 30 seconds. This was followed by 72°C for 7 minutes. Products were then mixed with an equal volume of stop buffer containing 95% formamide, 0.05% xylene cyanol, 0.05% bromophenol blue, and 20 mM EDTA. Samples were heat denatured at 94°C and 3- ⁇ l aliquots of each sample were loaded on 6% acrylamide gels containing 8.0 M urea. Gels were dried and exposed to Kodak XAR film. In this study, allelic loss using microsatellite analysis was determined according to criteria similar to those used in Southern analysis described above.
  • noncancerous prostate including areas of benign prostatic hypertrophy and normal prostate
  • Liver tissue from a single patient obtained at autopsy served as positive control for MSR staining.
  • Well preserved central and peripheral zone prostate tissue was obtained from the same patient at autopsy and stained for MSR protein. This patient had no evidence of malignancy at autopsy and prostate tissue was normal on gross examination and histologically.
  • Unfixed air-dried 6- ⁇ m frozen sections on glass slides were warmed to room temperature and fixed in 2%
  • one metastatic prostate cancer sample demonstrated homozygous deletion of MSR sequences.
  • Hybridization of the same blot with the DCC probe 15-65 establishes the presence of intact DNA of equivalent or larger size in the N2 tumor lane ( Figure 3) .
  • Repeat digestion of N2 DNA with Mspl, Taql, and EcoRI and probing for MSR has confirmed this finding, but at least one allele is present.
  • the boundary of the homozygous deletion is thus delimited by D8S163 and LPL.
  • loci telomeric and centromeric to this region are largely retained, with loss of one or more loci in only 9 of 48 (19%) of informative cases.
  • Distal loci studied on 8p23 are largely retained, with loss in only 4 of 38 (11%) of informative cases at D8S140 and in only 3 of 22 (14%) of cases at D8S201 (Table 1).
  • Loci studied on 8pll.2 and 8q24 are also infrequently deleted, with loss identified in 3 of 26 (12%) of informative cases at D8S194 and in 2 of 17 (12%) at D8S39.
  • Macrophage scavenger receptor protein was not detected among prostate cancer cells or noncancerous prostate epithelia. Scattered cells contained within the stroma of each of the prostate sections stained positively, consistent with staining in macrophages only.
  • Plasmid probe pABL4-2 detecting D8S21 was obtained from R. White and its preparation is disclosed in Tsui, L.C. et al. (1989). Its insert was partially sequenced by priming from E. coli amber suppressor tRNA Tyr using oligonucleotide 5'-GAATCCTTCCCCCAC-3', and two PCR primers were designed to create an STS (Table 2). Lambda phage CRI-R191 detecting D8S26 was obtained from the ATCC. A 4.2 kb EcoRI restriction fragment of this phage was subcloned and partially sequenced, from which an STS was designed (Table 2).
  • Cosmid CI8-487 detecting D8S233 was obtained from the Japanese Cancer Research Resources Bank. A 2.2 kb EcoRI restriction fragment of this cosmid was subcloned and partially sequenced to create an STS (Table 2). New STSs (Table 3) were created by partially sequencing random subclones of purified YAC DNA (see below). YAC end fragments (Table 3) were obtained by the inverse PCR method of Albertsen and Thliveris (Joslyn et al., 1991). PCR products were ligated into TA cloning vector (Invitrogen) and sequenced, from which STSs were made (Table 3). The remaining primer sequences were obtained from sources indicated in Tables 2 and 3.
  • DNA from a human chromosome 8 x CHO somatic cell hybrid mapping panel (Wagner et al., "A hybrid cell mapping panel for regional localization of probes to human chromosome 8.” Genomics 10:114-125 (1991)) was kindly provided by M. Wagner.
  • YAC clone stocks were streaked onto AHC-agar plates. Single pink colonies were picked and grown in 5 ml of YPD media at 30°C overnight, then expanded to 100 ml for an additional 24 hours. Yeast cells were
  • Genome-qualified low melting point agarose (1% in SE) and mineral oil were separately equilibrated to 45°C, and beakers containing 100 ml of ice-cold SE and a stir-bar were placed in ice buckets over magnetic stirrers at medium speed.
  • Five ml of agarose were added to cells in each flask and mixed.
  • Twenty ml of mineral oil were then added and the flask was swirled vigorously for 30 seconds to emulsify the contents, which were then poured immediately into an iced SE beaker. Beads were formed within 5 minutes.
  • the mixture in each flask was transferred to several 50 ml centrifuge tubes and spun at low speed to separate aqueous and oil layers.
  • a 0.6-cm thick, 1% agarose gel in 0.5X TBE was poured in a 20 cm wide x 14 cm long gel casting unit with 2 or 3 preparative wells. Wells were loaded with low- melt agarose beads containing YAC DNA and sealed with low-melt agarose.
  • Yeast chromosomes were separated on a BioRad CHEF-DR III PFGE apparatus running at 60-120 sec switch times ramped over 24 hours at 6 V/cm at a 120° angle in 0.5X TBE at 14°C. The gel was stained in 1 ⁇ g/ml ethidium bromide in 0.5X TBE for 30 minutes and chromosomes visualized by UV irradiation. Slots 5-7 mm wide were cut parallel to and in front of each YAC to be isolated, and the gel was replace on its platform.
  • YAC DNA ( ⁇ 50 ng) was digested with Bgl II and ligated into BamH I-digested pBluescript (Stratagene) by standard methods (Sambrook et al., "Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. pp. 1.98-1.104 (1989)). The ligation mix was redigested with BamH I to reduce nonrecombinant background and transformed into E. coliDH10B (GIBCO-BRL) with X-gal and IPTG for blue-white selection per
  • Plasmids derived from white colonies were screened for use as single-copy probes in Hind III-digested human genomic DNA (Sambrook et al., "Molecular cloning: a laboratory manual.” Cold Spring
  • Biotin-Labelling of YAC DNA Isolated YAC DNA (10 - 20 ⁇ l) was biotin- labelled by random primer extension in the presence of biotinylated dATP (BioPrime kit, BRL) in 50 ⁇ l volumes according to kit instructions. Successful labelling was verified by running 5 ⁇ l of reaction product on agarose gels and either visualizing a faint smear by ethidium bromide and UV irradiation or by transferring the DNA onto nylon membrane by standard methods. The membrane was blocked as for a Western blot and streptavidin- conjugated alkaline phosphate was added directly without primary or secondary antibodies. The biotin-labelled DNA smear was visualized by the BCIP/NBT substrate reaction.
  • Primer 2 was 5'-phosphorylated with ATP and T4 kinase (37°C, 30 min), heated to inactivate the enzyme, and annealed to
  • Poly-A + RNA was selected from total RNA with biotinylated oligo-dT primers and streptavidin-conjugated paramagnetic particles
  • Double-stranded cDNA was made from poly-A + RNAs and one sample of total RNA per manufacturer's instructions with random primers and M-MLV RT (RiboClone cDNA synthesis kit, Promega). A final step with T4 DNA polymerase yielded blunt-ended cDNAs. cDNA made from total RNA was set aside for later use as a probe for ribosomal DNA (rDNA). Excess linkers (see above) were ligated to the poly-A + -derived cDNAs with T4 DNA ligase. cDNA (1-5 ⁇ l) was amplified in 100 ⁇ l volumes using ⁇ 500 ng of Primer 1 and other PCR
  • PCR products were purified with Wizard PCR Prep spin columns (Promega) and eluted in 50 ⁇ l of 0.5 X TE. DNA was quantitated by DNA Dipstick (Invitrogen); typical yields were 500 ng of purified product per 100 ul reaction. Amplified cDNAs examined by agarose gel electrophoresis and ethidium bromide staining comprised a broad streak with maximal intensity at about 500 bp.
  • Purified amplified cDNA (1-2 ⁇ g) was mixed with equal amounts (w/w) of Cot 1 DNA (GIBCO-BRL) and reaction volumes were adjusted to 80 ⁇ g/ml in 120 mM NaPO 4 buffer pH 7 (e.g., 50 ⁇ l of cDNA was reduced to 21 ⁇ l by
  • Biotin-labelled YAC DNAs (100 ng or ⁇ 10 ⁇ l of labelling reaction per hybridization) were heat-denatured and loaded into Centricon 100 filter units with blocked cDNAs (1 ⁇ g excluding Cot 1 DNA) and 2 ml of 1 mM NaPO 4 , pH 7, and spun at 1000 x g for ⁇ 25 minutes. The phosphate buffer wash was repeated once, and the retentate (60-80 ⁇ l) was collected into microfuge tubes.
  • Streptavidin-conjugated paramagnetic particles (Promega) were prewashed twice with TE + 1 M NaCl then incubated with completed hybridization reactions in 200 ⁇ l of TE + 1 M NaCl at room temperature for 15 minutes. Particles were collected magnetically and supernatants were removed. Particles were washed 5 times with 15 minutes incubations in 200 ⁇ l of 0.1 X SSC + 0.1% SDS, two at room temperature, then three at 60°C, with
  • Bound cDNA was eluted from particles with 100 ⁇ l of 50 mM NaOH for 15 minutes, neutralized with 100 ⁇ l of 1 M Tris-HCl pH 7.5, and transferred to clean tubes. Supernatants were desalted and concentrated using Nal and silica matrices (Geneclean kit, Bio 101) per manufacturer's instructions into 20 ⁇ l volumes of TE. These cDNAs were re-amplified exactly as for the original libraries (see above) except that 5 ⁇ l of templates were used and PCR was carried out for 30 cycles. The resulting products were purified and blocked with Cot 1 DNA exactly as above. Selection with YAC DNA was also carried out a second time as above.
  • Second-round selected cDNAs were captured as above and amplified one more time.
  • Final PCR products were cloned directly into T-vector (Novagen), transformants of which were plated onto Tet + Amp LB-agar plates with X-gal and IPTG for blue-white selection per kit instructions. 12. Screening Recombinant Clones
  • nick-translated total human DNA Alu probe
  • random-primed rDNA ribosomal DNA probe
  • the CEPH megabase YAC library (-22,000 clones) was screened by PCR with primers for six simple tandem repeat polymorphisms (STRPs) and four RFLP-containing loci on several independent linkage maps (Fig. 5, Table 2) . About 30 YAC clones were identified and confirmed with the initial screens. Additional clone addresses were obtained by searching AluPCR and fingerprinting overlap tables (Cohen et al., Science 250:245-250 (1993)). These clones were integrated into the YAC map after being tested for STS content. A set of 31 markers was used to assemble the map, including the ten screening STSs, one additional published STRP (D8S206), two expressed
  • the YAC map was anchored to cytogenetic maps by the chromosomal location of the MSR and LPL (lipoprotein lipase) genes (8p22) (Mattei et al.,
  • probe El a random subclone of YAC 932_e_9, detected a Hind III RFLP in human DNA with two alleles, 12 kb and (8 kb + 4 kb).
  • YACs are subject to two kinds of rearrangement artifact, chimerism and internal deletion, which
  • the map was assembled based on standard mapping methodologies, including analysis of partial and double-enzyme digests.
  • cosmid CI8-2644, obtained from Dr. Y. Nakamura was located telomeric to the MSR gene rather than centromeric as suggested by Fujiwara et al. (Fujiwara et al., Genes Chromsom. Cancer 10:7-14 (1994)).
  • DNA from the single metastatic prostate tumor with a homozygous deletion of MSR was examined by Southern blotting analysis with numerous newly isolated genomic and selected cDNA probes in order to map the extent of this deletion. Probes found to be completely deleted in this tumor (boldface, Figure 8) begin with MSR and extend telomerically through probes 877-15 and CCI8-2644.
  • Markers Elc and 877-13 are the closest retained loci at the centromeric and telomeric ends, respectively. Based on the positions of lost and retained loci within mapped restriction fragments (Figure 8), the minimum and maximum sizes of the homozygous deletion in this tumor were determined at 740 kb and 920 kb, respectively. The target tumor suppressor gene was presumptively located within this region and was inactivated by this deletion. N33 was located within this region ( Figure 8).
  • mapping of cCI ⁇ -2644 to a position near the telomeric deletion boundary was significant because it suggests that the common region of allelic loss detected in colorectal, liver, and lung cancers found by Fujiwara et al. (Fujiwara et al., Genes Chromosom. Cancer 10:7-14 (1994) overlaps extensively with this region of
  • any gene within the homozygous deletion may also be important in these other cancers.
  • the size of the allelic loss region in the latter report must be larger than that stated in the paper (600 kb) and larger than the homozygous deletion in this tumor, ie., the homozygous deletion defines the smallest known critical region containing the putative tumor suppressor gene.
  • J12 contains sequences 95% identical to that of human protein phosphatase type 2C alpha subunit, i.e., a known gene that has not yet been localized. The sequence
  • J12 was a pseudogene for human protein phosphatase type 2C alpha subunit.
  • L21, ⁇ 21, N33, N36 and P14 overlap among each other and define a partial ORF with highly significant homology to a predicted gene in C. elegans identified by random sequencing of genomic cosmid or cDNA clones (SWISS-PROT P34669; GENBANK M88869, T01933,
  • cDNA clone N33 was used as a probe to screen a placenta lambda phage cDNA library (Clontech). Clone XN33C was isolated and its 1.3 kb EcoRI-EcoRI insert was subcloned into pBluescript to yield pBS- N33C(7). Sequencing revealed a 1342-bp insert flanked by EcoRI sites (Figs. 9, 10) and encoding a long ORF (nt 158 - 1202) (Fig. 11). Oligonucleotide primers N33GEX-f and N33GEX-r were synthesized based on this sequence (Fig.
  • RT-PCR products were cloned into pGEX-2T (Pharmacia) and two clones, A4 and A5, were isolated.
  • Clone A4 was colinear with pBS-N33C(7) whereas A5 lacked nt 1186 - 1250 (65 bp) compared to the other clones. Consequently, we presume that N33C(7) and A4 clones represent the longer (Form 1) mRNA whereas A5 represents the shorter Form 2 mRNA.
  • the ORFs encoded by the two forms differ over the last -20 bp and utilize different termination codons (Figs. 10-14).
  • the two ORFs are identical through residue 343 then encode 4 or 5 different C-terminal amino acids each.
  • N33C(7) was C in N33C(7) but T in A4 and A5 (Fig. 10). This change does not affect the Form 1 ORF encoded by N33C(7) because it occurs after stop codon 1. It is not known whether this difference represents a natural polymorphism, a cloning artifact, or a mutation in one or more of these clones. Both N33 predicted polypeptides were highly homologous (p ⁇ e -100 ) to the C. elegans predicted cDNA
  • N33 was not significantly related to any other sequences in GENBANK, PIR, SWISS-PROT or EMBL. 18. Expression of N33 in tissues, tumors and cultured cells.
  • Various selected cDNA clones were used to probe Northern blots containing mRNA from several normal human tissues, examples of which are shown in Fig. 16. A single mRNA of about 1.5 kb in size was detected with N33 probes in most tissues including heart, placenta, lung, liver, pancreas, prostate, testis, ovary and colon.
  • peripheral lymphocytes was low. Expression detected by another clone, J2, was seen mostly in skeletal muscle and testis, whereas two messages detected by clone J28 were found principally in placenta, testis and ovary.
  • adenocarcinoma was dissected from the colonic wall and tested for N33 mRNA, and specific expression was observed (Figure 18, lane 5). Finally, small amounts of total RNA were extracted from nine fresh prostate cancer samples (7 primary tumors and 2 metastases). Cryomicrotome-directed dissection was employed to reduce the numbers of
  • RNA, RT-PCR with N33-specific primers was employed to quantitate N33 expression.
  • Primers from Rb, p53 and G3PD were used to control for RNA quality and cDNA synthesis. Markedly decreased expression of N33 was observed in three cases (lanes 3, 6 and 9), where lane 6 RNA was obtained from Tumor N2 to verify the function of this assay.
  • Lane 3 and 9 RNAs were obtained from primary tumors, in which some quantity of N33 message is expected to be contributed by nonneoplastic cells.
  • N33 expression in colorectal tumor cells and prostate tumor tissues is unknown, but could be due to somatic mutations (e.g., affecting mRNA epxression or stability), methylation changes, or other epigenetic regulatory factors.
  • one prostatic tumor is known to have a large homozygous deletion in band 8p22
  • the genetic status of additional primary and metastatic prostate and colorectal tumors are determined by several methods, as follows: 1) Southern blots of tumor D ⁇ As are hybridized with ⁇ 33 cDNA probes and other 8p22 markers to detect homozygous deletions or genetic rearrangements 2) the structure of the PTSG locus is determined by cloning / sequencing at the genomic DNA level by standard techniques. For example, a PI clone containing the N33 gene has been isolated and is
  • PCR primers for amplifying each exon is synthesized.
  • Antibodies reactive to one form of N33 protein was made as follows: a conserved 16-amino acid peptide at the N33 C-terminus (Fig. 20) was coupled to KLH and used to immunize rabbits. After six weeks, serum was harvested and antibodies were affinity-purified against a peptide column. These polyclonal antibodies were tested in a Western blot of recombinant N33 fusion proteins expressed in E. coli. (Fig. 21). As described above, clones A4 and A5 (partial N33 proteins fused to the glutathiorie-S- transferase gene carried in expression vector pGEX-2T) were obtained representing form 1 and form 2 mRNAs, respectively.
  • the plasmid vector pLUSH containing segments of the telomeric end of the YAC4 vector, a bacterial Kan R gene, the yeast Lys2 auxotrophy gene, and the mammalian hygromycin R gene was kindly provided by D.
  • pLUSH DNA was linearized by Sal I digestion and 5-10 ⁇ g was used to transform YAC-containing yeast cells using an alkali cation yeast transformation kit (Bio 101, Inc.) per manufacturer's instructions. Cells were plated on
  • triple drop-out media (trp-ura-lys-) to select for clones containing both the YAC and the conversion vector. Colonies were picked after 3-4 days and grown overnight in 2 ml of YPD medium. Yeast DNA was prepared and tested for homologous integration of pLUSH by PCR with primers: 5'-CTTGAGATCGGGCGTTCGACTCGC-3' and 5'- TGAACGGTGATCCCCACCGGAATTG-3' (Hermanson et al., Nucl.
  • yeast cells grown by standard methods were pelleted, washed and resuspended in isotonic medium and cell walls digested with yeast lytic enzyme to produce yeast spheroplasts. These were layered on top of pelleted cultured mammalian cells such as NIH 3T3 cells or human tumor cells (50:1 numerical ratio) and incubated in the presence of polyethylene glycol 1500 (Boehringer-Mannheim) for 2 min at RT to induce fusion. Cells were diluted in tissue culture medium and incubated for 48 hr, after which selection with 300 ⁇ g/ml
  • the phenotype of tumor cells after transfer of tumor suppressor genes can be assessed by a common set of assays regardless of whether transfer method, e.g., microcell transfer, retrovirus-mediated gene transfer, transfection, cell fusion, etc.
  • Growth rate in vi tro ( 3 H- thymidine incorporation), growth of transformants in soft agar, and tumorigenicity in nude mice can be compared in modified and parental cells to assess for tumor
  • the tumor suppressor activity of PTSG is assessed in both in vitro cell culture conditions and in nude mouse animal models.
  • Any of the 13 N33-colon carcinoma cell lines listed in Figure 17 SW480, SW837, SW1417, HT-29, SW403, LS174T, DLD-1, CACO-2, EB, SK-CO-1, RKO, HCT116 and COLO-302 can be used to assess PTSG tumor suppressor activity.
  • the effect of PTSG on the 13 N33-colon carcinoma cell lines listed in Figure 17 SW480, SW837, SW1417, HT-29, SW403, LS174T, DLD-1, CACO-2, EB, SK-CO-1, RKO, HCT116 and COLO-302
  • ACN is a control adenoviral vector lacking a cDNA insert while AC-PTSG are adenoviral vectors expressing PTSG products under the control of the human CMV promoter.
  • Plasmid pBS-N33C(7) was tested for the ability to produce a 39 KD protein in the TNT Coupled
  • T7 promoter in the Bluescript vector allows for transcription and translation of the PTSG coding sequence by rabbit reticulocytes.
  • One microgram of mini-lysate DNA is added per TnT Reticulocyte reaction and is incubated for 1 hour at 30 degrees Celsius.
  • Ten microliters of the reaction is mixed with loading buffer and run on a 10% polyacrylamide gel (Novex) for 1 1/2 hour at 165 V. The gel is dried down and exposed to film overnight.
  • pcDNA3-N33 was digested with Xba I - BamH I, and the insert was directionally cloned into the Xba I - BamH I sites of pAdCMVb vector to yield pACN33-l.
  • pBS-N33C(7) was digested with ava III - EcoR I and the 5' half (nt 1 - 616) of N33 insert was purified.
  • Clone A5 was also digested with Ava III - EcoR I to release the 3' half of the N33 Form 2 insert (nt 617 - EcoRI).
  • the two gene halves were ligated and cloned into the EcoR I site of pcDNA3. Orientation of the reconstructed insert was agsin tested by Kpn I digestion and sequencing. An antisense orientation clone was then cut with Xba I and Bam HI and the insert cloned into pAdCMVb as above to yield pACN33-2.
  • the above plasmids are linearized with Nru I and are co-transfected with the large fragment of a Cla I digested d1309 mutants (Jones and Shenk, Cell, 17:683-689 (1979) which is incorporated herein by reference), using a CaPO 4 transfection kit (Stratagene). Viral plaques are isolated and recombinants are identified by both
  • colon carcinoma cell lines are infected with either the control or the PTSG- containing recombinant adenoviruses for a period of 24 hours at increasing multiplicities of infection (MOD of plaque forming units of virus/cell.
  • MOD multiplicities of infection
  • Cells are then washed once with PBS and harvested in lysis buffer (50mM Tris-Hcl Ph 7.5, 250 Mm NaCl, 0.1% NP40, 50mM NaF, 5mM EDTA, 10ug/ml aprotinin, 10 ug/ml leupeptin, and ImM PMSF).
  • lysis buffer 50mM Tris-Hcl Ph 7.5, 250 Mm NaCl, 0.1% NP40, 50mM NaF, 5mM EDTA, 10ug/ml aprotinin, 10 ug/ml leupeptin, and ImM PMSF.
  • Membranes are incubated with an anti-PTSG antibody followed by sheep anti-mouse IgG conjugated with horseradish peroxidase. Accurate expression of PTSG protein is visualized by chemiluminescence (ECL kit, Amersham) on Kodak XAR-5 film.
  • N33-negative colon cancer cells are seeded at 1x10 6 cells per 100 mm plate in Kaighn's F12/DME medium (Irvine Scientific) which is supplemented with 10% FBS and 0.2 IU insulin (Sigma). The plates are incubated overnight at 37°C in 7% CO 2 . The following day, the cells are refed with 10 mis of growth medium and are infected with either ACN control viral lysate (MOI 10) or with AC-PTSG viral lysates (MOI 10) and allowed to incubate at 37°C. After 3 days, the medium is removed and the cells are fixed with a 1:5 acetic acid-methanol solution. The cells are stained with a 20% methanol-0.5% crystal violet solution for 30 minutes and are rinsed with tap water to remove excess stain.
  • ACN control viral lysate MOI 10
  • AC-PTSG viral lysates MOI
  • Thymidine incorporation is also used to assess the effects of PTSG on cell proliferation. Briefly, approximately 3x10 3 cells are plated in each well of a 96-well plate (Costar) and allowed to incubate overnight (37°C, 7% CO 2 ). Serial dilutions of ACN or AC-PTSG are made in DME:F12/15% FBS/l% glutamine, and cells are infected at multiplicity of infection (MOI) of 10 and 100 (4 replicate wells at each MOI) with each adenovirus.
  • MOI multiplicity of infection
  • One-half of the cell medium volume is changed 24 hours after infection and every 48 hours until harvest. At 18 hours prior to harvest, 1 ⁇ Ci of 3 H-thymidine (Amersham) is added to each well. Cells are harvested onto glass- fiber filters 5 days after infection, and 3 H-thymidine incorporated into cellular nucleic acid is detected using liquid scintillation (TopCount®, Packard Instruments).
  • Approximately 2x10 7 cells are plated into T225 flasks, and cells are treated with sucrose buffer containing ACN or AC-PTSG adenoviruses at MOI of 3 or 30. Following overnight infections, cells are harvested and
  • Colon cancer cell lines are injected subcutaneously into female BALB/c athymic nude mice.
  • Tumors are allowed to develop for 32 days. At this point, a single injection of either ACN (control) or AC- PTSG adenoviruses are injected into the peritumoral space surrounding the tumor. Tumors are then excised at either Day 2 or Day 7 following the adenovirus injection, and poly-A+ RNA is isolated from each tumor. Reverse
  • transcriptase-PCR using PTSG specific primers are then used to detect PTSG RNA in the treated tumors.
  • Amplification with actin primers will serve as a control for the RT-PCR reaction while a plasmid containing the recombinant- (PTSG) sequence will serve as a positive control of the recombinant- (PTSG) specific band.
  • mice In a separate experiment, cells are injected into the subcutaneous space on the right flank of mice, and tumors are allowed to grow for 2 weeks. Mice receive peritumoral injections of buffer or recombinant virus twice weekly for a total of 8 doses. Tumor growth is monitored throughout treatment in the control animals receiving ACN and buffer and those animals receiving AC- PTSGs. Body weight and survival time is also monitored.

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Abstract

L'invention porte sur une nouvelle molécule d'acide nucléique codant un produit génique suppresseur des tumeurs de la prostate et du colon et sur des moyens et méthodes de détection des mutations et/ou pertes dudit gène suppresseur. Elle porte également sur des méthodes de suppression du phénotype néoplasique de cellules cancéreuses présentant un défaut dans ledit produit génique, et sur des moyens et méthodes de traitement du cancer par administration du susdit gène suppresseur de tumeurs de la prostate et du colon.
PCT/US1995/006593 1994-05-20 1995-05-22 Nouveau gene suppresseur des tumeurs de la prostate et du colon situe sur le chromosome humain 8 WO1995032214A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP95920649A EP0714401A4 (fr) 1994-05-20 1995-05-22 Nouveau gene suppresseur des tumeurs de la prostate et du colon situe sur le chromosome humain 8
SK246-96A SK24696A3 (en) 1994-05-20 1995-05-22 Prostate/colon tumor suppressor gene located on human chromosome 8
AU26034/95A AU686815B2 (en) 1994-05-20 1995-05-22 A novel prostate/colon tumor suppressor gene located on human chromosome 8
NO961539A NO961539L (no) 1994-05-20 1996-04-18 Et nytt prostata-/colontumorsuppressorgen lokalisert på humant kromosom 8
FI961723A FI961723A0 (fi) 1994-05-20 1996-04-19 Uusi eturauhas/paksunsuolikasvaimen suppressorigeeni

Applications Claiming Priority (2)

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US24660494A 1994-05-20 1994-05-20
US08/246,604 1994-05-20

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997015686A1 (fr) * 1995-10-23 1997-05-01 Imperial Cancer Research Technology Limited Diagnostic de predisposition au cancer et traitement approprie
WO1999000498A1 (fr) * 1997-06-27 1999-01-07 Human Genome Sciences, Inc. Gene-1 specifique de la prostate apparente au nk-3 humain
US6287854B1 (en) * 1996-10-22 2001-09-11 Imperial Cancer Research Technology Limited Diagnosis of susceptibility to cancer and treatment thereof
WO2003087408A2 (fr) * 2002-04-05 2003-10-23 University Court Of The University Of Edinburgh Genes associes a la schizophrenie
US6747555B2 (en) 2002-09-24 2004-06-08 International Business Machines Corporation Tracking apparatus and associated method for a radio frequency enabled reminder system
US6841350B2 (en) 1999-02-20 2005-01-11 The North West London Hospitals Nhs Trust Of Northwick Park Hospital Methods of diagnosing prostate cancer through the detection of the presence or absence of Pax 2 mRNA
US7223842B1 (en) 1986-08-11 2007-05-29 Massachusetts Eye And Ear Infirmary Detection of proteins whose absence is associated with a neoplasm
US7384735B1 (en) 1986-08-11 2008-06-10 Massachusetts Eye And Ear Infirmary Retinoblastoma nucleic acids

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AMERICAN JOURNAL OF PATHOLOGY, Vol. 144, No. 1, issued January 1994, CHANG et al., "Deletion Mapping of Chromosome 8p in Colorectal Carcinoma and Dysplasia Arising in Ulcerative Colitis, Prostatic Carcinoma, and Malignant Fibrous Histiocytomas", pages 1-6. *
CANCER RESEARCH, Vol. 54, No. 9, issued 01 May 1994, ICHIKAWA et al., "Suppression of Metastasis of Rat Prostatic Cancer by Introducing Human Chromosome 8", pages 2299-2302. *
See also references of EP0714401A4 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7223842B1 (en) 1986-08-11 2007-05-29 Massachusetts Eye And Ear Infirmary Detection of proteins whose absence is associated with a neoplasm
US7384735B1 (en) 1986-08-11 2008-06-10 Massachusetts Eye And Ear Infirmary Retinoblastoma nucleic acids
WO1997015686A1 (fr) * 1995-10-23 1997-05-01 Imperial Cancer Research Technology Limited Diagnostic de predisposition au cancer et traitement approprie
US6287854B1 (en) * 1996-10-22 2001-09-11 Imperial Cancer Research Technology Limited Diagnosis of susceptibility to cancer and treatment thereof
WO1999000498A1 (fr) * 1997-06-27 1999-01-07 Human Genome Sciences, Inc. Gene-1 specifique de la prostate apparente au nk-3 humain
US6617129B2 (en) 1997-06-27 2003-09-09 Human Genome Sciences, Inc. Human NK-3 related prostate specific gene-1
US7351694B2 (en) 1997-06-27 2008-04-01 Human Genome Sciences, Inc. Human NK-3 related prostate specific gene-1
US7892773B2 (en) 1997-06-27 2011-02-22 Human Genome Sciences, Inc. Antibodies to human NK3-related prostate specific gene 1 proteins
US6841350B2 (en) 1999-02-20 2005-01-11 The North West London Hospitals Nhs Trust Of Northwick Park Hospital Methods of diagnosing prostate cancer through the detection of the presence or absence of Pax 2 mRNA
WO2003087408A2 (fr) * 2002-04-05 2003-10-23 University Court Of The University Of Edinburgh Genes associes a la schizophrenie
WO2003087408A3 (fr) * 2002-04-05 2004-04-29 Univ Edinburgh Genes associes a la schizophrenie
US6747555B2 (en) 2002-09-24 2004-06-08 International Business Machines Corporation Tracking apparatus and associated method for a radio frequency enabled reminder system

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CA2170627A1 (fr) 1995-11-30
EP0714401A4 (fr) 1999-03-03
NO961539D0 (no) 1996-04-18
SK24696A3 (en) 1997-02-05
EP0714401A1 (fr) 1996-06-05
AU686815B2 (en) 1998-02-12
FI961723A (fi) 1996-04-19
NO961539L (no) 1996-04-18
FI961723A0 (fi) 1996-04-19
CZ53196A3 (en) 1997-05-14
AU2603495A (en) 1995-12-18

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