WO1999007893A1 - ISOLEMENT D'UN NOUVEAU GENE p23 DU FACTEUR DE SENESCENCE - Google Patents

ISOLEMENT D'UN NOUVEAU GENE p23 DU FACTEUR DE SENESCENCE Download PDF

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WO1999007893A1
WO1999007893A1 PCT/US1998/016343 US9816343W WO9907893A1 WO 1999007893 A1 WO1999007893 A1 WO 1999007893A1 US 9816343 W US9816343 W US 9816343W WO 9907893 A1 WO9907893 A1 WO 9907893A1
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cells
cell
seq
senescent
expression
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PCT/US1998/016343
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English (en)
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Karen Swisshelm
Suzanne Hosier
Manfred Kubbies
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University Of Washington
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Priority to KR1020007001338A priority Critical patent/KR20010022741A/ko
Priority to CA002296598A priority patent/CA2296598A1/fr
Priority to EP98938406A priority patent/EP1012333A4/fr
Priority to JP2000506375A priority patent/JP2001512698A/ja
Priority to AU86935/98A priority patent/AU8693598A/en
Priority to BR9811865-0A priority patent/BR9811865A/pt
Publication of WO1999007893A1 publication Critical patent/WO1999007893A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • 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
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the senescent phenotype appears to be controlled by more than one gene.
  • cell fusion experiments were performed with 40 different immortal human cell lines to determine whether the senescent phenotype could thus be restored. Based on the results, the cell lines were assigned to four different complementation groups, indicating that at least four genes or genetic pathways contribute to senescence (Smith and Pereira-Smith, Science 273:63-67, 1996). Other experiments have indicated that genes located on human chromosomes 1, 4, 6, 7, 1 1, 18, and X are involved in senescence (ibid.).
  • senescence may have evolved as a mechanism for tumor suppression, and that aging is an indirect effect of this circumstance. Because constraints on growth control are absent from tumor cells, such cells most likely have switched off the expression of genes whose products promote or maintain the senescent state. For example, in vitro studies have indicated that senescence can be partially circumvented by the inactivation of tumor suppressor proteins such as the retinoblastoma tumor suppressor gene RBI (Weinberg, Cell 81:323-330, 1995). This suggests the possibility that the loss of functional tumor suppressor genes in vivo could permit cells to gain a replicative advantage and eventually to undergo immortalization.
  • tumor suppressor proteins such as the retinoblastoma tumor suppressor gene RBI (Weinberg, Cell 81:323-330, 1995). This suggests the possibility that the loss of functional tumor suppressor genes in vivo could permit cells to gain a replicative advantage and eventually to undergo immortalization.
  • p23 The function of p23 is not known, but analysis of its deduced amino acid sequence (SEQ ID NO:2) suggests that it belongs to a family of transmembrane proteins known as the "PMP 22" family or "epithelial membrane protein” (EMP) family (e.g., see Taylor et al., J. Biol. Chem. 270:28824-28833, 1995; Lobsiger et al., Genomics 36:379-387, 1996; Taylor and Suter, Gene 175:115-120. 1996).
  • PMP 22 family
  • EMP epidermal membrane protein
  • p23 is expressed in several human tissues, including adult and fetal liver, pancreas, placenta, adrenals, prostate, and ovary, all of which are composed primarily of epithelial cells having endocrine or secretory function. It was noted further that p23 RNA is markedly decreased or absent from a number of human breast cancer cell lines, thus suggesting that the p23 polypeptide may play a role in suppressing the malignant phenotype in normal breast tissue.
  • p23-positive normal human mammary epithelial cells HMECs express reduced levels of p23 when cultured with retinoic acid, thus indicating that the p23 gene is transcriptionally regulated through a retinoic acid receptor pathway.
  • the present invention thus provides isolated p23 nucleic acid molecules that are involved in the senescence of human epithelial cells, as well as recombinant p23 polypeptides encoded by the nucleic acid molecules.
  • the invention provides vectors and host cells comprising the nucleic acid molecules, antibodies specific to the polypeptides, and methods of modulating senescence and of measuring the levels of p23 in a biological sample.
  • FIGURE 1 shows a comparison of the p23 polypeptide whose deduced amino acid sequence is shown in SEQ ID NO:2 with the coding sequence of the apoptosis- related rat ventral prostate gene 1 (RVP1) shown in SEQ ID NO:3.
  • FIGURE 2 shows structural features of the p23 polypeptide whose deduced amino acid sequence is shown in SEQ ID NO:2. These features were determined by analysis with the Motifs subroutine of the Genetics Computer Group (GCG) computer program for analyzing nucleotide and amino acid sequences. Triangles indicate hydrophobic regions, while ovals indicate hydrophilic regions. The "O" shown below the line between residues 50 and 100 indicates the position of a putative glycosylation site at amino acid residue 72.
  • GCG Genetics Computer Group
  • This invention provides a nucleic acid molecule encoding a polypeptide having the amino acid sequence shown in SEQ ID NO:2, which is a representative example of a p23 polypeptide.
  • a representative example of a nucleic acid molecule that encodes a p23 polypeptide comprises the nucleotide sequence shown in SEQ ID NO: 1, which corresponds to a cDNA encoding the polypeptide whose amino acid sequence is shown in SEQ ID NO:2.
  • SEQ ID NO: 1 The open reading frame in SEQ ID NO: 1 is located between nucleotides 221 and 853, thus the majority of this nucleotide sequence is untranslated.
  • a representative example of a p23 polypeptide is provided by the amino acid sequence shown in SEQ ID NO:2.
  • nucleic acid shown in SEQ ID NO: l depend on the ability of complementary nucleic acid strands to form duplexes, i.e., to hybridize with one another.
  • “Stringent hybridization conditions” means generally that the nucleic acid duplexes that form under these conditions are perfectly matched or nearly perfectly matched (Sambrook et al.. Molecular Cloning [2d ed.], 1989, which is hereby incorporated by reference).
  • stringent hybridization conditions means generally that the nucleic acid duplexes that form under these conditions are perfectly matched or nearly perfectly matched (Sambrook et al.. Molecular Cloning [2d ed.], 1989, which is hereby incorporated by reference).
  • complementary nucleic acid molecules derived from different allelic forms of a gene are expected to form stable hybrids, as allelic forms of a gene typically differ at very few nucleotide positions.
  • probes derived from a specific cDNA are expected to form stable hybrids under stringent conditions with cDNAs or genes corresponding to allelic forms or mutant forms of that same gene.
  • Stringent hybridization conditions for polynucleotide molecules >200 nucleotides in length typically involve hybridizing at a temperature 15°-25°C below the melting temperature (Tm) of the expected duplex, most preferably at 25°C below the Tm, and for oligonucleotide probes ( ⁇ 30 nucleotides), by hybridizing at 5°-10°C below the Tm (e.g., Sambrook et al., 1989; see Section 1 1.45).
  • conditions for stringent hybridization can be calculated by using the above formula or using some similar formula.
  • the Tm can be confirmed empirically by hybridizing the probe with a cloned p23 nucleic acid molecule, such as the one in SEQ ID NO:l, then incrementally increasing the temperature until the duplexes are melted.
  • the optimal hybridization temperature for a given probe likewise can be confirmed empirically by testing the rate of hybrid duplex formation at different temperatures.
  • probes that are at least 15 nucleotides in length are expected to hybridize specifically because sequences exceeding this length are extremely unlikely to be represented more than once in a mammalian genome (Sambrook et al., 1989, at Section 11.7).
  • hybridization conditions will be evident to one skilled in the art and will generally be guided by the purpose of the hybridization, the type of hybridization (DNA-DNA or DNA-RNA), and the level of desired relatedness between the sequences.
  • methods for hybridization and representative buffer formulations for high and low stringency hybridization are well established and are provided in the published literature (e.g., Sambrook et al., 1989; see also Hames and Higgins, eds., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington DC, 1985; Berger and Kimmel, eds., Methods in Enzymology, Vol.
  • Hybridization stringency can be altered by: adjusting the temperature of hybridization; adjusting the percentage of helix-destabilizing agents, such as formamide, in the hybridization mix; and adjusting the temperature and/or salt concentration of the wash solutions.
  • the stringency of hybridization is adjusted during the post-hybridization washes by varying the salt concentration and/or the temperature. Stringency of hybridization may be lowered by reducing the percentage of formamide in the hybridization solution or by decreasing the temperature of the wash solution.
  • Low stringency conditions may involve lower hybridization temperatures (e.g., 35-42°C in 20-50% formamide) with washes conducted at an intermediate temperature (e.g., 35-60°C) and in a wash solution having a relatively high salt concentration (e.g., 2-6 X SSC).
  • Moderate stringency conditions which may involve hybridization in 0.2-0.3 M NaCl at a temperature between 50°C and 65°C and washes in 0.1 X SSC, 0.1 % SDS at between 50°C and 55°C, may be used in conjunction with the disclosed polynucleotide molecules as probes to identify genomic or cDNA clones encoding related proteins, e.g., other members of the EMP family.
  • a nucleic acid molecule comprising the sequence shown in SEQ ID NO: 1 provides a tool that can be used to identify and isolate the entire gene encoding p23, as well as variants of the p23 gene, such as allelic variants or mutant forms of the gene.
  • p23 probe i.e., probes derived from all or subparts of SEQ ID NO: l, under stringent conditions with a phage or cosmid library of the human genome, DNA molecules corresponding to all or part of the p23 gene can be identified.
  • This invention also includes variant forms such as allelic variants and mutated forms of the p23 protein, gene, and cDNA.
  • Genes and cDNAs encoding variants of p23 are easily identified and subsequently isolated by using probes based on SEQ ID NO:l as a tool for screening cDNA or genomic libraries made from cells of interest, i.e., cells that may contain variant forms of the p23 gene or mRNA.
  • hybridizations are conducted under stringent conditions. Confirmation that clones thus isolated are p23 variants is accomplished by determining the nucleotide sequence of the cloned DNA and comparing the sequence, particularly the coding regions, with the p23 sequence shown in SEQ ID NO:l .
  • Variants of p23 are expected to share at least 90-95% of their nucleotide sequences.
  • cells may express a non-functional p23 protein or may contain no p23 protein due to genetic mutation or somatic mutations. Such cells, which may include genetically deficient cells or cancer cells, may thus escape the senescent state. For cancer cells having defects in p23, the cancer cells may be treated in a manner to cause the over-expression of wild-type p23 to force the cells to stabilize in G ⁇
  • the subject invention provides methods of inducing a senescent phenotype in a cell by introducing into the cell a nucleic acid molecule that encodes p23, such as, for example, the representative p23 sequence shown in SEQ ID NO:l .
  • Such methods for inducing senescence in a cell may involve introducing into non-senescent cells in vivo or in vitro a nucleic acid molecule that encodes the protein whose amino acid sequence is shown in SEQ ID NOS:l and 2, or that encodes an allelic form of the protein having essentially the same biological activity.
  • the untranslated regions of the p23 cDNA shown in SEQ ID NO: l may provide important regulatory functions that affect mRNA stability or processing, or other aspects of mRNA function.
  • recombinant expression vectors for expressing p23 in eukaryotic cells including yeast cells (e.g., retroviruses, Herpes simplex viruses, plasmids, vaccinia viruses, adenoviruses, defective parvoviruses, CMV, and the like), and plasmid or cosmid vectors for expressing p23 in prokaryotic cells.
  • yeast cells e.g., retroviruses, Herpes simplex viruses, plasmids, vaccinia viruses, adenoviruses, defective parvoviruses, CMV, and the like
  • plasmid or cosmid vectors for expressing p23 in prokaryotic cells.
  • Recombinant expression vectors of the invention are constructed, for example, by operably linking a nucleic acid molecule capable of encoding the p23 protein of SEQ ID NO:2 to suitable control sequences.
  • Nucleotides 221-853 of SEQ ID NO: l provide a representative nucleotide sequence having the requisite coding capacity.
  • "Operably linking” is used herein to mean ligating a p23 nucleic acid molecule to an expression vector nucleic acid in a manner that correctly positions the regulatory elements necessary for transcription and translation of p23, preferably under the predetermined positive (or negative) regulatory control exerted by control sequences in the expression vector (i.e., regulatory sequences capable of driving expression, over-expression, or constitutive expression of the p23 gene, e.g., promoter, enhancer, operator sequences, and the like).
  • the vector may contain an inducible promoter, for example, one that directs transcription only in the presence of a particular hormone, ion (e.g., zinc), growth-factor, co-factor, or metabolic substrate.
  • a particular hormone ion (e.g., zinc), growth-factor, co-factor, or metabolic substrate.
  • Selectable markers may also be present in the expression vector. Representative examples of such selectable markers include enzymes, antigens, drug resistance markers, or markers satisfying the growth requirements of the cell.
  • Regulatory elements may be present that exert control either in eukaryotic cells or in prokaryotic cells, or both types of regulatory elements may be present in a single vector.
  • the subject expression vectors are useful for transfecting or transducing cells to express transgenic p23 polypeptides, mutant p23 polypeptides, and antisense nucleic acids capable of forming duplexes with endogenous p23 mRNA.
  • Cells induced to express exogenous p23 are called "transgenic cells.”
  • the invention provides cell lines transformed by vectors that direct the expression of transgenic p23 polypeptide in the transformed cells.
  • the transgenic cells of the subject invention can be used to produce the p23 polypeptide in large quantities. To facilitate harvesting the p23 polypeptide from transgenic cells, the transgene.
  • Transgenic cells expressing p23 may be either eukaryotic or prokaryotic.
  • Another embodiment of the invention provides methods of inducing a senescent phenotype in a eukaryotic cell.
  • a p23 expression vector that constituitively, conditionally, or transiently over-expresses p23 is introduced into the cell.
  • the cell proliferates at a rate slower than its parent cell, or ceases proliferation altogether, i.e., the cell attains a senescent phenotype.
  • p23-transduced human diploid cells for example, will become arrested in G,.
  • Cultured cells or cells taken from a live host may be the target cells for the p23 expression vector.
  • the invention provides cell lines capable of expressing transgenic p23 polypeptide. If cells taken from a live host are transduced, these can be returned to the host or further studied in culture.
  • transgenic animals e.g., experimental and domestic animals
  • transgenic animals may be constructed that express p23 under the control of tissue-specific or inducible promoters, or that express antisense RNA for suppressing endogenous p23 expression.
  • nucleotide sequences encoding p23 may be used to obtain transient expression of p23 in cells by introducing cloned p23 nucleic acids into cells by such methods as electroporation, calcium phosphate precipitation, or in liposomes. Transient expression results when mRNA is transcribed from the initially introduced vector DNA prior to vector integration.
  • Antisense p23 nucleotide sequences that is, nucleotide sequences complementary to the transcribed or the non-transcribed strand of a p23 gene, may be used to block normal or mutant p23 expression in cancer cells or other proliferating cells.
  • Suitable antisense oligonucleotides are at least 1 1 nucleotides in length and may include untranslated (upstream or intron) and associated coding sequences.
  • the optimal length of an antisense oligonucleotide depends on the strength of the interaction between the antisense oligonucleotide and its complementary target sequence, the temperature and ionic environment in which translation takes place, the base sequence of the antisense oligonucleotide, and the presence of secondary and tertiary structure in the target mRNA and/or in the antisense oligonucleotide.
  • Suitable target sequences for antisense oligonucleotides include intron-exon junctions (to prevent proper splicing), regions in which DNA/RNA hybrids will prevent transport of mRNA from the nucleus to the cytoplasm, initiation factor binding sites, ribosome binding sites, and sites that interfere with ribosome progression.
  • a particularly preferred target region for antisense oligonucleotide is the 5' untranslated (promoter/enhancer) region of the gene of interest.
  • Antisense oligonucleotides may be prepared by the insertion of a DNA molecule containing the target DNA sequence into a suitable expression vector such that the DNA molecule is inserted downstream of a promoter in a reverse orientation as compared to the gene itself.
  • antisense oligonucleotides may be synthesized using standard manual or automated synthesis techniques. Synthesized oligonucleotides may be introduced into suitable cells by electroporation, calcium phosphate precipitation, liposomes, microinjection. or other means.
  • the stability of antisense oligonucleotide-mRNA hybrids may be increased, for example, by the addition of stabilizing agents to the oligonucleotide, such as intercalating agents covalently attached to one end of the oligonucleotide, or by incorporating phosphotriesters, phosphonates, phosphorothioates, phosphoroselenoates, phosphoramidates, or phosphorodithioates into the phosphodiester backbone.
  • stabilizing agents such as intercalating agents covalently attached to one end of the oligonucleotide, or by incorporating phosphotriesters, phosphonates, phosphorothioates, phosphoroselenoates, phosphoramidates, or phosphorodithioates into the phosphodiester backbone.
  • Protein harvested from transgenic cells expressing p23 can be used for a number of purposes, for example, for raising antiserum against p23.
  • the invention provides immunologic binding partners for p23 polypeptides such as polyclonal and monoclonal antibody molecules, and various antigen-binding fragments thereof, that are capable of specifically binding a p23 polypeptide such as the polypeptide whose amino acid sequence is shown in SEQ ID NO:2.
  • Antibodies may be raised against whole p23, or against fragments of the polypeptide.
  • the p23 used as an antigen may be denatured or in its native form prior to injection.
  • Antibodies against denatured proteins are often able to react with either native or denatured protein, and are often useful for Western blotting.
  • the antibodies can be used also for the identification of senescent cells in culture or in tissue biopsies using standard immunostaining protocols.
  • Immunospecific reagents capable of specifically binding p23 may be produced by hybridoma or by repeated injection of the purified protein or selected peptides derived from p23 in combination with an appropriate adjuvant (e.g., Freund's, ISCOMs, or the like) into a suitable animal such as a rabbit, sheep, or goat.
  • an appropriate adjuvant e.g., Freund's, ISCOMs, or the like
  • Therapeutic applications include binding partners that inhibit the binding of p23 to ligands that normally bind to it. thus promoting cell proliferation in the treated cell.
  • Representative examples of purification applications include immunochemical methods and immunoaffinity chromatography wherein the antibody is used as an affinity reagent to purify p23 from tissues in which it occurs naturally, or from cultured cells expressing transgenic p23.
  • Representative examples of diagnostic applications include enzyme-linked and radioisotopic immunoassays (i.e., ELISA and RIA), immunofluorescence, time-resolved fluorescence immunoassay and the like, to determine levels of p23 protein in tissue samples, such as tumor cells.
  • antibody against p23 could be used to selectively kill the senescent cells in a cell population.
  • p23 appears to be a transmembrane protein
  • antibody against p23 can be used to selectively lyse cells in whose membranes the protein is present.
  • an aging culture of cells could be rejuvenated by exposure to the antibody under conditions that permit the antibody to lyse cells expressing the protein, or by using anchored anti-p23 to cull p23 -expressing cells from a cell suspension.
  • these same procedures could be used to cull or enrich for senescent cells from tissue samples removed from patients. Young cells from such culled cell populations could be returned to the body, or if indicated, the senescent cells instead could be returned to the body.
  • the p23-specific immunologic binding partners further find general utility in diagnostic assays for detecting and quantitating levels (e.g., protein or antigen) of p23 in a cell such as a cultured cell, to provide an indicator of the remaining number of cell divisions that can be expected for that cell line.
  • levels e.g., protein or antigen
  • the measured level of p23 in the cell line is compared with the levels previously measured in early, intermediate, and late passage cultures of the same cell type.
  • the life expectancy of cultured epithelial cells can be predicted by comparison of p23 levels in the test culture with standard p23 levels measured after various numbers of passages in a representative epithelial cell line, e.g., HMECs.
  • an assay if greater than 90% of the cells are expressing high levels of p23 as compared with levels expressed during early passage cells, it can be assumed that the culture is senescent and will not substantially expand if replated.
  • an example of a compound that "modulates" the activity of a p23 protein is an inhibitor capable of decreasing the level of p23 expression following the administration of the compound to a cell expressing p23.
  • the functional activity of the p23 added to the cells can be assayed by measuring the recipient cell's restored responsiveness to mitogens.
  • Retinoic acid is an example of a compound that reduces p23 expression.
  • artificial p23 polypeptides include fragments of the p23 polypeptide, which can be produced from full length p23 by physical or enzymatic fragmentation or by use of recombinant DNA technology to express subportions of the p23 polypeptide.
  • the subject p23 polypeptides encompass normal p23 polypeptides (i.e., found in normal cells), mutant p23 polypeptide (e.g., resulting from mutagenesis. or found in tumor cells), and chemically modified p23 polypeptides (e.g., having one or more chemically altered amino acids, in which case a designated amino acid can be converted into another amino acid, or chemically substituted or derivatized and the like).
  • Functional sites in the p23 polypeptides are identified by constructing mutants of the p23 nucleic acid, e.g., and testing the constructs for expression products having altered functional properties such as failure to induce senescence when introduced into actively proliferating cultured cells.
  • mutant p23 nucleotide sequences may be constructed from the nucleotide sequence shown in SEQ ID NO: l .
  • Skilled artisans will recognize a variety of methods by which the sequence in SEQ ID NO:l may be mutated (e.g., with chemical agents or radiation or using recombinant DNA technology), and by which clones of cells containing the mutated p23 nucleotide sequences may be identified and/or selected.
  • the subject mutant p23 nucleotide sequences are useful for modulating or altering the activity of p23 in a cell.
  • the subject mutant p23 nucleotide sequences may be introduced into cells using vectors such as retroviral vectors, adenovirus vectors, or bacteriophage or plasmid vectors.
  • the subject invention includes assays for: a) detecting the absolute levels and activities of p23 expression in nonsynchronized cell populations (e.g., in tissue samples such as tumor biopsy specimens); b) comparing the levels and activities of p23 polypeptide or mRNA in synchronized or non-synchronized cell populations after various numbers of passages in culture; and c) determining the levels and activities of p23 expression products in biological fluids (i.e., blood, serum, plasma, mucus secretions. CNS fluid, cell extracts, and the like).
  • biological fluids i.e., blood, serum, plasma, mucus secretions. CNS fluid, cell extracts, and the like.
  • the absolute levels and activities of p23 expressed in malignant biological fluids may provide information on the aggressiveness of a tumor or may shed light on the likelihood that the tumor cells can be arrested in G ] by restoring p23.
  • the assayed levels and activities of p23 may serve as diagnostic markers for: a) staging tumors, since at least some types of malignant cells capable of metastasizing express little or no p23 ; b) determining prognosis, i.e., predicting patient survivability and time to recurrence of tumor, because rapidly growing malignant cells capable of metastasis may generally express less p23 than differentiated cells; and/or c) predicting therapeutic success, i.e., of a particular therapeutic regimen, because more slowly growing cells may express higher levels (or activities) of p23
  • the subject diagnostic assays may provide results that are useful to a physician in deciding how to stage a tumor, how to select an appropriate therapeutic regimen, how to evaluate the success of therapy, and how to evaluate patient risk or survivability.
  • the invention further provides methods for measuring the level of p23 expression in a biological sample.
  • the sample may be a cultured cell, a biological fluid, a patient tissue specimen, a tumor biopsy, or other sample.
  • the expression level can be measured, for example, by hybridizing RNA from the biological sample with a nucleic acid probe corresponding to an at least 15 nucleotide region of the nucleotide sequence of SEQ ID NO:l, and comparing the results with RNA standards from young and senescent cultured epithelial cells.
  • Probes are generally labeled, for example, with 32 P or biotin, using enzymes such as polynucleotide kinase, Klenow, or whole DNA polymerase, and using routine protocols (see, e.g., Sambrook et al., 1989).
  • enzymes such as polynucleotide kinase, Klenow, or whole DNA polymerase, and using routine protocols (see, e.g., Sambrook et al., 1989).
  • RNA is immobilized on a membrane filter, hybridized with the denatured labeled probe, and hybrids detected by autoradiography or chromogenic methods.
  • RNA standards e.g., RNA from young (i.e., low passage number) and senescent cultured epithelial cells
  • RNA standards provides a basis for determining whether the amount of p23 RNA in a test sample is "low” or "high,” i.e., the level in young standard cells from the selected standard cell line is defined as “low,” while the level in senescent standard cells is defined as “high.”
  • p23 expression levels can be determined by using antibody against p23 to measure the amount of p23 polypeptide. Again, amounts of p23 polypeptide in young and senescent epithelial cells provide a standard for comparison.
  • assays for p23 levels provide a valuable tool in managing use of scarce or valuable cell lines, such as cell lines established from unique tissue samples, or for maximizing the efficient use of non-immortalized cell lines whose passage history is not known.
  • assays could be used to characterize biopsy samples from normal or diseased tissue, e.g., tumor biopsies or tissue biopsies from degenerating tissues.
  • the invention provides assays for detecting chromosomal rearrangements in chromosome 3 in a human cell.
  • the chromosomal location of p23 has been mapped by computerized analysis (Unigene program; Boguski et al., Nature Genet. 10:369-371, 1995) to the distal long arm of chromosome 3, between bands q28 and q29.
  • the cloned p23 cDNA sequences provide a hybridization probe that can be used for in situ hybridization to visualize the p23 gene in metaphase chromosomes, thus enabling one to detect translocations involving this region of chromosome 3.
  • Translocation of the p23 gene may contribute to a phenotype of uncontrolled cell growth by removing p23 from regulatory control elements that ensure its expression and subsequent cell senescence.
  • rearrangement of a p23 gene in a cell may have dramatic results. If a rearrangement induces under- expression, the cell may acquire a malignant (i.e., uncontrolled) growth phenotype, and if a rearrangement induces over-expression, the cell may undergo premature senescence.
  • Screening cellular samples from individuals for chromosomal rearrangements involving p23 may provide information related to that patient's relative risk of developing specific types of cancer or other disease conditions, such as autosomal dominant optical atrophy (see below).
  • rearrangements in the long arm of chromosome 3 involving band q28 have been associated with at least one type of tumor, i.e., liposarcomas (Nature Genetics Special Issue, April, 1997. page 433).
  • chromosome 3 of the p23 gene is the same as that determined for a OPAl, an autosomal dominant genetic disease that is manifest by retinal ganglion cell or optic nerve degeneration. (Lunkes, A., Am. J. Hum. Genet., Oct., 1995; or Eiberg et al, Human Mol. Genetics 3:977-980, 1994). Both p23 and the OPAl trait map to the long arm of chromosome 3 between q28-q29, suggesting the possibility that optic atrophy could result from a mutation in p23 itself.
  • p23 Given its association with cell senescence, a mutation in p23 could well trigger premature or excessive expression of the gene, and the consequent premature entry of the affected cells into a senescent or aberrant state, thus manifesting as nerve cell degeneration. As this interband region of chromosome 3 is large enough to accommodate several genes, it remains possible that p23 is not directly responsible for OPAl, but rather is closely linked to the responsible gene, thus providing a genetic marker for the disease locus due to its proximity to the actual OPAl gene.
  • the p23 gene and its regulatory elements may serve as targets for gene therapy vectors that are designed to reactivate or to inactivate the rearranged gene, e.g., using in sz ' tw-directed recombination/mutagenesis or targeted integration to disrupt the translocated gene.
  • the primer used to transcribe the first DNA strand always hybridizes to a portion of the poly(A) tail of the mRNA template as well as to one or two non-(A) residues at the 3' end of the mRNA at the poly(A) junction.
  • primers are used that have a random sequence that is intended to be complementary to an internal sequence somewhere upstream (i.e., in a 5' direction) from the first primer. By varying the identity of the base or bases complementary to non-(A) residues for the first primer, different subsets of mRNA are targeted.
  • each primer pair will typically generate about 50-100 bands that range in size from 50-500 base pairs.
  • these mixtures of short cDNA sequences are displayed for comparison on a polyacrylamide sequencing gel.
  • the cDNAs that differ between the two populations can be recovered from the dried gel, reamplified with PCR, and subsequently cloned and further characterized. This method has been used successfully to identify a large number of senescence-related ESTs from fibroblasts (WO 96/13610).
  • HMECs normal human mammary epithelial cells
  • strains AG11132 and AG1 1134 were obtained from the Coriell Institute (National Institutes of Aging Cell Repository, Camden, New York).
  • HMECs were maintained in serum-free mammary essential basal medium (MEBM; Clonetics, San Diego, CA) supplemented with 0.4%) bovine pituitary extract (Clonetics), 10 mM HEPES (Sigma), 10 ng/ml human recombinant epidermal growth factor (EGF) (Upstate Biotechnology, Lake Placid, NY), 5 ⁇ g/ml human recombinant insulin (UBI), 0.5 ⁇ g/ml hydrocortisone (Sigma, H4001), and 10 -5 M isoproterenol (Sigma, 15627).
  • MEBM mammary essential basal medium
  • Clonetics bovine pituitary extract
  • 10 mM HEPES Sigma
  • 10 ng/ml human recombinant epidermal growth factor (EGF) Upstate Biotechnology, Lake Placid, NY
  • UBI human recombinant insulin
  • UBI human recombinant insulin
  • hydrocortisone Sigma, H400
  • AG11 134 is a line of normal HMECs that already had been serially passaged 6-8 at the time it was obtained from the Coriell Institute. The cells were passaged and expanded weekly by 1 :4 to 1 :2 dilutions, and cells were harvested for RNA preparation after 18 and 26 passages (pi 8 and p26). Total cellular RNA was purified as previously described (Swisshelm et al.. Cell Growth Differentiation 5: 133-141, 1994).
  • the cells had doubled about 60-65 times, and still proliferated rapidly, but by p26, corresponding to about 85 doublings, 80- 90%) of the cells failed to replicate when replated, thus had become senescent.
  • the senescent phenotype was verified by assaying for the presence of the pH-dependent ⁇ -galactosidase that is differentially expressed in senescent cells (Dimre et al., 1995). In the proliferating population (pi 8), about 12% of the cells stained positive for ⁇ -galactosidase, while in the p26 population, 99.4% of the cells stained positive for this enzyme.
  • Primers for the hybridization with the poly(A) end of the mRNA included three different primers having a 5' Hind III site to facilitate cloning of the amplified fragments (Liang et al, 1994; GenHunter Corp.. Brookline, MA). These three “H-Tt j " primers had the following sequences:
  • T 12 anchor primers obtained from Operon Technology, Alameda, CA
  • PCR reactions were conducted as follows: denaturation at 94°C, 30 seconds; annealing at 40°C, 2 minutes; extension at 72°C, 30 seconds. These steps were repeated for a total of 40 cycles, which were terminated with a 5-minute extension step.
  • each anchor primer was paired in a separate reaction tube with each of the random primers, except that the T
  • the cDNA band patterns corresponding to early passage and senescent cells were displayed and compared on DD gels, and a number of bands were excised from the dried gels that appeared to be either more abundant or less abundant in the senescent cells as compared with the young cells.
  • about fifty candidate cDNA fragments were extracted from the gels and reamplified by PCR.
  • Each of these amplified cDNA fragments was labeled with J P and used as a hybridization probe to analyze RNA from young and senescent AGI 1134 cells on Northern blots containing 5-10 ⁇ g/lane of whole cell RNA from each type of cell.
  • the Northern blots were hybridized at 37°C in buffer containing 0.25 M NaPO 4 , 0.25 M NaCl, 7% SDS, Im M EDTA, 5% dextran sulfate. lOO mg/ml salmon sperm DNA, and 50% formamide.
  • the Northern blots contained whole cell RNA from each of the cell cultures. Filters were washed at 37°C in a buffer containing 2 X SSC and 0.1% SDS. A probe corresponding to one of the excised DNA fragments, which was named "DD19,” was found to hybridize with a mRNA of approximately 4 kb in size that was present at much higher levels in senescent than in rapidly dividing cells.
  • the primer pair flanking this particular cDNA consisted of 5' GGAGGGTGTT 3' (SEQ ID NO: 19) (random primer OPB15, from Operon, Kit B) and 5' AAGCTTTTTTTTTTTC 3' (SEQ ID NO:6) (i.e., anchor primer H-T,,C).
  • the DD19 probe was labeled with 32 P-dCTP using a random primer kit (Boehringer - Mannheim).
  • DD19 corresponded to a mRNA elevated in senescent cells
  • Northern blot analysis was repeated using whole cell RNA from AGI 1 132 as well as from AGI 1134 cells, the former also being a line of normal HMECs.
  • Results of this experiment indicated that transcripts hybridizing with the DD19 probe were present at higher levels in senescent than in young cells for both strains of HMEC cells.
  • the most prominent band that hybridized with the probe had a size of about 4.0 kb, but a less abundant transcript with a size of about 3.0 kb was present also. It is possible that the 3.0 kb mRNA results from differential splicing of the primary p23 transcript.
  • the amplified DD19 DNA fragment which had a size of 326 bp, was cloned into the plasmid vector pCR (InVitrogen, Carlsbad, CA), using the TA cloning kit.
  • the insert from the cloned DD19 was sequenced both manually using SEQUENASE (USB) and by the fluorescence method using an ABI 377 automated sequencer (Murdock Laboratories, University of Montana). Sequencing was conducted using primers defined by the vector, i.e., T7 and SP6.
  • DD19 DNA cloned in the pCR vector was labeled and hybridized as described above with a panel of RNAs to confirm the initial observation that the 4.0 kb mRNA is elevated in senescent cells.
  • Sources of RNA for this panel were young, senescent, and quiescent AGI 1134 cells, as well as young, senescent, and quiescent AGI 1132 cells.
  • Quiescent cells are cells that have stopped dividing, but that retain the capacity to divide if placed under favorable conditions, e.g., if exposed to a mitogen, or if diluted and replated. Quiescent cells were prepared from early passage cells by allowing cells to become confluent, then maintaining them in culture with occasional feeding for an additional two weeks without further passage.
  • p23 was stripped from the filters, and the filters were rehybridized with labeled probe made from a cloned cDNA that corresponds to 36B4, which is a phosphoprotein present in ribosomes, and whose corresponding mRNA, which has a size of 1.5 Kb, is present at relatively constant levels in a wide variety of cell types (Masiakowski et al.. Nucl. Ac. Res. 10:7895-7903, 1982; Rio et al., Proc. Natl. Acad Sci. USA 84:9243-9247, 1987; Laborda, J., Nucl. Acids Res.
  • Hybridization experiments were conducted to ascertain the levels of expression of the 4.0 kb transcript in a panel of breast tumor cell lines. These were Hs578T, MCF7A, MDA-MD-435, MDA-MB-231, SKBR3, and T47D cells. These hybridizations were conducted using two different probes, one of which was the cloned DD19 DNA fragment, and the other of which was DD19.5 DNA, a cDNA clone corresponding to most or all of the 4.0 kb transcript (described below). Hybridization conditions were as described above, except that filters hybridized with DD19.5 probe were washed at 50°C instead of 37°C. Identical results were obtained with both probes.
  • Hybridization buffer contained 50% formamide, 5 X SSC, 100 mg/ml carrier DNA, 0.1%) SDS, 0.1% BSA, 0.1% polyvinylpyrrolidone, and 0.1% ficoll. Hybridizations were conducted at 37°C, and the filters were washed at 37°C in 2 X SSC and 0.1% SDS. Approximately 1.25 x 10 6 plaques were screened. Three positive clones were selected for further characterization.
  • Inserts from the three positive clones were sequenced both manually and with the ABI automated sequencer.
  • the cDNA cloned in DD19.5 was sequenced in its entirety using walking primers, and the cloned insert proved to be 3443 nucleotides in length.
  • Cloned DD19.5 was deposited on August 4, 1997, in accord with the terms of the Budapest Treaty at the American Type Culture Collection, located at 12301 Parklawn Drive, Rockville, MD, 20852, U.S.A., and was assigned the accession number .
  • the nucleotide sequence of DD19.5 was analyzed using the Wisconsin Package Version 9.0 (Genetics Computer Group, University Research Park, Madison. WI), hereafter referred to as the "GCG" package or program.
  • ESTs expressed sequence tags
  • GenBank accession number AC000088 with 45.3% identity in a 214 amino acid overlap
  • accession number AC000005 with 46.5% identity in a 198 amino acid overlap
  • accession number U19582 with 34.6% identity in a 188 amino acid overlap
  • accession number X94770 with a 24.3% identity in a 136 amino acid overlap
  • accession number XI 5436 with a 52.0%) identity in a 25 amino acid overlap
  • accession number M97881 with a 43.2% identity in a 37 amino acid overlap. No significant homology was detected between p23 and the senescence-related ESTs disclosed in WO 96/13610.
  • p23 is related to a gene known as "RVPl" that was cloned from a rat ventral prostate-androgen withdrawal cDNA library (Genbank accession No. A39484; Briehl and Miesfeld, Molec. Endocrinol. 5: 1381-1388, 1991).
  • RVPl rat ventral prostate-androgen withdrawal cDNA library
  • EMP proteins also are associated with cell growth arrest and degeneration, although it has been proposed that they play a dual role in development and differentiation.
  • PMP22 the prototype gene for this family, may be involved in both growth arrest and in differentiation in Schwann cells (Taylor et al., 1995; Taylor and Sutor, Gene 175: 115-120, 1996).
  • the putative transmembrane regions identified in p23 are located at amino acid residues 82-98 (76-108), 119-135 (115-141), 8-24 (3-28), and 170-186 (165-187) (the numbers in parentheses represent alternative overlapping possibilities).
  • This shared feature with EMP proteins supports the proposal that p23, like the EMP proteins it resembles, functions to suppress cell division.
  • the CPE-R protein encodes a 209 amino acid polypeptide, and also is predicted to contain four transmembrane regions.
  • the TMVCF protein had about a 46% identity and 55%> similarity with p23, while the CPE-R protein had a 46% identity and a 57% similarity.
  • the CPE-R and TMVCF genes give rise, respectively, to mRNAs of 1.8 and 1.4 kb.
  • EXAMPLE 2 Expression of p23 in Various Tissue Types Expression of p23 was further investigated by analyzing several different pre- made Northern blots (ClonTech, Palo Alto, CA) containing various panels of poly(A)+ mRNA. The hybridization probe used in these analyses was the cloned 326 bp DD19 fragment. Results of these hybridizations revealed that the gene is expressed in a wide variety of tissues at different levels. p23 expression was observed in heart, placenta, liver, fetal liver, lung, skeletal muscle, kidney, spleen, thymus, prostate, ovary, and small intestine.
  • a human endocrine tissue panel Northern blot showed abundant expression in the pancreas, and also in the adrenal gland, with somewhat lower levels in the thyroid, testis and thymus.
  • a human brain panel Northern blot showed expression from the occipital pole, lower levels of expression in the medulla, and very little expression elsewhere in the brain.
  • a human immune system Northern blot showed expression in spleen, lymph node, thymus, and appendix. Of all the tissues analyzed, the highest levels observed were in liver, pancreas, and fetal liver. Levels expressed in the other organs were about 10-50% lower than those seen in liver and pancreas.
  • ClonTech human cancer cell line panel which included HL-60 (promyelocytic leukemia), HeLa S3 (cervical carcinoma), K-562 (chronic myelogenous leukemia), MOLT-4 (lymphoblastic leukemia), Raji (Burkitt's lymphoma), SW480 (colorectal adenocarcinoma), A549 (lung carcinoma), and G361 (melanoma) cells.
  • Northern blot results indicated that p23 was expressed at low levels in the SW480 and A549 cells, but none was detected in the other cell lines in this panel. It may be significant that SW480 and A549 are epithelial cells.
  • EXAMPLE 3 p23 Expression in Presence of Retinoic Acid The response of p23 expression to retinoic acid was tested in senescent and early passage AGI 1 132 cells, early passage AGI 1 134 cells, MCF7 tumor cells (which express no detectable p23), and T47D tumor cells (which express levels of p23 comparable to senescent epithelial cells). Cells were cultured for 48 hours as described in Example 1, with the addition of l ⁇ M retinoic acid to the culture medium. Control cultures received no retinoic acid. Whole cell RNA was extracted from exposed and control cells, and was subjected to Northern blot analysis.
  • the probe used was cloned DD19, and the hybridization conditions were as described in Example 1 for initial Northern blot testing with RNA from young and senescent AGI 1134 cells. Signals on the resulting autoradiograms were quantified by densitometry, using an AGFA flatbed scanner and the program NIH Image. Results indicated a 25-50% reduction in the amount of p23 mRNA in all of the cells exposed to retinoic acid.
  • Dividing cells are transduced by adding the p23-encoding vector at a multiplicity of infection of 1 : 1 in the presence of 4 mg/ml POLYBRENE (Sigma). Cell selection is as described previously (Seewaldt et al., 1995). As controls, some of the cell cultures are transduced with the "empty" vector, i.e., vector not containing p23 coding sequences.
  • telomere sequence is verified by Northern blotting.
  • Total cell RNA is extracted with guanidinium hydrochloride, and analyzed after formaldehyde denaturation by electrophoresis in agarose gels, transferred to nylon membranes, and hybridized with probe made by labeling DD19 or DD19.5.
  • suitable synthetic probes are based on the nucleotide sequence of SEQ ID NO: 1, and specificity of the synthetic probes is verified by demonstrating that the probe hybridizes under stringent conditions to a 4.0 kb and a 3.0 kb mRNA expressed at higher levels in senescent than in young HMECs, and not with other mRNAs in those same cells.
  • Breast cancer cells transduced with a p23 expression vector are expected to divide less rapidly than control cells, and to enter a senescent state wherein they are arrested in G .
  • To determine whether cells are actively dividing cells are re-plated, and 3 H-thymidine is added to the medium at 1-10 ⁇ Ci/ml. After 1-6 hours, cells are harvested, and DNA is extracted and assessed for the amount of 3 H that was incorporated into DNA. Senescent cells do not incorporate 3 H-thymidine into their DNA, as they are arrested in Gi.
  • the effects of transduced p23 will be further assessed by evaluating cell doubling times in p23 -transduced and mock-transduced (i.e., cells infected with the empty vector) breast cancer cells.
  • Cells are plated at 5 x 10 4 cells per 35 mm tissue culture Petri dish and grown in standard medium containing 1 mg/ml G418. Individual plates are trypsinized at 24 to 48 hour intervals, and harvested cells are counted in duplicate. Doubling times are obtained by plotting cell number on a log scale against time on a linear scale.
  • Transgenic and mock-infected cells are injected subdermally into nude mice to assess tumorigenicity using 10 6 cells per injection intradermally, intra- peritoneally, or into the mammary fat pad, depending on the type of cells being injected.
  • transduced breast tumor cells are injected into the mammary fat pad. Tumor mean diameter is measured at weekly intervals following the inoculation. Reduced rate of tumor growth with p23 -transduced cells as compared with mock-transduced cells will indicate that induction of p23 expression can provide a therapeutic treatment for breast cancer or other types of cancer that involve epithelial cells.

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Abstract

L'invention concerne une molécule isolée d'acide nucléique relative à la sénescence codant un polypeptide de 23 kilodaltons (p23), des procédés servant à exprimer ce polypeptide de 23 kilodaltons dans des cultures de cellules, des polypeptides p23 de recombinaison, des vecteurs d'expression et des cellules hôtes exprimant p23, ainsi que des anticorps contre p23. Elle concerne également des procédés permettant de mettre en application p23 afin de moduler la sénescence, et de déterminer l'expression de p23 dans des spécimens biologiques.
PCT/US1998/016343 1997-08-08 1998-08-05 ISOLEMENT D'UN NOUVEAU GENE p23 DU FACTEUR DE SENESCENCE WO1999007893A1 (fr)

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KR1020007001338A KR20010022741A (ko) 1997-08-08 1998-08-05 신규 노화-인자 유전자, ｐ23의 단리
CA002296598A CA2296598A1 (fr) 1997-08-08 1998-08-05 Isolement d'un nouveau gene p23 du facteur de senescence
EP98938406A EP1012333A4 (fr) 1997-08-08 1998-08-05 ISOLEMENT D'UN NOUVEAU GENE p23 DU FACTEUR DE SENESCENCE
JP2000506375A JP2001512698A (ja) 1997-08-08 1998-08-05 新規な老化因子遺伝子ｐ２３の単離
AU86935/98A AU8693598A (en) 1997-08-08 1998-08-05 Isolation of a novel senescence-factor gene, p23
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WO2001053472A3 (fr) * 2000-01-21 2002-02-14 Polymun Scient Immunbio Forsch Marqueur de tumeur et de senescence
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WO2002024721A1 (fr) * 2000-09-20 2002-03-28 Human Genome Sciences, Inc. 21 proteines humaines secretees
US7846737B2 (en) 2001-06-13 2010-12-07 Millennium Pharmaceuticals, Inc. Genes, compositions, kits, and methods for identification, assessment, prevention and therapy of cervical cancer
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