EP0638126A1 - Facteur inhibiteur - Google Patents

Facteur inhibiteur

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Publication number
EP0638126A1
EP0638126A1 EP93910842A EP93910842A EP0638126A1 EP 0638126 A1 EP0638126 A1 EP 0638126A1 EP 93910842 A EP93910842 A EP 93910842A EP 93910842 A EP93910842 A EP 93910842A EP 0638126 A1 EP0638126 A1 EP 0638126A1
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EP
European Patent Office
Prior art keywords
inhibitory factor
cells
polypeptide
cell
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP93910842A
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German (de)
English (en)
Other versions
EP0638126A4 (fr
Inventor
Terry C. Johnson
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Kansas State University
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Kansas State University
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Publication date
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Publication of EP0638126A1 publication Critical patent/EP0638126A1/fr
Publication of EP0638126A4 publication Critical patent/EP0638126A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to factors that mediate mammalian cell cycle arrest and maintain mammalian cells in a viable state, the use of such factors, and to nucleic acid seguences encoding such factors.
  • the invention relates to these factors, to fragments and polypeptide analogs thereof and to DNA seguences encoding the same.
  • growth factors stimulateators of cell cycling
  • growth inhibitory factors inhibiting factors of cell cycling
  • TGF- ⁇ transforming growth factor- ⁇
  • the major inhibitory factors described include: 1) the 25 kDa homodimer transforming growth factor- ⁇ (TGF- ⁇ ) that has mitogenic activity with a variety of fibroblasts and yet expresses a potent inhibitory activity with normal human epithelial prokeratinocytes cultured in serum-free medium (Roberts et al., Proc. Natl. Acad. Sci. U.S.A., 82: 119-123, 1985; Coffey et al., Cancer Res.
  • TGF- ⁇ homodimer transforming growth factor- ⁇
  • a hydrophilic and active fragment of a larger glycoprotein inhibitory factor was released from intact cells that allowed purification by biochemical procedures (Sharifi et al., J. Chromat. 324: 173-180, 1985; Sharifi et al., Neurochem. 46: 461-469, 1986a).
  • the bovine inhibitory glycopeptide is composed of a single polypeptide chain of a molecular weight of approximately 18,000 that focuses by isoelectric focusing at about 3.0 (Sharifi et al, Neurochem. 46: 461-469, 1986; and Sharifi et al, J. Cell. Biochem. 31: 41-47, 1986).
  • the glycopeptide inhibits cellular protein and DNA synthesis, and arrests cells in the mitotic cycle at what appears to be a single block point near the G../S interphase (Fattaey et al., J. Cell. Physiol. 139: 269-274, 1989; and Fattaey et al, Exper. Cell Res. 194: 62-68, 1991).
  • the glycopeptide inhibitory factor reguires only a cell surface interaction to mediate its biological inhibitory activity (Sharifi et al., Biochem. Biophys. Res. Comm. 134: 1350-1357, 1986c), and the binding kinetics are consistent with a specific and saturable cell surface receptor (Bascom et al., J. Cell Physiol. 128: 202-208, 1986; Sharifi and Johnson, J. Biol. Chem. 262: 15752-15755, 1987).
  • the glycopeptide has been identified on the surfaces of 3T3 cells ( akshmanarao et al., Exper. Cell Res. 195: 412-415, 1991), and to be a potent antagonist of the tumor promoter 12-0-tetradecanoylphorbol-13- acetate (TPA) (Chou et al., Cancer Lett. 35: 119-128, 1987), epidermal growth factor (EGF) (Bascom et al., J. Cell. Biochem. 34: 283-291, 1987) and bombesin (Johnson and Sharifi, Biochem. Biophys. Res. Comm. 161: 468-474, 1989).
  • TPA tumor promoter 12-0-tetradecanoylphorbol-13- acetate
  • EGF epidermal growth factor
  • bombesin Johnson and Sharifi, Biochem. Biophys. Res. Comm. 161: 468-474, 1989.
  • glycopeptide was isolated from bovine cerebral cortex cells, its inhibitory action is effective on wide range of target cells.
  • Cells sensitive to its proliferative inhibitory action include vertebrate and invertebrate (insect) cells, fibroblast and epithelial-like cells, primary cells and established cell cultures, as well as a wide range of transformed cell lines (Fattaey et al., J. Cell. Physiol. 139: 269-274, 1989; and Fattaey et al, Exper. Cell Res. 194: 62-68, 1991).
  • HL-60 leukemic cells With the exception of one cell line, human HL-60 leukemic cells, all cells which were inhibited were reversibly inhibited by the glycopeptide in a nontoxic manner (Edson et al. Life Sci. 48: 1813-1820, 1991). HL-60 cells, however, were arrested in an irreversible fashion although they remained viable for at least 84 h. The glycopeptide mediated a terminal cellular differentiation, even after its removal.
  • glycopeptide is that the biological inhibitory activity clearly is Ca 2+ dependent, and possibly related to cellular Ca 2+ fluxes and/or intracellular Ca 2+ mobilization (Toole-Simms et al., J.
  • polypeptide factors having the ability to inhibit cell division or cell cycling.
  • inhibitor include purified naturally-occurring inhibitory factors.
  • the invention also relates to non-naturally occurring polypeptides having amino acid seguences sufficiently duplicative of that of naturally-occurring inhibitory factor to allow possession of a biological activity of naturally occurring inhibitory factor such as the ability to inhibit cell division.
  • the present invention also provides isolated nucleic acid seguences for use in securing expression in procaryotic or eucaryotic host cells of polypeptide products having amino acid seguences sufficiently duplicative of that of naturally- occurring inhibitory factors to allow possession of a biological activity of naturally occurring inhibitory factor.
  • DNA seguences include: a) DNA seguences encoding naturally occurring inhibitory factor disclosed in Example VII or their complementary strands; b) DNA seguences which hybridize to the DNA seguences defined in a) or fragments thereof; and c) DNA seguences which, but for the degeneracy of the genetic code, would hybridize to the DNA seguences defined in a) and b).
  • the invention also provides modified or substituted nucleic acid seguences (methyl phosphonate, thiolate, etc.) which bind to seguences either encoding inhibitory factor or compJLementary to those coding for inhibitory factor.
  • modified or substituted nucleic acid seguences methyl phosphonate, thiolate, etc.
  • vectors containing such DNA seguences and host cells transformed or transfected with such vectors.
  • methods of producing inhibitory factors by recombinant technigues, and methods of treating disorders are provided.
  • pharmaceutical compositions including inhibitory factors are provided.
  • Antibodies specifically binding inhibitory factors are also provided.
  • the invention also relates to a process for the efficient recovery of inhibitory factors from a material containing inhibitory factors.
  • FIGURE 1 shows an immunoblot analysis of components released from bovine brain cerebral cortex cell membrane. Egual aliguots of plasma membrane were incubated for 30 min. at 4°C with either isotonic buffer alone (0.154 M NaCl; 0.01 M potassium phosphate; 1 ⁇ g/ ⁇ l each of phosphora idon, pepstatin A, leupeptin and aprotinin; pH 7.2), or with isotonic buffer containing either 3 M NaCl or 3 M urea. After incubation the membranes were pelleted by centrifugation as described in the Materials and Methods and 100 ⁇ l of each supernatant fluid where tested for antigenicity as membrane released material (slots A, C and E). The membrane pellets were solubilized in 1% octyl- ⁇ -D-glucopyranoside (2.5 g protein/ ml) and 100 ⁇ l were tested for antigenicity as membrane bound material (slots B, D and F).
  • FIGURE 2 shows an SDS-PAGE analysis of bovine brain cerebral cortex cell membrane proteins during purification. Samples were separated by SDS-PAGE under reducing conditions, and the gels were then silver stained.— Original membrane preparation (100 ⁇ g protein, lane A), 3 M NaCl released membrane proteins (50 ⁇ g protein, lane B), preparative isoelectric focused pi 5.1 purified proteins (10 ⁇ g protein, lane C) and LPA affinity chromatography purified sample (5 ⁇ g protein, lane D) .
  • FIGURE 3 shows preparative isoelectric focusing analysis of 3 M NaCl released proteins from membranes of bovine brain cerebral cortex cells.
  • 1 mg membrane protein, released by 3 M NaCl was isoelectrofocused as described in the Materials and Methods, and the pH of the twenty 2 ml fractions was measured.
  • 50 ⁇ g protein from each fraction was analyzed by immunoblot using polyclonal antibody against the native bovine inhibitory factor. The relative amount of antigen in each fraction was guantified by densitometer scanning.
  • FIGURE 4 shows RB protein immunoprecipitation of inhibitory factor arrested HSBP and Swiss 3T3 fibroblasts.
  • FIGURE 5 shows RB protein immunoprecipitation of density-dependent guiescent HSBP and Swiss 3T3 fibroblasts.
  • HSBP and Swiss 3T3 cells were plated and allowed to grow to confluence as described in Example VIII. After reaching confluency the cultures were incubated an additional 24 hours, and the cells were then radiolabelled and immunoprecipitated.
  • Another set of HSBP and Swiss 3T3 cultures were plated on the same day at "1/3 the density. These cells were treated in the same fashion as the first set however, at the time of immunoprecipitation were subconfluent. Lane 1, subconfluent HSBP cultures; Lane 2, confluent HSBP cultures; Lane 3, subconfluent Swiss 3T3 cultures; and. Lane 4, confluent Swiss 3T3 cultures.
  • FIGURE 6 shows inhibitory factor cell proliferation inhibition assays carried out on the human osteosarcoma U20S (RB ) and SAOS-2 (RB ⁇ ) cell lines. Osteosarcoma cells grown in DMEM and 10% fetal calf serum, and either 9 x 10 —8M inhibitory factor (o) or an equal volume of PBS (•) was added at the time indicated by the arrows. Data are plotted as the average of duplicate wells.
  • FIGURE 7 shows inhibitory factor cell proliferation inhibition assays carried out on the human bladder carcinoma J82 (RB ⁇ ) and human prostate carcinoma DU145
  • RB ⁇ (RB ⁇ ) cell lines. Carcinoma cells grown in DMEM and 10% fetal calf serum, and either 9 x 10 —8M inhibitory factor
  • FIGURE 8 shows inhibitory factor cell proliferation inhibition assays carried out on the human keratinocyte HFK (normal) and human papillomavi us transformed (1321 and NCO) cell lines.
  • Cells were grown in KGM medium with appropriate growth factors (Clonetics, San Diego, CA), and either 9 x 10 —8 M inhibitory factor (o) or an equal volume of PBS (•) was added at the time indicated by the arrows.
  • FIGURE 9 shows inhibitory factor cell proliferation inhibition assays carried out on the adenovirus transformed human kidney epithelial cell line 293. Cells were grown in
  • FIGURE 10 shows inhibitory factor cell proliferation inhibition assays carried out on the murine fibroblast Swiss 3T3 (normal) and SV40 transformed (SVT-2 and F5B) cell lines. Fibroblasts were grown in DMEM and 10% calf serum, and either 9 x 10 —8 M inhibitory factor ( ) or an equal volume of PBA (•) were added at the time inclicated by the arrows. Data are plotted as the average of duplicate wells.
  • FIGURE 11 shows inhibition of hybridoma cell proliferation
  • the subject invention relates to purified naturally occurring factors and novel factors that inhibit cell growth, and to DNA seguences encoding such factors.
  • the invention also relates to the use of inhibitory factor as a research diagnostic or therapeutic agent. Additionally, the invention includes methods of purifying the inhibitory factor of the invention, Inhibitory factor is an inhibitor of cell division (or cell cycling) of a wide variety of cells including those derived from various tissues of mice, monkey, human, avian and insect-species. The factor inhibits cell division in a reversible and nontoxic fashion. It acts by binding a cell surface receptor and causing a variety of intracellular
  • inhibitory factory factor refers to naturally-occurring inhibitory factors
  • the subject invention provides non-naturally occurring polypeptides having a primary structural conformation (i.e., continuous seguence of amino acid residues) and glycosylation sufficiently duplicative of that of naturally occurring inhibitory factor to allow possession of a biological activity of naturally occurring inhibitory factor.
  • polypeptides include derivatives and analogs.
  • One embodiment of the invention is directed to an improved procedures to purify inhibitory factor, and the products of such purification. Although it was thought that the bovine glycopeptide had been purified to homogeneity, sensitive silver-stained gels of the final product exposed the presence of low molecular weight protein contaminants. It was found that the use of ion-exchange column high performance liguid chromatography (HPLC) removes these contaminating protein species and provides a glycopeptide purified to apparent homogeneity without protease activity.
  • HPLC high performance liguid chromatography
  • the subject invention includes a method to eliminate the protease activity and to obtain the 18 kDa inhibitory factor in a homogenous form. Such method includes the following steps:
  • protease selected from the group including: pronase, trypsin, chymotrypsin, B substilysin, serine proteases, thiolproteases, cathepsin D proteases, sulphydryl protease, metallo-proteases, trypsin like proteases, estrase and
  • the invention also includes a method of purifying the parental 66 kDa protein to apparent homogeneity. Such method includes the steps of:
  • DEAE chromatography can be performed by batch methods or by using gravity fed or a variety of pressurized columns.
  • Lectin affinity chromatography can be ,done in batches or columns using a variety of lectins to either bind the inhibitory factor (for example Limulus polyhemus agglutinin, LPA) or to bind contaminants while leaving inhibitory factor unbound (for example Wheat Germ Agglutinin, WGA) .
  • Multiple lectin affinity procedures optionally are substituted for the DEAE or isoelectric focussing.
  • Lectins are not useful in isolating material grown in E. coli but may be used in isolating materials from hosts capable of glycosylation.
  • novel inhibitory factors and DNA and RNA sequences coding for all or part of such inhibitory factors are provided.
  • the present invention includes DNA sequences which include: the incorporation of codons "preferred" for expression by selected nonmammalian hosts: the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences which facilitate construction of readily-expressed vectors, or production or purification of inhibitory factor.
  • the present invention also provides DNA sequences coding for polypeptide analogs or derivatives of inhibitory factor which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (i.e., deletion analogs containing less than all of the residues specified for inhibitory factor; substitution analogs, wherein one or more residues specified are replaced by other residues; and addition analogs wherein one or more amino acid residues is added to a terminal or medial portion of the polypeptide) and which share some or all of the properties of naturally-occurring forms.
  • the present invention specifically provides DNA seguences encoding the full length unprocessed amino acid seguence as well as DNA seguences encoding the processed form of inhibitory factor.
  • Novel DNA sequences of the invention include seguences useful in securing expression in procaryotic or eucaryotic host cells of polypeptide products having at least a part of the structural conformation and one or more of the biological properties of naturally-occurring inhibitory factor.
  • DNA seguences of the invention specifically comprise: (a) DNA seguences encoding inhibitory factor disclosed in Example VII or their complementary strands; (b) DNA seguences which hybridize (under the following hybridization conditions: 2xSSC, 40% formamide, at 37°C, 0.1% SDS; 5 x Denharts solution, 0.6 mg/ml yeast tRNA, 10 ⁇ g/ml sheared herring sperm DNA, 5.0% polyethylene glycol and 20 mM tris pH 7.5, or more stringent conditions) to the DNA seguences disclosed in Example VII or to fragments thereof; and (c) DNA seguences which, but for the degeneracy of the genetic code, would hybridize to the DNA ⁇ " seguences disclosed in Example VII.
  • genomic DNA sequences encoding allelic variant forms of inhibitory factor and/or encoding inhibitory factor from other mammalian species, and manufactured DNA sequences encoding inhibitory factor, fragments of inhibitory factor, and analogs of inhibitory factor.
  • the DNA sequences may incorporate condons facilitating transcription and translation of messenger RNA in microbial hosts.
  • Such manufactured seguences may readily be constructed according to the methods well known to those skilled in the art.
  • the DNA seguences described herein which encode polypeptides having inliibitory factor activity are valuable for the information which they provide concerning the amino acid sequence of the animal (including mammalian) proteins which have heretofore been unavailable.
  • the DNA sequences are also valuable as products useful in effecting the large scale synthesis of"inhibitory factor by a variety of recombinant techniques.
  • DNA seguences provided by the invention are useful in generating new and useful viral and circular plasmid DNA vectors, new and useful transformed and transfee ed procaryotic and eucaryotic host cells (including bacterial and yeast cells and mammalian cells grown in culture), and new and useful methods for cultured growth of such host cells capable of expression of inhibitory factor and its related products.
  • DNA seguences of the invention are also suitable materials for use as labeled probes in isolating human genomic DNA encoding inhibitory factor and other genes for related proteins as well as cDNA and genomic DNA seguences of other mammalian species.
  • DNA seguences are also be useful in various alternative methods of protein synthesis (e.g., in insect cells) or in genetic therapy in humans and other mammals.
  • ⁇ DNA seguences of the invention are expected to be useful in developing transgenic mammalian species which may serve as eucaryotic "hosts" for production of inhibitory factor and inliibitory factor products in guantity. See, generally, Palmiter et al.. Science 222, 809-813 (1983).
  • inhibitory factor is characterized by being the product of procaryotic or eucaryotic host expression (e.g., by bacterial, yeast, higher plant, insect and mammalian cells in culture) of exogenous DNA seguences obtained by genomic or cDNA cloning or by gene synthesis. That is, in an advantageous embodiment, inhibitory factor is "recombinant inhibitory factor.”
  • procaryotic or eucaryotic host expression e.g., by bacterial, yeast, higher plant, insect and mammalian cells in culture
  • inhibitory factor is "recombinant inhibitory factor.”
  • the products of expression in typical yeast e.g., Saccharomyces cerevisiae
  • procaryote e.g., E. coli
  • polypeptides of the invention may be glycosylated with mammalian or other eucaryotic carbohydrates or may be non-glycosylated.
  • Polypeptides of the invention optionally also include an initial methionine amino acid residue (at position -1).
  • inhibitory factor products such as polypeptide analogs of inhibitory factor.
  • analogs include fragments of inhibitory factor.
  • modifications of cDNA and genomic genes can be readily accomplished by well-known site-directed mutagenesis technigues and employed to generate analogs and derivatives of inhibitory factor.
  • Such products share at least one of the biological properties of inhibitory factor but may differ in others.
  • products of the invention include those which are foreshortened by e.g., deletions; or those which are more stable to hydrolysis (and, therefore, may have more pronounced or longer-lasting effects than naturally-occurring); or which have been altered to delete or to add one or more potential sites for 0-glyco.sylation and/or N-glycosylation or which have one or more cysteine. . residues deleted or replaced by, e.g., alanine or serine residues and are potentially more easily isolated in active form from microbial systems; or which have one or more tyrosine residues replaced by phenylalanine and bind more or less readily to target proteins or to receptors on target cells.
  • polypeptide fragments duplicating only a part of the continuous amino acid sequence or secondary conformations within inhibitory factor which fragments may possess one property of inhibitory factor, (e.g., receptor binding) and not others (e.g., cell inhibitory activity). It is noteworthy that activity is not necessary for any one or more of the products of the invention to have therapeutic utility [see, Weiland et al., Blut, 44, 173-175 (1982)] or utility in other contexts, such as in assays of inhibitory factor antagonism.
  • Competitive antagonists are useful in cases of overproduction of inhibitory factor or its receptor.
  • polypeptide analogs of the invention are reports of the immunological property of synthetic peptides which substantially duplicate the amino acid seguence extant in naturally-occurring proteins, glycoproteins and nucleoproteins. More specifically, relatively low molecular weight polypeptides have been shown to participate in immune reactions which are similar in duration and extent to the immune reactions of physiologically-significant proteins such as viral antigens, polypeptide hormones, and the like. Included among the immune reactions of such polypeptides is the provocation of the formation of specific antibodies in immunologically-active animals. See e.g., Lerner et al.. Cell. 23, 309-310 (1981) and Ross et al..
  • the present invention also includes that class of polypeptides coded for by portions of the DNA complementary to the protein-coding strand of the human cDNA or genomic DNA seguences of inhibitory factor, i.e., "complementary inverted proteins" as described by Tramontano et al. [Nucleic Acid Res., 12, 5049-5059 (1984)].
  • compositions comprising therapeutically effective amounts of polypeptide products of the invention together with suitable diluents, preservatives, solubilizers, emul ⁇ ifiers, adjuvants and/or carriers useful in inhibitory factor therapy.
  • suitable diluents preservatives, solubilizers, emul ⁇ ifiers, adjuvants and/or carriers useful in inhibitory factor therapy.
  • a "therapeutically effective amount” as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen.
  • compositions are liguids, gels, ointments, or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent adsorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g., glycerol, polyethylene glycol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material -into or onto particulate preparations
  • compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of inhibitory factor.
  • the choice of composition will depend on the physical and chemical properties of the protein having inhibitory factor activity. For example, a product derived from a membrane-bound form of inhibitory factor may reguire a formulation containing detergent.
  • Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils).
  • compositions coated with polymers e.g., poloxamers or poloxamines
  • inhibitory factor coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors.
  • Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitory factors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, topical (skin or mucosal) and oral.
  • compositions including one or more additional factors such as chemotherapeutic agents, TNF, cytokines (e.g., interleukins), antiproliferative drugs, 5FU, alkylating agents, antimetabolites, and drugs which interfere with DNA metabolism.
  • additional factors such as chemotherapeutic agents, TNF, cytokines (e.g., interleukins), antiproliferative drugs, 5FU, alkylating agents, antimetabolites, and drugs which interfere with DNA metabolism.
  • inhibitory factory factor is administered in conjunction with radiotherapy.
  • Polypeptides of the invention may be "labelled" by association with a detectable marker substance (e.g., radiolabeled with 125I, enzyme labelled, or biotinylated) to provide reagents useful in detection and guantification of inhibitory factor or its receptor bearing cells in solid tissue and fluid samples such as blood, urine, cerebral spinal fluid or culture media.
  • a detectable marker substance e.g., radiolabeled with 125I, enzyme labelled, or biotinylated
  • the subject invention also relates to antibodies specifically binding inhibitory factor.
  • One embodiment is polyclonal antibodies which bind inhibitory factor but not any proteases.
  • a further embodiment of the invention are stable hybridomas, i.e., hybridomas capable of being passaged repeatedly and cryopreservation, such hybridomas producing antibodies specifically binding inhibitory factor.
  • hybridomas capable of being passaged repeatedly and cryopreservation, such hybridomas producing antibodies specifically binding inhibitory factor.
  • monoclonal antibody is directed against a single determinant on the antigen.
  • Monoclonal antibodies are useful to improve the selectivity and specificity of diagnostic and analytical assay methods using antigen-antibody binding.
  • both monoclonal and polyclonal antibodies are used to neutralize or remove inhibitory factor from serum or from culture media or other liguids.
  • a second advantage of monoclonal antibodies is that they can be synthesized by hybridoma cells in culture, uncontaminated by other immunoglobulins.
  • Monoclonal antibodies may be prepared from supernatants of cultured hybridoma cells or from ascites induced by intraperitoneal inoculation of hybridoma cells into mice.
  • the hybridoma technique described originally by K ⁇ hler and Milstein [Eur. J. Immunol. 6, 511-519 (1976)] has been widely applied to produce hybrid cell lines that secrete high levels of monoclonal antibodies against many specific antigens.
  • the inhibitory factor of the subject invention is useful as a reagent to synchronize cell populations in culture for studies including, but not limited to, measuring specific biochemical events in specific stages of the cell cycle, receptor-ligand interactions that influence cell division, drug effects, effects of viruses, effects of transforming agents, effects of mutagens, and effects on the ability of cells to fuse with other cells or react to environmental stimuli (heat, cold, etc.), and signal transduction events that occur subsequent to receptor-ligand interaction.
  • the inhibitory factor is useful in studies with cells derived from mammalian and non-mammalian species, primary cultures and established cell lines and nontransformed and suitable tumorigenic cell lines.
  • the inhibitory factor is useful for examining various stages of the cell cycle. Adding, e.g., 1 to 10 x 10 —8 M of the inhibitory factor to exponentially dividing cell cultures, and incubating the cells for a period of time, allows all cells to come to arrest at one point in the cell cycle. Long incubation (over one generation time) provides the largest percentage of arrested cells. The incubation time varies according to the application. The inhibitory factor-containing medium is then removed or inactivated
  • control cultures containing: 1) control cultures never exposed to inhibitory factor; 2) control cultures 21
  • the polypeptide of the subject invention is useful for studying metabolic events of cells and effect of growth stimulators on confluent cells (density inhibited cells). Mitogens are added to non-growing cells to stimulate division of the cells and inhibitory factor e.g., 1 to 10 x 10 -8 M is added at the same time or at various times after the mitogens.
  • inhibitory factor e.g. 1 to 10 x 10 -8 M
  • Various metabolic events including, but not limited to, DNA synthesis, RNA synthesis, protein synthesis and posttranscriptional and posttranslational modifications of macromolecules can be studied as related to mitogen stimulated cell cycling.
  • this method offers a novel method to study the potential interactions between the inhibitory factor and various mitogenic substances.
  • Primary explants or cultures of tumors or some other cultures which are not in exponential growth or stable (confluent) state can be treated with inhibitory factor.
  • inhibitory factor synchronizes the vast majority of the cells in a culture. Inhibitory factor treated cells do not divide until approximately ten hours after removal of inhibitory factor; then, within one hour, greater than 95% enter and successfully complete mitosis, resulting in a striking doubling of cell number. This contrasts with the effects of currently used mitogens to stimulate division, for example following the stimulation of lymphocytes with PHA the number of mitosis gradually rises beginning at 40 hours, reaches a maximum and levels off at approximately 3% after 72 hours (Verma et al. Human Chromosomes, Pergamon Press, N.Y., N.Y. (1989)). Inhibitory factor reversibly inhibits over 90% of cultured human products of conception cells and cultures of many types of cancer cells.
  • inhibitory factor appears to halt the cells at a single highly discreet point in late Gl/GO (Fatteay et al., supra 1991) most arresting agents cause cells to grind to a halt when they run out of DNA precursors sometime in S. A higher degree of synchrony will be achieved using inhibitory factor.
  • the experimental data shows a very sharp increase in cell numbers and preservation of synchrony for more than one mitosis, a highly unusual property.
  • synchronization by inhibitory factor is less toxic than currently available methods.
  • inhibitory factor is expected to greatly increase the number of mitoses compared to untreated cells and even compared to cells subject to a typical metaphase block of a few hours, or PHA.
  • this method is used in combination with traditional metaphase blockers (e.g., Colcemid). This is especially important in solid tumors where the cells have a variety of doubling times.
  • metaphase blockers e.g., Colcemid
  • Inhibitory factor is useful for obtaining different groups of mitotic figures that represent subpopulations of cells with different rates of growth. This occurs since the.cells with the most rapid S phase enter mitoses before cells with a longer S phase. These cells are preferentially observed or isolated. Similarly cells with various length S phases can be isolated or observed. An easy way of observing such cells is with metaphase spreads and an easy way of isolating such cells is by shaking off the mitotic cells from the culture vessel.
  • the inhibitory factor is useful to experimentally induce cellular differentiation and the subsequent morphological and biochemical alterations that accompany this process. This includes cells obtained from solid and fluid tissues from mammalian and non-mammalian species.
  • Various cells in culture are treated with, e.g., 1 to 10 x 10 —8 M of the polypeptide inhibitory factor, and/or its peptide fragments and events associated with cellular differentiation, including but not limited to specific metabolic processes and morphological changes, are monitored during the culture period.
  • In vivo differentiation is useful to treat various types of cancers and other diseases (see below).
  • the inhibitory factor provides cell cycle arrest and cultures in "suspended animation" that subsequently permits an investigator to store the cultures without routine and laborious refeeding or subculturing the cells on as frequent a.schedule.
  • This application also provides a means to maintain cell cultures in "suspended animation" for purposes associated with shipping the cells over long distances, or maintaining the cultures outside of the culture facility for extended periods of time, without routine refeeding or exchanging cell culture medium. It can be especially difficult to refeed or perform maintenance or cells being prepared for transport to space or a large number of clones being analyzed for function. Embryos, fetuses and adult organisms can similarly be caused to suspend division temporarily by use of inhibitory factor.
  • the first embodiment of the invention for treatment of neoplastic diseases is the direct treatment to effect improved clinical state.
  • Inhibitory factor may be used alone or in combination with drugs to directly slow or stop unwanted proliferations.
  • Drugs most useful in combination with inhibitory factor to stop cancer cells are those that work throughout the cell cycle such as alkylating agents which inhibit glycolysis and respiration as well as effecting DNA. Examples of these are Busulfan, Chlorambucil, Cyclophosphamide, dacarbazine, Mechlorethahamine, Melphalan and Thiotepa.
  • Certain antitumor antibiotics such as the anthracycline and chromycins (Dactino ycin, Daunorubicin, Doxorabicin, Placamyccin, Mitomycin C) and the nitroureas and cytokines which are cell cycle nonspecific may similarly be used in combination with inhibitory factor to cause direct toxicity. Cancers are particularly dangerous, because the cancerous cells continue to proliferate and often metastasize (spread to and proliferate at distant sites). In general, "undifferentiated” or “embryonic” or “primitive” cells within the cancer are the most likely to proliferate and spread. Cancers that spontaneously regress often do so by undergoing differentiation. In addition, successful therapy often induces differentiation.
  • Animal cancers are subject to inhibitory factor therapy by causing differentiation.
  • An example of this is the differentiation and permanent irreversible inhibition of the human leukemia line H6-60 by inhibitory factor.
  • Inhibitory factor may be used in combination with known chemotherapy, as well as on its own to cause diffferentiation. Certain drugs are known to act by stimulation differentiation and slowing the growth of tumor cells. These includes androgens, estrogens, steroids and some cytokines. Inhibitory factor may be advantageously combined with drugs like Tamoxifin, Estradiol, Ethynl Estradiol, Diethylstibesterol, Premarin, Medrooxy progesterin, Megestrol, Hydroxyprogesterone, Testosterone, Floxymestrone, Methyl testosterone, Testolactorie and other androgens, and corticosteroids including Predsinsone, Hydroxycortisone and Dexamethasone to stimulate differentiation and slow tumor growth.
  • drugs like Tamoxifin, Estradiol, Ethynl Estradiol, Diethylstibesterol, Premarin, Medrooxy progesterin, Megestrol, Hydroxyprogesterone, Testosterone,
  • a second example of application in the class of direct therapy is treatment of any skin or squamous cancer or overproliferation of skin cells. It has been found that some cells — human keratinocytes — are especially sensitive to inhibitory factor. Basal Cell Epithelioma's (BCE), squamous carcinomas and a wide variety of proliferative skin lesions including various icthyosis and psoriasis are all treatable with inhibitory factor. Other proliferative diseases which are treated with inhibitory factor include easinophilia, benign reactive lymphocylic hyperplasia, lymphoproliferative diseases, adenomas and certain preneoplastic lesions like familiar polyposis.
  • BCE Basal Cell Epithelioma's
  • squamous carcinomas a wide variety of proliferative skin lesions including various icthyosis and psoriasis are all treatable with inhibitory factor.
  • Inhibitory factor is useful in the treatment of human and animal leukemic disease (feline leukemia, HTLV virus, etc.).
  • the second embodiment of the invention to treat proliferative lessions is in combination with other drugs.
  • the above diseases and other diseases may be treated thusly.
  • _in vitro inhibitory factor acts in a synergistic manner with other cell modulators.
  • preliminary experiments Wangs, et al., FASEB J, 5: 1463, 1991
  • inhibitory factor increases TNF cytotoxicity to certain tumor cells.
  • Other applications involve the inhibitory factor as adjuvant to increase the sensitivity of neoplastic cells to other agents. This permits the use of lower concentrations of anti-neqplastic agents to provide effective doses at less toxic levels.
  • inhibitory factor in multiple-drug therapy for neoplastic disease.
  • the inhibitory factor augments the efficacy of treatment by other compounds by a molecular mechanism that is separate but synergistic.
  • Inhibitory factor can be used alone or with one or more additional factors such as TNF and cytokines in the treatment of disorders.
  • inhibitory factor with other agents such as one or more other factors is temporally spaced or given together.
  • the route of administration may be intravenous, intraperitoneal sub-cutaneous, intramuscular, topical, oral or nasal.
  • a third embodiment of the invention relating to the use of inhibitory factor as a chemoprotectant for normal cells in combination with chemotherapy agents. This combination decreases side effects. It is dependent on the cancer being resistant to inhibitory factor. Since inhibitory factor 1) prevents Rb phosphorylation; 2) underphosphorylated Rb maintains cells in a quiescent state; 3) certain cancer cells have oncogene producers which complex Rb; and 4) complexed Rb is not be effected by inhibitory factor. Certain tumors are insensitive to inhibitory factor. Cell lines transformed with SV40 large T antigen were assayed for inhibition by inhibitory factor. These cells were not inhibited by inhibitory factor. Similarly, human fibroblasts transformed with Adenovirus are not inhibited.
  • Control 3T3 cells used in these experiments were inhibited as in previous presented experiments. All three of the cell lines chosen because they contain Rb binding oncoproteins were found to resist inhibition, while none of the randomly chosen lines previously .screened were resistant.
  • Prostate cancer, bone cancer and bladder cancer are examples of cancer types insensitive to inhibitory factor.
  • inhibitory factor acts as a chemoprotectant. Other mechanisms of resistance are also possible.
  • inhibitory factor human cancer cells not inhibited by inhibitory factor, can be killed by a variety of treatments that destroy dividing cells while normal cells which are reversibly inhibited by inhibitory factor would be protected from destruction.
  • inhibitory--factor- is very useful as a drug to decrease the side effects of chemotherapeutic agents. It is given along with or just prior to cytotoxic therapies.
  • the normal cells would respond to inhibitory factor by stopping in a physiologically "safe" Gl resting phase while the cancer cells would continue to grow and be susceptible to killing by cytotoxic agents such as drugs or radiation.
  • Certain drugs are known to specifically efffect cells in M or G2 phase of the cell cycle. These include Zinostatin Bleomycin and some other anti-tumor antibiotics. - In addition, the alkyloids such as vinblastin, vincristine, vindesine and others specifically act in M phase by blocking microtubule action.
  • the Epipodophyllotoxins, etoposide and teniposide also specifically act in M phase with some effect in G2 and S.
  • the antimetabolites such as Fluorouracil, Floxurridine, Cytarabine, purine antagonist (mercaptopurine, 6 thioguanine, azathioprine) and folate antagonist (methotrexate, dichloromethotrexate, triazinate), hydroxurea and hexamethylmelamine are also S sphase specific. Inhibitory factor which will keep cells in Gl and specifically chemoprotect the normal against the 'toxicity of agents in these classes.
  • Certain dangerous DNA viruses are believed to interfere with cellular control mechanisms by producing molecules that interact with RB or with other cell regulatory molecules controlled by phosphorylation or by mechanisms affected by inhibitory factor (e.g., p53 or cyclins). It is believed that certain types of Human Papilloma Virus play a major role in causing cervical cancer. Carcinogenic types of Human Papilloma Virus (HPV types 16 and 18) produce proteins inactivating RB (Dyson, et al., 1989) (similarly to cells transformed with Adeno E1A or SV40 large T) . This invention includes assays to determine if certain tumors have affected the RB mechanism, and the use of inhibitory factor as a protectant during therapy of these tumors.
  • inhibitory factor e.g., p53 or cyclins
  • a combination of inhibitory factor to protect adjacent normal tissue and a cytotoxic agent is useful for treatment.
  • Such combinations can be locally applied.
  • Current treatments are various surgical procedures which tend to leave some in situ cancer behind unless relatively large areas are removed.
  • Inhibitory factor without cytotoxic therapies are effective against the condylo a (wart) producing viruses that do not produce oncogene products reactive with RB (e.g., HPV Types 6/11, Dyson, et al., Science 243, 934-937, 1989).
  • the inhibitory factor (which provides mitotic arrest) has application in screening of drugs. Many drugs act specifically in one region of the cell cycle (see above). By comparing the putative anti-cancer drugs effect on parallel sets of cultures - one set proliferating and the other mitotically arrested with inhibitory factor - the relative action on stable versus multiplying populations is readily assessed. Since the mitotic arrest mediated by the polypeptide is totally reversible, future growth measured by colony formation as well as survival of inhibitory factor treated cells is easily assessed. Drugs which act at other specific stages of the cell cycle can also be advantageously sought and analyzed using ihihibitory factor. Inhibitory factor is used to place cells in a specific stage of the cell cycle as described in section A) above.
  • Inhibitory factor is also useful in the treatment of other diseases having abnormal proliferation such as psoriasis, other icthyosis, keloid or certain autoimmune diseases. Keratinocytes are especially susceptible to inhibitory factor. Inhibitory factor is useful in the treatment of psoriasis, keloids and other proliferative skin diseases.
  • Psoriasis results from the excess division of skin cells (as do other proliferative skin diseases called "icthyosis"). In patients with psoriasis, skin cells divide seven times faster than normal. This disorder is treatable with inhibitory factor. Warts
  • Warts also are the result of excess epthelial cell proliferation, as are several other skin pathologies. Warts are caused by Human Papilloma Viruses (HPV's). Since it is known that keratinocytes (the type of epithelial cells that overproliferate in these lesions) are especially sensitive (approximately 30-50 fold more sensitive than most cells) to inhibitory factor these lesions can be treated effectively with inhibitory factor.
  • HPV's Human Papilloma Viruses
  • Keloid is a disease caused by overproliferation of scar tissue, thus it also can be advantageously treated with inhibitory factor as it is known that human fibroblasts are inhibited.
  • Atherosclerosis involves the overproliferation of cells lining the blood vessels. Inhibitory factor reversibly prevents proliferation of such cells including smooth muscle cells and endothelial cells. Overproliferation leads to a variety of abnormalities including heart disease, strokes, renal disease and others. These diseases also can be prevented by decreasing atherosclerosis with inhibitory factor.
  • Proliferative diseases of the eye including retinopathy are treatable with inhibitory factor to stop unwanted proliferation.
  • Unwanted inflammatory states such as allergies and autoimmune disease, even some types of arthritis involve the proliferation of certain cells that can be stopped with appropriate inhibitory factor therapy.
  • Multiple sclerosis has been postulated to be either a viral or autoimmune (inflammatory disease)-.
  • TNF is known to be altered locally in M.S. and thus inhibitory factor can be used as therapy.
  • Temporary inhibition by inhibitory factory factor might also be extremely useful in situations where cell passages are difficult. For instance in eucaryotic cell cloning often a large number of clones are initially obtained but only a few will be useful. Growth and passage of many clones during the evaluation period (for example while assays-of the clone's ability to produce a biologic material, e.g., a monoclonal antibody, a biologic response modifier or an enzyme of interest) may be difficult. Inhibitory factor can be used to easily place cells in a safe but non-dividing state. This method preserves a much larger number of important clones during the evaluation period with less effort and chance of loss or contamination.
  • a biologic material e.g., a monoclonal antibody, a biologic response modifier or an enzyme of interest
  • Inhibitory factor reorients the protein synthetic mechanism of cells; initially it shuts off the synthesis of many proteins (total synthesis drops by 80%) however within hours the total synthesis is only 20% to 25% less than in exponentially growing cells. Thus, it is believed that certain structural proteins necessary for an increase in cell number are shut off but many proteins made in Gl are actually synthesized at a higher rate. Thus increased production of certain biologically useful proteins (e.g., monoclonal antibodies or other excreted proteins) is possible using inhibitory factor. Inhibitory factor increases production of monoclonal antibodies. Even if production per cell is not increased it may be very beneficial to have a metabolically active "bioreactor" with stable cell number in many instances. In theory, such bioreactors might even provide an entire metabolic pathway.
  • Inhibitory factor is helpful as an aid in karyotypic
  • chromosome chromosome analysis. It is especially important in situations where low numbers of mitotic cells are present, such as solid cancers, or in situations where low numbers of cells are available for analysis (some difficult amniotic fluid taps or the isolation of sub populations of cells) .
  • the inhibitory factor is useful in isolating viruses. Viruses often require activitely dividing cells. The most frequent reason for in laboratory failure to isolate viruses from adequate clinical specimens in plating of the virus on cells that have become too dense or too confluent. Overgrown cells are also the major cause of delay in isolation of viruses clinical labs most frequently.isolate. Inhibitory factor can be used to hold cells at the ideal density for virus growth and then initiating exponential growth (which is most helpful for growing viruses such as herpes virus, cytomegulovirus and many other viruses which require activily growing cells) by removing inhibitory factor as described above.
  • Some viruses are difficult to culture using current methods. However, if cells were infected at the optimum point in the cell cycle, for example, during S phase or M phase or G2 growth is much more reliable. The only methods currently available to achieve cultures with high amounts of S, M or G2 phase cells for this or any purpose is inhibitory factor.
  • inhibitory factor to allow growth of viruses which cannot currently be grown in the lab (e.g., HPV). It is believed that HPV and other viruses requires cells with certain differentiation properties. Since inhibitory factor will cause differentiation, it can facilitate growth of this class of viruses.
  • Nucleic acid products of the invention are useful when labeled with detectable markers (such as radiolabels and non-isotopic labels such as biotin) and employed in hybridization processes to locate the human inhibitory factor gene position and/or the position of any related gene family in a chromosomal map. They are also useful for identifying human inhibitory factor gene disorders at the DNA level and used as gene markers for identifying neighboring genes and their disorders. The identification of the genes and defects in them are important in diagnosis and prognosis of proliferative diseases and cancers. The protein from these genes is assayed by use of monoclonal or polyclonal antibodies in various formats including western blots, dots blots and ELISAs. The detection of protein facilitates diagnosis and prognosis of various diseases involving altered levels of cell proliferation.
  • detectable markers such as radiolabels and non-isotopic labels such as biotin
  • inhibitory factor should be administered in a range of 1 nanomolar to 1 micromolar, advantageously the factor is administered at a concentration from 1 to 10 x 10 —8 molar.
  • media with low calcium concentration increases the sensitivity to inhibitory factor.
  • a suspension of bovine cerebral cortex cells was prepared in Dulbecco's minimal essential medium (DMEM) containing 25 mM HEPES buffer (pH 7.1). The cells were pelleted by centrifugation at 2000 x g for 5 min, the cell pellet was suspended in HKM buffer (10 mM HEPES, 120 mM KCl, 5 mM MgCl-, pH 7.1) and incubated with 0.02 units/ml of proteinase from S. griseus ("pronase”) for 15 min at 37°C with constant mixing.
  • DMEM Dulbecco's minimal essential medium
  • HKM buffer 10 mM HEPES, 120 mM KCl, 5 mM MgCl-, pH 7.1
  • pronase proteinase from S. griseus
  • the cells were then removed by centrifugation at 2000 g for 5 min.
  • the supernatant fluids containing released molecules were then collected, the macromolecules were precipitated with ethanol overnight, and the resulting precipitate was collected by centrifugation, resuspended in 100 ml distilled water, extracted with chloroform/methanol (2:1, v/v), dialyzed against four liters of distilled water overnight, with at least six water changes, and the dialysate was then lyophilized to dryness.
  • the lyophilized material was resuspended in 2 ml of 0.05 M acetate buffer (pH 5.0), clarified by three subsequent centrifugations at 1,000 g for two minutes and applied to a DEAE-agarose gel.
  • Approximately 50 mg. protein of the chloroform/methanol-extracted material was incubated with DEAE-agarose gel (10 ml bed volume) at 4°C for 30 min with constant mixing.
  • the gel was washed three times with 3 ml of the acetate buffer and the biological inhibitory factor was eluted with 3 ml of 0.4 M NaCl in 0.05 M acetate buffer (pH 5.0). The eluate was then dried in a Savant speed-vac apparatus.
  • the DEAE-agarose purified samples were then further purified with agarose-bound wheat germ agglutinin (WGA) .
  • WGA agarose-bound wheat germ agglutinin
  • the WGA was previously equilibrated with phosphate buffered saline (PBS, pH 7-1), and the protein fraction was suspended in 1.5 ml of PBS and applied to a 1.0 ml WGA column. After incubation at 4°C for 30 minutes, the inhibitory factor-containing fraction that does not bind to the WGA column was removed by washing with 2 ml of PBS.
  • PBS phosphate buffered saline
  • the WGA-eluted fraction was then further purified by applying the protein to a HPLC TSK-3000 size exclusion column.
  • the elution buffer consisted of 0.1 M "sodium phosphate (pH 6.8), and the flow rate was adjusted to 0.1 ml/min.
  • the eluate was monitored for absorption at A,--, and the fractions associated with the major protein peak were pooled, dialyzed overnight at 4°C against four liters of dilute PBS.
  • the sample was then dried in a speed-vac apparatus, resuspended in 0.5 ml of distilled water and the protein content and the biological inhibitory activity were measured.
  • Protein synthesis was measured with cells from subconfluent cultures that were suspended in DMEM containing 25 mM HEPES buffer, pH 7.1 (Sharifi et al., Neurochem. 46: 461-469, 1986a; Bascom et al. , J. Cell Physiol. 128: 202-208, 1986). Either HKM buffer alone (controls), or HKM buffer with various concentrations of the 18 kD brain inhibitory factor (experimentals) were added to each reaction tube. The cells were incubated for
  • H-thymidine incorporation was measured with cultures in 24- or 48-well culture plates.
  • subconfluent cell monolayers were incubated with 0.2 ml of DMEM medium 3 containing 2.5% calf serum and H-thymidine (adjusted with non radioactive thymidine to a specific activity of 0.5
  • Macromolecules were precipitated with 10% TCA, after the addition of 0.1 ml of 1% BSA as a carrier.
  • Radioactive thymidine in the intracellular acid-soluble pools and in the cell DNA was measured by scintillation counting (Chou et al.. Cancer Lett. 35: 119-128, 1987;
  • sialoglycopeptide was studied as a potential antagonist to mitogens, that stimulate cell division, (e.g., EGF, TPA and bombesin) utilize confluent and guiescent cultures (See Bascom et al., J. Cell. Biochem. 34: 283-291, 1987; Chou et al., Cancer Lett. 35: 119-128, ⁇ 1987; ⁇ Johnson and Sharifi, Biochem. Biophys. Res. Comm. 161: 468-474, 1989).
  • Example I Although the purification procedure described in Example I appears to provide a 18 kDa glycopeptide product that was homogeneous, small molecular weight peptides contaminated the samples to various degrees from preparation to preparation. These contaminants were difficult to visualize when purified samples of the glycopeptide were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and stained by the Comassie Blue method of Sasse et al., in Current Protocols in Molecular Biology, (F.A. AuSabel, R. Brent, R.E. Scientific, D.D. Moore, J.G. Saidman, J.A. Smith and K. Struhl, eds.), pp. 10.6.1-10.6.2 (1991).
  • a simple, but effective, procedure was developed to provide a homogeneous glycopeptide preparation. This procedure involves the use of a HPLC/DEAE ion-exchange step as the final step of bioseparation.
  • a Protein-Pak DEAE (Waters) HPLC column was eguilibrated with 20 mM Tris-HCl (pH 8.2) or 40 mM NH 4 HC0_ (pH 8.0) and 10 mM NaCl.
  • the glycopeptide (20 to 60 micrograms) was added to the DEAE column and the glycoprotein was eluted by introducing over a 30 minute period a linear NaCl gradient that increased from 10 to 100 mM.
  • the eluant was monitored at A---, and the purified glycopeptide that eluted from the HPLC column as a single and sharp peak, at approximately 50 mM NaCl (as determined by refractometry), and well-separated from the contaminating small molecular weight peptides, was collected manually.
  • glycopeptide inhibitory factor was then lyophilized to dryness and resuspended in 2.0 ml of distilled water.
  • the sample was then desalted by five serial centrifugation ⁇ (each with 2.0 ml of distilled water) in microconcentrators (Amicon) fitted with a membrane that retained molecules over 10 kDa.
  • the retentate, containing the 18 kDa glycopeptide was lyophilized and stored frozen at -70°C.
  • a 12 microgram (600 pmoles) of inhibitory factor was prepared as in Example I and further purified as in Example III. Since the protein is both N-terminally blocked and glycosylated, sequencing and associated tasks were extremely difficult.
  • Cyanogen bromide was obtained from Sigma, sequencing grade trypsin, chymotrypsin, endoproteinase Asp-N and Olu-C from Boehringer Mannheim. Iodoacetic acid was purchased from Sigma, dithiothreitol from Calbiochem. HPLC grade trifluoroscetic acid was obtained from Applied Biosystems, Inc. (Foster City, California); HPLC-trade acetonitrile and water from Burdick & Jackson; 6N HCl from Pierce; and Vydac HPLC columns from the Nest Group (Southboro, Massachusetts). Automated sequencer and analyzer reagents were provided by the manufacturer. All other reagents were purchased from common commercial sources in the highest grade available.
  • Inhibitory factor destined for proteolytic cleavage was reduced and S-carboxymethylated as described by Stone et al, Techniques in Protein Chemistry (Hugli, ed. ) Academic Press, San Diego, pp. 377-391, (1989).
  • 6.0 ⁇ g (300 pmol) aliquots of bovine inhibitory factor were dissolved in 50 ⁇ l 8 M urea/0.4 M NH.HC0- and reduced with 5 ⁇ l of 45 mM dithiothreitol at 50°C for 15 min. Cysteine residues were alkylated by reaction with 5 ⁇ of 100 mM iodoacetic acid at room temperature for 15 min. Subsequent enzymic cleavage was carried out without , further desalting or transfer- as described below.
  • Peptides were chromatographed on a Hewlett-Packard 1090 HPLC eguipped with a 1040 diode array detector, using a Vydac 2.1 mm x 150 mm C18 column.
  • the gradient employed was a modification of that previously described by Stone et al, supra, (1989). Briefly, where buffer A was 0.06% trifluoroacetic acid/ H,0 and buffer B was 0.055% trifluoroacetic acid/acetonitrile, a gradient of 5%B at 0 min, 33%B at 63 min, 60%B at 95 min, and 80%B at 105 min with a flow rate of 150 ⁇ l/min was used.
  • Chromatographic data at 210 nm, 277 nm, 292 nm, and UV spectra from 209-321 nm of each peak were acguired. While monitoring absorbance at 210 nm, fractions were manually collected into 1.5 ml microfuge tubes and immediately stored without drying at -20°C in preparation for peptide seguence analysis.
  • inhibitory factor could not be cleaved with either trypsin or chymotrypsin, a series of analytical tests were run with bovine inhibitory factor, using 5 micrograms per assay, to determine if the 18 kDa bovine inhibitory factor could be proteolytically cleaved.
  • the inhibitory factor was first solubilized in phosphate buffered saline (PBS, pH 7.0), heated to 95°C for 5 min. to denature the polypeptide and then cooled to room temperature prior to the addition of the enzymes which were in 0.2 M ammonium bicarbonate (pH 8.0).
  • the two enzymes used were bovine pancreatic trypsin (TPCK-treated) (Sigma Chem. Co., catalog # T-8642), and endoproteinase Asp-N (Sigma Chem. Co., catalog # P-3303, suitable for sequencing and peptide mapping) .
  • Suitable enzymes may be chosen from the group including Olu-C and pronase, trypsin, chymotrypsin, B substilysin, serine proteases, thiolproteases, cathepsin D proteases, sulphydryl protease, metallo-proteases, trypsin like protease, estrase and carboxy proteases, non-specific proteases and other specific proteases.
  • the resulting fragments are then separated and sequenced.
  • the fragmentation of the purified 18 kDa bovine inhibitory factor (described in Example III), is carried out essentially as described above but with approximately 25 to 50 ⁇ g with an enzyme/protein ratio of at least 1:50.- This provides adequate quantities of the fragments for separation of the fragments by HPLC and subsequent sequencing by routine methods.
  • the DNA sequence encoding inhibitory factor can be obtained using routine procedures for synthesizing oligonucleotide probes using the amino acid sequence, and screening libraries. See Sambrook et al. , Molecular Cloning A Laboratory Manual, 2d Edition, Cold Spring Harbor Laboratory Press, Chapter 11, 1989; Heller et al., Biotechniques 12, No. 1, p. 30-35 (1992); Itakura et al. Annual Rev. of Biochem, 53, 323-356 (1984); or Wood et al, PNAS, 82, 1585-1588 (1985).
  • Rabbit polyclonal antibody against the native (nondenatured) form of the inhibitory factor was prepared by subcutaneous injection of New Zealand white rabbits with 200 ⁇ g of the 18 kDa bovine inhibitory factor purified as in Example 1 in an equal volume of Freund*s complete adjuvant or mixed with TitermaxTM adjuvant and as described by Lakshmanarao et al. Exptl. Cell Res. 195, 412-415, (1991).
  • the autoimmune offspring of a male Balb/c/J mouse and a female Balb/AJ mouse are used. Further boosting once a month with antigen in complete Fruends adjacent has resulted in higher avidity antibody.
  • Plasma membrane preparation and NaCl release - Plasma membranes were obtained from cell suspensions of bovine cerebral cortex tissue homogenized by 10 strokes in a Dounce homogenizer. The homogenate was centrifuged at 1,000 x g for 15 min, and the supernatant fluid" was collected and recentrifuged at 1,000 x g for 15 min.
  • the resulting supernatant fluid was centrifuged at 40,000 x g for 60 min to pellet the plasma membranes, and the membrane-associated proteins were released by resuspending' the pellet in 10 vol of a buffered 3M NaCl solution (3M NaCl, 0.1 M phosphate buffer, pH 7.2, containing l ⁇ g/ ⁇ l each of phospho amidon, pepstatin A, leupeptin and aprotinin) .
  • the membrane suspension was mixed for 30 min at 4°C, centrifuged at 104,000 x g for 60 min and the supernatant fluid was collected and dialyzed overnight at 4°C.
  • the samples were first dialyzed against 1 M NaCl, followed by three changes of double-distilled water and after dialysis protein determinations were carried out by the method of Bradford, Anal. Biochem. 72, 248-254 (1976).
  • Preparative isoelectric focusing The NaCl-released membrane proteins were resuspended in 40 ml of double-distilled water, and electrofocused at 12W for.4 h with 2% amphylines (pH 4-10, Pharmacia-LKB Biotechnology Inc., Gaithersburg, MD) in a BioRad Rotofor apparatus (Bio-Rad, Richmond, CA) . The resulting 20 (2 ml) fractions were dialyzed against three changes of dilute PBS and concentrated to dryness in a Savant Speedvac (Savant Instruments Inc., Hicksville, NY).
  • Lectin affinity chromatography The electrofocused samples were solubilized in working buffer (50 mM Tris-HCl, 10 mM CaCl_, pH 8.0) and then added to a column (1 ml bed-volume) of Limulus polyhemus agglutinin (LPA) (EY Laboratories, San Mateo, CA) that previously was equilibrated with working buffer. The samples were incubated with constant mixing for 1 h at room temperature, and the column was then washed with working buffer until no eluting protein (A___) could be detected.
  • working buffer 50 mM Tris-HCl, 10 mM CaCl_, pH 8.0
  • LPA Limulus polyhemus agglutinin
  • the bound proteins were eluted with elution buffer (50 mM Tris-HCl, 2 M EDTA, pH 8.0), and both the bound and unbound fractions were extensively dialyzed at 4°C against dilute PBS and lyophilized to dryness. Equal volume samples of the dialysis fluids were also lyophilized as controls for measurements of biologipal inhibitory activity.
  • elution buffer 50 mM Tris-HCl, 2 M EDTA, pH 8.0
  • Antibody affinity chromatography - IgG (1 mg) prepared to the native inhibitory factor was bound overnight at 4°C to 1 ml of prewashed Affi-Gel HZ beads (Bio-Rad, Richmond, CA) following the protocol provided by the commercial supplier.
  • the protein fraction that isoelectric focused at pi 5.1 ( ⁇ 1 mg protein) was added to the column, incubated overnight at 4°C and the column was then washed with column buffer until no eluting protein ( ,-- could be detected.
  • 3 M MgCl- (pH 7.1) was used to release the bound proteins, and the eluted protein fractions were collected, pooled, dialyzed overnight against dilute PBS at 4°C and lyophilized to dryness.
  • Protein synthesis inhibition assay At various stages of purification the ability of samples to inhibit protein synthesis was tested with Swiss 3T3 cells essentially as described by Sharifi et al., J. Neurochem. 46, 461-469, (1986).
  • Cell proliferation inhibition assay Cell proliferation inhibition was measured with exponentially dividing cultures of Swiss 3T3 cells propagated in 48-well plates as described by Fattaey et al., J. Cell Physiol. 139, 269-274, (1989). The total medium volume of all cultures was 300 ⁇ l, and one set of control cultures received 40 ⁇ l of PBS while another received 40 ⁇ l of the lyophilized dialysis fluids that were solubilized in 1 ml of sterile double-distilled water. Experimental cultures received complete culture medium with 40 ⁇ l containing various concentrations of LPA bound or unbound protein solubilized in 1 ml of sterile double-distilled water.
  • the NaCl-released membrane proteins were next subjected to preparative isoelectric focusing utilizing a BioRad Rotofor as described in the Materials and Methods. 85 mg of the NaCl-released proteins were introduced to the Rotofor unit, the material was focused for 4 h and 20 fractions (2 ml) were collected across a pH gradient from 4.0 to 12.0. The proteins were relatively equally distributed across the gradient with each fraction having somewhere between 3.5 to 4.0 mg of protein. Immunoblot analysis of each fraction revealed that the antigenically-reactive material primarily was associated with two fractions: a major reactive peak was found to be focused at a pi of 5.1 (fraction number 4); and, a minor reactive peak was focused at a pi of 7.2 (fraction number 10) (Fig. 3).
  • LPA Limulus polyhemus agglutinin
  • Both the purified 66 kDa membrane protein and the LPA unbound protein fraction were tested for biological inhibitory activity with exponentially dividing mouse Swiss 3T3 fibroblast cells.
  • the lyophilized 66 kDa protein was resuspended in 1 ml of distilled water, and 40 ⁇ l containing 1, 5 or 10 ⁇ g of protein were added to culture medium to provide a total volume of 300 ⁇ l, resulting in final concentrations of the parental inhibitory factor of 5 x 10 —8 M, 2.5 x 10-7 M and 5 x 10-7 M, " respectively.
  • the parental inhibitory factor of 5 x 10 —8 M, 2.5 x 10-7 M and 5 x 10-7 M, " respectively.
  • Immunoscreening of cDNA libraries with the polyclonal antibody prepared to the denatured inhibitory factor was carried out with commercially available cCNA libraries prepared from bovine cerebral cortex, human fetal brain and mouse kidney.
  • the cells were then centrifuged at 4000 x g for 10 minutes at room temperature and resuspended in 5 ml to 10 ml of LB medium supplemented as described above.
  • E. coli strain Y1090hsdR which is commonly used as the host for immunological screening of expression libraries constructed in ⁇ gtll as was the case for the MK library (see below), carries a plasmid (pMC9) that codes for the lac repressor and prevents synthesis of potentially toxic fusion proteins from the ⁇ -galactosidase promoter. This plasmid also carries a selectable marker (amp ). To ensure against loss of the plasmid, E. coli strain Y1090hsdR was grown in media containing 50 ⁇ g/ml ampicillin.
  • E. coli strains BB4 and XLl-Blue which were used for immunological screening of libraries constructed in ⁇ ZAP, carry a ⁇ acI 9 gene and a tetr marker on an Fi factor.
  • strains were therefore grown in media containing 12.5 ⁇ g/ml tetracycline.
  • a set of sterile tubes (13 mm x 100 mm) were arranged in a rack; a fresh tube was used for each plate infected.
  • 0.1 ml of the plating bacteria was mixed with 0.1 ml of sodium magnesium media (manniatis, supra) containing 3 x 10 4 pfu
  • Nitrocellulose filters were numbered. The filters were handled with gloved hands. The filters were soaked in a solution of isopropylthio- ⁇ -n-galactoside (IPTG) (10 mM in distilled water) for a few minutes. One set of plates were done without IPTG - treated nitrocellulose as a control. Using blunt-ended forceps (e.g;, Millipore forceps), the filters were removed from the solution, and allowed to dry at room temperature on a pad of Kimwipes.
  • IPTG isopropylthio- ⁇ -n-galactoside
  • the plates were removed from the incubator, and the agar quickly overlayed with the IPTG-impregnated nitrocellulose filters.
  • Each filter was marked in at least three asymmetric locations by stabbing through it and into the agar underneath with an 18-guage needle attached to a syringe containing waterproof black ink.
  • the filters were peeled off the plates and immediately immersed in a large volume of TNT. Any small remnants of agarose was rinsed away by gently agitating the filters in the buffer. The TNT was agitated to prevent the filters from sticking to one another.
  • the plates were wrapped in Saran Wrap, and stored at 4°C until the results of the immunological screening were available.
  • the filters When all of the filters are removed and rinsed, they are transferred one at a time to a fresh batch of TNT. When all of the filters have been transferred, the buffer is agitated gently for a further 30 minutes at room temperature.
  • the filters were transferred individually to glass trays or petri dishes containing blocking buffer (7.5 ml for each 82-mm filter; 15 ml for each 138-mm filter). When all of the filters had been submerged, the buffer was agitated slowly on a rotary platform for 30 minutes at room temperature.
  • the blocking buffer was stored at 4°C and reused several times.
  • Sodium azide was added to a final concentration of 0.05% to inhibit the growth of microorganisms.
  • the filters were transferred to fresh glass trays or petri dishes containing the primary antibody diluted in blocking buffer (7.5 ml for each 82-mm filter; 15 ml for each 138-mm filter). The highest dilution of antibody was used that gives acceptable background yet still allows detection of 50-100 pg of denatured antigen. When all of the filters had been submerged, the solutions were agitated gently on a rotary platform overnight at room temperature.
  • the antibody solution was stored at 4°C and reused several times.
  • Sodium azide was added to a final concentration of 0.05% to inhibit the growth of microorganisms.
  • the filters were washed for 10 minutes in each of the buffers below in the order given. The filters were transferred individually from one buffer to the next. 7.5 ml of each buffer was used for each 82-mm filter and 15 ml. for each 138-mm filter. ⁇ >e_
  • the antigen-antibody complexes were detected with the radiochemical reagents. ——
  • Radiolabeled protein A is available from commercial sources (sp. act.
  • Radioiodinated second antibody is prepared according to well known technigues. Radiolabeled ligands were diluted in blocking buffer (7.5 ml for each 82-mm filter; 15 ml for each 138-mm filter) . The filters were incubated 2 hrs. at room temperature, and then washed several times in TNT before autoradiographs were established.
  • the bacteriophage particles were allowed to elute from the agar plug for several hours at 4°C.
  • the titer of the bacteriophages in the eluate was determined, and then replated so as to obtain approximately 3000 plaques per 90-mm plate.
  • the plaques were rescreened as described above, and the process of screening and plating was repeated until a homogeneous population of immunopositive recombinant bacteriophages was obtained.
  • the clonal isolates were subcloned at least three times to provide a homogeneous positive population, and each time the plaques were tested with the polyclonal antibody probe to provide assurance of continued antigen product expression and the homogeneity of the final isolate.
  • Bovine Cerebral Cortex Library (BCC), lambda ZAPII phage/XL-1-Blue E. coli host, 2,000,000 plaques screened, five positive clones; the five postiive clones were pooled and labelled "B" and deported at the ATCC on April 27, 1992.
  • HLB Human Fetal Brain Library
  • MK Mouse Kidney Library
  • Two 20-ml starter cultures of bacteria were grown overnight in LB medium supplemented with maltose and magnesium sulfate as described above.
  • a lambda virus preparation with a titer of 10 plague forming units per ml was mixed with 200 microliters of the host cell starter culture, and the preparation was incubated at 37°C for 15 to 30 minutes. The preparation was then added to the 40 ml host culture and incubated at 37°C, with constant mixing, until lysis occurred (7 to 8 hours).
  • the DNA was then extracted with 1 volume of TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 7.4) saturated with phenol plus chloroform and isoamylalcohol (50:48:2).
  • TE buffer 10 mM Tris-HCl and 1 mM EDTA, pH 7.4
  • phenol plus chloroform and isoamylalcohol 50:48:2
  • the tubes were rocked gently for 1 minute, then centrifuged at 12,000 x g at 4°C for 5 minutes, and the initial extrarction was repeated.
  • the agueous phase was then removed and extracted once with chloroform/isoamylalcohol (24:1) with gentle rocking for 1 minute.
  • the mixture was centrifuged at 12,000 x g at 4°C for 5 minutes and the aqueous phase removed.
  • An equal volume of isopropanol was added, the tubes rocked gently, and the mixture was left at -70°C for at least 20 minutes.
  • the mixture was again centrifuged at 12,000 x g at 4°C for 10 minutes and the supernatant fluid was removed.
  • the pellet was rinsed by adding 1 ml of 70% ethanol, followed by immediate centrifugation at 12,000 x g at 4°C for 10 minutes.
  • the resulting pellet was air-dried and resuspended in 50 microliters of 50 mM Tris-HCl (pH 7.5).
  • This method provided 35 micrograms of lambda DNA.
  • the preparation of this example was an isolate with a 1.3 kilobase pair insert, obtained from the human fetal brain cDNA library.
  • the DNA inserts removed by restriction enzymes or per were then subcloned into the vector p-Flag system, available commercially from International Biotechnologies, Inc. (New Haven, CT), in either XLl-Blue or JM101 E. coli hosts. Each subclone was tested for inducibility, fusion proteins isolated from the microbial periplasm and identified by Western analysis. The purified fusion proteins are tested for biological inhibitory activity by assays described above.
  • Inhibitory factor mediated cell cycle arrest of both human diploid fibroblasts (HSBP) and mouse fibroblasts (Swiss 3T3) results in the maintenance of the RB protein in the hypophosphorylated state, consistent with a late Gl arrest site.- Although their normal nontransformed - counterparts are sensitive to cell cycle arrest mediated by inhibitory factor, cell lines lacking a functional RB protein, through either genetic mutation or DNA tumor virus oncoprotein interaction, are refractory.
  • sialoglycopeptide inhibitory factor inhibitor was released from intact bovine cerebral cortex cells by mild proteolysis and purified to apparent homogeneity as described above. Briefly, bovine cerebral cortex cells were treated with dilute protease, the released molecules precipitated with ethanol, the precipitates were extracted with chloroform/methanol (2:2), and inhibitory factor was purified by DEAE ion-exchange chromatography, lectin affinity chromatography and HPLC with a TSK-3000 size exclusion column. .
  • the samples were then dialyzed against distilled water, lyophilized and resuspended in phosphate buffered saline (PBS; 145 mM NaCl, 5 mM potassium phosphate, pH 7.2). Protein determinations were carried out by the method of Bradford, et al, Anal. Biochem. 72:248-254 (1976) using bovine serum albumin as a protein standard, and the purified inhibitory factor preparations were stored at -70°C.
  • PBS phosphate buffered saline
  • Human diploid foreskin fibroblasts (HSBP), human osteosarcoma cells (U20S and SAOS-2), human bladder carcinoma cells (J82), human prostate carcinoma cells (DU145), and adenovirus transformed human epithelial cells (293) and grown in DMEM with 10% fetal calf serum.
  • Human fibroblast keratinocytes (HFK), and HFK cells transformed with papillomaviruses (28-NC0 and 1321) Pietenpol, et al. Cell 61:777-785 (1990); Romanczuk, et al, J. Virol. 65:2739-2744 (1991) and grown in KGM media with growth factors (Clonetics, San Diego, CA) .
  • Protein synthesis inhibition assay Protein synthesis inhibition was tested essentially as described by Sharifi et al, J. Neurochem. 46:461-469 (1986). Various concentrations of the purified inhibitory factor were added
  • MEM/HEPES Eagle's medium
  • the cells were preincubated with the inhibitor for 30 min at 37°C to allow inhibitory factor to bind to the cell surface receptor, and then 2.0 ⁇ Ci of [ 35S]methionine, in 10 ⁇ l of methionine-free
  • MEM/HEPES were added, and the cells were incubated for an additional 15 min.
  • the cell proteins were precipitated with trichloroacetic acid (TCA), the precipitates were washed several times with 5% TCA, and the amount of radioactivity incorporated into acid-insoluble protein was measured in a liquid scintillation system Sharifi et al, J.
  • Cell proliferation assay Cells were plated in 48-well culture plates (Costar, Cambridge, MA) and allowed to attached for at least 4 h. Then -6-9 x 10 —8 M inhibitory factor, diluted in the appropriate culture medium, or medium alone, was added and the cell number determined at various times with a Coulter counter, model ZM Edson, et al. Life Sci. 48:1813-1820 (1991) and Fattaey, et al, J.
  • IgG 1 (PMG3-245, Pharminigen, San Diego, CA) . Due to the lower reactivity between the mouse RB product and the
  • Inhibitory factor binding assay Inhibitory factor was radioiodinated, and the binding studies were carried out as described by Bascom et al, J. Cell. Physiol. 128:202-208 (1986) . Briefly, radioiodination was by the chloramine T method (Sigma Chem. CO., St. Louis, MO) that resulted in a biologically active inhibitory factor with a specific
  • HSBP human diploid fibroblasts
  • Swiss 3T3 cells were used to study the potential effect of inhibitory factor mediated cell cycle arrest on the phosphorylation states of the RB gene product.
  • the cultures were incubated with or without inhibitory factor for 24 h, radiolabelled for 3.5 h with [ 35S]methionine, and the RB protein was immunoprecipitated with the monoclonal anti-human RB IgG as described above. Both exponentially growing cell cultures exhibited newly synthesized RB protein in both the hypo- and hyperphosphorylated states
  • both HSBP and Swiss 3T3 cells were plated and allowed to grow to confluence, and when the cultures reached confluence they were incubated for an additional 24 h to ensure that the majority of the cells were density-dependent arrested.
  • a second set of cultures were plated at the same time at ⁇ l/3 the density, and at the time of immunoprecipitation these cultures remained subconfIvien .
  • SAOS-2 cells were refractory to inhibition throughout the incubation period (Fig 6).
  • Human bladder carcinoma cell line J82 (RB-) and prostate carcinoma cell line DU145 (RB ⁇ ) also were resistant to the inhibitory action of inhibitory factor (Fig. 7). Even higher concentrations of the inhibitor also were ineffective in blocking cell cycling in
  • RB ⁇ cell lines Since cells that express a normal RB product can phenotypically act like RB cell lines when transformed with certain DNA tumor antigens, the possibility that these cell lines might also be resistant to the inhibitory influence of inhibitory factor was examined. While normal human keratinocytes were readily arrested by inhibitory factor, the 1321 and NCO papillomavirus E6/E7 protein transformed cell lines were totally refractory to the action of the inhibitor (Fig. 8). The adenovirus E1A protein transformed human epithelial cell line 293 also was resistant to the cell cycle arrest mediated by the inhibitory factor inhibitor (Fig. 9).
  • Swiss 3T3 cells were found to be sensitive to the inhibitory action of inhibitory factor, while consistent with the observations of papillomavirus and adenovirus transformed human cell lines, the proliferation of both the SV40 large T antigen transformed cell lines SVT2 and F5B were not inhibited by the sialoglycopeptide (Fig. 10).
  • the transformation of both human and mouse cells by the transforming antigens of several DNA oncogenic viruses, that are known to sequestered the nuclear RB product resulted in a refractory phenotype with regard to inhibitory factor action.
  • the cell lines used in this study also provided an examination of the potential role of a second tumor suppressor gene product, p53, with regard to the inhibitory action of inhibitory factor.
  • CeReS-18 inhibitor b v - Denotes presence of viral oncoproteins capable of sequestering RB and p53.
  • HL-60 cells are RB and p53 ⁇ and are sensitive target cells to the inhibitor.
  • Inhibitory factor derived from a parental cell surface component of bovine-cerebral cortex cells, has an unusually broad target cell range. It has the ability to mediate cell cycle arrest of cells obtained from mouse, human, rat, avian and insect species, all of which necessarily have specific cell surface receptors for the inhibitor. In addition, many tumorigenic cell lines, derived by mutation or retroviruses, have been shown to be highly sensitive to the proliferation inhibitor. For the most part, reversal experiments also have shown that this broad array of cells primarily are arrested in the Gl phase of the cell cycle. Studies with mouse and human cells confirm a Gl phase block by the presence of solely the underphosphorylated form of the RB in the inhibited cells.
  • the kinetics of reversal of DNA synthesis, cell doubling and the state of the RB protein are all consistent with the restriction (R) point, near the Gl/S interphase.
  • the one exception at the present time to this generality is the inhibitory factor mediated arrest of HL-60 cells. Unlike most others that appear to be synchronously released from cell cycle arrest when the inhibitor is removed, HL-60 cells are irreversibly arrested by the ⁇ sialoglycopeptide, and even after inhibitory factor is removed the cells progress through differentiation. This is of particular relevance to the present study since the HL-60 cells are p53 ⁇ and RB .
  • the human osteosarcoma SAOS-2 cell line is p53 ⁇ and RB ⁇ but it appears that the RB protein is the salient gene product with regard to inhibitory factor inhibition of cell cycling.
  • the central role of the RB product in inhibitory factor action was confirmed by the insensitivity of the human bladder J82 (RB ⁇ and p53 ) and human prostate DU145 (RB ⁇ and p53 ) carcinoma cell lines to the inhibitor (Fig. 7).
  • RB product is more than a casual player in the series of metabolic events that mediate cell cycle arrest by the inhibitor. Inhibitory factor arrests cells at a site where the RB unp os state is the dominant form of the tumor suppressor protein. Further, RB ⁇ cell lines are refractory to cell cycle inhibition by a sialoglycopeptide. Either its absence as a functional protein by mutation, or its being sequestered by transforming antigens of certain DNA oncoviruses, led to an insensitivity of cell cycle arrest by the sialoglycopeptide inhibitor (Table 4) . The maintenance of the RB product in the hypophosphorylated state alone, although readily seen in growth arrest cells (Fig.
  • Inhibitory factor is one of the few naturally occurring potential growth regulators that abrogates the phosphorylation of the RB protein.
  • the inhibitor is a cell surface component that influences cell cycling of a wide variety of cell types. Further, there is a similarity at a molecular level between the inhibitory factor arrested cells and those that naturally reach confluency and quiescence, and the reversibility of its inhibitory action.
  • Inhibitory factor represents a wide class of cell growth regulators that play a fundamental role in density-dependent growth inhibition. In this regard, the inhibitor is a valuable agent for studies of cell cycling, provides a controlled and synchronous population of cells in their progression through the cell cycle, and delineates the genetic and molecular events associated with the posttranslational modifications of the RB product that regulate cell proliferation.
  • Hybridoma cells (3G-10G-5) were plated into 96 well plates at 1 x 10 4 cells/well (100 ⁇ l medium). Hybridoma line, 3G-10G-5, produces monoclonal antibody to the budgerigar fledgling disease virus (BFDV) major capsid protein (VP1). Inhibitory factor treated (1.2 inhibitory units) experimental cultures (Fig. 11, open circles) had the inhibitor present at the time of plating (Fig. 11, arrow #1). Control cultures received medium without the inhibitor (Fig. 11, closed circles). Fresh medium was not added until seven days of culture. At the times indicated, the cells were pelleted by centrifugation, resuspended and counted, and the media were saved and used to quantitate the monoclonal antibody by ELISA.
  • BFDV budgerigar fledgling disease virus
  • VP1 major capsid protein
  • Inhibitory factor treated (1.2 inhibitory units) experimental cultures (Fig. 11, open circles) had the inhibitor present at the time of plating (Fig. 11, arrow #1).
  • ELISAs were performed with culture media from days 2, 7 and 11, in 96 well plates containing 90 ng/well of BFDV protein.
  • Hybridoma cells were effectively inhibited by inhibitory factor, and the inhibition is reversible. Antibody synthesis continued while cells were arrested by inhibitory factor and after reversal. [Note that the cell number was almost 30-times greater in control versus inhibitory factor treated cultures. ]
  • Hybridoma cells were incubated for seven days before media were changed and the inhibitory factor removed from the inhibited cultures.

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Abstract

L'invention concerne des facteurs inhibiteurs, des oligonucléotides les codant, et leurs procédés de production. Des compositions pharmaceutiques et les procédés de traitement de certains troubles sont également décrits.
EP93910842A 1992-04-27 1993-04-27 Facteur inhibiteur. Withdrawn EP0638126A4 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0315289A2 (fr) * 1987-11-06 1989-05-10 Oncogen Facteur inhibiteur de croissance cellulaire
EP0322084A2 (fr) * 1987-12-22 1989-06-28 Green Cross Corporation Facteur d'inhibition de cellules tumorales
EP0458673A1 (fr) * 1990-05-09 1991-11-27 Takeda Chemical Industries, Ltd. Facteur inhibiteur de la croissance et ADNc codant pour le facteur inhibiteur de la croissance
WO1992007938A1 (fr) * 1990-11-02 1992-05-14 Livio Mallucci Inhibiteurs de croissance cellulaire

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0315289A2 (fr) * 1987-11-06 1989-05-10 Oncogen Facteur inhibiteur de croissance cellulaire
EP0322084A2 (fr) * 1987-12-22 1989-06-28 Green Cross Corporation Facteur d'inhibition de cellules tumorales
EP0458673A1 (fr) * 1990-05-09 1991-11-27 Takeda Chemical Industries, Ltd. Facteur inhibiteur de la croissance et ADNc codant pour le facteur inhibiteur de la croissance
WO1992007938A1 (fr) * 1990-11-02 1992-05-14 Livio Mallucci Inhibiteurs de croissance cellulaire

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEM. BIOPHYS. RES. COMMUN., vol.124, no.1, 1984 pages 133 - 140 KINDERS ET AL. 'A monoclonal antibody to a unique cell surface growth regulatory glycopeptide' *
EXP. CELL. RES., vol.195, no.2, August 1991 pages 412 - 415 LAKSHMANARAO ET AL. 'Identification of a cell surface component of Swiss 3T3 cells associated with an inhibition of cell division' *
J. CELL BIOCHEM., vol.31, no.1, 1986 pages 41 - 47 SHARIFI ET AL. 'Relationship between protease activity and a sialoglycopeptide inhibitor isolated from bovine cortex' *
See also references of WO9322449A1 *

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