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WO1999035283A1 - Procedes et reactifs destines a moduler la motilite des cellules - Google Patents

Procedes et reactifs destines a moduler la motilite des cellules Download PDF

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Publication number
WO1999035283A1
WO1999035283A1 PCT/US1998/026720 US9826720W WO9935283A1 WO 1999035283 A1 WO1999035283 A1 WO 1999035283A1 US 9826720 W US9826720 W US 9826720W WO 9935283 A1 WO9935283 A1 WO 9935283A1
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Prior art keywords
cell
pkc
cells
integrin
camp
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PCT/US1998/026720
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English (en)
Inventor
Arthur M. Mercurio
Leslie M. Shaw
Margaret Lotz
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Beth Israel Deaconess Medical Center Inc
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Beth Israel Deaconess Medical Center Inc
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Priority to AU36350/99A priority Critical patent/AU3635099A/en
Publication of WO1999035283A1 publication Critical patent/WO1999035283A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Definitions

  • the field of the invention is regulation of cell motility and invasion in wound healing and cancer.
  • ⁇ 6 ⁇ 4 integrin is essential for the organization and maintenance of epithelial sheet architecture.
  • 6 ⁇ 4 integrin mediates the formation of stable adhesive structures, termed hemidesmosomes, which link the intermediate filament cytoskeleton with the extracellular matrix.
  • hemidesmosomes stable adhesive structures
  • the ability of ⁇ 6 ⁇ 4 integrin to associate with intermediate filaments distinguishes it from other mtegrins that interact primarily with the actin cytoskeleton.
  • epithelial cells are typically anchored in place via their cell-cell adhesion receptors and ⁇ 6 ⁇ 4 integrin-containing hemidesmosomes, epithelial cells also can lose their anchoring contacts and acquire a motile, mesenchymal phenotype.
  • the acquisition of motility is involved in both physiologically beneficial and pathological processes: for example, epithelial cell migration is an integral component of both wound healing and invasive carcinoma. Acquisition of a motile phenotype involves alterations in the expression and function of surface receptors that maintain the normal stationary epithelial phenotype.
  • invasive carcinoma is characterized by the loss of functional cell-cell adhesion receptors, such as cadherins.
  • ⁇ 6 ⁇ 4 integrin persists in many tumor cell types that exhibit a motile phenotype. This observation is counterintuitive, given that 6 ⁇ 4 integrin is found in hemidesmosomes, which are not commonly observed in motile cells such as invasive carcinoma cells.
  • PI 3-K phosphoinositide 3-hydroxyl kinase
  • PLC protein kinase C
  • the invention features methods for modulating the motility or invasiveness of a cell, and methods for identifying compounds that modulate the motility or invasiveness of a cell, by regulating the biological activity of the molecules comprising a PI 3-K-dependent signaling pathway that regulates cell motility and/or invasiveness, and by regulating PKC, cAMP phosphodiesterase, and/or ⁇ l integrin/actin interactions.
  • the invention features a method of identifying a compound that modulates the motility or invasiveness of a cell, comprising the steps of: (a) exposing a sample to a test compound, wherein the sample includes PI 3-K, PKC, cAMP-PDE, or ⁇ 4 integrin, and (b) assaying for altered biological activity of PI 3-K, PKC, cAMP-PDE, or ⁇ 4 integrin.
  • a decrease in the biological activity, relative to the biological activity of a sample not exposed to the compound indicates a compound that decreases cell motility or cell invasiveness
  • an increase in the biological activity, relative to the biological activity of a sample not exposed to the compound indicates a compound that increases cell motility or cell invasiveness.
  • the PI 3- K, PKC, or cAMP-PDE may be constitutively activated, the PI 3-K, PKC, cAMP-PDE, or ⁇ 4 integrin may be within a cell, the sample may comprise cell lysate or cell extract, the PKC may be an atypical PKC, or the method may further comprise a step in which the level of ⁇ 4 integrin or the biological activity of PI 3-K, PKC, cAMP-PDE, or ⁇ 4 integrin is increased or decreased prior to exposing the sample to the test compound.
  • the invention features a method of identifying a compound that modulates the motility or invasiveness of a cell, comprising the steps of: (a) modulating the biological activity of PI 3-K, PKC, cAMP-PDE, Akt, or ⁇ 4 integrin in a cell, (b) exposing the cell to a test compound, and (c) assaying for altered motility or invasiveness of the cell exposed to said compound.
  • a decrease in the motility or the invasiveness, relative to the motility or invasiveness of a cell not exposed to the compound, indicates a compound that decreases cell motility or cell invasiveness
  • an increase in the motility or the invasiveness, relative to a cell not exposed to said compound indicates a compound that increases cell motility or cell invasiveness
  • the biological activity may be increased by introducing constitutively active PI 3- K, PKC, or cAMP-PDE into the cell, the biological activity may be decreased by introducing dominant-negative PI 3-K, PKC, or cAMP-PDE into the cell, or the PKC may be an atypical PKC.
  • the invention features a method of decreasing the invasiveness of a cell or decreasing the predisposition to developing an invasive cell, comprising identifying the presence of at least one invasive cell or at least one cell with a predisposition to developing invasiveness, and exposing at least one invasive cell or at least one cell with a predisposition to developing invasiveness to a compound that decreases the biological activity of PI 3-K, PKC, cAMP-PDE, or ⁇ 4 integrin, or increases the biological activity of Akt.
  • the PKC may be an atypical PKC.
  • the compound may be: an antibody that specifically binds PI 3-K, PKC, cAMP-PDE, or ⁇ 4 integrin; antisense nucleic acid that specifically hybridizes with nucleic acid encoding PI 3-K, PKC, or cAMP- PDE; dominant-negative PI3-K, dominant-negative PKC, dominant-negative cAMP-PDE, or constitutively-activated Akt; wortmannin; or a substrate for PI 3-K, PKC, or cAMP-PDE.
  • the cell may be a neoplastic cell, such a colon carcinoma cell, a breast carcinoma cell, a prostate carcinoma cell, a cervical carcinoma cell, a uterine carcinoma cell, a testicular carcinoma cell, a liver carcinoma cell, an ovarian carcinoma cell, a renal carcinoma cell, a bladder carcinoma cell, a lung carcinoma cell, a laryngeal carcinoma cell, a squamous carcinoma cell, or a salivary gland carcinoma cell.
  • the invention features a method of increasing the motility of a cell, comprising exposing the cell to a compound that increases the biological activity of PI 3-K, PKC, cAMP-PDE, or ⁇ 4 integrin.
  • the PKC may be an atypical PKC
  • the cell may be an epithelial cell, such as an epidermal epithelial cell, an oral epithelial cell, a nasal epithelial cell, a gastrointestinal epithelial cell, a rectal epithelial cell, or an anal epithelial cell
  • the wound may result from a gastric ulcer, a duodenal ulcer, inflammatory bowel disease, ulcerative colitis, Crohn's disease, hemorrhoids, surgery, cancer, irradiation, exposure to toxic compounds, or physical trauma.
  • invasiveness and/or “motility” is meant the relative ability of a cell to migrate through a substratum.
  • the substratum may be artificial (such as Matrigel) or natural (such as an extracellular matrix laid down by cells). Invasiveness and/or motility are measured by motility assays known to those skilled in the art, such as the Matrigel invasion assay described herein.
  • motility assays known to those skilled in the art, such as the Matrigel invasion assay described herein.
  • the relative motility and/or invasiveness of a cell is expressed in comparison to a reference cell. For example, as shown in Fig. 2A, MDA-MB-435 cells expressing ⁇ 4 integrin are 3-4-fold more invasive than parental cells not expressing ⁇ 4 integrin.
  • Neoplastic is meant a cell or tissue multiplying or growing in an abnormal manner. Neoplastic growth is uncontrolled and progressive, and occurs under conditions that would not elicit, or would cause cessation of, multiplication of normal cells.
  • wound is meant an injury to the body (as from surgery, physical trauma, injury, illness, disease, or exposure to excessive radiation or toxic compounds) that involves an interruption or laceration of an epithelium (e.g., epidermal epithelium or the epithelial lining of the oral, nasal, or anal/rectal mucosa or gastrointestinal tract).
  • an epithelium e.g., epidermal epithelium or the epithelial lining of the oral, nasal, or anal/rectal mucosa or gastrointestinal tract.
  • wound healing is meant the biological processes, including epithelial cell migration, that occur during repair of an injury to the body.
  • phosphoinositide 3-hydroxyl kinase or "PI 3-K” is meant a lipid kinase, preferably derived from an animal, most preferably a mammal
  • protein kinase C or "PKC” is meant a member of a family of serine/threonine protein kinases, preferably derived from an animal, most preferably a mammal (such as a human or rodent), that are involved in signal transduction pathways that regulate many biological processes, including, but not limited to, growth and differentiation.
  • the biological activity (e.g., kinase activity) of "conventional" PKCs is known in the art to be activated by Ca +2 , phospholipid, diacylgycerol, and phorbol ester.
  • cAMP phosphodiesterase or "cAMP-PDE” is meant an enzyme, preferably derived from an animal, most preferably a mammal (such as a human or rodent), that hydrolyzes cyclic AMP, yielding AMP plus a proton.
  • biological activity of PI 3-K “biological activity of PKC,” or
  • biological activity of cAMP-PDE is meant, respectively, the enzymatic action of PI 3-K, PKC, or cAMP-PDE. Alterations in biological activity of PI 3-K, PKC, or cAMP-PDE may be determined by detecting or measuring enzymatic activity of PI 3-K, PKC, or cAMP-PDE in a test sample and comparing it to the analogous enzymatic activity in a reference sample, using assays that are known in the art or disclosed herein.
  • Alterations in biological activity of PI 3-K, PKC, or cAMP-PDE may also be determined by comparing PI 3-K, PKC, or cAMP-PDE polypeptide levels, mRNA levels, or reporter gene (under the regulation of a PI 3-K, PKC, or cAMP-PDE transcriptional control element) activity levels in a test sample to polypeptide levels, mRNA levels, or reporter gene levels in an appropriate reference sample.
  • biological activity of ⁇ 4 integrin is meant the interaction of ⁇ 4 integrin with actin.
  • Alterations in biological activity of ⁇ 4 integrin may be determined by detecting or measuring the amount of interaction between ⁇ 4 integrin and actin in a test sample and comparing it to the amount of ⁇ 4 integrin/actin interaction in an appropriate reference sample, using assays that are known in the art or disclosed herein.
  • the amount of PI 3-K, PKC, cAMP-PDE, or ⁇ 4 integrin biological activity may be modulated by increasing or decreasing the number of PI 3-K, PKC, cAMP-PDE, or ⁇ 4 polypeptide molecules present intracellularly, by stimulating or inhibiting upstream modulators or downstream effectors of PI 3-K, PKC, cAMP-PDE, or ⁇ 4 within the PI 3-K signal transduction pathway(s) that regulate(s) cell motility and/or invasiveness, or by post-translationally modifying PI 3-K, PKC, cAMP-PDE, or ⁇ 4.
  • phosphorylation of PI 3-K stimulates its biological activity.
  • Biological activity may be less than or greater than the activity of wild-type PI 3-K, PKC, cAMP-PDE, or ⁇ 4.
  • constitutively active PI 3-K has more than 100% of wild-type PI 3-K activity
  • dominant-negative PI 3-K has less than 100% of wild-type activity.
  • the absence of biological activity is defined by the presence of less than 10% of the biological activity that is found when assaying the wild-type protein in the same relevant assay.
  • high stringency conditions conditions that allow hybridization comparable with the hybridization that occurs during an overnight incubation using a DNA probe of at least 500 nucleotides in length, in a solution containing 0.5 M NaHP0 4 , pH 7.2, 7% SDS, 1 mM EDTA, 1% BSA (fraction V), and 100 ⁇ g/ml denatured, sheared salmon sperm DNA, at a temperature of 65° C, or a solution containing 48% formamide, 4.8X SSC (150 mM NaCl, 15 mM trisodium citrate), 0.2 M Tris-Cl, pH 7.6, IX Denhardt's solution, 10% dextran sulfate, 0.1 % SDS, and 100 ⁇ g/ml denatured, sheared salmon sperm DNA, at a temperature of 42° C (these are typical conditions for high stringency Northern or Southern, or colony hybridizations).
  • High stringency hybridization may be used for techniques such as high stringency PCR, DNA sequencing, single strand conformational polymorphism analysis, and in situ hybridization.
  • the immediately aforementioned techniques are usually performed with relatively short probes (e.g., usually 16 nucleotides or longer for PCR or sequencing, and 40 nucleotides or longer for in situ hybridization).
  • the high stringency conditions used in these techniques are well known to those skilled in the art of molecular biology, and may be found, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1997, hereby incorporated by reference.
  • probe or “primer” is meant a single-stranded DNA or RNA molecule of defined sequence that can base pair to a second DNA or RNA molecule that contains a complementary sequence (the “target”).
  • target a complementary sequence
  • the stability of the resulting hybrid depends upon the extent of the base pairing that occurs.
  • the extent of base-pairing is affected by parameters such as the degree of complementarity between the probe and target molecules, and the degree of stringency of the hybridization conditions.
  • the degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to one skilled in the art.
  • Probes or primers specific for nucleic acids e.g.
  • PI 3-K nucleic acid preferably will have at least 35% sequence identity, more preferably at least 45-55% sequence identity, still more preferably at least 60-75% sequence identity, still more preferably at least 80-90% sequence identity, and most preferably 100% sequence identity.
  • Probes may be detectably-labelled, either radioactively, or non-radioactively, by methods well-known to those skilled in the art. Probes are used for methods involving nucleic acid hybridization, such as: nucleic acid sequencing, nucleic acid amplification by the polymerase chain reaction, single stranded conformational polymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP) analysis, Southern hybridization, Northern hybridization, in situ hybridization, electrophoretic mobility shift assay (EMSA).
  • SSCP single stranded conformational polymorphism
  • RFLP restriction fragment polymorphism
  • pharmaceutically acceptable carrier means a carrier which is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered.
  • One exemplary pharmaceutically acceptable carrier is physiological saline.
  • Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (18 th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA.
  • identity is meant that a polypeptide or nucleic acid sequence possesses the same amino acid or nucleotide residue at a given position, compared to a reference polypeptide or nucleic acid sequence to which the first sequence is aligned.
  • Sequence identity is typically measured using sequence analysis software with the default parameters specified therein, such as the introduction of gaps to achieve an optimal alignment (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705).
  • substantially identical is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably 85%, more preferably 90%, and most preferably 95% identity to a reference amino acid or nucleic acid sequence.
  • the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids.
  • the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 1 10 nucleotides.
  • substantially pure polypeptide is meant a polypeptide that has been separated from the components that naturally accompany it.
  • the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the polypeptide is a polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure.
  • a substantially pure polypeptide may be obtained, for example, by extraction from a natural source (e.g., cultured cells) by expression of a recombinant nucleic acid encoding the polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • a protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state.
  • a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.
  • substantially pure polypeptides not only includes those derived from eukaryotic organisms but also those synthesized in E. coli or other prokaryotes.
  • substantially pure DNA DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • transformation is meant any method for introducing foreign molecules into a cell (e.g., a bacterial, yeast, fungal, algal, plant, insect, or animal cell).
  • a cell e.g., a bacterial, yeast, fungal, algal, plant, insect, or animal cell.
  • Lipofection, DEAE-dextran-mediated transfection, microinjection, protoplast fusion, calcium phosphate precipitation, transduction (e.g., bacteriophage, adenoviral, or retroviral delivery), electroporation, and biolistic transformation are just a few of the methods known to those skilled in the art which may be used.
  • transformed cell By “transfected cell,” or “transduced cells” is meant a cell (or a descendant of a cell) into which a DNA molecule encoding a polypeptide, for example, a dominant-negative PI 3-K, has been introduced, by means of recombinant DNA techniques.
  • positioned for expression is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., a polypeptide, a recombinant protein or a RNA molecule).
  • promoter is meant a minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell type-specific, tissue-specific, temporal-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' or intron sequence regions of the native gene.
  • operably linked is meant that a gene and one or more regulatory sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
  • detectably-labelled any means for marking and identifying the presence of a molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule.
  • Methods for detectably- labelling a molecule are well known in the art and include, without limitation, radioactive labelling (e.g., with an isotope such as 2 P or 35 S) and nonradioactive labelling (e.g., chemiluminescent labelling, e.g., fluorescein labelling).
  • antisense as used herein in reference to nucleic acids, is meant a nucleic acid sequence that is complementary to the coding strand of a gene, for example, a PI 3-K, PKC, or cAMP-PDE gene.
  • An antisense nucleic acid is capable of preferentially lowering the activity of a polypeptide encoded by a gene for which the antisense nucleic acid contains a complementary nucleic acid sequence.
  • telomere binding binds an antibody that recognizes and binds a human polypeptide (e.g., PI 3-K, PKC, cAMP-PDE, ⁇ 6 ⁇ 4 integrin) but that does not substantially recognize and bind other (non-PI 3-K) molecules in a sample, e.g., a biological sample, that naturally includes protein.
  • a human polypeptide e.g., PI 3-K, PKC, cAMP-PDE, ⁇ 6 ⁇ 4 integrin
  • neutralizing antibodies antibodies that interfere with any of the biological activities of a polypeptide (such as PI 3-K, PKC, cAMP- PDE, or ⁇ 6 ⁇ 4 integrin); for example, the ability of PI 3-K to catalyze the phosphorylation of phosphoinositide lipids.
  • the neutralizing antibody reduces the biological activity of PI 3-K, PKC, cAMP-PDE, or ⁇ 6 ⁇ 4 integrin preferably by at least 25%, more preferably by at least 50%, yet more preferably by at least 70%, and most preferably by at least 90%.
  • any standard assay for the biological activity of PI 3-K, PKC, cAMP-PDE, or ⁇ 6 ⁇ 4 integrin may be used to assess potentially neutralizing antibodies that are specific for these polypeptides.
  • exposure is meant to allow contact between an animal, cell, lysate or extract derived from a cell, or molecule derived from a cell, and a test compound.
  • treat is meant to submit or subject an animal (e.g. a human), cell, lysate or extract derived from a cell, or molecule derived from a cell to a test compound.
  • test compound is meant a chemical, be it naturally-occurring or artificially-derived, that is surveyed for its ability to modulate an alteration in reporter gene activity or protein levels, by employing one of the assay methods described herein.
  • Test compounds may include, for example, peptides, polypeptides, synthesized organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof.
  • test is meant analyzing the effect of a treatment, be it chemical or physical, administered to whole animals, cells derived therefrom, cell lysates or extracts, or partially- or fully-purified molecules from cell lysates or extracts.
  • the material being analyzed may be an animal, a cell, a lysate or extract derived from a cell, or a molecule derived from a cell.
  • the analysis may be for the purpose of detecting altered cell motility and/or invasiveness, altered protein biological activity (e.g., altered PI 3-K, PKC, or cAMP PDE enzymatic activity, or altered interactions of ⁇ 6 ⁇ 4 integrin with actin), altered protein stability, altered protein levels, altered mRNA levels, altered gene expression, or altered mRNA stability.
  • altered protein biological activity e.g., altered PI 3-K, PKC, or cAMP PDE enzymatic activity, or altered interactions of ⁇ 6 ⁇ 4 integrin with actin
  • altered protein stability e.g., altered protein levels, altered mRNA levels, altered gene expression, or altered mRNA stability.
  • the means for analyzing may include, for example, cell invasion assays, detection or quantification of the product of an enzymatic reaction (e.g., the formation of phosphorylated lipids as a result of PI 3-K activity), antibody-mediated detection or quantification of protein, immunoprecipitation, detection or quantification of mRNA by methods such as reverse transcription-polymerase chain reaction (RT-PCR) or filter hybridization (e.g., Northern or dot-blotting).
  • RT-PCR reverse transcription-polymerase chain reaction
  • filter hybridization e.g., Northern or dot-blotting
  • modulating is meant changing, either by decrease or increase, the relative invasiveness of a cell, or the biological activity of PI 3-K, PKC, cAMP PDE, or a polypeptide in the PI 3-K-dependent pathway that mediates ⁇ 6 ⁇ 4 integrin-stimulated cell invasiveness.
  • a decrease is meant an inhibition of the motility and/or invasiveness of a cell, or an inhibition of the level of biological activity of PI 3-K, PKC, cAMP PDE, ⁇ 6 ⁇ 4 integrin or any other polypeptide in the PI 3-K- dependent pathway that mediates ⁇ 6 ⁇ 4 integrin-stimulated cell, as measured by a lowering in: a) the motility or invasiveness of a cell in an assay known to those skilled in the art, e.g., a Matrigel invasion assay; b) biological activity of a polypeptide in the PI 3-K-dependent pathway (e.g., the phosphorylation of inositide lipids by PI 3-K); c) relative protein (or phosphorylated protein) levels, as measured by ELISA; d) reporter gene activity, as measured by reporter gene assay, for example, lacZ/ ⁇ -galactosidase, green fluorescent protein, luciferase, etc., under the transcriptional
  • an increase is meant a stimulation of the motility and/or invasiveness of a cell, or in the level of biological activity of PI 3-K, PKC, cAMP PDE, or a polypeptide in the PI 3-K-dependent pathway that mediates ⁇ 6 ⁇ 4 integrin-stimulated cell invasiveness, as measured by a rise in: a) the motility or invasiveness of a cell in an assay known to those skilled in the art, e.g., a Matrigel invasion assay; b) biological activity of a polypeptide in the PI 3-K-dependent pathway (e.g., the phosphorylation of inositide lipids by PI 3- K); c) relative protein (or phosphorylated protein) levels, as measured by ELISA; d) reporter gene activity, as measured by reporter gene assay, for example, lacZ/ ⁇ -galactosidase, green fluorescent protein, luciferase, etc., under the transcriptional regulation of a PI 3-
  • alteration in the level of gene expression is meant a change in gene activity such that the amount of a product of the gene, i.e., mRNA or polypeptide, is increased or decreased, or that the stability of the mRNA or the polypeptide is increased or decreased. Alterations in the level of gene expression may be detected or measured by comparing polypeptide levels, nucleic acid levels, or reporter gene (under the transcriptional regulation of transcriptional control elements from the gene of interest) activity in a test sample to that of a reference sample.
  • reporter gene any gene that encodes a product whose expression is detectable and/or quantitatable by immunological, chemical, biochemical or biological assays.
  • a reporter gene product may, for example, have one of the following attributes, without restriction: fluorescence (e.g., green fluorescent protein), enzymatic activity (e.g., lacZ/ ⁇ -galactosidase, luciferase, chloramphenicol acetyltransferase), toxicity (e.g., ricin A), or an ability to be specifically bound by a second molecule (e.g., biotin or a detectably-labelled antibody).
  • fluorescence e.g., green fluorescent protein
  • enzymatic activity e.g., lacZ/ ⁇ -galactosidase, luciferase, chloramphenicol acetyltransferase
  • toxicity e.g., ricin A
  • an ability to be specifically bound by a second molecule e
  • protein or “polypeptide” or “polypeptide fragment” is meant any chain of more than two amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally-occurring polypeptide or peptide, or constituting a non-naturally occurring polypeptide or peptide.
  • post-translational modification e.g., glycosylation or phosphorylation
  • FIG. 1 is a diagram showing a FACS analysis of surface expression of integrin subunits in MDA-MB-435 transfectants.
  • Fig. 2 A is a graph showing the relative invasiveness of MDA-MB- 435 ⁇ 4 and ⁇ 4- ⁇ CYT transfectants.
  • Fig. 2B is a graph showing the relative invasiveness of a MDA-MB- 435 ⁇ 4 transfectant.
  • Fig. 3 A is a graph showing an analysis of MAPK, PI 3-K, and p70 S6K involvement in MDA-MB-435 invasion.
  • Panel A shows a Matrigel invasion assay, and
  • Panel B shows a Western blot.
  • Fig. 3B is an autoradiogram showing MAPK activity of MDA-MB- 435 ⁇ 4 transfectants under various conditions.
  • Fig. 4A is an autoradiogram of a kinase assay showing PI 3-K activity in MDA-MB-435 ⁇ 4 transfectants.
  • Fig. 4B is a diagram showing PI 3-K activity in MDA-MB-435 ⁇ 4 and ⁇ 4- ⁇ CYT transfectants.
  • Figs. 5 A and 5B are graphs showing analyses of PI 3-K involvement in invasion of MDA-MB-435 cells by transient transfections.
  • Figs. 6A-6D are graphs showing analyses of downstream effectors in PI 3-K-dependent invasion of MDA-MB-435 cells.
  • Fig. 7A is a graph showing that ⁇ 6 ⁇ 4 integrin mediates invasiveness of an invasive carcinoma cell line.
  • Fig. 7B is a graph showing that PI 3-K mediates lamellar formation in an invasive colon carcinoma cell line.
  • Fig. 7C is a series of photomicrographs showing that lamellar formation of an invasive colon carcinoma cell line is dependent upon ⁇ 6 ⁇ 4 integrin and PI 3-K.
  • Figs. 8A and 8B are, respectively, an autoradiogram of a kinase assay and a graph depicting analyses of PI 3-K activity in Clone A cells.
  • Figs. 9A-9C are graphs showing that the PKC inhibitor Calphostin C inhibits the invasiveness of ⁇ 4 integrin-transfected MDA-MB-435 cells, whereas the tumor promoter PMA does not.
  • Fig. 10 is a graph showing that ⁇ 4 integrin-transfected MDA-MB- 435 clones (3A7, 5B3) have higher phosphodiesterase activity that mock- transfected cells (6D7).
  • Fig. 1 1 is a graph showing that the phosphodiesterase inhibitor IBMX blocks the LPA-stimulated chemotaxis of MDA-MB-435 cells.
  • Figs.l2A-12F are photomicrographs showing that ⁇ 6 ⁇ 4 integrin co- localized with F-actin in filapodia of clone A cells on laminin-1.
  • Figs. 13A-13D are photomicrographs of in i' tw-detergent-extracted cells, showing that ⁇ 6 ⁇ 1 integrin remains colocalized with F-actin.
  • Fig. 14 is an immunoprecipitation-western blot showing that ⁇ 6 ⁇ l integrin is released from permeabilized clone A cells by the actin-severing protein gelsolin.
  • Figs. 15A-15E are photomicrographs of immunostained cells showing that ⁇ 6 ⁇ 4 integrin is localized in actin-containing motility structures of various carcinoma cell lines.
  • Fig. 15F is a graph showing that an ⁇ 6 integrin-specific antibody inhibits formation of lamallae in carcinoma cells.
  • Fig. 16A-16F are photomicrographs showing that lamallar extension during T84 cell wound healing is wortmannin-sensitive.
  • Akt and S6K serine/threonine kinases do not contribute to the invasive process. This is surprising because both Akt and S6K are regulated by PI 3-K and are activated by the ⁇ 6 ⁇ 4 integrin. However, we found that activation of members of the protein kinase C serine/threonine kinase family, particularly the atypical isoforms, also are necessary for cell motility. We further noted increased cAMP phosphodiesterase activity in ⁇ 4 integrin-activated cell motility. Finally, we observed a novel interaction between ⁇ 4 integrin and F-actin in motile cells.
  • Invasion is a defining event in the progression of carcinoma.
  • the invasion process represents the ability of epithelial cells to acquire a mesenchymal phenotype characteristic of the breast and colon carcinoma cells used in our experiments.
  • integrins differ in their ability to activate PI 3-K, based on the preferential activation of PI 3-K by ⁇ 6 ⁇ 4, compared to ⁇ l integrins. This difference, observed in both MDA-MB-435 breast carcinoma cells and clone A colon carcinoma cells, is linked to a specific cellular response.
  • the amino acid sequence of the ⁇ 4 cytoplasmic domain is different from that of other integrin ⁇ subunits, yet it does not contain the consensus sequence for p85 binding via SH2 domains, YMXM, thus diminishing the possibility of a direct association with PI 3-K. In fact, we have not been able to detect such an association in our experiments. While we do not wish to bind our to a specific mechanism, we believe the involvement of signaling intermediates is more likely.
  • One downstream effector of PI 3-K that we demonstrate is involved in carcinoma invasion is Rac, a small GTP -binding protein. Interestingly, neither constitutively active Rac, nor constitutively active Rho and Cdc42, were able to increase invasion of MDA-MB-435 cells.
  • Akt kinase is activated by ⁇ 4 ligation, it is not required for invasion of the breast carcinoma cells that we studied.
  • the assays described herein can be used to test for compounds that modulate cell motility and or invasiveness via PI 3-K, PKC, cAMP phosphodiesterase, or ⁇ 4 integrin/actin interactions.
  • Other members of the PI 3-K-dependent pathway that modulate cell motility and/or invasiveness also may be used to test for such compounds.
  • Compounds identified using the methods provided herein may have therapeutic value in the treatment and prevention of cancer, particularly invasive carcinomas such as breast and colon carcinomas, and in the enhancement of wound healing.
  • Potentially useful therapeutic compounds that modulate (e.g. increase or decrease) cell motility or invasiveness via PI 3-K, its upstream modulators, or its downstream effectors may be isolated by various screens that are well-known to those skilled in the art. Such examples include, but are not limited to, the Matrigel invasion assay and the phosphoinositide phosphorylation assay described in the Examples below.
  • Useful compounds may, for example, modulate expression of PI 3-K or other molecules in the PI 3-K-dependent motility/invasiveness signaling pathway. Modulation of expression may be at the pre- or post-transcriptional level, or at the pre- or post-translational level.
  • Useful compounds also may modulate the biological activity of molecules in the PI 3-K-dependent pathway, e.g., by affecting post- translational modifications such as phosphorylation status. As shown in the Examples to follow, stimulation of PI 3-K biological activity enhances cell motility and/or invasiveness. This finding allows us to provide assays for drugs that modulate cell motility and/or invasiveness by modulating PI 3-K biological activity.
  • Such assays may measure, for example, PI 3-K biological activity by measuring changes in: (a) PI 3-K phosphorylation status; (b) PI 3-K association with cellular proteins; (c) levels of PI 3-K-induced cell motility or invasiveness; (d) levels of PI 3-K mRNA or gene expression, and (e) PI 3-K- induced lipid phophorylation.
  • Analogous assays that measure changes in PKC or cAMP phosphodiesterase (cAMP-PDE) activity also may be used.
  • assays that measure interactions between ⁇ 6 ⁇ 4 integrin and actin may be employed.
  • Such measurements may be made in vitro or in vivo and form the basis of assays which identify compounds that modulate cell motility and/or invasiveness.
  • Such identified compounds may have therapeutic value in the treatment and/or prevention of cancer, particularly invasive cancers such as invasive breast or colon carcinomas.
  • Other identified compounds i.e., those that stimulate cell motility by activating PI 3-K, or PKC, etc., may have therapeutic value in enhancing wound healing, particularly by enhancing epithelial cell migration.
  • Enzyme-linked immunosorbant assays are easily incorporated into high-throughput screens designed to test large numbers of compounds for their ability to modulate levels of a given protein.
  • changes in a given protein level of a sample, relative to a control reflect changes in the PI 3-K (or Rac, Akt, PKC, or other downstream effectors of PI 3-K) expression status of the cells within the sample.
  • Protocols for ELISA may be found, for example, in Ausubel et z ⁇ .,Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998.
  • Lysates from cells treated with potential modulators of, for example, PI 3-K expression are prepared (see, for example, Ausubel et al., supra), and are loaded onto the wells of microtiter plates coated with "capture" antibodies specific for PI 3-K. Unbound antigen is washed out, and a PI 3-K- specific antibody, coupled to an agent to allow for detection, is added.
  • Agents allowing detection include alkaline phosphatase (which can be detected following addition of colorimetric substrates such as -nitrophenolphosphate), horseradish peroxidase (which can be detected by chemiluminescent substrates such as ECL, commercially available from Amersham) or fluorescent compounds, such as FITC (which can be detected by fluorescence polarization or time-resolved fluorescence).
  • alkaline phosphatase which can be detected following addition of colorimetric substrates such as -nitrophenolphosphate
  • horseradish peroxidase which can be detected by chemiluminescent substrates such as ECL, commercially available from Amersham
  • fluorescent compounds such as FITC (which can be detected by fluorescence polarization or time-resolved fluorescence).
  • a positive assay result for example, identification of a compound that decreases PI 3-K expression, is indicated by a decrease in PI 3-K polypeptide within a sample, relative to the PI 3-K polypeptide level observed in cells which are not treated with a test compound.
  • Numerous variations of the basic assay may be employed. For example, in order to detect changes in PI 3-K phosphorylation status (and, hence, PI 3-K biological activity), duplicate ELISAS are used.
  • the capture antibody for both assays is specific for PI 3-K
  • the second (detection) antibody for the second assay is specific for phosphoprotein.
  • the first ELISA gives a measure of the absolute quantity of PI 3-K present in a sample
  • the second reveals the degree of PI 3-K phosphorylation in the sample.
  • Duplicate ELISAs also are performed for the appropriate control samples. For example, in testing the effect of a compound on PI 3-K phosphorylation, the duplicate ELISAs are also performed on control samples not treated with the test compound. Similar approaches may be used to isolate compounds that modulate interactions between ⁇ 4 integrin and actin, e.g., by employing a capture antibody for ⁇ 4 integrin, and a detection antibody for actin, or vice- versa.
  • Two-hybrid methods, and modifications thereof, are used to screen for polypeptides that physically interact with PI 3-K, PKC or Rac, and hence might be members of the PI 3-K-dependent pathway that modulates cell motility and/or invasiveness.
  • Regulators of PI 3-K e.g. proteins that interfere with the interaction between PI 3-K and other proteins, also are identified by the use of a three-hybrid system. Similar approaches may be used to find proteins that regulate the interaction between actin and ⁇ 4 integrin.
  • Such assays are well-known to skilled artisans, and may be found, for example, in Ausubel et al., supra.
  • Assays employing the detection of reporter gene products are extremely sensitive and readily amenable to automation, hence making them ideal for the design of high-throughput screens.
  • Assays for reporter genes may employ, for example, colorimetric, chemiluminescent, or fluorometric detection of reporter gene products.
  • Many varieties of plasmid and viral vectors containing reporter gene cassettes are easily obtained. Such vectors contain cassettes encoding reporter genes such as lacZ/ ⁇ -galactosidase, green fluorescent protein, and luciferase, among others.
  • Cloned DNA fragments encoding transcriptional control regions of interest e.g.
  • PI 3-K gene are easily inserted, by DNA subcloning, into such reporter vectors, thereby placing a vector-encoded reporter gene under the transcriptional control of any gene promoter of interest.
  • the transcriptional activity of a promoter operatively linked to a reporter gene can then be directly observed and quantitated as a function of reporter gene activity in a reporter gene assay.
  • Cells are transiently- or stably-transfected with PI 3-K control region/reporter gene constructs by methods that are well known to those skilled in the art. Transgenic mice containing PI 3-K control region/reporter gene constructs are used for late-stage screens in vivo.
  • Cells containing PI 3- K/reporter gene constructs are exposed to compounds to be tested for their potential ability to modulate PI 3-K expression.
  • cells are lysed and subjected to the appropriate reporter assays, for example, a colorimetric or chemiluminescent enzymatic assay for lacZ/ ⁇ -galactosidase activity, or fluorescent detection of GFP. Changes in reporter gene activity of samples treated with test compounds, relative to reporter gene activity of appropriate control samples, indicate the presence of a compound that modulates PI 3-K expression.
  • Analogous assays are used to screen for compounds that affect expression of other components of the PI 3-K-dependent pathway that regulates cell motility and/or invasiveness.
  • PCR polymerase chain reaction
  • rtPCR reverse transcription step
  • this assay is easily performed in a 96-well format, and hence is easily incorporated into a high-throughput screening assay.
  • Cells are treated with test compounds for the appropriate time course, lysed, the mRNA is reverse-transcribed, and the PCR is performed according to commonly used methods, (such as those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998), using oligonucleotide primers that specifically hybridize with PI 3-K nucleic acid.
  • Such primers are readily designed using well- known approaches such as software that can be used to design PCR primers. Changes in product levels of samples exposed to test compounds, relative to control samples, indicate test compounds that modulate PI 3-K expression.
  • test compounds that appear to modulate cell motility or invasiveness via a PI 3-K-dependent pathway are identified, it may be necessary or desirable to subject these compounds to further testing.
  • testing will be performed in vivo using animal models of invasive cancer or wound healing as appropriate, to confirm that the compounds initially identified to affect cell motility and/or invasiveness will have the predicted effect in vivo.
  • test Compounds In general, novel drugs (for the treatment and prevention of cancer, or for the enhancement of wound healing) that modulate the biological activity of PI 3-K, its upstream activators, or its downstream effectors, are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein.
  • extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI).
  • libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA).
  • Biotics Sussex, UK
  • Xenova Slough, UK
  • Harbor Branch Oceangraphics Institute Ft. Pierce, FL
  • PharmaMar, U.S.A. Chembridge, MA
  • natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods.
  • any library or compound is readily modified using standard chemical, physical, or biochemical methods.
  • Compounds identified using any of the methods disclosed herein may be administered to patients or experimental animals with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form.
  • a pharmaceutically-acceptable diluent, carrier, or excipient in unit dosage form.
  • Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such compositions to patients or experimental animals.
  • intravenous administration is preferred, any appropriate route of administration may be employed, for example, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration.
  • Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols. Methods well known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences.”
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for antagonists or agonists of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • the MDA-MB-435 breast carcinoma cell line was obtained from the Lombardi Breast Cancer Depository at Georgetown University.
  • the MDA-MB-435 cells were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal calf serum (Gibco) and 1% penicillin-streptomycin (Gibco).
  • DMEM Dulbecco's modified Eagle's medium
  • Clone A cells were grown in RPMI supplemented with 25 mM Hepes (RPMI-H), 10% fetal calf serum and 1 % penicillin-streptomycin.
  • Human T84 colon carcinoma cells were grown in DME-low glucose/Ham's F12 (GIBCO, Grand Island NY) supplemented with 6% normal calf serum, 2 mmol/L L-glutamine, 50 ⁇ g/ml streptomycin and 50 U/ml penicillin. Cells were cultured for 2 to 3 days after reaching confluency before use in assays.
  • Neomycin resistant cells were isolated by selective growth in medium containing G418 (0.6 mg/ml; Gibco). The stable transfectants were pooled and populations of cells that expressed the human ⁇ 4 subunit on the cell surface were isolated by FACS. A human ⁇ 4 integrin-specific monoclonal antibody (mAb), UM-A9 (obtained from Tom Carey, University of Michigan, Ann Arbor, MI), was used for this sorting and for subsequent analysis of the transfectants. The sorting was repeated sequentially to enrich for homogeneous populations of cells expressing high levels of the transfected ⁇ 4 and ⁇ 4- ⁇ CYT subunits on the cell surface. Subclones were isolated from these populations by FACS. Surface labeling and immunoprecipitation with A9 were done to confirm that the ⁇ 6 ⁇ 4 integrin heterodimer was expressed on these subclones. Analysis of Integrin Surface Expression
  • the relative surface expression of integrin subunits on the mock and ⁇ 4-transfectants of the MDA-MB-435 cells was assessed by flow cytometry. For this purpose, aliquots of cells (5 x 10 5 ) were incubated for 45 minutes at room temperature with RPMI-H and 0.2% BSA (RH/BSA) and the following integrin-specific antibodies: 2B7 (anti- ⁇ , etc.; prepared in our laboratory); mAb 13 (anti- ⁇ l ; provided by Stephen Akiyama, National Institutes of Health, Bethesda, MD); A9 (anti- ⁇ 4; provided by Thomas Carey, University of Michigan, Ann Arbor, MI), as well as mouse IgG (Sigma).
  • the cells were washed two times with RH/BSA and then incubated with goat F(ab')2 anti-mouse IgG coupled to fluorescein (Tago) for 45 minutes at room temperature. After washing two times with RH/BSA, the cells were resuspended in the same buffer and analyzed by flow cytometry.
  • the cells that had not invaded were removed from the upper face of the filters using cotton swabs and the cells that had invaded to the lower surface of the filters were fixed in methanol and then stained with a 0.2% solution of crystal violet in 2% ethanol. Invasion was quantitated by visual counting. The mean of five individual fields in the center of the filter where invasion was the highest was obtained for each well. In some assays, the cells were pre-incubated for 30 minutes before addition to the Matrigel-coated wells with either wortmannin (Ui et al, Trends in Biochemical Sciences 20:303-7, 1995), PD98059 (Dudley et al, Proc. Natl. Acad. Sci. USA 92:7686-9.
  • wortmannin Ui et al, Trends in Biochemical Sciences 20:303-7, 1995
  • PD98059 Dudley et al, Proc. Natl. Acad. Sci. USA 92:7686-9.
  • Cells were removed from their dishes with trypsin and washed twice with RPMI-H containing 0.2% heat-inactivated BSA. After washing, the cells were resuspended in the same buffer at a concentration of 2 x 10 6 cells/ml and incubated for 30 minutes with integrin-specific antibodies or in buffer alone. The cells were washed once, resuspended in the same buffer and added to plates that had been coated overnight with either anti-mouse IgG or laminin-1.
  • the cells were washed twice with cold PBS and solubilized at 4°C for 10 minutes in a 20 mM Tris buffer, pH 7.4, containing 0.14 M NaCl, 1% NP-40, 10% glycerol, 1 mM sodium orthovanadate, 2 mM phenylmethylsulfonylfluoride (PMSF), 5 ⁇ g/ml aprotinin, pepstatin and leupeptin. Nuclei were removed by centrifugation at 12,000 x g for 10 minutes.
  • the beads were resuspended in kinase buffer containing 10 ⁇ g of sonicated crude brain lipids (Sigma), 100 ⁇ M ATP, 25 mM MgCl 2 , and 10 ⁇ Ci g- 2 P-ATP and incubated for 10 minutes at room temperature. The reaction was stopped by the addition of 60 ⁇ l 2 N HC1 and 160 ⁇ l of a 1 : 1 mixture of chloroform and methanol. Lipids were resolved by thin layer chromatography plates coated with potassium oxalate.
  • MAP kinase activity To assay MAP kinase activity, total cell extracts, prepared as described above, were resolved by electrophoresis on SDS-polyacrylamide gels (10%), transferred to nitrocellulose, and blotted with a phospho-specific ERK polyclonal antibody that recognizes the phosphorylated isoforms of ERK- 1 and ERK-2 (New England BioLabs, Inc.). Immune complexes were detected using a secondary antibody conjugated to horseradish peroxidase and visualized by enhanced chemiluminescence (Amersham, Inc.). The blots were then stripped and re-probed with an ERK- 1 antibody (provided by John Blenis, Harvard Medical School, Boston, MA) that recognizes both ERK-1 and ERK-2.
  • ERK- 1 antibody Provided by John Blenis, Harvard Medical School, Boston, MA
  • Akt kinase activity To assay Akt kinase activity, total cell extracts containing equivalent amounts of protein were pre-cleared with a 1 : 1 mixture of Protein A/Protein G and then incubated with a polyclonal antibody that recognizes the carboxy-terminal end of Akt (provided by Thomas Franke, WHERE FROM) for 3 hours at 4°C. After a 1 hour incubation with the Protein A/Protein G mixture, the beads were washed 3 times with solubilization buffer, once with H 2 0, and once with a 20 mM Hepes buffer pH 7.4 containing 10 mM MgCl 2 and 10 mM MnCl 2 (kinase buffer).
  • the beads were resuspended in 30 ⁇ l of kinase buffer containing 5 ⁇ M ATP, 1 mM DTT, 10 ⁇ Ci g- 32 P-ATP, and 1.5 ⁇ g of Histone H2B (Boehringer Mannheim) and incubated for 20 minutes at room temperature. The reaction was stopped by the addition of 5X Laemmli sample buffer and resolved by electrophoresis on SDS-polyacrylamide gels ( 12%).
  • PKC protein kinase C
  • the constitutively active PI 3-K catalytic pi 10 subunit was a generous gift of Julian Downward, ICRF, London.
  • the small GTP-binding proteins V14Rho, V12Rac, V12Cdc42, N17Rac and N17Cdc42, in the pEBG vector, were a kind gift of Margaret Chou, University of Pennsylvania, Philadelphia, PA.
  • the constitutively active Akt was kindly provided by Philip Tsichlis, Fox Chase Cancer Center, PA.
  • the dominant-negative PI 3-K Dp85 subunit was kindly provided by Brian Schaffhausen, Tufts University.
  • the pCS2-(n) ⁇ -Gal was a gift from Sergei Sokol, Beth Israel Deaconess Medical Center.
  • Cells were co-transfected with 1 ⁇ g pCS2-(n) ⁇ -Gal and the specified cDNAs using Lipofectamine (Gibco) according to the manufacturer's instructions. Cells were harvested 24 hours after transfection and added to
  • the intracellular cAMP concentration was quantified using a cAMP enzyme-linked immunoabsorption assay (cAMP EIA; Cayman Biochemicals, Ann Arbor, MI) following the manufacturer's instructions, using non-acetylated cAMP as a standard and acetylcholine esterase-linked cAMP as a competitor. Values were corrected for cell number as determined from replicate plates. In some experiments, 50 ⁇ M forskolin, 12 mM IBMX or 100 nM LPA was added to cells 15 minutes prior to harvesting. For determination of cAMP content of cells under normal culture conditions, cells were plated in 35mm dishes in DMEM plus 10% FCS, incubated for 18 hrs and processed as described above.
  • cAMP EIA cAMP enzyme-linked immunoabsorption assay
  • the effect of integrin ligation on cAMP levels was determined by treating cells in suspension with 4 ⁇ g/ml antibody for 30 min. and then seeding them on plates (35mm) coated with 50 ⁇ g goat anti- mouse IgG antibody for 15 min. prior to harvesting for analysis of cAMP levels.
  • Phosphodiesterase Assays were performed according to the protocol of Sette et al., J. Biol. Chem. 269:9245-9252, 1994). Briefly, cells were harvested in PDE lysis buffer (20 mM Tris-HCl , pH 8.0, 1 mM EDTA, 0.2 mM EGTA, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 0.5 ⁇ g/ml leupeptin, 0.7 ⁇ g/ml pepstatin, 4 ⁇ g/ml aprotinin, and 2 mM PMSF) and sonicated.
  • PDE cAMP phosphodiesterase
  • PDE activity of cell extracts (2-4 ⁇ g protein) was assayed in cAMP-PDE assay buffer (20 mM Tris- HC1 , pH 8.0, 10 mM MgCl 2 , 1.25 mM ⁇ -mercaptoethanol, 0.14 mg/ml BSA, l ⁇ M cAMP and 0.2 ⁇ Ci [ 3 H]-(cAMP) for 10 min. at 34 °C.
  • the reaction was stopped by adding 40 mM Tris, pH 7.5, containing 10 mM EDTA and heating for 2 min. at 100°C.
  • reaction products were then digested with 50 ⁇ g Crotalus atrox snake venom (Sigma) for 30 min. at 34°C, separated from substrate using SpinZyme Acidic Alumina Devices (Pierce Biochemical), and quantified using a scintillation counter. Values were corrected for protein content and are reported as pmol cAMP hydrolyzed per min. per mg protein. Protein content of cell extracts was determined using the BioRad Protein Reagent with BSA as a protein standard.
  • T84 cells were dissociated with trypsin/EDTA and plated onto the permeable supports (8- ⁇ m pore size) of Transwell chambers (Costar, Cambridge, MA). Growth medium was placed in the upper and lower chambers and the cells were grown in a 37 °C, 5% C0 2 atmosphere until a confluent monolayer was formed. Subsequently, the monolayers were wounded by aspiration through a flame-polished pipette tip. Before immunostaining, some of the samples were treated for 15 minutes with 2 mmol/L EDTA in calcium- and magnesium-free phosphate -buffered saline (PBS) to ensure antibody access to the basal regions of the monolayers.
  • PBS calcium- and magnesium-free phosphate -buffered saline
  • the cells were treated with 1% Triton X-100 in PBS (pH 7.4) for 10 minutes before fixation with paraformaldehyde (4% in PBS for 10 minutes).
  • the permeable supports were cut from the Trans well chambers and incubated in a blocking solution (3% bovine serum albumin and 1% normal donkey serum in PBS) for 1 hour at room temperature.
  • a blocking solution 3% bovine serum albumin and 1% normal donkey serum in PBS
  • the permeable supports were washed in PBS (three times, 10 minutes each) and incubated for 1 hour at room temperature in fluorescein-conjugated goat anti-mouse IgG (1 : 100). All antibodies were diluted in blocking solution.
  • the permeable supports were washed in PBS and mounted in a mixture (8:2) of glycerol and PBS, pH 8.5, containing 1% propylgallate. Slides were examined by confocal imaging (MRC 600, BioRad Microsciences, Cambridge, MA) using a Zeiss Axiovert inverted microscope (Carl Zeiss, Thornwood, NY).
  • the clone A cell line was originally isolated from a human, poorly differentiated colon adenocarcinoma (Dexter, D.L., et al., Cancer Res. 39: 1020- 1025, 1979), and its in vitro properties and repertoire of integrin receptors have been described previously (Daneker, G.W., et al., Cancer Res. 49:681-686, 1989; Lee, E.C., et al., J. Cell Biol. 117:671-678, 1992; Lotz, M.M., et al., Cell Regul. 1 :249-257, 1990; and Tozeren, A., et al., J. Cell Sci. 107:3153-3163, 1994).
  • the CCL-228 colon carcinoma and the MDA-231 breast carcinoma cell lines were obtained from American Type Culture collection (Rockville, MD).
  • the MIP-101 colon carcinoma cell line has been described previously (Deneker, G.W., et al., Cancer Res. 49:681-686, 1989).
  • Cells were grown in RPMI 1640 medium containing 10 mM Hepes, penicillin (50 U/ ⁇ l), streptomycin (50 ⁇ g/ml), and 10% FBS.
  • mouse mAb 287 anti- integrin ⁇
  • rat mAB GoH3 anti-integrin ⁇
  • mouse mAb K20 anti-integrin ⁇ l
  • Steven Akiyama National Institutes of Health, Bethesda MD
  • mouse mAb A9 anti-integrin ⁇ 4
  • Thomas Carey Universal of Michigan, Ann Arbor, MI
  • mouse anti-pan-cytokeratin a mixture of antibodies that recognizes cytokeratins 1, 4, 5, 6, 8, 10, 13, and 19 from Sigma Chemical Co. (St. Louis, MO).
  • bacteriological dishes were coated with 10-100 ⁇ g of laminin-1 prepared from the EHS sarcoma as described (27 NEED REF) or collagen type I (Collaborative Research, Waltham, MA) for 2 h. at room temperature and then blocked with PB5 containing 1% BSA for 1 h.
  • Clone A cells in exponential growth were removed from culture dishes and resuspended in serum-free RPMI 1640 medium containing 10 mM Hepes and 0.1% BSA. The cells were then plated at low density (1 X 10 4 /cm 2 ) on the matrix-coated dishes and allowed to adhere for 30 min. in a humidified atmosphere with 5% C0 2 at 35 °C.
  • integrin-specific antibodies (2B7 or MC-13, 10 ⁇ g/ml) were added to the cells either before the cells were plated or after the cells had adhered for the 30 min..
  • the dishes were then sealed with parafilm and placed on a microscope stage heated to 37°C.
  • an inverted microscope (model Diaphol 300: Nikon, Inc., Melville, NY) with phase contrast optics was used. This microscope was connected to a CCD camera (Dage-MTI, Michigan City, IN), a frame-grabber (Scion), and a 7600 Power Macintosh computer (Cupertino, CA) to capture the images. Images were collected for 1 h. and analyzed with Iplab Spectrum image analysis software.
  • Migration speed was determined by following cell centroid displacements as a function of time for 1 h. at intervals of 15 min. For each individual experiment, 30-40 cells were analyzed. A frame-by- frame analysis of filopodia at intervals of 1 min. over the course of 1 h. was used to differentiate filopodia from retraction fibers and to monitor the formation and stabilization of filopodia. Lamellar area was determined by tracing lamellar contour and quantifying the area digitally.
  • Clone A cells were plated on matrix-coated dishes as described above and incubated for 1 h. in a humidified atmosphere with 5% C0 2 at 37 °C. The cells were then fixed for 20 min. at room temperature with a buffer containing 4% paraformaldehyde, 100 mM KC1 , 300 mM sucrose, 2 mM EGTA, 2 mM MgCl 2 , and 10 mM Pipes at pH 6.8. The cells in some experiments were extracted for 1 min.
  • the fixed cells were rinsed with PBS and incubated with a blocking solution that contained 1% albumin and 5% donkey serum in PBS for 30 min.
  • Primary antibodies GoH3, 1 :50: K20, 1 :50; pan-cytokeratin, 1 :200
  • FITC phalloidin 20 ⁇ g/ml
  • the cells were rinsed three times and either a fluorescein-conjugated donkey anti- mouse or a rhodamine-conjugated donkey anti-rat IgG (minimal cross-reactions interspecies, Jackson ImmunoResearch Labs, West Grove, PA) in blocking buffer (1 : 150) were used separately or in combination to stain the cells for 30 min.
  • Cells were rinsed with PBS and mounted in a mixture (8:2) of glycerol and PBS, pH 8.5, containing 1% propylgallate. The dishes were cut into slides and examined by confocal microscopy (model LSM; Carl Zeiss, Inc., Thorwood, NY).
  • a "low calcium" buffer (25 ⁇ m CaCl 2 , 100 mM KC1, 300 mM sucrose, 10 mM EGTA, 2 mM MgCl 2 , leupeptin (10 ⁇ g/ml), aprotinin (1 ⁇ g/ml), pepstatin (5 ⁇ g/ml), and 10 mM Pipes, pH 6.8) was used to remove the membrane buffer by washing the cells four times with gentle rocking. Subsequently, the low calcium buffer containing 200 nM gelsolin (kindly provided by Dr. Paul Janmey) and 50 ⁇ g/ml of GC-globulin (Calbiochem, La Jolla, CA) was added to the cells and incubated for 30 min.
  • Control cells were treated with the low calcium buffer alone. An equal volume of membrane buffer was added to the cells for 30 s. to terminate the reaction. The buffer was removed and collected in micro fiige tubes, cetrifuged at 12,000 rpm for 10 min. and immunoprecipitated with the 287 (anti- ⁇ 6) antibody. The immune complexes were resolved by SDS-PAGE and immunoblotted with an anti- ⁇ 4 integrin polyclonal antibody elicited against the last 20 amino acids of the ⁇ 4 cytoplasmic tail.
  • EXAMPLE II Expression of the ⁇ 6 ⁇ 4 integrin increases the invasiveness of MDA-MB-435 breast carcinoma cells.
  • the MDA-MB-435 cells used in this study do not express the ⁇ 6 ⁇ 4 integrin although they express the wild-type ⁇ l integrin. Stable subclones of these cells were generated that express either the ⁇ 4 integrin or a mutated ⁇ 6 ⁇ 4 integrin that lacks the ⁇ 4 cytoplasmic domain, with the exception of the four amino acids proximal to the transmembrane domain ( ⁇ 4- ⁇ CYT). Subclones of transfected MDA-MB-435 cells expressing ⁇ 4 on the cell surface were isolated by FACS using UM-A9, a mAb specific for the ⁇ 4 integrin subunit.
  • MDA-MB-435 cells transfected with vector alone (subclones 6D2 and 6D7) or the human ⁇ 4 integrin subunit (subclones 3 A7 and 5B3) were analyzed by flow cytometry using monoclonal antibodies specific for the indicated integrin subunits.
  • the relative surface expression of the ⁇ , ⁇ 4 and ⁇ l subunits on the subclones used in this study is shown in Fig. 1.
  • Expression of the ⁇ 4 subunit did not alter surface expression of the ⁇ subunit (Fig. 1) or other integrin ⁇ subunits on these cells.
  • a slight decrease in ⁇ 1 surface expression was observed in the ⁇ 4 transfectants that probably reflects a decrease in ⁇ l expression in response to ⁇ 6 ⁇ 4 expression (Fig. 1).
  • the rate of invasion of the ⁇ 4 transfectants was approximately 3-4 fold greater than that of the mock transfectants in a 4 hour assay.
  • the ⁇ 4- ⁇ CYT transfectants invaded at a slightly slower rate than that of the mock transfectants (Fig. 2A) indicating that the ⁇ 4 cytoplasmic domain is essential for stimulating invasion.
  • the rate of adhesion to laminin was not greater in the ⁇ 4 transfectants than in the mock transfectants.
  • EXAMPLE III Antibodies specific for the ⁇ 6 ⁇ 4 integrin stimulate invasion of MDA-MB-435 breast carcinoma cells.
  • integrin subunit-specific antibodies were pre-incubated for 30 minutes in the presence of antibodies before addition to the Matrigel-coated wells. After 4 hours at 37 °C, the cells that had not invaded were removed and the cells that had invaded to the lower surface of the filters were fixed, stained, and quantitated as described in experimental procedures.
  • IgG indicates non-specific antibody
  • ⁇ l indicates the anti- ⁇ l mAb 13
  • ⁇ indicates the anti- ⁇ mAb 2B7.
  • the data shown are from two individual subclones of each transfectant and are the mean values (+SEM) of a representative experiment done in triplicate.
  • the ⁇ l -specific antibody mAb 13 inhibited invasion of the mock and ⁇ 4-transfectants.
  • the ⁇ 6-specific mAb 2B7 inhibited invasion of the mock transfectants by approximately 60%, in agreement with our previous result that these cells use ⁇ l as a major laminin receptor.
  • the same antibody increased the rate of invasion of the ⁇ 4 transfectants by approximately 30%.
  • EXAMPLE IV Invasion of MDA-MB-435 breast carcinoma cells is dependent on PI 3-K.
  • MDA-MB-435 transfectants were assayed for their ability to invade Matrigel in the presence of either the MEK inhibitor PD98059 (25 ⁇ M), the PI 3-K inhibitor wortmannin ( 100 ⁇ M), or the p70 S6K inhibitor rapamycin (20 ng/ml).
  • Cells were preincubated for 10 minutes in the presence of the inhibitors before addition to the Matrigel-coated wells. After 4 hours at 37 °C, the cells that had not invaded were removed and the cells that had invaded to the lower surface of the filters were fixed, stained, and quantitated as described in experimental procedures.
  • the data shown are the mean values (+SD) of a representative experiment done in duplicate. "Mock” indicates MDA-MB-435 cells transfected with vector alone, and " ⁇ 4" indicates MDA-MB-435 cells transfected with full length ⁇ 4 subunit.
  • MDA-MB-435 transfectants were maintained in suspension (SUS) or incubated with ⁇ 6-specific antibodies and allowed to adhere to anti-mouse IgG coated plates for 30 minutes in the absence or presence of PD98059 or wortmannin (WT).
  • PI 3-K As a mediator of invasion because of its central involvement in multiple signaling pathways.
  • the PI 3-K inhibitor wortmannin (WT) inhibited invasion of both the mock and ⁇ 4 transfectants by 70-80% (Fig. 3A).
  • WT did not inhibit activation of MAPK by antibody-induced clustering of the ⁇ integrins in either the mock or ⁇ 4 transfectants (Fig. 3B).
  • EXAMPLE V Activation of PI 3-K by the ⁇ 6 ⁇ 4 integrin.
  • MDA-MB-435 breast carcinoma cells Fig. 4A
  • MDA-MB-435 transfectants were maintained in suspension or incubated with integrin-specific antibodies and allowed to adhere to anti-mouse IgG-coated plates or laminin- 1 coated plates for 30 minutes.
  • SUS indicates cells maintained in suspension
  • ⁇ indicates cells clustered with the ⁇ 6- specific antibody.
  • PI 3-K activity As shown in Fig. 4A, an increase in PI 3-K activity, indicated by the appearance of PtdIns-3,4,5-P3, was observed upon clustering the ⁇ l integrin in the mock transfectants and the ⁇ 6 ⁇ 4 integrin in the ⁇ 4 transfectants. More importantly, PI 3-K activity stimulated by clustering the ⁇ 4 integrin was markedly greater than that observed after clustering the ⁇ l receptor. This enhanced stimulation of PI 3-K was also seen using a ⁇ 4-specific mAb to ligate the ⁇ 6 ⁇ 4 integrin in the ⁇ 4 transfectants (Fig. 4B).
  • PI 3-K activity was higher in the ⁇ 4 transfectants ( ⁇ 4) than in the mock transfectants ( ⁇ l) after adhesion to laminin- 1 (Fig. 4A). This observation suggests that interactions with laminin through this receptor can stimulate PI 3-K activity even though ⁇ 4 is not used as an adhesion receptor in these cells. PI 3-K activity was not increased upon ligation of the ⁇ 4- ⁇ CYT receptor (Fig. 4B) and little PI 3-K activity was evident when the transfectants were maintained in suspension (Fig. 4A). Our data suggested that the ability of the ⁇ 4 integrin to activate PI
  • 3-K may be quantitatively greater than that of ⁇ l integrins in MDA-MB-435 cells. This possibility was examined by comparing PI 3-K activation in the ⁇ 4 transfectants in response to antibody ligation of either ⁇ l integrins or the ⁇ 6 ⁇ 4-integrin.
  • MDA-MB-435 cells were transfected with the full length ⁇ 4 subunit (cell clones 3A7 and 5B3), or the ⁇ 4 subunit lacking the cytoplasmic domain ( ⁇ 4- ⁇ CYT), and the cells were clustered with either the ⁇ l -specific antibody or the ⁇ 4-specific antibody.
  • Fig. 4B shows a densitometric analysis of the amount of radiolabeled PtdIns-3,4,5-P3 generated by the antibody-treated ⁇ 4 (3A7 and 5B3) and ⁇ 4- ⁇ CYT integrin transfectants.
  • the data shown are the mean values (+SD) from two representative experiments.
  • the results shown in Fig. 4B indicate that ligation of the ⁇ 4 integrin with ⁇ 4-specific antibodies stimulated PI 3-K activity approximately two-fold more than did ⁇ 1 integrin ligation, demonstrating that PI 3-K is activated preferentially by the ⁇ 6 ⁇ 4 integrin.
  • EXAMPLE VI Constitutively active PI-3K stimulates invasion of MDA-MB-435 breast carcinoma cells.
  • MDA-MB-435 cells were transiently transfected with 1 ⁇ g pCS2-(n) ⁇ -gal as an internal control for transfection efficiency and 4 ⁇ g of either the vector alone or a Myc-tagged, constitutively active form of the PI 3-K pi 10 catalytic subunit (Myr-pl 10) and assayed for their ability to invade Matrigel in the absence or presence of wortmannin (100 nM). Expression of the transiently expressed pi 10 subunit was confirmed by immunoblotting using a myc-specific antibody. The data shown are the mean values (+SD) of two
  • MDA-MB-435/ ⁇ 4 transfectants were transiently transfected with 1 ⁇ g pCS2-(n) ⁇ -gal and either the vector alone, 6 ⁇ g of a PI 3-K p85 subunit deleted in the pi 10 binding site ( ⁇ p85), or 6 ⁇ g of a wild-type PI 3-K p85 regulatory subunit (p85) and assayed for their ability to invade Matrigel (Fig. 5B).
  • EXAMPLE VIII The Akt/PKB kinase and p70 S6 kinase. downstream effectors of PI-3K. are not required for invasion.
  • Akt/PKB serine/threonine kinase (Akt) and the p70 S6 kinase (S6K) are activated downstream of PI 3-K, we hypothesized that these kinases could play important roles in invasion.
  • MDA-MB-435/ ⁇ 4 transfectants were maintained in suspension or incubated with ⁇ -specific antibodies and allowed to adhere to anti-mouse IgG coated plates for 30 minutes.
  • Cell extracts made from these samples were incubated with a polyclonal anti-Akt antibody and a 1 : 1 mixture of Protein A/Protein G, and the bead-coupled protein was subject to a kinase assay.
  • the phosphorylated substrate, histone H2B is shown in Fig. 6A.
  • rapamycin a specific inhibitor of S6K activation (Chung et al., Cell 69:1227-36, 1992; Kuo et al., Nature 358:70-3, 1992; Price et al., Science 251:913-1, 1992). As shown in Fig. 3, rapamycin did not decrease the invasion of either the mock or ⁇ 4 transfectants. Based on these results, we conclude that Akt and S6K are not required for MDA-MB-435 invasion.
  • EXAMPLE IX The small G-protein Rac is required for MDA-MB-435 invasion.
  • Rho family of small G-proteins is involved in the actin rearrangements that result in the formation of stress fibers, membrane ruffles and lamellae, and filopodia (Nobes and Hall, Cell 81 :53-62, 1995).
  • the ability of cells to form these actin-containing structures is linked to their motility and therefore could influence their invasive potential (Rabinovitz and Mercurio, Journal of Cell Biology (In Press), 1997; Sheetz, Seminars in Cell Biology 5:149-55, 1994).
  • constitutively active mutants of either Rho (V14Rho), Rac (V12Rac), or Cdc42 (V12Cdc42) were transiently expressed in the parental MDA-MB-435 cells.
  • MDA-MB-435 cells were transiently transfected with 1 ⁇ g of pCS2-(n) ⁇ -gal and either 4 ⁇ g of the vector alone, or 4 ⁇ g of constitutively active mutants of Akt (Myr-Akt), Rac (V12Rac), Cdc42 (V12Cdc42), or Rho (V14Rho) and assayed for their ability to invade Matrigel. After 5 hours at 37 °C, the cells that had not invaded were removed and the cells that had invaded to the lower surface of the filters were fixed and stained. Invasion was quantitated by counting the cells that stained positive for ⁇ -galactosidase expression.
  • Relative invasion was determined by comparing the amount of invasion obtained for the experimental transfections to that observed for the cells transfected with the vector alone, which was given the value of 1.
  • the data shown are the mean values (+SD) of 3 experiments done in triplicate.
  • independent expression of these constitutively active small G-proteins did not significantly alter the invasion of MDA-MB-435 cells indicating that they are not sufficient by themselves to increase invasion.
  • MDA-MB-435/ ⁇ 4 transfectants were transiently transfected with 1 ⁇ g of pCS2-(n) ⁇ -gal and either 4 ⁇ g of vector alone, or 4 ⁇ g of dominant- negative mutants of Rac (GST-N17Rac) or Cdc42 (N17Cdc42) and assayed for their ability to invade Matrigel as described above.
  • the data shown are the mean values (+SD) of a representative experiment done in triplicate. A significant reduction (50%) in invasion was observed when dominant-negative N17Rac was transiently expressed in the MDA-MB-435/ ⁇ 4 transfectants. In contrast, expression of dominant-negative N 17Cdc42 did not inhibit invasion significantly (Fig. 6C).
  • MDA-MB-435 cells were transiently transfected with 1 ⁇ g of pCS2-(n) ⁇ -gal and either 5 ⁇ g of the vector alone, 3 ⁇ g of the vector alone and 2 ⁇ g of a Myc-tagged constitutively active PI 3 -K pi 10 subunit (Myr-pl 10) or 2 ⁇ g of Myr-pl 10 and 3 ⁇ g N17Rac, and assayed for their ability to invade Matrigel as described above.
  • Myr-pl 10 Myr-pl 10
  • EXAMPLE X Involvement of PI 3-K in the ⁇ 6 ⁇ 4-dependent migration of invasive colon carcinoma cells.
  • clone A cells an invasive colon carcinoma cell line that we have characterized extensively.
  • Clone A cells express relatively high levels of the ⁇ 4 integrin and no ⁇ l integrin (Lee et al., J. Cell Biol. 1 17:671-8, 1992).
  • these cells use the ⁇ 4 integrin as an adhesion receptor for laminin- 1 (Lee et al., J. Cell Biol. 117:671-8, 1992; Lotz et al., Cell Regul. 1 :249-57, 1990), in contrast to the MDA-MB-435/ ⁇ 4 transfectants.
  • Fig. 8B shows a densitometric analysis of the amount of radiolabeled PtdIns-3,4,5-P3 for each experimental condition.
  • the adhesion-dependent levels of PtdIns-3,4,5-P3 were compared to the level observed for the cells that were maintained in suspension which was given the value of 1.
  • the value from this ratio was determined to be the relative PI 3-K activity stimulated by adhesion.
  • the data shown are the mean values (+SEM) from 2 experiments.
  • PI 3-K activity was increased after attachment to laminin- 1 but this increase was inhibited when the ⁇ 6 ⁇ 4 receptor was blocked by the ⁇ antibody.
  • ⁇ 6 ⁇ 4 is required for PI 3-K activation and formation of lamellae in response to laminin- 1 attachment in clone A colon carcinoma cells, functions that are required for the invasion of these cells.
  • Calphostin C a specific inhibitor of the PKC family, could inhibit carcinoma cell motility in the Matrigel invasion assay. As shown in Figure 9A, Calphostin C inhibits the invasion of both the mock (6D2) and ⁇ 4 (5B3) MDA-MB-435 breast carcinoma transfectants.
  • EXAMPLE XII ⁇ 6 ⁇ 4 integrin-dependent motility involves increased cAMP phosphodiesterase activity.
  • ⁇ 4 integrin-transfected MDA-MB-435 cells exhibited higher cAMP phosphodiesterase activity and lower cAMP levels than non- transfected cells.
  • ⁇ 4 integrin-enhanced motility was blocked by the phosphodiesterase inhibitors IBMX and rolipram, and by forskolin.
  • Fig. 10 shows the cAMP phosphodiesterase activity of MDA-MB-
  • MDA-MB-435 cells transfected with either full length ⁇ 4 integrin subunit (3A7, 5B3) or vector alone (6D7) in suspension or plated on laminin- 1 or collagen I were treated for 15 min. with 50 ⁇ M Forskolin or 100 nM LPA as noted. Cells were harvested and the cytosolic fraction was assayed for PDE activity as described in Example I. Bars represent standard error of at least 4 separate determinations. Phosphodiesterase activity is highest in ⁇ 4 integrin- expressing clones.
  • Fig. 11 shows that the phosphodiesterase inhibitor, IBMX, decreases LPA-stimulated chemotaxis of MDA-MB-435 cells.
  • Cells suspensions of MDA-MB-435 subclone 5B3 ( ⁇ 4 transfected; squares) or 6D7 (mock transfected; circles) were treated with the indicated concentration of IBMX for 30 min. prior to use in LPA chemotaxis assay as described in Example I. Bars represent standard deviation of triplicate determinations.
  • EXAMPLE XIII ⁇ 6 ⁇ 4 integrin functions in carcinoma cell migration by mediating the formation and stabilization of actin-containing mobility structures.
  • Clone A colon carcinoma cells which express ⁇ 4 integrin, but not ⁇ l integrin, exhibit dynamic adhesion and motility on laminin- 1. Their migration is characterized by filopodial extension and stabilization, followed by lamellae that extend in the direction of stabilized filopodia.
  • a functional blocking antibody for ⁇ 6 ⁇ 4 integrin inhibited clone A migration on laminin- 1, as well as inhibiting filopodial formation and stabilization and lamella formation, leading to the unexpected conclusion that ⁇ 6 ⁇ 4 integrin must be a component of the clone A cell motility apparatus.
  • Figure 12 shows that ⁇ 6 ⁇ 4 integrin co-localizes with F-actin in filopodia of clone A cells on laminin- 1.
  • Cells plated on either laminin- 1 (Fig. 12 A-D and F) or collagen I (Fig. 12E) at 35 °C for 1 h. were processed for double immunofluorescence using the rat GoH3 mAb followed by a rhodamine-conjugated anti-rat antibody and FITC-conjugated phalloidin.
  • the confocal images shown represent optical sections of the ventral surface of the cells (Fig. 12A and 12C) GoH3; (Figs. 12B-12F, Phalloidin).
  • FIG. 12A and 12B demonstrate co-localization of ⁇ 4 and F-actin in a group of filopodia.
  • Fig. 12D shows the formation of actin cables on the top lamella that project into filopodia. These filopodia are enriched in ⁇ 6 ⁇ 4 (Fig. 12C).
  • Fig. 12E shows the presence of polygonal actin cables in clone A cells plated on collagen I. In Fig. 12F, the cells were incubated with 2B7 antibody for 30 min. before fixation. Note the disappearance of actin cables (remaining protrusions are presumably retraction fibers). The bar shown at the bottom of Fig. 13F represents 10 ⁇ m.
  • ⁇ 4 integrin did not co-localize with cytokeratins in filopodia and distal sites of lamellae. Instead, cytokeratin staining was concentrated largely in the cell body and in proximal portions of lamellae.
  • ⁇ 4 did not co-localize with cytokeratins in filopodia and distal sites of many of the lamellae either in unextracted cells or after the Triton X-100 buffer extraction. These results suggest that ⁇ 6 ⁇ 4 is retained at the cell edges because of its association with actin and not with cytokeratins. In agreement with this possibility, these marginal areas of actin-associated ⁇ 4 integrin were removed by the Tween/deoxycholate buffer.
  • MIP-101 (Fig. 15A and 15B), MDA-MB-231 (Fig. 15C and 15D), and CCL-228 (Fig. 15E) carcinoma cells were analyzed by double immunostaining with the ⁇ 4-specific A9 antibody (Fig. 15 A, 15C and 15D) and FITC-phalloidin (Fig. 15B and 15C).
  • Fig. 15A, 15C and 15D MIP-101
  • Fig. 15C and 15D MDA-MB-231
  • CCL-228 Fig. 15E carcinoma cells were analyzed by double immunostaining with the ⁇ 4-specific A9 antibody (Fig. 15 A, 15C and 15D) and FITC-phalloidin (Fig. 15B and 15C).
  • concentration of ⁇ 4 in the actin nodes present in the filopodia of MIP-101 cells (Fig. 15A and 15B, arrowheads) and the distribution of ⁇ 4 in filopodia, retraction fibers, and lamellae of MDA-231 and CCL-2
  • an ⁇ 6-specific antibody inhibits formation of lamellae in CCL-228 cells.
  • Cells were plated on laminin- 1 in the presence or absence of 2B7 for 1 h. The cells were photographed, and their lamellar area ( ⁇ m 2 /cell) was determined by digital image analysis. Fifty cells were analyzed for each condition.
  • the bar in panel 15A represents 20 ⁇ m.
  • the error bar in Fig. 15F represents SEM.
  • EXAMPLE XIV Wound-induced motilitv of T84 colon carcinoma cells is dependent upon PI 3-K.
  • Disruptions in the mucosal lining of the gastrointestinal tract reseal by a process termed restitution This mode of wound healing results in the repair of superficial wounding in the mucosa by the process cell migration. More specifically, epithelial cells from the edges of wounds are thought to migrate as a sheet into the wound. When the cells are within the wound area, they rapidly reform cell contacts and re-establish barrier functions. Although the mechanism of intestinal epithelial restitution is poorly understood, it likely involves the altered regulation and expression of molecules involved in cell migration. Such molecules include cytoskeletal proteins, adhesion receptors, and extracellular matrix glycoproteins. Human T84 colon carcinoma cells provide a good model for the study of the biological processes, e.g., cell migration, that occur during wound healing.
  • Fig. 16A-16F are photomicrographs showing that lamellae extension during T84 cell wound healing is wortmannin-sensitive.
  • Fig. 16A is at time zero.
  • a wound is made in a confluent monolayer of T84 cells, a well- differentiated, polarized human colon carcinoma cell line. The wound was made by aspiration using a narrow gauge Pasteur pipet tip, and is 7577 ⁇ m 2 , equaling approximately 150 cells. These wounds heal by a process similar to that observed when rabbit stomach mucosa is damaged. Cells around the wound flatten and then extend lamellae, large thin protrusions involved in cell migration.
  • Fig. 16B is at 50 minutes post- wounding.
  • wortmannin an inhibitor of PI3 kinase
  • the wound is 2802 ⁇ m 2 in diameter.
  • the wound has sealed to 63% of its original diameter.
  • Fig. 16C is at 54 minutes post- wounding.
  • Fig. 16D is at 62 minutes post- wounding.
  • Lamellae continue to collapse, and the wound contains a prominent, thickened edge of a retracting lamellae (lower edge, approximately 6:00).
  • Fig. 16E is at 68 minutes post- wounding.

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Abstract

L'invention concerne un procédé pour moduler la motilité et/ou le pouvoir envahissant des cellules par la régulation des interactions entre l'intégrine α6β4 et l'actine, et par la régulation de l'activité biologique des molécules de signalisation comprenant une voie de signalisation phosphoïnositide 3-hydroxyl kinase (PI 3-K) telle que PI 3-K et par la régulation de l'activité biologique de la protéine kinase C (PKC) et de la phosphodiestérase cAMP. L'invention concerne également des procédés destinés à isoler des composés qui modulent le pouvoir envahissant des cellules par la régulation de l'activité biologique des interactions entre l'intégrine α6β4 et l'actine ainsi que des voies de signalisation PI 3-K, PKC et de la phosphodiestérase cAMP.
PCT/US1998/026720 1997-12-15 1998-12-15 Procedes et reactifs destines a moduler la motilite des cellules Ceased WO1999035283A1 (fr)

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JP2011140505A (ja) * 2000-07-31 2011-07-21 Univ Bar Ilan 創傷治療のための方法および薬学的組成物
EP1383540A4 (fr) * 2000-07-31 2004-06-30 Univ Bar Ilan Methodes et compositions pharmaceutiques de cicatrisation de lesions
US7402571B2 (en) 2000-07-31 2008-07-22 Bar-Ilan University Methods and pharmaceutical compositions for healing wounds
US8093211B2 (en) 2000-07-31 2012-01-10 Bar-Ilan University Methods and pharmaceutical compositions for healing wounds
WO2002058679A3 (fr) * 2001-01-25 2003-05-15 Univ British Columbia Composes anti-angiogeniques et dosage pour inhibiteurs d'invasion cellulaire
EP2526952A1 (fr) * 2003-07-15 2012-11-28 Bar-Ilan University Procédés et compositions pharmaceutiques servant à guérir des blessures
EP1648474A4 (fr) * 2003-07-15 2009-03-25 Univ Bar Ilan Methodes et compositions pharmaceutiques pour cicatriser des lesions
WO2005007072A2 (fr) 2003-07-15 2005-01-27 Bar-Ilan University Methodes et compositions pharmaceutiques pour cicatriser des lesions
EP2526953A1 (fr) * 2003-07-15 2012-11-28 Bar-Ilan University Procédés et compositions pharmaceutiques servant à guérir des blessures
EP2540302A1 (fr) * 2003-07-15 2013-01-02 Bar-Ilan University Procédés et compositions pharmaceutiques servant à guérir des blessures
EP2540301A3 (fr) * 2003-07-15 2013-04-10 Bar-Ilan University Procédés et compositions pharmaceutiques servant à guérir des blessures
EP1875906B1 (fr) * 2003-07-25 2011-06-29 Laboratorio Cifga, S.A. Utilisation thérapeutique de yessotaxine en tant qu'inhibiteur de croissance de cellule de tumeur humaine
US7638484B2 (en) 2003-08-07 2009-12-29 Healor Ltd. Methods for accelerating wound healing by administration of adipokines

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